INSULIN-SENSITIVE ASSOCIATION OF GLUT-4 WITH ENDOCYTIC CLATHRIN-COATED VESICLES REVEALED WITH THE USE OF BREFELDIN-A

The interaction of the adipocyte/skeletal muscle glucose transporter ( GLUT-4) with clathrin lattices may be important in maintaining its int racellular distribution. To better understand the role of clathrin lat tices in the sorting of GLUT-4, we have attempted to determine the cel lular origin of clathrin-coated vesicles (CCVs) that contain this tran sporter. The fungal toxin brefeldin A (BFA) causes the selective disas sembly of clathrin lattices at the trans-Gels network (TGN), but not a t the plasma membrane (PM), thus providing a way of estimating the pro portion of GLUT-4 in PM- versus TGN-derived clathrin lattices. Exposur e of 3T3-L1 adipocytes to BFA resulted in a rapid disassembly of clath rin lattices at the TGN, observed by optical sectioning microscopy, an d to a pronounced decrease in the yield of CCVs purified from these ce lls. Thus, CCVs isolated from BFA-treated cells are likely to be deriv ed from the PM. Immunoblotting experiments revealed the presence of GL UT-4 in such CCVs, suggesting that under basal conditions the transpor ter is continually retrieved from the PM through the CCV pathway. Expo sure of both BFA-treated or non-treated cells to insulin resulted in a 4-6-fold increase in the concentration of GLUT-4 at the PM. In parall el, the concentration of GLUT-4 in PM-derived CCVs decreased by 60%. T hese results suggest (a) that the effect of insulin to increase the ce ll surface concentration of GLUT-4 is not inhibited by BFA, and (b) th at a decreased association of GLUT-4 with endocytic CCVs may be import ant in facilitating its increased cell surface concentration in respon se to the hormone.