©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Heterotrimeric G-protein G Is Localized to the Insulin Secretory Granules of -Cells and Is Involved in Insulin Exocytosis (*)

Robert J. Konrad (1), Robert A. Young (1), Rae D. Record (1), Robert M. Smith (1), Paul Butkerait (2), David Manning (2), Leonard Jarett (1), Bryan A. Wolf (1)(§)

From the (1) Departments of Pathology and Laboratory Medicine and (2) Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Mastoparan, a tetradecapeptide found in wasp venom that stimulates G-proteins, increases insulin secretion from -cells. In this study, we have examined the role of heterotrimeric G-proteins in mastoparan-induced insulin secretion from the insulin-secreting -cell line -TC3. Mastoparan stimulated insulin secretion in a dose-dependent manner from digitonin-permeabilized -TC3 cells. Active mastoparan analogues mastoparan 7, mastoparan 8, and mastoparan X also stimulated secretion. Mastoparan 17, an inactive analogue of mastoparan, did not increase insulin secretion from permeabilized -TC3 cells. Mastoparan-induced insulin secretion from permeabilized -TC3 cells was inhibited by pretreatment of the cells with pertussis toxin, suggesting that mastoparan-induced insulin secretion is mediated through a pertussis toxin-sensitive G-protein present distally in exocytosis. Enriched insulin secretory granules (ISG) were prepared by sucrose/nycodenz ultracentrifugation. Western immunoblotting performed on -TC3 homogenate and ISG demonstrated that G was dramatically enriched in ISG. Levels of G and G were comparable in homogenate and ISG. Mastoparan stimulated ISG GTPase activity in a pertussis toxin-sensitive manner. Mastoparan 7 and mastoparan 8 also stimulated GTPase activity in the ISG, while the inactive analogue mastoparan 17 had no effect. Selective localization of G to ISG was confirmed with electron microscopic immunocytochemistry in -TC3 cells and -cells from rat pancreas. In contrast to G and G, G was clearly localized to the ISG. Together, these data suggest that mastoparan may act through the heterotrimeric G-protein G located in the ISG of -cells to stimulate insulin secretion.


INTRODUCTION

Insulin secretion from -cells can be stimulated by different types of secretagogues (1) . D-Glucose, a fuel secretagogue, is the major physiological stimulus (2, 3) . The mechanism by which glucose-induced insulin release occurs is not completely elucidated, although glucose oxidation is essential (3, 4, 5, 6) . Glucokinase is believed to act as a glucose sensor, with phosphorylation of glucose to glucose 6-phosphate serving as the rate-limiting step in glucose oxidation (7) . While inhibition of glucose oxidation inhibits insulin release, the details of the mechanism coupling glucose oxidation to insulin secretion are less clear. It is currently believed that oxidation of fuel secretagogues increases intracellular levels of ATP (8), although this view has been challenged by some groups (9) . An increased ATP/ADP ratio is believed to close K channels at the plasma membrane, resulting in decreased K efflux and subsequent depolarization of the -cell (10, 11, 12) . Depolarization then activates voltage-dependent Ca channels, causing an influx of extracellular Ca into the -cell and an increase in intracellular Ca levels (6, 13) . In addition, it has recently been shown that nutrient secretagogues increase -cell malonyl-CoA levels, which leads to increased cytosolic long-chain acyl-CoA esters that positively modulate insulin secretion (14-16). While increased intracellular Ca activates protein kinases such as the Ca- and calmodulin-dependent protein kinase (17, 18, 19, 20, 21) , the mechanism by which insulin exocytosis occurs is not completely elucidated. Over the past few years, factors other than calcium have been proposed to be involved, including GTP and small monomeric GTP-binding proteins such as Rab3A (22) .

In addition to small GTP-binding proteins, heterotrimeric GTP-binding proteins have also been implicated in regulating insulin secretion by pancreatic -cells (23, 24, 25) . Epinephrine is an adrenergic agonist that inhibits insulin secretion by acting through a heterotrimeric G-protein (23, 26, 27) . Although epinephrine acts through a G-protein-coupled adrenergic receptor located on the plasma membrane of the -cell to decrease cAMP accumulation, this is not thought to account entirely for its inhibition of insulin secretion (26) . The ability of epinephrine to inhibit insulin secretion is believed to be due to its action on the distal portion of the insulin exocytotic pathway, since epinephrine is capable of inhibiting calcium-induced insulin secretion from permeabilized -cells (27) . Epinephrine is thought to inhibit insulin release by acting through a pertussis toxin-sensitive G-protein. Epinephrine inhibition of insulin secretion is itself inhibited by pretreatment of cells with pertussis toxin, which ADP ribosylates pertussis toxin-sensitive G-protein subunits at a cysteine residue located four amino acids from the carboxyl terminus (26) . This ADP ribosylation renders the G-protein subunit incapable of interacting with its receptor.

