Pyruvate Dehydrogenase E1 Alpha Research Articles

Exogenous Lactate Treatment Immediately after Exercise Promotes Glycogen Recovery in Type-II Muscle in Mice.

Recent studies suggest that lactate intake has a positive effect on glycogen recovery after exercise. However, it is important to verify the effect of lactate supplementation alone and the timing of glycogen recovery. Therefore, in this study, we aimed to examine the effect of lactate supplementation immediately after exercise on glycogen recovery in mice liver and skeletal muscle at 1, 3, and 5 h after exercise. Mice were randomly divided into the sedentary, exercise-only, lactate, and saline-treated groups. mRNA expression and activation of glycogen synthesis and lactate transport-related factors in the liver and skeletal muscle were assessed using real-time polymerase chain reaction. Skeletal muscle glycogen concentration showed an increasing trend in the lactate group compared with that in the control group at 3 and 5 h after post-supplementation. Additionally, exogenous lactate supplementation significantly increased the expression of core glycogen synthesis enzymes, lactate transporters, and pyruvate dehydrogenase E1 alpha 1 in the skeletal muscles. Conversely, glycogen synthesis, lactate transport, and glycogen oxidation to acetyl-CoA were not significantly affected in the liver by exogenous lactate supplementation. Overall, these results suggest that post-exercise lactate supplement enables glycogen synthesis and recovery in skeletal muscles.

Baicalin improves the energy levels in the prefrontal cortex of mice exposed to chronic unpredictable mild stress

Depression is gradually becoming a primary mental disease threatening human health. Therefore, there is an urgent need to clarify the pathogenesis of depression and identify new effective natural antidepressants. This study aimed to investigate the antidepressant effects of baicalin and explore its potential mechanism in a mouse model of depression induced by chronic unpredictable mild stress (CUMS). Following a 6-week exposure to CUMS, mice were treated with baicalin (10 mg/kg) or fluoxetine (10 mg/kg) for 4 weeks by oral gavage. A sucrose preference test and a forced swimming test were performed to evaluate depression-like behaviors, and the levels of adenosine triphosphate (ATP) in the prefrontal cortex were measured. Moreover, gene expression and enzyme activities related to ATP production, and mitochondrial function, were monitored. The results indicated that baicalin and fluoxetine could alleviate CUMS-induced depression-like behaviors of mice. In addition, baicalin significantly elevated the ATP content and the expression of genes hexokinase 1 (Hk1), pyruvate dehydrogenase E1 alpha 1 (Pdha-1), isocitrate dehydrogenase (Idh), peroxisome proliferator-activated receptor, gamma, coactivator 1 alpha (Pgc-1α), and sirtuin-1 (Sirt1) in the prefrontal cortex. Furthermore, baicalin increased the activity of the respiratory chain complexes I and V as well as the mitochondrial membrane potential. In conclusion, baicalin may exert its antidepressant effect partly by upregulating the expression of some genes coding for enzymes involved in the glycolysis and the tricarboxylic acid cycle, and improving the mitochondrial function to enhance the ATP level in the brain.

Insulin reduces pyroptosis-induced inflammation by PDHA1 dephosphorylation-mediated NLRP3 activation during myocardial ischemia-reperfusion injury.

Previous studies proved that pyrin domain-containing protein 3 (NLRP3)-induced pyroptosis plays an important role in Myocardial ischemia-reperfusion injury (MIRI). Insulin can inhibit the activation of NLRP3 inflammasome, although the exact mechanism remains unclear. The aim of this study was to determine whether insulin reduces NLRP3-induced pyroptosis by regulating pyruvate dehydrogenase E1alpha subunit (PDHA1) dephosphorylation during MIRI. Rat hearts were subject to 30 min global ischemia followed by 60 min reperfusion, with or without 0.5 IU/L insulin. Myocardial ischemia-reperfusion injury was evaluated by measuring myocardial enzymes release, Cardiac hemodynamics, pathological changes, infarct size, and apoptosis rate. Cardiac aerobic glycolysis was evaluated by measuring ATP, lactic acid content, and pyruvate dehydrogenase complex (PDHc) activity in myocardial tissue. Recombinant adenoviral vectors for PDHA1 knockdown were constructed. Pyroptosis-related proteins were measured by Western blotting analysis, immunohistochemistry staining, and ELISA assay, respectively. It was found that insulin significantly reduced the area of myocardial infarction, apoptosis rate, and improved cardiac hemodynamics, pathological changes, energy metabolism. Insulin inhibits pyroptosis-induced inflammation during MIRI. Subsequently, Adeno-associated virus was used to knock down cardiac PDHA1 expression. Knockdown PDHA1 not only promoted the expression of NLRP3 but also blocked the inhibitory effect of insulin on NLRP3-mediated pyroptosis in MIRI. Results suggest that insulin protects against MIRI by regulating PDHA1 dephosphorylation, its mechanism is not only to improve myocardial energy metabolism but also to reduce the NLRP3-induced pyroptosis.

