Departments of 1 Medicine, 2 Physiology, and 3 Biochemistry, University of Toronto, Toronto M5S 1A8; and 4 Program in Cell Biology, Hospital for Sick Children Research Institute, Toronto, Ontario, Canada M5G 1X8
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ABSTRACT |
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Previous reports showed that cleavage of vesicle-associated
membrane protein-2 (VAMP-2) and synaptosomal-associated protein of 25 kDa (SNAP-25) by clostridial neurotoxins in permeabilized insulin-secreting -cells inhibited Ca2+-evoked insulin
secretion. In these reports, the soluble
N-ethylmaleimide-sensitive factor attachment protein target
receptor proteins might have formed complexes, which preclude full
accessibility of the putative sites for neurotoxin cleavage. In this
work, VAMP-2 and SNAP-25 were effectively cleaved before they formed
toxin-insensitive complexes by transient transfection of insulinoma HIT
or INS-1 cells with tetanus toxin (TeTx) or botulinum neurotoxin A
(BoNT/A), as shown by immunoblotting and immunofluorescence microscopy. This resulted in an inhibition of Ca2+ (glucose or
KCl)-evoked insulin release proportionate to the transfection
efficiency (40-50%) and an accumulation of insulin granules. With
the use of patch-clamp capacitance measurements, Ca2+-evoked exocytosis by membrane depolarization to
10
mV was abolished by TeTx (6% of control) but only moderately inhibited
by BoNT/A (30% of control). Depolarization to 0 mV to maximize
Ca2+ influx partially overcame BoNT/A (50% of control) but
not TeTx inhibition. Of note, cAMP activation potentiated
Ca2+-evoked secretion by 129% in control cells but only
55% in BoNT/A-transfected cells and had negligible effects in
TeTx-transfected cells. These results indicate that, whereas VAMP-2 is
absolutely necessary for insulin exocytosis, the effects of SNAP-25
depletion on exocytosis, perhaps on insulin granule pool priming or
mobilization steps, could be partially reversed by higher levels of
Ca2+ or cAMP potentiation.
vesicle-associated membrane protein-2; synaptosomal-associated protein of 25 kilodaltons; clostridial neurotoxins; insulin exocytosis; capacitance measurements; adenosine 3',5'-cyclic monophosphate
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INTRODUCTION |
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IN PANCREATIC ISLET
-CELLS the molecular basis for insulin granule exocytosis is
becoming clear (44). In regulated insulin secretion,
insulin is synthesized and stored in secretory vesicles, which are then
lodged beneath the plasma membrane of pancreatic islet
-cells. A
postprandial increase in plasma glucose causes membrane depolarization
by activation of voltage-dependent Ca2+ channels. This
results in a rise of cytosolic Ca2+, which causes the
fusion of insulin granules with the plasma membrane to release insulin.
Whereas glucose stimulates insulin secretion by this
Ca2+-dependent pathway (36), this pathway
becomes defective in diabetes (23). The cAMP-dependent
pathway of insulin secretion is of importance, particularly in light of
reports demonstrating that the glucagon-like peptide-1 agonists
(6) acting on this pathway can bypass the defective
Ca2+-activated pathways in diabetic islets (11,
14).
Much recent work has suggested that Ca2+-induced
exocytosis of insulin granules is controlled by a set of proteins
called soluble N-ethylmaleimide-sensitive factor attachment
protein target receptors (SNAREs) (45). SNAREs were
originally identified as the proteins required for synaptic vesicle
fusion (32, 38). These proteins are specific substrates
for proteolysis by the clostridial neurotoxins, enzymes known to
inhibit neurotransmitter release (22). A current view of
SNARE-mediated vesicle fusion is that the SNARE proteins on the donor
vesicle v-SNARE (vesicle-associated membrane protein or VAMP) and the
target membrane [t-SNAREs; synaptosomal-associated membrane protein of
25 kDa (SNAP-25) and syntaxin] interact to form a stable complex,
which provides the energy to drive membrane fusion (41).
