eEF1A2对胰岛素激活的骨骼肌糖代谢的作用与机制研究

发布时间：2018-08-26 07:12

【摘要】：背景和目的2型糖尿病(type 2 diabetes mellitus,T2DM)主要表现为骨骼肌、脂肪、肝脏等靶组织器官的胰岛素敏感性下降。骨骼肌是人体最大的胰岛素利用组织,对全身葡萄糖代谢起到了至关重要的作用。因此,从骨骼肌组织入手,通过筛查遗传性T2DM实验动物骨骼肌组织的异常表达基因或者突变基因,研究其对T2DM的作用,就成为探索T2DM发生机制和治疗靶点的一个有效途径。前期,我们在长爪沙鼠近交系培育过程中,偶然发现一个分支的长爪沙鼠空腹血糖较高,糖耐量受损,且这一性状具有遗传性。抑制消减杂交发现真核蛋白延长因子1α2(eukaryotic translation elongation factor 1 alpha 2,eEF1A2)在T2DM长爪沙鼠骨骼肌中表达显著降低。eEF1A2在真核细胞蛋白翻译阶段,以GTP依赖的方式促进翻译延长和密码子识别准确性。近期研究发现,胰岛素可调节eEF1A2表达和转位。eEF1A可促进酵母葡萄糖摄取,糖原合成和脂类转运。此外,eEF1A2通过活化PI3K/Akt通路参与多种细胞事件。这提示,在骨骼肌中eEF1A2可能能通过PI3K/Akt/GLUT4通路调节葡萄糖摄取和胰岛素敏感性,eEF1A2的异常表达与T2DM病理进程相关。为验证这一假说,本研究将在动物水平和细胞水平上研究eEF1A2对骨骼肌糖代谢的作用和分子机制。实验方法我们首先通过同源扩增和cDNA末端快速扩增技术(rapid amplification of cDNA ends,RACE)克隆长爪沙鼠eEF1A2 cDNA序列全长,并进行同源性分析。随后,利用三种T2DM动物模型,分析骨骼肌中eEF1A2表达及其甲基化变化。在正常肌管和两种胰岛素抵抗细胞模型中,探讨eEF1A2对胰岛素刺激后肌管的葡萄糖摄取,胰岛素信号活化和脂质生成利用相关基因表达的调节作用。最后,通过腺相关病毒注射实现db/db小鼠骨骼肌eEF1A2过表达,检测其在动物水平上对葡萄糖耐受和代谢相关基因的调节作用。实验结果1.利用RACE技术克隆长爪沙鼠eEF1A2基因cDNA全长1812 bp,CDs长1392 bp,编码463个氨基酸。同源性分析显示,长爪沙鼠eEF1A2核苷酸序列与人类、大鼠和小鼠的同源性分别为88.6%,94.0%和93.8%;其氨基酸序列与人类、大鼠和小鼠的同源性分别为100%,99.9%和99.9%。这提示长爪沙鼠可能是研究人类eEF1A2蛋白功能的良好实验动物。2.在自发性T2DM长爪沙鼠、db/db小鼠和高脂饮食(high fat diet,HFD)诱导的T2DM小鼠中,其骨骼肌eEF1A2表达均显著降低,且这一变化不依赖于eEF1A2启动子区域甲基化水平的改变。3.胰岛素可在60 min内显著上调正常饮食小鼠和高脂诱导T2DM小鼠骨骼肌eEF1A2表达,但高脂小鼠eEF1A2表达增加量低于正常饮食小鼠。这表明胰岛素可上调骨骼肌eEF1A2表达,且在T2DM下这一作用减弱。4.在分化的C2C12肌管细胞中,eEF1A2过表达显著增加胰岛素刺激的葡萄糖摄取,GLUT4膜转位,PI3K,Akt,ERK,m TOR,GSK-3β胰岛素信号分子的活化,以及脂质生成相关基因乙酰辅酶A羧化酶α(Acetyl CoA carboxylaseα,Acc-1),脂肪酸合酶(Fatty acid synthase,Fas),甘油三酯水解基因:脂蛋白脂酶(Lipoprotein lipase,Lpl),β-氧化基因:肉碱棕榈酰转移酶1(Carnitine palmitoyl transterase-1,Cpt-1)的表达。这一结果提示,在正常骨骼肌细胞中,eEF1A2促进胰岛素敏感性。5.分别利用高浓度胰岛素和棕榈酸构建胰岛素抵抗细胞模型。在高浓度胰岛素诱导的胰岛素抵抗模型中,eEF1A2过表达显著增加肌管中胰岛素刺激的葡萄糖摄取,GLUT4膜转位,PI3K,ERK,GSK-3β活化,且这一作用可被Akt抑制剂MK-2206 2HCl和PI3K抑制剂LY294002逆转。这表明在高浓度胰岛素诱导的胰岛素抵抗细胞模型中,eEF1A2能通过激活PI3K/Akt通路增强胰岛素敏感性。然而在棕榈酸诱导胰岛素抵抗模型中,eEF1A2过表达显著降低了葡萄糖摄取,GLUT4膜转位,以及Akt,ERK,mTOR的活化。这表明在棕榈酸诱导的胰岛素抵抗模型中,eEF1A2加剧了肌管的胰岛素抵抗。6.将eEF1A2过表达的腺相关病毒注射到db/db小鼠后肢骨骼肌中,实现eEF1A2骨骼肌过表达。注射两周后,与对照相比,eEF1A2过表达小鼠表现为更强的葡萄糖和胰岛素不耐受,且内质网应激基因CCAAT/增强子结合蛋白同源蛋白(CCAAT/enhancer-binding protein-homologous protein,Chop)和免疫球蛋白重链结合蛋白(Immunoglobulin heavy chain binding protein,Bip)表达显著增加。因此,eEF1A2加剧T2DM动物胰岛素抵抗。结论:T2DM下调骨骼肌eEF1A2表达,且不依赖于其启动子区域甲基化水平。eEF1A2表达受胰岛素调节,在正常肌管细胞中促进胰岛素刺激的葡萄糖摄取和信号活化;但在棕榈酸诱导的胰岛素抵抗细胞和db/db小鼠模型中,加剧胰岛素抵抗。这提示,eEF1A2有望成为治疗T2DM的新靶点,为临床治疗T2DM提供新思路和理论基础。
[Abstract]:BACKGROUND AND OBJECTIVE Type 2 diabetes mellitus (T2DM) is characterized by decreased insulin sensitivity in skeletal muscle, fat, liver and other target tissues and organs.Skeletal muscle is the largest insulin-utilizing tissue in the human body and plays an important role in glucose metabolism.Therefore, starting from skeletal muscle tissue, genetic screening is carried out. Abnormal expression genes or mutant genes in skeletal muscle tissue of experimental animals with sexual T2DM and their effects on T2DM are an effective way to explore the pathogenesis and therapeutic targets of T2DM. Inhibitory subtractive hybridization revealed that eukaryotic translation elongation factor 1 alpha 2 (eEF1A2) was significantly reduced in the skeletal muscle of T2DM Mongolian gerbils. In addition, eEF1A2 participates in many cellular events by activating the PI3K/Akt pathway. This suggests that eEF1A2 may regulate glucose uptake and insulin sensitivity through the PI3K/Akt/GLUT4 pathway in skeletal muscle. To verify this hypothesis, we will study the effect and molecular mechanism of eEF1A2 on glucose metabolism in skeletal muscle at animal and cell levels. Methods We first cloned eEF1A2 from Mongolian gerbils by homologous amplification and rapid amplification of cDNA ends (RACE). The expression of eEF1A2 and its methylation in skeletal muscle were analyzed by using three