Demonstration of insulin-responsive trafficking of GLUT4 and vptr in fibroblasts

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1 Journal of Cell Science 113, (2000) Printed in Great Britain The Company of Biologists Limited 2000 JCS Demonstration of insulin-responsive trafficking of GLUT4 and vptr in fibroblasts Michael A. Lampson 1, Attila Racz 3, Samuel W. Cushman 4 and Timothy E. McGraw 2, * 1 Program in Physiology, Biophysics and Molecular Medicine and 2 Department of Biochemistry, Weill Graduate School of Medical Sciences Cornell University, New York, NY 10021, USA 3 Department of Medicine, UCSF, San Francisco, CA, USA 4 Experimental Diabetes, Metabolism and Nutrition Section, Diabetes Branch, NIDDK, National Institutes of Health, Bethesda, Maryland, USA *Author for correspondence ( temcgraw@mail.med.cornell.edu) Accepted 7 September; published on WWW 31 October 2000 SUMMARY Insulin-responsive trafficking of the GLUT4 glucose transporter and the insulin-regulated aminopeptidase (IRAP) in adipose and muscle cells is well established. Insulin regulation of GLUT4 trafficking in these cells underlies the role that adipose tissue and muscle play in the maintenance of whole body glucose homeostasis. GLUT4 is expressed in a very limited number of tissues, most highly in adipose and muscle, while IRAP is expressed in many tissues. IRAP s physiological role in any of the tissues in which it is expressed, however, is unknown. The fact that IRAP, which traffics by the same insulin-regulated pathway as GLUT4, is expressed in non-insulin responsive tissues raises the question of whether these other cell types also have a specialized insulin-regulated trafficking pathway. The existence of an insulin-responsive pathway in other cell types would allow regulation of IRAP activity at the plasma membrane as a potentially important physiological function of insulin. To address this question we use reporter molecules for both GLUT4 and IRAP trafficking to measure insulin-stimulated translocation in undifferentiated cells by quantitative fluorescence microscopy. One reporter (vptr), a chimera between the intracellular domain of IRAP and the extracellular and transmembrane domains of the transferrin receptor, has been previously characterized. The other is a GLUT4 construct with an exofacial HA epitope and a C-terminal GFP. By comparing these reporters to the transferrin receptor, a marker for general endocytic trafficking, we demonstrate the existence of a specialized, insulinregulated trafficking pathway in two undifferentiated cell types, neither of which normally express GLUT4. The magnitude of translocation in these undifferentiated cells (approximately threefold) is similar to that reported for the translocation of GLUT4 in muscle cells. Thus, undifferentiated cells have the necessary retention and translocation machinery for an insulin response that is large enough to be physiologically important. Key words: Insulin action, Trafficking, GLUT4 INTRODUCTION An important component of whole body glucose homeostasis is the increased glucose uptake by adipose and muscle cells in response to insulin stimulation. These cells increase glucose uptake in large part by changing the distribution of GLUT4, a glucose transporter isoform. In adipocytes, for example, GLUT4 redistributes from less than 10% on the cell surface to about 50% on the cell surface in response to insulin (Cushman and Wardzala, 1980; Suzuki and Kono, 1980). In both the basal and insulin-stimulated states, GLUT4 continually cycles between the cell surface and intracellular compartments that have not been well characterized. GLUT4 is retained intracellularly in the basal state because the rate of recycling (or exocytosis) to the cell surface is slow compared to the internalization rate. Insulin stimulates translocation primarily by increasing the recycling rate (Jhun et al., 1992; Satoh et al., 1993; Yang and Holman, 1993). The molecular mechanisms that determine the slow recycling rate in the basal state and the insulin-stimulated increase are not understood. GLUT4 is thought to traffic through the general endosomal system, from which it is targeted to a specialized, insulin-regulated retention compartment (reviewed in Czech, 1995; Rea and James, 1997). Although a number of membrane proteins, such as the transferrin receptor (TR), colocalize to some degree with GLUT4, only the insulin-responsive aminopeptidase (IRAP) translocates to a similar extent in response to insulin. IRAP, which was cloned as a protein enriched in GLUT4-containing vesicles (Kandror and Pilch, 1994; Keller et al., 1995), has been shown to have similar trafficking characteristics to GLUT4 (Garza and Birnbaum, 2000; Keller et al., 1995; Malide et al., 1997b; Martin et al., 1997; Ross et al., 1998; Ross et al., 1996; Sumitani et al., 1997). As GLUT4 is almost exclusively expressed in adipose and muscle cells, studies of insulin-regulated trafficking have generally focused on these cell types. IRAP, however, is