In pancreatic islet -cells, the v-SNARES, VAMP-2 and VAMP-3, were
found on insulin granules (16, 28, 29, 43), and the
t-SNAREs, SNAP-25 and several syntaxin isoforms, were found on the
plasma membrane (10, 33, 43). We and others reported
(28, 29, 43) that addition of tetanus toxin light chain
(TeTx-LC) into permeabilized islets and insulinoma cells inhibited
Ca2+-evoked insulin secretion. Transient transfection of
HIT cells with VAMP mutants resistant to proteolytic cleavage by
TeTx-LC was able to rescue this toxin inhibition of
Ca2+-evoked insulin release (28). Syntaxin 1 was also required for Ca2+-induced insulin release, shown
by the fact that botulinum neurotoxin (BoNT)/C1 transfection of
insulinoma HIT cells inhibited KCl-evoked insulin secretion
(17). BoNT/C1, however, only minimally inhibited glucose-stimulated secretion (17). We recently showed that
SNAP-25 is also required for Ca2+-evoked insulin secretion
as well as for glucose-evoked insulin secretion, using the strategy of
transfecting botulinum neurotoxin A light chain (BoNT/A-LC) into HIT
cells to cleave and deplete endogenous SNAP-25 (15).
Whereas it is well established that Ca2+-evoked insulin
secretion requires these SNARE proteins, the roles of these SNARE
proteins are not clear within the distinct kinetic components (i.e.,
mobilization and release of insulin granule pools) of insulin
exocytosis. It is even less clear whether cAMP-dependent insulin
secretion also requires SNAREs. In support of a role for SNAREs in
cAMP-mediated secretion is the report that BoNT/B cleavage of VAMP-2
inhibited cAMP-dependent enzyme secretion in parotid acinar cells
(8). Exocrine acinar cells share similar properties with
islet -cells in utilizing both Ca2+- and cAMP-dependent
pathways to mediate secretion (44). In the present study,
we have further explored the role of VAMP-2 and SNAP-25 in
Ca2+- and cAMP-evoked insulin exocytosis from insulinoma
cell populations (by radioimmunoassay) and from single
-cells (by
patch-clamp capacitance measurements). We used the strategy of
transiently transfecting the TeTx-LC or BoNT/A-LC genes into insulinoma
cells, which effectively cleaves VAMP-2 (9, 19, 22) or
SNAP-25 (15). We demonstrated that both Ca2+
influx and elevated intracellular cAMP overcame BoNT/A but not TeTx-LC
inhibition of insulin exocytosis. We conclude that VAMP-2 and SNAP-25
might be involved in the different kinetic components of insulin
exocytosis. VAMP-2 is required for the insulin granule exocytotic
fusion, whereas SNAP-25 may be involved in the priming and mobilization
of insulin secretory granule pools in the exocytotic process, which is
mediated by Ca2+ and cAMP.
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MATERIALS AND METHODS |
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Materials. The HIT cell subclone T15 was a gift from Dr. Robert Santerre (Eli Lilly, Indianapolis, IN), and the INS-1 cell line was from American Type Culture Collection. A mouse monoclonal antibody generated against recombinant full-length TeTx-LC and TeTx-LC cDNA was a gift from H. Niemann (39) (Hanover, Germany), and anti-insulin antibody was from R. A. Pederson (University of British Columbia). A rabbit polyclonal antibody was generated against the NH2-terminal 16 amino acids of VAMP-2 (9). Forskolin and 3-isobutyl-1-methylxanthine (IBMX) were purchased from Sigma (St. Louis, MO).
Generation of constructs and transfections.
Plasmids that contained VAMP-2 (40) and TeTx-LC cDNAs
(7, 39) were subcloned into pcDNA3 as previously described
(15). The HIT-T15 cells were grown at 37°C in 5%
CO2-95% air in RPMI 1640 medium supplemented with 20 mM
glutamine, 10% fetal calf serum (GIBCO, Gaithersburg, MD), penicillin
(100 U/ml), and streptomycin (100 mg/ml). The cells were grown to a
density of 3 × 106 cells/well in 12 (20-mm)-well
plates. They were then transiently transfected with 2 µg of plasmid
DNA (in Optimum media) or empty vector as control using Lipofectamine
(GIBCO) and incubated for 4-6 h before normal RPMI was added. The
medium was discarded the following day, and fresh RPMI 1640 medium was
replaced. Transfection efficiency was determined by using
pcDNA3/His/Lac Z (Invitrogen, La Jolla, CA) as a control
plasmid for transfections, followed by determination of
-galactosidase expression and also by visualization of cells by
confocal microscopy.
