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Stimulus-specific inhibition of insulin release from rat pancreas by both rat and porcine galanin
Life Sciences, Vol. Printed in the USA
51, pp.
1945-1951
Pergamon
Press
STIMULUS-SPECIFIC INHIBITION OF INSULIN RELEASE FROM RAT PANCREAS BY BOTH RAT AND PORCINE GALANIN C. Bruce Verchere, Yin Nam Kwok and John C. Brown Medical Research Council of Canada Regulatory Peptide Group Department of Physiology, University of British Columbia Vancouver, B.C., Canada V6T 1Z3 (Received
in final
form October
8, 1992)
Summary_ The effect of the neuropeptide galanin on insulin and somatostatin secretion in the rat was studied under various conditions. In the perfused rat pancreas, insulin secretion stimulated by arginine, but not cholecystokinin-8 (CCK-8) or acetylcholine (ACh) was inhibited by both rat and porcine galanin, whereas ACh-stimulated somatostatin release was inhibited by rat but not porcine galanin. Neither arginine nor CCK-8 significantly "altered somatostatin secretion and galanin was without effect under those conditions. Gastric inhibitory polypeptide-stimulated insulin release from cultured mixtures of purified rat g- and non-B-cells was inhibited by rat and porcine galanin in a concentration-dependent and equipotent manner. The results suggest that the inhibitory effect of galanin on insulin and somatostatin secretion may be stimulusspecific and species-specific. Galanin is a 29 amino acid neuropeptide, isolated by Tatemoto et al (1), that is widely distributed in the central and peripheral nervous systems (2). The peptide may be an important neural regulator of islet hormone secretion (3). The presence of galanin in nerves in association with pancreatic islets has been demonstrated immunocytochemically in the dog (4) and rat (5) pancreas and the presence of galanin receptors has been demonstrated in a hamster pancreatic/3-cell line (6). Numerous studies have shown that galanin possesses a potent inhibitory effect on insulin secretion (3,7). Although its effects on glucagon and somatostatin secretion are less clear, the peptide has been shown to inhibit somatostatin and stimulate glucagon release from the dog (4). Studies in the rat (8-10) and mouse (11,12) have shown galanin to be a potent inhibitor of insulin secretion under a variety of conditions, suggesting that galanin interferes with an essential, common step in the insulin secretory mechanism (3,9). In contrast, other studies using B-cell lines (13) or perfused rat pancreas (14) have found that the inhibitory action of galanin was only observed in the presence of specific stimuli. Thus, galanin suppressed gastric inhibitory polypeptide (GIP)- but not carbamoylcholine-stimulated insulin secretion from Rinm5f g-cells (13), and the neuropeptide inhibited glucose- but not arginine- or potassium-stimulated insulin secretion from the perfused rat pancreas (14). The present experiments were undertaken to investigate these discrepancies more closely by examining the effect of galanin on insulin and somatostatin secretion from the perfused rat pancreas in the presence of different stimuli. Since previous studies in the rat have used porcine galanin, which might exhibit diminished biologic activity in rodent models, this study examined the inhibitory effects of both porcine and rat galanin. In addition, the potency of these two peptides in inhibiting GIP-stimulated B-cell secretion in vitro was compared using cultured B-cells obtained by fluorescence-activated cell-sorting.
Author for correspondence: Yin Nam Kwok, Ph.D., MRC Regulatory Peptide Group, University of British Columbia, 2146 Health Sciences Mall, Vancouver, B.C., Canada, V6T 1Z3
Copyright
0024-3205/92 $5.00 + .00 © 1992 Pergamon Press Ltd All rights
reserved.
1946
Galanin
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Methods Perfused pancreas. The procedure for surgical isolation and in situ vascular perfusion of the rat pancreas, modified from the technique of Penbos et al (15), has been previously described (16). Male Wistar rats weighing 250-350 g were used. The perfusate was a Krebs solution containing 0.2% bovine serum albumin (BSA; Sigma, RIA grade), 3% dextran (Sigma, clinical grade) and 4.4 mM or 8.9 mM glucose. It was heated to 37 °C and pumped through the pancreatic vasculature at a flow rate of 3 ml/min. Test substances were infused into the perfusate by a side-arm pump (0.1 ml/min). Venous effluent was collected (5 min/sample) via a portal vein cannula into tubes containing aprotinin (5000 K.I.U.). Aliquots (0.5 ml) were stored at -20 °C for radioimmunoassay (RIA); an additional 500 K.I.U. of aprotinin was added to samples for assay of somatostatin-like immunoreactivity (SLI) before freezing. Purified islet cells. Islets were isolated from male Wistar rats (250-350 g) by collagenase digestion and purification on a discontinuous dextran gradient (17). The islets from 4-8 rats (1000-5000 islets) were pooled and cultured overnight (37 °C, 5% CO2) in CMRL-1066 medium plus 10% Calf Supreme serum (Gibco), 2 mM L-glutamine, and an antibiotic-antimycotic mixture. Fractions of Bcells and non-B-cells were obtained from islets via the technique of Pipeleers et al (18). The islets were first dispersed in Ca++-free Earle's-HEPES (EH) medium containing 0.1% BSA, 2.75 mM glucose, 1 mM EGTA, 200 btg/ml trypsin and 2 p.g/ml DNase at 31 °C using a siliconized Pasteur pipet. When at least 50% of the cells were dissociated as single cells, the digestion was stopped by filtering the suspension through a 100 btm nylon screen into a 50 ml tube containing ice-cold EH plus Ca++. After centrifugation (5 rain at 500 X g) the cells were resuspended in 1 ml Ca++-free EH, and maintained at 17 °C for 15 rain prior to sorting on a FACS-IV (Becton-Dickinson, Sunny Vale, CA). Two cell fractions were obtained from the FACS and examined by immunocytochemical staining. The B-cell fraction of higher light scatter and autofluorescence consisted of more than 98% insulincontaining cells, while the non-B-cell fraction consisted of 40-60% a-cells, with a smaller proportion of g-, 8-, PP-, and unidentified cells. Since FACS-purified B-cells have been shown to be more responsive when reaggregated with a-cells (19), the B-cell and non-B-cell fractions were remixed (1:1) before culture. The ceils were seeded (6000 cells/well) in 200 gl culture medium (as for islets) in 96-well plates and cultured for 