Reduced Pyruvate Dehydrogenase Activity Research Articles

Exploring the potential for branched chain keto-acids to inhibit the mitochondrial pyruvate carrier

Metabolism of branched-chain amino acids (BCAAs) is often dysregulated in obesity and diabetes. To be oxidized, BCAAs are first converted to branched chain keto-acids (BCKAs), which are structurally similar to pyruvic acid. Previous studies have suggested that BCKAs inhibit the mitochondrial pyruvate carrier (MPC), particularly in hepatocytes, so we sought to investigate this further. Mitochondria were isolated from mouse liver and coupled pyruvate respiration was assessed before and after addition of either 100μM keto-leucine (alpha-ketoisocaproate), keto-isoleucine, keto-valine, an equimolar mixture of the 3 BCKAs, or NaCl control. Keto-leucine and the BCKA mixture reduced respiration by ~35%, but keto-isoleucine and keto-valine had no effect. This was repeated in mouse heart mitochondria, and again, only keto-leucine or the BCKA mixture reduced pyruvate respiration by ~30%. In comparison, 1μM of the synthetic MPC inhibitor UK-5099 reduced pyruvate respiration by 80%. In hepatocytes, most pyruvate is not oxidized but is carboxylated, which is an important first step for hepatic gluconeogenesis. Isolated hepatocytes were treated with pyruvate +/- BCKAs, and while UK-5099 reduced glucose production, none of the BCKAs had any effect. This experiment was repeated with 13C-pyruvate, and while UK-5099 reduced 13C enrichment into the hepatocyte TCA cycle, gluconeogenic intermediates, and glucose in the media, again none of the BCKAs had any effect. This lack of effect on gluconeogenesis may be due to the relatively mild inhibition and the ability of hepatocytes to perform pyruvate-alanine cycling to maintain some pyruvate carbon entry into the TCA cycle. Respiration was then assessed in MPC-deficient cardiac mitochondria, and keto-leucine and the BCKA mixture were no longer able to reduce pyruvate oxidation suggesting the effect is dependent on the MPC. Keto-leucine also had no effect on pyruvate dehydrogenase (PDH) activity of cardiac mitochondrial lysates, suggesting that the reduction in pyruvate oxidation is due to MPC inhibition and not reduced PDH activity. In a bioluminescent resonance energy transfer (BRET) assay which indicates inhibitor binding and MPC conformational change, while UK-5099 increased BRET, keto-leucine had no effect. Lastly, we hypothesized that rapid oxidation of the keto-leucine could explain the relatively mild inhibition of the MPC that we observe. Therefore, we repeated mitochondrial respiration studies in mitochondria isolated from PPM1K−/− mice which is the phosphatase that regulates the branched chain keto acid dehydrogenase (BCKDH) and BCKA oxidation. As expected, PPM1K−/− mitochondria displayed enhanced BCKDH phosphorylation, suggestive of inhibited BCKA oxidation. To our surprise, while pyruvate oxidation was inhibited by keto-leucine in WT mitochondria, this inhibition was completely lost in liver or heart mitochondria from PPM1K−/− mice. Altogether, these results suggest that a downstream metabolite of keto-leucine inhibits the MPC, via a mechanism that differs from synthetic MPC inhibitors. Ongoing studies are being performed to search for this downstream metabolite responsible for MPC inhibition. Saint Louis University School of Medicine internal sponsorship. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Evaluation of IP3R3 Gene Silencing Effect on Pyruvate Dehydrogenase (PDH) Enzyme Activity in Breast Cancer Cells with and Without Estrogen Receptor.

