Mitochondrial Pyruvate Dehydrogenase Research Articles

Pyruvate dehydrogenase kinase inhibition: Reversing the Warburg effect in cancer therapy

The poor efficacy of many cancer chemotherapeutics, which are often non-selective and highly toxic, is attributable to the remarkable heterogeneity and adaptability of cancer cells. The Warburg effect describes the up regulation of glycolysis as the main source of adenosine 5’-triphosphate in cancer cells, even under normoxic conditions, and is a unique metabolic phenotype of cancer cells. Mitochondrial suppression is also observed which may be implicated in apoptotic suppression and increased funneling of respiratory substrates to anabolic processes, conferring a survival advantage. The mitochondrial pyruvate dehydrogenase complex is subject to meticulous regulation, chiefly by pyruvate dehydrogenase kinase. At the interface between glycolysis and the tricarboxylic acid cycle, the pyruvate dehydrogenase complex functions as a metabolic gatekeeper in determining the fate of glucose, making pyruvate dehydrogenase kinase an attractive candidate in a bid to reverse the Warburg effect in cancer cells. The small pyruvate dehydrogenase kinase inhibitor dichloroacetate has, historically, been used in conditions associated with lactic acidosis but has since gained substantial interest as a potential cancer chemotherapeutic. This review considers the Warburg effect as a unique phenotype of cancer cells in-line with the history of and current approaches to cancer therapies based on pyruvate dehydrogenase kinase inhibition with particular reference to dichloroacetate and its derivatives.

Lack of mitochondria-generated acetyl-CoA by pyruvate dehydrogenase complex downregulates gene expression in the hepatic de novo lipogenic pathway.

During the absorptive state, the liver stores excess glucose as glycogen and synthesizes fatty acids for triglyceride synthesis for export as very low density lipoproteins. For de novo synthesis of fatty acids from glucose, the mitochondrial pyruvate dehydrogenase complex (PDC) is the gatekeeper for the generation of acetyl-CoA from glucose-derived pyruvate. Here, we tested the hypothesis that limiting the supply of PDC-generated acetyl-CoA from glucose would have an impact on expression of key genes in the lipogenic pathway. In the present study, although the postnatal growth of liver-specific PDC-deficient (L-PDCKO) male mice was largely unaltered, the mice developed hyperinsulinemia with lower blood glucose levels in the fed state. Serum and liver lipid triglyceride and cholesterol levels remained unaltered in L-PDCKO mice. Expression of several key genes (ACL, ACC1) in the lipogenic pathway and their upstream regulators (LXR, SREBP1, ChREBP) as well as several genes in glucose metabolism (Pklr, G6pd2, Pck1) and fatty acid oxidation (FAT, Cpt1a) was downregulated in livers from L-PDCKO mice. Interestingly, there was concomitant upregulation of lipogenic genes in adipose tissue from L-PDCKO mice. Although, the total hepatic acetyl-CoA content remained unaltered in L-PDCKO mice, modified acetylation profiles of proteins in the nuclear compartment suggested an important role for PDC-generated acetyl-CoA in gene expression in de novo fatty acid synthesis in the liver. This finding has important implications for the regulation of hepatic lipid synthesis in pathological states.

Requirements for Carnitine Shuttle-Mediated Translocation of Mitochondrial Acetyl Moieties to the Yeast Cytosol.

ABSTRACTIn many eukaryotes, the carnitine shuttle plays a key role in intracellular transport of acyl moieties. Fatty acid-grown Saccharomyces cerevisiae cells employ this shuttle to translocate acetyl units into their mitochondria. Mechanistically, the carnitine shuttle should be reversible, but previous studies indicate that carnitine shuttle-mediated export of mitochondrial acetyl units to the yeast cytosol does not occur in vivo. This apparent unidirectionality was investigated by constitutively expressing genes encoding carnitine shuttle-related proteins in an engineered S. cerevisiae strain, in which cytosolic acetyl coenzyme A (acetyl-CoA) synthesis could be switched off by omitting lipoic acid from growth media. Laboratory evolution of this strain yielded mutants whose growth on glucose, in the absence of lipoic acid, was l-carnitine dependent, indicating that in vivo export of mitochondrial acetyl units to the cytosol occurred via the carnitine shuttle. The mitochondrial pyruvate dehydrogenase complex was identified as the predominant source of acetyl-CoA in the evolved strains. Whole-genome sequencing revealed mutations in genes involved in mitochondrial fatty acid synthesis (MCT1), nuclear-mitochondrial communication (RTG2), and encoding a carnitine acetyltransferase (YAT2). Introduction of these mutations into the nonevolved parental strain enabled l-carnitine-dependent growth on glucose. This study indicates intramitochondrial acetyl-CoA concentration and constitutive expression of carnitine shuttle genes as key factors in enabling in vivo export of mitochondrial acetyl units via the carnitine shuttle.

