1 Department of Medicine, Gastrin-releasing peptide (GRP) stimulates
insulin secretion by a direct islet effect. In this study, we initially
demonstrated, by immunocytochemistry of the mouse pancreas, GRP
immunoreactive nerve fibers within exocrine tissue, islets, and
intrapancreatic ganglia. A more pronounced GRP innervation was found in
ganglia compared with in islets. We therefore studied whether indirect cholinergic mechanisms contribute to the insulinotropic action of GRP.
In mice, the insulinotropic response to GRP (4.25 nmol/kg iv) was
inhibited by the m3-selective,
muscarinic receptor antagonist 4-diphenylacetoxy-N-methyl piperidine
methobromide (4-DAMP, 0.21 mol/kg; by 68%,
P < 0.05) and by the ganglionic
blocker hexamethonium (28 mol/kg; by 98%,
P < 0.05). In contrast, in isolated
islets, 4-DAMP or hexamethonium (10 or 100 µM) did not inhibit GRP
(100 nM)-induced insulin secretion. Furthermore, afferent denervation by neonatal capsaicin did not affect the insulin response to GRP. We
conclude that the insulinotropic effect of GRP in the mouse is mediated
by both direct islet effects and through activation, at the ganglionic
level, of postganglionic cholinergic nerves. In vivo, the indirect
cholinergic mechanism predominates.
glucagon secretion; pancreatic nerves; islets of
Langerhans
SEVERAL PREVIOUS STUDIES have suggested that
gastrin-releasing peptide (GRP) might participate in the regulation of
insulin secretion as a pancreatic neurotransmitter exerting a direct
insulinotropic effect on the islet B cells. Thus GRP has been shown to
stimulate insulin secretion both under in vivo conditions (4, 20, 22, 23, 30) and in vitro from the perfused pancreas (6, 16) as well as in
isolated islets and insulin-producing B cell lines (5, 28, 29).
Furthermore, specific GRP-binding sites have been demonstrated in mouse
islets (28). In addition, in several species, GRP immunoreactivity has
been demonstrated in nerve fibers within the islets (2, 14, 21, 24).
GRP has also been demonstrated to be released from the porcine pancreas
upon electrical activation of the vagal nerve, and the insulin response
to electrical activation of the vagal nerve in pigs has been shown to
be inhibited by a GRP receptor antagonist (7, 14). This suggests that GRP contributes to vagally induced insulin secretion.
GRP has previously been shown to stimulate exocrine pancreatic
secretion through an indirect mechanism involving activation of
cholinergic nerves, since this effect of the peptide was inhibited by
cholinergic blockade (3, 15). Whether cholinergic mechanisms are
involved also in the effect of GRP on the endocrine pancreas is,
however, not established. In one previous study, in the perfused porcine pancreas, GRP-stimulated insulin secretion was unaffected by
muscarinic receptor blockade by atropine (16), whereas another study,
in the mouse, showed that methylatropine diminished both the insulin
and glucagon responses to intravenous GRP (22). In the present study,
we investigated in more detail the possible involvement of indirect
cholinergic mechanisms in GRP-stimulated insulin secretion in the
mouse.
Initially, we therefore, by immunocytochemistry, investigated the
occurrence of GRP immunoreactivity in sections of mouse pancreata. The
possible contribution of cholinergic and ganglionic mechanisms to
GRP-stimulated insulin secretion was investigated both under in vivo
conditions in the mouse and in isolated mouse pancreatic islets. We
thereby used the selective
m3-muscarinic receptor antagonist
4-diphenylacetoxy-N-methylpiperadine
methobromide (4-DAMP) and the ganglionic nicotinic receptor antagonist
hexamethonium. Moreover, to investigate the involvement of afferent
nerves, we examined whether the insulinotropic effect of GRP was
affected by neonatal capsaicin treatment, which induces a permanent
sensory denervation (9, 13).
Animals. Female mice of the Naval
Medicine Research Institute (NMRI) strain (Bolmholdtgaard
Breeding and Research Center, Ry, Denmark) weighing 25-35 g were
used throughout the study. The animals were fed a standard pellet diet
and tap water ad libitum. The study was approved by the Ethic Committee
for Animal Research of Lund University.
Immunocytochemical studies.
Immunocytochemistry for GRP immunoreactivity was undertaken as
described previously (21). In short, tissue specimens taken from mouse
pancreas were fixed overnight in Stefanini's fixative (15% saturated
picric acid and 2% formaldehyde in 0.1 M phosphate-buffered saline).
