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Insulin signaling leading to proliferation, survival, and membrane ruffling in C2C12 myoblasts
1Departmento de Bioquimica y Biologia Molecular, Facultad de Farmacia, Universidad Complutense, 28040-Madrid, Spain.
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.
Insights
Insulin promotes skeletal muscle cell growth and survival through PI3-kinase/AKT and p42/p44-MAPK pathways. It also triggers actin cytoskeleton changes via p38-MAPK signaling.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Insulin and IGF-I signaling pathways regulate cellular processes in skeletal muscle.
- C2C12 myoblasts express functional insulin receptors and are a relevant model for studying insulin's effects.
Purpose of the Study:
- To investigate insulin-signaling pathways governing mitogenesis, survival, and membrane ruffling in C2C12 myoblasts.
- To elucidate the specific roles of PI3-kinase, AKT, p70S6-kinase, and various MAPKs in insulin-mediated cellular responses.
Main Methods:
- Utilized C2C12 myoblasts expressing functional insulin receptors.
- Employed Western blotting to assess protein phosphorylation and interactions.
- Applied pharmacological inhibitors (e.g., rapamycin, LY294002, PD098059, SB203580) to dissect signaling pathways.
- Quantified cell proliferation, cell cycle progression, apoptosis, and actin cytoskeleton dynamics.
Main Results:
- Insulin rapidly activated the PI3-kinase/AKT/p70S6-kinase pathway and p42/p44-MAPK, while p38-MAPK was involved in membrane ruffling.
- Insulin-induced mitogenesis was dependent on p70S6-kinase and p42/p44-MAPK, but not p38-MAPK.
- Insulin promoted cell survival from serum deprivation in an AKT-dependent manner.
- Insulin stimulated membrane ruffling and decreased actin stress fibers, a process dependent on p38-MAPK.
Conclusions:
- Insulin exerts pleiotropic effects on C2C12 myoblasts, regulating proliferation, survival, and cytoskeletal organization through distinct signaling cascades.
- PI3-kinase/AKT/p70S6-kinase and p42/p44-MAPK pathways are crucial for insulin-driven mitogenesis.
- AKT signaling mediates insulin's anti-apoptotic effects.
- p38-MAPK plays a specific role in insulin-induced actin cytoskeleton remodeling.