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PubMed1972

Mechanisms regulating adipose-tissue pyruvate dehydrogenase.

1. Isolated rat epididymal fat-cell mitochondria showed an inverse relationship between ATP content and pyruvate dehydrogenase activity consistent with competitive inhibition of pyruvate dehydrogenase kinase by ADP. At constant ATP concentration pyruvate rapidly activated pyruvate dehydrogenase in fat-cell mitochondria, an observation consistent with inhibition of fat-cell pyruvate dehydrogenase kinase by pyruvate. Pyruvate dehydrogenase in fat-cell mitochondria was also activated by nicotinate (100mum) and by extramitochondrial Na(+) (replacing K(+)) but not by ouabain or insulin. 2. In rat epididymal fat-pads incubated in vitro pyruvate dehydrogenase was activated by addition of insulin in the absence of substrate or in the presence of glucose (10mm) or fructose (10mm). Glucose and fructose activated the dehydrogenase in the absence or in the presence of insulin, and pyruvate also activated in the absence of insulin. It is concluded that extracellular glucose, fructose and pyruvate may activate the dehydrogenase by raising intracellular pyruvate and that insulin may activate the dehydrogenase by some other mechanism. 3. Ouabain (300mum) and medium in which K(+) was replaced by Na(+), activated pyruvate dehydrogenase in epididymal fat-pads. Prostaglandin E(1) (1mug/ml), 5-methylpyrazole-3-carboxylate (10mum) and nicotinate (10mum), which are as effective as insulin as inhibitors of lipolysis and which like insulin lower tissue concentration of cyclic AMP (adenosine 3':5'-cyclic monophosphate), did not activate pyruvate dehydrogenase. Higher concentrations of prostaglandin E(1) (10mug/ml) and nicotinate (100mum) produced some activation of the dehydrogenase. 4. It is concluded that the activation of pyruvate dehydrogenase by insulin is not due to the antilipolytic effect of the hormone and that the action of insulin in lowering adipose-cell concentrations of cyclic AMP does not afford an obvious explanation for the effect of the hormone on pyruvate dehydrogenase. The possibility that the effects of insulin, ouabain and K(+)-free medium may be mediated by Ca(2+) is discussed.

Adenosine Diphosphate↗

PubMed1975

The mitochondrial pyruvate carrier. Kinetics and specificity for substrates and inhibitors.

1. Studies on the kinetics of pyruvate transport into mitochondria by an 'inhibitor-stop' technique were hampered by the decarboxylation of pyruvate by mitochondria even in the presence of rotenone. Decarboxylation was minimal at 6 degrees C. At this temperature the Km for pyruvate was 0.15 mM and Vmax. was 0.54nmol/min per mg of protein; alpha-cyano-4-hydroxycinnamate was found to be a non-competitive inhibitor, Ki 6.3 muM, and phenyl-pyruvate a competitive inhibitor, Ki 1.8 mM. 2. At 100 muM concentration, alpha-cyano-4-hydroxycinnamate rapidly and almost totally inhibited O2 uptake by rat heart mitochondria oxidizing pyruvate. Inhibition could be detected at concentrations of inhibitor as low as 1 muM although inhibition took time to develop at this concentration. Inhibition could be reversed by diluting out the inhibitor. 3. Various analogues of alpha-cyano-4-hydroxycinnamate were tested on rat liver and heart mitochondria. The important structural features appeared to be the alpha-cyanopropenoate group and the hydrophobic aromatic side chain. Alpha-Cyanocinnamate, alpha-cyano-5-phenyl-2,4-pentadienoate and compound UK 5099 [alpha-cyano-beta-(2-phenylindol-3-yl)acrylate] were all more powerful inhibitors than alpha-cyano-4-hydroxycinnamate showing 50% inhibition of pyruvate-dependent O2 consumption by rat heart mitochondria at concentrations of 200, 200 and 50 nM respectively. 4. The specificity of the carrier for its substrate was studied by both influx and efflux experiments. Oxamate, 2-oxobutyrate, phenylpyruvate, 2-oxo-4-methyl-pentanoate, chloroacetate, dichloroacetate, difluoroacetate, 2-chloropropionate, 3-chloropropionate and 2,2-dichloropropionate all exchanged with pyruvate, whereas acetate, lactate and trichloroacetate did not. 5. Pyruvate entry into the mitochondria was shown to be accompanied by the transport of a proton (or by exchange with an OH-ion). This proton flux was inhibited by alpha-cyano-4-hydroxycinnamate and allowed measurements of pyruvate transport at higher temperatures to be made. The activation energy of mitochondrial pyruvate transport was found to be 113 kJ (27 kcal)/mol and by extrapolation the rate of transport of pyruvate at 37 degrees C to be 42 nmol/min per mg of protein. The possibility that pyruvate transport into mitochondria may be rate limiting and involved in the regulation of gluconegenesis is discussed. 6. The transport of various monocarboxylic acids into mitochondria was studied by monitoring proton influx. The transport of dichloroacetate, difluoroacetate and oxamate appeared to be largely dependent on the pyruvate carrier and could be inhibited by pyruvate-transport inhibitors. However, many other halogenated and 2-oxo acids which could exchange with pyruvate on the carrier entered freely even in the presence of inhibitor.

