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FOXO1 and GSK-3β Are Main Targets of Insulin-Mediated Myogenesis in C2C12 Muscle Cells.

ABSTRACT: Myogenesis and muscle hypertrophy account for muscle growth and adaptation to work overload, respectively. In adults, insulin and insulin-like growth factor 1 stimulate muscle growth, although their links with cellular energy homeostasis are not fully explained. Insulin plays critical role in the control of mitochondrial activity in skeletal muscle cells, and mitochondria are essential for insulin action. The aim of this study was to elucidate molecular mechanism(s) involved in mitochondrial control of insulin-dependent myogenesis. The effects of several metabolic inhibitors (LY294002, PD98059, SB216763, LiCl, rotenone, oligomycin) on the differentiation of C2C12 myoblasts in culture were examined in the short-term (hours) and long-term (days) experiments. Muscle cell viability and mitogenicity were monitored and confronted with the activities of selected genes and proteins expression. These indices focus on the roles of insulin, glycogen synthase kinase 3 beta (GSK-3β) and forkhead box protein O1 (FOXO1) on myogenesis using a combination of treatments and inhibitors. Long-term insulin (10 nM) treatment in "normoglycemic" conditions led to increased myogenin expression and accelerated myogenesis in C2C12 cells. Insulin-dependent myogenesis was accompanied by the rise of mtTFA, MtSSB, Mfn2, and mitochondrially encoded Cox-1 gene expressions and elevated levels of proteins which control functions of mitochondria (kinase--PKB/AKT, mitofusin 2 protein--Mfn-2). Insulin, via the phosphatidylinositol 3-kinase (PI3-K)/AKT-dependent pathway reduced transcription factor FOXO1 activity and altered GSK-3β phosphorylation status. Once FOXO1 and GSK-3β activities were inhibited the rise in Cox-1 gene action and nuclear encoded cytochrome c oxidase subunit IV (COX IV) expressions were observed, even though some mRNA and protein results varied. In contrast to SB216763, LiCl markedly elevated Mfn2 and COX IV protein expression levels when given together with insulin. Thus, inhibition of GSK-3β activity by insulin alone or together with LiCl raised the expression of genes and some proteins central to the metabolic activity of mitochondria resulting in higher ATP synthesis and accelerated myogenesis. The results of this study indicate that there are at least two main targets in insulin-mediated myogenesis: notably FOXO1 and GSK-3β both playing apparent negative role in muscle fiber formation.

SUBMITTER: Litwiniuk A

PROVIDER: S-EPMC4718532 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:IntroductionChronic HCV infection results in insulin resistance (IR) through interaction of pathways for glucose homeostasis, insulin signaling, and autophagy. Not known is how soon the pathways are activated, and how IR is related to the signals generated by catabolic and anabolic conditions occurring in infected cells. We have extended our studies to a cell culture system mimicking acute infection and to downstream pathways involving energy-sensor AMPK and nutrient-sensor mTOR that are active in catabolic and anabolic processes within the infected cells.MethodsHuh7 liver cells were infected with HCV. We performed proteomics analysis of key proteins in infected cells by western blotting and IP experiments, with or without interferon-α exposure.ResultsWe show that IRS-1 Ser312, Beclin-1, protein conjugate Atg12-Atg5 or GS Ser641 are upregulated early in infection by activating the same pathways utilized for persistent infection; Bcl-XL, an inhibitor of both autophagy and apoptosis, is present in a core-complex with IRS-1 Ser312 and Beclin-1 during progression of IR; AMPK level remains the same in infected cells where it is activated by phosphorylation at Thr172 concomitant with increased autophagy, a hallmark of catabolic conditions; an mTOR level that promotes anabolism is increased rather than decreased under an expanded autophagy; hypophosphorylation of translational repressor 4E-BP1 downstream of mTOR is suggestive of reduced protein synthesis; and β-catenin is upregulated but not phosphorylated suggesting indirectly our previous contention that its kinase, GSK-3β, is mostly in an inactive state.Discussion/conclusionWe report that in the development of IR following chronic infection, anabolic and catabolic pathways are activated early, and the metabolic interaction occurs possibly in a core-complex with IRS-1 Ser312, Beclin-1 and autophagy inhibitor BcL-XL. Induction of autophagy is usually controlled by a two-edged mechanism acting in opposition under anabolic and catabolic conditions by AMPK/mTOR/4E-BP1 pathways with GSK-3β mediated feed-back loops. However, we observed that an up-regulation of mTOR along with an up-regulation of AMPK caused by HCV infection is a deviation from the normal scenario described above which might be of therapeutic interest.

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FOXO1 and GSK-3β Are Main Targets of Insulin-Mediated Myogenesis in C2C12 Muscle Cells.

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