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Pacific Northwest Research Institute, 720 Broadway, Seattle, Washington 98122, USA
1 Department of Pharmacology, University of Cambridge, Cambridge, UK
2 Department of Pharmacology, University of Chicago, Chicago, Illinois, USA
(Requests for offprints should be addressed to C J Rhodes; Email: cjr{at}pnri.org)
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Abstract |
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Introduction |
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Unfortunately, little is known about how increases in [Ca2+]i evoke specific protein components of the ß-cell to interact for triggering insulin exocytosis (Wollheim et al. 1996, Easom 2000). Analogies have been made to the mechanism of regulated synaptic vesicle exocytosis in neurons to help identify proteins that are necessary for the control of insulin exocytosis, and indeed there are similar protein isoforms involved (Burgoyne & Morgan 2003). However, there are significant differences in the mechanisms of synaptic-like vesicle exocytosis versus that of large dense-core secretory vesicles (LDCV), such as the insulin-containing ß-granules found in ß-cells (Martin 1994, Edwardson & Marciniak 1995). While common proteins catalyze both processes (e.g. members of the SNARE (soluble N-ethylmalemide-sensitive attachment receptors) protein family: syntaxins, SNAP-25 and synaptobrevins (Wheeler et al. 1996, Linial 1997, Easom 2000), and synaptotagmins (Gao et al. 2000)), there are likely other proteins that contribute to the mechanism of LDCV exocytosis that have yet to be defined (Burgoyne & Morgan 2003, Martin 2003). For the most part, proteins that interact to facilitate regulated LDCV exocytosis are located on the LDCV membrane, the plasma membrane and cytosol (Easom 2000, Burgoyne & Morgan 2003). However, studies in pancreatic exocrine cells have indicated that a soluble intragranular protein, syncollin, also plays a role in the regulation of exocrine large dense-core zymogen granule exocytosis, distinct from synaptic vesicle exocytosis (Wasle et al. 2005).
Syncollin was originally cloned as a Ca2+-dependent syntaxin-binding protein that associated with syntaxin at low [Ca2+]i to block exocytosis and when [Ca2+]i increased; syncollin disassociated from syntaxin allowing SNARE complex formation, LDCV docking with the plasma membrane and subsequently exocytosis (Edwardson et al. 1997). Consequently, at that time, syncollin was a prime candidate as a pivotal Ca2+-sensing protein in control LDCV exocytosis (Easom 2000). However, since then it has been found that syncollin has an N-terminal signal peptide sequence and is efficiently targeted as a soluble protein to the lumen of the zymogen granule in pancreatic exocrine cells (An et al. 2000). Initial observations in the syncollin knockout mouse suggested that syncollin did not play a role in exocrine cell-regulated secretion (Antonin et al. 2002); however, more recent studies using a more direct analysis of exocrine cell secretion have revealed that syncollin is required for efficient zymogen granule exocytosis (Wasle et al. 2005). Syncollin is currently thought to play a role in the mechanism of zymogen granule exocytosis in the formation of an exocytotic fusion pore acting from the inside lumen of the granule (Geisse et al. 2002).
In this study, we have examined whether an intragranular protein factor can influence the mechanism of insulin exocytosis in ß-cells. Although syncollin is not expressed in ß-cells, we have used adenoviral-mediated expression of syncollin in ß-cells as an experimental tool to investigate this possibility. Syncollin was efficiently targeted to the lumen of ß-granules, where it specifically inhibited the distal stages of insulin exocytosis without altering insulin production or generation of stimulus-coupling signals such as [Ca2+]i. This indicated that intragranular peptide factors may play a role in the control of insulin exocytosis. Intriguingly, a syncollin-green fluorescent protein (syncollinGFP) chimera was also efficiently targeted to ß-granules when expressed in ß-cells but, unlike native syncollin, did not affect insulin secretion. As such, syncollinGFP can be used as an experimental tool for tracking ß-granule movement and exocytosis in real time (Ma et al. 2004, Michael et al. 2004).
