Showing papers on "Glycolysis published in 1968"

Inhibition of Fatty Acid Stimulation of Gluconeogenesis by (+)-Decanoylcarnitine in Perfused Rat Liver

Abstract: Oleic acid produced a two- to three-fold stimulation of glucose production from lactate in perfused livers from fasted rats. This effect was completely blocked by (+)-decanoylcarnitine, a known inhibitor of (−)-carnitine palmityltransferase. Activation of fatty acids to the acyl CoA esters was not inhibited. Neither β-oxidation of octanoate, nor its stimulatory effect on gluconeogenesis was blocked by (+)-decanoylcarnitine. The results show that in the intact liver, the oxidation of long-chain but not short-chain fatty acids proceeds almost entirely through carnitine-dependent pathways. Changes in the tissue levels of metabolic intermediates after addition of oleic acid show that the increased rate of gluconeogenesis was caused by a stimulation of the pyruvate carboxylase step. Attendant increases in the levels of acetyl CoA and NADH are consistent with the proposal that the mitochondrial levels of these intermediates control carbohydrate metabolism by diverting pyruvate from oxidation towards the synthesis of glucose. A further control site was identified upon fatty acid addition in the region of the phosphofructokinase and fructose diphosphatase reactions. There was no evidence for control at either of these steps by adenine nucleotides, but a marked rise of citrate was consistent with a postulated inhibition of phosphofructokinase. All the changes in the levels of intermediates of the gluconeogenic pathway and citric acid cycle induced by oleic acid were blocked by (+)-decanoylcarnitine. The failure of oleic acid to increase acetyl CoA and citrate levels in the presence of (+)-decanoylcarnitine explains its failure to stimulate gluconeogenesis under these conditions. It is concluded that the effect of fatty acids on gluconeogenesis is determined by their rate of oxidation and not by their concentration in the perfusion fluid. The significance of these findings in relation to the interactions of carbohydrate and fatty acid metabolism in vivo in the normal, starved and diabetic states is discussed.

Early effects of phytohaemagglutinin on glucose metabolism of normal human lymphocytes.

Abstract: 1. Phytohaemagglutinin induced early changes in the catabolism of glucose by normal human lymphocytes suspended in a bicarbonate buffer. During 4hr. incubation glucose utilization was almost doubled. 2. The rates of several reactions in the metabolism of glucose were estimated. Total pyruvate formation, lactate production and fatty acid synthesis were stimulated to the same degree as was glucose utilization. The pentose cycle and the glycogen synthesis were also stimulated but less than was glucose utilization. The pentose cycle was found to account for 1.4% and 0.9% of the total glucose utilization without and with phytohaemagglutinin respectively. In these cells rates of triose phosphate iso-merization were at least six to seven times the rate of glucose phosphorylation. On an average 55-60% of the total carbon dioxide evolved was derived from decarboxylation of pyruvate, 25-30% from the tricarboxylic acid cycle and about 15% from the pentose cycle. Observed ratios of (14)C specific yields in glycogen from [1-(14)C]- and [6-(14)C]-glucose could possibly be explained by assuming the existence of two separate glucose 6-phosphate pools. 3. During 4hr. incubation in bicarbonate buffer (14)C from [U-(14)C]serine was incorporated into perchloric acid-insoluble material. This incorporation was stimulated by phytohaemagglutinin but was almost completely inhibited by puromycin. Puromycin also abolished phytohaemagglutinin-induced stimulation of glycolysis.

Adipose-tissue pyruvate kinase. Properties and interconversion of two active forms.

