Reduced GLP-1 and insulin responses and glucose intolerance after gastric glucose in GRP receptor-deleted mice

Kristin Persson1, Ronald L. Gingerich2, Sonali Nayak2, Keiji Wada3, Etsuko Wada3, and Bo Ahrén1

1 Department of Medicine, Lund University, Malmö, SE-205 02 Sweden; 2 Linco Research, St. Charles, Missouri 63301; 3 Department of Degenerative Neurological Diseases, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo 187-8502, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

By applying a newly developed ELISA technique for determining biologically active intact glucagon-like peptide [GLP-1, GLP-1-(7-36)amide] in mouse, plasma baseline GLP-1 in normal NMRI mice was found to be normally distributed (4.5 ± 0.3 pmol/l; n = 72). In anesthetized mice, gastric glucose (50 or 150 mg) increased plasma GLP-1 levels two- to threefold (P < 0.01). The simultaneous increase in plasma insulin correlated to the 10-min GLP-1 levels (r = 0.36, P < 0.001; n = 12). C57BL/6J mice deleted of the gastrin-releasing peptide (GRP) receptor by genetic targeting had impaired glucose tolerance (P = 0.030) and reduced early (10 min) insulin response (P = 0.044) to gastric glucose compared with wild-type controls. Also, the GLP-1 response to gastric glucose was significantly lower in the GRP receptor-deleted mice than in the controls (P = 0.045). In conclusion, this study has shown that 1) plasma levels of intact GLP-1 increase dose dependently on gastric glucose challenge in correlation with increased insulin levels in mice, and 2) intact GRP receptors are required for normal GLP-1 and insulin responses and glucose tolerance after gastric glucose in mice.

insulin secretion; glucose tolerance; knockout mice; glucagon-like peptide; gastrin-releasing peptide


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

INSULIN SECRETION IS REGULATED by circulating nutrients, such as glucose and amino acids, by paracrine factors produced by islet endocrine cells, by autonomic nerves innervating the islets, and by gut hormones released during food intake (2). The integrative action of these factors enables the beta -cells to secrete insulin in an optimal fashion, thereby preventing glucose from deviating to hyper- or hypoglycemic levels. This interplay between several factors is particularly important during food intake, when, besides the autonomic (parasympathetic) nerves and ingested and absorbed nutrients, gut hormones, the so-called incretin factors, are relevant (13). One important gut hormone in this respect is glucagon-like peptide 1 (GLP-1), which is released from the intestinal L cells during food intake and which augments glucose-stimulated insulin secretion (1).

GLP-1 is stored in the secretory granules of the L cells, which are located mainly in the distal portion of the small intestine and in the colon (9). The peptide is released into the bloodstream when the cells are activated. The most important stimulus for secretion of GLP-1 is ingestion of a mixed meal or oral ingestion of glucose (3, 24, 28). The main activator of the L cells is enteral glucose reaching the small intestine, because the secretion of GLP-1 correlates with the gastric emptying rate (28), and enteral glucose stimulates GLP-1 secretion in the isolated perfused canine ileum (36). However, intestinal hormones and neurotransmitters also affect GLP-1 secretion from the intestine. Thus it has been shown in rat L cells that GLP-1 secretion is stimulated by gastric inhibitory polypeptide (GIP), gastrin-releasing peptide (GRP), and the muscarinic agonist bethanechol and is inhibited by somatostatin (6, 16, 31). Furthermore, in the pig ileum, GLP-1 secretion is stimulated by GRP, substance P, and neurokinin A, whereas the secretion is inhibited by adrenergic nerve activation (17). The regulation of GLP-1 secretion is important to establish, because GLP-1 release seems to be impaired in fully developed type 2 diabetes (37). Furthermore, GLP-1 is required for normal glucose tolerance, because GLP-1 receptor antagonism by exendin results in glucose intolerance after oral glucose in rats and humans (8, 23, 38) and because mice lacking the GLP-1 receptor display glucose intolerance after oral glucose (32). Improvement of GLP-1 release, therefore, might be a feasible mode for the treatment of diabetes in conjunction with the use of exogenous administration of the peptide (1). To enforce such a strategy, however, a prerequisite is more detailed knowledge of the regulation of endogenous GLP-1 levels.

