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PubMed1991

On the mechanism of inhibition of gluconeogenesis and ureagenesis by sodium benzoate.

Synthesis of glucose from lactate and generation of urea from ammonia were inhibited when sodium benzoate was added to suspensions of rat hepatocytes. Assays with isolated mitochondria suggested pyruvate carboxylase and the N-acetyl-L-glutamate (NAG)-dependent carbamoylphosphate synthetase (CPS-I) as potential sites of inhibition for both pathways, owing to a shared dependency on aspartate efflux from the mitochondria and its subsequent conversion to oxaloacetate in the cytosol. Assays with isolated hepatocytes indicated inhibition to be initiated by accumulation of benzoyl CoA with a resultant depletion of free CoA and acetyl CoA. Measurements of adenine nucleotides showed that benzoate metabolism did not sufficiently alter energy status to account for the observed inhibition. Consistent with these interpretations, acceleration of the conversion of benzoyl CoA to hippurate by the addition of glycine restored the levels of free CoA and acetyl CoA and the rates of gluconeogenesis and ureagenesis. Reduction of the levels of aspartate and glutamate, presumably by interference with the anapleurotic function of pyruvate carboxylase, most likely accounted for inhibition of gluconeogenesis by benzoate. Whether reduced flux through the urea cycle also contributed to inhibition of gluconeogenesis (by diminishing cytosolic conversion of aspartate to oxaloacetate) requires further study. Depression of glutamate and acetyl CoA to levels at or below the Km for NAG synthetase probably accounted for the observed inhibition of ureagenesis. Rates of urea production were observed to vary with changes in the levels of NAG, suggesting NAG-dependent CPS-I to be the primary site of inhibition of ureagenesis by benzoate.

Acetyl Coenzyme A↗
PubMed1988

Pyruvate dehydrogenase activity in osmotically shocked rat brain mitochondria: stimulation by oxaloacetate.

Pyruvate dehydrogenase complex activity (PDHC) measured by CO2 release isotopic assay has generally been much lower than activity measured by the spectrophotometric arylamine acetyltransferase assay (ArAT). Decarboxylation of [1-14C]pyruvate was measured in osmotically shocked rat brain cortical mitochondria. Activity is dependent on the concentration of the substrate pyruvate. Activity of 74.6 units +/- 12.3 SD (n = 22) was observed at 4 mM pyruvate (1 unit = 1 nmol pyruvate decarboxylated/min/mg protein). Activity was dependent on added NAD, CoA, and thiamine pyrophosphate, implying increased mitochondrial permeability after osmotic shock. Freeze/thaw with sonication of the mitochondrial preparation reduced PDHC activity to 11.5 units +/- 3.0 SD (n = 4). Oxaloacetate produced a marked stimulation of activity. The optimal assay contained 3 mM oxaloacetate, and without oxaloacetate activity fell to 15.4 units +/- 9.9 SD (n = 8). These studies highlight the importance of optimal substrate concentrations in the CO2 release isotopic PDHC method. Higher PDHC activity is found with intact mitochondria and thus activity values should be interpreted in the light of the presence or absence of intact mitochondria in individual preparations.

Animals↗
PubMed1998

Inhibition of myocardial glucose uptake by cGMP.

Guanosine 3',5'-cyclic monophosphate (cGMP), a second messenger of nitric oxide (NO), regulates myocardial contractility. It is not known whether this effect is accompanied by a change in heart metabolism. We report here the effects of 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP), a cGMP analog, on regulatory steps of glucose metabolism in isolated working rat hearts perfused with glucose as the substrate. When glucose uptake was stimulated by increasing the workload, addition of the cGMP analog totally suppressed this stimulation and accelerated net glycogen breakdown. 8-BrcGMP did not affect pyruvate dehydrogenase activity but activated acetyl-CoA carboxylase, the enzyme that produces malonyl-CoA, an inhibitor of long-chain fatty acid oxidation. To test whether glucose metabolism could also be affected by altering the intracellular concentration of cGMP, we perfused hearts with NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase, or with S-nitroso-N-acetylpenicillamine (SNAP), a NO donor. Perfusion with L-NAME decreased cGMP and increased glucose uptake by 30%, whereas perfusion with SNAP resulted in opposite effects. None of these conditions affected adenosine 3',5'-cyclic monophosphate concentration. Limitation of glucose uptake by SNAP or 8-BrcGMP decreased heart work, and this was reversed by adding alternative oxidizable substrates (pyruvate, beta-hydroxybutyrate) together with glucose. Therefore, increased NO production decreases myocardial glucose utilization and limits heart work. This effect is mediated by cGMP, which is thus endowed with both physiological and metabolic properties.