Mitochondria‐Generated Acetyl‐CoA Regulates Gene Expression in Hepatic Lipid Synthesis

During the absorptive state the liver stores excess glucose as glycogen and synthesizes fatty acids for triglyceride synthesis for export as very low density lipoproteins. For de novo synthesis of fatty acids from glucose, the mitochondrial pyruvate dehydrogenase complex (PDC) plays a gatekeeper's role for generation of the carbon‐flux as acetyl‐CoA. We have shown previously that there was no incorporation of radioactive glucose‐carbon into newly synthesized fatty acids in livers from liver‐specific PDC‐deficient male mice (L‐PDHKO) due to the null mutation of the Pdha1 gene. In the present study, although the postnatal growth of L‐PDHKO mice was largely unaltered, they developed hyperinsulinemia with lower blood glucose levels in the fed state. Serum and liver lipid triglyceride and cholesterol levels remained unaltered in L‐PDHKO mice. To determine the effect of liver‐specific PDC deficiency on lipid and glucose metabolism, we investigated expression of several key genes as well as their upstream regulators in lipid and glucose metabolism. Interestingly, there was significant downregulation of expression of several key genes (such as ATP‐citrate lyase, acetyl‐CoA carboxylase) in the de novo fatty acid synthesis pathway and their upstream regulators (such as LXR, SREBP1, ChREBP). Downregulation of several genes in the metabolism of glucose (glucose‐6‐phosphate dehydrogenase, pyruvate kinase, phosphoenolpyruvate carboxykinase) and fatty acid oxidation (fatty acid transporter, carnitine‐palmitoyl‐CoA transferase‐1) were also observed in livers of L‐PDHKO mice. Interestingly, there was upregulation of lipogenic genes in epididymal adipose tissue from L‐PDHKO mice. Although, the total hepatic acetyl‐CoA content remained unaltered in these mice, modified acetylation profiles of several proteins in the nucleo‐cytoplasmic compartment (including increased level of acetylated histone3lysine9, H3K9) in livers of L‐PDHKO mice suggest an important role of mitochondria‐generated acetyl‐CoA from glucose‐derived pyruvate by PDC in gene expression in hepatic lipid metabolism. This finding has important implications for regulation of hepatic lipid biosynthesis in obesity, type 2 diabetes and non‐alcoholic fatty liver disease.Support or Funding Information(Supported in part by NIH Grant DK20478 and Department Fund, The Research Foundation of The State University of New York.)

MicroRNA-129-5p, a c-Myc negative target, affects hepatocellular carcinoma progression by blocking the Warburg effect.

Deregulation of microRNAs (miRNAs) and c-Myc (Myc) contributes to hepatocellular carcinoma (HCC) progression, but how miRNAs and Myc regulate each other in hepatocarcinogenesis is still poorly understood. Using a functional screen, we identified miR-129-5p as a miRNA that inhibits HCC cell growth. miR-129-5p targets the mitochondrial matrix protein pyruvate dehydrogenase kinase 4 (PDK4), which leads to decreased phosphorylation of the E1α subunit of pyruvate dehyrogenase (PDH) complex, inhibition of glycolysis, retarded tumor growth, and impaired lung colonization. Enforced expression of PDK4 refractory to inhibition by miR-129-5p rescued all of these phenotypes. Targeting PDK4 by shRNA recapitulated the effects caused by miR-129-5p. miR-129-5p is transcriptionally repressed by a complex comprised of Myc, histone deacetylase 3 (HDAC3), and enhancer of zeste 2 polycomb repressive complex 2 (EZH2). Levels of miR-129-5p negatively correlated with clinical stages in human HCC. Restoring miR-129-5p expression suppressed the diethylnitrosamine (DEN)-induced hepatocarcinogenesis in mice. Thus, we concluded that miR-129-5p, which is a negative target of Myc, blocks glycolysis to retard hepatocarcinogenesis via targeting PDK4. The critical link between miR-129-5p and PDK4 in the progression of HCC suggests potential points of therapeutic intervention for this disease.

