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PubMed1985

Pancreatic islets contain the M2 isoenzyme of pyruvate kinase. Its phosphorylation has no effect on enzyme activity.

To determine which of the major isoenzymes of pyruvate kinase pancreatic islet pyruvate kinase most resembled, it was compared to pyruvate kinase from other tissues in kinetic and immunologic studies. The pattern of activation by fructose bisphosphate and the patterns of inhibition by alanine and phenylalanine were most similar to those of the M2 isoenzyme from kidney and were dissimilar to those of the isoenzymes from skeletal muscle (type M1) and liver (type L). The islet pyruvate kinase was inhibited by anti-M1 pyruvate kinase serum (which crossreacts with the M2 isoenzyme), but not by anti-L pyruvate kinase. These results are most consistent with islets possessing predominantly, if not exclusively, the M2 isoenzyme of pyruvate kinase. We previously showed that rat pancreatic islet cytosol contains protein kinases that can catalyze a calcium-activated phosphorylation of an endogenous peptide that has properties, such as subunit molecular weight and isoelectric pH, that are identical to those of the M2 and M1 isoenzymes of pyruvate kinase, and that islet cytosol can catalyze phosphorylation of muscle pyruvate kinase. In the present study it was shown that incubating islet cytosol with ATP under conditions known to permit phosphorylation and inhibition of liver pyruvate kinase did not affect the islet pyruvate kinase activity. It is concluded that phosphorylation of the islet pyruvate kinase has no immediate effect on enzyme activity.

Adenosine Diphosphate↗
PubMed1989

Characterization of sodium and pyruvate interactions of the two carrier systems specific of mono- and di- or tricarboxylic acids by renal brush-border membrane vesicles.

The experiments reported in this paper aim at characterizing the carboxylic acid transport, the interactions of pyruvate and citrate with their transport sites and specificity. The study of these carriers was performed using isotopic solutes for the influx measurements in brush-border membrane vesicles under zero trans conditions where the membrane potential was abolished with KCl preloading with valinomycin or equilibrium exchange conditions and delta psi = 0. Under zero trans condition and delta psi = 0, the influence of pyruvate concentrations on its initial rates of transport revealed the existence of two families of pyruvate transport sites, one with a high affinity for pyruvate (Kt = 88 microM) and a low affinity for sodium (Kt = 57.7 mM) (site I), the second one with a low affinity for pyruvate (Kt = 6.1 mM) and a high affinity for sodium (Kt = 23.9 mM) (site II). The coupling factor [Na]/[pyruvate] stoichiometry were determined at 0.25 mM and 8 mM pyruvate and estimated at 1.8 for site I, and 3 when the first and the second sites transport simultaneously. Under chemical equilibrium (delta psi congruent to 0) single isotopic labeling, transport kinetics of pyruvate carrier systems have shown a double interaction of pyruvate with the transporter; the sodium/pyruvate stoichiometry also expressed according to a Hill plot representation was n = 1.7. The direct method of measuring Na+/pyruvate stoichiometry from double labeling kinetics and isotopic exchange, for a time course, gives a n = 1.67. Studies of transport specificity, indicate that the absence of inhibition of lactate transport by citrate and the existence of competitive inhibition of lactate and citrate transports by pyruvate leads to the conclusion that the low pyruvate affinity site can be attributed to the citrate carrier (tricarboxylate) and the high pyruvate affinity site to the lactate carrier (monocarboxylate).

Animals↗
PubMed1971

Metabolism of rat brain mitochondria. Studies on the potassium ion-stimulated oxidation of pyruvate.

