Showing papers on "Pyruvate dehydrogenase kinase published in 2001"
TL;DR: It is concluded that β‐amyloid can directly disrupt mitochondrial function, inhibits key enzymes and may contribute to the deficiency of energy metabolism seen in Alzheimer's disease.
Abstract: Disrupted energy metabolism, in particular reduced activity of cytochrome oxidase (EC 1.9.3.1), alpha-ketoglutarate dehydrogenase (EC 1.2.4.2) and pyruvate dehydrogenase (EC 1.2.4.1) have been reported in post-mortem Alzheimer's disease brain. beta-Amyloid is strongly implicated in Alzheimer's pathology and can be formed intracellularly in neurones. We have investigated the possibility that beta-amyloid itself disrupts mitochondrial function. Isolated rat brain mitochondria have been incubated with the beta-amyloid alone or together with nitric oxide, which is known to be elevated in Alzheimer's brain. Mitochondrial respiration, electron transport chain complex activities, alpha-ketoglutarate dehydrogenase activity and pyruvate dehydrogenase activity have been measured. Beta-amyloid caused a significant reduction in state 3 and state 4 mitochondrial respiration that was further diminished by the addition of nitric oxide. Cytochrome oxidase, alpha-ketoglutarate dehydrogenase and pyruvate dehydrogenase activities were inhibited by beta-amyloid. The K(m) of cytochrome oxidase for reduced cytochrome c was raised by beta-amyloid. We conclude that beta-amyloid can directly disrupt mitochondrial function, inhibits key enzymes and may contribute to the deficiency of energy metabolism seen in Alzheimer's disease.
454 citations
TL;DR: The close similarity of these crystal structures suggests the distinctive activity of these enzyme isoforms is likely to result directly from variation of charged surface residues peripheral to the active site, a hypothesis supported by electrostatic calculations based on each structure.
Abstract: Lactate dehydrogenase (LDH) interconverts pyruvate and lactate with concomitant interconversion of NADH and NAD(+). Although crystal structures of a variety of LDH have previously been described, a notable absence has been any of the three known human forms of this glycolytic enzyme. We have now determined the crystal structures of two isoforms of human LDH-the M form, predominantly found in muscle; and the H form, found mainly in cardiac muscle. Both structures have been crystallized as ternary complexes in the presence of the NADH cofactor and oxamate, a substrate-like inhibitor. Although each of these isoforms has different kinetic properties, the domain structure, subunit association, and active-site regions are indistinguishable between the two structures. The pK(a) that governs the K(M) for pyruvate for the two isozymes is found to differ by about 0.94 pH units, consistent with variation in pK(a) of the active-site histidine. The close similarity of these crystal structures suggests the distinctive activity of these enzyme isoforms is likely to result directly from variation of charged surface residues peripheral to the active site, a hypothesis supported by electrostatic calculations based on each structure. Proteins 2001;43:175-185.
259 citations
TL;DR: The mammalian pyruvate dehydrogenase complex (PDC) plays central and strategic roles in the control of the use of glucose-linked substrates as sources of oxidative energy or as precursors in the biosynthesis of fatty acids.
