Department of Biochemistry and Molecular Biology, Center for Diabetes Research, Indiana University School of Medicine, Indianapolis, Indiana 46202
Submitted 6 March 2003 ; accepted in final form 18 May 2003
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ABSTRACT |
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jasplakinolide; glucosylation; syntaxin; insulin granule; exocytosis
The rate-limiting step in glucose-stimulated insulin secretion is thought to be contained within the steps that include the trafficking/mobilization, priming, docking, and fusion of the insulin granules (35). The priming/docking/fusion steps of insulin granule exocytosis involve the specific pairing of the t-SNARE [target membrane SNAP (soluble N-ethylmaleimide-sensitive factor attachment protein) receptor] proteins syntaxin 1 and SNAP-25 with the granule v-SNARE (vesicle membrane SNAP receptor) protein VAMP-2 (vesicle-associated membrane protein) (16, 25, 28, 46, 52, 56, 68, 72). Thus, upon glucose stimulation, insulin granules bind with the syntaxin and SNAP-25 t-SNAREs on the plasma membrane via their VAMP-2 protein to dock/tether and become primed for fusion (66). Primed vesicles are incorporated into the plasma membrane after a fusion reaction, and insulin is secreted into the extracellular space.
The trafficking of the insulin granules to the cell surface requires components of the cytoskeletal framework of microtubules and actin filaments (27, 32, 34, 42). Microtubules have been shown to be required for directional guidance of the granule and for sustained insulin secretion (2, 17, 19, 45). By contrast, the role of actin filaments is less clear. For example, actin filaments have been proposed to function in some of the motive force behind granule transport (49, 64). However, data obtained using F-actin disruption agents showed enhanced secretagogue-induced insulin secretion, suggesting instead that F-actin blocks granule movement (37, 43, 49, 62, 69). Recently, we have shown that glucose transiently modulates cortical actin organization and disrupts the interaction of polymerized actin with the plasma membrane t-SNARE complex (62). However, the question of how glucose elicits the reorganization of F-actin still remains.
One endogenous islet cell factor that is involved in regulation of the
actin cytoskeleton and functions in a glucose-dependent and specific manner is
the Rho family GTPase Cdc42. Cdc42 is an upstream positive effector of F-actin
(see recent reviews, Refs. 23
and 67). Moreover, Cdc42 is
found localized with insulin secretory granules in pancreatic -cells
(30,
31). Cdc42 has been shown to
be required for glucose- but not KClstimulated insulin secretion in rat islets
(29). Cdc42 has also recently
been demonstrated to function in mastoparan-stimulated insulin release through
an indirect interaction with the exocytosis SNARE protein syntaxin
(15). However, the molecular
role of Cdc42 in glucose-stimulated insulin secretion remains unclear.
We have recently documented that glucose stimulation for 5 min results in a
diminished amount of phalloidin-stained cortical actin in MIN6 -cells
and in isolated rat islets
(62). Here we have
investigated the mechanism underlying this phenomenon. Specifically, using the
actin nucleating/stabilizing agent jasplakinolide, we have shown that cortical
actin reorganization induced by glucose occurs at a proximal step in the
stimulus-secretion pathway. Importantly, our data show that stimulation with
glucose results in alterations in the cycling of endogenous Cdc42 and show
correlations between Cdc42 glucosylation and a transient depolymerization of
cortical actin, all after 5 min of glucose stimulation. These data support a
model whereby cortical actin 1) is signaled to reorganize by a
glucose-stimulated regulatory signal, and 2) acts as a dynamic
chaperone of granules to maintain granule pool sizes to ensure that insulin
release is not rate-limited by depletion of primed granules.
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MATERIALS AND METHODS |
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Cell culture, insulin secretion, and insulin content assays. MIN6
cells were cultured in Dulbecco's modified Eagle's medium (DMEM; 25 mM
glucose) equilibrated with 5% CO2 at 37°C. The medium was
supplemented with 15% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml
streptomycin, L-glutamine, and 50 mM -mercaptoethanol, as
described previously (65).
