Increased Pyruvate Dehydrogenase Activity Research Articles

Effects of acute alcohol intoxication on the respiration and dehydrogenase activity of rat pancreatic acini

The exocrine function of pancreatic acini cells is a highly energy-intensive process. A cell always needs to maintain a stable level of ATP balancing between states of activation and rest. Low-energy is one of the possible mechanisms contributing to the development of pancreatic diseases. The most commonly encountered disease of the pancreas is acute pancreatitis. It is known that excessive alcohol consumption causes the development of pancreatitis. The pathogenesis of this disease is linked to the cellular loss of energy, but the mechanism of alcohol’s effect on the mitochondria in pancreatic acini is unclear. This study’s main aim is to assess the impact of acute alcohol administration on the mitochondrial function of rat pancreatic acini. Wistar rats were administered ethanol (6 g/kg body weight) by oral gavage for 3 h before the experiment. A suspension of isolated pancreatic acini was obtained following collagenase digestion. Respiration of isolated pancreatic acini was studied with a Clark electrode. The maximal respiration rate was studied at different concentrations of protonophore FCCP (0.5–2 μM) in solutions containing glucose combined with oxidative substrates (pyruvate and glutamine, monomethyl-succinate or dimethyl-α-ketoglutarate). Dehydrogenase activity was measured by colorimetric method. Ethanol administration caused a significant increase in the activity of pyruvate dehydrogenase. It was confirmed that FCCP induced an increase in the respiration rate of pancreatic acinar cells in each experimental group. The addition of 1.5 μM FCCP reduced the respiration rate of pancreatic acini during the oxidation of glucose and monomethyl succinate or dimethyl-α-ketoglutarate, but not during the oxidation of glucose, pyruvate and glutamine substrates. The administration of ethanol had no impact on the basal or FCCP-uncoupled respiration of isolated pancreatic acini. The observed data are consistent with the findings of other researchers. However, alcohol exposure is not sufficient to cause mitochondrial damage in pancreatic acinar cells. In conclusion, acute ethanol administration does not cause mitochondrial dysfunction in the pancreas of rats but causes an increase in pyruvate dehydrogenase activity.

MCUb is an inducible regulator of calcium-dependent mitochondrial metabolism and substrate utilization in muscle

Mitochondria use the electron transport chain to generate high-energy phosphate from oxidative phosphorylation, a process also regulated by the mitochondrial Ca2+ uniporter (MCU) and Ca2+ levels. Here, we show that MCUb, an inhibitor of MCU-mediated Ca2+ influx, is induced by caloric restriction, where it increases mitochondrial fatty acid utilization. To mimic the fasted state with reduced mitochondrial Ca2+ influx, we generated genetically altered mice with skeletal muscle-specific MCUb expression that showed greater fatty acid usage, less fat accumulation, and lower body weight. In contrast, mice lacking Mcub in skeletal muscle showed increased pyruvate dehydrogenase activity, increased muscle malonyl coenzyme A (CoA), reduced fatty acid utilization, glucose intolerance, and increased adiposity. Mechanistically, pyruvate dehydrogenase kinase 4 (PDK4) overexpression in muscle of Mcub-deleted mice abolished altered substrate preference. Thus, MCUb is an inducible control point in regulating skeletal muscle mitochondrial Ca2+ levels and substrate utilization that impacts total metabolic balance.

Real-time influence of intracellular acidification and Na+ /H+ exchanger inhibition on in-cell pyruvate metabolism in the perfused mouse heart: A 31 P-NMR and hyperpolarized 13 C-NMR study.

Disruption of acid-base balance is linked to various diseases and conditions. In the heart, intracellular acidification is associated with heart failure, maladaptive cardiac hypertrophy, and myocardial ischemia. Previously, we have reported that the ratio of the in-cell lactate dehydrogenase (LDH) to pyruvate dehydrogenase (PDH) activities is correlated with cardiac pH. To further characterize the basis for this correlation, these in-cell activities were investigated under induced intracellular acidification without and with Na+ /H+ exchanger (NHE1) inhibition by zoniporide. Male mouse hearts (n= 30) were isolated and perfused retrogradely. Intracellular acidification was performed in two ways: (1) with the NH4 Cl prepulse methodology; and (2) by combining the NH4 Cl prepulse with zoniporide. 31 P NMR spectroscopy was used to determine the intracellular cardiac pH and to quantify the adenosine triphosphate and phosphocreatine content. Hyperpolarized [1-13 C]pyruvate was obtained using dissolution dynamic nuclear polarization. 13 C NMR spectroscopy was used to monitor hyperpolarized [1-13 C]pyruvate metabolism and determine enzyme activities in real time at a temporal resolution of a few seconds using the product-selective saturating excitation approach. The intracellular acidification induced by the NH4 Cl prepulse led to reduced LDH and PDH activities (-16% and -39%, respectively). This finding is in line with previous evidence of reduced myocardial contraction and therefore reduced metabolic activity upon intracellular acidification. Concomitantly, the LDH/PDH activity ratio increased with the reduction in pH, as previously reported. Combining the NH4 Cl prepulse with zoniporide led to a greater reduction in LDH activity (-29%) and to increased PDH activity (+40%). These changes resulted in a surprising decrease in the LDH/PDH ratio, as opposed to previous predictions. Zoniporide alone (without intracellular acidification) did not change these enzyme activities. A possible explanation for the enzymatic changes observed during the combination of the NH4 Cl prepulse and NHE1 inhibition may be related to mitochondrial NHE1 inhibition, which likely negates the mitochondrial matrix acidification. This effect, combined with the increased acidity in the cytosol, would result in an enhanced H+ gradient across the mitochondrial membrane and a temporarily higher pyruvate transport into the mitochondria, thereby increasing the PDH activity at the expense of the cytosolic LDH activity. These findings demonstrate the complexity of in-cell cardiac metabolism and its dependence on intracellular acidification. This study demonstrates the capabilities and limitations of hyperpolarized [1-13 C]pyruvate in the characterization of intracellular acidification as regards cardiac pathologies.