Animals↗

PubMed1976

Studies on the influence of fatty acids on pyruvate dehydrogenase interconversion in rat-liver mitochondria.

1. The effect of fatty acids on the interconversion of pyruvate dehydrogenase between its active (nonphosphorylated) and inactive (phosphorylated) forms was measured in rat liver mitochondria respiring in state 3 with pyruvate plus malate and 2-oxoglutarate plus malate and during state 4 to state 3 transition in the presence of different substrates. The content of intramitochondrial adenine nucleotides was determined in the parallel experiments. 2. Decrease of the intramitochondrial ATP/ADP ratio with propionate and its increase with palmitoyl-L-carnitine in state 3 is accompanied by a shift of the steady-state of the pyruvate dehydrogenase system towards the active or the inactive form, respectively. 3. Transition from the high energy state (state4) to the active respiration (state3) in mitochondria oxidizing 2-oxoglutarate or plamitoyl-L-carnitine causes an increase of the amount of the active form of pyruvate dehydrogenase due to the decrease of ATP/ADP ratio in the matrix. 4. No change in ATP/ADP ratio can be observed in the presence of octanoate in mitochondria oxidizing pyruvate or 2-oxoglutarate in state 3 or during state 4 to state 3 transition. Simultanelusly, no significant change in phosphorylation state of pyruvate dehydrogenase occurs and a low amount of the enzyme in the active form is present with octanoate or octanoate plus 2-oxoglutarate. Pyruvate abolishes this effect of octanoate and shifts the steady-state of pyruvate dehydrogenase system towards the active form. 5. These results indicate that fatty acids influence the interconversion of pyruvate dehydrogenase mainly by changing intramitochondrial ATP/ADP ratio. However, the comparison of the steady-state level of the pyruvate dehydrogenase system in the presence of different substrates in various metabolic conditions provides some evidence that accumulation of acetyl-CoA and high level of NADH may promote the phosphorylation of pyruvate dehydrogenase. 6. Pyruvate exerts its protective effect against phosphorylation of pyruvate dehydrogenase in the presence of fatty acids of short, medium or long chain in a manner which depends on its concentration. It is suggested that in isolated mitochondria pyruvate counteracts the effect of acetyl-CoA and NADH on pyruvate dehydrogenase kinase.

Adenosine Diphosphate↗

PubMed1994

Mechanisms by which dichloroacetate lowers lactic acid levels: the kinetic interrelationships between lactate, pyruvate, alanine, and glucose.

Dichloroacetate (DCA) is gaining use as an alternative to bicarbonate therapy in the treatment of lactic acidosis. To determine the mechanism(s) by which DCA lowers blood lactate levels, we studied its effect on the kinetic interrelationships between pyruvate, lactate, alanine, and glucose in the hindlimb of dogs during hormonal stimulation of pyruvate production (Ra) and its conversion to lactate. Three groups of dogs (n = 6) were infused with 1-13C-pyruvate to measure whole body pyruvate Ra, and pyruvate Ra and utilization (Rd) across the hindlimb during either a 4-hr infusion of saline (controls), or somatostatin, glucagon, and epinephrine (SGE), or SGE plus dichloroacetate (SGE + DCA). Pyruvate Ra was used as an index of rate of glycolysis and Rd as an index of pyruvate oxidation. In the controls, all kinetic parameters were constant during the saline infusion. Hindlimb pyruvate Ra and Rd were almost equal, and lactate release negligible. Compared to controls, SGE administration significantly increased (P < 0.05) wholebody pyruvate Ra (48.5 +/- 6.2 vs 33.6 +/- 2.4 mumol/kg/min) and blood lactate levels (P < 0.05). Hindlimb pyruvate Ra increased by approximately 150%, but Rd remained unchanged resulting in marked increases in lactate and alanine effluxes. Adding DCA to the SGE infusion significantly reduced wholebody pyruvate Ra (P < 0.05) and blood lactate levels (P < 0.01). In the hindlimb, however, there was no decrease in lactate output, despite a 91% increase in pyruvate utilization because pyruvate Ra also increased. These results suggest that during stimulation of rate of glycolysis, DCA lowers lactate levels by reducing the overall availability of pyruvate for lactate synthesis. This is accomplished by suppressing the rate of glycolysis in tissues other than skeletal muscle and stimulating pyruvate oxidation.

