Glucose-Stimulated Insulin Secretion Is Coupled to the Interaction of Actin with the t-SNARE (Target Membrane Soluble N-Ethylmaleimide-Sensitive Factor Attachment Protein Receptor Protein) Complex
Debbie C. Thurmond,
Carmen Gonelle-Gispert1,
Megumi Furukawa1,
Philippe A. Halban and
Jeffrey E. Pessin
Department of Biochemistry and Molecular Biology (D.C.T.), Center for Diabetes Research, Indiana University School of Medicine, Indianapolis, Indiana 46202; Department of Physiology and Biophysics (M.F., J.E.P.), The University of Iowa, Iowa City, Iowa 52242; and Laboratoires Jeantet (C.G.-G., P.A.H.), University Medical Center, 1211 Geneva 4, Switzerland
Address all correspondence and requests for reprints to: Debbie C. Thurmond, Ph.D., Department of Biochemistry and Molecular Biology, Center for Diabetes Research, Indiana University School of Medicine, Indianapolis, Indiana 46202. E-mail: dthurmon{at}iupui.edu.
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ABSTRACT
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The actin monomer sequestering agent latrunculin B depolymerized ß-cell cortical actin, which resulted in increased glucose-stimulated insulin secretion in both cultured MIN6 ß-cells and isolated rat islet cells. In perifused islets, latrunculin B treatment increased both first- and second-phase glucose-stimulated insulin secretion without any significant effect on total insulin content. This increase in secretion was independent of calcium regulation because latrunculin B also potentiated calcium-stimulated insulin secretion in permeabilized MIN6 cells. Confocal immunofluorescent microscopy revealed a redistribution of insulin granules to the cell periphery in response to glucose or latrunculin B, which correlated with a reduction in phalloidin staining of cortical actin. Moreover, the t-SNARE [target membrane soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor] proteins Syntaxin 1 and SNAP-25 coimmunoprecipitated polymerized actin from unstimulated MIN6 cells. Glucose stimulation transiently decreased the amount of actin coimmunoprecipitated with Syntaxin 1 and SNAP-25, and latrunculin B treatment fully ablated the coimmunoprecipitation. In contrast, the actin stabilizing agent jasplakinolide increased the amount of actin coimmunoprecipitated with the t-SNARE complex and prevented its dissociation upon glucose stimulation. These data suggest a mechanism whereby glucose modulates ß-cell cortical actin organization and disrupts the interaction of polymerized actin with the plasma membrane t-SNARE complex at a distal regulatory step in the exocytosis of insulin granules.
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INTRODUCTION
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GLUCOSE STIMULATES INSULIN exocytosis by both fusion of a ready-releasable pool of plasma membrane-bound insulin granules (primed granules) and through the mobilization and trafficking of insulin granules to the cell surface from intracellular storage pools (1, 2). These events are triggered by increased glucose flux into ß-cells, resulting in increased glycolysis and an elevation in the intracellular ATP/ADP ratio (3). In turn, the increased ATP/ADP ratio results in the closure of KATP-channels (ATP-sensitive K+ channels) cell depolarization, opening of voltage-dependent calcium channels, and increased intracellular cytoplasmic calcium concentrations (4, 5, 6, 7). It is well established that this increase in intracellular calcium is a necessary trigger for insulin secretion and is thought to regulate both the initial exocytosis of primed granules and subsequent trafficking/mobilization of stored insulin granules (8, 9). However, the detailed molecular mechanisms controlling the trafficking/mobilization, docking, priming, and fusion of the insulin secretory granules have remained enigmatic (10).
In the case of intracellular vesicle trafficking, it is well established that targeting and fusion requires the pairing of vesicle compartment v-SNAREs [vesicle SNAP (soluble N-ethylmaleimide-sensitive factor attachment protein) receptors] with their cognate receptor complexes at the t-SNAREs (target membrane SNAP receptors; Refs. 11, 12, 13, 14). In the case of insulin secretion, several studies have demonstrated that Syntaxin 1A and SNAP-25 comprise the specific t-SNARE binary complex and VAMP2 (vesicle-associated membrane protein 2) functions as the requisite v-SNARE granule protein (15, 16, 17, 18, 19, 20, 21, 22). For example, toxin cleavage of either Syntaxin 1A, VAMP2, or SNAP-25 impairs ß-cell insulin secretion (10, 19, 20, 23, 24). Importantly, in vitro studies have demonstrated that the interaction of the Syntaxin 1/SNAP-25 complex with VAMP2 generates a coiled-coil bundle that can provide sufficient energy to drive the fusion reaction (25). Although these data provide a model by which fusion of primed vesicles can occur, the pathway accounting for the regulated targeting of vesicles to their cognate t-SNAREs has not been established.