1. Rat brain-cortex mitochondria were incubated in media containing 1, 5 or 100mm-K(+) in the presence of ADP, uncoupler (FCCP, carbonyl cyanide p-trifluoro-methoxyphenylhydrazone) or valinomycin while metabolizing pyruvate and malate, or acetylcarnitine and malate or glutamate and malate as substrates. Both the uptake of oxygen and disappearance of substrate were measured under these conditions. 2. With pyruvate and malate as substrate in the presence of both ADP and valinomycin, both the uptake of oxygen and disappearance of pyruvate increased markedly on increasing the K(+) content of the incubation medium from 5 to 100mm-K(+). However, in the presence of uncoupler (FCCP), although the oxygen uptake doubled little change was observed in the rate of disappearance of pyruvate on increasing the K(+) concentration. 3. Only small changes in uptake of substrate and oxygen were observed in the presence of ADP, uncoupler (FCCP) or valinomycin on increasing the K(+) concentration when acetylcarnitine+malate or glutamate+malate were used as substrates by brain mitochondria. 4. Further, increasing the K(+) concentration from 1 to 20mm when rat brain mitochondria were oxidizing a mixture of pyruvate and glutamate in the presence of malate and ADP caused a 30% increase in the respiration rate, 50% increase in the rate of disappearance of pyruvate and an 80% decrease in the rate of disappearance of glutamate. 5. Investigation of the redox state of the cytochromes and the nicotinamide nucleotides in various conditions with either pyruvate or acetylcarnitine as substrates suggested that the specific stimulation of metabolism of pyruvate by K(+) could not be explained by a general stimulation of the electron-transport system. 6. Low-amplitude high-energy swelling of rat brain mitochondria was investigated in both Na(+)- and K(+)-containing media. Swelling of brain mitochondria was much greater in the Na(+)-containing medium and in this medium, the addition of Mg(2+) caused a partial reversal of swelling together with an 85% decrease in the rate of utilization of pyruvate. However, in the K(+)-containing medium, the addition of Mg(2+), although also causing a reversal of swelling, did not affect the rate of disappearance of pyruvate. 7. Measurements of the ATP, NADH/NAD(+) and acetyl-CoA/CoA contents were made under various conditions and no evidence that K(+) concentrations affected these parameters was obtained. 8. The results are discussed in relationship to the physiological significance of the stimulation of pyruvate metabolism by K(+) in rat brain mitochondria. It is proposed that K(+) causes its effects by a direct stimulation of the pyruvate dehydrogenase complex.

Adenosine Diphosphate↗
PubMed1971

Metabolism of pyruvate and malate by isolated fat-cell mitochondria.

1. Metabolism of pyruvate and malate by isolated fat-cell mitochondria incubated in the presence of ADP and phosphate has been studied by measuring rates of pyruvate uptake, malate utilization or production, citrate production and oxygen consumption. From these measurements calculations of the flow rates through pyruvate carboxylase, pyruvate dehydrogenase and citrate cycle have been made under various conditions. 2. In the presence of bicarbonate, pyruvate was largely converted into citrate and malate and only about 10% was oxidized by the citrate cycle; citrate and malate outputs were linear after lag periods of 6-9min and 3min respectively, and no other end products of pyruvate metabolism were detected. On the further addition of malate or hydroxymalonate, the lag in the rate of citrate output was less marked but no net malate disappearance was detected. If, however, bicarbonate was omitted then net malate uptake was observed. Addition of butyl malonate was found to greatly inhibit the metabolism of pyruvate to citrate and malate in the presence of bicarbonate. 3. These results are in agreement with earlier conclusions that in adipose tissue acetyl units for fatty acid synthesis are transferred to the cytoplasm as citrate and that this transfer requires malate presumably for counter transport. They also support the view that oxaloacetate for citrate synthesis is preferentially formed from pyruvate through pyruvate carboxylase rather than malate through malate dehydrogenase and that the mitochondrial metabolism of citrate in fat-cells is restricted. The possible consequences of these conclusions are discussed. 4. Studies on the effects of additions of adenine nucleotides to pyruvate metabolism by isolated fat-cell mitochondria are consistent with inhibition of pyruvate carboxylase in the presence of ADP and pyruvate dehydrogenase in the presence of ATP.

Adenosine Diphosphate↗
PubMed1982

Long-term modulation of type L pyruvate kinase activity in young and mature rats.

The regulation of type L pyruvate kinase concentrations in liver of young (35-45 days old) and adult (60-85 days old) rats starved and re-fed a 71% sucrose diet was investigated. Re-feeding is accompanied by an increase in the enzyme level in liver determined kinetically and immunologically. A constant ratio of kinetic activity to immunological activity was observed under all conditions examined, indicating that activity changes are the result of a regulation of synthesis or degradation and not an interconversion between kinetically active and inactive forms of the enzyme. Synthesis of pyruvate kinase was directly examined by using hepatocytes isolated from starved and re-fed rats. A stimulation of pyruvate kinase synthesis is observed on re-feeding. This increase in synthesis of pyruvate kinase is retained by the isolated hepatocyte for up to 7h in the absence of hormonal stimuli. Administration of glucagon (1mum) to the isolated hepatocytes had no influence on synthesis of pyruvate kinase and no evidence for a glucagon-directed degradation of the enzyme was found. Re-feeding the rat was followed by a transient increase in the synthesis of pyruvate kinase. The peak rate of synthesis was observed before a detectable increase in the enzyme concentration. After a rapid synthesis period, a new steady-state level of the enzyme was achieved and synthesis rates declined. The time course and magnitude for the response to the sucrose diet was dependent on the age of the rat. In young rats, an increase in pyruvate kinase synthesis is observed within 6h and peak synthesis occurs at 11h after re-feeding sucrose. The peak synthesis rate for pyruvate kinase for young rats represents approx. 1% of total protein synthesis. With adult rats, increased pyruvate kinase synthesis is not observed for 11h, with peak synthesis occurring at 24h after re-feeding. In the older rats, peak pyruvate kinase synthesis constitutes greater than 4% of total protein synthesis. Continued re-feeding of the adult rat beyond 24h is accompanied by a decline of pyruvate kinase synthesis to approx. 1.5% of total protein synthesis. The concentration of the enzyme, however, does not decline during this period, suggesting that control of pyruvate kinase degradation as well as synthesis occurs.

