Glucose-Stimulated Insulin Secretion Is Coupled to

Glucose-Stimulated Insulin Secretion Is Coupled tothe Interaction of Actin with the t-SNARE (TargetMembrane Soluble N-Ethylmaleimide-SensitiveFactor Attachment Protein ReceptorProtein) Complex

DEBBIE C. THURMOND, CARMEN GONELLE-GISPERT*, MEGUMI FURUKAWA*,PHILIPPE A. HALBAN, AND JEFFREY E. PESSIN

Department of Biochemistry and Molecular Biology (D.C.T.), Center for Diabetes Research, IndianaUniversity School of Medicine, Indianapolis, Indiana 46202; Department of Physiology andBiophysics (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

The actin monomer sequestering agent latrunculinB depolymerized �-cell cortical actin, which re-sulted in increased glucose-stimulated insulin se-cretion in both cultured MIN6 �-cells and isolatedrat islet cells. In perifused islets, latrunculin B treat-ment increased both first- and second-phaseglucose-stimulated insulin secretion without anysignificant effect on total insulin content. This in-crease in secretion was independent of calciumregulation because latrunculin B also potentiatedcalcium-stimulated insulin secretion in permeabil-ized MIN6 cells. Confocal immunofluorescent mi-croscopy revealed a redistribution of insulin gran-ules to the cell periphery in response to glucose orlatrunculin B, which correlated with a reduction inphalloidin staining of cortical actin. Moreover, thet-SNARE [target membrane soluble N-ethylmale-imide-sensitive factor attachment protein (SNAP)

receptor] proteins Syntaxin 1 and SNAP-25 coim-munoprecipitated polymerized actin from unstimu-lated MIN6 cells. Glucose stimulation transientlydecreased the amount of actin coimmunoprecipi-tated with Syntaxin 1 and SNAP-25, and latrunculinB treatment fully ablated the coimmunoprecipita-tion. In contrast, the actin stabilizing agent jas-plakinolide increased the amount of actin coimmu-noprecipitated with the t-SNARE complex andprevented its dissociation upon glucose stimula-tion. These data suggest a mechanism wherebyglucose modulates �-cell cortical actin organiza-tion and disrupts the interaction of polymerizedactin with the plasma membrane t-SNARE com-plex at a distal regulatory step in the exocytosis ofinsulin granules. (Molecular Endocrinology 17:732–742, 2003)

GLUCOSE STIMULATES INSULIN exocytosis byboth fusion of a ready-releasable pool of plasma

membrane-bound insulin granules (primed granules)and through the mobilization and trafficking of insulingranules to the cell surface from intracellular storagepools (1, 2). These events are triggered by increasedglucose flux into �-cells, resulting in increased glyco-lysis and an elevation in the intracellular ATP/ADP ratio(3). In turn, the increased ATP/ADP ratio results in theclosure of KATP-channels (ATP-sensitive K� channels)cell depolarization, opening of voltage-dependent cal-cium channels, and increased intracellular cytoplas-mic calcium concentrations (4–7). It is well established

that this increase in intracellular calcium is a necessarytrigger for insulin secretion and is thought to regulateboth the initial exocytosis of primed granules and sub-sequent trafficking/mobilization of stored insulin gran-ules (8, 9). However, the detailed molecular mecha-nisms controlling the trafficking/mobilization, docking,priming, and fusion of the insulin secretory granuleshave remained enigmatic (10).

In the case of intracellular vesicle trafficking, it is wellestablished that targeting and fusion requires the pair-ing of vesicle compartment v-SNAREs [vesicle SNAP(soluble N-ethylmaleimide-sensitive factor attachmentprotein) receptors] with their cognate receptor com-plexes at the t-SNAREs (target membrane SNAP re-ceptors; Refs. 11–14). In the case of insulin secretion,several studies have demonstrated that Syntaxin 1Aand SNAP-25 comprise the specific t-SNARE binarycomplex and VAMP2 (vesicle-associated membraneprotein 2) functions as the requisite v-SNARE granuleprotein (15–22). For example, toxin cleavage of eitherSyntaxin 1A, VAMP2, or SNAP-25 impairs �-cell insu-

Abbreviations: FITC, Fluorescein isothiocyanate; KATP-channels, ATP-sensitive K� channels; KRB, Krebs-Ringer bi-carbonate; MKRBB, modified KRB buffer; PMA, phorbol 12-myristate 13-acetate; RITC, rhodamine isothiocyanate;SNAP, soluble N-ethylmaleimide-sensitive factor attachmentprotein; SNARE, SNAP receptor; TGN, trans-Golgi network;t-SNAREs, target membrane SNAP receptors; VAMP2, ves-icle-associated membrane protein 2; v-SNAREs, vesicleSNAP receptors.

