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Neuronostatin: Peripheral site of action in mouse stomach
Peptides 64 (2015) 8–13
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Neuronostatin: Peripheral site of action in mouse stomach Antonella Amato, Sara Baldassano, Gaetano Caldara, Flavia Mulè ∗ Dipartimento di Scienze e Tecnologie Molecolari e Biomolecolari (STEMBIO), Università di Palermo, 90128 Palermo, Italy
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Article history: Received 3 November 2014 Received in revised form 12 December 2014 Accepted 12 December 2014 Available online 22 December 2014 Keywords: Neuronostatin Food intake Gastric emptying Intestinal transit
a b s t r a c t Neuronostatin is a 13-amino acid peptide encoded by somatostatin gene. It is distributed in different organs including gastrointestinal tract and has been involved in the control of food intake and gastrointestinal motility, likely through an action in the brain. So far, there are no reports about the occurrence of peripheral action sites in the gut. Therefore, the purpose of the present study was to examine, in the mouse, the effects of peripheral administration of neuronostatin on food intake within 24 h and on gastrointestinal motility and to analyse neuronostatin actions on the gastric and intestinal mechanical activity in isolated preparations in vitro. When compared with PBS-treated mice, intraperitoneal neuronostatin reduced food intake in doses ranging from 1 to 15 ng/g b.w. only in the first hour postinjection with a maximum effect obtained at the dose of 15 ng/g b.w. (−46.9%). The peptide (15 ng/g b.w.) significantly reduced gastric emptying rate (−31.1%) and gastrointestinal intestinal transit. Non-amidated neuronostatin failed to affect food intake, gastric emptying and intestinal transit, suggesting the specificity of action. In vitro, neuronostatin induced concentration-dependent gastric relaxation, which was abolished by tetrodotoxin. Neuronostatin failed to affect the spontaneous mechanical activity or the evoked cholinergic contractions in duodenum. These results suggest that exogenous neuronostatin is able to reduce mouse gastric motility by acting peripherally in the stomach, through intramural nervous plexuses. This indirectly action could cause reduction of food intake in the short term. © 2014 Elsevier Inc. All rights reserved.
Introduction Neuronostatin is a recently described 13-amino acid peptide hormone; which is encoded by somatostatin gene and derived from the N-terminus of pro-somatostatin. Somatostatin is a peptide hormone; originally identified in hypothalamus and subsequently found in neuroendocrine organs; gastrointestinal tract; thyroid and adrenal glands; liver; kidney; inflammatory and immune cells. As expected, neuronostatin has been found to be expressed in the same tissues as somatostatin, including brain and gastrointestinal tissues with the highest level in the pancreas followed by cerebrum and hypothalamus [19]. As somatostatin, neuronostatin is considered a brain/gut peptide due to its site of production and its ability to induce early response genes c-Fos or c-Jun in neuronal, anterior pituitary gastrointestinal tissues [19] and cardiomyocytes
Abbreviations: EFS, electrical field stimulation; GE, gastric emptying; i.c.v., intracerebroventricular; i.p., intraperitoneal; PBS, phosphate-buffered saline; TTX, tetrodotoxin. ∗ Corresponding author at: Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farrmaceutiche (STEBICEF), Laboratorio di Fisiologia generale, Università di Palermo, Viale delle Scienze, 90128 Palermo, Italy. Tel.: +39 91 23897515; fax: +39 91 6577501. E-mail address: fl[email protected] (F. Mulè). http://dx.doi.org/10.1016/j.peptides.2014.12.003 0196-9781/© 2014 Elsevier Inc. All rights reserved.
[13]. Despite sharing common biological effects, such as inhibitory control of cardiac activity, pancreatic and gastrointestinal functions, neuronostatin and somatostatin possess biological activities that are distinct from each other [24]. Furthermore neuronostatin has been reported not to interact with the five putative somatostatin receptors [19]. Indeed, when injected centrally in rats neuronostatin leads to a dose-related inhibition of food and water intake and an increase in mean arterial pressure [25,27]. Intracerebroventricular (i.c.v.) injections of neuronostatin in mice produce antinociceptive effect via the central melanocortin and opioid systems [22] and a depression-like effect via the central melanocortin system [23]. Neuronostatin can modify the hypothalamic neuron firing and neuronal migration [10,19] and it is able to regulate cardiac and cardiomyocyte contractile function and cardiomyocyte survival [13,21,30]. Recent studies in isolated rat pancreatic islets or in the animals in vivo suggest also a role of neuronostatin in maintaining glucose homeostasis through inhibition of glucose-stimulated insulin secretion [18]. Neuronostatin has been also involved in the control of gastrointestinal motility. In fact, i.c.v. administration of neuronostatin in mice delays the gastric emptying and the gastrointestinal transit [20]. However, to date, there are no reports about the effects of peripheral administration of neuronostatin on food intake and
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gastrointestinal motility. It is likely to hypothesize that there are peripheral sites of action involved in the biological effects of the neuronostatin, similarly to other peptides involved in the control of food intake and gastrointestinal functions [2,5,9,17,29]. Unlikely, the neuronostatin receptor and its distribution are still unknown, although the orphan G protein-coupled receptor, GPR107, has been proposed [26]. Therefore the purpose of the present study was to examine if intraperitoneal administration of neuronostatin influences the food intake within 24 h and the gastrointestinal motility in mouse. Moreover, to better elucidate the gastrointestinal actions of neuronostatin, the effects of the exogenous peptide on the gastric and intestinal mechanical activities in vitro were examined and the mechanism of action responsible of the observed effects was studied. Materials and methods
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to precipitate proteins. After centrifugation (3000 rpm for 30 min at 4 ◦ C) the supernatant was added to 2 ml of 2 N NaOH to develop the maximum color intensity. The amount of phenol red was determined from the absorbency at 560 nm. This correlates with the concentration of phenol red in the stomach, which in turn depends on the gastric emptying. The gastric emptying (GE) rate was calculated as GE = (1 − X/Y)100, where X is absorbance of phenol red recovered from the stomach of animals sacrificed 20 min after test meal. Y is mean (n = 4) absorbance of phenol red recovered from the stomachs of animals killed at 0 min following test meal. Immediately after the excision of the stomach, the whole small intestine was grossly freed from its mesenteric attachments and its length (from the pyloric sphincter to the ileocecal junction) was measured. The intestine was opened at the level of the front of the test meal, which was revealed by few drops of 0.1 N NaOH. The rate of intestinal transit was expressed as the ratio between the distance traveled by the test meal and the total length of intestine, as previously described [16].