Age Factors↗
PubMed1999

Effects of pyruvate on intracellular Ca2+ regulation in cardiac myocytes from normal and diabetic rats.

1. Pyruvate has been shown to enhance the contractile performance of cardiac muscle when provided as an alternative substrate to glucose. The aims of the present study were to determine whether the inotropic effects of pyruvate are due to increased mobilization of intracellular Ca2+ ([Ca2+]i) and to compare the effects of pyruvate on [Ca2+]i levels in myocytes from normal and diabetic animals. 2. Fura-2 was used to monitor [Ca2+]i in ventricular myocytes isolated from control and streptozotocin-treated male Wistar rats. The experiments were performed at 25 degrees C, with an extracellular [Ca2+] of 1.5 mmol/L and either 10 mmol/L glucose or 10 mmol/L pyruvate as the substrate. 3. In myocytes from both control and diabetic rats, increasing the stimulus frequency from 0.33 to 2.0 Hz resulted in significant increases in resting and peak [Ca2+]i as well as in the amplitude of the [Ca2+]i transient, irrespective of substrate. Compared with glucose, pyruvate significantly increased resting and peak [Ca2+]i and the amplitude of the [Ca2+]i transient at each stimulus frequency in myocytes from both control and diabetic animals. However, the extent of potentiation of the [Ca2+]i transient amplitude produced by pyruvate was significantly less in myocytes from the diabetic rats. 4. The rate of restitution of the [Ca2+]i transient was used as an index of the rate of Ca2+ cycling by the sarcoplasmic reticulum (SR). Pyruvate enhanced the rate of restitution in control but not diabetic rat cells. 5. The time course of decay of the [Ca2+]i transient was analysed as a measure of the rate of removal of Ca2+ from the cytosol. Pyruvate tended to increase the rate of decay in cells from control but not diabetic animals. The rate of decay was slower in cells from diabetic animals compared with controls. 6. The data reveal that pyruvate increases SR Ca2+ cycling, leading to greater Ca2+ release and an increase in the amplitude of the [Ca2+]i transient. Therefore, it seems highly likely that increased [Ca2+]i mobilization is responsible for the previously reported positive inotropic actions of pyruvate. These effects of pyruvate are attenuated in diabetic rat cells, which may reflect an impaired capacity of mitochondria in diabetic hearts to oxidize pyruvate, thus limiting potential energetic benefits.

Animals↗
PubMed2003

Pyruvate-dependent preconditioning and cardioprotection in murine myocardium.

1. Whether pyruvate inhibits or can actually initiate myocardial preconditioning is unclear and whether pyruvate provides protection via its action as a 'cosubstrate' with glucose or via alternative mechanisms also remains controversial. We examined effects of a high concentration of pyruvate (10 mmol/L) alone or with 15 mmol/L glucose in mouse hearts subjected to 20 min ischaemia and 30 min reperfusion. 2. Provision of 10 mmol/L pyruvate alone or as a cosubstrate markedly reduced ischaemic contracture and enhanced postischaemic recovery. Time to contracture was increased from approximately 3 min to over 8 min, peak contracture was reduced from 90 mmHg to less than 60 mmHg and postischaemic pressure development was also improved. Effects on contracture were independent of the presence of pyruvate during ischaemia and improved postischaemic recovery was evident with pre-ischaemic pyruvate perfusion. 3. Cardioprotection did not require the presence of pyruvate during ischaemia or reperfusion and effects of pyruvate pretreatment could be mimicked by pretreatment with 1 mmol/L dichloroacetate (DCA), an activator of pyruvate dehydrogenase. 4. Myocardial adenosine efflux and Ca2+ content were elevated (by 215 and 65%, respectively) following pretreatment with pyruvate, potentially triggering a preconditioned state. A role for adenosine A1 receptors is supported by lack of added protection with pyruvate in hearts transgenically overexpressing adenosine A1 receptors. 5. Collectively, these observations demonstrate that pre-ischaemic treatment with pyruvate or DCA provides a beneficial preconditioning-like effect in ischaemic and postischaemic myocardium. The response appears unrelated to glycolytic inhibition, but may be mediated via transient changes in adenosine levels and/or cellular Ca2+.