In this study, we examined whether the neuropeptide GRP is important for the circulating levels of GLP-1. GRP is the mammalian homolog to the amphibian peptide bombesin and consists of a 27-amino acid residue that was initially isolated from the porcine gastrointestinal tract (26). The peptide has been shown to be a neuropeptide in the gastrointestinal tract and thereby is located in intrinsic neurons distributed from the fundus to the distal colon (29). In the gastrointestinal tract, a variety of actions of GRP have been documented, like stimulation of gastrin and pepsinogen secretion, and actions on motility (26, 33, 35). GRP seems also to be involved in the local regulation of GLP-1 secretion, because, in rat L cells and pig ileum preparations, the peptide stimulates the secretion of GLP-1 (6, 16, 17), and, in the anesthetized rat, GRP potentiates GLP-1 release induced by intraduodenal fat, and a GRP antagonist abolishes the GLP-1 response to duodenal fat (31). GRP is, however, also of relevance for insulin secretion and glucose tolerance as an islet neuropeptide, because it has been localized at islet nerve terminals to be released from the pancreas during vagal nerve activation and to stimulate insulin secretion both in vivo and in vitro through a direct action on the islet beta -cells (2, 11, 19, 20, 22, 30). Therefore, the insulinotropic action of GRP might be due to both a direct islet action of the peptide and an indirect action through the release of GLP-1.

The bombesin-like peptides, like GRP, bind to G protein-coupled receptors on the cell surface, of which three have been cloned: the GRP receptor, the neuromedin B receptor, and the bombesin receptor subtype 3 (4, 10, 40). Recently, GRP receptor-deleted mutant mice were generated by gene targeting (14, 39). These mice were shown to display increased locomotor activity during the dark period and increased social responses against an intruder but otherwise normal development (39). Furthermore, the mice exhibited loss of bombesin-induced suppression of feeding (14). Moreover, these mice displayed normal baseline values of glucose and insulin (39). This does not exclude, however, involvement of GRP and the GRP receptor in the homeostasis of insulin secretion and glucose tolerance after food intake. In this study, therefore, we have examined the relevance of the GRP receptor for GLP-1 and insulin responses to enteral administration of glucose by giving gastric glucose gavage to mice lacking the GRP receptor.

For these studies, we applied an ELISA technique for the nonradioactive quantification of the biologically active GLP-1 to studies in mouse plasma. It is known that active GLP-1 [i.e., GLP-1-(7-36)amide] is rapidly inactivated after its release from the intestinal L cells by the enzyme dipeptidyl peptidase IV (DPP IV) (15, 27). This enzyme truncates GLP-1 by liberating the two NH2-terminal amino acids (histidine-alanine), thereby yielding GLP-1-(9-36)amide, which is not only an inactive metabolite but may also function as an inhibitor of GLP-1 (15, 17, 21, 41). Due to this rapid inactivation, the half-life of active GLP-1-(9-36)amide is only 1-1.5 min (7), and only ~20% of total GLP-1 immunoreactivity consists of the active GLP-1 (12). Assays using an antibody directed to the NH2-terminal end of GLP-1, however, allow direct conclusions concerning the active form of GLP-1.

The aims of the study were, therefore, 1) to characterize the ELISA technique for measuring biologically active GLP-1 in mice and 2) to explore the relevance of the GRP receptor to GLP-1 and insulin responses and glucose tolerance after gastric glucose gavage in mice.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. For the characterization of the GLP-1 assay in vivo and the study on the dose-response relationship between gastric glucose and plasma GLP-1, normal female NMRI mice weighing 20-25 g were used. These mice were obtained from Bomholdtgaard Breeding and Research Center, Ry, Denmark. For the studies on the involvement of the GRP receptor in the regulation of plasma GLP-1, male GRP receptor-deleted mutant mice and their wild-type littermates were used. The GRP receptor gene is located on chromosome X in both mice and humans (25). Male mice of the C57BL/6J background hemizygous for deletion of the GRP receptor gene were generated by homologous recombination in embryonic stem cells as previously described (39). The disruption of the GRP receptor gene in mutant was confirmed by Southern hybridization with the use of tail DNA. As previously demonstrated, mutant (-/Y) and wild-type (+/Y) offspring were born at the same ratio, and GRP receptor-deficient mice were viable and fertile, with no abnormality observed on gross and routine histological analysis in the brain, lung, and gastrointestinal tract (39). All animals in the study were fed a standard pellet diet and tap water ad libitum. The study was approved by the Ethics Committe of Lund University.

Gastric glucose tolerance test. The mice were fasted for 2 h and then anesthetized with an intraperitoneal injection of midazolam (Dormicum, Hoffman-LaRoche, Basel, Switzerland, 0.4 mg/mouse) and a combination of fluanison (0.9 mg/mouse) and fentanyl (0.02 mg/mouse; Hypnorm, Janssen, Beerse, Belgium). At 30 min after induction of anesthesia, a blood sample (75 or 150 µl) was taken from the retrobulbar intraorbital capillary plexus, whereafter D-glucose (17, 50, or 150 mg/mouse dissolved in 0.5 ml saline) or saline was administered through a gavage tube (OD 1.2 mm) placed in the stomach. New blood samples were taken after 10, 30, and 60 min. The samples were taken in heparinized tubes and stored on ice. After centrifugation, plasma was separated and stored at -20°C until analysis.