Animals Functional studies in vitro The experimental procedures employed in the present study were in conformity with the Italian D.L. No. 116 of 27 January 1992 and subsequent variations and the recommendations of the European Economic Community (86/609/ECC). The studies were approved by Ministero della Sanità (Rome, Italy). Adult male C57BL/6J mice, purchased from Harlan Laboratories (San Pietro al Natisone, Udine, Italy) were singly housed under controlled environmental conditions (22 + 1 ◦ C, 55 + 15% relative humidity, 12 h light). Tap water and standard laboratory rodent chow (Mucedola, Settimo Milanese, MI, Italy) were provided ad libitum; except as otherwise stated. Food intake Fasted (16 h) mice were injected with intraperitoneal (i.p.) 100 l of either vehicle (phosphate-buffered saline, PBS) or neuronostatin (1, 6, 10, 15, 30 ng/g b.w.), in the early light phase (0800–0900 h). Prior to the initial study, mice received a daily i.p. injection of 100 l PBS for 7 days to habituate them to the procedure. Doses of neuronostatin were determined on the basis of the literature [30] and from preliminary experiments in our laboratory. A minimum of 72 h was allowed between each trial in the same mouse. Following injection, each mouse was returned to its home cage with a pre-weighed amount of chow. The food intake was determined at 1, 2, 4, 8, and 24 h following peptide or vehicle administration, by measuring the difference between the pre-weighed chow and the weight of chow at the end of each time interval. Any spillage was collected and weighed. Gastric emptying and intestinal transit To examine gastric emptying and small intestinal transit, the animals were deprived of food for 24 h before the experiments began. Then, mice were injected with i.p. 100 l of either PBS or neuronostatin (15 ng/g b.w.) 10 min before gastric load. This dose was chosen because it induced maximum effect in the food intake experiments. The experiments to assess gastric emptying and transit were conducted as previously reported [16]. Briefly, mice received by gavage 0.3 ml of test meal (a non-nutrient meal of 50 mg phenol red in 100 ml 1.5% carboxymethylcellulose) and were euthanized by cervical dislocation immediately (t = 0) or 20 min after gavaging. Under laparatomy, the stomach and the small intestine were excised after legation of the pylorus and the cardias. The stomach and its contents were homogenized in 25 ml of 0.1 N NaOH. The mixture was then kept for 1 h at room temperature. Then, 8 ml of the supernatant was added to 1 ml of 33% of trichloroacetic acid
After animal sacrifice, the abdomen was immediately opened, the esophagus was tied just below the lower oesophageal sphincter, and stomach and duodenum were excised. Isolated stomach As previously described [17], whole stomach was used in order to examine the muscle function under conditions where the influence of external factors is removed, but the muscle performs in a manner analogous to its in vivo capacity. The entire stomach was mounted in a custom designed organ bath, which was continuously perfused with oxygenated (95% O2 and 5% CO2 ) and heated (37 ◦ C) Krebs solution with the following composition (mM): NaCl 119; KCl 4.5; MgSO4 2.5; NaHCO3 25; KH2 PO4 1.2; CaCl2 2.5; and glucose 11.1. The pyloric end was tied around the mouth of a J-tube, which was connected to a standard pressure transducer (Statham Mod. P23XL; Grass Medical Instruments, Quincy, MA, USA) and the changes of endoluminal pressure were recorded on ink-writer polygraph (Grass model 7D). Preparations were allowed to equilibrate for about 60 min before starting the experiment. Under these conditions, mouse stomach exhibits spontaneous small rhythmic contractions and basal tone, allowing testing the relaxant activity directly without the use of a contractile agent. At the beginning of each experiment, the preparation was challenged with isoproterenol (1 M), until reproducible responses were obtained, to ensure that a stable and acceptable level of sensitivity had been reached before the experimental procedure was begun. Isoproterenol was tested also at the end of the experiment to assess the relaxant ability of the preparations. Isoproterenol was added into the bath after switching off the perfusion and left in contact with the preparation for 2 min. The responses to non-cumulative concentrations of neuronostatin (0.01–3 nM) were examined on the gastric basal tone. Neuronostatin was added into the bath at increasing concentrations in volumes of 50 l at 45-min intervals. Each concentration was left in contact with the tissue for 7 min. In some experiments, to confirm the specificity of the observed effect, non-amidated neuronostatin (0.01–3 nM) was tested. The response to neuronostatin was also tested in presence of tetrodotoxin (TTX) (1 M), a voltage-dependent Na+ -channel blocker, which was added to the perfusing solution at least 30 min before testing the peptide. TTX concentration was proven to be effective at blocking response induced by electrical field stimulation [15]. Duodenal segment The mechanical activity of duodenal preparations was recorded as previously described [2]. In brief, the distal end of each segment
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was tied around the mouth of a J-tube, which was connected via a T catheter to a pressure transducer (Statham Mod. P23XL; Grass Medical Instruments, Quincy, MA, USA) and to a syringe for filling the preparation with Krebs solution. Each preparation was distended with 0.2 ml Krebs solution. The ligated proximal end was secured with a silk thread and preloaded to 0.5 g. The ligated proximal end was secured with a silk thread to an isometric force transducer (Grass FT03; Grass Instruments Co., Quincy, MA, USA). Mechanical activity was detected as changes in intraluminal pressure, which are mainly generated by circular muscle, and recorded on ink-writer polygraph (Grass Model 7D). To provide electrical field stimulation (EFS), a pair of platinum plates was placed in parallel on either side of the duodenal segment. EFS was applied by an S88 square wave pulse generator (Grass Medical Instruments, Quincy, MA, USA) coupled via a stimulus isolation unit (Grass SIU5) to the electrodes. Preparations were allowed to equilibrate for 60 min before starting the experiment. After the equilibration time, neuronostatin was added noncumulatively to the bath at increasing concentrations (0.01–3 nM) and the effects on the baseline tone and amplitude of phasic contractions were recorded over a period of 10 min. The interval between single concentrations was 40 min. In another set of preparations the influence of neuronostatin (0.01–3 nM) on the electrically evoked contractions was evaluated. Trains of stimuli (duration 5 s, supramaximal voltage, 8 Hz and 0.5 ms pulse duration) were applied to duodenal preparations at intervals of 70 s and stable and reproducible responses for a time-period of 3 h were observed. Under basal condition, EFS induced a cholinergic muscular contraction which was abolished by atropine (1 M) and TTX (1 M). After stable control cholinergic contractions had been recorded, the responses evoked by EFS were analysed in the presence of increasing concentrations of neuronostatin. The contact time for each concentration was 7 min. Data analysis and statistical tests All data are mean values ± SEM. The letter n indicates the number of experimental animals. Relaxant responses were expressed as a percentage of the response produced by isoproterenol (1 M). Concentration–response curves were computer fitted to a sigmoidal curve using non-linear regression (Prism 4.0, GraphPad Software, San Diego, CA, USA). Statistical analysis was performed by means of paired Student’s t-test or analysis of variance (ANOVA) followed by Bonferroni-test, when appropriate. A probability value of less than 0.05 was regarded as significant. Drugs The following drugs were used: neuronostatin and nonamidated neuronostatin (Phoenix Europe GMBH, Karlsruhe, Germany), isoproterenol hydrochloride (Sigma-RBI, Milano, Italy), TTX (Alomone Labs, Jerusalem, Israel). Each compound was prepared as a stock solution in distilled water. The working solutions were prepared fresh the day of the experiments by diluting the stock solutions in Krebs. Results Neuronostatin and food intake I.p. neuronostatin (1, 6, 10, 15, 30 ng/g b.w.) reduced significantly the food intake in the first hour postinjection when compared with PBS-treated mice (Fig. 1). This inhibitory effect resulted dose-related within the range 1–15 ng/g b.w. The lowest dose of peptide that had an effect was 6 ng/g b.w. whereas the dose of 15 ng/g b.w. was the most efficacious. In fact, 30 ng/g
Fig. 1. Effects of peripherally administered doses of neuronostatin, on food intake in the first hour postinjection. Vehicle (PBS) or neuronostatin (1, 6, 10, 15, 30 ng/g b.w.) were injected i.p. in mice and food intake was measured at the intervals 0–1. Data are means ± SEM (n = 7). *P < 0.05 vs. PBS. # P < 0.05 vs. 15 ng/g b.w. dose. Table 1 Effects of neuronostatin on food intake at different temporal intervals. Food-intake (g/period) PBS Neuronostatin (15 ng/g b.w.)
