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Endocrinology Vol. 143, No. 10 3717-3726
Copyright © 2002 by The Endocrine Society

ARTICLE

Islet Function Phenotype in Gastrin-Releasing Peptide Receptor Gene-Deficient Mice

Kristin Persson, Giovanni Pacini, Frank Sundler and Bo Ahrén

Departments of Medicine and Physiological Sciences, Lund University, SE-221 84 Lund, Sweden; and Institute of Systems Science and Biomedical Engineering, 1-35127 Padova, Italy

Address all correspondence and requests for reprints to: Dr. Kristin Persson, Department of Medicine, Lund University, B11 BMC, SE-221 84 Lund, Sweden. E-mail: kristin.persson{at}med.lu.se.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Gastrin-releasing peptide (GRP) is an islet neuropeptide that stimulates insulin secretion. To explore whether islet GRP contributes to neurally mediated insulin secretion, we studied GRP receptor (GRPR)-deleted mice. By using RT-PCR we showed that GRPR mRNA is expressed in islets of wild-type mice, but is lost in GRPR-deleted mice. Functional studies revealed that GRP potentiates glucose-stimulated insulin secretion in wild-type animals, but not in GRPR-deleted mice. This shows that GRPR is the receptor subtype mediating GRP-induced insulin secretion and that GRPR-deleted mice are tools for studying the physiological role of islet GRP. We found that GRPR-deleted mice display 1) augmentation of the insulin response to glucose by a mechanism inhibited by ganglionic blockade; 2) increased insulin responsiveness also to the cholinergic agonist carbachol, but not to arginine; 3) impaired insulin and glucagon responses to autonomic nerve activation by 2-deoxyglucose; 4) normal islet adaptation to high fat-induced insulin resistance and fasting; and 5) normal islet cytoarchitecture, as revealed by immunocytochemistry of insulin and glucagon. In conclusion, 1) GRPR is the receptor subtype mediating the islet effects of GRP; 2) GRP contributes to insulin secretion induced by activation of the autonomic nerves; and 3) deletion of GRPR is compensated by increased cholinergic sensitivity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE 27-AMINO ACID neuropeptide, gastrin-releasing peptide (GRP), is the mammalian counterpart of bombesin, which is an amphibian peptide initially purified from the skin of the frog Bombina bombina in the early 1970s (1). In mammals, GRP was first extracted from the porcine gut in 1979 (2) and was found to be a highly conserved peptide containing N-terminal ends identical to those of bombesin (3). It is a widely distributed neuropeptide localized especially to neurons along the gastrointestinal tract, in the pancreas, and in the brain. It has a wide range of physiological effects on behavior, digestion, and metabolism and is a trophic factor for several organs (4).

In the pancreas GRP is localized to the parasympathetic nerve fibers and nerve cell bodies within the ganglia and, to a lesser extent, to nerve terminals within the islets (5, 6). As it has been shown in the pig pancreas, GRP is released upon vagal nerve activation (5), and although its functional role in the islets has not yet been fully established, GRP stimulates insulin secretion in vivo in different species, including mice and humans (6, 7, 8, 9, 10), as well as in vitro in perfused pancreas (11, 12) and in isolated islets (6, 13, 14, 15). The stimulation of insulin secretion involves both indirect activation of ganglionic neurons and direct effects on the islets (6). GRP also stimulates glucagon secretion, as demonstrated in man, mice, and rats (8, 9, 10). Four different receptors binding bombesin-like peptides have been cloned: the GRP receptor (GRPR), which shows a high affinity for GRP, but not for neuromedin B; the neuromedin B receptor, which shows a high affinity for neuromedin B, but not for GRP; and bombesin receptor subtypes 3 and 4, whose natural ligands have not yet been established (16, 17, 18, 19, 20). In human pancreas and in rat insulin-producing INS-1 cells, GRPR has been shown to be expressed (21, 22), and in human pancreas, bombesin receptor-3 is also expressed (23). However, the receptor subtype responsible for the islet effects of GRP has not been established.

