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Originally published In Press as doi:10.1074/jbc.M004284200 on August 9, 2000

J. Biol. Chem., Vol. 275, Issue 46, 36079-36085, November 17, 2000
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Synaptotagmin III/VII Isoforms Mediate Ca2+-induced Insulin Secretion in Pancreatic Islet beta -Cells*

Zhiyong GaoDagger, John Reavey-CantwellDagger, Robert A. Young, Patricia Jegier, and Bryan A. Wolf§

From the Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

Received for publication, May 18, 2000, and in revised form, July 24, 2000


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Synaptotagmins (Syt) play important roles in Ca2+-induced neuroexocytosis. Insulin secretion of the pancreatic beta -cell is dependent on an increase in intracellular Ca2+; however, Syt involvement in insulin exocytosis is poorly understood. Reverse transcriptase-polymerase chain reaction studies showed the presence of Syt isoforms III, IV, V, and VII in rat pancreatic islets, whereas Syt isoforms I, II, III, IV, V, VII, and VIII were present in insulin-secreting beta TC3 cell. Syt III and VII proteins were identified in rat islets and beta TC3 and RINm5F beta -cells by immunoblotting. Confocal microscopy showed that Syt III and VII co-localized with insulin-containing secretory granules. Two-fold overexpression of Syt III in RINm5F beta -cell (Syt III cell) was achieved by stable transfection, which conferred greater Ca2+ sensitivity for exocytosis, and resulted in increased insulin secretion. Glyceraldehyde + carbachol-induced insulin secretion in Syt III cells was 2.5-fold higher than control empty vector cells, whereas potassium-induced secretion was 6-fold higher. In permeabilized Syt III cells, Ca2+-induced and mastoparan-induced insulin secretion was also increased. In Syt VII-overexpressing RINm5F beta -cells, there was amplification of carbachol-induced insulin secretion in intact cells and of Ca2+-induced and mastoparan-induced insulin secretion in permeabilized cells. In conclusion, Syt III/VII are located in insulin-containing secretory granules, and we suggest that Syt III/VII may be the Ca2+ sensor or one of the Ca2+ sensors for insulin exocytosis of the beta -cell.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Insulin exocytosis from the beta -cell of the islets of Langerhans is stimulated by various physiological secretagogues that include glucose, amino acids, and receptor-mediated agonists such as acetylcholine, cholecystokinin, and glucagon like-peptide 1 (1-7). A common mechanism of action for these secretagogues is to cause an increase in cytosolic Ca2+. Elevation of intracellular Ca2+ is due to an influx of extracellular Ca2+ through voltage-dependent L-type Ca2+ channel and/or mobilization of intracellular Ca2+ from the endoplasmic reticulum (8-17). However, the mechanisms by which Ca2+ induces insulin granule fusion with the plasma membrane of beta -cell remain unclear (1, 16, 18, 20, 21).

