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Calcitonin gene-related peptide potently stimulates glucagon-like peptide-1 release in the isolated perfused rat ileum
Peptides 21 (2000) 431– 437
Calcitonin gene-related peptide potently stimulates glucagon-like peptide-1 release in the isolated perfused rat ileum C. Herrmann–Rinkea, G.P. McGregorb, B. Go¨kec,* a
Clinical Research Unit for Gastrointestinal Endocrinology, Department of Internal Medicine, Philipps University of Marburg, Marburg, Germany b Institute of Physiology, Philipps University of Marburg, Marburg, Germany c Department of Gastroenterology, Inselspital, University of Bern, Freiburgstr, 3010 Bern, Switzerland Received 19 April 1999; accepted 13 December 1999
Abstract The post-prandial release of glucagon-like peptide-1 (GLP-1) from the distal gut appears to involve a neural reflex that arises from the proximal gut. The neuropeptide calcitonin gene-related peptide (CGRP)’s potent stimulatory effect on GLP-1 release was characterized, using the isolated vascularly perfused rat ileum. CGRP, but not its homolog amylin, induced a dose-dependent and sustained release of GLP-1. This effect was greatly reduced in the presence of CGRP(8 –37), was abolished by galanin, potentiated by luminal glucose and unaffected by atropine. GIP enhanced, but did not potentiate, this effect. The results reveal how CGRP is involved in the complex regulation of GLP-1 release. © 2000 Elsevier Science Inc. All rights reserved. Keywords: CGRP; GLP-1; Perfused ileum
1. Introduction It is well established that postprandial insulin secretion involves the facilitatory effects of humoral signals (incretins) that arise from the gut. The insulinotropic peptide hormone, glucagon-like peptide-1 (GLP-1), is released rapidly after a meal and acts as a potent incretin [12,14,19]. GLP-1 arises from the endocrine L-cells, which are located in the epithelium of the distal intestines [12]. Therefore, the prompt postprandial increase of GLP-1 secretion is not likely to involve direct contact of luminal nutrients with the L-cells and is probably due to neuronal or humoral signals derived from the proximal gut [13,19]. The processes involved are probably complex and several candidate regulators of GLP-1 secretion have been identified. The peptide hormone, glucose-dependent insulinotropic peptide (GIP) is derived from the proximal gut and stimulates GLP-1 release [20,30]. Acetylcholine, via muscarinic receptors [1,5,21] and the neuropeptide, gastrin-release peptide (GRP) [29] are also potent GLP-1 secretagogues. We chose, in the studies described here, to further characterize the effect of calcito* Corresponding author. Tel.: ⫹41-31-632-8025; fax: ⫹41-31-6329765. E-mail address: [email protected] (B Goke)
nin gene related peptide (CGRP) that we have previously found to be a potent GLP-1 secretagogue [21]. In particular, how CGRP influences the effect of other GLP-1 secretagogues was investigated. CGRP exists as two forms—␣ and . CGRP-␣ is derived from the calcitonin gene by way of alternative post-translational processing that occurs in a cell-specific manner [31]. CGRP- is derived separately from the CGRP- gene [2]. In the rat, both forms have a primary structure of 37-residues that differs by a single amino acid substitution. Generally, both peptides appear to exert similar biologic actions and both react similarly with each of the two CGRPreceptor sub-types that have been defined pharmacologically [10]. Although both are present within the rat gastrointestinal tract, CGRP-␣ is confined to neurons that arise extrinsically from vagal or spinal sensory ganglia [6,26,32]. In contrast, CGRP- is confined to a population of neurons that arise from the intrinsic myenteric plexuses [26]. We have recently described the presence of CGRP binding sites throughout the rat gastrointestinal tract and within the mucosa, submucosa, and muscle layers [27], which is consistent with the peptide’s wide-ranging effects on gut functions including gastric mucosal blood flow, gastrointestinal secretion and motility [23]. It also has been found to potently
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influence endocrine cells [11,28,33–35] but possible effects on the intestinal L-cells have not been investigated in detail.
2. Materials and methods
EDTA out of the portal vein by dripping. The erythrocytes were rapidly separated by centrifugation and the supernatants were extracted with ethanol (1:2 v/v). The ethanol extracts were dried and kept at ⫺30°C for the subsequent determination of GLP-1 immunoreactivity (GLP-LI).
2.1. Materials
2.3. Experimental design, calculations, and statistics
All electrolytes, bovine serum albumin (BSA) Fraction V, atropine sulfate, and glucose were purchased from Sigma St. Louis, MO, USA. Gastric inhibitory polypeptide (GIP; synonym: glucose-dependent insulin-releasing polypeptide, porcine), galanin, ␣-CGRP (rat) and GLP-1 were obtained from Saxon Biochemical, Hannover, Germany. Bachem Feinchemikalien AG, Bubendorf, Switzerland, supplied amylin (rat), and CGRP [8 –37]. Azonutril 25 from Pharmacia Biotech Europe, Freiburg, Germany, served as amino acid source. The total amino acid content was 14.8 g/100 ml. The solution contains in detail: 3.4% isoleucine, 9.3% leucine, 8.5% lysine, 6.3% methionine, 8.3% phenylalanine, 3.4% threonine, 1.7% tryptophan, 8.4% valine, 2.7% aspartic acid, 3.4% glutamic acid, 6.4% alanine, 16.8% arginine, 1% cysteine, 6% glycine, 3.4% histidine, 5.4% proline, 0.9% serine, 0.2% tyrosine, 2% citrulline, and 1.5% ornithine.