After rinsing, the specimens were frozen on dry ice and were thereafter
cut at 10 µm in a cryostat and collected on chrome
alum-coated slides. The sections were then processed for
immunocytochemistry, using a rabbit anti-GRP antibody (code no. R6902;
dilution 1:640). For control sections, the antiserum was inactivated by
excess of synthetic GRP. The antiserum does not cross-react with
substance P, vasoactive intestinal polypeptide, Leu- and
Met-enkephalin, cholecystokinin-8, or neurotensin (21). Ganglionic
nerve cell bodies were identified by an antibody raised against the
neural cell soma marker protein gene product (PGP; Ultraclone,
Cambridge, UK; see Ref. 26) 9.5. Double staining for GRP and PGP was
used to discriminate between GRP immunoreactivity located to nerve cell
bodies versus to in nerve fibers of the ganglia.
In vivo experiments. Animals were
given an intravenous bolus injection of GRP at a dose level that
previously was shown to be maximal in regard to stimulation of insulin
secretion (4.25 nmol/kg; Peninsula, Belmont, CA; see Ref. 22). Controls
were given saline. At 15 min before the intravenous injection, animals were injected intraperitoneally with either the muscarinic
m3-selective receptor antagonist
4-DAMP (21 µmol/kg; Research Biochemicals, Natick, MA) or
hexamethonium (28 µmol/kg; Sigma Chemical, St. Louis, MO). Controls
were injected intraperitoneally with saline. The dose levels of these
antagonists were selected from previous studies in our laboratory.
Thus, at the dose level of 0.21 µmol/kg, 4-DAMP inhibits
carbachol-stimulated insulin and glucagon secretion in the mouse (11),
and hexamethonium inhibits neurally induced insulin and glucagon
secretion in the mouse at the dose level of 28 µmol/kg (10). At 2 min
after the intravenous injection, blood was sampled from the retrobulbar
venous plexus. At this time point, the plasma levels of insulin and
glucagon reach their peak after an intravenous injection of GRP as
previously demonstrated in the mouse (22). Plasma was separated by
centrifugation and was stored at Neonatal capsaicin treatment. Under
buprenorphine (Themgesic; Reckitt & Colmar, Hull, UK; 0.45 mg/kg)
analgesia, capsaicin (8-methyl-N-vanillyl-6-nonenamide; 50 mg/kg) was given as a subcutaneous injection at days
2 and 5 of life as
described previously (13). Capsaicin was dissolved in a vehicle
consisting of 10% ethanol and 10% Tween 80 in 0.9% NaCl. The volume
load was 20 µl/animal. Controls were treated with buprenorphine and
were given vehicle alone. This capsaicin treatment of neonatal mice has
previously been shown to induce a permanent destruction of sensory
nerves of the C-fiber type (8, 9) and to cause a permanent destruction of nerves immunoreactive for calcitonin gene-related peptide and a
substantial reduction in the number of substance P immunoreactive pancreatic nerves in the mouse, thus creating a permanent sensory denervation (13). We have previously demonstrated that neonatal capsaicin treatment of mice results in increased glucose tolerance (12). To verify that capsaicin, in the present study, acted as expected
from previous studies, an intravenous bolus injection of glucose (2.8 mmol/kg) was given to the animals used for the present experiments.
Thereby, basal plasma glucose levels did not differ between
vehicle-treated and capsaicin-treated animals, whereas at 10 min after
the glucose injection the plasma glucose levels were 13.8 ± 0.9 mmol/l in vehicle-treated animals and 9.5 ± 0.5 mmol/l in
capsaicin-treated animals (P < 0.01; n = 6 for each group),
verifying the increased glucose tolerance. For the subsequent
experiments on islet hormone secretion, female mice were used at the
age of 10-12 wk.
Isolation and incubation of islets.
