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PubMed1989

Pyruvate oxidation in vivo and in vitro in Dictyostelium discoideum.

The in vivo rate of the reaction catalyzed by the pyruvate dehydrogenase complex in Dictyostelium discoideum was measured by exposing cells to [1-14C]alanine and relating the rate of 14CO2 evolution to the specific radioactivity of the isolated [1-14C]pyruvate. The rate obtained, 0.33 mumol/min per ml packed cell volume, was very similar to that determined by independent calculations based upon the rate of utilization of endogenous protein as an energy source in this system.

Alanine↗
PubMed1996

Mammalian mitochondria possess homologous DNA recombination activity.

Mitochondrial protein extracts from normal and immortalized mammalian somatic cells catalyze homologous recombination of plasmid DNA substrates. Mitochondrial homologous recombination activity required exogenous adenosine triphosphate, although substantial activity remained when non-hydrolyzable analogs were used instead. There was no requirement for added nucleoside triphosphates, and the reaction was not inhibited by dideoxyadenosine triphosphate or aphidicolin. The majority of recombinant plasmid molecules result from a conservative process, indicating that nuclease-mediated strand-annealing is not responsible for the mitochondrial homologous recombination activity. Affinity-purified anti-recA antibodies inhibited the reaction, suggesting that activity is dependent on a mammalian mitochondrial homolog of the bacterial strand-transferase protein. The presence of homologous recombination activity within mammalian mitochondrial extracts suggests that this process is involved in mitochondrial DNA repair.

Animals↗
PubMed2006

Insights into the role of anaplerosis in insulin secretion: A 13C NMR study.

AIMS/HYPOTHESIS: Defining mechanisms and enzymatic paths critical to fuel-regulated insulin secretion are key goals of diabetes research. In this study, 13C-nuclear magnetic resonance spectroscopy and isotopomer analysis were used to investigate the link between insulin secretion and metabolic pathways associated with the tricarboxylic acid (TCA) cycle. MATERIALS AND METHODS: To this end, four insulinoma cell lines (betaTC3, betaTC-tet, INS-1 [832/13], R7T1) and porcine islets were examined under a variety of culture conditions (i.e. presence vs absence of amino acids and sera, and low vs high glucose). RESULTS: Glucose consumption, insulin release, and glutamate isotopomeric patterns were influenced by media complexity (e.g. PBS, plain culture media, fully supplemented culture media). The 13C-labelled metabolites increased with media complexity and increasing glucose concentration, with the notable exception of aspartate, which was always higher under low-glucose conditions. The 13C-glutamate isotopomeric fractions were fitted to metabolic models to estimate the relative metabolic fluxes to the TCA cycle through key enzymatic processes. These indices of metabolism were compared with insulin secretion to determine correlative links. A model containing a single pool of pyruvate, an entrance to the TCA cycle via the pyruvate dehydrogenase complex, and two anaplerotic entrances, one through pyruvate carboxylase and another through an undefined (by the modelling program) source, provided the best fit to the data under all conditions tested, for all cell lines. CONCLUSIONS/INTERPRETATION: On the basis of our findings, a strong correlation may exist between stimulated insulin secretion and non-pyruvate carboxylase anaplerosis for the four cell lines examined in this study.

Animals↗
PubMed1984

Catalytic and structural properties of the dihydrolipoyl transacylase component of bovine branched-chain alpha-keto acid dehydrogenase.

