Differential Activation of Mitogen-Activated Protein Kinase by Insulin and Epidermal Growth Factor in 3T3-L1 Adipocytes: A Possible Involvement of PI3-Kinase in the Activation of the MAP Kinase by Insulin

Mitogen-activated protein (MAP) kinase plays crucial roles in cell growth and differentiation. It has recently been shown that the MAP kinase cascade in growth factor signaling diverges and cross-talks with other signaling pathways. In the present study, we examined the effects of wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3-kinase), on the activation of Ras, Raf-1 kinase, and MAP kinase by insulin and epidermal growth factor (EGF). The effect of LY294002, a structurally distinct PI3-kinase inhibitor, on the activation of Raf-1 kinase by both ligands was also examined. In 3T3-L1 adipocytes, 25 nmol/l wortmannin inhibited the insulin-induced activation of Raf-1 kinase to the basal level, whereas the same dose of wortmannin had little effect on the EGF-induced activation of Raf-1 kinase. One hundred μmol/1 LY294002 blocked insulin-induced activation of Raf-1 kinase without affecting EGF-induced activation of this kinase. Twenty-five nmol/l wortmannin inhibited the insulin-induced activation of MAP kinase to the basal level with no effect on the EGF-induced activation of this kinase. But the same dose of wortmannin did not affect the formation of guanosine 5′-triphosphate (GTP)-bound Ras stimulated by either ligand. In KB cells, results similar to those in 3T3-L1 adipocytes were obtained. In contrast, in Chinese hamster ovary cells overexpressing the human insulin receptor (CHO-HIR cells), neither wortmannin nor LY294002 inhibited the insulin-induced activation of Raf-1 kinase, and wort-mannin had little effect on the activation of MAP kinase by insulin. These results indicate that 1) PI3- kinase or wortmannin-sensitive molecules are involved in the interaction between activated Ras and Raf-1 kinase in the insulin signaling in 3T3-L1 adipocytes, 2) the involvement of PI3-kinase or wortmannin-sensitive molecules in the insulin-induced activation of MAP kinase appears to be cell-type specific, and 3) differential mechanisms to activate Raf-1 kinase and MAP kinase by insulin and EGF exist.