2 4066 M. A. Lampson and others expressed in many tissues, which raises the possibility of insulin-regulated trafficking in cells other than adipose and muscle. The physiological function of IRAP is unknown, though it has been proposed to play a role in processing circulating peptide hormones (Herbst et al., 1997). If the substrates are extracellular, insulin could regulate IRAP activity by regulating its expression on the cell surface. Thus, the existence of an insulin-responsive trafficking pathway in cell types that do not express GLUT4 is an important question. Conflicting data have been published regarding this issue. A number of studies have shown that when GLUT4 is transfected into fibroblasts, either Chinese Hamster Ovary (CHO) or 3T3- L1 cells, it is retained in intracellular compartments, makes no contribution to glucose uptake, and does not translocate in response to insulin (Araki et al., 1996; Asano et al., 1992; Czech et al., 1993; Haney et al., 1991; Hudson et al., 1992; Shibasaki et al., 1992). Qualitative immunofluorescence of permeabilized cells was used to assess GLUT4 translocation in these studies. This is not a sensitive means of detecting changes in surface expression since the surface signal is diffusely distributed over a large area compared to the localized, and correspondingly brighter, intracellular signal. The failure to measure a contribution of GLUT4 to glucose uptake in fibroblasts may be because the contribution of GLUT4- mediated glucose uptake is not detected above glucose uptake mediated by the high levels of endogenous GLUT1 expressed in these cells. In contrast to those results, there is also evidence for insulin-responsive translocation of GLUT4 in fibroblasts, both 3T3-L1 and CHO cells, based on more sensitive assays (Dobson et al., 1996; Ishii et al., 1995; Kanai et al., 1993). In previous studies we used a chimeric reporter molecule for IRAP trafficking, vptr, which has the cytoplasmic domain of IRAP and the transmembrane and extracellular domains of the human TR (Johnson et al., 1998; Subtil et al., 2000). In CHO cells vptr translocates two- to threefold, compared to 1.3-fold for the TR, the established reporter for general endocytic trafficking, indicating that these cells are capable of a specific insulin response. vptr translocates to a larger extent in 3T3-L1 adipocytes, about fourfold compared to 1.3-fold for the TR, validating its use as a reporter for insulin-responsive trafficking. Further evidence of the specificity is provided by mutation of a dileucine sequence in the cytoplasmic domain of IRAP, which releases the retention of vptr and causes it to traffic like the TR in both CHO cells and 3T3-L1 adipocytes. In this study we use a GLUT4 construct with an exofacial HA epitope and a C-terminal GFP (HA-GLUT4-GFP), in addition to vptr, to rigorously test the capacity of undifferentiated cells for insulin-regulated trafficking. These molecules are reporters for the two proteins known to traffic by the insulin-responsive pathway in adipose and muscle cells. If the retention and translocation machinery in undifferentiated cells resembles that in adipose and muscle cells, it should recognize GLUT4 as well as the IRAP cytoplasmic domain. We have developed an assay that allows us to measure the translocation of HA-GLUT4-GFP, vptr, and the TR in 3T3- L1 fibroblasts and CHO cells by quantitative fluorescence microscopy. We find that HA-GLUT4-GFP and vptr translocate in response to insulin in all three cell types, and this translocation is specific in that the effect on the TR is relatively small. By directly comparing the trafficking of these reporters in the same cells and by the same method, we conclude that undifferentiated cells have the capacity for insulin-stimulated translocation. MATERIALS AND METHODS Ligands and chemicals Human Tf was obtained from Sigma (St Louis, MO, USA) and further purified by Sephacryl S-300 gel filtration. Diferric Tf and 125 I-Tf were prepared as described previously (Garippa et al., 1994). 125 I and 55 Fe 2 were purchased from NEN Life Science Products (Pittsburgh, PA, USA). Tf was labeled with the fluorescent dyes Cy3 (Biological Detection Systems, Pittsburgh, PA, USA) or Alexa-488 (Molecular Probes Inc., Eugene, OR, USA), according to the manufacturer s instructions. Fluorescently labeled antibodies were from Jackson ImmunoResearch (West Grove, PA, USA). All chemicals were from Sigma unless otherwise specified. Cell culture CHO cells were cultured in McCoy s 5A medium containing 5% fetal bovine serum, penicillin-streptomycin (Life Technologies, Inc., Gaithersburg, MD, USA), and 220 mm sodium bicarbonate. 3T3-L1 fibroblasts were cultured in Delbecco s modified Eagle s medium supplemented with 10% calf serum and penicillin-streptomycin. Transfected cell lines (both CHO and 3T3-L1) were carried in medium with 0.2 mg/ml G418 (Mediatech, Inc., Herndon, VA, USA). For experiments with adipocytes, 3T3-L1 fibroblasts were differentiated as described previously (Frost and Lane, 1985) and used 7-12 days after differentiation. For experiments with undifferentiated 3T3-L1 fibroblasts, cells were grown to confluence and carried for at least 2 days without fresh serum. At this point, referred to as day 0, the cells would be ready to start the differentiation process. Plasmids and transfection TRVb CHO cells, which do not express endogenous TR (McGraw et al., 1987), were cotransfected with HA-GLUT4-GFP and either vptr or the human TR using Lipofectamine (Life Technologies, Inc., Gaithersburg, MD, USA). Details of the transfection protocol were described previously (Johnson et al., 1998). The HA-GLUT4-GFP construct will be described elsewhere (A. Racz, D. Malide, A. Aviles- Hernandez and S. W. Cushman, unpublished). 3T3-L1 fibroblasts were transfected with HA-GLUT4-GFP, or the human TR, or vptr as described previously (Subtil et al., 2000). Cells expressing HA- GLUT4-GFP were selected for GFP fluorescence by FACS sorting and used as pooled populations. Clonal lines of 3T3-L1 fibroblasts expressing vptr or the TR were used. These lines were previously characterized as adipocytes (Subtil et al., 2000). HA-GLUT4-GFP translocation measured by fluorescence microscopy Cells, grown on coverslip bottom dishes, were pre-incubated at 37 C for at least 1 hour in McCoy s 5A medium, 220 mm sodium bicarbonate, 20 mm Hepes, ph 7.4 (med 1) for CHO cells, or in Dulbecco s modified Eagle s medium, 20 mm Hepes, ph 7.4 (med 2) for 3T3-L1 cells. Cells were incubated in the same medium with or without 170 nm insulin for 15 minutes, fixed for 10 minutes with 3.7% formaldehyde, and labeled with a mouse anti-ha monoclonal antibody (HA.11, Babco, Richmond, CA, USA). No detergent was used, so the antibody only had access to epitopes on the plasma membrane. The secondary antibody was a Cy3-goat anti-mouse or a Cy3-donkey anti-mouse. Fluorescence microscopy was done with a DMIRB inverted microscope (Leica Inc., Deerfield, IL, USA), with a cooled CCD camera (Princeton Instruments Inc., West Chester, PA, USA) and Metamorph (Universal Imaging Corporation, West Chester, PA, USA) image processing software. Images were acquired using a NA oil immersion objective, rather than a higher

