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Endocrinology Vol. 144, No. 10 4508-4518
Copyright © 2003 by The Endocrine Society

Mastoparan-Induced Insulin Secretion from Insulin-Secreting ßTC3 and INS-1 Cells: Evidence for Its Regulation by Rho Subfamily of G Proteins

Rajesh H. Amin, Hai-Qing Chen, Rajakrishnan Veluthakal, Robert B. Silver, Jingsong Li, GuoDong Li and Anjaneyulu Kowluru

Departments of Pharmaceutical Sciences (R.H.A., H.-Q.C., R.V., A.K.) and Pharmacology (R.B.S.), Physiology, Radiology, and Biomedical Engineering, Wayne State University, and ß Cell Biochemistry Research Laboratory (R.H.A., H.-Q.C., A.K.), John D. Dingell Veterans Affairs Medical Center, Detroit, Michigan 48201; and John D. Dingell Veterans Affairs Medical Center and Argonne National Laboratory (R.B.S.), and Cardiovascular Research Institute (J.L., G.L.), National University Medical Institutes, National University of Singapore, Singapore 117597

Address all correspondence and requests for reprints to: Anjan Kowluru, Ph.D., Department of Pharmaceutical Sciences, College of Pharmacy and Health Professions, Wayne State University, 259 Mack Avenue, Detroit, Michigan 48201. E-mail: akowluru{at}med.wayne.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mastoparan, a tetradecapeptide from wasp venom, stimulates insulin secretion from the islet ß-cells, presumably via activation of trimeric G proteins. Herein, we used Clostridial toxins, which selectively modify and inactivate the Rho subfamily of G proteins, to examine whether mastoparan-induced insulin secretion also involves activation of these signaling proteins. Mastoparan, but not mastoparan 17 (an inactive analog of mastoparan), significantly stimulated insulin secretion from ßTC3 and INS-1 cells. Preincubation of ßTC3 cells with either Clostridium difficille toxin B, which inactivates Rho, Cdc42, and Rac, or Clostridium sordellii toxin, which inactivates Ras, Rap, and Rac, markedly attenuated the mastoparan-induced insulin secretion, implicating Rac in this phenomenon. Mastoparan-stimulated insulin secretion was resistant to GGTI-2147, a specific inhibitor of geranylgeranylation of Rho G proteins (e.g. Rac), suggesting that mastoparan induces direct activation of Rac via GTP/GDP exchange. This was confirmed by a pull-down assay that quantifies the binding of activated (i.e. GTP-bound) Rac to p21-activated kinase. However, glucose-induced insulin secretion from these cells was abolished by toxin B or GGTI-2147, suggesting that the geranylgeranylation step is critical for glucose-stimulated secretion. Mastoparan significantly increased the translocation of cytosolic Rac and Cdc42 to the membrane fraction. Confocal light microscopy revealed a substantial degree of colocalization of Rac (and, to a lesser degree, Cdc42) with insulin in ß-cells exposed to mastoparan. Further, stable expression of a dominant negative (N17Rac) form of Rac into INS-1 cells resulted in a significant reduction in mastoparan-stimulated insulin secretion from these cells. Taken together, our findings implicate Rho G proteins, specifically Rac, in mastoparan-induced insulin release.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SEVERAL EARLIER STUDIES, including our own, have suggested a requirement for small molecular weight GTP-binding proteins (G proteins) in physiological insulin secretion (1, 2, 3, 4, 5, 6, 7, 8). Most of these studies have used specific pharmacological probes to arrive at these conclusions. For example, the majority of small molecular weight G proteins undergo a series of posttranslational modifications at their C-terminal cysteine (often referred to as the CAAX motif) that make them more hydrophobic and facilitate their translocation toward the membrane fraction for optimal interaction with their respective effector proteins (e.g. phospholipase C and adenylate cyclase) (2, 7, 8). Inhibitors of these posttranslational modifications, such as the 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase blockers (e.g. statins, which inhibit protein isoprenylation), acetyl farnesyl cysteine (an inhibitor of carboxyl methylation), or cerulenin (an inhibitor of fatty acylation), all seem to attenuate glucose-stimulated insulin secretion from pancreatic ß-cells, suggesting that such posttranslational modifications are important for physiological insulin secretion (1, 2, 4, 5, 6). More recently, we have synthesized novel inhibitors of protein prenylation and reported that protein prenylation plays an important role in both glucose- and calcium-mediated secretion (4).

In addition to the chemical inhibitors, we have used bacterial toxins to study the significance of small G proteins in calcium- and glucose-stimulated insulin secretion (3). The uniqueness of the Clostridial toxins lies in their ability to selectively glucosylate and inactivate specific G proteins (3). Using [14C]UDP-glucose, we have previously reported the specificity of these toxins in normal rat islets and clonal ß-cells. For example, Clostridium difficille toxin A or B monoglucosylates and inactivates Rho, Rac, and Cdc42. The Clostridium sordellii lethal toxin selectively monoglucosylates and inactivates Ras, Rap, and Rac. In addition to assigning regulatory roles for specific G proteins in glucose- and calcium-mediated insulin secretion (3), we have recently used these toxins to identify proapoptotic G proteins in isolated ß-cells (e.g. Ras) that play regulatory roles in cytokine-mediated dysfunction and demise of the pancreatic ß-cell (9, 10).