Islets were isolated by the collagenase digestion technique as
initially described by Lacy and Kostianovsky (17) with slight
modifications. In short, during pentobarbital sodium (120 mg/kg)
anesthesia, a catheter was inserted through the common bile duct, and
the duodenal papilla was occluded. Thereafter, the pancreas was
retrogradely filled with 3 ml of Hanks' balanced salt solution (Sigma
Chemical) supplemented with 0.3 mg/ml of collagenase (collagenase P;
Boehringer Mannheim). The pancreas was removed and incubated at
37°C for 20-22 min. Islets were thereafter hand picked under a
stereomicroscope and cultured overnight in a RPMI 1640 medium
(GIBCO-BRL, Paisley, Scotland) supplemented with 10% fetal calf serum
(Biological Industries, Kibbutz Beit Haemek, Israel), 2.06 mmol/l L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin (Biological Industries), and 2.5 µg/ml
amphotericin B (GIBCO). Single islets were incubated in a volume of 100 µl during 60 min at 37°C in an atmosphere of humidified air
saturated with 5% CO2. The
incubation buffer contained (in mM) 25 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (HEPES), 125 NaCl, 5.9 KCl, 1.28 CaCl2, and 1.1 MgCl2 and was supplemented with
0.1% human serum albumin and glucose (3.3 or 11.1 mM). The pH (7.36)
of the HEPES buffer was adjusted by the use of NaOH. During the
experiments, GRP (100 nM), carbachol (0.1 mM; Sigma), 4-DAMP (0.01 and
0.1 mM), or hexamethonium (0.01 and 0.1 mM) was added according to the
protocols. The concentration of hexamethonium used in the present
experiments has previously been demonstrated to induce ganglionic
blockade in vitro in the perfused rat pancreas (27). After the
incubation period, 25 µl of the medium were removed for analysis of
its insulin content by radioimmunoassay.
Determinations of insulin, glucagon, and
glucose. Insulin was determined by radioimmunoassay
using a guinea pig anti-rat insulin antibody,
125I-labeled human insulin as
tracer, and rat insulin as standard (Linco Research, St. Charles, MO).
The separation of free and bound radioactivity was performed by the
double antibody technique using a goat anti-guinea pig immunoglobulin G
antibody (Linco). Plasma glucagon levels were determined by
radioimmunoassay using an antibody specific for pancreatic glucagon,
125I-labeled glucagon, and
glucagon as standard (Milab, Malmö, Sweden). Plasma glucose
levels were determined with the glucose oxidase method.
Statistics. Data are presented as
means ± SE. One-way analysis of variance followed by Newman-Keuls
test or Student's t-test for unpaired observations were used for statistical evaluation. A
P value of <0.05 was considered
significant. For the immunohistochemical studies, at least five
sections from the pancreata of nine different mice were examined. The
relative frequency of GRP immunoreactivity in the exocrine parenchyma,
the islets, and the ganglia were estimated semiquantitatively by two
independent observers.
GRP immunoreactivity in mouse
pancreas. In sections of the mouse pancreas, GRP
immunoreactive varicose nerve fibers were found within the exocrine
parenchyma, the islets, and the intrapancreatic ganglia (Fig.
1). GRP immunoreactive nerve fibers were
more frequently observed in ganglia compared with in islets (Table
1). Double staining for GRP and the
neuronal cell soma marker PGP 9.5 revealed that the majority of the
ganglia showed GRP immunoreactivity confined only to nerve fibers.
However, rarely also, ganglionic nerve cell bodies showed a slight GRP
immunoreactivity (Fig. 1, B and
C). Thus GRP immunoreactive nerve
fibers occur in both the islets and ganglia of the mouse
pancreas.
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
20°C until assayed for its
content of insulin, glucagon, and glucose.
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Fig. 1.
Sections of mouse pancreas demonstrating fine varicose
gastrin-releasing peptide (GRP) immunoreactive nerve fibers in the
center and in the periphery of a pancreatic islet
(A).
B: ganglion with GRP immunoreactivity
confined to fine varicose nerve fibers (arrow) within the ganglion, and
also moderately stained GRP immunoreactive nerve cell bodies are
demonstrated. C: same ganglion as in
B is shown double stained for the
neuronal cell soma marker protein gene product (PGP) 9.5 to identify
the ganglionic nerve cell bodies; it is seen that two nerve cell bodies
are distinctly double stained for both GRP and PGP. Also in
C, PGP 9.5 immunoreactive nerve fibers
are seen scattered within the exocrine parenchyma. GRP immunoreactivity
was visualized by fluorescein isothiocyanate, and PGP immunoreactivity
was visualized by tetramethylrhodamine isothiocyanate. Magnification:
A, ×150;
B and
C, ×200.
Table 1.
