Effect of streptozotocin-induced diabetes mellitus on the turnover of rat liver pyruvate carboxylase and pyruvate dehydrogenase.

Immunochemical techniques were used to study the effect of streptozotocin-induced diabetes on the amounts of pyruvate carboxylase and pyruvate dehydrogenase and on their rates of synthesis and degradation. Livers from diabetic rats had twice the pyruvate carboxylase activity of livers from normal rats when expressed in terms of DNA or body weight. The changes in catalytic activity closely paralleled changes in immunoprecipitable enzyme protein. Relative rates of synthesis determined by pulse-labelling studies showed that the ratio of synthesis of pyruvate carboxylase to that of average mitochondrial protein was increased 2.0-2.5 times in diabetic animals over that of control animals. Other radioisotopic studies indicated that the rate of degradation of this enzyme was not altered significantly in diabetic rats, suggesting that the increase in this enzyme was due to an increased rate of synthesis. Similar experiments with pyruvate dehydrogenase, the first component of the pyruvate dehydrogenase complex, showed that livers from diabetic rats had approximately the same amount of immunoprecipitable enzyme protein as the control animals, but a larger proportion of the enzyme was in its inactive state. The rates of synthesis and degradation of pyruvate dehydrogenase were not affected significantly by diabetes.     

Pyruvate regulation of growth and differentiation in primary cultures of rat tracheal epithelial cells

These studies examined the effect of exogenous pyruvate on the growth and differentiation of primary cell cultures of rat tracheal epithelial cells. The cell cultures were derived from outgrowths of tracheal explants, and require pyruvate for survival and growth in the presence of 10% FBS. In pyruvate-supplemented (2 mM) medium, the number of cells attached to the dish increased rapidly, while exfoliation of cells into the medium as well as formation of cornified envelopes were relatively low. The growth response to pyruvate was concentration-dependent in these cell cultures. In the absence of pyruvate, the extent of terminal differentiation to keratinization gradually increased. This was characterized by a cessation of growth after one week, and an increase in exfoliation until all cells had sloughed from the dish. Accompanying these changes was a marked increase in the formation of cornified envelopes. Cells undergoing DNA synthesis were present throughout 2 weeks of culture in pyruvate-deprived medium, even as the total number of cells was diminishing. Several compounds, including other 2-oxocarboxylic acids, were ineffective growth substitutes for pyruvate. These results indicate that the requirement for pyruvate is quite stringent in these cultures and that one way pyruvate promotes the growth of tracheal epithelial cells is by inhibiting terminal differentiation.     

The effect of aging and acetyl-L-carnitine on the pyruvate transport and oxidation in rat heart mitochondria.

The effect of aging and acute treatment with acetyl-L-carnitine on the pyruvate transport and oxidation in rat heart mitochondria was studied. The activity of the pyruvate carrier as well as the rates of pyruvate-supported respiration were both depressed (around 40%) in heart mitochondria from aged rats, the major decrease occurring during the second year of life. Administration of acetyl-L-carnitine to aged rats almost completely restored the rates of these metabolic functions to the level of young control rats. This effect of acetyl-L-carnitine was not due to changes in the content of pyruvate carrier molecules. The heart mitochondrial content of cardiolipin, a key phospholipid necessary for mitochondrial substrate transport, was markedly reduced (approximately 40%) in aged rats. Treatment of aged rats with acetyl-L-carnitine reversed the age-associated decline in cardiolipin content. As the changes in cardiolipin content were correlated with changes in rates of pyruvate transport and oxidation, it is suggested that acetyl-L-carnitine reverses the age-related decrement in the mitochondrial pyruvate metabolism by restoring the normal cardiolipin content.     

Stimulation of mitochondrial pyruvate transport in rat renal cortex by phenylephrine

Phenylephrine effect on liver and kidney cortex mitochondrial pyruvate concentration was investigated. While in liver the alpha 1-adrenergic agent produced a decrease in pyruvate content, a significant increase was observed in kidney, even in the presence of 0.5 mM alpha-cyano-4-hydroxy-cinnamate. These changes were not observed when pyruvate was formed by intramitochondrial transamination of alanine, suggesting a role for the pyruvate transport across mitochondrial membranes in the regulation of mitochondrial pyruvate metabolism in kidney cortex. This was corroborated measuring the phenylephrine effect on pyruvate carboxylation.     

