SNAP23 promotes insulin-dependent glucose uptake in 3T3-L1 adipocytes: possible interaction with cytoskeleton

Leonard J. Foster, Karen Yaworsky, William S. Trimble, and Amira Klip

Cell Biology Programme, Hospital for Sick Children, Toronto, Ontario M5G 1X8; and Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada M5S 1A8


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The acute stimulation of glucose uptake by insulin in fat and muscle cells is primarily the result of translocation of facilitative glucose transporter 4 (GLUT-4) from an internal compartment to the plasma membrane. Here, we investigate the role of SNAP23 (a 23-kDa molecule resembling the 25-kDa synaptosome associated protein) in GLUT-4 translocation and glucose uptake in 3T3-L1 adipocytes. Microinjection of a polyclonal antibody directed to the carboxy terminus of SNAP23 inhibited GLUT-4 incorporation into the membrane in response to insulin, whereas microinjection of full-length recombinant SNAP23 enhanced the insulin effect. Introduction of recombinant SNAP23 into chemically permeabilized cells also enhanced insulin-stimulated glucose transport. These results indicate that SNAP23 is required for insulin-dependent, functional incorporation of GLUT-4 into the plasma membrane and that the carboxy terminus of the protein is essential for this process. SNAP23 is therefore likely to be a fusion catalyst along with syntaxin-4 and vesicle-associated membrane protein (VAMP)-2. Furthermore, the endogenous content of SNAP23 appears to be limiting for insulin-dependent GLUT-4 exposure at the cell surface. A measurable fraction of SNAP23 was sedimented with cytoskeletal elements when extracted with Triton X-100, unlike VAMP-2 and syntaxin-4, which were exclusively soluble in detergent. We hypothesize that SNAP23 and its interaction with the cytoskeleton may be targets for regulation of GLUT-4 traffic.

insulin action; SNARE; glucose transporter isoform 4 translocation; vesicle traffic


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ALTHOUGH IT IS WELL documented that membranes containing the glucose transporter of muscle and fat cells (isoform 4; GLUT-4) are translocated to the cell surface in response to insulin (4, 24), the mechanism of the interaction of this organelle with the plasma membrane is only partly understood. Recent work has revealed that the GLUT-4-containing organelle of fat cells contains a limited number of proteins (27), and some of these are common to other secretory organelles. Thus immunopurified GLUT-4 organelles (also called GLUT-4 vesicles) contain vesicle-associated membrane protein (VAMP)-2, VAMP-3/cellubrevin, a triplet called SCAMPs (secretory carrier membrane proteins), and an aminopeptidase (insulin-responsive aminopeptidase). Of these, only the VAMPs have been shown to be functionally involved in the process of membrane fusion in systems such as synaptic vesicles, secretory granules, and recycling endosomes. Work from our laboratory and those of others has also shown that integrity of VAMP-2 and/or VAMP-3/cellubrevin is required for incorporation of GLUT-4 into the plasma membrane of 3T3-L1 adipocytes (2, 13, 25).

VAMP-2 is one of three core proteins known as SNAREs [for soluble N-ethyl maleimide-sensitive fusion factor (NSF) attachment protein (SNAP) receptors] that are required for membrane fusion in events as distinct as intra-Golgi communication and synaptic vesicle exocytosis (15, 37). In neurons, its partners are the target membrane-associated proteins syntaxin-1 and SNAP25, a synaptosome-associated membrane protein of 25 kDa (21). 3T3-L1 adipocytes do not express either of these two gene products (34, 38). Instead, these cells express isoforms of these proteins, specifically syntaxin-4 (26, 34) and SNAP23 (35, 38), a SNAP25-like protein of 23 kDa. Both syntaxin-4 (17) and SNAP23 (6, 18) bind VAMP-2. Interestingly, SNAP23 appears to compete with VAMP-2 for binding to syntaxin-4 in vitro (6), in contrast to SNAP25, which promotes VAMP-2 binding to syntaxin-1 (17).