In addition to epinephrine, a variety of other compounds are thought to modulate insulin secretion by acting through heterotrimeric G-proteins (23). One class of such compounds consists of amphiphilic peptides. A well known member of this class is mastoparan, a tetradecapeptide found in wasp venom. Mastoparan stimulates insulin secretion from -cells (28, 29) and also stimulates secretion from many other cell types such as platelets (30, 31, 32) , neutrophils (33) , and pneumocytes (34) . Mastoparan has been shown to increase the GTPase activity of many G-proteins including G and G(35, 36) . Mastoparan is thought to stimulate these G-proteins by inserting itself into the membrane adjacent to G-proteins and mimicking the normal interaction that occurs between G-protein-coupled receptors and their respective G-proteins (37, 38) . Mastoparan is believed to bind to the carboxyl terminus of the G-protein subunit, resulting in and GDP dissociation from the subunit, causing the subunit to assume the active configuration (39, 40) . Mastoparan, however, unlike epinephrine, stimulates insulin secretion from -cells, suggesting that G-proteins, in addition to having a role in negatively modulating insulin secretion, may also positively modulate insulin secretion (28, 29) . The identity of the G-protein through which mastoparan stimulates insulin secretion from the -cell is unknown.

To better understand if mastoparan stimulates insulin secretion from the -cell by acting distally in exocytosis, we have examined the effects of mastoparan and its analogues on insulin secretion from digitonin-permeabilized -cells. We have also examined the effects of various mastoparan analogues on GTPase activity of insulin secretory granules (ISG)() prepared from -cells and have shown that the ability of mastoparan analogues to stimulate ISG GTPase activity correlates with their ability to stimulate insulin secretion. Finally, we have used G-protein antisera for Western blotting and electron microscopic immunocytochemistry in order to determine which G-protein is localized to the ISG of -cells and is a target for mastoparan.


EXPERIMENTAL PROCEDURES

Materials

Tissue culture medium (CMRL-1066) and 1 M HEPES were from Life Technologies, Inc. Newborn bovine serum was from Hazleton Biologics (Lenexa, KS). -TC3 cells (passage 34) were obtained through the University of Pennsylvania Diabetes Center from Dr. D. Hanahan (University of California, San Francisco). The following compounds were purchased from Sigma: D-glucose, carbachol, Hanks' balanced salt solution, penicillin, streptomycin, glutamine, Ficoll, bovine serum albumin, ovalbumin, ATP, GTP, NAD, thymidine, and creatine phosphate. Pertussis toxin was purchased from List Biological Laboratories (Campbell, CA). Mastoparan; its active analogues mastoparan 7, mastoparan 8, and mastoparan X; and its inactive analogue mastoparan 17 were purchased from Peninsula Laboratories (Belmont, CA). Creatine kinase and adenylyl imidodiphosphate were purchased from Boehringer Mannheim. Digitonin was obtained from Waco Pure Chemical Industries. [P]GTP (10 Ci/mmol) and [P]NAD (1000 Ci/mmol) were purchased from Amersham Corp. I-Protein A was purchased from ICN Radiochemicals (Irvine, CA). Antisera to selected G-proteins were raised by injecting New Zealand White rabbits with synthetic decapeptides corresponding to the carboxyl-terminal amino acid sequences of the respective G-proteins as described previously (41-43). In this study, the following antisera were used for Western blotting and electron microscopic immunocytochemical analysis of rat pancreas: anti-G 8730 (immunogen (KLH)KNNLKDCGLF), anti-G 9072 (immunogen (KLH)ANNLRGCGLY), and anti-G 946 (immunogen (KLH)QLNLKEYNLV). These same antisera, along with anti-G/G 5296 (immunogen bovine brain G) were used for electron microscopic immunocytochemical analysis of -TC3 cells.

Methods

-TC3 Cell Line Culture

-TC3 cells were cultured in 6-well plates or 15-cm dishes in the presence of RPMI 1640 (11 mM glucose) supplemented with 10% fetal bovine serum, penicillin (75 µg/ml), streptomycin (50 µg/ml), and 2 mML-glutamine. Cells were trypsinized and subcloned weekly. Medium was changed twice weekly and the day prior to an experiment. Insulin secretory capacity of the -TC3 cells in response to glucose and carbachol was monitored regularly. Cells were used between passages 40 and 55.

Pretreatment of -TC3 cells with Pertussis Toxin

Approximately 24 h prior to the experiment, -TC3 cells in either 6-well plates or 15-cm dishes were cultured in RPMI 1640 supplemented with 50 ng/ml pertussis toxin.

Permeabilization of -TC3 cells

-TC3 cells in 6-well plates were washed 3 times with 2 ml of Krebs-HEPES buffer (25 mM HEPES, pH 7.40, 115 mM NaCl, 24 mM NaHCO, 5 mM KCl, 2.5 mM CaCl, 1 mM MgCl, and 0.1% bovine serum albumin). Cells in monolayers were permeabilized in the same media supplemented with 20 µg/ml digitonin for 10 min at 37 °C under an atmosphere of 95% air, 5% CO(44) . Using this method, greater than 99% permeabilization of -TC3 cells was achieved as assessed by trypan blue exclusion. Lactate dehydrogenase release into the permeabilization medium was found to become maximal at a digitonin concentration of 20 µg/ml.

Incubation of Digitonin-permeabilized -TC3 for Insulin Secretion

Permeabilized cells were washed 3 times with 2 ml of TES buffer (50 mM TES, pH 7.40, 100 mM KCl, 1 mM EGTA, 2 mM MgCl, and 0.1% bovine serum albumin). Cells were incubated for 60 min at 37 °C under an atmosphere of 95% air, 5% CO with fresh buffer supplemented with 1 mM ATP and the appropriate secretagogue. In selected experiments, an ATP regenerating system consisting of 5 mM creatine phosphate and 0.2 mg/ml creatine phosphokinase was included. At the end of the incubation period, a sample of the supernatant was removed for insulin measurement by radioimmunoassay (45) .