All experiments started with a 20 min basal period followed by a 30 min stimulatory period. All ended with another 10 min phase under basal conditions. In some experiments, after the first 10 min of the basal period, another period of 20 min with atropine infusion was added. Thereafter, the stimulatory period followed with a combination of cGRP and atropine infused over 30 min. All intra-arterially infused drugs were dissolved in a Krebs–Henseleit buffer supplemented with 3% BSA and added in 0.25 ml/min into the perfusion system right next to the arterial inflow. Glucose (5 g in 100 ml ⫽ 5%) was dissolved in 0.9% saline and perfused into the gut lumen. In these experiments the glucose administration started with a 5 ml bolus to wash out saline before the luminal perfusion period started. The data in all figures are presented as means ⫹/⫺ SEM and is expressed as femtomoles (fmol) per time unit. Integrated responses, that represent the sum of additionally released GLP-LI during a stimulatory period of 30 min, were calculated by the substraction of the mean basal release GLP-LI during the stimulatory sequence from the total amount of the release peptide during the same time (calculated as difference of areas under the curves; AUC). The obtained data underwent a one-way ANOVA followed by procedures for multiple comparisons [20]. Students t-test for paired or unpaired data were used to compare data obtained under basal and stimulated conditions. P ⬍ 0.05 was considered as significant.
2.2. Surgical preparation The operative procedure and the viability of the isolated perfused rat ileum has been previously reported in detail [9,19]. Female Wistar rats (250 –300 g) were anesthetized with pentobarbital-sodium [50 mg/kg intraperitoneal (i.p.)]. After opening the abdomen with a midline incision the right and middle colic vessels were tied and cut off. The first silastic tube was inserted into the jejunal lumen, about 15 cm proximal the hindgut. The catheterized lumen was flushed out twice with prewarmed isotonic saline (15–20 ml). After that procedure the second silastic tube was inserted into the terminal ileum. By several ligatures on the supplying vessels the remaining upper small intestine was separated and removed. A metal cannula was quickly inserted into the mesenteric superior artery and a silastic one was introduced into the portal vein. The vascular perfusion started immediately at a rate of 2.5 ml/min. The perfusion solution was a Krebs–Henseleit buffer containing 25% washed bovine erythrocytes, 3% BSA, 8 mM glucose and 1% Azonutril (mixture of amino acids). The mixture was continuously gassed with 95% O2 and 5% CO2 and warmed at 37°C. The isolated segment was removed and transferred in a temperature stable box filled with isotonic saline at 37°C. Luminal catheters were conjuncted with a continuously luminal perfusion of isotonic saline at 37°C at a flow rate of 0.25 ml/min. The venous effluent was collected in two minute fractions in prechilled glass tubes filled with 250 l 200 mM
2.4. Radioimmunoassay of GLP-1 HPLC analysis of perfusate proved the specificity of the GLP-1 analysis as was reported earlier [20]. Immunoreactive GLP-1 was analyzed by a competitive radioimmunoassay with a specific polyclonal antibody (GA 1178, Affinity Research, Nottingham, UK.) The inter- and intra-assay coefficients of variation were 10.2 and 3.4%, respectively. The radioimmunoassay was performed after ethanol extraction for concentration of samples with a recovery of 84 ⫾ 6% [19 –21].
3. Results Viability studies on the model of the isolated perfused rat ileum are documented in detail [20]. Under basal conditions a stable release of GLP-1 was observed at a level of 15.6 ⫾ 3.2 fmol/2 min. The intra-arterial infusion of CGRP at 0.1
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Fig. 1. A, Immunoreactive GLP-1 response from the perfused ileum after intra-arterial infusion of CGRP. Arrows indicate onset and end of CGRPstimulation. Results are means ⫾ SEM (fmol/2 min) for eight experiments; B, Dose– dependence of the CGRP stimulated GLP-1 secretion. Demonstrated are integrated responses over 30 min in means ⫾ SEM.
Fig. 2. A, Effects of additional intra-arterial infusion of atropine (0.001 mM) and the neuropeptide galanin (0.1 m) on CGRP (0.1 m) stimulated GLP-1 secretion. Arrows indicate the onset and end of infusion (means ⫾ SEM, N ⫽ 6). Additionally galanin reduced GLP-1 release significantly (P ⬍ 0.05) from 22 to 48 min; B, Integrated responses of CGRP stimation alone and additional infused atropine and galanin on CGRP stimulated GLP-1 release. Demonstrated are means ⫾ SEM; n ⫽ 6.