Immunoblotting. SDS-PAGE and immunoblotting were performed as previously described (15, 43). Protein concentrations of each cell lysate sample were determined by the Bio-Rad (Hercules, CA) protein assay kit. The cell lysates were then dissolved in sample buffer, and proteins were separated on a 15% polyacrylamide gel, followed by transfer to a nitrocellulose membrane. The blots were then incubated with the primary antibody (anti-VAMP-2 antibody, 1:1,000; anti-TeTx-LC, 1:1,000; and anti-actin, 1:1,000 in 1% BSA and 0.05% Tween 20 in PBS) for 2 h at room temperature. Immunodetection was by enhanced chemiluminescence (Amersham ECL). Quantification of each band was determined by measuring the band area with NIH Image and Adobe Photoshop programs.
Insulin secretion. Forty-eight hours after transfection, the cells were cultured to ~80% confluence. As previously described (15), cells were washed and then incubated in 0.5 ml of low-K+ Krebs-Ringer bicarbonate (KRB) buffer (in mM: 4.8 KCl, 129 NaCl, 5 NaHCO3, 1.2 KH2PO4, 1 CaCl2, 1.2 MgSO4, 0.5 glucose, and 10 HEPES, pH 7.4) for 0.5 h. Each well was then exposed to basal conditions to obtain basal insulin secretion, followed by stimulatory conditions. The value of insulin released during the stimulatory conditions was subtracted from the value obtained from the basal conditions, which reflects the true stimulated secretion above basal levels (see Figs. 3, 4, and 6A). Specifically, for K+ stimulation studies, each well of cells was incubated with 0.5 ml of the same buffer containing low K+ (4.8 mM KCl and 0.5 mM glucose at 1 h) for basal secretion, and the media were collected after the incubation. This was followed by an exchange with 0.5 ml of a high-K+ buffer (30 mM KCl at 1 h), and the media were collected. For glucose stimulation, the wells were exposed to KRB buffer (with 4.8 mM KCl at 1 h) in which glucose was removed to determine basal secretion and then exchanged with a high-glucose buffer (11 mM with 4.8 mM KCl at 1 h). For cAMP stimulation, the cells were exposed to low-K+ buffer (0.5 mM glucose and 4.8 mM KCl) for basal level of insulin secretion, followed by an exchange with the same buffer containing 10 µM forskolin and 100 µM IBMX. We found that basal insulin secretion at 0.5 mM glucose was not different from that with no glucose, although for the cAMP-evoked secretion, the presence of low-glucose concentration (0.5 mM) was necessary. At the end of each incubation (basal and stimulated), the media collected were centrifuged at 3,000 g for 3 min to remove detached cells. The resulting top half of the supernatants was collected and determined for insulin release by radioimmunoassay (Linco, MO) according to the manufacturer's instructions. For the determination of total insulin content, the cell lysates remaining in the wells were scraped from the plates, and appropriate dilutions of the samples were carried out to allow the values to fall within the standard curve. As previously reported (15), we controlled the inherent variability of transfection efficiency and cell line passage by using the same plate of cells in each experiment, which included all the variables being tested. At least 3-5 independent experiments with triplicate or four replicate wells per experimental variable were performed. Values shown in the figures are the mean increases above basal levels, which were also determined in triplicate wells for each experiment. The data are expressed as means ± SE of the total number of wells and were analyzed by both Student's t-test and analysis of variance (ANOVA). A P value < 0.05 by both tests (shown in the text by ANOVA) indicates that the difference is considered significant.
Confocal immunofluorescence microscopy. Microscopy was performed as previously described (10, 15, 43). Briefly, the cells were plated on round polylysine-coated coverslips and placed in 12-well plates. Forty-eight hours after transfection, the cells were fixed in 2% formaldehyde for 0.5 h at room temperature, blocked with 5% normal goat serum with 0.1% saponin for 0.5 h at room temperature, and then immunolabeled with primary antibodies (1:100 anti-VAMP-2 and anti-TeTx-LC antibody) overnight at 4°C. After being rinsed with 0.1% saponin/PBS, the coverslips were then incubated with the appropriate rhodamine-labeled secondary antiserum for 1 h at room temperature, mounted on slides in a fading retarder 0.1% p-phenylenediamine in glycerol, and examined with a laser scanning confocal imaging system (Carl Zeiss). Transfected cells were identified by coexpression and visualization of the green fluorescence protein (GFP) (Clontech, Palo Alto, CA).
