固醇调节元件结合蛋白1c对骨骼肌细胞胰岛素受体底物1表达调控的影响

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

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.

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.