©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Expression and Functional Role of Syntaxin 1/HPC-1 in Pancreatic Cells
SYNTAXIN 1A, BUT NOT 1B, PLAYS A NEGATIVE ROLE IN REGULATORY INSULIN RELEASE PATHWAY (*)

(Received for publication, August 10, 1995; and in revised form, October 18, 1995)

Shinya Nagamatsu (1)(§) Tomonori Fujiwara (2) Yoko Nakamichi (1) Takashi Watanabe (3) Hiroshi Katahira (4) Hiroki Sawa (5) Kimio Akagawa (2)

From the  (1)Departments of Biochemistry and (2)Physiology and (3)Clinical Pathology and (4)Internal Medicine (III) and (5)Neurosurgery, Kyorin University School of Medicine, Mitaka, Tokyo 181, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Syntaxin 1/HPC-1 is an integral membrane protein, which is thought to be implicated in the regulation of synaptic neurotransmitter release. We investigated syntaxin 1 expression in pancreatic beta cells and the functional role of syntaxin 1 in the insulin release mechanism. Expression of syntaxin 1A, but not 1B, was detected in mouse isolated islets by the reverse transcriptase-polymerase chain reaction procedure. An immunoprecipitation study of metabolically labeled islets with an anti-rat syntaxin 1/HPC-1 antibody demonstrated syntaxin 1A protein with an apparent molecular mass of 35 kDa. Immunohistochemistry of the mouse pancreas demonstrated that syntaxin 1/HPC-1 was present in the plasma membranes of the islets of Langerhans. In order to determine the functional role of syntaxin 1 in pancreatic beta-cells, rat syntaxin 1A or 1B was overexpressed in mouse betaTC3 cells using the transient transfection procedure. Transfection of betaTC3 cells with either syntaxin 1 resulted in approximately 7-fold increases in their immunodetectable protein levels. Glucose-stimulated insulin release by syntaxin 1A-overexpressing cells was suppressed to about 50% of the level in control cells, whereas insulin release by syntaxin 1B-overexpressing and control cells did not differ. Next, we established stable betaTC3 cell lines that overexpressed syntaxin 1A and used them to evaluate the effect of syntaxin 1A on the regulatory insulin release pathway. Two insulin secretogogues, 4-beta-phorbol 12-myristate 13-acetate or forskolin, increased insulin release by untransfected betaTC3 cells markedly, but their effects were diminished in syntaxin 1A-overexpressing betaTC3 cells. Glucose-unstimulated insulin release and the proinsulin biosynthetic rate were not affected by syntaxin 1A overexpression, indicating a specific role of syntaxin 1A in the regulatory insulin release pathway. Finally, in vitro binding assays showed that syntaxin 1A binds to insulin secretory granules, indicating an inhibitory role of syntaxin 1A in insulin exocytosis via its interaction with vesicular proteins. These results demonstrate that syntaxin 1A is expressed in the islets of Langerhans and functions as a negative regulator in the regulatory insulin release pathway.


INTRODUCTION

There are two types of secretory pathway in eukaryotic cells. One is the constitutive secretory pathway which is involved in continuous exocytosis and the other is the regulated secretory pathway, in which soluble proteins and other substances are stored in secretory vesicles for later release. Neurons and endocrine cells exhibit regulated release, which mediates chemical signaling in these systems(1) . Regulated secretion occurs by docking and/or fusion of the secretory vesicles with the plasma membrane when chemical signals reach the targeted cells, which arises when membrane depolarization or a secretagogue stimulates a second messenger system. In the case of pancreatic beta cells, the membrane depolarization caused by high glucose stimulant activates voltage-dependent Ca channels (2, 3, 4) and triggers intracellular Ca release(5, 6) , initiating insulin secretion as a result of docking and/or fusion of insulin secretory granules with the plasma membrane. Although the molecular basis of vesicle fusion is poorly understood, a number of proteins involved in the targeted movement and fusion reactions of the vesicle membrane have recently been identified in neurons(7, 8) . The molecular components of secretion by pancreatic endocrine cells may share many attributes with those identified in neurons. In this study, we focused on the role of syntaxin 1/HPC-1 in the mechanism of insulin secretion by pancreatic beta cells. Syntaxin 1/HPC-1, originally isolated from rat hippocampus, is an integral membrane protein (9, 10) that has been postulated to act upon a family of vesicular transport receptors(11) , which suggests it plays a general role in protein traffic to the plasma membrane. Here, we demonstrated the syntaxin 1A expression in mouse pancreatic islets and determined its functional role in the insulin secretory pathway.