0888-8809/03/$15.00/0 Molecular Endocrinology 17(4):732–742Printed in U.S.A. Copyright © 2003 by The Endocrine Society

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lin secretion (10, 19, 20, 23, 24). Importantly, in vitrostudies have demonstrated that the interaction of theSyntaxin 1/SNAP-25 complex with VAMP2 generatesa coiled-coil bundle that can provide sufficient energyto drive the fusion reaction (25). Although these dataprovide a model by which fusion of primed vesiclescan occur, the pathway accounting for the regulatedtargeting of vesicles to their cognate t-SNAREs hasnot been established.

The cytoskeleton framework has been well recog-nized as an important component in the control ofvesicle trafficking and compartmentalization. Micro-filaments have been implicated in several key traffick-ing steps and are thought to function in both a positiveand negative manner depending upon the particularsystem. For example, depolymerization of filamentousactin (F-actin) dramatically inhibits insulin-stimulatedGLUT4 (glucose transporter 4) translocation in adipo-cytes and actin-based motility can provide the motiveforce driving several membrane transport processes(26–28). In contrast, although regulated secretion ofinsulin granules to the cell surface also involves com-ponents of the cytoskeleton (29–32), F-actin is thoughtto function as a negative regulator because depoly-merization of F-actin with either Clostridium botulinumC2 toxin or Cytochalasin B or E potentiated glucose-stimulated insulin secretion from pancreatic islets(33–35). Similarly, treatment with the actin filamentstabilizing compound jasplakinolide inhibited K�-stim-ulated insulin secretion (35). Consistent with this con-clusion, ultrastructural analysis indicated that F-actinwas organized as dense web beneath the plasmamembrane that was hypothesized to impede access ofinsulin granules (34, 36) and correlated with an in-crease in F-actin in islets from fasted hamsters (37).Furthermore, isolated insulin granules were found tocosediment with actin filaments, and this interactionwas decreased in the presence of calcium (38). How-ever, glucose stimulation was reported to increase thefraction of F-actin in pancreatic �-cell homogenatesprepared from mouse islets stimulated with glucose(39). These data can be reconciled if actin remodelingand not simply actin depolymerization is necessary forglucose-stimulated insulin secretion.

In this report, we demonstrate that the actin depo-lymerization agent latrunculin B markedly potentiatesglucose-stimulated insulin secretion in a �-cell lineand in both first and second phase insulin secretion inisolated rat islets. Actin depolymerization results in aredistribution of insulin granules to more peripheralregions of the cells in a manner that is independent ofcalcium channel function or calcium entry. Impor-tantly, in the basal state F-actin was associated withthe t-SNARE core complex, but this interaction is sub-stantially reduced after glucose stimulation or latrun-culin B treatment. These data suggest that the corticalactin network undergoes stimulus-dependent remod-eling that increases both the number of primed insulingranules as well as mobilization of insulin granules tothe plasma membrane.

RESULTS

Actin Depolymerization Potentiates Glucose-Stimulated Insulin Secretion in Cultured �-Cells

To examine the function of F-actin in the regulation ofinsulin secretion, the effect of glucose and latrunculinB in the transformed mouse �-cell line MIN6 was de-termined. Glucose stimulation of the control (vehicletreated) MIN6 cells displayed a typical time-dependentinsulin secretion that increased 2.5-fold over the un-stimulated cells by 10 min (Fig. 1A). After glucosestimulation, cells pretreated with latrunculin B dis-played a 4.2-fold increase in the rate of insulin secre-tion by 5 min. Although insulin secretion in the pres-ence of latrunculin B no longer increased at a linearrate by 10 min, even after 60 min the secretion ratewas considerably higher than that evoked by glucosealone (data not shown). This increase in secretion wasnot accompanied by any significant change in the totalcellular content of insulin (Fig. 1B). Because latrunculinB is a highly specific actin monomer sequesteringagent, these data suggest that actin depolymerizationpotentiates insulin secretion in MIN6 cells.

The effect of latrunculin B was also assessed usingphalloidin-fluorescein isothiocyanate (FITC) labeling.Untreated MIN6 cells displayed a strong distribution ofF-actin as a ring beneath the plasma membrane indic-ative of cortical actin (Fig. 2, panel 1). Glucose stimu-lation for 5 min resulted in a diminished amount ofcortical actin labeling that recovered within 30 min(Fig. 2, panels 2 and 3). As expected, latrunculin Btreatment abolished cortical actin staining at all timepoints examined and resulted in a cellular morphologysimilar to cells with glucose for 5 min (Fig. 2, comparepanels 4 and 2). MIN6 �-cells proliferate in clusters,often termed “pseudo-islets” (40), and the trans-Golginetwork (TGN) marker protein Syntaxin 6 was used todelineate individual cells in each cluster (41). Neitherglucose nor latrunculin B treatment had any effect onthe distribution or organization of the TGN (Fig. 2,panels 1–4). Taken together, these data suggest thatglucose stimulation results in the transient depolymer-ization of cortical actin that leads to the stimulation ofinsulin secretion.

