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目的探讨3T3-L1脂肪细胞水通道蛋白7(AQP7)的表达对胰岛素信号通路的影响,寻求改善胰岛素抵抗的新途径。方法体外培养3T3-L1脂肪细胞,以油红O染色鉴定分化成熟的脂肪细胞。以肿瘤坏死因子-α(TNF-α)处理脂肪细胞,检测葡萄糖代谢能力以及培养基中甘油浓度的变化;同时分别用荧光定量聚合酶链反应(Real-time PCR)和蛋白免疫印迹法(Western Blot)分析AQP7的表达水平。以腺病毒为载体高表达AQP7,检测胰岛素信号通路上胰岛素受体底物1(IRS-1)、蛋白激酶B(PKB)及其磷酸化蛋白水平的改变,从而阐明AQP7表达与胰岛素抵抗的关系。用SPSS 17.0软件进行统计学分析,两组计量资料的比较用独立样本t检验,多组计量资料的比较用单因素方差分析。结果 TNF-α作用分化成熟的脂肪细胞24 h构建胰岛素抵抗模型,与空白对照组比较,葡萄糖代谢水平下降50%,甘油浓度减少35%,而AQP7 m RNA及蛋白水平分别下调了75%和28%,差异均有统计学意义(P<0.01,P<0.05)。在胰岛素抵抗状态下,胰岛素刺激下的PKB磷酸化蛋白(p-PKB)、IRS-1酪氨酸632磷酸化蛋白(p-tyr632)及IRS-1总蛋白表达受到抑制,而IRS-1丝氨酸307磷酸化蛋白(p-ser307)异常升高,差异均有统计学意义(P<0.05)。以腺病毒为载体转染脂肪细胞高表达AQP7,随着AQP7表达上调,可逆转TNF-α的这些作用。结论 AQP7可能参与胰岛素抵抗的分子机制,高表达AQP7对改善胰岛素抵抗的基因治疗有一定的指导意义。
Objective To investigate the effect of AQP7 expression on insulin signaling pathway in 3T3-L1 adipocytes and seek new ways to improve insulin resistance. Methods 3T3-L1 adipocytes were cultured in vitro, and differentiated mature adipocytes were identified by oil red O staining. Adipocytes were treated with tumor necrosis factor-α (TNF-α), the glucose metabolism and the concentration of glycerol in the medium were measured. Real-time PCR and Western blotting Blot) analysis of AQP7 expression levels. AQP7 was overexpressed by adenoviruses and the changes of insulin receptor substrate 1 (IRS-1), protein kinase B (PKB) and phosphorylated proteins in insulin signaling pathway were detected to elucidate the relationship between AQP7 expression and insulin resistance . SPSS 17.0 software for statistical analysis, two sets of measurement data were compared with independent samples t test, multiple sets of measurement data were compared using one-way analysis of variance. Results The adipocytes differentiated into mature adipocytes induced by TNF-αinduced insulin resistance. Compared with the blank control group, the glucose metabolism level decreased by 50% and the glycerol concentration decreased by 35%, while the AQP7 mRNA and protein levels decreased by 75% and 28%, respectively %, The differences were statistically significant (P <0.01, P <0.05). Insulin-stimulated PKB phosphorylation (p-PKB), IRS-1 tyrosine 632 phosphorylation protein (p-tyr632) and IRS-1 total protein expression were inhibited in insulin resistance state, whereas IRS-1 serine 307 phosphorylation protein (p-ser307) abnormally increased, the difference was statistically significant (P <0.05). Adenovirus-mediated transfection of adipocytes with high expression of AQP7, with the up-regulation of AQP7, can reverse these effects of TNF-α. Conclusions AQP7 may be involved in the molecular mechanism of insulin resistance. High expression of AQP7 may be helpful in gene therapy to improve insulin resistance.