1 Division of Clinical Biochemistry, Department of Internal Medicine, University Medical Center, 1211 Geneva 4, Switzerland
2 Fukuda Initiative Research Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
* Author for correspondence (e-mail: claes.wollheim{at}medecine.unige.ch)
Accepted 24 February 2004
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Summary |
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Key words: RNA interference, ß-cell, Insulin secretion, INS-1E cells, Pancreatic islet, hGH secretion
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Introduction |
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Synaptotagmins (Syts) constitute a large family of proteins implicated in Ca2+-triggered exocytosis (Chapman, 2002; Südhof, 2002
). Vertebrates express at least 15 isoforms, which share a common structure composed of an intravesicular N-terminal domain and a single membrane-spanning region, followed by the two C-terminal C2A and C2B domains (Südhof and Rizo, 1996
; Rickman et al., 2004
). These domains mediate the Ca2+-dependent phospholipid binding that is essential for membrane interactions.
The archetypical closely related Syts I and II are localized to synaptic vesicles as well as secretory granules and they function as essential vesicular Ca2+ sensors in exocytosis (Geppert et al., 1994). However, these isoforms are not the only Ca2+ sensors because some Ca2+-dependent exocytosis remains in their absence (Geppert et al., 1994
). Our group has shown that Syt I and II are expressed in various pancreatic ß-cell lines but not in native ß-cells. Furthermore, both the C2A and C2B domains were required for Ca2+-triggered insulin exocytosis (Lang et al., 1997
).
Syt III is the most abundant synaptotagmin after Syts I and II. Syt III mRNA was detected in different insulin-secreting cell lines and in rat pancreatic islets (Gao et al., 2000; Gut et al., 2001
; Mizuta et al., 1994
). Using a monospecific antibody, endogenous Syt III was not observed in rat ß-cells but localized to the plasma membrane when overexpressed (Gut et al., 2001
). This contrasts with previous studies showing the association of Syt III with insulin-secretory granules in ß-cells and derived cell lines (Brown et al., 2000
; Gao et al., 2000
; Mizuta et al., 1997
).
Another isoform, Syt IV, is present in glucagon-secreting islet -cells and within the TGN in various ß-cell lines (Gut et al., 2001
). In addition, Syt VII and VIII mRNAs are present in different insulinoma cell lines and in rat pancreatic islets (Gao et al., 2000
; Gut et al., 2001
). The Syt VII protein was also detected in these cell preparations (Gao et al., 2000
). Indirect evidence suggested that Syt VII and VIII might be implicated in Ca2+-regulated exocytosis (Gut et al., 2001
).
The Syt V isoform has been reported to localize on dense-core vesicles in mouse brain and to control calcium-dependent exocytosis in PC12 cells (Saegusa et al., 2002). Moreover, the Syt V transcript was detected in rat pancreatic islets and in various insulin-secreting cell lines (Gao et al., 2000
; Gut et al., 2001
). However, the Syt V protein was only present in mouse pancreatic
-cells, constituting
20% of the islet endocrine cells (Saegusa et al., 2002
). Previous experiments in permeabilized primary ß-cells showed that the introduction of a recombinant peptide corresponding to the C2 domains of Syt V inhibited Ca2+-evoked insulin release. These findings suggested that Syt V might be one of the Ca2+ sensors for insulin exocytosis in the ß-cell (Gut et al., 2001
). Nonetheless, the subcellular localization of Syt V in insulin-secreting cells and its precise function in regulated exocytosis have not been clarified.
Syt IX, unfortunately also called Syt V, shares the highest sequence similarity with Syt I and Syt II (Rickman et al., 2004). It is synthesized at low abundance in non-neuronal tissues (such as rat kidney, adipose tissue, lung and heart) and at higher levels in brain and PC12 cells, where it is localized to synaptic vesicles (Craxton and Goedert, 1995
; Hudson and Birnbaum, 1995
). Syt IX was reported to have a role as a Ca2+ sensor in the release of norepinephrine from PC12 cells (Fukuda et al., 2002a
). In addition to exocytosis, Syt IX may also have a role in endocytosis. In fact, a recent study revealed that this isoform is involved in the transport of transferrin from the endocytic recycling compartment (ERC) to the cell surface (Haberman et al., 2003
). But to date there is no information regarding a putative involvement of Syt IX in insulin secretion.