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Materials and Methods |
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Fetal bovine serum was obtained from Hyclone Laboratories, Inc. (Logan, UT, USA). Antibodies obtained for these studies include anti-VAMP2 (vesicle-associated membrane protein 2) from Calbiochem (San Diego, CA, USA), anti-insulin from Sigma-Aldrich (St Louis, MO, USA), donkey anti-rabbit and goat anti-mouse IgG horseradish peroxidase from Upstate (Waltham, MA, USA), goat anti-rabbit Cy3 fluorophore and goat anti-guinea pig Cy5 fluorophore from Jackson Laboratories (West Grove, PA, USA). Syncollin polyclonal antiserum ‘B’ and monoclonal antiserum 87.1 have been described previously (An et al. 2000, Hodel & Edwardson 2000) and were kindly provided by Dr R Jahn (Max Planck Institute, Göttingen, Germany). ProMix [35S]protein labeling mix, from Amersham Biosciences (Arlington Heights, IL, USA), containing 75% L-[35S]methionine was used for islet protein synthesis radiolabeling. Uridine 5'-[
-32P]triphosphate (3000 Ci/mmol) was purchased from Amersham Biosciences. Glucagon-like peptide-1 (GLP-1)7–36 was purchased from Bachem Inc. (King of Prussia, PA, USA). Unless otherwise stated, all other chemical reagents were of the highest purity available and purchased from Sigma-Aldrich or Fisher Scientific (Santa Clara, CA, USA).
Islet isolation and INS-1 cell culture
INS-1 cells [832/13] were provided by Dr C B Newgard (Duke University, Durham, NC, USA) and were maintained in 11.1 mM glucose RPMI 1640 media (Invitrogen, Baltimore, MD, USA) containing 10% fetal bovine serum (FBS), 50 µM ß-mercaptoethanol, 100 units/ml penicillin and streptomycin, 0.5 M HEPES (pH 7.4), 102 mM glutamine and 50 mM sodium pyruvate (Hohmeier et al. 2000). Pancreatic rat islets were isolated from male Sprague–Dawley rats by collagenase digestion, followed by Histopaque–Ficoll gradient centrifugation as previously described (Alarcon et al. 1993).
Recombinant adenoviruses
Recombinant adenoviruses expressing GFP (Adv-GFP), syncollin-Met1 (Adv-Sync-Met1), syncollin-Met2 (Adv-Sync-Met2) and syncollinGFP (Adv-Sync-GFP) were generated, amplified and purified as previously described (He et al. 1998, Dickson et al. 2001). Syncollin (Met1) was cloned by RT-PCR from isolated rat pancreatic islets, which upon sequencing was equivalent to the published syncollin (Met1) sequence (Edwardson et al. 1997). The cloned syncollin (Met1) was subcloned into pACCMV.pLpA between EcoRI and SalI. Syncollin (Met2) was subcloned from pSyncollin-EGFP-N1 (Hodel & Edwardson 2000) into pAdTrack-CMV between BglII and HindIII. SyncollinGFP was cloned as previously described (Hodel & Edwardson 2000) and further subcloned into pShuttle-CMV between BglII and XbaI. The secreted alkaline phosphatase adenovirus (Adv-sAP) was generated as previously outlined (Molinete et al. 2000). For experiments with adenovirus-mediated protein expression, isolated islets were incubated with the adeno-viruses for 16–18 h at 1 x 107 plaque-forming units (pfu)/islet to 4 x 108 pfu/islet in 5.6 mM glucose RPMI 1640 media prior to analysis. INS-1 cells were incubated with 4 x 108 pfu/well for 2 h and cultured for 16–18 h in 5.6 mM glucose RPMI 1640 media prior to analysis. Adenovirus-mediated syncollin protein expression was determined by immunoblotting with a syncollin monoclonal antibody (87.1) on 10 µg protein. Unless otherwise stated, all functional experiments requiring Adv-Sync were performed with adenovirus titers at 4 x 108 pfu/islet, which exhibited good syncollin protein expression without adverse effects seen on cell viability.
In vitro insulin secretion and islet perifusion
Isolated rat islets or INS-1 [832/13] cells were infected with adenovirus and analyzed for insulin secretory activity by radioimmunoassay in both static and perifusion incubation experiments as described previously at either basal (2.8 mM) or stimulatory (16.7 mM) glucose ( ± 10 nM GLP-1 or 5µM glyburide as indicated) (Donelan et al. 2002).
Confocal microscopy
Adenovirus-infected INS-1 cells were grown on glass coverslips and fixed in 4% paraformaldehyde (Polysciences, Warrington, PA, USA). The cells were treated with 0.1% saponin in phosphate-buffered saline (PBS) for 30 min, blocked with 10% donkey serum in 5% bovine serum albumin (BSA)/PBS for 1 h, and subsequently probed with anti-syncollin polyclonal ‘B’ (1:100) and anti-insulin (1:100) antibodies in 5% BSA/PBS for 16–18 h at 4 °C. Secondary antibodies to goat anti-rabbit Cy3 (1:300) and goat anti-guinea pig Cy5 (1:300) fluorophores were incubated for 1 h in 5% BSA/PBS and the coverslips were mounted onto glass slides. The slides were viewed by confocal microscopy (Olympus FV500; Olympus, Melville, NY, USA) with a UPLAPO 100x objective at 568 and 670 nm using XYZ series sequential scans at 0.5µm z-sections. The images shown are digitally magnified to 300x and the XYZ scans are compressed into one image with Fluoview software (Olympus).
