Glucose deprivation enhances targeting of GLUT1 to lipid rafts in 3T3-L1 adipocytes

Abstract

. Glucose deprivation enhances targeting of GLUT1 to lipid rafts in 3T3-L1 adipocytes. Am J Physiol Endocrinol Metab 286: E568-E576, 2004. First published December 9, 2003 10.1152/ajpendo.00372.2003.-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 glucosedeprived, 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.glucose transporter 1; lipid rafts; stomatin MOST CELLS ARE DEPENDENT ON GLUCOSE UPTAKE and metabolism as a source of energy. Although the number of members of the transporter family is expanding, GLUT1 is ubiquitously expressed and is responsible for constitutive uptake in mammalian cells. The regulation of GLUT1 expression and activity has been intensively studied. Our focus has been on the nutrient-dependent regulation of glucose transport in 3T3-L1 adipocytes. Specifically, we (and others) have shown that a decrease in extracellular glucose concentration increases transport activity in 3T3-L1 adipocytes in a protein synthesisdependent fashion (27,39,55,56). Although complete glucose deprivation increases transport activity 10-to 15-fold, a change of fourfold is observed within the range of 2.5 to 25 mM, the limits of circulating glucose concentration in diabetic patients, suggesting physiological relevance. Although 3T3-L1 adipocytes express both GLUT1 and GLUT4, the insulin-stimulated transporter, the effec...

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