Insulin-regulated sorting of glucose transporters in 3T3-L1 adipocytes.

Insulin-regulated sorting in 3T3-LI adipocytes LINDA

J. ROBINSON

of glucose transporters

AND DAVID

E. JAMES

Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110 Robinson, Linda J., and David E. James. Insulin-regulated sorting of glucosetransporters in 3T3-Ll adipocytes.Am. J. Physiol. 263 (Endocrinol. Metab. 26): E383-E393, 1992.Two glucosetransporters (GLUT-4 and GLUT-l) move from within the cell to the plasma membrane (PM) when 3T3-Ll adipocytes are stimulated with insulin. To study the sorting of thesetwo molecules,vesiclescontaining GLUT-4 and GLUT-l wereimmunoadsorbedfrom basaland insulin-treated cells.Two different vesicle populations were isolated asfollows: 1) a compartment that contained the majority of intracellular GLUT-4 and GLUT-l and 2) a subpopulationof vesiclescontaining 43% of the intracellular GLUT-4 that was highly insulin regulatable and that contained relatively low levelsof GLUT-l. After incubation at 19OC,basal glucosetransport was slightly increased, whereasinsulin-dependent transport was blocked. Consistent with these observations, cell surface GLUT-l levels were increasedin the basal state, whereasinsulin-dependent translocation of GLUT-4 to the PM was blocked at 19OC.However, insulin-dependent sorting of GLUT-4 within the intracellular compartment wasstill evident at 19°C. Thesedata indicate that GLUT- 1 and GLUT-4 are heterogeneouslydistributed throughout the sameintracellular compartment in 3T3-Ll adipocytes. Furthermore, we have uncoupledtwo distinct stepsin the insulin-dependent movement of GLUT-4 to the cell surface. These include movement of GLUT-4 out of its storagecompartment and accumulation of GLUT-4 at the cell surface. Only the former step occurs in cellspreincubated at 19’C. GLUT-4; GLUT- 1; plasmamembrane;immunoadsorption STIMULATES GLUCOSE TRANSPORT in adipocytes, cardiac muscle, and skeletal muscle primarily by triggering the translocation of glucose transporters (GLUT-4) from an intracellular compartment to the plasma membrane (PM) (5, 26). With the use of immunocytochemistry it has been shown that, in the nonstimulated condition, GLUT-4 is localized to tubulovesicular elements that are clustered either in the trans-Golgi reticulum (TGR) or in the cytoplasm in adipocytes and myocytes (22, 24, 25). Cell surface GLUT-4 labeling, which is very low in the absence of stimulation, is markedly increased after insulin treatment. Furthermore, GLUT-4 labeling of both cell surface clathrin-coated pits (21,25) and the endosomal vacuole (25) is increased with insulin stimulation, suggesting that under these conditions the transporter constantly recycles between the PM and the intracellular compartment. Other proteins besides GLUT-4 also undergo insulindependent movement to the cell surface in adipocytes. Insulin stimulates the cell surface levels of GLUT-l, another glucose transporter isoform (2, 19), the transferrin receptor (6, 27), the insulin-like growth factor II (IGF-II)-mannose 6-phosphate receptor (18, 29), and the a2-macroglobulin receptor (4) by two- to threefold. In addition, fluid phase endocytosis (8) and secretion of adipsin (15), a serine protease, are increased by twofold after insulin stimulation of adipocytes. These data sugINSULIN

0193-1849/92

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Copyright

gest that insulin may have a generalized effect to promote recycling in the endosomal pathway. However, several properties unique to GLUT-4 indicate that the regulation of this protein is different from that of other proteins or markers that are targeted to the endosomal pathway. First, as noted above, GLUT-4 is virtually excluded from the PM in the basal cell. This is not the case for other insulin-sensitive proteins such as GLUT-l (19). Second, the magnitude of the insulindependent increase in cell surface GLUT-4 is more pronounced than that of the other insulin-sensitive processes described above. Because of the increase in GLUT-4 labeling of endosomal elements in the presence of insulin, we have previously suggested that GLUT-4 must be actively withdrawn from the recycling pathway in the absence of insulin (19, 25). In accordance with this proposal, we have recently shown that the NH2terminal of GLUT-4 contains targeting information that is sufficient to confer complete intracellular sequestration when transferred to the GLUT-l isoform (20). However, it is not clear whether this motif regulates endocytosis of GLUT-4 or retention within the cell. This is an important distinction in elucidating the potential locus of insulin action. A biochemical characterization of the intracellular sorting of GLUT-4 may lead to a clearer understanding of the unique properties of this protein and how they may relate to the insulin-dependent movement of GLUT-4 to the cell surface. Using immunoadsorption to isolate intracellular vesicles containing GLUT-4 previous studies have shown that GLUT-4 is localized to the same vesicle population that contains the transferrin receptor, the IGF-II-mannose 6-phosphate receptor, and GLUT-l in 3T3-Ll adipocytes (2, 19, 28). These data imply that insulin may not discriminate between individual proteins but rather may cause the exocytic movement of a vesicle population containing all of these proteins. In contrast, using a similar technique in rat adipocytes, it has been found that GLUT-l and GLUT-4 are segregated within separate intracellular vesicles (31). Based on studies previously performed in our own laboratory, we have proposed that this discrepancy may have arisen because the two transporters are localized together in separate intracellular compartments but with variable stoichiometry (19). We have shown that vesicles containing GLUT-l and GLUT-4 can be partially resolved using sucrose density gradient sedimentation of an intracellular fraction from 3T3-L1 adipocytes. In addition, regions of unique intracellular localization are observed using double-immunofluorescence labeling of whole cells. However, using both techniques, there is overlap between the intracellular compartments containing GLUT- 1 and GLUT-4

0 1992 the American

Physiological

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Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (143.088.066.066) on August 26, 2018. Copyright © 1992 American Physiological Society. All rights reserved.

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IN 3T3-L1

In the present study, to test the effect of insulin on the sorting of glucose transporters, we have isolated vesicles containing GLUT-4 and GLUT-l from 3T3-Ll adipocytes under both basal and insulin-stimulated conditions. We have isolated an intracellular GLUT-4 compartment that is highly insulin regulatable and that contains low levels of GLUT- 1. The sorting of glucose transporters has also been studied by preincubating cells at 19OC for 2 h. Although the insulin-induced translocation of GLUT-4 to the cell surface is inhibited at this temperature, we show that the insulin-stimulated intracellular sorting of GLUT-4, as measured by vesicle immunoadsorption, still occurs under these conditions. These data indicate that at least two steps are involved in the change in steady-state distribution of GLUT-4 with insulin. MATERIALS