Abstract: The mammalian pyruvate dehydrogenase complex (PDC) plays central and strategic roles in the control of the use of glucose-linked substrates as sources of oxidative energy or as precursors in the biosynthesis of fatty acids The activity of this mitochondrial complex is regulated by the continuous operation of competing pyruvate dehydrogenase kinase (PDK) and pyruvate dehydrogenase phosphatase (PDP) reactions The resulting interconversion cycle determines the fraction of active (nonphosphorylated) pyruvate dehydrogenase (E1) component Tissue-specific and metabolic state-specific control is achieved by the selective expression and distinct regulatory properties of at least four PDK isozymes and two PDP isozymes The PDK isoforms are members of a family of serine kinases that are not structurally related to cytoplasmic Ser/Thr/Tyr kinases The catalytic subunits of the PDP isoforms are Mg2+-dependent members of the phosphatase 2C family that has binuclear metal-binding sites within the active site The dihydrolipoyl acetyltransferase (E2) and the dihydrolipoyl dehydrogenase-binding protein (E3BP) are multidomain proteins that form the oligomeric core of the complex One or more of their three lipoyl domains (two in E2) selectively bind each PDK and PDP1 These adaptive interactions predominantly influence the catalytic efficiencies and effector control of these regulatory enzymes When fatty acids are the preferred source of acetyl-CoA and NADH, feedback inactivation of PDC is accomplished by the activity of certain kinase isoforms being stimulated upon preferentially binding a lipoyl domain containing a reductively acetylated lipoyl group PDC activity is increased in Ca2+-sensitive tissues by elevating PDP1 activity via the Ca2+-dependent binding of PDP1 to a lipoyl domain of E2 During starvation, the irrecoverable loss of glucose carbons is restricted by minimizing PDC activity due to high kinase activity that results from the overexpression of specific kinase isoforms Overexpression of the same PDK isoforms deleteriously hinders glucose consumption in unregulated diabetes
251 citations
TL;DR: Site specificity for phosphorylation of four PDKs with unique tissue distribution could contribute to the tissue-specific regulation of the pyruvate dehydrogenase complex in normal and pathophysiological states.
218 citations
TL;DR: The functional importance of PPARalpha for PDK4 expression during starvation is documented and an important role for elevated free fatty acids in the induction is suggested.
206 citations
TL;DR: Results reported here strongly suggest that the major determinants of the activity state of pyruvate dehydrogenase in mammalian tissues include the phosphorylation site specificity of isoenzymes of Kinase in addition to the absolute amounts of kinase and phosphatase protein expressed in mitochondria.
Abstract: The enzymic activity of the mammalian pyruvate dehydrogenase complex is regulated by the phosphorylation of three serine residues (sites 1, 2 and 3) located on the E1 component of the complex. Here we report that the four isoenzymes of protein kinase responsible for the phosphorylation and inactivation of pyruvate dehydrogenase (PDK1, PDK2, PDK3 and PDK4) differ in their abilities to phosphorylate the enzyme. PDK1 can phosphorylate all three sites, whereas PDK2, PDK3 and PDK4 each phosphorylate only site 1 and site 2. Although PDK2 phosphorylates site 1 and 2, it incorporates less phosphate in site 2 than PDK3 or PDK4. As a result, the amount of phosphate incorporated by each isoenzyme decreases in the order PDK1>PDK3>or=PDK4>PDK2. Significantly, binding of the coenzyme thiamin pyrophosphate to pyruvate dehydrogenase alters the rates and stoichiometries of phosphorylation of the individual sites. First, the rate of phosphorylation of site 1 by all isoenzymes of kinase is decreased. Secondly, thiamin pyrophosphate markedly decreases the amount of phosphate that PDK1 incorporates in sites 2 and 3 and that PDK2 incorporates in site 2. In contrast, the coenzyme does not significantly affect the total amount of phosphate incorporated in site 2 by PDK3 and PDK4, but instead decreases the rate of phosphorylation of this site. Furthermore, pyruvate dehydrogenase complex phosphorylated by the individual isoenzymes of kinase is reactivated at different rates by pyruvate dehydrogenase phosphatase. Both isoenzymes of phosphatase (PDP1 and PDP2) readily reactivate the complex phosphorylated by PDK2. When pyruvate dehydrogenase is phosphorylated by other isoenzymes, the rates of reactivation decrease in the order PDK4>or=PDK3>PDK1. Taken together, results reported here strongly suggest that the major determinants of the activity state of pyruvate dehydrogenase in mammalian tissues include the phosphorylation site specificity of isoenzymes of kinase in addition to the absolute amounts of kinase and phosphatase protein expressed in mitochondria.
206 citations
TL;DR: This review summarizes the recent developments on the regulation of human pyruvate dehydrogenase complex (PDC) by site-specific phosphorylation by four kinases by noting the presence of the multiple phosphorylated sites and isoenzymes of PDK is important for the tissue-specific regulation of PDC under different physiological conditions.