Branched-chain alpha-keto acid dehydrogenase is a multienzyme complex consisting of three catalytic components, i.e. branched-chain alpha-keto acid decarboxylase (E1), dihydrolipoyl transacylase (E2), and dihydrolipoyl dehydrogenase (E3). In this report the E2 component of highly purified branched-chain alpha-keto acid dehydrogenase from bovine kidney and liver was characterized with an independent radiochemical assay for this component. The assay uses the model reaction: R-14CO-S-CoA + Lip-(SH)2 in equilibrium R-14CO-S-Lip-SH + CoA-SH, which is similar to that catalyzed by the transacetylase component of the pyruvate dehydrogenase complex. In this reaction, exogenous dihydrolipoamide substitutes for the protein (E2)-bound dihydrolipoyl moiety, and [1-14C]acyl-CoA synthesized enzymatically is the acyl-CoA substrate. The thioester structure of the reaction product, S-acyldihydrolipoamide, was identified by mass spectrometry, its characteristic absorption at 232-245 nm and by formation of hydroxamate with hydroxylamine. Rates of the E2-catalyzed transacylation reaction with various [1-14C]acyl-CoAs are in the order of [1-14C]isobutyryl-CoA greater than [1-14C] isovaleryl-CoA greater than [1-14C]acetyl-CoA. The activity with acetyl-CoA is 15% of that with isobutyryl-CoA. The E2 activity is strongly inhibited by arsenite. Modification of the covalently bound lipoyl moiety through reductive acylation in the presence of N-ethylmaleimide is without effect on the transacylation reaction. These data, along with results of initial velocity and product inhibition suggest that the model reaction proceeds via a random Bi Bi mechanism. Limited proteolysis of purified bovine liver branched-chain alpha-keto acid dehydrogenase with trypsin results in complete loss of the overall activity catalyzed by the complex. Nonetheless the activity of the E2 component is not affected. The tryptic digestion cleaves E2 subunits (Mr = 52,600) into a major fragment of Mr = 25,700. By contrast, E1 alpha and E1 beta subunits of the complex are relatively resistant to proteolysis with trypsin. The results indicate that structural properties of the E2 component of branched-chain alpha-keto acid dehydrogenase are similar but not identical to those of the transacetylase component of the pyruvate dehydrogenase complex.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1999

The importance of pyruvate availability to PDC activation and anaplerosis in human skeletal muscle.

No studies have singularly investigated the relationship between pyruvate availability, pyruvate dehydrogenase complex (PDC) activation, and anaplerosis in skeletal muscle. This is surprising given the functional importance attributed to these processes in normal and disease states. We investigated the effects of changing pyruvate availability with dichloroacetate (DCA), epinephrine, and pyruvate infusions on PDC activation and accumulation of acetyl groups and tricarboxylic acid (TCA) cycle intermediates (TCAI) in human muscle. DCA increased resting PDC activity sixfold (P < 0.05) but decreased the muscle TCAI pool (mmol/kg dry muscle) from 1.174 +/- 0.042 to 0.747 +/- 0.055 (P < 0.05). This was probably a result of pyruvate being diverted to acetyl-CoA and acetylcarnitine after near-maximal activation of PDC by DCA. Conversely, neither epinephrine nor pyruvate activated PDC. However, both increased the TCAI pool (1.128 +/- 0.076 to 1.614 +/- 0.188, P < 0.05 and 1.098 +/- 0.059 to 1.385 +/- 0.114, P < 0.05, respectively) by providing a readily available pool of pyruvate for anaplerosis. These data support the hypothesis that TCAI pool expansion is principally a reflection of increased muscle pyruvate availability and, together with our previous work (J. A. Timmons, S. M. Poucher, D. Constantin-Teodosiu, V. Worrall, I. A. Macdonald, and P. L. Greenhaff. J. Clin. Invest. 97: 879-883, 1996), indicate that TCA cycle expansion may be of little functional significance to TCA cycle flux. It would appear therefore that the primary effect of DCA on oxidative ATP provision is to provide a readily available pool of acetyl groups to the TCA cycle at the onset of exercise rather than increasing TCA cycle flux by expanding the TCAI pool.

Adult↗
PubMed1998

Crystal structure of eucaryotic E3, lipoamide dehydrogenase from yeast.

The crystal structure of eucaryotic lipoamide dehydrogenase from yeast has been determined by an X-ray analysis at 2.7 (partially at 2.4) A resolution. The enzyme has two identical subunits related by a pseudo twofold symmetry. The tertiary structure is similar to those of other procaryotic enzymes. The active site, consisting of FAD, Cys44, and Cys49 from one subunit and His457' from the other subunit, is highly conserved. This enzyme is directly bound to the core protein E2 of the 2-oxoglutarate dehydrogenase complex, whereas it is bound to the pyruvate dehydrogenase complex through a protein X. The calculated electrostatic potential suggests two characteristic regions for binding with these two proteins.