Animals↗
PubMed2005

Methylcitrate synthase from Aspergillus fumigatus. Propionyl-CoA affects polyketide synthesis, growth and morphology of conidia.

Methylcitrate synthase is a key enzyme of the methylcitrate cycle and required for fungal propionate degradation. Propionate not only serves as a carbon source, but also acts as a food preservative (E280-283) and possesses a negative effect on polyketide synthesis. To investigate propionate metabolism from the opportunistic human pathogenic fungus Aspergillus fumigatus, methylcitrate synthase was purified to homogeneity and characterized. The purified enzyme displayed both, citrate and methylcitrate synthase activity and showed similar characteristics to the corresponding enzyme from Aspergillus nidulans. The coding region of the A. fumigatus enzyme was identified and a deletion strain was constructed for phenotypic analysis. The deletion resulted in an inability to grow on propionate as the sole carbon source. A strong reduction of growth rate and spore colour formation on media containing both, glucose and propionate was observed, which was coincident with an accumulation of propionyl-CoA. Similarly, the use of valine, isoleucine and methionine as nitrogen sources, which yield propionyl-CoA upon degradation, inhibited growth and polyketide production. These effects are due to a direct inhibition of the pyruvate dehydrogenase complex and blockage of polyketide synthesis by propionyl-CoA. The surface of conidia was studied by electron scanning microscopy and revealed a correlation between spore colour and ornamentation of the conidial surface. In addition, a methylcitrate synthase deletion led to an attenuation of virulence, when tested in an insect infection model and attenuation was even more pronounced, when whitish conidia from glucose/propionate medium were applied. Therefore, an impact of methylcitrate synthase in the infection process is discussed.

Acetyl Coenzyme A↗
PubMed1995

Effect of galactosamine-induced hepatitis on the aerobic and anaerobic metabolism of the rat exposed to high-altitude hypoxia.

Galactosamine-induced hepatitis caused a marked increase in plasma lactate and pyruvate, but completely abolished the increase in ketone bodies in the rat exposed to an 8000 m simulated altitude. Plasma free fatty acid as the precursor of ketone bodies was higher in the galactosamine-treated rats during and after an exposure to 8000 m altitude. Treatment of the rat with galactosamine markedly reduced activities of citrate synthase, fumarase, glutamate dehydrogenase and fructose 1,6-bisphosphatase, but increased hexokinase and glucose 6-phosphate dehydrogenase in the liver. The effect of galactosamine-induced hepatitis on the energy metabolism can be explained by a reduction of mitochondrial oxidative enzymes and gluconeogenesis, and involves a shift of the aerobic metabolism to anaerobic glycolysis at high altitude.

Aerobiosis↗
PubMed2006

Dual role of isocitrate lyase 1 in the glyoxylate and methylcitrate cycles in Mycobacterium tuberculosis.

The role of isocitrate lyase (ICL) in the glyoxylate cycle and its necessity for persistence and virulence of Mycobacterium tuberculosis has been well described. Recent reports have alluded to an additional role for this enzyme in M. tuberculosis metabolism, specifically for growth on propionate. A product of beta-oxidation of odd-chain fatty acids is propionyl-CoA. Clearance of propionyl-CoA and the by-products of its metabolism via the methylcitrate cycle is vital due to their potentially toxic effects. Although the genome of M. tuberculosis encodes orthologues of two of the three enzymes of the methylcitrate cycle, methylcitrate synthase and methylcitrate dehydratase, it does not appear to contain a distinct 2-methylisocitrate lyase (MCL). Detailed structural analysis of the MCL from Escherichia coli suggested that the differences in substrate specificity between MCLs and ICLs could be attributed to three conserved amino acid substitutions in the active site, suggesting an MCL signature. However, here we provide enzymatic evidence that shows that despite the absence of the MCL signature, ICL1 from M. tuberculosis can clearly function as a MCL. Furthermore, the crystal structure of ICL1 with pyruvate and succinate bound demonstrates that the active site can accommodate the additional methyl group without significant changes to the structure.