Inositol 1,4,5-trisphosphate receptor (IP3R), a critical calcium ion (Ca2+) regulator, plays a vital role in breast cancer (BC) metabolism. Dysregulated IP3R in BC cells can drive abnormal growth or cell death. Estradiol increases IP3R type 3 (IP3R3) levels in BC, promoting cell proliferation and metabolic changes, including enhanced pyruvate dehydrogenase (PDH) activity, which, when reduced, leads to cell apoptosis. The study silenced IP3R3 to assess its impact on PDH. The study used IP3R3 small interfering RNA (siRNA) to target Michigan Cancer Foundation-7 (MCF-7) and MDA-MB-231 cell lines. Transfection success was confirmed by flow cytometry. Cell viability and gene silencing were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and real-time quantitative polymerase chain reaction (PCR) assays. Protein expression and cellular activity were analyzed through western blotting and PDH activity measurement. Transfecting MCF-7 and MDA-MB-231 cells with IP3R3 siRNA achieved a 65% transfection rate without significant toxicity. IP3R3 gene silencing effectively reduced IP3R3 messenger RNA (mRNA) and protein levels in both cell lines, leading to decreased PDH enzyme activity, especially in MDA-MB-231 cells. The study highlights a link between high IP3R3 gene silencing and reduced PDH activity, with higher IP3R3 expression in estrogen-independent (MDA-MB-231) compared to estrogen-dependent (MCF-7) cell lines. This suggests a potential impact on BC metabolism and tumor growth via regulation of PDH activity.

Pyruvate dehydrogenase is a potential mitochondrial off-target for gentamicin based on in silico predictions and in vitro inhibition studies

During the drug development process, organ toxicity leads to an estimated failure of one-third of novel chemical entities. Drug-induced toxicity is increasingly associated with mitochondrial dysfunction, but identifying the underlying molecular mechanisms remains a challenge. Computational modeling techniques have proven to be a good tool in searching for drug off-targets. Here, we aimed to identify mitochondrial off-targets of the nephrotoxic drugs tenofovir and gentamicin using different in silico approaches (KRIPO, ProBis and PDID). Dihydroorotate dehydrogenase (DHODH) and pyruvate dehydrogenase (PDH) were predicted as potential novel off-target sites for tenofovir and gentamicin, respectively. The predicted targets were evaluated in vitro, using (colorimetric) enzymatic activity measurements. Tenofovir did not inhibit DHODH activity, while gentamicin potently reduced PDH activity. In conclusion, the use of in silico methods appeared a valuable approach in predicting PDH as a mitochondrial off-target of gentamicin. Further research is required to investigate the contribution of PDH inhibition to overall renal toxicity of gentamicin.

Abstract P2083: Erk-mediated Phosphorylation Of Grk2 Leads To Reduced Pyruvate Dehydrogenase Activity And Alters Pyroptotic Signaling

G protein-coupled receptors (GPCRs) are important regulators of cellular functions where agonist binding leads to receptor conformational changes and downstream activation of g-protein-mediated signaling cascades. Signal transduction is terminated by receptor phosphorylation mediated by G-protein coupled receptor kinases (GRKs). GPCR kinase 2 (GRK2) is a main GRK in the heart and is upregulated in heart failure (HF) patients. Recently, we and others have shown that GRK2 can translocate to cardiac mitochondria where it regulates metabolism. Notably, we found that phosphorylated GRK2 at S670 post- ischemia/reperfusion (IR) decreases glucose mitochondrial utilization and pyruvate dehydrogenase (PDH) activity. Furthermore, 2D-SDS PAGE followed by LC/MS/MS revealed a potential link between mitochondrial GRK2 and PDH phosphorylation. Using a novel PDHα KI CRISPR Cas HEK cell line, we tested the hypothesize that PDH phosphorylation of this novel site was important for PDH activity. Utilizing cytosolic and mitochondrially-targeted pyruvate indicators we measured pyruvate levels in WT and KI HEK cells. In parallel, using a novel mouse model GRK2-S670A, we investigated the role of ERK in mediating GRK2 mitochondrial translocation and pyroptotic signaling. Further experiments will determine long-term impact of mitochondrial GRK2 in cell survival in cardiac pathological conditions.

