Posts Tagged ‘Peroxisome proliferator-activated receptor’

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



ARTS protein and cancer

ARTS protein and cancer



Krebs cycle

Krebs cycle

Metabolic control analysis of respiration in human cancer tissue

Metabolic control analysis of respiration in human cancer tissue



(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.



Methylation of adenine

Methylation of adenine





(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



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

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





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

















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=”http://pharmaceuticalintelligence.com/wp-content/uploads/2014/04/1741-7007-11-65-s1-macromolecular-juggling-by-ubiquitylation-enzymes.mp4“][/video]

An Introduction to the Warburg Apparatus


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.



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



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”


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.






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.


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.


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.


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

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


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”.


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).


Baruj Benacerraf, Jean Dausset and George D. Snell 

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


Edmond H. Fischer and Edwin G. Krebs 

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


Alfred G. Gilman and Martin Rodbell –

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


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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Peroxisome proliferator-activated receptor (PPAR-gamma) Receptors Activation: PPARγ transrepression for Angiogenesis in Cardiovascular Disease and PPARγ transactivation for Treatment of Diabetes

Peroxisome proliferator-activated receptor (PPAR-gamma) Receptors Activation: PPARγ transrepression  for Angiogenesis in Cardiovascular Disease and PPARγ transactivation for Treatment of Diabetes


UPDATED on 11/27/2018

A new combination drug therapy for CVD patients with co-morbidity of DM2 is presented in the following article, representing different mechanism of actions, pathways and a novel treatment proposed in 2018:

Cardiovascular (CV) Disease and Diabetes: New ACC Guidelines for use of two major new classes of diabetes drugs — sodium-glucose cotransporter type 2 (SGLT2) inhibitors and glucagon-like peptide 1 receptor agonists (GLP-1RAs) for reduction of adverse outcomes


The title of this article

Peroxisome proliferator-activated receptor (PPAR-gamma) Receptors Activation: PPARγ transrepression  for Angiogenesis in Cardiovascular Disease and PPARγ transactivation for Treatment of Diabetes

represents an explanation for pathways and mechanism of actions of combination drug therapy novel in its conceptualization in 2013.



The research is presented in the following three parts. References for each part are at the end.


PART I:             Genetics and Biochemistry of Peroxisome proliferator-activated receptor

Reporter: Aviva Lev-Ari, PhD, RN

PART II:             Peroxisome proliferator-activated receptors as stimulants of angiogenesis in cardiovascular disease and diabetes

Reporter: Aviva Lev-Ari, PhD, RN

PART III:            PPAR-gamma Role in Activation of eNOS: The Cardiovascular Benefit

Author and Curator: Aviva Lev-Ari, PhD, RN



Genetics and Biochemistry of Peroxisome proliferator-activated receptor

PPAR -alpha and -gamma pathways

In the field of molecular biology, the peroxisome proliferator-activated receptors (PPARs) are a group of nuclear receptor proteins that function as transcription factors regulating the expression of genes.[1] PPARs play essential roles in the regulation of cellular differentiation, development, and metabolism (carbohydrate, lipid, protein), and tumorigenesis[2] of higher organisms.[3][4]

Three types of PPARs have been identified: alpha, gamma, and delta (beta):[3]

Physiological function

All PPARs heterodimerize with the retinoid X receptor (RXR) and bind to specific regions on the DNA of target genes. These DNA sequences are termed PPREs (peroxisome proliferator hormone response elements). The DNA consensus sequence is AGGTCANAGGTCA, with N being a random nucleotide. In general, this sequence occurs in the promotor region of a gene, and, when the PPAR binds its ligand, transcription of target genes is increased or decreased, depending on the gene. The RXR also forms a heterodimer with a number of other receptors (e.g., vitamin D and thyroid hormone).

The function of PPARs is modified by the precise shape of their ligand-binding domain (see below) induced by ligand binding and by a number of coactivator and corepressor proteins, the presence of which can stimulate or inhibit receptor function, respectively.[9]

Endogenous ligands for the PPARs include free fatty acids and eicosanoids. PPARγ is activated by PGJ2 (a prostaglandin). In contrast, PPARα is activated by leukotriene B4. PPARγ activation by agonist RS5444 may inhibit anaplastic thyroid cancer growth.[10]

Peroxisome proliferator-activated receptor

Peroxisome proliferator-activated receptor (Photo credit: Wikipedia)


The three main forms are transcribed from different genes:

  •                PPARα – chromosome 22q12-13.1 (OMIM 170998)
  •                PPARβ/δ – chromosome 6p21.2-21.1 (OMIM 600409)
  •                PPARγ – chromosome 3p25 (OMIM 601487).

Hereditary disorders of all PPARs have been described, generally leading to a loss in function and concomitant lipodystrophy, insulin resistance, and/or acanthosis nigricans.[11] Of PPARγ, a gain-of-function mutation has been described and studied (Pro12Ala) which decreased the risk of insulin resistance; it is quite prevalent (allele frequency 0.03 – 0.12 in some populations).[12] In contrast, pro115gln is associated with obesity. Some other polymorphisms have high incidence in populations with elevated body mass indexes.




Mechanism of action

Thiazolidinediones or TZDs act by activating PPARs (peroxisome proliferator-activated receptors), a group of nuclear receptors with greatest specificity for PPARγ (gamma). The endogenous ligands for these receptors are free fatty acids (FFAs) and eicosanoids. When activated, the receptor binds to DNA in complex with the retinoid X receptor (RXR), another nuclear receptor, increasing transcription of a number of specific genes and decreasing transcription of others.

PPARγ transactivation

Thiazolidinedione ligand dependent transactivation is responsible for the majority of anti-diabetic effects.