Amino Acid Sequence↗
PubMed1989

Interrelationships between glucose metabolism, energy state, and the cytosolic free calcium concentration in cortical synaptosomes from the guinea pig.

The stoichiometries of glycolysis and pyruvate oxidation were determined in cortical synaptosomes under varying rates of ATP consumption. Glycolysis was measured by using D-3-[3H]glucose as a marker and pyruvate oxidation by using D-3,4-[14C]glucose, which has to be metabolized to 1-[14C]pyruvate before being decarboxylated by the pyruvate dehydrogenase complex of intrasynaptosomal mitochondria. Cytosolic free Ca2+ concentration [( Ca2+]c) was determined in parallel and was manipulated by using EGTA in the incubation. The results show that in nonstimulated synaptosomes glycolysis and pyruvate oxidation are tightly coupled and stoichiometric. In the absence of Ca2+, when [Ca2+]c drops from 260 nM to 40 nM, glucose utilization increases, following the increase in energy demand, which has been shown to be due to elevated Na+ cycling. KCl depolarization, veratridine, and a mitochondrial uncoupler, carbonyl cyanide m-chlorophenylhydrazone, all stimulate glycolysis and pyruvate oxidation stoichiometrically, independently of the presence of external Ca2+. A rise in [Ca2+]c, therefore, is not required to regulate mitochondrial pyruvate metabolism. It is concluded that synaptosomes exhibit a high degree of respiratory control, that they rely on glucose oxidation for their energetics, and that stimulation of energy production can be achieved independently of changes in [Ca2+]c.

Adenosine Triphosphate↗
PubMed1987

Hepatic carbon flux after re-feeding. Hyperthyroidism blocks glycogen synthesis and the suppression of glucose output observed in response to carbohydrate re-feeding.

1. The work investigated hepatic glycogen synthesis and glucose output after the intragastric administration of glucose or glycerol or the provision of chow ad libitum to 48 h-starved euthyroid or hyperthyroid rats. 2. After glucose administration, glycogen synthesis via the indirect pathway [Newgard, Hirsch, Foster & McGarry (1983) J. Biol. Chem. 258, 8046-8052] occurred concomitantly with reversal of glucose flux across the liver and re-activation of pyruvate kinase in the euthyroid controls. Glycogen synthesis was decreased and net glucose output continued in the hyperthyroid rats, but normal re-activation of pyruvate kinase was observed. 3. Use of 3-mercaptopicolinate indicated that the glucose released from liver of hyperthyroid rats was synthesized from substrates metabolized via the gluconeogenic pathway. 4. Hepatic glycogen synthesis was also impaired in hyperthyroid rats after administration of glycerol or chow. Measurement of portal-minus-hepatovenous concentration differences and arterial glucose concentrations after the administration of glycerol in combination with 3-mercaptopicolinate indicated that flux from triose phosphate to glucose 6-phosphate was not decreased. 5. Inhibited glycogen synthesis after chow re-feeding was associated with accelerated re-activation of hepatic pyruvate dehydrogenase complex in the hyperthyroid rats. 6. The results indicate three distinct and independent actions of hyperthyroidism after re-feeding: (i) it inhibits the reversal of glucose flux across the liver normally observed in response to carbohydrate; (ii) it affects glycogen deposition at a site distal to glucose 6-phosphate; (iii) it allows more rapid re-activation of liver pyruvate dehydrogenase complex in response to a mixed diet.

Animals↗
PubMed2005

Mitochondrial dysfunction in a patient with Joubert syndrome.

