Constitutive Expression of Insulin Receptor Substrate (IRS)-1 Inhibits Myogenic Differentiation through Nuclear Exclusion of Foxo1 in L6 Myoblasts

Abstract

Insulin-like growth factors (IGFs) are well known to play essential roles in enhancement of myogenic differentiation. In this report we showed that initial IGF-I signal activation but long-term IGF-1 signal termination are required for myogenic differentiation. L6 myoblast stably transfected with myc-epitope tagged insulin receptor substrate-1, myc-IRS-1 (L6-mIRS1) was unable to differentiate into myotubes, indicating that IRS-1 constitutive expression inhibited myogenesis. To elucidate the molecular mechanisms underlying myogenic inhibition, IGF-I signaling was examined. IGF-I treatment of control L6 cells for 18 h resulted in a marked suppression of IGF-I stimulated IRS-1 association with the p85 PI 3-kinase and suppression of activation of Akt that correlated with a down regulation of IRS-1 protein. L6-mIRS1 cells, in contrast, had sustained high levels of IRS-1 protein following 18 h of IGF-I treatment with persistent p85 PI 3-kinase association with IRS-1, Akt phosphorylation and phosphorylation of the downstream Akt substrate, Foxo1. Consistent with Foxo1 phosphorylation, Foxo1 protein was excluded from the nuclei in L6-mIRS1 cells, whereas Foxo1 was localized in the nuclei in control L6 cells during induction of differentiation. In addition, L6 cells stably expressing a dominant-interfering form of Foxo1, Δ256Foxo1 (L6-Δ256Foxo1) were unable to differentiate into myotubes. Together, these data demonstrate that IGF-I regulation of Foxo1 nuclear localization is essential for the myogenic program in L6 cells but that persistent activation of IGF-1 signaling pathways results in a negative feedback to prevent myogenesis.

Developmental Stage, Muscle and Genetic Type Modify Muscle Transcriptome in Pigs: Effects on Gene Expression and Regulatory Factors Involved in Growth and Metabolism

“…Moreover, KLF1 was strongly upregulated in IBxDU pigs (25.61), which suggest an important role of this TRF. Other important TRFs identified in the performed analysis were FOS and FOXO1 , involved in muscle differentiation and metabolism [63], but also in the regulation of adipogenic genes (as PPARG ) expression and IMF accumulation [54, 62]. These TRFs were also previously identified in BF muscle of newborn piglets [30].…”

The AP-1 Complex Regulates Intracellular Localization of Insulin Receptor Substrate 1, Which Is Required for Insulin-Like Growth Factor I-Dependent Cell Proliferation

“…Retrovirus production and infection were performed as described previously (26). Infected cells were selected with G418 or puromycin.…”

“…DNA synthesis was measured as described previously (26). For cell proliferation assays, 4.0 ϫ 10 4 cells were grown in a 35-mm dish in DMEM supplemented with 1% fetal bovine serum (FBS) with or without 100 ng/ml of IGF-I.…”

Myoblasts With Higher IRS-1 Levels Are Eliminated From the Normal Cell Layer During Differentiation

“…L6 cells were maintained at 37 • C in a humidified 5% CO 2controlled atmosphere in DMEM supplemented with 10% FBS, 0.1% NaHCO 3 , 50 IU/mL penicillin, and 50 µg/mL streptomycin. L6 cell differentiation was induced as previously described (12). Passage number of cells used in experiments was 8∼15.…”

“…For example, IRS-1 knockdown C2C12 myoblasts had defects in myogenesis (11). On the other hand, we previously reported that IRS-1 overexpression inhibited myogenic differentiation in L6 myoblasts through continuous Foxo1 inhibition that might cause repression of MyHC at the late stage of differentiation (12).…”