Posts Tagged ‘Adenosine triphosphate’

Mitochondrial Metabolism and Cardiac Function

Curator: Larry H Bernstein, MD, FACP

This article is the SECOND in a four-article Series covering the topic of the Roles of the Mitochondria in Cardiovascular Diseases. They include the following;

The mitochondrion serves a critical role as a platform for
  • energy transduction,
  • signaling, and
  • cell death pathways
relevant to common diseases of the myocardium such as heart failure. This review focuses on the molecular regulatory events involved in mitochondrial energy metabolism.
This is followed by the derangements known to occur in the development of heart failure.

 Cardiac Energy Metabolism

All cellular processes are driven by ATP-dependent pathways. The heart has perpetually high energy demands related to
  • the maintenance of specialized cellular processes, including
    • ion transport,
    • sarcomeric function, and
    • intracellular Ca2+ homeostasis.
Myocardial workload (energy demand) and energy substrate availability (supply) are in continual flux. Thus, ATP-generating pathways must

  • respond proportionately to dynamic fluctuations in physiological demands and fuel delivery.
In order to support contractile activity, the human heart requires
  • a daily synthesis of approximately 30kg of ATP, via
    • oxidative phosphorylation at
    • the inner mitochondrial membrane.
These metabolic  processes are regulated, involving
  • allosteric control of enzyme activity,
  • signal transduction events, and
  • the activity of genes encoding
    • rate-limiting enzymes and proteins.
Catabolism of exogenous substrates ,such as
  • fatty acids,
  • glucose,
  • pyruvate,
  • lactate and
  • ketone bodies,
generates most of the reduced compounds,
  • NADH (nicotinamide adenine dinucleotide, reduced) and
  • FADH2 (ﬂavin adenine dinucleotide, reduced),
which are necessary for mitochondrial electron transport (Fig. 1).
Fig 1  Fatty acid beta-oxidation and the Krebs cycle produce
  1. nicotinamide adenine dinucleotide, reduced (NADH) and
  2. ﬂavin adenine dinucleotide, reduced(FADH2),
which are oxidized by complexes I and II, respectively, of
Electrons are transferred through the chain to the ﬁnal acceptor, namely oxygen(O2).
The free energy from electron transfer
  1. is used to pump hydrogen out of the mitochondria and
  2. generate an electrochemical gradient across the inner mitochondrial membrane.
This gradient is the driving force for ATP synthesis via the ATP synthase. Alternatively,
H can enter the mitochondria by a mechanism not coupled to ATP synthesis, via
  • the uncoupling proteins(UCPs), which results in the dissipation of energy.

[ANT, adenine nucleotide translocase; CoA, coenzymeA; FAT, fatty acid transporter; GLUT, glucose transporter;

NAD, nicotinamide adenine dinucleotide; TCA, tricarboxylic acid].

Cardiac Energy Metabolic Pathways

 Oxidation of free fatty acids (FFAs) and glucose in mitochondria
  • accounts for the vast majority of ATP generation in the healthy adult heart.
FFAs are the preferred substrate in the adult myocardium,
  • supplying 70-90% of total ATP.
FAs derived from circulating triglyceride-rich lipoproteins and albumin bound nonesterified FAs
  • are oxidized in the mitochondrial matrix by the process of beta-oxidation (FAO), whereas
pyruvate derived from glucose and lactate
  • is oxidized by the pyruvate-dehydrogenase (PDH) complex,
    • localized within the inner mitochondrial membrane.
Acetyl-CoA, derived from both pathways,
  • enters the tricarboxylic acid (TCA) cycle.
Reduced flavin adenine dinucleotide (FADH2) and NADH are generated, respectively, via
  • substrate flux through the
The reducing equivalents enter the electron transport (ET) chain,
  • producing an electrochemical gradient across the mitochondrial membrane
  • that drives ATP synthesis in the presence of molecular oxygen (oxidative phosphorylation).
The relative contributions of each of these substrates are determined
  • by their availability
  • cardiac workload and
  • hormonal status
In the healthy, normal heart, the ATP requirement is largely met in the actively metabolic mitochondria by
  • the catabolism of free fatty acids (FFAs) via beta-oxidation,
  • the tricarboxylic acid cycle and
  • oxidative phosphorylation
giving rise to a greater ATP yield per C2 unit than with glucose.
The relative contribution of glucose to the mitochondrial acetyl-coenzyme A (CoA) pool increases
  • during the postprandial period,
    • when the heart is insulin stimulated, and
  • during exercise
  • hypoxia, or
  • ischemia
when glucose is favored as a more oxygen-efﬁcient substrate than
  • FFAs (greater ATP yield per oxygen molecule consumed).
Substrate switching in the heart can also be achieved by
  • acute alterations in transcriptional regulation of key metabolic enzymes
  • in response to alterations in substrate levels and oxygen availability, or
  • indeed by the intracellular circadian clock.
This continual process of ﬁne adjustment in fuel selection
  • allows cardiac mitochondria to function
  • under a range of metabolic conditions to meet the high energy demands of the heart.
Mitochondrial enzymes are encoded by both nuclear and mitochondrial genes.
All of the enzymes of
  1. beta-oxidation and the TCA cycle, and
  2. most of the subunits of Electron Transfer/Oxidative Phosphorylation,
    • are encoded by nuclear genes.
The mitochondrial genome is comprised of
  • 1 circular double-stranded chromosome that encodes
  • 13 ET chain subunits within complexes I, III, and IV.
Since mitochondrial number and function require both nuclear and mitochondrial-encoded genes,
  • coordinated mechanisms exist to regulate the 2 genomes and
  • determine overall cardiac oxidative capacity.
In addition, distinct pathways exist to coordinately regulate
  • nuclear genes encoding component mitochondrial pathways.

Early Postnatal Low-protein Nutrition, Metabolic Programming and
the Autonomic Nervous System in Adult Life.

JC de Oliveira, S Grassiolli, C Gravena, PCF de Mathias  Nutr Metab. 2012;9(80)

The developmental origins of health and disease (DOHaD) hypothesis stipulates that adult metabolic disease

  • may be programmed during the perinatal stage.

A large amount of evidence suggests that the etiology of obesity is not only related to food abundance

  • but also to food restriction during early life.

Protein restriction during lactation has been used as a rat model of metabolic programming

  • to study the impact of perinatal malnutrition on adult metabolism.

In contrast to protein restriction during fetal life, protein restriction during lactation did not appear to cause

  • either obesity or the hallmarks of metabolic syndrome, such as hyperinsulinemia, when individuals reached adulthood.

Protein restriction provokes body underweight and hypoinsulinemia.
Hypoinsulinemia programs adult rats to maintain normoglycemia,

  • pancreatic β-cells are less sensitive to secretion stimuli:
  1.  glucose and
  2. cholinergic agents.

These pancreatic dysfunctions are attributed to an imbalance of ANS activity

  • recorded in adult rats that experienced maternal protein restriction

Several studies have reported that the ANS activity is altered in under- or malnourished organisms. After weaning,

  • rats fed a chronically protein-deficient diet exhibited low activity of the vagus nerve,
  • whereas high sympathetic activity was recorded

These data were in agreement with a low insulin response to glucose.
Pancreatic islets isolated from protein-restricted rats showed

  • weak glucose and cholinergic insulin tropic responses
  • suggesting that pancreatic β-cell dysfunction may be attributed to altered ANS activity

Food abundance or restriction with regard to body weight control involves changes in

  • metabolic homeostasis and ANS balance activity.

Although the secretion of insulin by the pancreatic β-cells is increased in people who were overweight,

  • it is diminished in people who were underweight.

Changes in the ANS activity may constitute the mechanisms underlying the β-cell dysfunction:

  • the high PNS tonus observed in obese individuals constantly potentiates insulin secretion,
  • whereas the low activity reported in underweight individuals is associated with a weak cholinergic insulin tropic effect.

Under Nutrition Early in Life and Epigenetic Modifications, Association With Metabolic Diseases Risk

relevant to this issue is the role of epigenetic changes in the increased risk of developing metabolic diseases,

  • such as type 2 diabetes and obesity, later in life.

Epigenetic mechanisms, such as DNA methylation and/or nucleoprotein acetylation/methylation, are

  • crucial to the normal/physiological development of several tissues in mammals, and
  • they involve several mechanisms to guarantee fluctuations of enzymes and other proteins that regulate the metabolism.

The intrauterine phase of development is particularly important for the genomic processes related to genes associated with metabolic pathways.
This phase of life may be particularly important for nutritional disturbance. In humans who experienced the Dutch famine Winter in 1944–1945 and
in rats that were deprived of food in utero, epigenetic modifications were detected in

  • the insulin-like growth factor 2 (IGF2) and
  • pancreatic and duodenal home box 1 (Pdx1),

the major factors involved in pancreas development and pancreatic β-cell maturation.
The pancreas and the pancreatic β-cells develop during the embryonic phase, but the postnatal life is also crucial for

  • the maintenance processes that control the β-cell mass:
  1. proliferation,
  2. neogenesis
  3. apoptosis.

Nutritional Restriction to the Fetus: A Risk of Obesity Onset

If an abundant diet is offered to people who have been undernourished during the perinatal life,

  • this opportunity induces a metabolic shift toward the storage of energy and high fat tissue accumulation

The concept of Developmental Origins of Health and Disease extends to any type of stressful situations that may

  • predispose babies or pups to develop metabolic disorders when they reach adulthood.

Programmed Metabolism and Insulin Secretion-coupling Process

What are the mechanisms involved in the low glucose insulin tropic response observed in low protein-programmed lean rats?
The pancreatic β-cells secrete insulin when stimulated mostly by glucose. However, several nutrients, such as

  • amino acids,
  • fatty acids,
  • and their metabolites,

stimulate cellular metabolism and increase ATP production.

ATP-sensitive potassium channels (KATP) are inactivated by an increased ATP/ADP ratio. This provokes

  • membrane depolarization and
  • the activation of voltage-dependent calcium channels.

These ionic changes increase the intracellular calcium concentration, which is involved in

  • the export of insulin to the bloodstream.

Glucose may also stimulate insulin secretion by alternative pathways involving KATP channels.

Programmed Metabolism and Insulin Tropic Effects of Neurotransmitters

Insulin release is modulated by non-nutrient secretagogues, such as neurotransmitters, which

  • enhance or inhibit glucose-stimulated insulin secretion.

Pancreatic β-cells contain several receptors for neurotransmitters and Neuropeptide, such as

  • adrenoceptors and cholinergic muscarinic receptors (mAChRs).

These receptors are stimulated by efferent signals from the central nervous system, including the ANS,

  • throughout their neural ends for pancreatic β-cells.

During blood glucose level oscillations, the β-cells receive inputs from

  • the parasympathetic and sympathetic systems to participate in glycemic regulation.

Overall, acetylcholine promotes the potentiation of glucose-induced insulin secretion,

  • whereas noradrenaline and adrenaline inhibit this response.

Functional studies of mAChR subtypes have revealed that M1 and particularly M3 are the receptors that are involved in

  • the insulin tropic effect of acetylcholine.

Interestingly, it was reported that M3mAChR gene knockout mice are

  • underweight,
  • hypophagic and
  • hypoinsulinemic,

as are adult rats that were protein-restricted during lactation.
The pancreatic islets from M3mAChR mice (-/-) showed a reduced secretory response to cholinergic agonists.
In studies using transgenic mice in which the pancreatic β-cell M3mAChRs are chronically stimulated,

  • an improvement of glycemic control has been observed

Adult male rat offspring from whose mothers were protein-restricted during lactation

  • exhibit a low PNS activity.