Bacterial Proteins↗
PubMed2000

The effects of cell ageing on metabolism in rainbow trout (Oncorhynchus mykiss) red blood cells.

The effects of cell age on metabolism in the nucleated red blood cells of rainbow trout (Oncorhynchus mykiss) were examined. Red blood cells were separated according to age using fixed-angle centrifugation. The mean erythrocyte haemoglobin concentration in old red blood cells was found to be 120 % of that in young red blood cells. In young red blood cells, the activities of the mitochondrial enzymes citrate synthase and cytochrome oxidase were 135-200 %, respectively, of those measured in old red blood cells. The activity of the glycolytic enzyme lactate dehydrogenase in young red blood cells was 170 % of that in old red blood cells, whereas the activity of the glycolytic enzyme pyruvate kinase was not significantly affected by cell age. In addition, young red blood cells consumed over twice as much O(2) and devoted 50 % more O(2) to protein synthesis and the activity of Na(+)/K(+)-ATPase than old red blood cells. Red blood cell age did not significantly affect the rate of lactate production. This study shows that ageing in rainbow trout nucleated red blood cells is accompanied by a significant decline in aerobic energy production and the processes it supports, as well as a corresponding increase in the glycolytic contribution to metabolism.

Animals↗
PubMedDate not supplied

[The enzyme of carbon metabolism in the thermotolerant sulfobacillus strain K1].

To determine enzymatic activities in the thermotolerant strain K1 (formerly "Sulfobacillus thermosulfidooxidans subsp. thermotolerans"), it was grown in a mineral medium with (1) thiosulfate and Fe2+ or pyrite (autotrophic conditions), (2) Fe2+, thiosulfate, and yeast extract or glucose (mixotrophic conditions), and (3) yeast extract (heterotrophic conditions). Cells grown mixo-, hetero-, and autotrophically were found to contain enzymes of the tricarboxylic acid (TCA) cycle, as well as malate synthase, an enzyme of the glyoxylate cycle. Cells grown organotrophically in a medium with yeast extract exhibited the activity of the key enzymes of the Embden-Meyerhof-Parnas and Entner-Doudoroff pathways. An increased content of carbon dioxide (up to 5 vol%) in the auto- and mixotrophic media enhanced the activity of the enzymes involved in the terminal reactions of the TCA cycle and the enzymes of the pentose phosphate pathway. Carbon dioxide was fixed in the Calvin cycle. The highest activity of ribulose bisphosphate carboxylase was detected in cells grown autotrophically at the atmospheric content of CO2 in the air used for aeration of the growth medium. The activities of pyruvate carboxylase, phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase, and phosphoenolpyruvate carboxytransphosphorylase decreased with the increasing content of CO2 in the medium.

Carbohydrate Metabolism↗
PubMed2006

Oral ingestion of mannose alters the expression level of deaminoneuraminic acid (KDN) in mouse organs.

Deaminoneuraminic acid (KDN) is a unique member of the sialic acid family. We previously demonstrated that free KDN is synthesized de novo from mannose as its precursor sugar in trout testis, and that the amount of intracellular KDN increases in mouse B16 melanoma cells cultured in mannose-rich media [Angata et al. (1999) J. Biol. Chem. 274, 22949-56; Angata et al. (1999) Biochem. Biophys. Res. Commun. 261, 326-31]. In the present study, we first demonstrated a mannose-induced increase in intracellular KDN in various cultured mouse and human cell lines. These results led us to examine whether KDN expression in mouse organs is altered by exogenously administered mannose. Under normal feeding conditions, intracellular free KDN was present at very low levels (19-48 pmol/mg protein) in liver, spleen, and lung, and was not detected in kidney or brain. Oral ingestion of mannose, both short-term (90 min) and long-term (2 wk), resulted in an increase of intracellular KDN up to 60-81 pmol/mg protein in spleen and lung and 6.9-18 pmol/mg protein in kidney and brain; however, no change was observed in liver. The level of KDN in organs appears not to be determined only by the KDN 9-phosphate synthase activity, but might also be affected by other enzymes that utilize mannose 6-phosphate as a substrate as well as the enzymes that breakdown KDN, like KDN-pyruvate lyase. In blood, the detectable amount of free KDN did not change on oral ingestion of mannose. These findings indicate that mannose in the diet affects KDN metabolism in various organs, and provide clues to the mechanism of altered KDN expression in some tumor cells and aged organs.

