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Metformin, thyroid-pituitary axis, diabetes mellitus, and metabolism


Metformin, thyroid-pituitary axis, diabetes mellitus, and metabolism

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

https://pharmaceuticalintelligence.com/9/27/2014/Metformin,_thyroid-pituitary_ axis,_diabetes_mellitus,_and_metabolism

The following article is a review of the central relationship between the action of
metformin as a diabetic medication and its relationship to AMPK, the important and
essential regulator of glucose and lipid metabolism under normal activity, stress, with
its effects on skeletal muscle, the liver, the action of T3 and more.

We start with a case study and a publication in the J Can Med Assoc.  Then we shall look
into key literature on these metabolic relationships.

Part I.  Metformin , Diabetes Mellitus, and Thyroid Function

Hypothyroidism, Insulin resistance and Metformin
May 30, 2012   By Janie Bowthorpe
The following was written by a UK hypothyroid patient’s mother –
Sarah Wilson.

My daughter’s epilepsy is triggered by unstable blood sugars. And since taking
Metformin to control her blood sugar, she has significantly reduced the number of
seizures. I have been doing research and read numerous academic medical journals,
which got me thinking about natural thyroid hormone and Hypothyroidism. My hunch
was that when patients develop hypothyroid symptoms, they are actually becoming
insulin resistant (IR). There are many symptoms in common between women with
polycystic ovaries and hypothyroidism–the hair loss, the weight gain, etc.
(http://insulinhub.hubpages.com/hub/PCOS-and-Hypothyroidism).

A hypothyroid person’s body behaves as if it’s going into starvation mode and so, to
preserve resources and prolong life, the metabolism changes. If hypothyroid is prolonged
or pronounced, then perhaps, chemical preservation mode becomes permanent even
with the reintroduction of thyroid hormones. To get back to normal, they need
a “jump-start” reinitiate a higher rate of metabolism. The kick start is initiated through
AMPK, which is known as the “master metabolic regulating enzyme.”
(http://en.wikipedia.org/wiki/AMP-activated protein kinase).

Guess what? This is exactly what happens to Diabetes patients when Metformin is
introduced. http://en.wikipedia.org/wiki/Metformin
Suggested articles: http://www.springerlink.com/content/r81606gl3r603167/  and
http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2265.2011.04029.x/pdf

Note the following comments/partial statements:
“Hypothyroidism is characterized by decreased insulin responsiveness”;
“the pivotal regulatory role of T3 in major metabolic pathways”.

The community knows that T3/NTH (natural thyroid hormone [Armour]) makes
hypothyroid patients feel better – but the medical establishment is averse to T3/NTH
(treating subclinical hypoT (T3/T4 euthyroid) with natural dessicated thyroid (NDT).
The medical establishment might find an alternative view about impaired metabolism
more if shown real proof that the old NDT **was/is** having the right result –i.e., the
T3 is jump-starting the metabolism by re-activating
 AMPK.

If NDT also can be used for hypothyroidism without the surmised “dangers” of NTH,
then they should consider it. [The reality in the choice is actually recombinant TH
(Synthroid)]. Metformin is cheap, stable and has very few serious side effects. I use the
car engine metaphor, and refer to glucose as our petrol, AMPK as the spark plug and
both T3 and Metformin as the ignition switches. Sometimes if you have flat batteries in
the car, it doesn’t matter how much you turn the ignition switch or pump the petrol
pedal, all it does is flatten the battery and flood the engine.

Dr. Skinner in the UK has been treating “pre-hypothyroidism” the way that some
doctors treat “pre-diabetes”. Those hypothyroid patients who get treated early
might not have had their AMPK pathways altered and the T4-T3 conversion still works.
There seems to be no reason why thyroid hormone replacement therapy shouldn’t
logically be given to ward off a greater problem down the line.

It’s my belief that there is clear and abundant academic evidence that the AMPK/
Metformin research should branch out to also look at thyroid disease.

Point – direct T3 is kicking the closed -down metabolic process back into life,
just like Metformin does for insulin resistance.
http://www.hotthyroidology.com/editorial_79.html
There is serotonin resistance! http://www.ncbi.nlm.nih.gov/pubmed/17250776

Metformin Linked to Risk of Low Levels of Thyroid Hormone

CMAJ (Canadian Medical Association Journal) 09/22/2014

Metformin, the drug commonly for treating type 2 diabetes,

  • is linked to an increased risk of low thyroid-stimulating hormone
    (TSH) levels
  • in patients with underactive thyroids (hypothyroidism),

according to a study in CMAJ (Canadian Medical Association Journal).

Metformin is used to lower blood glucose levels

  • by reducing glucose production in the liver.

previous studies have raised concerns that

  • metformin may lower thyroid-stimulating hormone levels.

Study characteristics:

  1. Retrospective  long-term
  2. 74 300 patient who received metformin and sulfonylurea
  3. 25-year study period.
  4. 5689 had treated hypothyroidism
  5. 59 937 had normal thyroid function.

Metformin and low levels of thyroid-stimulating hormone in
patients with type 2 diabetes mellitus

Jean-Pascal Fournier,  Hui Yin, Oriana Hoi Yun Yu, Laurent Azoulay  +
Centre for Clinical Epidemiology (Fournier, Yin, Yu, Azoulay), Lady Davis Institute,
Jewish General Hospital; Department of Epidemiology, Biostatistics and Occupational
Health (Fournier), McGill University; Division of Endocrinology (Yu), Jewish General
Hospital; Department of Oncology (Azoulay), McGill University, Montréal, Que., Cananda

CMAJ Sep 22, 2014,   http://dx.doi.org:/10.1503/cmaj.140688

Background:

  • metformin may lower thyroid-stimulating hormone (TSH) levels.

Objective:

  • determine whether the use of metformin monotherapy, when compared with
    sulfonylurea monotherapy,
  • is associated with an increased risk of low TSH levels(< 0.4 mIU/L)
  • in patients with type 2 diabetes mellitus.

Methods:

  • Used the Clinical Practice Research Datalink,
  • identified patients who began receiving metformin or sulfonylurea monotherapy
    between Jan. 1, 1988, and Dec. 31, 2012.
  • 2 subcohorts of patients with treated hypothyroidism or euthyroidism,

followed them until Mar. 31, 2013.

  • Used Cox proportional hazards models to evaluate the association of low TSH
    levels with metformin monotherapy, compared with sulfonylurea monotherapy,
    in each subcohort.

Results:

  • 5689 patients with treated hypothyroidism and 59 937 euthyroid patients were
    included in the subcohorts.

For patients with treated hypothyroidism:

  1. 495 events of low TSH levels were observed (incidence rate 0.1197/person-years).
  2. 322 events of low TSH levels were observed (incidence rate 0.0045/person-years)
    in the euthyroid group.
  • metformin monotherapy was associated with a 55% increased risk of low TSH
    levels 
    in patients with treated hypothyroidism (incidence rate 0.0795/person-years
    vs.0.1252/ person-years, adjusted hazard ratio [HR] 1.55, 95% confidence
    interval [CI] 1.09– 1.20), compared with sulfonylurea monotherapy,
  • the highest risk in the 90–180 days after initiation (adjusted HR 2.30, 95% CI
    1.00–5.29).
  • No association was observed in euthyroid patients (adjusted HR 0.97, 95% CI 0.69–1.36).

Interpretation: The clinical consequences of this needs further investigation.

 

Crude and adjusted hazard ratios for suppressed thyroid-stimulating hormone
levels (< 0.1 mIU/L) associated with the use metformin monotherapy, compared
with sulfonylurea monotherapy, in patients with treated hypothyroidism or
euthyroidism and type 2 diabetes
Variable No. events
suppressed
TSH levels
Person-years of
exposure
Incidence rate,
per 1000 person-years (95% CI)
Crude
HR
Adjusted HR*(95% CI)
Patients with treated hypothyroidism, = 5689
Sulfonylure,
= 762
18 503 35.8
(21.2–56.6)
1.00 1.00
(reference)
Metformin,
= 4927
130 3 633 35.8
(29.9–42.5)
1.05 0.99
(0.57–1.72)
Euthyroid patients, = 59 937
Sulfonylurea,
= 7980
12 8 576 1.4
(0.7–2.4)
1.00 1.00
(reference)
Metformin,
= 51 957
75 63 047 1.2
(0.9–1.5)
0.85 1.03
(0.52–2.03)

 

Part II. Metabolic Underpinning 
(Source: Wikipedia, AMPK and thyroid)

5′ AMP-activated protein kinase or AMPK or 5′ adenosine monophosphate-activated protein kinase
is an enzyme that plays a role in cellular energy homeostasis.
It consists of three proteins (subunits) that

  1. together make a functional enzyme, conserved from yeast to humans.
  2. It is expressed in a number of tissues, including the liver, brain, and skeletal
    muscle.
  3. The net effect of AMPK activation is stimulation of
    1. hepatic fatty acid oxidation and ketogenesis,
    2. inhibition of cholesterol synthesis,
    3. lipogenesis, and triglyceride synthesis,
    4. inhibition of adipocyte lipolysis and lipogenesis,
    5. stimulation of skeletal muscle fatty acid oxidation and muscle
      glucose uptake, and
    6. modulation of insulin secretion by pancreatic beta-cells.

The heterotrimeric protein AMPK is formed by α, β, and γ subunits. Each of these three
subunits takes on a specific role in both the stability and activity of AMPK.

  • the γ subunit includes four particular Cystathionine beta synthase (CBS) domains
    giving AMPK its ability to sensitively detect shifts in the AMP:ATP ratio.
  • The four CBS domains create two binding sites for AMP commonly referred to as
    Bateman domains. Binding of one AMP to a Bateman domain cooperatively
    increases the binding affinity of the second AMP to the other Bateman domain.
  • As AMP binds both Bateman domains the γ subunit undergoes a conformational
    change which exposes the catalytic domain found on the α subunit.
  • It is in this catalytic domain where AMPK becomes activated when
    phosphorylation takes place at threonine-172by an upstream AMPK kinase
    (AMPKK). The α, β, and γ subunits can also be found in different isoforms.

AMPK acts as a metabolic master switch regulating several intracellular systems

  1. the cellular uptake of glucose,
  2. the β-oxidation of fatty acids and
  3. the biogenesis of glucose transporter 4 (GLUT4) and
  4. mitochondria

The energy-sensing capability of AMPK can be attributed to

  • its ability to detect and react to fluctuations in the AMP:ATP ratio that take
    place during rest and exercise (muscle stimulation).

During muscle stimulation,

  • AMP increases while ATP decreases, which changes AMPK into a good substrate
    for activation.
  • AMPK activity increases while the muscle cell experiences metabolic stress
    brought about by an extreme cellular demand for ATP.
  • Upon activation, AMPK increases cellular energy levels by
    • inhibiting anabolic energy consuming pathways (fatty acid synthesis,
      protein synthesis, etc.) and
    • stimulating energy producing, catabolic pathways (fatty acid oxidation,
      glucose transport, etc.).

A recent JBC paper on mice at Johns Hopkins has shown that when the activity of brain
AMPK was pharmacologically inhibited,

  • the mice ate less and lost weight.

When AMPK activity was pharmacologically raised (AICAR see below)

  • the mice ate more and gained weight.

Research in Britain has shown that the appetite-stimulating hormone ghrelin also
affects AMPK levels.

The antidiabetic drug metformin (Glucophage) acts by stimulating AMPK, leading to

  1. reduced glucose production in the liver and
  2. reduced insulin resistance in the muscle.

(Metformin usually causes weight loss and reduced appetite, not weight gain and
increased appetite, ..opposite of expected from the Johns Hopkins mouse study results.)

Triggering the activation of AMPK can be carried out provided two conditions are met.

First, the γ subunit of AMPK

  • must undergo a conformational change so as to
  • expose the active site(Thr-172) on the α subunit.

The conformational change of the γ subunit of AMPK can be accomplished

  • under increased concentrations of AMP.

Increased concentrations of AMP will

  • give rise to the conformational change on the γ subunit of AMPK
  • as two AMP bind the two Bateman domains located on that subunit.
  • It is this conformational change brought about by increased concentrations
    of  AMP that exposes the active site (Thr-172) on the α subunit.

This critical role of AMP is further substantiated in experiments that demonstrate

  • AMPK activation via an AMP analogue 5-amino-4-imidazolecarboxamide
    ribotide (ZMP) which is derived fromthe familiar
  • 5-amino-4-imidazolecarboxamide riboside (AICAR)

AMPK is a good substrate for activation via an upstream kinase complex, AMPKK
AMPKK is a complex of three proteins,

  1. STE-related adaptor (STRAD),
  2. mouse protein 25 (MO25), and
  3. LKB1 (a serine/threonine kinase).

The second condition that must be met is

  • the phosphorylation/activation of AMPK on its activating loop at
    Thr-172of the α subunit
  • brought about by an upstream kinase (AMPKK).

The complex formed between LKB1 (STK 11), mouse protein 25 (MO25), and the
pseudokinase STE-related adaptor protein (STRAD) has been identified as

  • the major upstream kinase responsible for phosphorylation of AMPK
    on its activating loop at Thr-172

Although AMPK must be phosphorylated by the LKB1/MO25/STRAD complex,

  • it can also be regulated by allosteric modulators which
  • directly increase general AMPK activity and
  • modify AMPK to make it a better substrate for AMPKK
  • and a worse substrate for phosphatases.

It has recently been found that 3-phosphoglycerate (glycolysis intermediate)

  • acts to further pronounce AMPK activation via AMPKK

Muscle contraction is the main method carried out by the body that can provide
the conditions mentioned above needed for AMPK activation

  • As muscles contract, ATP is hydrolyzed, forming ADP.
  • ADP then helps to replenish cellular ATP by donating a phosphate group to
    another ADP,

    • forming an ATP and an AMP.
  • As more AMP is produced during muscle contraction,
    • the AMP:ATP ratio dramatically increases,
  • leading to the allosteric activation of AMPK

For over a decade it has been known that calmodulin-dependent protein kinase
kinase-beta (CaMKKbeta) can phosphorylate and thereby activate AMPK,

  • but it was not the main AMPKK in liver.

CaMKK inhibitors had no effect on 5-aminoimidazole-4-carboxamide-1-beta-4-
ribofuranoside (AICAR) phosphorylation and activation of AMPK.

  • AICAR is taken into the celland converted to ZMP,
  • an AMP analogthat has been shown to activate AMPK.

Recent LKB1 knockout studies have shown that without LKB1,

  • electrical and AICAR stimulation of muscleresults in very little
    phosphorylation of AMPK and of ACC, providing evidence that
  • LKB1-STRAD-MO25 is the major AMPKK in muscle.

Two particular adipokines, adiponectin and leptin, have even been demonstrated
to regulate AMPK. A main functions of leptin in skeletal muscle is

  • the upregulation of fatty acid oxidation.

Leptin works by way of the AMPK signaling pathway, and adiponectin also
stimulates the oxidation of fatty acids via the AMPK pathway, and

  • Adiponectin also stimulates the uptake of glucose in skeletal muscle.

An increase in enzymes which specialize in glucose uptake in cells such as GLUT4
and hexokinase II are thought to be mediated in part by AMPK when it is activated.
Increases in AMPK activity are brought about by increases in the AMP:ATP ratio
during single bouts of exercise and long-term training.

One of the key pathways in AMPK’s regulation of fatty acid oxidation is the

  • phosphorylation and inactivation of acetyl-CoA carboxylase.
  1. Acetyl-CoA carboxylase (ACC) converts acetyl-CoA (ACA) to malonyl-CoA
    (MCA), an inhibitor of carnitine palmitoyltransferase 1 (CPT-1).
  2. CPT-1 transports fatty acids into the mitochondria for oxidation.
  3. Inactivation of ACC results in increased fatty acid transport and oxidation.
  4. the AMPK induced ACC inactivation  and reduced conversion to MCA
    may occur as a result of malonyl-CoA decarboxylase (MCD)
  5. MCD as an antagonist to ACC, decarboxylatesmalonyl-CoA to acetyl-CoA
    (reversal of ACC conversion of ACA to MCA)
  6. This resultsin decreased malonyl-CoA and increased CPT-1 and fatty acid oxidation.

AMPK also plays an important role in lipid metabolism in the liver. It has long been
known that hepatic ACC has been regulated in the liver.

  1. It phosphorylates and inactivates 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR)
  2. acetyl-CoA(ACA) is converted to mevalonic acid (MVA) by ACC
    with inhibition of CPT-1
  3. HMGR converts 3-hydroxy-3-methylglutaryl-CoA, which is made from MVA
  4. which then travels down several more metabolic steps to become cholesterol.

Insulin facilitates the uptake of glucose into cells via increased expression and
translocation of glucose transporter GLUT-4. In addition, glucose is phosphorylated
by hexokinase wheni iot enters the cell. The phosphorylated form keeps glucose from
leaving the cell,

  • The decreasedthe concentration of glucose molecules creates a gradient for more
    glucose to be transported into the cell.
AMPK and thyroid hormone regulate some similar processes. Knowing these similarities,
Winder and Hardie et al. designed an experiment to see if AMPK was influenced by thyroid
hormone. They found that all of the subunits of AMPK were increased in skeletal muscle,
especially in the soleus and red quadriceps, with thyroid hormone treatment. There was
also an increase in phospho-ACC, a marker of AMPK activity.
  •  Winder WW, Hardie DG (July 1999). “AMP-activated protein kinase,
    a metabolic master switch: possible roles in type 2 diabetes”. J. Physiol. 277
    (1 Pt 1): E1–10. PMID 10409121.
  • Winder WW, Hardie DG (February 1996). “Inactivation of acetyl-CoA
    carboxylase and activation of AMP-activated protein kinase in muscle
    during exercise”. J. Physiol. 270 (2 Pt 1): E299–304. PMID 8779952.
  • Hutber CA, Hardie DG, Winder WW (February 1997). “Electrical stimulation
    inactivates muscle acetyl-CoA carboxylase and increases AMP-activated
    protein kinase”. Am. J. Physiol. 272 (2 Pt 1): E262–6. PMID 9124333
  • Durante PE, Mustard KJ, Park SH, Winder WW, Hardie DG (July 2002).
    “Effects of endurance training on activity and expression of AMP-activated
    protein kinase isoforms in rat muscles”. Am. J. Physiol. Endocrinol.
    Metab. 283 (1): E178–86. doi:10.1152/ajpendo.00404.2001. PMID 12067859
  • Corton JM, Gillespie JG, Hardie DG (April 1994). “Role of the AMP-activated
    protein kinase in the cellular stress response”. Curr. Biol. 4 (4):
    315–24. doi:10.1016/S0960-9822(00)00070-1. PMID 7922340
  • Winder WW (September 2001). “Energy-sensing and signaling by
    AMP-activated protein kinase in skeletal muscle”. J. Appl. Physiol. 91 (3):
    1017–28. PMID 11509493
  • Suter M, Riek U, Tuerk R, Schlattner U, Wallimann T, Neumann D (October
    2006). “Dissecting the role of 5′-AMP for allosteric stimulation, activation,
    and deactivation of AMP-activated protein kinase”.  J. Biol. Chem.
    281 (43): 32207–6. doi:10.1074/jbc.M606357200. PMID 16943194

 

Part III. Pituitary-thyroid axis and diabetes mellitus
The Interface Between Thyroid and Diabetes Mellitus

Leonidas H. Duntas, Jacques Orgiazzi, Georg Brabant   Clin Endocrinol. 2011;75(1):1-9.
Interaction of Metformin and Thyroid Function

Metformin acts primarily by

  • suppressing hepatic gluconeogenesis via activation of AMPK
  • It has the opposite effects on hypothalamic AMPK,
    • inhibiting activity of the enzyme.
  • the metformin effects on hypothalamic AMPK activity will
    • counteractT3 effects at the hypothalamic level.
  • AMPK therefore represents a direct target for dual regulation
    • in the hypothalamic partitioning of energy homeostasis.
  • metformin crossesthe blood–brain barrier and
    • levels in the pituitary gland are substantially increased.
  • It convincinglysuppresses TSH

A recent study recruiting 66 patients with benign thyroid nodules furthermore
demonstrated that metformin significantly decreases nodule size in patients with
insulin resistance.[76] The effect of metformin, which was produced over a
6-month treatment period, parallelled a fall in TSH concentrations and achieved a
shrinkage amounting to 30% of the initial nodule size when metformin was
administered alone and up to 55% when it was added to ongoing LT4 treatment.

These studies reveal a

  • suppressive effect of metformin on TSH secretion patterns in
    hypothyroid patients, an effect that is apparently
  • independent of T4 treatment and does not alter the TH profile.
  • A rebound of TSH secretion occurs at about 3 months following metformin
    withdrawal.

It appears that recommendations for more frequent testing, on an annual to
biannual basis, seems justified in higher risk groups like patients over 50 or 55,
particularly with suggestive symptoms, raised antibody titres or dylipidaemia.
We thus would support the suggestion of an initial TSH and TPO antibody testing
which, as discussed, will help to predict the development of hypothyroidism in
patients with diabetes.

Hypothalamic AMPK and fatty acid metabolism mediate thyroid
regulation of energy 
balance
M López,  L Varela,  MJ Vázquez,  S Rodríguez-Cuenca, CR González, …, & Vidal-Puig
Nature Medicine  29 Aug 2010; 16: 1001–1008 http://dx.doi.org:/10.1038/nm.2207

Thyroid hormones have widespread cellular effects; however it is unclear whether
their effects on the central nervous system (CNS) contribute to global energy balance.
Here we demonstrate that either

  • whole-body hyperthyroidism or central administration of triiodothyronine
    (T3) decreases

    • the activity of hypothalamic AMP-activated protein kinase (AMPK),
    • increases sympathetic nervous system (SNS) activity and
    • upregulates thermogenic markers in brown adipose tissue (BAT).

Inhibition of the lipogenic pathway in the ventromedial nucleus of the hypothalamus
(VMH) prevents CNS-mediated activation of BAT by thyroid hormone and reverses
the weight loss associated with hyperthyroidism. Similarly, inhibition of thyroid
hormone receptors in the VMH reverses the weight loss associated with hyperthyroidism.

This regulatory mechanism depends on AMPK inactivation, as genetic inhibition of this
enzyme in the VMH of euthyroid rats induces feeding-independent weight loss and
increases expression of thermogenic markers in BAT. These effects are reversed by
pharmacological blockade of the SNS. Thus, thyroid hormone–induced modulation
of AMPK activity and lipid metabolism in the hypothalamus is a major regulator of
whole-body energy homeostasis.

Metabolic Basis for Thyroid Hormone Liver Preconditioning:
Upregulation of AMP-Activated Protein Kinase Signaling
  
LA Videla,1 V Fernández, P Cornejo, and R Vargas
1Molecular and Clinical Pharmacology Program, Institute of Biomedical Sciences,
Faculty of Medicine, University of Chile, 2Faculty of Medicine, Diego Portales University,
Santiago, Chile
Academic Editors: H. M. Abu-Soud and D. Benke
The Scientific World Journal 2012; 2012, ID 475675, 10 pp
http://dx.doi.org/10.1100/2012/475675

The liver is a major organ responsible for most functions of cellular metabolism and

  • a mediator between dietary and endogenous sources of energy for extrahepatic tissues.
  • In this context, adenosine-monophosphate- (AMP-) activated protein kinase (AMPK)
    constitutes an intrahepatic energy sensor
  • regulating physiological energy dynamics by limiting anabolism and stimulating
    catabolism, thus increasing ATP availability.
  • This is achieved by mechanisms involving direct allosteric activation and
    reversible phosphorylation of AMPK, in response to signals such as

    • energy status,
    • serum insulin/glucagon ratio,
    • nutritional stresses,
    • pharmacological and natural compounds, and
    • oxidative stress status.

Reactive oxygen species (ROS) lead to cellular AMPK activation and

  • downstream signaling under several experimental conditions.

Thyroid hormone (L-3,3′,5-triiodothyronine, T3) administration, a condition
that enhances liver ROS generation,

  • triggers the redox upregulation of cytoprotective proteins
    • affording preconditioning against ischemia-reperfusion (IR) liver injury.

Data discussed in this work suggest that T3-induced liver activation of AMPK

  • may be of importance in the promotion of metabolic processes
  • favouring energy supply for the induction and operation of preconditioning
    mechanisms.

These include

  1. antioxidant,
  2. antiapoptotic, and
  3. anti-inflammatory mechanisms,
  4. repair or resynthesis of altered biomolecules,
  5. induction of the homeostatic acute-phase response, and
  6. stimulation of liver cell proliferation,

which are required to cope with the damaging processes set in by IR.

The liver functions as a mediator between dietary and endogenous sources
of energy and extrahepatic organs that continuously require energy, mainly
the brain and erythrocytes, under cycling conditions between fed and fasted states.

In the fed state, where insulin action predominates, digestion-derived glucose is
converted to pyruvate via glycolysis, which is oxidized to produce energy, whereas
fatty acid oxidation is suppressed. Excess glucose can be either stored as hepatic
glycogen or channelled into de novo lipogenesis.

In the fasted state, considerable liver fuel metabolism changes occur due to decreased
serum insulin/glucagon ratio, with higher glucose production as a consequence of
stimulated glycogenolysis and gluconeogenesis (from alanine, lactate, and glycerol).

Major enhancement in fatty acid oxidation also occurs to provide energy for liver
processes and ketogenesis to supply metabolic fuels for extrahepatic tissues. For these
reasons, the liver is considered as the metabolic processing organ of the body, and
alterations in liver functioning affect whole-body metabolism and energy homeostasis.

In this context, adenosine-monophosphate- (AMP-) activated protein kinase (AMPK)
is the downstream component of a protein kinase cascade acting as an

  • intracellular energy sensor regulating physiological energy dynamics by
  • limiting anabolic pathways, to prevent excessive adenosine triphosphate (ATP)
    utilization, and
  • by stimulating catabolic processes, to increase ATP production.

Thus, the understanding of the mechanisms by which liver AMPK coordinates hepatic
energy metabolism represents a crucial point of convergence of regulatory signals
monitoring systemic and cellular energy status

Liver AMPK: Structure and Regulation

AMPK, a serine/threonine kinase, is a heterotrimeric complex comprising

  1. a catalytic subunit α and
  2. two regulatory subunits β and γ .

The α subunit has a threonine residue (Thr172) within the activation loop of the kinase
domain, with the C-terminal region being required for association with β and γ subunits.
The β subunit associates with α and γ by means of its C-terminal region , whereas

  • the γ subunit has four cystathionine β-synthase (CBS) motifs, which
  • bind AMP or ATP in a competitive manner.

75675.fig.001 (not shown)

Figure 1: Regulation of AMP-activated protein kinase (AMPK) by
(A) direct allosteric activation and
(B) reversible phosphorylation and downstream responses maintaining
intracellular energy balance.

Regulation of liver AMPK activity involves both direct allosteric activation and
reversible phosphorylation. AMPK is allosterically activated by AMP through

  • binding to the regulatory subunit-γ, which induces a conformational change in
    the kinase domain of subunit α that protects AMPK from dephosphorylation
    of Thr172, probably by protein phosphatase-2C.

Activation of AMPK requires phosphorylation of Thr172 in its α subunit, which can be
attained by either

(i) tumor suppressor LKB1 kinase following enhancement in the AMP/ATP ratio, a
kinase that plays a crucial role in AMPK-dependent control of liver glucose and
lipid metabolism;

(ii) Ca2+-calmodulin-dependent protein kinase kinase-β (CaMKKβ) that
phosphorylates AMPK in an AMP-independent, Ca2+-dependent manner;

(iii) transforming growth-factor-β-activated kinase-1 (TAK1), an important
kinase in hepatic Toll-like receptor 4 signaling in response to lipopolysaccharide.

Among these kinases, the relevance of CaMKKβ and TAK1 in liver AMPK activation
remains to be established in metabolic stress conditions. Both allosteric and
phosphorylation mechanisms are able to elicit

  • over 1000-fold increase in AMPK activity, thus allowing
  • the liver to respond to small changes in energy status in a highly sensitive fashion.

In addition to rapid AMPK regulation through allosterism and reversible phosphorylation

  • long-term effects of AMPK activation induce changes in hepatic gene expression.

This was demonstrated for

(i) the transcription factor carbohydrate-response element-binding protein (ChREBP),

  • whose Ser568 phosphorylation by activated AMPK
  • blocks its DNA binding capacity and glucose-induced gene transcription
  • under hyperlipidemic conditions;(ii) liver sterol regulatory element-binding
    protein-1c (SREBP-1c), whose mRNA and protein expression and those of
    its target gene for fatty acid synthase (FAS)
  • are reduced by metformin-induced AMPK activation,
  • decreasing lipogenesis and increasing fatty acid oxidation due to
    malonyl-CoA depletion;

(iii) transcriptional coactivator transducer of regulated CREB activity-2 (TORC2),
a crucial component of the hepatic gluconeogenic program, was reported
to be phosphorylated by activated AMPK.

This modification leads to subsequent cytoplasmatic sequestration of TORC2 and
inhibition of gluconeogenic gene expression, a mechanism underlying

  • the plasma glucose-lowering effects of adiponectin and metformin
  • through AMPK activation by upstream LKB1.