Assay of GLP-1. An ELISA was recently developed to measure biologically active GLP-1 (GLP-1-(7-36)amide) in plasma (Linco Research, St. Charles, MO). The assay uses monoclonal guinea pig antibodies specific for the NH2- and COOH-terminal ends of GLP-1. The assay, therefore, does not react with its inactivation product, GLP-1-(9-36)amide. Synthetic GLP-1 (Peninsula Labs, Merseyside, UK) was used as a calibrator (2, 5, 10, 20, 50, and 100 pmol/l). The assay was performed as described in the kit protocol (Linco). Recovery of different amounts of synthetic GLP-1 (Peninsula; 2-100 pmol/l) added to a mouse plasma pool (with baseline GLP-1 concentration of 4 pmol/l) is shown in Table 1. Recoveries ranged from 85 to 103% over a range of 4 to 108 pmol/l. Furthermore, by use of 100, 50, and 25 µl of plasma in the assay, measures were revealed in the range from 67 to 120% of expected values (Table 2). Intra- and interassay variations were assessed by repeated analysis of four plasma samples containing from 4.8 to 72.3 pmol/l GLP-1. Coefficients of variation (CV) ranged from 6.4 to 7.7% within runs and from 1.5 to 9.1% between runs. In the assay in the following experiments reported, 50 µl of plasma were used.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Recovery of GLP-1 in mouse plasma


                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Dilution of mouse plasma and determination of GLP-1

Assay of insulin and glucose. Plasma insulin was determined radioimmunochemically with the use of a guinea pig anti-rat insulin antibody, 125I-labeled human insulin as tracer, and rat insulin as standard (Linco; 25 µl plasma). Free and bound radioactivity were separated by use of an anti-IgG (goat anti-guinea pig) antibody (Linco); the sensitivity of the assay is 17 pmol/l, and the CV is <3%. Plasma glucose was determined with the glucose oxidase method (10 µl plasma).

Statistics. Values shown are means ± SE unless otherwise stated. Statistical analyses were performed with the SPSS for Windows system. Statistical comparisons between groups were performed with Student's unpaired t-test with Bonferroni post hoc correction for multiple comparisons and with ANOVA. Pearson's product-moment correlation coefficients were obtained to estimate linear correlation between variables, and forward stepwise multiple linear regression was applied for determination of dependency of influences between significantly related variables. Normality of distribution was tested with the Kolmogorov-Smirnov goodness-of-fit test.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Baseline GLP-1, glucose, and insulin in mice. Figure 1 shows the pattern of distribution of baseline GLP-1 concentration in 72 female NMRI mice subjected to a standardized 2-h fasting period. It is seen that fasting GLP-1 displayed a normal distribution pattern with a tendency toward a distribution skewed to the right (P = 0.070 according to the Kolmogorov-Smirnov goodness-of-fit test). The mean of the distribution of plasma GLP-1 was 4.5 pmol/l (SD 2.1 pmol/l, SE 0.3 pmol/l). In 42 of the mice, glucose and insulin were also determined in the same samples. In these 42 mice, plasma GLP-1 was 4.5 ± 0.2 pmol/l, plasma glucose was 9.2 ± 0.2 mmol/l, and plasma insulin was 293 ± 25 pmol/l. There was no significant correlation between baseline GLP-1 and baseline insulin [r = -0.10, not significant (NS)] or between baseline GLP-1 and baseline glucose (r = -0.14, NS). Therefore, within the narrow range of baseline levels, GLP-1 did not correlate with insulin and glucose levels.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 1.   Distribution of baseline plasma glucagon-like peptide 1 (GLP-1) in 2-h-fasted female NMRI mice (n = 72).

