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Galparan: A Powerful Insulin-Releasing Chimeric Peptide Acting at a Novel Site¹

The Rolf Center for Diabetes Research, Department of Molecular Medicine, Endocrine and Diabetes Unit, Karolinska Hospital and Institute (C.-G.O., S.Z., P.-O.B., S.E.), S-171 76 Stockholm; and the Department of Neurochemistry and Neurotoxicology, Stockholm University (U.L., T.B.), S-106 91 Stockholm, Sweden

Address all correspondence and requests for reprints to: Dr. Claes-Göran Östenson, The Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, The Endocrine and Diabetes Unit, Karolinska Hospital, S-171 76 Stockholm, Sweden. E-mail: claesg{at}enk.ks.se

	Abstract

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Galparan is a 27-amino acid long chimeric peptide, GWTLNSAGYLLGP-INLKALAALAKKIL amide, consisting of galanin-(1–13) linked to mastoparan amide via a peptide bond to provide the mastoparan and galanin effector parts of the molecules. Galparan (10 µM) powerfully stimulates insulin secretion from isolated rat pancreatic islets in a reversible and dose-dependent manner; the stimulation is 26-fold at 3.3 mM glucose and 6-fold at 16.7 mM glucose. Galparan also enhances insulin secretion to a similar extent from islets of diabetic GK rats. The stimulatory effect of galparan on insulin release is not directly dependent on extracellular Ca²⁺, nor can it be explained only by changes in free cytosolic Ca²⁺ concentrations. Furthermore, galparan is effective in evoking insulin release in B cells depolarized by 25 mM KCl when ATP-sensitive K⁺ channels are kept open by diazoxide. Thus, galparan, like mastoparan, stimulates exocytosis of insulin at a distal site in the stimulus-secretion coupling of the B cell. This distal site is not identical to that used by mastoparan, as pertussis toxin pretreatment does not influence the insulinogenic effect of galparan. In conclusion, galparan evokes a large and reversible insulin secretion, acting at a yet unknown distal site and also promoting exocytosis in depolarized B cells from normal rats as well as diabetic GK rats.

	Introduction

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STIMULATION of insulin secretion by glucose involves metabolism of the hexose, leading to an increased ATP/ADP ratio, causing closure of ATP-regulated K⁺ channels, membrane depolarization, opening of L-type Ca²⁺ channels, an increase in the cytosolic free calcium concentration ([Ca²⁺]_i), and, eventually, exocytosis of insulin (1, 2). In addition, glucose has been shown to stimulate exocytosis directly by activating steps of the secretory machinery distal to the ion channels (3, 4, 5). Not only glucose, but also insulin-releasing drugs, such as glibenclamide (6) and imidazolines (7), and insulin-modulating peptides, such as galanin (8) and mastoparan (9, 10), may exert direct effects on the exocytotic machinery. In this context it is of interest that in the B cells of GK rats, a model of hereditary noninsulin-dependent diabetes mellitus (11, 12), not only the former but also the latter pathway of glucose signal transduction is severely impaired (5).

We have investigated the effects of galanin, mastoparan, as well as a chimeric galanin-mastoparan peptide, galparan, and its analogs on basal and glucose-stimulated insulin release from isolated pancreatic islets of normal rats and GK rats. Galanin is a 29/30-amino acid long peptide that inhibits glucose-induced insulin release by acting at a pertussis toxin (PTX)-sensitive, G protein-coupled seven-transmembrane domain receptor on the B cells (13, 14, 15, 16, 17). Several chimeric galanin receptor ligands have been described (18), among them galanin-mastoparan hybrids (19). The idea behind application of mastoparan in these peptide constructs originates from the special properties of mastoparan. The latter is a 14-amino acid peptide, which has been isolated from wasp venom and shown to stimulate insulin secretion (9, 10, 20, 21). Recent studies have suggested that mastoparan-induced insulin secretion is independent of changes in [Ca²⁺]_i and of protein kinase C activation, but dependent at least partly on activation of a PTX-sensitive G protein at a late stage in the secretory process, e.g. exocytosis (9, 10). The chimeric peptide galparan, or galanin-(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)-mastoparan, was demonstrated to activate Na⁺,K⁺-adenosine triphosphatase, an effect the opposite of inhibition of the enzyme by mastoparan (19). Such a behavior of galparan, being partly different from the effect initiated by its peptide components, fuels interest in the characterization of its effect on insulin secretion. Furthermore, in an attempt to elucidate the peptide’s mechanism of action, we studied the dependency of galparan’s effects on galanin receptor binding, [Ca²⁺]_i, and PTX-sensitive G proteins.

