INTRODUCTION

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The GLUT4 glucose transporter is expressed predominantly in adipose and muscle tissues, where it accounts for the bulk of insulin-stimulated glucose uptake (12, 84, 95). In the presence of insulin, GLUT4 is redistributed from an intracellular compartment to the plasma membrane, where it facilitates the diffusion of glucose into the cell (15, 35, 73, 77, 102). Another glucose transporter isoform, GLUT1, is also expressed in fat and muscle tissues and is present at high levels in many other cell types and in cultured cell lines. A large proportion of GLUT1 is present on the plasma membrane even in the absence of insulin. Thus, while both GLUT1 and GLUT4 recycle at the plasma membrane, only GLUT4 recycling is characterized by significant intracellular sequestration, resulting from a slow rate of exocytosis, in the absence of insulin. Insulin increases the rate of GLUT4 exocytosis, with little or no decrease in its rate of endocytosis, so that in adipocytes the proportion of GLUT4 at the cell surface increases from <10% in the absence of insulin to 35 to 50% in its presence (41, 55, 85, 113, 114).

Characterization of the intracellular insulin-responsive GLUT4-containing compartment is complicated by the fact that GLUT4 resides in several morphologically distinct locations within the cell. Ultrastructural studies have shown that GLUT4 is present in tubulovesicular structures distinct from lysosomes, as well as in a perinuclear compartment that is in close vicinity to the trans-Golgi network (39, 96-98). Recent work demonstrates that approximately 40 to 45% of intracellular GLUT4 localizes in a transferrin receptor (TfnR)-positive endosomal compartment, while 50 to 60% is in a second, TfnR-negative compartment; it is GLUT4 in this TfnR-negative compartment that is rapidly mobilized upon insulin addition (1, 32, 46, 55, 57, 65, 76). In primary adipocytes, this TfnR-negative compartment can be subdivided into separate storage and exocytic pools of GLUT4 (55, 56). Other data suggest that the TfnR-positive GLUT4 compartment is the precursor of the TfnR-negative, insulin-responsive compartment (109). Moreover, targeting motifs within GLUT4 mediate its distribution between TfnR-negative and TfnR-positive compartments (66). Studies using the cation-dependent mannose 6-phosphate receptor (CD-M6PR) as an endosomal marker also find a similar, highly insulin-responsive, M6PR-negative pool of intracellular GLUT4 in 3T3-L1 adipocytes (64). Thus, it appears that GLUT4 is sequestered out of endosomes and into a highly insulin-responsive compartment. We have recently shown that 3T3-L1 adipocytes also possess an insulin-regulated secretory compartment containing ACRP30, a tumor necrosis factor alpha-like protein produced exclusively by adipocytes (7, 87, 92). This regulated secretory compartment is distinct from the insulin-regulated compartment containing GLUT4 (7).

Kinetic studies are consistent with the notion that there are multiple compartments through which GLUT4 traffics in adipocytes. GLUT4 recycles between the plasma membrane and intracellular sites in both basal and insulin-stimulated states, yet the initial externalization of GLUT4 after insulin addition is too rapid to be explained by the steady-state rate constants for exocytosis and endocytosis in the presence of insulin (14, 85, 113). It has therefore been argued that a two-pool model, with one intracellular and one plasma membrane compartment, does not explain the observed kinetics GLUT4 externalization after insulin addition and that GLUT4 must traffic through three or more compartments (37, 85, 114). Among these compartments is postulated to be a highly insulin-responsive intracellular compartment from which GLUT4 is rapidly mobilized by insulin (37, 56, 114). The GLUT4 accumulated in this highly insulin-responsive compartment in the basal state is depleted upon insulin addition, and the steady-state exocytosis rate for GLUT4 in the continued presence of insulin becomes limited at some other step in the recycling pathway. This kinetically defined, highly insulin-responsive compartment in adipocytes corresponds well with the morphologically and biochemically defined, TfnR-negative compartments containing GLUT4 in adipocytes and myocytes, which are also depleted of GLUT4 after acute insulin stimulation (1, 32, 46, 55, 57, 65, 76).

The mechanisms controlling GLUT4 accumulation in this specialized insulin-sensitive compartment are poorly understood. The compartment is believed to be present only in muscle and fat and apparently develops early during 3T3-L1 adipocyte differentiation in cell culture (16, 19, 27, 34, 38, 39, 82, 89, 107). Some data indicate that a similar compartment to which GLUT4 is targeted is present in CHO cells, though this has been controversial (40, 44, 93, 105, 109). Kinetic studies suggest that even if some GLUT4 is targeted to a highly insulin-responsive compartment in CHO cells, sorting to this compartment is not efficient and does not constitute a major pathway by which GLUT4 traffics, in contrast to the case in adipocytes (3). Studies of the insulin-responsive aminopeptidase (IRAP), a protein of uncertain physiologic function that is thought to cotraffic with GLUT4, show that this protein participates in similar mechanisms for dynamic retention in the endosomal systems of CHO cells and 3T3-L1 adipocytes (43, 100). The assertion that the trafficking mechanisms employed by IRAP are identical those used by GLUT4 rests upon evidence that these proteins colocalize and cotraffic, with similar kinetics, under all conditions. Most data support this hypothesis (20, 23, 45, 62, 65, 81, 101). Of note, trafficking of IRAP, like that of GLUT4, is much more insulin-responsive in 3T3-L1 adipocytes than in CHO cells (43, 100). This is presumed to result from cell-type-specific differences, although, as with GLUT4, little is known about the mechanisms regulating IRAP accumulation and release from the highly insulin-responsive pool.