2–4
4–8
8–24
0.52 ± 0.03 0.48 ± 0.07
0.55 ± 0.08 0.59 ± 0.06
2.75 ± 0.41 2.64 ± 0.38
Data are means ± SEM of seven animals per group.
b.w. produced food intake inhibition significantly weaker than that produced by 15 ng/g b.w. However, the decrease in feeding was not maintained in the time. In fact, there was not observed any significant decrease in the food intake in the subsequent temporal intervals (Table 1). Non-amidated neuronostatin was used in order to verify the specificity of the effects observed. As shown in Table 2, i.p. non-amidated neuronostatin (15 ng/g b.w.) failed to affect significantly the food intake at the first hour (P > 0.05). Gastric emptying and intestinal transit To examine potential mechanisms underlying the peptide anorectic action, the rate of gastric emptying was determined in mice administered PBS or neuronostatin (15 ng/g b.w.). Mice injected with neuronostatin displayed a significant decrease in the rate of gastric emptying compared with PBS-treated mice (Fig. 2A). Intestinal transit rate, expressed as the ratio between the distance traveled by the phenol red meal and the total length of the small intestine was significantly decreased in mice treated with neuronostatin (15 ng/g b.w. i.p.) in comparison with PBS-treated animals (Fig. 2B). Once more, the animals treated with non-amidated neuronostatin (15 ng/g b.w.) did not show any significant difference in the gastric emptying rate or intestinal transit in comparison with the control mice (Table 2). In vitro studies: stomach As previously described [3,15] under the present experimental conditions gastric preparations showed spontaneous mechanical activity consisting of small phasic changes in endoluminal pressure (0.2–1 cm H2 0) with a frequency of about 6 contractions/min. Neuronostatin (0.01–3 nM) induced a relaxation which developed slowly, persisted throughout the contact time and were reversible after washing out (Fig. 3A). Disappearance of the spontaneous contractions was often observed depending on the preparations. The effect enhanced by increasing the concentration and the maximal response was obtained at the concentration of 1 nM (about 30% of the relaxation to 1 M isoproterenol) (Fig. 3B). Increasing
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Table 2 Effects of non-amidated neuronostatin on food intake, gastric emptying and intestinal transit.
PBS Non-amidated neuronostatin (15 ng/g b.w.)
Food-intake 0–1 h (g)
Gastric emptying (%)
Intestinal transit (%)
0.90 ± 0.06 (n = 4) 0.86 ± 0.03 (n = 4)
59.1 ± 5.6 (n = 5) 58.5 ± 1.6 (n = 5)
43.6 ± 1.2 (n = 5) 41.8 ± 0.9 (n = 5)
Fig. 2. Gastric emptying and small intestinal transit 20 min after gavage of 0.3 ml of phenol red meal in mice injected i.p. with 100 l of either vehicle (PBS) or neuronostatin (15 ng/g b.w.) 10 min before gastric load. Data are means ± SEM (n = 5). *P < 0.001.
concentrations of non-amidated neuronostatin (0.01–3 nM) did not produce any effect on the gastric spontaneous mechanical activity (Fig. 3B). Interestingly, the gastric relaxation induced by neuronostatin (1 nM) was completely abolished by pretreatment with TTX (1 M), a blocker of neural voltage-dependent Na+ channels. The TTX blocking action was reversible. The peptide (1 nM) was able to induce again gastric relaxation after washing out, even if smaller in amplitude (Fig. 4).
In vitro studies: duodenal segment As previously described [2], duodenal preparations showed spontaneous contractions, detected as changes in endoluminal pressure (1.3 ± 0.1 cm H2 O; n = 5). EFS (trains of 8 Hz for 5 s) induced muscular contractions with an amplitude of 2.9 ± 0.5 cm H2 O (n = 5) which was abolished by atropine (1 M) or TTX (1 M) suggesting their cholinergic origin. Neuronostatin (up to 3 nM) failed to affect the spontaneous mechanical activity (basal tone or the contraction amplitude) or the evoked contractions (Fig. 5).
Fig. 3. (A) Original tracings showing the relaxation induced by increasing concentrations of neuronostatin on isolated mouse gastric preparations. (B) Concentration–response curves for the relaxant effects induced by neuronostatin or non-amidated neuronostatin on isolated stomach. Data are expressed as a percentage of the relaxation induced by isoproterenol (ISO) (1 M) obtained in the same tissue. Data are means ± SEM (n = 5).
Discussion The results of the present study demonstrates that neuronostatin, peripherally administrated, is able to induce, in the mouse, reduction in food intake and gastrointestinal transit and to cause gastric relaxation through the enteric nervous system. Neuronostatin may exert multiple cardiovascular and metabolic actions in a variety of rodent tissues [13,19,25,30]. When injected into the lateral cerebroventricle of adult rats it induces a significant, dose-related, and biphasic increase in mean arterial pressure [19,25]. In addition, the peptide can directly alter the contractility of isolated cardiomyocytes and rat whole heart preparations [19,21]. Similarly, when centrally injected, neuronostatin leads to inhibition of food intake in rats [7,19,27] and gastrointestinal transit in mice [20], but, so far, experimental studies have not been performed to explore potential peripheral sites of action involved in the regulation of the gastrointestinal motility and food intake. In our experiments, neuronostatin, administered peripherally caused a significant decrease in food intake, confirming its
Fig. 4. Original tracings showing the effect of TTX (1 M) on the relaxation induced by neuronostatin (1 nM) in isolated mouse gastric preparations.
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Fig. 5. Typical recordings showing the lacking of effects of neuronostatin (1 nM) on: (A) spontaneous mechanical activity or (B) cholinergic contractile responses to EFS (train of 5 s, 0.5 ms, supramaximal voltage, 8 Hz) in circular muscle of mouse duodenal preparations.
ability to inhibit feeding. The effect induced by neuronostatin was dose-dependent, although 30 ng/g b.w. produced food intake inhibition weaker than that produced by 15 ng/g b.w., which could be interpreted as a bell-shaped dose–response curve. Although the mechanism responsible for this phenomenon is not clear, the observed bell-shaped dose–response curve for neuronostatin is similar to those described for other peptide biological activities, including antinociception [22], cardiovascular and gastrointestinal effects [19,20]. The administration of neuronostatin failed to alter locomotor activity (data not shown), therefore it is unlikely that the effect to suppress feeding was secondary to general malaise. Because the receptor for neuronostatin has not been identified yet, although the orphan G protein-coupled receptor, GPR107, has been proposed [26], to prove the specificity of the effect we injected mice with non-amidated neuronostatin. Indeed, different studies have demonstrated that C-terminal amidation of neuronostatin is essential for exerting hypertension activity, antinociceptive effects, depressive behavior and gastrointestinal motility regulation [20,22,23,25]. In our experimental protocols, non-amidated neuronostatin had no effect confirming the specificity of the neuronostatin action. However, inhibition of food intake after central administration of neuronostatin lasted through 4-h observation [19], while peripheral neuronostatin inhibitory effect on feeding appeared only in the first hour postinjection. In fact, neuronostatin failed to affect food intake in the long term, suggesting that the anorectic action is transient. The short duration of effect could be caused by a rapid degradation by endogenous peptidases, but unfortunately, up to date the half-life of the peptide is still unknown. Alternatively, it could be suggesting that exogenous peptide has an influence on the food intake in the short time. Because the regulation of the food intake in the short time relates primarily to gastric distension and more generally to gut motor function [8,12,14], we explored whether neuronostatin peripheral administration modifies gastric emptying and intestinal transit. Here, we found that mice treated with the peptide, at the same dose able to inhibit the food intake, showed delayed gastric emptying in comparison with control animals. This observation supports the hypothesis that the decrease in gastric emptying may contribute to the short-term reduction in food intake induced by neuronostatin. In fact, it is well known that delayed gastric emptying implies a prolonged distension of the stomach and hence a prolonged activation of mechanoreceptors,