The activated PPAR/RXR dimer binds to peroxisome proliferator hormone response elements upstream of target genes in complex with a number of coactivators such as nuclear receptor coactivator 1 and CREB binding protein, this causes upregulation of genes (for a full list see PPARγ:

TZDs also increase the synthesis of certain proteins involved in fat and glucose metabolism, which reduces levels of certain types of lipids, and circulating free fatty acids. TZDs generally decrease triglycerides and increase high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C). Although the increase in LDL-C may be more focused on the larger LDL particles, which may be less atherogenic, the clinical significance of this is currently unknown. Nonetheless, rosiglitazone, a certain glitazone, was suspended from allowed use by medical authorities in Europe, as it has been linked to an increased risk of heart attack and stroke.[3]

PPARγ transrepression

Thiazolidinedione ligand dependent transrepression mediates the majority of anti-inflammatory effects.

Binding of PPARγ to coactivators appears to reduce the levels of coactivators available for binding to pro-inflammatory transcription factors such as NF-κB, this causes a decrease in transcription of a number of pro inflammatory genes, including various interleukins and tumour necrosis factors.



 1. Waki H, Yamauchi T, Kadowaki T (February 2010). “[Regulation of differentiation and hypertrophy of adipocytes and adipokine network by PPARgamma]” (in Japanese). Nippon Rinsho 68 (2): 210–6. PMID 20158086.

2. Panigrahy D, Singer S, Shen LQ, et al. (2002). “PPARγ ligands inhibit primary tumor growth and metastasis by inhibiting angiogenesis”. J. Clin. Invest. 110 (7): 923–32. doi:10.1172/JCI15634. PMC 151148. PMID 12370270.

3. NHS: Avandia diabetes drug suspended, Friday 24th September 2010


Members of the class

The chemical structure of thiazolidinedione

Chemically, the members of this class are derivatives of the parent compound thiazolidinedione, and include:

  •                Rosiglitazone (Avandia), which was put under selling restrictions in the US and withdrawn from the market in            Europe due to an increased risk of cardiovascular events.
  •                Pioglitazone (Actos), France and Germany have suspended the sale of the diabetes drug Actos after a study suggested the drug, also known as pioglitazone, could raise the risk of bladder cancer.[4]
  •                Troglitazone (Rezulin), which was withdrawn from the market due to an increased incidence of drug-induced hepatitis.

Experimental agents include netoglitazone, an antidiabetic agent, rivoglitazone, and the early non-marketed thiazolidinedione ciglitazone.

Replacing one oxygen atom in a thiazolidinedione with an atom of sulfur gives a rhodanine.




Peroxisome proliferator-activated receptors as Stimulants of Angiogenesis in Cardiovascular Disease and Diabetes

In 2009 in Diabetes Metab Syndr Obes a seminal paper was published on the topic by  Desouza, Rentschler and Fonseca. (2009). This work constitutes Part II. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3048019/

Mechanisms by which PPARs may stimulate angiogenesis

PPARs seem to have a protective role in ischemic tissues, including brain, cardiac and skin. A part of this may be by stimulating angiogenesis and improving blood supply. Hypoxia is a trigger for the development of angiogenesis. One of the key mediators in hypoxia-induced angiogenesis is hypoxia inducible factor (HIF-1), which is induced in hypoxic cells and binds to hypoxia response element (HRE). HIF-1 mediates the transcriptional activation of several genes that promote angiogenesis, including VEGF, angiopoeitin (Ang-1, Ang-2), and matrix metalloproteinases (MMP-2, MMP-9).55 15-deoxy-delta(12, 14)-prostaglandin J(2) (15d-PGJ(2)), a PPAR-γ agonist, has been shown to induce HIF-1 expression and thereby angiogenesis (Figure 1).34 However pioglitazone has been shown to suppress the induction of HIF-1.56 Conditions that influence the stimulation or suppression of HIF activation by PPAR-γ are largely unknown.

Several studies suggest that eNOS synthase activation is required for angiogenesis that may be protective under certain conditions.5759 In one study pioglitazone reduced the myocardial infarct size in part via activation of eNOS.60 PPAR-α activation has also been shown to protect the type 2 diabetic rat myocardium against ischemia-reperfusion injury via the activation of the NO pathway (Table 1, Figure 1).61 However, stimulation of the inducible nitric oxide (iNOS) pathway can lead to undesirable angiogenesis that may be contribute to pathological states such as proliferative retinopathy. PPARs in fact have been shown to suppress iNOS expression, thereby suppressing undesirable angiogenesis.62,63 Here again the factors that allow for activation of eNOS and suppression of iNOS is largely unknown.

The most studied pathway by which PPARs may stimulate angiogenesis is the VEGF pathway. VEGF can stimulate angiogenesis via stimulation of the ERK1/2 pathway. PPAR-β/δ activation has been shown to increase VEGF expression and thereby stimulate angiogenesis (Figure 1).26 In some studies PPAR-α and PPAR-γ have also been shown to increase VEGF expression.47,48 However the majority of studies still show that PPAR activation suppresses VEGF expression. The end result of whether PPAR activation suppresses or stimulates VEGF expression seems to lie in the pathological condition in which its actions are observed (Figure 1). It is likely that PPAR activation results in increased VEGF expression in conditions where new blood vessel formation is required, such as ischemic skin flaps, brain, or cardiac tissue ischemia. On the other hand, pathological angiogenesis such as in the eye or within an atherosclerotic plaque is suppressed by PPAR activation via a suppression of VEGF (Figure 1).

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Mechanisms by which PPARs effect angiogenesis.

Table 1

Effect of PPARs on angiogenesis

Recently some studies indicate that PPARs may increase the expression and activation of the phosphatidylinositol-3-kinase (PI3K/AKT) pathway.61,64 The PI3K/AKT pathway stimulates angiogenesis.59,65 Again the majority of studies show that PPAR activation inhibits PI3K/AKT activation.