Animals↗
PubMed1996

Regulation of [15N]urea synthesis from [5-15N]glutamine. Role of pH, hormones, and pyruvate.

We have utilized both [5-15N]glutamine and [3-13C] pyruvate as metabolic tracers in order to: (i) examine the effect of pH, glucagon (GLU), or insulin on the precursor-product relationship between 15NH3, [15N]citrulline, and, thereby, [15N]urea synthesis and (ii) elucidate the mechanism(s) by which pyruvate stimulates [15N] urea synthesis. Hepatocytes isolated from rat were incubated at pH 6.8, 7.4, or 7.6 with 1 mM [5-15N]glutamine and 0.1 mM 14NH4Cl in the presence or the absence of [3-13C] pyruvate (2 mM). A separate series of experiments was performed at pH 7.4 in the presence of insulin or GLU. 15NH3 enrichment exceeded or was equal to that of [15N]citrulline under all conditions except for pH 7.6, when the 15N enrichment in citrulline exceeded that in ammonia. The formation of [15N]citrulline (atom % excess) was increased with higher pH. Flux through phosphate-dependent glutaminase (PDG) and [15N]urea synthesis were stimulated (p < 0.05) at pH 7.6 or with GLU and decreased (p < 0.05) at pH 6.8. Insulin had no significant effect on flux through PDG or on [15N]urea synthesis. Decreased [15N]urea production at pH 6.8 was associated with depleted aspartate and glutamate levels. Pyruvate attenuated this decrease in the aspartate and glutamate pools and stimulated [15N]urea synthesis. Production of Asp from pyruvate was increased with increasing medium pH. Approximately 80% of Asp was derived from [3-13C]pyruvate regardless of incubation pH or addition of hormone. Furthermore, approximately 20, 40, and 50% of the mitochondrial N-acetylglutamate (NAG) pool was derived from [3-13C]pyruvate at pH 6.8, 7.4, and 7.6, respectively. Both the concentration and formation of [13C]NAG from [3-13C]pyruvate were increased (p < 0.05) with glucagon and decreased (p < 0.05) with insulin or at pH 6.8. The data suggest a correlation between changes in [15N]urea synthesis and alterations in the level and synthesis of [13C]NAG from pyruvate. The current observations suggest that the stimulation of [15N]urea synthesis in acute alkalosis is mediated via increased flux through PDG and subsequent increased utilization of [5-15N] of glutamine for [15N]citrulline synthesis and/or increased synthesis of NAG from glutamate and pyruvate. The opposite may have occurred in acute acidosis. Glucagon, but not insulin, stimulated [15N]urea synthesis via increased flux through PDG and synthesis of NAG. Pyruvate stimulated urea synthesis via increased availability of aspartate and/or increased synthesis of NAG. The formation of NAG and aspartate from pyruvate are both pH-sensitive processes.

Amino Acids↗
PubMed1999

A mitochondrial pyruvate dehydrogenase bypass in the yeast Saccharomyces cerevisiae.

Spheroplasts of the yeast Saccharomyces cerevisiae oxidize pyruvate at a high respiratory rate, whereas isolated mitochondria do not unless malate is added. We show that a cytosolic factor, pyruvate decarboxylase, is required for the non-malate-dependent oxidation of pyruvate by mitochondria. In pyruvate decarboxylase-negative mutants, the oxidation of pyruvate by permeabilized spheroplasts was abolished. In contrast, deletion of the gene (PDA1) encoding the E1alpha subunit of the pyruvate dehydrogenase did not affect the spheroplast respiratory rate on pyruvate but abolished the malate-dependent respiration of isolated mitochondria. Mutants disrupted for the mitochondrial acetaldehyde dehydrogenase gene (ALD7) did not oxidize pyruvate unless malate was added. We therefore propose the existence of a mitochondrial pyruvate dehydrogenase bypass different from the cytosolic one, where pyruvate is decarboxylated to acetaldehyde in the cytosol by pyruvate decarboxylase and then oxidized by mitochondrial acetaldehyde dehydrogenase. This pathway can compensate PDA1 gene deletion for lactate or respiratory glucose growth. However, the codisruption of PDA1 and ALD7 genes prevented the growth on lactate, indicating that each of these pathways contributes to the oxidative metabolism of pyruvate.