Abstract 12092: IMB-1018792, an Investigational Agent Designed to Augment Cardiac Glucose Utlization and Energetics Reduces Cardiac Remodelling and Preserves Cardiac Function in a Model of Pressure Overload-Induced Heart Failure

Introduction: Energetic impairments play a key role in heart failure (HF). Contributors include reduced pyruvate dehydrogenase activity partially uncoupling glycolysis from glucose oxidation, increased dependence on fatty acid (FA) metabolism yielding less ATP than glucose per O 2 molecule consumed, a lower phosphate-to-O 2 ratio, mitochondrial uncoupling and futile cycling of FA intermediates. IMB-1018972 (IMB-101) is a novel investigational agent designed to increase cardiac glucose uptake to promote glucose oxidation (Randle effect) through partial inhibition of FA oxidation enhancing cardiac energetics and mechanical efficiency. Hypotheses: Administration of IMB-101 will: 1) increase cardiac glucose uptake and 2) attenuate LV hypertrophic remodelling and progression of HF resulting from chronic pressure overload. Methods: 1) Cardiac glucose metabolism was evaluated using dynamic 18 F-FDG PET/CT imaging in vivo in fasted normal rats following a subcutaneous dose of IMB-101 or saline. Each animal had 2 randomized sequenced scan sessions (n=16). 2) Male C57BL/6 mice aged 12 weeks underwent transverse aortic constriction (TAC) or sham surgery (n=15) and were treated with IMB-101 (10 mg/kg/day, n=15) or saline (n=15) from day 1 (D1) for 6 weeks by osmotic minipump. Cardiac function was assessed by conscious echocardiography (D1, week 3 and 6). Cardiac fibrosis was measured by Masson’s Trichrome staining. Results: IMB-101 robustly increased in vivo cardiac 18 F-FDG uptake vs saline in rats. In mice subjected to TAC, compared to saline controls, IMB-101 treatment: preserved LV systolic function (fractional shortening 46%±3% vs 34%±3%, at week 6, p<0.05); improved LV relaxation with less prolonged isovolumic relaxation time (32±1 ms vs 36±1 ms, p<0.05); reduced cardiac hypertrophy (LV mass 156±10 mg vs 195±12 mg, p<0.05; and heart:body weight ratio 7.4±0.3 mg/g vs. 9.1±0.5 mg/g, p<0.05); and attenuated cardiac fibrosis (6.6±0.6% vs 10.7±1%, p<0.01). Conclusions: IMB-101 increases cardiac glucose uptake and protects against LV dysfunction, hypertrophic remodelling and interstitial fibrosis after chronic pressure overload.

MondoA drives malignancy in B-ALL through enhanced adaptation to metabolic stress

Cancer cells are in most instances characterized by rapid proliferation and uncontrolled cell division. Hence, they must adapt to proliferation-induced metabolic stress through intrinsic or acquired antimetabolic stress responses to maintain homeostasis and survival. One mechanism to achieve this is reprogramming gene expression in a metabolism-dependent manner. MondoA (also known as Myc-associated factor X–like protein X-interacting protein [MLXIP]), a member of the MYC interactome, has been described as an example of such a metabolic sensor. However, the role of MondoA in malignancy is not fully understood and the underlying mechanism in metabolic responses remains elusive. By assessing patient data sets, we found that MondoA overexpression is associated with worse survival in pediatric common acute lymphoblastic leukemia (ALL; B-precursor ALL [B-ALL]). Using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) and RNA-interference approaches, we observed that MondoA depletion reduces the transformational capacity of B-ALL cells in vitro and dramatically inhibits malignant potential in an in vivo mouse model. Interestingly, reduced expression of MondoA in patient data sets correlated with enrichment in metabolic pathways. The loss of MondoA correlated with increased tricarboxylic acid cycle activity. Mechanistically, MondoA senses metabolic stress in B-ALL cells by restricting oxidative phosphorylation through reduced pyruvate dehydrogenase activity. Glutamine starvation conditions greatly enhance this effect and highlight the inability to mitigate metabolic stress upon loss of MondoA in B-ALL. Our findings give novel insight into the function of MondoA in pediatric B-ALL and support the notion that MondoA inhibition in this entity offers a therapeutic opportunity and should be further explored.

Difference in pyruvic acid metabolism between neonatal and adult mouse lungs exposed to hyperoxia.