GLP-1, insulin, and glucose responses to gastric glucose in mice. Figure 2 shows the GLP-1, glucose, and insulin responses to a gastric challenge of various amounts of glucose or saline in 2-h-fasted female NMRI mice. It is seen that, after gastric administration of glucose at 150 mg, plasma GLP-1 levels had significantly increased to 11 ± 0.8 pmol/l (n = 31, P < 0.001) after 10 min, i.e., approximately threefold (P = 0.009). Plasma GLP-1 levels were also significantly elevated at 30 min (P = 0.018) but had returned to baseline levels at 60 min. Like baseline GLP-1, stimulated GLP-1 levels also displayed a normal distribution pattern (P = 0.105 according to the Kolmogorov-Smirnov goodness-of-fit test). After the challenge by 50 mg of glucose, only the 10-min GLP-1 levels were significantly elevated above baseline (P = 0.002), and after the challenge by 17 mg of glucose, no significant changes in plasma GLP-1 levels were seen. Glucose levels were markedly increased after the challenge of 150 and 50 mg glucose (P < 0.001 for both) but were not significantly elevated after the 17-mg glucose challenge. Plasma insulin levels were markedly increased after the challenge of 150 and 50 mg of glucose and also increased at 10 min (640 ± 82 pmol/l vs. 282 ± 29 pmol/l at baseline, P = 0.003) after the challenge by 17 mg of glucose. GLP-1, glucose, or insulin levels were not significantly altered after administration of saline.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 2.   Plasma levels of GLP-1 (top), glucose (middle), and insulin (bottom) immediately before and at 10, 30, 60, and 120 min after gastric administration of glucose (17, 50, or 150 mg/mouse) or saline in 2-h-fasted anesthetized female NMRI mice. Values are means ± SE. Asterisks indicate the probability level of random difference for each value vs. baseline value: * P < 0.05, ** P < 0.01, *** P < 0.001; n = 12 animals in each group.

Comparison of GLP-1, glucose, and insulin responses to gastric glucose. Figure 3 compares the increase in the three variables at 10 min after administration of glucose at the three doses or saline in percentage of the maximal increase. It is seen that, except for the absence of any effect on plasma GLP-1 and glucose after administration of 17 mg of glucose, the three variables increased in parallel and in linearity to the logarithmically transformed glucose dose. To compare the relative importance of the increase in GLP-1 vs. glucose for the increase in insulin after gastric glucose, univariate and multiple correlations between these variables were performed. It was found that the increase in plasma insulin at 10 min (i.e., the Delta  value obtained by subtracting the baseline insulin value from the 10-min value) across all animals (n = 58) subjected to the gastric gavage (regardless of glucose dose or saline given) correlated significantly with the 10-min values of both glucose (r = 0.36, P = 0.011) and GLP-1 (r = 0.36; P = 0.017). This suggests that both glucose and GLP-1 are determinators of the increase in insulin after gastric glucose. To determine whether glucose and GLP-1 are independent predictors of the rise in plasma insulin after gastric glucose, a forward stepwise multiple linear regression model was performed, with the increase in plasma insulin at 10 min as the dependent variable and the 10-min values of plasma glucose and GLP-1 as independent variables. It was found that only the GLP-1 level was an independent predictor of insulin (r = 0.36; P = 0.017), whereas in this model, the 10-min glucose value was not significantly independently related to insulin (r = 0.22, NS).


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 3.   Change in plasma levels of GLP-1, glucose, and insulin during the first 10 min after gastric administration of glucose (17, 50, or 150 mg/mouse) or saline in 2-h-fasted anesthetized female NMRI mice in percentage of change after administration of 150 mg of glucose in relation to administered amount of glucose (x-scale is logarithimically transformed for the 3 glucose doses). Values are means ± SE.

