Insulin stimulated a concentration-dependent increase in protein synthesis in L6 myoblasts which was significant at 1 nM. This response was not prevented by the transcription inhibitor, actinomycin D. The protein kinase C (PKC) inhibitor, Ro-31-8220, and downregulation of PKC by prolonged incubation of cells with 12-O-tetradecanoylphorbol-13-acetate (TPA), had no effect on the ability of insulin to stimulate protein synthesis whilst completely blocking the response to TPA. In contrast, insulin failed to enhance protein synthesis significantly in the presence of either ibuprofen, a selective cyclooxygenase inhibitor or rapamycin, an inhibitor of the 70 kDa S6 kinase. When cell extracts were prepared and assayed for total myelin basic protein kinase activity, a stimulatory effect of insulin was not observed until the concentration approached 100-fold (i.e. 100 nM) that required to elicit increases in protein synthesis. Upon fractionation on a Mono-Q column, 100 nM insulin increased the activity of 3 peaks which phosphorylated myelin basic protein. Two of these peaks were identified as the 42 and 44 kDa forms of Mitogen Activated Protein (MAP) kinase by immunoblotting. In contrast, 1 nM insulin had no effect on the activity of these peaks. The data suggest that physiologically relevant concentrations of insulin do not stimulate translation in L6 cells through either PKC or the 42/44 kDa isoforms of MAP kinase and that this response is, at least in part, mediated through the activation of the 70 kDa S6 kinase by cyclooxygenase metabolites.
Abbreviations PLA2, phospholipase A2; PKC, protein kinase C; TPA, 12-O-tetradecanoyl-phorbol-13-acetate; PGF2α, prostaglandin F2α; IGF-1, Insulin-like Growth Factor-1; PMSF, phenylmethylsulphonoylfluoride; Ro-31-8220, protein kinase C inhibitor; MBP, Myelin Basin Protein; DMEM, Dulbecco's modified Eagles medium; EDL, Extensor Digitorum Longus; p70s6k, 70 kDa S6 kinase; p90rsk, 90 kDa ribosomal S6 kinase; MAP kinase, Mitogen Activated Protein kinase; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis;