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PubMed1983

Dual role of a single multienzyme complex in the oxidative decarboxylation of pyruvate and branched-chain 2-oxo acids in Bacillus subtilis.

The pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase activities of Bacillus subtilis were found to co-purify as a single multienzyme complex. Mutants of B. subtilis with defects in the pyruvate decarboxylase (E1) and dihydrolipoamide dehydrogenase (E3) components of the pyruvate dehydrogenase complex were correspondingly affected in branched-chain 2-oxo acid dehydrogenase complex activity. Selective inhibition of the E1 or lipoate acetyltransferase (E2) components in vitro led to parallel losses in pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complex activity. The pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complexes of B. subtilis at the very least share many structural components, and are probably one and the same. The E3 component appeared to be identical for the pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complexes in this organism and to be the product of a single structural gene. Long-chain branched fatty acids are thought to be essential for maintaining membrane fluidity in B. subtilis, and it was observed that the ace (pyruvate dehydrogenase complex) mutant 61142 was unable rapidly to take up acetoacetate, unlike the wild-type, indicative of a defect in membrane permeability. A single pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complex can be seen as an economical means of supplying two different sets of essential metabolites.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1989

The kinetics of transport of lactate and pyruvate into isolated cardiac myocytes from guinea pig. Kinetic evidence for the presence of a carrier distinct from that in erythrocytes and hepatocytes.

1. Time courses for the uptake of L-lactate, D-lactate and pyruvate into isolated cardiac ventricular myocytes from guinea pig were determined at 11 degrees C or 0 degrees C (for pyruvate) in a citrate-based buffer by using a silicone-oil-filtration technique. These conditions enabled initial rates of transport to be measured without interference from metabolism of the substrates. 2. At a concentration of 0.5 mM, transport of all these substrates was inhibited by approx. 90% by 5 mM-alpha-cyano-4-hydroxycinnamate; at 10 mM-L-lactate a considerable portion of transport could not be inhibited. 3. Initial rates of L-lactate and pyruvate uptake in the presence of 5 mM-alpha-cyano-4-hydroxycinnamate were linearly related to the concentration of the monocarboxylate and probably represented diffusion of the free acid. The inhibitor-sensitive component of uptake obeyed Michaelis-Menten kinetics, with Km values for L-lactate and pyruvate of 2.3 and 0.066 mM respectively. 4. Pyruvate and D-lactate inhibited the transport of L-lactate, with Ki values (competitive) of 0.077 and 6.6 mM respectively; the Ki for pyruvate was very similar to its Km for transport. The Ki for alpha-cyano-4-hydroxycinnamate as a non-competitive inhibitor was 0.042 mM. 5. These results indicate that L-lactate, D-lactate and pyruvate share a common carrier in guinea-pig cardiac myocytes; the low stereoselectivity for L-lactate over D-lactate and the high affinity for pyruvate distinguish it from the carrier in erythrocytes and hepatocytes. The metabolic roles for this novel carrier in heart are discussed.

Animals↗
PubMed1993

The contribution of pyruvate cycling to loss of [6-3H]glucose during conversion of glucose to glycogen in hepatocytes: effects of insulin, glucose and acinar origin of hepatocytes.

