Inhibition Of Pyruvate Dehydrogenase Complex Research Articles

Anti-Warburg Effect of Melatonin: A Proposed Mechanism to Explain its Inhibition of Multiple Diseases.

Glucose is an essential nutrient for every cell but its metabolic fate depends on cellular phenotype. Normally, the product of cytosolic glycolysis, pyruvate, is transported into mitochondria and irreversibly converted to acetyl coenzyme A by pyruvate dehydrogenase complex (PDC). In some pathological cells, however, pyruvate transport into the mitochondria is blocked due to the inhibition of PDC by pyruvate dehydrogenase kinase. This altered metabolism is referred to as aerobic glycolysis (Warburg effect) and is common in solid tumors and in other pathological cells. Switching from mitochondrial oxidative phosphorylation to aerobic glycolysis provides diseased cells with advantages because of the rapid production of ATP and the activation of pentose phosphate pathway (PPP) which provides nucleotides required for elevated cellular metabolism. Molecules, called glycolytics, inhibit aerobic glycolysis and convert cells to a healthier phenotype. Glycolytics often function by inhibiting hypoxia-inducible factor-1α leading to PDC disinhibition allowing for intramitochondrial conversion of pyruvate into acetyl coenzyme A. Melatonin is a glycolytic which converts diseased cells to the healthier phenotype. Herein we propose that melatonin’s function as a glycolytic explains its actions in inhibiting a variety of diseases. Thus, the common denominator is melatonin’s action in switching the metabolic phenotype of cells.

Dual Inhibition of Pyruvate Dehydrogenase Complex and Respiratory Chain Complex Induces Apoptosis by a Mitochondria-Targeted Fluorescent Organic Arsenical in vitro and in vivo.

Based on the potential therapeutic value in targeting mitochondria and the fluorophore tracing ability, a fluorescent mitochondria-targeted organic arsenical PDT-PAO-F16 was fabricated, which not only visualized the cellular distribution, but also exerted anti-cancer activity in vitro and in vivo via targeting pyruvate dehydrogenase complex (PDHC) and respiratory chain complexes in mitochondria. In details, PDT-PAO-F16 mainly accumulated into mitochondria within hours and suppressed the activity of PDHC resulting in the inhibition of ATP synthesis and thermogenesis disorder. Moreover, the suppression of respiratory chain complex I and IV accelerated the mitochondrial dysfunction leading to caspase family-dependent apoptosis. In vivo, the acute promyelocytic leukemia was greatly alleviated in the PDT-PAO-F16 treated group in APL mice model. Our results demonstrated the organic arsenical precursor with fluorescence imaging and target-anticancer efficacy is a promising anticancer drug.

Logical modelling reveals the PDC-PDK interaction as the regulatory switch driving metabolic flexibility at the cellular level

BackgroundMetabolic flexibility is the ability of an organism to switch between substrates for energy metabolism, in response to the changing nutritional state and needs of the organism. On the cellular level, metabolic flexibility revolves around the tricarboxylic acid cycle by switching acetyl coenzyme A production from glucose to fatty acids and vice versa. In this study, we modelled cellular metabolic flexibility by constructing a logical model connecting glycolysis, fatty acid oxidation, fatty acid synthesis and the tricarboxylic acid cycle, and then using network analysis to study the behaviours of the model.ResultsWe observed that the substrate switching usually occurs through the inhibition of pyruvate dehydrogenase complex (PDC) by pyruvate dehydrogenase kinases (PDK), which moves the metabolism from glycolysis to fatty acid oxidation. Furthermore, we were able to verify four different regulatory models of PDK to contain known biological observations, leading to the biological plausibility of all four models across different cells and conditions.ConclusionThese results suggest that the cellular metabolic flexibility depends upon the PDC-PDK regulatory interaction as a key regulatory switch for changing metabolic substrates.

Two-stage metabolic remodelling in macrophages in response to lipopolysaccharide and interferon-γ stimulation.