Synaptotagmin (Syt)1 is a family of membrane proteins initially found to be expressed in brain. At the present, 11 members of Syt have been identified (22, 23). The Syt molecule has a single transmembrane domain and two Ca2+ regulatory C2 domains. The C2 domains mediate Ca2+-dependent and Ca2+-independent interactions with target molecules that may regulate membrane fusion and membrane budding reactions (24, 25). Literature concerning the expression and functions of Syt in pancreatic beta -cell is very limited and contradictory. In an earlier study (26), Syt was found in the non-beta -cell of the islet mantle, but not in the beta -cell, using a non-isoform-specific antibody, and the mRNAs of Syt A and B were absent in mouse pancreatic beta -cell and RINm5F cells as demonstrated by in situ hybridization. Recently, the mRNA and protein of Syt isoforms I and II (27) were found in insulin-secreting beta -cell lines RINm5F, INS-1, and HIT-T15. It was reported that Syt I, II (27), and Syt III proteins (28) are localized mainly in insulin-containing secretory granules. However, only the mRNA but not the protein was detected in primary islet beta -cells (27). In other studies, Syt III mRNA was present in MIN6, RINm5F, HIT-T15, and beta TC6-f7 (29) cells and pancreatic islets (28), and the protein expression of Syt III in MIN6 cell and pancreatic islets was confirmed by one group (28) but not by another (29). The aims of the current study were to examine the expression of various Syt isoforms in pancreatic islets as well as insulin-secreting beta -cell lines, the subcellular localization of Syt, and the functional role of Syt in insulin exocytosis using a beta -cell line overexpressing Syt.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Insulin-secreting beta -Cell Lines and Islet Preparation-- The insulin-secreting mouse beta -cell line beta TC3 and rat beta -cell line RINm5F were maintained in culture as described previously (17, 30-33). In brief, cells were maintained in RPMI 1640 containing 11 mM glucose (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (HyClone, Logan, UT), 100 units/ml penicillin, 50 µg/ml streptomycin and incubated at 37 °C in a 5% CO2, 95% air humidified incubator. Isolated islets of Langerhans were prepared from Harlan Sprague-Dawley rats by collagenase digestion and Ficoll purification as described previously (34-36). In some experiments, single islet cells were prepared by digesting islets in Ca2+-free and Mg2+-free PBS containing 1 mM EGTA, 1% BSA, and trypsin at 30 °C for 10 min with continuous trituration (37).

Generation of Stable beta -Cells Overexpressing Synaptotagmin III or VII-- The plasmids with the insert of Syt III or Syt VII cDNA under the control of cytomegalovirus promoter were generously provided by Dr. Thomas Sudhof at University of Texas Southwestern Medical Center (38-40). RINm5F cells were transfected with the plasmid using cationic liposome reagent DMRIE-C (Life Technologies, Inc.) as described before (17). Transfected cells were selected with geneticin (Life Technologies, Inc.) for 4 weeks, and the surviving colonies (Syt III and Syt VII beta -cells) were individually picked and transferred to 24-well plates. Syt III and Syt VII protein levels were quantitated by immunoblotting. An empty vector control RINm5F beta -cell line (Neo) was also generated.

RT-PCR Detection of mRNA-- Total RNA was extracted from rat pancreatic islets and beta TC3 and RINm5F cells, using the micro-scale total RNA separator kit (CLONTECH) following the manufacturer's instructions, as described previously (41, 42). The purity and the yield of isolated RNA were determined by monitoring absorbance at 260 and 280 nm. The integrity of the RNA was confirmed by performing agarose denaturing gel electrophoresis. All RNA samples were treated with DNase to eliminate genomic DNA contamination before RT-PCR. The first strand cDNA from 2 µg of total RNA was synthesized using SuperscriptTM II Reverse Transcriptase (purchased from Life Technologies, Inc.) and oligo(dT) 15 primer (500 µg/ml, Life Technologies, Inc.). RNA was denatured at 70 °C for 10 min, followed by a reverse transcription at 42 °C for 60 min and then 75 °C for 10 min.

The primers were designed and synthesized based on the published gene sequence as shown in Table I. The PCR was carried out in a thermal cycler (model 480, PerkinElmer Life Sciences) with an initial denaturation step at 94 °C for 3 min, subjected to 35 cycles of denaturation (94 °C for 1 min), annealing (55 °C for 2 min), elongation (72 °C for 3 min), and a final elongation step at 72 °C for 7 min. PCR products were visualized by 1% agarose gel electrophoresis with ethidium bromide staining.

                              
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Table I
Primers for various Syt isoforms

Generation of Synaptotagmin C-terminal Peptides and Antibodies-- C-terminal peptides of Syt III (CP3) and Syt VII (CP7) were synthesized with the following amino acid sequences, CGGKGLSEKENSE and CSGPGEVKHWKDM, respectively. These peptides were synthesized with an additional cysteine at the N-terminal for cross-linking with m-maleimidobenzoyl-N-hydroxysuccinimide ester and purified by the Protein Chemistry Laboratory of the University of Pennsylvania School of Medicine. CP3 and CP7 were injected into three rabbits/peptide as antigen to generate polyclonal antibodies (Lampire Biological Laboratory). Immune serum was obtained and subjected to IgG Gradifrac purification by protein G. Antiserum was dialyzed, aliquoted, named as Anti-Syt III or Anti-Syt VII, respectively, and stored at -20 °C.