1. During conversion of [6-3H,U-14C]glucose to glycogen in liver, loss of 6-3H can occur either by cycling via pyruvate (between glycolysis and gluconeogenesis) or by other mechanisms. We used mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase, to determine the extent to which pyruvate cycling contributes to loss of 6-3H during glucose conversion to glycogen in hepatocytes. 2. Mercaptopicolinate increased the 3H/14C ratio in glycogen during incubation of rat, guinea pig, pig and human hepatocytes with [6-3H,U-14C]glucose. The increase in the 3H/14C ratio in glycogen caused by mercaptopicolinate was greater in periportal than in perivenous rat hepatocytes, indicating that cycling of glucose via pyruvate is more prominent in cells with a higher gluconeogenic relative to glycolytic capacity. 3. The effect of mercaptopicolinate on the 3H/14C ratio in glycogen was observed both in the absence and in the presence of insulin, indicating that stimulation of glycogen synthesis by insulin is not associated with inhibition of pyruvate cycling. In rat and guinea pig but not in pig hepatocytes, the effects of mercaptopicolinate on the 3H/14C ratio in glycogen were greater at 10-15 mM glucose than at 30 mM glucose, suggesting diminished cycling via pyruvate at high glucose concentrations. 4. Insulin increased the loss of 6-3H during stimulation of conversion of glucose to glycogen in hepatocytes from all species. This was due in part to an increase in pyruvate cycling and in part to other mechanisms that are not inhibited by mercaptopicolinate. 5. These results suggest that pyruvate cycling is a significant, but not exclusive, component of the loss of 6-3H in the hepatocyte during glucose conversion to glycogen. The extent of pyruvate cycling is dependent on the acinar origin of the hepatocytes and on the glucose concentration and presence of insulin.

Aged↗
PubMed2001

Dimethylsulfoxide promotes K+-independent activity of pyruvate kinase and the acquisition of the active catalytic conformation.

Pyruvate kinase requires K+ for maximal activity; the enzyme exhibits 0.02% of maximal activity in its absence [Kayne, F. J. (1971) Arch. Biochem. Biophys. 143, 232-239]. However, pyruvate kinase entrapped in reverse micelles exhibits an important K+-independent activity [Ramírez-Silva, L., Tuena de Gómez-Puyou, M., & Gómez-Puyou, A. (1993) Biochemistry 32, 5332-5338]. It is possible that the amount of water, as well as interactions of the protein with the micelles, can account for this behavior. We therefore explored the solvent effects on the catalytic properties of muscle pyruvate kinase. The enzyme exhibited an activity of 19.4 micromol x min(-1) x mg(-1) in 40% dimethylsulfoxide, compared with 280 and 0.023 micromol x min(1) x mg(-1) observed with and without K+ in water, respectively. pH activity profiles and kinetic constants for the substrates of pyruvate kinase in dimethylsulfoxide without K+ were similar to those in 100% water with K+, and differed from those in water without K+. The spectral center of mass of the emission spectrum of pyruvate kinase in 100% water exhibited a blue shift of 3.5 nm in the presence of Mg(2+), phosphenolpyruvate, and K+, ligands that induce the active conformation of the enzyme. The spectral center of mass of the apoenzyme in 30-40% dimethylsulfoxide coincided with that of the enzyme-Mg(2+)-phosphenolpyruvate-K+ complex in 100% water. The water relaxation rate enhancement factor and binding of phosphenolpyruvate to the pyruvate kinase-Mn(2+)-(CH3)4N+ complex in 30-40% dimethylsulfoxide were similar to those of the pyruvate kinase-Mn(2+)-K+ complex in water. The aforementioned results indicate that when muscle pyruvate kinase is without K+, 30-40% dimethylsulfoxide induces its active conformation.

Binding Sites↗
PubMed1977

Acyl group and electron pair relay system: a network of interacting lipoyl moieties in the pyruvate and alpha-ketoglutarate dehydrogenase complexes from Escherichia coli.