Effect of selectively knocking down key metabolic genes in Müller glia on photoreceptor health.

The importance of Müller glia for retinal homeostasis suggests that they may have vulnerabilities that lead to retinal disease. Here, we studied the effect of selectively knocking down key metabolic genes in Müller glia on photoreceptor health. Immunostaining indicated that murine Müller glia expressed insulin receptor (IR), hexokinase 2 (HK2) and phosphoglycerate dehydrogenase (PHGDH) but very little pyruvate dehydrogenase E1 alpha 1 (PDH-E1α) and lactate dehydrogenase A (LDH-A). We crossed Müller glial cell-CreER (MC-CreER) mice with transgenic mice carrying a floxed IR, HK2, PDH-E1α, LDH-A, or PHGDH gene to study the effect of selectively knocking down key metabolic genes in Müller glia cells on retinal health. Selectively knocking down IR, HK2, or PHGDH led to photoreceptor degeneration and reduced electroretinographic responses. Supplementing exogenous l-serine prevented photoreceptor degeneration and improved retinal function in MC-PHGDH knockdown mice. We unexpectedly found that the levels of retinal serine and glycine were not reduced but, on the contrary, highly increased in MC-PHGDH knockdown mice. Moreover, dietary serine supplementation, while rescuing the retinal phenotypes caused by genetic deletion of PHGDH in Müller glial cells, restored retinal serine and glycine homeostasis probably through regulation of serine transport. No retinal abnormalities were observed in MC-CreER mice crossed with PDH-E1α- or LDH-A-floxed mice despite Cre expression. Our findings suggest that Müller glia do not complete glycolysis but use glucose to produce serine to support photoreceptors. Supplementation with exogenous serine is effective in preventing photoreceptor degeneration caused by PHGDH deficiency in Müller glia.

Abstract PS17-50: Nuclear expression of acetyl-CoA producing enzymes and their roles in epigenetic reprogramming in breast cancer cells

Abstract Metabolic and epigenetic reprogramming are two key hallmarks of cancer. Rapidly proliferating cancer cells often exhibit a higher and differentially reprogrammed transcriptional activity than normal cells of the same tissue. To support transcriptional reprogramming and increased transcriptional load, sustained chromatin acetylation is required to loosen the chromatin and enable RNA transcription. Histone acetylation also requires a constant supply of acetyl-CoA, a labile, membrane impermeable metabolite that is the obligatory donor of the acetyl groups for histone acetylation. However, the pathways responsible for increased synthesis of acetyl-CoA inside the nucleus remain largely elusive. Using estrogen-responsive (MCF7) and their matched endocrine therapy resistant breast cancer cells (LCC9 and MCF7:5C), we show that blocking p300-mediated acetyltransferase activity is crucial for proliferation of endocrine therapy-resistant breast cancer cells. Furthermore, we found nuclear expression of two acetyl-CoA producing enzymes, i.e., pyruvate dehydrogenase complex (PDC), and ATP citrate lyase (ACLY), which implies their functional role inside the nucleus. PDC is a multimeric protein complex enzyme made up of five different subunits. Canonically, PDC is found in mitochondria and is responsible for the conversion of pyruvate to acetyl-CoA, linking glycolysis to the tricarboxylic acid (TCA) cycle. Our results show that multiple PDC enzyme subunits, e.g., pyruvate dehydrogenase (PDH) E1 alpha (E1 alpha), dihydrolipoal dehydrogenase (DLD), and PDHX (also known as E3BP) are expressed in the nucleus of MCF7, LCC9 and MCF7:5C cells. Moreover, expression of nuclear DLD and PDHX subunit is higher in endocrine resistant cells (LCC9 and MCF7:5C) than in their parental MCF7 cells, strongly suggesting a possible role in endocrine resistance. Notably, high expression of PDHX is correlated with poor relapse-free survival in estrogen-receptor positive breast cancer patients in four independent publicly available datasets. ACLY is a tetramer of identical subunits catalyzes conversion of citric acid and co-enzyme A to acetyl-CoA. ACLY is also overexpressed in the nucleus of endocrine-resistant breast cancer cells (LCC9 and MCF7:5C) when compared with their endocrine sensitive MCF7 controls. This study investigates the role of nuclear PDC and ACLY mediated acetyl-CoA synthesis and its impact on histone acetylation enabling reprogrammed transcriptional activity supporting proliferation of endocrine therapy-resistant breast cancer cells. Citation Format: Surojeet Sengupta, Shuait Nair, Lu Jin, Catherine M Sevigny, Brandon Jones, Robert Clarke. Nuclear expression of acetyl-CoA producing enzymes and their roles in epigenetic reprogramming in breast cancer cells [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-50.