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 of
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 with 95%
O2 for 30 min. Latrunculin B (10 µM) and jasplakinolide (5
µM) were solubilized in DMSO (vehicle) and added to the MKRBB for the 2-h
preincubation period as indicated. Cells were stimulated with the secretagogue
(20 mM glucose or 50 mM KCl) for the time indicated. The
Krebs-Ringer-bicarbonate buffers were collected, microcentrifuged for 5 min at
4°C to pellet cell debris, and stored at 20°C. Insulin secreted
into the supernatant was quantitated using a rat insulin immunoassay kit
(Linco Research, St. Charles MO). Cells were subsequently harvested in NP-40
detergent lysis buffer (25 mM Tris, pH 7.4, 1% NP-40, 10% glycerol, 50 mM
NaFl, 10 mM Na4P2O7, 137 mM NaCl, 1 mM
NaVO3, 1 mM PMSF, 10 µg/ml aprotinin, 1 µg/ml pepstatin, and
5 µg/ml leupeptin) and lysed for 10 min at 4°C, and lysates were
cleared of insoluble material by microcentrifugation for 10 min at 4°C.
Insulin content of the cells was measured by RIA.
Immunofluorescence and confocal microscopy. MIN6 cells at 40% confluency plated onto glass coverslips were incubated in MKRBB for 2 h, followed by stimulation with 20 mM glucose or 50 mM KCl, and were then fixed and permeabilized in 4% paraformaldehyde and 0.1% Triton X-100 for 10 min at 4°C. Fixed cells were blocked in 1% BSA and 5% donkey serum for 1 h at room temperature, followed by incubation with primary antibody (1:100) for 1 h. Cells were then washed with phosphate-buffered saline (PBS) and incubated with phalloidin-conjugated FITC (1:1,000) and/or Texas red or FITC secondary antibody for 1 h. Cells were washed again in PBS and overlayed with Vectashield mounting medium, and coverslips were mounted onto slides for confocal fluorescence microscopy using a Zeiss 510 confocal microscope. Because MIN6 cells proliferate as "pseudo-islets" in cell clusters, cells were randomly selected for imaging analysis based on similarity of cluster size as viewed with a x100 objective. Images presented were captured using identical settings unless otherwise specified.
Cdc42 activation assay and immunoblotting. A glutathione S-transferase (GST) fusion-protein, corresponding to the p21-binding domain of human p21-activated kinase (PAK1-PBD agarose) was used to specifically detect and interact with the GTP form of Cdc42 in MIN6 cell lysates as described previously (5). Briefly, cleared detergent lysates were prepared from cells incubated in MRKBB buffer for 2 h at 37°C and stimulated with glucose for 5 min. Freshly made cleared cell lysate (1 mg) was combined with 10 µg of PAK1-PDB agarose for 1 h at 4°C. After three washes with lysis buffer, proteins were eluted from the agarose beads and subjected to electrophoresis on 12% SDS-PAGE, followed by transfer to polyvinylidene difluoride membrane. Membranes were immunoblotted with rabbit anti-Cdc42 or mouse anti-Myc antibodies, and proteins were visualized by ECL.
Isolation, culture, and stimulation of insulin secretion of mouse islets. Pancreatic mouse islets of Langerhans were isolated using a modification of the previously described method (14, 33). Briefly, pancreata from 8- to 12-wk-old male C57B16J mice were digested with collagenase and purified using a Ficoll density gradient. After isolation, islets were cultured overnight in complete CMRL-1066 culture medium (CMRL-1066 medium supplemented with 2 mM glutamine, 10% heat-inactivated FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin). Fresh islets were hand picked and cultured in complete CMRL-1066 overnight. Islets were washed twice with Krebs-Ringer bicarbonate buffer (10 mM HEPES, pH 7.4, 134 mM NaCl, 5 mM NaHCO3, 4.8 mM KCl, 1 mM CaCl2, 1.2 mM MgSO4, and 1.2 mM KH2PO4) containing 2 mM glucose and 0.1% BSA. Groups of 10 islets were preincubated in Krebs-Ringer bicarbonate buffer in the presence or absence of 5 µM jasplakinolide for 1 h, followed by stimulation with 20 mM glucose in the continued absence or presence of jasplakinolide for 1 h. Media were collected to measure insulin secretion, and islets were harvested in NP-40 lysis buffer to determine cellular insulin content by RIA.
Adenoviral infection of MIN6 cells. The NH2-terminal Myctagged Cdc42 adenoviral particles were generated in collaboration with Dr. Jeffrey Pessin (SUNY Stony Brook, Stony Brook, NY) by using cDNA constructs previously described (4, 11) and purchased from The University of Iowa Gene Transfer Vector Core Facility (Iowa City, IA). MIN6 cells at 5060% confluence were infected in complete medium at a multiplicity of interest (MOI) of 100 for 2 h at 37°C, followed by two washes with PBS and 48 h of incubation at 37°C. Infection resulted in >90% of cells infected as determined by immunofluorescence using anti-Myc antibody to visually detect infected cells.