AND

METHODS

Materials

Sheepanti-mouseand sheep anti-rabbit magnetic beads were obtained from Dynal (Great Neck, NY). ““I-labeled goat antirabbit was obtained from ICN Radiochemicals (Irvine, CA). 3T3-LI fibroblasts were obtained from the American Type Culture Collection. All other chemicals were obtained from Sigma Chemical (St. Louis, MO). Antibodies The two GLUT-4 specific antibodies used in these studies have been described previously (19). The rabbit anti-GLUT-4 antiserum R820 was raised against a synthetic peptide corresponding to the COOH-terminal 12 residues of the rat GLUT-4 sequence. The monoclonal antibody 1F8 was raised against purified vesicles from rat adipocytes. The epitope of this antibody is within the COOH-terminal25 amino acids of GLUT-4 (R. C. Piper, unpublished observations). The rabbit anti-GLUT1 antiserum was raised against a synthetic peptide corresponding to the COOH-terminal 12 amino acids of the human GLUT-l sequence (19). The rabbit anti-Na+-K+-ATPase antiserum, kindly provided by Dr. Robert Mercer, was raised against the purified rat protein; the rabbit anti-adipsin antiserum, kindly provided by Dr. Bruce Spiegelman, was raised against purified recombinant baculovirus-expressed mouse adipsin (3). Cell Culture Murine-derived 3T3-Ll fibroblasts were cultured in Dulbecco’s modified Eagle’s medium on either loo-mm (for fractionation) or 35mm (for glucose transport) tissue culture dishes or on ethanol-washed glass cover slips (no. 1) and differentiated as described previously (19). All experiments were performed using adipocytes 8-12 days after withdrawal from differentiation media. Adipocytes were rinsed two times with Krebs-Ringer phosphate (KRP) buffer containing 2% bovine serum albumin and 2.5 mM glucose and incubated in KRP for 2 h at either 37°C or 19°C before experimentation. Subcellular Fractionation Differential centrifugation method. Cells were fractionated by a modification of a previously described protocol (19). After the 37 or 19OC preincubation (see above), cells were incubated in KRP containing either no additions (basal) or 10v7 M insulin for 30 min at either 19 or 37°C. Cells were then rinsed two times in (in mM) 20 N-2hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES), 1 EDTA, and 250 sucrose, pH 7.4 (HES buffer) at 4°C and scraped vigorously with a rubber policeman in HES buffer (3 ml). Cells were homogenized by passing 10

ADIPOCYTES

times through a Yamato LSC homogenizer. The homogenate was subjected to differential centrifugation, yielding a fraction enriched in PM markers, as well as a fraction enriched in intracellular GLUT-4 [low-density microsomal (LDM) fraction]. Membrane pellets were resuspended in HES buffer to a final protein concentration of l-5 mg/ml by pipetting up and down until no visible particles remained. The protein concentration in each fraction was determined using the bicinchoninic acid reagent (Pierce) according to the manufacturer’s instructions. Equivalent amounts of protein from each fraction were used for measurement of GLUT-l and GLUT-4 by immunoblotting. The LDM fraction was used as starting material for immunoadsorptions (see Immunoadsorption of Vesicles Containing GLUT-4 and GLUT-l). PM lawn technique. PM lawns were isolated using an adaptation of a previously described procedure (13, 17). The use of this procedure as an assay to measure GLUT-4 translocation has been well described and characterized (21). Adipocytes were grown on glass cover slips but otherwise cultured and treated with insulin as described above. Briefly, cover slips were rinsed in phosphate-buffered saline (PBS) and incubated for 10 s in PBS containing 0.5 mg poly(L-Lys)/ml followed by three 5-s incubations in hypotonic buffer [23 (in mM) KCl, 1.7 ethylene glycol-his@-aminoethyl ether)-N,iVJV’,N’-tetraacetic acid (EGTA), and 10 HEPES, pH 7.51. The cover slips were submerged in intracellular buffer [(in mM) 70 KCl, 5 MgCl,, 3 EGTA, 1 dithiothreitol, 0.1 phenylmethylsulfonyl fluoride, and 30 HEPES, pH 7.51 and sonicated for 2 s at a power setting of 15 (Kontes 115-V disrupter) by using a 3.2 x 48-mm tapered probe centered 0.3 cm above the cover slip. Cover slips were then immediately immersed in intracellular buffer containing 2% paraformaldehyde. Fixed cover slips were prepared for immunofluorescence microscopy as described previously (19)) with slight modifications. The primary antibody used was either 20 pg/ml protein A-purified anti-GLUT-4 antiserum (see Antibodies) or the anti-Na+-K+-ATPase antiserum at a 1:lOO dilution. After mounting cover slips on slides as described (19), the PM lawns were examined with a Zeiss Axioplan microscope equipped with a Bio-Rad MRC-500 laser confocal imaging system. Immunoisolation of GLUT+ and GLUT-l -Containing Vesicles Polyclonal method. The polyclonal antiserum R820 was adsorbed directly to the LDM starting material to purify only the antibodies that would bind to the LDM fraction from the antiserum. Appropriate concentrations (see RESULTS) of serum were added to 100 pg LDM in 0.5 ml PBS containing 0.1% bovine serum albumin (PBS/BSA) and incubated for 1 h at 4°C. Unbound antibody was separated from the bound fraction by layering membranes on top of a 2-ml cushion of 0.4 M sucrose in 20 mM HEPES-1 mM EDTA (pH 7.4) followed by centrifugation for 90 min at 200,000 g, The supernatant was removed, and the pellet was resuspended in a small volume of PBS. The amount of protein in the resuspended pellets was then determined. For each immunoadsorption, magnetic beads conjugated with sheep anti-rabbit antibodies (500 pg/50 ~1) were incubated in PBS-l% BSA (0.5 ml) for 30 min at room temperature. Beads were then washed 3 times in PBS by spinning them briefly in a microcentrifuge. Beads were resuspended in 50 ~1 PBS and added to the resuspended LDM pellet at a ratio of 50 ~1 beads/ 100 pg membrane protein. The beads were incubated with the LDM fraction for 4 h in 0.5 ml PBS/BSA at 4°C using endover-end rotation. Beads were isolated using a magnetic particle concentrator (Dynal), and the supernatant was removed and saved. Beads were then washed 3 times in PBS for 10 min at 4”C, pulling the beads to the magnet after each wash. Washed


SORTING

OF GLUT-4

beads were resuspended in Laemmli sample buffer (LSB). Supernatants were centrifuged at 200,000 g for 90 min. The pellet, representing nonadsorbed membranes, was resuspended in LSB. The nonadsorbed fraction and the bead fraction were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 10% resolving gel. Control immunoadsorptions were performed by incubating the LDM fraction with nonimmune serum. No detectable GLUT-l or GLUT-4 was immunoadsorbed in this fraction. Monoclonal method. The monoclonal antibody lF8 was directly coupled to magnetic beads conjugated with sheep antimouse antibodies before incubation of the coupled beads with the LDM fraction. Magnetic beads conjugated to sheep antimouse antibodies (750 pg/25 ~1) were incubated in PBS-l% BSA as described above. Beads were then incubated in 0.5 ml PBS/BSA containing 2.5 ~1 lF8 ascites for 2 h at 4”C, using end-over-end rotation. Beads were washed 3 times with PBS and incubated with LDM fraction (100 pg) diluted in PBS/BSA (0.5 ml) at 4°C for 4 h, using end-over-end rotation. The beads were washed, and the supernatant was harvested as described above for the polyclonal method. Controls were performed in which incubations were performed using beads not incubated with lF8. No detectable GLUT-4 or GLUT-l was immunoadsorbed in this fraction. When immunoadsorptions with lF8 were performed by first incubating the lF8 ascites directly with the LDM fraction, as described above for the polyclonal method, results similar to those obtained using the monoclonal method were obtained. In all cases, for both the polyclonal and monoclonal methods, quantitative recovery of either GLUT-l or GLUT-4 was obtained as determined by immunoblotting the adsorbed and nonadsorbed fractions. Furthermore, cell surface markers (Na+-K+-ATPase) were not detectable in the immunopurified fraction (data not shown).