	Materials and Methods

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Peptide synthesis
The peptides were assembled in a stepwise manner on a solid support using an Applied Biosystems model 431 A Peptide Synthesizer (Foster City, CA) with the standard dicyclohexylcarbodiimide/hydroxybenzotriazole solvent-activation strategy on a 0.1-mmol scale (small scale). The tert-Boc-amino acids were coupled to MBHA (Bachem Feinchemikalien, Bubendorf, Switzerland) resin as hydroxybenzotriazole esters. Synthesis, deprotection, cleavage, and purification of the peptides have been described previously (22). The purity of the individual peptides was checked by analytical Nucleosil 120–3 C₁₈ reverse phase HPLC column (0.4 x 10.0 cm) and determined to be more than 99%. Molecular weights of the peptides were determined using Plasma Desorption Mass Spectrometer (PDMS) Bioion 20 (Applied Biosystems).

Galanin receptor binding assay
Displacement of monoiodo-[¹²⁵I]Tyr²⁶-porcine galanin (0.2 nM) from galanin receptors by endogenous peptides and chimeric ligands was tested as previously described in membranes of the rat insulinoma cell line RINm5F (23).

Adenylate cyclase assay
The activity of adenylate cyclase was assayed in pancreatic RINm5F cell membranes in a buffer solution containing 0.1 mg/ml bacitracin, 0.5 mg/ml BSA, 1.0 mM ATP, 0.01 mM GTP, an ATP-regenerating system (10 mM phosphoenolpyruvate and 30 mg/ml pyruvate kinase), and the membrane fragments, with a final protein concentration of 0.03–0.05 mg/ml (24).

Incubations of pancreatic islets
Pancreatic islets were isolated aseptically from male Wistar and GK rats, weighing 200–250 g, using digestion of the pancreata with collagenase (Boehringer Mannheim, Mannheim, Germany). The isolated islets were then maintained for 24 h in tissue culture medium RPMI 1640 (SVA, Uppsala, Sweden), supplemented with 11.1 mM glucose and antibiotics. After culture, islets were preincubated for 30 min in Krebs-Ringer bicarbonate (KRb) buffer solution with 2 mg/ml BSA, 10 mM HEPES (Sigma Chemical Co., St. Louis, MO), and 3.3 mM glucose, pH 7.4, at 37 C with a gas phase of 5% CO₂-95% O₂. Batches of three islets were then incubated for 60 min in 300 µl KRb with the additions described above, except for a glucose concentration of either 3.3 or 16.7 mM and addition of peptides or the sulfonylurea glibenclamide, as given in the table. After incubations, aliquots of the medium were taken for RIA of insulin. Insulin secretion is expressed as microunits of hormone per islet per h.

In perifusion experiments, isolated rat islets were perifused at a flow rate of 0.2 mL/min in KRb buffer solution with 2 mg/ml BSA and 10 mM HEPES. In each experiment, 50 cultured islets were perifused and mixed with Bio-Gel P-4 polyacrylamide beads (200–400 mesh; Bio-Rad Laboratories, Richmond, CA) in a 0.5-mL column at 37 C. After an initial perifusion period with 3.3 mM glucose, the glucose concentration was raised to 16.7 mM as indicated, followed by a switch back to 3.3 mM glucose. Galparan (10 µM) was added together with the high glucose concentration. Fractions of the perifusate were collected every second minute and analyzed for insulin content.