Several earlier studies, including our own, have demonstrated that mastoparan, a tetradecapeptide from wasp venom, stimulates insulin secretion from normal rat islets, human islets, and clonal ß-cells (11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28). Although most studies demonstrated that mastoparan induces insulin secretion from these cells, the precise loci for its regulation remain only partially defined (11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28). Furthermore, the use of mastoparan (or mastoparan 7) as a probe to study ß-cell function is important because we have recently reported (24) that although glucose- and calcium-induced secretion of insulin is altered in islets derived from the Goto-Kakizaki rat, a model for noninsulin-dependent diabetes mellitus, mastoparan-induced insulin secretion remained intact in these islets, suggesting that a glucose- and calcium-independent, but mastoparan-sensitive, mechanism(s) might be operable in isolated islets. Therefore, in the present study we used Clostridial toxins to determine regulatory roles, if any, for small molecular mass G proteins in mastoparan-induced insulin release. Interestingly, our findings indicate that Rac activation is necessary for glucose-induced (3) as well as mastoparan-induced (current study) insulin secretion. However, in the present study we also present evidence to indicate that posttranslational geranylgeranylation of Rac may be required for glucose-induced, but not mastoparan-stimulated, secretion. The secretion data taken in concert with the confocal microscopic and transfection data implicate Rac as one of the key proteins involved in mastoparan-induced insulin secretion from the ß-cell.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Mastoparan, mastoparan 7, and mastoparan 17 were purchased from Biomol (Plymouth Meeting, PA). C. difficille toxin B and C. sordellii lethal toxins (LT-9048 and LT-82 variants) were provided by Dr. Michel Popoff (Pasteur Institute, Paris, France). GGTI-2147 was purchased from Calbiochem (San Diego, CA). Antisera directed against Rac, Cdc42, and Ras were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The Rac activation assay kit was purchased from Cytoskeleton, Inc. (Denver, CO). The rat insulin ELISA kit was purchased from American Laboratory Products Co. (Windham, NH). Antimouse insulin was purchased from Sigma-Aldrich (St. Louis, MO). Secondary antimouse conjugated to Alexa Fluor 488, antirabbit IgG conjugated to Alexa Fluor 660, and antimouse Golgin-97 were purchased from Molecular Probes (Eugene, OR). Anti-Myc monoclonal antibody was obtained from Roche (Indianapolis, IN).

Cell culture
ßTC3 cells were provided by Dr. Shimon Efrat (Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel). INS-1 cells were provided by Dr. Claes Wollheim (University of Geneva, Geneva, Switzerland). ßTC3 cells were cultured in DMEM containing 25 mM glucose supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, 100 IU/ml streptomycin, and 2 mM L-glutamine under 95% O2/5% CO2 (4). The medium was changed twice weekly, and cells were trypsinized and subcloned weekly. Cells were used between passages 20–65. INS-1 cells were cultured as described previously (29).

Insulin release studies
ßTC3 cells.
ßTC3 cells were cultured overnight in DMEM containing 5 mM glucose and 10% fetal calf serum in the presence of diluent alone or various concentrations of inhibitors as indicated in the text. After preincubation in the presence of 3.3 mM glucose, cells were incubated in the presence of 3.3 mM glucose, mastoparan, mastoparan 7, or mastoparan 17 (at indicated concentrations) for 45 min at 37 C. The supernatant was then removed, centrifuged at 300 x g for 10 min, and assayed for insulin. The amount of insulin was quantitated by ELISA (4).

INS-1 cells.
Insulin secretion from these cells was carried out as described previously (29). INS-1 cells, seeded in 24-well plates, were cultured for 2 d. After two washes in buffer, cells were preincubated for 30 min in the same buffer containing 2.8 mM glucose and 0.1% BSA. Subsequently, the cells were incubated in the absence or presence of mastoparan or mastoparan 17 (30 µM) for an additional 45 min. The supernatants were removed for measurements of secreted insulin, which was expressed as nanograms of insulin released per milliliter of incubation medium as described previously (4).

Translocation of small G proteins from the cytosolic to the membrane fraction
After incubation with diluent alone or mastoparan, cells were homogenized by sonication (three times, 15 sec each time) and subjected to a single step centrifugation at 105,000 x g for 60 min as described previously (2). Total membrane (pellet) and soluble (supernatant) fractions were separated and used for Western blotting for the detection of Rac and Cdc42. As a negative control, we also examined the relative degree of translocation of Ras in the membrane and soluble fractions, as we have reported no significant contributory roles for Ras in insulin secretion (3).

Rac activation assay
Rac activation was assayed in lysates derived from control and mastoparan-treated cells using a Rac activation assay kit. This assay is based on the principle that after activation (i.e. in their GTP-bound state), Rac or Cdc42 is specifically recognized by the CRIB interactive binding site of p21-activated kinase (PAK), an effector of Rac/Cdc42 (30, 31, 32, 33, 34). The interaction of Rac/Cdc42 with PAK results in a significant reduction in the rate of hydrolysis of GTP, making this an important tool for affinity purification and quantification of the GTP-bound (active) form of Rac/Cdc42 from the cell lysate. The amount of binding of Rac to PAK was quantitated according to the manufacturer’s instructions. In brief, ßTC3 cells were grown for 2 d in DMEM containing 25 mM glucose and 10% fetal calf serum. Cells were then exposed to DMEM containing 5 mM glucose and 5% serum for 24 h and lysis buffer (provided by the manufacturer) after stimulation with mastoparan (30 µM) for 30 min in Krebs-Ringer buffer medium as mentioned above. Lysates (4–5 mg/ml) were clarified by centrifugation for 5 min at 4800 x g, and PAK-PBD (p21-activated kinase-p21-binding domain) beads (20 µl) were added to the supernatant. The mixture was then rotated for 1 h at 4 C and pelleted by centrifuging at 4000 x g for 3 min. This pellet was washed once with lysis buffer, followed by a rinse in wash buffer [25 mM Tris (pH 7.5), 30 mM MgCl2, 40 mM NaCl, and 150 mM EDTA] and was then reconstituted in sample buffer. Proteins were resolved by SDS-PAGE and transferred to a nitrocellulose membrane, which was then blocked with 5% nonfat dry milk in TBST (20 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween) and washed twice (15 min each times) with TBST. The blot was then incubated with anti-Rac antibody (1:500) for 90 min at room temperature, washed three times with TBST for 10 min each time, and incubated with secondary antibody antirabbit IgG-horseradish peroxidase at room temperature for 1 h. Immune complexes were visualized using an enhanced chemiluminescence detection kit (Amersham, Piscataway, NJ).