Age- and brain region-specific differences in mitochondrial bioenergetics in Brown Norway rats

Mitochondria are central regulators of energy homeostasis and play a pivotal role in mechanisms of cellular senescence. The objective of the present study was to evaluate mitochondrial bioenergetic parameters in 5 brain regions (brain stem [BS], frontal cortex, cerebellum, striatum, hippocampus [HIP]) of 4 diverse age groups (1 month [young], 4 months [adult], 12 months [middle-aged], 24 months [old age]) to understand age-related differences in selected brain regions and their possible contribution to age-related chemical sensitivity. Mitochondrial bioenergetic parameters and enzyme activities were measured under identical conditions across multiple age groups and brain regions in Brown Norway rats (n = 5/group). The results indicate age- and brain region–specific patterns in mitochondrial functional endpoints. For example, an age-specific decline in ATP synthesis (State III respiration) was observed in BS and HIP. Similarly, the maximal respiratory capacities (State V1 and V2) showed age-specific declines in all brain regions examined (young > adult > middle-aged > old age). Amongst all regions, HIP had the greatest change in mitochondrial bioenergetics, showing declines in the 4, 12, and 24-months age groups. Activities of mitochondrial pyruvate dehydrogenase complex and electron transport chain complexes I, II, and IV enzymes were also age and brain region specific. In general, changes associated with age were more pronounced with enzyme activities declining as the animals aged (young > adult > middle-aged > old age). These age- and brain region-specific observations may aid in evaluating brain bioenergetic impact on the age-related susceptibility to environmental chemical stressors.

Increased mtPDH Activity Through Antisense Inhibition of Mitochondrial Pyruvate Dehydrogenase Kinase Enhances Inflorescence Initiation, and Inflorescence Growth and Harvest Index at Elevated CO2 in Arabidopsis thaliana.

Mitochondrial pyruvate dehydrogenase (mtPDH) is a key respiratory enzyme that links glycolysis and the tricarboxylic acid cycle, and it is negatively regulated by mtPDH kinase (mtPDHK). Arabidopsis lines carrying either a constitutive or seed-specific antisense construct for mtPDHK were used to test the hypothesis that alteration of mtPDH activity in a tissue- and dosage-dependent manner will enhance reproductive growth particularly at elevated CO2 (EC) through a combined enhancement of source and sink activities. Constitutive transgenic lines showed increased mtPDH activity in rosette leaves at ambient CO2 (AC) and EC, and in immature seeds at EC. Seed-specific transgenic lines showed enhanced mtPDH activity in immature seeds. A strong relationship existed between seed mtPDH activity and inflorescence initiation at AC, and at EC inflorescence stem growth, silique number and seed harvest index were strongly related to seed mtPDH activity. Leaf photosynthetic rates showed an increase in rosette leaves of transgenic lines at AC and EC that correlated with enhanced inflorescence initiation. Collectively, the data show that mtPDHK plays a key role in regulating sink and source activities in Arabidopsis particularly during the reproductive phase.

Primary biliary cholangitis in China.

Primary biliary cholangitis (PBC) is a chronic cholestatic liver disease characterized by immunomediated destruction of small and medium-sized intrahepatic bile ducts. In 1987, a cDNA for a 74 kDa mitochondrial autoantigen was cloned and identified as the E2 component of the mitochondrial pyruvate dehydrogenase complex, which improved the diagnosis and changed research directions in this field. In 1958, the first Chinese case of PBC was reported. But until 1990, a comprehensive description of the characteristics of Chinese PBC patients was published. In China we now know that PBC is not rare and usually does not progress to cirrhosis. The number of Chinese patients with PBC has increased each and every year. This increase may be associated with the changes of liver disease spectrum, the application of convenient autoantibody detection kits, and the comprehensive understanding of the disease. It may also reflect, however, a westernization change in environmental features with China. There is now significant and important basic and clinical research on PBC in China, with major contributions in diagnostic criteria, treatment, and on basic biology. This has led to exciting proposals based on Chinese PBC cohorts. Chinese hepatologists and scientists are now focusing their efforts on PBC. These efforts have led to new diagnostic biomarkers, novel therapeutic methods (stem cells and Chinese traditional medicine), and unique immunological mechanisms, including roles for T-follicular helper cells and monocyte subpopulations, both of which are involved in the breach of immune tolerance for PBC.