leading to sensations of fullness and satiety [12,28]. Moreover, we have ruled out that the reduction in gastric emptying is due to nonspecific action because once more non-amidated neuronostatin was without any effect. Previously, other anorexigenic peptides produced within the gastrointestinal system, such as cholecystokinin, glucagon like peptide 1 and glucagon like peptide 2, were shown to be able to regulate at the same time the ingestive behavior and the gastric motor function in order to control energy homeostasis and metabolism [1,4–6,11]. Interestingly, we found also that the intestinal distance traveled by the test meal was reduced in mice treated with neuronostatin, further confirming that the peptide may play a role in regulating motor gastrointestinal function. Our data strengthen the hypothesis that neuronostatin can play an inhibitory role in the control of gut motor functions, as previously described after central administration of the peptide [20]. On the basis of these results, we cannot conclude about the mechanism of action, i.e. whether the neuronostatin inhibitory effects on food intake and gut motility directly involves some of the brain centers regulating food intake or if it is indirect through peripheral sites. Indeed, participation of central melanocortin system has been reported to be responsible for anorexigenic and motor gastrointestinal effects caused by intracerebroventricular administration of the peptide [20,27]. Thus, we performed further experiments in vitro in mouse stomach and duodenum to analyze the effects of the exogenous peptide on gut smooth muscle function under conditions where the influence of external factors is removed and consequently to reveal the existence of peripheral action sites. Our results showed that neuronostatin induced a concentrationdependent reduction of mouse gastric tone in absence of extrinsic hormonal or neural influence suggesting that the peptide is able to act peripherically to control gastric motility in the mouse. This supports the hypothesis that neuronostatin can act also in the stomach to influence indirectly food intake. We cannot state if the range of effective concentrations of neuronostatin is physiological or pharmacological, because so far the murine plasma levels have not been determined. Indeed, higher concentrations of neurostatin are required to elicit significant effects on glucagon secretion in vitro [18] in agreement with the concentrations of neuronostatin in the pancreatic tissue [19]. It is also possible to speculate that the peptide acts with paracrine mechanism near its site of production. In fact, neuronostatin is expressed in gastric parietal cells of oxyntic mucosa and could serve as a paracrine chemical mediator [19]. In order to clarify whether neuronostatin induces gastric relaxation via a direct action on the smooth muscle cells and/or via an indirect action, involving intramural neural pathways, we tested the effects of neuronostatin after pre-treatment of the preparation with TTX. The observation that TTX abolished the gastric relaxation induced by the peptide suggests that the action of the peptide depends on an action potential-dependent mechanism in the gastric enteric neurons. The neuronostatin neural effect on the enteric nervous system is in agreement with the reported ability of neuronostatin to cause depolarization in cultured hypothalamic neurons [10,19]. Moreover, it is interesting to note that neuronostatin failed to affect the spontaneous or evoked mechanical activity of duodenal segment in vitro. A plausible explanation is that there are not peptide binding sites/receptors, which once activated are involved in the regulation of motility, in this intestinal segment. Therefore the hypothetical receptor could be expressed in a different manner depending on the gut region. Accordingly, previous studies were unable to detect the GPR107, the candidate orphan receptor for neuronostatin in rat duodenum [26]. The neuronostatin ability to evoke only gastric relaxation without any influence on duodenum contractility could support the hypothesis that neuronostatin acts as a paracrine factor at peripheral level. The reduction of the
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intestinal transit observed in vivo conditions could be the consequence of a weaker propulsive activity due to the reduced gastric emptying. In conclusion, our results show for the first time that the exogenous neuronostatin is able to act peripherally in mouse stomach to reduce gastric emptying. This action could cause indirectly reduction of food intake in the short term. Acknowledgements This work was supported by a grant from Palermo University (FFR 2009), Italy. References [1] Amato A, Baldassano S, Serio R, Mulè F. Glucagon-like peptide-2 relaxes mouse stomach through vasoactive intestinal peptide release. Am J Physiol: Gastrointest Liver Physiol 2009;296:G678–84. [2] Amato A, Cinci L, Rotondo A, Serio R, Faussone-Pellegrini MS, Vannucchi MG, et al. Peripheral motor action of glucagon-like peptide-1 through enteric neuronal receptors. Neurogastroenterol Motil 2010;22:664–72. [3] Amira S, Rotondo A, Mulè F. Relaxant effects of flavonoids on the mouse isolated stomach: structure–activity relationships. Eur J Pharmacol 2008;599: 126–30. [4] Baldassano S, Amato A. GLP-2: what do we know? What are we going to discover? Regul Pept 2014, http://dx.doi.org/10.1016/j.regpep.2014.09.002 [Epub ahead of print]. [5] Baldassano S, Bellanca A, Serio R, Mulè F. Food intake in lean and obese mice after peripheral administration of glucagon-like peptide-2. J Endocrinol 2012;213:277–84. [6] Bruen CM, O’Halloran F, Cashman KD, Giblin L. The effects of food components on hormonal signalling in gastrointestinal enteroendocrine cells. Food Funct 2012;3:1131–43. [7] Carlini VP, Ghersi M, Gabach L, Schiöth HB, Pérez MF, Ramirez OA, et al. Hippocampal effects of neuronostatin on memory, anxiety-like behaviour and food intake in rats. Neuroscience 2011;197:145–52. [8] Clegg M, Shafat A. Energy and macronutrient composition of breakfast affect gastric emptying of lunch and subsequent food intake, satiety and satiation. Appetite 2010;54:517–23. [9] Cummings DE, Overduin J. Gastrointestinal regulation of food intake. J Clin Invest 2007;117:13–23. [10] Dun SL, Brailoiu GC, Tica AA, Yang J, Chang JK, Brailoiu E, et al. Neuronostatin is co-expressed with somatostatin and mobilizes calcium in cultured rat hypothalamic neurons. Neuroscience 2010;166:455–63. [11] Janssen P, Rotondo A, Mulé F, Tack J. A comparison of glucagon-like peptides 1 and 2. Aliment Pharmacol Ther 2013;37:18–36.