It is very likely that a large amount of variation found in different studies is due to the use of agonists and antagonists of the PPAR receptors that exhibit direct PPAR-independent effects. Most study designs do not distinguish between direct effects and indirect effects of various pharmacological agonists/antagonist used. Fibrates and TZDs have both been shown to have direct independent effects on inflammation, proliferation and angiogenesis. Hence it is difficult to conclude that all the pro and antiangiogenic effects seen in various studies are a result of PPAR activation exclusively.

Clinical significance and conclusions

Some compounds such as TZDs and fibrates are routinely used in patients with diabetes, dyslipidemia, and cardiovascular disease. Other compounds such as partial agonists or dual agonists of PPAR-α and PPAR-γ are in development. The effects of these newer compounds, on angiogenesis and cardiovascular disease are yet to be determined. Current evidence from clinical trials suggest a mixed picture. TZD treatment in patients with type 2 diabetes has been shown to be associated with macular edema. On the other hand, the FIELD study using fenofibrate showed a decrease in the need for laser treatments in patients with diabetic retinopathy. The PROACTIVE study showed that pioglitazone trended to decrease certain cardiovascular endpoints. In some studies, rosiglitazone increased the risk of cardiovascular events. In other studies such as ACCORD and VADT, TZD treatment was not associated with increased cardiovascular event risk. Several factors, including the study design, PPAR receptor affinity, and the PPAR-independent actions of these compounds, possibly play a role in the differences in results seen. The duration of the pathological state and the vasculature of the effected organ likely play a role in whether PPARs prove beneficial or harmful. In conclusion it may be prudent to summarize that at this point the evidence suggests that PPARs can either stimulate or inhibit angiogenesis, depending on the biological context and pathological process.

Clinical Trials: Controversial Research Results

Peroxisome proliferator-activated receptors (PPARs) are a group of nuclear hormone receptors that regulate lipid and glucose metabolism. PPAR-α agonists such as fenofibrate and PPAR-γ agonists such as the thiozolidinediones have been used to treat dyslipidemia and insulin resistance in diabetes. Over the past few years research has discovered the role of PPARs in the regulation of inflammation, proliferation, and angiogenesis. Clinical trials looking at the effect of PPAR agonists on cardiovascular outcomes have produced controversial results. Studies looking at angiogenesis and proliferation in various animal models and cell lines have shown a wide variation in results. This may be due to the differential effects of PPARs on proliferation and angiogenesis in various tissues and pathologic states. This review discusses the role of PPARs in stimulating angiogenesis. It also reviews the settings in which stimulation of angiogenesis may be either beneficial or harmful.

affect inflammation, proliferation, immune function and angiogenesis.3 There are three PPAR isotypes, PPAR-α, PPAR-β/δ, and PPAR-γ. They form heterodimers with the retinoid X receptors and bind to specific DNA sequences, called peroxisome proliferator response elements (PPRE), in the promoter regions of their target genes. PPARs exhibit isotype-specific tissue expression patterns. PPAR-α is primarily expressed in organs with significant fatty acid catabolism. PPAR-β/δ is expressed in nearly all cell types and the level of expression seems to depend on the amount of angiogenesis, cell proliferation, and differentiation occurring in that specific tissue.4 PPAR-γ is found in adipose tissue and at lower levels in immune cells vascular tissue and some organs. PPAR-γ exists in two protein isoforms, PPAR-γ1 and PPAR-γ2, with different lengths of the N-terminal. The PPAR-γ2 isoform is predominantly expressed in adipose tissue, whereas PPAR-γ1 is relatively widely expressed.5 Expression of each isoform is driven by a specific promoter that confers the distinct tissue expression patterns. There are also two other mRNA variants of PPAR-γ, proteins identical to PPAR-γ1: PPAR-γ3, which is restricted to macrophages, adipose tissue, and colon, and PPAR-γ4, the tissue distribution of which is unclear at this time.5 Human PPAR-γ plays a critical physiological role as a central transcriptional regulator of both adipogenic and lipogenic programs. Its transcriptional activity is induced by the binding of endogenous and synthetic lipophilic ligands, which has led to the determination of many roles for PPAR-γ in pathological states such as type 2 diabetes, atherosclerosis, inflammation, and cancer.

The role of PPARs has traditionally been recognized as antiproliferative and antiangiogenic in a large number of disease states including cancer and cardiovascular disease.4 These studies have led to clinical trials with PPAR agonists to evaluate their benefits in cancer and cardiovascular disease. The results of some of these trials especially in cardiovascular disease have been mixed and hence controversial.

The results obtained with a PPAR-γ agonist pioglitazone do suggest a better impact on the lipid profile compared to rosiglitazone (the former lowers triglyceride significantly and has less adverse effects on low-density lipoprotein [LDL] cholesterol), and at least a mixed result (the primary composite endpoint was not reduced significantly but myocardial infarction, stroke, and death were reduced by 16%), in an outcome trial – PROspective pioglitAzone Clinical Trial In macroVascular Events (PROACTIVE).6 Rosiglitazone on the other hand was found to increase cardiovascular events in a large restrospective analysis study.7

This has led to a lot of recent research into PPARs that is contrary to the traditional literature in their role as inhibitors of angiogenesis. This review will examine the role and evidence of PPARs as promoters of angiogenesis, the mechanisms involved, and the implications thereof.


 Desouza, Rentschler and Fonseca. (2009).