Aldehyde Oxidoreductases↗
PubMed1999

Antioxidative properties of pyruvate and protection of the ischemic rat heart during cardioplegia.

Formation of oxygen free radicals during heart transplantation seems to be related to the alterations occurring during ischemia and reperfusion and could explain the short preservation time of donor hearts. The aim of our study was (a) to analyze the protective effects of pyruvate during cold cardioplegia and ischemia/reperfusion sequence, and (b) to investigate in vitro the radical scavenging properties of this compound. After 30 min of perfusion, isolated working rat hearts were arrested by cardioplegic solution, stored 4 h in B21 solutions at 4 degrees C, and reperfused with Krebs-Henseleit buffer for 45 min. Pyruvate (2 mM) was added to Krebs-Henseleit, cardioplegic, and storage solutions, and functional parameters were recorded throughout the experiments. In a second part, control hearts and hearts treated with pyruvate were cannulated via the aorta and perfused for 30 min by the Langendorff method, arrested by cardioplegic solution, stored 4 h in B21 solutions at 4 degrees C, and reperfused for 45 min by the Langendorff method. Malonedialdehyde and alpha-tocopherol levels were determined on heart homogenate. In situ detection of apoptotic cells also was performed on tissue samples (left ventricle) at the end of the ischemia/reperfusion sequence. To demonstrate in vitro the antioxidant effects of pyruvate, we monitored (a) its hydroxyl radical scavenging properties by using electron paramagnetic resonance (EPR) spectroscopy, and (b) the decrease of fluorescence of allophycocyanin, in the presence of a Fenton system (H2O2/Cu2+). Ischemia for 4 h, followed by myocardial reperfusion, resulted in substantially reduced mechanical function. Hearts subjected to this ischemia and pretreated with pyruvate showed a significant improvement in the function recovery. After the ischemia/reperfusion protocol, no significant decrease of malonedialdehyde levels was shown on hearts treated with pyruvate. However, alpha-tocopherol levels were higher in the pyruvate group compared with the control group. At the end of the reperfusion period, levels of apoptotic cells were significantly lower in hearts treated with pyruvate compared with control hearts. EPR studies showed that pyruvate was an efficient hydroxyl scavenger, with a median inhibitory concentration (IC50) of 8 mM. The allophycocyanin assay also showed a dose-dependent effect of pyruvate against hydroxyl radicals. In conclusion, these findings showed that pyruvate could prevent reperfusion injuries in the isolated heart, probably by its antioxidative properties. The application of pyruvate may contribute to the preservation of hearts for organ transplantation.

Animals↗
PubMed2005

Brain metabolism of exogenous pyruvate.

Pyruvate given in large doses may be neuroprotective in stroke, but it is not known to what degree the brain metabolizes pyruvate. Intravenous injection of [3-13C]pyruvate led to dose-dependent labelling of cerebral metabolites so that at 5 min after injection of 18 mmoles [3-13C]pyruvate/kg (2 g sodium pyruvate/kg), approximately 20% of brain glutamate and GABA were labelled, as could be detected by 13C nuclear magnetic resonance spectrometry ex vivo. Pyruvate, 9 mmoles/kg, was equivalent to glucose, 9 mmoles/kg, as a substrate for cerebral tricarboxylic acid (TCA) cycle activity. Inhibition of the glial TCA cycle with fluoroacetate did not affect formation of [4-13C]glutamate or [2-13C]GABA from [3-13C]pyruvate, but reduced formation of [4-13C]glutamine by 50%, indicating predominantly neuronal metabolism of exogenous pyruvate. Extensive formation of [3-13C]lactate from [2-13C]pyruvate demonstrated reversible carboxylation of pyruvate to malate and equilibration with fumarate, presumably in neurones, but anaplerotic formation of TCA cycle intermediates from exogenous pyruvate could not be detected. Too rapid injection of large amounts of pyruvate led to seizure activity, respiratory arrest and death. We conclude that exogenous pyruvate is an excellent energy substrate for neurones in vivo, but that care must be taken to avoid the seizure-inducing effect of pyruvate given in large doses.