Alanine↗

PubMed1989

Pyruvate metabolism by lymphocytes: evidence for an additional ketogenic tissue.

The rate of utilization of pyruvate (at various concentrations) was measured in lymphocytes prepared from rat mesenteric lymph nodes. The quantitative contribution of pyruvate to CO2, lactate, aspartate, alanine, citrate, acetate, acetyl-CoA and ketone bodies accounted for the pyruvate metabolized. Pyruvate utilization was depressed by increasing concentrations of pyruvate. The maximum catalytic activities and selected intracellular distributions of the following enzymes of pyruvate, citrate and acetyl-CoA metabolism were measured: citrate synthase, ATP-citrate lyase, lactate dehydrogenase, acetyl-CoA hydrolase, acetylcarnitine transferase, NAD+- and NADP+- isocitrate dehydrogenases, HMG-CoA lyase, HMG-CoA synthase, Pyruvate dehydrogenase, acetoacetyl-CoA thiolase, 3-oxoacid-CoA transferase, 3-hydroxybutyrate dehydrogenase and pyruvate carboxylase. Acetyl-CoA formed from pyruvate did not contribute to the respiratory energy metabolism of resting lymphocytes. Instead acetyl-CoA was converted to acetoacetate by reactions which may favour the pathway catalyzed by acetoacetyl-CoA thiolase and 3-oxoacid-CoA transferase. Acetate, acetyl- and palmitoyl-carnitine inhibited the decarboxylation of [1-14C] pyruvate. These observations may be connected with the suppression of pyruvate utilization by increased pyruvate substrate concentration. Only very small amounts of either pyruvate or acetate were incorporated into lipids in resting lymphocytes. The amounts incorporated were partitioned in approximately the same pattern into FFA, T.G., cholesterol and cholesterol esters. Taken together the data show that pyruvate metabolism is directed inter alia at the formation of acetoacetate which may serve as a lipid synthesis precursor. When pyruvate utilization and metabolism was enhanced by concanavalin A, then acetoacetate formation was not favoured and from this it is proposed that the acetyl units may then be directed into lipid synthesis and may also make a contribution to the energy metabolism of the activated lymphocyte.

Animals↗

PubMed1985

The effects of cyclopropane carboxylate on hepatic pyruvate metabolism.

The effects of cyclopropane carboxylate on gluconeogenesis and pyruvate decarboxylation from [1-14C]-labeled pyruvate and lactate were investigated in perfused livers from fasted rats. With high concentrations of pyruvate (greater than or equal to 0.5 mM) in the perfusion medium, infusion of cyclopropane carboxylate inhibited pyruvate decarboxylation and gluconeogenesis by 30 and 40%, respectively. With low, more physiological concentrations of pyruvate (50 microM) or with lactate (1 mM), cyclopropane carboxylate, at a concentration which elicits maximal inhibition of pyruvate decarboxylation from pyruvate (greater than or equal to 0.5 mM), did not affect either pyruvate decarboxylation or gluconeogenesis. Evidence is presented for the rapid formation of the coenzyme-A ester of cyclopropane carboxylate in perfused livers. Infusion of l-(-)carnitine (20 mM) prevented the inhibitory effects of cyclopropane carboxylate on pyruvate decarboxylation and gluconeogenesis from pyruvate (greater than or equal to 0.5 mM). Interestingly, no decrease in the tissue level of cyclopropanecarboxyl-CoA occurs under these conditions. The present study suggests that cyclopropane carboxylate, through a presently ill-defined mediator, inhibits pyruvate decarboxylation and gluconeogenesis by interfering with the pyruvate----oxalacetate----phosphoenolpyruvate----pyruvate cycle when pyruvate (greater than or equal to 0.5mM) supports gluconeogenesis.

Acyl Coenzyme A↗

PubMed1983

Effects of physiologic concentrations of lactate, pyruvate and ascorbate on glucose metabolism in unstressed and oxidatively stressed human red blood cells.