Patch-clamp capacitance measurements.
Insulinoma -cell lines HIT-T15 and INS-1 were studied under standard
whole cell patch-clamp conditions as previously described (34). To identify the TeTx-LC- and BoNT/A-LC-transfected
HIT or INS-1 cells, we coexpressed the GFP, which we previously
demonstrated to very reliably take up multiple plasmids
(15). All intracellular solution contained (in mM) 140 KCl, 10 HEPES, 1 EGTA, 1 MgCl2, and 3 MgATP, pH 7.3 with
KOH. Extracellular solution contained (in mM) 140 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2, and 10 HEPES, pH 7.3 with NaOH.
In experiments with cAMP stimulation (INS-1 cells), 0.5 mM glucose was
added to the above bath solution. Forskolin and IBMX were dissolved in
DMSO (1,000× stock solutions). They were added directly to the bath
solution to get a final concentration as indicated. Experiments were
performed at room temperature. Capacitance measurements
(Cm) were then performed similarly to that
described by Ammala et al. (1) and Renstrom et al.
(30). Specifically, the cells were held at
70 mV and
depolarized to
10 or 0 mV (as indicated in the figures) for 250 ms.
Immediately after depolarization, cells were exposed to a sine wave
centered at
70 mV, and the changes in Cm
stimulated by this depolarizing voltage pulse were observed. These
experiments were performed with a quick-response lock-in patch-clamp
amplifier (HEKA Electronics, Lambrecht, Germany) and a
high-speed and high-memory computer (PC, Pentium III) running
specialized patch-clamp Pulse software (HEKA). With the use of this
equipment configuration, very small changes (fF range) in whole cell
capacitance were monitored to indicate secretion granule (SG)
fusion. This analysis is based on the fact that the specific
capacitance of all biological membranes [including the plasma membrane
(PM) and SG membranes] is ~10 fF/µm2. Because
the fusion of an SG to the PM adds ~0.2 µm2 of membrane
to the PM, a corresponding incremental increase in Cm by 2 fF would result. To minimize the
possible influence of inherent variability of transfection and the
passage of the cell lines on capacitance, data from three to four cells
were averaged for each experiment (n = 1), and each
group had the same chance of coming from the same passage. Data were
presented as means ± SE. Data were compared by unpaired
Student's t-test for single comparisons and by ANOVA for
multiple comparisons. P < 0.05 was considered to be
statistically significant.
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RESULTS |
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Expression of TeTx-LC cleaves endogenous and overexpressed VAMP-2 in HIT-T15 cells. We and others reported that insulin secretion from permeabilized HIT-T15 is inhibited by TeTx-LC (29, 43). However, in those studies, only a modest inhibition was demonstrated (43). The lack of complete inhibition of insulin secretion could be due to the following: 1) VAMP-2 could be complexed with other proteins that render the cleavage site inaccessible to toxin cleavage (4, 12); 2) temporal limitations prevent the toxin from effectively acting on VAMP-2; and 3) inhibitory effects of cleavage products could be lost if these products leak out of the cell. To overcome these limitations, we transiently transfected HIT-T15 cells with the cDNA of the TeTx-LC (7, 39). This strategy should result in high levels of TeTx-LC within intact cells over a prolonged period of time, permitting VAMP-2 cleavage before it forms a complex with other SNAREs. The intact cell system would also permit the inhibitory effects on insulin secretion to be measured in response to a variety of secretagogues.
In Fig. 1A (top), HIT cells were transfected with TeTx-LC, and the anti-TeTx-LC antibody was used to detect expression of this protein. Recombinant TeTx-LC (9) used as a control (lane 3) was detected, and no TeTx-LC was detected in untransfected cells (lane 1). The lower part of the blot (bottom) was then probed with the anti-VAMP-2 antibody. This showed that endogenous VAMP-2 was cleaved by TeTx-LC (lane 2) compared with the intact VAMP-2 in untransfected cells (lane 1). The residual VAMP-2 (lane 1) comes from the untransfected cells, since transfection efficiency is ~40-50% based on cotransfection with the reporter
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Expression of TeTx-LC results in accumulation of insulin secretory
granules.