T2DM animal models. The glucose uptake, insulin signal activation and lipid production were studied in normal myotubes and two insulin resistant cell models. Finally, the over-expression of eEF1A2 in skeletal muscle of db/db mice was achieved by injection of adeno-associated virus, and the regulatory effect of eEF1A2 on glucose tolerance and metabolism-related genes was detected at animal level. Experimental results 1. The full-length cDNA of eEF1A2 gene of gerbil was cloned by RACE, and the CDs were 1392 BP long, encoding 463 amino acids. Homology analysis showed that the eEF1A2 nucleotide sequence of Mongolian gerbils was 88.6%, 94.0% and 93.8% homologous to human, and the amino acid sequence of Mongolian gerbils was 100%, 99.9% and 99.9% homologous to human, rat and mouse, respectively. The expression of eEF1A2 in skeletal muscle of male T2DM Mongolian gerbils, db/db mice and high fat diet (HFD) induced T2DM mice was significantly decreased, and this change was not dependent on the change of methylation level of eEF1A2 promoter region. 3. Insulin could significantly up-regulate the expression of eEF1A2 in skeletal muscle of normal diet mice and high fat-induced T2DM mice within 60 minutes. The expression of eEF1A2 in hyperlipidemia mice was lower than that in normal diet mice. This suggests that insulin can up-regulate the expression of eEF1A2 in skeletal muscle, and this effect is weakened under T2DM. 4. Overexpression of eEF1A2 in differentiated C2C12 myotube cells significantly increases insulin-stimulated glucose uptake, GLUT4 membrane translocation, PI3K, Akt, ERK, m TOR, GSK-3 beta insulin signaling. Activation of molecule No. 1, and activation of genes associated with lipid production, Acetyl CoA carboxylase alpha (Acc-1), Fatty acid synthase (Fas), triglyceride hydrolysis genes: Lipoprotein lipase (Lpl), beta-oxidation gene: Carnitine palmitoyl transterase-1 (Cpt-1) These results suggest that eEF1A2 promotes insulin sensitivity in normal skeletal muscle cells. 5. Insulin resistance cell models were constructed by high-concentration insulin and palmitic acid, respectively. PI3K, ERK, and GSK-3beta were activated, and this effect was reversed by Akt inhibitor MK-22062HCl and PI3K inhibitor LY294002. This suggests that eEF1A2 enhances insulin sensitivity by activating the PI3K/Akt pathway in insulin resistance cell model induced by high concentration of insulin. However, in palmitic acid-induced insulin resistance model, eEF1A2 is expressed by activating the PI3K/Akt pathway. This suggests that eEF1A2 exacerbates the insulin resistance in the myotube in a palmitic acid-induced insulin resistance model. 6. The overexpressed adeno-associated virus eEF1A2 was injected into the skeletal muscles of the hind limbs of db/db mice to achieve the overexpression of eEF1A2 in the skeletal muscles. Compared with the control group, the eEF1A2 overexpression mice showed stronger glucose and insulin intolerance, and the expression of CCAAT / enhancer binding protein homologous protein (Chop) and immunoglobulin heavy chain binding protein (Bip) increased significantly. Conclusion: T2DM down-regulates the expression of eEF1A2 in skeletal muscle and does not depend on its promoter methylation level. The expression of eEF1A2 is regulated by insulin and promotes insulin-stimulated glucose uptake and signal activation in normal myotube cells, but in palmitic acid-induced insulin resistance cells and db/d cells. This suggests that eEF1A2 may become a new target for the treatment of T2DM and provide new ideas and theoretical basis for the clinical treatment of T2DM.
【学位授予单位】：首都医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1

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