Glucose metabolism was studied in human red blood cells incubated in the presence of physiologic concentrations of ascorbate (0.1 mM) and/or lactate (2 mM) plus pyruvate (0.1 mM). The total flux through glycolysis, as measured by 14C-labeling of glycolytic intermediates, was increased about 15% by ascorbate, 30% by lactate plus pyruvate, and 40% by ascorbate plus lactate plus pyruvate. We found, however, that physiologic concentrations of ascorbate and/or lactate plus pyruvate had no effect on flux of glucose or recycling of pentoses through the hexose monophosphate shunt. Increased formation of lactate accounted for most of the observed increase in glycolysis with little change in pyruvate formation, indicating that the increased flux of reducing equivalents from glucose was stored as lactate rather than being consumed by red cell metabolism. In all experiments, there was a net increase with time in the absolute amount of both lactate and pyruvate in red cell suspensions, indicating that lactate or pyruvate present at zero time did not function as a stoichiometric source or sink for reducing equivalents. There was little effect on steady-state levels of ATP or 2,3-diphosphoglycerate. Equilibration of ascorbate between red cells and the medium was complete before the addition of 14C-labeled glucose to the medium. Glucose metabolism prevented net oxidation of ascorbate in the incubation medium. Physiologic concentrations of ascorbate, lactate and pyruvate appear to increase flux through glycolysis by increasing the turnover of ATP and/or 2,3-diphosphoglycerate. Red cells were exposed to mild oxidative stress by incubation with 0.27 mM 6-hydroxydopamine, 0.27 mM 6-aminodopamine, 0.13 mM 1,4-naphthoquinone-2-sulfonic acid or 0.27 mM phenylhydrazine. The metabolic response to oxidative stress was determined by measuring the formation of methemoglobin, pyruvate, lactate and CO2 in the presence and absence of physiologic concentrations of lactate, pyruvate and ascorbate. Lactate, pyruvate and ascorbate had no effect on the net methemoglobin accumulation but rather on the distribution of the metabolic sources of reducing equivalents and on the flux of reducing equivalents to oxygen. Physiologic lactate and pyruvate allowed increased flow of reducing equivalents from glycolysis to methemoglobin and ultimately oxygen without the necessity of increased flux through glycolysis. This was accomplished by a decrease in the ratio of newly formed lactate to newly formed pyruvate with no increase in total lactate plus pyruvate.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗

PubMedDate not supplied

Metabolic control analysis of eucaryotic pyruvate dehydrogenase multienzyme complex.

Metabolic control analysis (MCA) of pyruvate dehydrogenase multienzyme (PDH) complex of eucaryotic cells has been carried out using both in vitro and in vivo mechanistic models. Flux control coefficients (FCC) for the sensitivity of pyruvate decarboxylation rate to activities of various PDH complex reactions are determined. FCCs are shown to be strong functions of both pyruvate levels and various components of PDH complex. With the in vitro model, FCCs are shown to be sensitive to only the E1 component of the PDH complex at low pyruvate concentrations. At high pyruvate concentrations, the control is shared by all of the components, with E1 having a negative influence while the other three components, E2, X, and K, exert a positive control over the pyruvate decarboxylation rate. An unusual behavior of deactivation of the E1 component leading to higher net PDH activity is shown to be linked to the combined effect of protein X acylation and E1 deactivation. The steady-state analysis of the in vivo model reveals multiple steady state behavior of pyruvate metabolism with two stable and one unstable steady-states branches. FCCs also display multiplicity, showing completely different control distribution exerted by pyruvate and PDH components on three branches. At low pyruvate concentrations, pyruvate supply dominates the decarboxylation rate and PDH components do not exert any significant control. Reverse control distribution is observed at high pyruvate concentration. The effect of dilution due to cell growth on pyruvate metabolism is investigated in detail. While pyruvate dilution effects are shown to be negligible under all conditions, significant PDH complex dilution effects are observed under certain conditions. Comparison of in vitro and in vivo models shows that PDH components exert different degrees of control outside and inside the cells. At high pyruvate levels, PDH components are shown to exert a higher degree of control when reactions are taking place inside the cells as compared to the in vitro situation.

Cell Division↗

PubMed2002

Methyl pyruvate stimulates pancreatic beta-cells by a direct effect on KATP channels, and not as a mitochondrial substrate.

In pancreatic beta-cells, methyl pyruvate is a potent secretagogue and is widely used to study stimulus-secretion coupling. In contrast with pyruvate, which barely stimulates insulin secretion, methyl pyruvate was suggested to act as an effective mitochondrial substrate. We show that methyl pyruvate elicited electrical activity in the presence of 0.5 mM glucose, in contrast with pyruvate. Accordingly, methyl pyruvate increased the cytosolic free Ca(2+) concentration after an initial decrease, similar to glucose. The initial decrease was inhibited by thapsigargin, suggesting that methyl pyruvate stimulates ATP production. This assumption is supported by the observation that methyl pyruvate hyperpolarized the mitochondrial membrane potential, similar to glucose. However, in contrast with glucose, methyl pyruvate even slightly decreased NAD(P)H autofluorescence and did not influence ATP production or the ATP/ADP ratio. This observation questions the suggestion that methyl pyruvate acts as a powerful mitochondrial substrate. The finding that methyl pyruvate directly inhibited a cation current across the inner membrane of Jurkat T-lymphocyte mitochondria suggests that this metabolite may increase ATP production in beta-cells by activating the respiratory chains without providing reduction equivalents. We conclude that this mechanism may account for a slight and transient increase in ATP production. We further show that methyl pyruvate inhibited the K(ATP) current measured in the standard whole-cell configuration, an effect that was at least partly antagonized by diazoxide. Accordingly, single-channel currents in inside-out patches were blocked by methyl pyruvate. We conclude that inhibition of K(ATP) channels, and not activation of metabolism, mediates the induction of electrical activity in pancreatic beta-cells by methyl pyruvate.

