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Open Access Online Scientific Journal Site Statistics

Date	Views4/2012 – Present	# of articles	NIH Clicks	Nature Clicks
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Author Name	# articles	Views
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Stephen J Williams, PhD	0063	16,350
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Home page / Archives		175,938
Is the Warburg Effect the Cause or the Effect of Cancer: A 21st Century View? – Larry H Bernstein, MD		8,488
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Mesothelin: An early detection biomarker for cancer (By Jack Andraka) Tilda Barliya, PhD		3,249
Do Novel Anticoagulants Affect the PT/INR? The Cases of XARELTO (rivaroxaban) and PRADAXA (dabigatran) Aviva Lev-Ari, PhD, RN		3,130
Volume One: Perspectives on Nitric Oxide in Disease Mechanisms http://www.amazon.com/dp/B00DINFFYC		2,967
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AMPK Is a Negative Regulator of the Warburg Effect and Suppresses Tumor Growth In Vivo Stephen J Williams, PhD		2,278
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The mechanism of action of the drug ‘Acthar’ for Systemic Lupus Erythematosus (SLE)		1,989
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Problems of vegetarianism Sudipta Saha, PhD		1,883
Introduction to Transdermal Drug Delivery (TDD) system and nanotechnology Tilda Barliya, PhD		1,790
“The Molecular pathology of Breast Cancer Progression” Tilda Barliya, PhD		1,666
Confined Indolamine 2, 3 dioxygenase (IDO) Controls the Hemeostasis of Immune Responses for Good and Bad Demet Sag, PhD		1,543
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Innervation of Heart and Heart Rate

Posted in Ca2+ triggered activation, Calcium Signaling, Calmodulin Kinase and Contraction, Cardiovascular Research, Curation, Cytoskeleton, Diabetes Mellitus, Disease Biology, Electrophysiology, Frontiers in Cardiology and Cardiovascular Disorders, HTN, Innovations in Neurophysiology & Neuropsychology, Metabolism, Small Molecules in Development of Therapeutic Drugs, tagged cardiopulmonary physiology, HCN channels, Heart rate, Hypertension, Physical exercise, regulation of heart rhythm, sinoatrial node, treadmill test on February 15, 2015| Leave a Comment »

Innervation of Heart and Heart Rate

Writer and Curator: Larry H Bernstein, MD, FCAP

The heart is a four-chambered 350 gm semi-oval muscular organ composed of syncytial myocardium, innervated by the vagus nerve with a sino-atrial (SA) and a atrial ventricular (AV) node. The blood circulates through it by way of the pulmonary artery and aorta, carrying blood away from the ventricles, to the lungs and the systemic circulation, respectively, and two veins, the vena cava and pulmonary, carrying blood to the atria from the systemic circulation and lungs, respectively. The coronary arterial supply is the left anterior and left circumflex artery, and posteriorly, the right coronary artery, supplied by the aorta. Much of the pathology has been referred to in the introduction, except for the molecular pathology of atherosclerosis, which has been well covered in this journal. The chambers are divided centrally by the interventricular septum, which is not completely closed in the blue-baby syndrome, which was repaired surgically by Helen Taussig and Richard Bing. The piece that follows is primarily directed to the sympathetic innervation of the heart, variation in heart rate, and exercise or reaction to external threats.

What are the common observable events that stimulate or relax the heart:

Running or a treadmill test
Rowing or arm movement exercise
A whole body workout
Yoga or Ayurveda
Sleep – normal or disruptive

Some things that can cause a disruption of balance in integrated circulation, neural innervation, innate immune and hormonal response are:

Traumatic experience and/or Injuries
Climate and seasonal changes
Age
Emotions

The basis for the physiological distress has long been the primary basis for acupuncture, holistic and transcendental medicine, and stress management.

I shall here examine the experimental work that supports such an approach – in principle.

Seattle Heart Watch: Initial Clinical, Circulatory and Electrocardiographic Responses to Maximal Exercise

Robert A Bruce, G0 Gey, Jr., Mn Cooper, Ld Fisher, Dr Peterson
Amer J Cardiol 1974; 33(4): 459-469.

A network of 15 maximal exercise testing facilities in four teaching hospitals, 10 private offices and clinics and an industrial medical department was organized in July 1971 to study prospectively the antecedents of myocardial infarction and sudden cardiac death. Within 18 months 2,332 men were tested: 1,275 healthy “normal” subjects, 97 with prior myocardial infarction, 306 with angina pectoris, 193 with hypertension and 461 with various mutually exclusive combinations of these diagnoses; among these clinical groups were five patients who had had a prior episode of ventricular fibrillation.
Historical, physical and laboratory data were recorded on self-teaching printed forms, with normal, borderline and abnormal responses arranged in three columns. Classification with respect to “unlikely,” “questionable” or “likely” risk of future cardiac events was assessed from the highest tally of items in these columns.
Analysis showed computer-averaged S-T segment responses were more consistent and reliable predictors than visual interpretations. Cardiac manifestations in healthy men varled with age and risk assessment, and in patients with cardiovascular disease varied with diagnosis and natural history of disease. Many significant differences provided insights into mechanisms of impaired cardiac function in relation to type of clinical disease. Testing was responsible for one post-exertional cardiac arrest. Recovery was effected promptly by defibrillation; there was no mortality.

Normal and Abnormal Heart Rate Responses to Exercise

Kirk Hammond and Victor F. Froelicher
Prog Cardiovasc Dis 1985; XXVII(4) (January/February), pp 27l-296

Of the many factors ultimately important in determining the cardiac output, the heart rate is certainly the easiest to measure. By analysis of the heart rate response to exercise in a variety of disease states we felt that the interrelationships of inotropic state, stroke volume, autonomic dysfunction, and myocardial disease could be clarified. This paper reviews the normal and abnormal heart rate responses to exercise.

The normal heart rate is determined by the frequency of depolarization of specialized cells within the sino-atrial node (S-A node). The S-A node, the vestigal sinus venosus, lies in the posterior portion of the heart near the demarcation between the right atrium and the superior vena cava. In about 80% of humans it receives its primary source of blood from a branch of the right coronary artery. Unlike other myocardial cells, the specialized cells of the S-A node have a slow sodium channel and a low resting potential which give these cells their special property. The slowly rising diastolic depolarization (stage four) leads to a rhythmic slow rising action potential.

The autonomic nervous system plays a key role in the regulation of heart rate (Fig 1). The sympathetic nervous system input to the heart originates in a nucleus in the medulla oblongata. Stimulation of this area with implanted electrodes results in increased heart rate and systemic vascular resistance due to increased sympathetic output. Axons from these nuclei descend to the sympathetic trunk via the intermediolateral columns of the spinal cord. From their synapses in cervical ganglia, postganglionic fibers directly innervate the atrial and ventricular musculature, the S-A node, and the A-V node. The effector neurotransmitter is norepinephrine and the receptors are of the beta adrenergic type. There is evidence from competitive binding studies that the postganglionic fibers are predominantly associated with type I beta receptors. The parasympathetic influence to the S-A node and the myocardium originates from nuclei very near the origin of the sympathetic nerves. From the motor nuclei of the vagus and the nucleus solitarius come fibers that form part of the vagus nerve. These fibers terminate at ganglia in the wall of the heart. The postganglionic cholinergic fibers end mostly near the S-A node and the A-V node; there is little evidence for the distribution of parasympathetic nerves to the ventricular myocardium although cholinergic muscarinic receptors have been characterized. In normal conditions there exists a well balanced autonomic tone influencing the S-A node.

There is a complex interrelation among many systems to determine the autonomic tone at the S-A node (Fig 2). [Arterial mechanoreceptors of the carotid sinus and aortic arch respond to changes in arterial pressure and result in appropriate adjustment in the sympathetic and vagal outflow to the heart and resistance and capacitance vessels. (Reprinted with permission from Shepherd JT, Van Houlte PM: The Human Cardiovascular System, Facts and Concepts. New York, Raven Press, 1979).]

There are cortical inputs to the medullary centers; for example, fear results in tachycardia by this pathway. Visceral afferent inputs increase parasympathetic tone resulting in bradycardia. Several reflexes are present for homeostasis. For example, the baroreflex is important in sensing changes in blood pressure and increasing or decreasing the heart rate via autonomic influences at the S-A node to maintain appropriate cardiac output.

Arterial mechanoreceptors of the carotid sinus and aortic arch respond to changes in arterial pressure and result in appropriate adjustment in the sympathetic and vagal outflow to the heart and resistance and capacitance vessels. (Reprinted with permission from Shepherd JT, Van Houlte PM: The Human Cardiovascular System, Facts and Concepts. New York, Raven Press, 1979).

Although the importance of autonomic influence is well accepted in the usual cardioacceleration to exercise, the role of the recovery or deceleration of heart rate following exercise may not be influenced by autonomic input. Six men were studied after peak treadmill exercise. To assess the contribution of autonomic factors in heart rate recovery, the men were given atropine, propranolol, or both agents. It was found that exponential cardio-deceleration occurred under each experimental condition. They concluded that heart rate recovery after exercise is regulated by changes in venous return mediated through atrial stretch receptors of pacemaker tissue. This study implies that deceleration depends primarily on factors intrinsic to the intact circulation that are independent of autonomic control.

The control of heart rate is complex; autonomic tone, central and peripheral reflexes, hormonal influences, and factors intrinsic to the heart are all important. Although easily measured, the heart rate reflects an integrated physiologic response.

The physiologic response to exercise depends on the type of exercise performed; the two major types are isometric and isotonic. Creating muscle tension with no movement against resistance is a pure form of isometric exercise; this results in increased muscle mass and strength. Isotonic exercise is the repetitive, rhythmic movement of large muscle masses against little resistance, known also as dynamic or aerobic exercise. Although most activities involve degrees of both, running is predominantly dynamic, and weight lifting is predominantly isometric.

Bezucha and colleagues investigated the cardiovascular responses to isometric (static) exercise (leg extension) and compared these to those observed during static-dynamic exercise (one arm cranking) and dynamic exercise (leg cycling) in normal men. Heart rate responses to these three tasks were markedly different with static exercise (holding a 30% of maximum voluntary contraction for 3 minutes) resulting in a mean heart rate of 110 + 6 compared with 164 + 4 beats/min in bicycle exercise at 80% of V_o max. Cardiac outputs were raised in all three activities in a proportional manner: 6.8 + 0.7 for static, 10.8 f 0.7 for arm cranking, and 31.9 + 1.0 L/min for bicycling. Stroke volume did not significantly change in the static or combined static-dynamic exercises. The increases in cardiac output were primarily the result of increases in heart rate. This study demonstrates the predominant pressor response and modest cardio-acceleration of isometric exercise.

Longhurst and coworkers, examined the response to acute and chronic exercise in two groups of athletes who typify the two major types of exercise: long distance runners (dynamic) and weight lifters (isometric). The runners responded to isometric exercise with lower double products than the weight lifters. The end-diastolic volume index (evaluated by echocardiography) in the runners was greater than control subjects both at rest and with exercise. In contrast, the weight lifters’ responses were similar to weight matched controls. Not only is the type of exercise an important determinant of acute physiologic response, but chronic static exercise results in physiologic responses that are no different from the responses of sedentary men.

Dynamic exercise, also called isotonic or aerobic, involves the rapid movement of large muscle masses that results in the need for the body to respond with increased ventilation to increase oxygen consumption. Such exercise is called aerobic since it must be performed by using oxygen. The heart must increase its output and performs flow work rather than pressure work. The response to dynamic muscular exercise consists of a complex series of cardiovascular adjustments designed to:

(1) see that active muscles receive a blood supply appropriate to their metabolic needs;

(2) dissipate the heat generated by active muscles; and,

(3) maintain the blood supply to the brain and the heart.

The regulation of the circulation during exercise involves the four following adaptations?

Local
Nervous adaptations
Humoral adaptations
Mechanical adaptations

The relationship of pressure, flow, and resistance in rigid tubes is defined by Poiseuille’s law. This law states that resistance is proportional to pressure divided by flow. Peripheral resistance increases in the tissues that do not function in the performance of the ongoing exercise and decreases in active muscle. The result is a decrease in systemic vascular resistance. While pressure only increases mildly, flow can increase by as much as five times during dynamic exercise. Since flow increases much more than pressure, the result is a decrease in systemic resistance. Another mechanical adaptation occurs when the increasing venous return dilates the left ventricle and cardiac function is enhanced via the Frank-Starling mechanism.

There is a highly predictable relationship between total body oxygen consumption and both the cardiovascular and respiratory responses to exercise (Fig 4). [ (A) The linear relationship between heart rate and oxygen uptake. The data was collected from 86 adult male and female subjects. (B) The linear relationship between cardiac output and oxygen uptake. C The data was collected from 23 adult male and female subjects. (C) The linear relationship between minute ventilation and oxygen uptake. ] The data was collected from 225 subjects. (Reprinted with permission.) Both parameters increase linearly with increasing oxygen consumption until maximal oxygen consumption is approached.

In summary, the type of exercise is an important determinant of both acute and chronic cardiovascular responses. Isometric exercise can be viewed as a pressure load and dynamic exercise as a volume load to the left ventricle. The acute physiological adjustments to dynamic exercise include peripheral vasodilation in exercising muscle, neural mediated increases in sympathetic tone to the heart and the periphery, the release of catecholamines from the adrenal medulla, and changes in venous return due to mechanical and humoral factors. A linear relationship exists between the consumption of oxygen and cardiac output and minute ventilation such that the work performed is highly correlated with the amount of blood pumped and the oxygen consumed.

An increase in heart rate is a major factor contributing to the exercise-induced increased cardiac output. Bowditch demonstrated that the time interval between beats is a determinant of the force of myocardial contraction. This has been called the frequency-force relationship (Fig 5). [The frequency force relationship is demonstrated by a sudden increase in beat frequency in papillary muscle fixed for isometric contraction. A slow increase in isometric tension results from the change in rate implying in increased contractile state. Each vertical line represents an isometric contraction. (Reprinted with permission of W.B. Saunders.)] The increased tension that accompanies an increased heart rate is the result of increased contractility. Although the mechanism of this phenomenon is not known, it may have to do with calcium availability to contractile elements. Thus an increase in heart rate results in an increase in the force of contraction.

Variations in and Significance of Systolic Pressure During Maximal Exercise (Treadmill) Testing: Relation to Severity of Coronary Artery Disease and Cardiac Mortality

John B. Irving, Robert A. Bruce,, Timothy A. Derouen
Amer J Cardiol 1977; 39: 841-848.

Variations in clinical noninvasive systolic pressure at the point of symptom-limited exercise on a treadmill were examined in six groups of subjects: 5,459 men and 749 women classified into three categories each. Among the men, 2,532 were asymptomatic healthy, 592 were hypertensive and 1,586 had clinical manifestations of coronary heart disease (that is, typical angina pectoris, prior myocardial Infarction or sudden cardiac arrest with resuscitation). Among the women, 244, 158 and 347 were in the corresponding clinical categories. None had had cardiac surgery; all had follow-up status ascertained by periodic mail questionnaires.
Reported deaths were reviewed and classified by three cardiologists; 140 deaths were attributed to coronary heart disease, 118 of them in the men classified as having coronary heart disease. The majority of maximal systolic blood pressure readings were reported to the nearest centimeter rather than millimeter of pressure. Retesting of 156 persons from 1 to 32 months later showed that pressure values agreed within 10 percent in two thirds, the overall mean difference was only 8.6 mm Hg and the correlation at maximal exercise was superior to that of the resting observations just before exercise. Hypertensive patients had a significantly greater body weight than normotensive persons. Among men, the lowest maximal systolic pressure was observed in the group with coronary heart disease; among women, the lowest mean pressure was found in the healthy group. Patients with coronary heart disease were slightly older, and only the women showed a significant correlation in maximal pressure with age. Only 5 percent of the variation in maximal systolic pressure in the patients with coronary heart disease was due to a shortened duration of exercise. Maximal systolic pressures correlated fairly well (r = 0.46 to 0.68 for the various groups) with resting systolic pressure, and this relation was independent of the diagnosis of cardiovascular disease in both men and women. Relations between pressure and the number of stenotic coronary arteries and Impaired ejection fraction at rest were examined in 22 men without and 162 men with coronary artery disease. Lower maximal systolic pressures were often associated with two or three vessel disease or reduced ejection fraction, or both.

The prognostic value of maximal systolic pressure for subsequent death due to coronary heart disease was examined in the men with coronary heart disease. The annual rate of sudden cardiac death decreased from 97.9 per 1,000 men to 25.3 and 6.6 per 1,000 men as the range of maximal systolic pressure increased from less than 140 to 140 to 199 and to 200 mm Hg or more, respectively. Cardiomegaly, Q waves in the resting electrocardiogram and persistent postexertional S-T depression were more common in men with the lowest systolic pressure at maximal exercise.

Circulatory Adjustments to Dynamic Exercise and Effect of Physical Training in Normal Subjects and in Patients With Coronary Artery Disease

Jan Praetorius Clausen
Prog Cardiov Dis 1976; XVIII(6): 459-496

The present paper focuses upon the importance of peripheral circulatory alterations during adjustments to exercise and training. Although training results in central circulatory adaptations and may also improve left ventricular function, the prime importance of such adaptations as regards the circulatory and metabolic response to training will be questioned. The thesis that increased maximal exercise capacity can at least in part be attributed to local alterations in the trained muscles will be presented and analyzed. While it is accepted that maximal oxygen uptake is limited by the blood oxygen transport capacity, it will be postulated that the primary event normally responsible for an enhanced oxygen supply after training is an increased ability to reduce resistance to blood flow in exercising muscles rather than improved performance of the central pump.

adjustment to exercise is limited to factors pertinent to physical training of patients with CAD. More detailed accounts of the normal response to exercise can be found in recent books or reviews.

Astrand, P-O, Rodahl K: Textbook of Work Physiology. New York, McGraw-Hill, 1970
Ekblom B, Hermansen L: Cardiac outputs in athletes. J Appl Physiol 25:619, 1968
Christensen EH: Beitrlge zur Physiologie schwerer kijrperlicher Arbeit. Arbeits physiol 4:470, 1931
Saltin B, Blomqvist G, Mitchell JH, et al: Response to exercise after bed rest and after training. Circulation 38 (Suppl 7): 1, 1968
Clausen JP, Klausen K, Blomqvist G, et al. Central and peripheral circulatory changes after training of the arms or legs. Am J Physiol 225:675, 1973

In connection with patients with CAD, only one type of muscular work is of interest; namely, rhythmic or dynamic exercise, in which a considerable part of the skeletal muscle mass is active. This applies to naturally occurring physical activity. Only these types of activity will be referred to and only at work intensities that can be continued for 3-5 min or more.

Dynamic muscular exercise is characterized by a high metabolic rate in the muscle cells with the skeletal muscle functioning in a manner similar to the myocardium, with regularly alternating contraction and relaxation phases. The mechanical energy expended is grossly proportional to the force and the frequency of contraction, and it is derived from the breakdown of adenosine triphosphate (ATP) and creatine phosphate (CP). Only a limited number of a muscle’s fibers, and thus, of its maximal contractile power, can be used in dynamic work continuing for several minutes. During maximal exercise on a bicycle ergometer with a pedaling frequency of 60 rpm, about 15%-2% of the maximal isometric strength of the quadriceps muscle is mobilized. This is thought related to the fact that skeletal muscle, in contrast to myocardium, is composed of several types of fibers with different enzymatic characteristics.29 Some fibers are similar to cardiac muscle being rich in oxidative intramitochondrial enzymes connected to the citric acid cycle, the fatty acid cycle, and the respiratory chain. These are the classical “red” muscle fibers. At the other end of a continuous spectrum is the typical “white” muscle fiber, with a high content of enzymes necessary for anaerobic glycolysis, but containing few mitochondria. Due to their great capability for aerobic metabolism, red fibers sustain rhythmic contractions for long periods of time, whereas the anaerobic white fibers require longer restitution phases even after short periods of activity.

Oxygen extraction per milliliter of blood perfusing the muscle may increase three- to fourfold, and the enhanced muscle blood flow (MBF) is responsible for the remainder of the augmented oxygen uptake. In human muscle, maximal MBF is in the order of 70-100 ml X 100 g-r X min-^-1 against a resting value of 2-5 ml X 100 g-r X min-^-1. The increase in MBF is locally controlled by release of vasodilator metabolites and thereby closely geared to the metabolic demands. Muscle blood flow per unit weight of muscle is closely related to the relative work load; i.e., percentage of maximal work load. The metabolites responsible for the exercise-induced vasodilation and hyperemia in muscle are not yet conclusively identified. The finding that both MBF and ATP-CP depletion are related to the relative work load supports the speculation that split products from high energy phosphates may be involved.

During strenuous exercise, V_O2can attain individually varying maximal values, typically ranging from 2.0 to 6.0 1 02/min. The maximal oxygen consumption (V_{O2 max}) is a highly reproducible measure of a given subject’s capability to perform this type of exercise, and it constitutes a useful physiologic reference standard. The conditions required to obtain V_{O2 max,} and its physiologic implications have recently been reviewed in detail by Rowe and by Hermansen. The V_{O2 max} for a given type of work is normally achieved at a work intensity that can be sustained for at least 3 min, but will cause complete exhaustion within 5-10 min. At this intensity of exercise, the cardiovascular functional capacity with respect to increase in cardiac output (Q), widening of systemic arteriovenous oxygen difference (AVDO₂), and elevation of heart rate (HR) will be challenged maximally for the given type of exercise. However, the relative contribution of Q and AVDO_2.

The above description of the normal central and peripheral circulatory adjustment to exercise can be recapitulated as follows:

During dynamic exercise, Q increases in direct proportion to the augmentation of 30,. The increase in Q is directed to exercising skeletal muscles, to the myocardium and-if exercise is continued for more than approximately 5 min-also to the skin. Blood flow to most “nonexercising” tissues (SBF, RBF,
and noncontracting muscles) is reduced due to a general sympathetic vasoconstriction. At submaximal levels, muscle blood flow per unit tissue,
the degree of peripheral vasoconstriction, the acceleration of HR, and in consequence, the increase in myocardial blood flow and oxygen consumption are all functions of the relative V0₂ ; i.e., the actual VO₂ expressed as a percentage of the highest achievable V0₂ for the given type of exercise.

Most patients with CAD who have been included in exercise and training studies have had healed myocardial infarction and/or stable angina pectoris and have been between 35 and 65 years of age. Both the aging process and myocardial lesions contribute to the modification of the circulatory response to exercise in this group, as compared to healthy young people. In advanced age-especially after 60 years-the circulation tends to become hypokinetic; i.e., Q/VO₂ is reduced. The decline of Q in l/min is almost the same during submaximal exercise as at rest, and thus the increase in Q with VO₂ is essentially the same in older as in younger subjects. Stroke volume is lower at a given VO₂ , while arterial blood pressures are higher; Q, HR, and VO₂max decline with aging.

Although patients with angina pectoris often exhibit a more profound impairment of left ventricular function and of working capacity than patients with CAD without angina, there seems not to be any specific differences in their central or peripheral circulatory response to exercise. Accordingly, the abnormalities in hemodynamic adaptations in a patient with angina pectoris are present also at workloads that do not provoke angina pectoris.

From the point of view of an exercise physiologist, the patient with angina pectoris is peculiar in that his capacity for dynamic work is not limited by his total body VO₂max, but by VO₂max in myocardial regions supplied by narrowed coronary arteries. If pain is prevented by prophylactic administration of nitroglycerin, a patient with angina pectoris can exercise longer at a given work load or achieve higher workloads and thus obtain a higher VO₂ max.

The circulatory adjustment to exercise in patients with CAD typically differs from that of normal subjects in that the maximal values for Q (and thus for VO₂), for HR, and for blood pressures are lower. During submaximal exercise, the relation between Q and VO₂ tends to be reduced. Moreover, most of the patients with CAD exhibit signs of left ventricular failure during exercise, including a decrease in SV at higher workloads, reduced myocardial contractility, and increased LVEDp. Nonetheless, the peripheral circulatory regulation in patients with CAD corresponds in principle to that seen in healthy subjects of the same age.

Training changes the different local flows during exercise in such a way that, within the framework of an unchanged or reduced Q, its regional distribution at a given submaximal work load deviates less from that seen at rest: the perfusion of nonworking tissues is relatively greater and the flow to active muscles less elevated. However, this is only valid for exercise performed with trained muscles.

Although the precise mechanism mediating exercise hyperemia is unknown, it seems acceptable that enhanced content of oxidative enzymes enables a reduction in MBF at a given submaximal VO₂ . After training, due to the increased capacity for oxidative phosphorylation, ATP and CP in active muscles stabilize at a higher steady state level. At the same time glycolysis occurs at a slower rate, pH is relatively increased, and the concentration of multiple intermediate metabolic products may be lower. In consequence, the intra- and intercellular biochemical milieu-concentrations of electrolytes and osmolality included-is less disturbed as compared to the conditions at rest. Whatever substance or combinations of chemical alterations cause the vasodilation, their extent of change is probably reduced at a given respiratory rate in trained muscle tissue, and the vasodilation is thus diminished.

Training improves exercise tolerance in most patients with angina pectoris. The main part of this effect can be related to the training-induced reduction in HR and SBP that decreases myocardial O2 requirements at a given total body O2 uptake. However, at the same time, higher values for the product of HR and SBP are tolerated before pain is provoked after training, suggesting that training has additional economizing effects on myocardial function or directly improves myocardial O2 supply. As judged from the results obtained in exercise tests, training and nitroglycerin seem almost equally potent in alleviating or preventing angina pectoris on exertion. Beta receptor blockade may be somewhat less efficient, whereas aorto-coronary bypass surgery, when practicable, may be the most efficient treatment of exertional angina available today.

Physical training is efficient in improving exercise capacity in about two thirds of all patients with angina pectoris. Patients with angina pectoris provoked only by exercise will often respond favorably to training, even if their exercise capacity is low. In contrast, patients who suffer from angina at rest, especially nocturnal attacks, may be less likely to increase their exercise tolerance by training. Accordingly, Hellerstein reports that in patients with more severe coronary arteriosclerosis as assessed from coronary arteriograms and left ventricular function, physical fitness fails to improve from training.

Unfortunately, it appears that the patients who cannot be expected to respond favorably to training are also less likely to improve from other modes of treatment. According to Balcon, only younger patients with normal left ventricular function are prone to achieve substantial improvement in physical working capacity by vein graft surgery. Furthermore, the mortality from the operation is higher in patients with abnormal ventricular function. Thus, the appearance of an apparently efficient surgical intervention has not simplified the selection of treatment.

Characteristics of the Ventilatory Exercise Stimulus

F.M. Bennett and W.E. Fordyce
Respiration Physiology 1985; 59, 55-63

Simple mathematical models were used to quantitatively examine a number of hypotheses concerning the nature of the exercise stimulus. The modelling demonstrated the following for an exercise intensity of 5 times the resting metabolic rate.

(1) During the steady state, a deviation in the coupling between V_E and metabolic rate by + 25 % of the value necessary for isocapnia, results in a deviation of Pa_co2 of + 2 torr from isocapnia.

(2) In the transient phase, a mismatch between V_E and Q (and thus CO2 flow) of 50% results in a change of Pa_co2 of only 1 torr.

(3)When resting Pa_co2 is changed by 10 torr and it is assumed that the coupling between V_Eand Pa_co2 does not change, Pa_co2 deviates from isocapnia by less than 2 torr.

It is concluded that –

(1) to experimentally test hypotheses of the exercise stimulus requires resolution of small changes in Pa_co2;

(2) good regulation of Pa_co2 does not necessarily imply precise coupling between V_E and V_co2;

(3) the ventilatory exercise stimulus need not be a precise function of metabolic rate;

(4) in the steady state, the normal CO₂ controller will be very effective in minimizing changes in Pa_co2due to a mismatch between ventilation and metabolic rate.

Cardiorespiratory and Metabolic Responses to Positive, Negative and
Minimum-Load Dynamic Leg Exercise

Carl Magnus Hesser, Dag Linnarsson And Hilding Bjurstedt
Respiration Physiology 1977; 30, 5 I-67

Cardiorespiratory and metabolic responses to steady-state dynamic leg exercise were studied in seven male subjects who performed positive and negative work on a modified Krogh cycle ergometer at loads of 0. 16,33,49.98, and 147 W with a pedaling rate of60 rpm.
In positive work, O₂ uptake increased with the ergometric load in a parabolic fashion. Net O₂ uptake averaged 220 ml*min^{– 1} at 0 W (loadless pedaling), and was 75 ml* min^{– 1} lower at the point of physiological minimum load which occurred in negative work at approximately 9 W. The O₂ cost of loadless pedaling is for one-third attributed to the work of overcoming elastic and viscous resistance, the remaining part being due mainly to the work of antagonistic muscle contraction in the moving legs. Although at a given Vo₂ work rate was much higher in negative than in positive work, corresponding values for V_E were similar, suggesting that the mechanical tension in working muscles is of little or no importance in the control of ventilation in steady-state exercise.
Heart rate increased linearly with Vo₂ in both positive and negative work, with a steeper slope in negative work. Evidence is presented that none of the current definitions of muscular efficiency yields the true efficiency of muscular contraction in cycle ergometry, net efficiency calculation resulting in too low estimates, and work and delta efficiency calculations in overestimated values in the low-intensity work range, and in underestimated values in the high-intensity range.

The effect of exercise on left ventricular ejection time in patients with hypertension or angina pectoris

James R. Bowlby
Amer Heart J 1979; 97(3): 348-350

Using the method and regression equation of Lewis and associates, the present study confirms their findings in normal men up to the age of 65 years. Despite the significantly higher myocardial oxygen consumption, as measured by the double product, the hypertensive patients responded in a similar fashion. The patients with angina pectoris, however, showed a significantly prolonged post-exercise ejection time.

Cardiac Effects of Prolonged and Intense Exercise Training in Patients With Coronary Artery Disease

Ali A. Ehsani, Wade H. Martin Iii, Gregory W. Heath, Edward F. Coyle
Amer J Cardiol 1982; 50: 246-254

The effects of intense and prolonged exercise training on the heart were studied with echocardiography in eight men with coronary artery disease with a mean age (standard error of the mean) of 52 + 3 years. Training consisted of endurance exercise 3 times/week at 50 to 60 percent of the measured maximal oxygen uptake for 3 months followed by exercise 4 to 5 days/week at 70 to 60 percent of maximal oxygen uptake for 9 months. Maximal oxygen uptake capacity increased by 42 percent (26 + 1 versus 37 + 2 ml/kg per min; p <0.001). Heart rate at rest and submaximal heart rate and systolic blood pressure at a given work rate were significantly lower after training. Systolic blood pressure at the time of maximal exercise increased (145 + 9 before versus 166 + 6 mm Hg after training; probability [p] <0.01). Left ventricular end-diastolic diameter was increased after 12 months of training (from 47 + 1 to 51 + 1 mm; p <0.01. Left ventricular fractional shortening and mean velocity of circumferential shortening decreased progressively in response to graded iisometric handgrip exercise before training but not after training. At comparable levels of blood pressure during static exercise, mean velocity of circumferential shortening was significantly higher after training (0.76 + 0.04 versus 0.96 + 0.07 diameter/set, p <0.01). No improvement in echocardio-graphic or exercise variables was observed over a 12 month period in another group of five patients who did not exercise. Thus the data suggest that prolonged and vigorous exercise training in selected patients with coronary artery disease can elicit cardiac adaptations.

Physical activity and resting pulse rate in older adults: Findings from a randomized controlled trial

Bríain O’Hartaigh, Marco Pahor, Thomas W. Buford, John A. Dodson, et al.
Am Heart J 2014;168:597-604

Background Elevated resting pulse rate (RPR) is a well-recognized risk factor for adverse outcomes. Epidemiological evidence supports the beneficial effects of regular exercise for lowering RPR, but studies are mainly confined to persons younger than 65 years. We set out to evaluate the utility of a physical activity (PA) intervention for slowing RPR among older adults.
Methods A total of 424 seniors (ages 70-89 years) were randomized to a moderate intensity PA intervention or an education-based “successful aging” health program. Resting pulse rate was assessed at baseline, 6 months, and 12 months. Longitudinal differences in RPR were evaluated between treatment groups using generalized estimating equation models, reporting unstandardized β coefficients with robust SEs.
Results Increased frequency and duration of aerobic training were observed for the PA group at 6 and 12 months as compared with the successful aging group (P = 0.001). In both groups, RPR remained unchanged over the course of the 12-month study period (P = .67). No significant improvement was observed (β [SE] = 0.58 [0.88]; P = .51) for RPR when treatment groups were compared using the generalized estimating equation method. Comparable results were found after omitting participants with a pacemaker, cardiac arrhythmia, or who were receiving β-blockers.
Conclusions Twelve months of moderate intensity aerobic training did not improve RPR among older adults. Additional studies are needed to determine whether PA of longer duration and/or greater intensity can slow RPR in older persons.

Autonomic regulation and maze-learning performance in older and younger dults

Karen J. Mathewson, J Dywan, PJ Snyder, WJ Tays, SJ Segalowitz
Biological Psychology 88 (2011) 20– 27
http://dx.doi.org:/10.1016/j.biopsycho.2011.06.003

There is growing evidence that centrally modulated autonomic regulation can influence performance on complex cognitive tasks but the specificity of these influences and the effects of age-related decline in these systems have not been determined. We recorded pre-task levels of respiratory sinus arrhythmia (RSA; an index of phasic vagal cardiac control) and rate pressure produce (RPP; an index of cardiac workload) to determine their relationship to performance on a cumulative maze learning task. Maze performance has been shown to reflect executive error monitoring capacity and non-executive visuomotor processing speed. Error monitoring was predicted by RSA in both older and younger adults but by RPP only in the older group. Non-executive processes were unrelated to either measure. These data suggest that vagal regulation is more closely associated with executive than nonexecutive aspects of maze performance and that, in later life, pre-task levels of cardiac workload also influence executive control.

Sympathovagal Imbalance Contributes to Prehypertension Status and Cardiovascular Risks Attributed by Insulin Resistance, Inflammation, Dyslipidemia and Oxidative Stress in First Degree Relatives of Type 2 Diabetics

Gopal Krushna Pal, C Adithan, P Hariharan Ananthanarayanan, Pravati Pal, et al.
PLoS OME 2013; 8(11), e78072 http://dx.doi.org:/10.1371/journal.pone.0078072

Background: Though cardiovascular (CV) risks are reported in first-degree relatives (FDR) of type 2 diabetics, the pathophysiological mechanisms contributing to these risks are not known. We investigated the association of sympathovagal imbalance (SVI) with CV risks in these subjects.
Subjects and Methods: Body mass index (BMI), basal heart rate (BHR), blood pressure (BP), rate-pressure product (RPP), spectral indices of heart rate variability (HRV), autonomic function tests, insulin resistance (HOMA-IR), lipid profile, inflammatory markers, oxidative stress (OS) marker, rennin, thyroid profile and serum electrolytes were measured and analyzed in subjects of study group (FDR of type 2 diabetics, n = 72) and control group (subjects with no family history of diabetes, n = 104).
Results: BMI, BP, BHR, HOMA-IR, lipid profile, inflammatory and OS markers, renin, LF-HF (ratio of low-frequency to high frequency power of HRV, a sensitive marker of SVI) were significantly increased (p,0.0001) in study group compared to the control group. SVI in study group was due to concomitant sympathetic activation and vagal inhibition. There was significant correlation and independent contribution of markers of insulin resistance, dyslipidemia, inflammation and OS to LF-HF ratio. Multiple-regression analysis demonstrated an independent contribution of LF-HF ratio to prehypertension status (standardized beta 0.415, p,0.001) and bivariate logistic-regression showed significant prediction (OR 2.40, CI 1.128–5.326, p = 0.002) of LF-HF ratio of HRV to increased RPP, the marker of CV risk, in study group.
Conclusion: SVI in FDR of type 2 diabetics occurs due to sympathetic activation and vagal withdrawal. The SVI contributes to prehypertension status and CV risks caused by insulin resistance, dyslipidemia, inflammation and oxidative stress in FDR of type 2 diabetics.

Exercise prescription for patients with type 2 diabetes and pre-diabetes: A position statement from Exercise and Sport Science Australia

Matthew D. Hordern, DW Dunstan, JB Prins, MK Baker, et al.
Journal of Science and Medicine in Sport 15 (2012) 25–31
http://dx.doi.org:/10.1016/j.jsams.2011.04.005

Type 2 diabetes mellitus (T2DM) and pre-diabetic conditions such as impaired fasting glucose (IFG) and/or impaired glucose tolerance (IGT) are rapidly increasing in prevalence. There is compelling evidence that T2DM is more likely to develop in individuals who are insufficiently active. Exercise training, often in combination with other lifestyle strategies, has beneficial effects on preventing the onset of T2DM and improving glycaemic control in those with pre-diabetes. In addition, exercise training improves cardiovascular risk profile, body composition and cardiorespiratory fitness, all strongly related to better health outcomes. Based on the evidence, it is recommended that patients with T2DM or pre-diabetes accumulate a minimum of 210 min per week of moderate-intensity exercise or 125 min per week of vigorous intensity exercise with no more than two consecutive days without training. Vigorous intensity exercise is more time efficient and may also result in greater benefits in appropriate individuals with consideration of complications and contraindications. It is further recommended that two or more resistance training sessions per week (2–4 sets of 8–10 repetitions) should be included in the total 210 or 125 min of moderate or vigorous exercise, respectively. It is also recommended that, due to the high prevalence and incidence of comorbid conditions in patients with T2DM, exercise training programs should be written and delivered by individuals with appropriate qualifications and experience to recognise and accommodate comorbidities and complications.

Estimation of the Ejection Fraction in Patients with Myocardial Infarction Obtained from the Combined Index of Systolic and Diastolic Left Ventricular Function: A New Method

Jorge A. Lax, Alejandra M. Bermann, Tomás F. Cianciulli, Luis A. Morita, et al.
J Am Soc Echocardiogr 2000;13:116-23.

The index of myocardial performance combining systolic and diastolic time intervals (Index) is a useful method, already explained in past studies, that offers new values that have not been widely known among clinical cardiologists. The aim of this study is to obtain from this Index a measurement of the ejection fraction (EF), which is a very well-known value.
The study involved 97 patients with myocardial infarction, 55 of whom were studied retrospectively (group A, aged 46-62 years, 50 men) to obtain and test the formula EF = 60 – (34 × Index). The second group (group B, aged 47-63 years, 40 men) included 42 patients who were evaluated prospectively. The EF obtained was compared with that reached through the use of radionuclide angiography (EF-RNA).
The Index was obtained through the use of the formula (a – b)/b, where a is the interval between cessation and onset of the mitral inflow, and b is the ejection time. In group A the EF obtained by the Index (EF-Index) was 37.5% ± .8%, and the EF-RNA was 37.7% ± 11% (r = 0.76). In group B the EF-Index was 41.6% ± 7%, and the EF-RNA was 41.2% ± 10% (r = 0.75).
Conclusion: Through the new formula described here it is possible to obtain a reliable measurement of the EF in patients with myocardial infarction, a well known and extremely useful value, especially for those patients with poor acoustic windows.

HCN channels: new roles in sinoatrial node function

Christian Wahl-Schott, Stefanie Fenske and Martin Biel
Current Opinion in Pharmacology 2014, 15:83–90
http://dx.doi.org/10.1016/j.coph.2013.12.005

Hyperpolarization-activated cyclic nucleotide gated (HCN) channels pass a cationic current (I_h/I_f) that crucially contributes to the slow diastolic depolarization (SDD) of sinoatrial pacemaker cells and, hence, is a key determinant of cardiac automaticity and the generation of the heart beat. There is growing evidence, that HCN channel functions in the sinoatrial node (SAN) are not restricted to impulse formation but are also required for impulse propagation. In addition, HCN channels are involved in coordination and maintenance of sinoatrial network activity and, hence, are crucial for stabilizing cardiac rhythmicity. In the present review we will outline these new concepts.

In this review we will focus on HCN channel functions in the sinoatrial node beyond the established concepts described above. We will outline recent advances involving the characterization of the HCN1-deficient mouse line (HCN1^-/- mouse) which have provided evidence that HCN channels are required for impulse propagation and the precision of the heart beat [19**]. Furthermore, we show how these properties can be generalized across the other HCN channel subtypes in the sinoatrial node.

19** Fenske S, Krause SC, Hassan SI, Becirovic E, Auer F, Bernard R, Kupatt C, Lange P, Ziegler T, Wotjak CT et al.: Sick sinus syndrome in HCN1-deficient Mice. Circulation 2013. Epub 2013 Nov 11.
First demonstration of a functional relevance of HCN1 channels in the murine sinoatrial node. The authors demonstrate that mice lacking the pacemaker channel HCN1 display congenital sinoatrial node dysfunction characterized by bradycardia, sinus dysrhythmia, prolonged sinoatrial node recovery time, increased sinoatrial conduction time and recurrent sinus pauses. As a consequence of sinoatrial node dysfunction HCN1-deficient mice display a severely reduced cardiac output.

Recent studies indicate that the role of cardiac HCN channels extends well beyond generation of pacemaker potentials. In addition to being merely ‘pacemaker channels’, HCN channels are important for sinoatrial impulse propagation, cardiac excitability and for the precision of the heartbeat. Furthermore, cardiac HCN channels are involved in the repolarization process of heart ventricles [56**,57]. It will be important to consider the full spectrum of these diverse cardiac functions when exploring agents acting on HCN channels for a specific clinical purpose such as reduction of heart rate.

56.** Fenske S, Mader R, Scharr A, Paparizos C, Cao-Ehlker X, et al.: HCN3 contributes to the ventricular action potential waveform in the murine heart. Circ Res 2011, 109:1015-1023.
First study demonstrating a functional role of HCN3 channels in the heart. Using HCN3-deficient mouse line the authors show that HCN3 together with other members of the HCN channel family confers a depolarizing background current that regulates ventricular resting potential and counteracts the action of hyperpolarizing potassium currents in late repolarization.
57. Fenske S, Krause S, Biel M, Wahl-Schott C: The role of HCN channels in ventricular repolarization. Trends Cardiovasc Med 2011, 21:216-220.

Roles of HCN1 channels for sinoatrial impulse conduction (source-sink relation) The primary impulse initiating the heart beat is generated in the leading pacemaker cell(s) of the sinoatrial node. Once the leading pacemaker cell(s) reaches the threshold for L-type Ca2+ channels an action potential is generated. Since pacemaker cells are interconnected via gap junctions, the impulse is conducted through the sinoatrial network and to the atrium. During impulse propagation the source cell (the cell which first reached AP threshold and is firing the action potential) charges the neighboring cell (sink), in which the membrane potential is below threshold (Figure 1) [24*]. Impulse propagation depends on the source-sink relation [24*, 25–29]. HCN1 deletion increases the sinoatrial conduction time suggesting the existence of a source sink mismatch in the HCN1-deficient mouse [19**].

Role of HCN1 channels for impulse formation and impulse conduction in the sinoatrial node. Schematic pacemaker potential in sinoatrial node cells of wild type (a) and HCN1-/- mice.
(b) HCN channels contribute to the slow diastolic depolarization. In the absence of HCN1 the slope of SDD isdecreased and the time to threshold for an action potential increased. HCN channels decrease the maximal diastolic potential (MDP). In the absence of HCN1 the MDP is increased. This results in an increased distance and time to threshold for an action potential and a decrease in impulse propagation. [SDD: slow diastolic depolarization; MDD: maximal diastolic depolarization; Vthr: threshold potential for the generation of an action potential.]
(c) Direction of intracellular and extracellular current flow during propagation of an action potential from depolarized (source) to resting cells (sink).
(d)Source sink relationship in propagation. Charge from excited cells (source) flows into unexcited cell (sink) and provides the charge to depolarize them to activation threshold. Arrows and dotted lines indicate changes observed in HCN1-/- mice of parameter indicated leading to source sink mismatch and prolonged sinoatrial conduction. Modified from [24*].

24.* Spector P: Principles of cardiac electric propagation and their implications for re-entrant arrhythmias. Circ Arrhythm Electrophysiol 2013, 6:655-661.
The authors provide an excellent review of the principles of impulse propagation in relation to arrhythmia.

HCN1 channels increase the temporal and spatial precision of impulse formation in sinoatrial node

HCN1 channels increase the temporal and spatial precision of impulse formation in sinoatrial node.
(a) Schematic of the sinoatrial node. Atrial cells invaginate into the central sinoatrial node. Putative localization of HCN1 channels at contact interface between strands of atrial myocytes which extend into the central SAN and sinoatrial node pacemaker cells. Green: autonomous innervation. HCN1 channels dampen network noise generated by neighboring pacemaker cells in the sinoatrial network, by invading hyperpolarization of atrial cells and by autonomous regulation. SAN: sinoatrial node, RA: right atrium, CT: crista terminalis.
(b) Model of sinoatrial node function (for detail see text). Note that individual cells display different phases and slightly different periods.

Pharmacological inhibition of cardiac HCN channels

HCN channels have emerged as interesting targets for the development of drugs that lower the heart rate. Ivabradine is the first and currently the only clinically approved compound that specifically targets HCN channels. The therapeutic indication of ivabradine is the symptomatic treatment of chronic stable angina pectoris in patients with coronary artery disease with a normal sinus rhythm (for details see [48], the international trial on the treatment of angina with ivabradine vs. atenolol (INITIATIVE) trial (n = 939) [49] and the antianginal efficacy and safety of the association of the I_h/I_f current inhibitor ivabradine with a beta-blocker (ASSOCIATE) study (n = 889) [50]).

The Role of HCN Channels in Ventricular Repolarization

Stefanie Fenske, Stefanie Krause, Martin Biel, and Christian Wahl-Schott
Trends Cardiovasc Med 2011; 21:216-220
PII S1050-1738(12)00143-0

Hyperpolarization-activated cyclic nucleotide gated (HCN) channels pass a cationic current (Ih/If) that crucially contributes to the slow diastolic depolarization (SDD) of sinoatrial pacemaker cells and, hence, is a key determinant of cardiac automaticity and the generation of the heartbeat. However, there is growing evidence that HCN channels are not restricted to the spontaneously active cells of the sinoatrial node and the conduction system but are also present in ventricular cardiomyocytes that produce an action potential lacking SDD. This observation raises the question of the principal function(s) of HCN channels in working myocardium. Our recent analysis of an HCN3-deficient (HCN3–/–) mouse line has shed new light on this central question.

We propose that HCN channels contribute to the ventricular action potential waveform, specifically during late repolarization. In this review, we outline this new concept.

In the late 1970s, the hyperpolarization activated current (I_h/I_f) was discovered and characterized in sinoatrial node cells (Brown and Difrancesco 1980). This current displays several unique biophysical properties: activation upon hyperpolarization and deactivation by depolarization, with a small but substantial degree of activation at resting potentials typically observed in sinoatrial node pacemaker cells (–60 to –50 mV) and ventricular cells (–85 to –75 mV); shift of the activation curve to more positive potentials by cAMP; block by millimolar concentrations of external Cs⁺; and permeability for Na⁺ and K⁺ions with a reversal potential near –35 mV.

HCN3 Is a Component of Ventricular I_h
HCN Channels Prolong Action Potentials During Late Repolarization
HCN3 Forms Background Channels That Do Not Deactivate During the Action Potential
HCN channels need to be open at the resting membrane potential;
(2) HCN channels remain open during the entire time course of the action potential—de novo opening of HCN channels during the AP does not occur because these channels are activated by hyperpolarization and depolarization decreases open probability; and
(3) a driving force is needed to sustain an HCN-mediated current during the AP. A detailed analysis of the functional properties of heterologously expressed HCN3 channels revealed that these three prerequisites are met.

Neurophysiology of HCN channels: From cellular functions to multiple regulations

Chao He, Fang Chen, Bo Li, Zhian Hu
Progress in Neurobiology 112 (2014) 1–23
http://dx.doi.org/10.1016/j.pneurobio.2013.10.001

Hyperpolarization-activated cyclic nucleotide-gated (HCN) cation channels are encoded by HCN1-4 gene family and have four subtypes. These channels are activated upon hyperpolarization of membrane potential and conduct an inward, excitatory current Ih in the nervous system. Ih acts as pacemaker current to initiate rhythmic firing, dampen dendritic excitability and regulate presynaptic neurotransmitter release. This review summarizes recent insights into the cellular functions of I_h and associated behavior such as learning and memory, sleep and arousal. HCN channels are excellent targets of various cellular signals to finely regulate neuronal responses to external stimuli. Numerous mechanisms, including transcriptional control, trafficking, as well as channel assembly and modification, underlie HCN channel regulation. In the next section, we discuss how the intracellular signals, especially recent findings concerning protein kinases and interacting proteins such as cGKII, Ca2+/CaMKII and TRIP8b, regulate function and expression of HCN channels, and subsequently provide an overview of the effects of neurotransmitters on HCN channels and their corresponding intracellular mechanisms. We also discuss the dysregulation of HCN channels in pathological conditions. Finally, insight into future directions in this exciting area of ion channel research is provided.

The hyperpolarization-activated current, I_h, was first observed in sino-atrial node tissue in 1976 and later was identified in rod photoreceptors and hippocampal pyramidal neurons (Noma and Irisawa, 1976). Due to its unique properties, particularly the activation upon hyperpolarization of the membrane potential, Ih has been also termed I_f (f for funny) or I_q (q for queer). The hyperpolarization-activated cyclic nucleotide-gated (HCN) cation ion channels underlying I_h were discovered in the late 1990s and subsequently, the genes encoding these channels were identified, which enable the expression of HCN channels in heterologous systems.

HCN channels belong to the superfamily of voltage-gated pore loop channels with four pore-forming subunits (HCN1-4) encoded by the HCN1-4 gene family in mammals (Robinson and Siegelbaum, 2003). Each subunit has six transmembrane helices (S1–S6), with the positively charged voltage sensor (S4) and the pore region carrying the GYG motif between S5 and S6, which forms the ion selectivity filter (Macri et al., 2012). Following S6 is the 80-residue C-linker comprising six a-helices (A0–F0) and the cyclic nucleotide binding domain (CNBD). The CNBD consists of three a-helices (A–C) and a b-roll between the A- and B-helices (Fig. 1) (Biel et al., 2009; Wahl-Schott and Biel, 2009; Wicks et al., 2011). Together, the C-linker and CBND can be referred to as the ‘‘cAMP-sensing domain’’ (CSD) because they are of functional importance for the cAMP-induced positive shift of the voltage-dependent activation of HCN channels. The crystal structure of CSD has been elucidated at an atomic resolution, but a high-resolution structure of the transmembrane core remains unsolved.

Structure of HCN channels

Structure of HCN channels. Left: one subunit is composed of six transmembrane segments (S1–S6), with the positive charged voltage sensor (S4) and the pore region carrying the GYG motif between S5 and S6. The C-terminal of HCN channels is composed of the C-linker and the cyclic nucleotide-binding domain (CNBD) which mediates their responses to cAMP. The C-linker consists of six a-helices: A0 to F0 . The CNBD follows the C-linker domain and consists of a-helices A–C with a b-roll between the A- and B-helices. Right: the four subunits assemble in homomeric or heteromeric tetramer configurations in vivo.

Regulatory mechanisms of Ih function by the small molecules, protein kinases and interacting proteins.

Regulatory mechanisms of I_h function by the small molecules, protein kinases and interacting proteins. Black arrows indicate known sites of HCN channels interaction with small molecules, protein kinases and interacting proteins. Broken lines indicate the speculated interaction sites. Filamin A interacts with HCN1 via a region of 22 amino acids located downstream from the CNBD. Tamalin and Mint2 bind to the CNBD-downstream sequence of HCN2. The binding of the PDZ domain of S-SCAM occurs at the cyclic nucleotide-binding domain (CNBD) and the CNBD downstream sequence of the carboxy-terminal tail. CNBD, cyclic nucleotide binding domain; SNL, C-terminal tripeptide of HCN1, HCN2 and HCN4.

modulation of HCN channels by neurotransmitters and associated intracellular signal pathways

The modulation of HCN channels by neurotransmitters and associated intracellular signal pathways. Glutamate (Glu) activates N-methyl-D-aspartate receptors (NMDARs) and a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) which results in the Ca2+ influx and subsequently activates calcium calmodulin kinase (CaMKII). CaMKII increases channels surface expression through the interacting protein TRIP8b (1a-4) or reduces the HCN1 gene transcription via Neuronal Restrictive Silencing Factor (NRSF) in pathological conditions. Glu, norepinephrine (NE, in rats), 5-hydroxytryptamine (5-HT) and triphosphate (ATP) bind to specific G-coupled receptors and modulate the activity of HCN channels via the PLC-PKC or p38-MAPK signaling pathways. Activation of PKC suppresses the activation of HCN channels, whereas p38-MAPK causes a positive shift of HCN channels voltage-dependent activation. Adenosine, NE (in monkey), 5-HT, dopamine (DA) and Ach (acetylcholine) bind to Gs- or Gi coupled receptors. Gs or Gi oppositely control the activity of adenylate cyclase (AC), which catalyzes the ATP to cAMP. cAMP could shift the HCN channels voltage-dependent activation to positive direction and accelerate the kinetics of channels activation. Nitric oxide (NO) interacts with soluble guanylyl cyclase (GC) and thus increases the intracellular concentration of cGMP, which induces a positive shift of HCN channels voltage-dependent activation. Sharp and blunted arrows represent the positive and negative regulation, respectively. Broken lines indicate the speculated signal pathway.

Ultimately, the study of the HCN channels will provide an overall picture underlying the real-time in vivo regulation of the function and expression of HCN channels to fulfill complex functions in different contexts.

Oxygen uptake kinetics during high-intensity arm and leg exercise

Katrien Koppo, Jacques Bouckaert, Andrew M. Jones
Respiratory Physiology & Neurobiology 133 (2002) 241-250
PII: S1569 – 9048 ( 02 ) 00184 – 2

The purpose of the present study was to examine the oxygen uptake kinetics during heavy arm exercise using appropriate modelling techniques, and to compare the responses to those observed during heavy leg exercise at the same relative intensity. We hypothesized that any differences in the response might be related to differences in muscle fiber composition that are known to exist between the upper and lower body musculature. To test this, ten subjects completed several bouts of constant-load cycling and arm cranking exercise at 90% of the mode specific ˙V_O2peak. There was no difference in plasma [lactate] at the end of arm and leg exercise. The time constant of the fast component response was significantly longer in arm exercise compared to leg exercise (mean + S.D., 489 +12 vs. 219 + 5 sec; P < 0.01), while the fast component gain was significantly greater in arm exercise (12.19 + 1.0 vs. 9.29 + 0.5 ml min^-1 W^-1; P < 0.01). The ˙V_O2 slow component emerged later in arm exercise (1269 + 27 vs. 959 + 20 sec; P < 0.01) and, in relative terms, increased more per unit time (5.5 vs. 4.4% min^-1; P < 0.01). These differences between arm crank and leg cycle exercise are consistent with a greater and/or earlier recruitment of type II muscle fibers during arm crank exercise.

Probability and magnitude of response to cardiac resynchronization therapy according to QRS duration and gender in nonischemic cardiomyopathy and LBBB

Niraj Varma, Mahesh Manne, Dat Nguyen, …, Patrick Tchou
Heart Rhythm 2014; 11: 1139–1147
http://dx.doi.org/10.1016/j.hrthm.2014.04.001

BACKGROUND QRS morphology and QRS duration (QRSd) determine cardiac resynchronization therapy (CRT) candidate selection but criteria require refinement.
OBJECTIVE To assess CRT effect according to QRSd, treated by dichotomization vs a continuous function, and modulation by gender.
METHODS Patients selected were those with New York Heart Association classIII/IV heart failure and with left bundle branch block and nonischemic cardiomyopathy (totest “pure” CRT effect) with pre-and post- implant echocardiographic evaluations. Positive response was defined as increased left ventricular ejection fraction (LVEF) post-CRT.
RESULTS In 212 patients (LVEF 19% + 7.1%; QRSd 160 + 23 ms; 105 (49.5%) women), CRT increased LVEF to 30% + 15% (P < .001) during a median follow-up of 2 years. Positive response occurred in 150 of 212 (71%) patients. Genders did not differ for QRSd, pharmacotherapy, and comorbidities, but response to CRT among women was greater: incidence 84% (88of105) in women vs 58% (62of107) in men (P < .001); increase in LVEF 15%+ 14% vs 7.2% + 13%, respectively (P < .001). Overall, the response rate was 58% when QRSd <150 ms and 76% when QRSd > 150 ms (P <.009). This probability differed between genders: 86% in women vs 36% in men (P < .001) when QRSd <150 ms and 83% vs 69%, respectively, when QRSd >150 ms (P < .05). Thus, female response rates remained high whether QRSd was < 150 ms >150 ms (86% vs 83%; P = .77) but differed in men (36% vs 69%; P < .001). With QRSd as a continuum, the CRT-response relationship was nonlinear and significantly different between genders. Female superiority at shorter QRSd inverted with prolongation > 180 ms.
CONCLUSION The QRSd-CRT response relationship in patients with heart failure and with left bundle branch block and non-ischemic cardiomyopathy is better described by a sex-specific continuous function and not by dichotomization by 150ms, which excludes a large proportion of women with potentially favorable outcome.

Comparison of eterminants Myocardial Oxygen Consumption During Arm and Leg Exercise in Normal Persons

Gary J. Balady, et al. Am J Cardiol 1985; 57: 1385-87.

The effects of arm exercise on myocardiai oxygen consumption are not well understood; they may differ from the effects of leg exercise. Previous studies have shown that the ischemic threshold is higher in patients performing arm exercise and leg exercise at the same heart rate-blood pressure product. The contribution of other determinants of myocardiai oxygen consumption-left ventricular (LV) peak meridional systolic wail stress and contractility-to these observed differences were studied.
Thirty healthy subjects exercised to the same peak rate-pressure product during dynamic upper- and lower-extremity exercise. Peak workload was lower
during arm exercise (100 + 16 W) leg exercise (170 + 21 W, p < 0.001). LV wail stress did not differ during either form of exercise (197 + 44 vs 204 + 33 dynes/cm² X 103, arm vs leg, respectively). This was also true of contractility as assessed by the velocity of circumferential fiber shortening (2.6 + 0.6 vs 2.5 + 0.4 circ/s, arm vs leg, respectively) and the preejection period/LV ejection time ratio (0.33 + 0.11 vs 0.31 + 0.07, arm vs leg, respectively). Normal subjects exercising to a similar rate-pressure product showed the same levels at LV wail stress and contractility for arm and leg exercise despite the lower rkioad performed with arm exercise.

Anti-hypertensive effect of radiofrequency renal denervation in spontaneously hypertensive rats

Takeshi Machino, N Murakoshi, A Sato, …, T Hoshi, T Kimura, K Aonuma
Life Sciences 110 (2014) 86–92 http://dx.doi.org/10.1016/j.lfs.2014.06.015

Aims: We aimed to investigate the anti-hypertensive effect of radiofrequency (RF) renal denervation (RDN) in an animal model of hypertension. Materials and methods: RF energy was delivered to bilateral renal arteries through a 2 Fr catheter with opening abdomen in 8 spontaneously hypertensive rats (SHRs) and 8 Wistar–Kyoto rats (WKYs). Sham operation was performed in other 8 SHRs and 8 WKYs. Blood pressure (BP), heart rate (HR), and urinary norepinephrine excretion were followed up for 3 months. Plasma and renal tissue concentrations of norepinephrine and plasma renin activity were measured 3 months after the procedure. The RDN was confirmed by a decrease in renal tissue norepinephrine.
Key findings: RF-RDN restrained a spontaneous rise in systolic BP (46 ± 12% increase from 158 ± 8 to 230 ± 14 mmHg vs. 21 ± 18% increase from 165 ± 9 to 197 ± 20 mmHg, p= 0.01) and diastolic BP (55 ± 27% increase from 117 ± 9 to 179 ± 23 mmHg vs. 28 ± 13% increase from 120 ± 7 to 154 ± 13 mm Hg, p= 0.04) in SHRs; however, WKYs were not affected. Although there were no changes in HR and systemic norepinephrine, the renal tissue norepinephrine was decreased by RF-RDN in both SHR (302±41 vs. 159±44 ng/g kidney, p b 0.01) and WKY (203 ± 33 vs. 145 ± 26 ng/g kidney, p= 0.01). Plasma renin activity was reduced by the RF-RDN only in SHR (35.3 ± 9.5 vs. 21.4 ± 8.6 ng/mL/h, p < 0.01).
Significance: RF-RDN demonstrated an anti-hypertensive effect with a reduction of renal tissue norepinephrine and plasma renin activity in SHR.

Effectiveness of Renal Denervation Therapy for Resistant Hypertension: A Systematic Review and Meta-Analysis

Mark I. Davis, KB Filion, D Zhang, MJ Eisenberg, …, EL Schiffrin, D Joyal
J Am Coll Cardiol 2013; 62(3): 231-241.
http://dx.doi.org/10.1016/j.jacc.2013.04.010

Objectives This study sought to determine the current effectiveness and safety of sympathetic renal denervation (RDN) for resistant hypertension. Background RDN is a novel approach that has been evaluated in multiple small studies.
Methods We performed a systematic review and meta-analysis of published studies evaluating the effect of RDN in patients with resistant hypertension. Studies were stratified according to controlled versus uncontrolled design and analyzed using random-effects meta-analysis models. Results We identified 2 randomized controlled trials, 1 observational study with a control group, and 9 observational studies without a control group. In controlled studies, there was a reduction in mean systolic and diastolic blood pressure (BP) at 6 months of –28.9 mm Hg (95% confidence interval [CI]: –37.2 to –20.6 mm Hg) and –11.0 mm Hg (95% CI: –16.4 to –5.7 mm Hg), respectively, compared with medically treated patients (for both, p < 0.0001). In uncontrolled studies, there was a reduction in mean systolic and diastolic BP at 6 months of –25.0 mm Hg (95% CI: –29.9 to –20.1 mm Hg) and –10.0 mm Hg (95% CI: –12.5 to –7.5 mm Hg), respectively, compared with pre-RDN values (for both, p < 0.00001). There was no difference in the effect of RDN according to the 5 catheters employed. Reported procedural complications included 1 renal artery dissection and 4 femoral pseudoaneurysms.
Conclusions RDN resulted in a substantial reduction in mean BP at 6 months in patients with resistant hypertension. The decrease in BP was similar irrespective of study design and type of catheter employed. Large randomized controlled trials with long-term follow-up are needed to confirm the sustained efficacy and safety of RDN.

Effects of renal denervation on the development of post-myocardial infarction heart failure and cardiac autonomic nervous system in rats

Jialu Hu, Yan Yan, Qina Zhou, Meng Ji, Conway Niu, Yuemei Hou, Junbo Ge
Intl J Cardiol 172 (2014) e414–e416 http://dx.doi.org/10.1016/j.ijcard.2013.12.254

Prior studies indicated that radiofrequency renal denervation (RD) had beneficial effects on post-myocardial infarction (MI) heart failure (HF) in rats. In this study we aimed to assess its effects on cardiac autonomic nervous system (CANS) which might be one of the most important mechanisms of RD’s therapeutic effect on post-MI HF and determine the best timing for RD.

One hundred Wistar rats were randomly assigned into five experimental groups: MI group (n = 20), RD group (n = 20), MI-1d + RD group (RD performed one day post-MI, n = 20), MI-4w + RD group (RD performed four weeks post-MI, n = 20), and N group (control group, n = 20).MI was produced through ligation of the anterior descending artery. RD was performed through stripping of the renal nerves. The experimental design and implementation were conducted in accordance with animal welfare guidelines.

Eight weeks post-MI, significant improvements were observed in both MI-1d + RD and MI-4w + RD groups compared to the MI group, that include

(1) improved left ventricular (LV) function and hemodynamics with increased water and sodium excretion;
(2) decreased plasma and renal tissue norepinephrine levels while tissue norepinephrine content increased in myocardium;
(3) increased β1-receptor in myocardium and improved heart rate variability; (4) decreased plasma renin, angiotensin II, aldosterone, BNP and endothelin levels.

More therapeutic effects were found in the MI-1d + RD group than the MI-4w + RD group.

Firstly, our study showed that RD attenuated the remodeling of CANS and modulated its activities. RD leads to preservation of β1 receptors content along with the β1 mRNA expression in noninfarcted cardiac tissue in this HF model (Fig. 1). This correlated with an improvement in heart function and cardiac remodeling. HRV is a sensitive marker for the CANS. RD led to a slower HR and higher SDNN in both intervention groups.

Secondly, we found that RD blocked both peripheral and central RAAS and sympathetic nervous system (SNS) at the same time. And this may answer the question how RD exerted effect on CANS. In our study RD restores renin, angiotensin II, and aldosterone to near normal levels. This not only explains the increase in sodium and water excretion, but also confirms that RD blocks renal RAAS via blockage of the efferent renal sympathetic nerves which is consistent with our previous study.

Thirdly, early RD, performed one day post-MI, resulted in greater excretion of urinary sodium, lower circulating BNP and ET-1 levels compared to late interventions (four weeks post-MI). This suggests that RD performed in the acute phase of MI may not only reverse cardiac remodeling but also has a preventive effect against the development of HF, as what was observed with β-blockers. RD alleviated cardiac preload and afterload by increasing water and sodium retention, blocking cardiac sympathetic activation and decreasing a variety of vasomotor factors which may lead to alleviated acute and chronic ischemia of the heart.

RD improves hemodynamics, decreases neuro-hormonal activations, modulates cardiac autonomic activities, and attenuates LV remodeling in HF. Early intervention appears to have greater beneficial effects on cardiac functional recovery and reverse remodeling after myocardial injury. Circulating neuro-hormones may be effective indicators to evaluate the therapeutic effect of RD on HF. Our data suggested that RD is a safe, non-pharmaceutical treatment of HF after cardiac injury, with unique benefits in stabilizing cardiac autonomic activity and remodeling post-MI.

The cardiac pacemaker current

Mirko Baruscotti, Andrea Barbuti, Annalisa Bucchi
Journal of Molecular and Cellular Cardiology 48 (2010) 55–64
http://dx.doi.org:/10.1016/j.yjmcc.2009.06.019

In mammals cardiac rate is determined by the duration of the diastolic depolarization of sinoatrial node (SAN) cells which is mainly determined by the pacemaker If current. f-channels are encoded by four members of the hyperpolarization-activated cyclic nucleotide-gated gene (HCN1–4) family. HCN4 is the most abundant isoform in the SAN, and its relevance to pacemaking has been further supported by the discovery of four loss-of-function mutations in patients with mild or severe forms of cardiac rate disturbances. Due to its selective contribution to pacemaking, the If current is also the pharmacological target of a selective heart rate-reducing agent (ivabradine) currently used in the clinical practice. Albeit to a minor extent, the If current is also present in other spontaneously active myocytes of the cardiac conduction system (atrioventricular node and Purkinje fibres). In working atrial and ventricular myocytes f-channels are expressed at a very low level and do not play any physiological role; however in certain pathological conditions over-expression of HCN proteins may represent an arrhythmogenic mechanism. In this review some of the most recent findings on f/HCN channels contribution to pacemaking are described.

Cardiac pacemaking originates in the sinoatrial node (SAN) as a consequence of spontaneous firing of rhythmic action potentials generated by specialized myocytes. Although the electrical behavior of a typical SAN cell differs in several aspects from that of a working myocyte, the functional hallmark can be precisely identified in the events that take place during the diastolic interval. During this phase atrial and ventricular myocytes rest in a standby-like condition at a stable voltage (∼−80 mV); a quite different situation characterizes SAN cells, where the cell potential slowly creeps up from the
maximum diastolic potential of about −60 mV to the threshold for the ignition of a new action potential. Since this time interval sets the pace of the heart, this phase is named “pacemaker depolarization”. Given the large spectrum of heart rates observed in mammals the duration of this phase can vary substantially, however the voltage range encompassed is extremely constant and roughly extends from −60 to−40 mV . To sustain this phase several ionic currents and pumps enter in action at variable times and voltages, and this complexity allows for a highly flexible system since the chronotropic fine tuning operated by neuro-hormonal regulators can target different effectors.

In this review we will focus on the If current which is responsiblefor initiating the diastolic depolarization of SAN cells. Due to its fundamental role and its unusual characteristics of being activated in hyperpolarization, this current was named “pacemaker current” or “funny” (I_f) current. The unique property of a reverse voltage dependence, together with the inward nature of the current at diastolic potentials, makes this current apt to initiate and support the diastolic depolarization. In addition, the direct modulation of the current operated by the second messenger cAMP, represents one of the main pathways by which the autonomic nervous system controls cardiac chronotropism. Two recent clinical findings further confirm the role of f-channels in setting the cardiac rate: one is the evidence of a causative link between the presence of loss-of-function mutations found in these channels and the arrhythmic state of individuals carrying the mutations, and the other is the specific heart rate reduction observed in patients treated with ivabradine, a drug that at therapeutic doses selectively reduces the If current (see specific sections in this review).

Although originally discovered in the heart, the If current is also abundantly present in a large fraction of neuronal elements, where it contributes to rhythmic firing, synaptic integration, and dendritic integration.

Molecular and functional properties of SAN myocytes

Molecular and functional properties of SAN myocytes. (A) Spontaneous action potentials (left) and If current traces (right) recorded from typical rabbit SANmyocytes; currents were elicited by hyperpolarizing voltage steps in the range−45 to −75 mV. (B) Immunofluorescence analysis of rabbit SAN tissue slice labelled with anti-connexin 43 (Cx43, red) and anti-HCN4 (green) antibodies. HCN4 is strongly expressed in the central region of the SAN, while the opposite staining is observed for Cx43; crista terminalis (CT), interatrial septum (IS). (C) HCN4 labelling of single myocytes isolated from CT, SAN and IS (top), and representative current traces recorded at−125mV frommyocytes isolated from the same regions (bottom). Both If current density and HCN4 labelling are more abundant in the central nodal area. (Panels B and C from [61] with permission).

[61] Brioschi C, Micheloni S, Tellez JO, Pisoni G, Longhi R, Moroni P, et al. Distribution of the pacemaker HCN4 channel mRNA and protein in the rabbit sinoatrial node. J Mol Cell Cardiol 2009;47:221–7.

The search of new therapeutic tools consisting of gene- and/or cell-based intervention aimed to restore compromised cardiac functions has prompted researchers to exploit the use of HCN channels to alter cellular electrical activity in order to generate, in normally quiescent substrates, stable rhythmic activity similar to that of native pacemaker myocytes. The specific features of pacemaker channels and in particular the fact that they are activated only at diastolic potentials and do not contribute to other phases of the action potentials, make them particularly suitable for such purpose. Early in vitro studies demonstrated that virus-mediated over-expression of HCN2 channels induced a significant increase in the rate of spontaneously beating neonatal ventricular myocytes by causing an I_f-mediated increase of the diastolic depolarization slope. This approach was later confirmed in vivo by showing that direct injection of the HCN2-adenovirus in the left atrium or into the ventricular conduction system of dogs, was able to induce ectopic regular spontaneous activity after AV block. Similarly, adenovirus-mediated over-expression of HCN1 or HCN4 was sufficient to induce a regular rhythm in quiescent cardiomyocyte. Alternative cell-based strategies, aimed to avoid the use of viruses, have been developed by engineering cells in order to express high levels of HCN channels. Engineered human mesenchymal stem cells (hMSCs) expressing either HCN2 or HCN4 have been shown in vitro to properly connect to neonatal cardiomyocytes and to increase their intrinsic spontaneous rhythm. HCN2-expressing hMSCs have also been successfully transplanted in canine left ventricular wall where they were able to induce stable ectopic beats.

Currently, ivabradine is marketed for treatment of chronic stable angina in patients with normal sinus rhythm who have a contraindication or intolerance to β-blockers; clinical studies of patients with chronic stable angina have shown that ivabradine acts as a pure heart rate-reducing agent and has anti-ischemic and anti-anginal properties equivalent to β-blockers and Ca2+ channel blockers and presents a good safety and tolerability profile even during long-term treatment. Mild visual symptoms (phosphenes) were occasionally reported, but were generally well tolerated. Additional information comes from results from a recent large clinical trial (BEAUTIFUL) which indicate that ivabradine treatment of patients with stable coronary artery disease (CAD) and heart rate ≥70 bpm can reduce the incidence of some CAD outcomes such as hospitalization for myocardial infarction and coronary revascularization.

The beat goes on: Cardiac pacemaking in extreme conditions

Christopher M.Wilson, Georgina K. Cox, Anthony P. Farrell
Comparative Biochemistry and Physiology, Part A xxx (2014) xxx–xxx
http://dx.doi.org/10.1016/j.cbpa.2014.08.014

In order for an animal to survive, the heart beat must go on in all environmental conditions, or at least restart its beat. This review is about maintaining a rhythmic heartbeat under the extreme conditions of anoxia (or very severe hypoxia) and high temperatures. It starts by considering the primitive versions of the protein channels that are responsible for initiating the heartbeat, HCN channels, divulging recent findings from the ancestral craniate, the Pacific hagfish (Eptatretus stoutii). It then explores how a heartbeat can maintain a rhythm, albeit slower, for hours without any oxygen, and sometimes without autonomic innervation. It closes with a discussion of recent work on fishes, where the cardiac rhythm can become arrhythmic when a fish experiences extreme heat.

Sympathetic renal denervation: Hypertension beyond SYMPLICITY

Israel M. Barbash, Ron Waksman
Cardiovascular Revascularization Medicine 14 (2013) 229–235
http://dx.doi.org/10.1016/j.carrev.2013.02.004

Despite a wide range of drug treatment for hypertension, resistant hypertension rates remain high. The Symplicity™ Renal Denervation System (Medtronic, Santa Rosa, CA), which creates renal nerve denervation, has shown initial success in lowering blood pressure among patients with resistant hypertension. Given the enormous market for this treatment approach, an estimated two dozen other companies are pursuing technologies with alternative approaches. Despite this fact, very little has been published on preclinical and clinical experience with these new devices. The current review summarizes the most prominent technologies in the pipeline and provides insight into the mechanism of action, preclinical, and clinical experience with these new devices

A large body of evidence has established the central role of the kidneys in hypertension, both as an affector and effector of the central sympathetic system [9]. Renal efferent sympathetic activity initiates processes towards fluid retention, such as the release of renin and increased tubular sodium reabsorption. Moreover, afferent sympathetic activity increases central sympathetic drive, which plays a major role in sustaining hypertension. In fact, historic studies of surgical sympathectomy in patients with resistant hypertension or malignant hypertension uncontrolled by pharmacotherapy were shown to be effective in reducing blood pressure, albeit with severe side effects. Thus, with the introduction of more effective medications, this procedure was abandoned. Renal sympathetic nerves run alongside the renal artery adventitia to enter the hilus of the kidney. Thereafter, they divide into smaller nerve bundles following the anatomic course of the renal blood vessels, penetrating the cortical and juxtamedullary areas inside the kidneys. Based on these anatomic features, it was postulated that creating local nerve injury along the renal arteries may achieve effective denervation.

A key issue in accomplishing effective RDN is to target the sympathetic nerve bundles lying in the adventitia of the renal arteries. Because the vast majority of devices currently under development are percutaneous, RDN is performed from within the vessel lumen. Thus, one of the most important features of such a device is the ability to minimize the damage to the renal artery wall.

Ultrasound energy consists of high-frequency sound waves emitted by a transducer within the catheter. This high energy can pass through surrounding fluids and can generate frictional heating in tissues resulting in a temperature increase that is sufficient to cause injury to the surrounding tissue, specifically the renal nerves. Based on these principles, several systems were developed and are currently being evaluated. ReCor Medical’s (Ronkonkoma, NY) PARADISE™ Percutaneous Renal Denervation System is based on delivery of high ultrasonic energy to induce nerve tissue injury. The PARADISE system is composed of two components: a 6 F-compatible balloon catheter with a cylindrical ultrasound transducer that emits ultrasound energy circumferentially (Fig. 2A)[ Ultrasound based renal denervation systems: (A) Percutaneous Renal Denervation System (PARADISE™); (B) TIVUS system] and a portable generator which controls automated balloon inflation and deflation, and energy delivery. Energy is delivered in 3 different locations along the artery with 50 s inflation and delivery of ultrasound energy at each site. This device received CE mark in February 2012. For RDN, the PARADISE balloon catheter is positioned inside the renal artery and the generator automatically inflates the balloon, delivers the ultrasonic energy, and deflates the balloon. Endothelial thermal damage is prevented by cooled fluid in the balloon.

Radiofrequency based renal denervation systems: (A) Symplicity Renal Denervation System; (B) EnligHTN Renal Denervation System; (C) V2 bipolar balloon catheter; (D) OneShot Balloon catheter

Sample Entropy and Traditional Measures of Heart Rate Dynamics Reveal Different Modes of Cardiovascular Control During Low Intensity Exercise

Matthias Weippert, Martin Behrens, Annika Rieger and Kristin Behrens
Entropy 2014, 16, 5698-5711; http://dx.doi.org:/10.3390/e16115698

Biological time series like the normal heartbeat-to-heartbeat fluctuation demonstrate complex dynamics. Based on their potential to give additional information beyond traditional heart rate variability (HRV) indices, nonlinear parameters have been applied for investigating short and long term effects of exercise on heart rate (HR) control. However, despite their diagnosticity and their clinical significance, the physiological background of their behavior is not very well established. It is assumed that complexity and regularity measures are fundamentally different from traditional HRV indices and show no correlation to these measures. However, many researchers found at least modest correlations for some nonlinear measures and traditional HRV indices under different conditions. It has also been shown that complexity of short-term HRV is under control of the autonomic nervous system. Currently, there are only few studies available that compared the cardiovascular response pattern to different exercise modes at similar HR. Lindquist et al. found a stronger increase of systolic (SBP) and diastolic arterial blood pressure (DBP) during isometric handgrip compared to cycling at comparable HR of 90 bpm.

Nonlinear parameters of heart rate variability (HRV) have proven their prognostic value in clinical settings, but their physiological background is not very well established. We assessed the effects of low intensity isometric (ISO) and dynamic (DYN) exercise of the lower limbs on heart rate matched intensity on traditional and entropy measures of HRV. Due to changes of afferent feedback under DYN and ISO a distinct autonomic response, mirrored by HRV measures, was hypothesized. Five-minute inter-beat interval measurements of 43 healthy males (26.0 ± 3.1 years) were performed during rest, DYN and ISO in a randomized order. Blood pressures and rate pressure product were higher during ISO vs. DYN (p < 0.001). HRV indicators SDNN as well as low and high frequency power were significantly higher during ISO (p < 0.001 for all measures). Compared to DYN, sample entropy (SampEn) was lower during ISO (p < 0.001). Concluding, contraction mode itself is a significant modulator of the autonomic cardiovascular response to exercise. Compared to DYN, ISO evokes a stronger blood pressure response and an enhanced interplay between both autonomic branches. Non-linear HRV measures indicate a more regular behavior under ISO. Results support the view of the reciprocal antagonism being only one of many modes of autonomic heart rate control. Under different conditions; the identical “end product” heart rate might be achieved by other modes such as sympathovagal co-activation as well.

ANOVA revealed a significant effect of experimental condition on all cardiovascular measures and autonomic indices. Average HR raised moderately from 65 ± 9 bpm at baseline to 85 ± 9 bpm during both types of exercise. HR during the first exercise perfectly matched HR of the subsequent exercise; average difference was only 0.3 ± 1.5 bpm (range: −2.6 to 4.3 bpm). Accordingly, HR and average R-R interval did not differ between DYN and ISO. The traditional vagal modulation HRV measure RMSSD was also not affected by the exercise mode, whereas SDNN was. Natural log-transformed HRV spectral indices HFP and LFP, the normalized powers LF n. u. and HF n. u. as well SampEn (Figure 1) were significantly different between DYN and ISO. Interestingly, SampEn did not differ between REST and DYN. There was no difference of the LF/HF ratio between REST and ISO, whereas comparison of REST vs. DYN showed a statistical trend (p = 0.077). Further, there was a small effect of condition on the HF peak frequency (F(2; 84) = 4.959, p < 0.01, η² = 0.106). While HF peak significantly shifted from 0.22 ± 0.07 Hz during REST to 0.26 ± 0.09 Hz during DYN (p < 0.05), no difference was found between REST and ISO (0.23 ± 0. 07 Hz). Post-hoc pair wise comparison between DYN and ISO showed a statistical trend for the HF peak shift (p = 0.063). SBP and RPP were moderately, DBP and MAP largely affected by the type of exercise. In comparison to DYN, myocardial oxygen consumption, reflected by RPP, was about 5% higher under ISO. Correlation analysis revealed only modest associations between traditional HRV indices and entropy measures during the different experimental conditions. Consistent correlation coefficients across all conditions were found for SampEn and R-R length only.

Mean ± SD of sample entropy during REST, ISO, and DYN; N = 43.
*** = significantly different from rest on a p-level < 0.001;
§§§ = significantly different from the respective exercise condition on a p-level < 0.001.

Role of neurotensin and opioid receptors in the cardiorespiratory effects of [Ile9]PK20, a novel antinociceptive chimeric peptide

Katarzyna Kaczynska, M Szereda-Przestaszewska, P Kleczkowska, AW Lipkowski European Journal of Pharmaceutical Sciences 63 (2014) 8–13 http://dx.doi.org/10.1016/j.ejps.2014.06.018

Ile9PK20 is a novel hybrid of opioid–neurotensin peptides synthesized from the C-terminal hexapeptide of neurotensin and endomorphin-2 pharmacophore. This chimeric compound shows potent central and peripheral antinociceptive activity in experimental animals, however nothing is known about its influence on the respiratory and cardiovascular parameters.

The present study was designed to determine the cardiorespiratory effects exerted by an intravenous injection (i.v.) of [Ile9]PK20. Share of the vagal afferentation and the contribution of NTS1 neurotensin and opioid receptors were tested.

Intravenous injection of the hybrid at a dose of 100 lg/kg in the intact, anaesthetized rats provoked an increase in tidal volume preceded by a prompt short-lived decrease. Immediately after the end of injection brief acceleration of the respiratory rhythm appeared, and was ensued by the slowing down of breathing. Changes in respiration were concomitant with a bi-phasic response of the blood pressure: an immediate increase was followed by a sustained hypotension. Midcervical vagotomy eliminated the increase in tidal volume and respiratory rate responses. Antagonist of opioid receptors – naloxone hydrochloride eliminated only [Ile9]PK20-evoked decline in tidal volume response. Blockade of NTS1 receptors with an intravenous dose of SR 142,948, lessened the remaining cardiorespiratory effects. This study depicts that [Ile9]PK20 acting through neurotensin NTS1 receptors augments the tidal component of the breathing pattern and activates respiratory timing response through the vagal pathway. Blood pressure effects occur outside vagal afferentation and might result from activation of the central and peripheral vascular NTS1 receptors. In summary the respiratory effects of the hybrid appeared not to be profound, but they were accompanied with unfavorable prolonged hypotension.

Integrative regulation of human brain blood flow

Christopher K.Willie, Yu-Chieh Tzeng, Joseph A. Fisher and Philip N. Ainslie
J Physiol 2014; 592(5): pp 841–859
http://dx.doi.org:/10.1113/jphysiol.2013.268953

Herein, we review mechanisms regulating cerebral blood flow (CBF), with specific focus on humans. We revisit important concepts from the older literature and describe the interaction of various mechanisms of cerebrovascular control. We amalgamate this broad scope of information into a brief review, rather than detailing any one mechanism or area of research. The relationship between regulatory mechanisms is emphasized, but the following three broad categories of control are explicated:

the effect of blood gases and neuronal metabolism on CBF;
buffering of CBF with changes in blood pressure, termed cerebral autoregulation; and
the role of the autonomic nervous system in CBF regulation.

With respect to these control mechanisms, we provide evidence against several canonized paradigms of CBF control. Specifically, we corroborate the following four key theses:

(1) that cerebral autoregulation does not maintain constant perfusion through a mean arterial pressure range of 60–150 mmHg;
(2) that there is important stimulatory synergism and regulatory interdependence of arterial blood gases and blood pressure on CBF regulation;

(3) that cerebral autoregulation and cerebrovascular sensitivity to changes in arterial blood gases are not modulated solely at the pial arterioles; and
(4) that neurogenic control of the cerebral vasculature is an important player in autoregulatory function and, crucially, acts to buffer surges in perfusion pressure.
Finally, we summarize the state of our knowledge with respect to these areas, outline important gaps in the literature and suggest avenues for future research.

Integrative physiological and computational approaches to understand autonomic control of cerebral autoregulation

Can Ozan Tan and J. Andrew Taylor
Exp Physiol 99.1 (2014) pp 3–15 http://dx.doi.org:/10.1113/expphysiol.2013.072355

New Findings

What is the topic of this review?

This review focuses on the autonomic control of the cerebral vasculature in health and disease from an integrative physiological and computational perspective.

What advances does it highlight?

This review highlights recent studies exploring autonomic effectors of cerebral autoregulation as well as recent advances in experimental and analytical approaches to understand cerebral autoregulation.

The brain requires steady delivery of oxygen and glucose, without which neurodegeneration occurs within minutes. Thus, the ability of the cerebral vasculature to maintain relatively steady blood flow in the face of changing systemic pressure, i.e. cerebral autoregulation, is critical to neurophysiological health. Although the study of autoregulation dates to the early 20th century, only the recent availability of cerebral blood flow measures with high temporal resolution has allowed rapid, beat-by-beat measurements to explore the characteristics and mechanisms of autoregulation. These explorations have been further enhanced by the ability to apply sophisticated computational approaches that exploit the large amounts of data that can be acquired. These advances have led to unique insights. For example, recent studies have revealed characteristic time scales wherein cerebral autoregulation is most active, as well as specific regions wherein autonomic mechanisms are prepotent. However, given that effective cerebral autoregulation against pressure fluctuations results in relatively unchanging flow despite changing pressure, estimating the pressure–flow relationship can be limited by the error inherent in computational models of autoregulatory function. This review focuses on the autonomic neural control of the cerebral vasculature in health and disease from an integrative physiological perspective. It also provides a critical overview of the current analytical approaches to understand cerebral autoregulation.

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Neural Activity Regulating Endocrine Response

Posted in Curation, Disease Biology, Genetics & Innovations in Treatment, Genome Biology, Human Sensation and Cellular Transduction: Physiology and Therapeutics, Innovations in Neurophysiology & Neuropsychology, Neurohumoral Transmission, Neurological Diseases, Pain: Etiology, Genetics & Innovations in Treatment, Pharmaceutical Analytics, Pharmaceutical Discovery, Schizophrenia, Severe Autism, Signaling & Cell Circuits, Small Molecules in Development of Therapeutic Drugs, Social Development, tagged endocrine response, hypothalamus, neurophysiology, Stress, stress related disorders on February 13, 2015| Leave a Comment »

Neural Activity Regulating Endocrine Response

Writer and Curator: Larry H. Bernstein, MD, FCAP

Defensive responses of Brandt’s voles (Lasiopodomys brandtii) to chronic predatory stress

Ibrahim M. Hegab, Guoshen Shang, Manhong Ye, Yajuan, et al.
Physiology & Behavior 126 (2014) 1–7
http://dx.doi.org/10.1016/j.physbeh.2013.12.001

Predator odors are non-intrusive natural stressors of high ethological relevance. The objective of this study was to investigate the processing of a chronic, life-threatening stimulus during repeated prolonged presentation to Brandt’s voles. One hundred and twenty voles were tested by repeated presentation of cat feces in a defensive withdrawal apparatus. Voles exposed to feces for short periods showed more avoidance, more concealment in the hide box, less contact time with the odor source, more freezing behavior, less grooming, more jumping, and more vigilant rearing than did non-exposed voles, and those exposed for longer periods. Serum levels of adrenocortico-tropic hormone and corticosterone increased significantly when animals were repeatedly exposed to cat feces for short periods. The behavioral and endocrine responses habituated during prolonged presentation of cat feces. ΔfosB mRNA expression level was highest in voles exposed to cat feces for 6 and 12 consecutive days, and subsequently declined in animals exposed to cat feces for 24 days. We therefore conclude that the behavioral and endocrine responses to repeated exposure to cat feces undergo a process of habituation, while ΔfosB changes in the medial hypothalamic region exhibit sensitization. We propose that habituation and sensitization are complementary rather than contradictory processes that occur in the same individual upon repeated presentation of the same stressor.

Neuroendocrine changes upon exposure to predator odors

Ibrahim M. Hegab, Wanhong Wei
Physiology & Behavior 131 (2014) 149–155
http://dx.doi.org/10.1016/j.physbeh.2014.04.041

Predator odors are non-intrusive and naturalistic stressors of high ethological relevance in animals. Upon exposure to a predator or its associated cues, robust physiological and molecular anti-predator defensive strategies are

elicited thereby allowing prey species to recognize, avoid and defend against a possible predation threat. In this review, we will discuss the nature of neuroendocrine stress responses upon exposure to predator odors. Predator odors can have a profound effect on the endocrine system, including activation of the hypothalamic–pituitary–adrenal axis, and induction of stress hormones such as corticosterone and adrenocorticotropic hormone. On a neural level, short-term exposure to predator odors leads to induction of the c-fos gene, while induction of ΔFosB in a different brain region is detected under chronic predation stress. Future research should aim to elucidate the relationships between neuroendocrine and behavioral outputs to gage the different levels of antipredator responses in prey species.

Involvement of NR1, NR2A different expression in brain regions in anxiety-like behavior of prenatally stressed offspring

Hongli Sun, Ning Jia, Lixia Guan, Qing Su, et al.
Behavioural Brain Research 257 (2013) 1– 7
http://dx.doi.org/10.1016/j.bbr.2013.08.044

Prenatal stress (PS) has been shown to be associated with anxiety. However, the underlying neurological mechanisms are not well understood. To determine the effects of PS on anxiety-like behavior in the adult offspring, we evaluated anxiety-like behavior using open field test (OFT) and elevated plus maze (EPM) in the 3-month offspring. Both male and female offspring showed a significant reduction of crossing counts in the center, total crossing counts, rearing counts and time spent in the center in the OFT, and only male offspring showed a decreased percentage of open-arm entries and open-arm time in open arms in the EPM. Additionally, expression of NR1 and NR2A subunit of N-methyl-d-aspartate receptor (NMDAR) in the hippocampus (HIP), prefrontal cortex (PFC) and striatum (STR) was studied. Our results showed that PS reduced NR1 and NR2A expression in the HIP, NR2A expression in the PFC and STR in the offspring. The altered NR1 and NR2A could have potential impact on anxiety-like behavior in the adult offspring exposed to PS.

Acute serotonergic treatment changes the relation between anxiety and HPA-axis functioning and periaqueductal gray activation

Dietmar Hestermann, Yasin Temel, Arjan Bloklan, Lee Wei Lim
http://dx.doi.org/10.1016/j.bbr.2014.07.003

Serotonergic (5-HT) drugs are widely used in the clinical management of mood and anxiety disorders. However, it is reported that acute 5-HT treatment elicits anxiogenic-like behavior. Interestingly, the periaqueductal gray (PAG), a midbrain structure which regulates anxiety behavior – has robust 5-HT fibers and reciprocal connections with the hypothalamic–pituitary–adrenal (HPA) axis. Although the HPA axis and the 5-HT system are well investigated, the relationship between the stress hormones induced by 5-HT drug treatment
and the PAG neural correlates of the behavior remain largely unknown. In
this study, the effects of acute and chronic treatments with buspirone (BUSP)
and escitalopram (ESCIT) on anxiety related behaviors were tested in an open-
field (OF). The treatment effects on PAG c-Fos immunoreactivity (c-Fos-ir) and corticosterone (CORT) concentration were measured in order to determine the neural endocrine correlates of anxiety-related behaviors and drug treatments. Our results demonstrate that acute BUSP and ESCIT treatments induced anxiogenic behaviors with elevation of CORT compared to the baseline. A decrease of c-Fos-ir was found in the dorsomedial PAG region of both the treatment groups. Correlation analysis showed that the CORT were not associated with the OF anxiogenic behavior and PAG c-Fos-ir. No significant differences were found in behaviors and CORT after chronic treatment.
In conclusion, acute BUSP and ESCIT treatments elicited anxiogenic response with activation of the HPA axis and reduction of c-Fos-ir in the dorsomedial PAG. Although no correlation was found between the stress hormone and
the PAG c-Fos-ir, this does not imply the lack of cause-and-effect relationship between neuroendocrine effects and PAG function in anxiety responses. These correlation studies suggest that the regulation of 5-HT system was probably disrupted by acute 5-HT treatment.

Neuroendocrine mechanisms for immune system regulation during stress in fish

Gino Nardocci,, Cristina Navarro, Paula P. Cortes, Monica Imarai
Fish & Shellfish Immunology 40 (2014) 531e538
http://dx.doi.org/10.1016/j.fsi.2014.08.001

In the last years, the aquaculture crops have experienced an explosive and intensive growth, because of the high demand for protein. This growth has increased fish susceptibility to diseases and subsequent death. The constant biotic and abiotic changes experienced by fish species in culture are challenges that induce physiological, endocrine and immunological responses. These changes mitigate stress effects at the cellular level to maintain homeostasis. The effects of stress on the immune system have been studied for many years. While acute stress can have beneficial effects, chronic stress inhibits the immune response in mammals and teleost fish. In response to stress, a signaling cascade is triggered by the activation of neural circuits in the central nervous system because the hypothalamus is the central modulator of stress. This leads to the production of catecholamines, corticosteroid-releasing hormone, adrenocorticotropic hormone and glucocorticoids, which are the essential neuroendocrine mediators for this activation. Because stress situations are energetically demanding, the neuroendocrine signals are involved in metabolic support and will suppress the “less important” immune function. Understanding the cellular mechanisms of the neuroendocrine regulation of immunity in fish will allow the development of new pharmaceutical strategies and therapeutics for the prevention and treatment of diseases triggered by stress at all stages of fish cultures
for commercial production.

Stress and immune modulation in fish

Lluis Tort
Developmental and Comparative Immunology 35 (2011) 1366–1375
http://dx.doi.org:/10.1016/j.dci.2011.07.002

Stress is an event that most animals experience and that induces a number of responses involving all three regulatory systems, neural, endocrine and immune. When the stressor is acute and short-term, the response pattern is stimulatory and the fish immune response shows an activating phase that specially enhances innate responses. If the stressor is chronic the immune response shows suppressive effects and therefore the chances of an infection may be enhanced. In addition, coping with the stressor imposes an allostatic cost that may interfere with the needs of the immune response. In this paper the mechanisms behind these immunoregulatory changes are reviewed and the role of the main neuroendocrine mechanisms directly affecting the building of the immune response and their consequences are considered.

Stress is a general term proposed by Hans Selye in 1953 (Selye, 1953) applying to a situation in which a person or an animal is subjected to a challenge that may result in a real or symbolic danger for its integrity. The stress response applies to a wide range of physiological mechanisms, including gene and protein changes, metabolism, energetics, immune, endocrine, neural and even behavioral changes that will first try to overcome that situation and then compensate for the imbalances produced by either the stressor or the consequences generated by the first array of responses.

The stress response is a general and widespread reaction in animals and it
may be assumed that this response has common traits along the phylogenetic tree. Thus, responses such as the fight and flight reaction and therefore the repertoire of energetic arrangements to serve the surplus of activity are observed in all animals. For instance, in terms of molecular responses, the increase in heat shock proteins is observed from invertebrates to fish to humans; the induction of acute phase proteins is also a common trait.

Stress and immune response

Stress and immune response. Main events regarding the principal hormones and immune mechanisms involved in acute and chronic stress

A variety of immune changes have been described after applying different kinds of stressors in fish. Hence, both activating and suppressive processes have been described following stress episodes, although the majority of changes often result in deleterious effects. Immediate responses during the activation phase enhance innate humoral immunity such as increased levels of lysozyme and C3 proteins after acute stress or the increase of the number of myeloid-type leukocytes in the peritoneum after intraperitoneal bacterial injection. Moreover, glucocorticoid receptor sites increase in head kidney leukocytes after acute handling stress.

Longer term treatments normally show suppressive effects: Sea bass subjected to crowding stress show reduced immunocompetence, as shown by reduced rates of cytotoxicity and chemiluminescence. A decrease of complement activity, lysozyme levels, agglutination activity and antibody titers is observed after 3 days onwards after repeated stress in sea bream. Stress reduces the number of circulating B-lymphocytes, and decreases the antibody response after immunization in vivo.

Effects of cortisol on cell immune responses

Effects of cortisol on cell immune responses. The arrow indicates permissive and the cross indicates suppressive. Neuroendocrine response to stress after perception by the sensors of the nervous system involves the immediate secretion of corticosteroid releasing hormone (CRH) by the preoptic nucleus of the hypothalamus. The stimulated CRH receptors in the corticotropic cells of the pituitary gland induce release of adrenocorticotropic hormone (ACTH) into the circulation that subsequently stimulates release of cortisol by the head kidney interrenal cells. ACTH as well as melanocyte-stimulating hormone (α-MSH) are derived from cleavage of the pro-opiomelanocortin gene product. In most fishes this hormone releasing sequence is taking place in seconds for CRH, seconds to minutes for ACTH, and minutes for cortisol. Since the effect of corticosteroids is exerted in most tissues, a number of studies looking at the consequences of cortisol release on the immune system have been performed but less work has been done on its precursors.