EXPERIMENTAL PROCEDURES

Materials

[alpha-P]dCTP, I rat insulin assay kit, and Amplify(TM) were purchased from Amersham Corp. [S]Methionine-cysteine (Met-Cys) was from DuPont NEN. Protein A-Sepharose was purchased from Pierce, and the kits for DNA labeling and RNA PCR (^1)and Taq DNA polymerase were purchased from TakaRa Shuzo Ltd. Dulbecco's minimal essential medium (DMEM), RPMI 1640 medium, and fetal bovine serum (FBS) were obtained from Life Technologies, Inc., the nitrocellulose membranes were from Schleicher & Schuell, and the oligonucleotide primers were synthesized on an Applied Biosystem model 391 DNA synthesizer. All other reagents used were of the highest quality commercially available.

Islet Isolation and Cell Culture

Islets were isolated by collagenase digestion from the pancreases of mice fed a standard diet, as described previously(12) . The islets were collected by hand-picking under a dissection microscope, then were preincubated in RPMI 1640 containing 11 mM glucose and 10% (v/v) FBS for 1 h prior to the labeling experiment. The betaTC3 cell line was kindly provided by Dr. D. Hanahan (University of California, San Francisco) and grown at 37°C in DMEM containing 400 mg/dl glucose, 10% (v/v) FBS, 25 mM Hepes, and 1 mML-glutamine.

Reverse Transcriptase-PCR-Southern Blot Analysis

Total cellular RNA was isolated from mouse isolated islets or betaTC3 cells by the acid guanidinium thiocyanate-phenol-chloroform method(13) . Single-stranded cDNA was prepared using the total RNA (100 ng), reverse transcriptase, and random hexamers. A 340-bp DNA fragment coding for the syntaxin sequences was amplified using the PCR, as described previously(14) . The antisense and sense degenerate oligonucleotide primers used were EM-1 [5`-TCCAG(A/C)G(G/C)CAGCTGGAGATC-3`] and EM-2 [5`-C(T/G)IGCCTTGCTCTGGTA(T/C)TT-3`], which correspond to amino acid sequences IQRQLEI of Ile-Ile and KYQSKAR of Lys-Arg of the syntaxin 1 and 2 proteins, respectively, from several species(9, 15, 16) . The PCR was carried out for three initial cycles of 94 for 30 s, 42 for 1 min, and 65 for 1 min, followed by 30 cycles of 94 for 30 s, 55 for 1 min, and 72 for 90 s. The PCR products were run on a 1.2% agarose gel, transferred to a nitrocellulose filter, and hybridized with P-labeled rat syntaxin 1A(9) , or rat syntaxin 1B, which was prepared by the PCR using specific oligonucleotide primers based on GeneBank(TM)-entered nucleotide sequences (11, 16) as probes. Filters were washed under high stringency conditions (0.1 times SSC (1 times SSC = 0.15 M NaCl, 0.015 M sodium citrate), 0.1% SDS, 60 °C) and autoradiographed with intensifying screens.

Preparation of Antiserum to Syntaxin 1/HPC-1

A recombinant transmembrane deletion mutant of syntaxin 1A was produced in Escherichia coli using the pMAL-c vector/MBP (maltose-binding protein) fusion protein expression system (New England BioLab). Briefly, the bacterial host cells were cultured and then induced with 1 mM isopropyl-beta-D-thiogalactopyranoside, harvested by centrifugation, and the pellet was sonicated. The resulting cell lysate was applied to amirose resin, and the fusion protein was eluted with maltose. The resulting purified fusion protein was cleaved with restriction protease factor Xa, the recombinant syntaxin 1A was separated by 12% polyacrylamide gel electrophoresis (PAGE) and purified, as described previously(9) . The recombinant protein fraction was suspended in complete Freund's adjuvant and injected subcutaneously into rabbits to obtain syntaxin 1A antiserum. The specificity for polyclonal antibodies was tested by immunoblot analysis using the fusion protein. The antiserum against syntaxin 1A reacted with both syntaxin 1A and 1B proteins, due to their high homology (data not shown).