Imaging immunomodulatory treatment responses in a multiple sclerosis mouse model using hyperpolarized 13C metabolic MRI

BackgroundIn recent years, the ability of conventional magnetic resonance imaging (MRI), including T1 contrast-enhanced (CE) MRI, to monitor high-efficacy therapies and predict long-term disability in multiple sclerosis (MS) has been challenged. Therefore, non-invasive methods to improve MS lesions detection and monitor therapy response are needed.MethodsWe studied the combined cuprizone and experimental autoimmune encephalomyelitis (CPZ-EAE) mouse model of MS, which presents inflammatory-mediated demyelinated lesions in the central nervous system as commonly seen in MS patients. Using hyperpolarized 13C MR spectroscopy (MRS) metabolic imaging, we measured cerebral metabolic fluxes in control, CPZ-EAE and CPZ-EAE mice treated with two clinically-relevant therapies, namely fingolimod and dimethyl fumarate. We also acquired conventional T1 CE MRI to detect active lesions, and performed ex vivo measurements of enzyme activities and immunofluorescence analyses of brain tissue. Last, we evaluated associations between imaging and ex vivo parameters.ResultsWe show that hyperpolarized [1-13C]pyruvate conversion to lactate is increased in the brain of untreated CPZ-EAE mice when compared to the control, reflecting immune cell activation. We further demonstrate that this metabolic conversion is significantly decreased in response to the two treatments. This reduction can be explained by increased pyruvate dehydrogenase activity and a decrease in immune cells. Importantly, we show that hyperpolarized 13C MRS detects dimethyl fumarate therapy, whereas conventional T1 CE MRI cannot.ConclusionsIn conclusion, hyperpolarized MRS metabolic imaging of [1-13C]pyruvate detects immunological responses to disease-modifying therapies in MS. This technique is complementary to conventional MRI and provides unique information on neuroinflammation and its modulation.

Mitochondrial dysfunction and autophagy activation are associated with cardiomyopathy developed by extended methamphetamine self-administration in rats

The recent rise in illicit use of methamphetamine (METH), a highly addictive psychostimulant, is a huge health care burden due to its central and peripheral toxic effects. Mounting clinical studies have noted that METH use in humans is associated with the development of cardiomyopathy; however, preclinical studies and animal models to dissect detailed molecular mechanisms of METH-associated cardiomyopathy development are scarce. The present study utilized a unique very long-access binge and crash procedure of METH self-administration to characterize the sequelae of pathological alterations that occur with METH-associated cardiomyopathy. Rats were allowed to intravenously self-administer METH for 96 h continuous weekly sessions over 8 weeks. Cardiac function, histochemistry, ultrastructure, and biochemical experiments were performed 24 h after the cessation of drug administration. Voluntary METH self-administration induced pathological cardiac remodeling as indicated by cardiomyocyte hypertrophy, myocyte disarray, interstitial and perivascular fibrosis accompanied by compromised cardiac systolic function. Ultrastructural examination and native gel electrophoresis revealed altered mitochondrial morphology and reduced mitochondrial oxidative phosphorylation (OXPHOS) supercomplexes (SCs) stability and assembly in METH exposed hearts. Redox-sensitive assays revealed significantly attenuated mitochondrial respiratory complex activities with a compensatory increase in pyruvate dehydrogenase (PDH) activity reminiscent of metabolic remodeling. Increased autophagy flux and increased mitochondrial antioxidant protein level was observed in METH exposed heart. Treatment with mitoTEMPO reduced the autophagy level indicating the involvement of mitochondrial dysfunction in the adaptive activation of autophagy in METH exposed hearts. Altogether, we have reported a novel METH-associated cardiomyopathy model using voluntary drug seeking behavior. Our studies indicated that METH self-administration profoundly affects mitochondrial ultrastructure, OXPHOS SCs assembly and redox activity accompanied by increased PDH activity that may underlie observed cardiac dysfunction.