Confocal light microscopy
ßTC3 cells were plated onto glass coverslips and grown for 2 d, followed by overnight incubation with low glucose (5 mM)-containing DMEM. The following day cells were washed with Krebs-Ringer buffer and then incubated with mastoparan (30 µM) for 30 min. Cells were rinsed with cold PBS and fixed in an ice-cold 5% EGTA in methanol solution for 20 min, then rinsed twice more with PBS. Blocking solution consisting of 10% heat-inactivated horse serum was then applied for 30 min. Primary antimouse insulin or antimouse Golgin-97 was applied (1:1000) for 1 h. Cells were washed twice, and a second antibody against rabbit Rac-1 or Cdc42 was applied for another hour, followed by two washes in PBS. Secondary antimouse conjugated to Alexa Fluor 488 was applied (1:1000) for 1 h, followed by two washes in PBS. This was followed by application of an antirabbit conjugated to Alexa Fluor 660 for an additional hour. Coverslips were washed twice in PBS and once with distilled water, then mounted and photographed using a Zeiss LSM 510 confocal light microscope (Oberkochen, Germany) at the John D. Dingell Veterans Affairs Medical Center (Detroit, MI). The confocal microscope was operated with pinhole settings between 1.1 and 1.4 Airy units in each of the fluorescence channels; images were recorded at 2048 x 2048 pixels at 12 bits/channel.

Cell transfection studies
The plasmid with cloned Rac mutant (pEXVN17rac) was provided by Dr. Alan Hall (University College of London, London, UK). The plasmid was constructed by cloning the dominant negative form of N17Rac (substitution of threonine at 17 by asparagine) with the N-terminal c-Myc tagged into vector pIREShyg1. Control vector was constructed by deleting the inserts from above plasmid. Plasmids were amplified in XL1 Blue Escherichia coli. Transfections of INS-1 ß-cells with the purified plasmid DNA were performed using Superfect reagent (Qiagen, Hilden, Germany). Stably expressing cells were established by selecting transfected cells with 50 µg/ml hygromycin.

Determination of degree of expression of N17 Rac1
INS-1 cells transfected with empty (pIRES) or N17Rac1 vectors were homogenized as described above. Protein samples (20 µg protein each) were denatured in treatment buffer at 95 C for 5 min and separated by SDS-PAGE (15% acrylamide). The separated proteins were transferred to nitrocellulose membranes, and blots were probed with anti-Myc monoclonal antibody (2 µg/ml). After blocking with 5% fat-free milk, the membranes were incubated with horseradish peroxidase conjugated to antimouse IgG antibodies (1:1000). Immunoreactive bands were visualized using an enhanced chemiluminesce kit (Amersham).

Other methods
The protein concentration was quantified colorimetrically using a dye-binding method described previously (2, 3, 28). Unless otherwise specified, all values are presented as the mean ± SEM. Differences between groups with P < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Mastoparan and mastoparan 7, but not mastoparan 17 (an inactive analog of mastoparan), stimulate insulin secretion from ßTC3 and INS-1 cells
The data shown in Fig. 1Go demonstrate that mastoparan or mastoparan 7 stimulated, in a concentration-dependent manner, insulin secretion from ßTC3 cells under static incubation conditions. Significant stimulation of insulin secretion was demonstrable at concentrations as low as 10 µM. The structural specificity of mastoparan-induced effects on insulin secretion was examined using mastoparan 17, an inactive analog of mastoparan (17). The data shown in Fig. 2Go demonstrate that mastoparan stimulated insulin secretion from INS-1 (A) as well as ßTC3 (B) cells. However, unlike mastoparan, mastoparan 17 failed to exert any stimulatory effects on insulin secretion from either of the cell types, suggesting that mastoparan- or mastoparan 7-induced effects are structure specific (Fig. 2Go).



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  		FIG. 1. Stimulation by mastoparan or mastoparan 7 of insulin secretion from ßTC3 cells. Insulin release from ßTC3 cells was measured under static incubation conditions (for 45 min at 37 C) in the absence or presence of mastoparan (0–30 µM) or mastoparan 7 (0–30 µM) as indicated. Insulin released into the culture medium was quantitated by ELISA (see Materials and Methods for additional details). Data are the mean ± SEM from eight determinations in each case. *, P < 0.05 vs. control.

 


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  		FIG. 2. Stimulation by mastoparan, but not mastoparan 17, of insulin secretion from INS-1 and ßTC3 cells. Insulin release from INS-1 cells (A) or ßTC3 cells (B) was measured under static incubation conditions (for 45 min at 37 C) in the absence or presence of mastoparan (30 µM) or mastoparan 17 (30 µM). Insulin released into the culture medium was quantitated by ELISA (see Materials and Methods for additional details). Data are the mean ± SEM from 8–12 determinations in each case. *, P < 0.05 vs. control; **, not significantly different from basal secretion.

 
Removal of extracellular calcium causes a significant inhibition of mastoparan-stimulated insulin secretion from ßTC3 cells
Earlier studies with neuroblastoma cells from Suh et al. (35) indicated that mastoparan 7 increases the accumulation of intracellular calcium concentrations via increasing the calcium influx through membrane pores and via mobilization of calcium from stores mediated by phosphoinositide turnover. Therefore, we quantitated mastoparan-induced insulin secretion from ßTC3 cells in the absence or presence of extracellular calcium. The data shown in Fig. 3Go indicate a significant degree of inhibition of mastoparan-induced insulin secretion in the absence of extracellular calcium (50% inhibition in incremental response over the basal secretion). These data indicate that mastoparan-induced insulin secretion may in part be dependent upon extracellular calcium (see Discussion).



