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

J Cell Physiol. 2001 Apr;187(1):96-108. doi: 10.1002/1097-4652(2001)9999:9999<::AID-JCP1058>3.0.CO;2-V.

Abstract

We have recently shown that insulin induced myogenesis in the mouse C2C12 skeletal muscle cell line by activation of phosphatidylinositol (PI) 3-kinase/p70S6-kinase and p38-mitogen-activated protein kinase (MAPK) and downregulation of p42/p44-MAPK. This study investigated the insulin-signaling pathways involved in mitogenesis, survival, and membrane ruffling in C2C12 myoblasts, a cellular system that besides IGF-I receptors, expressed a high number of functional insulin receptors. Insulin (10 nM) rapidly stimulated beta-chain insulin receptor and IRS-1 tyrosine phosphorylation, IRS-2 being poorly and SHC not phosphorylated at all. However, an association of SHC with IRS-1 was found under insulin stimulation. Insulin stimulated IRS-1 association with p85alpha leading to the activation of PI3-kinase, and, subsequently AKT and p70S6-kinases. Moreover, both p42/p44- and p38-MAPKs resulted in phosphorylation after insulin stimulation. Insulin treatment for 24 h produced mitogenesis, as demonstrated by the increase in ((3)H)-thymidine incorporation, DNA content, the expression of PCNA and cyclin D1 proteins, and the 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 rapamycin or by the PI3-kinase inhibitor LY294002) as well as by inhibition of p44/p42-MAPK with PD098059, but was not affected by inhibition of p38-MAPK. 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 laddering or by increase in the percentage of hypodiploid cells. Insulin rescued serum-deprived cells from apoptosis in an AKT-dependent manner, as demonstrated by the inhibition of AKT-activity by the use of LY294002 and ML-9, meanwhile neither inhibition of p70S6-kinase, nor MAPK affected insulin-induced survival. Finally, we evaluated the capacity of insulin to modulate actin cytoskeleton rearrangement. Insulin stimulation of myoblasts produced membrane ruffling and decreased actin stress fibers; this biological response being dependent of p38-MAPK, as demonstrated by the use of the p38-MAPK inhibitors SB203580 or PD169316, but independent of PI3-kinase and p42/p44-MAPK.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing*
  • Adaptor Proteins, Vesicular Transport*
  • Animals
  • Apoptosis / drug effects
  • Cell Division / drug effects
  • Cell Line
  • Cell Membrane / ultrastructure
  • Cell Survival / drug effects
  • Insulin / pharmacology*
  • Insulin Receptor Substrate Proteins
  • Intracellular Signaling Peptides and Proteins
  • MAP Kinase Signaling System*
  • Mice
  • Mitogen-Activated Protein Kinase 1 / physiology
  • Mitogen-Activated Protein Kinase 3
  • Mitogen-Activated Protein Kinases / physiology
  • Muscle, Skeletal / cytology*
  • Muscle, Skeletal / metabolism
  • Muscle, Skeletal / ultrastructure
  • Phosphatidylinositol 3-Kinases / physiology
  • Phosphoproteins / metabolism
  • Phosphotyrosine / metabolism
  • Protein-Serine-Threonine Kinases*
  • Proteins / metabolism
  • Proto-Oncogene Proteins / physiology
  • Proto-Oncogene Proteins c-akt
  • Receptor, Insulin / metabolism
  • Ribosomal Protein S6 Kinases / physiology
  • Shc Signaling Adaptor Proteins
  • Src Homology 2 Domain-Containing, Transforming Protein 1
  • p38 Mitogen-Activated Protein Kinases

Substances

  • Adaptor Proteins, Signal Transducing
  • Adaptor Proteins, Vesicular Transport
  • Insulin
  • Insulin Receptor Substrate Proteins
  • Intracellular Signaling Peptides and Proteins
  • Irs1 protein, mouse
  • Irs2 protein, mouse
  • Phosphoproteins
  • Proteins
  • Proto-Oncogene Proteins
  • Shc Signaling Adaptor Proteins
  • Shc1 protein, mouse
  • Src Homology 2 Domain-Containing, Transforming Protein 1
  • Phosphotyrosine
  • Phosphatidylinositol 3-Kinases
  • Receptor, Insulin
  • Protein-Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-akt
  • Ribosomal Protein S6 Kinases
  • Mitogen-Activated Protein Kinase 1
  • Mitogen-Activated Protein Kinase 3
  • Mitogen-Activated Protein Kinases
  • p38 Mitogen-Activated Protein Kinases