Pyruvate Dehydrogenase A1 Phosphorylated by Insulin Associates with Pyruvate Kinase M2 and Induces LINC00273 through Histone Acetylation.

Insulin potently promotes cell proliferation and anabolic metabolism along with a reduction in blood glucose levels. Pyruvate dehydrogenase (PDH) plays a pivotal role in glucose metabolism. Insulin increase PDH activity by attenuating phosphorylated Ser293 PDH E1α (p-PDHA1) in normal liver tissue. In contrast to normal hepatocytes, insulin enhanced p-PDHA1 level and induced proliferation of hepatocellular carcinoma HepG2 cells. Here, we attempted to find a novel function of p-PDHA1 in tumorigenesis upon insulin stimulation. We found that p-Ser293 E1α, but not the E2 or E3 subunit of pyruvate dehydrogenase complex (PDC), co-immunoprecipitated with pyruvate kinase M2 (PKM2) upon insulin. Of note, the p-PDHA1 along with PKM2 translocated to the nucleus. The p-PDHA1/PKM2 complex was associated with the promoter of long intergenic non-protein coding (LINC) 00273 gene (LINC00273) and recruited p300 histone acetyl transferase (HAT) and ATP citrate lyase (ACL), leading to histone acetylation. Consequently, the level of transcription factor ZEB1, an epithelial–mesenchymal transition (EMT) marker, was promoted through increased levels of LINC00273, resulting in cell migration upon insulin. p-PDHA1, along with PKM2, may be crucial for transcriptional regulation of specific genes through epigenetic regulation upon insulin in hepatocarcinoma cells.

Abstract 10577: Dapagliflozin Improves Cardiac Function in Mice After Myocardial Infarction

Objective: We examined the effects of SGLT2 inhibition with Dapagliflozin (DAPA) on left ventricular ejection fraction (LVEF) and cardiomyocyte regeneration in mice after myocardial infraction (MI), a model for Heart Failure with reduced EF. Introduction: Neonatal mice can regenerate cardiomyocytes, but this ability is lost shortly after birth due in part to a switch from anaerobic glycolysis to fatty acid utilization. This switch is due to pyruvate dehydrogenase kinase 4 (PDK-4) inhibition of pyruvate dehydrogenase (PDH) dependent glycolysis. Empagliflozin use in mice after MI increases PDH activity and promotes glucose utilization. Hypothesis: DAPA use in mice after MI can improve LVEF by promoting cardiomyocyte regeneration secondary to increase PDH activity. Methods: A total of 49 mice, 8 weeks old, were randomized to LAD ligation (MI) or sham surgery (Sham). 72 hours after surgery, MI (n=19) and Sham (n=21) mice were randomized to PO DAPA (0.02 mg/ml) (MI-DAPA, Sham-DAPA) or vehicle (water) (MI-VEH, Sham-VEH). 20 mice were euthanized after each successive week (wk.) of treatment. Immunofluorescence, using Anti-phospho-Histone H3 (mitosis marker) (PH3)-Anti Cardiac Troponin T co-staining, was done at 2 wks. to assess regeneration. An echocardiogram was done at baseline, 72 hours after surgery, and weekly after treatment to assess LVEF. Results: At 1 wk. after MI, no significant change was observed between groups. At 2 wks., DAPA increased LVEF in MI mice (Sham-VEH 66.75%, SEM 1.18; Sham-DAPA 78.63%, SEM 2.82; MI-VEH 56.86%, SEM 4.80; MI-DAPA 74.3%, SEM 0.58%, p<0.05). At 2 wks., MI increased % of PH3 positive cardiomyocyte nuclei (Sham-VEH 0%, SEM 0; MI-VEH 0.4268%, SEM 0.1259; p<0.05). In DAPA treated mice no significant difference was observed as the % of PH3 positive cardiomyocyte increased in Sham-DAPA mice (Sham-DAPA 0.2253%, SEM 0.078; MI-DAPA 0.46%, SEM 0.07; p 0.47). Despite an increase in SHAM-VEH mice, no statistically significant difference was observed between SHAM-VEH and SHAM-DAPA. Conclusions: DAPA mitigate cardiac remodeling in mice after MI with increase LVEF after 2 wks. MI increases mitosis in cardiomyocytes after two wks. Despite a trend for increased mitosis with DAPA treatment for 2 wks., no significant change was seen.