Therefore, we have investigated the presence and the intracellular localization of Syt V and Syt IX in pancreatic endocrine cells and in a derived cell line (INS-1E). We also examine the functional impact of Syt V and Syt IX on insulin exocytosis in INS-1E cells, comparing their overexpression as well as suppression using RNA interference (RNAi).
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Materials and Methods |
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Cell culture
The INS-1E clone of the insulin-secreting cell line INS-1 (Merglen et al., 2004) was cultured in RPMI 1640 medium supplemented with 10% FCS and other additions as described (Asfari et al., 1992
). Cells were incubated in a humidified atmosphere of 5% CO2 at 37°C.
Pancreatic islets
Rat pancreatic islets were obtained by collagenase digestion and purified on a Ficoll gradient as described (Pralong et al., 1990). Islets were then homogenized and used for western blotting experiments as described below. Alternatively, just after Ficoll gradient, islet cells were prepared for immunofluorescence as described previously (Rouiller et al., 1991
). Briefly, the islets were dissociated by trypsin treatment, seeded on poly-ornithine-coated glass coverslips and maintained in culture 48 hours before the experiment.
Cell homogenates
The cells of the INS-1E clone or the pancreatic islets were washed twice in ice-cold phosphate-buffered saline (PBS) containing 5 mM EDTA, 5 µg/ml leupeptine, 5 µg/ml aprotinine and then disrupted by brief sonication (3x 1 second). The homogenates were stored at 20°C until use.
Brain crude membranes
A whole rat brain was homogenized in 2 ml ice cold sonication buffer containing 5 mM Tris-HCl pH 7.4, 1 mM EDTA, 1 mM dithiothreitol and a cocktail of protease inhibitors (Roche Diagnostica, Mannheim, Germany). The homogenate was briefly sonicated and centrifuged at 14,000 g for 30 minutes at 4°C. The obtained pellet was resuspended in 500 µl of 10 mM Tris-HCl pH 7.4, 1 mM EDTA, a cocktail of protease inhibitors (Roche Diagnostica, Mannheim, Germany) and then subjected to SDS-PAGE and immunoblotting.
Western blotting
The homogenates were suspended in Laemmli sample buffer, resolved by 10% SDS-PAGE under reducing conditions and transferred onto PVDF membrane. The transferred proteins were blocked in 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Tween 20, 5% milk and then incubated with the appropriate primary antibodies. Immunoreactive bands were revealed by enhanced chemiluminescence (Pierce, Lausanne, Switzerland) using horseradish peroxidase coupled secondary antibodies.
Immunofluorescence microscopy
INS-1E and primary pancreatic endocrine cells were cultured on glass coverslips coated with poly-ornithine (Sigma). Subconfluent monolayers were rinsed twice with PBS, fixed for 10 minutes in a 4% para-formaldehyde-PBS solution and permeabilized for 1 hour in PBS containing 0.1% saponin and 0.5% BSA. The cells were then incubated with primary antibodies overnight at 4°C. After rinsing with PBS, the cells were exposed to secondary antibodies for 1 hour at room temperature. Coverslips were mounted on glass slides with Vectastain (Reactolab, Lausanne, Switzerland). Samples were analysed using a Zeiss laser confocal microscope (LSM 510, Zurich, Switzerland). Images were taken with a 60x objective.
Subcellular fractionation of INS-1E cells
Subcellular fractionation of approximately 108 INS-1E cells was performed as described previously (Regazzi et al., 1996). Briefly, INS-1E cells were homogenized by nitrogen cavitation (9 bars for 30 minutes) in a solution containing 5 mM HEPES pH 7.4, 1 mM EGTA, 10 µg/ml leupeptin and 2 µg/ml aprotinin. The cell debris and the nuclei were eliminated by centrifuging the homogenate for 10 minutes at 3000 g. The obtained postnuclear supernatant was loaded on a continuous sucrose density gradient (0.45-2 M sucrose; 8 ml). After centrifugation for 18 hours at 110,000 g, 16 fractions of 0.5 ml were collected from the top of the tube. The concentration of sucrose in the fractions was determined by measuring the refractive index of the solution. The amount of insulin present in the fractions was measured by RIA. The distribution, throughout the gradient, of plasma membrane (Na+/K+-ATPase), synaptic-like microvesicles (synaptophysin) and insulin-containing secretory granules (carboxypeptidase H) was assessed by western blotting.
