 |
INTRODUCTION |
The ambient glucose level plays a central role in the regulation
of insulin secretion by pancreatic
cells. Although insulin release
is controlled by a complex array of nervous, nutritional, and hormonal
actions, glucose is the most important regulatory signal for insulin
secretion. The process of insulin release by pancreatic
cells
differs from most secretory mechanisms in other cell types in that a
nutrient rather than a hormone acts as the first messenger. A large
amount of evidence supports a hypothesis that glucose metabolism, in
part, inhibits ATP-sensitive K+ (KATP) channels
that are expressed in pancreatic
cells, resulting in membrane
depolarization and activation of voltage-sensitive L-type
Ca2+ channels. Finally, elevated intracellular
Ca2+ concentration
[Ca2+]i1
evokes insulin exocytosis (1-4). Thus, although the sensor system for
insulin release in pancreatic
cells differs from those in other
types of secretory cells (5), insulin exocytosis by pancreatic
cells is strictly controlled by Ca2+ ions (3, 6), as in
other secretory cells, including those of neuron origin (7). Therefore,
the later stages of the process of exocytotic insulin release by
pancreatic
cells after [Ca2+]i elevation have
been assumed to resemble, although not exactly, exocytotic mechanisms
in other secretory cell types, such as neurons.
It is now widely accepted that the fundamental components of the
machinery required for membrane fusion have been conserved evolutionally and that they are used in both constitutive and regulated
membrane trafficking pathways (8, 9). These components include
N-ethylmaleimide-sensitive factor (NSF) (10), the soluble NSF attachment proteins (
-,
-, and
-SNAPs) (11), and
membrane-associated SNAP receptors (SNAREs) (12). SNAREs are further
divided into two classes of proteins: v-SNAREs (vesicular SNAP
receptors) including synaptotagmins, vesicle-associated membrane
protein, and related proteins, and t-SNAREs (target SNAP receptors)
including syntaxin, SNAP-25, and related proteins. Recent experiments
using purified recombinant proteins have led to further
characterization of the protein-protein interactions of the SNAREs with
each other (13-16). The findings suggest that syntaxin is the major
receptor for
-SNAP that is able to stimulate the ATPase activity of
NSF (17). The role of NSF may be to act as a molecular chaperon to
modify the conformation of syntaxin (18). According to the original
SNARE hypothesis (19, 20), v-SNAREs bind t-SNAREs tightly to form a 7 S
complex, which is able to recruit
-SNAP and NSF to form a larger 20 S complex, and finally ATP hydrolysis by NSF then results in SNARE
complex disassembly, a late event in the steps leading to membrane
fusion during exocytosis. However, recent evidence in the yeast vacuole
system had shown that membrane fusion can occur in the presence of v-
and t-SNAREs alone (21, 22); thus, the physiological function of
-SNAP and NSF in exocytosis is now controversial.
Although the molecular machinery that triggers insulin exocytosis by
endocrine pancreatic
cells is not fully characterized, in
situ hybridization, immunoblotting, and Northern blotting studies have revealed that: 1) pancreatic
cells express v-SNAREs, vesicle membrane-associated protein-2, cellubrevin, and synaptotagmins (23-26); 2) t-SNAREs, SNAP-25, and syntaxin are localized in
pancreatic
cells, mainly on plasma membranes (27, 28); and 3)
ATPase NSF and
-SNAP are also expressed in insulinoma cells (29). Therefore, the SNARE hypothesis for neurotransmitter release may be
applicable, at least in part, to insulin exocytosis. However, the two
processes are not identical. Synaptic vesicles are recycled and
neurotransmitter is transported into them after the endocytosis of
fused granules (30), whereas there is no obvious evidence for a similar
recycling of insulin secretory granules. Also, endocrine pancreatic
cells, which are analogous with neuron, contain both large dense core
insulin secretory vesicles (LDCVs) (31) and synaptic-like microvesicles
(SLMVs) (32) which makes them a useful model to study the function of
individual SNARE proteins in possibly different forms of exocytosis. In
this study, we utilized a recombinant adenovirus-mediated gene
transduction system, which enabled expression of high levels of
-SNAP and mutant form in
cells in normal islets or insulinoma
cells, and exploited this methodology to evaluate the physiological
role of
-SNAP in insulin release via LDCVs and GABA release via SLMVs.
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EXPERIMENTAL PROCEDURES |
Antibody--
An affinity-purified anti-
- and
-SNAP
antibody, which was raised against synthetic peptide corresponding to
amino acid residues HYEQSADYYKGEE of rat
- and
-SNAP, was a
generous gift from Dr. M. Takahashi (Mitsubishi Kasei Institute of Life
Sciences, Tokyo, Japan). The characterization of this antibody and its
specificity have been described (33).
Islet Isolation and Cell Culture--
Pancreatic islets were
isolated from male Wistar rats (200-250 g) by collagenase digestion
and Ficoll gradient centrifugation, as described previously (34).
Isolated islets were placed in 1.5-ml Eppendorf tubes and cultured in
RPMI 1640 medium containing 11 mM glucose (Life
Technologies, Inc., Rockville, MD), supplemented with 10% fetal bovine
serum (Life Technologies, Inc.), 200 units/ml penicillin, and 200 µg/ml streptomycin at 37 °C, in an atmosphere of 5%
CO2. MIN6 cells (a gift from Dr. J-i. Miyazaki, Osaka
University, Osaka, Japan) at passage 15-25 and
TC3 cells (a gift
from Dr. D. Hanahan, University of California, San Francisco, CA) at
passage 40~50 were maintained in Dulbecco's modified Eagle's medium
containing 10% fetal bovine serum in an atmosphere of 5%
CO2 at 37 °C.
RT-PCR Southern Blot Analysis--
Total cellular RNA was
isolated from rat islets,
TC3 and MIN6 cells by the acid guanidinium
thiocyanate/phenol/chloroform method (35). Single-stranded cDNA was
prepared from the total RNA (~100 ng) by reverse transcriptase using
random 9-mers in the TaKaRa RT-PCR kit (TaKaRa Shuzo Ltd., Shiga,
Japan). The respective sense and antisense degenerate oligonucleotide
primers used were 5'-CAAGATGGCCAAGAACTGGA-3' and
5'-TTCTGTTCCTCATG(G/A)GCTTC-3', which correspond to amino acid
sequences KMAKNWS of Lys53-Ser59 and EAHEEQN of
Glu249-Gln255, respectively, of the rat
-
and
-SNAP protein sequences obtained from
GeneBankTM-entered nucleotide sequences. The PCR reaction
involved two initial cycles comprising 94 °C for 15 s, 42 °C
for 30 s, and 65 °C for 30 s, followed by 30 cycles
comprising 94 °C for 20 s, 55 °C for 30 s, and 72 °C
for 30 s. The 627-base pair PCR products were run on a 1.2%
agarose gel, transferred to a nitrocellulose filter, and hybridized
with 32P-labeled rat
- or
-SNAP cDNA, which was
prepared by PCR using specific oligonucleotide primers as probes.
Filters were washed under high stringency conditions (0.1 × SSC
(1 × SSC = 0.15 M NaCl, 0.015 M
sodium citrate), 0.1% SDS, 60 °C) and autoradiographed with
intensifying screens.
Immunofluorescence Microscopy--
Rat pancreas or isolated rat
islets was dissected in 4% paraformaldehyde in 0.1 M
phosphate buffer (pH 7.4), cryoprotected with graded concentrations of
sucrose in phosphate-buffered saline, 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 (28). Briefly, sections were incubated with rabbit
affinity-purified anti-
/
-SNAP antibody (diluted 1:50) in
phosphate-buffered saline containing 4% fetal calf serum, 0.1% sodium
azide, and 0.1% Triton X-100, followed by porcine 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. In order to demonstrate clearly the
morphological features of the pancreatic tissue or islet cells
subjected to immunostaining, each section was photographed using
Nomarski optics, as well as fluorescence optics.
Immunocytochemical Study of Recombinant Adenovirus-mediated
-SNAP Localization in
TC3 Cells--
TC3 cells were seeded on
eight-chamber Permanox slides (Lab Tek, Naperville, IL) coated with
poly-L-lysine, infected with recombinant adenoviruses, and
incubated for 2 days in an atmosphere of 5% CO2 at
37 °C. The media were removed, and the cells were fixed with 4%
paraformaldehyde, and then permeabilized with Triton X-100. Then they
were incubated with the primary antibody (diluted 1:50) in
phosphate-buffered saline at 4 °C for 2 h, washed with 2 mM glycine in phosphate-buffered saline, incubated with
fluorescence-labeled secondary antibody at room temperature for 4 h and finally, the cells on the coverslips were examined using
fluorescence microscopy.
Preparation of Recombinant Adenoviruses--
A 1.2-kilobase
cDNA fragment containing the entire coding sequence of bovine
-SNAP (a generous gift from Dr. Rothman, Sloan-Kettering Inst., New
York) was ligated into the pAdex1CA cosmid vector (36), which contains
the modified chicken
-actin promoter with cytomegalovirus-IE enhancer (CAG promoter) (a generous gift from Dr. Izumi Saito, Tokyo
University Institute of Medical Science, Tokyo, Japan). Then, the
recombinant adenovirus Adex1CA
-SNAP was prepared by homologous
recombination of the expression cosmid cassette and parental viral
genome (37, 38), and amplified to achieve a stock with a titer of
approximately 109 plaque-forming units/ml. For the
construction of
-SNAP mutant, C-terminal truncated
-SNAP mutant
(1-285 amino acid residue) was amplified by PCR from a plasmid
encoding full-length
-SNAP using the following primers: sense,
5'-GCTATGGACAACTCCGGGA-3'; antisense,
5'-CTCGTCACCCTGGATTTACTTCTTGATGCGCAG-3'. The PCR products were
confirmed to be correct by automated sequencing, ligated to the
pAdex1CA cosmid vector, and the recombinant adenovirus was constructed
as described above.
Adenovirus-mediated Gene Transduction--
Rat islets,
TC3
and MIN6 cells were incubated with Dulbecco's modified Eagle's medium
containing 5% fetal bovine serum and the required adenovirus for
2 h at 37 °C, after which, RPMI 1640 medium containing 11 mM glucose was added and 2 days later, the experiments were
performed. When
TC3 and MIN6 cells were infected with the adenovirus
Adex1CA Lac Z, Lac Z gene expression was observed in nearly 100% of
these cells (data not shown). In agreement with report from another
laboratory (39), rat islets infected with Adex1CA Lac Z and stained
with 5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside were a clear blue color in more than 70% of infected cells (data not
shown). Furthermore, insulin biosynthesis and secretion by rat islets
infected with Adex1w, which contains no foreign genes, were almost the
same as those by non-infected islets on post-infection day 2 (data not
shown). Therefore, in this study, rat islets and cultured cells
infected with Adex1w were used as controls.
Insulin Release--
Two days after infection, the incubation
media were removed, the islets or cultured cells were washed three
times with RPMI 1640 medium and preincubated with 2.2 mM
glucose for 2 h. Tissues were then challenged with 2.2 or 22 mM glucose alone, or 22 mM glucose + 20 µM forskolin for 1 h. The forskolin was added to evoke a near-maximal response. The media were collected at the end of
the challenge period, the cells were disrupted by sonication, and
aliquots of media and cell extracts were analyzed for immunoreactive insulin (IRI) by radioimmunoassay or enzyme-linked immunosorbent assay
(Medical Biology Laboratory (MBL), Nagoya, Japan). In some experiments,
insulin release was expressed as the fractional secretion rate per
hour, derived from the following equation: total IRI secreted/final IRI
in islets + total IRI secreted.
GABA HPLC Determinations--
Bioanalytical systems HPLC (System
Gold; Beckman instruments, Inc., Fullerton, CA) was used to analyze
GABA in MIN6-conditioned Hank's media by a modified isocratic
procedure with electrochemical detection (40). Briefly, 30 µl of
sample was mixed with 10 µl of derivatization reagent (4 mM o-phthalaldehyde and 2-mercaptoethanol) 2.5 min before injection onto a 4.6 × 150-mm MA-5ODS column (Eicom Co. Ltd., Kyoto, Japan). Mobile phase (pH 3.5) was 0.05 M
sodium phosphate buffer with 50% methanol delivered at a flow rate of 1.0 ml/min. Quantitation was by electrochemical detection, using a
glass carbon electrode set at 0.60 V.
Immunoblotting--
Rat islets,
TC3 and MIN6 cells were
disrupted by sonication, boiled in SDS sample buffer with 10 mM dithiothreitol, subjected to SDS-polyacrylamide gel
electrophoresis (PAGE), and then transferred onto nitrocellulose
filters. The protein concentrations were determined using a protein
assay kit (Bio-Rad). The filters were incubated with the required
primary antibody, followed by the appropriate horseradish
peroxidase-conjugated secondary antibody and the bands were visualized
using a chemiluminescence detection system (NEN Life Science Products
Inc., Boston, MA).
Syntaxin Binding Assay--
All the syntaxin binding samples
were run in duplicate. Maltose-binding protein-fused syntaxin 1A was
attached to amylose resin-agarose beads as described previously (28).
The beads were washed three times with binding buffer (10 mM Hepes-NaOH (pH 7.4), 0.15 µM NaCl, 2 mM MgCl2, 0.5% Triton X-100) and resuspended in 400 µl of binding buffer containing protease inhibitors (500 µg/liter pepstatin and 1 mM phenylmethylsulfonyl
fluoride) and approximately 50-150 ng
-SNAP and/or
-SNAP mutant
proteins and rotated (head over head) overnight at 4 °C. The beads
in the pellet fraction were washed four times with 1 ml of binding
buffer, the bound proteins were eluted in SDS sample buffer and
analyzed by SDS-PAGE followed by immunoblotting with anti-
/
-SNAP antibody.