[Ca²⁺]_imeasurements
[Ca²⁺]_i was measured in medium consisting of mM: glucose, 3.3 mM; NaCl, 125 mM; KCl, 5.9 mM; CaCl₂, 1.3 mM; MgCl₂, 1.2 mM; and HEPES, 25 mM, pH 7.4, supplemented with 2 mg/ml BSA and other additions of galparan and glucose, as shown in the figure. Single islets were loaded with 2 µM fura-2/AM for 1 h in medium containing 3.3 mM glucose. After loading, the single islet was placed under a microscopic grid in a custom-built open perifusion chamber for microscopic work and maintained at 37 C. Single islets were perifused at a flow rate of 0.15 mL/min, and measurements of the 340/380 nm fluorescence ratio, reflecting [Ca²⁺]_i, were made as previously described (25).

Dependency on PTX-sensitive G proteins
To elucidate the role of PTX-sensitive G proteins in insulin responses to galparan and mastoparan in rat pancreatic B cells, islets were cultured overnight in RPMI 1640 medium with 11 mM glucose and 10% (vol/vol) heat-inactivated FCS and 100 ng/ml PTX (Sigma) before experiments.

	Results

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The sequences of the chimeric peptide galparan [galanin-(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)-mastoparan amide] and its analogs are presented in Table 1 together with their binding affinities to rat galanin receptors on a pancreatic insulinoma cell line, RINm5F. These peptides include galparan, in which the active portion of galanin, galanin-(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13), is linked to the fully active sequence of mastoparan; Lys¹⁹,Leu²⁶-galparan, in which galanin-(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) is linked to Mas-17, which is an inactive analog of mastoparan; and Ala²-galparan, in which mastoparan is linked to an inactive analog of galanin-(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13), respectively. Galparan (K_d, 7 nM) had 18-fold lower affinity than galanin (K_d, 0.4 nM), yet it is still regarded as a high affinity galanin receptor ligand. Mastoparan, Mas-17, and Ala²-galparan all had negligible affinity for galanin receptors on RINm5F cells. The highest affinity (K_d, 0.2 nM) to galanin receptors in this series of chimeric peptides was shown by the Lys¹⁹,Leu²⁶-galparan.

View larger version (18K): [in this window] [in a new window]   Figure 1. Effect of galparan on insulin release from perifused rat islets. Batches of 50 rat islets were perifused at a flow rate of 0.2 ml/min. After an initial perifusion with 3.3 mM glucose, the glucose concentration was raised to 16.7 mM as indicated, followed by a switch back to 3.3 mM glucose. Galparan (10 µM) was added together with the high glucose concentration (•; n = 3). Control perifusions were performed in the absence of galparan (; n = 4). Fractions of the perifusate were collected every second minute and analyzed for insulin content. Shown are the mean ± SEM.

View larger version (15K): [in this window] [in a new window]   Figure 2. Influence of galanin (1 µM; Gal), mastoparan (100 µM; MP), and galparan (100 µM; Galp) on forskolin (1 µM; F)-stimulated activity of adenylate cyclase in the membranes from pancreatic RINm5F cells. Shown are the mean ± SEM of five experiments.

View larger version (10K): [in this window] [in a new window]   Figure 3. Effects of 10 µM galparan (a and c) or 16.7 mM glucose (G; b) on [Ca2+]i in single rat pancreatic islets. In c, CaCl2 was omitted, and 3 mM EGTA was added to the medium as indicated. a, A representative trace of 8 experiments; b, a representative trace of 24 experiments; c, a representative trace of 5 experiments.

	Discussion

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We have demonstrated that the chimeric peptide, galparan, exerts a powerful insulinogenic effect in pancreatic rat islets and that this effect is considerably stronger than that exerted by an equimolar concentration of one of its parent peptides, mastoparan, or by a high level of the sulfonylurea compound, glibenclamide. The insulin-stimulating effect by galparan is not likely to be due to B cell membrane perturbation and leakage of hormone, as the effect was reversible in perifused islets and did not affect subsequent stimulation of insulin release by KCl.

