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. 2004 Jul;24(14):6456-66.
doi: 10.1128/MCB.24.14.6456-6466.2004.

Insulin increases cell surface GLUT4 levels by dose dependently discharging GLUT4 into a cell surface recycling pathway

Roland Govers  1 , Adelle C F Coster, David E James
Affiliations

Insulin increases cell surface GLUT4 levels by dose dependently discharging GLUT4 into a cell surface recycling pathway

Roland Govers et al. Mol Cell Biol. 2004 Jul.

Abstract

The insulin-responsive glucose transporter GLUT4 plays an essential role in glucose homeostasis. A novel assay was used to study GLUT4 trafficking in 3T3-L1 fibroblasts/preadipocytes and adipocytes. Whereas insulin stimulated GLUT4 translocation to the plasma membrane in both cell types, in nonstimulated fibroblasts GLUT4 readily cycled between endosomes and the plasma membrane, while this was not the case in adipocytes. This efficient retention in basal adipocytes was mediated in part by a C-terminal targeting motif in GLUT4. Insulin caused a sevenfold increase in the amount of GLUT4 molecules present in a trafficking cycle that included the plasma membrane. Strikingly, the magnitude of this increase correlated with the insulin dose, indicating that the insulin-induced appearance of GLUT4 at the plasma membrane cannot be explained solely by a kinetic change in the recycling of a fixed intracellular GLUT4 pool. These data are consistent with a model in which GLUT4 is present in a storage compartment, from where it is released in a graded or quantal manner upon insulin stimulation and in which released GLUT4 continuously cycles between intracellular compartments and the cell surface independently of the nonreleased pool.