Syntaxin-4 is found predominantly in the plasma membrane of 3T3-L1 adipocytes, but a significant amount is also found associated with the GLUT-4 organelle (34). Introduction of anti-syntaxin-4 antibodies into oxygen-sensitive streptolysin toxin (SLO)-permeabilized 3T3-L1 adipocytes reduces the insulin-dependent GLUT-4 incorporation into the plasma membrane and its consequent increase in glucose uptake (13, 34). Immunoprecipitation of syntaxin-4 from cell lysates shows associated VAMP-2. Consistent with these observations, introduction of cytosolic fragments of VAMP-2 or syntaxin-4 by chemical permeabilization or transfection abrogates GLUT-4 incorporation into the cell surface (2, 13, 16). Thus, like VAMP-2, syntaxin-4 is also functionally required for this process.

Immunoprecipitation of SNAP23 from 3T3-L1 cell lysates shows associated VAMP-2 and syntaxin-4 (6). Furthermore, addition of the ancillary proteins alpha SNAP and NSF to lysates of rat adipocytes induced association among VAMP-2, syntaxin-4, and SNAP23 (22, 28). In the present study, we tested the possible participation of SNAP23 in GLUT-4 traffic by introduction of antibodies or recombinant proteins that would be expected to interfere with or mimic SNAP23 function. The results show that microinjected antibodies to the carboxy-terminal domain of SNAP23 reduce the insulin-dependent GLUT-4 association with the plasma membrane. Very recently, Rea et al. (19) described an inhibition of insulin-dependent GLUT-4 arrival at the membrane by an antibody directed to the amino terminus of SNAP23. The present observations complement and extend these results by showing that both the amino and carboxy termini of SNAP23 are required for vesicle-membrane interaction. We further show that full-length SNAP23 increases insulin action on GLUT-4 and glucose uptake in microinjected and chemically permeabilized cells, and we propose that SNAP23 may be necessary for insulin-dependent GLUT-4 incorporation into the membrane. This makes SNAP23 a likely target for regulation. In search of modes of regulation of SNAP23, we found that this protein sediments with cytoskeleton-containing fractions from 3T3-L1 adipocytes, unlike VAMP-2 or syntaxin-4.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Fusion proteins and antibodies. Polyclonal antibodies to GLUT-4 (33) and SNAP23 (termed alpha SN23.c12) were described previously (38). FITC-conjugated donkey anti-rabbit antibodies and rhodamine-conjugated dextran were obtained from Molecular Probes (Eugene, OR). Glutathione S-transferase (GST) and amino-terminal-fused GST-SNAP23 (38) were prepared as described previously. Horseradish peroxidase-conjugated protein A secondary antibodies were purchased from Bio-Rad.

Single-cell microinjection and plasma membrane lawns. 3T3-L1 cells were maintained as fibroblasts and induced to differentiate on 25-mm coverslips in six-well plates as described previously (34). Cells in a marked, 1-mm2 region of the coverslip were microinjected as described previously (32). Approximately 90% of the cells in the marked region (~100 cells) were microinjected with 20 µM GST or GST-SNAP23 or with 0.1 µg/µl alpha SN23.c12 or irrelevant IgG in a solution containing 1.1 µg/µl rhodamine-dextran (Mr 10,000), 110 mM potassium acetate, 10 mM HEPES (pH 7.2), and 1 mM EDTA. The volume microinjected was about one-tenth of the cell volume. This calculation is based on estimates of the volume of buffer delivered by the microinjector into oil-containing 1-µm-diameter latex beads for size comparison.

After microinjection, cells were incubated in DMEM for 2 h, followed by a 15-min stimulation with 100 nM insulin, when indicated. Plasma membrane lawns (sheets) were prepared as described by Volchuk et al. (32). Fluorescence images were obtained using a Leica inverted fluorescence microscope (model DM IRB) and quantitated and analyzed as described by Volchuk et al. (32). By this approach, >90% of the cells in the marked area can be microinjected. Any noninjected cells within the lawns quantitated as "microinjected" in these experiments will result in underestimation of the effects of the microinjected test material. Hence, all effects reported for microinjected material, whether increasing or decreasing GLUT-4 content, may be underestimated by ~10%.

It has been reported that insulin can increase the number of 3T3-L1 adipocytes that respond to insulin by translocating GLUT-4 (14). We therefore assessed whether the microinjected materials tested in the present study affect the number of responding cells. Insulin response was operationally defined as any plasma membrane GLUT-4 signal greater than X - sigma , where X and sigma  are the mean and SD, respectively, of the noninjected, insulin-treated cell population.