Because VAMP-2 is a v-SNARE protein involved in insulin granule
exocytosis, its cleavage by TeTx-LC might result in accumulation of
insulin granules. Indeed, in HIT cells lacking VAMP-2 (indicated by
arrows in Fig. 3A) as a result
of TeTx-LC expression, double labeling with anti-insulin antibodies
demonstrated an accumulation of cytoplasmic insulin puncta in
these cells (indicated by arrows in Fig. 3B) compared with
the untransfected cells, wherein VAMP-2 remains intact. Because the
immunofluorescence study in Fig. 3B suggested that
TeTx-LC-transfected cells appeared to contain more insulin than
nontransfected cells, we determined the total insulin content of these
cells by radioimmunoassay in Fig. 3C. Total insulin content
in cells expressing TeTx-LC was 322.3 ± 49 ng/well and in control
cells was 252.9 ± 14 ng/well, which was an increase of 27.4% (14 wells, 5 experiments, P = 0.15). In cells cotransfected with VAMP-2 and TeTx-LC, total insulin content was 243.6 ± 20.9 ng/well compared with cells transfected with VAMP-2 alone (203.1 ± 54.5 ng/well), which was an increase of 20% (n = 14 wells, 5 experiments; data not shown). These increases in total insulin content were, however, not statistically significant from control, but
they at least indicate that the inhibition of secretion shown in Figs.
4 to 6A was not due to the inhibition of insulin
synthesis. This apparent discrepancy could in part be explained by the
following inherent limitations of these assays. First, fluorescence
microscopy is not sufficiently quantitative and may overemphasize
differences. Second, the measurement of total insulin content in cell
lysates includes insulin in both transfected and untransfected cells, as well as insulin in subcellular compartments other than secretory granules. These cells may tend to minimize differences due to averaging. We have, in fact, observed similar immunofluorescence and
insulin content results with BoNT/A-LC transfection of HIT cells, which
we previously reported (15). In that report, we performed
electron microscopy and morphometric analysis and found that BoNT/A-LC
transfection resulted in accumulation of insulin granules at the plasma
membrane.
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Effects of TeTx-LC transfections on Ca2+-stimulated
insulin secretion.
We next examined the effects of VAMP-2 depletion by TeTx-LC
transfection on Ca2+-evoked insulin secretion. In
neuroendocrine islet -cells, Ca2+ influx across the
Ca2+ channel is brought about by membrane depolarization
caused by glucose-mediated closure of the ATP-sensitive K+
channel, which leads to K+ influx (27).
Membrane depolarization can therefore be directly affected by the
addition of high concentrations of K+ (30 mM) into a
Ca2+-containing buffer with low-K+
concentrations (4.8 mM) (15). We therefore examined the
effects of TeTx-LC transfection on glucose- and KCl-stimulated insulin secretion (Fig. 4).
Distinct effects of BoNT/A-LC and TeTx-LC on
Ca2+-evoked insulin secretion.
The results in Figs. 3 and 4 appear similar to those we have
reported for BoNT/A-LC transfection (15). It is,
therefore, intuitive to assume that cleavage of VAMP-2 and SNAP-25 by
these toxins would uniformly abrogate exocytic fusion. However, a more recent report on chromaffin cells using the patch-clamp capacitance measurements demonstrated that these neurotoxins exhibit distinct effects on the multiple components of exocytosis (47). The
acute addition of these toxins used in that report would not reliably effect complete cleavage of the SNARE proteins, particularly those that
already exist in the distinct SNARE complexes (12). In contrast, overexpression of the neurotoxin over a 24-h period ensures
both very high levels of the neurotoxin and full cycling of SNARE
complex assembly and disassembly. This ensures that the neurotoxin
gains full access to not only the endogenously but also to exogenously
expressed SNARE proteins (Figs. 1 and 2). We, therefore, use this
strategy of neurotoxin overexpression in HIT-T15 and INS-1 cell lines
to dissect the distinct effects of VAMP-2 and SNAP-25 proteolytic
depletion by these clostridial neurotoxins on Ca2+-evoked
secretion at the single cell level using patch-clamp capacitance measurements (1, 30) (Figs.
