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)

Biotin carboxylase from Escherichia coli catalyzes the ATP-dependent carboxylation of biotin and is one component of the multienzyme complex acetyl-CoA carboxylase, which catalyzes the committed step in long-chain fatty acid synthesis. For the carboxylation of biotin to occur, biotin must be deprotonated at its N1' position. Kinetic investigations, including solvent isotope effects and enzyme inactivation by N-ethylmaleimide, suggested a catalytic role for a cysteine residue and led to the proposal of a mechanism for the deprotonation of biotin. The proposed pathway suggests a catalytic base removes a proton from a nearby cysteine residue, forming a thiolate anion, which then abstracts the proton from biotin. Inactivation studies of pyruvate carboxylase, which has an analogous mode of action to biotin carboxylase, suggests the catalytic base in this reaction is a lysine residue. Using the crystal structure of biotin carboxylase, cysteine 230 and lysine 238 were identified as the likely active-site residues that act as this acid-base pair. To test the hypothesis that cysteine 230 and lysine 238 act as an acid-base pair to deprotonate biotin, site-directed mutagenesis was used to mutate cysteine 230 to alanine (C230A) and lysine 238 to glutamine (K238Q). Mutations at either residue resulted in a 50-fold increase in the K(m) for ATP. The C230A mutation had no effect on the formation of carboxybiotin, indicating that cysteine 230 does not play a role in the deprotonation of biotin. However, the K238Q mutation resulted in no formation of carboxybiotin, which showed that lysine 238 has a role in the carboxylation reaction. N-Ethylmaleimide was found to inactivate the C230A mutant but not the K238Q mutant, suggesting that N-ethylmaleimide is reacting with lysine 238 and not cysteine 230. The pH dependence of N-ethylmaleimide inactivation revealed that the pK value for lysine 238 was 9.4 or higher, suggesting lysine 238 is not a catalytic base. Thus, the results suggest that cysteine 230 and lysine 238 do not act as an acid-base pair in the deprotonation of biotin.
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PMID:Do cysteine 230 and lysine 238 of biotin carboxylase play a role in the activation of biotin? 1074 3

Lactate metabolism in the adult rat brain was investigated in relation with the concept of lactate trafficking between astrocytes and neurons. Wistar rats were infused intravenously with a solution containing either [3-(13)C]lactate (534 mM) or both glucose (750 mM) and [3-(13)C]lactate (534 mM). The time courses of both the concentration and (13)C enrichment of blood glucose and lactate were determined. The data indicated the occurrence of [3-(13)C]lactate recycling through liver gluconeogenesis. The yield of glucose labeling was, however, reduced when using the glucose-containing infusate. After a 20-min or 1-h infusion, perchloric acid extracts of the brain tissue were prepared and subsequently analyzed by (13)C- and (1)H-observed/(13)C-edited NMR spectroscopy. The (13)C labeling of amino acids indicated that [3-(13)C]lactate was metabolized in the brain. Based on the alanine C3 enrichment, lactate contribution to brain metabolism amounted to 35% under the most favorable conditions used. By contrast with what happens with [1-(13)C]glucose metabolism, no difference in glutamine C2 and C3 labeling was evidenced, indicating that lactate was metabolized in a compartment deprived of pyruvate carboxylase activity. This result confirms, for the first time from an in vivo study, that lactate is more specifically a neuronal substrate.
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PMID:The metabolism of [3-(13)C]lactate in the rat brain is specific of a pyruvate carboxylase-deprived compartment. 1089 22

