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 influence of glutamine on glucose oxidation was assessed in epithelial cells isolated from the mucosa of the proximal, mid-, and distal small intestine of young, fed, male rats. Glucose oxidation declined along the length of the small intestine, with values from the mid- and distal segments representing approximately 55% and 40%, respectively, of the value from the proximal segment. A gradient along the small intestine was noted also in the influence of glutamine on glucose oxidation: glutamine suppressed glucose oxidation approximately 60% in the proximal small intestine, 39% in the mid-intestine, and 31% in the distal small intestine. Glutamine suppressed the oxidation of glucose carbon that entered the tricarboxylic acid (TCA) cycle; this was determined using CO2 ratios derived from acetate and glucose isotopes. In cells from the proximal segment, the probability that carbon entering the cycle would complete one full turn was reduced by glutamine from 0.77 to 0.28. The entry of glucose-derived pyruvate into the TCA cycle did not appear to be influenced by the presence of glutamine, however. Glutamine had no influence on the proportion of glucose metabolism that occurred via the pentose phosphate pathway (which averaged 5% or less), but reduced flux of carbon through pyruvate carboxylase relative to flux through pyruvate dehydrogenase from 40% to 9% in cells from the proximal segment. These data suggest that, in the presence of glutamine, the fate of pyruvate carbon (derived from glucose or elsewhere) entering the TCA cycle is altered from that of oxidation to anaplerosis and subsequent efflux of TCA cycle intermediates into newly synthesized compounds.
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PMID:Oxidation of glucose carbon entering the TCA cycle is reduced by glutamine in small intestine epithelial cells. 761 9

Pyruvate carboxylase (EC 6.4.1.1; PC) catalyzes the formation of oxaloacetate by energy-dependent fixation of CO2 to pyruvate. The aim of the present work was to generate antibodies against PC and use them to localize PC in the cells of astroglia-rich and neuron-rich primary cultures derived from the brains of rats and mice. Mouse monoclonal antibodies raised against the enzyme were shown to be monospecific as indicated by immunoblotting. The staining of the cells for PC appeared in grains. These represent mitochondria, as PC is known as a mitochondrial enzyme. Immunocytochemical examination of astroglia-rich primary cultures of rat or mouse brain cells revealed a colocalization of PC with the astroglial marker glial fibrillary acidic protein (GFAP) in many cells. However, there were GFAP-positive cells showing no specific staining for PC, and vice versa. Also, in neuron-rich primary cultures PC was found only in the approximately 10% GFAP-expressing astroglial cells contaminating the neuron-rich primary culture, whereas it was absent from the neurons identified by antibodies against neuron-specific enolase. These results suggest that PC is predominantly an astroglial enzyme and that astroglial cells play an important role in the intermediary and the energy metabolism of the brain.
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PMID:Immunocytochemical examination of neural rat and mouse primary cultures using monoclonal antibodies raised against pyruvate carboxylase. 772 17

The metabolism of variously labeled [13C]- and [14C]glucoses, used at a physiological concentration (5 mM), has been studied in isolated rabbit kidney tubules both in the absence and the presence of NH4Cl. When present as sole exogenous substrate, glucose was metabolized at high rates and converted not only into CO2 and lactate but also, in contrast to a previous conclusion of Krebs (Krebs, H.A. (1935) Biochem. J. 29, 1951-1969), into glutamine. Absolute fluxes through enzymes of glycolysis and gluconeogenesis and of enzymes of three different cycles operating simultaneously were assessed by using a novel model describing reactions of glucose metabolism in conjunction with the 13C NMR and, to a lesser extent, the radioactive data obtained. The presence of NH4Cl (5 mM) caused a large stimulation of glucose removal and a large increase in lactate, glutamine, and glycerol 3-phosphate accumulation. Under this condition, the stimulation of glutamine synthesis was accompanied not by an activation of citrate synthesis but by an inhibition of flux through alpha-ketoglutarate dehydrogenase. The resulting depletion of citric acid cycle intermediates was compensated by anaplerosis at the level of pyruvate carboxylase. The "futile" cycle involving oxaloacetate, phosphoenolpyruvate, and pyruvate, which was intense in the presence of glucose alone, was greatly stimulated by the addition of NH4Cl.
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PMID:The rabbit kidney tubule utilizes glucose for glutamine synthesis. A 13C NMR study. 792 13