Preparation of crude membrane and cytosol fractions. Cytosol and crude membrane fractions from 3T3-L1 cells were prepared according to Wong et al. (38). For preparation of Triton X-100-soluble and -insoluble fractions, entire crude membrane pellets from two wells of a six-well plate of fully differentiated adipocytes were resuspended in lysis buffer containing 150 mM NaCl, 50 mM Tris · HCl (pH 7.2), 0.25% deoxycholate, 1.0% Triton X-100, 10 mM sodium pyrophosphate, 100 mM NaF, 2 mM EDTA, 10 µg/ml aprotinin, 2 µM leupeptin, 2 µM pepstatin A, 2 mM phenylmethylsulfonyl fluoride, and 1 mM NaVO4. After vigorous mixing, these suspensions were centrifuged at ~3,000 g for 10 min. The pellet of this step was considered to be the Triton-insoluble fraction, and the supernatant was considered to be the Triton-soluble fraction.

Glucose transport in SLO-permeabilized cells. The ability of insulin to stimulate glucose transport in SLO-permeabilized 3T3-L1 adipocytes was measured as described previously (34). The concentration of SLO used (0.675 µg/ml) creates pores large enough to allow immunoglobulin molecules to enter the cells (34). After permeabilization, the SLO solution was removed and replaced with potassium glutamate buffer and either 0.1 mg/ml alpha SN23.c12 or irrelevant IgG or 20 µM GST-SNAP23 or GST, and the preparation was incubated for 15 min at 37°C. Insulin (100 nM), when indicated, was added for a further 15 min in the presence of the proteins or antibodies. Stock transport solution {final concentrations 10 µM 2-[3H]deoxyglucose (1 µCi/ml) and 2 µM [14C]sucrose (0.2 µCi/ml)} was then added directly to the wells and incubated for a further 5 min at room temperature. All steps after and including permeabilization were performed in the presence of an ATP regeneration system. Cells were then lysed in 0.05 M NaOH, and associated radioactivity was measured by scintillation counting. The spillover of 14C into the 3H recording channel was subtracted. Permeabilization with SLO increased the diffusional component of glucose uptake by four- to fivefold, to represent ~25% of the total uptake. Facilitative transport was calculated by subtracting the diffusion-dependent 14C signal from the amount of 2-[3H]deoxyglucose associated with the cells (34). Statistical analysis was done using ANOVA.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Clostridial neurotoxins have proven to be valuable tools for the study of SNARE function in neuronal systems; however, many nonneuronal SNAREs are not affected by these proteases, including rodent forms of SNAP23 (12, 20). Therefore, other approaches have had to be developed to perturb SNARE proteins in nonneuronal systems.

The participation of the endogenous SNAP23 in insulin-dependent association of GLUT-4 with the cell membrane was first assessed by introducing into 3T3-L1 adipocytes antibodies raised to a hemocyanin-linked peptide comprising the carboxy-terminal 12 amino acids of SNAP23 (termed alpha SN23.c12). The antibody, in an isotonic potassium acetate buffer, was microinjected into 3T3-L1 adipocytes, and its effect on GLUT-4 content in membrane lawns was measured 90-120 min later (see Single-cell microinjection and plasma membrane lawns). Microinjection of alpha SN23.c12 or unrelated rabbit IgG did not alter the amount of plasma membrane GLUT-4 in the basal state (Fig. 1A, left). In contrast, microinjection of alpha SN23.c12 diminished the insulin-stimulated increase in GLUT-4 on the membrane lawns (Fig. 1A, middle). Unrelated IgG did not have any effect on insulin action (Fig. 1A, bottom). Digital quantitation of lawns from five independent experiments confirmed that, indeed, alpha SN23.c12 causes a statistically significant decrease in insulin-stimulated GLUT-4 incorporation into the membrane (Fig. 1B).