The present work began as a study of the cell type specificity of insulin-regulated GLUT4 trafficking. Using a novel reporter molecule to obtain detailed kinetic data, we find that GLUT4 participates in a highly insulin-responsive compartment not only in the fully differentiated 3T3-L1 adipocytes that we employ, but in undifferentiated 3T3-L1 preadipocytes as well. Such a compartment is not present in all cell types, since NIH 3T3 cells do not exhibit highly insulin-responsive trafficking. In CHO cells, we observe highly insulin-responsive trafficking only when the cells are cultured identically to 3T3-L1 adipocytes, in Dulbecco's modified Eagle's medium (DMEM). In standard F12 culture medium, the cells are less responsive. The highly insulin-responsive kinetics correlate with basal state redistribution of intracellular GLUT4 from the perinuclear region into punctate, peripheral structures. We pinpointed the amino acid content of these media as the relevant difference causing these trafficking characteristics: thus, given sufficient concentrations of essential amino acids, GLUT4 accumulates in a highly insulin-responsive compartment in CHO cells. Our data show that amino acids regulate GLUT4 accumulation in this compartment through a rapamycin-sensitive pathway. Finally, we demonstrate that amino acid sufficiency also modulates highly insulin-responsive GLUT4 trafficking in 3T3-L1 adipocytes and that this response is also rapamycin sensitive. Our data are consistent with the notion that both 3T3-L1 cells and CHO cells contain peripheral, highly insulin-responsive compartments through which GLUT4 traffics and that amino acid sufficiency modulates GLUT4 trafficking through these compartments in both cell types.

	MATERIALS AND METHODS

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Antibodies and reagents. Cell culture media and supplements were purchased from Life Technologies (Grand Island, N.Y.) and JRH Biosciences (Lenexa, Kans.). Anti-c-Myc monoclonal antibody (clone 9E10) was from Babco/Covance (Richmond, Calif.) and from Roche. An anti-insulin receptor -chain antibody was purchased from BD Transduction Laboratories. Normal donkey serum and R-phycoerythrin (PE)-conjugated donkey F(ab')₂ anti-mouse immunoglobulin G (IgG) secondary antibody were purchased from Jackson Immunoresearch (West Grove, Pa.). Restriction enzymes were from New England Biolabs (Beverly, Mass.) and Pfu and Taq DNA polymerases were from Stratagene (La Jolla, Calif.). Wortmannin, LY294002, and rapamycin were from Calbiochem (La Jolla, Calif.). Oil red O and other chemicals were from Sigma (St. Louis, Mo.).

Cell culture. Murine 3T3-L1 fibroblasts were cultured in DMEM containing 10% fetal bovine serum (or 10% calf serum, where noted), and differentiation was induced according to established protocols (7, 22). Briefly, cells were allowed to reach confluence at least 2 days prior to the induction of differentiation. Differentiation was induced (on day 0) with medium containing 0.25 µM dexamethasone, 160 nM insulin, and 500 µM methylisobutylxanthine. After 48 h (day 2), the cells were fed with medium containing 160 nM insulin. After an additional 48 h (day 4), the cells were refed every 2 days with DMEM-10% fetal bovine serum. All media were supplemented with 2 mM glutamine, 100 U of penicillin per ml, and 0.1 mg of streptomycin per ml. Differentiation was monitored by noting the accumulation of lipid droplets, which typically began by day 4 of differentiation. Cells were considered fully differentiated between days 8 and 12. Throughout this report, the terms day 0 3T3-L1 cells and confluent 3T3-L1 preadipocytes are used interchangeably.

Construction of a GLUT4 reporter. A human GLUT4 cDNA containing a c-Myc epitope tag in the first exofacial loop was kindly provided by Zhijun Luo and Joseph Avruch (Massachusetts General Hospital, Boston). This clone had been constructed as described by Kanai et al. (44). We fused the green fluorescent protein (GFP) coding sequence in frame to the carboxy terminus of this GLUT4 clone based on the results of Dobson et al. (18) that GLUT4-GFP appears to localize and traffic similarly to wild-type GLUT4. The GFP coding sequence from pEGFP-N1 (Stratagene) was first cloned into the pMX retroviral vector using EcoRI and NotI to generate plasmid pMX-GFP (68). PCR was done using primers 5'-GACATTTGACCAGATCTCGG-3' and 5'-GGCCCGCGGGTCATTCTCATCTGGCCC-3' to generate a ~110-bp BglII-SacII fragment from the 3' end of the rat GLUT4 cDNA (11). This PCR product and an EcoRI-BglII fragment containing most of the GLUT4myc cDNA were used in a three-way ligation with EcoRI- and SacII-digested pMX-GFP to generate pMX-GLUT4myc-GFP. Next, six additional myc epitope tags were added in tandem with the existing myc epitope tag, for a total of seven myc epitope tags. We first used PCR to amplify a ~240-bp EcoRI-HindIII fragment including the 5' end of the rat GLUT4 cDNA and part of a myc tag, using primers 5'-CCGGCCGAATTCATGCCGTCGGGTTTCCAGCAGATC-3' and 5'-CTTCAGAAATAAGCTTTTGCTCCTCTGCAGGACCCTGCCTACCCAGCCAAGTTGC-3'. This fragment was used to replace a corresponding fragment in pMX-GLUT4myc-GFP, creating a unique HindIII site within the myc epitope tag. A HindIII fragment containing six tandem myc epitope tags was amplified from plasmid pCS2+MT, a gift from Bill Schiemann (Whitehead Institute), using primers 5'-CCATCGATTTAAAGCTATGGAGCAAAAGCTTATTTCTGAAGAGG-3' and 5'-CAGAAATAAGCTTTTGCTCCTCTGCAGGCTCAAGAGGTCTTGAGTTCAAGTCCTCTTC-3'. This fragment was inserted into the HindIII site of pMX-GLUT4myc-GFP, creating pMX-GLUT4myc7-GFP. The entire coding regions of the pMX-GLUT4myc-GFP and pMX-GLUT4myc7-GFP plasmids were verified by sequencing. The GLUT4myc7-GFP coding sequence was also placed in the pB retrovirus vector in order to optimize the potential translation efficiency; pB is identical to pMX except that two point mutations were introduced to eliminate potential start codons 5' of the cloning site (J. S. Bogan, X. Liu, A. E. McKee, and H. F. Lodish, unpublished data). In this report, the GLUT4 reporter refers to that encoded by the GLUT4myc7-GFP sequence.