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  		FIG. 3. Mastoparan-dependent insulin secretion from ßTC3 cells in the absence or presence of extracellular calcium. Insulin release from ßTC3 cells was measured under static incubation conditions (for 45 min at 37 C) in the absence or presence of extracellular calcium as indicated. Insulin released into the culture medium was quantitated by ELISA (see Materials and Methodsfor additional details). Data are the mean ± SEM from 8–12 determinations in each case. *, P < 0.05 vs. control; **, P < 0.05 vs. in the presence of extracellular calcium.

 
Clostridial toxins markedly reduce mastoparan-induced insulin secretion from ßTC3 cells
Using normal rat islets and clonal ß-cells (HIT-T15), we have demonstrated previously that both glucose- and calcium-induced insulin secretion were inhibited significantly by preexposure of these cells to Clostridial toxins (3). We reported that these toxins specifically glucosylate candidate G proteins, resulting in their functional inactivation. For example, we have shown that C. difficille toxin B monoglucosylates and inactivates Rac, Cdc42, and Rho, in contrast to C. sordellii lethal toxin (LT), which monoglucosylates and inactivates Ras, Rap, and Rac. In the present study we used these two toxins (i.e. toxin B and LT) to examine their effects on mastoparan-induced insulin secretion.

The data shown in Fig. 4AGo indicate that pretreatment of ßTC3 cells with toxin B (200 ng/ml; overnight) markedly reduced (>50%) the ability of mastoparan to stimulate insulin secretion from these cells. Basal insulin secretion from control cells remained unaltered in the toxin-treated cells. Based on the specificity of this toxin, these data indicate that Cdc42, Rac, and Rho may be involved in mastoparan-induced insulin secretion from these cells. Next, we examined the effect of LT-9048 on mastoparan induced-insulin secretion (Fig. 4BGo). In a manner akin to toxin B, LT-9048 reduced mastoparan-stimulated insulin secretion. Under these conditions (200 ng/ml, overnight treatment), we observed more than 66% inhibition of mastoparan-induced insulin secretion from ßTC3 cells (Fig. 4BGo). In a limited number of studies we also observed a similar degree of inhibition of mastoparan-induced insulin release from cells after pretreatment with a second variant of LT, namely LT-82 (-70% inhibition; n = 4 determinations in each case; P = 0.03). Based on the specificity of LT, these data indicate that Ras, Rap, and Rac may be involved in mastoparan-induced insulin secretion. Together, data derived from the toxin experiments (Fig. 4Go, A and B) indicate that activation of Rac (a common substrate for toxin B and LT) may be necessary for mastoparan-induced insulin secretion, as treatment of these cells with either toxin B or LT resulted in a significant reduction of mastoparan-induced insulin secretion.



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  		FIG. 4. Inhibition by toxin B or LT-9048 of mastoparan-induced insulin secretion from ßTC3 cells. ßTC3 cells were cultured in the absence or presence of C. difficille toxin B (200 ng/ml; 18 h; A) or C. sordellii (200 ng/ml; 18 h; B) as described in Materials and Methods. After this, insulin release was quantitated under static conditions (45 min at 37 C) in the absence or presence of mastoparan (30 µM). Insulin in the medium was quantitated by ELISA. Data are the mean ± SEM from four separate experiments (n = 6 in each condition). *, P < 0.05 vs. secretion rates demonstrable in toxin-untreated cells in the presence of mastoparan.

 
Mastoparan-induced insulin secretion is resistant to GGTI-2147, a selective inhibitor of geranylgeranylation of G proteins
We next examined whether mastoparan-induced secretion of insulin, mediated via activation of Rac, requires geranylgeranylation of Rac. This study was based on our earlier observations (4) suggesting a regulatory role for such posttranslational modification steps in glucose- and calcium- mediated insulin secretion. To examine this, mastoparan-induced insulin secretion was quantitated in ßTC3 cells incubated in the absence or presence of GGTI-2147 (20 µM; overnight). The data shown in Fig. 5Go indicate that mastoparan-induced insulin secretion was completely resistant to GGTI-2147, indicating that mastoparan-induced activation of Rac may not require its posttranslational geranylgeranylation. These data are in contrast to our recent observations (4) indicating marked inhibition by GGTI-2147 of glucose- and KCl-induced insulin secretion from ßTC3 cells and imply that glucose- and calcium-induced, but not mastoparan-induced, insulin secretion requires posttranslational geranylgeranylation (see Discussion). These data appear to suggest that mastoparan-induced activation of Rac might involve direct exchange of GTP for GDP on Rac, as stimulatory effects of mastoparan on trimeric as well as monomeric G proteins have been reported to occur via GTP/GDP exchange (see Refs. 7 and 36 for recent reviews).



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  		FIG. 5. Mastoparan-induced insulin secretion from ßTC3 cells is resistant to GGTI-2147, a specific inhibitor of geranylgeranylation of Rho G proteins. ßTC3 cells were cultured in the absence or presence of GGTI-2147 (20 µM; 18 h) as described in Materials and Methods. After this, insulin release was quantitated under static conditions (45 min at 37 C) in the absence or presence of mastoparan (30 µM). Insulin in the medium was quantitated by ELISA. Data are the mean ± SEM from four separate experiments (n = 6 in each condition). *, Not significantly different from secretion rates demonstrable in the presence of mastoparan in GGTI-2147-untreated cells.

 
Mastoparan treatment results in the activation of Rac in ßTC3 cells
The above data prompted us to investigate whether stimulation of ßTC3 cells with mastoparan results in direct activation of Rac. During recent years, several activation mechanisms have been proposed for small G proteins, including those involving guanine nucleotide exchange factors, which facilitate replacement of a GTP for a GDP molecule on the candidate G protein. Such factors (e.g. Tiam 1) have been identified for Rac (37, 38). However, determination of Rac activation via Tiam 1 is difficult because numerous other exchange factors have also been identified for Rac that have been shown to activate Rac singly or through the intermediacy of other G proteins (39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51). Therefore, in the present study we quantitated the degree of mastoparan-induced activation of Rac (i.e. its GTP-bound form) in a pull-down assay using a PAK binding method (see Materials and Methods for additional details). The data shown in Fig. 6Go demonstrate a marked increase in the degree of binding of Rac to PAK in lysates of ß-cells following exposure to mastoparan. As a positive control, we used GTP{gamma}S, a nonhydrolyzable analog of GTP, to facilitate binding and activation of Rac endogenous to the ß-cell. As indicated in Fig. 6Go (middle band), we observed much higher binding of Rac to PAK. Together, these data appear to suggest that mastoparan-induced insulin secretion involves activation of Rac, presumably via stimulation of GTP/GDP exchange, independent of its prenylation step (Fig. 5Go).