Mutation of the Cardiac Sodium Channel Nav1.5 Leads to Sex Specific Arrhythmias

Introduction Cardiac Nav1.5 sodium channels, encoded by the SCN5A gene, are responsible for initiating the electrical action potential in the heart, resulting in coordinated muscular contraction. Channelopathies caused by mutations in Nav1.5 occur in 1/2500 individuals and lead to arrhythmic diseases, such as long QT syndrome, and cardiomyopathies1,2. Males with Nav1.5 mutations are at a higher risk for sudden cardiac arrest, indicative of an innate protection in females through an unknown mechanism. The gain of function Arg222Gln mutation affects the voltage sensing segment of Nav1.5, creating an aberrant pore leading to arrhythmia-inducing membrane ion leaks3. We sought to determine the sex-specific effects of this clinically relevant mutation, attempting to undercover the protective mechanisms present in females. Methods The Arg222Gln mutation was knocked into exon 6 of the mouse SCN5A gene to generate viable animals that phenocopy the human disease. Three-lead electrocardiograms (ECG) from adult male and female animals were recorded under anesthesia, including wild-type (WT) controls. Isoproterenol (β-adrenoreceptor agonist) was injected to examine electrophysiological changes under acute stress. Baseline echocardiograms were collected at four months and after 30 days of transverse aortic constriction (TAC). Differentially expressed genes from whole heart lysate were measured by chip-based analysis after TAC . Protein levels were quantified using Western blot and ELISA. Results ECG measurements revealed a prolonged QTc (0.064ms±0.006) and QRS interval (0.011ms±0.0003) in mutant males compared to WT males (0.036ms±0.008 and 0.0085ms±0.0004 respectively) (P<0.05). Mutant males had an observable sinus arrhythmia at baseline (RMSSD of beat to beat intervals of 4.60 vs 0.79 in WT males, P<0.05), whereas mutant and wild-type females had no evidence of adverse changes to the beating rhythm. The ejection fraction (EF) of mutant males decreased by 40%±9.9% 4 weeks post-TAC, whereas no significant changes in EF were observed in all other groups. We found that transcript and protein levels of pyruvate dehydrogenase kinase-4, which reduces pyruvate dehydrogenase (PDH) activity through phosphorylation, were 6-fold higher in mutant males compared to WT males (P<0.05), whereas no differences were observed between females. Despite this, phospho-PDH levels were lower and PDH activity higher in mutant males compared to WT males (P<0.01), with no differences between the female groups. Conclusion We found functional, electrophysiological, and metabolic enzyme abnormalities in male mice harboring the Arg222Gln Nav1.5 mutation. These differences did not exist in the female mutant mice. Our data illustrate a strong sex-specific phenotype, and suggest that metabolic alterations through increased PDH activity provide a protective mechanism against the arrhythmia and heart failure produced by Nav1.5-related cation leak. Further experiments will seek to determine if sex hormones alone can induce a protective effect in the context of the Arg222Gln mutation. 1Priori et al., NEJM, 2003 2Veerman et al., Gene, 2015 3Moreau et al., J Gen Physio, 2015

Very long chain fatty acid metabolism is required in acute myeloid leukemia

Acute myeloid leukemia (AML) cells have an atypical metabolic phenotype characterized by increased mitochondrial mass, as well as a greater reliance on oxidative phosphorylation and fatty acid oxidation (FAO) for survival. To exploit this altered metabolism, we assessed publicly available databases to identify FAO enzyme overexpression. Very long chain acyl-CoA dehydrogenase (VLCAD; ACADVL) was found to be overexpressed and critical to leukemia cell mitochondrial metabolism. Genetic attenuation or pharmacological inhibition of VLCAD hindered mitochondrial respiration and FAO contribution to the tricarboxylic acid cycle, resulting in decreased viability, proliferation, clonogenic growth, and AML cell engraftment. Suppression of FAO at VLCAD triggered an increase in pyruvate dehydrogenase activity that was insufficient to increase glycolysis but resulted in adenosine triphosphate depletion and AML cell death, with no effect on normal hematopoietic cells. Together, these results demonstrate the importance of VLCAD in AML cell biology and highlight a novel metabolic vulnerability for this devastating disease.

Insulin resistance induced by growth hormone is linked to lipolysis and associated with suppressed pyruvate dehydrogenase activity in skeletal muscle: a 2 × 2 factorial, randomised, crossover study in human individuals.

Growth hormone (GH) causes insulin resistance that is linked to lipolysis, but the underlying mechanisms are unclear. We investigated if GH-induced insulin resistance in skeletal muscle involves accumulation of diacylglycerol (DAG) and ceramide as well as impaired insulin signalling, or substrate competition between fatty acids and glucose. Nine GH-deficient male participants were randomised and examined in a 2 × 2 factorial design with and without administration of GH and acipimox (an anti-lipolytic compound). As-treated analyses were performed, wherefore data from three visits from two patients were excluded due to incorrect GH administration. The primary outcome was insulin sensitivity, expressed as the AUC of the glucose infusion rate (GIRAUC), and furthermore, the levels of DAGs and ceramides, insulin signalling and the activity of the active form of pyruvate dehydrogenase (PDHa) were assessed in skeletal muscle biopsies obtained in the basal state and during a hyperinsulinaemic-euglycaemic clamp (HEC). Co-administration of acipimox completely suppressed the GH-induced elevation in serum levels of NEFA (GH versus GH+acipimox, p < 0.0001) and abrogated GH-induced insulin resistance (mean GIRAUC [95% CI] [mgmin-1kg-1] during the HEC: control, 595 [493, 718]; GH, 468 [382, 573]; GH+acipimox, 654 [539, 794]; acipimox, 754 [618, 921]; GH vs GH+acipimox: p = 0.004). GH did not significantly change either the accumulation of DAGs and ceramides or insulin signalling in skeletal muscle, but GH antagonised the insulin-stimulated increase in PDHa activity (mean ± SEM [% from the basal state to the HEC]: control, 47 ± 19; GH, -15 ± 21; GH+acipimox, 3 ± 21; acipimox, 57 ± 22; main effect: p = 0.02). GH-induced insulin resistance in skeletal muscle is: (1) causally linked to lipolysis; (2) not associated with either accumulation of DAGs and ceramides or impaired insulin signalling; (3) likely to involve substrate competition between glucose and lipid intermediates. ClinicalTrials.gov NCT02782208 FUNDING: The work was supported by the Grant for Growth Innovation (GGI), which was funded by Merck KGaA, Darmstadt, Germany. Graphical abstract.

