Suppression of insulin-stimulated phosphatidylinositol 3-kinase activity by the β 3-adrenoceptor agonist CL316243 in rat adipocytes

Abstract

Insulin increased 2-deoxyglucose (2-DG) uptake via the translocation of glucose transporter (GLUT) 4 to the plasma membrane fraction in rat adipocytes. The stimulatory actions of insulin were accompanied by both an increase in the immunoreactive p85 subunit of phosphatidylinositol (PI) 3-kinase in the plasma membrane fractions and PI 3-kinase activation by tyrosine phosphorylation of the p85 subunit. The β 3-adrenoceptor agonist CL316243 (CL) suppressed all the insulin actions in adenosine deaminase (ADA)-treated cells, but was without effect in non-ADA-treated cells. The inhibitory effects of CL on GLUT 4 translocation and PI 3-kinase activation were abolished by the addition of N 6-phenylisopropyl adenosine. Cholera toxin treatment, which markedly increased intracellular cAMP levels, suppressed increases in the levels of GLUT 4 and PI 3-kinase in the plasma membrane fractions in response to insulin. In addition, dibutyryl (Bt 2) cAMP also impaired the activation of PI 3-kinase by insulin. These results indicated that CL suppressed insulin-stimulated glucose transport under conditions where cAMP levels were markedly increased (∼12-fold). The inhibitory actions of PI 3-kinase activation by insulin were exerted even when cAMP, 8-bromo-cAMP, or Bt 2 cAMP was added to immunoprecipitates of the p85 subunit of PI 3-kinase, after treating the cells with insulin. These results suggest that CL suppressed insulin-stimulated PI 3-kinase activity via a cAMP-dependent mechanism, at least in part, direct cAMP action in ADA-treated adipocytes, by which PI 3-kinase activation was inhibited, resulting in the decrease in GLUT 4 translocation and subsequent 2-DG uptake in response to insulin.