Animals↗
PubMed2003

Pyruvate modulates cardiac sarcoplasmic reticulum Ca2+ release in rats via mitochondria-dependent and -independent mechanisms.

The glycolytic product pyruvate has beneficial effects on cardiac contractile function. The postulated cellular mechanisms underlying the positive inotropic effect of pyruvate, however, are contradictory or have remained elusive. Therefore, we studied the effects of pyruvate on cardiac Ca2+ regulation, intracellular pH (pHi) and flavoprotein oxidation using fluorescence confocal microscopy in intact and permeabilized rat ventricular myocytes and single channel recordings from rat cardiac ryanodine receptors (RyRs) incorporated into planar lipid bilayers. In intact cells extracellular pyruvate (10 mM) elevated diastolic [Ca2+]i, which was due, at least in part, to a concomitant acidification of the cytosol. Furthermore, pyruvate increased the amplitude and slowed the kinetics of the electrically evoked [Ca2+]i transient, and augmented sarcoplasmic reticulum (SR) Ca2+ content. Recording of flavoprotein (FAD) fluorescence indicated that pyruvate caused a reduction of mitochondrial redox potential, which is proportional to an increase of the rate of ATP synthesis. Inhibitors of mitochondrial monocarboxylate transport (alpha-cyano-4-hydroxycinnamate, 0.5 mM), adenine nucleotide translocation (atractyloside, 0.3 mM) and the electron transport chain (cyanide, 4 mM) abolished or attenuated the pyruvate-mediated increase of the amplitude of the [Ca2+]i transient, but did not change the effect of pyruvate on diastolic [Ca2+]i. Results from experiments with permeabilized myocytes indicated a direct correlation between ATP/ADP ratio and SR Ca2+ content. Furthermore, pyruvate (4 mM) reduced the frequency of spontaneous Ca2+ sparks by approximately 50%. Single RyR channel recordings revealed a approximately 60% reduction of the open probability of the channel by pyruvate (1 mM), but no change in conductance. This effect of pyruvate on RyR channel activity was neither Ca2+ nor ATP dependent. Taken together, these findings suggest that, in cardiac tissue, pyruvate has a dual effect on SR Ca2+ release consisting of a direct inhibition of RyR channel activity and elevation of SR Ca2+ content. The latter effect was most probably mediated by an enhanced SR Ca2+ uptake due to an augmentation of mitochondria-dependent ATP synthesis.

Adenosine Diphosphate↗
PubMed2004

Ethyl pyruvate inhibits nuclear factor-kappaB-dependent signaling by directly targeting p65.

Ethyl pyruvate has been shown to have anti-inflammatory properties in numerous cell culture and animal studies. In this series of experiments, we tested the hypothesis that ethyl pyruvate inhibits signaling by the pro-inflammatory transcription factor, NF-kappaB. Ethyl pyruvate inhibited luciferase expression in lipopolysaccharide-stimulated murine macrophage-like RAW 264.7 cells transfected with an NF-kappaB-dependent luciferase reporter vector. Ethyl pyruvate also decreased NF-kappaB DNA-binding activity in lipopolysaccharide-stimulated RAW 264.7 cells and decreased lipopolysaccharide-induced expression of an NF-kappaB-dependent gene, inducible nitric oxide synthase. Ethyl pyruvate had no effect on the degradation of IkappaBalpha or IkappaBbeta in lipopolysaccharide-stimulated RAW 264.7 cells, suggesting that ethyl pyruvate acts distally to this step in the activation of NF-kappaB. In a cell-free system, binding of p50 homodimers to an NF-kappaB consensus oligonucleotide sequence was unaffected by ethyl pyruvate over a wide range of concentrations, indicating that ethyl pyruvate probably does not modify or interact with the p50 subunit of NF-kappaB. In contrast, ethyl pyruvate inhibited DNA binding by ectopically overexpressed wild-type p65 homodimers. However, ethyl pyruvate failed to inhibit the DNA-binding activity of homodimers of an overexpressed mutant form of a p65 with substitution of serine for cysteine 38. Taken together, these results suggest that ethyl pyruvate inhibits DNA-binding by covalently modifying p65 at Cys(38). We conclude that some of the beneficial anti-inflammatory effects of ethyl pyruvate may be due to modification of p65, thereby inhibiting signaling via the NF-kappaB pathway.

Animals↗
PubMed2004

Pyruvate shuttling during rest and exercise before and after endurance training in men.