Functional Consequences of PDK4 Deficiency in Doberman Pinscher Fibroblasts

A splice site mutation in the canine pyruvate dehydrogenase kinase 4 (PDK4) gene has been shown to be associated with the development of dilated cardiomyopathy (DCM) in Doberman Pinchers (DPs). Subsequent studies have successfully demonstrated the use of dermal fibroblasts isolated from DPs as models for PDK4 deficiency and have shown activation of the intrinsic (mitochondrial mediated) apoptosis pathway in these cells under starvation conditions. For this study, we sought to further explore the functional consequences of PDK4 deficiency in DP fibroblasts representing PDK4wt/wt, PDK4wt/del, and PDK4del/del genotypes. Our results show that starvation conditions cause increased perinuclear localization of mitochondria and decreased cell proliferation, altered expression levels of pyruvate dehydrogenase phosphatase (PDP) and pyruvate dehydrogenase (PDH), dramatically increased PDH activity, and an impaired response to mitochondrial stress in affected cells. In sum, these results show the broad impact of PDK4 deficiency and reveal mechanistic pathways used by these cells in an attempt to compensate for the condition. Our data help to elucidate the mechanisms at play in this extremely prevalent DP disorder and provide further support demonstrating the general importance of metabolic flexibility in cell health.

Mitochondrial substrate utilization regulates cardiomyocyte cell-cycle progression

The neonatal mammalian heart is capable of regeneration for a brief window of time after birth. However, this regenerative capacity is lost within the first week of life, which coincides with a postnatal shift from anaerobic glycolysis to mitochondrial oxidative phosphorylation, particularly towards fatty-acid utilization. Despite the energy advantage of fatty-acid beta-oxidation, cardiac mitochondria produce elevated rates of reactive oxygen species when utilizing fatty acids, which is thought to play a role in cardiomyocyte cell-cycle arrest through induction of DNA damage and activation of DNA-damage response (DDR) pathway. Here we show that inhibiting fatty-acid utilization promotes cardiomyocyte proliferation in the postnatatal heart. First, neonatal mice fed fatty-acid deficient milk showed prolongation of the postnatal cardiomyocyte proliferative window, however cell cycle arrest eventually ensued. Next, we generated a tamoxifen-inducible cardiomyocyte-specific, pyruvate dehydrogenase kinase 4 (PDK4) knockout mouse model to selectively enhance oxidation of glycolytically derived pyruvate in cardiomyocytes. Conditional PDK4 deletion resulted in an increase in pyruvate dehydrogenase activity and consequently an increase in glucose relative to fatty-acid oxidation. Loss of PDK4 also resulted in decreased cardiomyocyte size, decreased DNA damage and expression of DDR markers and an increase in cardiomyocyte proliferation. Following myocardial infarction, inducible deletion of PDK4 improved left ventricular function and decreased remodelling. Collectively, inhibition of fatty-acid utilization in cardiomyocytes promotes proliferation, and may be a viable target for cardiac regenerative therapies.

Increased pyruvate dehydrogenase activity in skeletal muscle of growth-restricted ovine fetuses.

Fetal sheep with placental insufficiency-induced intrauterine growth restriction (IUGR) have lower fractional rates of glucose oxidation and greater gluconeogenesis, indicating lactate shuttling between skeletal muscle and liver. Suppression of pyruvate dehydrogenase (PDH) activity was proposed because of greater pyruvate dehydrogenase kinase (PDK) 4 and PDK1 mRNA concentrations in IUGR muscle. Although PDK1 and PDK4 inhibit PDH activity to reduce pyruvate metabolism, PDH protein concentrations and activity have not been examined in skeletal muscle from IUGR fetuses. Therefore, we evaluated the protein concentrations and activity of PDH and the kinases and phosphatases that regulate PDH phosphorylation status in the semitendinosus muscle from placenta insufficiency-induced IUGR sheep fetuses and control fetuses. Immunoblots were performed for PDH, phosphorylated PDH (E1α), PDK1, PDK4, and pyruvate dehydrogenase phosphatase 1 and 2 (PDP1 and PDP2, respectively). Additionally, the PDH, lactate dehydrogenase (LDH), and citrate synthase (CS) enzymatic activities were measured. Phosphorylated PDH concentrations were 28% lower (P < 0.01) and PDH activity was 67% greater (P < 0.01) in IUGR fetal muscle compared with control. PDK1, PDK4, PDP1, PDP2, and PDH concentrations were not different between groups. CS and LDH activities were also unaffected. Contrary to the previous speculation, PDH activity was greater in skeletal muscle from IUGR fetuses, which parallels lower phosphorylated PDH. Therefore, greater expression of PDK1 and PDK4 mRNA did not translate to greater PDK1 or PDK4 protein concentrations or inhibition of PDH as proposed. Instead, these findings show greater PDH activity in IUGR fetal muscle, which indicates that alternative regulatory mechanisms are responsible for lower pyruvate catabolism.