Abstract: 1. Extraction of rat epididymal adipose tissue with buffer containing EDTA yields a pyruvate kinase, provisionally called PyK-A, the properties of which resemble in several respects those of the allosteric pyruvate kinase of liver. These properties include co-operative interactions with phosphoenolpyruvate, Mg(2+), K(+), NH(4) (+) and ATP, and sensitivity to activation by fructose 1,6-diphosphate. 2. Extraction in the absence of EDTA yields predominantly a form, PyK-B, that shows both normal Michaelis-Menten kinetics with phosphoenolpyruvate, Mg(2+) and ATP, and co-operative interactions with K(+) and NH(4) (+); this form is insensitive towards fructose 1,6-diphosphate. 3. Both forms yield simple kinetics with ADP, though K(m) values differ in the two systems. In all cases where co-operativity has been demonstrated, Hill-plot n values are between 1.4 and 2.0. 4. The conversion of PyK-A into PyK-B is mediated specifically by fructose 1,6-diphosphate; the reverse reaction is occasioned by EDTA, ATP or citrate. It is thought that a bivalent cation may be involved in this interconversion. 5. Attempts at partial purification have revealed that the enzyme resembles the pyruvate kinase of skeletal muscle, rather than that of liver, in its solubility in ammonium sulphate and elution from DEAE-cellulose. 6. The relevance of these properties in the regulation of pyruvate kinase activity in vivo in adipose tissue is discussed.

Glycolytic Intermediates and Co‐Factors in “Fast‐” and “Slow‐Glycolyzing” Muscles of the Pig

Abstract: SUMMARY— The Longissimus dorsi muscles from Chester White, Hampshire and Poland China animals were used to establish certain differences in metabolic intermediate patterns between muscles with “fast” and “slow” rates of post-mortem glycolysis. Metabolic intermediate patterns were consistent with the concept that phosphorylase is the primary control site of postmortem glycolysis. Adenine nucleotide levels appeared to be the primary regulatory factors for phosphorylase. The phosphofructokinase and pyruvate kinase enzymes were also involved in post-mortem glycolytic control. Levels of high-energy intermediates (adenosine triphosphate, phosphocreatine and pyridine nucleotides) were much higher in the “O” hr samples of “slow-glycolyzing” muscles than in similar samples from muscles having “fast” rates of post-mortem glycolysis. No significant differences in levels of lactate or glucose were observed among these three groups in blood samples taken either at or 24 hr prior to the time of exsanguination.

The effects of anoxia upon energy sources and selected metabolic intermediates in the brains of fish, frog and turtle.

Abstract: — The levels of the main cerebral energy reserves, ATP, P-creatine, glycogen and glucose, and of several glycolytic intermediates and lactate, were measured in the brains of fish (Carassius auratus), turtle (Pseudemys scripta elegans) and frog (Rana pipiens). The levels of glycogen in these brains were 2-9 times higher than those reported for mammals. In frog, cerebral glycogen levels were 35 per cent higher during the winter than in spring. The P-creatine: ATP ratios were 3 instead of the more usual (mammalian) value of 1. The levels of other intermediates were similar to those found in mammalian brain. When anoxia was produced by decapitation, changes in the various substances measured were similar to those in mammalian brain, but were much slower. The initial rate at which high-energy phosphate was used could be calculated from these changes. Values of 1.1 m-equiv./kg/min for fish and frog and of 0.46 m-equiv./kg/min for turtle were found, which are 1/20 and 1/50, respectively, of the rate in mouse brain. The rate of disappearance of high-energy phosphate reserves followed first-order kinetics for 4 hr in turtle and for at least an hour in the other species. Changes in metabolites as the experiment progressed were interpreted to indicate a progressively falling intracellular pH, prolonged inhibition of phosphofructokinase, and a long period of hexokinase inhibition.

Effect of glucose concentration on insulin and glucagon release from isolated islets of Langerhans of the rat.

Abstract: The isolated islets of Langerhans of the rat have been utilized to study simultaneous insulin and glucagon release. There is very little degradation of insulin or glucagon when a proteolytic enzyme inhibitor, Trasylol, is added to the incubation vessels. Glucose exerts an opposite effect on insulin and glucagon release. As the glucose concentration in the incubation media is raised from 30 to 300 mg. per 100 ml., there is a decreased secretion of glucagon and an increased secretion of insulin. The addition of 2-deoxyglucose to the media inhibited insulin release but had no effect on glucagon. The results are discussed in reference to the role of glucagon in insulinogenesis and in regard to the possible mechanisms of insulin and glucagon release.