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[12] Janssen P, Vanden Berghe P, Verschueren S, Lehmann A, Depoortere I, Tack J. The role of gastric motility in the control of food intake. Aliment Pharmacol Ther 2011;33:880–94. [13] Hua Y, Ma H, Samson WK, Ren J. Neuronostatin inhibits cardiac contractile function via a protein kinase A- and JNK-dependent mechanism in murine hearts. Am J Physiol: Regul Integr Comp Physiol 2009;297:R682–9. [14] Moran TH, Dailey MJ. Intestinal feedback signaling and satiety. Physiol Behav 2011;105:77–81. [15] Mulè F, Amato A, Baldassano S, Serio R. Involvement of CB1 and CB2 receptors in the modulation of cholinergic neurotransmission in mouse gastric preparations. Pharmacol Res 2007;56:185–92. [16] Mulè F, Amato A, Serio R. Gastric emptying, small intestinal transit and fecal output in dystrophic (mdx) mice. J Physiol Sci 2010;60:75–9. [17] Rotondo A, Amato A, Lentini L, Baldassano S, Mulè F. Glucagon-like peptide-1 relaxes gastric antrum through nitric oxide in mice. Peptides 2011;32:60–4. [18] Salvatori AS, Elrick MM, Samson WK, Corbett JA, Yosten GL. Neuronostatin inhibits glucose-stimulated insulin secretion via direct action on the pancreatic ␣-cell. Am J Physiol: Endocrinol Metab 2014;306:E1257–63. [19] Samson WK, Zhang JV, Avsian-Kretchmer O, Cui K, Yosten GLC, Klein C, et al. Neuronostatin encoded by the somatostatin gene regulates neuronal, cardiovascular, and metabolic functions. J Biol Chem 2008;283:31949–59. [20] Su SF, Yang AM, Yang SB, Wang NB, Lu SS, Wang HH, et al. Intracerebroventricular administration of neuronostatin delays gastric emptying and gastrointestinal transit in mice. Peptides 2012;35:31–5. [21] Vainio L, Perjes A, Ryti N, Magga J, Alakoski T, Serpi R, et al. Neuronostatin, a novel peptide encoded by somatostatin gene, regulates cardiac contractile function and cardiomyocyte survival. J Biol Chem 2012;287:4572–80. [22] Yang AM, Ge WW, Lu SS, Yang SB, Su SF, Mi ZY, et al. Central administration of neuronostatin induces antinociception in mice. Peptides 2011;32:1893–901. [23] Yang AM, Ji YK, Su SF, Yang SB, Lu SS, Mi ZY, et al. Intracerebroventricular administration of neuronostatin induces depression-like effect in forced swim test of mice. Peptides 2011;32:1948–52. [24] Yosten GL. Novel neuropeptides in the control of food intake: neuronostatin and nesfatin-1. Vitam Horm 2013;92:1–25. [25] Yosten GL, Pate AT, Samson WK. Neuronostatin acts in brain to biphasically increase mean arterial pressure through sympatho-activation followed by vasopressin secretion: the role of melanocortin receptors. Am J Physiol: Regul Integr Comp Physiol 2011;300:R1194–9. [26] Yosten GL, Redlinger LJ, Samson WK. Evidence for an interaction of neuronostatin with the orphan G protein-coupled receptor, GPR107. Am J Physiol: Regul Integr Comp Physiol 2012;303:R941–9. [27] Yosten GL, Samson WK. The melanocortins, not oxytocin, mediate the anorexigenic and antidipsogenic effects of neuronostatin. Peptides 2010;31:1711–4. [28] Wang GJ, Tomasi D, Backus W, Wang R, Telang A, Geliebter J, et al. Gastric distension activates satiety circuitry in the human brain. Neuroimage 2008;39:1824–31. [29] Wren AM, Bloom SR. Gut hormones and appetite control. Gastroenterology 2007;132:2116–30. [30] Zhu X, Hu N, Chen X, Zhu MZ, Dong H, Xu X, et al. Neuronostatin attenuates myocardial contractile function through inhibition of sarcoplasmic reticulum Ca2+ -ATPase in murine heart. Cell Physiol Biochem 2014;33:1921–32.
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Neuronostatin: Peripheral site of action in mouse stomach Antonella Amato, Sara Baldassano, Gaetano Caldara, Flavia Mulè ∗ Dipartimento di Scienze e Tecnologie Molecolari e Biomolecolari (STEMBIO), Università di Palermo, 90128 Palermo, Italy
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Article history: Received 3 November 2014 Received in revised form 12 December 2014 Accepted 12 December 2014 Available online 22 December 2014 Keywords: Neuronostatin Food intake Gastric emptying Intestinal transit
a b s t r a c t Neuronostatin is a 13-amino acid peptide encoded by somatostatin gene. It is distributed in different organs including gastrointestinal tract and has been involved in the control of food intake and gastrointestinal motility, likely through an action in the brain. So far, there are no reports about the occurrence of peripheral action sites in the gut. Therefore, the purpose of the present study was to examine, in the mouse, the effects of peripheral administration of neuronostatin on food intake within 24 h and on gastrointestinal motility and to analyse neuronostatin actions on the gastric and intestinal mechanical activity in isolated preparations in vitro. When compared with PBS-treated mice, intraperitoneal neuronostatin reduced food intake in doses ranging from 1 to 15 ng/g b.w. only in the first hour postinjection with a maximum effect obtained at the dose of 15 ng/g b.w. (−46.9%). The peptide (15 ng/g b.w.) significantly reduced gastric emptying rate (−31.1%) and gastrointestinal intestinal transit. Non-amidated neuronostatin failed to affect food intake, gastric emptying and intestinal transit, suggesting the specificity of action. In vitro, neuronostatin induced concentration-dependent gastric relaxation, which was abolished by tetrodotoxin. Neuronostatin failed to affect the spontaneous mechanical activity or the evoked cholinergic contractions in duodenum. These results suggest that exogenous neuronostatin is able to reduce mouse gastric motility by acting peripherally in the stomach, through intramural nervous plexuses. This indirectly action could cause reduction of food intake in the short term. © 2014 Elsevier Inc. All rights reserved.
Introduction Neuronostatin is a recently described 13-amino acid peptide hormone; which is encoded by somatostatin gene and derived from the N-terminus of pro-somatostatin. Somatostatin is a peptide hormone; originally identified in hypothalamus and subsequently found in neuroendocrine organs; gastrointestinal tract; thyroid and adrenal glands; liver; kidney; inflammatory and immune cells. As expected, neuronostatin has been found to be expressed in the same tissues as somatostatin, including brain and gastrointestinal tissues with the highest level in the pancreas followed by cerebrum and hypothalamus [19]. As somatostatin, neuronostatin is considered a brain/gut peptide due to its site of production and its ability to induce early response genes c-Fos or c-Jun in neuronal, anterior pituitary gastrointestinal tissues [19] and cardiomyocytes
Abbreviations: EFS, electrical field stimulation; GE, gastric emptying; i.c.v., intracerebroventricular; i.p., intraperitoneal; PBS, phosphate-buffered saline; TTX, tetrodotoxin. ∗ Corresponding author at: Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farrmaceutiche (STEBICEF), Laboratorio di Fisiologia generale, Università di Palermo, Viale delle Scienze, 90128 Palermo, Italy. Tel.: +39 91 23897515; fax: +39 91 6577501. E-mail address: fl[email protected] (F. Mulè). http://dx.doi.org/10.1016/j.peptides.2014.12.003 0196-9781/© 2014 Elsevier Inc. All rights reserved.
[13]. Despite sharing common biological effects, such as inhibitory control of cardiac activity, pancreatic and gastrointestinal functions, neuronostatin and somatostatin possess biological activities that are distinct from each other [24]. Furthermore neuronostatin has been reported not to interact with the five putative somatostatin receptors [19]. Indeed, when injected centrally in rats neuronostatin leads to a dose-related inhibition of food and water intake and an increase in mean arterial pressure [25,27]. Intracerebroventricular (i.c.v.) injections of neuronostatin in mice produce antinociceptive effect via the central melanocortin and opioid systems [22] and a depression-like effect via the central melanocortin system [23]. Neuronostatin can modify the hypothalamic neuron firing and neuronal migration [10,19] and it is able to regulate cardiac and cardiomyocyte contractile function and cardiomyocyte survival [13,21,30]. Recent studies in isolated rat pancreatic islets or in the animals in vivo suggest also a role of neuronostatin in maintaining glucose homeostasis through inhibition of glucose-stimulated insulin secretion [18]. Neuronostatin has been also involved in the control of gastrointestinal motility. In fact, i.c.v. administration of neuronostatin in mice delays the gastric emptying and the gastrointestinal transit [20]. However, to date, there are no reports about the effects of peripheral administration of neuronostatin on food intake and
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gastrointestinal motility. It is likely to hypothesize that there are peripheral sites of action involved in the biological effects of the neuronostatin, similarly to other peptides involved in the control of food intake and gastrointestinal functions [2,5,9,17,29]. Unlikely, the neuronostatin receptor and its distribution are still unknown, although the orphan G protein-coupled receptor, GPR107, has been proposed [26]. Therefore the purpose of the present study was to examine if intraperitoneal administration of neuronostatin influences the food intake within 24 h and the gastrointestinal motility in mouse. Moreover, to better elucidate the gastrointestinal actions of neuronostatin, the effects of the exogenous peptide on the gastric and intestinal mechanical activities in vitro were examined and the mechanism of action responsible of the observed effects was studied. Materials and methods
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to precipitate proteins. After centrifugation (3000 rpm for 30 min at 4 ◦ C) the supernatant was added to 2 ml of 2 N NaOH to develop the maximum color intensity. The amount of phenol red was determined from the absorbency at 560 nm. This correlates with the concentration of phenol red in the stomach, which in turn depends on the gastric emptying. The gastric emptying (GE) rate was calculated as GE = (1 − X/Y)100, where X is absorbance of phenol red recovered from the stomach of animals sacrificed 20 min after test meal. Y is mean (n = 4) absorbance of phenol red recovered from the stomachs of animals killed at 0 min following test meal. Immediately after the excision of the stomach, the whole small intestine was grossly freed from its mesenteric attachments and its length (from the pyloric sphincter to the ileocecal junction) was measured. The intestine was opened at the level of the front of the test meal, which was revealed by few drops of 0.1 N NaOH. The rate of intestinal transit was expressed as the ratio between the distance traveled by the test meal and the total length of intestine, as previously described [16].
