Showing papers on "Pyruvate dehydrogenase kinase published in 1995"
TL;DR: The current results suggest that during glucose-induced insulin secretion there is a shuttle operating across the mitochondrial membrane in which glucose-derived pyruvate is taken up by mitochondria and carboxylated to oxaloacetate by pyruVate carboxykinase.
282 citations
TL;DR: The deduced amino acid sequences of three isoenzymic forms of PDK found in humans suggest that PDK2 is a major isoenzyme responsible for regulation of pyruvate dehydrogenase in human tissues, and PDK3 appears to have the highest specific activity among the three isenzymes tested as recombinant proteins.
259 citations
TL;DR: The cloning of the pykA and pykF genes from Escherichia coli, which code for the two pyruvate kinase isoenzymes, were reported, which were insertionally inactivated with antibiotic resistance markers and utilized to interrupt one or both pyk genes in the E. coli chromosome.
Abstract: We report the cloning of the pykA and pykF genes from Escherichia coli, which code for the two pyruvate kinase isoenzymes (ATP:pyruvate 2-O-phosphotransferases; EC 2.7.1.40) in this microorganism. These genes were insertionally inactivated with antibiotic resistance markers and utilized to interrupt one or both pyk genes in the E. coli chromosome. With these constructions, we were able to study the role of these isoenzymes in pyruvate biosynthesis.
152 citations
TL;DR: The chemical mechanisms of the partial reactions and some of the roles played by amino acid residues of the enzyme in catalysing the reaction have been elucidated, and the domain structure of the yeast enzyme has been deduced by comparing its amino acid sequence with those of enzymes that have similar catalytic functions.
142 citations
TL;DR: Long-term exposure to fatty acids limits the conversion of pyruvate to acetyl-CoA through inhibition of PDH activity, which is linked to increased activity of a non-PDH-bound kinase.
Abstract: We previously found that long-term exposure to fatty acids impairs glucose-induced insulin release. In the present study, we investigated whether impairment is related to decreased pyruvate dehydrogenase (PDH) and increased PDH kinase activity. Rat pancreatic islets were cultured for 48 h in RPMI-1640 medium with or without 0.125 mmol/l palmitate. Potentiation of insulin responses to succinic acid monomethylester (SAM) by 10 mmol/l acetate and pyruvate were subsequently compared in order to assess whether generation of acetyl-coenzyme A (CoA) from pyruvate was deficient in the intact beta-cell. Potentiation by acetate was similar in control and palmitate-preexposed islets. In contrast, pyruvate potentiated SAM-induced response by 122% in control but by only 39% in palmitate-exposed islets (P < 0.001). In extracts of palmitate-exposed islets, the active (unphosphorylated) form of PDH was decreased by 50% and total PDH activity (assessed after phosphatase treatment) by 25%. The proportion of active form to total PDH activity was also reduced (42.7 +/- 2.6% after palmitate vs. 66.6 +/- 4.3% in control islets, P < 0.01). In the same preparations, PDH kinase activity was enhanced 1.7-fold by palmitate in terms of the rate constant of ATP-dependent inactivation of PDH (P < 0.05). To test for a role of free (not PDH-bound) kinase, a PDH-free mitochondrial fraction was prepared, and its kinase activity was tested against a pig heart PDH preparation. Free kinase activity was increased 1.9-fold in palmitate-treated islets (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)
88 citations
TL;DR: Tissue specific expression of these enzymes and/or control of their association with the complex will probably prove to be important for the long term regulation of the activity of the pyruvate dehydrogenase complex.
83 citations
TL;DR: This review uses information derived from calcium binding studies, analysis of conserved calcium binding motifs and comparison of amino acid sequences from calcium sensitive and non-sensitive enzymes to discuss how the recent cloning of several subunits from the four dehydrogenases enhances the authors' understanding of the ways in which these enzymes bind calcium.
Abstract: In mammalian cells, increases in calcium concentration cause increases in oxidative phosphorylation. This effect is mediated by the activation of four mitochondrial dehydrogenases by calcium ions; FAD-glycerol 3-phosphate dehydrogenase, pyruvate dehydrogenase, NAD-isocitrate dehydrogenase and oxoglutarate dehydrogenase. FAD-glycerol 3-phosphate dehydrogenase, being located on the outer surface of the inner mitochondrial membrane, is exposed to fluctuations in cytoplasmic calcium concentration. The other three enzymes are located within the mitochondrial matrix. While the kinetic properties of all of these enzymes are well characterised, the molecular basis for their regulation by calcium is not. This review uses information derived from calcium binding studies, analysis of conserved calcium binding motifs and comparison of amino acid sequences from calcium sensitive and non-sensitive enzymes to discuss how the recent cloning of several subunits from the four dehydrogenases enhances our understanding of the ways in which these enzymes bind calcium. FAD-glycerol 3-phosphate dehydrogenase binds calcium ions through a domain which is part of the polypeptide chain of the enzyme. In contrast, it is possible that the calcium sensitivity of the other three dehydrogenases may involve separate calcium binding subunits.