Immunofluorescence Staining

Mouse pancreases were dissected in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), cryoprotected with graded concentrations of sucrose in phosphate-buffered saline (PBS), embedded in OCT compound (Miles), and then frozen by immersion in liquid nitrogen. Frozen sections were cut with a Miles cryostat, transferred to poly-L-lysine-coated slides, and immunostained, as described previously(17) . Briefly, sections were incubated with anti-syntaxin 1/HPC-1 antibody (diluted 1: 500) in PBS containing 4% fetal calf serum, 0.1% sodium azide, and 0.1% Triton X-100, followed by swine anti-rabbit immunoglobulin coupled to fluorescein. The sections were examined using a Zeiss Axioplan fluorescence microscope and photographed with Tri-X film (Kodak) rated at 400 ASA.

Labeling of Cells

betaTC3 cells and islets were incubated in RPMI 1640 medium (10% (v/v) FBS) containing 11 mM glucose under the conditions described in the relevant figure legends. Then, 10^6 betaTC3 cells or 300400 islets were labeled with [S]Met-Cys (23.7 µCi/ml) in methionine- and cysteine-deficient RPMI 1640 medium (10% (v/v) dialyzed FBS), as described in the relevant figure legends, disrupted by sonication and dissolved in 0.1 ml of TAS buffer (0.1 M Tris-HCl, 0.05 M NaCl, 0.25% bovine serum albumin (pH 7.6)) containing proteinase inhibitors (1 mM phenylmethylsulfonyl fluoride and 50 µg/ml Trasylol).

Immunoprecipitations

Immunoprecipitation analysis was performed as described previously(12) . After the cell lysates had been absorbed with normal rabbit serum, they were incubated with anti-rat syntaxin 1/HPC-1 antibody and then pelleted with protein A-Sepharose beads. The immunoprecipitates were resolved on a 10% SDS-PAGE, treated with Amplify(TM), and dried. The radioactivity on the gel was visualized by autoradiography for 5 days -70 °C. Intensities of the syntaxin 1 bands were quantified by a laser scanning densitometric analyzer (model TIAS-2000, ACI Japan Co.)

Generation of Expression Construct and Transfection of Cells

The corresponding full-length rat HPC-1 (syntaxin 1A) cDNA was originally isolated as described previously(9) . A 2144-bp HindIII/BamHI fragment containing the entire coding sequence of rat HPC-1 (syntaxin 1A) plus 31 and 1330 bp of the 5`- and 3`- untranslated regions, respectively, was ligated into a pRC/RSV expression vector (Invitrogen). The full-length cDNA of rat syntaxin 1B was isolated from a rat brain cDNA library (Clonetech), and a pRC/RSV construct was produced as described above. betaTC3 cells that overexpressed either syntaxin 1 were produced by growing betaTC3 cells to subconfluence (5 times 10^5 cells/60-mm plate) in DMEM containing 10% FCS and were transfected transiently with the constructed vectors using a lipofectAMINE(TM) (GIBCO BRL), under the control of the Rous sarcoma virus long terminal repeat promoter. After 12 h, the medium was discarded, and the cells were incubated for a further 2 days in RPMI 1640 medium containing 11 mM glucose and then examined for insulin secretion and biosynthesis and syntaxin 1A and 1B expression.

In order to produce stable betaTC3 cell lines that overexpressed syntaxin 1A protein, the construct described above was introduced into betaTC3 cells by lipofectAMINE(TM), cultured in DMEM for 48 h, after which they were plated (10^5 cells/plate) on a 10-cm dishes and grown in the presence of 800 mg/ml G418 (Life Technologies, Inc.). Multiple, single stable clones of each of the constructs were isolated and expanded. In order to determine their syntaxin 1A contents, the cells were labeled with [S]Met-Cys, lysed, and immunoprecipitated with the anti-syntaxin 1/HPC-1 antibody, as described above. The amount of syntaxin 1A protein immunoprecipitated was estimated by densitometric analysis of the autoradiographic band. The stable transformants were selected and maintained in DMEM containing 10% FBS without G418.