3 days prior to insulin release experiments. Incubations were performed for 1 h at 37 °C in 250 btl Dulbecco's Modified Eagle's Medium plus 0.1% BSA, 17.8 mM glucose and the desired concentration of GIP and rat or porcine galanin. After collecting the incubation medium, the cells in each well were extracted in 200 gl acetic acid (2 M) and boiled for 10 min for the measurement of total insulin content. Incubation medium and cell extracts were stored at -20 °C until assay. Radioimmunoassays. Immunoreactive insulin (IRI) and somatostatin-like immunoreactivity (SLI) were measured using specific RIA's that have been previously described (16). Galanin has been shown not to interfere with the binding of insulin or somatostatin in these assays (10). In perfused pancreas experiments, IRI or SLI secretion was expressed as the percent change (mean +_S.E.M.) from the basal secretion rate (mean of 2 basal collection periods of 5 min each) in the presence of either 4.4 mM glucose (ACh and arginine experiments) or 8.9 mM glucose (CCK-8 experiments). For statistical comparison, the percent of basal release during a 10 rain infusion of rat or porcine galanin was compared to the percent of basal release during a 10 min infusion of a control vehicle using the Mann-Whitney U test. In B-cell culture experiments, IRI release was expressed as a percent of total cell IRI content for each well (mean +_S,E.M, of four triplicate determinations) and compared using one way analysis of variance with Dunnett's test. p<0.05 was considered significant. Results Perfused pancreas Effect of galanin on IRI release. In the presence of 4.4 mM glucose, arginine (20 mM) produced a marked stimulation of IRI secretion which was sustained for the duration of the arginine administration. When porcine galanin was introduced at a concentration of 10 nM, previously
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shown to potently inhibit GIP-stimulated IRI release in this preparation (10), a modest suppression of arginine-stimulated IRI secretion was observed (Fig. 1). The percent increase over basal (4.4 mM glucose alone) IRI secretion induced by arginine stimulation was significantly lower during the 10 min infusion of porcine galanin infusion when compared to a control vehicle infusion (2738+447 vs 4436+975%; p<0.05). Rat galanin (10 nM) inhibited arginine-stimulated IRI secretion to a similar extent (2419+718 vs 4436_+975%; p<0.05; Fig. 1).
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FIG. 1 Effect of porcine and rat galanin on arginine-stimulated IRI secretion from the perfused rat pancreas. Arginine (20 mM) was added to the perfusate during periods 3-9 and either 10 nM porcine galanin (-o-; n=6), 10 nM rat galanin (-o-; n=7) or a control vehicle (-13-; n=6) was infused during periods 6-7. Perfusate glucose concentration was 4.4 mM throughout.
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FIG. 2 Effect of porcine and rat galanin on ACh- and CCK-8-stimulated IRI secretion from the perfused rat pancreas. (A) ACh (5 I.tM) was added to the perfusate during periods 3-9. Perfusate glucose concentration was 4.4 mM throughout. (B) Perfusate glucose concentration was increased from 4.4 to 8.9 mM during periods 3-10 and CCK-8 (0.9 nM) was infused during periods 5-10. Either 10 nM porcine galanin (-o-), 10 nM rat galanin (-o-) or a control vehicle (-121-)was infused (n=5-7) during (A) periods 6-7 or (B) periods 7-8.
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In contrast, when IRI secretion was stimulated by 5 gM ACh in the presence of 4.4 mM glucose, neither rat nor porcine galanin (10 nM) significantly altered IRI secretion (Fig. 2A). The percent increase over basal IRI release during ACh stimulation in control experiments (12104_-297%) was not significantly different from that observed during porcine (1094+205%) or rat (1009+304%) galanin infusion. ACh-stimulated IRI release was also unaffected when rat galanin was administered at a concentration of 50 nM (data not shown). Similarly, when IRI secretion was stimulated by CCK-8 (0.9 nM) in the presence of 8.9 mM glucose, infusion of either porcine or rat galanin (10 nM) again failed to elicit a significant change in IRI release when compared to controls (Fig. 2B). Effect of galanin on SLI release. Of the insulin secretagogues tested, only ACh (5 gM) produced a significant increase in SLI release. SLI secretion increased during ACh infusion from basal levels of 149+29 pg/min to peak concentrations of 304+47 pg/min. Infusion of rat galanin (10 nM) inhibited the ACh-stimulated SLI secretion to approximately 40% of peak values (Fig. 3A); SLI release (percent of basal) was significantly lower in the presence of rat galanin when compared to controls (140-+24 vs 184-+11%; p<0.05). However, porcine galanin (10 nM) had no effect on AChstimulated SLI release (Fig. 3B; 194+23 vs 184+11%; p=NS). This concentration of porcine galanin was previously shown to inhibit gastric SLI release from the perfused rat stomach (10). Neither arginine (20 mM) nor CCK-8 (0.9 nM) plus glucose (8.9 mM) produced significant changes in SLI secretion, and addition of rat or porcine galanin was without effect under those conditions (data not shown).
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FIG. 3 Effect of rat and porcine galanin on ACh-stimulated SLI secretion from the perfused pancreas. ACh (5 #M) was infused during periods 3-9 and either (A) 10 nM rat galanin (rGal; n=7) or (B) 10 nM porcine galanin (pGal; n=6) was infused during periods 6-7. Perfusate glucose concentration was 4.4 mM throughout. Co-cultured 6- and non-6-cells A previous study showed that mixed cultures of FACS-purified 6- and non-13-cells were more responsive to glucose than g-cells alone (19). In the present study, 10 nM GIP (in the presence of 17.8 mM glucose) significantly (p<0.05) increased IRI secretion (4.1+0.4%) from this preparation compared to 17.8 mM glucose alone (2.4_+0.3%). Since porcine galanin was previously shown to potently inhibit GIP-stimulated insulin release (10), the effect of a range of concentrations (0.1 nM to 100 nM) of porcine and rat galanin on the IRI response to GIP (10 nM) plus glucose (17.8 raM) was examined. The two peptides inhibited GIP-stimulated IRI release in an equipotent and concentration-dependent manner (Fig. 4). The lowest concentration of either peptide to significantly (p<0.05) inhibit the IRI response to G1P was 1 nM.
