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Showing papers on "Glycolysis published in 1993"


Journal ArticleDOI
TL;DR: Acute loading of the liver with fructose causes sequestration of inorganic phosphate in fructose-1-phosphate and diminished ATP synthesis, and the inhibition by ATP of the enzymes of adenine nucleotide degradation is removed and uric acid formation accelerates with consequent hyperuricemia.

634 citations


Journal ArticleDOI
TL;DR: It is concluded that ACC is an important regulator of fatty acid oxidation in the heart and that acetyl-CoA supply is a key determinant of heart ACC-280 activity.

382 citations


Journal ArticleDOI
TL;DR: It is concluded that trehalose‐6‐P plays an important role in the regulation of the first steps of yeast glycolysis, mainly through the inhibition of hexokinase II.

342 citations


Journal ArticleDOI
27 Aug 1993-Cell
TL;DR: Strikingly, PCr levels decline normally during muscle exercise, suggesting that M-CK-mediated conversion is not the only route for PCr utilization in active muscle.

331 citations


Book
01 Dec 1993
TL;DR: The ornithine cycle for the production of urea: 'the urea cycle' is described.
Abstract: Introduction to metabolic pathways Biosynthesis of ATP, part I. ATP: the molecule that powers metabolism Biosynthesis of ATP, part II: the respiratory chain, and inhibitors and uncouplers of oxidative phosphorylation The oxidation of cytosolic NADH+H+: the malate/aspartate shuttle, and the glycerol phosphate shuttle Metabolism of glucose to give energy Metabolism of one molecule of glucose to form 38 molecules of ATP Metabolism of glucose to glycogen Anaerobic metabolism of glucose and glycogen to yield energy as ATP 2,3-Bisphosphoglycerate (2,3-BPG) and the red blood cell Metabolism of glucose to fat (triacylglycerol) Metabolism of glucose to fatty acids and triacylglycerol The pentose phosphate pathway and the production of NADPH+H+ The pyruvate/malate cycle and the production of NADPH+H+ Mammals cannot synthesize glucose from fatty acids Metabolism of triacylglycerol to provide energy as ATP The ornithine cycle for the production of urea: 'the urea cycle' Biosynthesis of the non-essential amino acids Catabolism of amino acids, part I Catabolism of amino acids, part II Metabolism of amino acids to glucose in starvation and during the period immediately after refeeding Metabolism of protein to fat Disorders of amino acid metabolism Amino acid metabolism, folate metabolism, and the 'l-carbon pool', part I: purine biosynthesis Amino acid metabolism, folate metabolism, and the 'l-carbon pool', part II: pyramidine biosynthesis Glycogen metabolism, part I Glycogen metabolism, part II Glycogen metabolism, part III: regulation of glycogen breakdown Glycogen synthesis, part IV: regulation of glycogen synthesis (Glycogen metabolism, part IV: regulation of glycogen synthesis) Regulation of glycolysis Regulation of Krebs cycle Regulation of gluconeogenesis Regulation of fatty acid oxidation, part I: mobilization of fatty acids from storage and adipose tissue Regulation of fatty acid oxidation, part II: the carnitine shuttle The ketone bodies Ketone body utilization B-Oxidation of unsaturated fatty acids Peroxisomal B-oxidation Elongation and desaturation of fatty acids Metabolism of ethanol Sorbitol, galactitol, glucuronate and xylitol Fructose metabolism Biochemistry of diabetes

308 citations


Journal ArticleDOI
TL;DR: It is demonstrated that the muscle glycogen content can be dramatically elevated by increasing the muscle Glut1 protein level and that glucose transport is a rate-limiting step for muscle glucose disposal in normal, resting mice.