In response to signals associated with infection or tissue damage, macrophages undergo a series of dynamic phenotypic changes. Here we show that during the response to LPS and interferon-γ stimulation, metabolic reprogramming in macrophages is also highly dynamic. Specifically, the TCA cycle undergoes a two-stage remodeling: the early stage is characterized by a transient accumulation of intermediates including succinate and itaconate, while the late stage is marked by the subsidence of these metabolites. The metabolic transition into the late stage is largely driven by the inhibition of pyruvate dehydrogenase complex (PDHC) and oxoglutarate dehydrogenase complex (OGDC), which is controlled by the dynamic changes in lipoylation state of both PDHC and OGDC E2 subunits and phosphorylation of PDHC E1 subunit. This dynamic metabolic reprogramming results in a transient metabolic state that strongly favors HIF-1α stabilization during the early stage, which subsides by the late stage; consistently, HIF-1α levels follow this trend. This study elucidates a dynamic and mechanistic picture of metabolic reprogramming in LPS and interferon-γ stimulated macrophages, and provides insights into how changing metabolism can regulate the functional transitions in macrophages over a course of immune response.

7-LB: Dichloroacetate Improves Cognitive Function during Hypoglycemia in T1DM Patients

Magnetic Resonance Spectroscopy (MRS) studies have suggested that exposure to recurrent hypoglycemia (RH) reduces brain cortical Pyruvate Dehydrogenase (PDH) flux in animal models. Examination of rat cortical RNA for inhibitory PDH kinase (PDK) isoform expression patterns identified a two-fold upregulation of PDK4 (p>0.01) after three episodes of hypoglycemia. To date no study has examined the functional role of PDH complex inhibition in the cognitive deficits associated with hypo in patients with T1DM. In a randomized controlled cross-over trial of 11 intensively treated T1DM subjects we examined whether PDK inhibition by dichloroacetate (DCA; 12.5mg/kg), which maintains PDH in its active state might restore cognitive function, counterregulatory hormonal responses and symptoms during clamped hypoglycemia (target 50mg/dL). Cognitive testing included measures of visual matching ability and short term visual and auditory memory as well as the ability to manage conflicting information while ignoring task-irrelevant information. Symptom ratings during hypoglycemia were measured using a modified Edinburgh scale. We found that DCA ingestion is associated with increased performance related to visual memory (DCA: 89% correct vs. Placebo: 69% correct, p=0.049), as well as a faster response time during the multitasking test (549ms DCA vs. 603ms placebo; p=0.028). On the Edinburgh scale, patients reported lower ratings of warmness during hypoglycemia with DCA vs. Placebo (2.5 vs. 3.6; p=0.075). Furthermore, we did not observe an effect of DCA on CRR hormonal responses. These results suggest that inhibition of PDH flux by enhanced PDK activity significantly contributes to cognitive impairment under hypoglycemia and may contribute to subjective symptoms and possibly hypoglycemia awareness. We therefore conclude that strategies focusing on reactivation of oxidative glucose metabolism in the brain may play an important role in mitigating the negative impact of hypoglycemia on cognitive function. Disclosure D. Groskreutz: None. D. Trico: None. N. Tai: None. R. Belfort-DeAguiar: Research Support; Self; Silver Palate Kitchens, Inc. J. Leventhal: None. W. Lam: None. M. Knight: None. J. Gannon: None. R.I. Herzog: Consultant; Spouse/Partner; Boehringer Ingelheim Pharmaceuticals, Inc. Funding JDRF

Protective effects of voltage-gated calcium channel antagonists against zinc toxicity in SN56 neuroblastoma cholinergic cells.

One of the pathological site effects in excitotoxic activation is Zn2+ overload to postsynaptic neurons. Such an effect is considered to be equivalent to the glutamate component of excitotoxicity. Excessive uptake of Zn2+ by active voltage-dependent transport systems in these neurons may lead to significant neurotoxicity. The aim of this study was to investigate whether and which antagonists of the voltage gated calcium channels (VGCC) might modify this Zn2+-induced neurotoxicity in neuronal cells. Our data demonstrates that depolarized SN56 neuronal cells may take up large amounts of Zn2+ and store these in cytoplasmic and mitochondrial sub-fractions. The mitochondrial Zn2+ excess suppressed pyruvate uptake and oxidation. Such suppression was caused by inhibition of pyruvate dehydrogenase complex, aconitase and NADP-isocitrate dehydrogenase activities, resulting in the yielding of acetyl-CoA and ATP shortages. Moreover, incoming Zn2+ increased both oxidized glutathione and malondialdehyde levels, known parameters of oxidative stress. In depolarized SN56 cells, nifedipine treatment (L-type VGCC antagonist) reduced Zn2+ uptake and oxidative stress. The treatment applied prevented the activities of PDHC, aconitase and NADP-IDH enzymes, and also yielded the maintenance of acetyl-CoA and ATP levels. Apart from suppression of oxidative stress, N- and P/Q-type VGCCs presented a similar, but weaker protective influence. In conclusion, our data shows that in the course of excitotoxity, impairment to calcium homeostasis is tightly linked with an excessive neuronal Zn2+ uptake. Hence, the VGCCs types L, N and P/Q share responsibility for neuronal Zn2+ overload followed by significant energy-dependent neurotoxicity. Moreover, Zn2+ affects the target tricarboxylic acid cycle enzymes, yields acetyl-CoA and energy deficits as well.