Neonatal lungs are more tolerant to hyperoxic injury than are adult lungs. This study investigated differences in the response to hyperoxic exposure between neonatal and adult mouse lungs using metabolomics analysis with capillary electrophoresis time-of-flight mass spectrometry (CE- TOFMS). Neonatal and adult mice were exposed to 21% or 95% O2 for four days. Subsequently, lung tissue samples were collected and analyzed by CE-TOFMS. Pyruvate dehydrogenase (PDH) enzyme activity was determined using a microplate assay kit. PDH kinase (Pdk) 1, Pdk2, Pdk3, and Pdk4 mRNA expression levels were determined using quantitative reverse transcription-polymerase chain reaction. Pdk4 protein expression was quantified by Western blotting and Pdk4 protein localization was evaluated by immunohistochemistry. Levels of 3-phosphoglyceric acid, 2-phosphoglyceric acid, phosphoenolpyruvic acid, and lactic acid were significantly elevated in the lungs of hyperoxia-exposed versus normoxia-exposed adult mice, whereas no significant differences were observed with hyperoxia exposure in neonatal mice. PDH activity was reduced in the lungs of adult mice only. Pdk4 mRNA expression levels after hyperoxic exposure were significantly elevated in adult mice compared with that in neonatal mice. Conversely, gene expression levels of Pdk1, Pdk2, and Pdk3 did not differ after hyperoxic exposure in either neonatal or adult mice. Pdk4 protein levels were also significantly increased in adult mouse lungs exposed to hyperoxia and were localized mainly to the epithelium of terminal bronchiole. Specific metabolites associated with glycolysis and gluconeogenesis were altered after hyperoxia exposure in the lungs of adult mice, but not in neonates, which was likely a result of reduced PDH activity due to Pdk4 mRNA upregulation under hyperoxia.

Brain metabolism modulates neuronal excitability in a mouse model of pyruvate dehydrogenase deficiency.

Glucose is the ultimate substrate for most brain activities that use carbon, including synthesis of the neurotransmitters glutamate and γ-aminobutyric acid via mitochondrial tricarboxylic acid (TCA) cycle. Brain metabolism and neuronal excitability are thus interdependent. However, the principles that govern their relationship are not always intuitive because heritable defects of brain glucose metabolism are associated with the paradoxical coexistence, in the same individual, of episodic neuronal hyperexcitation (seizures) with reduced basal cerebral electrical activity. One such prototypic disorder is pyruvate dehydrogenase (PDH) deficiency (PDHD). PDH is central to metabolism because it steers most of the glucose-derived flux into the TCA cycle. To better understand the pathophysiology of PDHD, we generated mice with brain-specific reduced PDH activity that paralleled salient human disease features, including cerebral hypotrophy, decreased amplitude electroencephalogram (EEG), and epilepsy. The mice exhibited reductions in cerebral TCA cycle flux, glutamate content, spontaneous, and electrically evoked in vivo cortical field potentials and gamma EEG oscillation amplitude. Episodic decreases in gamma oscillations preceded most epileptiform discharges, facilitating their prediction. Fast-spiking neuron excitability was decreased in brain slices, contributing to in vivo action potential burst prolongation after whisker pad stimulation. These features were partially reversed after systemic administration of acetate, which augmented cerebral TCA cycle flux, glutamate-dependent synaptic transmission, inhibition and gamma oscillations, and reduced epileptiform discharge duration. Thus, our results suggest that dysfunctional excitability in PDHD is consequent to reduced oxidative flux, which leads to decreased neuronal activation and impaired inhibition, and can be mitigated by an alternative metabolic substrate.

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.

Hyperpolarized 13 C magnetic resonance evaluation of renal ischemia reperfusion injury in a murine model.