IN 3T3-L1

ADIPOCYTES

2- Deoxyglucose

E385

Uptake

2-Deoxyglucose (2-DG) uptake was measured using a modification of a previously described assay (9). To show time dependence of the temperature block, we preincubated cells as follows: 1) 37°C for 2 h, 2) 37°C for 1 h followed by 19°C for 1 h, 3) 37°C for 1.5 h followed by 19°C for 30 min, or 4) at 19°C for 2 h. Cells were then incubated in the presence or absence of insulin (low7 M) for 20 min at the appropriate temperature. To measure the 2-DG uptake of cells incubated at 19OC without releasing the temperature block, all uptake measurements, regardless of the incubation temperature, were performed at 19OC. Therefore plates were washed once with KRP containing no glucose at 19°C and incubated for 1 min at 19°C in this buffer before the addition of 2-DG. Cells were incubated with 100 PM 2-[“H]DG (1 &i/ml) for 3 min at 19°C in the presence or absence of cytochalasin B (25 PM). Cells were washed three times in KRP at 4°C (without glucose or BSA), dried, and solubilized with 1% Triton X-100 (1 ml). The 2-[3H]DG in 0.5 ml from each dish was quantitated by scintillation counting, and protein assays were performed on the lysed cells. To show reversibility of the temperature block, cells were incubated at 19OC for 2 h, followed by incubation in the presence or absence of insulin at either 19 or 37°C for 10, 20, and 30 min; 2-DG uptake was then measured. Statistical

Analysis

Data are presented as means t SE. When more than one variable was compared within an experiment, data were initially analyzed by two- or three-way analyses of variance. Two-tailed paired or unpaired Student’s t tests were performed on all experiments where these tests were appropriate. RESULTS

SDS-PAGE

and Immunoblotting

Samples were subjected to SDS-PAGE, transferred to nitrocellulose sheets, and immunoblotted as previously described (19). Autoradiography was performed by exposing nitrocellulose sheets to X-Omat AR film (Kodak) and Cronex Lightning Plus enhancing screens at -80°C. Autoradiograms were quantified using an Epson scanner and Macintosh-based software. Adipsin Secretion After preincubation of cells at 19 or 37°C as described above, cells grown on loo-mm dishes were washed 2 times in KRP containing 1 mg/ml BSA at the appropriate temperature and incubated in this buffer (10 ml) in the presence or absence of insulin (lob7 M) at the appropriate temperature. Aliquots (0.5 ml) of media were removed at 5-min intervals over a period of 30 min and incubated on ice. The adipsin concentration in these aliquots was measured by immunoblotting. Fluid Phase Endocytosis Fluid phase endocytosis was measured using horseradish peroxidase (HRP) as a marker (30). After preincubation at the appropriate temperature, cells in 35-mm dishes were incubated in the presence or absence of insulin (10m7 M) for 10 min at the appropriate temperature. Cells were then incubated in KRP containing 4 mg/ml HRP in the continued presence or absence of insulin for 5 min at the same temperature. At the end of the incubations, dishes were transferred to a 500-ml beaker containing PBS-l% BSA (buffer A) at 37 or 19OC as appropriate for 1 min. Dishes were quickly transferred to another 500-ml beaker of buffer A at the same temperature, followed by three lo-min incubations in buffer A (500 ml) at 0°C. Cells were then lysed in 0.5 ml of 0.2% Triton X-100 for measurement of HRP activity (30) .

Immunoadsorption of Vesicles Containing GLUT-4 and GLUT-l

In this study, we have immunoadsorbed intracellular vesicles containing GLUT-4 and GLUT-1 from basal and insulin-treated 3T3-Ll adipocytes. We have utilized three antibodies for this purpose as follows: 1) R820, a polyclonal anti-GLUT-4 antibody, 2) R493, a polyclonal anti-GLUT-l antibody, and 3) lF8, a monoclonal antiGLUT-4 antibody. The purpose of this study was to determine the distributions of GLUT-4 and GLUT-l in all three of these vesicle fractions from basal and insulintreated cells. During the course of these studies, we found that the amount of antibody used for the immunoadsorptions was critical. Therefore, in Figs. l-3, we have titrated the immunoadsorption efficiencies vs. antibody concentrations used in the immunoadsorptions for all three of the antibodies used. The LDM fraction was used as the starting material for all immunoadsorption studies. We have previously shown that this fraction is highly enriched in intracellular organelles and deenriched in nuclei, mitochondria, and plasma membranes (19). Figures 1 and 2 show titration curves using the GLUT-4 antiserum (R820) and the GLUT-l antiserum (R493), respectively. In both cases, an optimal amount of GLUT-4 was immunoadsorbed using a similar ratio of antibody to LDM starting material. With the use of this optimal ratio, 93% of the intracellular GLUT-4 was immunopurified using R820, and 68% of the intracellular GLUT-4 was immunopurified using R493 (Figs. 1 and 2 and Table 1). When a higher or lower


E386

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GLUT4-+

/

OF GLUT-4

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ADIPOCYTES

\

GLUT4e

*

100 80 60 40 20 0 LDM

A

B

C

D

E

Fig. 1. Titration of amount of anti-GLUT-4 (R820) antibody required for optimal immunoadsorption of GLUT-4 vesicles from 3T3-Ll adipocytes. Intracellular (IC) low-density microsomal (LDM) fraction isolated from nonstimulated 3T3-Ll adipocytes was immunoadsorbed by incubating lOO-pg aliquots of LDM with 10 (A), 5 (B), 1 (C), 0.5 (D), or 0.05 (E) ~1 of R820 antiserum. Immunoadsorptions were performed as described in MATERIALS AND METHODS (polyclonal method), and adsorbed fractions were subject to SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotted with R820. LDM, 10 Kg of LDM starting material. Lanes A-E, l/10 of immunoadsorption reaction or amount of GLUT-4 immunoadsorbed using 10 pg LDM starting material. Immunolabeled bands were quantitated using densitometry. Percentage of IC GLUT-4 refers to amount of GLUT-4 in immunoadsorbed fraction as percentage of amount of GLUT-4 in LDM starting material, which is denoted as 100%.

ratio of antibody to LDM was used, there was a decrease in immunoadsorption efficiency. The basis for the decrease in immunoadsorption efficiency at higher antibody concentrations is unclear but may be related to inadequate separation of free from bound antibody at these very high serum concentrations. It is important to separate free antibody from the LDM fraction before the addition of beads, because otherwise the free and bound antibody compete for available binding sites on the beads. Figure 3 shows a titration curve using the anti-GLUT-4 monoclonal antibody lF8. With the use of an optimal concentration of lF8, 43% of the total GLUT-4 contained in the LDM fraction was immunoadsorbed (Fig. 3 and Table 1). In contrast to the titration curves with the polyclonal antibodies, the immunoadsorption efficiency using lF8 was saturable at higher ratios of antibody to LDM fraction. This is because lF8 is precoupled to the beads. This method was ineffective with the polyclonal antisera, presumably because the transporter-specific antibodies in the serum represent a low percentage of the total immunoglobulin G fraction. The effect of insulin on the distribution of transporters in these vesicle populations was examined by immunoadsorbing vesicles from both basal and insulin-treated cells, using the optimal antibody concentrations shown in Figs.