Activation of AMPK in the liver is a key regulatory mechanism controlling glucose
and lipid metabolism,

  1. inhibiting anabolic processes, and
  2. enhancing catabolic pathways in response to different signals, including
    1. energy status,
    2. serum insulin/glucagon ratio,
    3. nutritional stresses,
    4. pharmacological and natural compounds, and
    5. oxidative stress status

Reactive Oxygen Species (ROS) and AMPK Activation

The high energy demands required to cope with all the metabolic functions
of the liver are met by

  • fatty acid oxidation under conditions of both normal blood glucose levels and
    hypoglycemia, whereas
  • glucose oxidation is favoured in hyperglycemic states, with consequent
    generation of ROS.

Under normal conditions, ROS occur at relatively low levels due to their fast processing
by antioxidant mechanisms, whereas at acute or prolonged high ROS levels, severe
oxidation of biomolecules and dysregulation of signal transduction and gene expression
is achieved, with consequent cell death through necrotic and/or apoptotic-signaling
pathways.

Thyroid Hormone (L-3,3′,5-Triiodothyronine, T3), Metabolic Regulation,
and ROS Production

T3 is important for the normal function of most mammalian tissues, with major actions
on O2 consumption and metabolic rate, thus

  • determining enhancement in fuel consumption for oxidation processes
  • and ATP repletion.

T3 acts predominantly through nuclear receptors (TR) α and β, forming

  • functional complexes with retinoic X receptor that
  • bind to thyroid hormone response elements (TRE) to activate gene expression.

T3 calorigenesis is primarily due to the

  • induction of enzymes related to mitochondrial electron transport and ATP
    synthesis, catabolism, and
  • some anabolic processes via upregulation of genomic mechanisms.

The net result of T3 action is the enhancement in the rate of O2 consumption of target
tissues such as liver, which may be effected by secondary processes induced by T3

(i) energy expenditure due to higher active cation transport,

(ii) energy loss due to futile cycles coupled to increase in catabolic and anabolic pathways, and

(iii) O2 equivalents used in hepatic ROS generation both in hepatocytes and Kupffer cells

In addition, T3-induced higher rates of mitochondrial oxidative phosphorylation are
likely to induce higher levels of ATP, which are partially balanced by intrinsic uncoupling
afforded by induction of uncoupling proteins by T3. In agreement with this view, the
cytosolic ATP/ADP ratio is decreased in hyperthyroid tissues, due to simultaneous
stimulation of ATP synthesis and consumption.

Regulation of fatty acid oxidation is mainly attained by carnitine palmitoyltransferase Iα (CPT-Iα),

  • catalyzing the transport of fatty acids from cytosol to mitochondria for β-oxidation,
    and acyl-CoA oxidase (ACO),
  • catalyzing the first rate-limiting reaction of peroxisomal β-oxidation, enzymes that are
    induced by both T3 and peroxisome proliferator-activated receptor α (PPAR-α).

Furthermore, PPAR-α-mediated upregulation of CPT-Iα mRNA is enhanced by PPAR-γ
coactivator 1α (PGC-1α), which in turn

  • augments T3 induction of CPT-Iα expression.

Interestingly, PGC-1α is induced by

  1. T3,
  2. AMPK activation, and
  3. ROS,

thus establishing potential links between

  • T3 action, ROS generation, and AMPK activation

with the onset of mitochondrial biogenesis and fatty acid β-oxidation.

Liver ROS generation leads to activation of the transcription factors

  1. nuclear factor-κB (NF-κB),
  2. activating protein 1 (AP-1), and
  3. signal transducer and activator of transcription 3 (STAT3)

at the Kupffer cell level, with upregulation of cytokine expression (TNF-α, IL-1, IL-6),
which upon interaction with specific receptors in hepatocytes trigger the expression of

  1. cytoprotective proteins (Figure 3(A)).

These responses and the promotion of hepatocyte and Kupffer-cell proliferation
represent hormetic effects reestablishing

  1. redox homeostasis,
  2. promoting cell survival, and
  3. protecting the liver against ischemia-reperfusion injury.

T3 liver preconditioning also involves the activation of the

  1. Nrf2-Keap1 defense pathway
  • upregulating antioxidant proteins,
  • phase-2 detoxifying enzymes, and
  • multidrug resistance proteins, members of the ATP binding cassette (ABC)
    superfamily of transporters (Figure 3(B))

In agreement with T3-induced liver preconditioning, T3 or L-thyroxin afford
preconditioning against IR injury in the heart, in association with

  • activation of protein kinase C and
  • attenuation of p38 and
  • c-Jun-N-terminal kinase activation ,

and in the kidney, in association with

  • heme oxygenase-1 upregulation.

475675.fig.002

http://www.hindawi.com/journals/tswj/2012/floats/475675/thumbnails/475675.fig.002_th.jpg

Figure 2: Calorigenic response of thyroid hormone (T3) and its relationship with O2
consumption, reactive oxygen species (ROS) generation, and antioxidant depletion in the liver.
Abbreviations: CYP2E1, cytochrome P450 isoform 2E1; GSH, reduced glutathione; QO2, rate
of O2 consumption; SOD, superoxide dismutase.

475675.fig.003

genomic signaling in T3 calorigenesis and ROS production 475675.fig.003

genomic signaling in T3 calorigenesis and ROS production 475675.fig.003

http://www.hindawi.com/journals/tswj/2012/floats/475675/thumbnails/475675.fig.003_th.jpg

Figure 3: Genomic signaling mechanisms in T3 calorigenesis and liver reactive oxygen
species (ROS) production leading to
(A) upregulation of cytokine expression in Kupffer cells and hepatocyte activation of genes
conferring cytoprotection,
(B) Nrf2 activation controling expression of antioxidant and detoxication proteins, and
(C) activation of the AMPK cascade regulating metabolic functions.

Abbreviations: AP-1, activating protein 1; ARE, antioxidant responsive element; CaMKKβ,
Ca2+-calmodulin-dependent kinase kinase-β; CBP, CREB binding protein; CRC, chromatin
remodelling complex; EH, epoxide hydrolase; HO-1, hemoxygenase-1; GC-Ligase,
glutamate cysteine ligase; GPx, glutathione peroxidase; G-S-T, glutathione-S-transferase;
HAT, histone acetyltransferase; HMT, histone arginine methyltransferase; IL1,
interleukin 1; iNOS, inducible nitric oxide synthase; LKB1, tumor suppressor LKB1 kinase;
MnSOD, manganese superoxide dismutase; MRPs, multidrug resistance proteins; NF-κB,
nuclear factor-κB; NQO1, NADPH-quinone oxidoreductase-1; NRF-1, nuclear respiratory
factor-1; Nrf2, nuclear receptor-E2-related factor 2; PCAF, p300/CBP-associated
factor; RXR, retinoic acid receptor; PGC-1, peroxisome proliferator-activated receptor-γ
coactivator-1; QO2, rate of O2 consumption; STAT3, signal transducer and activator
of transcription 3; TAK1, transforming-growth-factor-β-activated kinase-1; TNF-α, tumor
necrosis factor-α; TR, T 3 receptor; TRAP, T3-receptor-associated protein; TRE,  T3 responsive element; UCP, uncoupling proteins; (—), reported mechanisms;
(- - - -), proposed mechanisms.

 

T3 is a key metabolic regulator coordinating short-term and long-term energy needs,
with major actions on liver metabolism. These include promotion of

(i) gluconeogenesis and hepatic glucose production, and

(ii) fatty acid oxidation coupled to enhanced adipose tissue lipolysis, with

  • higher fatty acid flux to the liver and
  • consequent ROS production (Figure 2) and
  • redox upregulation of cytoprotective proteins

affording liver preconditioning (Figure 3).

Thyroid Hormone and AMPK Activation: Skeletal Muscle and Heart

In skeletal muscle, T3 increases the levels of numerous proteins involved in

  1. glucose uptake (GLUT4),
  2. glycolysis (enolase, pyruvate kinase, triose phosphate isomerase),
  3. fatty acid oxidation (carnitine palmitoyl transferase-1, mitochondrial thioesterase I),
    and uncoupling protein-3,

effects that are achieved through enhanced transcription of TRE-containing genes

Skeletal muscle AMPK activation is characterized by

(i) being a rapid and transient response,

(ii) upstream activation by Ca2+-induced mobilization and CaMKKβ activation,

(iii) upstream upregulation of LKB1 expression, which requires association with STRAD
and MO25 for optimal phosphorylation/activation of AMPK, and

(iv) stimulation of mitochondrial fatty acid β-oxidation.

T3-induced muscle AMPK activation was found to trigger two major downstream

signaling pathways, namely,

(i) peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) mRNA
expression and phosphorylation, a transcriptional regulator for genes related to

  • mitochondrial biogenesis,
  • fatty acid oxidation, and
  • gluconeogenesis and

(ii) cyclic AMP response element binding protein (CREB) phosphorylation, which

  • in turn induces PGC-1α expression in liver tissue, thus
  • reinforcing mechanism (i).

These data indicate that AMPK phosphorylation of PGC-1α initiates many of the
important gene regulatory functions of AMPK in skeletal muscle.

In heart, hyperthyroidism increased glycolysis and sarcolemmal GLUT4 levels by the
combined effects of AMPK activation and insulin stimulation, with concomitant increase
in fatty acid oxidation proportional to enhanced cardiac mass and contractile function.

Thyroid Hormone, AMPK Activation, and Liver Preconditioning

Recent studies by our group revealed that administration of a single dose of 0.1 mg T3/kg
to rats activates liver AMPK (Figure 4; unpublished work).

  1. enhancement in phosphorylated AMPK/nonphosphorylated AMPK ratios in T3-
    treated rats over control values thatis significant in the time period of 1 to 48
    hours after hormone treatment
  2. Administration of a substantially higher dose (0.4 mg T3/kg) resulted in
    decreased liver AMPK activation at 4 h to return to control values at 6 h
    after treatment

Activation of liver AMPK by T3 may be of relevance in terms of

  • promotion of fatty acid oxidation for ATP supply,
  • supporting hepatoprotection against IR injury (Figure 3(C)).

This proposal is based on the high energy demands underlying effective liver
preconditioning for full operation of hepatic

  • antioxidant, antiapoptotic, and anti-inflammatory mechanisms,
  • oxidized biomolecules repair or resynthesis,
  • induction of the homeostatic acute-phase response, and
  • promotion of hepatocyte and Kupffer cell proliferation,

mechanisms that are needed to cope with the damaging processes set in by IR.
T3 liver preconditioning , in addition to that afforded by

  • n-3 long-chain polyunsaturated fatty acids given alone or
  • combined with T3 at lower dosages, or
  • by iron supplementation,

constitutes protective strategies against hepatic IR injury.

Studies on the molecular mechanisms underlying T3-induced liver AMPK
activation (Figure 4) are currently under assessment in our laboratory.

References

Fernández and L. A. Videla, “Kupffer cell-dependent signaling in thyroid hormone
calorigenesis: possible applications for liver preconditioning,” Current Signal
Transduction Therapy 2009; 4(2): 144–151.

Viollet, B. Guigas, J. Leclerc et al., “AMP-activated protein kinase in the regulation
of  hepatic energy metabolism: from physiology to therapeutic perspectives,” Acta
Physiologica 2009; 196(1): 81–98.

Carling, “The AMP-activated protein kinase cascade – A unifying system
for energy control,” Trends in Biochemical Sciences, 2004;. 29(1): 18–24.

E. Kemp, D. Stapleton, D. J. Campbell et al., “AMP-activated protein kinase,
super 
metabolic regulator,” Biochemical Society Transactions 2003; 31(1):
162–168
.

G. Hardie, “AMP-activated protein kinase-an energy sensor that
regulates all ;aspects of cell function,” Genes and Development,
2011; 25(18): 1895–1908.

Woods, P. C. F. Cheung, F. C. Smith et al., “Characterization of AMP-activated
protein kinase βandγ subunits Assembly of the heterotrimeric complex in vitro,”
Journal of Biological Chemistry 1996;271(17): 10282–10290.

Xiao, R. Heath, P. Saiu et al., “Structural basis for AMP binding to mammalian AMP-
activated protein kinase,” Nature 2007; 449(7161): 496–500.

more…

Impact of Metformin and compound C on NIS expression and iodine uptake in vitro and in vivo: a role for CRE in AMPK modulation of thyroid function.
Abdulrahman RM1, Boon MRSips HCGuigas BRensen PCSmit JWHovens GC.
Author information 
Thyroid. 2014 Jan;24(1):78-87.  Epub 2013 Sep 25.  PMID: 23819433
http://dx.doi.org:/10.1089/thy.2013.0041.

Although adenosine monophosphate activated protein kinase (AMPK) plays a crucial role
in energy metabolism, a direct effect of AMPK modulation on thyroid function has only
recently been reported, and much of its function in the thyroid is currently unknown.

The aim of this study was

  1. to investigate the mechanism of AMPK modulation in iodide uptake.
  2. to investigate the potential of the AMPK inhibitor compound C as an enhancer of
    iodide uptake by thyrocytes.

Metformin reduced NIS promoter activity (0.6-fold of control), whereas compound C
stimulated its activity (3.4-fold) after 4 days. This largely coincides with

  • CRE activation (0.6- and 3.0-fold).

These experiments show that AMPK exerts its effects on iodide uptake, at least partly,
through the CRE element in the NIS promoter. Furthermore, we have used AMPK-alpha1
knockout mice to determine the long-term effects of AMPK inhibition without chemical compounds.
These mice have a less active thyroid, as shown by reduced colloid volume and reduced
responsiveness to thyrotropin.

NIS expression and iodine uptake in thyrocytes

  • can be modulated by metformin and compound C.

These compounds exert their effect by

  • modulation of AMPK, which, in turn, regulates
  • the activation of the CRE element in the NIS promoter.

Overall, this suggests that AMPK modulating compounds may be useful for the
enhancement of iodide uptake by thyrocytes, which could be useful for the
treatment of thyroid cancer patients with radioactive iodine.

AMPK: Master Metabolic Regulator

© 1996–2013 themedicalbiochemistrypage.org, LLC | info
@ themedicalbiochemistrypage.org

AMPK-activating drugs metformin or phenformin might provide protection against cancer 1741-7007-11-36-5

AMPK-activating drugs metformin or phenformin might provide protection against cancer 1741-7007-11-36-5

 

AMPK and AMPK-related kinase (ARK) family  1741-7007-11-36-4

AMPK and AMPK-related kinase (ARK) family 1741-7007-11-36-4

 

central role of AMPK in the regulation of metabolism

 

 

AMP-activated protein kinase (AMPK) was first discovered as an activity that

AMPK induces a cascade of events within cells in response to the ever changing energy
charge of the cell. The role of AMPK in regulating cellular energy charge places this
enzyme at a central control point in maintaining energy homeostasis.

More recent evidence has shown that AMPK activity can also be regulated by physiological stimuli, independent of the energy charge of the cell, including hormones and nutrients.

 

Once activated, AMPK-mediated phosphorylation events

These events are rapidly initiated and are referred to as

  • short-term regulatory processes.

The activation of AMPK also exerts

  • long-term effects at the level of both gene expression and protein synthesis.

Other important activities attributable to AMPK are

  1. regulation of insulin synthesis and
  2. secretion in pancreatic islet β-cells and
  3. modulation of hypothalamic functions involved in the regulation of satiety.

How these latter two functions impact obesity and diabetes will be discussed below.

Regulation of AMPK

In the presence of AMP the activity of AMPK is increased approximately 5-fold.
However, more importantly is the role of AMP in regulating the level of phosphorylation
of AMPK. An increased AMP to ATP ratio leads to a conformational change in the γ-subunit
leading to increased phosphorylation and decreased dephosphorylation of AMPK.

The phosphorylation of AMPK results in activation by at least 100-fold. AMPK is
phosphorylated by at least three different upstream AMPK kinases (AMPKKs).
Phosphorylation of AMPK occurs in the α subunit at threonine 172 (T172) which

  • lies in the activation loop.

One kinase activator of AMPK is

  • Ca2+-calmodulin-dependent kinase kinase β (CaMKKβ)
  • which phosphorylates and activates AMPK in response to increased calcium.

The distribution of CaMKKβ expression is primarily in the brain with detectable levels
also found in the testes, thymus, and T cells. As described for the Ca2+-mediated
regulation of glycogen metabolism,

  • increased release of intracellular stores of Ca2+ create a subsequent demand for
    ATP.

Activation of AMPK in response to Ca fluxes

  • provides a mechanism for cells to anticipate the increased demand for ATP.

Evidence has also demonstrated that the serine-threonine kinase, LKB1 (also called
serine-threonine kinase 11, STK11) which is encoded by the Peutz-Jeghers syndrome
tumor suppressor gene, is required for activation of AMPK in response to stress.

The active LKB1 kinase is actually a complex of three proteins:

  1. LKB1,
  2. Ste20-related adaptor (STRAD) and
  3. mouse protein 25 (MO25).

Thus, the enzyme complex is often referred to as LKB1-STRAD-MO25. Phosphorylation
of AMPK by LKB1 also occurs on T172. Unlike the limited distribution of CaMKKβ,

  • LKB1 is widely expressed, thus making it the primary AMPK-regulating kinase.

Loss of LKB1 activity in adult mouse liver leads to

  • near complete loss of AMPK activity and
  • is associated with hyperglycemia.

The hyperglycemia is, in part, due to an increase in the transcription of gluconeogenic
genes. Of particular significance is the increased expression of

  • the peroxisome proliferator-activated receptor-γ (PPAR-γ) coactivator 1α
    (PGC-1α), which drives gluconeogenesis.
  • Reduction in PGC-1α activity results in normalized blood glucose levels in
    LKB1-deficient mice.

The third AMPK phosphorylating kinase is transforming growth factor-β-activated
kinase 1 (TAK1). However, the normal physiological conditions under which TAK1
phosphorylates AMPK are currently unclear.

The effects of AMP are two-fold:

  1. a direct allosteric activation and making AMPK a poorer substrate for
    dephosphorylation.

Because AMP affects both
the rate of AMPK phoshorylation in the positive direction and
dephosphorylation in the negative direction,

the cascade is ultrasensitive. This means that

  1. a very small rise in AMP levels can induce a dramatic increase in the activity of
    AMPK.

The activity of adenylate kinase, catalyzing the reaction shown below, ensures that

  • AMPK is highly sensitive to small changes in the intracellular [ATP]/[ADP] ratio.

2 ADP ——> ATP + AMP

Negative allosteric regulation of AMPK also occurs and this effect is exerted by
phosphocreatine. As indicated above, the β subunits of AMPK have a glycogen-binding domain, GBD. In muscle, a high glycogen content

  • represses AMPK activity and
  • this is likely the result of interaction between the GBD and glycogen,
  • the GBD of AMPK allows association of the enzyme with the regulation of glycogen metabolism
  • by placing AMPK in close proximity to one of its substrates glycogen synthase.

AMPK has also been shown to be activated by receptors that are coupled to

  • phospholipase C-β (PLC-β) and by
  • hormones secreted by adipose tissue (termed adipokines) such as leptinand adiponectin (discussed below).

Targets of AMPK

The signaling cascades initiated by the activation of AMPK exert effects on

  • glucose and lipid metabolism,
  • gene expression and
  • protein synthesis.

These effects are most important for regulating metabolic events in the liver, skeletal
muscle, heart, adipose tissue, and pancreas.

Demonstration of the central role of AMPK in the regulation of metabolism in response
to events such as nutrient- or exercise-induced stress. Several of the known physiologic
targets for AMPK are included as well as several pathways whose flux is affected by
AMPK activation. Arrows indicate positive effects of AMPK, whereas, T-lines indicate
the resultant inhibitory effects of AMPK action.

The uptake, by skeletal muscle, accounts for >70% of the glucose removal from the
serum in humans. Therefore, it should be obvious that this event is extremely important
for overall glucose homeostasis, keeping in mind, of course, that glucose uptake by
cardiac muscle and adipocytes cannot be excluded from consideration. An important fact
related to skeletal muscle glucose uptake is that this process is markedly impaired in
individuals with type 2 diabetes.

The uptake of glucose increases dramatically in response to stress (such as ischemia) and
exercise and is stimulated by insulin-induced recruitment of glucose transporters
to the plasma membrane, primarily GLUT4. Insulin-independent recruitment of glucose
transporters also occurs in skeletal muscle in response to contraction (exercise).

The activation of AMPK plays an important, albeit not an exclusive, role in the induction of
GLUT4 recruitment to the plasma membrane. The ability of AMPK to stimulate
GLUT4 translocation to the plasma membrane in skeletal muscle is by a different mechanism
than that stimulated by insulin and insulin and AMPK effects are additive.

Under ischemic/hypoxic conditions in the heart the activation of AMPK leads to the
phosphorylation and activation of the kinase activity of phosphofructokinase-2, PFK-2
(6-phosphofructo-2-kinase). The product of the action of PFK-2 (fructose-2,6-bisphosphate,
F2,6BP) is one of the most potent regulators of the rate of flux through
glycolysis and gluconeogenesis.

In liver the PKA-mediated phosphorylation of PFK-2 results in conversion of the
enzyme from a kinase that generates F2,6BP to a phosphatase that removes the
2-phosphate thus reducing the levels of the potent allosteric activator of the glycolytic
enzyme 6-phosphfructo-1-kinase, PFK-1 and the potent allosteric inhibitor
of the gluconeogenic enzyme fructose-1,6-bisphosphatase (F1,-6BPase).

It is important to note that like many enzymes, there are multiple isoforms of PFK-2
(at least 4) and neither the liver or the skeletal muscle isoforms contain the AMPK
phosphorylation sites found in the cardiac and inducible (iPFK2) isoforms of PFK-2.

Inducible PFK-2 is expressed in the monocyte/macrophage lineage in response to pro-
inflammatory stimuli. The ability to activate the kinase activity by phosphorylation of
PFK-2 in cardiac tissue and macrophages in response to ischemic conditions allows these
cells to continue to have a source of ATP via anaerobic glycolysis. This phenomenon is
recognized as the Pasteur effect: an increased rate of glycolysis in response to hypoxia.

Of pathological significance is the fact that the inducible form of PFK-2 is commonly
expressed in many tumor cells and this may allow AMPK to play an important role in
protecting tumor cells from hypoxic stress. Indeed, techniques for depleting AMPK in
tumor cells have shown that these cells become sensitized to nutritional stress upon loss
of AMPK activity.

Whereas, stress and exercise are powerful inducers of AMPK activity in skeletal muscle,
additional regulators of its activity have been identified.

Insulin-sensitizing drugs of the thiazolidinedione family (activators of PPAR-γ, see
below) as well as the hypoglycemia drug metformin exert a portion of their effects
through regulation of the activity of AMPK.

As indicated above, the activity of the AMPK activating kinase, LKB1, is critical for
regulating gluconeogenic flux and consequent glucose homeostasis. The action of
metformin in reducing blood glucose levels

  • requires the activity of LKB1 in the liver for this function.

Also, several adipokines (hormones secreted by adipocytes) either stimulate or inhibit
AMPK activation:

  1. leptin and adiponectin have been shown to stimulate AMPK activation, whereas,
  2. resistininhibits AMPK activation.

Cardiac effects exerted by activation of AMPK also include

AMPK-mediated phosphorylation of eNOS leads to increased activity and consequent
NO production and provides a link between metabolic stresses and cardiac function.

In platelets, insulin action leads to an increase in eNOS activity that is

  • due to its phosphorylation by AMPK.

Activation of NO production in platelets leads to

  • a decrease in thrombin-induced aggregation, thereby,
  • limiting the pro-coagulant effects of platelet activation.

The response of platelets to insulin function clearly indicates why disruption in insulin
action is a major contributing factor in the development of the metabolic syndrome

Activation of AMPK leads to a reduction in the level of SREBP

  • a transcription factor &regulator of the expression of numerous
    lipogenic enzymes

Another transcription factor reduced in response to AMPK activation is

  • hepatocyte nuclear factor 4α, HNF4α
    • a member of the steroid/thyroid hormone superfamily.
    • HNF4α is known to regulate the expression of several liver and
      pancreatic β-cell genes such as GLUT2, L-PK and preproinsulin.
  • Of clinical significance is that mutations in HNF4α are responsible for
    • maturity-onset diabetes of the young, MODY-1.

Recent evidence indicates that the gene for the carbohydrate-response-element-
binding protein (ChREBP) is a target for AMPK-mediated transcriptional regulation
in the liver. ChREBP is rapidly being recognized as a master regulator of lipid
metabolism in liver, in particular in response to glucose uptake.

The target of the thiazolidinedione (TZD) class of drugs used to treat type 2 diabetes is
the peroxisome proliferator-activated receptor γPPARγ which

  • itself may be a target for the action of AMPK.

The transcription co-activator, p300, is phosphorylated by AMPK

  • which inhibits interaction of p300 with not only PPARγ but also
  • the retinoic acid receptor, retinoid X receptor, and
  • thyroid hormone receptor.

PPARγ is primarily expressed in adipose tissue and thus it was difficult to reconcile how
a drug that was apparently acting only in adipose tissue could lead to improved insulin
sensitivity of other tissues. The answer to this question came when it was discovered that the TZDs stimulated the expression and release of the adipocyte hormone (adipokine),
adiponectin. Adiponectin stimulates glucose uptake and fatty acid oxidation in skeletal
muscle. In addition, adiponectin stimulates fatty acid oxidation in liver while inhibiting
expression of gluconeogenic enzymes in this tissue.

These responses to adiponectin are exerted via activation of AMPK. Another
transcription factor target of AMPK is the forkhead protein, FKHR (now referred to as
FoxO1). FoxO1 is involved in the activation of glucose-6-phosphatase expression and,
therefore, loss of FoxO1 activity in response to AMPK activation will lead to reduced
hepatic output of glucose.

This concludes a very complicated perspective that ties together the thyroid hormone
activity, the hypophysis, diabetes mellitus, and AMPK tegulation of metabolism in the
liver, skeletal muscle, adipose tissue, and heart.  I also note at this time that there
nongenetic points to be made here:

  1. The tissue specificity of isoenzymes
  2. The modulatory role of AMP:ATP ratio in phosphorylation/dephosphorylation
    effects on metabolism tied to AMPK
  3. The tie in of stress or ROS with fast reactions to protect harm to tissues
  4. The relationship of cytokine activation and release to the above metabolic events
  5. The relationship of effective and commonly used diabetes medications to AMPK
    mediated processes
  6. The preceding presentation is notable for the importance of proteomic and
    metabolomic invetigations in elucidation common chronic and nongenetic diseases

 

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The multi-step transfer of phosphate bond and hydrogen exchange energy

Curator: Larry H. Bernstein, MD, FCAP, Leaders in Pharmaceutical Intelligence

In this subtext of the series we expand on a tie between respiration and glycolysis, and the functioning of the mitochondrion to discover the key role played by oxidative phosphorylation, “acetyl coenzyme A, and electron transport.  This was crucial to understanding cellular energetics, which explains the high energy of fatty acid catabolism from stored adipose tissue, and the criticality of the multi-step sequence of reactions in energy transfer.

This portion considerably provides a response to the TWO points made by Jose EDS Rosallis:

  1. Just at the beginning, when phosphorylation of proteins is presented, I assume you must mention that some proteins are activated by phosphorylation. This is fundamental in order to present self –organization reflex upon fast regulatory mechanisms. This poiny needs further clarification, but he makes important observations here.
  • Even from an historical point of view. The first observation arrived from a sample due to be studied on the following day of glycogen synthetase. It was unintended left overnight out of the refrigerator. The result was it had changed from active form of the previous day to a non-active form.

The story could have being finished here, if the researcher did not decide to spent this day increasing substrate levels (it could be a simple case of denaturation of proteins that changes its conformation despite the same order of amino acids). He kept on trying and found restoration of maximal activity.

  • This assay was repeated with glycogen phosphorylase and the result was the opposite it increases its activity.

This led to the discovery of cAMP activated protein kinase and the assembly of a very complex system in the glycogen granule that is not a simple carbohydrate polymer. Instead

  • it has several proteins assembled and preserves the capacity to receive from a single event (rise in cAMP) two opposing signals with maximal efficiency,
  • stops glycogen synthesis, as long as levels of glucose 6 phosphate are low and
  • increases glycogen phosphorylation as long as AMP levels are high).

I did everything I was able to do by the end of 1970 in order to repeat these assays with

  • PK I, PKII and PKIII of M. Rouxii and Sutherland route to cAMP failed in this case.

I ask Leloir to suggest to my chief (SP) the idea of AA, AB, BB subunits as was observed in lactic dehydrogenase (tetramer)
(Nathan O. Kaplan discovery) indicating this as his idea. The reason was my “chief” (SP) more than once,  said to me: “Leave these great ideas for the Houssay, Leloir etc…We must do our career with small things. ” However, as she also had a faulty ability for recollection she also used to arrive some time later, with the very same idea but in that case, as her idea.

[This reminds me of when I was studying the emergence of lactic dehysrogenase isoenzyme patterns in the developing eye lens of cattle, I raised reservations about Elliott Vessells challenge to Nathan Kaplan, but that not being my primary problem, my brilliant mentor (H.M.), a very young full professor of anatomy said – leave that to NOK.}

Leloir, said to me: I will not offer your interpretation to her as mine. I think it is not phosphorylation, however I think it is

  • glycosylation that explains the changes in the isoenzymes with the same molecular weight preserved.