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RESULTS |
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To determine whether F-actin also functions in the control of insulin
secretion from primary cells, we preincubated isolated mouse islet cells
without or with jasplakinolide for 1 h before a 1-h incubation with or without
jasplakinolide under basal (2 mM glucose) or stimulatory conditions (20 mM
glucose). Glucose stimulation resulted in a 2.8-fold increase in insulin
release compared with unstimulated control cells
(Fig. 2A).
Jasplakinolide pretreatment had no significant effect on basal insulin release
but potentiated glucose-stimulated insulin secretion twofold. No significant
alterations of insulin content in islets incubated with jasplakinolide were
detected (Fig. 2B).
These data demonstrated that primary cells respond to jasplakinolide in a
manner similar to that of the MIN6 -cells. Having established that MIN6
cells were a useful model to study the effects of actin rearrangement on
insulin secretion, and given the limited number of primary mouse islet cells
readily available for study, we utilized the MIN6 cells to examine the
cellular and molecular changes in cortical actin correlated with
glucose-stimulated insulin secretion.
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Glucose alters cortical actin at a step proximal to the KATP channels. To determine whether actin rearrangement was important for non-nutrient-induced insulin secretion, we examined the effect of depolarizing levels of KCl (50 mM) on jasplakinolide-treated cells (Fig. 3A). Stimulation by KCl was expected to result in a faster rate of secretion in the initial phase than that induced by glucose, and the vehicle-treated MIN6 cells showed a 3.9-fold increase in insulin secretion after 10 min. However, cells pretreated with jasplakinolide failed to exhibit potentiated secretion, secreting slightly lower than vehicle-treated levels of insulin throughout the time period examined. Moreover, stimulation with secretagogues downstream of KCl similarly failed to show potentiated secretion (data not shown). Thus our data using KCl as the secretagogue agreed with earlier findings in RINm5F cells (69), indicating that glucose but not KCl was capable of mediating F-actin to initiate insulin secretion. However, this difference was not the result of differential insulin granule localization in jasplakinolide-treated cells stimulated with glucose or KCl, because all cells pretreated with jasplakinolide exhibited a marked increase in insulin granules localized to the periphery regardless of secretagogue (Fig. 3B). This increase in peripherally localized granules may be the result of jasplakinolide-induced nucleation of microfilaments or clearance of the microfilament "cell web," as has been described (9). Nevertheless, despite the increased availability of granules at the cell perimeter, KCl was incapable of eliciting an enhanced response in jasplakinolide-treated cells, which indicated that glucose and KCl had differential access to these granules.
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To investigate how jasplakinolide might confer differential responsiveness of cells to stimulation with glucose compared with KCl, we treated cells with vehicle or jasplakinolide and visually assessed cortical actin by phalloidin-FITC labeling in the MIN6 cells (Fig. 4A). Syntaxin 6 was colabeled using Texas red to demarcate the localization of the trans-Golgi network of individual cells within the cortical actin network. Visualization of the midplane view of cell clusters revealed the strong distribution of F-actin as a ring beneath the plasma membrane indicative of cortical actin, which was dramatically diminished after 5 min of stimulation with glucose in >80% of similarly sized cell clusters visualized (Fig. 4A, images 1 and 2). Stimulation with KCl was without effect on cortical actin (Fig. 4A, image 3). Because jasplakinolide competes with phalloidin for binding to F-actin and results in a dramatically reduced fluorescence emission, phalloidin-FITC was used at 1:10 to achieve visualization of F-actin in jasplakinolide-treated cells. Cells pretreated with jasplakinolide exhibited discernible cortical actin rings under basal conditions (Fig. 4A, image 4). By contrast, even with jasplakinolide pretreatment, glucose stimulation for 5 min resulted in markedly reduced cortical actin (Fig. 4A, image 5). However, jasplakinolide-treated cells stimulated with KCl exhibited cortical actin rings similar to those of the basal state (Fig. 4A, image 6). Even when assessed at much earlier time points, stimulation with KCl failed to result in alterations in cortical actin (data not shown). The effect of glucose is specific to D-glucose and is independent of general osmotic effects, because treatment of cells with L-glucose failed to induce alterations in cortical actin in these analyses (data not shown). In addition, green fluorescent protein (GFP)-actin-transfected cells treated with jasplakinolide showed similar alterations in cortical actin in response to glucose (data not shown). Thus both endogenous cortical actin and jasplakinolide-induced cortical actin was markedly reduced in response to stimulation by glucose but not by KCl. Moreover, this responsiveness of cortical actin to glucose correlated with the ability of glucose to initiate insulin secretion.