Antisense Phosphorothioate Oligonucleotide--
The antisense
phosphorothioate oligonucleotide (5'-TCCATGACGTCGCGCAGCTGC-3')
complementary to the mouse
-SNAP sequence surrounding the initiation
codon and the corresponding sense phosphorothioate oligonucleotide were
designed based on the
-SNAP cDNA sequence. They were synthesized
on a DNA synthesizer (Applied Biosystem Instruments) and purified by
reverse phase high-pressure liquid column chromatography. For antisense
experiments, MIN6 cells were treated with a mixture of
FuGeneTM (Boehringer-Mannheim) and 50 µM of
each oligonucleotide for 24 h. The medium was renewed and 20 µM of each oligonucleotide was added to the renewed
medium every 24 h. After 7 days, the treated MIN6 cells were
challenged by 2.2 or 22 mM glucose for 1 h and subjected to IRI and immunoblot analysis.
Statistical Analysis--
Results are presented as mean ± S.E. from at least three different experiments performed independently
on at least three different cell preparations, unless stated otherwise.
Statistical analysis was performed using Student's t test
and ANOVA in multiple comparisons.
 |
RESULTS |
-SNAP Expression and Localization in Pancreatic
-Cells--
We initially studied the expression of
/
-SNAP
mRNA in the islets of Langerhans, insulinoma
TC3 and MIN6 cells
using RT-PCR analysis. The cDNAs reverse-transcribed from the total
RNAs isolated from rat islets,
TC3 and MIN6 cells were amplified
with degenerate oligonucleotide primers, which were designed to bind to
- and
-SNAP cDNAs simultaneously, and the PCR products were
hybridized with the rat
- or
-SNAP cDNA probe. As shown in
Fig. 1A, the insulinoma
TC3
and MIN6 cells expressed both
- and
-SNAP mRNAs, whereas rat
islets expressed only
-SNAP mRNA. Although the EtBr-stained gel
showed a single band in all lanes (data not shown), the autoradiogram from the islets lane showed 2 bands. The upper band above the 627-base
pair DNA fragment may have been an unknown isoform of SNAP or an
alternatively spliced form, although Northern blot analysis of total
RNA extracted from 500 islets showed a weak single band (data not
shown). To verify whether rat islets express
-SNAP, we utilized
specific oligonucleotide primers based on the rat
-SNAP cDNA
sequence obtained by RT-PCR analysis. This method confirmed the lack of
expression of
-SNAP in the islets (data not shown). The
/
-SNAP
protein levels in the islets and insulinoma cell lines were evaluated
by performing immunoblot analysis of total cellular protein. Fig.
1B shows that MIN6 cells (105 cells) expressed
an appreciable amount of
-SNAP protein, although,
TC3
(105 cells) and rat islets (~109 cells/100
islets) expressed relatively low levels of this protein. A single band
of
-SNAP protein with a molecular mass of ~35 kDa was detected in
all the cell lysates, but the
-SNAP band was barely detectable in
TC3 and MIN6 cell lysates. Thus, pancreatic
cells predominantly
expressed
-SNAP. In order to determine the localization of
-SNAP
protein in rat pancreas, we performed an immunofluorescence study.
Immunohistochemical staining of rat pancreas sections revealed the
presence of
-SNAP in the cytoplasm of the islets of Langerhans (Fig.
2). There was little positive staining of
-SNAP in the exocrine cells.

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Fig. 1.
/ -SNAP
mRNA and protein expression in isolated rat islets and
insulinoma TC3 and MIN6 cells.
A, autoradiogram of RT-PCR-amplified / -SNAP cDNA
fragments. The cDNAs were reverse-transcribed from total RNAs
isolated from rat islets, TC3 and MIN6 cells and subjected to the
PCR, as described under "Experimental Procedures," then the PCR
products were separated by 1.2% agarose gel electrophoresis, blotted
onto a nitrocellulose filter, and hybridized with
32P-labeled - and -SNAP cDNA probes. The
predicted size of the amplified cDNA fragment is 627 base pairs.
B, immunoblot analysis of -SNAP proteins. The total
cellular proteins were extracted by sonication from islets (100 islets), TC3 (105 cells), and MIN6 (105
cells), subjected to SDS-polyacrylamide gel electrophoresis, and
immunoblotted with anti- / -SNAP antibody. Representative blots are
shown (n = 4).
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Fig. 2.
Immunofluorescence photomicrograph
of rat pancreas immunostained with anti- / -SNAP antibody.
Sections of rat pancreas were incubated with rabbit preimmune serum, or
affinity-purified anti- / -SNAP antibody, followed by
fluorescence-conjugated bovine anti-rabbit immunoglobulin.