m resulted in a prompt peak response of GLP-1 release at 73.2 ⫾ 17.8 fmol/2 min. This was followed by a sustained secretion of the peptide at a level of 42.8 ⫾ 15.0 fmol/2 min up to the cessation of the CGRP infusion. At the end of the perfusion experiments basal values of peptide secretion were reached again. During the whole period of CGRP administration, GLP-1 levels were significantly (P ⬍ 0.01) elevated above basal values. Fig. 1A shows the dose– dependent stimulation of GLP-1 secretion by CGRP. With reduced concentrations (0.01 and 0.001 m) of infused CGRP, there was a corresponding reduction in the measured increase of released GLP-1. Mean values under stimulation in comparison to mean basal values were significantly elevated at both concentrations (0.01 m: P ⬍ 0.005; 0.001 m: P ⬍ 0.01). Integrated responses, calculated as areas under the curve, showed a significant dose– dependent effect of CGRP infusion on GLP-1 release (P ⬍ 0.005; Fig. 1B). In previous experiments the intra-arterial infusion of the neuropeptide galanin (0.1 m) or atropine (0.1 mM) had no effects on the
GLP-1 release from the L-cells [20]. Likewise, the additional intra-arterial infusion of atropine (0.1 mM) was also without effect on the CGRP stimulated GLP-1 secretion. In contrast hereto, galanin reduced the amount of released GLP-1 obtained under CGRP stimulation (P ⬍ 0.05; Fig. 2A). The peak secretion was then reduced to 22.6 ⫾ 6.5 fmol/2 min. Calculating the integrated responses of secretion demonstrated clearly the strong inhibitory action of galanin on CGRP-induced release (P ⬍ 0.001; Fig. 2B). The combination of intra-arterially infused CGRP (0.1 m) and luminally infused glucose (5%) resulted in a clear potentiation of the stimulated GLP-1 release. The release GLP-1 levels reached a peak of 118.9 ⫾ 21.7 fmol/2 min above basal. This was followed by a sustained release at 82.7 ⫾ 17.9 fmol/2 min. This was significantly higher (P ⬍ 0.01) as compared with GLP-1 release obtained with one of the two secretagogues alone (Fig. 3A). Reducing the CGRP dose (0.01 m; n ⫽ 8) did not significantly alter the poten-
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Fig. 3. A, Effect of CGRP (0.1 m) infusion alone, intraluminally perfused glucose 5% alone and the combined stimulation on GLP-1 secretion. Arrows indicate the onset and end of stimulation. Demonstrated are means ⫾ SEM (n ⫽ 8); B, Comparison of integrated responses of GLP-1 secretion under stimulation of CGRP alone at the concentration of 0.01 m and 0.001 m or in combination with luminally perfused glucose 5%. Demonstrated are means ⫾ SEM (n ⫽ 8). For comparisons the calculated results are demonstrated on the right side without error bars. C, Comparison of integrated responses of GLP-1 secretion under a fixed concentration of CGRP (0.1 m) and different concentrations of luminally perfused glucose (5% and 2.5%, respectively). Demonstrated are means ⫾ SEM (n ⫽ 8). For comparisons the calculated results are demonstrated on the right side without error bars.
tiation of GLP-1 secretion, but, in contrast, reducing the luminal glucose load to 2.5% resulted in a clearly diminished effect (n ⫽ 6; Fig. 3B,C). Additional studies were performed with amylin because this peptide shows a 50% homology with CGRP. Amylin at a high concentration of 0.1 m had no effect on GLP-1
secretion. In separate experiments, CGRP [8 –37] (10⫺6 M) was found to significantly inhibit the stimulatory effect of CGRP (10⫺8 M) on GLP-1 release—the mean integrated GLP-1 response to CGRP (10⫺8 M) was 158 (⫹/⫺78 ⫽ SEM) fmol/30 min, compared to 56 (⫹/⫺28 ⫽ SEM) fmol/30 min for CGRP (10⫺8 M) in the presence of CGRP
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Fig. 4. A, Effects of intra-arterially infused CGRP or amylin at 0.1 m on the release of immunoreactive GLP-1. Arrows indicate the onset and end of the infusion (n ⫽ 6). Demonstrated are means ⫾ SEM. Significant differences (P ⬍ 0.005) were obtained for all measurements between 22 and 52 min; B, Integrated responses of GLP-1 release under stimulation of CGRP or amylin at 0.1 m (plain bars) or 0.01 m (hatched bars, n ⫽ 6). Demonstrated are means ⫾ SEM.
[8 –37] (10⫺6 M). The effect of combined infusion of CGRP with GIP was investigated in a further series of experiments. In these experiments, the mean integrated GLP-1 response to CGRP (10⫺8 M) was 97 (⫹/⫺48 ⫽ SEM) fmol/30 min, compared to 118 (⫹/⫺43 ⫽ SEM) fmol/30 min in response to GIP (10⫺8 M) and 295 (⫹/⫺59, SEM) fmol/30 min in response to CGRP (10⫺8 M) and GIP (10⫺8 M) combined. The secretory response to the combined peptides was greater as that obtained with each of the respective peptides alone, but this appears to be additive and not synergistic.
