罗小青, 曾晗, 谭伟, 杨萍, 陈压西, 阮雄中*
本研究旨在探讨在高脂饮食状态下CD36基因缺失对小鼠糖脂代谢的影响及作用机制。根据基因型将小鼠分为野生型小鼠(wild type, WT)及CD36基因敲除(CD36−/−)小鼠，给予高脂饮食喂养14周。小鼠腹腔注射葡萄糖(1 g/kg)或胰岛素(5 units/kg)进行葡萄糖耐量或胰岛素耐量测试。HE染色观察肝脏脂质变性，全自动生化分析仪测定小鼠血清甘油三酯(triglyceride, TG)、血清游离脂肪酸(free fatty acid, FFA)、天门冬氨酸转氨酶(aspartate aminotransferase, AST)和丙氨酸转氨酶(alanine aminotransferase, ALT)浓度。Real-time PCR和Western blot检测小鼠肝脏、肌肉组织胰岛素信号通路。Real-time PCR检测小鼠原代肝细胞中磷酸烯醇式丙酮酸羧激酶(phosphoenolpyruvate carboxykinase, PEPCK)的mRNA水平，葡萄糖检测试剂盒检测糖异生能力。免疫共沉淀(co-immunoprecipitation, Co-IP)及ELISA检测肌肉胰岛素受体β (insulin receptor β, IRβ)酪氨酸磷酸化水平。Real-time PCR和免疫荧光染色检测小鼠肌肉葡萄糖转运蛋白4 (glucose transporter 4, GLUT4)的表达和定位。结果显示，在高脂喂养后，CD36−/−小鼠血清FFA、TG、AST及ALT水平较WT小鼠明显升高(P < 0.05)，CD36−/−小鼠肝脏外观呈脂肪样变性，HE染色结果显示肝脏脂质积聚加重，提示CD36缺失促进脂肪肝的发生。然而，相对于WT小鼠，CD36−/−小鼠的空腹血糖水平降低、糖耐量升高，胰岛素耐量降低(P < 0.05)，提示在高脂饮食喂养条件下，CD36缺失并不会损害小鼠的糖耐量和胰岛素耐量。与WT小鼠相比，CD36−/−小鼠肝脏IR/IRS/AKT胰岛素信号通路无显著差异，两组小鼠原代肝细胞PEPCK表达水平及糖异生能力均无显著差异。而在CD36−/−小鼠肌肉组织中，Co-IP及ELISA实验显示IRβ酪氨酸磷酸化水平显著升高，p-AKT水平显著升高(P < 0.05)。免疫荧光染色实验提示肌肉GLUT4在细胞膜的定位增强，表明CD36−/−小鼠肌肉胰岛素敏感性及葡萄糖利用能力增强。以上结果提示，CD36基因缺失加重高脂饮食诱导的肝脏脂质积聚，对高脂饮食诱导的肝脏糖代谢无显著影响；CD36缺失主要通过提高肌肉组织胰岛素敏感性，促进GLUT4介导的葡萄糖利用以改善高脂饮食诱导的小鼠糖代谢异常。
Deletion of CD36 gene ameliorates glucose metabolism abnormality induced by high-fat diet and promotes liver lipid accumulation
LUO Xiao-Qing, ZENG Han, TAN Wei, YANG Ping, CHEN Ya-Xi, RUAN Xiong-Zhong*
Chongqing Key Laboratory of Lipid and Glucose Metabolism, Centre for Lipid Research, Chongqing Medical University, Chongqing 400016, China
This study aimed to investigate the effects and the underlying mechanism of CD36 gene on glucose and lipid metabolism disorder induced by high-fat diet in mice. Wild type (WT) mice and systemic CD36 knockout (CD36−/−) mice were fed with high-fat diet for 14 weeks (n = 12). Mice were intraperitoneally injected with glucose (1 g/kg) or insulin (5 units/kg) to perform glucose tolerance test (GTT) or insulin tolerance test (ITT). Liver lipid deposition was observed by HE staining, and the contents of total triglyceride (TG), free fatty acid (FFA), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum were determined by automatic biochemical analyzer. Real-time PCR and Western blot were used to detect insulin signaling pathways in liver and muscle tissues of mice. The mRNA levels of genes encoding phosphoenolpyruvate carboxykinase (PEPCK) in primary hepatocytes of mice were detected by real-time PCR, and glucose detection kit was used to detect gluconeogenesis. Co-immunoprecipitation (Co-IP) and ELISA were used to detect insulin receptor β (IRβ) tyrosine phosphorylation in mouse muscle. Real-time PCR and immunofluorescence staining (IF) were used to detect the expression and location of glucose transporter 4 (GLUT4) in muscle of mice. After high-fat diet feeding, serum FFA, TG, AST and ALT levels of CD36−/− mice were significantly higher than WT mice (P < 0.05). The appearance of CD36−/− mouse liver presented fatty degeneration, and HE staining results showed increased lipid accumulation in the liver, suggesting that CD36 knockout promoted the occurrence of fatty liver. However, CD36−/− mice showed decreased fasting glucose levels, increased glucose tolerance, and decreased insulin tolerance compared with WT mice (P < 0.05), suggesting that CD36 knockout protects against the abnormal glucose metabolism induced by high-fat diet. Compared with WT mice, there was no significant difference in insulin signaling pathway in CD36−/− mouse liver, and there were no significant differences in PEPCK expression and gluconeogenesis between the two groups of primary hepatocytes. In muscle tissue, Co-IP and ELISA experiments showed that the phosphorylation level of IRβ tyrosine was significantly increased in CD36−/− mice compared with that in WT mice. Besides, the levels of p-AKT in CD36−/− mouse muscle were significantly increased (P < 0.05). At the same time, IF experiment indicated that GLUT4 localization in cell membrane was enhanced in the muscle of CD36−/− mice, indicating that insulin sensitivity and glucose utilization ability were enhanced in CD36−/− mouse muscle. The results suggested that deletion of CD36 gene increased lipid accumulation in liver of mice with high-fat diet, but had no significant effect on liver gluconeogenesis. CD36 deficiency improves the abnormal glucose metabolism in mice with high-fat diet mainly through improving insulin sensitivity of muscle tissue and promoting GLUT4-mediated glucose utilization.
通讯作者：阮雄中 E-mail: email@example.com
罗小青, 曾晗, 谭伟, 杨萍, 陈压西, 阮雄中. CD36基因缺失改善高脂饮食诱导的糖代谢异常并促进肝脏脂质积聚[J]. 生理学报 2021; 73 (5): 805-812.
LUO Xiao-Qing, ZENG Han, TAN Wei, YANG Ping, CHEN Ya-Xi, RUAN Xiong-Zhong. Deletion of CD36 gene ameliorates glucose metabolism abnormality induced by high-fat diet and promotes liver lipid accumulation. Acta Physiol Sin 2021; 73 (5): 805-812 (in Chinese with English abstract).