长爪沙鼠eEF1A2通过活化PI3K/Akt通路参与胰岛素激活的骨骼肌糖代谢机制

Eukaryotic elongation factor 1A2（eEF1A2）is identified as an oncogene in many kinds of human cancers by activating PI3K/Akt pathway. PI3K/Akt pathway is also a pivotal mediator in insulin-stimulated glucose metabolism in skeletal muscle. A recent report showed that eEF1A2 can be regulated by insulin. These observations indicated that eEF1A2 may participate in glucose metabolism of skeletal muscle. Previously, we tested gene expression difference of skeletal muscle between genetic diabetes Mongolian gerbils and control, via suppression subtractive hybridization (SSH). Compared to control, eEF1A2 mRNA and protein levels decreased significantly in skeletal muscle of diabetes animals. And in skeletal muscle cell lines, insulin up-regulated cytoplasm eEF1A2 levels, which just coincided with insulin-induced Akt phosphorylation. Summarizing the above results, we speculate that eEF1A2 may function in glucose metabolism and improve insulin sensitivity of skeletal muscle. To test this hypothesis，our proposal aims to investigate whether eEF1A2 is involved in insulin-stimulated PI3K/Akt/Glut4 pathway activation and glucose uptake in skeletal muscle of Mongolian gerbils, by using gene overexpression and knockdown in skeletal muscle primary cells and cell lines, and genetic diabetes Mongolian gerbil models bred by ourselves. We hope that our project will deepen the understanding of development of genetic diabetes Mongolian gerbil models and Type 2 Diabetes Mellitus (T2DM) mechanisms, and provide a new drug target for T2DM.

真核延长因子1A2（eEF1A2）在多种癌症中通过活化PI3K/Akt通路发挥原癌基因特性；而骨骼肌中胰岛素通过活化PI3K/Akt途径调节血糖；近期研究证实，eEF1A2可受胰岛素调节，提示eEF1A2可能参与骨骼肌糖代谢。我们前期通过SSH技术对遗传性糖尿病长爪沙鼠骨骼肌进行差异表达基因筛选，结果发现：eEF1A2在糖尿病动物中较正常动物表达显著降低；且胰岛素上调骨骼肌胞质eEF1A2恰与胰岛素诱导Akt磷酸化同步发生。据此推测长爪沙鼠eEF1A2参与骨骼肌糖代谢和胰岛素敏感性调节。为验证这一假说，本研究拟利用自行培育的长爪沙鼠糖尿病动物模型、原代细胞和骨骼肌细胞系，应用过表达和敲减技术，研究长爪沙鼠骨骼肌eEF1A2参与胰岛素激活的PI3K/Akt/Glut4活化和葡萄糖摄取作用，为长爪沙鼠糖尿病模型的发生机制研究奠定基础，也为进一步认识T2DM发生机制和寻找新的药物靶点提供新思路。

近期研究显示，真核延长因子1A2（eEF1A2）可能在代谢中发挥重要作用。作为特异表达于骨骼肌、心脏和大脑的延长因子亚型，eEF1A2除具有经典的转录延长功能外，也转导多种信号事件。然而，eEF1A2是否参与骨骼肌葡萄糖代谢及其具体机制目前尚不清楚。通过同源扩增和cDNA末端快速扩增技术，本研究首先克隆了野生型长爪沙鼠eEF1A2 cDNA序列，全长1812 bp，编码区1392 bp，编码463个氨基酸。在自发长爪沙鼠糖尿病模型、db/db糖尿病小鼠模型和高脂饲喂诱导的糖尿病小鼠模型中，骨骼肌eEF1A2表达显著降低且不依赖于eEF1A2启动子区域的甲基化。同时，胰岛素可上调骨骼肌eEF1A2表达，但在糖尿病中这种上调作用受到抑制。在正常C2C12肌管细胞中，eEF1A2通过活化PI3K/Akt信号通路促进胰岛素刺激的葡萄糖摄取和葡萄糖转运蛋白4（GLUT4）膜转位。令人意外的是，在棕榈酸诱导的胰岛素抵抗肌管中，eEF1A2降低胰岛素刺激的葡萄糖摄取，GLUT4膜转位和Akt活化。在db/db小鼠中我们利用腺相关病毒介导骨骼肌过表达eEF1A2，也显著升高了db/db小鼠空腹血糖，加剧了动物的葡萄糖不耐和胰岛素不耐。进一步我们发现，在棕榈酸诱导的胰岛素抵抗肌管中，eEF1A2可与PKCβ在胞质结合并促进其活化，抑制PKCβ部分废除了eEF1A2对胰岛素敏感性的抑制作用；eEF1A2还加剧了骨骼肌内质网应激，抑制内质网应激部分改善eEF1A2加剧的胰岛素抵抗。因此，本研究表明在胰岛素抵抗骨骼肌中，eEF1A2通过活化PKCβ和增加内质网应激损伤胰岛素敏感性。本研究为长爪沙鼠糖尿病模型的发生机制研究奠定了基础，也为进一步认识T2DM发生机制和寻找新的药物靶点提供新思路。