Involvement of both phosphatidylinositol 3-kinase and p44/p42 mitogen-activated protein kinase pathways in the short-term regulation of pyruvate kinase L by insulin

Pyruvate kinase L (PK-L) is a key regulatory enzyme of the hepatic glycolyt ic/gluconeogenic pathway that can be dephosphorylated and activated in resp onse to insulin. However, the signaling cascades involved in this insulin e ffect have not been established. In this work we have investigated the pote ntial involvement of phosphatidylinositol 3-kinase (PI 3-K) and p44/p42 mit ogen-activated protein kinase (MAPK) pathways in the short-term modulation of PK-L by insulin in primary cultures of rat hepatocytes. Wortmannin, at a concentration of 100 nM, caused a marked inhibition of the PI 3-K/protein kinase B pathway, which became complete at 500 nw wortmannin. Likewise, wor tmannin at 100 and 500 nM, elicited partial and total inhibitions of insuli n-mediated activation of PK-L, respectively. However, this PI 3-K inhibitor also reduced insulin-mediated phosphorylation of p44/ p42 MAPK in cultured rat hepatocytes, indicating that both the PI 3-K and MAPK pathways could b e involved in PK-L activation by insulin. Three facts appear to reinforce t his hypothesis: 1) the selective and complete inhibition of the PI 3-K/prot ein kinase B pathway by LY294002 (50 muM) was accompanied by a partial bloc kade of insulin-induced PK-L activation; 2) when signaling through the MAPK cascade was selectively suppressed by the presence of PD98059 (50 mum), a 50% reduction of insulin-induced activation of PK-L was observed; and 3) th e effect of PD98059 (50 muM) on PK-L activation was reinforced by the addit ional presence of 100 nM wortmannin. We also observed that the blockade of p70 S6-kinase by rapamycin did not affect the activation of PK-L by insulin . From these findings it can be concluded that both PI 3-K and MAPK pathway s, but not p70 S6-kinase, are involved in the short-term activation of PK-L by insulin in rat hepatocytes.