We have previously demonstrated that the phosphatidylinositol-3 kinase (P13K)/Akt signaling is essential for pancreatic regeneration after partial pancreatectonny in mice. in the present study, we examined a role of P13K/Akt signaling for pancreatic duct cell differentiation into insulin-producing cells. Epithelial-like cells were isolated from mouse pancreas and confirmed to be positive for a duct cell marker cytokeratin-20 (CK-20) but negative for insulin. Incubation of these cells with epidermal growth factor, exhibited a gradual increase in Akt phosphorylation and expression of pancreatic duodenal homeobox-1 (PDX-1), a regulator of P-cell differentiation. Three weeks later, these CK-20-positive cells were noted to express insulin as determined by immunofluorescent double-staining. Akt phosphorylation, PDX-1 expression, and insulin production were effectively reduced by blocking the P13K/Akt pathway using siRNA to the p85 alpha regulatory subunit of P13K. Our results demonstrate that P13K(/Akt activation has a critical role for pancreatic duct cell differentiation into insulin-producing cells. (c) 2008 Elsevier Inc. All rights reserved.