We describe the isotopic exchange of lactate and pyruvate after arm vein infusion of [3-(13)C]lactate in men during rest and exercise. We tested the hypothesis that working muscle (limb net lactate and pyruvate exchange) is the source of the elevated systemic lactate-to-pyruvate concentration ratio (L/P) during exercise. We also hypothesized that the isotopic equilibration between lactate and pyruvate would decrease in arterial blood as glycolytic flux, as determined by relative exercise intensity, increased. Nine men were studied at rest and during exercise before and after 9 wk of endurance training. Although during exercise arterial pyruvate concentration decreased to below rest values (P < 0.05), pyruvate net release from working muscle was as large as lactate net release under all exercise conditions. Exogenous (arterial) lactate was the predominant origin of pyruvate released from working muscle. With no significant effect of exercise intensity or training, arterial isotopic equilibration [(IE(pyruvate)/IE(lactate)).100%, where IE is isotopic enrichment] decreased significantly (P < 0.05) from 60 +/- 3.1% at rest to an average value of 12 +/- 2.7% during exercise, and there were no changes in femoral venous isotopic equilibration. These data show that 1). the isotopic equilibration between lactate and pyruvate in arterial blood decreases significantly during exercise; 2). working muscle is not solely responsible for the decreased arterial isotopic equilibration or elevated arterial L/P occurring during exercise; 3). working muscle releases similar amounts of lactate and pyruvate, the predominant source of the latter being arterial lactate; 4). pyruvate clearance from blood occurs extensively outside of working muscle; and 5). working muscle also releases alanine, but alanine release is an order of magnitude smaller than lactate or pyruvate release. These results portray the complexity of metabolic integration among diverse tissue beds in vivo.

Adult↗
PubMed1977

Studies on the cyclic 3':5'-AMP-stimulated pig liver protein kinase reaction with pyruvate kinase as substrate.

The phosphorylation of pig liver pyruvate kinase by cyclic adenosine 3':5'-monophosphate-dependent protein kinase has been studied. For comparison, mixed histone and a synthetic heptapeptide were also used as substrates. Protein kinase was purified by chromatography on DEAE-cellulose, hydroxyapatite, and Sephadex G-200. The enzyme was stimulated by cyclic AMP with apparent Ka values of 2.5 and 0.8 x 10-7 M for pyruvate kinase and histone substrates, respectively. Divalent cations were essential for the activity of the protein kinase. Variation of the concentration of ATP resulted in approximately straight lines in Lineweaver-Burk plots for the phosphorylation of both pyruvate kinase and mixed histone. The apparent Km values for ATP were 21 and 11 muM, respectively. The phosphorylation rate increased with the concentration of pyruvate kinase even at a concentration of 2 muM pyruvate kinase. At a high ionic strength, the phosphorylation rate of both pyruvate kinase and histone decreased. The phosphorylation rate varied markedly with pH in imidazole/HC1 and Tris/HC1 buffers. At slightly alkaline pH values, pyruvate kinase was phosphorylated at a much higher rate than pH7, but this was not the case for histone. At pH 8.5, the phosphorylation rate of pyruvate kinase was 3.5 times the rate at pH 7, while the corresponding increase for the histone phosphorylation was 50 per cent. In potassium phosphate buffers, the phosphorylation rate of both substrates did not change significantly over the pH range studied. Arrhenius' plots of the protein kinase reaction resulted in a break at about 10 degrees when pyruvate kinase was used as substrate, whereas a straight line was obtained when using histone. The negative allosteric effectors of pyruvate kinase, alanine, and phenylalanine, increased the phosphorylation rate of pyruvate kinase at pH 8 by 50 and 120 per cent, respectively. The same effectors did not influence the phosphorylation rate of mixed histone or a synthetic heptapeptide. It is concluded that the conformations adopted by pyruvate kinase in the presence of allosteric inhibitors make it a better substrate for the protein kinase.

Adenosine Triphosphate↗
PubMed1979

Glucagon stimulation of liver mitochondrial CO2 fixation utilizing pyruvate generated inside the mitochondria.