This dialogue explains why during the Schroedinger’s “What is life?” reading with him he asked me if from biochemist in exile, to biochemist I expressed all of my thoughts to him. Since I had considered that Schrödinger did not confront Darlington & Haldane for being in exile. This may explain why Leloir could have answered a bad telephone call from P. Boyer, Editor of The Enzymes in a way that suggests the the pattern could be of covalent changes over a protein. Our FEBS and Eur J. Biochemistry papers on pyruvate kinase of M. Rouxii is wrongly quoted in this way on his review about pyruvate kinase of
that year(1971).

  1. show in detail with different colors what carbons belongs to CoA a huge molecule, in comparison with the single two carbons of acetate that will produce the enormous jump in energy yield in comparison with anaerobic glycolysis. The idea is how much must have being spent in DNA sequences to build that molecule in order to use only two atoms of carbon. Very limited aspects of biology could be explained in this way. In case we follow an alternative way of thinking, it becomes clearer that proteins were made more stable by interaction with other molecules (great and small). Afterwards, it rather easy to understand how the stability of protein-RNA complexes where transmitted to RNA (vibrational +solvational reactivity stability pair of conformational energy). Latter, millions of years, or as soon as, the information of interaction leading to activity and regulation could be found in RNA, proteins like reverse transcriptase move this information to a more stable form (DNA). In this way it is easier to understand the use of CoA to make two carbon molecules more reactive.

The outline of what I am presenting in series is as follows:

  1. Signaling and Signaling Pathways
    https://pharmaceuticalintelligence.com/2014/08/12/signaling-and-signaling-pathways/
  1. Signaling transduction tutorial.
    https://pharmaceuticalintelligence.com/2014/08/12/signaling-transduction-tutorial/
  1. Carbohydrate metabolism
    https://pharmaceuticalintelligence.com/2014/08/13/carbohydrate-metabolism/

3.1  Selected References to Signaling and Metabolic Pathways in Leaders in Pharmaceutical Intelligence

https://pharmaceuticalintelligence.com/2014/08/14/selected-references-to-signaling-and-metabolic-pathways-in-leaders-in-pharmaceutical-intelligence/

  1. Lipid metabolism

4.1  Studies of respiration lead to Acetyl CoA

https://pharmaceuticalintelligence.com/2014/08/18/studies-of-respiration-lead-to-acetyl-coa/

4.2 The multi-step transfer of phosphate bond and hydrogen exchange energy

  1. Protein synthesis and degradation
  2. Subcellular structure
  3. Impairments in pathological states: endocrine disorders; stress hypermetabolism; cancer.

Oxidation-Reduction Reactions

Rachel Casiday, Carolyn Herman, and Regina Frey
Department of Chemistry, Washington University
St. Louis, MO 63130

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/cytochromes.html

 

OX-Phos steps

OX-Phos steps

http://s1.hubimg.com/u/6583902_f496.jpg

 

Key Concepts:

  • ATP as Free-Energy Currency in the Body
  • Coupled Reactions
    • Standard Free-Energy Change for Coupled Reactions
    • ATP Dephosphorylation Coupled to Nonspontaneous Reactions
    • Coupled Reactions to Generate ATP
  • Structure and Function of the Mitochondria
  • Oxidation-Reduction Reactions in the Electron-Transport Chain
    • Electron-Carrier Proteins (NOTE: This section includes a separate link and an animation.)
    • Relationship Between Reduction Potentials and Free Energy
  • Proton Gradient as Means of Coupling Oxidative and Phosphorylation Components of Oxidative Phosphorylation
  • ATP Synthetase Uses Energy From Proton Gradient to Generate ATP

Every day, we build bones, move muscles, eat food, think, and perform many other activities with our bodies. All of these activities are based upon chemical reactions. However, most of these reactions are not spontaneous (i.e., they are accompanied by a positive change in free energy, DG>0) and do not occur without some other source of free energy. Hence, the body needs some sort of “free-energy currency,” (Figure 1) a molecule that can store and release free energy when it is needed to power a given biochemical reaction.

The four questions:

  1. How does the body “spend” free-energy currency to make a nonspontaneous reaction spontaneous? The answer, which is based on thermodynamics, is to use coupled reactions.
  2. How is food used to produce the reducing agents (NADH and FADH2) that can regenerate the free-energy currency? The answer, from biology, is found in glycolysis and the citric-acid cycle.
  3. How are the reducing agents (NADH and FADH2) able to generate the free-energy currency molecule (ATP)? Once again, coupled reactions are key.
  4. What mechanism does the body use to couple the reducing agent reactions and the generation of ATP? ATP is synthesized primarily by a two-step process consisting of an electron-transport chain and a proton gradient.  This process is based on electrochemistry and equilibrium, as well as thermodynamics.

The free-energy change (DG) for the net reaction is given by the sum of the free-energy changes for the individual reactions.  The phospholipids that form cell membranes are formed from glycerol with a phosphate group and two fatty-acid chains attached.This step actually consists of two reactions:

(1) the phosphorylation of glycerol, and

(2) the dephosphorylation of ATP (the free-energy-currency molecule). The reactions may be added as shown in Equations 2-4, below:

      Glycerol + HPO42- –>  (Glycerol-3-Phosphate)2- + H2O DGo= +9.2 kJ
(nonspontaneous)
(2)
+      ATP4- + H2O –>       ADP3- + HPO42- + H+ DGo30.5 kJ
(spontaneous)
(3)
     Glycerol + ATP4- –> (Glycerol-3-Phosphate)2- +ADP3- + H+ DGo21.3 kJ
(spontaneous)
(4)
   

ATP is the most important “free-energy-currency” molecule in living organisms (see Figure 2, below). Adenosine triphosphate (ATP) is a useful free-energy currency because the dephosphorylation reaction is very spontaneous; i.e., it releases a large amount of free energy (30.5 kJ/mol). Thus, the dephosphorylation reaction of ATP to ADP and inorganic phosphate (Equation 3) is often coupled with nonspontaneous reactions (e.g., Equation 2) to drive them forward. The body’s use of ATP as a free-energy currency is a very effective strategy to cause vital nonspontaneous reactions to occur.

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/ATP.jpg

structure of ATP

structure of ATP

This is the two-dimensional (ChemDraw) structure of ATP, adenosine triphosphate. The removal of one phosphate group (green) from ATP requires the breaking of a bond (blue) and results in a large release of free energy. Removal of this phosphate group (green) results in ADP, adenosine diphosphate.

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/ATP.jpg

flowchart of food energy

flowchart of food energy

This flowchart shows that the energy used by the body for its many activities ultimately comes from the chemical energy in our food. The chemical energy in our food is converted to reducing agents (NADH and FADH2). These reducing agents are then used to make ATP. ATP stores chemical energy, so that it is available to the body in a readily accessible form.

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/flowchart1.jpg

Glycolysis   Glucose + 2 HPO42- + 2 ADP3- + 2 NAD+ –>
2 Pyruvate + 2 ATP4- + 2 NADH + 2 H+ + 2 H2O
(5)
Intermediate Step   2(Pyruvate + Coenzyme A + NAD+ –>
Acetyl CoA + CO2 + NADH)
(6)
Citric-Acid Cycle 2(Acetyl CoA + 3 NAD++ FAD + GDP3-
+ HPO42- + 2H2O –> 2 CO2 + 3 NADH + FADH2
+ GTP4- + 2H+ + Coenzyme A)
(7)

The structures of the important molecules in Equations 5-7 are shown in Table 1, below.

How is Food Used to Make the Reducing Agents Needed for the Production of ATP?

To make ATP, energy must be absorbed. This energy is supplied by the food we eat, and then used to synthsize two reducing agents, NADH and FADH2 that are needed to produce ATP. One of the principal energy-yielding nutrients in our diet is glucose (see structure in Table 1 in the blue box below), a simple six-carbon sugar that can be broken down by the body. When the chemical bonds in glucose are broken, free energy is released. The complete breakdown of glucose into CO2 occurs in two processes: glycolysis and the citric-acid cycle. The reactions for these two processes are shown in the blue box below.

pyruvate

pyruvate

  Pyruvate

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/pyruvate.jpg

acetylCoA

acetylCoA

Acetyl CoA

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/acetylCoA.jpg

NADH

NADH

NADH

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/acetylCoA.jpg

 

FADH2

FADH2

FADH2

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/FADH2.jpg

two-dimensional representations of several important molecules in Equations 5-7.

As seen in Equations 5-7 in the blue box, glycolysis and the citric-acid cycle produce a net total of only four ATP or GTP molecules (GTP is an energy-currency molecule similar to ATP) per glucose molecule. This yield isfar below the amount needed by the body for normal functioning, and in fact is far below the actual ATP yield for glucose in aerobic organisms (organisms that use molecular oxygen). For each glucose molecule the body processes, the body actually gains approximately 30 ATP molecules! (See Figure 4, below.)  So, how does the body generate ATP?

The process that accounts for the high ATP yield is known as oxidative phosphorylation. A quick examination of Equations 5-7 shows that glycolysis and the citric-acid cycle generate other products besides ATP and GTP, namely NADH and FADH2 (blue). These products are molecules that are oxidized (i.e., give up electrons) spontaneously. The body uses these reducing agents (NADH and FADH2) in an oxidation-reduction reaction .  As you will see later in this tutorial, it is the free energy from these redox reactions that is used to drive the production of ATP.

flowchart - making of ATP

flowchart – making of ATP

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/flowchart2.jpg

This flowchart shows the major steps involved in breaking down glucose from the diet and converting its chemical energy to the chemical energy in the phosphate bonds of ATP, in aerobic (oxygen-using) organisms. Note: In this flowchart, red denotes a source of carbon atoms (originally from glucose),green denotes energy-currency molecules, and blue denotes the reducing agents that can be oxidized spontaneously.

In the discussion above, we see that glucose by itself generates only a tiny amount of ATP. However, during the breakdown of glucose, a large amount of NADH and FADHis produced; it is these reducing agents that dramatically increase the amount of ATP produced. How does this work?

How are the reducing agents (NADH and FADH2) able to generate the free-energy currency molecule (ATP)?

As discussed in an earlier section about coupling reactions, ATP is used as free-energy currency by coupling its (spontaneous) dephosphorylation (Equation 3) with a (nonspontaneous) biochemical reaction to give a net release of free energy (i.e., a net spontaneous reaction). Coupled reactions are also used to generate ATP by phosphorylating ADP. The nonspontaneous reaction of joining ADP to inorganic phosphate to make ATP (Equation 8, below, and Figure 2, above) is coupled to the oxidation reaction of NADH or FADH(Equation 9, below). (Recall, NADH and FADH2 are produced in glycolysis and the citric-acid cycle as described in the blue box). For simplicity, we shall henceforth discuss only the oxidation of NADH; FADH2 follows a very similar oxidation pathway.

The oxidation reaction for NADH has a larger, but negative, DG than the positive DG required for the formation of ATP from ADP and phosphate. This set of coupled reactions is so important that it has been given a special name: oxidative phosphorylation. This name emphasizes the fact that an oxidation (of NADH) reaction (Equation 9 and Figure 5, below) is being coupled to a phosphorylation (of ADP) reaction (Equation 8, below, and Figure 2, above). In addition, we must consider the reduction reaction (gaining of electrons) that accompanies the oxidation of NADH. (Oxidation reactions are always accompanied by reduction reactions, because an electron given up by one group must be accepted by another group.) In this case, molecular oxygen (O2) is the electron acceptor, and the oxygen is reduced to water (Equation 10, below) .

The individual reactions of interest for oxidative phosphorylation are:

Phosphorylation

ADP3- + HPO42- + H+ –>
ATP4- + H2O

DGo= +30.5 kJ
(nonspontaneous)
(8)
oxidation

NADH –> NAD+ + H+ +  2e

DGo158.2 Kj
(spontaneous)
(9)
reduction

1/2 O2 + 2H+ + 2e –> H2O

DGo61.9 kJ
(spontaneous)

                                                                       (10)                                    

The net reaction is obtained by summing the coupled reactions, as shown in Equation 11, below.

ADP3- + HPO42- + NADH + 1/2 O2 + 2H+ –>
ATP4- + NAD+ + 2 H2O
DGo= -189.6 kJ
(spontaneous)
(11)

The molecular changes that occur upon oxidation of NADH are shown:

NAD+_NADH

NAD+_NADH

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/NAD+_NADH.jpg

This is a two-dimensional (ChemDraw) representation showing the change that occurs when NADH is oxidized to NAD+. “R” represents the part of the structure that is shown in black in the drawing of NADH in Table 1, and does not change during the oxidation half-reaction. The molecular changes that occur upon oxidation are shown in red.

In this tutorial, we have seen that nonspontaneous reactions in the body occur by coupling them with a very spontaneous reaction (usually the ATP reaction shown in Equation 3). We have just seen that ATP is produced by coupling the phosphorylation reaction with NADH oxidation (a very spontaneous reaction). But we have not yet answered the question: by what mechanism are these reactions coupled?

Coupling Reactions in Biological Systems

Every day your body carries out many nonspontaneous reactions. As discussed earlier, if a nonspontaneous reaction is coupled to a spontaneous reaction, as long as the sum of the free energies for the two reactions is negative, the coupled reactions will occur spontaneously. How is this coupling achieved in the body? Living systems couple reactions in several ways, but the most common method of coupling reactions is to carry out both reactions on the same enzyme. Consider again the phosphorylation of glycerol (Equations 2-4). Glycerol is phosphorylated by the enzyme glycerol kinase, which is found in your liver. The product of glycerol phosporylation, glycerol-3-phosphate (Equation 2), is used in the synthesis of phospholipids.

Glycerol kinase is a large protein comprised of about 500 amino acids. X-ray crystallography of the protein shows us that there is a deep groove or cleft in the protein where glycerol and ATP attach (see Figure 6, below). Because the enzyme holds the ATP and the glycerol in place, the phosphate can be transferred directly from the ATP to glycerol. Instead of two separate reactions where ATP loses a phosphate (Equation 3) and glycerol picks up a phosphate (Equation 2), the enzyme allows the phosphate to move directly from ATP to glycerol (Equation 4).

The coupling in oxidative phosphorylation uses a more complicated (and amazing!) mechanism, but the end result is the same: the reactions are linked together, the net free energy for the linked reactions is negative, and, therefore, the linked reactions are spontaneous.

glyckin

glyckin

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/glyckin.jpg

This is a schematic representation of ATP and glycerol bound (attached) to glycerol kinase. The enzyme glycerol kinase is a dimer (consists of two identical subuits). There is a deep cleft between the subunits where ATP and glycerol bind. Since the ATP and phosphate are physically so close together when they are bound to the enzyme, the phosphate can be transferred directly from ATP to glycerol. Hence, the processes of ATP losing a phosphate (spontaneous) and glycerol gaining a phosphate (nonspontaneous) are linked together as one spontaneous process

Questions on ATP: The Body’s Free-Energy Currency (How Free-Energy Currency Works)

  • Biological systems involve many molecules containing phosphate groups, such as ATP. Although ATP is the most commonly used free-energy currency, any of these phosphorylated molecules could, in theory, be used as free-energy currency. The standard free-energy change (DGo) for the dephosphorylation (removal of a phosphate group) of several biological compounds is given below:
Acetyl phosphate DGo = -47.3 kJ/mol
Adenosine triphosphate (ATP) DGo = -30.5 kJ/mol
Glucose-6-phosphate DGo = -13.8 kJ/mol
Phosphoenolpyruvate (PEP) DGo = -61.9 kJ/mol
Phosphocreatine DGo = -43.1 kJ/mol

Neglecting any differences in difficulty synthesizing or accessing these molecules by biological systems, rank the molecules in order of their efficiency as a free-energy currency (i.e., the amount of nonspontaneous reactions enabled per phosphate removed from a molecule of free-energy currency) from the most efficient to the least efficient.

  • What, if any, changes are there in the shape of the ring as NADH is oxidized to NAD+(see Figure 5)? (Hint: Consider which atoms lie in the same plane in each structure.)

Mechanism of Coupling the Oxidative-Phosphorylation Reactions

In order to couple the redox and phosphorylation reactions needed for ATP synthesis in the body, there must be some mechanism linking the reactions together. In cells, this is accomplished through an elegant proton-pumping system that occurs inside special double-membrane-bound organelles (specialized cellular components) known as mitochondria. A number of proteins are required to maintain this proton-pumping system and catalyze the oxidative and phosphorylation reactions.

Synthesis of ATP (Equation 8) is coupled with the oxidation of NADH (Equation 9) and the reduction of O2 (Equation 10). There are three key steps in this process:

  1. Electrons are transferred from NADH, through a series of electron carriers, to O2. The electron carriers are proteins embedded in the inner mitochondrial membrane. (More detail about the structure of the mitochondria is presented in the next section.) (See Figure 7a.)
  2. Transfer of electrons by these carriers generates a proton (H+) gradient across the inner mitochondrial membrane. (See Figure 7b.)
  3. When Hspontaneously diffuses back across the inner mitochondrial membrane, ATP is synthesized. The large positive free energy of ATP synthesis is overcome by the even larger negative free energy associated with proton flow down the concentration gradient. (See Figure 7c.)

These steps are outlined below.

  1. Electron Transport (Oxidation-Reduction Reactions) Through a Series of Proteins in the Inner Membrane of the Mitochondria
e_transfer

e_transfer

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/e_transfer.jpg

Generation of H+(Proton) Concentration Gradient Across the Inner Mitochondrial Membrane During the Electron-Transport Process (via a Proton Pump)

. Generation of H+ (Proton) Concentration Gradient Across the Inner Mitochondrial Membrane

. Generation of H+ (Proton) Concentration Gradient Across the Inner Mitochondrial Membrane

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/gradient.jpg

Synthesis of ATP Using Free Energy Released From Spontaneous Diffusion of H+Back to the Matrix Inside the Inner Mitochondrial Membrane

. Synthesis of ATP Using Free Energy Released From Spontaneous Diffusion of H+

. Synthesis of ATP Using Free Energy Released From Spontaneous Diffusion of H+

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/ATP_produced.jpg

The three major steps in oxidative phosphorylation are

(a) oxidation-reduction reactions involving electron transfers between specialized proteins embedded in the inner mitochondrial membrane; 

(b) the generation of a proton (H+) gradient across the inner mitochondrial membrane (which occurs simultaneously with step (a)); and 

(c) the synthesis of ATP using energy from the spontaneous diffusion of electrons down the proton gradient generated in step (b).

Note: Steps (a) and (b) show cytochrome oxidase, the final electron-carrier protein in the electron-transport chain described above. When this protein accepts an electron (green) from another protein in the electron-transport chain, an Fe(III) ion in the center of a heme group (purple) embedded in the protein is reduced to Fe(II). The coordinates for the protein were determined using x-ray crystallography, and the image was rendered using SwissPDB Viewer and POV-Ray (see References).

Cells use a proton-pumping system made up of proteins inside the mitochondria to generate ATP. Before we examine the details of ATP synthesis, we shall step back and look at the big picture by exploring the structure and function of the mitochondria, where oxidative phosphorylation occurs.

Structure and Function of the Mitochondria

mitochondria

mitochondria

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/mitochondria.jpg

This is a schematic diagram showing the membranes of the mitochondrion. The purple shapes on the inner membrane represent proteins, which are described in the section below. An enlargement of the boxed portion of the inner membrane in this figure is shown in Figure.

The mitochondrial membranes are crucial for this organelle’s role in oxidative phosphorylation. As shown in Figure 8, mitochondria have two membranes, an inner and an outer membrane. The outer membrane ispermeable to most small molecules and ions, because it contains large protein channels called porins. The inner membrane is impermeable to most ions and polar molecules. The inner membrane is the site of oxidative phosphorylation. Although the membrane is mostly impermeable, it contains special H+ (proton) channels and pumps that enable the coupling of the redox reaction involving NADH and O2 (Equations 9-10) to the phosphorylation reaction of ADP (Equation 8), as described below (“Oxidation-Reduction Reactions and Proton Pumping in Oxidative Phosphorylation”). (Recall the discussion of protein channels in the “Maintaining the Body’s Chemistry: Dialysis in the Kidneys” Tutorial .)

As shown in Figure 8, inside the inner membrane is a space known as the matrix; the space between the two membranes is known as the intermembrane space. The matrix side of the inner membrane has a negative electrical charge relative to the intermembrane space due to an H+ gradient set up by the redox reaction (Equations 9 and 10). This charge difference is used to provide free energy (G) for the phosphorylation reaction (Equation 8).

Oxidation-Reduction Reactions and Proton Pumping in Oxidative Phosphorylation

Phosphorylation of ADP (Equation 8) is coupled to the oxidation-reduction reaction of NADH and O2 (Equations 9 and 10). Electrons are not transferred directly from NADH to O2, but rather pass through a series of intermediate electron carriers in the inner membrane of the mitochondrion. Why? This allows something very important to occur: the pumping of protons across the inner membrane of the mitochondrion. As we shall see, it is this proton pumping that is ultimately responsible for coupling the oxidation-reduction reaction to ATP synthesis.

Two major types of mitochondrial proteins (see Figure 9, below) are required for oxidative phosphorylation to occur. Both classes of proteins are located in the inner mitochondrial membrane.

  1. The electron carriers (NADH-Q reductase, ubiquinone (Q), cytochrome reductase, cytochrome c, and cytochrome oxidase shown in shades of purple in Figure 9 below) transport electrons in a stepwise fashion from NADH to O2.  Three of these carriers (NADH-Q reductase, cytochrome reductase, and cytochrome oxidase) are also proton pumps, and simultaneously pump H+ ions (protons) from the matrix to the intermembrane space. (Proton movement from one side of the membrane to the other is shown as blue arrows in Figure 9, below.) The protons that are pumped across the membrane complete the redox reaction (Equations 9 and 10). The creation of a proton gradient across the membrane is one way of storing free energy.
  2. ATP synthetase (shown in red in Figure 9 below) allows H+ ions to diffuse back into the matrix and uses the free energy released to synthesize ATP from ADP and HPO42-. The ATP synthetase is essential for the phosphorylation to occur (Equation 8). (Proton movement from one side of the membrane to the other is shown as blue arrows in Figure 9, below.)

The electron carriers can be divided into three protein complexes (NADH-Q reductase (1), cytochrome reductase (3), and cytochrome oxidase (5)) that pump protons from the matrix to the intermembrane space, and two mobile carriers (ubiquinone (2) and cytochrome c (4)) that transfer electrons between the three proton-pumping complexes. (Gold numbers refer to the labels on each protein in Figure 9, below.) Because electrons move from one carrier to another until they are finally transferred to O2, the electron carriers (shown in Figure 9,below) are said to form an electron-transport chain.

Figure  below, is a schematic representation of the proteins involved in oxidative phosphorylation. To see an animation of oxidative phosphorylation, click on “View the Movie.”

Proteins of inner space

Proteins of inner space

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/Proteins.jpg

This is a schematic diagram illustrating the transfer of electrons from NADH, through the electron carriers in the electron transport chain, to molecular oxygen. Please click on the pink button below to view a QuickTime animation of the functions of the proteins embedded in the inner mitochondrial membrane that are necessary for oxidative phosphorylation. Click the blue button below to download QuickTime 4.0 to view the movie.

NADH-Q reductase (1), cytochrome reductase (3) , and cytochrome oxidase (5) are electron carriers as well as proton pumps, using the energy gained from each electron-transfer step to move protons (H+) against a concentration gradient, from the matrix to the intermembrane space.Ubiquinone (Q) (2) and cytochrome c (Cyt C) (4) are mobile electron carriers. (Ubiquinone is not actually a protein.) All of the electron carriers are shown in purple, with lighter shades representing increasingly higher reduction potentials. Together, these electron carriers form a “chain” to transport electrons from NADH to O2. The path of the electrons is shown with the green dotted line.

ATP synthetase (red) has two components: a proton channel (allowing diffusion of protons down a concentration gradient, from the intermembrane space to the matrix), and a catalytic component to catalyze the formation of ATP.

For a more complete description of each step in oxidative phosphorylation (indicated by the gold numbers), click here.

view movie

view movie

http://www.apple.com/quicktime/

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/movie.jpg

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/QuickTime.jpg

Click here for a brief description of each of the electron carriers in the electron-transport chain. It is important to note that, although NADH donates two electrons and O2 ultimately accepts four electrons, each of the carriers can only transfer one electron at a time. Hence, there are several points along the chain where electrons can be collected and dispersed. For the sake of simplicity, these points are not described in this tutorial.

In the section above, we see that the oxidation-reduction process is a series of electron transfers that occurs spontaneously and produces a proton gradient. Why are the electron tranfers from one electron carrier to the next spontaneous?

What causes electrons to be transferred down the electron-transport chain?

As seen in Table 2, below, and Figure 7a, in these carriers, the species being oxidized or reduced is Fe, which is found either in a iron-sulfur (Fe-S) group or in a heme group. (Recall the heme group from the Chem 151 tutorial “Hemoglobin and the Heme Group: Metal Complexes in the Blood“.) The iron in these groups is alternately oxidized and reduced between Fe(II) (reduced) or Fe(III) (oxidized) states.

Table 2 shows that the electrons are transferred through the electron-transport chain because of the difference in the reduction potential of the electron carriers. As explained in the green box below, the higher the electrical potential (e) of a reduction half reaction is, the greater the tendency is for the species to accept an electron. Hence, in the electron-transport chain, electrons are transferred spontaneously from carriers whose reduction results in a small electrical potential change to carriers whose reduction results in an increasingly larger electrical potential change.

Reduction Potentials and Relationship to Free Energy

An oxidation-reduction reaction consists of an oxidation half reaction and a reduction half reaction. Every half reaction has an electrical potential (e). By convention, all half reactions are written as reductions, and the electrical potential for an oxidation half-reaction is equal in magnitude, but opposite in sign, to the electrical potential for the corresponding reduction (i.e., the opposite reaction). The electrical potential for an oxidation-reduction reaction is calculated by

erxn = eoxidation + ereduction (12)

For example, for the overall reaction of the oxidation of NADH paired with the reduction of O2, the potential can be calculated as shown below.

Reduction Potentials ereduction
NAD+ + 2H+ + 2e –> NADH + H+ -0.32 V
(1/2) O2 + 2H+ + 2e –> H2O +0.82 V

The overall reaction is

NADH + H–> NAD+ + 2H+ + 2e eoxidation = 0.32 V
(1/2) O2 + 2H+ + 2e –> H2O ereduction = 0.82 V
net: NADH + (1/2)O2 + H+ –>
H2O + NAD+
erxn = 1.14 V

The electrical potential (erxn) is related to the free energy (DG) by the following equation:

DG= -nFerxn (13)

where n is the number of electrons transferred (in moles, from the balanced equation), and F is the Faraday constant (96,485 Coulombs/mole). (Using this equation, DG is given in Joules; one Joule = 1 Volt x 1 Coulomb.)

Hence the overall reaction for the oxidation of NADH paired with the reduction of O2 has a negative change in free energy (DG =-220 kJ); i.e., it is spontaneous. Thus, the higher the electrical potential of a reduction half reaction, the greater the tendency for the species to accept an electron.

Just as in the box above, the electrical potential for the overall reaction (electron transfer) between two electron carriers is the sum of the potentials for the two half reactions. As long as the potential for the overall reaction is positive the reaction is spontaneous. Hence, from Table 2 below, we see that cytochrome c1 (part of the cytochrome reductase complex, #3 in Figure 9) can spontaneously transfer an electron to cytochrome c (#4 in Figure 9). The net reaction is given by Equation 16, below.

reduced cytochrome c–> oxidized cytochrome c+ e eoxidation = – .220 V (14)
oxidized cytochrome c + e –> reduced cytochrome c ereduction = .250 V (15)
NET: reduced cyt c1 + oxidized cyt c –>
oxidized cyt c+ reduced cyt c
erxn = 0.030 V (16) Spontaneous

We can also see from Table 2 that cytochrome c1 cannot spontaneously transfer an electron to cytochrome b (Equation 19):

reduced cyt c–> oxidized cyt c+ e eoxidation = – .220 V (17)
oxidized cyt b + e –> reduced cyt b ereduction = – 0.34 V (18)
NET: reduced cyt c1 + oxidized cyt c –>
oxidized cyt c+ reduced cyt c
erxn = – 0.56 V (19) NOT Spontaneous

Table 2 lists the reduction potentials for each of the cytochrome proteins (i.e., the last three steps in the electron-transport chain before the electrons are accepted by O2) involved in the electron-transport chain. Note that each electron transfer is to a cytochrome with a higher reduction potential than the previous cytochrome. As described in the box above and seen in Equations 14-19, an increase in potential leads to a decrease in DG (Equation 13), and thus the transfer of electrons through the chain is spontaneous.

Complex Name Half Reaction Reduction Potential
Cytochrome reductase

(also known as cytochrome b-c1 complex)

(3 in Figure 9)

Cytochrome b (Fe(III) center)
+ e –>
Cytochrome b (Fe(II) center)
-0.34 V
(at pH 7, T=30oC)
Cytochrome c1 (Fe(III) center)
+ e– –>
Cytochrome c1 (Fe(II) center)
+0.220 V
(at pH 7, T=30oC)
Cytochrome c

(4 in Figure 9)

Cytochrome c (Fe(III) center)
+ e– –>
Cytochrome c (Fe(II) center)
+0.250 V
(at pH 7, T=30oC)
Cytochrome oxidase

(5 in Figure 9)

Cytochrome oxidase
( Fe(III) center) + e– –>
Cytochrome oxidase
(Fe(II) center)
+0.285 V
(at pH 7.4, T=25oC)
Table 2

To view the cytochrome molecules interactively using RASMOL, please click on the name of the complex to download the pdb file.