Xenobiotics and loss of tolerance in primary biliary cholangitis.

Data from genome wide association studies and geoepidemiological studies established that a combination of genetic predisposition and environmental stimulation is required for the loss of tolerance in primary biliary cholangitis (PBC). The serologic hallmark of PBC are the presence of high titer anti-mitochondrial autoantibodies (AMA) that recognize the lipoyl domain of the mitochondrial pyruvate dehydrogenase E2 (PDC-E2) subunit. Extensive efforts have been directed to investigate the molecular basis of AMA. Recently, experimental data has pointed to the thesis that the breaking of tolerance to PDC-E2 is a pivotal event in the initial etiology of PBC, including environmental xenobiotics including those commonly found in cosmetics and food additives, suggesting that chemical modification of the PDC-E2 epitope may render its vulnerable to become a neo-antigen and trigger an immune response in genetically susceptible hosts. Here, we will discuss the natural history, genetics and immunobiology of PBC and structural constraints of PDC-E2 in AMA recognition which makes it vulnerable to chemical modification.

Leigh syndrome: Resolving the clinical and genetic heterogeneity paves the way for treatment options.

Leigh syndrome is a progressive neurodegenerative disorder, affecting 1 in 40,000 live births. Most patients present with symptoms between the ages of three and twelve months, but adult onset Leigh syndrome has also been described. The disease course is characterized by a rapid deterioration of cognitive and motor functions, in most cases resulting in death due to respiratory failure. Despite the high genetic heterogeneity of Leigh syndrome, patients present with identical, symmetrical lesions in the basal ganglia or brainstem on MRI, while additional clinical manifestations and age of onset varies from case to case. To date, mutations in over 60 genes, both nuclear and mitochondrial DNA encoded, have been shown to cause Leigh syndrome, still explaining only half of all cases. In most patients, these mutations directly or indirectly affect the activity of the mitochondrial respiratory chain or pyruvate dehydrogenase complex. Exome sequencing has accelerated the discovery of new genes and pathways involved in Leigh syndrome, providing novel insights into the pathophysiological mechanisms. This is particularly important as no general curative treatment is available for this devastating disorder, although several recent studies imply that early treatment might be beneficial for some patients depending on the gene or process affected. Timely, gene-based personalized treatment may become an important strategy in rare, genetically heterogeneous disorders like Leigh syndrome, stressing the importance of early genetic diagnosis and identification of new genes/pathways. In this review, we provide a comprehensive overview of the most important clinical manifestations and genes/pathways involved in Leigh syndrome, and discuss the current state of therapeutic interventions in patients.

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

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

Effects of orexin A on glucose metabolism in human hepatocellular carcinoma in vitro via PI3K/Akt/mTOR-dependent and -independent mechanism

Orexins are hypothalamic neuropeptides that regulate food intake, energy homeostasis, reward system and sleep/wakefulness states. The purpose of this study was to investigate the effects of orexin A on glucose metabolism in human hepatocellular carcinoma cell line, Hep3B, and determine the possible mechanisms. Hep3B cells were incubated with different concentrations of orexin A (10−9–10−7 M) in vitro in the presence or absence of the orexin receptor 1 (OX1R) inhibitor (SB334867), Akt inhibitor (PF-04691502) and mammalian target of rapamycin (mTOR) inhibitor (temsirolimus). Subsequently, OX1R protein expression, glucose transporter 1 (GLUT1) expression, glucose uptake, the mRNA expression of lactate dehydrogenase (LDHA), pyruvate dehydrogenase kinase 1 (PDK1) and pyruvate dehydrogenase B (PDHB), lactate generation and mitochondrial pyruvate dehydrogenase (PDH) enzyme activity were measured. The activity of phosphoinositide 3-kinase (PI3K)/Akt/mTOR signaling was also determined. OX1R was expressed in hepatoma tissues and Hep3B cells. Stimulation of the Hep3B cells with orexin A resulted in a dose-dependent increase of GLUT1 expression and glucose uptake, which was associated with the activation of PI3K/Akt/mTOR pathway. Further, orexin A increased PDHB expression and PDH enzyme activity, decreased LDHA, PDK1 mRNA levels and lactate generation independent of PI3K/Akt/mTOR pathway. Our results demonstrated that orexin A directed the cellular metabolism towards mitochondrial glucose oxidation rather than glycolysis. These findings provide functional evidence of the metabolic actions of orexin A in hepatocellular carcinoma cells.

