Truncated SNAP-25 (1197), Like Botulinum Neurotoxin A, Can Inhibit Insulin Secretion from HIT-T15 Insulinoma Cells
Xiaohang Huang,
Michael B. Wheeler,
You-hou Kang,
Laura Sheu,
Gergely L. Lukacs,
William S. Trimble and
Herbert Y. Gaisano
Departments of Medicine and Physiology University of Toronto
(X.H., M.B.W., Y.-h.K., L.S., H.Y.G.) and The Toronto Hospital
(H.Y.G., M.B.W.) Toronto, Ontario, Canada M5S 1A8
Divisions of Respirology (G.L.) and Cell Biology (W.S.T.)
Hospital for Sick Children Research Institute Toronto, Ontario,
Canada M5G 1X8
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ABSTRACT
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We and others have previously shown that
insulin-secreting cells of the pancreas express high levels of SNAP-25
(synaptosomal-associated protein of 25 kDa), a 206-amino acid t-SNARE
(target soluble N-ethylmaleimide-sensitive factor
attachment protein receptors) implicated in synaptic vesicle
exocytosis. In the present study, we show that SNAP-25 is required for
insulin secretion by transient transfection of Botulinum Neurotoxin A
(BoNT/A) into insulin-secreting HIT-T15 cells. Transient expression of
BoNT/A cleaved the endogenous as well as overexpressed SNAP-25 proteins
and caused significant reductions in K+ and
glucose-evoked secretion of insulin. To determine whether the
inhibition of release was due to the depletion of functional SNAP-25 or
the accumulation of proteolytic by-products, we transfected cells with
SNAP-25 proteins from which the C-terminal nine amino acids had been
deleted to mimic the effects of the toxin. This modified SNAP-25 (amino
acids 1197) remained bound to the plasma membrane but was as
effective as the toxin at inhibiting insulin secretion.
Microfluorimetry revealed that the inhibition of secretion was due
neither to changes in basal cytosolic Ca2+
levels nor in Ca2+ influx evoked by
K+-mediated plasma membrane depolarization.
Electron microscopy revealed that cells transfected with either BoNT/A
or truncated SNAP-25 contained significantly higher numbers of insulin
granules, many of which clustered close to the plasma membrane.
Together, these results demonstrate that functional SNAP-25 proteins
are required for insulin secretion and suggest that the inhibitory
action of BoNT/A toxin on insulin secretion is in part caused by the
production of the plasma membrane-bound cleavage product, which itself
interferes with insulin granule docking and fusion.
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INTRODUCTION
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In the neuroendocrine ß-cell, insulin granule exocytosis appears
to be regulated through the actions of a set of proteins, collectively
called SNAREs (soluble N-ethylmaleimide-sensitive factor
attachment protein receptors) (1), which were originally found in
neurons and yeasts (2). The SNARE hypothesis predicts that the cognate
vesicle (v-SNARE) and target (t-SNARE) membrane proteins interact to
form SNARE complexes that serve to target, dock, and fuse the vesicle
to the target membrane (2, 3). Each of these SNARE proteins possess
distinct domains that independently bind to their SNARE partners
(4, 5, 6). In the cell, the putative v-SNARE on the insulin secretion
granule is VAMP-2 (vesicle-associated membrane protein isoform 2) (7, 8), and the putative t-SNAREs on the plasma membrane include SNAP-25
(synaptosomal-associated protein of 25 kDa (8, 9) and several members
of the syntaxin family (8, 9, 10).
SNAP-25 proteins possess an amino-terminal domain that binds to
syntaxin, a carboxy-terminal that binds to VAMP-2, and a cysteine-rich
central domain that attaches this protein to the plasma membrane via
palmitoylation (4, 11). Specific cleavage of these SNARE proteins by
clostridial neurotoxins blocks neuroexocytosis (12). Botulinum
neurotoxin A (BoNT/A) and BoNT/E cleave SNAP-25 at
Gln197-Arg198 and
Arg180-Ile181, respectively, to remove
carboxyl-terminal 9- and 26-amino acid peptides, and this cleavage
results in inhibition of neurotransmitter release (13, 14) and insulin
secretion (9). In vitro studies have shown that cleavage of
SNAP-25 by BoNT/A does not prevent assembly of the SNARE complex, but
reduces its stability by inhibiting the acquisition of SDS resistance
(5, 6, 15). However, the relationship between these biochemical
properties and the observed blockade of secretion in vivo is
not clear. Four simple explanations for the actions of BoNT/A are: 1)
that the proteolytic action of the toxin depletes the cell of
functional SNAP-25 molecules; 2) that the nine-amino acid
carboxyl-terminal proteolytic fragment is inhibitory to SNARE complex
formation or function; 3) that the 197-amino acid amino-terminal
fragment of SNAP-25 forms SDS-sensitive complexes that are
nonfunctional, thereby inhibiting exocytosis; or 4) that the cleavage
products could interfere with Ca2+ entry into the cell and
indirectly block secretion.
The second hypothesis has been supported by experimental evidence.
Synthetic peptides of the carboxy-terminal 12 and 20 amino acids of
SNAP-25 interfered with Ca2+-evoked secretion in
permeabilized neuroendocrine chromaffin cells (16, 17). In the latter
report, secretion granules were observed to cluster close to the plasma
membrane, suggesting that SNAP-25 plays a role at the docking step in
Ca2+-mediated exocytosis. These studies suggested that the
mechanism of inhibition by these toxins may be through the generation
of the carboxy-terminal peptides released into the cytosol that could
then bind to VAMP to block the formation of the SNARE complexes.
In addition, evidence exists in support of the fourth hypothesis, that
SNAP-25 binds directly to the plasma membrane Ca2+ channel
(18). Coexpression of SNAP-25 and N-type or L-type Ca2+
influx channels in Xenopus oocytes caused an inhibition of
inward currents (19). A direct interaction between SNAP-25 and the
Ca2+ channel raises the possibility that the BoNT/A
cleavage products might inappropriately modulate Ca2+
channel activity in neuronal or neuroendocrine cells.