SIRT3 (Sirtuin-3) Prevents Ang II (Angiotensin II)-Induced Macrophage Metabolic Switch Improving Perivascular Adipose Tissue Function.

Infiltrated macrophages actively promote perivascular adipose tissue remodeling and represent a dominant population in the perivascular adipose tissue microenvironment of hypertensive mice. However, the role of macrophages in initiating metabolic inflammation remains uncertain. SIRT3 (sirtuin-3), a NAD-dependent deacetylase, is sensitive to metabolic status and mediates adaptation responses. In this study, we investigated the role of SIRT3-mediated metabolic shift in regulating NLRP3 (Nod-like receptor family pyrin domain-containing 3) inflammasome activation. Approach and Results: Here, we report that Ang II (angiotensin II) accelerates perivascular adipose tissue inflammation and fibrosis, accompanied by NLRP3 inflammasome activation and IL (interleukin)-1β secretion in myeloid SIRT3 knockout (SIRT3-/-) mice. This effect is associated with adipose tissue mitochondrial dysfunction. In vitro studies indicate that the deletion of SIRT3 in bone marrow-derived macrophages induces IL-1β production by shifting the metabolic phenotype from oxidative phosphorylation to glycolysis. Mechanistically, SIRT3 deacetylates and activates PDHA1 (pyruvate dehydrogenase E1 alpha) at lysine 83, and the loss of SIRT3 leads to PDH activity decrease and lactate accumulation. Knocking down LDHA (lactate dehydrogenase A) or using carnosine, a buffer against lactic acid, attenuates IL-1β secretion. Furthermore, the blockade of IL-1β from macrophages into brown adipocytes restores thermogenic markers and mitochondrial oxygen consumption. Moreover, NLRP3 knockout (NLRP3-/-) mice exhibited reduced IL-1β production while rescuing the mitochondrial function of brown adipocytes and alleviating perivascular adipose tissue fibrosis. SIRT3 represents a potential therapeutic target to attenuate NLRP3-related inflammation. Pharmacological targeting of glycolytic metabolism may represent an effective therapeutic approach.

Epigallocatechin-3-gallate downregulates PDHA1 interfering the metabolic pathways in human herpesvirus 8 harboring primary effusion lymphoma cells

Primary effusion lymphoma (PEL) is an aggressive neoplasm correlated with human herpesvirus 8 (HHV8). Metabolic reprogramming is a hallmark of cancers. The alterations in cellular metabolism are important to the survival of HHV8 latently infected cells. Pyruvate dehydrogenase (PDH) controls the flux of metabolites between glycolysis and the tricarboxylic acid cycle (TCA cycle) and is a key enzyme in cancer metabolic reprogramming. Glutaminolysis is required for the survival of PEL cells. Glutamate dehydrogenase 1 (GDH1) converts glutamate into α-ketoglutarate supplying the TCA cycle with intermediates to support anaplerosis. Previously we have observed that epigallocatechin-3-gallate (EGCG) can induce PEL cell death and N-acetyl cysteine (NAC) attenuates EGCG induced PEL cell death. In this study, results showed that EGCG upregulated the expression of glucose transporter GLUT3, and reduced the expression of pyruvate dehydrogenase E1-alpha (PDHA1), the major regulator of PDH, and GDH1. NAC could partially reverse the effects of EGCG in PEL cells. Overexpression of PDHA1 in PEL cells or supplement of α-ketoglutarate attenuated EGCG induced cell death. EGCG also reduced the levels of oncometabolite D-2-hydroxyglutarate (D2HG). These results suggest that EGCG may modulate the metabolism of PEL cells leading to cell death.

PDHA1 Gene Knockout In Human Esophageal Squamous Cancer Cells Resulted In Greater Warburg Effect And Aggressive Features In Vitro And In Vivo.

