©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cellubrevin Is a Resident Protein of Insulin-sensitive GLUT4 Glucose Transporter Vesicles in 3T3-L1 Adipocytes (*)

(Received for publication, November 8, 1994; and in revised form, February 1, 1995)

Allen Volchuk (§) Robert Sargeant (¶) Satoru Sumitani (**) Zhi Liu Lijing He Amira Klip (§§)

From the Division of Cell Biology, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Insulin stimulates glucose transport in muscle and fat cells by inducing translocation of GLUT4 glucose transporters from a storage site to the cell surface. The mechanism of this translocation and the identity of the storage site are unknown, but it has been hypothesized that transporters recycle between an insulin-sensitive pool, endosomes, and the cell surface. Upon cell homogenization and fractionation, the storage site migrates with light microsomes (LDM) separate from the plasma membrane fraction (PM). Cellubrevin is a recently identified endosomal protein that may be involved in the reexocytosis of recycling endosomes. Here we describe that cellubrevin is expressed in 3T3-L1 adipocytes and is more abundant in the LDM than in the PM. Cellubrevin was markedly induced during differentiation of 3T3-L1 fibroblasts into adipocytes, in parallel with GLUT4, and the development of insulin regulated traffic. In response to insulin, the cellubrevin content decreased in the LDM and increased in the PM, suggesting translocation akin to that of the GLUT4 glucose transporter. Vesicle-associated membrane protein 2 (VAMP-2)/synaptobrevin-II, a protein associated with regulated exocytosis in secretory cells, also redistributed in response to insulin. Both cellubrevin and VAMP-2 were susceptible to cleavage by tetanus toxin. Immunopurified GLUT4-containing vesicles contained cellubrevin and VAMP-2, and immunopurified cellubrevin-containing vesicles contained GLUT4 protein, but undiscernible amounts of VAMP-2. These observations suggest that cellubrevin and VAMP-2 are constituents of the insulin-regulated pathway of membrane traffic. These results are the first demonstration that cellubrevin is present in a regulated intracellular compartment. We hypothesize that cellubrevin and VAMP-2 may be present in different subsets of GLUT4-containing vesicles.


INTRODUCTION

Insulin stimulates glucose uptake into muscle and fat cells by recruiting glucose transporters (predominantly the GLUT4 isoform) from an intracellular storage site to the cell surface(1, 2) . In spite of the wide documentation of this phenomenon through biochemical and morphological techniques(3, 4) , the identity of the intracellular organelle endowed with glucose transporters and the mechanism of its incorporation into the plasma membrane remain largely unknown.

Several scenarios have been proposed to explain the intracellular traffic of glucose transporters. It has been proposed that regulation of GLUT4 intracellular traffic may share characteristics of the process of regulated secretion (a phenomenon involving fusion of specialized exocytic vesicles with the plasma membrane that occurs only in response to a stimulus). Support for the regulated exocytotic pathway is provided by the targeting of transfected GLUT4 glucose transporters to secretory granules in neuroendocrine PC12 cells(5) , by the abundant expression of Rab 3D in cells where glucose transport is regulated by insulin(6) , and by the colocalization of the adipocyte GLUT4 transporter with proteins that are thought to be exclusive to synaptic and secretory vesicles of neuroendocrine cells(7) . These proteins, named vesicle-associated membrane proteins or VAMPs(^1)/synaptobrevins, consist of two isoforms, I and II, both of 18 kDa, and are found in synaptic vesicles of neuronal cells(8) , secretory granules of pancreatic neuroendocrine (9) , and pancreatic exocrine cells(10, 11) . VAMPs are thought to be involved in docking/fusion of the vesicles/granules with the plasma membrane(8) . An antibody raised to the common domain of all VAMPs was recently shown to react with polypeptides of molecular mass of 17 and 18 kDa in GLUT4-containing membranes of rat white adipocytes(7) , and with yet a third band of lower molecular weight(12) .

It has also been suggested that glucose transporters recycle constitutively through the endocytic pathway (a phenomenon involving continuous formation of clathrin coated vesicles, their incorporation into endosomes and subsequent fusion of endosome-derived vesicles with the plasma membrane). The participation of the endocytic pathway in the intracellular traffic of glucose transporters is based on morphological and kinetic criteria. GLUT4 proteins can be detected by immunocytochemistry in clathrin-coated vesicles and early endosomes of white (13) and brown (4) adipocytes, although it is still debated whether insulin augments (13) or diminishes (14) the amount of GLUT4 in these structures. GLUT4 immunolabeling has also been demonstrated in endosomes filled with extracellular material(4) . The presence of the glucose transporter in endosomes could reflect its routing to the lysozomes, or alternatively its storage in a compartment capable of recycling. It has been shown that GLUT4 cycles to and from the cell membrane in the basal state(15, 16) , perhaps through endocytosis signals in its primary sequence(17, 18) . Consistent with this view, disruption of the clathrin coat by low K causes accumulation of GLUT4 polypeptides at the cell surface(19) . Insulin augments the rate of appearance of GLUT4 glucose transporters at the cell surface (15, 16) and reduces their rate of endocytosis(20, 21) . Upon insulin removal, surface photolabeled GLUT4 glucose transporters internalize, and a fraction of them can re-emerge at the cell surface upon a second exposure to insulin(22) . Collectively, these observations suggest that GLUT4 continuously recycles to and from the cell surface and that the rate of this process is altered by insulin (18) .

Recently, a protein belonging to the synaptobrevin family was purified and found to be localized in recycling endosomes(23) . This protein, named cellubrevin, is thought to be a marker of endocytic vesicles rather than of specialized secretory vesicles. In contrast to the circumscribed tissue expression of VAMP-1 and VAMP-2, the 17-kDa cellubrevin is widely distributed in a variety of tissues(23) . This protein is present in coated vesicles isolated from hepatocytes (23) and colocalizes with the transferrin receptor in CV-1 (23) and Chinese hamster ovary cells(24) .