To gain further insight into the mechanism of BoNT/A actions and
SNAP-25 functional domains, we have transfected insulinoma HIT-T15
cells with BoNT/A, full-length SNAP-25, or a SNAP-25 mutant in which
the C-terminal nine amino acids had been deleted to mimic the effects
of BoNT/A. We found that SNAP-25 proteins lacking the C-terminal nine
amino acids were as effective as BoNT/A at inhibiting agonist-evoked
insulin secretion from transfected cells, and that this effect was not
due to alterations in Ca2+ influx. Transfected cells had
significantly higher numbers of intracellular secretory granules, many
of which appeared to be in close proximity to the plasma membrane. This
suggests that the secretory blockade caused by BoNT/A is caused by the
production of the plasma membrane-bound amino-terminal 1197 fragment
of SNAP-25, which acts as an inhibitor of secretion.
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RESULTS
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Expression of BoNT/A or SNAP-25 (1197) Mutant in HIT-T15
Cells
To examine the effect of BoNT/A on insulin secretion, HIT-T15
cells were transiently transfected with the cDNA of the light chain of
BoNT/A, full-length SNAP-25 (amino acids 1206), or a SNAP-25 mutant
(amino acids 1197) truncated at the BoNT/A cleavage site in the
mammalian expression plasmid pcDNA3. Insulin secretion from HIT-T15 had
previously been shown to be inhibited by BoNT/A in permeabilized
preparations, but in those studies only modest inhibition was
demonstrated (9). The lack of complete inhibition of insulin secretion
observed in that study may be due, in part, to inaccessibility of the
cleavage site of SNAP-25 proteins that are already complexed to other
SNARE proteins (5, 6), temporal limitations of toxin action during
leakage of essential soluble factors such as SNAP (20, 21), or the loss
of potentially inhibitory peptide cleavage products through
permeabilization pores. We chose transfection experiments because they
would enable BoNT/A to effectively cleave SNAP-25 before it formed a
complex with other SNAREs and would permit the effect to be measured
without loss of essential factors or of the cleavage products.
Figure 1
demonstrates that the
transfected proteins are expressed at high levels in HIT-T15 cells.
Expression of BoNT/A results in the cleavage of the vast majority of
the endogenous SNAP-25 protein (upper panel). This residual
intact endogenous SNAP-25 is largely contributed by cells that were
either not transfected or expressed BoNT/A poorly. In fact, the
transfection efficiency based on the cotransfection with the
ß-galactosidase reporter gene ranges from 5080%. The inability to
detect the cleavage product is due, in part, to the lower
immunoreactivity of this cleavage product to our antibody, which was
generated against the full-length SNAP-25 (8). In support, when
exposure was extended (lower panel), the cleavage product
appeared (arrowhead). However, the faint appearance of the
cleaved fragment suggests the possibility that this fragment might have
further undergone accelerated proteolysis. To explore this possibility,
we probed the same HIT-T15 lysates expressing BoNT/A with an antibody
generated against the N-terminal amino acid 829 of SNAP-25
(Transduction Laboratories, Lexington, KY) (data not shown) which
showed identical results as those shown in Fig. 1
. These latter results
support the possibility of accelerated proteolytic depletion of SNAP-25
after BoNT/A cleavage.

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Figure 1. Immunoreactive SNAP-25 Proteins in Transiently
Transfected HIT-T15 Cell Lysates
Upper panel, HIT-T15 cells grown to 80% confluence
on 12-mm wells were transfected transiently with constructed expression
vectors for botulinum neurotoxin A-light-chain (BoNT/A), wild-type (WT,
SNAP-25, amino acids 1206), wild-type and BoNT/A (WT + BoNT/A), or
the SNAP-25 (amino acids 1197) mutant, compared with control
untransfected cells. Lower panel, the same blot in the
lower panel for the BoNT/A transfection but exposure was 5-fold longer
to visualize the cleaved protein (arrowhead). Ten
micrograms of protein of each sample were loaded, and blots were probed
with anti-SNAP-25 antisera. This is a representative of four
independent experiments.
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Figure 1
(upper panel) also shows that transfection with the
full-length SNAP-25 protein results in SNAP-25 levels significantly
higher than endogenous levels. Cotransfection with the full-length
SNAP-25 and BoNT/A resulted in virtually complete cleavage of the
expressed SNAP-25, as evidenced by the increased mobility of the
immunoreactive species. Complete cleavage of the cotransfected product
is not surprising since transfected cells typically coexpress both
plasmids (as in Fig. 2
). The antibody
used in these studies was able to detect both full-length and truncated
proteins. Transfection of the SNAP-25 (1197) mutant resulted in
equivalently high levels of expression compared with the full-length
protein, and the mobility of the product was identical to that
resulting from BoNT/A cleavage.

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Figure 2. Cellular Location of SNAP-25 Proteins of
Transiently Transfected HIT-T15 Cells Labeled with anti-SNAP-25
Antibody and Detected by Laser Scanning Confocal Microscopy
Untransfected HIT-T15 cells plated on coverslips were probed with
anti-SNAP-25 antibodies (a) alone or (b) preincubated with recombinant
GST-SNAP-25 proteins which completely blocked the SNAP-25 signal.
HIT-T15 cells were transfected with constructed vectors for (c)
wild-type SNAP-25 (amino acids 1206) or (e) BoNT/A and probed with
the anti-SNAP-25 antibody. Coexpression with the GFP (panels d and f,
indicated by arrows) shows the corresponding
increased SNAP-25 signal in cells overexpressing the
wild-type SNAP-25 protein (in c) and corresponding
decreased SNAP-25 signal in cells expressing the BoNT/A
toxin (in panel e). Panels c-f demonstrate that the transfected cells
(indicated by arrows) were able to take up both
plasmids. Cells transfected with (g) wild-type SNAP-25 + BoNT/A or (h)
SNAP-25 mutant (amino acids 1197) and were also probed with the
anti-SNAP-25 antibody, and the arrows point to the
transfected cells overexpressing the SNAP-25 proteins. Two days after
transfection, the coverslips were probed with rabbit anti-SNAP-25 antibody, fixed and labeled with
secondary antibodies, and then visualized on a laser scanning confocal
microscope as described in Materials and Methods.