Metabolic Connection of Inflammatory Pain: Pivotal Role of a Pyruvate Dehydrogenase Kinase-Pyruvate Dehydrogenase-Lactic Acid Axis

Pyruvate dehydrogenase kinases (PDK1-4) are mitochondrial metabolic regulators that serve as decision makers via modulation of pyruvate dehydrogenase (PDH) activity to convert pyruvate either aerobically to acetyl-CoA or anaerobically to lactate. Metabolic dysregulation and inflammatory processes are two sides of the same coin in several pathophysiological conditions. The lactic acid surge associated with the metabolic shift has been implicated in diverse painful states. In this study, we investigated the role of PDK-PDH-lactic acid axis in the pathogenesis of chronic inflammatory pain. Deficiency of Pdk2 and/or Pdk4 in mice attenuated complete Freund's adjuvant (CFA)-induced pain hypersensitivities. Likewise, Pdk2/4 deficiency attenuated the localized lactic acid surge along with hallmarks of peripheral and central inflammation following intraplantar administration of CFA. In vitro studies supported the role of PDK2/4 as promoters of classical proinflammatory activation of macrophages. Moreover, the pharmacological inhibition of PDKs or lactic acid production diminished CFA-induced inflammation and pain hypersensitivities. Thus, a PDK-PDH-lactic acid axis seems to mediate inflammation-driven chronic pain, establishing a connection between metabolism and inflammatory pain. The mitochondrial pyruvate dehydrogenase (PDH) kinases (PDKs) and their substrate PDH orchestrate the conversion of pyruvate either aerobically to acetyl-CoA or anaerobically to lactate. Lactate, the predominant end product of glycolysis, has recently been identified as a signaling molecule for neuron-glia interactions and neuronal plasticity. Pathological metabolic shift and subsequent lactic acid production are thought to play an important role in diverse painful states; however, their contribution to inflammation-driven pain is still to be comprehended. Here, we report that the PDK-PDH-lactic acid axis constitutes a key component of inflammatory pain pathogenesis. Our findings establish an unanticipated link between metabolism and inflammatory pain. This study unlocks a previously ill-explored research avenue for the metabolic control of inflammatory pain pathogenesis.

In recurrent primary biliary cirrhosis after liver transplantation, biliary epithelial cells show increased expression of mitochondrial proteins.

In biliary epithelial lesions in primary biliary cirrhosis (PBC), mitochondrial proteins associated with deregulated autophagy are abnormally expressed. We examined whether this could be used as a diagnostic marker for end-stage PBC and recurrent PBC after liver transplantation. We examined the expression of the mitochondrial protein pyruvate dehydrogenase complex-E2 component and cytochrome c oxidase, subunit I (CCO), the autophagy-related marker microtubule-associated protein-light chain 3 (LC3), and p62/sequestosome-1 and the senescence markers p16(Ink4a) and p21(WAF1/Cip1) in small bile ducts and bile ductules in explanted livers from patients with PBC (n = 20) in comparison with liver tissue from control patients (n = 21) and post-transplant samples including recurrent PBC and cellular rejection (n = 28). Intense granular expression of mitochondrial proteins was significantly more frequent in small bile ducts in explanted livers with PBC than in control livers (p < 0.05). Post-transplant samples comprised of three groups: group A (positive for mitochondrial proteins, n = 7), group B (positive for either autophagy-related or senescence markers but negative for mitochondrial proteins, n = 7), and group C (all negative, n = 14). All but one case of group A were clinically and histologically diagnosed as recurrent PBC. In contrast, all cases of group B were diagnosed as cellular rejection. This study suggests that the expression of mitochondrial proteins in small bile ducts may be a useful diagnostic marker for end-stage PBC and recurrent PBC after liver transplantation.

Mutations in the three Arabidopsis genes that encode the E2 subunit of the mitochondrial pyruvate dehydrogenase complex differentially affect enzymatic activity and plant growth.