Evidence suggests that ANS changes may contribute to the impairment of glycemic homeostasis in metabolically programmed rats.

Pathways involved in cardiac energy metabolism.

FA and glucose oxidation are the main ATP-generating pathways in the adult mammalian heart.
Acetyl-CoA derived from FA and glucose oxidation is
  • further oxidized in the TCA cycle to generate NADH and FADH2, which
  • enter the ET/oxidative phosphorylation pathway and drive ATP synthesis.
Genes encoding enzymes involved at multiple steps of these metabolic pathways
  1. uptake,
  2. esterification,
  3. mitochondrial transport,
  4. and oxidation
are transcriptionally regulated by PGC-1a
  • with its nuclear receptor partners, including PPARs and ERRs .
Glucose uptake/oxidation and electron transport/oxidative phosphorylation pathways are also regulated by PGC-1a via
  • other transcription factors, such as MEF-2 and NRF-1.

[Cyt c, cytochrome c]

 Fetal metabolism of carbohydrate utilization

This reviewer poses the question of whether the fetal cardiac metabolism, which is characterized by a (facultative) anaerobic glycolysis,
  • results in lactate production that is not redirected into the TCA cycle.
An unexamined, but related question is whether there is an associated change in the ratio of
  • mitochondrial to cytoplasmic malate dehydrogenase isoenzyme activity (m-MDH:c-MDH).
The fetal heart operates without oxygenation from a functioning lung, bathed in amniotic fluid.
An enzymatic feature might be expressed in a facultative anaerobic cytplasmic glycolytic pathway characterized by
  • a decrease in the h-type lactate dehydrogenase (LD) isoenzyme(s) (LD1, LD2) with a predominance of
  • the m-type LD isoenzymes (LD3, LD4, LD5).
The observation here is that the heart muscle is a syncytium, and it functions at a highly regulated rate,
  • not with the spurts of activity seen in skeletal muscle.
In another article in this series, there are morphological changes that occur in the heart mitochondria, and
  • there are three locations, as if the organelle itself were an organ.
The normal functioning myocardium can utilize lactic acid accumulated in the bloodstream during extreme exercise as fuel.
This is a virtue of mitochondrial function.  There is a significant functional difference between the roles of the h- and m-type LD isoenzymes.
The h-type is a regulatory enzyme that forms a complex as NADH is converted to NAD+ between the
  • LD (H4, H3M; LD1, LD2),
  • oxidized pyridine nucleotide coenzyme, and
  • pyruvate
The complex forms in 200 msec as observed in the Aminco-Morrow stop-flow analyzer.  This is not the case for the m-type isoenzyme.
I presume that it is not a factor in embryonic heart.  It would become a factor after birth with the expansion of the lungs.
This would also bring to the discussion the effect of severe restrictive lung disease on cardiac metabolism.

Related References:

LH Bernstein,  patents: Malate dehydrogenase method,  The lactate dehydrogenase method
LH Bernstein, J Everse. Determination of the isoenzyme levels of lactate dehydrogenase. Methods Enzymol 1975; 41 47-52    ICID: 825516
LH Bernstein, J Everse, N Shioura, PJ Russell. Detection of cardiac damage using a steady state assay for lactate dehydrogenase isoenzymes in serum.   J Mol Cell Cardiol 1974; 6(4):297-315  ICID: 825597
LH Bernstein, MB Grisham, KD Cole, J Everse . Substrate inhibition of the mitochondrial and cytoplasmic malate dehydrogenases. J Biol Chem 1978; 253(24):8697-8701. ICID: 825513
R Belding, L Bernstein, G Reynoso. An evaluation of the immunochemical LD1 method in routine clinical practice. Clin Chem 1981; 27(10):1027-1028.   ICID: 844981
J Adan, L H Bernstein, J Babb. Lactate dehydrogenase isoenzyme-1/total ratio: accurate for determining the existence of myocardial infarction. Clin Chem 1986; 32(4):624-628.  ICID: 825540
MB Grisham, LH Bernstein, J Everse. The cytoplasmic malate dehydrogenase in neoplastic tissues; presence of a novel isoenzyme? Br J Cancer 1983; 47(5):727-731. ICID: 825551
LH Bernstein, P Scinto. Two methods compared for measuring LD-1/total LD activity in serum. Clin Chem 1986; 32(5):792-796.   ICID: 825581

PGC-1a: an inducible integrator of transcriptional circuits

 The PPAR³ coactivator-1 (PGC-1) family of transcriptional coactivators is involved in regulating mitochondrial metabolism and biogenesis.
PGC-1a was the first member discovered through its functional interaction with the nuclear receptor PPAR³ in brown adipose tissue (BAT).
There are two PGC-1a related coactivators,
  1. PGC-1² (also called PERC) and
  2. PGC-1–related coactivator (PRC).
PRC coactivates transcription in mitochondrial biogenesis, with PGC-1a and PGC-1² . Both are expressed in tissues with high oxidative capacity, such as
  1. heart
  2. slow-twitch skeletal muscle, and
  3. BAT
They serve critical roles in the regulation of mitochondrial functional capacity. PGC-1a  also regulates
  • hepatic gluconeogenesis and
  • skeletal muscle glucose uptake.
PGC-1² appears to be important in regulating energy metabolism in the heart, but
  • PGC-1a is distinct from other PGC-1 family members, indeed from most coactivators, in its broad responsiveness to
  1. developmental alterations in energy metabolism and
  2. physiological and pathological cues at the level of expression and transactivation.
In the heart, PGC-1a expression increases at birth coincident with an increase in cardiac oxidative capacity and
  • a perinatal shift from reliance on glucose metabolism to the oxidation of fats for energy.
PGC-1a is induced by physiological stimuli that increase ATP demand and
  • stimulate mitochondrial oxidation, including
  1. cold exposure,
  2. fasting, and
  3. exercise.
Activation of this regulatory cascade increases cardiac mitochondrial oxidative capacity in the heart. In cardiac myocytes in culture, it
  1. increases mitochondrial number,
  2. upregulates expression of mitochondrial enzymes, and
  3. increases rates of FA oxidation and coupled respiration.
Thus, PGC-1a is an inducible coactivator that coordinately regulates
  • cardiac fuel selection and
  • mitochondrial ATP-producing capacity.
 PGC-1a activates expression of nuclear respiratory factor-1 (NRF-1) and NRF-2 and
  • directly coactivates NRF-1 on its target gene promoters.
NRF-1 and NRF-2 regulate expression of mitochondrial transcription factor A (Tfam),
  • a nuclear-encoded transcription factor that binds regulatory sites on mitochondrial DNA and is essential for
  1. replication,
  2. maintenance, and
  3. transcription of the mitochondrial genome.
Furthermore, NRF-1 and NRF-2 regulate the expression of nuclear genes encoding
  • respiratory chain subunits and other proteins required for mitochondrial function.
PGC-1a  also
  • coactivates the PPAR and ERR nuclear receptors, critical regulators of myocardial FFA utilization.
  • regulates genes involved in the cellular uptake and mitochondrial oxidation of FFAs.
  • is an integrator of the transcriptional network regulating mitochondrial biogenesis and function.
Numerous signaling pathways, by increasing either PGC-1a expression or activity, such as –
  • Ca2+-dependent,
  • NO,
  • MAPK, and
  • beta-adrenergic pathways (beta3/cAMP),
    • activate the PGC-1a directly
Additionally, the p38_MAPK pathway
  • selectively activates PPARa, which may bring about synergistic activation in the presence of PGC-1a,
  • whereas ERK-MAPK has the opposite effect.
These signaling pathways transduce physiological stimuli to the PGC-1a pathway:
  1. stress
  2. fasting
  3. exercise
PGC-1a, in turn, coactivates transcriptional partners,which regulate mitochondrial biogenesis and FA-oxidation pathways:
  • NRF-1 and -2,
  • ERRa, and
  • PPARa,
 Insights into the physiological responsiveness of the PGC-1a pathway come from
  • identification of signal transduction pathways that modulate the activity of PGC-1a and its downstream partners.
PGC-1a is upregulated in response to beta-adrenergic signaling, consistent with the involvement of this pathway in thermogenesis.
The stress-activated  p38_MAPK activates PGC-1a by increasing PGC-1a protein stability and promoting dissociation of a repressor.
p38 increases mitochondrial FAO through selective activation of the PGC-1a partner, PPARa. Conversely, the ERK-MAPK pathway
  • inactivates the PPARa/RXRa complex via direct phosphorylation.
Therefore, distinct limbs of the MAPK pathway exert
  • opposing regulatory influences on the PGC-1a cascade.
Recently, NO has emerged as a novel signaling molecule proposed to integrate pathways involved in
  • regulating mitochondrial biogenesis by inducing mitochondrial proliferation.

 A Paradox

Mitochondria are like little cells within our cells. They are the energy producing organelles of the body. The more energy a certain tissue requires
  • such as the brain and the heart
    • the more mitochondria those cells contain.
Conventional transmission electron microscopy of mammalian cardiac tissue reveals mitochondria to be
  1. elliptical individual organelles situated either in clusters beneath the sarcolemma (subsarcolemmal mitochondria, SSM) or
  2. in parallel, longitudinal rows ensconced within the contractile apparatus (interfibrillar mitochondria, IFM).
The two mitochondrial populations differ in their cristae morphology, with
  1. a lamelliform orientation in SSM, whereas
  2. the cristae orientation in IFM is tubular.
The morphology of mitochondria is responsive to changes in cardiomyocytes.
 Mitochondrial oxidative phosphorylation relies
  • not only on the activities of individual complexes, but also on
  • the coordinated action of supramolecular assemblies (respirasomes) of the electron transport chain (ETC) complexes
in both normal and failing heart.
Mitochondria have their own set of DNA and
  • the more energy they generate,
  • the more DNA-damaging free radicals they produce.
Mitochondrial DNA damage is incurred by generation of energy in ATP production, so that
  • the process that sustains life also is the source of toxic damage that causes the dysfunction and mitogeny in the cell.
In human mtDNA mutant cybrids with impaired mitochondrial respiration, the recovery of mitochondrial function
  • correlates with the formation of respirasomes suggesting that
  • respirasomes represent regulatory units of mitochondrial oxidative phosphorylation
    • by facilitating the electron transfer between the catalytic sites of the ETC.
We recently reported a decrease in mitochondrial respirasomes in CHF that fits in the category of a new mitochondrial cytopathy.
 ATP utilized by the heart is synthesized mainly by means of oxidative phosphorylation in the inner mitochondrial membrane,
  • a process that involves the coupling of electron transfer and oxygen consumption with phosphorylation of ADP to ATP.
The catabolism of exogenous substrates (FAs, glucose, pyruvate, lactate, and ketone bodies) provides the reduced intermediates,
  1. NADH (nicotinamide adenine dinucleotide, reduced) and
  2. FADH2 (flavin adenine dinucleotide, reduced),
as donors for mitochondrial electron transport.
The contribution of glucose to the acetyl CoA pool in the heart is
  • increased by insulin during the postprandial period and during exercise.
 All cells and tissues require
  • adenine,
  • pyridine, and
  • flavin nucleotides for energy
by way of Krebs cycle metabolism of fatty acids and carbohydrate substrates.
If DNA holds the blueprint for the proper function of a cell, then any change in the blueprint will change how the cell functions.
If the mitochondria do not function properly, then they cannot fulfill their role in producing energy:
  •  the cell will lose its ability to function adequately.

