Insulin Responsive Trafficking Of GLUT4 Vesicles Near The Plasma Membrane In C2C12 Cells

Type II diabetes is a metabolic disturbance disease of complicated pathomechanism, with insulin resistance as one of its major characters. In type II diabetes glucose uptake into muscle and fat is impaired and this is a major consequence of insulin resis -tance. When combined with defective pancreatic insulin secretion this results in a major dysregulation in blood glucose. The transport of glucose into muscle and fat tissue is the rate-limiting step for glucose utilisation and so defining the molecular nature of this process represents an important goal in diabetes research. In mammals the facilitative glucose transporter GLUT4 is of particular relevance to insulin action as its expression is confined to insulin sensitive cell types such as muscle, fat and cardiac tissue. The regulated entry of glucose into fat and muscle cells in response to insulin or contraction is mediated by the translocation of GLUT4 from intracellular membranes to the plasma membrane. As the final effector of several regulated pathways, GLUT4 plays a crucial role in keeping the glucose homostasis, which made it as an important research target of diabetes. Traditional researches were focusing on the field of insulin pathway; while nowadays the higher level real-time detection for GLUT4 trafficking is under requisition.Based on the fact that the major cellularmechanism for disposal of an exogenous glucose load is insulin-stimulated glucose transport into skeletal muscle and the mechanism of insulin regulation, this project chose C2C12 skeletal muscle cell line as the research target object, built the model at the cell and molecular level, and took the real-time observation of the GLUT4 trafficking in the scale of 200 nm above the plasma membrane in C2C12 cells for the first time. The object for this project is: 1. building the real-time observation of the fluorescence labled GLUT4 trafficking model based on Laser Scanning Confocal Microscopy and Total Internal Reflectional Fluorescence Microscopy; 2. measuring the spatio-timporal parameters involved in the GLUT4 trafficking and discussing about the GLUT4's distribution, translocation, docking and fusion, and defining the standards for docking, fusion's judgement; 3. exploring about the difference of the GLUT4's physical parameters between the basal condition and the insulin-stimulated condition.Through the project, we reached the conclusions as following: 1. under the basal condition, major GLUT4 concentrates at the area of TGN; 2. in C2C12 cells, GLUT4 and IRAP do not assemble in same vesicles, which is opposite with the conclusion from adipocytes; 3. collecting the real-time image information of the GLUT4 trafficking process, and summarizing the translocation characters: the vesicles moves along the same trajectors with different distance; the vesicles have the bi-directional movements; 4. under the stimulation of insulin, the translocation of GLUT4 vesicles becomes severe. This project built the real-time observation for GLUT4 translocation in C2C12 and discuessed its application, which supports and makes great contribution on searching the new drug-treatment target. And there are rare relative reports on the research of GLUT4 based on C2C12 cells.