In the present study, we investigate whether the endocytic marker cellubrevin is expressed in mouse 3T3-L1 cells, if its levels change during differentiation from fibroblasts into adipocytes, and whether this protein is involved in insulin-regulated intracellular traffic. We describe that, like GLUT4, cellubrevin translocates to the plasma membrane in response to insulin. Furthermore, cellubrevin is found to be a resident protein of GLUT4-containing vesicles, and immunopurified cellubrevin-containing vesicles contain GLUT4. Finally, we show that the VAMP isoform expressed in adipocytes is VAMP-2. These results suggest that there are GLUT4 vesicles containing cellubrevin and VAMP-2. The possibility that there are two types of intracellular GLUT4-containing vesicles is discussed, vis a vis the possible participation of the endosomal system and of vesicular exocytosis in the regulation of intracellular traffic of the GLUT4 transporter.


EXPERIMENTAL PROCEDURES

Materials

Cell culture medium, serum, supplements and reagents were obtained from Life Technologies, Inc.. Polyvinylidene difluoride (PVDF, 0.2-µm pore size) membranes were obtained from Bio-Rad. The immunoprecipitating anti-GLUT4 antiserum was prepared by immunizing rabbits with synthetic peptides corresponding to the 12 C-terminal amino acids of the GLUT4 protein as described by James et al.(2) . The following antibodies were used for immunoblotting. Anti-GLUT4 antiserum (East Acres Biologicals, Southbridge, MA) was diluted 1:1000. Affinity-purified anti-cellubrevin antiserum D204 (kind gift from Dr. T. C. Südhof, Howard Hughes Medical Institute, Dallas, TX) (23) was used at a 1:1000 dilution. The anti-cellubrevin antibody does not cross-react with either VAMP-1 or VAMP-2(23) . Two anti-VAMP-2 antibodies were used. One was raised to a peptide corresponding to the unique N-terminal sequence of VAMP-2 (kind gift from Dr. W. Trimble, University of Toronto) that was previously shown to react specifically and uniquely with this protein and not with either VAMP-1 or cellubrevin(25) , and one raised to a fusion protein encompassing residues 1-96, which includes the middle domain common to other synaptobrevins as well as the N terminus sequence unique to VAMP-2. As discussed in this study, this antibody reacted only very weakly with cellubrevin. Anti alpha1 Na/K-ATPase monoclonal antibody 6H (a kind gift from Dr. M. Caplan, Yale University, New Haven, CT), was used at a 1:500 dilution. Fluorescein isothiocyanate-conjugated anti-rabbit IgG was from Jackson Immunoresearch Laboratories Inc, West Grove, PA. Goat serum was from Life Technologies, Inc. Slow Fade(TM) was from Molecular Probes, Inc., Eugene, OR. Recombinant VAMP-1 and VAMP-2 (glutathione S-transferase derivatives of the cytosolic portion of each synaptobrevin) were a gift from Dr. W. Trimble, University of Toronto. Purified tetanus toxin light chain was a kind gift from Dr. Ernst Habermann, Justus-Leibig Universitat, Giesen, Germany.

Immunofluorescence

Indirect immunofluorescence was performed according to the method of Piper et al.(26) with slight modifications. 3T3-L1 cells were grown on glass coverslips, differentiated into adipocytes 2 days before reaching confluence, washed 2 times with serum-free Dulbecco's modified Eagle's medium, and fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. Excessive fixative was quenched by incubation in PBS containing 100 mM glycine for 15 min. Cells were then permeabilized with 0.1% Triton X-100 in PBS for 15 min at room temperature, washed 3 times with PBS, and incubated with 10% goat serum in PBS for 20 min. After three more washes in PBS, coverslips were incubated with anti-cellubrevin antibody (dilution 1:10 in PBS) at 4 °C overnight. Cells were then washed 3 times 15 min in PBS, incubated for 60 min with 6 µg/ml fluorescein isothiocyanate-conjugated donkey anti-rabbit IgG at room temperature, and mounted in Slow Fade(TM). Coverslips were examined with an Olympus VANOX AHBT3 fluorescence microscope.

Cell Culture and Subcellular Fractionation

3T3-L1 fibroblasts, kindly provided by Dr. D. Lane (Johns Hopkins School of Medicine, Baltimore, MD), were differentiated into adipocytes as described previously(27) . Cells from two 10-cm dishes/condition were pretreated for 3 h in serum-free Dulbecco's modified Eagle's medium and then incubated with or without 100 nM insulin for 20 min at 37 °C and fractionated according to Piper et al.(26) to obtain plasma membranes (PM), high density microsomes (HDM), and low density microsomes (LDM). The protein content of the fractions was determined by the Bio-Rad procedure. Total membranes (TM) from 3T3-L1 fibroblasts or adipocytes were prepared as follows. Monolayers were rinsed twice with ice-cold homogenization buffer (255 mM sucrose, 0.5 mM phenylmethylsulfonyl fluoride, 1 µM pepstatin A, 1 µM leupeptin, 10 µM E-64, 1 mM EDTA, and 20 mM Na-HEPES, pH 7.4), scraped vigorously with a rubber policeman into 4 ml of the same buffer, and homogenized with 30 strokes of a Teflon pestle in a glass homogenizer at 1200 rotations/min. The homogenate was centrifuged at 1000 times g for 3 min to pellet the nuclei and large cellular debris. The supernatant was centrifuged at 245,000 times g for 90 min to sediment the total membranes. The membrane samples were resuspended in homogenization buffer.