Three members of the E2 subunit of the Arabidopsis mitochondrial pyruvate dehydrogenase (mtPDC) complex differentially affect mtPDC activity and plant growth. In all organisms, the mitochondrial pyruvate dehydrogenase complex (mtPDC) consists of three components: E1, E2, and E3. In this multi-enzyme complex, the E2 subunits form the core structure. In Arabidopsis, the E2 subunits are encoded by three genes: mtE2-1, mtE2-2, and mtE2-3. The contribution of each mtE2 gene to total mtPDC activity, however, is unknown. In this study, we show that knockdown of the expression of the mtE2-1 gene to 17% of that in the wild type has only a slight effect on plant growth whereas knockout of mtE2-2 leads to an embryo-lethal phenotype. The nearly null mutation of mtE2-3 does not cause any developmental abnormality. Based on these results, we conclude that mtE2-2 plays a major role in determining the total activity of the mtPDC in Arabidopsis while mtE2-1 and mtE2-3 are more or less functionally redundant with mtE2-2 under normal growth condition. Our results provide genetic evidence for a recently proposed novel mechanism that regulates plant mtPDC activity.

Maternal stress predicts altered biogenesis and the profile of mitochondrial proteins in the frontal cortex and hippocampus of adult offspring rats

Currently, much attention is focused on the influence of mitochondrial disturbances at the onset of depression. The goal of this study was to investigate the impact of prenatal stress (an animal model of depression) on the mitochondrial biogenesis proteins and mitoproteome profile in the frontal cortex and hippocampus of adult 3-month-old male rats following a prenatal stress procedure.Our results show that rats that were exposed to prenatal stress stimuli displayed depression-like behaviors based on the sucrose preference and elevated plus maze tests. It has been found that the level of the PGC-1α protein was reduced in the frontal cortex and hippocampus of the adult offspring after the prenatal stress procedure. Moreover, in the frontal cortex, the level of the pro-apoptotic protein Bax was up-regulated. Two-dimensional electrophoresis coupled with mass spectrometry showed the statistically significant down-regulation of the mitochondrial ribosomal protein L12 (Mrpl12) and mitochondrial NADH dehydrogenase [ubiquinone] flavoprotein 2 (NDUFV2) as well as the up-regulation of the Tubulin Polymerization Promoting Proteins (Tppp/p25) in the frontal cortex. In contrast, in the hippocampus, the mitochondrial pyruvate dehydrogenase E1 component subunit beta, the voltage-dependent anion-selective channel protein 2 (VDAC2), and the GTP-binding nuclear protein RAN (RAN) were down-regulated and the expression of phosphatidylethanolamine-binding protein 1 (PEBP-1) was enhanced. These findings provide new evidence that stress during pregnancy may lead not only to behavioral deficits, but also to disturbances in the brain mitoproteome profile in adult rat offspring.

Identifying New Tumor Suppressor in Liver Cancer Using Unbiased High‐Throughput Approaches

PurposeTumor suppressor genes are often epigenetically silenced. This study identified a new tumor suppressor in hepatocellular cancer (HCC) using an unbiased high throughput approach.Methods/ResultsStudies from our laboratory has established nuclear receptor small heterodimer partner SHP as a tumor suppressor. We also established an inhibitory feedback regulatory loop between SHP and DNA methyltransferases (Dnmts). Methylated DNA‐IP‐on‐ChIP (MeDIP) revealed alterations in DNA methylation and RNA‐sequencing (RNA‐seq) revealed changes in gene expression. By integrating both studies using Bioinformatics tools and appropriate validation, we identified the mitochondrial protein pyruvate dehydrogenase kinase 4 (PDK4) as a putative tumor suppressor. PDK4 promoter was hypermethyalted and its expression downregualted in SHP‐/‐ mice. PDK4 was also silenced in human HCC. Treatment of HCC cell lines with the demethylating agent 5'‐aza ‐2'‐deoxycytidine (Aza) or histone deacetylase inhibitor trichostatin (TSA) induced PDK4 expression. The number of foci was decreased and the tumor sizes were reduced in HCC cells that stably overexpressing PDK4, whereas the opposite was found with PDK4 knockdown. PDK4 overexpression sensitized HCC cells to cell death induced by glucose deprivation by reducing the intracellular ATP contents.ConclusionsEpigenetic silencing of PDK4 is associated with the development of HCC.[Grant support] NIH DK080440, VA Merit Award 1I01BX002634.