Hence, the electron-transport chain (which works because of the difference in reduction potentials) leads to a large concentration gradient for H+. As we shall see below, this huge concentration gradient leads to the production of ATP.

Questions on Electron Carriers: Steps in the Electron-Transport Chain; Reduction Potentials and Relationship to Free Energy

  • Briefly, explain why electrons travel from NADH-Q reductase, to ubiquinone (Q), to cytochrome reductase, rather than in the opposite direction.
  • One result of the transfer of electrons from NADH-Q reductase down the electron transport chain is that the concentration of protons (H+ ions) in the intermembrane space is increased.  Could cells move protons (H+ ions) from the matrix to the intermembrane space without transporting electrons?  Why or why not?

 ATP Synthetase: Production of ATP

We have seen that the electron-transport chain generates a large proton gradient across the inner mitochondrial membrane. But recall that the ultimate goal of oxidative phosphorylation is to generate ATP to supply readily-available free energy for the body. How does this occur? In addition to the electron-carrier proteins embedded in the inner mitochondrial membrane, a special protein called ATP synthetase (Figure 9, the red-colored protein) is also embedded in this membrane. ATP synthetase uses the proton gradient created by the electron-transport chain to drive the phosphorylation reaction that generates ATP (Figure 7c).

ATP synthetase is a protein consisting of two important segments: a transmembrane proton channel, and a catalytic component located inside the matrix. The proton-channel segment allows H+ ions to diffuse from the intermembrane space, where the concentration is high, to the matrix, where the concentration is low. Recall from the Kidney Dialysis tutorial that particles spontaneously diffuse from areas of high concentration to areas of low concentration. Thus, since the diffusion of protons through the channel component of ATP synthetase is spontaneous, this process is accompanied by a negative change in free energy (i.e., free energy is released). The catalytic component of ATP synthetase has a site where ADP can enter. Then, using the free energy released by the spontaneous diffusion of protons through the channel segment, a bond is formed between the ADP and a free phosphate group, creating an ATP molecule. The ATP is then released from the reaction site, and a new ADP molecule can enter in order to be phosphorylated.

Questions on ATP Synthetase: Production of ATP

  • A scientist has created a phospholipid-bilayer membrane containing ATP-synthetase proteins. Instead of a proton gradient, this scientist has created a large Cs+ gradient (many Cs+ ions on the side of the membrane without the catalytic unit, and few Cs+ ions on the side of the membrane with the catalytic unit). Would you expect the ATP-synthetase proteins in this membrane to be able to generate ATP, given an abundant supply of ADP and phosphate? Briefly, explain your answer. (HINT: Draw on your knowledge of the structure of protein channels to predict what effect replacing H+ ions with Cs+ ions would have.)
  • Certain toxins allow H+ ions to move freely across the inner mitochondrial membrane (i.e., without needing to pass through the channel in ATP synthetase). What effect do you expect these toxins to have on the production of ATP? Briefly, explain your answer.

Summary

In this tutorial, we have learned that the ability of the body to perform daily activities is dependent on thermodynamic, equilibrium, and electrochemical concepts.   These activities, which are typically based on nonspontaneous chemical reactions, are performed by using free-energy currency. The common free-energy currency is ATP, which is a molecule that easily dephosphorylates (loses a phosphate group) and releases a large amount of free energy. In the body, the nonspontaneous reactions are coupled to this very spontaneous dephosphorylation reaction, thereby making the overall reaction spontaneous (DG < 0). As the coupled reactions occur (i.e., as the body performs daily activities), ATP is consumed and the body regenerates ATP by using energy from the food we eat (Figure 3). As seen in Figure 4, the breakdown of glucose (glycolysis) obtained from the food we eat cannot by itself generate the large amount of ATP that is needed for metabolic energy by the body. However, glycolysis and the subsequent step, the citric-acid cycle, produce two easily oxidized molecules: NADH and FADH2. These redox molecules are used in an oxidative-phosphorylation process to produce the majority of the ATP that the body uses. This oxidative-phosphorylation process consists of two steps: the oxidation of NADH (or FADH2) and the phosphorylation reaction which regenerates ATP. Oxidative phosphorylation occurs in the mitochondria, and the two reactions (oxidation of NADH or FADHand phosphorylation to generate ATP) are coupled by a proton gradient across the inner membrane of the mitochondria (Figure 9). As seen in Figures 7 and 9, the oxidation of NADH occurs by electron transport through a series of protein complexes located in the inner membrane of the mitochondria. This electron transport is very spontaneous and creates the proton gradient that is necessary to then drive the phosphorylation reaction that generates the ATP. Hence, oxidative-phosphorylation demonstrates that free energy can be easily transferred by proton gradients. Oxidative-phosphorylation is the primary means of generating free-energy currency for aerobic organisms, and as such is one of the most important subjects in the study of bioenergetics (the study of energy and its chemical changes in the biological world).

Additional Link:

  • This fun description of oxidative phosphorylation by Dr. E.J.Oakeley contains step-by-step animated illustrations of the redox reactions involved, as well as a quiz to test your understanding of the material.

References:

Alberts, B. et al. In Molecular Biology of the Cell, 3rd ed., Garland Publishing, Inc.: New York, 1994, pp. 653-684.

Becker, W.M. and Deamer, D.W. In The World of the Cell, 2nd ed., The Benjamin/Cummings Publishing Co., Inc.: Redwood City, CA, 1991, pp. 291-307.

Fasman, G.D. In Handbook of Biochemistry and Molecular Biology, 3rd ed., CRC Press, Inc.: Cleveland, OH, 1976, Vol. I (Physical and Chemical Data), pp. 132-137.

Guex, N. and Peitsch, M.C. Electrophoresis, 1997, 18, 2714-2723. (SwissPDB Viewer) URL: http://www.expasy.ch/spdbv/mainpage.htm.

Moa, C., Ozer, Z., Zhou, M. and Uckun, F. X-Ray Structure of Glycerol Kinase Complexed with an ATP Analog Implies a Novel Mechanism for the ATP-Dependent Gylcerol Phosphorylation by Glycerol Kinase.Biochemical and Biophysical Reaearch Communications. 1999, 259, 640-644.

Persistence of Vision Ray Tracer (POV-Ray). URL: http://www.povray.org.

Stryer, L. In Biochemistry, 4th. ed., W.H. Freeman and Co.: New York, 1995, pp. 490, 509, 513, 529-557.

Zubay, G. Biochemistry, 3rd. ed., Wm. C. Brown Publishers: Dubuque, IA, 1983, p. 42.

Acknowledgements:

The authors thank Dewey Holten (Washington University in St. Louis) for many helpful suggestions in the writing of this tutorial.

The development of this tutorial was supported by a grant from the Howard Hughes Medical Institute, through the Undergraduate Biological Sciences Education program, Grant HHMI# 71199-502008 to Washington University.

Copyright 1999, Washington University, All Rights Reserved.

 

 

 

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Epilogue: Envisioning New Insights in Cancer Translational Biology

Author and Curator: Larry H Bernstein, MD, FCAP

 

The foregoing  summary leads to a beginning as it is a conclusion.  It concludes a body of work in the e-book series,

Series C: e-Books on Cancer & Oncology

Series C Content Consultant: Larry H. Bernstein, MD, FCAP

 

VOLUME ONE 

Cancer Biology and Genomics for Disease Diagnosis

2014

Stephen J. Williams, PhD, Senior Editor

sjwilliamspa@comcast.net

Tilda Barliya, PhD, Editor

tildabarliya@gmail.com

Ritu Saxena, PhD, Editor

ritu.uab@gmail.com

Leaders in Pharmaceutical Business Intelligence 

that has been presented by the cancer team of professional experts, e-Book concept was conceived by Aviva Lev-Ari, PhD, RN, e-Series Editor-in-Chief and Founder of Leaders in Pharmaceutical Business Intelligence 

and the Open Access Online Scientific Journal

http://pharmaceuticalintelligence.com

Stephen J. Williams, PhD, Senior Editor, and other notable contributors in  various aspects of cancer research in the emerging fields of targeted  pharmacology,  nanotechnology, cancer imaging, molecular pathology, transcriptional and regulatory ‘OMICS’, metabolism, medical and allied health related sciences, synthetic biology, pharmaceutical discovery, and translational medicine.

This  volume and its content have been conceived and organized to capture the organized events that emerge in embryological development, leading to the major organ systems that we recognize anatomically and physiologically as an integrated being.  We capture the dynamic interactions between the systems under stress  that are elicited by cytokine-driven hormonal responses, long thought to be circulatory and multisystem, that affect the major compartments of  fat and lean body mass, and are as much the drivers of metabolic pathway changes that emerge as epigenetics, without disregarding primary genetic diseases.

The greatest difficulty in organizing such a work is in whether it is to be merely a compilation of cancer expression organized by organ systems, or whether it is to capture developing concepts of underlying stem cell expressed changes that were once referred to as “dedifferentiation”.  In proceeding through the stages of neoplastic transformation, there occur adaptive local changes in cellular utilization of anabolic and catabolic pathways, and a retention or partial retention of functional specificities.

This  effectively results in the same cancer types not all fitting into the same “shoe”. There is a sequential loss of identity associated with cell migration, cell-cell interactions with underlying stroma, and metastasis., but cells may still retain identifying “signatures” in microRNA combinatorial patterns.  The story is still incomplete, with gaps in our knowledge that challenge the imagination.

What we have laid out is a map with substructural ordered concepts forming subsets within the structural maps.  There are the traditional energy pathways with terms aerobic and anaerobic glycolysis, gluconeogenesis, triose phosphate branch chains, pentose shunt, and TCA cycle vs the Lynen cycle, the Cori cycle, glycogenolysis, lipid peroxidation, oxidative stress, autosomy and mitosomy, and genetic transcription, cell degradation and repair, muscle contraction, nerve transmission, and their involved anatomic structures (cytoskeleton, cytoplasm, mitochondria, liposomes and phagosomes, contractile apparatus, synapse.

Then there is beneath this macro-domain the order of signaling pathways that regulate these domains and through mechanisms of cellular regulatory control have pleiotropic inhibitory or activation effects, that are driven by extracellular and intracellular energy modulating conditions through three recognized structures: the mitochondrial inner membrane, the intercellular matrix, and the ion-channels.

What remains to be done?

  1. There is still to be elucidated the differences in patterns within cancer types the distinct phenotypic and genotypic features  that mitigate anaplastic behavior. One leg of this problem lies in the density of mitochondria, that varies between organ types, but might vary also within cell type of a common function.  Another leg of this problem has also appeared to lie in the cell death mechanism that relates to the proeosomal activity acting on both the ribosome and mitochondrion in a coordinated manner.  This is an unsolved mystery of molecular biology.

 

  1. Then there is a need to elucidate the major differences between tumors of endocrine, sexual, and structural organs, which are distinguished by primarily a synthetic or primarily a catabolic function, and organs that are neither primarily one or the other.  For example, tumors of the thyroid and paratnhyroids, islet cells of pancreas, adrenal cortex, and pituitary glands have the longest 5 year survivals.  They and the sexual organs are in the visceral compartment.  The rest of the visceral compartment would be the liver, pancreas, salivary glands, gastrointestinal tract, and lungs (which are embryologically an outpouching of the gastrointestinal tract), kidneys and lower urinary tract.  Cancers of these organs have a much less favorable survival (brain, breast and prostate, lymphatic, blood forming organ, skin).  The case  is intermediate for breast and prostate between the endocrine organs and GI tract, based on natural history, irrespective of the available treatments.  Just consider the dilemma over what we do about screening for prostate cancer in men over the age of 60 years age who have a 70 percent incident silent carcinoma of the prostate that could be associated with unrelated cause of death.  The very rapid turnover of the gastric and colonic GI epithelium, and of the  subepithelial  B cell mucosal lymphocytic structures  is associated  with a greater aggressiveness of the tumor.

 

  1. However, we  have to reconsider the observation by NO Kaplan than the synthetic and catabolic functions are highlighted by differences in the expressions of the balance of  the two major pyridine nucleotides – DPN (NAD) and TPN (NADP) – which also might be related to the density of mitochondria  which is associated with both NADP and synthetic activity, and  with efficient aerobic function.  These are in an equilibrium through the “transhydrogenase reaction” co-discovered by Kaplan, in Fritz Lipmann’s laboratory. There does  arise a conundrum involving the regulation of mitochondria in these high turnover epithelial tissues  that rely on aerobic energy, and generate ATP through TPN linked activity, when they undergo carcinogenesis. The cells  replicate and they become utilizers of glycolysis, while at the same time, the cell death pathway is quiescent. The result becomes the introduction of peripheral muscle and liver synthesized protein cannabolization (cancer cachexia) to provide glucose  from proteolytic amino acid sources.

 

  1. There is also the structural compartment of the lean body mass. This is the heart, skeletal  structures (includes smooth muscle of GI tract, uterus, urinary bladder, brain, bone, bone marrow).  The contractile component is associated with sarcomas.  What is most striking is that the heart, skeletal muscle, and inflammatory cells are highly catabolic, not anabolic.  NO Kaplan referred tp them as DPN (NAD) tissues. This compartment requires high oxygen supply, and has a high mechanical function. But again, we return to the original observations of enrgy requirements at rest being different than at high demand.  At work, skeletal muscle generates lactic acid, but the heart can use lactic acid as fuel,.

 

  1. The liver is supplied by both the portal vein and the hepatic artery, so it is not prone to local ischemic injury (Zahn infarct). It is exceptional in that it carries out synthesis of all the circulating transport proteins, has a major function in lipid synthesis and in glycogenesis and glycogenolysis, with the added role of drug detoxification through the P450 system.  It is not only the largest organ (except for brain), but is highly active both anabolically and catabolically (by ubiquitilation).
  2. The expected cellular turnover rates for these tissues and their balance of catabolic and anabolic function would have to be taken into account to account for the occurrence and the activities of oncogenesis. This is by no means a static picture, but a dynamic organism constantly in flux imposed by internal and external challenges.  It is also important to note the the organs have a concentration of mitochondria, associated with energy synthetic and catabolic requirements provided by oxygen supply and the electron transport mechanism for oxidative phosphorylation.  For example, tissues that are primarily synthetic do not have intermitent states of resting and high demand, as seen in skeletal muscle, or perhaps myocardium (which is syncytial and uses lactic acid generated from skeletal muscle when there is high demand).
  3. The existence of  lncDNA has been discovered only as a result of the human genome project (HGP). This was previously known only as “dark DNA”.  It has become clear that lncDNA has an important role in cellular regulatory activities centered in the chromatin modeling.  Moreover, just as proteins exhibit functionality in their folding, related to tertiary structure and highly influenced by location of –S-S- bridges and amino acid residue distances (allosteric effects), there is a less studied effect as the chromatin becomes more compressed within the nucleus, that should have a bearing on cellular expression.

According to Jose Eduardo de Salles Roselino , when the Na/Glucose transport system (for a review Silvermann, M. in Annu. Rev. Biochem.60: 757-794(1991)) was  found in kidneys as well as in key absorptive cells of digestive tract, it should be stressed its functional relationship with “internal milieu” and real meaning, homeostasis. It is easy to understand how the major topic was presented as how to prevent diarrheal deaths in infants, while detected in early stages. However, from a biochemical point of view, as presented in Schrödinger´s What is life?, (biochemistry offering a molecular view for two legs of biology, physiology and genetics). Why should it be driven to the sole target of understanding genetics? Why the understanding of physiology in molecular terms should be so neglected?

From a biochemical point of view, here in a single protein. It is found the transport of the cation most directly related to water maintenance, the internal solvent that bath our cells and the hydrocarbon whose concentration is kept under homeostatic control on that solvent. Completely at variance with what is presented in microorganisms as previously mentioned in Moyed and Umbarger revision (Ann. Rev42: 444(1962)) that does not regulates the environment where they live and appears to influence it only as an incidental result of their metabolism.

In case any attempt is made in order to explain why the best leg that supports scientific reasoning from biology for medical purposes was led to atrophy, several possibilities can be raised. However, none of them could be placed strictly in scientific terms. Factors that bare little relationship with scientific progress in general terms must also be taken into account.

One simple possibility of explanation can be found in one review (G. Scatchard – Solutions of Electrolytes Ann. Rev. Physical Chemistry 14: 161-176 (1963)).  A simple reading of it and the sophisticated differences among researchers will discourage one hundred per cent of biologists to keep in touch with this line of research. Biochemists may keep on reading.  However, consider that first: Complexity is not amenable to reductionist vision in all cases. Second, as coupling between scalar flows such as chemical reactions and vector flows such as diffusion flows, heat flows, and electrical current can occur only in anisotropic system…let them with their problems of solvents, ions and etc. and let our biochemical reactions on another basket. At the interface, for instance, at membrane level, we will agree that ATP is converted to ADP because it is far from equilibrium and the continuous replenishment of ATP that maintain relatively constant ATP levels inside the cell and this requires some non-stationary flow.

Our major point must be to understand that our biological limits are far clearer present in our limited ability to regulate the information stored in the DNA than in the amount of information we have in the DNA as the master regulator of the cells.

The amazing revelation that Masahiro Chiga   (discovery of liver adenylate kinase  distinct from that of muscle) taught  me (LHB) is – draw 2 circles  that intersect, one of which represents what we know, the other – what we don’t know.  We don’t teach how much we don’t know!  Even today, as much as 40 years ago, there is a lot we need to get on top of this.

 

The observation is rather similar to the presentations I  (Jose Eduardo de Salles Rosalino) was previously allowed to make of the conformational energy as made by R Marcus in his Nobel lecture revised (J. of  Electroanalytical Chemistry 438:(1997) p251-259. His description of the energetic coordinates of a landscape of a chemical reaction is only a two-dimensional cut of what in fact is a volcano crater (in three dimensions) ( each one varie but the sum of the two is constant. Solvational+vibrational=100% in ordinate) nuclear coordinates in abcissa. In case we could represent it by research methods that allow us to discriminate in one by one degree of different pairs of energy, we would most likely have 360 other similar representations of the same phenomenon. The real representation would take into account all those 360 representation together. In case our methodology was not that fine, for instance it discriminate only differences of minimal 10 degrees in 360 possible, will have 36 partial representations of something that to be perfectly represented will require all 36 being taken together. Can you reconcile it with ATGC? Yet, when complete genome sequences were presented they were described as we will know everything about this living being. The most important problems in biology will be viewed by limited vision always and the awareness of this limited is something we should acknowledge and teach it. Therefore, our knowledge is made up of partial representations.

 

Even though we may have complete genome data for the most intricate biological problems, they are not so amenable to this level of reductionism. However, from general views of signals and symptoms we could get to the most detailed molecular view and in this case the genome provides an anchor. This is somehow, what Houssay was saying to me and to Leloir when he pointed out that only in very rare occasions biological phenomena could be described in three terms: Pacco, the dog and the anesthetic (previous e-mail). The non-coding region, to me will be important guiding places for protein interactions.

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Introduction – The Evolution of Cancer Therapy and Cancer Research: How We Got Here?


Introduction – The Evolution of Cancer Therapy and Cancer Research: How We Got Here?

Author and Curator: Larry H Bernstein, MD, FCAP

The evolution of progress we have achieved in cancer research, diagnosis, and therapeutics has  originated from an emergence of scientific disciplines and the focus on cancer has been recent. We can imagine this from a historical perspective with respect to two observations. The first is that the oldest concepts of medicine lie with the anatomic dissection of animals and the repeated recurrence of war, pestilence, and plague throughout the middle ages, and including the renaissance.  In the awakening, architecture, arts, music, math, architecture and science that accompanied the invention of printing blossomed, a unique collaboration of individuals working in disparate disciplines occurred, and those who were privileged received an education, which led to exploration, and with it, colonialism.  This also led to the need to increasingly, if not without reprisal, questioning long-held church doctrines.

It was in Vienna that Rokitansky developed the discipline of pathology, and his student Semelweis identified an association between then unknown infection and childbirth fever. The extraordinary accomplishments of John Hunter in anatomy and surgery came during the twelve years war, and his student, Edward Jenner, observed the association between cowpox and smallpox resistance. The development of a nursing profession is associated with the work of Florence Nightengale during the Crimean War (at the same time as Leo Tolstoy). These events preceded the work of Pasteur, Metchnikoff, and Koch in developing a germ theory, although Semelweis had committed suicide by infecting himself with syphilis. The first decade of the Nobel Prize was dominated by discoveries in infectious disease and public health (Ronald Ross, Walter Reed) and we know that the Civil War in America saw an epidemic of Yellow Fever, and the Armed Services Medical Museum was endowed with a large repository of osteomyelitis specimens. We also recall that the Russian physician and playwriter, Anton Checkov, wrote about the conditions in prison camps.

But the pharmacopeia was about to open with the discoveries of insulin, antibiotics, vitamins, thyroid action (Mayo brothers pioneered thyroid surgery in the thyroid iodine-deficient midwest), and pitutitary and sex hormones (isolatation, crystal structure, and synthesis years later), and Karl Landsteiner’s discovery of red cell antigenic groups (but he also pioneered in discoveries in meningitis and poliomyelitis, and conceived of the term hapten) with the introduction of transfusion therapy that would lead to transplantation medicine.  The next phase would be heralded by the discovery of cancer, which was highlighted by the identification of leukemia by Rudolph Virchow, who cautioned about the limitations of microscopy. This period is highlighted by the classic work – “Microbe Hunters”.

John Hunter

John Hunter

Walter Reed

Walter Reed

Robert Koch

Robert Koch

goldberger 1916 Pellagra

goldberger 1916 Pellagra

Louis Pasteur

Louis Pasteur

A multidisciplinary approach has led us to a unique multidisciplinary or systems view of cancer, with different fields of study offering their unique expertise, contributions, and viewpoints on the etiology of cancer.  Diverse fields in immunology, biology, biochemistry, toxicology, molecular biology, virology, mathematics, social activism and policy, and engineering have made such important contributions to our understanding of cancer, that without cooperation among these diverse fields our knowledge of cancer would never had evolved as it has. In a series of posts “Heroes in Medical Research:” the work of researchers are highlighted as examples of how disparate scientific disciplines converged to produce seminal discoveries which propelled the cancer field, although, at the time, they seemed like serendipitous findings.  In the post Heroes in Medical Research: Barnett Rosenberg and the Discovery of Cisplatin (Translating Basic Research to the Clinic) discusses the seminal yet serendipitous discoveries by bacteriologist Dr. Barnett Rosenberg, which eventually led to the development of cisplatin, a staple of many chemotherapeutic regimens. Molecular biologist Dr. Robert Ting, working with soon-to-be Nobel Laureate virologist Dr. James Gallo on AIDS research and the associated Karposi’s sarcoma identified one of the first retroviral oncogenes, revolutionizing previous held misconceptions of the origins of cancer (described in Heroes in Medical Research: Dr. Robert Ting, Ph.D. and Retrovirus in AIDS and Cancer).   Located here will be a MONTAGE of PHOTOS of PEOPLE who made seminal discoveries and contributions in every field to cancer   Each of these paths of discovery in cancer research have led to the unique strategies of cancer therapeutics and detection for the purpose of reducing the burden of human cancer.  However, we must recall that this work has come at great cost, while it is indeed cause for celebration. The current failure rate of clinical trials at over 70 percent, has been a cause for disappointment, and has led to serious reconsideration of how we can proceed with greater success. The result of the evolution of the cancer field is evident in the many parts and chapters of this ebook.  Volume 4 contains chapters that are in a predetermined order:

  1. The concepts of neoplasm, malignancy, carcinogenesis,  and metastatic potential, which encompass:

(a)     How cancer cells bathed in an oxygen rich environment rely on anaerobic glycolysis for energy, and the secondary consequences of cachexia and sarcopenia associated with progression

invasion

invasion

ARTS protein and cancer

ARTS protein and cancer

Glycolysis

Glycolysis

Krebs cycle

Krebs cycle

Metabolic control analysis of respiration in human cancer tissue

Metabolic control analysis of respiration in human cancer tissue

akip1-expression-modulates-mitochondrial-function

akip1-expression-modulates-mitochondrial-function

(b)     How advances in genetic analysis, molecular and cellular biology, metabolomics have expanded our basic knowledge of the mechanisms which are involved in cellular transformation to the cancerous state.

nucleotides

nucleotides

Methylation of adenine

Methylation of adenine

ampk-and-ampk-related-kinase-ark-family-

ampk-and-ampk-related-kinase-ark-family-

ubiquitylation

ubiquitylation

(c)  How molecular techniques continue to advance our understanding  of how genetics, epigenetics, and alterations in cellular metabolism contribute to cancer and afford new pathways for therapeutic intervention.

 genomic effects

genomic effects

LKB1AMPK pathway

LKB1AMPK pathway

mutation-frequencies-across-12-cancer-types

mutation-frequencies-across-12-cancer-types

AMPK-activating drugs metformin or phenformin might provide protection against cancer

AMPK-activating drugs metformin or phenformin might provide protection against cancer

pim2-phosphorylates-pkm2-and-promotes-glycolysis-in-cancer-cells

pim2-phosphorylates-pkm2-and-promotes-glycolysis-in-cancer-cells

pim2-phosphorylates-pkm2-and-promotes-glycolysis-in-cancer-cells

pim2-phosphorylates-pkm2-and-promotes-glycolysis-in-cancer-cells

2. The distinct features of cancers of specific tissue sites of origin

3.  The diagnosis of cancer by

(a)     Clinical presentation

(b)     Age of onset and stage of life

(c)     Biomarker features

hairy cell leukemia

hairy cell leukemia

lymphoma leukemia

lymphoma leukemia

(d)     Radiological and ultrasound imaging

  1. Treatments
  2. Prognostic differences within and between cancer types

We have introduced the emergence of a disease of great complexity that has been clouded in more questions than answers until the emergence of molecular biology in the mid 20th century, and then had to await further discoveries going into the 21st century.  What gave the research impetus was the revelation of

1     the mechanism of transcription of the DNA into amino acid sequences

Proteins in Disease

Proteins in Disease

2     the identification of stresses imposed on cellular function

NO beneficial effects

NO beneficial effects

3     the elucidation of the substructure of the cell – cell membrane, mitochondria, ribosomes, lysosomes – and their functions, respectively

pone.0080815.g006  AKIP1 Expression Modulates Mitochondrial Function

AKIP1 Expression Modulates Mitochondrial Function

4     the elucidation of oligonucleotide sequences

nucleotides

nucleotides

dna-replication-unwinding

dna-replication-unwinding

dna-replication-ligation

dna-replication-ligation

dna-replication-primer-removal

dna-replication-primer-removal

dna-replication-leading-strand

dna-replication-leading-strand

dna-replication-lagging-strand

dna-replication-lagging-strand

dna-replication-primer-synthesis

dna-replication-primer-synthesis

dna-replication-termination

dna-replication-termination

5     the further elucidation of functionally relevant noncoding lncDNA

lncRNA-s   A summary of the various functions described for lncRNA

6     the technology to synthesis mRNA and siRNA sequences

RNAi_Q4 Primary research objectives

Figure. RNAi and gene silencing

7     the repeated discovery of isoforms of critical enzymes and their pleiotropic properties

8.     the regulatory pathways involved in signaling

signaling pathjways map

Figure. Signaling Pathways Map

This is a brief outline of the modern progression of advances in our understanding of cancer.  Let us go back to the beginning and check out a sequence of  Nobel Prizes awarded and related discoveries that have a historical relationship to what we know.  The first discovery was the finding by Louis Pasteur that fungi that grew in an oxygen poor environment did not put down filaments.  They did not utilize oxygen and they produced used energy by fermentation.  This was the basis for Otto Warburg sixty years later to make the comparison to cancer cells that grew in the presence of oxygen, but relied on anaerobic glycolysis. He used a manometer to measure respiration in tissue one cell layer thick to measure CO2 production in an adiabatic system.

video width=”1280″ height=”720″ caption=”1741-7007-11-65-s1 Macromolecular juggling by ubiquitylation enzymes.” mp4=”https://pharmaceuticalintelligence.files.wordpress.com/2014/04/1741-7007-11-65-s1-macromolecular-juggling-by-ubiquitylation-enzymes.mp4“][/video]

An Introduction to the Warburg Apparatus

http://www.youtube.com/watch?v=M-HYbZwN43o

Lavoisier Antoine-Laurent and Laplace Pierre-Simon (1783) Memoir on heat. Mémoirs de l’Académie des sciences. Translated by Guerlac H, Neale Watson Academic Publications, New York, 1982.

Instrumental background 200 years later:   Gnaiger E (1983) The twin-flow microrespirometer and simultaneous calorimetry. In Gnaiger E, Forstner H, eds. Polarographic Oxygen Sensors. Springer, Heidelberg, Berlin, New York: 134-166.

otto_heinrich_warburg

otto_heinrich_warburg

Warburg apparatus

The Warburg apparatus is a manometric respirometer which was used for decades in biochemistry for measuring oxygen consumption of tissue homogenates or tissue slices.

The Warburg apparatus has its name from the German biochemist Otto Heinrich Warburg (1883-1970) who was awarded the Nobel Prize in physiology or medicine in 1931 for his “discovery of the nature and mode of action of the respiratory enzyme” [1].