The aim of this study was first to establish whether it is the GRPR that mediates the insulinotropic action of GRP, and second to explore whether GRP has a physiological role in the regulation of islet function. Specifically, because GRP is localized to islet nerves and released from the pancreas after nerve stimulation (5, 6), we hypothesized that GRP contributes to neurally mediated insulin secretion. To that end, after establishing that mRNA for GRPR is expressed in islets, we studied genetically modified mice lacking the GRPR gene. The gene coding for GRPR is located on the X-chromosome, and a C57BL/6J strain of mice with deletion of this gene has been developed by Wada et al. (24). We examined the islet function phenotype in these mice by studying the islet cytoarchitecture and measuring the islet hormone responses to glucose, arginine, the cholinergic agonist carbachol, and the glucose analog, 2-deoxyglucose (2-DG), the latter being a model of activation of the autonomic nervous system (25). Finally, we explored the importance of GRPR for the islet adaptation to both insulin resistance, by feeding the mice a high fat diet, and to fasting.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Male mice of the C57BL/6J background hemizygous for deletion of the GRPR gene, which is located on the X chromosome, were generated by homologous recombination in embryonic stem cells as previously described (24). Disruption of the GRPR gene in mutants was confirmed by Southern hybridization with the use of tail DNA. Mutant (-/Y) and wild-type (+/Y) offspring were born at the same ratio, and GRPR-deficient mice were viable and fertile, with no abnormality observed on gross and routine histological analysis of brain, lung, and gastrointestinal tract (24). The mice were fed a standard pellet diet and tap water ad libitum. Body weight and food intake were monitored continuously. The study was approved by the ethics committee at Lund University.

RT-PCR of GRPR gene expression
Total RNA was extracted from 100 freshly isolated islets using an RNeasy mini kit (QIAGEN, Valencia, CA), and cDNA was synthesized with an enhanced avian reverse transcriptase system (Sigma, St. Louis, MO) following the supplier’s protocol. GRPR gene expression was verified by RT, followed by PCR analysis. The forward primer on exon 1 had the sequence 5'-TTC ATC TAT GTC ATCC CT GCA-3', and the reverse primer on exon 2 had the sequence 5'-ATT GTA GGC ACT CTG AAT CA-3'(MWG Biotech, Ebersberg, Germany) (24). PCR consisted of an initial denaturation cycle at 94 C for 3 min, followed by 40 cycles of annealing at 94 C for 30 sec, elongation at 60 C for 2 min, and denaturation at 72 C for 3 min. A further cycle at 72 C for 7 min finished the amplification process. PCR products were examined by electrophoresis on 2% agarose gels and assessed as a 0.6-kb band after ethidium bromide staining, compared with a 1-kb DNA ladder (Life Technologies, Inc., Invitrogen, Lidingo, Sweden).

Immunohistochemistry
Sections of pancreas from 6-month-old mice were fixed overnight by immersion in Stefanini’s solution (2% formaldehyde and 0.2% picric acid in 0.1 M PBS, pH 7.2) and thereafter rinsed repeatedly in sucrose-enriched (10%) buffer. The preparations were then frozen on dry-ice and stored at -80 C until being cut (10 µm) in a cryostat and mounted on slides. The sections were incubated with a primary antibody overnight at 4 C. The primary antibodies were an antiunconjugated human proinsulin antibody raised in guinea pigs (code 9003; dilution, 1:1280; Euro-Diagnostica, Malmo, Sweden) for insulin detection and an antiprotein-conjugated glucagon antibody raised in rabbits (code 7811; dilution, 1:5120; Euro-Diagnostica) for glucagon detection. The sections were then incubated for 1 h at room temperature with a secondary antibody, coupled to fluorescein isothiocyanate with specificity for IgG (Euro-Diagnostica; dilution, 1:80) of the primary antibody, and examined in a fluorescence microscope. Pancreata from a total of 6 mice were examined in each group, and in each pancreas 20–30 islets were examined.

In vitro assays
Pancreatic islets were isolated by collagenase digestion in Hanks’ balanced salt solution (HBSS;Sigma). In brief, after a midline laparotomy the common bile duct was ligated at the papilla vateri and cannulated. The pancreas was filled with 3 ml ice-cold HBSS supplemented with 0.4 mg/ml collagenase P (Roche Molecular Biochemicals, Mannheim, Germany) before removal and incubation at 37 C for 19 min. After washing three times in HBSS, the islets were handpicked under a stereomicroscope. The islets were incubated overnight in RPMI medium supplemented with 2.06 mmol/liter L-glutamine (Life Technologies, Inc., Taby, Sweden), 10% fetal bovine serum, 100 U/ml penicillin, and 0.5 mg/ml streptomycin (all from Kebo Laboratory, Spanga, Sweden) at 37 C in 5% CO2 air. The islets were then handpicked into HEPES medium (pH 7.36) supplemented with 0.1% human serum albumin (Sigma) and 3.3 mmol/liter glucose and preincubated for 60 min at 37 C. The medium consisted of the following: 125 mmol/liter NaCl, 5.9 mmol/liter KCl, 1.2 mmol/liter MgCl2, 1.28 mmol/liter CaCl2 (all from Sigma), and 25 mmol/liter HEPES (Roche). Groups of three islets were transferred to separate chambers containing 200 µl of the medium supplemented with glucose alone or with synthetic porcine GRP (Sigma) at various concentrations. After incubation at 37 C for 60 min, 25 µl of the medium were collected from each chamber and stored at -20 C until analysis.