It is assumed that the nervous system plays a principal role in stress episodes as the main center for sensing the challenge and developing fight-or-flight responses. At the same time, endocrine networks are responsible for a number of responses related to the subsequent reorganization of energetic resources and modification of metabolism. Finally, the immune system is not only activated very early in the time course response but it has been shown to appear as a main partner in the regulatory network that is able to modulate non-specific immediate responses and modify hormonal activity. Therefore, in summary

all three regulatory systems have a role in the building of a stress response
(b) their interaction modulates and fine tunes the initial response to avoid excessive activation and adapting resources to the specific challenge.
These interactions will not only serve for any particular stress episode but also for adapting and preparing the response for future challenges.

Neural Input Is Critical for Arcuate Hypothalamic Neurons to Mount Intracellular Signaling Responses to Systemic Insulin and Deoxyglucose Challenges in Male Rats: Implications for Communication Within Feeding and Metabolic Control Networks

Arshad M. Khan, Ellen M. Walker, Nicole Dominguez, and Alan G. Watts
Endocrinology 155: 405–416, 2014
http://dx.doi.org:/10.1210/en.2013-1480

The hypothalamic arcuate nucleus (ARH) controls rat feeding behavior in part through peptidergic

neurons projecting to the hypothalamic paraventricular nucleus (PVH). Hindbrain catecholaminergic

(CA) neurons innervate both the PVH and ARH, and ablation of CA afferents to PVH neuroendocrine

neurons prevents them from mounting cellular responses to systemic metabolic challenges such as insulin or 2-deoxy-D-glucose (2-DG). Here, we asked whether ablating CA afferents also limits their ARH responses to the same challenges or alters ARH connectivity with the PVH. We examined ARH neurons for three features:

(1) CA afferents, visualized by dopamine-β-hydroxylase (DBH)– immunoreactivity;

(2) activation by systemic metabolic challenge, as measured by increased numbers of neurons immunoreactive (ir) for phosphorylated ERK1/2 (pERK1/2);

(3) density of PVH-targeted axons immunoreactive for the feeding control peptides Agouti-related peptide and α-melanocyte-stimulating hormone (αMSH).
Loss of PVH DBH immunoreactivity resulted in concomitant ARH reductions of DBH-ir and pERK1/2-ir neurons in the medial ARH, where AgRP neurons are enriched. In contrast, pERK1/2 immunoreactivity after systemic metabolic challenge was absent in αMSH-ir ARH neurons. Yet surprisingly, axonal αMSH immune-reactivity in the PVH was markedly increased in CA-ablated animals. These results indicate that

(1) intrinsic ARH activity is insufficient to recruit pERK1/2-ir ARH neurons during systemic metabolic challenges (rather, hindbrain-originating CA neurons are required); and

(2) rats may compensate for a loss of CA innervation to the ARH and PVH by increased expression of αMSH.
These findings highlight the existence of a hierarchical dependence for ARH responses to neural and humoral signals that influence feeding behavior and metabolism.

Acute hypernatremia dampens stress-induced enhancement of long-term potentiation in the dentate gyrus of rat hippocampus

Chiung-Chun Huang, Chiao-Yin Chu, Che-Ming Yeh , Kuei-Sen Hsu
Psychoneuroendocrinology (2014) 46, 129—140
http://dx.doi.org/10.1016/j.psyneuen.2014.04.016

Stress often occurs within the context of homeostatic threat, requiring integration of physiological and psychological demands to trigger appropriate behavioral, autonomic and endocrine responses. However, the neural mechanism underlying stress integration remains elusive. Using an acute hypernatremic challenge (2.0 M NaCl subcutaneous), we assessed whether physical state may affect subsequent responsiveness to psychogenic stressors. We found that experienced forced swimming (FS, 15 min in 25 8C), a model of psychogenic stress, enhanced long-term potentiation (LTP) induction in the dentate gyrus (DG) of the rat hippocampus ex vivo. The effect of FS on LTP was prevented when the animals were adrenalectomized or given mineralocorticoid receptor antagonist RU28318 before experiencing stress. Intriguingly, relative to normonatremic controls, hypernatremic challenge effectively elevated plasma sodium concentration and dampened FS-induced enhancement of LTP, which was prevented by adrenalectomy. In addition, acute hypernatremic challenge resulted in increased extracellular signal regulated kinase (ERK)1/2 phosphorylation in the DG and occluded the subsequent activation of ERK1/2 by FS. Moreover, stress response dampening effects by acute hypernatremic challenge remained intact in conditional oxytocin receptor knockout mice. These results suggest that acute hypernatremic challenge evokes a sustained increase in plasma corticosterone concentration,

Long-term dysregulation of brain corticotrophin and glucocorticoid receptors and stress reactivity by single early-life pain experience in male and female rats

Nicole C. Victoria, Kiyoshi Inoue, Larry J. Young, Anne Z. Murphy
Psychoneuroendocrinology (2013) 38, 3015—3028
http://dx.doi.org/10.1016/j.psyneuen.2013.08.013

Inflammatory pain experienced on the day of birth (postnatal day 0: PD0) significantly dampens behavioral responses to stress- and anxiety-provoking stimuli in adult rats. However, to date, the mechanisms by which early life pain permanently alters adult stress responses remain unknown. The present studies examined the impact of inflammatory pain, experienced on the day of birth, on adult expression of receptors or proteins implicated in the activation and termination of the stress response, including corticotrophin releasing factor receptors (CRFR1 and CRFR2) and glucocorticoid receptor (GR). Using competitive receptor autoradiography, we show that Sprague Dawley male and female rat pups administered 1% carrageenan into the intraplantar surface of the hindpaw on the day of birth have significantly decreased CRFR1 binding in the basolateral amygdala and midbrain periaqueductal gray in adulthood. In contrast, CRFR2 binding, which is associated with stress termination, was significantly increased in the lateral septum and cortical amygdala. GR expression, measured with in situ hybridization and immunohistochemistry, was significantly increased in the paraventricular nucleus of the hypothalamus and significantly decreased in the hippocampus of neonatally injured adults. In parallel, acute stress-induced corticosterone release was significantly attenuated and returned to baseline more rapidly in adults injured on PD0 in comparison to controls.
Collectively, these data show that early life pain alters neural circuits that regulate responses to and neuroendocrine recovery from stress, and suggest that pain experienced by infants in the Neonatal Intensive Care Unit may permanently alter future responses to anxiety- and stress provoking stimuli.

The Impact of Ventral Noradrenergic Bundle Lesions on Increased IL-1 in the PVN and Hormonal Responses to Stress in Male Sprague Dawley Rats

Peter Blandino Jr, CM Hueston, CJ Barnum, C Bishop, and Terrence Deak
Endocrinology 154: 2489–2500, 2013
http://dx.doi.org:/10.1210/en.2013-1075

The impact of acute stress on inflammatory signaling within the central nervous system is of interest because these factors influence neuroendocrine function both directly and indirectly. Exposure to certain stressors increases expression of the proinflammatory cytokine, Il-1 in the hypothalamus. Increased IL-1 is reciprocally regulated by norepinephrine (stimulatory) and corticosterone (inhibitory), yet neural pathways underlying increased IL-1 have not been clarified.
These experiments explored the impact of bilateral lesions of the ventral noradrenergic bundle (VNAB) on IL-1 expression in the paraventricular nucleus of the hypothalamus (PVN) after foot shock. Adult male Sprague Dawley rats received bilateral 6-hydroxydopamine lesions of the VNAB (VNABx) and were exposed to intermittent foot shock. VNABx depleted approximately 64% of norepinephrine in the PVN and attenuated the IL-1 response produced by foot shock. However, characterization of the hypothalamic-pituitary-adrenal response, a crucial prerequisite for interpreting the effect of VNABx on IL-1 expression, revealed a profound dissociation between ACTH and corticosterone.

Specifically, VNABx blocked the intronic CRH response in the PVN and the increase in plasma ACTH, whereas corticosterone was unaffected at all time points examined. Additionally, foot shock led to a rapid and profound increase in cyclooxygenase-2 and IL-1 expression within the adrenal glands, whereas more subtle effects were observed in the pituitary gland.

Together the findings were

1) demonstration that exposure to acute stress increased expression of inflammatory factors more broadly throughout the hypothalamic-pituitary-adrenal axis;

2) implication of a modest role for norepinephrine-containing fibers of the VNAB as an upstream regulator of PVN IL-1; and

3) suggestion of an ACTH-independent mechanism controlling the release of corticosterone in VNABx rats.

Stress and trauma: BDNF control of dendritic-spine formation and regression

M.R. Bennett, J. Lagopoulos
Progress in Neurobiology 112 (2014) 80–99
http://dx.doi.org/10.1016/j.pneurobio.2013.10.005

Chronic restraint stress leads to increases in brain derived neurotrophic factor (BDNF) mRNA and protein in some regions of the brain, e.g. the basal lateral amygdala (BLA) but decreases in other regions such as the CA3 region of the hippocampus and dendritic spine density increases or decreases in line with these changes in BDNF. Given the powerful influence that BDNF has on dendritic spine growth, these observations suggest that the fundamental reason for the direction and extent of changes in dendritic spine density in a particular region of the brain under stress is due to the changes in BDNF there. The most likely cause of these changes is provided by the stress initiated release of steroids, which readily enter neurons and alter gene expression, for example that of BDNF. Of particular interest is how glucocorticoids and mineralocorticoids tend to have opposite effects on BDNF gene expression offering the possibility that differences in the distribution of their receptors and of their downstream effects might provide a basis for the differential transcription of the BDNF genes. Alternatively, differences in the extent of methylation and acetylation in the epigenetic control of BDNF transcription
are possible in different parts of the brain following stress. Although present evidence points to changes in BDNF transcription being the major causal agent for the changes in spine density in different parts of the brain following stress, steroids have significant effects on downstream pathways from the TrkB receptor once it is acted upon by BDNF, including those that modulate the density of dendritic spines. Finally, although glucocorticoids play a canonical role in determining BDNF modulation of dendritic spines, recent studies have shown a role for corticotrophin releasing factor (CRF) in this regard. There is considerable improvement in the extent of changes in spine size and density in rodents with forebrain specific knockout of CRF receptor 1 (CRFR1) even when the glucocorticoid pathways are left intact. It seems then that CRF does have a role to play in determining BDNF control of dendritic spines.

Structure of the rodent BDNF gene

Structure of the rodent BDNF gene. Exons are represented as boxes and the introns as lines. Numbers of the exons are indicated in Roman numerals. The coding exon (exon IX) contains two polyadenylation sites (poly A). The start codon (ATG) that marks the initiation of transcription is indicated. The red box shows the region of exon IX coding for the pro-BDNF protein. Some exons, like exon II and IX, contain different transcript variants with alternative splice-donor sites. Also shown is part of the BDNF exon IV sequence in adults with adverse infant experiences showing cytosine methylation (M) at three of the 12 CG dinucleotide sites (numbered with superscripts). See Boulle et al. (2012).

Epigenetic mechanism associated with repression and activation of BDNF exon IV transcription.

Epigenetic mechanism associated with repression and activation of BDNF exon IV transcription. The BDNF exon IV displays 12 distinct CpG sites, which can be methylated and interact selectively with MeCp2 to form complexes that repress gene transcription (see also Fig. 1). Histone methyltransferases (HMT) are responsible for adding methyl groups at histone tails (Panel A), whereas histone deacetylases (HDAC) remove acetylation at histone tails (Panel B), both processes that repress gene transcription. Moreover, low levels of nicotinamine adenine dinucleotide (NAD) promote DNA methylation at the BDNF locus. BDNF gene activation is associated with increased histone H3 and H4 acetylation, which is mediated by histone acetyl transferase (HAT) activity. DNA demethylation might be facilitated by growth arrest and DNA damage proteins such as Gadd45b. An increased binding of CREB to its specific binding protein, CREB binding protein (CBP), is also associated with an increase in BDNF gene transcription. See Boulle et al. (2012).

signaling and epigenetic pathways in granule neurons of the dentate gyrus

Schematic representation of the signaling and epigenetic pathways in granule neurons of the dentate gyrus thought to be involved in the consolidation process of memory formation after a psychologically stressful challenge. Activation of NMDAR results in stimulation of the MAPK/ERK signaling cascade, the AC /PKA cascade and the CaMKII cascade. In conjunction with activated GR these signaling cascades result in the activation of MSK and ERK leading to the formation of dual histone acetylation marks along the c-Fos promoter and subsequently induction of gene transcription. Signaling via CREB also leads to the same outcome. The induction of gene transcription is thought to be instrumental in the consolidation of memory formation in various stressful learning events. See Trollope et al. (2012).

Model for G9a-GLP complex transcriptional activity in the hippocampus

Model for G9a/GLP complex transcriptional activity in the hippocampus during fear memory consolidation. Shown (panels A and B) is the role of G9a/GLP in the regulation of chromatin remodeling during long-term memory consolidation. Regulation of histone lysine methylation mediates active and repressive transcriptional regulation of genes in the hippocampus. The
changes in chromatin structure results in transcriptional gene silencing in the hippocampus. H3K9me2 dimethylation is associated with transcriptional silencing (not shown). The G9a/GLP complex methyltransferase is specific for producing this modification. Abbreviations: Ac, acetylation; M, methylation; MLLI, histone H3 lysine 4 methyltransferase (which regulates memory formation); H3K9me2, histone H3 lysine 9 dimethylation; HAT, histone acetyltransferase; G9a/GLP, G9a/G9a-like protein (GLP) complex methyltransferase.

Modification of serotonin reuptake transport, with inhibitors such as fluoxetine, augments BDNF exon I mRNA levels in the BLA as well as in the hippocampus. This augmentation is lost and replaced by a decrease in BDNF levels if the mice are homozygous for the BDNF Val66Met SNP. A better outcome is obtained for erasing fear memories in PTSD subjects than using D-cycloserine if a combination is used of extinction training with chronic fluoxetine treatment that augments BDNF exon I mRNA.

Conclusion

The following points are suggested by the present review on identifying the changes in dendritic spine synapses in neural networks under stress, the mechanisms that drive these, and how these networks can be reinstated to normality.

Dendritic spines and BDNF

Activation of BDNF leads to the sprouting of dendrites in many areas of the brain, such as CA1 in the hippocampus. As glucocorticoids decrease BDNF expression they decrease dendritic spine density in these areas . Thus activation of both GR and MR with corticosterone leads to an increase in dendritic spine turnover on pyramidal neurons in these areas. In other areas of the brain glucocorticoids do not have this. Extinction of a fear memory, such as, of the negative effects of opiate withdrawal, involves increases of BDNF mRNA and protein in the ventromedial prefrontal cortex, through the action of CREB at histone H3 of the BDNF exon I transcript promoter with acetylation of the histone. This could be enhanced before extinction training with histone deacetylase inhibitors such as trichostatin A or inhibitors such as U0126 of ERK.
Major risk factors for PTSD are low levels of cortisol in the blood immediately after the trauma occasion; and before the trauma, in peripheral blood mononuclear cells, the presence of high GR numbers, low FKBP5 expression, and high levels of GILZ mRNA. All of these risk factors are involved in the action of cytoplasmic GR in modulating gene transduction, including most likely that for the BDNF gene, as well as regulating the capacity for BDNF itself to act. This emphasis on GR in PTSD is enforced by the observations that there is an association between two polymorphisms in the GR gene (N363S and Bcl1) and PTSD as there is between that of FKBP5 and GILZ on the one hand and the capacity of GR to modulate gene function on the other.

Brain-derived neurotrophic factor in the amygdala mediates susceptibility to fear conditioning

Dylan Chou, Chiung-Chun Huang, Kuei-Sen Hsu
Experimental Neurology 255 (2014) 19–29
http://dx.doi.org/10.1016/j.expneurol.2014.02.016

Fear conditioning in animals has been used extensively tomodel clinical anxiety disorders. While individual animals exhibit marked differences in their propensity to undergo fear conditioning, the physiologically relevant mediators have not yet been fully characterized. Here, we demonstrate that C57BL/6 inbred mouse strain subjected to a regimen of chronic social defeat stress (CSDS) can be separated into susceptible and resistant subpopulations that display different levels of fear responses in an auditory fear conditioning paradigm. Susceptible mice had significantly more c-Fos protein expression
in neurons of the basolateral amygdala (BLA) following CSDS and showed exaggerated conditioned fear responses, while there were no significant differences between groups in innate anxiety- and depressive-like behaviors. Through the use of conditional brain-derived neurotrophic factor (BDNF) knockout strategies, we find that elevated BLA BDNF level following fear conditioning training is a key mediator contributing to determine the levels of conditioned fear responses. Our results also show that relative to susceptible mice, resistant mice had a much faster recovery from conditioned stimuli-induced cardiovascular and corticosterone responses. Systemic administration of norepinephrine reuptake inhibitor atomoxetine increased c-Fos protein expression in BLA neurons following fear conditioning training and promoted the expression of conditioned fear in resistant mice. Conversely, administration of β-adrenergic receptor antagonist propranolol reduced fear conditioning training-induced c-Fos protein expression in BLA neurons and reduced conditioned fear responses in susceptible mice. These findings reveal a novel role for the BDNF signaling within the BLA in mediating individual differences in autonomic, neuroendocrine and behavioral reactivity to fear conditioning.

Melanocortin-4 receptor in the medial amygdala regulates emotional stress-induced anxiety-like behavior, anorexia and corticosterone secretion

Jing Liu, Jacob C. Garza, Wei Li and Xin-Yun Lu
Intl J Neuropsychopharmacology (2013), 16, 105–120.
http://dx.doi.org:/10.1017/S146114571100174X

The central melanocortin system has been implicated in emotional stress-induced anxiety, anorexia and activation of the hypothalamo-pituitary-adrenal (HPA) axis. However, the underlying neural substrates have not been identified. The medial amygdala (MeA) is highly sensitive to emotional stress and expresses high levels of the melanocortin-4 receptor (MC4R). This study investigated the effects of activation and blockade of MC4R in the MeA
on anxiety-like behavior, food intake and corticosterone secretion. We demonstrate that MC4R-expressing neurons in the MeA were activated by acute restraint stress, as indicated by induction of c-fos mRNA expression. Infusion of a selective MC4R agonist into the MeA elicited anxiogenic-like effects in the elevated plus-maze test and decreased food intake. Local MeA infusion of SHU 9119, an MC4R antagonist, on the other hand, blocked restraint stress-induced anxiogenic and anorectic effects. Moreover, plasma corticosterone levels were increased by intra-MeA infusion of the MC4R agonist under non-stressed conditions and restraint stress-induced elevation of plasma corticosterone levels was attenuated by pretreatment with SHU 9119 in the MeA. Thus, stimulating MC4R in the MeA induces stress-like anxiogenic and anorectic effects as well as activation of the HPA axis, whereas antagonizing MC4R in this region blocks such effects induced by restraint stress. Together, our results implicate MC4R signaling in the MeA in behavioral and endocrine responses to stress.

The neuroendocrine functions of the parathyroid hormone 2 receptor

Arpád Dobolyi, Eugene Dimitrov, Miklós Palkovits and Ted B. Usdin
Front in Endocr Oct 2012 | Volume 3 | Article 121, 1-10
http://dx.doi.org:/10.3389/fendo.2012.00121

The G-protein coupled parathyroid hormone 2 receptor (PTH2R) is concentrated in endocrine and limbic regions in the forebrain. Its endogenous ligand, tuberoinfundibular peptide of 39 residues (TIP39), is synthesized in only two brain regions, within the posterior thalamus and the lateral pons.TIP39-expressing neurons have a widespread projection pattern, which matches the PTH2R distribution in the brain. Neuroendocrine centers including the preoptic area, the periventricular, paraventricular, and arcuate nuclei contain the highest density of PTH2R-positive networks. The administration of TIP39 and an antagonist of the PTH2R as well as the investigation of mice that lack functional TIP39 and PTH2R revealed the involvement of the PTH2R in a variety of neural and neuroendocrine functions. TIP39 acting via the PTH2R modulates several aspects of the stress response. It evokes corticosterone release by activating corticotropin-releasing hormone-containing neurons in the hypothalamic paraventricular nucleus. Block of TIP39 signaling elevates the anxiety state
of animals and their fear response, and increases stress-induced analgesia.

TIP39 has also been suggested to affect the release of additional pituitary hormones including arginine-vasopressin and growth hormone. A role of the TIP39-PTH2R system in thermoregulation was also identified. TIP39 may play
a role in maintaining body temperature in a cold environment via descending excitatory pathways from the preoptic area. Anatomical and functional studies also implicated the TIP39-PTH2R system in nociceptive information processing. Finally, TIP39 induced in postpartum dams may play a role in the release of prolactin during lactation. Potential mechanisms leading to the activation ofTIP39 neurons and how they influence the neuroendocrine system are also described. The unique TIP39-PTH2R neuromodulator system provides the possibility for developing drugs with a novel mechanism of action to control neuroendocrine disorders.

Interaction of the Serotonin Transporter-Linked Polymorphic Region and Environmental Adversity: Increased Amygdala-Hypothalamus Connectivity as a Potential Mechanism Linking Neural and Endocrine Hyperreactivity

Nina Alexander, T Klucken, G Koppe, R Osinsky, B Walter, et al.
Biol Psychiatry 2012;72:49–56
http://dx.doi.org:/10.1016/j.biopsych.2012.01.030

Background: Gene by environment (GE) interaction between genetic variation in the promoter region of the serotonin transporter gene (serotonin transporter-linked polymorphic region [5-HTTLPR]) and stressful life events (SLEs) has been extensively studied in the context of depression. Recent findings suggest increased neural and endocrine stress sensitivity as a possible mechanism conveying elevated vulnerability to psychopathology. Furthermore, these GE mediated alterations very likely reflect interrelated biological processes. Methods: In the present functional magnetic resonance imaging study, amygdala reactivity to fearful stimuli was assessed in healthy male adults (n[1]44),who were previously found to differ with regard to endocrine stress reactivity as a function of 5-HTTLPRSLEs. Furthermore, functional connectivity between the amygdala and the hypothalamus was measured as a potential mechanism linking elevated neural and endocrine responses during stressful/threatening situations. The study sample was carefully preselected regarding 5-HTTLPR genotype and SLEs. Results: We report significant GE interaction on neural response patterns and functional amygdala-hypothalamus connectivity. Homozygous carriers of the 5-HTTLPR S’ allele with a history of SLEs (S’S’/high SLEs group) displayed elevated bilateral amygdala activation in response to fearful faces. Within the same sample, a comparable GE interaction effect has previously been demonstrated regarding increased cortisol reactivity, indicating a cross-validation of heightened biological stress sensitivity. Furthermore, S’S’/high SLEs subjects were characterized by an increased functional coupling between the right amygdala and the hypothalamus, thus indicating a potential link between neural and endocrine hyperreactivity.

Amygdala reactivity to fearful faces as a function of the serotonin transporter-linked polymorphic region (5-HTTLPR)

Amygdala reactivity to fearful faces as a function of the serotonin transporter-linked polymorphic region (5-HTTLPR) stressful life events (SLEs). The color bar depicts t values for the gene by environment interaction effect. For illustration reasons, the data were thresholded with a t value at 2.5 (see color bar for exact t values).

We report a significant 5-HTTLPRxSLEs interaction effect on bilateral amygdala reactivity to fearful faces in a sample of healthy male adults. As hypothesized, S’S’/high SLEs individuals appeared to be most reactive, which can be interpreted in terms of elevated amygdala reactivity to briefly presented (phasic) aversive stimuli. Interestingly, we have observed a similar response pattern regarding cortisol reactivity to acute stress within the same sample, indicating a cross-validation of neuroendocrine hyperreactivity to threatening/stressful stimuli as a function of 5-HTTLPRxSLEs.

Thus, our results are in line with findings from a small sample sized (n = 15) study reporting a positive association between amygdala reactivity to fearful faces and SLEs in S allele carriers during an unconscious fear processing condition. In contrast, a study using a comparable paradigm and sample size (n = 44) to our own found amygdala activity in the contrast neutral faces versus fixation to be negatively associated with SLEs in S allele carriers. The authors interpret the latter finding in support of a tonic model, by which SLEs interact with 5-HTTLPR on amygdala resting activation. Similar inconsistencies have been reported regarding the association of 5-HTTLPR and amygdala activation independent of environmental adversity, with studies supporting either a phasic or tonic model. Likewise, increased resting blood perfusion in S allele carriers has been reported in independent studies, whereas the largest study
to date could not replicate these findings.

Functional connectivity between the right amygdala as the seed region

Functional connectivity between the right amygdala as the seed region

(blue circle, right figure) and the hypothalamus (red circles). The middle figure depicts significant differences in activation patterns between the S’S’/high stressful life events (SLEs) and the L’/low SLEs groups and the left figure displays significant differences between S’S’/high SLEs and S’S’/high SLEs subjects. For illustration reasons, threshold was t =2.5 b (below).
(B) Surface plot of functional connectivity at the z-slice location of the peak coordinate. Voxel intensities are given in t values. 5-HTTLPR, serotonin-transporter-linked polymorphic region.

In conclusion, we report increased amygdala responsivity to aversive stimuli in healthy S’S’/high SLEs subjects who have previously been shown to display elevated cortisol secretion in response to psychosocial stress. Thus, our findings contribute to the current debate on potential mechanisms mediating susceptibility for the development of psychiatric disorders as a function of 5-HTTLPRxSLEs. Moreover, the present study extends previous findings by demonstrating altered functional coupling between the amygdala and the hypothalamus, thus indicating a potential link between threat/stress related neural and endocrine alterations associated with 5-HTTLPR x SLEs.

Identifying Molecular Substrates in a Mouse Model of the Serotonin Transporter Environment Risk Factor for Anxiety and Depression

Valeria Carola, Giovanni Frazzetto, Tiziana Pascucci, Enrica Audero, et al.
Biol Psychiatry 2008;63:840–846
http://dx.doi.org:/10.1016/j.biopsych.2007.08.013

Background: A polymorphism in the serotonin transporter (5-HTT) gene modulates the association between adverse early experiences and risk for major depression in adulthood. Although human imaging studies have begun to elucidate the neural circuits involved in the 5-HTT environment risk factor, a molecular understanding of this phenomenon is lacking. Such an understanding might help to identify novel targets for the diagnosis and therapy of mood disorders. To address this need, we developed a gene-environment screening paradigm in the mouse.

Methods: We established a mouse model in which a heterozygous null mutation in 5-HTT moderates the effects of poor maternal care on adult anxiety and depression-related behavior. Biochemical analysis of brains from these animals was performed to identify molecular substrates of the gene, environment, and gene environment effects.

Results: Mice experiencing low maternal care showed deficient ϒ-aminobutyric acid–A receptor binding in the amygdala and 5-HTT heterozygous null mice showed decreased serotonin turnover in hippocampus and striatum. Strikingly, levels of brain-derived neurotrophic factor (BDNF) messenger RNA in hippocampus were elevated exclusively in 5-HTT heterozygous null mice experiencing poor maternal care, suggesting that developmental programming of hippocampal circuits might underlie the 5-HTT environment risk factor.

Conclusions: These findings demonstrate that serotonin plays a similar role in modifying the long-term behavioral effects of rearing environment in diverse mammalian species and identifies BDNF as a molecular substrate of this risk factor. In summary, we have produced a mouse model of the 5-HTT environment risk factor for human depression and have used this model to identify molecular substrates underlying this risk factor.

Elevated GABA-A receptor expression in amygdala, decreased 5-HT turnover in hippocampus, and enhanced BDNF expression in hippocampus each correlated significantly with the behavioral phenotype seen in our mice. In particular, increased expression of BDNF in CA1 pyramidal neurons was found in mice with reduced 5-HTT function and exposed to low maternal care. This defect was accompanied by an increased bias in the response to threatening cues as assessed by ambiguous cue fear conditioning.

Our data suggest that alterations in hippocampal gene expression and function underlie at least part of the interaction between 5-HTT and rearing environment and point to a role for this structure in the increased anxiety and depression-related behavior that is a risk factor for major depression.

Gene—environment interactions predict cortisol responses after acute stress: Implications for the etiology of depression

Nina Alexander, Yvonne Kuepper, Anja Schmitz, Roman Osinsky, et al.
Psychoneuroendocrinology (2009) 34, 1294—1303
http://dx.doi.org:/10.1016/j.psyneuen.2009.03.017

Background: Growing evidence suggests that the serotonin transporter polymorphism (5-HTTLPR) interacts with adverse environmental influences to produce an increased risk for the development of depression while the underlying mechanisms of this association remain largely unexplored. As one potential intermediate phenotype, we investigated alterations of hypothalamic—pituitary—adrenal (HPA) axis responses to stress in individuals with no history of psychopathology depending on both 5-HTTLPR and stressful life events.

Methods: Healthy male adults (N = 100) were genotyped and completed a questionnaire on severe stressful life events (Life Events Checklist). To test for gene-by-environment interactions on endocrine stress reactivity, subjects were exposed to a standardized laboratory stress task (Public Speaking). Saliva cortisol levels were obtained at 6 time points prior to the stressor and during an extended recovery period.

Results: Subjects homozygous for the s-allele with a significant history of stressful life events exhibited markedly elevated cortisol secretions in response to the stressor compared to all other groups, indicating a significant gene-by-environment interaction on endocrine stress reactivity. No main effect of either 5-HTTLPR (biallelic and triallelic) or stressful life events on cortisol secretion patterns appeared.

Conclusion: This is the first study reporting that 5-HTTLPR and stressful life events interact to predict endocrine stress reactivity in a non-clinical sample. Our results underpin the potential moderating role of HPA-axis hyper-reactivity as a premorbid risk factor to increase the vulnerability for depression in subjects with low serotonin transporter efficiency and a history of severe life events.

The immune system and developmental programming of brain and behavior

Staci D. Bilbo, Jaclyn M. Schwarz
Frontiers in Neuroendocrinology 33 (2012) 267–286
http://dx.doi.org/10.1016/j.yfrne.2012.08.006

The brain, endocrine, and immune systems are inextricably linked. Immune molecules have a powerful impact on neuroendocrine function, including hormone–behavior interactions, during health as well as sickness. Similarly, alterations in hormones, such as during stress, can powerfully impact immune function or reactivity. These functional shifts are evolved, adaptive responses that organize changes in behavior and mobilize immune resources, but can also lead to pathology or exacerbate disease if prolonged or exaggerated. The developing brain in particular is exquisitely sensitive to both endogenous and exogenous signals, and increasing evidence suggests the immune system has a critical role in brain development and associated behavioral outcomes for the life of the individual. Indeed, there are associations between many neuropsychiatric disorders and immune dysfunction, with a distinct etiology in neurodevelopment. The goal of this review is to describe the important role of the immune system during brain development, and to discuss some of the many ways in which immune activation during early brain development can affect the later-life outcomes of neural function, immune function, mood and cognition.

Neuroplasticity signaling pathways linked to the pathophysiology of schizophrenia

Darrick T. Balua, Joseph T. Coyle
Neuroscience and Biobehavioral Reviews 35 (2011) 848–870
http://dx.doi.org:/10.1016/j.neubiorev.2010.10.005

Schizophrenia is a severe mental illness that afflicts nearly 1% of the world’s population. One of the cardinal pathological features of schizophrenia is perturbation in synaptic connectivity. Although the etiology of schizophrenia is unknown, it appears to be a developmental disorder involving the interaction of a potentially large number of risk genes, with no one gene producing a strong effect except rare, highly penetrant copy number variants. The purpose of this review is to detail how putative schizophrenia risk genes (DISC-1, neuregulin/ErbB4, dysbindin, Akt1, BDNF, and the NMDA receptor) are involved in regulating neuroplasticity and how alterations in their expression may contribute to the disconnectivity observed in schizophrenia. Moreover, this review highlights how many of these risk genes converge to regulate common neurotransmitter systems and signaling pathways. Future studies aimed at elucidating the functions of these risk genes will provide new insights into the pathophysiology of schizophrenia and will likely lead to the nomination of novel therapeutic targets for restoring proper synaptic connectivity in the brain in schizophrenia and related disorders.

Glutamate receptor composition of the post-synaptic density is altered in genetic mouse models of NMDA receptor hypo- and hyperfunction

Darrick T. Balu, Joseph T. Coyle
Brain Research 1392 (2011 ) 1–7
http://dx.doi.org:/10.1016/j.brainres.2011.03.051

The N-methyl-D-aspartate receptor (NMDAR) and α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (AMPAR) are ionotropic glutamate receptors responsible for excitatory neurotransmission in the brain. These excitatory synapses are found on dendritic spines, with the abundance of receptors concentrated at the postsynaptic density (PSD).
We utilized two genetic mouse models, the serine racemase knockout (SR−/−) and the glycine transporter subtype 1 heterozygote mutant (GlyT1+/−), to determine how constitutive NMDAR hypo- and hyperfunction, respectively, affect the glutamate receptor composition of the PSD in the hippocampus and prefrontal cortex (PFC).

Using cellular fractionation, we found that SR−/− mice had elevated protein levels of NR1 and NR2A NMDAR subunits specifically in the PSD-enriched fraction from the hippocampus, but not from the PFC. There were no changes in the amounts of AMPAR subunits (GluR1, GluR2), or PSD protein of 95 kDa (PSD95) in either brain region. GlyT1+/− mice also had elevated protein expression of NR1 and NR2A subunits in the PSD, as well as an increase in total protein. Moreover, GlyT1+/− mice had elevated amounts of GluR1 and GluR2 in the PSD, and higher total amounts of GluR1. Similar to SR−/− mice, there were no protein changes observed in the PFC. These findings illustrate the complexity of synaptic adaptation to altered NMDAR function.

Interleukin-1 (IL-1): A central regulator of stress responses

Inbal Goshen, Raz Yirmiya
Frontiers in Neuroendocrinology 30 (2009) 30–45
http://dx.doi.org:/10.1016/j.yfrne.2008.10.001

Ample evidence demonstrates that the pro-inflammatory cytokine interleukin-1 (IL-1), produced following exposure to immunological and psychological challenges, plays an important role in the neuroendocrine and behavioral stress responses. Specifically, production of brain IL-1 is an important link in stress induced activation of the hypothalamus-pituitary-adrenal axis and secretion of glucocorticoids, which
mediate the effects of stress on memory functioning and neural plasticity, exerting beneficial effects at low levels and detrimental effects at high levels. Furthermore, IL-1 signaling and the resultant glucocorticoid secretion mediate the development of depressive symptoms associated with exposure to acute and chronic stressors, at least partly via suppression of hippocampal neurogenesis. These findings indicate
that whereas under some physiological conditions low levels of IL-1 promote the adaptive stress responses necessary for efficient coping, under severe and chronic stress conditions blockade of IL-1 signaling can be used as a preventive and therapeutic procedure for alleviating stress-associated neuropathology
and psychopathology.

IL-1 mediates stress-induced activation of the HPA axis

IL-1 mediates stress-induced activation of the HPA axis. Immunological and
psychological stressors increase the levels of IL-1 in various brain areas, including
several brain stem nuclei, the hypothalamus and the hippocampus. In turn, IL-1
induces the secretion of CRH from the hypothalamic paraventricular nucleus (PVN),
ACTH from the pituitary and glucocorticoids from the adrenal. Following immunological
stressors, peripheral IL-1 can directly influence brain stem nuclei, such as
the nucleus tractus solitarius (NTS) and ventrolateral medulla (VLM) as well as the
hypothalamus via penetration to adjacent circumventricular organs, (the area
postrema (AP) and the organum vasculosum of the lamina terminalis (OVLT),
respectively). Concomitantly, IL-1 in the periphery can activate vagal afferents,
which innervate and activate the NTS and VLM. These nuclei project to the
hypothalamus, in which the secretion of NE induces further elevation of IL-1 levels,
possibly by microglial activation. Psychological stressors can also activate the NTS
and VLM, either by intrinsic brain circuits or via vagal feedback from physiological
systems (e.g., the cardiovascular system) that are stimulated by the sympathetic
nervous system. Similarly to their role in immunological stress, the NTS and VLM
then elevate hypothalamic IL-1 levels, stimulating the CRH neurons.

The inverted U-shaped effect of IL-1 on memory and plasticity is mediated by glucocorticoids

The inverted U-shaped effect of IL-1 on memory and plasticity is mediated by glucocorticoids. The influence of IL-1 on memory and plasticity follows an inverted Ushape pattern, i.e., learning-associated increase in IL-1 levels is needed for memory formation (green), whereas any deviation from the physiological range, either by excess elevation in IL-1 levels or by blockade of IL-1 signaling, results in memory and plasticity impairment (red). Low dose GCs can also facilitate memory, whereas chronic or severe stressors, as well as high GC levels, can impair memory and neural plasticity. Studies on the implications of the interaction between stress, IL-1 and GCs on memory
and plasticity show that IL-1 mediates the detrimental effects of stress on memory, and that GCs are involved in both the detrimental and the beneficial effects of IL-1 on memory formation. Based on these studies, the following model is proposed: stressful stimuli induce an increase in brain IL-1 levels, which in turn contributes to the activation of the HPA axis. Subsequently, the secretion of GCs affects memory and plasticity processes in an inverted U-shaped pattern.

Immune modulation of learning, memory, neural plasticity and neurogenesis

Raz Yirmiya ⇑, Inbal Goshen
Brain, Behavior, and Immunity 25 (2011) 181–213
http://dx.doi.org:/10.1016/j.bbi.2010.10.015

Over the past two decades it became evident that the immune system plays a central role in modulating learning, memory and neural plasticity. Under normal quiescent conditions, immune mechanisms are activated by environmental/psychological stimuli and positively regulate the remodeling of neural circuits, promoting memory consolidation, hippocampal long-term potentiation (LTP) and neurogenesis.
These beneficial effects of the immune system are mediated by complex interactions among brain cells with immune functions (particularly microglia and astrocytes), peripheral immune cells (particularly T cells and macrophages), neurons, and neural precursor cells. These interactions involve the responsiveness of non-neuronal cells to classical neurotransmitters (e.g., glutamate and monoamines) and hormones
(e.g., glucocorticoids), as well as the secretion and responsiveness of neurons and glia to low levels of inflammatory cytokines, such as interleukin (IL)-1, IL-6, and TNFa, as well as other mediators, such as prostaglandins and neurotrophins. In conditions under which the immune system is strongly activated by infection or injury, as well as by severe or chronic stressful conditions, glia and other brain immune cells change their morphology and functioning and secrete high levels of pro-inflammatory
cytokines and prostaglandins. The production of these inflammatory mediators disrupts the delicate balance needed for the neurophysiological actions of immune processes and produces direct detrimental effects on memory, neural plasticity and neurogenesis. These effects are mediated by inflammation induced neuronal hyper-excitability and adrenocortical stimulation, followed by reduced production of neurotrophins and other plasticity-related molecules, facilitating many forms of neuropathology
associated with normal aging as well as neurodegenerative and neuropsychiatric diseases.

It is now firmly established that the immune system can modulate brain functioning and behavioral processes. This modulation is exerted by plasticity are among the most important aspects of brain functioning that are modulated by immune mechanisms. The aim of the present review is to present a comprehensive and integrative view of the complex dual role of the immune system in learning,memory, neural plasticity and neurogenesis. The first part of the review will focus on the physiological
beneficial effects of the immune system under normal, quiescent conditions. Under such conditions, immune mechanisms are activated by environmental/psychological stimuli and positively regulate neuroplasticity and neurogenesis, promoting learning, memory, and hippocampal long-term potentiation (LTP). The second part of the review will focus on the detrimental effects of inflammatory conditions induced by infections and injury as well as severe or chronic stress, demonstrating that under such
conditions the delicate physiological balance between immune and neural processes is disrupted, resulting in neuronal hyperexcitability, hormonal aberrant ions, reduced neurotrophic factors production and suppressed neurogenesis, leading to impairments in learning, memory and neuroplasticity.

A systemic model of the beneficial role of immune processes in behavioral and neural plasticity

A systemic model of the beneficial role of immune processes in behavioral and neural plasticity. Learning, memory and synaptic plasticity involve neural activation of hippocampal circuits by glutamatergic inputs that originate mainly in multiple cortical areas. Long-term memory consolidation also requires emotional (limbic) activation (particularly of the amygdala and hypothalamus), inducing a mild stressful condition, which in turn results in HPA axis and sympathetic nervous system (SNS) stimulation. The peripheral organs that are the targets of these systems (e.g., the adrenal glad, heart, blood vessels and gastrointestinal (GI) tract), in turn, send afferent inputs to the brain that culminate in stimulation of receptors for glucocorticoids, norepinephrine, dopamine and serotonin on hippocampal cells. These inputs are critical for memory consolidation, neural plasticity and neurogenesis. Furthermore, these inputs induce the production of IL-1, and possibly other cytokines, chemokines and immune mediators in the hippocampus, as well as in other brain areas (such as the hypothalamus and brain stem) that are critically important for neurobehavioral plasticity. Moreover, these cytokines, in turn further activate the HPA axis and SNS, thus participating in a brain-to-body-to-brain reverberating feedback loops.

Chemokines and the hippocampus: A new perspective on hippocampal plasticity and vulnerability

Lauren L. Williamson, Staci D. Bilbo
Brain, Behavior,and Immunity 30(2013)186–194
http://dx.doi.org/10.1016/j.bbi.2013.01.077

Chemokines roles within the hippocampus

Chemokines have important roles within the hippocampus and may modulate plasticity and vulnerability within this unique structure. Neuroimmune signaling can occur across the blood-brain-barrier (BBB) via endothelial cells, astrocytes, and microglia within the BBB that recapitulate the immune signal from the periphery by secreting their own cohort of cytokines into the brain. Chemokines recruit cells to sites of injury as well . Microglia receive input from neurons via several membrane-bound and secreted factors, including neuronal CX3CL1 (fractalkine) and its receptor, CX3CR1, on microglia, which allow direct neuroimmune interaction. CXCL12 is released from vesicles concomitantly with GABA from basket cells onto immature neurons in the DG granule cell layer. In the healthy brain, chemokines may modulate neuronal signaling during behavior, though this phenomenon remains to be explored. The spatial and temporal signaling and cellular sources of chemokines and their receptors are critical for understanding

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Endocrine Action on Midbrain

Posted in Biological Networks, Calcium Signaling, Cell Biology, Cerebrovascular and Neurodegenerative Diseases, Chemical Biology and its relations to Metabolic Disease, Curation, Cytoskeleton, Developmental biology, Diabetes Mellitus, Disease Biology, Embryology, Endocrine Diseases, Genomic Endocrinology, Innovations in Neurophysiology & Neuropsychology, Lipid metabolism, Metabolism, Metabolomics, Neurodegenerative Diseases, Neurological Diseases, Nutrition, Pharmaceutical Analytics, Pharmaceutical Discovery, Pharmacologic toxicities, Preimplantation Genetic Diagnosis and Reproductive Genomics, Proteomics, Reproductive Andrology, Embryology, Genomic Endocrinology, Preimplantation Genetic Diagnosis and Reproductive Genomics, Signaling, Signaling & Cell Circuits, Small Molecules in Development of Therapeutic Drugs, tagged brain, cannabinoids, Cognition, glutamate, gonadotrophs, GPCRs, midbrain, neurodegeneration, neuroendocrinology, neuronal plasticity, sexual dimorphism, sterois hormones on February 12, 2015| Leave a Comment »

Endocrine Action on Midbrain

Writer and Curator: Larry H. Bernstein, MD, FCAP

Brain’s Role in Browning White Fat
Insulin and leptin act on specialized neurons in the mouse hypothalamus to promote conversion of white to beige fat.

By Anna Azvolinsky | January 15, 2015

JUSTIN HEWLETT, MNHS MULTIMEDIA, MONASH UNIVERSITY

Ever since energy-storing white fat has been shown to convert to metabolically active beige fat, through a process called browning, scientists have been trying to understand how this switch occurs. The immune system has been shown to contribute to activation of brown fat cells. Now, researchers from Monash University in Australia and their colleagues have shown that insulin and leptin—two hormones that regulate glucose metabolism and satiety and hunger cues—activate “satiety” neurons in the mouse hypothalamus to promote the conversion of white fat to beige. The results are published today (January 15) in Cell.

Hypothalamic appetite-suppressing proopiomelanocortin (POMC) neurons are known to relay the satiety signals in the bloodstream to other parts of the brain and other tissues to promote energy balance. “What is new here is that one way that these neurons promote calorie-burning is to stimulate the browning of white fat,” said Xiaoyong Yang, who studies the molecular mechanisms of metabolism at the Yale University School of Medicine, but was not involved in the work. “The study identifies how the brain communicates to fat tissue to promote energy dissipation.”

“The authors show that [insulin and leptin] directly interact in the brain to produce nervous-system signaling both to white and brown adipose tissue,” said Jan Nedergaard, a professor of physiology at Stockholm University who also was not involved in the study. “This is a nice demonstration of how the acute and chronic energy status talks to the thermogenic tissues.”

Although the differences between beige and brown fat are still being defined, the former is currently considered a metabolically active fat—which converts the energy of triglycerides into heat—nestled within white fat tissue. Because of their energy-burning properties, brown and beige fat are considered superior to white fat, so understanding how white fat can be browned is a key research question. Exposure to cold can promote the browning of white fat, but the ability of insulin and leptin to act in synergy to signal to the brain to promote browning was not known before this study, according the author Tony Tiganis, a biochemist at Monash.

White fat cells steadily produce leptin, while insulin is produced by cells of the pancreas in response to a surge of glucose into the blood. Both hormones are known to signal to the brain to regulate satiety and body weight. To explore the connection between this energy expenditure control system and fat tissue, Garron Dodd, a postdoctoral fellow in Tiganis’s laboratory, and his colleagues deleted one or both of two phosphatase enzymes in murine POMC neurons. These phosphatase enzymes were previously known to act in the hypothalamus to regulate both glucose metabolism and body weight, each regulating either leptin or insulin signaling. When both phosphatases were deleted, mice had less white fat tissue and increased insulin and leptin signaling.

“These [phosphatase enzymes] work in POMC neurons by acting as ‘dimmer switches,’ controlling the sensitivity of leptin and insulin receptors to their endogenous ligands,” Dodd told The Scientist in an e-mail. The double knockout mice also had an increase in beige fat and more active heat-generating brown fat. When fed a high-fat diet, unlike either the single knockout or wild-type mice, the double knockout mice did not gain weight, suggesting that leptin and insulin signaling to POMC neurons is important for controlling body weight and fat metabolism.

The researchers also infused leptin and insulin directly into the hypothalami of wild-type mice, which promoted the browning of white fat. But when these hormones were infused but the neuronal connections between the white fat and the brain were physically severed, browning was prevented. Moreover, hormone infusion and cutting the neuronal connection to only a single fat pad resulted in browning only in the fat pad that maintained signaling ties to the brain. “This really told us that direct innervation from the brain is necessary and that these hormones are acting together to regulate energy expenditure,” said Tiganis.

These results are “really exciting as, perhaps, resistance to the actions of leptin and insulin in POMC neurons is a key feature underlying obesity in people,” said Dodd.

Another set of neurons in the hypothalamus, the agouti-related protein expressing (AgRP) or “hunger” neurons, are activated by hunger signals and promote energy storage. Along with Tamas Horvath, Yale’s Yang recently showed that fasting activates AgRP neurons that then suppress the browning of white fat. “These two stories are complimentary, providing a bigger picture: that the hunger and satiety neurons control browning of fat depending on the body’s energy state,” said Yang. Activation of POMC neurons during caloric intake protects against diet-induced obesity while activation of AgRP neurons tells the body to store energy during fasting.

Whether these results hold up in humans has yet to be explored. Expression of the two phosphatases in the hypothalamus is known to be higher in obese people, but it is not clear whether this suppresses the browning of white fat.

“One of the next big questions is whether this increased expression and prevention of insulin plus leptin signaling, and conversion of white to brown fat perturbs energy balance and promotes obesity,” said Tiganis. Another, said Dodd, is whether other parts of the brain are involved in signaling to and from adipose tissue.

Dodd et al., “Leptin and insulin act on POMC neurons to promote the browning of white fat,”

Cell, 2015. http://dx.doi.org:/10.1016/j.cell.2014.12.022 http://medicine.yale.edu/lab/horvath/index.aspx

Our main interest is the neuroendocrine regulation of homeostasis with particular emphasis on metabolic disorders, such as obesity and diabetes, and the effect of metabolic signals on higher brain functions and neurodegeneration. We have active research programs to pursue the role of synaptic plasticity in the mediation of peripheral hormones’ effects on the central nervous system.

We also study the role of mitochondrial membrane potential in normal and pathological brain functions with particular emphasis on the acute effect of mitochondria in neuronal transmission and neuroprotection. We combine classical neurobiological approaches, including electrophysiology and neuroanatomy, with endocrine and genetic techniques to better understand biological events at the level of the organism.

Leptin and Insulin Act on POMC Neurons to Promote the Browning of White Fat

Garron T. Dodd, Stephanie Decherf, Kim Loh, Stephanie E. Simonds, Florian Wiede, Eglantine Balland, Troy L. Merry, et al.

http://dx.doi.org/10.1016/j.cell.2014.12.022

Highlights

Insulin and leptin act synergistically on POMC neurons to promote WAT browning
Increased POMC-mediated WAT browning prevents diet-induced obesity
PTP1B and TCPTP attenuate leptin and insulin signaling in POMC neurons
Combined PTP1B and TCPTP deficiency in POMC neurons promotes white fat browning

The primary task of white adipose tissue (WAT) is the storage of lipids. However, “beige” adipocytes also exist in WAT. Beige adipocytes burn fat and dissipate the energy as heat, but their abundance is diminished in obesity. Stimulating beige adipocyte development, or WAT browning, increases energy expenditure and holds potential for combating metabolic disease and obesity. Here, we report that insulin and leptin act together on hypothalamic neurons to promote WAT browning and weight loss. Deletion of the phosphatases PTP1B and TCPTP enhanced insulin and leptin signaling in proopiomelanocortin neurons and prevented diet-induced obesity by increasing WAT browning and energy expenditure. The coinfusion of insulin plus leptin into the CNS or the activation of proopiomelanocortin neurons also increased WAT browning and decreased adiposity. Our findings identify a homeostatic mechanism for coordinating the status of energy stores, as relayed by insulin and leptin, with the central control of WAT browning. http://www.cell.com/cms/attachment/2023992410/2043906325/fx1.jpg

Light on the Brain

Researchers find that photoreceptors expressed in zebrafish hypothalamus contribute to light-dependent behavior.

By Sabrina Richards | September 20, 2012

A 21 day old zebrafish. Their optical clarity and relatively easy maintenance make them a favorite for geneticists and developmental biologists. In this fish, the muscles can be seen as chevron shapes in the tail, the swim bladder as a “bubble” just behind the head, and the food that the fish has been eating as a brown patch just below the swim bladder.

Juvenile zebrafish. Shawn Burgess, NHGRI

Zebrafish larvae without eyes or pineal glands can still respond to light using photopigments located deep within their brains. Published today (September 20) in Current Biology, the findings are the first to link opsins, photoreceptors in the hypothalamus and other brain areas, to increased swimming in response to darkness, a behavior researchers hypothesize may help the fish move toward better-lit environments.

“[It’s a] strong demonstration that opsin-dependent photoreceptors in deep brain areas affect behaviors,” said Samer Hattar, who studies light reception in mammals at Johns Hopkins University but did not participate in the research.

Photoreceptors in eyes enable vision, and photoreceptors in the pineal gland, a small endocrine gland located in the center of the vertebrate brain, regulate circadian rhythms. But photoreceptors are also found in other brain areas of both invertebrates and vertebrate lineages. The function of these extraocular photoreceptors has been best studied in birds, where they regulate seasonal reproduction, explained Harold Burgess, a behavioral neurogeneticist at the Eunice Kennedy Shriver National Institute for Child Health and Human Development. Many opsins have been reported in the brains of tiny and transparent larval zebrafish, raising the possibility that light could be stimulating the photoreceptors even deep in the brain. To test for behaviors that may be regulated by deep brain photoreceptors, Burgess and his colleagues in Wolfgang Driever’s lab at the University of Freiburg removed the eyes of zebrafish larvae, and compared their behavior to larvae that retained their eyes. Although most light-dependent behavior required eyes, the eyeless larvae did respond when the lights were turned off, increasing their activity for a several minutes, though to a somewhat lesser extent than control larvae. But the fact that they responded at all suggests that non-retinal photoreceptors contributed to the behavior.

To confirm the role of the deep brain photoreceptors, the researchers also tested eyeless larvae that had been genetically modified to block expression of photoreceptors in the pineal gland. This fish still showed this jump in activity for several minutes after entering darkness.

Two different types of opsins—melanopsin and multiple tissue opsin—are expressed in the same type of neuron in zebrafish hypothalamus. Burgess and his colleagues looked at zebrafish missing the transcription factor Orthopedia, which is unique to these neurons, and found that the darkness-induced activity boost is nearly absent in these fish. To further narrow the search for the responsible photoreceptors, the researchers overexpressed melanopsin in hypothalamus neurons that co-express Orthopedia and melanopsin, and found that it increased the sensitivity of eyeless zebrafish to reductions in light. The results point to both melanopsin and Orthopedia as key players in modulating this behavior and pinpoint the location to neurons that coexpress these factors in the zebrafish hypothalamus.

Interestingly, the hypothalamus is one of the oldest parts of the vertebrate brain, said Detlev Arendt, a developmental biologist at the European Molecular Biology Laboratory in Heidelberg. “It’s very possible that this is one of the oldest functions”—one that evolved in “non-visual organisms” that had no eyes but still needed to sense light.

Although not as directed and efficient as eye-dependent behaviors that help fish swim toward light, Burgess speculates that deep brain opsins can still benefit zebrafish larvae. “You could imagine situation where it can’t see light, if a leaf falls on it and it doesn’t know where to swim. I think this behavior puts it in a hyperactive state where it swims wildly for several minutes until it reaches enough light for eyes to take over,” he explained, noting that such behavior is common in invertebrates.

It remains to be seen whether these deep brain opsins regulate other behaviors, perhaps in similar fashion to seasonal hormonal regulation in birds, but Hattar believes it is likely. “It’s beyond reasonable doubt there are many functions for these deep brain photoreceptors.”

Fernandes et al., “Deep brain photoreceptors control light-seeking behavior in zebrafish larvae,” Current Biology, 22:1-6, 2012.

Neuroendocrine basis of sexuality, mood, anxiety, social consciousness

Physiology, signaling, and pharmacology of galanin peptides and receptors: Three decades of emerging diversity

Lang, R., Gundlach, A.L., Holmes, F.E., (…), Hökfelt, T., Kofler, B.
Pharmacological Reviews 2015: 67 (1), pp. 118-175
http://dx.doi.org:/10.1124/pr.112.006536

Galanin was first identified 30 years ago as a “classic neuropeptide,” with actions primarily as a modulator of neurotransmission in the brain and peripheral nervous system. Other structurally-related peptides—galanin-like peptide and alarin—with diverse biologic actions in brain and other tissues have since been identified, although, unlike galanin, their cognate receptors are currently unknown. Over the last two decades, in addition to many neuronal actions, a number of nonneuronal actions of galanin and other galanin family peptides have been described. These include actions associated with neural stem cells, nonneuronal cells in the brain such as glia, endocrine functions, effects on metabolism, energy homeostasis, and paracrine effects in bone. Substantial new data also indicate an emerging role for galanin in innate immunity, inflammation, and cancer. Galanin has been shown to regulate its numerous physiologic and pathophysiological processes through interactions with three G protein–coupled receptors, GAL1, GAL2, and GAL3, and signaling via multiple transduction pathways, including inhibition of cAMP/PKA (GAL1, GAL3) and stimulation of phospholipase C (GAL2). In this review, we emphasize the importance of novel galanin receptor–specific agonists and antagonists. Also, other approaches, including new transgenic mouse lines (such as a recently characterized GAL3 knockout mouse) represent, in combination with viral-based techniques, critical tools required to better evaluate galanin system physiology. These in turn will help identify potential targets of the galanin/galanin-receptor systems in a diverse range of human diseases, including pain, mood disorders, epilepsy, neurodegenerative conditions, diabetes, and cancer.

Estradiol regulates responsiveness of the dorsal premammillary nucleus of the hypothalamus and affects fear- and anxiety-like behaviors in female rats

Litvin, Y., Cataldo, G., Pfaff, D.W., Kow, L.-M.
European Journal of Neuroscience 2014; 40 (2), pp. 2344-2351
10.1111/ejn.12608

Research suggests a causal link between estrogens and mood. Here, we began by examining the effects of estradiol (E2) on rat innate and conditioned defensive behaviors in response to cat odor. Second, we utilized whole-cell patch clamp electrophysiological techniques to assess noradrenergic effects on neurons within the dorsal premammillary nucleus of the hypothalamus (PMd), a nucleus implicated in fear reactivity, and their regulation by E2. Our results show that E2 increased general arousal and modified innate defensive reactivity to cat odor. When ovariectomized females treated with E2 as opposed to oil were exposed to cat odor, they showed elevations in risk assessment and reductions in freezing, indicating a shift from passive to active coping. In addition, animals previously exposed to cat odor showed clear cue + context conditioning 24 h later. However, although E2 persisted in its effects on general arousal in the conditioning task, its effects on fear disappeared. In the patch clamp experiments noradrenergic compounds that typically induce fear clearly excited PMd neurons, producing depolarizations and action potentials. E2 treatment shifted some excitatory effects of noradrenergic agonists to inhibitory, possibly by differentially affecting α- and β-adrenoreceptors. In summary, our results implicate E2 in general arousal and fear reactivity, and suggest these may be governed by changes in noradrenergic responsivity in the PMd. These effects of E2 may have ethological relevance, serving to promote mate seeking even in contexts of ambiguous threat and shed light on the involvement of estrogen in mood and its associated disorders.

Endogenous opiates and behavior: 2013

Richard J. Bodnar
Peptides 62 (2014) 67–136
http://dx.doi.org/10.1016/j.peptides.2014.09.013

This paper is the thirty-sixth consecutive installment of the annual review of research concerning the endogenous opioid system. It summarizes papers published during 2013 that studied the behavioral effects of molecular, pharmacological and genetic manipulation of opioid peptides, opioid receptors, opioid agonists and opioid antagonists. The particular topics that continue to be covered include the molecular-biochemical effects and neurochemical localization studies of endogenous opioids and their receptors related to behavior, and the roles of these opioid peptides and receptors in pain and analgesia; stress and social status; tolerance and dependence; learning and memory; eating and drinking; alcohol and drugs of abuse; sexual activity and hormones, pregnancy, development and endocrinology; mental illness and mood; seizures and neurologic disorders; electrical-related activity and neurophysiology; general activity and locomotion; gastrointestinal, renal and hepatic functions; cardiovascular responses; respiration and thermoregulation; and immunological responses.

Brain aromatase (cyp19a1b) and gonadotropin releasing hormone (gnrh2 and gnrh3) expression during reproductive development and sex change in black sea bass (Centropristis striata)

Timothy S Breton, Matthew A DiMaggio, Stacia A Sowe, David L Berlinsky, et al.
Comparative Biochemistry and Physiology, Part A 181 (2015) 45–53
http://dx.doi.org/10.1016/j.cbpa.2014.11.020

Teleost fish exhibit diverse reproductive strategies, and some species are capable of changing sex. The influence of many endocrine factors, such as gonadal steroids and neuropeptides, has been studied in relation to sex change, but comparatively less research has focused on gene expression changes within the brain in temperate grouper species with non-haremic social structures. The purpose of the present study was to investigate gonadotropin releasing hormone (GnRH) and brain aromatase (cyp19a1b) gene expression patterns during reproductive development and sex change in protogynous (female to male) black sea bass (Centropristis striata). Partial cDNA fragments for cyp19a1b and eef1a (a reference gene) were identified, and included with known gnrh2 and gnrh3 sequences in real time quantitative PCR. Elevated cyp19a1b expression was evident in the olfactory bulbs, telencephalon, optic tectum, and hypothalamus/
midbrain region during vitellogenic growth, which may indicate changes in the brain related to neurogenesis or sexual behavior. In contrast, gnrh2 and gnrh3 expression levels were largely similar among gonadal states, and all three genes exhibited stable expression during sex change. Although sex change in black sea bass is not associated with dramatic changes in GnRH or cyp19a1b gene expression among brain regions, these genes may mediate processes at other levels, such as within individual hypothalamic nuclei, or through changes in neuron size, that warrant further research.

Evaluation for roles of neurosteroids in modulating forebrain mechanisms controlling vasopressin secretion and related phenomena in conscious rats

Ken’ichi Yamaguchi
Neuroscience Research xxx (2015) xxx–xxx
http://dx.doi.org/10.1016/j.neures.2015.01.002

Anteroventral third ventricular region (AV3V) regulates autonomic functions through a GABAergic mechanism that possesses neuroactive steroid (NS)-synthesizing ability. Although NS can exert effects by acting on a certain type of GABAA-receptor (R), it is not clear whether NS may operate to modulateAV3V GABAergic activity for controlling autonomic functions. This study aimed to investigate the issue.AV3V infusion with a GABAA antagonist bicuculline increased plasma vasopressin (AVP), glucose, blood pressure (BP), and heart rate in rats. These events were abolished by preinjecting its agonist muscimol, whereas the infusion with allopregnanolone, a NS capable of potentiating GABAA-R function, affectednone of the variables in the absence or presence of such bicuculline actions. Similarly, AV3V infusion with pregnanolone sulfate, a NS capable of antagonizing GABAA-R, produced no effect on those variables.AV3V infusion with muscimol was effective in inhibiting the responses of plasma AVP or glucose, orBP to an osmotic loading or bleeding. However, AV3V infusion with aminoglutethimide, a NS synthesis inhibitor, did not affect any of the variables in the absence or presence of those stimuli. These results suggest that NS may not cause acute effects on the AV3V GABAergic mechanism involved in regulating AVP release and other autonomic function.

Novel receptor targets for production and action of allopregnanolone in the central nervous system: a focus on pregnane xenobiotic receptor

Cheryl A. Frye, Carolyn J. Koonce, and Alicia A. Walf
Front in Cell Neurosci Apr 2014; 8(106)
http://dx.doi.org:/10.3389/fncel.2014.00106

Neurosteroids are cholesterol-based hormones that can be produced in the brain, independent of secretion from peripheral endocrine glands, such as the gonads and adrenals. A focus in our laboratory for over 25 years has been how production of the pregnane neurosteroid, allopregnanolone, is regulated and the novel (i.e., non steroid receptor) targets for steroid action for behavior. One endpoint of interest has been lordosis, the mating posture of female rodents. Allopregnanolone is necessary and sufficient for lordosis, and the brain circuitry underlying it, such as actions in the midbrain ventral tegmental area (VTA), has been well-characterized. Published and recent findings supporting a dynamic role of allopregnanolone are included in this review. First, contributions of ovarian and adrenal sources of precursors of allopregnanolone, and the requisite enzymatic actions for de novo production in the central nervous system will be discussed.
Second, how allopregnanolone produced in the brain has actions on behavioral processes that are independent of binding to steroid receptors, but instead involve rapid modulatory actions via neurotransmitter targets (e.g., g-amino butyric acid-GABA, Nmethyl-D-aspartate- NMDA) will be reviewed.
Third, a recent focus on characterizing the role of a promiscuous nuclear receptor, pregnane xenobiotic receptor (PXR), involved in cholesterol metabolism and expressed in the VTA, as a target for allopregnanolone and how this relates to both actions and production of allopregnanolone will be addressed. For example, allopregnanolone can bind PXR and knocking down expression of PXR in the midbrain VTA attenuates actions of allopregnanolone via NMDA and/or GABAA for lordosis. Our understanding of allopregnanolone’s actions in the VTA for lordosis has been extended to reveal the role of allopregnanolone for broader, clinically-relevant questions, such as neurodevelopmental processes, neuropsychiatric disorders, epilepsy, and aging.

Long-term dysregulation of brain corticotrophin and glucocorticoid receptors and stress reactivity by single early-life pain experience in male and female rats

Nicole C. Victoria, Kiyoshi Inoue, Larry J. Young, Anne Z. Murphy
Psychoneuroendocrinology (2013) 38, 3015—3028
http://dx.doi.org/10.1016/j.psyneuen.2013.08.013

Inflammatory pain experienced on the day of birth (postnatal day 0: PD0) significantly dampens behavioral responses to stress- and anxiety-provoking stimuli in adult rats. However, to date, the mechanisms by which early life pain permanently alters adult stress responses remain unknown. The present studies examined the impact of inflammatory pain, experienced on the day of birth, on adult expression of receptors or proteins implicated in the activation and termination of the stress response, including corticotrophin releasing factor receptors (CRFR1 and CRFR2) and glucocorticoid receptor (GR). Using competitive receptor autoradiography, we show that Sprague Dawley male and female rat pups administered 1% carrageenan into the intraplantar surface of the hindpaw on the day of birth have significantly decreased CRFR1 binding in the basolateral amygdala and midbrain periaqueductal gray in adulthood. In contrast, CRFR2 binding, which is associated with stress termination, was significantly increased in the lateral septum and cortical amygdala. GR expression, measured with in situ hybridization and immunohistochemistry, was significantly increased in the paraventricular nucleus of the hypothalamus and significantly decreased in the hippocampus of neonatally injured adults. In parallel, acute stress-induced corticosterone release was significantly attenuated and returned to baseline more rapidly in adults injured on PD0 in comparison to controls. Collectively, these data show that early life pain alters neural circuits that regulate responses to and neuroendocrine recovery from stress, and suggest that pain experienced by infants in the Neonatal Intensive Care Unit may permanently alter future responses to anxiety- and stress provoking stimuli.

Dysruption of Corticotropin Releasing Factor in hypocampal region

Stress and trauma: BDNF control of dendritic-spine formation and regression

M.R. Bennett, J. Lagopoulos
Progress in Neurobiology 112 (2014) 80–99
http://dx.doi.org/10.1016/j.pneurobio.2013.10.005

Chronic restraint stress leads to increases in brain derived neurotrophic factor (BDNF) mRNA and protein in some regions of the brain, e.g. the basal lateral amygdala (BLA) but decreases in other regions such as the CA3 region of the hippocampus and dendritic spine density increases or decreases in line with these changes in BDNF. Given the powerful influence that BDNF has on dendritic spine growth, these observations suggest that the fundamental reason for the direction and extent of changes in dendritic spine density in a particular region of the brain under stress is due to the changes in BDNF there.
The most likely cause of these changes is provided by the stress initiated release of steroids, which readily enter neurons and alter gene expression, for example that of BDNF. Of particular interest is how glucocorticoids and mineralocorticoids tend to have opposite effects on BDNF gene expression offering the possibility that differences in the distribution of their receptors and of their downstream effects might provide a basis for the differential transcription of the BDNF genes. Alternatively, differences in the extent of methylation and acetylation in the epigenetic control of BDNF transcription are possible in different parts of the brain following stress.
Although present evidence points to changes in BDNF transcription being the major causal agent for the changes in spine density in different parts of the brain following stress, steroids have significant effects on downstream pathways from the TrkB receptor once it is acted upon by BDNF, including those that modulate the density of dendritic spines.
Finally, although glucocorticoids play a canonical role in determining BDNF modulation of dendritic spines, recent studies have shown a role for corticotrophin releasing factor (CRF) in this regard. There is considerable improvement in the extent of changes in spine size and density in rodents with forebrain specific knockout of CRF receptor 1 (CRFR1) even when the glucocorticoid pathways are left intact. It seems then that CRF does have a role to play in determining BDNF control of dendritic spines.

Central CRF system perturbation in an Alzheimer’s disease knockin mouse model

Qinxi Guo, Hui Zheng, Nicholas John Justice
Neurobiology of Aging 33 (2012) 2678–2691
http://dx.doi.org:/10.1016/j.neurobiolaging.2012.01.002

Alzheimer’s disease (AD) is often accompanied by changes in mood as well as increases in circulating cortisol levels, suggesting that regulation of the stress responsive hypothalamic-pituitary-adrenal (HPA) axis is disturbed. Here, we show that amyloid precursor protein (APP) is endogenously expressed in important limbic, hypothalamic, and midbrain nuclei that regulate hypothalamic-pituitary-adrenal axis activity. Furthermore, in a knockin mouse model of AD that expresses familial AD (FAD) mutations of both APP with humanized amyloid beta (hA), and presenilin 1 (PS1), in their endogenous patterns (APP/hA/PS1 animals), corticotropin releasing factor (CRF) levels are increased in key stress-related nuclei, resting corticosteroid levels are elevated, and animals display increased anxiety-related behavior. Endocrine and behavioral phenotypes can be normalized by loss of 1 copy of CRF receptor type-1 (Crfr1), consistent with a perturbation of central CRF signaling in APP/hA/PS1 animals. However, reductions in anxiety and corticosteroid levels conferred by heterozygosity of CRF receptor type-1 do not improve a deficit in working memory observed in APP/hA/PS1 mice, suggesting that perturbations of the CRF system are not the primary cause of decreased cognitive performance.

Alzheimer’s disease-like neuropathology of gene-targeted APP-SLxPS1mut mice expressing the amyloid precursor protein at endogenous levels

Christoph Kohler, Ulrich Ebert, Karlheinz Baumann, and Hannsjorg Schroeder
Neurobiology of Disease 20 (2005) 528 – 540
http://dx.doi.org:/10.1016/j.nbd.2005.04.009

Most transgenic mice used for preclinical evaluation of potential disease-modifying treatments of Alzheimer’s disease develop major histopathological features of this disease by several-fold overexpression of the human amyloid precursor protein. We studied the phenotype of three different strains of gene-targeted mice which express the amyloid precursor protein at endogenous levels. Only further crossing with transgenic mice overexpressing mutant human presenilin1 led to the deposition of extracellular amyloid, accompanied by the deposition of apolipoprotein E, an astrocyte and microglia reaction, and the occurrence of dilated cholinergic terminals in the cortex. Features of neurodegeneration, however, were absent. The pattern of plaque development and deposition in these mice was similar to that of amyloid precursor protein overproducing strains if crossed to presenilin1-transgenics. However, plaque development started much later and developed slowly until the age of 18 months but then increased more rapidly.

Central Cholinergic Functions In Human Amyloid Precursor Protein Knock-In/Presenilin-1 Transgenic Mice

Hartmann, C. Erb, U. Ebert, K. H. Baumann, A. Popp, G. Koenig, J. Klein
Neuroscience 125 (2004) 1009–1017
http://dx.doi.org:/10.1016/j.neuroscience.2004.02.038

Alzheimer’s disease is characterized by amyloid peptide formation and deposition, neurofibrillary tangles, central cholinergic dysfunction, and dementia; however, the relationship between these parameters is not well understood. We studied the effect of amyloid peptide formation and deposition on central cholinergic function in knock-in mice carrying the human amyloid precursor protein (APP) gene with the Swedish/London double mutation (APP-SL mice) which were crossbred with transgenic mice overexpressing normal (PS1wt) or mutated (M146L; PS1mut) human presenilin-1. APP-SLxPS1mut mice had increased levels of Aβ peptides at 10 months of age and amyloid plaques at 14 months of age while APP-SLPS1wt mice did not have increased peptide levels and did not develop amyloid plaques. We used microdialysis in 15–27 months old mice to compare hippocampal acetylcholine (ACh) levels in the two mouse lines and found that extracellular ACh levels were slightly but significantly reduced in the APP-SLPS1mut mice (-26%; P=0.044). Exploratory activity in the open field increased hippocampal ACh release by two-fold in both mouse lines; total and relative increases were not significantly different for the two strains under study. Similarly, infusion of scopolamine (1 µM) increased hippocampal ACh release to a similar extent (3–5-fold) in both groups. High-affinity choline uptake, a measure of the ACh turnover rate, was identical in both mouse lines. Neurons expressing choline acetyltransferase were increased in the septum of APP-SLPS1mut mice (26%; P =0.046). We conclude that amyloid peptide production causes a small decrease of extracellular ACh levels. The deposition of amyloid plaques, however, does not impair stimulated ACh release and proceeds without major changes of central cholinergic function.

Glutamate Neurotoxicity

Glutamate Neurotoxicity and Diseases of the Nervous System

Dennis W. Choi
Neuron. Oct, 1988; 1: 623-634

A growing number of studies now suggest that the cellular mechanisms which normally participate in signaling in the central nervous system (CNS) can be transformed by disease into instruments of neuronal cell destruction. Excitatory synaptic transmission in the mammalian CNS is principally mediated by L-glutamate. In fact, glutamate excites virtually all central neurons and is present in nerve terminals at millimolar levels (Curtis and Johnston, 1974). Normally, the extracellular levels of glutamate rise to high levels only in the brief and spatially localized fashion appropriate to synaptic transmission. This is fortunate, because as Lucas and Newhouse first showed in 1957, sustained exposure to glutamate can destroy retinal neurons. In a subsequent set of pioneering experiments, Olney (Olney and Sharpe, 1969; Olney et al., 1971) established that this toxicity, which he later called excitotoxicity, was not unique to glutamate or to retinal neurons, but was a feature common to the actions of all excitatory amino acids on central neurons. He postulated therefore that glutamate, or related compounds, might be the cause of the neuronal cell loss found in certain neurological diseases. In recent years, this hypothesis has gathered considerable support, fueled by new insights into glutamate receptor function and the development of effective glutamate antagonist drugs. The evidence is most convincing in diseases involving an acute insult to the brain, as occurs in a stroke, with abrupt deprivation of blood supply. But neurotoxicity due to excitatory amino acids may also be involved in slowly progressive degenerative diseases such as Huntington’s disease. Although the detailed molecular basis of glutamate neurotoxicity is not known, it appears that Ca2+ influx may play a critical role.
Glutamate interacts with at least three classes of membrane receptors, each commonly referred to by preferred pharmacological agonists: N-methyl-o-aspartate (NMDA), quisqualate, and kainate (Watkins and Olverman, 1987) (Figure I). These three classes are linked to membrane cation channels. A second type of quisqualate receptor has been additionally linked to a second messenger system (see below). It has been suggested that all three classes might actually be substates of a single molecular complex, but binding studies and newer physiological studies favor separate structures.

Quisqualate NMDA Kainate

Three Classes of Glutamate Receptors

One type of quisqualate receptor stimulates the formation of inositol 1,4,5-trisphosphate UPS) and diacylglycerol (DAG) from phosphatidylinositol-4,5-biphosphate (PIP,); the other is linked directly to a Na+ ionophore. Activation of the quisqualate receptor-ionophore complex can be potentiated by Zn2+. The NMDA receptor opens a channel permeable to Ca2+ as well as Na+; this receptor-channel complex has several modulatory sites discussed in the text. The kainate receptor opens an ionophore permeable to Na+.

Best defined is the NMDA receptor. This receptor opens a distinctive membrane channel characterized by high conductance (main state about 50 pS), voltage dependent Mgz+ blockade and permeability to both Ca2+ and Na+. The NMDA receptor can be selectively activated by several endogenous compounds, including L-aspartate, homocysteate, and quinolinate. Activation requires the coavailability of glycine in near micromolar concentrations. The action of glutamate at the NMDA receptor can be selectively antagonized: competitively by 2-amino-5-phosphonovalerate (APV) and 2-amino-5-phosphonoheptanoate (APH), or noncompetitively by drugs that bind to the phencyclidine site within the open channel (such as phencyclidine, MK-801, dextrorphan, or ketamine. The NMDA receptor-activated channel can also be blocked noncompetitively by Znz+, most likely at a site different from that which binds Mg2.
Although glutamate has high affinity for all three classes of postsynaptic receptors, it is not easy to demonstrate its neurotoxicity in vivo. Even when directly injected into brain, bypassing the blood-brain barrier, extremely high doses of glutamate are required to create lesions. Mangano & Schwartz found that they could infuse 0.5 crl/hr of a 300 mM glutamate solution into the hippocampus of a rat for 2 weeks without producing neuronal injury. This apparent low in vivo neurotoxic potency of glutamate may represent one reason why Olney’s “glutamate hypothesis” of neurological disease did not initially achieve a more widespread following. However, in fact, glutamate is a potent and rapidly acting neurotoxin; its neurotoxicity in vivo is likely masked by the efficiency of normal cellular uptake mechanisms in removing glutamate from the extracellular space. Glutamate neurotoxicity can be most directly studied in cell culture where bath exposure is not limited by cellular uptake.
The toxic changes produced by glutamate or related excitatory amino acids in vivo are of two sorts:

acute swelling of neuronal dendrites and cell bodies and a
more slowly evolving neuronal degeneration (Olney, 1986).

Axons and glia are relatively spared, although high levels of excitatory amino acids can produce some swelling of glia. A hallmark of excitatory amino acid neurotoxicity is its cellular selectivity, with distinctive patterns of neuronal loss produced by different excitatory amino acids and different routes of administration. For example, Nadler and co-workers (1978) found that intraventricular kainate preferentially destroys hippocampal CA3 neurons but spares dentate granule neurons. Different neuronal subpopulations
may differ in their intrinsic vulnerability to damage.

Possible Mechanisms Involved in Glutamate Neurotoxicity

How Ca*+ may mediate glutamate-induced neuronal degeneration. Glutamate acts on NMDA, non-NMDA, and “metabotropic” receptors (the quisqualate receptor linked to a second messenger system) to produce an increase in cytosolic free Ca*+. This cytosolic Ca *+, in concert with diacylglycerol liberated by the quisqualate-triggered second messenger system, activates protein kinase C, which acts via a number of mechanisms (primarily by altering membrane ion channels) to increase neuronal excitability and further increase cytosolic Ca*+. Elevated cytosolic Ca2+ then activates several enzymes capable of either directly or indirectly (through free radical formation) destroying cellular structure. Glutamate released from synaptic terminals or leaking nonspecifically from ruptured neurons contributes to additional injury propagation.

Glutamate Neurotoxicity in Perspective

The hypothesis that excitatory amino acids may specifically mediate pathological neuronal injury gives new form to this age-old enemy and raises the tantalizing possibility that current molecular and cellular insights into excitatory amino acid transmitter systems might be harnessed to develop an efficacious clinical therapy. Some points of attack are already apparent; others will likely be defined as the biology of excitatory amino acids continues to be unraveled. An intriguing area for investigation is the relationship between excitatory amino acid neurotoxicity and normal neuronal processes such as maturation, neurite outgrowth, and synaptic plasticity.

Glutamate Toxicity in a Neuronal Cell line Involves Inhibition of Cystine Transport Leading to Oxidative Stress

Timothy H. Murphy, M Miyamoto, A Sastre, R Schnaar and JT Coyle
Neuron 1989: 2: 1547-88.

Glutamate binds to both excitatory neurotransmitter binding sites and a W-dependent, quisqualate- and cystine-inhibited transport site on brain neurons. The neuroblastoma-primary retina hybrid cells (NWRE-105) are susceptible to glutamate-induced cytotoxicity. The Cl–dependent transport site to which glutamate and quisqualate (but not kainate or NMDA) bind has a higher affinity for cystine than for glutamate. Towering cystine concentrations in the cell culture medium results in cytotoxicity similar to that induced by glutamate addition in its morphology, kinetics, and CaZ+ dependence. Glutamate-induced cytotoxicity is directly proportional to its ability to inhibit cystine uptake. Exposure to glutamate (or lowered cystine) causes a decrease in glutathione levels and an accumulation of intracellular peroxides. Like NW-RE-105 cells, primary rat hippocampal neurons (but not glia) in culture degenerate in medium with lowered cystine concentration. Thus, glutamate-induced cytotoxicity in N18-RE-105 cells is due to inhibition of cystine uptake, resulting in lowered glutathione levels leading to oxidative stress and cell death.

Mechanism of glutamate-induced neurotoxicity in HT22 mouse hippocampal cells

Masayuki Fukui, Ji-Hoon Song, Jinyoung Choi, Hye Joung Choi, Bao Ting Zhu
European Journal of Pharmacology 617 (2009) 1–11
http://dx.doi.org:/10.1016/j.ejphar.2009.06.059

Glutamate is an endogenous excitatory neurotransmitter. At high concentrations, it is neurotoxic and contributes to the development of certain neurodegenerative diseases. There is considerable controversy in the literature with regard to whether glutamate-induced cell death in cultured HT22 cells (an immortalized mouse hippocampal cell line) is apoptosis, necrosis, or a new form of cell death. The present study focused on investigating the mechanism of glutamate-induced cell death. We found that glutamate induced, in a time dependent manner, both necrosis and apoptosis in HT22 cells. At relatively early time points (8–12 h), glutamate induced mostly necrosis, whereas at late time points (16–24 h), it induced mainly apoptosis. Glutamate-induced mitochondrial oxidative stress and dysfunction were crucial early events required for the induction of apoptosis through the release of the mitochondrial apoptosis-inducing factor (AIF), which catalyzed DNA fragmentation (an ATP-independent process). Glutamate-induced cell death proceeded independently of the Bcl-2 family proteins and caspase activation. The lack of caspase activation likely resulted from the lack of intracellular ATP when the mitochondrial functions were rapidly disrupted by the mitochondrial oxidative stress. In addition, it was observed that activation of JNK, p38, and ERK signaling molecules was also involved in the induction of apoptosis by glutamate. In conclusion, glutamate-induced apoptosis is AIF-dependent but caspase-independent, and is accompanied by DNA ladder formation but not chromatin condensation.

Understanding Low Reliability of Memories for Neutral Information Encoded under Stress: Alterations in Memory-Related Activation in the Hippocampus and Midbrain

Shaozheng Qin, EJ Hermans, HJF van Marle, and G Fernandez, et al.
The Journal of Neuroscience, Mar 21, 2012; 32(12): 4032–4041
http://dx.doi.org:/10.1523/JNEUROSCI.3101-11.2012

Exposure to an acute stressor can lead to unreliable remembrance of intrinsically neutral information, as exemplified by low reliability of eyewitness memories, which stands in contrast with enhanced memory for the stressful incident itself. Stress-sensitive neuromodulators (e.g., catecholamines) are believed to cause this low reliability by altering neurocognitive processes underlying memory formation. Using event-related functional magnetic resonance imaging, we investigated neural activity during memory formation in 44 young, healthy human participants while incidentally encoding emotionally neutral, complex scenes embedded in either a stressful or neutral context.
We recorded event-related pupil dilation responses as an indirect index of phasic noradrenergic activity. Autonomic, endocrine, and psychological measures were acquired to validate stress manipulation. Acute stress during encoding led to a more liberal response bias (more hits and false alarms) when testing memory for the scenes 24 h later. The strength of this bias correlated negatively with pupil dilation responses and positively with stress-induced heart rate increases at encoding. Acute stress, moreover, reduced subsequent memory effects (SMEs; items later remembered vs forgotten) in hippocampus and midbrain, and in pupil dilation responses.
The diminished SMEs indicate reduced selectivity and specificity in mnemonic processing during memory formation. This is in line with a model in which stress-induced catecholaminergic hyperactivation alters phasic neuromodulatory signaling in memory-related circuits, resulting in generalized (gist-based) processing at the cost of specificity. Thus, one may speculate that loss of specificity may yield less discrete memory representations at time of encoding, thereby causing a more liberal response bias when probing these memories.

Neuroendocrinology – Signaling, neuron plasticity and memory

Leptin Signaling Modulates the Activity of Urocortin 1 Neurons in the Mouse Nonpreganglionic Edinger-Westphal Nucleus

Lu Xu, Wim J. J. M. Scheenen, Rebecca L. Leshan, Christa M. Patterson, et al.
Endocrinology 152(3): 979–988, 2011
http://dx.doi.org:/10.1210/en.2010-1143

A recent study systematically characterized the distribution of the long form of the leptin receptor (LepRb) in the mouse brain and showed substantial LepRb mRNA expression in the nonpreganglionic Edinger-Westphal nucleus (npEW) in the rostroventral part of the midbrain. This nucleus hosts the majority of urocortin 1 (Ucn1) neurons in the rodent brain, and because Ucn1 is a potent satiety hormone and electrical lesioning of the npEW strongly decreases food intake, we have hypothesized a role of npEW-Ucn1 neurons in leptin-controlled food intake. Here, we show by immunohistochemistry that npEW-Ucn1 neurons in the mouse contain LepRb and respond to leptin administration with induction of the Janus kinase 2-signal transducer and activator of transcription 3 pathway, both in vivo and in vitro. Furthermore, systemic leptin administration increases the Ucn1 content of then pEW significantly, whereas in mice that lack LepRb (db/db mice), then pEW contains considerably reduced amount of Ucn1. Finally, we reveal by patch clamping of midbrain Ucn1 neurons that leptin administration reduces the electrical firing activity of the Ucn1 neurons. In conclusion, we provide ample evidence for leptin actions that go beyond leptin’s well-known targets in the hypothalamus and propose that leptin can directly influence the activity of the midbrain Ucn1 neurons.

Leptin regulation of hippocampal synaptic function in health and disease

Andrew J. Irving and Jenni Harvey
Trans. R. Soc. B 369: 20130155 http://dx.doi.org/10.1098/rstb.2013.0155

The endocrine hormone leptin plays a key role in regulating food intake and body weight via its actions in the hypothalamus. However, leptin receptors are highly expressed in many extra-hypothalamic brain regions and evidence is growing that leptin influences many central processes including cognition. Indeed, recent studies indicate that leptin is a potential cognitive enhancer as it markedly facilitates the cellular events underlying hippocampal-dependent learning and memory, including effects on glutamate receptor trafficking, neuronal morphology and activity-dependent synaptic plasticity. However, the ability of leptin to regulate hippocampal synaptic function markedly declines with age and aberrant leptin function has been linked to neurodegenerative disorders such as Alzheimer’s disease (AD). Here, we review the evidence supporting a cognitive enhancing role for the hormone leptin and discuss the therapeutic potential of using leptin-based agents to treat AD.

The Y2 receptor agonist PYY3–36 increases the behavioral response to novelty and acute dopaminergic drug challenge in mice

Ulrike Stadlbauer, Elisabeth Weber, Wolfgang Langhans and Urs Meyer
International Journal of Neuropsychopharmacology (2014), 17, 407–419
http://dx.doi.org:/10.1017/S1461145713001223

The gastrointestinal hormone PYY3–36 is a preferential Y2 neuropeptide Y (NPY) receptor agonist. Recent evidence indicates that PYY3–36 acts on central dopaminergic pathways, but its influence on dopamine-dependent behaviors remains largely unknown. We therefore explored the effects of peripheral PYY3–36 treatment on the behavioral responses to novelty and to dopamine-activating drugs in mice. In addition, we examined whether PYY3–36 administration may activate distinct dopamine and γ-aminobutyric acid (GABA) cell populations in the mesoaccumbal and nigrostriatal pathways. We found that i.p. PYY3–36 injection led to a dose-dependent increase in novel object exploration. The effective dose of PYY3–36 (1 μg/100 g body weight) also potentiated the locomotor reaction to the indirect dopamine receptor agonist amphetamine and increased stereotyped climbing/leaning responses following administration of the direct dopamine receptor agonist apomorphine. PYY3–36 administration did not affect activity of midbrain dopaminergic cells as evaluated by double immuno-enzyme staining of the neuronal early gene product c-Fos with tyrosine hydroxylase. PYY3–36 did, however, lead to a marked increase in the number of cells co-expressing c-Fos with glutamic acid decarboxylase in the nucleus accumbens and caudate putamen, indicating activation of GABAergic cells in dorsal and ventral striatal areas. Our results support the hypothesis that acute administration of the preferential Y2 receptor agonist PYY3–36 modulates dopamine-dependent behaviours. These effects do not seem to involve direct activation of midbrain dopamine cells but instead are associated with neuronal activation in the major input areas of the mesoaccumbal and nigrostriatal pathways.

Somatostatin and nociceptin inhibit neurons in the central nucleus of amygdala that project to the periaqueductal grey

Billy Chieng, MacDonald J. Christie
Neuropharmacology 59 (2010) 425e430
http://dx.doi.org:/10.1016/j.neuropharm.2010.06.001

The central nucleus of amygdala (CeA) plays an important role in modulation of the descending antinociceptive pathways. Using whole-cell patch clamp recordings from brain slices, we found that CeA neurons responded to the endogenous ligands somatostatin (SST) and nociceptin/orphanin FQ (OFQ) via an increased K-conductance. Co-application with selective antagonists suggested that SST and OFQ act on SSTR2 and ORL1 receptors, respectively. Taking account of anatomical localisation of recorded neurons, the present study showed that many responsive neurons were located within the medial subdivision of CeA and all CeA projection neurons to the midbrain periaqueductal grey invariably responded to these peptides. Randomly selected agonist-responsive neurons in CeA predominantly classified physiologically as low-threshold spiking neurons. The similarity of SST, OFQ and, as previously reported, opioid responsiveness in a sub-population of CeA neurons suggests converging roles of these peptides to inhibit the activity of projections from CeA to vlPAG, and potentially similar antinociceptive actions in this pathway.

In vitro identification and electrophysiological characterization of dopamine neurons in the ventral tegmental area

Tao A. Zhang, Andon N. Placzek, John A. Dani
Neuropharmacology 59 (2010) 431e436
http://dx.doi.org:/10.1016/j.neuropharm.2010.06.004

Dopamine (DA) neurons in the ventral tegmental area (VTA) have been implicated in brain mechanisms related to motivation, reward, and drug addiction. Successful identification of these neurons in vitro has historically depended upon the expression of a hyperpolarization-activated current (I_h) and immunohistochemical demonstration of the presence of tyrosine hydroxylase (TH), the rate-limiting enzyme for DA synthesis. Recent findings suggest that electrophysiological criteria may be insufficient for distinguishing DA neurons from non-DA neurons in the VTA. In this study, we sought to determine factors that could potentially account for the apparent discrepancies in the literature regarding DA neuron identification in the rodent brain slice preparation. We found that confirmed DA neurons from the lateral VTA generally displayed a larger amplitude I_h relative to DA neurons located in the medial VTA. Measurement of a large amplitude I_h (>100 pA) consistently indicated a dopaminergic phenotype, but non-dopamine neurons also can have I_h current. The data also showed that immunohistochemical TH labeling of DA neurons can render false negative results after relatively long duration (>15 min) wholecell patch clamp recordings. We conclude that whole-cell patch clamp recording in combination with immunohistochemical detection of TH expression can guarantee positive but not negative DA identification in the VTA.

Dopamine Enables In Vivo Synaptic Plasticity Associated with the Addictive Drug Nicotine

Jianrong Tang and John A. Dani
Neuron, Sept 10, 2009; 63, 673–682
http://dx.doi.org:/10.1016/j.neuron.2009.07.025

Addictive drugs induce a dopamine signal that contributes to the initiation of addiction, and the dopamine signal influences drug-associated memories that perpetuate drug use. The addiction process shares many commonalities with the synaptic plasticity mechanisms normally attributed to learning and memory. Environmental stimuli repeatedly linked to addictive drugs become learned associations, and those stimuli come to elicit memories or sensations that motivate continued drug use. Applying in vivo recording techniques to freely moving mice, we show that physiologically relevant concentrations of the addictive drug nicotine directly cause in vivo hippocampal synaptic potentiation of the kind that underlies learning and memory. The drug-induced long-term synaptic plasticity required a local hippocampal dopamine signal. Disrupting general dopamine signaling prevented the nicotine-induced synaptic plasticity and conditioned place preference. These results suggest that dopaminergic signaling serves as a functional label of salient events by enabling and scaling synaptic plasticity that underlies drug-induced associative memory.

NCS-1 in the Dentate Gyrus Promotes Exploration, Synaptic Plasticity, and Rapid Acquisition of Spatial Memory

Bechara J. Saab, John Georgiou, Arup Nath, Frank J.S. Lee, et al.
Neuron, Sept 10, 2009; 63, 643–656
http://dx.doi.org:/10.1016/j.neuron.2009.08.014

The molecular underpinnings of exploration and its link to learning and memory remain poorly understood. Here we show that inducible, modest overexpression of neuronal calcium sensor 1 (Ncs1) selectively in the adult murine dentate gyrus (DG) promotes a specific form of exploratory behavior. The mice also display a selective facilitation of longterm potentiation (LTP) in the medial perforant path and a selective enhancement in rapid-acquisition spatial memory, phenotypes that are reversed by direct application of a cell-permeant peptide (DNIP) designed to interfere with NCS-1 binding to the dopamine type-2 receptor (D2R). Moreover, the DNIP and the D2R-selective antagonist L-741,626 attenuated exploratory behavior, DG LTP, and spatial memory in control mice. These data demonstrate a role for NCS-1 and D2R in DG plasticity and provide insight for understanding how the DG contributes to the origin of exploration and spatial memory acquisition.

Neuroligin 2 Drives Postsynaptic Assembly at Perisomatic Inhibitory Synapses through Gephyrin and Collybistin

Alexandros Poulopoulos, Gayane Aramuni, Guido Meyer, Tolga Soykan, et al.
Neuron 63, 628–642, Sept 10, 2009
http://dx.doi.org:/10.1016/j.neuron.2009.08.023

In the mammalian CNS, each neuron typically receives thousands of synaptic inputs from diverse classes of neurons. Synaptic transmission to the postsynaptic neuron relies on localized and transmitter-specific differentiation of the plasma membrane with postsynaptic receptor, scaffolding, and adhesion proteins accumulating in precise apposition to presynaptic sites of transmitter release. We identified protein interactions of the synaptic adhesion molecule neuroligin 2 that drive postsynaptic differentiation at inhibitory synapses. Neuroligin 2 binds the scaffolding protein gephyrin through a conserved cytoplasmic motif and functions as a specific activator of collybistin, thus guiding membrane tethering of the inhibitory postsynaptic scaffold. Complexes of neuroligin 2, gephyrin and collybistin are sufficient for cell-autonomous clustering of inhibitory neurotransmitter receptors. Deletion of neuroligin 2 in mice perturbs GABAergic and glycinergic synaptic transmission and leads to a loss of postsynaptic specializations specifically at perisomatic inhibitory synapses.

A Subset of Ventral Tegmental Area Neurons is Inhibited by Dopamine, 5-Hydroxytryptamine and Opioids

L. Cameron, M. W. Wessendorf and J. T. Williams
Neuroscience 1997; 77(1), pp. 155–166 PII: S0306-4522(96)00444-7

Neurons originating in the ventral tegmental area are thought to play a key role in the formation of addictive behaviors, particularly in response to drugs such as cocaine and opioids. In this study we identified different populations of ventral tegmental area neurons by the pharmacology of their evoked synaptic potentials and their response to dopamine, 5-hydroxytryptamine and opioids. Intracellular recordings were made from ventral tegmental area neurons in horizontal slices of guinea-pig brain and electrical stimulation was used to evoke synaptic potentials. The majority of cells (61.3%) hyperpolarized in response to dopamine, depolarized to 5-hydroxytryptamine, failed to respond to [Met]5enkephalin and exhibited a slow GABAB-mediated inhibitory postsynaptic potential. A smaller proportion of cells (11.3%) hyperpolarized in response to [Met]5enkephalin, depolarized to 5-hydroxytryptamine, failed to respond to dopamine and did not exhibit a slow inhibitory postsynaptic potential. These two groups of cells corresponded to previously described ‘‘principal’’ and ‘‘secondary’’ cells, respectively. A further group of cells (27.4%) was identified that, like the principal cells, hyperpolarized to dopamine.

However, these ‘‘tertiary cells’’ also hyperpolarized to both 5-hydroxytryptamine and [Met]5enkephalin and exhibited a slow, cocaine-sensitive 5-hydroxytryptamine1A-mediated inhibitory postsynaptic potential. When principal and tertiary cells were investigated immuno-histochemically, 82% of the principal cells were positive for tyrosine hydroxylase compared
with only 29% of the tertiary cells. The 5-hydroxytryptamine innervation of both these cell types was investigated and a similar density of putative contacts was observed near the somata and dendrites in both groups. This latter finding suggests that the existence of a 5-hydroxytryptamine-mediated inhibitory postsynaptic potential in the tertiary cells may be determined by the selective expression of 5-hydroxytryptamine receptors, rather than the distribution or density of the 5-hydroxytryptamine innervation.
We conclude that tertiary cells are a distinct subset of ventral tegmental area neurons where cocaine and μ-opioids both mediate inhibition.