Glucose Metabolism Drives Histone Acetylation Landscape Transitions that Dictate Muscle Stem Cell Function

SUMMARYThe impact of glucose metabolism on muscle regeneration remains unresolved. We identify glucose metabolism as a crucial driver of histone acetylation and myogenic cell fate. We use single-cell mass cytometry (CyTOF) and flow cytometry to characterize the histone acetylation and metabolic states of quiescent, activated, and differentiating muscle stem cells (MuSCs). We find glucose is dispensable for mitochondrial respiration in proliferating MuSCs, so that glucose becomes available for maintaining high histone acetylation via acetyl-CoA. Conversely, quiescent and differentiating MuSCs increase glucose utilization for respiration and have consequently reduced acetylation. Pyruvate dehydrogenase (PDH) activity serves as a rheostat for histone acetylation and must be controlled for muscle regeneration. Increased PDH activity in proliferation increases histone acetylation and chromatin accessibility at genes that must be silenced for differentiation to proceed, and thus promotes self-renewal. These results highlight metabolism as a determinant of MuSC histone acetylation, fate, and function during muscle regeneration.

The antianginal ranolazine mitigates obesity-induced nonalcoholic fatty liver disease and increases hepatic pyruvate dehydrogenase activity.

Obese individuals are often at risk for nonalcoholic fatty liver disease (NAFLD), insulin resistance, type 2 diabetes (T2D), and cardiovascular diseases such as angina, thereby requiring combination therapies for their comorbidities. Ranolazine is a second-line antianginal agent that also improves glycemia, and our aim was to determine whether ranolazine modifies the progression of obesity-induced NAFLD. Twelve-week-old C57BL/6J male mice were fed a low-fat or high-fat diet for 10 weeks and then treated for 30 days with either vehicle control or ranolazine (50 mg/kg via daily s.c. injection). Glycemia was monitored via glucose/pyruvate/insulin tolerance testing, whereas in vivo metabolism was assessed via indirect calorimetry. Hepatic triacylglycerol content was quantified via the Bligh and Dyer method. Consistent with previous reports, ranolazine treatment reversed obesity-induced glucose intolerance, which was associated with reduced body weight and hepatic steatosis, as well as increased hepatic pyruvate dehydrogenase (PDH) activity. Ranolazine's actions on hepatic PDH activity may be directly mediated, as ranolazine treatment reduced PDH phosphorylation (indicative of increased PDH activity) in HepG2 cells. Therefore, in addition to mitigating angina, ranolazine also reverses NAFLD, which may contribute to its documented glucose-lowering actions, situating ranolazine as an ideal antianginal therapy for obese patients comorbid for NAFLD and T2D.

Abstract 5232: 1,25-Dihydroxyvitamin D3 mitigates lung cancer cell mediated mitochondrial dysfunction in human skeletal muscle

Abstract To assess cellular mechanisms that could contribute to muscle weakness in the context of cancer cachexia, we evaluated the effect of conditioned medium (CM) from highly tumorigenic Lewis lung carcinoma (LLC1) cells, CM from less tumorigenic MLE12 cells and non-CM on human skeletal muscle mitochondrial morphology and function. We assessed mitochondrial oxygen consumption, morphology, enzyme activity and gene expression in human skeletal muscle cells in the presence of tumor conditioned medium or control medium. We examined the secretome of LLC1 cells for mediators of skeletal muscle changes. We demonstrate that LLC1 CM inhibits cellular O2 consumption rate (OCR) and increases the proton production rate in human skeletal muscle cells. Mitochondrial fragmentation and the expression of the fission mediator, FIS1, are increased, whereas the expression of fusion mediators, OPA1 and MFN2 are reduced by LLC1 CM. LLC1 CM reduces pyruvate dehydrogenase (PDH) activity thereby limiting the production of acetyl CoA. Reduced PDH activity is associated with reduced PDHA1 sub-unit expression. The amount inactive phospho-PDH is increased in association with reduced PDP2 expression and increased PDK4 and SIRT4 expression in human skeletal muscle cells treated with LLC1 CM. The expression of several signaling pathways is altered by LLC1 CM in human skeletal muscle cells. When compared to MLE12 cells, LLC1 cells show increased expression of mRNAs encoding several potential mediators of altered muscle function such as Gdf15, TGFβ-induced protein, IL11, Il27, Il33, Il34, and PTH-like protein. We show that the active vitamin D metabolite, 1α,25(OH)2D3, that we previously demonstrated increases myoblast mitochondrial oxygen consumption and PDH activity, mitigates LLC1 CM effects on hSkMC OCR, mitochondrial fragmentation and mediators thereof. Mitochondrial oxygen consumption, morphology and enzyme activity in human skeletal muscle cells are inhibited or altered by tumor cell conditioned medium, and are reversed by 1α,25(OH)2D3, the active metabolite of vitamin D. Citation Format: Zachary C. Ryan, Theodore A. Craig, Xuewei Wang, Philippe Delmotte, Jeffrey L. Salisbury, Ian R. Lanza, Gary C. Sieck, Rajiv Kumar. 1,25-Dihydroxyvitamin D3 mitigates lung cancer cell mediated mitochondrial dysfunction in human skeletal muscle [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5232.