Animals Functional studies in vitro The experimental procedures employed in the present study were in conformity with the Italian D.L. No. 116 of 27 January 1992 and subsequent variations and the recommendations of the European Economic Community (86/609/ECC). The studies were approved by Ministero della Sanità (Rome, Italy). Adult male C57BL/6J mice, purchased from Harlan Laboratories (San Pietro al Natisone, Udine, Italy) were singly housed under controlled environmental conditions (22 + 1 ◦ C, 55 + 15% relative humidity, 12 h light). Tap water and standard laboratory rodent chow (Mucedola, Settimo Milanese, MI, Italy) were provided ad libitum; except as otherwise stated. Food intake Fasted (16 h) mice were injected with intraperitoneal (i.p.) 100 l of either vehicle (phosphate-buffered saline, PBS) or neuronostatin (1, 6, 10, 15, 30 ng/g b.w.), in the early light phase (0800–0900 h). Prior to the initial study, mice received a daily i.p. injection of 100 l PBS for 7 days to habituate them to the procedure. Doses of neuronostatin were determined on the basis of the literature [30] and from preliminary experiments in our laboratory. A minimum of 72 h was allowed between each trial in the same mouse. Following injection, each mouse was returned to its home cage with a pre-weighed amount of chow. The food intake was determined at 1, 2, 4, 8, and 24 h following peptide or vehicle administration, by measuring the difference between the pre-weighed chow and the weight of chow at the end of each time interval. Any spillage was collected and weighed. Gastric emptying and intestinal transit To examine gastric emptying and small intestinal transit, the animals were deprived of food for 24 h before the experiments began. Then, mice were injected with i.p. 100 l of either PBS or neuronostatin (15 ng/g b.w.) 10 min before gastric load. This dose was chosen because it induced maximum effect in the food intake experiments. The experiments to assess gastric emptying and transit were conducted as previously reported [16]. Briefly, mice received by gavage 0.3 ml of test meal (a non-nutrient meal of 50 mg phenol red in 100 ml 1.5% carboxymethylcellulose) and were euthanized by cervical dislocation immediately (t = 0) or 20 min after gavaging. Under laparatomy, the stomach and the small intestine were excised after legation of the pylorus and the cardias. The stomach and its contents were homogenized in 25 ml of 0.1 N NaOH. The mixture was then kept for 1 h at room temperature. Then, 8 ml of the supernatant was added to 1 ml of 33% of trichloroacetic acid
After animal sacrifice, the abdomen was immediately opened, the esophagus was tied just below the lower oesophageal sphincter, and stomach and duodenum were excised. Isolated stomach As previously described [17], whole stomach was used in order to examine the muscle function under conditions where the influence of external factors is removed, but the muscle performs in a manner analogous to its in vivo capacity. The entire stomach was mounted in a custom designed organ bath, which was continuously perfused with oxygenated (95% O2 and 5% CO2 ) and heated (37 ◦ C) Krebs solution with the following composition (mM): NaCl 119; KCl 4.5; MgSO4 2.5; NaHCO3 25; KH2 PO4 1.2; CaCl2 2.5; and glucose 11.1. The pyloric end was tied around the mouth of a J-tube, which was connected to a standard pressure transducer (Statham Mod. P23XL; Grass Medical Instruments, Quincy, MA, USA) and the changes of endoluminal pressure were recorded on ink-writer polygraph (Grass model 7D). Preparations were allowed to equilibrate for about 60 min before starting the experiment. Under these conditions, mouse stomach exhibits spontaneous small rhythmic contractions and basal tone, allowing testing the relaxant activity directly without the use of a contractile agent. At the beginning of each experiment, the preparation was challenged with isoproterenol (1 M), until reproducible responses were obtained, to ensure that a stable and acceptable level of sensitivity had been reached before the experimental procedure was begun. Isoproterenol was tested also at the end of the experiment to assess the relaxant ability of the preparations. Isoproterenol was added into the bath after switching off the perfusion and left in contact with the preparation for 2 min. The responses to non-cumulative concentrations of neuronostatin (0.01–3 nM) were examined on the gastric basal tone. Neuronostatin was added into the bath at increasing concentrations in volumes of 50 l at 45-min intervals. Each concentration was left in contact with the tissue for 7 min. In some experiments, to confirm the specificity of the observed effect, non-amidated neuronostatin (0.01–3 nM) was tested. The response to neuronostatin was also tested in presence of tetrodotoxin (TTX) (1 M), a voltage-dependent Na+ -channel blocker, which was added to the perfusing solution at least 30 min before testing the peptide. TTX concentration was proven to be effective at blocking response induced by electrical field stimulation [15]. Duodenal segment The mechanical activity of duodenal preparations was recorded as previously described [2]. In brief, the distal end of each segment
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was tied around the mouth of a J-tube, which was connected via a T catheter to a pressure transducer (Statham Mod. P23XL; Grass Medical Instruments, Quincy, MA, USA) and to a syringe for filling the preparation with Krebs solution. Each preparation was distended with 0.2 ml Krebs solution. The ligated proximal end was secured with a silk thread and preloaded to 0.5 g. The ligated proximal end was secured with a silk thread to an isometric force transducer (Grass FT03; Grass Instruments Co., Quincy, MA, USA). Mechanical activity was detected as changes in intraluminal pressure, which are mainly generated by circular muscle, and recorded on ink-writer polygraph (Grass Model 7D). To provide electrical field stimulation (EFS), a pair of platinum plates was placed in parallel on either side of the duodenal segment. EFS was applied by an S88 square wave pulse generator (Grass Medical Instruments, Quincy, MA, USA) coupled via a stimulus isolation unit (Grass SIU5) to the electrodes. Preparations were allowed to equilibrate for 60 min before starting the experiment. After the equilibration time, neuronostatin was added noncumulatively to the bath at increasing concentrations (0.01–3 nM) and the effects on the baseline tone and amplitude of phasic contractions were recorded over a period of 10 min. The interval between single concentrations was 40 min. In another set of preparations the influence of neuronostatin (0.01–3 nM) on the electrically evoked contractions was evaluated. Trains of stimuli (duration 5 s, supramaximal voltage, 8 Hz and 0.5 ms pulse duration) were applied to duodenal preparations at intervals of 70 s and stable and reproducible responses for a time-period of 3 h were observed. Under basal condition, EFS induced a cholinergic muscular contraction which was abolished by atropine (1 M) and TTX (1 M). After stable control cholinergic contractions had been recorded, the responses evoked by EFS were analysed in the presence of increasing concentrations of neuronostatin. The contact time for each concentration was 7 min. Data analysis and statistical tests All data are mean values ± SEM. The letter n indicates the number of experimental animals. Relaxant responses were expressed as a percentage of the response produced by isoproterenol (1 M). Concentration–response curves were computer fitted to a sigmoidal curve using non-linear regression (Prism 4.0, GraphPad Software, San Diego, CA, USA). Statistical analysis was performed by means of paired Student’s t-test or analysis of variance (ANOVA) followed by Bonferroni-test, when appropriate. A probability value of less than 0.05 was regarded as significant. Drugs The following drugs were used: neuronostatin and nonamidated neuronostatin (Phoenix Europe GMBH, Karlsruhe, Germany), isoproterenol hydrochloride (Sigma-RBI, Milano, Italy), TTX (Alomone Labs, Jerusalem, Israel). Each compound was prepared as a stock solution in distilled water. The working solutions were prepared fresh the day of the experiments by diluting the stock solutions in Krebs. Results Neuronostatin and food intake I.p. neuronostatin (1, 6, 10, 15, 30 ng/g b.w.) reduced significantly the food intake in the first hour postinjection when compared with PBS-treated mice (Fig. 1). This inhibitory effect resulted dose-related within the range 1–15 ng/g b.w. The lowest dose of peptide that had an effect was 6 ng/g b.w. whereas the dose of 15 ng/g b.w. was the most efficacious. In fact, 30 ng/g
Fig. 1. Effects of peripherally administered doses of neuronostatin, on food intake in the first hour postinjection. Vehicle (PBS) or neuronostatin (1, 6, 10, 15, 30 ng/g b.w.) were injected i.p. in mice and food intake was measured at the intervals 0–1. Data are means ± SEM (n = 7). *P < 0.05 vs. PBS. # P < 0.05 vs. 15 ng/g b.w. dose. Table 1 Effects of neuronostatin on food intake at different temporal intervals. Food-intake (g/period) PBS Neuronostatin (15 ng/g b.w.)