Western Blot Analysis-- Cell lysates from pancreatic islets and beta TC3 and RINm5F cells were prepared, and immunoblotting was performed essentially as described previously using anti-Syt III and anti-Syt VII antibodies described above (17, 43).

Confocal Microscopy-- Islets were dispersed into single cells, washed once with 1 ml of RPMI 1640 medium, and then plated on coverslips and cultured at 37 °C for 2 days before use. RINm5F cells were plated on coverslips and cultured for 1-2 days. Coverslips with islet cells or RINm5F cells were washed twice with ice-cold PBS containing 1% BSA. One ml of -20 °C-precooled 70% methanol was added to the coverslip and incubated at -20 °C for 30 min to permeabilize and fix the cells. Cells were washed twice again with ice-cold PBS, incubated with primary antibody, washed twice, incubated with secondary fluorescent-labeled antibody (Jackson ImmunoResearch Laboratory Inc., West Grove, PA), washed twice, and fixed on glass slides before analysis by confocal laser scan microscopy (Biomedical Imaging Core, DERC, University of Pennsylvania).

Insulin Secretion in Intact beta -Cells-- RINm5F cells were plated in 6-well dishes. On the day of experiment, they were washed 3 times with Krebs-Ringer buffer (KRB: 115 mM NaCl, 24 mM NaHCO3, 5 mM KCl, 1 mM MgCl2, 2.5 mM CaCl2, and 25 mM HEPES, pH 7.4) and preincubated with 1 ml of KRB for 30 min at 37 °C. Cells were then incubated for additional 30 min with 1 ml of fresh KRB containing the tested agents. Samples were taken for insulin measurement by radioimmunoassay (Diabetes Endocrinology Research Center, University of Pennsylvania) (30).

Insulin Secretion in Permeabilized beta -Cells-- Control and Syt III or Syt VII RINm5F cells were plated in 24-well dishes. On the day of experiment, they were washed once with KRB without glucose and Ca2+ and washed once with TES buffer (50 mM TES, 100 mM KCl, 2 mM MgCl2, 1 mM ATP, 0.1% BSA, 3 mM glucose, 1 mM EGTA, pH 7.40). Cells were incubated for 15 min with 0.5 ml of TES buffer containing digitonin as described previously (33, 44). In another set of experiments, cells were washed once with KRB and once with KG buffer (140 mM potassium glutamate, 5 mM ATP, 5 mM NaCl, 7 mM MgSO4, 0.4 mM EGTA, 1% BSA, and 20 mM HEPES, pH 7.4). They were incubated for 15 min with 0.5 ml of KG containing streptolysin O. Trypan blue exclusion test demonstrated that more than 90% of the cells were permeabilized using either method. Cells were then washed and incubated with various agents for 30 min. Samples were taken for insulin measurement by radioimmunoassay.

Data Analysis-- Student's t test was performed when two groups were compared. Analysis of variance was used, followed by the Dunnett test when multiple groups were compared. Differences were considered significant for p < 0.05.

Materials-- All other chemicals were from Sigma and molecular biological reagents were from Life Technologies, Inc.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Identification of Synaptotagmin Isoforms in Islets and Insulin-secreting beta -Cells-- The expression of synaptotagmin mRNA (isoforms I through VIII) in pancreatic islet cells and insulin-secreting cell lines beta TC3 and RINm5F was examined by RT-PCR with specific primers (Fig. 1). mRNAs of Syt isoform III (positive with both sets of primers), IV, V, and VII were found in pancreatic islet cells, whereas isoform I (positive with both sets of primers), II, III (positive with both sets of primers), IV, V, VII, and VIII were found in beta TC3 cells. The rest of the isoforms were not found in islets or beta TC3 cells. Because the affinities for Ca2+ binding of isoforms III and VII are in the low micromolar range (23, 24), they were chosen for further investigation. It was also demonstrated that these two isoforms were present in RINm5F beta -cells.


