Difference in the Effect of Glucagon and Starvation upon l‐Type Pyruvate Kinase from Rat Liver

Abstract

1. The mechanism of the glucagon action upon pyruvate kinase in isolated hepatocytes from fed or starved rats has been investigated. Glucagon incubation of hepatocytes increases the reaction responsible for pyruvate kinase inactivation. This inactivation reaction is completely dependent upon the presence of adenosine 3': 5'-monophosphate (CAMP), (half maximal activation at 0.1 pM CAMP) with cells incubated without glucagon, while with cells incubated with glucagon CAMP addition only has a marginal effect. It is concluded that glucagon acts by activation of the protein kinase responsible for pyruvate kinase inactivation.2. Glucagon addition to isolated hepatocytes from fed and starved rats causes a similar decrease in pyruvate kinase activity of 40.7 2.5% and 36.6 f 3.6%, respectively, when measured at suboptimal phosphoenolpyruvate concentrations. Although optimal concentrations of glucagon (1 pM) are used during the cell incubations, the pyruvate kinase activity can be inactivated further in vitro by incubation under conditions for optimal protein kinase activity. From the kinetic curves for the substrate phosphoenolpyruvate, obtained before and after further incubation in vitro, it can be concluded that pyruvate kinase by its presence inside the cell is protected against a complete inactivation by glucagon.3. The difference in apparent affinity of pyruvate kinase for phosphoenolpyruvate from glucagonincubated cells versus cells incubated without glucagon disappears after further incubation of the supernatants of these cells in vitro under optimal conditions for protein kinase activity (plus 10 pM CAMP) and an enzyme with a low affinity for phosphoenolpyruvate is obtained (Ko.5 = 4.4 mM). These results indicate that the glucagon-induced change in kinetic properties of pyruvate kinase can be adequately explained by an increased phosphorylation state of pyruvate kinase.4. The difference in apparent affinity of pyruvate kinase for phosphoenolpyruvate from fed liver versus starved liver (P < 0.001) still persists after further incubation of the liver supernatants under optimal conditions for protein kinase activity in vitro ( P < 0.001). For the completely inactivated enzyme from livers of fed and starved rats K0.5 values for phosphoenolpyruvate of respectively 2.8 and 4.4 mM are obtained. These results indicate that the nature of the effect of starvation differs from that of glucagon. It is suggested that the effect of starvation might be caused by partial proteolysis of the enzyme or oxidation of essential thiol groups.L-type pyruvate kinase has been shown to be a site of glucagon action in the hepatic gluconeogenic pathway [l-61. The mechanism of glucagon action has been investigated with isolated hepatocytes [7] and liver slices [8]. The glucagon-inhibited enzyme in hepatocytes can be reactivated in vitro, which reactivation is promoted by Mg2' and blocked by F- [7]. These properties are most consistent with a protein phosphatase mediated process implying that the glucagon-induced inactivation is the consequen...

Mechanism of glucagon stimulation of fructose‐1,6‐bisphosphatase in rat hepatocytes

“…Protein concentrations were determined by the method of Lowry et al [22]. L-type pyruvate kinase activity was determined as in [23]. Collagenase type I, bovine serum albumin fraction V, fructose 1,6-bisphosphate and fructose 2,6-bisphosphate were from Sigma.…”