Compartment ablation analysis of the insulin-responsive glucose transporter (GLUT4) in 3T3-L1 adipocytes

Abstract

The translocation of a unique facilitative glucose transporter isoform (GLUT4) from an intracellular site to the plasma membrane accounts for the large insulin-dependent increase in glucose transport observed in muscle and adipose tissue. The intracellular location of GLUT4 in the basal state and the pathway by which it reaches the cell surface upon insulin stimulation are unclear. Here, we have examined the colocalization of GLUT4 with the transferrin receptor, a protein which is known to recycle through the endosomal system. Using an anti-GLUT4 monoclonal antibody we immunoisolated a vesicular fraction from an intracellular membrane fraction of 3T3-L1 adipocytes that contained 90 % of the immunoreactive GLUT4 found in this fraction, but only 40 % of the transferrin receptor (TfR). These results suggest only a limited degree of colocalization of these proteins. Using a technique to cross-link and render insoluble (' ablate ') intracellular compartments containing the TfR by means of a transferrin-horseradish peroxidase conjugate (Tf-HRP), we further examined the relationship between the endosomal recycling pathway and the intracellular compartment containing GLUT4 in these cells. Incubation of non-stimulated cells with Tf-HRP for 3 h at 37 mC resulted in quantitative ablation of the intracellular TfR, GLUT1 and mannose-6-phosphate receptor and a shift in the density of Rab5-positive membranes. In contrast, only 40 % of intracellular GLUT4 was ablated under the same conditions. Ablation was specific for the endosomal system as there was no significant

Trafficking of Glut4–Green Fluorescent Protein chimaeras in 3T3-L1 adipocytes suggests distinct internalization mechanisms regulating cell surface Glut4 levels

“…As shown in Figure 2, these compounds were effectively internalized in cells expressing GFP-Glut4. We, and others, have suggested that a significant proportion of intracellular Glut4 resides in a compartment spatially distinct from TfR in both adipocytes and muscle [1,5,15,30,39]. The only partial co-localization of GFPGlut4 or Glut4-GFP with Texas-Red transferrin (Figure 2) provides support for these observations and further suggests that the localization of the GFP-Glut4 chimaeras studied in the present work is accurately reflecting the localization of the Trafficking of green fluorescent protein-Glut4 in adipocytes…”

“…This recycling has been shown to involve, at least in part, clathrin-coated pits [12,13] via a dynamin\amphiphysin-dependent mechanism [14], and is consistent with the observed localization of Glut4 to clathrin lattices in the plasma membrane and the partial overlap of Glut4 with the transferrin receptor (TfR) itinerary [15][16][17]. After insulin withdrawal, Glut4 is rapidly internalized from the plasma membrane and is effectively sequestered within the cell until such time as insulin is re-introduced [18][19][20].…”

GLUT4 vesicle dynamics in living 3T3 L1 adipocytes visualized with green-fluorescent protein

“…Insulin caused a substantial re-distribution of GFP-GLUT4, such that an almost continuous line of fluorescence was observed around the plasma membrane after 60 min (Figure 1b). Insulin did not cause a complete translocation of GLUT4, as would be expected from studies on the redistribution of native GLUT4 in these cells [3,4,6,18,19].…”

“…Immunofluorescence and electron-microscopic analysis of non-stimulated cells has revealed that GLUT4-containing vesicles are clustered adjacent to early or late endosomes, in the trans-Golgi reticulum, or in the cytoplasm often close to the plasma membrane [1][2][3][4]. A small proportion of GLUT4 is also localized within the constitutively recycling endocytic system, but mounting evidence favours the existence of a unique pool of insulin-regulatable GLUT4 vesicles [5,6].…”

V-type ATPase is involved in biogenesis of GLUT4 vesicles

“…The nature of the "GLUT4 pathway" in insulin-sensitive cells is not completely understood. It has been shown, however, that internalized GLUT4 passes from early endosomes (31) to recycling endosomes (17,37,40) and then to specialized insulin-responsive storage vesicles, or IRVs, that represent the final target of insulin regulation (19,22,36). Transport of GLUT4 from early to recycling endosomes may proceed via a distinct population of vesicles that are marked by the presence of cellugyrin, which is absent from the IRVs (14, 15).…”