The Effect of Alpha Lipoic Acid (ALA) on <i>S. cerevisiae </i>Cell Proliferation

This experiment investigates what effect ALA may have on the growth of S. cerevisiae, based on extensive studies that have been conducted previously on the therapeutic capabilities of ALA (α-lipoic acid) in various cancers because of its potent antioxidant and apoptotic effects. It manages this because it serves a significant role in enzymatic processes crucial to energy production in cells. In energy metabolism, ALA is responsible for generating acetyl-CoA from pyruvate, and it thus plays a large role in the PDC (pyruvate dehydrogenase complex), an important link connecting glucose metabolism with the citric acid cycle [1]. It helps to shuttle carbon into aerobic respiration, specifically for converting pyruvate into acetyl-CoA and the formation of NADH and carbon dioxide. The process is regulated by enzymes called PDKs, which serve to catalyze suppression of the PDC when the cellular environment becomes nutrient-limited, or glucose scarce. PDKs are speculated to be able to inactivate the PDC via phosphorylation, thus preserving pyruvate for a different metabolic pathway. It is known that overexpression of PDKs blocks oxidative decarboxylation of pyruvate, so targeting the inhibition of PDKs could upregulate PDC activity [2]. Studies have revealed that PDC inhibition is associated with the mechanisms of many metabolic disorders, even cancer (Stacpoole, 2017). In S.cerevisiae cells, which are model organisms for cancer studies, energy is generated typically anaerobically through alcohol fermentation, until this is impacted and it switches to metabolism controlled by the citric acid cycle. This slows down cell proliferation, so it is predicted that as ALA concentrations are increased, cell proliferation will decrease, as indicated by an average cell count of each culture. After experimentation, results revealed that each ALA concentration, 0.125% (2.37×10-20), 0.25% (1.49×10-16), 0.5% (6.41×10-16), and 1% (9.36×10-18), significantly reduced the cell count of their respective cell culture, as compared to the control of 0% ALA. Also, alcohol test strips revealed that with higher concentrations, the cells switched to aerobic respiration as no alcohol was being produced, so fermentation was stopped. This supports the hypothesis that increasing concentrations of ALA will lead to a decrease in cell proliferation, and may indicate that ALA upregulates PDC activity via inhibition of PDKs, which makes it a potential candidate in adjuvant therapies for cancer treatment.

FRI-516 - Inhibition of pyruvate dehydrogenase complex and lactate dehydrogenase for therapy of acute liver failure

FRI-516 - Inhibition of pyruvate dehydrogenase complex and lactate dehydrogenase for therapy of acute liver failure

Pyruvate dehydrogenase complex and lactate dehydrogenase are targets for therapy of acute liver failure