Distribution of GRP immunoreactive nerve fibers in the mouse pancreas
Muscarinic and ganglionic antagonism in vivo. Intravenous injection of GRP (4.25 nmol/kg) elevated plasma insulin levels to 246 ± 60 pmol/l compared with 72 ± 18 pmol/l in controls (P < 0.05). The muscarinic receptor antagonist 4-DAMP (0.21 µmol/kg) inhibited this response by ~68% (P < 0.05), whereas hexamethonium (28 µmol/kg) inhibited the insulinotropic effect of GRP by 92% (P < 0.05). Also, plasma glucagon levels were increased by GRP (to 805 ± 70 pg/ml compared with 309 ± 13 pg/ml in controls; P < 0.001). This response was not significantly affected by 4-DAMP but was partially (by 48%) inhibited by hexamethonium (P < 0.05). The plasma glucose levels were not significantly affected by GRP or 4-DAMP, whereas pretreatment with hexamethonium resulted in a slight lowering of the plasma glucose levels in both controls (from 8.8 ± 0.4 to 7.3 ± 0.3 mmol/l; P < 0.01) and GRP-injected animals (from 8.9 ± 0.3 to 7.2 ± 0.3 mmol/l; P < 0.01; Fig. 2).
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These results show that GRP-stimulated insulin secretion under in vivo conditions is critically dependent on muscarinic m3-receptors and ganglionic mechanisms, whereas GRP-stimulated glucagon secretion is partially dependent on ganglionic but not on muscarinic (m3) mechanisms.
Effects of muscarinic (m3) and ganglionic blockade on insulin release from isolated pancreatic islets. To investigate whether the inhibition of GRP-stimulated insulin secretion by 4-DAMP or hexamethonium observed under the in vivo conditions was due to interaction of the receptor antagonists with islet receptors, experiments were performed on isolated mouse islets. Thereby, GRP (100 nM) was found to stimulate insulin secretion in the presence of 11.1 mM glucose (P < 0.05). 4-DAMP at the concentrations of 10 or 100 µM did not affect glucose (11.1 mM) or GRP-stimulated insulin secretion (Table 2), whereas carbachol (0.1 mM)-stimulated insulin secretion was totally abolished by the antagonist. Thus carbachol increased insulin release from 0.16 ± 0.04 to 0.75 ± 0.07 pmol/islet (P < 0.01), and 4-DAMP (100 µM) decreased this response to 0.12 ± 0.04 pmol/islet (P < 0.001). Also, hexamethonium at 10 and 100 µM did not affect the insulin response to glucose (11.1 mM) or to GRP (100 nM; Table 2). Furthermore, at 11.1 mM glucose, hexamethonium did not affect the insulinotropic effect of carbachol (0.67 ± 0.11 pmol/islet after incubation with carbachol vs. 0.67 ± 0.1 pmol/islet after incubation with carbachol + 100 µM of hexamethonium; not significant). Thus the inhibition of the insulinotropic response to GRP by 4-DAMP or hexamethonium observed under in vivo conditions was not due to a nonspecific action of these antagonists on islet GRP receptors. Furthermore, the direct islet insulinotropic effect of GRP does not require intact muscarinic (m3) or nicotinic receptors.
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DISCUSSION |
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The present study demonstrates that GRP, in addition to exerting a direct effect on the islet B cells, also stimulates insulin secretion by an indirect mechanism that is critically dependent on muscarinic and ganglionic nicotinic mechanisms but not on capsaicin-sensitive afferent nerves. We also revealed that GRP immunoreactive nerve fibers, in addition to innervating the islets also, and in fact more markedly, innervate intrapancreatic ganglia.
The involvement of muscarinic receptors in the insulinotropic effect of GRP in the mouse under in vivo conditions has previously been implicated (22). The present study confirms these previous findings and further demonstrates that the muscarinic receptor subtype involved is of the m3-subtype, which is the muscarinic receptor subtype present on the pancreatic B cells (1, 11). The fact that the insulinotropic response to GRP was found to be inhibited by ganglionic blockade by hexamethonium suggests that GRP acts at the ganglionic level to activate postganglionic cholinergic nerves innervating the islets, since hexamethonium did not affect GRP-induced insulin secretion in vitro in isolated islets.
The present study also confirmed previous studies demonstrating that GRP acts directly on the islets to stimulate insulin secretion (5, 28, 29) and further demonstrated that this effect is unaffected by muscarinic receptor blockade with 4-DAMP or by the nicotinic ganglionic receptor antagonist hexamethonium used at a concentration that has previously been shown to induce ganglionic blockade in the endocrine pancreas (27). This shows that the effects of these receptor antagonists on GRP-stimulated insulin secretion observed in vivo are not due to nonspecific actions on islet GRP receptors and that the action of GRP does not require intact muscarinic receptors at the level of the B cell plasma membrane. The failure of GRP to exert any insulinotropic effect under the in vivo conditions in the presence of hexamethonium, in spite of stimulated insulin secretion in vitro, is probably due to the islets being affected by a high concentration of GRP for a longer period of time in vitro than in vivo. Hence, the major part of the insulinotropic response to GRP under these in vivo conditions of the present study is explained by a ganglionic activation. Such a hypothesis is further supported by our immunohistochemical findings that intrapancreatic ganglia contained GRP immunoreactive nerve terminals, suggesting that the ganglia are innervated by GRP-containing nerves. Therefore, in the mouse, GRP seems to participate in the regulation of insulin release by acting as an intrapancreatic neurotransmitter both at the islet and at the ganglionic level. Previously, the pancreatic ganglia have been demonstrated to participate in the regulation of insulin release (25), and our present results show that GRP is a neurotransmitter involved in this neural regulation of insulin release.
