Showing papers on "Glycolysis published in 1976"

Aerobic glycolysis during lymphocyte proliferation

Abstract: GLYCOLYSIS, for most mammalian cells, is only a prelude to the complete respiratory oxidation of glucose. Lactate production is usually barely, if at all, detectable in aerobic conditions1. Consequently, when Warburg2,3 observed that various tumours showed active aerobic glycolysis, he postulated that defective tumour cell respiration was the reason and was, moreover, the basic difference between normal and cancer cells. Although aerobic glycolysis by tumour tissue has been confirmed many times, defective respiration in cancer cells has not been established4. The failure to uncover a respiratory defect in cancer cells has led to other explanations. It has been suggested, for example, that aerobic glycolysis is linked to cell growth rather than to malignancy5, and for several hepatomas a correlation could be made between the amount of lactate produced and the cell doubling time6,7. It follows that if aerobic glycolysis is related to cell growth, it might be possible as much in normal as malignant cells. There is some evidence for this. Aerobic glycolysis has been noted in proliferating fibroblasts during the period of maximum increase in cell numbers8,9. Human lymphocytes have shown an increase in respiration and also produced lactate when stimulated by the mitogen phytohaemagglutinin10,11. In chick embryo and skeletal muscle fibroblasts12,13, glycolysis increased during log phase growth, but several factors seemed to influence lactate production. Medium composition, aggregation state of cells, culture pH and cell density were all considered more important in determining glycolytic activity than growth rate. It should be noted, however, that these factors are also important to the proliferative rate of these cells. We believe that the important question is not whether culture conditions can influence lactate production, but rather whether glycolysis is linked to cell division. So far no systematic study of the relationship of the appearance of aerobic glycolysis to cell proliferation or the phases of the cell cycle has been reported. This report presents evidence in normal lymphocytes that aerobic glycolysis not only is associated with cellular proliferation, but more specifically is temporally related to DNA synthesis.

Anaerobic metabolism in bivalvia (Mollusca) Characteristics of anaerobic metabolism

Abstract: 1. 1. The role of carbohydrates (glycogen, trehalose, glucose), lipids and proteins as forms of energy storage during aerobiosis and anaerobiosis is discussed. 2. 2. Fatty acid and amino acid conversions cannot be ruled out during anaerobiosis, but this is accompanied by a simultaneous mobilization of carbohydrates. 3. 3. Carbohydrates are therefore the main energy reserve stores under this condition. A Pasteur effect has been shown only in a few studies. The consumed carbohydrates form only a fraction of the pool size. This may be the result of a reduction of the metabolic rate during anaerobiosis. 4. 4. Calculations of ATP production and consumption reveal that the lowest possible energy demand is far below that of the standard rate. 5. 5. Anaerobiosis may lead to the accumulation of pyruvate, lactate, octopine, succinate, alanine, acetate, propionate and CO 2 . The ratio in which these compounds accumulate alters with time. Alanine and lactate are probably initial end products. 6. 6. Stoichiometric studies between consumed fuel substrates and accumulated compounds are discussed. 7. 7. There is a relationship between pH changes and shell movements. During shell closure there is a gradual decrease in pH. 8. 8. ATP levels decrease during anaerobiosis.

Effects of regional ischemia on metabolism of glucose and fatty acids. Relative rates of aerobic and anaerobic energy production during myocardial infarction and comparison with effects of anoxia.

Abstract: The rate of coronary flow reaching the oxygen-linited heart appears to be crucial in determining the myocardial tissue metabolic response. The tissue metabolic response to anoxia, well studied in hearts perfused with anoxic media, differs in many important ways from the response to ischemia. In regional ischemia (developing infarction) there is still a residual oxygen uptake which is reduced approximately to the same extent as the delivery of O2; there is also decreased delivery of substrates and decreased removal of CO2, H+, and lactate, with increased concentrations of these metabolites. Contents of hexose monophosphates rise rather than fall in anoxia. Measurements of glycolytic intermediates show an initial burst of accelerated glycolytic flux lasting less than 1 minute after coronary artery ligation; thereafter rates of flux decrease to control values or even less at 120 minutes. Relative inhibition of phosphofructokinase (PFK) activity may be explained by a slow rate of fall of ATP and a developing intracellular acidosis. In this model, glucose accounts for a greater part of the residual oxidative metabolism than does free fatty acid (FFA).