5 and
6, B and C). To
identify the neurotoxin-transfected cells, we coexpressed GFP in the
cells in which we have shown to reliably take up multiple plasmids
(15). The cells with the highest GFP fluorescence were
studied, which contained the corresponding high concentration of the
coexpressed neurotoxins. Figure 5 shows the effects of TeTx-LC
or BoNT/A-LC expression on membrane depolarization-evoked exocytosis in
HIT-T15 cells compared with the control cells. Figure 5A
shows representative capacitance recordings, and Fig. 5B
shows a summary of these recordings. These cells were evoked by a
depolarization to 10 mV (Fig. 5A, top, and Fig.
5B, open bars) or 0 mV (Fig. 5A,
bottom, and Fig. 5B, filled bars). These levels
of depolarization were selected on the basis of our previous study of
HIT-T15 cells on voltage-evoked Ca2+ currents in which we
showed that a depolarization to 0 mV resulted in a 41% increase in
Ca2+ current over depolarization to
10 mV
(34). At
10 mV, TeTx-LC expression virtually abolished
(1.7 ± 0.7 fF, or 5.6% of control cells) the capacitive response
compared with the control cells (30.3 ± 1.7 fF). A depolarization
to 0 mV, which further increases Ca2+ influx and the
capacitive response in the control cells (40.7 ± 1.9 fF), did not
reverse the inhibition by TeTx-LC at all (1.6 ± 0.6 fF). In
contrast, at
10 mV, BoNT/A-LC expression inhibited the capacitive
response to 30% of control (9.52 ± 0.9 fF), demonstrating that
BoNT/A-LC caused a smaller inhibition than TeTx-LC. More interestingly,
at 0 mV, this inhibition by BoNT/A-LC was greatly reversed to ~50%
of control cells (20.4 ± 3.1 fF), with the actual capacitive
change at 0 mV to double that at
10 mV (Fig. 5B).
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Distinct effects of TeTx-LC and BoNT/A-LC on cAMP-stimulated
potentiation of Ca2+-evoked insulin secretion.
Glucose is the physiological agonist for the islet -cells and
activates Ca2+-dependent pathways to stimulate insulin
secretion, which may become defective in the diabetic states
(23). cAMP pathways, including those evoked by the
incretin hormone, glucagon-like peptide-1 (GLP-1), can potentially
bypass or potentiate glucose-mediated pathways (6, 11,
14). We therefore examined whether transient transfection of
TeTx-LC or BoNT/A-LC might also affect cAMP-mediated insulin release
(Fig. 6A) and potentiation of Ca2+-evoked
secretion (Fig. 6B). The cAMP pathway was stimulated by forskolin and IBMX at the concentrations indicated. Figure
6A shows that, in the TeTx-LC-transfected HIT cells in the
presence of the permissive glucose concentrations (0.5 mM), insulin
secretion (above the basal levels) was 2.27 ± 0.29 ng/well
(P = 0.02, n = 12 wells, 4 experiments)
and 3.05 ± 0.22 ng/well in the control cells. TeTx caused a
significant inhibition of 25.4% (P = 0.03) compared
with the control. In BoNT/A-LC toxin-transfected cells, insulin
secretion from HIT cells was 1.91 ± 0.29 ng/well, an inhibition of 37% (P = 0.02, n = 12 wells, 4 experiments) compared with the control, which was not significantly
different from that caused by TeTx-LC (P = 0.31).