The affinity of galparan to galanin receptors can be ascribed to the galanin-(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) portion of galparan. This is indicated by the loss of affinity in the galparan analog, Ala²-galparan, in which the major pharmacophore of the galanin portion of galparan Trp² is substituted with Ala² (28). The high affinity found for galparan suggests that galanin receptors were always fully occupied by galparan in the present insulin release experiments in isolated islets, as concentrations of 1 and 10 µM were used. Galanin is known to inhibit adenylate cyclase via a PTX-sensitive GTP-binding (G_i) protein (29). For agonist action at the pancreatic galanin receptor, only the N-terminal portion-(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) of galanin, which is present in galparan, is required (28). We now show that supramaximal concentrations of galanin (1 µM) and galparan (100 µM) produced inhibitory effects on adenylate cyclase activity, suggesting that although galparan, like galanin, inhibits adenylate cyclase activity, its stimulatory effect on insulin secretion cannot be exerted through an increased B cell level of cAMP.

It is well known that [Ca²⁺]_i has an important function in insulin secretion, but also that [Ca²⁺]_i plays a permissive, rather than a direct regulatory, role in this context (1). Galanin inhibits insulin secretion both by activation of low conductance sulfonylurea-insensitive K⁺-channels, resulting in repolarization of membrane potential, closure of voltage-gated L-type Ca²⁺ channels, and a lowering of [Ca²⁺]_i, and by direct interference with the exocytotic machinery; both effects are mediated by a PTX-sensitive G protein (8, 30). The reversible effect on the [Ca²⁺]_i signal in the presence of PTX cannot be explained by raised levels of cAMP, as galanin still lowers [Ca²⁺]_i in the presence of activators of adenylate cyclase (30). It should be noted that galanin also inhibits insulin release under conditions where [Ca²⁺]_i is increased subsequent to direct activation of the voltage-gated L-type Ca²⁺ channel by K⁺-induced depolarization of the B cell membrane potential. This indicates that the direct inhibitory effect of the peptide on exocytosis is distal to the site activated by an increase in [Ca²⁺]_i (31). It is also an example of a dissociation between an increase in [Ca²⁺]_i and activation of insulin exocytosis. In this context, it is of interest to note that the marked stimulatory effect of galparan on insulin secretion cannot be explained by changes in [Ca²⁺]_i. Although galparan per se increased [Ca²⁺]_i, this increase was less than that induced by 16.7 mM glucose. Hence, galparan shows a dual stimulatory effect on insulin release, a property described previously for some imidazoline and sulfonylurea compounds (6, 7). In addition to its action on [Ca²⁺]_i, galparan stimulated insulin secretion by effects at a late stage in the secretory pathway, i.e. exocytosis. The latter mechanism was clearly demonstrated in pancreatic islets, which were depolarized by the addition of 25 mM KCl, and their ATP-regulated K⁺ channels were kept open by diazoxide. It is also of interest to note that the stimulatory effects of galparan (10 µM) on [Ca²⁺]_i and insulin release from pancreatic islets incubated in Ca²⁺-free medium supplemented with 3 mM EGTA and 3.3 mM glucose were less pronounced, although the fold increase in insulin secretion was similar to the increase in the presence of extracellular Ca²⁺. Thus, the amount of secreted insulin seems to be determined by the effect of galparan on both [Ca²⁺]_i and the exocytotic machinery.

Depolarization by the addition of 25 mM KCl at 3.3 mM glucose elicited similar insulin responses in islets from healthy control rats and GK rats, a spontaneous model of noninsulin-dependent diabetes mellitus that is characterized by a markedly impaired insulin response to glucose (12). Such an effect by KCl in GK rat islets is in agreement with previous observations and supports a defect in the stimulus-secretion coupling for glucose in GK rat B cells (5, 32). Interestingly, also galparan induced similar insulin responses in control and GK rat islets, suggesting intact exocytotic mechanisms related to the galparan effect in B cells of the diabetic rat.