Insulin Biosynthesis and Secretion

Immunoprecipitation of proinsulin was performed using guinea pig anti-insulin antiserum, as described elsewhere(12) , and the amount of insulin in the medium was assayed using rat insulin standards by radioimmunoassay (IRI kit, Amersham Corp.)

Subcellular Fractionation and Immunoblotting

Cells were grown to confluence on 10-cm dishes and fractionated using the method of Klip et al.(18) with slight modifications to obtain plasma membranes, as follows. The cells were rinsed twice with ice-cold PBS, scraped vigorously with a rubber policeman into 2 ml of homogenization buffer (225 mM sucrose, 20 mM Tris-HCl (pH 7.4), 1 mM EDTA) 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 resulting supernatant was centrifuged at 12,000 times g for 15 min to sediment the total membranes, and the pellet was resuspended in homogenization buffer and processed for sucrose density gradient centrifugution at 25,000 times g for 1 h. The band that appeared between 45 and 40% sucrose was collected and pelleted by centrifugation at 100,000 times g for 30 min to obtain the plasma membranes, which were assayed to determine their protein content using the Bio-Rad assay (Bio-Rad), subjected to SDS-PAGE in 10% polyacrylamide gel, and transferred to nitrocellulose membranes. Immunoblotting was carried out as described previously(19) . The bound anti-syntaxin 1/HPC-1 antibodies were visualized by alkaline phosphatase-coupled goat anti-rabbit IgG secondary antibody (DAKO) using nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate (Wako Ltd.) as substrates.

In Vitro Protein Binding Studies

Twenty nmol of MBP-syntaxin 1A was incubated for 2 h at 4 (head-over-head rotation) with 50 µl of amino resin-agarose slurry in binding buffer (10 mM Tris (pH 7.4), 250 mM NaCl, 1 mM EDTA), washed six times with binding buffer, and the MBP fusion syntaxin 1A bound to amirose resin-agarose beads was resuspended in 1 ml of binding buffer. In order to analyze syntaxin 1A binding, betaTC3 cells were solubilized in buffer A (10 mM Hepes-NaOH (pH 7.4), 0.15 M NaCl, 2 mM MgCl(2), 0.5% Triton X-100) containing protease inhibitors (0.5 mgl pepstatin and 1 mM phenylmethylsulfonyl fluoride), and the insoluble proteins were removed by centrifugation. The solubilized betaTC3 cell extract was incubated with the MBP fusion syntaxin 1A attached to amirose resin-agarose beads at 4°C with 1 mM Ca under gentle agitation. The beads were washed three times with PBS, and the bound proteins were analyzed by SDS-PAGE followed by immunoblotting using polyclonal antibodies against insulin and PC2. The anti-PC2 antibody was a gift from Dr. D. F. Steiner (University of Chicago, IL).

Statistical Analysis

The data are expressed as means ± S.E. and were analyzed using analysis of variance analysis (one-way analysis). Differences at p values of less than 0.05 were considered to be significant.


RESULTS AND DISCUSSION

As the regulated secretory pathways of neurons and endocrine cells show similarities(20) , and there are several similarities between secretory mechanisms in yeast and mammalian cells(21, 22) , the molecular components involved in secretion from pancreatic beta cells may share common features with those identified in neurons. In neuronal cells, syntaxin 1/HPC-1 binds to many exocytosis-related proteins, such as VAMP, NSF, alpha-SNAP, SNAP-25(7) , synaptotagmins, and N-type-Ca channels(23, 24) , suggesting that it is an essential part of the fusion apparatus involved in forming a molecular machinery complex(25) . The results of our immunohistochemical study showed clearly that syntaxin 1/HPC-1 is localized in the plasma membranes of the islets of Langerhans (Fig. 1), indicating that it may function in insulin exocytosis as a part of the molecular machinery complex for docking and/or fusion apparatus. This led us to investigate the expression and functional role of syntaxin 1/HPC-1 in pancreatic beta cells.


Figure 1: Immunofluorescence photomicrograph of the mouse pancreas stained for syntaxin 1/HPC-1. Sections of the mouse pancreas were incubated with anti-syntaxin 1 antiserum followed by swine anti-rabbit immunoglobulin coupled to fluorescein. The islets of Langerhans and exocrine tissues were immunostained. Immunoreactivity of syntaxin 1 can be seen in the plasma membranes of many cells in the islets of Langerhans, but little is evident in the exocrine tissues.