Production of retroviral supernatant and isolation of infected cell populations. Phoenix or VE23 ecotropic packaging cells were transfected with the pMX-GLUT4myc-GFP, pMX-GLUT4myc7-GFP, or pB-GLUT4myc7-GFP plasmid using calcium phosphate as described (51, 99, 103). In some instances, Phoenix cells were transfected using Fugene 6 (Roche) per the manufacturer's protocol. Media containing recombinant retroviruses were harvested 48 or 72 h after transfection and used to infect dividing 3T3-L1 preadipocytes, NIH 3T3 cells, or CHO cells expressing the murine ecotropic receptor. For 3T3-L1 preadipocytes infected with pMX-GLUT4myc7-GFP, flow cytometry demonstrated the presence of GFP in >90% of cells after infection. We isolated stable populations of infected cells expressing large, medium, and small amounts of the reporter by flow sorting cells falling within narrow ranges of GFP fluorescence. The sorted cells were expanded and differentiated into adipocytes, and insulin-stimulated GLUT4 trafficking (stimulated/basal) was measured by flow cytometry in all cases. We reasoned that there might be a trade-off between signal/noise (with small amounts of the reporter) and potential saturation of a trafficking mechanism (at large amounts of the reporter). In general, we chose medium- or high-expressing cells for use in subsequent experiments, since these had the greatest fold increase in cell surface GLUT4 after insulin treatment. These cells generally contained 5 to 10 times as much reporter protein as native GLUT4 in the mature 3T3-L1 adipocytes, as judged by immunoblotting with antibodies directed against the N and C termini of GLUT4 (kindly provided by Maureen Charron, Albert Einstein College of Medicine, Bronx, N.Y.). Similar optimization of reporter protein expression levels was carried out in NIH 3T3 and CHO cells.

Measurement of plasma membrane GLUT4 trafficking by flow cytometry. Confluent cells were reseeded on the indicated day of differentiation to six-well plates (Corning; Costar no. 3506) 1 day before use in experiments and starved in DMEM without fetal bovine serum for at least 3 h before insulin stimulation. Insulin was generally used at 160 nM; we occasionally used 80 or 200 nM and noted no difference between these concentrations in either 3T3-L1 or CHO cells. Insulin was added directly to the wells from a 100× stock. After treatment in the presence or absence of insulin for the times indicated in each figure, cells were quickly transferred to 4°C and washed with cold phosphate-buffered saline (PBS) containing 0.9 mM Ca²⁺ and 0.5 mM Mg²⁺ (PBS⁺⁺). All subsequent steps were carried out at 4°C, and staining of externalized myc epitope was done on adherent cells. Cells were incubated with a 1:200 dilution of anti-Myc (9E10) ascites or with purified 9E10 (25 µg/ml) in PBS⁺⁺-2% bovine serum albumin (BSA)-4% donkey serum for 1.5 h. Cells were then washed twice in PBS⁺⁺ for 5 min each time. They were then incubated for 45 min in 12.5 to 25 µg of PE-conjugated donkey F(ab')₂ anti-mouse IgG secondary antibody per ml, diluted in PBS⁺⁺-2% BSA-4% donkey serum. Cells were rinsed twice in PBS⁺⁺, then washed three times in PBS⁺⁺ for 10 min each, and resuspended by gentle scraping in PBS⁺⁺-2% BSA or PBS⁺⁺-5% fetal bovine serum for flow cytometry. For experiments involving insulin removal, cells were chilled as above after insulin stimulation and then washed twice with 5 mM sodium acetate-150 mM NaCl (pH 4.0) (48, 112). Cells were rewarmed in 37°C DMEM for the times indicated, restimulated with insulin or not restimulated, then returned to 4°C, and chilled with cold PBS⁺⁺ before staining as above.