VOI.
51, No.
25,
1992
Galanin
Inhibition
of I n s u l i n
1949
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[Galanin] (nM) FIG. 4 Comparison of the effect of rat and porcine galanin on GIP-stimulated IRI secretion from cocultured 13- and non-B-cells. IRI secretion is expressed as a percent of total cell IRI content, after 1 h incubation in the presence of 17.8 mM glucose, 10 nM GIP, and indicated concentration of rat (-o-) or porcine (-e-) galanin (n=4 of triplicate determinations). Discussion The conditions under which galanin influences insulin and somatostatin secretion in the rat were evaluated. In this and a previous study (10) using the in situ perfused rat pancreas, only GIP- and arginine-stimulated insulin release were inhibited by galanin, while the insulin responses to CCK-8 and ACh were unaffected by this neuropeptide. The results suggest that the inhibitory action of galanin on the B-cell is stimulus-specific. The lack of effect of galanin on ACh- and CCK-8-stimulated insulin release may provide insight into the mechanism of action of galanin on the B-cell. Both CCK-8 and cholinergic agonists stimulate insulin secretion by increasing 13-cell phosphoinositol metabolism and intracellular calcium levels (20). These data suggest that galanin does not influence those intracellular pathways in the rat B-cell. This idea was supported by the recent observation that porcine galanin potently inhibited forskolinand GIP-stimulated insulin secretion and cAMP production in the Rinm5f B-cell line, but had no effect on carbamoylcholine-stimulated insulin secretion (13). It was suggested that galanin inhibited B-cell secretion via inhibition of cAMP production. In this study, the inhibitory effect of galanin on GIP- and arginine-stimulated insulin secretion may have occurred via an inhibition of B-cell cAMP production, since both of these B-cell secretagogues have been shown to increase cAMP levels in isolated rat islets (21,22). In apparent conflict with these results, Miralles et al found that CCK-8-stimulated insulin secretion was suppressed by porcine galanin in the perfused rat pancreas (8). However, in those experiments a priming dose of galanin was given prior to the introduction of CCK-8 followed by infusion of galanin throughout the stimulation, whereas in this study galanin was infused for only a 10 min period during the second phase of CCK-8-stimulated insulin secretion. It is possible that the
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experimental protocol of this study masked an inhibitory effect of galanin on CCK-8- (or ACh)stimulated secretion, that would only be evident when the presence of galanin precedes the stimulation. Indeed, it has been shown that galanin infusion inhibited the first phase of glucosestimulated insulin release only when commenced 4 rain prior to the glucose stimulation, suggesting that galanin acts on early events of the stimulus-secretion coupling in the B-cell (14). It is also possible that an inhibitory effect of galanin on ACh- or CCK-8-stimulated insulin release may have been observed if higher concentrations of the neuropeptide were used. However, 50 nM rat galanin failed to inhibit ACh-stimulated insulin release in this study, and this concentration was 5 fold greater than that required to suppress GIP- (10) or arginine-stimulated insulin secretion in the perfused rat pancreas. This observation strongly supports the idea that the inhibitory action of galanin is more selective for certain B-cell secretagogues in the rat. A stimulus-specific inhibitory effect of galanin was also observed by Yoshimura et al (14), who found that porcine galanin inhibited glucose-, but not arginine- or potassium-stimulated insulin release in the perfused rat pancreas. Although the present study found galanin to inhibit arginine-stimulated insulin secretion, the concentration of galanin used in this study (10 riM) was 10 fold the concentration employed by Yoshimura et al (14). The results of this study also conflict with previous studies in the dog (23) and mouse (11,12), in which galanin was found to inhibit the insulin response to a variety of secretagogues, including CCK-8 (12,23) and carbachol (11). This discrepancy may be due to the existence of species differences in the sensitivity of the B-cell to the action of galanin. In humans, infusions of high doses of porcine galanin failed to alter serum insulin levels (24), suggesting a lack of g-cell sensitivity to galanin in humans, while in the perfused pig pancreas, porcine galanin stimulated insulin release (25). Together, these studies suggest that the contribution of galanin to the normal regulation of B-cell secretion may vary widely between species. In these experiments, porcine and rat galanin had identical effects on insulin secretion from rat Bcells under every condition tested and the two peptides were equipotent inhibitors of GIP-stimulated insulin release in vitro. These results confirm that homologous galanin inhibits insulin secretion in the rat (26,27). The data also support the recent demonstration that the two peptides were equipotent in their inhibition of the insulin response to glucose in the anesthetized rat (28). Further, the results are in accordance with recent suggestions that the C-terminal part of the galanin molecule is not necessary for its inhibitory action on the B-cell, since the 3 amino acid differences between the porcine and rodent peptides reside near the C-terminus (29,30). In the present study, porcine galanin had no effect on pancreatic somatostatin release stimulated by ACh. The concentration of porcine galanin used in these experiments (10 nM) was previously shown to inhibit gastric somatostatin release from the perfused rat stomach (10), suggesting that gastric and pancreatic 8-cells differ in their sensitivity to galanin. Previous studies using the perfused rat pancreas also found no effect of porcine galanin on pancreatic somatostatin release under either basal or stimulated conditions (8-10). In contrast, rat galanin suppressed AChstimulated somatostatin secretion in this study. Since ACh-stimulated insulin secretion was not affected by galanin, the peptide may exert its inhibitory influence on B- and 8-cells by different mechanisms. A powerful inhibitory effect of rat galanin but not porcine galanin on somatostatin secretion from the perfused rat pancreas was recently observed in another study (26). The results indicate that the pancreatic 8-cell of the rat, unlike the B-cell, is only sensitive to homologous galanin and support the idea that the C-terminal region of the galanin molecule is essential for its inhibitory action on the 8-cell (26,29). Homologous galanin has also been shown to be a potent inhibitor of pancreatic somatostatin secretion in the pig (25) suggesting that galanin is an important neural regulator of islet somatostatin release. However, since neither CCK-8 nor arginine (unlike ACh) were found to significantly stimulate somatostatin secretion in this study, it could not be ascertained whether rat galanin inhibits arginine- or CCK-8-stimulated 8-cell secretion. A recent study using the perfused rat pancreas found that rat galanin abolished arginine-, glucose-, and vasoactive intestinal polypeptide (VIP)-stimulated somatostatin secretion, and inhibited basal somatostatin secretion by 15% (26). In summary, the results of this study indicate that the inhibitory action of galanin on the rat B-cell depends upon the secretagogue present. The physiologic importance of this finding, and whether it exists in other species, remains to be determined.
