Insulin Regulated Trafficking of Glucose Transporter 4: A Kinetic Perspective
Regulation of blood glucose homeostasis is a critical process; defects in this process can lead to insulin resistance, type II diabetes, and metabolic syndrome. Glucose homeostasis is regulated in part by insulin action on peripheral tissues, especially muscle, fat, and the liver. In muscle and adipose tissue, insulin increases glucose transport by increasing the amount of glucose transporter isoform 4 (Glut4) in the plasma membrane by recruiting the transporter from intracellular storage depots. Insulin regulation of Glut4 trafficking has been extensively studied, and although Glut4 partially overlaps with the general trafficking pathways, it is uniquely regulated by insulin. Few studies have determined if experimental perturbations to the system affect important rate-limiting regulatory steps unique to the trafficking of Glut4. Our lab has developed a series of high-throughput quantitative flow cytometric methods for analytical measurement of Glut4 trafficking kinetics. In order to provide mechanistic information about the roles of proteins regulating Glut4 traffic, we used these methods to develop trafficking assays for Glut4 in addition to other well characterized markers of general endosomal pathways. We demonstrated that insulin promotes Glut4 translocation by increasing the exocytosis of Glut4 and releasing Glut4 from basal storage compartments. We illustrated that insulin does not regulate the rate of Glut4 endocytosis and demonstrated that previously measured decreases of endocytosis may be an artifact of ignoring the effects of exocytosis on internalization assays. We also showed that Glut4 traffics very differently from a marker of the early endosomal pathway and similarly to a late endosomal marker. In order to identify important regulatory mechanisms expressed in adipocytes, we characterized trafficking of the three endosomal proteins in undifferentiated fibroblasts as well as in adipocytes, and identified the minimum number of regulatory steps required for the unique traffic of Glut4. We studied an important regulator of Glut4 - AS160, and demonstrated that the Rab GAP is required for retention of Glut4 in sequestered compartments under basal conditions, and does not directly regulate vesicle exocytosis as previously suggested. Using inhibitors of Akt, we identified a novel Akt-dependent rate limiting step that lies downstream of AS160 but upstream of the insertion of Glut4 into the plasma membrane. To determine if the Rab substrates of AS160 also regulate sequestration, we characterized Glut4 traffic in cells depleted of Rabs 8a, 8b, 10, and 14, and found that Rab10 does regulate release from sequestration, while Rab14 regulates movement of proteins out of early endosomes. This identified a novel AS160-dependent step in Glut4 trafficking. Finally, we characterized the role of Sortilin, and demonstrated that many proteins reported to regulate Glut4 trafficking simply regulate Glut4 stability at a posttranslational level.
endocytosis, exocytosis, Glut4, Insulin, mathematical modeling, membrane trafficking
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