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  		Fig. 1.   Synaptotagmin isoform expression in pancreatic islet and beta TC3 and RINm5F beta -cells. Total RNA was extracted from isolated rat pancreatic islets (panel a) and beta TC3 cells (panel b) and RINm5F beta -cells (panel c). After DNase treatment of RNA, reverse transcription and polymerase chain reaction was performed with specific Syt primers (I-VIII). PCR products were analyzed on 2% agarose gel and identified with ethidium bromide staining. Representative results are from three experiments.

C-terminal peptides of III (CP3) and VII (CP7) isoforms were then synthesized and used as antigen to generate specific antibodies anti-Syt III and anti-Syt VII for protein studies (Fig. 2). Cell lysate of rat brain (indicated by B), pancreatic islets (I), beta TC3 (beta ), and RINm5F (R) beta -cells was prepared and analyzed by immunoblotting. The Syt III protein was present in rat brain with two different molecular masses of about 66 and 50 kDa. The Syt III protein of the same molecular mass was also present in rat islet (panel c), RINm5F (panels a and c), and beta TC3 (panel a) cells with about equal amount of proteins in islet and RINm5F beta -cells but less protein in beta TC3 cells. The protein band of Syt III detected in this study was considered specific for the following reasons. 1) It had the expected molecular weight. 2) The anti-Syt III antibody binding was blocked by the specific peptide CP3, as shown by the comparison between the control in panel e and the dose-dependent (1-100 µg/10 ml) inhibition in panels f---i. 3) The band was not inhibited by a peptide of another isoform, 100 µg/10 ml CP7 (panel j). The Syt VII protein was also present in rat islet (panel d), RINm5F (panels b and d), and beta TC3 (panel b) cells with about equal amounts of protein in islet, RINm5F, and beta TC3 cells.


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  		Fig. 2.   Western blot analysis of synaptotagmin III and VII isoforms in pancreatic islets and beta TC3 and RINm5F beta -cells. C-terminal peptides of synaptotagmin III (CP3) and VII (CP7) isoforms were synthesized for generation of isoform-specific antibodies. Cell lysate of rat brain (indicated by B), pancreatic islets (I), beta TC3 (beta ), and RINm5F (R) beta -cells was prepared and analyzed by SDS-PAGE. Protein was transferred to cellulose membrane, blotted with anti-Syt III (panels a and c) or anti-Syt VII (panels b and d) antibodies, and subsequently with 125I-labeled protein-A. Protein bands were visualized and quantified using a PhosphorImager. In panels e-j, the primary antibody anti-Syt III was incubated in the absence of CP3 (panel e) or in the presence of 1, 10, 50, and 100 µg/10 ml CP3 (panels f-i), or 100 µg/10 ml CP7 (panel j). Figure is representative of at least three experiments.

Subcellular Localization of Synaptotagmin III and VII in beta -Cells-- The subcellular localization of Syt III and VII in pancreatic islet cells and RINm5F beta -cells was examined by confocal microscopy (Fig. 3). Insulin was stained in an intracellular vesicle pattern in RINm5F beta -cells (red in row A). The staining of Syt III (green in row A) was found to also have a similar pattern. Superimposition of the insulin and Syt III images indicated that the two proteins were co-localized in RINm5F beta -cells. Similarly Syt VII was found to be co-localized with insulin in a vesicle pattern (row B). Such co-localization of Syt III (row C) and Syt VII (row D) with insulin was also demonstrated in primary rat pancreatic islet cells. These results indicate that Syt III and VII may have important roles in insulin secretion of beta -cells.


