Evidence for Involvement of Protein Kinase C (PKC)- 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)-z in L6 myotubes, and the dependence of this acti- vation on phosphatidylinositol (PI) 3-kinase. We also evaluated a number of issues that are relevant to the question of whether diac- ylglycerol (DAG)-dependent PKCs or DAG-insensitive PKCs, such as PKC-z, are more likely to play a role in insulin-stimulated glucose transport in L6 myotubes and other insulin-sensitive cell types. We found that insulin increased the enzyme activity of immunoprecipi- table PKC-z in L6 myotubes, and this effect was blocked by PI 3- kinase inhibitors, wortmannin and LY294002; this suggested that PKC-z operates downstream of PI 3-kinase during insulin action. We also found that treatment of L6 myotubes with 5 mM tetradecanoyl phorbol-13-acetate (TPA) for 24 h led to 80 -100% losses of all DAG- dependent PKCs (a, b1, b2, d, e) and TPA-stimulated glucose transport (2-deoxyglucose uptake); in contrast, there was full retention of PKC-z, as well as insulin-stimulated glucose transport and translo- cation of GLUT4 and GLUT1 to the plasma membrane. Unlike what has been reported in BC3H-1 myocytes, TPA treatment did not elicit increases in PKCb2 messenger RNA or protein in L6 myotubes, and selective retention of this PKC isoform could not explain the retention of insulin effects on glucose transport after prolonged TPA treatment. Of further interest, TPA acutely activated membrane-associated PI 3-kinase in L6 myotubes, and acute effects of TPA on glucose trans- port were inhibited, not only by the PKC inhibitor, LY379196, but also by both wortmannin and LY294002; this suggested that DAG-sensi- tive PKCs activate glucose transport through cross-talk with phos- phatidylinositol (PI) 3-kinase, rather than directly through PKC. Also, the cell-permeable, myristoylated PKC-z pseudosubstrate in- hibited insulin-stimulated glucose transport both in non-down-reg- ulated and PKC-depleted (TPA-treated) L6 myotubes; thus, the PKC-z pseudosubstrate appeared to inhibit a protein kinase that is required for insulin-stimulated glucose transport but is distinct from DAG-sensitive PKCs. In keeping with the latter dissociation of DAG- sensitive PKCs and insulin-stimulated glucose transport, LY379196, which inhibits PKC-b (preferentially) and other DAG-sensitive PKCs at relatively low concentrations, inhibited insulin-stimulated glucose transport only at much higher concentrations, not only in L6 myo- tubes, but also in rat adipocytes, BC3H-1 myocytes, 3T3/L1 adipo- cytes and rat soleus muscles. Finally, stable and transient expression of a kinase-inactive PKC-z inhibited basal and insulin-stimulated glucose transport in L6 myotubes. Collectively, our findings suggest that, whereas PKC-z is a reasonable candidate to participate in in- sulin stimulation of glucose transport, DAG-sensitive PKCs are un- likely participants. (Endocrinology 138: 4721- 4731, 1997)