281 citations


Journal ArticleDOI
TL;DR: It is concluded that glycochenodeoxycholate causes a bioenergetic form of lethal cell injury dependent on ATP depletion analogous to thelethal cell injury of anoxia.
Abstract: Chenodeoxycholate is toxic to hepatocytes, and accumulation of chenodeoxycholate in the liver during cholestasis may potentiate hepatocellular injury. However, the mechanism of hepatocellular injury by chenodeoxycholate remains obscure. Our aim was to determine the mechanism of cytotoxicity by chenodeoxycholate in rat hepatocytes. At a concentration of 250 microM, glycochenodeoxycholate was more toxic than either chenodeoxycholate or taurochenodeoxycholate. Cellular ATP was 86% depleted within 30 min after addition of glycochenodeoxycholate. Fructose, a glycolytic substrate, maintained ATP concentrations at 50% of the initial value and protected against glycochenodeoxycholate cytotoxicity. ATP depletion in the absence of a glycolytic substrate suggested impairment of mitochondrial function. Indeed, glycochenodeoxycholate inhibited state 3 respiration in digitonin-permeabilized cells in a dose-dependent manner. After ATP depletion, a sustained rise in cytosolic free calcium (Cai2+) was observed. Removal of extracellular Ca2+ abolished the rise in Cai2+, decreased cellular proteolysis, and protected against cell killing by glycochenodeoxycholate. The results suggest that glycochenodeoxycholate cytotoxicity results from ATP depletion followed by a subsequent rise in Cai2+. The rise in Cai2+ leads to an increase in calcium-dependent degradative proteolysis and, ultimately, cell death. We conclude that glycochenodeoxycholate causes a bioenergetic form of lethal cell injury dependent on ATP depletion analogous to the lethal cell injury of anoxia.

254 citations


Journal ArticleDOI
TL;DR: Under stressful conditions, increased secretion of other hormones such as adrenaline, cortisol and growth hormone, and increased activity of the sympathetic nervous system, come into play; their actions to increase hepatic glucose output and to suppress tissue glucose uptake are partly mediated by increases in tissue fatty acid oxidation.
Abstract: Maintenance of plasma glucose concentrations within a narrow range despite wide fluctuations in the demand (e.g. vigorous exercise) and supply (e.g. large carbohydrate meals) of glucose results from coordination of factors that regulate glucose release into and removal from the circulation. On a moment-to-moment basis these processes are controlled mainly by insulin and glucagon, whose secretion is reciprocally influenced by the plasma glucose concentration. In the resting postabsorptive state, release of glucose from the liver (equally via glycogenolysis and gluconeogenesis) is the key regulated process. Glycogenolysis depends on the relative activities of glycogen synthase and phosphorylase, the latter being the more important. The activities of fructose-1,6-diphosphatase, phosphoenolpyruvate carboxylkinase and pyruvate dehydrogenase regulate gluconeogenesis, whose main precursors are lactate, glutamine and alanine. In the postprandial state, suppression of liver glucose output and stimulation of skeletal muscle glucose uptake are the most important factors. Glucose disposal by insulin-sensitive tissues is regulated initially at the transport step and the mainly by glycogen synthase, phosphofructokinase and pyruvate dehydrogenase. Hormonally induced changes in intracellular fructose 2,6-bisphosphate concentrations play a key role in muscle glycolytic flux and both glycolytic and gluconeogenic flux in the liver. Under stressful conditions (e.g. hypoglycaemia, trauma, vigorous exercise), increased secretion of other hormones such as adrenaline, cortisol and growth hormone, and increased activity of the sympathetic nervous system, come into play; their actions to increase hepatic glucose output and to suppress tissue glucose uptake are partly mediated by increases in tissue fatty acid oxidation. In diabetes, the most common disorder of glucose homeostasis, fasting hyperglycaemia, results primarily from excessive release of glucose by the liver due to increased gluconeogenesis; postprandial hyperglycaemia results from both impaired suppression of hepatic glucose release and impaired skeletal muscle glucose uptake. These abnormalities are usually due to the combination of impaired insulin secretion and tissue resistance to insulin, the causes of which remain to be determined.

227 citations


Journal ArticleDOI
TL;DR: It is concluded that the intermediates of anaerobic glycolysis between fructose 1,6-diphosphate and phosphoenolpyruvate are essential for beta-cell glucose sensing and, therefore, glucokinase acts as the glucose sensor.
Abstract: The beta cells of the pancreatic islets of Langerhans respond to changes in glucose concentration by varying the rate of insulin synthesis and secretion. Beta cells sense glucose concentration by the levels of the products of glucose catabolism. Distinctive beta-cell proteins glucose transporter 2 and glucokinase catalyze the first two steps in beta-cell glucose catabolism. To test whether either protein controls the sensitivity of the beta cell to glucose by controlling the rate of glucose catabolism, we used gene-transfer techniques to express the isoenzymes glucose transporter 1 and hexokinase I in beta cells and measured the response to glucose of the insulin gene promoter. Cells expressing glucose transporter 1 do not differ significantly from control cells, but in cells expressing hexokinase I, insulin promoter activity increases, reaches a maximum by 1 mM glucose, and does not respond to changes in glucose concentration within the physiologic range. We conclude that glucokinase catalyzes the rate-limiting step of glucose catabolism in beta cells and, therefore, acts as the glucose sensor. Pyruvate, the end product of anaerobic glycolysis, is readily oxidized by mitochondria in normal beta cells but cannot substitute for glucose as a stimulator of insulin synthesis and secretion. We found that pyruvate can stimulate the insulin promoter in cells expressing the bacterial gluconeogenic enzyme phosphoenolpyruvate carboxykinase, which allows the conversion of pyruvate to phosphoenolpyruvate and the earlier intermediates of glycolysis. We conclude that the intermediates of anaerobic glycolysis between fructose 1,6-diphosphate and phosphoenolpyruvate are essential for beta-cell glucose sensing.