Regulation of mitochondrial pyruvate carboxylation and gluconeogenesis in rat hepatocytes via an alpha-adrenergic, adenosine 3':5'-monophosphate-independent mechanism.

Experiments were performed to determine if catecholamines can regulate control points in the gluconeogenic pathway, such as mitochondrial pyruvate carboxylation and pyruvate kinase activity, via an alpha-adrenergic, adenosine 3':5'-monophosphate-independent mechanism. Of a number of alpha agonists tested, only norepinephrine, epinephrine, and phenylephrine caused an increase in mitochondrial pyruvate metabolism. The effects of catecholamines on pyruvate carboxylation were not attenuated by 1-propranolol which abolishes changes in cyclic nucleotide levels but were blocked by alpha antagonists such as ergotamine, phenoxybenzamine, and phentolamine. Time course experiments demonstrated that the effects of catecholamines on the mitochondria and on carbohydrate metabolism correlated temporally with the concentration of epinephrine in the medium but not with the small changes in adenosine 3':5'-monophosphate. The effects of catecholamines appeared to require extracellular Ca2+ ion. The observation that catecholamines do not increase gluconeogenesis to the same extent as glucagon was not due to a differential effect on mitochondrial CO2 fixation. Rather, catecholamines caused a smaller inhibition of pyruvate kinase activity than did glucagon. The effects of catecholamines on pyruvate kinase also appeared to be mediated by an alpha-adrenergic, adenosine 3':5'-monophosphate-independent mechanism.     

Regulation of pyruvate kinase expression and growth in mastocytoma cells : I. Initial observations

The specific activities of pyruvate kinase and phosphofructokinase but not lactate dehydrogenase increase as P-815 mastocytoma cells approach the stationary phase. During this growth period, the rates of uptake of labelled precursors into DNA, RNA and total protein decreases. On the other hand, the pyruvate kinase protein level changes in parallel with activity. Although the K-isozyme is the primary form of pyruvate kinase expressed, some M-type subunit is also present and both forms undergo an increase in specific activity. In addition, pyruvate kinase expression is also elevated by adding cAMP analogues with theophylline, butyrate or conditioned media. This increased level of expression is hypothesized to be a secondary event associated with a differentiation-like-induced expression of the mast cell phenotype.     

Biological studies on the effects of some therapeutic procedures used in psychiatry.

The glucose, lactate, and pyruvate levels, the lactate/pyruvate ratio and pH were studied in serum and CSF of patients with schizophrenia, reactive psychosis, symptomatic or circular psychosis under the effect of atropine coma, ES and pentetrazole convulsions, tranquilizer treatment and combined therapy. Convulsive therapy caused a disorder in cerebral carbohydrate metabolism while no similar changes were induced by atropine coma. Anaerobic carbohydrate metabolism was stimulated by combined therapy. This treatment had the best effect and the changes caused by it were slighter than in the case of convulsive therapy. The changes in EEG frequency corresponded to the biochemical changes.     

Metabolic control of hepatic gluconeogenesis during exercise.

Prolonged exercise increased the concentrations of the hexose phosphates and phosphoenolpyruvate and depressed those of fructose 1,6-bisphosphate, triose phosphates and pyruvate in the liver of the rat. Since exercise increases gluconeogenic flux, these changes in metabolite concentrations suggest that metabolic control is exerted, at least, at the fructose 6-phosphate/fructose 1,6-bisphosphate and phosphoenolpyruvate/pyruvate substrate cycles. Exercise increased the maximal activities of glucose 6-phosphatase, fructose 1,6-bisphosphatase, pyruvate kinase and pyruvate carboxylase in the liver, but there were no changes in those of glucokinase, 6-phosphofructokinase and phosphoenolpyruvate carboxykinase. Exercise changed the concentrations of several allosteric effectors of the glycolytic or gluconeogenic enzymes in liver; the concentrations of acetyl-CoA, ADP and AMP were increased, whereas those of ATP, fructose 1,6-bisphosphate and fructose 2,6-bisphosphate were decreased. The effect of exercise on the phosphorylation-dephosphorylation state of pyruvate kinase was investigated by measuring the activities under conditions of saturating and subsaturating concentrations of substrate. The submaximal activity of pyruvate kinase (0.5 mM-phosphoenolpyruvate), expressed as percentage of Vmax., decreased in the exercised animals to less than half that found in the controls. These changes suggest that hepatic pyruvate kinase is less active during exercise, possibly owing to phosphorylation of the enzyme, and this may play a role in increasing the rate of gluconeogenesis.     