GLP-1, glucose, and insulin responses to gastric glucose in GRP receptor deleted mice. To study whether intact GRP receptors are required for the GLP-1, glucose, and insulin responses to enteral administration of glucose, glucose (150 mg) was administered gastrically to GRP receptor-deleted C57BL/6J mice and their wild-type controls. It was found (Fig. 4) that the GLP-1 response to gastric glucose administration was reduced compared with the controls, because the 10-min GLP-1 value was significantly lower (P = 0.045). Glucose levels were higher in GRP receptor-deleted mice than in controls at 30 min after glucose administration (P = 0.030), whereas plasma insulin levels were significantly lower at 10 min (P = 0.044). Thus GRP receptor-deficient mice displayed inhibited GLP-1 response to gastric glucose in conjunction with impaired insulin secretion and glucose intolerance. Analyzing the entire data pool with ANOVA revealed significantly lower GLP-1 (P = 0.036) and insulin (P = 0.022) responses to gastric glucose in the GRP receptor-deleted mice than in the controls.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 4.   Plasma levels of GLP-1 (top), glucose (middle), and insulin (bottom) immediately before and at 10, 30, and 60 min after gastric administration of glucose (150 mg/mouse) in 2-h-fasted anesthetized male GRP receptor-deleted C57BL/6J mice or their wild-type controls. Values are means ± SE. Asterisks indicate the probability level of random difference between the groups: * P < 0.05; n = 10 GRP receptor-deleted animals, n = 9 controls.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we have applied a newly developed ELISA technique for measurement of biologically active GLP-1 in mice. This assay will allow investigations of factors involved in the regulation of this important incretin hormone in experimental mouse models. A monoclonal antibody used in the assay is reacting specifically with the NH2-terminal portion of GLP-1, and therefore, the antibody recognizes the biologically intact GLP-1-(7-36)amide, but not the main metabolite of the peptide, GLP-1-(9-36)amide, which is formed by truncation of GLP-1 through activity of the enzyme DPP IV (15, 17, 27). It has previously been shown that active intact GLP-1 contributes only by ~20% to total immunoreactive GLP-1 (12). Therefore, NH2-terminally directed assays determine the biologically active form of GLP-1, although they measure only a fraction of the total GLP-1 in plasma. The ELISA assay that we used in the present study is not only specific for the intact GLP-1 because it uses an NH2-terminally directed antibody; it also shows high recovery (>90%) and sensitivity (2 pmol/l), enabling mouse GLP-1 to be determined both under baseline conditions and after stimulation of GLP-1 secretion. The assay also shows low intra- and interassay variability. We found that the plasma concentration of active GLP-1 in the basal state in the mice was 4.5 pmol/l, which is within the same range as that previously determined in humans (3). We also found that plasma GLP-1 was increased when glucose was administered through a gastric tube. A twofold increase was observed after administration of 50 mg of glucose and a threefold increase when 150 mg of glucose were administered. The maximal increase was observed at 10 min after gastric glucose administration, illustrating the rapid secretory response in the L-cells to ingested nutrients. The glucose levels at 10 min after glucose administration did not differ in mice given 50 vs. 150 mg of glucose, yet the GLP-1 levels were more markedly elevated after 150 mg of glucose. This shows that plasma glucose levels do not contribute to the degree of the GLP-1 response, which confirms previous results that the main stimulus for GLP-1 secretion is the enteric presentation of glucose (1, 28, 36). The rapid GLP-1 response is, however, not due to the presence of nutrients close to the L cells, because passage of nutrients to distally located cells requires a longer period of time. Rather, the rapid GLP-1 response seems mediated by nerves that are activated by nutrient ingestion, which innervate the L cells (1). The impairment of the GLP-1 response to gastric glucose in the GRP receptor-deleted mice indicates, furthermore, that GRP nerves are involved in this action. In fact, previous studies in rat L cells and in the perfused pig ileum have shown that exogenous administration of GRP stimulates the secretion of GLP-1 (6, 16, 17), and, in the anesthetized rat, a GRP antagonist abolishes the GLP-1 response to duodenally introduced fat (31). Thus, because GRP is a gut neurotransmitter (29), a neural GRP-ergic action seems to be responsible for at least part of the rapid GLP-1 response to food intake, and our study also shows that this is executed through the GRP receptors.

The increase in plasma insulin after gastric glucose administration was directly proportional to the glucose load, displaying a straight linear relation with the logarithmic transformation of the glucose dose. The 10-min increase in plasma insulin correlated with the 10-min values in both GLP-1 and glucose, showing that both of these variables contribute to the insulin response. Interestingly, however, a forward stepwise multiple regression model including both GLP-1 and glucose levels as independent variables showed that the increase in insulin was significantly dependent only on the GLP-1 levels and not on the glucose levels. This shows that, under these conditions, GLP-1 is of greater importance than glucose for eliciting a graded insulin response. This is also evident considering that the increase in plasma glucose at 10 min after glucose administration was the same regardless of administration of 50 or 150 mg of glucose, yet the insulin response was augmented at 150 vs. the response at 50 mg of glucose (as was the GLP-1 response), illustrating that GLP-1 is an important incretin factor in mice. It may be hypothesized that glucose initiates secretion of insulin by the beta -cells but that the fine tuning of the degree of stimulation is governed by the increase in GLP-1 levels. Previously, an important incretin action of GLP-1 was suggested in rats and humans, because exendin, a specific GLP-1 receptor antagonist, prevents the insulin response to oral glucose (8, 23, 38) and in GLP-1 receptor-deleted mice, which exhibit impaired insulin response to oral glucose (32).

In this study, we also found that glucose tolerance after gastric glucose administration was slightly impaired in GRP receptor-deleted mice, accompanied by an impaired early (10 min) insulin response. This shows that intact GRP receptors are required for a normal insulin response and glucose tolerance after enteral glucose, supporting a role for GRP in the prandial insulin response. Previously, GRP was shown to be localized at nerve terminals in the islets (29) and to be released from the pancreas by vagal nerve activation (22). GRP has also been shown to stimulate insulin secretion in mice (19, 30). This shows that GRP is a parasympathetic islet neurotransmitter, and because the parasympathetic nerves have been shown to be important for the normal insulin response to food intake, i.e., the so-called cephalic phase of insulin secretion (5, 34), the impaired insulin response to gastric glucose in our present study would at first impression indicate that GRP participates in this process by being released from islet nerve terminals and then directly stimulating insulin secretion from the islet beta -cells. However, the present study offers an alternative suggestion, because the GRP receptor-deleted mice were found to exhibit impaired GLP-1 response to the gastric glucose administration. Hence, the impairment of the GLP-1 response to gastric glucose in the GRP receptor-deleted mice may explain the impaired insulin response. The relative contribution of this effect vs. impairment of local islet neurotransmitter action for the impaired insulin response to gastric glucose in GRP receptor-deleted mice remains to be established.