LDM

A

B

C

D

E

Fig. 2. Titration of amount of anti-GLUT-l (R493) antibody required for optimal immunoadsorption of GLUT-4. LDM (100 pg) from nonstimulated 3T3-Ll adipocytes was immunoadsorbed using 10 (A), 5 (B), 1 (C), 0.5 (D), or 0.05 (E) ~1 of R493 antiserum. Fractions were subject to SDS-PAGE and immunoblotted with R820 as described in Fig. 1.

l-3. In these studies, the level of GLUT-4 or GLUT-l in the LDM fraction from either basal or insulin-treated cells has been designated a value of lOO%, despite the fact that, after insulin treatment, the amount of transporter in the LDM fraction is decreased due to recruitment of transporters to the cell surface (see Table 3). This normalization procedure allows us to discern the sorting of glucose transporters within immunoisolated vesicles relative to the entire intracellular compartment (i.e., LDM) and thus overcomes the necessity to consider those transporters that are present in the PM fraction. Therefore we have compared the amounts of immunoisolated GLUT-4 and GLUT-l to the total amount of GLUT-4 and GLUT-l in the LDM fraction, regardless of whether this fraction was isolated from basal or insulin-treated cells. These data are summarized in Table 1. Vesicles immunoadsorbed with R820 from either basal or insulintreated cells contained >90% of the intracellular GLUT-4 and >80% of the intracellular GLUT-l (Fig. 4 and Table 1). However, the amount of GLUT-4 in the R493 vesicles immunopurified from insulin-treated cells was largely depleted relative to the amount of GLUT-l in these vesicles (Fig. 4). Thus the GLUT-4-to-GLUT-1 ratio in R493 vesicles isolated from insulin-treated cells was 3.3fold lower than that in the corresponding LDM fraction (Table 1). In contrast to the immunoadsorption efficiencies observed with either R820 or R493, vesicles immunopurified from basal cells using optimal concentrations of lF8 (25 ~1 lF8 beads/100 pg LDM fraction) contained only 43% of intracellular GLUT-4 and 18% of the GLUT-l (Fig. 5 and Table 1). Thus the GLUT-4-to-GLUT-1 ratio in this vesicle fraction was significantly higher than that


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ADIPOCYTES

100 80 Insulin

60

Ab

40 20 0 LDM

A

6

C

D

E

Fig. 3. Titration of amount of anti-GLUT-4 (lF8) antibody required for optimal immunoadsorption of GLUT-4. LDM (100 pg) aliquots were incubated with 100 (A), 50 (B), 25(C), 12.5 (D), or 6.25 (E) ~1 of sheep anti-mouse magnetic beads coated with lF8. Immunoadsorptions were performed as described in MATERIALS AND METHODS (monoclonal method). Immunoisolated fractions were subjected to SDS-PAGE and immunoblotted with R820 as described in Fig. 1. Percentage of IC GLUT-4 was calculated as described in Fig. 1.

Table 1. Percentages of GLUT-4 and GLUT-l immunoadsorbed from basal and insulin-stimulated cells using antibodies to GLUT-4 and GLUT-l SIC B%3l

GLUT-4 Insulin

‘SIC Basal

GLUT-l Insulin

GLUT-4/GLUT-1 Basal

Insulin

LDM 100 100 100 100 1.O:l.O 1.O:l.O R820 93f4 1.o:l.O 1.O:l.O 82+8 95+4 83+13 R493 68klO 24-c8 6557 83211 1.1:l.O 0.3:l.O lF8 43+6 25f4 18_t3 3f2 2.4:l.O 8.3:l.O Values are means + SE of 3-9 separate experiments. Low-density microsomal (LDM) fractions from basal or insulin-stimulated 3T3-Ll adipocytes were immunoadsorbed with optimal concentrations (see text) of R820, R493. or lF8 antibodies as described in MATERIALS AND METHODS. Amount of GLUT-4 or GLUT-1 in these fractions was quantitated as described in Fig. 1. Percentage of intracellular (IC) GLUT-4 or GLUT-l in each vesicle fraction was calculated relative to LDM fraction, which is denoted as 100% in each case, as described in Fig. 1.

in the LDM fraction (Table 1). With insulin, there was a significant reduction in the amount of GLUT-4 and GLUT-l in the lF8 vesicle by 18 or 15%, respectively (Fig. 5). Thus the GLUT-4-to-GLUT-1 ratio in vesicles from insulin-stimulated cells was 8.3-fold higher than that observed in either the LDM fraction or in vesicles immunoadsorbed with R820 (Table 1). These data clearly indicate the presence of a vesicle subpopulation in 3T3-Ll adipocytes that is selectively recognized by lF8 and that is relatively enriched in GLUT-4 compared with the GLUT-l isoform. Furthermore, this vesicle population appears to be highly regulatable by insulin.

-

+ R820

-

+ R493

Fig. 4. Immunoadsorption of GLUT-4 (R820) and GLUT-l (R493) vesicles from 3T3-Ll adipocytes incubated in presence (+) and absence (-) of insulin. LDM fractions from cells treated in presence or absence of insulin were immunoadsorbed with anti-GLUT-4 antiserum (R820) or anti-GLUT-l antiserum (R493) using optimal concentrations of both antibodies as determined in Figs. 1 and 2. Immunoadsorbed fractions were subjected to SDS-PAGE and immunoblotted with R820 as described in Fig. 1. Percentage of IC GLUT-4 was calculated as described in Fig. 1. Data are means + SE of 4 separate experiments.

Effects of 19’C Preincubation on Constitutive Secretion in 3T3-Ll Adipocytes

Previous studies have identified temperature-specific blocks on membrane traffic between various cellular organelles (7,10,12,16,23). Because GLUT-4 is targeted to a TGR-like compartment in insulin-sensitive cells and because movement out of the TGR is blocked at temperatures I 2O”C, we studied the effects of prolonged incubation of the cells at 19°C on insulin-dependent movement of GLUT-4. The temperature block was effective in 3T3-Ll cells because constitutive secretion of adipsin, a serine protease released from adipocytes (3), was inhibited by 83% after incubation of the cells at 19°C for 2 h. (Table 2). Similarly, insulin-stimulated adipsin secretion was blocked at 19°C (Table 2). Effects of 19OC Preincubation on Fluid Phase Endocytosis

To further characterize the effects of the 19°C preincubation on adipocytes, we examined the uptake of the fluid phase marker HRP (Fig. 6). Consistent with previous findings (8), insulin stimulated fluid phase endocytosis by approximately twofold at 37°C. When adipocytes were preincubated at 19”C, there was a 20-30% decrease in basal HRP uptake, as well as a 60% inhibition in the insulin-stimulated component of HRP uptake (Fig. 6). Effects of 19OC Preincubation on 2-DG Transport

To investigate the effect of this block on glucose transporter localization and translocation, we first examined the effect of a 19°C preincubation on insulin-stimulated 2-DG uptake. In cells preincubated at 37”C, insulin stimulated 2-DG uptake by 25fold (Fig. 7). During preincubation at 19”C, there was an inhibition of insulin-stimulated glucose uptake, as well as an increase in basal 2-DG uptake (Fig. 7). The effects of low temperature on 2-DG


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Basal GLUT4

/,

GLUT 1

/

”

0

Insulin

123

123

123

-

123

+

-

GLUT4

+

GLUT 1

Fig. 5. Immunoadsorption of GLUT-4 vesicles from basal and insulin-stimulated cells using lF8 or R820. A: LDM fractions (lane 1) were isolated from 3T3-Ll adipocytes incubated in presence (insulin) or absence (basal) of insulin. Aliquots of LDM fraction (100 pg membrane protein) were immunoadsorbed with lF8 (Zone 2) or R820 (lane 3) under optimal conditions. Immunoadsorbed and LDM fractions were immunoblotted with either anti-GLUT-4 (R820) or anti-GLUT-l (R493) as indicated. B: lF8 vesicles were immunoadsorbed from LDM fractions obtained from cells treated in presence (+) or absence (-) of insulin at 37°C using 25 ~1 lF8 beads/100 pg LDM. Immunoisolated fractions were subjected to SDS-PAGE and immunoblotted with R820 (GLUT-4) or R493 (GLUT-l) as indicated, and autoradiographs were quantitated by densitometry. Percentage of IC transporter in lF8 vesicle refers to amount of GLUT-4 or GLUT-1 in immunoadsorbed fraction as percentage of amount of GLUT-4 or GLUT-l in LDM starting material, which is denoted as 100% for both transporters. Data represent means zt SE of 9 (GLUT-4) or 7 (GLUT-l) separate experiments.