Acute liver failure is a rapidly progressive deterioration of hepatic function resulting in high mortality and morbidity. Metabolic enzymes can translocate to the nucleus to regulate histone acetylation and gene expression. Levels and activities of pyruvate dehydrogenase complex (PDHC) and lactate dehydrogenase (LDH) were evaluated in nuclear fractions of livers of mice exposed to various hepatotoxins including CD95-antibody, α-amanitin, and acetaminophen. Whole-genome gene expression profiling by RNA-seq was performed in livers of mice with acute liver failure and analyzed by gene ontology enrichment analysis. Cell viability was evaluated in cell lines knocked-down for PDHA1 or LDH-A and in cells incubated with the LDH inhibitor galloflavin after treatment with CD95-antibody. We evaluated whether the histone acetyltransferase inhibitor garcinol or galloflavin could reduce liver damage in mice with acute liver failure. Levels and activities of PDHC and LDH were increased in nuclear fractions of livers of mice with acute liver failure. The increase of nuclear PDHC and LDH was associated with increased concentrations of acetyl-CoA and lactate in nuclear fractions, and histone H3 hyper-acetylation. Gene expression in livers of mice with acute liver failure suggested that increased histone H3 acetylation induces the expression of genes related to damage response. Reduced histone acetylation by the histone acetyltransferase inhibitor garcinol decreased liver damage and improved survival in mice with acute liver failure. Knock-down of PDHC or LDH improved viability in cells exposed to a pro-apoptotic stimulus. Treatment with the LDH inhibitor galloflavin that was also found to inhibit PDHC, reduced hepatic necrosis, apoptosis, and expression of pro-inflammatory cytokines in mice with acute liver failure. Mice treated with galloflavin also showed a dose-response increase in survival. PDHC and LDH translocate to the nucleus, leading to increased nuclear concentrations of acetyl-CoA and lactate. This results in histone H3 hyper-acetylation and expression of damage response genes. Inhibition of PDHC and LDH reduces liver damage and improves survival in mice with acute liver failure. Thus, PDHC and LDH are targets for therapy of acute liver failure. Acute liver failure is a rapidly progressive deterioration of liver function resulting in high mortality. In experimental mouse models of acute liver failure, we found that two metabolic enzymes, namely pyruvate dehydrogenase complex and lactic dehydrogenase, translocate to the nucleus resulting in detrimental gene expression. Treatment with an inhibitor of these two enzymes was found to reduce liver damage and to improve survival.

Therapeutic Targeting of the Pyruvate Dehydrogenase Complex/Pyruvate Dehydrogenase Kinase (PDC/PDK) Axis in Cancer.

The mitochondrial pyruvate dehydrogenase complex (PDC) irreversibly decarboxylates pyruvate to acetyl coenzyme A, thereby linking glycolysis to the tricarboxylic acid cycle and defining a critical step in cellular bioenergetics. Inhibition of PDC activity by pyruvate dehydrogenase kinase (PDK)-mediated phosphorylation has been associated with the pathobiology of many disorders of metabolic integration, including cancer. Consequently, the PDC/PDK axis has long been a therapeutic target. The most common underlying mechanism accounting for PDC inhibition in these conditions is post-transcriptional upregulation of one or more PDK isoforms, leading to phosphorylation of the E1α subunit of PDC. Such perturbations of the PDC/PDK axis induce a "glycolytic shift," whereby affected cells favor adenosine triphosphate production by glycolysis over mitochondrial oxidative phosphorylation and cellular proliferation over cellular quiescence. Dichloroacetate is the prototypic xenobiotic inhibitor of PDK, thereby maintaining PDC in its unphosphorylated, catalytically active form. However, recent interest in the therapeutic targeting of the PDC/PDK axis for the treatment of cancer has yielded a new generation of small molecule PDK inhibitors. Ongoing investigations of the central role of PDC in cellular energy metabolism and its regulation by pharmacological effectors of PDKs promise to open multiple exciting vistas into the biochemical understanding and treatment of cancer and other diseases.

Defective Branched-Chain Amino Acid Catabolism Disrupts Glucose Metabolism and Sensitizes the Heart to Ischemia-Reperfusion Injury

Elevated levels of branched-chain amino acids (BCAAs) have recently been implicated in the development of cardiovascular and metabolic diseases, but the molecular mechanisms are unknown. In amouse model of impaired BCAA catabolism (knockout [KO]), we found that chronic accumulation of BCAAs suppressed glucose metabolism and sensitized the heart to ischemic injury. High levels of BCAAs selectively disrupted mitochondrial pyruvate utilization through inhibition of pyruvate dehydrogenase complex (PDH) activity. Furthermore, downregulation of the hexosamine biosynthetic pathway in KO hearts decreased protein O-linked N-acetylglucosamine (O-GlcNAc) modification and inactivated PDH, resulting in significant decreases in glucose oxidation. Although the metabolic remodeling in KO did not affect baseline cardiac energetics or function, it rendered the heart vulnerable to ischemia-reperfusion injury. Promoting BCAA catabolism or normalizing glucose utilization by overexpressing GLUT1 in the KO heart rescued the metabolic and functional outcome. These observations revealed a novel role of BCAA catabolism in regulating cardiac metabolism and stress response.

Specific inhibition by synthetic analogs of pyruvate reveals that the pyruvate dehydrogenase reaction is essential for metabolism and viability of glioblastoma cells.