Adenosine Diphosphate↗

PubMed1978

The effect of fatty acids on the regulation of pyruvate dehydrogenase in perfused rat liver.

The effect of fatty acids on the rate of pyruvate decarboxylation was studied in perfused livers from fed rats. The production of 14CO2 from infused [1-14C]pyruvate was employed as a monitor of the flux through the pyruvate dehydrogenase reaction. A correction for other decarboxylation reactions was made using kinetic analyses. Fatty acid (octanoate or oleate) infusion caused a stimulation of pyruvate decarboxylation at pyruvate concentrations in the perfusate below 1 mM (up to 3-fold at 0.05 mM pyruvate) but decreased the rate to one-third of control rates at pyruvate concentrations near 5 mM. These effects were half-maximal at fatty acid concentrations below 0.1 mM. Infusion of 3-hydroxybutyrate also caused a marked stimulation of pyruvate decarboxylation at low pyruvate concentrations. The data suggest that the mechanism by which fatty acids stimulate the flux through the pyruvate dehydrogenase reaction in perfused liver at low (limiting) pyruvate concentrations involves an acceleration of pyruvate transport into the mitochondrial compartment due to an exchange with acetoacetate. Furthermore, it is proposed that a relationship exists between ketogenesis and the regulation of pyruvate oxidation at pyruvate concentrations approximating conditions in vivo.

Acetoacetates↗

PubMed1995

Growth of Methanosarcina barkeri (Fusaro) under nonmethanogenic conditions by the fermentation of pyruvate to acetate: ATP synthesis via the mechanism of substrate level phosphorylation.

A mutant of Methanosarcina barkeri (Fusaro) is able to grow on pyruvate as the sole carbon and energy source. During growth, pyruvate is converted to CH4 and CO2, and about 1.5 mol of ATP per mol of CH4 is formed (A.-K. Bock, A. Prieger-Kraft, and P. Schönheit, Arch. Microbiol. 161:33-46, 1994). The pyruvate-utilizing mutant of M. barkeri could also grow on pyruvate when methanogenesis was completely inhibited by bromoethanesulfonate (BES). The mutant grew on pyruvate (80 mM) in the presence of 2 mM BES with a doubling time of about 30 h up to cell densities of about 400 mg (dry weight) of cells per liter. During growth on pyruvate, the major fermentation products were acetate and CO2 (about 0.9 mol each per mol of pyruvate). Small amounts of acetoin, acetolactate, alanine, leucine, isoleucine, and valine were also detected. CH4 was not formed. The molar growth yield (Yacetate) was about 9 g of cells (dry weight) per mol of acetate, indicating an ATP yield of about 1 mol/mol of acetate formed. Growth on pyruvate in the presence of BES was limited; after six to eight generations, the doubling times increased and the final cell densities decreased. After 9 to 11 generations, growth stopped completely. In the presence of BES, suspensions of pyruvate-grown cells fermented pyruvate to acetate, CO2, and H2. CH4 was not formed. Conversion of pyruvate to acetate, in the complete absence of methanogenesis, was coupled to ATP synthesis. Dicyclohexylcarbodiimide, an inhibitor of H(+)-translocating ATP synthase, did not inhibit ATP formation. In the presence of dicyclohexylcarbodiimide, stoichiometries of up to 0.9 mol of ATP per mol of acetate were observed. The uncoupler arsenate completely inhibited ATP synthesis, while the rates of acetate, CO2, and H2 formation were stimulated up to fourfold. Cell extracts of M. barkeri grown on pyruvate under nonmethenogenic conditions contained pyruvate: ferredoxin oxidoreductase (0.5 U/mg), phosphate acetyltransferase (12 U/mg), and acetate kinase (12 U/mg). From these data it is concluded that ATP was synthesized by substrate level phosphorylation during growth of the M. barkeri mutant on pyruvate in the absence of methanogenesis. This is the first report of growth of a methanogen under nonmethanogenic conditions at the expense of a fermentative energy metabolism.

Acetates↗

PubMed1975

Control of pyruvate dehydrogenase activity in intact cardiac mitochondria. Regulation of the inactivation and activation of the dehydrogenase.