Table 2. Adipsin Temperature, “C

secretion is blocked at 19°C Insulin

Adipsin Secretion, U/min

37 4.2kO.5 + 37 5.2kO.7 19 0.7kO.3 19 + 0.5rto.3 Values are means + SE from 3 separate experiments. Adipocytes were incubated with Krebs-Ringer phosphate buffer containing low (1 mg/ml) bovine serum albumin at appropriate temperature in the presence (+) or absence (-) of insulin for 30 min. Media samples were collected at 5-min intervals for measurement of adipsin by immunoblotting. Immunoblots were quantitated by densitometry, and data are expressed as rate of secretion with time. In all cases, rates of adipsin secretion were linear over 30-min period measured.

Insulin

-

+

+

37-c 19-c Fig. 6. Horseradish peroxidase (HRP) uptake in 3T3-Ll cells incubated in presence (+) or absence (-) of insulin at 37°C or 19°C. 3T3-Ll adipocytes were preincubated at appropriate temperature for 2 h, followed by 10 min in presence or absence of insulin at indicated temperature. Cells were then incubated with HRP (4 mg/ml) for 5 min in presence or absence of insulin at appropriate temperature. Data represent amount of HRP activity in solubilized cell extracts per milligram protein. All data points were performed in triplicate and are representative of 2 separate experiments. Temperature

transport were fully reversed after a 20-min incubation 37°C (data not shown).

at

Effects of 19OC Preincubation on Distribution of GLUT-4 and GLUT-l by Subcellular Fractionation The above changes in glucose transport were consistent with changes in the subcellular distribution of GLUT-4 and GLUT-l. PM fractions were isolated from cells incubated under various conditions by two different methods as follows: 1) the PM lawn method (21) and 2) differential centrifugation. LDM fractions were also isolated from cells using the differential centrifugation method. With the use of both fractionation methods, we established that insulin-dependent translocation of GLUT-4 to the cell surface was significantly inhibited after preincubation of the cells at 19°C (Figs. 8 and 9 and Table 3). Incubation of cells at 19°C per se caused an increase in cell surface levels of GLUT-l, as well as a decrease in LDM levels of GLUT-l, to almost the same extent as that

observed with insulin at 37°C. However, there was neither a significant increase in cell surface GLUT-4 levels, nor a significant decrease in LDM GLUT-4 levels, when cells were incubated at 19°C (Figs. 8 and 9 and Table 3). Therefore the inhibition in insulin-induced translocation of GLUT-4 to the PM at 19°C presumably accounts for the inhibition in insulin-induced 2-DG transport at this temperature, whereas the increase in the basal levels of GLUT-l at the PM at 19°C probably accounts for the increase in basal levels of 2-DG uptake at 19°C.


SORTING

U +

OF GLUT-4

IN 3T3-Ll

ADIPOCYTES

E389

observed at 37°C (see Fig. 5). Therefore, even though the insulin-induced translocation of GLUT-4 to the PM was blocked at 19”C, insulin-induced intracellular sorting of GLUT-4 still occurred at this temperature.

basal insulin

DISCUSSION

0

30

60

time

at

DO

19°C

120

(min)

Fig. 7. Effect of insulin on 2-deoxyglucose (2-DG) uptake in 3T3-Ll cells incubated for various times at 19°C. 3T3-Ll adipocytes were incubated at 37 and/or 19°C for a total of 2 h as described in MATERIALS AND METHODS. Cells were then incubated in presence or absence of insulin at appropriate temperature, and 2-DG uptake was measured at 19°C for all incubation conditions. All data points were performed in triplicate, and are representative of 2 separate experiments. I

GLUT4

#&

GLUT 1

Insulin:

-

+ A

-

+ B

-

+ C

Fig. 8. Effect of insulin on translocation of GLUT-4 and GLUT-l to plasma membrane (PM) at 37 and 19°C as analyzed by differential centrifugation. 3T3-Ll adipocytes were preincubated at 37°C (A) or 19°C (B and C) for 2 h. Cells were then incubated in presence (+) or absence (-) of insulin at 37°C (A and B) or 19°C (C) for 20 min. PM fractions were isolated by differential centrifugation, and lo-pg samples were subjected to SDS-PAGE and immunoblotted with R820 (GLUT-I) or R493 (GLUT-l).

Effects of 19OC Preincubation on Immunoadsorption of GLUT-4-Containing Vesicles

As shown in Figs. 7,8, and 9 and Table 3, the insulininduced movement of GLUT-4 to the plasma membrane that occurs at 37°C does not occur after cells have been preincubated at 19°C. Based on these results, we were interested in determining the effects of low temperature on the GLUT-4 content in immunoisolated vesicle fractions. Cells were preincubated at 19°C for 2 h, followed by a 20-min incubation in the presence or absence of insulin. Vesicles were immunoadsorbed from the intracellular membrane (LDM) fraction using lF8. As shown in Fig. 10, insulin caused a significant decrease in the GLUT-4 content in these vesicles, comparable with the decrease

We have previously shown that, in 3T3-Ll adipocytes, GLUT-l and GLUT-4 populate the same intracellular vesicles, but at variable stoichiometry, such that some vesicles contained a high ratio of GLUT-l to GLUT-4, whereas others contained a higher ratio of GLUT-4 to GLUT-l. In the present studies we have confirmed this observation using a vesicle immunoadsorption procedure. By isolating vesicles from an intracellular microsomal fraction using antibodies specific for the cytoplasmic tail of either GLUT-l or GLUT-4, it was possible to quantify the amount of both transporters in each vesicle population. Immunoadsorption of intracellular vesicles from basal 3T3-Ll adipocytes using either a polyclonal antibody specific for GLUT-l (R493) or GLUT-4 (R820) resulted in purification of vesicle populations containing ~65% of both transporters. Therefore these data support previous conclusions (2,19) that there is an overlap in the distribution of both GLUT-l and GLUT-4 within the intracellular compartment(s) of these cells. However, using the monoclonal antibody lF8, we have been able to further subfractionate these intracellular vesicles, thus uncovering the heterogenous distribution of both transporters within individual vesicles derived from the infracellular compartments. The subpopulation of vesicles isolated using lF8 was relatively enriched in GLUT-4 compared with GLUT-l. We believe that this compartment may serve a specialized function with respect to the sorting of GLUT-4, both in terms of its targeting under basal conditions and the ability of insulin to recruit GLUT-4 to the cell surface. While insulin is known to stimulate the translocation of both GLUT-l and GLUT-4 to the cell surface in adipocytes (see Table 3), little is known about the effects of insulin on the intracellular distribution of these transporters. Commensurate with the insulin-dependent increase in cell surface transporters, there is a 30-40% decrease in transporter levels within the intracellular compartments (i.e., the LDM fraction). To determine whether there is a selective effect of insulin on the distribution of transporters within intracellular subcompartments, we have nominally assigned the levels of GLUT-l and GLUT-4 in the LDM from basal and insulin-treated cells a value of one. This permits an assessment of the redistribution of transporters within intracellular subcompartments independently of their movement to the cell surface. Vesicles immunoadsorbed from both basal and insulin-treated cells with the polyclonal antibody R820 had the same characteristics as the LDM fraction; the GLUT-l-to-GLUT-4 ratio maintained a value of one, and there was no change in the levels of each transporter in these vesicles relative to the LDM after insulin treatment. In contrast, we observed a large insulin-induced decrease in GLUT-4 and GLUT-l levels in the vesicle population recognized by lF8, relative to transporter levels in the LDM. We also found that,