3 Insulin-regulated trafficking 4067 magnification, to increase the depth of field at the expense of some resolution of intracellular structures. For quantification, nonspecific fluorescence was estimated either from cells not expressing HA-GLUT4-GFP (for GFP), or from cells labeled with only the secondary antibody (for Cy3). Total fluorescence of each fluorophore was summed over all cells in a field using Metamorph software, and nonspecific fluorescence was subtracted. The ratio of Cy3 fluorescence to GFP fluorescence (Cy3/GFP) was calculated and averaged over multiple fields. This ratio is a measure of surface HA-GLUT4-GFP normalized for the total construct expressed. Differentiated 3T3-L1 adipocytes have high levels of autofluorescence. At the levels of HA-GLUT4-GFP expressed in these cells, the Cy3 fluorescence in the basal state is difficult to distinguish from the background fluorescence because of the low signal-to-noise ratio, thereby making precise quantitative measurements of translocation impossible at this time. To estimate the degree of translocation of HA-GLUT4-GFP in adipocytes, we have calculated the smallest signal of surface HA-GLUT4-GFP (i.e. Cy3 fluorescence) that would be distinguishable from the background. To calculate this value we used the square root of the sum of the squares of the standard deviations in the background fluorescence and the Cy3 surface fluorescence, using data from three independent experiments on 3T3-L1 adipocytes in the basal state. Using this value as the surface expression of HA-GLUT4-GFP in the basal state and the measured value of surface HA-GLUT4-GFP in the presence of insulin (which is readily detected above background), we determined that translocation in adipocytes is at least sixfold. Since basal surface expression could be lower (although not higher) than what we estimate, the translocation in adipocytes could be greater than the estimate of sixfold. vptr or TR translocation measured by fluorescence microscopy 3T3-L1 fibroblasts expressing either vptr or TR, but not HA- GLUT4-GFP, or CHO cells coexpressing HA-GLUT4-GFP and either vptr or TR, were used for these experiments. In 3T3-L1 fibroblasts the endogenous mouse TR was downregulated as described previously (Subtil et al., 2000). 3T3-L1 fibroblasts were incubated with Alexa 488-Tf in med 2 for 1 hour at 37 C, with or without 170 nm insulin for the last 15 minutes, fixed for 10 minutes in 3.7% formaldehyde, and labeled with a mouse monoclonal antibody to the human TR extracellular domain, which also recognizes vptr (B3/25, Boehringer Manheim, Chicago, IL, USA). No detergent was used, so the antibody had access only to epitopes exposed on the cell surface. The secondary antibody was a Cy3-goat anti-mouse or a Cy3-donkey anti-mouse. CHO cells were treated the same way, but with Cy3-Tf in med 1 and a Cy5-donkey anti-mouse secondary antibody (because the cells express GFP so Alexa-488 cannot be used). Images were collected and processed as above. The Cy3/Alexa 488 ratio was calculated for 3T3-L1 fibroblasts, and the Cy5/Cy3 ratio for CHO cells. These ratios are measures of surface vptr or TR normalized for expression level. Colocalization of vptr or TR and HA-GLUT4-GFP CHO cells coexpressing HA-GLUT4-GFP and either vptr or TR were loaded for 2 hours with Cy3-Tf in med 1, washed and fixed in 3.7% formaldehyde. Images were collected with an Axiovert 100M inverted microscope equipped with an LSM 510 laser scanning unit and a NA plan Apochromat objective (Carl Zeiss, Inc., Thornwood, NY, USA). 543 and 488 nm light were used to stimulate Cy3 and GFP fluorescence, respectively. Emissions were selected with a 560-nm long pass filter for Cy3, or a nm band pass filter for GFP, and collected sequentially to prevent crossover. Steady-state distribution of vptr or TR measured with 125 I-Tf The surface-to-internal distribution of TR or vptr was determined using a previously described assay (Garippa et al., 1996). CHO cells expressing HA-GLUT4-GFP and either vptr or TR were used for these experiments. RESULTS HA-GLUT4-GFP construct The HA-GLUT4-GFP construct is shown schematically in Fig. 1. The HA-GLUT4 has been previously used (Quon et al., 1996), and the C-terminal GFP was added more recently. The construct has been characterized in rat adipose cells and shown to colocalize with IRAP, to be properly retained in the basal state, and to translocate to the surface with insulin (A. Racz, A. Aviles-Hernandez, S. W. Cushman and D. Malide, unpublished). The advantage of this construct is that ratiometric methods are used to compare surface amounts of GLUT4 among cells with different expression levels. Binding of fluorescently labeled antibodies to the HA epitope is proportional to surface protein, while the GFP fluorescence is proportional to the total protein expressed. The ratio of surface fluorescence to total fluorescence is a measure of surface HA- GLUT4-GFP normalized for the total construct expressed, so it does not depend on expression level. This ratio does not give any indication of the fraction of the total