Dopamine reward circuitry: Two projection systems from the ventral midbrain to the nucleus accumbens–olfactory tubercle complex

Satoshi Ikemoto
Brain Research Reviews 56 (2007) 27–78
http://:dx.doi.org:/10.1016/j.brainresrev.2007.05.004

Anatomical and functional refinements of the meso-limbic dopamine system
of the rat are discussed. Present experiments suggest that dopaminergic neurons localized in the posteromedial ventral tegmental area (VTA) and central linear nucleus raphe selectively project to the ventromedial striatum (medial olfactory tubercle and medial nucleus accumbens shell), whereas
the anteromedial VTA has few if any projections to the ventral striatum,
and the lateral VTA largely projects to the ventrolateral striatum (accumbens
core, lateral shell and lateral tubercle). These findings complement the recent behavioral findings that cocaine and amphetamine are more rewarding when administered into the ventromedial striatum than into the ventrolateral striatum. Drugs such as nicotine and opiates are more rewarding when administered into the posterior VTA or the central linear nucleus than into
the anterior VTA. A review of the literature suggests that
(1) the midbrain has corresponding zones for the accumbens core and medial shell;
(2) the striatal portion of the olfactory tubercle is a ventral extension of the nucleus accumbens shell; and
(3) a model of two dopamine projection systems from the ventral midbrain to the ventral striatum is useful for understanding reward function.
The medial projection system is important in the regulation of arousal characterized by affect and drive and plays a different role in goal directed learning than the lateral projection system, as described in the variation–selection hypothesis of striatal functional organization.

Metabolic hormones, dopamine circuits, and feeding

Nandakumar S. Narayanan, Douglas J. Guarnieri, Ralph J. DiLeone
Frontiers in Neuroendocrinology 31 (2010) 104–112
http://dx.doi.org:/10.1016/j.yfrne.2009.10.004

Recent evidence has emerged demonstrating that metabolic hormones such as ghrelin and leptin can act on ventral tegmental area (VTA) midbrain dopamine neurons to influence feeding. The VTA is the origin of mesolimbic dopamine neurons that project to the nucleus accumbens (NAc) to influence behavior. While blockade of dopamine via systemic antagonists or targeted gene delete can impair food intake, local NAc dopamine manipulations have little effect on food intake. Notably, non-dopaminergic manipulations in the VTA and NAc produce more consistent effects on feeding and food choice. More recent genetic evidence supports a role for the substantia nigra-striatal dopamine pathways in food intake, while the VTA–NAc circuit is more likely involved in higher-order aspects of food acquisition, such as motivation and cue associations. This rich and complex literature should be considered in models of how peripheral hormones influence feeding behavior via action on the midbrain circuits.

Control of brain development and homeostasis by local and systemic insulin signaling

Liu, P. Speder & A. H. Brand
Diabetes, Obesity and Metabolism 16 (Suppl. 1): 16–20, 2014

Insulin and insulin-like growth factors (IGFs) are important regulators of growth and metabolism. In both vertebrates and invertebrates, insulin/IGFs are made available to various organs, including the brain, through two routes: the circulating systemic insulin/IGFs act on distant organs via endocrine signaling, whereas insulin/IGF ligands released by local tissues act in a paracrine or autocrine fashion. Although the mechanisms governing the secretion and action of systemic insulin/IGF have been the focus of extensive investigation, the significance of locally derived insulin/IGF has only more recently come to the fore. Local insulin/IGF signaling is particularly important for the development and homeostasis of the central nervous system, which is insulated from the systemic environment by the blood–brain barrier. Local insulin/IGF signaling from glial cells, the blood–brain barrier and the cerebrospinal fluid has emerged as a potent regulator of neurogenesis. This review will address the main sources of local insulin/IGF and how they affect neurogenesis during development. In addition, we describe how local insulin/IGF signaling couples neural stem cell proliferation with systemic energy state in Drosophila and in mammals.

Pharmacology, Physiology, and Mechanisms of Action of Dipeptidyl Peptidase-4 Inhibitors

Erin E. Mulvihill and Daniel J. Drucker
Endocrine Reviews 35: 992–1019, 2014
http://dx.doi.org/10.1210/er.2014-1035

Dipeptidyl peptidase-4 (DPP4) is a widely expressed enzyme transducing actions through an anchored transmembrane molecule and a soluble circulating protein. Both membrane-associated and soluble DPP4 exert
catalytic activity, cleaving proteins containing a position 2 alanine or proline. DPP4-mediated enzymatic cleavage alternatively inactivates peptides or generates new bioactive moieties that may exert competing or novel activities. The widespread use of selective DPP4 inhibitors for the treatment of type 2 diabetes has heightened interest in the molecular mechanisms through which DPP4 inhibitors exert their pleiotropic actions. Here we review the biology ofDPP4with a focus on:
1) identification of pharmacological vs physiological DPP4 substrates; and
2) elucidation of mechanisms of actions of DPP4 in studies employing genetic elimination or chemical reduction of DPP4 activity.
We review data identifying the roles of key DPP4 substrates in transducing the glucoregulatory, anti-inflammatory, and cardiometabolic actions of DPP4 inhibitors in both preclinical and clinical studies. Finally, we highlight experimental pitfalls and technical challenges encountered in studies designed to understand the mechanisms of action and downstream targets activated by inhibition of DPP4.
Dipeptidyl peptidase-4 (DPP4) is a multifunctional protein that exerts biological activity through pleiotropic actions including:

protease activity (1),
association with adenosine deaminase (ADA) (2),
interaction with the extracellular matrix (3),
cell surface coreceptor activity mediating viral entry (4), and
regulation of intracellular signal transduction coupled to control of cell migration and proliferation (5).

The complexity of DPP4 action is amplified by the panoply of bioactive DPP4 substrates, which in turn act as elegant biochemical messengers in multiple tissues, including the immune and neuroendocrine systems.

DPP4 transmits signals across cell membranes and interacts with other membrane proteins (Figure). Remarkably, most of the protein is extracellular, including the C-terminal catalytic domain, a cysteine-rich area, and a large glycosylated region linked by a flexible stalk to the transmembrane segment. Only six N-terminal amino acids are predicted to extend into the cytoplasm. The active site, Ser 630, is flanked by the classic serine peptidase motif Gly-Trp-Ser630-Tyr-Gly-Gly-Tyr-Val.

Membrane-bound DPP4

Membrane-bound DPP4 contains residues 1–766, whereas sDPP4 contains residues 39–766. sDPP4 is lacking the cytoplasmic domain [residues 1–6], transmembrane domain [residues 7–28], and the flexible stalk [residues 29–39]. Both membrane-bound and circulating sDPP4 share many domains including the glycosylated region [residues 101–535, specific residues 85,92, 150], ADA binding domain [340–343], fibronectin binding domain [468–479], cysteine-rich domain [351–506, disulfide bonds are formed from 385–394, 444–472, and 649–762], and the catalytic domain [507–766 including residues composing the catalytic active site 630, 708, and 740].

DPP4 activity is subject to regulation at many levels, including control of gene and protein expression, interaction with binding partners, and modulation of enzyme activity. The DPP4 gene does not contain conventional TATAA or CCAAT promoter sequences but is characterized by a cytosine/guanine-rich promoter region.
DPP4 contains eight to 11 potential N-glycosylation sites, which can contribute to its folding and stability. Although glycosylation may contribute approximately 18–25% of the total molecular weight, mutational analysis has determined that the glycosylation sites are not required for dimerization, catalytic activity, or ADA binding. However, N-terminal sialylation facilitates trafficking of DPP4 to the apical membrane. Interestingly, molecular analysis of DPP4 isoforms isolated from the rat kidney brush border membrane reveals extensive heterogeneity in the oligosaccharides of DPP4.DPP4 was first investigated for its role in hydrolysis of dietary prolyl peptides (58); subsequent studies using DPP4 isolated using immunoaffinity chromatography and ADA binding identified DPP4 as the primary enzyme responsible for the generation of Gly-Prop-nitroanilide substrates in human serum. It is now known that DPP4 can cleave dozens of peptides, including chemokines, neuropeptides, and regulatory peptides, most containing a proline or alanine residue at position 2 of the amino-terminal region. Despite the preference for a position 2 proline, alternate residues (hydroxyproline, dehydroproline > alanine >, glycine, threonine, valine, or leucine) at the penultimate position are also cleaved by DPP4, suggesting a required stereochemistry. The DPP4 cleavage at postproline peptide bonds inactivates peptides and/or generates new bioactive peptides (see Figure), thereby regulating diverse biological processes.

DPP4 cleavage regulates substrate-receptor interactions

DPP4 cleavage regulates substrate/receptor interactions. A, DPP4 cleaves NPY [1–36] and PYY [1–36]. The intact forms of these peptides signal through Y1R-Y5R. After DPP4 cleavage, NPY [3–36] and PYY [3–36] are generated and preferentially signal through the Y2R and Y5R. B, DPP4 cleaves SP [1–11], which signals through the NK1R receptor to generate SP [5–11], which can signal through (NK1R, -2R, -3R).

GHRH and IGF-1

GHRH [1–44] and [1–40] are produced in the arcuate nucleus of the hypothalamus and bind its receptor on the anterior pituitary to stimulate the release of GH, and in turn, GH stimulates hepatic IGF-1 release. GHRH was among the first peptides to be identified as a DPP4 substrate; it is rapidly degraded in rodent and human plasma to GHRH [3–44]/GHRH [3–40], and this cleavage was blocked upon incubation of human plasma with the DPP4 inhibitor, diprotin A (99).GHRH[1–44] or [1–40] exhibits a very short half-life (6 min) andDPP4 cleavage was initially perceived to be a critical regulator of GHRH bioactivity and, in turn, the GH-IGF-1 axis. IGF-1, the downstream effector of GHRH and GH, is a 105-amino acid protein produced mainly by the liver.
IGF-1 was identified as a pharmacological DPP4 substrate by matrix-assisted laser desorption/ionization-time of flight analysis of molecular forms of IGF-1 generated after incubation with DPP4 purified from baculovirus-infected insect cells. However, studies in pigs treated with sitagliptin at doses inhibiting 90% of DPP4 activity failed to demonstrate an increase in active intact IGF-1.
Clinically, treatment of healthy human male subjects with sitagliptin (25–600 mg) for 10 days did not produce increased concentrations of serum IGF-1 or IGF-binding protein 3 as measured by ELISA. Furthermore, Dpp4/ mice or rats do not exhibit increased organ growth or body size. Hence, the available data suggest that although DPP4 cleaves and inactivates both GHRH and IGF-1, enzymatic inactivation by DPP4 is not the major mechanism regulating the bioactivity of the GHRH-IGF-1 axis.

The role of acute cortisol and DHEAS in predicting acute and chronic PTSD symptoms

Joanne Mouthaan, Marit Sijbrandij, Jan S.K. Luitse
Psychoneuroendocrinology (2014) 45, 179—186
http://dx.doi.org/10.1016/j.psyneuen.2014.04.001

Background: Decreased activation of the hypothalamus—pituitary—adrenal (HPA) axis in response to stress is suspected to be a vulnerability factor for posttraumatic stress disorder (PTSD). Previous studies showed inconsistent findings regarding the role of cortisol in predicting PTSD. In addition, no prospective studies have examined the role of dehydroepiandrosterone (DHEA), or its sulfate form DHEAS, and the cortisol-to-DHEA(S) ratio in predicting PTSD. In this study, we tested whether acute plasma cortisol, DHEAS and the cortisol-to-DHEAS ratio predicted PTSD symptoms at 6 weeks and 6 months post-trauma. Methods: Blood samples of 397 adult level-1 trauma center patients, taken at the trauma resuscitation room within hours after the injury, were analyzed for cortisol and DHEAS levels. PTSD symptoms were assessed at 6 weeks and 6 months post-trauma with the Clinician Administered PTSD Scale. Results: Multivariate linear regression analyses showed that lower cortisol predicted PTSD symptoms at both 6 weeks and 6 months, controlling for age, gender, time of blood sampling, injury, trauma history, and admission to intensive care. Higher DHEAS and a smaller cortisol-to-DHEAS ratio predicted PTSD symptoms at 6 weeks, but not after controlling for the same variables, and not at 6 months. Conclusions: Our study provides important new evidence on the crucial role of the HPA-axis in response to trauma by showing that acute cortisol and DHEAS levels predict PTSD symptoms in survivors of recent trauma.
Neurobiology of DHEA and effects on sexuality, mood and cognition

Pluchino, P.Drakopoulos, F.Bianchi-Demicheli, J.M.Wenger
J Steroid Biochem & Molec Biol 145 (2015) 273–280
http://dx.doi.org/10.1016/j.jsbmb.2014.04.012

Dehydroepiandrosterone (DHEA) and its sulfate ester, DHEAS, are the most abundant steroid hormones in the humans. However, their physiological significance, their mechanisms of action and their possible roles as treatment are not fully clarified. Biological actions of DHEA(S) in the brain involve neuroprotection, neurite growth, neurogenesis and neuronal survival, apoptosis, catecholamine synthesis and secretion, as well as anti-oxidant, anti- inflammatory and antiglucocorticoid effects. In addition, DHEA affects neurosteroidogen is and endorphin synthesis/release. We also demonstrated in a model of ovariectomized rats that DHEA therapy increases proceptive behaviors, already after 1 week of treatment, affecting central function of sexual drive. In women, the analyses of clinical outcomes are far from being conclusive and many issues should still be addressed. Although DHEA preparations have been available in the market since the 1990s, there are very few definitive reports on the biological functions of this steroid. We demonstrate that 1 year DHEA administration at the dose of 10mg provided a significant improvement in comparison with vitamin D in sexual function
and in frequency of sexual intercourse in early postmenopausal women. Among symptomatic women, the spectrum of symptoms responding to DHEA requires further investigation, to define the type of sexual symptoms (e.g. decreased sexual function or hypoactive sexual desire disorder) and the degree of mood/cognitive symptoms that could be responsive to hormonal treatment.
In this regard, our findings are promising, although they need further exploration with a larger and more representative sample size.
Although adrenal cortex is considered to be the primary source of DHEAS in the brain, it was reported that DHEAS did not dis- appear or decrease in the brain 15 days neither after orchiectomy, adrenalectomy, or both, nor after the inhibition of adrenal secretion by dexamethasone. DHEA and DHEAS were among the first neurosteroids identified in rat brains. Cytochrome P450c17 was found in a subset of neurons of embryonic rodent brains. While P450c17 protein was readily detected in the brain, the abundance of P450c17 mRNA transcripts in the embryonic mouse brain or hippocampus of adult male rats was low, and was approximated to be 1/200^th of the expression in testis.
DHEAS may be synthesized in the brain from DHEA. Sulfation of DHEA has been observed in the brains of rhesus monkeys in vivo and in human fetal brain slices in vitro. DHEA sulfotransferase (HSTor SULT2A1) is an enzyme that sulfonates DHEA (in addition to pregnenolone).Western blotting and immune-histochemistry showed protein expression of an HST in adult Wistar rat brain. In addition SULT2A1 mRNA expression has been shown in rat brains. DHEAS is predominately transported out of the brain across the blood–brain barrier and DHEAS found in the brain is most likely due to local synthesis . DHEA(S) may mediate some of its actions through conversion into more potent sex steroids and activation of androgen or estrogen receptors in tissue.
According to existing assumption of the biology of depression, DHEA(S) ability to modulate many neurobiological actions could underlie relationships between endogenous and/or exogenously- supplemented DHEA(S) concentrations and depression. There is evidence that DHEAS concentrations are negatively correlated with ratings of depressed mood. However, the remaining literature examining plasma and serum DHEA(S) concentrations in depression is still inconsistent and other plasma indexes were studied in order to more accurately discriminate depressed from nondepressed individuals. Hypothalamic–pituitary–adrenal axis (HPA) hyperactivity has
been demonstrated in chronic diseases affecting nervous system disorders like depression. The end products of HPA axis, glucocorticoids (GCs), regulate many physiological functions and play an important role in affective regulation and dysregulation. Despite DHEAS levels which markedly decrease throughout adulthood, an increase in circulating cortisol with advanced age has been observed in human and nonhuman primates.
The most relevant aspect meriting attention is certainly the controversial finding among the studies that investigate the correlation of the endogenous DHEA sulfate (DHEAS) level, the aging process or organ illness with the results coming from studies focusing on the effects of exogenous DHEAS administration on brain function, sexuality, cardiovascular health and metabolic syndrome. Indeed, the marked age-related decline in serum DHEA and DHEAS has suggested that a deficiency of these steroids may be causally related to the development of a series of diseases that are generally associ- ated with aging. The postulated consequences of low DHEA levels include insulin resistance, obesity, cardiovascular disease, cancer, reduction of the immune defense system as well as psychosocial problems such as depression and a general deterioration in the sensation of well-being and cognitive function, DHEA replacement may seem an attractive treatment opportunity. Nevertheless, the analyses of clinical outcomes are far from being conclusive.

Dehydroepiandrosterone, its metabolites and ion channels

Hill, M. Dusková, L. Stárka
J Steroid Biochem & Molec Biology 145(2015)293–314
http://dx.doi.org/10.1016/j.jsbmb.2014.05.006

This review is focused on the physiological and pathophysiological relevance of steroids influencing the activities of the central and peripheral nervous systems with regard to their concentrations in body fluids and tissues in various stages of human life like the fetal development or pregnancy. The data summarized in this review shows that DHEA and its unconjugated and sulfated metabolites are physiologically and pathophysiologically relevant in modulating numerous ion channels and participate in vital functions of the human organism. DHEA and its unconjugated and sulfated metabolites including 5 _/ _-reduced androstane steroids participate in various physiological and pathophysiological processes like the management of GnRH cyclic release, regulation of glandular and neurotransmitter secretions, maintenance of glucose homeostasis on one hand and insulin insensitivity on the other hand, control of skeletalmuscle and smooth muscle activities including vasoregulation, promotion of tolerance to ischemia and other neuroprotective effects. In respect of prevalence of steroid sulfates over unconjugated steroids in the periphery and the opposite situation in the CNS, the sulfated androgens and androgen metabolites reach relevance in peripheral organs. The unconjugated androgens and estrogens are relevant in periphery and so much the more in the CNS due to higher concentrations of most unconjugated steroids in the CNS tissues than in circulation and peripheral organs.

Neurotrophins are proteins found within a broad range of cell types in the brain and periphery that facilitate neuronal growth, survival, and plasticity. The neurotrophin ‘‘superfamily’’ includes nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), neurotrophin-4/5 (NT4/5), and neurotrophin-6. Target tissues are hypothesized to regulate neuron survival by making neurotrophins available in limited amounts, resulting in selection of neurons with the best connectivity to the target tissue. NGF, in particular, is released by the target tissue and taken up in responsive neurons by receptor-mediated endocytosis. It is then transported retrogradedly into the cell where it exerts trophic effects. Lu et al. proposed a ‘‘Yin and Yang model,’’ whereby neurotrophic action is mediated by two principal classes of transmembrane receptor systems: the tyrosine kinase (Trk) receptors (including TrkA [selective for NGF], TrkB [selective for BDNF and NT4/5], and TrkC [selective for NT3]) and the neurotrophin receptor p75NTR. Each receptor type binds mature neurotrophins and/or neurotrophin precursors (proneurotrophins), creating a complex ‘‘balance’’ that then causes neuronal survival or death.
DHEA has been shown to evoke NGF mRNA expression in target cells. In a study of pregnant women, Schulte-Herbrüggen et al. showed no relationships between serum DHEAS and NGF. In contrast, we showed that DHEAS independently associated with salivary NGF (sNGF) in military men under baseline conditions, while DHEA did not. We now know that both DHEA(S) and NGF respond affirmatively to stressful insult, yet the association between these analytes during stress exposure is not understood. Characterization of this relationship has implications for prevention and treatment of traumatic stress and injury, degenerative disease management, and nerve repair. In this report, we extended our prior study of neuroprotective properties of DHEAS in men under baseline conditions to a prospective paradigm involving intense stress exposure in both men and women. We hypothesized that

(a) robust associations would prevail between total output of DHEAS and sNGF across the stress trajectory and at each time point,
(b) changes in DHEAS would predict corresponding changes in sNGF, and
(c) baseline DHEAS would positively predict total sNGF output across the stress trajectory.
We also explored the roles of testosterone and cortisol. In light of less definitive prior literature, directional hypotheses were not stated regarding these analytes.

In the first regression model, total hormone output (AUCG) of the independent variables (DHEAS, testosterone, and cortisol) combined to explain 63.7% of variance in sNGF output (F = 65.4, p < 0.001). Standardized beta coefficients revealed that testosterone exerted an independent effect (b = 0.80, p < 0.001), while the other predictors were not significant. In light of this unexpected finding, we then used regression-based causal steps modeling to evaluate whether testosterone mediated a hypothesized direct effect of DHEAS on sNGF. Following this approach, DHEAS predicted sNGF in an initial regression model (b = 0.45, p < 0.001). When testosterone was added, the direct effect of DHEAS (path c0) on sNGF was nearly eradicated and no longer significant (b = .04, p = .57), thus suggesting a mediated effect. An alternate statistical test (Sobel Test; 34) evaluating the hypothesized difference between the total effect (path c) and the direct effect (path c0) of DHEAS on sNGF produced a similar result (test statistic = 4.0, p < 0.001). Fig. 1 depicts positive association of DHEAS to sNGF, while Fig. 2 depicts Positive association of testosterone to sNGF.

Positive association of DHEAS total output and sNGF total output

Positive association of testosterone total output and sNGF total output

Positive association of testosterone total output and sNGF total output.
The models were then decomposed at each time point. At baseline, the independent variables (DHEAS, testosterone, and cortisol) combined to account for 10.2% of variance in sNGF (F = 5.3, p < 0.01). Standardized beta coefficients showed that DHEAS exerted an independent effect on sNGF (b = 0.39, p < 0.001), while the other predictors were not significant. During stress exposure, the independent variables combined to account for 28.0% of variance in NGF (F = 15.8, p < 0.001). Again, DHEAS exerted an independent effect (b = 0.56, p < 0.001) while the other predictors were not significant. During recovery, the predictor set accounted for 18.0% of variance in sNGF (F = 9.2, p < 0.001), and DHEAS exerted an independent effect (b = 0.47, p < 0.001) while the other predictors did not.
The models were then decomposed relative to each change index. In terms of reactivity, the independent variables (DHEAS, testosterone, and cortisol reactivity) and covariate (sex) combined to account for 20.3% of variance in sNGF reactivity (F = 8.2, p < 0.001). Standardized beta coefficients revealed that DHEAS reactivity exerted an independent effect (b = 0.39, p < 0.001), while the other predictors were not significant. In terms of recovery, the predictors combined to account for 28.2% of variance in sNGF recovery (F = 15.5, p < 0.001); DHEAS recovery exerted an independent effect (b = 0.52, p < 0.001), as did testosterone recovery (b = [1]0.27, p < 0.01). In terms of residual elevation/depression, the independent variables explained 12.4% of variance in sNGF residual elevation (F = 6.2, p < 0.001). DHEAS residual elevation exerted an independent effect (b = 0.35, p < 0.001), while the other predictors did not.

Endocrine-Disrupting Chemicals and Human Growth and Maturation: A Focus on Early Critical Windows of Exposure

Julie Fudvoye, Jean-Pierre Bourguignon, Anne-Simone Parent
Vitamins and Hormones, 2014; 94: Chapt 1. 1-25.
http://dx.doi.org/10.1016/B978-0-12-800095-3.00001-8

Endocrine-disrupting chemicals (EDCs) are exogenous substances that interfere with hormone synthesis, metabolism, or action. In addition, some of them could cause epigenetic alterations of DNA that can be transmitted to the following generations. Because the developing organism is highly dependent on sex steroids and thyroid hormones for its maturation, the fetus and the child are very sensitive to any alteration of their hormonal environment. An additional concern about that early period of life comes from the shaping of the homeostatic mechanisms that takes place also at that time with involvement of epigenetic mechanisms along with the concept of fetal origin of health and disease. In this chapter, we will review the studies reporting effects of EDCs on human development. Using a translational approach, we will review animal studies that can shed light on some mechanisms of action of EDCs on the developing organism. We will focus on the major hormone-dependent stages of development: fetal growth, sexual differentiation, puberty, brain development, and energy balance. We will also discuss the possible epigenetic effects of EDCs on human development.

Several studies have reported that prenatal or early postnatal exposure to some EDCs is associated with alterations of cognitive or motor functions in children. Knowing the fundamental role played by thyroid hormones and sex steroids in cortex development, one can hypothesize that disruption of those hormones could cause alteration of the development of the cerebral cortex and of its functions later in life. We will review here the human data suggesting a causal effect for endocrine disrupters on impairment of cortical functions and approach some EDC mechanisms of action using animal models.

Thyroid hormones are known to be essential for brain development. They regulate progenitor proliferation and differentiation, neuron migration, and dendrite outgrowth (Parent, Naveau, Gerard, Bourguignon, & Westbrook, 2011). Even mild thyroid hormone insufficiency in humans can produce measurable deficits in cognitive functions (Zoeller & Rovet, 2004). Thyroid hormone action is mediated by two classes of nuclear receptors (Forrest & Vennstro¨m, 2000) that exhibit differential spatial and temporal expressions in the brain, suggesting that thyroid hormones have variable functions during brain development. This differential expression of thyroid hormone receptors explains the critical period of thyroid hormone action on brain development as suggested by models of maternal hypothyroidism or congenital hypothyroidism.

Depending on the timing of onset of hypothyroidism, the offspring will display problems of visual attention, gross or fine motor skills, or language and memory skills. Similarly, one can hypothesize that disruption of thyroid function by EDCs will have different effects based on the timing of exposure. However, few studies focused on that aspect. Polychlorinated biphenyls (PCBs) form a group of widespread environmental contaminants composed of 209 different congeners used in a wide variety of applications. Their production was banned in the 1970s but PCBs are still present in the environment due to their high stability. PCBs were among the first EDCs identified as responsible for alterations of cognitive functions. Indeed, impaired memory and altered learning abilities have been associated with prenatal exposure to EDCs in humans and In animal models, perinatal exposure to PCBs has been consistently associated with a decrease of thyroid hormone concentration in maternal serum as well as pup serum. Some but not all epidemiological studies in human have found an association between PCB body burden and thyroid hormone levels. This disruption of thyroid function could explain some of the effects of PCBs on the developing brain. Indeed, animal models have shown that the ototoxic effects of PCBs could be partially ameliorated by thyroxin replacement and PCBs seem to alter some of the developmental processes in the cortex and the cerebellum that are dependent on thyroid hormones. However, recent publications raise important issues.

As it is the case for other EDCs, some windows of susceptibility have been identified during pre- and postnatal brain development. Recent studies have shown that exposure to PBDEs causes alteration of thyroid hormone levels in pregnant women and infants as it is the case in rodents. Only very few studies, however, have focused on the molecular or cellular effects of perinatal exposure to PBDEs in vivo. Viberg et al. have reported a decrease of cholinergic nicotinic receptors in the hippocampus after exposure to BDE-99 and BDE-153. However, the link between such a decrease and the behavioral effects of PBDEs is still unclear. Other teams have reported that exposure to PBDEs reduced hippocampal long term potentiation and decreased brain-derived neurotrophic factor expression in the brain. While several studies have reported negative effect of PBDEs on brain development and cognitive function in animals, there is relatively little information about adverse health effects of PBDEs in humans. Some very recent studies have identified a correlation between prenatal exposure to PBDEs and alteration of cognitive functions.

Endocrine-Disrupting Chemicals: Elucidating Our Understanding of Their Role in Sex and Gender-Relevant End Points

Cheryl A. Frye
Vitamins and Hormones, 2014; 94: 41-98
http://dx.doi.org/10.1016/B978-0-12-800095-3.00003-1

Endocrine-disrupting chemicals (EDCs) are diverse and pervasive and may have significant consequence for health, including reproductive development and expression of sex-/gender-sensitive parameters. This review chapter discusses what is known about common EDCs and their effects on reproductively relevant end points. It is proposed that one way that EDCs may exert such effects is by altering steroid levels (androgens or 17-estradiol, E2) and/or intracellular E2 receptors (ERs) in the hypothalamus and/or hippocampus. Basic research findings that demonstrate developmentally sensitive end points to androgens and E2 are provided. Furthermore, an approach is suggested to examine differences in EDCs that diverge in their actions at ERs to elucidate their role in sex-/gender-sensitive parameters.

Reproductive dysfunction among adults and emotional, attentional, and behavioral disorders among children are on the rise. Sperm counts and fertility have declined in the last 50 years . Incidence of attention-deficit hyperactivity disorder (ADHD) and autism has increased in the last 30 years. These increases in reproductive dysfunction and developmental disorders may be due to increased exposure to environmental contaminants, although there is controversy about the relationship between exposure and these effects.
Many contaminants in the environment, including polychlorinated biphenyls (PCBs), dioxins, and metals, accumulate in exposed individuals and may have adverse consequences due to effects as endocrine-disrupting chemicals (EDCs). EDCs may have effects by altering steroid levels (androgens or 17β-estradiol, E2) and/or intracellular E2 receptors (ERs) in the hypothalamus and/or hippocampus.
Steroid hormones, during critical periods of development, organize sexual dimorphisms in brain and behavior and give rise to sex differences in later responses to steroid hormones. EDCs can profoundly disrupt reproductive responses following adult exposure and result in pervasive effects that extend throughout the life of their offspring. Many nonreproductive behaviors, such
as spatial performance, activity, and arousal, are also sexually dimorphic and organized and activated by steroid hormones. Thus, EDCs may affect reproductive and the aforementioned nonreproductive parameters by altering E2 levels and/or ER binding in the hypothalamus and/or hippocampus.
Results from the literature and preliminary data will be presented that demonstrate our use of a whole-animal model to begin to investigate effects of exposure (in adulthood and/or development) to EDCs on steroid levels (androgens and E2), actions at ERs (in hypothalamus and hippocampus), and reproductive-sensitive measures (anogenital distance, accessory structure weight, onset of puberty and sexual maturity, and reproductive behavior) and nonreproductive behaviors (spatial performance, play behavior, and arousal) throughout development.

A common feature of many environmental contaminants is their estrogenic effects. Some contaminants can alter production of E2 and/or androgens or act as agonists or antagonists for intracellular or membrane ERs. Thus, the term “endocrine-disrupting chemicals” (EDCs) in this chapter is used to refer to contaminants with these effects. An important question considered here is the extent to which EDCs’ actions to alter E2 levels and/or ER binding in the hypothalamus or hippocampus mitigates effects on reproductive or nonreproductive processes. There are potential pervasive, negative effects of endocrine disrupters on steroid sensitive tissues, which may confer risk to disease states, such as cancer, heart disease, and neurodegenerative disorders. The following discussion provides evidence that exposure to EDCs during development may result in permanent, lifelong differences in sexual function and reproductive ability, as well as cognitive function and/or emotional reactivity/arousal. Gonad development, sex determination, and reproductive success of offspring are highly dependent on sex hormone systems. The developing organism is exquisitely sensitive to alterations in hormone function. In the early embryonic state, the gonads of human males and females are morphologically identical. Sexual differentiation begins under hormonal influence during the fifth and sixth weeks of fetal development, and thus, alterations in hormones during this highly sensitive period can have profound consequences. Disruption of the sex steroid system during fetal stages of life results in profound adverse developmental reproductive effects, as is well known from the effects of DES. The balance of estrogens and androgens is critical for normal development, growth, and functioning of the reproductive system. Although especially important during development, this balance is important throughout life for the preservation of normal feminine or masculine traits, as well as the expression of some sexually dimorphic behaviors (sex, spatial performance, and arousal).

Proposed negative effects of exposure to endocrine disrupters during development in people and in animals. The focus here is on vulnerability to sexually dimorphic processes that are estrogen-sensitive, such as reproductive, cognitive, and emotional development and associated behavioral processes

The existing data clearly indicate that developmental exposure to EDCs can adversely affect sexual development of people and animals; however, there are different effects depending upon the EDCs and when in development exposure occurs. Therefore, we consider the next effects of EDCs exposure at different point in development and the consequences for reproductive development and behavior, as well as E2 levels and hypothalamic ER binding.
Steroid hormones also play a critical role in neurodevelopment that influences not only reproductive but also nonreproductive behaviors that show sex differences. Specific behavioral differences in nonreproductive behaviors between males and females include differences in spatial learning, play, exploration, activity levels, novelty-seeking behavior, and emotional reactivity. These sex dimorphisms are thought to reflect adaptive differences for behavioral strategies in coping as a result of sexual selection. Moreover, these sexually dimorphic behaviors may be relevant for concerns regarding increased developmental, cognitive, or emotional disabilities over the past 30 years. Also, behaviors are particularly sensitive measures of effects of EDCs.
EDCs can alter cognitive development. Some, but not all, studies have shown a predictive relationship between prenatal PCB exposure and cognitive development in infancy through preschool years. EDCs have direct effects on nervous system function. Long-term potentiation (LTP), a form of synaptic plasticity used as a model system for study of cognitive potential, is altered by PCBs and lead. The protein kinase C (PKC)-signaling pathway is involved in the modulation of learning, memory, and motor behavior and may be a target of E2’s actions. PCBs also alter PKC signaling. Although findings provide evidence that EDCs can alter cognitive performance, these measures of cognition are neither sexually dimorphic nor E2- or ER-dependent.
There are sex-specific effects of perinatal PCB and dioxin exposure on spatial learning. Yu-Cheng boys that were prenatally exposed to high levels of PCBs and PCDFs when their mothers were accidentally exposed to these contaminants in rice oil show more disrupted cognitive development, mainly spatial function, than did exposed girls. In animal studies, spatial learning that favors males is mediated by perinatal exposure to androgens. Gestational and lactational exposure to ortho-substituted PCBs produces spatial deficits at adolescence in male mice or adulthood in male rats. The sparse data suggest that developmental exposure to EDCs disrupts spatial memory. Furthermore, Exposure during adulthood to EDCs can also have activational effects on spatial memory. Females exposed to a phytoestrogen-rich diet exhibit “masculinized” spatial performance in a radial arm maze, while males fed with a phytoestrogen-free diet show “feminized” performance.
An important question is what are the mechanisms by which developmental and/or adult exposure to EDCs alters spatial performance? There is evidence for sex differences in spatial performance and activational effects of E2 in adulthood to alter spatial performance of rats. Systemic or intrahippocampal administration of E2 improves spatial performance of female rats. Further, E2’s actions at intracellular ERs in the hippocampus of adults do not seem to be required to mediate these effects on spatial performance.
EDCs may have effects on E2 metabolism in a number of ways. First, some EDCs can alter serum lipid concentrations. Cholesterol is the precursor for the production of E2 and other steroid hormones (see Fig. 3.3). Second, there is also evidence that some EDCs can alter metabolism enzymes that are necessary for converting cholesterol to steroid hormones. Induction of CYP occurs when EDCs, such as TCDD, bind the aromatic hydrocarbon receptor (AhR). There is a firm link between PCBs, enzyme induction, and AhR. The binding of EDCs with AhR can result in antiestrogenic activity through increased metabolism and depletion of endogenous E2. Elevated levels of CYP enzymes, primarily expressed not only in the liver but also in the brain and other tissues, result in increased E2 metabolism and excretion. Alternatively, compounds that are metabolized by P450s may result in a net estrogenic effect if they inhibit endogenous estrogens from being metabolized.
Steroid hormones are lipid molecules with limited solubility in plasma and are accordingly carried through the plasma compartment to target cells by specific plasma transporter proteins. Each transporter protein has a specific ligand-binding domain for its associated hormone. It is generally accepted that the “free” formof the steroid hormone, and not the conjugate of the hormone with its plasma transport protein, enters target cells and binds with the appropriate receptor. Receptors for the steroid hormones are proteins located primarily in the cell nucleus or partitioned between the cytoplasm and the nucleus. The unoccupied steroid receptors may reside in the cell as heterodimeric complexes with the 90 kDa heat-shock protein, which prevents the receptor from binding with the DNA until the receptor has first bound with its steroid hormone. Once the hormone binds to the receptor, the hormone receptor complexes with the heterodimeric heat-shock protein and undergoes a conformational change and is activated. The activated receptor binds with DNA at a specific site, initiating gene transcription.

Traditional effects of steroid hormones at their cognate steroid receptors

Traditional effects of steroid hormones at their cognate steroid receptors, which act as transcription factors. In this example, effects of steroid hormones, such as estradiol, to bind estrogen receptor (ER) subtypes, referred to as ERa and ERb, are shown.

Beyond traditional actions solely through intracellular cognate estrogen receptors (ERs; ERa and ERb), steroids, such as estradiol, and estradiol-mimetics (endocrine disrupters) may have novel actions involving membrane bound ERs, other neurotransmitter systems (e.g., NMDA receptor), and signal transduction cascades (e.g., growth factors, MAPK).

To date, there has been little investigation in a whole-animal model of the effects of EDCs on E2 levels and/or activity at intracellular ERs in the brain. Thus, changes in E2 levels and ER activity in the hypothalamus and hippocampus, concomitant with alterations in endocrine parameters and reproductive behavior and nonreproductive behavior, respectively, are
needed to elucidate tissue specificity of EDCs’ functions and mechanisms.

Low-Dose Effects of Hormones and Endocrine Disruptors

Laura N. Vandenberg
Vitamins and Hormones, 2014; 94: 129-165
http://dx.doi.org/10.1016/B978-0-12-800095-3.00005-5

Endogenous hormones have effects on tissue morphology, cell physiology, and behaviors at low doses. In fact, hormones are known to circulate in the part-per-trillion and part-per-billion concentrations, making them highly effective and potent signaling molecules.

Many endocrine-disrupting chemicals (EDCs) mimic hormones, yet there is strong debate over whether these chemicals can also have effects at low doses. In the 1990s, scientists proposed the “low-dose hypothesis,” which postulated that EDCs affect humans and animals at environmentally relevant doses. This chapter focuses on data that support and refute the low-dose hypothesis. A case study examining the highly controversial example of bisphenol A and its low-dose effects on the prostate is examined through the lens of endocrinology. Finally, the chapter concludes with a discussion of factors that can influence the ability of a study to detect and interpret low-dose effects appropriately.

Since EDCs began to be studied in depth in the 1990s, there has been intense debate over whether the public should be concerned about low level exposures to these chemicals. The low-dose hypothesis, proposed at that time, has steadily accumulated evidence that EDCs have actions at low doses, and these effects are not necessarily predicted from high-dose toxicology testing. In 2002, the NTP expert panel reported evidence for low-dose effects for a small number of EDCs and estradiol. In 2012, an updated approach identified several dozen additional EDCs with evidence for low-dose effects. Further, epidemiology studies continue to find relationships between EDC exposure levels and diseases in the general public, which has raised concerns because the general public is exposed to a large number of environmental chemicals at low doses. For decades, hormones have been known to produce striking changes in tissue morphology, physiology, and behaviors at exceedingly low doses.

A relatively large body of evidence suggests that EDCs, and in particular those environmental chemicals that mimic endogenous hormones, have similar effects at low doses. Although there is still no consensus about the universality of “low-dose effects” in the toxicology community, the Endocrine Society (Diamanti-Kandarakis et al., 2009; Zoeller et al., 2012) believes not only that there is sufficient evidence in support of this phenomenon but also that it is time for public health agencies to make changes to risk assessment paradigms and give greater consideration to studies that specifically identify low-dose effects when considering risks from chemical exposures.

Bisphenol A interferes with synaptic remodeling

Tibor Hajszan, Csaba Leranth
Frontiers in Neuroendocrinology 31 (2010) 519–530
http://dx.doi.org:/10.1016/j.yfrne.2010.06.004

The potential adverse effects of Bisphenol A (BPA), a synthetic xenoestrogen, have long been debated. Although standard toxicology tests have revealed no harmful effects, recent research highlighted what was missed so far: BPA-induced alterations in the nervous system. Since 2004, our laboratory has been investigating one of the central effects of BPA, which is interference with gonadal steroid-induced synaptogenesis and the resulting loss of spine synapses. We have shown in both rats and nonhuman primates that BPA completely negates the ~70–100% increase in the number of hippocampal and prefrontal spine synapses induced by both estrogens and androgens. Synaptic loss of this magnitude may have significant consequences, potentially causing cognitive decline, depression, and schizophrenia, to mention those that our laboratory has shown to be associated with synaptic loss. Finally, we discuss why children may particularly be vulnerable to BPA, which represents future direction of research in our laboratory.

Bisphenol-A rapidly promotes dynamic changes in hippocampal dendritic morphology through estrogen receptor-mediated pathway by concomitant phosphorylation of NMDA receptor subunit NR2B

Xiaohong Xu ⁎, Yinping Ye, Tao Li, Lei Chen, Dong Tian, Qingqing Luo, Mei Lu
Toxicology and Applied Pharmacology 249 (2010) 188–196
http://dx.doi.org:/10.1016/j.taap.2010.09.007

Bisphenol-A (BPA) is known to be a potent endocrine disrupter. Evidence is emerging that estrogen exerts a rapid influence on hippocampal synaptic plasticity and the dendritic spine density, which requires activation of NMDA receptors. In the present study, we investigated the effects of BPA (ranging from 1 to 1000 nM), focusing on the rapid dynamic changes in dendritic filopodia and the expressions of estrogen receptor (ER) β and NMDA receptor, as well as the phosphorylation of NMDA receptor subunit NR2B in the cultured hippocampal neurons. A specific ER antagonist ICI 182,780 was used to examine the potential involvement of ERs. The results demonstrated that exposure to BPA (ranging from 10 to 1000 nM) for 30 min rapidly enhanced the motility and the density of dendritic filopodia in the cultured hippocampal neurons, as well as the phosphorylation of NR2B (pNR2B), though the expressions of NMDA receptor subunits NR1, NR2B, and ERβ were not changed. The antagonist of ERs completely inhibited the BPA-induced increases in the filopodial motility and the number of filopodia extending from dendrites. The increased pNR2B induced by BPA (100 nM) was also completely eliminated. Furthermore, BPA attenuated the effects of 17β-estradiol (17β-E2) on the dendritic filopodia outgrowth and the expression of pNR2B when BPA was co-treated with 17β-E2. The present results suggest that BPA, like 17β-E2, rapidly results in the enhanced motility and density of dendritic filopodia in the cultured hippocampal neurons with the concomitant activation of NMDA receptor subunit NR2B via an ER-mediated signaling pathway. Meanwhile, BPA suppressed the enhancement effects of 17β-E2 when it coexists with 17β-E2. These results provided important evidence suggesting the neurotoxicity of the low levels of BPA during the early postnatal development of the brain.

Bisphenol-A rapidly enhanced passive avoidance memory and phosphorylation of NMDA receptor subunits in hippocampus of young rats

Xiaohong Xu⁎, Tao Li, Qingqing Luo, Xing Hong, Lingdan Xie, Dong Tian
Toxicology and Applied Pharmacology 255 (2011) 221–228
http://dx.doi.org:/10.1016/j.taap.2011.06.022

Bisphenol-A (BPA), an endocrine disruptor, is found to influence development of brain and behaviors in rodents. The previous study indicated that perinatal exposure to BPA impaired learning-memory and inhibited N-methyl-D-aspartate receptor (NMDAR) subunits expressions in hippocampus during the postnatal development in rats; and in cultured hippocampal neurons, BPA rapidly promotes dynamic changes in dendritic morphology through estrogen receptor-mediated pathway by concomitant phosphorylation of NMDAR subunit NR2B. In the present study, we examined the rapid effect of BPA on passive avoidance memory and NMDAR in the developing hippocampus of Sprague–Dawley rats at the age of postnatal day 18. The results showed that BPA or estradiol benzoate (EB) rapidly extended the latency to step down from the platform 1 h after foot shock and increased the phosphorylation levels of NR1, NR2B, and mitogen-activated extracellular signal-regulated kinase (ERK) in hippocampus within 1 h. While 24 h after BPA or EB treatment, the improved memory and the increased phosphorylation levels of NR1, NR2B, ERK disappeared. Furthermore, pre-treatment with an estrogen receptors (ERs) antagonist, ICI182, 780, or an ERK-activating kinase inhibitor, U0126, significantly attenuated EB- or BPA-induced phosphorylations of NR1, NR2B, and ERK within 1 h. These data suggest that BPA rapidly enhanced short-term passive avoidance memory in the developing rats. A non-genomic effect via ERs may mediate the modulation of the phosphorylation of NMDAR subunits NR1 and NR2B through ERK signaling pathway.

Bisphenol A promotes dendritic morphogenesis of hippocampal neurons through estrogen receptor-mediated ERK1/2 signal pathway

Xiaohong Xu, Yang Lu, Guangxia Zhang, Lei Chen, Dong Tian, et al.
Chemosphere 96 (2014) 129–137
http://dx.doi.org/10.1016/j.chemosphere.2013.09.063

Bisphenol A (BPA), an environmental endocrine disruptor, has attracted increasing attention to its adverse effects on brain developmental process. The previous study indicated that BPA rapidly increased motility and density of dendritic filopodia and enhanced the phosphorylation of N-methyl-D-aspartate (NMDA) receptor subunit NR2B in cultured hippocampal neurons within 30 min. The purpose of the present study was further to investigate the effects of BPA for 24 h on dendritic morphogenesis and the underlying mechanisms. After cultured for 5 d in vitro, the hippocampal neurons from 24 h-old rat were infected by AdV-EGFP to indicate time-lapse imaging of living neurons. The results demonstrated that the exposure of the cultured hippocampal neurons to BPA (10, 100 nM) or 17β-estradiol (17β-E2, 10 nM) for 24 h significantly promoted dendritic development, as evidenced by the increased total length of dendrite and the enhanced motility and density of dendritic filopodia. However, these changes were suppressed by an ERs antagonist, ICI182,780, a non-competitive NMDA receptor antagonist, MK-801, and a mitogen activated ERK1/2-activating kinase (MEK1/2) inhibitor, U0126. Meanwhile, the increased F-actin (filamentous actin) induced by BPA (100 nM) was also completely eliminated by these blockers. Furthermore, the result of western blot analyses showed that, the exposure of the cultures to BPA or 17β-E2 for 24 h promoted the expression of Rac1/Cdc42 but inhibited that of RhoA, suggesting Rac1 (Ras related C3 botulinum toxinsubstrate 1)/Cdc42 (cell divisioncycle 42) and RhoA (Ras homologous A), the Rho family of small GTPases, were involved in BPA- or 17β-E2-induced changes in the dendritic morphogenesis of neurons. These BPA- or 17b-E2-induced effects were completely blocked by ICI182,780, and were partially suppressed by U0126. These results reveal that, similar to 17β-E2, BPA exerts its effects on dendritic morphogenesis by eliciting both nuclear actions and extranuclear-initiated actions that are integrated to influence the development of dendrite in hippocampal neurons.

Tyreoliberin (Trh) – The Regulatory Neuropeptide Of Cns Homeostasis
Danuta Jantas
Advances In Cell Biology 2;(4)/2010 (139–154)
http://dx.doi.org:/10.2478/v10052-010-0008-4

The physiological role of thyreoliberin (TRH) is the preservation of homeostasis within four systems
(i) the hypothalamic-hypophsysiotropic neuroendocrine system,
(ii) the brain stem/midbrain/spinal cord system,
(iii) the limbic/cortical system, and
(iv) the chronobiological system.

Thus TRH, via various cellular mechanisms, regulates a wide range of biological processes (arousal, sleep, learning, locomotive activity, mood) and possesses the potential for unique and widespread applications for treatment of human illnesses. Since the therapeutic potential of TRH is limited by its pharmacological profile (enzymatic instability, short half-life, undesirable effects), several synthetic analogues of TRH were constructed and studied in mono- or adjunct therapy of central nervous system (CNS) disturbances. The present article summarizes the current state of understanding of the physiological role of TRH and describes its putative role in clinical indications in CNS maladies with a focus on the action of TRH analogues.

Breakthrough in neuroendocrinology by discovering novel neuropeptides and neurosteroids: 2. Discovery of neurosteroids and pineal neurosteroids

Kazuyoshi Tsutsui, Shogo Haraguchi
General and Comparative Endocrinology 205 (2014) 11–22
http://dx.doi.org/10.1016/j.ygcen.2014.03.008

Bargmann–Scharrer’s discovery of ‘‘neurosecretion’’ in the first half of the 20th century has since matured into the scientific discipline of neuroendocrinology. Identification of novel neurohormones, such as neuropeptides and neurosteroids, is essential for the progress of neuroendocrinology. Our studies over the past two decades have significantly broadened the horizons of this field of research by identifying novel neuropeptides and neurosteroids in vertebrates that have opened new lines of scientific investigation in neuroendocrinology. We have established de novo synthesis and functions of neurosteroids in the brain of various vertebrates. Recently, we discovered 7α-hydroxypregnenolone (7α-OH PREG), a novel bioactive neurosteroid that acts as a key regulator for inducing locomotor behavior by means of the dopaminergic system. We further discovered that the pineal gland, an endocrine organ located close to the brain, is an important site of production of neurosteroids de novo from cholesterol (CHOL). The pineal gland secretes 7α-OH PREG and 3α,5α-tetrahydroprogesterone (3α,5α-THP; allopregnanolone) that are involved in locomotor rhythms and neuronal survival, respectively. Subsequently, we have demonstrated their mode of action and functional significance. This review summarizes the discovery of these novel neurosteroids and its contribution to the progress of neuroendocrinology.

Mechanisms of crosstalk between endocrine systems: Regulation of sex steroid hormone synthesis and action by thyroid hormones

Paula Duarte-Guterman, Laia Navarro-Martín, Vance L. Trudeau
General and Comparative Endocrinology 203 (2014) 69–85
http://dx.doi.org/10.1016/j.ygcen.2014.03.015

Thyroid hormones (THs) are well-known regulators of development and metabolism in vertebrates. There is increasing evidence that THs are also involved in gonadal differentiation and reproductive function. Changes in TH status affect sex ratios in developing fish and frogs and reproduction (e.g., fertility), hormone levels, and gonad morphology in adults of species of different vertebrates. In this review, we have summarized and compared the evidence for cross-talk between the steroid hormone and thyroid axes and present a comparative model. We gave special attention to TH regulation of sex steroid synthesis and action in both the brain and gonad, since these are important for gonad development and brain sexual differentiation and have been studied in many species. We also reviewed research showing that there is a TH system, including receptors and enzymes, in the brains and gonads in developing and adult vertebrates. Our analysis shows that THs influences sex steroid hormone synthesis in vertebrates, ranging from fish to pigs. This concept of crosstalk and conserved hormone interaction has implications for our understanding of the role of THs in reproduction, and how these processes may be dysregulated by environmental endocrine disruptors.

Insights into the structure of class B GPCRs

Kaspar Hollenstein, Chris de Graaf, Andrea Bortolato, Ming-Wei Wang, et al.
Trends in Pharmacological Sciences, Jan 2014; 35(1)
http://dx.doi.org/10.1016/j.tips.2013.11.001

The secretin-like (class B) family of G protein-coupled receptors (GPCRs) are key players in hormonal homeostasis and are interesting drug targets for the treatment of several metabolic disorders (such as type 2 diabetes, osteoporosis, and obesity) and nervous system diseases (such as migraine, anxiety, and depression). The recently solved crystal structures of the transmembrane domains of the human glucagon receptor and human corticotropin-releasing factor receptor 1 have opened up new opportunities to study the structure and function of class B GPCRs. The current review shows how these structures offer more detailed explanations to previous biochemical and pharmacological studies of class B GPCRs, and provides new insights into their interactions with ligands.

Class B G protein-coupled receptors (GPCRs), also referred to as the secretin family of GPCRs, include receptors for 15 peptide hormones, which can be grouped into five subfamilies based on their physiological role (see Table 1 for an overview) [1]. These receptors are important drug targets in many human diseases, including diabetes, osteoporosis, obesity, cancer, neurodegeneration, cardiovascular disease, headache, and psychiatric disorders. However, the identification of small-molecule oral drugs for this family has proved extremely challenging.

(A,B) Crystal structures of the class B G protein-coupled receptors corticotropin-releasing factor receptor 1 (CRF1) [Protein Data Bank (PDB) identifier: 4K5Y] and glucagon receptor (PDB identifier: 4L6R) are shown in blue and orange ribbons, respectively, in two different views from within the membrane. Transmembrane (TM) helices and helix 8 are labelled. The disulfide bond tethering extracellular loop 2 (ECL2) to the tip of TM3 is shown as purple sticks. In CRF1 the small-molecule antagonist CP-376395 is shown in stick representation with carbon, nitrogen, and oxygen atoms colored magenta, blue, and red, respectively, and as skeletal formula in an inset. (C) Superposition of the two structures, with insets highlighting regions of particular interest. To highlight the structural differences in the extracellular halves of CRF1 and glucagon receptor, the distance of approximately 11 A° between the Ca-atoms of residues 7.33b at the N-terminal end of TM7 is indicated with a red arrow. The small molecule binding pocket is shown as a superposition of the two receptors around CP-376395, illustrating the antagonist binding mode and the substantial structural differences observed for TM6 of the two receptors.

Overview of NMR and crystal structures of class B G protein-coupled

receptor (GPCR) extracellular domains (ECDs; magenta) and their complexes with peptide ligands (different colors). A complete overview of corresponding Protein Data Bank identifiers is presented in Table 1 (not shown). (B) Structure-based sequence alignment of representative peptide ligands of class B GPCR, adopted from Parthier et al. [6]. The residues of the peptide ligands solved in ECD–ligand complex crystal structures are marked using the same colour as in Figure 2A. The residues that are boxed black are found in an α-helical conformation in the complex. Peptide ligand residues that covalently bind receptors in photo-crosslinking or cysteine-trapping studies are colored and boxed green, whereas peptide ligand residues that have been mutated and studied in combination with receptor mutants are colored and boxed red. Note that the first residue of glucagon-like peptide-1 (GLP-1) is His7. A complete overview of all ECD structures and important peptide ligands for all class B GPCRs is presented in Table 1. Putative helix-capping residues [6] are coloured blue and cysteines involved in a disulfide-bridge (calcitonin) are coloured orange. D-phenylalanine (f), and norleucine (m) residues are indicated in stressin and astressin. The last 41 and 99 residues of parathyroid hormone (PTH) and PTH-related protein. (PTHrP), respectively, are not displayed. Abbreviations: CGRP, calcitonin gene-related peptide; CLR, calcitonin receptor-like receptor; CRF, corticotropin-releasing factor; CT, calcitonin; Ext-4, exendin-4; GHRHR, growth hormone releasing hormone receptor; GIP, glucose-dependent insulinotropic peptide; PAC, pituitary adenylate cyclase; PACAP, pituitary adenylate cyclase activating polypeptide; RAMP, receptor-activity modifying proteins; SCTR, secretin receptor; Ucn, urocortin; VPAC, vasoactive pituitary adenylate cyclase.

Figure 3. (not shown) (A) The spatial correspondence between residues in transmembrane (TM) helices of class A and class B G protein-coupled receptors (GPCRs) makes it possible to align the most conserved residues in class A (designated X.50, Ballesteros–Weinstein numbering) and class B (designated X.50b, Wootten numbering) for comparisons between GPCR classes (Box 1). (B) Structural alignment of corticotropin-releasing factor receptor 1 (CRF1; blue) and glucagon receptor (GCGR; orange) to two representative class A GPCRs, histamine H1 receptor (H1R; Protein Data Bank identifier: 3RZE) and CXC-chemokine receptor 4 (CXCR4; Protein Data Bank identifier: 3ODU/3OE0) (in grey). Helices are depicted as cylinders, and the ligands glucagon (for GCGR), CP-376395 (for CRF1), doxepin (for H1R), and IT1t and CVX15 (for CXCR4) are shown as sticks. The

location of the Ca-atoms of the most conserved residues of TM1–3 and TM5 among class A and class B GPCRs (Box 1) are indicated by spheres (TM4 is not depicted for clarity).

The GCGR and CRF1 crystal structures show distinct structural features and different binding pockets compared to class A GPCRs, and give new insights into the molecular details of peptide and small-molecule binding to class B GPCRs. The first two crystal structures of the TM domains of class B GPCRs provide a structural framework that will enable the design of biochemical and biophysical experiments detailing the complex structure of this class of receptors, and facilitate the design of stabilized constructs that might lead to the solution of full-length class B GPCR–ligand complexes. The structures furthermore present more reliable structural templates for the design of specific and potent small molecules for the treatment of type 2 diabetes (GCGR) and depression (CRF1) in particular, and open new avenues for structure-based small-molecule drug discovery for class B GPCRs as a whole.

Novel receptor targets for production and action of allopregnanolone in the central nervous system: a focus on pregnane xenobiotic receptor

Cheryl A. Frye, Carolyn J. Koonce and Alicia A. Walf
Front in Cell Neurosci Apr 2014; 8(106): 1-13.
http://dx.doi.org:/10.3389/fncel.2014.00106

Neurosteroids are cholesterol-based hormones that can be produced in the brain,

independent of secretion from peripheral endocrine glands, such as the gonads and

adrenals. A focus in our laboratory for over 25 years has been how production of the

pregnane neurosteroid, allopregnanolone, is regulated and the novel (i.e., non steroid

receptor) targets for steroid action for behavior. One endpoint of interest has been lordosis, the mating posture of female rodents. Allopregnanolone is necessary and sufficient for lordosis, and the brain circuitry underlying it, such as actions in the midbrain ventral tegmental area (VTA), has been well-characterized. Published and recent findings supporting a dynamic role of allopregnanolone are included in this review.
First, contributions of ovarian and adrenal sources of precursors of allopregnanolone, and the requisite enzymatic actions for de novo production in the central nervous system will be discussed.
Second, how allopregnanolone produced in the brain has actions on behavioral processes that are independent of binding to steroid receptors, but instead involve rapid modulatory actions via neurotransmitter targets (e.g., g-amino butyric acid-GABA, N methyl-D-aspartate- NMDA) will be reviewed.
Third, a recent focus on characterizing the role of a promiscuous nuclear receptor, pregnane xenobiotic receptor (PXR), involved in cholesterol metabolism and expressed in the VTA, as a target for allopregnanolone and how this relates to both actions and production of allopregnanolone will be addressed. For example, allopregnanolone can bind PXR and knocking down expression of PXR in the midbrain VTA attenuates actions of allopregnanolone via NMDA and/or GABAA for lordosis. Our understanding of allopregnanolone’s actions in the VTA for lordosis has been extended to reveal the role of allopregnanolone for broader, clinically-relevant questions, such as neurodevelopmental processes, neuropsychiatric disorders, epilepsy, and aging.

Genetically Encoded Chemical Probes in Cells Reveal the Binding Path of Urocortin-I to CRF Class B GPCR

Irene Coin, Vsevolod Katritch, Tingting Sun, Zheng Xiang, Fai Yiu Siu
Cell Dec 2013; 155, 1258–1269
http://dx.doi.org/10.1016/j.cell.2013.11.008

Molecular determinants regulating the activation of class B G-protein-coupled receptors (GPCRs) by native peptide agonists are largely unknown. We have investigated here the interaction between the corticotropin releasing factor receptor type 1 (CRF1R) and its native 40-mer peptide ligand Urocortin- I directly in mammalian cells. By incorporating unnatural amino acid photochemical and new click chemical probes into the intact receptor expressed in the native membrane of live cells, 44 intermolecular spatial constraints have been derived for the ligand-receptor interaction. The data were analyzed in the context of the recently resolved crystal structure of
CRF1R transmembrane domain and existing extracellular domain structures, yielding a complete conformational model for the peptide-receptor complex. Structural features of the receptor-ligand complex yield molecular insights
on the mechanism of receptor activation and the basis for discrimination between agonist and antagonist function.

Investigation of GPCR-Ligand Interactions under Native Conditions Using Genetically Encoded Chemical Probes GPCRs are integral membrane proteins containing multiple domains and various posttranslational modifications. To understand GPCR-ligand interactions by crystallography, receptors have to be extracted from the cell membrane and modified with a series of expedients such as deglycosylation, therm-stabilizing mutations, fusions with soluble proteins, or complexes with stabilizing nanobodies. We present here a method to investigate GPCR-ligand interactions at the intact fully posttranslationally modified receptor bound to its WT ligand on the membrane of the live cell, which mimics the native conditions for GPCR function. We first genetically incorporated into the receptor the photocrosslinking Uaa Azi, which served as
a proximity probe to provide an overall map of the ligand binding sites on the receptor. We then determined the relative position of the ligand in the binding pocket using a residue-specific chemical crosslinking reaction between Ffact genetically incorporated into the receptor and Cys introduced into the ligand. The derived intermolecular spatial constraints served eventually to build a detailed conformational model for the receptor-ligand complex.

Glutamate Neurons within the Midbrain Dopamine Regions

Morales and D. H. Root
Neuroscience 282 (2014) 60–68
http://dx.doi.org/10.1016/j.neuroscience.2014.05.032

Midbrain dopamine (DA) neurons are hypothesized to play roles in reward-based behavior and addiction, reward prediction and learning by error detection, effort-based decision making, flexible reward-directed behaviors,

incentive salience, stimulus salience (e.g., prediction of rewarding and aversive events), aversion, depression, and fear. The extensive, divergent behavioral roles of midbrain dopamine neurons, predominantly from the ventral tegmental area (VTA), indicate that this system is highly heterogeneous.
This heterogeneity may be reflected in part by the diverse phenotypic characteristics among DAergic neurons and their interactive brain structures.

Midbrain dopamine systems play important roles in Parkinson’s disease, schizophrenia, addiction, and depression. The participation of midbrain dopamine systems in diverse clinical contexts suggests these systems are highly complex. Midbrain dopamine regions contain at least three neuronal phenotypes: dopaminergic, GABAergic, and glutamatergic. Here, we review the locations, subtypes, and functions of glutamatergic neurons within midbrain dopamine regions. Vesicular glutamate transporter 2 (VGluT2) mRNA-expressing neurons are observed within each midbrain dopamine system. Within rat retrorubral field (RRF), large populations of VGluT2 neurons are observed throughout its anteroposterior extent. Within rat substantia nigra pars compacta (SNC), VGluT2 neurons are observed centrally and caudally, and are most dense within the laterodorsal subdivision. RRF and SNC rat VGluT2 neurons lack tyrosine hydroxylase (TH), making them an entirely distinct population of neurons from dopaminergic neurons. The rat ventral tegmental area (VTA) contains the most heterogeneous populations of VGluT2 neurons. VGluT2 neurons are found in each VTA subnucleus but are most dense within the anterior midline subnuclei. Some subpopulations of rat VGluT2 neurons co-express TH or glutamic acid decarboxylase (GAD), but most of the VGluT2 neurons lack TH or GAD. Different subsets of rat VGluT2-TH neurons exist based on the presence or absence of vesicular monoamine transporter 2, dopamine transporter, or D2 dopamine receptor. Thus, the capacity by which VGluT2-TH neurons may release dopamine will differ based on their capacity to accumulate vesicular dopamine, uptake extracellular dopamine, or be autoregulated by dopamine. Rat VTA VGluT2 neurons exhibit intrinsic VTA projections and extrinsic projections to the accumbens and to the prefrontal cortex. Mouse VTA VGluT2 neurons project to accumbens shell, prefrontal cortex, ventral pallidum, amygdala, and lateral habenula. Given their molecular diversity and participation in circuits involved in addiction, we hypothesize that individual VGluT2 subpopulations of neurons play unique roles in addiction and other disorders. This article is part of a Special Issue entitled: Ventral Tegmentum & Dopamine. Published by Elsevier Ltd. On behalf of IBRO.

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Gastrointestinal Endocrinology

Posted in Cell Biology, Curation, Disease Biology, Endocrine Diseases, Genomic Endocrinology, Human Immune System in Health and in Disease, Imaging-based Cancer Patient Management, Innovations, Liver & Digestive Diseases Research, Microbiology, Neurological Diseases, Nutrition, Signaling & Cell Circuits, Small Molecules in Development of Therapeutic Drugs, tagged cancer treatment, enteric peptides, Gastroenterology, somatostatin on February 10, 2015| Leave a Comment »

Gastrointestinal Endocrinology

Writer and Curator: Larry H Bernstein, MD, FCAP

The Gut Microbial Endocrine Organ: Bacterially Derived Signals Driving Cardiometabolic DiseasesMark Brown and Stanley L. Hazen

Annual Review of Medicine Jan 2015; 66: 343-359
http://dx.doi.org:/10.1146/annurev-med-060513-093205

The human gastrointestinal tract is home to trillions of bacteria, which vastly outnumber host cells in the body. Although generally overlooked in the field of endocrinology, gut microbial symbionts organize to form a key endocrine organ that converts nutritional cues from the environment into hormone-like signals that impact both normal physiology and chronic disease in the human host. Recent evidence suggests that several gut microbial-derived products are sensed by dedicated host receptor systems to alter cardiovascular disease (CVD) progression. In fact, gut microbial metabolism of dietary components results in the production of proatherogenic circulating factors that act through a meta-organismal endocrine axis to impact CVD risk. Whether pharmacological interventions at the level of the gut microbial endocrine organ will reduce CVD risk is a key new question in the field of cardiovascular medicine. Here we discuss the opportunities and challenges that lie ahead in targeting meta-organismal endocrinology for CVD prevention.

Exogenous glucagon-like peptide 1 reduces contractions in human colon circular muscle

Antonella Amato, Sara Baldassano, Rosa Liotta1, Rosa Serio and Flavia Mulè
J Endocrinol April 1, 2014 221 29-37
http://dx.doi.org:/10.1530/JOE-13-0525

Glucagon-like peptide 1 (GLP1) is a naturally occurring peptide secreted by intestinal L-cells. Though its primary function is to serve as an incretin, GLP1 reduces gastrointestinal motility. However, only a handful of animal studies have specifically evaluated the influence of GLP1 on colonic motility. Consequently, the aims of this study were to investigate the effects induced by exogenous GLP1, to analyze the mechanism of action, and to verify the presence of GLP1 receptors (GLP1Rs) in human colon circular muscular strips. Organ bath technique, RT-PCR, western blotting, and immunofluorescence were used. In human colon, exogenous GLP1 reduced, in a concentration-dependent manner, the amplitude of the spontaneous contractions without affecting the frequency and the resting basal tone. This inhibitory effect was significantly reduced by exendin (9–39), a GLP1R antagonist, which per se significantly increased the spontaneous mechanical activity. Moreover, it was abolished by tetrodotoxin, a neural blocker, or Nω-nitro-L-arginine – a blocker of neuronal nitric oxide synthase (nNOS). The biomolecular analysis revealed a genic and protein expression of the GLP1R in the human colon. The double-labeling experiments with anti-neurofilament or anti-nNOS showed, for the first time, that immunoreactivity for the GLP1R was expressed in nitrergic neurons of the myenteric plexus. In conclusion, the results of this study suggest that GLP1R is expressed in the human colon and, once activated by exogenous GLP1, mediates an inhibitory effect on large intestine motility through NO neural release.

The impact of dipeptidyl peptidase 4 inhibition on incretin effect, glucose tolerance, and gastrointestinal-mediated glucose disposal in healthy subjects

N A Rhee, S H Østoft, J J Holst, C F Deacon, T Vilsbøll and F K Knop
Eur J Endocrinol September 1, 2014 171 353-36
http://dx.doi.org:/10.1530/EJE-14-0314

Objective Inhibition of dipeptidyl peptidase 4 (DPP4) is thought to intensify the physiological effects of the incretin hormones. We investigated the effects of DPP4 inhibition on plasma levels of glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide 1 (GLP1), incretin effect, glucose tolerance, gastrointestinal-mediated glucose disposal (GIGD) and gastric emptying in healthy subjects. Design A randomised, controlled and open-labelled study. Methods Ten healthy subjects (six women; age, 40±5 years (mean±S.E.M.); BMI, 24±3 kg/m2; fasting plasma glucose, 5.1±0.2 mmol/l and HbA1c, 34±1 mmol/mol (5.3±0.1%)) were randomised to two-paired study days comprising a 4-h 50 g oral glucose tolerance test (OGTT) with paracetamol (A) and an isoglycaemic intravenous (i.v.) glucose infusion (B), with (A1+B1) and without (A2+B2) preceding administration of the DPP4 inhibitor sitagliptin. Results Isoglycaemia was obtained in all subjects on the paired study days. Significant increases in fasting levels and OGTT-induced responses of active GLP1 and GIP were seen after DPP4 inhibition. No significant impact of DPP4 inhibition on fasting plasma glucose (5.1±0.1 vs 4.9±0.1 mmol/l, P=0.3), glucose tolerance (area under the curve (AUC) for plasma glucose, 151±35 vs 137±26 mmol/l×min, P=0.7) or peak plasma glucose during OGTT (8.5±0.4 vs 8.1±0.3 mmol/l, P=0.3) was observed. Neither incretin effect (40±9% (without DPP4 inhibitor) vs 40±7% (with DPP4 inhibitor), P=1.0), glucagon responses (1395±165 vs 1223±195 pmol/l×min, P=0.41), GIGD (52±4 vs 56±5%, P=0.40) nor gastric emptying (Tmax for plasma paracetamol: 86±9 vs 80±12 min, P=0.60) changed following DPP4 inhibition. Conclusions These results suggest that acute increases in active incretin hormone levels do not affect glucose tolerance, GIGD, incretin effect, glucagon responses or gastric emptying in healthy subjects.

Morphology and Tissue Distribution of Four Kinds of Endocrine Cells in the Digestive Tract of the Chinese Yellow Quail (Coturnix japonica)

He, M., Liang, X., Wang, K., (…), Li, X., Liu, L.
Analytical and Quantitative Cytology and Histology 2014; 36 (4), pp. 199-205

Objective: To describe the tissue distribution, density, and the morphological characteristics of 4 kinds of endocrine cells in the digestive tract of the Chinese yellow quail (Coturnix japonica). Study design: The streptavidin-biotin-peroxidase complex immunohistochemical method was used to identify the distribution of somatostatin (SS), serotonin (5-HT), gastrin and neuropeptide Y (NPY) in digestive tracts including proventriculus, duodenum, jejunum, ileum, and rectum. SPSS 19.0 software was used to perform biological statistical analysis. Results: The results showed that the SS and 5-HT secreting cells were mainly distributed in the proventriculus (19.2±6.9 and 16.1±3.4 cfu/mm2) and duodenum (2.9±2.0 and 1.9±0.6 cfu/mm2). Gastrin and NPY were not detected in each section of the digestive tract. Moreover, there was no significant difference in the quantitative distribution and morphological characteristics of SS and 5-HT secreting cells in the digestive tract between male and female quails. Conclusion: The distribution and morphological characteristics of endocrine cells were closely related to the physiological functions of different parts in the digestive tract. The preferential location of endocrine cells provides additional information for future studies on the physiological roles of gastrointestinal peptides in the gastrointestinal tract of the Chinese yellow quail

GEP-NETS update: Functional localisation and scintigraphy in neuroendocrine tumours of the gastrointestinal tract and pancreas (GEP-NETs)

Wouter W de Herder
Eur J Endocrinol May 1, 2014 170 R173-R183
http://dx.doi.org:/10.1530/EJE-14-0077

For patients with neuroendocrine tumours (NETs) of the gastrointestinal tract and pancreas (GEP) (GEP-NETs), excellent care should ideally be provided by a multidisciplinary team of skilled health care professionals. In these patients, a combination of nuclear medicine imaging and conventional radiological imaging techniques is usually mandatory for primary tumour visualisation, tumour staging and evaluation of treatment. In specific cases, as in patients with occult insulinomas, sampling procedures can provide a clue as to where to localise the insulin-hypersecreting pancreatic NETs. Recent developments in these fields have led to an increase in the detection rate of primary GEP-NETs and their metastatic deposits. Radiopharmaceuticals targeted at specific tumour cell properties and processes can be used to provide sensitive and specific whole-body imaging. Functional imaging also allows for patient selection for receptor-based therapies and prediction of the efficacy of such therapies. Positron emission tomography/computed tomography (CT) and single-photon emission CT/CT are used to map functional images with anatomical localisations. As a result, tumour imaging and tumour follow-up strategies can be optimised for every individual GEP-NET patient. In some cases, functional imaging might give indications with regard to future tumour behaviour and prognosis.

An immunohistochemical study on the distribution of endocrine cells in the digestive tract of gray goose (Anser anser)

Jun YANG1, Lei ZHANG,, Xin LI, , Leii ZHANG, , Xiangjiang LIU, , Kemei PENG

Turk. J. Vet. Anim. Sci. 2012; 36(4): 373-379
http://dx.doi.org:/10.3906/vet-1101-654

The objective of this study was to investigate the morphology and the distribution of 5-hydroxytryptamine (5-HT), somatostatin (SS), gastrin (Gas), glucagon (Glu), and substance P immunoreactive (IR) cells in the digestive tract of gray goose by the immunohistochemical streptavidin-peroxidase method.

The samples were taken from 10 healthy adult gray geese. Th e results showed that 5 kinds of IR cells were mainly distributed between the mucous epithelium and intestinal gland. The number of 5-HT-IR cells was highest in the rectum and duodenum, but none were observed in the pylorus. SS-IR cells appeared in great numbers in the pylorus, duodenum, and cecum; however, they were not found in esophagus. Gas-IR cells were mainly distributed in the glandular stomach and jejunum. Glu-IR cells appeared in small numbers in the glandular stomach, duodenum, and jejunum, but were not detected in other tissues. Substance P-IR cells were located in the jejunum, cecum, and rectum. Analysis of the present study showed that the distribution and morphological features of these 5 different endocrine cells were related to the feeding habits and metabolism in the digestive tract of the gray goose

Chapter 154 – Somatostatin

Mathias Guggera, Jean-Claude Meunierb

Handbook of Biologically Active Peptides 2006, Pages 1123–1130
http:/dx.doi.org:/10.1016/B978-012369442-3/50157-4

Somatostatin is abundant in the mucosa and in the enteric nervous system of the gastrointestinal tract and in the pancreas. In these tissues, it exerts a broad range of mainly inhibitory physiological actions in multiple targets, including endocrine glands, exocrine glands, smooth muscles, blood vessels, and immune cells, mediated by up to six somatostatin receptor subtypes. Several diseases of the gastrointestinal tract are characterized by disturbances in the somatostatin production or by overexpression of somatostatin receptors. In particular, somatostatin receptors have been found to be overexpressed in neuroendocrine gastroenteropancreatic tumors. These tumors can be diagnostically and therapeutically targeted with somatostatin analogs. In addition, various nonneoplastic diseases, including bleeding in the upper gastrointestinal tract, fistulas, and diarrhea can also be treated with somatostatin analogs.

Immunocytochemical study of the distribution of endocrine cells in the pancreas of the Brazilian sparrow species Zonotrichia Capensis Subtorquata (Swaison, 1837)

Nascimento, AA.*; Sales, A.; Cardoso, TRD.; Pinheiro, NL.; Mendes, RMM.
Braz. J. Biol. Nov. 2007; 67(4): São Carlos

In the present study, we investigated types of pancreatic endocrine cells and its respective peptides in the Brazilian sparrow species using immunocytochemistry. The use of polyclonal specific antisera for somatostatin, glucagon, avian pancreatic polypeptide (APP), YY polypeptide (PYY) and insulin, revealed a diversified distribution in the pancreas. All these types of immunoreactive cells were observed in the pancreas with different amounts. Insulin- Immunoreactive cells to (B cells) were most numerous, preferably occupying the central place in the pancreatic islets. Somatostatin, PPA, PYY and glucagon immunoreactive cells occurred in a lower frequency in the periphery of pancreatic islets.

Immunolocalisation of the serotonin in the fundus ventriculi and duodenum of the Asia minor ground squirrel: (Spermophilus xanthoprymnus)

Timurkaan, S., Özkan, E., Ilgün, R., Gür, F.M
Veterinarski Arhiv 2009; 79 (1), pp. 69-76

Serotonin immunoreactive cells were located and distributed in the fundus and duodenum with variable frequencies. They were spherical or spindle-shaped and the highest frequency serotonin immunoreactive cells were detected in the whole fundic region. The regional distribution of the endocrine cells in the fundus and duodenum of the citellus resembled other mammalian species.

An Immunohistochemical Study of Gastrointestinal Endocrine Cells in the BALB/c Mouse

Ku, S.K., Lee, H.S., Lee, J.H.
J Vet Med Series C: Anatomia Histologia Embryologia 2004; 33 (1), pp. 42-48

The distributions and frequencies of some endocrine cells in the eight portions of the gastrointestinal tract (GIT) of BALB/c mouse were studied. Endocrine cells were stained using immunohistochemical method with seven types of anti-sera against bovine chromogranin (BCG), serotonin, gastrin, cholecystokinin (CCK)-8, somatostatin, glucagon and human pancreatic polypeptide (HPP), and the regional distributions and their relative frequencies were observed in the eight portions of the GIT of BALB/c mice. All seven types of immunoreactive (IR) cells were identified. Most of the IR cells in the intestinal portion were generally spherical or spindle in shape (open type cell) while round-shaped cells (closed type cell) were found in the intestinal gland and stomach regions occasionally. Their relative frequencies varied according to each portion of the GIT. BCG-IR cells were observed throughout the whole GIT except for the rectum and they were most predominant in the pylorus. Serotonin-IR cells were detected throughout the whole GIT and they showed the highest frequency in the fundus. Gastrin- and CCK-IR cells were restricted to the pylorus and duodenum with a majority in the pylorus and rare or a few frequencies in the duodenum. Compared with other mammals, somatostatin-IR cells were restricted to the fundus and pylorus with a few frequencies, respectively. In addition, glucagon- and HPP-IR cells were restricted to the fundus and duodenum, respectively, with relative low frequencies. Some species-dependent unique distributions and frequencies of endocrine cells were observed in the GIT of BALB/c mouse compared with other rodents.

Immunohistochemical study of the distribution of serotonin in the gastrointestinal tract of the porcupines (Hystrix cristata)

Timurkaan, S., Karan, M., Aydin, A.
Revue de Medecine Veterinaire 2005; 156 (11), pp. 533-536

Serotonin immunoreactive cells were located in the gastric glands and in the intestinal epithelium with variable frequencies. They were spherical or spindle-shaped. Serotonin immuno-reactive cells were detected in almost all regions of the gastrointestinal tract and they showed highest frequency in the stomach and colon.

Effects of carbachol on gastrin and somatostatin release in rat antral tissue culture

Wolfe, M.M., Jain, D.K., Reel, G.M., McGuigan, J.E.
Gastroenterology 1984; 87 (1), pp. 86-93

Recent studies have demonstrated that somatostatin-containing cells are in close anatomic proximity to gastrin-producing cells in antral mucosa, suggesting a potential local regulatory role for somatostatin. The purpose of this study was to examine further the relationships between gastrin and somatostatin and the effects of the cholinergic agonist carbachol on content and release of gastrin and somatostatin using rat antral mucosa in tissue culture. Antral mucosa was cultured at 37 °C in KrebsHenseleit buffer, pH 7.4, gassed with 95% O2-5% CO2. After 1 h, the culture medium was decanted and the tissue was boiled to extract mucosal gastrin and somatostatin. Inclusion of carbachol 2.5 × 10-6 M in the culture medium decreased medium somatostatin from 1.91 ± 0.28 (SEM) ng/mg tissue protein to 0.62 ± 0.12 ng/mg (p < 0.01), extracted mucosal somatostatin from 2.60 ± 0.30 to 1.52 ± 0.16 ng/mg (p < 0.001), and percentage of somatostatin released from 42% ± 2.6% to 27% ± 2.2% (p < 0.01). Carbachol also increased culture media gastrin from 14 ± 2.5 to 27 ± 3.0 ng/mg protein (p < 0.01). Tissue content and release of gastrin and somatostatin were also examined during culture of rat antral mucosa in culture media containing antibodies to somatostatin in the presence and in the absence of carbachol. Incubation with somatostatin antisera, both with and without carbachol, markedly increased culture media concentrations of somatostatin, all of which was effectively bound by antibodies present in the media. Antibody binding of somatostatin was accompanied by significant increases in culture media gastrin concentrations, both in the presence and in the absence of carbachol. Results of these studies support the hypothesis that antral somatostatin exerts a local regulatory effect on gastrin release and that cholinergic stimulation of gastrin release is mediated, at least in part, through inhibition of somatostatin synthesis and release.

Endogenous somatostatin-28 modulates postprandial insulin secretion. Immunoneutralization studies in baboons

J W Ensinck, R E Vogel, E C Laschansky, D J Koerker, et al.
J Clin Invest. 1997; 100(9):2295–2302.
http://dx.doi.org:/10.1172/JCI119767

Somatostatin-28 (S-28), secreted into the circulation from enterocytes after food, and S-14, released mainly from gastric and pancreatic D cells and enteric neurons, inhibit peripheral cellular functions. We hypothesized that S-28 is a humoral regulator of pancreatic B cell function during nutrient absorption. Consistent with this postulate, we observed in baboons a two to threefold increase in portal and peripheral levels of S-28 after meals, with minimal changes in S-14. We attempted to demonstrate a hormonal effect of these peptides by measuring their concentrations before and after infusing a somatostatin-specific monoclonal antibody (mAb) into baboons and comparing glucose, insulin, and glucagon-like peptide-1 levels before and for 4 h after intragastric nutrients during a control study and on 2 d after mAb administration (days 1 and 2). Basal growth hormone (GH) and glucagon levels and parameters of insulin and glucose kinetics were also measured. During immunoneutralization, we found that (a) postprandial insulin levels were elevated on days 1 and 2; (b) GH levels rose immediately and were sustained for 28 h, while glucagon fell; (c) basal insulin levels were unchanged on day 1 but were increased two to threefold on day 2, coincident with decreased insulin sensitivity; and (d) plasma glucose concentrations were similar to control values. We attribute the eventual rise in fasting levels of insulin to its enhanced secretion in compensation for the heightened insulin resistance from increased GH action. Based on the elevated postmeal insulin levels after mAb administration, we conclude that S-28 participates in the enteroinsular axis as a decretin to regulate postprandial insulin secretion.

The Therapeutic Value of Somatostatin and Its Analogues

Sadaf Farooqi, John S. Bevan, Michael C. Shepperd, John A. H. Wass
Pituitary June 1999; 2(1), pp 79-88
http:/dx.doi.org:/10.1023/A:1009978106476

In this review we discuss the physiological effects of somatostatin, which are mediated by specific receptor subtypes on different tissues. These observations have suggested new therapeutic possibilities for the use of the synthetic somatostatin analogues in the treatment of acromegaly as well as a number of other endocrine and non-endocrine disorders.

Somatostatin and Somatostatin Receptors

Ujendra Kumar, Michael Grant
Cellular Peptide Hormone Synthesis and Secretory Pathways
(Results and Problems in Cell Differentiation) 2010; 50: pp 97-120
http://dx.doi.org:/10.1007/400_2009_29

The biological effects of somatostatin (SST) were first encountered unexpectedly in the late 1960s in two unrelated studies, one by Krulich et al. (1968) who reported on a growth hormone (GH)-releasing inhibitory substance from hypothalamic extracts, and the other, by Hellman and Lernmark (1969), on the presence of a potent insulin inhibitory factor from the extracts of pigeon pancreatic islets. However, the inhibitory substance was not officially identified until 1973 by Guillemin’s group (Brazeau et al. 1973). In both synthetic and naturally occurring forms, this tetradecapeptide, originally coined as somatotropin release-inhibitory factor (SRIF, SST-14) was shown by Brazeau et al. to be the substance controlling hypothalamic GH release. This single achievement not only pioneered SST research but was also duly recognized, as Guillemin shared the 1977 Nobel Prize in Medicine. The following years bequeathed an exponential increase in SST-related studies. It soon became clear that SST-synthesis was not restricted to the hypothalamus. Its production is widely distributed throughout the central nervous system (CNS), peripheral neurons, the gastrointestinal tract, and the pancreatic islets of Langerhans (Luft et al. 1974; Arimura et al. 1975; Dubois 1975; Hokfelt et al. 1975; Orci et al. 1975; Pelletier et al. 1975; Polak et al. 1975; Patel and Reichlin 1978). In fact, SST-like immunoreactivity can be found throughout various tissues of vertebrates and invertebrates, including the plant kingdom (Patel 1992; Tostivint et al. 2004). Given its broad anatomical distribution, it is no wonder that SST produces a wide spectrum of biological effects. Generally regarded as an inhibitory factor, SST can function either locally on neighboring cells or distantly through the circulation, to regulate such physiological processes as glandular secretion, neurotransmission, smooth muscle contractility, nutrient absorption, and cell division (Reichlin 1983a, b; Patel 1992, 1999; Patel et al. 2001; Barnett 2003).

Receptor-Mediated Tumor Targeting with Radiopeptides. Part 1. General Concepts and Methods: Applications to Somatostatin Receptor-Expressing Tumors

Alex N. Eberle, Gabriele Mild, and Sylvie Froidevaux
Journal of Receptors and Signal Transduction 2004; 24(4) , Pages 319-455
http://dx.doi.org:/10.1081/RRS-200040939

Radiolabeled peptides have become important tools in nuclear oncology, both as diagnostics and more recently also as therapeutics. They represent a distinct sector of the molecular targeting approach, which in many areas of therapy will implement the old “magic bullet” concept by specifically directing the therapeutic agent to the site of action. In this three-part review, we present a comprehensive overview of the literature on receptor-mediated tumor targeting with the different radiopeptides currently studied. Part I summarizes the general concepts and methods of targeting, the selection of radioisotopes, chelators, and the criteria of peptide ligand development. Then, the >400 studies on the application to somatostatin/somatostatin-release inhibiting factor receptor-mediated tumor localization and treatment will be reviewed, demonstrating that peptide radiopharmaceuticals have gained an important position in clinical medicine.

The somatostatin neuroendocrine system: physiology and clinical relevance in gastrointestinal and pancreatic disorders

Malcolm J. Low
Best Practice & Res Clin Endocr & Metab, 2004; 18(4), pp. 607–622
http://dx.doi.org:/10.1016/j.beem.2004.08.005

The physiologic functions of hypothalamic somatostatin in the regulation of pituitary hormone secretion and the clinical use of somatostatin analogs for the treatment of pituitary adenomas have been reviewed. Similarly, the distribution, normal function and potential pathogenic roles of somatostatin in the central nervous system have been reported in detail. This review will focus exclusively on the physiologic actions of somatostatin and its receptors in the gastrointestinal tract, pancreas and immune system. Diagnostic and therapeutic roles of somatostatin analogs in a diverse catalog of neoplastic, inflammatory and autoimmune conditions affecting peripheral systems are outlined, with an emphasis on both well-established indications and current areas of exploration.

Somatostatin is produced in enteroendocrine D cells and intrinsic neurons of the stomach, intestines and pancreas. Its physiologic actions are mediated primarily by somatostatin receptors type 2 and 5, and include the inhibition of secretion of most endocrine and exocrine factors. Diseases directly attributable to somatostatin excess or deficiency are rare, although there is a complex pathogenic relationship between persistent Helicobacter pylori infection and reduced somatostatin in chronic gastritis. Abundant somatostatin receptors on many neoplastic and inflammatory cells are the basis for sensitive in vivo imaging with radiolabeled somatostatin analogs and provide a therapeutic target. Current indications for somatostatin therapy include hormone-expressing neuroendocrine tumors, intractable diarrhea and variceal bleeding secondary to portal hypertension. Exciting advances are being made in the development of high-affinity nonpeptide analogs with receptor-subtype selectivity and increased bioavailability. Somatostatin analogs coupled to high-energy radionuclides show promise as novel cytotoxic agents for certain metastatic tumors.

Evolution of the somatostatin gene family Both forms of mammalian somatostatin are derived post-translationally from a common pro-hormone by the action of specific pro-protein convertases (PCs). Genetic studies indicate a primary role for PC2 in the generation of SST147, which is the predominant form of somatostatin produced in the brain and most other tissues. SST28 is found in its highest concentrations in the gastrointestinal tract, especially the mucosal epithelial cells of the intestines.
A revised evolutionary concept of the somatostatin gene family is that a primordial gene underwent duplication during or before the advent of chordates and that the two resulting genes subsequently underwent differing rates of mutation to produce the distinct prepro-somatostatin and prepro-cortistatin genes in mammals. A second gene duplication event likely occurred in teleosts to generate PSS1 and PSS-II from the ancestral somatostatin gene.
It is possible that additional related genes have not yet been identified. Recent studies utilizing unique polyclonal antisera and a strain of somatostatin-deficient mouse have demonstrated the existence of a novel gastrointestinal peptide with homology to the amino acid sequence of SST28(1–13) that has been named thrittene.
Somatostatin gene organization and regulation The mammalian PPS1 (or SMST) gene has a relatively simple organization consisting of two coding exons separated by one intron. A single promoter directs transcription of the PPS1 gene in all tissues, and there are no known alternative mRNA splicing events. The molecular mechanisms underlying the developmental and hormonal regulation of somatostatin gene transcription have been most extensively studied in pancreatic islets and islet-derived cell lines. The proximal enhancer elements in the somatostatin gene promoter that bind complexes of homeodomain-containing transcription factors (PAX6, PBX, PREP1) to upregulate transcription in pancreatic islets may actually represent gene silencer elements in neurons (promoter elements TSEII and UE-A). Conversely, another related cis-element in the somatostatin gene (promoter element TSEI) apparently binds a homeodomain transcription factor PDX1 (also called STF1/ IDX1/IPF1) that is common to developing brain, pancreas and foregut, and regulates gene expression in both the CNS and gut.
Enteroendocrine cells of the gut mucosa differentiate from pluripotential stem cells in the crypts, share molecular phenotypes and retain close paracrine interactions among the daughter cells. Similarly, pancreatic islet cells share common precursors. Recent studies have demonstrated that bone marrow contains a stem cell population capable of producing islet-like cell clusters in vitro that contain somatostatin-positive cells together with the other cell types found in normally differentiated islets.
Somatostatin Receptors There are five somatostatin receptor subtypes (SSTR1–5) encoded by separate genes located on different chromosomes. Alternative mRNA splicing generates SSTR2α and SSTR2β from heteronuclear RNA after transcription from the single SSTR2 gene. SSTRs are members of the rhodopsin-like G protein-coupled receptor superfamily and are most closely related structurally to the opioid receptors. The unique amino acid signature of SSTRs is contained in a seven-element fingerprint of peptide sequences located in conserved regions of the N and C termini, extra- and intra-cellular loops, and transmembrane domains. SSTRs are expressed in discrete or partially overlapping distributions in multiple target organs and differ in their coupling to second messenger signaling molecules, and therefore in their range and mechanism of intracellular actions. The subtypes also differ in their binding affinity to specific somatostatin-like ligands. Some of these differences have important implications for the use of somatostatin analogs in diagnostic imaging and in pharmacotherapy.
All SSTR subtypes are coupled to pertussis toxin-sensitive G proteins and bind SST14 and SST28 with high affinity in the low nanomolar range, although SST28 has a modestly higher affinity for SSTR5. All the subtypes are expressed in brain and pituitary to varying degrees with different distributions, but SSTR2 and SSTR5 are clearly the most abundant in peripheral tissues. These two subtypes are also the most physiologically important in pancreatic islets. SSTR5 is responsible for the inhibition of insulin secretion from b-cells, and SSTR2 is essential for the inhibition of glucagon from a-cells. SSTR1 is expressed at low levels in gastrointestinal structures. The binding of somatostatin to its receptors leads to the activation of one or more inhibitory G proteins (Gi/o), which in turn decrease adenylyl cyclase activity and the concentration of intracellular cAMP. Other G protein-mediated actions common to all The somatostatin neuroendocrine system 609 SSTRs are activation of a vanadate-sensitive phosphotyrosine phosphatase (PTP) and modulation of mitogen-activated protein kinases (MAPKs).
Inhibition of endocrine and exocrine secretion Somatostatin has diverse biologic activities in the gastrointestinal system. It is secreted from D cells into the extracellular space to act as a paracrine factor on nearby endocrine cells and as an autocrine factor to inhibit its own secretion. Most of the circulating hormonal somatostatin originates from the stomach and intestines. Basal plasma levels are in the range of 30–100 pg/ml and increase postprandially by as much as 100% over baseline for a duration up to 2 hours. The release of somatostatin from enteric D cells is regulated by a combination of nutritional, humoral, neural and paracrine signals.

The modulatory role of somatostatin in gastric acid secretion by parietal cells illustrates the typical complexity of hormonal, paracrine and neural integration within the gastrointestinal tract.
Somatostatin secreted from gastric D cells modulates the gastrin-enterochromaffin-like cell—parietal cell axis. Gastrin, secreted from G cells, stimulates the release of histamine from enterochromaffin-like cells (ECL), which is in turn a major secretagog of hydrochloric acid (HCl) from gastric parietal (P) cells. Somatostatin (SST14) inhibits secretion from each of these cell types, although the predominant actions are on the G and ECL cells. Food intake mediates gastric acid secretion by activating both vagal nerves and intrinsic gastric neurons. D cells are stimulated by the autocrine release of amylin, the paracrine release of bombesin and atrial natiuretic peptide (ANP), the enteric neuron release of pituitary adenylate cyclase-activating peptide (PACAP) and cholecystokinin (CCK), and the T lymphocyte release of interleukin-4 (IL-4).D cells are inhibited by histamine acting on H3 receptors in a negative paracrine feedback loop from ECL cells and by other factors, including gamma-aminobutyric acid (GABA) and opioid peptides. The pathways illustrated are not all-inclusive but represent many of the key regulatory steps.

Practice points

† long-acting somatostatin analogs are primary therapeutic tools for the symptomatic treatment of the excessive hormone and monoamine secretion from carcinoids and other neuroendocrine tumors

† somatostatin and long-acting somatostatin analogs are effective first-line
medical treatment for upper gastrointestinal bleeding from esophageal varices associated with hepatic cirrhosis and portal hypertension but are not indicatedfor the treatment of bleeding from gastric varices or duodenal ulcers

† radiolabeled somatostatin analogs provide a sensitive imaging technique for a wide range of neoplastic and inflammatory disorders, including neuroendocrine tumors, meningiomas and sarcoidosis because of their high level expression of somatostatin receptors.

The role(s) of somatostatin, structurally related peptides and somatostatin receptors in the gastrointestinal tract: a review

J Van Op den bosch, D Adriaensen, L Van Nassauw, Jean-Pierre Timmermans
Regulatory Peptides 156 (2009) 1–8
http://dx.doi.org:/10.1016/j.regpep.2009.04.003

Extensive functional and morphological research has demonstrated the pivotal role of somatostatin (SOM) in the regulation of a wide variety of gastrointestinal activities. In addition to its profound inhibitory effects on gastrointestinal motility and exocrine and endocrine secretion processes along the entire gastrointestinal tract, SOM modulates several organ-specific activities. In contrast to these well-known SOM-dependent effects, knowledge on the SOM receptors (SSTR) involved in these effects is much less conclusive. Experimental data on the identities of the SSTRs, although species- and tissue-dependent, point towards the involvement of multiple receptor subtypes in the vast majority of gastrointestinal SOM-mediated effects. Recent evidence demonstrating the role of SOM in intestinal pathologies has extended the interest of gastrointestinal research in this peptide even further. More specifically, SOM is supposed to suppress intestinal inflammatory responses by interfering with the extensive bidirectional communication between mucosal mast cells and neurons. This way, SOM not only acts as a powerful inhibitor of the inflammatory cascade at the site of inflammation, but exerts a profound anti-nociceptive effect through the modulation of extrinsic afferent nerve fibers. The combination of these physiological and pathological activities opens up new opportunities to explore the potential of stable SOM analogues in the treatment of GI inflammatory pathologies.

Schematic overview of the distribution of the SSTRs 1–5

Schematic overview of the distribution of the SSTRs 1–5 in the murine small intestine under control conditions (left panel) and during intestinal schistosomiasis (right panel). In non-inflamed conditions, SSTR1, SSTR2A and SSTR4 are expressed in non-neuronal (glial cells, enterocytes…) and neuronal cells, both from intrinsic and extrinsic origin. SSTR3 and SSTR5 are undetectable. In response to intestinal schistosomiasis, profound sprouting of nerve fibres expressing SSTR1, SSTR3 and SSTR4 is observed, in addition to the expression of SSTR1 and SSTR3 in mucosal mast cells (MMC).

Somatostatin and Its Receptor Family

Yogesh C. Patel
Frontiers in Neuroendocrinology 1999; 20, 157–198 Article ID frne.1999.0183

Somatostatin (SST), a regulatory peptide, is produced by neuroendocrine, inflammatory, and immune cells in response to ions, nutrients, neuropeptides, neurotransmitters, thyroid and steroid hormones, growth factors, and cytokines. The peptide is released in large amounts from storage pools of secretory cells, or in small amounts from activated immune and inflammatory cells, and acts as an endogenous inhibitory regulator of the secretory and proliferative responses of target cells that are widely distributed in the brain and periphery. These actions are mediated by a family of seven transmembrane (TM) domain G-protein-coupled receptors that comprise five distinct subtypes (termed SSTR1–5) that are endoded by separate genes segregated on different chromosomes. The five receptor subtypes bind the natural SST peptides, SST-14 and SST-28, with low nanomolar affinity. Short synthetic octapeptide and hexapeptide analogs bind well to only three of the subtypes, 2, 3, and 5. Selective nonpeptide agonists with nanomolar affinity have been developed for four of the subtypes (SSTR1, 2, 3, and 4) and putative peptide antagonists for SSTR2 and SSTR5 have been identified. The ligand binding domain for SST ligands is made up of residues in TMs III–VII with a potential contribution by the second extracellular loop. SSTRs are widely expressed in many tissues, frequently as multiple subtypes that coexist in the same cell. The five receptors share common signaling pathways such as the inhibition of adenylyl cyclase, activation of phosphotyrosine phosphatase (PTP), and modulation of mitogen-activated protein kinase (MAPK) through G-protein-dependent mechanisms.