The maximum activities of hexokinase, phosphorylase, phosphofructokinase, glycerol phosphate dehydrogenases, lactate dehydrogenase, octopine dehydrogenase, phosphoenolpyruvate carboxykinase, nucleoside diphosphatekinase, glutamate-oxaloacetate transaminase and arginine kinase in relation to carbohydrate utilization in muscles from marine invertebrates.

Abstract: Comparison of the activities of hexokinase, phosphorylase and phosphofructokinase in muscles from marine invertebrates indicates that they can be divided into three groups. First, the activities of the three enzymes are low in coelenterate muscles, catch muscles of molluscs and muscles of echinoderms; this indicates a low rate of carbohydrate (and energy) utilization by these muscles. Secondly, high activities of phosphorylase and phosphofructokinase relative to those of hexokinase are found in, for example, lobster abdominal and scallop snap muscles; this indicates that these muscles depend largely on anaerobic degradation of glycogen for energy production. Thirdly, high activities of hexokinase are found in the radular muscles of prosobranch molluscs and the fin muscles of squids; this indicates a high capacity for glucose utilization, which is consistent with the high activities of enzymes of the tricarboxylic acid cycle in these muscles [Alp, Newsholme & Zammit (1976) Biochem. J. 154, 689-700]. 2. The activities of lactate dehydrogenase, octopine dehydrogenase, phosphoenolpyruvate carboxykinase, cytosolic and mitochondrial glycerol 3-phosphate dehydrogenase and glutamate-oxaloacetate transaminase were measured in order to provide a qualitative indication of the importance of different processes for oxidation of glycolytically formed NADH. The muscles are divided into four groups: those that have a high activity of lactate dehydrogenase relative to the activities of phosphofructokinase (e.g. crustacean muscles); those that have high activities of octopine dehydrogenase but low activities of lactate dehydrogenase (e.g. scallop snap muscle); those that have moderate activities of both lactate dehydrogenase and octopine dehydrogenase (radular muscles of prosobranchs), and those that have low activities of both lactate dehydrogenase and octopine dehydrogenase, but which possess activities of phosphoenolpyruvate carboxykinase (oyster adductor muscles). It is suggested that, under anaerobic conditions, muscles of marine invertebrates form lactate and/or octopine or succinate (or similar end product) according to the activities of the enzymes present in the muscles (see above). The muscles investigated possess low activities of cytosolic glycerol 3-phosphate dehydrogenase, which indicates that glycerol phosphate formation is quantitatively unimportant under anaerobic conditions, and low activities of mitochondrial glycerol phosphate dehydrogenase, which indicates that the glycerol phosphate cycle is unimportant in the re-oxidation of glycolytically produced NADH in these muscles under aerobic conditions. Conversely, high activities of glutamate-oxaloacetate transaminase are present in some muscles, which indicates that the malate-aspartate cycle may be important in oxidation of glycolytically produced NADH under aerobic conditions. 3. High activities of nucleoside diphosphate kinase were found in muscles that function for prolonged periods under anaerobic conditions (e.g...

Hormonal control of pyruvate kinase activity and of gluconeogenesis in isolated hepatocytes.