Administration, Oral↗
PubMed2001

Effect of metformin on fatty acid and glucose metabolism in freshly isolated hepatocytes and on specific gene expression in cultured hepatocytes.

The short-term effect of metformin on fatty acid and glucose metabolism was studied in freshly incubated hepatocytes from 24-hr starved rats. Metformin (5 or 50 mM) had no effect on oleate or octanoate oxidation rates (CO(2)+ acid-soluble products), whatever the concentration used. Similarly, metformin had no effect on oleate esterification (triglycerides and phospholipid synthesis) regardless of whether the hepatocytes were isolated from starved (low esterification rates) or fed rats (high esterification rates). In contrast, metformin markedly reduced the rates of glucose production from lactate/pyruvate, alanine, dihydroxyacetone, and galactose. Using crossover plot experiments, it was shown that the main effect of metformin on hepatic gluconeogenesis was located upstream of the formation of dihydroxyacetone phosphate. Increasing the time of exposure to metformin (24 hr instead of 1 hr) led to significant changes in the expression of genes involved in glucose and fatty acid metabolism. Indeed, when hepatocytes were cultured in the presence of 50 to 500 microM metformin, the expression of genes encoding regulatory proteins of fatty acid oxidation (carnitine palmitoyltransferase I), ketogenesis (mitochondrial hydroxymethylgltaryl-CoA synthase), and gluconeogenesis (glucose 6-phosphatase, phosphoenolpyruvate carboxykinase) was decreased by 30 to 60%, whereas expression of genes encoding regulatory proteins involved in glycolysis (glucokinase and liver-type pyruvate kinase) was increased by 250%. In conclusion, this work suggests that metformin could reduce hepatic glucose production through short-term (metabolic) and long-term (genic) effects.

Animals↗
PubMed2001

Enzyme-assisted preparation of isotope-labeled 1-deoxy-d-xylulose 5-phosphate.

Recombinant 1-deoxy-D-xylulose 5-phosphate synthase of Bacillus subtilis was used for the preparation of isotope-labeled 1-deoxy-D-xylulose 5-phosphate using isotope-labeled glucose and/or isotope-labeled pyruvate as starting materials. The simple one-pot methods described afford almost every conceivable isotopomer of 1-deoxy-D-xylulose 5-phosphate carrying (13)C or (14)C from commercially available precursors with an overall yield around 50%.

Bacillus subtilis↗
PubMed1998

Morphometric and biochemical characteristics of ventricular hypertrophy in male rainbow trout (Oncorhynchus mykiss).

We examined the morphometric and biochemical effects of ventricular hypertrophy in male rainbow trout (Oncorhynchus mykiss) during sexual maturation. Our investigation focused on characterizing the growth of ventricular layers, on cardiomyocyte dimensions (length, cross-sectional area and cell volume) and on the activities of enzymes involved in intermediary metabolism. Relative ventricle mass (100 x ventricle mass/body mass) increased by as much as 2.4-fold during sexual maturation [as defined by an increasing gonadosomatic index (100 x gonad mass/body mass)], and this resulted in an increased proportion of epicardium relative to endocardium. Ventricular enlargement was associated with increased length (+31 %) and transverse cross-sectional area (+83 %) of cardiomyocytes, which resulted in an expansion of up to 2.2-fold in mean myocyte volume (from 1233 to 2751 micron3). These results indicate that sexual maturation induces ventricular enlargement through myocyte hypertrophy. Cell length and cross-sectional area were similar in both myocardial layers, and myocytes were elliptical rather than circular in transverse cross section. Ventricular hypertrophy did not alter transverse cell shape, perhaps reflecting the maintenance of short diffusion distances for small molecules as cells hypertrophy. Myocyte hypertrophy could not account entirely for the sevenfold range of ventricle masses from different-sized fish, indicating that myocyte hyperplasia contributes substantially to ventricular growth as trout grow. Measurements of the maximal activities of metabolic enzymes demonstrated that ventricular hypertrophy was associated with (1) higher epicardial but not endocardial activities of citrate synthase (by 23 %) and beta-hydroxyacyl-CoA dehydrogenase (by 20 %); (2) lower activities of hexokinase (by 50 %) in both layers, and (3) no change in lactate dehydrogenase or pyruvate kinase activities, which were also similar between layers. These results suggest that the energetic needs of the hypertrophied trout ventricle may be met through increased reliance on fatty acid oxidation, particularly by the endocardium, but decreased reliance on glucose as a metabolic fuel in both layers.