2–4
4–8
8–24
0.52 ± 0.03 0.48 ± 0.07
0.55 ± 0.08 0.59 ± 0.06
2.75 ± 0.41 2.64 ± 0.38
Data are means ± SEM of seven animals per group.
b.w. produced food intake inhibition significantly weaker than that produced by 15 ng/g b.w. However, the decrease in feeding was not maintained in the time. In fact, there was not observed any significant decrease in the food intake in the subsequent temporal intervals (Table 1). Non-amidated neuronostatin was used in order to verify the specificity of the effects observed. As shown in Table 2, i.p. non-amidated neuronostatin (15 ng/g b.w.) failed to affect significantly the food intake at the first hour (P > 0.05). Gastric emptying and intestinal transit To examine potential mechanisms underlying the peptide anorectic action, the rate of gastric emptying was determined in mice administered PBS or neuronostatin (15 ng/g b.w.). Mice injected with neuronostatin displayed a significant decrease in the rate of gastric emptying compared with PBS-treated mice (Fig. 2A). Intestinal transit rate, expressed as the ratio between the distance traveled by the phenol red meal and the total length of the small intestine was significantly decreased in mice treated with neuronostatin (15 ng/g b.w. i.p.) in comparison with PBS-treated animals (Fig. 2B). Once more, the animals treated with non-amidated neuronostatin (15 ng/g b.w.) did not show any significant difference in the gastric emptying rate or intestinal transit in comparison with the control mice (Table 2). In vitro studies: stomach As previously described [3,15] under the present experimental conditions gastric preparations showed spontaneous mechanical activity consisting of small phasic changes in endoluminal pressure (0.2–1 cm H2 0) with a frequency of about 6 contractions/min. Neuronostatin (0.01–3 nM) induced a relaxation which developed slowly, persisted throughout the contact time and were reversible after washing out (Fig. 3A). Disappearance of the spontaneous contractions was often observed depending on the preparations. The effect enhanced by increasing the concentration and the maximal response was obtained at the concentration of 1 nM (about 30% of the relaxation to 1 M isoproterenol) (Fig. 3B). Increasing
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Table 2 Effects of non-amidated neuronostatin on food intake, gastric emptying and intestinal transit.
PBS Non-amidated neuronostatin (15 ng/g b.w.)
Food-intake 0–1 h (g)
Gastric emptying (%)
Intestinal transit (%)
0.90 ± 0.06 (n = 4) 0.86 ± 0.03 (n = 4)
59.1 ± 5.6 (n = 5) 58.5 ± 1.6 (n = 5)
43.6 ± 1.2 (n = 5) 41.8 ± 0.9 (n = 5)
Fig. 2. Gastric emptying and small intestinal transit 20 min after gavage of 0.3 ml of phenol red meal in mice injected i.p. with 100 l of either vehicle (PBS) or neuronostatin (15 ng/g b.w.) 10 min before gastric load. Data are means ± SEM (n = 5). *P < 0.001.
concentrations of non-amidated neuronostatin (0.01–3 nM) did not produce any effect on the gastric spontaneous mechanical activity (Fig. 3B). Interestingly, the gastric relaxation induced by neuronostatin (1 nM) was completely abolished by pretreatment with TTX (1 M), a blocker of neural voltage-dependent Na+ channels. The TTX blocking action was reversible. The peptide (1 nM) was able to induce again gastric relaxation after washing out, even if smaller in amplitude (Fig. 4).
In vitro studies: duodenal segment As previously described [2], duodenal preparations showed spontaneous contractions, detected as changes in endoluminal pressure (1.3 ± 0.1 cm H2 O; n = 5). EFS (trains of 8 Hz for 5 s) induced muscular contractions with an amplitude of 2.9 ± 0.5 cm H2 O (n = 5) which was abolished by atropine (1 M) or TTX (1 M) suggesting their cholinergic origin. Neuronostatin (up to 3 nM) failed to affect the spontaneous mechanical activity (basal tone or the contraction amplitude) or the evoked contractions (Fig. 5).
Fig. 3. (A) Original tracings showing the relaxation induced by increasing concentrations of neuronostatin on isolated mouse gastric preparations. (B) Concentration–response curves for the relaxant effects induced by neuronostatin or non-amidated neuronostatin on isolated stomach. Data are expressed as a percentage of the relaxation induced by isoproterenol (ISO) (1 M) obtained in the same tissue. Data are means ± SEM (n = 5).
Discussion The results of the present study demonstrates that neuronostatin, peripherally administrated, is able to induce, in the mouse, reduction in food intake and gastrointestinal transit and to cause gastric relaxation through the enteric nervous system. Neuronostatin may exert multiple cardiovascular and metabolic actions in a variety of rodent tissues [13,19,25,30]. When injected into the lateral cerebroventricle of adult rats it induces a significant, dose-related, and biphasic increase in mean arterial pressure [19,25]. In addition, the peptide can directly alter the contractility of isolated cardiomyocytes and rat whole heart preparations [19,21]. Similarly, when centrally injected, neuronostatin leads to inhibition of food intake in rats [7,19,27] and gastrointestinal transit in mice [20], but, so far, experimental studies have not been performed to explore potential peripheral sites of action involved in the regulation of the gastrointestinal motility and food intake. In our experiments, neuronostatin, administered peripherally caused a significant decrease in food intake, confirming its
Fig. 4. Original tracings showing the effect of TTX (1 M) on the relaxation induced by neuronostatin (1 nM) in isolated mouse gastric preparations.
