REGULATION OF INSULIN-STIMULATED GLUCOSE-TRANSPORTER GLUT4 TRANSLOCATION AND AKT KINASE-ACTIVITY BY CERAMIDE

The sphingomyelin derivative ceramide is a signaling molecule implicat ed in numerous physiological events. Recently published reports indica te that ceramide levels are elevated in insulin-responsive tissues of diabetic animals and that agents which trigger ceramide production inh ibit insulin signaling. In the present series of studies, the short-ch ain ceramide analog C-2-ceramide inhibited insulin-stimulated glucose transport by similar to 50% in 3T3-L1 adipocytes, with similar reducti ons in hormone-stimulated translocation of the insulin-responsive gluc ose transporter (GLUT4) and insulin-responsive aminopeptidase. C-2-cer amide also inhibited phosphorylation and activation of Akt, a molecule proposed to mediate multiple insulin-stimulated metabolic events. C-2 -ceramide, at concentrations which antagonized activation of both gluc ose uptake and Akt, had no effect on the tyrosine phosphorylation of i nsulin receptor substrate 1 (IRS-1) or the amounts of p85 protein and phosphatidylinositol kinase activity that immunoprecipitated with anti -IRS-l or antiphosphotyrosine antibodies. Moreover, C-2-ceramide also inhibited stimulation of Akt by platelet-derived growth factor, an eve nt that is IRS-1 independent. C-2-ceramide did not inhibit insulin-sti mulated phosphorylation of mitogen-activated protein kinase or pp70 S6 -kinase, and it actually stimulated phosphorylation of the latter in t he absence of insulin. Various pharmacological agents, including the i mmunosuppressant rapamycin, the protein synthesis inhibitor cyclohexim ide, and several protein kinase C inhibitors, were without effect on c eramide's inhibition of Akt. These studies demonstrate ceramide's capa city to inhibit activation of Akt and imply that this is a mechanism o f antagonism of insulin-dependent physiological events, such as the pe ripheral activation of glucose transport and the suppression of apopto sis.