Latrunculin B Potentiates Insulin Secretion at aStep Distal to the Calcium Channels

Actin has been reported to interact with L-type cal-cium channels, and calcium channels have been re-ported to interact with Syntaxin 1 (42–44). To deter-mine whether the latrunculin B potentiation of insulinsecretion was specific for glucose-stimulation or af-fected other pathways, we evaluated the effect of actindepolymerization on several secretagogues (Fig. 3).After 30-min stimulation, glucose, K�, and phorbol12-myristate 13-acetate (PMA) stimulated insulin se-cretion above baseline by 2.3-, 4.7-, and 3.7-fold.Pretreatment with latrunculin B resulted in a significant

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potentiation of insulin secretion for all three agonistscompared with vehicle-treated cells. These data indi-cate that the effect of latrunculin B was not specific forglucose stimulation and therefore probably modulatesa distal step in the insulin granule fusion/mobilizationprocess.

To determine more directly whether latrunculin Bpotentiated insulin secretion through alterations in cal-cium channel function, MIN6 cells were permeabilizedwith Streptolysin-O and incubated in a high calciumbuffer to increase intracellular calcium levels indepen-dent of the calcium channel (Fig. 4). Incubation of thepermeabilized cells with 10 �M calcium resulted inincreased insulin secretion compared with cells main-tained at approximately resting calcium levels (0.1 �M).Importantly, pretreatment with latrunculin B resulted inan approximately 5-fold increase of insulin secretion inthe permeabilized cells stimulated with 10 �M calcium.A similar latrunculin B potentiation of calcium inducedinsulin secretion was observed using the calcium iono-phore ionomycin (data not shown). Together, thesedata demonstrate that the latrunculin B potentiation ofinsulin secretion occurs at one or more steps down-stream of calcium channel function and calcium influx.

Actin Depolymerization Potentiates Glucose-Stimulated Insulin Secretion in Pancreatic Islets

To determine whether F-actin also functions in thecontrol of insulin secretion from primary cells, isolatedrat islet cells in monolayer were incubated without(control) or with latrunculin B (10 �M) for 20 min beforea 1-h incubation with or without latrunculin underbasal (2 mM glucose) or stimulatory conditions (20 mM

glucose; Fig. 5). Glucose stimulation resulted in anapproximately 10-fold increase in insulin release com-pared with unstimulated control cells (Fig. 5A). Latrun-culin B pretreatment had no significant effect on basalinsulin release but significantly potentiated glucose-stimulated insulin secretion by an additional 2.2-fold.The effectiveness of latrunculin B to depolymerize ac-tin was assessed by phalloidin-RITC (rhodamine iso-thiocyanate) labeling in these same cells (Fig. 5B).Untreated cells displayed a strong distribution of F-actin as a ring beneath the plasma membrane indica-tive of cortical actin (Fig. 5B, panel 1). In the samecells, insulin granules were dispersed throughout thecells with only a small amount localized at the plasmamembrane (Fig. 5B, panel 5). Latrunculin B treatmentdisrupted this continuous ring of F-actin that becamefragmented and concentrated into small clusters scat-tered throughout the cell (Fig. 5B, panel 2). In parallel,insulin granules became more peripherally distributedand concentrated juxtaposed to the plasma mem-brane (Fig. 5B, panel 6). Although not as effective aslatrunculin B, glucose stimulation also resulted in adepolymerization of cortical actin and redistribution ofinsulin granules to the cell periphery (Fig. 5B, panels 3and 7). Similarly, the combination of latrunculin B andglucose stimulation had a marked effect on both actinorganization and insulin granule distribution (Fig. 5B,panels 4 and 8). In sum, the alterations in insulin gran-ule distribution and actin organization correlated withthe stimulation of insulin secretion.

To determine the effects of latrunculin on the twophases of glucose-stimulated insulin secretion, iso-

Fig. 1. Actin Depolymerization by Latrunculin B PotentiatesGlucose-Stimulated Insulin Secretion from MIN6 �-Cells,without Affecting Cellular Insulin Content

MIN6 �-cells were incubated in glucose-free MKRBB in thepresence of 10 �M latrunculin B (LAT) or DMSO vehicle control(VEH) for 2 h followed by stimulation with 20 mM glucose over a10-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 ofexperiments, vs. vehicle-treated cells after 5-min [P � 0.0005(‡)] and 10-min [P � 0.01 (*)] glucose stimulation. B, Whole-celldetergent lysates were prepared from unstimulated cells andinsulin content quantitated by RIA. Data shown are the aver-age � SE of at three independent sets of experiments.