HA-GLUT4-GFP on the surface; it is only meaningful in comparisons between cells that have received different treatments, such as basal versus insulin-stimulated. HA-GLUT4-GFP translocation in differentiated 3T3- L1 adipocytes Insulin-responsive translocation of GLUT4 from intracellular compartments to the cell surface is well established in differentiated 3T3-L1 adipocytes, so we used these cells to test the HA-GLUT4-GFP construct. Proliferative 3T3-L1 fibroblasts were stably transfected with the construct and used as a pooled population. Surface HA epitopes were labeled by indirect immunofluorescence, and images were taken with a objective (instead of a higher magnification) to increase the depth of field at the expense of some resolution of intracellular structures. In these experiments we are interested in total fluorescence rather than details of the intracellular distribution. Examples of cells from independent experiments are shown in Fig. 2. Only a few cells in each field express observable amounts of HA-GLUT4-GFP. In the basal state the majority of HA-GLUT4-GFP is localized to a pericentriolar compartment, which is typical of endogenous GLUT4; smaller cytoplasmic structures are not resolved. We have not measured colocalization of HA-GLUT4- GFP with endogenous GLUT4 because antibodies to GLUT4 will recognize both proteins. Insulin does not dramatically change the morphology of the intracellular GLUT4 compartment, but a redistribution to the cell surface is apparent from the GFP fluorescence in some cells (Fig. 2G). The surface labeling of the HA epitope is a much more sensitive indicator of this redistribution. In insulin-stimulated cells the surface HA-GLUT4-GFP is clearly visible as a bright ring around the cell perimeter. For cells with similar levels of GFP (Fig. 2A,C or E,G), there is a large increase of surface labeling in cells treated with insulin, indicating a translocation of HA-GLUT4- GFP to the cell surface during the insulin treatment.

4 4068 M. A. Lampson and others Fig. 1. Schematic diagram of HA-GLUT4-GFP construct. Details of the construct will be reported elsewhere (A. Racz, D. Malide, A. Aviles-Hernandez and S. W. Cushman, unpublished). HA-GLUT4 has been previously characterized (Quon et al., 1996). HA-GLUT4-GFP translocation in undifferentiated 3T3-L1 fibroblasts The translocation in adipocytes confirms that HA-GLUT4- GFP is a good reporter for insulin-stimulated trafficking. We used this reporter to test translocation in undifferentiated, confluent 3T3-L1 fibroblasts (day 0, described in Materials and Methods). For these experiments we used the same pooled population as for the differentiated cells. Images from translocation assays are shown in Fig. 3. The pericentriolar distribution of GLUT4 in these cells is similar to the distribution in differentiated cells. Smaller structures are also resolved in the undifferentiated cells because the cells are flatter, so there is less out-of-plane fluorescence. Cell surface labeling of the HA epitope is apparent, particularly in insulintreated cells. Because the cells are relatively flat, fluorescence from the bottom of the cell as well as the perimeter is in the focal plane. HA-GLUT4-GFP on the bottom surface of the cell appears in a punctate pattern; a similar pattern is seen in focal planes at the bottom of differentiated 3T3-L1 adipocytes (not shown), and has been previously observed in rat adipose cells (Malide et al., 1997a). It is not clear why these aggregate structures are present. For cells with comparable levels of GFP fluorescence, the cell surface labeling is clearly greater in insulin-stimulated cells. We quantified the extent of translocation by summing the GFP and cell surface fluorescence for each cell and taking the ratio of surface to total. Results from a single experiment are shown in Fig. 3; surface labeling increased 3.6±0.8-fold in insulin-stimulated cells versus basal cells, demonstrating that undifferentiated cells have the capacity for insulin-regulated trafficking. We have not quantified the translocation in adipocytes (shown in Fig. 2) because the surface labeling in the basal state, at the levels of HA-GLUT4-GFP expressed in these cells, is difficult to distinguish from the background fluorescence of the cell (which is generally high in adipocytes). We estimate that insulin stimulates at least a sixfold translocation of HA-GLUT4-GFP to the surface of 3T3-L1 adipocytes (explained in Materials and Methods). Therefore, the magnitude of translocation is clearly larger than in fibroblasts, which is clearly supported by the images (compare Figs 2 and 3). One characteristic of GLUT4 translocation in adipocytes is that wortmannin, a phosphatidylinositol 3 -kinase inhibitor, blocks the insulin response (Clarke et al., 1994; Egert et al., 1997; Evans et al., 1995; Ross et al., 1996; Shimizu and Shimazu, 1994). We tested wortmannin in 3T3-L1 fibroblasts Fig. 2. Translocation of HA-GLUT4-GFP in 3T3-L1 adipocytes. Cells from a pooled population of 3T3-L1 adipocytes expressing HA-GLUT4- GFP were incubated for at least 1 hour in serum-free medium, with insulin or without (basal) for the last 15 minutes. Cells were fixed, and surface HA-GLUT4-GFP was labeled with Cy3 by indirect immunofluorescence. Paired images (A and B, C and D, E and F, G and H) are either GFP (A,C,E,G) or Cy3 (B,D,F,H) fluorescence from identical focal planes. Images are shown from experiments on different days, but each set of four images (A-D and E-H) is from a single experiment. Cells in A and C have similar levels of GFP expression, as do cells in E and G. For each fluorophore, acquisition times were identical for all images within an experiment. Asterisks in B and F mark the location of cells visible in A and E. Scale bar, 10 µm.