Previously, the GRP receptor was suggested to be involved in behavioral physiology, because GRP receptor-deleted mice were shown to display increased locomotor activity during the dark period and increased social responses against an intruder but showed otherwise normal development (39). Furthermore, GRP receptor-deleted mice have also been shown to exhibit abnormal feeding behavior, because the bombesin-induced suppression of feeding was absent (14). The present study suggests that another function of the GRP receptor is its involvement in the regulation of the GLP-1 and insulin responses to enteral administration of glucose. In fact, based on the results of this study of lowered plasma GLP-1 and insulin levels and glucose intolerance after gastric glucose in the GRP receptor-deleted mice, we suggest that GRP is involved in the regulation of glucose intolerance. This may be caused both by a local islet neural effect and by regulation of GLP-1 secretion. The relative contribution of the impaired GLP-1 response vs. impairment by intra-islet neural action of GRP in the GRP receptor-deficient mice remains to be established.

In conclusion, besides showing that the ELISA GLP-1 assay is possible to use in studies on the regulation of circulating GLP-1 in mice, this study shows that 1) enteral presentation of glucose increases plasma levels of intact GLP-1 in mice; 2) a normal GLP-1 response to enteral glucose requires intact GRP receptors; and 3) a normal early insulin response to enteral glucose requires intact GRP receptors in mice.


    ACKNOWLEDGEMENTS

The authors are grateful to Lilian Bengtsson and Ragnar Alm for expert technical assistance in the determination of GLP-1, to Lena Kvist for expert and active participation in the execution of the in vivo experiments, and to Ulrika Gustavsson for expert technical assistance in the determinations of glucose.


    FOOTNOTES

The study was supported by the Swedish Medical Research Council (Grant no. 14X-6834), the Ernhold Lundström, Albert Påhlsson, and Novo Nordisk Foundations, the Swedish Diabetes Association, Malmö University Hospital, and the Faculty of Medicine, Lund University.

Address for reprint requests and other correspondence: B. Ahrén, Dept. of Medicine, Malmö Univ. Hospital, SE-205 02 Malmö, Sweden (E-mail: Bo.Ahren{at}medforsk.mas.lu.se).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 10 February 2000; accepted in final form 23 May 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Ahrén, B. Glucagon-like peptide 1 (GLP-1)---a gut hormone of potential interest in the treatment of diabetes. Bioessays 20: 642-651, 1998[ISI][Medline].

2.   Ahrén, B. Potentiators and inhibitors of insulin secretion. In: Advances in Molecular and Cell Biology, vol. 29: The Biology of the Pancreatic beta -Cell, edited by Howell SL. Greenwich, CT: Jai, 1999, p. 175-197.

3.   Ahrén, B, Larsson H, and Holst JJ. Reduced gastric inhibitory polypeptide but normal glucagon-like peptide 1 response to oral glucose in postmenopausal women with impaired glucose tolerance. Eur J Endocrinol 137: 127-131, 1997[ISI][Medline].

4.   Battey, JF, Way J, Corjay MH, Shapira H, Kusano K, Harkins R, Wu JM, Slattery T, Mann E, and Feldman RI. Molecular cloning of the bombesin/gastrin releasing peptide receptor from Swiss 3T3 cells. Proc Natl Acad Sci USA 88: 395-399, 1991[Abstract].

5.   Berthoud, HR, Bereiter DA, Trimble ER, Siegel EG, and Jeanrenaud B. Cephalic phase, reflex insulin secretion. Neuroanatomical and physiological characterization. Diabetologia 20: 393-401, 1981[ISI][Medline].

6.   Brubaker, PL. Regulation of intestinal proglucagon-derived peptide secretion by intestinal regulatory peptides. Endocrinology 128: 3175-3182, 1991[Abstract].

7.   Deacon, CF, Pridal L, Klarskov L, Olesen M, and Holst JJ. Glucagon-like peptide 1 undergoes differential tissue-specific metabolism in the anesthetized pig. Am J Physiol Endocrinol Metab 271: E458-E464, 1996[Abstract/Free Full Text].