Studies on Respiration and Glycolysis in Transplanted Hepatic Tumors of the Rat

Abstract: Summary In a continuation of studies on glycolytic and respiratory interrelationships in a series of rat hepatomas ranging widely in growth rate and degree of differentiation, whole, fortified homogenates of these tissues were incubated at 28°C in the presence of fructose-1,6-diphosphate (FDP), 2-deoxyglucose (2-DG), and exogenous hexokinase; respiration, lactate formation, and uptake of 2-DG were measured. Preliminary studies with this model system established that uptake of 2-DG was a valid measure of ATP formation. On the assumption that each mole of lactate formed from FDP leads to production of 2 moles of ATP via phosphoglycerate kinase and pyruvate kinase, glycolytic phosphorylation was estimated as twice that of lactate, and respiratory phosphorylation was calculated as the difference between total ATP and glycolytic ATP formation. Without exogenous substrate, respiration was high in homogenates of well-differentiated tumors, and low in those of poorly differentiated tumors. In both tumor types respiration was coupled with ATP formation, yielding P/O ratios of 1 to 2. Addition of FDP to liver homogenates resulted in moderate lactate and glycolytic ATP formation, the latter being formed largely at the expense of respiratory phosphorylation. In homogenates of well-differentiated tumors, lactate formation and glycolytic phosphorylation were low, and neither respiration nor respiratory phosphorylation was decreased. In contrast, homogenates of the poorly differentiated hepatomas exhibited high lactate formation, and though respiration was increased somewhat by FDP addition, essentially all of the ATP was formed via glycolysis. It thus appears that, in this system, transphosphorylating enzymes of glycolysis are a major site of glycolytic control, presumably through competition with the respiratory ADP acceptors for the available ADP. Further evidence for such competition was obtained by intermixing the supernatant and particulate fractions. Replacement of particles from a low-respiring, poorly differentiated tumor by particles from a high-respiring, well-differentiated tumor resulted in a pronounced Pasteur effect; respiration was increased, together with respiratory ATP production, while glycolysis was markedly decreased. However, when particles of a high-respiring tumor were replaced with particles of a low-respiring tumor, respiration and respiratory phosphorylation were decreased and glycolysis was markedly increased. These findings provide evidence for the suggestion offered many years ago by Johnson and by Lynen that the Pasteur effect may reflect competition for ADP and inorganic phosphate (P 1 ) at the transphosphorylating sites of glycolysis and respiration. They also suggest that the high aerobic glycolysis which is, in general, characteristic of highly dedifferentiated tumors may be, in part, a resultant of their low respiratory activity and high levels of glycolytic transphosphorylating enzymes.

Biochemical energetics of simulated platelet plug formation: Effect of thrombin, adenosine diphosphate, and epinephrine on intra- and extracellular adenine nucleotide kinetics

Abstract: Washed human platelets were incubated in a modified Ringer's solution, pH 7.1, at 37°C for 1 hr. Intracellular basal levels for glycogen, adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and orthophosphate were 31.1, 2.52, 1.39, 0.36, and 1.2 μmoles/ml of platelets, respectively. Extracellular ATP, ADP, and AMP remained fairly constant and represented 4, 2, and 4% of total adenine nucleotide content. Total adenine nucleotide content remained unchanged during the period of control incubation. Glycogen depletion was 17.8 μmoles/ml at the end of 1 hr; lactate production was 20.7 μmoles/ml per hr. In the presence of glucose, lactate production increased 100%, and glycogen depletion was spared 13%. Approximately 55% of glucose or glycogen fuel was converted to lactate. The agglutinating agents, thrombin, ADP, and epinephrine, resulted in increased glycogen depletion and lactate production both in the presence and absence of glucose. The effect of thrombin was greater than epinephrine. The effect of epinephrine was greater than ADP. All three agglutinating agents resulted in loss of high energy phosphates (net decline in adenine nucleotides) with release of adenine nucleotides into the extracellular environment. The effect of thrombin was greater than ADP. The effect of ADP was greater than epinephrine. In experiments with ADP addition, significant quantities of ADP were converted to AMP extracellularly. In experiments with thrombin and epinephrine appreciable quantities of extracellular orthophosphate were taken up by plateletes and could not be accounted for by changes in intracellular orthophosphate or adenine nucleotide. Sufficient ADP was released during exposure to thrombin and epinephrine to account for platelet agglutination. Changes in intracellular adenine nucleotides and orthophosphate could be correlated with the activation of regulator glycogenolytic and glycolytic enzymes.