Abstract: This review summarizes the recent developments on the regulation of human pyruvate dehydrogenase complex (PDC) by site-specific phosphorylation by four kinases. Mutagenic analysis of the three phosphorylation sites of human pyruvate dehydrogenase (E1) showed the site-independent mechanism of phosphorylation as well as site-independent dephosphorylation of the three phosphorylation sites and the importance of each phosphorylation site for the inactivation of E1. Both the negative charge and size of the group introduced at site 1 were involved in human E1 inactivation. Mechanism of inactivation of E1 was suggested to be site-specific. Phosphorylation of site 1 affected E1 interaction with the lipoyl domain of dihydrolipoamide acetyltransferase, whereas phosphorylation site 3 appeared to be closer to the thiamine pyrophosphate (TPP)-binding region affecting coenzyme interaction with human E1. Four isoenzymes of pyruvate dehydrogenase kinase (PDK) showed different specificity for the three phosphorylation sites of E1. All four PDKs phosphorylated sites 1 and 2 in PDC with different rates, and only PDK1 phosphorylated site 3. PDK2 was maximally stimulated by the reduction/acetylation of the lipoyl groups of E2. Presence of the multiple phosphorylation sites and isoenzymes of PDK is important for the tissue-specific regulation of PDC under different physiological conditions.
172 citations
TL;DR: The results suggest that the continuing increase in PDK activity over the 3-day HF/LC diet is not due to increasing PDK protein beyond 1 day, and could be due to the contribution of another isoform to the totalPDK activity or to a continual increase inPDK4 or PDK2 specific activity.
Abstract: The increase in skeletal muscle pyruvate dehydrogenase kinase (PDK) activity was measured in skeletal muscle of six healthy males after a eucaloric high-fat/low-carbohydrate (HF/LC; 5% carbohydrate...
161 citations
TL;DR: In this article, the authors showed that ThTDP and ThTTDP bound to the parent E1 via a two-step mechanism, but that ThTP degraded the performance of the Y177A and Y177F variants.
110 citations
TL;DR: It is suggested that the conversion of alanine to releasable lactate proceeds at the expense of flux of glycolytic pyruvate through lactate dehydrogenase, which is used for ammonia fixation byAlanine synthesis in the cytosol and for mitochondrial TCA cycle activity.
Abstract: After incubation of glial cells with both 13C-labeled and unlabeled glucose and alanine, 13C isotopomer analysis indicates two cytosolic pyruvate compartments in astrocytes. One pyruvate pool is in an exchange equilibrium with exogenous alanine and preferentially synthesizes releasable lactate. The second pyruvate pool, which is of glycolytic origin, is more closely related to mitochondrial pyruvate, which is oxidized via tri carbonic acid (TCA) cycle activity. In order to provide 2-oxoglutarate as a substrate for cytosolic alanine aminotransferase, glycolytic activity is increased in the presence of exogenous alanine. Furthermore, in the presence of alanine, glutamate is accumulated in astrocytes without subsequent glutamine synthesis. We suggest that the conversion of alanine to releasable lactate proceeds at the expense of flux of glycolytic pyruvate through lactate dehydrogenase, which is used for ammonia fixation by alanine synthesis in the cytosol and for mitochondrial TCA cycle activity. In addition, an intracellular trafficking occurs between cytosol and mitochondria, by which these two cytosolic pyruvate pools are partly connected. Thus, exogenous alanine modifies astrocytic glucose metabolism for the synthesis of releasable lactate disconnected from glycolysis. The data are discussed in terms of astrocytic energy metabolism and the metabolic trafficking via a putative alanine-lactate shuttle between astrocytes and neurons. GLIA 34:200–212, 2001. © 2001 Wiley-Liss, Inc.
104 citations
TL;DR: Results suggest a transcriptional mechanism for decreased fatty oxidation and increased reliance of the heart for glucose during hypoxia, and PPARalpha-regulated gene expression is decreased in two models of Hypoxia in vivo.
TL;DR: Analysis of the structure of mitochondrial pyruvate dehydrogenase kinase isozyme 2 reveals this C-terminal domain to be very similar to the nucleotide-binding domain of bacterial histidine kinases, but the catalytic mechanism appears similar to that of the eukaryotic serine kinases and ATPases.
