Evidence that protein kinase C and mitogen activated protein kinase are not involved in the mechanism by which insulin stimulates translation in L6 myoblasts.
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.
Thompson
M G
MG
Rowett Research Institute, Aberdeen, UK.
Pascal
M
M
Mackie
S C
SC
Thom
A
A
Morrison
K S
KS
Backwell
F R
FR
Palmer
R M
RM
D000595:Amino Acid Sequence; D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002460:Cell Line; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D015536:Down-Regulation / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D038362:Glycogen Synthase Kinase 3; D007052:Ibuprofen / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology*; D008969:Molecular Sequence Data; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000378:metabolism*; D011090:Polyenes / Q000494:pharmacology; D014176:Protein Biosynthesis / Q000187:drug effects*; D011493:Protein Kinase C / Q000378:metabolism*; D020123:Sirolimus; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000935:Antifungal Agents; D016861:Cyclooxygenase Inhibitors; D004791:Enzyme Inhibitors; D007211:Indoles; D007328:Insulin; D011090:Polyenes; D003609:Dactinomycin; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D038362:Glycogen Synthase Kinase 3; D013755:Tetradecanoylphorbol Acetate; D020123:Sirolimus; C064758:Ro 31-8220; D007052:Ibuprofen