The metabolism of [1,2-13C2]glucose and [U-13C4]3-hydroxybutyrate was studied in rat brain with in vivo and in vitro 13C NMR spectroscopy, taking advantage, in particular, of homonuclear 13C-13C spin coupling patterns. After infusion of [1,2-13C2]glucose or [U-13C4]3-hydroxybutyrate into rats, the uptake of the substrates in brain and their metabolism to [1-13C]bicarbonate could be detected with in vivo 13C NMR spectroscopy. At the end of the infusion experiment, methanol/HCl/HClO4 extracts of the brain tissue were further analysed by high resolution 13C NMR spectroscopy. The 13C spin coupling patterns revealed entirely different isotopomer distributions for the closely related cerebral metabolites glutamate, glutamine and 4-aminobutyric acid. A quantitative analysis of the 13C spectra demonstrated (i) the existence of two kinetically distinct pools of glutamate, (ii) a pronounced CO2 fixation via pyruvate carboxylase in the glial cells accounting for as much as 38% of the oxaloacetate synthesis in the glial tricarboxylic acid cycle, (iii) a cerebral pyruvate recycling system contributing maximally 17% of the pyruvate metabolism through the pyruvate dehydrogenase in neurons, and (iv) a predominant production of 4-aminobutyric acid from glutamate synthesized in the neurons. In addition, the labelling pattern of N-acetyl aspartate upon infusion of labelled glucose or 3-hydroxybutyrate provided insight into the synthesis of this compound in mammalian brain. While the acetyl moiety originates from the metabolic equivalent of the C-1-C-2 part of cerebral glutamate, the aspartyl moiety is not in direct contact with the intermediates of glycolysis or of the tricarboxylic acid cycles.
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PMID:Cerebral metabolism of [1,2-13C2]glucose and [U-13C4]3-hydroxybutyrate in rat brain as detected by 13C NMR spectroscopy. 810 58

Carbonic anhydrase (CA) was examined in two adipocyte cell lines, 3T3-L1 and 3T3-F442A. Both CA III and non-CA III activities, measured by 18O mass spectrometry, were present in 3T3-L1 and 3T3-F442A adipocytes; however, no CA activity was detected in 3T3 preadipocytes of either line. These observations were supported by immunoblot experiments employing CA III and CA II isoform-specific antisera. CA III, a major protein in rodent and murine adipocytes, and CA II, another isoform known to be present in adipose tissue, were observed only in the differentiated 3T3 adipocytes. The differentiation-dependent expression of these isozymes may imply an adipocyte-related role for CA. Compared with cultures maintained in the absence of insulin, 3T3 adipocytes maintained in the presence of insulin exhibited 65-90% lower concentrations of CA III. CA II was unaffected. This negative effect of insulin on CA III may explain the metabolic regulation of adipose CA III observed in vivo. After media changes, 3T3 adipocyte cultures rapidly lower media pH, which in turn lowers the bicarbonate/CO2 of bicarbonate/CO2-buffered media. Cultures maintained at low pH displayed 50-90% lower concentrations of CA II and CA III. Similarly, cultures maintained in a low bicarbonate/CO2 media (GibCO2-I medium containing 1 mM bicarbonate under an atmosphere of 100% humidified air) displayed 30-50% lower CA II and CA III concentrations. Thus CA II and CA III concentrations are influenced by pH and bicarbonate/CO2. Neither effect, the pH or the GibCO2-I media effect, was associated with changes in the concentration of pyruvate carboxylase or ATP citrate lyase (2 markers of adipocyte differentiation). Because the regulation by pH and bicarbonate/CO2 may be relatively selective for CA in adipocytes, a simple method for reducing the concentration/activity of CA in 3T3 adipocytes is described that may be a useful tool for studies on the physiological role of the enzyme.
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PMID:Differentiation-dependent expression of carbonic anhydrase II and III in 3T3 adipocytes. 833 33

A simple model describing reactions of alanine metabolism in isolated hepatocytes from fasted rats is proposed and applied to radioactive data obtained in experiments in which L-[1-14C]-, L-[2-14C]-, L-[3-14C]-, and L-[U-14C]alanine as well as L-alanine plus NaH14CO3 were used as substrates in parallel. Measurements of the rates of incorporation of the label into glucose and CO2 and of accumulation of [1-14C]pyruvate, [1-14C]lactate, [1-14C]alanine and [1-14C]glutamate plus [1-14C]glutamine from the different substrates used allows to calculate flux of alanine carbon through the various metabolic steps taken into account in the model. The validity of this model is indicated by the agreement found between calculations and measurement of the 14CO2 released from [1-14C]alanine as well as between the values of flux through pyruvate carboxylase calculated in two different ways. It is shown that the oxaloacetate synthesized by pyruvate carboxylase enters into the Krebs cycle and into the pathway of phosphoenolpyruvate synthesis in about equal proportions and that about 40% of the oxaloacetate synthesized as a result of alanine metabolism is derived from the Krebs cycle operation. These results, together with the conclusion that flux of alanine carbon through pyruvate dehydrogenase is negligible, are in agreement with known characteristics of hepatic alanine metabolism in the fasted state and, therefore, provide further evidence for the validity of the model proposed in the present study.
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PMID:A simple model for alanine metabolism in isolated rat hepatocytes. 841 95

Whole-cell CO2 fixation and ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) activity were determined in Rhodobacter sphaeroides wild-type and mutant strains. There is no obvious difference in the levels of whole-cell CO2 fixation for the wild type, a form I RubisCO deletion mutant, and a form II RubisCO deletion mutant. No ribulose 1,5-bisphosphate-dependent CO2 fixation was detected in a form I-form II RubisCO double-deletion mutant (strain 16) or strain 16PHC, a derivative from strain 16 which was selected for the ability to grow photoheterotrophically with CO2 as an electron acceptor. However, significant levels of whole-cell CO2 fixation were detected in both strains 16 and 16PHC. Strain 16PHC exhibited CO2 fixation rates significantly higher than those of strain 16; the rates found for strain 16PHC were 30% of the level found in photoheterotrophically grown wild-type strain HR containing both form I and form II RubisCO and 10% of the level of the wild-type strain grown photolithoautotrophically. Strain 16PHC could not grow photolithoautotrophically in a CO2-H2 atmosphere; however, CO2 fixation catalyzed by photoheterotrophically grown strain 16PHC was repressed by addition of the alternate electron acceptor dimethyl sulfoxide. Dimethyl sulfoxide addition also influenced RubisCO activity under photolithoautotrophic conditions; 40 to 70% of the RubisCO activity was reduced without significantly influencing growth. Strain 16PHC and strain 16 contain nearly equivalent but low levels of pyruvate carboxylase, indicating that CO2 fixation enzymes other than pyruvate carboxylase contribute to the ability of strain 16PHC to grow with CO2 as an electron acceptor.
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PMID:Reductive pentose phosphate-independent CO2 fixation in Rhodobacter sphaeroides and evidence that ribulose bisphosphate carboxylase/oxygenase activity serves to maintain the redox balance of the cell. 850 Oct 41

The incorporation of radioactivity from 14C-labeled compounds into metabolic intermediates and total lipids was examined in 3T3 adipocytes. The heterocyclic sulfonamide carbonic anhydrase inhibitor (SCAI) 6-ethoxyzolamide (ETZ) caused a decrease (42+/-7% of control, IC50 = 2.2+/-1.1 x 10(-7) M) in the incorporation of [14C] bicarbonate into several Krebs cycle intermediates in 3T3-F442A adipocytes. This decrease in pyruvate carboxylase-mediated [14C] carbon fixation was associated with a reduction in fluorometrically determined [citrate] and [malate]. The ability of ETZ to decrease both the incorporation of radioactivity into and the concentrations of Krebs cycle intermediates was not of sufficient magnitude to lower [ATP], but was associated with a decrease in de novo lipogenesis from [14C]glucose. De novo lipogenesis was also inhibited to a similar extent by trifluormethanesulfonamide, an aliphatic SCAI, which suggests that the effects are mediated by carbonic anhydrase. ETZ did not inhibit de novo lipogenesis from [14C]glutamine (12.38+/-1.068 nmol/mg protein, ETZ; 12.5+/-0.846 nmol/mg protein, DMSO). This suggests that ETZ inhibition of lipogenesis involves an inhibitory effect on pyruvate carboxylase as opposed to acetyl CoA carboxylase, because the incorporation of glutamine into lipids does not involve pyruvate carboxylase. Decreased de novo lipogenesis was also observed by incubating cultures in media that contained 1 mM bicarbonate (atmosphere:100% humidified air) rather than 25 mM bicarbonate (atmosphere: 95% humidified air/5% CO2). This suggests that exogenous CO2/bicarbonate may be required to sustain maximal rates of de novo lipogenesis. Because these results implied that CA V, the mitochondrial isoform of carbonic anhydrase, might be present in adipocytes, CA V levels were measured by immunoblotting. Mitochondrial preparations of adipocytes and liver were found to contain similar concentrations of CA V. Unlike adipocyte CA III, CA V concentrations were not significantly different in lean and obese Zucker rats. However, CA V levels were ninefold higher in differentiated 3T3-F442A adipocytes compared to undifferentiated adipoblasts. Our data indicate that CA V is relatively abundant in adipocyte mitochondria and exhibits differentiation-dependent expression like pyruvate carboxylase and the cytosolic isozymes CA II and CA III. The possible roles of CA II and CA V in pyruvate carboxylation are discussed.
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PMID:Differentiation-dependent expression of CA V and the role of carbonic anhydrase isozymes in pyruvate carboxylation in adipocytes. 864 47