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  		Fig. 3.   Immunofluorescent analysis of synaptotagmin III and VII isoforms in pancreatic islet cells and RINm5F beta -cells. Permeabilized and fixed RINm5F beta -cells (panels A and B) and pancreatic islet cells (panels C and D) were first incubated with either rabbit anti-Syt III (panels A and C) or rabbit anti-Syt VII (panels B and D) in the presence of bovine anti-insulin antibody. They were then incubated with both Cy2-conjugated goat anti-rabbit and Texas Red-conjugated goat anti-bovine antibodies. Cells were scanned with a laser confocal microscope using two sets of excitation (488 and 596 nm) and emission (520 and 620 nm) filters.

Insulin Secretion in Intact Stable beta -Cells Overexpressing Synaptotagmin III-- In order to examine the functional role of Syt, overexpression of Syt III was achieved by stable transfection of Syt III in RINm5F beta -cells (designated as Syt III beta -cell). A 2.3-fold increase of Syt III protein level was demonstrated in the transfected cell line by Western blot (Fig. 4, panel A). Since RINm5F cells do not respond to glucose stimulation, the effects of other stimuli on insulin secretion were examined. In empty vector-transfected beta -cells (Neo), 15 mM glyceraldehyde stimulated insulin secretion by about 2-fold, and the increase was about 2.4-fold in Syt III beta -cells. However, the small difference between Neo and Syt III beta -cells was not statistically significant (Fig. 4, panel B). Carbachol-induced insulin secretion in Neo (154 ± 16) and Syt III (177 ± 13) cells was not significantly different (>0.05). In contrast, 500 µM carbachol + 15 mM glyceraldehyde increased insulin secretion by about 3.5-fold in Syt III beta -cell, which was significantly higher than the 2-fold increase in Neo beta -cells (Fig. 4, panel C, p < 0.05). 50 mM potassium increased insulin secretion by about 30-fold in Syt III beta -cell, which was significantly higher then the 6-fold increase obtained in Neo beta -cells (Fig. 4, panel D, p < 0.05). These data show that increased cytosolic Ca2+ induced by carbachol or potassium is more effective for insulin exocytosis in beta -cells overexpressing Syt III.


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  		Fig. 4.   Insulin secretion in intact RINm5F beta -cells overexpressing synaptotagmin III. Panel A, immunoblot analysis of control Neo beta -cells and Syt III overexpressing beta -cells with Syt III antibody. Panels B-D, control Neo beta -cells and Syt III overexpressing beta -cells were plated in 24-well dishes. Cells were washed, preincubated, and incubated in 1 ml of KRB containing no glucose (control, open bars), with 15 mM glyceraldehyde (Gly, solid bars) (panel B), with glyceraldehyde + 500 µM carbachol (Gly + carbachol (CCH), solid bars) (panel C), with 30 mM KCl (K30, solid bars) (panel D) for 30 min. Results are shown as the mean ± S.E. of insulin secretion expressed as a percent of control (no secretagogue, open bars) from 12 observations per condition.

Insulin Secretion in Permeabilized Stable beta -Cells Overexpressing Synaptotagmin III-- This increased sensitivity of Syt III beta -cells to Ca2+ was also demonstrated in permeabilized cells. Neo and Syt III beta -cells were permeabilized by digitonin treatment. Intracellular Ca2+ level was then buffered to various levels with EGTA (Fig. 5). In Neo beta -cells, increasing intracellular Ca2+ from 0 to 200 µM did not affect the rate of insulin secretion (100% versus 118 ± 27%, p > 0.05), whereas it increased insulin secretion significantly from 100% to 157 ± 20% in Syt III cells (p < 0.05). An increase of intracellular Ca2+ from 0 to 1000 µM also did not affect the rate of insulin secretion in Neo beta -cells (100% versus 154 ± 42%, p > 0.05) but increased insulin secretion significantly from 100% to 231 ± 35% in Syt III beta -cells (p < 0.05). When stimulated with 200 µM Ca2+ plus 10 µM mastoparan, the rate of insulin secretion was increased significantly from 100% to 180 ± 27% in Neo beta -cells (p < 0.05), whereas it was increased significantly from 100% to 295 ± 31% in Syt III beta -cells (p < 0.05). Thus, mastoparan-induced insulin secretion in Syt III was higher than in control Neo beta -cells. Similarly higher insulin secretion in Syt III beta -cells was also observed when 1000 µM Ca2+ + 10 µM mastoparan was used. In another set of experiment, cells were permeabilized by streptolysin O treatment. Insulin secretion in the presence of 10 µM intracellular Ca2+ was about 3.5-fold that of the basal secretion with 0.1 µM Ca2+ in Syt III cells. This stimulation was significantly more potent than the 2-fold increase in Neo beta -cells (p < 0.05). GTPgamma S-induced insulin secretion in streptolysin O-permeabilized cells was the same between control and overexpressing cells (Fig. 6).


