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 intracellular distribution. To better understand the role of clathrin lattices in the sorting of GLUT-4, we have attempted to determine the cellular origin of clathrin-coated vesicles (CCVs) that contain this transporter. The fungal toxin brefeldin A (BFA) causes the selective disassembly of clathrin lattices at the trans-Golgi network (TGN), but not at the plasma membrane (PM), thus providing a way of estimating the proportion of GLUT-4 in PM- versus TGN-derived clathrin lattices. Exposure of 3T3-L1 adipocytes to BFA resulted in a rapid disassembly of clathrin lattices at the TGN, observed by optical sectioning microscopy, and to a pronounced decrease in the yield of CCVs purified from these cells. Thus, CCVs isolated from BFA-treated cells are likely to be derived from the PM. Immunoblotting experiments revealed the presence of GLUT-4 in such CCVs, suggesting that under basal conditions the transporter is continually retrieved from the PM through the CCV pathway. Exposure 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 parallel, the concentration of GLUT-4 in PM-derived CCVs decreased by 60%. These results suggest (a) that the effect of insulin to increase the cell surface concentration of GLUT-4 is not inhibited by BFA, and (b) that a decreased association of GLUT-4 with endocytic CCVs may be important in facilitating its increased cell surface concentration in response to the hormone.
Chakrabarti
R
R
Program in Molecular Medicine, University of Massachusetts Medical School, Worcester 01655.
Buxton
J
J
Joly
M
M
Corvera
S
S
D016475:3T3 Cells; D017667:Adipocytes / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D020126:Brefeldin A; D002458:Cell Fractionation; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002966:Clathrin / Q000187:drug effects / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000187:drug effects / Q000378:metabolism*; D003517:Cyclopentanes / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis; D005455:Fluorescent Antibody Technique; D051275:Glucose Transporter Type 4; D007328:Insulin / Q000494:pharmacology*; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism; D008565:Membrane Proteins / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins / Q000187:drug effects / Q000378:metabolism*; D009124:Muscle Proteins; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D013347:Subcellular Fractions / Q000378:metabolism
D002966:Clathrin; D003517:Cyclopentanes; D051275:Glucose Transporter Type 4; D007328:Insulin; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D011500:Protein Synthesis Inhibitors; C495693:Slc2a4 protein, mouse; D020126:Brefeldin A