Bar, 10 µm.
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To determine the localization of transfected SNAP-25 proteins and the
effect of BoNT/A expression on that localization, laser confocal
immunofluorescence microscopy was performed using antibodies specific
to SNAP-25 and coexpression of the green fluorescence protein (GFP). In
untransfected control HIT-T15 cells, SNAP-25 was found on the plasma
membrane consistent with its role as a plasma membrane t-SNARE for
insulin granule exocytosis (Fig. 2A
). Specificity of this antibody
staining was demonstrated by the complete blockade of this signal when
the antibody was preincubated with recombinant GST-SNAP-25 protein
(Fig. 2B
). Coexpression of the wild-type SNAP-25 (amino acids 1206)
protein along with the GFP shown in Fig. 2
, C and D, confirmed that the
transfected cells (indicated by arrows) expressed both
plasmids. In all experiments performed throughout these studies,
transformation frequency was quantitated, either as a measure of the
number of cells overexpressing the SNAP-25 proteins, those expressing
cotransfected GFP, or cotransfection of ß-galactosidase in control
wells, to be between 50% and 80%. The intensity of the SNAP-25 signal
was not altered when the GFP alone was expressed (data not shown).
Transient transfection with BoNT/A along with the GFP in Fig. 2
, E and
F, demonstrated a diminished intensity in the SNAP-25 signal in the
transfected cells (indicated by arrows), which corresponds
to the profound proteolysis of SNAP-25 as measured by Western blot
(Fig. 1
). However, there was no effect on the plasma membrane
localization of the cleaved SNAP-25 protein (Fig. 2E
). Similarly,
transfection of both BoNT/A and the wild-type SNAP-25 (1206) in Fig. 2G
shows that the cleavage of the overexpressed SNAP-25 proteins by
BoNT/A (indicated by arrows) did not alter their plasma
membrane localization. This result was identical to that obtained in
Fig. 2H
with cells transfected with the SNAP-25 (1197) mutant protein
(indicated by arrows), which also did not result in
mislocalization (Fig. 2G
). In Fig. 2
, CG, cells that were not
transfected showed a similar low intensity of plasma membrane staining
equivalent to the control untransfected cells (Fig. 2A
), whereas cells
that were inefficiently transfected showed intermediate signal
intensity. These results, taken together, indicate that overexpression
of the wild-type or mutant form of SNAP-25, or of BoNT/A, did not
affect the fidelity of targeting of these SNAP-25 proteins to their
normal destination, the plasma membrane. These modified SNAP-25
proteins should therefore be able to interact with plasma membrane
proteins, including their SNARE partners and the L-type
Ca2+ channel, located in the plasma membrane.
Effects of BoNT/A or SNAP-25 (1197) Mutant Transfections on
Potassium- or Glucose-Stimulated Insulin Secretion
K+ depolarization of the endocrine cell plasma
membrane activates the distal secretory machinery by directly inducing
Ca2+ influx, which in turn evokes insulin exocytosis (22).
To examine the effect of the treatments used above on evoked insulin
secretion, we treated control and transfected HIT-T15 cells with
Ca2+-containing high K+ (30 mM)
buffer or a control buffer with low K+ (4.8 mM)
in a manner similar to that described by Boyd et al. (23).
Figure 3
shows the insulin secretion
evoked by the high K+ buffer above basal insulin release
caused by low K+ buffer. In control untransfected cells,
the high K+ buffer evoked insulin release of 2- to 4-fold
that released by the low K+ buffer (data not shown). To
minimize inter- and intraassay variations and possible variation in
transfection efficiency, the following controls were performed. As a
control for the effect of transfection on release, cells transfected
with the empty vector had no effect on insulin release compared with
untransfected cells (data not shown). Cells in each set of experiments
were from a single plate of cells of the same passage, plated in
identical manner into each well. Triplicate wells for each variable and
control were performed for each experiment, and at least three
independent experiments were performed. The viability and confluence of
the cells in each well were confirmed to be uniform by visual
examination. Transfection efficiencies in the range of 5080% were
confirmed by coexpression of the ß-galactosidase gene in a control
well performed in every set of experiments.

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Figure 3. Potassium-Stimulated Insulin Release from HIT-T15
Cells
HIT-T15 cells were transfected as described in Materials and
Methods with BoNT/A in panel A, wild-type (WT, SNAP-25 amino
acids 1206) + BoNT/A in panel B, or SNAP-25 (1197) mutant in panel
C. Each experiment was performed and compared with control
untransfected cells. In panel B, cells transfected with the wild-type
SNAP-25 protein alone were also compared. The release of insulin from
the cells during a 1-h incubation period with 30 mM
K+ or 4.8 mM K+ (basal level) was
determined by RIA as described in Materials and Methods.
Each value shown is the means ± SEM of the insulin
released above basal levels. The number of wells for each value and
their statistical significance are indicated in
Results.
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Figure 3A
demonstrates that transient expression of BoNT/A reduced the
high K+-evoked insulin release to 0.796 ± 0.203
ng/well compared with 1.984 ± 0.261 ng/well for untransfected
cells (n = 9 wells, 3 experiments, P = 0.001), an
approximately 60% reduction. Figure 3B
demonstrates that
cotransfection of BoNT/A, along with the wild-type native SNAP-25
protein, resulted in a high K+-evoked insulin release of
1.005 ± 0.18 ng/well (n = 13 wells, 4 experiments) which was
a significant reduction of
65% and
62%, respectively, compared
with the insulin released from control (2.905 ± 0.458 ng/well,
P = 0.002) or wild-type SNAP-25 transfected cells
(2.656 ± 0.63 ng/well, P = 0.002).