E390

SORTING

OF GLUT-4

Basal

IN 3T3-Ll

ADIPOCYTES

Insulin

37O

19”

Fig. 9. Effect of insulin on translocation of GLUT-4 to PM at 37 and 19°C as analyzed by PM lawn method. 3T3-Ll adipocytes were preincubated at 37 or 19°C for 2 h, followed by incubation at same temperature in presence or absence of insulin for 20 min. PM lawns were isolated and prepared for immunofluorescence microscopy by incubation with R820 followed by fluorescein isothiocyanate-conjugated goat anti-rabbit antibody. Fields are representative of quantitative data shown in Table 3.

whereas the amount of GLUT-l in vesicles immunoisolated with the anti-GLUT-l polyclonal antibody (R493) from basal and insulin-treated cells remained constant, there was a large insulin-induced decrease in the amount of GLUT-4 in this vesicle population. The inability of R493 to adsorb a portion of the intracellular GLUT-4 only from insulin-treated cells is consistent with the observation that the levels of GLUT-l in the vesicles immunoisolated with lF8 were markedly diminished after insulin treatment. Thus it is likely that the anti-GLUT-l polyclonal antibody R493 fails to immunoadsorb the same vesicles specifically adsorbed with the GLUT-4 monoclonal antibody lF8 in insulin-treated cells, due to the selective loss of GLUT-l from this subcompartment. Our interpretation of these data is based on the notion that the ability of an antibody to adsorb a vesicle from a membrane fraction is a function of the antigen density per vesicle and/or the antibody concentration used during

immunoisolation. Previous studies performed by Gruenberg and Howell (11) support this concept. By titrating the amount of antigen present in erythrocyte PMs after fusion of known amounts of vesicular stomatitis virus, it was shown that an antigen density of X0 molecules/pm* membrane surface area was required for immunoadsorption of vesicles containing VSV G protein (11). It is unlikely that this selectivity is due to epitope masking because we have observed similar data with two different antibodies (lF8 and R493). Furthermore, the selectivity of lF8 is not a special feature of this antibody because we have previously observed a similar selectivity using R820 at suboptimal concentrations (19). Subpopulations of intracellular vesicles containing varying concentrations of GLUT-l and GLUT-4 clearly exist in 3T3-Ll adipocytes. By performing double-label immunofluorescence microscopy in 3T3-Ll adipocytes, we have observed that the intracellular distribution of GLUT-l and GLUT-4 do


SORTING

OF GLUT-4

Table 3. Effect of 19% preincubation on GLUT-l and GLUT-4 translocation Fractionation Method PM lawn

Fraction PM

Temperature, “C 37

IuS

Multiples of Change in Transporter GLUT-1

IN 3T3-Ll

E391

ADIPOCYTES

A 1 16kD--)f=

.a?

GLUT-4

+ + + + + -

ND 1.0 ND 6.7kO.9 1.6+0.4 19 ND ND 2.6kO.5 1.0 1.0 Differential PM 37 centrifugation 2.5t0.6 3.7rtl.O 2.0&0.4 1.520.2 19 2.7t0.8 1.7kO.2 LDM 1.0 1.0 37 0.6kO.2 0.7kO.l 19 0.6kO.2 1.OrtO.l 0.7kO.l O&O.1 Values are means + SE. For plasma membrane (PM) lawn method, GLUT-4 levels in PM fragments from 5 separate experiments were quantified as described in MATERIALS AND METHODS. Measurement of translocation of GLUT-4 and GLUT-l by differential centrifugation method was performed as described in Fig. 8. For differential centrifugation method, immunoblots of PM and LDM fractions from 3 to 9 separate experiments were quantitated by densitometry. In all cases, data were normalized to 37°C basal condition, which is indicated as 1.0 for each condition. Temperature is for 2-h preincubation as well as 20smin incubation in presence or absence of insulin (Ins); +, presence or -, absence of insulin (lo-’ M). ND, not determined.

overlap. However, using enhanced computer subtraction imaging, we were able to resolve compartments that were relatively enriched in GLUT-l and deenriched in GLUT-4 and vice-versa (19). Hence we feel that it is very likely that this graded distribution constitutes the basis for the selectivity of immunoadsorption as reported here. The hypothetical model shown in Fig. 11 illustrates relative intracellular distribution of GLUT-l and the GLUT-4 in basal and insulin-treated cells. Both GLUT-l and GLUT-4 have been immunolocalized to the TGRendosomal system (1,25). This system is represented as a contiguous column (Fig. 1 l), but it could also be depicted as a series of vesicles or organelles. It is noted that the distribution of GLUT-l and GLUT-4 within the column overlap, consistent with the fact that both transporters can be quantitatively immunoadsorbed using a polyclonal antibody (R820) specific for GLUT-4. However, the distribution of each transporter forms a gradient within the column, due to clustering of the transporters in separate microdomains or subcompartments. In the case of GLUT-4, this microdomain is represented by the lF8 subcompartment. Relative to GLUT-4, GLUT-l is excluded from this compartment, accounting for the high GLUT-4-to-GLUT-1 ratio in vesicles immunoadsorbed with lF8. This subcompartment of GLUT-4 is depicted at the base of the column, reflecting withdrawal of GLUT-4 from the recycling pathway in the absence of insulin (25). In a previous study, using both sucrose density gradient analysis of intracellular vesicles from 3T3-Ll adipocytes and double-label immunofluorescence microscopy, we have observed that the bulk of intracellular GLUT-l is segregated from the bulk of intracellular GLUT-4 (19). This supports the notion that both transporters are clustered at different positions within the TGR-endosomal system. It is assumed that GLUT-l is

35kD-b’

B Q, 60 ‘;; “;; Q, 50 > :

40 c ; 30 5 = 20 u-

g

10

w 0

Insulin

-

+

Fig. 10. Immunoisolation of GLUT-4 vesicles using lF8 from 3T3-Ll cells treated in presence (+) or absence (-) of insulin at 19°C. A: after preincubation at 19°C for 2 h, cells were incubated in presence or absence of insulin for 20 min at 19°C. Vesicles were then immunoisolated from LDM fractions using lF8 as described in Fig. 5. LDM fractions (10 pg; lanes 1 and 3) and corresponding amounts of fractions immunoisolated with lF8 (lanes 2 and 4) from basal (lanes 1 and 2) and insulin-stimulated (lanes 3 and 4) cells were analyzed by SDS-PAGE and immunoblotted with R820. Immunoblot is representative of data shown in B. B: quantitation of percentage of GLUT-4 immunoadsorbed with lF8 in cells incubated in absence or presence of insulin at 19°C. Data are means + SE of 5 separate experiments.