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Fig. 1.   Microinjection of SNAP23 antibodies reduces glucose transporter isoform 4 (GLUT-4) incorporation into plasma membrane. Approximately one-tenth of cell volume of 0.1 µg/µl alpha SN23.c12 or irrelevant IgG were microinjected into ~90% of all cells in an outlined region of coverslip. Coverslips were then serum starved for 2 h and incubated a further 15 min in absence (A, left) or presence (A, right) of insulin. Plasma membrane lawns were prepared as described and stained for GLUT-4. Indirect immunofluorescence images of GLUT-4 on plasma membrane lawns from noninjected (A, top) and injected (A, middle and bottom) cells were collected. Representative images from 1 of 5 experiments are shown. B: images such as those in A were digitally quantitated by measuring average intensities for individual lawns. Values from individual experiments were averaged and normalized to basal values. Values are means ± SE of effect of insulin relative to its corresponding basal control from 5 independent experiments. * Significantly different from either control (P < 0.05, ANOVA).

To more directly prove the participation of SNAP23 in GLUT-4 organelle fusion, we tested the effect of exogenous full-length SNAP23 in this process. The protein is naturally soluble but attaches to membranes via palmitoylation and association with other proteins such as syntaxin-4. Therefore, it was conceivable that introduction of an excess full-length SNAP23 might increase membrane fusion events if free syntaxin-4 and incoming vesicles were present. Recombinant GST-SNAP23 was able to interact with the cytosolic domains of VAMP-2 and syntaxin-4 in vitro in a manner previously demonstrated with recombinant SNAP23 binding to VAMP-2 and GST-syntaxin-4 (results not shown and Ref. 6).

GST-SNAP23 was microinjected into a clearly identified area of serum-deprived 3T3-L1 adipocytes. On the basis of the volume microinjected and the calculated cell volume, the final concentration of SNAP23 introduced may have reached 2 µM. The cells were incubated in culture medium for 90 min, followed by stimulation with 100 nM insulin for 15 min. Membrane lawns were immediately generated as described in Single-cell microinjection and plasma membrane lawns, and GLUT-4 was detected on these lawns by indirect immunofluorescence. The signals from cells in the area defined for microinjection were compared with those of adjacent, noninjected cells. As observed previously (34), insulin caused an increase in GLUT-4 labeling on plasma membrane lawns from noninjected cells (Fig. 2). Microinjection of GST-SNAP23 had no effect on the amount of GLUT-4 detected in the basal state, compared with adjacent, noninjected cells. Digital analysis of four independent experiments of unstimulated (basal) cells showed that microinjection of GST-SNAP23 did not alter the GLUT-4 signal relative to vicinal noninjected cells. On average, 36 lawns were quantitated within each experiment. The GLUT-4 signal in the noninjected cells was assigned a value of 1.00 within each experiment, and the effect of GST-SNAP23 was calculated relative to this value. In the four experiments, the microinjected cells had a GLUT-4 signal of 0.99 ± 0.14 (mean ± SE). Therefore, GST-SNAP23 did not affect the basal amount of GLUT-4 present at the plasma membrane (Fig. 2).


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Fig. 2.   Microinjection of exogenous SNAP23 protein improves GLUT-4 incorporation into plasma membrane. Approximately one-tenth of cell volume of 20 µM glutathione S-transferase (GST)-SNAP23 or GST were microinjected into ~90% of all cells in an outlined region of coverslip. Coverslips were then serum starved for 2 h and incubated a further 15 min in absence or presence of insulin. Plasma membrane lawns were prepared as described and stained for GLUT-4. Indirect immunofluorescence images of GLUT-4 on plasma membrane lawns from noninjected and injected cells were collected. Intensity of GLUT-4 staining in plasma membranes was digitally quantitated by measuring average intensities for individual lawns. Values from individual experiments were averaged and normalized to basal values. Values are means ± SE of effect of insulin relative to its corresponding basal control from 4 independent experiments. * Significantly different from either control (P < 0.05, ANOVA).

We then examined the effect of insulin on GLUT-4 levels on membrane lawns. The GLUT-4 signal in the basal state was assigned a value of 1.00 in each experiment, and the effect of insulin was calculated in relative units. In four independent experiments with noninjected cells, insulin caused a 2.53 ± 0.07-fold (mean ± SE) increase in GLUT-4 over basal values. In lawns from cells microinjected with GST-SNAP23, the insulin response rose to 3.20 ± 0.30-fold (mean ± SE) relative to unstimulated, microinjected cells (Fig. 2). This gain of 26% in insulin response caused by GST-SNAP23 was statistically significant vis-à-vis both noninjected and GST-injected cells (P < 0.05, ANOVA; Fig. 2).

