CD36基因缺失通过上调PTP1B基因表达降低小鼠肌肉胰岛素敏感性

CD36 gene deletion reduces muscle insulin sensitivity in mice by up-regulating PTP1B expression

目的探讨在正常饮食状态下,CD36基因缺失对小鼠肌肉胰岛素信号通路的影响及作用机制。方法野生型小鼠(WT)和CD36基因敲除小鼠(CD36^(-/-))给予正常饮食喂养14周(n=12)。小鼠禁食4 h,腹腔注射胰岛素(1 U/kg)进行胰岛素耐量实验。Real-timePCR检测小鼠肌肉胰岛素受体(IR)、胰岛素受体底物1/2(IRS1/2)、蛋白酪氨酸磷酸酶1B(PTP1B)基因表达。Western blot检测小鼠肌肉蛋白激酶B(AKT)、IR、IRS1/2和PTP1B的蛋白表达。免疫共沉淀(Co-IP)检测肌肉IR和IRS1的酪氨酸磷酸化程度。染色质免疫共沉淀(ChIP)技术检测肌肉PTP1B启动子组蛋白乙酰化水平。结果在正常饮食状态下,与WT小鼠相比,CD36^(-/-)小鼠的胰岛素耐量显著增强(P<0.05),血清胰岛素浓度升高(P<0.01),胰岛素抵抗指数(HOMA-IR)升高(P<0.05)。在肌肉组织中,CD36^(-/-)小鼠与WT小鼠相比,p-AKT/AKT蛋白表达比值显著降低(P<0.01)。Real-timePCR和Westernblot结果表明,肌肉组织IR,IRS1,IRS2的mRNA和蛋白水平在WT和CD36^(-/-)小鼠间无显著差异(P>0.05)。Co-IP实验显示IR和IRS1的酪氨酸磷酸化水平在CD36^(-/-)小鼠中显著降低(P<0.05)。CD36^(-/-)小鼠肌肉中PTP1B mRNA和蛋白表达均高于WT小鼠(P<0.05),ChIP实验显示PTP1B基因启动子的组蛋白乙酰化水平显著升高(P<0.01)。腹腔注射PTP1B的抑制剂可改善CD36^(-/-)小鼠的胰岛素敏感性。结论在正常饮食条件下,CD36基因对于维持生理肌肉胰岛素敏感性十分重要,小鼠CD36基因缺失通过上调PTP1B基因表达,诱导IR、IRS1去酪氨酸磷酸化而使肌肉胰岛素信号传导受损。

Objective To investigate the effect CD36 deficiency on muscle insulin signaling in mice fed a normal-fat diet and explore the possible mechanism.Methods Wild-type(WT)mice and systemic CD36 knockout(CD36^(-/-))mice with normal feeding for 14 weeks(n=12)were subjected to insulin tolerance test(ITT)after intraperitoneal injection with insulin(1 U/kg).Real-time PCR was used to detect the mRNA expressions of insulin receptor(IR),insulin receptor substrate 1/2(IRS1/2)and protein tyrosine phosphatase 1B(PTP1B),and Western blotting was performed to detect the protein expressions of AKT,IR,IRS1/2 and PTP1B in the muscle tissues of the mice.Tyrosine phosphorylation of IR and IRS1 and histone acetylation of PTP1B promoter in muscle tissues were detected using co-immunoprecipitation(Co-IP)and chromatin immunoprecipitation(ChIP),respectively.Results CD36^(-/-)mice showed significantly lowered insulin sensitivity with obviously decreased area under the insulin tolerance curve in comparison with the WT mice(P<0.05).CD36^(-/-)mice also had significantly higher serum insulin concentration and HOMA-IR than WT mice(P<0.05).Western blotting showed that the p-AKT/AKT ratio in the muscle tissues was significantly decreased in CD36^(-/-)mice as compared with the WT mice(P<0.01).No significant differences were found in mRNA and protein levels of IR,IRS1 and IRS2 in the muscle tissues between WT and CD36^(-/-)mice(P>0.05).In the muscle tissue of CD36^(-/-)mice,tyrosine phosphorylation levels of IR and IRS1 were significantly decreased(P<0.05),and the mRNA and protein levels of PTP1B(P<0.05)and histone acetylation level of PTP1B promoters(P<0.01)were significantly increased as compared with those in the WT mice.Intraperitoneal injection of claramine,a PTP1B inhibitor,effectively improved the impairment of insulin sensitivity in CD36^(-/-)mice.Conclusion CD36 is essential for maintaining muscle insulin sensitivity under physiological conditions,and CD36 gene deletion in mice causes impaired insulin sensitivity by up-regulating muscle PTP1B expression,which results in detyrosine phosphorylation of IR and IRS1.