Subcellular Fractionation of -TC3 Cells

-TC3 cells were fractionated by sequential centrifugation using a sucrose/nycodenz gradient as described previously (46) . 10-20 confluent 15-cm dishes of -TC3 cells were washed 3 times with ice-cold 50 mM MES, pH 7.20, containing 0.25 M sucrose and 1 mM EGTA. Cells were scraped and resuspended in 1 ml of the same buffer. Cells were then mechanically homogenized with 10 strokes of a motor-driven Teflon pestle in a Potter-Elvehjem homogenizing tube. The homogenate was centrifuged (600 g, 5 min) to remove intact cells and nuclear debris. The supernatant was saved, and the pellet was homogenized twice more as above. Supernatants were combined and transferred to a 13-ml Beckman ultracentrifuge tube already underlaid with stepwise gradients (4 ml) of nycodenz stock/homogenization buffer in the following ratios: 16:84, 32:68, 64:36. Tubes were centrifuged at 100,000 g (SW-40 rotor, Beckman TL-100 centrifuge) for 60 min to yield a band at the lower interface corresponding to ISG. Granules were harvested, resuspended in a 15-ml Corex tube in 14 ml of 10 mM MES, pH 6.50 containing 0.25 M sucrose, and centrifuged at 27,000 g (Beckman J2-21 centrifuge, JA-20 rotor) for 20 min. ISG were resuspended in 100-200 µl of 10 mM MES, pH 7.40, supplemented with 1 mM EDTA. Insulin enrichment of the ISG relative to the respective homogenate was systematically checked. Enzyme marker studies were performed as described previously (47) .

Western Blotting of -TC3 Fractions with Antibodies to G-Proteins

For these experiments, 2-25 µg of protein in 20 µl of loading buffer (70 mM Tris, pH 6.7, 16 M urea, 6.0% SDS, 100 mM dithiothreitol, 0.005% bromphenol blue) were loaded onto 10% SDS-polyacrylamide gel electrophoresis gels. Colored rainbow molecular weight markers (Amersham Corp.) were also run on each gel. Proteins were separated for 1 h at 175 V at room temperature using a Bio-Rad Mini-PROTEAN II dual slab cell. Proteins were transferred to nitrocellulose paper (Hybond C, Amersham Corp.) for 1.5 h at 100 V at 4 °C using a Bio-Rad mini Trans-Blot electrophoretic transfer cell. Nitrocellulose blots were blocked overnight in blocking buffer (1% ovalbumin, 3% bovine serum albumin, 10 mM Tris, pH 7.40, 150 mM NaCl, and 0.1% sodium azide) at 4 °C. The next day, blots were probed with appropriate antisera at a dilution of 1:100 in blocking buffer for 3 h at room temperature. Blots were washed twice with 10 mM Tris, pH 7.40, 150 mM NaCl, and 0.1% sodium azide (TNA) for 10 min, once with TNA supplemented with 0.05% Nonidet P-40 for 5 min, and twice more with TNA for 10 min. Blots were incubated with 5 µCi of I-protein A in 10 ml of blocking buffer for 1 h at room temperature. Blots were washed again as above, air dried, and exposed on a PhosphorImager cassette (Molecular Dynamics) overnight at room temperature. The following day, radioactivity bound to the blot was quantitated using a Molecular Dynamics PhosphorImager and ImageQuant software.

Measurement of GTPase Activity

The reaction was initiated by the addition of 5 µg of sample protein to 100 µl of 25 mM HEPES, pH 7.20, supplemented with 1 mM dithiothreitol, 1 mM EGTA, 20 µM MgCl, 1 mM ATP, 1 mM adenylyl imidodiphosphate, 5 mM creatine phosphate, 0.2 mg/ml creatine phosphokinase, 0.2% bovine serum albumin, 100 nM [-P]GTP, and varying concentrations of mastoparan or mastoparan analogues in a 1.5-ml snap-top Eppendorf tube. Tubes were incubated at 30 °C in a shaking water bath for 5 min. The reaction was stopped by the addition of 900 µl of ice-cold trichloroacetic acid, pH 2.0, supplemented with 5% charcoal by weight, which was kept continuously stirring on ice. Tubes were centrifuged at 10,000 g for 30 min at 4 °C, and P in the supernatant was quantitated by liquid scintillation spectrophotometry. Blanks determined in the presence of 50 µM GTP were routinely subtracted, and GTPase activity was calculated as pmol of GTP hydrolyzed per mg of protein/min. Under these conditions, GTPase activity was linear at 30 min.

Pertussis Toxin-catalyzed ADP-ribosylation Assay

The reaction was initiated by the addition of 5 µg of protein to 100 µl of 25 mM HEPES, pH 7.20, supplemented with 1 mM EDTA, 1 mM ATP, 0.1 mM GTP, 0.1% Lubrol, 0.02% bovine serum albumin, 10 mM thymidine, 10 µM [P]NAD, and 5 µg/ml activated pertussis toxin. Tubes were incubated at 30 °C in a shaking water bath for 30 min. The reaction was stopped by the addition of 900 µl of ice-cold 10% trichloroacetic acid, pH 2.0. Tubes were centrifuged at 10,000 g for 20 min at 4 °C, and membrane pellets were washed once with 1 ml of 10% trichloroacetic acid, pH 2.0. Membrane pellets were then suspended in 1 ml of ice-cold diethyl ether. Ether was evaporated in a Savant concentrator connected to a -90 °C cold trap, and membrane pellets were resuspended in 30 µl of loading buffer supplemented with 15 mg/ml dithiothreitol. Samples were then run on 10% SDS-polyacrylamide gels as described above. Gels were dried and exposed on a PhosphorImager cassette overnight. The following day, radioactivity bound to the blot was quantitated using a Molecular Dynamics PhosphorImager and ImageQuant software.