4. Discussion Currently, the exact physiological regulation of intestinal secretion of proglucagon-derived peptides, including GLP-1, is not fully understood. Circulating concentrations of GLP-1 increase promptly after a meal [13] although the majority of the GLP-1-releasing L-cells are located in distal
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segments of the gut [12]. Therefore, the rapid increase of GLP-1 after a meal implies a signal from the proximal gut. The results of some studies have already suggested that entry of nutrients into the duodenum plays a key role in the secretion of proglucagon derived peptides and that the direct effects on the L-cell of luminal factors are less important [4,13–15,29]. In a previous study using this model, various gut-derived neuropeptides and neurotransmitters were screened for their effects on GLP-1 release, and CGRP was hinted to be one of a number of effective secretogogues [21]. It is present throughout the gut and is expressed by neurons of the myenteric nervous system but also by a population of extrinsic sensory neurons that supplies the gut [32]. CGRPcontaining nerve fibers are found within the gut mucosa and may supply the epithelial layer. Although there is evidently a high density of CGRP binding sites within the rat intestinal mucosa [27], the possible existence of CGRP receptors on intestinal epithelial cells will only be resolved when specific antibodies become available for more precise immunocytochemical analyses. CGRP seems to be involved in the regulation of several different functions in the gut, including gut hormone release. An effect on intestinal L-cells that synthesize and release the peptide hormones GLP-1 and PYY, was indicated earlier in studies with isolated fetal rat intestinal cells [5] and gained some support in preliminary studies employing the isolated vascularly perfused rat ileum [21]. This effect of the peptide on the L-cells is now characterized, here, in more detail. We show that its effect is dose– dependent and is sustained for the duration of the period of administration. Also this effect of CGRP is blocked with the CGRP-specific antagonist, CGRP [8 –37], which indicates that CGRP is acting here through the CGRP-receptor 1 subtype. The GLP-1 plasma levels attained in the presence of CGRP were very similar to those attained in the presence of equimolar concentrations of GIP, which is a potent GLP-1 secretogogue that is considered of physiological relevance. Further investigations are required to establish whether CGRP is acting directly on the L-cell. However, the lack of effect of atropine on CGRP-induced GLP-1 release indicates that it is not acting via a cholinergic pathway as has been suggested to be part of CGRP’s mechanism of action in influencing gastric acid secretion [28]. The neuropeptide galanin was able to abolish the effect of CGRP as it does GIP-induced GLP-1 secretion [20]. The convergence of the actions of these three peptides on GLP-1 release indicates the complexity of regulated secretion from the L-cell. Furthermore, these findings strongly suggest that both CGRP and GIP act either directly on the L-cell or indirectly via similar pathways. Gastrin-releasing peptide (GRP) is another potent GLP-1 secretagogue that appears to act downstream of GIP [29] because, in vivo, GRP antagonists completely block GIP-evoked GLP-1 release. Further similar studies using CGRP antagonists are required to resolve the
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role of CGRP within this complex interaction of neural and endocrine signals. Rocca and Brubacher [30] have proposed a scheme in which enteric and vagal nerve pathways converge, along with the endocrine signal, GIP, on the L-cell to regulate postprandial GLP-1 release. CGRP was not included in this scheme and nor was the anti-secretogogue, galanin. CGRP, GRP and galanin are found within the separate subpopulations of neurones supplying the rat intestinal mucosa [33] and may be the local neuroeffectors that mediate the different proximal signals that converge on the L-cell. The combined effect of vascular CGRP and luminal glucose was a dramatic augmentation of the amount of GLP-1 released. This interesting effect is consistent with the notion that the effect of CGRP on GLP-1 release is of physiological significance. Cooperative effects of luminal and peptidergic factors have been described before in the regulation of gut endocrine cells. For example, similar effects were observed in the regulation of neurotensin secretion where different peptide combinations resulted in potentiation or additive effects [18]. In the isolated vascularly perfused rat ileum, taurocholic acid was capable of potentiating the effect of neuropeptides on intestinal N-cells to induce neurotensin secretion [19]. This observation of a potentiated GLP-1 release by luminal glucose combined with CGRP may have a therapeutic implication because a mobilization of endogenous GLP-1 reserves may offer a new therapeutic approach in the treatment of diabetes mellitus. Because CGRP is a potent vasodilator and increases local blood flow [24], the effect of the peptide on the L-cells might be a secondary phenomenon. In the gastric mucosa, its dilatory effect on sub-mucosal blood vessels is thought to be a vital component of mucosal protective mechanisms aimed to maintain mucosal integrity [24]. However, a similar role for CGRP in the intestine could not, so far, be demonstrated [4] and other vasodilators that increase local blood flow, such as substance P or serotonin, did not alter released GLP-1 levels in this model [21]. CGRP is known to influence hormone release from gut endocrine cells. For example, there is considerable evidence that CGRP stimulates somatostatin release from the gastric and pancreatic D-cell and, via this route, can reduce gastrin and insulin release, respectively [11]. Our results add support to the notion that the release of GLP-1 into the circulation is regulated in a complex manner that involves both the autonomic nervous system and gut hormones, which are activated by presence of nutrients in the lumen of the proximal gut. Although luminal nutrients are capable of directly stimulating GLP-1 release, a neuroenteroendocrine axis that connects the upper to the lower intestine is proposed to exist and to be responsible for the early post-prandial release of GLP-1. In fact, recent in vivo studies [3,30] have added significant support to this notion, and details of how the neural and endocrine pathways interact in regulating the endocrine L-cell is beginning to
emerge. CGRP is known to be present in both vagal and enteric nerve pathways that are implicated in the control of the post-prandial release of GLP-1. Our findings suggest that CGRP should be considered a crucial mediator of these complex interactions.