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Acknowledgements This work was supported by a grant from the Medical Research Council of Canada, the British Columbia Health Research Foundation, and a Diabetes Canada studentship (C.B.V.). The technical assistance of Herminia Sy is gratefully acknowledged. References 1. K. TATEMOTO, A. ROKAEUS, H. JORNVALL, T.J. MCDONALD and V. MUTI', FEBS Lett. 164124-128 (1983). 2. A. ROKAEUS, Trends Neurosci. 10158-164 (1987). 3. B.E. DUNNING and G.J. TABORSKY, JR., Diabetes 3__2_71157-1162 (1988). 4. B.E. DUNNING, B. AHREN, R.C. VEITH, G. BOTI'CHER, F. SUNDLER and G.J. TABORSKY, JR., Am. J. Physiol. 251 E127-E133 (1986). 5. S. LINDSKOG, B. AHREN, B.E. DUNNING, and F. SUNDLER, Cell Tiss. Res. 264363368 (1991). 6. B. AMIRANOFF, A.L. SERVIN, C. ROUYER-FESSARD, A. COUVINEAU, K. TATEMOTO and M. LABURTHE, Endocrinology 121284-289 (1987). 7. B. AHREN, P. RORSMAN and P.-O. BERGGREN, FEBS Lett. 2233-237 (1988). 8. P. MIRALLES, E. PEIRO, R.A. SILVESTRE, M.L. VILLANUEVA and J. MARCO, Metabolism 37766-770 (1988). 9. R.A. SILVESTRE, P. MIRALLES, L. MONGE, P. MORENO, L. VILLANUEVA and J. MARCO, Endocrinology 121378-383 (1987). 10. Y.N. KWOK, C.B. VERCHERE, C.H.S. MCINTOSH and J.C. BROWN, Eur. J. Pharm. 14549-54 (1988). 11. S. LINDSKOG and B. AHREN, Acta Physiol. Scand. 129305-309 (1987). 12. S. LINDSKOG and B. AHREN, Hormone Res. 29237-240 (1988). 13. B. AMIRANOFF, A.-M. LORINET, I. LAGNY-POURMIR and M. LABURTHE, Eur. J. Biocbem. 177147-152 (1988). 14. T. YOSHIMURA, J. ISHIZUKA, G.H. GREELEY, JR. and J.C. THOMPSON, Am. J. Physiol. 256 E619-E623 (1989). 15. J.C. PENHOS, C.-H. WU, J.C. BASABE, N. LOPEZ, and F.W. WOLFF, Diabetes 1_.88733738 (1969). 16. J. TAKEMURA, Y.N. KWOK, and J.C. BROWN, Am. J. Med. 81 (suppl 6B) 65-73 (1986). 17. J.A. VAN DER VLIET, R.M. MELOCHE, M.J. FIELD, D.J. CHEN, D.B. KAUFMAN, and D.E.R. SUTHERLAND, Transplantation 45493-495 (1988). 18. D.G. PIPELEERS, P.A. IN'T VELD, M. VAN DE WINKEL, E. MAES, F.C. SCHUIT, and W. GEPTS, Endocrinology 117816-823 (1985). 19. D.G. PIPELEERS, F.C. SCHUIT, P.A. IN'T VELD, E. MAES, E.L. HOOGHE-PETERS, M. VAN DE WINKEL, and W. GEPTS, Endocrinology 117824-833 (1985). 20. M. PRENTKI and F.M. MATSCHINSKY, Physiol. Reviews 671185-1248 (1987). 21. E.G. SIEGEL and W. CREUTZFELDT, Diabetologia 28857-861 (1985). 22. P. GARCIA-MORALES, S.P. DUFRANE, A. SENER, I. VALVERDE, and W.J. MALAISSE, Biosci. Rep. 4_511-521 (1984). 23. I.M. HRAMIAK, J. DUPRE, and T.J. MCDONALD, Endocrinology 1222486-2491 (1988). 24. S.G. GILBEY, J. STEPHENSON, D.J. O'HALLORAN, J.M. BURRIN, and S.R. BLOOM, Diabetes 381114-1116 (1989). 25. T. MESSELL, H. HARLING, G. BO'I'FCHER, A.H. JOHNSEN, and J.J. HOLST, Regul. Pept. 2._88161-176 (1990). 26. P. MIRALLES, E. PEIRO, P. DEGANO, R.A. SILVESTRE, and J. MARCO, Diabetes 3._99 996-1001 (1990). 27. S. LINDSKOG, B.E. DUNNING, H. MARTENSSON, A. AR'RAJAB, G.J. TABORSKY, JR., and B. AHREN, Acta Physiol. Scand. 139591-596 (1990). 28. E.M. SCHNUERER, A. ROKAEUS, M. CARLQUIST, T. BERGMAN, J. DUPRE and T.J. MCDONALD, Pancreas 570-74 (1990). 29. K. HERMANSEN, N. YANAIHARA and B. AHREN, Acta Endo. (Copen) 121545-550 (1989). 30. B. AMIRANOFF, A.-M. LORINET, N. YANAIHARA and M. LABURTHE, Eur. J. Pharm. 163205-207 (1989).