Angiogenesis is described as the formation of new capillaries from the existing vasculature. This process involves the breakdown of the extracellular matrix and formation of an endothelial tube. Angiogenesis is an important physiologic process in the female reproductive cycle, wound healing, and bone formation. Angiogenesis is also a crucial step in several disease states including cancer, diabetic retinopathy, rheumatoid arthritis, stroke, and ischemic coronary artery disease.810 Neoangiogenesis has harmful as well as beneficial effects in the setting of type 2 diabetes and cardiovascular disease.10 In the setting of diabetes, there is abnormal regulation and signaling of vascular endothelial growth factor (VEGF) and its receptor Flk-1.11 This may lead to increased levels of circulating VEGF, resulting in increased permeability of vascular structures throughout the body. In the retina, this results in the formation of protein-rich exudates containing VEGF that induces a local inflammatory response resulting in capillary sprouting. A similar process might take place in the arterial wall, thereby promoting capillary sprouting and plaque destabilization.12 At the same time, the lack of Flk-1 activation in endothelial cells and abnormal VEGF-dependent activation of monocytes impair the arteriogenic response that requires monocyte recruitment, and monocyte and endothelial cell migration and proliferation.11 This could lead to a deficient angiogenic response in ischemic tissue. VEGF/Flk-1 signaling may also be required for bone marrow release of circulating endothelial progenitor cells that play a role in endothelial function and arteriogenesis.13 The abnormal release of endothelial progenitors could further reduce arteriogenic response. This has therapeutic implications in terms of vascularization and survival of skin grafts in patients with diabetes as well as vascularization of the ischemic myocardium. An important mechanism by which PPARs seem to regulate angiogenesis is via VEGF.11,12 It would therefore appear that PPARs have a role in regulating both beneficial and harmful effects of angiogenesis thereby leading to controversial results (Figure 1).

The other factor influencing the results of angiogenesis studies is the use of PPAR agonists that have pleotropic effects. PPAR-α agonists such as fibrates stimulate pathways that do not depend on PPAR-α.14 PPAR-γ agonists such as thiozolidinediones (TZDs) have PPARγ independent actions on proliferative and inflammatory pathways.14 Therefore to conclude that the effects of commonly used PPAR agonists on angiogenesis are specifically due to PPAR activation is at best controversial.15

Although the majority of studies point towards the antiproliferative, antiangiogenic properties of PPAR-α, this may be due to the use of fibrates as agonists in these experiments. A lot more research needs to be done using methods such as spontaneous PPAR-α activation, overexpression, silencing and knockout mice, rather than using chemical agonists and antagonists which might have pleotropic effects unrelated to PPAR-α.


PPAR-γ and angiogenesis

PPAR-γ is probably the most studied PPAR, likely due to the use and development of several PPAR-γ agonists such as thiozolidinediones in the treatment of type 2 diabetes. Endogenous ligands for PPAR-γ include long chain polyunsaturated fatty acids and their derivatives, 15-deoxy-Δ12, 14-prostaglandin J2 (15d-PGJ2).4 Other natural ligands include nitrolinoleic acids. 15d-PGJ2 has been found to upregulate the expression of PPAR-γ and also the DNA binding and transcriptional activity.34 Synthetic ligands include TZDs and various nonsteroidal anti-inflammatory drugs.35

Studies supporting antiproliferative properties of PPAR-γ

PPAR-γ has widespread effects involving, inflammation, atherosclerosis, obesity, diabetes, and cancer.36 PPAR-γ agonists directly inhibit tumor cell growth, induce cell differentiation, and apoptosis in various cancer types (Table 1).37 TZDs have been shown to decrease post angioplasty neointimal hyperplasia in both animals and humans (Table 1).38,39 PPAR-γ ligands have been shown to inhibit and stimulate angiogenesis (Table 1). Inhibition by PPAR-γ ligands can occur through direct effects on the endothelium or through indirect effects on the net balance of proangiogenic and antiangiogenic mediators.37 PPAR-γ expressed in choroidal endothelial cells inhibits the differentiation and proliferation of those cells.38,39 Rosiglitazone inhibited endothelial cell proliferation and migration and decreased VEGF-induced tubule formation in human umbilical vein endothelial cells.40,41 In another study PPAR-γ ligands stimulated endothelial cell caspase-mediated apoptosis.42 15d-PGJ2, an endogenous ligand of PPAR-γ, induces growth inhibition, differentiation, and apoptosis of tumor cells.43 PPAR-γ activation interrupts NF-kβ signaling with subsequent blockade of proinflammatory gene expression.43 Pioglitazone and rosiglitazone inhibit the effects of growth factors such as bFGF and VEGF. Endothelial cell migration is also inhibited by both compounds.44 Thus natural and synthetic ligands of PPAR-γ exhibit antiangiogenic properties under certain conditions.

Studies supporting proangiogenic role of PPAR-γ

However, PPAR- ligands have also been shown to stimulate the angiogenic pathway (Table 1). In bovine aortic endothelial cells, prolonged treatment with troglitazone increased VEGF and endothelial nitric oxide (NO) production with no change in endothelial nitric oxide synthase (eNOS) expression.45 In cultured rat myofibroblasts, activation of PPAR-γ by troglitazone and 15-dPGJ2 induced VEGF expression and augmented tubule formation.46 In mice treated with rosiglitazone, angiogenesis was stimulated in adipose tissue with increased expression of VEGF and angiopoeitin-4 (Ang-4). Ang-4 stimulated endothelial cell growth and tubule formation. 47 In rats with focal cerebral ischemia, rosiglitazone treatment enhanced neurologic improvement and reduced the infarct size by reducing caspase-3 activity, increasing the number of endothelial cells, and increasing eNOS expression.48 In the setting of diabetes, PPAR-γ agonists may promote revascularization of ischemic tissue. Diabetic mice with induced unilateral hind limb ischemia, when treated with pioglitazone showed normalization of VEGF, upregulation of eNOS activity, and partial restoration of blood flow recovery.49 In mice treated with pioglitazone, VEGR-receptor-2 positive EPCs were upregulated and migratory capacity was increased. In vivo angiogenesis was increased 2-fold.50 In an endothelial/interstitial cell co-culture assay, treatment with PPAR-γ agonists stimulated production of VEGF. In the same study, corneas treated with the same PPAR-γ agonists increased phosphorylation of eNOS.20

