Discordant effects of glucosamine on insulin-stimulated glucose metabolism and phosphatidylinositol 3-kinase activity.
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The impact of increased GlcN availability on insulin-stimulated p85/p110 phosphatidylinositol 3-kinase (PI3K) activity in skeletal muscle was examined in relation to GlcN-induced defects in peripheral insulin action. Primed continuous GlcN infusion (750 micromol/kg bolus; 30 micromol/kg.min) in conscious rats limited both maximal stimulation of muscle PI3K by acute insulin (I) (1 unit/kg) bolus (I + GlcN = 1.9-fold versus saline = 3.3-fold above fasting levels; p < 0.01) and chronic activation of PI3K following 3-h euglycemic, hyperinsulinemic (18 milliunits/kg.min) clamp studies (I + GlcN = 1.2-fold versus saline = 2.6-fold stimulation; p < 0.01). To determine the time course of GlcN-induced defects in insulin-stimulated PI3K activity and peripheral insulin action, GlcN was administered for 30, 60, 90, or 120 min during 2-h euglycemic, hyperinsulinemic clamp studies. Activation of muscle PI3K by insulin was attenuated following only 30 min of GlcN infusion (GlcN 30 min = 1.5-fold versus saline = 2.5-fold stimulation; p < 0.05). In contrast, the first impairment in insulin-mediated glucose uptake (Rd) developed following 110 min of GlcN infusion (110 min = 39.9 +/- 1.8 versus 30 min = 42.8 +/- 1.4 mg/kg.min, p < 0.05). However, the ability of insulin to stimulate phosphatidylinositol 3,4, 5-trisphosphate production and to activate glycogen synthase in skeletal muscle was preserved following up to 180 min of GlcN infusion. Thus, increased GlcN availability induced (a) profound and early inhibition of proximal insulin signaling at the level of PI3K and (b) delayed effects on insulin-mediated glucose uptake, yet (c) complete sparing of insulin-mediated glycogen synthase activation. The pattern and time sequence of GlcN-induced defects suggest that the etiology of peripheral insulin resistance may be distinct from the rapid and marked impairment in insulin signaling. (+info)
Role of energy metabolism in the preconditioned heart--a possible contribution of mitochondria.
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A brief period of ischemia and reperfusion has been shown to protect the myocardium against subsequent sustained ischemia and reperfusion injury, which is called "preconditioning". A great number of investigators have explored the mechanisms underlying this preconditioning-induced cardioprotection. This article dealt with possible mechanisms of energy metabolism and mitochondrial activity for preconditioning-induced cardioprotection. Particularly, the contribution of energy metabolites produced during a brief period of ischemia and reperfusion injury, as well as mitochondrial function that is modified by changes in mitochondrial ATPase activity, opening of mitochondrial ATP-dependent potassium channels and production of free radicals in mitochondria, to ischemic preconditioning is discussed. (+info)
Glucokinase overexpression restores glucose utilization and storage in cultured hepatocytes from male Zucker diabetic fatty rats.
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Zucker diabetic fatty rats develop type 2 diabetes concomitantly with peripheral insulin resistance. Hepatocytes from these rats and their control lean counterparts have been cultured, and a number of key parameters of glucose metabolism have been determined. Glucokinase activity was 4.5-fold lower in hepatocytes from diabetic rats than in hepatocytes from healthy ones. In contrast, hexokinase activity was about 2-fold higher in hepatocytes from diabetic animals than in healthy ones. Glucose-6-phosphatase activity was not significantly different. Despite the altered ratios of glucokinase to hexokinase activity, intracellular glucose 6-phosphate concentrations were similar in the two types of cells when they where incubated with 1-25 mM glucose. However, glycogen levels and glycogen synthase activity ratio were lower in hepatocytes from diabetic animals. Total pyruvate kinase activity and its activity ratio as well as fructose 2,6-bisphosphate concentration and lactate production were also lower in cells from diabetic animals. All of these data indicate that glucose metabolism is clearly impaired in hepatocytes from Zucker diabetic fatty rats. Glucokinase overexpression using adenovirus restored glucose metabolism in diabetic hepatocytes. In glucokinase-overexpressing cells, glucose 6-phosphate levels increased. Moreover, glycogen deposition was greatly enhanced due to the activation of glycogen synthase. Pyruvate kinase was also activated, and fructose-2,6-bisphosphate concentration and lactate production were increased in glucokinase-overexpressing diabetic hepatocytes. Overexpression of hexokinase I did not increase glycogen deposition. In conclusion, hepatocytes from Zucker diabetic fatty rats showed depressed glycogen and glycolytic metabolism, but glucokinase overexpression improved their glucose utilization and storage. (+info)
Markers of capacity to utilize fatty acids in human skeletal muscle: relation to insulin resistance and obesity and effects of weight loss.