Acute kidney injury (AKI) is a major risk factor for the development of chronic kidney disease (CKD). Persistent oxidative stress and mitochondrial dysfunction are implicated across diverse forms of AKI and in the transition to CKD. In this study, we applied hyperpolarized (HP) 13 C dehydroascorbate (DHA) and 13 C pyruvate magnetic resonance spectroscopy (MRS) to investigate the renal redox capacity and mitochondrial pyruvate dehydrogenase (PDH) activity, respectively, in a murine model of AKI at baseline and 7days after unilateral ischemia reperfusion injury (IRI). Compared with the contralateral sham-operated kidneys, the kidneys subjected to IRI showed a significant decrease in the HP 13 C vitamin C/(vitamin C+DHA) ratio, consistent with a decrease in redox capacity. The kidneys subjected to IRI also showed a significant decrease in the HP 13 C bicarbonate/pyruvate ratio, consistent with impaired PDH activity. The IRI kidneys showed a significantly higher HP 13 C lactate/pyruvate ratio at day 7 compared with baseline, although the 13 C lactate/pyruvate ratio was not significantly different between the IRI and contralateral sham-operated kidneys at day 7. Arterial spin labeling magnetic resonance imaging (MRI) demonstrated significantly reduced perfusion in the IRI kidneys. Renal tissue analysis showed corresponding increased reactive oxygen species (ROS) and reduced PDH activity in the IRI kidneys. Our results show the feasibility of HP 13 C MRS for the non-invasive assessment of oxidative stress and mitochondrial PDH activity following renal IRI.

Impaired hippocampal glucose metabolism during and after flurothyl-induced seizures in mice: Reduced phosphorylation coincides with reduced activity of pyruvate dehydrogenase.

To determine changes in glucose metabolism and the enzymes involved in the hippocampus ictally and postictally in the acute mouse flurothyl seizure model. [U-13 C]-Glucose was injected (i.p.) prior to, or following a 5 min flurothyl-induced seizure. Fifteen minutes later, mice were killed and the total metabolite levels and % 13 C enrichment were analyzed in the hippocampal formation using gas chromatography-mass spectrometry. Activities of key metabolic and antioxidant enzymes and the phosphorylation status of pyruvate dehydrogenase were measured, along with lipid peroxidation. During seizures, total lactate levels increased 1.7-fold; however, [M + 3] enrichment of both lactate and alanine were reduced by 30% and 43%, respectively, along with a 28% decrease in phosphofructokinase activity. Postictally the % 13 C enrichments of all measured tricarboxylic acid (TCA) cycle intermediates and the amino acids were reduced by 46-93%. At this time, pyruvate dehydrogenase (PDH) activity was 56% of that measured in controls, and there was a 1.9-fold increase in the phosphorylation of PDH at ser232. Phosphorylation of PDH is known to decrease its activity. Here, we show that the increase of lactate levels during flurothyl seizures is from a source other than [U-13 C]-glucose, such as glycogen. Surprisingly, although we saw a reduction in phosphofructokinase activity during the seizure, metabolism of [U-13 C]-glucose into the TCA cycle seemed unaffected. Similar to our recent findings in the chronic phase of the pilocarpine model, postictally the metabolism of glucose by glycolysis and the TCA cycle was impaired along with reduced PDH activity. Although this decrease in activity may be a protective mechanism to reduce oxidative stress, which is observed in the flurothyl model, ATP is critical to the recovery of ion and neurotransmitter balance and return to normal brain function. Thus we identified promising novel strategies to enhance energy metabolism and recovery from seizures.

CBIO-42DOWN REGULATION OF PYRUVATE DEHYDROGENASE ACTIVITY IS ESSENTIAL FOR CELL PROLIFERATION IN IDH1 MUTANT GLIOMA CELLS

Somatic mutations in the isocitrate dehydrogenase 1 (IDH1) gene occur in 70-90% of low-grade gliomas. The mutation leads to the production of 2-hydroxyglutarate (2-HG), a novel “oncometabolite” that alters the epigenetic profile of DNA and inhibits cellular differentiation. Recent studies indicate that metabolic changes in IDH1 mutant cells extend beyond 2-HG production. We recently demonstrated that steady state levels of glutamate, lactate and phosphocholine were reduced in IDH1 mutant cells using two genetically engineered models, a U87 glioblastoma-based model and a normal human astrocyte (NHA) model. In this study, we explored the mechanism behind the reduction in glutamate levels in IDH1 mutant cells. We discovered that pyruvate dehydrogenase (PDH) activity was significantly reduced in U87 and NHA IDH1 mutant cells compared to wild-type cells. Reduced PDH activity was associated with increased inhibitory phosphorylation of PDH in both models. Examination of mRNA and protein levels for the pyruvate dehydrogenase kinases (PDKs) revealed that PDK3 expression was up-regulated in IDH1 mutant cells in both models. Levels of hypoxia inducible factor-1α (HIF-1α), an established regulator of PDK3 expression in cancer cells, was also higher in IDH1 mutant cells. Importantly, treatment of U87 and NHA IDH1 wild-type cells with 2-HG recapitulated the effects of the IDH1 mutation, with higher levels of HIF-1α and PDK3, and reduced PDH activity, in 2-HG treated cells. Importantly, pharmacological activation of PDH activity by treatment with dichloroacetate, a PDK inhibitor, completely abrogated the increase in clonogenicity conferred by the IDH1 mutation. Treatment of patient-derived mutant IDH1 neurosphere models (BT142 and BT54 cell lines) with dichloroacetate further confirmed that PDH activity is essential for cell proliferation. Thus, our results indicate that reversing the down regulation of PDH activity in IDH1 mutant glioma cells adversely affects their proliferation, a finding with therapeutic implications.

