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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.

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”.

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.

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

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.