Animal preparation and injected doses
The mice were anesthetized with an ip injection of midazolam (0.14 mg/mouse; Dormicum, Hoffmann-La Roche, Basel, Switzerland) and a combination of fluanison (0.9 mg/mouse) and fentanyl (0.02 mg/mouse; Hypnorm, Jansen, Beerse, Belgium). Thereafter, glucose (0.25, 0.5, or 1 g/kg; British Drug Houses, Poole, UK) alone or with addition of synthetic porcine GRP (20 nmol/kg), arginine (0.25 g/kg), the cholinergic agonist carbachol (0.53 µg/kg), the ganglionic antagonist hexamethonium (28 µmol/kg), or the glucose analog 2-DG (0.5 g/kg; all from Sigma) was injected iv in a tail vein. Blood samples were taken from the retrobulbar capillary plexus and immediately centrifuged, and plasma was removed and stored at -20 C until analysis.

Data analysis
To determine glucose tolerance, insulin and glucagon secretion, insulin sensitivity, and glucose effectiveness, the insulin, glucagon and glucose responses to iv challenges were evaluated in wild-type and GRPR-deleted mice by sampling immediately before and at various time points after injection. The net glucose elimination rate after glucose injection (KG; the glucose tolerance index) was calculated as the slope for the interval 1–20 min after glucose injection of the logarithmic transformation of the individual plasma glucose values. Insulin and glucagon secretion were evaluated from plasma insulin or glucagon concentrations either as the area under the 20 or 50 min insulin or glucagon curves (AUC; trapezoid rule) or the insulin response the first 5 min after injection of the secretagogue [acute insulin response (AIR); mean suprabasal 1- and 5-min insulin levels), or in vitro by comparing medium insulin concentrations. To verify possible relative influences on hyperinsulinemia by increased insulin secretion or reduced insulin clearance, separate experiments were performed where glucose was injected iv at 1 g/kg in anesthetized mice in which C peptide was determined instead of insulin. The rationale behind this experiment is that whereas insulin to a large degree is extracted during its first passage through the liver, hepatic C peptide extraction is negligible (26). From these experiments the acute C peptide release was calculated (mean suprabasal 1- and 5-min C peptide levels). Insulin sensitivity and glucose effectiveness were analyzed from the seven-sample iv glucose tolerance test, where glucose and insulin concentration data were analyzed with the minimal model technique. Details of the performance in mice of this widely used method and of its validation have been reported previously (27). This method provides the parameter SI (insulin sensitivity index), which is defined as the ability of insulin to enhance glucose disappearance and inhibit glucose production (28), and the parameter SG, which is the glucose effectiveness, representing glucose disappearance per se from plasma without any change in dynamic insulin (27, 29).

Islet adaptation to insulin resistance and fasting
To examine whether the adaptive islet response to insulin resistance induced by a high fat diet is dependent on GRPR, wild-type and GRPR-deleted mice were fed a high fat diet with an energy content of 23.4 kJ/g derived from 16.4% protein, 25.6% carbohydrates, and 58.0% fat. Comparison was made with mice fed a regular diet (12.6 kJ/g; 25.8% protein, 62.8% carbohydrates, and 11.4% fat; both diets from Research Diets, New Brunswick, NJ). Body weight and plasma levels of insulin, glucose, and glucagon were monitored regularly, and an iv glucose (1 g/kg) challenge was performed after 3 months in anesthetized mice as described above.

Analysis of insulin, glucagon, C peptide and glucose
Insulin was determined by RIA using guinea pig antirat insulin, 125I-labeled human insulin, and rat insulin standard (Linco Research, Inc., St. Charles, MO). Glucagon was determined by RIA using guinea pig antiglucagon specific for pancreatic glucagon, [125I]glucagon, and glucagon standard (Linco Research, Inc.). C Peptide was determined by RIA using guinea pig antirat C peptide, [125I]rat C peptide, and purified recombinant rat C peptide standard (Linco Research, Inc.). In all three RIAs, free and bound radioactivity were separated using an anti-IgG (goat antiguinea pig) antibody (Linco Research, Inc.). Plasma glucose was determined by the glucose oxidase method.