 Related articles


Mitochondrial dynamics and cardiovascular diseases    Ritu Saxena
Mitochondrial Damage and Repair under Oxidative Stress   larryhbern
Mitochondria: Origin from oxygen free environment, role in aerobic glycolysis, metabolic adaptation   larryhbern
http://pharmaceuticalintelligence.com/2012/09/26/mitochondria-origin-from-oxygen-free-environment-role-in-aerobic-glycolysis-metabolic-adaptation/ Ca2+ signaling: transcriptional control     larryhbern
http://pharmaceuticalintelligence.com/2013/03/06/ca2-signaling-transcriptional-control/ MIT Scientists on Proteomics: All the Proteins in the Mitochondrial Matrix identified  Aviva Lev-Ari
Ubiquinin-Proteosome pathway, autophagy, the mitochondrion, proteolysis and cell apoptosis  larryhbern
Low Bioavailability of Nitric Oxide due to Misbalance in Cell Free Hemoglobin in Sickle Cell Disease – A Computational Model   Anamika Sarkar
The rationale and use of inhaled NO in Pulmonary Artery Hypertension and Right Sided Heart Failure    larryhbern
Mitochondrial Dysfunction and Cardiac Disorders, Larry H Bernstein, MD, FACP
Clinical Trials Results for Endothelin System: Pathophysiological role in Chronic Heart Failure, Acute Coronary Syndromes and MI – Marker of Disease Severity or Genetic Determination? Aviva Lev-Ari, PhD, RN 10/19/2012
Endothelin Receptors in Cardiovascular Diseases: The Role of eNOS Stimulation, Aviva Lev-Ari, PhD, RN 10/4/2012
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, Aviva Lev-Ari, PhD, RN 10/4/2012
Genomics & Genetics of Cardiovascular Disease Diagnoses: A Literature Survey of AHA’s Circulation Cardiovascular Genetics, 3/2010 – 3/2013, L H Bernstein, MD, FACP and Aviva Lev-Ari,PhD, RN  3/7/2013
Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production, Aviva Lev-Ari, PhD, RN 7/19/2012
Cardiovascular Risk Inflammatory Marker: Risk Assessment for Coronary Heart Disease and Ischemic Stroke – Atherosclerosis. Aviva Lev-Ari, PhD, RN 10/30/2012
Cholesteryl Ester Transfer Protein (CETP) Inhibitor: Potential of Anacetrapib to treat Atherosclerosis and CAD, Aviva Lev-Ari, PhD, RN 4/7/2013
Hypertriglyceridemia concurrent Hyperlipidemia: Vertical Density Gradient Ultracentrifugation a Better Test to Prevent Undertreatment of High-Risk Cardiac Patients, Aviva Lev-Ari, PhD, RN  4/4/2013 https://pharmaceuticalintelligence.com/2013/04/04/hypertriglyceridemia-concurrent-hyperlipidemia-vertical-density-gradient-ultracentrifugation-a-better-test-to-prevent-undertreatment-of-high-risk-cardiac-patients/
Fight against Atherosclerotic Cardiovascular Disease: A Biologics not a Small Molecule – Recombinant Human lecithin-cholesterol acyltransferase (rhLCAT) attracted AstraZeneca to acquire AlphaCore, Aviva Lev-Ari, PhD, RN 4/3/2013
High-Density Lipoprotein (HDL): An Independent Predictor of Endothelial Function & Atherosclerosis, A Modulator, An Agonist, A Biomarker for Cardiovascular Risk, Aviva Lev-Ari, PhD, RN 3/31/2013 

Peroxisome proliferator-activated receptor (PPAR-gamma) Receptors Activation: PPARγ transrepression for Angiogenesis in Cardiovascular Disease and PPARγ transactivation for Treatment of Diabetes, Aviva Lev-Ari, PhD, RN 11/13/2012
Sulfur-Deficiciency and Hyperhomocysteinemia, L H Bernstein, MD, FACP



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Author: Larry H. Bernstein, MD, FCAP  

Is the Warburg Effect the Cause or the Effect of Cancer A 21st Century View?

Word Cloud by Daniel Menzin

A Critical Review

What is the Warburg effect?

“Warburg Effect” describes the preference of glycolysis and lactate fermentation rather than oxidative phosphorylation for energy production in cancer cells. Mitochondrial metabolism is an important and necessary component in the functioning and maintenance of the organelle, and accumulating evidence suggests that dysfunction of mitochondrial metabolism plays a role in cancer. Progress has demonstrated the mechanisms of the mitochondrial metabolism-to-glycolysis switch in cancer development and how to target this metabolic switch.
In vertebrates, food is digested and supplied to cells mainly in the form of glucose. Glucose is broken down further to make Adenosine Triphosphate (ATP) by two pathways. One is via anaerobic metabolism occurring in the cytoplasm, also known as glycolysis. The major physiological significance of glycolysis lies in making ATP quickly, but in a minuscule amount. The breakdown process continues in the mitochondria via the Krebs’s cycle coupled with oxidative phosphorylation, which is more efficient for ATP production. Cancer cells seem to be well-adjust to glycolysis. In the 1920s, Otto Warburg first proposed that cancer cells show increased levels of glucose consumption and lactate fermentation even in the presence of ample oxygen (known as “Warburg Effect”). Based on this theory, oxidative phosphorylation switches to glycolysis which promotes the proliferation of cancer cells. Many studies have demonstrated glycolysis as the main metabolic pathway in cancer cells.
Why cancer cells prefer glycolysis, an inefficient metabolic pathway?

It is now accepted that glycolysis provides cancer cells with the most abundant extracellular nutrient, glucose, to make ample ATP metabolic intermediates, such as ribose sugars, glycerol and citrate, nonessential amino acids, and the oxidative pentose phosphate pathway, which serve as building blocks for cancer cells.
Since, cancer cells have increased rates of aerobic glycolysis, investigators argue over the function of mitochondria in cancer cells. Mitochondrion, a one of the smaller organelles, produces most of the energy in the form of ATP to supply the body. In Warburg’s theory, the function of cellular mitochondrial respiration is dampened and mitochondria are not fully functional. There are many studies backing this theory. A recent review on hypoxia nicely summarizes some current studies and speculates that the “Warburg Effect” provides a benefit to the tumor not by increasing glycolysis but by decreasing mitochondrial activity.
Glycolysis is enhanced and beneficial to cancer cells. The mammalian target of rapamycin (mTOR) has been well discussed in its role to promote glycolysis; recent literature has revealed some new mechanisms of how glycolysis is promoted during skin cancer development.
On the other hand, Akt is not only involved in the regulation of mitochondrial metabolism in skin cancer but also of glycolysis. Activation of Akt has been found to phosphorylate FoxO3a, a downstream transcription factor of Akt, which promotes glycolysis by inhibiting apoptosis in melanoma. In addition, activated Akt is also associated with stabilized c-Myc and activation of mTOR, which both increase glycolysis for cancer cells.
Nevertheless, ras mutational activation prevails in skin cancer. Oncogenic ras induces glycolysis. In human squamous cell carcinoma, the c-Jun NH(2)-terminal Kinase (JNK) is activated as a mediator of ras signaling, and is essential for ras-induced glycolysis, since pharmacological inhibitors if JNK suppress glycolysis. CD147/basigin, a member of the immunoglobulin superfamily, is high expressed in melanoma and other cancers.
Glyoxalase I (GLO1) is a ubiquitous cellular defense enzyme involved in the detoxification of methylglyoxal, a cytotoxic byproduct of glycolysis. In human melanoma tissue, GLO1 is upregulated at both the mRNA and protein levels.
Knockdown of GLO1 sensitizes A375 and G361 human metastatic melanoma cells to apoptosis.
The transcription factor HIF-1 upregulates a number of genes in low oxygen conditions including glycolytic enzymes, which promotes ATP synthesis in an oxygen independent manner. Studies have demonstrated that hypoxia induces HIF-1 overexpression and its transcriptional activity increases in parallel with the progression of many tumor types. A recent study demonstrated that in malignant melanoma cells, HIF-1 is upregulated, leading to elevated expression of Pyruvate Dehydrogenase Kinase 1 (PDK1), and downregulated mitochondrial oxygen consumption.
The M2 isoform of Pyruvate Kinase (PKM2), which is required for catalyzing the final step of aerobic glycolysis, is highly expressed in cancer cells; whereas the M1 isoform (PKM1) is expressed in normal cells. Studies using the skin cell promotion model (JB6 cells) demonstrated that PKM2 is activated whereas PKM1 is inactivated upon tumor promoter treatment. Acute increases in ROS inhibited PKM2 through oxidation of Cys358 in human lung cancer cells. The levels of ROS and stage of tumor development may be pivotal for the role of PKM2.

Mitochondrial metabolism and glycolysis targeting for cancer drug delivery
In cancer cells including skin cancer cells, the metabolic shift is composed of increased glycolysis, activation of anabolic pathways including amino acid and pentose phosphate production, and increased fatty acid biosynthesis. More and more studies have converged on particular glycolytic and mitochondrial metabolic targets for cancer drug discovery.
A marker for increased glycolysis in melanoma is the elevated levels of Lactate Dehydrogenase (LDH) in the blood of patients with melanoma, which has proven to be an accurate predictor of prognosis and response to treatments. LDH converts pyruvate, the final product of glycolysis, to lactate when oxygen is absent. High concentrations of lactate, in turn, negatively regulate LDH. Therefore, targeting acid excretion may provide a feasible and effective therapeutic approach for melanoma. For instance, JugloSne, a main active component in walnut, has been used in traditional medicines. Studies have shown that Juglone causes cell membrane damage and increased LDH levels in a concentration-dependent manner in cultured melanoma cells. As one of the rate-limiting enzyme of glycolysis, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isozyme 3 (PFKFB3) is activated in neoplastic cells. Studies have confirmed that an inhibitor of PFKFB3, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), suppresses glycolysis in neoplastic cells. In melanoma cell lines, the concentrations of Fru-2, 6-BP, lactate, ATP, NAD+, and NADH are diminished by 3PO. Therefore, targeting PFKFB3 using 3PO and other PFKFB3 specific inhibitors could be effective in melanoma chemotherapy.
A new NO (nitric oxide) donating compound [(S,R)-3-phenyl-4,5-dihydro-5-isoxazole acetic acid–nitric oxide (GIT-27NO)] has been tested in treating melanoma cells. The results suggest that GIT-27/NO causes a dose-dependent reduction of mitochondrial respiration in treated A375 human melanoma cells.

At least two mitochondrial enzymes are affected by angiostatin which include malate dehydrogenase, a member of the Kreb’s cycle enzymes; and adenosine triphosphate synthase. Both are identified potential angiostatin-binding partners. Treated with angiostatin, the ATP concentrations of A2058 cells were decreased. Meanwhile, using siRNA of these two enzymes also inhibited the ATP production. PKM2 is up regulated in the early stage of skin carcinogenesis, therefore, targeting PKM2 could serve as a new approach for skin cancer prevention and therapy.
The signaling pathways critical for this glycolytic activation could serve as preventive and therapeutic targets for human skin cancer.

The Historical Challenge posed by the Warburg Hypothesis.

