Application of stress induced fluctuations in metabolite levels to the study of brain glycolytic control in vivo

Abstract

The project was undertaken to gain an understanding of glycolytic control in rat brain in vivo. The method involved stressing rats to disturb the metabolic steady state of the brain and then allowing a recovery period so that the steady state might be re-established. The concentrations of glycolytic and related metabolites, were measured at various times during both the stress and recovery periods. Relationships among the metabolites were then studied and conclusions drawn on the subject of glycolytic control. Stressing the rats involved gently tumbling them for 2 min and this was followed by the recovery period which lasted up to 36 min but was 32 min in the case of most metabolites examined. At the required time, the animals were killed by immersion in liquid nitrogen, extracts made of the whole brains and these extracts assayed. The metabolites measured were glycogen, glucose, glucose-6-phosphate, fructose-6- phosphate, fructose-1,6-bisphosphate, dihydroxyacetone phosphate, glyceraldehyde-3-phosphate, 1,3-diphosphoglycerate, 3-phosphoglycerate, 2-phosphoglycerate, phospho-enol-pyruvate, pyruvate, ATP, ADP, AMP, NAD+, phosphocreatine, blood glucose, blood haemoglobin, brain haemoglobin and NADH (normal steady state value only). Results were also available for lactate. The values for blood glucose and the haemoglobin concentrations in blood and brain enabled a correction to be made to the total brain glucose for a contribution from blood in the brain. In most cases, the metabolites showed oscillatory behaviour. The oscillations were usually irregular but damped, and most metabolites regained their normal (pre-tumbling) levels within a recovery period at 32 minutes.Of the main energy sources, glucose, ATP and phosphocreatine showed increased usage after the onset of stressing. Glycogen was the last potential energy source to show a change in net concentration. This occurred after about one minute of stressing. This delay before a net change in glycogen concentration was observed, indicated that the breakdown of glycogen was regulated. Comparison of the changes with time in the concentrations of the metabolites was used to identify enzymic reactions which were or were not at equilibrium. Application of mass-action ratios to the various enzymic reactions and a calculation of ?G’ where applicable also helped to pinpoint enzymic reactions which were not close to equilibrium and hence, potential control sites. The enzymic reactions of glycolysis not using cofactors include phosphoglucoisomerase, aldolase, triosephosphate isomerase, phosphoglyceromutase and enolase. Of these, the reactions catalysed by phosphoglucoisomerase and phosphoglyceromutase were always close to equilibrium. Enolase was somewhat removed from equilibrium and apparently acted as a rate limiting step, at least during periods of increased flux. The existence of a disequilibrium at triosephosphate isomerase allowed a qualitative measure of glycolytic flux change to be calculated. It was proposed that the value of the mass-action ratio for this reaction varied inversely as the flux change. The reaction at aldolase was assumed to be close to equilibrium and on this basis, it was determined that only about 1% of the total fructose-1,6-bisphosphate was free in the cytoplasm. The relationship between the free and the non-free fructose-1,6-bispiosphate was determined over the experimental period. Of the enzymic reactions using cofactors, those catalysed by glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerokinase and lactate dehydrogenase have been assumed to be close to equilibrium on the basis of experimental and literature data. It was possible that glyceraldehyde-3-phosphate dehydrogenase may have been transiently rate-limiting in the first 3.5 minutes. The mass-action ratios of these enzymic reactions were used to calculate values for the cytoplasmic level of inorganic phosphate, and also, values for the cytoplasmic ratios of [ATP] / [ADP] and [NAD+] / [NADH]. The remaining three reactions, catalysed by hexokinase, 6-phosphofructckinase and pyruvate kinase, were non-equilibrium as indicated by their mass-action ratios, calculated with both observed and cytoplasmic values of [ATP] / [ADP]. However, the situation at the hexokinase step was obscure, and the data did not permit separation of the reaction catalysed by hexokinase from glucose entry into the cells when possible regulation was considered. The problem concerned the lack of knowledge of the intracellular glucose concentration. Only the reactions catalysed by 6-phosphofructokinase and pyruvate kinase were unequivocally displaced far from equilibrium. A comparison of the concentrations of fructose-6-phosphate and phosphoenolpyruvate with the mass-action ratio for the triosephosphate isomerase reaction revealed that both enzymes underwent activation and deactivation. It appeared that 6-phosphofructokinase was controlled at least by ATP and/or ADP, as well as possibly by citrate. The changes in activity of pyruvate kinase were compatible with activation by fructose-1,6-bisphosphate, thus allowing co-ordination between the two enzymes. Evidence was presented for the existence of an homeostatic mechanism controlled by brain glucose concentration and designed to maintain an adequate supply of blood glucose to the brain. A broad view of the relationship between metabolism and energy requirements was given by a calculation of the Adenylate Energy Charge for the whole brain. This parameter did not show the rapid variations associated with the qualitative measure of cytoplasmic flux change.