Effects of fatty acids, ketone bodies, lactate and pyruvate on glucose utilization by guinea-pig cerebral cortex slices.
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ABSTRACT:
SUBMITTER: Rolleston FS
PROVIDER: S-EPMC1270614 | biostudies-other | 1967 Aug
REPOSITORIES: biostudies-other
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Project description:By the use of 1mm-iodoacetate to inhibit glycolysis in guinea-pig cerebral tissue slices, the kinetics of the uptake of monosaccharides on transfer of tissue from 0 degrees to 37 degrees were studied. d-Ribose, d-galactose, d-mannose, l-sorbose, and d-fructose showed diffusion kinetics, whereas 2-deoxy-d-glucose, d-glucose, d-arabinose and d-xylose showed saturation kinetics.
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Project description:1. When guinea-pig cerebral-cortex slices were incubated with [U-(14)C]glutamate as substrate, the specific radioactivities of the citric acid-cycle intermediates were lower than that of the aspartate isolated from the same vessels. 2. Aspartate was significantly labelled when [5-(14)C]glutamate was used as substrate and the aspartate contained almost no label when [1-(14)C]glutamate was present as substrate. 3. When specifically labelled glutamate was used as substrate, the label was found in the isolated aspartate in the position that would be predicted by citric acid-cycle mechanisms. 4. The results are consistent with the theory of ;compartmentation' of amino acid metabolism.
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Project description:The concentrations of ATP and the ATP/AMP concentration ratios were maintained in thioglycollate-elicited mouse peritoneal macrophages incubated in vitro for 90 min in the presence or absence of added substrate: rates of glycolysis, lactate formation and glutamine utilization were approximately linear with time for at least 60 min of incubation. The rate of oxygen consumption by macrophages was only increased above the basal rate (i.e. that in the absence of added substrate) by addition of succinate or pyruvate, or by addition of the uncoupling agent carboxyl cyanide m-chlorophenylhydrazone ('CCCP'); it was decreased by 75% by the addition of KCN. These findings suggest that metabolism of endogenous substrate can provide most, if not all, of the energy requirement of these cells, at least for a short period. The rates of glucose and glutamine utilization by incubated macrophages were approx. 300 and 100 nmol/min per mg of protein respectively. A large proportion of the glutamine that is utilized is converted into glutamate and aspartate, and very little (perhaps less than 10%) is oxidized. Similarly almost all of the glucose that is utilized is converted into lactate and very little is oxidized. This characteristic is similar to that of resting lymphocytes and rapidly dividing cells; in non-proliferating macrophages it may be a mechanism to provide precision in control of the rate of biosynthetic processes that utilize intermediates of these pathways, e.g. purines and pyrimidines for mRNA for the synthesis of secretory proteins and glycerol 3-phosphate for phospholipid synthesis for membrane recycling. No utilization of acetoacetate or 3-hydroxybutyrate by macrophages was detected. In contrast, both butyrate and oleate were oxidized. The rate of [14C]oleate conversion into 14CO2 (1.3 nmol/h per mg of protein) could account for most of the oxygen consumption by incubated macrophages, suggesting that long-chain fatty acids might provide an important fuel in situ. This may be one explanation for the secretion of lipoprotein lipase by these cells, to provide fatty acids for oxidation from the degradation of local triacylglycerol.
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Project description:1. The effect of various agents on the turnover of protein-bound phosphorus in respiring slices of cerebral cortex was studied. 2. Confirming previous work turnover was increased by the application of electrical pulses for 10s to the tissue. 3. Turnover was also increased by exposure of the slices for 10min to noradrenaline (0.5mm), 5-hydroxytryptamine (1mum) and histamine (0.1mm). 4. When slices were stimulated by electrical pulses in the presence of histamine the increase in turnover was the sum of the responses given by each agent above, suggesting that different phosphorylating systems were involved. 5. Tetrodotoxin (0.5mum) blocked the increased turnover due to electrical pulses, but not that due to histamine. Tetrodotoxin also prevented the increase in tissue cyclic AMP content caused by the application of electrical pulses. 6. Phosphoprotein turnover was not affected by adenosine, despite the increase in tissue cyclic AMP content given by this agent. 7. Adenosine blocked the phosphoprotein response to histamine, but did not affect the response to electrical pulses. 8. The results are discussed in relation to the hypothesis that the stimulation of protein phosphorus turnover by electrical pulses is secondary to the release of cyclic AMP in the tissue.