The dihydrolipoyl transacetylase component of the Escherichia coli pyruvate dehydrogenase complex [pyruvate:lipoate oxidoreductase (decarboxylating and acceptor-acetylating), EC 1.2.4.1] bears two sites on each of its 24 polypeptide chains that undergo reductive acetylation by [2-(14)C]pyruvate and thiamin pyrophosphate, acetylation by [1-(14)C]acetyl-CoA in the presence of DPNH, and reaction with N-ethyl[2,3-(14)C]maleimide in the presence of pyruvate and thiamin pyrophosphate. The data strongly imply that these sites are covalently bound lipoyl moieties. The results of similar experiments with the E. coli alpha-ketoglutarate dehydrogenase complex [2-oxoglutarate:lipoate oxidoreductase (decarboxylating and acceptor-succinylating), EC 1.2.4.2] indicate that its dihydrolipoyl transsuccinylase component bears only one lipoyl moiety on each of its 24 chains. Charging of the 48 acetyl acceptor sites on the transacetylase or the 24 succinyl acceptor sites on the transsuccinylase by pyruvate or alpha-ketoglutarate, respectively, and thiamin pyrophosphate was observed in the presence of only a few functionally active pyruvate dehydrogenase or alpha-ketoglutarate dehydrogenase chains. Extensive crosslinking of the transacetylase chains was observed when the pyruvate dehydrogenase complex was treated with pyruvate and thiamin pyrophosphate or with DPNH in the presence of N,N'-o- or N,N'-p-phenylenedimaleimide, respectively. When the alpha-ketoglutarate dehydrogenase complex was treated with DPNH in the presence of N,N'-p-phenylenedimaleimide, only transsuccinylase monomers and crosslinked transsuccinylase dimers were detected. It appears that the 48 lipoyl moieties in the transacetylase and the 24 lipoyl moieties in the transsuccinylase comprise an interacting network that functions as an acyl group and electron pair relay system through thiol-disulfide and acyl-transfer reactions among all of the lipoyl moieties.

Acetyl Coenzyme A↗
PubMed1978

Thiamin deficiency effects on rat leukocyte pyruvate decarboxylation rates.

Thiamin status usually is assessed by urinary excretion of thiamin or by exogenous thiamin pyrophosphate (TPP) stimulation of erythrocyte transketolase activity. Because of the possible great utility of a biologically and chemically sensitive alternative method for thiamin status assessment, studies were made of rat leukocyte pyruvate decarboxylation activity in thiamin deficiency. Pyruvate decarboxylation rates were determined by assaying 14CO2 produced by leukocytes from 1-14C-pyruvic acid in vitro. Reaction conditions were 5 mumoles pyruvic acid, 2.2 X 10(4) DPM 1-14C-pyruvic acid, leukocytes from 5 ml whole blood, 50 mumoles NaH2PO4, 5 mumoles MgSO4, and 1 mumole MnSO4 at pH 7.4 in 1 ml reaction volume at 25 C. Four weeks of thiamin deficiency decreased leukocyte pyruvate decarboxylation rates and markedly increased the TPP effect on this reaction. Dual weekly assays in the same rats showed that 21 days of thiamin deficiency significantly increased the TPP effect on leukocyte pyruvate decarboxylation rates. In contrast, the TPP effect on erythrocyte transketolase activity was significantly increased after only 7 days of thiamin deficiency. Erythrocyte transketolase is more sensitive than leukocyte pyruvate decarboxylation rate to early thiamin deficiency in rats.

Animals↗
PubMed1976

Properties and subunit structure of pig heart pyruvate dehydrogenase.

Pyruvate dehydrogenase [EC 1.2.4.1] was separated from the pyruvate dehydrogenase complex and its molecular weight was estimated to be about 150,000 by sedimentation equilibrium methods. The enzyme was dissociated into two subunits (alpha and beta), with estimated molecular weights of 41,000 (alpha) and 36,000 (beta), respectively, by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The subunits were separated by phosphocellulose column chromatography and their chemical properties were examined. The subunit structure of the pyruvate dehydrogenase was assigned as alpha2beta2. The content of right-handed alpha-helix in the enzyme molecule was estimated to be about 29 and 28% by optical rotatory dispersion and by circular dichroism, respectively. The enzyme contained no thiamine-PP, and its dehydrogenase activity was completely dependent on added thiamine-PP and partially dependent on added Mg2+ and Ca2+. The Km value of pyruvate dehydrogenase for thiamine diphosphate was estimated to be 6.5 X 10(-5) M in the presence of Mg2+ or Ca2+. The enzyme showed highly specific activity for thiamine-PP dependent oxidation of both pyruvate and alpha-ketobutyrate, but it also showed some activity with alpha-ketovalerate, alpha-ketoisocaproate, and alpha-ketoisovalerate. The pyruvate dehydrogenase activity was strongly inhibited by bivalent heavy metal ions and by sulfhydryl inhibitors; and the enzyme molecule contained 27 moles of 5,5'-dithiobis(2-nitrobenzoic acid)-reactive sulfhydryl groups and a total of 36 moles of sulfhydryl groups. The inhibitory effect of p-chloromercuribenzoate was prevented by preincubating the enzyme with thiamine-PP plus pyruvate. The structure of pyruvate dehydrogenase necessary for formation of the complex is also reported.