clustered either in or close to the recycling pathway, toward the top of the column. As shown in Fig. 11, right, insulin triggers two distinct events. GLUT-4 moves out of its clustered microdomain (i.e., it moves up the length of the column) and it accumulates at the PM. Both GLUT-l and GLUT-4 probably move up the column with insulin because GLUT-l also leaves the lF8 subcompartment when treated with insulin. Furthermore, we observed an insulin-induced decrease in GLUT-4 levels in the compartment isolated with R493, indicating that GLUT-l has moved out of a GLUT-4-containing subcompartment. We propose that the GLUT-4 microdomain isolated using lF8, indicated at the base of the column, is a compartment that is constructed primarily as a storage depot for GLUT-4 by virtue of some specific targeting event. However, other proteins such as GLUT-l may


E392

SORTING

OF GLUT-4

Plasma

IN 3T3-Ll

ADIPOCYTES

membrane (fJ+-

A

Recycling Recycling

f

/

BASAL Fig. 11. Model denoting sorting of GLUT-4 and GLUT1 in basal and insulin-stimulated 3T3-Ll adipocytes. Shown is distribution of GLUT-l (1) and GLUT-4 (4) within PM and intracellular compartment (denoted as a continuous column beneath PM) of basal and insulin-treated adipocytes. GLUT-l-to-GLUT-4 ratios in these locations are based on previous quantitation of 2 isoforms in 3T3-Ll cells (2,19). In basal state, recycling to PM occurs predominantly from top of column, and GLUT-4 is shown to be withdrawn from this pathway. Insulin facilitates entry of GLUT-4 into recycling pathway (lower arrow) in addition to accelerating overall rate of recycling (top arrow).

enter this compartment, albeit at a much reduced efficiency compared with GLUT-4. In the presence of insulin, as the recycling rate is increased, the ability of GLUT-l to enter this specialized GLUT-4 compartment may be markedly reduced. The insulin-dependent accumulation of GLUT- 1 and GLUT-4 at the top of the column probably reflects movement of these transporters into recycling endosomes. This is supported by the observation that there is a marked increase in internalized HRP, a fluid phase marker, in vesicles immunoadsorbed with R820 from insulin-treated cells (unpublished observations). Furthermore, electron microscopy immunocytochemical analysis of GLUT-4 distribution in brown adipose tissue has indicated that levels of GLUT-4 in early endosomes are increased with insulin treatment (25). These data are consistent with a model in which, in the absence of insulin, GLUT-4 is withdrawn from the recycling pathway in a compartment that is selectively identified by immunoisolation with lF8. Insulin somehow overcomes this withdrawal such that GLUT-4 now participates in the endosomal recycling pathway. Hence this model implicates at least two intracellular trafficking events in recruiting GLUT-4 to the cell surface with insulin; first, GLUT-4 must leave the intracellular compartment identified by lF8, and, second, GLUT-4 must recycle between the early endosomes and the PM. Further evidence in support of a two-step model of insulin action on GLUT-4 movement to the PM was obtained by studying cells at low temperature. Although the insulin-dependent accumulation of GLUT-4 at the PM was blocked at 19”C, there was still a significant decrease in the amount of GLUT-4 found in the intracellular subcompartment isolated with lF8 under these conditions. These data can be explained by a tempera-

ture-induced block in the endocytic recycling pathway. Hence, at this low temperature, we have uncoupled two distinct events that occur during the insulin-stimulated recruitment of GLUT-4 to the PM. We have considered two hypotheses to explain the inhibition in insulin-stimulated accumulation of GLUT-4 at the PM at 19OC. First, GLUT-4 may move from its storage compartment (the lF8 vesicle) and fuse directly with the PM at 19OC. Because of its efficient internalization rate, GLUT-4 may recycle to early endosomes but may be prevented from returning to the PM at 19°C because of a specific block in the exocytic leg of the recycling pathway. This hypothesis, however, is not supported by the accumulation of GLUT-l at the cell surface at 19OC or by the inhibition in fluid phase endocytosis at this temperature. Nevertheless, it is conceivable that GLUT-4 has a very efficient propensity for endocytosis compared with other molecules, such that it is not affected even at reduced temperatures. A second hypothesis is that vesicles containing GLUT-4 may undergo one or more sorting steps before fusion with the PM, resulting in a change in the density of GLUT-4 per vesicle. Such a sorting step would involve the unclustering of GLUT-4 from the IF8 subcompartment. This could result from either a specific change in the oligomerit state of GLUT-4 with insulin (14) or fusion of the GLUT-4 storage compartment with another compartment before movement to the PM. We are unable to distinguish between these possi .bilities from the present data. However, the low-temperature block may provide a useful model system for studying these changes. We thank Suhong Pang and Zuhaira Razzack for tance, Dr. Dan Studelska for assistance with statistics, preparation of figures, the Diabetes Research Training eration of peptides, and Dr. Robert Piper, Diana Harris, Hanpeter for helpful discussions and critical reading of

technical assisKerri James for Center for genand Dr. David the manuscript.


SORTING

OF GLUT-4

IN 3T3-Ll

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-42503. D. E. James was a recipient of a Career Development Award from the Juvenile Diabetes Foundation. Address for reprint requests: D. E. James, Dept. of Cell Biology and Physiology, Washington Univ. School of Medicine, Box 8228, 660 S. Euclid Ave., St. Louis, MO 63110. Received

20 May

1992; accepted

in final

form

15 June

1992.

16.

17.

18.

REFERENCES 1. Blok, J., E. M. Gibbs, G. E. Lienhard, J. W. Slot, and H. J. Geuze. Insulin-induced translocation of glucose transporters from post-Golgi compartments to the plasma membrane of 3T3-Ll adipocytes. J. Cell Biol. 106: 69-76, 1988. 2. Calderhead, D. M., K. Kitagawa, L. I. Tanner, G. D. Holman, and G. E. Lienhard. Insulin regulation of the two glucose transporters in 3T3-Ll adipocytes. J. Biol. Chem. 265: 1380013808, 1990. 3. Cook, K. S., H. Y. Min, D. Johnson, R. J. Chaplinsky, J. S. Flier, C. R. Hunt, and B. M. Spiegelman. Adipsin: a circulating serine protease homolog secreted by adipose tissue and sciatic nerve. Science Wash. DC 237: 402-405, 1987. 4. Corvera, S., D. F. Graver, and R. M. Smith. Insulin increases the cell surface concentration of aZ-macroglobulin receptors in 3T3-Ll adipocytes. J. BioL. Chem. 264: 10133-10138, 1989. 5. Cushman, S. W., and L. J. Wardzala. Potential mechanism of insulin action on glucose transport in the isolated rat adipose cell: apparent translocation of intracellular transport systems to the plasma membrane. J. Biol. Chem. 255: 4758-4762, 1980. 6. Davis, R. J., S. Corvera, and M. P. Czech. Insulin stimulates cellular iron uptake and causes the redistribution of intracellular transferrin receptors to the plasma membrane. J. Biol. Chem. 261: 8708-8711, 1986. 7. Dunn, W. A., A. L. Hubbard, and N. N. Aronson, Jr. Low temperature selectively inhibits fusion between pinocytic vesicles and lysosomes during heterophagy of V-asialofetuin by the perfused rat liver. J. Biol. Chem. 255: 5971-5978, 1980. 8. Gibbs, E. M., G. E. Lienhard, J. R. Appleman, D. M. Lane, and S. C. Frost. Insulin stimulates fluid-phase endocytosis and exocytosis in 3T3-Ll adipocytes. J. BioZ. Chem. 261: 3944-3951, 1986. 9. Gibbs, E. M., G. E. Lienhard, and G. W. Gould. Insulin-induced translocation of glucose transporters to the plasma membrane precedes full stimulation of hexose transport. Biochemistry 27: 6681-6685, 1988. 10. Griffiths, G., S. Pfeiffer, K. Simons, and K. Matlin. Exit of newly synthesized membrane proteins from the trans cisterna of the Golgi complex to the plasma membrane. J. Cell Biol. 101: 949-964, 1985. 11. Gruenberg, J., and K. E. Howell. Immuno-isolation of vesicles using antigenic sites either located on the cytoplasmic or the exoplasmic domain of an implanted viral protein. A quantitative analysis. Eur. J. Cell Biol. 38: 312-321, 1985. 12. Haylett, T., and L. Thilo. Endosome-lysosome fusion at low temperature. J. Biol. Chem. 266: 8322-8327, 1991. 13. Heuser, J. E., and R. G. W. Anderson. Hypertonic media inhibits receptor-mediated endocytosis by blocking clathrincoated pit formation. J. Cell Biol. 108: 389-400, 1989. 14. Jacobs, D. B., C. J. Berenski, R. A. Spangler, and C. Y. Jung. Radiation inactivation target size of rat adipocyte glucose transporters in the plasma membrane and intracellular pools. J., Biol. Chem. 262: 8084-8087, 1987. 15. Kitagawa, K., B. S. Rosen, B. M. Spiegelman, G. L. Lienhard, and L. I. Tanner. Insulin stimulates the acute release of