Treatment with Tetanus Toxin

Membrane fractions derived from 3T3-L1 cells were freeze dried and resuspended in 20 µl of potassium glutamate buffer (138 mM potassium glutamate, 20 mM HEPES, 8 mM MgCl(2), 0.285 mM CaCl(2), 1 mM EGTA, 1 mM dithiothreitol, pH 7.15) containing 0.5% Triton X-100 with or without the indicated concentrations of tetanus toxin light chain prepared as in (28) . The samples were incubated at 37 °C in a water bath under constant agitation for 1 h; a brief vortex was given at the 30-min interval. The samples were then boiled for 3 min in 20 µl of 2 times concentrated Laemmli sample buffer and immediately resolved by SDS-PAGE. Proteins were then electrotransferred and immunoblotted.

Immunoprecipitation of GLUT4-containing Vesicles and of Cellubrevin-containing Vesicles

GLUT4-containing vesicles were immunoprecipitated by a modification of the protocol of Laurie et al.(12) . Briefly, 6 µl of anti-GLUT4 serum or preimmune serum were incubated with 100 µl of sheep anti-rabbit IgG magnetic beads (M-280 Dynabeads, Dynal Inc, Great Neck, NY) in 100 mM potassium phosphate buffer, pH 7.4 (KP) containing 5 mg/ml bovine serum albumin for 5-6 h at 4 °C under constant rotation then washed 3 times with 0.5 ml of KP-bovine serum albumin. Adipocyte monolayers were fractionated as above using 5 ml of homogenization buffer/10-cm diameter dish to remove nuclei, mitochondria, plasma membranes, and heavy microsomes but without sedimenting the LDM. The supernatant (500 µl containing approximately 350 µg protein of LDM), adjusted to 100 mM KP, was added to the beads and incubated 16-18 h at 4 °C under constant rotation. The supernatant was removed, the beads were washed 3 times with KP without bovine serum albumin, and the resulting supernatants were combined with the first one. Pooled supernatants were centrifuged at 200,000 times g for 60 min, and the sedimented membranes were resuspended in 2 times concentrated Laemmli sample buffer (29) containing 8 M urea (sample named Supernatant). The material bound to the pelleted magnetic beads was equally solubilized in Laemmli sample buffer containing 8 M urea (sample named Pellet). The entire volumes of Supernatant and Pellet were resolved by SDS-PAGE. For immunopurification of cellubrevin-containing vesicles, a similar procedure was employed except that the anti-cellubrevin antiserum was used as primary antibody and an irrelevant serum was used for the nonimmune serum control.

PAGE and Immunoblotting

Membrane proteins were separated by SDS-PAGE (29) in 13 and 14% polyacrylamide gels (as indicated in the figure legends) and transferred electrophoretically to PVDF membranes. Immunoblotting was carried out as described previously(25) . Bound monoclonal antibodies were detected with I-labeled sheep-anti-mouse IgG. Polyclonal antibodies with I-labeled protein A or Enhanced Chemiluminescence (Amersham Corp.) as indicated in the figure legends. Scanning of x-ray films was done using a PDI model DNA35 scanner with the version 1.3 of the Discovery Series one-dimensional gel analysis software.

RNA Isolation and Northern Blot Analysis

Total RNA was extracted from 3T3-L1 fibroblasts or adipocytes and from rat brain (as control), using the single-step RNA isolation with acid guanidinium thiocyanate-phenol chloroform(30) , quantitated by its 260/280 UV absorbance and electrophoresed under denaturing conditions in 1.2% (w/v) agarose gels containing 8% (v/v) formaldehyde. RNA was then transferred onto nytron membranes and baked for 90 min at 80 °C in a vacuum oven. Equal loading of all samples containing 20 µg of RNA (3T3-L1 fibroblasts and adipocytes) was confirmed by the ethidium bromide fluorescence incorporated into nucleic acids. Where indicated, mRNA was isolated using the Dynabeads mRNA direct kit (Dynal, Oslo, Norway) according to the manufacturer's instructions. The nytron membranes containing RNA or mRNA samples were pre-hybridized overnight at 42 °C with 200 µg/ml salmon sperm DNA in 6 times SSPE (1 times SSPE: 0.15 M NaCl, 10 mM NaH(2)PO(4), 1 mM EDTA, pH 7.4), 10 times Denhardt's solution, 0.5% SDS. Hybridization was carried out in 50% formamide, 6 times SSPE, 0.5% SDS, 5% dextran sulfate, 100 µg/ml salmon sperm DNA for 16 h at 37 °C by adding [alpha-P]dCTP-labeled VAMP-1 cDNA (5.5 times 10^8 cpm/µg DNA) or VAMP-2 cDNA (5.7 times 10^8 cpm/µg DNA), each labeled by the random primer method. The VAMP-1 probe corresponded to nucleotides 1-1470 of the rat VAMP-1 sequence(31) , and the VAMP-2 probe corresponded to nucleotides 1-432. Both were the kind gift of Dr. W. Trimble, University of Toronto. Following hybridization, nytron membranes were washed 3 times 5 min with 1 times SSC, 0.1% SDS at room temperature and 2 times 1 h with 0.1 times SSC, 0.5% SDS at 65 °C prior to exposure to DuPont autoradiographic film at -70 °C.


