Increased Pyruvate Dehydrogenase Activity Research Articles

Abstract 2148: Phenyl butyrate inhibits pyruvate dehydrogenase kinase 1 and contributes to its anti-cancer effects

Abstract In cancer cells, the metabolic features are significantly different from those of normal ones, which are hallmarks of several malignancies. Recent studies brought atypical cellular metabolism, such as aerobic glycolysis or the Warburg effect, into the scientific limelight. Pyruvate dehydrogenase kinase 1 (PDK1), a key enzyme in the pathway of glucose metabolism, could inactivate the pyruvate dehydrogenase (PDH) by phosphorylating it and preserving the substrates pyruvate, lactate and alanine for gluconeogenesis. Overexpression of PDK1 could block the oxidative decarboxylation of pyruvate to satisfy high oxygen demand in cancer cells, while inhibition of PDK1 could upregulate the activity of PDH and rectify the balance between the demand and supply of oxygen, which could lead to cancer cell death. Thus, inhibitors targeting PDK1 might be a promising strategy for cancer treatment by acting on glycolytic tumors while showing minimal side effects on the oxidative healthy organs. Phenyl butyrate (PB) has been proved to decrease PDH phosphorylation level and increase PDH activity by inhibiting PDK1 in fibroblast cells, PDH deficiency zebrafish and wild type mice. PB has also shown efficacy in many cancers and so far, all of its anti-tumor activity has been attributed to the histone deacetylase (HDAC) inhibitor mode of action. As PDK1/PDH controls the critical switch between oxidative phosphorylation and glycolysis in cancer cells, PDK1 is a key target in tumor metabolism for anti-cancer treatment. We hypothesize that the therapeutic effects of PB in cancers might depend on suppressing PDKs and promoting PDH activity, in addition to its proposed role as HDAC inhibitor. We showed that PB directly inhibited the kinase activity of PDK1 in a purified system. In several different cancer cell lines, PB reduced the phosphorylation level of PDH, increased the mitochondrial respiration, decreased glycolysis in cytoplasm, reversed mitochondrial hyperpolarization, activated some proteins in apoptotic signalling pathway and then induced the apoptosis of cells. In summary, this is the first study indicated that PB could exert its anti-cancer effects through inhibiting PDK1, altering the mitochondrial bioenergetics and inducing apoptosis. Note: This abstract was not presented at the meeting. Citation Format: Wen Zhang, Kin Yip Tam. Phenyl butyrate inhibits pyruvate dehydrogenase kinase 1 and contributes to its anti-cancer effects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2148. doi:10.1158/1538-7445.AM2017-2148

Phenyl butyrate inhibits pyruvate dehydrogenase kinase 1 and contributes to its anti-cancer effect

Phenyl butyrate (PB) has been proved to decrease pyruvate dehydrogenase (PDH) phosphorylation level and increase PDH activity by inhibiting pyruvate dehydrogenase kinase 1 (PDK1) in fibroblast cells, PDH deficiency zebrafish and wild type mice. PB has also shown efficacy in many cancers and so far, all of its anti-tumor activity has been attributed to the histone deacetylase (HDAC) inhibition. As PDK1/PDH controls the critical switch between oxidative phosphorylation and glycolysis in cancer cells, PDK1 is a key target in tumor metabolism for anti-cancer treatment. We hypothesize that the therapeutic effects of PB in cancers might also depend on suppressing PDK1 and promoting PDH activity, in addition to its proposed role as HDAC inhibitor. We showed that PB directly inhibited the kinase activity of PDK1 and increased the activity of PDH in an enzyme assay. In several different cancer cell lines, PB reduced the phosphorylation level of PDH, increased the mitochondrial respiration, decreased glycolysis in cytoplasm, reversed mitochondrial hyperpolarization, activated several proteins in apoptotic signaling pathway and then induced the apoptosis of cells. In summary, this is the first study indicated that PB could exert its anti-cancer effects through inhibiting PDK1, altering the mitochondrial bioenergetics and inducing apoptosis.

Inhibition of sarcolemmal FAT/CD36 by sulfo-N-succinimidyl oleate rapidly corrects metabolism and restores function in the diabetic heart following hypoxia/reoxygenation.