Abstract: Treatment of isolated rat hepatocytes with saturating concentrations of glucagon caused several modifications properties of pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40): S0.5 (substrate concentration at half maximum velocity) for phosphoenolpyruvate was about doubled, whereas Vmax was not changed; the activity measured at 0.15 mM phosphoenolpyruvate (physiological concentration) was reduced 65-80%; and there was also an increase in the Hill coefficient and in the affinity of the enzyme for the inhibitors Mg-ATP and alanine. Glucagon, 3':5'-cyclic AMP, and epinephrine caused an inactivation of pyruvate kinase together with a sitmulation of gluconeogenesis. Insulin (10 nM) antagonized the effect of suboptimal doses of glucagon or cyclic AMP and of even maximal doses of epinephrine, on both pyruvate kinase activity and on gluconeogenesis. These observations can be explained by a phosphorylation of pyruvate kinase by cyclic-AMP-dependent protein kinase, as described by Ljungstrom et al. [(1974) Biochim. Biophys. Acta 358, 289-298] in a reconstructed system. They offer a molecular explanation for the hormonal control of gluconeogenesis. Glucose caused an inhibition of gluconeogenesis with no corresponding change in pyruvate kinase activity.

Glucose metabolism in perfused skeletal muscle. Effects of starvation, diabetes, fatty acids, acetoacetate, insulin and exercise on glucose uptake and disposition

Abstract: 1. The regulation of glucose uptake and disposition in skeletal muscle was studied in the isolated perfused rat hindquarter. 2. Insulin and exercise, induced by sciatic-nerve stimulation, enhanced glucose uptake about tenfold in fed and starved rats, but were without effect in rats with diabetic ketoacidosis. 3. At rest, the oxidation of lactate (0.44 mumol/min per 30 g muscle in fed rats) was decreased by 75% in both starved and diabetic rats, whereas the release of alanine and lactate (0.41 and 1.35 mumol/min per 30 g respectively in the fed state) was increased. Glycolysis, defined as the sum of lactate+alanine release and lactate oxidation, was not decreased in either starvation or diabetes. 4. In all groups, exercise tripled O2 consumption (from approximately 8 to approximately 25 mumol/min per 30 g of muscle) and increased the release and oxidation of lactate five- to ten-fold. The differences in lactate release between fed, starved and diabetic rats observed at rest were no longer apparent; however, lactate oxidation was still several times greater in the fed group. 5. Perfusion of the hindquarter of a fed rat with palmitate, octanoate or acetoacetate did not alter glucose uptake or lactate release in either resting or exercising muslce; however, lactate oxidation was significantly inhibited by acetoacetate, which also increased the intracellular concentration of acetyl-CoA. 6. The data suggest that neither that neither glycolysis nor the capacity for glucose transport are inhbitied in the perfused hindquarter during starvation or perfusion with fatty acids or ketone bodies. On the other hand, lactate oxidation is inhibited, suggesting diminished activity of pyruvate dehydrogenase. 7. Differences in the regulation of glucose metabolism in heart and skeletal muscle and the role of the glucose/fatty acid cycle in each tissue are discussed.

Energy metabolism in respiration-deficient and wild type Chinese hamster fibroblasts in culture.

Abstract: This paper presents a comparison of energy metabolism in wild type and respiration-deficient Chinese hamster cells. From previous work (DeFrancesco et. al., '75) it was concluded that the mutant satisfies essentially all of its energy requirements from glycolysis and in this study we measure precisely the amount of glucose consumed and lactate produced per milligram increment of protein in exponentially growing cultures. From these measurements we calculate the amount of ATP derived from glycolysis (and hence the total energy requirement for normal proliferation) to be 105 +/- 15 mumoles ATP/delta mg protein in the mutant. It is 63 +/- 10 mumoles ATP/delta mg protein derived from glycolysis in wild type cells. We present evidence that the total energy requirement of wild type cells is similar to that of the mutant suggesting that approximately 40% of the energy requirement is derived from respiration. The oxidation of glutamine appears to be more significant than the complete oxidation of glucose to CO2 in these Chinese hamster fibroblasts. The amount of ATP required by the mutant cells per milligram increment of protein is relatively independent of pH.