Amino Acids↗
PubMed2003

Ethyl pyruvate: a novel anti-inflammatory agent.

Pyruvate plays a central role in intermediary metabolism. Pyruvate, however, is also a potent antioxidant and free radical scavenger, and numerous studies have shown that treatment with this compound can be salutary in numerous pathologic conditions that are thought to be mediated, at least in part, by redox-dependent phenomena. Unfortunately, aqueous solutions of pyruvate rapidly undergo an aldol-like condensation reaction to form 2-hydroxy-2-methyl-4-ketoglutarate (parapyruvate), a compound that is a potent inhibitor of a critical step in the mitochondrial tricarboxylic acid cycle. To circumvent this issue, our laboratory formulated a derivative of pyruvic acid, ethyl pyruvate, in a calcium- and potassium-containing balanced salt solution. We showed that treatment with this fluid could ameliorate much of the structural and functional damage to the intestinal mucosa caused by mesenteric ischemia and reperfusion in rats. In subsequent studies, we showed that treatment with ethyl pyruvate solution could improve survival in rodent models of hemorrhagic shock and resuscitation and also down-regulate a number of proinflammatory genes. Recently, ethyl pyruvate was also shown to improve survival in murine models of acute endotoxemia and bacterial peritonitis. Although the biochemical basis for the anti-inflammatory actions of pyruvate remain to be elucidated, this simple compound warrants further evaluation as a treatment for a number of conditions commonly encountered in the practice of critical care medicine.

Animals↗
PubMed1989

Molecular and immunological characterization of plastid and cytosolic pyruvate kinase isozymes from castor-oil-plant endosperm and leaf.

1. Monospecific antiserum was raised in rabbits to homogeneous cytosolic pyruvate kinase isolated from 5-day-old germinating endosperm of the castor oil plant, Ricinus communis. An earlier study demonstrated that the purified enzyme is putatively heterotetrameric, composed of two subunits which migrate as 57-kDa and 56-kDa proteins upon sodium dodecyl sulfate/polyacrylamide gel electrophoresis [Plaxton, W. C. (1988) Plant Physiol. (Bethesda) 86, 1065-1069]. Both proteins were detected on Western blots of extracts prepared under denaturing conditions from 4-8-day-old, but not 0-3-day-old, germinating-endosperm tissue. This suggests that both subunits exist in vivo, and that the large increase in pyruvate kinase activity which occurs around the fourth day of germination is due to an increase in pyruvate kinase concentration. 2. The cytosolic and plastidic pyruvate kinase isozymes (termed PKc and PKp, respectively) from castor-oil-plant developing endosperm and expanding leaf tissue were separated by anion-exchange chromatography on Q-Sepharose. The antigenic reaction of the partially purified enzyme preparations to rabbit polyclonal antibodies raised against homogeneous germinating-castor-bean PKc was tested by immunoprecipitation and Western blotting. Although developing-endosperm and leaf PKc appeared to be antigenically very similar to germinating-endosperm PKc, they differed from the heterotetrameric germinating-endosperm enzyme by being composed of a single type of subunit with a molecular mass of about 56 kDa. No cross-reactivity of the PKc antibodies was observed with either developing-endosperm or leaf PKp, nor with rabbit muscle or Bacillus stearothermophilus pyruvate kinase. Conversely, none of the castor-oil-plant pyruvate kinase preparations showed significant cross-reactivity with antibodies raised against purified yeast or rabbit muscle pyruvate kinases. 3. To investigate the structural relationship between the two germinating-endosperm-PKc subunits, each polypeptide was characterized by amino acid composition analysis and peptide mapping by CNBr fragmentation. The amino acid compositions and CNBr cleavage patterns of the two subunits were similar, but not identical, suggesting that these polypeptides are related, but distinct, proteins. Mild tryptic attack of native enzyme led to an approximate 6-kDa reduction in the apparent molecular mass of both subunits, further indicating sequence similarity between the two polypeptides. 4. Native molecular masses of the various castor-oil-plant pyruvate kinases were estimated by Superose-6 gel-filtration chromatography.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acids↗
PubMed1994