The cytoskeleton framework has been well recognized as an important component in the control of vesicle trafficking and compartmentalization. Microfilaments have been implicated in several key trafficking steps and are thought to function in both a positive and negative manner depending upon the particular system. For example, depolymerization of filamentous actin (F-actin) dramatically inhibits insulin-stimulated GLUT4 (glucose transporter 4) translocation in adipocytes and actin-based motility can provide the motive force driving several membrane transport processes (26, 27, 28). In contrast, although regulated secretion of insulin granules to the cell surface also involves components of the cytoskeleton (29, 30, 31, 32), F-actin is thought to function as a negative regulator because depolymerization of F-actin with either Clostridium botulinum C2 toxin or Cytochalasin B or E potentiated glucose-stimulated insulin secretion from pancreatic islets (33, 34, 35). Similarly, treatment with the actin filament stabilizing compound jasplakinolide inhibited K+-stimulated insulin secretion (35). Consistent with this conclusion, ultrastructural analysis indicated that F-actin was organized as dense web beneath the plasma membrane that was hypothesized to impede access of insulin granules (34, 36) and correlated with an increase in F-actin in islets from fasted hamsters (37). Furthermore, isolated insulin granules were found to cosediment with actin filaments, and this interaction was decreased in the presence of calcium (38). However, glucose stimulation was reported to increase the fraction of F-actin in pancreatic ß-cell homogenates prepared from mouse islets stimulated with glucose (39). These data can be reconciled if actin remodeling and not simply actin depolymerization is necessary for glucose-stimulated insulin secretion.
In this report, we demonstrate that the actin depolymerization agent latrunculin B markedly potentiates glucose-stimulated insulin secretion in a ß-cell line and in both first and second phase insulin secretion in isolated rat islets. Actin depolymerization results in a redistribution of insulin granules to more peripheral regions of the cells in a manner that is independent of calcium channel function or calcium entry. Importantly, in the basal state F-actin was associated with the t-SNARE core complex, but this interaction is substantially reduced after glucose stimulation or latrunculin B treatment. These data suggest that the cortical actin network undergoes stimulus-dependent remodeling that increases both the number of primed insulin granules as well as mobilization of insulin granules to the plasma membrane.
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RESULTS
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Actin Depolymerization Potentiates Glucose-Stimulated Insulin Secretion in Cultured ß-Cells
To examine the function of F-actin in the regulation of insulin secretion, the effect of glucose and latrunculin B in the transformed mouse ß-cell line MIN6 was determined. Glucose stimulation of the control (vehicle treated) MIN6 cells displayed a typical time-dependent insulin secretion that increased 2.5-fold over the unstimulated cells by 10 min (Fig. 1A
). After glucose stimulation, cells pretreated with latrunculin B displayed a 4.2-fold increase in the rate of insulin secretion by 5 min. Although insulin secretion in the presence of latrunculin B no longer increased at a linear rate by 10 min, even after 60 min the secretion rate was considerably higher than that evoked by glucose alone (data not shown). This increase in secretion was not accompanied by any significant change in the total cellular content of insulin (Fig. 1B
). Because latrunculin B is a highly specific actin monomer sequestering agent, these data suggest that actin depolymerization potentiates insulin secretion in MIN6 cells.

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Figure 1. Actin Depolymerization by Latrunculin B Potentiates Glucose-Stimulated Insulin Secretion from MIN6 ß-Cells, without Affecting Cellular Insulin Content
MIN6 ß-cells were incubated in glucose-free MKRBB in the presence of 10 µM latrunculin B (LAT) or DMSO vehicle control (VEH) for 2 h followed by stimulation with 20 mM glucose over a 10-min period. A, Insulin secreted into the media over the 10-min period was measured by RIA (nanograms/milliliter media). Data shown are the average ± SE of three independent sets of experiments, vs. vehicle-treated cells after 5-min [P < 0.0005 ( )] and 10-min [P < 0.01 (*)] glucose stimulation. B, Whole-cell detergent lysates were prepared from unstimulated cells and insulin content quantitated by RIA. Data shown are the average ± SE of at three independent sets of experiments.
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The effect of latrunculin B was also assessed using phalloidin-fluorescein isothiocyanate (FITC) labeling. Untreated MIN6 cells displayed a strong distribution of F-actin as a ring beneath the plasma membrane indicative of cortical actin (Fig. 2
, panel 1). Glucose stimulation for 5 min resulted in a diminished amount of cortical actin labeling that recovered within 30 min (Fig. 2
, panels 2 and 3). As expected, latrunculin B treatment abolished cortical actin staining at all time points examined and resulted in a cellular morphology similar to cells with glucose for 5 min (Fig. 2
, compare panels 4 and 2). MIN6 ß-cells proliferate in clusters, often termed "pseudo-islets" (40), and the trans-Golgi network (TGN) marker protein Syntaxin 6 was used to delineate individual cells in each cluster (41). Neither glucose nor latrunculin B treatment had any effect on the distribution or organization of the TGN (Fig. 2
, panels 14). Taken together, these data suggest that glucose stimulation results in the transient depolymerization of cortical actin that leads to the stimulation of insulin secretion.