In a landmark review, six essential alterations in cell physiology could underlie malignant cell growth. These six alterations were described as the hallmarks of nearly all cancers and included,

  • self-sufficiency in growth signals,
  • insensitivity to growth inhibitory (antigrowth) signals,
  • evasion of programmed cell death (apoptosis),
  • limitless replicative potential,
  • sustained vascularity (angiogenesis), and
  • tissue invasion and metastasis.

Genome instability, leading to increased mutability, was considered the essential enabling characteristic for manifesting the six hallmarks. The loss of genomic “caretakers” or “guardians”, involved in sensing and repairing DNA damage, was proposed to explain the increased mutability of tumor cells. The loss of these caretaker systems would allow genomic instability thus enabling pre-malignant cells to reach the six essential hallmarks of cancer.
In addition to the six recognized hallmarks of cancer, aerobic glycolysis or the Warburg effect is also a robust metabolic hallmark of most tumors. Aerobic glycolysis in cancer cells involves elevated glucose uptake with lactic acid production in the presence of oxygen. This metabolic phenotype is the basis for tumor imaging using labeled glucose analogues and has become an important diagnostic tool for cancer detection and management. Genes for glycolysis are overexpressed in the majority of cancers examined.
Warburg proposed with considerable certainty and insight that irreversible damage to respiration was the prime cause of cancer. Warburg’s biographer, Hans Krebs, mentioned that Warburg’s idea on the primary cause of cancer, i.e., the replacement of respiration by fermentation (glycolysis), was only a symptom of cancer and not the cause. While there is renewed interest in the energy metabolism of cancer cells, it is widely thought that the Warburg effect and the metabolic defects expressed in cancer cells arise primarily from genomic mutability selected during tumor progression. Emerging evidence, however, questions the genetic origin of cancer and suggests that cancer is primarily a metabolic disease.
Abnormal metabolism of tumors, a selective advantage
The initial observation of Warburg 1916 on tumor glycolysis with lactate production is still a crucial observation. Two fundamental findings complete the metabolic picture:

  • the discovery of the M2 pyruvate kinase (PK) typical of tumors
  • and the implication of tyrosine kinase signals and subsequent phosphorylations in the M2 PK blockade.

A typical feature of tumor cells is a glycolysis associated to an inhibition of apoptosis. Tumors overexpress the high affinity hexokinase 2, which strongly interacts with the mitochondrial ANT-VDAC-PTP complex. In this position, close to the ATP/ADP exchanger (ANT), the hexokinase receives efficiently its ATP substrate. As long as hexokinase occupies this mitochondria site, glycolysis is efficient. However, this has another consequence, hexokinase pushes away from the mitochondria site the permeability transition pore (PTP), which inhibits the release of cytochrome C, the apoptotic trigger. The site also contains a voltage dependent anion channel (VDAC) and other proteins. The repulsion of PTP by hexokinase would reduce the pore size and the release of cytochrome C. Thus, the apoptosome-caspase proteolytic structure does not assemble in the cytoplasm. The liver hexokinase or glucokinase, is different it has less interaction with the site, has a lower affinity for glucose; because of this difference, glucose goes preferentially to the brain.
Further, phosphofructokinase gives fructose 1-6 bisphosphate; glycolysis is stimulated if an allosteric analogue, fructose 2-6 bis phosphate increases in response to a decrease of cAMP. The activation of insulin receptors in tumors has multiple effects, among them; a decrease of cAMP, which will stimulate glycolysis. Another control point is glyceraldehyde P dehydrogenase that requires NAD+ in the glycolytic direction. If the oxygen supply is normal, the mitochondria malate/aspartate (MAL/ASP) shuttle forms the required NAD+ in the cytosol and NADH in the mitochondria. In hypoxic conditions, the NAD+ will essentially come via lactate dehydrogenase converting pyruvate into lactate. This reaction is prominent in tumor cells; it is the first discovery of Warburg on cancer.
At the last step of glycolysis, pyruvate kinase (PK) converts phospho-enolpyruvate (PEP) into pyruvate, which enters in the mitochondria as acetyl- CoA, starting the citric acid cycle and oxidative metabolism. To explain the PK situation in tumors we must recall that PK only works in the glycolytic direction, from PEP to pyruvate, which implies that gluconeogenesis uses other enzymes for converting pyruvate into PEP. In starvation, when cells need glucose, one switches from glycolysis to gluconeogenesis and ketogenesis; PK and pyruvate dehydrogenase (PDH) are off, in a phosphorylated form, presumably following a cAMP-glucagon-adrenergic signal. In parallel, pyruvate carboxylase (Pcarb) becomes active.
Moreover, in starvation, much alanine comes from muscle protein proteolysis, and is transaminated into pyruvate. Pyruvate carboxylase first converts pyruvate to OAA and then, PEP carboxykinase converts OAA to PEP etc…, until glucose. The inhibition of PK is necessary, if not one would go back to pyruvate. Phosphorylation of PK, and alanine, inhibit the enzyme.
PK and a PDH of tumors are inhibited by phosphorylation and alanine, like for gluconeogenesis, in spite of an increased glycolysis! Moreover, in tumors, one finds a particular PK, the M2 embryonic enzyme [2,9,10] the dimeric, phosphorylated form is inactive, leading to a “bottleneck “. The M2 PK has to be activated by fructose 1-6 bis P its allosteric activator, whereas the M1 adult enzyme is a constitutive active form. The M2 PK bottleneck between glycolysis and the citric acid cycle is a typical feature of tumor cell glycolysis.
Above the bottleneck, the massive entry of glucose accumulates PEP, which converts to OAA via mitochondria PEP carboxykinase, an enzyme requiring biotine-CO2-GDP. This source of OAA is abnormal, since Pcarb, another biotin-requiring enzyme, should have provided OAA. Tumors may indeed contain “morule inclusions” of biotin-enzyme suggesting an inhibition of Pcarb, presumably a consequence of the maintained citrate synthase activity, and decrease of ketone bodies that normally stimulate Pcarb. The OAA coming via PEP carboxykinase and OAA coming from aspartate transamination or via malate dehydrogenase condenses with acetyl CoA, feeding the elevated tumoral citric acid condensation starting the Krebs cycle.
Thus, tumors have to find large amounts of acetyl CoA for their condensation reaction; it comes essentially from lipolysis and β oxidation of fatty acids, and enters in the mitochondria via the carnitine transporter. This is the major source of acetyl CoA. It is as if the mechanism switching from gluconeogenesis to glycolysis was jammed in tumors, PK and PDH are at rest, like for gluconeogenesis, but citrate synthase is on. Thus, citric acid condensation pulls the glucose flux in the glycolytic direction, which needs NAD+; it will come from the pyruvate to lactate conversion by lactate dehydrogenase (LDH) no longer in competition with a quiescent Pcarb.
Since the citrate condensation consumes acetyl CoA, ketone bodies do not form; while citrate will support the synthesis of triglycerides via ATP citrate lyase and fatty acid synthesis… The cytosolic OAA drives the transaminases in a direction consuming amino acid. The result of these metabolic changes is that tumors burn glucose while consuming muscle protein and lipid stores of the organism. In a normal physiological situation, one mobilizes stores for making glucose or ketone bodies, but not while burning glucose!
The 21st Century Genomic Challenge?
According to the modern understanding of cancer, it is a disease caused by genetic and epigenetic alterations. Although this is now widely accepted, perhaps more emphasis has been given to the fact that cancer is a genetic disease. Numerous studies, including our earlier works, have supported the notion that carcinogenesis involves the activation of tumor-promoting oncogenes and the inactivation of growth-inhibiting tumor suppressor genes. It should be noted that in the post-genome sequencing project period of the 21st century, an in depth investigation of the factors associated with tumorigenesis is required for achieving it. Extensive research is warranted in two areas, namely, tumor bioenergetics and the cancer stem cell (CSC) hypothesis, neither of which received the required attention after the success of the genome sequencing project. An investigation of these two concepts would give rise to a new era in the study of cancer biology. Indeed, recent studies have indicated that the two apparently distinct fields might be related to each other and can converge more rapidly than previously recognized.
Warburg Effect Revisited
Cancer cells rarely depend on mitochondria for respiration and obtain almost half of their ATP by directly metabolizing glucose to lactic acid, even in the presence of oxygen. However, with the discovery that tumors do not show any shift to glycolysis, Warburg’s cancer theory (high lactate production and low mitochondrial respiration in tumor under normal oxygen pressure) was gradually discredited. Otto Warburg won a Nobel Prize in 1931 for the discovery of tumor bioenergetics, which is now commonly used as the basis of positron emission tomography (PET), a highly sensitive noninvasive technique used in cancer diagnosis. The increasing number of recent reports on the Warburg effect has reestablished the significance of this effect in tumorigenesis, indicating that bioenergetics may play a critical role in malignant transformation. Furthermore, it has been reported that TP53, which is one of the most commonly mutated genes in cancer, can trigger the Warburg effect. Glycolytic conversion is initiated in the early stages in cells that are genetically engineered to become cancerous, and the conversion was enhanced as the cells became more malignant. Therefore, the Warburg effect might directly contribute to the initiation of cancer formation not only by enhanced glycolysis but also via decreased respiration in the presence of oxygen, which suppresses apoptosis. This effect may also produce a metabolic shift to enhanced glycolysis and play a role in the early stages of multistep tumorigenesis in vivo.

Cancer Stem Cells (CSC) and Embryonic Stem Cells (ESC)
The importance of the cancer stem cell (CSC) hypothesis in therapy-related resistance and metastasis has been recognized during the past 2 decades. Accumulating evidence suggests that tumor bioenergetics plays a critical role in CSC regulation; this finding has opened up a new era of cancer medicine, which goes beyond cancer genomics.

Embryonic stem (ES) cells and immortalized primary and cancerous cells show a common concerted metabolic shift, including:

  • enhanced glycolysis,
  • decreased apoptosis, and
  • reduced mitochondrial respiration.

This finding reinforces the use of somatic stem cells or metastatic tumor cells in hypoxic niches. Hypoxia appears to regulate the functions of hematopoietic stem cells in the bone marrow and metastatic tumor cells by preserving important stem cell functions, such as:

  • cell cycle control,
  • survival,
  • metabolism, and
  • protection against oxidative stress.

Several companies and laboratories are now attempting to evaluate the bioenergetics associated with tumorigenesis by testing and challenging the available anticancer drugs.

A small population of cancer-initiating cells plays a very important role in current investigations. These CSCs may cause resistance to chemotherapy or radiation therapy or lead to post-therapy recurrence even when most of the cancer cells appear to be dead. In addition to their genetic alterations, CSCs are believed to mimic normal adult stem cells with regard to properties like self-renewal and undifferentiated status, which eventually leads to the formation of differentiated cells. Unlike well-differentiated daughter cells, small populations of CSCs are believed to be more resistant to toxic injuries and chemoradiotherapy. Indeed, the conventional cancer therapies have always been targeted toward proliferating cells. The control of CSCs, which is often exercised in the dormant phase of the cell cycle, can now be applied to achieve complete tumor regression.
Identification of cancer-specific markers
Due to their potential use in clinical applications, the surface markers of CSCs have been studied and identified. Adult stem cells and their malignant counterparts share similar intrinsic and extrinsic factors that regulate the

  • self renewal,
  • differentiation, and
  • proliferation pathways.

