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目的探讨线粒体解耦联机制在NYGGF4基因致胰岛素抵抗(IR)中的作用。方法以3T3-L1前体脂肪细胞为工具,建立NYGGF4基因稳定过表达细胞株,以空载质粒转染的细胞为对照,将前体脂肪细胞诱导分化为成熟脂肪细胞,采用液闪仪检测成熟脂肪细胞对[3H]-2-脱氧葡萄糖的摄取率,采用荧光定量反转录-PCR技术检测成熟脂肪细胞中解耦联蛋白(UCP)2 mRNA、UCP4mRNA表达。采用线粒体特异性染料MitoTracker Red预染成熟脂肪细胞,激光共聚焦显微镜下观察线粒体膜电位,进一步应用流式细胞仪对荧光强度进行定量分析。结果 1.NYGGF4基因过表达脂肪细胞胰岛素刺激状态下的葡萄糖摄取率显著低于对照空载脂肪细胞,NYGGF4基因可显著降低成熟脂肪细胞胰岛素刺激下的葡萄糖摄取率;2.NYGGF4过表达脂肪细胞线粒体UCP2 mRNA、UCP4 mRNA表达显著高于对照空载脂肪细胞;3.NYGGF4过表达脂肪细胞线粒体膜电位低于空载脂肪细胞,但定量分析差异无统计学意义。结论 NYGGF4基因可以上调脂肪细胞线粒体UCP2 mRNA、UCP4 mRNA表达,线粒体解耦联异常可能参与NYGGF4致脂肪细胞线粒体功能障碍及IR的机制。
Objective To investigate the role of mitochondrial uncoupling mechanism in insulin resistance (IR) induced by NYGGF4 gene. Methods 3T3-L1 preadipocytes were used as tools to establish stable over-expressing NYGGF4 cell lines. The transfected cells were used as control to differentiate preadipocytes into mature adipocytes, The uptake of [3H] -2-deoxyglucose by adipocytes was measured by fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) to detect the uncoupling protein (UCP) 2 mRNA and UCP4 mRNA expression in mature adipocytes. MitoTracker Red was used to pre-stain mature adipocytes. The mitochondrial membrane potential was observed under confocal laser scanning microscope. The fluorescence intensity was further analyzed by flow cytometry. Glucose uptake rate in insulin-stimulated state of overexpression adipocytes was significantly lower than that of control, and NYGGF4 gene could significantly reduce glucose uptake rate in mature adipocytes stimulated by insulin.2.NYGGF4 overexpression adipocytes mitochondria UCP2 mRNA and UCP4 mRNA expression was significantly higher than that of blank control adipocytes.3. The mitochondrial membrane potential ofNYGGF4 overexpression adipocytes was lower than that of no-load adipocytes, but there was no statistical difference in quantitative analysis. Conclusion NYGGF4 gene can up-regulate mitochondrial UCP2 mRNA and UCP4 mRNA expression in adipocytes, and abnormal mitochondrial uncoupling may play a role in mitochondrial dysfunction and IR in adipocytes induced by NYGGF4.