5 Insulin-regulated trafficking 4069 Fig. 3. Translocation of HA-GLUT4-GFP in undifferentiated 3T3-L1 fibroblasts. (A-H) Cells from the same pooled population used in Fig. 2 were incubated for at least 1 hour in serum-free medium, with insulin or without (basal) for the last 15 minutes. Cells were fixed, and surface HA-GLUT4-GFP was labeled with Cy3 by indirect immunofluorescence. Paired images (A and B, C and D, E and F, G and H) are either GFP (A,C,E,G) or Cy3 (B,D,F,H) fluorescence from identical focal planes. Images are shown from experiments on different days, but each set of four images (A-D and E-H) is from a single experiment. Cells in A and C have similar levels of GFP expression, as do cells in E and G. Scale bar, 10 µm. (I) Data were quantified for a single experiment (means ± s.e.m., N>10 fields) by summing the Cy3 and GFP fluorescence in each field, taking the ratio of Cy3/GFP, and averaging over multiple fields. For experiments with wortmannin (images not shown), cells were incubated with 100 nm wortmannin for 10 minutes before and during the insulin treatment. and found that it blocks the insulin-stimulated translocation of HA-GLUT4-GFP (Fig. 3), indicating that the insulin response is similar in both differentiated and undifferentiated cells in its requirement for phosphatidylinositol 3 -kinase activity. TR and vptr translocation in undifferentiated 3T3- L1 fibroblasts The insulin-stimulated translocation of GLUT4 is only meaningful in comparison to general endocytic trafficking. In 3T3-L1 adipocytes, for example, the five- to tenfold translocation of GLUT4 is large compared to that of the TR or GLUT1 (Tanner and Lienhard, 1987; Yang et al., 1992), indicating that the effect of insulin is specific and not a general mobilization of endocytic compartment proteins to the plasma membrane. To test this specificity in undifferentiated 3T3-L1 fibroblasts, we used the same fluorescence-based assay to measure translocation of the TR in cells expressing the human TR. In Fig. 4A-D intracellular receptors are labeled by transferrin uptake, and cell surface receptors are labeled by indirect immunofluorescence. The pericentriolar recycling compartment and smaller cytoplasmic structures are apparent by transferrin uptake. The total transferrin fluorescence is unchanged by insulin treatment, which demonstrates that uptake of labeled transferrin is constant with or without insulin (data not shown). In addition, there is no large increase in surface TR following insulin treatment. We quantified the extent of translocation from the fluorescence ratio (surface label over transferrin label) for each field. In the experiment shown, the ratio increases 1.2±0.1-fold in response to insulin, as compared to the more than threefold increase for GLUT4. These data demonstrate that the insulin response measured for GLUT4 is specific and not a general effect on endocytic trafficking. GLUT4 is one of two molecules known to traffic by the insulin-responsive pathway; the other is IRAP. If undifferentiated cells have this pathway, then IRAP translocation should also be stimulated by insulin. To test this response, we used vptr as a reporter for IRAP trafficking and measured translocation by the same assay used for the TR. In 3T3-L1 fibroblasts expressing vptr, surface labeling increases

6 4070 M. A. Lampson and others Fig. 4. Translocation of vptr and TR in undifferentiated 3T3-L1 cells. (A-H) Cells expressing either the human TR (A-D) or vptr (E-H) were loaded for 1 hour with Alexa 488-Tf in serum-free medium, with insulin or without (basal) for the last 15 minutes. Cells were fixed, and surface receptors labeled with Cy3 by indirect immunofluorescence. Paired images (A and B, C and D, E and F, G and H) are either Alexa 488 (A,C,E,G) or Cy3 (B,D,F,H) fluorescence from identical focal planes. Scale bar, 10 µm. (I) Data were quantified (means ± s.e.m., N=5 fields, approximately 20 cells per field) by summing the Cy3 and Alexa 488 fluorescence in each field, taking the ratio, and averaging across multiple fields. All images and quantitation are from a single experiment. markedly with insulin, while total transferrin uptake is constant (Fig. 4E-H). This finding demonstrates that the increase in surface fluorescence is due to a redistribution of internal vptr to the cell surface. The 3.3±0.6-fold translocation measured in this experiment is consistent with that of GLUT4 in undifferentiated cells, showing that the translocation of both reporters is specifically regulated by insulin. HA-GLUT4-GFP and vptr translocation in CHO cells The insulin-responsive translocation of HA-GLUT4-GFP and vptr in 3T3-L1 fibroblasts demonstrates that differentiation (i.e. expression of GLUT4 and accumulation of lipid droplets) is not necessary for insulin-responsive trafficking. 