©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Effect of Botulinum Neurotoxins on the Release of Insulin from the Insulinoma Cell Lines HIT-15 and RINm5F (*)

(Received for publication, March 31, 1995; and in revised form, May 26, 1995)

Robert S. Boyd (§) Michael J. Duggan Clifford C. Shone (1) Keith A. Foster

From the From Speywood Pharmaceuticals Ltd., Porton House, 1 Bath Road, Maidenhead, Berkshire SL4 4UH, United Kingdom and Division of Biologics, Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire SP4 OJG, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Western blotting of the insulin-secreting beta-cell lines HIT-15 and RINm5F with anti-SNAP-25 (synaptosomal associated protein of 25 kDa), anti-synaptobrevin, and anti-syntaxin 1 antibodies revealed the presence of proteins with the same electrophoretic mobility as found in neural tissue. Permeabilization of both of these insulinoma cell lines to botulinum neurotoxin A by electroporation resulted, after 3 days of culture, in the loss of 90% of SNAP-25 immunoreactivity. A similar permeabilization of these cells with botulinum neurotoxin B resulted in the cleavage of 90% of the synaptobrevin-like immunoreactivities. Botulinum neurotoxin F also cleaved 90% of the synaptobrevin-like immunoreactivity in RINm5F cells. The permeabilization of both insulinoma cells to neurotoxin A resulted in a >90% inhibition of potassium-stimulated, calcium-dependent insulin release. By contrast, permeabilization of the insulinoma cell lines to neurotoxin B resulted in only a 60% inhibition of potassium-stimulated insulin release in HIT-15 cells, and neither neurotoxin B nor F caused inhibition in RINm5F cells. Thus HIT-15 and RINm5F cells contain the components of the putative exocytotic docking complex described in cells derived from the neural crest. In HIT-15 cells both SNAP-25 and synaptobrevin appear to be involved in calcium-dependent insulin secretion, whereas in RINm5F cells SNAP-25 but not synaptobrevin is involved.


INTRODUCTION

The clostridial neurotoxins, botulinum and tetanus, inhibit neurotransmitter release from presynaptic nerve endings (1) by Zn-dependent proteolysis of specific synaptic proteins(2) . The different botulinum neurotoxin serotypes specifically cleave one of three proteins, synaptobrevin, syntaxin, or SNAP-25. (^1)A recent model of synaptic vesicle fusion proposes that the vesicle protein synaptobrevin, termed a v-SNARE, and the plasma membrane-associated proteins syntaxin and SNAP-25, t-SNARES, interact to form a receptor for the assembly of a fusion complex involving the N-ethylmaleimide-sensitive fusion protein(3, 4, 5) . Botulinum neurotoxin type A (BoNT/A) selectively cleaves SNAP-25(6) , whereas botulinum neurotoxin type B (BoNT/B), in common with tetanus neurotoxin (7) and botulinum neurotoxin F (BoNT/F), cleave synaptobrevin(8, 9) . Two forms of synaptobrevin, synaptobrevin 1 and 2, have been described in mammals. In most species both forms are substrates for BoNT/B; however, in the rat synaptobrevin 1 is not a substrate(10) . Synaptobrevin 1 and 2 in the rat are substrates for BoNT/F(9) . A homologue of synaptobrevin, cellubrevin, has been identified in non-neuronal cells and is a toxin substrate(11) .

The specificity of clostridial neurotoxins for nerve terminals, particularly the cholinergic terminals of the neuromuscular junction, derives from a selective uptake mechanism(12, 13) . When the uptake barrier is removed clostridial neurotoxins inhibit secretion from not only neuronal cells but also from PC12 (14) and chromaffin cells(15, 16) . Synaptobrevin, cellubrevin, SNAP-25, and syntaxin are found in PC12 and chromaffin cells(16, 17, 18) , and inhibition of noradrenaline release from chromaffin cells has been associated with cleavage of SNAP-25 by BoNT/A (17) and cleavage of synaptobrevin and cellubrevin by BoNT/B(16) .