The control of pyruvate dehydrogenase activity by inactivation and activation was studied in intact mitochondria isolated from rabbit heart. Pyruvate dehydrogenase could be completely inactivated by incubating mitochondria with ATP, oligomycin, and NaF. This loss in dehydrogenase activity was correlated with the incorporation of 32P from [gamma-32P]ATP into mitochondrial protein(s) and with a decrease in the mitochondrial oxidation of pyruvate. ATP may be supplied exogenously, generated from endogenous ADP during oxidative phosphorylation, or formed from exogenous ADP in carbonyl cyanid p-trifluoromethoxyphenylhydrazone-uncoupled mitochondria. With coupled mitochondria the concentration of added ATP required to half-inactivate the dehydrogenase was 0.24 mM. With uncoupled mitochondria the apparent Km was decreased to 60 muM ATP. Inactivation of pyruvate dehydrogenase by exogenous ATP was sensitive to atractyloside, suggesting that pyruvate dehydrogenase kinase acts internally to the atractyloside-sensitive barrier. The divalent cation ionophore, A23187, enhanced the loss of dehydrogenase activity. Pyruvate dehydrogenase activity is regulated additionally by pyruvate, inorganic phosphate, and ADP. Pyruvate, in the presence of rotenone, strongly inhibited inactivation. This suggests that pyruvate facilitates its own oxidation and that increases in pyruvate dehydrogenase activity by substrate may provide a modulating influence on the utilization of pyruvate via the tricarboxylate cycle. Inorganic phosphate protected the dehydrogenase from inactivation by ATP. ADP added to the incubation mixture together with ATP inhibited the inactivation of pyruvate dehydrogenase. This protection may result from a direct action on pyruvate dehydrogenase kinase, as ADP competes with ATP, and an indirect action, in that ADP competes with ATP for the translocase. It is suggested that the intramitochondrial [ATP]:[ADP] ratio effects the kinase activity directly, whereas the cytosolic [ATP]:[ADP] ratio acts indirectly. Mg2+ enhances the rate of reactivation of the inactivated pyruvate dehydrogenase presumably by accelerating the rate of dephosphorylation of the enzyme. Maximal activation is obtained with the addition of 0.5 mM Mg2+..

Adenosine Diphosphate↗

PubMed2001

The effects of feed and intracellular pyruvate levels on the redistribution of metabolic fluxes in Escherichia coli.

In a previous study, an Escherichia coli strain lacking the key enzymes (acetate kinase and phosphotransacetylase, ACK-PTA) of the major acetate synthesis pathways reduced acetate accumulation. The ackA-pta mutant strain also exhibits an increased lactate synthesis rate. Metabolic flux analysis suggested that the majority of excessive carbon flux was redirected through the lactate formation pathway rather than the ethanol synthesis pathway. This result indicated that lactate dehydrogenase may be competitive at the pyruvate node. However, a 10-fold overexpression of the fermentative lactate dehydrogenase (ldhA) gene in the wild-type parent GJT001 was not able to divert carbon flux from acetate. The carbon flux through pyruvate and all its end products increases at the expense of flux through biosynthesis and succinate. Intracellular pyruvate measurements showed that strains overexpressing lactate dehydrogenase (LDH) depleted the pyruvate pool. This observation along with the observed excretion of pyruvate in the ackA-pta strain indicates the significance of intracellular pyruvate pools. In the current study, we focus on the role of the intracellular pyruvate pool in the redirection of metabolic fluxes at this important node. An increasing level of extracellular pyruvate leads to an increase in the intracellular pyruvate pool. This increase in intracellular pyruvate affects carbon flux distribution at the pyruvate node. Partitioning of the carbon flux to acetate at the expense of ethanol occurs at the acetyl-CoA node while partitioning at the pyruvate node favors lactate formation. The increased competitiveness of the lactate pathway may be due to the allosteric activation of LDH as a result of increased pyruvate levels. The interaction between the reactions catalyzed by the enzymes PFL (pyruvate formate lyase) and LDH was examined.

Acetyltransferases↗

PubMed1976

Dual divalent cation requirement for activation of pyruvate kinase; essential roles of both enzyme- and nucleotide-bound metal ions.