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  		FIG. 6. Mastoparan induces Rac-1 activation in ßTC3 cells, as evidenced by increased binding to p21-activated kinase. Lysates from ßTC3 cells (control or mastoparan treated; 30 µM for 30 min) were incubated with PAK-PBD beads, and bound proteins were sedimented by centrifugation (see Materials and Methods for additional details). The proteins in the pellet were resolved by SDS-PAGE and transferred to a nitrocellulose membrane, then probed for Rac-1 using an antirabbit serum for Rac-1. Immune complexes were identified using an enhanced chemiluminescence kit as described in Materials and Methods. In addition, lysates from control cells were preincubated with GTP{gamma}S (200 µM for 10 min) to facilitate the formation of ß-cell endogenous Rac-1-GTP{gamma}S complex for use as a positive control in the pull-down assays using PAK-PBD (as described above). As expected, data from these experiments indicate a substantially higher degree of Rac-1 being pulled down under these conditions compared with the mastoparan-treated cell lysates. A representative blot from three experiments yielding comparable results is shown here.

 
Glucose-induced insulin secretion is sensitive to Clostridial toxins and GGTI-2147
We then examined putative regulatory roles of Rac activation in glucose-stimulated insulin secretion from ßTC3 cells. To verify this, we studied the effects of Clostridial toxin B as well as GGTI-2147 on glucose-induced insulin secretion from these cells. The data shown in Fig. 7Go demonstrate marked inhibition of glucose-stimulated insulin secretion by toxin B or GGTI-2147. These findings suggest that, in contrast to mastoparan-stimulated secretion, glucose-stimulated insulin secretion might require geranylgeranylation of Rac, compatible with our earlier observations (4). Taken together, our findings indicate the requirement for Rac activation in mastoparan- as well as glucose-stimulated insulin secretion. The principal difference in the two signaling steps appears to be at the level of posttranslational prenylation of Rac (see Discussion).



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  		FIG. 7. Inhibition by toxin B or GGTI-2147 of glucose-stimulated insulin secretion from ßTC3 cells. ßTC3 cells were cultured in the absence or presence of toxin B (200 ng/ml) or GGTI-2147 (20 µM; 18 h) as described in Materials and Methods. After this, insulin release was quantitated under static conditions (45 min at 37 C) in the presence of either low (5 mM) or high (25 mM) glucose as indicated in the figure. Insulin in the medium was quantitated by ELISA. Data are the mean ± SEM from at least six determinations in each case. No significant effect of either toxin B or GGTI-2147 on basal secretion was demonstrable under the current experimental conditions. *, P < 0.05 vs. basal secretion rates demonstrable in the presence of 5 mM glucose. **, Not significantly different from basal secretion demonstrable in the presence of 5 mM glucose.

 
Mastoparan induces translocation of Rac and Cdc42, but not Ras, from the cytosolic to the membrane compartment
In the next series of experiments, we examined whether exposure of ßTC3 cells to mastoparan results in translocation of specific G proteins from the cytosolic fraction to the membrane fraction for interaction with their effector proteins. For this purpose, ßTC3 cells were incubated with mastoparan (30 µM; 45 min), as in the case of insulin secretion studies (described above). Total membrane and soluble fractions were isolated by a single step centrifugation method (see Materials and Methods for additional details). The relative abundance of small molecular weight G proteins in each of these fractions was determined by Western blotting. The data shown in Fig. 8Go (top panel) indicate a significant increase in the membrane-associated Rac after exposure of these cells to mastoparan. Further, mastoparan induced translocation of Cdc42, a small G protein belonging to Rho subfamily (Fig. 8Go, middle panel). On the other hand, mastoparan had no demonstrable effect on translocation of Ras, another small G protein, to the membrane fraction (Fig. 8Go, bottom panel) under conditions whether it stimulated translocation of Rac and Cdc42 to the membrane fraction. These data are compatible with our previous studies in which we have demonstrated regulatory roles for Cdc42 and Rac, but not Ras, in insulin secretion (3).



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  		FIG. 8. Mastoparan induces translocation of Rac and Cdc42, but not Ras, from the soluble to the particulate fraction in ßTC3 cells. After exposure to mastoparan (30 µM; for 45 min), ßTC3 cells were homogenized, and total particulate (marked as M) and soluble (marked as C) fractions were isolated by single step centrifugation as described in Materials and Methods. Proteins from each fraction were separated by SDS-PAGE, and the relative abundance of Rac, Cdc42, and Ras in the cytosolic and membrane fractions was determined by Western blotting. Data are representative of three experiments with comparable results.