Acknowledgments This work was supported by the Deutsche Forschungsgemeinschaft (DFG).
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Calcitonin gene-related peptide potently stimulates glucagon-like peptide-1 release in the isolated perfused rat ileum C. Herrmann–Rinkea, G.P. McGregorb, B. Go¨kec,* a
Clinical Research Unit for Gastrointestinal Endocrinology, Department of Internal Medicine, Philipps University of Marburg, Marburg, Germany b Institute of Physiology, Philipps University of Marburg, Marburg, Germany c Department of Gastroenterology, Inselspital, University of Bern, Freiburgstr, 3010 Bern, Switzerland Received 19 April 1999; accepted 13 December 1999
Abstract The post-prandial release of glucagon-like peptide-1 (GLP-1) from the distal gut appears to involve a neural reflex that arises from the proximal gut. The neuropeptide calcitonin gene-related peptide (CGRP)’s potent stimulatory effect on GLP-1 release was characterized, using the isolated vascularly perfused rat ileum. CGRP, but not its homolog amylin, induced a dose-dependent and sustained release of GLP-1. This effect was greatly reduced in the presence of CGRP(8 –37), was abolished by galanin, potentiated by luminal glucose and unaffected by atropine. GIP enhanced, but did not potentiate, this effect. The results reveal how CGRP is involved in the complex regulation of GLP-1 release. © 2000 Elsevier Science Inc. All rights reserved. Keywords: CGRP; GLP-1; Perfused ileum
1. Introduction It is well established that postprandial insulin secretion involves the facilitatory effects of humoral signals (incretins) that arise from the gut. The insulinotropic peptide hormone, glucagon-like peptide-1 (GLP-1), is released rapidly after a meal and acts as a potent incretin [12,14,19]. GLP-1 arises from the endocrine L-cells, which are located in the epithelium of the distal intestines [12]. Therefore, the prompt postprandial increase of GLP-1 secretion is not likely to involve direct contact of luminal nutrients with the L-cells and is probably due to neuronal or humoral signals derived from the proximal gut [13,19]. The processes involved are probably complex and several candidate regulators of GLP-1 secretion have been identified. The peptide hormone, glucose-dependent insulinotropic peptide (GIP) is derived from the proximal gut and stimulates GLP-1 release [20,30]. Acetylcholine, via muscarinic receptors [1,5,21] and the neuropeptide, gastrin-release peptide (GRP) [29] are also potent GLP-1 secretagogues. We chose, in the studies described here, to further characterize the effect of calcito* Corresponding author. Tel.: ⫹41-31-632-8025; fax: ⫹41-31-6329765. E-mail address: [email protected] (B Goke)
nin gene related peptide (CGRP) that we have previously found to be a potent GLP-1 secretagogue [21]. In particular, how CGRP influences the effect of other GLP-1 secretagogues was investigated. CGRP exists as two forms—␣ and . CGRP-␣ is derived from the calcitonin gene by way of alternative post-translational processing that occurs in a cell-specific manner [31]. CGRP- is derived separately from the CGRP- gene [2]. In the rat, both forms have a primary structure of 37-residues that differs by a single amino acid substitution. Generally, both peptides appear to exert similar biologic actions and both react similarly with each of the two CGRPreceptor sub-types that have been defined pharmacologically [10]. Although both are present within the rat gastrointestinal tract, CGRP-␣ is confined to neurons that arise extrinsically from vagal or spinal sensory ganglia [6,26,32]. In contrast, CGRP- is confined to a population of neurons that arise from the intrinsic myenteric plexuses [26]. We have recently described the presence of CGRP binding sites throughout the rat gastrointestinal tract and within the mucosa, submucosa, and muscle layers [27], which is consistent with the peptide’s wide-ranging effects on gut functions including gastric mucosal blood flow, gastrointestinal secretion and motility [23]. It also has been found to potently
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influence endocrine cells [11,28,33–35] but possible effects on the intestinal L-cells have not been investigated in detail.
2. Materials and methods
EDTA out of the portal vein by dripping. The erythrocytes were rapidly separated by centrifugation and the supernatants were extracted with ethanol (1:2 v/v). The ethanol extracts were dried and kept at ⫺30°C for the subsequent determination of GLP-1 immunoreactivity (GLP-LI).
2.1. Materials
2.3. Experimental design, calculations, and statistics
All electrolytes, bovine serum albumin (BSA) Fraction V, atropine sulfate, and glucose were purchased from Sigma St. Louis, MO, USA. Gastric inhibitory polypeptide (GIP; synonym: glucose-dependent insulin-releasing polypeptide, porcine), galanin, ␣-CGRP (rat) and GLP-1 were obtained from Saxon Biochemical, Hannover, Germany. Bachem Feinchemikalien AG, Bubendorf, Switzerland, supplied amylin (rat), and CGRP [8 –37]. Azonutril 25 from Pharmacia Biotech Europe, Freiburg, Germany, served as amino acid source. The total amino acid content was 14.8 g/100 ml. The solution contains in detail: 3.4% isoleucine, 9.3% leucine, 8.5% lysine, 6.3% methionine, 8.3% phenylalanine, 3.4% threonine, 1.7% tryptophan, 8.4% valine, 2.7% aspartic acid, 3.4% glutamic acid, 6.4% alanine, 16.8% arginine, 1% cysteine, 6% glycine, 3.4% histidine, 5.4% proline, 0.9% serine, 0.2% tyrosine, 2% citrulline, and 1.5% ornithine.
