Insulin-induced GLUT4 recycling in rat adipose cells by a pathway insensitive to brefeldin A

Brefeldin A induces a rapid redistribution of coat proteins associated with clathrin-coated vesicles that bud from the trans-Golgi network but not of those associated with vesicles derived from the plasma membrane. Its effect on glucose transport and the subcellular distribution of the glucose transporter GLUT4 were therefore investigated in the absence or in the presence of insulin in order to characterize the mechanism of insulin-induced GLUT4 recycling. Exposure of rat adipose cells to brefeldin A in the absence of insulin resulted in a slight increase both in 3-O-methyl-D-glucose transport and in the amount of GLUT4 in the plasma membrane as determined by immunoblot analysis. In contrast, it had no effect on the insulin-induced increase in 3-O-methyl-glucose transport and plasma-membrane-associated GLUT4. Immunohistochemical analysis revealed clathrin immunoreactivity in the juxtanuclear region and in the cell periphery, and the staining in the perinuclear region decreased after exposure of cells to 35 mu M brefeldin A. However, it had no effect on the basal pattern of GLUT4 staining or on the insulin-induced redistribution of GLUT4 staining to the cell periphery. These results suggest that basal recycling of GLUT4 responsible for maintaining the intracellular location of the transporter may include a membrane budding step sensitive to brefeldin A, possibly associated with clathrin-coated vesicles, but that the insulin-stimulated GLUT4 recycling pathway is mediated predominantly by a brefeldin A-insensitive mechanism.