In order to determine whether mouse pancreatic islets express syntaxin 1A and/or 1B, we utilized the reverse transcriptase-PCR technique. Total islet RNA was subjected to the reverse transcriptase-PCR using degenerate oligonucleotide primers and syntaxin 1A, but no 1B mRNA, was detected in mouse isolated islets (Fig. 2). Northern blot analysis of the total RNA (15 µg) from mouse isolated islets detected no syntaxin 1A transcripts (data not shown), indicating that the expression level of syntaxin 1A mRNA in beta cells was quite low. Therefore, we attempted to detect syntaxin 1A protein in mouse isolated islets. For this purpose, about 400 islets were labeled metabolically with [S]Met-Cys for 3 h in RPMI 1640 medium containing 11 mM glucose. Autoradiography of the protein immunoprecipitated from the labeled cell extract with rabbit anti-rat syntaxin 1/HPC-1 antiserum after SDS-PAGE revealed a protein band with a molecular mass of approximately 35 kDa (Fig. 2). This protein was identified as syntaxin 1A on the basis of the following criteria: 1) it migrated as a 35-kDa protein, as expected on the basis of the predicted molecular mass; 2) it was not immunoprecipitated from a nonimmune control serum; 3) the addition of 50 µg of unlabeled rat syntaxin 1A displaced this band (data not shown); and 4) reverse transcriptase-PCR analysis of mouse isolated islets detected only syntaxin 1A, not 1B mRNA. It is of interest that only syntaxin 1A was expressed in mouse isolated islets. As immunohistochemical and in situ hybridization studies of the rat brain have shown that the localization of their proteins is not identical, (^2)the biological role of syntaxin 1A may be different from that of syntaxin 1B. Our findings suggest that syntaxin 1A expressed in pancreatic beta cells plays an important role in the docking and/or fusion of insulin secretory granules with the plasma membrane.


Figure 2: Expression of syntaxin 1/HPC-1 mRNA and protein in mouse isolated islets. Upper panel, autoradiogram of PCR-amplified syntaxin cDNA fragments from mouse isolated islets. cDNAs, reverse-transcribed from total RNAs (100 ng) of islets, were subjected to PCR, as described under ``Experimental Procedures.'' The PCR products were separated on a 1.2% agarose gel, blotted onto a nitrocellulose filter, and hybridized with syntaxin 1A or 1B cDNA probe. The predicted size of the amplified fragment is 340 bp. Lower panel, identification of the syntaxin 1/HPC-1 protein band in mouse isolated islets. Mouse islets (300400) isolated by collagenase digestion were labeled for 3 h with [S]Met-Cys in RPMI 1640 (10% (v/v) dialyzed FBS) containing 11 mM glucose. The cells were disrupted and immunoprecipitated with rabbit anti-rat syntaxin 1/HPC-1 antiserum (lane 1), or rabbit normal serum (lane 2), as described under ``Experimental Procedures.'' The immunoprecipitates were analyzed using 10% SDS-PAGE, followed by fluorography with Amplify(TM) and autoradiography.



