Impaired phosphorylation and insulin-stimulated translocation to the plasma membrane of protein kinase B/Akt in adipocytes from Type II diabetic subjects

Aims/hypothesis. To examine protein kinase B/Akt distribution and phosphory lation in response to insulin in different subcellular fractions of human f at cells from healthy subjects and subjects with Type II (noninsulin-depend ent) diabetes mellitus. Methods. We prepared subcellular fractions of plasma membranes (PM), low de nsity microsomes and cytosol and examined gene and protein expression as we ll as serine and threonine phosphorylation in response to insulin, Results. Protein kinase B/Akt mRNA as well as total protein kinase B/Akt pr otein in whole-cell lysate and cytosol were similar in both groups. Insulin increased protein kinase B/Akt translocation to the the plasma membrane ab out twofold [(p < 0.03) in non-diabetic cells but this effect was impaired in diabetic cells (similar to 30 %; p > 0.1)]. In both groups, protein kina se B/Akt threonine phosphorylation considerably increased in low density mi crosomes and cytosol whereas serine phosphorylation was predominant in the plasma membrane. Phosphatidylinositol-dependent kinase 1, which partially a ctivates and phosphorylates protein kinase B/Akt on the specific threonine site, was predominant in cytosol but it was also recovered in low density m icrosomes. Serine phosphorylation in response to insulin was considerably r educed (50-70%; p < 0.05) in diabetic cells but threonine phosphorylation w as less reduced (similar to 20%). Wortmannin inhibited these effects of ins ulin supporting a role for PI3-kinase activation. Conclusion/interpretation. Insulin stimulates a differential subcellular pa ttern of phosphorylation of protein kinase B/Akt. Furthermore, insulin-stim ulated translocation of protein kinase B/Akt to the plasma membrane, where serine phosphorylation and full activation occurs, is impaired in Type II d iabetes. Threonine phosphorylation was much less reduced. This discrepancy may be related to differential activation of phosphatidylinositol 3-kinase in the different subcellular compartments and phosphatidylinositol-dependen t kinase 1 having high affinity for phosphatidylinositol phosphate 3.