t  1

t  1

Subcellular fractionation. Four 10-cm plates of 3T3-L1 adipocytes were used per condition to isolate low-density microsomal and plasma membrane fractions (Fig. 1b). Eight and ten plates per condition were used to isolate low-density microsomes for analysis by sucrose density gradient centrifugation (Fig. 1c) and in vesicle immunopurification experiments (Fig. 1d), respectively. Five plates of adipocytes and 10 plates of preadipocytes were used per condition in the experiments presented in Fig. 3b and 4b, respectively. For experiments presented in Fig. 1b and 1c, cells were serum starved overnight, treated in the presence or absence of insulin (160 nM, 10 min), then transferred to 4°C, and washed with cold PBS⁺⁺. For the vesicle immunopurification (Fig. 1d), cells were starved overnight, then transferred to 4°C, and washed with cold PBS⁺⁺ without insulin stimulation. For kinetic studies (Fig. 3b and 4b), 480 nM insulin was used (added from a prewarmed 3× stock) in order to maximally and simultaneously stimulate all of the cells on each 10-cm dish. Cell were transferred to 4°C and washed with cold PBS⁺⁺ at the indicated times. In all cases, cells were next washed once with cold 250 mM sucrose-10 mM Tris (pH 7.4)-0.5 mM EDTA (buffer A). Cells were resuspended by scraping in cold buffer A with Complete protease inhibitors (Roche) and then homogenized using 16 strokes (four plates) or 25 strokes (eight plates) in a Dounce-type Teflon tissue grinder (Kontes no. 22; VWR). All subsequent steps were performed at 4°C. The homogenate was centrifuged at 11,500 rpm in an SS-34 rotor (16,000 × g) for 20 min. The pellet was resuspended in buffer A and then layered on top of 1.12 M sucrose-10 mM Tris (pH 7.4)-0.5 mM EDTA in a ~2-ml centrifuge tube. The samples were centrifuged in a TLS-55 rotor at 36,000 rpm (158,000 × g) for 20 min. The interface was removed using a syringe, diluted in buffer A, and centrifuged in a TLA-100.2 rotor at 37,000 rpm (74,000 × g) for 9 min. The pellet was resuspended in buffer A and centrifuged again under identical conditions. The pellet from this centrifugation, designated PM, was resuspended in TNET (1% Triton X-100, 150 mM NaCl, 20 mM Tris [pH 8.0], 2 mM EDTA) or in radioimmunoprecipitation assay (RIPA) buffer (PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS]) and stored at 20°C until needed.

Other methods. Immunofluorescence microscopy of permeablized cells was done essentially as described (7). Cells were fixed with 4% paraformaldehyde in PBS for 40 min, permeablized with 0.2% Triton X-100 for 5 min, and then washed extensively with PBS⁺⁺. Staining of CHO cells expressing the GLUT4 reporter was done in order to increase the fluorescent signal from that due to GFP alone and employed a monoclonal anti-Myc antibody and a fluorescein isothiocyanate (FITC)-conjugated secondary antibody.

	RESULTS

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Novel assay for GLUT4 trafficking at the plasma membrane. To assay changes in the proportion of GLUT4 that is present at the plasma membrane, we constructed a cDNA encoding a GLUT4 reporter protein. This protein contains seven c-Myc epitope tags in the first exofacial loop of GLUT4 and GFP fused in-frame at the carboxy terminus. As shown in Fig. 1a, expression of this protein in cells enables us to measure changes in the proportion of GLUT4 at the cell surface as changes in a ratio of fluorescence intensities. We use an anti-Myc monoclonal antibody followed by a PE-conjugated secondary antibody to detect reporter protein present at the surface of living cells. Green (GFP) fluorescence indicates the total amount of the reporter present in each cell. Thus, the ratio of PE to GFP fluorescence intensities corresponds to the proportion of total GLUT4 that is present at the plasma membrane. We employ flow cytometry to measure these fluorescence intensities simultaneously and on a cell-by-cell basis.

