EVIDENCE FOR INVOLVEMENT OF PROTEIN-KINASE-C (PKC)-ZETA AND NONINVOLVEMENT OF DIACYLGLYCEROL-SENSITIVE PKCS IN INSULIN-STIMULATED GLUCOSE-TRANSPORT IN L6 MYOTUBES

We examined the question of whether insulin activates protein kinase C (PKC)-zeta in L6 myotubes, and the dependence of this activation on p hosphatidylinositol (PI) 3-kinase. We also evaluated a number of issue s that are relevant to the question of whether diacylglycerol (DAG)-de pendent PKCs or DAG-insensitive PKCs, such as PKC-zeta, are more likel y to play a role in insulin-stimulated glucose transport in L6 myotube s and other insulin-sensitive cell types. We found that insulin increa sed the enzyme activity of immunoprecipitable PKC-zeta in L6 myotubes, and this effect was blocked by PI 3-kinase inhibitors, wortmannin and LY294002; this suggested that PKC-zeta operates downstream of PI 3-ki nase during insulin action. We also found that treatment of L6 myotube s with 5 mu M tetradecanoyl phorbol-13-acetate (TPA) for 24 h led to 8 0-100% losses of all DAG-dependent PKCs (alpha, beta(1), beta(2), delt a, epsilon) and TPA-stimulated glucose transport (2-deoxyglucose uptak e); in contrast, there was full retention of PKC-zeta, as well as insu lin-stimulated glucose transport and translocation of GLUT4 and GLUT1 to the plasma membrane. Unlike what has been reported in BC3H-1 myocyt es, TPA treatment did not elicit increases in PKC beta 2 messenger RNA or protein in L6 myotubes, and selective retention of this PKC isofor m could not explain the retention of insulin effects on glucose transp ort after prolonged TPA treatment. Of further interest, TPA acutely ac tivated membrane-associated PI 3-kinase in L6 myotubes, and acute effe cts of TPA on glucose transport were inhibited, not only by the PKC in hibitor, LY379196, but also by both wortmannin and LY294002; this sugg ested that DAG-sensitive PKCs activate glucose transport through cross -talk with phosphatidylinositol (PI) 3-kinase, rather than directly th rough PKC. Also, the cell-permeable, myristoylated PKC-zeta pseudosubs trate inhibited insulin-stimulated glucose transport both in non-down- regulated and PKC-depleted (TPA-treated) L6 myotubes; thus, the PKC-ze ta pseudosubstrate appeared to inhibit a protein kinase that is requir ed for insulin-stimulated glucose transport but is distinct from DAG-s ensitive PKCs. In keeping with the latter dissociation of DAG-sensitiv e PKCs and insulin-stimulated glucose transport, LY379196, which inhib its PKC-beta (preferentially) and other DAG-sensitive PKCs at relative ly low concentrations, inhibited insulin-stimulated glucose transport only at much higher concentrations, not only in L6 myotubes, but also in rat adipocytes, BC3H-1 myocytes, 3T3/L1 adipocytes and rat soleus m uscles. Finally, stable and transient expression of a kinase-inactive PKC-zeta inhibited basal and insulin-stimulated glucose transport in L 6 myotubes. Collectively, our findings suggest that, whereas PKC-zeta is a reasonable candidate to participate in insulin stimulation of glu cose transport, DAG-sensitive PKCs are unlikely participants.