221 citations


Journal ArticleDOI
TL;DR: It is concluded that there was no correlation between lactic acid content and acidosis for these tumors derived from ras-transfected fibroblasts, providing evidence that the production of lactic Acid via glycolysis is not the only mechanism responsible for the development of an acidic environment within solid tumors.
Abstract: Solid tumors have been observed to develop an acidic extracellular environment, which is believed to occur as a result of lactic acid accumulation produced during aerobic and anaerobic glycolysis. Experiments using glycolysis-deficient ras-transfected Chinese hamster lung fibroblasts have been performed to test the hypothesis that lactic acid production within solid tumors is responsible for the development of tumor acidity. The variant cells have defects in glucose transport and in the glycolytic enzyme phosphoglucose isomerase with 1% activity compared to parental cells. Consequently, the in vitro rate of lactic acid production by variant cells was < 4% compared to parental cells. An in vitro correlation between lactic acid production and acidification of exposure medium was observed for parental and variant cells. Implantation of both cell lines into nude mice led to tumors with minimal difference in growth rate. As expected, variant cells died when exposed to hypoxic conditions in culture, and parental tumors were observed to have a larger fraction of cells resistant to radiation due to hypoxia (27%) than variant tumors (2%). Using pH microelectrodes, parental (n = 12) and variant (n = 12) tumors were observed to have extracellular pH (pHe) values of 6.65 +/- 0.07 and 6.78 +/- 0.04 (mean +/- SE, P = 0.13), respectively, whereas normal muscle had a pHe of 7.29 +/- 0.06 (P < 0.0001 for both cell lines). The lactic acid content of variant tumors was found to be similar to that in serum, whereas parental tumors had lactic acid content that was higher than in serum (P < 0.0001). We conclude that there was no correlation between lactic acid content and acidosis for these tumors derived from ras-transfected fibroblasts. These results provide evidence that the production of lactic acid via glycolysis is not the only mechanism responsible for the development of an acidic environment within solid tumors.

208 citations


Journal ArticleDOI
TL;DR: The characterization of mutations in glucokinase that are associated with a distinct and readily recognizable form of NIDDM has led to the identification of key amino acids involved in gluckinase catalysis and localized functionally important regions of the glucokin enzyme molecule.
Abstract: The glycolytic enzyme glucokinase plays an important role in the regulation of insulin secretion and recent studies have shown that mutations in the human glucokinase gene are a common cause of an autosomal dominant form of non-insulin-dependent (type 2) diabetes mellitus (NIDDM) that has an onset often during childhood The majority of the mutations that have been identified are missense mutations that result in the synthesis of a glucokinase molecule with an altered amino acid sequence To characterize the effect of these mutations on the catalytic properties of human beta-cell glucokinase, we have expressed native and mutant forms of this protein in Escherichia coli All of the missense mutations show changes in enzyme activity including a decrease in Vmax and/or increase in Km for glucose Using a model for the three-dimensional structure of human glucokinase based on the crystal structure of the related enzyme yeast hexokinase B, the mutations map primarily to two regions of the protein One group of mutations is located in the active site cleft separating the two domains of the enzyme as well as in surface loops leading into this cleft These mutations usually result in large reductions in enzyme activity The second group of mutations is located far from the active site in a region that is predicted to undergo a substrate-induced conformational change that results in closure of the active site cleft These mutations show a small approximately 2-fold reduction in Vmax and a 5- to 10-fold increase in Km for glucose The characterization of mutations in glucokinase that are associated with a distinct and readily recognizable form of NIDDM has led to the identification of key amino acids involved in glucokinase catalysis and localized functionally important regions of the glucokinase molecule

Journal ArticleDOI
TL;DR: The enzymatic activities studied dropped significantly by 10–30% in brain cortex and hippocampus 3 and 6 weeks after icv STZ injection, resembling metabolic abnormalities such as have been found in noninsulin‐dependent diabetes mellitus.