View larger version (151K): [in this window] [in a new window]   FIG. 1.   Assay for changes in proportion of GLUT4 at the plasma membrane. A GLUT4 reporter containing Myc epitope tags in the first exofacial loop as well as GFP fused in frame at the carboxy terminus was constructed as described in Materials and Methods. As shown in panel a, this reporter enables measurement of changes in the proportion of GLUT4 at the plasma membrane as changes in the ratio of fluorescence intensities corresponding to cell surface and total amounts of the reporter. Cell surface GLUT4 reporter is detected using an anti-Myc primary antibody and PE-conjugated secondary antibody. Total GLUT4 reporter is proportional to GFP fluorescence. (b) Low-density microsome (LDM) and plasma membrane (PM) fractions were isolated from 3T3-L1 adipocytes expressing the reporter and analyzed by SDS-PAGE and immunoblotting. Equal amounts of protein were loaded in each lane. Immunoblotting was done using an antibody directed against the carboxyl terminus of GLUT4 and demonstrates that both the reporter (95 kDa) and native GLUT4 (50 kDa) are redistributed from the LDM fraction to the PM after acute insulin treatment. The amount of translocation is quantitatively similar. (c) The LDM fractions from basal and insulin-stimulated 3T3-L1 adipocytes expressing the reporter and from control cells were further separated by sedimentation on a 10 to 30% linear sucrose gradient, as described in Materials and Methods. Equal volumes of each gradient fraction were analyzed by to determine total protein and by SDS-PAGE and immunoblotting to detect native GLUT4 (in control cells, using an anti-GLUT4 antibody) and the GLUT4 reporter (using an anti-Myc antibody). As shown in the top panels, the reporter and endogenous GLUT4 cosediment in both basal (left) and insulin-stimulated (right) cells. Densitometry was used to quantify the bands (middle panels), and data are plotted as the percentage of the total reporter or native GLUT4 present in each gradient fraction; these profiles are quite similar. As a control, the percentage of total protein present in each gradient fraction is plotted (lower panels); these profiles are similar to each other and distinct from those of the GLUT4 reporter and endogenous GLUT4. (d) LDM fractions from unstimulated 3T3-L1 adipocytes expressing the reporter and from control cells not expressing the reporter were used in vesicle immunopurification experiments. LDM fractions were incubated with two pooled anti-GFP monoclonal antibodies, followed by protein G-Sepharose beads. After pelleting and washing of the beads, the immunopurified material was eluted in sample buffer and analyzed by SDS-PAGE and immunoblotting with an anti-GLUT4 rabbit polyclonal antibody. As shown in the upper two panels, native GLUT4 is detected in the immunopurified material. The lower panels present a similar immunoblot of the supernatants and demonstrate that even though the immunopurification did not quantitatively remove all of the GLUT4 reporter, the endogenous GLUT4 is depleted, as expected. (e) Flow cytometry is used to quantify the insulin-stimulated change in the proportion of GLUT4 at the plasma membrane of 3T3-L1 adipocytes expressing the reporter protein. Serum-starved cells were treated or not with insulin, chilled, stained for externalized Myc epitope tag, and analyzed by FACS as described in Materials and Methods. PE and GFP fluorescence intensities are plotted on the vertical and horizontal axes, respectively, of the dotplots presented. Note that both scales are logarithmic. Compared to the background fluorescence of cells not expressing the reporter (yellow), cells expressing the reporter (blue) have increased GFP fluorescence (leftmost panel). Among cells expressing the reporter, unstained cells (blue) and basal (stained for cell surface Myc, shown in red) and insulin-stimulated (stained for cell surface Myc, shown in green) populations have progressively increasing PE fluorescence with no change in GFP fluorescence. Control experiments show that the background staining is negligible (not shown). The four panels allow direct comparison of pairs of samples. In this experiment, insulin caused a fourfold increase in the ratio of median fluorescence intensities attributable to externalized Myc epitope and to GFP expression, corresponding to a fourfold increase in the proportion of total GLUT4 present at the cell surface. (f) Flow cytometry was used to measure insulin-stimulated GLUT4 translocation in confluent CHO cells. As in panel e, PE fluorescence (proportional to cell surface GLUT4 reporter) is plotted on the vertical axis and GFP fluorescence (proportional to total GLUT4 reporter) is plotted on the horizontal axis; both scales are logarithmic. Background (unstained) cells expressing the reporter are shown in blue, and basal and insulin-stimulated populations are shown in red and green, respectively. The three panels allow direct comparison between each pair of samples. There is a minor population of unstained cells (blue) within the first decade of each scale; these cells do not express the GLUT4 reporter and conveniently demonstrate that the flow cytometer is properly adjusted to compensate for fluorophore bleedthrough. Compared to 3T3-L1 adipocytes, the background fluorescences (both PE and GFP) account for much less of the total fluorescence intensities in CHO cells, and the signal-noise ratio is correspondingly increased (compare panels e and f). In this experiment, insulin stimulated a 3.5-fold increase in the proportion of total GLUT4 present at the cell surface.

Insulin stimulates GLUT4 translocation similarly in undifferentiated 3T3-L1 cells and throughout 3T3-L1 adipose differentiation. One difficulty in working with 3T3-L1 cells is that if the undifferentiated fibroblasts are allowed to become confluent, they must be induced to undergo adipose differentiation or they will lose that capacity. This characteristic makes the introduction of exogenous proteins by stable transfection technically difficult, since the cells invariably become confluent during clonal selection. We circumvented this difficulty by isolating a pool of cells infected with a replication-deficient retrovirus encoding the reporter protein. Over 90% of the target 3T3-L1 fibroblasts were infected, and those falling within a narrow window of GFP fluorescence were isolated by FACS; individual cells in this sorted population express similar amounts of the reporter. Figure 2a demonstrates that these sorted cells undergo normal 3T3-L1 adipose differentiation, as assessed by Oil red O staining to highlight the development of intracellular lipid droplets.

View larger version (177K): [in this window] [in a new window]   FIG. 2.   Adipose differentiation and GLUT4 translocation in 3T3-L1 cells. 3T3-L1 preadipocytes were infected with a retrovirus containing the GLUT4 reporter, and flow sorting was used to isolate a population of cells falling within a narrow range of GFP fluorescence intensities. These cells were expanded and used in experiments; because the retrovirus integrates into the genome, the population is stable. These cells undergo normal 3T3-L1 adipose differentiation, as demonstrated by staining lipid with Oil red O. (a) Phase-contrast (upper left) and bright-field (upper right and lower left and right) microscopy of cells at the indicated days of differentiation is shown. Scale bar, 50 µm. (b) Confluent 3T3-L1 preadipocytes (day 0) or 3T3-L1 cells that had undergone differentiation for various lengths of time were stimulated or not with insulin (160 nM, 10 min), and changes in the proportion of GLUT4 reporter present at the cell surface were measured using flow cytometry as described in the text. Some samples were treated with either 100 nM wortmannin (42) or 50 µM LY294002 (8, 13) for 40 min prior to insulin addition, as noted. The amount of the reporter within each cell varies during 3T3-L1 differentiation and is increased approximately threefold on days 2 and 4 (not shown). We attribute this to increased activity of the retroviral promoter as the cells undergo clonal expansion at the onset of adipocyte differentiation, especially since we also observed increased expression of the reporter in preconfluent, dividing cells (91). Because the assay measures changes in the ratio of cell surface to total GLUT4 rather than in the absolute amount of cell surface GLUT4, the data presented are internally controlled for this variation, and data from different days of differentiation can be meaningfully compared. The numbering on the vertical scale indicates a relative measure of GLUT4 at the cell surface, and these arbitrary units cannot be compared in absolute terms to those in other figures. In all instances, insulin stimulates GLUT4 exocytosis, and this effect is blocked by either of the two phosphatidylinositol-3-kinase inhibitors.