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Figure 2. Incubation of MIN6 ß-Cells with Glucose Dynamically Reduces Cortical Actin Staining
MIN6 ß-cells were incubated in glucose-free MKRBB in the presence of DMSO vehicle for 2 h followed by stimulation with 20 mM glucose for 0, 5, or 30 min (panels 13) or 10 µM latrunculin B followed by stimulation with glucose for 5 min (panel 4). Cells were then fixed and permeabilized with 4% paraformaldehyde and 0.1% Triton X-100 and incubated with monoclonal anti-Syntaxin 6 primary antibody for 1 h, followed by the addition of antimouse Texas Red-conjugated secondary antibody and phalloidin conjugated to FITC for an additional 1 h. The FITC and Texas Red staining was visualized by confocal microscopy (x100) at the mid-plane of the cell cluster. The images are representative of four independent sets of cells.
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Latrunculin B Potentiates Insulin Secretion at a Step Distal to the Calcium Channels
Actin has been reported to interact with L-type calcium channels, and calcium channels have been reported to interact with Syntaxin 1 (42, 43, 44). To determine whether the latrunculin B potentiation of insulin secretion was specific for glucose-stimulation or affected other pathways, we evaluated the effect of actin depolymerization on several secretagogues (Fig. 3
). After 30-min stimulation, glucose, K+, and phorbol 12-myristate 13-acetate (PMA) stimulated insulin secretion above baseline by 2.3-, 4.7-, and 3.7-fold. Pretreatment with latrunculin B resulted in a significant potentiation of insulin secretion for all three agonists compared with vehicle-treated cells. These data indicate that the effect of latrunculin B was not specific for glucose stimulation and therefore probably modulates a distal step in the insulin granule fusion/mobilization process.

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Figure 3. Potentiation of Secretagogue-Stimulated Insulin Secretion by Latrunculin B
MIN6 ß-cells were incubated in glucose-free MKRBB in the presence of 10 µM Latrunculin B or DMSO vehicle control for 2 h followed by stimulation with 20 mM glucose, 50 mM KCl, or 200 nM phorbol ester (PMA) for 30 min. Insulin secreted into the media after the 30-min period was measured by RIA. Data shown are the average ± SE of four to seven independent sets of experiments: vs. glucose-stimulated vehicle-treated cells, P < 0.0002 ( ) and vs. K+ or PMA-stimulated vehicle-treated cells. P < 0.05 (*).
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To determine more directly whether latrunculin B potentiated insulin secretion through alterations in calcium channel function, MIN6 cells were permeabilized with Streptolysin-O and incubated in a high calcium buffer to increase intracellular calcium levels independent of the calcium channel (Fig. 4
). Incubation of the permeabilized cells with 10 µM calcium resulted in increased insulin secretion compared with cells maintained at approximately resting calcium levels (0.1 µM). Importantly, pretreatment with latrunculin B resulted in an approximately 5-fold increase of insulin secretion in the permeabilized cells stimulated with 10 µM calcium. A similar latrunculin B potentiation of calcium induced insulin secretion was observed using the calcium ionophore ionomycin (data not shown). Together, these data demonstrate that the latrunculin B potentiation of insulin secretion occurs at one or more steps downstream of calcium channel function and calcium influx.

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Figure 4. Latrunculin Potentiates KATP Channel Independent Calcium-Stimulated Insulin Secretion in Permeabilized MIN6 ß-Cells
Cells were incubated in glucose-free modified permeabilization buffer for 2 h with DMSO vehicle control or latrunculin B (10 µM), Streptolysin O-permeabilized as described in Materials and Methods, and stimulated with 0.1 µM Ca2+ or 10 µM Ca2+ for 8 min. Insulin secreted into the media was assessed by RIA. Fold stimulation was calculated by setting secretion from nonstimulated (0.1 µM Ca2+) permeabilized cells equal to 1. Data shown are the average ± SE of four independent sets of experiments, P < 0.05.
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Actin Depolymerization Potentiates Glucose-Stimulated Insulin Secretion in Pancreatic Islets
To determine whether F-actin also functions in the control of insulin secretion from primary cells, isolated rat islet cells in monolayer were incubated without (control) or with latrunculin B (10 µM) for 20 min before a 1-h incubation with or without latrunculin under basal (2 mM glucose) or stimulatory conditions (20 mM glucose; Fig. 5
). Glucose stimulation resulted in an approximately 10-fold increase in insulin release compared with unstimulated control cells (Fig. 5A
). Latrunculin B pretreatment had no significant effect on basal insulin release but significantly potentiated glucose-stimulated insulin secretion by an additional 2.2-fold. The effectiveness of latrunculin B to depolymerize actin was assessed by phalloidin-RITC (rhodamine isothiocyanate) labeling in these same cells (Fig. 5B
). Untreated cells displayed a strong distribution of F-actin as a ring beneath the plasma membrane indicative of cortical actin (Fig. 5B
, panel 1). In the same cells, insulin granules were dispersed throughout the cells with only a small amount localized at the plasma membrane (Fig. 5B
, panel 5). Latrunculin B treatment disrupted this continuous ring of F-actin that became fragmented and concentrated into small clusters scattered throughout the cell (Fig. 5B
, panel 2). In parallel, insulin granules became more peripherally distributed and concentrated juxtaposed to the plasma membrane (Fig. 5B
, panel 6). Although not as effective as latrunculin B, glucose stimulation also resulted in a depolymerization of cortical actin and redistribution of insulin granules to the cell periphery (Fig. 5B
, panels 3 and 7). Similarly, the combination of latrunculin B and glucose stimulation had a marked effect on both actin organization and insulin granule distribution (Fig. 5B
, panels 4 and 8). In sum, the alterations in insulin granule distribution and actin organization correlated with the stimulation of insulin secretion.