AimsThe type 2 diabetic heart oxidizes more fat and less glucose, which can impair metabolic flexibility and function. Increased sarcolemmal fatty acid translocase (FAT/CD36) imports more fatty acid into the diabetic myocardium, feeding increased fatty acid oxidation and elevated lipid deposition. Unlike other metabolic modulators that target mitochondrial fatty acid oxidation, we proposed that pharmacologically inhibiting fatty acid uptake, as the primary step in the pathway, would provide an alternative mechanism to rebalance metabolism and prevent lipid accumulation following hypoxic stress.Methods and resultsHearts from type 2 diabetic and control male Wistar rats were perfused in normoxia, hypoxia and reoxygenation, with the FAT/CD36 inhibitor sulfo-N-succinimidyl oleate (SSO) infused 4 min before hypoxia. SSO infusion into diabetic hearts decreased the fatty acid oxidation rate by 29% and myocardial triglyceride concentration by 48% compared with untreated diabetic hearts, restoring fatty acid metabolism to control levels following hypoxia-reoxygenation. SSO infusion increased the glycolytic rate by 46% in diabetic hearts during hypoxia, increased pyruvate dehydrogenase activity by 53% and decreased lactate efflux rate by 56% compared with untreated diabetic hearts during reoxygenation. In addition, SSO treatment of diabetic hearts increased intermediates within the second span of the Krebs cycle, namely fumarate, oxaloacetate, and the FAD total pool. The cardiac dysfunction in diabetic hearts following decreased oxygen availability was prevented by SSO-infusion prior to the hypoxic stress. Infusing SSO into diabetic hearts increased rate pressure product by 60% during hypoxia and by 32% following reoxygenation, restoring function to control levels.ConclusionsDiabetic hearts have limited metabolic flexibility and cardiac dysfunction when stressed, which can be rapidly rectified by reducing fatty acid uptake with the FAT/CD36 inhibitor, SSO. This novel therapeutic approach not only reduces fat oxidation but also lipotoxicity, by targeting the primary step in the fatty acid metabolism pathway.

Pyruvate dehydrogenase alpha 1 as a target of omega-3 polyunsaturated fatty acids in human prostate cancer through a global phosphoproteomic analysis.

Prostate cancer is one of the leading cancers in men. Taking dietary supplements, such as fish oil (FO), which is rich in n-3 polyunsaturated fatty acids (PUFAs), has been employed as a strategy to lower prostate cancer risk and control disease progression. In this study, we investigated the global phosphoproteomic changes induced by FO using a combination of phosphoprotein-enrichment strategy and high-resolution tandem mass spectrometry. We found that FO induces many more phosphorylation changes than oleic acid when they both are compared to control group. Quantitative comparison between untreated group and FO- or oleic acid-treated groups uncovered a number of important protein phosphorylation changes induced by n-3PUFAs. This phosphoproteomic discovery study and the follow-up Western Blot validation study elucidate that phosphorylation levels of the two regulatory serine residues in pyruvate dehydrogenase alpha 1 (PDHA1), serine-232 and serine-300, are significantly decreased upon FO treatment. As expected, increased pyruvate dehydrogenase activity was also observed. This study suggests that FO-induced phosphorylation changes in PDHA1 is more likely related to the glucose metabolism pathway, and n-3 PUFAs may have a role in controlling the balance between lipid and glucose oxidation.

Fusarium graminearum pyruvate dehydrogenase kinase 1 (FgPDK1) Is Critical for Conidiation, Mycelium Growth, and Pathogenicity.

Pyruvate dehydrogenase kinase (PDK) is an important mitochondrial enzyme that blocks the production of acetyl-CoA by selectively inhibiting the activity of pyruvate dehydrogenase (PDH) through phosphorylation. PDK is an effectively therapeutic target in cancer cells, but the physiological roles of PDK in phytopathogens are largely unknown. To address these gaps, a PDK gene (FgPDK1) was isolated from Fusarium graminearum that is an economically important pathogen infecting cereals. The deletion of FgPDK1 in F. graminearum resulted in the increase in PDH activity, coinciding with several phenotypic defects, such as growth retardation, failure in perithecia and conidia production, and increase in pigment formation. The ΔFgPDK1 mutants showed enhanced sensitivity to osmotic stress and cell membrane-damaging agent. Physiological detection indicated that reactive oxygen species (ROS) accumulation and plasma membrane damage (indicated by PI staining, lipid peroxidation, and electrolyte leakage) occurred in ΔFgPDK1 mutants. The deletion of FgPDK1 also prohibited the production of deoxynivalenol (DON) and pathogenicity of F. graminearum, which may resulted from the decrease in the expression of Tri6. Taken together, this study firstly identified the vital roles of FgPDK1 in the development of phytopathogen F. graminearum, which may provide a potentially novel clue for target-directed development of agricultural fungicides.