Somatostatin receptors

Lars Neisig Møller, Carsten Enggaard Stidsen, Bolette Hartmann, Jens Juul Holst
Biochimica et Biophysica Acta 1616 (2003) 1 – 84
http://dx.doi.org:/10.1016/S0005-2736(03)00235-9

In 1972, Brazeau et al. isolated somatostatin (somatotropin release-inhibiting factor, SRIF), a cyclic polypeptide with two biologically active isoforms (SRIF-14 and SRIF-28). This event prompted the successful quest for SRIF receptors. Then, nearly a quarter of a century later, it was announced that a neuropeptide, to be named cortistatin (CST), had been cloned, bearing strong resemblance to SRIF. Evidence of special CST receptors never emerged, however. CST rather competed with both SRIF isoforms for specific receptor binding. And binding to the known subtypes with affinities in the nanomolar range, it has therefore been acknowledged to be a third endogenous ligand at SRIF receptors. This review goes through mechanisms of signal transduction, pharmacology, and anatomical distribution of SRIF receptors. Structurally, SRIF receptors belong to the superfamily of G protein-coupled (GPC) receptors, sharing the characteristic seven-transmembrane-segment (STMS) topography. Years of intensive research have resulted in cloning of five receptor subtypes (sst1-sst5), one of which is represented by two splice variants (sst2A and sst2B). The individual subtypes, functionally coupled to the effectors of signal transduction, are differentially expressed throughout the mammalian organism, with corresponding differences in physiological impact. It is evident that receptor function, from a physiological point of view, cannot simply be reduced to the accumulated operations of individual receptors. Far from being isolated functional units, receptors co-operate. The total receptor apparatus of individual cell types is composed of different-ligand receptors (e.g. SRIF and non-SRIF receptors) and co-expressed receptor subtypes (e.g. sst2 and sst5 receptors) in characteristic proportions. In other words, levels of individual receptor subtypes are highly cell-specific and vary with the co-expression of different-ligand receptors. However, the question is how to quantify the relative contributions of individual receptor subtypes to the integration of transduced signals, ultimately the result of collective receptor activity. The generation of knock-out (KO) mice, intended as a means to define the contributions made by individual receptor subtypes, necessarily marks but an approximation. Furthermore, we must now take into account the stunning complexity of receptor co-operation indicated by the observation of receptor homo- and heterodimerisation, let alone oligomerisation. Theoretically, this phenomenon adds a novel series of functional megareceptors/super-receptors, with varied pharmacological profiles, to the catalogue of monomeric receptor subtypes isolated and cloned in the past. SRIF analogues include both peptides and non-peptides, receptor agonists and antagonists. Relatively long half lives, as compared to those of the endogenous ligands, have been paramount from the outset. Motivated by theoretical puzzles or the shortcomings of present-day diagnostics and therapy, investigators have also aimed to produce subtype-selective analogues. Several have become available.

Somatostatin And Its Analogues In The Therapy Of Gastrointestinal Disease

Wynick, J. M. Polak And S. R. Bloom
Pharmac. Ther. 1989; 41, pp. 353-370

During the course of efforts to determine the distribution of growth hormone-releasing factor (GHRF) in rat hypothalamus a substance that inhibited growth hormone release was unexpectedly detected by Krulich et aL (1968). Their findings led them to hypothesize that the secretion of growth hormone from the pituitary was regulated by two different interacting neurohumoral factors–one stimulatory, the other inhibitory–each under the control of the nervous system. At about the same time Hellman and Lernmark (1969) found a factor in extracts of pigeon pancreatic islet-cells that inhibited insulin release in vivo from cultured pancreatic islet-cells. These two observations, seemingly unrelated, were ultimately to converge with the chemical identification of somatostatin, as an inhibitory peptide found in both the hypothalamus and pancreas.

Growth hormone-release inhibitory activity was re-discovered in 1972 by Brazeau et al. (1973). A concentrated effort to isolate and sequence the active principal was successful and it proved to be a cyclic peptide, to which the term ‘somatostatin’ (somatotrophin release inhibitory factor) was applied. Subsequent work (Reichlin, 1982a,b, 1983a,b; Iverson, 1983; Guillemin, 1978a,b) has considerably expanded the initially simple concept of somatostatin as a 14 amino-acid containing peptide (tetradecapeptide), bridged by a sulphur-sulphur bond whose main function was the regulation of growth-hormone secretion (Bonfils, 1985). Somatostatin related peptides are now known to constitute a family that includes the original identified peptide (designated somatostatin 14), an N-terminal extended somatostatin (somatostatin 28), several species specific variants and larger prohormone forms.
The name somatostatin may now be considered to be inappropriate because this compound is distributed widely in cells that have nothing to do with growth-hormone regulation or release. Tissues where somatostatin may be found include the nervous system, the gut and endocrine glands.
Somatostatin is present in every vertebrate class and even in primitive invertebrates (Vale et al., 1976; Falkmer et al., 1978; Jackson, 1978). This would suggest that this molecule and its controlling gene or genes evolved before the appearance on earth of differentiated cell-cell and nerve-cell communication (Roth et al., 1982). The evolutionary paths of mammals and fish are thought to have diverged at least 400 million years ago. The fact that the phenotype of somatostatin 14 is so well conserved (as to a lesser degree is that of somatostatin 28) suggests that throughout evolutionary history the specific configuration of somatostatin 14 has endowed a selective advantage on the animal kingdom, and its absence is not compatible with life.
Though widely distributed in cells throughout the body of vertebrates somatostatin does not in Guillemin’s words (1978a), “inhibit secretion of everything and anything” (since, for example it has no effect on the release of LH and FSH). Despite this it has certainly earned itself the nickname ‘endocrine cyanide’ (Bloom and Polak, 1987). The peptide is found in most but not all organs and displays specific and selective functions depending on its location. Within the nervous system somatostatinergic neurons are found in the cortex, limbic system, anterior pituitary, brain stem and spinal cord.
The various biological effects of somatostatin seem to be mediated through its specific high affinity receptors found in the brain, pituitary, adrenal, pancreas and gastrointestinal tract. Not only normal target tissue, but also tumors from the same endocrine tissues i.e. human pituitary adenomas, human and hamster pancreatic insulinomas, glucagonomas and VIPomas all bear somatostatin receptors (Reubi et al., 1981, 1982a, 1984a, 1985a, 1987a,b). Interestingly, tumors from tissues which are not established targets for somatostatin also seem to bear somatostatin receptors (Goodman et al., 1982; Reubi et al., 1986). Reubi et al. (1987b) demonstrated that many endocrine tumors including meningiomas, breast, pancreatic and pituitary tumors all have somatostatin receptors however, they demonstrated no receptors in prostatic carcinomas, ovarian carcinomas, endometrial carcinomas, primary liver cell carcinomas, pheochromocytomas, aldosterone secreting tumors, medullary carcinoma of the thyroid and a number of pulmonary carcinoids. Somatostatin receptors were also found in benign or malignant tumors originating from tissues not primarily known as somatostatin target organs, the biological function of such receptors is therefore unknown though it may be that they mediate the anti-proliferative effect of somatostatin and may therefore potentially be of therapeutic interest (Blankenstein et al., 1983, 1984).

Review article: somatostatin analogues in the treatment of gastroenteropancreatic neuroendocrine (carcinoid) tumoursModlin,

M. Pavel[1], M. Kidd & B. I. Gustafsson
Aliment Pharmacol Ther 2009; 31, 169–188
http://dx.doi.org:/10.1111/j.1365-2036.2009.04174.x

Background

The discovery of somatostatin (SST) and the synthesis of a variety of analogues constituted a major therapeutic advance in the treatment of gastroenteropancreatic neuroendocrine (carcinoid) tumours (GEP-NETs). They currently provide the most efficient treatment to achieve symptomatic relief and have recently been demonstrated to inhibit tumour growth.

Aim To review 35 years of experience regarding the clinical application and

efficacy of SST analogues. Methods The PubMed database (1972–2009) was searched using somatostatin as a search term with combinations of terms including ‘treatment’; ‘neuroendocrine’; ‘carcinoid’; ‘tumor’; ‘octreotide’; ‘lanreotide’ and ‘pasireotide’. Results In a review of 15 studies including 481 patients, the slow-release formulations Sandostatin LAR and Somatuline SR⁄ Autogel achieved symptomatic relief in 74.2% (61.9–92.8%) and 67.5% (40.0–100%), biochemical response in 51.4% (31.5–100%) and 39.0% (17.9–58%), and tumor response in 69.8% (47.0–87.5%) and 64.4% (48.0–87.0%) respectively. New SST analogues like SOM230 (pasireotide) that exhibit pan SST receptor activity and analogues with high affinity to specific somatostatin receptor (sstr) subtypes show promise. Conclusion As more precise understanding of NET cell biology evolves and molecular biological tools advance, more accurate identification of individual tumours sstr profile will probably facilitate a more precise delineation of SST analogue treatment.

Novel Autonomic Neurotransmitters And Intestinal Function

S. Taylor and R. A. R. Bywater
Pharmac. Ther. 1989; 40, pp. 401 to 438

In this review we will discuss some of the difficulties encountered in ascribing a neurotransmitter function to the more recently discovered peptides and other substances within the intestine. We will also provide a brief (and of necessity incomplete) account of some of the properties of intestinal putative neurotransmitters, and their possible roles in the functions of the small and large intestine.
The Enteric Nervous System The diverse intestinal functions associated with transit, digestion and absorption rely upon an intact enteric nervous system. The enteric nervous system essentially consists of those neurons whose cell bodies lie within the walls of the gastrointestinal tract. In the small and large intestine the cell bodies lie within the myenteric and submucous plexuses; their processes ramify throughout the majority of the intestinal wall and in many areas give rise to additional plexuses (Furness and Costa, 1987; Gabella, 1987). Functionally, these neurons can be divided into sensory neurons, interneurons and motor neurons. Some enteric neurons receive projections from extrinsic neurons and/or send projections centrally; we will not consider these projections further here.
The early observations of the co-existence of peptides in the enteric nervous system (Schultzberg et al., 1980) have now been extended and these studies demonstrate that the co-existence of two or more peptides is the rule rather than the exception (H6kfelt et al., 1987). The mix of peptides within neurons does not appear to be random; rather, there appears to be a systematic grouping of peptides in neurons with particular projections. This has led to the concept of “chemical coding” of enteric neurons. According to this concept, particular combinations of peptides are associated with particular neural pathways and perhaps with particular functions. For example, in the guinea pig small intestine, two chemically coded groups of submucous neurons have projections of different lengths running to the mucosa. Cell bodies with longer projections show immunoreactivity for dynorphin (DYN) and VIP. The other group shows immunoreactivity for choline acetyltransferase (CHAT), cholecystokinin, (CCK), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY) and somatostatin (SOM) (Costa et al., 1986a; Furness et al., 1987a). More recently it has been demonstrated that both groups of neurons show immunoreactivity for galanin (GAL) (Furness et al., 1987a,b). As for the neurotransmitter roles in the gut, the key question then becomes; “How does the presence of specific combinations of chemical substances (including peptides) relate to neuronal function?” It has been known for several years that “classical” transmitter substances can coexist in combination with various peptides (H6kfelt et al., 1980; Gilbert and Emson, 1983).
The above commentary upon the possible co-existence of several putative transmitter substances highlights the complex neurochemistry of the enteric nervous system. A corresponding degree of complexity appears to exist for the neuronal circuitry that ultimately directs the differing, but highly organized, patterns of motility and the secretory/absorptive functions of the intestinal tract. In vitro electrophysiological studies of the myenteric and submucous plexuses have indicated that several different types of neurons are present, each with their own biophysical characteristics. Furthermore, neurotransmission through, and probably between, the plexuses involves synaptic potentials which have time courses ranging from several milliseconds up to several minutes, depending upon the characteristics (stimulus strength, frequency and train length, etc.) and location of the applied electrical stimulus (see Wood, 1987, for references). Intracellular recordings from smooth muscle cells have also shown that excitatory and inhibitory junction potentials (EJPs and IJPS) of varying time courses can be evoked at various locations along the intestine during transmural electrical stimulation in response to selective stimulus regimens (see, for instance, Bywater and Taylor, 1986).
A number of authors have proposed criteria which should be fulfilled in order that transmitter status can be bestowed upon a particular substance (see Furness and Costa, 1982, for references). These criteria were developed with reference to the classical transmitter substances such as ACh, using the paradigm of a single transmitter per neuron. Regardless of the coexistence of several putative transmitters, status can only be granted to those substances that are found to be released from that nerve terminal. In the enteric nervous system a particular putative transmitter may be contained in several different functional pathways. However, in general, the methods used for eliciting release of putative transmitter substances (e.g. transmural electrical stimulation) are not specific for particular projections. Thus, for any substance, the association of demonstrated release with a given transmitter role is not facile.

New roles of the multidimensional adipokine: Chemerin

Syeda Sadia Fatima, Rehana Rehman, Mukhtiar Baig, Taseer Ahmed Khan
Peptides 62 (2014) 15–20
http://dx.doi.org/10.1016/j.peptides.2014.09.019

The discovery of several adipokines with diverse activities and their involvement in regulation of various pathophysiological functions of human body has challenged the researchers. In the family of adipokine, chemerin is a novel and unique addition. Ever since the first report on chemerin as a chemo-attractant protein, there are numerous studies showing a multitasking capacity of chemerin in the maintenance of homeostasis, for the activation of natural killer cells, macrophages and dendritic cells in both innate and adaptive immunity. Its diversity ranges from generalized inflammatory cascades to being explicitly involved in the manifestation of arthritis, psoriasis and peritonitis. Its association with certain cancerous tissue may render it as a potential tumor marker. In present review, we aim to consolidate recent data of investigations on chemerin in context to functional characteristics with a special reference to its role as a metabolic signal in inflammation and non-metabolic syndromes.

Neuropeptide Y is expressed in subpopulations of insulin- and non-insulin-producing islet cells in the rat after dexamethasone treatment: a combined immunocytochemical and in situ hybridisation study

Myrs6n a, *, B. Ahr6n b, F. Sundler
Regulatory Peptides 1995; 60, 19-31

Neuropeptide Y (NPY) is known to occur in adrenergic and non-adrenergic nerves in rat pancreatic islets. Analysis of islet extracts has revealed local NPY synthesis after glucocorticoid treatment. The cellular localization of NPY expression in rat islets following dexamethasone treatment (2 mg/kg daily, for 12 days), was investigated by a combination of immunocytochemistry (ICC) and in situ hybridization (ISH). NPY-immunoreactive nerve fibers were seen in pancreatic islets of both control and dexamethasone-treated rats. In the controls weak NPY immunoreactivity but no NPY mRNA was observed in occasional i:dets. After dexamethasone treatment, clusters of islet cells distributed both centrally and peripherally displayed intense NPY immunoreactivity and NPY mRNA labelling. Immunocytochemical double staining and ISH combined with ICC for NPY and islet hormones revealed that most NPY expressing cells were identical with insulin cells; a few cells were identical[ with somatostatin or pancreatic polypeptide (PP) cells. In contrast, glucagon cells seemed to be devoid of NPY immunoreactivity and NPY mRNA labelling. Thus, in the rat, glucocorticoids cause a marked upregulation of NPY expression in islet cells, preferentially the insulin cells. The expression of NPY might represent an islet adaptation mechanism to the reduced peripheral insulin sensitivity.

Neuropeptide Y is expressed in islet somatostatin cells of the hamster pancreas: a combined immunocytochemical and in situ hybridization study

Ulrika Myrsrn, Frank Sundler
Regulatory Peptides 1995; 57, 65-76

Neuropeptide Y (NPY) is known to occur in the autonomic nervous system, including the pancreatic islet innervation. We now present evidence that NPY is also expressed in endocrine islet cells in hamster pancreas. Thus, NPY-immunoreactivity and gene expression were detected in peripheral islet cells, using immunocytochemistry (ICC), in situ hybridization (ISH), and a combination of these techniques. Double immunostaining for NPY and somatostatin enabled localization of NPY to the vast majority of the somatostatin cells. However, a few somatostatin cells were devoid of NPY immunoreactivity and an occasional NPY-immunoreactive cell was devoid of somatostatin. ISH with an NPY mRNA specific probe, showed labelling of cells in the islet periphery. Furthermore, combined ISH for NPY mRNA and ICC for somatostatin showed autoradiographic labelling of somatostatin cells to a varying degree. Both somatostatin and NPY are inhibitors of insulin and/or glucagon secretion. Thus, in the islets these two peptides may be coreleased and cooperate in the, regulation of islet hormone secretion. The role for NPY emanating from islet cells is probably paracrine rather than endocrine.

Neuropeptide Y and Peptide YY Immunoreactivities in the Pancreas of Various Vertebrates

Wei-Guang Ding, Hiroshi Kimura, Masaki Fujimura And Mineko Fujimiya
Peptides, 1997; 18(10), pp. 1523–1529 PII S0196-9781(97)00237-4

NPY-like immunoreactivity was observed in nerve fibers and endocrine cells
in pancreas of all species examined except the eel, which showed no NPY innervation. The density of NPY-positive nerve fibers was higher in mammals than in the lower vertebrates. These nerve fibers were distributed throughout the parenchyma, and were particularly associated with the pancreatic duct
and vascular walls. In addition, the density of NPY-positive endocrine cells was found to be higher in lower vertebrates than mammals; in descending order; eel 5 turtle 5 chicken . bullfrog . mouse 5 rat 5 human . guinea pig 5 dog. These NPY-positive cells in the eel and certain mammals tended to be localized throughout the islet region, whereas in the turtle and chicken they were mainly scattered in the exocrine region. PYY-immunoreactivity was only present in the pancreatic endocrine cells of all species studied, and localized similarly to NPY. Thus these two peptides may play endocrine or paracrine roles in the regulation of islet hormone secretion in various vertebrate species.

Inhibitory effect of somatostatin on inflammation and nociception

Erika Pintér, Zsuzsanna Helyes, János Szolcsányi
Pharmacology & Therapeutics 112 (2006) 440–456

Somatostatin is released from capsaicin-sensitive, peptidergic sensory nerve endings in response to noxious heat and chemical stimuli such as vanilloids, protons or lipoxygenase products. It reaches distant parts of the body via the circulation and exerts systemic anti-inflammatory and analgesic effects. Somatostatin binds to G-protein coupled membrane receptors (sst1–sst5) and diminishes neurogenic inflammation by prejunctional action on sensory-efferent nerve terminals, as well as by postjunctional mechanisms on target cells. It decreases the release of pro-inflammatory neuropeptides from sensory nerve endings and also acts on receptors of vascular endothelial, inflammatory and immune cells. Analgesic effect is mediated by an inhibitory action on peripheral terminals of nociceptive neurons, since circulating somatostatin cannot exert central action.
Somatostatin itself is not suitable for drug development because of its broad spectrum and short elimination half-life, stable, receptor-selective agonists have been synthesized and investigated. The present overview is aimed at summarizing the physiological importance of somatostatin and sst receptors, pharmacological significance of synthetic agonists and their potential in the development of novel anti-inflammatory and analgesic drugs. These compounds might provide novel perspectives in the pharmacotherapy of acute and chronic painful inflammatory diseases, as well as neuropathic conditions.

the sources, target cells and effects of somatostatin (SST) involved in inflammatory and nociceptive processes

This schematic drawing demonstrates the sources, target cells and effects of somatostatin (SST) involved in inflammatory and nociceptive processes

Characterization, detection and regulation of somatostatin receptors

The physiological actions of SST are initiated by its binding to membrane receptors. Five human somatostatin receptors (sst), have been cloned and characterized and referred to as sst1-5 receptors using the nomenclature suggested by Hoyer et al. (1995). Structurally, sst receptors are 7 transmembrane domain glycoproteins, comprised of 7 membrane spanning α helical domains connected by short loops, an N-terminal extracellular domain and a C-terminal intracellular domain. On the basis of binding studies using synthetic somatostatin analogs, sst receptors can be divided into 2 different subgroups: SRIF1 group comprising sst2, sst3 and sst5 are able to bind octapeptide analogs, whereas SRIF2 group comprising sst1 and sst4 have negligible affinity for these compounds. Within sst2 receptors, sst2A and sst2B are encoded on the same chromosome 17 and generated through alternative splicing of sst2 mRNA (Patel et al., 1993). None of the peptide analogs bind exclusively to only one of the sst subtypes, although new approaches might yield subtype-selective agonists and antagonists (Hofland et al., 1995; Hoyer et al., 1995; Patel et al., 1995; Reisine & Bell, 1995; Florio & Schettini, 1996; Patel, 1997; Meyerhof, 1998; Janecka et al., 2001). Somatostatin receptors are linked to multiple cellular effector systems via G-proteins. They mediate the inhibition of adenylate cyclase activity (Jakobs et al., 1983; Patel et al., 1995), reduce the conductance of voltage-dependent Ca2+ channels (Schally, 1988; Patel et al., 1995) and activate K+ channels (Mihara et al., 1987; Moore et al., 1988; Wang et al., 1989). Somatostatin receptors also mediate the stimulation of tyrosine phosphatase activity, induce a reduction of cell proliferation and inhibit a Na+/H+ exchanger (NHE1) (Barber et al., 1989; Buscail et al., 1994; Patel et al., 1995). Sst receptors represent a major class of inhibitory receptors which play an important role in modulating higher brain functions, secretory processes, cell proliferation and apoptosis.
Endogenous Somatostatin-28 Modulates Postprandial Insulin Secretion Immunoneutralization Studies in Baboons

John W. Ensinck, Robin E. Vogel, Ellen C. Laschansky, Donna J. Koerker, et al.
J Clin Invest 1997. 100: 2295–2302.). http://dx.doi.org/10.1172/JCI119767

Somatostatin-28 (S-28), secreted into the circulation from enterocytes after food, and S-14, released mainly from gastric and pancreatic δ cells and enteric neurons, inhibit peripheral cellular functions. We hypothesized that S-28 is a humoral regulator of pancreatic β cell function during nutrient absorption. Consistent with this postulate, we observed in baboons a two to threefold increase in portal and peripheral levels of S-28 after meals, with minimal changes in S-14. We attempted to demonstrate a hormonal effect of these peptides by measuring their concentrations before and after infusing a somatostatin-specific monoclonal antibody (mAb) into baboons and comparing glucose, insulin, and glucagon-like peptide-1 levels before and for 4 h after intragastric nutrients during a control study and on 2 d after mAb administration (days 1 and 2). Basal growth hormone (GH) and glucagon levels and parameters of insulin and glucose kinetics were also measured. During immunoneutralization, we found that
(a) postprandial insulin levels were elevated on days 1 and 2;
(b) GH levels rose immediately and were sustained for 28 h, while glucagon fell; (c) basal insulin levels were unchanged on day 1 but were increased two to threefold on day 2, coincident with decreased insulin sensitivity; and
(d) plasma glucose concentrations were similar to control values.
We attribute the eventual rise in fasting levels of insulin to its enhanced secretion in compensation for the heightened insulin resistance from increased GH action. Based on the elevated postmeal insulin levels after mAb administration, we conclude that S-28 participates in the enteroinsular axis as a decretin to regulate postprandial insulin secretion.

Effects of glucagon-like peptide 1 on appetite and body weight: focus on the CNS

L van Bloemendaal, J S ten Kulve, S E la Fleur, R G Ijzerman and M Diamant
Journal of Endocrinology 2014; 221, T1–T16
http://dx.doi.org:/10.1530/JOE-13-0414

The delivery of nutrients to the gastrointestinal tract after food ingestion activates the secretion of several gut-derived mediators, including the incretin hormone glucagon-like peptide 1 (GLP-1). GLP-1 receptor agonists (GLP-1RA), such as exenatide and liraglutide, are currently employed successfully in the treatment of patients with type 2 diabetes mellitus. GLP-1RA improve glycaemic control and stimulate satiety, leading to reductions in food intake and body weight. Besides gastric distension and peripheral vagal nerve activation, GLP-1RA induce satiety by influencing brain regions involved in the regulation of feeding, and several routes of action have been proposed. This review summarises the evidence for a physiological role of GLP-1 in the central regulation of feeding behavior and the different routes of action involved. Also, we provide an overview of presently available data on pharmacological stimulation of GLP-1 pathways leading to alterations in CNS activity, reductions in food intake and weight loss.

Critical role for peptide YY in protein-mediated satiation and body-weight regulation

Rachel L. Batterham, Helen Heffron, Saloni Kapoor, Joanna E. Chivers, et al.
Cell Metab 2006; 4, 223–233 http://dx.doi.org:/10.1016/j.cmet.2006.08.001

Dietary protein enhances satiety and promotes weight loss, but the mechanisms by which appetite is affected remain unclear. We investigated the role of gut hormones, key regulators of ingestive behavior, in mediating the satiating effects of different macronutrients. In normal-weight and obese human subjects, high-protein intake induced the greatest release of the anorectic hormone peptide YY (PYY) and the most pronounced satiety. Long-term augmentation of dietary protein in mice increased plasma PYY levels, decreased food intake, and reduced adiposity. To directly determine the role of PYY in mediating the satiating effects of protein, we generated PYY null mice, which were selectively resistant to the satiating and weight-reducing effects of protein and developed marked obesity that was reversed by exogenous PYY treatment. Our findings suggest that modulating the release of endogenous satiety factors, such as PYY, through alteration of specific diet constituents could provide a rational therapy for obesity.

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Parathyroids and Bone Metabolism

Posted in Biological Networks, Bone Disease and Musculoskeletal Disease, Calcium, Cell Biology, Chemical Biology and its relations to Metabolic Disease, Curation, Diagnostic Immunology, Diagnostics and Lab Tests, Endocrine Diseases, Liver & Digestive Diseases Research, Metabolism, Nephrology, Nutrition, Population Health Management, Signaling & Cell Circuits, Translational Science, tagged Aging, bone mineral density, bone resorption, hypoparathyroid, osteitis fibrosa cystica (hyperparathyroid), osteoblasst activity decline, osteoclast, osteoporosis, parathyroid glands, phosphate, thyroid surgery complication on February 10, 2015| Leave a Comment »

Parathyroids and Bone Metabolism

Writer and Curator: Larry H. Bernstein, MD, FCAP

Parathyroid hormone (PTH), parathormone or parathyrin, is secreted by the chief cells of the parathyroid glands as a polypeptide containing 84 amino acids. It acts to increase the concentration of calcium (Ca2+) in the blood, whereas calcitonin (a hormone produced by the parafollicular cells (C cells) of the thyroid gland) acts to decrease calcium concentration. PTH acts to increase the concentration of calcium in the blood by acting upon the parathyroid hormone 1 receptor (high levels in bone and kidney) and the parathyroid hormone 2 receptor (high levels in the central nervous system, pancreas, testis, and placenta). PTH half-life is approximately 4 minutes.[2] It has a molecular mass of 9.4 kDa.

hPTH-(1-34) crystallizes as a slightly bent, long helical dimer. Analysis reveals that the extended helical conformation of hPTH-(1-34) is the likely bioactive conformation.[4] The N-terminal fragment 1-34 of parathyroid hormone (PTH) has been crystallized and the structure has been refined to 0.9 Å resolution.

The_ribbon_cartoon_structure – hPTH helical dimer

http://upload.wikimedia.org/wikipedia/commons/1/1e/The_ribbon_cartoon_structure.png

Regulation of serum calcium

PTH was one of the first hormones to be shown to use the G-protein, adenylyl cyclase second messenger system.

Normal total plasma calcium level ranges from 8.5 to 10.2 mg/dL (2.12 mmol/L to 2.55 mmol/L).

Region	Effect
bone	It enhances the release of calcium from the large reservoir contained in the bones.^[7] Bone resorption is the normal destruction of bone by osteoclasts, which are indirectly stimulated by PTH. Stimulation is indirect since osteoclasts do not have a receptor for PTH; rather, PTH binds to osteoblasts, the cells responsible for creating bone. Binding stimulates osteoblasts to increase their expression of RANKL and inhibits their expression of Osteoprotegerin (OPG). OPG binds to RANKL and blocks it from interacting with RANK, a receptor for RANKL. The binding of RANKL to RANK (facilitated by the decreased amount of OPG available for binding the excess RANKL) stimulates these osteoclast precursors to fuse, forming new osteoclasts, which ultimately enhances bone resorption
kidney	It enhances active reabsorption of calcium and magnesium from distal tubules and the thick ascending limb. As bone is degraded, both calcium and phosphate are released. It also decreases the reabsorption of phosphate, with a net loss in plasma phosphate concentration. When the calcium:phosphate ratio increases, more calcium is free in the circulation
intestine via kidney	It enhances the absorption of calcium in the intestine by increasing the production of activated vitamin D. Vitamin D activation occurs in the kidney. PTH up-regulates25-hydroxyvitamin D₃ 1-alpha-hydroxylase, the enzyme responsible for 1-alpha hydroxylation of 25-hydroxy vitamin D, converting vitamin D to its active form (1,25-dihydroxy vitamin D). This activated form of vitamin D increases the absorption of calcium (as Ca²⁺ ions) by the intestine via calbindin.

http://en.wikipedia.org/wiki/Parathyroid_hormone

Development of Present Concepts of the Parathyroid –
The Parathyroids – Progress, problems and practice,
in Current Problems in Surgery, 1971; 8(8): 3-64.
Leon Goldman, Gilbert Gordon, Betty S. Roof
http://dx.doi.org/10.1016/S0011-3840(71)80008-4

The parathyroid gland first achieved clinical significance because of hypoparathyroid tetany. Tetany: a syndrome manifested by painful muscle spasms or rigors; is derived from the Greek: tetanos, past participle of the verb teinein, meaning “to stretch,” Tetany : stretched, or spastic, in modern terms “up tight.,’ When the word was used by Hippocrates, no differentiation was made between the types of muscular spasms caused by neurotoxins (e.g., lockjaw) and those of metabolic causes. The word ~ went through the Latin, tetanus, and to French. Te’tanie, where the attribute of intermittent muscular spasm was added.

Owea’s drawing of parathyroid gland of Indian rhinoceros

According to file Oxford English Dictionary, the relation of tetany to surgical operations was noted in tile year 1805 in The Medical Journal XIV, 304: “tetanie affections very often to|low the great operations. . .” It is not clear from this reference what type of operations were invo]ved. The relationship of tetany to thyroidectomy was recognized as early as 1878 when WoIfler described convulsions in one of the patients on whom Billroth had performed a total thyroidectomy. The great surgeon WilIiam Stewart Halsted suggested that postoperative hypoparathyroidism had not been reported earlier because before that time total thyroidectomy had always been fatal, leaving insufficient time for tetany to develop. In 1883 Weiss collected 13 cases of tetany, all following total thyroidcctomy. The relation to total thyroidectomy became historically significant later when postoperative tetany was misinterpreted as the acute form of thyroid insufficiency, while myxedema was correctly recognized as the chronic form.
Anatomically, the parathyroid glands had been noted fleetingly by Remak (1855), by Virchow (1863) and probably by others in the course of human dissection. Perhaps better publicized was the description by Sir Richard Owen, published in 1852. As Hunterian Professor and Conservator of the Museum in the Royal College of Surgeons, Owen anatomized animals that died at the London Zoo. In 1849, while performing an autopsy on tile Great Indian rhinoceros, Owen clearly noted, drew and named the parathyroid gland (Fig. 1). However, microscopic examination was not reported, and it was not known at that time whether the parathyroid gland was separate.
The causal relationship of the parathyroid gland to post-thyroidectomy tetany was clarified by the French physiologist Eugdne Gley in 1891. He showed that, in the rabbit, removal of the thyroid gland was not responsible for these seizures but that removal of the parathyroid glands caused fatal convulsions.
Very soon after this, a parallel discovery was made in Berkeley, California, by Jacques Loeb. Loeb noticed that the rhythmic contractions of a frog muscle in a saline medium were stopped by the addition of calcium. He concluded that calcium has the important function of inhibiting excessive neuromuscular
irritability. Loeb’s studies led MacCallum, in 1909, to investigate the possibility that a low blood calcium level might be responsible for the increased excitability of the muscles, in hypoparathyroid tetany. He and Voegtlin removed the parathyroids from dogs and showed that tetany ensued when the serum calcium level fell. They also showed that administration of calcium promptly relieved tetany. Less well known is their publication in the following
year, which entirely recanted the earlier view. Their observations that calcium, magnesium and strontium immediately abolish tetany, and the report of Joseph and Sleltzer that infusion of hypertonic sodium chloride slowly relieves this kind of tetany, led MacCallum to believe that the effect of calcium was nonspecific.
By this time thyroid surgery was being performed widely. The Reverdin brothers in Geneva noted what they considered complex nervous manifestations following total thyroidectomv, Moussu’ s observations in animals were confirmed in patients; post-thyroidectomy convulsions were not necessarily fatal.
Thyroid surgery was now sufficiently improved so that Kocher was able to find symptoms of tetany–and these were transient ….. in only 1 of his 18 cases of total thyroidectomy. How many more would have been identified as victims of hypoparathyroidism by appropriate chemical examination can only be conjectured. By 1907 Halsted had recognized the importance of the parathyroids and how essential the intimate knowledge of their anatomy is to the goiter surgeon. Halsted put a bright young medical student to work on this project as a penalty for delinquent attendance at lectures. The sketch of the beautiful dissection by the student, Herbert McLean Evans, was used by Halsted to illustrate his monograph on The Operative History of Goiter. On the basis of this knowledge, of anatomy, it was established that the parathyroids are usually related to the posterior capsule and that leaving this capsule intact greatly reduces the risk of tetany.
In 1923 the distinguished Norwegian physician-physiologist, Harald Salvesen published beautiful, imaginative and thorough studies in which he showed, that complete parathyroid ablation invariably lowered the blood calcium, that the blood sugar level was not altered and that guanidine accumulation occurred only terminally during agonal convulsions. He further found that parathyroid tetany could be prevented by calcium feeding and confirmed MacCallum’s earlier observation that it could be promptly corrected by calcium infusion. He also noted that one of his dogs with parathyroid tetany developed a cataract. In our opinion, the relation of the parathyroid gland to calcium metabolism was first firmly established by Salvesen in 1923.
Consider the knowledge and use of endocrines in 1923. Desiccated thyroid, which Osler had praised as the miracle of modern metabolic therapy, was the only orally effective endocrine preparation. ]nsulin had just been discovered. Another potent preparation was the hydrochloric acid extract of parathyroid glands made by Adolph Hanson. That it was an effective preparation is perhaps best attested by the fact that it is still used, under the name Parathyroid Extract USP, and that much of the work on the actions of parathyroid hormone has been carried out with this crude extract. In 1925 Collip, who had been of such immeasurable help to Banting, Best and McLeod in preparing a clean, potent insulin extract from normal pancreas, applied his genius to the parathyroid with an equally satisfactory result. His relatively clean parathyroid extract made it possible for the first time to elucidate the actions of the parathyroid glands in man.
Using this preparation, Albright and Ellsworth in 1929 clarified the two fundamental actions of parathyroid hormone (PTH) identical with those obtained nowadays with the most highly purified preparations. These two actions are:
(1) elevation of serum calcium and
(2) excretion of phosphate by the kidneys, with a consequent lowering of the serum phosphate.
It will later be shown that the action that raises serum calcium levels is, for the most part, an increase in the rate of bone breakdown. It remained for Copp and associates to show in 1961 that another horrnone, calcitonin, with an opposite action, is necessary for maintenance of calcium homeostasis. And still later Chase and Aurbach showed in 1968 that the phosphaturic action of PTH is mediated by the enzyme adenyl cyclase, which stimulates production of cyclic 3’5′-adenosine monophosphate (AMP).
It is now clear that hypophosphatemia predisposes to hyperealcemia and that hyperphosphatemia can actually abolish hypercalcemia. However, numerous experiments, one of them by Albright’s collaborators, Ellsworth and Futeher in 1935 showed that parathyroid extract raised the serum calcium level in the absence of the kidneys. Clearly, therefore, the calcium-mobilizing effect of PTH is not the result of the phosphate diuretic action only. Conclusive evidence was obtained by Barnicot of Cambridge in 1948. …
The brilliant group at the Massachusetts General Hospital, led by Aub and including two young men destined to make brilliant records in American medicine Fuller Albright and Waiter Bauer soon showed that the kind of hyperparathyroidism described by Recklinghausen, Mandl and Askanazy is, in fact, the end stage of a series of chemical events predictable from the known actions of PTH. Starting with the famous case of Captain Charles Martell, a mariner with severe bone disease, who shrank in stature in 10 years, Albright soon clarified the most significant feature of hyperparathyroidism: the hypercalcemia that is found in at least 99% of patients with proved primary hyperparathyroidism.
It was not until 1953 that Jonas Shota directly demonstrated the other action of excess PTH in hyperparathyroidism: a low rate of tubular reabsorption of phosphate (TRP), as fifteen years later, in 1968, Chase and Aurbach would show that this action is mediated by renal adenyl cyclase and cyclic AMP. Meanwhile, in 1935, Pappenheimer and Wilens had described another form of hyperparathyroidism arising not as a primary tumor, but as a secondary or compensatory response to the metabolic abnormalities of uremia. Goldman independently described this phenomenon. It .is noteworthy that hyperparathyroidism secondary to lack of dietary calcium had already been described by Erdheiqm and that these 2 causes of secondary hyperparathyroidism, Uremia and intestinal malabsorption, have subsequently been shown, to have in comrnon inadequate intestinal absorption of calcium.
Since the classic studies of Sandstrom, Gley, Loeb, Salvesen, Cotlip, Aub, Bauer and Albright, enormous strides have advanced our knowledge of parathyroid physiology. Isolation, purification, and characterization of the hormone and development of a highly sensitive radioimmunoassay for PTH. Almost slmultaneously in1959, Aurbach, Rasmussen and Craig obtained a purified bovine PTH. These two groups of investigators identified a similar peptide with a molecular weight of about 8,500 and with biological activity of about 3.000 units/mg. This peptide contains 84 amino acid residfies the first 30-45 are necessary for biologic and immunologic activity. A tentative molecular structure reported by Potts, Aurbach and Sherwood in 1965 has subsequently been modified by Brewer and Ronan, with confirmation by Niall et aI. in Potts’s laboratory. The heterogeneous nature of circulating PTH was first: shown by Berson and Yalow using two antisera prepared from beef PTH but showing quantitative differences in reaction to circulating PTH. They were able to detect two parathormones, one with a half-life of only 10-20 minutes, and another with a half-life of about 1.5 hours.
The parathyroid hormone-regulated transcriptome in osteocytes: Parallel actions with 1,25-dihydroxyvitamin D3 to oppose gene expression changes during differentiation and to promote mature cell function

Hillary C. St. John, MB Meyer, NA Benkusky, AH Carlson, M Prideaux, et al.
Bone 72 (2015) 81–91
http://dx.doi.org/10.1016/j.bone.2014.11.010

Although localized to the mineralized matrix of bone, osteocytes are able to respond to systemic factors such as the calciotropic hormones 1, 25-(OH)₂ D3 and PTH. In the present studies, we examined the transcriptomic response to PTH in an osteocyte cell model and found that this hormone regulated an extensive panel of genes. Surprisingly, PTH uniquely modulated two cohorts of genes, one that was expressed and associated with the osteoblast to osteocyte transition and the other a cohort that was expressed only in the mature osteocyte. Interestingly, PTH’s effects were largely to oppose the expression of differentiation-related genes in the former cohort, while potentiating the expression of osteocyte-specific genes in the latter cohort. A comparison of the transcriptional effects of PTH with those obtained previously with 1, 25-(OH)₂D3 revealed a subset of genes that was strongly overlapping. While 1, 25-(OH)₂D3 potentiated the expression of osteocyte-specific genes similar to that seen with PTH, the overlap between the two hormones was more limited. Additional experiments identified the PKA-activated phospho-CREB (pCREB) cistrome, revealing that while many of the differentiation-related PTH regulated genes were apparent targets of a PKA-mediated signaling pathway, a reduction in pCREB binding at sites associated with osteocyte-specific PTH targets appeared to involve alternative PTH activation pathways. That pCREB binding activities positioned near important hormone-regulated gene cohorts were localized to control regions of genes was reinforced by the presence of epigenetic enhancer signatures exemplified by unique modifications at histones H3 and H4. These studies suggest that both PTH and 1, 25-(OH)₂D3 may play important and perhaps cooperative roles in limiting osteocyte differentiation from its precursors while simultaneously exerting distinct roles in regulating mature osteocyte function. Our results provide new insight into transcription factor-associated mechanisms through which PTH and 1, 25-(OH)₂D3 regulate a plethora of genes important to the osteoblast/osteocyte lineage.

Bone, a dynamic and integrating tissue

The guest editors Bram C.J. van der Eerden, Anna Teti, Willian F. Zambuzzi
Archives of Biochemistry and Biophysics 561 (2014) 1–2
http://dx.doi.org/10.1016/j.abb.2014.08.012

The special issue ‘Bone, a dynamic and integrating tissue’ provides the most recent information regarding the interacting nature of bone cells with their immediate neighboring cells within the skeleton as well as with distant target cells in other organs, using different types of both cellular and non-cellular communication. It should appeal to any scientist or clinician in the field, given the wide variety of topics, covering molecular, experimental cell and animal biology, biomechanics and -physics, genetics and medicine.

This special issue arose from a collaboration between the guest editors within ‘INTERBONE’, a European Union funded Marie Curie Actions – People – International Research Staff Scheme (PIRSESGA-2011-295181) on the interplay among bone cells, matrices and systems.

Over the recent years, many developments have paved new avenues to study signaling pathways and mechanisms in bone in much greater detail. Genetic progress has been made, which has provided us with novel genes behind already known as well as hitherto idiopathic bone diseases. The enormous expansion of specific animal models has enabled us to study new mechanisms and pathways in vivo in great spatial and temporal detail. As a consequence, novel treatment modalities have seen the light, which are predominantly focusing on bone anabolic therapies. These advances will not cease to exist and an exciting biological era lies ahead of us, with many discoveries to be made.

In this special issue of Archives in Biochemistry and Biophysics, experts in the field of bone metabolism have addressed the recent developments in which special attention is paid to the concept that bone is not just a static, isolated organ, but a dynamic and integrating tissue. Over the last decade, discoveries have led to the notion that bone cells are interacting with many other cell types within bone. Besides this intraskeletal communication, bone cells produce factors that are capable of controlling cell types and organs elsewhere in the organism, which are now being recognized as bona fide hormones.

All contributors have explored the recent advances made in their research area. The latest progress in osteoblast/osteocyte and osteoclast biology is revisited with special focus on bone morphogenetic proteins, microRNAs and extracellular vesicles as illustrative examples of different levels of communication between cell types. In separate chapters, the interaction of osteoblasts and osteoclasts, as well as their cross-talk with endothelial cells, fat cells, immune cells, hematopoietic stem cells and different types of cancer cells is discussed extensively, further emphasizing the interactive nature of bone cells in their microenvironment. Beside cell–cell interaction, attention has been paid to the osteointegration of bone cells in a non-cellular context, including extracellular matrix and metal devices, combining main components for bone bioengineering. Finally, the endocrine role of bone is discussed in great detail by several contributors, focusing on the control of bone cell function by the brain as well as the role of bone-produced factors in, amongst others, phosphate homeostasis, energy metabolism and fertility.

The Great Beauty of the osteoclast

Alfredo Cappariello, Antonio Maurizi, Vimal Veeriah, Anna Teti
Archives of Biochemistry and Biophysics 561 (2014) 13–21
http://dx.doi.org/10.1016/j.abb.2014.08.009

Much has been written recently on osteoclast biology, but this cell type still astonishes scientists with its multifaceted functions and unique properties. The last three decades have seen a change in thinking about the osteoclast, from a cell with a single function, which just destroys the tissue it belongs to, to an ‘‘orchestrator’’ implicated in the concerted regulation of bone turnover. Osteoclasts have unique morphological features, organelle distribution and plasma membrane domain organization. They require polarization to cause extracellular bone breakdown and release of the digested bone matrix products into the circulation. Osteoclasts contribute to the control of skeletal growth and renewal. Alongside other organs, including kidney, gut, thyroid and parathyroid glands, they also affect calcemia and phosphatemia. Osteoclasts are very sensitive to pro-inflammatory stimuli, and studies in the ‘00s ascertained their tight link with the immune system, bringing about the question why bone needs a cell regulated by the immune system to remove the extracellular matrix components. Recently, osteoclasts have been demonstrated to contribute to the hematopoietic stem cell niche, controlling local calcium concentration and regulating the turnover of factors essential for hematopoietic stem cell mobilization. Finally, osteoclasts are important regulators of osteoblast activity and angiogenesis, both by releasing factors stored in the bone matrix, and secreting ‘‘clastokines’’ that regulate the activity of neighboring cells. All these facets will be discussed in this review article, with the aim of underscoring The Great Beauty of the osteoclast.

Osteoclasts: more than ‘bone eaters’

Julia F. Charles and Antonios O. Aliprantis
Trends in Molecular Medicine, Aug 2014; 20(8): 449-459
http://dx.doi.org/10.1016/j.molmed.2014.06.001

As the only cells definitively shown to degrade bone, osteoclasts are key mediators of skeletal diseases including osteoporosis. Bone-forming osteoblasts, and hematopoietic and immune system cells, each influence osteoclast formation and function, but the reciprocal impact of osteoclasts on these cells is less well appreciated. We highlight here the functions that osteoclasts perform beyond bone resorption.
First, we consider how osteoclast signals may contribute to bone formation by osteoblasts and to the pathology of bone lesions such as fibrous dysplasia and giant cell tumors.
Second, we review the interaction of osteoclasts with the hematopoietic system, including the stem cell niche and adaptive immune cells. Connections between osteoclasts and other cells in the bone microenvironment are discussed within a clinically relevant framework.

Bone is a composite tissue of protein and mineral which undergoes continual remodeling to grow, heal damage, and regulate calcium and phosphate metabolism. This remodeling process is executed by the concerted and sequential effort of bone-resorbing osteoclasts and bone-forming osteoblasts, acting in what has been termed the basic multicellular unit (BMU) (Figure 1A). Osteocytes, long-lived osteoblast-derived cells that reside within the bone matrix, monitor bone quality and stress, and coordinate remodeling through membrane-bound and secreted factors. Skeletal integrity is maintained throughout the life-span by matching bone formation and resorption, a process referred to as osteoclast:osteoblast ‘coupling.’ Coupling is thoroughly summarized in recent excellent reviews and in Figure 1.

Coupling: how osteoclasts ‘talk back’ to cells of the osteoblast lineage Coupling of bone formation to resorption is likely achieved through multiple mechanisms, including signals that stimulate the proliferation of pre-osteoblasts, their recruitment to resorption lacunae, and their differentiation into bone-forming cells. Cellular mediators of coupling include osteoclasts, osteoblasts, osteocytes, macrophages, and T cells, which produce a variety of factors including Wnt pathway regulators, such as sclerostin, and cytokines such as oncostatin M

Osteoclasts–osteoblast interactions in the basic multicellular unit (BMU).

Osteoclasts–osteoblast interactions in the basic multicellular unit (BMU).
Cell–cell contact mechanisms may also mediate OC-OB communication. Bidirectional signaling from OC ephrins and OB Eph receptors, and reverse signaling through RANKL on OBs, have both been invoked.

Box 1. Usurping local resources: osteoclasts feed bone invaders

Liberation of growth factors embedded in bone matrix by osteoclasts may promote metastatic tumor growth in bone. Reciprocal stimulation of osteoclasts by cancer cell derived parathyroid hormone related protein (PTHrP), and other factors, could potentiate growth factor release in what has been termed the ‘vicious cycle’ ]. Xenograft experiments utilizing breast cancer cells expressing a TGFβ responsive reporter demonstrated osteolytic metastases had high TGFβ activity. Inhibition of osteoclastic bone resorption with pamidronate reduced TGFβ activity and osteolytic lesions, suggesting that matrix resorption is a relevant source of TGFβ for skeletal metastasis in vivo. Although prophylactic pamidronate treatment decreased frequency of bone metastasis, the drug did not decrease disease progression if administered after tumor cell inoculation. Thus, whether inhibiting the release of matrix growth factors by osteoclasts has a substantive effect on tumor growth is unclear. Several bisphosphonates, as well as the anti-RANKL antibody denosumab, reduce skeletal events in metastatic cancer, but data on whether they prevent bone metastasis are inconsistent.

Immunoregulation by osteoclasts. Osteoclast precursors (OCPs) and osteoclasts (OCs) inhibit CD4 and CD8 T cell proliferation via nitric oxide (NO) production in response to T cell derived interferon g (IFNg). IFNg in turn inhibits differentiation of OCPs into mature OCs. OCs also present antigen through major histocompatibility complex class I (MHCI) to skew CD8+ T cells toward an induced Treg phenotype termed OC-iTcreg. OC-iTcreg cells in turn inhibit OCP differentiation to mature OC through IFNg, interleukin 10 (IL10), and IL6.

In mouse models, we suggest that systems for the temporal deletion of conditional alleles in osteoclasts and their precursors be established. Moreover, clinical research in humans with emerging therapeutics which specifically target key regulators of bone remodeling, such as RANKL, cathepsin K, and sclerostin, could include nested translational studies that specifically address their effects on the immune system, HSCs, and tumor growth, where appropriate. In these ways, a clear picture of osteoclast biology beyond their role as ‘bone eaters’ will emerge.

Leukemia inhibitory factor: A paracrine mediator of bone metabolism

Natalie A. Sims & Rachelle W. Johnson
Growth Factors, April 2012; 30(2): 76–87
http://dx.doi.org:/10.3109/08977194.2012.656760

Leukemia inhibitory factor (LIF) is a soluble interleukin-6 family cytokine that regulates a number of physiologic functions, including normal skeletal remodeling. LIF signals through the cytokine co-receptor glycoprotein-130 in complex with its cytokine-specific receptor [LIF receptor (LIFR)] to activate signaling cascades in cells of the skeletal system, including stromal cells, chondrocytes, osteoblasts, osteocytes, adipocytes, and synovial fibroblasts. LIF action on skeletal cells is cell-type specific, and frequently dependent on the state of cell differentiation. This review describes the expression patterns of LIF and LIFR in bone, their regulation by physiological and inflammatory agents, as well as cell-specific influences of LIF on osteoblast, osteoclast, chondrocyte, and adipocyte differentiation. The actions of LIF in normal skeletal growth and maintenance, in pathological states (e.g. autocrine tumor cell signaling and growth in bone) and inflammatory conditions (e.g. arthritis) will be discussed, as well as the signaling pathways activated by LIF and their importance in bone formation and resorption.

In vivo evidence of IGF-I–estrogen crosstalk in mediating the cortical bone response to mechanical strain

Subburaman Mohan, CG Bhat, JE Wergedal and C Kesavan
Bone Research (2014) 2, 14007 http://dx.doi.org:/10.1038/boneres.2014.7

Although insulin-like growth factor-I (IGF-I) and estrogen signaling pathways have been shown to be involved in mediating the bone anabolic response to mechanical loading, it is not known whether these two signaling pathways crosstalk with each other in producing a skeletal response to mechanical loading. To test this, at 5 weeks of age, partial ovariectomy (pOVX) or a sham operation was performed on heterozygous IGF-I conditional knockout (HIGF-I KO) and control mice generated using a Cre-loxP approach. At 10 weeks of age, a 10 N axial load was applied on the right tibia of these mice for a period of 2 weeks and the left tibia was used as an internal non-non-loaded control. At the cortical site, partial estrogen loss reduced total volumetric bone mineral density (BMD) by 5% in control pOVX mice (P50.05, one-way ANOVA), but not in the H IGF-I KO pOVX mice. At the trabecular site, bone volume/total volume (BV/TV) was reduced by 5%–6% in both control pOVX (P,0.05) and H IGF-I KO pOVX (P50.05) mice. Two weeks of mechanical loading caused a 7%–8% and an 11%–13%(P,0.05 vs. non-loaded bones) increase in cortical BMD and cortical thickness (Ct.Th), respectively, in the control sham, control pOVX and H IGF-I KO sham groups. By contrast, the magnitude of cortical BMD (4%, P50.13) and Ct.Th (6%, P,0.05) responses were reduced by 50% in the H IGF-I KO pOVX mice compared to the other three groups. The interaction between genotype and estrogen deficiency on the mechanical loading-induced cortical bone response was significant (P,0.05) by two-way ANOVA. Two weeks of axial loading caused similar increases in trabecular BV/TV (13%–17%) and thickness (17%–23%) in all four groups of mice. In conclusion, partial loss of both estrogen and IGF-I significantly reduced cortical but not the trabecular bone response to mechanical loading, providing in vivo evidence of the above crosstalk in mediating the bone response to loading.

Role of FGF/FGFR signaling in skeletal development and homeostasis: learning from mouse models

Nan Su, Min Jin and Lin Chen
Bone Research (2014) 2, 14003; http://dx.doi.org:/10.1038/boneres.2014.3

Fibroblast growth factor (FGF)/fibroblast growth factor receptor (FGFR) signaling plays essential roles in bone development and diseases. Missense mutations in FGFs and FGFRs in humans can cause various congenital bone diseases, including chondrodysplasia syndromes, craniosynostosis syndromes and syndromes with dysregulated phosphate metabolism. FGF/FGFR signaling is also an important pathway involved in the maintenance of adult bone homeostasis. Multiple kinds of mouse models, mimicking human skeleton diseases caused by missense mutations in FGFs and FGFRs, have been established by knock-in/out and transgenic technologies. These genetically modified mice provide good models for studying the role of FGF/FGFR signaling in skeleton development and homeostasis. In this review, we summarize the mouse models of FGF signaling-related skeleton diseases and recent progresses regarding the molecular mechanisms, underlying the role of FGFs/FGFRs in the regulation of bone development and homeostasis. This review also provides a perspective view on future works to explore the roles of FGF signaling in skeletal development and homeostasis.

Osteoporosis in men: a review

Robert A Adler
Bone Research (2014) 2, 14001; http://dx.doi.org:/10.1038/boneres.2014.1

Osteoporosis and consequent fracture are not limited to postmenopausal women. There is increasing attention being paid to osteoporosis in older men. Men suffer osteoporotic fractures about 10 years later in life than women, but life expectancy is increasing faster in men than women. Thus, men are living long enough to fracture, and when they do the consequences are greater than in women, with men having about twice the 1-year fatality rate after hip fracture, compared to women. Men at high risk for fracture include those men who have already had a fragility fracture, men on oral glucocorticoids or those men being treated for prostate cancer with androgen deprivation therapy. Beyond these high risk men, there are many other risk factors and secondary causes of osteoporosis in men. Evaluation includes careful history and physical examination to reveal potential secondary causes, including many medications, a short list of laboratory tests, and bone mineral density testing by dual energy X-ray absorptiometry (DXA) of spine and hip. Recently, international organizations have advocated a single normative database for interpreting DXA testing in men and women. The consequences of this change need to be determined. There are several choices of therapy for osteoporosis in men, with most fracture reduction estimation based on studies in women.

From skeletal to non skeletal: The intriguing roles of BMP-9: A literature review

Leblanc, G. Drouin, G. Grenier, N. Faucheux, R. Hamdy
Advances in Bioscience and Biotechnology, 2013; 4: 31-46
http://dx.doi.org/10.4236/abb.2013.410A4004

In the well-known superfamily of transforming growth factors beta (TGF-), bone morphogenetic proteins (BMPs) are one of the most compelling cytokines for their major role in regulation of cell growth and differentiation in both embryonic and adult tissues. This subfamily was first described for its ability of potentiating bone formation, but nowadays, the power of BMPs is well beyond the bone healing scope. Some of the BMPs have been well studied and described in the literature, but the BMP9 is still worthy of attention. It has been shown by many authors that it is the most potent osteogenic BMP. Moreover, it has been de- scribed as one of the rare circulating BMPs. In this paper, we will review the recent literature on BMP9 and the different avenues for future research in that field. Our primary scope is to review its relation to bone formation and to elaborate on the available literature on other systems.

Fong et al. recently demonstrated in vitro that rhBMP9 can also augment bone resorption. This increase was shown to be functional and not related to osteoclast formation. Furthermore, rhBMP9 could alter the intrinsic apoptosis pathway and increase survival of osteoclasts. The effect of rhBMP9 on osteoclast was explained by the presence of ALK1 and BMPRII co-receptors and their activation of the Smad 1/5/8 and non-smad MAPK/ERK pathways. These results show for the first time that BMP9 can directly affect human osteoclasts, acting on their function and their survival.

Insulin resistance is a systemic multifactorial impairment of glucose uptake. Muscle, a glucose consuming organ, needs Akt2 to be able to activate insulin-induced glucose uptake and this pathway seems to be severely impaired in insulin resistance. Interestingly, a combination of bioinformatic and high- throughput functional analyses have shown BMP9 to be the first hepatic factor to regulate blood glucose concencentration. Moreover, this effect was thought to be mediated by activation of Akt kinase in differentiated myotubes. Then, it has been demonstrated that recombinant BMP9 (1 and 5 mg/kg) improves glucose homeostasis in vivo in diabetic and non-diabetic rodents. The mechanism relied on the upregulation of Smad5 and Akt2 in differentiated rats myotubes. On the opposite side, Smad5 was downregulated in myotubes by de xamethasone, a well known hyperglycemia inducer and Smad5 knockdown in rats decreased Akt2 expression and phosphorylation leading to a decrease in insulin-induced glucose uptake by myotubes. It was then hypothesized that Smad5 regulated glucose uptake in skeletal muscle through Akt2 expression and phosphorylation. These findings also revealed Smad5 as a potential target for the treatment of type 2 diabetes. Hence, BMP9 could be seen as a potential activator of Smad5 for that purpose.

BMP9 is a major member of the TGF- superfamily that is implied in many fundamental developmental and pa- thologic processes. Future research will certainly bring answers to the many questions left open, and those an- swers will unquestionably lead to clinical applications.

Understanding Bone Loss

Max Stanley Chartrand, PhD.
DigiCare® Behavioral Research

During their lifetimes, at least half of those over age 50 will be at risk of developing osteoporosis. When we speak of bone loss we are primarily speaking of three diagnostic stages: Osteoarthritis (1-2% loss per annum), Osteopenia (3% per annum), and Osteoporosis (4-5%+ per annum) that are caused almost entirely by diet, hydration, lifestyle, medications, and environ-mental stressors.

Human bones are highly vascularized and mineralized tissues that are constantly being shaped and developed by cells called osteoblasts and torn down and resorbed by cells called osteoclasts. Recent research confirms that throughout one’s lifespan it is osteoblast activity that controls and dictates osteoclast activity as long as the body receives the nutrients it requires to maintain homeostasis. Growing children, for instance, have a far greater abundance of osteoblasts than of osteoclasts. By the time they reach young adulthood (at about age 26 for men, 22 for women) osteoclasts increase while osteoblasts slow down. Even so, humans of any age can increase osteoblast activity and slow the formation of osteoclasts through weight bearing exercise and other methods.

Long bone

Long bone
The problem of bone shrinkage and decline in strength presents most often in health states involving:

Sedentary Lifestyle, making weight bearing exercise a frontline defense against bone loss for everyone.
Acidosis (low pH), from a diet that is nutritionally lacking, genetically modified, degerminated, irradiated, laden with toxins & over-processed.
Chronic dehydration from too much caffeine and high fructose corn syrup (a GMO) and not enough water that is both ionized and alkalized.
Lacking in calcium that is live, ionically charged, as well as phosphorus, magnesium, boron, and other minerals comprised in human bones. On the other hand, commercially available calcium causes atherosclerosis, kidney stones, bone spurs, cataracts, and yet MORE bone loss!
Taking prescription medications, especially acid reflux meds, NSAIs and steroids. These and more interfere with osteoblast activity and weaken immunology. Osteoporosis meds prevent living bone mass!
Unhealed injuries and deterioration of the spine, such as compression fractures (>50% of the US adult population), spinal stenosis, kyphosis, and scoliosis. These cause even more rapid loss of bone mass.
Subclinical infections: tooth and gum sepsis, around artificial joints, keratosis obturans, kidney and bladder infections, neuropathies, and osteomyelitis as a result of injuries and/or shock to the bones.
Heavy metal accumulations: lead, mercury, cadmium, arsenic, formaldehyde, cyanide, etc. found in the drinking water, fresh foods, cosmetics, paints, fuels, and a host of commonly used products.
 Lifestyle Substances– Smoking, alcohol, excess coffee, marijuana, opium (including opiate pain killers), diet sodas, caffeine drinks.

The Kinetics of Skeletal Remodeling

Jan 1, 1966 by Lent C. Johnson
Semin Musculoskelet Radiol. 2000;4(1):1-15.

Bone tumor dynamics: an orthopedic pathology perspective.
Johnson LC1, Vinh TN, Sweet DE.

The diagnosis and classification of primary bone tumors remains as much a challenge today as it has for the last 80 plus years. Although pathology is invariably equated with the image of a diagnostic microscope, the vast majority of diagnoses are made grossly with the unaided eye, as are the tissue specimens selected for microscopic “confirmation.” Radiologic studies, particularly plain radiographs, remain the gold standard in gross pathologic diagnosis of the skeleton. Today, confirmation and final classification continue as the pathologist’s domain, but perhaps not for long, considering the evolving ancillary imaging techniques and progressive sophistication of magnetic resonance (MR) imaging. The bone tumor cases collected and compiled by Ernest Codman, M.D. during the second through fourth decades of this century formed the basis of the first tumor registry. The Codman Bone Sarcoma Registry demonstrated among other things the importance of radiographic/pathologic correlation, underscoring the reliability of a bone tumor’s location, margin (host bone/tumor interface), periosteal reaction, and matrix patterns as an accurate guide to classification and likely future biologic behavior. “A General Theory of Bone Tumors,” written by Lent C. Johnson nearly 50 years ago and published in the Bulletin of The New York Academy of Medicine (February 1953, second series, vol. 29, no. 2, pp. 164-171), provided a conceptual cellular approach to the understanding bone tumor dynamics reinforcing radiologic/pathologic correlation as a reliable diagnostic tool. At the time of Dr. Lent C. Johnson’s death (1910-1998), he was literally working on an updated version of his original article, the latter of which is being reprinted as the core of this illustrated revision. Our continued experience with bone tumors over the past five decades has only served to validate, on a daily basis, the fundamental principles outlined in Johnson’s original article. In like fashion, it is important to keep in mind that terminology and nomenclature has also evolved since 1953, despite a continued inability to achieve complete consensus.
PMID: 11061688 http://www.ncbi.nlm.nih.gov/pubmed/11061688

Interactions between adrenal-regulatory and calcium-regulatory hormones in human health

Brown, J.M., Vaidya, A.

Curr Opinion in Endocr, Diabetes and Obesity 2014; 21 (3), pp. 193-201

Purpose of review: To summarize the evidence characterizing the interactions between adrenal-regulating and calcium-regulating hormones, and the relevance of these interactions to human cardiovascular and skeletal health. Recent findings: Human studies support the regulation of parathyroid hormone (PTH) by the renin-angiotensin-aldosterone system (RAAS): angiotensin II may stimulate PTH secretion via an acute and direct mechanism, whereas aldosterone may exert a chronic stimulation of PTH secretion.
Studies in primary aldosteronism, congestive heart failure, and chronic
kidney disease have identified associations between hyperaldosteronism, hyperparathyroidism, and bone loss, which appear to improve when
inhibiting the RAAS. Conversely, elevated PTH and insufficient vitamin D
status have been associated with adverse cardiovascular outcomes, which
may be mediated by the RAAS. Studies of primary hyperparathyroidism implicate PTH-mediated stimulation of the RAAS, and recent evidence shows that the vitamin D-vitamin D receptor complex may negatively regulate renin expression and RAAS activity. Ongoing human interventional studies are evaluating the influence of RAAS inhibition on PTH and the influence of vitamin D receptor agonists on RAAS activity. Summary: Although previously considered independent endocrine systems, emerging evidence supports a complex web of interactions between adrenal-regulating and calcium-regulating hormones, with implications for human cardiovascular and
skeletal health.

Backbone modification of a polypeptide drug alters duration of action in vivo

Cheloha, R.W., Maeda, A., Dean, T., Gardella, T.J., Gellman, S.H.

Nature Biotechnology 2014; 32 (7), pp. 653-655 http://dx.doi.org/doi:10.1038/nbt.2920

Systematic modification of the backbone of bioactive polypeptides through amino acid residue incorporation could provide a strategy for generating molecules with improved drug properties, but such alterations can result in lower receptor affinity and potency. Using an agonist of parathyroid hormone receptor-1 (PTHR1), a G protein-coupled receptor in the B-family, we present an approach for residue replacement that enables both high activity and improved pharmacokinetic properties in vivo.

Mouse and human BAC transgenes recapitulate tissue-specific expression
of the vitamin D receptor in mice and rescue the VDR-null phenotype

Lee, S.M., Bishop, K.A., Goellner, J.J., O’Brien, C.A., Pike, J.W.
Endocrinology 2014; 155 (6), pp. 2064-2076
http://dx.doi.org/10.1210/en.2014-1107

The biological actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) are mediated by the vitamin D receptor (VDR), which is expressed in numerous target tissues in a cell type-selective manner. Recent studies using genomic analyses and recombined bacterial artificial chromosomes (BACs) have defined the specific features of mouse and human VDR gene loci in vitro. In the current study, we introduced recombined mouse and human VDR BACs as transgenes into mice and explored their expression capabilities in vivo. Individual transgenic mouse strains selectively expressed BAC derived mouse or human VDR proteins in appropriate vitamin D target tissues, thereby recapitulating the tissue-specific expression of endogenous mouse VDR. The mouse VDR transgene was also regulated by 1,25(OH)2D3 and dibutyryl-cAMP. When crossed into a VDR-null mouse background, both transgenes restored wild-type basal as well as 1,25(OH)2D3-inducible gene expression patterns in the appropriate tissues. This maneuver resulted in the complete rescue of the aberrant phenotype noted in the VDR-null mouse, including systemic features associated with altered calcium and phosphorus homeostasis and disrupted production of parathyroid hormone and fibroblast growth factor 23, and abnormalities associated with the skeleton, kidney, parathyroid gland, and the skin. This study suggests that both mouse and human VDR transgenes are capable of recapitulating basal and regulated expression of the VDR in the appropriate mouse tissues and restore 1,25(OH)2D 3 function. These results provide a baseline for further dissection of mechanisms integral to mouse and human VDR gene expression and offer the potential to explore the consequence of selective mutations in VDR proteins in vivo.

The sclerostin-independent bone anabolic activity of intermittent PTH treatment is mediated by T-cell-produced Wnt10β

Li, J.-Y., Walker, L.D., Tyagi, A.M., (…), Neale Weitzmann, M., Pacifici, R
Journal of Bone and Mineral Research 2014; 29 (1), pp. 43-54
http://onlinelibrary.wiley.com/doi/10.1002/jbmr.2044/pdf

Both blunted osteocytic production of the Wnt inhibitor sclerostin (Scl) and increased T-cell production of the Wnt ligand Wnt10b contribute to the bone anabolic activity of intermittent parathyroid hormone (iPTH) treatment. However, the relative contribution of these mechanisms is unknown. In this study, we modeled the repressive effects of iPTH on Scl production in mice by treatment with a neutralizing anti-Scl antibody (Scl-Ab) to determine the contribution of T-cell-produced Wnt10b to the Scl-independent modalities of action of iPTH. We report that combined treatment with Scl-Ab and iPTH was more potent than either iPTH or Scl-Ab alone in increasing stromal cell production of OPG, osteoblastogenesis, osteoblast life span, bone turnover, bone mineral density, and trabecular bone volume and structure in mice with T cells capable of producing Wnt10b. In T-cell-null mice and mice lacking T-cell production of Wnt10b, combined treatment increased bone turnover significantly more than iPTH or Scl-Ab alone. However, in these mice, combined treatment with Scl-Ab and iPTH was equally effective as Scl-Ab alone in increasing the osteoblastic pool, bone volume, density, and structure. These findings demonstrate that the Scl-independent activity of iPTH on osteoblasts and bone mass is mediated by T-cell-produced Wnt10b. The data provide a proof of concept of a more potent therapeutic effect of combined treatment with iPTH and Scl-Ab than either alone.

N-cadherin restrains PTH activation of Lrp6/β-catenin signaling and osteoanabolic action

Revollo, L., Kading, J., Jeong, S.Y., (…), Mbalaviele, G., Civitelli, R.
Journal of Bone and Mineral Research 2015; 30 (2), pp. 274-28

Interaction between parathyroid hormone/parathyroid hormone-related peptide receptor 1 (PTHR1) and low-density lipoprotein receptor-related protein 6 (Lrp6) is important for parathyroid hormone (PTH) signaling and anabolic action. Because N-cadherin has been shown to negatively regulate canonical Wnt/β-catenin signaling, we asked whether N-cadherin alters PTH signaling and stimulation of bone formation. Ablation of the N-cadherin gene (Cdh2) in primary osteogenic lineage cells resulted in increased Lrp6/PTHR1 interaction in response to PTH1-34, associated with enhanced PTH-induced PKA signaling and PKA-dependent β-catenin C-terminus phosphorylation, which promotes β-catenin transcriptional activity. β-catenin C-terminus phosphorylation was abolished by Lrp6 knockdown. Accordingly, PTH1-34 stimulation of Tcf/Lef target genes, Lef1 and Axin2, was also significantly enhanced in Cdh2-deficient cells. This enhanced responsiveness to PTH extends to the osteo-anabolic effect of PTH, as mice with a conditional Cdh2 deletion in Osx+ cells treated with intermittent doses of PTH1-34 exhibited significantly larger gains in trabecular bone mass relative to control mice, the result of accentuated osteoblast activity. Therefore, N-cadherin modulates Lrp6/PTHR1 interaction, restraining the intensity of PTH-induced β-catenin signaling, and ultimately influencing bone formation in response to intermittent PTH administration.

EphrinB2 signaling in osteoblasts promotes bone mineralization by preventing apoptosis

Tonna, S., Takyar, F.M., Vrahnas, C., (…), Martin, T.J., Sims, N.A.
FASEB Journal 2014; 28 (10), pp. 4482-4496 10.1096/fj.14-254300

Cells that form bone (osteoblasts) express both ephrinB2 and EphB4, and previous work has shown that pharmacological inhibition of the ephrinB2/ EphB4 interaction impairs osteoblast differentiation in vitro and in vivo. The purpose of this study was to determine the role of ephrinB2 signaling in the osteoblast lineage in the process of bone formation. Cultured osteoblasts from mice with osteoblast-specific ablation of ephrinB2 showed delayed expression of osteoblast differentiation markers, a finding that was reproduced by ephrinB2, but not EphB4, RNA interference. Microcomputed tomography, histomorphometry, and mechanical testing of the mice lacking ephrinB2 in osteoblasts revealed a 2-fold delay in bone mineralization, a significant reduction in bone stiffness, and a 50% reduction in osteoblast differentiation induced by anabolic parathyroid hormone (PTH) treatment, compared to littermate sex- and age-matched controls. These defects were associated with significantly lower mRNA levels of late osteoblast differentiation markers and greater levels of osteoblast and osteocyte apoptosis, indicated by TUNEL staining and transmission electron microscopy of bone samples, and a 2-fold increase in annexin V staining and 7-fold increase in caspase 8 activation in cultured ephrinB2 deficient osteoblasts. We conclude that osteoblast differentiation and bone strength are maintained by antiapoptotic actions of ephrinB2 signaling within the osteoblast lineage.-
Bone involvement in primary hyperparathyroidism and changes after parathyroidectomy

Rolighed, L., Rejnmark, L., Christiansen, P.
European Endocrinology 2014; 10 (1), pp. 84-87

Parathyroid hormone (PTH) is produced and secreted by the parathyroid glands and has primary effects on kidney and bone. During the pathological growth of one or more parathyroid glands, the plasma level of PTH increases and causes primary hyperparathyroidism (PHPT). This disease is normally characterized by hyperparathyroid hypercalcemia. In PHPT a continuously elevated PTH stimulates
the kidney and bone causing a condition with high bone turnover, elevated plasma calcium and increased fracture risk. If bone resorption is not followed by a balanced formation of new bone, irreversible bone loss may occur in these patients. Medical treatment can help to minimize the loss of bone but the cure of PHPT is by parathyroidectomy. After operation, bone mineral density increases during the return to normal bone metabolism. Supplementation with calcium and vitamin D after operation may improve the normalization to normal bone metabolism with a secondary reduction in fracture risk.

Primary hyperparathyroidism and the skeleton

Mosekilde, L.
Clinical Endocrinology 2008; 69 (1), pp. 1-19
http://dx.doi.org:/10.1111/j.1365-2265.2007.03162.x

Today, primary hyperparathyroidism (PHPT) in the developed countries is typically a disease with few or no obvious clinical symptoms. However, even in the asymptomatic cases the endogenous excess of PTH increases bone turnover leading to an insidious reversible loss of cortical and trabecular bone because of an expansion of the remodelling space and an irreversible loss of cortical bone due to increased endocortical resorption. In contrast trabecular bone structure and integrity to a large extent is maintained and there may be a slight periosteal expansion. Most studies have reported decreased bone mineral density (BMD) in PHPT mainly located at cortical sites, whereas sites rich in trabecular bone only show a modest reduction or even a slight increase in BMD. The frequent occurrence of vitamin D insufficiency and deficiency in PHPT and increased plasma FGF23 levels may also contribute to the decrease in BMD. The effect of smoking is unsolved. Epidemiological studies have shown that the relative risk of spine and nonspine fractures is increased in untreated PHPT starting up to 10 years before the diagnosis is made. Successful surgery for PHPT normalizes bone turnover, increases BMD and decreases fracture risk based on larger epidemiological studies. However, 10 years after surgery fracture risk appears to increase again due to an increase in forearm fractures. There are no randomized controlled studies (RCTs) demonstrating a protective effect of medical treatment on fracture risk in PHPT. Less conclusive studies suggest that vitamin D supplementation may have a beneficial effect on plasma PTH and BMD in vitamin D deficient PHPT patients. Hormone replacement therapy (HRT) and maybe SERM appear to reduce bone turnover and increase BMD. However, their nonskeletal side-effects preclude their use for this purpose. Bisphosphonates reduce bone turnover and increase BMD in PHPT as in osteoporosis and may be a therapeutical option in selected patients with low BMD. Obviously, there is a need for larger RCTs with fractures as end-points that appraise this possibility. Calcimimetics reduce plasma calcium and PTH in PHPT but has no beneficial effect on bone turnover or BMD. In symptomatic hypercalcemic PHPT with low BMD where curative surgery is impossible or contraindicated a combination of a calcimimetic and a bisphosphonate may be an undocumented therapeutical option that needs further evaluation.

Current Issues in the Presentation of Asymptomatic Primary Hyperparathyroidism: Proceedings of the Fourth International Workshop

Shonni J. Silverberg, Bart L. Clarke, Munro Peacock, Francisco Bandeira, et al. The Journal of Clinical Endocrinology & Metabolism 2014; 99(10) http://dx.doi.org/10.1210/jc.2014-1415

Objective: This report summarizes data on traditional and nontraditional manifestations of primary hyperparathyroidism (PHPT) that have been published since the last International Workshop on PHPT.

Participants: This subgroup was constituted by the Steering Committee to address key questions related to the presentation of PHPT. Consensus was established at a closed meeting of the Expert Panel that followed.

Evidence: Data from the 5-year period between 2008 and 2013 were
presented and discussed to determine whether they support changes in recommendations for surgery or nonsurgical follow-up.

Consensus Process: Questions were developed by the International Task
Force on PHPT. A comprehensive literature search for relevant studies was undertaken. After extensive review and discussion, the subgroup came to agreement on what changes in the recommendations for surgery or nonsurgical follow-up of asymptomatic PHPT should be made to the Expert Panel.

Conclusions:

1) There are limited new data available on the natural history of
asymptomatic PHPT. Although recognition of normocalcemic PHPT
(normal serum calcium with elevated PTH concentrations; no secondary
cause for hyperparathyroidism) is increasing, data on the clinical
presentation and natural history of this phenotype are limited.
2) Although there are geographic differences in the predominant
phenotypes of PHPT (symptomatic, asymptomatic, normocalcemic),
they do not justify geography-specific management guidelines.
3) Recent data using newer, higher resolution imaging and analytic
methods have revealed that in asymptomatic PHPT, both trabecular
bone and cortical bone are affected.
4) Clinically silent nephrolithiasis and nephrocalcinosis can be detected
by renal imaging and should be listed as a new criterion for surgery.
5) Current data do not support a cardiovascular evaluation or surgery
for the purpose of improving cardiovascular markers, anatomical or
functional abnormalities.
6) Some patients with mild PHPT have neuropsychological complaints
and cognitive abnormalities, and some of these patients may benefit
from surgical intervention. However, it is not possible at this time to
predict which patients with neuropsychological complaints or cognitive
issues will improve after successful parathyroid surgery.

Sclerosing Bone Dysplasias: Leads Toward Novel Osteoporosis Treatments

Igor Fijalkowski, Eveline Boudin, Geert Mortier, Wim Van Hul
Current Osteoporosis Reports Sept 2014; 12(3), pp 243-251
http://dx.doi.org:/10.1007/s11914-014-0220-5

Sclerosing bone dysplasias are a group of rare, monogenic disorders characterized by increased bone density resulting from the disturbance in the fragile equilibrium between bone formation and resorption. Over the last decade, major contributions have been made toward better understanding of the pathogenesis of these conditions. These studies provided us with important insights into the bone biology and yielded the identification of numerous drug targets for the prevention and treatment of osteoporosis. Here, we review this heterogeneous group of disorders focusing on their utility in the development of novel osteoporosis therapies.

Clinical development of neridronate: potential for new applications

Gatti D, Rossini M, Viapiana O, Idolazzi L, Adami S
Ther & Clin Risk Manag Apr 2013; 2013(9): Pages 139—147

Neridronate is an aminobisphosphonate, licensed in Italy for the treatment
of osteogenesis imperfecta (OI) and Paget’s disease of bone (PDB). A characteristic property of neridronate is that it can be administered both intravenously and intramuscularly, providing a useful system for administration in homecare. In this review, we discuss the latest clinical results of neridronate administration in OI and PDB, as well as in osteoporosis and other conditions. We will focus in particular on the latest evidence of the effect of neridronate on treatment of complex regional pain syndrome type I.

Disorders of bone remodeling

Feng, X., McDonald, J.M.
Ann Rev of Pathol: Mechanisms of Disease 2011; 6, pp. 121-145
http://dx.doi.org:/10.1146/annurev-pathol-011110-130203

The skeleton provides mechanical support for stature and locomotion, protects vital organs, and controls mineral homeostasis. A healthy skeleton must be maintained by constant bone modeling to carry out these crucial functions throughout life. Bone remodeling involves the removal of old or damaged bone by osteoclasts (bone resorption) and the subsequent replacement of new bone formed by osteoblasts (bone formation). Normal bone remodeling requires a tight coupling of bone resorption to bone formation to guarantee no alteration in bone mass or quality after each remodeling cycle. However, this important physiological process can be derailed by a variety of factors, including menopause-associated hormonal changes, age-related factors, changes in physical activity, drugs, and secondary diseases, which lead to the development of various bone disorders in both women and men. We review the major diseases of bone remodeling, emphasizing our current understanding of the underlying pathophysiological mechanisms.

Paget’s disease and hypercalcemia: Coincidence or causal relationship?

Green, I., Altman, A.
Harefuah 2009; 148 (10), pp. 708-710

Paget’s disease is a chronic disease in which osteoclast mediated bone resorption precedes imperfect osteoblast mediated bone repair. Symptoms include bone pain, pathological fractures, osteoarthritis and neurological symptoms. There is evidence that genetic and viral component are involved in the etiology. Hypercalcemia is rare and when it is diagnosed, primary hyperparathyroidism should be ruled out. The authors present a patient with Paget’s disease and concomitant hypercalcemia. Evaluation for hypercalcemia revealed an adenoma of the parathyroid. However, despite the removal of the adenoma, the symptoms persisted. Previous studies
showed that hyperparathyroidism causes hypercalcemia in Paget’s disease patients. Removal of the adenoma led to improvement in calcium and alkaline phosphatase (ALP) levels but clinical improvement is seen only in patients with high calcium level prior to the operation. This leads to the assumption that symptoms of Paget’s disease are due to osteoclast hypersensitivity to parathyroid hormone (PTH) and by removing the adenoma the osteoclast activity is also reduced. In summary, the most common cause of hypercalcemia in Paget’s disease patients is hyperparathyroidism and adenectomy may improve the biochemical and sometimes also the clinical symptoms of Paget’s disease.

Signaling networks that control the lineage commitment and differentiation of bone cells

Soltanoff, C.S., Yang, S., Chen, W., Li, Y.-P.
Critical Reviews in Eukaryotic Gene Expression 2009; 19 (1), pp. 1-46

Osteoblasts and osteoclasts are the two major bone cells involved in the bone remodeling process. Osteoblasts are responsible for bone formation while osteoclasts are the bone-resorbing cells. The major event that triggers osteogenesis and bone remodeling is the transition of mesenchymal stem cells into differentiating osteoblast cells and monocyte/macrophage precursors into differentiating osteoclasts. Imbalance in differentiation and function of these two cell types will result in skeletal diseases such as osteoporosis, Paget’s disease, rheumatoid arthritis, osteopetrosis, periodontal disease, and bone cancer metastases.
Osteoblast and osteoclast commitment and differentiation are controlled by complex activities involving signal transduction and transcriptional regulation of gene expression. Recent advances in molecular and genetic studies using gene targeting in mice enable a better understanding of the multiple factors and signaling networks that control the differentiation process at a molecular level.
This review summarizes recent advances in studies of signaling transduction pathways and transcriptional regulation of osteoblast and osteoclast cell lineage commitment and differentiation. Understanding the signaling networks that control the commitment and differentiation of bone cells will not only expand our basic understanding of the molecular mechanisms of skeletal development but will also aid our ability to develop therapeutic means of intervention in skeletal diseases.

Salmon calcitonin: a review of current and future therapeutic indications

H. Chesnut III, M. Azria, S. Silverman, M. Engelhardt, M. Olson, L. Mindeholm Osteoporosis International 2008; 19(4), pp 479-491
http://dx.doi.org:/10.1007/s00198-007-0490-1

Salmon calcitonin, available as a therapeutic agent for more than 30 years, demonstrates clinical utility in the treatment of such metabolic bone diseases as osteoporosis and Paget’s disease, and potentially in the treatment of osteoarthritis. This review considers the physiology and pharmacology of salmon calcitonin, the evidence based research demonstrating efficacy and safety of this medication in postmenopausal osteoporosis with potentially an effect on bone quality to explain its abilities to reduce the risk of spine fracture, the development of an oral salmon calcitonin preparation, and the therapeutic rationale for this preparation’s chondroprotective effect in osteoarthritis.

Pharmacotherapies to Manage Bone Loss-Associated Diseases: A Quest for the Perfect Benefit-to-Risk Ratio

Valverde

Current Medicinal Chemistry : 15 (3): Pages 284-304
http://dx.doi.org:/10.2174/092986708783497274

In this review, benefits and side-effects of current and emerging therapies to treat and prevent pathological bone loss are described. Bisphosphonates are the antiresorptive compounds most widely used in the treatment of bone-loss associated diseases. They are generally well-tolerated although have recently been associated with osteonecrosis of the jaw and other complications. Therapies modulating estrogen receptor activation are indicated in the prevention and treatment of either breast cancer or osteoporosis in postmenopausal women. Thus, hormone replacement therapy is effective in prevention of osteoporosis, but its long-term use can increase the risk of breast cancer, stroke and embolism. Tamoxifen benefits all stages of breast cancer, but its use may lead to uterine cancer and thromboembolism. Raloxifene is approved in prevention of breast cancer and treatment of postmenopausal osteoporosis, but its use can increase the risk of fatal stroke. Aromatase inhibitors are superior to tamoxifen at advanced stages of disease and as adjuvants, but their use increase fracture incidence. Fulvestrant is as effective as aromatase inhibitors in the treatment of advanced breast cancer and does not cause bone fractures. Another antiresorptive available for the treatment of postmenopausal osteoporosis, Pagets disease and hypercalcemia is calcitonin, which also exhibits analgesic effects. A promising antiresorptive agent currently in clinical trials is denosumab. Aditional therapies for osteoporosis that decrease fracture risk consist of PTH-like anabolic agents and the dual action bone agent strontium ranelate. Antiseptics and antibiotics are used extensively in periodontal disease intervention to target bacterial biofilm, although hostdirected therapies are also being developed. – See more at: http://www.eurekaselect.com/66301/article#sthash.EGNCH4Eu.dpuf

Parathyroid Hormone An Anabolic Treatment for Osteoporosis

Paul Morley, James F. Whitfield and Gordon E. Willick
Current Pharmaceutical Design Pages 671-687
http://dx.doi.org:/10.2174/1381612013397780

Osteoporosis is a disease characterised by low bone mass, structural deterioration of bone and increased risk of fracture. The prevalence, and cost, of osteoporosis is increasing dramatically with our ageing population and the World Health Organization now considers it to be the second-leading healthcare problem. All currently approved therapies for osteoporosis (eg., estrogen, bisphosphonates, calcitonin and selective estrogen receptor modulators) are anti-resorptive agents that act on osteoclasts to prevent further bone loss. A new class of bone anabolic agent capable of building mechanically strong new bone in patients with established osteoporosis is
in development. While the parathyroid hormone (PTH) is classically considered to be a bone catabolic agent, when delivered intermittently at low doses PTH potently stimulates cortical and trabecular bone growth in animals humans. The native hPTH-(1-84) and its osteogenic fragment, hPTH-(1-34), have already entered Phase III clinical trials. Understanding the mechanism of PTHs osteogenic actions has led to the development of smaller PTH analogues which can also build mechanically normal bone in osteopenic rats. These new PTH analogues are promising candidates for treating osteoporosis in humans as they are as efficacious as hPTH-(1-84) and hPTH-(1-34), but there is evidence that they may have considerably less ability to induce hypercalcemia, the major side effect of PTH therapy. In addition to treating osteoporosis, PTHs may be used to promote fracture healing, to restore bone loss in immobilized patients, or following excessive glucocorticoid or prolonged spaceflight, and to treat psoriasis. http://www.eurekaselect.com/65008/article#sthash.FWa67NrB.dpuf

Effects of Parathyroid Hormone on Cancellous Bone Mass and Structure in Osteoporosis

Naohisa Miyakoshi
Current Pharmaceutical Design ;10(21): Pages 2615-2627
http://dx.doi.org:/10.2174/1381612043383737

Parathyroid hormone (PTH) is the major hormonal regulator of calcium homeostasis. PTH is a potent stimulator of bone formation and can restore bone to an osteopenic skeleton, when administered intermittently. Osteoblasts are the primary target cells for the anabolic effects of PTH in bone tissue. Anabolic effects of PTH on bone have been demonstrated in animals and humans, by numerous measurement techniques including bone mineral density and bone histomorphometry. Clinically, the most important aspect of treatment for osteoporosis is prevention of fractures. Microstructural alterations, such as loss of trabecular connectivity, have been implicated in increased propensity for fracture. Recent two-dimensional (2D) and three-dimensional (3D) assessments of cancellous bone structure have shown that PTH can re-establish lost trabecular connectivity in animals and humans.
These results provide new insight into the positive clinical effects of PTH in osteoporosis. In recent randomized controlled clinical trials of intermittent
PTH treatment, PTH decreased incidence of vertebral and non-vertebral fractures
in postmenopausal women. Thus, PTH shows strong potential as therapy for osteoporosis. However, 2D and 3D structural analysis of advanced osteopenia in animals has shown that there is a critical limit of trabecular connectivity and bone strength below which PTH cannot completely reverse the condition. Given that PTH treatment fails to completely restore trabecular connectivity and bone strength in animals with advanced osteopenia, early treatment of osteoporosis appears important and efficacious for preventing fractures caused by decreased bone strength resulting from decreased trabecular connectivity. – See more at: http://www.eurekaselect.com/62780/article#sthash.OnoaRPyh.dpuf

Clinical applications of RANK-ligand inhibition

Romas, E.
Internal Medicine Journal 2009; 39 (2), pp. 110-116
http://dx.doi.org:/10.1111/j.1445-5994.2008.01732.x

An enhanced rate of bone remodelling fuelled by osteoclastogenesis mediates diseases such as osteoporosis, arthritic bone destruction, Paget’s disease and malignancy-induced bone loss. Thus, the control of osteoclastogenesis is of major clinical importance. The receptor activator of nuclear factor κB (RANK); its ligand, RANKL and decoy receptor, osteoprotegerin, are critical determinants of osteoclastogenesis, and increased RANK signalling is involved in several bone diseases, providing the rationale for RANKL inhibition. The effects of RANKL inhibition are being witnessed in clinical trials of neutralizing fully human monoclonal antibodies that target RANKL (e.g. denosumab) and which induce profound and sustained inhibition of bone resorption. The relative efficacy, cost-effectiveness and side-effects of targeted RANKL inhibition compared with conventional antiresorptive drugs (i.e. bisphosphonates) should be resolved by clinical trials in coming years.

Clinical development of neridronate: potential for new applications

Davide Gatti, M Rossini, O Viapiana, L Idolazzi, SAdami
Therapeutics and Clinical Risk Management 2013:9 139–147
http://dx.doi.org/10.2147/TCRM.S35788

Neridronate is an aminobisphosphonate, licensed in Italy for the treatment of osteogenesis imperfecta (OI) and Paget’s disease of bone (PDB). A characteristic property of neridronate is that it can be administered both intravenously and intramuscularly, providing a useful system for administration in homecare. In this review, we discuss the latest clinical results of neridronate administration in OI and PDB, as well as in osteoporosis and other conditions. We will focus in particular on the latest evidence of the effect of neridronate on treatment of complex regional pain syndrome type I.

The Sclerostin‐Independent Bone Anabolic Activity of Intermittent PTH Treatment Is Mediated by T‐Cell–Produced Wnt10β

Jau‐Yi Li, Lindsey D Walker, Abdul Malik Tyagi, Jonathan Adams, et al.
Journal of Bone and Mineral Research, Jan 2014; 29(1): pp 43–54
http://dx.doi.org:/10.1002/jbmr.2044

Both blunted osteocytic production of the Wnt inhibitor sclerostin (Scl) and increased T‐cell production of the Wnt ligand Wnt10β contribute to the bone anabolic activity of intermittent parathyroid hormone (iPTH) treatment. However, the relative contribution of these mechanisms is unknown. In this study, we modeled the repressive effects of iPTH on Scl production in mice by treatment with a neutralizing anti‐Scl antibody (Scl‐Ab) to determine the contribution of T‐cell–produced Wnt10β to the Scl‐independent modalities of action of iPTH. We report that combined treatment with Scl‐Ab and iPTH was more potent than either iPTH or Scl‐Ab alone in increasing stromal cell production of OPG, osteoblastogenesis, osteoblast life span, bone turnover, bone mineral density, and trabecular bone volume and structure in mice with T cells capable of producing Wnt10β. In T‐cell–null mice and mice lacking T‐cell production of Wnt10β, combined treatment increased bone turnover significantly more than iPTH or Scl‐Ab alone. However, in these mice, combined treatment with Scl‐Ab and iPTH was equally effective as Scl‐Ab alone in increasing the osteoblastic pool, bone volume, density, and structure. These findings demonstrate that the Scl‐independent activity of iPTH on osteoblasts and bone mass is mediated by T‐cell–produced Wnt10β. The data provide a proof of concept of a more potent therapeutic effect of combined treatment with iPTH and Scl‐Ab than either alone.

Treatment of Paget’s disease with hypercalcemia

Donald H. Gutteridge – Letter to the Editor
Bone 12 Jan 2006; 39(668)
http://dx.doi.org:/10.1016/j.bone.2006.01.165

Selby et al. [7] “Guidelines on the management of Paget’s disease of bone” produced a very helpful review, with 139 references. I take issue however with their approach to the clinical problem of concurrent Paget’s and hypercalcemia.
Firstly, the combination is not rare. Of 1836 literature and personally reported unselected patients with Paget’s disease, 90 had concurrent hypercalcemia due to primary hyperparathyroidism [PHPT], i.e., 4.9% [4]. The number with unspecified hypercalcemia would have exceeded 5%. Secondly, the authors give similar weight to immobilization and PHPT as causes. Immobilization as a cause of hypercalcemia in Paget’s disease is rare [4,3]. The former paper studied 184 consecutive new referrals with Paget’s disease over 15 years. Hypercalcemia was present in 21: two had malignancy (multiple myeloma, secondary cancer); the remaining 19 had biochemical PHPT with most confirmed by neck exploration; none had hypercalcemia of immobilization. Gillespie [3] reported two patients who died following pagetic fractures with immobilization. One was diagnosed and treated as immobilization hypercalcemia; both had large parathyroid adenomas at autopsy.
Thirdly, they have recommended that “patients with Paget’s disease and hypercalcemia should be treated with bisphosphonate”. Since most patients with this combination have PHPT, since bisphosphonate treatment of Paget’s disease is associated with parathyroid hormone (PTH) stimulation [5] and since activation of Paget’s disease occurs with increased PTH [2], it seems reasonable to exclude PHPT (and other causes— e.g., milk alkali syndrome and vitamin D toxicity) and consider neck exploration before bisphosphonate treatment. The response to parathyroidectomy can be profound—and is predictable. In those with PHPT there is a significant linear relationship between preoperative severity (plasma calcium corrected for plasma albumin) and postoperative improvement in bone turnover (%fall in plasma alkaline phosphatase) [4]. In those 7 patients with a preoperative calcium >3.0 mmol/l, the postoperative mean fall in plasma alkaline phosphatase was 68%. Bisphosphonate treatment may be an option in those with PHPT and mild asymptomatic hypercalcemia; likewise following a reasonable interval (say 6 months) after successful neck exploration, should increased bone turnover and pagetic symptoms persist.

In those rare cases with the combination of Paget’s disease, hypercalcemia and immobilized pagetic fracture, where other causes of hypercalcemia have been excluded [1,6], bisphosphonate treatment is eminently reasonable.

[1] Bannister P, Roberts M, Sheridan P. Recurrent hypercalcaemia in a young man with mono-ostotic Paget’s disease. Postgrad Med J 1986;62:481–3.
[2] Genuth SM, Klein L. Hypoparathyroidism and Paget’s disease: the effect of parathyroid hormone administration. J Clin Endocrinol Metab 1972;35: 693–9.
[3] Gillespie WJ. Hypercalcaemia in Paget’s disease of bone. Aust N Z J Surg 1979;49:84–6.
[4] Gutteridge DH, Gruber HE, Kermode DG, Worth GK. Thirty cases of concurrent Paget’s disease and primary hyperparathyroidism: sex distribution, histomorphometry, and prediction of the skeletal response to parathyroidectomy. Calcif Tissue Int 1999;65:427–35.
[5] Harinck HIJ, Bijvoet OLM, Blanksma HJ, Dahlinghaus-Nienhuys PJ. Efficacious management with aminobisphosphonate (APD) in Paget’s disease of bone. Clin Orthop Relat Res 1987;217:79–98.
[6] Nathan AW, Ludlam HA, Wilson DW, Dandona P. Hypercalcaemia due to immobilization of a patient with Paget’s disease of bone. Postgrad Med J 1982;58:714–5.
[7] Selby PL, Davie MWJ, Ralston SH, Stone MD. Guidelines on the management of Paget’s disease of bone. Bone 2002;31:10–9.

The authors of the article entitled “Guidelines on the management of Paget’s disease of bone” published in BONE 2002:31:10–9, have elected not to respond to the above letter to the Editor.

Safety of Bisphosphonates in the Treatment of Osteoporosis

Robert R. Recker, E. Michael Lewiecki, Paul D. Miller, James Reiffel
The American Journal of Medicine (2009) 122, S22–S32
http://dx.doi.org:/10.1016/j.amjmed.2008.12.004

In this review 4 experts consider the major safety concerns relating to bisphosphonate therapy for osteoporosis. Specific topics covered are skeletal safety (particularly with respect to atypical fractures and delayed healing), gastrointestinal intolerance, hypocalcemia, acute-phase (i.e., postdose) reactions, chronic musculoskeletal pain, renal safety, and cardiovascular safety (specifically, atrial fibrillation).