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Fig. 5. Typical recordings showing the lacking of effects of neuronostatin (1 nM) on: (A) spontaneous mechanical activity or (B) cholinergic contractile responses to EFS (train of 5 s, 0.5 ms, supramaximal voltage, 8 Hz) in circular muscle of mouse duodenal preparations.
ability to inhibit feeding. The effect induced by neuronostatin was dose-dependent, although 30 ng/g b.w. produced food intake inhibition weaker than that produced by 15 ng/g b.w., which could be interpreted as a bell-shaped dose–response curve. Although the mechanism responsible for this phenomenon is not clear, the observed bell-shaped dose–response curve for neuronostatin is similar to those described for other peptide biological activities, including antinociception [22], cardiovascular and gastrointestinal effects [19,20]. The administration of neuronostatin failed to alter locomotor activity (data not shown), therefore it is unlikely that the effect to suppress feeding was secondary to general malaise. Because the receptor for neuronostatin has not been identified yet, although the orphan G protein-coupled receptor, GPR107, has been proposed [26], to prove the specificity of the effect we injected mice with non-amidated neuronostatin. Indeed, different studies have demonstrated that C-terminal amidation of neuronostatin is essential for exerting hypertension activity, antinociceptive effects, depressive behavior and gastrointestinal motility regulation [20,22,23,25]. In our experimental protocols, non-amidated neuronostatin had no effect confirming the specificity of the neuronostatin action. However, inhibition of food intake after central administration of neuronostatin lasted through 4-h observation [19], while peripheral neuronostatin inhibitory effect on feeding appeared only in the first hour postinjection. In fact, neuronostatin failed to affect food intake in the long term, suggesting that the anorectic action is transient. The short duration of effect could be caused by a rapid degradation by endogenous peptidases, but unfortunately, up to date the half-life of the peptide is still unknown. Alternatively, it could be suggesting that exogenous peptide has an influence on the food intake in the short time. Because the regulation of the food intake in the short time relates primarily to gastric distension and more generally to gut motor function [8,12,14], we explored whether neuronostatin peripheral administration modifies gastric emptying and intestinal transit. Here, we found that mice treated with the peptide, at the same dose able to inhibit the food intake, showed delayed gastric emptying in comparison with control animals. This observation supports the hypothesis that the decrease in gastric emptying may contribute to the short-term reduction in food intake induced by neuronostatin. In fact, it is well known that delayed gastric emptying implies a prolonged distension of the stomach and hence a prolonged activation of mechanoreceptors,
leading to sensations of fullness and satiety [12,28]. Moreover, we have ruled out that the reduction in gastric emptying is due to nonspecific action because once more non-amidated neuronostatin was without any effect. Previously, other anorexigenic peptides produced within the gastrointestinal system, such as cholecystokinin, glucagon like peptide 1 and glucagon like peptide 2, were shown to be able to regulate at the same time the ingestive behavior and the gastric motor function in order to control energy homeostasis and metabolism [1,4–6,11]. Interestingly, we found also that the intestinal distance traveled by the test meal was reduced in mice treated with neuronostatin, further confirming that the peptide may play a role in regulating motor gastrointestinal function. Our data strengthen the hypothesis that neuronostatin can play an inhibitory role in the control of gut motor functions, as previously described after central administration of the peptide [20]. On the basis of these results, we cannot conclude about the mechanism of action, i.e. whether the neuronostatin inhibitory effects on food intake and gut motility directly involves some of the brain centers regulating food intake or if it is indirect through peripheral sites. Indeed, participation of central melanocortin system has been reported to be responsible for anorexigenic and motor gastrointestinal effects caused by intracerebroventricular administration of the peptide [20,27]. Thus, we performed further experiments in vitro in mouse stomach and duodenum to analyze the effects of the exogenous peptide on gut smooth muscle function under conditions where the influence of external factors is removed and consequently to reveal the existence of peripheral action sites. Our results showed that neuronostatin induced a concentrationdependent reduction of mouse gastric tone in absence of extrinsic hormonal or neural influence suggesting that the peptide is able to act peripherically to control gastric motility in the mouse. This supports the hypothesis that neuronostatin can act also in the stomach to influence indirectly food intake. We cannot state if the range of effective concentrations of neuronostatin is physiological or pharmacological, because so far the murine plasma levels have not been determined. Indeed, higher concentrations of neurostatin are required to elicit significant effects on glucagon secretion in vitro [18] in agreement with the concentrations of neuronostatin in the pancreatic tissue [19]. It is also possible to speculate that the peptide acts with paracrine mechanism near its site of production. In fact, neuronostatin is expressed in gastric parietal cells of oxyntic mucosa and could serve as a paracrine chemical mediator [19]. In order to clarify whether neuronostatin induces gastric relaxation via a direct action on the smooth muscle cells and/or via an indirect action, involving intramural neural pathways, we tested the effects of neuronostatin after pre-treatment of the preparation with TTX. The observation that TTX abolished the gastric relaxation induced by the peptide suggests that the action of the peptide depends on an action potential-dependent mechanism in the gastric enteric neurons. The neuronostatin neural effect on the enteric nervous system is in agreement with the reported ability of neuronostatin to cause depolarization in cultured hypothalamic neurons [10,19]. Moreover, it is interesting to note that neuronostatin failed to affect the spontaneous or evoked mechanical activity of duodenal segment in vitro. A plausible explanation is that there are not peptide binding sites/receptors, which once activated are involved in the regulation of motility, in this intestinal segment. Therefore the hypothetical receptor could be expressed in a different manner depending on the gut region. Accordingly, previous studies were unable to detect the GPR107, the candidate orphan receptor for neuronostatin in rat duodenum [26]. The neuronostatin ability to evoke only gastric relaxation without any influence on duodenum contractility could support the hypothesis that neuronostatin acts as a paracrine factor at peripheral level. The reduction of the
A. Amato et al. / Peptides 64 (2015) 8–13
intestinal transit observed in vivo conditions could be the consequence of a weaker propulsive activity due to the reduced gastric emptying. In conclusion, our results show for the first time that the exogenous neuronostatin is able to act peripherally in mouse stomach to reduce gastric emptying. This action could cause indirectly reduction of food intake in the short term. Acknowledgements This work was supported by a grant from Palermo University (FFR 2009), Italy. References [1] Amato A, Baldassano S, Serio R, Mulè F. Glucagon-like peptide-2 relaxes mouse stomach through vasoactive intestinal peptide release. Am J Physiol: Gastrointest Liver Physiol 2009;296:G678–84. [2] Amato A, Cinci L, Rotondo A, Serio R, Faussone-Pellegrini MS, Vannucchi MG, et al. Peripheral motor action of glucagon-like peptide-1 through enteric neuronal receptors. Neurogastroenterol Motil 2010;22:664–72. [3] Amira S, Rotondo A, Mulè F. Relaxant effects of flavonoids on the mouse isolated stomach: structure–activity relationships. Eur J Pharmacol 2008;599: 126–30. [4] Baldassano S, Amato A. GLP-2: what do we know? What are we going to discover? Regul Pept 2014, http://dx.doi.org/10.1016/j.regpep.2014.09.002 [Epub ahead of print]. [5] Baldassano S, Bellanca A, Serio R, Mulè F. Food intake in lean and obese mice after peripheral administration of glucagon-like peptide-2. J Endocrinol 2012;213:277–84. [6] Bruen CM, O’Halloran F, Cashman KD, Giblin L. The effects of food components on hormonal signalling in gastrointestinal enteroendocrine cells. Food Funct 2012;3:1131–43. [7] Carlini VP, Ghersi M, Gabach L, Schiöth HB, Pérez MF, Ramirez OA, et al. Hippocampal effects of neuronostatin on memory, anxiety-like behaviour and food intake in rats. Neuroscience 2011;197:145–52. [8] Clegg M, Shafat A. Energy and macronutrient composition of breakfast affect gastric emptying of lunch and subsequent food intake, satiety and satiation. Appetite 2010;54:517–23. [9] Cummings DE, Overduin J. Gastrointestinal regulation of food intake. J Clin Invest 2007;117:13–23. [10] Dun SL, Brailoiu GC, Tica AA, Yang J, Chang JK, Brailoiu E, et al. Neuronostatin is co-expressed with somatostatin and mobilizes calcium in cultured rat hypothalamic neurons. Neuroscience 2010;166:455–63. [11] Janssen P, Rotondo A, Mulé F, Tack J. A comparison of glucagon-like peptides 1 and 2. Aliment Pharmacol Ther 2013;37:18–36.
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