Animals↗
PubMed2004

Differential hepatic gene expression in a polygenic mouse model with insulin resistance and hyperglycemia: evidence for a combined transcriptional dysregulation of gluconeogenesis and fatty acid synthesis.

New Zealand obese (NZO) mice exhibit severe insulin resistance of hepatic glucose metabolism. In order to define its biochemical basis, we studied the differential expression of genes involved in hepatic glucose and lipid metabolism by microarray analysis. NZOxF1 (SJLxNZO) backcross mice were generated in order to obtain populations with heterogeneous metabolism but comparable genetic background. In these backcross mice, groups of controls (normoglycemic/normoinsulinemic), insulin-resistant (normoglycemic/hyperinsulinemic) and diabetic (hyperglycemic/hypoinsulinemic) mice were identified. At 22 weeks, mRNA was isolated from liver, converted to cDNA, and used for screening of two types of cDNA arrays (high-density filter arrays and Affymetrix oligonucleotide microarrays). Differential gene expression was ascertained and assessed by Northern blotting. The data indicate that hyperinsulinemia in the NZO mouse is associated with: (i) increased mRNA levels of enzymes involved in lipid synthesis (fatty acid synthase, malic enzyme, stearoyl-CoA desaturase) or fatty acid oxidation (cytochrome P450 4A14, ketoacyl-CoA thiolase, acyl-CoA oxidase), (ii) induction of the key glycolytic enzyme pyruvate kinase, and (iii) increased mRNA levels of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase. These effects were enhanced by a high-fat diet. In conclusion, the pattern of gene expression in insulin-resistant NZO mice appears to reflect a dissociation of the effects of insulin on genes involved in glucose and lipid metabolism. The data are consistent with a hypothetical scenario in which an insulin-resistant hepatic glucose production produces hyperinsulinemia, and an enhanced insulin- and substrate-driven lipogenesis further aggravates the deleterious insulin resistance of glucose metabolism.

Animals↗
PubMed1999

Acetate stimulates flux through the tricarboxylic acid cycle in rabbit renal proximal tubules synthesizing glutamine from alanine: a 13C NMR study.

Although glutamine synthesis has a major role in the control of acid-base balance and ammonia detoxification in the kidney of herbivorous species, very little is known about the regulation of this process. We therefore studied the influence of acetate, which is readily metabolized by the kidney and whose metabolism is accompanied by the production of bicarbonate, on glutamine synthesis from variously labelled [(13)C]alanine and [(14)C]alanine molecules in isolated rabbit renal proximal tubules. With alanine as sole exogenous substrate, glutamine and, to a smaller extent, glutamate and CO(2), were the only significant products of the metabolism of this amino acid, which was removed at high rates. Absolute fluxes through the enzymes involved in alanine conversion into glutamine were assessed by using a novel model describing the corresponding reactions in conjunction with the (13)C NMR, and to a smaller extent, the radioactive and enzymic data. The presence of acetate (5 mM) led to a large stimulation of fluxes through citrate synthase and alpha-oxoglutarate dehydrogenase. These effects were accompanied by increases in the removal of alanine, in the accumulation of glutamate and in flux through the anaplerotic enzyme pyruvate carboxylase. Acetate did not alter fluxes through glutamate dehydrogenase and glutamine synthetase; as a result, acetate did not change the accumulation of ammonia, which was negligible under both experimental conditions. We conclude that acetate, which seems to be an important energy-provider to the rabbit renal proximal tubule, simultaneously traps as glutamate the extra nitrogen removed as alanine, thus preventing the release of additional ammonia by the glutamate dehydrogenase reaction.