The pyruvate dehydrogenase complex (PDHC) and its phosphorylation are considered essential for oncotransformation, but it is unclear whether cancer cells require PDHC to be functional or silenced. We used specific inhibition of PDHC by synthetic structural analogs of pyruvate to resolve this question. With isolated and intramitochondrial PDHC, acetyl phosphinate (AcPH, KiAcPH = 0.1 μM) was a much more potent competitive inhibitor than the methyl ester of acetyl phosphonate (AcPMe, KiAcPMe = 40 μM). When preincubated with the complex, AcPH also irreversibly inactivated PDHC. Pyruvate prevented, but did not reverse the inactivation. The pyruvate analogs did not significantly inhibit other 2-oxo acid dehydrogenases. Different cell lines were exposed to the inhibitors and a membrane-permeable precursor of AcPMe, dimethyl acetyl phosphonate, which did not inhibit isolated PDHC. Using an ATP-based assay, dependence of cellular viability on the concentration of the pyruvate analogs was followed. The highest toxicity of the membrane-permeable precursor suggested that the cellular action of charged AcPH and AcPMe requires monocarboxylate transporters. The relevant cell-specific transcripts extracted from Gene Expression Omnibus database indicated that cell lines with higher expression of monocarboxylate transporters and PDHC components were more sensitive to the PDHC inhibitors. Prior to a detectable antiproliferative action, AcPH significantly changed metabolic profiles of the investigated glioblastoma cell lines. We conclude that catalytic transformation of pyruvate by pyruvate dehydrogenase is essential for the metabolism and viability of glioblastoma cell lines, although metabolic heterogeneity causes different cellular sensitivities and/or abilities to cope with PDHC inhibition.

Differential effects of lipopolysaccharide on energy metabolism in murine microglial N9 and cholinergicSN56 neuronal cells

There are significant differences between acetyl-CoA and ATP levels, enzymes of acetyl-CoA metabolism, and toll-like receptor 4 contents in non-activated microglial N9 and non-differentiated cholinergic SN56 neuroblastoma cells. Exposition of N9 cells to lipopolysaccharide caused concentration-dependent several-fold increases of nitrogen oxide synthesis, accompanied by inhibition of pyruvate dehydrogenase complex, aconitase, and α-ketoglutarate dehydrogenase complex activities, and by nearly proportional depletion of acetyl-CoA, but by relatively smaller losses in ATP content and cell viability (about 5%). On the contrary, SN56 cells appeared to be insensitive to direct exposition to high concentration of lipopolysaccharide. However, exogenous nitric oxide resulted in marked inhibition pyruvate dehydrogenase and aconitase activities, depletion of acetyl-CoA, along with respective loss of SN56 cells viability. These data indicate that these two common neurodegenerative signals may differentially affect energy-acetyl-CoA metabolism in microglial and cholinergic neuronal cell compartments in the brain. Moreover, microglial cells appeared to be more resistant than neuronal cells to acetyl-CoA and ATP depletion evoked by these neurodegenerative conditions. Together, these data indicate that differential susceptibility of microglia and cholinergic neuronal cells to neurotoxic signals may result from differences in densities of toll-like receptors and degree of disequilibrium between acetyl-CoA provision in mitochondria and its utilization for energy production and acetylation reactions in each particular group of cells. There are significant differences between acetyl-CoA and ATP levels and enzymes of acetyl-CoA metabolism in non-activated microglial N9 and non-differentiated cholinergic SN56 neuroblastoma cells. Pathological stimulation of microglial toll-like receptors (TLRs) triggered excessive synthesis of microglia-derived nitric oxide (NO)/NOO radicals that endogenously inhibited pyruvate dehydrogenase complex (PDHC), aconitase, and α-ketoglutarate dehydrogenase complex. However, it caused none or small suppressions of acetyl-CoA and microglial viability, respectively. Microglia-derived NO inhibited same enzymes in cholinergic neuronal cells causing marked viability loss because of acetyl-CoA deficits evoked by its competitive consumption by energy producing and acetylcholine/N-acetyl-l-aspartate (NAA) synthesizing pathways.