The aqueous phase is vigorously shaken to equilibrate with a gas phase, from which oxygen is consumed while the evolved carbon dioxide is trapped, such that the pressure in the constant-volume gas phase drops proportional to oxygen consumption. The Warburg apparatus was introduced to study cell respiration, i.e. the uptake of molecular oxygen and the production of carbon dioxide by cells or tissues. Its applications were extended to the study of fermentation, when gas exchange takes place in the absence of oxygen. Thus the Warburg apparatus became established as an instrument for both aerobic and anaerobic biochemical studies [2, 3].

The respiration chamber was a detachable glass flask (F) equipped with one or more sidearms (S) for additions of chemicals and an open connection to a manometer (M; pressure gauge). A constant temperature was provided by immersion of the Warburg chamber in a constant temperature water bath. At thermal mass transfer equilibrium, an initial reading is obtained on the manometer, and the volume of gas produced or absorbed is determined at specific time intervals. A limited number of ‘titrations’ can be performed by adding the liquid contained in a side arm into the main reaction chamber. A Warburg apparatus may be equipped with more than 10 respiration chambers shaking in a common water bath.   Since temperature has to be controlled very precisely in a manometric approach, the early studies on mammalian tissue respiration were generally carried out at a physiological temperature of 37 °C.

The Warburg apparatus has been replaced by polarographic instruments introduced by Britton Chance in the 1950s. Since Chance and Williams (1955) measured respiration of isolated mitochondria simultaneously with the spectrophotometric determination of cytochrome redox states, a water chacket could not be used, and measurements were carried out at room temperature (or 25 °C). Thus most later studies on isolated mitochondria were shifted to the artifical temperature of 25 °C.

Today, the importance of investigating mitochondrial performance at in vivo temperatures is recognized again in mitochondrial physiology.  Incubation times of 1 hour were typical in experiments with the Warburg apparatus, but were reduced to a few or up to 20 min, following Chance and Williams, due to rapid oxygen depletion in closed, aqueous phase oxygraphs with high sample concentrations.  Today, incubation times of 1 hour are typical again in high-resolution respirometry, with low sample concentrations and the option of reoxygenations.

“The Nobel Prize in Physiology or Medicine 1931”. Nobelprize.org. 27 Dec 2011 www.nobelprize.org/nobel_prizes/medicine/laureates/1931/

  1. Oesper P (1964) The history of the Warburg apparatus: Some reminiscences on its use. J Chem Educ 41: 294.
  2. Koppenol WH, Bounds PL, Dang CV (2011) Otto Warburg’s contributions to current concepts of cancer metabolism. Nature Reviews Cancer 11: 325-337.
  3. Gnaiger E, Kemp RB (1990) Anaerobic metabolism in aerobic mammalian cells: information from the ratio of calorimetric heat flux and respirometric oxygen flux. Biochim Biophys Acta 1016: 328-332. – “At high fructose concen­trations, respiration is inhibited while glycolytic end products accumulate, a phenomenon known as the Crabtree effect. It is commonly believed that this effect is restric­ted to microbial and tumour cells with uniquely high glycolytic capaci­ties (Sussman et al, 1980). How­ever, inhibition of respiration and increase of lactate production are observed under aerobic condi­tions in beating rat heart cell cultures (Frelin et al, 1974) and in isolated rat lung cells (Ayuso-Parrilla et al, 1978). Thus, the same general mechanisms respon­sible for the integra­tion of respiration and glycolysis in tumour cells (Sussman et al, 1980) appear to be operating to some extent in several isolated mammalian cells.”

Mitochondria are sometimes described as “cellular power plants” because they generate most of the cell’s supply of adenosine triphosphate (ATP), used as a source of chemical energy.[2] In addition to supplying cellular energy, mitochondria are involved in other tasks such as signalingcellular differentiationcell death, as well as the control of the cell cycle and cell growth.[3]   The organelle is composed of compartments that carry out specialized functions. These compartments or regions include the outer membrane, the intermembrane space, the inner membrane, and the cristae and matrix. Mitochondrial proteins vary depending on the tissue and the species. In humans, 615 distinct types of proteins have been identified from cardiac mitochondria,[9   Leonor Michaelis discovered that Janus green can be used as a supravital stain for mitochondria in 1900.  Benjamin F. Kingsbury, in 1912, first related them with cell respiration, but almost exclusively based on morphological observations.[13] In 1913 particles from extracts of guinea-pig liver were linked to respiration by Otto Heinrich Warburg, which he called “grana”. Warburg and Heinrich Otto Wieland, who had also postulated a similar particle mechanism, disagreed on the chemical nature of the respiration. It was not until 1925 when David Keilin discovered cytochromes that the respiratory chain was described.[13]    

The Clark Oxygen Sensor

Dr. Leland Clark – inventor of the “Clark Oxygen Sensor” (1954); the Clark type polarographic oxygen sensor remains the gold standard for measuring dissolved oxygen in biomedical, environmental and industrial applications .   ‘The convenience and simplicity of the polarographic ‘oxygen electrode’ technique for measuring rapid changes in the rate of oxygen utilization by cellular and subcellular systems is now leading to its more general application in many laboratories. The types and design of oxygen electrodes vary, depending on the investigator’s ingenuity and specific requirements of the system under investigation.’Estabrook R (1967) Mitochondrial respiratory control and the polarographic measurement of ADP:O ratios. Methods Enzymol. 10: 41-47.   “one approach that is underutilized in whole-cell bioenergetics, and that is accessible as long as cells can be obtained in suspension, is the oxygen electrode, which can obtain more precise information on the bioenergetic status of the in situ mitochondria than more ‘high-tech’ approaches such as fluorescent monitoring of Δψm.” Nicholls DG, Ferguson S (2002) Bioenergetics 3. Academic Press, London.

Great Figures in Cancer

Dr. Elizabeth Blackburn,

Dr. Elizabeth Blackburn,

j_michael_bishop onogene

j_michael_bishop onogene

Harold Varmus

Harold Varmus

Potts and Habener (PTH mRNA, Harvard MIT)  JCI

Potts and Habener (PTH mRNA, Harvard MIT) JCI

JCI Fuller Albright and hPTH AA sequence

JCI Fuller Albright and hPTH AA sequence

Dr. E. Donnall Thomas  Bone Marrow Transplants

Dr. E. Donnall Thomas Bone Marrow Transplants

Dr Haraldzur Hausen  EBV HPV

Dr Haraldzur Hausen EBV HPV

Dr. Craig Mello

Dr. Craig Mello

Dorothy Hodgkin  protein crystallography

Lee Hartwell - Hutchinson Cancer Res Center

Lee Hartwell – Hutchinson Cancer Res Center

Judah Folkman, MD

Judah Folkman, MD

Gertrude B. Elien (1918-1999)

Gertrude B. Elien (1918-1999)

The Nobel Prize in Physiology or Medicine 1922   

Archibald V. Hill, Otto Meyerhof

AV Hill –

“the production of heat in the muscle” Hill started his research work in 1909. It was due to J.N. Langley, Head of the Department of Physiology at that time that Hill took up the study on the nature of muscular contraction. Langley drew his attention to the important (later to become classic) work carried out by Fletcher and Hopkins on the problem of lactic acid in muscle, particularly in relation to the effect of oxygen upon its removal in recovery. In 1919 he took up again his study of the physiology of muscle, and came into close contact with Meyerhof of Kiel who, approaching the problem differently, arrived at results closely analogous to his study. In 1919 Hill’s friend W. Hartree, mathematician and engineer, joined in the myothermic investigations – a cooperation which had rewarding results.

Otto Meyerhof

otto-fritz-meyerhof

otto-fritz-meyerhof

lactic acid production in muscle contraction Under the influence of Otto Warburg, then at Heidelberg, Meyerhof became more and more interested in cell physiology.  In 1923 he was offered a Professorship of Biochemistry in the United States, but Germany was unwilling to lose him.  In 1929 he was he was placed in charge of the newly founded Kaiser Wilhelm Institute for Medical Research at Heidelberg.  From 1938 to 1940 he was Director of Research at the Institut de Biologie physico-chimique at Paris, but in 1940 he moved to the United States, where the post of Research Professor of Physiological Chemistry had been created for him by the University of Pennsylvania and the Rockefeller Foundation.  Meyerhof’s own account states that he was occupied chiefly with oxidation mechanisms in cells and with extending methods of gas analysis through the calorimetric measurement of heat production, and especially the respiratory processes of nitrifying bacteria. The physico-chemical analogy between oxygen respiration and alcoholic fermentation caused him to study both these processes in the same subject, namely, yeast extract. By this work he discovered a co-enzyme of respiration, which could be found in all the cells and tissues up till then investigated. At the same time he also found a co-enzyme of alcoholic fermentation. He also discovered the capacity of the SH-group to transfer oxygen; after Hopkins had isolated from cells the SH bodies concerned, Meyerhof showed that the unsaturated fatty acids in the cell are oxidized with the help of the sulfhydryl group. After studying closer the respiration of muscle, Meyerhof investigated the energy changes in muscle. Considerable progress had been achieved by the English scientists Fletcher and Hopkins by their recognition of the fact that lactic acid formation in the muscle is closely connected with the contraction process. These investigations were the first to throw light upon the highly paradoxical fact, already established by the physiologist Hermann, that the muscle can perform a considerable part of its external function in the complete absence of oxygen.

But it was indisputable that in the last resort the energy for muscle activity comes from oxidation, so the connection between activity and combustion must be an indirect one, and observed that in the absence of oxygen in the muscle, lactic acid appears, slowly in the relaxed state and rapidly in the active state, disappearing in the presence of oxygen. Obviously, then, oxygen is involved when muscle is in the relaxed state. http://upload.wikimedia.org/wikipedia/commons/e/e1/Glycolysis.jpg

The Nobel Prize committee had been receiving nominations intermittently for the previous 14 years (for Eijkman, Funk, Goldberger, Grijns, Hopkins and Suzuki but, strangely, not for McCollum in this period). Tthe Committee for the 1929 awards apparently agreed that it was high time to honor the discoverer(s) of vitamins; but who were they? There was a clear case for Grijns, but he had not been re-nominated for that particular year, and it could be said that he was just taking the relatively obvious next steps along the new trail that had been laid down by Eijkman, who was also now an old man in poor health, but there was no doubt that he had taken the first steps in the use of an animal model to investigate the nutritional basis of a clinical disorder affecting millions. Goldberger had been another important contributor, but his recent death put him out of consideration. The clearest evidence for lack of an unknown “something” in a mammalian diet was presented by Gowland Hopkins in 1912. This Cambridge biochemist was already well known for having isolated the amino acid tryptophan from a protein and demonstrated its essential nature. He fed young rats on an experimental diet, half of them receiving a daily milk supplement, and only those receiving milk grew well, Hopkins suggested that this was analogous to human diseases related to diet, as he had suggested already in a lecture published in 1906. Hopkins, the leader of the “dynamic biochemistry” school in Britain and an influential advocate for the importance of vitamins, was awarded the prize jointly with Eijkman. A door was opened. Recognition of work on the fat-soluble vitamins begun by McCollum. The next award related to vitamins was given in 1934 to George WhippleGeorge Minot and William Murphy “for their discoveries concerning liver therapy in cases of [then incurable pernicious] anemia,” The essential liver factor (cobalamin, or vitamin B12) was isolated in 1948, and Vitamin B12  was absent from plant foods. But William Castle in 1928 had demonstrated that the stomachs of pernicious anemia patients were abnormal in failing to secrete an “intrinsic factor”.

1937   Albert von Szent-Györgyi Nagyrápolt

” the biological combustion processes, with special reference to vitamin C and the catalysis of fumaric acid”

http://www.biocheminfo.org/klotho/html/fumarate.html

structure of fumarate

Szent-Györgyi was a Hungarian biochemist who had worked with Otto Warburg and had a special interest in oxidation-reduction mechanisms. He was invited to Cambridge in England in 1927 after detecting an antioxidant compound in the adrenal cortex, and there, he isolated a compound that he named hexuronic acid. Charles Glen King of the University of Pittsburgh reported success In isolating the anti-scorbutic factor in 1932, and added that his crystals had all the properties reported by Szent-Györgyi for hexuronic acid. But his work on oxidation reactions was also important. Fumarate is an intermediate in the citric acid cycle used by cells to produce energy in the form of adenosine triphosphate (ATP) from food. It is formed by the oxidation of succinate by the enzyme succinate dehydrogenase. Fumarate is then converted by the enzyme fumarase to malate. An enzyme adds water to the fumarate molecule to form malate. The malate is created by adding one hydrogen atom to a carbon atom and then adding a hydroxyl group to a carbon next to a terminal carbonyl group.

In the same year, Norman Haworth from the University of Birmingham in England received a Nobel prize from the Chemistry Committee for having advanced carbohydrate chemistry and, specifically, for having worked out the structure of Szent-Györgyi’s crystals, and then been able to synthesize the vitamin. This was a considerable achievement. The Nobel Prize in Chemistry was shared with the Swiss organic chemist Paul Karrer, cited for his work on the structures of riboflavin and vitamins A and E as well as other biologically interesting compounds. This was followed in 1938 by a further Chemistry award to the German biochemist Richard Kuhn, who had also worked on carotenoids and B-vitamins, including riboflavin and pyridoxine. But Karrer was not permitted to leave Germany at that time by the Nazi regime. However, the American work with radioisotopes at Lawrence Livermore Laboratory, UC Berkeley, was already ushering in a new era of biochemistry that would enrich our studies of metabolic pathways. The importance of work involving vitamins was acknowledged in at least ten awards in the 20th century.

1.   Carpenter, K.J., Beriberi, White Rice and Vitamin B, University of California Press, Berkeley (2000).

2.  Weatherall, M.W. and Kamminga, H., The making of a biochemist: the construction of Frederick Gowland Hopkins’ reputation. Medical History vol.40, pp. 415-436 (1996).

3.  Becker, S.L., Will milk make them grow? An episode in the discovery of the vitamins. In Chemistry and Modern Society (J. Parascandela, editor) pp. 61-83, American Chemical Society,

Washington, D.C. (1983).

4.  Carpenter, K.J., The History of Scurvy and Vitamin C, Cambridge University Press, New York (1986).

Transport and metabolism of exogenous fumarate and 3-phosphoglycerate in vascular smooth muscle.

D R FinderC D Hardin

Molecular and Cellular Biochemistry (Impact Factor: 2.33). 05/1999; 195(1-2):113-21.  http://dx.doi.org/10.1023/A:1006976432578

The keto (linear) form of exogenous fructose 1,6-bisphosphate, a highly charged glycolytic intermediate, may utilize a dicarboxylate transporter to cross the cell membrane, support glycolysis, and produce ATP anaerobically. We tested the hypothesis that fumarate, a dicarboxylate, and 3-phosphoglycerate (3-PG), an intermediate structurally similar to a dicarboxylate, can support contraction in vascular smooth muscle during hypoxia. 3-PG improved maintenance of force (p < 0.05) during the 30-80 min period of hypoxia. Fumarate decreased peak isometric force development by 9.5% (p = 0.008) but modestly improved maintenance of force (p < 0.05) throughout the first 80 min of hypoxia. 13C-NMR on tissue extracts and superfusates revealed 1,2,3,4-(13)C-fumarate (5 mM) metabolism to 1,2,3,4-(13)C-malate under oxygenated and hypoxic conditions suggesting uptake and metabolism of fumarate. In conclusion, exogenous fumarate and 3-PG readily enter vascular smooth muscle cells, presumably by a dicarboxylate transporter, and support energetically important pathways.

Comparison of endogenous and exogenous sources of ATP in fueling Ca2+ uptake in smooth muscle plasma membrane vesicles.

C D HardinL RaeymaekersR J Paul

The Journal of General Physiology (Impact Factor: 4.73). 12/1991; 99(1):21-40.   http://dx.doi.org:/10.1085/jgp.99.1.21

A smooth muscle plasma membrane vesicular fraction (PMV) purified for the (Ca2+/Mg2+)-ATPase has endogenous glycolytic enzyme activity. In the presence of glycolytic substrate (fructose 1,6-diphosphate) and cofactors, PMV produced ATP and lactate and supported calcium uptake. The endogenous glycolytic cascade supports calcium uptake independent of bath [ATP]. A 10-fold dilution of PMV, with the resultant 10-fold dilution of glycolytically produced bath [ATP] did not change glycolytically fueled calcium uptake (nanomoles per milligram protein). Furthermore, the calcium uptake fueled by the endogenous glycolytic cascade persisted in the presence of a hexokinase-based ATP trap which eliminated calcium uptake fueled by exogenously added ATP. Thus, it appears that the endogenous glycolytic cascade fuels calcium uptake in PMV via a membrane-associated pool of ATP and not via an exchange of ATP with the bulk solution. To determine whether ATP produced endogenously was utilized preferentially by the calcium pump, the ATP production rates of the endogenous creatine kinase and pyruvate kinase were matched to that of glycolysis and the calcium uptake fueled by the endogenous sources was compared with that fueled by exogenous ATP added at the same rate. The rate of calcium uptake fueled by endogenous sources of ATP was approximately twice that supported by exogenously added ATP, indicating that the calcium pump preferentially utilizes ATP produced by membrane-bound enzymes.

Evidence for succinate production by reduction of fumarate during hypoxia in isolated adult rat heart cells.

C HohlR OestreichP RösenR WiesnerM Grieshaber

Archives of Biochemistry and Biophysics (Impact Factor: 3.37). 01/1988; 259(2):527-35. http://dx.doi.org:/10.1016/0003-9861(87)90519-4   It has been demonstrated that perfusion of myocardium with glutamic acid or tricarboxylic acid cycle intermediates during hypoxia or ischemia, improves cardiac function, increases ATP levels, and stimulates succinate production. In this study isolated adult rat heart cells were used to investigate the mechanism of anaerobic succinate formation and examine beneficial effects attributed to ATP generated by this pathway. Myocytes incubated for 60 min under hypoxic conditions showed a slight loss of ATP from an initial value of 21 +/- 1 nmol/mg protein, a decline of CP from 42 to 17 nmol/mg protein and a fourfold increase in lactic acid production to 1.8 +/- 0.2 mumol/mg protein/h. These metabolite contents were not altered by the addition of malate and 2-oxoglutarate to the incubation medium nor were differences in cell viability observed; however, succinate release was substantially accelerated to 241 +/- 53 nmol/mg protein. Incubation of cells with [U-14C]malate or [2-U-14C]oxoglutarate indicates that succinate is formed directly from malate but not from 2-oxoglutarate. Moreover, anaerobic succinate formation was rotenone sensitive.

We conclude that malate reduction to succinate occurs via the reverse action of succinate dehydrogenase in a coupled reaction where NADH is oxidized (and FAD reduced) and ADP is phosphorylated. Furthermore, by transaminating with aspartate to produce oxaloacetate, 2-oxoglutarate stimulates cytosolic malic dehydrogenase activity, whereby malate is formed and NADH is oxidized.

In the form of malate, reducing equivalents and substrate are transported into the mitochondria where they are utilized for succinate synthesis.

1953 Hans Adolf Krebs –

 ” discovery of the citric acid cycle” and In the course of the 1920’s and 1930’s great progress was made in the study of the intermediary reactions by which sugar is anaerobically fermented to lactic acid or to ethanol and carbon dioxide. The success was mainly due to the joint efforts of the schools of Meyerhof, Embden, Parnas, von Euler, Warburg and the Coris, who built on the pioneer work of Harden and of Neuberg. This work brought to light the main intermediary steps of anaerobic fermentations.

In contrast, very little was known in the earlier 1930’s about the intermediary stages through which sugar is oxidized in living cells. When, in 1930, I left the laboratory of Otto Warburg (under whose guidance I had worked since 1926 and from whom I have learnt more than from any other single teacher), I was confronted with the question of selecting a major field of study and I felt greatly attracted by the problem of the intermediary pathway of oxidations.

These reactions represent the main energy source in higher organisms, and in view of the importance of energy production to living organisms (whose activities all depend on a continuous supply of energy) the problem seemed well worthwhile studying.   http://www.johnkyrk.com/krebs.html

Interactive Krebs cycle

There are different points where metabolites enter the Krebs’ cycle. Most of the products of protein, carbohydrates and fat metabolism are reduced to the molecule acetyl coenzyme A that enters the Krebs’ cycle. Glucose, the primary fuel in the body, is first metabolized into pyruvic acid and then into acetyl coenzyme A. The breakdown of the glucose molecule forms two molecules of ATP for energy in the Embden Meyerhof pathway process of glycolysis.

On the other hand, amino acids and some chained fatty acids can be metabolized into Krebs intermediates and enter the cycle at several points. When oxygen is unavailable or the Krebs’ cycle is inhibited, the body shifts its energy production from the Krebs’ cycle to the Embden Meyerhof pathway of glycolysis, a very inefficient way of making energy.  

Fritz Albert Lipmann –

 “discovery of co-enzyme A and its importance for intermediary metabolism”.

In my development, the recognition of facts and the rationalization of these facts into a unified picture, have interplayed continuously. After my apprenticeship with Otto Meyerhof, a first interest on my own became the phenomenon we call the Pasteur effect, this peculiar depression of the wasteful fermentation in the respiring cell. By looking for a chemical explanation of this economy measure on the cellular level, I was prompted into a study of the mechanism of pyruvic acid oxidation, since it is at the pyruvic stage where respiration branches off from fermentation.

For this study I chose as a promising system a relatively simple looking pyruvic acid oxidation enzyme in a certain strain of Lactobacillus delbrueckii1.   In 1939, experiments using minced muscle cells demonstrated that one oxygen atom can form two adenosine triphosphate molecules, and, in 1941, the concept of phosphate bonds being a form of energy in cellular metabolism was developed by Fritz Albert Lipmann.

In the following years, the mechanism behind cellular respiration was further elaborated, although its link to the mitochondria was not known.[13]The introduction of tissue fractionation by Albert Claude allowed mitochondria to be isolated from other cell fractions and biochemical analysis to be conducted on them alone. In 1946, he concluded that cytochrome oxidase and other enzymes responsible for the respiratory chain were isolated to the mitchondria. Over time, the fractionation method was tweaked, improving the quality of the mitochondria isolated, and other elements of cell respiration were determined to occur in the mitochondria.[13]

The most important event during this whole period, I now feel, was the accidental observation that in the L. delbrueckii system, pyruvic acid oxidation was completely dependent on the presence of inorganic phosphate. This observation was made in the course of attempts to replace oxygen by methylene blue. To measure the methylene blue reduction manometrically, I had to switch to a bicarbonate buffer instead of the otherwise routinely used phosphate. In bicarbonate, pyruvate oxidation was very slow, but the addition of a little phosphate caused a remarkable increase in rate. The phosphate effect was removed by washing with a phosphate free acetate buffer. Then it appeared that the reaction was really fully dependent on phosphate.

A coupling of this pyruvate oxidation with adenylic acid phosphorylation was attempted. Addition of adenylic acid to the pyruvic oxidation system brought out a net disappearance of inorganic phosphate, accounted for as adenosine triphosphate.   The acetic acid subunit of acetyl CoA is combined with oxaloacetate to form a molecule of citrate. Acetyl coenzyme A acts only as a transporter of acetic acid from one enzyme to another. After Step 1, the coenzyme is released by hydrolysis to combine with another acetic acid molecule and begin the Krebs’ Cycle again.

Hugo Theorell

the nature and effects of oxidation enzymes”

From 1933 until 1935 Theorell held a Rockefeller Fellowship and worked with Otto Warburg at Berlin-Dahlem, and here he became interested in oxidation enzymes. At Berlin-Dahlem he produced, for the first time, the oxidation enzyme called «the yellow ferment» and he succeeded in splitting it reversibly into a coenzyme part, which was found to be flavin mononucleotide, and a colourless protein part. On return to Sweden, he was appointed Head of the newly established Biochemical Department of the Nobel Medical Institute, which was opened in 1937.

Succinate is oxidized by a molecule of FAD (Flavin Adenine Dinucleotide). The FAD removes two hydrogen atoms from the succinate and forms a double bond between the two carbon atoms to create fumarate.

1953

double-stranded-dna

double-stranded-dna

crick-watson-with-their-dna-model.

crick-watson-with-their-dna-model.

Watson & Crick double helix model 

A landmark in this journey

They followed the path that became clear from intense collaborative work in California by George Beadle, by Avery and McCarthy, Max Delbruck, TH Morgan, Max Delbruck and by Chargaff that indicated that genetics would be important.

1965

François Jacob, André Lwoff and Jacques Monod  –

” genetic control of enzyme and virus synthesis”.

In 1958 the remarkable analogy revealed by genetic analysis of lysogeny and that of the induced biosynthesis of ß-galactosidase led François Jacob, with Jacques Monod, to study the mechanisms responsible for the transfer of genetic information as well as the regulatory pathways which, in the bacterial cell, adjust the activity and synthesis of macromolecules. Following this analysis, Jacob and Monod proposed a series of new concepts, those of messenger RNA, regulator genes, operons and allosteric proteins.

Francois Jacob

Having determined the constants of growth in the presence of different carbohydrates, it occurred to me that it would be interesting to determine the same constants in paired mixtures of carbohydrates. From the first experiment on, I noticed that, whereas the growth was kinetically normal in the presence of certain mixtures (that is, it exhibited a single exponential phase), two complete growth cycles could be observed in other carbohydrate mixtures, these cycles consisting of two exponential phases separated by a-complete cessation of growth.

Lwoff, after considering this strange result for a moment, said to me, “That could have something to do with enzyme adaptation.”

“Enzyme adaptation? Never heard of it!” I said.

Lwoff’s only reply was to give me a copy of the then recent work of Marjorie Stephenson, in which a chapter summarized with great insight the still few studies concerning this phenomenon, which had been discovered by Duclaux at the end of the last century.  Studied by Dienert and by Went as early as 1901 and then by Euler and Josephson, it was more or less rediscovered by Karström, who should be credited with giving it a name and attracting attention to its existence.

Lwoff’s intuition was correct. The phenomenon of “diauxy” that I had discovered was indeed closely related to enzyme adaptation, as my experiments, included in the second part of my doctoral dissertation, soon convinced me. It was actually a case of the “glucose effect” discovered by Dienert as early as 1900.   That agents that uncouple oxidative phosphorylation, such as 2,4-dinitrophenol, completely inhibit adaptation to lactose or other carbohydrates suggested that “adaptation” implied an expenditure of chemical potential and therefore probably involved the true synthesis of an enzyme.

With Alice Audureau, I sought to discover the still quite obscure relations between this phenomenon and the one Massini, Lewis, and others had discovered: the appearance and selection of “spontaneous” mutants.   We showed that an apparently spontaneous mutation was allowing these originally “lactose-negative” bacteria to become “lactose-positive”. However, we proved that the original strain (Lac-) and the mutant strain (Lac+) did not differ from each other by the presence of a specific enzyme system, but rather by the ability to produce this system in the presence of lactose.  This mutation involved the selective control of an enzyme by a gene, and the conditions necessary for its expression seemed directly linked to the chemical activity of the system.

1974

Albert Claude, Christian de Duve and George E. Palade –

” the structural and functional organization of the cell”.

I returned to Louvain in March 1947 after a period of working with Theorell in Sweden, the Cori’s, and E Southerland in St. Louis, fortunate in the choice of my mentors, all sticklers for technical excellence and intellectual rigor, those prerequisites of good scientific work. Insulin, together with glucagon which I had helped rediscover, was still my main focus of interest, and our first investigations were accordingly directed on certain enzymatic aspects of carbohydrate metabolism in liver, which were expected to throw light on the broader problem of insulin action. But I became distracted by an accidental finding related to acid phosphatase, drawing most of my collaborators along with me. The studies led to the discovery of the lysosome, and later of the peroxisome.

In 1962, I was appointed a professor at the Rockefeller Institute in New York, now the Rockefeller University, the institution where Albert Claude had made his pioneering studies between 1929 and 1949, and where George Palade had been working since 1946.  In New York, I was able to develop a second flourishing group, which follows the same general lines of research as the Belgian group, but with a program of its own.

1968

Robert W. Holley, Har Gobind Khorana and Marshall W. Nirenberg –

“interpretation of the genetic code and its function in protein synthesis”.

1969

Max Delbrück, Alfred D. Hershey and Salvador E. Luria –

” the replication mechanism and the genetic structure of viruses”.

1975 David Baltimore, Renato Dulbecco and Howard Martin Temin –

” the interaction between tumor viruses and the genetic material of the cell”.

1976

Baruch S. Blumberg and D. Carleton Gajdusek –

” new mechanisms for the origin and dissemination of infectious diseases” The editors of the Nobelprize.org website of the Nobel Foundation have asked me to provide a supplement to the autobiography that I wrote in 1976 and to recount the events that happened after the award. Much of what I will have to say relates to the scientific developments since the last essay. These are described in greater detail in a scientific memoir first published in 2002 (Blumberg, B. S., Hepatitis B. The Hunt for a Killer Virus, Princeton University Press, 2002, 2004).

1980

Baruj Benacerraf, Jean Dausset and George D. Snell 

” genetically determined structures on the cell surface that regulate immunological reactions”.

1992

Edmond H. Fischer and Edwin G. Krebs 

“for their discoveries concerning reversible protein phosphorylation as a biological regulatory mechanism”

1994

Alfred G. Gilman and Martin Rodbell –

“G-proteins and the role of these proteins in signal transduction in cells”

2011

Bruce A. Beutler and Jules A. Hoffmann –

the activation of innate immunity and the other half to Ralph M. Steinman – “the dendritic cell and its role in adaptive immunity”.