Liver Cell Heterogeneity. The Distribution of Pyruvate Kinase and Phosphoenolpyruvate Carboxykinase (GTP) in the Liver Lobule of Fed and Starved Rats

Abstract: Pyruvate kinase and phosphoenolpyruvate carboxykinase activities were determined in microdissected freeze-dried liver cells from the periportal and pericentral area of the liver lobule. Pyruvate kinase activity was measured by a microfluorimetric procedure adapted to 20-200 ng tissue dry weight. In livers from fed rats, its activity was twice as high in the central zone as in the periportal cells; starvation reduced this gradient by decreasing central activities. Phosphoenolpyruvate carboxykinase activity was measured by a microradiochemical technique in 100-300 ng tissue dry weight. In livers from fed rats, this enzyme was nearly 3 times more active in the periportal cells than in the central area. Starvation increased this enzyme in both zones with a more pronounced change in the central cells. The results indicate a heterogeneous distribution of enzymes of carbohydrate metabolism in the liver lobule. Gluconegenesis seems to be localized preferentially in periportal hepatocytes, whereas the glycolytic enzyme was found to be more active in cells surrounding the pericentral liver cells.

Autoregulatory shift from fructolysis to lactate gluconeogenisis in rat hepatocyte suspensions. The problem of metabolic zonation of liver parenchyma.

Abstract: Hepatocytes were isolated from fed rats with glucose and insulin and freom fasted rats with glucagon in all media in an attempt to obtain cells which might be fixed preferentially in either the glycolytic or gluconeogenic state. When tested enzymatically, both "fed" and fasted" cells catalyzed glucose formation from lactate (gluconeogenesis) and lactate formation from fructose (fructolysis); lactate formation from glucose may have occurred in "fed" cells. Thus it was impossible, at least in the C3 part of the metabolic pathways between triosephosphate and pyruvate, to fix the hepatocytes in either metabolic state. The shift from glycolysis to gluconeogenesis could be investigated for the C3 part in "fasted" cells with fructose as the glycolytic and lactate as the gluconeogenic substrate. Lactate was first formed from fructose and later reutilized to a large extent. This reconsumption was blocked by the gluconeogenesis inhibitor quinolinate, both when tested enzymatically and radiochemically. Thus fructolysis was shifted to lactate gluconeogenesis. This shift at the assumed phosphoenolpyruvate/pyruvate cycle was autoregulatory, i.e. dependent on substrates and independent of circulating horomes. Maximal velocities and half saturating concentrations were determined for fructose and for lactate as substrates. The kinetic data obtained, especially the sigmoidal pattern of fructolysis, could nicely explain phenomenologically the rather sudden slow-down of lactate production and the shift to lactate consumption. The levels of the metabolites ATP, ADP, AMP, fructose bisphosphate and alanine, which control the enzymes of the assumed phosphoenolypyruvate/pyruvate cycle, were determined in the cytosol and in the mitochondria before and after the shift from fructose glycolysis to lactate gluconeogenesis. The changes observed could not explain the shift. Experiments with [14C] fructose plus unlabelled lactate and reciprocally, with unlabelled fructose plus [14C] lactate, clearly reveled that within the C3 part, glycolysis and gluconeogenesis were catalyzed simultaneously. The simultaneity of and the shift between fructolysis and gluconeogenesis by the liver cell suspension can best be explained by assuming two metabolically different types of hepatocytes rather than one type of hepatocyte with metabolically equal or different cell compartment. In vivo, the different types of hepatocytes would form a gluconeogenic and a glycolytic zone within the liver parenchyma. Since, under normal conditions, the size of these metabolic zones should remain unaltered, the shift from net glycolysis to net gluconeogenesis would be dependent primarily on substrate concentrations (autoregulation).

