BREFELDIN-A INHIBITS INSULIN-INDUCED GLUCOSE-TRANSPORT STIMULATION AND GLUT4 RECRUITMENT IN RAT ADIPOCYTES

GLUT4, the major insulin-responsive glucose transporter isoform in rat adipocytes, rapidly recycles between an intracellular pool and the pl asma membrane in the basal and insulin-stimulated states. To gain insi ght into the route of this GLUT4 recycling, we studied the effects of brefeldin A (BFA) on glucose transport and glucose transporter subcell ular distribution in rat adipocytes in the absence and in the presence of insulin. 3-O-Methyl-D-glucose equilibrium exchange measurements re vealed that BFA inhibits insulin-stimulated glucose transport by as mu ch as 80%, whereas the inactive BFA analog, B36, was without effect. T he inhibition was reversible and was a saturable function of BFA conce ntration with an apparent K-i of less than 1 mu M. In the absence of i nsulin, on the other hand, BFA caused a slight (up to 2-fold) increase in glucose transport. Subcellular fractionation and semiquantitative immunoblotting analysis revealed that BFA inhibits insulin-induced red istribution of GLUT4 from microsomes to the plasma membranes, with a d ose dependence similar to that for glucose transport inhibition. BFA a lso caused a slight increase in the plasma membrane GLUT4 level in the absence of insulin. BFA did not affect the subcellular distribution o f GLUT1 in these experiments. These findings strongly suggest that GLU T4 recycling in rat adipocytes involves a BFA-sensitive, coat protein- mediated, membrane budding step, which is distinct between the constit utive and the insulin induced pathways.