-TC3 Cell Processing for Immunoelectron Microscopy

-TC3 cells were plated on Costar Transwell cell culture inserts 5 days before use and cultured as described above. Medium was changed the day prior to fixation. On the day of fixation, the medium was aspirated, and 2 ml of ice-cold phosphate-buffered saline (PBS), pH 7.40, was added to wash the cells. Inserts were removed and transferred to a culture plate containing PBS for 1-2 min. PBS was aspirated, and 2 ml of GPA fixative containing 1% glutaraldehyde and 0.2% picric acid in pH 7.40 PBS at 4 °C was added. Inserts were transferred to culture plates containing fixative. The cells were allowed to fix for 2-18 h at 4 °C. Cells were dehydrated with 70, 80, and 90% ethanol at 4 °C for 30 min. Cells were stained with 1% eosin B (to permit visualization of the cells) in 100% ethanol for 8 min at 24 °C and washed in 100% ethanol 3-4 times.

Inserts were transferred to LR White, 100% ethanol (1:1) for 2 h and through three changes of 100% LR White (Electron Microscopy Sciences, Fort Washington, PA) for 1 h, overnight, and for 1 h the next morning. The membrane on which the cells were attached was removed from the plastic insert with a scalpel. The membrane was gently rolled, with cells on the inside, into a roll approximately 2 mm in diameter. The membrane was secured in two places by tying a short length of hair around the roll. The roll was cut between the two ties and excess membrane on the outer side of the ties was removed and discarded. Membranes were placed in gelatin capsules partially filled with LR White resin with the cut end oriented toward the bottom of the capsule. Capsules were filled with resin, capped, and cured for 18 h at 60 °C. Thin sections (600-700 Å thick) were cut perpendicular to the plane of the membrane and mounted on nickel grids.

Tissue Processing for Immunoelectron Microscopy

Pancreata were excised from male Sprague-Dawley rats and minced in the GPA fixative. Tissue was fixed 2-18 h, dehydrated, infiltrated, and embedded in LR White resin as described above. Thick sections were cut from randomly selected tissue blocks and stained with 1% toluidine blue in 1% sodium borate to select blocks with islets. Thin sections (600-700 Å thick) were cut from the selected blocks and mounted on nickel grids.

Immunostaining and Electron Microscopy

Thin sections of -TC3 cells and rat pancreas were floated section-side down on small drops of 1% ovalbumin, 0.2% cold water fish skin gelatin in PBS for 1 h at 24 °C to reduce nonspecific immunoglobulin absorption. Sections of -TC3 cells were incubated with G-protein antisera diluted one-tenth to one-thousandth with PBS overnight at 4 °C in a humidified chamber. Sections were washed by transferring the grids through small drops of 20 mM Tris-HCl-buffered saline with 0.02% Tween-20, pH 7.40, (TBST) four times for 5 min, each at 24 °C. -TC3 cells were incubated with 15-nm diameter gold-labeled protein A prepared as described (48, 49, 50) for 1 h at 24 °C to detect bound immunoglobulin. Two staining procedures were used to identify -cells and -cells and, by deduction, -cells, in islets. In the first, serial sections were cut, and different sections were stained with anti-insulin, anti-glucagon, or anti-G-protein antisera, and the antibodies were detected with gold-labeled protein A as described above. Alternatively, sections were co-incubated overnight at 4 °C with anti-G-protein Ig complexed to 18-nm diameter colloidal gold and anti-insulin or anti-glucagon Ig complexed to 8-nm diameter colloidal gold. All gold-labeled immunoglobulins were prepared as described previously (49, 50) . All sections were washed twice with TBST, twice with deionized water, and then stained with 2% aqueous uranyl acetate for 3 min.

Other Methods

Insulin radioimmunoassay was performed by the University of Pennsylvania Diabetes Endocrine Research Center. Protein concentrations of -TC3 cell granule fractions and homogenates were calculated using a bicinchoninic acid microassay with bovine serum albumin as the standard (51) .

Data Analysis

Results are expressed as the mean ± S.E. Statistical analysis was performed using version 5.0 of SPSS for Windows. Data were analyzed by one-way analysis of variance or analysis of covariance as appropriate followed by multiple comparisons between means using the least significant difference test. A probability of p < 0.05 was considered statistically significant.


RESULTS

Choice of -TC3 Cells

An insulinoma cell line was used to study mastoparan-induced insulin secretion and the effects of mastoparan on secretory granule GTPase activity because of the large amount of cells necessary to perform these experiments. Between 30 and 60 rats would need to be sacrificed to obtain the same number of cells provided by 20 15-cm dishes of -TC3 cells. In addition, -TC3 cells provide a pure population of -cells (which respond to glucose and carbachol with robust insulin secretion) (25) , whereas islets obtained from rats constitute a mixed population of endocrine and other cell types. Finally, -TC3 cells grown in monolayers can be more uniformly permeabilized than islets, which consist of aggregates of thousands of cells.

