The distribution of hepatic metabolites and the control of the pathways of carbohydrate metabolism in animals of different dietary and hormonal status

Abstract

A method is described by which the cytoplasmic and mitochondrial content of malate, oxaloacetate, aspartate, glutamate, 2-oxoglutarate, isocitrate, and citrate can be calculated. The values so obtained confirm that oxaloacetate occurs mainly in the cytosol. Aspartate, glutamate, and 2-oxoglutarate appear to be mainly located in the cytosol. Considerable redistribution of these metabolites occurs in the different nutritional and hormonal states. The redox state of the nicotinamide nucleotides in the two compartments has been calculated using the compartmented values. The mitochondrial redox state of the NADP couple appears to be far more reduced than has hitherto been thought. Control of the glycolytic pathway is vested in phosphofructokinase, pyruvate kinase, and glyceraldehyde-3-phosphate dehydrogenase/3-phosphoglycerate kinase. The most important modifier of hepatic phosphofructokinase seems to be fructose-6-phosphate, which may act by changing the K i ; for citrate, thus permitting a sufficient concentration of citrate to be present in the cytosol for fatty acid synthesis without inhibition of phosphofructokinase. This overcomes the difficulty of the requirement for a rapid glycolytic flux simultaneously with lipid synthesis from citrate. Ultimate control of glycolysis may rest with glucokinase. The extent of deviation of triose phosphate isomerase from equilibrium is suggested as an index of glycolytic pathway flux and direction. Compartmentation of metabolites in the span pyruvate to phosphoenolpyruvate provided additional evidence for an increased flux through the control enzymes pyruvate carboxylase and phosphoenolpyruvate carboxykinase in gluconeogenesis. The possibility that cAMP may be a positive effector of phosphoenolpyruvate carboxykinase is considered. The source of reducing equivalents for gluconeogenesis is examined. It is concluded that transfer of carbon occurs both as malate and aspartate, and that the requirement for reducing equivalents is met in part by the transfer of malate to the cytosol and in part by NADH generated by the fumarate cycle geared to urea production. Calculations of the compartmentation of tricarboxylic acid cycle intermediates suggested that a very different environment may exist in the mitochondria from that deduced from total cell metabolites. Gluconeogenic situations were characterized by increased mitochondrial 2-oxoglutarate and small changes of oxaloacetate, while the total cell contents of both were substantially decreased. Calculations of mitochondrial oxaloacetate by two independent procedures gave similar values, with no evidence for any significant fall below control level. Control of the tricarboxylic acid cycle by the redox state of the nicotinamide nucleotides and by the acetyl CoA CoA ratio is also discussed. Evidence from mass-action ratios and metabolite profiles point to control of the oxidative segment of the pentose phosphate pathway being located at glucose-6-phosphate dehydrogenase, the role of the NADP + NADPH ratio being emphasized. The advantages of using the reactants of 6-phosphogluconate dehydrogenase in the determination of the cytoplasmic NADP redox state are shown. The close agreement between the two different procedures for the calculation of the compartmentation of such key metabolites as malate and citrate lends credence to the validity of such procedures.