View larger version (92K): [in this window] [in a new window]   FIG. 3.   Kinetics of GLUT4 trafficking in 3T3-L1 cells. (a) Confluent 3T3-L1 preadipocytes (day 0) or 3T3-L1 cells at various stages of adipocyte differentiation (as indicated) were treated with insulin for various lengths of time, and changes in the proportion of GLUT4 reporter present at the cell surface were analyzed. Data are plotted for basal cells and for cells treated with 80 nM insulin for 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 10, 12, 15, 20, or 30 min. Membrane trafficking was stopped by washing with cold PBS, cells were stained at 4°C for externalized Myc epitope tag using a PE-conjugated secondary antibody, and PE and GFP fluorescence intensities were measured using flow cytometry as described in the text. Regardless of the state of differentiation, insulin causes a rapid externalization of GLUT4 that peaks 4 to 5 min after insulin addition. Subsequently, there is a net internalization, so that a steady state in the presence of insulin is reached 20 min after insulin addition. The numbering on the vertical scale indicates a relative measure of GLUT4 at the cell surface, and these arbitrary units cannot be compared in absolute terms to those in other figures. (b) Insulin-stimulated translocation of endogenous GLUT4 to the plasma membrane of 3T3-L1 adipocytes was analyzed by subcellular fractionation. Cells were starved overnight and then stimulated with 480 nM insulin (added from a prewarmed, 3× stock) for various amounts of time. Cells were transferred to 4°C, washed with cold PBS, and fractionated as described in Materials and Methods. Plasma membrane (PM) and low-density microsomal (LDM) fractions were analyzed by immunoblotting. Equal amounts of protein were loaded in each lane. GLUT4 translocates to the plasma membrane in a biphasic pattern, with a peak at 7 min after insulin addition, followed by a subsequent decrease. A reciprocal pattern is observed in the LDM fraction. As a control, the PM immunoblot was reprobed with an anti-insulin receptor (-subunit) antibody, which demonstrates similar amounts of insulin receptor at the plasma membrane at all time points. The experiment was performed twice, with similar results each time. (c) 3T3-L1 preadipocytes (day 0) or cells at various times during adipocyte differentiation were stimulated with 80 nM insulin for 20 min, then placed at 4°C, and washed with an acidic buffer to remove insulin. Cells were rewarmed in serum-free medium to allow GLUT4 reinternalization for 6, 12, 18, 24, 30, 40, 60, 90, or 120 min; some cells that had been rewarmed for 120 min were restimulated with 80 nM insulin for 5, 10, or 15 min. Cells were stained for cell surface Myc epitope and analyzed by flow cytometry as described in the text. In all instances, the GLUT4 reporter was reinternalized after removal of insulin and recycles upon readdition of insulin.

View larger version (75K): [in this window] [in a new window]   FIG. 4.   Kinetics of GLUT4 trafficking in 3T3-L1 preadipocytes and NIH 3T3 cells. (a) Confluent 3T3-L1 preadipocytes and NIH 3T3 cells expressing similar amounts of the reporter were treated with 160 nM insulin for various lengths of time, chilled, and analyzed by FACS to measure changes in the proportion of GLUT4 reporter present at the cell surface. Insulin caused a much more marked redistribution of GLUT4 to the plasma membrane of 3T3-L1 preadipocytes than of NIH 3T3 cells. (b) Translocation of the GLUT4 reporter in 3T3-L1 preadipocytes was assayed by subcellular fractionation and immunoblotting. After serum starvation, cells were stimulated with 480 nM insulin (added from a prewarmed 3× stock) for various amounts of time, then washed with cold PBS++, and fractionated as described in Materials and Methods. Equal amounts of protein were loaded in each lane. Insulin caused an increase in the amount of GLUT4 reporter in the plasma membrane (PM) fraction, with a peak response at 8 min after insulin addition and a subsequent decrease at 20 min. A reciprocal pattern is apparent in the low-density microsomal (LDM) fraction. The experiment was performed twice, with similar results each time. (c) Confluent 3T3-L1 preadipocytes were cultured for 3 days in either 10% fetal bovine serum or 10% calf serum. Cells were then starved, stimulated with 160 nM insulin for various amounts of time, and analyzed by flow cytometry. Samples were measured in duplicate (control samples were in triplicate or quadruplicate). Insulin caused similar increases in the fraction of GLUT4 reporter at the plasma membrane under both conditions. The experiment was done twice, with similar results each time.