A, anti- / -SNAP antibody; B, preimmune
serum.
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Adenovirus-mediated
-SNAP Overexpression in Isolated
Islets--
Overexpression of
-SNAP in rat islets was achieved
utilizing an adenovirus-mediated gene transduction system, as
demonstrated by immunoblotting with antibodies against
/
-SNAP
(Fig. 3). Infecting islets with the
Adex1CA
-SNAP recombinant adenovirus resulted in a 10-20-fold
increase in the level of the protein relative to that of control islets
infected with the Adex1w control adenovirus. The efficiency of the
recombinant adenovirus system for gene transfer into mammalian cells
and isolated rat islets was previously evaluated (39). In agreement
with those results, more than 70% of the islet cells infected with
Adex1CA
-GAL were stained blue which was a slightly lower level than
that of insulinoma cells (MIN6 and
TC3), which were near 100% (data
not shown). To confirm the efficiency of the adenovirus treatment in
isolated islets, rat islets infected with the recombinant adenovirus
were examined by immunofluorescence studies. Fig.
4 shows
-SNAP immunostaining of islet
cells infected with Adex1w or Adex1CA
-SNAP. Strong immunostaining
for AdexlCA
-SNAP was observed in more than 90% of the cells in
islets infected with Adex 1CA
-SNAP. By contrast, only weak
immunostaining was observed in Adex1w-infected islets. Phase-contrast
studies showed similar results. To show that adenovirus-mediated overexpression does not affect protein targeting or sorting, we compared the immunofluorescence of endogeneous
-SNAP protein to that
of
-SNAP overexpressed in
TC3 cells. As shown in Fig. 5, in cells infected with Adex1CA
-SNAP, the immunoreactivity for
-SNAP was observed only in the
cytoplasm similar to the distribution of endogenous
-SNAP in
uninfected cells.

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Fig. 3.
Immunoblot analysis of
recombinant adenovirus-mediated overexpression
of -SNAP in isolated rat islets.
Islet proteins (20-30 islets/lane for experiment 1 and 2, 80 islets/lane for experiment 3) were extracted 2 days after treatment
with the indicated adenovirus, subjected to SDS-PAGE, and immunoblotted
with anti- / -SNAP antibody. The -SNAP protein band in the lane
of Adex1w-treated islets from experiments 1 and 2 was too weak to
be visualized by this chemiluminescence detection.
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Fig. 4.
Immunofluorescence photomicrograph of
adenovirus-infected isolated rat islets stained with anti- / -SNAP
antibody. Two days after infection with adenovirus, rat islets
were processed for immunofluorescence with anti- / -SNAP
antibody as described under "Experimental Procedures."
A, infected with Adex1w; B, infected with Adex1CA
-SNAP. Left panels, immunofluorescence; right
panels, phase-contrast.
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Fig. 5.
Immunocytochemical photomicrographs of
adenovirus-infected TC3 cells stained with
anti- / -SNAP
antibody. Mouse TC3 cells were infected with adenoviruses, then
2 days later fixed on coverslide glasses and immunostained with
anti- / -SNAP antibody. A, infected with Adex1w;
B, infected with Adex1CA -SNAP. Left panels,
immunofluorescence; right panels, phase-contrast.
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Effect of
-SNAP Overexpression on Insulin Release by Rat Islets
and Insulinoma Cells--
Two days post-infection, islets
overexpressing
-SNAP were incubated with 2.2 mM glucose
or 22 mM glucose alone, or 22 mM glucose + 20 µM forskolin for 1 h. Basal insulin release in the presence of 2.2 mM glucose by islets infected with Adex1CA
-SNAP almost doubled that of the Adex 1w-infected control (1.9 ± 0.5 versus 3.9 ± 0.8%/h) (Fig.
6). Insulin release by islets infected with Adex1CA
-SNAP in response to 22 mM glucose alone or
22 mM glucose plus 20 µM forskolin increased
by approximately 128 or 144% Adex1w-infected control levels,
respectively, (22 mM glucose, 6.7 ± 2.0 versus 8.6 ± 1.6%/h; 22 mM glucose + 20 µM forskolin, 9.4 ± 1.6 versus 13.5 ± 1.7%/h) (Fig. 6). The insulin content of islets infected with
Adex1CA
-SNAP tended to decrease, but did not differ significantly
from the control value (data not shown). We also examined the effect of
-SNAP overexpression on insulin release by insulinoma MIN6 cells.
MIN6 cells infected with Adex1CA
-SNAP showed about a 5-10-fold
increase in the level of
-SNAP protein detected by immunoblot
analysis, relative to the control level (data not shown). Since MIN6
cells responded to glucose within the range of physiological
concentrations during a short incubation period, these cells were
challenged with 2.2, 5.5, 11, and 22 mM glucose for 1 h. As shown in Fig. 7, insulin release by
MIN6 cells was stimulated by glucose, but was not affected by
-SNAP
overexpression.