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  		Fig. 5.   Insulin secretion in digitonin-permeabilized RINm5F beta -cells overexpressing synaptotagmin III. Control Neo beta -cells and Syt III overexpressing beta -cells were plated in 6-well dishes. beta -Cells were permeabilized with digitonin as described under "Experimental Procedures." Permeabilized cells were washed, preincubated, and incubated in 1 ml of intracellular buffer with no Ca2+ (Ca0, open bars) or 0.2 mM Ca2+ (Ca0.2, solid bars, panel A), with 1 mM Ca2+ (Ca1, solid bars, panel B), with 0.2 mM Ca2+ + 10 µM mastoparan (Ca0+MP, solid bars, panel C), with 1 mM Ca2+ + MP (Ca1+MP, solid bars, panel D) for 30 min. Results are shown as the mean ± S.E. of insulin secretion expressed as a percent of control (no secretagogue, open bars) from 6 to 26 observations per condition.


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  		Fig. 6.   Insulin secretion in streptolysin O-permeabilized RINm5F cells overexpressing synaptotagmin III. Control Neo beta -cells and Syt III overexpressing beta -cells were plated in 6-well dishes. beta -Cells were permeabilized with streptolysin O as described under "Experimental Procedures." Permeabilized cells were washed, preincubated, and incubated in 1 ml of intracellular buffer with 0.1 (Low Ca or control, open bars) or 10 µM Ca2+ (High Ca, solid bars, left panel) or 100 µM GTPgamma S (solid bars, right panel). Results are shown as the mean ± S.E. of insulin secretion expressed as a percent of control (no secretagogue, open bars) from 12 observations per condition.

Insulin Secretion in beta -Cells Overexpressing Synaptotagmin VII-- In contrast to the lack of amplification in Syt III cells, carbachol-induced insulin secretion in intact Syt VII cell (354 ± 86%) was amplified compared with Neo cells (154 ± 16%) as shown in Table II. The effect of glyceraldehyde in Syt VII cell (336 ± 35%) was greater than those in Syt III (242 ± 20%) or Neo cells (198 ± 17%). The combination of carbachol and glyceraldehyde caused a greater effect in Syt VII (468 ± 59%) than in Syt III (340 ± 33%) or Neo (203 ± 24%) cells. The effects of mastoparan or 30 mM K+ were the same between Syt VII and Neo cells. In permeabilized cells, the effects of 200 or 1000 µM Ca2+ were significantly higher in Syt VII than in Neo cells. Mastoparan plus high intracellular Ca2+ (either 200 or 1000 µM) also caused more secretion in Syt VII cells than in Neo cells.