GRP78 is implicated in the modulation of tumor aerobic glycolysis by promoting autophagic degradation of IKKβ

Compared with normal differentiated cells, cancer cells take up much more glucose and metabolize it mainly via aerobic glycolysis. This metabolic phenotype is characterized with high expression of glucose transporters (Gluts) and pyruvate kinase M2 (PKM2). Glucose regulated protein 78 (GRP78) is a glucose-sensing protein and frequently up-regulated in cancer cells, however, whether it is directly implicated in glucose metabolism remains to be elucidated. Here we report that upon glucose deficiency, the induction of GRP78 resulted in enhanced HIF-1α transcription, accompanied by a transient increased expression of Glut-1. In addition, GRP78 was likely to facilitate the membrane translocation of Glut-1 via protein–protein interaction. Glucose starvation-stimulated GRP78 also impaired the expression of PKM2 but promoted the expression of mitochondrial pyruvate dehydrogenase A (PDHA) and B (PDHB), resulting in the metabolic shift from glycolysis to the TCA cycle. Interestingly, the inhibition of PKM2 by GRP78 was abrogated when glucose supply was restored, suggesting that GRP78 and PKM2 expressions are adaptable to the nutritional levels in the microenvironment. Further mechanistic study indicated that GRP78 overexpression activated the Class III PI3K-mediated autophagy pathway and induced autophagic degradation of IKKβ, which caused inactivation of NF-κB pathway and subsequently altered the expression of PKM2 and HIF-1α. Our study establishes GRP78 and PKM2 as the crucial molecular links between cancer cell glucose metabolism and tumor microenvironment alterations.

A comprehensive proteomic approach to identifying capacitation related proteins in boar spermatozoa.

BackgroundMammalian spermatozoa must undergo capacitation, before becoming competent for fertilization. Despite its importance, the fundamental molecular mechanisms of capacitation are poorly understood. Therefore, in this study, we applied a proteomic approach for identifying capacitation-related proteins in boar spermatozoa in order to elucidate the events more precisely. 2-DE gels were generated from spermatozoa samples in before- and after-capacitation. To validate the 2-DE results, Western blotting and immunocytochemistry were performed with 2 commercially available antibodies. Additionally, the protein-related signaling pathways among identified proteins were detected using Pathway Studio 9.0.ResultWe identified Ras-related protein Rab-2, Phospholipid hydroperoxide glutathione peroxidase (PHGPx) and Mitochondrial pyruvate dehydrogenase E1 component subunit beta (PDHB) that were enriched before-capacitation, and NADH dehydrogenase 1 beta subcomplex 6, Mitochondrial peroxiredoxin-5, (PRDX5), Apolipoprotein A-I (APOA1), Mitochondrial Succinyl-CoA ligase [ADP-forming] subunit beta (SUCLA2), Acrosin-binding protein, Ropporin-1A, and Spermadhesin AWN that were enriched after-capacitation (>3-fold) by 2-DE and ESI-MS/MS. SUCLA2 and PDHB are involved in the tricarboxylic acid cycle, whereas PHGPx and PRDX5 are involved in glutathione metabolism. SUCLA2, APOA1 and PDHB mediate adipocytokine signaling and insulin action. The differentially expressed proteins following capacitation are putatively related to sperm functions, such as ROS and energy metabolism, motility, hyperactivation, the acrosome reaction, and sperm-egg interaction.ConclusionThe results from this study elucidate the proteins involved in capacitation, which may aid in the design of biomarkers that can be used to predict boar sperm quality.

Chronic PFOS exposure alters the expression of neuronal development-related human homologues in Eisenia fetida

PFOS is a toxic, persistent environmental pollutant which is widespread worldwide. PFOS contamination has entered the food chain and is interfering with normal development in man and is neurotoxic, hepatotoxic and tumorigenic. The earthworm, Eisenia fetida is one of the organisms which can help to diagnose soil health and contamination at lower levels in the food chain. Studying the chronic effects of sub-lethal PFOS exposure in such an organism is therefore appropriate. As PFOS bioaccumulates and is not easily biodegraded, it is biomagnified up the food chain. Gene expression studies will give us information to develop biomarkers for early diagnosis of soil contamination, well before this contaminant passes up the food chain. We have carried out mRNA sequencing of control and chronically PFOS exposed E. fetida and reconstructed the transcripts in silico and identified the differentially expressed genes. Our findings suggest that PFOS up/down regulates neurodegenerative-related human homologues and can cause neuronal damage in E. fetida. This information will help to understand the links between neurodegenerative disorders and environmental pollutants such as PFOS. Furthermore, these up/down regulated genes can be used as biomarkers to detect a sub-lethal presence of PFOS in soil. Neuronal calcium sensor-2, nucleoside diphosphate kinase, polyadenylate-binding protein-1 and mitochondrial Pyruvate dehydrogenase protein-X component, could be potential biomarkers for sub lethal concentrations of PFOS.