3T3-L1 cells are pre-adipocytes, however, so they may have some specialized machinery that other cells lack. We have previously shown that insulin specifically stimulates translocation of vptr in CHO cells (Johnson et al., 1998), indicating that CHO cells also have the necessary machinery. If both of these undifferentiated cell types have the same capacity for insulinregulated trafficking, it would suggest that the mechanism is the same and may exist in many cells. To directly compare the insulin response in CHO cells and 3T3-L1 fibroblasts, we used the same fluorescence-based assay in TRVb CHO cells, which do not express functional endogenous TR (McGraw et al., 1987), stably transfected with HA-GLUT4-GFP and either vptr or the human TR. The intracellular distribution of HA-GLUT4-GFP in these cells is shown by confocal microscopy in Fig. 5. The GLUT4 construct is highly colocalized with vptr, both in the pericentriolar region and in more peripheral structures. GLUT4 also colocalizes to a large extent with the TR in the pericentriolar region and in peripheral structures, although there are small punctate structures in the cell periphery that contain TR (red) but little GLUT4 (green). Results from an HA-GLUT4-GFP translocation assay are shown in Fig. 6. In this experiment, cell surface GLUT4 increased 3.5±0.3-fold with insulin, and this increase was blocked by wortmannin. The results shown in Fig. 6 are for cells that coexpress HA-GLUT4-GFP and vptr; a similar insulin-responsive translocation of HA-GLUT4-GFP was measured in cells coexpressing the TR (data not shown). We next measured translocation of vptr and the TR in the CHO cells coexpressing HA-GLUT4-GFP. Surface labeling of the vptr increases with insulin treatment, but the TR is

7 Insulin-regulated trafficking 4071 Fig. 5. Colocalization of HA-GLUT4-GFP and either vptr or TR in CHO cells. CHO cells coexpressing HA-GLUT4-GFP and either vptr (A-C) or the TR (D-F) were loaded for 2 hours with Cy3-Tf in serumfree medium and fixed. Projections of several planes from the confocal microscope are shown. For each set of three images (A-C, D-F), green is GFP fluorescence, red is Cy3 from an identical projection, and the merged image is on the right. relatively constant (Fig. 7). In the experiment shown, surface vptr and TR increase 2.4±0.4-fold and 1.2±0.1-fold, respectively. Similar translocation of the TR and vptr in CHO cells has been reported by surface binding of iodinated Tf (Johnson et al., 1998); we repeated these measurements in cells coexpressing HA-GLUT4-GFP and either vptr or the TR, and found results identical to those measured by fluorescence. We do not observe any correlation between the magnitude of translocation of vptr or GLUT4 and the expression level of either protein, indicating that the levels are not high enough to saturate either the retention or translocation mechanism. A summary of multiple translocation experiments performed in CHO and 3T3-L1 fibroblasts is presented in Fig. 8. Cell surface GLUT4 and vptr increase approximately threefold in both cell types, while surface TR increases only 1.2-fold. Agreement between the two methods shown in Fig. 8 (fluorescently labeled versus iodinated Tf) validates use of the fluorescence-based assay. These data demonstrate that insulin specifically translocates both vptr and GLUT4 in two undifferentiated cell types. The magnitudes of the responses are similar in both cell types, suggesting that the same mechanism is responsible. 3T3-L1 fibroblasts express more insulin receptors than CHO cells: 7,000-8,400 versus 2,800 receptors per cell (Ebina et al., 1985; Reed et al., 1981; Rubin et al., 1978), but this difference in expression is not reflected in an increased translocation, which suggests that insulin receptor signaling is not limiting the extent of translocation. The insulin response is reversible Data from experiments performed in CHO cells and in 3T3-L1 adipocytes indicate that GLUT4 and vptr redistribution is achieved by changing the ratio of the efflux rate to the internalization rate in response to insulin (Jhun et al., 1992; Johnson et al., 1998; Satoh et al., 1993; Subtil et al., 2000; Yang and Holman, 1993). A key aspect of this process is that following insulin removal, the distribution must return to the basal state as the rates return to their basal values. We tested this hypothesis in CHO cells by incubating cells with insulin for 15 minutes, followed by 1 hour without insulin before fixation. The redistribution of both HA-GLUT4-GFP and vptr is fully reversible (Fig. 9). This experiment demonstrates that both GLUT4 and vptr are internalized in CHO cells after insulin stimulation, as expected for a dynamic retention mechanism. A more detailed characterization of the internalization of HA-GLUT4-GFP will be published separately (A. Racz, A. Aviles-Hernandez, S. W. Cushman and D. Malide, unpublished). We have shown that the cytoplasmic domain of IRAP promotes constitutive internalization by clathrin-coated pits, in both CHO cells and 3T3-L1 adipocytes ((Johnson et al., 1998; Subtil et al., 2000; our unpublished observations). Others groups have measured the kinetics of internalization of endogenous IRAP in 3T3-L1 adipocytes (Garza and Birnbaum, 2000; Ross et al., 1997). There is a single report that IRAP is not internalized after insulin stimulation in rat adipocytes (Kandror, 1999). This finding is inconsistent with all other published reports (see Garza and Birnbaum, 2000 for a discussion). It is not clear why IRAP internalization was not detected in that study. DISCUSSION In this study we use two reporters for insulin-regulated endocytic trafficking: HA-GLUT4-GFP and vptr. Insulin-