The bone-remodeling cycle

The bone-remodeling cycle.
Remodeling of bone in a multicellular bone unit starts with osteoblastic activation of osteoclast differentiation, fusion, and activation (A and B).
When resorption lacunae are formed, the osteoclasts leave the area and mononucleated cells of uncertain origin appear and “clean up” the organic matrix remnants left by the osteoclast, also possibly forming the cement line (dotted line) at the bottom of the lacunae
(C). During the resorption process, coupling factors, including insulin-like growth factor–I and transforming growth factor–β, are released from the bone-extracellular matrix, and these growth factors contribute to the recruitment of osteoblasts to the resorption lacunae and their activation.
(D). The osteoblasts will then fill the lacunae with new bone; when the same amount of bone is formed as is being resorbed, the remodeling process is finished, and the mineralized extracellular matrix will be covered by osteoid and a single-cell layer of osteoblasts
(E). (Reprinted with permission from J Dent Res.6)

SUMMARY

Persistent, long-term antifracture efficacy has been demonstrated for bisphosphonates, and there is no evidence that the antifracture efficacy declines during treatment periods lasting as long as 10 years. Bisphosphonate-induced oversuppression of remodeling and return of fracturing remains a theoretical possibility.
It is likely that a few patients who are potential candidates for bisphosphonate treatment have preexisting oversuppression of bone remodeling. Treatment with a bisphosphonate in these cases would not be helpful and might even be harmful. The problem when encountering a patient with fractures and deciding whether to recommend treatment with a bisphosphonate is that no reliable diagnostic method exists that allows detection of the rare instance of preexisting oversuppression of remodeling. When pretreatment BMD is not particularly low, that is, not lower than normal or mildly osteopenic, the persistence of fracturing during treatment may mean that oversuppression of remodeling was already present and a change in medication would be appropriate. There is no evidence that bisphosphonate treatment impairs fracture healing. Indeed, there are a substantial number of reports involving animal models, as well as a few human case reports, to suggest that bisphosphonate treatment actually improves fracture healing. In general, it is important to bear in mind the positive benefit-to-risk ratio for this therapeutic class when making treatment recommendations for patients with osteoporosis.

Bisphosphonate Safety:

1. Gastrointestinal Intolerance,2. Hypocalcemia,

3. Acute-Phase Reaction, and

4. Chronic Bone and Muscle Pain

PTH: Potential role in management of heart failure

Gruson, A. Buglioni, J.C. Burnett Jr.
Clinica Chimica Acta 433 (2014) 290–296
http://dx.doi.org/10.1016/j.cca.2014.03.029

Biomarkers play an important role for the diagnosis and prognosis of heart failure (HF), a disease with high morbidity and mortality as well as a huge impact on healthcare budgets. Parathyroid hormone (PTH) is a major systemic calcium-regulating hormone and an important regulator of bone and mineral homeostasis. PTH testing is important for differential diagnosis of calcemia related disorders and for the management of patients with chronic kidney disease. As secondary hyperparathyroidism has been evidenced in HF patients, PTH testing might be relevant in HF patients for risk stratification and more personalized selection of treatment.

Heart failure and neurohormonal activation

Heart failure is a syndrome characterized by increasing prevalence, high morbidity, elevated hospital readmission rate and high mortality. The continuing improvement of diagnosis, prognosis, treatment and management of HF requires a better understanding of the different sub-phenotypes and heterogeneity of this syndrome at the cellular, organ, and systemic level. Neurohormonal activation, one of the hallmarks of HF, plays a significant role in the myocardial and multi-organ adaptation. The comprehensive understanding of neurohormonal activation has allowed the identification of several biomarkers, such as natriuretic peptides, which are now playing an important role in HF management. Beside their contribution to the diagnosis of HF, natriuretic peptides are also relevant for follow-up and prognosis of HF patients. Nevertheless, natriuretic peptides are more related to ventricular stretch, and biomarkers from other biological pathways like cardiac remodeling might provide additional value for the risk stratification of HF patients. The integration of biomarkers from several pathophysiological pathways along with imaging and genetic testing, might therefore be used to define HF subtypes, responding differently to specific therapeutic actions and contributing to more tailored based approaches.
Abnormalities of bone and mineral metabolism are also found in HF. Secondary hyperparathyroidism has been evidenced in this context and several recent reports have documented the potential use of parathyroid hormone (PTH) testing for a more personalized management of HF patients. The aim of this article is therefore to review some of the cardiac effects of PTH and the potential role of PTH testing in HF.

Parathyroid hormone: biology and cardiac effects
PTH is one of the major regulators of the bone and mineral metabolism and its secretion is modulated by changes in concentration of calcium in the blood; decreased calcium concentrations stimulating PTH secretion via calcium-sensing receptors in the parathyroid gland. In response to hypocalcemia,
PTH has different targets to increase circulating calcium concentration. A fundamental target is the renal tubule where PTH will increase phosphorus excretion in the proximal tract and will enhance calcium reabsorption from the ascending limb of the loop of Henle to the collecting duct. The proximal renal tubule is also a target where PTH will stimulate the 1-α hydroxylation of 25-hydroxyvitamin D to 1,25-dihydroxyvitamin D: this biologically active form of vitamin D acts on the gut to increase absorption of both dietary calcium and phosphorus. Another target of PTH is the osteoclasts, leading to increased bone resorption with release of phosphorus and calcium in the blood.
PTH is a polypeptide containing 84 amino acids secreted by the parathyroid glands after cleavage from preproparathyroid hormone to proparathyroid hormone to the mature hormone. However, it displays several circulating forms and related fragments. PTH is secreted predominantly as an intact molecule, but it is rapidly cleaved in peripheral tissues to amino terminus and carboxy terminus fragments. The amino terminus fragment is biologically active and has a relatively short circulating half-life. The carboxy-terminal species include a 7-84 peptide and a variety of shorter fragments. These fragments can have a longer half-life and accumulate in renal failure. PTH assays measure not only the full-length form of PTH but also isoforms as well as fragments and differences can be observed between assays depending on the antibody specificities.

Cardiac effects of PTH
Primary hyperparathyroidism has been associated with heart diseases, underlying the potential cardiac consequences of increased circulating levels of PTH. Furthermore, as the heart is one of the target organs of PTH, the involvement of this hormone in the pathogenesis of cardiovascular diseases was previously suggested. PTH has different effects on the heart and can stimulate hypertrophy, arrhythmias and a pro-oxidative state. PTH has a direct hypertrophic action on cardiomyocytes. PTH is able, through a direct effect mediated through its receptors, to activate protein kinase C which further stimulates hypertrophic growth and reexpression of fetal type proteins in cardiomyocytes. PTH was also reported as a potent activator of protein kinase A (PKA) and several other downstream effectors related to cardiomyocyte hypertrophy. The hypertrophic effect of PTH on cardiac cells is also reinforced by its ability to stimulate an anti-hypertrophic response, including the natriuretic peptide gene transcription and by the increased of plasma concentrations of N-terminal pro-B-type natriuretic peptide (NT-proBNP) observed in patients with primary hyperparathyroidism. The hypertrophic effect of PTH on the heart was also evidence by a close relation between PTH levels and left ventricular mass.
In addition to its hypertrophic action, PTH can stimulate cardiac arrhythmias. PTH was documented as a chronotropic agent able to cause early death ofmyocytes in rat. Importantly, Bogin et al. showed in cultured heart cells of rat, that both amino-terminal PTH 1–34 and intact PTH 1–84 produced an immediate, sustained and significant rise in beats per minute and that the cells died earlier than control cardiomyocytes. Similar bservations were obtained by Shimoyama et al. In human, recent data showed that chronic renal failure and hyperparathyroidism are associated with a sympathetic over-activity. In that case, PTH 1–34 was able to facilitate norepinephrine release in human heart atria by activating the PTH-receptors. Therefore, highly increased PTH levels that can be observed in HF patients can participate to the elevated sympathetic nerve activity and the associated cardiovascular mortality.
The cardiac impact of PTH is also related to calcium overloading in myocardial cells. This cytoplasmic calcium overloading induced by PTH in myocardial cells appears as a paradox for hyperparathyroidism states. The mechanisms behind the increase of intracellular calcium involve a receptor-mediated rise in L-type calcium channel at the plasma membrane level leading to a secondary entry of calcium into cardiomyocyte and mobilization of calcium from sarcoplasmic reticulum. Both PTH 1–34 and PTH 1–84 produced such a dose dependent increase of intracellular calcium in cardiomyocytes. This increase of cytosolic calcium can be prevented by the calcium channel blocker verapamil.
Hyperparathyroidism has also been documented to trigger oxidative stress. When PTH levels are increased, a higher H₂O₂ production is observed in peripheral blood mononuclear cells. The increase in intracellular calcium induced by PTH might impair the mitochondrial function and ATP production, inducing the production of reactive oxygen species and leading to oxidative stress as well as inflammation and, at the end, to cardiomyocyte necrosis.
Lastly, increased circulating concentrations of PTH might stimulate adrenal aldosterone synthesis, initiating a vicious circle between hyperparathyroidism and hyperaldosteronism and leading to more proinflammatory, pro-oxidant and pro-fibrotic actions.

The rise of PTH in HF
Through its cardiac effects PTH can participate to the pathophysiology of cardiovascular diseases and a chronic excess to high circulating levels of PTH can have some deleterious consequences for the HF patients. Several factors might explain the increase of circulating PTH levels in HF patients.
First of all, impaired cation homeostasis and calcium loss should be considered. Alteration in electrolyte equilibrium is frequent in HF patients as a consequence of hormonal changes in this pathological condition (hyperadrenergic state and secondary hyperaldosteronism). Calcium wasting is also triggered by diuretics, used to treat HF patients.
A second important factor can be a deficiency of vitamin D. Low vitamin D levels are frequently observed in HF patients and can lead to a rise in PTH levels.
Another documented factor is the interrelationship between hemodynamic state and serum intact PTH levels in patients with HF. Indeed, in a cross-sectional study including 105 patients with chronic HF, log-transformed intact PTH levels were positively and significantly correlated with pulmonary capillary wedge pressure and inversely correlated with stroke volume index after adjusting for variables associated with PTH.

The cross talk between PTH and aldosterone
The cross talk between PTH and FGF-23
Circulating levels of PTH and heart failure
PTH levels in HF patients
PTH testing and heart failure: conclusions and perspectives
PTH testing: assay matters

secondary hyperparathyroidism

Potential involvement of secondary hyperparathyroidism in the worsening course of heart failure significant correlations were observed, through generation assays, between PTH and natriuretic peptides aswell as galectin-3. Importantly, the different immunoreactivities might impact on the value of PTH testing in treatment and prognosis of HF.

The measurement of PTH concentrations in HF can, like in patients with chronic kidney disease, help to monitor the efficiency of the treatment (drugs as well as medical devices). The use of PTH testing in HF patients might also allow the selection of more personalized and tailored therapies. HF patients with higher PTH levels could be relevant candidates for vitamin D supplementation or other pharmacological treatment. Based on the positive relationship between aldosterone and PTH, higher PTH levels can be an additional reason to use aldosterone blockers in HF patients.

Parathyroid hormone and cardiovascular disease events: A systematic review and meta-analysis of prospective studies

Adriana J. van Ballegooijen, I Reinders, M Visser, and IA Brouwer
Am Heart J 2013;165:655-664.e5
http://dx.doi.org/10.1016/j.ahj.2013.02.014

The parathyroid hormone (PTH) is a key hormone for the maintenance of calcium homeostasis. Low serum calcium triggers the secretion of PTH from the parathyroid glands.1 This results in a raise in serum calcium by promoting the release of calcium from bone, reduces calcium excretion by the kidneys, and increases the calcium absorption by the small intestine. In turn, the increase in calcium inhibits PTH secretion from the parathyroid glands.
In addition to traditionally known target organs, PTH is of interest for its potential impact on cardiovascular disease (CVD) risk. Observational studies have demonstrated that chronic PTH elevation is linked to hypertension, cardiac hypertrophy, and myocardial dysfunction. Furthermore, PTH receptors are present in the myocardium and exert hypertrophic effects on cardiomyocytes. Taken together, these associations suggest plausible mechanisms whereby elevated PTH concentrations may be involved in pathological processes that lead to CVD.

Background Parathyroid hormone (PTH) excess might play a role in cardiovascular health. We therefore conducted a systematic review and meta-analysis to evaluate the association between PTH and cardiovascular disease (CVD) events, and intermediate outcomes.
Methods We conducted a systematic and comprehensive database search using MEDLINE and Embase between 1947 and October 2012. We included English-language prospective studies that reported risk estimates for PTH and CVD events, and intermediate outcomes. The characteristics of study populations, exposure, and outcomes of total CVD events, fatal and non-fatal CVD events were reported, and a quality assessment was conducted. Results were extracted for the highest versus lowest PTH concentrations, and meta-analyses were carried out using random effects models.
Results The systematic literature search yielded 5770 articles, and 15 studies were included. Study duration ranged between 2 and 14 years. All studies were performed primarily in whites with a mean age between 55 and 75 years. The metaanalyses included 12 studies, of which 10 investigated total CVD events; 7, fatal CVD events; and 3, non-fatal CVD events. PTH excess indicated an increased risk for total CVD events: pooled HR (95% CI), 1.45 (1.24-1.71). The results for fatal CVD events and non-fatal CVD events were: HR 1.50 (1.18-1.91) and HR 1.48 (1.14-1.92). Heterogeneity was moderately present; however, sensitivity analyses for follow-up duration, prior CVD, or PTH as dichotomous values showed similar results.
Conclusions The meta-analysis indicates that higher PTH concentrations are associated with increased risk of CVD events.

Impact of estrogen on mechanically stimulated cells in vitro

Jörg Neunzehn, Ulrich Meyer and Hans-Peter Wiesman
Int.J.Curr.Microbiol.App.Sci (2014) 3(5) 898-906
Estrogen deficiency and decreased exercise known to be major causes for osteoporosis in elderly patients are assumed on important role in implant failure. Hormone replacement therapy and exercise are established methods to prevent the accompanying bone loss, thereby improving the conditions for implant osseointegration. Whereas the clinical effects of estrogen on bone are well documented, less is known about estrogen effects on loaded and unloaded osteoblasts on a cellular level. This study was aimed at investigating the effects of estrogen on mechanically stimulated osteoblast like cells in culture. Mechanically unstimulated cultures served as controls. Our investigations revealed that estrogen had a suppressive effect on the proliferative response of osteoblasts towards mechanical strain. Estrogen increased the synthesis of bone specific proteins in mechanically stimulated cultures whereas estrogen had no effect on unstimulated cells. The differentiation effects significant altered at estrogen doses of 10nmol and 10 μmol. Our data suggest a positive effect of hormone substitution on the composition of the extracellular matrix in loaded bones. In the context of implant dentistry, hormone repaints therapy should be regarded as a medical tool to improve the conditions for an undisturbed implant healing.

Normal bone physiology, remodelling and its hormonal regulation

Jennifer S Walsh
Surgery 2014; 33:1

The skeleton has structural and locomotor functions, and is a mineral reservoir. Bone turnover by osteoclasts and osteoblasts is a lifelong process, incorporating growth, modelling and remodeling to repair microdamage and access the mineral reservoir.
Bone formation and resorption are the basis of growth, modeling and remodeling. The bone remodeling cycle is an ongoing process that renews bone to repair microdamage and maintain strength. It also maintains serum calcium in the normal physiological range by release of mineral from the bone matrix as required. About 5-10% of the adult skeleton is replaced by remodeling each year.
On trabecular bone and at the endocortical surface, remodeling takes place on the surface of bone, but within cortical bone the osteoclasts form a cutting cone through the bone matrix. The signal to initiate remodeling may be endocrine (such as increased parathyroid hormone (PTH) in response to hypocalcaemia), which leads to generalized increases in osteoclast activation. Localized remodeling is initiated in response to microdamage, probably by signals from osteocytes. During a remodeling cycle, osteoclasts on the bone surface become activated and resorb bone matrix, creating a defect which is filled in by osteoblasts. The cycle usually takes about 200 days to complete. The bone remodeling cycle is highly regulated, and resorption and formation are closely coupled.
Signaling between bone cells is essential for the coordination of these processes. Osteoblasts regulate osteoclast activity through the receptor activator of nuclear factor-kB (RANK)/RANK ligand/osteoprotegerin system, and osteocytes regulate osteoblast activity through sclerostin secretion. If resorption and formation are balanced there is no net change in bone mass after each cycle, but with ageing and some disease states resorption exceeds formation leading to remodeling imbalance, decreased bone mass and loss of microstructural integrity. The rate of remodeling is determined by loading and endocrine influences. The most important endocrine regulator of bone turnover is probably estrogen, but other hormones regulating bone metabolism include insulin-like growth factor-1, parathyroid hormone and gut and adipocyte hormones.

Differential Diagnosis, Causes, and Management of Hypercalcemia

Fredriech K. W. Chan, et al.
Current Problems In Surgery June 1997; 34(6)

Hypercalcemia is a challenging clinical syndrome, both in diagnosis and therapy. The two most common causes of hypercalcemia, primary hyperparathyroidism and malignancy, account for approximately 90% of all patients with an elevated calcium level. In the general population, primary hyperparathyroidism is more common than malignancy. In a hospitalized population, malignancy is by far the more common. The differential diagnosis of hypercalcemia should be focused initially on the distinction between primary hyperparathyroidism and malignancy.

Primary hyperparathyroidism is caused by excessive, abnormally regulated secretion of parathyroid hormone from one or more adenomatous or hyperplastic parathyroid glands. In 80% of cases, primary hyperparathyroidism is due to a single adenoma. In 15% to 20% of patients, all four glands are enlarged as a result of hyperplasia. Parathyroid hyperplasia is also encountered in patients with Multiple Endocrine Neoplasia, Type I or II. Rarely, in fewer than 0.5% of patients, primary hyperparathyroidism is due to parathyroid carcinoma. The clinical features of primary hyperparathyroidism result from the hypercalcemia and the excessive output of parathyroid hormone (PTH).
The major target organs are the bones and the kidneys. The classic but rare bone disease of primary hyperparathyroidism is osteitis fibrosa cystica. Since the advent of the multichannel autoanalyzer in the early 1970s, an era marked by a great increase in incidence of primary hyperparathyroidism, the prevalence of radiologically apparent bone disease in patients with primary hyperparathyroidism has declined from 10% to 15% to a vanishingly small 1% to 2%. Sensitive technologies such as bone densitometry and bone histomorphometry, however, have revealed skeletal involvement with preferential reduction of cortical bone mass and relative preservation of cancellous bone mass. Although the incidence of nephrolithiasis in primary hyperparathyroidism has also decreased markedly, from approximately 60% in the 1940s and 1950s to 15% to 20% now, nephrolithiasis is still the most frequent complication of primary hyperparathyroidism.
Primary hyperparathyroidism also can be associated with neuropsychiatric, gastrointestinal, and cardiovascular manifestations. However, evidence that these features are pathophysiologically linked to the hyperparathyroid process or are reversible after successful parathyroidectomy is not compelling.

Management of Skeletal Health in Patients With Asymptomatic Primary Hyperparathyroidism

Michael Lewiecki
J Clin Densitometry: Assessment of Skeletal Health, 2010; 13(4), 324e334.
http://dx.doi.org:/10.1016/j.jocd.2010.06.004

Asymptomatic primary hyperparathyroidism (PHPT) may cause adverse skeletal effects that include high bone remodeling, reduced bone mineral density (BMD), and increased fracture risk. Parathyroid surgery, the definitive treatment for PHPT, has been shown to increase BMD and appears to reduce fracture risk. Current guidelines recommend parathyroid surgery for patients with symptomatic PHPT or asymptomatic PHPT with serum calcium > 1 mg/dL above the upper limit of normal, calculated creatinine clearance < 60 mL/min, osteoporosis, previous fracture, or age > 50 yr. The type of operation performed (parathyroid exploration or minimally invasive procedure) and localizing studies to identify the abnormal parathyroid glands preoperatively should be individualized according to the skills of the surgeon and the resources of the institution. In patients who choose not to be treated surgically or who have contraindications for surgery, medical therapy should include a daily calcium intake of at least 1200 mg and maintenance of serum 25-hydroxyvitamin D levels of at least 20 ng/mL (50 nmol/L). Bisphosphonates and estrogens have been shown to provide skeletal benefits that appear to be similar to parathyroid surgery. Cinacalcet reduces serum calcium in PHPT patients with intractable hypercalcemia but has not been shown to improve BMD. It is not known whether any medical intervention reduces fracture risk in patients with PHPT. There are insufficient data on the natural history and treatment of normocalcemic PHPT to make recommendations for management of this disorder.

Hyperparathyroidism

William D Fraser
thelancet July 11, 2009; 374: 145-158 – Seminar

Hyperparathyroidism is due to increased activity of the parathyroid glands, either from an intrinsic abnormal change altering excretion of parathyroid hormone (primary or tertiary hyperparathyroidism) or from an extrinsic abnormal change affecting calcium homoeostasis stimulating production of parathyroid hormone (secondary hyperparathyroidism). Primary hyperparathyroidism is the third most common endocrine disorder, with the highest incidence in postmenopausal women. Asymptomatic disease is common, and severe disease with renal stones and metabolic bone disease arises less frequently now than it did 20–30 years ago. Primary hyperparathyroidism can be cured by surgical removal of an adenoma, increasingly by minimally invasive parathyroidectomy. Medical management of mild disease is possible with bisphosphonates, hormone replacement therapy, and calcimimetics. Vitamin D deficiency is a common cause of secondary hyperparathyroidism, particularly in elderly people. However, the biochemical definition of vitamin D deficiency and its treatment are subject to much debate. Secondary hyperparathyroidism as the result of chronic kidney disease is important in the genesis of renal bone disease, and several new treatments could help achieve the guidelines set out by the kidney disease outcomes quality initiative.

Table 1: Changing clinical presentation of primary hyperparathyroidism
	1930–1970	1970–2000
Nephrolithiasis	51–57%	17–37%
Hypercalciuria	36%	40%
Overt skeletal disease	10–23%	4–14%
Asymptomatic	6–18%	22–80%
Modified from reference 12

Panel 1: Recommendations for surgery from the National Institutes of Health
consensus conference on primary hyperparathyroidism in 1990 and 2002• Serum albumin-adjusted calcium greater than 0·25 mmol/L
above the upper limit of local laboratory reference range

• Urine calcium greater than 10 mmol per 24 h

• Creatinine clearance reduced by 30% or more

• Bone mineral density T score less than –2·5 (at any site)

• Age younger than 50 years

• Patient request; adequate follow-up unlikely

Aldosterone and parathyroid hormone interactions as mediators of metabolic and cardiovascular disease

Andreas Tomaschitz, Eberhard Ritz, Burkert Pieske, Jutta Rus-Machan
Metabolism Clinical and Experimental 2014; 63: 20 31
http://dx.doi.org/10.1016/j.metabol.2013.08.016

Several studies demonstrated a strong link between dysregulation of the aldosterone and parathyroid hormone (PTH) axes on the one hand and CV pathology on the other hand. Such evidence documents clinically relevant interactions between aldosterone and PTH and a resulting impact on CV health. This review provides an up to date overview discussing the mechanisms and the clinical relevance underlying the interactions between aldosterone and PTH.

Inappropriate aldosterone and parathyroid hormone (PTH) secretion is strongly linked with development and progression of cardiovascular (CV) disease. Accumulating evidence suggests a bidirectional interplay between parathyroid hormone and aldosterone. This interaction may lead to a disproportionally increased risk of CV damage, metabolic and bone diseases.

This review focuses on mechanisms underlying the mutual interplay between aldosterone and PTH as well as their potential impact on CV, metabolic and bone health. PTH stimulates aldosterone secretion by increasing the calcium concentration in the cells of the adrenal zona glomerulosa as a result of binding to the PTH/PTH-rP receptor and indirectly by potentiating angiotensin 2 induced effects. This may explain why after parathyroidectomy lower aldosterone levels are seen in parallel with improved cardiovascular outcomes.

Aldosterone mediated effects are inappropriately pronounced in conditions such as chronic heart failure, excess dietary salt intake (relative aldosterone excess) and primary aldosteronism.

PTH is increased as a result of
(1) the MR (mineralocorticoid receptor)mediated calciuretic and magnesiuretic effects with a trend of hypocalcemia and hypomagnesemia; the resulting secondary hyperparathyroidism causes myocardial fibrosis and disturbed bone metabolism; and

(2) direct effects of aldosterone on parathyroid cells via binding to the MR. This adverse sequence is interrupted by mineralocorticoid receptor blockade and adrenalectomy.

Hyperaldosteronism due to klotho deficiency results in vascular calcification, which can be mitigated by spironolactone treatment. In view of the documented reciprocal interaction between aldosterone and PTH as well as the potentially ensuing target organ damage, studies are needed to evaluate diagnostic and therapeutic strategies to address this increasingly recognized pathophysiological phenomenon.

The classical view that aldosterone acts exclusively on the electrolyte transport in epithelial cells has been broadened after the mineralocorticoid receptor (MR) has been identified in non-epithelial cells as well, e.g. vascular smooth muscle cells and cardiomyocytes. Apart from classical genomic effects, non-genomic aldosterone mediated effects have been identified in various tissues and organs outside of the kidneys and colon, e.g. inner ear, choroid plexus, endothelial cells and cardiomyocytes.

In the past it had been documented that primary aldosteronism (PA; absolute aldosterone excess) contributed to the development of CVD. Several studies suggested, however, that “absolute aldosterone excess” is only the tip of the iceberg leading to the concept of “relative aldosterone excess” . Several large cross-sectional and prospective studies demonstrated a consistent relationship between circulating aldosterone levels, CV risk factors and mortality risk.

Such recent studies also document that even circulating aldosterone concentrations in the “normal” range may result in inappropriate aldosterone–MR interaction which may be reversed by MR blockade.
The identification of PTH receptors within the CV system e.g. in cardiomyocytes, vascular smooth muscle, and endothelial cells, indicates that inappropriate PTH secretion may impact on the CV health beyond the dysregulation of calcium and phosphate homeostasis.

Application of PTH after myocardial infarction attenuates ischaemic cardiomyopathy by increasing migration of bone marrow-derived stem cells to the ischaemic myocardium. On the other hand the PTH excess in primary hyperparathyroidism (pHPT) is linked in the long-term to a spectrum of adverse effects e.g. bone loss and increased fracture risk, coronary microvascular dysfunction, derangement of lipid and glucose metabolism, subclinical aortic valve calcification, increased aortic stiffness, endothelial dysfunction and arterial hypertension.

Interactions between vitamin D, klotho and aldosterone
Increased activity of systemic or local renin–angiotensin systems (RAS) is linked to increased target organ damage. The organ and tissue protective effects of vitamin D have in part been explained by vitamin D induced modulation of RAS activity.

In landmark experiments Li et al. documented markedly elevated renin mRNA expression in the juxtaglomerular apparatus of vitamin D receptor (VDR) knock-out mice compared to wild type mice. Furthermore, 1,25-dihydroxy vitamin D (1,25(OH2)D3) modulated renin gene transcription and renin synthesis and this was independent of serum calcium, PTH and angiotensin 2. Angiotensin 2 in turn reduces renal klotho expression resulting in modulations of FGF-23-signaling and of 1-α hydroxylase activity. Klotho is a membrane (and circulating) protein which is highly expressed in the kidney and modulates the inhibitory effects of FGF-23 on calcitriol formation; klotho contributes to the regulation of renal tubular calcium and phosphate reabsorption. The modulatory effects of vitamin D on the RAS might result in a lower risk of development and progression of CV morbidity and mortality.

Evidence for stimulating effects of PTH on adrenal aldosterone secretion Aldosterone synthesis is mainly initiated by angiotensin 2 and potassium via activating the Ca2+-messenger system in zona glomerulosa (ZG) cells to stimulate the steroidogenic cascade within the mitochondria. The Ca2+-messenger system further participates in the initiation of steroidogenesis by facilitating the cholesterol transfer into the mitochondria. Findings from experimental, mechanistic, observational and interventional studies suggest that PTH contributes to the regulation of aldosterone secretion in the ZG of the adrenal glands.

The interaction between aldosterone and Klotho and its relationship to vascular osteoinduction

Estradiol determines the effects of PTH on ERa-dependent transcription in MC3T3-E1 cells

Monika H.E. Christensen, IS Fenne, MH Flågeng, B Almås, et al.
Biochemical and Biophysical Research Communications 450 (2014) 360–365
http://dx.doi.org/10.1016/j.bbrc.2014.05.109

Bone remodeling is a continuous process regulated by several hormones such as estrogens and parathyroid hormone (PTH). Here we investigated the influence of PTH on estrogen receptor alpha (ERa)-dependent transcriptional activity in MC3T3-E1 osteoblasts. Cells that were transfected with an ER-responsive reporter plasmid and treated with PTH showed increased luciferase activity. However, in the presence of 17b-estradiol, we observed that PTH inhibited ERa-mediated transcription. cAMP mimicked the effects by PTH, and the findings were confirmed in COS-1 cells transfected with expression vector encoding the catalytic subunit of cAMP-dependent protein kinase (PKA). Furthermore, PTH exhibited specific effects on the mRNA expression of the decoy receptor osteoprotegerin (OPG) and the receptor activator of NF kappa-B ligand (RANKL) in MC3T3-E1 osteoblasts. In the absence of 17b-estradiol, PTH and cAMP enhanced the OPG/RANKL ratio, whereas, OPG/RANKL was suppressed when estradiol was present. In conclusion, our results indicate that the presence of estradiol determines whether PTH and cAMP stimulates or inhibits ERa-dependent activity and the OPG/RANKL mRNA expression in an osteoblastic cell line.

Ginsenoside-Rb2 displays anti-osteoporosis effects through reducing oxidative damage and bone-resorbing cytokines during osteogenesis

Qiang Huang, Bo Gao, Qiang Jie, Bo-Yuan Wei, et al.
Bone 66 (2014) 306–314
http://dx.doi.org/10.1016/j.bone.2014.06.010

Reactive oxygen species (ROS) are a significant pathogenic factor of osteoporosis. Ginsenoside-Rb2 (Rb2), a 20(S)-protopanaxadiol glycoside extracted from ginseng, is a potent antioxidant that generates interest regarding the bone metabolism area. We tested the potential anti-osteoporosis effects of Rb2 and its underlying mechanism in this study. We produced an oxidative damage model induced by hydrogen peroxide (H2O2) in osteoblastic MC3T3-E1 cells to test the essential anti-osteoporosis effects of Rb2 in vitro. The results indicated that treatment of 0.1 to 10 μMRb2 promoted the proliferation of MC3T3-E1 cells, improved alkaline phosphatase (ALP) expression, elevated calcium mineralization and mRNA expressions of Alp, Col1a1, osteocalcin (Ocn) and osteopontin (Opn) against oxidative damage induced by H2O2. Importantly, Rb2 reduced the expression levels of receptor activator of nuclear factor kappa-B ligand (RANKL) and IL-6 and inhibited the H2O2-induced production of ROS. The in vivo study indicated that the Rb2 administered for 12 weeks partially decreased blood malondialdehyde (MDA) activity and elevated the activity of reduced glutathione (GSH) in ovariectomized (OVX)mice. Moreover, Rb2 improved the micro-architecture of trabecular bones and increased bone mineral density (BMD) of the 4th lumbar vertebrae (L4) and the distal femur. Altogether, these results demonstrated that the potential anti-osteoporosis effects of Rb2 were linked to a reduction of oxidative damage and bone-resorbing cytokines, which suggests that Rb2 might be effective in preventing and alleviating osteoporosis.

Inflammatory cytokines in Paget’s disease of bone

GRW de Castro, Z Buss, JS Da Rosa, TS Fröde
International Immunopharmacology 18 (2014) 277–281
http://dx.doi.org/10.1016/j.intimp.2013.12.003

This study was undertaken to evaluate the expression of inflammatory cytokines in patients with Paget’s disease of bone (PDB). Serum levels of tumoral necrosis factor-α, interleukin 1β, interleukin-6 and interleukin-17
were measured in 51 patients with PDB and in 24 controls with primary osteoarthritis. Compared to controls, patients with Paget’s disease of bone presented higher levels of interleukin 6 and reduced interleukin 17, but levels of tumoral necrosis factor α and interleukin 1 β did not differ significantly. We found no significant differences when patients were compared according to disease activity or current treatment. There were no correlations between cytokine levels and bone-specific alkaline phosphatase or extension of Paget’s disease of bone on bone scintigraphs. In conclusion, patients with PDB present significant differences on levels of certain cytokines in comparison to primary osteoarthritis patients, but these alterations did not appear to have a clear correlation with parameters of disease activity or severity.

Development and validation of a novel cell-based assay for potency determination of human parathyroid hormone (PTH)
Axel Hohenstein, Meike Hebell, Heidi Zikry, Maria El Ghazaly, et al.
Journal of Pharmaceutical and Biomedical Analysis 98 (2014) 345–350
http://dx.doi.org/10.1016/j.jpba.2014.06.004

Disorders of bone metabolism
Orthopaedics I: General Principles

Nicola Peel
Surgery 33:1

Bone remodeling is critical to bone health. Alterations in the normal processes and regulation of remodeling may impact on bone mass and bone strength. Changes may be generalized or focal and underlie many of the common disorders of bone metabolism. This article focuses on the changes in bone remodeling which underlie both the development and treatment of osteoporosis. Osteomalacia, as an example of a mineralization disorder and Paget’s disease as an example of a focal disorder of bone remodeling, are also briefly reviewed.

There are many causes of increased bone turnover with the most common being the loss of estrogen at menopause. Increased bone turnover is initiated by increased activation frequency of osteoclasts. The consequent increase in remodeling space leads to bone loss which is, at least in part, reversible. Increased bone turnover is also associated with an increased risk of trabecular perforation with the increased number of remodeling sites acting as stress risers within the trabecular architecture. Bone loss within the trabecular compartment occurs preferentially from the horizontal, non-weight bearing plates resulting in disproportionate loss of bone strength for the reduction in bone mass.
Alterations in bone turnover also have potential to affect bone.

strength by changing the degree of mineralization. Primary mineral apposition occurs early after production of bone matrix by osteoblasts. After completion of the cycle, secondary mineral apposition occurs over many months. Increased bone turnover leads to reduced mineralization as the time between remodeling cycles reduces. Conversely, decreased bone turnover rates reduce the average time between remodeling at any site and hence lead to a greater degree of mineralization. Biomechanical principles indicate that the yield strength (stiffness) of highly mineralized bone increases but that it will withstand less deformation before fracture and therefore becomes brittle. A reduced degree of mineralization results in greater pliability but a reduction in bone strength.
Alterations in bone remodeling underpin changes in bone mass and bone strength. The impact of these changes is manifest in the development and clinical presentation of osteoporosis.

Paget’s disease

Paget’s disease: (a) increased uptake on nuclear medicine scanning in the right hemipelvis, sacrum and left femur and (b) left femur showing radiological changes of Paget’s including a fissure fracture in the proximal lateral cortex

Paget’s disease is an example of a localised disorder of bone turnover. Its aetiology remains unclear. Paget’s disease is not uncommon but is often asymptomatic and diagnosed coincidentally. It is estimated to affect approximately 2% of adults over the age of 55 in the UK but the prevalence varies markedly between populations. It is increasingly prevalent with increasing age and affects men more frequently than women. In 80% of cases more than one bone is involved, characteristically in an asymmetric distribution.
Pagetic bone is characterized by the presence of giant multinucleated osteoclasts resulting in dramatic increases in bone resorption in the affected bones. These regions undergo a lytic phase followed by a compensatory increase in bone formation. Rapid bone formation results in an accumulation of woven bone, which is mechanically abnormal resulting in loss of bone strength.
The typical clinical manifestation is of bone pain, which may be associated with bone expansion and deformity. Complications of Paget’s disease include the development of secondary osteoarthritis, fissure fractures and very rarely, osteosarcomatous change (<1% of cases).

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Pancreatic Islets

Posted in Curation, Diabetes Mellitus, Diagnostics and Lab Tests, Disease Biology, Endocrine Diseases, Epigenetics and Cardiovascular Risks, Fatty acids, Frontiers in Cardiology and Cardiovascular Disorders, Gene Regulation, Genomic Endocrinology, Lipid metabolism, Lipids, Metabolism, Nephrology, Neurodegenerative Diseases, Nitric Oxide in Health and Disease, Nutrition, Nutritional Supplements: Atherogenesis, Origins of Cardiovascular Disease, tagged alpha cell, Apoptosis, Beta cell, beta cell amyloid, endocrinology, insulin and glucagon, oligomerization, Pancreas, peptides on February 8, 2015| Leave a Comment »

Pancreatic Islets

Writer and Curator: Larry H. Bernstein, MD, FCAP

Part I. Endocrine Pancreas

The eclipse and rehabilitation of JJR Macleod, Scotland’s insulin laureate

Bliss, M
Journal of the Royal College of Physicians of Edinburgh 2013; 43(4): 1-8

John JR Macleod (1876-1935,) an Aberdonian Scot who had emigrated to North America, shared the 1923 Nobel Prize with Frederick Banting for their discovery of insulin at the University of Toronto in 1921-22. Macleod finished his career as Regius Professor of Physiology at the University of Aberdeen from 1928 to 1935.Macleod’s posthumous reputation was deeply tarnished by the campaigns against him carried out by his fellow laureate, Banting, and by Banting’s student assistant during the insulin research, Charles Best. Banting’s denigration of Macleod was based on their almost total personality conflict; Best’s was based on a hunger for personal recognition. New research indicates how scarred both men were in their obsessions.The rehabilitation of Macleod’s reputation, begun in 1982 with my book, The Discovery of Insulin, has continued in both scholarly and popular circles. By 2012, the ninetieth anniversary of the discovery of insulin, it had become complete both at the University of Toronto and in Canada.

Almost famous: E. Clark Noble, the common thread in the discovery of insulin and vinblastine

Wright Jr., J.R.
CMAJ 2002; 167 (12), pp. 1391-1396

CLARK NOBLE WAS ONE OF THE FIRST members of the University of Toronto insulin team and came within a coin toss of replacing Charles Best as Frederick Banting’s assistant during the summer of 1921. Noble performed important early studies helping to characterize insulin’s action, and he co-authored many of the original papers describing insulin. Because mass production of insulin from livestock pancreata had proved elusive throughout 1922, J.J.R. Macleod hired Noble during the summer of 1923 to help him test and develop a new method for producing commercial quantities of insulin that Macleod believed would revolutionize insulin production. However, commercial production of insulin from fish proved impractical and was dropped by 1924, as methods to produce large quantities of mammalian insulin had improved very rapidly. Noble later played a small but critical role in the most important Canadian contribution to cancer chemotherapy research: the discovery of vinca alkaloids by his brother Robert Laing Noble. Although one might expect that a physician involved in 2 of Canada’s most important medical discoveries during the 20th century must be famous, such was not Clark Noble’s fate. He died without so much as an obituary in CMAJ.

The Pathophysiology of Diabetes and Cardiovascular Disease

Larry H. Bernstein, MD, FCAP, Reviewer and Curator
and Aviva Lev-Ari, PhD, RN, Curator

http://pharmaceuticalintelligence.com/2014/01/15/pathophysiological-effects-of-diabetes-on-ischemic-cardiovascular-disease-and-on-chronic-obstructive-pulmonary-disease-copd/

This is a multipart article that develops the pathological effects of type-2 diabetes in the progression of a systemic inflammatory disease with a development of neuropathy, and fully developing into cardiovascular disease. It also identifies a systemic relationship to the development of chronic obstructive pulmonary disease. In medical school we were taught that syphilis is the great masquerader. The more we learn about diabetes, we learn about its generalized systemic effects.

Part 1. Role of Autonomic Cardiovascular Neuropathy in Pathogenesis

This article is an abstract only of a related publication of the pathogenesis of autonomic neuropathy in diabetics leading to ischemic heart disease.

The role of autonomic cardiovascular neuropathy in pathogenesis of ischemic heart disease in patients with diabetes mellitus

Subjects: Medicine (General), Medicine, Medicine (General), Health Sciences
Authors: Popović-Pejičić Snježana, Todorović-Đilas Ljiljana, Pantelinac Pavle
Publisher: Društvo lekara Vojvodine Srpskog lekarskog društva
Publication: Medicinski Pregled 2006; 59(3-4): Pp 118-123 (2006)
http://dx.doi.org/10.2298/MPNS0604118P

http://www.doiserbia.nb.rs/img/doi/0025-8105/2006/0025-81050604118P.pdf

Keywords: diabetes mellitus, autonomic nervous system diseases, heart diseases, myocardial ischemia, comorbidity

Introduction. Diabetes is strongly associated with macrovascular complications, among which ischemic heart disease is the major cause of mortality. Autonomic neuropathy increases the risk of complications, which calls for an early diagnosis. The aim of this study was to determine both presence and extent of cardiac autonomic neuropathy, in regard to the type of diabetes mellitus, as well as its correlation with coronary disease and major cardiovascular risk factors. Material and methods. We have examined 90 subjects, classified into three groups, with 30 patients each: those with type 1 diabetes, type 2 diabetes and control group of healthy subjects. All patients underwent cardiovascular tests (Valsalva maneuver, deep breathing test, response to standing, blood pressure response to standing sustained, handgrip test), electrocardiogram, treadmill exercise test and filled out a questionnaire referring to major cardiovascular risk factors: smoking, obesity, hypertension, and dyslipidemia. Results. Our results showed that cardiovascular autonomic neuropathy was more frequent in type 2 diabetes, manifesting as autonomic neuropathy. In patients with autonomic neuropathy, regardless of the type of diabetes, the treadmill test was positive, i.e. strongly correlating with coronary disease. In regard to coronary disease risk factors, the most frequent correlation was found for obesity and hypertension. Discussion. Cardiovascular autonomic neuropathy is considered to be the principal cause of arteriosclerosis and coronary disease. Our results showed that the occurrence of cardiovascular autonomic neuropathy increases the risk of coronary disease due to dysfunction of autonomic nervous system. Conclusions. Cardiovascular autonomic neuropathy is a common complication of diabetes that significantly correlates with coronary disease. Early diagnosis of cardiovascular autonomic neuropathy points to increased cardiovascular risk, providing a basis for preventive and therapeutic measures.

Part 2. a longitudinal cohort study of the cardiovascular experience of individuals at high risk for diabetes

Protocol for ADDITION-PRO: a longitudinal cohort study of the cardiovascular experience of individuals at high risk for diabetes recruited from Danish primary care

Subjects: Public aspects of medicine, Medicine, Public Health, Health Sciences
Authors: Johansen Nanna B, Hansen Anne-Louise S, Jensen Troels M, Philipsen Annelotte, Rasmussen Signe S, Jørgensen Marit E, Simmons Rebecca K, Lauritzen Torsten, Sandbæk Annelli, Witte Daniel R
Publisher: BioMed Central Date of publication: 2012 December
Published in: BMC Public Health 2012; 12(1): 1078
ISSN(s): 1471-2458 Added to DOAJ: 2013-03-12 http://dx.doi.org/10.1186/1471-2458-12-1078 http://www.biomedcentral.com/1471-2458/12/1078

Keywords: Diabetes, Cardiovascular disease, Primary care, Complications, Microvascular, Impaired fasting glucose, Impaired glucose intolerance, Aortic stiffness, Physical activity, Body composition.

Background: Screening programmes for type 2 diabetes inevitably find more individuals at high risk for diabetes than people with undiagnosed prevalent disease. While well established guidelines for the treatment of diabetes exist, less is known about treatment or prevention strategies for individuals found at high risk following screening. In order to make better use of the opportunities for primary prevention of diabetes and its complications among this high risk group, it is important to quantify diabetes progression rates and to examine the development of early markers of cardiovascular disease and microvascular diabetic complications. We also require a better understanding of the mechanisms that underlie and drive early changes in cardiometabolic physiology. The ADDITION-PRO study was designed to address these issues among individuals at different levels of diabetes risk recruited from Danish primary care. Methods/Design: ADDITION-PRO is a population-based, longitudinal cohort study of individuals at high risk for diabetes. 16,136 eligible individuals were identified at high risk following participation in a stepwise screening programme in Danish general practice between 2001 and 2006. All individuals with impaired glucose regulation at screening, those who developed diabetes following screening, and a random sub-sample of those at lower levels of diabetes risk were invited to attend a follow-up health assessment in 2009–2011 (n = 4,188), of whom 2,082 (50%) attended. The health assessment included detailed measurement of anthropometry, body composition, biochemistry, physical activity and cardiovascular risk factors including aortic stiffness and central blood pressure. All ADDITION-PRO participants are being followed for incident cardiovascular disease and death. Discussion: The ADDITION-PRO study is designed to increase understanding of cardiovascular risk and its underlying mechanisms among individuals at high risk of diabetes. Key features of this study include (i) a carefully characterised cohort at different levels of diabetes risk; (ii) detailed measurement of cardiovascular and metabolic risk factors; (iii) objective measurement of physical activity behaviour; and (iv) long-term follow-up of hard clinical outcomes including mortality and cardiovascular disease. Results will inform policy recommendations concerning cardiovascular risk reduction and treatment among individuals at high risk for diabetes. The detailed phenotyping of this cohort will also allow a number of research questions concerning early changes in cardiometabolic physiology to be addressed.

Part 3. Clinical significance of cardiovascular dysmetabolic syndrome

This third part is a description of a longitudinal cohort study of individuals at high-risk for diabetes. Unlike the SSA study, the study is not focused on protein-energy malnutrition. This study also addresses the issue of diabetes insulin resistance leading to cardiovascular dysmetabolic syndrome.

Subjects: Diseases of the circulatory (Cardiovascular) system, Specialties of internal medicine, Internal medicine, Medicine, Cardiovascular, Medicine (General), Health Sciences
Authors: Deedwania Prakash C
Publisher: BioMed Central Date of publication: 2002 January
Published in: Trials 2002; 3: 1(2)
ISSN(s): 1468-6708
Added to DOAJ: 2004-06-03
http://dx.doi.org/10.1186/1468-6708-3-2
Full text: http://cvm.controlled-trials.com/content/3/1/2

Keywords: cardiovascular dysmetabolic syndrome, coronary heart disease, diabetes mellitus, hyperinsulinemia, insulin resistance

Although diabetes mellitus is predominantly a metabolic disorder, recent data suggest that it is as much a vascular disorder. Cardiovascular complications are the leading cause of death and disability in patients with diabetes mellitus. A number of recent reports have emphasized that many patients already have atherosclerosis in progression by the time they are diagnosed with clinical evidence of diabetes mellitus. The increased risk of atherosclerosis and cardiovascular complications in diabetic patients is related to the frequently associated dyslipidemia, hypertension, hyperglycemia, hyperinsulinemia, and endothelial dysfunction.

The evolving knowledge regarding the variety of metabolic, hormonal, and hemodynamic abnormalities in patients with diabetes mellitus has led to efforts designed for early identification of individuals at risk of subsequent disease. It has been suggested that insulin resistance, the key abnormality in type II diabetes, often precedes clinical features of diabetes by 5–6 years.

Careful attention to the criteria described for the cardiovascular dysmetabolic syndrome should help identify those at risk at an early stage. The application of nonpharmacologic as well as newer emerging pharmacologic therapies can have beneficial effects in individuals with cardiovascular dysmetabolic syndrome and/or diabetes mellitus by improving insulin sensitivity and related abnormalities. Early identification and implementation of appropriate therapeutic strategies would be necessary to contain the emerging new epidemic of cardiovascular disease related to diabetes.

Part 4. Waist circumference a good indicator of future risk for type 2 diabetes and cardiovascular disease

Subjects: Public aspects of medicine, Medicine, Public Health, Health Sciences Authors: Siren Reijo, Eriksson Johan G, Vanhanen Hannu
Publisher: BioMed Central Date of publication: 2012 August
Published in: BMC Public Health 2012; 12: 1(631)
ISSN(s): 1471-2458
Added to DOAJ: 2013-03-12
http://dx.doi.org/10.1186/1471-2458-12-631
http://www.biomedcentral.com/1471-2458/12/631

Keywords: Waist circumference, Type 2 diabetes, Cardiovascular disease, Middle-aged men.

Background: Abdominal obesity is a more important risk factor than overall obesity in predicting the development of type 2 diabetes and cardiovascular disease. From a preventive and public health point of view it is crucial that risk factors are identified at an early stage, in order to change and modify behaviour and lifestyle in high risk individuals. Methods: Data from a community based study was used to assess the risk for type 2 diabetes, cardiovascular disease and prevalence of metabolic syndrome in middle-aged men. In order to identify those with increased risk for type 2 diabetes and/or cardiovascular disease sensitivity and specificity analysis were performed, including calculation of positive and negative predictive values, and corresponding 95% CI for eleven different cut-off points, with 1 cm intervals (92 to 102 cm), for waist circumference. Results: A waist circumference ≥94 cm in middle-aged men, identified those with increased risk for type 2 diabetes and/or for cardiovascular disease with a sensitivity of 84.4% (95% CI 76.4% to 90.0%), and a specificity of 78.2% (95% CI 68.4% to 85.5%). The positive predictive value was 82.9% (95% CI 74.8% to 88.8%), and negative predictive value 80.0%, respectively (95% CI 70.3% to 87.1%). Conclusions: Measurement of waist circumference in middle-aged men is a reliable test to identify individuals at increased risk for type 2 diabetes and cardiovascular disease. This measurement should be used more frequently in daily practice in primary care in order to identify individuals at risk and when planning health counselling and interventions.

Part 5. Chronic obstructive pulmonary disease and glucose metabolism: a bitter sweet symphony

Subjects: Diseases of the circulatory (Cardiovascular) system, Specialties of internal medicine, Internal medicine, Medicine, Cardiovascular, Medicine (General), Health Sciences
Authors: Mirrakhimov Aibek E
Publisher: BioMed Central
Date of publication: Oct 2012
ISSN(s): 1475-2840
ADDED to DOAJ: 2013-03-12
Published in: Cardiovascular Diabetology 2012; 11(1):132
Journal Language(s): English Country of publication: United Kingdom
http://dx.doi.org:/10.1186/1475-2840-11-132
Full text: http://www.cardiab.com/content/11/1/132

Keywords: COPD, Dysglycemia, Insulin resistance, Obesity, Metabolic syndrome, Diabetes mellitus endothelial dysfunction, Vasculopathy

Chronic obstructive pulmonary disease, metabolic syndrome and diabetes mellitus are common and underdiagnosed medical conditions. It was predicted that chronic obstructive pulmonary disease will be the third leading cause of death worldwide by 2020. The healthcare burden of this disease is even greater if we consider the significant impact of chronic obstructive pulmonary disease on the cardiovascular morbidity and mortality.

Chronic obstructive pulmonary disease may be considered as a novel risk factor for new onset type 2 diabetes mellitus via multiple pathophysiological alterations such as: inflammation and oxidative stress, insulin resistance, weight gain and alterations in metabolism of adipokines.

On the other hand, diabetes may act as an independent factor, negatively affecting pulmonary structure and function. Diabetes is associated with an increased risk of pulmonary infections, disease exacerbations and worsened COPD outcomes. On the top of that, coexistent OSA may increase the risk for type 2 DM in some individuals.

The current scientific data necessitate a greater outlook on chronic obstructive pulmonary disease and chronic obstructive pulmonary disease may be viewed as a risk factor for the new onset type 2 diabetes mellitus. Conversely, both types of diabetes mellitus should be viewed as strong contributing factors for the development of obstructive lung disease. Such approach can potentially improve the outcomes and medical control for both conditions, and, thus, decrease the healthcare burden of these major medical problems.

The Economic Costs of Diabetes: Is It Time for a New Treatment Paradigm?

Commentary: William H. Herman
Diabetes Care Apr 2013; 36: 775-776

In a series of rigorous and exhaustive descriptive cost analyses conducted over the past decade, the American Diabetes Association (ADA) has documented an inexorable increase in the cost of diabetes in the U.S. and its detrimental impact on productivity. For the 2012 study, the ADA estimated that there were 22.3 million Americans diagnosed with diabetes. These patients incurred $306 billion in direct medical costs, more than 1 of 5 dollars spent on medical care in the U.S. The direct medical costs attributed to diabetes, that is, the costs of medical care for people with diabetes in excess of those that would be expected in the absence of diabetes, were $176 billion or approximately 1 of 8 dollars spent on medical care in the U.S. Americans with diagnosed diabetes have annual medical expenditures that are $7,900 or approximately 2.3 times higher than they would be in the absence of diabetes ($13,700 vs. $5,800). Americans with diabetes also incur $69 billion in costs related to absenteeism, reduced productivity while at work or at home, diabetes-related disability, and premature mortality. The increasing economic burden of diabetes is due in large part to the increase in the number of people with diagnosed diabetes.

Randomized controlled clinical trials have demonstrated that intensive glycemic management can delay the onset of microvascular, neuropathic, and cardiovascular complications in people with both type 1 and type 2 diabetes, and that the benefits of early intensive treatment persist over time. Randomized controlled clinical trials have also demonstrated that blood pressure management (target blood pressure 135/80 mmHg) and lipid management using statin medications can delay or prevent the development of adverse cardiovascular outcomes.

The growing economic and societal burden of diabetes as documented by the ADA in this issue of Diabetes Care highlights the urgent need to implement interventions to delay the development of type 2 diabetes. Both intensive lifestyle and pharmacologic interventions are proven effective and cost-effective. Health policy should support their implementation.

Complimentary societal interventions to delay the onset of type 2 diabetes include school-based health promotion programs and interventions that address advertising, food availability and price, the built and workplace environment, and even tax policy. In addition, early aggressive management of glycemia and cardiovascular risk factors must be implemented for persons diagnosed with diabetes. Increasing access to care, including self management education and nutritional counseling, and ensuring access to necessary treatments and supplies are critical, especially in light of the proven value of early intensive treatment in preventing chronic complications. The cost estimates provided by the ADA from 2002, 2007, and 2012 show that the economic and societal burden of diabetes is growing in the U.S. This trend underscores the importance of prevention and interventions to mitigate the complications of diabetes.

Insulin regulates carboxypeptidase E by modulating translation initiation scaffolding protein eIF4G1 in pancreatic β cells

Liew, C.W., Assmann, A., Templin, A.T., (…), Urano, F., Kulkarni, R.N
2014 Proc National Academy of Sciences USA 111 (22), pp. E2319-E2328

Insulin resistance, hyperinsulinemia, and hyperproinsulinemia occur early in the pathogenesis of type 2 diabetes (T2D). Elevated levels of proinsulin and proinsulin intermediates are markers of β-cell dysfunction and are strongly associated with development of T2D in humans. However, the mechanism(s) underlying β-cell dysfunction leading to hyperproinsulinemia is poorly understood. Here, we show that disruption of insulin receptor (IR) expression in β cells has a direct impact on the expression of the convertase enzyme carboxypeptidase E (CPE) by inhibition of the eukaryotic translation initiation factor 4 gamma 1 translation initiation complex scaffolding protein that is mediated by the key transcription factors pancreatic and duodenal homeobox 1 and sterol regulatory element-binding protein 1, together leading to poor proinsulin processing. Reexpression of IR or restoring CPE expression each independently reverses the phenotype. Our results reveal the identity of key players that establish a previously unknown link between insulin signaling, translation initiation, and proinsulin processing, and provide previously unidentified mechanistic insight into the development of hyperproinsulinemia in insulin-resistant states.

Disruption of growth factor receptor-binding protein 10 in the pancreas enhances β-cell proliferation and protects mice from streptozotocin-induced β-cell apoptosis

Zhang, J., Zhang, N., Liu, M., (…), Lu, X.-Y., Liu, F.
2014 Environmental Science and Technology 48 (9), pp. 5179-5186

It has been reported that organotin compounds such as triphenyltin or tributyltin (TBT) induce diabetes and insulin resistance. However, histopathological effects of organotin compounds on the Islets of Langerhans and exocrine pancreas are still unclear. In the present study, male KM mice were orally administered with TBT (0.5, 5, and 50 µg/kg) once every 3 days. The fasting plasma glucose levels significantly elevated, and the levels of serum insulin or glucagon decreased in the animals treated with TBT for 60 days. In animals treated for 45 days, the number of apoptotic cells in the islets and exocrine pancreas was elevated in a dose-dependent manner. The percentage of proliferating (PCNA-positive) cells was decreased in the islets, while it was increased in exocrine acinar cells. Immunohistochemistry analysis showed that estrogen receptor (ER) and androgen receptor (AR) were present in vascular endothelium, ductal cells, and islet cells, but absent from pancreatic exocrine cells. TBT exposure decreased the production of estradiol and triiodothyronine and elevated the concentration of testosterone, and resulted in a decrease of ERβ expression and an elevation of AR in the pancreas measured by Western blotting. The results suggested that TBT inhibited the proliferation and induced the apoptosis of islet cells via multipathways, causing a decrease of relative islet area in the animals treated for 60 days, which could result in a disruption of glucose homeostasis. The different presence of ERs and AR between the islets and exocrine pancreas might be one of reasons causing different effects on cell proliferation

Pancreatic alpha-cell dysfunction contributes to the disruption of glucose homeostasis and compensatory insulin hypersecretion in glucocorticoid-treated rats

Rafacho, A., Gonçalves-Neto, L.M., Santos-Silva, J.C., (…), Nadal, A., Quesada, I.
2014 Journal of Biological Chemistry 289 (9), pp. 6028-604

In α-cells, syntaxin (Syn)-1A interacts with SUR1 to inhibit ATP-sensitive potassium channels (KATP channels). PIP2 binds the Kir6.2 subunit to open KATP channels. PIP2 also modifies Syn-1A clustering in plasma membrane (PM) that may alter Syn-1A actions on PM proteins like SUR1. Here, we assessed whether the actions of PIP 2 on activating KATP channels is contributed by sequestering Syn-1A from binding SUR1. In vitro binding showed that PIP 2 dose-dependently disrupted Syn-1A·SUR1 complexes, corroborated by an in vivo Forster resonance energy transfer assay showing disruption of SUR1-(-EGFP)/Syn-1A(-mCherry) interaction along with increased Syn-1A cluster formation. Electrophysiological studies of rat α-cells, INS-1, and SUR1/Kir6.2-expressing HEK293 cells showed that PIP2 dose-dependent activation of KATP currents was uniformly reduced by Syn-1A. To unequivocally distinguish between PIP2 actions on Syn-1A and Kir6.2, we employed several strategies. First, we showed that PIP 2-insensitive Syn-1A-5RK/A mutant complex with SUR1 could not be disrupted by PIP2, consequently reducing PIP2 activation of KATP channels. Next, Syn-1A·SUR1 complex modulation of KATP channels could be observed at a physiologically low PIP 2 concentration that did not disrupt the Syn-1A·SUR1 complex, compared with higher PIP2 concentrations acting directly on Kir6.2. These effects were specific to PIP2 and not observed with physiologic concentrations of other phospholipids. Finally, depleting endogenous PIP 2 with polyphosphoinositide phosphatase synaptojanin-1, known to disperse Syn-1A clusters, freed Syn-1A from Syn-1A clusters to bind SUR1, causing inhibition of KATP channels that could no longer be further inhibited by exogenous Syn-1A. These results taken together indicate that PIP2 affects islet β-cell KATP channels not only by its actions on Kir6.2 but also by sequestering Syn-1A to modulate Syn-1A availability and its interactions with SUR1 on PM.

Aging and sleep deprivation induce the unfolded protein response in the pancreas: Implications for metabolism

Naidoo, N., Davis, J.G., Zhu, J., (…), Agarwal, B., Baur, J.A.
2014 Aging Cell 13 (1), pp. 131-141

Sleep disruption has detrimental effects on glucose metabolism through pathways that remain poorly defined. Although numerous studies have examined the consequences of sleep deprivation (SD) in the brain, few have directly tested its effects on peripheral organs. We examined several tissues in mice for induction of the unfolded protein response (UPR) following acute SD. In young animals, we found a robust induction of BiP in the pancreas, indicating an active UPR. At baseline, pancreata from aged animals exhibited a marked increase in a pro-apoptotic transcription factor, CHOP, that was amplified by SD, whereas BiP induction was not observed, suggesting a maladaptive response to cellular stress with age. Acute SD increased plasma glucose levels in both young and old animals. However, this change was not overtly related to stress in the pancreatic beta cells, as plasma insulin levels were not lower following acute SD. Accordingly, animals subjected to acute SD remained tolerant to a glucose challenge. In a chronic SD experiment, young mice were found to be sensitized to insulin and have improved glycemic control, whereas aged animals became hyperglycemic and failed to maintain appropriate plasma insulin concentrations. Our results show that both age and SD cooperate to induce the UPR in pancreatic tissue. While changes in insulin secretion are unlikely to play a major role in the acute effects of SD, CHOP induction in pancreatic tissues suggests that chronic SD may contribute to the loss or dysfunction of endocrine cells and that these effects may be exacerbated by normal aging

Bayesian total internal reflection fluorescence correlation spectroscopy reveals hIAPP-induced plasma membrane domain organization in live cells

Guo, S.-M., Bag, N., Mishra, A., Wohland, T., Bathe, M.
2014 Biophysical Journal 106 (1), pp. 190-200

Amyloid fibril deposition of human islet amyloid polypeptide (hIAPP) in pancreatic islet cells is implicated in the pathogenesis of type II diabetes. A growing number of studies suggest that small peptide aggregates are cytotoxic via their interaction with the plasma membrane, which leads to membrane permeabilization or disruption. A recent study using imaging total internal reflection-fluorescence correlation spectroscopy (ITIR-FCS) showed that monomeric hIAPP induced the formation of cellular plasma membrane microdomains containing dense lipids, in addition to the modulation of membrane fluidity. However, the spatial organization of microdomains and their temporal evolution were only partially characterized due to limitations in the conventional analysis and interpretation of imaging FCS datasets. Here, we apply a previously developed Bayesian analysis procedure to ITIR-FCS data to resolve hIAPP-induced microdomain spatial organization and temporal dynamics. Our analysis enables the visualization of the temporal evolution of multiple diffusing species in the spatially heterogeneous cell membrane, lending support to the carpet model for the association mode of hIAPP aggregates with the plasma membrane. The presented Bayesian analysis procedure provides an automated and general approach to unbiased model-based interpretation of imaging FCS data, with broad applicability to resolving the heterogeneous spatial-temporal organization of biological membrane systems.

SMAD2 disruption in mouse pancreatic beta cells leads to islet hyperplasia and impaired insulin secretion due to the attenuation of ATP-sensitive K + channel activity

Nomura, M., Zhu, H.-L., Wang, L., (…), Takayanagi, R., Teramoto, N.
2014 Diabetologia 57 (1), pp. 157-166

Aims/hypothesis: The TGF-β superfamily of ligands provides important signals for the development of pancreas islets. However, it is not yet known whether the TGF-β family signalling pathway is required for essential islet functions in the adult pancreas. Methods: To identify distinct roles for the downstream components of the canonical TGF-β signalling pathway, a Cre-loxP system was used to disrupt SMAD2, an intracellular transducer of TGF-β signals, in pancreatic beta cells (i.e. Smad2-β- knockout [KO] mice). The activity of ATP-sensitive K+ channels (KATP channels) was recorded in mutant beta cells using patch-clamp techniques. Results: The Smad2-β-KO mice exhibited defective insulin secretion in response to glucose and overt diabetes. Interestingly, disruption of SMAD2 in β-cells was associated with a striking islet hyperplasia and increased pancreatic insulin content, together with defective glucose-responsive insulin secretion. The activity of KATP channels was decreased in mutant β-cells. Conclusions/interpretation: These results suggest that in the adult pancreas, TGF-β signalling through SMAD2 is crucial for not only the determination of beta cell mass but also the maintenance of defining features of mature pancreatic beta cells, and that this involves modulation of KATP channel activity.

Disruption of protein-tyrosine phosphatase 1B expression in the pancreas affects β-cell function

Liu, S., Xi, Y., Bettaieb, A., (…), Kulkarni, R.N., Haj, F.G.
2014 Endocrinology 155 (9), pp. 3329-3338

Protein-tyrosine phosphatase 1B (PTP1B) is a physiological regulator of glucose homeostasis and energy balance. However, the role of PTP1B in pancreatic endocrine function remains largely unknown. To investigate the metabolic role of pancreatic PTP1B, we generated mice with pancreas PTP1B deletion (panc-PTP1B KO). Mice were fed regular chow or a high-fat diet, and metabolic parameters, insulin secretion and glucose tolerance were determined. On regular chow, panc-PTP1B KO and control mice exhibited comparable glucose tolerance whereas aged panc-PTP1B KO exhibited mild glucose intolerance. Furthermore, high-fat feeding promoted earlier impairment of glucose tolerance and attenuated glucose-stimulated insulin secretion in panc-PTP1B KO mice. The secretory defect in glucose-stimulated insulin secretion was recapitulated in primary islets ex vivo, suggesting that the effects were likely cell-autonomous. At the molecular level, PTP1B deficiency in vivo enhanced basal and glucose-stimulated tyrosyl phosphorylation of EphA5 in islets. Consistently, PTP1B overexpression in the glucose-responsive MIN6 β-cell line attenuated EphA5 tyrosyl phosphorylation, and substrate trapping identified EphA5 as a PTP1B substrate. In summary, these studies identify a novel role forPTP1Bin pancreatic endocrine function.

Fluorescence recovery after photobleaching reveals regulation and distribution of connexin36 gap junction coupling within mouse islets of Langerhans

Farnsworth, N.L., Hemmati, A., Pozzoli, M., Benninger, R.K.P.
2014 Journal of Physiology 592 (20), pp. 4431-4446

Key points: Gap junctions provide electrical coupling that is critical to the function of pancreatic islets. Disruptions to connexin36 (Cx36) have been suggested to occur in diabetes. No accurate and non-invasive technique has yet been established to quantify changes in Cx36 gap junction coupling in the intact islet. This study developed fluorescence recovery after photobleaching (FRAP) as a non-invasive technique for quantifying Cx36 gap junction coupling in living islets. The study identified treatments that modulate gap junction coupling, confirmed that the cellular distribution of coupling throughout the islet is highly heterogeneous and confirmed that β-cells and β-cells do not form functional Cx36 gap junctions. This technique will enable future studies to examine the regulation of Cx36 gap junction coupling and its disruption in diabetes, and to uncover potential novel therapeutic targets associated with gap junction coupling. The pancreatic islets are central to the maintenance of glucose homeostasis through insulin secretion. Glucose-stimulated insulin secretion is tightly linked to electrical activity in β-cells within the islet. Gap junctions, composed of connexin36 (Cx36), form intercellular channels between β-cells, synchronizing electrical activity and insulin secretion. Loss of gap junction coupling leads to altered insulin secretion dynamics and disrupted glucose homeostasis. Gap junction coupling is known to be disrupted in mouse models of pre-diabetes. Although approaches to measure gap junction coupling have been devised, they either lack cell specificity, suitable quantification of coupling or spatial resolution, or are invasive. The purpose of this study was to develop fluorescence recovery after photobleaching (FRAP) as a technique to accurately and robustly measure gap junction coupling in the islet. The cationic dye Rhodamine 123 was used with FRAP to quantify dye diffusion between islet β-cells as a measure of Cx36 gap junction coupling. Measurements in islets with reduced Cx36 verified the accuracy of this technique in distinguishing between distinct levels of gap junction coupling. Analysis of individual cells revealed that the distribution of coupling across the islet is highly heterogeneous. Analysis of several modulators of gap junction coupling revealed glucose- and cAMP-dependent modulation of gap junction coupling in islets. Finally, FRAP was used to determine cell population specific coupling, where no functional gap junction coupling was observed between β-cells and β-cells in the islet. The results of this study show FRAP to be a robust technique which provides the cellular resolution to quantify the distribution and regulation of Cx36 gap junction coupling in specific cell populations within the islet. Future studies utilizing this technique may elucidate the role of gap junction coupling in the progression of diabetes and identify mechanisms of gap junction regulation for potential therapies.

Glucocorticoid treatment and endocrine pancreas function: Implications for glucose homeostasis, insulin resistance and diabetes

Rafacho, A., Ortsäter, H., Nadal, A., Quesada, I.
2014 Journal of Endocrinology 223 (3), pp. R49-R62

Glucocorticoids (GCs) are broadly prescribed for numerous pathological conditions because of their anti-inflammatory, antiallergic and immunosuppressive effects, among other actions. Nevertheless, GCs can produce undesired diabetogenic side effects through interactions with the regulation of glucose homeostasis. Under conditions of excess and/or long-term treatment, GCs can induce peripheral insulin resistance (IR) by impairing insulin signalling, which results in reduced glucose disposal and augmented endogenous glucose production. In addition, GCs can promote abdominal obesity, elevate plasma fatty acids and triglycerides, and suppress osteocalcin synthesis in bone tissue. In response to GC-induced peripheral IR and in an attempt to maintain normoglycaemia, pancreatic β-cells undergo several morphofunctional adaptations that result in hyperinsulinaemia. Failure of β-cells to compensate for this situation favours glucose homeostasis disruption, which can result in hyperglycaemia, particularly in susceptible individuals. GC treatment does not only alter pancreatic β-cell function but also affect them by their actions that can lead to hyperglucagonaemia, further contributing to glucose homeostasis imbalance and hyperglycaemia. In addition, the release of other islet hormones, such as somatostatin, amylin and ghrelin, is also affected by GC administration. These undesired GC actions merit further consideration for the design of improved GC therapies without diabetogenic effects. In summary, in this review, we consider the implication of GC treatment on peripheral IR, islet function and glucose homeostasis.

β-Cell failure in type 2 diabetes: Postulated mechanisms and prospects for prevention and treatment

Halban, P.A., Polonsky, K.S., Bowden, D.W., (…), Sussel, L., Weir, G.C.
2014 Journal of Clinical Endocrinology and Metabolism 99 (6), pp. 1983-1992

OBJECTIVE: This article examines the foundation of β-cell failure in type 2 diabetes (T2D) and suggests areas for future research on the underlying mechanisms that may lead to improved prevention and treatment. RESEARCH DESIGN AND METHODS: A group of experts participated in a conference on 14-16 October 2013 cosponsored by the Endocrine Society and the American Diabetes Association. A writing group prepared this summary and recommendations. RESULTS: The writing group based this article on conference presentations, discussion, and debate. Topics covered include genetic predisposition, foundations of β-cell failure, natural history of β-cell failure, and impact of therapeutic interventions. CONCLUSIONS: β-Cell failure is central to the development and progression of T2D. It antedates and predicts diabetes onset and progression, is in part genetically determined, and often can be identified with accuracy even though current tests are cumbersome and not well standardized. Multiple pathways underlie decreased β-cell function and mass, some of which may be shared and may also be a consequence of processes that initially caused dysfunction. Goals for future research include to 1) impact the natural history of β-cell failure; 2) identify and characterize genetic loci for T2D; 3) target β-cell signaling, metabolic, and genetic pathways to improve function/mass; 4) develop alternative sources of β-cells for cell-based therapy; 5) focus on metabolic environment to provide indirect benefit to β-cells; 6) improve understanding of the physiology of responses to bypass surgery; and 7) identify circulating factors and neuronal circuits underlying the axis of communication between the brain and β-cells.

Metabolic effects of sleep disruption, links to obesity and diabetes

Nedeltcheva, A.V., Scheer, F.A.J.L
2014 Current Opinion in Endocrinology, Diabetes and Obesity 21 (4), pp. 293-298

Purpose of Review: To highlight the adverse metabolic effects of sleep disruption and to open ground for research aimed at preventive measures. This area of research is especially relevant given the increasing prevalence of voluntary sleep curtailment, sleep disorders, diabetes, and obesity. Recent Findings: Epidemiological studies have established an association between decreased self-reported sleep duration and an increased incidence of type 2 diabetes (T2D), obesity, and cardiovascular disease. Experimental laboratory studies have demonstrated that decreasing either the amount or quality of sleep decreases insulin sensitivity and decreases glucose tolerance. Experimental sleep restriction also causes physiological and behavioral changes that promote a positive energy balance. Although sleep restriction increases energy expenditure because of increased wakefulness, it can lead to a disproportionate increase in food intake, decrease in physical activity, and weight gain. SUMMARY: Sleep disruption has detrimental effects on metabolic health. These insights may help in the development of new preventive and therapeutic approaches against obesity and T2D based on increasing the quality and/or quantity of sleep. Video abstract http://links.lww.com/COE/A6.

Impaired proteostasis: Role in the pathogenesis of diabetes mellitus

Jaisson, S., Gillery, P.
2014 Diabetologia 57 (8), pp. 1517-1527

In living organisms, proteins are regularly exposed to ‘molecular ageing’, which corresponds to a set of non-enzymatic modifications that progressively cause irreversible damage to proteins. This phenomenon is greatly amplified under pathological conditions, such as diabetes mellitus. For their survival and optimal functioning, cells have to maintain protein homeostasis, also called ‘proteostasis’. This process acts to maintain a high proportion of functional and undamaged proteins. Different mechanisms are involved in proteostasis, among them degradation systems (the main intracellular proteolytic systems being proteasome and lysosomes), folding systems (including molecular chaperones), and enzymatic mechanisms of protein repair. There is growing evidence that the disruption of proteostasis may constitute a determining event in pathophysiology. The aim of this review is to demonstrate how such a dysregulation may be involved in the pathogenesis of diabetes mellitus and in the onset of its long-term complications.

Influence of miRNA in insulin signaling pathway and insulin resistance: Micro-molecules with a major role in type-2 diabetes

Chakraborty, C., Doss, C.G.P., Bandyopadhyay, S., Agoramoorthy, G.
2014 Wiley Interdisciplinary Reviews: RNA 5 (5), pp. 697-712

The prevalence of type-2 diabetes (T2D) is increasing significantly throughout the globe since the last decade. This heterogeneous and multifactorial disease, also known as insulin resistance, is caused by the disruption of the insulin signaling pathway. In this review, we discuss the existence of various miRNAs involved in regulating the main protein cascades in the insulin signaling pathway that affect insulin resistance. The influence of miRNAs (miR-7, miR-124α, miR-9, miR-96, miR-15α/β, miR-34α, miR-195, miR-376, miR-103, miR-107, and miR-146) in insulin secretion and beta (β) cell development has been well discussed. Here, we highlight the role of miRNAs in different significant protein cascades within the insulin signaling pathway such as miR-320, miR-383, miR-181β with IGF-1, and its receptor (IGF1R); miR-128α, miR-96, miR-126 with insulin receptor substrate (IRS) proteins; miR-29, miR-384-5p, miR-1 with phosphatidylinositol 3-kinase (PI3K); miR-143, miR-145, miR-29, miR-383, miR-33α/β miR-21 with AKT/protein kinase B (PKB) and miR-133α/β, miR-223, miR-143 with glucose transporter 4 (GLUT4). Insulin resistance, obesity, and hyperlipidemia (high lipid levels in the blood) have a strong connection with T2D and several miRNAs influence these clinical outcomes such as miR-143, miR-103, and miR-107, miR-29α, and miR-27β. We also corroborate from previous evidence how these interactions are related to insulin resistance and T2D. The insights highlighted in this review will provide a better understanding on the impact of miRNA in the insulin signaling pathway and insulin resistance-associated diagnostics and therapeutics for T2D

Genetic disruption of sod1 gene causes glucose intolerance and impairs β-cell function

Muscogiuri, G., Salmon, A.B., Aguayo-Mazzucato, C., (…), Van Remmen, H., Musi, N.
2013 Diabetes 62 (12), pp. 4201-4207

Oxidative stress has been associated with insulin resistance and type 2 diabetes. However, it is not clear whether oxidative damage is a cause or a consequence of the metabolic abnormalities present in diabetic subjects. The goal of this study was to determine whether inducing oxidative damage through genetic ablation of superoxide dismutase 1 (SOD1) leads to abnormalities in glucose homeostasis. We studied SOD1-null mice and wild-type (WT) littermates. Glucose tolerance was evaluated with intraperitoneal glucose tolerance tests. Peripheral and hepatic insulin sensitivity was quantitated with the euglycemic-hyperinsulinemic clamp. β-Cell function was determined with the hyperglycemic clamp and morphometric analysis of pancreatic islets. Genetic ablation of SOD1 caused glucose intolerance, which was associated with reduced in vivo β-cell insulin secretion and decreased b-cell volume. Peripheral and hepatic insulin sensitivity were not significantly altered in SOD1-null mice. High-fat diet caused glucose intolerance in WT mice but did not further worsen the glucose intolerance observed in standard chow-fed SOD1-null mice. Our findings suggest that oxidative stress per se does not play a major role in the pathogenesis of insulin resistance and demonstrate that oxidative stress caused by SOD1 ablation leads to glucose intolerance secondary to β-cell dysfunction.

VHL-mediated disruption of Sox9 activity compromises β-cell identity and results in diabetes mellitus

Puri, S., Akiyama, H., Hebrok, M.
2013 Genes and Development 27 (23), pp. 2563-2575

Precise functioning of the pancreatic β cell is paramount to whole-body glucose homeostasis, and β-cell dysfunction contributes significantly to diabetes mellitus. Using transgenic mouse models, we demonstrate that deletion of the von Hippel-Lindau (Vhlh) gene (encoding an E3 ubiquitin ligase implicated in, among other functions, oxygen sensing in pancreatic β cells) is deleterious to canonical β-cell gene expression. This triggers erroneous expression of factors normally active in progenitor cells, including effectors of the Notch, Wnt, and Hedgehog signaling cascades. Significantly, an up-regulation of the transcription factor Sox9, normally excluded from functional β cells, occurs upon deletion of Vhlh. Sox9 plays important roles during pancreas development but does not have a described role in the adult β cell. β-Cell-specific ectopic expression of Sox9 results in diabetes mellitus from similar perturbations in β-cell identity. These findings reveal that assaults on the β cell that impact the differentiation state of the cell have clear implications toward our understanding of diabetes mellitus

Second generation antipsychotic-induced type 2 diabetes: A role for the muscarinic M3 receptor

Weston-Green, K., Huang, X.-F., Deng, C.
2013 CNS Drugs 27 (12), pp. 1069-1080

Second generation antipsychotics (SGAs) are widely prescribed to treat various disorders, most notably schizophrenia and bipolar disorder; however, SGAs can cause abnormal glucose metabolism that can lead to insulin-resistance and type 2 diabetes mellitus side-effects by largely unknown mechanisms. This review explores the potential candidature of the acetylcholine (ACh) muscarinic M3 receptor (M3R) as a prime mechanistic and possible therapeutic target of interest in SGA-induced insulin dysregulation. Studies have identified that SGA binding affinity to the M3R is a predictor of diabetes risk; indeed, olanzapine and clozapine, SGAs with the highest clinical incidence of diabetes side-effects, are potent M3R antagonists. Pancreatic M3Rs regulate the glucose-stimulated cholinergic pathway of insulin secretion; their activation on β-cells stimulates insulin secretion, while M3R blockade decreases insulin secretion. Genetic modification of M3Rs causes robust alterations in insulin levels and glucose tolerance in mice. Olanzapine alters M3R density in discrete nuclei of the hypothalamus and caudal brainstem, regions that regulate glucose homeostasis and insulin secretion through vagal innervation of the pancreas. Furthermore, studies have demonstrated a dynamic sensitivity of hypothalamic and brainstem M3Rs to altered glucometabolic status of the body. Therefore, the M3R is in a prime position to influence glucose homeostasis through direct effects on pancreatic β-cells and by potentially altering signaling in the hypothalamus and brainstem. SGA-induced insulin dysregulation may be partly due to blockade of central and peripheral M3Rs, causing an initial disruption to insulin secretion and glucose homeostasis that can progressively lead to insulin resistance and diabetes during chronic treatment.

Islet amyloid polypeptide toxicity and membrane interactions

Cao, P., Abedini, A., Wang, H., (…), Schmidt, A.M., Raleigh, D.P.
2013 Proc National Academy of Sciences USA 110 (48), pp. 19279-19284

Islet amyloid polypeptide (IAPP) is responsible for amyloid formation in type 2 diabetes and contributes to the failure of islet cell transplants, however the mechanisms of IAPP-induced cytotoxicity are not known. Interactions with model anionic membranes are known to catalyze IAPP amyloid formation in vitro. Human IAPP damages anionic membranes, promoting vesicle leakage, but the features that control IAPP-membrane interactions and the connection with cellular toxicity are not clear. Kinetic studies with wild type IAPP and IAPP mutants demonstrate that membrane leakage is induced by prefibrillar IAPP species and continues over the course of amyloid formation, correlating additional membrane disruption with fibril growth. Analyses of a set of designed mutants reveal that membrane leakage does not require the formation of α-sheet or α-helical structures. A His-18 to Arg substitution enhances leakage, whereas replacement of all of the aromatic residues via a triple leucine mutant has no effect. Biophysical measurements in conjunction with cytotoxicity studies show that nonamyloidogenic rat IAPP is as effective as human IAPP at disrupting standard anionic model membranes under conditions where rat IAPP does not induce cellular toxicity. Similar results are obtained with more complex model membranes, including ternary systems that contain cholesterol and are capable of forming lipid rafts. A designed point mutant, I26P-IAPP; a designed double mutant, G24P, I26P-IAPP; a double N-methylated variant; and pramlintide, a US Food and Drug Administration-approved IAPP variant all induce membrane leakage, but are not cytotoxic, showing that there is no one-to-one relationship between disruption of model membranes and induction of cellular toxicity.

Diabetes and beta cell function: From mechanisms to evaluation and clinical implications

Cernea, S., Dobreanu, M.
2013 Biochemia Medica 23 (3), pp. 266-280

Diabetes is a complex, heterogeneous condition that has beta cell dysfunction at its core. Many factors (e.g. hyperglycemia/glucotoxicity, lipotoxicity, autoimmunity, inflammation, adipokines, islet amyloid, incretins and insulin resistance) influence the function of pancreatic beta cells. Chronic hyperglycemia may result in detrimental effects on insulin synthesis/secretion, cell survival and insulin sensitivity through multiple mechanisms: gradual loss of insulin gene expression and other beta-cell specific genes; chronic endoplasmic reticulum stress and oxidative stress; changes in mitochondrial number, morphology and function; disruption in calcium homeostasis. In the presence of hyperglycemia, prolonged exposure to increased free fatty acids result in accumulation of toxic metabolites in the cells (“lipotoxicity”), finally causing decreased insulin gene expression and impairment of insulin secretion. The rest of the factors/mechanisms which impact on the course of the disease are also discusses in detail. The correct assessment of beta cell function requires a concomitant quantification of insulin secretion and insulin sensitivity, because the two variables are closely interrelated. In order to better understand the fundamental pathogenetic mechanisms that contribute to disease development in a certain individual with diabetes, additional markers could be used, apart from those that evaluate beta cell function. The aim of the paper was to overview the relevant mechanisms/factors that influence beta cell function and to discuss the available methods of its assessment. In addition, clinical considerations are made regarding the therapeutical options that have potential protective effects on beta cell function/mass by targeting various underlying factors and mechanisms with a role in disease progression.

The PACAP-regulated gene selenoprotein T is abundantly expressed in mouse and human β-cells and its targeted inactivation impairs glucose tolerance

Prevost, G., Arabo, A., Jian, L., (…), Pattou, F., Anouar, Y
2013 Endocrinology 154 (10), pp. 3796-3806

Selenoproteins are involved in the regulation of redox status, which affects several cellular processes, including cell survival and homeostasis. Considerable interest has arisen recently concerning the role of selenoproteins in the regulation of glucose metabolism. Here, we found that selenoprotein T (SelT), a new thioredoxin-like protein of the endoplasmic reticulum, is present at high levels in human and mouse pancreas as revealed by immunofluorescence and quantitative PCR. Confocal immunohistochemistry studies revealed that SelT is mostly confined to insulin- and somatostatin-producing cells in mouse and human islets. To elucidate the role of SelT in β-cells, we generated, using a Cre-Lox strategy, a conditional pancreatic β-cell SelT-knockout C57BL/6J mice (SelT-insKO) in which SelT gene disruption is under the control of the rat insulin promoter Cre gene. Glucose administration revealed that male SelT-insKO mice display impaired glucose tolerance. Although insulin sensitivity was not modified in the mutant mice, the ratio of glucose to insulin was significantly higher in the SelT-insKO mice compared with wild-type littermates, pointing to a deficit in insulin production/secretion in mutant mice. In addition, morphometric analysis showed that islets from SelT-insKO mice were smaller and that their number was significantly increased compared with islets from their wild-type littermates. Finally, we found that SelT is up-regulated by pituitary adenylate cyclase-activating polypeptide (PACAP) in β-pancreatic cells and that SelT could act by facilitating a feed-forward mechanism to potentiate insulin secretion induced by the neuropeptide. Our findings are the first to show that the PACAP-regulated SelT is localized in pancreatic α- and β-cells and is involved in the control of glucose homeostasis

SIRT1 deacetylates FOXA2 and is critical for Pdx1 transcription and β-cell formation

Wang, R.-H., Xu, X., Kim, H.-S., Xiao, Z., Deng, C.-X.
2013 International Journal of Biological Sciences 9 (9), pp. 934-946

Pancreas duodenum homeobox 1 (PDX1) is essential for pancreas development and β-cell formation; however more studies are needed to clearly illustrate the precise mechanism regarding spatiotemporal regulation of Pdx1 expression during β-cell formation and development. Here, we demonstrate that SIRT1, FOXA2 and a number of proteins form a protein complex on the promoter of the Pdx1 gene. SIRT1 and PDX1 are expressed in the same set of cells during β-cell differentiation and maturation. Pancreas-specific disruption of SIRT1 diminished PDX1 expression and impaired islet development. Consequently, SIRT1 mutant mice develop progressive hyperglycemia, glucose intolerance, and insulin insufficiency, which directly correlate with the extent of SIRT1 deletion. We further show that SIRT1 interacts with and deacetylates FOXA2 on the promoter of the Pdx1gene, and positively regulates its transcription. These results uncover an essential role of SIRT1 in β-cell formation by maintaining expression of PDX1 and its downstream genes, and identify pancreas-specific SIRT1 mutant mice as a relevant model for studying insulin insufficiency.

NOX, NOX who is there? The contribution of NADPH oxidase one to beta cell dysfunction

Taylor-Fishwick, D.A.
2013 Frontiers in Endocrinology 4 (APR), Article 40

Predictions of diabetes prevalence over the next decades warrant the aggressive discovery of new approaches to stop or reverse loss of functional beta cell mass. Beta cells are recognized to have a relatively high sensitivity to reactive oxygen species (ROS) and become dysfunctional under oxidative stress conditions. New discoveries have identified NADPH oxidases in beta cells as contributors to elevated cellular ROS. Reviewed are recent reports that evidence a role for NADPH oxidase-1 (NOX-1) in β-cell dysfunction. NOX-1 is stimulated by inflammatory cytokines that are elevated in diabetes. First, regulation of cytokine-stimulated NOX-1 expression has been linked to inflammatory lipid mediators derived from 12-lipoxygenase activity. For the first time in beta cells these data integrate distinct pathways associated with beta cell dysfunction. Second, regulation of NOX-1 in
β-cells involves feed-forward control linked to elevated ROS and Src-kinase activation. This potentially results in unbridled ROS generation and identifies candidate targets for pharmacologic intervention. Third, consideration is provided of new, first-in-class, selective inhibitors of NOX-1. These compounds could have an important role in assessing a disruption of NOX-1/ROS signaling as a new approach to preserve and protect beta cell mass in diabetes.

Retinoblastoma tumor suppressor protein in pancreatic progenitors controls α- and β-cell fate

Cai, E.P., Wu, X., Schroer, S.A., (…), Zacksenhaus, E., Woo, M.
2013 Proc National Academy of Sciences USA 110 (36), pp. 14723-14728

Pancreatic endocrine cells expand rapidly during embryogenesis by neogenesis and proliferation, but during adulthood, islet cells have a very slow turnover. Disruption of murine retinoblastoma tumor suppressor protein (Rb) in mature pancreatic β-cells has a limited effect on cell proliferation. Here we show that deletion of Rb during embryogenesis in islet progenitors leads to an increase in the neurogenin 3-expressing precursor cell population, which persists in the postnatal period and is associated with increased β-cell mass in adults. In contrast, Rb-deficient islet precursors, through repression of the cell fate factor aristaless related homeobox, result in decreased β-cell mass. The opposing effect on survival of Rb-deficient β- and β-cells was a result of opposing effects on p53 in these cell types. As a consequence, loss of Rb in islet precursors led to a reduced α- to β-cell ratio, leading to improved glucose homeostasis and protection against diabetes.

Statin therapy and new-onset diabetes: Molecular mechanisms and clinical relevance

Banach, M., Malodobra-Mazur, M., Gluba, A., (…), Rysz, J., Dobrzyn, A.
2013 Current Pharmaceutical Design 19 (27), pp. 4904-4912

Despite positive effects on the plasma lipid profile and vascular events, statin use is associated with various side effects. Among these, statins might cause a disruption of a number of regulatory pathways including insulin signaling. This may affect insulin sensitivity, pancreatic beta-cell function and adipokine secretion. The statin-associated risk of new-onset diabetes (NOD) appears to be a dose-dependent class effect. It still remains unclear whether statin treatment is associated with increased risk of NOD in the general population or if there are groups of individuals at particular risk. However, according to the available data it seems that cardiovascular (CV) benefits in high-risk individuals strongly favor statin therapy since it outweighs other risks. Whether statins should be used for primary prevention among patients with a relatively low baseline CV risk is still questionable, however the results of primary prevention trials have shown reductions in mortality in this population. Thus, there is a need for randomized, placebo-controlled statin studies with carefully selected groups of patients and NOD as a key end point in order to resolve queries concerning this issue.

Basement membrane extract preserves islet viability and activity in vitro by up-regulating α3 integrin and its signal

Miao, G., Zhao, Y., Li, Y., (…), Li, J., Wei, J
2013 Pancreas 42 (6), pp. 971-976

OBJECTIVE: Survival of transplanted islets is limited partly because of the disruption of the islet basement membrane (BM) occurring during isolation. We hypothesized that the embedment of BM extract (BME) could induce a viable cell mass and prolong islet functionality before transplantation. METHODS: A special reconstituted BME that solidifies into a gel at 37 C was used to embed isolated islets in this study. The strategy was used to re-establish the interaction between the islets and peri-islet BM. RESULTS: Islets embedded in BME showed lower caspase-3 levels and higher Akt activity than those in suspension. Moreover, we found for the first time that the expression of β3 integrin and focal adhesion kinase (FAK) and FAK activity was up-regulated in islets after BME embedment. The reverse effect was observed on islet apoptosis when islets rescued from a 24-hour suspension culture were embedded in BME for the next 24 hours. In addition, expression of pancreatic duodenal homeobox factor-1 and phospho-extracellular signal-regulated kinase 1/2 was partially preserved, suggesting the positive effect of BME on islet development. CONCLUSIONS: These results indicate that BME embedment of islets can up-regulate the expression of β3 integrin and its signal transduction, which may improve islet viability.

Involvement of the Clock Gene Rev-erb alpha in the Regulation of Glucagon Secretion in Pancreatic Alpha-Cells

Vieira, E., Marroquí, L., Figueroa, A.C., (…), Gomis, R., Quesada, I.
2013 PLoS ONE 8 (7), e6993

Disruption of pancreatic clock genes impairs pancreatic β-cell function, leading to the onset of diabetes. Despite the importance of pancreatic α-cells in the regulation of glucose homeostasis and in diabetes pathophysiology, nothing is known about the role of clock genes in these cells. Here, we identify the clock gene Rev-erbα as a new intracellular regulator of glucagon secretion. Rev-erbα down-regulation by siRNA (60-70% inhibition) in alphaTC1-9 cells inhibited low-glucose induced glucagon secretion (p<0.05) and led to a decrease in key genes of the exocytotic machinery. The Rev-erbα agonist GSK4112 increased glucagon secretion (1.6 fold) and intracellular calcium signals in αTC1-9 cells and mouse primary alpha-cells, whereas the Rev-erbα antagonist SR8278 produced the opposite effect. At 0.5 mM glucose, alphaTC1-9 cells exhibited intrinsic circadian Rev-erbα expression oscillations that were inhibited by 11 mM glucose. In mouse primary alpha-cells, glucose induced similar effects (p<0.001). High glucose inhibited key genes controlled by AMPK such as Nampt, Sirt1 and PGC-1 alpha in alphaTC1-9 cells (p<0.05). AMPK activation by metformin completely reversed the inhibitory effect of glucose on Nampt-Sirt1-PGC-1 alpha and Rev-erb alpha. Nampt inhibition decreased Sirt1, PGC-1 alpha and Rev-erb alpha mRNA expression (p<0.01) and glucagon release (p<0.05). These findings identify Rev-erb alpha as a new intracellular regulator of glucagon secretion via AMPK/Nampt/Sirt1 pathway.

Bmal1 and β-cell clock are required for adaptation to circadian disruption, and their loss of function leads to oxidative stress- induced β-cell failure in mice

Lee, J., Moulik, M., Fang, Z., (…), Moore, D.D., Yechoor, V.K.
2013 Molecular and Cellular Biology 33 (11), pp. 2327-2338

Circadian disruption has deleterious effects on metabolism. Global deletion of Bmal1, a core clock gene, results in β-cell dysfunction and diabetes. But it is unknown if this is due to loss of cell-autonomous function of Bmal1 in β cells. To address this, we generated mice with β-cell clock disruption by deleting Bmal1 in β cells (β-Bmal1-/-). β-Bmal1-/- mice develop diabetes due to loss of glucose-stimulated insulin secretion (GSIS). This loss of GSIS is due to the accumulation of reactive oxygen species (ROS) and consequent mitochondrial uncoupling, as it is fully rescued by scavenging of the ROS or by inhibition of uncoupling protein 2. The expression of the master antioxidant regulatory factor Nrf2 (nuclear factor erythroid 2-related factor 2) and its targets, Sesn2, Prdx3, Gclc, and Gclm, was decreased in β-Bmal1-/- islets, which may contribute to the observed increase in ROS accumulation. In addition, by chromatin immunoprecipitation experiments, we show that Nrf2 is a direct transcriptional target of Bmal1. Interestingly, simulation of shift work-induced circadian misalignment in mice recapitulates many of the defects seen in Bmal1-deficient islets.

Thus, the cell-autonomous function of Bmal1 is required for normal β-cell function by mitigating oxidative stress and serves to preserve β-cell function in the face of circadian misalignment.

A common landscape for membraneactive peptides

Last, N.B., Schlamadinger, D.E., Miranker, A.D.
2013 Protein Science 22 (7), pp. 870-882

Three families of membrane-active peptides are commonly found in nature and are classified according to their initial apparent activity. Antimicrobial peptides are ancient components of the innate immune system and typically act by disruption of microbial membranes leading to cell death. Amyloid peptides contribute to the pathology of diverse diseases from Alzheimer’s to type II diabetes. Preamyloid states of these peptides can act as toxins by binding to and permeabilizing cellular membranes. Cell-penetrating peptides are natural or engineered short sequences that can spontaneously translocate across a membrane. Despite these differences in classification, many similarities in sequence, structure, and activity suggest that peptides from all three classes act through a small, common set of physical principles. Namely, these peptides alter the Brownian properties of phospholipid bilayers, enhancing the sampling of intrinsic fluctuations that include membrane defects. A complete energy landscape for such systems can be described by the innate membrane properties, differential partition, and the associated kinetics of peptides dividing between surface and defect regions of the bilayer. The goal of this review is to argue that the activities of these membrane-active families of peptides simply represent different facets of what is a shared energy landscape.

Membrane disordering is not sufficient for membrane permeabilization by islet amyloid polypeptide: Studies of IAPP(20-29) fragments

Brender, J.R., Heyl, D.L., Samisetti, S., (…), Pesaru, R.R., Ramamoorthy, A.
2013 Physical Chemistry Chemical Physics 15 (23), pp. 8908-8915

A key factor in the development of type II diabetes is the loss of insulin-producing beta-cells. Human islet amyloid polypeptide protein (human-IAPP) is believed to play a crucial role in this process by forming small aggregates that exhibit toxicity by disrupting the cell membrane. The actual mechanism of membrane disruption is complex and appears to involve an early component before fiber formation and a later component associated with fiber formation on the membrane. By comparing the peptide-lipid interactions derived from solid-state NMR experiments of two IAPP fragments that cause membrane disordering to IAPP derived peptides known to cause significant early membrane permeabilization, we show here that membrane disordering is not likely to be sufficient by itself to cause the early membrane permeabilization observed by IAPP, and may play a lesser role in IAPP membrane disruption than expected.
Downregulation of Fas activity rescues early onset of diabetes in c-KitWv/+ mice

Feng, Z.-C., Riopel, M., Li, J., Donnelly, L., Wang, R.
2013 American Journal of Physiology – Endocrinology and Metabolism 304 (6), pp. E557-E565

c-Kit and its ligand stem cell factor (SCF) are important for β-cell survival and maturation; meanwhile, interactions between the Fas receptor (Fas) and Fas ligand are capable of triggering β-cell apoptosis. Disruption of c-Kit signaling leads to severe loss of β-cell mass and function with upregulation of Fas expression in c-KitWv/++ mouse islets, suggesting that there is a critical balance between c-Kit and Fas activation in β-cells. In the present study, we investigated the interrelationship between c-Kit and Fas activation that mediates β-cell survival and function. We generated double mutant, c-KitWv/++;Faslpr/lpr (Wv-/-), mice to study the physiological and functional role of Fas with respect to β-cell function in c-KitWv/++ mice. Isolated islets from these mice and the INS-1 cell line were used. We observed that islets in c-KitWv/++ mice showed a significant increase in β-cell apoptosis along with upregulated p53 and Fas expression. These results were verified in vitro in INS-1 cells treated with SCF or c-Kit siRNA combined with a p53 inhibitor and Fas siRNA. In vivo, Wv-/- mice displayed improved β-cell function, with significantly enhanced insulin secretion and increased β-cell mass and proliferation compared with Wv+/+ mice. This improvement was associated with downregulation of the Fas-mediated caspase-dependent apoptotic pathway and upregulation of the cFlip/NF-?B pathway. These findings demonstrate that a balance between the c-Kit and Fas signaling pathways is critical in the regulation of β-cell survival and function.
Study Suggests Genetic Susceptibility to T2D May Have Shifted with Human Migration

May 24, 2013 By a GenomeWeb staff reporter

NEW YORK (GenomeWeb News) – The apparent genetic risk for type 2 diabetes seems to vary between human populations from different parts of the world, new research suggests, with populations in Africa and East Asia showing particularly pronounced differences in T2D susceptibility.

