Estimation of the Relative Contributions of Enhanced Production of Oxalacetate and Inhibition of Pyruvate Kinase to Acute Hormonal Stimulation of Gluconeogenesis in Rat Hepatocytes AN ANALYSIS OF THE EFFECTS OF GLUCAGON, ANGIOTENSIN 11, AND DEXAMETHASONE ON GLUCONEOGENIC FLUX

Hepatocytes, isolated from fasted rats, were incubated with graded concentrations of lactate and pyruvate, at a mean constant ratio of 10-13:1, to alter systematically the concentrations of gluconeogenic intermediate metabolites and rates of glucose production. By analyzing glucose production rates as a function of corresponding concentrations of extracellular pyruvate, cytosolic oxalacetate, and cellular 3-phosphoglycerate in the presence and bsence of hormones and assuming no primary activation of phosphoenolpyruvate carboxykinase, estimates were made of the relative contributions of stimulation of formation of cytosolic oxalacetate and inhibition of pyruvate kinase to hormonal stimulations of gluconeogenesis. Addition of dexamethasone, glucagon, or angiotensin I1 did not cause a shift in the relationship between cellular 3-phosphoglycerate concentrations and rates of glucose production, indicating that there was no effect of these agents on the reactions involved in conversion of phosphoenolpyruvate to glucose. All three agents shifted the relationships between rates of glucose production and both cytosolic oxalacetate and extracellular pyruvate. The following conclusions were drawn from computer analyses of these results. 1) At low concentrations of pyruvate, stimulation of oxalacetate production and pyruvate kinase inhibition were approximately equally contributory to the overall stimulations of gluconeogenesis by angiotensin I1 and dexamethasone. 2) At higher pyruvate concentrations, pyruvate kinase inhibition by angiotensin 11 played a greater role, accounting for 90% of the overall stimulation. 3) For dexamethasone, as the pyruvate concentration was increased, stimulation of gluconeogenesis resulting from enhanced formation of oxalacetate diminished as did overall stimulation of gluconeogenesis. 4) Glucagon addition resulted in an inhibition of pyruvate kinase flux that accounted for 75% of the hormone’s overall effect at low pyruvate concentrations; this increased to 95% at high pyruvate concentrations.