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DISCUSSION |
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SNARE proteins VAMP-2, SNAP-25, and syntaxin constitute the
"minimal" machinery for membrane fusion (41), and,
therefore, it would be intuitive to assume that cleavage of these
proteins by BoNT should have identical effects on exocytosis. However, TeTx-LC (this study) and BoNT/A-LC (15), which cleave
VAMPs and SNAP-25, respectively, possess distinct (18) and
broader effects on the signaling pathways (Ca2+, cAMP,
glucose) mediating insulin secretion than BoNT/C1 (17), which cleaves the syntaxins. This suggests that each of these SNARE
proteins has additional distinct actions within the insulin secretory
process that are differentially coupled to these signaling pathways. In
fact, these SNARE proteins are potential substrates for phosphorylation
by Ca2+/calmodulin-dependent protein kinase II, PKA, and
protein kinase C (PKC) (13, 31, 37). Activation of protein
kinases and inhibition of protein phosphatases play a central role in
the regulation of insulin exocytosis (1). However,
mutations of Ca2+/calmodulin-dependent protein kinase II
phosphorylation sites in VAMP-2 had no effect on
Ca2+-stimulated insulin secretion (28). If
these kinases act directly on the SNARE proteins to induce exocytosis,
then SNAREs other than VAMP-2 must be the substrates (13, 31,
37). In support of this possibility, SNAP-25 in the insulinoma
RIN cells was phosphorylated via an undefined tyrosine kinase mechanism
after glucose or GLP-1 stimulation (49). SNAP-25 also
contains consensus sequences for PKC and PKA phosphorylation (13,
31, 37). However, mutations of putative phosphorylation sites
within SNAP-25 and syntaxins were recently shown to have little effect
on SNARE complex assembly in vitro (31). Nonetheless,
these researchers indicated that phosphorylation of SNARE proteins may
still affect SNARE complex function through their actions on other
proteins, such as synaptotagmins (31), which are also
present in -cells (43). Functional assays are,
therefore, necessary to demonstrate how these SNARE proteins are
actually coupled to these signaling pathways.
We have indeed demonstrated that cleavage of VAMP-2 and SNAP-25
(15) by TeTx-LC and BoNT/A-LC, respectively, inhibited
insulin secretion evoked by glucose, Ca2+ (by KCl-evoked
membrane depolarization), and cAMP. The residual insulin secretion
observed with the wells transfected with TeTx-LC or BoNT/A-LC were
likely due to cells that were either not transfected or that expressed
lower levels of TeTx-LC. The levels of reduction in evoked insulin
secretion of 45-60% in these population cell studies are
remarkably comparable to the transfection efficiency of 40-50%,
based on the reported -galactosidase gene assays and the confocal
microscopy studies (Fig. 2). An inhibition of secretion could be due to
blockade of the secretion step or an alteration in the biosynthesis of
secretory vesicles. It has been shown previously that SNARE proteins
participate in vesicle formation at the stage of vesicle coating
(20, 35). However, we do not believe that the reduction of
secretion is related to an inhibition of granule synthesis since, as
shown in Fig. 3, TeTx-expressing cells possess more insulin-containing
granules, consistent with a block in release. To more precisely
determine the extent and distinct kinetic components of insulin
exocytosis affected, we have employed the method of patch-clamp
capacitance measurements on cells expressing the highest levels of
neurotoxins. With the use of single cell capacitance measurements, we
showed that VAMP-2 and SNAP-25 might act at distinct sites within the
Ca2+-evoked insulin exocytic machinery.
Differences between the inhibitory effects of BoNT/A and TeTx on neurotransmitter release have been observed many times over the past two decades in a variety of systems (2, 5, 18, 21, 24, 25 47). With the use of permeabilized chromaffin cells, Lawrence et al. (18) showed that BoNT/A partially inhibited MgATP-primed neurotransmitter release, whereas TeTx completely abolished it. Moreover, the inhibition by BoNT/A seen in autaptic hippocampal cultures resembled that obtained by reduced extracellular Ca2+ (24). Interestingly, reduced Ca2+ levels reduced the probability of release in these systems. More recently, by combining flash photolysis with capacitance measurements in chromaffin cells, Xu and colleagues (47) showed that BoNT/A affected the rate of both the exocytic burst phase and slow phase of catecholamine release. Together, these results suggest that the COOH-terminal nine amino acids of SNAP-25 appear to couple to the Ca2+ sensor to facilitate rapid exocytosis. Here we show that in the insulinoma HIT-T15 cells, BoNT/A and TeTx also inhibit insulin secretion differently. As in neurons and chromaffin cells, BoNT/A intoxication can be overcome by a stronger depolarization that results in more Ca2+ influx across the Ca2+ channels. In contrast, TeTx-LC inhibition of secretion was complete and could not be overcome by the stronger depolarization. BoNT/A-LC and TeTx-LC expression, rather than injection into cells (25, 47), ensures complete cleavage of SNAP-25 and VAMP-2, and, therefore, any lack of inhibition cannot be simply attributed to the uncleaved SNAREs in toxin-insensitive complexes.