Abstract 4972: PDK inhibition sensitizes bladder tumors to cisplatin

Abstract Pyruvate Dehydrogenase Kinase-4 (PDK4) is a member of the PDK enzyme family. PDKs phosphorylate and inactivate the pyruvate dehydrogenase (PDH) complex, shunting pyruvate into the cytoplasm for oxidative metabolism, a phenomenon known as aerobic glycolysis or the Warburg Effect. This confers both growth advantages and resistance to chemotherapy. We previously noted that high grade human bladder tumors have substantial upregulation of PDK4, in the absence of other PDKs. We hypothesized PDK4 would be upregulated in bladder tumors, and inhibition of PDK4 would reduce bladder tumor growth and sensitize cells to cisplatin. We explored tested this hypothesis in cell culture and xenograft and carcinogen induced mouse models. Substantial overexpression of PDK4 (20-120 fold) was confirmed by qPCR in multiple bladder cancer cell lines as compared to UROtsa cells, a benign urothelial cell line. Treatment with dichloroacetate (DCA), a pan-specific PDK inhibitor, significantly increased PDH activity and reduced cellular proliferation in HTB-9 and HTB-5 cancer cells. In line with previous studies in other tumors, DCA also sensitized HTB-9 and HTB-5 cells to cisplatin induced cell death. Notably, this was not through enhanced apoptosis, as caspase activity did not increase, but rather co-treatment resulted in significant increases in LDH release, a marker of necrosis, and increased PI/Annexin V positive cells in DCA plus cisplatin treated cells compared to DCA or cisplatin alone. Inhibition of apoptosis with z-VAD-fmk confirmed the increased necrosis, as DCA plus cisplatin treated cells still had significant increases in cell death compared to DCA or cisplatin alone. Treatment of HTB-9 tumor xenografts with DCA or cisplatin did not reduce tumor volumes; however, co-treatment with both compounds resulted in significant reductions in viable tumor weights, along with increased TUNEL staining. As DCA is a pan-specific PDK inhibitor (PDK1-4), we also used the recently generated PS10 compound with equal specificity for PDK2 and PDK4. PS10 also significantly increased PDH activity, sensitized UM-UC3 cells to cisplatin and reduced cellular proliferation at concentrations at, and above 20µM. To further validate PDK inhibition as a therapeutic target, C57Bl/6J WT mice were treated with 0.05% BBN in the drinking water for 14 weeks to initiate tumorigenesis, and then treated with 70mg/kg PS10 daily +/- weekly[B1] ip cisplatin for 3 weeks. PS10 alone had no effect. While cisplatin reduced bladder weights, only PS10 plus cisplatin resulted in significant reduction in bladder tumor weight. Inhibition of PDKs sensitizes bladder cancers to cisplatin both in vitro and in vivo, as well as having direct anti-proliferative effects in vitro. PDK inhibition may be a useful and novel way to enhance cisplatin based therapies in bladder cancer. Citation Format: Sambantham Shanmugam, Erika Abbott, Justin Penticuff, Dharamainder Choudhary, Benjamin L. Woolbright, John A. Taylor. PDK inhibition sensitizes bladder tumors to cisplatin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4972.

Pyruvate Dehydrogenase PDH-E1β Controls Tumor Progression by Altering the Metabolic Status of Cancer Cells

Downregulation of pyruvate dehydrogenase (PDH) is critical for the aberrant preferential activation of glycolysis in cancer cells under normoxic conditions. Phosphorylation-dependent inhibition of PDH is a relevant event in this process, but it is not durable as it relies on PDH kinases that are activated ordinarily under hypoxic conditions. Thus, it remains unclear how PDH is durably downregulated in cancer cells that are not hypoxic. Building on evidence that PDH activity depends on the stability of a multi-protein PDH complex, we found that the PDH-E1β subunit of the PDH complex is downregulated to inhibit PDH activity under conditions of prolonged hypoxia. After restoration of normoxic conditions, reduced expression of PDH-E1β was sustained such that glycolysis remained highly activated. Notably, PDH-E1β silencing in cancer cells produced a metabolic state strongly resembling the Warburg effect, but inhibited tumor growth. Conversely, enforced exogenous expression of PDH-E1β durably increased PDH activity and promoted the malignant growth of breast cancer cells in vivo Taken together, our results establish the specific mechanism through which PDH acts as an oncogenic factor by tuning glycolytic metabolism in cancer cells.Significance: This seminal study offers a mechanistic explanation for why glycolysis is aberrantly activated in normoxic cancer cells, offering insights into this long-standing hallmark of cancer termed the Warburg effect. Cancer Res; 78(7); 1592-603. ©2018 AACR.