Journal ArticleDOI
TL;DR: In rat thymocytes, after mitogen stimulation, the ATP turnover has increased twofold in these proliferating cells, indicating a profound Crabtree effect which is not present in resting cells as discussed by the authors, which may be the result of the 8-10fold increase in glycolytic enzyme activities which occurs with proliferation.
Abstract: Rat thymocytes have been used to characterize the changes in energy metabolism that occur as cells undergo a resting/proliferation transition. In the resting state, anaerobic ATP production accounts for only 4% of ATP turnover. The remainder is fueled by the oxidation of a mixture of an unidentified endogenous fuel (62%), glucose (18%) and glutamine (16%). 48 h after mitogen stimulation, the ATP turnover has increased twofold. In these proliferating cells, glucose inhibits oxygen consumption by 58%, indicating a profound Crabtree effect which is not present in resting cells. Consequently, proliferating cells, in the presence of glucose and glutamine, fuel the majority (61%) of ATP turnover anaerobically, producing lactate from glucose. The development of a Crabtree effect may be the result of the 8-10-fold increase in glycolytic enzyme activities which occurs with proliferation. Possible advantages of such a proliferative metabolism are a sparing of endogenous fuel, and a minimizing of oxidative metabolism, with its concurrent production of free radicals.

Journal ArticleDOI
TL;DR: It is concluded that anaerobic glycolysis and glycogenolysis is halted momentarily on termination of contraction and that PCr is not resynthesized during ischaemic recovery, which clearly indicates that parameters other than PCr, ATP, Pi, calculated pH, free ADP and free AMP regulate glyCOlytic flow of human skeletal muscle very efficiently under ischaemia conditions.
Abstract: Changes in the metabolites phosphocreatine (PCr), Pi and ATP were quantified by 31P n.m.r. spectroscopy in the human calf muscle during isometric contraction and recovery under ischaemic conditions. Time resolution of the measurements was 10 s. During a 30-60 s ischaemic isometric contraction, PCr decreased linearly at a rate of 1.17%/s (relative to the resting value) at a contraction strength equivalent to 70% of the maximal voluntary contraction (MVC) and at a rate of 2.43%/s at 90% MVC. There was a corresponding increase in Pi but the concentration of ATP did not change. pH decreased linearly during contraction by 4.22 and 8.23 milli-pH units/s at 70 and 90% MVC respectively. During a subsequent 5 min interval of ischaemic recovery, PCr, Pi, ATP, phosphomonoesters and calculated free ADP, free AMP and pH retained the value they had attained by the end of contraction with no significant recovery. Thus it is concluded that anaerobic glycolysis and glycogenolysis is halted momentarily on termination of contraction and that PCr is not resynthesized during ischaemic recovery. This paradoxical arrest of glycolytic flow in spite of the very significantly elevated concentration of potent activators such as Pi and free AMP clearly indicates that parameters other than PCr, ATP, Pi, calculated pH, free ADP and free AMP regulate glycolysis and glycogenolysis of human skeletal muscle very efficiently under ischaemic conditions.

Journal ArticleDOI
TL;DR: It is concluded that the extreme N terminus of band 3 can bind and inhibit glycolytic enzymes in vivo and that it probably participates in control of red cell glycoleysis.