Pancreatic endocrine cells also contain SNAP-25, synaptobrevin, cellubrevin, and syntaxin 1A, 4, and 5(19) . If these proteins are involved in Ca-dependent secretion from these cells, then their cleavage by botulinum toxins should inhibit Ca-dependent release of hormones or enzymes from pancreatic cells. Indeed cleavage of synaptobrevin by tetanus toxin inhibits enzyme secretion from rat pancreatic acinar cells(20) .

We have confirmed the presence of SNAP-25, syntaxin, and synaptobrevin immunoreactivities in the beta-pancreatic cell lines, HIT-15 and RINm5F, and determined the effect of BoNT/A, -B, and -F both on these proteins and on the release of insulin. To study the effects of the toxins in these cells it was necessary to introduce them into the cell cytoplasm, and electroporation was used for this purpose(21) .


EXPERIMENTAL PROCEDURES

Materials

BoNT/A, BoNT/B, and BoNT/F were purified as described previously(22, 23, 24) . The rat insulin radioimmunoassay kit and ECL reagents were obtained from Amersham International plc. The rabbit polyclonal antiserum used to detect SNAP-25 immunoreactivity was raised using a synthetic peptide of sequence CANGRATKMLGSG following published methods(25, 26) . A mouse monoclonal antibody against SNAP-25, SMI 81, was obtained from Affiniti Research Products Ltd. (Nottingham, UK). The polyclonal antibody used to detect synaptobrevin immunoreactivity was raised in guinea pig against a synthetic peptide (HV62) comprising amino acids 33-94 from the conserved cytoplasmic domain of human synaptobrevin 2(24) . This antibody has been shown to recognize sequences common to both synaptobrevin I and II. The anti-syntaxin antibody, 10H5, was a gift from Prof. Takahashi (Mitsubishi Kasei Institute, Tokyo, Japan). HIT-15 and RINm5F cells were a gift from Dr. Irene Green (Sussex University, Brighton, UK). Electroporation cuvettes were from Bio-Rad. The SDS-polyacrylamide gel electrophoresis gels and nitrocellulose were from R& Systems Europe Ltd. (Abingdon, UK). All other reagents and chemicals used in this study were obtained from Sigma Chemical Co. Ltd. (Fancy Road, Poole, UK).

Cell Culture

HIT-15 cells and RINm5F cells were cultured in 80-cm^2 flasks in DMEM containing 2% (v/v) fetal calf serum (HIT-15 cells) or 10% (v/v) fetal calf serum (RINm5F cells). Cultures were maintained at 37 °C in a humidified atmosphere of 95% air and 5% CO(2).

Electroporation

Cells (HIT-15 or RINm5F) were selected when they were subconfluent and harvested using cell dissociation reagent (Sigma). After washing in phosphate-buffered saline the cells were resuspended in DMEM and 0.1% (w/v) bovine serum albumin at a density of 2-5 million cells/ml, and 0.8 ml was added to each of a number of 0.4-cm electroporation cuvettes. Toxins were added as required, and the cells were then immediately electroporated at 960 microfarads and 0.28 kV using a Bio-Rad electroporator(21) . After electroporation the cells were washed three times in DMEM and bovine serum albumin by centrifugation (1000 g for 5 min). The cells were then resuspended in the appropriate medium containing antibiotics and plated out at a density of 0.3-0.6 million cells/well in a 24-multiwell plate. The cells were cultured for 3 days at 37 °C in a humidified atmosphere of 95% air and 5% CO(2).