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Fig. 6.
Insulin release by rat islets
overexpressing -SNAP. Rat islets were
infected with Adex1w or Adex1CA -SNAP, then 2 days later challenged
with 2.2 mM glucose or 22 mM glucose alone, or
22 mM glucose + 20 µM forskolin for 1 h.
The amount of IRI in the media and cells were measured and fractional
insulin release was calculated as described under "Experimental
Procedures" (n = 16).
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Fig. 7.
Insulin release by MIN6 cells
overexpressing -SNAP. MIN6 cells were
infected with Adex1CA -SNAP or Adex1w (control) in 12-well multiwell
plates at a density of 5 × 105 cells per well and the
experiment was carried out 2 days later. MIN6 cells were stimulated
with the indicated concentrations of glucose for 1 h and the
amounts of IRI in the media were measured (n = 4).
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Binding of Truncated
-SNAP Mutant (1-285) to Syntaxin
1A--
In order to further clarify the function of
-SNAP in
insulin exocytosis, we attempted to produce and express
dominant-negative mutant forms of
-SNAP. For this purpose, we
produced C-terminal deletion mutant
-SNAP (1-285), which has
reduced ability to stimulate NSF ATPase activity to less than 30% that
of wild-type
-SNAP in vitro (41). The binding of
wild-type
-SNAP and truncated
-SNAP mutant (1-285) to syntaxin
1A immobilized on agarose bead was first examined. As shown in Fig.
8, both wild-type
-SNAP and
-SNAP
mutant (1-285) proteins bound to syntaxin 1A, although the binding
capacity of mutant protein was slightly decreased, in agreement with
previous results (41), indicating that overexpression of this mutant
may competitively inhibit the cellular binding of wild-type
-SNAP to
syntaxin 1A.

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Fig. 8.
Binding of wild-type
-SNAP and truncated -SNAP
mutant (1-285) to syntaxin 1A immobilized on agarose bead.
Amirose resin-agarose bead preloaded with MBP-fused syntaxin 1A were
incubated with 50-150 ng of wild-type -SNAP and C-terminal deleted
-SNAP mutant (1-285), for 16 h at 4 °C, then the unbound
(S) and bound materials (P) were analyzed by
SDS-PAGE followed by immunoblotting with anti- / -SNAP antibody.
The bands were visualized by chemiluminescence detection.
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Effects of Overexpressed
-SNAP Mutant (1-285) on Insulin
Release by Rat Islets and MIN6 Cells--
As shown in Fig.
9, immunoblot analysis revealed that rat
islets infected with Adex1CA
-SNAP mutant (1-285) expressed high levels of
-SNAP mutant protein. Figs. 9 and
10 show the diminished insulin release
by
-SNAP mutant overexpression in rat islets and MIN6 cells,
respectively. In particular, a marked reduction in glucose-stimulated
insulin release (approximately 50% of control levels) was observed
with this mutant in both islets and MIN6 cells (islets, 8.4 ± 2.0 versus 4.6 ± 1.4 ng/islet/h; MIN6, 1765 ± 310 versus 950 ± 220 ng/ml/105 cells). By
contrast, the change in insulin release at low glucose was small in any
condition.

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Fig. 9.
Effects of truncated
-SNAP mutant (1-285) overexpression in rat islets
on insulin release. Two days after infecting islets with Adex1w
(control) or Adex1CA -SNAP mutant (1-285), the islets were
stimulated with 2.2 or 22 mM glucose plus 20 µM forskolin. The media were harvested and IRI levels
assayed as before (n = 8~10). Outer panel,
islet proteins (100 islets/lane for Adex1w, 20 islets/lane for Adex1CA
-SNAP mutant) extracted from the indicated adenovirus-treated islets
were subjected to SDS-PAGE followed by immunoblot analysis with the
anti- / -SNAP antibody, as described under "Experimental
Procedures." Representative blot is shown.
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Fig. 10.
Inhibition of the glucose-stimulated insulin
release by overexpression of -SNAP mutant in
MIN6 cells. MIN6 cells were infected with Adex1w ( ) or Adex1CA
-SNAP mutant (1-285) ( ). Two days later, MIN6 cells were
challenged with 2.2 or 22 mM glucose for 1 h and the
amounts of IRI in the media measured (n = 8).