Intracellular redistribution of acetyl-CoA, the pivotal point in differential susceptibility of cholinergic neurons and glial cells to neurodegenerative signals

Intramitochondrial decarboxylation of glucose-derived pyruvate by PDHC (pyruvate dehydrogenase complex) is a principal source of acetyl-CoA, for mitochondrial energy production and cytoplasmic synthetic pathways in all typesof brain cells. The inhibition of PDHC, ACO (aconitase) and KDHC (ketoglutarate dehydrogenase complex) activities by neurodegenerative signals such as aluminium, zinc, amyloid β-peptide, excess nitric oxide (NO) or thiamine pyrophosphate deficits resulted in much deeper losses of viability, acetyl-CoA and ATP in differentiated cholinergic neuronal cells than in non-differentiated cholinergic, and cultured microglial or astroglial cell lines. In addition, in cholinergic cells, such conditions caused inhibition of ACh (acetylcholine) synthesis and its quantal release. Furthermore, cholinergic neuronal cells appeared to be resistant to high concentrations of LPS (lipopolysaccharide). In contrast, in microglial cells, low levels of LPS caused severalfold activation of NO, IL-6 (interleukin 6) and TNFα (tumour necrosis factor α) synthesis/release, accompanied by inhibition of PDHC, KDHC and ACO activities, and suppression of acetyl-CoA, but relatively small losses in their ATP contents and viability parameters. Compounds that protected these enzymes against inhibitory effects of neurotoxins alleviated acetyl-CoA and ATP deficits, thereby maintaining neuronal cell viability. These data indicate that preferential susceptibility of cholinergic neurons to neurodegenerative insults may result from competition for acetyl-CoA between mitochondrial energy-producing and cytoplasmic ACh-synthesizing pathways. Such a hypothesis is supported by the existence of highly significant correlations between mitochondrial/cytoplasmic acetyl-CoA levels and cell viability/transmitter functions respectively.

Tyr-94 Phosphorylation Inhibits Pyruvate Dehydrogenase Phosphatase 1 and Promotes Tumor Growth

Many cancer cells rely more on aerobic glycolysis (the Warburg effect) than mitochondrial oxidative phosphorylation and catabolize glucose at a high rate. Such a metabolic switch is suggested to be due in part to functional attenuation of mitochondria in cancer cells. However, how oncogenic signals attenuate mitochondrial function and promote the switch to glycolysis remains unclear. We previously reported that tyrosine phosphorylation activates and inhibits mitochondrial pyruvate dehydrogenase kinase (PDK) and phosphatase (PDP), respectively, leading to enhanced inhibitory serine phosphorylation of pyruvate dehydrogenase (PDH) and consequently inhibition of pyruvate dehydrogenase complex (PDC) in cancer cells. In particular, Tyr-381 phosphorylation of PDP1 dissociates deacetylase SIRT3 and recruits acetyltransferase ACAT1 to PDC, resulting in increased inhibitory lysine acetylation of PDHA1 and PDP1. Here we report that phosphorylation at another tyrosine residue, Tyr-94, inhibits PDP1 by reducing the binding ability of PDP1 to lipoic acid, which is covalently attached to the L2 domain of dihydrolipoyl acetyltransferase (E2) to recruit PDP1 to PDC. We found that multiple oncogenic tyrosine kinases directly phosphorylated PDP1 at Tyr-94, and Tyr-94 phosphorylation of PDP1 was common in diverse human cancer cells and primary leukemia cells from patients. Moreover, expression of a phosphorylation-deficient PDP1 Y94F mutant in cancer cells resulted in increased oxidative phosphorylation, decreased cell proliferation under hypoxia, and reduced tumor growth in mice. Together, our findings suggest that phosphorylation at different tyrosine residues inhibits PDP1 through independent mechanisms, which act in concert to regulate PDC activity and promote the Warburg effect.

Abstract 997: Oncogenic tyrosine kinases are localized to mitochondria and regulate cancer metabolism by phosphorylating key components of pyruvate dehydrogenase kinase complex