Journal ArticleDOI
TL;DR: In NIDDM, marked hyperinsulinemia normalizes glycogen synthesis and total flux through glycolysis, but does not restore a normal distribution between oxidation and nonoxidative gly colysis; (d) hyperglycemia cannot overcome the defects in glucose oxidation andnonoxidatives; and (e) lipid oxidation is elevated and is suppressed only with hyperinsulininemia.
Abstract: Seven non-insulin-dependent diabetes mellitus (NIDDM) patients participated in three clamp studies performed with [3-3H]- and [U-14C]glucose and indirect calorimetry: study I, euglycemic (5.2 +/- 0.1 mM) insulin (269 +/- 39 pM) clamp; study II, hyperglycemic (14.9 +/- 1.2 mM) insulin (259 +/- 19 pM) clamp; study III, euglycemic (5.5 +/- 0.3 mM) hyperinsulinemic (1650 +/- 529 pM) clamp. Seven control subjects received a euglycemic (5.1 +/- 0.2 mM) insulin (258 +/- 24 pM) clamp. Glycolysis and glucose oxidation were quantitated from the rate of appearance of 3H2O and 14CO2; glycogen synthesis was calculated as the difference between body glucose disposal and glycolysis. In study I, glucose uptake was decreased by 54% in NIDDM vs. controls. Glycolysis, glycogen synthesis, and glucose oxidation were reduced in NIDDM patients (P < 0.05-0.001). Nonoxidative glycolysis and lipid oxidation were higher. In studies II and III, glucose uptake in NIDDM was equal to controls (40.7 +/- 2.1 and 40.7 +/- 1.7 mumol/min.kg fat-free mass, respectively). In study II, glycolysis, but not glucose oxidation, was normal (P < 0.01 vs. controls). Nonoxidative glycolysis remained higher (P < 0.05). Glycogen deposition increased (P < 0.05 vs. study I), and lipid oxidation remained higher (P < 0.01). In study III, hyperinsulinemia normalized glycogen formation, glycolysis, and lipid oxidation but did not normalize the elevated nonoxidative glycolysis or the decreased glucose oxidation. Lipid oxidation and glycolysis (r = -0.65; P < 0.01), and glucose oxidation (r = -0.75; P < 0.01) were inversely correlated. In conclusion, in NIDDM: (a) insulin resistance involves glycolysis, glycogen synthesis, and glucose oxidation; (b) hyperglycemia and hyperinsulinemia can normalize total body glucose uptake; (c) marked hyperinsulinemia normalizes glycogen synthesis and total flux through glycolysis, but does not restore a normal distribution between oxidation and nonoxidative glycolysis; (d) hyperglycemia cannot overcome the defects in glucose oxidation and nonoxidative glycolysis; (e) lipid oxidation is elevated and is suppressed only with hyperinsulinemia.

Journal ArticleDOI
TL;DR: 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, and revealed entirely different isotopomer distributions for the closely related cerebral metabolites glutamate, glutamine and 4‐aminobutyric acid.
Abstract: 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.

Journal ArticleDOI
TL;DR: A possible requirement of trehalose synthesis for a metabolic balance of sugar phosphates and free inorganic phosphate during the transition from derepressed to fermentative metabolism is discussed.
Abstract: Yeast cells defective in the GGS1 (FDP1/BYP1) gene are unable to adapt to fermentative metabolism. When glucose is added to derepressed ggs1 cells, growth is arrested due to an overloading of glycolysis with sugar phosphates which eventually leads to a depletion of phosphate in the cytosol. Ggs1 mutants lack all glucose-induced regulatory effects investigated so far. We reduced hexokinase activity in ggs1 strains by deleting the gene HXK2 encoding hexokinase PII. The double mutant ggs1Δ, hxk2Δ grew on glucose. This is in agreement with the idea that an inability of the ggs1 mutants to regulate the initiation of glycolysis causes the growth deficiency. However, the ggs1Δ, hxk2Δ double mutant still displayed a high level of glucose-6-phosphate as well as the rapid appearance of free intracellular glucose. This is consistent with our previous model suggesting an involvement of GGS1 in transport-associated sugar phosphorylation. Glucose induction of pyruvate decarboxylase, glucoseinduced cAMP-signalling, glucose-induced inactivation of fructose-1,6-bisphosphatase, and glucose-induced activation of the potassium transport system, all deficient in ggs1 mutants, were restored by the delection of HXK2. However, both the ggs1Δ and the ggs1Δ, hk2Δ mutant lack detectable trehalose and trehalose-6-phosphate synthase activity. Trehalose is undetectable even in ggs1Δ strains with strongly reduced activity of protein kinase A which normally causes a very high trehalose content. These data fit with the recent cloning of GGS1 as a subunit of the trehalose-6-phosphate synthase/phosphatase complex. We discuss a possible requirement of trehalose synthesis for a metabolic balance of sugar phosphates and free inorganic phosphate during the transition from derepressed to fermentative metabolism.