51, pp.
1945-1951
Pergamon
Press
STIMULUS-SPECIFIC INHIBITION OF INSULIN RELEASE FROM RAT PANCREAS BY BOTH RAT AND PORCINE GALANIN C. Bruce Verchere, Yin Nam Kwok and John C. Brown Medical Research Council of Canada Regulatory Peptide Group Department of Physiology, University of British Columbia Vancouver, B.C., Canada V6T 1Z3 (Received
in final
form October
8, 1992)
Summary_ The effect of the neuropeptide galanin on insulin and somatostatin secretion in the rat was studied under various conditions. In the perfused rat pancreas, insulin secretion stimulated by arginine, but not cholecystokinin-8 (CCK-8) or acetylcholine (ACh) was inhibited by both rat and porcine galanin, whereas ACh-stimulated somatostatin release was inhibited by rat but not porcine galanin. Neither arginine nor CCK-8 significantly "altered somatostatin secretion and galanin was without effect under those conditions. Gastric inhibitory polypeptide-stimulated insulin release from cultured mixtures of purified rat g- and non-B-cells was inhibited by rat and porcine galanin in a concentration-dependent and equipotent manner. The results suggest that the inhibitory effect of galanin on insulin and somatostatin secretion may be stimulusspecific and species-specific. Galanin is a 29 amino acid neuropeptide, isolated by Tatemoto et al (1), that is widely distributed in the central and peripheral nervous systems (2). The peptide may be an important neural regulator of islet hormone secretion (3). The presence of galanin in nerves in association with pancreatic islets has been demonstrated immunocytochemically in the dog (4) and rat (5) pancreas and the presence of galanin receptors has been demonstrated in a hamster pancreatic/3-cell line (6). Numerous studies have shown that galanin possesses a potent inhibitory effect on insulin secretion (3,7). Although its effects on glucagon and somatostatin secretion are less clear, the peptide has been shown to inhibit somatostatin and stimulate glucagon release from the dog (4). Studies in the rat (8-10) and mouse (11,12) have shown galanin to be a potent inhibitor of insulin secretion under a variety of conditions, suggesting that galanin interferes with an essential, common step in the insulin secretory mechanism (3,9). In contrast, other studies using B-cell lines (13) or perfused rat pancreas (14) have found that the inhibitory action of galanin was only observed in the presence of specific stimuli. Thus, galanin suppressed gastric inhibitory polypeptide (GIP)- but not carbamoylcholine-stimulated insulin secretion from Rinm5f g-cells (13), and the neuropeptide inhibited glucose- but not arginine- or potassium-stimulated insulin secretion from the perfused rat pancreas (14). The present experiments were undertaken to investigate these discrepancies more closely by examining the effect of galanin on insulin and somatostatin secretion from the perfused rat pancreas in the presence of different stimuli. Since previous studies in the rat have used porcine galanin, which might exhibit diminished biologic activity in rodent models, this study examined the inhibitory effects of both porcine and rat galanin. In addition, the potency of these two peptides in inhibiting GIP-stimulated B-cell secretion in vitro was compared using cultured B-cells obtained by fluorescence-activated cell-sorting.
Author for correspondence: Yin Nam Kwok, Ph.D., MRC Regulatory Peptide Group, University of British Columbia, 2146 Health Sciences Mall, Vancouver, B.C., Canada, V6T 1Z3
Copyright
0024-3205/92 $5.00 + .00 © 1992 Pergamon Press Ltd All rights
reserved.
1946
Galanin
Inhibition
of I n s u l i n
Vol.
51, No.
25,
1992
Methods Perfused pancreas. The procedure for surgical isolation and in situ vascular perfusion of the rat pancreas, modified from the technique of Penbos et al (15), has been previously described (16). Male Wistar rats weighing 250-350 g were used. The perfusate was a Krebs solution containing 0.2% bovine serum albumin (BSA; Sigma, RIA grade), 3% dextran (Sigma, clinical grade) and 4.4 mM or 8.9 mM glucose. It was heated to 37 °C and pumped through the pancreatic vasculature at a flow rate of 3 ml/min. Test substances were infused into the perfusate by a side-arm pump (0.1 ml/min). Venous effluent was collected (5 min/sample) via a portal vein cannula into tubes containing aprotinin (5000 K.I.U.). Aliquots (0.5 ml) were stored at -20 °C for radioimmunoassay (RIA); an additional 500 K.I.U. of aprotinin was added to samples for assay of somatostatin-like immunoreactivity (SLI) before freezing. Purified islet cells. Islets were isolated from male Wistar rats (250-350 g) by collagenase digestion and purification on a discontinuous dextran gradient (17). The islets from 4-8 rats (1000-5000 islets) were pooled and cultured overnight (37 °C, 5% CO2) in CMRL-1066 medium plus 10% Calf Supreme serum (Gibco), 2 mM L-glutamine, and an antibiotic-antimycotic mixture. Fractions of Bcells and non-B-cells were obtained from islets via the technique of Pipeleers et al (18). The islets were first dispersed in Ca++-free Earle's-HEPES (EH) medium containing 0.1% BSA, 2.75 mM glucose, 1 mM EGTA, 200 btg/ml trypsin and 2 p.g/ml DNase at 31 °C using a siliconized Pasteur pipet. When at least 50% of the cells were dissociated as single cells, the digestion was stopped by filtering the suspension through a 100 btm nylon screen into a 50 ml tube containing ice-cold EH plus Ca++. After centrifugation (5 rain at 500 X g) the cells were resuspended in 1 ml Ca++-free EH, and maintained at 17 °C for 15 rain prior to sorting on a FACS-IV (Becton-Dickinson, Sunny Vale, CA). Two cell fractions were obtained from the FACS and examined by immunocytochemical staining. The B-cell fraction of higher light scatter and autofluorescence consisted of more than 98% insulincontaining cells, while the non-B-cell fraction consisted of 40-60% a-cells, with a smaller proportion of g-, 8-, PP-, and unidentified cells. Since FACS-purified B-cells have been shown to be more responsive when reaggregated with a-cells (19), the B-cell and non-B-cell fractions were remixed (1:1) before culture. The ceils were seeded (6000 cells/well) in 200 gl culture medium (as for islets) in 96-well plates and cultured for 3 days prior to insulin release experiments. Incubations were performed for 1 h at 37 °C in 250 