Abstract The Warburg effect describes the observation that cancer cells take up more glucose than normal tissue and favor aerobic glycolysis. Many tumor cells rely on aerobic glycolysis instead of mitochondrial oxidative phosphorylation for their continued proliferation and survival. This may be, in part, due to attenuated mitochondrial function, which is suggested to be achieved through inhibition of pyruvate dehydrogenase complex (PDC) in cancer cells. Pyruvate dehydrogenase (PDH) is the first and most important enzyme component of PDC, which catalyzes the conversion of pyruvate to acetyl-CoA that subsequently enters into the Krebs cycle to produce ATP and electron donors including NADH. PDH activity is negatively regulated by phosphorylation at several serine sites. Phosphorylation of PDH by PDH kinase (PDHK) results in the inactivation of PDC, while dephosphorylation by pyruvate dehydrogenase phosphatase (PDP) restores PDC activity. Thus, PDHK and PDP coordinate to regulate the phosphorylation and consequently activation status of PDH. In cancer cells, c-Myc and HIF-1alpha are believed to promote inhibition of PDC by, in part, upregulating gene expression of PDHK. However, how oncogenic signals regulate PDH, and/or coordinate PDHK and PDP activity to inhibit PDH and ultimately attenuate PDC activity in cancer metabolism still remains unclear. We recently reported that, surprisingly, functional PDC can form in mitochondria outside of matrix in some cancer cells. Moreover, several oncogenic tyrosine kinases including FGFR1, FGFR1 fusion tyrosine kinase, and FLT3-ITD mutant, as well as BCR-ABL and JAK2 V617F mutants, are localized to different mitochondrial compartments including outer membrane and matrix, respectively, in cancer cells, where they phosphorylate PDHK1, PDP1 and PDHA. Our studies demonstrate that tyrosine phosphorylation enhances PDHK1 kinase activity by promoting ATP and PDC binding, which promotes cancer cell metabolism, proliferation and tumor growth. In consonance with these findings, we found that tyrosine phosphorylation attenuates PDP1 phosphatase activity but inhibits PDHA enzyme activity. These data suggest that mitochondrial oncogenic tyrosine kinases regulate PDC activity by inhibiting PDHA1 enzyme activity directly through tyrosine phosphorylation and indirectly by enhancing and attenuating PDHK1 and PDP1 activity, respectively. These findings represent an acute mechanism underlying the Warburg effect, in addition to the long-term regulation that is believed to be regulated by c-Myc and HIF-1alpha. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 997. doi:1538-7445.AM2012-997

The Role of FOXO and PPAR Transcription Factors in Diet-Mediated Inhibition of PDC Activation and Carbohydrate Oxidation During Exercise in Humans and the Role of Pharmacological Activation of PDC in Overriding These Changes

High-fat feeding inhibits pyruvate dehydrogenase complex (PDC)–controlled carbohydrate (CHO) oxidation, which contributes to muscle insulin resistance. We aimed to reveal molecular changes underpinning this process in resting and exercising humans. We also tested whether pharmacological activation of PDC overrides these diet-induced changes. Healthy males consumed a control diet (CD) and on two further occasions an isocaloric high-fat diet (HFD). After each diet, subjects cycled for 60 min after intravenous infusion with saline (CD and HFD) or dichloroacetate (HFD+DCA). Quadriceps muscle biopsies obtained before and after 10 and 60 min of exercise were used to estimate CHO use, PDC activation, and mRNAs associated with insulin, fat, and CHO signaling. Compared with CD, HFD increased resting pyruvate dehydrogenase kinase 2 (PDK2), PDK4, forkhead box class O transcription factor 1 (FOXO1), and peroxisome proliferator–activated receptor transcription factor α (PPARα) mRNA and reduced PDC activation. Exercise increased PDC activation and whole-body CHO use in HFD, but to a lower extent than in CD. Meanwhile PDK4 and FOXO1, but not PPARα or PDK2, mRNA remained elevated. HFD+DCA activated PDC throughout and restored whole-body CHO use during exercise. FOXO1 appears to play a role in HFD-mediated muscle PDK4 upregulation and inhibition of PDC and CHO oxidation in humans. Also, pharmacological activation of PDC restores HFD-mediated inhibition of CHO oxidation during exercise.