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To quantitate these visible alterations in cortical actin with glucose, we used a coimmunoprecipitation assay. We recently reported (62) that F-actin and not monomeric actin specifically coimmunoprecipitates with the plasma membrane t-SNARE protein syntaxin 1 in MIN6 cells. Jasplakinolide treatment increased the amount of actin coimmunoprecipitated, whereas treatment with the F-actin-depolymerizing agent latrunculin B (61) abolished the interaction, indicating that syntaxin 1 coimmunoprecipitated specifically F-actin. This interaction was used to evaluate and compare the effects of glucose with KCl on cytoskeletal rearrangement. Glucose stimulation for 5 min resulted in a twofold decrease in the amount of actin coimmunoprecipitated from vehicle-treated cells (Fig. 4B, lanes 1 and 2). Similarly, jasplakinolide-treated cell lysates showed a twofold decrease in actin coimmunoprecipitated with anti-syntaxin 1 (Fig. 4B, lanes 4 and 5). By contrast, stimulation with KCl for 5 min had no effect on the quantity of actin coimmunoprecipitated from cells treated with or without jasplakinolide (Fig. 4B, lanes 3 and 6). Thus these data indicated that glucose had the capacity to rearrange cytoskeletal actin, even in cells treated with jasplakinolide. Taken together, these data suggested that glucose signals alterations in the cytoskeleton early in the secretory pathway, possibly in parallel with or upstream of the KATP channels.
Glucose-stimulated insulin secretion requires Cdc42 cycling. Cdc42 is a Rho family small GTPase that associates with specific downstream targets to affect changes in the actin cytoskeleton and has been shown to be preferentially important in glucose-stimulated insulin secretion (29). Because glucose induces alterations of cortical actin by 5 min, we sought to determine whether the activation state of Cdc42 was differentially affected by glucose or KCl stimulation within the initial 5-min period. MIN6 cells were infected with CsCl-purified adenoviral particles (MOI = 100 to achieve infection of >90% of cell population) encoding wild-type (WT), dominant negative GDP-bound (T17N), and constitutively active GTP-bound (Q61L) forms of Cdc42. The PAK1-PBD interaction assay was used to confirm the GTP activation state of the recombinant proteins, utilizing the NH2-terminal myc epitope tag on the recombinant proteins to distinguish them from endogenous Cdc42 (Fig. 5A). All three forms of Cdc42 were present in cleared cell lysates made from infected MIN6 cells (Fig. 5A, lanes 13), although the Q61L form of Cdc42 expressed poorly compared with WT and T17N. As expected, the WT and Q61L proteins that bind GTP were able to interact with PAK1-PBD, whereas the GDP-bound T17N form failed to interact with PAK1 (Fig. 5A, lanes 46).
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To determine whether expression of these various forms of Cdc42 would
impact the ability of glucose to affect cortical actin in -cells, we
visualized F-actin by using phalloidin-FITC to compare the T17N- or
Q61L-infected cells with control cells
(Fig. 5B). The level
of infection was assessed to be 90% or greater, as shown by immunofluorescent
labeling of the Myc-tagged recombinant Cdc42 proteins
(Fig. 5B, images
1, 3, 5, and 7). As expected, the Myc-tagged T17N protein
localized cytosolically and the cells showed strong cortical actin staining
(Fig. 5B, images
1 and 2). The Myc-tagged Q61L protein was correctly localized to
the plasma membrane and also showed strong cortical actin staining
(Fig. 5B, images
3 and 4). Like noninfected cells or cells infected with the WT
construct (data not shown), T17N-infected cells were responsive to glucose as
visualized by disruption of cortical actin after 5 min of stimulation
(Fig. 5B, image
6). In contrast, the cortical actin of Q61L-infected cells was unaffected
by glucose (Fig. 5B,
image 8). In contrast to either mutant form of Cdc42, endogenous
Cdc42 is reportedly punctate and present in both the cytosol and periphery in
isolated islets (31). Cdc42
was localized similarly in MIN6 cells (Fig.