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A number of biochemical defects have been identified in glucose metabolism within skeletal muscle in obesity, and positive effects of weight loss on insulin resistance are also well established. Less is known about the capacity of skeletal muscle for the metabolism of fatty acids in obesity-related insulin resistance and of the effects of weight loss, though it is evident that muscle contains increased triglyceride. The current study was therefore undertaken to profile markers of human skeletal muscle for fatty acid metabolism in relation to obesity, in relation to the phenotype of insulin-resistant glucose metabolism, and to examine the effects of weight loss. Fifty-five men and women, lean and obese, with normal glucose tolerance underwent percutaneous biopsy of vastus lateralis skeletal muscle for determination of HADH, CPT, heparin-releasable (Hr) and tissue-extractable (Ext) LPL, CS, COX, PFK, and GAPDH enzyme activities, and content of cytosolic and plasma membrane FABP. Insulin sensitivity was measured using the euglycemic clamp method. DEXA was used to measure FM and FFM. In skeletal muscle of obese individuals, CPT, CS, and COX activities were lower while, conversely, they had a higher or similar content of FABP(C) and FABP(PM) than in lean individuals. Hr and Ext LPL activities were similar in both groups. In multivariate and simple regression analyses, there were significant correlations between insulin resistance and several markers of FA metabolism, notably, CPT and FABP(PM). These data suggest that in obesity-related insulin resistance, the metabolic capacity of skeletal muscle appears to be organized toward fat esterification rather than oxidation and that dietary-induced weight loss does not correct this disposition. (+info)
AMP deamination and purine exchange in human skeletal muscle during and after intense exercise.
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1. The present study examined the regulation of human skeletal muscle AMP deamination during intense exercise and quantified muscle accumulation and release of purines during and after intense exercise. 2. Seven healthy males performed knee extensor exercise at 64.3 W (range: 50-70 W) to exhaustion (234 s; 191-259 s). In addition, on two separate days the subjects performed exercise at the same intensity for 30 s and 80 % of exhaustion time (mean, 186 s; range, 153-207 s), respectively. Muscle biopsies were obtained from m.v. lateralis before and after each of the exercise bouts. For the exhaustive bout femoral arterio-venous concentration differences and blood flow were also determined. 3. During the first 30 s of exercise there was no change in muscle adenosine triphosphate (ATP), inosine monophosphate (IMP) and ammonia (NH3), although estimated free ADP and AMP increased 5- and 45-fold, respectively, during this period. After 186 s and at exhaustion muscle ATP had decreased (P < 0.05) by 15 and 19 %, respectively, muscle IMP was elevated (P < 0. 05) from 0.20 to 3.65 and 5.67 mmol (kg dry weight)-1, respectively, and muscle NH3 had increased (P < 0.05) from 0.47 to 2.55 and 2.33 mmol (kg d.w.)-1, respectively. The concentration of H+ did not change during the first 30 s of exercise, but increased (P < 0.05) to 245.9 nmol l-1 (pH 6.61) after 186 s and to 374.5 nmol l-1 (pH 6. 43) at exhaustion. 4. Muscle inosine and hypoxanthine did not change during exercise. In the first 10 min after exercise the muscle IMP concentration decreased (P < 0.05) by 2.96 mmol (kg d.w.)-1 of which inosine and hypoxanthine formation could account for 30 %. The total release of inosine and hypoxanthine during exercise and 90 min of recovery amounted to 1.07 mmol corresponding to 46 % of the net ATP decrease during exercise or 9 % of ATP at rest. 5. The present data suggest that AMP deamination is inhibited during the initial phase of intense exercise, probably due to accumulation of orthophosphate, and that lowered pH is an important positive modulator of AMP deaminase in contracting human skeletal muscle in vivo. Furthermore, formation and release of purines occurs mainly after intense exercise and leads to a considerable loss of nucleotides. (+info)
Reversal of metabolic block in glycolysis by enzyme replacement in triosephosphate isomerase-deficient cells.