The Role of PDK4 in High Fat Diet - Induced Insulin Resistance

Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) is a potential main regulator of the metabolic program that is shown to be activated by exercise and down-regulated by high fat feeding and sedentary lifestyle. It is shown that PGC-1α coactivates the pyruvate dehydrogenase kinase (PDK4) gene expression via the estrogen related receptor (ERRα). The expression of PDK4 is shown to be elevated with diabetes, fasting and other conditions associated with the switch from the utilization of glucose to fatty acids as an energy source. PURPOSE: Our aim in this study was to investigate the effects of high fat (HF) diet and physical activity in the expression levels of the above mentioned genes in the skeletal muscles of C57BL/6J mice. METHODS: In this experiment we induced insulin resistance by feeding high fat diet for 19 weeks to C57BL/6J mice. After 9 and 18 weeks on the diet, the fasting glucose and insulin levels were measured. The total cholesterol, free fatty acids and triglycerides were measured after the 19 - week experiment. The expression levels of the genes PGC-1α, ERRα and PDK4 were measured with quantitative RT-PCR and by Western blotting from the quadriceps femoris muscle. RESULTS:The HF animals had significantly higher diet - induced fasting glucose and insulin levels compared to low fat (LF) fed animals. The HOMA index indicated that after 18 weeks HF mice were more insulin resistant than LF mice, but no running effect could be seen. The HF animals showed an altered blood lipid profile suggesting changes in the overall lipid metabolism. Chronic HF feeding and voluntary running training did not have significant effects on PGC-1α mRNA or protein levels. Upregulation of ERRα and PDK4 was seen after HF diet and this combined with running had even more profound effects on the expression levels. CONCLUSIONS: We observed an up-regulated PDK4 expression along with more modest changes in PGC-1α and ERRα expression in response to both high fat diet and voluntary exercise. We speculate that this increased PDK4 expression is due to previous increase of the PGC-1α and ERRα expression, which subsequently blunts cellular glucose oxidation. In this scenario, PGC-1α is the master regulator of energy metabolism by regulating the increase of fatty acid oxidation and decreasing glucose oxidation via PGC-1α/ERRα pathway. Our data further suggests that the inhibition of pyruvate dehydrogenase complex by PDK4 must be contemplated as a possible mechanism or contributor to insulin resistance. The research was funded by the Finnish Ministry of Education and Culture.

Augmented cerebellar lactate in copper deficient rat pups originates from both blood and cerebellum

Copper (Cu) is essential for proper brain development, particularly the cerebellum, and functions as a cofactor for enzymes including mitochondrial cytochrome c oxidase (CCO). Cu deficiency severely limits CCO activity. Augmented lactate in brain of Cu deficient (Cu-) humans and cerebella of Cu- rats is though to originate from impaired mitochondria. However, brain lactate may also originate from elevated blood lactate. The hypothesis that cerebellar lactate originates from elevated blood lactate in Cu- rat pups was tested. Analysis of Cu- and Cu adequate (Cu+) rat pups (experiment I) revealed blood lactate was elevated in Cu- rat pups and cerebellar lactate levels were closely correlated to blood lactate concentration. A second rat experiment (experiment II) assessed Cu- cerebellar lactate without the confounding factor of elevated blood lactate. Blood lactate levels of Cu- rat pups in experiment II were equal to those of controls; however, Cu- cerebellar lactate was still elevated, suggesting mitochondrial impairment by Cu deficiency. Treatment of rat pups with dichloroacetate (DCA), an activator of mitochondrial pyruvate dehydrogenase complex (PDC), lowered Cu- cerebellar lactate to control levels suggesting PDC inhibition is a site of mitochondrial impairment in Cu- cerebella. Results suggest Cu- cerebellar lactate originates from blood and cerebellum.

Pyruvate Dehydrogenase Complex Activity Controls Metabolic and Malignant Phenotype in Cancer Cells

High lactate generation and low glucose oxidation, despite normal oxygen conditions, are commonly seen in cancer cells and tumors. Historically known as the Warburg effect, this altered metabolic phenotype has long been correlated with malignant progression and poor clinical outcome. However, the mechanistic relationship between altered glucose metabolism and malignancy remains poorly understood. Here we show that inhibition of pyruvate dehydrogenase complex (PDC) activity contributes to the Warburg metabolic and malignant phenotype in human head and neck squamous cell carcinoma. PDC inhibition occurs via enhanced expression of pyruvate dehydrogenase kinase-1 (PDK-1), which results in inhibitory phosphorylation of the pyruvate dehydrogenase alpha (PDHalpha) subunit. We also demonstrate that PDC inhibition in cancer cells is associated with normoxic stabilization of the malignancy-promoting transcription factor hypoxia-inducible factor-1alpha (HIF-1alpha) by glycolytic metabolites. Knockdown of PDK-1 via short hairpin RNA lowers PDHalpha phosphorylation, restores PDC activity, reverts the Warburg metabolic phenotype, decreases normoxic HIF-1alpha expression, lowers hypoxic cell survival, decreases invasiveness, and inhibits tumor growth. PDK-1 is an HIF-1-regulated gene, and these data suggest that the buildup of glycolytic metabolites, resulting from high PDK-1 expression, may in turn promote HIF-1 activation, thus sustaining a feed-forward loop for malignant progression. In addition to providing anabolic support for cancer cells, altered fuel metabolism thus supports a malignant phenotype. Correction of metabolic abnormalities offers unique opportunities for cancer treatment and may potentially synergize with other cancer therapies.