Thiamine as a neuroprotective agent after cardiac arrest

AimsReduction of pyruvate dehydrogenase (PDH) activity in the brain is associated with neurological deficits in animals resuscitated from cardiac arrest. Thiamine is an essential co-factor of PDH. The objective of this study was to examine whether administration of thiamine improves outcomes after cardiac arrest in mice. Secondarily, we aimed to characterize the impact of cardiac arrest on PDH activity in mice and humans. MethodsAnimal study: Adult mice were subjected to cardiac arrest whereupon cardiopulmonary resuscitation was performed. Thiamine or vehicle was administered 2min before resuscitation and daily thereafter. Mortality, neurological outcome, and metabolic markers were evaluated.Human study: In a convenience sample of post-cardiac arrest patients, we measured serial PDH activity from peripheral blood mononuclear cells and compared them to healthy controls. ResultsAnimal study: Mice treated with thiamine had increased 10-day survival (48% versus 17%, P<0.01) and improved neurological function when compared to vehicle-treated mice. In addition, thiamine markedly improved histological brain injury compared to vehicle. The beneficial effects of thiamine were accompanied by improved oxygen consumption in mitochondria, restored thiamine pyrophosphate levels, and increased PDH activity in the brain at 10 days.Human study: Post-cardiac arrest patients had lower PDH activity in mononuclear cells than did healthy volunteers (estimated difference: ⿿5.8O.D./min/mg protein, P<0.001). ConclusionsThe provision of thiamine after cardiac arrest improved neurological outcome and 10-day survival in mice. PDH activity was markedly depressed in post-cardiac arrest patients suggesting that this pathway may represent a therapeutic target.

Abstract 18488: Metabolic Modulation of the Diseased Human Lung; A Translational, Novel ex vivo Lung Perfusion Model

Introduction: Pulmonary arterial hypertension (PAH) and idiopathic pulmonary fibrosis (IPF) are deadly diseases that often require lung transplantation. Preclinical evidence suggests that mitochondrial suppression underlies the pro-proliferative and anti-apoptotic phenotype of pulmonary artery smooth muscle cells and pulmonary fibroblasts, respectively. Two therapies to improve mitochondrial function are a) dichloroacetate (DCA), an activator of pyruvate dehydrogenase (PDH), the gate-keeping enzyme for glucose oxidation, and b) 4-phenylbutyrate (4-PBA), an inhibitor of endoplasmic reticulum stress, a response that suppresses mitochondrial function during cellular stress. Although these drugs can be studied in vitro or in animals, their metabolic effects on the whole human lung may only by studied through ex vivo lung perfusion (EVLP). Methods: We used a custom-made EVLP system to study explanted, diseased human lungs of patients undergoing lung transplant for PAH (n=2), or IPF (n=4). The bronchi were connected to a ventilator, while a pump-driven system perfused the pulmonary artery (PA) with modified Krebs-Henseleit buffer or STEEN solution™ in recirculation. Lung biopsies were collected to measure mitochondrial respiration (using a Seahorse analyzer) and PDH activity at baseline, and after drug intervention with DCA or 4-PBA for 1 hour. Results: In the PAH lungs, hypoxic pulmonary vasoconstriction, a validated marker of lung health, was absent. However administrating the prostacyclin analogue treprostinil reduced PA pressure by 31.6% (at a level of flow corresponding to the patient’s cardiac output), confirming abnormal but present vasoreactivity in the model. DCA caused a 61.1% increase in oxygen consumption, and a 34-fold increase in PDH activity. In IPF lungs, PBA increased oxygen consumption by 62.0%. Conclusions: EVLP is a feasible model to study the direct effects of drugs on diseased human lungs. Since before-after biopsies are not possible in clinical trials, this is a valuable model to facilitate translation of preclinical therapies to clinical trials. Our results show, for the first time in human lungs, that the metabolic remodeling of the PAH and IPF can be reversed by clinically available small molecules like DCA and 4-PBA.

Abstract 13675: New Treatments for Myocardial Stunning: Creating an Optimal Drug Cocktail for CPR

Introduction: Myocardial stunning following sudden cardiac arrest resuscitation increases morbidity and mortality. Previous work suggests that intra-ischemic cooling protects against stunning by enhancing Akt survival signaling, with related enhancement of glucose utilization and NAD+ recovery. New or existing drugs that mimic these effects could improve cardiac arrest outcomes. Here we test agents known to activate PI3K/Akt signaling, increase pyruvate dehydrogenase activity, and enhance NAD+ content. They were tested for their ability to improve contractile recovery in a mouse cardiomyocyte stunning model. Methods: Primary neonatal cardiomyocytes were exposed to 30 min ischemia and 90 min reperfusion followed by 10 min isoproterenol (1μM). Therapeutic hypothermia (TH) using intra-ischemic cooling (32 ± 0.5°C), the PTEN inhibitor VO-OHpic (VO, 1μM), insulin (0.01U/ml), intralipid (ILE, 0.25%), dichloracetate (DCA, 2 mM), and the NAD+ augmentation agents NAM (10 μM), and Nampt peptide (1-200 nM) were administered at reperfusion. Akt phosphorylation was detected by Western blot. ATP content was measured by a fluorescent assay kit. Results: Compared to control cells, spontaneous contractions ceased within 5 min of ischemia and upon reperfusion recovered to less than 40% of baseline contractions despite isoproterenol treatment. Comparable to TH, the use of VO, DCA, NAM, insulin or ILE all induced a rapid and strong contractile recovery with the most significant sustained effect seen with VO and ILE. Both VO and TH improved recovery of ATP after reperfusion to comparable levels. NAM resulted in a rapid recovery that was not sustained with one dose, while Nampt had no effect. The most synergistic cocktail of two-drug combinations tested was VO plus NAM. VO increased Akt phosphorylation while NAM treatment did not, suggesting that the complementary protective effect of NAM was downstream of Akt activation. Conclusions: Treatment of contractile stunning after cardiac ischemia can be achieved by drugs that mimic critical aspects of cooling protection, including Akt activation and NAD+ supplementation. Further work is needed to test drug combinations during cardiopulmonary resuscitation (CPR) that improve myocardial recovery.