79 citations
TL;DR: Using a model reaction system consisting of varying mixtures of purified lactate and pyruvate dehydrogenases, it was found that the presence of oxamate, a competitive inhibitor of the lactate dehydrogensase, allowed the measurement of a linear rate of pyruve dehydrogenase activity without interference from lactate dehydration.
75 citations
TL;DR: A defect in an enzyme called glucose-6-phosphate dehydrogenase causes red blood cells to break down prematurely, which results in the destruction ofRed blood cells, which carry oxygen from the lungs to tissues throughout the body.
72 citations
TL;DR: It is suggested that transfer of tightly bound kinase between E2 domains occurs by a direct interchange mechanism without formation of free kinase (model presented), which would explain how a kinase molecule can rapidly phosphorylate a large complement of pyruvate dehydrogenase tetramers which are bound throughout the surface of the E2 oligomer.
66 citations
Journal Article•
TL;DR: The current results support the idea that carboxylation of glucose-derived pyruvate, as well as decarboxylating of pyruVate, is important for glucose-induced insulin secretion.
TL;DR: A kinase-mediated decrease in PDH activity in islets of GK rats may in part account for the decreased ratio of oxidized to utilized glucose and impaired insulin release in these islets; and impaired diabetes release in the GK Rats is not linked to an inhibitory influence of islet fatty acid oxidation.
Abstract: We investigated the role of islet pyruvate dehydrogenase (PDH) enzyme activity and fatty acid oxidation in the impaired insulin secretion in spontaneously diabetic GK rats. Blood glucose levels were elevated in 2- to 3-month-old GK rats (8.7 +/- 0.5 vs. 6.5 +/- 0.3 mM in control Wistar rats; P < 0.01), whereas serum insulin levels were comparable to those in control rats. Insulin and DNA contents were similar in freshly isolated islets from GK and control rats, whereas insulin responses to 27 mM glucose from GK islets were reduced by 52%. The effect of acetate or pyruvate on insulin responses evoked by succinate monomethylester (SAM) were compared to indirectly assess deficient generation of acetyl-coenzyme A from pyruvate. Acetate potentiated SAM-induced insulin secretion similarly in GK and control islets, whereas 10 mM pyruvate (which supplies acetyl-coenzyme A through PDH enzyme activity) failed to normally potentiate insulin secretion in GK islets (92% of SAM-induced response in GK vs. 154% in control islets). The PDH activity (active form) was decreased in GK islets by 35% (P < 0.001). The proportion of active form PDH to total PDH activity was reduced in GK islets (56% vs. 71% in control islets; P < 0.01). The activity of PDH kinase (which inactivates PDH by phosphorylation) was increased in GK islets, the rate of ATP-dependent inactivation of PDH was -0.29 +/- 0.02 vs. -0.19 +/- 0.02/min in control islets (P < 0.05). Culturing GK islets for 48 h at 5.5 mM glucose failed to correct the impaired insulin response to glucose and the decreased PDH activity. Serum FFA levels and islet triglyceride contents did not differ between GK and control rats. Etomoxir (1.0 and 10 microM), a carnitine palmitoyl transferase I inhibitor, failed to enhance glucose-induced insulin release in GK islets. The following conclusions were reached: 1) a kinase-mediated decrease in PDH activity in islets of GK rats may in part account for the decreased ratio of oxidized to utilized glucose and impaired insulin release in these islets; and 2) impaired insulin release in the GK rats is not linked to an inhibitory influence of islet fatty acid oxidation.
TL;DR: A series of mutants are constructed in the mobile, active site loop of the Escherichia coli malate dehydrogenase that incorporate the complementary change, conversion of arginine 81 to glutamine, to evaluate the role of charge distribution and conformational flexibility within this loop in defining the substrate specificity of these enzymes.
Journal Article•
TL;DR: Chimeric constructs in which the normal and mutant pyruvate dehydrogenase E1 alpha targeting sequences were attached to the mitochondrial matrix protein ornithine transcarbamylase were synthesized in a cell free translation system, and mitochondrial import of normal and Mutant proteins was compared in vitro.