Relationship between oxygen and glucose consumption by transplanted tumors in vivo

Abstract: Summary The in vivo consumption of oxygen and glucose was studied in relation to growth in Walker Carcinoma 256, Hepatoma 5123, and Fibrosarcoma 4956 transplanted in rats. Glucose not eliminated as lactate or carbon dioxide in the efferent blood was presumed to be retained by the tumor. The retention was so high that growth alone could not account for it. As an alternative, the elimination of glucose by the tumors in some unknown manner is suggested. The weight-doubling times of the tumors were independent of oxygen consumption and lactate production. The tumors needed oxygen to survive; there was no indication, however, that in vivo tumor metabolism shifted from respiration to glycolysis when the supply of oxygen was deficient. The opposite was found to be true: glucose consumption and lactate elimination were in direct proportion to the oxygen utilized and a lack of oxygen blocked both of them. The fraction of glucose transformed into lactate was maximal during insulin-induced hypoglycemia. These glucose-starved tumors did not produce lactate during glucose refeeding despite a large glucose utilization. Neither lack of oxygen nor large glucose consumption appeared to be the dominant causes of in vivo lactate production by tumors. Experimental increases in the lactate content of subcutaneous tissue could be obtained in the absence of any tumor. The possibility that glycolysis is related to changes of cellular components not necessarily involved in the neoplastic process is suggested.

Glucose induced inactivation of malate dehydrogenase in intact yeast cells.

Abstract: The glucose-dependent inactivation of malate dehydrogenase (l-malate: NAD oxidoreductase, EC 1.1.1.37) in acetate-grown yeast cells was studied in vivo. Among different yeast species inactivation was observed only in strains of the genus Saccharomyces. The phenomenon is initiated by the addition of glucose or related hexoses to the medium and is reversibly interrupted at 0°. By use of a yeast mutant requiring tryptophan, it was shown that malate dehydrogenase inactivation is not influenced by inhibition of protein synthesis, whereas recovery of enzyme activity in inactivated cells requires the presence of an energy source and tryptophan and is presumably due to de novo protein synthesis. The reported results provide evidence that a metabolite formed in the first steps of glycolysis might be responsible for initiation of malate dehydrogenase inactivation.

Compartmentation between glycolysis and gluconeogenesis in rat liver

Abstract: 1. The specific radioactivity–time relationships of glucose, glucose 6-phosphate, glycerol 1-phosphate and UDP-glucose were determined in rat liver after the intravenous injection of [U-14C]fructose, and a kinetic analysis was carried out. The glucose 6-phosphate pool was found to be compartmented into gluconeogenic and glycolytic components, and evidence was obtained that the triose phosphates were similarly compartmented. The glycolytic pathway was fed by glycogenolysis and glucose phosphorylation. There was no direct evidence that glycogenolysis fed only the glycolytic pathway, but this interpretation would make the liver resemble other organs in this respect. 2. UDP-glucose was not formed solely from gluconeogenic glucose 6-phosphate, as there was some dilution of label in the intervening glucose 1-phosphate pool, probably from glycogenolysis, though other pathways cannot be excluded. 3. The data cannot be explained by isotopic exchange.

Exercise: Effects on Hexokinase Activity in Red and White Skeletal Muscle

Abstract: Single bouts of exercise increase hexokinase activity in red and white skeletal muscle of guinea pigs. Multiple bouts of exercise cause two-fold increases. In contrast to other enzymes associated with glycolysis, hexokinase activity is higher in red than in white skeletal muscle.