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Inherited deficiency of the housekeeping enzyme triosephosphate isomerase (TPI) is the most severe clinical disorder of glycolysis. Homozygotes manifest congenital hemolytic anemia and progressive neuromuscular impairment, which in most cases pursues an inexorable course with fatal outcome in early childhood. No effective therapy is available. Hitherto specific enzyme replacement has not been attempted in disorders of glycolysis. Primary skeletal muscle myoblasts and Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines generated from homozygous TPI-deficient patients were cultured in the presence of exogenous enzyme or cocultured with human K562 erythroleukemia cells as an exogenous source of TPI. Uptake of active enzyme by TPI-deficient cells resulted in reversal of intracellular substrate accumulation, with a reduction in dihydroxyacetone phosphate (DHAP) concentration to levels seen in TPI-competent cells. Evidence of successful metabolic correction of TPI deficiency in vitro establishes the feasibility of enzyme replacement therapy, and has important implications for the potential role of allogeneic bone marrow transplantation and gene therapy as a means of sustained delivery of functional enzyme in vivo. (+info)
Iron-dependent changes in cellular energy metabolism: influence on citric acid cycle and oxidative phosphorylation.
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Iron modulates the expression of the critical citric acid cycle enzyme aconitase via a translational mechanism involving iron regulatory proteins. Thus, the present study was undertaken to investigate the consequences of iron perturbation on citric acid cycle activity, oxidative phosphorylation and mitochondrial respiration in the human cell line K-562. In agreement with previous data iron increases the activity of mitochondrial aconitase while it is reduced upon addition of the iron chelator desferrioxamine (DFO). Interestingly, iron also positively affects three other citric acid cycle enzymes, namely citrate synthase, isocitric dehydrogenase, and succinate dehydrogenase, while DFO decreases the activity of these enzymes. Consequently, iron supplementation results in increased formation of reducing equivalents (NADH) by the citric acid cycle, and thus in increased mitochondrial oxygen consumption and ATP formation via oxidative phosphorylation as shown herein. This in turn leads to downregulation of glucose utilization. In contrast, all these metabolic pathways are reduced upon iron depletion, and thus glycolysis and lactate formation are significantly increased in order to compensate for the decrease in ATP production via oxidative phosphorylation in the presence of DFO. Our results point to a complex interaction between iron homeostasis, oxygen supply and cellular energy metabolism in human cells. (+info)
Role of CcpA in regulation of the central pathways of carbon catabolism in Bacillus subtilis.
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The Bacillus subtilis two-dimensional (2D) protein index contains almost all glycolytic and tricarboxylic acid (TCA) cycle enzymes, among them the most abundant housekeeping proteins of growing cells. Therefore, a comprehensive study on the regulation of glycolysis and the TCA cycle was initiated. Whereas expression of genes encoding the upper and lower parts of glycolysis (pgi, pfk, fbaA, and pykA) is not affected by the glucose supply, there is an activation of the glycolytic gap gene and the pgk operon by glucose. This activation seems to be dependent on the global regulator CcpA, as shown by 2D polyacrylamide gel electrophoresis analysis as well as by transcriptional analysis. Furthermore, a high glucose concentration stimulates production and excretion of organic acids (overflow metabolism) in the wild type but not in the ccpA mutant. Finally, CcpA is involved in strong glucose repression of almost all TCA cycle genes. In addition to TCA cycle and glycolytic enzymes, the levels of many other proteins are affected by the ccpA mutation. Our data suggest (i) that ccpA mutants are unable to activate glycolysis or carbon overflow metabolism and (ii) that CcpA might be a key regulator molecule, controlling a superregulon of glucose catabolism. (+info)