Abstract: A mutation in the mitochondrial targeting sequence was characterized in a male patient with X chromosome-linked pyruvate dehydrogenase E1 alpha deficiency. The mutation was a base substitution of G by C at nucleotide 134 in the mitochondrial targeting sequence of the PDHA1 gene, resulting in an arginine-to-proline substitution at codon 10 (R10P). Pyruvate dehydrogenase activity in cultured skin fibroblasts was 28% of the control value, and immunoblot analysis revealed a decreased level of pyruvate dehydrogenase E1 alpha immunoreactivity. Chimeric constructs in which the normal and mutant pyruvate dehydrogenase E1 alpha targeting sequences were attached to the mitochondrial matrix protein ornithine transcarbamylase were synthesized in a cell free translation system, and mitochondrial import of normal and mutant proteins was compared in vitro. The results show that ornithine transcarbamylase targeted by the mutant pyruvate dehydrogenase E1 alpha sequence was translocated into the mitochondrial matrix at a reduced rate, suggesting that defective import is responsible for the reduced pyruvate dehydrogenase level in mitochondria. The mutation was also present in an affected brother and the mildly affected mother. The clinical presentations of this X chromosome-linked disorder in affected family members are discussed. To our knowledge, this is the first report of an amino acid substitution in a mitochondrial targeting sequence resulting in a human genetic disease.
TL;DR: A monoclonal antibody is isolated that recognizes the E1α subunit of the plant mitochondrial pyruvate dehydrogenase complex and will be a useful tool for study of plant mitochondria, yeast, or bacteria.
Patent•
09 Jan 1995TL;DR: A mutant of the genus Escherichia is described in this article, where α-ketoglutarate dehydrogenase activity is either deficient or reduced, and/or the phosphoenol pyruvate carboxylase and glutamate de-hydrogenase activities are amplified.
Abstract: A mutant of the genus Escherichia is described, the α-ketoglutarate dehydrogenase activity of which is deficient or reduced, and/or the phosphoenol pyruvate carboxylase and/or glutamate dehydrogenase activities of which are amplified. The mutant is useful in the fermentative production of L-glutamic acid.
TL;DR: The results indicate that this physiological state has serious metabolic consequences and ultimately leads to cell death; thereby identifying the pyruvate carrier as a possible target for chemotherapeutic intervention.
Abstract: The pyruvate produced by glycolysis in the bloodstream form of the trypanosome is excreted into the host bloodstream by a facilitated diffusion carrier. The sensitivity of pyruvate transport for alpha-cyano-4-hydroxycinnamate and the compound UK5099 [alpha-cyano-beta-(1-phenylindol-3-yl)acrylate], which are known to be selective inhibitors of pyruvate (monocarboxylate) transporters present in mitochondria and the plasma membrane of eukaryotic cells, was examined. The trypanosomal pyruvate carrier was found to be rather insensitive to inhibition by alpha-cyano-4-hydroxycinnamate (Ki = 17 mM) but could be completely blocked by UK5099 (Ki = 49 microM). Inhibition of pyruvate transport resulted in the retention, and concomitant accumulation, of pyruvate within the trypanosomes, causing acidification of the cytosol and osmotic destabilization of the cells. Our results indicate that this physiological state has serious metabolic consequences and ultimately leads to cell death; thereby identifying the pyruvate carrier as a possible target for chemotherapeutic intervention.
TL;DR: Both enantiomers of lipoic acid are inhibitors of the overall reaction of the bovine pyruvate dehydrogenase complex, but stimulate the respective enzyme complexes from rat as well as from Escherichia coli.
TL;DR: The results indicate that pyruvate-dehydrogenase kinase activity in oxidative skeletal muscle is a target for longer-term regulation by high-fat feeding and that the fatty acid composition of the diet, rather than the fat content, is a key influence.
Abstract: The provision of a diet high in saturated and monounsaturated fat for 28 days evoked a significant (1.9-fold) increase in pyruvate-dehydrogenase kinase activity measured in isolated mitochondria from representative slow-twitch (oxidative) skeletal muscles (pooled soleus and adductor longus muscles) from adult rats. The increase observed in response to 28 days of high-fat feeding in slow-twitch skeletal muscle mitochondria was similar in magnitude to that observed in heart mitochondria. Pyruvate-dehydrogenase kinase activity was not increased in response to the provision of the high-fat diet in mitochondria prepared from a representative fast-twitch muscle (tibialis anterior), while the increases evoked by 28 days of high-fat feeding in cardiac and slow-twitch skeletal muscle were prevented by the replacement of 7% of the dietary fatty acids with long-chain ω–3 fatty acids from marine oil. Cardiac myocytes prepared from the high-fat-fed rats showed impaired responses of this enzyme to n -octanoate (1 mM) and N6,2-O -dibutyryl-adenosine 3′,5′-monophosphate (50 μM) individually in cultured cardiac myocytes and of glucose uptake to insulin at low concentrations in freshly prepared cardiac myocytes, compared with control rats maintained on standard low-fat/high-carbohydrate diet. These impairments in responses to agonists were substantially improved by the inclusion of long-chain ω–3 fatty acids in the high-fat diet. The results indicate that pyruvate-dehydrogenase kinase activity in oxidative skeletal muscle is a target for longer-term regulation by high-fat feeding and that the fatty acid composition of the diet, rather than the fat content, is a key influence.