Journal ArticleDOI
TL;DR: Results implicate Ca(2+)-independent phospholipase A2 as a putative glucose sensor which can couple alterations in glycolytic metabolism to the generation of biologically active eicosanoids and thereby facilitate glucose-induced insulin secretion.
Abstract: The recent demonstration that myocardial Ca(2+)-independent phospholipase A2 exists as a complex of catalytic and regulatory polypeptides that is modulated by ATP has suggested a novel mechanisms through which alterations in glycolytic flux can be coupled to the generation of eicosanoids which facilitate insulin secretion. To determine the potential relevance of this mechanism, we examined the kinetic characteristics, substrate specificities, and cellular locus of phospholipase A2 activity in pancreatic islets. Rat pancreatic islets contain a Ca(2+)-independent phospholipase A2 activity which is optimal at physiologic pH, preferentially hydrolyzes phospholipid substrates containing a vinyl ether linkage at the sn-1 position, and prefers arachidonic acid compared to oleic acid in the sn-2 position. Rat islet Ca(2+)-independent phospholipase A2 activity is inhibited by the mechanism-based inhibitor (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one and is stimulated by ATP. Purification of beta-cells from dispersed pancreatic islet cells by fluorescence-activated cell sorting demonstrated that beta-cells (but not non-beta-cells) contain Ca(2+)-independent, ATP-stimulated phospholipase A2 activity. Remarkably, clonal RIN-m5f insulinoma cells, which possess a defect in glucose-induced insulin secretion, contain a Ca(2+)-independent phospholipase A2 which is not modulated by alterations in ATP concentration. Collectively, these results and those of an accompanying paper [Ramanadham et al. (1993) Biochemistry (following paper in this issue)] implicate Ca(2+)-independent phospholipase A2 as a putative glucose sensor which can couple alterations in glycolytic metabolism to the generation of biologically active eicosanoids and thereby facilitate glucose-induced insulin secretion.

Journal ArticleDOI
TL;DR: The model was used for calculating metabolic fluxes in a rat tumor cell line, the C6 glioma, incubated with [1-13C]glucose, and the results emphasize different metabolic characteristics of C6 cells when compared to astrocytes, their normal counterpart.
Abstract: A mathematical model of mammalian cell intermediary metabolism is presented It describes the distribution of the carbon-13 isotope (13C) at the different carbon positions of metabolites in cells fed with 13C-enriched substrates The model allows the determination of fluxes through different metabolic pathways from 13C- and 1H-NMR spectroscopy and mass spectrometry data The considered metabolic network includes glycolysis, gluconeogenesis, the citric acid cycle and a number of reactions corresponding to protein or fatty acid metabolism The model was used for calculating metabolic fluxes in a rat tumor cell line, the C6 glioma, incubated with [1-13C]glucose After evolution to metabolic and isotopic steady states, the intracellular metabolites were extracted with perchloric acid The specific enrichments of glutamate, aspartate and alanine carbons were determined from 13C-, 1H-NMR spectroscopy, or mass spectrometry data Taking into account the rate of glucose consumption and of lactate formation, determined from the evolution of glucose and lactate contents in the cell medium, and knowing the activity of the hexose monophosphate shunt, it was possible to estimate the absolute values of all the considered fluxes From the analysis the following results were obtained (a) Glucose accounts for about 78% of the pyruvate and 57% of the CoASAc (b) A metabolic channelling occurs at the citric acid cycle level; it favours the conversion of carbons 2, 3, 4, and 5 of 2-oxoglutarate into carbons 1, 2, 3, and 4 of oxaloacetate, respectively The percentage of channelled metabolites amounts to 39% (c) The pyruvate carboxylase activity and the efflux from the citric acid cycle are estimated to be very low, suggesting a lack of glutamine production in C6 cells The results emphasize different metabolic characteristics of C6 cells when compared to astrocytes, their normal counterpart

Journal ArticleDOI
01 Jun 1993-Diabetes
TL;DR: An important role is suggested for glucose phosphorylation rates in regulation of the β-cell insulin secretory response to glucose in β-cells derived from insulinomas arising in transgenic mice expressing SV40 Tag.
Abstract: Pancreatic βTC lines derived from insulinomas arising in transgenic mice expressing SV40 Tag under control of the insulin promoter manifest a differentiated β-cell phenotype and secrete insulin in response to glucose. Previously reported βTC lines respond to subphysiological extracellular glucose levels compared with normal β-cells. Recently, several βTC lines were developed with normal glucose-regulated insulin secretion from insulinomas obtained by breeding of the RIP-Tag transgene from the original C57BI/6 mouse strain into the C3HeB/FeJ strain. One of these βTC lines, βTC7, was characterized in detail. βTC7 cells express GLUT2 and have levels of glucokinase and hexokinase activity similar to those of normal islets. As a result these cells exhibit a normal glucose concentration dependency for glycolysis and insulin secretion, thus representing an accurate model of β-cell function. On continuous propagation in culture, βTC7 cells acquired a response to lower extracellular glucose levels. This change was associated with a fourfold increase in hexokinase activity, without significant changes in glucokinase activity and glucose uptake rates. These findings suggest an important role for glucose phosphorylation rates in regulation of the β-cell insulin secretory response to glucose.