Few studies have evaluated angiogenesis in humans. Pioglitazone treatment has been shown to increase serum VEGF, IL-8, and angiogenin levels in patients with type 2 diabetes.51 In another study thiozolidinedione use in patients with type 2 diabetes was associated with diabetic macular edema.52

PGC-1α and angiogenesis

Peroxisome proliferator-activated receptor (PPAR)-gamma coactivator 1alpha (PGC-1α) is a nuclear transcriptional coactivator that regulates several important metabolic processes, including mitochondrial biogenesis, adaptive thermogenesis, respiration, insulin secretion and gluconeogenesis. 53 PGC-1α also co-activates PPAR-α, PPAR-β/δ, and PPAR-γ which are important transcription factors of genes regulating lipid and glucose metabolism.53 Recently Arany and colleagues have shown that PGC-1α stimulates angiogenesis in ischemic tissues. Using a combination of muscle cell assays and genetically modified mice that over or underexpess PGC-1α, they showed that PGC-1α is a powerful inducer of VEGF expression. PGC-1α did not involve HIF-1 but activated the nuclear receptor, estrogen-related receptor-α (ERR-α).33 PGC-1α−/− mice are viable, suggesting that PGC-1α is not essential in embryonic vascularization but they show a striking failure to reconstitute blood flow in a normal manner to the limb after an ischaemic insult.54 Transgenic expression of PGC-1α in skeletal muscle is protective against ischemic insults. This suggests that PGC-1α plays a more important role in a disease state rather than a physiologically healthy state.


PART III: PPAR-gamma Role in Activation of eNOS: The Cardiovascular Benefit

Author and Curator: Aviva Lev-Ari, PhD, RN


Mechanism of Action (MOA) for ElectEagle‘s component 3

Treatment Regime with PPAR-gamma agonists (TZDs)

For ElectEagle‘s component 1:


Lev-Ari, A., (2012 X). Clinical Trials Results for Endothelin System: Pathophysiological role in Chronic Heart Failure, Acute Coronary Syndromes and MI – Marker of Disease Severity or Genetic Determination?



Lev-Ari, A., (2012W). Endothelin Receptors in Cardiovascular Diseases: The Role of eNOS Stimulation



Lev-Ari, A., (2012V). Inhibition of ET-1, ETA and ETA-ETB, Induction of NO production, stimulation of eNOS and Treatment Regime with PPAR-gamma agonists (TZD): cEPCs Endogenous Augmentation for Cardiovascular Risk Reduction – A Bibliography


For ElectEagle‘s component 2:


Lev-Ari, A. (2012L).. Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production


Lev-Ari, A. (2012i). Bystolic’s generic Nebivolol – positive effect on circulating Endothelial Proginetor Cells endogenous augmentation


Three indications for PPAR-gamma agonist (TZD): Experimental agents include netoglitazone, an antidiabetic agent, rivoglitazone, and the early non-marketed thiazolidinedione ciglitazone

  •                Antisclerosis, angiogenic progenitor cell differentiation and endogenous augmentation of cEPCs
  •                Stimulation of eNOS
  •                Decrease insulin resistance

Classic indication: action to decrease insulin resistance. PPAR-gamma receptors are complex and modulate the expression of the genes involved in lipid and glucose metabolism, insulin signal transduction and adipocyte and other tissue differentiation. TZDs have significant effects on vascular endothilium, the immune system, the ovaries, and tumor cells. Some of these responses may be independent of the PPAR-gamma pathway (Nolte and Karam, 2004). TZDs are ligands of PPAR-gamma receptors part of the steroid (estrogen receptor ligands) and thyroid superfamily of nuclear receptors found in muscle, liver and adipocytes. In the gold standard of all pharmacology books, the cardinal indication for TZDs is action to decrease insulin resistance (Nolte and Karam, 2004 in Katzung). However, the recent research has proposed two new indications for Rosiglitazone in addition to the original insulin sensitivity reduction indication.

As implied in Part I of the ElectEagle Project, TDZs were selected for a new indication in the domain of modulation of atherosclerosis (Verma and Szmitko, 2006), (Li et al., 2004)and facilitation of the differentiation of angiogenic progenitor cells, inhibition of vascular smooth muscle, proliferation and migration to improve endothelial function (Wang et al., 2004).

The following three seminal papers on the function of TDZs in modulation of vascular disease served as an inspiration for our extension of their new indication for TDZs in the anti-atherosclerosis domain into the cEPCs endogenous augmentation proposed treatment area.

Verma S, Szmitko, PE, (2006). The vascular biology of peroxisome proliferator-activated receptors: Modulation of atherosclerosis. Can J Cardiol, 22 (Suppl B):12B-17B.

Wang C-H, Ciliberti N, Li S-H, Szmitko PE, Weisel RD, Fedak PWM, Al-Omran M, Cherng W-J, Li R-K, Stanford WL, Verma S., (2004). Rosiglitazone facilitates angiogenic progenitor cell differentiation toward endothelial lineage: a new paradigm in glitazone pleiotropy. Circulation, 109:1392-1400.

Li AC, Binder CJ, Gutierrez A, Brown KK, Plotkin CR, Pattison JW, Valledor AF, Davis RA, Wilson TM, Wizttum JL, Palinski W, Glass CK., (2004). Differential inhibition of macrophage foam-cell formation and atherosclerosis in mice by PPAR alpha, beta/delta, and gamma. J. Clin. Invest., 114:1564-1576.

