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
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
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