5C, image 1). This distribution did not markedly
change in response to glucose or KCl (Fig.
5C, images 2 and 3). Because Cdc42
localizes to the plasma membrane upon activation and only a small percentage
of Cdc42 is activated in a given cellular pool in response to stimuli
(59), this finding was not
unexpected. Moreover, colabeling of endogenous Cdc42 and F-actin (using
phalloidin-FITC) showed that although there is some colocalization of Cdc42
with cortical actin, much of the Cdc42 was in the cytosol
(Fig. 5C, images
46).
To correlate the qualitative changes in cortical actin in response to
glucose in the T17N-infected cells with quantitative alterations in F-actin,
we compared the amount of actin that coimmunoprecipitated with endogenous
Cdc42 vs. the T17N form of Cdc42 in the absence and presence of glucose
stimulation (Fig. 5D).
Cdc42 is known to interact with F-actin via binding with N-WASp (neuronal
Wiskott-Aldrich syndrome protein) and the Arp2/3 (actin-related protein)
complex (see recent review, Ref.
18). In MIN6 cell lysates,
anti-Cdc42 antibody coimmunoprecipitated actin, and this association was
markedly reduced in cells stimulated with glucose for 5 min
(Fig. 5D, lanes
1 and 2) and was fully ablated by latrunculin B pretreatment of
the cells (data not shown). In the same manner, myc-T17N Cdc42
coimmunoprecipitated actin from unstimulated cells, and this association was
completely absent in glucose-stimulated cells
(Fig. 5D, lanes
3 and 4). However, the relative G:F-actin ratio was relatively
unchanged after 5 min of glucose stimulation such that the cells contained
8386% G-actin and 1417% F-actin (data not shown),
consistent with the ratio derived from rat islets
(24). In addition, expression
of the T17N or Q61L forms of Cdc42 had no statistically significant effect on
the G:F-actin ratio, consistent with another study using Cdc42 mutants
(36). Thus the decreased
cortical actin visualized in glucose-stimulated T17N-expressing cells
correlated with decreased interaction between Cdc42 and F-actin.
The effects of expressing T17N and Q61L forms of Cdc42 upon glucose-stimulated insulin secretion were determined. Cells overexpressing the T17N form of Cdc42 showed an approximately twofold stimulation within 5 min of glucose addition (Fig. 6A), similar to noninfected cells (Fig. 1A). In contrast, cells overexpressing the Q61L form of Cdc42 showed an ablated response to glucose (Fig. 6A), correlating with a lack of responsiveness of cortical actin in Q61L-infected cells to glucose (Fig. 5B, image 8). Consistent with this finding, Q61L-infected cells treated with latrunculin B for 2 h before stimulation showed a significant stimulation by glucose without affecting basal secretion (Fig. 6B). Combined with data demonstrating that Q61L- and WT-expressing cells are equally responsive to KCl (Fig. 6C), these data indicated that the expression of Q61L protein does not inhibit general secretory function of the cells. Taken together, these data suggested that cycling of Cdc42 to the GDP-bound state was required for glucose-stimulated insulin secretion.
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To correlate changes in the Cdc42 activation state with the changes
observed in cortical actin and insulin secretion over time, we next determined
the endogenous level of GTP-bound Cdc42 in cleared cell lysates prepared from
MIN6 cells stimulated with glucose over a 5-min time period
(Fig. 7). The amount of Cdc42
interacting with the PAK1-PBD (indicating GTP bound) was 7% of total
cellular Cdc42, similar to the level of Cdc42-GTP reported for other cell
types (5,
38). The cellular content of
Cdc42-GTP rose to
14% after 3 min of glucose stimulation, falling to
levels below that of the basal state after 5 min of stimulation
(Fig. 7A). This peak
in Cdc42-GTP level correlated with the peak rate of glucose-stimulated insulin
secretion between 1 and 3 min (Fig.
7B). The amount of detergent-soluble Cdc42 protein was
unchanged in glucose-stimulated compared with unstimulated lysates, and there
was no alteration in Cdc42 activation in cells stimulated with KCl over the
same time period (data not shown). These data suggested that stimulation with
glucose induces changes in the ability of Cdc42 to bind PAK1-PBD or effector
molecules. Importantly, the lowest level of Cdc42-GTP was detected after 5
min, the same time that glucose was found to stimulate depolymerization of
cortical actin.