IDH1 Mutation Induces Reprogramming of Pyruvate Metabolism.

Mutant isocitrate dehydrogenase 1 (IDH1) catalyzes the production of 2-hydroxyglutarate but also elicits additional metabolic changes. Levels of both glutamate and pyruvate dehydrogenase (PDH) activity have been shown to be affected in U87 glioblastoma cells or normal human astrocyte (NHA) cells expressing mutant IDH1, as compared with cells expressing wild-type IDH1. In this study, we show how these phenomena are linked through the effects of IDH1 mutation, which also reprograms pyruvate metabolism. Reduced PDH activity in U87 glioblastoma and NHA IDH1 mutant cells was associated with relative increases in PDH inhibitory phosphorylation, expression of pyruvate dehydrogenase kinase-3, and levels of hypoxia inducible factor-1α. PDH activity was monitored in these cells by hyperpolarized (13)C-magnetic resonance spectroscopy ((13)C-MRS), which revealed a reduction in metabolism of hyperpolarized 2-(13)C-pyruvate to 5-(13)C-glutamate, relative to cells expressing wild-type IDH1. (13)C-MRS also revealed a reduction in glucose flux to glutamate in IDH1 mutant cells. Notably, pharmacological activation of PDH by cell exposure to dichloroacetate (DCA) increased production of hyperpolarized 5-(13)C-glutamate in IDH1 mutant cells. Furthermore, DCA treatment also abrogated the clonogenic advantage conferred by IDH1 mutation. Using patient-derived mutant IDH1 neurosphere models, we showed that PDH activity was essential for cell proliferation. Taken together, our results established that the IDH1 mutation induces an MRS-detectable reprogramming of pyruvate metabolism, which is essential for cell proliferation and clonogenicity, with immediate therapeutic implications.

ATP6AP2/(Pro)renin Receptor Contributes to Glucose Metabolism via Stabilizing the Pyruvate Dehydrogenase E1 β Subunit

Aerobic glucose metabolism is indispensable for metabolically active cells; however, the regulatory mechanism of efficient energy generation in the highly evolved mammalian retina remains incompletely understood. Here, we revealed an unsuspected role for (pro)renin receptor, also known as ATP6AP2, in energy metabolism. Immunoprecipitation and mass spectrometry analyses identified the pyruvate dehydrogenase (PDH) complex as Atp6ap2-interacting proteins in the mouse retina. Yeast two-hybrid assays demonstrated direct molecular binding between ATP6AP2 and the PDH E1 β subunit (PDHB). Pdhb immunoreactivity co-localized with Atp6ap2 in multiple retinal layers including the retinal pigment epithelium (RPE). ATP6AP2 knockdown in RPE cells reduced PDH activity, showing a predilection to anaerobic glycolysis. ATP6AP2 protected PDHB from phosphorylation, thus controlling its protein stability. Down-regulated PDH activity due to ATP6AP2 knockdown inhibited glucose-stimulated oxidative stress in RPE cells. Our present data unraveled the novel function of ATP6AP2/(P)RR as a PDHB stabilizer, contributing to aerobic glucose metabolism together with oxidative stress.

