EFFECTS OF INSULIN ON PROTEIN-KINASE-C (PKC) IN HIRC-B CELLS - SPECIFIC ACTIVATION OF PKC-EPSILON AND ITS RESISTANCE TO PHORBOL ESTER-INDUCED DOWN-REGULATION

We evaluated the role of protein kinase-C (PKC) during insulin action in HIRC-B cells. Insulin provoked rapid increases in 1) diacylglycerol ; 2) translocation of PKC epsilon, but not PKC alpha, PKC delta, or PK C zeta, from the cytosol to the membrane fraction; 3) membrane PKC enz yme activity; and 4) phosphorylation of immunoprecipitable 80-kilodalt on (kDa) myristylated alanine-rich C-kinase substrate (MARCKS) protein and heat-stable 80-kDa protein (also probably MARCKS). Phorbol esters stimulated the translocation of PKC alpha and PKC delta as well as PK C epsilon, but not PKC zeta. The effects of phorbol esters on 80-kDa M ARCKS phosphorylation were approximately 4 times as strong as those of insulin. Treatment of HIRC-B cells with phorbol esters for 20-24 h re sulted in complete loss of immunoreactive PKC alpha and PKC delta in c ytosol and membrane fractions, but substantial amounts of PKC epsilon were persistently translocated to the membrane fraction of downregulat ed cells. This persistently translocated, residual PKC epsilon in down regulated cells was associated with increased basal hexose uptake, but this was not due to PKC activation, as it was not inhibited by the PK C inhibitor, RO 31-8220. Acute insulin treatment, on the other hand, i ncreased hexose uptake in down-regulated cells, and this insulin-stimu lated uptake was inhibited by RO 31-8220 in down-regulated cells as we ll as in nondown-regulated cells. Insulin also stimulated the phosphor ylation of the heat-stable 80-kDa protein in down-regulated cells, sug gesting that the residual PKC epsilon in these cells can be activated by insulin.