BackgroundOne of the remarkable metabolic characteristics of cancer cells is that they prefer glycolysis rather than oxidative phosphorylation (OXPHOS). Pyruvate dehydrogenase E1 alpha subunit (PDHA1) is an important prerequisite for OXPHOS. Our previous studies have shown that low level of PDHA1 protein expression in esophageal squamous cell cancer (ESCC) was correlated with poor prognosis. However, the effect of PDHA1 inhibition on metabolism and biological behavior of esophageal cancer cells remains unclear.Methods And ResultsIn this study, a KYSE450 PDHA1 knockout (KO) cell line of esophageal cancer was established by CRISPR/Cas9 technology. Then, the glycose metabolism, cell proliferation and migration abilities, chemotherapeutic tolerance and angiogenesis of the PDHA1 KO cells were investigated in vitro and in vivo. In the PDHA1 KO cells, the glycolysis and the consumption of glucose and glutamine were significantly enhanced, while the OXPHOS was significantly suppressed, implying Warburg effect in the PDHA1 KO cells. Furthermore, it was also proved in vitro experiments that the PDHA1 KO cell obtained proliferation advantage, as well as significantly greater chemotherapy tolerance and migration ability. Xenograft experiments discovered not only larger tumors but also increased angiogenesis in the PDHA1 KO cell group.ConclusionInhibition of PDHA1 gene expression in human ESCC leads to metabolic reprogramming of Warburg effect and increased malignancies. Targeting ESCC metabolic reprogramming may become a potential therapeutic target.

Potential regulation by miRNAs on glucose metabolism in liver of common carp (Cyprinus carpio) at different temperatures

Water temperature can affect the metabolism of fish. Common carp (Cyprinus carpio) is a representative eurythermic fish that can survive at a wide range of ambient temperatures, allowing it to live in an extensive geographical range. The goal of this work was to study the glucose metabolism of common carp at different temperatures and determine the miRNAs involved in the regulation of glucose metabolism. We determined the indicators related to glucose metabolism after long-term temperature stress and constructed nine small RNA libraries of livers under different temperature stress (5 °C, 17 °C, and 30 °C, with three biological replicates for each temperature), and subjected these samples to high-throughput sequencing. A positive relationship was observed between weight gain rate (WGR) and temperature increase after 18 days of temperature stress. However, the glucose level in the plasma maintained a gentle decrease. Unexpectedly, liver lactic acid levels were elevated in HTG (high temperature group) and LTG (low temperature group). Six down-regulated miRNAs (miR-122, miR-30b, miR-15b-5p, miR-20a-5p, miR-1, and miR-7b) were identified as involved in the regulation of glycolysis. Twelve genes were predicted as targets of these miRNAs, and these genes are in pathways related to pyruvate metabolism, glycolysis/gluconeogenesis, and the citrate cycle (TCA cycle). The results allowed prediction of a potential regulatory network of miRNAs involved in the regulation of glycolysis. The target genes of six down-regulated miRNAs were up-regulated under temperature stress, including Aldolase C, fructose-bisphosphate, b (ALDOCB), multiple inositol-polyphosphate phosphatase 1 (MINPP1), phosphoenolpyruvate carboxykinase 1 (PCK1), pyruvate dehydrogenase E1 alpha 1 (PDHA1), aldehyde dehydrogenase 9 family member A1a (ALDH9A1A), Acetyl-coenzyme A synthetase (ACSS), lactate dehydrogenase b (LDH-b), and glyoxylate reductase/hydroxypyruvate reductase (GRHPR). Other key genes of glycolysis, glucose transporter 1 (GLUT-1), pyruvate kinase PKM (PKM), and mitochondrial pyruvate carrier (MPC) were significantly up-regulated in LTG and HTG. Overall, the results suggest that miRNAs maintain their energy requirements by regulating glycolysis and play an important role in the molecular response to cold and heat stress of common carp. These data provide the foundation for further studies of the role of miRNAs in environmental adaptation in fish.

SIRT3-MEDIATED MACROPHAGE PYRUVATE DEHYDROGENASE ACTIVITY IMPROVES PERIVASCULAR ADIPOSE TISSUE REMODELING IN HYPERTENSIVE MICE