Kinetics of insulin-stimulated GLUT4 translocation in CHO cells are medium dependent. To determine if the kinetics of insulin-stimulated GLUT4 externalization are similar in CHO cells and in 3T3-L1 cells, we assayed changes in the proportion of GLUT4 at the cell surface of CHO cells stimulated for various amounts of time. To parallel the conditions used for 3T3-L1 cells, some CHO cells were placed in DMEM 2 days before the experiment; others were left in standard CHO medium (F12). As shown in Fig. 5, CHO cells cultured in DMEM respond to insulin with a dramatic, biphasic redistribution of GLUT4 to the cell surface. In contrast, CHO cells cultured in F12 medium redistributed GLUT4 less dramatically and with no overshoot of the final steady-state proportion of GLUT4 at the cell surface in the presence of insulin. As with 3T3-L1 cells, GLUT4 externalization in DMEM-cultured CHO cells peaks at 4 to 5 min after insulin addition and then decreases to reach a steady state by 20 min after insulin addition. The peak fraction of GLUT4 at the cell surface is 5.5-fold more than that in unstimulated cells, and in several experiments 50 to 60% of this increase is eliminated in the subsequent decrease. For comparison, the peak response of 3T3-L1 adipocytes in Fig. 3a was 5.4-fold over basal (average of days 8 and 10), though the subsequent decrease to steady state was only ~20% of this peak response.

View larger version (18K): [in this window] [in a new window]   FIG. 5.   Culture conditions modulate the kinetics of insulin-stimulated GLUT4 translocation in CHO cells. Two days before the experiment, confluent CHO cells were placed in DMEM identical to that used for 3T3-L1 adipocytes or left in F12 culture medium. Cells were starved overnight before the experiment. On the day of the experiment, cells were stimulated with 80 nM insulin for 0, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or 30 min, then transferred to 4°C, and washed with cold PBS. Staining and flow cytometry were done as described in the text. For cells cultured in DMEM, insulin stimulated a rapid increase in GLUT4 reporter present at the cell surface, peaking at 4 to 5 min after insulin addition. Subsequently, the proportion of GLUT4 on the plasma membrane decreases, and a steady state in the presence of insulin is reached 20 min after insulin addition. In contrast, cells cultured in F12 medium externalized GLUT4 with monophasic kinetics, characterized by no overshoot of the final steady-state response in the presence of insulin. Both the initial and final proportions of GLUT4 at the cell surface are essentially unaffected by the culture conditions, despite the marked effect on intermediate time points. For cells cultured in DMEM, the peak response is a 5.5-fold increase in the proportion of GLUT4 at the cell surface compared to the basal state. The kinetics and the amplitude of the peak response are similar in CHO cells cultured in DMEM and in 3T3-L1 cells (compare to Fig. 3a, 4a, and 4c).

View larger version (110K): [in this window] [in a new window]   FIG. 6.   Culture conditions modulate the subcellular distribution of GLUT4 in CHO cells. CHO cells expressing the GLUT4 reporter were plated on coverslips, allowed to reach confluence, and then treated as described in the legend to Fig. 5. Two days before microscopy, cells were changed to DMEM or continued in F12 culture medium. On the day of microscopy, cells were serum starved for 3 h and then stimulated with 160 nM insulin for the times indicated. (a) Cells were chilled and stained without fixation or permeablization to detect externalized Myc epitope tag. A red (Alexa594-conjugated) secondary antibody was used, and the images are shown in the first (F12) and third (DMEM) rows. GFP was used to detect the total cellular GLUT4 reporter, and images are shown in green in the second (F12) and fourth (DMEM) rows. Cells cultured in F12 medium have the greatest amount of GLUT4 at the cell surface at 20 min, whereas those cultured in DMEM have more at the cell surface at 5 min. Scale bar, 10 µm. (b) The subcellular distribution of the GLUT4 reporter is more closely examined. These cells were fixed, permeablized, and stained with anti-Myc antibody and a FITC-conjugated secondary antibody in order to increase the total green fluorescent signal, which is then due to the combination of GFP and FITC. In cells cultured in F12, there is prominent staining of the GLUT4 reporter in the perinuclear region; this does not change significantly with short-term insulin treatment (bottom panels, arrowheads). In contrast, the GLUT4 reporter is absent from the perinuclear region in unstimulated cells cultured in DMEM and is present more prominently in punctate, peripheral structures (top left panel). Insulin treatment for 5 min results in a dramatic accumulation of GLUT4 at the plasma membrane in the cells cultured in DMEM (top center panel, arrows). In contrast, cells cultured in F12 medium have much less plasma membrane GLUT4 after 5 min of insulin treatment (bottom center panel). By 20 min after insulin addition, plasma membrane GLUT4 is similar in cells cultured in the two culture media (top right and bottom right panels) and is less prominent than at 5 min in the cells cultured in DMEM (compare to top center panel). The changes observed at the plasma membrane by microscopy correlate well with those quantified by flow cytometry (Fig. 5). Additionally, microscopy demonstrates that when the cells are cultured in DMEM rather than F12 medium, GLUT4 is distributed away from the perinuclear region and into punctate structures in the periphery. Scale bar, 10 µm.