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Figure 5. Latrunculin B Potentiates Glucose-Stimulated Insulin Release from Isolated Rat Islet Cells
A, Islets cells were preincubated for 20 min with or without latrunculin B (LAT, 10 µM) followed by an incubation under basal (2 mM glucose, Basal) or stimulated conditions (20 mM glucose, Gluc) for 1 h. Insulin secretion was measured by RIA. Data are mean ± SE, n = 4 replicates for one out of two independent experiments: vs. basal or basal + LAT P < 0.00001 ( ), and vs. glucose-stimulated P < 0.001 (*). B, At the end of these incubations, the cells were fixed and permeabilized with 4% paraformaldehyde and 0.1% Triton X-100 and incubated with monoclonal antiinsulin primary antibody for 1 h (panels 58), followed by the addition of antimouse Alexa Fluor (FITC)-conjugated secondary antibody and phalloidin conjugated to RITC for an additional 1 h (panels 14). The FITC and RITC staining was visualized by confocal microscopy (x100) at the mid-plane of the cell cluster. The images are representative of two independent sets of cells.
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To determine the effects of latrunculin on the two phases of glucose-stimulated insulin secretion, isolated rat islets were perifused and insulin secretion monitored (Fig. 6
). Islets were incubated with 2 mM glucose for 5 min, then for an additional 20 min in the absence or presence of latrunculin B, followed by 20 mM glucose stimulation for 30 min. Although insulin secretion was unaffected during the initial 20-min incubation with latrunculin B, perifusion with 20 mM glucose significantly increased insulin secretion in the latrunculin B-pretreated cells (filled squares). This marked potentiation of insulin secretion occurred throughout the time course examined and was clearly present during both the first and second phases of insulin secretion compared with untreated cells (open diamonds). Furthermore, the subsequent removal of glucose resulted in a sharp decrease in insulin secretion to near basal levels within 10 min, in the absence or presence of latrunculin B. These results indicate that depolymerization of F-actin facilitates the fusion of the readily-releasable pool of insulin granules as well as increasing the mobilization of granules to the cell surface, although secretion of insulin into the media is entirely dependent upon the presence of glucose.

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Figure 6. Latrunculin B Potentiates Both Phases of Glucose-Stimulated Insulin Secretion
After preincubation in 2 mM glucose containing KRB-HEPES buffer (basal condition), islets were loaded into perifusion chambers. Perifusion medium fractions were collected from 10 min after the beginning of the perifusion. Experimental design: 5-min basal condition (2 mM glucose, Gluc), 20-min basal condition with or without latrunculin B (2 mM Gluc ± LAT), and then 30 min under stimulatory conditions with or without latrunculin B (20 mM Gluc ± LAT). Finally islets were perifused for 15 min under basal conditions (2 mM Gluc ± LAT). The amount of insulin released from islets as well as their insulin content was determined by RIA. Insulin secretion is expressed as a percentage of insulin content. The results are mean ± SE, n = 3 replicates for one representative experiment from a total of three experiments performed, P < 0.05.
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Glucose Mediates Interaction between Filamentous Actin and t-SNARE Proteins
Having established that MIN6 cells are a useful model to study the effects of depolymerization of F-actin on insulin secretion and given the limited number of primary rat islet cells readily available for study, we used the MIN6 cells to examine the molecular interactions of actin with the SNARE complex involved in insulin granule fusion. As expected, immunoprecipitation of Syntaxin 1 from MIN6 cell extracts demonstrated the immunoprecipitation of Syntaxin 1A and the coimmunoprecipitation of SNAP-25 (Fig. 7A
, lane 1). In addition, actin was also found to coimmunoprecipitate with the Syntaxin 1 antibody. Glucose stimulation for 5 or 10 min resulted in a marked decrease in the amount of coimmunoprecipitated actin with no significant change in SNAP-25 (Fig. 7A
, lanes 2 and 3). At longer times after glucose stimulation (20 and 30 min), the amount of coimmunoprecipitated actin began to recover to near control values (Fig. 7A
, lanes 4 and 5). This interaction was shown to be specific, as IgG alone failed to immunoprecipitate actin and the LD-type calcium channel antibody was capable of immunoprecipitating SNAP-25 from unstimulated MIN6 cells as has been described (45), but no actin was associated with this complex (Fig. 7A
, lanes 7 and 8). In addition, Munc18a but not Munc18c was coimmunoprecipitated with Syntaxin 1, showing specificity for the Syntaxin 1-based complex (data not shown). No other plasma membrane or Golgi/TGN syntaxins (isoforms 2, 3, 4, or 6) were detected as being part of this complex. Moreover, the kinetics of these interactions correlate with the sharp increase rate of insulin secretion within the initial 10 min with glucose, after which the rate decreases (Fig. 7B
). In all, these data indicate that Syntaxin 1A associates with the actin cytoskeleton in ß-cell lines, and that this association is dramatically reduced precisely during the time period that we observe a sharp loss in cortical actin staining.