Statistics
The mean ± SEM are shown. Statistical comparisons for differences between GRPR-deleted and wild-type controls were performed by unpaired t test. The regression coefficient was determined by Pearson analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of GRPR in islets
GRPR expression in mouse islets was studied by RT-PCR. A 0.6-kb PCR product between two primers on exons 1 and 2 was detected in RNA extracted from wild-type mouse islets (Fig. 1Go), but not in islets from GRPR-deleted mice (not shown in Fig. 1Go). This shows that GRPR is expressed in islets, and that GRPR-deleted mice are tools for establishing the importance of GRPR for islet physiology.



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  		Figure 1. Expression of GRPR in islets of wild-type mice. Aliquots of total RNA extracted from wild-type mice were submitted to RT-PCR analysis using primers specific for GRPR. Portions of PCR products were resolved by agarose gel electrophoresis and stained with ethidium bromide. The migration of the product was compared with a standard 1-kB DNA ladder (left lane). The size of the product was 0.6 kb (right lane), confirming the expression of GRPR in islets.

 
Body weight, food intake, and basal plasma insulin, glucose, and glucagon
GRPR-deleted and wild-type mice were followed until 6 months of age with regular determinations of body weight, food intake, and baseline levels of insulin, glucose, and glucagon. No significant difference was observed in any of these variables between the two groups. Table 1Go shows the results in the 4-month-old animals.


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  		Table 1. Body weight; basal plasma levels of insulin, glucose, and glucagon; and food and energy intake in conscious mice, aged 4 months

 
Immunohistochemistry
The islet cytoarchitecture of GRPR-deleted and wild-type animals was examined by immunohistochemistry for visualization of insulin- and glucagon-producing cells. The topographical cytoarchitecture of the islet and islet size were the same in GRPR-deleted mice and wild-type mice (Fig. 2Go). Thus, insulin-producing ß-cells were clustered in the center of the islets surrounded by a rim of glucagon-producing {alpha}-cells in both groups of mice.



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  		Figure 2. Immunohistochemistry of insulin (top) and glucagon (bottom) cells. Right, Wild-type mice; left, GRPR-deleted mice. Similar cellular distributions of insulin and glucagon cells are evident for the two groups. Bar, 50 µm.

 
Effect of GRP on insulin secretion
In the wild-type mice (n = 8), iv GRP potentiated the insulin response to glucose, resulting in enhanced glucose elimination, whereas GRP had no effect on either insulin secretion or glucose elimination in the GRPR-deleted mice (n = 8;Fig. 3Go, A–D). Similarly, GRP potentiated glucose-stimulated insulin secretion in vitro almost 3-fold in islets from wild-type mice, but had no effect in islets isolated from GRPR-deleted mice (Fig. 3EGo). This shows that GRP-stimulated insulin secretion is mediated by the GRPR, verifying that GRPR-deleted mice are good tools for establishing the role of GRP in islet physiology.



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  		Figure 3. Effect of GRP on insulin secretion. Plasma levels of insulin and glucose before and for 50 min after the iv administration of glucose alone (1 g/kg; {circ}) or glucose with synthetic porcine GRP (20 nmol/kg; {bullet}) in wild-type mice (A and B) or GRPR-deleted mice (C and D). There were 8 mice in each group. E, Medium insulin concentration after 60-min incubation of islets isolated from wild-type ({square}) or GRPR-deleted ({blacksquare}) mice. Islets were incubated at 11.1 mmol/liter glucose with synthetic porcine GRP at different concentrations. The mean ± SEM are shown. There were 24 incubations of 3 islets each in each experimental group. Asterisks indicate the probability level of random difference between the two groups: *, P < 0.05;***, P < 0.001.

 
Glucose-stimulated insulin secretion and glucose tolerance in GRPR-deleted mice
Exogenous glucose administration induced a significantly higher insulin response in the GRPR-deleted mice (n = 50) than in the wild-type mice (n = 50;Fig. 4BGo). By 1 min after iv glucose administration, plasma insulin had risen to 1381 ± 155 pmol/liter in GRPR-deleted mice compared with only 951 ± 122 pmol/liter in wild-type animals (P =0.008), and the AUCinsulin during the 50-min study period was augmented in GRPR-deleted mice (20.7 ± 1.3 nmol/liter·50 min in GRPR-deleted mice vs. 16.1 ±1.5 nmol/liter·50 min in wild-type mice; P = 0.002). At the same time, GRPR-deleted mice displayed a markedly increased net glucose elimination rate, as shown by increased KG (2.7 ± 0.3% in GRPR-deleted mice vs. 1.8 ± 0.3%/min in wild-type mice;P = 0.009) and lowered 50 min plasma glucose levels (11.1 ± 0.5 mmol/liter in GRPR-deleted mice vs. 15.7 ± 0.9 mmol/liter in wild-type mice; P =0.006). When the seven-sample iv glucose tolerance test with minimal model analyses of the data was used in GRPR-deleted mice (n = 19) and wild-type mice (n = 16), it was found that neither the insulin sensitivity index [1.84 ± 0.32 in GRPR-deleted mice vs. 1.60 ± 0.16 10-4 min-1/(pmol/liter) in wild-type mice;P = 0.53] nor the glucose effectiveness (0.0038 ± 0.004 in GRPR-deleted mice vs. 0.052 ± 0.007 min-1 in wild-type mice; P =0.092) was significantly different between the groups. This indicates that the increased glucose tolerance in GRPR-deleted mice is due solely to an enhanced insulin response and not to changes in insulin sensitivity. The link between the increased insulin response and the increased glucose tolerance is further supported by a significant correlation between AIR and KG across all 35 animals (r = 0.535; P = 0.01;Fig. 5Go).