A pair of papers appearing online — both led by investigators at Stanford University — outline the approaches and analyses used to reach that conclusion.

For the first study, published in PLOS Genetics, researchers trolled through data on more than 1,000 individuals from around the world who’d been genotyped for the Human Genome Diversity Panel project. Patterns in that data revealed geography or population-related differentiation in the genetic risk associated with certain diseases.

“We demonstrated that differences in genetic risk for multiple diseases go well beyond what is expected by genetic drift,” the study authors noted. “In addition, using a human population phylogenetic tree allowed us to elucidate a substructure of worldwide relationships.”

In the East Asian population, for instance, the team saw diminished genetic risk for both T2D and pancreatic cancer. On the other hand, individuals of African ancestry appeared to be more apt to carry T2D risk alleles, results of the analysis suggest, pointing to possible migration-related shifts in genetic susceptibility to T2D.

For their PLOS Genetics analysis, the researchers used data for 1,043 individuals genotyped for the HGDP to delve into the genetic risk associated with more than 100 diseases, including T2D.

Because the individuals hailed from 51 different populations around the world, the group was able to get a glimpse at relationships between these genetic risk contributors and human migration and population patterns.

From that data, investigators saw at least 11 conditions for which risk variant profiles differed across human populations, researchers reported, including ulcerative colitis, bladder cancer, lupus, and inflammatory bowel disease.

For T2D, that genetic differentiation appeared to correspond with population patterns stemming from human migrations out of Africa and into other parts of the world. For instance, the analysis indicated that genetic risk for T2D dips in East Asian populations but tends to be elevated in populations from Africa — particularly the Mandinka population, which appeared to be at highest genetic risk of T2D.

“East Asians definitely get diabetes,” Stanford University’s Atul Butte, senior author on the study, said in a statement.

Nevertheless, he added, it’s possible that there are population-specific differences in the risk alleles and genetic pathways involved, potentially producing somewhat distinct forms of the disease.

Those involved in the study noted that additional, follow-up research is needed, including whole-genome sequencing analysis, which can offer a look at larger structural variants contributing to disease risk in different populations, for instance.

But if findings from the current analysis hold in future studies, that may ultimately prompt a shift in researchers’ understanding of T2D and the factors contributing to it.

“Other fields of medicine have undergone a radical rethinking in disease taxonomy,” Butte said in a statement, “but this has not happened yet for diabetes, one of the world’s public health menaces.”

“If these are separate diseases at a molecular level, we need to try to understand that,” he added.

A related study in the journal Diabetes Care, also by Stanford’s Butte and his colleagues, touched on the consequences of such genetic differences. That work highlighted apparent clinical differences in T2D-related traits — particularly in insulin resistance and insulin response — in African, East Asian, and Caucasian populations.

More generally, Butte and his colleagues put together a so-called “Genetic Risk World Map” to tie together the information generated from their study of disease risk genetics in the context of human migration. The resource is available online through a Stanford website.
Use of pioglitazone in the treatment of diabetes: effect on cardiovascular risk

Authors: Zou C, Hu H
Published Date: 25 July 2013; 9: 429 – 433
DOI: http://dx.doi.org/10.2147/VHRM.S34421

Pioglitazone and other thiazolidinediones (TZDs) initially showed great promise as unique receptor-mediated oral therapy for type 2 diabetes, but a host of serious side effects, primarily cardiovascular, have limited their utility. It is crucial at this point to perform a risk–benefit analysis to determine what role pioglitazone should play in our current treatment of type 2 diabetes and where the future of this class of drugs is headed. This review provides a comprehensive overview of the present literature. Clinical data currently available indicate that pioglitazone is an effective and generally well-tolerated treatment option for use in patients with type 2 diabetes. Pioglitazone can still reduce adverse cardiovascular risk.

Glucophage, Glucophage XR

In a US double-blind clinical study of GLUCOPHAGE in patients with type 2 diabetes, a total of 141 patients received GLUCOPHAGE therapy (up to 2550 mg per day) and 145 patients received placebo. Adverse reactions reported in greater than 5% of the GLUCOPHAGE patients, and that were more common in GLUCOPHAGE- than placebo-treated patients are reported.

The following adverse reactions were reported in ≥ 1.0% to ≤ 5.0% of GLUCOPHAGE patients and were more commonly reported with GLUCOPHAGE than placebo:

abnormal stools, – myalgia, – lightheaded, – dyspnea,

the following adverse reactions were reported in ≥ 1.0% to ≤ 5.0% of GLUCOPHAGE XR patients and were more commonly reported with GLUCOPHAGE XR than placebo

dizziness, – More common

Metabolic side effects have included lactic acidosis, which is a potentially fatal metabolic complication. The incidence of lactic acidosis has been about 1.5 cases per 10,000 patient years. The risk of lactic acidosis has been particularly high in patients with underlying renal insufficiency. Cases of lactic acidosis occurring in patients with normal renal function have been rarely reported.

Signs and symptoms of severe acidosis may include bradycardia (lactic acidosis)
lactic acid concentration, serum electrolytes, blood pH

High-Fructose Corn Syrup Linked to Diabetes

By Brenda Goodman, MA WebMD Health News
Reviewed By Louise Chang, MD

In a study published in the journal Global Health, researchers compared the average availability of high-fructose corn syrup to rates of diabetes in 43 countries.

About half the countries in the study had little or no high-fructose corn syrup in their food supply. In the other 20 countries, high-fructose corn syrup in foods ranged from about a pound a year per person in Germany to about 55 pounds each year per person in the United States.

The researchers found that countries using high-fructose corn syrup had rates of diabetes that were about 20% higher than countries that didn’t mix the sweetener into foods. Those differences remained even after researchers took into account data for differences in body size, population, and wealth.

But couldn’t that mean that people in countries that used more high-fructose corn syrup were just eating more sugar or more total calories?

The researchers say no: There were no overall differences in total sugars or total calories between countries that did and didn’t use high-fructose corn syrup, suggesting that there’s an independent relationship between high-fructose corn syrup and diabetes.

“It raises a lot of questions about fructose,” says researcher Michael I. Goran, PhD, co-director of the Diabetes and Obesity Research Institute at the Keck School of Medicine at the University of Southern California, in Los Angeles. Although the study found an association, it doesn’t establish a cause/effect relationship.
Genetic association of ADIPOQ gene variants with type 2 diabetes, obesity and serum adiponectin levels in south Indian population.

Ramya K; Ayyappa KA; Ghosh S; Mohan V; Radha V
Gene 2013 Dec 15;532(2):253-62 (ISSN: 1879-0038)

OBJECTIVE: To investigate the genetic association of eight variants of the adiponectin gene with type 2 diabetes mellitus (T2DM), obesity and serum adiponectin level in the south Indian population. METHODS: The study comprised of 1100 normal glucose tolerant (NGT) and 1100 type 2 diabetic, unrelated subjects randomly selected from the Chennai Urban Rural Epidemiology Study (CURES), in southern India. Fasting serum adiponectin
levels were measured by radioimmunoassay. The variants were screened by polymerase chain reaction-restriction fragment length polymorphism. Linkage disequilibrium was estimated from the estimates of haplotype frequencies. RESULTS: Of the 8 variants, four SNPs namely, +276 G/T (rs1501299), -4522 C/T (rs822393), -11365 C/G (rs266729), and +712 G/A (rs3774261) were significantly associated with T2DM in our study population. The -3971 A/G (rs822396) and -11391 G/A (rs17300539) SNPs’ association with T2DM diabetes was mediated through obesity (where the association with type 2 diabetes was lost after adjusting for BMI). There was an independent
association of +276 G/T (rs1501299) and -3971 A/G (rs822396) SNPs with generalized obesity and +349 A/G (rs2241767) with central obesity. Four SNPs, -3971 A/G (rs822396), +276 G/T (rs1501299), -4522 C/T (rs822393) and Y111H T/C (rs17366743) were significantly associated with hypoadiponectinemia. The haplotypes GCCATGAAT and AGCGTGGGT conferred lower risk of T2DM in this south Indian population. CONCLUSION: The adiponectin gene variants and haplotype contribute to the genetic risk towards the development of type 2 diabetes, obesity and hypoadiponectinemia in the south Indianpopulation. [ 2013.].

Association of family history of type 2 diabetes mellitus with markers of endothelial dysfunction in South Indian population.

Dhananjayan R; Malati T; Brindha G; Kutala VK
Indian J Biochem Biophys 2013 Apr;50(2):93-8 (ISSN: 0301-1208)

Studies indicate that risk for type 2 diabetes mellitus (T2D) or cardiovascular disease is detectable in childhood, though these disorders may not emerge until adulthood. This study was aimed to assess the markers of endothelial dysfunction in patients with the family history of T2D from South Indian population. A total of 450 subjects were included in the study comprising Group I (n = 200) of T2D, Group II (n = 200) of age- and sex-matched healthy controls, Group III (n = 25) of children of T2D patients and Group IV (n = 25) of children of healthy controls. Results showed that intimal medial thickening (IMT) was significantly higher in T2D patients, compared with control subjects with no family history of diabetes. The fasting plasma glucose, glycated hemoglobin, serum total cholesterol, triglyceride, LDL-cholesterol, apolipoprotein B (ApoB) and high-sensitive C-reactive protein (hsCRP) levels were significantly increased, whereas HDL-cholesterol and serum nitrite levels were significantly decreased in T2D patients. However, children of T2D patients who were not diabetic did not show significant increase in the IMT, as compared to those of healthy controls. In conclusion, the present study demonstrate that IMT was significantly higher in the T2D patients and increased with age and family history. The increased levels of lipids, hsCRP, IMT and decreased nitrite levels might contribute to the risk of endothelial dysfunction in patients with T2D. However, further studies are warranted with other biomarkers of endothelial dysfunction in T2D patients with increased sample size.

Hemoglobin A1c variability as an independent correlate of cardiovascular disease in patients with type 2 diabetes: a cross-sectional analysis of the renal insufficiency and cardiovascular events (RIACE) Italian multicenter study.

Penno G; Solini A; Zoppini G; Orsi E; Fondelli C; Zerbini G; Morano S; and
Renal Insufficiency and Cardiovascular Events (RIACE) Study Group.
Cardiovasc Diabetol 2013;12:98 (ISSN: 1475-2840)

BACKGROUND: Previous reports have clearly indicated a significant relationship between hemoglobin (Hb) A1c change from one visit to the next and microvascular complications, especially nephropathy (albuminuria and albuminuric chronic kidney disease, CKD). In contrast, data on macrovascular disease are less clear. This study was aimed at examining the association of HbA1c variability with cardiovascular disease (CVD) in the large cohort of subjects with type 2 diabetes from the Renal Insufficiency and Cardiovascular Events (RIACE) Italian Multicenter Study. METHODS: Serial (3-5) HbA1c values obtained during the 2-year period preceding recruitment, including that obtained at the enrolment, were available from 8,290 subjects from 9 centers (out of 15,773 patients from 19 centers). Average HbA1c and HbA1c variability were calculated as the intra-individual mean (HbA1c-MEAN) and standard deviation (HbA1c-SD), respectively, of 4.52 0.76 values. Prevalent CVD, total and by vascular bed, was assessed from medical history by recording previous documented major acute events. Diabetic retinopathy (DR) was assessed by dilated fundoscopy. CKD was defined based on albuminuria, as measured by immunonephelometry or immunoturbidimetry, and estimated glomerular filtration rate, as calculated from serum creatinine. RESULTS: HbA1c-MEAN, but not HbA1c-SD, was significantly higher (P <0.0001) in subjects with history of any CVD (n. 2,133, 25.7%) than in those without CVD (n. 6,157, 74.3%). Median and interquartile range were 7.78 (7.04-8.56) and 7.49 (6.81-8.31), respectively, for HbA1c-MEAN, and 0.47 (0.29-0.75) and 0.46 (0.28-0.73), respectively, for HbA1c-SD. Logistic regression analyses showed that HbA1c-MEAN, but not HbA1c-SD (and independent of it), was a significant correlate of any CVD. Similar findings were observed in subjects with versus those without any coronary or cerebrovascular event or myocardial infarction. Conversely, none of these measures were associated with stroke, whereas both correlated with any lower limb vascular event and HbA1c-SD alone with ulceration/gangrene. All these associations were independent of known CVD risk factors and microvascular complications (DR and CKD). CONCLUSIONS: In patients with type 2 diabetes, HbA1c variability has not a major impact on macrovascular complications, at variance with average HbA1c, an opposite finding as compared with microvascular disease, and particularly nephropathy. TRIAL REGISTRATION: ClinicalTrials.Gov NCT00715481.

Genetic association of adiponectin gene polymorphisms (+45T/G and +10211T/G) with type 2 diabetes in North Indians.

Saxena M; Srivastava N; Banerjee M
Diabetes Metab Syndr 2012 Apr-Jun;6(2):65-9 (ISSN: 1878-0334)

Adiponectin (ADIPOQ) is an abundant protein hormone which belongs to a family of so-called adipokines. It is expressed mostly by adipocytes and is an important regulator of lipid and glucose metabolism. It was shown that decreased serum adiponectin concentration indicated insulin resistance and type 2 diabetes (T2DM) with the risk of cardiovascular complications. The fact that adiponectin is an insulin-sensitizing hormone with anti-diabetic, anti-inflammatory and anti-atherogenic properties, we proposed to study the association of ADIPOQ gene polymorphisms in subjects with T2DM. DNA was isolated from venous blood samples, quantified and subjected to Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) using suitable primers and restriction endonucleases. Adiponectin levels were measured in serum using ELISA. The genotypic, allelic and carriage rate frequencies distribution in patients and controls were analyzed by PSAW software (ver. 17.0). Odd ratios (OR) with 95% confidence interval (CI) were determined to describe the strength of association by logistic regression model. Out of the two polymorphisms studied, +10211T/G showed significant association (P=0.042), the ‘G’ allele association being highly significant (P=0.022). Further analysis showed that individuals with ‘GG’ haplotype were at increased risk of T2DM up to 15.5 times [P=0.015, OR (95% CI); 15.558 (1.690-143.174)]. The present study showed that the ‘G’ allele of ADIPOQ gene (+10211T/G) plays a prominent role with respect to T2DM susceptibility in North-Indian population. [Copyright 2012 Diabetes India. Published by Elsevier Ltd. All rights reserved.].

Association of RAGE gene polymorphism with vascular complications in Indian type 2 diabetes mellitus patients [In Process Citation]

Tripathi AK; Chawla D; Bansal S; Banerjee BD; Madhu SV; Kalra OP
Diabetes Res Clin Pract 2014 Mar;103(3):474-81 (ISSN: 1872-8227)

AIMS: The study was designed to evaluate the association of -374T/A and -429T/C polymorphism in the promoter region and Gly82Ser polymorphism in exon 3 region of RAGE gene with diabetic vascular complications in Indian population. METHODS: We screened 603 subjects which includes 176 healthy controls, 140 type 2 diabetes mellitus (T2DM) subjects without any vascular complications (DM), 152 T2DM subjects with microvascular complications (DM-micro) and 135 T2DM subjects with macrovascular complications (DM-macro) for -374T/A, -429T/C and Gly82Ser polymorphisms of RAGE gene. DNA isolated from the enrolled subjects were genotyped by PCR-RFLP. Logistic regression analysis was used to evaluate the association of single nucleotide polymorphisms (SNPs). RESULTS: The -429 T/C and Gly82Ser RAGE polymorphisms were found to be significantly associated with the development of macrovascular and microvascular complications, respectively, in T2DM subjects while -374A allele showed reduced risk towards the development of macrovascular complications. Further, -429T/C, -374T/A and Gly82Ser haplotype analysis revealed association of CTG haplotype with development of macrovascular complications while haplotype TAG was observed to be significantly protective towards development of macrovascular complications in T2DM subjects (OR=0.617, p=0.0202). CONCLUSIONS: Our data indicates significant association of RAGE SNPs and haplotypes with vascular complications in North Indian T2DM subjects.
Clinical profile and complications of childhood- and adolescent-onset type 2 diabetes seen at a diabetes center in south India.

Amutha A; Datta M; Unnikrishnan R; Anjana RM; Mohan V
Diabetes Technol Ther 2012 Jun;14(6):497-504 (ISSN: 1557-8593)

OBJECTIVE: This study describes the clinical characteristics of childhood- and adolescent-onset type 2 diabetes mellitus (CAT2DM) seen at a diabetes center in southern India. RESEARCH DESIGN AND METHODS: Between January 1992 and December 2009, 368 CAT2DM patients were registered. Anthropometric measurements were done using standardized techniques. Biochemical investigations included C-peptide measurements and glutamic acid decarboxylase antibody assay wherever feasible. Retinopathy was diagnosed by retinal photography; microalbuminuria, if urinary albumin excretion was between 30 and 299vmg/1/4g of creatinine; nephropathy, if urinary albumin excretion was (yen)300vmg/1/4g; and neuropathy, if vibration perception threshold on biothesiometry was (yen)20vV. RESULTS: The proportion of CAT2DM patients, expressed as percentage of total patients registered at our center, rose from 0.01% in 1992 to 0.35% in 2009 (P <0.001). Among the 368 cases of CAT2DM, 96 (26%) were diagnosed before the age of 15 years. The mean age at first visit and age at diagnosis of the CAT2DM subjects were 22.29.7 and 16.12.5 years, respectively. Using World Health Organization growth reference charts, 56% of boys and 50.4% of girls were > 85(th) percentile of body mass index for age. Prevalence rates of retinopathy, microalbuminuria, nephropathy, and neuropathy were 26.7%, 14.7%, 8.4%, and 14.2%, respectively. Regression analysis revealed female gender, body mass index > 85(th) percentile, parental history of diabetes, serum cholesterol, and blood pressure to be associated with earlier age at onset of CAT2DM. CONCLUSIONS: CAT2DM appears to be increasing in urban India, and the prevalence of microvascular complications is high. Female predominance is seen at younger ages.

Variants of the adiponectin gene and diabetic microvascular complications in patients with type 2 diabetes.

Choe EY; Wang HJ; Kwon O; Kim KJ; Kim BS; Lee BW; Ahn CW; et al.
Metabolism 2013 May;62(5):677-85 (ISSN: 1532-8600)

OBJECTIVE: The aim of this study was to examine the association between common polymorphisms of the adiponectin gene (ADIPOQ) and microvascular complications in patients with type 2 diabetes mellitus (T2DM). RESEARCH DESIGN AND METHODS: Rs2241766 and rs1501299 of ADIPOQ were genotyped in 708 patients with T2DM. Fundus photography, nerve conducting velocity, and urine analysis were performed to check for the presence of microvascular complications including diabetic nephropathy, retinopathy and neuropathy. RESULTS: The prevalence of diabetic nephropathy tended to be different according to rs2241766 genotype (p=0.057) and the GG genotype of rs2241766 was associated with diabetic nephropathy [urine albumin/creatinine ratio (UACR) greater than 30 mg/g] after adjusting for age, sex, body mass index, duration of diabetes, HDL-cholesterol, smoking status, and blood pressure (odds ratio=1.96; 95% confidence interval=1.01-3.82, p=0.049). Also, the G allele of rs2241766 demonstrated a trend to be associated with an increase in UACR (p=0.087). Rs2241766 genotype was not associated with diabetic retinopathy (p=0.955) and neuropathy (p=0.104) or any diabetic microvascular complications (p=0.104). There was no significant association between the rs1501299 genotype of ADIPOQ and the prevalence of diabetic retinopathy and neuropathy or any diabetic microvascular complications even after adjustment. CONCLUSION: These data suggest that the GG genotype at rs2241766 is implicated in the pathogenesis of risk for diabetic nephropathy defined as UACR greater than 30 mg/day in patients with T2DM. [Copyright 2013 Elsevier Inc. All rights reserved.].

The prevalence of presarcopenia in Asian Indian individuals with and without type 2 diabetes.

Anbalagan VP; Venkataraman V; Pradeepa R; Deepa M; Anjana RM; Mohan V
Diabetes Technol Ther 2013 Sep;15(9):768-75 (ISSN: 1557-8593)

OBJECTIVE: This study compared the skeletal muscle mass and prevalence of presarcopenia between Asian Indian individuals with and without type 2 diabetes. SUBJECTS AND METHODS: Participants with type 2 diabetes (n=76) and age- and sex-matched controls without diabetes (n=76) were drawn from the Chennai Urban Rural Epidemiological Study (CURES), which was carried out on a representative sample of Chennai City in South India. Skeletal muscle mass was estimated by dual-energy X-ray absorptiometry, and skeletal muscle mass index (SMI) was calculated by dividing the appendicular skeletal muscle mass by the square of the individual’s height in meters and expressed as kg/m. Presarcopenia was defined as an SMI of 7.26 kg/m² for males and 5.5 kg/m² for females. Biochemical and anthropometric measurements were done using standardized procedures. RESULTS: The 152 participants included 68 women (44.7%). Mean age was 449 years (range, 28-67 years), and the mean body mass index (BMI) was 25.73.8 kg/m². The prevalence rates of presarcopenia among individuals with and without diabetes were 39.5% and 15.8%, respectively (P=0.001). The mean SMI values were significantly lower in those with diabetes (6.841.02 kg/m² compared with participants without diabetes (7.281.01 kg/m²) (P=0.009). SMI showed a positive correlation with BMI and waist circumference but a negative correlation with age, fasting plasma glucose, glycated hemoglobin, and low-density lipoprotien cholesterol in the total study population. Logistic regression analysis showed that diabetes was independently associated with presarcopenia (P=0.001). CONCLUSIONS: Prevalence of presarcopenia is higher among Asian Indian subjects with type 2 diabetes compared with age- and sex-matched participants without diabetes.

Increased risk of type 2 diabetes with ascending social class in urban South Indians is explained by obesity: The Chennai urban rural epidemiology study (CURES-116).

Skar M; Villumsen AB; Christensen DL; Petersen JH; Deepa M; Anjana RM; et al.
Indian J Endocrinol Metab 2013 Nov;17(6):1084-9 (ISSN: 2230-8210)

AIM: The aim of this study is to determine the factors responsible for differences in the prevalence of diabetes mellitus (DM) in subjects of different social class in an urban South Indian population. MATERIALS AND METHODS: Analyses were based on the cross-sectional data from the Chennai Urban Rural Epidemiology Study of 1989 individuals, aged (yen)20 years. Entered in the analyses were information obtained by self-report on (1) household income; (2) family history of diabetes; (3) physical activity; (4) smoking status; (5) alcohol consumption. Biochemical, clinical and anthropometrical measurements were performed and included in the analyses. Social class was classified based on income as low (Rs. <2000) intermediate (Rs. 2000-5000`) and high (Rs. 5000-20000). RESULTS: The prevalence rates of DM were 12.0%, 18.4% and 21.7% in low, intermediate and high social class, respectively (P < 0.001). A significant increase in the risk of diabetes was found with ascending social class (Intermediate class: Odds ratio [OR], 1.7 [confidence interval [CI], 1.2-2.3]; High class: OR, 2.0 [CI-1.4-2.9]). The multivariable adjusted logistic regression analysis revealed that the effect of social class on the risk of diabetes remained significant (P = 0.016) when age, family history of diabetesand blood pressure were included. However, with the inclusion of abdominal obesity in the model, the significant effect of social class disappeared (P = 0.087). CONCLUSION: An increased prevalence of DM was found in the higher social class in this urban South Indian population, which is explained by obesity.

Prevalence of inflammatory markers (high-sensitivity C-reactive protein, nuclear factor-(ordM)B, and adiponectin) in Indian patients with type 2 diabetes mellitus with and without macrovascular complications.

Misra DP; Das S; Sahu PK
Metab Syndr Relat Disord 2012 Jun;10(3):209-13 (ISSN: 1557-8518)

BACKGROUND: Atherosclerosis is more prevalent in subjects with diabetes mellitus. Recent evidence suggests that diabetic atherosclerosis is not simply a disease of hyperlipidemia, but is also an inflammatory disorder. Our aim was to study the prevalence of inflammatory markers such as high-sensitivity C-reactive protein (hsCRP), adiponectin, and nuclear factor-(ordM)B (NF-(ordM)B) expression, in peripheral blood mononuclear cells in Indian patients with type 2 diabetes mellitus (T2DM) with and without macrovascular disease (MVD). METHODS: A total of 29 consecutive cases of T2DM with proven MVD (group A), 28 matched cases without MVD (group B), and 14 healthy controls (group C) were evaluated for the clinical parameters fasting blood glucose (FBG), 2-h postprandial blood glucose (PPBG), glycosylated hemoglobin (HbA1c), lipid profile, and the above-mentioned inflammatory markers. RESULTS: Diabetic subjects with T2DM had higher hsCRP and NF-(ordM)B expression and lower values of adiponectin compared to healthy controls. Group A had significantly higher serum hsCRP than group B (P=0.0001) despite comparable values of BMI, FBG, 2-h PPBG, HbA1c, and lipid parameters. Group A had significantly higher serum hsCRP and NF-(ordM)B expression and significantly lower levels of adiponectin than group C (P=0.0001, 0.007, and 0.02, respectively). In Group A, serum adiponectin negatively correlated with NF-(ordM)B expression. In Group B, adiponectin values correlated negatively with both FBG and 2-h PPBG. CONCLUSIONS: Indian subjects with T2DM with or without MVD had higher hsCRP and lower adiponectin values as compared to healthy controls, whereas hsCRP was significantly higher in those with MVD, suggesting that our patients with T2DM were in a proinflammatory state.

Adiponectin G276T gene polymorphism is associated with cardiovascular disease in Japanese patients with type 2 diabetes.

Katakami N; Kaneto H; Matsuoka TA; Takahara M; Maeda N; Shimizu I; et al.
Atherosclerosis 2012 Feb;220(2):437-42 (ISSN: 1879-1484)

OBJECTIVE: Adiponectin has anti-atherogenic properties and reduced serum adiponectin levels are associated with cardiovascular disease (CVD). In this study, we examined the relationship between CVD and adiponectin (ADIPOQ) gene G276T polymorphism that is associated with serum adiponectin level in a large cohort of type 2 diabetic patients. RESEARCH DESIGN AND METHODS: We enrolled 2637 Japanese type 2 diabetic subjects (males, 61.1%; age, 54.97.9 years old), determined their genotypes regarding ADIPOQ G276T polymorphisms, and evaluated the association between this polymorphism and the prevalence of CVD (myocardial infarction and/or cerebral infarction). RESULTS: The prevalence of CVD tended to be higher as the number of G alleles increased [GG (9.5%), GT (6.8%), TT (5.6%), p value for trend=0.0059] and was significantly higher in the subjects with GG genotype compared to those with GT or TT genotype (9.5% vs. 6.6%, p=0.0060). Multiple logistic regression analyses revealed that the number of G alleles (Odds ratio (OR)=1.49 with 95%CI 1.09-2.05, p=0.0125) and GG genotype (OR=1.66 with 95%CI 1.13-2.43, p=0.0098) were significantly associated with CVD even after adjustment for conventional risk factors. Interestingly, the presence of obesity further and significantly increased the risk of CVD in the subjects with GG genotype (OR=1.67 with 95%CI 1.14-2.44, p=0.0090) but not in the subjects with TT or GT genotype (OR=1.17 with 95%CI 0.73-1.89, NS). CONCLUSIONS: It is likely that the G allele of the ADIPOQ G276T polymorphism is a susceptibility allele for CVD in Japanese type 2 diabetic patients, especially when they accompany obesity. [Copyright 2011 Elsevier Ireland Ltd. All rights reserved.].

A comprehensive investigation of variants in genes encoding adiponectin (ADIPOQ) and its receptors (ADIPOR1/R2), and their association with serum adiponectin, type 2 diabetes, insulin resistance and the metabolic syndrome.

Peters KE; Beilby J; Cadby G; Warrington NM; Bruce DG; Davis WA; et al.
BMC Med Genet 2013;14:15 (ISSN: 1471-2350)

BACKGROUND: Low levels of serum adiponectin have been linked to central obesity, insulin resistance, metabolic syndrome, and type 2 diabetes. Variants in ADIPOQ, the gene encoding adiponectin, have been shown to influence serum adiponectin concentration, and along with variants in theadiponectin receptors (ADIPOR1 and ADIPOR2) have been implicated in metabolic syndrome and type 2 diabetes. This study aimed to comprehensively investigate the association of common variants in ADIPOQ, ADIPOR1 and ADIPOR2 with serum adiponectin and insulin resistance syndromes in a large cohort of European-Australian individuals. METHODS: Sixty-four tagging single nucleotide polymorphisms in ADIPOQ, ADIPOR1 and ADIPOR2 were genotyped in two general population cohorts consisting of 2,355 subjects, and one cohort of 967 subjects with type 2 diabetes. The association of tagSNPs with outcomes were evaluated using linear or logistic modelling. Meta-analysis of the three cohorts was performed by random-effects modelling. RESULTS: Meta-analysis revealed nine genotyped tagSNPs in ADIPOQ significantly associated with serum adiponectinacross all cohorts after adjustment for age, gender and BMI, including rs10937273, rs12637534, rs1648707, rs16861209, rs822395, rs17366568, rs3774261, rs6444175 and rs17373414. The results of haplotype-based analyses were also consistent. Overall, the variants in the ADIPOQ gene explained <5% of the variance in serum adiponectin concentration. None of the ADIPOR1/R2 tagSNPs were associated with serum adiponectin. There was no association between any of the genetic variants and insulin resistance or metabolic syndrome. A multi-SNP genotypic risk score for ADIPOQ alleles revealed an association with 3 independent SNPs, rs12637534, rs16861209, rs17366568 and type 2 diabetes after adjusting foradiponectin levels (OR=0.86, 95% CI=(0.75, 0.99), P=0.0134). CONCLUSIONS: Genetic variation in ADIPOQ, but not its receptors, was associated with altered serum adiponectin. However, genetic variation in ADIPOQ and its receptors does not appear to contribute to the risk of insulin resistance or metabolic syndrome but did for type 2
diabetes in a European-Australian population.
Autophagy: Protection Against T2D?

By Salynn Boyles, Contributing Writer,
MedPage Today Published: Jul 27, 2014 | Updated: Jul 28, 2014

The cellular regulatory system known as autophagy appeared to play a key role in preventing type 2 diabetes by protecting insulin-secreting beta cells from the accumulation of toxic amylin oligomers, researchers reported.

Findings from three independent research teams, published online in the Journal of Clinical Investigation, suggested autophagy boosting therapies could prove to be a novel approach for type 2 diabetes prevention.

Autophagy — derived from the Greek words for “self” (auto) and “to eat” (phagein) — describes the controlled disposal of damaged organelles within the cell. This cell-cleaning process is increasingly being recognized as a potential protective mechanism against many diseases, including Parkinson’s disease, amyotrophic lateral sclerosis, and Alzheimer’s disease.

Earlier studies found autophagy to be important for normal beta-cell functionand autophage activity to be increased in beta cells from patients with type 2 diabetes.

The studies provide new insight into how beta cells are normally protected against amylin (IAPP) toxic oligomers, wrote Dhananjay Gupta, PhD, and Jack L. Leahy, MD, of the University of Vermont in Burlington in an accompanying editorial.

Action Points:

Autophagy appeared to play a key role in preventing type 2 diabetes by protecting insulin-secreting beta cells from the accumulation of toxic amylin oligomers.
Note that the studies suggest that autophagy — controlled disposal of damaged organelles within the cell — boosting therapies could prove to be a novel approach for type 2 diabetes prevention.

Autophagy – continued

IAPP: Co-Expressed With Insulin

Type 2 diabetes is characterized by loss of beta-cell, beta-cell dysfunction, and increased beta-cell apoptosis. Islet pathology in type 2 diabetes is also characterized by accumulation of extracellular islet amyloid derived from islet amyloid polypeptide (IAPP).

“IAPP is a 37-amino acid protein co-expressed and secreted by pancreatic [beta cells] along with insulin,” wrote Peter Butler, MD, from the University of California Los Angeles, and colleagues. “While the extracellular islet amyloid is relatively inert, intracellular membrane-permeant toxic oligomers of IAPP that form within [beta cells in type 2 diabetes] are thought to induce [beta-cell dysfunction and apoptosis].”

In contrast to the human form of IAPP (h-IAPP), which forms toxic membrane-permeant oligomers, the rodent form of IAPP (r-IAPP) is nonamyloidogenic and nontoxic due to proline substitutions. Transgenic expression of h-IAPP in [beta cells] of rodents may lead to development of diabetes as a consequence of [beta-cell] apoptosis and formation of intracellular IAPP oligomers comparable to those found in humans with type 2 diabetes.

In earlier in vitro studies, the authors reported that enhancement of autophagy was protective while attenuated lysosomal degradation rendered beta cells more vulnerable to h-IAPP-induced apoptosis.

In the current study, the researchers determined that beta-cell IAPP content is regulated by autophagy through p62-dependent lysosomal degradation.

“Induction of high levels of human IAPP in mouse [beta cells] resulted in accumulation of this amyloidogenic protein as relatively inert fibrils with cytosolic p62-positive inclusions, which temporarily averts formation of toxic oligomers,” they wrote.

Mice hemizygous for transgenic expression of human IAPP did not develop diabetes. But the loss of beta cell-specific autophagy in the mice induced diabetes as a result of the accumulation of toxic human IAPP oligomers and loss of beta-cell mass, the researchers noted.

“In human IAPP-expressing mice that lack [beta-cell] autophagy, increased oxidative damage and loss of an antioxidant-protective pathway appeared to contribute to increased [beta- cell] apoptosis,” they wrote. “These findings indicate that autophagy/lysosomal degradation defends [beta cells] against proteotoxicity induced by oligomerization-prone human IAPP.”

‘Enhance the Toxic Potential of h-IAPP’

In a separate study, Yoshio Fujitani, PhD, of Juntendo University, Tokyo, and colleagues, examined the pathogenic role of human-IAPP and its relation to autophagy in h-IAPP-knock-in mice.

In animals fed a standard diet, h-IAPP had no toxic effects on beta-cell function. However, h-IAPP-knock-in mice did not exhibit a high-fat diet-induced compensatory increase in beta-cell mass, which was due to limited beta-cell proliferation and enhanced beta-cell apoptosis, the researchers wrote.

Expression of h-IAPP in mice with a beta-cell-specific autophagy defect resulted in substantial deterioration of glucose tolerance and dispersed cytoplasmic expression of p62-associated toxic oligomers, which were otherwise sequestrated within p62-positive inclusions.

“Together, our results indicate that increased insulin resistance in combination with reduced autophagy may enhance the toxic potential of h-IAPP and enhance [beta-cell] dysfunction and progression of type 2 diabetes,” the researchers noted.

Autophagy Enhancers

In the third paper, Myung-Shik Lee, MD, PhD, of the Sungkyunkwan University School of Medicine in Seoul, and colleagues, studied transgenic mice with beta cell-specific expression of h-IAPP to evaluate the contribution of autophagy in type 2 diabetes-associated accumulation of h-IAPP.

In mice with beta-cell-specific expression of h-IAPP, a deficiency in autophagy resulted in development of overt diabetes, which was not observed in mice expressing h-IAPP alone or lacking autophagy alone. Lack of autophagy in h-IAPP-expressing animals also resulted in h-IAPP oligomer and amyloid accumulation in pancreatic islets, leading to increased death and decreased mass of beta cells.

“Expression of h-IAPP in purified monkey islet cells or a murine [beta cell] line resulted in pro-h-IAPP dimer formation, while dimer formation was absent or reduced dramatically in cells expressing either nonamyloidogenic mouse-IAPP or nonfibrillar mutant h-IAPP,” the researchers wrote. “In autophagy-deficient cells, accumulation of pro-h-IAPP dimers increased markedly, and pro-h-IAPP trimers were detected in the detergent-insoluble fraction.”

Enhancement of autophagy also improved the metabolic profile of h-IAPP-expressing mice fed a high-fat diet.

“These results suggest that autophagy promotes clearance of amyloidogenic h-IAPP, autophagy deficiency exacerbates pathogenesis of human [type 2 diabetes], and autophagy enhancers have therapeutic potential for islet amyloid accumulation-associated human [type 2 diabetes],” the researchers concluded.

Building on Previous Work

Gupta and Leahy noted that all three research teams generated human IAPP-expressing mice with a beta-cell-specific deficiency of the autophagy indicator ATG7, and all three found that autophagy-dependent packaging of monomeric or unprocessed IAPP dimers or trimers into p62-associated vacuoles allowed autophagosomes to dispose of these molecules, keeping them nontoxic.

Each team showed the activity of this detoxification system to be increased when a high-fat diet was fed to the mice with hyperexpression of h-IAPP.

The studies build on previous work and the findings that don’t discern – “how and when during the course of type 2 diabetes development this autophagy-dependent detoxification system might be overcome, allowing toxic IAPP oligomers to form.”

“There are many additional mechanisms that have been proposed for [beta-cell] dysfunction and death in type 2 diabetes, including ER stress, oxidative stress, and autoimmune damage, all of which have been linked to IAPP toxicity,” they wrote. “While it is tempting to try and connect the dots through a single, unified mechanism, all of these proposed pathways of [beta-cell] dysfunction have been recapitulated and extensively studied in rodent models of diabetogenic systems, such as high-fat feeding and partial pancreatectomy, or through genetic modification.”

Given the absence of rodent IAPP oligomerization, these mechanisms of reduced beta-cell function clearly do not require IAPP activation, they noted.

These papers have implications for the study of target therapies for type 2 diabetes based on the common link to T2D and IAPP oligomerization.

“Patients with type 2 diabetes have an increased risk of Alzheimer’s disease, suggesting a common pathogenesis,” they wrote. Disordered neuronal autophagy, described in Alzheimer’s, with alteration in the clearance of amyloidogenic proteins may be a tie between these two diseases

They concluded that acceptance of the hypothesis that IAPP oligomer formation and subsequent plaque development are a major cause of type 2 diabetes will require a better understanding of

when this mechanism is activated and
what modulates its destructive potential.

“These current studies may shift the focus away from

the biology of how IAPP oligomers cause [beta cell] destruction
to probing for defects within the protective system against the formation of toxic IAPP oligomers,” they wrote.

Part 2. Pancreatic Islet Cell Dysfunction
N-terminal fragment of probrain natriuretic peptide is associated with diabetes microvascular complications in type 2 diabetes

Kumiko Hamano, Ikue Nakadaira, Jun Suzuki, Megumi Gonai
Vascular Health and Risk Management 2014:10 585–589
http://dx.doi.org/10.2147/VHRM.S67753

Aim/introduction: Circulating levels of N-terminal fragment of probrain natriuretic peptide (NT-proBNP) are established as a risk factor for cardiovascular disease and mortality in patients with diabetes, as well as in the general population. We sought to examine the possibility of NT-proBNP as a biomarker of microvascular complications in patients with type 2 diabetes. Materials and methods: In total, 277 outpatients with type 2 diabetes were consecutively enrolled as a hospital cohort. Two hundred and seventeen of these patients (132 males; mean age, 63.4 years) were designated as cases with any of the diabetic complications (retinopathy, neuropathy, nephropathy, ischemic heart disease, strokes, peripheral artery disease), and 60 (42 males; mean age, 54.1 years) were set as controls without clinical evidence of diabetic complications. Diabetic complications were evaluated by medical record and routine laboratory examinations. NT-proBNP was measured and investigated with regard to the associations with diabetic complications. Results: Mean NT-proBNP levels were significantly higher in patients with any of the diabetic complications (59 versus 33 pg/mL; P,0.0001). In logistic regression analysis, NT-proBNP levels .79 pg/mL, which was the highest tertile, were independently associated with a 5.04 fold increased risk of all complications (P,0.0051) compared to the lowest tertile (NT-proBNP levels ,31 pg/mL). Odd ratios of cardiovascular disease and nephropathy, neuropathy, and retinopathy were 9.33, 6.23, 6.6 and 13.78 respectively, in patients with NT-proBNP values in the highest tertile (.79 pg/mL), independently of age, sex, duration of diabetes or other risk factors, such as body mass index or hemoglobin A1c. In addition, NT-proBNP levels were associated with surrogate markers of atherosclerosis, such as brachial-ankle pulse wave velocity (r=0.449, P,0.0001) and left ventricular hypertrophy (r=0.212, P,0.001). Conclusion: In this hospital-based cohort of type 2 diabetes, the NT-proBNP levels were associated with systemic atherosclerosis and comorbid diabetic microvascular as well as macrovascular complications. It is useful to stratify high-risk diabetic patients by measuring NT-proBNP and to start comprehensive care for preventing the progression of diabetic complications. It is necessary to elucidate the underlying mechanism for the progression of diabetic complications represented by an elevation of NT-proBNP and to demonstrate the ability of NT-proBNP as a predictive global biomarker for diabetic complications in Japanese type 2 diabetic patients.
How are patients with type 2 diabetes and renal disease monitored and managed? Insights from the observational OREDIA study

Alfred Penfornis, J F Blicklé, B Fiquet, S Quéré, S Dejager
Vascular Health and Risk Management 2014:10 341–352
http://dx.doi.org/10.2147/VHRM.S60312

Background and aim: Chronic kidney disease (CKD) is frequent in type 2 diabetes mellitus (T2DM), and therapeutic management of diabetes is more challenging in patients with renal impairment (RI). The place of metformin is of particular interest since most scientific societies now recommend using half the dosage in moderate RI and abstaining from use in severe RI, while the classic contraindication with RI has not been removed from the label. This study aimed to assess the therapeutic management, in particular the use of metformin, of T2DM patients with CKD in real life. Methods: This was a French cross-sectional observational study: 3,704 patients with T2DM diagnosed for over 1 year and pharmacologically treated were recruited in two cohorts (two-thirds were considered to have renal disease [CKD patients] and one-third were not [non-CKD patients]) by 968 physicians (81% general practitioners) in 2012. Results: CKD versus non-CKD patients were significantly older with longer diabetes history, more diabetic complications, and less strict glycemic control (mean glycated hemoglobin [HbA1c] 7.5% versus 7.1%; 25% of CKD patients had HbA1c $8% versus 15% of non-CKD patients). Fifteen percent of CKD patients had severe RI, and 66% moderate RI. Therapeutic management of T2DM was clearly distinct in CKD, with less use of metformin (62% versus 86%) but at similar mean daily doses (∼2 g/d). Of patients with severe RI, 33% were still treated with metformin, at similar doses. For other oral anti-diabetics, a distinct pattern of use was seen across renal function (RF): use of sulfonylureas (32%, 31%, and 20% in normal RF, moderate RI, and severe RI, respectively) and DPP4-i (dipeptidyl peptidase-4 inhibitors) (41%, 36%, and 25%, respectively) decreased with RF, while that of glinides increased (8%, 14%, and 18%, respectively). CKD patients were more frequently treated with insulin (40% versus 16% of non-CKD patients), and use of insulin increased with deterioration of RF (19%, 39%, and 61% of patients with normal RF, moderate RI, and severe RI, respectively). Treatment was modified at the end of the study-visit in 34% of CKD patients, primarily to stop or reduce metformin. However, metformin was stopped in only 40% of the severe RI patients. Conclusion: Despite a fairly good detection of CKD in patients with T2DM, RI was insufficiently taken into account for adjusting anti-diabetic treatment.

Efficacy and safety of insulin glargine added to a fixed-dose combination of metformin and a dipeptidyl peptidase-4 inhibitor: results of the GOLD observational study

Jochen Seufert, Katrin Pegelow, Peter Bramlage
Vascular Health and Risk Management 2013:9 711–717
http://dx.doi.org/10.2147/VHRM.S54362

Background: For patients with type 2 diabetes who are uncontrolled on a combination of two oral antidiabetic agents, addition of the long-acting basal insulin glargine is a well established treatment option. However, data on the efficacy and safety of a combination of metformin, a dipeptidyl peptidase-4 (DPP-4) inhibitor, and insulin glargine are limited in real-world settings. Therefore, the aim of this study was to analyze blood glucose control, rates of hypoglycemia and body weight in a large cohort of patients with type 2 diabetes treated with this combination therapy in real practice. Methods: This noninterventional, multicenter, prospective, observational trial with a follow-up of 20 weeks enrolled insulin-naïve patients who had been on a stable fixed dose of metformin and a DPP-4 inhibitor for at least 3 months, and had a glycosylated hemoglobin (HbA1c) between 7.5% and 10%. Patients were selected at the investigators’ discretion for initiation of insulin glargine at baseline. A total of 1,483 patients were included, of whom 1,262 were considered to be the efficacy set. Primary efficacy parameters were HbA1c and fasting plasma glucose. Secondary outcome measures included achievement of glycemic targets, body weight, rates of hypoglycemia, and other safety parameters, as well as resource consumption. Results: Upon initiation of insulin glargine, mean HbA1c decreased from 8.51% to 7.36% (−1.15%±0.91%; 95% confidence interval [CI] −1.20 to −1.10). An HbA1c level ,6.5% was achieved in 8.2% of patients and a level ,7.0% in 31.5%. Mean fasting plasma glucose decreased from 174±47 mg/dL to 127±31 mg/dL (−47.3±44.1 mg/dL; 95% CI −49.8 to −44.8). In 11.9% of patients, a fasting plasma glucose level ,100 mg/dL was achieved. Bodyweight decreased on average by 0.98±3.90 kg (95% CI 1.19–0.76). Hypoglycemia (blood glucose #70 mg/dL) was observed in 29 patients (2.30%), of whom six (0.48%) had nocturnal hypoglycemia and four (0.32%) had documented severe events (blood glucose ,56 mg/dL). Conclusion: The results of this observational study show that insulin glargine, when added to a fixed-dose combination of metformin and a DPP-4 inhibitor, resulted in a significant and clinically relevant improvement of glycemic control. Importantly, this intervention did not interfere with the action of the DPP-4 inhibitors, resulting in neutral effects on weight and low rates of hypoglycemia. We conclude that this treatment intensification approach may be useful, efficient, and safe in daily clinical practice for patients with type 2 diabetes.

Long-term insulin glargine therapy in type 2 diabetes mellitus: a focus on cardiovascular outcomes

Joshua J Joseph, Thomas W Donner
Vascular Health and Risk Management 2015:11 107–116
http://dx.doi.org/10.2147/VHRM.S50286

Cardiovascular disease is the leading cause of mortality in type 2 diabetes mellitus. Hyperinsulinemia is associated with increased cardiovascular risk, but the effects of exogenous insulin on cardiovascular disease progression have been less well studied. Insulin has been shown to have both cardioprotective and atherosclerosis-promoting effects in laboratory animal studies. Long-term clinical trials using insulin to attain improved diabetes control in younger type 1 and type 2 diabetes patients have shown improved cardiovascular outcomes. Shorter trials of intensive diabetes control with high insulin use in higher risk patients with type 2 diabetes have shown either no cardiovascular benefit or increased all cause and cardiovascular mortality. Glargine insulin is a basal insulin analog widely used to treat patients with type 1 and type 2 diabetes. This review focuses on the effects of glargine on cardiovascular outcomes. Glargine lowers triglycerides, leads to a modest weight gain, causes less hypoglycemia when compared with intermediate-acting insulin, and has a neutral effect on blood pressure. The Outcome Reduction With Initial Glargine Intervention (ORIGIN trial), a 6.2 year dedicated cardiovascular outcomes trial of glargine demonstrated no increased cardiovascular risk.

Visceral obesity is not an independent risk factor of mortality in subjects over 65 years

Frédérique Thomas, Bruno Pannier, Athanase Benetos, Ulrich M Vischer
Vascular Health and Risk Management 2013:9 739–745
http://dx.doi.org/10.2147/VHRM.S49922

The aim of the study was to determine the role of obesity evaluated by body mass index (BMI), waist circumference (WC), and their combined effect on all-cause mortality according to age and related risk factors. This study included 119,090 subjects (79,325 men and 39,765 women), aged from 17 years to 85 years, who had a general health checkup at the Centre d’Investigations Préventives et Cliniques, Paris, France. The mean follow-up was 5.6±2.4 years. The prevalence of obesity, defined by WC and BMI categories, was determined according to age groups (< 55, 55–65, > 65 years). All-cause mortality according to obesity and age was determined using Cox regression analysis, adjusted for related risk factors and previous cardiovascular events.
For the entire population, WC adjusted for BMI, an index of central obesity, was strongly associated with mortality, even after adjustment for hypertension, dyslipidemia, and diabetes. The prevalence of obesity increased with age, notably when defined by WC. Nonetheless, the association between WC adjusted for BMI and mortality was not observed in subjects .65 years old (hazard ratio [HR] =1.010, P=NS) but was found in subjects < 55 (HR =1.030,
P < 0.0001) and 55–65 years old (HR =1.023, P,0.05). By contrast, hypertension
(HR =1.31, P < 0.05), previous cardiovascular events (HR =1.98, P < 0.05), and smoking (HR =1.33, P < 0.05) remained associated with mortality even after
age 65.
In conclusion, WC adjusted for BMI is strongly and independently associated with all-cause mortality before 65 years of age, after taking into account the associated risk factors. This relationship disappears in subjects
> 65 years of age, suggesting a differential impact of visceral fat deposition according to age.

Insulin degludec/insulin aspart combination for the treatment of type 1 and type 2 diabetes

Angela Dardano, Cristina Bianchi, Stefano Del Prato, Roberto Miccoli
Vascular Health and Risk Management 2014:10 465–475
http://dx.doi.org/10.2147/VHRM.S40097

Glycemic control remains the major therapeutic objective to prevent or delay the onset and progression of complications related to diabetes mellitus. Insulin therapy represents a cornerstone in the treatment of diabetes and has been used widely for achieving glycemic goals. Nevertheless, a large portion of the population with diabetes does not meet the internationally agreed glycemic targets. Moreover, insulin treatment, especially if intensive, may be associated with emergency room visits and hospitalization due to hypoglycemic events. Therefore, fear of hypoglycemia or hypoglycemic events represents the main barriers to the attainment of glycemic targets. The burden associated with multiple daily injections also remains a significant obstacle to initiating and maintaining insulin therapy. The most attractive insulin treatment approach should meet the patients’ preference, rather than demanding patients to change or adapt their lifestyle. Insulin degludec/insulin aspart (IDegAsp) is a new combination, formulated with ultra-long-acting insulin degludec and rapid-acting insulin aspart, with peculiar pharmacological features, clinical efficacy, safety, and tolerability. IDegAsp provides similar, noninferior glycemic control to a standard basal–bolus regimen in patients with type 1 diabetes mellitus, with additional benefits of significantly lower episodes of hypoglycemia (particularly nocturnal) and fewer daily insulin injections. Moreover, although treatment strategy and patients’ viewpoint are different in type 1 and type 2 diabetes, trial results suggest that IDegAsp may be an appropriate and reasonable option for initiating insulin therapy in patients with type 2 diabetes inadequately controlled on maximal doses of conventional oral agents. This paper will discuss the role of IDegAsp combination as a novel treatment option in diabetic patients.

UCP2 Regulates the Glucagon Response to Fasting and Starvation

Emma M. Allister, Christine A. Robson-Doucette, Kacey J. Prentice, et al.
Diabetes Feb 22, 2013; p 1-11. http://dx.doi.org:/10.2337/db12-0981
http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-0981/-/DC1

Glucagon is important for maintaining euglycemia during fasting/starvation, and abnormal glucagon secretion is associated with type 1 and type 2 diabetes; however, the mechanisms of hypoglycemia-induced glucagon secretion are poorly understood. We previously demonstrated that global deletion of mitochondrial uncoupling protein 2 (UCP22/2) in mice impaired glucagon secretion from isolated islets. Therefore, UCP2 may contribute to the regulation of hypoglycemia-induced glucagon secretion, which is supported by our current finding that UCP2 expression is increased in nutrient-deprived murine and human islets. Further to this, we created a-cell–specific UCP2 knockout (UCP2AKO) mice, which we used to demonstrate that blood glucose recovery in response to hypoglycemia is impaired owing to attenuated glucagon secretion. UCP2-deleted a-cells have higher levels of intracellular reactive oxygen species (ROS), due to enhanced mitochondrial coupling, which translated into defective stimulus/secretion coupling. The effects of UCP2 deletion were mimicked by the UCP2 inhibitor genipin on both murine and human islets and also by application of exogenous ROS, confirming that changes in oxidative status and electrical activity directly reduce glucagon secretion. Therefore, a-cell UCP2 deletion perturbs the fasting/hypoglycemic glucagon response and shows that UCP2 is necessary for normal a-cell glucose sensing and the maintenance of euglycemia.

Main points:

UCP2 is efficiently deleted specifically from islet a-cells of UCP2AKO mice.
α-Cell UCP2 deletion reduces glucagon secretion in vivo
UCP2AKO mice display normal glucose tolerance and GLP-1 secretion
α-Cell UCP2 deletion reduces the gluconeogenic response of the liver and switches fatty acid usage during a prolonged fast
UCP2 expression is increased after nutrient depletion and glucagon secretion from UCP2AKO islets was impaired.
UCP2AKO α-cells display enhanced hyperpolarization of ΔψC_m and increased superoxide levels
UCP2AKO α-cells have more depolarized plasma membranes and reduced intracellular calcium
UCP2 is required for normal glucagon secretion in response to hypoglycemia

Management of Diabetes Mellitus: Could Simultaneous Targeting of Hyperglycemia and Oxidative Stress Be a Better Panacea?

Omotayo O. Erejuwa

Int. J. Mol. Sci. 2012, 13, 2965-2972; http://dx.doi.org:/10.3390/ijms13032965

Oxidative stress is defined as an “imbalance between oxidants and antioxidants in favor of the oxidants, potentially leading to damage”. It is implicated in the pathogenesis and complications of diabetes mellitus. The role of oxidative stress is more definite in the pathogenesis of type 2 diabetes mellitus than in type 1 diabetes mellitus. In regard to diabetic complications, there is compelling evidence in support of the role of oxidative stress in both types of diabetes mellitus. Evidence suggests that elevated reactive oxygen species (ROS), which causes oxidative stress, accumulate in certain micro milieu or tissues (such as retina and kidney) where they cause damage or toxicity. In diabetes mellitus, oxidative stress is enhanced through various sources such as hyperglycemia, dyslipidemia, hyperinsulinemia, insulin resistance, impaired antioxidant defense network, uncoupling of ROS-generating enzymes, elevated level of leptin and sedentary lifestyle.

A number of mechanisms or pathways by which hyperglycemia, the major contributing factor of increased ROS production, causes tissue damage or diabetic complications have been identified. These include: hyperglycemia-enhanced polyol pathway; hyperglycemia-enhanced formation of advanced glycation endproducts (AGEs); hyperglycemia-activated protein kinase C (PKC) pathway; hyperglycemia-enhanced hexosamine pathway; and hyperglycemia-activated Poly-ADP ribose polymerase (PARP) pathway. These pathways are activated or enhanced by hyperglycemia-driven mitochondrial superoxide overproduction. Even though oxidative stress plays an important role in its pathogenesis and complications, unlike other diseases characterized by oxidative stress, diabetes mellitus is unique. Its cure (restoration of euglycemia, e.g., via pancreas transplants) does not prevent oxidative stress and diabetic complications. This is very important because hyperglycemia exacerbates oxidative stress which is linked to diabetic complications]. Theoretically, restoration of euglycemia should prevent oxidative stress and diabetic complications. However, this is not the case.

The primary aim of the current management of diabetes mellitus is to achieve and/or maintain a glycated hemoglobin level of ≤6.5%. However, recent evidence indicates that intensive treatment of hyperglycemia is characterized by increased weight gain, severe hypoglycemia and higher mortality. Besides, evidence suggests that it is difficult to achieve and/or maintain optimal glycemic control in many diabetic patients; and that the benefits of intensively-treated hyperglycemia are restricted to microvascular complications only. In view of these adverse effects and limitations of intensive treatment of hyperglycemia in preventing diabetic complications, which is linked to oxidative stress, this commentary proposes a hypothesis that “simultaneous targeting of hyperglycemia and oxidative stress” could be more effective than “intensive treatment of hyperglycemia” in the management of diabetes mellitus.

The Relationship between Inflammation, Oxidative Stress, and Metabolic Risk Factors in Type 2 Diabetic Patients

Fatemeh Azizi Soleiman, N Pahlavani, H Rasad, O Sadeghi, MR Gohari
Iranian Journal Of Diabetes And Obesity 2013; 5(4): 151-156

Increased production of free radicals due to the imbalance between free radicals and antioxidants load may reduce antioxidants levels, partial clearing of free radicals, and cause oxidation of lipids, sugars, proteins and nucleic acids which eventually leads to widespread pathological consequences of diabetes. One of the factors that facilitate formation of atherosclerosis in diabetes is oxidative stress.

Objective: Globally, 3-5.2 percent of people suffer from diabetes which is one of the most serious metabolic disorders resulting in an increase in inflammatory biomarkers e.g. interleukin-6, tumor necrosis factor-alpha, and C-reactive protein. The aim of this study was to investigate the relationship between inflammation, oxidative stress and fasting blood glucose, lipid profile and anthropometric parameters in patients with type 2 diabetes. Material and methods: This study was conducted as a cross sectional study in Tehran through 2009-2010 on 45 men and women aged 35-65 years old with type 2 diabetes. Blood glucose, lipid profile, C-reactive protein, and malonedialdehyde were measured. Independent sample T-test and linear regression analysis were used. Results: Fasting blood glucose, malonedialdehyde, total cholesterol and body mass index were higher in women than in men; but there was no difference between two sexes in other factors. Malonedialdehyde, neither directly or after adjustment for sex was related to fasting blood glucose, total cholesterol, triglycerides and anthropometric indices (weight, body mass index, and body fat mass). Conclusion: This study showed that oxidative stress had no relationship with blood glucose, lipid profile, and anthropometric index, but inflammation was related to glycemia, body mass index, and fat mass. Control of inflammation and oxidative stress is necessary for accelerating treatment process and preventing complications due to them.

This study showed that in diabetic patients, oxidative stress which was measured by MDA, was not significantly associated with fasting blood glucose, lipid profile and anthropometric parameters. However, fasting plasma glucose, body mass index and body fat mass were significant predictors of the inflammatory factor, CRP.

Oxidative Stress as an Underlying Contributor in the Development of Chronic Complications in Diabetes Mellitus

Suziy de M. Bandeira, Lucas José S. da Fonseca, Glaucevane da S. Guedes, et al.
Int. J. Mol. Sci. 2013, 14, 3265-3284; http://dx.doi.doi:/10.3390/ijms14023265

The high prevalence of diabetes mellitus and its increasing incidence worldwide, coupled with several complications observed in its carriers, have become a public health issue of great relevance. Chronic hyperglycemia is the main feature of such a disease, being considered the responsible for the establishment of micro and macrovascular complications observed in diabetes. Several efforts have been directed in order to better comprehend the pathophysiological mechanisms involved in the course of this endocrine disease. Recently, numerous authors have suggested that excess generation of highly reactive oxygen and nitrogen species is a key component in the development of complications invoked by hyperglycemia. Overproduction and/or insufficient removal of these reactive species result in vascular dysfunction, damage to cellular proteins, membrane lipids and nucleic acids, leading different research groups to search for biomarkers which would be capable of a proper and accurate measurement of the oxidative stress (OS) in diabetic patients, especially in the presence of chronic complications.
In the face of this scenario, the present review briefly addresses the role of hyperglycemia in OS, considering basic mechanisms and their effects in diabetes mellitus, describes some of the more commonly used biomarkers of oxidative/nitrosative damage and includes selected examples of studies which evaluated OS biomarkers in patients with diabetes, pointing to the relevance of such biological components in general oxidative stress status of diabetes mellitus carriers.
The role of FOXO1 in β‑cell failure and type 2 diabetes mellitus

Tadahiro Kitamura
Nat. Rev. Endocrinol. 2013; 9, 615–623
http://dx.doi.org:/10.1038/nrendo.2013.157

Over the past two decades, insulin resistance has been considered essential to the etiology of type 2 diabetes mellitus (T2DM). However, insulin resistance does not lead to T2DM unless it is accompanied by pancreatic β‑cell dysfunction, because healthy β cells can compensate for insulin resistance by increasing in number and functional output. Furthermore, β‑cell mass is decreased in patients with diabetes mellitus, suggesting a primary role for β‑cell dysfunction in the pathogenesis of T2DM. The dysfunction of β cells can develop through various mechanisms, including oxidative, endoplasmic reticulum or hypoxic stress, as well as via induction of cytokines; these processes lead to apoptosis, uncontrolled autophagy and failure to proliferate. Transdifferentiation between β cells and α cells occurs under certain pathological conditions, and emerging evidence suggests that β‑cell dedifferentiation or transdifferentiation might account for the reduction in β‑cell mass observed in patients with severe T2DM. FOXO1, a key transcription factor in insulin signaling, is implicated in these mechanisms. This Review discusses advances in our understanding of the contribution of FOXO1 signaling to the development of β‑cell failure in T2DM.

Selective peroxisome proliferator-activated receptor g (PPARg) modulation as a strategy for safer therapeutic PPARg activation

Linda Slanec Higgins and Alex M DePaoli
Am J Clin Nutr 2010;91(suppl):267S–72S.
http://dx.doi.org:/10.3945/ajcn.2009.28449E

Peroxisome proliferator-activated receptor c (PPARc) is a clinically validated target for treatment of insulin resistance. PPARc activation by full agonists such as thiazolidinediones has shown potent and durable glucose-lowering activity in patients with type 2 diabetes without the concern for hypoglycemia or gastrointestinal toxicities associated with some other medications used to treat this disease. However, thiazolidinediones are linked to safety and tolerability issues such as weight gain, fluid retention, edema, congestive heart failure, and bone fracture. Distinctive properties of PPARc provide the opportunity for selective modulation of the receptor such that desirable therapeutic effects may be attained without the unwanted effects of full activation. PPARc is a nuclear receptor that forms a complex with coreceptor RXR and a cell type– and cell state– specific array of coregulators to control gene transcription. PPARc affinity for these components, and hence transcriptional response, is determined by the conformational changes induced by ligand binding within a complex pocket with multiple interaction points. This molecular mechanism thereby offers the opportunity for selective modulation. A desirable selective PPARc modulator profile would include high-affinity interaction with the PPARc-binding pocket in a manner that leads to retention of the insulin-sensitizing activity that is characteristic of full agonists as well as mitigation of the effects leading to increased adiposity, fluid retention, congestive heart failure, and bone fracture. Examples of endogenous and synthetic selective PPARc modulator (SPPARM) ligands have been identified. SPPARM drug candidates are being tested clinically and provide support for this strategy.

Predicting response to incretin-based therapy

Sanjay Kalra, Bharti Kalra, Rakesh Sahay, Navneet Agrawal
Research and Reports in Endocrine Disorders 2011:1 11–19
http://dx.doi.org:/10.2147/RRED.S16282

There are two important incretin hormones, glucose-dependent insulin tropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1). The biological activities of GLP-1 include stimulation of glucose-dependent insulin secretion and insulin biosynthesis, inhibition of glucagon secretion and gastric emptying, and inhibition of food intake. GLP-1 appears to have a number of additional effects in the gastrointestinal tract and central nervous system. Incretin based therapy includes GLP-1 receptor agonists like human GLP-1 analogs (liraglutide) and exendin-4 based molecules (exenatide), as well as DPP-4 inhibitors like sitagliptin, vildagliptin and saxagliptin. Most of the published studies showed a significant reduction in HbA1c using these drugs. A critical analysis of reported data shows that the response rate in terms of target achievers of these drugs is average. One of the first actions identified for GLP-1 was the glucose-dependent stimulation of insulin secretion from islet cell lines. Following the detection of GLP-1 receptors on islet beta cells, a large body of evidence has accumulated illustrating that GLP-1 exerts multiple actions on various signaling pathways and gene products in the β cell. GLP-1 controls glucose homeostasis through well-defined actions on the islet β cell via stimulation of insulin secretion and preservation and expansion of β cell mass. In summary, there are several factors determining the response rate to incretin therapy. Currently minimal clinical data is available to make a conclusion. Key factors appear to be duration of diabetes, obesity, presence of autonomic neuropathy, resting energy expenditure, plasma glucagon levels and plasma free fatty acid levels. More clinical evidence is required to identify the factors affecting response rate to incretin therapy.

Regulation of Large Conductance Ca2⁺-activated K⁺ (BK) Channel β1 Subunit Expression by Muscle RING Finger Protein 1 in Diabetic Vessels

Fu Yi, Huan Wang, Qiang Chai, Xiaoli Wang, et al.
J. Biol. Chem. 2014, 289: 10853-10864
http://dx.doi.org:/10.1074/jbc.M113.520940

Background: Impaired BK channel function in diabetic vessels is associated with decreased BK channel[1]1 subunit (BK-β1) expression. Results: Muscle RING finger protein 1 (MuRF1) physically interacts with BK-β1 and accelerates BK-β1 proteolysis. Conclusion: Increased MuRF1 expression is a novel mechanism underlying diabetic BK channelopathy and vasculopathy. Significance: MuRF1 is a potential therapeutic target of BK channel dysfunction and vascular complications in diabetes.

The large conductance Ca2⁺-activated K⁺ (BK) channel, expressed abundantly in vascular smooth muscle cells (SMCs), is a key determinant of vascular tone. BK channel activity is tightly regulated by its accessory β1 subunit (BK-β1). However, BK channel function is impaired in diabetic vessels by increased ubiquitin/proteasome-dependent BK-β1 protein degradation. Muscle RING finger protein 1 (MuRF1), a muscle-specific ubiquitin ligase, is implicated in many cardiac and skeletal muscle diseases. However, the role of MuRF1 in the regulation of vascular BK channel and coronary function has not been examined. In this study, we hypothesized that MuRF1 participated in BK-β1 proteolysis, leading to the down-regulation of BK channel activation and impaired coronary function in diabetes. Combining patch clamp and molecular biological approaches, we found that MuRF1 expression was enhanced, accompanied by reduced BK-β1 expression, in high glucose-cultured human

coronary SMCs and in diabetic vessels. Knockdown of MuRF1 by siRNA in cultured human SMCs attenuated BK-β1 ubiquitination and increased BK-β1 expression, whereas adenoviral expression of MuRF1 in mouse coronary arteries reduced BK-β1 expression and diminished BK channel-mediated vasodilation. Physical interaction between the N terminus of BK-β1 and the coiled-coil domain of MuRF1 was demonstrated by pulldown assay. Moreover, MuRF1 expression was regulated by NF-κB. Most importantly, pharmacological inhibition of proteasome and NF-κB activities preserved BK-β1 expression and BK-channel-mediated coronary vasodilation in diabetic mice. Hence, our results provide the first evidence that the up-regulation of NF-κB-dependent MuRF1 expression is a novel mechanism that leads to BK channelopathy and vasculopathy in diabetes.
The origin of circulating CD36 in type 2 diabetes

MJ Alkhatatbeh, AK Enjeti, S Acharya, RF Thorne, and LF Lincz
Nutrition and Diabetes (2013) 3, e59; http://dx.doi.org:/10.1038/nutd.2013.1

Objective: Elevated plasma levels of the fatty acid transporter, CD36, have been shown to constitute a novel biomarker for type 2 diabetes mellitus (T2DM). We recently reported such circulating CD36 to be entirely associated with cellular microparticles (MPs) and aim here to determine the absolute levels and cellular origin(s) of these CD36 + MPs in persons with T2DM. Design: An ex vivo case-control study was conducted using plasma samples from 33 obese individuals with T2DM (body mass index (BMI) =39.9±6.4 kgm²; age=57±9 years; 18 male:15 female) and age- and gender-matched lean and obese non-T2DM controls (BMI =23.6±1.8 kgm² and 33.5±5.9 kgm², respectively). Flow cytometry was used to analyse surface expression of CD36 together with tissue-specific markers: CD41, CD235α, CD14, CD105 and phosphatidyl serine on plasma MPs. An enzyme-linked immunosorbent assay was used to quantify absolute CD36 protein concentrations. Results: CD36 + MP levels were significantly higher in obese people with T2DM (P<0.00001) and were primarily derived from erythrocytes (CD235α + = 35.8±14.6%); although this did not correlate with hemoglobin A1c. By contrast, the main source of CD36 + MPs in non-T2DM individuals was endothelial cells (CD105 + = 40.9±8.3% and 33.9±8.3% for lean and obese controls, respectively). Across the entire cohort, plasma CD36 protein concentration varied from undetectable to 22.9 µgml^-1 and was positively correlated with CD36 +MPs measured by flow cytometry (P=0.0006) but only weakly associated with the distribution of controls and T2DM (P=0.021). Multivariate analysis confirmed that plasma CD36 + MP levels were a much better biomarker for diabetes than CD36 protein concentration (P=0.009 vs P=0.398, respectively). Conclusions: Both the levels and cellular profile of CD36 + MPs differ in T2DM compared with controls, suggesting that these specific vesicles could represent distinct biological vectors contributing to the pathology of the disease.
A Novel High-Throughput Assay for Islet Respiration Reveals Uncoupling of Rodent and Human Islets

Jakob D. Wikstrom, Samuel B. Sereda, Linsey Stiles, Alvaro Elorza, et al.
PLoS ONE 7(5): e33023. http://dx.doi.org:/10.1371/journal.pone.0033023

Background: The pancreatic beta cell is unique in its response to nutrient by increased fuel oxidation. Recent studies have demonstrated that oxygen consumption rate (OCR) may be a valuable predictor of islet quality and long term nutrient responsiveness. To date, high-throughput and user-friendly assays for islet respiration are lacking. The aim of this study was to develop such an assay and to examine bioenergetic efficiency of rodent and human islets. Methodology/Principal Findings: The XF24 respirometer platform was adapted to islets by the development of a 24-well plate specifically designed to confine islets. The islet plate generated data with low inter-well variability and enabled stable measurement of oxygen consumption for hours. The F1F0 ATP synthase blocker oligomycin was used to assess uncoupling while rotenone together with myxothiazol/antimycin was used to measure the level of non-mitochondrial respiration. The use of oligomycin in islets was validated by reversing its effect in the presence of the uncoupler FCCP. Respiratory leak averaged to 59% and 49% of basal OCR in islets from C57Bl6/J and FVB/N mice, respectively. In comparison, respiratory leak of INS-1 cells and C2C12 myotubes was measured to 38% and 23% respectively. Islets from a cohort of human donors showed a respiratory leak of 38%, significantly lower than mouse islets. Conclusions/Significance: The assay for islet respiration presented here provides a novel tool that can be used to study islet mitochondrial function in a relatively high-throughput manner. The data obtained in this study shows that rodent islets are less bioenergetically efficient than human islets as well as INS1 cells.

Refeeding and metabolic syndromes: two sides of the same coin

OA Obeid, DH Hachem and JJ Ayoub
Nutrition & Diabetes (2014) 4, e120; http://dx.doi.org:/10.1038/nutd.2014.21

Refeeding syndrome describes the metabolic and clinical changes attributed to aggressive rehabilitation of malnourished subjects. The metabolic changes of refeeding are related to hypophosphatemia, hypokalemia, hypomagnesemia, sodium retention and hyperglycemia, and these are believed to be mainly the result of increased insulin secretion following high carbohydrate intake. In the past few decades, increased consumption of processed food (refined cereals, oils, sugar and sweeteners, and so on) lowered the intake of several macrominerals (mainly phosphorus, potassium and magnesium). This seems to have compromised the postprandial status of these macrominerals, in a manner that mimics low grade refeeding syndrome status. At the pathophysiological level, this condition favored the development of the different components of the metabolic syndrome. Thus, it is reasonable to postulate that metabolic syndrome is the result of long term exposure to a mild refeeding syndrome.

HSP72 protects against obesity-induced insulin resistance

Jason Chung, Anh-Khoi Nguyen, Darren C. Henstridge, Anna G. Holmes, et al.
PNAS Feb 5, 2008; 105(5): 1739–1744
http://www.pnas.org/cgi/doi/10.1073/pnas.0705799105

Patients with type 2 diabetes have reduced gene expression of heat shock protein (HSP) 72, which correlates with reduced insulin sensitivity. Heat therapy, which activates HSP72, improves clinical parameters in these patients. Activation of several inflammatory signaling proteins such as c-jun amino terminal kinase (JNK), inhibitor of B kinase, and tumor necrosis factor-β, can induce insulin resistance, but HSP 72 can block the induction of these molecules in vitro. Accordingly, we examined whether activation of HSP72 can protect against the development of insulin resistance. First, we show that obese, insulin resistant humans have reduced HSP72 protein expression and increased JNK phosphorylation in skeletal muscle. We next used heat shock therapy, transgenic overexpression, and pharmacologic means to overexpress HSP72 either specifically in skeletal muscle or globally in mice. Herein, we show that regardless of the means used to achieve an elevation in HSP72 protein, protection against diet- or obesity induced hyperglycemia, hyperinsulinemia, glucose intolerance, and insulin resistance was observed. This protection was tightly associated with the prevention of JNK phosphorylation. These findings identify an essential role for HSP72 in blocking inflammation and preventing insulin resistance in the context of genetic obesity or high-fat feeding.

pH-responsive modulation of insulin aggregation and structural transformation of the aggregates

Ekaterina Smirnova, I Safenkova, V Stein-Margolina, V Shubin, et al.
Biochimie 109 (2015) 49e59
http://dx.doi.org/10.1016/j.biochi.2014.12.006

Over the past two decades, much information has appeared on electrostatically driven molecular mechanisms of protein self-assembly and formation of aggregates of different morphology, varying from soluble amorphous structures to highly-ordered amyloid-like fibrils. Protein aggregation represents a special tool in biomedicine and biotechnology to produce biological materials for a wide range of applications. This has awakened interest in identification of pH-triggered regulators of transformation of aggregation-prone proteins into structures of higher order. The objective of the present study is to elucidate the effects of low-molecular-weight biogenic agents on aggregation and formation of supramolecular structures of human recombinant insulin, as a model therapeutic protein. Using dynamic light scattering, turbidimetry, circular dichroism, fluorescence spectroscopy, atomic force microscopy, transmission electron microscopy, and nuclear magnetic resonance, we have demonstrated that the amino acid L-arginine (Arg) has the striking potential to influence insulin aggregation propensity. It was shown that modification of the net charge of insulin induced by changes in the pH level of the incubation medium results in dramatic changes in the interaction of the protein with Arg. We have revealed the dual effects of Arg, highly dependent on the pH level of the solution e suppression or acceleration of the aggregation of insulin at pH 7.0 or 8.0, respectively. These effects can be regulated by manipulating the pH of the environment. The results of this study may be of interest for development of appropriate drug formulations and for the more general insight into the functioning of insulin in living systems, as the protein is known to release by exocytosis from pancreatic beta cells in a pH-dependent manner.
Human β-cell proliferation by promoting Wnt signaling

Carol Wilson
Original article Aly, H. et al. A novel strategy to increase the proliferative potential of adult human β-cells while maintaining their differentiated phenotype. PLoS ONE 2013; 8, e66131
Nature Reviews Endocrinology 2013; 9, 502
http://dx.doi.org:/10.1038/nrendo.2013.130

Islet transplantation for patients with type 1 diabetes mellitus typically requires 2–4 donors for one recipient, whereas use of one donor would minimize the risk of immune rejection. Proliferation of adult β cells in vitro could hold the key to providing one donor for one recipient.

“In previous studies, we found that activation of the Wnt/GSK-3/β-catenin pathway by pharmacologic inhibition of GSK-3 in combination with nutrient activation of mTOR, modestly enhanced human β-cell proliferation in vitro,” says lead researcher Haytham Aly of the Washington University School of Medicine in St. Louis, MO, USA. “However, expansion of human islets was associated with a loss of insulin content and secretory function.”

In the current study, the researchers aimed to engage canonical and noncanonical Wnt signalling at the receptor level to increase the proliferation of human β cells in vitro, without losing the capacity of the cells to produce and secrete insulin.

The researchers treated cadaver-derived intact human islets with a conditioned medium from L cells that constitutively produce Wnt-3a, R-spondin-3 and Noggin. A similar medium had previously enabled successful proliferation of mouse colonic intestinal epithelial cells. The researchers added inhibitors of ROCK and RhoA to this medium to augment cell survival.

The conditioned medium with the inhibitors lead to ~20-fold proliferation of the human β cells above that with glucose alone. Crucially, treatment with this conditioned medium did not impair glucose-stimulated insulin secretion or decrease insulin content of the cells.

“This novel strategy has clear potential for use in the in vitro expansion of human islets and the subsequent treatment of impaired β-cell functional mass in type 1 diabetes mellitus and type 2 diabetes mellitus,” concludes Aly.

Betatrophin—inducing β-cell expansion to treat diabetes mellitus?

Elisabeth Kugelberg
Original article Yi, P. et al. Betatrophin: a hormone that controls pancreatic β cell proliferation. Cell http://dx.doi.org:/10.1016/j.cell.2013.04.008
Nature Reviews Endocrinology 2013; 9, 379; http://dx.doi.org:/10.1038/nrendo.2013.98

Betatrophin, a newly identified hormone, increases the production and expansion of insulin-secreting β cells in mice, research from Harvard University suggests.

When insulin resistance develops, pancreatic β cells undergo an expansion in mass and proliferation to compensate for increasing insulin needs. To date, the mechanisms regulating β-cell replication are unclear.

Yi et al. developed a mouse model of insulin resistance using the insulin receptor antagonist S961. Subcutaneous injections of the S961 peptide into mice led to dose-dependent, instant β-cell proliferation and hyperglycemia.

Microarray analysis revealed that a highly conserved mammalian gene, betatrophin, was upregulated fourfold in liver and threefold in white adipose tissue cells in response to the acute peripheral insulin resistance induced by S961.

Yi and coworkers found that Betatrophin encodes a secreted protein that can be detected in human plasma. Intravenous injection of betatrophin-expressing constructs into mice resulted in a 17-fold higher β-cell proliferation rate compared with control vectors, and ultimately led to increased islet size and insulin content, with improvements in glucose tolerance, in betatrophin-injected animals.

The mechanisms of action of betatrophin are still unknown, and the next step is to test the effects of recombinant betatrophin protein on β-cell mass. The authors conclude that the identification of betatrophin and its control of β-cell proliferation opens a new door to possible diabetes therapy.

Blocking RANKL signaling might prevent T2DM

Carol Wilson
Original article Kiechl, S. et al. Blockade of receptor activator of nuclear factor-κB (RANKL) signaling improves hepatic insulin resistance and prevents development of diabetes mellitus. Nat. Med.
http://dx.doi.org:/10.1038/nm.3084

Nature Reviews Endocrinology 2013; 9, 188;
http://dx.doi.org:/10.1038/nrendo.2013.43

Blockade of receptor activator of nuclear factor κB ligand (RANKL) signaling in hepatocytes protects against type 2 diabetes mellitus (T2DM), report researchers.

“It is well known that activation of nuclear factor κB (NF-κB) in the liver is a crucial event in the development of hepatic insulin resistance and T2DM,” explains lead author Stefan Kiechl of the Medical University of Innsbruck, Austria. “RANKL, a member of the tumour necrosis factor superfamily, is a potent activator of NF-κB, and its receptor RANK is expressed on liver cells. We, thus, hypothesized that RANKL is involved in hepatic NF-κB activation, leading to T2DM.”

The researchers studied the association between serum levels of soluble RANKL and osteoprotegerin and subsequent risk of developing T2DM in 844 men and women without T2DM aged 40–79 years. Soluble RANKL was assessed because it has been shown to be functionally active.

During follow-up, between 1990 and 2005, 78 individuals of the cohort developed T2DM. Baseline levels of soluble RANKL between individuals who had and had not developed T2DM differed considerably: risk of T2DM was elevated in the group with the top tertile T2DM of concentrations of soluble RANKL compared with the group with the bottom tertile (OR 4.06, 95% CI 2.01–8.20). Adjustment for lifestyle factors and body composition did not significantly affect the risk association. Interestingly, although concentrations of osteoprotegerin were not elevated preceding T2DM onset, as they were for soluble RANKL, increased levels were found in individuals after disease occurrence.

In a series of mouse models in which RANKL signaling was downregulated systemically or in the liver, the investigators showed that hepatic insulin sensitivity and plasma glucose concentrations improved with blockade of RANKL signaling. In one such experiment, mice with a hepatocyte-specific Rank knockout were fed a high-fat diet for 4 weeks. These mice did not develop insulin resistance, whereas control mice did.

The investigators note that medications for T2DM already available, such as metformin, lower RANKL activity in bone and might also lower RANKL activity in the liver. They speculate that RANKL antagonism could be a yet unknown.

SFRP4—a biomarker for islet dysfunction?

Carol Wilson
Original article Mahdi, T. et al. Secreted frizzled-related protein 4 reduces insulin secretion and is overexpressed in type 2 diabetes. Cell Metab. http://doi.org:/10.1016/j.cmet.2012.10.009

Secreted frizzled-related protein 4 (SFRP4) reduces insulin secretion and is a potential biomarker for islet dysfunction in type 2 diabetes mellitus (T2DM), report researchers.

Mahdi et al. discovered these insights into the pathophysiology of T2DM by the analysis of global gene expression in human pancreatic islets. The researchers identified a group of co-expressed genes (also called a gene co-expression module) associated with T2DM, reduced insulin secretion and elevated HbA1c levels after analysing global microarray expression data from human islets of 48 individuals, including 10 with T2DM. This module was enriched for IL-1-related genes.

The investigators identified SFRP4 as a gene highly expressed in islets from patients with T2DM. The protein encoded by SFRP4 is an extracellular regulator of the Wnt pathway, and has roles in tissue development, cancer and phosphate metabolism. Further study revealed that the expression and release of SFRP4 from islets was stimulated by IL-1β. Furthermore, elevated systemic SFRP4 levels led to reduced glucose tolerance as a result of decreased islet expression of voltage-gated Ca2+ channels and supressed insulin exocytosis.

Interestingly, levels of SFRP4 were elevated in serum of patients a few years before they developed T2DM, which indicates that this protein has potential to be used as a biomarker for T2DM. The researchers also point out that their data suggest that SFRP4 could be a therapeutic target for the treatment of islet dysfunction.

Add-on to metformin in T2DM —linagliptin or glimepiride?

Mikkel Christensen and Filip K. Knop
Nat. Rev. Endocrinol. 2012; 8, 576–578 http://dx.doi.org:/10.1038/nrendo.2012.163

Dipeptidyl peptidase 4 (DPP4) inhibitors, also known as gliptins, are a rapidly expanding class of oral antidiabetic drugs for the treatment of type 2 diabetes mellitus (T2DM). Since 2006, five DPP4 inhibitors have reached the market and, because they can be administered orally and have an almost impeccable safety profile, these drugs have gained widespread use in the treatment of T2DM. The DPP4 inhibitor linagliptin was approved in 2011 by the FDA and the European Medicines Agency (EMA) for use in patients with T2DM as second-line therapy to add on to metformin either alone or in combination with another second-line treatment.

The UK Prospective Diabetes Study trial showed that sulphonylurea treatment was more effective than metformin treatment after 1 year in terms of reducing HbA1c levels; however, after 6 years of treatment, the effectiveness of sulphonylurea treatment declined and metformin treatment was more effective. A decline in the effectiveness of the sulphonylurea treatment over time could be due to sulphonylureas inducing stress and possibly causing apoptosis in β cells. However, in the trial by Gallwitz et al. the sustained efficacies of the add-on treatments with linagliptin and glimepiride were similar after 2 years.

The inhibitors of DPP4 enhance glucose-dependent insulin secretion and could even augment the counter-regulatory glucagon response to hypoglycemia. DPP4 inhibition generally has a neutral effect upon body weight.

The study by Gallwitz et al. included patients whose plasma glucose levels were near-normal whilst they were receiving metformin monotherapy (baseline level 6–7 mmol/l), which could result in increased occurrence of hypoglycemia. Treating patients whose blood glucose levels were, by many standards, already adequately controlled with metformin with a drug known to be associated with inducing hypoglycemia would be expected to increase the frequency of hypoglycemia in this group, inflating the differences in the frequency of this event between the group receiving linagliptin and that receiving glimepiride.

The most groundbreaking findings in the study by Gallwitz et al. are related to cardiovascular outcomes. Although the study was not adequately powered to detect subtle differences in cardiovascular event frequency, significantly fewer patients who received linagliptin than glimepiride experienced major cardiovascular events (12 versus 26 individuals, respectively). This difference was driven by fewer patients experiencing nonfatal myocardial infarctions and nonfatal strokes in the linagliptin-treated group than in the glimepiride-treated group (9 versus 21 individuals, respectively).

Clinicians are responsible for selecting a suitable second-line treatment for patients with type 2 diabetes mellitus when metformin monotherapy fails. New evidence could aid clinicians in deciding between one of the most commonly used second-line agents, glimepiride, and the recently approved dipeptidyl peptidase 4 inhibitor linagliptin.

Relation of Mitochondrial Oxygen Consumption in Peripheral Blood Mononuclear Cells to Vascular Function in Type 2 Diabetes Mellitus

Mor-Li Hartman, Orian S. Shirihai, Monika Holbrook, Guoquan Xu, et al.
Vasc Med. 2014 February ; 19(1): 67–74. http://dx.doi.org:/10.1177/1358863X14521315.

Recent studies have shown mitochondrial dysfunction and increased production of reactive

oxygen species in peripheral blood mononuclear cells (PBMC’s) and endothelial cells from patients with diabetes mellitus. Mitochondria oxygen consumption is coupled to ATP production and also occurs in an uncoupled fashion during formation of reactive oxygen species by components of the electron transport chain and other enzymatic sites. We therefore hypothesized that diabetes would be associated with higher total and uncoupled oxygen consumption in PBMC’s that would correlate with endothelial dysfunction. We developed a method to measure oxygen consumption in freshly isolated PBMC’s and applied it to 26 patients with type 2 diabetes mellitus and 28 non-diabetic controls. Basal (192±47 vs. 161±44 pMoles/min, P=0.01), uncoupled (64±16 vs. 53±16 pMoles/min, P=0.007), and maximal (795±87 vs. 715±128 pMoles/min, P=0.01) oxygen consumption rates were higher in diabetic patients compared to controls. There were no significant correlations between oxygen consumption rates and endothelium-dependent flow-mediated dilation measured by vascular ultrasound. Non-endothelium-dependent nitroglycerin-mediated dilation was lower in diabetics (10.1±6.6 vs. 15.8±4.8%, P=0.03) and correlated with maximal oxygen consumption (R= −0.64, P=0.001). In summary, we found that diabetes mellitus is associated with a pattern of mitochondrial oxygen consumption consistent with higher production of reactive oxygen species. The correlation between oxygen consumption and nitroglycerin-mediated dilation may suggest a link between mitochondrial dysfunction and vascular smooth muscle cell dysfunction that merits further study. Finally, the described method may have utility for assessment of mitochondrial function in larger scale observational and interventional studies in humans.

Musashi expression in b-cells coordinates insulin expression, apoptosis and proliferation in response to endoplasmic reticulum stress in diabetes

M Szabat, TB Kalynyak, GE Lim, KY Chu, YH Yang, A Asadi, BK Gage, et al.
Cell Death and Disease (2011) 2, e232
http://dx.doi.org:/10.1038/cddis.2011.119

Diabetes is associated with the death and dysfunction of insulin-producing pancreatic b-cells. In other systems, Musashi genes regulate cell fate via Notch signaling, which we recently showed regulates b-cell survival. Here we show for the first time that human and mouse adult islet cells express mRNA and protein of both Musashi isoforms, as well Numb/Notch/Hes/neurogenin-3 pathway components. Musashi expression was observed in insulin/glucagon double-positive cells during human fetal development and increased during directed differentiation of human embryonic stem cells (hESCs) to the pancreatic lineage. De-differentiation of b-cells with activin A increased Msi1 expression. Endoplasmic reticulum (ER) stress increased Msi2 and Hes1, while it decreased Ins1 and Ins2 expression, revealing a molecular link between ER stress and b-cell dedifferentiation in type 2 diabetes. These effects were independent of changes in Numb protein levels and Notch activation. Overexpression of MSI1 was sufficient to increase Hes1, stimulate proliferation, inhibit apoptosis and reduce insulin expression, whereas Msi1 knockdown had the converse effects on proliferation and insulin expression. Overexpression of MSI2 resulted in a decrease in MSI1 expression. Taken together, these results demonstrate overlapping, but distinct roles for Musashi-1 and Musashi-2 in the control of insulin expression and b-cell proliferation. Our data also suggest that Musashi is a novel link between ER stress and the compensatory b-cell proliferation and the loss of b-cell gene expression seen in specific phases of the progression to type 2 diabetes.

Cooperation between brain and islet in glucose homeostasis and diabetes

Michael W. Schwartz, RJ Seeley, MH Tschöp, SC Woods, et al.
Nature 7 Nov 2013; 503: 59–66 http://dx.doi.org/10.1038/nature12709

Although a prominent role for the brain in glucose homeostasis was proposed by scientists in the nineteenth century, research throughout most of the twentieth century focused on evidence that the function of pancreatic islets is both necessary and sufficient to explain glucose homeostasis, and that diabetes results from defects of insulin secretion, action or both. However, insulin-independent mechanisms, referred to as ‘glucose effectiveness’, account for roughly 50% of overall glucose disposal, and reduced glucose effectiveness also contributes importantly to diabetes pathogenesis. Although mechanisms underlying glucose effectiveness are poorly understood, growing evidence suggests that the brain can dynamically regulate this process in ways that improve or even normalize glycaemia in rodent models of diabetes. Here we present evidence of a brain-centred glucoregulatory system (BCGS) that can lower blood glucose levels via both insulin-dependent and -independent mechanisms, and propose a model in which complex and highly coordinated interactions between the BCGS and pancreatic islets promote normal glucose homeostasis. Because activation of either regulatory system can compensate for failure of the other, defects in both may be required for diabetes to develop. Consequently, therapies that target the BCGS in addition to conventional approaches based on enhancing insulin effects may have the potential to induce diabetes remission, whereas targeting just one typically does not.

The traditional view holds that diabetes arises as a consequence of damage to, and ultimately failure of, beta-cell function. We propose a two-component model in which failure of glucose homeostasis can begin after initial impairment.

Schematic illustrations of brain- and islet-centred glucoregulatory systems

Schematic illustrations of brain- and islet-centred glucoregulatory systems
The BCGS is proposed to regulate tissue glucose metabolism and plasma glucose levels via mechanisms that are both insulin dependent (for example, by regulating tissue insulin sensitivity) and insulin independent

Proposed contributions of defective brain- and islet-centred glucoregulatory systems to T2D pathogenesis

Insulin’s discovery: New insights on its ninetieth birthday

Jesse Roth, Sana Qureshi, Ian Whitford, Mladen Vranic, et al.
Diabetes Metab Res Rev 2012; 28: 293–304
http://dx.doi.org:/10.1002/dmrr.2300

2012 marks the 90th year since the purification of insulin and the miraculous rescue from death of youngsters with type 1 diabetes. In this review, we highlight several previously unappreciated or unknown events surroundingthe discovery.
(i) We remind readers of the essential contributions of each of the four discoverers – Banting, Macleod, Collip, and Best.
(ii) Banting and Best (each with his own inner circle) worked not only to accrue credit for himself but also to minimize credit to the other discoverers.
(iii) Banting at the time of the insulin research was very likely suffering from post-traumatic stress disorder (PTSD) that originated during his heroic service as a surgeon in World War I on the Western Front in 1918, including an infected shrapnel wound that threatened amputation of his arm. His war record along with the newly discovered evidence of a suicide threat goes along with his paranoia, combativeness, alcohol excess, and depression, symptoms we associate with PTSD.
(iv) Banting’s eureka idea, ligation of the pancreatic duct to preserve the islets, while it energized the early research, was unnecessary and was bypassed early.
(v) Post discovery,Macleod uncovered many features of insulin action that he summarized in his 1925 Nobel Lecture.Macleod closed by raising the question – what is the mechanism of insulin action in the body? – a challenge that attracted many talented investigators but remained unanswered until the latter third of the 20th century.

Genetic Variants Associated With Glycine Metabolism and Their Role in Insulin Sensitivity and Type 2 Diabetes

Weijia Xie, Andrew R. Wood, Valeriya Lyssenko, Michael N. Weedon, et al.
Diabetes 2013; 62:2141–2150 http://dx.doi.org:/10.2337/db12-0876

Circulating metabolites associated with insulin sensitivity may represent useful biomarkers, but their causal role in insulin sensitivity and diabetes is less certain. We previously identified novel metabolites correlated with insulin sensitivity measured by the hyperinsulinemic-euglycemic clamp. The top-ranking metabolites were in the glutathione and glycine biosynthesis pathways. We aimed to identify common genetic variants associated with metabolites in these pathways and test their role in insulin sensitivity and type 2 diabetes. With 1,004 nondiabetic individuals from the RISC study, we performed a genome-wide association study (GWAS) of 14 insulin sensitivity–related metabolites and one metabolite ratio. We replicated our results in the Botnia study (n = 342). We assessed the association of these variants with diabetes-related traits in GWAS meta-analyses (GENESIS [including RISC, EUGENE2, and Stanford], MAGIC, and DIAGRAM). We identified four associations with three metabolites—glycine (rs715 at CPS1), serine (rs478093 at PHGDH), and betaine (rs499368 at SLC6A12; rs17823642 at BHMT)—and one association signal with glycine-to-serine ratio (rs1107366 at ALDH1L1). There was no robust evidence for association between these variants and insulin resistance or diabetes. Genetic variants associated with genes in the glycine biosynthesis pathways do not provide consistent evidence for a role of glycine in diabetes related traits.