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Figure 7. Filamentous Actin Associates with SNARE Core Complexes, and this Association Is Dynamically Regulated by Glucose
MIN6 ß-cells were incubated in glucose-free MKRBB for 2 h followed by stimulation with 20 mM glucose from 560 min. Clarified whole-cell detergent extracts were prepared and immunoprecipitated with (A) anti-syntaxin 1 (12 µg/mg lysate protein) and (B) media removed for quantitation of insulin by RIA. Immunoprecipitates were immunoblotted with anti-syntaxin 1A, SNAP-25, and actin antibodies. Control immunoprecipitations were performed similarly using mouse IgG (lane 7) or LDCC antibodies (lane 8, HC: heavy chain). C, Detergent lysates prepared from MIN6 cells stimulated for 510 min with 20 mM glucose were immunoprecipitated with anti-SNAP-25 monoclonal antibodies and immunoblotted as described above with the addition of VAMP2 (Syntaxin 1 antibody used recognized A and B isoforms). These results are representative of 36 sets of lysates prepared from independent passages of MIN6 cells. D, MIN6 ß-cells were incubated in glucose-free MKRBB for 2 h in the presence of 10 µM latrunculin B (L) or 5 µM jasplakinolide (J) for 2 h followed by stimulation with 20 mM glucose for 5 min. Clarified whole-cell detergent extracts were prepared and immunoprecipitated with anti-Syntaxin 1 antibody as described in (A). Lysates and immunoprecipitates were immunoblotted with anti-Syntaxin 1A, SNAP-25, and actin antibodies. These results are representative of three sets of lysates prepared from independent passages of MIN6 cells.
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To confirm that the regulated interaction of actin with Syntaxin 1 was reflective of the SNARE complex, cell extracts were also immunoprecipitated with a SNAP-25 antibody (Fig. 7C
). In control cell extracts, Syntaxin 1, VAMP2, and actin were coimmunoprecipitated with SNAP-25 (Fig. 7C
, lane 1). Similar to Syntaxin 1 immunoprecipitates, glucose stimulation for 5 and 10 min resulted in a decreased coimmunoprecipitated actin with no change in the amount of coimmunoprecipitated Syntaxin 1 or VAMP2 (Fig. 7C
, lanes 2 and 3). In addition, there was no significant difference in the total cellular content or detergent extractability of Syntaxin 1, SNAP-25, VAMP2, or actin (Fig. 7C
, lanes 46). It should be noted that the SNAP-25 antibody coimmunoprecipitated both Syntaxin 1A (35 kDa) and 1B (32 kDa) isoforms, recognizable by the Syntaxin 1 antibody.
The actin polymerization state is in dynamic equilibrium between monomeric G-actin and filamentous F-actin states. To discern which form of actin was associating with the SNARE complex, cells were pretreated with either latrunculin B to decrease the amount of polymerized actin, or jasplakinolide to create and stabilize small filamentous actin oligomers (46). In addition, jasplakinolide has been previously demonstrated to inhibit calcium-stimulated insulin secretion (35). In the absence of either actin-modifying agent, actin was coimmunoprecipitated with Syntaxin 1 in unstimulated cells (Fig. 7D
, lanes 1 and 5). As previously described, glucose stimulation for 5 min resulted in a marked decrease in the amount of coimmunoprecipitated actin (Fig. 7D
, lanes 3 and 7). Depolymerization of F-actin with latrunculin B resulted in a near-complete loss of actin that could be coimmunoprecipitated with Syntaxin 1 (Fig. 7D
, lanes 2 and 4). In contrast, cells pretreated with jasplakinolide had an increased amount of actin coimmunoprecipitated with Syntaxin 1 and blunted the glucose-stimulated decrease in the coassociation of actin with Syntaxin 1A (Fig. 7D
, lanes 6 and 8). Together, these data demonstrate that F-actin and not G-actin associates with the SNARE complex, and further suggests that the glucose dependent depolymerization of F-actin accounts for the loss of this interaction.
