| Objective: To study the regulatory effects of insulin on sterol regulatory element bingding protein 1c(SREBP-1c) in skeletal muscle cells.Methods: 30 SD male rats(140-160g) were induced to be insulin resistance by fed with high-fat diet for 12 weeks and then were divided into 3 groups randomly: negative control group(NC), high-fat diet group(HF), high-fat diet treated with metformin group(HF+MET). Then IPGTT was done at week 4, 8, 10. The rats were killed and skeletal muscle tissue collected at week 10. Rat L6 muscle cells were differentiated into myotubes, which were divided into negative control group(NC), palmitic acid group(PA), then treated with100 n M insulin for 1h, 2h, 3h, 4h, 5h, 6h, 9h, 12h; or treated with 1n M, 10 n M, 100 n M insulin for 12 h, respectively. Rat L6 myotubes were differentiated into the following 8 groups: NC(not given drugs as control), Ins(treated with insulin), Ins+Wort(treated with insulin plus wortmannin), Ins+CC(treated with insulin plus compound C), PA(given palmic acid), PA+Ins(treated with insulin under given plamitic acid), PA+Ins+Wort(treated with insulin plus wortmannin under given plamitic acid), PA+Ins+CC(treated with insulin plus compound C under given plamitic acid). Quantitative real-time(RT-PCR) and Western blot were performed to observe the expressions of SREBP-1c, insulin receptor substrate 1(IRS-1), protein kinase B(AKT), AMP-activated protein kinase(AMPK), mammalian target of rapamycin(m TOR). ANOVA or LSD test were used for data analysis.Results:(1) Compared with NC groups, the gene and protein levels of IRS-1 in HF groups were significantly decreased(P<0.05), while the gene and protein levels of SREBP-1c were significantly increased(P<0.05), p-IRS-1(Tyr608/612) protein levels were decreased(P<0.05). Compared with HF groups, the gene and protein levels of IRS-1 in HF+MET groups were increased(P<0.05), while the gene and protein levels of SREBP-1c were decreased(P < 0.05), p-IRS-1(Tyr608/612) protein levels were increased(P<0.05).(2) Compared with NC group, the gene and protein levels of SREBP-1c in insulin treated groups were increased in a time-dependent manner(P<0.05), and the protein levels of p-AKT(Ser473) were increased(P<0.05). Compared with PA group, the gene levels of SREBP-1c in PA plus insulin groups were first increased and then decreased(P<0.05), while the protein levels of SREBP-1c were decreased in a time-dependent manner(P<0.05), the protein levels of p-AKT(Ser473) were increased(P<0.05).(3) Compared with NC group, the protein levels of SREBP-1c and p-AKT(Ser473) in 100 n M insulin treated group were increased(P<0.05), while the protein levels of pAMPK(Thr172) in 1n M, 10 n M, 100 n M insulin treated groups were all increased(P<0.05). Compared with PA group, the protein levels of p-AKT(Ser473) in 10 n M and 100 n M insulin treated groups were increased(P<0.05),while in 1n M, 10 n M, 100 n M insulin treated groups, the protein levels of p-AMPK(Thr172) were increased(P<0.05), and the protein levels of SREBP-1c were decreased(P<0.05).(4) Compared with NC group, the protein levels of SERBP-1c, p-AKT(Ser473), p-AKT(Tyr308), p-AMPK(Thr172), p-m TOR(Ser2448) were increased in Ins group(P<0.05). Compared with Ins group, the protein levels of SREBP-1c were decreased in Ins+Wortgroup(P<0.05). Compared with PA group, the protein levels of SREBP-1c, p-mTOR(Ser2448) were decreased in PA+Ins group(P<0.05), while the protein levels of p-AKT(Ser473), p-AKT(Tyr308), p-AMPK(Thr172) increased(P<0.05). Compared with PA+Ins group, the protein levels of SREBP-1c were increased in PA+Ins+CC group(P<0.05).Conclusions: Metformin may play a role in the alleviating of insulin resistance(IR) in high-fat diet induced IR rats through upregulating IRS-1 and downregulating SREBP-1c. Under physiological condition of skeletal muscle, the effect of insulin on the upregulation of SREBP-1c may mainly work through AKT-m TOR pathway. Under PA induced IR condition, the effect of insulin on the downregulation of SREBP-1c may work mainly through AMPK-m TOR pathway. |
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