Analysis of the co-localization of the insulin-responsive glucose transporter (GLUT4) and the trans Golgi network marker TGN38 within 3T3-L1 adipocytes.
The exposure of isolated adipocytes to insulin results in an approximately 20-fold increase in the rate of glucose transport into the cell. This increase is mediated by the movement of a pool of intracellular vesicles containing the so-called insulin-responsive glucose transporter (GLUT4) to the cell surface. In the resting state, most of the GLUT4 molecules are sequestered inside the adipocyte in an as yet unidentified intracellular compartment. TGN38 is an integral membrane protein which has been shown to be predominantly localized to the trans Golgi network [Luzio, Brake, Banting, Howell, Braghetta and Stanley (1990) Biochem. J. 270, 97-102]. Here we investigate whether GLUT4 and TGN38 are co-localized in the murine 3T3-L1 adipocyte cell line. Immuno-adsorption of intracellular vesicles containing GLUT4 with an anti-peptide antibody specific for this isoform did not deplete the low-density microsomal fraction of TGN38 in these cells; moreover, no TGN38 was detected in the GLUT4-containing vesicles by immunoblotting with a TGN38-specific antiserum. Immuno-adsorption of TGN38-containing vesicles and subsequent analysis of the proteins in these vesicles revealed that a detectable amount of GLUT4 (5-10%) did co-localise with TGN38. The amount of GLUT4 in the TGN38-containing vesicles did not change in response to insulin. Immunofluorescence analysis of TGN38 and GLUT4 in these cells revealed markedly different staining patterns. Reversal of insulin-stimulated glucose transport and subsequent analysis of the TGN38-containing vesicles demonstrated that during the re-cycling of GLUT4 to the intracellular storage site there was no increase in the amount of GLUT4 co-localized with TGN38. Taken together, these results suggest that the trans Golgi network is not the major site of the intracellular GLUT4 pool within 3T3-L1 adipocytes.
Department of Biochemistry, University of Glasgow, Scotland, U.K.
S
S
Martin
B
B
Reaves
G
G
Banting
G W
GW
Gould
D016475:3T3 Cells; D017667:Adipocytes / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D002451:Cell Compartmentation; D005455:Fluorescent Antibody Technique; D005947:Glucose / Q000378:metabolism; D051275:Glucose Transporter Type 4; D006023:Glycoproteins; D006056:Golgi Apparatus / Q000378:metabolism; D066298:In Vitro Techniques; D007328:Insulin / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008565:Membrane Proteins; D051379:Mice; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009124:Muscle Proteins
D051275:Glucose Transporter Type 4; D006023:Glycoproteins; D007328:Insulin; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; C495693:Slc2a4 protein, mouse; C513047:Tgoln1 protein, mouse; C513046:Tgoln2 protein, mouse; D005947:Glucose