Acetates↗
PubMed1975

Positional information and pattern regulation in hydra: enzyme profiles.

Certain key enzymes of alternative pathways of glucose metabolism, of amino acid metabolism and of redox systems have been measured in hydra and this profile compared with mammalian differentiated tissues with a view to locating pathways of specific importance in hydra. There was a marked constant proportionality in the major part of the enzymes investigated, the profile suggested a metabolic pattern geared to utilization of amino acids as a carbon source for biosynthesis and energy production and to the production and conservation of pyruvate. The importance of conversion to ionized forms was noted. The most notable specific proportion changes were the exceptionally low lactate dehydrogenase, malic enzyme and the relatively high citrate synthase. The proximal-distal gradients in hydra were examined and these gradients suggested a switch to a more anaerobic type of metabolism and an elevation of the pentose phosphate pathway as the basal region was approached. Measurements of the formation of 14CO2 from specifically labelled glucose provided additional evidence for the functional activity and polarity of the pentose phosphate pathway in hydra. The effect of oligomycin, which can reverse polarity in hydra, had a significant effect on gradients of enzymes eliminating all except that observed for G6P dehydrogenase. The profile suggested a movement towards a more anaerobic type of metabolism, in keeping with the known biochemical action of this inhibitor. It is suggested that redox states and/or phosphorylation states may be featured in the positional information of cells in hydra.

Alanine Transaminase↗
PubMed1983

Fatty acid oxidation intermediates and the effect of fasting on oxidation in red and white skeletal muscle.

In vitro oxidation of U-[14C]glucose-6-phosphate, 2-[14C]pyruvate, and 1-[14C]pyruvate was significantly reduced in red skeletal muscle from fasting rats. Over the same time interval of fasting, 1-[14C]palmitate oxidation remained unchanged. Pyruvate dehydrogenase activity, assayed before in vitro activation, was also reduced in red muscle. Long chain acylcarnitine and long chain acyl CoA increased over the same time period in red muscle. Changes in long chain fatty acid derivatives and pyruvate oxidation were much less dramatic in white muscle. We propose that the rise in levels of long chain fatty acid derivatives may be directly related to the inhibition of carbohydrate oxidation during fasting in red skeletal muscle.

Animals↗
PubMed2002

Immunohistochemical localization of pyruvate carboxylase and carbamyl-phosphate synthetase I in normal and neoplastic human pancreatic tissues.

INTRODUCTION: It has been suggested that pyruvate carboxylase (PC) and carbamyl-phosphate synthetase I (CPS I) might be colocalized with carbonic anhydrase V (CA V), which is generally considered to provide HCO3- ions for PC and CPS I. AIM: To examine the immunohistochemical staining of endogenous biotin; of three mitochondrial biotin-binding enzymes (namely, PC, CPS I, and propionyl CoA-carboxylase); and of cytosolic acetyl CoA-carboxylase in pancreatic tissues. METHODOLOGY: Immunohistochemical analysis was performed on 23 samples of normal pancreas and 63 samples of neoplastic pancreatic tissues. RESULTS: It was found that the distribution of PC, CPS I, and endogenous biotin was not related to that of CA V but was similar to that of CA II in normal centroacinar cells, intercalated duct cells, and intralobular duct cells. In addition, PC was detected unexpectedly in delta-cells of islets. CONCLUSION: It seems likely that CA II plays a major role in the secretion of NaHCO3 into the pancreatic juice. Hence, it is possible that PC and CPS I in the centroacinar cells, intercalated duct cells, and intralobular duct cells are strongly activated and might use HCO3- ions provided by CA II and not by CA V. Among the pancreatic neoplasms examined, ductal adenocarcinomas exhibited significantly elevated immunoreactivity specific for the four biotin-binding enzymes.

Acetyl-CoA Carboxylase↗