Insulin signaling leading to proliferation, survival, and membrane ruffling in C2C12 myoblasts

We have recently shown that insulin induced myogenesis in the mouse C2C12 s keletal muscle cell line by activation of phosphatidylinositol (PI) 3-kinas e/p70S6-kinase and p38-mitogen-activated protein kinase (MAPK) and downregu lation of p42/p44-MAPK. This study investigated the insulin-signaling pathw ays involved in mitogenesis, survival, and membrane ruffling in C2C12 myobl asts, a cellular system that besides IGF-I receptors, expressed a high numb er of functional insulin receptors. Insulin (10nM) rapidly stimulated beta -chain insulin receptor and IRS-1 tyrosine phosphorylation, IRS-2 being poo rly and SHC not phosphorylated at all. However, an association of SHC with IRS-1 was found under insulin stimulation. Insulin stimulated IRS-1 associa tion with p85 alpha leading to the activation of P13-kinase, and, subsequen tly AKT and p70S6-kinases. Moreover, both p42/p44- and p38-MAPKs resulted i n phosphorylation after insulin stimulation. Insulin treatment for 24 h pro duced mitogenesis, as demonstrated by the increase in (H-3)thymidine incorp oration, DNA content, the expression of PCNA and cyclin D1 proteins, and th e proportion of cells in S + G2/M phases of the cell cycle. This mitogenic effect of insulin was precluded by inhibition of p70S6-kinase (either by ra pamycin or by the P13-kinase inhibitor LY294002) as well as by inhibition o f p44/p42-MAPK with PD098059, but was not affected by inhibition of p38-MAP K. Serum deprivation of C2C12 myoblasts resulted in growth arrest at the GO /G1 phases of the cell cycle and apoptosis, as detected either by DNA ladde ring or by increase in the percentage of hypodiploid cells. Insulin rescued serum-deprived cells from apoptosis in an AKT-dependent manner, as demonst rated by the inhibition of AKT-activity by the use of LY294002 and ML-9, me anwhile neither inhibition of p70S6-kinase, nor MAPK affected insulin-induc ed survival. Finally, we evaluated the capacity of insulin to modulate acti n cytoskeleton rearrangement. Insulin stimulation of myoblasts produced mem brane ruffling and decreased actin stress fibers; this biological response being dependent of p38-MAPK, as demonstrated by the use of the p38-MAPK inh ibitors SB203580 or PD169316, but independent of P13-kinase and p42/p44-MAP K.). (C) 2001 Wiley-Liss. Inc.