Identification of the dihydrolipoamide acetyltransferase subunit of the human pyruvate dehydrogenase complex as an autoantigen in halothane hepatitis. Molecular mimicry of trifluoroacetyl-lysine by lipoic acid.

Trifluoroacetylated (CF3CO-) proteins, elicited upon exposure of animals or humans to halothane, were recognized by anti-CF3CO antibody, monospecific for the hapten derivative N6-trifluoroacetyl-L-lysine. Anti-CF3CO antibodies cross-reacted with the dihydrolipoamide acetyltransferase (E2 subunit) of pyruvate dehydrogenase, indicating that epitopes on the E2 subunit of pyruvate dehydrogenase molecularly mimic those on CF3CO-proteins. Lipoic acid, the prosthetic group of the E2 subunit of pyruvate dehydrogenase was essential in this process, in that only the lipoylated form of the recombinantly expressed inner lipoyl domain of the human E2 subunit of pyruvate dehydrogenase, but not the unlipolyated form, was recognized by anti-CF3CO antibody. Furthermore, based on a high degree of structural relatedness, both CF3CO-Lys and (6RS)-lipoic acid, as well as the lipoylated peptide ETDK(lipoyl)ATIG specifically inhibited the recognition by anti-CF3CO antibody of the E2 subunit of pyruvate dehydrogenase, of trifluoroacetylated rabbit serum albumin and of human liver CF3CO-proteins. In sera of patients with halothane hepatitis, autoantibodies with properties identical to those of anti-CF3CO antibody were identified which could not discriminate between CF3CO-proteins and the E2 subunit of pyruvate dehydrogenase. These data suggest that the E2 subunit pyruvate of dehydrogenase is an autoantigen in halothane hepatitis and that molecular mimicry of CF3CO-proteins by the E2 subunit of pyruvate dehydrogenase is due to the similar structures of CF3CO-Lys and lipoic acid.

Acetyltransferases↗
PubMed2006

The crystal structure of pyruvate decarboxylase from Kluyveromyces lactis. Implications for the substrate activation mechanism of this enzyme.

The crystal structure of pyruvate decarboxylase from Kluyveromyces lactis has been determined to 2.26 A resolution. Like other yeast enzymes, Kluyveromyces lactis pyruvate decarboxylase is subject to allosteric substrate activation. Binding of substrate at a regulatory site induces catalytic activity. This process is accompanied by conformational changes and subunit rearrangements. In the nonactivated form of the corresponding enzyme from Saccharomyces cerevisiae, all active sites are solvent accessible due to the high flexibility of loop regions 106-113 and 292-301. The binding of the activator pyruvamide arrests these loops. Consequently, two of four active sites become closed. In Kluyveromyces lactis pyruvate decarboxylase, this half-side closed tetramer is present even without any activator. However, one of the loops (residues 105-113), which are flexible in nonactivated Saccharomyces cerevisiae pyruvate decarboxylase, remains flexible. Even though the tetramer assemblies of both enzyme species are different in the absence of activating agents, their substrate activation kinetics are similar. This implies an equilibrium between the open and the half-side closed state of yeast pyruvate decarboxylase tetramers. The completely open enzyme state is favoured for Saccharomyces cerevisiae pyruvate decarboxylase, whereas the half-side closed form is predominant for Kluyveromyces lactis pyruvate decarboxylase. Consequently, the structuring of the flexible loop region 105-113 seems to be the crucial step during the substrate activation process of Kluyveromyces lactis pyruvate decarboxylase.