It is possible that BoNT/A inhibition of insulin release may be
mediated by its proteolytic effects on undefined substrates other than
SNAP-25. We have therefore examined the effects on insulin secretion
resulting from the transfection of a SNAP-25 mutant (amino acids
1197) in which the terminal nine amino acids corresponding to the
BoNT/A cleavage site are truncated (Fig. 3C
). This strategy further
allows us to isolate the distinct effects of amino acids 1197 domain
of SNAP-25 independent of the C-terminal nine-amino acid fragment
resulting from BoNT/A proteolytic cleavage. Insulin release evoked by
the high K+ was 1.007 ± 0.23 ng/well (n = 12
wells, 4 experiments) for the SNAP-25 mutant and 3.09 ± 0.76
ng/well for the control wells, which was a significant reduction of
67% (P = 0.006).
These studies were also conducted using a physiological secretagogue,
glucose, at a concentration known to maximally stimulate insulin
secretion (24, 25). As in Fig. 3
, cells were transfected with BoNT/A
(Fig. 4A
) or the SNAP-25 (1197) mutant
protein (Fig. 4B
) compared with control cells transfected with the
empty vector. To evoke secretion, cells were incubated in Krebs-Ringer
bicarborate (KRB) media containing 10 mM glucose (1 h, 37
C), which released insulin that was generally 2-fold of basal insulin
secretion. Figure 4
shows evoked insulin release that was subtracted
from basal insulin release at 0 glucose concentration. We found that
transfection of BoNT/A reduced insulin release from the control value
of 1.70 ± 0.09 ng/well to 0.83 ± 0.08 ng/well (n = 15
wells, 4 experiments, P = 0.0002), or a reduction of
51%
(Fig. 4A
). Overexpression of the SNAP-25 (1197) mutant (Fig. 4B
)
caused a reduction of insulin release from a control value of 1.53
± 0.49 ng/well to 0.26 ± 0.06 ng/well (n = 9 wells, 3
experiments, P = 0.027), or a reduction of
83%. The
more marked inhibition observed with these latter
studies is due largely to the higher transfection rate in this set of
experiments. These results, demonstrating similar inhibition of acute
glucose-evoked insulin release by transient expression of either BoNT/A
or of the SNAP-25 (1197) mutant, are consistent with the results
obtained with acute high K+-evoked insulin release in Fig. 3
.

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Figure 4. Glucose Stimulation of Insulin Release from HIT-T15
Cells
HIT-T15 cells were grown and transiently transfected with constructed
vectors of (A) BoNT/A or (B) SNAP-25 (amino acids 1197) mutant as
described in Fig. 3 . Control cells were transfected with the vector
only. The release of insulin from the cells during a 1-h incubation
period with no glucose (basal levels) or 10 mM glucose was
determined by RIA as described in Materials and Methods.
Each value shown is the means ± SEM of the insulin
released above basal levels. The number of wells for each value and
their statistical significance are indicated in
Results.
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The residual insulin secretion of
2040% observed with the wells
transfected with either the BoNT/A (Fig. 3
, A and B) or SNAP-25
(1197) mutant (Fig. 3C
) genes is likely to be due to cells that were
either not transfected or that express lower levels of cleaved or
mutant SNAP-25 proteins. Nonetheless, the levels of reduction in evoked
insulin secretion of
5080% in these studies are remarkably
comparable to the efficiency of transfection of 5080% based on the
ß-galactosidase assay and
67% based on confocal microscopy
studies (mean of 17 randomly chosen micrographs as in Fig. 2
). Taken
together, these studies strongly suggest that high K+- or
glucose-evoked insulin secretion from the transfected cells were
markedly, if not totally, inhibited. Furthermore, the uniform
inhibition of evoked insulin secretion from either the BoNT/A- or
SNAP-25 (1197) mutant-transfected cells demonstrate that expression
of the SNAP-25 (1197) mutant proteins could account for the entire
BoNT/A-mediated inhibition of evoked insulin release.
We also compared the basal insulin release in the studies performed in
Figs. 3
and 4
and found no statistical difference between the cells
expressing BoNT/A or truncated SNAP-25 mutant and control cells (data
not shown), suggesting no effects of these treatments on constitutive
insulin exocytosis.
Effects of BoNT/A or SNAP-25 (1197) Mutant Transfections on
Potassium-Mediated Plasma Membrane Depolarization-Evoked
Ca2+ Influx
Because of the recent reports that SNAP-25 could directly modulate
Ca2+ channel kinetics, including the neuroendocrine L-type
channel (19), it is possible that these transfections could affect
Ca2+ influx via this or other Ca2+ influx
channels to inhibit the insulin secretion, as observed in Figs. 3
and 4
. We have therefore performed fluorescent video-imaging of transfected
HIT-T15 cells loaded with the Ca2+ indicator dye fura 2AM
and perfused with buffers containing different concentrations of
Ca2+ and K+ (Fig. 5
). The HIT-T15 cells studied were
transfected with the empty vector (control, Fig. 5A
), BoNT/A (Fig. 5B
),
or the SNAP-25 (1197) mutant protein (Fig. 5C
). Transfection
efficiency was again confirmed by coexpression of GFP performed as a
control in each experiment. Fluorescent ratio intensity values at 340
nm and 380 nm were obtained for each of the cells visualized in the
field, and the data are expressed as the ratio of 340 nm to 380 nm
(Fig. 5
). There was no detectable difference in the ratio values
between the cells within each coverslip during exposure to low
K+ or high K+ buffers. We therefore randomly
chose a field within each coverslip, and the ratio values obtained from
all of the cells within the field were analyzed together as described
in Materials and Methods. Basal Ca2+ levels were
first determined with a low K+, 1 mM
Ca2+ buffer, and the ratio values (mean ±
SEM) obtained for the control (0.75 ± 0.01), BoNT/A
(0.75 ± 0.01), and SNAP-25 (1197) mutant (0.73 ± 0.01)
transfected cells as indicated in Table 1
were not significantly different. To better assess Ca2+
influx activity, ratio values (mean ± SEM) were
obtained at 0.5 min and 3 min after stimulation (indicated by the
first arrows) with the high K+, 1 mM
Ca2+ buffer. As shown in Table 1
, ratio values at each of
these time points were not significantly different. Upon removal of the
extracellular Ca2+ (in 10 mM EGTA, indicated by
the second arrows), the intracellular Ca concentration
([Ca2+ ]i) uniformly and abruptly returns to basal
levels. This latter result, together with a control experiment using a
high K+, nominal free Ca2+ (no added
Ca2+ in 10 mM EGTA) buffer that gave no rise in
[Ca2+]i (not shown), indicate that the rise in
[Ca2+]i evoked by the high K+ buffer was
generated entirely from an extracellular source coming through
voltage-dependent Ca2+ influx channels. These results,
taken together, indicate that the inhibition of insulin secretion
caused by these transfections does not seem to involve a direct effect
on the Ca2+ influx into the ß-cell.