The above results are based on the averaged GLUT-4 signal from multiple lawns per field. To assess whether GST-SNAP23 could have changed the number of insulin-responding cells, the signal from each lawn was counted as positive or negative using the cutoff value described under Single-cell microinjection and plasma membrane lawns. By this analysis, 95.6 ± 3.1% (mean ± SE) of cells microinjected with GST-SNAP23 were found to respond to insulin, compared with 84.6 ± 2.3% of noninjected cells. The difference between the values was statistically significant at the P < 0.05 level (Student's t-test). However, the number of responding cells increased by only 10%, whereas the average gain in GLUT-4 per lawn was 26%. Taken together, these results suggest that microinjection of GST-SNAP23 not only increased the number of responding cells but also stimulated the magnitude of the response within each cell.

One limitation of the quantitation of GLUT-4 levels in membrane lawns is that it may not distinguish between GLUT-4 that is functionally incorporated into the plasma membrane and GLUT-4 that is merely closely associated with it (or incompletely incorporated into the membrane). To determine whether the changes in insulin-stimulated GLUT-4 translocation observed using GST-SNAP23 actually resulted in increased GLUT-4 exposure at the cell surface, we measured glucose uptake in permeabilized cells exposed to GST or GST-SNAP23. As shown previously by us and others, limited chemical permeabilization of 3T3-L1 adipocytes with bacterial toxins still allows for measurement of 2-deoxyglucose uptake (9, 25, 34). Under these conditions, a reduced but reproducible insulin-dependent stimulation of glucose uptake is observed, allowing for the introduction of macromolecules to study their effect on insulin action. GST-SNAP23 was introduced into SLO-permeabilized 3T3-L1 adipocytes for 15 min before addition of insulin for another 15 min, and then glucose uptake was measured as described in Glucose transport in SLO-permeabilized cells. Table 1 shows that introduction of the fusion protein caused a significant enhancement of insulin-stimulated glucose uptake relative to cells exposed to GST or buffer alone. This supports the results in Fig. 2 showing that microinjection of GST-SNAP23 increases the level of GLUT-4 on membrane lawns. In addition, it demonstrates that this GLUT-4 is functionally incorporated into the plasma membrane lawns.

                              
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Table 1.   Full-length SNAP23 protein improves insulin-dependent glucose uptake in SLO-permeabilized 3T3-L1 adipocytes

For microinjected GST-SNAP23 to potentiate insulin-dependent incorporation of GLUT-4 into the membrane, it must be able to reach its target sites. Recently, it was reported that the endogenous SNAP23 of mast cells is able to relocate intracellularly, possibly due to association with cytoskeletal elements (7). Triton X-100 insolubility is frequently used to predict interactions with cytoskeletal elements (3, 7). To examine whether SNAP23 partitions with cytoskeletal elements in 3T3-L1 adipocytes, Triton X-100-soluble and -insoluble fractions were purified from isolated crude membrane fractions, and the presence of SNAP23 was determined by immunoblotting. The results indicate that endogenous SNAP23 partitions into both Triton X-100-soluble and -insoluble components (Fig. 3). Analysis of the total protein yields in each fraction revealed that ~5% of the membrane-bound SNAP23 originally isolated was recovered in the Triton-insoluble fraction. As expected, this fraction was shown to contain actin (Fig. 3) and is therefore likely to contain the insoluble cytoskeleton. Actin present in the soluble fraction is likely to represent the nonfilamentous form of this protein. In contrast to SNAP23, the SNAREs syntaxin-4 and VAMP-2 were found exclusively in the Triton-soluble fractions (Fig. 3), highlighting that the SNAP23 association with the cytoskeleton is a particular property of this SNARE.


