Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:6.4.1.1 (pyruvate carboxylase)
1,516 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effects of troglitazone and pioglitazone on glucose and fatty acid metabolism were studied in hepatocytes isolated from 24-h-starved rats. These thiazolidinediones inhibited long-chain fatty acid (oleate) oxidation and produced a very oxidized mitochondrial redox state. By contrast, thiazolidinediones did not affect the rate of medium-chain fatty acid (octanoate) oxidation or the activity of mitochondrial carnitine palmitoyltransferase (CPT) I. Thiazolidinediones inhibited selectively triglyceride synthesis but not phospholipid synthesis. The combined inhibition of oleate oxidation and esterification by troglitazone was due to a noncompetitive inhibition of mitochondrial and microsomal long-chain acyl-CoA synthetase (ACS) activities. It was suggested that troglitazone must be metabolized into its sulfo-conjugate derivative in liver cells to inhibit mitochondrial and microsomal ACS activities. Thiazolidinediones inhibited glucose production from lactate/pyruvate or from alanine. Analysis of gluconeogenic metabolite concentrations suggested that troglitazone would inhibit gluconeogenesis at the level of pyruvate carboxylase and glyceraldehyde-3-phosphate dehydrogenase reactions. It was concluded that 1) at a similar concentration, troglitazone was more efficient than pioglitazone to inhibit fatty acid metabolism and gluconeogenesis and 2) the inhibition of gluconeogenesis by troglitazone could be the result of the inhibition of long-chain fatty acid oxidation (decrease in acetyl-CoA, NADH-to-NAD+, and ATP-to-ADP ratios).
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PMID:Troglitazone inhibits fatty acid oxidation and esterification, and gluconeogenesis in isolated hepatocytes from starved rats. 886 61

Gluconeogenesis, or the formation of glucose from mainly lactate/ pyruvate, glycerol and alanine, plays an essential role in the maintenance of normoglycaemia during fasting. Inborn deficiencies are known of each of the four enzymes of the glycolytic-gluconeogenic pathway that ensure a unidirectional flux from pyruvate to glucose: pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose-1,6-bisphosphatase, and glucose-6-phosphatase. In this paper, the clinical picture, pathophysiology, diagnostic tests, genetics, treatment and prognosis of the deficiencies of fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxykinase are reviewed.
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PMID:Disorders of gluconeogenesis. 888 71

Metabolism of [U-13C5]glutamine was studied in primary cultures of cerebral cortical astrocytes in the presence or absence of extracellular glutamate. Perchloric acid extracts of the cells as well as redissolved lyophilized media were subjected to nuclear magnetic resonance and mass spectrometry to identify 13C-labeled metabolites. Label from glutamine was found in glutamate and to a lesser extent in lactate and alanine. In the presence of unlabeled glutamate, label was also observed in aspartate. It could be clearly demonstrated that some [U-13C5]glutamine is metabolized through the tricarboxylic acid cycle, although to a much smaller extent than previously shown for [U-13C5]glutamate. Lactate formation from tricarboxylic acid cycle intermediates has previously been demonstrated. It has, however, not been demonstrated that pyruvate, formed from glutamate or glutamine, may reenter the tricarboxylic acid cycle after conversion to acetyl-CoA. The present work demonstrates that this pathway is active, because [4,5-13C2]glutamate was observed in astrocytes incubated with [U-13C5]-glutamine in the additional presence of unlabeled glutamate. Furthermore, using mass spectrometry, mono-labeled alanine, glutamate, and glutamine were detected. This isotopomer could be derived via the action of pyruvate carboxylase using 13CO2 produced within the mitochondria or from labeled intermediates that had stayed in the tricarboxylic acid cycle for more than one turn.
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PMID:Metabolism of [U-13C5] glutamine in cultured astrocytes studied by NMR spectroscopy: first evidence of astrocytic pyruvate recycling. 893 91

Chemical modification of Escherichia coli phosphoenolpyruvate carboxylase (P-pyruvate carboxylase) by 2,4,6-trinitrobenzene sulfonate, a specific reagent for amino groups, causes desensitization to allosteric inhibitors, L-aspartate and L-malate, as well as inactivation. When L-malate is included in the modification mixture, P-pyruvate carboxylase was markedly protected from both desensitization and inactivation [Naide, A., Izui, K., Yoshinaga, T. & Katsuki, H. (1979) J. Biochem. (Tokyo) 85, 423-432]. To determine the lysine residue(s) involved in allosteric inhibition, the lysine residues that were protected from modification by L-malate were investigated by analyzing trinitrophenylated peptides liberated by digestion with glutamyl endopeptidase (V8-protease). The identified residues were Lys491, Lys620, Lys650, and Lys773. Each of these residues was individually replaced with an alanine or serine residue by site-directed mutagenesis to produce mutant enzymes. The mutant enzyme whose lysine residue was replaced with serine ([Ser620]P-pyruvate carboxylase) showed a marked desensitization to L-aspartate and L-malate, while retaining almost the same maximal catalytic activity as the wild-type P-pyruvate carboxylase. Essentially no changes in enzymatic properties were observed for the [Ala491]- and [Ala650]P-pyruvate carboxylases, while for the [Ala620]- and [Ala773]P-pyruvate carboxylases the polypeptides of the expected size were not significantly accumulated in the transformed E. coli cells, presumably due to intracellular degradation.
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PMID:The replacement of Lys620 by serine desensitizes Escherichia coli phosphoenolpyruvate carboxylase to the effects of the feedback inhibitors L-aspartate and L-malate. 924 11