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Figures

FIG. 1.
FIG. 1.
Characterization of GLUT4 mutants. (A) Schematic representation of HA-tagged wild-type GLUT4 (WT), GLUT4 Tail mutant, GLUT4 L489,490A, and GLUT4 F5A. (B) 3T3-L1 adipocytes expressing HA-tagged wild-type GLUT4 were immunolabeled with anti-HA and anti-GLUT4 for the detection of HA-GLUT4 and total cellular GLUT4 content, respectively. Arrow indicates a cell that does not express HA-GLUT4. Bar, 30 μm. (C) 3T3-L1 fibroblasts (F) and 3T3-L1 adipocytes (A) expressing the indicated HA-tagged GLUT4 molecules were lysed, and equal amounts of protein were analyzed by Western blotting with the indicated antibodies. The syntaxin 4 immunoblot serves as an internal control. (D) The blots in C were analyzed as described in Materials and Methods, and GLUT4 overexpression was compared to endogenous GLUT4 expression in noninfected adipocytes. The anti-HA immunoblot was used to determine the relative expression of GLUT Tail, as this molecule is not recognized by the anti-GLUT4 antibody. (E) Nonstimulated 3T3-L1 fibroblasts and adipocytes expressing the indicated HA-GLUT4 molecules were immunolabeled with anti-HA. Bar, 10 μm.
FIG. 2.
FIG. 2.
Analysis of insulin-induced GLUT4 translocation with a novel assay. Noninfected (A) or HA-GLUT4-expressing 3T3-L1 adipocytes (B) were grown in 96-well plates and stimulated for the indicated periods with 200 nM insulin. After fixation, cells were incubated with or without saponin to allow the labeling of all cellular GLUT4 (total) and PM-localized GLUT4 (PM), respectively. Subsequently, cells were incubated with nonrelevant (NR) or anti-HA (HA) antibody and with Alexa 488-conjugated goat anti-mouse antibody, after which fluorescence was measured with a fluorescence plate reader. (C) With the fluorescence values from panel B, anti-HA-specific labeling at the plasma membrane was expressed as percentage of total anti-HA-specific labeling. (D) 3T3-L1 adipocytes were incubated for 20 min with insulin as indicated, in the absence or presence of 100 nM wortmannin, and analyzed as described for panel C. Representative experiments are shown.
FIG. 3.
FIG. 3.
Insulin-induced GLUT4 translocation in 3T3-L1 fibroblasts and adipocytes. Fibroblasts and adipocytes expressing the indicated GLUT4 molecules were incubated with 200 nM insulin for up to 25 min, and the percentage of GLUT4 at the PM was determined as in Fig. 2.
FIG. 4.
FIG. 4.
Internalization kinetics of GLUT4 in 3T3-L1 adipocytes. Adipocytes expressing the indicated GLUT4 molecules were incubated for 20 min with 200 nM insulin at 37°C and for 1 h with anti-HA antibody on ice. Excess antibody was washed away, and cells were incubated for the indicated periods at 37°C in the presence of either 100 nM wortmannin, to measure GLUT4 internalization in the basal state, or 200 nM insulin. Cells were exposed to fixative and incubated with fluorescent secondary antibody in the absence of permeabilizing agent to allow measurement of the time-dependent disappearance of anti-HA-labeled GLUT4 from the cell surface.
FIG. 5.
FIG. 5.
GLUT4 recycling in 3T3-L1 fibroblasts and adipocytes. HA-GLUT4-expressing adipocytes were incubated for 2 h with 200 nM insulin and subsequently for 2 h without insulin and for 20 min in the absence (A) or presence (B) of 200 nM insulin. Parallel cultures were incubated for 2 h with 200 nM insulin and anti-HA antibody, followed by a 2-h incubation without insulin and anti-HA and a 20-min incubation in the absence (C) or presence (D) of 200 nM insulin. HA-GLUT4 (A and B) and anti-HA antibody (C and D) were visualized by immunolabeling and confocal microscopy. Bar, 10 μm. (E) For quantitation of antibody uptake, fibroblasts and adipocytes expressing the indicated HA-GLUT4 molecules were incubated with or without 200 nM insulin for 20 min, after which anti-HA antibody was added. Cells were incubated further for up to 180 min, fixed, permeabilized, and incubated with fluorescent secondary antibody. The amount of anti-HA antibody taken up by the cells was expressed as a percentage of total postfixation anti-HA labeling.
FIG. 6.
FIG. 6.
Analysis of GLUT4 recycling during the differentiation of 3T3-L1 fibroblasts into adipocytes. Cells were analyzed at different stages during differentiation as indicated. After incubation for 18 h in medium containing fetal bovine serum and for 2 h in the absence of serum, the cells were incubated in the continuous presence of anti-HA antibody as described for Fig. 5. Parallel cultures were incubated similarly but analyzed by immunofluorescence confocal microscopy (left microscopy panels). Noninfected cells were analyzed for endogenous GLUT4 (green) and lipid droplet content (red) during differentiation (right microscopy panels). The bottom right microscopy panels show a Z-section image of the cells. White dotted lines mark the contours of the cells. Bar, 20 μm.
FIG. 7.
FIG. 7.
Correlation between insulin concentration and the size of the recycling GLUT4 pool in 3T3-L1 adipocytes. (A) 3T3-L1 adipocytes expressing HA-tagged wild-type GLUT4 and HA-GLUT4 Tail were incubated at 37°C with anti-HA antibody and the indicated concentrations of insulin and analyzed as described. (B) 3T3-L1 adipocytes expressing HA-tagged wild-type GLUT4 and GLUT4 Tail were incubated for 20 min at 37°C with 0.032, 0.24, 3.2, or 200 nM insulin, and the amounts of GLUT4 at the PM were determined and expressed as a percentage of maximal insulin-induced GLUT4 translocation. (C) HA-GLUT4-expressing 3T3-L1 adipocytes were incubated for 3 h with anti-HA antibody and the indicated concentrations of insulin. Cells were fixed, permeabilized, incubated with fluorescent secondary antibody, and analyzed by confocal immunofluorescence microscopy. Bar, 20 μm.
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