Cell Secretion Experiments

After 3 days in culture the cells were washed three times with 1 ml of DMEM. Secretion of insulin was then assessed by incubation of the cells for 30 min at 37 °C in a humidified atmosphere of 95% air and 5% CO(2) with 0.3 ml of Krebs-Ringer bicarbonate (KRB) buffer containing 129 mM NaCl, 5 mM NaHCO(3), 4.8 mM KCl, 1.2 mM KH(2)PO(4), 1.0 mM CaCl(2), 1.2 mM MgSO(4), 2.8 mM glucose, and 10 mM HEPES, pH 7.4 (27) or in HK-KRB in which KCl was increased to 30 mM and NaCl was reduced to 103.8 mM. In some experiments calcium was omitted from the KRB buffer, and EGTA was added to a final concentration of 0.1 mM, Ca-free KRB. At the end of the experimental incubation the supernatants were removed and centrifuged at 13,000 g for 4 min. The supernatants and cells were frozen at -80 °C until analysis. Insulin was assayed using the Amersham International plc rat insulin radioimmunoassay kit according to the manufacturer's instructions.

Western Blot Analysis

Hydrophobic proteins were extracted from the cells remaining after the secretion experiment by a modification of the method of Bordier(28) . The cells in each well were treated with 0.5 ml of 0.2 M NaOH for 10 min at room temperature before being neutralized with 0.5 ml of 0.2 M HCl. The mixture was then cooled to 4 °C, and 100 µl of Triton X-114 (10%, v/v) was added, mixed three times, and left for 1 h at 4 °C. The resulting solution was then centrifuged at 13,000 g for 10 min at 4 °C, and the supernatant was incubated in a water bath at 37 °C for 30 min until two distinct phases formed. After separation by centrifugation the upper phase was removed and the lower phase containing hydrophobic proteins was retained. The samples were prepared for SDS-polyacrylamide gel electrophoresis by precipitation with chloroform/methanol, and the precipitate was dissolved in 20 µl of sample buffer.

The proteins were resolved on a 4-20% Tris/glycine polyacrylamide gel and then transferred to nitrocellulose membranes. The blots were probed with antibodies and the immunoreactivities visualized using chemiluminescent detection essentially as described previously(26) .

Data was quantified from Hyperfilm ECL (Amersham International plc) using a Molecular Dynamics personal densitometer, and the effect of any treatment was expressed as a percentage of the control value for each experiment. The results were within the linear range of the technique.


RESULTS

The efficiency of electroporation was determined by the use of a fluorescein-dextran molecule of comparable molecular weight to the toxin molecule. Electroporation was found to permeabilize >95% of both the HIT-15 and RINm5F cells with only limited effects on cell viability and no significant difference between the basal levels of insulin release and protein in control and electroporated cells.

Both HIT-15 and RINm5F cells were found to contain a SNAP-25-immunoreactive protein of M(r) 30,000 as determined by two separate antibodies. Both insulinoma cells contained two synaptobrevin-immunoreactive proteins of M(r) 16,000-17,000. The lower synaptobrevin immunoreactivity was less intense in both cell lines, and in the HIT-15 cells it was not always clearly visible. A protein of M(r) 36,000 with syntaxin immunoreactivity was also detected in both cell lines (Fig. 1).


Figure 1: Western blot analysis of cells electroporated in the presence of botulinum neurotoxins. Duplicate Triton X-114 extracts of HIT-15 (HIT) and RINm5F (RIN) cells that had been electroporated in the absence of neurotoxin (C) or in the presence of BoNT/A (A) or BoNT/B (B) were subjected to Western blot analysis as detailed under ``Experimental Procedures.'' The same blots were consecutively probed with anti-synaptobrevin (SYB), anti-SNAP-25 (SNAP), and 10H5 (SYNT). The relative electrophoretic mobilities (determined by comparison to prestained protein standards) were 16,000-17,000, 30,000, and 36,000, respectively.