Continuous succinic acid fermentation by Escherichia coli KJ122 with cell recycle

High cell densities were obtained by separating the cells with an external hollow fibre filter. Extreme product inhibition at high succinate titres resulted in cell death and subsequent lysis. Accordingly, the highest succinate titre obtained during continuous fermentation was 25gL−1 at a dilution rate of 0.05h−1. The highest volumetric productivity of 3gL−1h−1 and the highest succinate yield (0.77gg−1) were obtained at the highest dilution rate (0.15h−1). The improved yield was caused by increased pyruvate dehydrogenase activity, leading to a decrease in pyruvate and formate excretion and an increase in the reductive flux towards succinate as additional reduction power was produced. The oxidative tricarboxylic acid cycle flux was determined to be minimal, with most of the acetyl coenzyme A (acetyl-CoA) culminating as acetate. Although comparative batch fermentations exhibited a fivefold lower volumetric productivity than the maximum obtained in the cell recycle runs, higher succinate titres (56gL−1) and yields (0.85gL−1) were obtained. The higher batch yields were attributed to pyruvate and formate consumption after the termination of cell growth.

PD33-11 METABOLIC MODULATION WITH DICHLOROACETATE REVERSES THE WARBURG EFFECT AND REDUCES THE GROWTH OF CLEAR CELL RENAL CELL CARCINOMA IN VITRO AND IN VIVO

You have accessJournal of UrologyKidney Cancer: Basic Research III1 Apr 2015PD33-11 METABOLIC MODULATION WITH DICHLOROACETATE REVERSES THE WARBURG EFFECT AND REDUCES THE GROWTH OF CLEAR CELL RENAL CELL CARCINOMA IN VITRO AND IN VIVO Adam Kinnaird, Peter Dromparis, Roxane Paulin, Bruno Saleme, Trevor Stenson, Desmond Pink, John Lewis, and Evangelos Michelakis Adam KinnairdAdam Kinnaird More articles by this author , Peter DromparisPeter Dromparis More articles by this author , Roxane PaulinRoxane Paulin More articles by this author , Bruno SalemeBruno Saleme More articles by this author , Trevor StensonTrevor Stenson More articles by this author , Desmond PinkDesmond Pink More articles by this author , John LewisJohn Lewis More articles by this author , and Evangelos MichelakisEvangelos Michelakis More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.2109AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Clear Cell Renal Cell Carcinoma (ccRCC) exhibits a distinct metabolic phenotype using cytoplasmic glycolysis over a suppressed glucose oxidation (GO) even in normoxia (Warburg effect; WE). This offers growth advantages including suppressed apoptosis and increased proliferation and angiogenesis. Most ccRCC have constitutively active Hypoxia Inducible Factor (HIF) (due to mutation or silencing of the von Hippel Lindau tumor factor), which induces the WE by upregulating Pyruvate Dehydrogenase kinase (PDK) and thus inhibiting Pyruvate Dehydrogenase (PDH), the gate keeping enzyme in GO. PDK inhibitors like the clinically available small molecule Dichloroacetate (DCA) have been shown to increase GO and reverse the WE, decreasing growth of solid tumors. The resulting increase in mitochondrial signals (mitochondrial-derived reactive oxygen species and α-ketoglutarate) can inhibit the redox-sensitive HIF transcriptional activity (PMID: 22614004). We hypothesized that DCA will decrease ccRCC growth even in the presence of constitutively active HIF. METHODS The 786-O ccRCC cell line was used in vitro, in ovo (cells injected into the chorioallantoic membrane of fertilized chicken eggs) and in vivo in Nu/Nu mice, following both a prevention and reversal protocol and at doses similar to those achieved in the serum of DCA treated patients. RESULTS In vitro DCA increased PDH activity and GO, increased apoptosis, decreased proliferation and decreased HIF activity, including decreasing VEGF levels. siRNA inhibition of FIH prevented most of the DCA-induced decrease in HIF activity in an α-ketoglutarate dependent manner. DCA both prevented tumor growth and decreased tumor size in the reversal protocol: in ovo mean tumor weight 74mg control, 18mg DCA, n=20 and 29 eggs respectively, p<0.05; in vivo mean tumor volume 271mm3 control, 136mm3 DCA, n=10 and 20 respectively, p<0.01. DCA significantly decreased angiogenesis in vivo, measured by lectin imaging. CONCLUSIONS DCA can reverse the WE in ccRCC and decrease tumor growth and angiogenesis even though HIF is constitutively expressed. The DCA-induced inhibition of angiogenesis may be superior to VEGF inhibitors since it suppresses the HIF axis proximal to VEGF. We suggest that DCA should be tested in early phase clinical trials in ccRCC. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e713 Peer Review Report Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Adam Kinnaird More articles by this author Peter Dromparis More articles by this author Roxane Paulin More articles by this author Bruno Saleme More articles by this author Trevor Stenson More articles by this author Desmond Pink More articles by this author John Lewis More articles by this author Evangelos Michelakis More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