In order to investigate the functional role of syntaxin 1A in pancreatic beta cells, we induced overexpression of rat syntaxin 1A or 1B in mouse betaTC3 cells, which possess both regulated and constitutive insulin secretion pathways(26, 27) , because manipulation of protein expression levels has proved to be a powerful tool for studying biological activities of proteins(28, 29) . Mouse betaTC3 cells were transfected transiently with rat syntaxin 1A or 1B using lipofectAMINE(TM) under the control of the Rous sarcoma virus long terminal repeat promoter. As shown in Fig. 3, the transfection of betaTC3 cells with either syntaxin 1 resulted in approximately 7-fold increases in their respective immunoprecipitable proteins, which were quantified by densitometric scanning of the blots, relative to untreated betaTC3 cell levels. The impact of syntaxin 1 overexpression was evaluated by incubating the cells under glucose-stimulated conditions (11 mM glucose) for 2 days posttransfection and measuring insulin secretion into the medium. As shown in Fig. 4A, glucose-stimulated insulin release by syntaxin 1A-overexpressing betaTC3 cells was reduced to about half the level in control cells. Although the data from the independent transfection experiments are not shown, the inhibitory effect of syntaxin 1A overexpression on insulin secretion varied among the independent transfection experiments. This is probably due to the different transfection efficiency in each experiment (see Fig. 3), because the levels of syntaxin 1A overexpression were compatible with the degrees of insulin release inhibition (data not shown). In contrast, insulin release by syntaxin 1B-overexpressing cells did not differ from that by control cells. The biosynthetic rate of immunopurified proinsulin was also measured by labeling the cells with [S]Met-Cys for 30 min. There were no difference in the glucose-stimulated proinsulin biosynthetic rates between the syntaxin 1-overexpressing and control cells (Fig. 4B). In agreement with the above results, insulin content in syntaxin 1A-overexpressing cells was elevated about 2-fold relative to control cells (Table 1). Thus, syntaxin 1A overexpression in betaTC3 cells inhibited glucose-stimulated insulin release, and this inhibitory effect was specific for the insulin release pathway.


Figure 3: Immunoprecipitation analysis of syntaxin 1 expression in transiently transfected mouse betaTC3 cells. betaTC3 cells were transfected transiently with rat syntaxin 1A or 1B constructed expression vectors using lipofectAMINE(TM) under the control of the Rous sarcoma virus long terminal repeat promoter. Two days later, the cells were labeled with [S]Met-Cys and processed for immunoprecipitation analysis, as described in the legend to Fig. 2. The figure represents four independent transfection experiments for each syntaxin 1 isoform. The syntaxin 1A and 1B expression levels were determined by densitometric scanning of the blots.




Figure 4: A, inhibitory effect of syntaxin 1A overexpression in mouse betaTC3 cells on glucose-stimulated insulin release. betaTC3 cells transfected with the indicated vector, or left untreated, were incubated in RPMI 1640 (10% (v/v) FBS) containing 11 mM glucose for 2 days, after which the media were collected and insulin secretion was analyzed by IRI. The insulin secretion data were calculated after pooling the data obtained from all of transfection experiments, as described in the legend to Fig. 3. B, proinsulin biosynthesis stimulated by 11 mM glucose in mouse betaTC3 cells. Transfected and untransfected betaTC3 cells were labeled with [S]Met-Cys for 30 min and then processed for measurement of the proinsulin biosynthetic rate as described under ``Experimental Procedures.''





To address the issue of the effect of syntaxin 1A on the regulatory insulin release pathway in detail, we established the stable betaTC3 cell lines that overexpressed syntaxin 1A and examined the effect of syntaxin 1A in the regulatory insulin secretion pathway by utilizing two insulin secretogogues, TPA (4-beta-phorbol 12-myristate 13-acetate) and forskolin, which activate protein kinase C and protein kinase A, respectively(30, 31, 32, 33) . We obtained three different clones that overexpressed syntaxin 1A protein, in all of which glucose-stimulated insulin release was inhibited (data not shown). We investigated further by subjecting a stable clone designated betaTC3-hpc1, which expressed approximately 20 times more syntaxin 1A protein than control betaTC3 cells, shown by immunoprecipitation analysis, and immunoblot analysis showed that the protein was present in the plasma membrane fraction (Fig. 5), indicating that the overproduced syntaxin 1A was processed correctly in the cells. In agreement with the result of the transient transfection experiments, glucose-stimulated insulin release by betaTC3-hpc1 cells was lower than that by control cells (Fig. 6). The inhibitory effect of syntaxin 1A on insulin secretion was only observed when betaTC3-hpc1 cells were incubated under glucose-stimulated conditions (11 mM glucose); it was not apparent under glucose-unstimulated conditions (0 mM glucose), indicating that syntaxin 1A affects only the regulatory insulin release pathway. We further examined the effect of syntaxin 1A overproduction on the regulatory insulin release pathway by performing experiments using potent secretogogues. As described previously(27) , the incubation of betaTC3 cells with TPA for 1 h under either the absense or presence of glucose increased insulin release markedly (Fig. 7). Forskolin increased insulin release only in the presence of glucose, in agreement with the previous report(34, 35) . On the other hand, the insulin release by betaTC3-hpc1 cells was completely abolished when cells were incubated with TPA in the absence of glucose or in the presence of glucose with forskolin. However, when betaTC3-hpc1 cells were incubated with TPA in the presence of glucose, insulin release was stimulated even by syntaxin 1A-overproducing betaTC3-hpc1 cells, although the levels of insulin release by betaTC3-hpc1 were only half of the levels of control betaTC3 cells. The reason why only the combination of both glucose and TPA stimulated the insulin release by betaTC3-hpc1 cells is presently unknown. A possible explanation is that this combination increases the efficiency of alpha-SNAP biosynthesis or its stabilization in the cells, which is known to stimulate the Ca-dependent exocytosis(36) , thereby overcoming the inhibitory effect of overproducing syntaxin 1A protein. Taken together with these results, the insulin release stimulated by three different insulin secretogogues, such as glucose, TPA, and forskolin, was inhibited by syntaxin 1A protein overproduction in betaTC3 cells, suggesting that syntaxin 1A plays an essential role as a negative regulator in the regulatory insulin release pathway, which is probably associated with the docking and/or fusion of insulin secretory granules, as suggested for neurotransmitter release(8, 37) .