Renato L. Baserga, M.D.

Kimmel Cancer Center and Keck School of Medicine

Dr. Baserga’s research focuses on the multiple roles of the type 1 insulin-like growth factor receptor (IGF-IR) in the proliferation of mammalian cells. The IGF-IR activated by its ligands is mitogenic, is required for the establishment and the maintenance of the transformed phenotype, and protects tumor cells from apoptosis. It, therefore, serves as an excellent target for therapeutic interventions aimed at inhibiting abnormal growth. In basic investigations, this group is presently studying the effects that the number of IGF-IRs and specific mutations in the receptor itself have on its ability to protect cells from apoptosis.

This investigation is strictly correlated with IGF-IR signaling, and part of this work tries to elucidate the pathways originating from the IGF-IR that are important for its functional effects. Baserga’s group has recently discovered a new signaling pathway used by the IGF-IR to protect cells from apoptosis, a unique pathway that is not used by other growth factor receptors. This pathway depends on the integrity of serines 1280-1283 in the C-terminus of the receptor, which bind 14.3.3 and cause the mitochondrial translocation of Raf-1.

Another recent discovery of this group has been the identification of a mechanism by which the IGF-IR can actually induce differentiation in certain types of cells. When cells have IRS-1 (a major substrate of the IGF-IR), the IGF-IR sends a proliferative signal; in the absence of IRS-1, the receptor induces cell differentiation. The extinction of IRS-1 expression is usually achieved by DNA methylation.

Janardan Reddy, MD

Northwestern University

The central effort of our research has been on a detailed analysis at the cellular and molecular levels of the pleiotropic responses in liver induced by structurally diverse classes of chemicals that include fibrate class of hypolipidemic drugs, and phthalate ester plasticizers, which we designated hepatic peroxisome proliferators. Our work has been central to the establishment of several principles, namely that hepatic peroxisome proliferation is associated with increases in fatty acid oxidation systems in liver, and that peroxisome proliferators, as a class, are novel nongenotoxic hepatocarcinogens.

We introduced the concept that sustained generation of reactive oxygen species leads to oxidative stress and serves as the basis for peroxisome proliferator-induced liver cancer development. Furthermore, based on the tissue/cell specificity of pleiotropic responses and the coordinated transcriptional regulation of fatty acid oxidation system genes, we postulated that peroxisome proliferators exert their action by a receptor-mediated mechanism. This receptor concept laid the foundation for the discovery of

  • a three member peroxisome proliferator-activated receptor (PPARalpha-, ß-, and gamma) subfamily of nuclear receptors.
  •  PPARalpha is responsible for peroxisome proliferator-induced pleiotropic responses, including
    • hepatocarcinogenesis and energy combustion as it serves as a fatty acid sensor and regulates fatty acid oxidation.

Our current work focuses on the molecular mechanisms responsible for PPAR action and generation of fatty acid oxidation deficient mouse knockout models. Transcription of specific genes by nuclear receptors is a complex process involving the participation of multiprotein complexes composed of transcription coactivators.  

Jose Delgado de Salles Roselino, Ph.D.

Leloir Institute, Brazil

Warburg effect, in reality “Pasteur-effect” was the first example of metabolic regulation described. A decrease in the carbon flux originated at the sugar molecule towards the end metabolic products, ethanol and carbon dioxide that was observed when yeast cells were transferred from anaerobic environmental condition to an aerobic one. In Pasteur´s works, sugar metabolism was measured mainly by the decrease of sugar concentration in the yeast growth media observed after a measured period of time. The decrease of the sugar concentration in the media occurs at great speed in yeast grown in anaerobiosis condition and its speed was greatly reduced by the transfer of the yeast culture to an aerobic condition. This finding was very important for the wine industry of France in Pasteur time, since most of the undesirable outcomes in the industrial use of yeast were perceived when yeasts cells took very long time to create a rather selective anaerobic condition. This selective culture media was led by the carbon dioxide higher levels produced by fast growing yeast cells and by a great alcohol content in the yeast culture media. This finding was required to understand Lavoisier’s results indicating that chemical and biological oxidation of sugars produced the same calorimetric results. This observation requires a control mechanism (metabolic regulation) to avoid burning living cells by fast heat released by the sugar biological oxidative processes (metabolism). In addition, Lavoisier´s results were the first indications that both processes happened inside similar thermodynamics limits.

In much resumed form, these observations indicates the major reasons that led Warburg to test failure in control mechanisms in cancer cells in comparison with the ones observed in normal cells. Biology inside classical thermo dynamics poses some challenges to scientists. For instance, all classical thermodynamics must be measured in reversible thermodynamic conditions. In an isolated system, increase in P (pressure) leads to decrease in V (volume) all this in a condition in which infinitesimal changes in one affects in the same way the other, a continuum response. Not even a quantic amount of energy will stand beyond those parameters. In a reversible system, a decrease in V, under same condition, will led to an increase in P.

In biochemistry, reversible usually indicates a reaction that easily goes from A to B or B to A. This observation confirms the important contribution of E Schrodinger in his What´s Life: “This little book arose from a course of public lectures, delivered by a theoretical physicist to an audience of about four hundred which did not substantially dwindle, though warned at the outset that the subject-matter was a difficult one and that the lectures could not be termed popular, even though the physicist’s most dreaded weapon, mathematical deduction, would hardly be utilized. The reason for this was not that the subject was simple enough to be explained without mathematics, but rather that it was much too involved to be fully accessible to mathematics.”

Hans Krebs describes the cyclic nature of the citrate metabolism. Two major research lines search to understand the mechanism of energy transfer that explains how ADP is converted into ATP. One followed the organic chemistry line of reasoning and therefore, searched how the breakdown of carbon-carbon link could have its energy transferred to ATP synthesis. A major leader of this research line was B. Chance who tried to account for two carbon atoms of acetyl released as carbon dioxide in the series of Krebs cycle reactions. The intermediary could store in a phosphorylated amino acid the energy of carbon-carbon bond breakdown. This activated amino acid could transfer its phosphate group to ADP producing ATP. Alternatively, under the possible influence of the excellent results of Hodgkin and Huxley a second line of research appears.

The work of Hodgkin & Huxley indicated the storage of electrical potential energy in transmembrane ionic asymmetries and presented the explanation for the change from resting to action potential in excitable cells. This second line of research, under the leadership of P Mitchell postulated a mechanism for the transfer of oxide/reductive power of organic molecules oxidation through electron transfer as the key for energetic transfer mechanism required for ATP synthesis. Paul Boyer could present how the energy was transduced by a molecular machine that changes in conformation in a series of 3 steps while rotating in one direction in order to produce ATP and in opposite direction in order to produce ADP plus Pi from ATP (reversibility). Nonetheless, a victorious Peter Mitchell obtained the correct result in the conceptual dispute, over the B. Chance point of view, after he used E. Coli mutants to show H gradients in membrane and its use as energy source.

However, this should not detract from the important work of Chance. B. Chance got the simple and rapid polarographic assay method of oxidative phosphorylation and the idea of control of energy metabolism that bring us back to Pasteur. This second result seems to have been neglected in searching for a single molecular mechanism required for the understanding of the buildup of chemical reserve in our body. In respiring mitochondria the rate of electron transport, and thus the rate of ATP production, is determined primarily by the relative concentrations of ADP, ATP and phosphate in the external media (cytosol) and not by the concentration of respiratory substrate as pyruvate. Therefore, when the yield of ATP is high as is in aerobiosis and the cellular use of ATP is not changed, the oxidation of pyruvate and therefore of glycolysis is quickly (without change in gene expression), throttled down to the resting state. The dependence of respiratory rate on ADP concentration is also seen in intact cells. A muscle at rest and using no ATP has very low respiratory rate.

I have had an ongoing discussion with Jose Eduardo de Salles Roselino, inBrazil. He has made important points that need to be noted.

  1. The constancy of composition which animals maintain in the environment surrounding their cells is one of the dominant features of their physiology. Although this phenomenon, homeostasis, has held the interest of biologists over a long period of time, the elucidation of the molecular basis for complex processes such as temperature control and the maintenance of various substances at constant levels in the blood has not yet been achieved. By comparison, metabolic regulation in microorganisms is much better understood, in part because the microbial physiologist has focused his attention on enzyme-catalyzed reactions and their control, as these are among the few activities of microorganisms amenable to quantitative study. Furthermore, bacteria are characterized by their ability to make rapid and efficient adjustments to extensive variations in most parameters of their environment; therefore, they exhibit a surprising lack of rigid requirements for their environment, and appears to influence it only as an incidental result of their metabolism. Animal cells on the other hand have only a limited capacity for adjustment and therefore require a constant milieu. Maintenance of such constancy appears to be a major goal in their physiology (Regulation of Biosynthetic Pathways H.S. Moyed and H EUmbarger Phys Rev,42 444 (1962)).
  2. A living cell consists in a large part of a concentrated mixture of hundreds of different enzymes, each a highly effective catalyst for one or more chemical reactions involving other components of the cell. The paradox of intense and highly diverse chemical activity on the one hand and strongly poised chemical stability (biological homeostasis) on the other is one of the most challenging problems of biology (Biological feedback Control at the molecular Level D.E. Atkinson Science vol. 150: 851, 1965). Almost nothing is known concerning the actual molecular basis for modulation of an enzyme`s kinetic behavior by interaction with a small molecule. (Biological feedback Control at the molecular Level D.E. Atkinson Science vol. 150: 851, 1965). In the same article, since the core of Atkinson´s thinking seems to be strongly linked with Adenylates as regulatory effectors, the previous phrases seems to indicate a first step towards the conversion of homeostasis to an intracellular phenomenon and therefore, one that contrary to Umbarger´s consideration could be also studied in microorganisms.
  3.  Most biochemical studies using bacteria, were made before the end of the third upper part of log growth phase. Therefore, they could be considered as time-independent as S Luria presented biochemistry in Life an Unfinished Experiment. The sole ingredient on the missing side of the events that led us into the molecular biology construction was to consider that proteins, a macromolecule, would never be affected by small molecules translational kinetic energy. This, despite the fact that in a catalytic environment and its biological implications S Grisolia incorporated A K Balls observation indicating that the word proteins could be related to Proteus an old sea god that changed its form whenever he was subjected to inquiry (Phys Rev v 4,657 (1964).
  1. In D.E. Atkinson´s work (Science vol 150 p 851, 1965), changes in protein synthesis acting together with factors that interfere with enzyme activity will lead to “fine-tuned” regulation better than enzymatic activity regulation alone. Comparison of glycemic regulation in granivorous and carnivorous birds indicate that when no important nutritional source of glucose is available, glycemic levels can be kept constant in fasted and fed birds. The same was found in rats and cats fed on high protein diets. Gluconeogenesis is controlled by pyruvate kinase inhibition. Therefore, the fact that it can discriminate between fasting alone and fasting plus exercise (carbachol) requirement of gluconeogenic activity (correspondent level of pyruvate kinase inhibition) the control of enzyme activity can be made fast and efficient without need for changes in genetic expression (20 minute after stimulus) ( Migliorini,R.H. et al Am J. Physiol.257 (Endocrinol. Met. 20): E486, 1989). Regrettably, this was not discussed in the quoted work. So, when the control is not affected by the absorption of nutritional glucose it can be very fast, less energy intensive and very sensitive mechanism of control despite its action being made in the extracellular medium (homeostasis).

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 Curator: Ritu Saxena, Ph.D.

Vitamin C or Ascorbic acid (AA) or Ascorbate

Biochemical role: AA serves a basic biochemical role of accelerating hydroxylation in several biochemical reactions. It provides electrons to metal ions, the reduced forms of which are required for the full enzymatic activity of some enzymes. Most emphasized role of AA is as a cofactor for the enzyme required for the biosynthesis of collagen.

Molecular structure and the oxidized form of AA, dihydroascorbic acid, bear similarity to that of glucose.

Biological role: AA is an essential vitamin for humans and its deficiency leads to disease called Scurvy characterized by initial symptoms of malaise and lethargy, followed by formation of spots on the skin, spongy gums, and bleeding from the mucous membranes. As scurvy advances, there can be open, suppurating wounds, loss of teeth, jaundice, fever, neuropathy and death. AA is water soluble and found in high concentrations in several tissues including eye lens, WBCs, adrenal glad and pituitary gland. Some of the roles of ascorbate include:

  1. Carnitine synthesis from lysine
  2. Neurotransmitter synthesis,
  3. Cytochrome P-450 activity,
  4. Cholesterol metabolism,
  5. Detoxification of exogenous compounds,
  6. Antioxidant
  7. Possibly an ergogenic aid (Ergogenic aids are substances, devices, or practices that enhance an individual’s energy use, production, or recovery.)

Vitamin C and Cancer

As early as in 1949, vitamin C was implicated in cancer therapy. Since then, several research articles have been published exploring the role of ascorbate in cancer therapy. Among the plethora of literature discussing the relationship between vitamin C and cancer, one of the very significant and comprehensive reviews was published in 1979 in Cancer Research (2).

Mechanisms of action of AA (1) with respect to cancer have been divided and subdivided into the following:

  1. Primary mechanisms
  2. Secondary mechanisms
  • Preventive mechanism

Ascorbate acts as a cancer preventive agent by virtue of its strong antioxidant activities. Being one of the strongest reductants and radical scavenger, it absorbs unstable oxygen, nitrogen, and sulphur-centered radicals. AA can prevent biomembranes from peroxidative damage from peroxyl radicals. Ascorbate can trap peroxyl radicals and lead to their peroxidation in the aqueous phase before they reach the lipid rich biomembranes and cause damage. Ascorbate has been speculated to have a biomembrane protective action by its synergistic antioxidant activity with vitamin E (tocopherol).  Vitamin E is lipid-soluble and tocopheroxyl radical is generated in the cell membranes as a result of its antioxidant activity.  Ascorbate reacts with the tocopheroxyl radical and regenerates tocopherol transferring the oxidative challenge to the aqueous phase. At this point, the less active ascorbate radical might be reduced to AA by an NADP-dependent system. The probably mechanism might explain the reduction of nitrates via ascorbate to prevent the formation of carcinogenic nitrosamines.

  • Anticancer mechanisms

1. Primary anticancer mechanisms

i.     Oxidative, oxidant and pro-oxidant properties: Ascorbate has been reported to be cytotoxic at high concentrations, which has been demonstrated in a number of malignant cell lines. Transcription factor NFkB is potentially activated via ascorbate and its radicals leading to the inhibition of cell growth. Also, ascorbate inhibits certain prostaglandins leading to decrease in cell proliferation.

ii.     Hydrogen peroxide: On oxidation with oxygen, ascorbate produces a hydrogen peroxide, a reactive oxygen species. Hydrogen peroxide can generate several other reactive species and can have several damaging effects on cells including decrease in cell viability by damaging cell membranes of malignant cells. The amount of these reactive species produced via oxidation is limited in healthy cells unlike that in malignant cells where they exist in large amounts. The amount of hydrogen peroxide generated has been correlated to the amount of ascorbate in the cells. The reactive species can lead to multiple negative effects on cells including DNA strand breaks, lipid peroxidation leading to membrane function disruption, cellular ATP depletion.

Authors state that “the failure to maintain high ATP production may be a consequence of oxidative inactivation of key enzymes especially those related to the Krebs cycle and the electron transport chain.” This might result in alteration of transmembrane potential and distortion of mitochondrial function, suggestive of the important role of mitochondria in the process of carcinogenesis. In this paper, vitamin C has been correlated with cancer with the involvement of altered mitochondrial function. In addition, ascorbate has been detected in mitochondria where it is also regenerated. Different aspects of mitochondrial involvement in cancer have been discussed in several posts published earlier (3-8).

iii.     Other oxidation products of AA: Other oxidation products of AA include 2,3-diketoglutonic acid, and 5-methyl 1-3, 4-dehydrotetrone and other degradation products, have demonstrated antitumor activity. Additionally, some degradation and oxidation products of AA, gamma-cronolactone and 3-hydroxyl-2-pyrone, have been found to inhibit tumor growth. The mechanism of their antitumor actions is complex and might involve multitude of steps, including generation of reactive oxygen species, lipid peroxidation, inducing structural changes in important cellular proteins, inhibition of mitosis and so on.

iv.     Intracellular transport of ascorbate and its tumor specificity: Oxidized ascorbate, dihydroascorbic acid, is transported intracellularly where it is reduced back to ascorbate. Owing to its structural similarity with glucose, dihydroascorbic transport is facilitated via glucose transporters (GLUTs). Ascrobate in its reduced form is transported through a sodium-dependent cotransporter in some cells. Tumor cells require large amounts of glucose, which leads to an increase in the number of GLUTs, hence, resulting in an increase in ascorbate concentration within cancer cells. Because of this selective increased uptake of ascorbate and its cytotoxic effects in cancer cells (generation of hydrogen peroxide, DNA damage, other cytotoxic effects), AA has become a selective, nontoxic chemotherapeutic agent. The difference in the levels of catalase enzyme has been found to lead to intracellular tumor selectivity in cancer cells.

Ascorbate induced cytotoxicity in cancer cells involves its final electron acceptor, oxygen, which interferes with the anaerobic respiration within malignant cells. This gives an important clue for the involvement of mitochondria in malignant cells.

v.     Intravenous AA: High concentrations of AA in plasma (>200mg/dL) have been found to be cytotoxic to cancer cells. Clinically high plasma concentrations of AA can be achieved by its intravenous administration. It was observed that 60g infusion of AA given to cancer patients for 60 minutes followed by 20g given over the next 60 minutes resulted in a 240 minutes high plasma AA concentration of >400mg/dL, that is known to be cytotoxic.

Lipoic acid when administered with AA, is able to reduce the high-dose requirement of AA for its cytotoxic activity reducing it from 700mg/dL to 120mg/dL. Lipoic acid can recycle vitamin C, mediate the reduction of dihydroascorbic acid and improves mitochondrial function. Thus, energy intermediates such as coenzyme Q, vitamin K3, B-complex vitamins, alpha-ketoglutarate aspartate, magnesium might aid in cancer therapy by intercting with ascorbate, directly or indirectly, thereby stimuating/interacting/correcting aerobic mitochondrial respiration.

Hence, the pro-oxidant activity of vitamin C is being referred to as the primary mechanism of anticancer action.

2. Secondary anticancer mechanisms

i.     AA and intracellular matrix: Collagen is an important constituent of the matrix and its concentration determines the strength of the tissue along with its resistance to the infiltration of malignant cancer cells. In Scurvy, a disease resulting from a chronic deficiency of vitamin C, there is generalized tissue disintegration, dissolution of intercellular ground substance and the disruption of collagen bundles. This disintegration leads to ulceration; bacterial colonization and general undifferentiated cellular proliferation with specialized cells reverting back to their primitive form, very much like cancer.  Lack of ascorbate causes a reduction in the hydroxylation of prolyl and lysyl residues into hydroxyproline and hydroxylysine, leading to instability of the collagen triple helix, a common feature in scurvy and also in cancer. Thus, a secondary mechanism of ascorbic acid anticancer mechanism would be to repair these sites, which is emphasized by its role in wound healing, including surgical recovery and other traumatic injuries.

ii.     Ascorbate and immunocompetence: Ascorbate plays several roles for the efficient functioning of immune system in ways that are invoved in both humoral and cell-mediated.  Ascorbate provides humoral immunocompetence as it is essential for immunoglobulin synthesis. In addition, lymphocytes, seminal cells involved in cell-mediated immunity have been found to contain high concentrations of ascorbate. Other immune system roles include, aid in active phagocytosis and enhancing of interferon production.

Classical vitamin C and Cancer controversy-A possible explanation

Conflicting results were obtained from the studies performed by Pauling (Pauling Institute) and Cameron (Mayo Clinic) with vitamin C and its effect on cancer, the issue was debated a few decades ago. Both the studies, however, used oral doses of ascorbate (10g). Gonzalez et al, authors of the review on which the post is based, analyzed and expressed their views on the controversy. They state that the plasma concentration cannot be replicated when the dose is given orally as opposed to when the dose is given intravenously. According to their research, when AA is administered intravenously, higher plasma levels of ascorbate are achieved that could be retained for longer time periods. Also, the authors advocate the use of substantially higher doses (25-200g) to be given intravenously for selective toxicity towards cancer cells.

Modern vitamin C and Cancer controversy-Chemotherapy and radiation

A recent concern regarding the antioxidants like vitamin C is that they might reduce the effectiveness of chemotherapy and radiation by reducing the potency of free radicals necessary for killing cells. A publication by Agus et al (13) has a major role to play in this misconception. The authors describe how cancer cells acquire and concentrate vitamin C providing malignant cells with metabolic advantage. However, details or explanations regarding the theory are missing. Some studies, on the other hand, explain that high concentrations of AA in cancer cells is cytotoxic and is achieved because of similarity in structure between AA and glucose. Cancer cells uptake AA derivative, dehydroascorbic acid via glucose transporters (GLUTs).

In a case report published in PNAS in 1985 (12), two patients with ovarian cancer stage IIIC were found to respond positively to chemotherapy along with high-dose of antioxidants. Antioxidant, AA was administered intravenously to maintain a high plasma dose of 200 mg/dL. The two patients didn’t show disease recurrence after three years of chemotherapy and vitamin C administration. Vast literature exists on the topic indicating that antioxidants, including ascorbate, provide beneficial effects in several cancers without reducing the efficacy of chemotherapy or radiation during treatment of these cancers. Some data, in fact, suggests increase in effectiveness of chemotherapy when supplemented with antioxidants along with an increase in adverse effects. The topic has been summarized and discussed in a series of articles by Lawson and Brignall (9-11).

REFERENCES

The post is primarily based on the following two review articles:

1. González MJ et al. Orthomolecular oncology review: ascorbic acid and cancer 25 years later.  Integr Cancer Ther. 2005 Mar;4(1):32-44.

2. Cameron E, Pauling L, Leibovitz B. Ascorbic acid and cancer: a review. Cancer Res. 1979 Mar;39(3):663-81.

Other articles  on Mitochondria and Cancer were published on this Open Source Online Scientific Journal

3. Ritu Saxena. Mitochondria and Cancer: An overview of mechanisms

4. Ritusaxena. β Integrin emerges as an important player in mitochondrial dysfunction associated Gastric Cancer.

5. Larry H Bernstein. Mitochondria: Origin from oxygen free environment, role in aerobic glycolysis, metabolic adaptation

6. Ritu Saxena. Mitochondria and Cancer: An overview of mechanisms

7. Larry H Bernstein. Mitochondrial Damage and Repair under Oxidative Stress

8. Larry H Bernstein. What can we expect of tumor therapeutic response?

Research articles:

9. Lamson DW, Brignall MS. Antioxidants and cancer, part 3: quercetin. Altern Med Rev. 2000 Jun;5(3):196-208. Review.

10. Lamson DW, Brignall MS. Antioxidants and cancer therapy II: quick reference guide. Altern Med Rev. 2000 Apr;5(2):152-63.

11. Lamson DW, Brignall MS. Antioxidants in cancer therapy; their actions and interactions with oncologic therapies. Altern Med Rev. 1999 Oct;4(5):304-29.

12. Bensch KG, Fleming JE, Lohman W. The role of ascorbic acid in senile cataracts. Proc Natl Acad Sci USA 1985;82:7193-7196.

13. Agus DB, Vera JG, Golde DW. Stand allocation: a mechanism by which tumors obtain vitamin C. Cancer Res. 1999;59:4555-4558.

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Mitochondrial Damage and Repair under Oxidative Stress

Curator: Larry H Bernstein, MD, FCAP

 


Keywords: Mitochondria, mitochondrial dysfunction, electron transport chain, mtDNA, oxidative stress, oxidation-reduction, NO, DNA repair, lipid peroxidation, thiols, ROS, RNS, sulfur,base excision repair, ferredoxin.
Summary: The mitochondrion is the energy source for aerobic activity of the cell, but it also has regulatory functions that will be discussed. The mitochondrion has been discussed in other posts at this site. It has origins from organisms that emerged from an anaerobic environment, such as the bogs and marshes, and may be related to the chloroplast. The aerobic cell was an advance in evolutionary development, but despite the energetic advantage of using oxygen, the associated toxicity of oxygen abundance required adaptive changes. Most bacteria that reduce nitrate (producing nitrite, nitrous oxide or nitrogen) are called facultative anaerobes use electron acceptors such as ferric ions, sulfate or carbon dioxide which become reduced to ferrous ions, hydrogen sulfide and methane, respectively, during the oxidation of NADH (reduced nicotinamide adenine dinucleotide is a major electron carrier in the oxidation of fuel molecules).

The underlying problem we are left with is oxidation-reduction reactions that are necessary for catabolic and synthetic reactions, and that cumulatively damage the organism associated with cancer, cardiovascular disease, neurodegerative disease, and inflammatory overload. Aerobic organisms tolerate have evolved mechanisms to repair or remove damaged molecules or to prevent or deactivate the formationof toxic species that lead to oxidative stress and disease. However, the normal balance between production of pro-oxidant species and destruction by the antioxidant defenses is upset in favor of overproduction of the toxic species, which leads to oxidative stress and disease. How this all comes together is the topic of choice.

Schematic diagram of the mitochondrial .

The transformation of energy is central to mitochondrial function. The system of energetics includes:

  • the enzymes of the Kreb’s citric acid or TCA cycle,
  • some of the enzymes involved in fatty acid catabolism (β-oxidation), and
  • the proteins needed to help regulate these systems,

central to mitochondrial physiology through the production of reducing equivalents. Reducing equivalents are also used for anabolic reactions.
Electron Transport Chain
It also houses the protein complexes involved in the electron transport component of oxidative phosphorylation and proteins involved in substrate and ion transport. The chemical energy contained in both fats and amino acids can also be converted into NADH and FADH2 through mitochondrial pathways. The major mechanism for harvesting energy from fats is β-oxidation; the major mechanism for harvesting energy from amino acids and pyruvate is the TCA cycle. Once the chemical energy has been transformed into NADH and FADH2, these compounds are fed into the mitochondrial respiratory chain.
Under physiological conditions, electrons generally enter either through complex I (NADH-mediated, examined in vitro using substrates such as glutamate/malate) or complex II (FADH2-mediated, examined in vitro using succinate).

Electrons are then sequentially passed through a series of electron carriers.

The progressive transfer of electrons (and resultant proton pumping) converts the chemical energy stored in carbohydrates, lipids, and amino acids into potential energy in the form of the proton gradient. The potential energy stored in this gradient is used to phosphorylate ADP forming ATP.
Redox-Cycling

In redox cycling the reductant is continuously regenerated, thereby providing substrate for the “auto-oxidation” reaction.

When partially oxidized compounds are enzymatically reduced, the auto-oxidative generation of superoxide and other ROS to start again. Several enzymes

  •  NADPH-cytochrome P450 reductase,
  • NADPH-cytochrome b5 reductase [EC 1.6.2.2]
  • NADPH-ubiquinone oxidoreductase [EC 1.6.5.3], and
  • xanthine oxidase [EC 1.2.3.2]),

can reduce quinones into semiquinones in a single electron process.

The semiquinone can then reduce dioxygen to superoxide during its oxidation to a quinone.

Redox cycling is thought to play a role in carcinogenesis. The naturally occurring estrogen metabolites (the catecholestrogens) have been implicated in hormone-induced cancer, possibly as a result of their redox cycling and production of ROS. It is thought that diethylstilbestrol causes the production of the mutagenic lesion 8-hydroxy-2’deoxyguanosine. It can also cause DNA strand breakage.

Another oxidative reaction that is associated with H2O2 is a significant problem for living organisms as a consequence of the reaction between hydrogen peroxide and oxidizable metals, the Fenton reaction [originally described in the oxidation of an α-hydroxy acid to an α-keto acid in the presence of hydrogen peroxide (or hypochlorite) and low levels of iron salts (Fenton (1876, 1894)).
Chemical Reactions and Biological Significance

The hydroxyl free radical is so aggressive that it will react within 5 (or so) molecular diameters from its site of production. The damage caused by it, therefore, is very site specific. Biological defenses have evolved that reduce the chance that the hydroxyl free radical will be produced to repair damage. An antioxidant would have to occur at the site of hydroxyl free radical production and be at sufficient concentration to be effective.

Some endogenous markers have been proposed as a useful measures of total “oxidative stress” e.g., 8-hydroxy-2’deoxyguanosine in urine. The ideal scavenger

  • must be non-toxic,
  • have limited or no biological activity,
  • readily reach the site of hydroxyl free radical production,
  • react rapidly with the free radical, be specific for this radical, and
  • neither the scavenger nor its product(s) should undergo further metabolism.

Unlike oxygen, nitrogen does not possess unpaired electrons and is therefore considered diamagnetic. Nitrogen does not possess available d orbitals so it is limited to a valency of 3. In the presence of oxygen, nitrogen can produce Nitric oxide which occurs physiologically with the immune system which, when activated, can produce large quantities of nitric oxide.

Nitric oxide is produced by stepwise oxidation of L-arginine catalyzed by nitric oxide synthase (NOS). Nitric oxide is formed from the guanidino nitrogen of the L-arginine in a reaction that

  • consumes five electrons and
  • requires flavin adenine dinucleotide (FAD),
  • flavin mononucleotide (FMN) tetrahydrobiopterin (BH4), and
  • iron protoporphyrin IX as cofactors.