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The activation state of Cdc42 has been reported to be altered by several posttranslational modifications, including carboxyl methylation and glucosylation (29, 31). However, whereas carboxyl methylation requires GTP and results in Cdc42 activation, glucosylation is not known to be GTP dependent and results in Cdc42 inactivation. To determine whether changes in the activation state were correlated with changes in the glucosylation state of Cdc42, we immunoprecipitated Cdc42 from detergent cell lysates made from cells stimulated with or without glucose for 5 min (Fig. 8A, lanes 1 and 2). Although equivalent levels of Cdc42 were immunoprecipitated, 2.3-fold more glucosylated (O-linked) Cdc42 protein was immunoprecipitated from glucose-stimulated cells than from unstimulated cells as determined by immunoblotting using the O-linked N-acetylglucosamine (O-GlcNAc)-specific antibody RL2 (Fig. 8A, inset). This finding represented an increase in glucosylated Cdc42 from approximately <2% in the basal state to 4% after glucose stimulation for 5 min. The reciprocal immunoprecipitation using the RL2 antibody confirmed this finding, as shown by an increase in glucosylation of Cdc42 after glucose stimulation for 5 min (Fig. 8A, lanes 3 and 4). To determine whether Cdc42 was a specific target of glucosylation, we examined the glucosylation state of syntaxin 1, an indirect binding partner of Cdc42 (15). Although syntaxin 1 was present in the cell lysates (Fig. 8B, lanes 1 and 2), it was not immunoprecipitated with the RL2 antibody (Fig. 8B, lanes 3 and 4). In addition, the insulin granule v-SNARE protein VAMP2 was present in cell lysates but was not glucosylated (data not shown), consistent with studies of glucosylated SNARE proteins in 3T3-L1 adipocytes (10). A more thorough time course of Cdc42 glucosylation revealed that the small but significant increase in glucosylated Cdc42 protein was evident only after 3 min (Fig. 8C) and was reversibly reduced back to basal levels by 30 min of static glucose incubation (Fig. 8D). This rate of reversibility could be increased by removal of glucose at the 5-min time point, followed by incubation in glucose-free MKRBB for 10 min. Taken together, these data suggested a correlation between the timing of inactivation of Cdc42 by selective glucosylation and the timing of cortical actin rearrangement in the initiation of insulin secretion stimulated by glucose.
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DISCUSSION |
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The rate of replenishment of granule pools was recently demonstrated to be
a critical factor in nutrient-stimulated insulin secretion as opposed to the
transient secretion elicited by KCl
(3,
48). Because Cdc42 is a key
upstream regulator of F-actin that colocalized with insulin secretory granules
(30,
31), it appeared to be a
perfect target for modification by glucose to exert a large effect in a short
time frame. Consistent with the initial transient depolymerization of F-actin
5 min after glucose addition to the media, expression of constitutively active
Cdc42 abolished glucose- but not KClstimulated insulin secretion within 5 min,
suggesting that Cdc42 cycling to the GDP-bound form leads to actin
depolymerization. Expression of dominant negative Cdc42 had a small
stimulatory but statistically insignificant effect on insulin secretion,
suggesting that interruption of Cdc42 cycling overall does not inhibit insulin
secretion. To investigate the importance of cycling further studies using
Cdc42Hs (F28L), which can undergo constitutive GTP/GDP exchange without
interfering with GTP hydrolysis
(39), will be needed.
Importantly, the inhibition of secretion by the constitutively active Cdc42
was reversed by treatment of cells with latrunculin B, indicating that this
mutant protein did not impair the glucose signaling pathway or overall cell
function. The specificity of responsiveness of Cdc42 to glucose and not KCl
places Cdc42 in the proximal signaling pathway, perhaps concurrent at the
level of KATP channel closure. Therefore, we propose that glucose
triggers insulin secretion while simultaneously initiating the cascade of
events necessary to transiently induce actin depolymerization. Because neither
cortical actin depolymerization nor granule mobilization is an instantaneous
process, it seems advantageous for the -cell to initiate this process in
advance of the need to elicit second-phase insulin secretion.
