title: Testosterone enables growth and hypertrophy in fusion impaired myoblasts that display myotube atrophy: deciphering the role of androgen and IGF-I receptors.
abstract: We have previously highlighted the ability of testosterone (T) to improve differentiation and myotube hypertrophy in fusion impaired myoblasts that display reduced myotube hypertrophy via multiple population doublings (PD) versus their parental controls (CON); an observation which is abrogated via PI3K/Akt inhibition (Deane et al. 2013). However, whether the most predominant molecular mechanism responsible for T induced hypertrophy occurs directly via androgen receptor or indirectly via IGF-IR/PI3K/Akt pathway is currently debated. PD and CON C2C12 muscle cells were exposed to low serum conditions in the presence or absence of T (100 nM) ± inhibitors of AR (flutamide/F, 40 μm) and IGF-IR (picropodophyllin/PPP, 150 nM) for 72 h and 7 days (early/late muscle differentiation respectively). T increased AR and Akt abundance, myogenin gene expression, and myotube hypertrophy, but not ERK1/2 activity in both CON and PD cell types. Akt activity was not increased significantly in either cell type with T. Testosterone was also unable to promote early differentiation in the presence of IGF-IR inhibitor (PPP) yet still able to promote appropriate later increases in myotube hypertrophy and AR abundance despite IGF-IR inhibition. The addition of the AR inhibitor powerfully attenuated all T induced increases in differentiation and myotube hypertrophy with corresponding reductions in AR abundance, phosphorylated Akt, ERK1/2 and gene expression of IGF-IR, myoD and myogenin with increases in myostatin mRNA in both cell types. Interestingly, despite basally reduced differentiation and myotube hypertrophy, PD cells showed larger T induced increases in AR abundance vs. CON cells, a response abrogated in the presence of AR but not IGF-IR inhibitors. Furthermore, T induced increases in Akt abundance were sustained despite the presence of IGF-IR inhibition in PD cells only. Importantly, flutamide alone reduced IGF-IR mRNA in both cell types across time points, with an observed reduction in activity of ERK and Akt, suggesting that IGF-IR was transcriptionally regulated by AR. However, where testosterone increased AR protein content there was no increases observed in IGF-IR gene expression. This suggested that sufficient AR was important to enable normal IGF-IR expression and downstream signalling, yet elevated levels of AR due to testosterone had no further effect on IGF-IR mRNA, despite testosterone increasing Akt abundance in the presence of IGF-IR inhibitor. In conclusion, testosterones ability to improve differentiation and myotube hypertrophy occurred predominately via increases in AR and Akt abundance in both CON and PD cells, with fusion impaired cells (PD) showing an increased responsiveness to T induced AR levels. Finally, T induced increases in myotube hypertrophy (but not early differentiation) occurred independently of upstream IGF-IR input, however it was apparent  that normal AR function in basal conditions was required for adequate IGF-IR gene expression and downstream ERK/Akt activity.
authors.affiliation: Stem Cells, Ageing & Molecular Physiology Unit, Research Institute for Sport and Exercise Sciences (RISES), School of Sport and Exercise Sciences, Liverpool John Moores University, Liverpool, UK.|Department of Neurobiology, Physiology and Behavior, University of California, Davis, CA, USA.
authors.forename: David C
authors.initials: DC
authors.lastname: Hughes
authors.affiliation: Stem Cells, Ageing & Molecular Physiology Unit, Research Institute for Sport and Exercise Sciences (RISES), School of Sport and Exercise Sciences, Liverpool John Moores University, Liverpool, UK.
authors.forename: Claire E
authors.initials: CE
authors.lastname: Stewart
authors.affiliation: Institute for Clinical Exercise and Health Science (ICEHS), University of the West of Scotland, Hamilton, Scotland, UK.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Sculthorpe
authors.affiliation: Stem Cells, Ageing & Molecular Physiology Unit, Research Institute for Sport and Exercise Sciences (RISES), School of Sport and Exercise Sciences, Liverpool John Moores University, Liverpool, UK.
authors.forename: Hannah F
authors.initials: HF
authors.lastname: Dugdale
authors.affiliation: Stem Cells, Ageing & Molecular Physiology Unit, Research Institute for Sport and Exercise Sciences (RISES), School of Sport and Exercise Sciences, Liverpool John Moores University, Liverpool, UK.
authors.forename: Farzad
authors.initials: F
authors.lastname: Yousefian
authors.affiliation: National Centre for Sport and Exercise Medicine, School of Sport, Exercise and Health Science, Loughborough University, Loughborough, UK.
authors.forename: Mark P
authors.initials: MP
authors.lastname: Lewis
authors.affiliation: Stem Cells, Ageing & Molecular Physiology Unit, Research Institute for Sport and Exercise Sciences (RISES), School of Sport and Exercise Sciences, Liverpool John Moores University, Liverpool, UK. a.sharples@ljmu.ac.uk.
authors.forename: Adam P
authors.initials: AP
authors.lastname: Sharples
mesh_terms: D000818:Animals; D002459:Cell Fusion; D002460:Cell Line; D049109:Cell Proliferation / Q000502:physiology; D006984:Hypertrophy; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000378:metabolism* / Q000473:pathology*; D032446:Myoblasts / Q000378:metabolism* / Q000473:pathology*; D017526:Receptor, IGF Type 1 / Q000378:metabolism*; D011944:Receptors, Androgen / Q000378:metabolism*; D013739:Testosterone / Q000378:metabolism*
chemical_list: D011944:Receptors, Androgen; D013739:Testosterone; D017526:Receptor, IGF Type 1
keywords: Aging; Akt; Androgens; IGF-I; Muscle; Satellite cell; Testosterone