734 Mol Endocrinol, April 2003, 17(4):732–742 Thurmond et al. • Glucose Regulates Actin-SNARE AssociationD

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lated rat islets were perifused and insulin secretionmonitored (Fig. 6). Islets were incubated with 2 mM

glucose for 5 min, then for an additional 20 min in theabsence or presence of latrunculin B, followed by 20mM glucose stimulation for 30 min. Although insulinsecretion was unaffected during the initial 20-min in-cubation with latrunculin B, perifusion with 20 mM

glucose significantly increased insulin secretion in thelatrunculin B-pretreated cells (filled squares). Thismarked potentiation of insulin secretion occurredthroughout the time course examined and was clearlypresent during both the first and second phases ofinsulin secretion compared with untreated cells (opendiamonds). Furthermore, the subsequent removal ofglucose resulted in a sharp decrease in insulin secre-tion to near basal levels within 10 min, in the absenceor presence of latrunculin B. These results indicatethat depolymerization of F-actin facilitates the fusion

of the readily-releasable pool of insulin granules as wellas increasing the mobilization of granules to the cellsurface, although secretion of insulin into the media isentirely dependent upon the presence of glucose.

Glucose Mediates Interaction betweenFilamentous Actin and t-SNARE Proteins

Having established that MIN6 cells are a useful modelto study the effects of depolymerization of F-actin oninsulin secretion and given the limited number of pri-mary rat islet cells readily available for study, we usedthe MIN6 cells to examine the molecular interactionsof actin with the SNARE complex involved in insulingranule fusion. As expected, immunoprecipitation ofSyntaxin 1 from MIN6 cell extracts demonstrated theimmunoprecipitation of Syntaxin 1A and the coimmu-noprecipitation of SNAP-25 (Fig. 7A, lane 1). In addi-

Fig. 2. Incubation of MIN6 �-Cells with Glucose Dynamically Reduces Cortical Actin StainingMIN6 �-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 1–3) or 10 �M latrunculin B followed by stimulation with glucose for 5 min (panel 4). Cellswere 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 andphalloidin conjugated to FITC for an additional 1 h. The FITC and Texas Red staining was visualized by confocal microscopy(�100) at the mid-plane of the cell cluster. The images are representative of four independent sets of cells.


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tion, actin was also found to coimmunoprecipitate withthe Syntaxin 1 antibody. Glucose stimulation for 5 or10 min resulted in a marked decrease in the amount ofcoimmunoprecipitated actin with no significantchange in SNAP-25 (Fig. 7A, lanes 2 and 3). At longertimes after glucose stimulation (20 and 30 min), theamount of coimmunoprecipitated actin began to re-cover to near control values (Fig. 7A, lanes 4 and 5).This interaction was shown to be specific, as IgG alonefailed to immunoprecipitate actin and the LD-type cal-cium channel antibody was capable of immunopre-cipitating SNAP-25 from unstimulated MIN6 cells ashas been described (45), but no actin was associatedwith this complex (Fig. 7A, lanes 7 and 8). In addition,Munc18a but not Munc18c was coimmunoprecipi-tated with Syntaxin 1, showing specificity for the Syn-taxin 1-based complex (data not shown). No otherplasma membrane or Golgi/TGN syntaxins (isoforms2, 3, 4, or 6) were detected as being part of thiscomplex. Moreover, the kinetics of these interactionscorrelate with the sharp increase rate of insulin secre-tion within the initial 10 min with glucose, after whichthe rate decreases (Fig. 7B). In all, these data indicatethat Syntaxin 1A associates with the actin cytoskele-ton in �-cell lines, and that this association is dramat-ically reduced precisely during the time period that weobserve a sharp loss in cortical actin staining.

To confirm that the regulated interaction of actin withSyntaxin 1 was reflective of the SNARE complex, cellextracts were also immunoprecipitated with a SNAP-25antibody (Fig. 7C). In control cell extracts, Syntaxin 1,VAMP2, and actin were coimmunoprecipitated withSNAP-25 (Fig. 7C, lane 1). Similar to Syntaxin 1 immu-

noprecipitates, glucose stimulation for 5 and 10 min re-sulted in a decreased coimmunoprecipitated actin withno change in the amount of coimmunoprecipitated Syn-taxin 1 or VAMP2 (Fig. 7C, lanes 2 and 3). In addition,there was no significant difference in the total cellularcontent or detergent extractability of Syntaxin 1, SNAP-25, VAMP2, or actin (Fig. 7C, lanes 4–6). It should benoted that the SNAP-25 antibody coimmunoprecipitatedboth Syntaxin 1A (35 kDa) and 1B (32 kDa) isoforms,recognizable by the Syntaxin 1 antibody.