SIRT3 deacetylates and increases pyruvate dehydrogenase activity in cancer cells

Pyruvate dehydrogenase E1α (PDHA1) is the first component enzyme of the pyruvate dehydrogenase (PDH) complex that transforms pyruvate, via pyruvate decarboxylation, into acetyl-CoA that is subsequently used by both the citric acid cycle and oxidative phosphorylation to generate ATP. As such, PDH links glycolysis and oxidative phosphorylation in normal as well as cancer cells. Herein we report that SIRT3 interacts with PDHA1 and directs its enzymatic activity via changes in protein acetylation. SIRT3 deacetylates PDHA1 lysine 321 (K321), and a PDHA1 mutant mimicking a deacetylated lysine (PDHA1K321R) increases PDH activity, compared to the K321 acetylation mimic (PDHA1K321Q) or wild-type PDHA1. Finally, PDHA1K321Q exhibited a more transformed in vitro cellular phenotype compared to PDHA1K321R. These results suggest that the acetylation of PDHA1 provides another layer of enzymatic regulation, in addition to phosphorylation, involving a reversible acetyllysine, suggesting that the acetylome, as well as the kinome, links glycolysis to respiration.

Abstract 3268: Targeting leukemia metabolism: augmentation of anti-leukemic activity of arsenic trioxide (As2O3) against non-APL AML cell lines in presence of dichloroacetate (DCA).

Abstract Background: DCA increases pyruvate dehydrogenase activity via inhibition of pyruvate dehydrogenase kinase, which shunts pyruvate away from lactate production into the mitochondrial respiration, resulting in enhanced mitochondrial activity. As2O3 is approved for treatment of relapsed/refractory acute promyelocytic leukemia (APL). As2O3 as a single agent or in combination with cytarabine is ineffective for treatment of non-APL acute myeloid leukemia (AML) by most studies. As2O3 affects several different molecular targets and cellular functions such as decreasing mitochondrial membrane potential by opening permeability transition pore complex in membranes, reactive oxygen species production, and caspase pathways activation. We hypothesized that combination of DCA, as a mitochondrial respiration enhancer, and As2O3, which impairs mitochondrial activity, would have anti-leukemic synergistic effects and potentially overcome drug resistance particularly in relapsed/refractory AML. Methods: IC50s for As2O3 and DCA were generated for two AML cell lines, MOLM-14 and MV4-11, the latter carries FLT3-ITD mutation. Cells were treated with DCA at IC30 and As2O3 either concurrently for 72 h or sequentially with 48 h exposure to DCA followed by the addition of As2O3 (100 nM - 5μM) for 48 h. Each experiment was terminated with WST-1 (Roche). Viability of cells was assessed by trypan blue exclusion. Experiments were performed in triplicate. Results: IC50s for As2O3 and DCA were 0.88 ± 0.02 μM and 15.87 ± 1.22 mM for MOLM-14, 0.54 ± 0.03 μM and 19.08 ± 2.25 mM for MV4-11, respectively. When treated concurrently, the potentiation factors were 1.8 and 1.3 for MOLM-14 and MV4-11, respectively. When the cells were pretreated or "primed" with DCA for 48 h and then exposed to As2O3 for 48 h, the potentiation factors were 2 and 1.8 for MOLM-14 and MV4-11, respectively. Viability tests showed a 33-50% increase in cell kill when DCA and As2O3 were used in combination compared to As2O3 or DCA alone. The number of viable cells remaining after combination treatment was significantly lower than the number of viable cells after either treatment alone when treated sequentially or concurrently (p&amp;lt;0.05). Further mechanistic studies and preclinical models are ongoing to best predict the future clinical dose and schedule. Conclusion: Combination of DCA (IC30, 11 mM) and As2O3 whether used sequentially or concurrently in AML cells caused significantly more cellular damage as compared to either drug alone. To the best of our knowledge, this is the first report of the combination of DCA+As2O3 against AML cells. These data suggest that targeting cellular metabolism in leukemia may provide a therapeutic option for AML patients with unfavorable risk, such as relapsed/refractory, FLT3-ITD or who are not candidates for cytotoxic agents and warrant further clinical investigation. Citation Format: Ashkan Emadi, Mariola Sadowska, Brandon A. Carter-Cooper, Rena G. Lapidus, Edward A. Sausville. Targeting leukemia metabolism: augmentation of anti-leukemic activity of arsenic trioxide (As2O3) against non-APL AML cell lines in presence of dichloroacetate (DCA). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3268. doi:10.1158/1538-7445.AM2013-3268