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DISCUSSION
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Previous studies have hypothesized that the actin cytoskeleton functions as a negative regulator of insulin secretion by providing a physical barrier that blocks the access of insulin granules to the plasma membrane. This was based upon the visualization of a dense cortical actin network beneath the plasma membrane and that disruption of polymerized F-actin by various pharmacological agents facilitated glucose-stimulated insulin secretion (33, 34, 35, 36). Conversely, noradrenaline inhibition of insulin secretion occurred concomitant with a redistribution of actin to the cortical matrix (47). A negative role for cortical actin structure has also been implicated in several other secretory processes involving dense core granules. For example, depolymerization of cortical F-actin enhances granule release in chromaffin, mast, and parotid acinar cells (48, 49, 50). In addition, the sites of active secretion appear to occur in regions of the plasma membrane with relatively decreased densities of cortical actin (51). Although these data provide strong evidence for an inhibitory function of cortical actin, this has not been universally observed. Actin depolymerization appears to inhibit glucose-stimulated insulin secretion from the HIT (hamster) ß-cell line as it does the insulin-stimulated translocation of glucose transporter 4 in 3T3L1 adipocytes (26, 27, 28). Actin polymerization has also been implicated in Golgi membrane trafficking and in intracellular sorting decisions between apical and basolateral membranes in polarized epithelial cells (52). Thus, it appears that the role of actin in membrane trafficking may be cell type and/or compartment specific. Moreover, the molecular mechanisms responsible for the dynamic changes and structural elements controlling cortical F-actin have remained elusive.
Our data clearly demonstrate that depolymerization of F-actin in cultured MIN6 ß-cells and primary rat ß-cells results in a marked potentiation of glucose-stimulated insulin secretion. It is important to emphasize that latrunculin B does not initiate insulin secretion in the absence of glucose. This potentiation of glucose-stimulated insulin secretion occurs without any significant effect on basal insulin release and appears to result from an enhancement of both first and second phase insulin secretion. Although the molecular events accounting for biphasic insulin secretion are not completely resolved, it is generally believed that the first phase results from the fusion of primed granules prebound at the plasma membrane, whereas the second phase primarily reflects the recruitment of new granules to the plasma membrane (1, 2). Under these conditions, the potentiation of first phase insulin secretion by actin depolymerization could have resulted from either an increase in the efficiency of granule fusion and/or due to an increased number of granules in the primed readily releasable state. However, the potentiation of second phase insulin secretion could result from only an increase in the recruitment of new granules from intracellular storage sites. Consistent with an increase in the number of primed readily releasable granule, latrunculin B potentiation of insulin secretion occurred in conjunction with a decrease in cortical actin and a redistribution of insulin granules to the cell periphery. Although not as dramatic, glucose also induced a similar decrease in cortical actin and localization of insulin granules to the plasma membrane.
Despite these correlations, it is unclear how cortical actin actually prevents insulin granule trafficking to the plasma membrane and how glucose might be involved. However, our data provide several important clues to this process. First, it has been reported that Syntaxin 1A can interact with both the L-type calcium channel and actin, suggesting that this might result in the tether of channel function to the sites of insulin secretion (42, 43, 44). However, under these conditions depolymerization of actin would be expected to inhibit rather than potentiate Ca2+-stimulated secretion. The ability of latrunculin B to potentiate insulin secretion induced by several agonists, including elevation of intracellular Ca2+, strongly suggests events downstream of calcium channel function. However, it remains formally possible that Ca2+ entry activates an F-actin severing protein such as Scinderin or gelsolin (50, 53, 54).
Alternatively or additionally, our data demonstrate that the t-SNARE complex responsible for insulin granule plasma membrane docking and fusion (Syntaxin 1A and SNAP-25) is associated with F-actin. Importantly, glucose stimulation results in a decrease in the amount of F-actin associated with this t-SNARE complex. The temporal relationship between the potentiation of insulin secretion, peripheral redistribution of insulin granules and dissociation of F-actin from the SNARE complex provides strong evidence that this interaction is responsible for limiting the rate and extent of insulin release. In contrast, it is unclear from our results as to whether actin binds directly or indirectly to a particular protein in this SNARE core complex. At any given time the t-SNARE complex is also associated with v-SNARE protein, VAMP2. Previous studies have also observed that purified insulin granules can be copurified along with F-actin (39). Thus, it is unclear whether the association of F-actin reflects direct binding to one of the SNARE proteins, the core complex and/or indirectly through another actin binding adaptor. For example, the SNAP-25-interacting protein colocalizes with SNAP-25 and actin, and myosin V can bind to VAMP2 (55, 56). In any case, based upon these data we propose that glucose signals the transient reduction of F-cortical actin, disengaging actin tethering to the SNARE complex to promote granule exocytosis.