Glucose metabolism in the mucosa of the small intestine. A study of hexokinase activity.

Abstract: 1. The intracellular distribution of hexokinase activity was studied in the mucosa of rat and guinea-pig small intestine. In the rat 60% and in the guinea pig 45% of the hexokinase activity of homogenates were recovered in a total particulate fraction that contained only 5–17% of the homogenate activity of hexose phosphate isomerase, pyruvate kinase, lactate dehydrogenase and overall glycolysis (formation of lactate from glucose). 2. Fractionation of homogenates from guineapig small intestine showed that the particulate hexokinase activity was chiefly in the mitochondrial fraction with a small proportion in the nuclei plus brush-border fraction. 3. After chromatography of the particle-free supernatants on DEAE-cellulose, hexokinase types I and II were determined quantitatively. No evidence was obtained for the presence of hexokinase type III or glucokinase. In the preparations from guinea pigs, hexokinase types I and II amounted to 69% and 31% respectively of the eluted activity; the corresponding values for preparations from rats were 5·8% and 94·2%. 4. Total and specific hexokinase activities decreased significantly in homogenates and particle-free supernatants prepared from the intestinal mucosa of rats starved for 36hr. and increased again after re-feeding. The decrease in hexokinase activity in the particle-free supernatant from starved rats was chiefly due to a decrease in the type II enzyme.

Metabolism of infarcted heart muscle during tissue repair.

Abstract: The metabolism of infarcted canine heart muscle was studied by examining the enzyme profile of infarcted and normal tissue; oxidative and glycolytic metabolism, as expressed in ATP and lactate levels of the infarcted muscle; and lipid synthesis and fatty degeneration of the infarcted muscle. The activities of heart muscle enzymes were assayed during tissue repair following experimental myocardial infarction. The activities of 10 enzymes were determined in normal heart muscle and in the center and periphery of infarcted muscle. A significant increase in hexosemonophosphate (HMP)-shunt activity was observed in infarcted tissue 5 hours after infarction. the activities of glucose-6-phosphate dehydrogenase (g-6-PDH) and 6-phosphogluconate dehydrogenase (6-PGDH) increase 31–34-fold 10 days after infarction. In contrast to increased activity of the HMP-shunt, there was a rapid decline in oxidative and glycolytic enzyme activities in infarcted tissue. Oxidative enzymes reached the lowest level in center of infarcted tissue 10 days after infarction with 8 to 13 per cent of control activities. Glycolytic enzymes showed a significant fall in activities with the greatest diminution in activity of aldolase to 14 per cent of control but a relatively smaller fall in glyceraldehyde phosphate dehydrogenase (GAPDH) activity. The increased activity of the HMP-shunt in infarcted tissue was also expressed in the relative oxidation of glucose-1-C14 and glucose-6-C14 to C14o2. The diminution in oxidative metabolism was also expressed in a marked fall (85 to 90 per cent decrease) in adenosine triphosphate (ATP) content of the infarcted tissue, and a significant rise in lactate content two days after infarction. ten days after coronary occlusion the cellular ATP level increased again with a parallel decrease in lactate content. The incorporation of acetate-1-C14 into lipids was significantly increased in both infarcted and uninfarcted areas of the injured muscle, compared to normal heart muscle without coronary occlusion. The distribution of labeled acetate in the lipid fractions indicated a marked increase in triglyceride formation in the infarcted tissue. The rate of fatty acid and glyceride synthesis was found to be regulated by the atp level, stimulated at low ATP levels (1 to 2 mM) but markedly inhibited at physiological ATP levels (5 to 6 mM). The conditions most favorable for lipid synthesis in cardiac muscle are those of a hypoxic or ischemic muscle with diminished ATP level.