All experiments started with a 20 min basal period followed by a 30 min stimulatory period. All ended with another 10 min phase under basal conditions. In some experiments, after the first 10 min of the basal period, another period of 20 min with atropine infusion was added. Thereafter, the stimulatory period followed with a combination of cGRP and atropine infused over 30 min. All intra-arterially infused drugs were dissolved in a Krebs–Henseleit buffer supplemented with 3% BSA and added in 0.25 ml/min into the perfusion system right next to the arterial inflow. Glucose (5 g in 100 ml ⫽ 5%) was dissolved in 0.9% saline and perfused into the gut lumen. In these experiments the glucose administration started with a 5 ml bolus to wash out saline before the luminal perfusion period started. The data in all figures are presented as means ⫹/⫺ SEM and is expressed as femtomoles (fmol) per time unit. Integrated responses, that represent the sum of additionally released GLP-LI during a stimulatory period of 30 min, were calculated by the substraction of the mean basal release GLP-LI during the stimulatory sequence from the total amount of the release peptide during the same time (calculated as difference of areas under the curves; AUC). The obtained data underwent a one-way ANOVA followed by procedures for multiple comparisons [20]. Students t-test for paired or unpaired data were used to compare data obtained under basal and stimulated conditions. P ⬍ 0.05 was considered as significant.
2.2. Surgical preparation The operative procedure and the viability of the isolated perfused rat ileum has been previously reported in detail [9,19]. Female Wistar rats (250 –300 g) were anesthetized with pentobarbital-sodium [50 mg/kg intraperitoneal (i.p.)]. After opening the abdomen with a midline incision the right and middle colic vessels were tied and cut off. The first silastic tube was inserted into the jejunal lumen, about 15 cm proximal the hindgut. The catheterized lumen was flushed out twice with prewarmed isotonic saline (15–20 ml). After that procedure the second silastic tube was inserted into the terminal ileum. By several ligatures on the supplying vessels the remaining upper small intestine was separated and removed. A metal cannula was quickly inserted into the mesenteric superior artery and a silastic one was introduced into the portal vein. The vascular perfusion started immediately at a rate of 2.5 ml/min. The perfusion solution was a Krebs–Henseleit buffer containing 25% washed bovine erythrocytes, 3% BSA, 8 mM glucose and 1% Azonutril (mixture of amino acids). The mixture was continuously gassed with 95% O2 and 5% CO2 and warmed at 37°C. The isolated segment was removed and transferred in a temperature stable box filled with isotonic saline at 37°C. Luminal catheters were conjuncted with a continuously luminal perfusion of isotonic saline at 37°C at a flow rate of 0.25 ml/min. The venous effluent was collected in two minute fractions in prechilled glass tubes filled with 250 l 200 mM
2.4. Radioimmunoassay of GLP-1 HPLC analysis of perfusate proved the specificity of the GLP-1 analysis as was reported earlier [20]. Immunoreactive GLP-1 was analyzed by a competitive radioimmunoassay with a specific polyclonal antibody (GA 1178, Affinity Research, Nottingham, UK.) The inter- and intra-assay coefficients of variation were 10.2 and 3.4%, respectively. The radioimmunoassay was performed after ethanol extraction for concentration of samples with a recovery of 84 ⫾ 6% [19 –21].
3. Results Viability studies on the model of the isolated perfused rat ileum are documented in detail [20]. Under basal conditions a stable release of GLP-1 was observed at a level of 15.6 ⫾ 3.2 fmol/2 min. The intra-arterial infusion of CGRP at 0.1
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Fig. 1. A, Immunoreactive GLP-1 response from the perfused ileum after intra-arterial infusion of CGRP. Arrows indicate onset and end of CGRPstimulation. Results are means ⫾ SEM (fmol/2 min) for eight experiments; B, Dose– dependence of the CGRP stimulated GLP-1 secretion. Demonstrated are integrated responses over 30 min in means ⫾ SEM.
Fig. 2. A, Effects of additional intra-arterial infusion of atropine (0.001 mM) and the neuropeptide galanin (0.1 m) on CGRP (0.1 m) stimulated GLP-1 secretion. Arrows indicate the onset and end of infusion (means ⫾ SEM, N ⫽ 6). Additionally galanin reduced GLP-1 release significantly (P ⬍ 0.05) from 22 to 48 min; B, Integrated responses of CGRP stimation alone and additional infused atropine and galanin on CGRP stimulated GLP-1 release. Demonstrated are means ⫾ SEM; n ⫽ 6.