Joubert syndrome is a genetically heterogeneous disorder. The diagnostic criteria include episodic hyperventilation, abnormal eye movements, psychomotor retardation, hypotonia, ataxia, and the characteristic neuro-imaging findings (molar-tooth sign). Many of these clinical features have been observed in new-borns with mitochondrial disorders as well. Congenital brain malformations, including cerebellar hypoplasia, have been described in pyruvate dehydrogenase deficiency. Malformations of the vermis and the cerebellar peduncles, with the lack of axonal decussations, however, are characteristic for Joubert syndrome but unique in patients with mitochondrial disorders. Here, we describe a child with Joubert syndrome presenting with primary lactic acidemia, decreased pyruvate oxidation rates, decreased ATP production, and a mildly decreased pyruvate dehydrogenase complex activity measured in a fresh muscle biopsy. Sequence analysis of the PDHc E1 alpha gene and the PDHX genes revealed no mutations. The patient received continuous feeding through a feeding tube for two years and showed a significant clinical improvement with a complete resolution of the chronic lactic acidemia. A second muscle biopsy revealed significantly decreased pyruvate oxidation rates and ATP production, but a normal pyruvate dehydrogenase complex activity. We suggest that the described mitochondrial dysfunction in our patient is secondary to an underlying mutation leading to Joubert syndrome.

Abnormalities, Multiple↗
PubMed1995

Holeboard maze-learning deficits and brain monoaminergic neurotransmitter concentrations in rats after intracerebroventricular injection of 3-bromopyruvate.

3-Bromopyruvate is a suicide inhibitor of pyruvate dehydrogenase complex in brain homogenates, and after intracerebral injection reduces acetylcholine tissue content and muscarinic cholinergic receptors in brain cortex and hippocampus for extended periods of time. A stereotaxic injection of 0.2 mumol 3-bromopyruvate was given twice into the cerebral ventricles of male Wistar rats. Ten weeks later, the animals were tested for learning deficits in a food-motivated complex holeboard test. 3-Bromopyruvate-treated rats showed an increased number of visits to nonfood-baited holes over a 5-day testing period (four trials per day) compared to sham-operated control rats, an increased number of visits to food-baited holes over the first 2 days of the testing period and an increased time for completing the task. There were no changes in brain monoaminergic neurotransmitter concentrations compared to controls. The results indicate that long-term learning deficits in a spatial discrimination paradigm are caused by 3-bromopyruvate, which might be related to a cholinergic deficit induced by a primary inhibition of brain glucose metabolism at the step of pyruvate dehydrogenase complex. This animal model may be useful for behavioral studies in relation to neurodegenerative diseases like dementia of Alzheimer type.

Animals↗
PubMed1996

Chemical modification of the essential arginine residues of pyruvate dehydrogenase prevents its phosphorylation by kinase.

The mechanism of regulatory phosphorylation of the pyruvate dehydrogenase component (E1) of muscle pyruvate dehydrogenase complex was studied. Chemical modification of the arginine residues essential for substrate binding was shown to prevent incorporation of 32P from [gamma-32P]ATP into E1 catalyzed by kinase and to exclude completely the interaction of holo-E1 with pyruvate. It is proposed that negatively charged phosphoseryl residues may compete with pyruvate for the active site arginine and thereby block the substrate binding.

Animals↗
PubMed1981

Energy metabolism in rat brain: inhibition of pyruvate decarboxylation by 3-hydroxybutyrate in neonatal mitochondria.