Figure 5: Comparison of the amounts of syntaxin 1A protein in betaTC3-hpc1 and betaTC3 cells and its presence in the plasma membranes. Stable betaTC3 cells that overexpressed syntaxin 1A protein were produced by transfecting betaTC3 cells with a rat syntaxin 1A expression vector, as described under ``Experimental Procedures.'' Stable transformants were cloned by selection in 800 mg/ml G418 and designated betaTC3-hpc. betaTC3-hpc1 and control betaTC3 cells were labeled with [S]Met-Cys for 3 h, and immunoprecipitation analysis with an anti-syntaxin 1/HPC-1 antibody was performed as described. The intensity of the syntaxin 1A protein band was estimated by densitometric scanning. In order to investigate whether syntaxin 1A protein was present in plasma membranes, the plasma membrane fractions were isolated from cells. Samples of protein (10 µg) were analyzed by SDS-PAGE in 10% polyacrylamide gel and electrotransferred to a nitrocellulose filter, immunoblotted with the anti-syntaxin 1/HPC-1 antibody, and detected by alkaline-phosphatase method.




Figure 6: Insulin release from betaTC3-hpc1 cells. betaTC3-hpc1 cells were incubated in RPMI 1640 (10% (v/v) dialyzed FBS) with and without 11 mM glucose for 2 days, after which the media were collected and the amount of insulin secreted into each medium was analyzed by IRI. *, p < 0.0001 (versus betaTC3 cells)




Figure 7: Effect of TPA and forskolin on insulin release by syntaxin 1A-overexpressed cells (betaTC3-hpc1 cells). betaTC3-hpc1 and betaTC3 cells were cultured in DMEM on 60-mm plates to 80% confluence, then incubated with 50 nM TPA or 10 uM forskolin under either the absence or presence of glucose for 1 h at 37 °C, after which the media were collected and insulin release was measured by IRI. *, p < 0.0001 (versus betaTC3 cells)



Although the biochemical mechanism underlying the inhibitory effect of syntaxin 1A on the regulatory insulin release pathway is unknown, we hypothesize that syntaxin 1A acts as a suppressor of exocytosis via interaction with synaptotagmins, which are expressed not only in brain but also in pancreatic beta cells(38) , and have been suggested to inhibit neurotransmitter exocytosis in neurons by unknown mechanism (39, 40) . Indeed, syntaxin 1 is known to bind to synaptotagmins(41) , which are major components of vesicular proteins(37) , probably regulating the Ca-stimulated exocytosis process by undergoing Ca-dependent conformational changes(42) . Therefore, in the present study, we characterized the interaction of syntaxin 1A with insulin secretory granules, which may possess vesicular proteins, such as synaptotagmins, synaptotagmin-like molecules, or VAMP, by in vitro binding assays. After purified syntaxin 1A fused to maltose-binding protein (MBP-syntaxin) were coupled to agarose beads, they were incubated with betaTC3 cell homogenates. The bound proteins were eluted, electrophoresed, and immunoblotted with anti-insulin or anti-PC2 antibodies. As shown in Fig. 8, the eluted proteins were proinsulin, insulin, and PC2 (a converting enzyme present in the insulin secretory granules) (43, 44) , indicating that syntaxin 1A may interact with insulin secretory granules via vesicular proteins. Thus, in pancreatic beta cells as well as in neurons, syntaxin 1A is thought to bind to secretory vesicles, thereby overproduced syntaxin 1A may suppress insulin exocytosis via the interaction with vesicular proteins, probably by mechanisms similar to those observed in neurons(39, 40) .