Occurrence of the Malate-Aspartate Shuttle in Various Tumor Types

Abstract: The activity of the malate-aspartate shuttle for the reoxidation of cytoplasmic reduced nicotinamide adenine dinucleotide (NADH) by mitochondria was assessed in six lines of rodent ascites tumor cells (two strains of Ehrlich ascites carcinoma, Krebs II carcinoma, Novikoff hepatoma, AS-30D hepatoma, and L1210 mouse leukemia). All the tumor cells examined showed mitochondrial reoxidation of cytoplasmic NADH, as evidenced by the accumulation of pyruvate when the cells were incubated aerobically with L-lactate. Reoxidation of cytoplasmic NADH thus generated was completely inhibited by the transaminase inhibitor aminooxyacetate. The involvement of the respiratory chain in the reoxidation of cytoplasmic NADH was demonstrated by the action of cyanide, rotenone, and antimycin A, which strongly inhibited the formation of pyruvate from added L-lactate. Compounds that inhibit the carrier-mediated entry of malate into mitochondria, such as butylmalonate, benzenetricarboxylate, and iodobenzylmalonate, also inhibited the accumulation of pyruvate from added L-lactate by the tumor cells. The maximal rate of the malate-aspartate shuttle was established by addtion of arsenite to inhibit the mitochondrial oxidation of the pyruvate formed from added lactate. The capacity of the various tumor lines for the reoxidation of cytoplasmic NADH via the malate-aspartate shuttle approaches 20% of the total respiratory rate of the cells and thus appears to be sufficient to account for the mitochondrial reoxidation of that fraction of glycolytic NADH not reoxidized by pyruvate and lactate dehydrognenase in the cytoplasm.

Effects of mechanical activity and hormones on myocardial glucose and fatty acid utilization.

Abstract: The effects of increased cardiac work on glycolysis, the citric acid cycle, and oxidation of fatty acids were studied in isolated rat hearts Glycolysis was stimulated by increased work in heart perfused with glucose alone or with glucose, high levels of insulin, and low levels of palmitate With glucose alone, stimulation was associated with a rapid decrease in phosphate potential and rapid activation of phosphofructokinase, but an apparently slower activation of glucose transport With glucose, insulin, and palmitate present, stimulation of glucose utilization was rapid and correlated with activation of phosphofructokinase

ATP pools and transients in the blue-green alga, Anabaena cylindrica

Abstract: Anabaena cylindrica grown in steady state continuous culture has an extractable ATP pool, measured on the basis of the luciferin-luciferase assay of 165±35 nmoles ATP mg chla-1. This pool is maintained by a dynamic balance between the rate of ATP synthesis and the rate of ATP utilization. Phosphorylating mechanisms which can maintain the pool in the short term are total photophosphorylation, cyclic photophosphorylation and oxidative phosphorylation. The alga can maintain its ATP pool by switching rapidly from one of these phosphorylating mechanisms to another depending on the environmental conditions. At each switch-over there is a transient drop in the ATP pool for a few seconds. On switching to conditions where only substrate level phosphorylation operates, the ATP pool falls immediately, but takes several hours to recover. The apparent rates of ATP synthesis by total photophosphorylation and by cyclic photophosphorylation are both much higher (210±30 and 250±13 μmoles ATP mg chla-1 h-1 respectively) than the apparent rate of ATP synthesis by oxidative phosphorylation (22±3 μmoles ATP mg chla-1 h-1). In long term experiments the ATP pool is maintained when total photophosphorylation is operating. It cannot be maintained in the long term by cyclic photophosphorylation alone in the absence of photosystem II activity or endogenous carbon compounds, or by oxidative phosphorylation in the absence of endogenous carbon compounds. Measurements of ATP, ADP and AMP show that the total pool of adenylates is similar in the light and in the dark in the short term. There is only limited production of ATP under dark anaerobic conditions when glycolysis and substrate phosphorylation can operate which suggests that these processes are of limited significance in providing ATP in Anabaena cylindrica.

Biochemical adaptations for flight in the insect.