The effect of 3-hydroxybutyrate on pyruvate decarboxylation by neonatal rat brain mitochondria and synaptosomes was investigated. The rate of [1-14C]pyruvate decarboxylation (1 mM final concentration) by brain synaptosomes derived from 8-day-old rats was inhibited by 10% in the presence of 2 mM-D,L-3-hydroxybutyrate and by more than 20% in the presence of 20 mM D,L-3-hydroxybutyrate. The presence of 2 mM-D,L-3-hydroxybutyrate did not affect the rate of [1-14C]pyruvate decarboxylation (1 mM final concentration) by brain mitochondria; however, at a concentration of 20 mM-D,L-3-hydroxybutyrate, a marked inhibition was seen in preparations from both 8-hydroxybutyrate, a marked inhibition was seen in preparations from both 8-day-old (35% inhibition) and 21-day-old (24% inhibition) but not in those from adult rats. Although the presence of 100 mM-K+ in the incubation medium stimulated the rate of pyruvate decarboxylation by approximately 50% compared with the rate in presence of 1 mM-K+, the presence of 20 mM-D,L-3-hydroxybutyrate still caused a marked inhibition in both media (1 and 100 mM-K+). The presence of 20 mM-D,L-3-hydroxybutyrate during the incubation caused an approximately 20% decrease in the level of the active form of the pyruvate dehydrogenase complex in brain mitochondria from 8-day-old rats. The concentrations of ATP, ADP, NAD+, NADH, acetyl CoA, and CoA were measured in brain mitochondria from 8-day-old rats incubated in the presence of 1 mM-pyruvate alone or 1 mM-pyruvate plus 20 mM-D,L-3-hydroxybutyrate. Neither the APT/ADP nor the NADH/NAD+ ratio showed significant changes. The acetyl CoA/CoA ratio was significantly increased by more than twofold in the presence of 3-hydroxybutyrate. The possible mechanisms and physiological significance of 3-hydroxybutyrate inhibition of pyruvate decarboxylation in neonatal rat brain rat mitochondria are discussed.

3-Hydroxybutyric Acid↗
PubMed1983

Regulation of fatty acid oxidation in heart muscle. Effects of pyruvate and dichloroacetate.

The possibility of a mutual regulation between carbohydrate and fatty acid oxidation was studied in isolated perfused rat hearts. Infusions of pyruvate and/or dichloroacetate were employed to convert fully the pyruvate dehydrogenase complex into its active form during [1-14C]octanoate oxidation, the rate of which was measured by the production of 14CO2. It was found that 5 mM dichloroacetate suppressed the oxidation of 0.1 mM octanoate by 58%, but 10 mM external pyruvate was without effect. Dichloroacetate reduced the tissue malate concentration by 41% and the citrate concentration by 76% at a 0.1 mM octanoate concentration, but had no effect on the metabolite concentrations or fatty acid oxidation rate in perfusions with 1 mM octanoate. Metabolite depletion was probably partly due to inhibition of the carboxylation of pyruvate, as verified by determination of the metabolite labelling kinetics from [1-14C]pyruvate. It is, therefore, possible that the dichloroacetate-induced inhibition of octanoate oxidation could also be partly due to inhibition of tricarboxylic acid cycle secondary to metabolite depletion. Since both dichloroacetate and pyruvate converted pyruvate dehydrogenase to its active form, but only dichloroacetate inhibited fatty acid oxidation, the latter effect could not be due to oxidation of a competing substrate, but instead may result from an inhibition of fatty acid uptake, activation or transport, as also indicated by the observed decrease in the acid-soluble acyl-CoA concentration. This interpretation is also supported by the mitochondrial redox effects of dichloroacetate observed during octanoate oxidation in the perfused heart.

Acetates↗
PubMed1980

Pyruvate dehydrogenase activity in group N streptococci.

Pyruvate dehydrogenase activity was detected in whole cells but not in cell-free extracts of Streptococcus lactis. However, the three component enzymes (pyruvate decarboxylase, lipoate acetyltransferase and lipoyl dehydrogenase) of the pyruvate dehydrogenase complex were identified in the cell-free extracts. Whole cells of the three species of group N streptococci formed acetoin and diacetyl only after the pathway forming acetate had become saturated. S. lactis subsp. diacetylactis DRC2 formed more acetoin and diacetyl and less acetate from pyruvate than did S. lactis C10. Strains C10 and DRC2 were able to form acetoin via alpha-acetolactate or diacetyl and to convert acetoin to butane-2,3-diol. S. cremoris HP was able to form acetoin only via alpha-acetolactate and could not convert acetoin to butane-2,3-diol.

Acetyltransferases↗
PubMed2003

Glycolysis and pyruvate oxidation in cardiac hypertrophy--why so unbalanced?