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Figure 5. Effects on Ca2+ Influx Evoked by
Potassium-Mediated Plasma Depolarization in Single HIT-T15 Cells
HIT-T15 cells were plated on coverslips and transfected with the empty
vector (Control) in panel A, BoNT/A in panel B, or SNAP-25 (amino acids
1197) mutant in panel C as described in Materials and
Methods. After loading, the cells were with fura-2AM, the
coverslip was mounted onto a perfusion chamber and onto the microscope,
and a field of cells was randomly chosen. Fluorescent images of
cytosolic Ca2+ levels of single cells were then obtained as
described in Materials and Methods. The coverslips were
first perfused with a low K+ (4.8 mM), 1
mM Ca2+ buffer until a steady baseline was
obtained. The coverslip was then perfused with a high K+
(30 mM), 1 mM Ca2+ buffer as
indicated by the first arrows, followed by removal of the
Ca2+ (30 mM K+, 0 mM
Ca2+) as indicated by the second arrows. Each
value is expressed as mean ± SEM of the mean values
of each field of cells (1525 cells per field) randomly picked from a
coverslip, and at least seven coverslips from two to three independent
experiments were analyzed as also described in Materials and
Methods. The Ca2+ values were not significantly
different (see Table 1 ).
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BoNT/A or SNAP-25 (1197) Mutant Transfections Resulted in
Accumulation of Insulin Granule Near the Plasma Membrane
Another possible explanation for the decreased insulin release
from cells expressing either BoNT/A or SNAP-25(1197) could be that
the transfected cells simply produced less insulin. To examine the
quantity and subcellular location of insulin-containing granules, we
performed electron microscopy of these cells (Fig. 6
). Since not all cells were transfected
in any treatment, experiments were performed in double blind
conditions, and 23 micrographs were randomly taken from three
independent experiments for each vector. Parallel control transfections
were performed to confirm that the transfection efficiency was more
than 50%. Transfection with the ß-galactosidase reporter gene did
not affect the number of granules compared with control untransfected
cells (not shown). In all cases, the number of insulin granules in the
HIT-T15 insulinoma cell line was relatively sparse compared with normal
islet ß-cells (our unpublished observation). To accurately determine
the cellular distribution of the accumulated granules in these
transfected cells, we performed morphometric analysis of these randomly
chosen micrographs as a function of the distance from the plasma
membrane to the nucleus, and data were analyzed and expressed as
frequency distribution histograms of the number of granules per
µm2 of cytosolic space (Fig. 6
). Only 16 of the 23
micrographs for each of the vector-contained cells with the appropriate
distance of at least 3.6 µm of cytosolic space between the plasma
membrane and nuclei were analyzed. In the control cells, the insulin
granules were randomly distributed at a density of about 1
granule/µm2 from the nucleus to 0.45 µm from the plasma
membrane, but within 0.45 µm of the plasma membrane that density
approximately doubled. The granule distribution immediately adjacent to
the nucleus in BoNT/A and SNAP-25 (1197) mutant transfected cells
appeared similar to the control cells. However, in contrast to control
cells, the granule densities in BoNT/A and SNAP-25 mutant transfected
cells rise progressively and uniformly as the plasma membrane was
approached, and were highest at within 0.45 µm from the plasma
membrane, where they were 2-fold higher than in control cells. These
results demonstrate that the long-term effects of the BoNT/A or
truncated SNAP-25 (amino acid 1197) expression caused an accumulation
of insulin secretion granules. This pattern of accumulation of the
insulin granules suggests not only an exocytotic blockade at the plasma
membrane, but might also affect additional more proximal steps in the
mobilization of the insulin secretion granules. Alternatively, the
blockade of exocytosis might cause some feedback in the granule
translocation process.

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Figure 6. Subcellular Distribution of Insulin Granules in
HIT-T15 Cells
HIT-T15 cells were grown in RPMI 1640 and transiently transfected with
either the empty vector for control (in panel A) or the constructed
vectors of BoNT/A (in panel B) or SNAP-25 (amino acids 1197) mutant
(in panel C) as described in Figs. 3 and 4 . The cells were then
processed for ultrastructural study as described in Materials
and Methods. The micrographs obtained at a uniform original
magnification of 6,600x for each vector were subjected to morphometric
analysis using the NIH Image analysis program. This figure shows the
frequency distribution histograms of the number of insulin granules as
a function of the distance from the plasma membrane (0 µm) to the
nucleus (3.6 µm). The surface area within each 0.45 µm bin width
from the plasma membrane was determined and the number of insulin
granules within the bin counted. The data are then expressed as the
number of granules per µm2 of cytosolic surface area of
each 0.45 µm bin width. Of the 23 random micrographs obtained from
three independent experiments for each vector, only 16 micrographs per
vector contained cells with the appropriate cytosolic space between the
plasma membrane and the nucleus for analysis. Each bin value is
expressed as the mean ± SEM (n = 16
micrographs).