Regulation and Function of Lactate Oxidation in Streptococcus faecium

Abstract: Regulation of the synthesis and function of an l(+)-specific lactate-oxidizing enzyme system found in a homofermentative Streptococcus was investigated. With the exception of fructose, aerobic growth at the expense of a variety of substrates resulted in the formation of a lactate oxidation system; anaerobic growth resulted in a marked reduction or complete loss of lactate-oxidizing activity. Growth on fructose, under aerobic and anaerobic conditions, invariably produced a decrease in the activity of the lactate oxidation system. A negative control, activated by an early intermediate product of glycolysis, appeared to be responsible for repression of the lactate-oxidizing enzyme(s). The enzyme system confers upon the organism the ability to grow aerobically at the expense of l(+)-lactic acid.

Factors affecting the glucose 6-phosphate inhibition of hexokinase from cerebral cortex tissue of the guinea pig.

Abstract: 1. The inhibition of hexokinase by glucose 6-phosphate has been investigated in crude homogenates of guinea-pig cerebral cortex by using a sensitive radio-chemical technique for the assay of hexokinase activity. 2. It was observed that 44% of cerebral-cortex hexokinase activity did not sediment with the microsomal or mitochondrial fractions (particulate fraction), and this is termed soluble hexokinase. The sensitivities of soluble and particulate hexokinase, and hexokinase in crude homogenates, to the inhibitory actions of glucose 6-phosphate were measured; 50% inhibition was produced by 0·023, 0·046 and 0·068mm-glucose 6-phosphate for soluble, particulate and crude homogenates respectively. 3. The optimum Mg2+ concentration for the enzyme was about 10mm, and this appeared to be independent of the ATP concentration. In the presence of added glucose 6-phosphate, raising the Mg2+ concentration to 5mm increased the activity of hexokinase, but above this concentration Mg2+ potentiated the glucose 6-phosphate inhibition. When present at a concentration above 1mm, Ca2+ ions inhibited the enzyme in the presence or absence of glucose 6-phosphate. 4. When the ATP/Mg2+ ratio was 1·0 or below, variations in the ATP concentration had no effect on the glucose 6-phosphate inhibition; above this value ATP inhibited hexokinase in the presence of glucose 6-phosphate. ATP had an inhibitory effect on soluble hexokinase similar to that on a whole-homogenate hexokinase, so that the ATP inhibition could not be explained by a conversion of particulate into soluble hexokinase (which is more sensitive to inhibition by glucose 6-phosphate). It is concluded that ATP potentiates glucose 6-phosphate inhibition of cerebral-cortex hexokinase, whereas the ATP–Mg2+ complex has no effect. Inorganic phosphate and l-α-glycerophosphate relieved glucose 6-phosphate inhibition of hexokinase; these effects could not be explained by changes in the concentration of glucose 6-phosphate during the assay. 5. The inhibition of hexokinase by ADP appeared to be independent of the glucose 6-phosphate effect and was not relieved by inorganic phosphate. 6. The physiological significance of the ATP, inorganic phosphate and α-glycerophosphate effects is discussed in relation to the control of glycolysis in cerebral-cortex tissue.

The effect of collagen on platelet glycolysis and nucleotide metabolism.

Abstract: Summary. Glycolytic activity and nucleotide metabolism were studied in normal resting platelets and platelets exposed to collagen. Lactate production was found to be a more sensitive index of glycolytic activity than glucose consumption. In the presence of added glucose, the rate of lactate production was linear for at least 60 minutes. In the absence of glucose, lactate production continued for 60 minutes and appeared to be independent of glucose for the first 10–15 minutes. Addition of both collagen and pure tropocollagen to the platelet suspension produced a significant and constant increase in glycolytic rate. This was associated with release of ATP and ADP and a small but significant increase in total ATP. Addition of ADP in concentrations similar to those released by collagen had no effect on glycolytic activity. Addition of calcium ions produced release of nucleotides but no increase in glycolytic rate. EDTA inhibited glycolysis both in the resting platelets and in the presence of collagen. It is suggested that the stimulation of glycolysis by collagen may be due to the action of collagen on one or more rate-limiting glycolytic enzymes.