Phosphorylation of rat skeletal muscle pyruvate dehydrogenase phosphatase in response to insulin stimulation

Pyruvate dehydrogenase phosphatase (PDP) regulates carbohydrate oxidation through the mitochondrial pyruvate dehydrogenase (PDH) complex. PDP dephosphorylates and activates PDH, enabling increased carbohydrate flux towards oxidative energy production. In cell culture, both PDP1 and 2 appear to undergo covalent activation in direct response to insulin–stimulation by PKCδ. Our objective was to examine the effect of insulin on PDP phosphorylation and PDH activation. Intact rat extensor digitorum longus muscles were incubated (oxygenated at 25°C, fixed to 1g of resting tension) tendon–to–tendon for 30min in basal or insulin–stimulated (10mU/mL) media. PDH activity increased 58% following insulin stimulation, (p=0.057, n=11). Serine phosphorylation of PDP1 (p=0.037) and PDP2 (p=0.027) increased by 44% and 54% respectively following stimulation (n=4), and PKCδ protein content was enriched in the mitochondrial fraction by 45% in response to insulin stimulation (p=0.0009, n=8). These data suggest that the insulin–stimulated increase in PDH activity in whole tissue is mediated through mitochondrial migration of PKCδ and subsequent PDP phosphorylation.Research supported by NSERC, Canada.

Synergetic Neuroprotection of Normobaric Oxygenation and Ethanol in Ischemic Stroke Through Improved Oxidative Mechanism

Normobaric oxygenation (NBO) and ethanol both provide neuroprotection in stroke. We evaluated the enhanced neuroprotective effect of combining these 2 treatments in a rat stroke model. Sprague-Dawley rats were subjected to middle cerebral artery occlusion for 2 hours. Reperfusion was then established and followed by treatment with either (1) an intraperitoneal injection of ethanol (1.0 g/kg), (2) NBO treatment (2-hour duration), or (3) NBO plus ethanol. The extent of brain injury was determined by infarct volume and motor performance. Oxidative metabolism was determined by ADP/ATP ratios, reactive oxygen species levels, nicotinamide adenine dinucleotide phosphate oxidase activity, and pyruvate dehydrogenase activity. Protein expression of major nicotinamide adenine dinucleotide phosphate oxidase subunits (p47(phox), gp91(phox), and p67(phox)) and the enzyme pyruvate dehydrogenase was evaluated through Western immunoblotting. NBO and ethanol monotherapies each demonstrated reductions as compared to stroke without treatment in infarct volume (36.7% and 37.9% vs 48.4%) and neurological deficits (score of 6.4 and 6.5 vs 8.4); however, the greatest neuroprotection (18.8% of infarct volume and 4.4 neurological deficit) was found in animals treated with combination therapy. This neuroprotection was associated with the largest reductions in ADP/ATP ratios, reactive oxygen species levels, and nicotinamide adenine dinucleotide phosphate oxidase activity, and the largest increase in pyruvate dehydrogenase activity. Combination therapy with NBO and ethanol enhances the neuroprotective effect produced by each therapy alone. The mechanism behind this synergistic action is related to changes in cellular metabolism after ischemia reperfusion. NBO plus ethanol is attractive for clinical study because of its ease of use, tolerability, and tremendous neuroprotective potential in stroke.

Inhibition of Carnitine Palmitoyltransferase-1 Activity Alleviates Insulin Resistance in Diet-Induced Obese Mice

Impaired skeletal muscle fatty acid oxidation has been suggested to contribute to insulin resistance and glucose intolerance. However, increasing muscle fatty acid oxidation may cause a reciprocal decrease in glucose oxidation, which might impair insulin sensitivity and glucose tolerance. We therefore investigated what effect inhibition of mitochondrial fatty acid uptake has on whole-body glucose tolerance and insulin sensitivity in obese insulin-resistant mice. C57BL/6 mice were fed a high-fat diet (60% calories from fat) for 12 weeks to develop insulin resistance. Subsequent treatment of mice for 4 weeks with the carnitine palmitoyltransferase-1 inhibitor, oxfenicine (150 mg/kg i.p. daily), resulted in improved whole-body glucose tolerance and insulin sensitivity. Exercise capacity was increased in oxfenicine-treated mice, which was accompanied by an increased respiratory exchange ratio. In the gastrocnemius muscle, oxfenicine increased pyruvate dehydrogenase activity, membrane GLUT4 content, and insulin-stimulated Akt phosphorylation. Intramyocellular levels of lipid intermediates, including ceramide, long-chain acyl CoA, and diacylglycerol, were also decreased. Our results demonstrate that inhibition of mitochondrial fatty acid uptake improves insulin sensitivity in diet-induced obese mice. This is associated with increased carbohydrate utilization and improved insulin signaling in the skeletal muscle, suggestive of an operating Randle Cycle in muscle.