Abstract: 1. Flight by insects is characterized by the most intense respiration known in biology and also the most controlled. Thus insect flight muscle may be the tissue of choice for the study of biochemical adaptation in the control of catabolism and biological oxidations, and many of the results obtained with insects have a significance and a relevance that transcend the boundaries between classes. In insects, such as the blowfly, flight is distinguished additionally by high wingbeat frequencies and an asynchronous type of excitation-contraction coupling. In spite of this intense muscular work, metabolic processes are not limited by the availability of oxygen. Also of importance is the morphological organization of the flight muscle and mitochondria, which have evolved ultrastructurally and biochemically into an effective catabolic machine. 2. In the fly, carbohydrate, principally glycogen, is the sole metabolic fuel; fats are not used in flight and enzymes concerned with fatty acid utilization are virtually lacking. Glycogenolysis does not lead to lactic acid; instead, the end products of glycolysis are pyruvate and alpha-glycerophosphate. The alpha-glycerophosphate cycle provides a mechanism not only for the reoxidation of glycolytically produced NADH but also for the stoicheiometric formation from each molecule of hexose equivalent of two molecules of pyruvate, which are then available for oxidation via the tricarboxylate cycle. The absence of dicarboxylate and tricarboxylate carriers from the mitochondria ensures that tricarboxylate-cycle intermediates do not exit from the mitochondrion but that pyruvate is oxidized to completion. On initiation of flight, mitochondrial oxidation of pyruvate is impeded by the lack of tricarboxylate-cycle intermediates for the generation of oxaloacetate. This is circumvented by the oxidation of proline. 3. The controls on metabolism in flight muscle, i.e. (1) glycogenolysis at phosphorylase and phosphorylase kinase, (2) glycolysis at phosphofructokinase, (3) alpha-glycerophosphate dehydrogenase, (4) proline dehydrogenase and (5) tricarboxylate cycle at isocitrate dehydrogenase, are effected by the phosphate potential and/or Ca2+. It is suggested that the metabolic changes, such as those seen in the rest-to-flight transition, are achieved by the concerted actions of these effectors at the different loci.

Effects of Thyroid Hormone Deficiency on the Distribution of Hepatic Metabolites and Control of Pathways of Carbohydrate Metabolism in Liver and Adipose Tissue of the Rat

Abstract: 1 Measurements have been made of the hepatic metabolites in normal and thyroidectomized rats and of enzymes of the glycolytic route, the pentose phosphate pathway, the tricarboxylic acid cycle and of lipogenesis together with the flux of glucose through alternative pathways of glucose metabolism and into lipid. 2 There is a significant fall in the content of ADP, AMP, citrate, long-chain acyl-CoA derivatives and a rise of 3-phosphoglycerate, 2-phosphoglycerate and phosphoenolpyruvate following thyroidectomy. The observed changes in the glycolytic intermediates may be correlated with the increased activity of phosphofructokinase relative to pyruvate kinase and fructose bisphosphatase. 3 The NAD+/NADH quotient of the mitochondrial compartment, calculated from the reactants and Keq of 3-hydroxybutyrate dehydrogenase, becomes significantly more oxidized in hypothyroid animals. The redox state of the cytosolic NAD and NADP couples remains relatively unchanged. 4 The changes in the hepatic content of CoA derivatives and citrate and in the mitochondrial redox state are interpreted as indicating a depressed rate of lipid oxidation. 5 Calculations of the compartmentation of metabolites between the cytosol and mitochondria indicate a very marked decrease in mitochondrial citrate, 2-oxoglutarate and glutamate with smaller changes in aspartate and malate. These changes are interpreted as providing evidence for the importance of modifications in the malate-aspartate shuttle in hypothyroidism; this is further supported by measured changes in the distribution and activities of the component enzymes of the hydrogen shuttles. 6 There is a diminished activity of glucokinase and of enzymes of the glycolytic pathway below phosphofructokinase in livers from hypothyroid rats. The oxidative enzymes of the pentose phosphate pathway, ATP-citrate lyase, ‘malic’ enzyme and fatty acid synthetase also decrease markedly. There is a striking parallelism between the changes of enzyme profile of liver and adipose tissue in hypothyroidism. 7 Adrenal glands from hypothyroid rats showed a generalized decline in enzyme activity in parallel with the fall in tissue weight. Cytosolic glycerol-3-phosphate dehydrogenase decreased sharply. 8 The present results are discussed in relation to data in the literature on the increased activities of cyclic AMP and cyclic GMP phosphodiesterases in hypothyroidism, observations which integrate the relationships between anabolic and catabolic pathways of carbohydrate and lipid metabolism with alterations in hormone sensitivity in hypothyroidism.