Reverse transcription and PCR amplification of Syt IX cDNA
Total RNA was extracted from rat brain, rat islets and INS-1E cells by the use of TRIzolTM Reagent (Gibco Life Sciences, Basel, Switzerland). cDNA was synthesized for 45 minutes at 48°C followed by 2 minutes at 94°C using AMV reverse transcriptase (Promega, Catalys AG, Walisellen, Switzerland). The primers used for reverse transcription and PCR amplification of cDNAs were: 5' CAGGATCCTTCCCGGAACCCCCGACC 3' (sense strand, positions 128-145 of Syt IX cDNA sequence) and 5' TCATAATCCAGTGAATACTG 3' (reverse strand, positions 460-480 of Syt IX cDNA sequence). The PCR program involved 40 cycles of denaturation at 94°C for 30 seconds, annealing at 60°C for 1 minute and elongation at 68°C for 2 minutes. A final elongation step of 7 minutes at 68°C allowed the truncated product to extend to full length. PCR reactions were performed using Tfl DNA polymerase (Promega, Catalys AG, Walisellen, Switzerland). Control experiments showed that amplification products were absent after reactions omitting reverse transcriptase.
Construction of myc-tagged Syt IX cDNA
The Syt IX cDNA was amplified by PCR using the following primers: 5' CAG GATCCTTCCCGGAACCCCCGACC 3' and 5' GCGAATTCAATCAGGGTG CAGGTATTGG 3' and then cloned in the pGEM® -T Easy Vector (Promega, Catalys AG, Walisellen, Switzerland). The DNA fragment was next purified by agarose gel electrophoresis and ligated into BamHI/NotI sites of the pcDNA3 vector (Invitrogen, San Diego, CA) in frame with the N-terminal myc epitope. The construct was verified by DNA sequencing.
Generation of Syt V and Syt IX silencing vectors
Mammalian expression vectors directing the synthesis of small interfering RNAs targeted against Syt V and Syt IX were prepared according to Ambion's guidelines (Huntingdon, Cambridgeshire, UK). Briefly, using the `siRNA Target Finder and Design Tool' web page, the mRNA sequence of each protein was scanned for AA dinucleotides and recorded for the 19 nucleotides downstream of the AA. The potential siRNA target sequence was then compared with the genome database to eliminate any sequences with significant homology to other genes. On the basis of the chosen target transcript, two complementary DNA fragments encoding a 19-nucleotide sequence and separated from its reverse 19-nucleotide complement by a short spacer were synthesized by Microsynth (Balgach, Switzerland). The two complementary DNA fragments were annealed and cloned into EcoRI/ApaI sites of the pSilencerTM 1.0-U6 siRNA Expression Vector (Ambion) The Syt V silencer was generated using the sequence corresponding to nucleotides 94-112 of rat Syt V cDNA; the Syt IX silencer was constructed using the nucleotide sequence 158-176 of rat Syt IX cDNA.
Cell transfection
Transient transfection of INS-1E cells was performed using the Lipofectamine 2000 reagent (Invitrogen, Groningen, Switzerland), according to the manufacturer's instructions. In all the experiments the DNA/Lipofectamine ratio was 1/2.
Secretion from transfected INS-1E cells
INS-1E cells were transiently cotransfected with a plasmid encoding human growth hormone (hGH) as a reporter for secretion, and with plasmids containing either the RNAi silencers or the cDNAs expression constructs. Three days after transfection the cells were incubated at 37°C for 2 hours in complete medium without glucose. They were preincubated for 30 minutes in modified Krebs-Ringer-Bicarbonate buffer (KRB) (Regazzi et al., 1996) containing 2.5 mM glucose and then incubated during 30 minutes either in the same buffer (basal conditions), or in KRB supplemented with either 15 mM glucose or 100 nM PMA (stimulated conditions). After incubation, secretion from transfected cells was assessed by measuring the amount of hGH released into the medium during the incubation period by enzyme-linked immunoabsorbent assay (ELISA) (Roche, Diagnostica, Mannheim, Germany).