Namely, in the ElectEagleProject, a finely tuned interpretation is provided. We assume that TZDs may have a potential therapeutic effect on augmentation of cEPCs in a significant way should a combination drug therapy be designed to include Rosiglitazoneand two other drugsonewhich inhibits receptors ETA and ETA-ETB and the other which induces eNOS. TDZs were selected for a new indication related to anti-atherosclerosis, however, we extend and emphasize TZDs function in cell differentiation and cell migration of EPCs following encouraging results by Wang et al., (2004). Thus, we are shifting the indication from atherosclerosis and peripheral vascular disease to cardiovascular and CAD.

In 2005 a new indication for TZDs emerged from new finding about the PPAR-gamma receptors function in cell nitric oxide (NO) release without increasing the expression of endothelial nitric oxide synthase (eNOS) (Polikandriotis et al., 2005). This is an important finding for the drug combination components selected for ElectEagleProject. This subject is covered in the following section, Role of PPAR-gamma in eNOS stimulation.

Mechanism of action (MOA) for ElectEagle‘s components 2 & 3

Role of PPAR-gamma in eNOS stimulation

Polikandriotis et al. (2005), recently reported that the peroxisome proliferator-activated receptor gamma (PPARgamma) ligands 15-deoxy-Delta(12,14)-prostaglandin J2 (15d-PGJ2) and ciglitazone increased cultured endothelial cell nitric oxide (NO) release without increasing the expression of endothelial nitric oxide synthase (eNOS). Their study was designed to characterize further the molecular mechanisms underlying PPARgamma-ligand-stimulated increases in endothelial cell NO production.

Their methods and Results: Treating human umbilical vein endothelial cells (HUVEC) with PPARgamma ligands (10 micromol/L 15d-PGJ2, ciglitazone, or rosiglitazone) for 24 hours increased NOS activity and NO release. In selected studies, HUVEC were treated with PPARgamma ligands and with the PPARgamma antagonist GW9662 (2 micromol/L), which fully inhibited stimulation of a luciferase reporter gene, or with small interfering RNA to PPARgamma, which reduced HUVEC PPARgamma expression. Treatment with either small interfering RNA to PPARgamma or GW9662 inhibited 15d-PGJ2-, ciglitazone-, and rosiglitazone-induced increases in endothelial cell NO release. Rosiglitazone and 15d-PGJ2, but not ciglitazone, increased heat shock protein 90-eNOS interaction and eNOS ser1177 phosphorylation. The heat shock protein 90 inhibitor geldanamycin attenuated 15d-PGJ2- and rosiglitazone-stimulated NOS activity and NO production. Their Conclusion: These findings further clarify mechanisms involved in PPARgamma-stimulated endothelial cell NO release and emphasize that individual ligands exert their effects through distinct PPARgamma-dependent mechanisms

Originally, Rosiglitazone was indicated as an adjunct to diet and exercise to improve glycemic control in patients with type 2 diabetes mellitus who are already treated with combination rosiglitazone and metformin or who are not adequately controlled on metformin alone. As a result of the FDA drug recall of Rosiglitazone, we suggest here several alternatives: Experimental agents include netoglitazone, an antidiabetic agent, rivoglitazone, and the early non-marketed thiazolidinedione ciglitazone

In the ElectEagle project, Rosiglitazone was identified for a new indication – as a PPAR-gamma agonist implicated with efficacy for endogenous augmentation of cEPCs which serves as a biomarker for CVD risk reduction — an extension of the anti-atherosclerosis indication or the confinement to perileral vascular endothelium (Verma & Szmitko), (Wang et al., 2004), (Li et al., 2004).

Polikandriotis et al. (2005) is a very import publication for ElectEagle project for the following critical five reasons:

  •                Polikandriotis et al. (2005) clarify the mechanism of action of PPAR-gamma agonists at the protein level in a set of novel experiments, thus contributes to the understanding of the physiological process of the mechanism of action of PPAReceptor-gamma and its relations to L-arginine: NO pathway and its impact in many areas of research, notably vascular biology.
  •                Polikandriotis et al. (2005) compare two PPAR-gamma agonist agents and confirm Rosiglitazone to be the more potent among the two for the experiments described above
  •                Polikandriotis et al. (2005) identify Rosiglitazone capability to stimulate endothelial cell NO release, which is a third indication for Rosiglitazone.
  •                The combination drug therapy selected in May 2006, for the ElectEagle project involved three drugs. Two of which where a PPAR-gamma agonist, specifically, Rosiglitazone. The other drug was an eNOS agonist to stimulate NO production and reuptake. By identifying Rosiglitazone capability to stimulate endothelial cell NO release, Polikandriotis et al. (2005) offer reassurance for the selection of Rosiglitazone in the first place, and further more we became aware that it will exert synergies with the drug chosen as an eNOS agonist.
  •                In the ElectEagle project, a new experiment is called for following Polikandriotis et al. (2005) findings on Rosiglitazone impact on NO release. It will be needed to measure the incremental induction of NO release resulting from a combination therapy which includes an eNOS agonist and a PPAR-gamma agonist implicated in 2005 with stimulant effects on the release NO.

