Inactivation of phosphofructokinase by glucagon in rat hepatocytes.

Kinetic evidence of a time- and dose-dependent inactivation of phosphofructokinase by glucagon in isolated rat hepatocytes is reported. This inactivation, which persists after gel filtration of a cell-free extract on Sephadex G-25 and after 400-fold purification of the enzyme on agarose-ATP, is observed when the enzyme activity is measured at subsaturating concentrations of fructose 6-phosphate, while there is no change in Vmax. Phosphofructokinase inactivation by glucagon parallels the known inactivation of pyruvate kinase L and activation of glycogen phosphorylase alpha. Exogenous cyclic AMP mimics the effect of this hormone. Half-maximal effect for both phosphofructokinase and pyruvate kinase L is caused by a similar dose of glucagon (1 x 10(-10) M). The inactivation of phosphofructokinase by nonsaturating concentration of glucagon is reversed spontaneously within 40 min of incubation and this reversion is accelerated by insulin.


Inactivation
of Phosphofkuctokinase by Glucagon in Rat Hepatocytes* (Received for publication, January 30, 1979, and  A decrease in the metabolic flux through phosphofructokinase by the action of glucagon has been deduced from isotopic studies in isolated rat hepatocytes (1,2). Siiling and co-workers (3, 4) have concluded from their experiments that rat liver phosphofructokinase can be phosphorylated by a CAMP-independent protein kinase and dephosphorylated by a phosphatase with, respectively, increase and decrease of the V,,,,, of the enzyme. These results cannot easily explain the decrease in the metabolic flux through phosphofructokinase by glucagon as this hormone acts mainly by a CAMP-dependent mechanism. Furthermore, a clear evidence of a change in the kinetic properties of the liver enzyme produced by glucagon has not been reported (5, 6).
We have found a modification of the kinetic regulatory properties of phosphofructokinase from isolated rat hepatocytes treated with glucagon which produces the inactivation of the enzyme measured at subsaturating concentrations of fructose-6-P. This inactivation, antagonized by insulin and mimicked by CAMP, could account for the decrease in the metabolic flux through phosphofrvctokinase by glucagon.  .fold dilution is achieved in the reaction mixture. Care was taken to maintain the final concentration of phosphate at 5 mM in the assay of the partially purified enzyme. Protein was measured according to Lowry et al. (12). Specific activity of the enzymes is expressed as nanomoles of substrate transformed per min per mg of protein, at 25°C. Biochemicals were from Sigma or Boehringer. Other reagents were of analytical reagent grade.
Results are expressed as means + SD. from three experiments, unless otherwise indicated.

Treatment
of isolated hepatocytes with glucagon (lo-" M) causes a marked inactivation of phosphofructokinase measured at 0.25 mM fructose-6-P (Fig. l), while no change was observed at saturating concentration (5 mM). V,,,,, was 12 f 2 nmol X min-' X mg of protein-', both in control and glucagontreated hepatocytes. The inactivation of phosphofructokinase parallels the inactivation of pyruvate kinase and activation of glycogen phosphorylase a (Fig. 1). The addition of CAMP (1 X 10m4 M) to the isolated hepatocytes also inactivates phosphofructokinase, decreasing the ratio ( u0.2JVmax) X 100 from 50 f 3 to 9 + 2 after 10 min of incubation and also without affecting V,,,. Gel filtration on Sephadex G-25 of cell-free extracts from control or glucagon-treated hepatocytes does not affect either  (Table   I). Glucagon treatment of hepatocytes changes the kinetic properties of phosphoh-uctokinase, decreasing the affinity for fructose-6-P and raising the cooperativity of the enzyme for this substrate, both in crude extracts and in the partially purified enzyme (Fig. 2).
The dose-dependent inactivation of phosphofructokinase and pyruvate kinase is shown in Fig. 3 is SpOntaneOUSly reversible without change in V,,,,,. Addition of insulin (lo-* M) clearly accelerates this reactivation (Fig. 4). Insulin alone (lo-" M) has no effect on the enzyme activity either measured at 0.25 or 5 mM fructose-6-P (Fig. 4). This reversion has also been reported for pyruvate kinase L inactivation by glucagon (10). DISCUSSION It seems clear that the pair fructose-1,6-bisphosphatasephosphofructokinase is one of the sites of action of glucagon in the regulation of hepatic gluconeogenesis (1, 2). We have found a short term regulation of phosphofructokinase activity of isolated hepatocytes by glucagon, which involves an increase in S& and nn for fructose-6-P without change in V,,,,,.
As a result of these kinetic changes, an inactivation of phosphofructokinase is detected when the enzyme activity is assayed at subsaturating concentration of fructose-6-P (0.25 mM).
The fact that this inactivation persists after gel filtration of a cell-free extract on Sephadex G-25 and that a 400-fold purified phosphofructokinase preparation, from control and glucagon-treated hepatocytes, maintains kinetic properties similar to their respective crude extracts, clearly indicates that the changes observed by the action of glucagon on phosphofructokinase activity is due to a stable modification of the enzyme and not to changes in metabolite concentrations or in other enzymes.
As shown in Fig. 4, the inactivation of phosphofructokinase is spontaneously reversible; therefore, a proteolytic mecha- The inactivation of phosphofructokinase by glucagon that can attain 80% in our assay conditions would diminish the recycling between fructose-6-P and fructose-1,6-Pz, raising the net gluconeogenic flux. The glucagon-insulin antagonism that is implicated in the short term regulation of hepatic gluconeogenesis is also manifested in the regulation of phosphofructokinase activity. These facts support a relevant role of phosphofructokinase in the control of the gluconeogenic pathway as it has been shown for pyruvate kinase (10). The coordinate hormonal control of these two major regulatory glycolytic enzymes could explain, at least in part, the increase of hepatic gluconeogenesis by glucagon, both for substrates entering at the pyruvate and the triose phosphate levels.