The actin polymerization state is in dynamic equilib-rium between monomeric G-actin and filamentous F-actin states. To discern which form of actin was associ-ating with the SNARE complex, cells were pretreatedwith either latrunculin B to decrease the amount of po-lymerized actin, or jasplakinolide to create and stabilizesmall filamentous actin oligomers (46). In addition, jas-plakinolide has been previously demonstrated to inhibitcalcium-stimulated insulin secretion (35). In the absenceof either actin-modifying agent, actin was coimmunopre-cipitated with Syntaxin 1 in unstimulated cells (Fig. 7D,lanes 1 and 5). As previously described, glucose stimu-lation for 5 min resulted in a marked decrease in theamount of coimmunoprecipitated actin (Fig. 7D, lanes 3and 7). Depolymerization of F-actin with latrunculin Bresulted in a near-complete loss of actin that could becoimmunoprecipitated with Syntaxin 1 (Fig. 7D, lanes 2and 4). In contrast, cells pretreated with jasplakinolidehad an increased amount of actin coimmunoprecipitatedwith Syntaxin 1 and blunted the glucose-stimulated de-

Fig. 3. Potentiation of Secretagogue-Stimulated Insulin Se-cretion by Latrunculin B

MIN6 �-cells were incubated in glucose-free MKRBB in thepresence of 10 �M Latrunculin B or DMSO vehicle control for2 h followed by stimulation with 20 mM glucose, 50 mM KCl,or 200 nM phorbol ester (PMA) for 30 min. Insulin secretedinto the media after the 30-min period was measured by RIA.Data shown are the average � SE of four to seven indepen-dent sets of experiments: vs. glucose-stimulated vehicle-treated cells, P � 0.0002 (‡) and vs. K� or PMA-stimulatedvehicle-treated cells. P � 0.05 (*).

Fig. 4. Latrunculin Potentiates KATP Channel IndependentCalcium-Stimulated Insulin Secretion in Permeabilized MIN6�-Cells

Cells were incubated in glucose-free modified permeabi-lization buffer for 2 h with DMSO vehicle control or latrunculinB (10 �M), Streptolysin O-permeabilized as described in Ma-terials and Methods, and stimulated with 0.1 �M Ca2� or 10�M Ca2� for 8 min. Insulin secreted into the media wasassessed by RIA. Fold stimulation was calculated by settingsecretion from nonstimulated (0.1 �M Ca2�) permeabilizedcells equal to 1. Data shown are the average � SE of fourindependent sets of experiments, P � 0.05.


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crease in the coassociation of actin with Syntaxin 1A(Fig. 7D, lanes 6 and 8). Together, these data demon-strate that F-actin and not G-actin associates with theSNARE complex, and further suggests that the glucosedependent depolymerization of F-actin accounts for theloss of this interaction.

DISCUSSION

Previous studies have hypothesized that the actin cy-toskeleton functions as a negative regulator of insulin

secretion by providing a physical barrier that blocksthe access of insulin granules to the plasma mem-brane. This was based upon the visualization of adense cortical actin network beneath the plasmamembrane and that disruption of polymerized F-actinby various pharmacological agents facilitated glu-cose-stimulated insulin secretion (33–36). Conversely,noradrenaline inhibition of insulin secretion occurredconcomitant with a redistribution of actin to the corti-cal matrix (47). A negative role for cortical actin struc-ture has also been implicated in several other secre-tory processes involving dense core granules. For

Fig. 5. Latrunculin B Potentiates Glucose-Stimulated Insulin Release from Isolated Rat Islet CellsA, Islets cells were preincubated for 20 min with or without latrunculin B (LAT, 10 �M) 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 aremean � 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 5–8),followed by the addition of antimouse Alexa Fluor (FITC)-conjugated secondary antibody and phalloidin conjugated to RITC foran additional 1 h (panels 1–4). The FITC and RITC staining was visualized by confocal microscopy (�100) at the mid-plane of thecell cluster. The images are representative of two independent sets of cells.


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example, depolymerization of cortical F-actin en-hances granule release in chromaffin, mast, and pa-rotid acinar cells (48–50). In addition, the sites of activesecretion appear to occur in regions of the plasmamembrane with relatively decreased densities of cor-tical actin (51). Although these data provide strongevidence for an inhibitory function of cortical actin, thishas not been universally observed. Actin depolymer-ization appears to inhibit glucose-stimulated insulinsecretion from the HIT (hamster) �-cell line as it doesthe insulin-stimulated translocation of glucose trans-porter 4 in 3T3L1 adipocytes (26–28). Actin polymer-ization has also been implicated in Golgi membranetrafficking and in intracellular sorting decisions be-tween apical and basolateral membranes in polarizedepithelial cells (52). Thus, it appears that the role ofactin in membrane trafficking may be cell type and/orcompartment specific. Moreover, the molecular mech-anisms responsible for the dynamic changes andstructural elements controlling cortical F-actin haveremained elusive.

Our data clearly demonstrate that depolymerizationof F-actin in cultured MIN6 �-cells and primary rat�-cells results in a marked potentiation of glucose-stimulated insulin secretion. It is important to empha-size that latrunculin B does not initiate insulin secretionin the absence of glucose. This potentiation of glu-cose-stimulated insulin secretion occurs without anysignificant effect on basal insulin release and appearsto result from an enhancement of both first and sec-ond phase insulin secretion. Although the molecularevents accounting for biphasic insulin secretion arenot completely resolved, it is generally believed that

the first phase results from the fusion of primed gran-ules prebound at the plasma membrane, whereas thesecond phase primarily reflects the recruitment of newgranules to the plasma membrane (1, 2). Under theseconditions, the potentiation of first phase insulin se-cretion by actin depolymerization could have resultedfrom either an increase in the efficiency of granulefusion and/or due to an increased number of granulesin the primed readily releasable state. However, thepotentiation of second phase insulin secretion couldresult from only an increase in the recruitment of newgranules from intracellular storage sites. Consistentwith an increase in the number of primed readily re-leasable granule, latrunculin B potentiation of insulinsecretion occurred in conjunction with a decrease incortical actin and a redistribution of insulin granules tothe cell periphery. Although not as dramatic, glucosealso induced a similar decrease in cortical actin andlocalization of insulin granules to the plasmamembrane.

