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