Journal ArticleDOI
TL;DR: It is indicated that glycolysis plays a crucial role during early reperfusion in the functional and metabolic recovery of post-ischemic myocardium.

Journal ArticleDOI
TL;DR: It is proposed that the interference of glycogenolysis with glycolysis in pancreatic islets from glucose-infused rats participates in the paradoxical changes in insulin output which represent a typical feature of B-cell glucotoxicity.
Abstract: When pancreatic islets isolated from rats infused for 48-72 h with a hypertonic solution of D-glucose were incubated for two successive periods of 10 min each, in the presence first of 16.7 mM and then 2.8 mM D-[U-14C]glucose, the total output of L-lactic acid during the second incubation was as high as that recorded during the first incubation, while the specific radioactivity of L-lactic acid dramatically decreased during the second incubation. In islets from normoglycemic rats, however, the total output of L-lactic acid decreased and its specific radioactivity modestly increased as the concentration of D-glucose was lowered from 16.7 to 2.8 mM. Such contrasting results indicate that in the glycogen-rich islets isolated from glucose-infused rats, the fall in extracellular D-glucose concentration was not accompanied by a parallel fall in glycolytic flux, the decreased utilization of exogenous D-[U-14C]glucose coinciding with stimulation of glycogenolysis. This unusual metabolic situation also coincided with a transient and paradoxical stimulation of insulin release in response to the decrease in extracellular D-glucose concentration. It is proposed, therefore, that the interference of glycogenolysis with glycolysis in pancreatic islets from glucose-infused rats participates in the paradoxical changes in insulin output which represent a typical feature of B-cell glucotoxicity.

Journal ArticleDOI
01 Oct 1993-Diabetes
TL;DR: The results strongly suggest that the step responsible for the metabolic dysfunction of diabetic β-cells is located within the glycolytic pathway before glyceraldehyde-3-phosphate or in the glycerol phosphate shuttle.
Abstract: In the Goto-Kakizaki rat, a new genetic model of NIDDM, insulin response to glucose is selectively impaired. To elucidate the mechanism of this abnormality, we studied the properties of ATP-sensitive K+ channels, the inhibition of which is a key step of insulin secretion induced by fuel substrates, using the patch-clamp technique. The glucose-sensitivity of KATP channels was considerably reduced in GK rats. However, the inhibitory effects of ATP on channel activity and unitary conductance were not significantly different between control and GK rats. Thus, it appears that the impaired insulinotropic action of glucose in beta-cells of GK rats is attributable to insufficient closure of the KATP channels, probably because of deficient ATP production by impaired glucose metabolism. KATP-channel activities in both control and diabetic beta-cells were found to be equally suppressed by glyceraldehyde and 2-ketoisocaproate. These results strongly suggest that the step responsible for the metabolic dysfunction of diabetic beta-cells is located within the glycolytic pathway before glyceraldehyde-3-phosphate or in the glycerol phosphate shuttle.

Journal ArticleDOI
TL;DR: Pancreatic islets were cultured for 24 h in the presence of 1 mM glucose, which renders islets incapable of responding to glucose with insulin release, and decarboxylation and car boxylation of pyruvate were about equally suppressed in incapacitated islets and that direct inhibition of reactions of the cycle was unlikely.

Journal ArticleDOI
01 Jul 1993-Yeast
TL;DR: Increase in the level of fructose‐2, 6‐bisphosphate is demonstrated to depend on an internal metabolite upstream of the phosphoglucose isomerase reaction, indicating an adaptational mechanism.
Abstract: The glycolytic pathway in Saccharomyces cerevisiae is activated by fermentable sugars at several steps. Mutants with deletions of genes coding for enzymes of the upper part of glycolysis were used to characterize the triggering mechanisms. Synthesis of fructose-2,6-bisphophate is catalysed by two 6-phosphofructo-2-kinase isoenzymes, one of which is activated by fermentable sugars while synthesis of the second enzyme is induced (Kretschmer and Fraenkel, 1991). Increase in the level of fructose-2,6-bisphosphate is demonstrated to depend on an internal metabolite upstream of the phosphoglucose isomerase reaction. The signalling process correlates with distinct temporal changes in the concentration of glucose-6-phosphate but not with its absolute level, indicating an adaptational mechanism. It is independent of the uptake and phosphorylation systems used by different sugars. Interestingly, this increase, although delayed, could also be observed in strains lacking the rapid cAMP increase after sugar addition which is thought to be responsible for the activating process. Synthesis of glucose-6-P and fructose-6-P is needed for the complete induction of pyruvate kinase and inactivation of fructose-1,6-bisphosphatase. On the other hand, induction of pyruvate decarboxylase depends mainly on a signal in the lower part of glycolysis.