|
|
It is conceivable that the long-term effects of BoNT/A or truncated
SNAP-25 expression might affect processes other than exocytosis of
insulin secretion granules, including insulin synthesis and insulin
vesicular loading. We have therefore determined the total insulin
content of these cells (Fig. 7
) and
compared the results to the total number of insulin granules in these
cells. The total insulin content cells expressing BoNT/A and truncated
SNAP-25 mutant were 236.84 ± 19.63 ng/well and 244.64 ±
17.36 ng/well, respectively, compared with 188.75 ± 11.99 ng/well
in the control cells (n = 15 wells, 5 experiments). This was a
small but significant increase of only 25% for BoNT/A
(P = 0.004) and 30% for truncated SNAP-25 mutant
(P = 0.0004) transfected cells compared with control
cells. We then counted the total number of granules from each bin in
Fig. 6
as a function of the total cytosolic surface occupied by these
granules. There were 1.03 ± 0.13 granules/µm2
(n = 16 micrographs) for the control cells, which was
significantly different from BoNT/A (1.81 ± 0.18
granules/µm2, n = 16 micrographs, P
= 0.001) and SNAP-25 (1197) mutant (1.86 ± 0.23
granules/µm2, n = 16 micrographs, P
= 0.004) transfected cells. The granule density and total insulin
content between BoNT/A and SNAP-25 (1197) mutant transfected cells
were similar. This granule density ratio of 2:1 of either BoNT/A or
SNAP-25 (1197) mutant transfected cells compared with the control
cells was preserved when the rest of the 23 micrographs per vector were
accounted for (data not shown). The incremental increase in insulin
granule density is about 3-fold that of the incremental increase in
total insulin content in these cells. Taken together, these results
suggest that the small increase in total insulin content effected by
the long-term treatments with the BoNT/A or SNAP-25 mutant are likely
to be contained mostly, if not entirely, in the insulin secretion
granule compartment. Furthermore, these treatments are unlikely to have
caused a significant effect on insulin synthesis or nascent insulin
content in other cellular compartments.

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|
Figure 7. Total Insulin Content in Transiently Transfected
HIT-T15 Cells
HIT-T15 cells were treated and transiently transfected with either the
empty vector for control (in panel A) or the constructed vectors of
BoNT/A (in panel B) or SNAP-25 (amino acids 1197) mutant (in panel C)
as in Fig. 6 . The cells were then harvested in 1 M acetic
acid, lysed by two freeze-thaw cycles, and appropriately diluted, and
insulin was determined by RIA.
|
|
 |
DISCUSSION
|
---|
Insulin exocytosis in the islet cell shares many attributes with
neurotransmitter release including the presence of neuronal SNAREs
VAMP-2, SNAP-25, and syntaxin 1 in islets and insulinoma cells, each of
which have all been shown to participate in regulating insulin
secretion (7, 8, 9, 10, 26). Although the use of
Clostridial neurotoxins has been instrumental in
gaining insights into the functions of these SNARE proteins in the
cell, the precise mechanism by which these toxins block secretion had
not been clear. We have therefore attempted to examine the molecular
basis for BoNT/A actions in the cell by transient expression of BoNT/A
or the SNAP-25 (1197) mutant in which the C-terminal nine amino acids
are deleted.
Our work has demonstrated that transient transfection of the truncated
version of the SNAP-25 protein (amino acids 1197) identical to the
larger fragment produced by BoNT/A cleavage results in a major
inhibition of insulin secretion, which is at least equivalent to that
caused by transfection of BoNT/A itself. Therefore, the inhibition of
both K+- and glucose-stimulated insulin secretion by BoNT/A
may be due, in part, to the accumulation of the N-terminal amino acid
1197 fragment of SNAP-25, which remains bound to the plasma membrane.
This does not preclude a role of accelerated degradation of the BoNT/A
cleaved N-terminal amino acid 1197 fragment of SNAP-25 as shown in
Fig. 1
, or a role for the smaller C-terminal nine-amino acid cleavage
product release into the cytosol (17), but indicates that these
are not solely responsible for the BoNT/A inhibition. These three
mechanisms could cooperate in vivo to cause inhibition of
insulin release.
The remarkable similarity between the degree of inhibition of insulin
secretion and the frequency of transfection suggests that within the
individual transfected cells, secretion may have been completely
inhibited. This inhibition of insulin release is considerably greater
than that observed with BoNT/A by Sadoul et al. (9) using a
permeabilized assay evoked by Ca2+ stimulation, but similar
to that reported by Boyd et al. (23) using the
electroporation technique. The likely explanation for these differences
is that electroporation causes only a transient permeabilization of the
cells, thereby avoiding a rundown of cytosolic proteins, and, in
addition, allows a more prolonged and effective action of the
internalized neurotoxin. Taken together, these results demonstrate that
the N-terminal 1197 domain of SNAP-25 acting at its native t-SNARE
location on the plasma membrane is capable of inhibiting insulin
secretion.
Several lines of evidence suggest that components of the SNARE complex
may interact directly with the Ca2+ channels. Bokvist
et al. (27), using the patch clamp technique, suggested that
the insulin granules and the L-type channel are in close proximity, if
not colocalized, in the ß-cell. This is analogous to the situation in
the neuron where the SNARE core complex has been shown to directly
interact with the neuronal N-type channel (18). Furthermore, Wiser
et al. (19) found coexpression of either N-type or L-type
Ca2+ channels with SNAP-25 in Xenopus oocytes
resulted in inhibition of inward Ca2+ current. Thus, the
proximity between SNAP-25 and the L-type channel raises the possibility
that the products of BoNT/A action may disrupt Ca2+ channel
activation. However, our microfluorimetry studies demonstrated that
basal cytosolic Ca2+ levels, as well as
K+-evoked Ca2+ influx determined over at least
3 min of continued perfusion, were identical between cells expressing
either BoNT/A or SNAP-25 (1197) mutant constructs, and control
HIT-T15 cells. Taken together, these studies indicate that the
inhibitory effects on insulin secretion by the modified SNAP-25
proteins are likely to be independent of the ß-cell Ca2+
influx channels.