Statistical analysis
Results are presented as mean±s.e.m. from experiments performed independently on at least three different cell preparations.
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Results |
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Subcellular distribution of Syt V and Syt IX in insulin-secreting cells
To determine the subcellular localization of Syt V, INS-1E cells were immunostained and analysed by confocal microscopy. Double labelling experiments showed that Syt V perfectly colocalizes with insulin-containing secretory granules (Fig. 2A). Additional immunofluorescence studies performed in cultured primary pancreatic endocrine cells revealed that Syt V was not expressed in the ß-cells but was present in the glucagon-containing islet -cells. (Fig. 2B).
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The intracellular distribution of Syt IX in INS-1E cells and native ß-cells was also analysed by confocal microscopy. The overlapping images in Fig. 3 show that Syt IX colocalizes with the insulin-containing granules in both INS-1E and primary ß-cells.
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To further characterize the association of Syt V and Syt IX with insulin-containing granules, we performed subcellular fractionation by continuous sucrose density gradient (0.45-2 M) (Reetz et al., 1991; Regazzi et al., 1996
). INS-1E cells contain a large number of secretory granules and are therefore appropriate for this type of biochemical characterization. As illustrated in the top panel of Fig. 4, when homogenates of INS-1E cells are centrifuged at equilibrium, insulin-containing granules are recovered in the fractions 11-17 corresponding to 1.4-1.8 M sucrose. The lower panels in Fig. 4 show that the plasma membrane marker Na+/K+-ATPase was detected in fractions 9-13; synaptophysin, a marker of GABA-containing synaptic-like microvesicles, was found in fractions 7-10, while carboxypeptidase H, a resident protein in the insulin-granule, was concentrated in fractions 11-17. Finally, Syt V and Syt IX were also enriched in fractions 11-17, consistent with an association with insulin-containing secretory granules. These results confirm the data obtained by immunofluorescence experiments.
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The intracellular localization of Syt V and Syt IX was also investigated after transient transfection of INS-1E cells. To monitor the expression of the transfected proteins, a myc epitope was inserted at the amino terminus of each Syt isoform. Immunolabelling followed by confocal microscopy analysis revealed that the myc-tagged Syt V and Syt IX proteins colocalize with insulin-containing granules (Fig. 5A,B, upper images), confirming the data obtained with the sucrose gradient. When the expression of the Syt proteins was further increased (2 µg, not shown; 5 µg, Fig. 5A,B, lower images), the Syts distributed with insulin to plasma membrane-near regions in a dose-dependent manner. Similar localization has also been reported for secretory granules in nontransfected INS-1E cells (Waselle et al., 2003). Taken together, these results are consistent with the findings that both endogenous and extrinsic Syt V and Syt IX are associated with insulin-containing granules.
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Functional role of Syt V and Syt IX in insulin exocytosis
To study the potential involvement of Syt V and Syt IX in insulin exocytosis, these proteins were overexpressed in INS-1E cells. Syt V or Syt IX was transiently cotransfected with a plasmid encoding human growth hormone (hGH). hGH is targeted to insulin-containing granules and can be used to monitor exocytosis as an insulin substitute in the subpopulation of transfected cells (Iezzi et al., 2000). Using this approach, we observed that an increase in the glucose concentration from 2.5 mM to 15 mM caused a 4.3-fold rise in hGH release (Fig. 6, lower panel). The overexpression of Syt V did not significantly affect basal secretion, whereas Syt IX slightly reduced it. Remarkably, exocytosis stimulated by glucose was not affected by the overexpression of the two Syts. Moreover, neither of the Syt constructs affected the hGH cellular content that ranged from 400-500 ng/ml. As estimated by immunoblotting compared with the endogenous protein, Syt V and Syt IX were overexpressed approximately fivefold (Fig. 6, upper panels). The two Syt isoforms were expressed at similar levels, as revealed by blotting the membranes with the anti-myc antibody. It should be noted that in Syt IX-overexpressing cells, the anti-Syt IX antibody recognizes both the larger and smaller forms of the protein (see Discussion).
