Glut4突变调控脂肪重分布及肌纤维重塑的机制研究

旨在探讨Glut4基因突变后骨骼肌能量代谢及肌纤维转化的分子机制。本研究利用CRISPR/Cas9技术构建Glut4^Q177L突变鼠。选取22周龄健康的野生雄鼠和Glut4^Q177L突变雄鼠，即试验分为两组，每组3只，3个重复，进行表型评价和糖耐量、胰岛素耐量试验；荧光定量检测两组小鼠腓肠肌葡萄糖转运蛋白、脂代谢、肌纤维类型相关调控基因的表达差异；Western blot测定腓肠肌AMPK及其磷酸化蛋白含量。结果表明，突变鼠皮下脂肪和附睾脂重量显著低于对照组（P<0.05）；突变鼠糖耐量曲线下面积极显著增加（P<0.01），表明其糖耐量受损；突变鼠血清中甘油三酯的含量显著下降（P<0.05）。相较于野生型小鼠，Glut4^Q177L突变鼠腓肠肌中Glut4、Glut1和Glut12等多个葡萄糖转运蛋白表达量显著升高（P<0.05）；葡萄糖摄取受限导致调控能量代谢关键基因AMPK在mRNA、蛋白和磷酸化修饰水平均显著升高（P<0.05），同时促进与脂肪酸摄取与合成代谢相关的CD36和ATGL等基因表达上调（P<0.05）；慢速氧化型肌纤维（I型）和快速氧化型肌纤维（IIa型）相关基因表达极显著升高（P<0.01），而快速酵解型纤维（IIb型）相关基因表达显著降低（P<0.05）。本研究结果显示，Glut4葡萄糖结合关键位点突变降低了骨骼肌和脂肪葡萄糖摄取能力，通过上调其它转运蛋白增加葡萄糖摄取；激活AMPK信号通路及分泌肌细胞因子调控脂肪分解以满足能量需求；促进线粒体丰富的氧化型肌纤维生成以提高能量利用效率。Glut4突变不仅可以为骨骼肌胰岛素抵抗提供有效的动物模型，还可以为家畜生物育种提供基因编辑参考位点。

Research of the Mechanism of Glut4 Mutation Regulating Fat Redistribution and Muscle Fiber Remodeling

The purpose of this study was to find out the molecular mechanism of skeletal muscle energy metabolism and muscle fiber transformation due to Glut4 gene mutation. Glut4^Q177L mutant mice were constructed by CRISPR/Cas9. The animals were divided into 2 groups including 22-week-old healthy wild male mice and Glut4^Q177L mutant male mice, 3 in each group, repeated 3 times. Subsequently, the phenotypic evaluation, glucose tolerance and insulin tolerance tests were performed. The expression of genes related to glucose transporters, lipid metabolism and muscle fiber type in gastrocnemius muscle of mice in the two groups were detected by qPCR; Western blot was applied to detect the content of AMPK and its phosphorylation level in gastrocnemius muscle. The results showed that the weight of subcutaneous fat and epididymal fat in mutant mice was significantly lower than that in the control group (P<0.05), and the area under the glucose tolerance curve of mutant mice significantly increased (P<0.01), indicated the glucose tolerance was impaired. The content of triglyceride in serum of mutant mice significantly decreased(P<0.05). The expression of several glucose transporters such as Glut4, Glut1 and Glut12 in gastrocnemius muscle of mutant mice was significantly higher than that in the control group (P<0.05). The limitation of glucose uptake resulted in a significant increase in the mRNA, protein and phosphorylation modification expression level of AMPK(P<0.05), which was the key gene regulating energy metabolism. At the same time, the expression of CD36 and ATGL, which promote fatty acid uptake and lipolysis, were significantly up-regulated (P<0.05). The expression of slow oxidation fiber type (type I) and fast oxidation fiber type (type IIa) related genes significantly increased(P<0.01), while the expression of fast glycolysis fiber type (type IIb) related gene significantly decreased(P<0.05). The results of this study showed that Glut4^Q177Lmutation reduced glucose uptake in skeletal muscle and fat tissue, resulting in compensating glucose uptake by up-regulating other glucose transporters. Besides, the insufficiency of energy could activate AMPK signaling pathway and secrete myokines to meet energy requirement by facilitating lipolysis. In addition, Glut4 mutation promoted the expression of slow oxidation fiber type related gene, rich in mitochondria, to improve energy efficiency. Glut4^Q177Lmice can not only provide an effective animal model of skeletal muscle insulin resistance, but also provide gene editing reference sites for animal breeding.