The following are the examples of candidate markers: musashi-1 (Msi-1), hairy and enhancer of split homolog-1 (Hes-1), CD133 (prominin-1, Prom1), epithelial cellular adhesion molecule (EpCam), claudin-7,29 CD44 variant isoforms, Lgr5,30Hedgehog (Hh), bone morphogenic protein (Bmp), Notch, and Wnt.
Is cancer a metabolic disease and genomic instability a secondary effect?
Bioenergetics of Cancer Stem Cells
The bioenergetics associated with the adaptation of CSCs to their micro-environment still requires extensive research. Although numerous studied suggested the association between Warburg effect and reduced oxidative stress in cancer, the relevant molecular mechanism was not known until very recently when Ruckenstuhl, et al. reported their findings in a yeast model.

How cancer cells achieve one of the most common phenotypes, namely, the “Warburg effect,” i.e., elevated glycolysis in the presence of oxygen, is still a topic of hypothesis, unless the involvement of glycolysis genes is considered.
The Warburg effect has been observed in differentiating cancer cells (e.g., cells that undergo epithelial-to-mesenchymal and mesenchymal-to-amoeboid transition), cells resistant to anoikis, and cells which interact with the stromal components of the metastatic niche. The epithelial-to-mesenchymal transition is involved in the resistance to chemotherapy in gastrointestinal cancer cells.

Cancer metastasis can be regarded as an integrated “escape program” triggered by redox changes. These alterations might be associated with avoiding oxidative stress in the niche of the tumor cells, or presumably with the response to treatments aimed at genetic targets, such as chemotherapy and radiation.
The introduction of induced pluripotent stem (iPS) cell genes was necessary for inducing the expression of immature status-related proteins in gastrointestinal cancer cells, and that the induced pluripotent cancer (iPC) cells were distinct from natural cancer cells with regard to their sensitivity to differentiation inducing treatment. For the complete eradication of cancer, however, future efforts should be directed toward improving translational research.
Cancer metabolism.
Glycolysis is elevated in tumors, but a pyruvate kinase (PK) “bottleneck” interrupts phosphoenol pyruvate (PEP) to pyruvate conversion. Thus, alanine following muscle proteolysis transaminates to pyruvate, feeding lactate dehydrogenase, converting pyruvate to lactate, (Warburg effect) and NAD+ required for glycolysis. Cytosolic malate dehydrogenase also provides NAD+ (in OAA to MAL direction). Malate moves through the shuttle giving back OAA in the mitochondria. Below the PK-bottleneck, pyruvate dehydrogenase (PDH) is phosphorylated (second bottleneck). However, citrate condensation increases: acetyl-CoA, will thus come from fatty acids b-oxydation and lipolysis, while OAA sources are via PEP carboxy kinase, and malate dehydrogenase, (pyruvate carboxylase is inactive). Citrate quits the mitochondria, (note interrupted Krebs cycle). In the cytosol, ATP citrate lyase cleaves citrate into acetyl CoA and OAA.
Arginine gives ornithine via arginase, ornithine is decarboxylated into putrescine by ornithine decarboxylase. Putrescine and SAM form polyamines (spermine spermidine) via SAM decarboxylase. The other product 5-methylthioadenosine provides adenine. Arginine deprivation should affect tumors. The SAM destruction impairs methylations, particularly of PP2A, removing the “signaling kinase brake”, PP2A also fails to dephosphorylate PK and PDH, forming the “bottlenecks”.

Insulin or IGF actions boost the cellular influx of glucose and glycolysis. However, if the signaling pathway gets out of control, the tyrosine kinase phosphorylations may lead to a parallel PK blockade explaining the tumor bottleneck at the end of glycolysis. Since an activation of enyme kinases may indeed block essential enzymes (PK, PDH and others); in principle, the inactivation of phosphatases may also keep these enzymes in a phosphorylated form and lead to a similar bottleneck and we do know that oncogenes bind and affect PP2A phosphatase. In sum, a perturbed MAP kinase pathway, elicits metabolic features that would give to tumor cells their metabolic advantage.

Warburg effect and the prognostic value of stromal caveolin-1 as a marker of a lethal tumor microenvironment
Cancer cells show a broad spectrum of bioenergetic states, with some cells using aerobic glycolysis while others rely on oxidative phosphorylation as their main source of energy. In addition, there is mounting evidence that metabolic coupling occurs in aggressive tumors, between epithelial cancer cells and the stromal compartment, and between well-oxygenated and hypoxic compartments. We recently showed that oxidative stress in the tumor stroma, due to aerobic glycolysis and mitochondrial dysfunction, is important for cancer cell mutagenesis and tumor progression. More specifically, increased autophagy/mitophagy in the tumor stroma drives a form of parasitic epithelial-stromal metabolic coupling. These findings explain why it is effective to treat tumors with either inducers or inhibitors of autophagy, as both would disrupt this energetic coupling. We also discuss evidence that glutamine addiction in cancer cells produces ammonia via oxidative mitochondrial metabolism.

Ammonia production in cancer cells, in turn, could then help maintain autophagy in the tumor stromal compartment. In this vicious cycle, the initial glutamine provided to cancer cells would be produced by autophagy in the tumor stroma. Thus, we believe that parasitic epithelial-stromal metabolic coupling has important implications for cancer diagnosis and therapy, for example, in designing novel metabolic imaging techniques and establishing new targeted therapies. In direct support of this notion, we identified a loss of stromal caveolin-1 as a marker of oxidative stress, hypoxia, and autophagy in the tumor microenvironment, explaining its powerful predictive value. Loss of stromal caveolin-1 in breast cancers is associated with early tumor recurrence, metastasis, and drug resistance, leading to poor clinical outcome.
The conventional ‘Warburg effect’ versus oxidative mitochondrial metabolism
Warburg’s original work indicated that while glucose uptake and lactate production are greatly elevated, a cancer cell’s rate of mitochondrial respiration is similar to that of normal cells. He, however, described it as a ‘respiratory impairment’ due to the fact that, in cancer cells, mitochondrial respiration is smaller, relative to their glycolytic power, but not smaller relative to normal cells. He recognized that oxygen consumption is not diminished in tumor cells, but that respiration is disturbed because glycolysis persists in the presence of oxygen. Unfortunately, the perception of his original findings was simplified over the years, and most subsequent papers validated that cancer cells undergo aerobic glycolysis and produce lactate, but did not measure mitochondrial respiration, and just presumed decreased tricarboxylic acid (TCA) cycle activity and reduced oxidative phosphorylation [1,2]. It is indeed well documented that, as a consequence of intra-tumoral hypoxia, the hypoxia-inducible factor (HIF)1α pathway is activated in many tumors cells, resulting in the direct up-regulation of lactate dehydrogenase (LDH) and increased glucose consumption.
It is now clear that cancer cells utilize both glycolysis and oxidative phosphorylation to satisfy their metabolic needs. Experimental assessments of ATP production in cancer cells have demonstrated that oxidative pathways play a signifi cant role in energy generation, and may be responsible for about 50 to 80% of the ATP generated. several studies now clearly indicate that mitochondrial activity and oxidative phosphorylation support tumor growth. Loss-of-function mutations in the TCA cycle gene IDH1 (isocitrate dehydrogenase 1) are found in about 70% of gliomas, but, interestingly, correlate with a better prognosis and improved survival, suggesting that severely decreased activity in one of the TCA cycle enzymes does not favor tumor aggressiveness. The mitochondrial protein p32 was shown to maintain high levels of oxidative phosphorylation in human cancer cells and to sustain tumorigenicity in vivo. In addition, STAT3 is known to enhance tumor growth and to predict poor prognosis in human cancers. Interestingly, a pool of STAT3 localizes to the mitochondria, to sustain high levels of mitochondrial respiration and to augment transformation by oncogenic Ras. Similarly, the mitochondrial transcription factor A (TFAM), which is required for mitochondrial DNA replication and oxidative phosphorylation, is also required for K-Ras induced lung tumorigenesis.
There is also evidence that pro-oncogenic molecules regulate mitochondrial function. Cyclin D1 inhibits mitochondrial function in breast cancer cells. Overexpression of cyclin D1 is observed in about 50% of invasive breast cancers and is associated with a good clinical outcome, indicating that inhibition of mitochondrial activity correlates with favorable prognosis. Importantly, it was shown that the oncogene c-Myc stimulates mitochondrial biogenesis, and enhances glutamine metabolism by regulating the expression of mitochondrial glutaminase, the first enzyme in the glutamine utilization pathway. Glutamine is an essential metabolic fuel that is converted to alpha-ketoglutarate and serves as a substrate for the TCA cycle or for glutathione synthesis, to promote energy production and cellular biosynthesis, and to protect against oxidative stress. Interestingly, pharmacological targeting of mitochondrial glutaminase inhibits cancer cell transforming activity, suggesting that glutamine metabolism and its role in fueling and replenishing the TCA cycle are required for neoplastic transformation.
Reverse Warburg Effect.
It is increasingly apparent that the tumor microenvironment regulates neoplastic growth and progression. Activation of the stroma is a critical step required for tumor formation. Among the stromal players, cancer associated fi broblasts (CAFs) have recently taken center stage [25]. CAFs are activated, contractile        fibroblasts that display features of myo-fibroblasts, express muscle specific actin, and show an increased ability to secrete and remodel the extracellular matrix. They are not just neutral spectators, but actively support malignant transformation and metastasis, as compared to normal resting fibroblasts.

Importantly, the tumor stroma dictates clinical outcome and constitutes a source of potential biomarkers. Expression profiling has identified a cancer-associated stromal signature that predicts good and poor clinical prognosis in breast cancer patients, independently of other factors.

A loss of caveolin-1 (Cav-1) in the stromal compartment is a novel biomarker for predicting poor clinical outcome in all of the most common subtypes of human breast cancer, including the more lethal triple negative subtype. A loss of stromal Cav-1 predicts early tumor recurrence, lymph node metastasis, tamoxifen-resistance, and poor survival.

Overall, breast cancer patients with a loss of stromal Cav-1show a 20% 5-year survival rate, compared to the 80% 5-year survival of patients with high stromal Cav-1 expression. In triple negative patients, the 5-year survival rate is 75.5% for high stromal Cav-1 versus 9.4% for absent stromal Cav-1. A loss of stromal Cav-1 also predicts progression to invasive disease in ductal carcinoma in situ patients, suggesting that a loss of Cav-1 regulates tumor progression. Similarly, a loss of stromal Cav-1 is associated with advanced disease and metastasis, as well as a high Gleason score, in prostate cancer patients.

The autophagic tumor stroma model of cancer metabolism.
Cancer cells induce oxidative stress in adjacent cancer-associated fibroblasts (CAFs). This activates reactive oxygen species (ROS) production and autophagy. ROS production in CAFs, via the bystander eff ect, serves to induce random mutagenesis in epithelial cancer cells, leading to double-strand DNA breaks and aneuploidy. Cancer cells mount an anti-oxidant defense and upregulate molecules that protect them against ROS and autophagy, preventing them from undergoing apoptosis. So, stromal fibroblasts conveniently feed and mutagenize cancer cells, while protecting them against death. See the text for more details. A+, autophagy positive; A-, autophagy negative; AR, autophagy resistant.