RESULTS

Expression of Cellubrevin during Differentiation of 3T3-L1 Cells

Fig. 1examines the expression of cellubrevin in total membranes of undifferentiated 3T3-L1 fibroblasts and differentiated 3T3-L1 adipocytes. The first lane (Br) shows that the anti-cellubrevin antibody does not react with proteins present in rat brain homogenates, suggesting that it does not cross-react with either VAMP-1 or VAMP-2, abundant proteins of brain stem and brain, respectively(32) . Lanes labeled FTM (fibroblast total membranes) contained total membranes of 3T3-L1 fibroblasts, in which the 17 kDa band of cellubrevin was not detectable. Overexposure of the x-ray films revealed a very faint band in this fraction in the absence of tetanus toxin light chain (lane ``[minus]''), and this weak band totally disappeared in fibroblast total membranes treated with the toxin in vitro (lane ``+''). Lanes labeled ATM (adipocyte total membranes) show the significantly higher abundance of the 17 kDa band in total membranes of fully differentiated 3T3-L1 adipocytes compared with undifferentiated fibroblasts. Treatment with tetanus toxin light chain in vitro completely eliminated the immunoreactive 17 kDa band. This strongly supports the notion that the reactive band is indeed cellubrevin, since the clostridium endopeptidase cleaves VAMPs/synaptobrevins and cellubrevin but not other known proteins(8, 32) . The endopeptidase did not alter the protein profile of the membranes as assessed by Coomassie Blue staining or the content of other membrane proteins such as the GLUT4 glucose transporter (results not shown). These results demonstrate that the abundance of cellubrevin, per mg of protein of total membranes, sharply increases during differentiation of 3T3-L1 fibroblasts into adipocytes (also shown more clearly in the last two lanes of Fig. 3).


Figure 1: Detection of cellubrevin and its sensitivity to tetanus toxin. The following membrane fractions were isolated from 3T3-L1 cells: fibroblast total membranes (FTM), adipocyte total membranes (ATM), adipocyte light density microsomes (LDM), and adipocyte plasma membranes (PM). Samples of 30 µg of protein (FTM and ATM) or 20 µg of protein (LDM and PM) were treated without (lanes labeled ``[minus]'') or with 90 nM tetanus toxin light chain (lanes labeled ``+'') as described under ``Experimental Procedures.'' These fractions as well as 10 µg of brain microsomes (Br) were analyzed by SDS-PAGE in 14% polyacrylamide gels and electrotransferred to PVDF filters for immunoblotting with anti-cellubrevin antibody. Detection was by the enhanced chemiluminescence method.




Figure 3: Effect of insulin on the subcellular distribution of alpha1 Na/K-ATPase, GLUT4 glucose transporters, and cellubrevin in 3T3-L1 adipocytes. The following membrane fractions were isolated from control (C) or insulin-treated (I) 3T3-L1 cells as described under ``Experimental Procedures'': adipocyte high density microsomes (HDM), adipocyte plasma membranes (PM), adipocyte low density microsomes (LDM), and adipocyte (A) or fibroblast (F) total membranes (TM). Twenty µg of HDM, PM or LDM, and 40 µg of TM were resolved by SDS-PAGE on 13% polyacrylamide gels and electrotransferred to PVDF filters. A, the top portion of the filters was immunoblotted with antibodies to the alpha1 subunit of the Na/K-ATPase (apparent M(r) 105,000), and the bottom part was immunoblotted with antibodies to the GLUT4 glucose transporter (apparent M(r) 53,000), followed by detection with I-labeled sheep-anti-mouse IgG or protein A, respectively. B, parallel samples were resolved on a 14% polyacrylamide gel, electrotransferred, and immunoblotted with anti-cellubrevin antibody detected by the enhanced chemiluminescence procedure.



The last four lanes analyze the subcellular distribution of cellubrevin in purified LDM and PM from 3T3-L1 adipocytes. Lanes labeled ``[minus]'' show the amount of the 17-kDa protein in untreated membranes, and lanes labeled ``+'' show its complete disappearance upon in vitro treatment with tetanus toxin light chain. The 17-kDa protein was present in both the LDM and PM fractions, but per mg of protein it was enriched in the LDM. Scans of six independent experiments show that the relative enrichment per mg of protein in LDM to PM was 2.64 ± 0.54 to 1.00. On average, the subcellular fractionation of one confluent 10-cm dish of 3T3-L1 adipocytes yields approximately 160 µg of protein of PM, 40 µg of protein of HDM, and 225 µg of protein of LDM. When this yield of protein in the LDM and PM fractions was considered, the relative recovery of cellubrevin in these fractions was 3.7 to 1.00. Cellubrevin was located largely in these two fractions, since analysis of HDM of these cells showed only minute amounts of the protein (shown later in Fig. 3). Finally, the anti-cellubrevin antibody did not detect any bands in samples of recombinant VAMP-2 or VAMP-1, respectively (results not shown). This confirms the specificity of the antibody, and its tetanus toxin sensitivity underscores the identity of the 17 kDa band seen in 3T3-L1 cell membranes as cellubrevin.

This affinity-purified antibody was therefore used to immunolocalize cellubrevin in permeabilized 3T3-L1 adipocytes. The antibody was previously used to detect cellubrevin by immunofluorescence in CV-1 cells(23) . Fig. 2shows a representative micrograph of the distribution of cellubrevin in a 3T3-L1 adipocyte. Panela shows the fluorescence image when focussing near the cell surface, and panelb shows the fluorescence image when focussing at the level of the nucleus. The two images show that immunoreactive cellubrevin was distributed in a pole around the nucleus as well as in discrete structures (punctate fluorescence) throughout the available cytoplasmic space and outlining the cell boundaries. Neither the nuclei nor the fat droplets were stained by the antibody, which appear as dark circular structures clearly seen in panelb. Control experiments performed without primary antibody showed no detectable fluorescence (not shown).


Figure 2: Immunolocalization of cellubrevin in 3T3-L1 adipocytes. Indirect immunofluorescence was used to detect the presence of cellubrevin in 3T3-L1 adipocytes grown on coverslips as described under ``Experimental Procedures.'' Panelsa and b show images from the same cell detecting cellubrevin with the affinity-purified antibody and fluorescein-conjugated secondary antibody. Panela shows an image that focuses near the cell surface, and panelb shows an image that focuses at the level of the nucleus. n in panelb denotes the nucleus.