TL;DR: The results show that the size rather than negative charge of the substituted amino acid residue affects the active site of E1 and that modification of each of the three serine residues affect theactive site in a site-specific manner for its ability to bind the cofactor and substrates.
TL;DR: The role of the intracellular pyruvate pool in the redirection of metabolic fluxes at this important node is focused on and the increased competitiveness of the lactate pathway may be due to the allosteric activation of LDH as a result of increased pyruve levels.
TL;DR: In muscle and liver cells, insulin causes activation and mitochondrial translocation of PKCδ, accompanied by PDP phosphorylation and activation, which are necessary for insulin activation of the PDH complex in these cells.
Patent•
19 Jan 2001TL;DR: In this paper, a combination of a DPP-IV inhibitor and at least one further antidiabetic compound, preferably selected from the group consisting of insulin signalling pathway modulators, is presented for the cosmetic treatment of a mammal in order to effect a cosmetically beneficial loss of body weight.
Abstract: The invention relates to a combination which comprises a DPP-IV inhibitor and at least one further antidiabetic compound, preferably selected from the group consisting of insulin signalling pathway modulators, like inhibitors of protein tyrosine phosphatases (PTPases), non-small molecule mimetic compounds and inhibitors of glutamine-fructose-6-phosphate amidotransferase (GFAT), compounds influencing a dysregulated hepatic glucose production, like inhibitors of glucose-6-phosphatase (G6Pase), inhibitors of fructose-1,6-bisphosphatase (F-1,6-BPase), inhibitors of glycogen phosphorylase (GP), glucagon receptor antagonists and inhibitors of phosphoenolpyruvate carboxykinase (PEPCK), pyruvate dehydrogenase kinase (PDHK) inhibitors, insulin sensitivity enhancers, insulin secretion enhancers, α-glucosidase inhibitors, inhibitors of gastric emptying, insulin, and α2-adrenergic antagonists, for simultaneous, separate or sequential use in the prevention, delay of progression or treatment of conditions mediated by dipeptidylpeptidase - IV (DPP-IV), in particular diabetes, more especially type 2 diabetes mellitus, conditions of impaired glucose tolerance (IGT), conditions of impaired fasting plasma glucose, metabolic acidosis, ketosis, arthritis, obesity and osteoporosis; and the use of such combination for the cosmetic treatment of a mammal in order to effect a cosmetically beneficial loss of body weight.
TL;DR: Fiber type specificity for expression of all three rat skeletal muscle pyruvate dehydrogenase kinase ( PDK) isoforms (PDK1, 2, and 4) was determined in fed and 24-h fasted rats, finding that PDK activity and PDK1 and 4 protein and mRNA were lower in glycolytic vs. oxidative muscles from fed and fasted Rats.
Abstract: Fiber type specificity for expression of all three rat skeletal muscle pyruvate dehydrogenase kinase (PDK) isoforms (PDK1, 2, and 4) was determined in fed and 24-h fasted rats. PDK activity and iso...
TL;DR: The present study has unequivocally demonstrated a quantitatively important pyruvate carboxylation in astrocytes but it was not possible to demonstrate the presence of such carboxYLation in neurons, and based on the present results it may be safely concluded that neuronal pyruVate car boxylation is unlikely to be of quantitative significance.
Abstract: Pyruvate carboxylation was studied in cerebellar astrocytes and granule neurons. The cells were incubated in medium containing [U-(13)C]glucose (2.5 mM) and [U-(13)C]lactate (1 mM) and varying amounts of 3-nitropropionic acid (3-NPA) plus/minus aspartate. 3-NPA alone clearly stopped tricarboxylic acid (TCA) cycle activity at the succinate dehydrogenase step in both culture types as evidenced by a buildup of succinate. Labeling of aspartate and glutamate was abolished in neurons in the presence of 3-NPA. In astrocytes, however, labeled glutamate and glutamine derived from pyruvate carboxylation was detected. Unchanged glucose and lactate metabolism in the absence of a functioning malate aspartate shuttle indicates the importance of the glycerol-3-phosphate shuttle in brain cells. To compensate for the loss of oxaloacetate in the presence of 3-NPA, unlabeled aspartate (0.25 mM) was added. In this case [1,2-(13)C] and [3,4-(13)C]aspartate were observed in neurons but not in astrocytes. This labeling pattern in aspartate occurs after a full turn of the TCA cycle and thus indicates only partial inhibition by 3-NPA in the neurons when aspartate is present. In astrocytes, however, aspartate derived from uniformly labeled pyruvate was observed clearly indicating pyruvate carboxylation. The present study has unequivocally demonstrated a quantitatively important pyruvate carboxylation in astrocytes but it was not possible to demonstrate the presence of such carboxylation in neurons. Based on the present results it may be safely concluded that neuronal pyruvate carboxylation is unlikely to be of quantitative significance.
TL;DR: This work was undertaken to clarify the role of acetaldehyde dehydrogenases in Saccharomyces cerevisiae metabolism during growth on respiratory substrates and constructed mutants in two parental strains, finding that the mutation effects are strain-dependent.
Abstract: This work was undertaken to clarify the role of acetaldehydedehydrogenases in Saccharomyces cerevisiae metabolismduring growth on respiratory substrates. Until now, there hasbeen little agreement concerning the ability of mutantsdeleted in gene ALD4, encoding mitochondrial acetaldehydedehydrogenase, to grow on ethanol. Therefore we con-structed mutants in two parental strains (YPH499 andW303-1a). Some differences appeared in the growthcharacteristics of mutants obtained from these two parentalstrains. For these experiments we used ethanol, pyruvate orlactate as substrates. Mitochondria can oxidize lactate intopyruvate using an ATP synthesis-coupled pathway. Theald4Dmutant derived from the YPH499 strain failed togrow on ethanol, but growth was possible for the ald4Dmutant derived from the W303-1a strain. The co-disruptionof ALD4 and PDA1 (encoding subunit E1a of pyruvatedehydrogenase) prevented the growth on pyruvate for bothstrains but prevented growth on lactate only in the doublemutant derived from the YPH499 strain, indicating that themutation effects are strain-dependent. To understand thesedifferences, we measured the enzyme content of thesedifferent strains. We found the following: (a) the activity ofcytosolic acetaldehyde dehydrogenase in YPH499 wasrelatively low compared to the W303-1a strain; (b) it waspossible to restore the growth of the mutant derived fromYPH499 either by addition of acetate in the media or byintroduction into this mutant of a multicopy plasmidcarrying the ALD6 gene encoding cytosolic acetaldehydedehydrogenase. Therefore, the lack of growth of the mutantderived from the YPH499 strain seemed to be related to thelow activity of acetaldehyde oxidation. Therefore, whencultured on ethanol, the cytosolic acetaldehyde dehydro-genase can partially compensate for the lack of mitochon-drial acetaldehyde dehydrogenase only when the activity ofthe cytosolic enzyme is sufficient. However, when culturedon pyruvate and in the absence of pyruvate dehydrogenase,the cytosolic acetaldehyde dehydrogenase cannot compen-sate for the lack of the mitochondrial enzyme because themitochondrial form produces intramitochondrial NADH andconsequently ATP through oxidative phosphorylation.Keywords: Saccharomycescerevisiae; acetaldehydedehydro-genase; pyruvate dehydrogenase.This work was undertaken to clarify the role of acetaldehydedehydrogenases in Saccharomyces cerevisiae metabolismduring growth on respiratory substrates. Three conversionpathways of pyruvate into acetyl-CoA have been describedin yeast (Fig. 1). The pyruvate dehydrogenase complexlocated inside the mitochondrial matrix converts pyruvateinto acetyl-CoA with the production of NADH. This com-plex has been purified from S. cerevisiae [1,2] and its expres-sion is independent of the carbon source used for growth [3].Another metabolic pathway occurs via the cytosolic pyru-vate dehydrogenase bypass [4] and requires the followingenzymes: pyruvate decarboxylase, cytosolic acetaldehydedehydrogenase and acetyl-CoA synthase. All these enzymesare located in the cytosol. This appears to be an alternativepathway for the production of cytoplasmic acetyl-CoA forbiosynthesis. Recently, a mitochondrial pyruvate dehydro-genase bypass was described, showing that pyruvate can beoxidized inside mitochondria by a pathway involving mito-chondrial acetaldehyde dehydrogenase [5]. Pyruvate is firstdecarboxylated to acetaldehyde in the cytosol by pyruvatedecarboxylase and is then oxidized by mitochondrial acet-aldehyde dehydrogenase, leading to the reduction of NAD
TL;DR: Results indicate that the underlying defect in patients with fatal mitochondrial disease manifesting soon after birth is under the control of a nuclear gene, the locus of which is on chromosome 2.