Allosteric Regulation↗
PubMed2006

A molecular mechanism of pyruvate protection against cytotoxicity of reactive oxygen species in osteoblasts.

We demonstrated previously that exogenous pyruvate has a protective action against cell death by hydrogen peroxide in cultured osteoblasts through a mechanism associated with its antioxidative property. In the present study, we have evaluated possible participation of monocarboxylate transporters (MCTs) responsible for the bidirectional membrane transport of pyruvate in the cytoprotective property in osteoblasts. Expression of the MCT2 isoform was found in cultured rat calvarial osteoblasts and in osteoblasts located on mouse tibia at both mRNA and protein levels. The accumulation of [14C]pyruvate occurred in a temperature- and pH-dependent manner in osteoblasts cultured for 7 days with high sensitivity to a specific MCT inhibitor, whereas pyruvate was released into extracellular spaces from cultured osteoblasts in a fashion sensitive to the MCT inhibitor. Transient overexpression of the MCT2 isoform led to reduced vulnerability to the cytotoxicity of hydrogen peroxide with an increased activity of [14C]pyruvate accumulation in murine osteoblastic MC3T3-E1 cells. Ovariectomy significantly decreased the content of pyruvate in femoral bone marrows in mice in vivo, whereas daily i.p. administration of pyruvate at 0.25 g/kg significantly prevented alterations of several histomorphometric parameters as well as cancellous bone loss in femurs by ovariectomy on 28 days after the operation. These results suggest that MCTs may be functionally expressed by osteoblasts to play a pivotal role in mechanisms related to the cytoprotective property of pyruvate.

Animals↗
PubMed1979

Nutritional and hormonal regulation of pyruvate metabolism in the liver.

The effect of fasting, glucose, and glucagon injection on pyruvate metabolism of rat liver mitochondria was studied. Fasting for 24 h caused a) a twofold increase in mitochondrial pyruvate uptake, b) fivefold increase in CO2 fixation, and c) no change in pyruvate decarboxylation. Injection of glucose to fasted rats 2 h prior to preparation suppressed by one-half the increase in mitochondrial pyruvate uptake and CO2 fixation and increased hepatic pyruvate content. Injection of glucagon together with glucose abolished the depression of pyruvate uptake by glucose but did not prevent the decrease in mitochondrial CO2 fixation or hepatic ketone content caused by glucose alone. The effects of insulin injection resembled that of glucose in decreasing hepatic ketone content, but differed by increasing pyruvate uptake without much change in CO2 fixation. It is concluded that the increase in gluconeogenesis induced by fasting is due to an increase in pyruvate uptake and carboxylation by hepatic mitochondria. The latter is due to the increased mobilization and oxidation of fatty acids induced by reciprocal changes in insulin and glucagon.

Animals↗
PubMed2005

Pyruvate-fortified cardioplegia suppresses oxidative stress and enhances phosphorylation potential of arrested myocardium.

Cardioplegic arrest for bypass surgery imposes global ischemia on the myocardium, which generates oxyradicals and depletes myocardial high-energy phosphates. The glycolytic metabolite pyruvate, but not its reduced congener lactate, increases phosphorylation potential and detoxifies oxyradicals in ischemic and postischemic myocardium. This study tested the hypothesis that pyruvate mitigates oxidative stress and preserves the energy state in cardioplegically arrested myocardium. In situ swine hearts were arrested for 60 min with a 4:1 mixture of blood and crystalloid cardioplegia solution containing 188 mM glucose alone (control) or with additional 23.8 mM lactate or 23.8 mM pyruvate and then reperfused for 3 min with cardioplegia-free blood. Glutathione (GSH), glutathione disulfide (GSSG), and energy metabolites [phosphocreatine (PCr), creatine (Cr), P(i)] were measured in myocardium, which was snap frozen at 45 min arrest and 3 min reperfusion to determine antioxidant GSH redox state (GSH/GSSG) and PCr phosphorylation potential {[PCr]/([Cr][P(i)])}. Coronary sinus 8-isoprostane indexed oxidative stress. Pyruvate cardioplegia lowered 8-isoprostane release approximately 40% during arrest versus control and lactate cardioplegia. Lactate and pyruvate cardioplegia dampened (P < 0.05 vs. control) the surge of 8-isoprostane release following reperfusion. Pyruvate doubled GSH/GSSG versus lactate cardioplegia during arrest, but GSH/GSSG fell in all three groups after reperfusion. Myocardial [PCr]/([Cr][P(i)]) was maintained in all three groups during arrest. Pyruvate cardioplegia doubled [PCr]/([Cr][P(i)]) versus control and lactate cardioplegia after reperfusion. Pyruvate cardioplegia mitigates oxidative stress during cardioplegic arrest and enhances myocardial energy state on reperfusion.

Acid-Base Equilibrium↗
PubMed2003

Profiling substrate fluxes in the isolated working mouse heart using 13C-labeled substrates: focusing on the origin and fate of pyruvate and citrate carbons.

The availability of genetically modified mice requires the development of methods to assess heart function and metabolism in the intact beating organ. With the use of radioactive substrates and ex vivo perfusion of the mouse heart in the working mode, previous studies have documented glucose and fatty acid oxidation pathways. This study was aimed at characterizing the metabolism of other potentially important exogenous carbohydrate sources, namely, lactate and pyruvate. This was achieved by using (13)C-labeling methods. The mouse heart perfusion setup and buffer composition were optimized to reproduce conditions close to the in vivo milieu in terms of workload, cardiac functions, and substrate-hormone supply to the heart (11 mM glucose, 0.8 nM insulin, 50 microM carnitine, 1.5 mM lactate, 0.2 mM pyruvate, 5 nM epinephrine, 0.7 mM oleate, and 3% albumin). The use of three differentially (13)C-labeled carbohydrates and a (13)C-labeled long-chain fatty acid allowed the quantitative assessment of the metabolic origin and fate of tissue pyruvate as well as the relative contribution of substrates feeding acetyl-CoA (pyruvate and fatty acids) and oxaloacetate (pyruvate) for mitochondrial citrate synthesis. Beyond concurring with the notion that the mouse heart preferentially uses fatty acids for energy production (63.5 +/- 3.9%) and regulates its fuel selection according to the Randle cycle, our study reports for the first time in the mouse heart the following findings. First, exogenous lactate is the major carbohydrate contributing to pyruvate formation (42.0 +/- 2.3%). Second, lactate and pyruvate are constantly being taken up and released by the heart, supporting the concept of compartmentation of lactate and glucose metabolism. Finally, mitochondrial anaplerotic pyruvate carboxylation and citrate efflux represent 4.9 +/- 1.8 and 0.8 +/- 0.1%, respectively, of the citric acid cycle flux and are modulated by substrate supply. The described (13)C-labeling strategy combined with an experimental setup that enables continuous monitoring of physiological parameters offers a unique model to clarify the link between metabolic alterations, cardiac dysfunction, and disease development.

Acetyl Coenzyme A↗
PubMed1984

Regulation of pyruvate dehydrogenase complex in ischemic rat heart.