Our data support a molecular mechanism wherein Cdc42 becomes glucosylated
upon stimulation with glucose, which correlates with the observed transient
loss of cortical actin induced by glucose. The endogenous modification of
Cdc42 by glucosylation in response to stimulation of -cells with glucose
for only 5 min is a novel finding. O-linked glucosylation is highly dynamic,
with rapid cycling in response to cellular signals analogous to
phosphorylation. Previous studies have shown that Cdc42 is glucosylated on
Thr35, which lies in the effector domain of the GTPase and is
involved in coordinating the
-phosphate of bound GTP
(26). Glucosylation by
Clostridium difficile toxin targets this residue and decreases the
GTPase activity by 85% and also inhibits the GTPase-activating protein (GAP)
stimulation of Cdc42 GTPase activity
(58). In islets and
-cells, glucosylation by this toxin specifi-cally inhibited glucose- but
not KCl-induced insulin secretion within 1 h
(29). The abundance of
O-GlcNAc in islet
-cells markedly and transiently increases in response
to 20 mM glucose (40).
O-GlcNAc transferase is highly enriched in
-cells relative to other
known cell types and is thought to be of vital importance for
-cell
response to glucose loads
(21). In fact, hyperglycemia
leads to the rapid and reversible accumulation of OGlcNAc specifically in
-cells in vivo (40).
Moreover, in a recent study glucosylation was blocked by incubation of mouse
islets with the glutamine:fructose-6-phosphate amidotransferase (GFAT)
inhibitor azaserine, resulting in inhibition of glucose-stimulated insulin
secretion (73), and inhibition
was reversed by the addition of GlcNAc. Intriguingly, many regulatory proteins
that interact with actin contain O-GlcNAc, as well as proteins thought to
bridge the cytoskeleton to the plasma membrane such as vinculin, talin, and
the synapsins (12,
20,
41). In addition, we have
recently reported the specific glucosylation of Munc18c, a SNARE accessory
protein that regulates GLUT-4 vesicle fusion, suggesting that glucosylation
may also exert distal effects on the exocytosis machinery
(10). Further studies are
required to investigate the potential regulation of insulin granule exocytosis
by O-GlcNAc at these various steps in the stimulus-secretion pathway.
Recently, it was reported
(15) that Cdc42 indirectly
interacts with syntaxin 1, and this interaction is promoted by the
secretagogue mastoparan or by constitutively activating Cdc42 using the Q61L
mutation. Mastoparan, a hormone receptor-mimetic peptide isolated from wasp
venom, stimulates insulin release from pancreatic -cells independently
of a requirement for increased [Ca2+]i
(15). In support of this, we
report that jasplakinolide cannot potentiate secretion elicited by
secretagogues downstream of glucose or in Streptolysin-O-permeabilized cells
stimulated under high-Ca2+ buffer conditions (data not
shown). Thus our data are consistent with the notion that Cdc42 is involved in
glucose-stimulated insulin secretion and further suggest that Cdc42 may be
part of the protein complex that bridges to the SNARE protein complexes at the
plasma membrane.
Syntaxin 1A has also been demonstrated to interact with L-type
Ca2+ channels, and it may be that this localization is
sufficient to expedite granule fusion after stimulation by secretagogue
(6,
70,
71). Although it has also been
shown that actin can tether to channels, latrunculin B depolymerization of
F-actin resulted in potentiation rather than inhibition of
Ca2+ induced secretion
(62). However, it is possible
that Ca2+ entry activates F-actin-severing proteins such
as scinderin, which clears F-actin. In fact, in mouse pancreatic -cells,
scinderin-derived actin-binding peptides reduced
Ca2+-dependent exocytosis of insulin granules
(8). Furthermore,
immunofluorescence studies in chromaffin cells show a stimulus-initiated
spatial and temporal correlation between scinderin relocalization and actin
depolymerization (63). Because
it has been shown that exocytosis in these cells occurs in cortical areas
devoid of F-actin (47), it
seems possible that redistribution of scinderin to locations near
KATP and/or Ca2+ channels could facilitate
granule movement to channel-localized syntaxin 1A SNARE complexes.
Nearly 30 years ago it was discovered that F-actindepolymerizing agents
potentiate the secretion of insulin stimulated by glucose. Our results
demonstrate that glucose can induce transient actin cytoskeletal
rearrangements in isolated mouse islets and in MIN6 -cells. We propose
that glucose signals the transient reduction of filamentous cortical actin to
coordinate granule mobilization and pool refilling that will ultimately
promote granule exocytosis.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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