Rabbit muscle pyruvate kinase requires two divalent cations per active site for catalysis of the enolization of pyruvate in the presence of adenosine 5'-triphosphate (ATP). One divalent cation is bound directly to the enzyme and forms a second sphere complex with the bound ATP (site 1). The second divalent cation is directly coordinated to the phosphoryl groups of ATP and does not interact with the enzyme (site 2). The essential role of the divalent cation at site 1 is shown by the requirement for Mg2+ or Mn2+ for the enolization of pyruvate in the presence of the substitution inert Cr3+-ATP complex. The rate of detritiation of pyruvate shows a hyperbolic dependence of Mn2+ concentration in the presence of high concentrations of enzyme and Cr3+-ATP. A dissociation constant for Mn2+ from the pyruvate kinase-Mn2+-ATP-Cr3+-pyruvate complex of 1.3 +/- 0.5 muM is determined by the kinetics of detritiation of pyruvate and by parallel Mn2+ binding studies using electron paramagnetic resonance. The essential role of the divalent cation at site 2 is shown by the sigmoidal dependence of the rate of detritiation of pyruvate on Mn2+ concentration in the presence of high concentrations of enzyme and ATP yielding a dissociation constant of 29 +/- 9 muM for Mn2+ from site 2. This value is similar to the dissociation constant of the binary Mn-ATP complex (14 +/- 6 muM) determined under similar conditions. The rate of detritiation of pyruvate is proportional to the concentration of the pyruvate kinase-Mn2+-ATP-Mn2+-pyruvate complex, as determined by parellel kinetic and binding studies. Variation of the nature of the divalent cation at site 1 in the presence of CrATP causes only a twofold change in the rate of detritiation of pyruvate which does not correlate with the pKa of the metal-bound water. Variation of the nature of the divalent cation at both sites in the presence of ATP causes a sevenfold variation in the rate of detritiation or pyruvate that correlates with the pKa of the metal-bound water. The greater rate of enolization observed with CrATP fits this correlation, indicating that the electrophilicity of the nucleotide bound metal (at site 2) determines the rate of enolization of pyruvate.

Adenosine Triphosphate↗

PubMed1971

Regulation of adipose tissue pyruvate dehydrogenase by insulin and other hormones.

1. In epididymal adipose tissue synthesizing fatty acids from fructose in vitro, addition of insulin led to a moderate increase in fructose uptake, to a considerable increase in the flow of fructose carbon atoms to fatty acid, to a decrease in the steady-state concentration of lactate and pyruvate in the medium, and to net uptake of lactate and pyruvate from the medium. It is concluded that insulin accelerates a step in the span pyruvate-->fatty acid. 2. Mitochondria prepared from fat-cells exposed to insulin put out more citrate than non-insulin-treated controls under conditions where the oxaloacetate moiety of citrate was formed from pyruvate by pyruvate carboxylase and under conditions where it was formed from malate. This suggested that insulin treatment of fat-cells led to persistent activation of pyruvate dehydrogenase. 3. Insulin treatment of epididymal fat-pads in vitro increased the activity of pyruvate dehydrogenase measured in extracts of the tissue even in the absence of added substrate; the activities of pyruvate carboxylase, citrate synthase, glutamate dehydrogenase, acetyl-CoA carboxylase, NADP-malate dehydrogenase and NAD-malate dehydrogenase were not changed by insulin. 4. The effect of insulin on pyruvate dehydrogenase activity was inhibited by adrenaline, adrenocorticotrophic hormone and dibutyryl cyclic AMP (6-N,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate). The effect of insulin was not reproduced by prostaglandin E(1), which like insulin may lower the tissue concentration of cyclic AMP (adenosine 3':5'-cyclic monophosphate) and inhibit lipolysis. 5. Adipose tissue pyruvate dehydrogenase in extracts of mitochondria is almost totally inactivated by incubation with ATP and can then be reactivated by incubation with 10mm-Mg(2+). In this respect its properties are similar to that of pyruvate dehydrogenase from heart and kidney where evidence has been given that inactivation and activation are catalysed by an ATP-dependent kinase and a Mg(2+)-dependent phosphatase. Evidence is given that insulin may act by increasing the proportion of active (dephosphorylated) pyruvate dehydrogenase. 6. Cyclic AMP could not be shown to influence the activity of pyruvate dehydrogenase in mitochondria under various conditions of incubation. 7. These results are discussed in relation to the control of fatty acid synthesis in adipose tissue and the role of cyclic AMP in mediating the effects of insulin on pyruvate dehydrogenase.

Adipose Tissue↗

PubMed2001

Ringer's ethyl pyruvate solution ameliorates ischemia/reperfusion-induced intestinal mucosal injury in rats.