Effect of Leucine Administration to Female Rats During Pregnancy and Lactation on Oxidative Stress and Enzymes Activities of Phosphoryltransfer Network in Cerebral Cortex and Hippocampus of the Offspring

Maple Syrup Urine Disease is an inborn error of metabolism caused by severe deficiency in the activity of branched-chain α-keto acid dehydrogenase complex. Neurological disorder is common in patients with maple syrup urine disease. Although leucine is considered the main toxic metabolite, the mechanisms underlying the neuropathology of brain injury are poorly understood. In the present study, we evaluated the possible preventive effect of the co-administration of creatine plus pyruvate on the effects elicited by leucine administration to female Wistar rats during pregnancy and lactation on some oxidative stress parameters as well as the activities of some enzymes involved in the phosphoryltransfer network in the brain cortex and hippocampus of the offspring at 21 days of age. Leucine administration induced oxidative stress and altered the activities of pyruvate kinase, adenylate kinase, mitochondrial and cytosolic creatine kinase. Co-administration of creatine plus pyruvate was partially effective in the prevention of some alterations provoked by leucine administration on the oxidative stress but not in the enzymes of phosphoryltransfer network. These results suggest that non-treated maternal hyperleucinemia may be toxic to the brain of the offspring.     

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.     

Tellurite-induced carbonylation of the Escherichia coli pyruvate dehydrogenase multienzyme complex

The soluble tellurium oxyanion, tellurite, is toxic for most organisms. At least in part, tellurite toxicity involves the generation of oxygen-reactive species which induce an oxidative stress status that damages different macromolecules with DNA, lipids and proteins as oxidation targets. The objective of this work was to determine the effects of tellurite exposure upon the Escherichia coli pyruvate dehydrogenase (PDH) complex. The complex displays two distinct enzymatic activities: pyruvate dehydrogenase that oxidatively decarboxylates pyruvate to acetylCoA and tellurite reductase, which reduces tellurite (Te⁴⁺) to elemental tellurium (Te^o). PDH complex components (AceE, AceF and Lpd) become oxidized upon tellurite exposure as a consequence of increased carbonyl group formation. When the individual enzymatic activities from each component were analyzed, AceE and Lpd did not show significant changes after tellurite treatment. AceF activity (dihydrolipoil acetyltransferase) decreased ~30% when cells were exposed to the toxicant. Finally, pyruvate dehydrogenase activity decreased >80%, while no evident changes were observed in complex′s tellurite reductase activity.     

Pyruvate reduces DNA damage during hypoxia and after reoxygenation in hepatocellular carcinoma cells

Pyruvate is located at a crucial crossroad of cellular metabolism between the aerobic and anaerobic pathways. Modulation of the fate of pyruvate, in one direction or another, can be important for adaptative response to hypoxia followed by reoxygenation. This could alter functioning of the antioxidant system and have protective effects against DNA damage induced by such stress. Transient hypoxia and alterations of pyruvate metabolism are observed in tumors. This could be advantageous for cancer cells in such stressful conditions. However, the effect of pyruvate in tumor cells is poorly documented during hypoxia/reoxygenation. In this study, we showed that cells had a greater need for pyruvate during hypoxia. Pyruvate decreased the number of DNA breaks, and might favor DNA repair. We demonstrated that pyruvate was a precursor for the biosynthesis of glutathione through oxidative metabolism in HepG2 cells. Therefore, glutathione decreased during hypoxia, but was restored after reoxygenation. Pyruvate had beneficial effects on glutathione depletion and DNA breaks induced after reoxygenation. Our results provide more evidence that the alpha-keto acid promotes the adaptive response to hypoxia followed by reoxygenation. Pyruvate might thus help to protect cancer cells under such stressful conditions, which might be harmful for patients with tumors.     