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Project description:Inhibition of receptor-coupled adenylate cyclase by hormones is proposed to be associated with GTP hydrolysis. Since adenosine inhibits cerebral-cortical adenylate cyclase via A1 adenosine receptors, the present study attempts to verify this mechanism for A1-selective adenosine derivatives. In guinea-pig cortical membranes N6-(phenylisopropyl)adenosine (PIA) increased the Vmax. of the low-Km GTPase, with an EC50 (concentration causing 50% of maximal stimulation) of about 0.1 microM, and the stimulatory effect was competitively antagonized by 5 microM-8-phenyltheophylline. The rank order of potency of the stereoisomers of PIA and of 5-(N-ethylcarboxamido)adenosine (NECA) to stimulate GTPase correlated with their ability to inhibit adenylate cyclase activity (R-PIA greater than NECA greater than S-PIA). Competition binding studies with (-)-N6- ([125I]iodo-4-hydroxyphenylisopropyl)adenosine suggest that adenylyl imidodiphosphate (p[NH]ppA), an essential component of the GTPase assay system, is a more potent A1-receptor agonist than ATP, with an IC50 (concentration giving half-maximal displacement of radioligand binding) of 7.9 microM. On the basis of the p[NH]ppA concentration used in the GTPase assay (1.25 mM), enzyme stimulation by adenosine seems to be highly underestimated. Nevertheless, adenosine-induced GTP hydrolysis reflects an increased turnover of guanine nucleotides at the Ni regulatory site and appears to be a crucial step in the sequence of events processing the inhibitory signal to adenylate cyclase.
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Project description:1. Hexokinase activities were estimated in primary subcellular fractions from guinea-pig cerebral cortex and in sucrose-density-gradient subfractions of the mitochondrial and microsomal fractions. 2. Appreciable activities were observed in mitochondrial, microsomal and soluble fractions. The activity in the mitochondrial fraction was associated with the mitochondria rather than with myelin or nerve endings and that in the microsomal fraction was associated with membrane fragments. 3. Most of the mitochondrial activity was extracted in soluble form by osmotic ;shock'. The activity of the mitochondrial extract differed from the soluble activity in kinetic properties and in electrophoretic behaviour. 4. No evidence was obtained for the presence of a high-K(m) glucokinase in the brain. 5. The results are discussed in terms of relevance to considerations of glucose utilization by the brain.
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Project description:The contribution of pyruvate to ketogenesis was determined in rat hepatocyte suspensions by using [14C]pyruvate. The rates of conversion of pyruvate into ketone bodies in hepatocytes from fed and 24 h-starved rats were 10 and 17 mumol/h per g wet wt. respectively, and accounted for 50 and 29% of the total ketone bodies formed. In hepatocytes from fed rats, the addition of palmitate (0.25-1 mM) increased the rate of conversion of pyruvate into ketone bodies (80-140%), but decreased the relative contribution of pyruvate to total ketogenesis. In hepatocytes from starved rats, palmitate did not increase pyruvate conversion into ketone bodies.
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Project description:1. Intracellular concentrations of intermediates and cofactors of glycolysis were measured in guinea-pig cerebral cortex slices incubated under varying conditions. 2. Comparison of mass-action ratios with apparent equilibrium constants for the reactions of glycolysis showed that hexokinase, phosphofructokinase and pyruvate kinase catalyse reactions generally far from equilibrium, whereas phosphoglucose isomerase, aldolase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, adenlyate kinase and creatine phosphokinase are generally close to equilibrium. The possibility that glyceraldehyde 3-phosphate dehydrogenase may catalyse a ;non-equilibrium' reaction is discussed. 3. Correlation of changes in concentrations of substrates for enzymes catalysing ;non-equilibrium' reactions with changes in rates of glycolysis caused by alteration of the conditions of incubation showed that hexokinase, phosphofructokinase, pyruvate kinase and possibly glyceraldehyde 3-phosphate dehydrogenase are subject to metabolic control in cerebral cortex slices. 4. It is suggested that the glycolysis is controlled by two regulatory systems, the hexokinase-phosphofructokinase system and the glyceraldehyde 3-phosphate dehydrogenase-pyruvate kinase system. These are discussed. 5. It is concluded that the rate of glycolysis in guinea-pig cerebral cortex slices is limited either by the rate of glucose entry into the slices or by the hexokinase-phosphofructokinase system. 6. It is concluded that addition of 0.1mm-ouabain to guinea-pig cerebral cortex slices causes inhibition of either glyceraldehyde 3-phosphate dehydrogenase or phosphoglycerate kinase or both, in a manner independent of the known action of ouabain on the sodium- and potassium-activated adenosine triphosphatase.
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