Electroporation of the HIT-15 and RINm5F cells with BoNT/A (500 nM) resulted in a reduction of SNAP-25 immunoreactivity after 3 days of culture as assessed using the anti-peptide antibody (Fig. 1). In HIT-15 cells 6.4 ± 5.3% of the protein remained compared with control (n = 3), and in RINm5F cells 14.5 ± 13.5% of the protein remained compared with control (n = 3). Use of the monoclonal antibody, SMI 81, revealed a small shift in electrophoretic mobility to M(r) 29,000; this is consistent with the known site of proteolysis of SNAP-25 by BoNT/A, which results in the loss of a 9-residue peptide from the C terminus of the protein(6) . Electroporation of the HIT-15 and RINm5F cells with BoNT/B (500 nM) resulted in a reduction of both synaptobrevin-like immunoreactivities after 3 days of culture (Fig. 1). In HIT-15 cells 7.8 ± 2.9% of the protein remained compared with control (n = 3), and in RINm5F cells 12.3 ± 4.8% of the protein remained compared with control (n = 4).

In Ca-containing KRB, high K treatment significantly stimulated insulin release from both the HIT-15 and RINm5F cells ( Fig. 2and Fig. 3). High K did not stimulate insulin release from RINm5F cells in Ca-free KRB (94.8 ± 7% of basal release; means ± range of two determinations).


Figure 2: The effect of botulinum toxins A and B on calcium-dependent insulin release from HIT-15 cells. HIT-15 cells were untreated (Con), electroporated (EP-Con), electroporated in the presence of 500 nM BoNT/A (EP-A), or electroporated in the presence of 500 nM BoNT/B (EP-B). The cells were subsequently cultured for 3 days. The release of insulin from the cells during a 30-min incubation period with 4.8 mM KCl-KRB (LK-KRB) or 30 mM KCl-KRB (HK-KRB) was determined by specific radioimmunoassay. The secretion data presented are the means ± S.E. of three experiments, each performed in duplicate; one star indicates p < 0.05 and two stars indicate p < 0.02 for differences from control, determined by a two-tailed, unpaired t test.




Figure 3: The effect of botulinum toxins A and B on calcium-dependent release of insulin from RINm5F cells. RINm5F cells were untreated (Con), electroporated (Con-EP), electroporated in the presence of 500 nM BoNT/A (EP-A), electroporated in the presence of 500 nM BoNT/B (EP-B), or electroporated in the presence of 250 nM of BoNT/F (EP-F). The cells were subsequently cultured for 3 days. The release of insulin from the cells during a 30-min incubation period with 4.8 mM KCl-KRB (LK-KRB) or 30 mM KCl-KRB (HK-KRB) was then determined by specific radioimmunoassay. The secretion data presented are the means ± S.E. of n separate experiments, each performed in duplicate; three stars indicate p < 0.001 for differences from control, determined by a two-tailed, unpaired t test.



After 3 days in culture following electroporation in the presence of BoNT/A (500 nM), high K-stimulated insulin release from HIT-15 cells was reduced by 95.0% (p < 0.02; n = 4; Fig. 2). Basal or high K-stimulated insulin release from HIT-15 cells was not significantly affected by electroporation perse. Electroporation of HIT-15 cells with heat-treated BoNT/A did not significantly affect high K-stimulated insulin release (data not shown). After 3 days of culture following electroporation of BoNT/B (500 nM) into HIT-15 cells, high K-stimulated insulin release was reduced by 63% (p < 0.05; n = 3). This partial inhibition of high K-stimulated insulin release was not increased by electroporation of cells with higher levels of BoNT/B (2.5 µM; data not shown).

As with the HIT-15 cells, electroporation of RINm5F cells did not significantly affect subsequent basal or high K-stimulated insulin release (Fig. 3). Electroporation of RINm5F cells with BoNT/A (500 nM) resulted, after 3 days in culture, in the inhibition of insulin release by 91.3% (p < 0.001; n = 5). However, after 3 days in culture following electroporation of the RINm5F cells with BoNT/B (500 nM), there was no significant block of high K-stimulated insulin release nor did electroporation of the cells in the presence of a much higher concentration of BoNT/B (2.5 µM) have any effect (data not shown).