8.   Edwards, CM, Todd JF, Mahmoudi M, Wang Z, Wang RM, Ghatei MA, and Bloom SR. Glucagon-like peptide 1 has a physiological role in the control of postprandial glucose in humans: studies with the antagonist exendin 9-39. Diabetes 48: 86-93, 1999[Abstract].

9.   Eissele, R, Göke R, Willemer S, Harthus HP, Vermeer H, Arnold R, and Göke B. Glucagon-like peptide 1 cells in the gastrointestinal tract and pancreas of rat, pig, and man. Eur J Clin Invest 22: 283-291, 1992[ISI][Medline].

10.   Fathi, A, Corjay MH, Shapira H, Wada E, Benya R, Jensen R, Viallet J, Sausville EA, and Battey JF. A novel bombesin receptor subtype selectively expressed in testis and lung carcinoma cells. J Biol Chem 268: 5979-5984, 1993[Abstract/Free Full Text].

11.   Gregersen, S, and Ahrén B. Studies on the mechanisms by which gastrin releasing peptide stimulates insulin secretion from mouse islets. Pancreas 12: 48-57, 1996[ISI][Medline].

12.   Gutniak, MK, Larsson H, Heiber SJ, Juneskans OT, Holst JJ, and Ahrén B. Potential therapeutic levels of glucagon-like peptide-1 achieved in humans by a buccal tablet. Diabetes Care 19: 843-848, 1996[Abstract].

13.   Habener, JF. The incretin concept and its relevance to diabetes. Endocrinol Metab Clin North Am 22: 775-794, 1993[ISI][Medline].

14.   Hampton, LL, Ladenheim EE, Akeson M, Way JM, Weber HC, Sutliff VE, Jensen RT, Wine LJ, Arnheiter H, and Battey JF. Loss of bombesin-induced feeding suppression in gastrin-releasing peptide receptor-deficient mice. Proc Natl Acad Sci USA 95: 3188-3192, 1998[Abstract/Free Full Text].

15.   Hansen, L, Deacon CF, Ørskov C, and Holst JJ. Glucagon-like peptide-1-(7-36)amide is transformed to glucagon-like peptide-1-(9-36)amide by dipeptidyl peptidase IV activity in the capillaries supplying the L-cells of the porcine intestine. Endocrinology 140: 5356-5363, 1999[Abstract/Free Full Text].

16.   Herrman-Rinke, C, Voge A, Hess M, and Göke B. Regulation of glucagon-like peptide-1 secretion from rat ileum by neurotransmitter and peptides. J Endocrinol 147: 25-31, 1995[Abstract].

17.   Holst, JJ. Enteroglucagon. Annu Rev Physiol 59: 257-271, 1997[ISI][Medline].

18.   Holst, JJ, and Deacon CF. Inhibition of the activity of dipeptidyl-peptidase IV as a treatment for type 2 diabetes. Diabetes 47: 1663-1670, 1998[Abstract].

19.   Karlsson, S, Sundler F, and Ahrén B. Insulin secretion by gastrin-releasing peptide in mice: ganglionic versus direct islet effect. Am J Physiol Endocrinol Metab 274: E124-E129, 1998[Abstract/Free Full Text].

20.   Kloss, H, Wahl MA, Neye H, and Verspohl EJ. Modulation of gastrin-releasing peptide (GRP) receptors in insulin secreting cells. Cell Biochem Funct 17: 229-236, 1999[ISI][Medline].

21.   Knudsen, LB, and Pridal L. Glucagon-like peptide-1 (9-36)amide is a major metabolite of glucagon-like peptide-1 (7-36)amide after in vivo administration to dogs, and it acts as an antagonist on the pancreatic receptor. Eur J Pharmacol 318: 429-435, 1996[ISI][Medline].

22.   Knuhtsen, S, Holst JJ, Baldissera FG, Skak-Nielsen T, Poulsen SS, Jensen SL, and Nielsen OV. Gastrin-releasing peptide in the porcine pancreas. Gastroenterology 92: 1153-1158, 1987[ISI][Medline].

23.   Kolligs, F, Fehmann HC, Göke R, and Göke B. Reduction of the incretin effect in rats by the glucagon-like peptide 1 receptor antagonist exendin (9-39)amide. Diabetes 44: 16-19, 1995[Abstract].

24.   Kreyman, B, Ghatei MA, Williams G, and Bloom SR. Glucagon-like peptide-1 7-36: a physiological incretin in man. Lancet 2: 1300-1303, 1987[ISI][Medline].

25.   Maslen, GL, and Boyd Y. Comparative mapping of the Grpr locus on the X chromosomes of man and mouse. Genomics 17: 106-109, 1993[ISI][Medline].

