Insulin produces myogenesis in C2C12 myoblasts by induction of NF-kappa B and downregulation of AP-1 activities

In the present study, we have examined the insulin-signaling pathways invol ved in myogenesis in mouse C2C12 skeletal muscle cell line, a cellular syst em that expresses high number of high affinity insulin receptors. Insulin ( 50 nM) rapidly (5 min) stimulated beta -chain insulin receptor, activated t he phosphatidylnositol (PI 3-kinase/Akt/p70S6-kinase signaling pathway, as well as phosphorylated both p44/p42- and p38-mitogen-activated protein kina ses (MAPKs). Preconfluent cells were differentiated in a serum-free medium in response to 50 nM insulin for 72 h, as revealed by the formation of mult inucleated myotubes and the induction of the creatine kinase activity. This differentiation process was also monitored by the inhibition of the PCNA c ontent and induction of the cell cycle inhibitor p21. Furthermore, insulin induced nuclear factor-kappaB (NF-kappaB) DNA binding activity and down-reg ulated activating protein-1 (AP-1) DNA binding activity throughout the diff erentiation process. The use of specific inhibitors of the insulin-signalin g pathways indicated that myogenesis was precluded by treatment for 72 h wi th LY294002 tan inhibitor of PI 3-kinase), rapamycin (a p70S6-kinase blocke r), and SB203580 or PD169316 (p38-MAPK inhibitors). These inhibitors abolis hed insulin induction of NF-betaB DNA binding activity and kappaB-chloramph enicol acetyltransferase (CAT) promoter activity, maintaining expressed cyt osolic I kappaB-alpha protein, and increased AP-l DNA binding activity and TRE-CAT promoter activity. These data suggest that insulin induces myogenes is in C2C12 through PI 3-kinase/ p70S6-kinase and p38-MAPK pathways, the si gnaling through p44/p42-MAPK being inhibited. J. Cell. Physiol. 186:82-94, 2001. (C) 2001 Wiley-Liss, Inc.