Abstract: We have studied cultured skin fibroblasts from three siblings and one unrelated individual, all of whom had fatal mitochondrial disease manifesting soon after birth. After incubation with 1 mM glucose, these four cell strains exhibited lactate/pyruvate ratios that were six times greater than those of controls. On further analysis, enzymatic activities of the pyruvate dehydrogenase complex, the 2-oxoglutarate dehydrogenase complex, NADH cytochrome c reductase, succinate dehydrogenase, and succinate cytochrome c reductase were severely deficient. In two of the siblings the enzymatic activity of cytochrome oxidase was mildly decreased (by ∼50%). Metabolite analysis performed on urine samples taken from these patients revealed high levels of glycine, leucine, valine, and isoleucine, indicating abnormalities of both the glycine-cleavage system and branched-chain α-ketoacid dehydrogenase. In contrast, the activities of fibroblast pyruvate carboxylase, mitochondrial aconitase, and citrate synthase were normal. Immunoblot analysis of selected complex III subunits (core 1, cyt c 1 , and iron-sulfur protein) and of the pyruvate dehydrogenase complex subunits revealed no visible changes in the levels of all examined proteins, decreasing the possibility that an import and/or assembly factor is involved. To elucidate the underlying molecular defect, analysis of microcell-mediated chromosome-fusion was performed between the present study's fibroblasts (recipients) and a panel of A9 mouse:human hybrids (donors) developed by Cuthbert et al. (1995). Complementation was observed between the recipient cells from both families and the mouse:human hybrid clone carrying human chromosome 2. These results indicate that the underlying defect in our patients is under the control of a nuclear gene, the locus of which is on chromosome 2. A 5-cM interval has been identified as potentially containing the critical region for the unknown gene. This interval maps to region 2p14-2p13.
TL;DR: It is proposed that specific up-regulation of renal PDK4 protein expression in starvation, by maintaining PDC activity relatively low, facilitates pyruvate carboxylation to oxaloacetate and therefore entry of acetyl-CoA derived from FA beta-oxidation into the TCA cycle, allowing adequate ATP production for brisk rates of gluconeogenesis.
TL;DR: Increased blood levels of thyroid hormone are proposed to be responsible for increased expression of branched-chain alpha-keto acid dehydrogenase kinase in animals starved for protein.
Abstract: Branched-chain amino acids are toxic in excess but have to be conserved for protein synthesis. This is accomplished in large part by control of the activity of the branched-chain alpha-keto acid dehydrogenase complex by phosphorylation/dephosphorylation. Regulation of the activity of the hepatic enzyme appears particularly important, at least in rats, since an exceptional high activity of the complex in this tissue makes the liver the primary clearing house for excess branched-chain alpha-keto acids released by other tissues. The degree to which the branched-chain alpha-keto acid dehydrogenase complex is inactivated by phosphorylation is determined by the activity of the branched-chain alpha-keto acid dehydrogenase kinase, which is itself regulated by allosteric effectors as well as factors that affect its level of expression. Well established among these are the alpha-keto acid produced by leucine transamination, which is a potent inhibitor of the kinase, and starvation for dietary protein, which causes increased expression of the branched-chain alpha-keto acid dehydrogenase kinase. The latter finding resulted in the working hypothesis that nutrients and hormones regulate expression of the branched-chain alpha-keto acid dehydrogenase kinase. Evidence has been obtained for the involvement of thyroid hormone, glucocorticoids and ligands for peroxisome proliferator-activated receptor alpha. Thyroid hormone induces, whereas glucocorticoids and peroxisome proliferator-activated receptor alpha ligands repress, expression of the kinase. Increased blood levels of thyroid hormone are proposed to be responsible for increased expression of branched-chain alpha-keto acid dehydrogenase kinase in animals starved for protein.