Effect of Mastoparan and Mastoparan Analogues on Insulin Secretion from Digitonin-permeabilized -TC3 Cells and on -TC3 Cell Insulin Secretory Granule GTPase Activity

In this study, digitonin-permeabilized -TC3 cells were used as a model system to study insulin exocytosis. Mastoparan is a peptide that mimics an activated receptor (35, 36, 37, 38) by causing exchange of GDP for GTP on the G-protein subunit concomitant with dissociation of from (35, 39, 40). The subunit, having assumed an active configuration, can interact with the appropriate effector, as can in certain instances. Mastoparan stimulated insulin secretion from digitonin-permeabilized -TC3 cells in a dose-dependent manner (Fig. 1A). Mastoparan-induced insulin secretion was largely dependent on ATP being present. Removal of 1 mM ATP from the incubation buffer decreased mastoparan-induced insulin secretion from 493.10 ± 32.65% of control to 209.16 ± 8.50% of control (n = 6). Inclusion of an ATP regenerating system (18) in addition to the presence of 1 mM ATP did not significantly affect mastoparan-induced secretion (n = 6). Mastoparan-induced insulin secretion was not significantly increased or decreased by the addition of 2.5 mM CaCl to the incubation buffer (n = 6). The active mastoparan analogues mastoparan 7, mastoparan 8, and mastoparan X stimulated insulin secretion to 517 ± 29, 501 ± 55, and 412 ± 57% of control, respectively (p < 0.05 versus control), while mastoparan 17, an inactive analogue of mastoparan, had no significant effect (Fig. 1B). The inclusion of 1 mM GTP or 10 µM GTPS in the incubation buffer did not significantly augment mastoparan-induced insulin secretion (n = 6). This could be due to the possibility that permeabilization of the cells does not completely remove GTP from cellular membranes. Alternatively, the GTP hydrolyzed by the G-protein(s) stimulated by mastoparan could reside inside an intracellular structure, such as the insulin secretory granule, which is not permeabilized by digitonin (18) .


Figure 1: Effect of mastoparan and its analogues on insulin secretion from digitonin-permeabilized -TC3 cells and GTPase activity in -TC3 insulin secretory granules. Panel A, dose curve of mastoparan on insulin secretion from digitonin-permeabilized -TC3 cells. -TC3 cells were grown in 6-well plates in RPMI, and the medium was changed the day prior to the experiment. On the day of the experiment, cells were washed 3 times with Krebs-HEPES buffer and permeabilized for 10 min with 20 µg/ml digitonin in the same buffer. Cells were washed twice in 50 mM TES buffer, pH 7.40, supplemented with 100 mM KCl, 2 mM MgCl, 1 mM EGTA, and 0.1% bovine serum albumin. Cells were incubated for 60 min in the same buffer further supplemented with 1 mM ATP and 0-50 µM mastoparan. At the end of the incubation, a sample of the supernatant was removed for insulin assay. Results are shown as the mean ± S.E. of insulin secretion expressed as a percent of control from eight observations. Basal insulin secretion was 60.8 ± 3.7 microunits insulin/well/min. Panel B, effect of mastoparan analogues on insulin secretion from digitonin-permeabilized -TC3 cells. -TC3 cells grown in 6-well plates were permeabilized as in A. Cells were washed twice in 50 mM TES, pH 7.40, supplemented with 100 mM KCl, 2 mM MgCl, 1 mM EGTA, and 0.1% bovine serum albumin. Cells were incubated for 60 min in the same buffer further supplemented with 1 mM ATP and 10 µM mastoparan or mastoparan analogues. At the end of the incubation, a sample of the supernatant was removed for insulin assay. Results are shown as the mean ± S.E. of insulin secretion expressed as a percent of control from 8-20 observations. Panel C, stimulation of GTPase activity in -TC3 cell-enriched insulin secretory granule fraction with mastoparan and mastoparan analogues. Enriched ISG fractions were prepared from -TC3 cells grown in 15-cm dishes. GTPase activity in ISG prepared from -TC3 cells was measured by incubating 5 µg of granule protein with 100 nM [P]GTP in the presence or absence of 50 µM mastoparan or various mastoparan analogues for 5 min as described under ``Experimental Procedures.'' Blanks determined in the presence of 50 µM GTP were routinely subtracted. Basal ISG GTPase activity was 1.66 ± 0.45 pmol/mg of protein/min. Results are shown as the mean ± S.E. of GTPase activity expressed as a percent of control from six observations.



In order to determine if mastoparan stimulated a G-protein present in ISG, highly enriched ISG were prepared by sucrose/nycodenz ultracentrifugation. ISG fractions were significantly enriched 6.0 ± 0.96-fold in insulin content relative to homogenate (p < 0.05, n = 5) and had no significant contamination with mitochondria or endoplasmic reticulum as assessed by enzyme markers. ISG fractions, however, were also enriched with the plasma membrane 5` nucleotidase. Electron microscopy of the prepared ISG fraction (not shown) demonstrated that it consisted mostly of insulin secretory granules with few scattered organelles present. GTPase activity in ISG was measured in the presence or absence of mastoparan and mastoparan analogues. ISG were highly enriched in GTPase activity compared with the homogenate. GTPase activity in the homogenate was 0.12 pmol/mg/min compared with 1.66 pmol/mg/min in the ISG. Mastoparan increased homogenate GTPase activity to 0.49 pmol/mg/min and increased ISG GTPase activity to 10.09 pmol/mg/min (n = 6). Mastoparan increased GTPase activity of ISG in a dose-dependent manner at concentrations of 0-50 µM, with maximal stimulation at 50 µM (n = 6). Similar to their effects on secretion, mastoparan, mastoparan 7, and mastoparan 8 stimulated ISG GTPase activity 497 ± 56, 770 ± 40, and 908 ± 48%, respectively (p < 0.05 versus control). The inactive mastoparan analogue mastoparan 17 had no significant effect on ISG GTPase activity (Fig. 1C). Importantly, in control experiments, mastoparan did not promote lysis of the isolated insulin secretory granules as assessed by measuring insulin release directly from the granules (n = 3) as well as by electron microscopic analysis of the granules (n = 3).