Fractalkine (CX3CL1), a new factor protecting b-cells against TNFa

Sabine Rutti, Caroline Arous, Domitille Schvartz, Katharina Timper, et al.
MOLMET164_proof ■ 14 Aug 2014 ■ 1/11
http://dx.doi.org/10.1016/j.molmet.2014.07.007

Objective: We have previously shown the existence of a muscleepancreas intercommunication axis in which CX3CL1 (fractalkine), a CX3C chemokine produced by skeletal muscle cells, could be implicated. It has recently been shown that the fractalkine system modulates murine β-cell function. However, the impact of CX3CL1 on human islet cells especially regarding a protective role against cytokine-induced apoptosis remains to be investigated. Methods: Gene expression was determined using RNA sequencing in human islets, sorted β- and non-β-cells. Glucose-stimulated insulin secretion (GSIS) and glucagon secretion from human islets was measured following 24 h exposure to 1e50 ng/ml CX3CL1. GSIS and specific protein phosphorylation were measured in rat sorted β-cells exposed to CX3CL1 for 48 h alone or in the presence of TNFα (20 ng/ml). Rat and human β-cell apoptosis (TUNEL) and rat β-cell proliferation (BrdU incorporation) were assessed after 24 h treatment with increasing concentrations of CX3CL1. Results: Both CX3CL1 and its receptor CX3CR1 are expressed in human islets. However, CX3CL1 is more expressed in non-β-cells than in b-cells while its receptor is more expressed in β-cells. CX3CL1 decreased human (but not rat) β-cell apoptosis. CX3CL1 inhibited human islet glucagon secretion stimulated by low glucose but did not impact human islet and rat sorted β-cell GSIS. However, CX3CL1 completely prevented the adverse effect of TNFa on GSIS and on molecular mechanisms involved in insulin granule trafficking by restoring the phosphorylation (Akt, AS160, paxillin) and expression (IRS2, ICAM-1, Sorcin, PCSK1) of key proteins involved in these processes. Conclusions: We demonstrate for the first time that human islets express and secrete CX3CL1 and CX3CL1 impacts them by decreasing glucagon secretion without affecting insulin secretion. Moreover, CX3CL1 decreases basal apoptosis of human β-cells. We further demonstrate that CX3CL1 protects β-cells from the adverse effects of TNFa on their function by restoring the expression and phosphorylation of key proteins of the insulin secretion pathway.
Heart Failure, Saxagliptin and Diabetes Mellitus: Observations from the SAVOR – TIMI 53 Randomized Trial

Benjamin M. Scirica; Eugene Braunwald; Itamar Raz, and SAVOR-TIMI 53 Steering Committee and Investigators
Circulation. Sept 4, 2014 http://dx.doi.org:/10.1161/CIRCULATIONAHA.114.010389
Background—Diabetes and heart failure frequently coexist. However, few diabetes trials have prospectively evaluated and adjudicated heart failure as an endpoint. Methods and Results—16,492 patients with type 2 diabetes and a history of, or at risk for, cardiovascular events were randomized to saxagliptin or placebo (mean followup-2.1 years). The primary endpoint was the composite of cardiovascular death, myocardial infarction, or ischemic stroke. Hospitalization for heart failure was a predefined component of the secondary endpoint. Baseline NT-proBNP was measured in 12,301 patients. More patients treated with saxagliptin (289, 3.5%) were hospitalized for heart failure compared to placebo (228, 2.8%) (HR 1.27; 95%CI 1.07-1.51; p=0.007). Corresponding rates at 12-months were 1.9% vs.1.3% (HR 1.46, 95%CI 1.15-1.88, p=0.002, with no significant difference thereafter time-varying interaction
p=0.017). Subjects at greatest risk for hospitalization for heart failure had prior heart failure, EGFR < 60 ml/min and/or elevated baseline levels of NT-proBNP. There was no evidence of heterogeneity between NT-proBNP and saxagliptin (p for interaction=0.46), though the absolute risk excess for heart failure with saxagliptin was greatest in the highest NT-proBNP quartile (2.1%). Even in patients at high-risk for hospitalization for heart failure, the risk of the primary and secondary endpoints were similar between treatment groups. Conclusions—In the context of balanced primary and secondary endpoints, saxagliptin treatment was associated with an increased risk for hospitalization for heart failure. This increase in risk was highest among patients with elevated levels of natriuretic peptides, prior heart failure, or chronic kidney disease.
Angiotensin 1–7 improves insulin sensitivity by increasing skeletal muscle glucose uptake in vivo

Omar Echeverría-Rodríguez, Leonardo Del Valle-Mondragón, Enrique Hong
Peptides 51 (2014) 26– 30 http://dx.doi.org/10.1016/j.peptides.2013.10.022

The renin–angiotensin system (RAS) regulates skeletal muscle insulin sensitivity through different mechanisms. The overactivation of the ACE (angiotensin-converting enzyme)/Ang (angiotensin) II/AT1R (Ang IItype 1 receptor) axis has been associated with the development of insulin resistance, whereas the stimulation of the ACE2/Ang 1–7/MasR (Mas receptor) axis improves insulin sensitivity. The in vivo mechanismsby which this axis enhances skeletal muscle insulin sensitivity are scarcely known. In this work, we investigated whether rat soleus muscle expresses the ACE2/Ang 1–7/MasR axis and determined the effect ofAng 1–7 on rat skeletal muscle glucose uptake in vivo. Western blot analysis revealed the expression ofACE2 and MasR, while Ang 1–7 levels were detected in rat soleus muscle by capillary zone electrophoresis. The euglycemic clamp exhibited that Ang 1–7 by itself did not promote glucose transport, but itincreased insulin-stimulated glucose disposal in the rat. In a similar manner, captopril (an ACE inhibitor) enhanced insulin-induced glucose uptake and this effect was blocked by the MasR antagonist A-779. Our results show for the first time that rat soleus muscle expresses the ACE2/Ang 1–7/MasR axis of the RAS,and Ang 1–7 improves insulin sensitivity by enhancing insulin-stimulated glucose uptake in rat skeletal muscle in vivo. Thus, endogenous (systemic and/or local) Ang 1–7 could regulate insulin-mediated glucose transport in vivo.

Evolving concepts in advanced glycation, diabetic nephropathy, and diabetic vascular disease

George Jerums, S Panagiotopoulos, J Forbes, T Osicka, and Mark Cooper
Archives of Biochemistry and Biophysics 419 (2003) 55–62
http://dx.doi.org:/10.1016/j.abb.2003.08.017

Advanced glycation endproducts (AGEs) have been postulated to play a role in the development of both nephropathy and large vessel disease in diabetes. However, it is still not clear which AGE subtypes play a pathogenetic role and which of several AGE receptors mediate AGE effects on cells. This review summarises the renoprotective effect of inhibitors of AGE formation, including aminoguanidine, and of cross-link breakers, including ALT-711, on experimental diabetic nephropathy and on mesenteric vascular hypertrophy. It also demonstrates similar effects of aminoguanidine and ramipril (an angiotensin converting enzyme inhibitor) on fluorescent and immunoassayable AGE levels, renal protein kinase C activity, nitrotyrosine expression, lysosomal function, and protein handling in experimental diabetes. These findings indicate that inhibition of the renin angiotensin system blocks both upstream and downstream pathways leading to tissue injury. We postulate that the chemical pathways leading to advanced glycation endproduct formation and the renin angiotensin systems may interact through the generation of free radicals, induced both by glucose and angiotensin II. There is also evidence to suggest that AGE-dependent pathways may play a role in the development of tubulointerstitial fibrosis in the diabetic kidney. This effect is mediated through RAGE and is TGF-b and CTGF-dependent.

Preconditioning with Associated Blocking of Ca2+ Inflow Alleviates Hypoxia-Induced Damage to Pancreatic β-Cells

Zuheng Ma, Noah Moruzzi, Sergiu-Bogdan Catrina, Ingrid Hals, et al.
PLoS ONE 8(7): e67498. http://dx.doi.org:/10.1371/journal.pone.0067498

Objective: Beta cells of pancreatic islets are susceptible to functional deficits and damage by hypoxia. Here we aimed to characterize such effects and to test for and pharmacological means to alleviate a negative impact of hypoxia. Methods and Design: Rat and human pancreatic islets were subjected to 5.5 h of hypoxia after which functional and viability parameters were measured subsequent to the hypoxic period and/or following a 22 h re-oxygenation period. Preconditioning with diazoxide or other agents was usually done during a 22 h period prior to hypoxia. Results: Insulin contents decreased by 23% after 5.5 h of hypoxia and by 61% after a re-oxygenation period. Preconditioning with diazoxide time-dependently alleviated these hypoxia effects in rat and human islets. Hypoxia reduced proinsulin biosynthesis (3H-leucine incorporation into proinsulin) by 35%. Preconditioning counteracted this decrease by 91%. Preconditioning reduced hypoxia-induced necrosis by 40%, attenuated lowering of proteins of mitochondrial complexes I–IV and enhanced stimulation of HIF-1-alpha and phosphorylated AMPK proteins. Preconditioning by diazoxide was abolished by co-exposure to tolbutamide or elevated potassium (i.e. conditions which increase Ca2+ inflow). Preconditioning with nifedipine, a calcium channel blocker, partly reproduced effects of diazoxide. Both diazoxide and nifedipine moderately reduced basal glucose oxidation whereas glucose-induced oxygen consumption (tested with diazoxide) was unaffected. Preconditioning with diaxoxide enhanced insulin contents in transplants of rat islets to nondiabetic rats and lowered hyperglycemia vs. non-preconditioned islets in streptozotocin-diabetic rats. Preconditioning of human islet transplants lowered hyperglycemia in streptozotocin-diabetic nude mice.
Conclusions:
1) Prior blocking of Ca2+ inflow associates with lesser hypoxia-induced damage,
2) preconditioning affects basal mitochondrial metabolism and accelerates activation of hypoxia-reactive and potentially protective factors,
3) results indicate that preconditioning by K+-ATP-channel openers has therapeutic potential for islet transplantations.

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Adrenal Cortex

Posted in Curation, Disease Biology, Endocrine Diseases, tagged adrenal gland, aldosterone, cortex, corticosteroid, medulla, pregnenalone on February 7, 2015| Leave a Comment »

Adrenal Cortex

Writer and Curator: Larry H Bernstein, MD, FCAP

Anatomy of the Adrenal Glands

The adrenal glands are two, triangular-shaped organs that measure about 1.5 inches in height and 3 inches in length. They are located on top of each kidney. Their name directly relates to their location (ad—near or at; renes—kidneys).

Each adrenal gland is comprised of two distinct structures—the outer part of the adrenal glands is called the adrenal cortex. The inner region is known as the adrenal medulla.

Hormones of the Adrenal Glands

The adrenal cortex and the adrenal medulla have very different functions. One of the main distinctions between them is that the hormones released by the adrenal cortex are necessary for life; those secreted by the adrenal medulla are not.

Adrenal Cortex Hormones

Typically, endocrinologists classify steroid hormones into five groups of molecules, based primarily on the receptor to which they bind:

Glucocorticoids; cortisol is the major representative in most mammals

Mineralocorticoids; aldosterone being most prominent

Androgens such as testosterone

Estrogens, including estrodiol and estrone

Progestogens (also known a progestins) such as progesterone

Corticosteroids: The class of chemicals that includes the steroid hormones that are produced in the adrenal cortex of vertebrates, and synthetic analogues of these hormones. They are involved in the stress response, immune response, and regulation of inflammation, carbohydrate metabolism, protein catabolism, blood electrolyte levels, and behavior.

The adrenal cortex produces two main groups of corticosteroid hormones—glucocorticoids and mineralocorticoids. The release of glucocorticoids is triggered by the hypothalamus and pituitary gland. Mineralocorticoids are mediated by signals triggered by the kidney.

When the hypothalamus produces corticotrophin-releasing hormone (CRH), it stimulates the pituitary gland to release adrenal corticotrophic hormone (ACTH). These hormones, in turn, alert the adrenal glands to produce corticosteroid hormones.

Glucocorticoids such as cortisol control carbohydrate, fat and protein metabolism, and are anti-inflammatory.

Glucocorticoids released by the adrenal cortex include:

Hydrocortisone: Commonly known as cortisol regulates how the body converts fats, proteins, and carbohydrates to energy. It also helps regulate blood pressure and cardiovascular function.

Corticosterone: This hormone works with hydrocortisone to regulate immune response and suppress inflammatory reactions.

Mineralocorticoids such as aldosterone control electrolyte and water levels, mainly by promoting sodium retention in the kidney, thereby maintaining the right balance of salt and water while helping control blood pressure.

There is a third class of hormone released by the adrenal cortex in small amounts is sex steroids or sex hormones – primarily released by the testes or ovaries
(estrogen and testosterone) under the influence of gonadotrophic hormone.
http://www.endocrineweb.com/endocrinology/overview-adrenal-glands

Class of Steroid	Major Representative	Physiologic Effects
Mineralocorticoids	Aldosterone	Na+, K+ and water homeostasis
Glucocorticoids	Cortisol	Glucose homeostasis and many others

Adrenal Corticosteroids Animation
http://www.muschealth.com/video/Default.aspx?cId=5

Like all steroids, adrenal “corticosteroids” are synthesized from cholesterol through a series of enzyme-mediated transformations. The details of these pathways are presented elsewhere, but the major branches are easy to understand.

steroid synthesis

http://www.vivo.colostate.edu/hbooks/pathphys/endocrine/adrenal/stersyn.gif

http://en.wikipedia.org/wiki/Corticosteroid

Steroidogenesis

The basic cyclopentanoperhydrophenanthrene ring structure and carbon numbering system of all steroid hormones is depicted to the right, using pregnenolone as an example. Pregnenolone is an example of what is called a “C-21 steroid” because it has 21 carbons. Similarly, a steroid such as testosterone (see below) is referred to as a “C-19 steroid”.

pregnenolone

http://www.vivo.colostate.edu/hbooks/pathphys/endocrine/basics/pregnenolone.gif

Common name	“Old” name	Current name
Side-chain cleavage enzyme; desmolase	P450SCC	CYP11A1
3 beta-hydroxysteroid dehydrogenase	3 beta-HSD	3 beta-HSD
17 alpha-hydroxylase/17,20 lyase	P450C17	CYP17
21-hydroxylase	P450C21	CYP21A2
11 beta-hydroxylase	P450C11	CYP11B1
Aldosterone synthase	P450C11AS	CYP11B2
Aromatase	P450aro	CYP19

Biosynthesis of steroid hormones requires a battery of oxidative enzymes located in both mitochondria and endoplasmic reticulum. The rate-limiting step in this process is the transport of free cholesterol from the cytoplasm into mitochondria. Within mitochondria, cholesterol is converted to pregnenolone by an enzyme in the inner membrane called CYP11A1. Pregnenolone itself is not a hormone, but is the immediate precursor for the synthesis of all of the steroid hormones. The following table delineates the enzymes required to synthesize the major classes of steroid hormones.

The biosynthetic pathways for major representatives of these classes of steroid hormones is depicted in the following diagram.

major pathways of steroid synthesis

http://www.vivo.colostate.edu/hbooks/pathphys/endocrine/basics/steroidogenesis.gif

Target-specific utilization of transcriptional regulatory surfaces by the glucocorticoid receptor

Inez Rogatsky, Jen-Chywan Wang, Mika K. Derynck, Daisuke F. Nonaka, et al.
PNAS [1] Nov 25, 2003; 100(24): 13845–13850
http://www.pnas.org/cgi/doi/10.1073/pnas.2336092100

The glucocorticoid receptor (GR) activates or represses transcription depending on the sequence and architecture of the glucocorticoid response elements in target genes and the availability and activity of interacting cofactors. Numerous GR cofactors have been identified, but they alone are insufficient to dictate the specificity of GR action. Furthermore, the role of different functional surfaces on the receptor itself in regulating its targets is unclear, due in part to the paucity of known target genes. Using DNA microarrays and real-time quantitative PCR, we identified genes transcriptionally activated by GR, in a translation-independent manner, in two human cell lines. We then assessed in U2OS osteosarcoma cells the consequences of individually disrupting three GR domains, the N-terminal activation function (AF) 1, the C-terminal AF2, or the dimer interface, on activation of these genes. We found that GR targets differed in their requirements for AF1 or AF2, and that the dimer interface was dispensable for activation of some genes in each class. Thus, in a single cell type, different GR surfaces were used in a gene-specific manner. These findings have strong implications for the nature of gene response element signaling, the composition and structure of regulatory complexes, and the mechanisms of context-specific transcriptional regulation.

Promoter-specific relevance of histone modifications induced by dexamethasone during the regulation of pro-inflammatory mediators

Linda Palma, Stefano Amatori, Ivan Cruz Chamorroa, Mirco Fanelli, Mauro Magnani
Biochimica et Biophysica Acta 1839 (2014) 571–578
http://dx.doi.org/10.1016/j.bbagrm.2014.05.006

Glucocorticosteroids (GCs) are widely used to treat different kinds of chronic inflammatory and immune diseases through transcriptional regulation of inflammatory genes.
Modulation of gene expression by GCs is known to occur through diverse mechanisms of varying relevance to specific classes of genes. Epigenetic modifications are indeed a pivotal regulatory feature of glucocorticoid receptor and other transcription factors.
In this study, histone post-translational modifications were investigated for their involvement in the regulation of selected pro-inflammatory genes – expressed in human monocyte-derived macrophages – in response to treatment with synthetic GC dexamethasone (DEX). We show that histone tail acetylation status is modified following DEX administration, through distinct and alternative mechanisms at the promoters of interleukin-8 and interleukin-23. In addition to histone H3 acetylation, our results demonstrate that H3 lysine 4 trimethylation is affected following drug treatment.
The CYP11B subfamily

Lina Schiffer, S Anderko, F Hannemann, A Eiden-Plach, R Bernhardt
Journal of Steroid Biochemistry & Molecular Biology xxx (2014) xxx–xxx
http://dx.doi.org/10.1016/j.jsbmb.2014.10.011

The biosynthesis of steroid hormones is dependent on P450-catalyzed reactions. In mammals, cholesterol is the common precursor of all steroid hormones, and its conversion to pregnenolone is the initial and rate-limiting step in hormone biosynthesis in steroidogenic tissues such as gonads and adrenal glands. The production of glucocorticoids and mineralocorticoids takes place in the adrenal gland and the final steps are catalyzed by 2 mitochondrial cytochromes P450, CYP11B1 (11b-hydroxylase or P45011b) and CYP11B2 (aldosterone synthase or P450aldo). The occurrence and development of these 2 enzymes in different species, their contribution to the biosynthesis of steroid hormones as well as their regulation at different levels (gene expression, cellular regulation, regulation on the level of proteins) is the topic of this chapter
Two-pore domain potassium channels in the adrenal cortex

Sascha Bandulik, Philipp Tauber, Enzo Lalli, Jacques Barhanin & Richard Warth
Pflugers Arch – Eur J Physiol Oct 2014
http://dx.doi.org:/10.1007/s00424-014-1628-6

The physiological control of steroid hormone secretion from the adrenal cortex depends on the function of potassium channels. The “two-pore domain K+ channels” (K2P) TWIK-related acid sensitive K+ channel 1 (TASK1), TASK3, and TWIK-related K+ channel 1 (TREK1) are strongly expressed in adrenocortical cells. They confer a background K+ conductance to these cells which is important for the K+ sensitivity as well as for angiotensin II and adrenocorticotropic hormone-dependent stimulation of aldosterone and cortisol synthesis. Mice with single deletions of the Task1 or Task3 gene as well as Task1/Task3 double knockout mice display partially autonomous aldosterone synthesis. It appears that TASK1 and TASK3 serve different functions: TASK1 affects cell differentiation and prevents expression of aldosterone synthase in the zona fasciculata, while TASK3 controls aldosterone secretion in glomerulosa cells. TREK1 is involved in the regulation of cortisol secretion in fasciculata cells. These data suggest that a disturbed function of K2P channels could contribute to adrenocortical pathologies in humans.

Visinin-like peptide 1 in adrenal gland of the rat. Gene expression andits hormonal control

Marcin Trejter, A Hochol, M Tyczewska, A Ziolkowska, K Jopek, et al.
Peptides 63 (2015) 22–29
http://dx.doi.org/10.1016/j.peptides.2014.10.017

tVSNL1 encodes the calcium-sensor protein visinin-like 1 and was identified previously as an upregulated gene in a sample set of aldosterone-producing adenomas. Recently, by means of microarray studies we demonstrated high expression of Vsnl1 gene in rat adrenal zona glomerulosa (ZG). Only scanty data are available on the role of this gene in adrenal function as well as on regulation of its expression by factors affecting adrenal cortex structure and function. Therefore we performed relevant studies aimed at clarifying some of the above issues. By Affymetrix®Rat Gene 1.1 ST Array Strip, QPCR and immunohistochemistry we demonstrated that expression levels of Vsnl1 in the rat adrenal ZG are notably higher than in the fasciculata/reticularis zone. In QPCR assay this difference was approximately 10 times higher. Expression of this gene in the rat adrenal gland or adrenocortical cells was acutely down regulated by ACTH, while chronic administration of corticotrophin or dexamethasone did not change Vsnl1 mRNAlevels. In enucleation-induced adrenocortical regeneration expression levels of both Vsnl1 and Cyp11β2 were notably lowered and positively correlated. Despite these findings, the physiological significance of adrenal Vsnl1 remains unclear, and requires further investigation.
Sox2 Acts in a Dose-Dependent Fashion to Regulate Proliferation of Cortical Progenitors

Daniel W. Hagey, Jonas Muhr
Hagey & Muhr, 2014, Cell Reports 9, 1908–1920
http://dx.doi.org/10.1016/j.celrep.2014.11.013

Hagey and Muhr show that high levels of Sox2 maintain stem cells of the developing cortex in a slowly self-renewing state by directly repressing cell-cycle genes. They further demonstrate that proneural protein-induced commitment to differentiation induces a rapidly dividing state via the reduction of Sox2 expression levels.

A slow-cycling stem cell state is demarcated by high levels of Sox2 in the cortex

Proneural bHLHs repress Sox2 levels as a prerequisite to commit cells to proliferation

Regulatory DNA regions interpret Sox2 levels via high- and low affinity motifs

Sox2 represses Cyclin D1 in synergy with Tcf/Lef through the recruitment of Gro/Tle
reduction of Sox2 expression

reduction of Sox2 expression

Silencing diacylglycerol kinase-theta expression reduces steroid hormone biosynthesis and cholesterol metabolism in human adrenocortical cells

Kai Cai, Natasha C. Lucki, Marion B. Sewer
Biochimica et Biophysica Acta 1841 (2014) 552–562
http://dx.doi.org/10.1016/j.bbalip.2013.12.005

Diacylglycerol kinase theta (DGKθ) plays a pivotal role in regulating adrenocortical steroidogenesis by synthesizing the ligand for the nuclear receptor steroidogenic factor 1 (SF1). In response to activation of the cAMP signaling cascade nuclear DGK activity is rapidly increased, facilitating PA-mediated, SF1-dependent transcription of genes required for cortisol and dehydroepiandrosterone (DHEA) biosynthesis. Based on our previous work identifying DGKθ as the enzyme that produces the agonist for SF1, we generated a tetracycline-inducible H295R stable cell line to express a short hairpin RNA (shRNA) against DGKθ and characterized the effect of silencing DGKθ on adrenocortical gene expression. Genome-wide DNA microarray analysis revealed that silencing DGKθ expression alters the expression of multiple genes, including steroidogenic genes, nuclear receptors and genes involved in sphingolipid, phospholipid and cholesterol metabolism. Interestingly, the expression of sterol regulatory element binding proteins (SREBPs) was also suppressed. Consistent with the suppression of SREBPs, we observed a down-regulation of multiple SREBP target genes, including 3-hydroxy-3-methylglutaric coenzyme A reductase (HMG-CoA red) and CYP51, concomitant with a decrease in cellular cholesterol. DGKθ knockdown cells exhibited a reduced capacity to metabolize PA, with a down-regulation of lipin and phospholipase D (PLD) isoforms. In contrast, suppression of DGKθ increased the expression of several genes in the sphingolipid metabolic pathway, including acid ceramidase (ASAH1) and sphingosine kinases (SPHK). In summary, these data demonstrate that DGKθ plays an important role in steroid hormone production in human adrenocortical cells.
Origin of the response to adrenal and sex steroids: Roles of promiscuity and co-evolution of enzymes and steroid receptors

Michael E. Baker, David R. Nelson, Romain A. Studer
Journal of Steroid Biochemistry & Molecular Biology xxx (2015) xxx–xxx
http://dx.doi.org/10.1016/j.jsbmb.2014.10.020

Many responses to adrenal and sex steroids are mediated by receptors that belong to the nuclear receptor family of transcription factors. We investigated the co-evolution of these vertebrate steroid receptors and the enzymes that synthesize adrenal and sex steroids through data mining of genomes from cephalochordates [amphioxus], cyclostomes [lampreys, hagfish], chondrichthyes [sharks, rays, skates], actinopterygii [ray-finned fish], sarcopterygii [coelacanths, lungfishes and terrestrial vertebrates]. An ancestor of the estrogen receptor and 3-ketosteroid receptors evolved in amphioxus. A corticoid receptor and a progesterone receptor evolved in cyclostomes, and an androgen receptor evolved in gnathostomes. Amphioxus contains CYP11, CYP17, CYP19, 3b/D5–4-HSD and 17b-HSD14, which suffice for the synthesis of estradiol and D5-androstenediol. Amphioxus also contains CYP27, which catalyzes the synthesis of 27-hydroxy-cholesterol, another estrogen. Lamprey contains, in addition, CYP21, which catalyzes the synthesis of 11-deoxycortisol. Chondrichthyes contain, in addition, CYP11A, CYP11C, CYP17A1, CYP17A2. Coelacanth also contains CYP11C1, the current descendent from a common ancestor with modern land vertebrate CYP11B genes, which catalyze the synthesis of cortisol, corticosterone and aldosterone. Interestingly, CYP11B2, aldosterone synthase, evolved from separate gene duplications in at least old world monkeys and two suborders of rodents. Sciurognathi (including mice and rats) and Hystricomorpha (including guinea pigs). Thus, steroid receptors and steroidogenic enzymes co-evolved at key transitions in the evolution of vertebrates. Together, this suite of receptors and enzymes through their roles in transcriptional regulation of reproduction, development, homeostasis and the response to stress contributed to the evolutionary diversification of vertebrates. This article is part of a Special Issue entitled ‘Steroid/Sterol signaling’.
Stress and glucocorticoid receptor-dependent mechanisms in long-term memory: From adaptive responses to psychopathologies

Charles Finsterwald, Cristina M. Alberini
Neurobiology of Learning and Memory 112 (2014) 17–29
http://dx.doi.org/10.1016/j.nlm.2013.09.017

A proper response against stressors is critical for survival. In mammals, the stress response is primarily mediated by secretion of glucocorticoids via the hypothalamic–pituitary–adrenocortical (HPA) axis and release of catecholamines through adrenergic neurotransmission. Activation of these pathways results in a quick physical response to the stress and, in adaptive conditions, mediates long-term changes in the brain that lead to the formation of long-term memories of the experience. These long-term memories are an essential adaptive mechanism that allows an animal to effectively face similar demands again. Indeed, a moderate stress level has a strong positive effect on memory and cognition, as a single arousing or moderately stressful event can be remembered for up to a lifetime. Conversely, exposure to extreme, traumatic, or chronic stress can have the opposite effect and cause memory loss, cognitive impairments, and stress-related psychopathologies such as anxiety disorders, depression and post-traumatic stress disorder (PTSD). While more effort has been devoted to the understanding of the negative effects of chronic stress, much less has been done thus far on the identification of the mechanisms engaged in the brain when stress promotes long-term memory formation. Understanding these mechanisms will provide critical information for use in ameliorating memory processes in both normal and pathological conditions. Here, we will review the role of glucocorticoids and glucocorticoid receptors (GRs) in memory formation and modulation. Furthermore, we will discuss recent findings on the molecular cascade of events underlying the effect of GR activation in adaptive levels of stress that leads to strong, long-lasting memories. Our recent data indicate that the positive effects of GR activation on memory consolidation critically engage the brain-derived neurotrophic factor (BDNF) pathway. We propose and will discuss the hypothesis that stress promotes the formation of strong long-term memories because the activation of hippocampal GRs after learning is coupled to the recruitment of the growth and pro-survival BDNF/cAMP response element-binding protein (CREB) pathway, which is well-known to be a general mechanism required for long-term memory formation. We will then speculate about how these results may explain the negative effects of traumatic or chronic stress on memory and cognitive functions.

Autophagy in the endocrine glands

Andrea Weckman, Antonio Di Ieva, Fabio Rotondo, Luis V Syro, Leon D Ortiz, et al.
Journal of Molecular Endocrinology (2014) 52, R151–R163
http://dx.doi.org:/10.1530/JME-13-0241

Autophagy is an important cellular process involving the degradation of intracellular components. Its regulation is complex and while there are many methods available, there is currently no single effective way of detecting and monitoring autophagy. It has several cellular functions that are conserved throughout the body, as well as a variety of different physiological roles depending on the context of its occurrence in the body. Autophagy is also involved in the pathology of a wide range of diseases. Within the endocrine system, autophagy has both its traditional conserved functions and specific functions. In the endocrine glands, autophagy plays a critical role in controlling intracellular hormone levels. In peptide-secreting cells of glands such as the pituitary gland, crinophagy, a specific form of autophagy, targets the secretory granules to control the levels of stored hormone. In steroid-secreting cells of glands such as the testes and adrenal gland, autophagy targets the steroid-producing organelles. The dysregulation of autophagy in the endocrine glands leads to several different endocrine diseases such as diabetes and infertility. This review aims to clarify the known roles of autophagy in the physiology of the endocrine system, as well as in various endocrine diseases.
Angiotensin IV stimulates high atrial stretch-induced ANP secretionvia insulin regulated aminopeptidase

Byung Mun Park, Seung Ah Cha, Bo Ram Han, Suhn Hee Kim
Peptides 63 (2015) 30–37
http://dx.doi.org/10.1016/j.peptides.2014.10.016

tAngiotensin IV (Ang IV) is formed by aminopeptidase N (APN) from angiotensin III (Ang III) by remov-ing the first N-terminal amino acid. Previouslt, we reported that angiotensin II (Ang II) inhibits atrialnatriuretic peptide (ANP) secretion via angiotensin II type 1 receptor (AT1R). In contrast, angiotensin-(1–7) [Ang-(1–7)] and Ang III stimulate ANP secretion via Mas receptor (Mas R) and angiotensin II type 2receptor (AT2R), respectively. However, it is not known whether there is any relationship between AngIV and ANP secretion. Therefore, the aim of the present study was to determine the effect of Ang IV onANP secretion and to find its downstream signaling pathway using in isolated perfused beating atria. AngIV (0.1, 1 and 10 [1]M) stimulated high atrial stretch-induced ANP secretion and ANP concentration in adose-dependent manner. The augmented effect of Ang IV (1 [1]M) on high atrial stretch-induced ANP secre-tion and concentration was attenuated by pretreatment with insulin-regulated aminopeptidase (IRAP)antagonist but not by AT1R or AT2R antagonist. Pretreatment with inhibitors of downstream signalingpathway including phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt) and mammalian targetof rapamycin (mTOR) blocked Ang IV-induced ANP secretion and concentration. Therefore, these results suggest that Ang IV stimulates ANP secretion and concentration via IRAP and PI3K-Akt-mTOR pathway.
Adrenal cortex tissue homeostasis and zonation: A WNT perspective

Coralie Drelon, Annabel Berthon, Mickael Mathieu, Antoine Martinez, Pierre Val
Molecular and Cellular Endocrinology ■■ (2015) ■■–■■
http://dx.doi.org/10.1016/j.mce.2014.12.014

The adrenal cortex plays essential roles in the control of sodium and water homeostasis, stress response, inflammation and metabolism, through secretion of glucocorticoids and mineralocorticoids. Coordinated production of these hormones relies on functional zonation of the cortex, characterized by expression of Cyp11b2 under the control of angiotensin II and plasma potassium level in zona glomerulosa (ZG) and Cyp11b1 under the control of ACTH in zona fasciculata (ZF). The mechanisms involved in the establishment of functional zonation and its maintenance during centripetal cortex cell renewal are still poorly understood. Here, we hypothesize that the hormonal and signaling pathways that control adrenal cortex function are also involved in cortical zonation. In particular, we summarize evidence on the role of WNT/β-catenin signaling in ZG differentiation and how tight control of its activity is required to shape the adult cortex. In this context, we discuss the potential role of known WNT regulators and the possibility of a reciprocal cross-talk between PKA and WNT signaling.
Acute and chronic stress induced changes in sensitivity of peripheral inflammatory pathways to the signals of multiple stress systems — 2011 Curt Richter Award Winner

Nicolas Rohleder
Psychoneuroendocrinology (2012) 37, 307—316
http://dx.doi.org:/10.1016/j.psyneuen.2011.12.015

Exposure to psychosocial stress has been associated with increasing rates of morbidity in humans and in animal models, but the underlying mechanisms are not completely understood. Major stress responsive systems, such as the hypothalamus-pituitary adrenal (HPA) axis and the autonomic nervous system (ANS) are under investigation as underlying pathways, but although acute stress reliably activates these systems, findings of long-term alternations in baseline activity are inconsistent at present. Emerging evidence suggests that stress-related changes in the sensitivity of target systems toward glucocorticoid (GC) regulation, i.e. development of GC resistance, might help explain inflammatory disinhibition and development of disease related to inflammation. More recent findings further show that the autonomic nervous system might play an important role in the regulatory control of the inflammatory cascade. The major argument put forward in this manuscript is that target tissues for stress system modulation, such as the inflammatory cascade, vary in their ability to respond to stress system signaling, and that assessing alterations in this stress signal sensitivity which can be caused by stress or disease processes, might be necessary to understand and explain stress effects on health. This review focuses on the inflammatory system in particular, because anti-inflammatory effects of most stress systems have been documented, but the general assumption might have to be generalized to other target systems. The main conclusion to be made is that reduction in glucocorticoid sensitivity of target tissues is the most consistent finding at present, and that assessing such changes in glucocorticoid sensitivity might be necessary to understand many stress-related changes in physiology.

Adrenal insufficiency

Evangelia Charmandari, Nicolas C Nicolaides, George P Chrousos
The Lancet Jun 21, 2014; 383: 2152-2167
http://dx.doi.org/10.1016/S0140-6736(13)61684-0

Adrenal insufficiency is the clinical manifestation of deficient production or action of glucocorticoids, with or without deficiency also in mineralocorticoids and adrenal androgens. It is a life-threatening disorder that can result from primary adrenal failure or secondary adrenal disease due to impairment of the hypothalamic–pituitary axis. Prompt diagnosis and management are essential. The clinical manifestations of primary adrenal insufficiency result from deficiency of all adrenocortical hormones, but they can also include signs of other concurrent autoimmune conditions. In secondary or tertiary adrenal insufficiency, the clinical picture results from glucocorticoid deficiency only, but manifestations of the primary pathological disorder can also be present. The diagnostic investigation, although well established, can be challenging, especially in patients with secondary or tertiary adrenal insufficiency. We summarize knowledge at this time on the epidemiology, causal mechanisms, pathophysiology, clinical manifestations, diagnosis, and management of this disorder.

Molecular immunopathogenesis of primary adrenal insufficiency

A persistent subclinical viral infection or an aberrant response to inflammatory stressors could cause adrenocortical cell apoptosis or necrosis, leading to dendritic-cell activation by cellular components, including peptides derived from 21-hydroxylase. After activation, dendritic cells transport and present adrenocortical antigens to CD4-positive T-helper-1 (Th1) cells within the local draining lymph node. Activated specific CD4-positive Th1 cells could provide help for the activation and clonal expansion of cytotoxic lymphocytes and autoreactive B cells producing anti-21-hydroxylase and other antibodies. The continuing progressive destruction of adrenal cortex is mediated by several different mechanisms: direct cytotoxicity by apoptosis-inducing cytotoxic lymphocytes via perforin and granzyme B or by the FasL-Fas pathway; direct cytotoxicity by interferon-γ and lymphotoxin-α secreted by CD4-positive Th1 cells; autoantibody-induced activation of the complement system or antibody dependent cellular cytotoxicity; cytotoxic effects of inflammatory cytokines (tumor necrosis factor-α [TNFα], interleukin-1β) and free radicals (nitric oxide, superoxide) released by monocytes and macrophages or by the adrenocortical cells themselves.

1-Phenylsulfinyl-3-(pyridin-3-yl)naphthalen-2-ols: A new class of potent and selective aldosterone synthase inhibitors

Cornelia M. Grombein, Qingzhong Hu, Ralf Heim, Sabrina Rau, Christina Zimmer
European Journal of Medicinal Chemistry 89 (2015) 597e605
http://dx.doi.org/10.1016/j.ejmech.2014.10.027

1-Phenylsulfinyl-3-(pyridin-3-yl)naphthalen-2-ols and related compounds were synthesized and evaluated for inhibition of aldosterone synthase (CYP11B2), a potential target for cardiovascular diseases associated with elevated plasma aldosterone levels like congestive heart failure and myocardial fibrosis. Introduction of substituents at the phenylsulfinyl moiety and changes of the substitution pattern at the naphthalene core were examined. Potent compounds were further examined for selectivity versus other important steroidogenic CYP enzymes, i.e. the highly homologous 11b-hydroxylase (CYP11B1), CYP17 and CYP19. The most potent compound (IC50 ¼ 14 nM) discovered was the meta-trifluoromethoxy derivative 11, which also exhibited excellent selectivity toward CYP11B1 (SF ¼ 415), and showed no inhibition of CYP17 and CYP19.

Structural bisphenol analogues differentially target steroidogenesis in murine MA-10 Leydig cells as well as the glucocorticoid receptor

Maarke J.E. Roelofs, M van den Berg, TFH Bovee, AH Piersma, MBM van Duursen
Toxicology 329 (2015) 10–20
http://dx.doi.org/10.1016/j.tox.2015.01.003

Although much information on the endocrine activity of bisphenol A (BPA) is available, a proper human hazard assessment of analogues that are believed to have a less harmful toxicity profile is lacking. Here the possible effects of BPA, bisphenol F (BPF), bisphenol S (BPS), as well as the brominated structural analogue and widely used flame retardant tetrabromobisphenol A (TBBPA) on human glucocorticoid and androgen receptor (GR and AR) activation were assessed. BPA, BPF, and TBBPA showed clear GR and AR antagonism with IC50 values of 67 mM, 60 mM, and 22 nM for GR, and 39mM, 20 mM, and 982 nM for AR, respectively, whereas BPS did not affect receptor activity. In addition, murine MA-10 Leydig cells exposed to the bisphenol analogues were assessed for changes in secreted steroid hormone levels. Testicular steroidogenesis was altered by all bisphenol analogues tested. TBBPA effects were more directed towards the male end products and induced testosterone synthesis, while BPF and BPS predominantly increased the levels of progestogens that are formed in the beginning of the steroidogenic pathway. The MA-10 Leydig cell assay shows added value over the widely used H295R steroidogenesis assay because of its fetal-like characteristics and specificity for the physiologically more relevant testicular D4 steroidogenic pathway. Therefore, adding an in-vitro assay covering fetal testicular steroidogenesis, such as the MA-10 cell line, to the panel of tests used to screen potential endocrine disruptors, is highly recommendable.
Preclinical Assessment for Selectively Disrupting a Traumatic Memory via Postretrieval Inhibition of Glucocorticoid Receptors

Stephen M. Taubenfeld, Justin S. Riceberg, Antonia S. New, and Cristina M. Alberini
Biol Psychiatry 2009; 65:249–257
http://dx.doi.org:/10.1016/j.biopsych.2008.07.005

Background: Traumatic experiences may lead to debilitating psychiatric disorders including acute stress disorder and posttraumatic stress disorder. Current treatments for these conditions are largely ineffective, and novel therapies are needed. A cardinal symptom of these pathologies is the reexperiencing of the trauma through intrusive memories and nightmares. Studies in animal models indicate that memories can be weakened by interfering with the postretrieval restabilization process known as memory reconsolidation. We previously reported that, in rats, intraamygdala injection of the glucocorticoid receptor antagonist RU38486 disrupts the reconsolidation of a traumatic memory. Here we tested parameters important for designing novel clinical protocols targeting the reconsolidation of a traumatic memory with RU38486.
Methods: Using rat inhibitory avoidance, we tested the efficacy of postretrieval systemic administration of RU38486 on subsequent memory retention and evaluated several key preclinical parameters.
Results: Systemic administration of RU38486 before or after retrieval persistently weakens inhibitory avoidance memory retention in a dose-dependent manner, and memory does not reemerge following a footshock reminder. The efficacy of treatment is a function of the intensity of the initial trauma, and intense traumatic memories can be disrupted by changing the time and number of interventions. Furthermore, one or two treatments are sufficient to disrupt the memory maximally. The treatment selectively targets the reactivated memory without interfering with the retention of another nonreactivated memory.
Conclusions: RU38486 is a potential novel treatment for psychiatric disorders linked to traumatic memories. Our data provide the parameters for designing promising clinical trials for the treatment of flashback-type symptoms of PTSD.

Maternal PTSD associates with greater glucocorticoid sensitivity in offspring of Holocaust survivors

Amy Lehrner, Linda M. Bierer, Vincent Passarelli, et al.
Psychoneuroendocrinology (2014) 40, 213—220
http://dx.doi.org/10.1016/j.psyneuen.2013.11.019

Intergenerational effects of trauma have been observed clinically in a wide range of populations, and parental PTSD has been associated with an increased risk for psychopathology in offspring. In studies of Holocaust survivor offspring, parental PTSD, and particularly maternal PTSD, has been associated with increased risk for PTSD, low basal urinary cortisol excretion and enhanced cortisol suppression in response to dexamethasone. Such findings implicate maternally derived glucocorticoid programming in the intergenerational transmission of trauma-related consequences, potentially resulting from in utero influences or early life experiences. This study investigated the relative influence of Holocaust exposure and PTSD in mothers and fathers on glucocorticoid sensitivity in offspring. Eighty Holocaust offspring and 15 offspring of non-exposed Jewish parents completed evaluations and provided blood and urine samples. Glucocorticoid sensitivity was evaluated using the lysozyme suppression test (LST), an in vitro measure of glucocorticoid receptor sensitivity in a peripheral tissue, the dexamethasone suppression test (DST), and 24-h urinary cortisol excretion. Maternal PTSD was associated with greater glucocorticoid sensitivity in offspring across all three measures of glucocorticoid function. An interaction of maternal and paternal PTSD on the DSTand 24-h urinary cortisol showed an effect of decreased glucocorticoid sensitivity in offspring with paternal, but not maternal, PTSD. Although indirect, these findings are consistent with the hypothesis that epigenetic programming may be involved in the intergenerational transmission of trauma-related effects on glucocorticoid regulation.

Marked Cortisol Production by Intracrine ACTH in GIP Treated Cultured Adrenal Cells in Which the GIP Receptor Was Exogenously Introduced

Hiroko Fujii, M Tamamori-Adachi, K Uchida, T Susa, T Nakakura, et al.
PLOS ONE Oct 2014;9(10):e110543
http://dx.doi.org:/10.1371/journal.pone.0110543

The ectopic expression of the glucose-dependent insulinotropic polypeptide receptor (GIPR) in the human adrenal gland causes significant hypercortisolemia after ingestion of each meal and leads to Cushing’s syndrome, implying that human GIPR activation is capable of robustly activating adrenal glucocorticoid secretion. In this study, we transiently transfected the human GIPR expression vector into cultured human adrenocortical carcinoma cells (H295R) and treated them with GIP to examine the direct link between GIPR activation and steroidogenesis. Using quantitative RT-PCR assay, we examined gene expression of steroidogenic related proteins, and carried out immunofluorescence analysis to prove that forced GIPR overexpression directly promotes production of steroidogenic enzymes CYP17A1 and CYP21A2 at the single cell level. Immunofluorescence showed that the transfection efficiency of the GIPR gene in H295R cells was approximately 5%, and GIP stimulation enhanced CYP21A2 and CYP17A1 expression in GIPR-introduced H295R cells (H295R-GIPR). Interestingly, these steroidogenic enzymes were also expressed in the GIPR (–) cells adjacent to the GIPR (+) cells. The mRNA levels of a cholesterol transport protein required for all steroidogenesis, StAR, and steroidogenic enzymes, HSD3b2, CYP11A1, CYP21A2, and CYP17A1 increased 1.2-2.1-fold in GIP-stimulated H295R-GIPR cells. These changes were reflected in the culture medium in which 1.5-fold increase in the cortisol concentration was confirmed. Furthermore, the levels of adenocorticotropic hormone (ACTH) receptor and ACTH precursor proopiomelanocortin (POMC) mRNA were upregulated 2- and 1.5-fold, respectively. Immunofluorescence showed that ACTH expression was detected in GIP-stimulated H295RGIPR cells. An ACTH-receptor antagonist significantly inhibited steroidogenic gene expression and cortisol production. Immunostaining for both CYP17A1 and CYP21A2 was attenuated in cells treated with ACTH receptor antagonists as well as with POMC siRNA. These results demonstrated that GIPR activation promoted production and release of ACTH, and that steroidogenesis is activated by endogenously secreted ACTH following GIP administration, at least in part, in H295R cells.

Improvements in body composition, cardiometabolic risk factors and insulin sensitivity with trenbolone in normogonadic rats

Daniel G. Donner, BR Beck, AC Bulmer, AK Lam, EF Du Toit, et al.
Steroids 94 (2015) 60–69
http://dx.doi.org/10.1016/j.steroids.2014.12.017

Trenbolone (TREN) is used for anabolic growth-promotion in over 20 million cattle annually and continues to be misused for aesthetic purposes in humans. The current study investigated TREN’s effects on body composition and cardiometabolic risk factors; and its tissue-selective effects on the cardiovascular system, liver and prostate. Male rats (n = 12) were implanted with osmotic infusion pumps delivering either cyclodextrin vehicle (CTRL) or 2 mg/kg/day TREN for 6 weeks. Dual-energy X-ray Absorptiometry assessment of body composition; organ wet weights and serum lipid profiles; and insulin sensitivity were assessed. Cardiac ultrasound examinations were performed before in vivo studies assessed myocardial susceptibility to ischemia–reperfusion (I/R) injury. Circulating sex hormones and liver enzyme activities; and prostate and liver histology were examined. In 6 weeks, fat mass increased by 34 ± 7% in CTRLs (p < 0.01). Fat mass decreased by 37 ± 6% and lean mass increased by 11 ± 4% with TREN (p < 0.05). Serum triglycerides, HDL and LDL were reduced by 62%, 57% and 78% (p < 0.05) respectively in TREN rats. Histological examination of the prostates from TREN-treated rats indicated benign hyperplasia associated with an increased prostate mass (149% compared to CTRLs, p < 0.01). No evidence of adverse cardiac or hepatic effects was observed. In conclusion, improvements in body composition, lipid profile and insulin sensitivity (key risk factors for cardiometabolic disease) were achieved with six-week TREN treatment without evidence of adverse cardiovascular or hepatic effects that are commonly associated with traditional anabolic steroid misuse. Sex hormone suppression and benign prostate hyperplasia were confirmed as adverse effects of the treatment.
Gender-dependent changes in hematological parameters in patients with Cushing’s disease before and after remission

Ambrogio, A.G., De Martin, M., Ascoli, P., Cavagnini, F., Giraldi, F.P

European Journal of Endocrinology Mar 2014; 170(3): 393-400

Objective: Glucocorticoids stimulate several steps in red blood cell (RBC) development; however, little is known on changes in erythroid parameters in patients with Cushing’s disease. The aim of this study was to assess both RBC and white blood cell (WBC) parameters in a large cohort of patients with Cushing’s disease and report on alterations in the active phase and after surgical remission. Design and methods: A total of 80 patients with Cushing’s disease (63 women and 17 men) were studied before and for up to 254 months’ follow-up (mean follow-up 65.8±6.71 months) after pituitary/adrenal surgery. Details of blood counts were reviewed and compared with data obtained from a database of healthy subjects. Results: The RBC counts and hemoglobin levels were low in men with active Cushing’s disease (over 80% of values in the lowest quartile) and four patients were overtly anemic, whereas erythrocyte counts and hemoglobin levels were evenly distributed across the normal range in women with active Cushing’s disease. Low erythroid parameters were linked to hypogonadism in men with Cushing’s disease. Recovery in erythroid parameters occurred slowly after remission of hypercortisolism in men, in parallel with improvements in testosterone levels. Over 50% of patients with active disease presented increased WBC counts, irrespective of gender, and prompt normalization within 1 month after surgery. Conclusions: Male patients with Cushing’s disease present reduced RBC counts and hemoglobin levels, associated with low testosterone concentrations, which resolve over time after remission of hypercortisolism. Anemia should therefore be regarded as another unfavorable feature in men with Cushing’s disease.
Myopathies of endocrine disorders: A prospective clinical and biochemical study

Sharma, V., Borah, P., Basumatary, L.J., (…), Goswami, M., Kayal, A.K
Annals of Indian Academy of Neurology 2014; 17 (3), pp. 298-302
http://dx.doi.org:/10.4103/0972-2327.138505

Introduction: Major categories of endocrine myopathy include those associated with: Adrenal dysfunction (as in Cushing’s disease or steroid myopathy); thyroid dysfunction (as in myxedema coma or thyrotoxic myopathy); vitamin D deficiency; parathyroid dysfunction; and pituitary dysfunction. Steroid myopathy is the most common endocrine myopathy. Objective: To study the etiology, varied presentations, and outcome after therapy of patients with endocrine myopathies. Materials and Methods: Myopathy was evaluated by the standard clinical procedures: Detailed clinical history, manual muscle strength testing, and creatine phosphokinase (CPK). Endocrine disorders were diagnosed as per clinical features and biochemical parameters. The treatment was given to patients as per underlying endocrine disease. Myopathy was assessed before and after treatment. Results: Out of the 37 patients who were diagnosed with endocrine myopathies, thyroid dysfunction was the most common cause (17 cases), followed by vitamin D deficiency in nine, adrenal dysfunction in six, parathyroid dysfunction in three, and pituitary dysfunction in two. Some patients had atypical presentation (repeated falls in one, tongue fasciculations in one, neck weakness in five, one with ptosis and facial weakness, asymmetrical onset in one, and calf hypertrophy in one. The serum creatine kinase (CK) concentration did not correlate with muscle weakness. Following the treatment regimen which was specific for a given myopathy, 26 patients recovered fully. Conclusion: We found varied clinical presentations of endocrine myopathies. All the patients with neuromuscular complaints should be investigated for endocrine causes because significant number of them recovers fully with specific treatment.
Human Sterol Regulatory Element-Binding Protein 1a Contributes Significantly to Hepatic Lipogenic Gene Expression

Andreas Bitter, Andreas K. Nüssler, Wolfgang E. Thasler, Kathrin Klein, et al.
Cell Physiol Biochem 2015;35:803-815
http://dx.doi.org:/10.1159/000369739

Background/Aims: Sterol regulatory element-binding protein (SREBP) 1, the master regulator of lipogenesis, was shown to be associated with non-alcoholic fatty liver disease, which is attributed to its major isoform SREBP1c. Based on studies in mice, the minor isoform SREBP1a is regarded as negligible for hepatic lipogenesis. This study aims to elucidate the expression and functional role of SREBP1a in human liver. Methods: mRNA expression of both isoforms was quantified in cohorts of human livers and primary human hepatocytes. Hepatocytes were treated with PF-429242 to inhibit the proteolytic activation of SREBP precursor protein. SREBP1a-specific and pan-SREBP1 knock-down were performed by transfection of respective siRNAs. Lipogenic SREBP-target gene expression was analyzed by real-time RT-PCR. Results: In human liver, SREBP1a accounts for up to half of the total SREBP1 pool. Treatment with PF-429242 indicated SREBP-dependent auto-regulation of SREBP1a, which however was much weaker than of SREBP1c. SREBP1a-specific knock-down also reduced significantly the expression of SREBP1c and of SREBP-target genes. Regarding most SREBP-target genes, simultaneous knock-down of both isoforms resulted in effects of only similar extent as SREBP1a-specific knock-down. Conclusion: We here showed that SREBP1a is significantly contributing to the human hepatic SREBP1 pool and has a share in human hepatic lipogenic gene expression.
Greater glucocorticoid receptor activation in hippocampus of aged rats sensitizes microglia

Ruth M. Barrientos, Vanessa M. Thompson, Meagan M. Kitt, Jose Amat, et al.
Neurobiology of Aging xxx (2015) 1e13
http://dx.doi.org/10.1016/j.neurobiolaging.2014.12.003

Healthy aging individuals are more likely to suffer profound memory impairments following an immune challenge than are younger adults. These challenges produce a brain inflammatory response that is exaggerated with age. Sensitized microglia found in the normal aging brain are responsible for this amplified response, which in turn interferes with processes involved in memory formation. Here, we examine factors that may lead aging to sensitize microglia. Aged rats exhibited higher corticosterone levels in the hippocampus, but not in plasma, throughout the daytime (diurnal inactive phase). These elevated hippocampal corticosterone levels were associated with increased hippocampal 11β-hydroxysteroid dehydrogenase type 1 protein expression, the enzyme that catalyzes glucocorticoid formation and greater hippocampal glucocorticoid receptor (GR) activation. Intracisternal administration of mifepristone, a GR antagonist, effectively reduced immune-activated proinflammatory responses, specifically from hippocampal microglia and prevented Escherichia coli induced memory impairments in aged rats. Voluntary exercise as a therapeutic intervention significantly reduced total hippocampal GR expression. These data strongly suggest that increased GR activation in the aged hippocampus plays a critical role in sensitizing microglia.
Glucocorticoid Receptor-Mediated Cell Cycle Arrest Is Achieved through Distinct Cell-Specific Transcriptional Regulatory Mechanisms

Inez Rogatsky, Janet M. Trowbridge, and Michael J. Garabedian
Molecular and Cellular Biology, Jun 1997; 17(6): 3181–3193

Glucocorticoids inhibit proliferation of many cell types, but the events leading from the activated glucocorticoid receptor (GR) to growth arrest are not understood. Ectopic expression and activation of GR in human osteosarcoma cell lines U2OS and SAOS2, which lack endogenous receptors, result in a G1 cell cycle arrest. GR activation in U2OS cells represses expression of the cyclin-dependent kinases (CDKs) CDK4 and CDK6 as well as their regulatory partner, cyclin D3, leading to hypophosphorylation of the retinoblastoma protein (Rb). We also demonstrate a ligand-dependent reduction in the expression of E2F-1 and c-Myc, transcription factors involved in the G1-to-S-phase transition. Mitogen-activated protein kinase, CDK2, cyclin E, and the CDK inhibitors (CDIs) p27 and p21 are unaffected by receptor activation in U2OS cells. The receptor’s N-terminal transcriptional activation domain is not required for growth arrest in U2OS cells. In Rb-deficient SAOS2 cells, however, the expression of p27 and p21 is induced upon receptor activation. Remarkably, in SAOS2 cells that express a GR deletion derivative lacking the N-terminal transcriptional activation domain, induction of CDI expression is abolished and the cells fail to undergo ligand-dependent cell cycle arrest. Similarly, murine S49 lymphoma cells, which, like SAOS2 cells, lack Rb, require the N-terminal activation domain for growth arrest and induce CDI expression upon GR activation. These cell-type-specific differences in receptor domains and cellular targets linking GR activation to cell cycle machinery suggest two distinct regulatory mechanisms of GR-mediated cell cycle arrest: one involving transcriptional repression of G1 cyclins and CDKs and the other involving enhanced transcription of CDIs by the activated receptor.
Glucocorticoid receptor binds half sites as a monomer and regulates specific target genes

Benjamin J Schiller, R Chodankar, LC Watson, MR Stallcup and KR Yamamoto
Genome Biology 2014; 15:418 http://genomebiology.com/2014/15/7/418

Background: Glucocorticoid receptor (GR) is a hormone-activated, DNA-binding transcriptional regulatory factor that controls inflammation, metabolism, stress responses, and other physiological processes. In vitro, GR binds as an inverted dimer to a motif consisting of two imperfectly palindromic 6 bp half sites separated by 3 bp spacers. In vivo, GR employs different patterns of functional surfaces of GR to regulate different target genes. The relationships between GR genomic binding and functional surface utilization have not been defined. Results: We find that A477T, a GR mutant that disrupts the dimerization interface, differs from wild-type GRα in binding and regulation of target genes. Genomic regions strongly occupied by A477T are enriched for a novel half site motif. In vitro, GRα binds half sites as a monomer. Through the overlap between GRα- and A477T-bound regions, we identify GRα-bound regions containing only half sites. We further identify GR target genes linked with half sites and not with the full motif. Conclusions: Genomic regions bound by GR differ in underlying DNA sequence motifs and in the GR functional surfaces employed for regulation. Identification of GR binding regions that selectively utilize particular GR surfaces may discriminate sub-motifs, including the half site motif, that favor those surfaces. This approach may contribute to predictive models for GR activity and therapy.
Cortisol responses to chronic stress in adult macaques: Moderation by a polymorphism in the serotonin transporter gene

Dongdong Qina, Joshua Rizaka, Xiaoli Feng, Shangchuan Yang, Lichuan Yang
Behavioural Brain Research 278 (2015) 280–285
http://dx.doi.org/10.1016/j.bbr.2014.10.001

Accumulating evidence has shown that a polymorphism in the promoter region of the serotonin transporter gene (5-HTTLPR) moderates the association between stress and depressive symptoms. However, the exact etiologies underlying this moderation are not well understood. Here it is reported that among adult female rhesus macaques, an orthologous polymorphism (rh5-HTTLPR) exerted an influence on cortisol responses to chronic stress. It was found that females with two copies of the short allele were associated with increased cortisol responses to chronic stress in comparison to their counterparts who have one or two copies of the long allele. In the absence of stress, no differences related to genotype were observed in these females. This genetic moderation was found without a genetic influence on exposure to stressful situations. Rather it was found to be a genetic modulation of cortisol responses to chronic stress. These findings indicate that the rh5-HTTLPR polymorphism is closely related to hypothalamus–pituitary–adrenal (HPA) axis reactivity, which may increase susceptibility to depression in females with low serotonin transporter efficiency and a history of stress.
Downregulation of the glucocorticoid-induced leucine zipper (GILZ) promotes vascular inflammation

Rebecca T. Hahn, Jessica Hoppstädter, Kerstin Hirschfelder, Nina Hachenthal, et al.
Atherosclerosis 234 (2014) 391e400
http://dx.doi.org/10.1016/j.atherosclerosis.2014.03.028

Objective: Glucocorticoid-induced leucine zipper (GILZ) represents an anti-inflammatory mediator, whose downregulation has been described in various inflammatory processes. Aim of our study was to decipher the regulation of GILZ in vascular inflammation. Approach and results: Degenerated aortocoronary saphenous vein bypass grafts (n = 15), which exhibited inflammatory cell activation as determined by enhanced monocyte chemoattractrant protein 1 (MCP-1, CCL2) and Toll-like receptor 2 (TLR2) expression, showed significantly diminished GILZ protein and mRNA levels compared to healthy veins (n = 23). GILZ was also downregulated in human umbilical vein endothelial cells (HUVEC) and macrophages upon treatment with the inflammatory cytokine TNF-α in a tristetraprolin (ZFP36, TTP)- and p38 MAPK-dependent manner. To assess the functional implications of decreased GILZ expression, we determined NF-kB activation after GILZ knockdown by siRNA and found that NF-kB activity and inflammatory gene expression were significantly enhanced. Importantly, ZFP36 is induced in TNF-α-activated HUVEC as well as in degenerated vein bypasses. When atheroprotective laminar shear stress was employed, GILZ levels in HUVEC increased on mRNA and protein level. Laminar flow also counteracted TNF-α-induced ZFP36 expression and GILZ downregulation. MAP kinase phosphatase 1 (MKP-1, DUSP1), a negative regulator of ZFP36 expression, was distinctly upregulated under laminar shear stress conditions and downregulated in degenerated vein bypasses. Conclusion: Our data show a diminished expression of the anti-inflammatory mediator GILZ in the inflamed vasculature and indicate that GILZ downregulation requires the mRNA binding protein ZFP36. We suggest that reduced GILZ levels play a role in cardiovascular disease.
Effects of Angiotensin Converting Enzyme Inhibition or Angiotensin Receptor Blockade in Dialysis Patients: A Nationwide Data Survey and Propensity Analysis

Cho-Kai Wu, Yao-Hsu Yang, Jyh-Ming Jimmy Juang, et al.
Medicine Jan 2015; 94(3): 1-7 Medicine 94(3):e424)
http://dx.doi.org:/10.1097/MD.0000000000000424

Long-term benefit of using a renin–angiotensin–aldosterone system blocker (an angiotensin-converting enzyme inhibitor (ACEI), or an angiotensin II receptor blocker (ARB)) for patients already receiving dialysis remains undetermined. The aim of this study is to assess the efficacy and safety of ACEI or ARB use in dialysis patients. We performed a population-based cohort study with time-to-event analyses to estimate the relation between the use of ACEI/ARB and their outcomes. We used a nationwide database (Registry for Catastrophic Illnesses) for Taiwan, which has data from 1995 to 2008 nearly of all patients who received dialysis therapy. The records of all dialysis patients aged 18 with no evidence of cardiovascular (CV) events in 1997 and 1998 (133,564 patients) were examined. Users (n=50,961) and nonusers (n=59,913) of an ACEI/ARB were derived. We then used propensity score matching and Cox proportional hazards regression models to estimate adjusted hazard ratios (HRs) for all-cause mortality and CV events in users and nonusers of an ACRI/ARB. The 15,182 patients, who used an ACEI/ARB, and the 15,182 nonusers had comparable baseline characteristics during the 14 years of follow-up. The mortality was significantly greater in patients who did not use an ACEI/ARB (HR=0.90, 95% CI=0.86–0.93). Subgroup analysis of 3 tertiles of patients who used different total amounts of ACEI/ARB during the study period indicated that CV events were more common in patients who used an ACEI/ARB for a short duration (tertile 1: HR=1.63), but less common in those who used an ACEI/ARB for long durations (tertile 2: HR=1.05; tertile 3: HR=0.94; trend for declining HR from tertile 1 to 3: P<0.001). The mortality benefit provided by use of an ACEI/ARB was consistent across most patient subgroups, as was the benefit of ARB monotherapy rather than ACEI monotherapy. Independent of traditional risk factors, overall mortality was significantly lower in dialysis patients who used an ACEI/ARB. In addition, subjects who used an ACEI/ARB for longer durations were significantly less likely to experience CV events.
Adrenal CYP11B1/2 expression in primary aldosteronism: Immunohistochemical analysis using novel monoclonal antibodies

Yasuhiro Nakamura, Takashi Maekawa, Saulo J.A. Felizola, Fumitoshi Satoh, et al.
Biochimica et Biophysica Acta 1842 (2014) 2601–2609
http://dx.doi.org/10.1016/j.bbadis.2014.05.036

CYP11B1 and CYP11B2 play pivotal roles in adrenocorticosteroids synthesis. We performed semiquantitative immunohistochemical analysis of these proteins in adrenals from patients with primary aldosteronism using novel monoclonal antibodies. Clusters of cortical cells positive for CYP11B2 were detected in the zona glomerulosa (ZG) of normal adrenal gland (NA), idiopathic hyperaldosteronism (IHA) and the adjacent adrenal of aldosterone-producing adenoma (APA). In APA, heterogenous immunolocalization of CYP11B2 and diffuse immunoreactivity of CYP11B1 were detected in tumor cells, respectively. The relative immunoreactivity of CYP11B2 in the ZG of adjacent adrenal of APA was significantly lower than that of NA, IHA and APA tumor cells, suggestive of suppressed aldosterone biosynthesis in these cells. These findings did indicate the regulatory mechanisms of aldosterone biosynthesis were different between normal/hyperplastic and neoplastic aldosterone-producing cells in human adrenals. CYP11B2 immunoreactivity in the ZG could also serve as a potential immunohistochemical marker differentiating morphologically hyperplastic ZG of IHA and APA adjacent adrenal.

Aldosterone signaling and soluble adenylyl cyclase—A nexus for the kidney and vascular endothelium

Boris Schmitz, Stefan-Martin Brand, Eva Brand
Biochimica et Biophysica Acta 1842 (2014) 2601–2609
http://dx.doi.org/10.1016/j.bbadis.2014.05.036

The steroid hormone aldosterone regulates the reabsorption of water and ions in the kidney and plays a central role in blood pressure regulation and homeostasis. In recent years, the vascular endothelium has been established as an important aldosterone target organ with major implications in renal and cardiovascular health and disease. Different lines of evidence suggest that the calcium- and bicarbonate-activated soluble adenylyl cyclase (sAC) is a novel mediator of aldosterone signaling in both the kidney and vascular endothelium. This review summarizes our current understanding of the molecular mechanisms of sAC gene expression regulation in the kidney and vascular endothelium and outlines the potential clinical implications of sAC in chronic kidney disease and cardiovascular disease. This review is part of a special issue entitled: The role of soluble adenylyl cyclase in health and disease. This article is part of a Special Issue entitled: The role of soluble adenylyl cyclase in health and disease.

Trenbolone enhances myogenic differentiation by enhancing β-catenin signaling in muscle-derived stem cells of cattle

J.-X. Zhao, J. Hu, M.-J. Zhu, M. Du

Domestic Animal Endocrinology 40 (2011) 222–229

http://dx.doi.org:/10.1016/j.domaniend.2011.01.004

Testosterone is a key hormone regulating animal growth and development, which promotes skeletal muscle growth and inhibits fat deposition; however, the underlying mechanisms remain poorly defined. Because canonical Wingless and Int/β-catenin signaling promotes myogenesis, we hypothesized that testosterone regulates myogenesis through enhancing the β-catenin signaling pathway and the expression of its targeted genes. Muscle-derived stem cells were prepared from the skeletal muscle of fetal calf at day 180 of gestation and treated with or without trenbolone (10 nM), a synthetic analog of testosterone, in a myogenic medium. Trenbolone treatment increased the protein levels of MyoD and myosin heavy chain, as well as the androgen receptor content. The myogenic effect of trenbolone was blocked by cyproterone acetate, a specific inhibitor of androgen receptor, showing that the myogenic effect of trenbolone was mediated by the androgen receptor. Immunoprecipitation showed that androgen receptor and -catenin formed a complex, which was increased by trenbolone treatment. Trenbolone activated adenosine monophosphate–activated protein kinase, which might phosphorylate β-catenin at Ser552, stabilizing -catenin. Indeed, both cytoplasmic and nuclear Testosterone is a key hormone regulating animal growth and development, which promotes skeletal muscle growth and inhibits fat deposition; however, the underlying mechanisms remain poorly defined. Because canonical Wingless and Int/β-catenin signaling promotes myogenesis, we hypothesized that testosterone regulates myogenesis through enhancing the β-catenin signaling pathway and the expression of its targeted genes. Muscle-derived stem cells were prepared from the skeletal muscle of fetal calf at day 180 of gestation and treated with or without trenbolone (10 nM), a synthetic analog of testosterone, in a myogenic medium. Trenbolone treatment increased the protein levels of MyoD and myosin heavy chain, as well as the androgen receptor content. The myogenic effect of trenbolone was blocked by cyproterone acetate, a specific inhibitor of androgen receptor, showing that the myogenic effect of trenbolone was mediated by the androgen receptor. Immunoprecipitation showed that androgen receptor and β-catenin formed a complex, which was increased by trenbolone treatment. Trenbolone activated adenosine monophosphate–activated protein kinase, which might phosphorylate β-catenin at Ser552, stabilizing β-catenin. Indeed, both cytoplasmic and nuclear β-catenin levels were increased after trenbolone treatment. As a result, β-catenin–mediated transcriptional activity was enhanced by trenbolone treatment. In conclusion, these data provide evidence that testosterone increases cellular β-catenin content which promotes the expression of β-catenin–targeted genes and myogenesis in the muscle-derived stem cells of cattle. β-catenin levels were increased after trenbolone treatment. As a result, β-catenin–mediated transcriptional activity was enhanced by trenbolone treatment. In conclusion, these data provide evidence that testosterone increases cellular β-catenin content which promotes the expression of β-catenin–targeted genes and myogenesis in the muscle-derived stem cells of cattle.
Strategic combination therapy overcomes tyrosine kinase coactivation in adrenocortical carcinoma

Chi-Iou Lin, Edward E. Whang, Jacob Moalem, and Daniel T. Ruan
Surgery 2012; 152:1045-50.
http://dx.doi.org/10.1016/j.surg.2012.08.035

Background. Coactivation of tyrosine kinase limits the efficacy of tyrosine kinase inhibitors. We hypothesized that a strategic combination therapy could overcome tyrosine kinase coactivation and compensatory oncogenic signaling in patients with adrenocortical carcinoma (ACC). Methods. We profiled 88 tyrosine kinases before and after treatment with sunitinib in H295R and SW13 ACC cells. The effects of monotherapy and strategic combination regimens were determined by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (ie, MTS) assay. Results. The minimum inhibitory concentrations (ICmin) of sunitinib quenched its primary targets: FLT-3, VEGFR-2, and RET. In contrast, ERK, HCK, Chk2, YES, CREB, MEK, MSK, p38, FGR, and AXL were hyperactivated. Monotherapy with sunitinib or PD98059 at their ICmin reduced proliferation by 23%and 19%, respectively, in H295R cells and by 25% and 24%, respectively, in SW13 cells. Sunitinib and PD98059 in combination decreased proliferation by 68% and 64% in H295R and in SW13 cells, respectively (P < .05 versus monotherapy). The effects of combination treatment exceeded the sum of the effects observed with each individual agent alone. Conclusion. We describe the first preclinical model to develop strategic combination therapy to overcome tyrosine kinase coactivation in ACC. Because many tyrosine kinase inhibitors are readily available, this model can be immediately tested in clinical trials for patients with advanced ACC.
Silencing diacylglycerol kinase-theta expression reduces steroid hormone biosynthesis and cholesterol metabolism in human adrenocortical cells

Kai Cai, Natasha C. Lucki, Marion B. Sewer
Biochimica et Biophysica Acta 1841 (2014) 552–562
http://dx.doi.org/10.1016/j.bbalip.2013.12.005

Diacylglycerol kinase theta (DGKθ) plays a pivotal role in regulating adrenocortical steroidogenesis by synthesizing the ligand for the nuclear receptor steroidogenic factor 1 (SF1). In response to activation of the cAMP signaling cascade nuclear DGK activity is rapidly increased, facilitating PA-mediated, SF1-dependent transcription of genes required for cortisol and dehydroepiandrosterone (DHEA) biosynthesis. Based on our previous work identifying DGKθ as the enzyme that produces the agonist for SF1, we generated a tetracycline-inducible H295R stable cell line to express a short hairpin RNA (shRNA) against DGKθ and characterized the effect of silencing DGKθ on adrenocortical gene expression. Genome-wide DNA microarray analysis revealed that silencing DGKθ expression alters the expression of multiple genes, including steroidogenic genes, nuclear receptors and genes involved in sphingolipid, phospholipid and cholesterol metabolism. Interestingly, the expression of sterol regulatory element binding proteins (SREBPs) was also suppressed. Consistent with the suppression of SREBPs, we observed a down-regulation of multiple SREBP target genes, including 3-hydroxy-3-methylglutary coenzyme A reductase (HMG-CoA red) and CYP51, concomitant with a decrease in cellular cholesterol. DGKθ knockdown cells exhibited a reduced capacity to metabolize PA, with a down-regulation of lipin and phospholipase D (PLD) isoforms. In contrast, suppression of DGKθ increased the expression of several genes in the sphingolipid metabolic pathway, including acid ceramidase (ASAH1) and sphingosine kinases (SPHK). In summary, these data demonstrate that DGKθ plays an important role in steroid hormone production in human adrenocortical cells.
RRM1 modulates mitotane activity in adrenal cancer cells interfering with its metabolization

Antonina Germano, I Rapa, M Volante, S De Francia, C Migliore, et al.
Molecular and Cellular Endocrinology 401 (2015) 105–110
http://dx.doi.org/10.1016/j.mce.2014.11.027

The anti-proliferative activity of mitotane (o,p′DDD) in adrenocortical cancer is mediated by its metabolites o,p′DDE and o,p′DDA. We previously demonstrated a functional link between ribonucleotide reductase M1 (RRM1) expression and o,p′DDD activity, but the mechanism is unknown. In this study we assessed the impact of RRM1 on the bioavailability and cytotoxic activity of o,p′DDD, o,p′DDE and o,p′DDA in SW13 and H295R cells. In H295R cells, mitotane and its metabolites showed a similar cytotoxicity and RRM1 expression was not influenced by any drug. In SW13 cells, o,p′DDA only showed a cytotoxic activity and did not modify RRM1 expression, whereas the lack of sensitivity to o,p′DDE was associated to RRM1 gene up-modulation, as already demonstrated for o,p′DDD. RRM1 silencing in SW13 cells increased the intracellular transformation of mitotane into o,p′DDE and o,p′DDA. These data demonstrate that RRM1 gene interferes with mitotane metabolism in adrenocortical cancer cells, as a possible mechanisms of drug resistance.
New options in the treatment of Cushing’s disease: a focus on pasireotide

Anne-Gaëlle Poullot, Nicolas Chevalier
Research and Reports in Endocrine Disorders 2013:3 31–38
http://dx.doi.org/10.2147/RRED.S30972

Cushing’s disease is caused by a corticotroph pituitary adenoma secreting adrenocorticotropin and can be fatal in the absence of adequate treatment. Transsphenoidal surgery remains the treatment of choice in almost all cases. However, remission rates are relatively low, and recurrence is usual and can be diagnosed up to decades after the initial diagnosis. Repeat surgery or radiation can be useful in these cases, although both have clear limitations with respect to efficacy and/or side effects. Hence, there is a clear unmet need for an effective medical treatment in patients with recurrent or persistent Cushing’s disease. Pasireotide is a novel multireceptor-targeted somatostatin analog with a high affinity for somatostatin receptor (sstr)-1, sstr-2, sstr-3, and sstr-5. Compared with octreotide, pasireotide has an in vitro binding affinity 40-fold higher for sstr-5, which is the major receptor subtype expressed by corticotroph pituitary adenoma. Recent studies have suggested a role for this new multireceptor somatostatin analog in Cushing’s disease. We review in this article the current data available regarding pharmacokinetics, clinical efficiency, and tolerance of pasireotide in patients with de novo, persistent, or recurrent Cushing’s disease, with a special focus on the disturbances of glucose metabolism induced by such a treatment.

Optimal management of Cushing syndrome

Edgar G Durán-Pérez, OT Moreno-Loza, G Carrasco-Tobón, A Segovia-Palomo
Research and Reports in Endocrine Disorders 2012:2 19–30
http://dx.doi.org/10.2147/RRED.S25345

Cushing syndrome (CS) caused by endogenous hypercortisolism is a diagnostic challenge. The most common cause is Cushing disease. Surgical treatment is the first-line therapy for Cushing disease. However, due to the often clinical instability of the patient’s condition, which needs acute treatment of hypercortisolism or inoperable tumors, initial surgery is often not possible. It is therefore important to provide appropriate initial medical treatment. Following surgery, the patient needs to be evaluated and confirmed for disease resolution based on standard criteria, and treated with appropriate supportive measures for the rest of life if necessary. This article reviews the current data and treatment options for Cushing syndrome and proposes a therapeutic algorithm for its optimal management.

Cytotoxic activity of gemcitabine, alone or in combination with mitotane, in adrenocortical carcinoma cell lines

Antonina Germano, Ida Rapa, Marco Volante, Nicola Lo Buono, et al.
Molecular and Cellular Endocrinology 382 (2014) 1–7
http://dx.doi.org/10.1016/j.mce.2013.08.023

We aimed at investigating in vitro the cytotoxic activity (determined using WST-1, apoptosis and cell cycle assays) of gemcitabine, alone or in combination with mitotane, in mitotane-sensitive H295R and mitotane-insensitive SW-13 cells. Results of these experiments were compared with drug-induced modulation of RRM1 gene, the specific target of gemcitabine. In H295R cells, mitotane and gemcitabine combinations showed antagonistic effects and interfered with the gemcitabine-mediated inhibition of the S phase of the cell cycle. By contrast, in SW-13 cells, except when mitotane was sequentially administered prior to gemcitabine, the combination of the two drugs was synergistic. Such opposite effects were associated with opposite expression profiles of the target gene, with significant up-modulation in H295R but not in SW-13 under gemcitabine and mitotane combination treatment.

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Sex Hormones

Posted in Biological Networks, CANCER BIOLOGY & Innovations in Cancer Therapy, Cancer Screening, Clinical Diagnostics, Curation, Developmental biology, Diagnostics and Lab Tests, Disease Biology, Endocrine Diseases, Gene Regulation and Evolution, Genetics & Pharmaceutical, Genomic Endocrinology, Genomic Testing: Methodology for Diagnosis, Imaging-based Cancer Patient Management, Medical and Population Genetics, Metabolism, Mutant Gene Expression, tagged Cholesterol, endocrinology, Estrogen, ovary, Progesterone, Prostate, testes, testosterone, uterus on February 7, 2015| Leave a Comment »

Sex Hormones

Author: Larry H Bernstein, MD, FCAP

A steroid hormone is a steroid that acts as a hormone. Steroid hormones can be
grouped into five groups by the receptors to which they bind:

glucocorticoids,
mineralocorticoids,
androgens,
estrogens, and
progestogens.
Vitamin D derivatives, are a sixth closely related hormone system with homologous receptors. They have some of the characteristics of true steroids as receptor ligands.

Steroid hormones help control metabolism, inflammation, immune functions, salt
and water balance, development of sexual characteristics, and the ability to withstand
illness and injury. The term steroid describes both hormones produced by the body
and artificially produced medications that duplicate the action for the naturally occurring steroids

The natural steroid hormones are generally synthesized from cholesterol in the gonads and adrenal glands. These forms of hormones are lipids. They can pass through the cell membrane as they are fat-soluble,[4] and then bind to steroid hormone receptors (which may be nuclear or cytosolic depending on the steroid hormone) to bring about changes within the cell. Steroid hormones are generally carried in the blood, bound to specific carrier proteins such as sex hormone-binding globulin or corticosteroid-binding globulin. Further conversions and catabolism
occurs in the liver, in other “peripheral” tissues, and in the target tissues.

Synthetic steroids and sterols

A variety of synthetic steroids and sterols have also been contrived. Most are
steroids, but some non-steroidal molecules can interact with the steroid receptors
because of a similarity of shape. Some synthetic steroids are weaker or stronger
than the natural steroids whose receptors they activate.

Some examples of synthetic steroid hormones:
Glucocorticoids: alclometasone, prednisone, dexamethasone, triamcinolone
Mineralocorticoid: fludrocortisone
Vitamin D: dihydrotachysterol
Androgens: apoptone, oxandrolone, oxabolone, testosterone, nandrolone (also
known as anabolic steroids)
Estrogens: diethylstilbestrol (DES)
Progestins: danazol, norethindrone, medroxyprogesterone acetate,
17-Hydroxyprogesterone caproate.

Some steroid antagonists:
Androgen: cyproterone acetate
Progestins: mifepristone, gestrinone
http://www.en.wikipedia.org/wiki/Steroid

Steroid-Hormone-Synthesis

Steroidogenesis

http://www.gfmer.ch/Books/Reproductive_health/Image171.gif

The regulation of spermatogenesis by androgens

Lee B. Smith, William H. Walker
Seminars in Cell & Developmental Biology 30 (2014) 2–13
http://dx.doi.org/10.1016/j.semcdb.2014.02.012

Testosterone is essential for maintaining spermatogenesis and male fertility.
However, the molecular mechanisms by which testosterone acts have not
begun to be revealed until recently. With the advances obtained from the use
of transgenic mice lacking or overexpressing the androgen receptor, the cell
specific targets of testosterone action as well as the genes and signaling pathways
that are regulated by testosterone are being identified. In this review, the critical
steps of spermatogenesis that are regulated by testosterone are discussed as well
as the intracellular signaling pathways by which testosterone acts. We also review
the functional information that has been obtained from the knock out of the androgen
receptor from specific cell types in the testis and the genes found to be regulated
after altering testosterone levels or androgen receptor expression.

The essence of female–male physiological dimorphism: Differential Ca2+-homeostasis
enabled by the interplay between farnesol-like endogenous sesquiterpenoids and
sex-steroids? The Calcigender paradigm

Arnold De Loof
General and Comparative Endocrinology 211 (2015) 131–146
http://dx.doi.org/10.1016/j.ygcen.2014.12.003

Ca2+ is the most omnipresent pollutant on earth, in higher concentrations a real
threat to all living cells. When [Ca2+]i rises above 100 nM (=resting level), excess
Ca2+ needs to be confined in the SER and mitochondria, or extruded by the different
Ca2+-ATPases. The evolutionary origin of eggs and sperm cells has a crucial, yet
often overlooked link with Ca2+-homeostasis. Because there is no goal whatsoever
in evolution, gametes did neither originate ‘‘with the purpose’’ of generating a progeny
nor of increasing fitness by introducing meiosis. The explanation may simply be that
females ‘‘invented the trick’’ to extrude eggs from their body as an escape strategy
for getting rid of toxic excess Ca2+ resulting from a sex-hormone driven increased
influx into particular cells and tissues.
The production of Ca2+-rich milk, seminal fluid in males and all secreted proteins
by eukaryotic cells may be similarly explained. This view necessitates an upgrade
of the role of the RER-Golgi system in extruding Ca2+. In the context of insect
metamorphosis, it has recently been (re)discovered that (some isoforms of) Ca2+-
ATPases act as membrane receptors for some types of lipophilic ligands, in
particular for endogenous farnesol-like sesquiterpenoids (FLS) and, perhaps, for
some steroid hormones as well.
A novel paradigm, tentatively named ‘‘Calcigender’’ emerges. Its essence is: gender-
specific physiotypes ensue from differential Ca2+-homeostasis enabled by genetic
differences, farnesol/FLS and sex hormones. Apparently the body of reproducing
females gets temporarily more poisoned by Ca2+ than the male one, a selective
benefit rather than a disadvantage.

Sex differences in the expression of estrogen receptor alpha within noradrenergic
neurons in the sheep brain stem

J.L. Rose, A.S. Hamlin, C.J. Scott
Domestic Animal Endocrinology 49 (2014) 6–13
http://dx.doi.org/10.1016/j.domaniend.2014.04.003

In female sheep, high levels of estrogen exert a positive feedback action
on gonadotropin releasing hormone (GnRH) secretion to stimulate a surge in
luteinizing hormone (LH) secretion. Part of this action appears to be via brain
stem noradrenergic neurons. By contrast, estrogen action in male sheep has
a negative feedback action to inhibit GnRH and LH secretion. To investigate
whether part of this sex difference is due to differences in estrogen action in
the brain stem, we tested the hypothesis that the distribution of estrogen
receptor a (ERα) within noradrenergic neurons in the brain stem differs
between rams and ewes. To determine the distribution of ERα, we used
double-label fluorescence immunohistochemistry for dopamine b-Hydroxylase,
as a marker for noradrenergic and adrenergic cells, and ERα. In the ventro-
lateral medulla (A1 region), most ERα-immunoreactive (-ir) cells were
located in the caudal part of the nucleus. Overall, there were more ERα-ir
cells in rams than ewes, but the proportion of double-labeled cells was did
not differ between sexes. Much greater numbers of ERα–ir cells were
found in the nucleus of the solitary tract (A2 region), but <10% were double
labeled and there were no sex differences. The majority of ERα-labeled cells
in this nucleus was located in the more rostral areas. Erα labeled cells were
found in several rostral brain stem regions but none of these were double
labeled and so were not quantified. Because there was no sex difference
in the number of ERα-ir cells in the brain stem that were noradrenergic,
the sex difference in the action of estrogen on gonadotropin secretion in
sheep is unlikely to involve actions on brain stem noradrenergic cells.

Androgens, estrogens, and second messengers

William Rosner, DJ Hryb, MS Khan, AM Nakhla, and NA Romas
Steroids 1998; 63:278 –281 PII S0039-128X(98)00017-8

Over the course of the last four decades, a detailed understanding of the
molecular mechanisms by which steroid hormones exert their effects has
evolved, and continues to evolve. The major focus of research in this area
has been on the manner in which steroid receptors activate transcription.
Pathways of steroid action other than by direct interaction with intracellular
receptors have received relatively little attention. However, there is a growing
body of evidence that steroid hormones exert effects through mechanisms
in addition to those involving their classic intracellular receptors. One such
mechanism is based on the observation that a number of cells have receptors
on their plasma membranes for the plasma protein, sex hormone binding
globulin (SHBG). It is the purpose of this review to briefly describe our current
knowledge of this system.

SHBG binds to a receptor (RSHBG) on cell membranes cAMP and the steroid-SHBG-RSHBG system
Biology of the SHBG-RSHBG system

Relation between the affinity of steroid for SHBG and its potency in inhibiting
the binding of SHBG to RSHBG.

KA (SHBG) = Association constant for SHBG and the indicated steroid.
Ki SHBG-RSHBG = The inhibition constant for the indicated steroid on the
binding of SHBG to RSHBG.

PSA secretion was stimulated by DHT. Although estradiol alone had no effect
on PSA secretion, it caused an increase equal to that seen with DHT if the
prostate tissue was first loaded with SHBG, e.g., if RSHBG was occupied by
SHBG. Because estradiol-SHBG increases intracellular cAMP, we ascertained
whether other compounds that raise cAMP (forskolin), or cAMP itself, could
increase PSA secretion. Such was the case. cAMP begins its signal cascade
by activating protein kinase A (PKA) so that if estradiol-SHBG increases PSA
secretion by a mechanism involving cAMP, inhibition of PKA should block
estradiol-SHBG-initiated PSA secretion. Estradiol-SHBG failed to stimulate
PSA when PKA was inhibited with PKI. On the other hand, DHT-stimulated
PSA secretion, which does not involve PKA, was not inhibited by PKI. That
the effect of estradiol-SHBG was independent of the estrogen receptor was
shown by the lack of inhibition of estrogen-stimulated PSA secretion by two
anti-estrogens, tamoxifen and ICI 164,284. The promoter of the PSA gene
has an androgen response element, and both PSA secretion and the
expression of PSA mRNA are androgen-regulated. We investigated the
effect of hydroxyflutamide and cyproterone acetate. Both potent anti-
androgens, on the E2-SHBG-mediated increase in PSA secretion. secretion.
They also blocked the effect of E2-SHBG on PSA secretion. Since E2 is
not exerting its effect by binding to the AR, e.g., it is not its cognate ligand,
the E2-induced secretion of PSA observed in this study reflects ligand-
independent activation of the AR.26 Thus, estradiol activates a typical
AR-mediated event, PSA synthesis and secretion, by activating SHBG-
RSHBG. These data make clear the fact that there is cross-talk between a
steroid hormone-engendered event at the cell membrane and a classic
intracellular steroid hormone receptor.
Abbreviations: PSA, prostate specific antigen; DHT, dihydrotestosterone;
E2, estradiol; PKI, inhibitor of protein kinase A; ICI 164,384 (a pure anti-
estrogen); 2MeOE2, 2 methoxyestradiol; Cypro, cyproterone acetate,
OHFlut, hydroxyflutamide.

Role of G protein-coupled estrogen receptor 1, GPER, in inhibition of oocyte
maturation by endogenous estrogens in zebrafish

Yefei Pang, Peter Thomas
Developmental Biology 342 (2010) 194–206
http://dx.doi.org:/10.1016/j.ydbio.2010.03.027

Estrogen inhibition of oocyte maturation (OM) and the role of GPER (formerly
known as GPR30) were investigated in zebrafish. Estradiol-17β (E2) and G-1,
a GPER-selective agonist, bound to zebrafish oocyte membranes suggesting
the presence of GPER which was confirmed by immunocytochemistry using
a specific GPER antibody. Incubation of follicle-enclosed oocytes with an
aromatase inhibitor, ATD, and enzymatic and manual removal of the ovarian
follicle cell layers significantly increased spontaneous OM which was partially
reversed by co-treatment with either 100 nM E2 or G-1. Incubation of
denuded oocytes with the GPER antibody blocked the inhibitory effects of
estrogens on OM, whereas microinjection of estrogen receptor alpha (ERα)
antisense oligonucleotides into the oocytes was ineffective. The results
suggest that endogenous estrogens produced by the follicle cells inhibit or
delay spontaneous maturation of zebrafish oocytes and that this estrogen
action is mediated through GPER. Treatment with E2 and G-1 also attenuated
the stimulatory effect of the teleost maturation-inducing steroid, 17,20 β-
dihyroxy-4-pregnen-3-one (DHP), on OM. Moreover, E2 and G-1 down-
regulated the expression of membrane progestin receptor alpha (mPRα),
the intermediary in DHP induction of OM. Conversely DHP treatment caused
a N50% decline in GPER mRNA levels. The results suggest that estrogens
and GPER are critical components of the endocrine system controlling
the onset of OM in zebrafish. A model is proposed for the dual control of the
onset of oocyte maturation in teleosts by estrogens and progestins acting
through GPER and mPRα, respectively, at different stages of oocyte
development.
Reprint of ’’GPR30 mediates estrogen rapid signaling and neuroprotection’’

Hui Tang, Q Zhang, L Yang, Y Dong, M Khan, F Yang, DW Brann, R Wang
Molecular and Cellular Endocrinology 389 (2014) 92–98
http://dx.doi.org/10.1016/j.mce.2014.01.024
http://dx.doi.org/10.1016/j.mce.2014.05.005

G-protein-coupled estrogen receptor-30 (GPR30), also known as G-protein
estrogen receptor-1 (GPER1), is a putative extranuclear estrogen receptor
whose precise functions in the brain are poorly understood. Studies using
exogenous administration of the GPR30 agonist, G1 suggests that GPR30
may have a neuroprotective role in cerebral ischemia. However, the
physiological role of GPR30 in mediating estrogen (E2)-induced neuro-
protection in cerebral ischemia remains unclear. Also unclear is whether
GPR30 has a role in mediating rapid signaling by E2 after cerebral ischemia,
which is thought to underlie its neuroprotective actions. To address these
deficits in our knowledge, the current study examined the effect of antisense
oligonucleotide (AS) knockdown of GPR30 in the hippocampal CA1 region
upon E2-BSA induced neuroprotection and rapid kinase signaling in a rat
model of global cerebral ischemia (GCI). Immunohistochemistry demonstrated
that GPR30 is strongly expressed in the hippocampal CA1 region and
dentate gyrus, with less expression in the CA3 region. E2-BSA exerted
robust neuroprotection of hippocampal CA1 neurons against GCI, an effect
abrogated by AS knockdown of GPR30. Missense control oligonucleotides had
no effect upon E2-BSA-induced neuroprotection, indicating specificity of the
effect. The GPR30 agonist, G1 also exerted significant neuroprotection against
GCI. E2-BSA and G1 also rapidly enhanced activation of the prosurvival
kinases, Akt and ERK, while decreasing proapototic JNK activation. Importantly,
AS knockdown of GPR30 markedly attenuated these rapid kinase signaling
effects of E2-BSA. As a whole, the studies provide evidence of an important
role of GPR30 in mediating the rapid signaling and neuroprotective actions
of E2 in the hippocampus.
Regulation of brain microglia by female gonadal steroids

Pardes Habib, Cordian Beyer
Journal of Steroid Biochemistry & Molecular Biology 2015; 146: 3–14
http://dx.doi.org/10.1016/j.jsbmb.2014.02.018

Microglial cells are the primary mediators of the CNS immune defense system
and crucial for shaping inflammatory responses. They represent a highly
dynamic cell population which is constantly moving and surveying their
environment. Acute brain damage causes a local attraction and activation of
this immune cell type which involves neuron-to-glia and glia-to-glia interactions.
The prevailing view attributes microglia a “negative” role such as defense and
debris elimination. More topical studies also suggest a protective and “positive”
regulatory function. Estrogens and progestins exert anti-inflammatory and
neuroprotective effects in the CNS in acute and chronic brain diseases.
Recent work revealed that microglial cells express subsets of classical and
non-classical estrogen and progesterone receptors in a highly dynamic way.
In this review article, we would like to stress the importance of microglia for
the spreading of neural damage during hypoxia, their susceptibility to functional
modulation by sex steroids, the potency of sex hormones to switch microglia
from a pro-inflammatory M1 to neuroprotective M2 phenotype, and the
regulation of pro-and anti-inflammatory properties including the inflammasome.
We will further discuss the possibility that the neuroprotective action of sex
steroids in the brain involves an early and direct modulation of local microglia
cell function. Neuroprotection by gonadal steroid hormones in acute brain
damage requires cooperation with astroglia and microglia

Sonja Johann, Cordian Beyer
http://dx.doi.org/10.1016/j.jsbmb.2012.11.006

The neuroactive steroids 17β-estradiol and progesterone control a broad
spectrum of neural functions. Besides their roles in the regulation of classical
neuroendocrine loops, they strongly influence motor and cognitive systems,
behavior, and modulate brain performance at almost every level. Such a
statement is underpinned by the widespread and lifelong expression pattern
of all types of classical and non-classical estrogen and progesterone receptors
in the CNS. The life-sustaining power of neurosteroids for tattered or seriously
damaged neurons aroused interest in the scientific community in the past years
to study their ability for therapeutic use under neuropathological challenges.
Documented by excellent studies either performed in vitro or in adequate animal
models mimicking acute toxic or chronic neuro-degenerative brain disorders,
both hormones revealed a high potency to protect neurons from damage
and saved neural systems from collapse. Unfortunately, neurons, astroglia,
microglia, and oligodendrocytes are comparably target cells for both steroid
hormones. This hampers the precise assignment and understanding of
neuroprotective cellular mechanisms activated by both steroids. In this article,
we strive for a better comprehension of the mutual reaction between these
steroid hormones and the two major glial cell types involved in the maintenance
of brain homeostasis, astroglia and microglia, during acute traumatic brain
injuries such as stroke and hypoxia. In particular, we attempt to summarize
steroid-activated cellular signaling pathways and molecular responses in these
cells and their contribution to dampening neuroinflammation and neural
destruction.

Photoperiod influences the ontogenetic expression of aromatase
and estrogen receptor α in the developing tilapia brain.

Li-Hsueh Wang, Ching-Lin Tsai
General and Comparative Endocrinology 2006; 145: 62–66
http://dx.doi.org:/10.1016/j.ygcen.2005.07.004

Neural development is determined not only by genetic regulation, but also
by environmental cues. Central estrogen-forming/estrogen-sensitive systems
play an important role in the neural development of the brain. In the present
study, the quantitative reverse transcription-polymerase chain reaction method
was used to investigate the effects of photoperiod on the ontogenetic
expression of aromatase and estrogen receptor a (ERα) in the developing
tilapia brain. Before day 5 post-hatch, brain aromatase mRNA expression was
significantly decreased by constant light but not influenced by constant darkness.
During this period, brain ERα mRNA expression was significantly increased
under both constant light and constant darkness. Between days 5 and 10, and
between days 10 and 15, neither brain aromatase nor brain ERα expression
was altered under constant darkness and constant light. These results indicate
that the ontogenetic expression of brain aromatase and brain ERα is not via a
light-inducing process but influenced by a light-entraining signal during the
very early period of development.

Orphanin FQ-ORL-1 Regulation of Reproduction and Reproductive Behavior in
the Female

Kevin Sinchak, Lauren Dalhousay, Nayna Sanathara
Vitamins and Hormones 187-220. http://dx.doi.org/10.1016/bs.vh.2014.11.002

Orphanin FQ (OFQ/N) and its receptor, opioid receptor-like receptor-1 (ORL-1),
are expressed throughout steroid-responsive limbic and hypothalamic circuits
that regulate female ovarian hormone feedback and reproductive behavior
circuits. The arcuate nucleus of the hypothalamus (ARH) is a brain region
that expresses OFQ/N and ORL-1 important for both sexual behavior and
modulating estradiol feedback loops. Within the ARH, the activation of the
OFQ/N-ORL-1 system facilitates sexual receptivity (lordosis) through the
inhibition of β-endorphin neuronal activity. Estradiol initially activates ARH
β-endorphin neurons to inhibit lordosis. Simultaneously, estradiol upregulates
coexpression of OFQ/N and progesterone receptors and ORL-1 in ARH
β-endorphin neurons. Ovarian hormones regulate pre- and postsynaptic
coupling of ORL-1 to its G protein-coupled signaling pathways. When the
steroid-primed rat is nonreceptive, estradiol acts pre- and postsynaptically
to decrease the ability of the OFQ/N-ORL-1 system to inhibit ARH β-endorphin
neurotransmission. Conversely, when sexually receptive, ORL-1 signaling is
restored to inhibit β-endorphin neurotransmission. Although steroid signaling
that facilitates lordosis converges to deactivate ARH.
Estradiol Activates the Prostate Androgen Receptor and Prostate specific Antigen
Secretion through the Intermediacy of Sex Hormone-binding Globulin

Atif M. Nakhla, Nicholas A. Romas, and William Rosner
J Biol Chem Mar 14, 1997; 272(11): 6838–6841 http://www-jbc.stanford.edu/jbc/

These experiments were designed to examine the relationship between the
effects of steroid hormones mediated by classic intracellular steroid hormone
receptors and those mediated by a signaling system subserved at the plasma
membrane by a receptor for sex hormone binding globulin. It is known that
unliganded sex hormone-binding globulin (SHBG) binds to a receptor (RSHBG)
on prostate membranes. The R_SHBG*SHBG complex is rapidly activated by
estradiol to stimulate adenylate cyclase, with a resultant increase in intracellular
cAMP. In this paper we examine the effect of this system on a prostate gene
product known to be activated by androgens, prostate-specific antigen.
We have shown previously that estradiol (E2) participates in a signaling
system that originates, not within the cell, but at the plasma membrane.
Through the intermediacy of the plasma protein, sex hormone-binding
globulin (SHBG), it causes the generation of cAMP. In brief, unliganded
SHBG binds to a receptor (R_SHBG) on certain cell surfaces and the
R_SHBG*SHBG complex is rapidly activated by E2 to stimulate adenylate cyclase,
with a resultant increase in intracellular cAMP. There is a paucity of information
on events subsequent to the generation of cAMP by this system. In this paper
we examine the effect of E2-SHBG-R_SHBG on an androgen responsive gene.
The gene for prostate-specific antigen (PSA) contains an androgen response
element. After binding its cognate ligand, the androgen receptor (AR) interacts
with this response element to initiate PSA mRNA transcription and secretion.
We show that, in the absence of androgens, E2 in concert with SHBG*R_SHBG,
acts at the cell membrane to cause secretion of PSA and that this effect is
blocked by anti-androgens. This observation provides a first functional link
between a classic steroid hormone receptor and a cell membrane-mediated
steroidal effect. In serum-free organ culture of human prostates,
dihydrotestosterone caused an increase in prostate specific antigen secretion.
This event was blocked by the anti-androgens cyproterone acetate and
hydroxyflutamide. In the absence of androgens, estradiol added to prostate
tissue, whose RSHBG was occupied by SHBG, reproduced the results seen
with dihydrotestosterone. Neither estradiol alone nor SHBG alone duplicated
these effects. The estradiol*SHBG-induced increase in prostate-specific
antigen was not blocked by anti-estrogens, but was blocked both by anti-
androgens and a steroid (2-methoxyestradiol) that prevents the binding of
estradiol to SHBG. Furthermore, an inhibitor of protein kinase A prevented
the estradiol*SHBG-induced increase in prostate-specific antigen but not
that which followed dihydrotestosterone. These data indicate that there is a
signaling system that amalgamates steroid-initiated intracellular events
with steroid-dependent occurrences generated at the cell membrane and
that the latter signaling system proceeds by a pathway that involves protein
kinase A.
Mechanisms of crosstalk between endocrine systems: Regulation of sex steroid
hormone synthesis and action by thyroid hormones

Paula Duarte-Guterman, Laia Navarro-Martín, Vance L. Trudeau
General and Comparative Endocrinology 203 (2014) 69–85
http://dx.doi.org/10.1016/j.ygcen.2014.03.015

Thyroid hormones (THs) are well-known regulators of development and
metabolism in vertebrates. There is increasing evidence that THs are also
involved in gonadal differentiation and reproductive function. Changes in TH
status affect sex ratios in developing fish and frogs and reproduction
(e.g., fertility), hormone levels, and gonad morphology in adults of species of
different vertebrates. In this review, we have summarized and compared the
evidence for cross-talk between the steroid hormone and thyroid axes and
present a comparative model. We gave special attention to TH regulation of
sex steroid synthesis and action in both the brain and gonad, since these are
important for gonad development and brain sexual differentiation and have
been studied in many species. We also reviewed research showing that
there is a TH system, including receptors and enzymes, in the brains and
gonads in developing and adult vertebrates. Our analysis shows that THs
influences sex steroid hormone synthesis in vertebrates, ranging from fish
to pigs. This concept of crosstalk and conserved hormone interaction has
implications for our understanding of the role of THs in reproduction, and
how these processes may be dysregulated by environmental endocrine
disruptors.
Inverse relationship between hSHBG affinity for testosterone and hSHBG
concentration revealed by surface plasmon resonance

Laurence Heinrich-Balard, Wael Zeinyeh, Henri Déchaud, Pascaline Rivory, et al.
Molecular and Cellular Endocrinology 399 (2015) 201–207
http://dx.doi.org/10.1016/j.mce.2014.10.002

A wide range of human sex hormone-binding globulin (hSHBG) affinity constants
for testosterone (KA__hSHBG) has been reported in literature. To bring new insight
on the KA__hSHBG value, we implemented a study of the molecular interactions
occurring between testosterone and its plasma transport proteins by using
surface plasmon resonance. The immobilization on the sensor-chip of a
testosterone derivative was performed by an oligoethylene glycol linker.
For different plasmas with hSHBG concentrations, an assessment of the
KA__hSHBG was obtained from a set of sensor-grams and curve-fitting these
data.We observed that KA__hSHBG decreased, from at least two decades,
when the plasma hSHBG concentration increased from 4.4 to 680 nmol/L.
Our study shows a wide biological variability of KA__hSHBG that is related
to the hSHBG concentration.
These unexpected results may have a physiological significance and question
the validity of current methods that are recommended for calculating free
testosterone concentrations to evaluate androgen disorders in humans.
Intracrinology in action: Importance of extragonadal sex steroid biosynthesis
and inactivation in peripheral tissues in both women and men.