Figure 8: Binding of syntaxin 1A to insulin secretory granules. The MBP fusion syntaxin 1A bound to amirose resin-agarose beads were incubated with the total betaTC3 cell extract at 4 °C for 24 h. Proteins bound to washed beads were analyzed by SDS-PAGE, following by immunoblotted with antibodies recognizing 9-kDa precursor and 6-kDa mature insulin and 65-kDa PC2.



Binding of syntaxin 1 to munc 18 homologues(45, 46) , which is also expressed in MIN6 insulinoma cell line(46) , was reported to inhibit its interaction with SNAP-25(47) , resulting in inhibition of the formation of the core complex (syntaxin 1-synaptotagmin-SNAP-25), which serves as a receptor for SNAP and NSF (called SNARE, SNAP/NSF receptor) (48) . Since the overproduced syntaxin 1A protein binds tightly not only to vesicular proteins, such as synaptotagmins and VAMP, but also to munc 18 homologues and SNAP-25, the ``core complex'' may not be assembled effectively during the final steps between vesicle docking and fusion, and eventually the fusion reaction may be disturbed. An inhibitory role of syntaxin 1A in the exocytosis process was also observed in our recent studies, which showed that 1) inhibition of the function of syntaxin 1 protein as a result of microinjection of an anti-rat syntaxin 1 antibody into PC12 h cells stimulated norepinephrine release in the regulatory pathway(49) ; 2) syntaxin 1A proteins translated from rat syntaxin 1A capped cRNAs injected into the embryonic cells of Japanese newt inhibited secretion of the extracellular matrix(50) . In contrast, insulin release under glucose-unstimulated conditions was unaffected by syntaxin 1A protein overproduction, suggesting that syntaxin 1A is not obligatory for the constitutive pathway in pancreatic beta cells. Taken together with these results, our data indicate that syntaxin 1A contributes to the pathway involved in the regulation of insulin release, probably as a negative regulator of regulatory exocytosis.

In conclusion, syntaxin 1A, but not 1B, is expressed in pancreatic beta cells and plays an essential role in the regulatory insulin release pathway as a negative regulator. Therefore, any dysfunction in the expression, function, or regulation of syntaxin 1A may result in impaired insulin release and may be involved in the pathogenesis of non-insulin-dependent diabetes mellitus.


FOOTNOTES

*
This study was supported in part by a grant-in-aid for Japan Private School Promotion Foundation (to S. N.) and by the Ciba-Geigy Foundation (Japan) for the Promotion of Science (to K. A.). 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.

§
To whom correspondence should be addressed: Dept. of Biochemistry, Kyorin University School of Medicine, Shinkawa 6-20-2, Mitaka, Tokyo 181, Japan. Fax: 81-422-41-6865.

(^1)
The abbreviations used are: PCR, polymerase chain reaction; DMEM, Dulbecco's minimal essential medium; PAGE, polyacrylamide gel electrophoresis; SSC, standard saline citrate; VAMP, vesicle-associated membrane protein; SNAP-25, synaptosomal-associated protein of 25 kDa; NSF, N-ethylmaleimide-sensitive fusion protein; SNAP, soluble NSF attachment protein; IRI, insulin radioimmunoassay; FBS, fetal bovine serum; bp, base pair(s); PBS, phosphate-buffered saline; MBP, maltose-binding protein.

(^2)
Y. Kushima, T. Fujiwara, and K. Akagawa, unpublished data.

(^3)
S. Nagamatsu, Y. Nakamichi, and H. Sawa, unpublished data.


ACKNOWLEDGEMENTS

We thank S. Matsushima for her assistance with the immunohistochemical work and A. Nishikatsu for her assistance in the preparation of this manuscript.


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