Despite these correlations, it is unclear how corticalactin actually prevents insulin granule trafficking to theplasma membrane and how glucose might be in-volved. However, our data provide several importantclues to this process. First, it has been reported thatSyntaxin 1A can interact with both the L-type calciumchannel and actin, suggesting that this might result inthe tether of channel function to the sites of insulinsecretion (42–44). However, under these conditionsdepolymerization of actin would be expected to inhibitrather than potentiate Ca2�-stimulated secretion. Theability of latrunculin B to potentiate insulin secretioninduced by several agonists, including elevation of

Fig. 6. Latrunculin B Potentiates Both Phases of Glucose-Stimulated Insulin SecretionAfter 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 30min under stimulatory conditions with or without latrunculin B (20 mM Gluc � LAT). Finally islets were perifused for 15 min underbasal conditions (2 mM Gluc � LAT). The amount of insulin released from islets as well as their insulin content was determinedby RIA. Insulin secretion is expressed as a percentage of insulin content. The results are mean � SE, n � 3 replicates for onerepresentative experiment from a total of three experiments performed, P � 0.05.


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intracellular Ca2�, strongly suggests events down-stream of calcium channel function. However, it re-mains formally possible that Ca2� entry activates anF-actin severing protein such as Scinderin or gelsolin(50, 53, 54).

Alternatively or additionally, our data demonstrate thatthe t-SNARE complex responsible for insulin granuleplasma membrane docking and fusion (Syntaxin 1A andSNAP-25) is associated with F-actin. Importantly, glu-cose stimulation results in a decrease in the amount ofF-actin associated with this t-SNARE complex. The tem-poral relationship between the potentiation of insulin se-cretion, peripheral redistribution of insulin granules anddissociation of F-actin from the SNARE complex pro-vides strong evidence that this interaction is responsiblefor limiting the rate and extent of insulin release. In con-trast, it is unclear from our results as to whether actinbinds directly or indirectly to a particular protein in thisSNARE core complex. At any given time the t-SNAREcomplex is also associated with v-SNARE protein,

VAMP2. Previous studies have also observed that puri-fied insulin granules can be copurified along with F-actin(39). Thus, it is unclear whether the association of F-actinreflects direct binding to one of the SNARE proteins, thecore complex and/or indirectly through another actinbinding adaptor. For example, the SNAP-25-interactingprotein colocalizes with SNAP-25 and actin, and myosinV can bind to VAMP2 (55, 56). In any case, based uponthese data we propose that glucose signals the transientreduction of F-cortical actin, disengaging actin tetheringto the SNARE complex to promote granule exocytosis.

MATERIALS AND METHODS

Materials

The RIA-grade BSA, D-glucose, and phalloidin-conjugatedFITC were obtained from Sigma (St. Louis, MO). Monoclonalanti-Syntaxin 6 and anti-SNAP-25 (used for immunofluores-

Fig. 7. Filamentous Actin Associates with SNARE Core Complexes, and this Association Is Dynamically Regulated by GlucoseMIN6 �-cells were incubated in glucose-free MKRBB for 2 h followed by stimulation with 20 mM glucose from 5–60 min.

Clarified whole-cell detergent extracts were prepared and immunoprecipitated with (A) anti-syntaxin 1 (1–2 �g/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 LDCCantibodies (lane 8, HC: heavy chain). C, Detergent lysates prepared from MIN6 cells stimulated for 5–10 min with 20 mM glucosewere immunoprecipitated with anti-SNAP-25 monoclonal antibodies and immunoblotted as described above with the addition ofVAMP2 (Syntaxin 1 antibody used recognized A and B isoforms). These results are representative of 3–6 sets of lysates preparedfrom independent passages of MIN6 cells. D, MIN6 �-cells were incubated in glucose-free MKRBB for 2 h in the presence of 10�M latrunculin B (L) or 5 �M jasplakinolide (J) for 2 h followed by stimulation with 20 mM glucose for 5 min. Clarified whole-celldetergent extracts were prepared and immunoprecipitated with anti-Syntaxin 1 antibody as described in (A). Lysates andimmunoprecipitates were immunoblotted with anti-Syntaxin 1A, SNAP-25, and actin antibodies. These results are representativeof three sets of lysates prepared from independent passages of MIN6 cells.