Effect of Pertussis Toxin Pretreatment on Mastoparan-induced Insulin Secretion from Digitonin-permeabilized -TC3 Cells and on Mastoparan-induced Increases in -TC3 Cell Insulin Secretory Granule GTPase Activity

The above data strongly suggested that the effect of mastoparan to increase insulin secretion from permeabilized -cells was specific and not attributable to lytic effects of the peptide that have been reported at higher concentrations on intact cells (52, 53, 54) . Furthermore, pertussis toxin pretreatment decreased mastoparan-induced insulin secretion by permeabilized -TC3 cells from 347 ± 17% of control (p < 0.05 versus control) to 219 ± 9% (p < 0.05 versus control, p < 0.05 versus mastoparan-stimulated insulin secretion) (Fig. 2A). Pretreatment of -TC3 cells with pertussis toxin (24 h) reduced mastoparan-induced ISG GTPase activity from 479 ± 141% of control (p < 0.05 versus control) to 129 ± 33% (Fig. 2A). ISG from untreated -TC3 cells contained a 40-41-kDa substrate that was clearly ADP ribosylated in vitro by pertussis toxin. In contrast, when -TC3 cells had been incubated 24 h with pertussis toxin, ADP-ribosylation of this substrate was markedly diminished (Fig. 2B), verifying that pertussis toxin pretreatment of -TC3 cells resulted in ADP ribosylation of a pertussis toxin-sensitive ISG G-protein.


Figure 2: Effect of pertussis toxin on mastoparan-induced insulin secretion from digitonin-permeabilized -TC3 cells and on mastoparan-induced increases in ISG GTPase activity. Panel A, effect of pertussis toxin pretreatment on mastoparan-induced insulin secretion from digitonin-permeabilized -TC3 cells and on mastoparan-induced insulin secretory granule GTPase activity. -TC3 cells in 6-well plates were incubated 24 h ± 50 ng/ml pertussis toxin. Cells were permeabilized as in Fig. 1 and washed twice in 50 mM TES buffer, pH 7.40, supplemented with 100 mM KCl, 2 mM MgCl, 1 mM EGTA, and 0.1% bovine serum albumin. Cells were incubated for 60 min in the same buffer further supplemented with 1 mM ATP and 10 µM mastoparan. At the end of the incubation, a sample of the supernatant was removed for insulin assay. Results are shown as the mean ± S.E. from 15 observations. For GTPase measurements, enriched ISG fractions were prepared from -TC3 cells in 15-cm dishes incubated 24 h with or without 50 ng/ml pertussis toxin. GTPase activity in ISG prepared from -TC3 cells was measured as in Fig. 1. Data represent the mean ± S.E. from six observations. Panel B, pertussis toxin-catalyzed ADP-ribosylation of a G-protein present in -TC3 cell granule fraction. Pertussis toxin-catalyzed ADP ribosylation of G-proteins was measured in -TC3 cell ISG as described above. The reaction was initiated by the addition of 5 µg of granule protein to 100 µl of 25 mM HEPES, pH 7.40, supplemented with 1 mM EDTA, 1 mM ATP, 0.1 mM GTP, 0.1% Lubrol, 0.02% bovine serum albumin, 10 mM thymidine, 10 µM [P]NAD, and 5 µg/ml activated pertussis toxin. Following a 30-min incubation at 30 °C, membranes were analyzed by SDS-polyacrylamide gel electrophoresis. PTX, pertussis toxin pretreatment.



G-protein Localization in Insulin Secretory Granules of -TC3 Cells

The data shown in Fig. 1and Fig. 2 suggested that mastoparan-induced insulin secretion from permeabilized -TC3 cells was mediated through a pertussis toxin-sensitive heterotrimeric G-protein located in the ISG. In order to determine if the pertussis toxin-sensitive G-proteins G or G were enriched in the ISG, Western blotting was performed on homogenate and ISG with antibodies directed against G and G, as well as G. Western blotting with quantitative PhosphorImager analysis demonstrated highly significant enrichment of G in ISG (6.5 ± 1.6-fold enrichment, p < 0.05 versus homogenate), but not of G (2.7 ± 0.5-fold enrichment, nonsignificant compared with homogenate) and G (1.9 ± 0.3-fold enrichment, nonsignificant compared with homogenate) (Fig. 3).