Objective: Infiltrated macrophages are a dominant population in the perivascular adipose tissue (PVAT) microenvironment and actively promote PVAT remodeling in hypertensive mice. However, the molecular mechanism underlying the role of macrophages in initiating metabolic inflammation remains uncertain. Here, we investigate whether Situin-3(SIRT3) is determinant in macrophage activation. Design and method: 10-week-old male WT, SIRT3 KO, and NLRP3 KO mice were infused with Angiotension II (Ang II, 1000ng/kg/min) or normal saline (NS) for 7 days. The morphological and functional changes of thoracic aorta and PVAT were determined by histological analysis and immunofluorescence staining. In vitro studies were performed on bone marrow-derived macrophages (BMDMs) and brown adipocytes isolated from WT or SIRT3 KO mice. Results: We report that Ang II accelerates PVAT inflammation and fibrosis in SIRT3-deficient mice. This effect is associated with attenuating browning markers and lipid droplet-associated protein expression. Immunofluorescence analysis confirmed that there is an increase in NLRP3 inflammasome and secretion of interleukin-1β (IL-1β). In vitro studies indicate that BMDMs from SIRT3 KO mice induce interleukin-IL-1β production by shifting metabolic phenotype from oxidative phosphorylation towards glycolysis. Mechanically, SIRT3 deacetylates and activates pyruvate dehydrogenase E1 alpha (PDHE1a) at lysine 83, loss of SIRT3 lead to decreased PDH activity, and accumulation of pyruvate and lactate metabolites. The PDHK inhibitor dichloroacetate (DCA) suppresses both pro-IL-1β maturation and caspase-1 activation in macrophages in response to Ang II. The blockade of IL-1β from macrophage into brown adipocytes restores expression of brown adipocyte specific markers. Coincidentally, NLRP3 KO mice exhibited a reduction in infiltrated macrophages in PVAT, while partially rescuing brown adipocytes mitochondrial function and collagen accumulation. Conclusions: Taken together, these findings reveal that SIRT3-mediated activity of PDHE1a inhibits macrophage activation. Pharmacological targeting of glycolysis metabolism may therefore represent an effective approach.

Shengui Sansheng San Ameliorates Cerebral Energy Deficiency via Citrate Cycle After Ischemic Stroke.

Cerebral energy deficiency is a key pathophysiologic cascade that results in neuronal injury and necrosis after ischemic stroke. Shengui Sansheng San (SSS) has been used to treat stroke for more than 300 years. In present study, we investigated the therapeutic efficacy and mechanism of SSS extraction on cerebral energy deficiency post-stroke. In permanent middle cerebral artery occlusion (pMCAo) model of rats, it suggested that SSS extraction in dose-dependent manner improved neurological function, cerebral blood flow (CBF), 18F-2-deoxy-glucose uptake and the density and diameter of alpha smooth muscle actin (α-SMA) positive vasculature in ipsilateral area, as well as decreased infarcted volume. Meanwhile, the metabolomics study in cerebrospinal fluid (CSF) was performed by using 5-(diisopropylamino)amylamine (DIAAA) derivatization-UHPLC-Q-TOF/MS approach. Eighty-eight endogenous metabolites were identified, and mainly involved in citrate cycle, fatty acid biosynthesis, aminoacyl-tRNA biosynthesis, amino acids metabolism and biosynthesis, etc. The remarkable increase of citrate in CSF after treatment with three dosages indicated that the therapeutic mechanism of SSS extraction might be related with citrate cycle. Simultaneously, it showed that high dosage group significantly increased peripheral blood glucose level, the expressions of glucose transporter (GLUT) 1, GLUT3, and monocarboxylic acid transporter 1 (MCT1), which contributed to the transportation of glucose and lactate. By the regulations of phosphorylated pyruvate dehydrogenase E1-alpha (p-PDHA1), acetyl CoA synthetase and citrate synthetase (CS), the levels of citrate and its upstream molecules (pyruvate and acetyl CoA) in peri-infarction zone further enhanced, which ultimately caused the massive yield of adenosine triphosphate (ATP). Our study first demonstrated that SSS extraction could ameliorate cerebral energy deficiency after ischemia by citrate cycle, which is characterized by the enhancements of glucose supply, transportation, utilization, and metabolism.

Characterization of Mycoplasma gallisepticum pyruvate dehydrogenase alpha and beta subunits and their roles in cytoadherence.

