Our main objectives are 2-fold: 1) to define the mechanisms regulating rat liver pyruvate kinase in vivo; and 2) to correlate the changes in liver pyruvate kinase activity with the changes in overall carbohydrate flux between glucose and pyruvate in rat liver. To achieve the first objective we will examine normal animals, alloxan diabetic animals, and isolated, perfused livers which have been subjected to dietary and hormonal stresses (insulin, glucagon, and the glucocorticoids) for evidence supporting or refuting five postulated mechanisms of in vivo control of liver pyruvate kinase: 1) protein turnover; 2) covalent alteration of the enzyme molecule; 3) variations in effector concentrations; 4) induction of a regulatory peptide specific for the enzyme; and 5) the intracellular compartmentation of substrate and enzyme. To achieve the second objective we will determine the rate of carbohydrate flux between glucose and pyruvate in the isolated, perfused rat liver as a function of dietary and hormonal stresses so that we may correlate the changes in liver pyruvate kinase with the overall changes in carbohydrate flux.