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  		Figure 4. Insulin and C peptide responses to iv glucose. A and B, Plasma glucose and insulin levels before and for 50 min after the iv injection of glucose (1 g/kg) in wild-type ({circ}) and GRPR-deleted mice ({bullet}). There were 25 mice in each group. The AIR (C) and acute C peptide response (ACR; D) to iv administration of glucose at 0.25, 0.5, or 1 g/kg in wild-type ({square}) and GRPR-deleted mice ({blacksquare}). C, The number of animals was 14–15 (0.25 g/kg), 9–10 (0.5 g/kg), and 50 (1 g/kg) in each group, respectively; D, the number of animals was 4–7 in each group. The mean ± SEM are shown. Asterisks indicate the probability level of random difference between the groups: *, P < 0.05;**, P < 0.01; ***, P < 0.001. The bottom of C and D displays the mean 1-min glucose levels.

 


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  		Figure 5. Linear regression between the AIR in the first 5 min after iv administration of glucose (1 g/kg) vs. the KG in GRPR-deleted mice and wild-type mice (n = 35). The regression coefficient is shown.

 
In Fig. 4DGo, the results of the experiments in which C peptide was measured instead of insulin are shown. The C peptide response to glucose was significantly higher in GRPR-deleted mice than in wild-type mice [the 5-min C peptide response was 1.35 ± 0.14 nmol/liter in GRPR-deleted mice (n = 7) vs. 0.89 ± 0.13 nmol/liter in wild-type mice (n = 6); P = 0.03]. This indicates that the hyperinsulinemia seen after iv glucose in GRPR-deleted mice is due to a potentiated glucose-stimulated insulin secretion. In contrast, when glucose was administered at the lower doses of 0.25 or 0.5 g/kg (Fig. 4CGo), or when insulin secretion was studied in isolated islets incubated in different glucose concentrations (Fig. 6Go), there was no difference in the insulin response between GRPR-deleted and control mice. This shows that the potentiated insulin secretion is an in vivo phenomenon and that a high glucose load is required for its detection.



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  		Figure 6. Islet hormone secretion in vitro. The medium insulin concentration after 60-min incubation of islets isolated from wild-type ({circ}) or GRPR-deleted mice ({bullet}) is shown. Islets were incubated in glucose at different concentrations. The mean ± SEM are shown. There were 24 incubations of 3 islets each in each experimental group.

 
Nonglucose-stimulated insulin secretion
Whereas arginine-induced insulin secretion was not significantly different between the groups [AIR, 599 ± 102 pmol/liter in GRPR-deleted mice (n = 16) vs. 525 ± 80 pmol/liter in wild-type mice (n = 16); P = 0.47], carbachol-stimulated insulin secretion was increased in GRPR-deleted mice (n = 12) compared with wild-type mice (n = 16;Fig. 7AGo). This shows that the potentiated insulin secretion in GRPR-deleted mice is not a general ß-cell phenomenon nor is it restricted to activation by glucose, but also occurs after carbachol. Plasma glucagon levels at 1 min after iv arginine was 52 ± 7 ng/liter in GRPR-deleted mice vs. 44 ± 9 ng/liter in wild-type mice (P = 0.52), and no difference was observed in response to carbachol either (Fig. 6AGo), indicating that glucagon secretion does not seem to be perturbed in GRPR-deleted mice.



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  		Figure 7. Islet hormone secretion in vivo. The AUCinsulin and AUCglucagon for 50 min after iv administration of carbachol (0.53 µg/kg; A), AIR in the first 5 min after the iv administration of glucose (1 g/kg) with concomitant administration of hexamethonium (0.28 µmol/kg; B), and AUCinsulin and AUCglucagon for 20 min after the iv administration of 2-DG (0.5 g/kg) in wild-type ({square}) and GRPR-deleted mice ({blacksquare}; C). The mean ± SEM are shown. There were 12–24 animals in each group. Asterisks indicate the probability level of random difference: *, P < 0.05; **, P < 0.01.