 
Mastoparan induces differential subcellular distribution of Rac and Cdc42 in ßTC3 cells
As the total membrane fraction derived from the single step centrifugation technique employed in the above studies would be comprised of membranes derived from various intracellular organelles (e.g. secretory granules and Golgi complex), we studied the effects of mastoparan on subcellular distribution (and association of) Rac in ßTC3 cells by confocal light microscopy. In the first set of experiments, we examined association of Rac with Golgin-97, a known marker for the Golgi complex. The data shown in Fig. 9Go demonstrate significant colocalization of Rac with Golgin-97 under basal conditions, as evidenced by the bright yellow color in the merged image (A3) of Rac (in red) and Golgin-97 (in green). However, mastoparan treatment caused significant dissociation of Rac from the Golgi complex, as evidenced by only a modest intensity of yellow color demonstrable in Fig. 9GoB3. These data prompted us to determine the association of Rac with insulin under the same experimental conditions (as described in Fig. 9Go). The underlying premise for these experiments is based on experimental evidence from several laboratories, including our own, indicating the involvement of small G proteins in vesicular transport and their fusion with the plasma membrane (7, 8, 36, 41, 45, 52, 53, 54). In these experiments we examined the degree of colocalization of Rac with insulin in control and mastoparan-treated cells to verify whether this G protein would specifically colocalize with insulin under stimulatory conditions. In these experiments colocalization of insulin (in red) with Rac (in green) was verified in control and mastoparan-treated ßTC3 cells. Data derived from the control cells are depicted in Fig. 10Go, A1–A3 (see Fig. 10AGo for additional details). They indicate that Rac and insulin are not colocalized under basal conditions (Fig. 9A3Go). However, after mastoparan stimulation (Fig. 10Go, B1–B3), we observed a significant association of Rac with insulin, as evidenced by the bright yellow color (Fig. 10B3Go). These data indicate significant association of Rac with insulin-containing secretory granules after stimulation with mastoparan. We also investigated colocalization of Cdc42 with insulin under the above experimental conditions to determine whether there is any specificity with regard to mastoparan-induced effects on the association of candidate G proteins with insulin. Data derived from control cells are given in Fig. 10BGo (C1–C3). They indicate modest, if any, colocalization of Cdc42 with insulin (Fig. 10C3Go) under basal conditions. Interestingly, however, in contrast to its more pronounced effects on colocalization of Rac with insulin, mastoparan exerted only modest effects on colocalization of Cdc42 with insulin (Fig. 10D3Go). Together, the data described in Fig. 8Go (Western blotting) and Figs. 9Go and 10Go (confocal light microscopy) indicate substantial, but differential, distribution of Rac and Cdc42 within the ß-cell after stimulation with mastoparan (see Discussion).



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  		FIG. 9. Mastoparan induces alterations in association of Rac with the Golgi complex. A1–A3, Control cells. A1, Immunolocalization of Rac (red) in control ßTC3 cells. A2, Immunolocalization of Golgin-97 (green), which is a marker for the Golgi complex, in the same cells as in A1. A3, Overlay image of A1 and A2; the bright yellow is indicative of a considerable degree of association of Rac with the Golgi complex under basal conditions. B1–B3, Mastoparan-treated cells. B1, Immunolocalization of Rac (red) in mastoparan-treated cells (30 µM; 30 min). B2, The Golgi complex (green) in the same cells as in B1. B3, Overlay image of B1 and B2. A much less intense yellow may indicate probable movement or dissociation of Rac from the Golgi after mastoparan stimulation (see text for additional details). Data are representative of three experiments with comparable results. Total magnification of images, approximately x400.

 


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  		FIG. 10. A, Association of Rac with insulin in ßTC3 cells. Effects of mastoparan. A1–A3, Control cells. A1, Immunolocalization of insulin (red). A2, Immunolocalization of Rac (green). A3, Overlay of A1 and A2. These data suggest Rac and insulin are not colocalized under basal conditions. B1–B3, Mastoparan-treated cells. B1, Immunolocalization of insulin (red). B2, Immunolocalization of Rac (green). B3, Overlay of B1 and B2. The bright yellow (B3) suggests a significant degree of association of Rac with insulin after stimulation with mastoparan. Data are representative of three experiments with comparable results. B, Association of Cdc42 with insulin in ßTC3 cells; effects of mastoparan. C1–C3, Control cells. C1, Insulin (red). C2: Rac (green). C3, Overlay of C1 and C2, suggesting very little, if any, association of Cdc42 with insulin under basal conditions. D1–D5, Mastoparan-treated cells. D1, Insulin (red). D2, Rac (green). D3, Overlay of D1 and D2, indicating a modest association of Cdc42 with insulin after mastoparan stimulation. Data are representative of three experiments with comparable results.

 
Stable expression of dominant negative Rac markedly reduces mastoparan-induced insulin secretion
To further confirm that Rac is a key regulatory protein in mastoparan-induced insulin secretion, we studied mastoparan-stimulated insulin secretion from INS-1 cells stably expressing vector alone or the dominant negative form of Rac (see Materials and Methods for additional details). Using an antiserum directed against the epitope tag (e.g. Myc), we confirmed the expression of dominant negative Rac into ß-cells (Fig. 11AGo). The data shown in Fig. 11BGo demonstrate that mastoparan (30 µM for 45 min) significantly stimulated insulin secretion. Further, we observed a significant reduction in mastoparan-induced insulin secretion from INS-1 cells expressing dominant negative (N17Rac) Rac (Fig. 11BGo). These findings further support our toxin experiments (Fig. 4Go, A and B), indicating that Rac represents one of the regulatory proteins involved in mastoparan-induced insulin secretion from isolated ß-cells. It must be kept in mind that the degree of inhibition of mastoparan-induced insulin secretion from cells expressing dominant negative Rac is less than the degree of inhibition demonstrable in toxin-treated cells (-30% in transfected cells vs. more than -50% in toxin-treated cells). Therefore, it is very likely that mastoparan-induced insulin secretion could involve the intermediacy of other proteins (e.g. Cdc42; see Discussion). Alternatively, some residual endogenous Rac may still be functional, although we failed to detect a significant translocation of Rac from the cytosolic to the membrane fraction in transfected cells (55).