Metabolic evidence was sought to explain the reduced body fat and increased body protein observed in Atlantic salmon fed diets supplemented with L-carnitine. By stimulating fatty acid oxidation, dietary carnitine might increase flux through pyruvate carboxylase and decrease flux through the branched-chain alpha-keto acid dehydrogenase complex, by increasing regulatory ratios of acetyl CoA:free enzyme A (CoA-SH) and ATP:ADP. Such changes could conserve nitrogen by providing more carbon for amino acid biosynthesis and by blocking oxidative loss of the branched-chain amino acids. Consistent with this hypothesis, salmon fed carnitine (23 mmol/kg diet) for 9 wk exhibited greater metabolic rates than cohorts fed a carnitine-free diet (P < 0.05) for the following: 1) 1-[14C] palmitate oxidation by liver cubes (48%) and by isolated hepatocytes (151%), 2) pyruvate-dependent [14 CO2]-fixation by isolated mitochondria (81%), 3) incorporation of 1-[14C] lactate into glucose by liver cubes (120%) and by isolated hepatocytes (210%), and 4) incorporation of [35S]-methionine into the acid-insoluble fraction of liver cubes (59%) and isolated hepatocytes (89%). Hepatic concentrations of seven amino acids, including the branched-chain amino acids, were greater (7-112%), as were the plasma concentrations of three of these (45-130%). However, 230% more enzyme in the mitochondria of carnitine-fed fish, and not a difference in the ratios of acetyl CoA:CoA-SH or ATP:ADP, appeared to account for accelerated flux through pyruvate carboxylase; flux through the dehydrogenase complex was unchanged. These results implicate induction of pyruvate carboxylase (or a reduction in turnover) and enhanced protein synthesis in the mechanism for carnitine-induced changes in gluconeogenesis and nitrogen metabolism.
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PMID:Atlantic salmon (Salmo salar) fed L-carnitine exhibit altered intermediary metabolism and reduced tissue lipid, but no change in growth rate. 875 66

To gain insight into the regulation of pancreatic beta-cell mitochondrial metabolism, the direct effects on respiration of different mitochondrial substrates, variations in the ATP/ADP ratio and free Ca2+ were examined using isolated mitochondria and permeabilized clonal pancreatic beta-cells (HIT). Respiration from pyruvate was high and not influenced by Ca2+ in State 3 or under various redox states and fixed values of the ATP/ADP ratio; nevertheless, high Ca2+ elevated pyridine nucleotide fluorescence, indicating activation of pyruvate dehydrogenase by Ca2+. Furthermore, in the presence of pyruvate, elevated Ca2+ stimulated CO2 production from pyruvate, increased citrate production and efflux from the mitochondria and inhibited CO2 production from palmitate. The latter observation suggests that beta-cell fatty acid oxidation is not regulated exclusively by malonyl-CoA but also by the mitochondrial redox state. alpha-Glycerophosphate (alpha-GP) oxidation was Ca(2+)-dependent with a half-maximal rate observed at around 300 nM Ca2+. We have recently demonstrated that increases in respiration precede increases in Ca2+ in glucose-stimulated clonal pancreatic beta-cells (HIT), indicating that Ca2+ is not responsible for the initial stimulation of respiration [Civelek, Deeney, Kubik, Schultz, Tornheim and Corkey (1996) Biochem. J. 315, 1015-1019]. It is suggested that respiration is stimulated by increased substrate (alpha-GP and pyruvate) supply together with oscillatory increases in ADP [Nilsson, Schultz, Berggren, Corkey and Tornheim (1996) Biochem. J. 314, 91-94]. The rise in Ca2+, which in itself may not significantly increase net respiration, could have the important functions of (1) activating the alpha-GP shuttle, to maintain an oxidized cytosol and high glycolytic flux; (2) activating pyruvate dehydrogenase, and indirectly pyruvate carboxylase, to sustain production of citrate and hence the putative signal coupling factors, malonyl-CoA and acyl-CoA; and (3) increasing mitochondrial redox state to implement the switch from fatty acid to pyruvate oxidation.
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PMID:Regulation of pancreatic beta-cell mitochondrial metabolism: influence of Ca2+, substrate and ADP. 880 55


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