(Received for publication, November 8, 1994; and in revised form, February 1, 1995)
From the
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.
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()/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.
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 1 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
1 subunit of the
Na
/K
-ATPase (apparent M
105,000), and the bottom part was immunoblotted
with antibodies to the GLUT4 glucose transporter (apparent M
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.
In contrast
to the 1 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) .
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.
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 (GLUT4), pre-immune
serum (
PI) or anti-cellubrevin antibody (
Cellub). 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.
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.
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.
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.
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.