The primary product of NOS activity may be the nitroxyl anion that is then converted to nitric oxide by electron acceptors.

NOS cDNAs show homology with the cytochrome P450 reductase family. Based on molecular genetics there appears to be at least three distinct forms of NOS:

  • A Ca2+/calmodulin-requiring constitutive enzyme (c-NOS; ncNOS or type I)
  • A calcium-independent inducible enzyme (i-NOS; type II), which is primarily involved in the mediation of the cellular immune response; and
  • A second Ca2+/calmodulin-requiring constitutive enzyme found in aortic and umbilical endothelia (ec-NOS or type III)

This has been discussed extensively in this series of posts. Recently, a mitochondrial form of the enzyme, which appears to be similar to the endothelial form, has been found in brain and liver tissue. Although the exact role of nitric oxide in the mitochondrion remains elusive, it may play a role in the regulation of cytochrome oxidase.
Nitric Oxide
Nitric oxide appears to regulate its own production through a negative feedback loop. The binding of nitric oxide to the heme prosthetic group of NOS inhibits this enzyme, and c-NOS and ec-NOS are much more sensitive to this regulation than i-NOS. It appears that in the brain, NO can regulate its own synthesis and therefore the neurotransmission process.

  • On the one hand, inhibition of ec-NOS will prevent the cytotoxicity associated with excessive nitric oxide production.
  • On the other, the insensitivity of i-NOS to nitric oxide will enable high levels of nitric oxide to be produced for cytotoxic effects.

Endogenous inhibitors of NOS (guanidino-substituted derivatives of arginine) occur in vivo as a result of post-translational modification of protein contained arginine residues by S-adenosylmethionine. The dimethylarginines (NG,NG-dimethyl-L-arginine and NG,N’G-dimethyl-L-arginine) occurs in tissue proteins, plasma, and urine of humans and they are thought to act as both regulators of NOS activity and reservoirs of arginine for the synthesis of nitric oxide.
It has been calculated that even though membrane makes up about 3% of the total tissue volume, 90% of the reaction of nitric oxide with oxygen occurs within this compartment. Thus the membrane is an important site for nitric oxide chemistry.
There are two major aspects to nitric oxide chemistry.

  • It can undergo single electron oxidation and reduction reactions producing nitrosonium and nitroxyl
  • Having a single unpaired electron in its π*2p molecular orbital it will react readily with other molecules that also have unpaired electrons, such as free radicals and transition metals.

Examples of the reaction of nitric oxide with radical species include:

  • Nitric oxide will react with oxygen to form the peroxynitrite (nitrosyldioxyl) radical (ONO2)
  • and with superoxide to form the powerful oxidizing and nitrating agent, peroxynitrite anion (ONO2-). Peroxynitrite causes damage to many important biomolecules

Importance:

  • nitrosothiols that are important in the regulation of blood pressure terminates lipid peroxidation
  • 3-nitrosotyrosine and/or 4-O-nitrosotyrosine can affect the activity of enzymes that utilize tyrosyl radicals
  • rapidly reacts with oxyhemoglobin, the primary route of its destruction in vivo
  • the reaction between nitric oxide and transition metal complexes

During the last reaction a “ligand” bond is formed (the unpaired electron of nitric oxide is partially transferred to the metal cation),

 resulting in a nitrosated (nitrosylated) complex.

For example, such complexes can be formed with free iron ions,

iron bound to heme or iron located in iron-sulfur clusters.

Ligand formation allows nitric oxide to act as a signal, activating some enzymes while inhibiting others. Thus, the binding of nitric oxide to the Fe (II)-heme of guanylate (guanalyl) cyclase [GTP-pyrophosphate lyase: cyclizing] is the signal transduction mechanism. Guanylate cyclase exists as cytosolic and membrane-bound isozymes.
Thiol-Didulfide Redox Couple

The thiol-disulfide redox couple is very important to oxidative metabolism. For example, GSH is a reducing cofactor for glutathione peroxidase, an antioxidant enzyme responsible for the destruction of hydrogen peroxide.

The importance of the antioxidant role of the thiol-disulfide redox couple:

Thiols and disulfides can readily undergo exchange reactions, forming mixed disulfides. Thiol-disulfide exchange is biologically very important. For example,

  • GSH can react with protein cystine groups and influence the correct folding of proteins.
  • GSH may also play a direct role in cellular signaling through thiol-disulfide exchange reactions with membrane bound receptor proteins
  •                        the insulin receptor complex)
  •                        transcription factors (e.g., nuclear factor κB)
  •                        and regulatory proteins in cells

Conditions that alter the redox status of the cell can have important consequences on cellular function.

The generation of ROS by redox cycling is only one possible explanation for the action of many drugs. Rifamycin not only owes its activity to ROS generation but also to its ability to block bacterial RNA synthesis as well. Quinones (and/or semiquinones) can also form adducts with nucleophiles, especially thiols. These adducts may act as toxins directly or indirectly through the inhibition of key enzymes (e.g., by reacting with essential cysteinyl residues) or the depletion of GSH.
DNA Adduct Formation

By far the most intense research in this field has been directed towards the chemistry and biology of DNA adduct formation. Attack of the free bases and nucleosides by pro-oxidants can yield a wide variety of adducts and DNA-protein cross-links. Such attack usually occurs

  • at the C-4 and C-8 position of purines and
  • C-5 and C-6 of pyrimidines.

Hydroxyl free radical-induced damage to purine bases and nucleosides can proceed through a C-8-hydroxy N-7 radical intermediate, and then either undergo oxidation with the production of an 8-hydroxy purine, or reduction, probably by cellular thiols, followed by ring opening and the formation of FAPy (formamido-pyrimidine) metabolites (hydroxyl free radical-induced damage to guanosine). Although most research has focused on 8-hydroxy-purine adducts a growing number of publications are attempting to measure the FAPy derivative.

Nitrosation of the Amines of the Nucleic Acid Bases.

Primary aromatic amines produce deaminated products, while secondary amines form N-nitroso compounds.
Formation of Peroxynitrite from Nitric Oxide.

Peroxynitrite shows complex reactivity

  • with DNA initiating DNA strand breakage, oxidation (e.g., formation of 8-hydroxyguanine, 8-OH2’dG, (5-hydroxymethyl)-uracil, and FAPyGua),
  • nitration (e.g., 8-nitroguanine), and
  • deamination of bases.

Peroxynitrite can also promote the production of lipid peroxidation related active carbonyls and cause the activation of NAD+ ADP-ribosyltransferase.

Modification of Guanine
Although all DNA bases can be oxidatively damaged, it is the modification of guanine that is the most frequent. 8OH2’dG is the most abundant DNA adduct. This can affect its hydrogen bonding between base-pairs. These base-pair substitutions are usually found clustered into areas called “hot spots”. Guanine normally binds to cytosine.

8OH2’dG, however, can form hydrogen bonds with adenine. The formation of 8OH2’dG in DNA can therefore result in a G→T transversion.

8-Hydroxyguanine was also shown to induce codon 12 activation of c-Ha-ras and K-ras in mammalian systems. G→T transversions are also the most frequent hot spot mutations found in the p53 supressor gene which is associated with human tumors.

Other mechanisms by which ROS/RNS can lead to mutations have been
proposed. Direct mechanisms include:

  • conformational changes in the DNA template that reduces the accuracy of replication by DNA polymerases
  • altered methylation of cytosine that affects gene control

Indirect mechanisms include:

  • Oxidative damage to proteins, including DNA polymerases and repair enzymes.
  • Damage to lipids causes the production of mutagenic carbonyl compounds
  • Misalignment mutagenesis (“slippery DNA”)
DNA Mismatch Repair 5

DNA Mismatch Repair 5 (Photo credit: Allen Gathman)

Repair of ROS/RNS-induced DNA Damage
The repair of damaged DNA is an ongoing and continuous process involving a
number of repair enzymes. Damaged DNA appears to be mended by two major mechanisms:

  1. base excision repair (BER) and
  2. nucleotide excision repair (NER)

Isolated DNA is found to contain low levels of damaged bases, so it appears that these repair processes are not completely effective.
Base Excision Repair

BER is first started by DNA glycosylases which recognize specific base
modifications (e.g., 8OH2’dG). For example,

  • Formamido-pyrimidine-DNA glycosylase (Fpg protein) recognizes damaged purines such as 8-oxoguanine and FAPyGua.
  • Damaged pyrimidines are recognized by endonuclease III, which acts as both a glycosylase and AP endonuclease.
  • Glycosylases cleave the N-glycosylic bond between the damaged base and the sugar

Following the glycosylase step, AP endonucleases then remove the 3′-deoxyribose moiety by cleavage of the phosphodiester bonds thereby generating a 3’-hydroxyl group that can then be extended by DNA polymerase.

The final step in mending damaged DNA is the rejoining of the free ends of DNA by a DNA ligase. It also appears that the presence of 8-oxoguanine modified bases in DNA is not only a result of ROS attack on this macromolecule. Oxidized nucleosides and nucleotides from free cellular pools can also be incorporated into DNA by polymerases and cause AT to CG base substitution mutations.

Mitochondrial DNA Repair

The mitochondrion genome encodes the various complexes of the electron transport chain, but contains no genetic information for DNA repair enzymes. These enzymes must be obtained from the nucleus. As mitochondria are continuously producing DNA damaging pro-oxidant species, effective DNA repair mechanisms must exist within the mitochondrial matrix in order for these organelles to function. Mitochondria have a short existence, and excessively damaged mitochondria will be quickly removed. Mitochondria contain many BER enzymes and are proficient at repair, but they do not appear to repair damaged DNA by NER mechanisms.

Single Strand DNA Damage and PARP Activation

Single strand DNA breakage activates NAD+ ADP-ribosyltransferase (PARP). PARP is a protein-modifying, nucleotide-polymerizing enzyme and is found at high levels in the nucleus. Activated PARP

  1. cleaves NAD+ into ADP-ribose and nicotinamide
  2. then attaches the ADP-ribose units to a variety of nuclear proteins (including histones and its own automodification domain).
  3. then polymerizes the initial ADP-ribose modification with other ADP-ribose units to form the nucleic acid-like polymer, poly (ADP) ribose.

PARP only appears to be involved with BER and not NER. In BER PARP does not appear to play a direct role but rather it probably helps by keeping the chromatin in a conformation that enables other repair enzymes to be effective. It may also provide temporary protection to DNA molecules while it is being repaired. Conflicting evidence suggests that PARP may not be an important DNA repair enzyme as cells from a PARP knockout mouse model have normal repair characteristics.

Activation of PARP can be dangerous to the cell. For each mole of ADP-ribose transferred, one mole of NAD+ is consumed, and through the regeneration of NAD+ four ATP molecules are wasted. Thus the activation of PARP can rapidly deplete a cell’s energy store and even lead to cell death. Some researchers suggest that this may be one mechanism whereby cells with excessive DNA damage are effectively removed. However, a variety of diseases may involve PARP overactivation including

  • circulatory shock,
  • CNS injury,
  • diabetes,
  • drug-induced cytotoxicity, and
  • inflammation.

The Indirect Pathway.
This (mutation) pathway does not involve oxidative damage to the protein per se. This process involves oxidative damage to the DNA molecule encoding the protein. Thus pro-oxidants can cause changes in the base sequence of the DNA molecule. If such base modification is in a coding region of DNA (exon) and not corrected, the DNA molecule may be transcribed incorrectly. Translation of the mutant mRNA can result in a mutant protein containing a wrong amino acid in its primary sequence. If this modified amino acid occurs in an essential part of the protein (e.g., the active site of an enzyme or a portion that alters folding), the function of that protein may be impaired. Fortunately, unlike modified DNA
that can pass from cell to cell during mitosis thereby continuing the production of mutant protein, damage to a protein is non-replicating and stops with its destruction.

The Direct Pathway

This (post-translational) pathway involves the action of a pro-oxidant on a protein resulting in

  • modification of amino acid residues,
  • the formation of carbonyl adducts,
  • cross-linking and
  • polypeptide chain fragmentation.

Such changes often result in altered protein conformation and/or activity. Proteins will produce a variety of carbonyl products when exposed to metal-based systems (metal/ascorbate and metal/hydrogen peroxide) in vitro. For example, histidine yields aspartate, asparagine and 2-oxoimidazoline, while proline produces glutamate, pyroglutamate, 4-hydroxyproline isomers, 2-pyrrolidone and γ-aminobutyric acid. Metal-based systems and other pro-oxidant conditions can oxidize methionine to its sulfoxide.

This portion of the presentation is endebted to THE HANDBOOK OF REDOX
BIOCHEMISTRY, Ian N. Acworth, August 2003, esa. (inacworth@esainc.com).
We shall now identify more recent work related to this presentation.

Oxygen and Oxidative Stress

The reduction of oxygen to water proceeds via one electron at a time. In the mitochondrial respiratory chain, Complex IV (cytochrome oxidase) retains all partially reduced intermediates until full reduction is achieved. Other redox centres in the electron transport chain, however, may leak electrons to oxygen, partially reducing this molecule to superoxide anion (O2_•). Even though O2_• is not a strong oxidant, it is a precursor of most other reactive oxygen species, and it also becomes involved in the propagation of oxidative chain reactions. Despite the presence of various antioxidant defences, the mitochondrion appears to be the main intracellular source of these oxidants. This review describes the main mitochondrial sources of reactive species and the antioxidant defences that evolved to prevent oxidative damage in all the mitochondrial compartments.

Reactive oxygen species (ROS) is a phrase used to describe a variety of molecules and free radicals (chemical species with one unpaired electron) derived from molecular oxygen. Molecular oxygen in the ground state is a bi-radical, containing two unpaired electrons in the outer shell (also known as a triplet state).

Since the two single electrons have the same spin, oxygen can only react with one electron at a time and therefore it is not very reactive with the two electrons in a chemical bond.

On the other hand, if one of the two unpaired electrons is excited and changes its spin, the resulting species (known as singlet oxygen) becomes a powerful oxidant as the two electrons with opposing spins can quickly react with other pairs of electrons, especially double bonds.

The formation of OH• is catalysed by reduced transition metals, which in turn may be re-reduced by O2 -•, propagating this process. In addition, O2-• may react with other radicals including nitric oxide (NO•) in a reaction controlled by the rate of diffusion of both radicals. The product, peroxynitrite, is also a very powerful oxidant. The oxidants derived from NO• have been recently called reactive nitrogen species (RNS).

‘Oxidative stress’ is an expression used to describe various deleterious processes resulting from an imbalance between the excessive formation of ROS and/or RNS and limited antioxidant defences.

  • Whilst small fluctuations in the steady-state concentration of these oxidants may actually play a role in intracellular signalling,
  • uncontrolled increases in the steady-state concentrations of these oxidants lead to free radical mediated chain reactions

which indiscriminately target

  • proteins,
  • lipids,
  • polysaccharides.

In vivo, O2-• is produced both enzymatically and nonenzymatically.

Enzymatic sources include

  • NADPH oxidases located on the cell membrane of
  • polymorphonuclear cells,
  • macrophages and
  • endothelial cells and
  • cytochrome P450-dependent oxygenases.

The proteolytic conversion of xanthine dehydrogenase to xanthine oxidase provides another enzymatic source of both O2 -• and H2O2 (and therefore constitutes a source of OH•) and has been proposed to mediate deleterious processes in vivo.

Given the highly reducing intramitochondrial environment, various respiratory components, including flavoproteins, iron–sulfur clusters and ubisemiquinone, are thermodynamically capable of transferring one electron to oxygen. Moreover, most steps in the respiratory chain involve single-electron reactions, further favouring the monovalent reduction of oxygen. On the other hand, the mitochondrion possesses various antioxidant defences designed to eliminate both O2- • and H2O2.

The rate of O2 -• formation by the respiratory chain is controlled primarily by mass action, increasing both when electron flow slows down (increasing the concentration of electron donors, R•) and when the concentration of oxygen increases (eqn (1); Turrens et al. 1982).

d[O2]/dt = k [O2] [R•].

The energy released as electrons flow through the respiratory chain is converted into a H+ gradient through the inner mitochondrial membrane (Mitchell, 1977). This gradient, in turn, dissipates through the ATP synthase complex (Complex V) and is responsible for the turning of a rotor-like protein complex required for ATP synthesis. In the absence of ADP,

  • the movement of H+ through ATP synthase ceases and
  • the H+ gradient builds up
  • causing electron flow to slow down and
  • the respiratory chain to become more reduced (State IV respiration).

Mitochondrial Antioxidant Defences

The deleterious effects resulting from the formation of ROS in the mitochondrion are, to a large extent, prevented by various antioxidant systems. Superoxide is enzymatically converted to H2O2 by a family of metalloenzymes called superoxide dismutases (SOD). Since O2-• may either reduce transition metals, which in turn can react with H2O2 producing OH• or spontaneously react with NO• to produce peroxynitrite, it is important to maintain the steady-state concentration of O2-• at the lowest possible level. Thus, although the dismutation of O2-• to H2O2 and O2 can also occur spontaneously, the role of SODs is to increase the rate of the reaction to that of a diffusion-controlled process.

The mitochondrial matrix contains a specific form of SOD, with manganese in the active site, which eliminates the O2 -• formed in the matrix or on the inner side of the inner membrane. The expression of MnSOD is further induced by agents that cause oxidative stress, including radiation and hyperoxia, in a process mediated by the oxidative activation of the nuclear transcription factor NFkB .

Turrens JF. Mitochondrial formation of reactive oxygen species. J Physiol 2003; 552(2): 335–344. DOI: 10.1113/jphysiol.2003.049478. http://www.jphysiol.org

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Reactive Oxygen Species and Control of Apoptosis

Reactive oxygen species (ROS) are products of normal metabolism and xenobiotic exposure, and depending on their concentration, ROS can be beneficial or harmful to cells and tissues.

  • At physiological low levels, ROS function as “redox messengers” in intracellular signaling and regulation, whereas
  • excess ROS induce oxidative modification of cellular macromolecules, inhibit protein function, and promote cell death.

Additionally, various redox systems, such as

  • the glutathione,
  • thioredoxin, and
  • pyridine nucleotide redox couples,
  • NADPH and antioxidant defense
  • NAD+ and the function of sirtuin proteins

participate in cell signaling and modulation of cell function, including apoptotic cell death. Cell apoptosis is initiated by extracellular and intracellular signals via two main pathways,

  1. the death receptor and
  2. the mitochondria-mediated pathways.

ROS and JNK-mediated apoptotic signaling

              GSH redox status and apoptotic signaling

Various pathologies can result from oxidative stress-induced apoptotic signaling that is consequent to

  • ROS increases and/or antioxidant decreases,
  • disruption of intracellular redox homeostasis, and
  • irreversible oxidative modifications of lipid, protein, or DNA.

We focus on several key aspects of ROS and redox mechanisms in apoptotic signaling and highlight the gaps in knowledge and potential avenues for further investigation. A full understanding of the redox control of apoptotic initiation and execution could underpin the development of therapeutic interventions targeted at oxidative stress-associated disorders.

Circu, M. L.; Aw, T. Y., Reactive oxygen species, cellular redox systems, and apoptosis, Free Radic. Biol. Med. 2010. FRB-10057; pp 14. doi:10.1016/j.freeradbiomed.2009.12.022

Assembly of Iron-sulfur (FeyS) Clusters

Iron-sulfur (FeyS) cluster-containing proteins catalyze a number of electron transfer and metabolic reactions. The components and molecular mechanisms involved in the assembly of the FeyS clusters have been identified only partially. In eukaryotes, mitochondria have been proposed to execute a crucial task in the generation of intramitochondrial and extramitochondrial FeyS proteins. Herein, we identify the essential ferredoxin Yah1p of Saccharomyces cerevisiae mitochondria as a central component of the FeyS protein biosynthesis machinery. Depletion of Yah1p by regulated gene expression resulted in a

30-fold accumulation of iron within mitochondria,

similar to what has been reported for other components involved in FeyS protein biogenesis. Yah1p was shown to be required for the assembly of FeyS proteins both inside mitochondria and in the cytosol. Apparently, at least one of the steps of FeyS cluster biogenesis within mitochondria requires reduction by ferredoxin. Our findings lend support to the idea of a primary function of mitochondria in the biosynthesis of FeyS proteins outside the organelle. To our knowledge, Yah1p is the first member of the ferredoxin family for which a function in FeyS cluster formation has been established. A similar role may be predicted for the bacterial homologs that are encoded within iron-sulfur cluster assembly (isc) operons of prokaryotes.
H Lange, A Kaut, G Kispal, and R Lill. A mitochondrial ferredoxin is essential for biogenesis of cellular iron-sulfur proteins. PNAS 2000; 97(3): 1050–1055.

DNA Charge Transport

Damaged bases in DNA are known to lead to errors in replication and transcription, compromising the integrity of the genome. The authors proposed a model where repair proteins containing redoxactive [4Fe-4S] clusters utilize DNA charge transport (CT) as a first step in finding lesions. In this model, the population of sites to search is reduced by a localization of protein in the vicinity of lesions. Here, we examine this model using single-molecule atomic force microscopy (AFM). XPD, a 5′-3′ helicase involved in nucleotide
excision repair, contains a [4Fe-4S] cluster and exhibits a DNA bound redox potential that is physiologically relevant.

In AFM studies, they observe the redistribution of XPD onto kilobase DNA strands containing a single base mismatch, which is not a specific substrate for XPD but, like a lesion, inhibits CT. They also provide evidence for DNA-mediated signaling between XPD and Endonuclease III (EndoIII), a base excision repair glycosylase that also contains a [4Fe-4S] cluster.

  • When XPD and EndoIII are mixed together, they coordinate in relocalizing onto the mismatched strand.
  • However, when a CT-deficient mutant of either repair protein is combined with the CT-proficient repair partner, no relocalization occurs.

The data presented here indicate that XPD, an archaeal protein from the NER pathway, may cooperate with other proteins that are proficient at DNA CT to localize in the vicinity of damage. XPD, a superfamily 2 DNA helicase with 5′-3′ polarity, is a component of TFIIH that is essential for repair of bulky lesions generated by exogenous sources such as UV light and chemical carcinogens. XPD contains a conserved [4Fe-4S] cluster suggested to be conformationally controlled by ATP binding and hydrolysis.

Mutations in the iron-sulfur domain of XPD can lead to diseases including TTD and XP, yet the function of the [4Fe-4S] cluster appears to be unknown.

Electrochemical studies have shown that when BER proteins MutY and EndoIII bind to DNA, their [4Fe-4S] clusters are activated toward one electron oxidation. XPD exhibits a DNA-bound midpoint potential similar to that of EndoIII and MutY when bound to DNA (approximately 80 mV vs. NHE), indicative of a possible role for the [4Fe-4S] cluster in DNA-mediated CT.

For EndoIII we have also already determined a direct correlation between the ability of proteins to redistribute in the vicinity of mismatches as measured by AFM, and the CT proficiency of the proteins measured electrochemically. Thus, we may utilize single-molecule AFM as a tool to probe the redistribution of proteins in the vicinity of base lesions and in so doing, the proficiency of the protein to carry out DNA CT.

Here we show that, like the BER protein EndoIII, XPD, involved both in transcription and NER, redistributes in the vicinity of a lesion. Importantly, this ability to relocalize is associated with the ability of XPD to carry out DNA CT. The mutant L325V is defective in its ability to carry out DNA CTand this XPD mutant also does not redistribute effectively onto the mismatched strand.

These data not only indicate a general link between the ability of a repair protein to carry out DNA CT and its ability to redistribute onto DNA strands near lesions but also provide evidence for coordinated DNA CT between different repair proteins in their search for damage in the genome. These data also provide evidence that two different repair proteins, each containing a [4Fe-4S] cluster at similar DNA bound potential, can communicate with one another through DNA-mediated CT.

Sontz PA, Mui TP, Fuss JO, Tainer JA, and Barton JK. DNA charge transport as a first step in coordinating the detection of lesions by repair proteins. PNAS 2012; 109(6):1856–1861. doi:10.1073/pnas.1120063109/-/ DCSupplemental. http://www.pnas.org/lookup/suppl/

Janus Bifron 

The signaling function of mitochondria is considered with a special emphasis on their role in the regulation of redox status of the cell, possibly determining a number of pathologies including cancer and aging. The review summarizes the transport role of mitochondria in energy supply to all cellular compartments (mitochondria as an electric cable in the cell), the role of mitochondria in plastic metabolism of the cell including synthesis of

  • heme,
  • steroids,
  • iron-sulfur clusters, and
  • reactive oxygen and nitrogen species.

Mitochondria also play an important role in the Ca2+-signaling and the regulation of apoptotic cell death. Knowledge of mechanisms responsible for apoptotic cell death is important for the strategy for prevention of unwanted degradation of postmitotic cells such as cardiomyocytes and neurons.

In accordance with P. Mitchell’s chemiosmotic concept, vectorial transmembrane transfer of electrons and protons is accompanied by generation of electrochemical difference of proton electrochemical potential on the inner mitochondrial membrane; its utilization by ATP synthase induces conformational rearrangements resulting in ATP synthesis from ADP and inorganic phosphate. Details of the mechanism responsible for ATP synthesis are given elsewhere.

Membrane potential (DY) generated across the inner mitochondrial membrane is the component of the transmembrane electrochemical potential of H+ ions (DμH+), which provides ATP synthesis together with the concentration component (DpH). Maintenance of constant membrane potential is a vitally important precondition for functioning of mitochondria and the cell. Under conditions of limited supply of the cell with oxygen (hypoxia) and inability to carry out aerobic ATP synthesis, mitochondria become ATP consumers (rather than generators) and ATP is hydrolyzed by mitochondrial ATPase, and this is accompanied by generation of membrane potential.

Redox homeostasis, i.e. the sum of redox components (including proteins, low molecular weight redox components such as NAD/NADH, flavins, coenzymes Q, oxidized and reduced substrates, etc.) is one of important preconditions for normal cell functioning.

Single-strand and double-strand DNA damage

Single-strand and double-strand DNA damage (Photo credit: Wikipedia)

Mitochondria generate such potent regulators of redox potential as

  • superoxide anion,
  • hydrogen peroxide,
  • nitric oxide,
  • peroxynitrite, etc.

They are actively involved in regulation of cell redox potential and consequently

  • control proteolysis,
  • activation of transcription,
  • changes in mitochondrial DNA (mDNA),
  • cell metabolism, and
  • cell differentiation.

Zorov DB, Isaev NK, Plotnikov EY, Zorova LD, et al. The Mitochondrion as Janus Bifrons. Biochemistry (Moscow) 2007; 72(10): 1115-1126. ISSN 0006-2979.
DOI: 10.1134/S0006297907100094

Structure of the human mitochondrial genome.

Structure of the human mitochondrial genome. (Photo credit: Wikipedia)

Gene Expression Associated with Oxidoreduction and Mitochondria
The naked mole-rat (Heterocephalus glaber) is a long-lived, cancer resistant rodent and there is a great interest in identifying the adaptations responsible for these and other of its unique traits. We employed RNA sequencing to compare liver gene expression profiles between naked mole-rats and wild-derived mice. Our results indicate that genes associated with oxidoreduction and mitochondria were expressed at higher relative levels in naked mole-rats. The largest effect is nearly

300-fold higher expression of epithelial cell adhesion molecule (Epcam), a tumour-associated protein.

Also of interest are the

  • protease inhibitor, alpha2-macroglobulin (A2m), and the
  • mitochondrial complex II subunit Sdhc,

both ageing-related genes found strongly over-expressed in the naked mole-rat.

These results hint at possible candidates for specifying species differences in ageing and cancer, and in particular suggest complex alterations in mitochondrial and oxidation reduction pathways in the naked mole-rat. Our differential gene expression analysis obviated the need for a reference naked mole-rat genome by employing a combination of Illumina/Solexa and 454 platforms for transcriptome sequencing and assembling transcriptome contigs of the non-sequenced species. Overall, our work provides new research foci and methods for studying the naked mole-rat’s fascinating characteristics.

C Yu, Y Li, A Holmes, K Szafranski, CG Faulkes, et al. RNA Sequencing Reveals Differential Expression of Mitochondrial and Oxidation reduction Genes in the Long-Lived Naked Mole-Rat When Compared to Mice. PLoS ONE 2011; 6(11): 1-9. e26729. http://www.plosone.org

The complete set of viable deletion strains in Saccharomyces cerevisiae was screened for sensitivity of mutants to five oxidants to identify cell functions involved in resistance to oxidative stress. This screen identified a unique set of mainly constitutive functions providing the first line of defense against a particular oxidant; these functions are very dependent on the nature of the oxidant. Most of these functions are distinct from those involved in repair and recovery from damage, which are generally induced in response to stress, because there was little correlation between mutant sensitivity and
the reported transcriptional response to oxidants of the relevant gene. The screen identified 456 mutants sensitive to at least one of five different types of oxidant, and these were ranked in order of sensitivity. Many genes identified were not previously known to have a role in resistance to reactive oxygen species. These encode functions including

  • protein sorting,
  • ergosterol metabolism,
  • autophagy, and
  • vacuolar acidification.

two mutants were sensitive to all oxidants examined,
12 were sensitive to at least four,

Different oxidants had very different spectra of deletants that were sensitive. These findings highlight the specificity of cellular responses to different oxidants:

  • No single oxidant is representative of general oxidative stress.
  • Mitochondrial respiratory functions were overrepresented in mutants sensitive to H2O2, and
  • vacuolar protein-sorting mutants were enriched in mutants sensitive to diamide.