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  		FIG. 11. Stable expression of dominant negative form of Rac markedly attenuates mastoparan-induced insulin secretion from INS-1 cells. A, INS-1 cells transfected with empty (pIRES) or N17Rac1 vectors were homogenized as described above. Protein samples (20 µg protein each) were denatured in treatment buffer at 95 C for 5 min and separated by SDS-PAGE (15% acrylamide). The separated proteins were transferred to nitrocellulose membranes, and blots were probed with anti-Myc monoclonal antibody (2 µg/ml). After blocking with 5% fat-free milk, the membranes were incubated with horseradish peroxidase conjugated to antimouse IgG antibodies (1:1000). Immunoreactive bands were visualized using an enhanced chemiluminescence kit. B, INS-1 cells (control or transfected with dominant negative Rac) were preincubated (in 24-well plates) in Krebs-Ringer buffer with 2.8 mM glucose for 30 min. The cells were then stimulated with 30 µM mastoparan for 45 min. Results are the mean ± SEM from three separate experiments performed in triplicate. *, P < 0.05 vs. secretion demonstrable in control cells stimulated with mastoparan.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
One of the main objectives of the current study is to verify whether mastoparan-induced insulin secretion from isolated ß-cells is under the control of small molecular mass G proteins. Salient features of the current study include the following: 1) mastoparan or mastoparan 7, but not mastoparan 17 (an inactive analog), stimulation of insulin secretion from ßTC3 cells and INS-1 cells; 2) a marked attenuation of mastoparan-induced insulin secretion by two types of Clostridial toxins, which selectively inactivate the Rho subfamily of G proteins (e.g. Rac and Cdc42); 3) a complete resistance of mastoparan-induced insulin secretion to a specific inhibitor of geranylgeranylation of G proteins (e.g. Rac and Cdc42); 4) a significant inhibition of glucose-stimulated insulin secretion by Clostridial toxin as well as GGTI-2147; 5) a significant translocation of Rac and Cdc42, but not Ras, from the cytosolic to the membrane compartment in cells treated with mastoparan; 6) a significant colocalization of Rac (and, to a lesser degree, Cdc42) with insulin in cells treated with mastoparan; and 7) a significant reduction in mastoparan-induced insulin secretion in cells expressing the dominant negative form of Rac. Taken together, these data provide evidence to indicate that mastoparan-induced insulin secretion involves activation of specific small G proteins, such as Rac and Cdc42.

Our findings also suggest that the effects of mastoparan on insulin secretion from ßTC3 cells and INS-1 cells are structure specific, as we failed to observe any stimulatory effect of mastoparan 17, the inactive analog of mastoparan, on insulin secretion from these cells. We also observed a significant inhibition of mastoparan-induced insulin secretion in the absence of extracellular calcium. These observations are in contrast to earlier reports by Straub et al. (in rat and human islets) (23) and Jones et al. (in electrically permeabilized rat islets) (14) that demonstrate minimal effects of extracellular calcium on mastoparan-stimulated insulin secretion. Reasons for these differences among these observations remain unclear at this time, but could include differences among the cell types used in these studies. These differences may not lie at the level of activation of these G proteins (e.g. Rac and Cdc42), as we have demonstrated previously (3) that Clostridial toxins (e.g. toxin B and lethal toxin) inhibited glucose-stimulated insulin secretion from normal rat islets as well as clonal ß-cells. Additional studies are needed to explain these differences between cell types with regard to the requirement for extracellular calcium.

In a recent study Daniel et al. (27) demonstrated that expression of wild-type Cdc42 into an insulin-secreting cell line (ßHC9) resulted in a 2-fold increase in mastoparan-induced insulin release compared with those cells expressing the vector alone. These investigators also provided evidence to indicate that such an increase was not demonstrable in the presence of other secretagogues, such as glucose, that stimulate insulin secretion via the calcium-dependent pathway. Lastly, they used pertussis toxin (Ptx), which modifies the inhibitory type (e.g. Gi or Go) of trimeric G proteins, and they ruled out a possible involvement of a heterotrimeric protein(s) in mastoparan-induced, Cdc42-mediated stimulation of insulin secretion from these cells (27) (see below). Our present study complements that report, in that low molecular weight G proteins are involved in mastoparan-induced insulin secretion.

Our Western blot data indicate that mastoparan induces the translocation of Rac and Cdc42, but not Ras, to the membrane fraction. These data are also compatible with the confocal light microscopy data suggestive of an increase in the association of Rac and, to a lesser degree, Cdc42 with insulin. This raises the interesting possibility that Rac may be involved in the transport of insulin-containing secretory granules to the plasma membrane for the fusion and release of granular contents. It may also be involved in cytoskeletal organization, as proposed in other cell types (52, 53, 54), to facilitate the movement of granules to the plasma membrane fraction. Compatible with this postulation are our current observations indicating a significant association of Rac with insulin-containing secretory granules after exposure of insulin-secreting cells to mastoparan. In addition, our data provide clear evidence for the association of this G protein with the Golgi complex (Golgin-97 used as a marker protein) under basal conditions and its subsequent dissociation from the Golgi complex after stimulation with mastoparan, suggesting significant alterations in the distribution of Rac in mastoparan-treated cells. Further, in support of these findings are recent observations by Li et al. (55) demonstrating significant morphological as well as glucose-sensitive insulin secretory abnormalities in INS-1 cells after the transfection of dominant negative Rac1 in these cells. On the other hand, mastoparan-induced translocation of Cdc42 to the membrane fraction could promote interaction between Cdc42 and syntaxin, as recently demonstrated by Daniel et al. (27). It is also likely that translocation of Cdc42 to the membrane fraction results in its activation of phospholipase C, as we have demonstrated in rat islets and clonal ß-cells (2). Therefore, mastoparan-induced insulin secretion might involve its effects on more than one G protein (e.g. Rac and Cdc42), subserving different functions in the stimulus-secretion coupling cascade.