Thus, the expression of either BoNT/A or the truncated SNAP-25 (1197)
cleavage product may lead to the same result: the accumulation of
products that compete with the endogenous SNAP-25 for binding to
syntaxin and VAMP during the processes of docking and/or fusion.
Syntaxin binds to a region within the N-terminal 100 amino acids of
SNAP-25, whereas the C terminus of SNAP-25 binds to VAMP-2 (5, 6, 15).
In vitro studies have demonstrated that either a
BoNT/A-cleaved SNAP-25 protein or a recombinant truncated SNAP-25
protein corresponding to our SNAP-25 (amino acids 1197) mutant
weakened the binding of these truncated SNAP-25 proteins to VAMP-2
compared with the native SNAP-25 but did not affect their binding to
syntaxin 1. These truncated SNAP-25 proteins therefore remained able to
assemble into a ternary complex with VAMP-2 and syntaxin 1, but this
complex had a reduced ability to assemble in an SDS-resistant form. The
SDS-resistant property of the SNARE complex may reflect a low-energy
state for the protein complex required for subsequent functional
activation by the action of
-soluble
N-ethylmaleimide-sensitive attachment protein (
SNAP) and
N-ethylmaleimide-sensitive factor (NSF). Biophysical studies
are now required to determine how the loss of the C-terminal nine amino
acids of SNAP-25 can alter its function so profoundly.
 |
MATERIALS AND METHODS
|
---|
Materials
The HIT cell subclone T15 was a gift from Dr. P. Robertson
(University of Minnesota, Duluth, MN). Plasmid cDNAs for full-length
BoNT/A-light chain and SNAP-25 were generous gifts from H. Niemann
(Hanover, Germany) and M. Wilson (La Jolla, CA), respectively.
Generation of Expression Construct and Transfection of Cells
For the SNAP-25 mutant, a sense oligonucleotide primer
(5'-ACTACCATGGCCGAGGACGCAGACATG-3') and antisense primer
(5'-CTATTGGTTGGCTTCATCAATTCTGGT-3') were generated according to the
published sequence. The PCR products were cloned into PCRII vector
(Invitrogen, San Diego, CA) and then subcloned into a pcDNA3 vector.
The identity of the PCR products of the predicted size was confirmed by
DNA sequencing using T7 polymerase (Pharmacia, Piscataway, NJ).
Plasmids containing the corresponding full length SNAP-25 wild-type
(1206) and Botulinum A-light-chain cDNAs were subcloned into pcDNA3
as with the SNAP25 mutant (amino acids 1197). The HIT-T15 cells were
grown at 37 C in 5% CO2/95% air in RPMI 1640 medium
supplemented with 20 mM glutamine, 10% FCS (GIBCO,
Gaithersburg, MD), penicillin (100 U/ml), and streptomycin (100 mg/ml).
After the cells were grown to a density of 3 x 106
cells per well in 12-well (20 mm) plates, these cells were transiently
transfected with 2 µg plasmid DNA in Optimum medium the following day
using Pfx-1 lipid (Perfect Lipid, Invitrogen) at a DNA/lipid ratio of
1:6 (wt/wt). After a 4-h incubation at 37 C in 5% CO2,
normal RPMI 1640 medium was added. The medium was discarded the
following day and replaced with fresh RPMI 1640 medium. Transfection
efficiency was determined by using pcDNA3/His/Lac Z (Invitrogen) as a
control plasmid for transfections followed by determination of
ß-galactosidase expression, and by counting the cells that
overexpressed the transfected proteins in the confocal microscopy
studies.
Immunoblotting
Affinity-purified rabbit antisera generated against the
recombinant full-length SNAP-25 was a generous gift from M. Bennett
(University of California, Berkeley, CA). This antibody has been
previously reported and validated to be specific for SNAP-25 (8, 28).
SDS-PAGE and immunoblotting were performed as previously described (8).
Protein concentrations of each cell lysate sample were initially
determined by the Bio-Rad (Richmond, CA) protein assay kit. The cell
lysates were then dissolved in sample buffer and the proteins separated
on a 15% polyacrylamide gel. These proteins were then transferred to a
nitrocellulose membrane, and the blots were then incubated with the
primary antibody (anti-SNAP 25 antibody, 1:1000 dilution in 1% BSA,
0.05% Tween-20 in PBS) for 1.52 h at room temperature.
Immunodetection was by enhanced chemiluminescence (ECL) (Amersham,
Arlington Heights, IL).
Insulin Secretion
Twenty four hours after transfection, the media were changed and
then further incubated for an additional 24 h. These cells were
cultured to
80% confluence before stimulation.
Potassium (K+) and Glucose
Stimulation
K+ stimulation was conducted according to a slightly
modified method described by Boyd et al. (23). The
transfected or control cells were washed and then incubated in 0.5 ml
low K+-KRB buffer containing 4.8 mM KCl, 129
mM NaCl, 5 mM NaHCO3, 1.2
mM KH2PO4, 1 mM
CaCl2, 1.2 mM MgSO4, 0.5
mM glucose, and 10 mM HEPES, pH 7.4, for
0.5 h. The media were then discarded and then exchanged with 0.5
ml of the same buffer containing either low K+ (4.8
mM KCl, for basal levels) or high K+ in which
the KCl concentration was increased to 30 mM and NaCl was
reduced to 104 mM for 1 h at 37 C. For glucose
stimulation, the wells were exposed to the KRB buffer (with 4.8
mM KCl) in which glucose was either removed (for basal
levels) or increased to 10 mM, and incubation was carried
out for 1 h at 37 C. At the end of incubation, the media were
collected and then centrifuged at 3000 x g for 3 min
to remove detached cells, and the resulting upper half of the
supernatants were collected. Both supernatants and some the cells
(collected by scraping the plates) were frozen at -20 C. Insulin in
the samples was then assayed within 1 week using a rat insulin RIA kit
(Linco, St. Charles, MO) according to manufacturers instructions.
