Glucose deprivation enhances targeting of GLUT1 to lipid rafts in 3T3-L1 adipocytes.
Glucose deprivation dramatically increases glucose transport activity in 3T3-L1 adipocytes without changing the concentration of GLUT1 in the plasma membrane (PM). Recent data suggest that subcompartments within the PM, specifically lipid rafts, may sequester selected proteins and alter their activity. To evaluate this possibility, we examined the distribution of GLUT1 in Triton X-100-soluble and -insoluble fractions. Our data show that 77% of the GLUT1 pool in PMs isolated from control 3T3-L1 adipocytes was extracted by 0.2% Triton X-100. After glucose deprivation for 12 h, only 56% of GLUT1 was extracted by detergent. In contrast, there was a twofold increase in the GLUT1 content of the detergent-resistant fraction. To evaluate whether GLUT1 interacts with a specific protein within lipid rafts, we focused on stomatin, recently shown to interact with and inhibit GLUT1 activity. Stomatin is distributed about equally between the PM and the biosynthetic compartments, and its expression is not affected by glucose deprivation. Nearly 90% of the PM pool of stomatin is in detergent-resistant lipid rafts. In normal 3T3-L1 adipocytes, we were unable to demonstrate an interaction between GLUT1 and stomatin in coimmunoprecipitation experiments. However, in stomatin-overexpressing cells, there was clear coprecipitation of stomatin with GLUT1 antibodies. Glucose deprivation increased this interaction threefold, which may reflect the increase of GLUT1 in lipid rafts. Despite this, there was little change in transport activity in glucose-deprived, stomatin-overexpressing cells vs. that in control cells. Thus GLUT1 interacts with stomatin in lipid rafts, but this interaction per se does not alter transport activity. Rather, stomatin may serve as an anchor for GLUT1 in lipid rafts, the environment of which favors activation.
Kumar
Anil
A
Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 32610, USA.
Xiao
Yu-Ping
YP
Laipis
Philip J
PJ
Fletcher
Bradley S
BS
Frost
Susan C
SC
D016475:3T3 Cells; D017667:Adipocytes / Q000378:metabolism*; D000818:Animals; D001798:Blood Proteins / Q000037:antagonists & inhibitors / Q000276:immunology; D015152:Blotting, Northern; D015153:Blotting, Western; D002460:Cell Line; D005947:Glucose / Q000502:physiology*; D051272:Glucose Transporter Type 1; D021962:Membrane Microdomains / Q000378:metabolism*; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000276:immunology; D051379:Mice; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism
D001798:Blood Proteins; C488964:Stom protein, mouse; D051272:Glucose Transporter Type 1; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; C495678:Slc2a1 protein, mouse; D005947:Glucose