中心体周蛋白Pericentrin通过调控GLUT4转位参与骨骼肌胰岛素抵抗的机制研究

Pericentrin(PCNT) deficiency contributes to severe IR and early onset of type 2 diabetes. Glucose transporter 4(GLUT4) is a key protein in taking part in glucose transport in skeletal muscle, and its translocation defects can lead to IR. On the bases of previous clinic case, we found that declining expression of Pcnt in C2C12 cell decreased glucose uptake, while Glut4 expression was normal, which suggested that PCNT deficiency bring about IR in skeletal muscle by influencing GLUT4 translocation. Via siRNA and so on, We plan to establish C2C12-GLUT4myc cell line, further to lower or elevate expression of Pcnt protein. By using confocal, electron microscope and co-immunoprecipitation, we will further to evaluate glusoce uptake, to observe the structures of microtubule, to test the expression and distribution of GLUT4 and key molecules in its translocation(Akt, AS160, Rab and so on), to detect the transport of GLUT4 vesicle and the interaction between Pcnt and KIF3. Also, we plan to raise skeletal muscle specific pcnt gene knockout mice by using Cre/Loxp, to observe the phenotypes, and explore the impact on GLUT4 translocation in cellular level. Our study will be helpful to explore the mechanism and role of Pcnt on IR in muscle, provide the target for early treatment for diabetes.

中心体周蛋白（PCNT）缺陷可导致严重胰岛素抵抗（IR）和早发2型糖尿病。葡萄糖转运蛋白4（GLUT4）在骨骼肌葡萄糖转运中至关重要，其转位异常可导致IR。课题组前期研究发现：下调Pcnt可导致小鼠C2C12骨骼肌细胞葡萄糖摄取减少，而对GLUT4表达量无显著影响，推测PCNT缺陷可能通过影响GLUT4转位导致骨骼肌IR。本项目拟构建Pcnt下调/过表达小鼠C2C12-GLUT4myc细胞模型，通过激光共聚焦、电镜、免疫共沉淀等方法，研究Pcnt变化对葡萄糖摄取、微管结构和GLUT4及其转位关键分子(Akt、AS160、Rab等）的影响；并探索GLUT4囊泡转运变化及Pcnt与KIF3之间的相互作用；利用Cre/Loxp构建骨骼肌特异性Pcnt敲除小鼠模型，观察表型变化，培养骨骼肌原代细胞验证Pcnt缺陷对GLUT4转位影响，阐明Pcnt参与IR机制，为糖尿病早期干预提供靶点。

胰岛素抵抗（IR）是 T2DM 的主要病理生理学特征及核心问题之一，早期减轻 IR 是预防和延缓 T2DM 发生、发展的重点。因此，研究 IR 的调控机制是 T2DM 防治的关键。课题组前期临床研究发现PCNT缺陷可导致严重IR和早发T2DM，进一步细胞学实验结果提示PCNT 缺陷通过影响 GLUT4 的转位进而导致骨骼肌 IR。第二部分利用Cre/LoxP系统构建骨骼肌特异性Pcnt基因敲除小鼠模型。骨骼肌特异性Pcnt敲除小鼠在 12周即出现四肢肌力明显受损和病理改变：肌纤维排列紊乱，部分核膜消失，电镜结构未见明显中心体结构，且该病理改变随着年龄增加病变加重；另肌酸激酶水平稍高于对照组。追踪至 4 -6个月龄模型小鼠空腹血糖及糖耐量均受损，且进一步进行胰岛素刺激试验，显示其模型小鼠血糖下降缓慢，提示存在胰岛素抵抗表现。本项目首次聚焦于PCNT 调控骨骼肌 GLUT4 转位机制，将骨骼肌 IR 的调控机制研究拓展到细胞骨架等亚细胞结构领域。该研究部分阐明Pcnt参与IR机制，为糖尿病早期干预提供靶点。