Subcellular Distribution of other Membrane Proteins: Effect of Insulin and Differentiation

Fig. 3A demonstrates the purity of the isolated membranes and the specificity of insulin action by analyzing the subcellular distribution of the 105-kDa alpha1 subunit of the Na/K-ATPase (toppanel) and of the GLUT4 glucose transporter (lowerpanel). The alpha1 Na/K-ATPase polypeptide was largely circumscribed to the PM, with only trace amounts showing up in either the HDM or the LDM. Insulin did not alter the distribution of the alpha1 subunit of the Na/K-ATPase, and the abundance of this polypeptide expressed per mg of protein actually decreased during 3T3-L1 cell differentiation.

In contrast to the alpha1 Na/K-ATPase, the 53-kDa polypeptide of GLUT4 was completely induced upon differentiation of 3T3-L1 fibroblasts into adipocytes, as documented previously (33, 34) and shown in the lasttwolanes of the lowerpanel. Confirming previous reports, the glucose transporter was found predominantly in the LDM, and its content was markedly reduced in this fraction in response to an acute (20 min) challenge of the cells with insulin. Simultaneously, the GLUT4 content increased markedly in the PM. Insulin had no significant effect on the content of GLUT4 protein in the HDM. In nine separate experiments, the increase in the PM was 2.18 ± 0.26-fold, and the decrease in the LDM was 40 ± 3%. These changes are in good agreement with those in other studies with 3T3-L1 adipocytes(14, 35, 36, 37) .

Effect of Insulin on the Subcellular Distribution of Cellubrevin

Fig. 3B demonstrates that cellubrevin is a protein regulated by insulin; after a 20-min treatment of 3T3-L1 adipocytes with insulin, the content of cellubrevin in the LDM was reduced, concomitant with a marked elevation in the PM fraction. The lasttwolanes of Fig. 3B show again the marked induction in cellubrevin expression during 3T3-L1 cell differentiation from fibroblasts into adipocytes, comparing equal amounts of protein of total membrane fractions. In six independent experiments, insulin caused a reduction in cellubrevin content in the LDM of 33 ± 6% and a gain in the PM of 2.24 ± 0.33-fold. Like GLUT4, cellubrevin was barely detectable in the HDM fraction, and its content in these membranes was not significantly altered by insulin treatment.

VAMP-2 in 3T3-L1 Adipocytes: Subcellular Distribution and Effect of Insulin

A previous study detected the presence of VAMP-like proteins in LDM of rat adipocytes, which translocated to the PM in response to insulin and co-purified with GLUT4-containing vesicles. Because of the need to differentiate between the regulated exocytic and the recycling endocytic pathways in insulin-dependent translocation of GLUT4 transporters, it is important to determine whether the VAMP-like protein is VAMP-1, VAMP-2, cellubrevin, or even a novel isoform specific to the insulin-regulated pathway. The antibody used in the rat adipocyte study was raised to the central domain common to all VAMPs and therefore could not differentiate among these possibilities(7, 12) . In the present study, we used one antibody raised to the cytosolic portion of VAMP-2 that reacts with both VAMP-1 and VAMP-2 (the anti-fusion protein antibody) and one that reacts exclusively with VAMP-2 (the anti-peptide antibody). Both antibodies are expected to react exclusively with VAMP-2 in 3T3-L1 adipocytes, since Northern blot analysis failed to detect the presence of VAMP-1 transcripts in these cells (Fig. 4). Unavailability of antibodies specific to VAMP-1 precluded the demonstration of absence of this polypeptide. In contrast, VAMP-2 mRNA was readily detectable in total RNA of 3T3-L1 fibroblasts or adipocytes, to a level comparable with that detected previously in skeletal muscle(25) .


Figure 4: Search for VAMP-1 and VAMP-2 mRNA transcripts in RNA from brain and 3T3-L1 cells. Total RNA was isolated from rat brain (Br), 3T3-L1 fibroblasts (F), 3T3-L1 adipocytes (A), or rat skeletal muscle (SM). mRNA was also isolated from 3T3-L1 adipocytes (A(m)). For the VAMP-1 Northern blot, 10 µg of brain RNA and 20 µg of fibroblast or adipocyte RNA were analyzed. For the VAMP-2 Northern blots, the toppanel contained 20 µg of total RNA from rat brain, 3T3-L1 fibroblasts, 3T3-L1 adipocytes, or skeletal muscle. The lowerpanel of VAMP-2 Northern blot contained 1 µg of total RNA from rat brain, 25 µg of total RNA from 3T3-L1 adipocytes, 5 µg of purified mRNA from 3T3-L1 adipocytes, and 25 µg of total RNA from 3T3-L1 fibroblasts. The size of the transcripts in kilobases is indicated in each blot.



Using the antibody raised to the glutathione S-transferase-fusion protein containing the N terminus and middle portions of the VAMP-2 polypeptide, we detected a single 18 kDa band in membrane fractions from 3T3-L1 adipocytes (Fig. 5A). Per unit protein, the 18 kDa band was much more enriched in the LDM than in the the PM. In both fractions, the protein was totally cleaved by tetanus toxin light chain in vitro. Only a very low reactivity was seen in the region of migration of cellubrevin (corresponding to 17 kDa), and this required overexposure of the x-ray film, suggesting that the antibody reacts very poorly, if at all, with cellubrevin. The antibody also detected a single band of 18 kDa in rat brain homogenates. The majority of this band was susceptible to proteolysis by tetanus toxin light chain, confirming that VAMP-2 is detected by the antibody. The remaining immunoreactivity of the brain sample may be ascribed to VAMP-1, which in rat cells is not susceptible to hydrolysis by tetanus toxin.