Lipoic acid metabolism in Trypanosoma cruzi as putative target for chemotherapy

Lipoic acid (LA) is a cofactor of relevant enzymatic complexes including the glycine cleave system and 2-ketoacid dehydrogenases. Intervention on LA de novo synthesis or salvage could have pleiotropic deleterious effect in cells, making both pathways attractive for chemotherapy. We show that Trypanosoma cruzi was susceptible to treatment with LA analogues. 8-Bromo-octanic acid (BrO) inhibited the growth of epimastigote forms of both Dm28c and CL Brener strains, although only at high (chemotherapeutically irrelevant) concentrations. The methyl ester derivative MBrO, was much more effective, with EC50 values one order of magnitude lower (62–66 μM). LA did not bypass the toxic effect of its analogues. Small monocarboxylic acids appear to be poorly internalized by T. cruzi: [14C]-octanoic acid was taken up 12 fold less efficiently than [14C]-palmitic acid. Western blot analysis of lipoylated proteins allowed the detection of the E2 subunits of pyruvate dehydrogenase (PDH), branched chain 2-ketoacid dehydrogenase and 2-ketoglutarate dehydrogenase complexes. Growth of parasites in medium with 10 fold lower glucose content, notably increased PDH activity and the level of its lipoylated E2 subunit. Treatment with BrO (1 mM) and MBrO (0.1 mM) completely inhibited E2 lipoylation and all three dehydrogenases activities. These observations indicate the lack of specific transporters for octanoic acid and most probably also for BrO and LA, which is in agreement with the lack of a LA salvage pathway, as previously suggested for T. brucei. They also indicate that the LA synthesis/protein lipoylation pathway could be a valid target for drug intervention. Moreover, the free LA available in the host would not interfere with such chemotherapeutic treatments.

Enhanced pyruvate dehydrogenase activity improves cardiac outcomes in a murine model of cardiac arrest.

RationalePost-ischemic changes in cellular metabolism alter myocardial and neurological function. Pyruvate dehydrogenase (PDH), the limiting step in mitochondrial glucose oxidation, is inhibited by increased expression of PDH kinase (PDK) during ischemia/reperfusion injury. This results in decreased utilization of glucose to generate cellular ATP. Post-cardiac arrest (CA) hypothermia improves outcomes and alters metabolism, but its influence on PDH and PDK activity following CA are unknown. We hypothesized that therapeutic hypothermia (TH) following CA is associated with the inhibition of PDK activity and increased PDH activity. We further hypothesized that an inhibitor of PDK activity, dichloroacetate (DCA), would improve PDH activity and post-CA outcomes.Methods and resultsAnesthetized and ventilated adult female C57BL/6 wild-type mice underwent a 12-minute KCl-induced CA followed by cardiopulmonary resuscitation. Compared to normothermic (37°C) CA controls, administering TH (30°C) improved overall survival (72-hour survival rate: 62.5% vs. 28.6%, P<0.001), post-resuscitation myocardial function (ejection fraction: 50.9±3.1% vs. 27.2±2.0%, P<0.001; aorta systolic pressure: 132.7±7.3 vs. 72.3±3.0 mmHg, P<0.001), and neurological scores at 72-hour post CA (9.5±1.3 vs. 5.4±1.3, P<0.05). In both heart and brain, CA increased lactate concentrations (1.9-fold and 3.1-fold increase, respectively, P<0.01), decreased PDH enzyme activity (24% and 50% reduction, respectively, P<0.01), and increased PDK protein expressions (1.2-fold and 1.9-fold, respectively, P<0.01). In contrast, post-CA treatment with TH normalized lactate concentrations (P<0.01 and P<0.05) and PDK expressions (P<0.001 and P<0.05), while increasing PDH activity (P<0.01 and P<0.01) in both the heart and brain. Additionally, treatment with DCA (0.2 mg/g body weight) 30 min prior to CA improved both myocardial hemodynamics 2 hours post-CA (aortic systolic pressure: 123±3 vs. 96±4 mmHg, P<0.001) and 72-hour survival rates (50% vs. 19%, P<0.05) in normothermic animals.ConclusionsEnhanced PDH activity in the setting of TH or DCA administration is associated with improved post-CA resuscitation outcomes. PDH is a promising therapeutic target for improving post-CA outcomes.