The effect of flow-induced ischemia on the rate of pyruvate decarboxylation and the activation state of the pyruvate dehydrogenase multienzyme complex was investigated in the isolated, perfused rat heart. Pyruvate dehydrogenase activity in the heart decreased significantly during flow-induced ischemia and was a function of changes in the activation state (i.e., active/total activity) of the enzyme complex. In the absence of pyruvate, the activation state of pyruvate dehydrogenase decreased from nearly 100% active at the normal flow rate (10 ml/min) to 20% active as the flow was reduced to 0.5 ml/min. At high pyruvate levels (5 mM), the activation state increased from nearly 70% active at control flow rates to 100% active during ischemia. At an intermediate pyruvate concentration (0.5 mM), the enzyme complex was maintained at a relatively low activation state (30-35% active) throughout the range of flow rates tested. Ischemia caused elevated perfusate lactate concentrations only when the flow rates were less than 5.0 ml/min. The activation state of the pyruvate dehydrogenase complex in hearts perfused with glucose was also decreased during ischemia.

Adenine Nucleotides↗
PubMed1998

In vitro and ex vivo 13C-NMR spectroscopy studies of pyruvate recycling in brain.

Pyruvate recycling is a well established pathway in the liver, but in the brain, the cellular localization of pyruvate recycling remains controversial and its physiological significance is unknown. In cultured cortical astrocytes, pyruvate formed from [U-13C]glutamate was shown to re-enter the TCA cycle after conversion to acetyl-CoA, as demonstrated by the labelling patterns in aspartate C-2 and C-3, lactate C-2, and glutamate C-4, which provides evidence for pyruvate recycling in astrocytes. This finding is in agreement with previous studies of astrocytic cultures, in which pyruvate recycling has been described from [U-13C]glutamine, in the presence of glutamate, and from [U-13C]aspartate. Pyruvate recycling in brain was studied in fasted rats receiving either an intraperitoneal or a subcutaneous injection of [1,2-13C]acetate followed by decapitation 30 min later. Extracts of cortical tissue were analysed with 13C-NMR spectroscopy and total amounts of amino acids quantified by HPLC. Plasma extracts were analysed with 1H- and 13C-NMR spectroscopy, and showed a significantly larger amount of [1, 2-13C]acetate in the intraperitoneal group compared to the subcutaneous group. Furthermore, a small amount of label was detected in glucose in both groups. In the subcutaneously injected rats, [4-13C]glutamate and [2-13C]GABA were less enriched than plasma glucose, which might have been the precursor. In the intraperitoneally injected rats, however, pyruvate formation from [1, 2-13C]acetate, and re-entry of this pyruvate into the TCA cycle was demonstrated by the presence of greater 13C enrichment in [4-13C]glutamate and [4-13C]glutamine compared to the subcutaneous group, probably resulting from the significantly higher [1, 2-13C]acetate concentration in brain and plasma.

Acetates↗
PubMed1970

A defect in pyruvate decarboxylase in a child with an intermittent movement disorder.

A patient with an intermittent movement disorder has been found to have an inherited defect in pyruvate decarboxylase ((2-oxo-acid carboxy-lyase, E.C. 4.1.1.1.). The patient is a 9 yr old boy who since infancy has had repeated episodes of a combined cerebellar and choreoathetoid movement disorder. He has an elevated level of pyruvic acid in his blood, an elevated urinary alanine content, and less marked elevations in blood alanine and lactate. Methods were developed to study his metabolic abnormality in dilute suspensions of white blood cells and cultured skin fibroblasts, as well as in cell-free sonicates of fibroblasts. Oxidation of pyruvic acid-1-(14)C and pyruvic acid-2-(14)C by his cells and pyruvate decarboxylase activity in sonicates of his cells were less than 20% of those in cells from control subjects. Oxidation of glutamic acid-U-(14)C, acetate-1-(14)C, and palmitate-1-(14)C was normal, as was incorporation of alanine-U-(14)C into protein. The rate of oxidation of pyruvic acid by the father's cells and the activity of pyruvate decarboxylase in the father's sonicated fibroblasts were intermediate between those of the patient and those of controls. Values for the mother were at or just below the lower limits of the ranges in controls. Kinetic data suggested the posibility of several forms of pyruvate decarboxylase in this family. Possible mechanisms relating the chemical abnormality and the clinical symptoms in this patient are discussed.

Alanine↗