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 b ut not of those associated with vesicles derived from the plasma membr ane. Its effect on glucose transport and the subcellular distribution of the glucose transporter GLUT4 were therefore investigated in the ab sence or in the presence of insulin in order to characterize the mecha nism of insulin-induced GLUT4 recycling. Exposure of rat adipose cells to brefeldin A in the absence of insulin resulted in a slight increas e 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-gluco se transport and plasma-membrane-associated GLUT4. Immunohistochemical analysis revealed clathrin immunoreactivity in the juxtanuclear regio n and in the cell periphery, and the staining in the perinuclear regio n decreased after exposure of cells to 35 mu M brefeldin A. However, i t had no effect on the basal pattern of GLUT4 staining or on the insul in-induced redistribution of GLUT4 staining to the cell periphery. The se results suggest that basal recycling of GLUT4 responsible for maint aining the intracellular location of the transporter may include a mem brane budding step sensitive to brefeldin A, possibly associated with clathrin-coated vesicles, but that the insulin-stimulated GLUT4 recycl ing pathway is mediated predominantly by a brefeldin A-insensitive mec hanism.