8 4072 M. A. Lampson and others Fig. 6. Translocation of HA-GLUT4-GFP in CHO cells. (A-H) CHO cells coexpressing HA-GLUT4-GFP and vptr were incubated in serumfree medium for at least 1 hour, with insulin or without (basal) for the last 15 minutes. Cells were fixed, and surface receptors labeled with Cy3 by indirect immunofluorescence. Wortmannin-treated cells (E-H) were incubated with 100 nm wortmannin for 10 minutes before and during the insulin treatment. Paired images (A and B, C and D, E and F, G and H) are either GFP (A,C,E,G) or Cy3 (B,D,F,H) fluorescence from identical focal planes. Scale bar, 10 µm. (I) Data were quantified (means ± s.e.m., N=5 fields, >20 cells per field) by summing the Cy3 and GFP fluorescence in each field, taking the ratio, and averaging over multiple fields. All images and quantitation are from a single experiment. stimulated translocation of vptr has previously been reported in 3T3-L1 adipocytes (Subtil et al., 2000), and here we show the same for HA-GLUT4-GFP, establishing that both molecules are good reporters for insulin-regulated trafficking. We measure insulin-stimulated translocation in 3T3-L1 and CHO fibroblasts using both of these surrogate reporters as well as the TR. Because we use the same quantitative method for all measurements, the results are directly comparable. Cell surface HA-GLUT4-GFP and vptr increase about 200% in both fibroblast cell types, while the TR increases only about 20%. These results demonstrate that undifferentiated cells have the necessary machinery to retain GLUT4 and vptr in an intracellular compartment and to redistribute both proteins to the plasma membrane in response to insulin. The response is specific for these proteins because the effect on the TR, a marker for general endocytic trafficking, is relatively small. We have previously documented that in CHO cells and 3T3- L1 adipocytes, vptr is dynamically retained in the basal state by rapid internalization from the plasma membrane and slow exocytosis from endosomal recycling compartments (Johnson et al., 1998; Subtil et al., 2000). We find that the trafficking parameters of vptr in undifferentiated 3T3-L1 cells are similar to those in CHO cells (M. A. Lampson and T. E. McGraw, unpublished observations). There is no reason to expect that CHO cells would specifically have an insulinregulated trafficking pathway. Consequently, our data suggest that cell types other than adipose and muscle may have an insulin-regulated endocytic recycling pathway capable of dynamically retaining proteins in the basal state and translocating these proteins to the surface following insulin treatment. Since IRAP is expressed in many tissues, the existence of a specialized, insulin-regulated trafficking pathway in many cell types might be expected. This pathway would provide a way for insulin to regulate hormonal activity via IRAP s peptidase activity on circulating substrates (Herbst et al., 1997). As the physiological function of IRAP is not known, the significance of insulin regulation of IRAP expression on the cell surface, whether in adipose, muscle or fibroblast cells, remains to be determined. Our data raise the question of how closely the mechanism of insulin-regulated trafficking in fibroblasts resembles that in adipose and muscle cells. The magnitude of the response in fibroblasts is similar to what has been reported in muscle, twoto threefold in skeletal muscle and L6 muscle cells (Hansen et al., 1998; Lund et al., 1997; Ryder et al., 1999; Wang et al., 1998), though some studies have reported a larger response

9 Insulin-regulated trafficking 4073 Fig. 7. Translocation of vptr or TR in CHO cells expressing HA-GLUT4-GFP. (A-H) CHO cells expressing HA-GLUT4-GFP and either the TR (A-D) or vptr (E-H) were loaded for 1 hour with Cy3-Tf in serum-free medium, incubated with insulin or without (basal) for 15 minutes, fixed, and surface receptors labeled with Cy5 by indirect immunofluorescence. Paired images (A and B, C and D, E and F, G and H) are either Cy3 (A,C,E,G) or Cy5 (B,D,F,H) fluorescence from identical focal planes. Scale bar, 10 µm. (I) Data were quantified (means ± s.e.m., N=5 fields, >20 cells per field) by summing the Cy3 and Cy5 fluorescence in each field, taking the ratio, and averaging over multiple fields. All images and quantitation are from a single experiment. (Lund et al., 1993; Wilson and Cushman, 1994). The magnitude is larger in adipocytes, but other aspects of the process are similar. Translocation of endogenous GLUT4 in adipocytes is achieved primarily by increasing the efflux rate, and the same principle applies to translocation of vptr in adipocytes (Subtil et al., 2000), undifferentiated 3T3-L1 fibroblasts (M. A. Lampson and T. E. McGraw, unpublished data), and in CHO cells (Johnson et al., 1998). It is difficult to draw further conclusions about mechanistic similarities because the magnitude of the response is not necessarily a good indicator of the underlying mechanism, as suggested by the varying levels of translocation observed in insulin-responsive tissues. In primary rat adipose cells, for example, the magnitude of GLUT4 translocation depends on the physiological state of the animal, such as diet, age and obesity (Hissin et al., 1982a; Hissin et al., 1982b; Kahn et al., 1988). Regardless of the magnitude relative to adipocytes, however, fibroblasts clearly have a bona fide mechanism for insulinregulated trafficking. Moreover, GLUT4 translocation in fibroblasts is quantitatively similar to that in muscle, which is responsible for a large part of the whole-body glucose disposal Fold increase on surface (Insulin/basal) Fluorescence CHO cells 125 I -Tf CHO cells Fluorescence 3T3-L1 fibroblasts GLUT4 vptr TR vptr Fig. 8. Summary of translocation data for CHO cells and undifferentiated 3T3-L1 fibroblasts. The fold increase is the surface label in the insulin-stimulated state divided by the surface label in the basal state. Data were quantified as in previous figures for fluorescence, or by labeling with 125 I-Tf. Values are means ± s.e.m. over multiple experiments. TR