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Fig. 3.   SNAP23, but not vesicle-associated membrane protein (VAMP)-2 or syntaxin-4, associates with cytoskeleton. Crude membranes were prepared from serum-starved 3T3-L1 adipocytes as described in MATERIALS AND METHODS. Membranes were then solubilized in Triton X-100-containing buffer and centrifuged to pellet insoluble material (I), leaving soluble proteins (S) in supernatant. Proteins in soluble and insoluble fractions were then separated by 13% SDS-PAGE and analyzed by immunoblotting. Shown are representative blots from 4 independent experiments. Entire pellet of insoluble material was run next to one-twentieth of total protein from soluble fraction.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The SNARE hypothesis of synaptic vesicle exocytosis initially suggested that SNAP25 is a target (t)-SNARE required for docking and/or fusion of incoming vesicles. Along with VAMP-2 and syntaxin-1, they constitute the minimum number of proteins that can lead to membrane fusion (37). In yeast, homologues of VAMP and syntaxin are required for vacuolar fusion (15), and at least two proteins with domains resembling SNAP25 have been described as participating in this function (1, 29). Subsets of mammalian and yeast SNAREs form very stable ternary complexes via coiled-coil interactions, and the energy released from this tight binding is thought to drive membrane fusion (5, 8, 11, 37). In contrast to VAMP-2 and syntaxin-1, which are transmembrane proteins, SNAP25 does not penetrate the membrane but associates with it through palmitoyl moieties attached to a set of four cysteine residues in the middle of the molecule (30) and by a secondary mechanism involving coiled-coil interactions with syntaxin-1.

SNAP25 is expressed only in neuronal and neuroendocrine cells. However, regulated membrane fusion events occur at the surface of many cells, notably exocrine secretion from vesicles and granules and regulated exocytosis of membrane proteins such as glucose transporters in muscle and fat cells, water channels in kidney cells, and proton pumps in gastric parietal cells. SNAP23 is the only homologue of SNAP25 so far identified in mammals. The rodent SNAP23 has 57.8% identity with rodent SNAP25 (35). Both the human and rodent SNAP23 lack the sites required for proteolysis by botulinum toxins E or A (18, 20). SNAP23 is expressed in a variety of secretory cells and is also present in muscle and fat cells (38). In the latter cells, its distribution is largely restricted to the plasma membrane.

Recently, the function of SNAP23 was tested in two studies. In one, transient expression of this protein in pancreatic islet beta -cells rescued the loss of insulin-secretion caused by proteolysis of the endogenous SNAP25 with botulinum toxin E (20). This indicates that exogenous SNAP23 has the ability to participate in membrane traffic events. In the second study, proteolysis of the canine SNAP23, a species isoform that is sensitive to botulinum toxin E, prevented transferrin recycling in Madin-Darby canine kidney cells (10). However, differences in the behavior of SNAP25 and SNAP23 have been noted in their interaction with other SNAREs in vitro. Thus, whereas SNAP25 potentiated binding of VAMP-2 to syntaxin-1 (17), SNAP23 appeared to decrease the association of VAMP-2 to syntaxin-4 (6). Moreover, the SDS resistance displayed by in vitro complexes of recombinant proteins encoding the soluble segments of syntaxin-1, VAMP-2, and SNAP25 was not reproduced by the equivalent segments of syntaxin-4, VAMP-2, and SNAP23 (6). This indicates that the stability of the two tripartite complexes differs. On the other hand, a possible role of SNAP23 as a partner of syntaxin-4 and VAMP-2 in nonneuronal cells is supported by recent findings that all three coprecipitate from 3T3-L1 adipocyte cell lysates (6) and from alpha SNAP- and NSF-enriched lysates of rat fat cells (22).

In both 3T3-L1 adipocytes and rat fat cells, GLUT-4-containing organelles incorporate into the plasma membrane in response to insulin. This phenomenon has been shown to be sensitive to hydrolysis of VAMP-2 and/or VAMP-3/cellubrevin with botulinum toxins (2, 25) and to neutralization of syntaxin-4 with specific antibodies (26, 34). Similar to observations made with antibodies directed to other SNARE proteins, microinjection of antibodies directed to the carboxy-terminal domain of SNAP23 reduced the insulin-dependent arrival of GLUT-4 at the cell surface (Fig. 1). A similar observation was recently made by Rea et al. (19), using antibodies directed to the amino-terminal domain of SNAP23. The fact that both the carboxy terminus and the amino terminus of SNAP23 appear to be involved in the incorporation of GLUT-4 into the membrane is, by analogy, consistent with recent reports that both the amino terminus and the carboxy terminus of SNAP25 are part of the interacting core of the SNARE complex (23).

