Transcriptional regulation of phosphoenolpyruvate carboxykinase (PEPCK), the rate-limiting enzyme in hepatic gluconeogenesis, by insulin was investigated with the use of adenovirus vectors encoding various mutant signaling proteins. Insulin inhibited transcription induced by dexamethasone and cAMP of a chloramphenicol acetyltransferase (CAT) reporter gene fused with the PEPCK promoter sequence in HL1C cells stably transfected with this construct. A dominant negative mutant of phosphoinositide (PI) 3-kinase blocked insulin inhibition of transcription of the PEPCK-CAT fusion gene, whereas a constitutively active mutant of PI 3-kinase mimicked the effect of insulin. Although a constitutively active mutant of Akt (protein kinase B) inhibited PEPCK-CAT gene transcription induced by dexamethasone and cAMP, a mutant Akt (Akt-AA) in which the phosphorylation sites targeted by insulin are replaced by alanine did not affect the ability of insulin to inhibit transcription of the fusion gene. Akt-AA almost completely inhibited insulin-induced activation of both endogenous and recombinant Akt in HL1C cells. Furthermore, neither a kinase-defective mutant protein kinase Clambda (PKClambda), which blocked insulin-induced activation of endogenous PKClambda, nor a dominant negative mutant of the small GTPase Rac prevented inhibition of PEPCK-CAT gene transcription by insulin. These data suggest that phosphoinositide 3-kinase is important for insulin-induced inhibition of PEPCK gene transcription and that a downstream effector of phosphoinositide 3-kinase distinct from Akt, PKClambda, and Rac may exist for mediating the effect of insulin.