Glut4 trafficking: Roles of LRP1, endosomal pH and Rab GTPases

Insulin regulates Glut4 trafficking between the intracellular storage compartments and the PM of fat and muscle cells, which is a key process in glucose homeostasis. Insulin is released in response to post-prandial excess blood sugar and facilitates glucose clearance by stimulating Glut4 translocation. The detailed Glut4 trafficking itinerary and how insulin regulates this process is not known. Our research was focused in understanding the effect of insulin in Glut4 trafficking and the pathways Glut4 take during its trafficking between the Glut4 storage compartments (GSVs) and the PM. Using a flow cytometric based assays, we show that insulin increased Glut4 exocytic constant (kex) and the amount of Glut4 cycling consistent with previous studies, but also showed that insulin does not affect the Glut4 endocytic constant (ken). Previous studies have estimated ken values but in this study we measured it directly using multiple experimental approaches and showed it is not inhibited by insulin. We also showed all the AS160 substrate Rab GTPases found on Glut4 vesicles are involved in Glut4 trafficking. Even though Rab10 was already previously shown to be involved in Glut4 trafficking, we show here that Rabs 8A, 8B and 14 are also involved in Glut4 trafficking in adipocytes. These knockdowns also affected LRP1 trafficking. Using the Rab knockdown's effect on Glut4 and LRP1 trafficking, we modeled their effect in membrane trafficking in the constitutive and regulated pathways. We also show evidence that one of the major cargo proteins of GSVs, LRP1, might be involved in Glut4 trafficking using its luminal domain. Endosomal pH is required for receptor mediated endocytosis but in this study we showed for the first time that it is also required for Glut4 trafficking. We show evidence that there are two Glut4 trafficking pathways: pH-dependent and pH-independent trafficking

Enhanced fasting glucose turnover in mice with disrupted action of TUG protein in skeletal muscle

Insulin stimulates glucose uptake in 3T3-L1 adipocytes in part by causing endoproteolytic cleavage of TUG (tether containing a ubiquitin regulatory X (UBX) domain for glucose transporter 4 (GLUT4)). Cleavage liberates intracellularly sequestered GLUT4 glucose transporters for translocation to the cell surface. To test the role of this regulation in muscle, we used mice with muscle-specific transgenic expression of a truncated TUG fragment, UBX-Cter. This fragment causes GLUT4 translocation in unstimulated 3T3-L1 adipocytes. We predicted that transgenic mice would have GLUT4 translocation in muscle during fasting. UBX-Cter expression caused depletion of PIST (PDZ domain protein interacting specifically with TC10), which transmits an insulin signal to TUG. Whereas insulin stimulated TUG proteolysis in control muscles, proteolysis was constitutive in transgenic muscles. Fasting transgenic mice had decreased plasma glucose and insulin concentrations compared with controls. Whole-body glucose turnover was increased during fasting but not during hyperinsulinemic clamp studies. In muscles with the greatest UBX-Cter expression, 2-deoxyglucose uptake during fasting was similar to that in control muscles during hyperinsulinemic clamp studies. Fasting transgenic mice had increased muscle glycogen, and GLUT4 targeting to T-tubule fractions was increased 5.7-fold. Whole-body oxygen consumption (VO2), carbon dioxide production (VCO2), and energy expenditure were increased by 12-13%. After 3 weeks on a high fat diet, the decreased fasting plasma glucose in transgenic mice compared with controls was more marked, and increased glucose turnover was not observed; the transgenic mice continued to have an increased metabolic rate. We conclude that insulin stimulates TUG proteolysis to translocate GLUT4 in muscle, that this pathway impacts systemic glucose homeostasis and energy metabolism, and that the effects of activating this pathway are maintained during high fat diet-induced insulin resistance in mice

Kinetic Evidence That Glut4 Follows Different Endocytic Pathways than the Receptors for Transferrin and α2-Macroglobulin*

Insulin regulates glucose uptake through effects on the trafficking of the glucose transporter Glut4. To investigate the degree of overlap between Glut4 and the general endocytic pathways, the kinetics of trafficking of Glut4 and the receptors for transferrin (Tf) and α2-macroglobulin (α-2-M; LRP-1) were compared using quantitative flow cytometric assays. Insulin increased the exocytic rate constant (kex) for both Glut4 and Tf. However, the kex of Glut4 was 5–15 times slower than Tf in both basal and insulin-stimulated cells. The endocytic rate constant (ken) of Glut4 was also five times slower than Tf. Insulin did not affect the ken of either protein. In basal cells, the ken for α-2-M/LRP-1 was similar to Glut4 but 5-fold slower than Tf. Insulin increased ken for α-2-M/LRP-1 by 30%. In contrast, the kex for LRP-1 was five times faster than Glut4 in basal cells, and insulin did not increase this rate constant. Thus, although there is overlap in the protein machineries/compartments utilized, the differences in trafficking kinetics indicate that Glut4, the Tf receptor, and LRP-1 are differentially processed both within the cell and at the plasma membrane. It has been reported that insulin decreases the ken of Glut4 in adipocytes. However, the effect of exocytosis on the “internalization” assays was not considered. Because it is counterintuitive, the effect of exocytosis on these assays is often overlooked in endocytosis studies. Using mathematical modeling and simulation, we show that the reported decrease in Glut4 ken can be entirely accounted for by the well established increase in Glut4 kex