Moncada & Higgs, (2006) explain that the low concentrations of NO generated by eNOS protect against atherosclerosis by promoting vasodilatation, inhibiting leucocyte and platelet adhesion and/or aggregation and smooth muscle cell proliferation. However, higher concentrations of NO generated by iNOS promote atherosclerosis, either directly or via the formation of NO adducts, such as peroxynitrite. Such a paradox in the action of NO was apparent from their experiments some years ago, in which they found that the acute vascular injury in the ileum and colon following administration of lipopolysaccharide is aggravated by early treatment with a NO synthase inhibitor, whereas delayed administration of such a compound provides protection against the damage to the intestinal vasculature (Laszlo et al., 1994). A prominent example of this comes from experiments in Apo-Emutant mice in which the concomitant knocking out of eNOS leads to an increase in atherosclerosis, while the knocking out of iNOS reduces atherosclerosis (Moncada, 2005).

Research Goals in characterization of ElectEagle Version I


Provided rationale for agent selection for

ElectEagle Version I – Component 3: Treatment Regime with PPAR-gamma agonists (TZD)

agent selection: Rosiglitazone

As a result of the FDA drug recall of Rosiglitazone, we suggest here several alternatives: Experimental agents include netoglitazone, an antidiabetic agentrivoglitazone, and the early non-marketed thiazolidinedione ciglitazone


Retionale:            See discussion on TZDs MOA, above

a-priori postulates presented in Part I for Component 3: PPAR-gamma

  • dose concentration dependence on PPAReceptor-gamma – confirmed by a study for Rosiglitazone and a study for Ciglitazone
PPAReceptor-gamma agonists time concentration dependence manner dose concentration dependencemanner time and dose dose 
Rosiglitazone Polikandriotis et al., (2005) maximum recommended daily dose of 8 mg to 2,000 mg.
Ciglitazone   Polikandriotis et al., (2005)


Proposed integration plan for ElectEagle’s Version I with CVD patients current medication regimen for selective medical diagnoses

Blood Pressure Medicine:

Beta blockers, Verapamil (Calan), Reserpine (Hydropes), Clonidine (Catapres), Methyldopa (Aldomet)


Thiazides, Spironolactone (Aldactone), Hydralazine


Prozac, Lithium, MOA’s, Tricyclics

Stomach Medicine:

Tagamet and Zantac, plus other compounds containing Cimetidine and Ranitidine or associated compounds in Anticholesterol Drugs


Chlorpromazine (Thorazine), Pimozide (Orap), Thiothixine (Navane), Thiordazine (Mellaril), Sulpiride, Haloperidol (haldol), Fluphenazine (Modecate, Prolixin)

Heart Medicine:

Clofibrate (Atromid), Gemfibrozil, Diagoxin


Estrogen, Progesterone, Proscar, Casodex, Eulexin, Corticosteroids Gonadotropin releasing antagonists: Zoladex and Lupron

Cytotoxic agents:

Cyclophosphamide, Methotrexate, Roferon Non-steroidal anti-inflammatories


Alprazolam, Amoxapine, Chlordiazepoxide, Sertraline, Paroxetine, Clomipramine, Fluvoxamine, Fluoxetine, Imipramine, Doxepine, Desipramine, Clorprothixine, Bethanidine, Naproxen, Nortriptyline, Thioridazine, Tranylcypromine, Venlafaxine, Citalopram.

INTERACTIONS for Nebivolol – Component 2

Calcium Antagonists:

Caution should be exercised when administering beta-blockers with calcium antagonists of the verapamil or diltiazem type because of their negative effect on contractility and atrio-ventricular conduction. Exaggeration of these effects can occur particularly in patients with impaired ventricular function and/or SA or AV conduction abnormalities. Neither medicine should therefore be administered intravenously within 48 hours of discontinuing the other.


Caution should be exercised when administering beta-blockers with Class I anti-arrhythmic drugs and amiodarone as their effect on atrial conduction time and their negative inotropic effect may be potentiated. Such interactions can have life threatening consequences.


Beta-blockers increase the risk of rebound hypertension after sudden withdrawal of chronic clonidine treatment.


Digitalis glycosides associated with beta-blockers may increase atrio-ventricular conduction times. Nebivolol does not influence the kinetics of digoxin & clinical trials have not shown any evidence of an interaction.

Special note: Digitalisation of patients receiving long term beta-blocker therapy may be necessary if congestive cardiac failure is likely to develop. The combination can be considered despite the potentiation of the negative chronotropic effect of the two medicines. Careful control of dosages and of individual patient’s response (notably pulse rate) is essential in this situation.

Insulin & Oral Antidiabetic drugs:

Glucose levels are unaffected, however symptoms of hypoglycemia may be masked.


Concomitant use of beta-blockers & anaesthetics e.g. ether, cyclopropane & trichloroethylene may attenuate reflex tachycardia & increase the risk of hypotension

Medical Diagnoses Current medication regiment ET-1, ETA and ETA-ETBinhibition eNOS agonistsproduction stimulation of NO PPAR-gamma agonist (TZD) PPAR-gamma agonist (TZD) as eNOS stimulant
CAD patients Beta blockers, ACEI, ARB, CCB, Diagoxin, Coumadin yes yes yes
Endothelial Dysfunction in DM patients with or without Erectile Dysfunction Insulin yes yes yes yes
Atherosclerosis patients: Arteries and or veins AntihypertensiveCoumadin yes yes yes yes
pre-stenting treatment phase Beta blockers, Verapamil (Calan), Reserpine (Hydropes), Clonidine (Catapres), Methyldopa (Aldomet) yes yes yes
post-stenting treatment phase Antiplatelets yes yes
if stent is a Bare Mesh stent (BMS) CoumadinBeta blockers yes yes
if stent is Drug Eluting stent (DES) antibiotics yes
if stent is EPC antibody coated yes yes
post CABG patients CoumadinBeta blockers, Verapamil(Calan), Reserpine (Hydropes), Clonidine (Catapres), Methyldopa (Aldomet) yes yes
CVD patients on blood thinner Coumadin yes yes yes