Mycoplasma gallisepticum is a causative agent of chronic respiratory disease in chickens, typically causing great economic losses. Cytoadherence is the critical stage for mycoplasma infection, and the associated proteins are important for mycoplasma pathogenesis. Many glycolytic enzymes are localized on the cell surface and can bind the extracellular matrix of host cells. In this study, the M. gallisepticum pyruvate dehydrogenase E1 alpha subunit (PDHA) and beta subunit (PDHB) were expressed in Escherichia coli, and their enzymatic activities were identified based on 2,6-dichlorophenol indophenol reduction. When recombinant PDHA (rPDHA) and recombinant PDHB (rPDHB) were mixed at a 1:1 molar ratio, they exhibited strong enzymatic activity. Alone, rPDHA and rPDHB exhibited no or weak enzymatic activity. Further experiments indicated that both PDHA and PDHB were surface-exposed immunogenic proteins of M. gallisepticum. Bactericidal assays showed that the mouse anti-rPDHA and anti-rPDHB sera killed 48.0% and 75.1% of mycoplasmas respectively. A combination of rPDHA and rPDHB antisera had a mean bactericidal rate of 65.2%, indicating that rPDHA and rPDHB were protective antigens, and combining the two sera did not interfere with bactericidal activity. Indirect immunofluorescence and surface display assays showed that both PDHA and PDHB adhered to DF-1 chicken embryo fibroblast cells and adherence was significantly inhibited by antisera against PDHA and PDHB. Adherence inhibition of M. gallisepticum to DF-1 chicken embryo fibroblast cells was 30.2% for mouse anti-rPDHA serum, 45.1% for mouse anti-rPDHB serum and 72.5% for a combination of rPDHA and rPDHB antisera, suggesting that rPDHA and rPDHB antisera may have synergistically interfered with M. gallisepticum cytoadherence. Plasminogen (Plg)-binding assays further demonstrated that both PDHA and PDHB were Plg-binding proteins, which may have contributed to bacterial colonization. Our results clarified the enzymatic activity of M. gallisepticum PDHA and PDHB and demonstrated these compounds as Plg-binding proteins involved in cytoadherence.

PGC1\u03b1 promotes cholangiocarcinoma metastasis by upregulating PDHA1 and MPC1 expression to reverse the Warburg effect

PGC1α acts as a central regulator of mitochondrial metabolism, whose role in cancer progression has been highlighted but remains largely undefined. Especially, it is completely unknown about the effect of PGC1α on cholangiocarcinoma (CCA). Here we showed that PGC1α overexpression had no impact on CCA growth despite the decreased expression of PGC1α in CCA compared with adjacent normal tissue. Instead, PGC1α overexpression-promoted CCA metastasis both in vitro and in vivo. Mechanistically, for the first time, we illuminated that PGC1α reversed the Warburg effect by upregulating the expression of pyruvate dehydrogenase E1 alpha 1 subunit and mitochondrial pyruvate carrier 1 to increase pyruvate flux into the mitochondria for oxidation, whereas simultaneously promoting mitochondrial biogenesis and fusion to mediate the metabolic switch to oxidative phosphorylation. On the one hand, enhanced mitochondrial oxidation metabolism correlated with elevated reactive oxygen species (ROS) production; on the other hand, increased PGC1α expression upregulated the expression levels of mRNA for several ROS-detoxifying enzymes. To this end, the ROS levels, which were elevated but below a critical threshold, did not inhibit CCA cells proliferation. And the moderately increased ROS facilitated metastatic dissemination of CCA cells, which can be abrogated by antioxidants. Our study suggests the potential utility of developing the PGC1α-targeted therapies or blocking PGC1α signaling axis for inhibiting CCA metastasis.

Clinical manifestations in two patients with pyruvate dehydrogenase deficiency and long-term survival

Pyruvate dehydrogenase E1-alpha deficiency (PDHAD) results in lactic acidosis and hyperpyruvatemia. Two patients with PDHAD, a man with a p.R263Q mutation, and a girl with a p.C145del mutation in PDHE1α, presented with lactic acidosis with neurological disorder. These patients were able to survive for a long period under careful nursing care. Herein, we discuss the factors contributing to their relatively stable clinical course, albeit with intellectual disability.

Elevated miR-130a/miR130b/miR-152 expression reduces intracellular ATP levels in the pancreatic beta cell

MicroRNAs have emerged as important players of gene regulation with significant impact in diverse disease processes. In type-2 diabetes, in which impaired insulin secretion is a major factor in disease progression, dysregulated microRNA expression in the insulin-secreting pancreatic beta cell has been widely-implicated. Here, we show that miR-130a-3p, miR-130b-3p, and miR-152-3p levels are elevated in the pancreatic islets of hyperglycaemic donors, corroborating previous findings about their upregulation in the islets of type-2 diabetes model Goto-Kakizaki rats. We demonstrated negative regulatory effects of the three microRNAs on pyruvate dehydrogenase E1 alpha (PDHA1) and on glucokinase (GCK) proteins, which are both involved in ATP production. Consequently, we found both proteins to be downregulated in the Goto-Kakizaki rat islets, while GCK mRNA expression showed reduced trend in the islets of type-2 diabetes donors. Overexpression of any of the three microRNAs in the insulin-secreting INS-1 832/13 cell line resulted in altered dynamics of intracellular ATP/ADP ratio ultimately perturbing fundamental ATP-requiring beta cell processes such as glucose-stimulated insulin secretion, insulin biosynthesis and processing. The data further strengthen the wide-ranging influence of microRNAs in pancreatic beta cell function, and hence their potential as therapeutic targets in type-2 diabetes.