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cence and immunoblotting) antibodies were obtained fromBD Transduction Laboratories (Lexington, KY). The monoclo-nal SNAP-25 antibody used for immunoprecipitation wasobtained from Sternberger Monoclonals, Inc. (Lutherville,MD). The monoclonal VAMP2 and rabbit polyclonal actinantibodies were purchased from Synaptic Systems (Gottin-gen, Germany) and Sigma, respectively. Monoclonal anti-Syntaxin antibody was obtained from Upstate Biotechnology,Inc. (Lake Placid, NY) for use in immunoprecipitation, andmonoclonal anti-Syntaxin 1A was purchased from StressGen(Victoria, British Columbia, Canada) for immunoblotting. La-trunculin B and jasplakinolide were purchased from Calbio-chem (La Jolla, CA) and Molecular Probes, Inc. (Eugene, OR),respectively. Streptolysin-O and PMA were purchased fromSigma. Texas Red-conjugated secondary antibody and con-trol mouse IgG for immunoprecipitation were purchased fromJackson ImmunoResearch Laboratories, Inc. (West Grove,PA). Vectashield was obtained from Vector Laboratories, Inc.(Burlingame, CA). The MIN6 cells were a gift from Dr. JohnHutton (University of Colorado Health Sciences Center). ECLkit and Hyperfilm-MP were obtained from Amersham Bio-sciences (Piscataway, NJ).

Isolation of Rat Islets and Monolayer Culture ofIslet Cells

All animal experimentation described was conducted in ac-cord with accepted standards of humane animal care, andstudies were approved by the authors’ institutional commit-tee on animal care. Pancreatic rat islets of Langerhans wereobtained as described previously (57). Briefly, pancreata fromeight rats were digested with collagenase in Ca2�-containingHanks’ buffer and islets of Langerhans purified from exocrinetissue by discontinuous density-gradient centrifugation (His-topaque 1077 from Sigma). For monolayer culture, freshlyisolated islets were trypsinized for 6 min to obtain a single cellsuspension and seeded overnight in nonadherent Petridishes in DMEM containing 11.2 mM glucose. The next daycells were established in monolayer using Petri dishes coatedwith 804G matrix (produced by a rat bladder carcinoma cellline) and used 6 h later for insulin secretion measurementsand 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 inKrebs-Ringer bicarbonate buffer (KRB-HEPES: 134 mM NaCl;4.8 mM KCl; 1 mM CaCl2; 1.2 mM MgSO4; 1.2 mM KH2PO4; 5mM NaHCO3; 0.1% BSA; and 10 mM HEPES, pH 7.4) con-taining 2 mM glucose (basal condition). Isolated islets (30–45)were hand picked and then perifused for an additional 15 minin basal conditions at a flow rate of 0.5 ml/min to stabilizesecretion and to establish basal insulin secretory rates. Mediawere collected after the first 10 min of perifusion. After thestabilization period, islets were perifused under basal condi-tions with or without latrunculin B (10 �M) for 20 min, followedby incubation with or without latrunculin under stimulatoryconditions (20 mM glucose) for 30 min. All perifusate solutionswere maintained at 37 C. The amount of insulin released intothe media as well as the insulin content of islets was deter-mined by RIA.

Cell Culture, Insulin Secretion, and InsulinContent Assays

MIN6 cells were cultured in DMEM (25 mM glucose) equili-brated with 5% CO2 at 37 C. The medium was supplementedwith 15% fetal bovine serum, 100 U/ml penicillin, and 100�g/ml streptomycin, L-glutamine, and 50 mM �-mercapto-ethanol, as described (58). MIN6 cells were used between

passages 49 and 56. Insulin secretion was determined usingcells grown in 35-mm wells to 60–80% confluence. Cellswere washed twice with and incubated for 2 h in 1 ml mod-ified Kreb’s ringer bicarbonate buffer (MKRBB: 5 mM KCl; 120mM NaCl; 15 mM HEPES, pH 7.4; 24 mM NaHCO3; 1 mM

MgCl2; 2 mM CaCl2; and 1 mg/ml BSA (RIA grade) that wasgassed in 95% O2 for 30 min. Latrunculin B (10 �M) andjasplakinolide (5 �M) were solubilized in dimethylsulfoxide(DMSO; vehicle) and added to the MKRBB for the 2-h pre-incubation period where described in the figure legend. Cellswere stimulated with the secretagogue (20 mM glucose, 50mM KCl, or 200 nM PMA) for the time listed in the figurelegend, after which the media were collected, microcentri-fuged for 5 min at 4 C to pellet cell debris, and stored at �20C. Insulin secreted into the medium was quantitated using arat insulin immunoassay kit (Linco Research, Inc., St. Charles,MO). Insulin content was quantitated in cleared cell detergentlysates made from cells incubated in MKRBB containinglatrunculin B (10 �M) or DMSO control for 2 h. Media con-taining secreted insulin was removed, cells were harvested inNonidet 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 phenylmethylsulfonylfluoride; 10 �g/ml aprotinin; 1 �g/ml pepstatin; and 5 �g/mlleupeptin) and lysed for 10 min at 4 C, and lysates cleared ofinsoluble material by microcentrifugation for 10 min at 4 C.Insulin content of the cells was measured by RIA. For theinsulin secretion measurements from primary islet cells, cellswere cultured in monolayer as described above. Cells werewashed and preincubated for 30 min in KRB-HEPES contain-ing 2 mM glucose (basal condition). Cells were then incubatedfurther for 20 min in basal conditions with or without latrun-culin B (10 �M) followed by incubation for 1 h in the basal (2mM glucose) or stimulatory condition (20 mM glucose) with orwithout latrunculin. Media were collected and centrifuged toremove any detached cells. The amount of insulin releasedunder basal and stimulated conditions was determined usingan ultrasensitive rat insulin ELISA Kit (Mercodia, Uppsala,Sweden).