                              
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Table II
Effects of various conditions on insulin secretion of Neo and SytVII cells
Control Neo and SytVII-overexpressing beta -cells were plated in 24 wells for intact cell insulin secretion and 6-well dishes for permeabilized cell insulin secretion. Intact cells were incubated with carbachol (0.5 mM), glyceraldehyde (Gly, 15 mM), mastoparan (MP, 10 µM), or K30 (30 mM). beta -Cells were permeabilized with digitonin as described under "Experimental Procedures." Permeabilized cells were washed, preincubated, and incubated in 1 ml of intracellular buffer with no Ca2+ (Ca0) or 0.2 mM Ca2+ (Ca0.2), with 1 mM Ca2+ (Ca1), with 0.2 mM Ca2+ + 10 µM mastoparan (Ca0 + MP), with 1 mM Ca2+ + MP (Ca1 + MP) for 30 min. Results are shown as mean ± S.E. of insulin secretion expressed as a percent of control (no secretagogue) from 6 to 30 observations per condition.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Increased cytosolic Ca2+ is required for secretagogue-induced insulin secretion from pancreatic beta -cells. The various isoforms of synaptotagmins are known to play major roles in regulated secretion of neurotransmitters in presynaptic terminals (22, 23). Synaptotagmin has been shown to bind Ca2+ in a phospholipid-dependent manner and undergoes a Ca2+-dependent conformational change that enables it to bind syntaxin on the plasma membrane for granule fusion. The expression of Syt I and II mRNA and protein in pancreatic beta -cells has recently been shown in insulin-secreting beta -cell lines RINm5F, INS-1, and HIT-T15 (27). An involvement of Syt I or II in insulin secretion of tumor cell lines is supported by the inhibition of Ca2+-induced insulin secretion in permeabilized cells using antibodies directed against the Ca2+-dependent phospholipid-binding site of the first C2 domain of Syt I or II. However, overexpression of Syt II in HIT-T15 beta -cell failed to affect insulin secretion (27). Moreover, this hypothesis may not be applicable to normal pancreatic beta -cells, because of the lack of Syt I and II expression in primary islet beta -cell as shown previously and in this study (27). These observations suggest that Syt I and II do not have a physiological role in insulin secretion and imply the presence of alternative signaling Syt proteins in the pancreatic beta -cell. Another argument against the role of Syt I in insulin secretion is that it requires levels of free Ca2+ that are at least 1 to 2 orders of magnitude higher than those observed during insulin exocytosis in the beta -cells (22, 45, 46).

Our study has discovered new Syt isoforms (IV, V, VII, and VIII) as well as confirmed previously reported Syt isoform (I, II and III) expression in beta TC3 (I, II, III, IV, V, VII, and VIII), RINm5F cells (III and VII), and rat primary islet beta -cells (III, IV, V, and VII). The expression of Syt VII protein in beta TC3, RIMm5F, and primary rat islet is a novel finding. The absence of RT-PCR product for synaptotagmin isoforms I, II, VI, and VIII in primary rat islets suggests they are either not expressed or expressed at very low levels. The difference of isoform expression between tumor cell line and primary rat islet cell cannot be easily explained. However, this may indicate that isoforms III and VII are more physiologically important than isoforms I and II in insulin secretion. Further investigation is required to address the roles of Syt IV and V in pancreatic beta -cell.

An important candidate for insulin exocytosis is the Syt III isoform (47, 48). Its Ca2+ sensitivity is submicromolar, which is the physiological range present in stimulated beta -cells. Previous studies have mainly focused on Syt III identification in beta -cells. Syt III has been found in the insulin-secreting cell line MIN6 as well as in primary islet cells (48). Syt III and VII have been localized to insulin granules in various beta -cell lines and islet cells, which implicates that they may play important roles in granule fusion and exocytosis (47). Our study shows that by confocal microscopy, synaptotagmin III and VII co-localize with insulin secretory granules. Thus, all studies so far have localized Syt III in the beta -cell and in particular to the insulin-containing secretory granule.

Because of the localization of Syt III and Syt VII to the insulin secretory granule, it is conceivable that they may have a major role in insulin exocytosis, by analogy with their known role in neurotransmitter release. Treatment of permeabilized MIN6 cells with anti-Syt III antibody inhibited Ca2+-triggered insulin secretion (48). Another study reported that synaptotagmin III antibodies inhibited Ca2+-induced changes in beta -cell membrane capacitance (47). Although indirect, these experiments implicate a role of Syt III in insulin secretion. We have directly addressed this issue by engineering insulin-secreting beta -cells that overexpress Syt III or Syt VII and compared them to an empty vector Neo control beta -cell line.