 
Neurally stimulated insulin secretion
The ganglionic blocker, hexamethonium, prevented the augmentation of insulin secretion that is evident in the GRPR-deleted mice, because the insulin response to glucose was not significantly different between the two groups when hexamethonium had been added [AIR, 170 ± 54 pmol/liter in GRPR-deleted mice (n = 16) vs. 157 ± 51 pmol/liter in wild-type mice (n = 16); P =0.86;Fig 7BGo]. This suggests that the augmentation of insulin secretion in GRPR-deleted mice is dependent on the autonomic nerves. When 2-DG was administered, there was an impairment in the release of both insulin and glucagon in GRPR-deleted mice (n = 24) compared with wild-type mice (n = 20), as judged by the reduced AUCinsulin (34 ± 9 nmol/liter·20 min in GRPR-deleted mice vs. 63 ± 14 nmol/liter·20 min in wild-type mice; P = 0.042) and AUCglucagon (4.1 ± 0.5 in GRPR-deleted mice vs. 7.2 ± 0.8 ng/liter·20 min in wild-type mice; P = 0.01;Fig. 7CGo). This suggests that GRP is a neurotransmitter of importance for an adequate release of insulin and glucagon after autonomic neural activation.

Islet adaptation to insulin resistance and fasting
Insulin secretion is up-regulated in insulin resistance, and we have previously shown that in the insulin resistance model of high fat-fed mice, this augmented insulin secretion is partially dependent on increased cholinergic activity (30, 31). This would imply a role of islet nerves in the islet compensation to insulin resistance. To examine whether GRP is involved in this islet compensation, we fed GRPR-deleted and wild-type mice a high fat diet to induce insulin resistance, followed by studies of the insulin response to glucose. If the augmented insulin response to insulin resistance is dependent on GRP nerves, then the insulin response to glucose should be reduced in GRPR-deleted mice fed a high fat diet. We found that the increase in body weight produced by a high fat diet was similar in GRPR-deleted mice (n = 9) and wild-type mice (n = 9;Fig. 8AGo). However, we found that also when glucose was administered iv to these mice, glucose-stimulated insulin secretion was potentiated in GRPR- deleted vs. wild-type mice. Thus, the plasma insulin level at 1 min after iv glucose was 2635 ± 466 pmol/liter in GRPR-deleted mice vs. only 1195 ± 187 pmol/liter in wild-type mice (P =0.03). By subtracting baseline insulin from these values, the 1 min insulin response to glucose was calculated. This response was augmented in GRPR-deleted mice vs. wild-type mice. This augmentation was accentuated after the high fat diet compared with that after the control diet. Thus, in mice given high fat diet, the 1-min insulin response was augmented by 362 ± 52% in GRPR-deleted mice compared with that in wild-type mice. The corresponding figure in mice given a regular diet was only 68 ± 27% (P =0.03). This shows that the GRPR is not required for an islet adaptive compensation for insulin resistance, but, instead, that islet perturbations after GRPR deletion result in augmented adaptive capacity.



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  		Figure 8. Response to iv glucose in high fat-fed mice. A, Body weight in GRPR-deleted ({bullet}) and wild-type ({circ}) mice given either the regular or high fat diet for 3 months. B, Plasma glucose (top) and insulin (bottom) levels after iv injection of glucose (1 g/kg). The mice given a regular diet are shown to the left, and those given a high fat diet are shown to the right. There were nine animals in each group.

 
Finally, the glucose, insulin, and glucagon responses to fasting were also examined in GRPR-deleted mice (n = 15) and wild-type mice (n = 15) fasted for 24 h. Table 2Go shows that changes in glucose, insulin, and glucagon levels were not different between GRPR-deleted and wild-type animals. Hence, the GRPR is not required for the islet adaptation to fasting.


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  		Table 2. Body weight and plasma levels of insulin, glucose, and glucagon before and after 24-h fasting in GRPR-deleted or wild-type mice