10 4074 M. A. Lampson and others GLUT vptr Basal Insulin Insulin reversed Fig. 9. The effect of insulin on GLUT4 and vptr translocation in CHO cells is reversible. Translocation experiments were performed as in Figs 6 and 7, with cells coexpressing vptr and HA-GLUT4- GFP, except that some cells were incubated for 15 minutes with insulin, then for 60 minutes further without insulin before fixation ( insulin reversed ). (Left) Surface HA-GLUT4-GFP was labeled with Cy3. (Right) Surface vptr was labeled with Cy5 in cells preloaded with Cy3-Tf. Data were quantified (means ± s.e.m., N=5 fields) by summing the Cy3 and either Cy5 or GFP fluorescence in each field, taking the ratio (Cy3/GFP or Cy5/Cy3), and averaging over multiple fields. All data are from a single experiment. stimulated by insulin (De Fronzo et al., 1985; De Fronzo et al., 1981). Thus, fibroblasts have the necessary retention and translocation machinery for an insulin response that is large enough to be physiologically important. Previous studies have addressed the necessity of GLUT4 expression for the insulin response. Several groups have measured endogenous GLUT1 and IRAP translocation in 3T3- L1 cells during the differentiation process to test the existence of an insulin-regulated retention compartment prior to GLUT4 expression. The fraction of GLUT1 on the cell surface decreases about 75% from preconfluent to confluent 3T3-L1 fibroblasts, with a twofold insulin-stimulated increase in confluent cells, indicating development of an insulinresponsive retention compartment (Weiland et al., 1990; Yang et al., 1992). The magnitude of this translocation increases as differentiation proceeds, though not to the same extent as GLUT4 translocation, indicating that some fraction of the GLUT1 traffics through an insulin-responsive pathway. Another group found that GLUT1 and IRAP cosediment in an insulin-responsive compartment by day 3 of differentiation, before expression of GLUT4 (El-Jack et al., 1999). These studies conclude that an insulin-regulated retention compartment is developed at an early stage of differentiation, before GLUT4 expression, and GLUT4 is targeted to this preexisting compartment. Our data, which demonstrate that the translocation of vptr in CHO and 3T3-L1 fibroblasts is independent of GLUT4 expression (this report; Johnson et al., 1998), provide further evidence that GLUT4 is cargo in the insulin-regulated pathway and not directly required for formation of the compartment. Existence of the insulin-regulated trafficking pathway in undifferentiated cells has important implications for GLUT4 trafficking and endocytic trafficking in general. One view has been that genes turned on during differentiation of adipose and muscle cells are essential for insulin-stimulated translocation. Based on our results, the process appears to be similar in undifferentiated cells, though the efficacy of the insulin response in adipocytes increases with differentiation, suggesting that differentiation may involve a subtle modulation of the translocation machinery rather than development of a completely new mechanism. Thus, the many genes turned on during adipocyte differentiation may be related to other aspects of adipocyte function, such as triglyceride storage and metabolism. Many cell types perform various types of specialized trafficking. In collecting duct cells of the kidney, for example, vasopressin increases levels of aquaporin-2 on the cell surface by recruiting an intracellular pool of transporters, like the effects of insulin on GLUT4 (Knepper and Inoue, 1997). As we propose for insulin-regulated trafficking, other specialized pathways may arise from relatively slight modifications of the general endocytic system, rather than development of completely new machinery. A high degree of mechanistic conservation is known to exist in cells as disparate as yeast and neurons, so it is not surprising that cells would use existing machinery to accomplish specialized tasks such as regulated glucose transport. Using the tools presented here, we hope to determine the relationship between the specialized, insulinregulated pathway and the general endocytic pathway. One important question to be addressed in future studies is whether fibroblasts and adipocytes retain vptr and GLUT4 by the same mechanism. The current model is that adipose and muscle cells target GLUT4 and IRAP to a specialized, insulinresponsive recycling compartment. In CHO cells, based on colocalization studies, vptr and GLUT4 appear to be retained within the TR-containing endosomal system. During differentiation adipocytes may develop a specialized compartment, derived from the endosomal system, that retains GLUT4 more efficiently than in fibroblasts and is more responsive to insulin. We thank David Erstejn for technical assistance, and Alona Cohen and Amy Johnson for helpful comments on the manuscript. This work was supported by National Institutes of Health Grant DK52852 to T.E.M. REFERENCES Araki, S., Yang, J., Hashiramoto, M., Tamori, Y., Kasuga, M. and Holman, G. D. (1996). Subcellular trafficking kinetics of GLU4 mutated at the N- and C-terminal. Biochem. J. 315, Asano, T., Takata, K., Katagiri, H., Tsukuda, K., Lin, J. L., Ishihara, H., Inukai, K., Hirano, H., Yazaki, Y. and Oka, Y. (1992). Domains responsible for the differential targeting of glucose transporter isoforms. J. Biol. Chem. 267, Clarke, J. F., Young, P. W., Yonezawa, K., Kasuga, M. and Holman, G. D. (1994). Inhibition of the translocation of GLUT1 and GLUT4 in 3T3-L1 cells by the phosphatidylinositol 3-kinase inhibitor, wortmannin. Biochem. J. 300, Cushman, S. W. and Wardzala, L. J. (1980). Potential mechanism of insulin action on glucose transport in the isolated rat adipose cell. 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