Histone Deacetylase Inhibitors Protect Against Pyruvate Dehydrogenase Dysfunction in Huntington's Disease.

Transcriptional deregulation and changes in mitochondrial bioenergetics, including pyruvate dehydrogenase (PDH) dysfunction, have been described in Huntington's disease (HD). We showed previously that the histone deacetylase inhibitors (HDACIs) trichostatin A and sodium butyrate (SB) ameliorate mitochondrial function in cells expressing mutant huntingtin. In this work, we investigated the effect of HDACIs on the regulation of PDH activity in striatal cells derived from HD knock-in mice and YAC128 mice. Mutant cells exhibited decreased PDH activity and increased PDH E1alpha phosphorylation/inactivation, accompanied by enhanced protein levels of PDH kinases 1 and 3 (PDK1 and PDK3). Exposure to dichloroacetate, an inhibitor of PDKs, increased mitochondrial respiration and decreased production of reactive oxygen species in mutant cells, emphasizing PDH as an interesting therapeutic target in HD. Treatment with SB and sodium phenylbutyrate, another HDACI, recovered cell viability and overall mitochondrial metabolism in mutant cells. Exposure to SB also suppressed hypoxia-inducible factor-1 (HIF-1α) stabilization and decreased the transcription of the two most abundant PDK isoforms, PDK2 and PDK3, culminating in increased PDH activation in mutant cells. Concordantly, PDK3 knockdown improved mitochondrial function, emphasizing the role of PDK3 inactivation on the positive effects achieved by SB treatment. YAC128 mouse brain presented higher mRNA levels of PDK1-3 and PDH phosphorylation and decreased energy levels that were significantly ameliorated after SB treatment. Furthermore, enhanced motor learning and coordination were observed in SB-treated YAC128 mice. These results suggest that HDACIs, particularly SB, promote the activity of PDH in the HD brain, helping to counteract HD-related deficits in mitochondrial bioenergetics and motor function.SIGNIFICANCE STATEMENT The present work provides a better understanding of mitochondrial dysfunction in Huntington's disease (HD) by showing that the pyruvate dehydrogenase (PDH) complex is a promising therapeutic target. In particular, the histone deacetylase inhibitor sodium butyrate (SB) may indirectly (through reduced hypoxia-inducible factor 1 alpha stabilization) decrease the expression of the most abundant PDH kinase isoforms (e.g., PDK3), ameliorating PDH activity and mitochondrial metabolism and further affecting motor behavior in HD mice, thus constituting a promising agent for HD neuroprotective treatment.

Abstract 1858: Development of a multiplex panel of biomarkers to assess energy metabolism in cancer.

Abstract Background: Cancer cells have an altered glucose metabolism, which not only permits them to survive under hypoxic conditions but confers a distinct growth advantage by sustaining higher proliferation rates, invasiveness, and subsequent distant metastasis. This unique difference in glucose metabolism from healthy cells opens up a selective mechanism by which to kill cancer cells, bypassing normal healthy cells. Several enzymes in glucose metabolism are attractive targets for development of first-in-class therapeutic agents. We have undertaken development of a multiplex panel involving biomarkers involved in glucose metabolism to support drug development and to interrogate glycolytic pathway in cancer patients. Methods: The panel includes quantitative determinations of protein levels of hexokinase-2 (HK2), pyruvate kinase isoform-2 (PKM2), lactate dehydrogenase alpha (LDHa), and phosphorylated pyruvate dehydrogenase E1-alpha (phospho-PDHE1a). The sandwich immunoassays were built on the Luminex® xMAP technology platform using MagPlex magnetic beads and cloned proteins are employed as calibrators. The panel is designed to measure protein concentrations in fractionated cell extracts from flash frozen tumor biopsies. Results: We have completed development of three sandwich immunoassays for HK2, PK-M2, and LDHa on Luminex platform. Suitable capture antibodies as well as phycoerythrin labeled detection antibodies have been identified and tested for sandwich formation and multiplexing using recombinant calibrators and cell lysates. The multiplex is currently standardized for optimal assay performance. Known pharmacological modulators (3-bromopyruvate, bortezumib, oxamate, dichloroacetate) of HK2, PKM2, LDHa, and PDHE1a were used in vitro to demonstrate expected directional changes in biomarkers in the panel. Conclusions: We have developed a multiplex panel to measure protein levels of three key molecules involved in energy metabolism in cancer cells. The multiplex panel provides a critical tool, in the clinical setting, to support ongoing therapeutic strategies aimed at targeting the energy provision pathway in cancer patients. Importantly, the panel can be used to survey metabolic phenotype of different tumors to identify type and stage of cancer most susceptible to inhibition of glycolysis. Funded by NCI Contract No HHSN261200800001E. Citation Format: Meenakshi P. Balakrishnan, Ralph E. Parchment, Melinda G. Hollingshead, Joseph E. Tomaszewski, Robert J. Kinders, James H. Doroshow, Apurva K. Srivastava. Development of a multiplex panel of biomarkers to assess energy metabolism in cancer. [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 1858. doi:10.1158/1538-7445.AM2013-1858