Decreased mitochondrial bioenergetics and calcium buffering capacity in the basal ganglia correlates with motor deficits in a nonhuman primate model of aging

Altered mitochondrial function in the basal ganglia has been hypothesized to underlie cellular senescence and promote age-related motor decline. We tested this hypothesis in a nonhuman primate model of human aging. Six young (6–8 years old) and 6 aged (20–25 years old) female Rhesus monkeys (Macaca mulatta) were behaviorally characterized from standardized video records. Additionally, we measured mitochondrial bioenergetics along with calcium buffering capacity in the substantia nigra and putamen (PUT) from both age groups. Our results demonstrate that the aged animals had significantly reduced locomotor activity and movement speed compared with younger animals. Moreover, aged monkeys had significantly reduced ATP synthesis capacity (in substantia nigra and PUT), reduced pyruvate dehydrogenase activity (in PUT), and reduced calcium buffering capacity (in PUT) compared with younger animals. Furthermore, this age-related decline in mitochondrial function in the basal ganglia correlated with decline in motor function. Overall, our results suggest that drug therapies designed to enhance altered mitochondrial function may help improve motor deficits in the elderly.

Growth hormone‐induced insulin resistance in human subjects involves reduced pyruvate dehydrogenase activity

Insulin resistance induced by growth hormone (GH) is linked to promotion of lipolysis by unknown mechanisms. We hypothesized that suppression of the activity of pyruvate dehydrogenase in the active form (PDHa) underlies GH-induced insulin resistance similar to what is observed during fasting. Eight healthy male subjects were studied four times in a randomized, single-blinded parallel design: Control, GH, Fasting (36h) and GH+Fasting. GH (30ng×kg(-1)×min(-1)) or saline was infused throughout the metabolic study day. Substrate metabolism and insulin sensitivity were assessed by indirect calorimetry and isotopically determined rates of glucose turnover before and after a hyperinsulinemic euglycemic clamp. PDHa activity, PDH-E1α phosphorylation, PDK4 expression and activation of insulin signalling proteins were assessed in skeletal muscle. Both fasting and GH promoted lipolysis, which was associated with ≈50% reduction in insulin sensitivity compared with the control day. PDHa activity was significantly reduced by GH as well as fasting. This was associated with increased inhibitory PDH-E1α phosphorylation on site 1 (Ser(293)) and 2 (Ser(300)) and up-regulation of PDK4 mRNA, while canonical insulin signalling to glucose transport was unaffected. Competition between intermediates of glucose and fatty acids seems to play a causal role in insulin resistance induced by GH in human subjects.

ANG II causes insulin resistance and induces cardiac metabolic switch and inefficiency: a critical role of PDK4

The renin-angiotensin system (RAS) may alter cardiac energy metabolism in heart failure. Angiotensin II (ANG II), the main effector of the RAS in heart failure, has emerged as an important regulator of cardiac hypertrophy and energy metabolism. We studied the metabolic perturbations and insulin response in an ANG II-induced hypertrophy model. Ex vivo heart perfusion showed that hearts from ANG II-treated mice had a lower response to insulin with significantly reduced rates of glucose oxidation in association with increased pyruvate dehydrogenase kinase 4 (PDK4) levels. Palmitate oxidation rates were significantly reduced in response to insulin in vehicle-treated hearts but remained unaltered in ANG II-treated hearts. Furthermore, phosphorylation of Akt was also less response to insulin in ANG II-treated wild-type (WT) mice, suggestive of insulin resistance. We evaluated the role of PDK4 in the ANG II-induced pathology and showed that deletion of PDK4 prevented ANG II-induced diastolic dysfunction and normalized glucose oxidation to basal levels. ANG II-induced reduction in the levels of the deacetylase, SIRT3, was associated with increased acetylation of pyruvate dehydrogenase (PDH) and a reduced PDH activity. In conclusion, our findings show that a combination of insulin resistance and decrease in PDH activity are involved in ANG II-induced reduction in glucose oxidation, resulting in cardiac inefficiency. ANG II reduces PDH activity via acetylation of PDH complex, as well as increased phosphorylation in response to increased PDK4 levels.

