Glucagon-like peptide-1 (GLP-1) increases -cell function and growth through PKA- and PI3-K/PKB, respectively. GLP-1 acts via a G protein-coupled receptor, and PI3-K is known to be activated by G_. The role of PI3-K in the chronic effects of GLP-1 on the -cell was therefore investigated using PI3-K knock-out (KO) mice treated with the GLP-1 receptor agonist, exendin-4 (Ex4; 1 nmol/kg s.c q24 h for 14 d). In vivo, glucose and insulin responses were similar in PBS- and Ex4-treated KO and wild-type (WT) mice. However, glucose-stimulated insulin secretion was markedly impaired in islets from PBS-KO mice (P < 0.05), and this was partially normalized by chronic Ex4 treatment (P < 0.05). In contrast, insulin content was increased in PBS-KO islets, and this was paradoxically decreased by Ex4 treatment, compared with the stimulatory effect of Ex4 on WT islets (P < 0.05-0.01). Transfection of INS-1E -cells with siRNA for PI3-K similarly decreased glucose-stimulated insulin secretion (P < 0.01) and increased insulin content. Basal values for -cell mass, islet number and proliferation, GLUT2, glucokinase and IRS-2 were increased in PBS-KO mice (P < 0.05-0.001) and, although they were increased by Ex4 treatment of WT animals (P < 0.05), they were decreased in Ex4-KO mice (P < 0.05-0.01). These findings indicate that PI3-K deficiency impairs insulin secretion, resulting in compensatory islet growth to maintain normoglycemia. Chronic Ex4 treatment normalizes the secretory defect, thereby relieving the pressure for expansion of -cell mass. These studies reveal a new role for PI3-K as a positive regulator of insulin secretion, and reinforce the importance of GLP-1 for the maintenance of normal -cell function.