Alkaline phosphatase activity
Isolated islets were incubated for 16–18 h with 1 x 108 pfu/islet Adv-sAP with either Adv-GFP (control) or Adv-Sync-Met2. Alkaline phosphatase activity was then measured from both the media and cell lysate as previously described (Molinete et al. 2000).
Proinsulin biosynthesis and preproinsulin mRNA analysis
Isolated rat islets were infected with 4 x 108 pfu/islet of purified Adv-Sync or Adv-GFP for 16–18 h and incubated for 1 h with 2.8 mM or 16.7 mM glucose in 200 µl Krebs–Ringer buffer (KRB) (pH 7.4). Immunoprecipitation analysis of proinsulin biosynthesis in adenovirus-infected isolated islets pulse-radiolabeled with [35S]methionine was as previously described (Alarcon et al. 1995). Preproinsulin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were analyzed by RNase protection assay as described (Wicksteed et al. 2001).
Standard wide-field epifluorescence imaging
Dual-wavelength excitation microspectrophotometry was used to measure [Ca2+]i as described (Yaekura et al. 2003). Adenovirus-infected islets were loaded with Fura-2 and perifused in 2 mM glucose KRB. Imaging was obtained utilizing a fluorescence imaging system as described previously (Yaekura et al. 2003).
Other methods
Where appropriate, data are presented as the means ± S.E. Statistical significance is determined by unpaired Student’s t-test where indicated, where P<0.05 was considered statistically significant. Protein concentrations were measured by bicinchoninic acid assay (Pierce, Rockford, IL, USA).
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Results |
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Syncollin is predominantly expressed in the exocrine pancreas, but not in endocrine pancreatic islets (Tan & Hooi 2000). However, because of the role syncollin plays in facilitating regulated exocytosis from inside a zymogen granule in pancreatic exocrine cells, we examined whether syncollin could be targeted to ß-granules and influence insulin secretion when adenovirally expressed in endocrine pancreatic ß-cells. Adenoviruses to the two possible isoforms of syncollin (Met1 and Met2) and a syncollinGFP chimera containing a C-terminal GFP tag were generated. Initial cloning of syncollin identified a potential start site 31 amino acids upstream of the N-terminus of native syncollin (Met1) (Edwardson et al. 1997). However, further characterization of syncollin revealed a second alternative start site (Met2) 12 amino acids downstream from the original start site (Fig. 1A
) (Tan & Hooi 2000). It has been unclear whether both these syncollin proforms can generate the soluble 13 kDa native from of syncollin found in the exocrine pancreas (An et al. 2000). Theoretically, unprocessed syncollin (Met1) would result in a protein of a predicted molecular weight (MW) of 15 kDa, whereas unprocessed syncollin (Met2) would have a predicted MW of 14 kDa. Amino terminal sequencing of syncollin and in vitro transcription/translation ± pancreatic microsomes of syncollin (Met2) revealed that native syncollin has a MW of 13 kDa, and indicated that the N-terminal hydrophobic sequence is proteolitically cleaved as a signal pre-peptide (An et al. 2000). Immunoblot analysis of both Adv-Sync-Met1 and Adv-Sync-Met2 adenoviruses in isolated rat islets indicated that adenoviral-mediated expression of both forms of syncollin yielded an identical 13 kDa mature protein (Fig. 1B), as found in exocrine cells. As such, (Met1) and (Met2) syncollins possess a leader signal peptide that is cleaved C-terminally of Gly32 in pre-syncollin (Met1) or Gly21 in pre-syncollin (Met2) to yield the same mature syncollin protein (Fig. 1A) (An et al. 2000).
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) or Adv-Sync-Met2 (data not shown) showed that mature syncollin (green) was localized in a punctate pattern with that of insulin-positive (red) ß-granules of 0.2–0.3µm in diameter (Fig. 2). Co-localization of syncollin-positive and insulin-positive ß-granules was demonstrated by the orange/yellow color in the merged image (Fig. 2). Likewise, adenoviral-mediated expression of syncollinGFP in INS-1 [832/13] cells showed that it too was localized to ß-granules (Fig. 2). These data indicated that syncollin and syncollinGFP were efficiently targeted to the lumen of ß-granules in pancreatic ß-cells similar to secretory granule localization of syncollin expressed in anterior pituitary AtT20 cells (Hodel & Edwardson 2000), as is native syncollin in exocrine cells (An et al. 2000).
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It was investigated whether adenoviral-mediated expression of syncollin or syncollinGFP in isolated pancreatic rat islets and INS-1 [832/13] cells affected regulated insulin secretion. Experiments were performed using both Adv-Sync-Met1 and Adv-Sync-Met2 vectors which yielded similar results, not surprisingly since both yield the same mature 13 kDa native syncollin (Fig. 1
). For a majority of studies the Adv-Sync-Met2 construct was used, and it is these data that are presented. Adenoviral-mediated expression of syncollin in INS-1 [832/13] cells had no effect on basal insulin secretion at 2.8 mM glucose, but significantly inhibited 16.7 mM glucose-stimulated insulin secretion by 48 ± 8% (P<0.05, n=4) compared with control Adv-GFP-infected INS-1 [832/13] cells (Fig. 3A). Likewise, syncollin expression in isolated rat islets did not affect basal insulin secretion yet inhibited glucose-induced insulin secretion by 65 ± 8% (P<0.05, n=6) compared with control Adv-GFP-infected islets (Fig. 4A). Intriguingly, expression of syncollinGFP in both INS-1 1 [832/13] cells (Fig. 3A) and isolated rat islets (Fig. 3B) had no significant effect on glucose-stimulated insulin secretion.
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). GLP-1 did not potentiate insulin secretion at 2.8 mM basal glucose as expected due to the glucose-dependent nature of GLP-1 as an insulin secretagogue (Schmidt et al. 1985), and syncollin expression did not affect basal insulin secretion from islets (Fig. 4B). The sulfonylurea, glyburide, augments insulin secretion independently of glucose by inhibiting the KATP channel thereby depolarizing the ß-cell causing L-type Ca2+ channels to open, instigating a rise in cytosolic [Ca2+]i (Sturgess et al. 1985, Nelson et al. 1987). Adenoviral-mediated expression of syncollin significantly inhibited glyburide-induced insulin secretion at 2.8 mM basal glucose by 82 ± 17%, and that at the 16.7 mM stimulatory glucose by 60 ± 19% compared with that in control Adv-GFP-infected islets (P<0.05, n=4; Fig. 4B).
Perifusion studies of isolated rat islets examined the effect of syncollin on the kinetics of glucose-induced insulin secretion (Fig. 4C). In control Adv-GFP-infected islets there was a characteristic biphasic insulin secretory response to 16.7 mM stimulatory glucose, consisting of a rapid first phase of insulin secretion over the first 10 min, followed by a more sustained second phase of insulin release (Gingerich et al. 1979). In contrast, the first phase of glucose-stimulated insulin secretion was significantly blunted by 53.4 ± 12.4% (P<0.05, n=5) in perifused Adv-Sync-infected isolated islets compared with control Adv-GFP-infected islets (Fig. 4C and D). The second phase of glucose-stimulated insulin secretion in Adv-Sync-infected islets was also observed to be reduced, although this did not reach statistical significance (Fig. 4C and D), and was most likely reflective of the diminished first phase of insulin secretion. Again, basal insulin secretion was unaffected in Adv-Sync-infected islets (Fig. 4C).
Syncollin does not adversely affect insulin production
A possible explanation for the inhibitory effect of syncollin expression on secretagogue-induced insulin secretion was that insulin production in islet ß-cells was being compromised. However, preproinsulin mRNA levels were comparable in Adv-Sync-infected islets with that in control Adv-GFP-infected islets incubated for 1 h at either 2.8 mM basal or 16.7 mM stimulatory glucose concentrations (Fig. 5A). Likewise, translational regulation of glucose-induced proinsulin biosynthesis was similar in both Adv-GFP- and Adv-Sync-infected islets (Fig. 5B). Hence, proinsulin production was unaffected by syncollin expression. Total protein synthesis was also normal in Adv-Sync-infected islets compared with control Adv-GFP-infected islets (data not shown). Insulin content in Adv-Sync-infected (67 ± 11 ng/islet, n=6) was also comparable with that in control Adv-GFP-infected islets (66 ± 11 ng/islet, n=6). These data indicated that the inhibition of regulated insulin secretion in syncollin-infected islets was not due to any adverse affects on insulin production.