1. Recycled Nutrients
2. Random Mutagenesis
3. Protection Against Apoptosis
The clinical use of PET is well established in Hodgkin’s lymphomas which are composed of less than 10% tumor cells, the rest being stromal and inflammatory cells. Yet, Hodgkin’s lymphomas are very PET avid tumors, suggesting that 2-deoxy-glucose uptake may be associated with the tumor stroma. That the fibrotic component may be glucose avid is further supported by the notion that PET is clinically used to assess the therapeutic response in gastrointestinal stromal tumors (GIST), which are a subset of tumors of mesenchymal origin.
The reverse Warburg effect can be described as ‘metabolic coupling’ between supporting glycolytic stromal cells and oxidative tumor cells. Metabolic cooperativity between adjacent cell-compartments is observed in several normal physiological settings.
The reverse Warburg effect.
Via oxidative stress, cancer cells activate two major transcription factors in adjacent stromal fibroblasts (hypoxia-inducible factor (HIF)1α and NFκB).
This leads to the onset of both autophagy and mitophagy, as well as aerobic glycolysis, which then produces recycled nutrients (such as lactate, ketones, and glutamine).
These high-energy chemical building blocks can then be transferred and used as fuel in the tricarboxylic acid cycle (TCA) in adjacent cancer cells.
The outcome is high ATP production in cancer cells, and protection against cell death. ROS, reactive oxygen species.
The methylation hypothesis and the role of PP2A phosphatase
Diethanolamine decreased choline derivatives and methyl donors in the liver, like seen in a choline deficient diet. Such conditions trigger tumors in mice, particularly in the B6C3F1 strain. Again, the historical perspective recalled by Newberne’s comment brings us back to insulin. Indeed, after the discovery of insulin in 1922, Banting and Best were able to keep alive for several months depancreatized dogs, treated with pure insulin. However, these dogs developed a fatty liver and died. Unlike pure insulin, the total pancreatic extract contained a substance that prevented fatty liver: a lipotropic substance identified later as being choline. Like other lipotropes, (methionine, folate, B12) choline supports transmethylation reactions, of a variety of substrates, that would change their cellular fate, or action, after methylation. In the particular case concerned here, the removal of triglycerides from the liver, as very low-density lipoprotein particles (VLDL), requires the synthesis of lecithin, which might decrease if choline and S-adenosyl methionine (SAM) are missing. Hence, a choline deficient diet decreases the removal of triglycerides from the liver; a fatty liver and tumors may then form. In sum, we have seen that pathways exemplified by the insulin-tyrosine kinase signaling pathway, which control anabolic processes, mitosis, growth and cell death, are at each step targets for oncogenes; we now find that insulin may also provoke fatty liver and cancer, when choline is not associated to insulin.

We know that after the tyrosine kinase reaction, serine-threonine kinases take over along the signaling route. It is thus highly probable that serine-threonine phosphatases will counteract the kinases and limit the intensity of the insulin or insulin like signals. One of the phosphatases involved is PP2A, itself the target of DNA viral oncogenes (Polyoma or SV40 antigens react with PP2A subunits and cause tumors). We found a possible link between the PP2A phosphatase brake and choline. the catalytic C subunit of PP2A is associated to a structural subunit A. When C receives a methyl, the dimer recruits a regulatory subunit B. The trimer then targets specific proteins that are dephosphorylated. choline, via SAM, methylates PP2A, which is targeted toward the serine-threonine kinases that are counteracted along the insulin-signaling pathway.

The choline dependent methylation of PP2A is the brake, the “antidote”, which limits “the poison” resulting from an excess of insulin signaling. Moreover, it seems that choline deficiency is involved in the L to M2 transition of PK isoenzymes. The negative regulation of Ras/MAP kinase signals mediated by PP2A phosphatase seems to be complex. The serine-threonine phosphatase does more than simply counteracting kinases; it binds to the intermediate Shc protein on the signaling cascade, which is inhibited. The targeting of PP2A towards proteins of the signaling pathway depends of the assembly of the different holoenzymes.

The relative decrease of methylated PP2A in the cytosol, not only cancels the brake over the signaling kinases, but also favors the inactivation of PK and PDH, which remain phosphorylated, contributing to the metabolic anomaly of tumor cells. In order to prevent tumors, one should then favor the methylation route rather than the phosphorylation route for choline metabolism. This would decrease triglycerides, promote the methylation of PP2A and keep it in the cytosol, reestablishing the brake over signaling kinases. Moreover, PK, and PDH would become active after the phosphatase action. One would also gain to inhibit their kinases as recently done with dichloroacetate for PDH kinase. The nuclear or cytosolic targeting of PP2A isoforms is a hypothesis also inspired by several works.
Hypoxic adaptations in the presence of oxygen
Through different biochemical and biophysical pathways, which are characteristic to cancer cells, tumor cells adopt this phenotype, i.e., high glycolysis and decreased respiration, in the presence of oxygen. It has been shown that although the induction of hypoxia and cellular proliferation engage entirely different cellular pathways, they often coexist during tumor growth. The ability of cells to grow during hypoxia results, in part, from the crosstalk between hypoxia-inducible factors (Hifs) and the proto-oncogene c-Myc. These genes partially regulate the development of complex adaptations of tumor cells growing in low O2, and contribute to fine tuning the adaptive responses of cells to hypoxic environments.

Hypoxic conditions seem to trigger back the expression of the fetal gene packet via HIF1-Von-Hippel signals. The mechanism would depend of a double switch since not all fetal genes become active after hypoxia. First, the histones have to be in an acetylated form, opening the way to transcription factors, this depends either of histone eacetylase (HDAC) inhibition or of histone acetyltransferase (HAT) activation, and represents the main switch

Growth hormone-IGF actions, the control of asymmetrical mitosis
When IGF – Growth hormone operate, the fatty acid source of acetyl CoA takes over. Indeed, GH stimulates a triglyceride lipase in adipocytes, increasing the release of fatty acids and their b oxidation. In parallel, GH would close the glycolytic source of acetyl CoA, perhaps inhibiting the hexokinase interaction with the mitochondrial ANT site. This effect, which renders apoptosis possible, does not occur in tumor cells. GH mobilizes the fatty acid source of acetyl CoA from adipocytes, which should help the formation of ketone bodies. Since citrate synthase activity is elevated in tumors, ketone bodies do not form. This result silences several genes like PETEN, P53, or methylase inhibitory genes. It is probable that the IGFBP gene gets silent as well.

Uncoupling Proteins in Cancer



A small increase in membrane potential gives rise to a large stimulation of ROS production, whereas a small decrease in membrane potential (10 mV) is able to inhibit ROS production by 70% . Therefore, mild uncoupling, i.e., a small decrease in membrane potential, has been suggested to have a natural antioxidant effect.

Consistent with such a proposal, the inhibition of UCPs by GDP in mitochondria has been shown to increase membrane potential and mitochondrial ROS production. The loss of UCP2 or UCP3 in knockouts yielded increased ROS production concurrent with elevated membrane potential specifically in those tissues normally expressing the missing protein.
ROS and Cancer
ROS are thought to play multiple roles in tumor initiation, progression and maintenance, eliciting cellular responses that range from proliferation to cell death. In normal cells, ROS play crucial roles in several biological mechanisms including phagocytosis, proliferation, apoptosis, detoxification and other biochemical reactions. Low levels of ROS regulate cellular signaling and play an important role in normal cell proliferation. During initiation of cancer, ROS may cause DNA damage and mutagenesis, while ROS acting as second messengers stimulate proliferation and inhibit apoptosis, conferring growth advantage to established cancer cells. Cancer cells have been found to have increased ROS levels.

One of the functional roles of these elevated ROS levels during tumor progression is constant activation of transcription factors such as NF-kappaB and AP-1 which induce genes that promote proliferation and inhibit apoptosis. In addition, oxidative stress can induce DNA damage which leads to genomic instability and the acquisition of new mutations, which may contribute to cancer progression.

Role of ROS in control of proliferation and apoptosis
ROS are also essential mediators of apoptosis which eliminates cancer and other cells that threaten our health [81–86]. Many chemotherapeutic drugs and radiotherapy are aimed at increasing ROS levels to promote apoptosis by stimulating pro-apoptotic singaling molecules such as ASK1, JNK and p38. Because of the pivotal role of ROS in triggering apoptosis, antioxidants can inhibit this protective mechanism by depleting ROS. Thus, antioxidant mechanisms are thought to interfere with the therapeutic activity of anticancer drugs that kill advanced stage cancer cells by apoptosis.


Uncoupling-to-survive hypothesis (proposed by Brand)



Roles of UCPs in Cancer Progression
The growth of a tumor from a single genetically altered cell is a stepwise progression requiring the alterations of several genes which contribute to the acquisition of a malignant phenotype. Such genetic alterations are positively selected when in the tumor, they confer a proliferative, survival or treatment resistance advantage for the host cell. In addition, several mutations, such as those silencing tumour suppressor genes, trigger the probability of accumulating new mutations, so the process of malignant transformation is progressively self-accelerated.


Increased uncoupled respiration may be a mechanism to lower cellular oxygen concentration and, thus, alter molecular pathways of oxygen sensing such as those regulated by hypoxia-inducible factor (HIF). In normoxia, the alpha subunit of HIF-1 is a target for prolyl hydroxylase, which makes HIF-1alpha a target for degradation by the proteasome. During hypoxia, prolyl hydroxylase is inhibited since it requires oxygen as a cosubstrate. Thus, hypoxia allows HIF to accumulate and translocate into the nucleus for induction of target genes regulating glycolysis, angiogenesis and hematopoiesis. By this mechanism, UCPs activity may contribute to increase the expression of genes related to the formation of blood vessels, and thus promote tumor growth.

Roles of UCPs in Cancer Energy Metabolism

The ability of UCPs to uncouple ATP synthesis from respiration and the fact that UCP2 is overexpressed in several chemoresistant cancer cell lines and primary human colon cancers have lead to speculate about the existence of a link between UCPs and the Warburg effect. As mentioned above, uncoupling induced by overexpression of UCP2 has been shown to prevent ROS formation, and, in turn, increase apoptotic threshold in cancer cells, providing a pro-survival advantage and a resistance mechanism to cope with ROS-inducing chemo-therapeutic agents.

Mitochondrial Krebs cycle is one of the sources for these anabolic precursors. The export of these metabolites to cytoplasm for anabolic purposes involves the replenishment of the cycle intermediates by anaplerotic substrates such as pyruvate and glutamate. Thus, glycolysis-derived pyruvate, as well as alpha-ketoglutarate derived from glutaminolysis, may be necessary to sustain anaplerotic reactions. At the same time, to keep Krebs cycle functional, the reduced cofactors NADH and FADH2 would have to be re-oxidized, a function which relies on the mitochondrial respiratory chain. Once again, uncoupling may be crucial for cancer cell mitochondrial metabolism, allowing Krebs cycle to be kept functional to meet the vigorous biosynthetic demand of cancer cells.

Several cancer cells resistant to chemotherapeutics and radiation often exhibit higher rates of fatty acid oxidation and it has been observed that inhibition of fatty acid oxidation potentiates apoptotic death induced by chemotherapeutic agents. These findings are in agreement with the proposed need of fatty acid for the activity of UCPs, suggesting that the lack of these potential substrates or activators would decrease uncoupling activity, subsequently increasing membrane potential, ROS production and therefore lowering apoptotic threshold.

Roles of UCPs in Cancer Cachexia
Cachexia is a wasting syndrome characterized by weakness, weight and fat loss, and muscle atrophy which is often seen in patients with advanced cancer or AIDS. Cachexia has been suggested to be responsible for at least 20 % of cancer deaths and also plays an important part in the compromised immunity leading to death from infection. The imbalance between energy intake and energy expenditure underlying cachexia cannot be reversed nutritionally.