Figure 3: Western blotting showing the relative enrichment of G in the enriched insulin secretory granule fractions of -TC3 cells. To determine the cellular localization of G, G, and G, subcellular fractions were prepared from -TC3 cells using a sucrose/nycodenz-based sequential centrifugation procedure. Secretory granule fractions were prepared that were routinely enriched 6.0 ± 0.96-fold in insulin content relative to the homogenate. G-proteins were detected by Western blotting using antisera directed against their respective subunits. Equivalent amounts (2.5-25 µg) of granule and homogenate protein were separated, transferred to nitrocellulose, and probed with the respective antibodies. Blots were washed and incubated with I-protein A. Radioactivity bound to the blot was quantitated with PhosphorImager analysis. The enrichment of various G-proteins in secretory granules was calculated relative to the homogenate. A representative analysis from five independent experiments is shown in panelA (G, granule fraction; H, homogenate). PanelB shows the -fold enrichment in insulin and G-proteins in ISG compared with homogenate, expressed as mean ± S.E. from five independent experiments. ISG were consistently significantly enriched in insulin content and G, but not in G and G.



Immunoelectron Microscopy of -TC3 Cells and -Cells from Rat Islets for Selected G-proteins

Although the ISG fractions were consistently highly enriched in insulin, biochemically we could not exclude some degree of contamination with plasma membrane. In order to address this issue, electron microscopic immunocytochemistry of -TC3 cells and of rat endocrine pancreas was performed. Sections of rat pancreas or -TC3 cells grown on filters were fixed, and electron microscopic immunocytochemistry was performed using rabbit polyclonal antisera directed against the subunits of the respective G-proteins. In selected instances, electron microscopic immunocytochemical analysis included identification of -cells and -cells with anti-glucagon or anti-insulin antibodies, respectively, as described under ``Methods.'' In rat pancreatic islets, -cell secretory granules that stained positively for insulin also stained positively for G (antisera 8730) as shown in Fig. 4A. The majority of gold particles complexed to the anti-G Ig appeared to be at the periphery of the ISG. In contrast, neither anti-G or anti-G antisera (9072 and 946 respectively) were detected in the -cell secretory granules (not shown). No G was detected in the granules of cells presumed to be -cells based on the lack of insulin or glucagon immunostaining (Fig. 4B).


Figure 4: Electron microscopic immunocytochemistry of -cells from rat pancreas and -TC3 cells for G and insulin. Minced rat pancreatic tissue was fixed, dehydrated, infiltrated, and embedded. Thick sections were cut and stained with 1% toluidine blue in 1% sodium borate to select blocks with islets. A and B, rat pancreas was co-incubated with anti-G (8730) Ig complexed to 18-nm diameter colloidal gold and anti-insulin Ig complexed to 8-nm colloidal gold as described under ``Methods.'' The majority of the large gold particles (G) colocalized to the ISG, which were also stained with colloidal gold-labeled anti-insulin Ig (small particles). B, in the same section as A, immunostaining of the -cells was insignificant. Sections of -TC3 cells were incubated with anti-G/G (5296) (C), anti-G (9072) (D), or anti G (946) (E) and detected with 15-nm diameter colloidal gold-labeled protein A as described under ``Methods.'' G and G (D and E, respectively) were localized to the plasma membranes and microvilli and dispersed throughout the -TC3 cells. The anti-G/G antibody labeled the ISG of the -TC3 cells. Because anti-G failed to stain the granules, this suggests that the anti-G/G antibody recognized G in the ISG.



In -TC3 cells, immunolabeling with anti-insulin and anti-glucagon antibodies demonstrated only insulin-containing granules in all cells examined (not shown), therefore, dual labeling, as shown in Fig. 4A, was not necessary. Antiserum 5296, which recognizes both G and G equally, localized to the ISG of the -TC3 cells (Fig. 4C). We believe this demonstrates primarily G in the ISG because antibodies to G only(9072) did not associate with the granules as shown in Fig. 4D. Both G and G (Fig. 4, D and E, respectively) were found on the membranes and dispersed throughout the -TC3 cells.


DISCUSSION

In this study, we have presented data that strongly suggest that mastoparan stimulates insulin secretion from permeabilized -TC3 cells by activating the heterotrimeric G-protein G, which is localized to the ISG. Mastoparans constitute a family of peptides whose activities are measured in terms of their ability to accelerate guanine nucleotide exchange; regulatory activity requires that the peptides be both amphiphilic and cationic (55) . Helix-breaking residues or charged residues on the hydrophobic face of the helix severely diminish activity, as is the case for mastoparan 17, which has a lysyl residue on the hydrophobic side of the helix (55) . Mastoparan has previously been shown to cause secretion from a variety of cell types (28, 30-34, 56). The exact mechanism of action of the tetradecapeptide is not elucidated. Mastoparan is believed to exert its effects by acting through G-proteins present in membranes (35, 36) . It is thought that mastoparan intercalates into the membrane and mimics the receptor-G-protein interaction (37, 38) . In this manner, mastoparan binds to the carboxyl terminus of the G-protein and activates it by causing dissociation of the subunits and GDP from the subunit (35, 39, 40) . The subunit assumes the active configuration and interacts with its effector. Supportive data for this mechanism of action stem mainly from experiments demonstrating that mastoparan increases the rate of guanine nucleotide exchange, while not affecting the intrinsic rate of GTP hydrolysis by G-protein subunits (35, 40) .