OBJECTIVE: Pyruvate has been shown to be protective in numerous in vitro and in vivo models of oxidant-mediated cellular or organ system injury. Unfortunately, the usefulness of pyruvate as a therapeutic agent is abrogated by its very poor stability in solution. In an effort to take advantage of the ability of pyruvate to scavenge reactive oxygen species while avoiding the problems associated with the instability of pyruvate in solution, we sought to determine whether a simple derivative, ethyl pyruvate, would be protective in an animal model of reactive oxygen species-mediated tissue injury, namely mesenteric ischemia and reperfusion in rats. DESIGN: Prospective, randomized trial. SETTING: Animal research center. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: Under general anesthesia, rats were subjected to 60 mins of mesenteric ischemia followed by 60 mins of reperfusion. Controls (n = 6) received intravenous lactated Ringer's solution according this dosing schedule: 1.5 mL/kg bolus before ischemia, 3.0 mL/kg bolus before resuscitation, and 1.5 mL.kg-1.hr-1 by continuous infusion. Two experimental groups received similar volumes of either pyruvate (n = 6 each) or ethyl pyruvate (n = 9) solution made up exactly like lactated Ringer's solution except for the substitution of either pyruvate or ethyl pyruvate for lactate, respectively. MEASUREMENTS AND MAIN RESULTS: To obtain tissues for assessing mucosal permeability and histology, five 10-cm long segments of small intestine were obtained at the following time points: baseline, after 30 and 60 mins of ischemia, and after 30 and 60 mins of reperfusion. Mucosal permeability to fluorescein isothiocyanate dextran (molecular weight 4000 Da) was assessed ex vivo by using an everted gut sac method. Compared with controls, treatment of rats with either pyruvate solution or ethyl pyruvate solution significantly ameliorated the development of intestinal mucosal hyperpermeability during the reperfusion. Treatment with ethyl pyruvate solution also significantly decreased the extent of histologic mucosal damage after mesenteric reperfusion. CONCLUSIONS: Treatment with Ringer's ethyl pyruvate solution ameliorated structural and functional damage to the intestinal mucosa in a rat model of mesenteric ischemia/reperfusion. Ethyl pyruvate solution warrants further evaluation as a novel therapeutic agent for preventing oxidant-mediated injury in various disease states.

Animals↗

PubMed1990

Phosphorylation of pyruvate kinase type K in human gliomas by a cyclic adenosine 5'-monophosphate-independent protein kinase.

In recent years, we reported the isozyme shift of pyruvate kinase from the M- toward the K-type in human neuroectodermal tumors. To investigate whether this shift enables phosphorylation of pyruvate kinase in these tumors, we studied 29 different specimens of human brain tumors for endogenous pyruvate kinase phosphorylation. While in normal human brain no phosphorylation of pyruvate kinase was detected, in all brain tumors pyruvate kinase became phosphorylated. There was no correlation between the extent of the pyruvate kinase phosphorylation and the histological classification and grading or the pyruvate kinase isozyme composition of the tumors. Only pyruvate kinase type K, and not type M, served as a substrate in the phosphorylation reaction. The phosphorylation of pyruvate kinase could be completely inhibited by addition of fructose 1,6-bisphosphate, a positive effector of pyruvate kinase type K; alanine, however, a negative effector, and phospho-enol-pyruvate, a substrate in the pyruvate kinase reaction, had no effect. While pyruvate kinase type L in liver is phosphorylated by a cyclic AMP-dependent protein kinase, the incorporation of phosphate into pyruvate kinase in human brain tumors appeared to be cyclic AMP independent and occurred exclusively on serine residues.

Biopsy↗

PubMed1985

The effects of alpha-adrenergic stimulation on the regulation of the pyruvate dehydrogenase complex in the perfused rat liver.

The regulation of the pyruvate dehydrogenase multienzyme complex was investigated during alpha-adrenergic stimulation with phenylephrine in the isolated perfused rat liver. The metabolic flux through the pyruvate dehydrogenase reaction was monitored by measuring the production of 14CO2 from infused [1-14C] pyruvate. In livers from fed animals perfused with a low concentration of pyruvate (0.05 mM), phenylephrine infusion significantly inhibited the rate of pyruvate decarboxylation without affecting the amount of pyruvate dehydrogenase in its active form. Also, phenylephrine caused no significant effect on tissue NADH/NAD+ and acetyl-CoA/CoASH ratios or on the kinetics of pyruvate decarboxylation in 14CO2 washout experiments. Phenylephrine inhibition of [1-14C]pyruvate decarboxylation was, however, closely associated with a decrease in the specific radioactivity of perfusate lactate, suggesting that the pyruvate decarboxylation response simply reflected dilution of the labeled pyruvate pool due to phenylephrine-stimulated glycogenolysis. This suggestion was confirmed in additional experiments which showed that the alpha-adrenergic-mediated inhibitory effect on pyruvate decarboxylation was reduced in livers perfused with a high concentration of pyruvate (1 mM) and was absent in livers from starved rats. Thus, alpha-adrenergic agonists do not exert short term regulatory effects on pyruvate dehydrogenase in the liver. Furthermore, the results suggest either that the rat liver pyruvate dehydrogenase complex is insensitive to changes in mitochondrial calcium or that changes in intramitochondrial calcium levels as a result of alpha-adrenergic stimulation are considerably less than suggested by others.

Ammonium Chloride↗