Recent studies in the insulin-secreting cell lines RINm5F and HIT have demonstrated that mastoparan increases [Ca²⁺]_i by closure of ATP-regulated K⁺ channels, leading to B cell depolarization and activation of voltage-gated Ca²⁺ channels (33). However, mastoparan has also been shown to stimulate insulin secretion in pancreatic islets and RINm5F cells in the absence of extracellular Ca²⁺ (9, 10) as well as in permeabilized islets when the ambient free Ca²⁺ concentration was clamped at a substimulatory concentration (10). These data support the idea that also mastoparan-induced insulin release can be dissociated from changes in [Ca²⁺]_i. The failure of galanin to suppress mastoparan-induced insulin secretion may be accounted for by an effect of mastoparan at a step distal to the site of action of galanin in the B cell stimulus-secretion coupling (34). The extent to which this site is responsible for the pronounced stimulatory effect of galparan on insulin exocytosis remains unclear.

G Proteins are thought to be involved in the process of exocytosis, and the actions of certain G proteins are inhibited by PTX-catalyzed ADP ribosylation. In previous studies in RINm5F cells, PTX pretreatment led to enhanced insulin response to mastoparan, suggesting the involvement of PTX-sensitive G protein in the mechanism of peptide action (9). In contrast to this finding, mastoparan-induced insulin secretion in another insulin-secreting cell line, ß-TC3 cells, was inhibited by PTX pretreatment (35). In view of the demonstration that the heterotrimeric G protein G_i was abundantly located to the insulin secretory granules, it was proposed that mastoparan acts through G_i to stimulate insulin secretion. Our findings in PTX-pretreated islets are in concert with the above-discussed study in ß-TC3 cells regarding a possible effect of mastoparan on a heterotrimeric G_i protein (35). However, the strong insulinogenic effects of galparan and Ala²-galparan are obviously not dependent on PTX-sensitive pathways.

It is clear that at the concentrations employed, galparan saturates all galanin receptors. These are not the only sites to which galparan binds, however, as galparan could produce an effect in addition to that of a supramaximal concentration of galanin in the inhibition of adenylate cyclase. These alternative pathways are probably also crucial for the insulinogenic effect of galparan, as galanin per se did not interfere with the insulin-releasing effect of galparan. In addition, Ala²-galparan, which is not recognized by galanin receptors, is also a 3- to 5-fold more potent insulin-secreting agent than mastoparan, showing that N-terminal elongation of mastoparan, without recognition of galanin receptors, is already sufficient for increasing the insulinogenic efficacy of mastoparan analogs. However, it would be wrong to regard galparan as just one of the more efficient, N-terminally elongated mastoparan analogs. As its insulinogenic action, unlike that of mastoparan, was not PTX sensitive in B cells, galparan must use also other sites in the excytotic mechanism than mastoparan.

Chimeric agents that, like galparan, embody an "address" to B cells, such as the galanin receptor recognizing the N-terminal fragment, galanin-(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13), and a "message," such as the mastoparan portion of galparan, may be useful in exploring the molecular mechanisms of insulin exocytosis in both normal and diabetic states.

	Acknowledgments

 
The skillful technical assistance of Marianne Sundén, Anita Nylén, and Homa Hasavan is gratefully acknowledged.

	Footnotes

 
¹ This work was supported by grants from the Swedish Board of Technical Development, the Swedish Medical Research Council (00034 and 09890), the Swedish Research Council for Engineering Sciences, the Ivar Bendixsons Foundation, the Pharmacia Research Foundation, the Novo Nordisk Foundation, the Swedish Diabetes Association, and Stockholm University (bilateral collaboration).

² Permanent address: Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119899, Russia.

Received November 15, 1996.

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