In the above context we speculate, at least based on known properties of mastoparan, that activation by mastoparan of Rac primarily involves GDP/GTP exchange, since we failed to observe any effects of GGTI-2147 an inhibitor of geranylgeranylation of Rac and Cdc42 on mastoparan-induced insulin secretion. Data from our pull-down assays for PAK-Rac1 binding are suggestive of mastoparan-induced activation (i.e. formation of the GTP-bound form) of Rac in ßTC3 cells. These data thus imply no direct regulation by mastoparan of this modification step in isolated ß-cells. In support of this, we have demonstrated previously (1) that mastoparan-induced insulin release from normal rat islets was resistant to either lovastatin (an inhibitor of protein prenylation) or acetyl farnesyl cysteine (an inhibitor of C-terminal methylation). Interestingly, in these studies we observed that absolute insulin released by mastoparan was potentiated by lovastatin and acetyl farnesyl cysteine, suggesting that, in addition to its stimulatory effects, mastoparan might simultaneously activate one (or more) inhibitory G proteins, which require isoprenylation and carboxyl methylation. In addition, recent data from the laboratory of Aizawa (26) have demonstrated resistance of mastoparan-induced insulin release from ß-cells to cerulenin, an inhibitor of fatty acylation of proteins, including G proteins (1, 7, 8). Taken together, these data appear to suggest that mastoparan-induced insulin secretion does not require posttranslational modification of G proteins at their C-terminal cysteines. Interestingly, we have also recently characterized a novel histidine kinase activity in human islets, rat islets, and clonal ß-cells, which is stimulated by mastoparan (28). In that report we hypothesized that such a kinase could subserve the function of facilitating the conversion of the GDP-bound inactive form of G proteins to their GTP-bound active configuration (28, 36, 56). It remains to be determined whether such a mastoparan-sensitive histidine kinase is involved in the activation of Rac, by converting its GDP-bound inactive form to its GTP-bound active status. Our current data also indicate significant differences between mastoparan-induced and glucose-induced insulin secretion from these cells. For example, we have shown that unlike mastoparan, glucose-induced insulin secretion requires a geranylgeranylation step, as GGTI-2147 abolished glucose-stimulated insulin secretion from ßTC3 cells. Based on these data, we hypothesize that this posttranslational modification may be necessary for optimal interaction of Rac with the putative effector protein(s) required for glucose-stimulated insulin secretion.

Several earlier studies have demonstrated involvement of Ptx-sensitive G proteins in mastoparan-induced insulin release (1, 11, 12, 16, 19). Along these lines, we have also characterized a mastoparan-sensitive, high affinity guanosine triphosphatase activity that is associated with the insulin-containing secretory granule fraction derived from normal rat islets and human islets. Such an activity was stimulated by prostaglandin E2 and was sensitive to Ptx (16). Mastoparan-activated, Ptx-sensitive, trimeric G proteins in the ß-cell have been identified by other investigators as well (19). Therefore, our current data do not exclude the possibility that there may be an intermediary involvement of heterotrimeric G protein, which, in turn, regulates a low molecular weight G protein or vice versa. Indeed, using human embryonic kidney cells, Schmidt et al. (57) demonstrated inhibition by Clostridium toxin B of phospholipase D activity stimulated by aluminum fluoride. As the latter stimulates heterotrimeric G proteins, but only rarely small G proteins, these data suggest that toxin B interferes with the signaling pathway to phospholipase D somewhere downstream of the receptor-mediated activation of heterotrimeric G proteins. In view of the recent observations by Daniels et al. (27), and based on our current observations, we also do not exclude the possibility that a cascade of several small G proteins (e.g. Rac and Cdc42) is involved in mastoparan-induced insulin secretion. Indeed, a similar signal transduction mechanism(s), involving a cascade of G proteins (e.g. Cdc42->Rho->Rac), has been proposed in other cell types for regulation of the actin cytoskeleton (3).

Our current studies may also have direct relevance to mechanisms underlying abnormal insulin secretion from islets from diabetic animal models. We recently reported (24) that despite intact contents of insulin and protein, islets derived from the Goto-Kakizaki (GK) rat, a model for type 2 diabetes, had markedly deficient insulin release in response to glucose as well as to mitochondrial fuels or a nonnutrient, membrane-depolarizing stimulus (40 mM KCl). In contrast, in GK islets, mastoparan completely circumvented any secretory defect. The basal and stimulated levels of adenine and guanine nucleotides, the activation of phospholipase C by Ca2+ or glucose, the secretory response to Ptx, and the activation of select small G proteins (e.g. Cdc42) were not impaired. However, we observed significant defects in the autophosphorylation and catalytic activity of cytosolic nucleoside diphosphokinase, which may provide compartmentalized GTP pools to activate G proteins (24, 56). Based on these data, we proposed (24, 56) that defects late in signal transduction in the islets from GK rats, possibly occurring at the site of activation by nucleoside diphosphokinase of a mastoparan-sensitive G protein-dependent step in exocytosis. Although our present studies clearly demonstrate a requirement for Rac activation in mastoparan- or glucose-stimulated insulin secretion, they also provide unique differences in their requirements (i.e. geranylgeranylation step). Therefore, it may also be probable that the functional status of protein-prenylating enzymes is significantly impaired in the GK islet. In this context, we have recently obtained immunological evidence for localization of farnesyl and geranylgeranyl transferases in isolated ß-cells (Kowluru, A., H.-Q. Chen, and M. Tannous, submitted). Additional studies are needed to identify these critical signaling steps to precisely determine alterations in the diabetic islet leading to abnormal insulin secretion.


   Footnotes
 
This work was supported by funds from the Department of Veterans Affairs (Merit Review and Research Enhancement Award Program, to A.K.), the National Institutes of Health (DK-56005, to A.K.), the Grodman Cure Foundation (to A.K.), the American Diabetes Association (to A.K.), the National Medical Research Council of Singapore (R-364-000-004-213 and NMRC/0540/2001, to G.D.L.), and a Research Career Scientist Award from the Department of Veterans Affairs (to A.K.).

R.H.A. and H.-Q.C. contributed equally to this work.

Abbreviations: GK, Goto-Kakizaki; GTP{gamma}S, a nonhydrolyzable GTP analog; LT, lethal toxin; PAK, p21-activated kinase; PBD, p21-binding domain; Ptx, pertussis toxin.

Received January 22, 2003.

Accepted for publication June 10, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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