Glucagon administration to the intact rat has been shown to stimulate pyruvate metabolism in liver mitochondria, presumably by increasing pyruvate transport into the organelle. In this report, we used alanine in place of pyruvate to examine the possibility that glucagon might stimulate pyruvate carboxylation per se independent of its postulated action on pyruvate transport. In agreement with previous reports, injection of a low dose of glucagon (50 micrograms/kg of rat) increased respiration, ATP synthesis, pyruvate decarboxylation, and CO2 fixation in liver mitochondria subsequently isolated. When alanine was used as a substrate, CO2 fixation, but not decarboxylation, was increased in liver mitochondria isolated from glucagon-treated rats. Pyruvate accumulation under these conditions was significantly lower in the glucagon-treated rat preparation. When mitochondria were incubated in a HCO3- -deficient buffer, pyruvate accumulation was identical in both preparations. The addition of a pyruvate transport inhibitor, alpha-cyanohydroxycinnamate (0.5 mM), inhibited CO2 fixation with pyruvate by 70%, but had no effect when alanine was used. Our data therefore suggest that glucagon stimluates mitochondrial pyruvate carboxylation independent of its possible action on pyruvate transport.

Alanine↗
PubMed1983

Induction of pyruvate dehydrogenase in 3T3-L1 cells during differentiation.

The activity of the pyruvate dehydrogenase complex and the content and turnover of the pyruvate dehydrogenase component were measured during the differentiation of 3T3-L1 preadipocytes into 3T3-L1 adipocytes. The specific activity of "total" pyruvate dehydrogenase complex increased approximately 7-fold in 3T3-L1 adipocytes differentiated with a treatment of insulin plus dexamethasone plus 1-methyl-3-isobutyl xanthine. The ratio of "active" pyruvate dehydrogenase complex to total pyruvate dehydrogenase complex remained unaltered in both 3T3-L1 preadipocytes and adipocytes. A specific goat antibody to bovine kidney pyruvate dehydrogenase quantitatively precipitated both alpha and beta subunits of pyruvate dehydrogenase from solubilized 3T3-L1 adipocytes. Using immunoprecipitation and gel electrophoresis techniques, we demonstrated an approximate 6-fold increase in pyruvate dehydrogenase content in 3T3-L1 adipocytes as compared to 3T3-L1 preadipocytes. Pulse labeling experiments revealed an approximately 5-fold increase in the rates of synthesis of both alpha and beta subunits of pyruvate dehydrogenase in 3T3-L1 adipocytes after 6 days of the hormonal treatment compared to those observed in 3T3-L1 preadipocytes. In contrast, the half-lives of alpha and beta subunits of pyruvate dehydrogenase were not significantly altered in 3T3-L1 preadipocytes (41 h) and adipocytes (49 h). The 6-8-fold increment in the specific activity of the pyruvate dehydrogenase complex in 3T3-L1 adipocytes therefore results from increased rates of synthesis of alpha and beta subunits of pyruvate dehydrogenase.

Adipose Tissue↗
PubMed1977

Pyruvate metabolism in bovine epididymal spermatozoa.

Treating bovine epididymal spermatozoa with rutamycin or rotenone inhibited both respiration and motility supported by endogenous substrates. When oxidative phosphorylation had been blocked with various inhibitors, pyruvate was metabolized to yield ATP and restored motility. Fructose, which is metabolized via glycolysis to yield ATP, was also able to resuscitate the cells. Other substrates tested (lactate, acetate, alpha-ketoglutarate, or glyoxylate) were unable to restore motility in rutamycin-treated cells. In the presence of pyruvate, the phosphorylation uncoupler, carbonylcyanide-p-trifluoromethyoxphenylhydrazone, reduced motility and ATP to common levels in untreated cells or cells treated with rutamycin or rotenone. Pyruvate is thus metabolized to produce ATP by a pathway independent of oxidative phosphorylation associated with the electron transport chain. 5-Methoxyindole-2-carboxylic acid, an inhibitor of lipoyldehydrogenase, prevented the increase of motility and ATP in rutamycin-treated cells, indicating that alpha-keto acid oxidation is involved in the production of ATP from pyruvate when rutamycin is present. With pyruvate present, bongkrekic acid, antimycin A, and anaerobiosis eliminated motility, reduced ATP to low levels, and also significantly reduced the rate of pyruvate metabolism. Acetate was produced from pyruvate only when cellular ATP concentrations were low. Decreases in free carnitine concentrations showed that pyruvate initially used was converted to acetylcarnitine. The results indicate that the intramitochondrial lactate dehydrogenase X, which is unique to spermatozoa, allows the NADH resulting from pyruvate oxidation to reduce other pyruvate molecules to lactate. Pyruvate thus competes with, and can substitute for, the NADH dehydrogenase of the electron transport chain. Pyruvate rapidly repletes the acetylcarnitine pool under a variety of conditions.

Adenosine Diphosphate↗