CAJ | 학술논문

目的探讨固醇调节元件结合蛋白1c(SREBP-1c)对大鼠骨骼肌细胞胰岛素受体底物1(IRS-1)表达调控的影响.方法采用酶联合消化法取2～3 d SPF级雄性SD大鼠原代骨骼肌细胞,将原代细胞分为对照组(C)、对照+胰岛素组(C+I)、高脂组(PA)及高脂+胰岛素组(PA+I).将表达SREBP-1c腺病毒转染L6细胞,根据感染复数(MOI)分为含绿色荧光蛋白阴性载体(GFP)组、MOI值为5、50、100、200组.将靶基因为SREBP-1c的干扰RNA (siRNA)转染L6细胞,并分为空白对照组、阴性siRNA组及SREBP-1c siRNA组.Western blotting和实时定量聚合酶链反应(RT-PCR)检测SREBP-1c、IRS-1、蛋白激酶B(Akt)基因及蛋白表达,油红O染色法检测细胞内脂质沉积情况.多组资料比较采用方差分析,两两比较采用最小显著差异法.结果与C组相比,PA组SREBP-1c基因和蛋白水平升高(分别为2.72±0.08比1.00±0.18,3.02 ±0.19比1.00±0.05,t=15.240、18.289,均P＜0.05),IRS-1基因和蛋白水平降低(分别为0.71 ±0.04比1.00 ±0.05,0.82 ±0.04比1.00±0.04,t=-7.960、-6.052,均P＜0.05),丝氨酸磷酸化IRS-1蛋白表达升高,丝氨酸磷酸化Akt(p-Akt)蛋白表达下降(t=20.987、-5.869,均P＜0.05).与GFP组相比,MOI值为50、100和200组的SREBP-1c基因和蛋白表达呈剂量依赖性上升(均P＜0.05),IRS-1基因和蛋白表达水平呈剂量依赖性下降(均P＜0.05).与空白对照组和阴性siRNA组相比,SREBP-1c siRNA组SREBP-1c基因和蛋白水平降低,IRS-1蛋白表达升高(均P＜0.05).结论 SREBP-1c可抑制骨骼肌IRS-1胰岛素信号通路,参与肌细胞胰岛素抵抗的发生.
목적 탐토고순조절원건결합단백1c(SREBP-1c)대대서골격기세포이도소수체저물1(IRS-1)표체조공적영향.방법 채용매연합소화법취2～3 d SPF급웅성SD대서원대골격기세포,장원대세포분위대조조(C)、대조+이도소조(C+I)、고지조(PA)급고지+이도소조(PA+I).장표체SREBP-1c선병독전염L6세포,근거감염복수(MOI)분위함록색형광단백음성재체(GFP)조、MOI치위5、50、100、200조.장파기인위SREBP-1c적간우RNA (siRNA)전염L6세포,병분위공백대조조、음성siRNA조급SREBP-1c siRNA조.Western blotting화실시정량취합매련반응(RT-PCR)검측SREBP-1c、IRS-1、단백격매B(Akt)기인급단백표체,유홍O염색법검측세포내지질침적정황.다조자료비교채용방차분석,량량비교채용최소현저차이법.결과 여C조상비,PA조SREBP-1c기인화단백수평승고(분별위2.72±0.08비1.00±0.18,3.02 ±0.19비1.00±0.05,t=15.240、18.289,균P＜0.05),IRS-1기인화단백수평강저(분별위0.71 ±0.04비1.00 ±0.05,0.82 ±0.04비1.00±0.04,t=-7.960、-6.052,균P＜0.05),사안산린산화IRS-1단백표체승고,사안산린산화Akt(p-Akt)단백표체하강(t=20.987、-5.869,균P＜0.05).여GFP조상비,MOI치위50、100화200조적SREBP-1c기인화단백표체정제량의뢰성상승(균P＜0.05),IRS-1기인화단백표체수평정제량의뢰성하강(균P＜0.05).여공백대조조화음성siRNA조상비,SREBP-1c siRNA조SREBP-1c기인화단백수평강저,IRS-1단백표체승고(균P＜0.05).결론 SREBP-1c가억제골격기IRS-1이도소신호통로,삼여기세포이도소저항적발생.
Objective To study the regulatory effects of sterol regulatory element-binding protein-1 c (SREBP-1 c) 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-1 c 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-1 c siRNA group (all P ＜0.05),while the protein expression of IRS-1 was increased (all P ＜0.05).Conclusion SREBP1c can inhibit IRS-1 signaling pathway and play an important role in the development of insulin resistance in skeletal muscle cells.