Abstract
Glucose deprivation increases the rate of glucose transport in 3T3-L1 adipocytes in a protein synthesis-dependent fashion. To determine if translocation of either GLUT1 or GLUT4 is responsible for this phenomenon, we adapted existing fractionation procedures toward isolating 3T3-L1 adipocyte membranes. By Western blot analysis of equal protein, GLUT1 was distributed between plasma membranes, high density "microsomal" membranes, and low density "microsomal" membranes isolated from control cells. GLUT4 comigrated with high density and low density membranes. Glucose deprivation for 12 h did not alter the distribution of either GLUT1 or GLUT4, despite an 8-10-fold increase in glucose transport activity in intact cells. Importantly, increased transport activity was retained in plasma membrane vesicles isolated from glucose-deprived cells. These data show for the first time that the increase in transport activity associated with glucose deprivation does not result from the translocation of either of the glucose transporters known to exist in 3T3-L1 adipocytes. As GLUT4 is excluded from the plasma membrane, these data provide evidence for activation of GLUT1.
Highlights
The 3T3-L1 cell line has provided an important tool in the study of glucose transport because this adipocyte model system allows the investigation of chronic treatments
We demonstrate that about 20% of GLUT1 resides at the cell surface in control cells and that its distribution does not change in response to glucose deprivation
We [11] and others [1,2,3] have shown previously that glucose deprivation increases the rate of glucose transport in 3T3-L1 adipocytes
Summary
Materials—Dulbecco’s modified Eagle’s medium and glucose-free Dulbecco’s modified Eagle’s medium were obtained from Life Technologies, Inc. (Gaithersburg, MD). Materials—Dulbecco’s modified Eagle’s medium and glucose-free Dulbecco’s modified Eagle’s medium were obtained from Life Technologies, Inc. Calf serum (J13605) and fetal bovine serum (LB95508) were purchased from Intergen (Milford, MA). Rabbit polyclonal antibodies for GLUT1 and GLUT4 were generated against the C-terminal 13 amino acids, respectively. The anti-GRP78 antibody was generated against a peptide containing the first 12 amino acids in the N terminus. Anti-sialyltransferase was a gift from Dr William Dunn (University of Florida). Anti-Naϩ,Kϩ-ATPase was a gift from Dr Michael Caplan (Yale University). NHS-LC1 biotin was purchased from Pierce (Rockford, IL). The steel block, ball bearing homogenizer was made by Auburn Tool and Die (Warwick, RI). All other reagents used were of analytical grade from commercial sources.
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