TL;DR: The relative roles of pyruvate kinase and malic enzyme in substrate cycling between pyruVate and oxaloacetate were examined in perfused livers of 24-hour-fasted normal and triiodothyronine (T3)-treated rats using an inhibitor of malic enzymes (hydroxymalonate).
Abstract: The relative roles of pyruvate kinase and malic enzyme in substrate cycling between pyruvate and oxaloacetate were examined in perfused livers of 24-hour—fasted normal and triiodothyronine (T3)-treated rats using an inhibitor of malic enzyme (hydroxymalonate). Livers were perfused for 60 minutes in a recirculating system with [3-13C]alanine (10 mmol/L, 99% 13C-enriched). The combined flux through pyruvate kinase plus malic enzyme relative to pyruvate carboxylase flux was assessed by the 13C-enrichment ratio of alanine C2 to glucose C5 in the perfusate, determined with 13C and 1H nuclear magnetic resonance (NMR) spectroscopy. In normal rat livers, the relative carbon flux through pyruvate kinase plus malic enzyme to pyruvate carboxylase was 0.18 ± 0.04, and increased to 0.44 ± 0.08 (P < .05) in the T3-treated group. After addition of hydroxymalonate, this relative carbon flux was unchanged in normal rat livers, but decreased to 0.15 ± 0.04 (P < .01) in the T3-treated group, suggesting that the increased carbon flux in T3-treated livers was caused by increased flux through malic enzyme. Malic enzyme activity increased from 0.36 ± 0.05 U/g liver in normal livers to 2.51 ± 0.50 U/g liver (P < .05) in the T3-treated group, whereas there was no effect of T3 treatment on pyruvate kinase activity. We conclude that (1) carbon flux through malic enzyme relative to pyruvate carboxylase flux is minimal in the liver of normal 24-hour—fasted rats, (2) T3 treatment stimulates substrate cycling between pyruvate and oxaloacetate by increasing carbon flux through malic enzyme, and (3) under hyperthyroid conditions, substrate cycling between pyruvate carboxylase and malic enzyme accounts for a major fraction of the gluconeogenic flux.
TL;DR: It is suggested that L-carnitine stimulates pyruvate dehydrogenase complex activity and enhances non-oxidative glucose metabolism by increasing the mitochondrial acetylc Carnitine efflux in the absence of exogenous fatty acids.
TL;DR: Results are presented that indicate that the prolonged consumption of a diet high in saturated fat is associated with a stable enhancement of pyruvate dehydrogenase (PDH) kinase activity at least in two oxidative tissues--liver and heart.
Abstract: This review examines the molecular mechanisms underlying substrate competition between glucose and lipid in starvation and in insulin-resistant states We demonstrate that lipid-derived substrates are oxidized in preference to glucose by skeletal muscle in vivo during prolonged starvation An accelerated and exaggerated lipolytic and ketogenic response to starvation in late pregnancy is associated with more rapid suppression of glucose oxidation by the maternal skeletal-muscle mass These benign adaptations to changes in lipid availability (which occur secondarily to changes in carbohydrate supply and demand) contrast with the well-documented detrimental effects to health of an inappropriately high supply of dietary lipid We present results that indicate that the prolonged consumption of a diet high in saturated fat is associated with a stable enhancement of pyruvate dehydrogenase (PDH) kinase activity at least in two oxidative tissues--liver and heart This long-term enhancement of PDH kinase activity is concomitant with the development of whole-body insulin resistance and adds a new dimension to the potential role of dietary composition in the pathogenesis of insulin resistance
TL;DR: A series of halogenated acetophenones with potent activity against PDH‐Kinase (IC50 1–3 μM) and inactive towards other protein kinases was identified and the lead compound displayed non‐linear kinetics and was shown to be an uncompetitive inhibitor with respect to ATP.
