Insulin resistance is a major cause of type 2 diabetes mellitus. To understand the molecular basis of insulin resistance, we tried to clarify the molecular mechanisms or insulin action. The major metabolic actions of insulin is regulated by phosphatidylinositol (PI)3-kinase. However, the downstream effectors of the various singaling pathways that emanate from PI 3-kinase remain unclear. Akt (protein kinase B), a serine-threonine kinase with a pleckstrin homology domain, is thought to be one such downswtream effector. A mutant Akt (Akt-AA) in which the phosphorylation sites (Thr308 and Ser473) by insulin are replaced by alanine was found to lack protein kinase activity and, when expressed in 3T3-L1 adipocytes with the use of an adenovirus vector, to inhibit insulin- induced activation of endogenous Akt. Insulin-induced phosphorylation and activation of PDE3B in 3T3-L1 adipocytes were inhibited by expression of an Akt-AA. Serine-273 of mouse PDE3B is located within a motif (RXRXXS) that is preferentially phosphorylated by Akt. A mutant PDE3B in which serine-273 was replaced by alanine was not phosphorylated either in response to insulin in intact cells or by purified Akt in vitro. Moreover, the serine-273 mutant of PDE3B was not aactivated by insulin when expressed in adipocytes. These results suggest that PDE3B is a physiological substrate of Akt, and that Akt-mediated phosphorylation of PDE3B on serine-273 is important for insulin- induced activation of PDE3B. Insulin-induced phosphorylation of PHAS-1 and activation of glycogen synthase were partially inhibited by expression of an Akt-AA. However, insulin- stimulated glucose uptake in 3T3-L1 adipocytes was not affected by expression of an Akt-AA. These data indicate that Akt acts as a downstream effector in some, but not all, of the signaling pathways downstream of Pi 3-kinase. Atypical protein kinase C (PKCζ and PKOλ) is also though to be one such downstream effector of PI 3-kinase. Expression of a kinase-deficient mutants PKCλ(λKD), resulted in inhibition of insulin activation of PKCλ. Insulin-stimulated glucose uptake and translocation of the gluscose transporter GLUT4 to the plasma membrane were inhibited by IKD in a dose-dependent manner. These mutants did not inhibit insulin-induced activation of Akt. Furthermore, expression of an Akt-AA resulted in inhibition of insulin-induced activation of Akt but not of PKCλ. These results suggest that insulin-elicited signals that pass through PI 3-kinase subsequently diverge into at least two independent pathways, an Akt pathway and a PKCλ pathway, and that the latter pathway contributes, at least in pan, to insulin stimulation of glucose uptake in 3T3-L1 adipocytes. The expression of phos-phoenolpyruvate carboxykinase (PEPCK), a rate-controlling enzyme of gluconeogenesis, is regulated by insulin. Insulin inhibited the transcription induced by dexamethasone and cAMP in HLIC cells of a construct containing the chloramphenicol acetyltrans-ferase (CAT) reporter gene fused to the PEPCK promoter. Expression of ᅀp85 (dominant negative mutant of PI 3-kinase) blocked this effects of insulin on expression of the PEPCK-CAT fusion gene, suggesting that PI 3-kitiase is required for the insulin-induced inhibition of PEPCK gene transcription. However, neither Akt-AA nor the kinase- deficient mutant of PKCλ affected the inhibitory action or insulin on PEPCK-CAT gene transcription. These data suggest that a downstream effector of PI 3-kinase distinct from Akt and PKCλ may mediate the effect of insulin on PEPCK gene transcription.