In addition to its specific effect on G-proteins at relatively low concentrations, mastoparan has also been shown to increase secretion at much higher concentrations due to a toxic effect ascribed to membrane lysis (52, 53) . At very high concentrations, mastoparan has been claimed to be toxic to -cells due to lysis of the plasma membrane (54). In our experiments, several observations demonstrated that mastoparan acts selectively through heterotrimeric G-proteins to stimulate insulin secretion from -TC3 cells rather than merely causing insulin release due to lytic toxicity. First, the effect of mastoparan was evident at relatively low concentrations of the tetradecapeptide. Second, the ability of mastoparan to stimulate insulin secretion from permeabilized -TC3 cells was attenuated by pretreatment of the cells with pertussis toxin. Third, no effect on insulin secretion from permeabilized -TC3 cells was seen with the inactive analogue of mastoparan, mastoparan 17, while active analogues of mastoparan such as mastoparan 7, mastoparan 8, and mastoparan X caused robust insulin secretion. Fourth, only mastoparan analogues that stimulated GTPase activity in the enriched insulin secretory granule fraction were able to stimulate insulin secretion from permeabilized -TC3 cells. Fifth, the heterotrimeric G-protein G, a target of mastoparan, was localized to ISG both by Western blotting and electron microscopic immunocytochemistry. Sixth, mastoparan had no direct lytic effect on ISG as assessed by electron microscopic analysis and insulin release. Taken together, these observations suggest that nonspecific lytic properties of the tetradecapeptide are not responsible for its ability to increase insulin secretion from permeabilized -TC3 cells. Rather, these data suggest that the ability of the tetradecapeptide to increase insulin secretion is due to its stimulation of GTPase activity in ISG. The above data suggest that mastoparan-induced increases in GTPase activity and insulin exocytosis are attributable to G activity. However, other G-proteins associated with ISG, the plasma membrane, or other intracellular loci could contribute to the GTPase activity since pertussis toxin pretreatment diminution of mastoparan-induced insulin secretion was less than its diminution of mastoparan-induced ISG GTPase activity.

Although heterotrimeric G-proteins are believed to have a role in modulating insulin secretion from pancreatic -cells, their exact localization within the -cell and their mechanism of action has remained uncertain (57) . Our observation by immunoelectron microscopy that G is selectively localized to the ISG of -cells represents the first direct detection of G in the insulin secretory granule. G-proteins have multiple roles in insulin secretion. In addition to the classical receptor-coupling roles at the plasma membrane, there is increasing evidence that heterotrimeric G-proteins are also involved in the distal steps of insulin exocytosis, both as inhibitors and activators of insulin secretion. Thus, epinephrine, galanin, and somatostatin inhibit insulin secretion and activate an unidentified pertussis toxin-sensitive G-protein present distally to the generation of intracellular messengers (26, 27, 58, 59) . In contrast, guanine nucleotides have been shown to stimulate insulin secretion from permeabilized -cells independent of calcium (60). Furthermore, it was recently suggested that the putative stimulatory G-protein of exocytosis may be activated by mastoparan (29). Our data, which localize G to the ISG and strongly suggest mastoparan-induced activation of G, support a role for G in insulin exocytosis. These observations are consistent with the known roles of G in secretion and vesicular trafficking (61, 62, 63) .

Recently, Olszewski et al.(22) have shown that the small monomeric GTP-binding protein Rab3A induces insulin exocytosis independent of calcium by interacting with specific cytosolic proteins and that this interaction may be part of a preexocytotic protein complex (22) . Rab3A binds to these inhibitory proteins, called REEP-1 and REEP-2, causing them to dissociate from the complex and allowing exocytosis to proceed (22) . In our study, the effects of mastoparan to stimulate exocytosis are independent of cytosolic proteins, since digitonin-permeabilization of -cells results in the loss of most cytosolic proteins (18) . It is difficult at the present time to identify at which level of the cascade of insulin exocytosis G functions, however, it is reasonable to suggest that it may be involved in a very distal step of exocytosis. Further work will be required to understand what effectors are coupled to G in the ISG. One possible effector could be phospholipase A. It has been shown that vesicle fusion with plasma membrane requires phospholipase A activity and the presence of unsaturated fatty acids such as arachidonic acid (64, 65) . Of interest, arachidonic acid has also been shown to inhibit GTPase activity in ISG (66) . Thus, a possible effector of G could be a phospholipase A isoform localized to the ISG. G-protein activation of this enzyme would produce arachidonic acid, which could then, by feedback, inhibit granule GTPase activity.

The true effectors coupled to G in ISG remain to be identified. Whatever they may be, our data suggest that activation of the heterotrimeric G-protein G in the ISG is a crucial and distal event of insulin exocytosis that is important in the fusion of the insulin secretory granule with the -cell plasma membrane.


FOOTNOTES

*
This work was supported by a Juvenile Diabetes Foundation Fellowship (to R. J. K.), National Institutes of Health Research Grants RDK43354 (to B. A. W.), R01 GM34781 (to D. M.), R01 DK28143 (to L. J.), a National Institutes of Health Research Career Development Award K04 DK02217 (to B. A. W.), the William Pepper Fund of the University of Pennsylvania (to B. A. W.), and the Diabetes Endocrine Research Center (DK19525). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: University of Pennsylvania School of Medicine, Dept. of Pathology and Laboratory Medicine, 217 John Morgan, Philadelphia, PA 19104-6082. Tel.: 215-898-0025; Fax: 215-573-2266.

The abbreviations used are: ISG, insulin secretory granule; TES, 2-([2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino)ethanesulfonic acid; PBS, phosphate-buffered saline; MES, 2-(N-morpholino)ethanesulfonic acid; GTPS, guanosine 5`-O-(thiotriphosphate).


ACKNOWLEDGEMENTS

We thank Donna Berner and Frances Chilsholm for help in performing the insulin RIA (Diabetes Endocrine Research Center). We also thank Chris Major and Misha Amagasu for excellent technical assistance.


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