Appropriate dilutions of the samples were carried out to allow the
values to fall into the standard curve.
Because of the inherent variability of transfection efficiency and the
effects of the passage of the cell lines, cells from all wells in each
experiment performed were always from the same plate of cells. Every
experiment always included all of the variables indicated in each
figure being compared, and at least three to five independent
experiments with triplicate or four replicate wells per experimental
variable were performed. Furthermore, the protein concentrations of
each well, determined to be 392 ± 10.4 (n = 8 independent
experiments), and confluence of cells in each well (
80%) were
nearly identical within and between experiments. Values shown in the
figures are the mean increase above basal levels, which were also
determined in triplicate wells for each experiment. The data are
expressed as means ± SEM of the total number of wells
and analyzed by Students t test. A P value
<0.05 indicates that the difference is significant.
Confocal Immunofluorescence Microscopy
Microscopy was performed as previously described (8, 28).
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-SNAP-25 antibody) overnight at 4 C. After
rinsing with 0.1% saponin/PBS, the coverslips were then incubated with
the appropriate rhodamine-labeled secondary antiserum for 1 h at
room temperature and then mounted on slides in a fading retarder, 0.1%
p-phenylenediamine in glycerol, and examined using a laser
scanning confocal imaging system (Zeiss, Oberkochen, Germany).
Transfected cells were identified by coexpression and visualization of
the GFP (Clontech, Palo Alto, CA).
Single-Cell [Ca2+]i Determinations
Cells plated on polylysine-coated coverslips were loaded with
0.5 µM fura-2 AM in serum-free medium for 20 min at 37 C,
washed, and then further incubated in dye-free medium for an additional
10 min at 37 C. The coverslip was then mounted on a chamber and
perfused continuously at 25 C with the test media, which uniformly
contain 5 mM NaHCO3, 1.2 mM
MgSO4, 2.8 mM glucose, 10 mM HEPES,
pH 7.4, but with variable concentrations of
K+/Na+ [4.8 mM KCl with 129
mM NaCl for low K+ buffer, and 30
mM KCl/103.8 mM NaCl for high K+
buffer] and Ca2+ [0 or 1 mM
CaCl2]. Quantitative fluorescence imaging of single cell
[Ca2+]i at low light levels was then performed in a
manner similar to what we had previously described for pancreatic
acinar cells (29) using an Axiovert 100 inverted microscope (Zeiss)
coupled to a cooled CCD camera (Princeton Instruments Inc., Princeton,
NJ). Fluorescent images (excitation, 340 and 380 nm; emission, 510 nm)
were acquired every 2030 sec, and ratios of fluorescence intensities
were calculated for each cell (1520 cells per field per coverslip)
after subtracting the background fluorescence using Metafluor software
(Universal Imaging Corp., West Chester, PA). For each condition, two to
three independent experiments with three to four separate coverslips
per experiment or a total of at least seven coverslips per condition
were performed. The fluorescent ratio values of the cells in each
coverslip (1525 cells per field) were integrated, and the mean of
these values was determined at basal, 0.5 min, and 3 min after
stimulation with the high K+ medium. The final data
expressed are the mean ± SEM of the mean values
obtained from each coverslip. Transfection efficiency for each
experiment was confirmed to be
5080% by coexpression and
visualization of either the GFP or ß-galactosidase protein in a
control coverslip.
 |
ACKNOWLEDGMENTS
|
---|
Electron Microscopy
Forty eight hours after transfection, both transfected cells and
control cells (untransfected or transfected with pcDNA3/His/Lac Z) were
fixed with 0.5% glutaraldehyde/4% paraformaldehyde in 0.1
M phosphate buffer (pH 7.2) for 20 min at room temperature,
followed by osmication with 1% aqueous OsO4 for 30 min.
The samples were then dehydrated according to standard procedures and
embedded in Epon 812 (Electron Microscopy Sciences, Fort Washington,
PA). Thin sections were stained with uranyl acetate for 5 min and then
treated with lead citrate for 5 min and observed with a H600
transmission electron microscope (Hitachi, Tokyo, Japan) at 80 kV at a
uniform magnification of 6600x. Twenty three micrographs were randomly
taken for each condition from three independent experiments by a
morphology technician not involved with our laboratory. To determine
the granule density distribution within each cell, a method similar to
that described by Gutierrez et al. (17) in chromaffin
cells using the NIH Image analysis program and Adobe Photoshop (Adobe
Systems Inc., San Jose, CA) software were used. Insulin granules,
identified as a dense core granule with a distinct membrane, were
counted within a bin width of 0.45 µm from the plasma membrane to the
nucleus, with the plasma membrane at 0 µm and the nuclear membrane at
3.6 µm. In our study, we further determined the surface area within
each of the 0.45 µm bin width of cytosolic space using the same NIH
Image program, and the number of granules was expressed as a function
of this cytosolic surface area. A secretory granule density
distribution histogram was then constructed from these data in Fig. 6
.
Significance was measured by Students t test.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Herbert Y. Gaisano, Room 7226 Medical Sciences Building, University of Toronto, Toronto, Ontario, Canada M5S 1A8. E-mail: herbert.gaisano{at}utoronto.ca
Supported by grants from Canadian Diabetes Association, Juvenile
Diabetes Foundation, Eli Lilly-Banting and Best Diabetes Research
Program at the University of Toronto (to H.Y.G. and M.B.W.) and the
Medical Research Council of Canada (MT-13169 to H.Y.G. and W.S.T.). H.
Gaisano is a recipient of the American Gastroenterology
Association/Industry (Pharmacia and Upjohn) Research Scholar Award.
Received for publication September 30, 1997.
Revision received January 22, 1997.
Accepted for publication March 9, 1998.
 |
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