1g l

2. l

21 l

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ADIPOCYTES

E393

adipsin from 3T3-Ll adipocytes. Biochim. Biophys. Acta 1014: 83-89, 1989. Matlin, K. S., and K. Simons. Reduced temperature prevents transfer of a membrane glycoprotein to the cell surface but does not prevent terminal glycosylation. Cell 34: 233-243, 1983. Moore, M. S., D. T. Mahaffey, F. M. Brodsky, and R. G. W. Anderson. Assembly of clathrin-coated pits onto purified plasma membranes. Science Wash. DC 236: 558-563, 1987. Oka, Y., C. Mottola, C. L. Oppenheimer, and M. P. Czech. Insulin activates the appearance of insulin-like growth factor II receptors on the adipocyte cell surface. Proc. Natl. Acad. Sci. USA 81: 4028-4032, 1984. Piper, R. C., L. J. Hess, and D. E. James. Differential sorting of two glucose transporters expressed in insulin-sensitive cells. Am. J. Physiol. 260 (Cell Physiol. 29): C570-C580, 1991. Piper, R. C., C. T. Tai, J. W. Slot, C. S. Hahn, C. Rice, H. Huang, and D. E. James. The efficient intracellular sequestration of the insulin-regulatable glucose transporter (GLUT4) is conferred by the NH* terminus. J. Cell Biol. 117: 729-743, 1992. Robinson, L. J., S. Pang, D. S. Harris, J. Heuser, and D. E. James. Translocation of the glucose transporter (GLUT4) to the cell surface in permeabilized 3T3-Ll adipocytes: effects of ATP, insulin and GTPyS and localization of GLUT4 to clathrin lattices. J. Cell Biol. 117: 1181-1196, 1992. Rodnick, K. J., J. W. Slot, D. R. Studelska, D. E. Hanpeter, L. J. Robinson, H. J. Geuze, and D. E. James. Immunocytochemical and biochemical studies of GLUT4 in rat skeletal muscle. J. BioZ. Chem. 267: 6278-6285, 1992. Saraste, J., G. E. Palade, and M. G. Farquhar. Temperature-sensitive steps in the transport of secretory proteins through the Golgi complex in exocrine pancreatic cells. Proc. Natl. Acad. Sci. USA 83: 6425-6429, 1986. Slot, J. W., H. J. Geuze, S. Gigengack, D. E. James, and G. E. Lienhard. Translocation of the glucose transporter GLUT4 in cardiac myocytes of the rat. Proc. Natl. Acad. Sci. USA 88: 78157819, 1991. Slot, J. W., H. J. Geuze, S. Gigengack, G. E. Lienhard, and D. E. James. Immuno-localization of the insulin regulatable glucose transporter in brown adipose tissue of the rat. J. Cell Biol. 113: 123-135, 1991. Suzuki, K., and T. Kono. Evidence that insulin causes the translocation of glucose transport activity to the plasma membrane from an intracellular storage site. Proc. Natl. Acad. Sci. USA 77: 2542-2545, 1980. Tanner, L. I., and G. E. Lienhard. Insulin elicits a redistribution of transferrin receptors in 3T3-Ll adipocytes through an increase in the rate constant for receptor externalization. J. Biol. Chem. 262: 8975-8980, 1987. Tanner, L. I., and G. E. Lienhard. Localization of transferrin receptors and insulin-like growth factor II receptors in vesicles from 3T3-Ll adipocytes that contain intracellular glucose transporters. J. Cell Biol. 108: 1537-1545, 1989. Wardzala, L. J., I. A. Simpson, M. M. Rechler, and S. W. Cushman. Potential mechanism of the stimulatory action of insulin on insulin-like growth factor II binding to the isolated rat adipocyte cell. J. Biol. Chem. 259: 8378-8383, 1984. West, M. A., M. S. Bretscher, and C. Watts. Distinct endocytic pathways in epidermal growth factor-stimulated human carcinoma A431 cells. J. CeZl Biol. 109: 2731-2739, 1989. Zorzano, A., W. Wilkinson, N. Kotliar, G. Thoidis, B. E. Wadzinkski, A. E. Ruoho, and P. F. Pilch. Insulin-regulated glucose uptake in rat adipocytes is mediated by two transporter isoforms present in at least two vesicle populations. J. Biol. Chem. 264: 12358-12363, 1989.


muscle glucose transporters in 3T3L1 adipocytes. Effect of chronic glucose deprivation.

Cultured 3T3L1 adipocytes dispose of excess medium glucose as lactate under abundant oxygen availability.

Suppressed intrinsic catalytic activity of GLUT1 glucose transporters in insulin-sensitive 3T3-L1 adipocytes.

Sex-sorting of boar spermatozoa does not influence the localization of glucose transporters.

Insulin-responsive glucose transporters are concentrated in a cell surface-derived membrane fraction of 3T3-L1 adipocytes.

Regulation of glucose transporters in diabetes.

Distribution of glucose transporters in renal diseases.

Association of Metformin's effect to increase insulin-stimulated glucose transport with potentiation of insulin-induced translocation of glucose transporters from intracellular pool to plasma membrane in rat adipocytes.

Glucose Transporters in Cardiac Metabolism and Hypertrophy.

Corticotrophin-Releasing Factor (CRF) and the urocortins are potent regulators of the inflammatory phenotype of human and mouse white adipocytes and the differentiation of mouse 3T3L1 pre-adipocytes.

Functional architecture of MFS D-glucose transporters.

The molecular biology of mammalian glucose transporters.

Regulation of glucose transport in isolated adipocytes by levamisole.

Glucose transporters serve as water channels.

Avian and Mammalian Facilitative Glucose Transporters.

Facilitative glucose transporters: an expanding family.

Glucose transporters: structure, function, and regulation.

Brain glucose transporters: implications for neurologic disease.

Regulation of dipeptidyl peptidase 4 production in adipocytes by glucose.

Osteopontin upregulates the expression of glucose transporters in osteosarcoma cells.

Hydrogen sulfide promotes adipogenesis in 3T3L1 cells.

Isoform-specific subcellular targeting of glucose transporters in mouse fibroblasts.

Developmental regulation in the expression of rat heart glucose transporters.

SIK2 regulates CRTCs, HDAC4 and glucose uptake in adipocytes.