Editorial
Journal of Steroid Biochemistry & Molecular Biology 145 (2015) 131–132
http://dx.doi.org/10.1016/j.jsbmb.2014.09.012

It seems appropriate, as introduction, to summarize the mechanisms at the
basis of the new paradigm of steroid biosynthesis in the human peripheral
tissues, namely intracrinology. While the first clinical proof of the role of
extragonadal sex steroid biosynthesis was obtained with combined androgen
blockade in men treated for prostate cancer, the first demonstration of the
efficacy of DHEA replacement therapy was on the symptoms of vulvovaginal
atrophy in postmenopausal women; (Archer, this issue).
DHEA is transformed specifically in each cell of each peripheral tissue into
the proper amounts of estrogens and/or androgens, depending upon the
local expression of the appropriate steroid forming enzymes; (Labrie, this issue).
Most importantly, the sex steroids synthesized and acting intracellularly in
peripheral tissues are also inactivated locally before being released in the
extracellular space, thus maintaining the serum levels of estradiol and
testosterone at biologically inactive concentrations, thus avoiding systemic
exposure to sex steroids during menopause as well illustrated by atrophy
of the endometrium.
As mentioned above, that extragonadal androgen biosynthesis is clinically
important became obvious in 1982 when the addition of the antiandrogen
flutamide to castration provided very exciting and unexpected beneficial results
(Labrie, this issue). In fact, combining a pure anti-androgen to castration has
been the first treatment shown to prolong life in prostate cancer and very clearly
confirmed by the prolongation of life of 2.2–4.8 months observed following
addition of MDV-3100 or abiraterone to castration resistant prostate cancer
patients (Grist et al., this issue). (Mizokami et al., this issue) very competently
complement the mechanisms potentially involved in extragonadal steroid
biosynthesis. A repeated observation is the association between serum DHEA
levels and increased longevity, a subject reviewed by Ohlsson et al., this issue.
Most importantly, a subject which remains to be supported by long-term clinical
trials but which shows very promising preclinical data is the possibility of a
beneficial effect of DHEA on brain functions, especially cognition, memory
and delayed development of mild cognitive impairment and Alzheimer’s
disease (see Starka et al.; Soma et al; Pluchino et al; Maggio et al.; Hill et al.,
this issue). The information summarized in the very up-to-date manuscripts
of this special JSBMB issue has the potential of opening the way to a prodrug
replacement therapy already well illustrated on the symptoms and signs of
vulvovaginal atrophy and sexual dysfunction (Archer, this issue). The
administration to sex steroid deficient women of an appropriate amount of
DHEA able to correct the symptoms of vulvovaginal atrophy (mostly estrogen-
sensitive) and sexual dysfunction (androgen-sensitive), and potentially, in the
future, other problems of menopause, does permit to the sex steroid-deficient
women to benefit from a normal/sufficient level of sex steroids in specific tissues
using the enzymes developed over 500 million years to permit a better quality
of life during the menopausal years.

Inactivation of androgens by UDP-glucuronosyltransferase enzymes in humans

Alain Belanger, Georges Pelletier, Fernand Labrie, Olivier Barbier and Sarah Chouinard
TRENDS in Endocrinology and Metabolism 2003; 14(10):473-78
http://dx.doi.org:/10.1016/j.tem.2003.10.005

In humans, 3b-hydroxysteroid dehydrogenase (3β-HSD), 17β-HSD and
5α-reductase activities in androgen target tissues, such as the prostate and
skin, convert dehydroepiandrosterone, androstenedione and testosterone into
the most potent natural androgen dihydrotestosterone (DHT). This androgen
is converted mainly in situ into two phase I metabolites, androsterone (ADT)
and androstane-3α,17β-diol (3α-DIOL), which might be back converted to DHT.
Here, we discuss the recent findings regarding the characterization of specific
UDP glucuronosyltransferases (UGTs), UGT2B7, B15 and B17, responsible for
the glucuronidation of these metabolites. The tissue distribution and cellular
localization of the UGT2B transcripts and proteins in humans clearly indicate
that these enzymes are synthesized in androgen-sensitive tissues. It is
postulated that the conjugating activity of UGT enzymes is the main mechanism
for modulating the action of steroids and protecting the androgen-sensitive
tissues from deleteriously high concentrations of DHT, ADT and 3α-DIOL.
Synthesis and Evaluation of Potential Radioligands for the Progesterone Receptor

R.M. Hoyte, W. Rosner, I.S. Johnson, J. Zielinski, and R. B. Hochberg
J. Med. Chem. 1985; 28: 1695-1699

Several steroidal analogues were synthesized as potential y-emitting radioligands
for the progesterone receptor. Each of these compounds was tested as an inhibitor
of the specific binding of [³H]-17α,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione
(R5020) to the progesterone receptor in rabbit uterine cytosol. R5020 is a well-
known progestin with high affinity for the receptor. Of the compounds synthesized,
aromatic N-substituted (2-17 steroidal carboxamides inhibited the binding only
poorly. Three compounds, 16α-iodo-4-estren-17β-ol-3-one, 17α-[2(E)-iodovinyl]
-4-estren-17β-ol-3-one, and 17α-[2(Z)-iodovinyl]-4-estren-l7β-ol-3-one are
excellent competitors, each having a K_iless than or equal to that of the natural
progestin, progesterone. Since similar iodinated analogues of estrogens
have been shown to be extremely stable both in vivo and in vitro, these compounds
are potentially useful ligands for the progesterone receptor.

Estradiol concentration and the expression of estrogen receptors in the testes of
the domestic goose (Anser anser f. domestica) during the annual reproductive cycle

Leska, J. Kiezun, B. Kaminska, L. Dusza
Domestic Animal Endocrinology 51 (2015) 96–104
http://dx.doi.org/10.1016/j.domaniend.2014.12.002

Seasonal fluctuations in the activity of bird testes are regulated by a complex mechanism
where androgens play a key role. Until recently, the role played by estrogens in males has
been significantly underestimated. However, there is growing evidence that the proper
functioning of the testes is associated with optimal estradiol (E2) concentration
in both the plasma and testes of many mammalian species. Estrogens are
gradually emerging as very important players in hormonal regulation of
reproductive processes in male mammals. Despite the previously mentioned,
it should be noted that estrogenic action is limited by the availability of
specific receptors – estrogen receptor alpha (ERα) and estrogen receptor beta
(ERβ). Interestingly, there is a general scarcity of information concerning the
estrogen responsive system in the testes of male birds, which is of particular
interest in exploring the phenomenon of seasonality of reproduction. To address
this question, we have investigated for the first time the simultaneous
expression of testicular ERα and ERβ genes and proteins with the
accompanying plasma and testicular E2 concentrations during the annual
reproductive cycle of male bird. The research model was the domestic
goose (Anser anser f. domestica), a species whose annual reproductive
cycle can be divided into 3 distinct phases characterized by changes
in testicular activity. It has been revealed that the stable plasma E2 profile
did not correspond to changing intratesticular E2 profile throughout the
experiment. The expression of ERα and ERβ genes and proteins was detected
in gander testes and it fluctuated on a seasonal basis with lower level in
breeding and sexual reactivation stages and higher level during the
nonbreeding stage. Our results demonstrated changes in testicular sensitivity
to estrogens in male domestic goose during the annual reproductive cycle.
The seasonal pattern of estrogen receptors (ERs) expression was analyzed
against the hormonal background and a potential mechanism of ERs regulation
in bird testes was proposed. The present study revealed seasonal variations
in the estrogen responsive system, but further research is needed to fully
explore the role of estrogens in the reproductive tract of male birds.

Effects of 5α-dihydrotestosterone on expression of genes related to steroidogenesis
and spermatogenesis during the sex determination and differentiation periods of
the pejerrey, Odontesthes bonariensis

Anelisa González, Juan I. Fernandino, Gustavo M. Somoza
Comparative Biochemistry and Physiology, Part A 182 (2015) 1–7
http://dx.doi.org/10.1016/j.cbpa.2014.12.003

Sex steroid hormones are important players in the control of sex differentiation
by regulating gonadal development in teleosts. Although estrogens are clearly
associated with the ovarian differentiation in teleosts, the effects of androgens
on early gonadal development are still a matter of debate. Traditionally,
11-ketotestosterone (11-KT) is considered themajor androgen in fish; however,
5α-dihydrotestosterone (5α-DHT), the most potent androgen in tetrapods, was
recently found in fish testis and plasma, but its physiological role is still unknown.
In this context, the expression of genes associated with steroidogenesis and
spermatogenesis, body growth and sex differentiation were assessed in
Odontesthes bonariensis larvae fed with food supplemented with two doses of
5α-DHT (0.1 and 10 μg/g of food) from hatching to 6 weeks of age. At the lowest
dose, 5α-DHT treated larvae showed an estrogenic gene expression pattern, with
low hsd11β2 and high cyp19α1α and er2 expression levels with no differences
in sex ratio. At the highest dose, 5α-DHT produced a male-shifted sex ratio and
the larvae exhibited a gene expression profile characteristic of an advancement
of spermatogenesis, with inhibition of amh and stimulation of ndrg3. No
differences were observed in somatic growth. These results suggest that in
this species, 5α-DHT could have a role on sex differentiation and its effects
can differ according to the dose.
Do androgens link morphology and behavior to produce phenotype-specific
behavioral strategies?

Douglas G. Barron, Michael S. Webster, Hubert Schwabl
Animal Behaviour 100 (2015) 116e124
http://dx.doi.org/10.1016/j.anbehav.2014.11.016

Morphological and behavioral traits often covary with each other, and the links
between them may arise from shared physiological mechanisms. In particular,
androgens such as testosterone have emerged as prime candidates for linking
behaviour and morphology due to the environmental sensitivity and pleiotropic
effects of these hormones. In this study we investigated the hypothesis that
androgens simultaneously relate to morphological and behavioral variation,
thereby producing the integrated reproductive phenotypes of male red-backed
fairy-wrens, Malurus melanocephalus. Males of this species can adopt one of
three discrete breeding phenotypes: breeding in red/black plumage, breeding
in brown plumage, or remaining as nonbreeding brown natal auxiliaries. Although
the expression of morphological traits in this species is regulated by androgens
and phenotypes differ in baseline androgen levels (red/black breeder > brown
breeder > auxiliary), injection with GnRH failed to expose phenotype specific
constraints on androgen production. Observations of territoriality, nestling
feeding and extraterritorial forays revealed phenotype-specific patterns of mating
and parental effort, yet these were largely related to age and were not correlated
with baseline or GnRH-induced androgen levels, or the androgen change between
these points. While these findings support the idea that morphological and
behavioral traits are linked via phenotypic correlations, they do not support
the hypothesis that behavioral differences arise from variation in circulating
androgens or the capacity to produce them.
Effects of sex steroids on expression of genes regulating growth-related
mechanisms in rainbow trout (Oncorhynchus mykiss)

Beth M. Cleveland, Gregory M. Weber
General and Comparative Endocrinology xxx (2015) xxx–xxx
http://dx.doi.org/10.1016/j.ygcen.2014.11.018

Effects of a single injection of 17b-estradiol (E2), testosterone (T), or
5b-dihydrotestosterone (DHT) on expression of genes central to the
growth hormone (GH)/insulin-like growth factor (IGF) axis, muscle
regulatory factors, transforming growth factor-beta (TGFβ) superfamily
signaling cascade, and estrogen receptors were determined in rainbow
trout (Oncorhynchus mykiss) liver and white muscle tissue. In liver in
addition to regulating GH sensitivity and IGF production, sex
steroids also affected expression of IGF binding proteins, as E2, T,
and DHT increased expression of igfbp2β and E2 also increased
expression of igfbp2 and igfbp4. Regulation of this system also occurred
in white muscle in which E2 increased expression of igf1, igf2, and
igfbp5β1, suggesting anabolic capacity may be maintained in white
muscle in the presence of E2. In contrast, DHT decreased expression
of igfbp5β1. DHT and T decreased expression of myogenin, while other
muscle regulatory factors were either not affected or responded similarly
for all steroid treatments. Genes within the TGFβ superfamily signaling
cascade responded to steroid treatment in both liver and muscle,
suggesting a regulatory role for sex steroids in the ability to transmit
signals initiated by TGFβ superfamily ligands, with a greater number
of genes responding in liver than in muscle. Estrogen receptors were
also regulated by sex steroids, with era1 expression increasing for all
treatments in muscle, but only E2- and T-treatment in liver. E2 reduced
expression of erb2 in liver. Collectively, these data identify how
physiological mechanisms are regulated by sex steroids in a manner
that promotes the disparate effects of androgens and estrogens on
growth in salmonids.
Distribution and function of 3′,5′-Cyclic-AMP phosphodiesterases in the human ovary

T.S. Petersen, S.G. Kristensen, J.V. Jeppesen, .., K.T. Macklon, C.Y. Andersen
Molecular and Cellular Endocrinology 403 (2015) 10–20
http://dx.doi.org/10.1016/j.mce.2015.01.004

The concentration of the important second messenger cAMP is regulated by
phosphodiesterases (PDEs) and hence an attractive drug target. However,
limited human data are available about the PDEs in the ovary. The aim of the
present study was to describe and characterise the PDEs in the human ovary.
Results were obtained by analysis of mRNA microarray data from follicles and
granulosa cells (GCs), combined RT-PCR and enzymatic activity analysis in GCs,
immunohisto-chemical analysis of ovarian sections and by studying the effect
of PDE inhibitors on progesterone production from cultured GCs. We found that
PDE3, PDE4, PDE7 and PDE8 are the major families present while PDE11A
was not detected. PDE8B was differentially expressed during folliculogenesis.
In cultured GCs, inhibition of PDE7 and PDE8 increased basal progesterone
secretion while PDE4 inhibition increased forskolin-stimulated progesterone
secretion. In conclusion, we identified PDE3, PDE4, PDE7 and PDE8 as
the major PDEs in the human ovary.
Diethylstilbestrol can effectively accelerate estradiol-17-O-glucuronidation, while
potently inhibiting estradiol-3-O-glucuronidation

Liangliang Zhu, Ling Xiao, Yangliu Xia, .., Yan Wu, Ganlin Wu, Ling Yang
Toxicology and Applied Pharmacology 283 (2015) 109–116
http://dx.doi.org/10.1016/j.taap.2015.01.003

This in vitro study investigates the effects of diethylstilbestrol (DES), a widely
used toxic synthetic estrogen, on estradiol-3- and 17-O- (E2-3/17-O)
glucuronidation, via culturing human liver microsomes (HLMs) or
recombinant UDP-glucuronosyl-transferases (UGTs) with DES and E2.
DES can potently inhibit E2-3-O-glucuronid-ation in HLM, a probe reaction
for UGT1A1. Kinetic assays indicate that the inhibition follows a competitive
inhibition mechanism, with the Ki value of 2.1 ± 0.3 μM, which is less than
the possible in vivo level. In contrast to the inhibition on E2-3-O-glucuronidation,
the acceleration is observed on E2-17-O-glucuronidation in HLM, in which
cholestatic E2-17-O-glucuronide is generated. In the presence of DES
(0–6.25 μM), Km values for E2-17-Oglucuronidation are located in the
range of 7.2–7.4 μM, while Vmax values range from 0.38 to 1.54 nmol/min/mg.
The mechanism behind the activation in HLM is further demonstrated by
the fact that DES can efficiently elevate the activity of UGT1A4 in catalyzing
E2-17-O-glucuronidation. The presence of DES (2 μM) can elevate Vmax from
0.016 to 0.81 nmol/min/mg, while lifting Km in a much lesser extent from 4.4 to
11 μM. Activation of E2-17-O-glucuronidation is well described by a two binding
site model, with KA, α, and β values of 0.077 ± 0.18 μM, 3.3 ± 1.1 and 104 ± 56,
respectively. However, diverse effects of DES towards E2-3/17-O-glucuronidation
are not observed in liver microsomes from several common experimental animals.
In summary, this study issues new potential toxic mechanisms for DES: potently
inhibiting the activity of UGT1A1 and powerfully accelerating the formation of
cholestatic E2-17-O-glucuronide by UGT1A4.
Dehydroepiandrosterone: A neuroactive steroid

Luboslav Stárka, Michaela Dusková, Martin Hill
Journal of Steroid Biochemistry & Molecular Biology 145 (2015) 254–260
http://dx.doi.org/10.1016/j.jsbmb.2014.03.008

Dehydroepiandrosterone (DHEA) and its sulfate bound form (DHEAS) are important
steroids of mainly adrenal origin. They are produced also in gonads and in the brain.
Dehydroepiandrosterone easily crosses the brain–blood barrier and in part is also
produced locally in the brain tissue. In the brain, DHEA exerts its effects after
conversion to either testosterone and dihydrotestosterone or estradiol via androgen
and estrogen receptors present in the most parts of the human brain, through
mainly non-genomic mechanisms, or eventually indirectly via the effects of its
metabolites formed locally in the brain. As a neuroactive hormone, DHEA in
cooperation with other hormones and transmitters significantly affects some
aspects of human mood, and modifies some features of human emotions and
behavior. It has been reported that its administration can increase feelings of well-
being and is useful in ameliorating atypical depressive disorders. It has
neuroprotective and antiglucocorticoid activity and modifies immune reactions,
and some authors have also reported its role in degenerative brain diseases.
Here we present a short overview of the possible actions of dehydroepiandrosterone
and its sulfate in the brain, calling attention to various mechanisms of their action
as neurosteroids and to prospects for the knowledge of their role in brain disorders.
Androgens and mammalian male reproductive tract development

Aki Murashima, Satoshi Kishigami, Axel Thomson, Gen Yamada
Biochimica et Biophysica Acta 1849 (2015) 163–170
http://dx.doi.org/10.1016/j.bbagrm.2014.05.020

One of the main functions of androgen is in the sexually dimorphic development of
the male reproductive tissues. During embryogenesis, androgen determines the
morphogenesis of male specific organs, such as the epididymis, seminal vesicle,
prostate and penis. Despite the critical function of androgens in masculinization,
the downstream molecular mechanisms of androgen signaling are poorly
understood. Tissue recombination experiments and tissue specific androgen
receptor (AR) knockout mouse studies have revealed epithelial or mesenchymal
specific androgen-AR signaling functions. These findings also indicate that
epithelial–mesenchymal interactions are a key feature of AR specific activity,
and paracrine growth factor action may mediate some of the effects of androgens.
This review focuses on mouse models showing the interactions of androgen and
growth factor pathways that promote the sexual differentiation of reproductive organs.
Recent studies investigating context dependent AR target genes are also discussed.
This article is part of a Special Issue entitled: Nuclear receptors in animal development.

All sex steroids are made intracellularly in peripheral tissues by the mechanisms of
intracrinology after menopause

Fernand Labrie
Journal of Steroid Biochemistry & Molecular Biology 145 (2015) 133–138
http://dx.doi.org/10.1016/j.jsbmb.2014.06.001

Following the arrest of estradiol secretion by the ovaries at menopause, all estrogens
and all androgens in postmenopausal women are made locally in peripheral target
tissues according to the physiological mechanisms of intracrinology. The locally
made sex steroids exert their action and are inactivated intracellularly without
biologically significant release of the active sex steroids in the circulation.The
level of expression of the steroid-forming and steroid-inactivating enzymes is
specific to each cell type in each tissue, thus permitting to each cell/tissue to
synthesize a small amount of androgens and/or estrogens in order to meet the
local physiological needs without affecting the other tissues of the organism.
Achieved after 500 million years of evolution, combination of the arrest of ovarian
estrogen secretion, the availability of high circulating levels of DHEA and the
expression of the peripheral sex steroid-forming enzymes have permitted the
appearance of menopause with a continuing access to intra-tissular sex steroids
for the individual cells/tissues without systemic exposure to circulating estradiol.
In fact, one essential condition of menopause is to maintain serum estradiol at
biologically inactive (subthreshold) concentrations, thus avoiding stimulation of the
endometrium and risk of endometrial cancer. Measurement of the low levels of
serum estrogens and androgens in postmenopausal women absolutely requires
the use of MS/MS-based technology in order to obtain reliable accurate, specific
and precise assays. While the activity of the series of steroidogenic enzymes can
vary, the serum levels of DHEA show large individual variations going from barely
detectable to practically normal “premenopausal” values, thus explaining the
absence of menopausal symptoms in about 25% of women. It should be added
that the intracrine system has no feedback elements to adjust the serum levels
of DHEA, thus meaning that women with low DHEA activity will not be improved
without external supplementation. Exogenous DHEA, however, follows the same
intracrine rules as described for endogenous DHEA, thus maintaining serum
estrogen levels at subthreshold or biologically inactive concentrations. Such blood
concentrations are not different from those observed in normal postmenopausal
women having high serum DHEA concentrations. Androgens, on the other hand,
are practically all made intracellularly from DHEA by the mechanisms of intracrinology
and are always maintained at very low levels in the blood in both pre- and
postmenopausal women. Proof of the importance of intracrinology is also provided,
among others, by the well-recognized benefits of aromatase inhibitors and
anti-estrogens used successfully for the treatment of breast cancer in
postmenopausal women where all estrogens are made locally. Each medical
indication for the use of DHEA, however, requires clinical trials performed
according to the FDA guidelines and the best rules of clinical medicine.
A multi-step, dynamic allosteric model of testosterone’s binding to sex hormone
binding globulin

Mikhail N. Zakharov, Shalender Bhasin, Thomas G. Travison, Ran Xue, et al.
Molecular and Cellular Endocrinology 399 (2015) 190–200
http://dx.doi.org/10.1016/j.mce.2014.09.001

Purpose: Circulating free testosterone (FT) levels have been used widely in the
diagnosis and treatment of hypogonadism in men. Due to experimental
complexities in FT measurements, the Endocrine Society has recommended
the use of calculated FT (cFT) as an appropriate approach for estimating FT.
We show here that the prevailing model of testosterone’s binding to SHBG,
which assumes that each SHBG dimer binds two testosterone molecules
and that the two binding sites on SHBG have similar binding affinity is
erroneous and provides FT values that differ substantially from those
obtained using equilibrium dialysis.
Methods: We characterized testosterone’s binding to SHBG using
binding isotherms, ligand depletion curves, and isothermal titration
calorimetry (ITC). We derived a new model of testosterone’s binding to
SHBG from these experimental data and used this model to determine
FT concentrations and compare these values with those derived from
equilibrium dialysis.
Results: Experimental data on testosterone’s association with SHBG
generated using binding isotherms including equilibrium binding, ligand
depletion experiments, and ITC provide evidence of a multi-step dynamic
process, encompassing at least two inter-converting microstates in unliganded
SHBG, readjustment of equilibria between unliganded states upon binding
of the first ligand molecule, and allosteric interaction between two binding
sites of SHBG dimer. FT concentrations in men determined using the new
multistep dynamic model with complex allostery did not differ from those
measured using equilibrium dialysis. Systematic error in calculated FT
vales in females using Vermeulen’s model was also significantly reduced.
In European Male Aging Study, the men deemed to have low FT (<2.5th
percentile) by the new model were at increased risk of sexual symptoms
and elevated LH.
Conclusion: Testosterone’s binding to SHBG is a multi-step dynamic
process that involves complex allostery within SHBG dimer. FT values
obtained using the new model have close correspondence with those
measured using equilibrium dialysis.

Cohesin modulates transcription of estrogen-responsive genes

Jisha Antony, Tanushree Dasgupta, Jenny M. Rhodes, Miranda V. McEwan, et al.
Biochimica et Biophysica Acta 1849 (2015) 257–269
http://dx.doi.org/10.1016/j.bbagrm.2014.12.011

The cohesin complex has essential roles in cell division, DNA damage repair
and gene transcription. The transcriptional function of cohesin is thought to
derive from its ability to connect distant regulatory elements with gene promoters.
Genome-wide binding of cohesin in breast cancer cells frequently coincides
with estrogen receptor alpha (ERα), leading to the hypothesis that cohesin
facilitates estrogen-dependent gene transcription. We found that cohesin
modulates the expression of only a subset of genes in the ER transcription
program, either activating or repressing transcription depending on the gene
target. Estrogen-responsive genes most significantly influenced by cohesin
were enriched in pathways associated with breast cancer progression such
as PI3K and ErbB1. In MCF7 breast cancer cells, cohesin depletion enhanced
transcription of TFF1 and TFF2, and was associated with increased ER binding
and increased interaction between TFF1 and its distal enhancer situated
within TMPRSS3. In contrast, cohesin depletion reduced c-MYC mRNA and
was accompanied by reduced interaction between a distal enhancer of c-MYC
and its promoters. Our data indicates that cohesin is not a universal facilitator
of ER-induced transcription and can even restrict enhancer–promoter communication.
We propose that cohesion modulates transcription of estrogen-dependent genes
to achieve appropriate directionality and amplitude of expression.
Angiogenesis in Breast Cancer and its Correlation with Estrogen, Progesterone
Receptors and other Prognostic Factors

Jyotsna Naresh Bharti, Poonam Rani, Vinay Kamal, Prem Narayan Agarwal
Journal of Clinical and Diagnostic Research. 2015 Jan, Vol-9(1): EC05-EC07
http://dx.doi.org:/10.7860/JCDR/2015/10591.5447

Purpose: The aim of study is to evaluate angiogenesis using CD34, in
estrogen, progesterone positive and negative breast cancer and to correlate
the microvessel density with known histological prognostic factors,
morphological type of breast carcinoma and lymph node metastasis.
Materials and Methods: Twenty eight untreated cases of breast cancer were
included in the study and paraffin embedded sections were obtained
from representative mastectomy specimen of breast cancer patient. The sections
were stained with hematoxylin and eosin stain and immunohistochemistry was
performed using CD34, estrogen, progesterone, cytokeratin and epithelial
membrane antigen antibody. Angiogenesis was analyzed using CD 34 antibody.
For statistical analysis, cases were grouped into estrogen, progesterone positive
and negative receptors.
Results: Mean microvessel density in ER-/PR-, ER-/ PR+, ER+/PR-, ER+/PR+
was 15.45, 14.83, 11, 10.89 respectively. A significant correlation was found
between ER receptors and mean vascular density with p-value (< 0.05).
A significant difference was observed in mean vascular density between
the four groups comprising (p-value < 0.05). Infiltrating duct carcinoma
(NOS) grade III has got the highest mean microvessel density (14.17)
followed by grade II (12.93) and grade I (12.33).
Conclusion: Information about prognostic factors in breast cancer
patients may lead to better ways to identify those patients at high risk
who might benefit from adjuvant therapies.

Combined blockade of testicular and locally made androgens in prostate cancer:
A highly significant medical progress based upon intracrinology

Fernand Labrie
Journal of Steroid Biochemistry & Molecular Biology 145 (2015) 144–156
http://dx.doi.org/10.1016/j.jsbmb.2014.05.012

Recently two drugs, namely the antiandrogen MDV-3100 and the inhibitor
of 17β-hydroxylase abiraterone have been accepted by the FDA for the
treatment of castration-resistant prostate cancer (CRPC) with or without
previous chemotherapy, with a prolongation of overall survival of 2.2–4.8months.
While medical (GnRH agonist) or surgical castration reduces the serum levels
of testosterone by about 97%, an important concentration of testosterone and
dihydrotestosterone remains in the prostate and activates the androgen receptor
(AR), thus offering an explanation for the positive data obtained in CRPC. In fact,
explanation of the response observed with MDV-3100 or enzalutamide in CRPC
is essentially a blockade of the action or formation of intraprostatic androgens.
In addition to the inhibition of the action or formation of androgens made locally
by the mechanisms of intracrinology, increased AR levels and AR mutations can
be involved, especially in very advanced disease.

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Thyroid Function and Disorders

Posted in Amino acids, Calcium Signaling, Clinical Diagnostics, Curation, Developmental biology, Disease Biology, Endocrine Diseases, Genomic Endocrinology, Metabolism, Neurological Diseases, Proteins, Translational Science, tagged blood brain barrier, brain, hyperthyroidism, hypothyroid, MCT8, retinoids, T3, T4, thyroglobulin, Thyroid, thyroid nodule on February 5, 2015| Leave a Comment »

Thyroid Function and Disorders

Writer and Curator: Larry H. Bernstein, MD, FCAP

Normal thyroid function is maintained by endocrine interactions between the hypothalamus, anterior pituitary and thyroid gland [Matfin, 2009]. Iodide is transported across the basement membrane of the thyroid cells by an intrinsic membrane protein called the Na/I symporter (NIS). At the apical border, a second iodide transport protein called pendrin moves iodide into the colloid, where it is involved in hormono-genesis. Once inside the follicle, most of the iodide is oxidized by the enzyme thyroid peroxidase (TPO) in a reaction that facilitates combination with a tyrosine molecule to ultimately form thyroxine (T4) and triiodothyronine (T3). Thyroxine is the major thyroid hormone secreted into the circulation (90%, with T3 composing the other 10%). There is evidence that T3 is the active form of the hormone and that T4 is converted into T3 before it can act physiologically.

All of the major organs in the body are affected by altered levels of thyroid hormone. These actions are mainly mediated by T3. In the cell, T3 binds to a nuclear receptor, resulting in transcription of specific thyroid hormone response genes.

Maternal thyroid hormones are essential for neural development in zebrafish.

Marco A Campinho, João Saraiva, Claudia Florindo, Deborah M Power Molecular endocrinology (Baltimore, Md.) 05/2014;
http://dx.doi.org:/10.1210/me.2014-1032

ABSTRACT Teleost eggs contain an abundant store of maternal thyroid hormones (THs) and early in zebrafish embryonic development all the genes necessary for TH signalling are expressed. Nonetheless the function of THs in embryonic development remains elusive. To test the hypothesis that THs are fundamental for zebrafish embryonic development an MCT8 knockdown strategy was deployed to prevent maternal TH uptake. Absence of maternal THs did not affect early specification of the neural epithelia but profoundly modified later dorsal specification of the brain and spinal cord as well as specific neuron differentiation. Maternal THs acted upstream of pax2a, pax7 and pax8 genes but downstream of shha and fgf8a signalling. The lack of inhibitory spinal cord interneurons and increased motorneurons in the MCT8 morphants is consistent with their stiff axial body and impaired mobility. MCT8 mutations are associated with X-linked mental retardation in humans and the cellular and molecular consequences of MCT8 knockdown during embryonic development in zebrafish provides new insight into the potential role of THs in this condition.
Relationship between thyroid status and renal function in a general population of unselected outpatients

Giuseppe Lippi, Martina Montagnana, Giovanni Targher, Gian Luca Salvagno, Gian Cesare Guidi
Clin Biochem May 2008; 41(7–8): 625-627

When compared with euthyroid subjects, those with TSH < 0.2 mIU/L and > 2.5 mIU/L had increased and decreased estimated glomerular filtration rate (e-GFR), respectively. TSH levels were an independent predictor of e-GFR.

Serum Thyroid-Stimulating Hormone Measurement for Assessment of Thyroid Function and Disease

Douglas S. Ross
Endocr and Metab Clinics of N Am, Jun 2001; 30(2, 1): 245-264

Thyrotropin, or thyroid-stimulating hormone (TSH), is one of a family of glycoprotein hormones including luteinizing hormone (LH), follicle-stimulating hormone (FSH), and human chorionic gonadotropin (hCG) that share a common α-subunit and a unique β-subunit. Pituitary TSH regulates the secretion of the thyroid hormones T4 (thyroxine) and T3 (triiodothyronine). TSH secretion, in turn, is controlled through negative feedback by thyroid hormone on the pituitary thyrotrope. This relationship is negative log-linear. Small changes in serum free thyroid hormone concentrations result in large changes in serum TSH concentrations, and even subtle changes in thyroid hormone production are best assessed by measurement of serum TSH . Until the late 1980s, the detection limit of TSH assays was within the normal range, and these first-generation TSH assays were useful only for the detection of hypothyroidism. Free T4 measurements were primarily used for assessing thyroid function despite the technical difficulties in free thyroid hormone measurements owing to abnormal binding proteins, changes in binding protein concentrations, and the effects of drugs and illness on thyroid hormone binding. With the use of sensitive second- and third-generation TSH assays, TSH measurement has emerged as the single most useful test of thyroid function. It is widely and appropriately used as a screening test. Unfortunately, the trend has been to rely on TSH measurements alone for the assessment of complicated thyroid disease and patients undergoing treatment for thyroid dysfunction. This article focuses on the potential and real limitations of TSH measurement.
Correlation of creatinine with TSH levels in overt hypothyroidism — A requirement for monitoring of renal function in hypothyroid patients?

Vandana Saini, Amita Yadav, Megha Kataria Arora, Sarika Arora, Ritu Singh, Jayashree Bhattacharjee
Clin Biochem Feb 2012; 45(3): 212-214

Highlights
► Increase serum creatinine levels in both subclinical and overt hypothyroidism. ► Creatinine levels progressively increase with increasing degree of hypothyroidism. ► Increase in creatinine correlated with TSH levels in overt hypothyroid subjects. ► Regular monitoring of renal function is required in hypothyroid patients.

Renal function is influenced by thyroid status. Therefore, this study was done to determine the relationship between renal function and different degrees of thyroid dysfunction.
Design and methods
Thyroid and kidney function tests were analyzed in 47 patients with overt (TSH ≥ 10.0 μIU/L) and 77 patients with subclinical hypothyroidism (TSH 6.0–9.9 μIU/L) in a cross-sectional study. These were compared with 120 age- and sex-matched euthyroid controls.
Results
Overt hypothyroid subjects showed significantly raised serum urea, creatinine and uric acid levels as compared to controls whereas subclinical hypothyroid patients showed significant increased levels of serum urea and creatinine levels. TSH showed significant positive correlation with creatinine and uric acid values and, fT4 had a negative correlation with uric acid in overt hypothyroidism.
Conclusion
Hypothyroid state is associated with significant derangement in biochemical parameters of renal function. Hence the renal function should be regularly monitored in hypothyroid patients.

Ability of Serum Thyroid-Stimulating Hormone Levels to Reflect Peripheral and Central Thyroid Hormone Action Appropriately

Uncertainty Owing to Heterogeneity of T4 Deiodinases
Uncertainty Owing to Heterogeneity of T3 Receptors
Uncertainty Owing to Resetting of the Threshold for Negative Feedback

Clinical Utility of Thyroid-Stimulating Hormone Measurement
Screening for Thyroid Disease and Assessment of Patients Suspected of Having Thyroid Disease

Limitations of Thyroid-Stimulating Hormone Testing in Patients with Known Thyroid Disease Central Hypothyroidism
Thyrotoxicosis Owing to Inappropriate Thyroid-Stimulating Hormone Secretion
Monitoring Thyroid Hormone Therapy
Patients Treated for Hyperthyroidism

The Pituitary-Thyroid Axis in Nonthyroidal Illness

Measurement of Thyroid-Stimulating Hormone
Drugs that Affect Serum Thyroid-Stimulating Hormone Concentrations

Investigations into the etiology of elevated serum T3 levels in protein-malnourished rats

Robert C. Smallridge, Allan R. Glass, Leonard Wartofsky, Keith R. Latham, Kenneth D. Burman
Metabolism, V June 1982; 31(6): 538-542

Thyroid function studies and the peripheral metabolism of thyroid hormone were examined in rats fed a low protein diet (9% casein) for 4–8 wk. Compared to animals fed a normal protein diet ad libitum, both the low protein rats and a pair-fed control group weighed less at the end of the study. However, serum total T3 levels were significantly higher only in the protein deficient rats. The elevated serum T3 was not explainable by enhanced peripheral T4 to T3 conversion, as there was no evidence of any change in hepatic or renal 5′-deiodinase activity when homogenates were examined for conversion of T4 to T3, reverse T3 to 3,3′-diiodothyronine, or 3′,5′-diiodothyronine to 3′-monoiodothyronine. Neither was there an effect on hepatic T3 receptor maximal binding capacity (204 ± 24 versus 168 ± 15 fmol/mg DNA control) or binding affinity (2.07 ± 0.38 versus 2.49 ± 0.24 × 10−10 M control). In two separate experiments the dialyzable fraction of T3 was significantly lower in the low protein group while free T3 concentrations were unchanged or reduced. In contrast, serum total and free T4 were either normal or reduced and dialyzable T4 was unaffected by protein deficiency. We conclude that while serum total T3 is elevated in rats chronically fed a low protein diet, this elevation is not due to enhanced T4 to T3 conversion. Rather, the increased T3 levels can be accounted for by a striking alteration in protein binding to T3. Moreover, the failure to demonstrate similar changes in serum total and dialyzable T4 suggests that in the rat, protein deficiency has different effects on binding to the two major thyroid hormones. Dietary induced changes in serum thyroid hormone binding must be kept in mind in nutrition studies in the rat.

Role of thyrotropin in metabolism of thyroid hormones in nonthyroidal tissues

Udaya M. Kabadi
Metabolism, Jun 2006; 55(6): 748-750

T4 conversion into T3 in peripheral tissues is the major source of circulating T3. However, the exact mechanism of this process is ill defined. Several in vitro studies have demonstrated that thyrotropin facilitates deiodination of T4 into T3 in liver and kidneys. However, there is a paucity of in vitro studies confirming this activity of thyrotropin. Therefore, this study was conducted to examine the influence of thyrotropin on thyroid hormone metabolism in nonthyroidal tissues. We assessed T4, T3, reverse T3 (rT3), and T3 resin uptake (T3RU) responses up to 12 hours at intervals of 4 hours in 6 thyroidectomized female mongrel dogs rendered euthyroid with LT4 replacement therapy before and after subcutaneous (SC) administration of bovine thyrotropin (5 U) on one day and normal saline (0.5 mL) on another in a randomized sequence between 08:00 and 09:00 am. Euthyroid state after LT4 replacement was confirmed before thyrotropin administration. Serum T4, T3, rT3, and T3RU all remained unaltered after SC administration of normal saline. No significant alteration was noted in serum T3RU values on SC administration of thyrotropin. However, serum T3 rose progressively reaching a peak at 12 hours with simultaneous declines being noted in both serum T4 and rT3 concentrations (P < .05 vs prethyrotropin values for all determinations). The changes after SC administration were significantly different (P < .001) in comparison to those noted on SC administration of normal saline. Thyrotropin may promote both the conversion of T4 to T3 and metabolism of rT3 into T2 in nonthyroidal tissues via enhancement of the same monodeionase.

Effects of growth hormone administration on fuel oxidation and thyroid function in normal man

Jens Møller, Jens O.L. Jørgensen, Niels Møller, Jens S. Christiansen, Jørgen Weeke
Metabolism, Jul 1992; 41(7): 728-731

In a randomized, double-blind, placebo-controlled, cross-over study, we examined the effects of 14 days of growth hormone (GH) administration (12 IU/d subcutaneously) on energy expenditure (EE), respiratory exchange ratio (RER), and thyroid function in 14 normal adults of normal weight (eight men and six women). EE (kcal/24 h) was significantly elevated after GH administration (2,073 ± 392, [GH], 1,900 ± 310, [placebo], P = .01). RER was significantly lowered during GH administration (0.73 ± 0.04 v 0.78 ± 0.06, P = .02), reflecting increased oxidation of lipids. Total triiodothyronine (TT3) (nmol/L) and free T3 (FT3) (pmol/L) increased significantly during GH (TT3: 1.73 ± 0.06 [GH], 1.48 ± 0.08 [placebo], P = .01; FT3: 6.19 ± 0.56 [GH], 5.49 ± 0.56 [placebo], P = .01). Concomitantly, an insignificant decrease in reverse T3 (rT3) (nmol/L) was observed (0.07 ± 0.01 [GH], 0.15 ± 0.01 [placebo], P = .08). GH caused a highly significant increase in T3/thyroxine (T4 (×100) ratio (1.84 ± 0.12 [GH], 1.37 ± 0.06 [placebo]). Serum thyrotropin (TSH) was not significantly changed by GH. No changes in total thyroxine (TT4) (nmol/L) (98 ± 6 [GH], 111 ± 8 [placebo], P = .40) and free thyroxine (FT4) (pmol/L) (17.4 ± 1.3 [GH], 18.6 ± 1.1 [placebo], P = .37) after 14 days of GH administration were observed. In conclusion, 2 weeks of GH administration increases EE and lipidoxidation. This finding may partly be mediated by an increase in peripheral T4 to T3 conversion.

Studies on the deiodination of thyroid hormones in Xenopus laevis tadpoles

Helen Robinson, Valerie Anne Galton
Gen Compar Endocr, Sept 1976; 30(1): 83-90

Liver and tail tissues from Xenopus laevis tadpoles possess deiodinating systems capable of degrading both thyroxine (T4) and 3,5,3′-triiodothyronine (T3). Deiodinating activity in liver remains at a constant level throughout late development and metamorphosis with the exception of a transient increase at stage 59, the onset of metamorphosis. Tail activity remains constant during development but rises sharply during metamorphosis when the tail is undergoing regression. In contrast to these findings on spontaneously metamorphosing tadpoles, tail tips induced to regress in vitro do not exhibit any rise in deiodinating activity, even when the tail tips are undergoing extensive autolysis. These results indicate that, while a rise in deiodinating activity may coincide temporarily with hormone action during metamorphosis, the two phenomena may be separated. The deiodinating activity present in tadpole tissues appears to be enzymic and possesses properties characteristic of peroxidase activity. The reaction catalyzed by this mechanism does not appear to involve monodeiodination and hence cannot be considered a mechanism for the peripheral conversion of T4 to T3.

Mechanisms governing the relative proportions of thyroxine and 3,5,3′-triiodothyronine in thyroid secretion

Peter Laurberg
Metabolism, Apr 1984; 33(4): 379-392

In subjects with normal thyroid function only a minor part of circulating 3,5,3′-triiodothyronine (T3) originates directly from the thyroid; the majority is produced in the peripheral tissues by deiodination of thyroxine (T4). However, T3 of thyroidal origin constitutes a relatively high fraction of the total T3 produced in many patients with thyroid hyperfunction or hypofunction. Such a relatively high T3 content in the secretion of the thyroid could be caused by a low T4T3 ratio in thyroglobulin. Severe iodine deficiency is a well-known inducer of a low T4T3 ratio, but a low T4T3 ratio can also be produced independent of the iodine content. This is seen in in vitro studies of thyroglobulin iodination when small amounts of DIT are added to the incubation mixture and in vivo in TSH-treated animals and in patients with Graves’ disease. Another mechanism for high thyroidal secretion of T3 could be an enhanced fractional deiodination of T4 to T3 in the thyroid. In vitro thyroid perfusion studies have shown that the T3 content of thyroid secretions is higher than would be expected from the T4T3 ratio of thyroid hydrolysate and that the major mechanism is deiodination of T4 to T3. Thyroxine deiodinases are also present in the human thyroid, and the amount of T4 deiodinase is enhanced in the thyroids from patients with medically treated Graves’ disease and in the hyperstimulated thyroids of rats. Other factors of possible importance for the mixture of T3 and T4 secreted by the thyroid are a relatively faster liberation of T3 than of T4 from thyroglobulin during partial hydrolysis (this faster release of T3 is probably the mechanism behind the more “rapid” secretion of T3 than of T4), or some kind of thyroid heterogeneity leading to pinocytosis and hydrolysis of thyroglobulin with a lower T4T3 ratio than that of average thyroglobulin.

Starvation-induced alterations of circulating thyroid hormone concentrations in man

Thomas J. Merimee, E.S. Fineberg
Metabolism Jan 1976; 25(1): 79-83

Serum concentrations of triiodothyronine (T3), thyroxine (T4), and TSH were examined in seven men and seven women of normal weight during a 60-hr fast. Similar studies were conducted in two women who received daily for 1 mo before and during a similar fast, 0.4 mg and 0.5 mg of l-thyroxine.
The serum concentrations of T3 decreased in each of the untreated normal subjects (sign test of significance, p < 0.001). The mean control concentration of T3 in women was 152 ± 9 ng100 ml (X ± SEM); after 24 hr of fasting, 131 ± 31 ng100 ml; and at the termination of the fast, 90 ± 15 ng100 ml. The latter value differed from the control value with a p value of < 0.01. Similar changes of T3 concentration occurred in men (mean basal T = 160 ± 11 ng100 ml; mean at termination of fast = 87 ± 16 ng100 ml). The range of decrease for T3 in all subjects varied from 24% to 55%.
The mean T4 concentration at the beginning of the fast was 6.9 ± 0.9, and at the termination of the fast, 7.5 ± 0.6 (p = NS). TSH concentrations remained unchanged (Control, 3.8 ± 0.45 μU/ml; at 60 hr, 4.0 ± 0.26 μU/ml, p = NS).
Studies in two women who received, before and during a fast, T4, indicate that a decreased peripheral conversion of T4 to T3 is the most likely mechanism responsible for this change.

Effect of estrogens on thyroid function. II. Alterations in plasma thyroid hormone levels and their metabolism

Ramesh C. Sawhney, Indra Rastogi, Gopal K. Rastogi
Metabolism Mar 1978; 27(3): 279-288

The circulating levels of total triiodothyronine (TT3), thyroxine (TT4, and T4-bbinding globulin (TBG) and the kinetics of T3 and T4 were studied in five menstruating rhesus monkeys before, during, and after prolonged treatment with estradiol monobenzoate (E2B, 50 μg/kg body weight/day subcutaneously). A significant increase over pretreatment (p < 0.01) plasma TT3, TT4, and TBG was recorded on day 6 of E2B therapy. A further significant stepwise increase in these parameters was noted up to day 19 of E2B, when the levels plateaued for the rest of the period of E2B treatment. Two weeks after discontinuation of E2B, plasma TT3, TT4, and TBG had returned to the pretreatment range and remained so up to 40 days of observation. Although the percent free T3 and percent free T4 were significantly decreased (p < 0.01) during E2B therapy, the absolute concentrations of free T3 and free T4 were not altered. After prolonged E2B treatment the metabolic clearance rate, distribution space, and production rate (PR) of both T3 and T4 were decreased (p < 0.01). The extrathyroidal T4 pool (ETT4P) was significantly increased (p < 0.01), whereas ETT3P did not show any significant alterations (p > 0.05). The decreased PR of T4 might have been due to a direct inhibitory effect of E2B on the thyroid, whereas the decrease in PR of T3 might have been due to either decreased conversion of T4 to T3, to decreased secretion by the thyroid, or both.
Zebrafish as a model to study peripheral thyroid hormone metabolism in vertebrate development

Marjolein Heijlen, Anne M. Houbrechts, Veerle M. Darras
Gen Compar Endocr 1 Jul 2013; 188: 289-296

To unravel the role of thyroid hormones (THs) in vertebrate development it is important to have suitable animal models to study the mechanisms regulating TH availability and activity. Zebrafish (Danio rerio), with its rapidly and externally developing transparent embryo has been a widely used model in developmental biology for some time. To date many of the components of the zebrafish thyroid axis have been identified, including the TH transporters MCT8, MCT10 and OATP1C1, the deiodinases D1, D2 and D3, and the receptors TRα and TRβ. Their structure and function closely resemble those of higher vertebrates. Interestingly, due to a whole genome duplication in the early evolution of ray-finned fishes, zebrafish possess two genes for D3 (dio3 and dio3a) and for TRα (thraa and thrab). Transcripts of all identified genes are present during embryonic development and several of them show dynamic spatio-temporal distribution patterns. Transient morpholino-knockdown of D2, D3 or MCT8 expression clearly disturbs embryonic development, confirming the importance of each of these regulators during early life stages. The recently available tools for targeted stable gene knockout will further increase the value of zebrafish to study the role of peripheral TH metabolism in pre- and post-hatch/post-natal vertebrate development.

The consequences of inappropriate treatment because of failure to recognize the syndrome of pituitary and peripheral tissue resistance to thyroid hormone

Samuel Refetoff, Angel Salazar, Terry J. Smith, Neal H. Scherberg
Metabolism Aug 1983; 32(8); 822-834

Since the description of the syndrome of global (peripheral tissues and pituitary) resistance to thyroid hormone, new cases are being recognized with increasing frequency. The patient described herein had a markedly elevated serum TSH concentration of 260 μU/mL at the time of diagnosis. Studies suggest that elevations of serum TSH levels in this and other patients with the syndrome are most likely iatrogenic in origin. The patient was 312 years old when a goiter and a high serum T4 concentration were detected. Despite subtotal thyroidectomy, antithyroid drugs were required to maintain her T4 level in the normal range. She was referred at age 1112 years because of recurrent goiter. Her parents and five older siblings had normal thyroid function. Off therapy, her serum T4 level was 14.9 μg/dL, FT4I was 17.0, T3 was 362 ng/dL, TSH was 260 μU/mL, and antibodies were negative. There were no signs of thyrotoxicosis, her bone age was 7 years, her growth was stunted (third percentile), her intellectual quotient (IQ) was 67, and there was a 30–50 dB sensorineural hearing loss. The presence of a pituitary adenoma was ruled out. Her TSH had normal bioreactivity and rose to 540 μU/mL in response to TRH. Triiodothyronine was given in incremental doses of 50, 100, 200, and 400 μg/d over 28 days. The log concentrations of serum TSH showed an inverse linear correlation with serum T3. While receiving the highest dose of T3, on which the level of serum T3 ranged from 1400 to 2500 ng/dL, the TSH response to TRH normalized (basal 4.2 and peak 20 μU/mL), as did the high levels of serum cholesterol, carotene, and T4. Her BMR rose from +5 to +22%, her IQ rose to 77, and she gained weight without an increase in caloric intake. Only minimal changes were observed in levels of urinary cAMP, hydroxyproline, magnesium, and nitrogen. All values, with the exception of the weight gain, returned to baseline 2 months after T3 treatment was discontinued. The TSH level was suppressed by l-dopa and by prednisone. Long-term therapy with equivalent doses of T4 (from 300 to 1000 μg/d) produced a growth of 3 cm during the initial 6 weeks, 10.5 cm over the ensuring year (above the 10th percentile), and regression of goiter without thyrotoxicosis. The patient exhibited resistance to thyroid hormone in pituitary and peripheral tissues. The optimal dose of T4 replacement could be predicted by studying tissue responses to incremental doses of T3. The marked elevation in serum TSH concentration, stunted growth, and laboratory evidence of hypothyroidism were due to the limited thyroidal reserve caused by thyroidectomy. All patients with an impaired ability to compensate for the defect as a result of inappropriate treatment should be given thyroid hormone in amounts short of producing catabolic effects. Such a dose is expected to normalize the basal serum TSH concentration and its response to TRH.

Solving the mystery of iodine uptake

Valda Vinson
Science 20 Jun 2014; 344(6190), p. 1355
http://dx.doi.org:/10.1126/science.344.6190.1355-a

The thyroid gland produces iodine-containing hormones that regulate metabolism. The cell membrane protein NIS (sodium/iodine symporter) transports iodine into thyroid cells, but because iodine concentrations outside of the cell are so low, how it does so is a mystery. The key? Moving two sodium ions along with the iodine ion, Nicola et al found. NIS also does not bind sodium very tightly, but the high concentrations of sodium outside the cell allow one sodium ion to bind. This binding increases the affinity of NIS for a second sodium ion and also for iodine. With the three ions bound, NIS changes its conformation so that it opens to the inside of the cell, where the sodium concentration is low enough for NIS to release its sodium ions. When the sodium goes away, so does NIS’s affinity for iodine, leading NIS to release it.

Unliganded Thyroid Hormone Receptor α Regulates Developmental Timing via Gene Repression in Xenopus tropicalis

Jinyoung Choi, Ken-ichi T. Suzuki, Tetsushi Sakuma, Leena Shewade, Takashi Yamamoto, and Daniel R. Buchholz
Endocr Feb 2015; 156(2): 735–744 http://dx.doi.org:/10.1210/en.2014-1554

Thyroid hormone (TH) receptor (TR) expression begins early in development in all vertebrates when circulating TH levels are absent or minimal, yet few developmental roles for unliganded TRs have been established. Unliganded TRs are expected to repress TH-response genes, increase tissue responsivity to TH, and regulate the timing of developmental events. Here we examined the role of unliganded TRα in gene repression and development in Xenopus tropicalis. We used transcription activator-like effector nuclease gene disruption technology to generate founder animals with mutations in the TRα gene and bred them to produce F1 offspring with a normal phenotype and a mutant phenotype, characterized by precocious hind limb development. Offspring with a normal phenotype had zero or one disrupted TRα alleles , and tadpoles with the mutant hind limb phenotype had two truncated TRα alleles with frame shift mutations between the two zinc fingers followed by 40–50 mutant amino acids and then an out-of-frame stop codon. We examined TH-response gene expression and early larval development with and without exogenous TH in F1 offspring. As hypothesized, mutant phenotype tadpoles had increased expression of TH-response genes in the absence of TH and impaired induction of these same genes after exogenous TH treatment, compared with normal phenotype animals. Also, mutant hind limb phenotype animals had reduced hind limb and gill responsivity to exogenous TH. Similar results in methimazole-treated tadpoles showed that increased TH-response gene expression and precocious development were not due to early production of TH. These results indicate that unliganded TRα delays developmental progression by repressing TH-response genes.
The discovery of thyroid replacement therapy. Part 2: The critical 19th century
Conceptualizing the link between the thyroid and myxoedema

Stefan Slater
R Soc Med 2011; 104: 59–63. http://dx.doi.org:/10.1258/jrsm.2010.10k051

Sir William Withey Gull (1816–1890)

Frederik Ruysch, anatomist in Leyden around 1690, adopted, according to Albrecht von Haller in 1766, the opinion that a peculiar fluid was elaborated in the gland and poured into the veins’. The 19th century thus began with thyroidology at best in embryo; but during that century endocrinology was born and the thyroid was its standard bearer. In 1836, Thomas Wilkinson King of Guys Hospital, regarded by some as the ‘Father of Endocrinology’, anticipated on the basis of observation and experiment the internal secretion of the thyroid. In a meticulous paper on its anatomy: he wrote of the thyroid gland that ‘its absorbent vessels carry its peculiar secretion to the great veins of the body’. This language is almost identical to that of Ruysch and Haller more than a century earlier. The idea was prompted by the thyroid’s disproportionately large vascular supply in the absence of any evident mechanical or other local function and also at what he described as its ‘peculiar’ fluid. King notes that his view ‘has been indirectly surmised by Morgagni [probably in 1761] and others’.
In 1850, at a meeting of the Royal Medical and Chirurgical Society of London, chaired by Thomas Addison, Thomas Blizzard Curling, surgeon at the London Hospital, provided a clear clinicopathological correlate in a paper entitled ‘Two cases of absence of the thyroid body and symmetrical swellings of fat tissue at the sides of the neck, connected with defective cerebral development’. Postmortem examination in each revealed no trace of thyroid tissue and that the swellings consisted only of fat. Curling’s important observation was not pursued until 1871 when, at another meeting of the Society, Curling himself then in the chair, Charles Hilton Fagge, a physician at Guy’s Hospital, presented a paper on sporadic cretinism. He described four living cases and noted that none of them had a goiter and that one had been well up to the age of eight and, although now physically cretinous at age 16, she remained very intelligent. He referred to Curling’s paper and reached the same conclusion that the ‘healthy thyroid body is capable of exerting a counteracting influence [on cretinism]’.
Two years later, in 1873, Fagge’s senior colleague at Guy’s, Sir William Withey Gull, presented before the Clinical Society of London two of the five cases he had seen of what he called ‘A Cretinoid State supervening in Adult Life in Women’. He described their cretin-like appearance, drawing particular attention to the broad and thick tongue and the guttural voice and its pronunciation ‘as if the tongue were too large for the mouth’. He acknowledged his remarks were tentative, hence, he said, his use of the word ‘cretinoid’, but he had no doubt this was a ‘substantive’ condition and not one of cardiac or renal origin.
Gull was an interesting personality with apparently a remarkable presence, resembling Napoleon in face, form and manner (Figure). In the 1970s, 80 years after his death in 1890, he was the subject of a theory, quickly discredited, that he had been ‘Jack the Ripper’, the killer in the still unsolved murders and mutilations of at least five Whitechapel prostitutes in 1888. He figured in the 1988 TV film series, Jack the Ripper, starring Michael Caine as the detective. Gull is credited with the first description of hypothyroidism in adults and his paper was important in defining a recognizable clinical syndrome.
Then, in 1877, William Miller Ord, read his paper before the Royal Medical and Chirurgical Society of London and proposed the term ‘myxoedema’ for the adult condition. He described the non-pitting, ‘mucous edema’. He also presented an engaging theory to explain the lethargy, inertia and slow responses associated with the disease. He suggested that these might result from the sheathing and insulation of the body in a ‘jelly-like’, mucin-laden integument that interfered with sensory perceptions and stimulation. Six years later, he chaired the committee set up by the Clinical Society of London to investigate the whole matter. He also later undertook some of the earliest metabolic studies of the effects of treating myxoedema with thyroid extract, showing the rapid weight loss and rise in temperature and in urinary volume and nitrogen excretion that occurred.
The key papers, which advanced these English authors observations, were those of the Swiss surgeons, Jaques-Louis Reverdin in Geneva and Emil Theodor Kocher in Bern, Kocher later receiving the Nobel Prize for his work on the thyroid. How fitting it is that it should be two Swiss doctors whose practices unlocked an understanding of the importance of the thyroid. For they each identified the late effects of total ablation (extirpation) of goiters. they

noted the great similarity of Gull’s and Ord’s myxoedema cases with their affected postoperative patients, referring to the comparison as a ‘rapprochement complet’, clearly making the connection. They acknowledged Gull’s primacy in describing the clinical manifestations and Ord’s ‘christening’ the condition ‘myxoedema’, and proposed that surgical cases be known as ‘myxoedème opératoire’. In light of his findings in 1882, Reverdin thereafter sought to conserve a part of the gland during thyroidectomy for goiter, speculating that its complete removal may have been responsible for these late effects. He had noticed that no such problems followed a just unilateral lobectomy. Kocher called the disease picture in his affected cases ‘cachexia strumipriva’ – literally, a bad condition due to the removal of a struma (goiter) without reference to the earlier work of Reverdin. Halsted noted in his monumental review of goiter surgery: ‘It is interesting to follow the argumentation of a mind so exceptionally keen and sane as Kocher’s in its futile efforts to explain insufficiently illuminated phenomena’. In reading Kocher’s 1909 Nobel Prize Lecture (in English translation), one gets the impression that Kocher was aware in 1883 of Gull’s and Ord’s reports, despite not referring to them, and he dismisses Reverdin’s contribution.
There ensued a competition over the contribution to the thyroid discovery. When post-thyroidectomy myxedema wsas brought to the attention of Kocher, he agreed it was analogous to his cases of cachexia strumipriva. It is also obvious that Kocher, like many surgeons of the time, cannot have engaged in routine postoperative outpatient follow-up, for otherwise the ensuing problems in his goiter-operated patients would have been detected years earlier. In respect of this key moment in the history of the thyroid, Reverdin could be said to hold the intellectual property. The thought has been expressed that perhaps he should have shared the 1909 Nobel Prize with Kocher.
The Emerging Roles of Thyroglobulin

Yuqian Luo, Yuko Ishido, Naoki Hiroi, Norihisa Ishii, and Koichi Suzuki
Adv in Endocr 2014, Article ID 189194, 7 pp http://dx.doi.org/10.1155/2014/189194

Thyroglobulin (Tg), the most important and abundant protein in thyroid follicles, is well known for its essential role in thyroid hormone synthesis. In addition to its conventional role as the precursor of thyroid hormones, we have uncovered a novel function of Tg as an endogenous regulator of follicular function over the past decade. The newly discovered negative feedback effect of Tg on follicular function observed in the rat and human thyroid provides an alternative explanation for the observation of follicle heterogeneity. Given the essential role of the regulatory effects of Tg, we consider that dysregulation of normal Tg function is associated with multiple human thyroid diseases including autoimmune thyroid disease and thyroid cancer. Additionally, extrathyroid Tg may serve a regulatory function in other organs. Further exploration of Tg action, especially at the molecular level, is needed to obtain a better understanding of both the physiological and pathological roles of Tg.

The Surgical Management of Thyroid Cancer

Sara A. Morrison, Hyunsuk Suh, and Richard A. Hodin
Rambam Maimonides Med J 2014; 5(2):e0008. http://dx.doi.org:/10.5041/RMMJ.10142

There are approximately 63,000 reported cases of thyroid carcinoma annually in the United States, representing roughly 4% of all documented malignancies.1 Diagnosis typically stems from work-up of a thyroid nodule. Data from the Framingham study suggests that palpable thyroid nodules are present in 4% of the US population,2 but non-palpable nodules may exist in up to 67% of the population. Such nodules are often found incidentally secondary to the rising use of imaging modalities in medical settings. The large majority of thyroid nodules are benign, with an overall reported risk of malignancy from 5% to 15%.
Thyroid cancer has been increasing in incidence, with the number of reported cases in the US rising by 25% over the last 3 years. With growing technological advances in the field and improved contributions of diagnostics, surgical decision-making and operative planning have taken on new challenges. Herein, we review the current clinical practice recommendations and active areas of surgical controversy, reflective of the most recently published professional consensus guidelines and a systematic review of the literature.
The use of FNA in current clinical practice has resulted in post-surgical pathology findings of malignancy in over 50% of specimens.7 The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) was developed in order to allow pathologists among varying institutions to communicate results to clinical care-takers with widely under-stood descriptors. Results of FNA biopsies are broken down into the following categories with the corresponding risks of malignancy: non-diagnostic or unsatisfactory (1%–4%), benign (0%–3%), atypia of undetermined significance or follicular lesion of undetermined significance (AUS/FLUS; 5%–15%), follicular neoplasm or suspicious for a follicular neoplasm (FN/sFN; 15%–30%), suspicious for malignancy (60%–75%), and malignant (97%–99%).
Mutational Panels.
AsuragenmiR Inform (Austin, TX, USA) mutation analysis assay and Thyroid Cancer Mutation Panel by Quest Diagnostics (Madison, NJ, USA) are the two main commercially available mutational tests which test for known genetic alterations such as BRAF, RAS, RET/PTC, and PAX8/PPAR. These mutational panels are highly specific for malignancy; however, due to the low overall frequency of these mutations in thyroid cancers, negative results do not rule out cancer. Therefore, mutational panel tests are considered a “rule-in” test. If a preoperative mutational test is positive, the nodule should be considered malignant, and total thyroidectomy should be recommended.
Gene Expression Profiling.
The most widely known gene expression profiling test is Afirma Gene Expression Classifier (Veracyte, San Francisco, CA, USA), and, with its recent clinical validation by Alexander et al., Afirma is already being utilized in many clinical settings. The Afirma Gene Expression Classifier (GEC) is an RNA-based assay that utilizes FNA samples to evaluate 167 molecular genes associated with benign nodules based on their proprietary algorithm. Unlike the mutational panel testing, Afirma testing is considered a “rule-out” test since the test has a high negative predictive value in distinguishing benign nodules. However, a positive result reported as “suspicious” carries only 38% risk of malignancy.
In all, these molecular tests should be utilized judiciously and should be considered as a complementary diagnostic tool in the management of thyroid nodules. In the future, molecular testing could become more cost-effective and accurate as a diagnostic tool while providing prognostic and therapeutic information.
Papillary Thyroid Cancer.
Total thyroidectomy is the gold standard for patients with a preoperative diagnosis of papillary thyroid cancer when the nodule is greater than 1 cm in size. Completion thyroidectomy is indicated in patients who have undergone prior lobectomy and are found on final pathology to have papillary thyroid cancer that is larger than 1 cm. The completion thyroidectomy should generally be performed within 6 months of the original procedure in order to minimize the risk of lymph node metastasis.
Involvement of cervical lymph nodes in papillary thyroid cancer is frequent, reported to occur in up to 50% of patients. The role of neck dissection at the time of total thyroidectomy is somewhat controversial, however, since most of the nodal involvement is microscopic and does not affect overall survival. It is generally agreed upon that a therapeutic neck dissection should be pursued in the setting of well-differentiated thyroid cancer patients with clinically positive lymph nodes, whether in the central or lateral neck compartments. Prophylactic neck dissection is not done for follicular thyroid cancer, as the rates of lymph node metastasis are typically less than 10%.
Medullary thyroid cancer (MTC) comprises 4% of all thyroid malignancies. The majority of cases are sporadic in nature; approximately 20%–25% represent familiar/hereditary syndromes. Diagnosis is commonly made by FNA biopsy with specific staining for the presence of calcitonin in the tissue specimen. All patients with a diagnosis of medullary thyroid cancer must be evaluated for multiple endocrine neoplasia (MEN) 2 and be ruled out for the synchronous presence of pheochromocytoma prior to scheduling thyroid surgery.
Effects of Dose Level of Anti-thyroid Drug Carbimazole on Thermoregulation and Blood Constituents in Male Rabbits (Oryctolagus cuniculus)

Intisar H. Saeed, Abdalla M. Abdelatif and Mohamed E. Elnageeb
Adv in Research 2014; 2(3): 129-144. Article no. AIR.2014.002

Carbimazole (CBZ) is an anti-thyroid drug commonly used in the treatment of hyperthyroidism. The objective of this study was to evaluate the effects of dose level of CBZ on thermoregulation and blood constituents in mature male rabbits. Twenty animals were assigned to 4 groups (A, B, C, D) of 5 each. Group A served as control and treated animals in groups B,C,D, received daily orally CBZ doses of 10, 15 and 20 mg/animal for 3 weeks, respectively.
The values of rectal temperature (Tr,), respiration rate (RR) and heart rate (HR) decreased in treated rabbits and the mean values of HR decreased with increase in the dose level of CBZ. The packed cell volume (PCV), Hb concentration and total leukocyte count (TLC) were lower in CBZ treated rabbits. Serum levels of total protein and globulins increased and serum albumin level decreased in treated groups of rabbits. Serum urea level was lower in CBZ treated groups and there was an increase in serum urea level with increase in CBZ dose level. Serum cholesterol level was higher in treated groups and there was an increase in serum cholesterol level with increase in CBZ dose level. Plasma glucose level decreased significantly in CBZ treated groups compared with the control and the mean values decreased with increase in the dose level of CBZ. The results indicate that the responses of basic physiological parameters were almost dose dependent in the range adopted in this study.
Phosphatase Inhibitor Calyculin A Activates TRPC2 Channels in Thyroid FRTL-5 Cells

Pramod Sukumaran, MY Asghar, C Löf, T Viitanen, and Kid Törnquist
Calcium Signaling Jun 2014; 1(2) http://www.researchpub.org/journal/cs/cs.html

We have previously shown that rat thyroid FRTL-5 cells express a calcium entry pathway regulated by a phosphatase. The nature of the calcium entry pathway is presently unknown. We have also shown that FRTL-5 cells express only the TRPC2 channel of the TRPC family of cation channels. In the present investigation we show, using pharmacological inhibitors, the measurement of sodium and calcium entry, stable TRPC2 knock-down cells, and transfection with a non-conducting form of TRPC2, that the calcium entry pathway regulated by a phosphatase is, in fact, the TRPC2 channel. Our data thus point to a novel mechanism by which the TRPC2 channels can be regulated.

Thyroxine Uptake by Perfused Rat Liver
No Evidence for Facilitation by Five Different Thyroxine-binding Proteins

Carl M. Mendel and Richard A. Weisiger
J. Clin. Invest. 1990; 86: 1840-1847

For each of the five protein-hormone complexes studied, the rate of hepatic uptake of T4 (measured under conditions expected to result in dissociation-limited uptake) closely approximated the rate of spontaneous dissociation of the protein-hormone complex within the hepatic sinusoids. These findings indicate an absence of special cellular mechanisms that facilitate the hepatic uptake of T4 from its plasma binding proteins, and support the view that uptake occurs from the free T4 pool after spontaneous dissociation of T4 from its binding proteins.
Thyroxine Transport and Distribution in Nagase Analbuminemic Rats

Carl M. Mendel, RR Cavalieri, LA Gavin, T Pettersson, and M Inoue
J. Clin. Invest. 1989; 83: 143-148

The postulate that thyroxine (T4) in plasma enters tissues by protein-mediated transport or enhanced dissociation from plasma-binding proteins leads to the conclusion that almost all T4 uptake by tissues in the rat occurs via the pool of albumin bound T4 (Pardridge, W. M., B. N. Premachandra, and G. Fierer. 1985. Am. J. Physiol. 248:G545-G550).
To directly test this postulate, and to test more generally whether albumin might play a special role in T4 transport in the rat, we performed in vivo kinetics studies in six Nagase analbuminemic rats and in six control rats, all of whom had similar serum T4 concentrations and percent free T4 values.
Evaluation of the plasma disappearance curves of simultaneously injected ¹²⁵I-T4 and ^I31I-albumin indicated that the flux of T4 from the extracellular compartment into the rapidly exchangeable intracellular compartment was similar in the analbuminemic rats (51±21 ng/min, mean±SD) and in the control rats (54±15 ng/min), as was the size of the rapidly exchangeable intracellular pool of T4 (1.13±0.53 vs. 1.22±036 Mg). This latter finding was confirmed by direct analysis of tissue samples (liver, kidney, and brain). We also performed in vitro kinetics studies using the isolated perfused rat liver. The single-pass fractional extraction by normal rat liver of T4 in pooled analbuminemic rat serum was indistinguishable from that of T4 in pooled control rat serum (10.9±3.3%, n = 3, vs. 11.4±3.4%). When > 98% of the albumin was removed from normal rat serum by chromatography with Affi-Gel blue, the single-pass fractional extraction of T4 (measured by a bolus injection method) did not change (16.3±2.1%, n = 5, vs. 15.2±2.5%). These data provide the first valid experimental test of the enhanced

dissociation hypothesis and indicate that there is no special, substantive role for albumin in T4 transport in the rat.
Influence of thyroid receptors on breast cancer cell proliferation

Conde, R. Paniagua, J. Zamora, M. J. Blanquez, B. Fraile, A. Ruiz & M. I. Arenas
Ann Oncol 2005; http://dx.doi.org:/10.1093/annonc/mdj040

Background: The involvement of thyroid hormones in the development and differentiation of normal breast tissue has been established. However, the association between breast cancer and these hormones is controversial. Therefore, the objective of the present study was to determine the protein expression pattern of thyroid hormone receptors in different human breast pathologies and to evaluate their possible relationship with cellular proliferation.
Patients and methods: The presence of thyroid hormone receptors was evaluated by immunohistochemistry and western blot analysis in 84 breast samples that included 12 cases of benign proliferative diseases, 20 carcinomas in situ and 52 infiltrative carcinomas.
Results: TR-α was detected in the nuclei of epithelial cells from normal breast ducts and acini, while in any pathological type this receptor was located in the cytoplasm. However, TR-b presented a nuclear location in benign proliferative diseases and carcinomas in situ and a cytoplasmatic location in normal breast and infiltrative carcinomas. The highest proliferation index was observed in carcinomas in situ, although in infiltrative carcinomas an inverse correlation between this index and the TR-α expression was encountered.
Conclusions: The results of this study reveal substantial changes in the expression profile of thyroid hormone.
Zebrafish as a model for monocarboxyl transporter 8-deficiency

GD Vatine, D Zada, T Lerer-Goldshtein, A Tovin, G Malkinson, K Yaniv and L Appelbaum
J Biol Chem Nov 2012; Manuscript M112.413831
http://dx.doi.org:/10.1074/jbc.M112.413831

Background: Mutations in the thyroid hormone transporter MCT8 are associated with psychomotor retardation AHDS.
Results: In zebrafish, as in humans, mct8 is expressed primarily in the nervous system. Elimination of MCT8 causes severe neural impairment.
Conclusion: MCT8 is a crucial regulator during zebrafish embryonic development. Significance: Establishment of the first vertebrate model for MCT8-deficiency, which exhibits a neurological phenotype.
Unusual Ratio between Free Thyroxine and Free Triiodothyronine in a Long-Lived Mole-Rat Species with Bimodal Ageing

Yoshiyuki Henning, Christiane Vole, Sabine Begall, Martin Bens, et al.
PlusOne Nov 2014; 9(11),e113698. http://dx.doi.org:/10.1371/journal.pone.0113698

Ansell’s mole-rats (Fukomys anselli) are subterranean, long-lived rodents, which live in eusocial families, where the maximum lifespan of breeders is twice as long as that of non-breeders. Their metabolic rate is significantly lower than expected based on allometry, and their retinae show a high density of S-cone opsins. Both features may indicate naturally low thyroid hormone levels.
In the present study, we sequenced several major components of the thyroid hormone pathways and analyzed free and total thyroxine and triiodothyronine in serum samples of breeding and non-breeding F. anselli to examine whether
a) their thyroid hormone system shows any peculiarities on the genetic level,
b) these animals have lower hormone levels compared to euthyroid rodents (rats and guinea pigs), and
c) reproductive status, lifespan and free hormone levels are correlated.
Genetic analyses confirmed that Ansell’s mole-rats have a conserved thyroid hormone system as known from other mammalian species. Interspecific comparisons revealed that free thyroxine levels of F. anselli were about ten times lower than of guinea pigs and rats, whereas the free triiodothyronine levels, the main biologically active form, did not differ significantly amongst species. The resulting fT4:fT3 ratio is unusual for a mammal and potentially represents a case of natural hypothyroxinemia.
Comparisons with total thyroxine levels suggest that mole-rats seem to possess two distinct mechanisms that work hand in hand to downregulate fT4 levels reliably. We could not find any correlation between free hormone levels and reproductive status, gender or weight. Free thyroxine may slightly increase with age, based on subsignificant evidence. Hence, thyroid hormones do not seem to explain the different ageing rates of breeders and nonbreeders. Further research is required to investigate the regulatory mechanisms responsible for the unusual proportion of free thyroxine and free triiodothyronine.
Transthyretin Regulates Thyroid Hormone Levels in the Choroid Plexus, But Not in the Brain Parenchyma: Study in a Transthyretin-Null Mouse Model

JA Palha, R Fernandes, GM De Escobar, V Episkopou, M Gottesman, and MJ Saraiva
Endocr 2000; 141(9): 3267–3272.

Transthyretin (TTR) is the major T4-binding protein in rodents. Using a TTR-null mouse model we asked the following questions.
1) Do other T4 binding moieties replace TTR in the cerebrospinal fluid (CSF)?
2) Are the low whole brain total T4 levels found in this mouse model associated with hypothyroidism, e.g. increased 59-deiodinase type 2 (D2) activity and RC3-neurogranin messenger RNA levels?
3) Which brain regions account for the decreased total whole brain T4 levels?
4) Are there changes in T3 levels in the brain?
Our results show the following.
1) No other T4-binding protein replaces TTR in the CSF of the TTR-null mice.
2) D2 activity is normal in the cortex, cerebellum, and hippocampus, and total brain RC3-neurogranin messenger RNA levels are not altered.
3) T4 levels measured in the cortex, cerebellum, and hippocampus are normal. However T4 and T3 levels in the choroid plexus are only 14% and 48% of the normal values, respectively.
4) T3 levels are normal in the brain parenchyma.
The data presented here suggest that TTR influences thyroid hormone levels in the choroid plexus, but not in the brain. Interference with the blood-choroid-plexus-CSF-TTR-mediated route of T4 entry into the brain caused by the absence of TTR does not produce measurable features of hypothyroidism. It thus appears that TTR is not required for T4 entry or for maintenance of the euthyroid state in the mouse brain.
Identification of monocarboxylate transporter 8 as a specific thyroid hormone transporter

E.C.H. Friesema, S Ganguly, A. Abdalla, J.E.M. Fox, AP. Halestrap, and TJ. Visser
J Biol Chem 2003; Manuscript M300909200
http://dx.doi.org/10.1074/jbc.M300909200

Transport of thyroid hormone across the cell membrane is required for its action and

metabolism. Recently, a T-type amino acid transporter was cloned which transports aromatic amino acids but not iodothyronines. This transporter belongs to the monocarboxylate transporter (MCT) family, and is most homologous with MCT8 (SLC16A2). Therefore, we cloned rat MCT8, and tested it for thyroid hormone transport in Xenopus laevis oocytes. Oocytes were injected with rat MCT8 cRNA, and after 3 days immunofluorescence microscopy demonstrated expression of the protein at the plasma membrane. MCT8 cRNA induced a ~10-fold increase in uptake of 10 nM ¹²⁵I-labeled thyroxine (T4), 3,3′,5-triiodothyronine (T3), 3,3′,5′-triiodothyronine (rT3) and 3,3′-diiodothyronine. Due to the rapid uptake of the ligands, transport was only linear with time for <4 min. MCT8 did not transport Leu, Phe, Trp or Tyr. [¹²⁵I]T4 transport was strongly inhibited by L-T4, D-T4, L-T3, D-T3, 3,3’,5-triiodothyroacetic acid, N-bromoacetyl-T3, and bromosulfophthalein. T3 transport was less affected by these inhibitors. Iodothyronine uptake in uninjected oocytes was reduced by albumin but the stimulation induced by MCT8 was markedly increased. Saturation analysis provided apparent Km values of 2-5 μM for T4, T3 and rT3. Immunohistochemistry showed high expression in liver, kidney, brain and heart. In conclusion, we have identified MCT8 as a very active and specific thyroid hormone transporter.
Thyroid hormones,T3 andT4, in the brain
Amy C. Schroeder and Martin L. Privalsky
Front Endocr Mar 2014; 5 article 40. http://dx.doi.org:/10.3389/fendo.2014.00040

Thyroid hormones (THs) are essential for fetal and post-natal nervous system development and also play an important role in the maintenance of adult brain function. Of the two major THs, T4 (3,5,30,50-tetraiodo-l-thyronine) is classically viewed as an pro-hormone that must be converted toT3 (3,5,30-tri-iodo-l-thyronine) via tissue-level deiodinases for biological activity. THs primarily mediate their effects by binding to thyroid hormone receptor (TR) isoforms, predominantly TRα1 and TRβ1, which are expressed in different tissues and exhibit distinctive roles in endocrinology. Notably, the ability to respond toT4 and toT3 differs for the two TR isoforms, with TRα1 generally more responsive to T4 than TRβ1. TRα1 is also the most abundantly expressed TR isoform in the brain, encompassing 70–80% of all TR expression in this tissue. Conversion of T4 into T3 via deiodinase 2 in astrocytes has been classically viewed as critical for generating local T3 for neurons. However, deiodinase-deficient mice do not exhibit obvious defectives in brain development or function. Considering that TRα1 is well-established as the predominant isoform in brain, and that TRα1 responds to both T3 and T4, we suggest T4 may play a more active role in brain physiology than has been previously accepted.
Thyroid hormone action: astrocyte–neuron communication

Beatriz Morte and Juan Bernal
Front Endocr May 2014; 5, Article 82 http://dx.doi.org:/10.3389/fendo.2014.00082

Thyroid hormone (TH) action is exerted mainly through regulation of gene expression by binding of T3 to the nuclear receptors.T4 plays an important role as a source of intracellular T3 in the central nervous system via the action of the type 2 deiodinase (D2), expressed in the astrocytes. A model of T3 availability to neural cells has been proposed and validated. The model contemplates that brain T3 has a double origin: a fraction is available directly from the circulation, and another is produced locally from T4 in the astrocytes by D2. The fetal brain depends almost entirely on theT3 generated locally. The contribution of systemic T3 increases subsequently during development to account for approximately 50% of total brain T3 in the late postnatal and adult stages. In this article, we review the experimental data in support of this model, and how the factors affectingT3 availability in the brain, such as deiodinases and transporters, play a decisive role in modulating local TH action during development.
The Significance of Thyroid Hormone Transporters in the Brain

Juan Bernal
Endocr Apr 2005; 146(4):1698–1700. http://dx.doi.org:/10.1210/en.2005-0134

The MCT family comprises up to 14 members, some of which are involved in the transport of important substrates for the brain such as lactate and pyruvate. MCT8 has been shown to act as a specific transporter for T4 and T3 and displays slightly higher affinity for T3. Heuer et al. have also studied the regional expression of MCT8 mRNA. In addition to high expression levels in the choroid plexus, they found that MCT8 is expressed in neurons of the neocortex, hippocampus, basal ganglia, amygdala, hypothalamus, and the Purkinje cells of the cerebellum, all regions known to be sensitive to thyroid hormones. Expression of MCT8 in neurons suggests that neuronal uptake of the T3 produced in astrocytes is facilitated by this transporter.
The physiological significance ofMCT8 as a transporter for thyroid hormone is supported by the finding of mutations in humans by Dumitrescu et al. and Friesema et al. The syndrome affects children from an early age and consists of severe developmental delay and neurological damage together with an unusually altered pattern of thyroid hormone levels in blood. The patients presented low total and free T4, high total and free T3, and low rT3. TSH was moderately elevated in two of the patients and normal or slightly elevated in the other five. Inactivating mutations of the MCT8 transporter could result in the altered thyroid hormone levels. In vitro uptake of T4 and T3 by fibroblasts isolated from affected males was strongly reduced, and intracellular D2 was increased 6- to 8-fold. It is thus hypothesized that the resulting increase in intracellularly generated T3 accumulates in blood because of its poor reuptake into cells.
The second trimester is also the period when thyroid hormone receptors increase in concentration in the brain. If MCT8 is needed at this stage of development for T3 entry into neurons, mutations of the transporter could interfere with T3-dependent developmental processes. Knowledge of the ontogenetic patterns of MCT8 in the human fetal brain would certainly be helpful. On the other hand, there is also the possibility that MCT8 mutations interfere with transport of other substrates for brain metabolism that could be even more important than T3 in determining the severity and outcome of the syndrome. Other members of the family transport metabolic substrates such as pyruvate and lactate, but MCT8 so far appears to be specific for iodothyronines

Peripheral markers of thyroid function: The effect of T4 monotherapy versus T4/T3 combination therapy in hypothyroid subjects: A randomized cross-over study

Ulla Schmidt, B Nygaard, EW Jensen, J Kvetny, A Jarløv, and Jens Faber
Endocrine Connections Jan 10, 2013 http://dx.doi.org:/10.1530/EC-12-0

Background: A recent randomized controlled trial suggests that hypothyroid subjects may find L-T4 and L-T3 combination therapy to be

superior to L-T4 monotherapy in terms of quality of life, suggesting that the brain registered increased T3 availability during the

combination therapy.

Hypothesis: Peripheral tissue might also be stimulated during T4/T3 combination therapy compared to T4 monotherapy.
Methods: Serum levels of Sex Hormone-Binding Globulin (SHBG), pro-collagen-1-N-terminal peptide (PINP), and N-terminal pro-brain natriuretic peptide (NT-proBNP) (representing hepatocyte, osteoblast, and cardiomyocyte stimulation, respectively) were measured in 26 hypothyroid subjects in a double blind, randomized, cross-over trial, which compared the replacement therapy with T4/T3 in combination (50 Fg T4 was substituted with 20 Fg T3) to T4 alone (once daily regimens). This was performed to obtain unaltered serum thyroid stimulating hormone (TSH) levels during the trial and between the two treatment groups. Blood sampling was performed 24 hours after the last intake of thyroid hormone medication.
Results: TSH remained unaltered between the groups ((median) 0.83 vs. 1.18 mU/l in T4/T3 combination and T4 mono-therapy, respectively; p=0.534). SHBG increased from (median) 75 nmol/l at baseline to 83 nmol/l in the T4/T3 group (p=0.015), but remained unaltered in the T4 group (67 nmol/l); thus, it was higher in the T4/T3 vs. T4 group (p=0.041). PINP levels were higher in the T4/T3 therapy (48 vs. 40 Fg/l (p<0.001)). NT-proBNP did not differ between the groups. Conclusions: T4/T3 combination therapy in hypothyroidism seems to have more metabolic effects than the T4 monotherapy.
Stimulatory effects of thyroid hormone on brain angiogenesis in vivo and in vitro

Liqun Zhang, CM Cooper-Kuhn, U Nannmark, K Blomgren and HG Kuhn
J Cereb Blood Flow & Metab 2010; 30:323–335. http://dx.doi.org:/10.1038/jcbfm.2009.216

Thyroid hormone is critical for the proper development of the central nervous system. However, the specific role of thyroid hormone on brain angiogenesis remains poorly understood. Treatment of rats from birth to postnatal day 21 (P21) with propylthiouracil (PTU), a reversible blocker of triiodothyronine (T3) synthesis, resulted in decreased brain angiogenesis, as indicated by reduced complexity and density of microvessels. However, when PTU was withdrawn at P22, these parameters were fully recovered by P90. These changes were paralleled by an altered expression of vascular endothelial growth factor A (Vegfa) and basic fibroblast growth factor (Fgf2). Physiologic concentrations of T3 and thyroxine (T4) stimulated proliferation and tubulogenesis of rat brain derived endothelial (RBE4) cells in vitro. Protein and mRNA levels of VEGF-A and FGF-2 increased after T3 stimulation of RBE4 cells. The thyroid hormone receptor blocker NH-3 abolished T3-induced Fgf2 and Vegfα upregulation, indicating a receptor-mediated effect. Thyroid hormone inhibited the apoptosis in RBE4 cells and altered mRNA levels of apoptosis-related genes, namely Bcl2 and Bad. The present results show that thyroid hormone has a substantial impact on vasculature development in the brain. Pathologically altered vascularization could, therefore, be a contributing factor to the neurologic deficits induced by thyroid hormone deficiency.

Molecules important for thyroid hormone

synthesis and action – known facts and future perspectives

Klaudia Brix, Dagmar Führer, Heike Biebermann
Thyroid Research 2011, 4(Suppl 1):S9 http://www.thyroidresearchjournal.com/content/4/S1/S9

Thyroid hormones are of crucial importance for the functioning of nearly every organ. Remarkably, disturbances of thyroid hormone synthesis and function are among the most common endocrine disorders affecting approximately one third of the working German population. Over the last ten years our understanding of biosynthesis and functioning of these hormones has increased tremendously. This includes the identification of proteins involved in thyroid hormone biosynthesis like Thox2 and Dehal where mutations in these genes are responsible for certain degrees of hypothyroidism. One of the most important findings was the identification of a specific transporter for triiodothyronine (T3), the monocarboxylate transporter 8 (MCT8) responsible for directed transport of T3 into target cells and for export of thyroid hormones out of thyroid epithelial cells. Genetic disturbances of MCT8 in patients result in a biochemical constellation of high T3 levels in combination with low or normal TSH and thyroxine levels leading to a new syndrome of severe X-linked mental retardation. Importantly mice lacking MCT8 presented only with a mild phenotype, indicating that compensatory mechanisms exist in mice. Moreover, it has become clear that not only genomic actions of T3 exist. T3 is also capable to activate adhesion receptors and it signals via activation of PI3K and MAPK pathways. Most recently, thyroid hormone derivatives were identified, the thyronamines which are decarboxylated thyroid hormones initiating physiological actions like lowering body temperature and heart rate, thereby acting in opposite direction to the classical thyroid hormones. So far it is believed that thyronamines function via the activation of a G-protein coupled receptor, TAAR1. The objective of this review is to summarize the recent findings in thyroid hormone synthesis and action and to discuss their implications for diagnosis of thyroid disease and for treatment of patients.

Retinoic Acid Induces Expression of the Thyroid Hormone Transporter, Monocarboxylate Transporter 8 (Mct8)

T Kogai, Yan-Yun Liu, LL Richter, K Mody, H Kagechika, and GA Brent
J Biol Chem Jun 2010. Manuscript M110.123158
http://www.jbc.org/cgi/doi/10.1074/jbc.M110.123158

Retinoic acid (RA) and thyroid hormone are critical for differentiation and organogenesis in the embryo. The monocarboxylate transporter-8 (Mct8), expressed predominantly in brain and placenta, mediates thyroid hormone uptake from the circulation and is required for normal neural development. RA induces differentiation of F9 mouse teratocarcinoma cells towards neurons as well as extraembryonal endoderm. We hypothesized that Mct8 is functionally expressed in F9 cells and induced by RA. All trans RA (tRA), and other RA receptor (RAR) agonists, dramatically (> 300-fold) induced Mct8. tRA treatment significantly increased uptake of triiodothyronine and thyroxine (4.1 fold and 4.3 fold, respectively), which was abolished by a selective Mct8 inhibitor, bromosulfophthalein. Sequence inspection of the Mct8 promoter region and
5′-rapid amplification of cDNA ends (5’-RACE) PCR analysis in F9 cells identified
11 transcription start sites and a proximal Sp1 site, but no TATA-box. tRA significantly enhanced Mct8 promoter activity through a consensus RA responsive element located 6.6 kilobases upstream of the coding region. Chromatin immunoprecipitation assay demonstrated binding of RAR and retinoid-X receptor (RXR) to the RA response element. The promotion of thyroid hormone uptake through the transcriptional up-regulation of Mct8 by RAR is likely to be important for extraembryonic endoderm development and neural differentiation. This finding demonstrates crosstalk between RA signaling and thyroid hormone signaling in early development at the level of the thyroid hormone transporter.
Abnormal thyroid hormone metabolism in mice lacking the monocarboxylate transporter 8

Marija Trajkovic, Theo J. Visser, Jens Mittag, Sigrun Horn, et al.
J. Clin. Invest. 2007; 117:627–635. http://dx.doi.org:/10.1172/JCI28253

In humans, inactivating mutations in the gene of the thyroid hormone transporter monocarboxylate transporter 8 (MCT8; SLC16A2) lead to severe forms of psychomotor retardation combined with imbalanced thyroid hormone serum levels. The MCT8-null mice described here, however, developed without overt deficits but also exhibited distorted 3,5,3′-triiodothyronine (T3) and thyroxine (T4) serum levels, resulting in increased hepatic activity of type 1 deiodinase (D1). In the mutants’ brains, entry of T4 was not affected, but uptake of T3 was diminished. Moreover, the T4 and T3 content in the brain of MCT8-null mice was decreased, the activity of D2 was increased, and D3 activity was decreased, indicating the hypothyroid state of this tissue. In the CNS, analysis of T3 target genes revealed that in the mutants, the neuronal T3 uptake was impaired in an area-specific manner, with strongly elevated thyrotropin-releasing hormone transcript levels in the hypothalamic paraventricular nucleus and slightly decreased RC3 mRNA expression in striatal neurons; however, cerebellar Purkinje cells appeared unaffected, since they did not exhibit dendritic outgrowth defects and responded normally to T3 treatment in vitro.
In conclusion, the circulating thyroid hormone levels of MCT8-null mice closely resemble those of humans with MCT8 mutations, yet in the mice, CNS development is only partially affected.
3-Monoiodothyronamine: the rationale for its action as an endogenous adrenergic-blocking neuromodulator

HS Gompf, JH Greenberg, G Aston-Jones, A Ianculescu, TS Scanlan, and MB Dratman
Brain Res. 2010 Sep 10; 1351: 130–140. http://dx.doi.org:/10.1016/j.brainres.2010.06.067

The investigations reported here were designed to gain insights into the role of
3-monoiodothyronamine (T1AM) in the brain, where the amine was originally identified and characterized.
Extensive deiodinase studies indicated that T1AM was derived from the T4 metabolite, reverse triiodothyronine (revT3), while functional studies provided well-confirmed evidence that T1AM has strong adrenergic blocking effects. Because a state of adrenergic overactivity prevails when triiodothyronine (T3) concentrations becomes excessive, the possibility that T3’s metabolic partner, revT3, might give rise to an antagonist of those T3 actions was thought to be reasonable.
All T1AM studies thus far have required use of pharmacological doses.
Therefore we considered that choosing a physiological site of action was a priority and focused on the locus coeruleus (LC), the major noradrenergic control center in the brain. Site-directed injections of T1AM into the LC elicited a significant, dose-dependent neuronal firing rate change in a subset of adrenergic neurons with an EC50=2.7 μM, a dose well within the physiological range. Further evidence for its physiological actions came from autoradiographic images obtained following intravenous carrier-free 125I-labeled T1AM injection. These showed that the amine bound with high affinity to the LC and to other selected brain nuclei, each of which is both an LC target and a known T3 binding site. This new evidence points to a physiological role for T1AM as an endogenous adrenergic-blocking neuromodulator in the central noradrenergic system.

Thyroid hormones are transported through the blood-brain barrier

Thyroid hormones are transported through the blood-brain barrier (OATP) or the blood-CSF barrier (OATP and MCT8). In the astrocytes and tanycytes T4 is converted to T3 which then enters the neurons through MCT8. In the neurons both T4 and T3 are degraded by D3. T3 from the tanycytes may reach the portal vessels in the median eminence. Other transporters may be present on the astrocyte or tanycyte membranes. In most cases the transport could be bidirectional, although only one direction is shown.
Juan Bernal – Instituto de Investigaciones Biomedicas – 28029 Madrid, Spain

the interactions of maternal, placental and fetal thyroid

Old and new concepts of thyroid hormone action.

A: Old concept of thyroid hormone action. In former times it was assumed that thyroid hormones are able to pass the plasma membrane by passive transport. Once in the cytosol T4 is deiodinated to T3 which exerts genomic effects by binding to the thyroid hormone receptor (TR). After hetero-dimerization with other nuclear receptors like retinoic X receptor (RXR), transcriptional regulation is initiated resulting in activation or inactivation of target genes.
B: New concepts of thyroid hormone action. Thyroid hormones enter a target cell via specific transporters, e.g. T3 uses the monocarboxylate transporter MCT8 while T4 entry is mediated by Lat2 or Oatp14. Moreover, T3 can interact with avb3 integrins to induce ERK1/2 signalling. Cytosolic T3 exerts genomic effects but can additionally also act by non-genomic means after TR binding and activation of down-stream PI-3 kinase. Likewise, the naturally occurring iodothyronine T2 is believed to stimulate metabolic rates via mitochondrial pathways, thereby bypassing genomic regulation. Besides thyroid hormones, derivatives like the thyronamines T1AM or T0AM, modulate the action of T3, e.g. counter-acting its effects in certain target cells. Thyronamines (TAMs) bind to and activate G-protein coupled receptors (GPCRs) of the trace amine associated receptor (TAAR) family. So far, it is only known that TAAR1 is activated by TAMs and signals via adenylylcyclase (AC) activation with subsequent rise of cAMP levels. However other GPCRs are likely targets for thyroid hormone derivatives

Brix et al.: Molecules important for thyroid hormone synthesis and action – known facts and future perspectives. Thyroid Research 2011 4(Suppl 1):S9.
http://dx.doi.org:/10.1186/1756-6614-4-S1-S9

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