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Involvement of
-SNAP in Insulin Exocytosis--
To further
examine the physiological function of
-SNAP, we attempted to
suppress the expression of endogeneous
-SNAP in MIN6 cells by
treatment with its antisense oligonucleotide. An antisense
phosphorothioate oligonucleotide complementary to the
-SNAP cDNA
sequence surrounding the initiation codon was added to the serum-free
medium at a concentration of 40 µM and again at 20 µM every 24 h in medium containing 5% fetal bovine
serum. The corresponding sense oligonucleotide was used in parallel as the control. When MIN6 cells were treated with the antisense
oligonucleotide for 7 days, the expression of
-SNAP was reduced as
observed by immunoblot analysis (Fig.
11). However, treatment of MIN6 cells with the sense oligonucleotide did not affect the expression level of
-SNAP protein. Antisense oligonucleotide treatment decreased the
insulin release to less than 50% the level of
oligonucleotide-untreated control values in both basal and stimulated
conditions (basal 40 ± 13%, and stimulated 48 ± 16%
versus oligonucleotide untreated control values).

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Fig. 11.
Inhibition of insulin release by an
antisense phosphorothioate oligonucleotide complementary to
-SNAP sequence in MIN6 cells. Upper
panel, immunoblot analysis. Total cellular proteins of MIN6 cells
were subjected to immunoblot analysis using anti- / -SNAP antibody.
Sense, sense oligonucleotide-treated cells ( );
antisense, antisense oligonucleotide-treated cells ( ).
Lower panel, insulin release. MIN6 cells were treated with
sense-, antisense-, or non-oligonucleotide (control) for 7 days, then
challenged with 2.2 or 22 mM glucose for 1 h, and IRI
levels in the media were measured. Insulin release was expressed as % of control (n = 6).
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Lack of
-SNAP Function in GABA Release--
Although pancreatic
cells are known to release GABA from synaptic-like microvesicles
(32), it is still controversial if the release is regulated by glucose
(42, 43). Fig. 12 shows that GABA
release by MIN6 cells was not affected by glucose, although insulin
release was markedly stimulated by glucose as shown in Fig. 7. Thus,
our data indicate that GABA is released via a constitutive pathway in
pancreatic
cells, in agreement with the report from Smismans
et al. (43). Overexpression of wild-type
-SNAP in MIN6
cells did not affect GABA release under basal and stimulated conditions, similar to what was seen for insulin release (Fig. 7).
Overexpression of mutant
-SNAP (1-285) in MIN6 cells also did not
affect GABA release under both basal and glucose-stimulated conditions
(Fig. 12), although insulin release was markedly inhibited (Fig.
10).

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Fig. 12.
GABA release from recombinant
adenovirus-treated MIN6 cells. MIN6 cells were treated with a
virus containing a wild-type -SNAP cDNA (Adex1CA -SNAP, ),
-SNAP mutant (1-285) (Adex1CA -SNAP mutant (1-285), ), or a
control virus (Adex1w, ). Fourty-eight hours after viral treatment,
cells were preincubated in RPMI 1640 containing 2.2 mM
glucose for 3 h, and then switched to 400 µl of Hank's medium
containing 2.2 or 22 mM glucose for 2 h.
Thirty-microliter aliquots of media were assayed for GABA content by
HPLC-electrochemical detection. Data are presented as
picomole/105 cells and represent the mean ± S.E. from
triplicate wells (n = 6).
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DISCUSSION |
In the docking/fusion of secretory vesicles, the v- and t-SNAREs
bind each other in a pairwise, cognate fashion (44-46), followed by
binding of SNAPs and NSF to the SNARE complex and resulting in membrane
fusion (19, 20). However, recent evidence has indicated that in at
least some systems, SNAPs and NSF are not essential for fusion (21,
22). Lin and Scheller (47) have recently proposed a new SNARE model,
suggesting that the major function of SNARE proteins is in the
endocytotic reuptake of fused granules (47). On the other hand, there
are several reports that
-SNAP function is associated with the final
fusion step in vivo (48-51), but most of the evidence has
been obtained from experiments with permeabilized cells, which may not
fully reproduce the normal localization and/or physiological function
of this protein. Function of
-SNAP may appear to differ in various
systems, probably because the components themselves, their biochemical characterization, and/or the nature and size of secretory granules in
the cells are heterogeneous, but details remain obscure. In the present
study, we explored the function of
-SNAP in exocytosis of both
insulin and GABA from pancreatic
cells. We analyzed the effect of
overexpression of wild-type and mutant
-SNAP on the release of
insulin and GABA via LDCVs and SLMVs, respectively, using pancreatic
islets and insulinoma
cells.