btl Dulbecco's Modified Eagle's Medium plus 0.1% BSA, 17.8 mM glucose and the desired concentration of GIP and rat or porcine galanin. After collecting the incubation medium, the cells in each well were extracted in 200 gl acetic acid (2 M) and boiled for 10 min for the measurement of total insulin content. Incubation medium and cell extracts were stored at -20 °C until assay. Radioimmunoassays. Immunoreactive insulin (IRI) and somatostatin-like immunoreactivity (SLI) were measured using specific RIA's that have been previously described (16). Galanin has been shown not to interfere with the binding of insulin or somatostatin in these assays (10). In perfused pancreas experiments, IRI or SLI secretion was expressed as the percent change (mean +_S.E.M.) from the basal secretion rate (mean of 2 basal collection periods of 5 min each) in the presence of either 4.4 mM glucose (ACh and arginine experiments) or 8.9 mM glucose (CCK-8 experiments). For statistical comparison, the percent of basal release during a 10 rain infusion of rat or porcine galanin was compared to the percent of basal release during a 10 min infusion of a control vehicle using the Mann-Whitney U test. In B-cell culture experiments, IRI release was expressed as a percent of total cell IRI content for each well (mean +_S,E.M, of four triplicate determinations) and compared using one way analysis of variance with Dunnett's test. p<0.05 was considered significant. Results Perfused pancreas Effect of galanin on IRI release. In the presence of 4.4 mM glucose, arginine (20 mM) produced a marked stimulation of IRI secretion which was sustained for the duration of the arginine administration. When porcine galanin was introduced at a concentration of 10 nM, previously
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shown to potently inhibit GIP-stimulated IRI release in this preparation (10), a modest suppression of arginine-stimulated IRI secretion was observed (Fig. 1). The percent increase over basal (4.4 mM glucose alone) IRI secretion induced by arginine stimulation was significantly lower during the 10 min infusion of porcine galanin infusion when compared to a control vehicle infusion (2738+447 vs 4436+975%; p<0.05). Rat galanin (10 nM) inhibited arginine-stimulated IRI secretion to a similar extent (2419+718 vs 4436_+975%; p<0.05; Fig. 1).
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FIG. 1 Effect of porcine and rat galanin on arginine-stimulated IRI secretion from the perfused rat pancreas. Arginine (20 mM) was added to the perfusate during periods 3-9 and either 10 nM porcine galanin (-o-; n=6), 10 nM rat galanin (-o-; n=7) or a control vehicle (-13-; n=6) was infused during periods 6-7. Perfusate glucose concentration was 4.4 mM throughout.
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FIG. 2 Effect of porcine and rat galanin on ACh- and CCK-8-stimulated IRI secretion from the perfused rat pancreas. (A) ACh (5 I.tM) was added to the perfusate during periods 3-9. Perfusate glucose concentration was 4.4 mM throughout. (B) Perfusate glucose concentration was increased from 4.4 to 8.9 mM during periods 3-10 and CCK-8 (0.9 nM) was infused during periods 5-10. Either 10 nM porcine galanin (-o-), 10 nM rat galanin (-o-) or a control vehicle (-121-)was infused (n=5-7) during (A) periods 6-7 or (B) periods 7-8.
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In contrast, when IRI secretion was stimulated by 5 gM ACh in the presence of 4.4 mM glucose, neither rat nor porcine galanin (10 nM) significantly altered IRI secretion (Fig. 2A). The percent increase over basal IRI release during ACh stimulation in control experiments (12104_-297%) was not significantly different from that observed during porcine (1094+205%) or rat (1009+304%) galanin infusion. ACh-stimulated IRI release was also unaffected when rat galanin was administered at a concentration of 50 nM (data not shown). Similarly, when IRI secretion was stimulated by CCK-8 (0.9 nM) in the presence of 8.9 mM glucose, infusion of either porcine or rat galanin (10 nM) again failed to elicit a significant change in IRI release when compared to controls (Fig. 2B). Effect of galanin on SLI release. Of the insulin secretagogues tested, only ACh (5 gM) produced a significant increase in SLI release. SLI secretion increased during ACh infusion from basal levels of 149+29 pg/min to peak concentrations of 304+47 pg/min. Infusion of rat galanin (10 nM) inhibited the ACh-stimulated SLI secretion to approximately 40% of peak values (Fig. 3A); SLI release (percent of basal) was significantly lower in the presence of rat galanin when compared to controls (140-+24 vs 184-+11%; p<0.05). However, porcine galanin (10 nM) had no effect on AChstimulated SLI release (Fig. 3B; 194+23 vs 184+11%; p=NS). This concentration of porcine galanin was previously shown to inhibit gastric SLI release from the perfused rat stomach (10). Neither arginine (20 mM) nor CCK-8 (0.9 nM) plus glucose (8.9 mM) produced significant changes in SLI secretion, and addition of rat or porcine galanin was without effect under those conditions (data not shown).
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FIG. 3 Effect of rat and porcine galanin on ACh-stimulated SLI secretion from the perfused pancreas. ACh (5 #M) was infused during periods 3-9 and either (A) 10 nM rat galanin (rGal; n=7) or (B) 10 nM porcine galanin (pGal; n=6) was infused during periods 6-7. Perfusate glucose concentration was 4.4 mM throughout. Co-cultured 6- and non-6-cells A previous study showed that mixed cultures of FACS-purified 6- and non-13-cells were more responsive to glucose than g-cells alone (19). In the present study, 10 nM GIP (in the presence of 17.8 mM glucose) significantly (p<0.05) increased IRI secretion (4.1+0.4%) from this preparation compared to 17.8 mM glucose alone (2.4_+0.3%). Since porcine galanin was previously shown to potently inhibit GIP-stimulated insulin release (10), the effect of a range of concentrations (0.1 nM to 100 nM) of porcine and rat galanin on the IRI response to GIP (10 nM) plus glucose (17.8 raM) was examined. The two peptides inhibited GIP-stimulated IRI release in an equipotent and concentration-dependent manner (Fig. 4). The lowest concentration of either peptide to significantly (p<0.05) inhibit the IRI response to G1P was 1 nM.