Figure 5: A, detection of VAMP-2 in membranes from 3T3-L1 adipocytes. Two µg of protein of brain microsomes (Br), and 20 µg of protein of LDM or PM isolated from 3T3-L1 adipocytes were incubated without(-) or with (+) 90 nM tetanus toxin light chain in vitro as indicated under ``Experimental Procedures.'' The fractions were then analyzed by SDS-PAGE in 14% polyacrylamide gels, electrotransferred to PVDF filters, immunoblotted with antiserum to the VAMP-2 fusion protein, and detected by the enhanced chemiluminescence procedure. B, the effect of insulin on the subcellular distribution of VAMP-2 in 3T3-L1 adipocytes using the anti-fusion protein antibody. HDM, LDM, and PM were isolated from control (C) or insulin-treated (I) 3T3-L1 adipocytes as described under ``Experimental Procedures.'' Twenty µg of each fraction, as well as 5 µg of brain microsomes were analyzed by SDS-PAGE in 14% polyacrylamide gels, electrotransferred onto PVDF filters, immunoblotted with antiserum to VAMP-2 fusion protein, and followed by I-coupled protein A.



Fig. 5B shows that the subcellular localization of the VAMP-2 band is altered by insulin treatment of 3T3-L1 adipocytes. The content of VAMP-2 increased in the PM and diminished in the LDM, without changing in the HDM where it was also abundant. Scanning of four experiments showed that insulin decreased this protein in the LDM by 36 ± 6% and increased it in the PM by 1.76 ± 0.08-fold. A similar result was obtained when an antibody raised to the N terminus portion specific of VAMP-2 was used. This antibody does not cross-react with either VAMP-1 or cellubrevin(25) . Scanning of five independent experiments using the latter antibody revealed that the reduction caused by insulin was of 48% in the LDM and the gain in the PM was 1.8-fold. These results demonstrate that VAMP-2 is indeed the isoform regulated by insulin in 3T3-L1 adipocytes, as was seen for cellubrevin (see Fig. 3B) when using the cellubrevin-specific antibody.

Localization of Cellubrevin and VAMP-2 in Immunopurified GLUT4-containing Vesicles

The change in subcellular localization of cellubrevin and VAMP-2 in response to insulin clearly parallels that of the GLUT4 protein, but it does not prove whether these two proteins are present on the same vesicles as the glucose transporter or whether insulin causes the translocation of multiple vesicles, some containing GLUT4 and others containing cellubrevin and/or VAMP-2. In order to test these possibilities, GLUT4-containing vesicles were immunopurified using anti-GLUT4 antibodies coupled to magnetic beads, and their content of immunoreactive VAMP-2 or cellubrevin was examined. The antibody reacts with the C terminus of the GLUT4 protein, which in the normal configuration of the intracellular membrane structure faces the cytosol. Membranes containing GLUT4 polypeptides were sedimented by this procedure only when immunoprecipitated with anti-GLUT4 antiserum but not with preimmune serum (Fig. 6A, firstpanel). In seven independent experiments, the anti-GLUT4 antiserum sedimented 70 ± 4% of the total GLUT4 protein contained in the starting material. This suggests that only a small fraction of the GLUT4 protein escapes access to the antibody, either blocked through aggregation or interaction with other proteins, or the reacting epitope facing intraluminal sites as a result of cell homogenization prior to isolation of the supernatant fraction containing LDM. Since the majority of the GLUT4 protein did immunoprecipitate, we explored the possible presence of cellubrevin and VAMP-2 in these vesicles.


Figure 6: Distribution of cellubrevin, VAMP-2 and GLUT4 in purified GLUT4-containing vesicles (column A) and in purified cellubrevin-containing vesicles (column B). Vesicles were isolated as described under ``Experimental Procedures'' using either GLUT4-specific antiserum (alphaGLUT4), pre-immune serum (alphaPI) or anti-cellubrevin antibody (alphaCellub). The entire pellet (P) and sedimented supernatant (S, i.e. nonadsorbed membranes) were analyzed by SDS-PAGE and probed by immunoblotting for the presence of GLUT4 (top), cellubrevin (middle), and VAMP-2 (bottom). Detection of all GLUT4 and VAMP-2 (columnB) immunoreactivity was done by I-labeled protein A; detection of VAMP-2 (columnA) and of cellubrevin was done by the ECL procedure. Equal amounts of specific and preimmune sera were used, and this was confirmed by scanning the intensity of the IgG bands in the respective immunoblots (in three similar experiments the ratio of intensities of the IgG band in the anti-GLUT4 pellets to preimmune pellets was 0.98).



Fig. 6A, secondpanel, shows that indeed a substantial fraction of the total cellubrevin copurified with the immunoprecipitated GLUT4-containing vesicles. In contrast, no cellubrevin was found when preimmune serum was used for the immunoprecipitation. Of the total cellubrevin, 55.1 ± 1.4% co-precipitated with GLUT4 vesicles compared with 72.7 ± 5.3% of the total GLUT4 protein in the same experiments, indicating that the majority of the cellubrevin is in GLUT4-containing vesicles.

Fig. 6A, thirdpanel, shows the presence of VAMP-2 in the immunopurified GLUT4-containing vesicles detected by the anti-fusion protein antibody. There was only minimal precipitation of VAMP-2 in parallel assays using preimmune antiserum instead of anti-GLUT4 serum during the vesicle precipitation. In three similar experiments, this nonspecific sedimentation was subtracted from the amount of VAMP-2 brought down by the anti-GLUT4 antibody to calculate the specific sedimentation of VAMP-2. From the total VAMP-2 content of the supernatant containing all of the LDM, 16.3 ± 3.3% co-precipitated specifically with the GLUT4-containing vesicles. This suggests that the majority of VAMP-2 is not associated with GLUT4 vesicles, although a small fraction does indeed copurify with them.