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MATERIALS AND METHODS
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Materials
The RIA-grade BSA, D-glucose, and phalloidin-conjugated FITC were obtained from Sigma (St. Louis, MO). Monoclonal anti-Syntaxin 6 and anti-SNAP-25 (used for immunofluorescence and immunoblotting) antibodies were obtained from BD Transduction Laboratories (Lexington, KY). The monoclonal SNAP-25 antibody used for immunoprecipitation was obtained from Sternberger Monoclonals, Inc. (Lutherville, MD). The monoclonal VAMP2 and rabbit polyclonal actin antibodies were purchased from Synaptic Systems (Gottingen, Germany) and Sigma, respectively. Monoclonal anti-Syntaxin antibody was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY) for use in immunoprecipitation, and monoclonal anti-Syntaxin 1A was purchased from StressGen (Victoria, British Columbia, Canada) for immunoblotting. Latrunculin B and jasplakinolide were purchased from Calbiochem (La Jolla, CA) and Molecular Probes, Inc. (Eugene, OR), respectively. Streptolysin-O and PMA were purchased from Sigma. Texas Red-conjugated secondary antibody and control mouse IgG for immunoprecipitation were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Vectashield was obtained from Vector Laboratories, Inc. (Burlingame, CA). The MIN6 cells were a gift from Dr. John Hutton (University of Colorado Health Sciences Center). ECL kit and Hyperfilm-MP were obtained from Amersham Biosciences (Piscataway, NJ).
Isolation of Rat Islets and Monolayer Culture of Islet Cells
All animal experimentation described was conducted in accord with accepted standards of humane animal care, and studies were approved by the authors institutional committee on animal care. Pancreatic rat islets of Langerhans were obtained as described previously (57). Briefly, pancreata from eight rats were digested with collagenase in Ca2+-containing Hanks buffer and islets of Langerhans purified from exocrine tissue by discontinuous density-gradient centrifugation (Histopaque 1077 from Sigma). For monolayer culture, freshly isolated islets were trypsinized for 6 min to obtain a single cell suspension and seeded overnight in nonadherent Petri dishes in DMEM containing 11.2 mM glucose. The next day cells were established in monolayer using Petri dishes coated with 804G matrix (produced by a rat bladder carcinoma cell line) and used 6 h later for insulin secretion measurements and fixation for immunofluorescence studies.
Islet Perifusion
Islets were allowed to recover overnight in culture at 37 C. Islets were washed twice and preincubated for 45 min in Krebs-Ringer bicarbonate buffer (KRB-HEPES: 134 mM NaCl; 4.8 mM KCl; 1 mM CaCl2; 1.2 mM MgSO4; 1.2 mM KH2PO4; 5 mM NaHCO3; 0.1% BSA; and 10 mM HEPES, pH 7.4) containing 2 mM glucose (basal condition). Isolated islets (30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45) were hand picked and then perifused for an additional 15 min in basal conditions at a flow rate of 0.5 ml/min to stabilize secretion and to establish basal insulin secretory rates. Media were collected after the first 10 min of perifusion. After the stabilization period, islets were perifused under basal conditions with or without latrunculin B (10 µM) for 20 min, followed by incubation with or without latrunculin under stimulatory conditions (20 mM glucose) for 30 min. All perifusate solutions were maintained at 37 C. The amount of insulin released into the media as well as the insulin content of islets was determined by RIA.
Cell Culture, Insulin Secretion, and Insulin Content Assays
MIN6 cells were cultured in DMEM (25 mM glucose) equilibrated with 5% CO2 at 37 C. The medium was supplemented with 15% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin, L-glutamine, and 50 mM ß-mercaptoethanol, as described (58). MIN6 cells were used between passages 49 and 56. Insulin secretion was determined using cells grown in 35-mm wells to 6080% confluence. Cells were washed twice with and incubated for 2 h in 1 ml modified Krebs ringer bicarbonate buffer (MKRBB: 5 mM KCl; 120 mM NaCl; 15 mM HEPES, pH 7.4; 24 mM NaHCO3; 1 mM MgCl2; 2 mM CaCl2; and 1 mg/ml BSA (RIA grade) that was gassed in 95% O2 for 30 min. Latrunculin B (10 µM) and jasplakinolide (5 µM) were solubilized in dimethylsulfoxide (DMSO; vehicle) and added to the MKRBB for the 2-h preincubation period where described in the figure legend. Cells were stimulated with the secretagogue (20 mM glucose, 50 mM KCl, or 200 nM PMA) for the time listed in the figure legend, after which the media were collected, microcentrifuged for 5 min at 4 C to pellet cell debris, and stored at -20 C. Insulin secreted into the medium was quantitated using a rat insulin immunoassay kit (Linco Research, Inc., St. Charles, MO). Insulin content was quantitated in cleared cell detergent lysates made from cells incubated in MKRBB containing latrunculin B (10 µM) or DMSO control for 2 h. Media containing secreted insulin was removed, cells were harvested in Nonidet P-40 detergent lysis buffer (25 mM Tris, pH 7.4; 1% Nonidet P-40; 10% glycerol; 50 mM NaF; 10 mM Na4P2O7; 137 mM NaCl; 1 mM Na3VO4; 1 mM phenylmethylsulfonyl fluoride; 10 µg/ml aprotinin; 1 µg/ml pepstatin; and 5 µg/ml leupeptin) and lysed for 10 min at 4 C, and lysates cleared of insoluble material by microcentrifugation for 10 min at 4 C. Insulin content of the cells was measured by RIA. For the insulin secretion measurements from primary islet cells, cells were cultured in monolayer as described above. Cells were washed and preincubated for 30 min in KRB-HEPES containing 2 mM glucose (basal condition). Cells were then incubated further for 20 min in basal conditions with or without latrunculin B (10 µM) followed by incubation for 1 h in the basal (2 mM glucose) or stimulatory condition (20 mM glucose) with or without latrunculin. Media were collected and centrifuged to remove any detached cells. The amount of insulin released under basal and stimulated conditions was determined using an ultrasensitive rat insulin ELISA Kit (Mercodia, Uppsala, Sweden).