Roles of UCPs in Chemoresistance

Cancer cells acquire drug resistance as a result of selection pressure dictated by unfavorable microenvironments. Although mild uncoupling may clearly be useful under normal conditions or under severe or chronic metabolic stress such as hypoxia or anoxia, it may be a mechanism to elude oxidative stress-induced apoptosis in advanced cancer cells. Several anti-cancer treatments are based on promotion of ROS formation, to induce cell growth arrest and apoptosis. Thus, increased UCP levels in cancer cells, rather than a marker of oxidative stress, may be a mechanisms conferring anti-apoptotic advantages to the malignant cell, increasing their ability to survive in adverse microenvironments, radiotherapy and chemotherapy. UCPs appear to play a permissive role in tumor cell survival and growth.



Hypoxia-inducible factor-1 in tumour angiogenesis
HIF-b subunits, is a heterodimeric transcriptional activator. In response to

  • stimulation of growth factors, and
  • activation of oncogenes as well as carcinogens,

HIF-1a is overexpressed and/or activated and targets those genes which are required for angiogenesis, metabolic adaptation to low oxygen and promotes survival.

Several dozens of putative direct HIF-1 target genes have been identified on the basis of one or more cis-acting hypoxia-response elements that contain an HIF-1 binding site. Activation of HIF-1 in combination with activated signaling pathways and regulators is implicated in tumour progression and prognosis.
In order for a macroscopic tumour to grow, adequate oxygen delivery must be effected via tumor angiogenesis that results from an increased synthesis of angiogenic factors and a decreased synthesis of anti-angiogenic factors. The metabolic adaptation of tumor cells to reduced oxygen availability by increasing glucose transport and glycolysis to promote survival are important consequences in response to hypoxia.

Hypoxia and HIF-1
Hypoxia is one of the major drivers to tumour progression as hypoxic areas form in human tumours when the growth of tumour cells in a given area outstrips local neovascularization, thereby creating areas of inadequate perfusion. Although several transcriptional factors have been reported to be involved
in the response to hypoxic stress such as AP-1, NF-kB and HIF-1, HIF-1 is the most potent inducer of the expression of genes such as those encoding for glycolytic enzymes, VEGF and erythropoietin.

HIF-a subunit exists as at least three isoforms, HIF-1a, HIF-2a and HIF-3a. HIF-1a and HIF-2a can form heterodimers with HIF-b. Although HIF-b subunits are constitutive nuclear proteins, both HIF-1a andHIF-2a subunits are strongly induced by hypoxia in a similar manner. HIF-1a is up-regulated in hypoxic tumour cells and activates the transcription of target genes by binding to cis-acting enhancers, hypoxic responsive element (HRE) close to the promoters of these genes with a result of tumour cellular adaptation to hypoxia and tumour angiogenesis, and promotion of further growth of the primary tumour. Studies have shown HIF-1a to be over-expressed by both tumour cells and such stromal cells as macrophages in many forms of human malignancy.

Regulation of HIF-1
The first regulator of HIF-1 is oxygen. HIF-1α appears to be the HIF-1 subunit regulated by hypoxia. The oxygen sensors in the HIF-1α pathway are two kinds of oxygen dependent hydroxylases. One is prolyl hydroxylase which could hydroxylize the proline residues 402 and 564 at the oxygen dependent domain (ODD) of HIF-1 in the presence of oxygen and iron with a result of HIF-α degradation. The other is hydroxylation of Asn803 at the C-terminal transactivation domain (TAD-C) by FIH-1, which could inhibit the interaction of HIF-1α with co-activator p300 with a subsequent inhibition of HIF-1α transactivity. The hydroxylation of proline 564 at ODD of HIF-1α under normoxia was shown using a novel hydroxylation-specific antibody to detect hydroxylized HIF-1α.

Oncogene comes as the second regulator. Many oncogenes have effects on HIF-1α. Among them, some function in regulation of HIF-1α protein stability or degradation, others play roles in several activated signaling pathways. Tumor suppressor genes as p53 and von Hippel-Lindau (VHL) influence the levels and functions of HIF-1. The wild type (wt) form of p53 protein was involved in inhibiting HIF-1 activity by targeting the HIF-1a subunit for Mdm2-mediated ubiquitination and proteasomal degradation, and in inducing inhibitors of angiogenesis such as thrombospondin-1, while loss of wt p53 (by gene deletion or mutation) could enhance HIF-1α accumulation in hypoxia.

The third regulator is a battery of growth factors and cytokines from stromal and parenchymal cells such as

  • EGF,
  • transforming growth factor-α,
  • insulin-like growth factors 1 and 2,
  • heregulin, and interleukin-1b

via autocrine and paracrine pathways. These regulators not only induce the expression of HIF-1α protein, HIF-1 DNA binding activity and transactivity, but also make HIF-1 target gene expression under normoxia or hypoxia.
The fourth one is a group of reactive oxygen species (ROS) resulting from carcinogens such as Vanadate and Cr (VI) or stimulation of cytokines such as angiotensin and TNFa. However, it seems controversial when it comes to the production of ROS under hypoxia and their individual role in regulation of HIF-1a. It is well known that ROS plays an important role in carcinogenesis induced by a variety of carcinogens.

Signaling Pathways Involved in Regulation of HIF-1α
HIF-1 is a phosphorylated protein and its phosphorylation is involved in HIF-1a subunit expression and/or stabilization as well as in the regulation of HIF-1 transcriptional activity. Three signaling pathways involved in the regulation of HIF-1α have been reported to date.

  • The PI-3k pathway has been mainly and frequently implicated in regulation of HIF-1α protein expression and stability.
  • Akt is also activated by hypoxia. Activated Akt initiates two different pathways in regulation of HIF-1α. The function of these two pathways appears to show consistent impact on HIF-1α activation.
  • Signal transduction pathway in HIF-1α regulation.Oncogenes, growth factors and hypoxia have been documented to regulate HIF-1α protein and increase its transactivity. GSK and mTOR were two target events of Akt and could contribute to decreasing HIF-1α degradation and increasing HIF-1α protein synthesis. Activated ERK1/2 could mainly up-regulate.

HIF-1a, Angiogenesis and Tumour Prognosis
Hypoxia, oncogenes and a variety of growth factors and cytokines increase HIF-1α stability and/or synthesis and transactivation to initiate tumour angiogenesis, metabolic adaptation to hypoxic situation and promote cell survival or anti-apoptosis resulting from a consequence of more than sixty putative direct HIF-1 target gene expressions.

The crucial role of HIF-1 in tumour angiogenesis has sparked scientists and clinical researchers to try their best to understand the whole diagram of HIF-1 so as to find out novel approaches to inhibit HIF-1 overexpression. Indeed, the combination of anti-angiogenic agent and inhibitor of HIF-1 might be particularly efficacious, as the angiogenesis inhibitor would cut off the tumour’s blood supply and HIF-1 inhibitor would reduce the ability of tumour adaptation to hypoxia and suppress the proliferation and promote apoptosis. Screens for small-molecule inhibitors of HIF-1 are underway and several agents that inhibit HIF-1, angiogenesis and xenograft growth have been identified.

Hypoxia, autophagy, and mitophagy in the tumor stroma
Metabolomic profiling reveals that Cav-1(-/-) null mammary fat pads display a highly catabolic metabolism, with the increased release of several metabolites, such as amino acids, ribose and nucleotides, and a shift towards gluconeogenesis, as well as mitochondrial dysfunction. These changes are consistent with increased autophagy, mitophagy and aerobic glycolysis, all processes that are induced by oxidative stress. Autophagy or ‘self-eating’ is the process by which cells degrade their own cellular components to survive during starvation or to eliminate damaged organelles after oxidative stress. Mitophagy, or mitochondrial-autophagy, is particularly important to remove damaged ROS-generating mitochondria.

An autophagy/mitophagy program is also triggered by hypoxia. Hypoxia is a common feature of solid tumors, and promotes cancer progression, invasion and metastasis. Interestingly, via induction of autophagy, hypoxia is sufficient to induce a dramatic loss of Cav-1 in fibroblasts. The hypoxia-induced loss of Cav-1 can be inhibited by the autophagy inhibitor chloroquine, or by pharmacological inhibition of HIF1α. Conversely, small interfering RNA-mediated Cav-1 knock-down is sufficient to induce pseudo-hypoxia, with HIF1α and NFκB activation, and to promote autophagy/mitophagy, as well as a loss of mitochondrial membrane potential in stromal cells. These results indicate that a loss of stromal Cav-1 is a marker of hypoxia and oxidative stress.

In a co-culture model, autophagy in cancer-associated fibroblasts was shown to promote tumor cell survival via the induction of the pro-autophagic HIF1α and NFκB pathways in the tumor stromal microenvironment. Finally, the mitophagy marker Bnip3L is selectively upregulated in the stroma of human breast cancers lacking Cav-1, but is notably absent from the adjacent breast cancer epithelial cells.

Metabolome profiling of several types of human cancer tissues versus corresponding normal tissues have consistently shown that cancer tissues are highly catabolic, with the significant accumulation of many amino acids and TCA cycle metabolites. The levels of reduced glutathione were decreased in primary and metastatic prostate cancers compared to benign adjacent prostate tissue, suggesting that aggressive disease is associated with increased oxidative stress. Also, these data show that the tumor microenvironment has increased oxidative-stress-induced autophagy and increased catabolism.

Taken together, all these findings suggest an integrated model whereby
A loss of stromal Cav-1 induces autophagy/mitophagy in the tumor stroma, via oxidative stress.

This creates a catabolic micro-environment with the local accumulation of chemical building blocks and recycled nutrients (such as amino acids and nucleotides), directly feeding cancer cells to sustain their survival and growth.
This novel idea is termed the ‘autophagic tumor stroma model of cancer’ .
This new paradigm may explain the ‘autophagy paradox’, which is based on the fact that both the systemic inhibition and systemic stimulation of autophagy prevent tumor formation.

What is presented suggests that vectorial energy transfer from the tumor stroma to cancer cells directly sustains tumor growth, and that interruption of such metabolic coupling will block tumor growth. Autophagy inhibitors (such as chloroquine) functionally block the catabolic transfer of metabolites from the stroma to the tumor, inducing cancer cell starvation and death. Conversely, autophagy inducers (such as rapamycin) promote autophagy in tumor cells and induce cell death. Thus, both inhibitors and inducers of autophagy will have a similar effect by severing the metabolic coupling of the stroma and tumor cells, resulting in tumor growth inhibition (cutting ‘off ’ the fuel supply).

This model may also explain why enthusiasm for antiangiogenic therapy has been dampened. In most cases, the clinical benefits are short term, and more importantly, new data suggest an unexpected link between anti-angiogenic treatments and metastasis. In pre-clinical models, anti-vascular endothelial growth factor (anti-VEGF) drugs (sunitinib and anti-VEGFR2 blocking antibodies) were shown to inhibit localized tumor formation, but potently induced relapse and metastasis. Thus, by inducing hypoxia in the tumor microenvironment, antiangiogenic drugs may create a more favorable metastatic niche.
Glutamine, glutaminolysis.
In direct support that cancer cells use mitochondrial oxidative metabolism, many investigators have shown that cancer cells are ‘addicted’ to glutamine. Glutamine is a non-essential amino acid that is metabolized to glutamate and enters the TCA cycle as α-ketoglutarate, resulting in high ATP generation via oxidative phosphorylation. Recent studies also show that ammonia is a by-product of glutaminolysis. In addition, ammonia can act as a diffusible inducer of autophagy. Given these observations, glutamine addiction in cancer cells provides another mechanism for driving and maintaining autophagy in the tumor micro-environment .

