PC-PLC介导长时间胰岛素刺激促进GLUT4降解的分子机制研究

Disorder of glucose transporter 4 (GLUT4) translocation is one of the hot topics in the study of the pathogenesis of type 2 diabetes. Although insulin acutely stimulates glucose uptake by promotion of GLUT4 translocation from intracellular compartments to the plasma membrane in adipocytes and muscles, long term insulin stimulation causes a depletion of GLUT4 protein, which is particularly prominent in the highly insulin-responsive GLUT4 storage vesicles (GSVs). We previously found that prolonged insulin stimulation down-regulates GLUT4 through oxidative stress-mediated retromer complex inhibition but not dependent on the activity of PI3K and Erk1/2 signaling. Preliminary experiments indicated that PC-PLC plays an essential role in this insulin signaling down regulates GLUT4. Thus, this study aims to assesse the effects of PC-PLC and its products p-Cho and DAG on GLUT4 degradation by examing the subcellular distribution of GLUT4 and retromer complex using density gradient centrifugation, examine their effects on ROS generation by pHyPer-Cyto, furthermore investigates activation of PC-PLC and Nox4 during prolonged insulin stimulation in 3T3-L1 adipocytes by quantitative analyze of lipids and FRET, respectively.

GLUT4转位障碍是目前2型糖尿病发病机制研究的热点之一。研究证实长时间胰岛素刺激可促进 GLUT4 降解，但确切机制尚不明确。申请人前期研究发现长时间胰岛素刺激通过Nox4途径引起的氧化应激可抑制retromer复合体从内吞循环途径中分选回收GLUT4，而促进其降解；且不依赖于PI3K和Erk1/2等信号。预实验显示PC-PLC介导了这一过程。基于此，本项目以3T3-L1脂肪细胞为研究对象，用密度梯度离心法检测PC-PLC及其产物p-Cho、DAG对GLUT4和retromer复合体亚细胞分布的影响；用pHyPer-Cyto检测其对活性氧生成的影响；通过脂质测定等实验检测PC-PLC活性，明确胰岛素对PC-PLC的活化；Western、FRET等技术检测Nox4活性，明确PC-PLC对Nox4的活化。揭示PC-PLC为胰岛素负性调控GLUT4的新信号分子，为2型糖尿病新药研发提供线索。