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Next, we directly investigated the functional implication of Syt V and Syt IX in insulin exocytosis. For this purpose, we generated a plasmid that directs the synthesis of small interfering RNAs (siRNAs) targeting the sequences of the two Syt isoforms. As illustrated in Fig. 7A, transient expression of the Syt V and Syt IX silencers in INS-1E cells selectively abolished the production of these proteins, but did not affect other endogenous unrelated proteins (Fig. 7A, arrow in b). To verify the silencing effect on the endogenous protein, INS-1E cells were transfected with GFP together with the Syt V or Syt IX silencers. Homogenates of FACS-enriched GFP-expressing cells were analysed by western blotting. Fig. 7B shows that Syt V silencer strongly reduced the amount of Syt V protein (panel a) but did not affect Syt IX (panel b); similarly Syt IX silencer almost completely eliminated the Syt IX protein (panel c) without changing the amount of Syt V (panel d). These results emphasize the selectivity of the designed Syt siRNAs.
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The efficacy of the two silencers was further verified by immunofluorescence studies. Transfected cells identified by the expression of GFP were found to contain low levels of Syt V (Fig. 8A) and Syt IX (Fig. 8B), confirming the silencing of the given endogenous protein.
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Finally, INS-1E cells were transfected with the hGH together with Syt V or Syt IX silencers. Selective knockdown of Syt proteins by RNA interference did not alter basal secretion. When hormone secretion was stimulated by 15 mM glucose, there was strong inhibition reaching 79% and 74% for Syt V and Syt IX, respectively (Fig. 9, upper panel). To distinguish whether the inhibitory effects of the silencers are exerted on Ca2+-dependent or -independent exocytosis, the experiments were repeated in the presence of the phorbol ester PMA. This compound acts by potentiating insulin exocytosis distal to the increase in cytosolic Ca2+ (Regazzi et al., 1989). A decrease in Syt V or Syt IX expression neither affected basal nor the marked potentation of exocytosis (fourfold relative to 15 mM glucose alone) in the presence of PMA (Fig. 9, lower panel). Again, the silencers did not change the cellular content of hGH (not shown).
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Discussion |
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Our data prove that Syt V (70 kDa) is expressed at the protein level in rat pancreatic islets and in the clonal ß-cell line INS-1E. These results are in accordance with the presence of Syt V mRNA in various insulinoma cell lines and in rat islets (Gut et al., 2001
) and the protein level in mouse pancreatic islets (Saegusa et al., 2002
). We assessed the subcellular distribution of Syt V by confocal microscopy and continuous sucrose density gradient in INS-1E cells. The latter method permits the separation of insulin-containing granules from synaptic-like microvesicles (Iezzi et al., 1999
). Both approaches show that Syt V colocalizes with insulin-granules but is absent from synaptic-like microvesicles. This is consistent with the targeting of Syt V to dense-core vesicles but not to synaptic-like microvesicles in PC12 cells, and agrees with the location in mouse brain (Saegusa et al., 2002
). Further immunofluorescence studies performed in rat pancreatic endocrine cells revealed that Syt V is expressed in the glucagon-secreting
-cells but not in ß-cells, confirming the data obtained in mouse pancreatic islets (Saegusa et al., 2002
). Taken together, the fascinating observation that Syt V is associated with insulin-granules in INS-1E cells suggests a mixed
-ß cell phenotype that is more closely related to neurons than native ß-cells. The tumor origin of INS-1E cells may explain their islets progenitor cell phenotype (Asfari et al., 1992
; Merglen et al., 2004
). (N.B: mature rodent islet contains 70% ß-cells, 20%
-cells and 5% each of somatostatin and pancreatic-polypeptide cells.)