m resulted in a prompt peak response of GLP-1 release at 73.2 ⫾ 17.8 fmol/2 min. This was followed by a sustained secretion of the peptide at a level of 42.8 ⫾ 15.0 fmol/2 min up to the cessation of the CGRP infusion. At the end of the perfusion experiments basal values of peptide secretion were reached again. During the whole period of CGRP administration, GLP-1 levels were significantly (P ⬍ 0.01) elevated above basal values. Fig. 1A shows the dose– dependent stimulation of GLP-1 secretion by CGRP. With reduced concentrations (0.01 and 0.001 m) of infused CGRP, there was a corresponding reduction in the measured increase of released GLP-1. Mean values under stimulation in comparison to mean basal values were significantly elevated at both concentrations (0.01 m: P ⬍ 0.005; 0.001 m: P ⬍ 0.01). Integrated responses, calculated as areas under the curve, showed a significant dose– dependent effect of CGRP infusion on GLP-1 release (P ⬍ 0.005; Fig. 1B). In previous experiments the intra-arterial infusion of the neuropeptide galanin (0.1 m) or atropine (0.1 mM) had no effects on the
GLP-1 release from the L-cells [20]. Likewise, the additional intra-arterial infusion of atropine (0.1 mM) was also without effect on the CGRP stimulated GLP-1 secretion. In contrast hereto, galanin reduced the amount of released GLP-1 obtained under CGRP stimulation (P ⬍ 0.05; Fig. 2A). The peak secretion was then reduced to 22.6 ⫾ 6.5 fmol/2 min. Calculating the integrated responses of secretion demonstrated clearly the strong inhibitory action of galanin on CGRP-induced release (P ⬍ 0.001; Fig. 2B). The combination of intra-arterially infused CGRP (0.1 m) and luminally infused glucose (5%) resulted in a clear potentiation of the stimulated GLP-1 release. The release GLP-1 levels reached a peak of 118.9 ⫾ 21.7 fmol/2 min above basal. This was followed by a sustained release at 82.7 ⫾ 17.9 fmol/2 min. This was significantly higher (P ⬍ 0.01) as compared with GLP-1 release obtained with one of the two secretagogues alone (Fig. 3A). Reducing the CGRP dose (0.01 m; n ⫽ 8) did not significantly alter the poten-
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Fig. 3. A, Effect of CGRP (0.1 m) infusion alone, intraluminally perfused glucose 5% alone and the combined stimulation on GLP-1 secretion. Arrows indicate the onset and end of stimulation. Demonstrated are means ⫾ SEM (n ⫽ 8); B, Comparison of integrated responses of GLP-1 secretion under stimulation of CGRP alone at the concentration of 0.01 m and 0.001 m or in combination with luminally perfused glucose 5%. Demonstrated are means ⫾ SEM (n ⫽ 8). For comparisons the calculated results are demonstrated on the right side without error bars. C, Comparison of integrated responses of GLP-1 secretion under a fixed concentration of CGRP (0.1 m) and different concentrations of luminally perfused glucose (5% and 2.5%, respectively). Demonstrated are means ⫾ SEM (n ⫽ 8). For comparisons the calculated results are demonstrated on the right side without error bars.
tiation of GLP-1 secretion, but, in contrast, reducing the luminal glucose load to 2.5% resulted in a clearly diminished effect (n ⫽ 6; Fig. 3B,C). Additional studies were performed with amylin because this peptide shows a 50% homology with CGRP. Amylin at a high concentration of 0.1 m had no effect on GLP-1
secretion. In separate experiments, CGRP [8 –37] (10⫺6 M) was found to significantly inhibit the stimulatory effect of CGRP (10⫺8 M) on GLP-1 release—the mean integrated GLP-1 response to CGRP (10⫺8 M) was 158 (⫹/⫺78 ⫽ SEM) fmol/30 min, compared to 56 (⫹/⫺28 ⫽ SEM) fmol/30 min for CGRP (10⫺8 M) in the presence of CGRP
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Fig. 4. A, Effects of intra-arterially infused CGRP or amylin at 0.1 m on the release of immunoreactive GLP-1. Arrows indicate the onset and end of the infusion (n ⫽ 6). Demonstrated are means ⫾ SEM. Significant differences (P ⬍ 0.005) were obtained for all measurements between 22 and 52 min; B, Integrated responses of GLP-1 release under stimulation of CGRP or amylin at 0.1 m (plain bars) or 0.01 m (hatched bars, n ⫽ 6). Demonstrated are means ⫾ SEM.
[8 –37] (10⫺6 M). The effect of combined infusion of CGRP with GIP was investigated in a further series of experiments. In these experiments, the mean integrated GLP-1 response to CGRP (10⫺8 M) was 97 (⫹/⫺48 ⫽ SEM) fmol/30 min, compared to 118 (⫹/⫺43 ⫽ SEM) fmol/30 min in response to GIP (10⫺8 M) and 295 (⫹/⫺59, SEM) fmol/30 min in response to CGRP (10⫺8 M) and GIP (10⫺8 M) combined. The secretory response to the combined peptides was greater as that obtained with each of the respective peptides alone, but this appears to be additive and not synergistic.