26.   McDonald, TJ, Jörnvall H, Nilsson G, Vagne M, Ghatei M, and Bloom SR. Characterization of a gastrin releasing peptide from porcine non-antral gastric tissue. Biochem Biophys Res Commun 90: 227-233, 1979[ISI][Medline].

27.   Mentlein, R. Dipeptidyl-peptidase IV (CD26)---role in the inactivation of regulatory peptides. Regul Pept 85: 9-24, 1999[ISI][Medline].

28.   Miholic, J, Ørskov C, Holst JJ, Kotzer H, and Meyer HJ. Emptying of the gastric substitute, glucagon-like peptide-1 (GLP-1), and reactive hypoglycemia after total gastrectomy. Dig Dis Sci 36: 1361-1370, 1991[ISI][Medline].

29.   Moghimzadeh, E, Ekman R, Håkanson R, Yanaihara N, and Sundler F. Neuronal gastrin-releasing peptide in the mammalian gut and pancreas. Neuroscience 10: 553-563, 1983[ISI][Medline].

30.   Pettersson, M, and Ahrén B. Gastrin-releasing peptide (GRP): effects on basal and stimulated insulin and glucagon secretion in the mouse. Peptides 8: 55-60, 1987[ISI][Medline].

31.   Roberge, JN, Gronau KA, and Brubaker PL. Gastrin-releasing peptide is a novel mediator of proximal nutrient-induced proglucagon-derived peptide secretion from the distal gut. Endocrinology 137: 2383-2388, 1996[Abstract].

32.   Scrocchi, LA, Brown TJ, MacLusky N, Brubaker PL, Auerbach AB, Joyner AL, and Drucker DJ. Glucose intolerance but normal satiety in mice with a null mutation in the glucagon-like peptide 1 receptor gene. Nat Med 2: 1254-1258, 1996[ISI][Medline].

33.   Skak-Nielsen, T, Holst JJ, Christensen JD, and Fjalland B. Role of gastrin-releasing peptide in pepsinogen secretion from the isolated perfused rat stomach. Regul Pept 23: 95-104, 1988[ISI][Medline].

34.   Strubbe, JH. Parasympathetic involvement in rapid meal-associated conditioned insulin secretion in the rat. Am J Physiol Regulatory Integrative Comp Physiol 263: R615-R618, 1992[Abstract/Free Full Text].

35.   Sugano, K, Park J, Soll AH, and Yamada T. Stimulation of gastrin release by bombesin and canine gastrin-releasing peptides. Studies with isolated canine G cells in primary culture. J Clin Invest 79: 935-942, 1987[ISI][Medline].

36.   Sugiyama, K, Manaka H, Kato T, Yamatani K, Tominaga M, and Sasaki H. Stimulation of truncated glucagon-like peptide-1 release from the isolated perfused canine ileum by glucose absorption. Digestion 55: 24-28, 1994[ISI][Medline].

37.   Toft-Nielsen, MB, Damholt MB, Hilsted L, Hughes T, Krarup T, Madsbad S, and Holst JJ. Glucagon-like peptide-1 (GLP-1) secretion is decreased in type II diabetic patients compared to matched control subjects with normal glucose tolerance (Abstract). Diabetes 48, Suppl1: 22, 1999.

38.   Tseng, CC, Zhang XY, and Wolfe MM. Effect of GIP and GLP-1 antagonists on insulin release in the rat. Am J Physiol Endocrinol Metab 276: E1049-E1054, 1999[Abstract/Free Full Text].

39.   Wada, E, Watase K, Yamada K, Ogura H, Yamano M, Imomata Y, Eguchi J, Yamamoto K, Sunday ME, Maeno H, Mikoshiba K, Ohki-Hamazaki H, and Wada K. Generation and characterization of mice lacking gastrin-releasing peptide receptor. Biochem Biophys Res Commun 239: 28-33, 1997[ISI][Medline].

40.   Wada, E, Way J, Shapira H, Kusano K, Lebaq-Verheyden AM, Coy D, Jensen R, and Battey J. cDNA cloning, characterization and brain region-specific expression of neuromedin B-preferring bombesin receptor. Neuron 6: 421-430, 1991[ISI][Medline].

41.   Wettergren, A, WØjdemann M, and Holst JJ. Glucagon-like peptide-1 (7-36)amide's inhibitory effect on antral motility is antagonised by its N-terminally truncated primary metabolite GLP-1 (9-36)amide. Peptides 19: 877-882, 1998[ISI][Medline].


Am J Physiol Endocrinol Metab 279(5):E956-E962
0193-1849/00 $5.00 Copyright © 2000 the American Physiological Society