As further evidence of a functional role of SNAP23 in insulin-stimulated GLUT-4 translocation, microinjection of full-length SNAP23 into 3T3-L1 adipocytes enhanced the amount of GLUT-4 present at the cell surface upon an insulin challenge (Fig. 2). Microinjected recombinant SNAP23, in the presence of insulin, also led to an increase in the number of cells responding to this challenge. A change in the number of insulin-responding cells has been shown to occur in response to activated phosphatidylinositol 3'-kinase (14). Similarly, introduction of SNAP23 by chemical permeabilization of the cells allowed for a higher insulin-dependent stimulation of glucose uptake (Table 1). This strongly suggests that the exogenous SNAP23 promotes the functional incorporation of GLUT-4 proteins into the membrane, presumably by facilitating fusion of the GLUT-4-containing organelle with the cell surface lipid bilayer.

Whereas the amount of endogenous SNAP23 is not known, it is likely to be in lower molar yield than the amount of its partner t-SNARE syntaxin-4. This is concluded from the observation that immunoprecipitation of SNAP23 from 3T3-L1 adipocyte cell lysates brings along syntaxin-4 but immunoprecipitation of syntaxin-4 does not show detectable SNAP23 (6). Presumably, a large fraction of the endogenous syntaxin-4 is available to interact with the exogenous SNAP23. Our results are consistent with the interpretation that SNAP23 is in limiting amounts for the fusion of GLUT-4 vesicles, so that increasing the cellular content of SNAP23 facilitates this process.

The ability of exogenous SNAP23 to enhance the effect of insulin is in contrast to the inhibitory effects of the cytoplasmic domains of syntaxin-4 or VAMP-2 (2, 13, 16), which presumably act as competitive inhibitors of the endogenous, membrane-bound syntaxin-4 and VAMP-2. In those studies, both molecules were missing their transmembrane domains, suggesting that this link with the appropriate membranes is critical for their function in incorporating GLUT-4 compartments into the plasma membrane. In contrast, full-length SNAP23 is thought to be membrane associated via cysteine palmitoylation and protein-protein interactions, by analogy to SNAP25 (31). We hypothesize that the microinjected SNAP23 protein assists in incorporating the GLUT-4 organelle into the plasma membrane by binding to its natural partners syntaxin-4 and VAMP-2. The exogenous SNAP23 would act additively to the (limiting) amount of endogenous SNAP23.

The observation that SNAP23 can associate with the cytoskeleton (Fig. 3) raises the possibility that this interaction could facilitate the positioning of the incoming vesicles, improving their access to either insulin-signaling molecules and/or to the fusion machinery. Indeed, there is emerging evidence that an intact actin cytoskeleton is required for the correct targeting of insulin-stimulated phosphatidylinositol 3-kinase to intracellular compartments containing GLUT-4 in 3T3-L1 adipocytes (36) and that activated phosphatidylinositol 3-kinase associates with the cytoskeleton (3).

Collectively, the results presented here suggest that SNAP23 may be a target of insulin-dependent regulation of vesicle docking/fusion in 3T3-L1 adipocytes. The molecular mechanisms of the regulation of SNAP23 by the cytoskeleton require further elucidation. Our results, combined with those of Rea et al. (19), suggest that SNAP23 binds other proteins via regions analogous to those in SNAP25, leading to GLUT-4 vesicle docking/fusion in response to insulin. Furthermore, our results show that SNAP23 is important for the functional incorporation of GLUT-4 leading to an increase in glucose transport activity.


    ACKNOWLEDGEMENTS

We thank Dr. Zhi Lui for assistance with preparation of fusion proteins, Peggy Wong and Nick Daneman for help with antibody preparation, Dr. Allen Volchuk for continued advice, and Dr. Philip Bilan for comments on the manuscript.


    FOOTNOTES

This work was supported by a Juvenile Diabetes Foundation grant (to A. Klip and W. S. Trimble). L. J. Foster was supported by a Medical Research Council of Canada studentship. K. Yaworsky was supported by a Restracomp studentship from the Research Institute of the Hospital for Sick Children.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: A. Klip, Cell Biology Programme, Hospital for Sick Children, Toronto, ON, Canada M5G 1X8 (E-mail: amira{at}sickkids.on.ca).

Received 12 November 1998; accepted in final form 3 February 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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