Immunofluorescence and Confocal Microscopy
For whole-cell time courses, MIN6 cells at 40% confluency plated onto glass coverslips were incubated in MKRBB for 2 h followed by stimulation with either 20 mM glucose or 50 mM KCl over the time courses shown in the figure and then fixed and permeabilized in 4% paraformaldehyde and 0.1% Triton X-100 for 10 min at 4 C. Primary rat islet cells were similarly fixed and permeabilized before or after stimulation by glucose with or without latrunculin as indicated. Fixed cells were blocked in 1% BSA and 5% donkey serum for 1 h at room temperature, followed by incubation with Syntaxin 6 antibody (1:100) for 1 h. MIN6 cells were then washed with PBS and incubated with phalloidin-conjugated FITC (1:1000) and antimouse Texas Red secondary antibody for 1 h. Islet cells were incubated with the monoclonal insulin antibody at 1:100 for 1 h followed by donkey antimouse IgG-Alexa Fluor (Molecular Probes, Inc.) incubation at 1:1000 for 40 min. F-actin was stained with 100 nM RITC-labeled phalloidin in 1% BSA/PBS solution with the secondary antibody. All cells were washed again in PBS, overlayed with Vectashield mounting medium and mounted for confocal fluorescence microscopy using a Carl Zeiss (Jena, Germany) 510 confocal microscope.
Streptolysin-O Permeabilization
MIN6 cells at 60% confluence were washed twice with MKRBB and incubated for 2 h at 37 C in the presence of latrunculin (10 µM) or DMSO vehicle control. Cells were then permeabilized in low calcium glutamate buffer for permeabilization (128 mM potassium glutamate; 5 mM sodium ATP; 5 mM MgSO4; 10.2 mM EGTA; 20 mM HEPES, pH 7.4, plus 0.5 mM CaCl2) containing freshly prepared Streptolysin-O solution (12.5 µg/ml), as described (23, 59). After 7 min of permeabilization at 37 C buffer was aspirated and replaced with either low-calcium (0.1 µM) or high-calcium (10 µM) glutamate buffer for 8 min at 37 C, after which media was quickly removed for insulin RIA analysis as described above. Propidium iodide staining was used to confirm permeabilization in every experiment.
Coimmunoprecipitation and Immunoblotting
Cleared detergent lysates were prepared as described above from cells incubated in MRKBB buffer, in the absence or presence of actin-modifying reagents as indicated, for 2 h at 37 C and stimulated with glucose for the times indicated in figure. Lysate (0.91.4 mg) was combined with 1 µg of Syntaxin (Upstate Biotechnology) or SNAP-25 (Sternberger Monoclonals) monoclonal antibody for 2 h at 4 C followed by a second incubation with protein G Plus Sepharose for 2 h. The resulting immunoprecipitates were subjected to electrophoresis on 12% SDS-PAGE followed by transfer to PVDF membrane. Membranes were blocked in 5% milk/TBS-Tween for 1 h at room temperature, followed by immunoblotting with primary antibodies for 1 h (Syntaxin 1A, SNAP-25, and actin diluted at 1:1000; VAMP2 at 1:10,000). Membranes were rinsed in TBS-Tween and further incubated in secondary antibodies conjugated to horseradish peroxidase diluted at 1:5000 for 1 h, rinsed again, and proteins visualized by enhanced chemiluminescence.
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ACKNOWLEDGMENTS
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We wish to thank Dr. John Hutton for the MIN6 cells. We also are grateful to Diana Boeglin and Angela Nevins for technical expertise.
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FOOTNOTES
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1 C.G.-G. and M.F. contributed equally to the study 
This work was supported by research grants from the Indiana University School of Medicine Diabetes Research and Training Center Pilot project grant P60-DK-20542 from the NIH, the Indiana University School of Medicine Biomedical Research Fund, and from the Swiss National Science Fund Grant 3200-06177.00.
Abbreviations: FITC, Fluorescein isothiocyanate; KATP- channels, ATP-sensitive K+ channels; KRB, Krebs-Ringer bicarbonate; MKRBB, modified KRB buffer; PMA, phorbol 12-myristate 13-acetate; RITC, rhodamine isothiocyanate; SNAP, soluble N-ethylmaleimide-sensitive factor attachment protein; SNARE, SNAP receptor; TGN, trans-Golgi network; t-SNAREs, target membrane SNAP receptors; VAMP2, vesicle-associated membrane protein 2; v-SNAREs, vesicle SNAP receptors.
Received for publication September 25, 2002.
Accepted for publication January 10, 2003.
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