title: 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.
authors.affiliation: Department of Neuroendocrinology, Centre of Postgraduate Medical Education, Marymoncka 99/103, 01-813, Warsaw, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Litwiniuk
authors.affiliation: Department of Molecular and Cellular Neurobiology, Laboratory of Neurobiology, Nencki Institute of Experimental Biology PAS, Pasteura 3, 02-093, Warsaw, Poland.
authors.forename: Barbara
authors.initials: B
authors.lastname: Pijet
authors.affiliation: Department of Dermatology, Medical University of Warsaw, Koszykowa 82A, 02-008, Warsaw, Poland.
authors.forename: Maja
authors.initials: M
authors.lastname: Pijet-Kucicka
authors.affiliation: Department of Physiological Sciences, Warsaw University of Life Sciences-SGGW, Nowoursynowska 159, 02-776, Warsaw, Poland.
authors.forename: Małgorzata
authors.initials: M
authors.lastname: Gajewska
authors.affiliation: Department of Physiological Sciences, Warsaw University of Life Sciences-SGGW, Nowoursynowska 159, 02-776, Warsaw, Poland.|Electron Microscopy Platform, Mossakowski Medical Research Centre PAS, Pawińskiego 5, 02-106, Warsaw, Poland.
authors.forename: Beata
authors.initials: B
authors.lastname: Pająk
authors.affiliation: Department of Physiological Sciences, Warsaw University of Life Sciences-SGGW, Nowoursynowska 159, 02-776, Warsaw, Poland.|Electron Microscopy Platform, Mossakowski Medical Research Centre PAS, Pawińskiego 5, 02-106, Warsaw, Poland.
authors.forename: Arkadiusz
authors.initials: A
authors.lastname: Orzechowski
mesh_terms: D000818:Animals; D002460:Cell Line; D003576:Electron Transport Complex IV / Q000235:genetics / Q000378:metabolism; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors / Q000235:genetics / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000235:genetics / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D007328:Insulin / Q000378:metabolism*; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D032342:Muscle Cells / Q000166:cytology / Q000378:metabolism*; D024510:Muscle Development; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D015290:Second Messenger Systems
chemical_list: D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; C467200:Foxo1 protein, mouse; D007328:Insulin; D003576:Electron Transport Complex IV; D019869:Phosphatidylinositol 3-Kinases; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; D051057:Proto-Oncogene Proteins c-akt; D038362:Glycogen Synthase Kinase 3; D020558:GTP Phosphohydrolases; C488751:Mfn2 protein, mouse