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Adenovirally mediated expression of syncollin in ß-cells resulted in a specific targeting of syncollin to ß-granules (Fig. 2), as found in anterior pituitary cells (Hodel & Edwardson 2000). However, at the trans-Golgi network, newly synthesized syncollin could be sorted to either the constitutive or regulated secretory pathways (Arvan & Castle 1998). Consequently, we examined whether syncollin expression in rat pancreatic islets might also affect the constitutive secretory pathway. sAP was used as a marker of constitutive secretion as previously described (Molinete et al. 2000). Isolated rat islets were co-infected with Adv-sAP and either Adv-GFP (as a control) or Adv-Sync, and the amount of alkaline phosphatase secreted over a 1-h incubation period was measured in parallel to insulin secreted from the same islets. The same degree of sAP activity was secreted (12–15% of content) at 2.8 mM basal or 16.7 mM stimulatory glucose concentrations, confirming that sAP was acting as a suitable marker of constitutive secretion (Molinete et al. 2000) (Table 1). In contrast, in Adv-Sync-infected islets glucose-induced insulin secretion was significantly inhibited by 59 ± 7% (P>0.05, n=5) compared with that in control Adv-GFP-infected islets (Table 1). These data demonstrated that syncollin specifically inhibited regulated insulin secretion, but not constitutive secretion in isolated rat islets, which is complementary to the specific targeting of syncollin to ß-granules in islet ß-cells.
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Downstream of increased glucose metabolism, an increase in cytosolic [Ca2+]i is an important secondary stimulus-coupling factor for glucose-induced triggering of insulin exocytosis (Wollheim et al. 1996). We examined whether syncollin-mediated inhibition of insulin secretion was due to an adverse effect on the ability of glucose to cause a rise in cytosolic [Ca2+]i. Changes in cytosolic [Ca2+]i levels in Adv-Sync- and control Adv-ßGalactosidase-infected islet ß-cells was by Fura-2 fluorescence imaging using both 14 mM glucose- and 30 mM KCl-induced depolarization as stimuli (Yaekura et al. 2003). An increase from basal 2 mM to 14 mM glucose gave a rapid increase in cytosolic [Ca2+]i that was equivalent in control Adv-ßGal- and Adv-Sync-infected islets (Fig. 6). A 30 mM KCl-induced depolarization of islet ß-cells at 2 mM basal glucose also gave a rapid increase in cytosolic [Ca2+]i, which was similar in Adv-Sync- and control Adv- ßGal-infected islets (Fig. 6). The mean peak increase in apparent cytosolic [Ca2+]i levels in response to 14 mM glucose and 30 mM KCl was calculated. There was no significant difference in cytosolic [Ca2+]i responses between Adv-Sync- and control Adv-ßGal-infected islet ß-cells (Fig. 6B). Hence, syncollin likely mediated its inhibitory effect on regulated insulin secretion downstream of generating secondary signals, at the level of exocytosis.
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Discussion |
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In contrast to syncollinGFP, expression of native syncollin in pancreatic islet ß-cells specifically inhibited secretagogue-stimulated insulin release. This effect of syncollin was specific to the regulated secretory pathway in ß-cells since there was no effect of syncollin on the constitutive secretory pathway. This is consistent with the specific localization of syncollin protein expression in ß-cells to ß-granules. Syncollin-mediated inhibition of insulin secretion was not at the level of insulin production, since preproinsulin mRNA levels, translational control of proinsulin biosynthesis or islet insulin content were unaffected in Adv-Sync-infected islet ß-cells. Moreover, as syncollin expression in islet ß-cells did not affect glucose- or K+ depolarization-stimulated increases in cytosolic [Ca2+]i (which is a prerequisite to trigger insulin exocytosis from ß-cells (Wollheim et al. 1996)), syncollin-induced inhibition of insulin secretion was not due to impairing the generation of stimulus-coupling signals needed to trigger exocytosis. This suggested that syncollin was mediating its inhibitory effect downstream of secondary signals at the level of exocytosis. Consistent with this notion is that syncollin expression in ß-cells also inhibited stimulation of insulin secretion by other secretagogues, GLP-1 and glyburide.