Effects of Cyanide and Ethylene on the Respiration of Cyanide-sensitive and Cyanide-resistant Plant Tissues

Abstract: The effects of cyanide and ethylene, respectively, were studied on the respiration of a fully cyanide-sensitive tissue-the fresh pea, a slightly cyanide-sensitive tissue-the germinating pea seedling, and a cyanide-insensitive tissue-the cherimoya fruit. Cyanide inhibition of both fresh pea and pea seedling respiration was attended by a conventional Pasteur effect where fermentation was enhanced with an accumulation of lactate and ethanol and a change in the level of glycolytic intermediates indicative of the activation of phosphofructokinase and pyruvate kinase accompanied by a sharp decline in ATP level. In these tissues, ethylene had little or no effect on the respiration rate, or on the level of glycolytic intermediates or ATP. By contrast, ethylene as well as cyanide enhanced both respiration and aerobic glycolysis in cherimoya fruits with no buildup of lactate and ethanol and with an increase in the level of ATP. The data support the proposition that for ethylene to stimulate respiration the capacity for cyanide-resistant respiration must be present.

The stimulus-secretion coupling of glucose-induced insulin release. XXII. Qualitative and quantitative aspects of glycolysis in isolated islets.

Abstract: When isolated islets of Langerhans are suddenly exposed to glucose, the entry of the hexose into islet cells first occurs at a high rate resulting in rapid equilibration of free glucose across the cell membrane; thereafter, the rate of net glucose uptake depends on its metabolism. More than 95% of the glucose taken up by the islets is converted to triosephosphate. The fractional contribution of the sorbitol and pentose-phosphate pathways to such a process does not exceed 10%. The output of lactate from the islets accounts for approximately half of the glycolytic flux. At increasing glucose concentrations up to 4.3 mM, the rate of glycolysis increases towards a first asymptotic value; at higher glucose levels (up to 27.8 mM), a sigmoidal pattern is seen tending towards a second saturation value. The total ATP content of the islets does not correlate with their insulin-secretory activity. It is suggested that, in the process of glucose-induced insulin release, glycolysis may regulate physiological processes possibly located in the micro-environment of the cell boundary.

Transport of calcium ions by Ehrlich ascites-tumour cells

Abstract: Ehrlich ascites-tumour cells accumulate Ca2+ when incubated aerobically with succinate, phosphate and rotenone, as revealed by isotopic and atomic-absorption measurements. Ca2+ does not stimulate oxygen consumption by carefully prepared Ehrlich cells, but des so when the cells are placed in a hypo-osmotic medium. Neither glutamate nor malate support Ca2+ uptake in ‘intact’ Ehrlich cells, nor does the endogenous NAD-linked respiration. Ca2+ uptake is completely dependent on mitochondrial energy-coupling mechansims. It was an unexpected finding that maximal Ca2+ uptake supported by succinate requires rotenone, which blocks oxidation of enogenous NAD-linked substrates. Phosphate functions as co-anion for entry of Ca2+. Ca2+ uptake is also supported by extra-cellular ATP; no other nucleoside 5′-di- or tri-phosphate was active. The accumulation of Ca2+ apparently takes place in the mitochondria, since oligomycin and atractyloside inhibit ATP-supported Ca2+ uptake. Glycolysis does not support Ca2+ uptake. Neither free mitochondria released from disrupted cells nor permeability-damaged cells capable of absorbing Trypan Blue were responsible for any large fraction of the total observed energy-coupled Ca2+ uptake. The observations reported also indicate that electron flow through energy-conserving site 1 promotes Ca2+ release from Ehrlich cells and that extra-cellular ATP increase permeability of the cell membrane, allowing both ATP and Ca2+ to enter the cells more readily.