Presence of GLUT4 and VAMP-2 in Immunopurified Cellubrevin-containing Vesicles

Fig. 6B shows the analysis of proteins present in immunopurified cellubrevin-containing vesicles. The secondpanel demonstrates for the first time the ability of this antibody to isolate cellubrevin-containing vesicles. In three separate experiments, an average 30 ± 2% of the total cellubrevin precipitated with the antibody. This yield could not be improved even when a higher amount of antibody was used, suggesting hindrance of a large proportion of the cellubrevin antigenic sites from recognition by the antibody. Almost no cellubrevin-containing vesicles were sedimented with nonimmune irrelevant antibody. In the specifically immunoprecipitated cellubrevin-containing vesicles, the presence of GLUT4 was tested (Fig. 6B, firstpanel). A commensurate amount of GLUT4 was detected in these vesicles. In four experiments, this amounted to 27 ± 6% of the total GLUT4 content of the unsedimented LDM. These results suggest that cellubrevin-containing vesicles are endowed with GLUT4, just like cellubrevin is a resident protein of GLUT4-containing vesicles (Fig. 6A). A quantitative analysis of the colocalization of cellubrevin and GLUT4 is not possible given the incomplete immunoprecipitation of vesicles, especially by the cellubrevin antibody under the conditions tested. However, the present experiments support the colocalization of these proteins in vesicles present in the LDM.

Fig. 6B, thirdpanel, shows attempts to detect VAMP-2 in the cellubrevin-containing vesicles. Within the fraction of cellubrevin-containing vesicles sedimented by the anti-cellubrevin antibody, there was no specific detection of VAMP-2 beyond the background level obtained using nonimmune IgG. With the caveat that only a fraction of the total cellubrevin-containing vesicles is sampled by the immunoprecipitation procedure, this result suggests that there is no colocalization of VAMP-2 and cellubrevin within the same vesicles.


DISCUSSION

Expression of Cellubrevin during Differentiation of 3T3-L1 Cells

The presence of cellubrevin in 3T3-L1 adipocytes was determined by its reactivity with a cellubrevin-specific antibody, by its typical molecular size of 17 kDa and by its susceptibility to hydrolysis by tetanus toxin light chain. Cellubrevin was found in both plasma membranes and intracellular light microsomes, although per unit protein it was much more concentrated in the latter. This is consistent with the immunofluorescence image, which showed a preferential localization in the perinuclear region, in punctate structures throughout the cytoplasm, and to a lesser extent in the periphery. By immunofluorescence it is difficult to discern whether the peripheral staining is on the plasma membrane, likely because this technique cannot distinguish unambiguously cell surface localization of proteins that are distributed in endosomal vesicles. It is possible that upon subcellular fractionation, some of these structures could migrate with the purified PM, contributing to the signal in this subcellular fraction. The cellular localization of the endogenous cellubrevin observed by immunofluorescence in 3T3-L1 adipocytes is similar to that observed in CV-1 cells transfected with cellubrevin cDNA by McMahon et al.(23) and of the endogenous protein in Chinese hamster ovary cells observed by Galli et al.(24) . In both studies, cellubrevin was found in punctate bodies and concentrated around the nucleus. The nature of the perinuclear organelle has not been defined, but it could be part of the Golgi complex or more likely represent the intracellular recycling vesicles in the region of the centriole, which have been shown to internalize transferrin(38) .

Interestingly, the amount of cellubrevin in total membranes increased substantially during differentiation of 3T3-L1 fibroblasts into adipocytes. This result was surprising, since endocytic activity is not a function exclusive to adipocytes. A typical function that matures with cell differentiation is the stimulation of glucose transport by insulin, mediated by recruitment of glucose transporters to the cell surface(33, 34) . The lack of the insulin response in fibroblasts is due in part to the absence of GLUT4 expression at the fibroblast stage, but it has also been argued that the required pathway of intracellular traffic is missing in fibroblasts. Indeed, transfection of GLUT4 into 3T3-L1 fibroblasts does not result in translocation of the protein to the cell surface in response to insulin, in spite of the presence of functional insulin receptors(39, 40) . Thus, differentiation-associated proteins such as cellubrevin may play a key role in the translocation of GLUT4 glucose transporters to the cell surface in response to insulin in differentiated 3T3-L1 adipocytes.

Cellubrevin Translocates to the Plasma Membrane in Response to Insulin

A second major observation of this study is the insulin-dependent relocalization of cellubrevin. In principle, this translocation could be linked to the recruitment of GLUT4 proteins. To examine this possibility, we probed for the colocalization of these proteins in immunopurified vesicles. GLUT4-containing vesicles showed a marked content of cellubrevin, and conversely, cellubrevin-containing vesicles showed detectable amounts of GLUT4 protein. Due to incomplete vesicle sedimentation by each antibody, it is difficult to provide an accurate quantitative estimate of the proportion of colocalization of these proteins. The nonassociated cellubrevin may be present in other vesicles devoid of GLUT4 protein. This is expected since cellubrevin is an endosomal protein expected to be present in a variety of recycling vesicles. Whether the small fraction of cellubrevin-containing vesicles devoid of GLUT4 protein also respond to insulin remains to be determined.

A further observation is that the -fold increase in cellubrevin content in the PM in response to insulin is comparable with the -fold increase in GLUT4 protein. An unrelated protein has also been reported to parallel quantitatively the response of GLUT4 to insulin, a glycoprotein of molecular weight 160,000 in rat adipocytes(41, 42) . This is in contrast to other membrane proteins that translocate to the PM to a much lesser extent in response to the hormone, such as the GLUT1 glucose transporter, the mannose 6-phosphate receptor, and the transferrin receptor(43, 44, 45) . It has been suggested that the latter three proteins recycle through a constitutive endosomal pathway and that GLUT4 recycles through a ``regulated endosomal pathway''(18) . Our results would suggest that, in 3T3-L1 adipocytes, cellubrevin resides in the ``regulated endosomal pathway'' and that in response to insulin it accompanies the translocating GLUT4 complement.