Our results clearly show that beta -cells overexpressing Syt III secrete more insulin in response to stimuli that increase intracellular Ca2+. This effect was consistently observed in intact beta -cells as well as permeabilized beta -cells. In normal beta -cells, depolarization with K+ causes an influx of extracellular Ca2+ and subsequent insulin secretion. However, in the beta -cells overexpressing Syt III, K+-induced insulin secretion was further amplified 6-fold. In the case of glyceraldehyde- and carbachol-induced insulin secretion, the amplification obtained was only 2.5-fold. This is probably due to the fact that cytosolic Ca2+ elevation is the sole mechanism for K+-induced insulin secretion, whereas glyceraldehyde and carbachol stimulate insulin secretion through increased intracellular Ca2+ as well as other mechanisms, such as activation of protein kinase C (8-17). Similarly, the lack of amplification of glyceraldehyde-induced secretion in Syt III beta -cells is probably due to the even lower contribution of Ca2+-dependent signaling in this pathway. Increased Ca2+-induced insulin secretion was also directly observed in permeabilized Syt III beta -cells. Mastoparan, which directly triggers insulin granule fusion and exocytosis (33), also increased insulin secretion. The findings in our study suggest that Syt III and Syt VII may be the Ca2+ sensor or one of the Ca2+ sensors for insulin exocytosis of the beta -cell and may be a potentially interesting target for cellular and pharmacological therapies aimed at increasing insulin secretion. The possibility that Syt IV or V isoforms play a role in Ca2+ sensing for insulin exocytosis cannot be excluded.

It is interesting that both Syt III and Syt VII beta -cells had increased insulin secretion induced by direct elevation of intracellular Ca2+ concentration in permeabilized cells. However, they secreted insulin differently in response to carbachol and high K+. The amplified response to 30 mM K+ was larger in Syt III cells and not significant in Syt VII cells. On the other hand, insulin secretion triggered by carbachol was larger in Syt VII cells but was not significant in Syt III cells. The explanation of these differences is currently unclear. One possibility is that the two Syt isoforms sense changes of cytosolic Ca2+ in different subcellular locations. For example, Syt III may sense an increase in intracellular Ca2+ due to influx of extracellular Ca2+, whereas Syt VII may be relevant when intracellular Ca2+ elevation is induced by intracellular mobilization through inositol 1,4,5-trisphosphate receptors. It has been demonstrated that synaptotagmin directly interacts with N-type (49-57) or P/Q-type (19, 54, 55) Ca2+ channels in neurons and Lc-type Ca2+ channels (20). Inositol 1,4,5-trisphosphate receptors are present in pancreatic beta -cells; however, it is currently unclear whether they associate with synaptotagmin.

In conclusion, we have shown that isoforms III, IV, V, and VII of synaptotagmin instead of isoforms I and II are expressed in islet beta -cells and that Syt III and Syt VII may have an important physiological role in insulin exocytosis of the pancreatic beta -cell. The physiological roles of the other isoforms remain to be investigated.

    ACKNOWLEDGEMENT

The Diabetes Endocrinology Research Center Radioimmunoassay Core and the Biomedical Imaging Core are supported by National Institutes of Health Grant DK19525.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants DK43354 and DK49814.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.

Dagger Both authors should be considered first authors.

§ To whom correspondence should be addressed: University of Pennsylvania School of Medicine, Dept. of Pathology & Laboratory Medicine, 230 John Morgan Bldg., 3620 Hamilton Walk, Philadelphia, PA 19104-6082. Tel.: 215-898-0025; Fax: 215-573-2266; E-mail: wolfb@mail. med.upenn.edu.

Published, JBC Papers in Press, August 9, 2000, DOI 10.1074/jbc.M004284200

    ABBREVIATIONS

The abbreviations used are: Syt, synaptotagmin; beta TC3, beta TC3 insulin-secreting beta -cell; CP3, C-terminal peptide of synaptotagmin III; CP7, C-terminal peptide of synaptotagmin VII; RIN, RINm5F beta -cell; Neo, empty vector-transfected RINm5F beta -cell; BSA, bovine serum albumin; RT-PCR, reverse transcriptase-polymerase chain reaction; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate; PBS, phosphate-buffered saline.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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