Inactivation of the HIF-1α/PDK3 Signaling Axis Drives Melanoma toward Mitochondrial Oxidative Metabolism and Potentiates the Therapeutic Activity of Pro-Oxidants

Cancer cells can undergo a metabolic reprogramming from oxidative phosphorylation to glycolysis that allows them to adapt to nutrient-poor microenvironments, thereby imposing a selection for aggressive variants. However, the mechanisms underlying this reprogramming are not fully understood. Using complementary approaches in validated cell lines and freshly obtained human specimens, we report here that mitochondrial respiration and oxidative phosphorylation are slowed in metastatic melanomas, even under normoxic conditions due to the persistence of a high nuclear expression of hypoxia-inducible factor-1α (HIF-1α). Pharmacologic or genetic blockades of the HIF-1α pathway decreased glycolysis and promoted mitochondrial respiration via specific reduction in the expression of pyruvate dehydrogenase kinase-3 (PDK3). Inhibiting PDK3 activity by dichloroacetate (DCA) or siRNA-mediated attenuation was sufficient to increase pyruvate dehydrogenase activity, oxidative phosphorylation, and mitochondrial reactive oxygen species generation. Notably, DCA potentiated the antitumor effects of elesclomol, a pro-oxidative drug currently in clinical development, both by limiting cell proliferation and promoting cell death. Interestingly, this combination was also effective against BRAF V600E-mutant melanoma cells that were resistant to the BRAF inhibitor vemurafenib. Cotreatment of melanomas with DCA and elesclomol in vivo achieved a more durable response than single agent alone. Our findings offer a preclinical validation of the HIF-1/PDK3 bioenergetic pathway as a new target for therapeutic intervention in metastatic melanoma, opening the door to innovative combinations that might eradicate this disease.

Mitochondrial activation by inhibition of PDKII suppresses HIF1a signaling and angiogenesis in cancer

Most solid tumors are characterized by a metabolic shift from glucose oxidation to glycolysis, in part due to actively suppressed mitochondrial function, a state that favors resistance to apoptosis. Suppressed mitochondrial function may also contribute to the activation of hypoxia-inducible factor 1α (HIF1α) and angiogenesis. We have previously shown that the inhibitor of pyruvate dehydrogenase kinase (PDK) dichloroacetate (DCA) activates glucose oxidation and induces apoptosis in cancer cells in vitro and in vivo. We hypothesized that DCA will also reverse the 'pseudohypoxic' mitochondrial signals that lead to HIF1α activation in cancer, even in the absence of hypoxia and inhibit cancer angiogenesis. We show that inhibition of PDKII inhibits HIF1α in cancer cells using several techniques, including HIF1α luciferase reporter assays. Using pharmacologic and molecular approaches that suppress the prolyl-hydroxylase (PHD)-mediated inhibition of HIF1α, we show that DCA inhibits HIF1α by both a PHD-dependent mechanism (that involves a DCA-induced increase in the production of mitochondria-derived α-ketoglutarate) and a PHD-independent mechanism, involving activation of p53 via mitochondrial-derived H(2)O(2), as well as activation of GSK3β. Effective inhibition of HIF1α is shown by a decrease in the expression of several HIF1α regulated gene products as well as inhibition of angiogenesis in vitro in matrigel assays. More importantly, in rat xenotransplant models of non-small cell lung cancer and breast cancer, we show effective inhibition of angiogenesis and tumor perfusion in vivo, assessed by contrast-enhanced ultrasonography, nuclear imaging techniques and histology. This work suggests that mitochondria-targeting metabolic modulators that increase pyruvate dehydrogenase activity, in addition to the recently described pro-apoptotic and anti-proliferative effects, suppress angiogenesis as well, normalizing the pseudo-hypoxic signals that lead to normoxic HIF1α activation in solid tumors.