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Galanin
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[Galanin] (nM) FIG. 4 Comparison of the effect of rat and porcine galanin on GIP-stimulated IRI secretion from cocultured 13- and non-B-cells. IRI secretion is expressed as a percent of total cell IRI content, after 1 h incubation in the presence of 17.8 mM glucose, 10 nM GIP, and indicated concentration of rat (-o-) or porcine (-e-) galanin (n=4 of triplicate determinations). Discussion The conditions under which galanin influences insulin and somatostatin secretion in the rat were evaluated. In this and a previous study (10) using the in situ perfused rat pancreas, only GIP- and arginine-stimulated insulin release were inhibited by galanin, while the insulin responses to CCK-8 and ACh were unaffected by this neuropeptide. The results suggest that the inhibitory action of galanin on the B-cell is stimulus-specific. The lack of effect of galanin on ACh- and CCK-8-stimulated insulin release may provide insight into the mechanism of action of galanin on the B-cell. Both CCK-8 and cholinergic agonists stimulate insulin secretion by increasing 13-cell phosphoinositol metabolism and intracellular calcium levels (20). These data suggest that galanin does not influence those intracellular pathways in the rat B-cell. This idea was supported by the recent observation that porcine galanin potently inhibited forskolinand GIP-stimulated insulin secretion and cAMP production in the Rinm5f B-cell line, but had no effect on carbamoylcholine-stimulated insulin secretion (13). It was suggested that galanin inhibited B-cell secretion via inhibition of cAMP production. In this study, the inhibitory effect of galanin on GIP- and arginine-stimulated insulin secretion may have occurred via an inhibition of B-cell cAMP production, since both of these B-cell secretagogues have been shown to increase cAMP levels in isolated rat islets (21,22). In apparent conflict with these results, Miralles et al found that CCK-8-stimulated insulin secretion was suppressed by porcine galanin in the perfused rat pancreas (8). However, in those experiments a priming dose of galanin was given prior to the introduction of CCK-8 followed by infusion of galanin throughout the stimulation, whereas in this study galanin was infused for only a 10 min period during the second phase of CCK-8-stimulated insulin secretion. It is possible that the
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experimental protocol of this study masked an inhibitory effect of galanin on CCK-8- (or ACh)stimulated secretion, that would only be evident when the presence of galanin precedes the stimulation. Indeed, it has been shown that galanin infusion inhibited the first phase of glucosestimulated insulin release only when commenced 4 rain prior to the glucose stimulation, suggesting that galanin acts on early events of the stimulus-secretion coupling in the B-cell (14). It is also possible that an inhibitory effect of galanin on ACh- or CCK-8-stimulated insulin release may have been observed if higher concentrations of the neuropeptide were used. However, 50 nM rat galanin failed to inhibit ACh-stimulated insulin release in this study, and this concentration was 5 fold greater than that required to suppress GIP- (10) or arginine-stimulated insulin secretion in the perfused rat pancreas. This observation strongly supports the idea that the inhibitory action of galanin is more selective for certain B-cell secretagogues in the rat. A stimulus-specific inhibitory effect of galanin was also observed by Yoshimura et al (14), who found that porcine galanin inhibited glucose-, but not arginine- or potassium-stimulated insulin release in the perfused rat pancreas. Although the present study found galanin to inhibit arginine-stimulated insulin secretion, the concentration of galanin used in this study (10 riM) was 10 fold the concentration employed by Yoshimura et al (14). The results of this study also conflict with previous studies in the dog (23) and mouse (11,12), in which galanin was found to inhibit the insulin response to a variety of secretagogues, including CCK-8 (12,23) and carbachol (11). This discrepancy may be due to the existence of species differences in the sensitivity of the B-cell to the action of galanin. In humans, infusions of high doses of porcine galanin failed to alter serum insulin levels (24), suggesting a lack of g-cell sensitivity to galanin in humans, while in the perfused pig pancreas, porcine galanin stimulated insulin release (25). Together, these studies suggest that the contribution of galanin to the normal regulation of B-cell secretion may vary widely between species. In these experiments, porcine and rat galanin had identical effects on insulin secretion from rat Bcells under every condition tested and the two peptides were equipotent inhibitors of GIP-stimulated insulin release in vitro. These results confirm that homologous galanin inhibits insulin secretion in the rat (26,27). The data also support the recent demonstration that the two peptides were equipotent in their inhibition of the insulin response to glucose in the anesthetized rat (28). Further, the results are in accordance with recent suggestions that the C-terminal part of the galanin molecule is not necessary for its inhibitory action on the B-cell, since the 3 amino acid differences between the porcine and rodent peptides reside near the C-terminus (29,30). In the present study, porcine galanin had no effect on pancreatic somatostatin release stimulated by ACh. The concentration of porcine galanin used in these experiments (10 nM) was previously shown to inhibit gastric somatostatin release from the perfused rat stomach (10), suggesting that gastric and pancreatic 8-cells differ in their sensitivity to galanin. Previous studies using the perfused rat pancreas also found no effect of porcine galanin on pancreatic somatostatin release under either basal or stimulated conditions (8-10). In contrast, rat galanin suppressed AChstimulated somatostatin secretion in this study. Since ACh-stimulated insulin secretion was not affected by galanin, the peptide may exert its inhibitory influence on B- and 8-cells by different mechanisms. A powerful inhibitory effect of rat galanin but not porcine galanin on somatostatin secretion from the perfused rat pancreas was recently observed in another study (26). The results indicate that the pancreatic 8-cell of the rat, unlike the B-cell, is only sensitive to homologous galanin and support the idea that the C-terminal region of the galanin molecule is essential for its inhibitory action on the 8-cell (26,29). Homologous galanin has also been shown to be a potent inhibitor of pancreatic somatostatin secretion in the pig (25) suggesting that galanin is an important neural regulator of islet somatostatin release. However, since neither CCK-8 nor arginine (unlike ACh) were found to significantly stimulate somatostatin secretion in this study, it could not be ascertained whether rat galanin inhibits arginine- or CCK-8-stimulated 8-cell secretion. A recent study using the perfused rat pancreas found that rat galanin abolished arginine-, glucose-, and vasoactive intestinal polypeptide (VIP)-stimulated somatostatin secretion, and inhibited basal somatostatin secretion by 15% (26). In summary, the results of this study indicate that the inhibitory action of galanin on the rat B-cell depends upon the secretagogue present. The physiologic importance of this finding, and whether it exists in other species, remains to be determined.