Abstract: The activity of pyruvate dehydrogenase (PDH), and hence of glucose oxidation, is regulated by reversible phosphorylation. We have searched for novel PDH-Kinase inhibitors using a high-throughput microtiter plate assay. A series of halogenated acetophenones with potent activity against PDH-Kinase (IC50 1–3 μM) and inactive towards other protein kinases was identified. The lead compound displayed non-linear kinetics and was shown to be an uncompetitive inhibitor with respect to ATP. These novel inhibitors of PDH-Kinase may be potential new drug leads for the treatment of diabetes or ischaemic conditions. © 1995 Wiley-Liss, Inc.
TL;DR: The effects of anoxia or freezing exposure in vivo on the activities of five enzymes of carbohydrate metabolism were assessed in foot muscle and hepatopancreases of the marine periwinkle Littorina littorea, suggesting that a primary contributor to freezing survival is the implementation of anaerobic metabolism and metabolic arrest mechanisms that also promote anoxian survival in marine molluses.
Abstract: The effects of anoxia (N2 atmosphere at 5 °C) or freezing (at-8 °C) exposure in vivo on the activities of five enzymes of carbohydrate metabolism were assessed in foot muscle and hepatopancreases of the marine periwinkle Littorina littorea. Changes in glycogen phosphorylase, glycogen synthetase, pyruvate kinase and pyruvate dehydrogenase under either stress were generally consistent with covalent modification of the enzymes to decrease enzyme activity and/or convert the enzyme to a less active form. However, no evidence for a similar covalent modification of phosphofructokinase was found. The metabolic effects of freezing and anoxia were generally similar, suggesting that a primary contributor to freezing survival is the implementation of anaerobic metabolism and metabolic arrest mechanisms that also promote anoxia survival in marine molluses. However, in hepatopancreas phosphorylase was activated and pyruvate kinase remained in two enzyme forms in freezing-exposed snails, contrary to the results for anoxic animals. Ion exchange chromatography on DE-52 Sephadex revealed the presence of two forms of pyruvate kinase in both tissues of control L. littorea, eluting at 30–50 mmol·1-1 KCl (peak I) or 90–110 mmol·1-1 KCl (peak II). Anoxia exposure converted pyruvate kinase in both tissues to the peak I form, as did freezing for foot muscle pyruvate kinase. Kinetic analysis showed that peak I pyruvate kinase had lower affinities for substrates, phosphoenolpyruvate and ADP, and was very strongly inhibited by l-alanine compared with the peak II enzyme. Peak I pyruvate kinase had an I50 value for l-alanine of 0.38 mmol·1-1, whereas peak II pyruvate kinase was unaffected by l-alanine evenat 40 mmol·1-1. In vitro incubation of extracts from control foot muscle under conditions promoting phosphorylation or dephosphorylation identified the peak I and II forms as the low and high phosphate forms, respectively. This result for L. littorea pyruvate kinase was highly unusual and contrary to the typical effect of anoxia on pyruvate kinase in marine molluscs which is to stimulate the phosphorylation of pyruvate kinase and, thereby, convert the enzyme to a less active form.
TL;DR: It is demonstrated that hyperthyroidism leads to a stable increase in the activity of cardiomyocyte PDH kinase, a response that is mimicked by T3 in vitro.
TL;DR: PDC activity in isolated yeast mitochondria could be inactivated in an ATP‐dependent fashion and reactivated in the presence of Ca2+ ions.
TL;DR: Monitoring of the effect of phosphorylation of E1 of the muscle pyruvate dehydrogenase complex (PDC) by E1‐kinase inhibits substrate conversion both in oxidative and non‐oxidative reactions.
TL;DR: Results agree with the previous observations that pyruvate markedly prevents the loss of cytosolic and mitochondrial glutathione after ischemia orIschemia followed by reperfusion and exerts a protective effect when calcium + phosphate-induced swelling is exacerbated by hypoxic conditions.
Abstract: Pyruvate prevents the permeability transition of rat heart mitochondria induced by the system calcium ions + phosphate or by the dithiol reagent phenylarsenoxide and measured as swelling. Since swelling induced by the latter is relieved by the dithiol 2,3-dimercaptopropanol (BAL), it is inferred that the effect of pyruvate might be mediated by the reduction of lipoic acid. In isolated mitochondria, pyruvate also exerts a protective effect when calcium + phosphate-induced swelling is exacerbated by hypoxic conditions. These results agree with our previous observations that pyruvate markedly prevents the loss of cytosolic and mitochondrial glutathione after ischemia or ischemia followed by reperfusion.