Stimulation of glucose oxidation protects against acute myocardial infarction and reperfusion injury

During reperfusion of the ischaemic myocardium, fatty acid oxidation rates quickly recover, while glucose oxidation rates remain depressed. Direct stimulation of glucose oxidation via activation of pyruvate dehydrogenase (PDH), or secondary to an inhibition of malonyl CoA decarboxylase (MCD), improves cardiac functional recovery during reperfusion following ischaemia. However, the effects of such interventions on the evolution of myocardial infarction are unknown. The purpose of this study was to determine whether infarct size is decreased in response to increased glucose oxidation. In vivo, direct stimulation of PDH in mice with the PDH kinase (PDHK) inhibitor, dichloroacetate, significantly decreased infarct size following temporary ligation of the left anterior descending coronary artery. These results were recapitulated in PDHK 4-deficient (PDHK4-/-) mice, which have enhanced myocardial PDH activity. These interventions also protected against ischaemia/reperfusion injury in the working heart, and dichloroacetate failed to protect in PDHK4-/- mice. In addition, there was a dramatic reduction in the infarct size in malonyl CoA decarboxylase-deficient (MCD-/-) mice, in which glucose oxidation rates are enhanced (secondary to an inhibition of fatty acid oxidation) relative to their wild-type littermates (10.8 ± 3.8 vs. 39.5 ± 4.7%). This cardioprotective effect in MCD-/- mice was associated with increased PDH activity in the ischaemic area at risk (1.89 ± 0.18 vs. 1.52 ± 0.05 μmol/g wet weight/min). These findings demonstrate that stimulating glucose oxidation via targeting either PDH or MCD decreases the infarct size, validating the concept that optimizing myocardial metabolism is a novel therapy for ischaemic heart disease.

Growth factor withdrawal in combination with sodium butyrate addition extends culture longevity and enhances antibody production in CHO cells

The effect of growth factor (GF) and sodium butyrate (NaBu) on Chinese hamster ovary (CHO) cell growth, cell viability and antibody production was investigated using shaking flasks in GF-containing and GF-deficient medium containing 0, 1 and 3mM NaBu. The withdrawal of GF and the addition of NaBu suppressed cell growth, but they significantly increased specific antibody productivity, q(Ab). Interestingly, the withdrawal of GF in combination with the addition of NaBu markedly retarded cell death, leading to extended culture longevity. For instance, at 3mM NaBu, cell viability fell below 80% after day 4 in GF-containing medium, but it remained over 80% until day 18 in GF-deficient medium. Due to the enhanced q(Ab) and the extended culture longevity, approximately 2-fold increase in total antibody production was achieved in pseudo-perfusion culture with 1mM NaBu in GF-deficient medium, compared to the culture in GF-containing medium. The effect of GF and NaBu on the change in the expression and activity of cellular proteins, c-Myc, Bcl-2 and pyruvate dehydrogenase (PDH), was also investigated. Both the withdrawal of GF and the addition of NaBu decreased the expression of c-Myc. The expression of Bcl-2 was enhanced by the addition of NaBu in a dose-dependent manner while it was not affected by the withdrawal of GF. In addition, both the withdrawal of GF and the addition of NaBu reduced metabolic rates, q(Glc), q(Lac) and Y(Lac/Glc), and increased PDH activity while not affecting PDH expression, suggesting that they may reduce the glycolytic rates, but enhance the conversion rates of pyruvate to TCA intermediates. Taken together, the withdrawal of GF in combination with the addition of NaBu can be considered as a relevant strategy for alleviating NaBu-induced cell apoptosis and enhancing antibody production since it can be easily implemented as well as enhance q(Ab) and extend culture longevity.

Intracerebroventricular Leptin Administration Differentially Alters Cardiac Energy Metabolism in Mice Fed a Low-fat and High-fat Diet

Leptin directly acts on peripheral tissues and alters energy metabolism in obese mice. It also has acute beneficial effects on these tissues via its hypothalamic action. However, it is not clear what effect chronic intracerebroventrical (ICV) leptin administration has on cardiac energy metabolism. We examined the effects of chronic ICV leptin on glucose and fatty acid metabolism in isolated working hearts from high-fat-fed and low-fat-fed mice. Mice were fed a high-fat (60% calories from fat) or low-fat (10% calories from fat) diet for 8 weeks before ICV leptin (5 [mu]g/d) for 7 days. In low-fat-fed mice, leptin increased glucose oxidation rates in isolated working hearts when compared with control [203 +/- 21 vs. 793 +/- 93 nmol[middle dot](g dry weight)-1[middle dot]min-1]. In high-fat-fed mice leptin inhibited fatty acid oxidation [476 +/- 73 vs. 251 +/- 38 nmol[middle dot](g[middle dot]dry[middle dot]wt)-1[middle dot]min-1]. The increase in glucose oxidation in low-fat-fed mice was accompanied by increased pyruvate dehydrogenase activity. In high-fat-fed mice, leptin increased cardiac malonyl coenzyme A levels, secondary to a decrease in malonyl coenzyme A decarboxylase expression. These results suggest that ICV leptin alters cardiac energy metabolism opposite to its peripheral effects and that these effects differ depending on energy substrate supply to the mice.