Carbon metabolism was investigated in cerebellar and cortical astrocytes cultured for 15 or 35 days. The consumption rates of exogenous carbon sources--amino acids and glucose--and the production rates of exported metabolites--citrate, lactate, alanine and glutamine--were determined. The specific 13C-enrichment of lactate and glutamine carbons were determined after cell incubation with [1-13C]glucose. These data were used to evaluate the fluxes through metabolic pathways using a monocompartmental model of the cell metabolism including glycolysis and tricarboxylic acid cycle related pathways. The model concluded to a very large contribution of fatty acids as an endogenous carbon source of acetyl-CoA. As a consequence of the high fatty acid turn-over, there was an important recycling (via pyruvate) of the oxaloacetate molecules generated by citrate lyase activity. This recycling represented in fact the major part of the pyruvate carboxylase activity, which therefore was not directly related to metabolite export. Comparing the data from cerebellar and cortical astrocytes evidenced, on the other hand, some differences in metabolite contents which could be related to different cell maturation stages linked to their different tissular origins.
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PMID:Analysis of carbon metabolism in cultured cerebellar and cortical astrocytes. 929 87

In isolated rabbit renal kidney-cortex tubules 2 mM glycerol, which is a poor gluconeogenic substrate, does not induce glucose formation in the presence of alanine, while it activates gluconeogenesis on substitution of alanine by aspartate, glutamate or proline. The addition of either 5 mM 3-hydroxybutyrate or 5 mM acetoacetate to renal tubules incubated with alanine + glycerol causes a marked induction of glucose production associated with inhibition of glutamine synthesis. In contrast, the rate of the latter process is not altered by ketones in the presence of glycerol and either aspartate, glutamine or proline despite the stimulation of glucose formation. Acceleration of gluconeogenesis by ketone bodies in the presence of amino acids and glycerol is probably due to (i) stimulation of pyruvate carboxylase activity, (ii) activation of malate-aspartate shuttle as concluded from elevated intracellular levels of malate, aspartate and glutamate, as well as (iii) diminished supply of ammonium for glutamine synthesis from alanine resulting from a decrease in glutamate dehydrogenase activity.
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PMID:Ketone bodies activate gluconeogenesis in isolated rabbit renal cortical tubules incubated in the presence of amino acids and glycerol. 936 Jul 22

CO2 fixation was measured in cultured astrocytes isolated from neonatal rat brain to test the hypothesis that the activity of pyruvate carboxylase influences the rate of de novo glutamate and glutamine synthesis in astrocytes. Astrocytes were incubated with 14CO2 and the incorporation of 14C into medium or cell extract products was determined. After chromatographic separation of 14C-labelled products, the fractions of 14C cycled back to pyruvate, incorporated into citric acid cycle intermediates, and converted to the amino acids glutamate and glutamine were determined as a function of increasing pyruvate carboxylase flux. The consequences of increasing pyruvate, bicarbonate, and ammonia were investigated. Increasing extracellular pyruvate from 0 to 5 mM increased pyruvate carboxylase flux as observed by increases in the 14C incorporated into pyruvate and citric acid cycle intermediates, but incorporation into glutamate and glutamine, although relatively high at low pyruvate levels, did not increase as pyruvate carboxylase flux increased. Increasing added bicarbonate from 15 to 25 mM almost doubled CO2 fixation. When 25 mM bicarbonate plus 0.5 mM pyruvate increased pyruvate carboxylase flux to approximately the same extent as 15 mM bicarbonate plus 5 mM pyruvate, the rate of appearance of [14C] glutamate and glutamine was higher with the lower level of pyruvate. The conclusion was drawn that, in addition to stimulating pyruvate carboxylase, added pyruvate (but not added bicarbonate) increases alanine aminotransferase flux in the direction of glutamate utilization, thereby decreasing glutamate as pyruvate + glutamate --> alpha-ketoglutarate + alanine. In contrast to previous in vivo studies, the addition of ammonia (0.1 and 5 mM) had no effect on net 14CO2 fixation, but did alter the distribution of 14C-labelled products by decreasing glutamate and increasing glutamine. Rather unexpectedly, ammonia did not increase the sum of glutamate plus glutamine (mass amounts or 14C incorporation). Low rates of conversion of alpha-[14C]ketoglutarate to [14C]glutamate, even in the presence of excess added ammonia, suggested that reductive amination of alpha-ketoglutarate is inactive under conditions studied in these cultured astrocytes. We conclude that pyruvate carboxylase is required for de novo synthesis of glutamate plus glutamine, but that conversion of alpha-ketoglutarate to glutamate may frequently be the rate-limiting step in this process of glutamate synthesis.
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PMID:Role of pyruvate carboxylase in facilitation of synthesis of glutamate and glutamine in cultured astrocytes. 937 62