In support of this idea, a loss of Cav-1 in the stroma is sufficient to drive autophagy, resulting in increased glutamine production in the tumor micro-environment. Thus, this concept defines a new vicious cycle in which autophagy in the tumor stroma transfers glutamine to cancer cells, and the by-product of this metabolism, ammonia, maintains autophagic glutamine production. This model fits well with the ‘autophagic tumor stroma model of cancer metabolism’, in which energy rich recycled nutrients (lactate, ketones, and glutamine) fuel oxidative mitochondrial metabolism in cancer cells.

Glutamine utilization in cancer cells and the tumor stroma. Oxidative mitochondrial metabolism of glutamine in cancer cells produces ammonia. Ammonia production is sufficient to induce autophagy. Thus, autophagy in cancer-associated fibroblasts provides cancer cells with an abundant source of glutamine. In turn, the ammonia produced maintains the autophagic phenotype of the adjacent stromal fibroblasts.

Lessons from other paradigms

an infectious parasitic cancer cell that metastasizes and captures mitochondrial DNA from host cells
Cancer cells behave like ‘parasites’, by inducing oxidative stress in normal host fibroblasts, resulting in the production of recycled nutrients via autophagy.

This is exactly the same mechanism by which infectious parasites (such as malaria) obtain nutrients and are propagated by inducing oxidative stress and autophagy in host cells. In this regard, malaria is an ‘intracellular’ parasite, while cancer cells may be thought of as ‘extracellular’ parasites. This explains why chloroquine is both an effective antimalarial drug and an effective anti-tumor agent, as it functions as an autophagy inhibitor, cutting off the ‘fuel supply’ in both disease states.

Human cancer cells can ‘steal’ live mitochondria or mitochondrial DNA from adjacent mesenchymal stem cells in culture, which then rescues aerobic glycolysis in these cancer cells. This is known as mitochondrial transfer. Interestingly, metastatic breast cancer cells show the up-regulation of numerous mitochondrial proteins, specifically associated with oxidative phosphorylation, as seen by unbiased proteomic analysis.

Thus, increased mitochondrial oxidative metabolism may be a key driver of tumor cell metastasis. In further support of this argument, treatment of MCF7 cancer cells with lactate is indeed sufficient to induce mitochondrial biogenesis in these cells.

To determine if these findings may be clinically relevant, a lactate-induced gene signature was recently generated using MCF7 cells. This gene signature shows that lactate induces ‘stemness’ in cancer cells, and this lactate induced gene signature predicts poor clinical outcome (including tumor recurrence and metastasis) in breast cancer patients.


Li W and Zhao Y. “Warburg Effect” and Mitochondrial Metabolism in Skin Cancer. Epidermal Pigmentation, Nucleotide Excision Repair and Risk of Skin Cancer. J Carcinogene Mutagene 2012; S4:002 doi:10.4172/2157-2518.
Seyfried TN, Shelton LM. Cancer as a metabolic disease. Nutrition & Metabolism 2010; 7:7(22 pg). doi:10.1186/1743-7075-7-7
Israël M, Schwartz L. The metabolic advantage of tumor cells. Molecular Cancer 2011, 10:70-82. http://www.molecular-cancer.com/content/10/1/70
Valle A, Oliver J, Roca P. Role of Uncoupling Proteins in Cancer. Cancers 2010, 2, 567-591; doi:10.3390/cancers2020567
Ishii H, Doki Y, Mori M. Perspective beyond Cancer Genomics: Bioenergetics of Cancer Stem Cells. Yonsei Med J 2010; 51(5):617-621. DOI 10.3349/ymj. 2010.51.5.617 pISSN: 0513-5796, eISSN: 1976-2437
Sotgia F, Martinez-Outschoorn, Pavlides S,Howell A . Understanding the Warburg effect and the prognostic value of stromal caveolin-1 as a marker of a lethal tumor microenvironment. Breast Cancer Research 2011, 13:213-26. http://breast-cancer-research.com/content/13/4/213
Yong-Hong Shi, Wei-Gang Fang. Hypoxia-inducible factor-1 in tumour angiogenesis. World J Gastroenterol 2004; 10(8): 1082-1087. http:// wjgnet.com /1007-9327/10/1082.asp

English: Glycolysis pathway overview.

English: Glycolysis pathway overview. (Photo credit: Wikipedia)

Adenosine triphosphate





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Warburg Effect, glycolysis, pyruvate kinase, PKM2, PIM2, mtDNA, complex I, NSCLC

Ribeirão Preto Area, Brazil|Government Relations
Current- University, USP-FMRP Physiology – Biochemistry
Previous- Imperial Cancer Research Fund Lab, Instituto de Investigaciones Bioquimicas, Luta armada e CIA
Education – FMRP-USP
While exile interrupted ny graduation in Medicine, started a port grade at Leloir´s Instituto Investigaciones bioquímicas Buenos Aires Argentina Jan 1970 – jan 1972. PhD from 1972- jan 1976 FMRP-USP . Post Doc ICRF London England 1979 -1980

“Those…..whose acquitance with scientific research is derived chiefly from its practical results easily develop a completely false notion of the { scientific } mentality.”

My goal and previous experience besides laboratory work was dedicated to solve this apparent conflict.

When Pasteur did his work studying the chemistry outside yeast-cells, he was able to perceive that in anaerobiosis yeast cells were able to convert sugar at a great velocity to its end products. While the same yeast cells, therefore with the same genome did it at a very slow speed in aerobiosis. Warburg tested tumor cells for the same and found that while normal cells from the organ in which he has found tumors presented a similar metabolic regulatory response to anaerobic/aerobic transition tumor cells did not. Tumos cells continued to display strong acidification (producing great amounts of lactate in the culture media) in aerobiosis.

Tumor cells displayed a failure in this regulatory mechanism that, he O. Warburg, named Pasteur effect. He also noticed that this defect continues from old cancer cells to newer ones. Therefore, for him, it is a genetic defect. Furthermore, as mitochondria generates newer mitochondria in the cells, the genetic component is in the mitochondria that were the site of core metabolism in aerobiosis.

Larry Bernstein
Part of what you describe is in Warburg biography by Hans Krebs (out of print). He also refers to a Meyerhof Quotient to express the degree of metabolic anaerobiosis. I don’t recall a reference to a mitochondrial “genetic” defect. That is farsighted. He did conclude that once the cancer cells were truly anaplastic, metastatic behavior was irreversible. It was only later that another Nobelist who described fatty acid synthesis, I think, concluded that the synthetic process tied up the same aerobic pathway so that anaerobic glycolysis became essential for the cells energetics. You can correct me if I’m in error. Where does the substrate come from? Lean body mass breaks down to provide gluconeogenic precursors. Points of concern are – deamination of branch chain AAs, the splitting of a 6 carbon sugar into 2 3-carbon chains, and the conversion of pyruvate to lactate with the reverse reaction blocked. There is also evidence that there is an impairment in the TCA cycle at the point of fumarase. Then there is never any consideration of the flow of substrates back and forth across the mitochondrial membrane (malate, aspartate), and the redox potentials.

Jose: For reasons independent of my will, the copy of Science,123(3191):309-14,1956 translation of O Warburg original article is not in my hands. Some that do not find any alternative form to respond to my critics concerning molecular biology distortion of biochemistry left on purpose, almost all of my older reprints on the rain. Anyway, O. Warburg refers to mitochondria using the expression of “grana” or “grains” if my recollections are correct. The original work of O Warburg is one of 1924 – Biochemisch ZeiTschrift.,152:51-60.1924. “Weberssert method zur messing der atmung un glycolyse, drese zeitschr.” Other aspects of your interesting comment I will try to comment latter.

By Jose Eduardo de Salles Roselino
Larry Bernstein, not as a correction but , following my line of reasoning about carbono fluxes…It is almost impossible to figure out what was really inside the mind-set of a scientific researcher at the time he has performed his work. Anyway, by the knowledge available at his time, we may conjecture about, even when we acknowledge that, what he may have had in mind at the start could be quite different from what he have latter published from his results.
In case, we try to present the ideas behind Warburg´s works we may take into account the following pre-existing knowledge: Lavoisier (done by 1779-1784) measured very carefully the amount of heat released by respiration and chemical oxidative processes. Reached the conclusion that respiration was slower but essentially similar to carbon combustion by chemical oxidative processes. By early XIX caloric values for gram of sugar, lipids and proteins where made clear. T Schwann recognized that yeast cells convert sugar in ethanol plus a volatile acid (carbon dioxide). Pasteur-effect was seen just as a change in the velocity of product production from a same sugar and/or a decrease in sugar concentration in the growth medium. This is a change in carbon flux velocity in the oxidative process calorimetrically measured by Lavoisier.
In Germany, however, the preponderance of organic chemical point of view has great scientists as Berzelius, Liebig etc. to consider that yeast where similar to inorganic catalysts. The oxidative process was caused by oxygen only. You may amuse yourself reading, how they attack T. Schwann proposal in Annalen, 29: 100 (1839). When O Warburg paid tribute to L. Pasteur, we can see clearly on which side he was. Furthermore, his apparatus, in which I was introduced to this line of research, a rather modern version at that time (1960s) that look like a very futuristic robotic version of the original with a pair of blinking red and green round lights as two eyes on a metallic head, offers a very important clue about the change in carbon flow.
Inside a Warburg glass flask, kept at very finely controlled temperature to avoid changes in pressure derived from changes in the temperature, you would certainly find a central well in which the volatile acid must be trapped in order to determine a pressure reduction only due to the decrease in oxygen pressure. Inside this central well, you could determine one product (carbon dioxide) and in the outside of it where tissues or cells where in the media you could also determine the other end product of the oxidative combustion of sugar (lactate). This has provide him with the result that indicate not only a change in the velocity as originally found by Pasteur in yeast cells but in the organic flow of sugar carbons.

comment –  E-mail: vbungau11@yahoo.com

-About carcinogenesis- Electronegativity is a nucleus of an atom’s ability to attract and maintain a cloud of electrons. Copper atom electronegativity is higher than the Iron atom electronegativity. Atom with lower electronegativity (Iron),remove the atom with higher electronegativity (Copper) of combinations. This means that,in conditions of acidosis, we have cytochrome oxidase with iron (red, neoplastic), instead of cytochrome oxidase with copper (green, normal).I think this is the key to carcinogenesis. Siincerely, Dr. Viorel Bungau

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English: A diagram of cellular respiration inc...


English: Figure from Journal publication of sc...

English: Diagram showing regulation of the enz...

Reporter and Curator: Larry H Bernstein, MD, FACP



Function in aerobic and anaerobic metabolism





Biogenesis of mitochondrial structures from aerobically grown S. cerevisiae




  • Aerobic yeast contained more cardiolipin than anaerobic yeast .
  • A chromosomal mutant lacking respiratory function had normal cardiolipin content .


Mitochondria, hydrogenosomes and mitosomes



In the 1970s and 1980s the unequivocal demonstration of mitochondrial DNA occurred




Mentel and Martin. Anaerobic mitochondria: more common all the time. BMC Biology 2010; 8:32. BioMed Central Ltd. http://www.biomedcentral.com/1741-7007/8/32.

Anaerobic mitochondrial enzymes


Mitochondrial redox status






  • leading to a new approach to pharmaceutical targeted drug design.

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