We next showed the presence of Syt IX mRNA in INS-1E cells and, albeit at moderate levels, in pancreatic endocrine cells. The lower abundance of Syt IX protein in the endocrine tissue was confirmed by immunoblotting experiments. Syt IX was also detected in the hamster ß-cell line HIT-T15 cells (M.F., unpublished). Syt IX (50 kDa) was also identified in INS-1E cells by the use of a rabbit polyclonal antibody directed against the C-terminal region of the protein. Moreover, the immunoreactive band of
50 kDa was abolished when the immunizing peptide was included in the blotting solution (results not shown). These data strongly support the specificity of the Syt IX N-terminal monoclonal antibody used in this study. We also performed immunofluorescence and sucrose subcellular fractionation experiments showing that Syt IX is associated with insulin-granules but not with synaptic-like microvesicles in both INS-1E and native ß-cells. Similarly, immunocytochemical studies showed the presence of Syt IX on dense-core vesicles in nerve growth factor-differentiated PC12 cells (Fukuda et al., 2002a
). The participation of Syt IX in insulin-containing granule secretion may be more important than that of Syt I and II, which are also associated with synaptic like microvesicles (Lang et al., 1997
).
Further immunofluorescence experiments on overexpressed myc-tagged Syt V and Syt IX in INS-1E cells confirm the association of the Syt proteins with insulin-granules. As shown by immunoblotting, the specificity of Syt V and Syt IX antibodies was further confirmed by overexpressing the recombinant Syts in INS-IE (Fig. 6, upper panels) and COS-7 cells (data not shown). In particular, in Syt IX-overexpressing cells, the anti-Syt IX antibody recognizes two forms of the protein that could represent post-translational modifications (e.g. palmitoylation and O-glycosylation), which may affect Syt size and immunoreactivity. Similar results were recently obtained in Syt IX-overexpressing rat basophilic leukaemia (RBL) cells (Haberman et al., 20003). Our previous study reported that transient expression of Syt II did not alter exocytosis in HIT-T15 cells (Lang et al., 1997). Likewise, the overexpressed Syt V and Syt IX had no effect on stimulated insulin secretion in INS-1E cells. This indicates that the Syt proteins are not interfering with the stoichiometry of the SNARE proteins whose overexpression inhibits insulin exocytosis (Nagamatsu et al., 1999
). By contrast, stable clones expressing Syt III and VII showed enhanced Ca2+-induced insulin secretion in RINm5F ß-cells (Gao et al., 2000
). However, the long-term expression of the proteins may affect other components of the secretory pathway. For this reason it is more attractive to suppress a given protein to elucidate its function.
In a recent study RNAi was initiated in insulin-secreting cells and efficiently employed to investigate ß-cell function (Waselle et al., 2003). Here, we also used the RNAi approach to directly analyse the role of Syt V and Syt IX in insulin exocytosis. Silencing of each of the Syts in INS-1E cells did not affect basal secretion but decreased the glucose-induced insulin release. Therefore, it seems that the inhibition is caused by impairment of the exocytotic process rather than by interference with granule transport or endocytic events (Haberman et al., 2003
). The strong attenuation of hormone release suggests that Syt V and Syt IX act as positive modulators of exocytosis. This effect is remarkably selective for the Ca2+-dependent release, as PMA-induced secretion was unchanged (Lang, 1999
). As deficiency of both Syt V and Syt IX inhibits Ca2+-dependent exocytosis, it is possible that this process requires the formation of oligomeric complexes, implicating several Syt isforms in agreement with previous studies (Sudhof, 2002
; Fukuda et al., 1999
; Fukuda et al., 2002b
). By contrast, PMA may not require such oligomerization, as it can stimulate insulin exocytosis in the complete absence of Ca2+ (Vallar et al., 1987
). The exact mode of action of PMA on exocytosis is unclear. The compound not only activates protein kinase C but also interacts with Munc 13-1 (Brose and Rosenmund, 2002
), which participates in the regulation of insulin exocytosis (Sheu et al., 2003
).
In conclusion, our data show that Syt V and Syt IX are localized on insulin-containing granules and directly control Ca2+-mediated insulin exocytosis. In the endocrine pancreas Syt V and Syt IX segregate respectively to - and ß-cell granules. This suggests that Syt IX may be the Ca2+ sensor for insulin secretion in the ß-cell. Verification of this hypothesis requires specific suppression of Syt IX in islets.
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Acknowledgments |
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