Journal ArticleDOI
TL;DR: Data suggest the existence of a lactic acid carrier in mammalian neuronal and astrocytic plasma membranes, which might serve an acid-scavenging function under conditions of altered pH homeostasis, and in the setting of in vivo cerebral ischemia, this carrier may promote the efflux of lactic Acid from astroCytes, redistributing it among less metabolically active neurons.
Abstract: The glycolytic end product lactic acid induced a rapid transient decrease in cytosolic pH in cultured neurons and astrocytes, as measured by microspectrofluorometry using the fluorescent indicator dye 2',7'-bis-(2-carboxyethyl)-5-(and-6) carboxyfluorescein acetoxymethyl ester. Over a physiological range of pH, the initial rate of cellular acidification was a saturable function of the extracellular lactate concentration, suggesting that a saturable transport system mediated lactic acid permeation across the plasma membrane. This transport process displayed stereoselectivity, with a threefold higher rate of intracellular acidification by L-lactic acid than by its D-isomer. Lactic acid-induced acidification occurred in the absence of intracellular ATP, suggesting that transport proceeded independently of the cellular energy charge. These data suggest the existence of a lactic acid carrier in mammalian neuronal and astrocytic plasma membranes, which might serve an acid-scavenging function under conditions of altered pH homeostasis. In the setting of in vivo cerebral ischemia, this carrier may promote the efflux of lactic acid from astrocytes, redistributing it among less metabolically active neurons.

Journal ArticleDOI
TL;DR: The results indicate that in established lines of hepatoma cells the biochemical properties of the bifunctional enzyme, PFK-2/FBPase-2, involved in the synthesis and degradation of fructose 2,6-bisphosphate, differ from those of the enzyme from normal liver.

Journal ArticleDOI
TL;DR: It is hypothesized that sarcolemma-associated glycolytic enzymes may be important in maintaining a high local cytosolic ATP/ADP ratio in the vicinity of KATP channels, where sarcolemmal ATPases are tending to depress the local ATP/ ADP ratio.
Abstract: Activation of ATP-sensitive K+ (KATP) channels has been implicated as a cause of increased cellular K+ efflux and action potential duration (APD) shortening during myocardial ischemia, hypoxia, and selective glycolytic inhibition, since selective KATP channel antagonists partially or completely block increased cellular K+ efflux and APD shortening under these conditions. During substrate-free hypoxia or myocardial ischemia in intact rabbit ventricle, unidirectional K+ efflux rate during systole approximately doubled and APD decreased by ≈40% after 10 minutes. In patch-clamped guinea pig ventricular myocytes, similar changes could be produced by activation of <0.5% of the maximal KATP channel conductance. Furthermore, from studying the desensitizing effects of ADPi on the ATP sensitivity of KATP channels in excised inside-out patches, it was estimated that the rapid changes in the cytosolic ATP/ADP ratio during ischemia and hypoxia were of sufficient magnitude to activate KATP channels to this degree. During selective glycolytic inhibition, however, the global cytosolic ATP/ADP ratio in intact heart remained normal despite an increase in cellular K+ efflux comparable to ischemia and hypoxia. In patch-clamped saponin-permeabilized ventricular myocytes, KATP channels were preferentially suppressed by glycolytic ATP production compared to ATP generated by mitochondria or by the creatinine kinase reaction, and functional glycolytic enzymes were found to be associated with KATP channels in excised membrane patches. We hypothesize that sarcolemma-associated glycolytic enzymes may be important in maintaining a high local cytosolic ATP/ADP ratio in the vicinity of KATP channels, where sarcolemmal ATPases are tending to depress the local ATP/ADP ratio.

Journal ArticleDOI
TL;DR: Kinetic properties of the enzymes involved in gluconeogenesis indicate that they operate in the direction of sugar synthesis, whereas sugar degradation to pyruvate proceeds via a modified “non-phosphorylated” Entner-Doudoroff pathway.
Abstract: The hyperthermophilic archaeon Pyrococcus furiosus was grown on pyruvate as carbon and energy source. The enzymes involved in gluconeogenesis were investigated. The following findings indicate that glucose-6-phosphate formation from pyruvate involves phosphoenolpyruvate synthetase, enzymes of the Embden-Meyerhof pathway and fructose-1,6-bisphosphate phosphatase.