Electroporation of RINm5F cells with BoNT/F (250 nM) caused a reduction in the synaptobrevin immunoreactivities equivalent to that caused by BoNT/B; 6.2 ± 4% of the protein remained compared with control (n = 2, mean ± range). As observed for BoNT/B, insulin secretion was unaffected by the BoNT/F treatment (Fig. 3).


DISCUSSION

A docking/fusion complex composed of SNAP-25, syntaxin, and synaptobrevin has been proposed as an essential step in exocytosis(3) . In this study we show that all these proteins are present in the pancreatic cell lines studied. The detection of a syntaxin-like immunoreactive band is consistent with the reported expression of syntaxin 1A in cells of this type (19) and the known specificity of this monoclonal antibody(30) . BoNT/A, which cleaves SNAP-25 in both neurons and chromaffin cells, also cleaves the SNAP-25 immunoreactive protein in the HIT-15 and RINm5F insulinoma cells. None of the other proteins studied was found to be cleaved by BoNT/A. In addition to cleavage of SNAP-25, electroporation with BoNT/A was found to inhibit high K-stimulated insulin secretion in both cell lines, which is in agreement with the recent findings in pancreatic cells (29) . The inhibition of calcium-dependent insulin release was almost complete in both cell lines, consistent with the observation that the majority of the SNAP-25 immunoreactivity had disappeared from the cells. These results are consistent with the involvement of a SNARE-based mechanism involving SNAP-25 in insulin secretion.

Two synaptobrevin-like immunoreactivities were observed in this study. Both were equally sensitive to proteolysis by BoNT/B or BoNT/F. Given that one of the cell lines, RINm5F, is of rat origin, the similar extent of cleavage of both immunoreactivities by BoNT/B or BoNT/F is consistent with this cell line containing synaptobrevin 2 and cellubrevin but not synaptobrevin 1, a conclusion in agreement with a recent report(19) . The identification of the two synaptobrevin immunoreactivities as synaptobrevin 2 and cellubrevin is consistent with the electrophoretic mobilities observed.

The degree of cleavage of synaptobrevin in bovine chromaffin cells has been found to be closely correlated with the degree of inhibition of calcium-stimulated secretion(16) . In this study, however, despite the almost total cleavage of synaptobrevin and cellubrevin seen, only in HIT-15 cells was a blockade of insulin release detected, and this was only partial. As discussed above, insensitivity of Ca-dependent insulin secretion from RINm5F cells to BoNT/B is not due to the presence of synaptobrevin 1 in these cells nor would this explain the lack of inhibition by BoNT/F.

Thus data for SNAP-25 supports the involvement of a SNARE complex in insulin secretion; however, for synaptobrevin or cellubrevin to be the v-SNARE in RINm5F cells would require them to be present in at least a 10-fold excess. Alternatively, there could be an unidentified v-SNARE in these cells, which is not a substrate for either BoNT/B or BoNT/F and which is not recognized by the HV62 antiserum. Whichever explanation proves to be the case, the results presented show that there is no simple relationship between the presence of synaptobrevin 2 and SNAP-25 in cells and the mechanism of Ca-dependent secretion.


FOOTNOTES

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

§
To whom correspondence should be addressed. Tel.: 44 628 771447; Fax: 44 628 770199.

^1
The abbreviations used are: SNAP-25, synaptosomal associated protein of 25 kDa; BoNT, botulinum neurotoxin; DMEM, Dulbecco's modified Eagle's medium; HK-KRB, high potassium Krebs-Ringer bicarbonate buffer; LK-KRB, low potassium Krebs-Ringer bicarbonate buffer; v-SNARE and t-SNARE, vesicle and target-soluble, respectively, N-ethylmaleimide-sensitive factor-associated protein receptor.


ACKNOWLEDGEMENTS

We are grateful to Prof. Masami Takahashi for the anti-syntaxin antibody, 10H5, to Dr. I. Green for providing the insulinoma cell lines, and to A. Jen for culturing them. We thank Dr. J. R. North for critically reading the manuscript and Prof. J. O. Dolly and Prof. B. Gomperts for helpful discussions.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.