Three live-imaging techniques for comprehensively understanding the initial trigger for insulin-responsive intracellular GLUT4 trafficking

Quantitative features of GLUT4 glucose transporter’s behavior deep inside cells remain largely unknown. Our previous analyses with live-cell imaging of intracellular GLUT4 trafficking demonstrated two crucial early events responsible for triggering insulin-responsive translocation processes, namely, heterotypic fusion and liberation. To quantify the regulation, interrelationships, and dynamics of the initial events more accurately and comprehensively, we herein applied three analyses, each based on our distinct dual-color live-cell imaging approaches. With these approaches, heterotypic fusion was found to be the first trigger for insulin-responsive GLUT4 redistributions, preceding liberation, and to be critically regulated by Akt substrate of 160 kDa (AS160) and actin dynamics. In addition, demonstrating the subcellular regional dependence of GLUT4 dynamics revealed that liberated GLUT4 molecules are promptly incorporated into the trafficking itinerary of transferrin receptors. Our approaches highlight the physiological significance of endosomal “GLUT4 molecule trafficking” rather than “GLUT4 vesicle delivery” to the plasma membrane in response to insulin.