The Regulatory Effects Of Sterol Regulatory Element Binding Protein-1c On IRS-1in Skeletal Muscle

Objective:To study the regulatory effects of sterol regulatory element-binding protein-lc (SREBP-1c) on insulin receptor substrate1(IRS-1) in rat skeletal muscle cells.Methods:Primary rat skeletal muscle cells were obtained by mixed enzymatic digestion, cells were divided into the following4groups:control (C), control+Insulin (C+I), palmitic acid (PA), palmitic acid+Insulin (PA+I). L6cell line was induced to differentiation until myotubes could be observed, then divided into the following5groups:control (C), control+Insulin (C+I), palmitic acid (PA), palmitic acid+Insulin (PA+I), palmitic acid+Metformin (PA+MET).L6myotubes were transfected with adenoviral vectors expressing SREBP-1c with increasing multiplicity of infection (MOI), and divided into groups of GFP, MOI5, MOI50, MOI100, and MOI200. L6myotubes were transfected with SREBP-1c siRNA, and divided into groups of control, negative control siRNA, SREBP-1c siRNA. Skeletal muscle cells were identified by immunofluorescence method. Cells were stained with Oil Red O to display intracytoplasmic lipid. Western blotting and quantitative real-time (RT-PCR) were performed to observe the expressions of SREBP-1c, IRS-1, protein kinase B (Akt), mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK). ANOVA or LSD test were used for data analysis.Results:(1) Immunofluorescent staining shows more than90%of the cells are positively stained for Desmin actin, which proves that the obtained cells are skeletal muscle cells.Lipid droplet accumulation was examined by stained with Oil red O, after cells were incubated with PA for24hours.(2) Compared with C groups, the gene and protein levels of SREBP-lc in PA groups were increased significantly (all P<0.05), while the gene and protein expressions of IRS-1were decreased (all P<0.05), p-IRS-1(Ser636/639) protein levels were increased, p-IRS-1(Tyr608/612) and p-Akt (Ser473) protein levels were decreased (all P<0.05).(3) Compared with GFP group, the gene and protein levels of SREBP-1c were increased in a dose-dependent manner in MOI50,100and200groups(all P<0.05), while the gene and protein expression of IRS-1were decreased(all P<0.05). p-IRS-1(Tyr608/612) and p-Akt (Ser473) protein levels were both decreased significantly in MOI50and100groups(all P<0.05).(4) Compared with control and negative control siRNA groups, the gene nd proteinlevels of SREBP-1c were decreased in SREBP-1c siRNA group(all P<0.05), while the protein expression of IRS-1was increased (P<0.05).(5) Compared with PA group, p-AMPK (Thrl72),p-IRS-1(Tyr608/612) and p-Akt (Ser473) protein levels were decreased significantly in PA+I group and PA+MET group (all P<0.05), while the protein expression of p-mTOR (Ser2448) and SREBP-1c were decreased (all P<0.05).Conclusions:SREBP-lc can decrease IRS-1signaling pathway in skeletal muscle and play an important role in the occurrence of insulin resistance; and insulin and metformin could suppress mTOR-SREBP-1c signaling pathway in skeletal muscle and improve insulin resistance induced by lipid toxicity.