ASSESSMENT OF THE ROLES OF MITOGEN-ACTIVATED PROTEIN-KINASE, PHOSPHATIDYLINOSITOL 3-KINASE, PROTEIN-KINASE-B, AND PROTEIN-KINASE-C IN INSULIN-INHIBITION OF CAMP-INDUCED PHOSPHOENOLPYRUVATE CARBOXYKINASE GENE-TRANSCRIPTION

Transcription of the phosphoenolpyruvate carboxykinase (PEPCK) gene is induced by glucagon, acting through cAMP and protein kinase A, and th is induction is inhibited by insulin. Conflicting reports have suggest ed that insulin inhibits induction by cAMP by activating the Ras/mitog en-activated protein kinase (MAPK) pathway or by activating the phosph atidylinositol 3-kinase (PI3-kinase), but not MAPK, pathway. Insulin a ctivated PI3-kinase phosphorylates Lipids that activate protein kinase B (PKB) and Ca2+/diacylglycerol-insensitive forms of protein kinase C (PKC). We have assessed the roles of these pathways in insulin inhibi tion of cAMP/PKA-induced transcription of PEPCK by using dominant nega tive and dominant active forms of regulatory enzymes in the Ras/MAPK a nd PKB pathways and chemical inhibitors of PKC isoforms. Three indepen dently acting inhibitory enzymes of the Ras/MAPK pathway, blocking SOS , Ras, and MAPK had no effect upon insulin inhibition. However, domina nt active Ras prevented induction of PEPCK and also stimulated transcr iption mediated by Elk, a MAPK target. Insulin did not stimulate Elk-m ediated transcription, indicating that insulin did not functionally ac tivate the Ras/MAPK pathway. Inhibitors of PI3-kinase, LY294002 and wo rtmannin, abolished insulin inhibition of PEPCK gene transcription. Ho wever, inhibitors of PKC and mutated forms of PKB, both of which are k nown downstream targets of PI3-binase, had no effect upon insulin inhi bition. Dominant negative forms of PKB did not interfere with insulin inhibition and a dominant active form of PKB did not prevent induction by PKA. Phorbol eater-mediated inhibition of PEPCK transcription was blocked by bisindole maleimide and by staurosporine, but insulin-media ted inhibition was unaffected. Thus, insulin inhibition of PKA-induced PEPCK expression does not require MAPK activation but does require ac tivation of PI3-binase, although this signal. is not transmitted throu gh the PKB or PKC pathways.