Cloning of the pyruvate kinase gene (pyk) of Corynebacterium glutamicum and site-specific inactivation of pyk in a lysine-producing Corynebacterium lactofermentum strain.

The pyruvate kinase gene pyk from Corynebacterium glutamicum was cloned by applying a combination of PCR, site-specific mutagenesis, and complementation. A 126-bp DNA fragment central to the C. glutamicum pyk gene was amplified from genomic DNA by PCR with degenerate oligonucleotides as primers. The cloned DNA fragment was used to inactivate the pyk gene in C. glutamicum by marker rescue mutagenesis via homologous recombination. The C. glutamicum pyk mutant obtained was unable to grow on minimal medium containing ribose as the sole carbon source. Complementation of this phenotype by a gene library resulted in the isolation of a 2.8-kb PstI-BamHI genomic DNA fragment harboring the C. glutamicum pyk gene. Multiple copies of plasmid-borne pyk caused a 20-fold increase of pyruvate kinase activity in C. glutamicum cell extracts. By using large internal fragments of the cloned C. glutamicum gene, pyk mutant derivatives of the lysine production strain Corynebacterium lactofermentum 21799 were generated by marker rescue mutagenesis. As determined in shake flask fermentations, lysine production in pyk mutants was 40% lower than that in the pyk+ parent strain, indicating that pyruvate kinase is essential for high-level lysine production. This finding questions an earlier hypothesis postulating that redirection of carbon flow at the phosphoenol pyruvate branch point of glycolysis through elimination of pyruvate kinase activity results in an increase of lysine production in C. glutamicum and its close relatives.     

Pyruvate prevents hydrogen peroxide-induced apoptosis

Studies were carried out to investigate the protective effects of pyruvate, a key glycolytic intermediate and α-keto-monocarboxylate, against oxidative stress-induced apoptosis. Oxidative stress was induced by treating mouse thymocytes with 25 μM hydrogen peroxide for 15 min at 37°C under 5% CO₂ in air. Pre- and post-treatment of cells with 10 mM pyruvate inhibited morphological changes, internucleosomal DNA fragmentation, and translocation of phosphatidylserine to the plasma membrane surface, which are characteristic features of apoptosis. L-lactate (10 mM) and acetate (10 mM) were ineffective in inhibiting apoptosis and appeared to be toxic to the cells under similar conditions. The results suggest that pyruvate has therapeutic potential for use in the treatment of oxidative stress-induced disorders associated with increased apoptosis.     

Effect of long-chain fatty acyl-CoA on mitochondrial and cytosolic ATP/ADP ratios in the intact liver cell.

The effect of long-chain acyl-CoA on subcellular adenine nucleotide systems was studied in the intact liver cell. Long-chain acyl-CoA content was varied by varying the nutritional state (fed and starved states) or by addition of oleate. Starvation led to an increase in the mitochondrial and a decrease in the cytosolic ATP/ADP ratio in liver both in vivo and in the isolated perfused organ as compared with the fed state. The changes were reversed on re-feeding glucose in liver in vivo or on infusion of substrates (glucose, glycerol) in the perfused liver, respectively. Similar changes in mitochondrial and cytosolic ATP/ADP ratios occurred on addition of oleate, but, importantly, not with a short-chain fatty acid such as octanoate. It is concluded that long-chain acyl-CoA exerts an inhibitory effect on mitochondrial adenine nucleotide translocation in the intact cell, as was previously postulated in the literature from data obtained with isolated mitochondria. The physiological relevance with respect to pyruvate metabolism, i.e. regulation of pyruvate carboxylase and pyruvate dehydrogenase by the mitochondrial ATP/ADP ratio, is discussed.     

The stimulation of mitochondrial pyruvate carboxylation after dexamethasone treatment of rats.