cAMP and downstream PKA activation are known to increase the release
probability of insulin secretory granules in the readily releasable
pool and also increase the rate of refilling of this pool by
accelerated mobilization of insulin secretory granules from the reserve
pool (30). This is the postulated mechanism by which
cAMP/PKA activation amplifies both the exocytic burst and sustained
phase of insulin secretion (30), the latter of which
involves the refilling of the readily releasable pool. These enhancements of the insulin exocytotic machinery by cAMP/PKA
activation, even in the presence of maximal depolarization-induced
Ca2+ influx to increase priming and mobilization of insulin
granules, could not reverse the inhibitory effects of TeTx-LC
expression in -cells. SNAP-25 and its ability to form SNARE
complexes also plays a critical role in the sustained phase of
secretion in chromaffin cells, which involves priming and granule pool
mobilization steps (42, 47, 48). Here we show that
cAMP/PKA activation is able to overcome the BoNT/A-LC inhibition,
suggesting that a PKA-mediated mechanism may be able to rescue the
unstable SNARE complex that is perhaps formed by the truncated SNAP-25
and enable it to still mediate exocytic membrane fusion (albeit a less
efficient process). SNAP-25 indeed possesses PKA
phosphorylation sites within this truncated segment (13, 31,
37), which might positively alter its binding to the cognate
SNAREs to form the putative complex(es) that specifically affect these
exocytic steps. Alternatively, another protein substrate for PKA, such
as
-SNAP (13), could modulate the kinetics of priming
and mobilization of insulin granule pools (46). Together,
our results demonstrate that increased Ca2+ influx and/or
cAMP/PKA activation can overcome the inhibitory effects of BoNT/A-LC
cleavage of SNAP-25 but not TeTx-LC cleavage of VAMP-2.
Our recent report that levels of SNARE proteins, including VAMP-2,
SNAP-25, and syntaxin 1 were diminished in islets of obese diabetic
fa/fa rats, a model of type 2 diabetes, led us to postulate that this could contribute to the basal hypersecretion as well as the
dampened insulin secretion in response to the high-glucose concentration (3). Patients with type 2 diabetes exhibit a similar presentation of basal hyperinsulinemia and low-insulin secretory responses to the postprandial high-glucose concentrations. GLP-1, likely by PKA-dependent mechanisms, was able to bypass these
secretory defects in these patients and normalized insulin secretion
(11, 14). It therefore behooves us and others to determine
which steps within the dysregulated insulin exocytotic machinery (i.e.,
exocytotic fusion, priming, and mobilization of insulin granule pools)
in these diabetic islet -cells, which are coupled to the
Ca2+- and cAMP-signaling pathways, are specifically
affected by the depletion of SNARE proteins. Such novel insights may
lead to the identification of new therapeutic targets.
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ACKNOWLEDGEMENTS |
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This work was supported by a grant from the Juvenile Diabetes Research Foundation, National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-55160, and by grants from the Canadian Diabetes Association (to H. Gaisano) and the Canadian Institutes for Health Research (to H. Gaisano and A. M. Salapatek).
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FOOTNOTES |
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* X. Huang, Y. Kang, and E. A. Pasyk contributed equally to this work.
H. Gaisano is a Research Scholar of the American Gastroenterology Association/Pharmacia and Upjohn. A. M. Salapatek is a recipient of a scholarship jointly funded by Eli Lilly, Banting and Best Diabetes Centre of the University of Toronto, and the Canadian Institutes of Health Research. W. S. Trimble is a Canadian Institutes of Health Research Scientist.
Address for reprint requests and other correspondence: H. Y. Gaisano, Rm. 7226, Medical Sciences Bldg., Univ. of Toronto, Toronto, Ontario, Canada M5S 1A8 (E-mail: herbert.gaisano{at}utoronto.ca).
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. Section 1734 solely to indicate this fact.
Received 14 March 2000; accepted in final form 8 April 2001.
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