Infant pigs (8.5 kg) were fasted for 16 h and infused for 6 h with [U-13C]glucose. The fractional abundances of all 13C mass isotopomers of plasma glucose, lactate, and pyruvate and of plasma, hepatic, and very low density lipoprotein apolipoprotein B-100 (apoB-100) alanine, glutamate, and aspartate were measured. The ratios of [13C3]aspartate. [13C3]glutamate, and [13C3]alanine in apoB-100 were used to estimate the positional equilibrium of [13C3]oxaloacetate, the fractional contribution of pyruvate carboxylase to the hepatic oxaloacetate flux, and the activity of hepatic pyruvate dehydrogenase. The values were compared with those based on glucose labeling and previously published equations. The two methods [Katz and Lee method (J. Katz, P.A. Wals., and W.-N. P. Lee. J. Biol. Chem. 264: 12994-13001, 1989) and apoB method] gave similar estimates of the positional equilibrium of [13C3]oxaloacetate (0.59, Katz and Lee method; 0.61, apoB method) but slightly different estimates of the contribution of pyruvate carboxylase to the oxaloacetate flux (0.36, Katz and Lee; 0.31 apoB). Gluconeogenesis apparently contributed between 71 (Katz and Lee method) and 80% (apoB method) of the glucose entry rate (25 mumol.kg-1.min-1), and pyruvate dehydrogenase contributed 20% of the hepatic acetyl-CoA. We conclude that the labeling of aspartate in apoB-100 provides a good estimate of the isotopomer distribution in hepatic oxaloacetate but may underestimate the absolute isotopic enrichment by 50%.
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PMID:Gluconeogenesis measured with [U-13C]glucose and mass isotopomer analysis of apoB-100 amino acids in pigs. 948 70

Pyruvate carboxylase (PC) is a biotinylated mitochondrial enzyme that catalyzes the conversion of pyruvate to oxaloacetate. Children with inborn errors of PC metabolism have lactic acidosis, hypoglycemia, and mental retardation. The variable severity of the clinical phenotype is dependent on both genetic and environmental factors. Two consanguineous families with moderate forms of PC deficiency were characterized at the biochemical and molecular levels. In both families, the probands were found to have low PC activity (range, 2-25% of control) in blood lymphocytes and skin fibroblasts associated with either diminished or normal protein levels. In the first case, sequencing of patient-specific PC cDNA demonstrated a T to C substitution at nucleotide 434, which causes a valine to alanine change at amino acid residue 145. Direct sequencing of the parents showed that they are heterozygous for this mutation. In the second family, a brother and sister had mental retardation and episodes of severe lactic/ketoacidosis in early childhood. In these cases, a C to T substitution at nucleotide 1351 results in a cysteine for arginine substitution at amino acid residue 451; the parents were also found to be heterozygous for this mutation. In both families, no other mutations were found, and both substitutions occurred in relatively conserved amino acid residues. These mutations, located in the biotin carboxylase domain, provide a unique opportunity to analyze how natural occurring mutations affect PC function.
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PMID:Molecular characterization of pyruvate carboxylase deficiency in two consanguineous families. 958 2

Kreb's tricarboxylic (TCA) cycle was studied in Halobacterium salinarum cells grown in the presence of glucose or alanine. The cells were incubated with 13C-labeled substrate and the labeling pattern of various carbon positions in glutamate was monitored by 13C-NMR spectroscopy. [2-13C]pyruvate, when used as a substrate, led mainly to signals for C-1 and C-5 glutamate, with some C-3 glutamate. [3-13C]pyruvate as a substrate produced signals, mainly C-2, C-3, and C-4 glutamate, with some C-1 and C-5 glutamate. The multiplicity of the signals and observation of a C-1 signal in this case indicates extensive cycling of the label in the TCA cycle. Isotopomer analysis of glutamate labeling suggested that of the total pyruvate entering the TCA cycle, the flux through pyruvate:ferredoxin oxidoreductase was 90% while that through pyruvate carboxylase was 10%. Only 53% of the total acetyl-CoA was produced from the added labeled pyruvate, the rest being generated endogenously. In the presence of nitrogen, mainly transamination reaction products were formed in the case of both these substrates.
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PMID:Kreb's TCA cycle in Halobacterium salinarum investigated by 13C nuclear magnetic resonance spectroscopy. 982 32


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