Abstract： Objective To observe the effects of insulin and β-adrenergic agonists on the expression and secretion of adiponectin in rat adipocytes. To investigate the PI3-kinase-PDE3B-cAMP pathway in mediating the effects of insulin on the production of adiponectin. Methods Adipocytes were prepared from epididymal fat pads of male rats. The adipocytes were incubated in the presence of inhibitors including β-agonists, insulin, c-AMP analogus, specific PDE3 inhibitor and PI3-kinase inhibitors. Using real-time PCR assays, we analyzed the expression of adiponectin gene in rat adipocytes. Results(1) The β-agonist isoproterenol decreased adiponectin secretion and expression of rat adipocytes in a dose-dependent manner.Isoproterenol at 400 nmol/L inhibited the release of adiponectin by approximately 75% (P<0.05). Such inhibitory effect could be blocked by insulin. At 400μmol/L, 8-Bromo-cAMP and N6-monobutyryl-cAMP inhibited adiponectin secretion by 53% and 45% respectively (both P<0.05). Insulin blocked the inhibitory effects of 8-Bromo-cAMP (hydrolyzable by PDE), but not the effects of N6-monobutyryl-cAMP (resistant to hydrolysis by PDE) on the secretion of adiponectin. (2) The 10μmol/L milrinone (a specific PDE3 inhibitor) completely blocked the effects of insulin,restoring isoproterenol-suppressed secretion of adiponectin. Neither wortmannin (20nmol/L) nor LY294002 (10μmol/L) influenced the inhibitory effects of isoproterenol on the secretion of adiponectin. However, these PI3-kinase inhibitors completely eliminated the ability of insulin to restore the secretion of adiponectin. Conclusions (1) In vitro, insulin and β-agonists act directly at the adipocytes in opposing fashions to regulate the production of adiponectin. Cyclic-AMP is one of the most important second messengers regulating the secretion and expression of adiponectin. (2) The PI3-kinase-PDE3B-cAMP pathway mediates the effects of insulin on restoring β-agonist/cAMP-suppressed secretion and expression of adiponectin.