In Saccharomyces cerevisiae, the existence of PYC1 and PYC2 encoding cytosolic pyruvate carboxylase isoform I and II is rather puzzling, owing to the lack of potent differential gene regulation by the carbon sources. We report several findings indicating that these two genes are differentially regulated by the nature of the nitrogen source. In wild-type cells, the activity of pyruvate carboxylase, which is the sum of pyruvate carboxylase isoform I and II, was two- to fivefold lower in carbon medium containing aspartate, asparagine, glutamate or glutamine instead of ammonium as the nitrogen source, whereas it was 1.5- to threefold higher when the ammonium source was substituted by arginine, methionine, threonine or leucine. These enzymatic changes were independent of the nature of the carbon source and closely correlated to the changes in beta-galactosidase from PYC1-lacZ gene fusion and in PYC1 transcripts. Transfer of exponentially growing cells of the pyc2 mutant from an aspartate or a glutamate medium to an ammonium medium caused a fivefold increase in PYC1 mRNA in less than 30 min, whereas in the inverse experiment, PYC1 transcripts returned within 30 min to the low levels found in aspartate/glutamate medium. By contrast, these conditions affected neither the pyruvate carboxylase activity encoded by PYC2 nor PYC2 mRNA. Considering that changes in PYC1 expression inversely correlated with changes in alpha-ketoglutarate concentration or in alpha-ketoglutarate/glutamate ratio following the nitrogen shift experiments, and taking into account the pivotal role of this metabolite in ammonium assimilation, it is suggested that changes in alpha-ketoglutarate or in the alpha-ketoglutarate/glutamate ratio might be implicated in triggering the nitrogen effects on PYC1 expression. The physiological significance of the differential sensitivity of PYC1 and PYC2 genes with respect to the nitrogen source in the growth medium is also discussed.
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PMID:Regulation of pyc1 encoding pyruvate carboxylase isozyme I by nitrogen sources in Saccharomyces cerevisiae. 1108 92

In order to address the question whether lactate in blood can serve as a precursor for cerebral metabolites, fully awake rats were injected intravenously with [U-(13)C]lactate or [U-(13)C]glucose followed 15 min later by decapitation. Incorporation of label from [U-(13)C]glucose was seen mainly in glutamate, GABA, glutamine, aspartate, alanine and lactate. More label was found in glutamate than glutamine, underscoring the predominantly neuronal metabolism of pyruvate from [U-(13)C]glucose. It was estimated that the neuronal metabolism of acetyl CoA from glucose accounts for at least 66% and the glial for no more than 34% of the total glucose consumption. When [U-(13)C]lactate was the precursor, label incorporation was similar to that observed from [U-(13)C]glucose, but much reduced. Plasma analysis revealed the presence of approximately equal amounts of [1,2,3-(13)C]- and [1,2-(13)C]glucose, showing gluconeogenesis from [U-(13)C]lactate. It was thus possible that the labeling seen in the cerebral amino acids originated from labeled glucose, not [U-(13)C]lactate. However, the presence of significantly more label in [U-(13)C]- than in [2,3-(13)C]alanine demonstrated that [U-(13)C]lactate did indeed cross the blood-brain barrier, and was metabolized further in the brain. Furthermore, contributions from pyruvate carboxylase (glial enzyme) were detectable in glutamine, glutamate and GABA, and were comparatively more pronounced in the glucose group. This indicated that relatively more pyruvate from lactate than glucose was metabolized in neurons. Surprisingly, the same amount of lactate was synthesized via the tricarboxylic acid cycle in both groups, indicating transfer of neurotransmitters from the neuronal to the astrocytic compartment, as previous studies have shown that this lactate is synthesized primarily in astrocytes. Taking into consideration that astrocytes take up glutamate more avidly than GABA, it is conceivable that neuronal lactate metabolism was more prominent in glutamatergic neurons.
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PMID:(13)C MR spectroscopy study of lactate as substrate for rat brain. 1111 Nov 59

Tight glycemic control during diabetic pregnancy has been shown to significantly reduce the occurrence of congenital malformations and other effects of maternal diabetes on the offspring. However, intensive insulin therapy often causes recurring acute maternal hypoglycemia, which has been found to be harmful to the developing fetus, although the mechanisms involved are not clear. The aim of our work was to study the effect of acute insulin-induced maternal hypoglycemia on glucose metabolism in the fetal brain. To this end, near-term pregnant New Zealand rabbits were rendered hypoglycemic, and [U-(13)C]glucose was infused into maternal circulation. The metabolic fate of the (13)C-labeled glucose was then studied in fetal brain extracts by (13)C NMR isotopomer analysis, together with conventional biochemical assays of glucose and lactate levels in both plasma and brain. For comparison [U-(13)C]glucose was also administered to insulin-induced hypoglycemic young adult rabbits. Our results showed that while plasma glucose levels were significantly reduced (approximately 70%) relative to controls, no changes in cerebral glucose levels could be detected. Lactate levels were found to be significantly decreased in hypoglycemic fetal plasma and brain. No differences in lactate levels between control and hypoglycemic young rabbit plasma and brain were observed. These differences were attributed to the utilization of lactate as an energy substrate in the fetal brain, but not in the adult brain. Higher relative (13)C enrichments of most glucose metabolites, except lactate, in the hypoglycemic fetal and young rabbit brains, observed by (13)C NMR, stem from reduced endogenous plasma glucose pools, thereby diluting the labeled glucose to a lower extent. The relative glucose (or glucose-derived lactate) flux via the pyruvate carboxylase and pyruvate dehydrogenase pathways (PC/PDH ratio) was not altered under hypoglycemic conditions in the fetal brain for both glutamine and glutamate, but significantly increased in the adult brain for both glutamine and glutamate. The presented data indicate the ability of the fetal brain to maintain energy metabolism during acute hypoglycemia, via lactate utilization. The increase in the adult PC/PDH ratio was suggested by us to stem from increased PC activity, in order to replenish TCA cycle intermediates.
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PMID:Effect of acute insulin-induced hypoglycemia on fetal versus adult brain fuel utilization, assessed by (13)C MRS isotopomer analysis of [U-(13)C]glucose metabolites. 1111 Nov 61

The principle substrate for brain metabolism is glucose, which provides both energy and the carbon skeletons of glutamate and glutamine, via the TCA cycle. The existence of two distinct cerebral metabolic compartments, neurons and glia, involved in glutamate and glutamine synthesis, respectively, is a widely accepted concept. In previous work, the relative glucose flux via pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC) in adult rabbit brain, using 13C NMR isotopomer analysis of glutamate and glutamine, was quantified. In this work, manifestation of cerebral compartmentation in the near-term fetal rabbit was investigated, using the above approach. Following infusion of [U-13C]glucose into maternal circulation (1 mg/kg per min) for 60-70 min, fetal brains were excised and brain extracts were studied by 13C NMR. The labelling patterns of fetal cerebral metabolites differed from those observed in the young adult brain. The most significant differences were found for glutamine labelling patterns. We suggested that these differences are a result of increased utilization of non-labeled fuels, mainly beta-hydroxybutyrate (beta-HBA) in the glia, the site of glutamine synthesis. In addition, we have shown that acute exposure to elevated beta-HBA levels leads to increased uptake, but not utilization, into the fetal rabbit brain; no increase in uptake is observed in the adult brain. We have also demonstrated that during short-term starvation, although no changes are detected in plasma and cerebral glucose levels in the fetal and young adult brain, amino acid levels and energy metabolism are altered in the young adult brain.
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PMID:Energy fuel utilization by fetal versus young rabbit brain: a 13C MRS isotopomer analysis of [U-(13)C]glucose metabolites. 1127 79

The neurological consequences of diabetes mellitus have recently been receiving greater attention in both clinical and experimental settings. The deleterious effect of hyperglycemia and altered oxidative substrate availability on the diabetic brain is the subject of many studies. The aim of the present study was to examine the effect of the altered metabolic environment, namely, hyperglycemia and hyperketonemia, on glucose metabolism in the diabetic brain. More specifically, we examined the effect of diabetes on the glucose flux via the pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC) pathways and subsequent metabolism in the tricarboxylic acid cycles in neurons and glia. To this end, [U-(13)C]glucose was infused into the circulation of alloxan-induced diabetic young adult rabbits, and the [(13)C]glucose metabolites were subsequently studied in brain extracts by (13)C-NMR. Significantly elevated brain glucose levels were found. In the hyperketonemic rabbits, elevated cerebral levels of beta-hydroxybutyrate (beta-HBA) were found. Alterations in the labeling patterns of glutamine in the hyperketonemic group lead to the conclusion that the elevated beta-HBA levels inhibit glucose metabolism, mostly in glia. This results in accumulation of glucose in the diabetic brain. In addition, altered levels of glutamine, glutamate, and GABA were also attributed to the effect of beta-HBA on brain metabolism. The possible role of these metabolic perturbations in causing neurological damage remains to be investigated.
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PMID:Effect of endogenous beta-hydroxybutyrate on glucose metabolism in the diabetic rabbit brain: a (13)C-magnetic resonance spectroscopy study of [U-(13)C]glucose metabolites. 1128 49

