抄録
Insulin stimulates glucose transport into adipocyte and muscle cells by inducing the translocation of the insulin responsive glucose transporter GLUT4 from intracellular storage compartments to the plasma membrane. GLUT4 translocation is a complicated process involving budding and fission at the storage compartment, trafficking to the plasma membrane, and fusion at the plasma membrane. Here, we have established a new method to visualize the movement of single GLUT4 molecule in living cells. 3T3L1 adipocyte expressing exofacial-myc-GLUT4-eCFP was labeled with Qdot-conjugated Myc antibody in the presence of insulin. Qdot-GLUT4 complex was then endocytosed by washing out of insulin. Observation was performed under video-rate confocal microscope equipped with high sensitivity EMCCD camera so that movement of GLUT4 molecule can be tracked for ∼10sec. Movement of GLUT4 molecules was obtained before or after the 2nd insulin stimulation for 5, 15, and 30 min. Analysis of the diffusion of GLUT4 molecules showed that movements can be classified in either 1) free diffusion, 2) confined diffusion, or 3) transport and all these movement existed both at basal state and after insulin stimulation. Overall movement tended to be in confined diffusion at the basal state, but fraction of transported GLUT4 increased by insulin stimulation. Diffusion coefficient of GLUT4 was higher after insulin stimulation than basal state. Together, insulin increases the mobility of GLUT4 and enhances its translocation to the plasma membrane. [J Physiol Sci. 2006;56 Suppl:S112]