4. Discussion Currently, the exact physiological regulation of intestinal secretion of proglucagon-derived peptides, including GLP-1, is not fully understood. Circulating concentrations of GLP-1 increase promptly after a meal [13] although the majority of the GLP-1-releasing L-cells are located in distal
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segments of the gut [12]. Therefore, the rapid increase of GLP-1 after a meal implies a signal from the proximal gut. The results of some studies have already suggested that entry of nutrients into the duodenum plays a key role in the secretion of proglucagon derived peptides and that the direct effects on the L-cell of luminal factors are less important [4,13–15,29]. In a previous study using this model, various gut-derived neuropeptides and neurotransmitters were screened for their effects on GLP-1 release, and CGRP was hinted to be one of a number of effective secretogogues [21]. It is present throughout the gut and is expressed by neurons of the myenteric nervous system but also by a population of extrinsic sensory neurons that supplies the gut [32]. CGRPcontaining nerve fibers are found within the gut mucosa and may supply the epithelial layer. Although there is evidently a high density of CGRP binding sites within the rat intestinal mucosa [27], the possible existence of CGRP receptors on intestinal epithelial cells will only be resolved when specific antibodies become available for more precise immunocytochemical analyses. CGRP seems to be involved in the regulation of several different functions in the gut, including gut hormone release. An effect on intestinal L-cells that synthesize and release the peptide hormones GLP-1 and PYY, was indicated earlier in studies with isolated fetal rat intestinal cells [5] and gained some support in preliminary studies employing the isolated vascularly perfused rat ileum [21]. This effect of the peptide on the L-cells is now characterized, here, in more detail. We show that its effect is dose– dependent and is sustained for the duration of the period of administration. Also this effect of CGRP is blocked with the CGRP-specific antagonist, CGRP [8 –37], which indicates that CGRP is acting here through the CGRP-receptor 1 subtype. The GLP-1 plasma levels attained in the presence of CGRP were very similar to those attained in the presence of equimolar concentrations of GIP, which is a potent GLP-1 secretogogue that is considered of physiological relevance. Further investigations are required to establish whether CGRP is acting directly on the L-cell. However, the lack of effect of atropine on CGRP-induced GLP-1 release indicates that it is not acting via a cholinergic pathway as has been suggested to be part of CGRP’s mechanism of action in influencing gastric acid secretion [28]. The neuropeptide galanin was able to abolish the effect of CGRP as it does GIP-induced GLP-1 secretion [20]. The convergence of the actions of these three peptides on GLP-1 release indicates the complexity of regulated secretion from the L-cell. Furthermore, these findings strongly suggest that both CGRP and GIP act either directly on the L-cell or indirectly via similar pathways. Gastrin-releasing peptide (GRP) is another potent GLP-1 secretagogue that appears to act downstream of GIP [29] because, in vivo, GRP antagonists completely block GIP-evoked GLP-1 release. Further similar studies using CGRP antagonists are required to resolve the
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role of CGRP within this complex interaction of neural and endocrine signals. Rocca and Brubacher [30] have proposed a scheme in which enteric and vagal nerve pathways converge, along with the endocrine signal, GIP, on the L-cell to regulate postprandial GLP-1 release. CGRP was not included in this scheme and nor was the anti-secretogogue, galanin. CGRP, GRP and galanin are found within the separate subpopulations of neurones supplying the rat intestinal mucosa [33] and may be the local neuroeffectors that mediate the different proximal signals that converge on the L-cell. The combined effect of vascular CGRP and luminal glucose was a dramatic augmentation of the amount of GLP-1 released. This interesting effect is consistent with the notion that the effect of CGRP on GLP-1 release is of physiological significance. Cooperative effects of luminal and peptidergic factors have been described before in the regulation of gut endocrine cells. For example, similar effects were observed in the regulation of neurotensin secretion where different peptide combinations resulted in potentiation or additive effects [18]. In the isolated vascularly perfused rat ileum, taurocholic acid was capable of potentiating the effect of neuropeptides on intestinal N-cells to induce neurotensin secretion [19]. This observation of a potentiated GLP-1 release by luminal glucose combined with CGRP may have a therapeutic implication because a mobilization of endogenous GLP-1 reserves may offer a new therapeutic approach in the treatment of diabetes mellitus. Because CGRP is a potent vasodilator and increases local blood flow [24], the effect of the peptide on the L-cells might be a secondary phenomenon. In the gastric mucosa, its dilatory effect on sub-mucosal blood vessels is thought to be a vital component of mucosal protective mechanisms aimed to maintain mucosal integrity [24]. However, a similar role for CGRP in the intestine could not, so far, be demonstrated [4] and other vasodilators that increase local blood flow, such as substance P or serotonin, did not alter released GLP-1 levels in this model [21]. CGRP is known to influence hormone release from gut endocrine cells. For example, there is considerable evidence that CGRP stimulates somatostatin release from the gastric and pancreatic D-cell and, via this route, can reduce gastrin and insulin release, respectively [11]. Our results add support to the notion that the release of GLP-1 into the circulation is regulated in a complex manner that involves both the autonomic nervous system and gut hormones, which are activated by presence of nutrients in the lumen of the proximal gut. Although luminal nutrients are capable of directly stimulating GLP-1 release, a neuroenteroendocrine axis that connects the upper to the lower intestine is proposed to exist and to be responsible for the early post-prandial release of GLP-1. In fact, recent in vivo studies [3,30] have added significant support to this notion, and details of how the neural and endocrine pathways interact in regulating the endocrine L-cell is beginning to
emerge. CGRP is known to be present in both vagal and enteric nerve pathways that are implicated in the control of the post-prandial release of GLP-1. Our findings suggest that CGRP should be considered a crucial mediator of these complex interactions.
Acknowledgments This work was supported by the Deutsche Forschungsgemeinschaft (DFG).
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