Recently, GRP-stimulated amylase release from isolated rat pancreatic lobules was demonstrated to partially involve a cholinergic mechanism inhibited by hexamethonium, atropine, as well as tetrodotoxin (3). GRP was in that study also shown to induce the release of [3H]acetylcholine from the lobules, although at concentrations above those required for amylase release. A similar mechanism might underlie the cholinergic dependence of GRP-stimulated insulin release, i.e., GRP might facilitate the release of acetylcholine at the presynaptic ganglionic level or alternatively increase the postsynaptic sensitivity to acetylcholine, as was recently demonstrated for cholecystokinin-8 in intracellular recordings from cat pancreatic ganglion cells (19). However, the exact mechanism by which GRP affects the pancreatic ganglia has to await further studies.
Afferent neural mechanisms have previously been demonstrated to be involved in the regulation of glucose homeostasis, glucose-stimulated insulin secretion, and neuroglycopenia-induced glucagon secretion (12, 13). Furthermore, activation of capsaicin-sensitive afferent nerves has previously been suggested to be a mechanism involved in the satiety effect induced by the GRP-like peptide bombesin (18). However, the insulin or glucagon responses to GRP were in the present study demonstrated to be unaffected by capsaicin treatment. Therefore, our results demonstrate that the mechanism of GRP-stimulated insulin release does not involve capsaicin-sensitive afferent nerves.
GRP is known also to stimulate glucagon release under in vivo conditions in several species, including the mouse (22). In the present study, GRP-stimulated glucagon secretion was not affected by muscarinic antagonism by the m3-selective antagonist 4-DAMP but was partially inhibited by hexamethonium. This is in contrast to a previous study in the mouse in which methylatropine was shown to slightly diminish GRP-stimulated glucagon secretion (22). The lack of effect of 4-DAMP in the present study is unlikely to be due to the use of too low a dose level since, in the mouse, 4-DAMP inhibits carbachol-stimulated glucagon secretion at lower dose levels than insulin secretion (11). Instead, the results might imply that the cholinergic receptor involved in GRP-stimulated glucagon secretion is not of the m3 subtype. Furthermore, the fact that GRP-stimulated glucagon release is not affected by m3-blockade, whereas GRP-stimulated insulin release is, shows that the insulinotropic effect of GRP is not secondary to increased glucagon secretion. The partial inhibition by hexamethonium and the lack of effect of 4-DAMP on the glucagon response to GRP imply, besides an indirect ganglionic mechanism, a major contribution of a direct islet effect of GRP in glucagon secretion in the mouse. Indeed, GRP has previously been demonstrated to potently stimulate glucagon secretion from isolated mouse pancreatic islets (29).
We conclude that, in the mouse, GRP is confined to intrapancreatic nerves of the islets and the ganglia and that the peptide acts as an intrapancreatic neurotransmitter, stimulating insulin release both directly through an action on the islet B cell and indirectly through activation of postganglionic cholinergic nerves, presumably by an action at the ganglionic level. The findings imply that activation of the cholinergic mechanisms is a major determinant for the effect of GRP on insulin secretion under in vivo conditions.
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ACKNOWLEDGEMENTS |
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We are grateful to L. Bengtsson, L. Kvist, and I. Reimertz for technical assistance.
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FOOTNOTES |
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The work was financially supported by grants from The Swedish Medical Research Council (Grants 14X-6834 and 14X-4499), The Swedish Diabetes Association, Ernhold Lundström, Novo Nordisk, The Emma Ekstrand, Hildur and Jan Tegger, Albert Påhlssson and Crafoord Foundations, Malmö University Hospital, and by the Faculty of Medicine, Lund University, Sweden.
Address for reprint requests: S. Karlsson, The Wallenberg Laboratory, Dept. of Medicine, Malmö Univ. Hospital, S-205 02 Malmö, Sweden.
Received 18 November 1996; accepted in final form 18 September 1997.
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