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
GRP is a neuropeptide in pancreatic ganglia and islets, is released from the pancreas upon vagal nerve activation, and stimulates insulin and glucagon secretion, as shown in different experimental models (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15). In the present study we used mice that were genetically deleted for the GRPR gene to identify the physiological role of GRP. The GRPR is one of the four known receptor subtypes with affinity for peptides of the bombesin peptide family (16, 17, 18, 19, 20), which, beside bombesin and GRP, also includes neuromedin B (32). Only the GRPR, however, shows a high affinity for GRP, but not for the other peptides in the family (16, 17). Previously, the GRP binding capacity of the islets has been documented (14), and the GRPR has been shown to be expressed in human pancreas (21) and clonal rat insulinoma cells (22). GRP has also previously been shown to stimulate insulin secretion by increasing cytoplasmic calcium (33, 34) through an effect dependent on protein kinase C (13, 35). However, which of the receptor subtypes mediates the insulinotropic action of GRP has not been established. We found that the GRPR is expressed in pancreatic islets. As we also found that GRP does not stimulate insulin release in GRPR-deleted mice both in vivo and in vitro, a novel conclusion from this work is that the insulinotropic action of GRP is mediated by the GRPR. The main findings in the phenotype characterization of GRPR-deleted mice were that GRPR-deleted mice displayed islets that, as shown by immunocytochemistry, had a similar cytoarchitecture topography and size as islets from wild-type mice, that GRP appears to contribute to neurally mediated insulin secretion, and that GRPR deletion is compensated by augmented insulin secretion in response to glucose.

GRPR-deleted mice showed a potentiated in vivo insulin response to iv glucose compared with wild-type mice. The increased hyperinsulinemia was due to elevated insulin secretion because the C peptide response to iv glucose was also enhanced in GRPR-deleted mice. The potentiation of glucose-stimulated insulin secretion in GRPR-deleted mice was not seen in isolated islets and was seen only when the mice were challenged by high levels of glucose. Therefore, GRPR gene deletion seems to induce compensatory mechanisms that result in augmented glucose-stimulated insulin secretion in vivo. To examine whether the augmented insulin secretion in GRPR-deleted mice is specific for stimulation by glucose, insulin secretion stimulated by the amino acid arginine or the cholinergic agonist carbachol was also examined. Both of these secretagogues stimulate insulin secretion by direct islet actions through other mechanisms than does glucose. Thus, whereas a primary effect of glucose is to close ATP-regulated K+ channels, these channels are bypassed by arginine, which, instead, directly depolarizes the plasma membrane to elicit an opening of voltage-sensitive Ca2+ channels (36). Furthermore, activation of muscarinic receptors signals a stimulated insulin secretion through activation of phospholipase C, with subsequent formation of inositol-1,4,5-trisphosphate and release of Ca2+ from intracellular stores (37). Both arginine and carbachol also stimulate glucagon secretion (38); thus, their effects on {alpha}-cell release have also been investigated. We demonstrate that in contrast to the augmented glucose-stimulated insulin secretion in GRPR-deleted mice, the insulin response to iv arginine was not increased. The potentiated insulin response to iv glucose is thus not a generally increased ß-cell responsiveness to stimulation, but is caused by specific actions elicited by glucose. However, the insulin response to carbachol, like that to glucose, was augmented in GRPR-deleted mice. This indicates that after GRPR deletion, an up-regulation of cholinergic responsiveness evolves. This hypothesis was tested by a direct approach using hexamethonium, which blocks the ganglionic activation of postganglionic nerves. Hexamethonium has previously been used in studies of islet hormone secretion in mice (6, 39). Hexamethonium prevents glucose from augmenting insulin secretion in GRPR-deleted mice if the augmentation is due to increased cholinergic activity. We found that the potentiation of glucose-stimulated insulin secretion in GRPR-deleted mice was abolished by hexaethonium. Therefore, the potentiated glucose-stimulated insulin secretion in these mice is caused by enhanced cholinergic responsiveness.

The augmented insulin secretion in insulin resistance induced by high fat feeding of mice is partially explained by increased cholinergic sensitivity (30, 31). This might be dependent on GRP acting as a neuropeptide in cholinergic nerves. To explore this possibility, we fed the GRPR-deleted mice and their wild-type counterparts a high fat diet, which previously has been shown to induce insulin resistance with islet compensation in mice (27). The novel finding here is that the potentiation of glucose-stimulated insulin secretion in GRPR-deleted mice was more pronounced after high fat feeding than in normal diet-fed mice. This shows that the islet adaptation to insulin resistance with the compensatory increase in insulin secretion is not dependent on GRPR. Instead, the results are compatible with the view that additively increased cholinergic responsiveness had occurred in the two models (GRPR deletion and high fat feeding) when they were combined. Although the mechanism of this addition needs to be examined in more detail, the results suggest that a similar mechanism has evolved in the two models.