Unilateral periventricular leukomalacia in association with pyruvate dehydrogenase deficiency

Pyruvate dehydrogenase (PDH) deficiency is a major cause of primary lactic acidosis and neurological dysfunction in infancy and early childhood. A deficiency of PDH E1 alpha, a subunit of the PDH complex, is a prominent cause of congenital lactic acidosis. We describe a female infant born at term and delivered by emergency Caesarean section because of fetal distress. There was no parental consanguinity. She presented at 5 months of age with failure to thrive, microcephaly, hypertonia, and developmental impairment. Her plasma and cerebrospinal fluid lactate were raised. She had raised plasma pyruvate with a normal lactate-pyruvate ratio. Magnetic resonance imaging of the brain showed a focal dilatation of the right lateral ventricle with unilateral periventricular leukomalacia (PVL) with subependymal cyst. Skin fibroblast culture assay revealed PDH deficiency, confirmed by mutation analysis of the E1 alpha subunit. At 18 months of age, she has hypertonia and global impairment and is making slow progress. Denver II assessment showed delay in gross motor, fine motor, adaptive, personal, social, and language categories. She has been treated with dichloroacetate and a ketogenic diet since the age of 10 and 13 months respectively, without any side effects. To our knowledge, unilateral PVL as a neuroradiological feature has not been described in children with PDH deficiency. PDH deficiency should be considered as a differential diagnosis if PVL is unilateral and if the perinatal history is not typical of PVL.

MRI Features of 4 Female Patients With Pyruvate Dehydrogenase E1 alpha Deficiency

Pyruvate dehydrogenase complex is a key intramitochondrial multienzyme complex required for the conversion of pyruvate to acetyl-CoA. Most patients with pyruvate dehydrogenase deficiency have a defect in the E1 alpha subunit, associated with mutations in the PDHA1 gene. In this report, we submit detailed magnetic resonance images in 4 affected female patients with PDHA1 mutations who had with severe cortical atrophy, dilated ventricles, and an incomplete corpus callosum. In one of these patients, the magnetic resonance imaging pattern prompted molecular diagnostic testing when enzymatic testing was normal. We underscore that this constellation of features, which may be misdiagnosed as periventricular leukomalacia, illustrates a pattern highly suggestive of a deficiency of pyruvate dehydrogenase E1 alpha in female patients and should trigger appropriate diagnostic investigations.

A quantitative mass spectrometry-based approach for identifying protein kinase clients and quantifying kinase activity

The Homo sapiens and Arabidopsis thaliana genomes are believed to encode more than 500 and 1000 protein kinases, respectively. Despite this abundance, few bona fide kinase-client relationships have been described in detail. Here we describe a quantitative mass spectrometry (MS)-based approach for identifying kinase-client proteins. During method development, we used the dedicated kinase pyruvate dehydrogenase kinase (PDK) for the in vitro assays. As kinase substrate, we used synthetic peptide cocktails and, in the process, demonstrated that the assay is both sensitive and specific. The method is also useful for characterizing protein kinase-substrate kinetics once the peptide substrate is detected. Applying a label-free spectral counting method, the activity of PDK was determined using the peptide substrate YHGH(292)SMSDPGSTYR derived from the pyruvate dehydrogenase E1alpha subunit sequence. The utility of spectral counting was further validated by studying the negative effect of Met oxidation on peptide phosphorylation. We also measured the activity of the unrelated calcium-dependent protein kinase 3 (CPK3), demonstrating the utility of the method in protein kinase screening applications.