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Acknowledgements This work was supported by a grant from the Medical Research Council of Canada, the British Columbia Health Research Foundation, and a Diabetes Canada studentship (C.B.V.). The technical assistance of Herminia Sy is gratefully acknowledged. References 1. K. TATEMOTO, A. ROKAEUS, H. JORNVALL, T.J. MCDONALD and V. MUTI', FEBS Lett. 164124-128 (1983). 2. A. ROKAEUS, Trends Neurosci. 10158-164 (1987). 3. B.E. DUNNING and G.J. TABORSKY, JR., Diabetes 3__2_71157-1162 (1988). 4. B.E. DUNNING, B. AHREN, R.C. VEITH, G. BOTI'CHER, F. SUNDLER and G.J. TABORSKY, JR., Am. J. Physiol. 251 E127-E133 (1986). 5. S. LINDSKOG, B. AHREN, B.E. DUNNING, and F. SUNDLER, Cell Tiss. Res. 264363368 (1991). 6. B. AMIRANOFF, A.L. SERVIN, C. ROUYER-FESSARD, A. COUVINEAU, K. TATEMOTO and M. LABURTHE, Endocrinology 121284-289 (1987). 7. B. AHREN, P. RORSMAN and P.-O. BERGGREN, FEBS Lett. 2233-237 (1988). 8. P. MIRALLES, E. PEIRO, R.A. SILVESTRE, M.L. VILLANUEVA and J. MARCO, Metabolism 37766-770 (1988). 9. R.A. SILVESTRE, P. MIRALLES, L. MONGE, P. MORENO, L. VILLANUEVA and J. MARCO, Endocrinology 121378-383 (1987). 10. Y.N. KWOK, C.B. VERCHERE, C.H.S. MCINTOSH and J.C. BROWN, Eur. J. Pharm. 14549-54 (1988). 11. S. LINDSKOG and B. AHREN, Acta Physiol. Scand. 129305-309 (1987). 12. S. LINDSKOG and B. AHREN, Hormone Res. 29237-240 (1988). 13. B. AMIRANOFF, A.-M. LORINET, I. LAGNY-POURMIR and M. LABURTHE, Eur. J. Biocbem. 177147-152 (1988). 14. T. YOSHIMURA, J. ISHIZUKA, G.H. GREELEY, JR. and J.C. THOMPSON, Am. J. Physiol. 256 E619-E623 (1989). 15. J.C. PENHOS, C.-H. WU, J.C. BASABE, N. LOPEZ, and F.W. WOLFF, Diabetes 1_.88733738 (1969). 16. J. TAKEMURA, Y.N. KWOK, and J.C. BROWN, Am. J. Med. 81 (suppl 6B) 65-73 (1986). 17. J.A. VAN DER VLIET, R.M. MELOCHE, M.J. FIELD, D.J. CHEN, D.B. KAUFMAN, and D.E.R. SUTHERLAND, Transplantation 45493-495 (1988). 18. D.G. PIPELEERS, P.A. IN'T VELD, M. VAN DE WINKEL, E. MAES, F.C. SCHUIT, and W. GEPTS, Endocrinology 117816-823 (1985). 19. D.G. PIPELEERS, F.C. SCHUIT, P.A. IN'T VELD, E. MAES, E.L. HOOGHE-PETERS, M. VAN DE WINKEL, and W. GEPTS, Endocrinology 117824-833 (1985). 20. M. PRENTKI and F.M. MATSCHINSKY, Physiol. Reviews 671185-1248 (1987). 21. E.G. SIEGEL and W. CREUTZFELDT, Diabetologia 28857-861 (1985). 22. P. GARCIA-MORALES, S.P. DUFRANE, A. SENER, I. VALVERDE, and W.J. MALAISSE, Biosci. Rep. 4_511-521 (1984). 23. I.M. HRAMIAK, J. DUPRE, and T.J. MCDONALD, Endocrinology 1222486-2491 (1988). 24. S.G. GILBEY, J. STEPHENSON, D.J. O'HALLORAN, J.M. BURRIN, and S.R. BLOOM, Diabetes 381114-1116 (1989). 25. T. MESSELL, H. HARLING, G. BO'I'FCHER, A.H. JOHNSEN, and J.J. HOLST, Regul. Pept. 2._88161-176 (1990). 26. P. MIRALLES, E. PEIRO, P. DEGANO, R.A. SILVESTRE, and J. MARCO, Diabetes 3._99 996-1001 (1990). 27. S. LINDSKOG, B.E. DUNNING, H. MARTENSSON, A. AR'RAJAB, G.J. TABORSKY, JR., and B. AHREN, Acta Physiol. Scand. 139591-596 (1990). 28. E.M. SCHNUERER, A. ROKAEUS, M. CARLQUIST, T. BERGMAN, J. DUPRE and T.J. MCDONALD, Pancreas 570-74 (1990). 29. K. HERMANSEN, N. YANAIHARA and B. AHREN, Acta Endo. (Copen) 121545-550 (1989). 30. B. AMIRANOFF, A.-M. LORINET, N. YANAIHARA and M. LABURTHE, Eur. J. Pharm. 163205-207 (1989).