Since endocytotic reuptake and recycling of insulin secretory granules
is not likely to occur in pancreatic
cells, our results showing
enhanced insulin release by
-SNAP overexpression indicate that
-SNAP functions in the initial docking and fusion of insulin secretory granules consistent with the originally proposed SNARE hypothesis. The mechanism underlying
-SNAP induced increase in insulin release may be as follows. As
-SNAP stimulates the ATPase activity of NSF in vitro (17),
-SNAP overexpression may
cause efficient disassembly of the SNARE complex, resulting in
efficient membrane fusion. Since the amount of endogeneous
-SNAP in
MIN6 cells may already be at saturating levels for the disassembly reaction,
-SNAP overexpression in MIN6 cells is thought to induce no
significant changes in insulin release. Although NSF protein expression
in rat islets was found to be extremely low based on immunofluorescence
and immunoblotting studies,2
it appears from in vitro studies that
-SNAP and NSF
functions are coordinated in islets. Indeed, in permiabilized HIT T15
cells, the addition of these proteins into the medium restored the
Ca2+-dependent insulin release (51). In the
present study, we could not resolve the question as to why the
expression of
-SNAP is so low in islets relative to insulinoma cell
lines, despite the evidence that
TC3 and MIN6 cells contain much
lower insulin and a much smaller population of secretory granules
(52-54). Although the other type of SNAP may mainly function in
islets, one possible explanation is that since
-SNAP and NSF may be
rate-limiting factors for the docking/fusion process, lower expression
levels are more adequate for the tightly controlled regulation of
secretion characteristics of the islets.
To establish the physiological function of
-SNAP in insulin
exocytosis, we overexpressed the
-SNAP mutant to induce a
dominant-negative effect on insulin release.
-SNAP, which is
composed of 295 amino acids (11), has at least 4 coiled-coil domains
(55) and is involved in several protein-protein interactions (12).
-SNAP has the proposed syntaxin-binding domain in the N- and
C-terminal portion and has the NSF-binding domain in the C-terminal
region (55). Indeed,
-SNAP binds to syntaxin 1A, SNAP-25, and NSF (15). In an attempt to disrupt such interactions by introducing
-SNAP mutant into cells, we produced a C-terminal truncated
-SNAP mutant (1-285), which was effective as a dominant-negative inhibitor as shown in Figs. 9 and 10. Overexpression of
-SNAP mutant (1-285), which can bind to syntaxin 1A but lacks the ability to stimulate NSF
ATPase activity (41, 55), decreased markedly insulin release, but,
overexpression of the deletion mutant
-SNAP (1-199), which can
neither bind to syntaxin 1A nor stimulate NSF ATPase activity (41), has
only marginal effect on insulin release (data not shown). Thus, our
data indicate that
-SNAP plays a key role in glucose-stimulated
insulin exocytosis, probably via the interaction with syntaxin 1A, and
suggest that
-SNAP functions in the docking/fusion of insulin
secretory granules with the plasma membrane. It may also be speculated
that
-SNAP acts as the priming for insulin secretory granules to be
targeted to the docking site on the plasma membrane.
In additional studies, we performed experiments using the antisense
oligonucleotide in order to suppress the endogeneous
-SNAP expression in MIN6 cells. Antisense-treated MIN6 cells exhibited a
marked decrease in both basal and stimulated insulin release, while a
dominant-negative effect of
-SNAP mutant (1-285) overexpression was
mainly observed in the stimulated insulin release. It is conceivable that antisense oligonucleotide treatment of MIN6 cells inhibited the
expression of not only
-SNAP, but also of
-SNAP since the sequence used in the antisense oligonucleotide for
-SNAP was identical to that of
-SNAP. However,
- and
-SNAPs have been reported to have interchangeable roles in regulated exocytosis (50) and
hence may function coordinately in insulinoma cells. Further studies on
function of
-SNAP in exocytosis will be necessary to settle this issue.
The question was then addressed as to whether
-SNAP also functions
in GABA release. GABA is believed to be released via SLMVs, which may
also be recycled in pancreatic
cells as is well documented in
neuronal cells. We found that GABA release by MIN6 cells was not
affected by glucose, suggesting that GABA is not present in insulin
secretory granules. We also examined the effect of wild-type
-SNAP
and
-SNAP mutant (1-285) overexpression on GABA release from MIN6
cells. Since the GABA levels in the media of isolated rat islets was
very low (data not shown), we could not utilize islets for this
experiment. Our data with MIN6 cells demonstrate clearly that
overexpression of both wild-type and mutant
-SNAP did not affect
GABA release, indicating no requirement of
-SNAP in the
docking/fusion of SLMVs. Thus,
-SNAP appears to work differently in
different types of secretory granules in even the same cell.
In conclusion, our data demonstrate that
-SNAP plays a key role in
insulin release via LDCVs, but not in GABA release via SLMVs in
pancreatic
cells. Thus, the functional role of
-SNAP in
cells depends on the nature and types of secretory granules.