TL;DR: Pyruvate carboxylation through malic enzyme is active during energy deficiency and leads to an increase in the level of dicarboxylates that can be metabolized through the tricar boxylic acid cycle for ATP production, and may be one mechanism through which treatment is effective.
Abstract: Carboxylation of pyruvate in the brain was for many years thought to occur only in glia, an assumption that formed much of the basis for the concept of the glutamine cycle. It was shown recently, however, that carboxylation of pyruvate to malate occurs in neurons and that it supports formation of transmitter glutamate. The role of pyruvate carboxylation in neurons is to ensure tricarboxylic acid cycle activity by compensating for losses of alpha-ketoglutarate that occur through release of transmitter glutamate and GABA; these amino acids are alpha-ketoglutarate derivatives. Available data suggest that neuronal pyruvate carboxylation is quantitatively important. But because there is no net CO(2) fixation in the brain, pyruvate carboxylation must be balanced by decarboxylation of malate or oxaloacetate. Such decarboxylation occurs in both neurons and astrocytes. Several in vitro studies have shown a neuroprotective effect of pyruvate supplementation. Pyruvate carboxylation may be one mechanism through which such treatment is effective, because pyruvate carboxylation through malic enzyme is active during energy deficiency and leads to an increase in the level of dicarboxylates that can be metabolized through the tricarboxylic acid cycle for ATP production.
TL;DR: Although no changes in islet PPar-alpha expression were observed after the starvation protocol, activation of PPAR-alpha in vivo may be a potential mechanism underlying upregulation of islet PDK4 protein expression in starvation.
Abstract: The pyruvate dehydrogenase complex (PDC) has a pivotal role in islet metabolism. The pyruvate dehydrogenase kinases (PDK1–4) regulate glucose oxidation through inhibitory phosphorylation of PDC. Starvation increases islet PDK activity (AmJ Physiol Endocrinol Metab 270:E988–E994, 1996). In this study, using antibodies against PDK1, PDK2, and PDK4 (no sufficiently specific antibodies are as yet available for PDK3), we identified the PDK isoform profile of the pancreatic islet and delineated the effects of starvation (48 h) on protein expression of individual PDK isoforms. Rat islets were demonstrated to contain all three PDK isoforms, PDK1, PDK2, and PDK4. Using immunoblot analysis with antibodies raised against the individual recombinant PDK isoforms, we demonstrated increased islet protein expression of PDK4 in response to starvation (2.3-fold; P
TL;DR: This study provides the first detailed functional characterization of human erythrocyte pyruvate kinase, and will allow the establishment of a fine correlation between molecular abnormalities and the clinical expression of the disease.
TL;DR: The findings support the hypothesis that pyruvate protection against H2O2 apoptosis is mediated in part via the mitochondrial matrix compartment, and possible mediators include anti-apoptotic bcl-2 and/or products of mitochondrial pyruVate metabolism such as citrate that affect metabolic regulation and anti-oxidant status in the cytoplasm.