Immunofluorescence and Confocal Microscopy

For whole-cell time courses, MIN6 cells at 40% confluencyplated onto glass coverslips were incubated in MKRBB for2 h followed by stimulation with either 20 mM glucose or 50mM KCl over the time courses shown in the figure and thenfixed and permeabilized in 4% paraformaldehyde and 0.1%Triton X-100 for 10 min at 4 C. Primary rat islet cells weresimilarly fixed and permeabilized before or after stimulationby glucose with or without latrunculin as indicated. Fixedcells were blocked in 1% BSA and 5% donkey serum for 1 hat room temperature, followed by incubation with Syntaxin 6antibody (1:100) for 1 h. MIN6 cells were then washed withPBS and incubated with phalloidin-conjugated FITC (1:1000)and antimouse Texas Red secondary antibody for 1 h. Isletcells were incubated with the monoclonal insulin antibody at1:100 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 in1% BSA/PBS solution with the secondary antibody. All cellswere washed again in PBS, overlayed with Vectashieldmounting medium and mounted for confocal fluorescencemicroscopy using a Carl Zeiss (Jena, Germany) 510 confocalmicroscope.

Streptolysin-O Permeabilization

MIN6 cells at 60% confluence were washed twice withMKRBB and incubated for 2 h at 37 C in the presence oflatrunculin (10 �M) or DMSO vehicle control. Cells were thenpermeabilized in low calcium glutamate buffer for permeabi-lization (128 mM potassium glutamate; 5 mM sodium ATP; 5


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mM MgSO4; 10.2 mM EGTA; 20 mM HEPES, pH 7.4, plus 0.5mM CaCl2) containing freshly prepared Streptolysin-O solu-tion (12.5 �g/ml), as described (23, 59). After 7 min of per-meabilization at 37 C buffer was aspirated and replaced witheither low-calcium (0.1 �M) or high-calcium (10 �M) glutamatebuffer for 8 min at 37 C, after which media was quicklyremoved for insulin RIA analysis as described above. Pro-pidium iodide staining was used to confirm permeabilizationin every experiment.

Coimmunoprecipitation and Immunoblotting

Cleared detergent lysates were prepared as described abovefrom cells incubated in MRKBB buffer, in the absence orpresence of actin-modifying reagents as indicated, for 2 h at37 C and stimulated with glucose for the times indicated infigure. Lysate (0.9–1.4 mg) was combined with 1 �g of Syn-taxin (Upstate Biotechnology) or SNAP-25 (SternbergerMonoclonals) monoclonal antibody for 2 h at 4 C followed bya second incubation with protein G Plus Sepharose for 2 h.The resulting immunoprecipitates were subjected to electro-phoresis on 12% SDS-PAGE followed by transfer to PVDFmembrane. Membranes were blocked in 5% milk/TBS-Tween for 1 h at room temperature, followed by immunoblot-ting with primary antibodies for 1 h (Syntaxin 1A, SNAP-25,and actin diluted at 1:1000; VAMP2 at 1:10,000). Membraneswere rinsed in TBS-Tween and further incubated in second-ary antibodies conjugated to horseradish peroxidase dilutedat 1:5000 for 1 h, rinsed again, and proteins visualized byenhanced chemiluminescence.

Acknowledgments

We wish to thank Dr. John Hutton for the MIN6 cells. Wealso are grateful to Diana Boeglin and Angela Nevins fortechnical expertise.

Received September 25, 2002. Accepted January 10,2003.

Address all correspondence and requests for reprints to:Debbie C. Thurmond, Ph.D., Department of Biochemistry andMolecular Biology, Center for Diabetes Research, IndianaUniversity School of Medicine, Indianapolis, Indiana 46202.E-mail: [email protected].

* C.G.-G. and M.F. contributed equally to the studyThis work was supported by research grants from the

Indiana University School of Medicine Diabetes Researchand Training Center Pilot project grant P60-DK-20542 fromthe NIH, the Indiana University School of Medicine Biomed-ical Research Fund, and from the Swiss National ScienceFund Grant 3200-06177.00.

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