Cardiac hypertrophy, induced by chronic pressure or volume overload, is associated with abnormalities in energy metabolism as well as characteristic increases in muscle mass and alterations in the structure of the heart. Hypertrophied hearts display increased rates of glycolysis and overall glucose utilization, but rates of pyruvate oxidation do not rise in step with rates of pyruvate generation. Glycolysis and glucose oxidation, therefore, become markedly less 'coupled' in hypertrophied hearts than in non-hypertrophied hearts. Because the pyruvate dehydrogenase complex (PDC) contributes so powerfully to the control of glucose oxidation, we set out to test the hypothesis that the function of PDC is impaired in cardiac hypertrophy. In this review we describe evidence indicating that the alterations in glucose metabolism in hypertrophied hearts cannot be explained simply by changes in PDC expression or control. Additional mechanisms that may lead to an altered balance of pyruvate metabolism in cardiac hypertrophy are discussed, with commentaries on possible changes in pyruvate transport, NADH shuttles, lactate dehydrogenase, and amino acid metabolism.

Animals↗
PubMed2001

Redirection of pyruvate catabolism in Lactococcus lactis by selection of mutants with additional growth requirements.

Based on requirements for acetate or lipoic acid for aerobic (but not anaerobic) growth, Lactococcus lactis subsp. lactis mutants with impaired pyruvate catabolism were isolated following classical mutagenesis. Strains with defects in one or two of the enzymes, pyruvate formate-lyase (PFL), lactate dehydrogenase (LDH) and the pyruvate dehydrogenase complex (PDHC) were obtained. Growth and product formation of these strains were characterized. A PFL-defective strain (requiring acetate for anaerobic growth) displayed a two-fold increase in specific lactate production compared with the corresponding wild-type strain when grown anaerobically. LDH defective strains directed 91-96% of the pyruvate towards alpha-acetolactate, acetoin and diacetyl production when grown aerobically in the presence of acetate and absence of lipoic acid (a similar characteristic was observed in an LDH and PDHC defective strain in the presence of both acetate and lipoic acid) and more than 65% towards formate, acetate and ethanol production under anaerobic conditions. Another strain with defective PFL and LDH was strictly aerobic. However, a variant with strongly enhanced diacetyl reductase activities (NADH/NAD+ dependent diacetyl reductase, acetoin reductase and butanediol dehydrogenase activities) was selected from this strain under anaerobic conditions by supplementing the medium with acetoin. This strain is strictly aerobic, unless supplied with acetoin.

Acetyltransferases↗
PubMed1984

Parallel stimulation by Ca2+ of inotropism and pyruvate dehydrogenase in perfused heart.

The effects of extracellular Ca2+ (0.375-3.75 mM) on pyruvate oxidation and active form of the pyruvate dehydrogenase complex (PDCa) were quantitated in perfused guinea pig hearts in relation to inotropism, hydraulic work performance, and myocardial oxygen uptake (MVO2). The effects of afterload and norepinephrine (NE), alone or combined with the Ca2+ channel blocker D 600, were also examined. Hearts utilized 1-5 mM pyruvate in presence of 5 mM DL-3-hydroxybutyrate as substrates. Pyruvate oxidation and MVO2 increased essentially in parallel regardless of whether inotropism and energy metabolism were stimulated by increasing the Ca2+ concentration [( Ca2+]), the NE concentration [( NE]), or the afterload. PDCa activity was also directly related to [Ca2+], [NE], and afterload, respectively. Elevated [Ca2+] failed, however, to stimulate pyruvate oxidation and PDCa activity when MVO2 was held constant by an appropriate decrease in afterload at constant preload. Compound D 600, theophylline, and dibutyryl adenosine 3',5'-cyclic monophosphate also produced parallel alterations in cardiac mechanics, pyruvate oxidation, and MVO2. The striking proportionality between PDCa parameters, MVO2, and cardiac mechanics during the various alterations in cellular Ca2+ metabolism seemed to suggest that the observed Ca2+ stimulation of the PDC might be mainly secondary to increased myocardial energy utilization and myocyte respiration. Evidence for an additional direct effect of Ca2+ on the intact PDC system was not obtained.

Animals↗