Amino acid sufficiency modulates highly insulin-responsive GLUT4 trafficking in CHO cells and in 3T3-L1 adipocytes. DMEM and F12 medium differ in several respects. Though DMEM has greater glucose and calcium concentrations, neither of these components alone or in combination proved necessary or sufficient to cause highly insulin-responsive (i.e., biphasic) kinetics (data not shown). We noted that many essential amino acids are present at markedly higher concentrations in DMEM than in F12 (see above) and tested the possibility that these are required for highly insulin-responsive GLUT4 trafficking. After culture for 24 to 36 h in various media, we examined the kinetics of insulin-stimulated GLUT4 translocation in CHO cells. As shown in Fig. 7a, the degree of overshoot of the final steady-state response in the presence of insulin correlates quite well with the concentration of most essential amino acids in different media. DMEM has twofold the concentrations of most amino acids present in MEM, which in turn has 2- to 12-fold greater concentrations of most amino acids than F12. In MEM made without any amino acids, we observed no overshoot of the final, steady-state proportion of GLUT4 at the plasma membrane in the presence of insulin. We also tested the kinetics of GLUT4 externalization in CHO cells cultured in MEM with 2×, 1×, 0.2×, or no amino acids, as shown in Fig. 7b. Higher concentrations of amino acids result in a greater overshoot of the final steady-state response in the presence of insulin. Since glutamine was held constant, we surmise that flux through the glucosamine pathway is not likely to be responsible for this effect.

View larger version (23K): [in this window] [in a new window]   FIG. 7.   Amino acid concentrations regulate the amount of rapidly insulin-mobilized GLUT4 in CHO cells. (a) CHO cells expressing the reporter were cultured in the indicated media for 36 h and serum starved during the last 12 h of this period. Cells were stimulated with 160 nM insulin for various amounts of time, then chilled, stained for externalized Myc epitope tag, and analyzed by FACS as described in the text to determine the relative proportion of GLUT4 at the cell surface in each sample. Compared to the amino acid concentrations in standard MEM (defined as 1× amino acids), concentrations of most amino acids in DMEM are twofold higher, and concentrations of most amino acids in F12 are only 0.08 to 0.5 times as high (depending on the particular amino acid; see Materials and Methods). All media contained 2 mM glutamine. The degree to which insulin stimulates a transient overshoot of the final, steady-state proportion of GLUT4 at the plasma membrane correlates well with the concentrations of essential amino acids in the various media. The data shown are from two separate experiments (mean ± standard deviation) and are normalized to the steady-state response in the presence of insulin (30-min time point). (b) A similar experiment was performed with cells cultured in MEM containing various concentrations of essential amino acids except for glutamine, which was held constant (see text). Higher amino acid concentrations cause a greater overshoot of the final, steady-state fraction of GLUT4 at the plasma membrane after insulin addition.

View larger version (28K): [in this window] [in a new window]   FIG. 8.   Rapamycin treatment diminishes the amount of rapidly insulin-mobilized GLUT4 in CHO cells. CHO cells expressing the GLUT4 reporter were cultured in DMEM, treated with the indicated concentrations of rapamycin for 36 h, and serum starved during the last 12 h of this period. Cells were stimulated with 160 nM insulin for the indicated times, then chilled, stained for externalized Myc epitope, and subjected to flow cytometry to determine the relative proportion of GLUT4 at the cell surface in each sample. Increasing concentrations of rapamycin caused a progressive diminution of the first phase of GLUT4 externalization (i.e., the overshoot before the steady-state response). Simultaneously, the proportion of GLUT4 at the cell surface in the basal state increased slightly but progressively with increasing rapamycin concentration. Together with the data presented in Fig. 7, these data show that amino acid abundance regulates the amount of rapidly insulin-translocated GLUT4 in CHO cells through a rapamycin-sensitive mechanism.

View larger version (22K): [in this window] [in a new window]   FIG. 9.   Amino acid sufficiency modulates insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. 3T3-L1 adipocytes expressing the GLUT4 reporter were cultured in MEM with the indicated concentrations of amino acids for 36 h and serum starved during the last 12 h. Cells were stimulated with 160 nM insulin for the indicated times, then chilled, stained for externalized Myc epitope tag, and analyzed by flow cytometry to determine the relative proportion of GLUT4 at the cell surface in each sample. MEM with 2× amino acids approximates the amino acid concentrations found in DMEM (see Materials and Methods). Culture of the cells in media with poor amino acid availability results in reduced translocation of GLUT4 to the cell surface after insulin stimulation.

View larger version (22K): [in this window] [in a new window]   FIG. 10.   Rapamycin treatment diminishes insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. 3T3-L1 adipocytes expressing the GLUT4 reporter were cultured in DMEM with the indicated concentrations of rapamycin for 36 h and starved for the final 12 h of this period. Cells were then stimulated with 160 nM insulin for the indicated amounts of time, chilled, stained to detect externalized Myc epitope, and subjected to FACS to determine the relative proportion of GLUT4 at the cell surface in each sample. In the presence of increasing concentrations of rapamycin, GLUT4 was translocated to progressively fewer degrees by insulin stimulation.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

We have shown that insulin triggers rapid exocytosis of GLUT4 in 3T3-L1 adipocytes and preadipocytes and in CHO cells, but not in NIH 3T3 cells. This conclusion is based on studies using a novel, FACS-based assay to measure changes in the proportion of GLUT4 present at the plasma membrane and is supported by subcellular fractionation data. This action of insulin is blocked by either of two structurally dissimilar phosphatidylinositol-3-kinase inhibitors in 3T3-L1 cells at all times during differentiation, suggesting that identical signaling mechanisms are involved. Moreover, insulin stimulates GLUT4 externalization with identical, biphasic kinetics at all times during 3T3-