The effects of brefeldin A on the glucose transport system in rat adipocytes. Implications regarding the intracellular locus of insulin-sensitive Glut4.
Insulin activates glucose transport by recruiting Glut4 glucose transporters from an intracellular pool to plasma membrane (PM). To localize intracellular translocating Glut4, we studied the effects of brefeldin A (BFA), which disassembles Golgi and prevents trans-Golgi vesicular budding, on the glucose transport system. Isolated rat adipocytes were treated with and without both BFA (10 micrograms/ml) and insulin. BFA did not affect maximal rates of either 2-deoxyglucose or 3-O-methyl-glucose transport or the insulin:glucose transport dose-response curve but did increase basal transport by approximately 2-fold (p < 0.05). We also measured Glut4 in PM, low (LDM) and high density microsome subfractions. In basal cells, BFA increased PM Glut4 by 58% concomitant with a 18% decrease in LDM (p < 0.05). Insulin alone increased PM Glut4 by 3-fold concomitant with a 56% decrease in LDM. BFA did not affect insulin-induced changes in Glut4 levels in PM or LDM. Most intracellular Glut4 was localized to sub-PM vesicles by immunoelectron microscopy in basal cells, and BFA did not affect insulin-mediated recruitment of immunogold-labeled Glut4 to PM. In summary, 1) in basal cells, BFA led to a small increase in glucose transport activity and redistribution of a limited number of transporters from LDM to PM; 2) BFA did not affect insulin's ability to stimulate glucose transport or recruit normal numbers of LDM Glut4 to PM; and 3) insulin action is predominantly mediated by a BFA-insensitive pool of intracellular Glut4, which localizes to sub-PM vesicles. Thus, the major translocating pool of Glut4 in rat adipocytes does not involve trans-Golgi.
Bao
S
S
Department of Medicine, Medical University of South Carolina, Charleston 29425, USA.
Smith
R M
RM
Jarett
L
L
Garvey
W T
WT
D019325:3-O-Methylglucose; D017667:Adipocytes / Q000187:drug effects / Q000378:metabolism*; D000273:Adipose Tissue / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D020126:Brefeldin A; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D003517:Cyclopentanes / Q000494:pharmacology*; D003847:Deoxyglucose / Q000378:metabolism; D004822:Epididymis; D005947:Glucose / Q000378:metabolism*; D051275:Glucose Transporter Type 4; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D008297:Male; D008757:Methylglucosides / Q000378:metabolism; D009004:Monosaccharide Transport Proteins / Q000096:biosynthesis / Q000378:metabolism*; D009124:Muscle Proteins; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000494:pharmacology
D003517:Cyclopentanes; D051275:Glucose Transporter Type 4; D007328:Insulin; D008757:Methylglucosides; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D011500:Protein Synthesis Inhibitors; D011994:Recombinant Proteins; C495692:SLC2A4 protein, human; C495694:Slc2a4 protein, rat; D019325:3-O-Methylglucose; D020126:Brefeldin A; D003847:Deoxyglucose; D005947:Glucose