长爪沙鼠Nedd4通过泛素化eEF1A2改善糖尿病骨骼肌胰岛素敏感性作用和机制研究

As a HECT-type ubiquitin ligase, neural precursor cell expressed developmentally down-regulated (Nedd4) plays pivotal roles in many physiological and pathological events. A recent report shows that Nedd4 haploinsufficient mice display moderate insulin resistance, and impaired insulin signal transduction, suggesting the involvement of Nedd4 in glucose metabolism. However, the underlying mechanisms are not clear. Previously, by using diabetic Mongolian gerbil models and cell culture, we found that Nedd4 expression was significantly down-regulated in skeletal muscle of diabetic animals; eEF1A2 aggravated skeletal muscle insulin resistance and animal glucose intolerance by activating PKCβ and endoplasmic reticulum stress; Ubiquitin-mediated proteasomal degradation of eEF1A2 was observed, and eEF1A2 directly bound with Nedd4, and they were also colocalized in cells. Summarizing the above results, we speculate that decreased Nedd4 in diabetic skeletal muscle may induce abnormal eEF1A2 ubiquitination, which impaires insulin sensitivity and glucose homeostasis in Mongolian gerbils. To test this hypothesis, our proposal aims to investigate whether Nedd4 improves glucose metabolism by ubiquitination of eEF1A2, by using gene overexpression and knockdown in diabetic Mongolian gerbil inbred models bred by ourselves, conditional knockout mice, cell lines and in vitro experiments. We hope that our project will deepen the understanding of development of genetic diabetic Mongolian gerbil models and provide a new drug target for type 2 diabetes mellitus (T2DM).

神经前体细胞表达发育下调基因4（Nedd4）为含HECT结构域的泛素化E3连接酶，在多种生理和病理过程发挥重要功能。近期研究证实，Nedd4全身敲除杂合子小鼠表现为中度胰岛素抵抗，胰岛素信号转导障碍，提示Nedd4参与机体糖代谢，但具体机制不清。我们前期通过糖尿病长爪沙鼠和细胞模型发现，糖尿病骨骼肌Nedd4低表达；eEF1A2通过活化PKCβ和内质网应激加剧骨骼肌胰岛素抵抗和机体葡萄糖不耐；eEF1A2存在泛素化水解，且与Nedd4结合共表达。据此推测糖尿病长爪沙鼠骨骼肌Nedd4低表达引发eEF1A2泛素化异常，进而损伤胰岛素敏感性和机体葡萄糖稳态。为验证这一假说，本研究拟利用自行培育的糖尿病长爪沙鼠近交系、条件敲除小鼠、细胞和体外实验，应用过表达和敲减技术，研究Nedd4通过泛素化eEF1A2改善机体糖代谢作用，为长爪沙鼠糖尿病模型发生机制研究奠定基础，也为寻找新药物靶点提供新思路。

在前期项目中，我们利用自行培育的糖尿病长爪沙鼠近交系和细胞模型发现，真核延长因子eEF1A2通过PKCβ和内质网应激加剧糖尿病骨骼肌胰岛素抵抗和机体葡萄糖不耐。然而，在糖尿病骨骼肌中eEF1A2上游有何调节机制，生理条件下骨骼肌eEF1A2对机体糖代谢有何影响，目前均不清楚。近期研究显示泛素化E3连接酶Nedd4可能参与机体糖代谢，但具体机制不清。本研究中，我们通过IPMS筛选发现Nedd4与eEF1A2互作。Nedd4下调eEF1A2蛋白而非mRNA，降低其蛋白稳定性，促进其泛素化修饰，这表明Nedd4通过泛素化-蛋白酶水解途径下调eEF1A2蛋白稳定性。进一步，我们建立了eEF1A2骨骼肌特异性敲除小鼠，并发现该小鼠空腹血糖降低，葡萄糖和胰岛素耐量增强，脂肪特别是内脏脂肪含量降低，快肌纤维向慢肌纤维转化，显示出在生理状态下eEF1A2可能通过肌纤维类型转换促进葡萄糖利用和脂肪酸代谢。同时，我们通过给予近交系糖尿病长爪沙鼠骨骼肌注射腺相关病毒载体构建骨骼肌Nedd4过表达及敲减糖尿病沙鼠模型，结果显示过表达和敲减Nedd4分别改善和加剧了动物葡萄糖和胰岛素耐量，降低和升高了空腹胰岛素水平，显示出Nedd4缓解糖尿病沙鼠胰岛素抵抗的作用。在胰岛素抵抗肌管中，我们发现Nedd4通过促进eEF1A2下游PI3K-mTOR-GSK3β通路活化和抑制内质网应激改善胰岛素敏感性，表明eEF1A2介导了Nedd4改善胰岛素敏感性的分子机制。因此，本研究提示Nedd4通过泛素化下调eEF1A2蛋白稳定性，促进PI3K-mTOR-GSK3β通路活化抑制内质网应激，从而改善糖尿病动物葡萄糖代谢。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。