Treatment of rats for 3 h with dexamethasone was shown to stimulate both pyruvate carboxylation and decarboxylation in the subsequently isolated mitochondria. The effect of hormone treatment on pyruvate carboxylation was also apparent in liver homogenates assayed within minutes of killing the animal and was independent of the temperature at which the assay was performed, suggesting that it was not an artifact of the mitochondrial preparation procedure. The stimulation of both aspects of pyruvate metabolism in the intact organelle was independent of the induction of either pyruvate carboxylase or pyruvate dehydrogenase. Similarly, there was no change in the percentage of pyruvate dehydrogenase in the active form, indicating that the effect of steroid treatment on pyruvate oxidation was not via changes in the degree of phosphorylation of the enzyme. Adrenalectomizing the animals for a period of 14 days before the experiment had no effect on either parameter. Glucocorticoid treatment of the animals increased the rate of pyruvate uptake into the mitochondria, as measured by the titration of pyruvate metabolism with alpha-cyano-4-hydroxycinnamate, a specific inhibitor of the pyruvate translocator. It also increased the intramitochondrial concentrations of acetyl-CoA and ATP and led to an elevated [ATP]/[ADP] ratio within the mitochondria. It is suggested that both enzymes of pyruvate metabolism exist in the mitochondria under considerable restraint and that glucocorticoids act to relieve this restraint by alterations in substrate supply and the intramitochondrial concentrations of effector molecules.     

Decreased expression of cytosolic pyruvate kinase in potato tubers leads to a decline in pyruvate resulting in an in vivo repression of the alternative oxidase

The aim of this work was to investigate the effect of decreased cytosolic pyruvate kinase (PKc) on potato (Solanum tuberosum) tuber metabolism. Transgenic potato plants with strongly reduced levels of PKc were generated by RNA interference gene silencing under the control of a tuber-specific promoter. Metabolite profiling showed that decreased PKc activity led to a decrease in the levels of pyruvate and some other organic acids involved in the tricarboxylic acid cycle. Flux analysis showed that this was accompanied by changes in carbon partitioning, with carbon flux being diverted from glycolysis toward starch synthesis. However, this metabolic shift was relatively small and hence did not result in enhanced starch levels in the tubers. Although total respiration rates and the ATP to ADP ratio were largely unchanged, transgenic tubers showed a strong decrease in the levels of alternative oxidase (AOX) protein and a corresponding decrease in the capacity of the alternative pathway of respiration. External feeding of pyruvate to tuber tissue or isolated mitochondria resulted in activation of the AOX pathway, both in the wild type and the PKc transgenic lines, providing direct evidence for the regulation of AOX by changes in pyruvate levels. Overall, these results provide evidence for a crucial role of PKc in the regulation of pyruvate levels as well as the level of the AOX in heterotrophic plant tissue, and furthermore reveal that these parameters are interlinked in vivo.     

Stimulation of alanine metabolism by ammonia in the perfused rat liver. Quantitative analysis by means of a mathematical model

The effect of ammonia on the catabolism of alanine was studied in the perfused rat liver. Addition of 0.5 mM NH4Cl to the perfusion medium containing 5 mM alanine plus 0.1 mM octanoate produced drastic changes in the metabolite concentrations in the efflux medium. Not only the rate of ureogenesis was activated, but also the formation of glucose, lactate and pyruvate. Additionally, respiration was stimulated, the output of ketone bodies decreased, and the redox ratios lactate/pyruvate as well as 3-hydroxybutyrate/acetoacetate became more oxidized. To interpret the causes of these metabolic changes, a mathematical model was developed. It contains kinetic equations by which fluxes through essential pathways of alanine catabolism, gluconeogenesis and energy metabolism were related to the intracellular concentrations of pyruvate, oxaloacetate and ammonia, as well as to the redox ratios lactate/pyruvate and 3-hydroxybutyrate/acetoacetate. Using a nonlinear regression procedure, the model was suitable to be fitted to the data found in the experiments. The consistency of the model and experiment allowed the changes caused by ammonia to be explained. Primarily, ammonia stimulated ureogenesis hence accelerating the deamination of alanine which led to the increased formation of pyruvate, lactate and glucose. The enhanced energetic load resulting from ureogenesis and gluconeogenesis shifted the mitochondrial and cytosolic NAD systems towards more oxidized states which additionally modified the flux rates. The results demonstrate that there is a high degree of cooperativity between the metabolic pathways.