At variance with the current view that only liver and kidney are gluconeogenic organs, because both are the only tissues to express glucose-6-phosphatase (Glc6Pase), we have recently demonstrated that the Glc6Pase gene is expressed in the small intestine in rats and humans and that it is induced in insulinopenic states such as fasting and diabetes. We used a combination of arteriovenous balance and isotopic techniques, reverse transcription-polymerase chain reaction, Northern blot analysis, and enzymatic activity assays. We report that rat small intestine can release neosynthesized glucose in mesenteric blood in insulinopenia, contributing 20-25% of total endogenous glucose production. Like liver glucose production, small intestine glucose production is acutely suppressed by insulin infusion. In the small intestine, glutamine and, to a much lesser extent, glycerol are the precursors of glucose, whereas alanine and lactate are the main precursors in liver. Accounting for these metabolic fluxes: 1) the phosphoenolpyruvate carboxykinase gene (required for the utilization of glutamine) is strongly induced at the mRNA and enzyme levels in insulinopenia; 2) the glycerokinase gene is expressed, but not induced; 3) the pyruvate carboxylase gene (required for the utilization of alanine and lactate) is repressed by 80% at the enzyme level in insulinopenia. These studies identify small intestine as a new insulin-sensitive tissue and a third gluconeogenic organ, possibly involved in the pathophysiology of diabetes.
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PMID:Rat small intestine is an insulin-sensitive gluconeogenic organ. 1128 37

Anaplerosis, or de novo formation of intermediates of the tricarboxylic acid (TCA) cycle, compensates for losses of TCA cycle intermediates, especially alpha-ketoglutarate, from brain cells. Loss of alpha-ketoglutarate occurs through release of glutamate and GABA from neurons and through export of glutamine from glia, because these amino acids are alpha-ketoglutarate derivatives. Anaplerosis in the brain may involve four different carboxylating enzymes: malic enzyme, phosphoenopyruvate carboxykinase (PEPCK), propionyl-CoA carboxylase, and pyruvate carboxylase. Anaplerotic carboxylation was for many years thought to occur only in glia through pyruvate carboxylase; therefore, loss of transmitter glutamate and GABA from neurons was thought to be compensated by uptake of glutamine from glia. Recently, however, anaplerotic pyruvate carboxylation was demonstrated in glutamatergic neurons, meaning that these neurons to some extent can maintain transmitter synthesis independently of glutamine. Malic enzyme, which may carboxylate pyruvate, was recently detected in neurons. The available data suggest that neuronal and glial pyruvate carboxylation could operate at as much as 30% and 40-60% of the TCA cycle rate, respectively. Cerebral carboxylation reactions are probably balanced by decarboxylation reactions,, because cerebral CO2 formation equals O2 consumption. The finding of pyruvate carboxylation in neurons entails a major revision of the concept of the glutamine cycle.
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PMID:Carboxylation and anaplerosis in neurons and glia. 1141 79

The effect of adenosine A(2) receptor agonist 2-[p-(2-carboxyethyl)phenylethylamino]-5'-ethylcarboxamidoadenosine (CGS 21680) and antagonist 3,7-dimethyl-1-propargylxanthine (DMPX) on [1-(13)C]glucose and [1,2-(13)C]acetate metabolism was studied in rats by (13)C magnetic resonance (MR) spectroscopy and HPLC. In the cortex a significant reduction was observed in the amounts of [2-(13)C]GABA and [3-(13)C]aspartate from [1-(13)C]glucose in CGS 21680. In the subcortex the concentration of labelled [4-(13)C]glutamate was increased in both treatment groups. The amounts of [2 + 3-(13)C]succinate and [3-(13)C]lactate were increased in the CGS 21680 group compared to control, and the DMPX group showed an increase in the total amount of [6-(13)C]N-acetyl aspartate compared to control in the subcortex. Astrocyte metabolism was only affected in the cortex as shown by a decrease in the pyruvate carboxylase/pyruvate dehydrogenase ratio in glutamate and glutamine in the treatment groups. Labelling from [1,2-(13)C]acetate was not much affected by CGS 21680 or DMPX. However, the amount of [1,2-(13)C]acetate in cortex and subcortex was reduced in the DMPX group. In the cortex a reduction in the labelling of [3-(13)C]GABA in the DMPX group compared to control and an increase in the total amount of taurine in both treatment groups was detected. The present study shows that A(2) receptor agonist and antagonist have similar effects; however, in cortex GABAergic neurones and astrocytes were affected in contrast to subcortex, where glutamatergic neurones showed the greatest changes.
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PMID:In vivo effects of adenosine A(2) receptor agonist and antagonist on neuronal and astrocytic intermediary metabolism studied with ex vivo (13)C MR spectroscopy. 1172 81


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