We previously reported that GRPR-deleted mice display glucose intolerance after gastric glucose in conjunction with impaired secretion of insulin caused by a low response of the incretin hormone, glucagon-like peptide-1 (40). This long-term glucose intolerance may also be a stimulus for a compensatory mechanism to maintain normal glucose levels. It may thus be speculated that increased cholinergic hyperresponsiveness in GRPR-deleted mice is due to the long-standing glucose intolerance after feeding, and that this is an adaptive process for compensation of glucose intolerance. This would be similar in GRPR-deleted mice and other models of impaired glucose tolerance, e.g. high fat-fed mice, ob/ob mice, and fa/fa mice, which all display glucose intolerance with compensated insulin secretion and increased cholinergic sensitivity (30, 31, 41, 42, 43, 44, 45, 46, 47).

As the glucose elimination rate was augmented in GRPR-deleted mice, we also calculated insulin sensitivity and glucose effectiveness using the iv glucose tolerance test. To that end, we exploited the minimal model analysis of net glucose disposal, the use of which in mice has previously been described and validated against the hyperinsulinemic euglycemic glucose clamp (27). We found that it is the increased insulin response to iv glucose that explains the elevated glucose disposal in GRPR-deleted mice because insulin sensitivity and glucose effectiveness were not altered by GRPR deletion. This was also supported by the linear relation between AIR and KG. Hence, the importance of GRP to peripheral glucose metabolism seems to be entirely explained by its ability to stimulate insulin secretion.

A main novel finding of this study is that it appears that GRPRs are essential for normal islet hormone release after autonomic activation, which implies a role for GRP in the autonomic neural transmission involved in the regulation of islet function. We tested the hypothesis by iv administration of the glucose analog, 2-DG. Intravenous 2-DG is a model for autonomic nerve activation because the glucose analog creates neuroglycopenia by competing for glucose uptake and intracellular glucose phosphorylation (48), which leads to an activation of both sympathetic and parasympathetic nerves, the net effect being stimulation of both insulin and glucagon secretion in mice (6, 39). The results show that both insulin and glucagon responses to 2-DG were reduced in GRPR-deleted mice, which suggests that GRPR partially mediates the islet hormone response after autonomic activation. The site of action of GRP to contribute to islet hormone secretion after autonomic nerve activation cannot be established in this study, because it may be either a ganglionic or an islet effect, or even a potential central effect, where GRP may be involved in the integration of neural responses. Further studies are required to establish this mechanism.

Although not central to the present study, our results also imply that GRP is not essential for the long-term regulation of body weight or food intake, as neither of these parameters was different between GRPR-deleted and control mice. Hamptton et al. (49) previously reported that GRPR-deleted mice display loss of bombesin-induced satiety after acute administration, suggesting that the GRPR might be involved in bombesin-induced satiety. However, on a more long-term basis, as in the present study, GRPR deletion does not seem to affect food intake. This may be due to redundant mechanisms involved in the regulation of food intake, similar to the results of a study in baboons showing that chronic administration of bombesin only transiently suppressed food intake (50).

In conclusion, this phenotype characterization of GRPR-deleted mice has shown that the GRPR is the subtype of GRP-binding receptors that is of importance for GRP-stimulated insulin secretion. In addition, this receptor type is important for normal regulation of islet function, because GRPR-deleted mice display 1) augmentation of the insulin response to glucose in vivo, which is prevented by ganglionic blockade; 2) increased insulin responsiveness to carbachol, but not to arginine; and 3) and impaired insulin and glucagon responses to autonomic nerve activation. At the same time, GRPR-deleted mice display normal body weight, food intake, and adaptation to high fat-induced insulin resistance, and fasting and a topographical cytoarchitecture not different from that in wild-type mice. The results suggest that GRP contributes to neurally mediated islet hormone secretion and that deletion of GRPR is compensated by increased cholinergic sensitivity.


   Acknowledgments
 
We thank Drs. E. Wada and K. Wada (Tokyo, Japan) for providing the animal model. We are grateful to Lilian Bengtsson, Kerstin Knutsson, and Lena Kvist for expert technical assistance.


   Footnotes
 
This work was supported by grants from the Swedish Research Council (Grant 6834), the Albert Påhlsson, Crafoordska and Novo Nordisk Foundations, the Swedish Diabetes Association, and research funds at University Hospital of Lund and Faculty of Medicine, Lund University. The results of this study were partially presented at the 36th Annual Meeting of the European Association for the Study of Diabetes, Jerusalem, Israel, September 2000, and at the 37th Annual Meeting of the European Association for the Study of Diabetes, Glasgow, Scotland, September 2001.

Abbreviations: AIR, Acute insulin response; AUC, area under the curve; 2-DG, 2-deoxyglucose; GRP, gastrin-releasing peptide; GRPR, gastrin-releasing peptide receptor; HBSS, Hanks’ balanced salt solution; KG, net glucose elimination rate after glucose injection.

Received April 3, 2002.

Accepted for publication June 5, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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