The regulatory effects of sterol regulatory element binding protein-1c on insulin receptor substrate-1 in skeletal muscle

Abstract

Objective To study the regulatory effects of sterol regulatory element-binding protein-1c (SREBP-1c) on insulin receptor substrate-1 (IRS-1) in rat skeletal muscle cells. Methods Primary rat skeletal muscle cells were obtained by mixed enzymatic digestion, cells were divided into the following 4 groups: control (C), control+ insulin (C+ I), palmitic acid (PA), palmitic acid+ insulin (PA+ I). L6 myotubes were transfected with adenoviral vectors expressing SREBP-1c with increasing multiplicity of infection (MOI), and divided into groups of green fluorescent protein (GFP), MOI 5, MOI 50, MOI 100 and MOI 200. L6 myotubes were transfected with SREBP-1c siRNA, and divided into groups of control, negative control siRNA, SREBP-1c siRNA.Western blotting and quantitative real-time polymerase chain reaction (RT-PCR) were performed to observe the expressions of SREBP-1c, IRS-1 and protein kinase B (Akt). Cells were stained with Oil Red O to display intracytoplasmic lipid. ANOVA or LSD test were used for data analysis. Results Compared with C and C+ I groups, the gene and protein levels of SREBP-1c in PA and PA+ I groups were increased significantly (2.72±0.08 vs 1.00±0.18, 3.02±0.19 vs 1.00±0.05, t=15.240, 18.289, all P<0.05), while the gene and protein expressions of IRS-1 were decreased (0.71±0.04 vs 1.00±0.05, 0.82±0.04 vs 1.00±0.04, t=-7.960, -6.052, all P<0.05), p-IRS-1 (Ser636/639) protein levels were increased, and p-Akt (Ser473) protein levels were decreased (t=20.987, -5.869, all P<0.05). Compared with GFP group, the gene and protein levels of SREBP-1c were increased in a dose-dependent manner in MOI 50, 100 and 200 groups(all P<0.05), while the gene and protein expression of IRS-1 were decreased. P-IRS-1 (Tyr608), and p-Akt (Ser473) protein levels were both decreased significantly in MOI 50 and 100 groups (all P<0.05). Compared with control and negative control siRNA groups, the gene and protein levels of SREBP-1c were decreased in SREBP-1c siRNA group(all P<0.05), while the protein expression of IRS-1 was increased (all P<0.05). Conclusion SREBP-1c can inhibit IRS-1 signaling pathway and play an important role in the development of insulin resistance in skeletal muscle cells. Key words: Sterol regulatory element-binding protein-1c; Insulin receptor substrate 1; Insulin resistance; Skeletal muscle