Recently, another protein typical of recycling membranes, secretory carrier associated membrane protein (SCAMP) (46) also refered to as GTV3 (47) was found in GLUT4-containing vesicles of rat adipocytes(12, 47) . In contrast to the pattern of cellubrevin, the content of SCAMP did not increase appreciably in the PM upon insulin treatment. Based on these observations, it was proposed that the SCAMP proteins may approach the cell surface but may not become incorporated into the plasma membrane or alternatively that SCAMPs may endocytose faster than the GLUT4 polypeptide or other proteins present on GLUT4-containing vesicles. Our results suggest that cellubrevin behaves differently from SCAMPs; although both proteins are found in GLUT4-containing vesicles, only cellubrevin may actually fuse with the plasma membrane and become an integral part of it. One may speculate that cellubrevin could accompany GLUT4 in this process, and we would like to hypothesize that potential roles of cellubrevin might be participation in the endocytosis of GLUT4 proteins upon removal of insulin and/or in the fusion of endosome-derived vesicles with the plasma membrane.

VAMP-2 Translocates to the PM in Response to Insulin and Is Present in GLUT4-containing Vesicles: Redundance with Cellubrevin?

The third conclusion of this study is the identification of the VAMP isoform, which is present in GLUT4-containing vesicles and translocates to the PM in response to insulin. A previous study in rat adipocytes had shown this behavior for members of the VAMP family, detected with a generic antibody that reacts with the region of the polypeptide common to all VAMPs, including cellubrevin(7) . By Northern blot analysis, we failed to detect VAMP-1 mRNA, indicating that this isoform is not expressed in these cells. Our study also shows that, in addition to cellubrevin, VAMP-2 is found in 3T3-L1 adipocytes, as detected with an antibody specific to this isoform (which does not react with either VAMP-1 or cellubrevin). However, the proportion of VAMP-2 that was found to be associated with GLUT4-containing vesicles was significantly less than the proportion of associated cellubrevin.

The subcellular redistribution of both VAMP-2 and cellubrevin in response to insulin and the presence of both proteins in GLUT4-containing vesicles poses the question of whether each protein plays a specific role in intracellular traffic, and especially raises the interesting possibility that each protein may be present in a different subset of GLUT4-containing vesicles. Definitive answers to these questions will require demonstration of separate morphological localization or the physical separation of pools of GLUT4-containing vesicles, one endowed with VAMP-2 and one with cellubrevin. We have obtained preliminary evidence that cellubrevin-containing vesicles populated with GLUT4 protein do not contain detectable amounts of VAMP-2. The converse experiment, i.e. the measurement of cellubrevin content in immunopurified VAMP-containing vesicles, cannot be performed at present given the ineffectiveness of the available antibodies to immunoprecipitate these vesicles. The current evidence suggests that there may indeed exist two different pools of GLUT4-containing vesicles, one endowed with cellubrevin and the other supplied with VAMP-2. One may speculate that the GLUT4 vesicles containing cellubrevin originate from the endosomal compartment and that those containing VAMP-2 originate from the regulated exocytic compartment described in the Introduction. It is conceivable that, in the unstimulated state, GLUT4 recycles between the endosomal pool and the cell surface. In the insulin-stimulated state, the exocytic pool containing VAMP-2 might be mobilized to feed directly into the cell surface and/or into the endosomal pool; simultaneously, the endosomal pool might be stimulated to segregate GLUT4 and cellubrevin proteins to be efficiently recruited to the cell surface. This hypothetical scheme reconciles most of the current evidence favoring either the regulated exocytic model or the regulated recycling model (for review, see (18) ).

In conclusion, the present results demonstrate the redistribution of cellubrevin in response to insulin in 3T3-L1 adipocytes. This suggests that cellubrevin may play a role in regulated intracellular traffic in addition to its proposed role in constitutive endosomal recycling. Furthermore, the results establish the presence of both VAMP-2 and cellubrevin in GLUT4-containing vesicles and raise the possibility that each protein may be a marker of a distinct pool of vesicles endowed with glucose transporters. In this way, the proteins may provide the handle to separate the pools biochemically. The present study advances further the previous knowledge derived from studies with rat adipocytes by identifying that both cellubrevin and VAMP-2 are insulin-responsive proteins and by unraveling an increase in expression of cellubrevin associated with the differentiated phenotype. These studies may lead to the purification of different pools of glucose transporters and may aid in characterizing the itinerary of glucose transporters in response to insulin.


FOOTNOTES

*
This work was supported by a grant from the Medical Research Council of Canada (to A. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a studentship from the Medical Research Council of Canada.

Supported by an Ontario graduate studentship.

**
Supported by a postdoctoral fellowship from the Hospital for Sick Children.

§§
To whom correspondence should be addressed: Div. of Cell Biology, 555 University Ave., Toronto, Ontario M5G 1X8, Canada. Tel.: 416-813-6392; Fax: 416-813-5028.

(^1)
The abbreviations used are: VAMP, vesicle-associated membrane protein; PVDF, polyvinylidine difluoride; PBS, phosphate-buffered saline; PM, plasma membranes; HDM, high density microsomes; LDM, low density microsomes; TM, total membrane; PAGE, polyacrylamide gel electrophoresis; SCAMP, secretory carrier associated membrane protein.


ACKNOWLEDGEMENTS

We thank Dr. Thomas Südhof for the anticellubrevin antibody, Dr. William Trimble for the anti-peptide antibody to VAMP-2 and the synaptobrevin fusion protein standards, Dr. Alec Hinek for help with the immunofluorescence study, Dr. Ernst Habermann for the tetanus toxin light chain, and Dr. Michael Caplan for the anti-alpha1 Na/K-ATPase. We also thank Dr. Yasuhide Mitsumoto for helpful discussions and Theos Tsakiridis for careful reading of this manuscript.


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