Abstract: In the hydrogen peroxide (H2O2) apoptosis model of the murine thymocyte, redox reactant and antioxidant pyruvate prevents programmed cell death. We tested the hypothesis that such protection was mediated, at least in part, via pyruvate handling by mitochondrial metabolism. Cultured bovine pulmonary artery endothelial cells were incubated for 30 min with 0.5 mM H2O2 in the absence and presence of 0.5 mM α-cyano-3-hydroxycinnamate, as a selective inhibitor of the mitochondrial pyruvate transporter. In controls H2O2 decreased cell viability by 30% within 24 h; this was associated with apoptosis-like bodies, nuclear condensation, and biochemical DNA damage consistent with programmed cell death. Pyruvate (0.1–20 mM) enhanced cell viability in a dose-dependent manner, with ≥ 85% viable cells at ≥ 3 mM and no DNA laddering, no positive nick-end labeling (TUNEL), and no detectable Annexin V or propidium iodide staining. In contrast, using ≥ 5 mM L-lactate as a cytosolic reductant or acetate as a redox-neutral substrate, cell death increased to ≈ 40%, which was associated with intense DNA laddering, positive TUNEL and Hoechst 33258 assays. α-Cyano-3-hydroxycinnamate alone did not significantly decrease endothelial viability but reduced viability from 85 ± 3 to 71 ± 4% (p = 0.023) in presence of 3 mM pyruvate plus H2O2; pathological cell morphology and DNA laddering under the same conditions suggested loss of pyruvate protection against apoptosis. Since α-cyano-3-hydroxycinnamate re-distributed medium pyruvate and L-lactate consistent with selective blockade of pyruvate uptake into the mitochondria, the findings support the hypothesis that pyruvate protection against H2O2 apoptosis is mediated in part via the mitochondrial matrix compartment. Possible mediators include anti-apoptotic bcl-2 and/or products of mitochondrial pyruvate metabolism such as citrate that affect metabolic regulation and anti-oxidant status in the cytoplasm.
TL;DR: The data demonstrate that carbon substrate availability to the mitochondria is not the only cause of the reduction of myocardial phosphocreatine (PCr)/ATP that occurs at high workstates.
Abstract: This study was performed to determine whether the fall in myocardial high-energy phosphates (HEP) that occurs during high workstates can be ascribed to either inadequate glycolytic pyruvate generat...
TL;DR: Intracellular metabolite profiles indicated that the in vivo glyceraldehyde-3-phosphate dehydrogenase activity was no longer flux limiting in the Deltaldh strain, and a shift toward mixed acid fermentation was correlated with the lower intracellular trioses phosphate concentration and diminished allosteric inhibition of pyruvate formate lyase.
TL;DR: It is concluded that MP-induced beta-cell membrane depolarization or insulin release does not relate directly to mitochondrial ATP production, but instead MP may exert a direct extramitochondrial effect, or it may stimulate beta- cell mitochondria to produce coupling factors different from ATP to initiate insulin release.
Abstract: The role of mitochondria in stimulus-secretion coupling of pancreatic beta-cells was examined using methyl pyruvate (MP). MP stimulated insulin secretion in the absence of glucose, with maximal effect at 5 mM. K+ (30 mM) alone, or in combination with diazoxide (100 microM), failed to enhance MP-induced secretion. Diazoxide (100 microM) inhibited MP-induced insulin secretion. MP depolarized the beta-cell in a concentration-dependent manner (5-20 mM). The sustained depolarization induced by 20 mM MP was not influenced by 100 microM diazoxide, but the continuous spiking activity was suppressed by 500 microM diazoxide. Pyruvate failed to initiate insulin release (5-20 mM) or to depolarize the membrane potential. ATP production in isolated beta-cell mitochondria was detected as accumulation of ATP in the medium during incubation in the presence of malate or glutamate in combination with pyruvate or MP. There was no difference in ATP production induced by pyruvate/malate or MP/malate in isolated beta-cell mitochondria. ATP production by MP/glutamate was higher than that induced by pyruvate/glutamate, but it was much lower than that induced by alpha-ketoisocaproate/glutamate. Pyruvate (5 mM) or MP (5 mM) had no effect on the ATP/ADP ratio in whole islets, whereas glucose (20 mM) significantly increased the whole islet ATP/ADP ratio. It is concluded that MP-induced beta-cell membrane depolarization or insulin release does not relate directly to mitochondrial ATP production. Instead MP may exert a direct extramitochondrial effect, or it may stimulate beta-cell mitochondria to produce coupling factors different from ATP to initiate insulin release.