Role of N-terminal protein formylation in central metabolic processes in Staphylococcus aureus

BackgroundBacterial protein biosynthesis usually depends on a formylated methionyl start tRNA but Staphylococcus aureus is viable in the absence of Fmt, the tRNAMet formyl transferase. fmt mutants exhibit reduced growth rates indicating that the function of certain proteins depends on formylated N-termini but it has remained unclear, which cellular processes are abrogated by the lack of formylation.ResultsIn order to elucidate how global metabolic processes are affected by the absence of formylated proteins the exometabolome of an S. aureus fmt mutant was compared with that of the parental strain and the transcription of corresponding enzymes was analyzed to identify possible regulatory changes. The mutant consumed glucose and other carbon sources slower than the wild type. While the turnover of several metabolites remained unaltered fmt inactivation led to increases pyruvate release and, concomitantly, reduced pyruvate dehydrogenase activity. In parallel, the release of the pyruvate-derived metabolites lactate, acetoin, and alanine was reduced. The anaerobic degradation of arginine was also reduced in the fmt mutant compared to the wild-type strain. Moreover, the lack of formylated proteins caused increased susceptibility to the antibiotics trimethoprim and sulamethoxazole suggesting that folic acid-dependant pathways were perturbed in the mutant.ConclusionsThese data indicate that formylated proteins are crucial for specific bacterial metabolic processes and they may help to understand why it has remained important during bacterial evolution to initiate protein biosynthesis with a formylated tRNAMet.

Regulation of Islet β-Cell Pyruvate Metabolism: Interactions of Prolactin, Glucose, and Dexamethasone

Prolactin (PRL) induces beta-cell proliferation and glucose-stimulated insulin secretion (GSIS) and counteracts the effects of glucocorticoids on insulin production. The mechanisms by which PRL up-regulates GSIS are unknown. We used rat islets and insulinoma (INS-1) cells to explore the interactions of PRL, glucose, and dexamethasone (DEX) in the regulation of beta-cell pyruvate carboxylase (PC), pyruvate dehydrogenase (PDH), and the pyruvate dehydrogenase kinases (PDKs), which catalyze the phosphorylation and inactivation of PDH. PRL increased GSIS by 37% (P < 0.001) in rat islets. Glucose at supraphysiological concentrations (11 mm) increased PC mRNA in islets; in contrast, PRL suppressed PC mRNA levels in islets and INS-1 cells, whereas DEX was without effect. Neither PRL nor DEX altered PC protein or activity levels. In INS-1 cells, PRL increased PDH activity 1.4- to 2-fold (P < 0.05-0.001) at glucose concentrations ranging from 2.5-11 mm. DEX reduced PDH activity; this effect was reversed by PRL. PDK1, -2, -3, and -4 mRNAs were detected in both islets and insulinoma cells, but the latter expressed trivial amounts of PDK4. PRL reduced PDK2 mRNA and protein levels in rat islets and INS-1 cells and PDK4 mRNA in islets; DEX increased PDK2 mRNA in islets and INS-1 cells; this effect was reversed by PRL. Our findings suggest that PRL induction of GSIS is mediated by increases in beta-cell PDH activity; this is facilitated by suppression of PDKs. PRL counteracts the effects of DEX on PDH and PDK expression, suggesting novel roles for the lactogens in the defense against diabetes.

Thiamine limitation determines the transition from aerobic to fermentative-like metabolism inRhizobium etliCE3

Both thiamine and biotin when added to minimal medium subcultures reversed the fermentative-like metabolism exhibited by Rhizobium etli CE3. Thiamine auxotrophs lacking thiCOGE genes were used to investigate the role of thiamine in this medium. A thiC1169::miniTn5lacZ1 thiamine auxotroph subjected to the above subcultures resulted in growth arrest, reduced pyruvate-dehydrogenase activity, and a smaller amount of poly-beta-hydroxybutyrate compared with the CE3 strain. Moreover, thiC and thiEb genes were overexpressed as result of thiamine limitation. The absence of classical thi genes suggests that thiamine is synthesized with low efficiency by an alternative pathway. Low levels of thiamine cause the CE3 strain to exhibit a fermentative-like metabolism.