The stimulus-secretion coupling of glucose-induced insulin release. XX. Fasting: A model for altered glucose recognition by the B-cell

Abstract: The rate of glucose uptake and oxidation, the output of lactate, the net uptake of calcium, the release of preformed or newly synthesized insulin and, possibly to a lesser extent, the biosynthesis of proinsulin are all diminished in islets removed from fasted rats and exposed to glucose. Theophylline and dibutyryl-adenosine-3',5'-cyclic monophosphate fail to fully restore a normal secretory response to glucose, despite the fact that they increase lactate production by the islets from fasted animals. The insulinotropic action of other secretagogues, including glyceraldehyde, leucine, beta-hydroxybutyrate, and sulfonylurea is unaffected by prior fasting of the donor rats. The islets metabolism of glyceraldehyde is also unaffected by fasting. These data indicate that fasting is associated with a block in glucose metabolism in the early steps of glycolysis, prior to the triose-phosphate level, and suggest that the insulin secretory response to glucose may be closely dependent on the rate at which the hexose is metabolized by islet tissue.

The stimulus-secretion coupling of glucose-induced insulin release. Does glycolysis control calcium transport in the B-cell?

Abstract: 1. The metabolism of glucose and the exchangeable Ca2+ pool were measured in rat pancreatic islets, in order to assess the possible significance of glycolysis in the process of glucose-induced insulin release. 2. At high glucose concentration (16.7 mM), glucose was metabolized at the following rate (pmol of glucose residue/h per islet +/- S.E.M.): 131 +/- 11 for glucose uptake, 129+/-8 for glucose utilization, as judged by the conversion of [5-3H]glucose into 3H2O,60+/-2 for lactate output and 25+/-2 for glucose oxidation. 3. The secretory pattern usually correlated with the metabolic data. For instance, the ability of different sugars (glucose, mannose, fructose, galactose, D-glyceraldehyde) to stimulate lactate output closely paralleled their relative insulinotropic capacity. A disparity between metabolic and secretory responses was, however, encountered in the presence of dibutyryl cyclic AMP and theophylline. 4. Despite this contrasting behaviour, the size of the Ca2+- exchangeable pool (net uptake of 45Ca2+) was invariably proportional to the rate of lactate output under all experimental conditions examined. It is concluded that glycolysis usually exerts a tight control on the rate constant for Ca2+ transport across the B-cell membrane.

A comparison of the enzyme levels and the in vitro utilization of various substrates for lipogenesis in pair-fed lean and obese pigs.

Abstract: In this study of spontaneous obesity of pigs, specific metabolic shifts were observed, which explain an increase in fat deposition. Liver tissue utilization of pyruvate and glucose for oxidation and lipogenesis showed no significant difference between lean and obese pigs. Adipose tissue utilization of glucose, acetate and glycerol for triglyceride and fatty acid synthesis was greater in obese pigs than lean pigs (P less than 0.01). No significant difference in leucine incorporation into lipid fractions was found. Of the substrates utilized, glucose supplied 86 and 94% of the glyceride-glycerol synthesized in lean and obese pigs, respectively. Glycerol was not a major contributor to glyceride-glycerol synthesis (3.5 to 5.5%), in spite of the presence of adipose tissue glycerokinase. An increase (P less than 0.05) in alanine incorporation into glucose was observed in liver tissue from obese pigs. In general, the levels of enzymes activities associated with gluconeogenesis, glycolysis, and lipogenesis supported the findings of in vitro utilization of these substrates.