Core functions required for a broad range of oxidative-stress resistance include

  • transcription,
  • protein trafficking, and
  • vacuolar function.

GW Thorpe, CS Fong, N Alic, VJ Higgins, and IW Dawes. Cells have distinct mechanisms to maintain protection against different reactive oxygen species: Oxidative-stress-response genes. PNAS 2004;101: 6564–6569. http://www.pnas.org cgi doi 10.1073 pnas.0305888101
Subcellular Thiol Redox State in Complex I Deficiency

Isolated complex I deficiency is the most common enzymatic defect of the oxidative phosphorylation (OXPHOS) system, causing a wide range of clinical phenotypes. Th authers reported before that the rates at which reactive oxygen species (ROS)-sensitive dyes are converted into their fluorescent oxidation products are markedly increased in cultured skin fibroblasts of patients with nuclear-inherited isolated complex I deficiency.

Using videoimaging microscopy we show here that these cells also display a marked increase in NAD(P)H autofluorescence. Linear regression analysis revealed a negative correlation with the residual complex I activity and a positive correlation with the oxidation rates of the ROS sensitive dyes (5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein and hydroethidine for a large cohort of 10 patient cell lines.

On the other hand, video-imaging microscopy of cells selectively expressing reduction-oxidation sensitive GFP1 in either the mitochondrial matrix or cytosol showed the absence of any detectable change in thiol redox state. In agreement with this result, neither the glutathione nor the glutathione disulfide content differed significantly between patient and healthy fibroblasts.

Finally, video-rate confocal microscopy of cells loaded with C11-BODIPY581/591 demonstrated that the extent of lipid peroxidation, which is regarded as a measure of oxidative damage, was not altered in patient fibroblasts. Our results indicate that fibroblasts of patients with isolated complex I deficiency maintain their thiol redox state despite marked increases in ROS production.

S Verkaart, WJH Koopman, J Cheek, SE van Emst-de Vries. Mitochondrial and cytosolic thiol redox state are not detectably altered in isolated human NADH:ubiquinone oxidoreductase deficiency. Biochimica et Biophysica Acta (BBA) – Molecular Basis of Disease 2007; 1772(9): 1041. DOI : 10.1016/j.bbadis.2007.05.004

  • Mitochodrial mtDNA and Cancer
  • Mitochondrial research has recently been driven by the

identification of mitochondria-associated diseases and 
the role of mitochondria in apoptosis.

Moreover, mitochondria have been implicated in the process of carcinogenesis because of their vital role in

  • energy production,
  • nuclear-cytoplasmic signal integration and
  • control of metabolic pathways.

At some point during neoplastic transformation, there is an increase in reactive oxygen species (ROS), which damage the mitochondrial genome. This accelerates the somatic mutation rate of mitochondrial DNA.

Mitochondrial characteristics

There are several biological characteristics which cast mitochondria and, in particular, the mitochondrial genome, as a biological tool for early detection and monitoring of neoplasia and its potential progression. These vital characteristics are important in cancer research, as not all neoplasias become malignant. Mitochondria are archived in the cytoplasm of the ovum and as such do not recombine.

This genome has an accelerated mutation rate, by comparison with the nucleus, and accrues somatic mutations in tumour tissue. Moreover, mitochondrial DNA (mtDNA) has a high copy number in comparison with the nuclear archive of DNA. There are potentially thousands of mitochondrial genomes per cell, which enables detection of important biomarkers, even at low levels. In addition, mtDNA can be heteroplasmic, which means that disease-associated mutations occur in a subset of the genomes.

The presence of heteroplasmy is an indication of disease and is found in many human tumours. Identification of low levels of heteroplasmy may allow unprecedented early identification and monitoring of neoplastic progression to malignancy.

Coding for just 13 enzyme complex subunits, 22 transfer RNAs and two ribosomal RNAs, the mitochondrial genome is packaged in a compact 16,569 base pair (bp) circular molecule. These products participate in the critical electron transport process of ATP production. Collectively, mitochondria generate 80 per cent of the chemical fuel which fires cellular metabolism.

As a result, nuclear investment in the mitochondria is high — that is, several thousand nuclear genes control this organelle in order to accomplish the complex interactions required to maintain a network of pathways, which coordinate energy demand and supply.

It has been proposed that these mutations may serve as an early indication of potential cancer development and may represent a means for tracking tumour progression.

Does this provide a potential utility in that these mutations may be used for the identification and monitoring of neoplasia and malignant transformation where appropriate body fluids or non-invasive tissue access is available for mtDNA recovery? Specifically discussed are:

  • prostate,
  • breast,
  • colorectal,
  • skin and
  • lung cancers

There are many important questions yet to be addressed: such as

  • the relationship between mtDNA and the actual disease;
  • are mutations causative or merely a reflection of nuclear instability?
  • And, are these processes independent events?

Alterations in the non-coding D-loop suggest genome instability;
however, as studies focus more on the coding regions of the
mitochondrial genome,

Particularly in the case of nonsynonymous mutations in the genes
contributing products to the electron transport process, metabolic
implications are evident. Moreover, mutations in mitochondrial
transfer RNAs indicate the possibility of a global mitochondrial
translational shut down.

RL Parr, GD Dakubo, RE Thayer, K McKenney, MA Birch-Machin. Mitochondrial DNA as a potential tool for early cancer detection. HUMAN GENOMICS 2006; 2(4). 252–257.
Mitochondrial DNA (mtDNA) is particularly prone to oxidation due to the lack of histones and a deficient mismatch repair system. This explains an increased mutation rate of mtDNA that results in heteroplasmy, e.g., the coexistence of the mutant and wild-type mtDNA molecules within the same mitochondrion. Hyperglycemia is a key risk factor not only for diabetes-related disease, but also for cardiovascular and all-cause mortality. One can assume an increase in the risk of cardiovascular disease by 18% for each unit (%) glycated hemoglobin HbA1c. In the Glucose Tolerance in Acute Myocardial Infarction study of patients with acute coronary syndrome, abnormal glucose tolerance was the strongest independent predictor of subsequent cardiovascular complications and death. In the Asian Pacific Study, fasting plasma glucose was shown to be an independent predictor of cardiovascular events up to a level of 5.2 mmol/L.

Glucose level fluctuations and hyperglycemia are triggers for inflammatory responses via increased mitochondrial superoxide production and endoplasmic reticulum stress. Inflammation leads to insulin resistance and β-cell dysfunction, which further aggravates hyperglycemia. The molecular pathways that integrate hyperglycemia, oxidative stress, and diabetic vascular complications have been most clearly described in the pathogenesis of endothelial dysfunction, which is considered as the first step in atherogenesis according to the response to injury hypothesis.

  • In diabetes mellitus,
  • glycotoxicity,
  • advanced oxidative stress,
  • collagen cross-linking, and
  • accumulation of lipid peroxides

in foam macrophage cells and arterial wall cells may significantly

  • decrease the mutation threshold,
  • endothelial dysfunction,
  • promoting atherosclerosis.

Alterations in mitochondrial DNA (mtDNA), known as homoplasmic and heteroplasmic mutations, may influence mitochondrial OXPHOS capacity, and in turn contribute to the magnitude of oxidative stress in micro- and macrovascular networks in diabetic patients.
The authors critically consider the impact of mtDNA mutations on the pathogenesis of cardiovascular diabetic complications.

Mutation Threshhold

Although cells may harbor mutant mtDNA, the expression of disease is dependent on the percent of alleles bearing mutations. Modeling confirms that an upper threshold level might exist for mutations beyond which the mitochondrial population collapses, with a subsequent decrease in ATP. This decrease in ATP results in the phenotypic expression of disease. It is estimated that in many patients with clinical manifestations of mitochondrial disorders, the proportion of mutant DNA exceeds 50%.

For the MELAS (mitochondrial encephalopathy, lactic acidosis and stroke-like syndrome)-causing mutation m.3243 A>G in the mitochondrial gene encoding tRNALeu, which is also associated with diabetes plus deafness, a strong correlation between the level of mutational heteroplasmy and documented disease has been found. Increased percentages of mutant mtDNA in muscle cells (up to 71%) can lead to mitochondrial myopathy. Levels of heteroplasmy of over 80% may lead to recurrent stroke and mutation levels of 95% have been associated with MELAS.

Regardless of the type of mutation or the level of heteroplasmy in affected mitochondria, unrepaired damage leads to a decrease in ATP, which in turn causes the phenotypic manifestation of disease. The manifestation of disease not only depends on the ATP level but also on the tissue affected. Various tissues have differing levels of demand on OXPHOS capacity. To evaluate a tissue threshold, Leber’s hereditary optic neuropathy can be used as a model for mitochondrial neurodegenerative disease. For neural and skeletal muscle tissues, the tissue threshold should be as high as or higher than 90% of
damaged (mutated) mtDNA. To induce mitochondrial malfunctions, the tissue threshold of the cardiac muscle is estimated to be significantly lower (approximately 64%-67%). In chronic vascular disease such as atherosclerosis, a mutation threshold in the affected vessel wall (e.g., in the postmortem aortic atherosclerotic plaques) was observed to be significantly lower. For example, for mutations m.3256 C>T, m.12315 G>A, m.15059 G>A, and m.15315 G>A, the heteroplasmy range of 18%-66% in the atherosclerotic lesions was 2-3.5-fold that in normal vascular tissue.

Mitochondrial stress and insulin resistance

  • Mitochondrial damage precedes the development of atherosclerosis and tracks the extent of the lesion in apoE-null mice, and
  • mitochondrial dysfunction caused by heterozygous deficiency of a superoxide dismutase increases atherosclerosis and vascular mitochondrial damage in the same model.

Blood vessels destined to develop atherosclerosis may be characterized by inefficient ATP production due to the uncoupling of respiration and OXPHOS. Blood vessels have regions of hypoxia, which lower the ratio of state 3 (phosphorylating) to state 4 (nonphosphorylating) respiration. Human atherosclerotic lesions have been known for decades to be deficient in essential fatty acids, a condition that causes respiratory uncoupling and atherosclerosis.

The finding by Kokaze et al.  helps to explain, at least in part, the anti-atherogenic effect of the allele m. 5178A due to its relation with the favorable lipid profile. The nucleotide change causes leucine-to-methionine substitution at codon 237 (Leu-237Met) of the NADH dehydrogenase subunit 2 located in the loop between 7th and 8th transmembrane domains of the mitochondrial protein. Given that this methionine residue is exposed at the surface of respiratory Complex I, this residue may be available as an efficient oxidant scavenger. Complex I

  • accepts electrons from NADH,
  • transfers them to ubiquinone, and
  • uses the energy released to pump protons across the mitochondrial inner membrane.

Thus, the Leu237Met replacement in the ND2 subunit might have a protective effect against oxidative damage to mitochondria.

Most fatty acid oxidation, which is promoted by peroxisome proliferator-activated receptor α (PPARα) activation, occurs in the mitochondria. Mitochondrial effects could explain why PPARα- deficient mice are protected from diet-induced insulin resistance and atherosclerosis as well as glucocorticoid induced insulin resistance and hypertension. Caloric restriction,

  • improves features of insulin resistance,
  • increases mitochondrial biogenesis and, surprisingly,
  • enhances the efficiency of ATP production.

Dysfunctional mitochondria in cultured cells can be rescued by transfer of mitochondria from adult stem cells, raising the possibility of restoration of normal bioenergetics in the vasculature to treat atherosclerosis associated with insulin resistance.
Chistiakov DA, Sobenin IA, Bobryshev YV, Orekhov AN. Mitochondrial dysfunction and mitochondrial DNA mutations in atherosclerotic complications in diabetes. World J Cardiol 2012; 4(5): 148-156. ISSN 1949-8462 (online). doi:10.4330/wjc.v4.i5.148. http://www.wjgnet.com/1949-8462/full/v4/i5/148.htm

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Author and Curator: Ritu Saxena, Ph.D.

Consultants: Aviva Lev-Ari, PhD, RN and Pnina G. Abir-Am, PhD

CONTENT:

Section I   : Mitochondrial diseases and molecular understanding

Section II  : Diagnosis and therapy of mitochondrial diseases

Section III: Mitochondria, metabolic syndrome and research

I. MITOCHONDRIAL DISEASES and MOLECULAR UNDERSTANDING

Mitochondrial cytopathy in adults – current understanding:

Mitochondrial cytopathies are a diverse group of inherited and acquired disorders that result in inadequate energy production leading to illnesses. Several syndromes have been linked to mutations in mitochondrial DNA. Some key features common to mitochondrial diseases are listed as follows:

  • Diverse manifestations of mitochondrial diseases: Although all mitochondrial diseases have the same characteristic of inadequate energy production as compared to the demand, they seem to show diverse manifestations in the form of organs being affected, age of onset and the rate of progression. Reason lies in the unique genetic makeup of mitochondria. The percentage of mtDNA carrying defects varies when the ovum divides and one daughter cells receiving more defective mtDNA and the other receiving less. Hence, successive divisions may lead to accumulation of defects in one of the developing organs or tissues. Since the process in which defective mtDNA becomes concentrated in an organ is random, this may account for the differing manifestations among patients with the same genetic defect. Also, somatic mutations and mutations occurring as a result of exposure to environmental toxins may cause mitochondrial diseases.

As stated by Robert K. Naviaux, founder and co-director of the Mitochondrial and Metabolic Disease Center (MMDC) at the University of California, San Diego;  

“It is a hallmark of mitochondrial diseases that identical mtDNA mutations may not produce identical diseases…the converse is also true, different mutations can lead to the same diseases.”

  • Postmitotic tissues are more vulnerable to mitochondrial diseases: Postmitotic tissues such as those in the brain, muscles, nerves, retinas, and kidneys, are vulnerable for several reasons. Apart from the fact that these tissues have high-energy demands, healthier neighboring cells unlike that observed in skin cannot replace the diseased cells. Thus, mutations in mtDNA accumulate over a period of time resulting in progressive dysfunction of individual cells and hence the organ itself.
  • High rate of mtDNA mutation: MtDNA mutates at rate that is six-seven times higher than the rate of mutation of nuclear DNA. First reason is the absence of histones on mtDNA and second is the exposure of mtDNA to free radicals due to their close proximity to electron transport chain. Additionally, lack of DNA repair enzymes results in mutant tRNA, rRNA and protein transcripts

Spectrum of mitochondrial diseases:

Following is the list of mitochondrial diseases occurring as a result of either mtDNA mutations, alteration in mitochondrial function or those diseases that sometimes might be associated with mitochondrial dysfunction.

  • Disorders associated with mtDNA mutations-

MELAS, MERRF, NARP, Myoneurogastrointestinal disorder and encephalopathy (MNGIE), Pearson Marrow syndrome Kearns-Sayre-CPEO, Leber hereditary optic neuropathy (LHON), Aminoglycoside-associated deafness, Diabetes with deafness

  • Mendelian disorders of mitochondrial function related to fuel homeostasis-

Luft disease, Leigh syndrome (Complex I, COX, PDH), Alpers Disease, MCAD, SCAD, SCHAD, VLCAD, LCHAD, Glutaric aciduria II, Lethal infantile cardiomyopathy, Friedreich ataxia, Maturity onset diabetes of young Malignant hyperthermia, Disorders of ketone utilization, mtDNA depletion syndrome, Reversible COX deficiency of infancy, Various defects of the Krebs Cycle, Pyruvate dehydrogenase deficiency, Pyruvate carboxylase deficiency, Fumarase deficiency, Carnitine palmitoyl transferase deficiency

  • Disorders sometimes associated with mitochondrial function-

Hemochromatosis, Wilson disease, Batten disease, Huntington disease, Menkes disease, Lesch-Nyhan syndrome, Aging, Type II diabetes mellitus, Atherosclerotic heart disease, Parkinson disease, Alzheimer dementia, Congestive heart failure, Niacin-responsive hypercholesterolemia, Postpartum cardiomyopathy, Alcoholic myopathy, Cancer metastasis, Irritable bowel syndrome Gastroparesis-GI dysmotility, Multiple sclerosis, Systemic lupus erythematosis, Rheumatoid arthritis.

II. DIAGNOSIS AND THERAPY OF MITOCHONDRIAL DISEASES

Diagnosis:

Owing to the diversity of symptoms, there is no accepted criterion for diagnosis. Also, due to overlapping symptoms of several diseases with those of mitochondrial dysfunction illnesses, it is important to evaluate the patient for other conditions. A diagnosis could involve combination of molecular genetic, pathologic, or biochemical data in a patient who has clinical features consistent with the diagnosis including mutational analysis on blood lymphocytes and possibly muscle biopsy for visual and biochemical analysis.

The two main biochemical features in most mtDNA disorders are:

  1. Respiratory chain deficiency and
  2. Lactic acidosis.

Skeletal muscle is chosen to study the pathogenic consequence of mtDNA mutations because of the formation of ragged-red fibers (RRF) through mitochondrial proliferation and massive mitochondrial accumulation in many pathogenic situations. RRF can be detected in two ways. Mitochondrial fibers in a subset of these fibers are shown by red or purple stained area by Gomori trichrome stain; the normal or less-affected fibers stain blue or turquoise. Deep purple areas show accumulations of mitochondria as activity of succinate dehydrogenase (SDH) in the case of mitochondrial mutation.

The primary care physician should remember this relatively simple rule of thumb: “When a common disease has features that set it apart from the pack, or involves 3 or more organ systems, think mitochondria.”

Treatment:

There are no cures for mitochondrial diseases; therefore, the treatment is focused on alleviating symptoms and enabling normal functioning of the affected organs. Most patients have used cofactor and vitamins; however, there is no overwhelming evidence that they are helpful in most patients.

  • Coenzyme Q10 (CoQ10) is the best-known cofactor used in treating mitochondrial cytopathies with no known side effects. CoQ10, residing in the inner mitochondrial membrane, functions as the mobile electron carrier and is a powerful antioxidant with benefits such as reduction in lactic acid levels, improved muscle strength, decreased muscle fatigue and so on.
  • Levocarnitine (L-carnitine, carnitine), is a cofactor required for the metabolism of fatty acids. Levocarnitine therapy improves strength, reversal of cardiomyopathy, and improved gastrointestinal motility, which can be a major benefit to those with poor motility due to their disease. Intestinal cramping and pain are the major side effects.
  • Creatine phosphate, synthesized from creatine can accumulate in small amounts in the body, and can act as storage for a high-energy phosphate bond. Muscular creatine may be depleted in mitochondrial cytopathy, and supplemental creatine phosphate has been shown to be helpful in some patients with weakness due to their disease.
  • B Vitamin, are necessary for the function of several enzymes associated with energy production. The need for supplemental B vitamin therapy is not proven, aside from rare cases of thiamine (vitamin B1)-responsive pyruvate dehydrogenase deficiency.

Research – Restriction enzyme for gene therapy of Mitochondria diseases:

Mitochondrial DNA (mtDNA) is the only extrachromosomal DNA in humans and defects in this genome are now recognized as important causes of various diseases. Presently, there is no effective treatment for patients suffering from diseases that harbor mutations in mtDNA.

Tanaka et al discovered a gene therapy method to treat a mitochondrial disease associated with mtDNA heteroplasmy. Heteroplasmy is where mutant and wild-type mtDNA molecules co-exist within cells. This syndrome of neurogenic muscle weakness, ataxia and retinitis pigmentosa (NARP) is caused by mutations in mtDNA leading to amino acid replacement in the resulting protein that codes for a subunit of mitochondrial ATP synthase. Level of mutant mtDNA is crucial for the disease as above a certain threshold level of mtDNA, the disease becomes biochemically and clinically apparent. Authors hypothesized that a possible method to treat patients was by selectively destroying mutant mtDNA, thereby only allowing propagation of wild-type mtDNA. Since restriction endonucleases can recognize highly specific sequences, they were utilized for gene therapy. Tanaka et al utilized Sma1, a restriction endonuclease to destroy mutant mtDNA, leading to increase in wild-type mtDNA levels.

Thus, authors concluded, “ the present results indicate that the use of a mitochondrion-targeted restriction enzyme which specifically recognizes a mutant mtDNA provides a novel strategy for gene therapy of mitochondrial diseases.”

III. MITOCHONDRIA, METABOLIC SYNDROME & RESEARCH

Mitochondria:

Mitochondria are double-membrane organelles located in the cytoplasm and often referred to as the “powerhouse” of the cell. In simple terms, they convert energy into forms that are usable by the cell. Mitochondria are semi-autonomous in that they are only partially dependent on the cell to replicate and grow. They have their own DNA, ribosomes, and can make their own proteins. They are the sites of cellular respiration that generates fuel for the cell’s activities. Mitochondria are also involved in other cell processes such as cell division, cellular growth and cell death. Multiple essential cellular functions are mediated by thousands of mitochondrial-specific proteins, encoded by both the nuclear and mitochondrial genomes.

Interestingly, mitochondria take on many different shapes and along with serving several different metabolic functions. In fact, each mitochondrion’s shape is characteristic of the specialized cell in which it resides. The number of mitochondria too varies in difference cell types, with as high as 500-2000 in some nucleated cells and as low as zero in RBCs and 2-6 in platelets.

The standard sequence to which all human mtNDNA is compared is referred to as the “Cambridge Sequence.” It was sequenced from several different human mtDNAs by a Medical Research Council (MRC) labora- tory based at Cambridge, UK, in 1981 and as a part of this work, Fred Sanger, the received his second Nobel Prize. Several variations in the form of polymorphisms are observed from the Cambridge sequence in the mtDNA of different individuals.

Metabolic syndrome:

Metabolic syndrome is a cluster of conditions — increased blood pressure, a high blood sugar level, excess body fat around the waist or abnormal cholesterol levels — that occur together, increasing your risk of heart disease, stroke and diabetes. Metabolic syndrome is becoming more and more common in the United States. In the future, it may overtake smoking as the leading risk factor for heart disease. In general, a person who has metabolic syndrome is twice as likely to develop heart disease and five times as likely to develop diabetes as someone who doesn’t have metabolic syndrome.

The five conditions described below are metabolic risk factors. You must have at least three metabolic risk factors to be diagnosed with metabolic syndrome.

  • A large waistline. This also is called abdominal obesity or “having an apple shape.” Excess fat in the stomach area is a greater risk factor for heart disease than excess fat in other parts of the body, such as on the hips.
  • A high triglyceride level (or you’re on medicine to treat high triglycerides). Triglycerides are a type of fat found in the blood.
  • A low HDL cholesterol level (or you’re on medicine to treat low HDL cholesterol). HDL sometimes is called “good” cholesterol. This is because it helps remove cholesterol from your arteries. A low HDL cholesterol level raises your risk for heart disease.
  • High blood pressure (or you’re on medicine to treat high blood pressure). Blood pressure is the force of blood pushing against the walls of your arteries as your heart pumps blood. If this pressure rises and stays high over time, it can damage your heart and lead to plaque buildup.
  • High fasting blood sugar (or you’re on medicine to treat high blood sugar). Mildly high blood sugar may be an early sign of diabetes.

Role of Mitochondria in Metabolic Syndrome & Diabetes:

Impaired mitochondrial function has recently emerged as a potential causes of insulin resistance and/or diabetes progression, risk factors of metabolic syndrome.

Mitochondria plays several key functions including generation of ATP, and generating metabolites via Tricarboxylic acid cycle that function in cytosolic pathways, oxidative catabolism of amino acids, ketogenesis, urea cycle; the generation of reactive oxygen species (ROS); the control of cytoplasmic calcium; and the synthesis of all cellular Fe/S clusters, protein cofactors essential for cellular functions such as protein translation and DNA repair. These roles define the mitochondria to be involved in metabolic homeostasis and hence, a major candidate for metabolic syndrome and its associated risk factor including diabetes, obesity and insulin resistance.

Research and Therapeutic relevance:

Understanding the underlying molecular mechanism of aberrant role of mitochondria is important in developing therapeutic agents for mitochondria-associated diseases. In the recent issue of Mitonews, several papers have been published using the products of MitoSciences, which describe research pertaining to the importance of mitochondria in obesity and diabetes. Some recent research articles based on mitochondrial research (also mentioned in MitoNews) have been briefly discussed here:

  • Metabolic inflexibility and Metabolic syndrome: Metabolic inflexibility is defined as the failure of insulin-resistant patients to appropriately adjust mitochondrial fuel selection in response to nutritional cues. Although the phenomenon has been emphasized an important aspect of metabolic syndrome, the molecular mechanisms have not yet been fully deciphered. In a recent article by Muoio et al, published in Cell Metabolism journal, essential role of the mitochondrial matrix enzyme, carnitine acetyltransferase (CrAT) has been identified in regulating substrate switching and glucose tolerance. CrAT regulates mitochondrial and intracellular Carbon trafficking by converting acetyl-CoA to its membrane permeant acetylcarnitine ester. Using muscle muscle-specific Crat knockout mice, primary human skeletal myocytes, and human subjects undergoing L-carnitine supplementation, authors have suggested a model wherein CrAT combats nutrient stress, promotes metabolic flexibility, and enhances insulin action by permitting mitochondrial efflux of excess acetyl moieties that otherwise inhibit key regulatory enzymes such as pyruvate dehydrogenase. These findings offer therapeutically relevant insights into the molecular basis of metabolic inflexibility.
  • Rosiglitazone and obesity: Eepicardial adipose tissue (EAT) has been described in humans as a functioning brown adipose tissue (BAT) and has been shown in animal models to have a lower glucose oxidation rate and higher fatty acid (FA) metabolism. In obese individuals, epicardial adipose tissue (EAT) is “hypertrophied”. EAT is a source of BAT may be a source of proinflamatory cytokines. Distel et al published their studies using a rat model of obesity and insulin resistance treated with rosiglitazone. They observed that rosiglitazone, promoted a BAT phenotype in the EAT depot characterized by an increase in the expression levels of genes encoding proteins involved in mitochondrial processing and density PPARγ coactivator 1 alpha (PGC-1α), NADH dehydrogenase 1 and cytochrome oxidase (COX4) resulting in significant up-regulation of PGC1-α and COX4 protein. The authors concluded that PPAR-γ agonist could induce a rapid browning of the EAT that probably contributes to the increase in lipid turnover. Thus, important insights into the mechanism of fat metabolism and involvement of mitochondrial proteins with a therapy were presented in the article.
  • Mitochondrial dysfunction and diabetic neuropathy: Animal models of diabetic neuropathy show that mitochondrial dysfunction occurs in sensory neurons that may contribute to distal axonopathy. The adenosine monophosphate-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) signalling axis senses the metabolic demands of cells and regulates mitochondrial function. Studies in muscle, liver and cardiac tissues have shown that the activity of AMPK and PGC-1α is decreased under hyperglycaemia. Chowdhury et al using type 1 and type 2 diabetic rat and mice models studied the hypothesis that deficits in AMPK/PGC-1 signalling in sensory neurons underlie impaired axonal plasticity, suboptimal mitochondrial function and development of neuropathy. The authors have shown there is a significant reduction in phospho-AMPK, phopho-ACC, total PGC-1α, NDUFS3and COXIV in sensory neurons of the dorsal root ganglia of 14 week old diabetic mice with marked signs of thermal hypoalgesia. These results were associated with an impaired neuronal bioenergetic profile and a decrease in the activity of mitochondrial complex I, complex IV and citrate synthase. The fact that resveratrol treatment reversed the changes observed in vitro and in vivo suggest that the development of distal axonopathy in diabetic neuropathy is linked to nutrient excess and mitochondrial dysfunction via defective signalling of the AMPK/PGC-1α pathway.
  • ROS and diabetes: Mitochondria generated reactive oxygen species (ROS) has been associated with kidney damage occurring in diabetes. Rosca et al, published an article investigating the source and site of ROS production by kidney cortical tubule mitochondria in streptozotocin-induced type 1 diabetes in rats. The authors observed that in diabetic mitochondria, the fatty acid oxidation enzymes were elevated with increased oxidative phosphorylation and increased ROS production. The authors observed ROS production with fatty acid oxidation remained unchanged by limiting electron flow in ETC complexes, changes in ETC substrate processing and that the ROS supported by pyruvate also remained unaltered. The authors hence concluded that mitochondrial fatty acid oxidation is the source of increased ROS production in kidney cortical tubules in early diabetes

Sources:

http://www.ncbi.nlm.nih.gov/pubmed/11453081

http://health.cat/open.php?url=http://biochemgen.ucsd.edu/mmdc/ep-3-10.pdf

http://findarticles.com/p/articles/mi_go2827/is_n6_v27/ai_n28687375/

http://www.columbiamitodiagnostics.org/images/Mitobrochure.pdf

http://www.ncbi.nlm.nih.gov/pubmed?term=12372991

http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0004546/

http://www.mayoclinic.com/health/metabolic%20syndrome/DS00522

http://www.nhlbi.nih.gov/health/health-topics/topics/ms/

http://www.ncbi.nlm.nih.gov/pubmed?term=22560225

http://www.ncbi.nlm.nih.gov/pubmed?term=%20%20%20%2022575275

http://www.ncbi.nlm.nih.gov/pubmed?term=%20%20%20%2022561641

http://www.mitosciences.com/mitonews_08_06.html

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