  •       Most favorable and unexpected to us was finding in the literature new indications for TDZs as stimulators of eNOS, in addition to the new indication for atherosclerosis besides the classic indication in pharmacology books, being in the reduction of insulin resistance. Reassuring our selection of Rosiglitazone. As a result of the FDA recoll, the drug substitute will be an Experimental agents include netoglitazone, an antidiabetic agentrivoglitazone, and the early non-marketed thiazolidinedione ciglitazone 
  •       Most favorable and unexpected to us was finding in the literature new indications for beta blockers as NO stimulant, nebivolol, a case in point, thus, fulfilling two indications in one drug along the direction of the study to identify eNOS agonists.
  •       The following combination of drugs was selected for ElectEagle Version I

Bosentan (Tracleer), Oral: 62.5 mg tablets

Nebivolol, Oral: 5mg once daily

Experimental agents include netoglitazone, an antidiabetic agent, rivoglitazone, and the early non-marketed thiazolidinedione ciglitazone

  •       We confirmed time and dose concentrations postulating apriori in most cases. Additional literature searches will benefit the project for the three drugs selected
  •       We have identified Inhibition of ET-1, ETA and ETA-ETB as one of the agent in the drug combination. The entire literature on cEPCs does not implicate Endothelin with impact on eEPCs while it is known that mechanical stress increase its secretion, this type of stress is implicated with hypertension. To leave out ET-1 from the cEPCs function in CVD risk equates to leaving out Thrombin from the coagulation cascade. ElectEagle Version I corrects that ommission.


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Part II is based on the following:

Cyrus V Desouza, Lindsey Rentschler, and Vivian Fonseca

Peroxisome proliferator-activated receptors as stimulants of angiogenesis in cardiovascular disease and diabetes Diabetes Metab Syndr Obes. 2009; 2: 165–172. Published online 2009 September 25 PMCID: PMC3048019


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Inhibition of ET-1, ETA and ETA-ETB, Induction of NO production, stimulation of eNOS and Treatment Regime with PPAR-gamma agonists (TZD): cEPCs Endogenous Augmentation for Cardiovascular Risk Reduction – A Bibliography



Additional References to Studies on PPAR-Gamma


Repository on BioInfoBank Library on Peroxisome proliferator-activated receptor



Repository on Science.gov on Peroxisome proliferator-activated receptor



On this Open Access OnLine Scientific Journal, Dr. Lev-Ari’s research on Pharmaco-Therapy of Cardiovascular Diseases includes the following:


Lev-Ari, A., (2012 X). Clinical Trials Results for Endothelin System: Pathophysiological role in Chronic Heart Failure, Acute Coronary Syndromes and MI – Marker of Disease Severity or Genetic Determination?



Lev-Ari, A., (2012W). Endothelin Receptors in Cardiovascular Diseases: The Role of eNOS Stimulation



Lev-Ari, A., (2012V). Inhibition of ET-1, ETA and ETA-ETB, Induction of NO production, stimulation of eNOS and Treatment Regime with PPAR-gamma agonists (TZD): cEPCs Endogenous Augmentation for Cardiovascular Risk Reduction – A Bibliography



Lev-Ari, A., (2012U). Cardiovascular Outcomes: Function of circulating Endothelial Progenitor Cells (cEPCs): Exploring Pharmaco-therapy targeted at Endogenous Augmentation of cEPCs


Lev-Ari, A., (2012T). Endothelial Dysfunction, Diminished Availability of cEPCs, Increasing CVD Risk for Macrovascular Disease – Therapeutic Potential of cEPCs


Lev-Ari, A., (2012S). Vascular Medicine and Biology: CLASSIFICATION OF FAST ACTING THERAPY FOR PATIENTS AT HIGH RISK FOR MACROVASCULAR EVENTS Macrovascular Disease – Therapeutic Potential of cEPCs


Lev-Ari, A. (2012L).. Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production


Lev-Ari, A. (2012a). Resident-cell-based Therapy in Human Ischaemic Heart Disease: Evolution in the PROMISE of Thymosin beta4 for Cardiac Repair


Lev-Ari, A. (2012b). Triple Antihypertensive Combination Therapy Significantly Lowers Blood Pressure in Hard-to-Treat Patients with Hypertension and Diabetes


Lev-Ari, A. (2012h). Macrovascular Disease – Therapeutic Potential of cEPCs: Reduction Methods for CV Risk


Lev-Ari, A. (2012j) Mitochondria Dysfunction and Cardiovascular Disease – Mitochondria: More than just the “powerhouse of the cell”


Lev-Ari, A. (2012i). Bystolic’s generic Nebivolol – positive effect on circulating Endothelial Proginetor Cells endogenous augmentation



Electronic versions NOT available for:

Lev-Ari, A. & Abourjaily, P. (2006a) “An Investigation of the Potential of circulating Endothelial Progenitor Cells (cEPC) as a Therapeutic Target for Pharmacologic Therapy Design for Cardiovascular Risk Reduction.”Part I: Macrovascular Disease – Therapeutic Potential of cEPCs – Reduction methods for CV risk. Part II: (2006b) Therapeutic Strategy for cEPCs Endogenous Augmentation: A Concept-based Treatment Protocol for a Combined Three Drug Regimen. Part III: (2006c) Biomarker for Therapeutic Targets of Cardiovascular Risk Reduction by cEPCs Endogenous Augmentation using New Combination Drug Therapy of Three Drug Classes and Several Drug Indications. Northeastern University, Boston, MA 02115

Lev-Ari, A. (2007) Heart Vasculature Regeneration and Protection of Coronary Artery Endothelium and Smooth Muscle: A Concept-based Pharmacological Therapy of a Combined Three Drug Regimen. Bouve College of Health Sciences, Northeastern University, Boston, MA 02115




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