Under normal basal conditions there are around 10 000 ß-granules per pancreatic ß-cell, mostly in a storage compartment, with only around 50–100 docked at the plasma membrane in a so-called ready releasable pool (Rorsman & Renstrom 2003). The biphasic nature of glucose-stimulated insulin secretion is thought to reflect the initial release of ß-granules from the ready releasable pool (first phase) followed by ß-granules being brought in to replenish that pool and subsequently undergo exocytosis (second phase) (Daniel et al. 1999, Bratanova-Tochkova et al. 2002). Syncollin appeared to preferentially inhibit the first phase of glucose-induced insulin secretion, although the second phase of insulin secretion was also reduced. This also suggested that syncollin was mediating its effect at the level of ß-granule exocytosis, perhaps at the distal docking and/or fusion steps. However, it should be noted that the inhibitory effect of syncollin on insulin exocytosis was only partial. This cannot be satisfactorily explained by a limitation in adenoviral-mediated gene transfer to isolated islets, which in our hands is 
80% efficient, to rat islet ß-cells. A more plausible explanation might be that syncollin is hampering ß-granule transit in and out of the ready releasable pool in ß-cells. This would be consistent with the observation that syncollin expression in anterior pituitary AtT20 cells reduced the number of secretory granules in the tips of cellular processes which, in turn, resulted in an inhibition of regulation of adrenocorticotropin secretion (Waesle et al. 2004). Since the INS-1 and islet ß-cells used in this study do not show as many cellular processes and are more populated with large dense-core granules than AtT20 cells, an identifiable distribution of ß-granules in a ready releasable pool is difficult using conventional microscopy. As such, alternative methods of fluorescently tagging different pools of ß-granules in ß-cells, perhaps using dsRed-E5(‘TIMER’) (Duncan et al. 2003), will be needed in future studies to better outline ß-granules in the ready releasable pool and see if syncollin expression alters their distribution.
Although syncollin is not normally expressed in pancreatic ß-cells, its adenoviral-mediated expression in rat islets and specific inhibition of regulated insulin secretion have revealed it as a potentially useful experimental tool to gain novel insight into the mechanism of insulin exocytosis. To date only ß-granule membrane proteins that have domains exposed on the cytosolic surface (e.g. VAMP-2 (Hua & Scheller 2001), synaptogamin-3 and -7 (Gao et al. 2000), Rab3A (Yaekura et al. 2003)) have been shown to play a role in promoting insulin exocytosis by transiently interacting with certain cytosolic and plasma membrane proteins (Rorsman & Renstrom 2003). Syncollin expression in ß-cells is localized to the lumen ß-granules and, to our knowledge, this study is the first example of a peptide having influence on the insulin exocytotic mechanism from within the ß-granule lumen. However, the mechanism as to how syncollin inhibits regulated insulin secretion is unresolved. It is noted that syncollin is able to specifically bind Ca2+ (Edwardson et al. 1997) and that intragranular Ca2+ may contribute to control of insulin secretion (Mitchell et al. 2001). Thus, one possibility is that syncollin is chelating low molecular weight and/or ionic constituents of a ß-granule lumen that in turn adversely affect insulin exocytosis. However, this seems unlikely, since syncollinGFP, which should have similar chelating ability to native syncollin, failed to inhibit insulin release. Endogenous syncollin in pancreatic exocrine cells is required for efficient regulated exocytosis (Wasle et al. 2005), so another possibility might be that syncollin expression is interfering with a ‘syncollin-like’ protein in ß-granules (as well as AtT20 cell secretory granules (Waesle et al. 2004)) that has a similar action to native syncollin in exocrine cells, rendering regulated insulin exocytosis less efficient. However, further experimentation will be required to identify a ‘syncollin-like’ protein in ß-cells (and/or AtT20 cells (Waesle et al. 2004)).
Notwithstanding, regardless of the means by which syncollin inhibits regulated insulin secretion, the evidence presented in this study unveils two novel tools that can be used to investigate the mechanism of insulin exocytosis in ß-cells: syncollinGFP, which can be utilized in real-time imaging to examine the dynamics of insulin exocytosis (Ma et al. 2004, Michael et al. 2004), and sycollin which might be used to better investigate how intragranular factors can influence regulated insulin exocytosis.
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Acknowledgements |
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Received 24 January 2005
Accepted 27 January 2005
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0.05 in Adv-Sync-Met2-infected islets versus the equivalent Adv-GFP control islets. (B) Adv-Sync-GFP-and Adv-GFP (control)-infected rat islets were analyzed for glucose-induced insulin secretion as described (Materials and Methods). The data are presented as means ± S.E. of the percentage of insulin cellular content secreted in five individual experiments.
