The Prokinetic Face of Ghrelin

This review evaluated published data regarding the effects of ghrelin on GI motility using the PubMed database for English articles from 1999 to September 2009. Our strategy was to combine all available information from previous literature, in order to provide a complete structured review on the prokinetic properties of exogenous ghrelin and its potential use for treatment of various GI dysmotility ailments. We classified the literature into two major groups, depending on whether studies were done in health or in disease. We sub-classified the studies into stomach, small intestinal and colon studies, and broke them down further into studies done in vitro, in vivo (animals) and in humans. Further more, the reviewed studies were presented in a chronological order to guide the readers across the scientific advances in the field. The review shows evidences that ghrelin and its (receptor) agonists possess a strong prokinetic potential to serve in the treatment of diabetic, neurogenic or idiopathic gastroparesis and possibly, chemotherapy-associated dyspepsia, postoperative, septic or post-burn ileus, opiate-induced bowel dysfunction and chronic idiopathic constipation. Further research is necessary to close the gap in knowledge about the effect of ghrelin on the human intestines in health and disease.


Introduction
Ghrelin is a 28-amino acid motilin-related peptide hormone mainly secreted by the X/A-like enteroendocrine cells of the oxyntic (parietal) mucosa of the gastric fundus. It is the endogenous ligand for the growth-hormone secretagogue receptor (GHS-R) 1a, recently discovered by two independent research groups [1,2]. The discovery of ghrelin bore great importance in the scientific media for the following reasons: (1) ghrelin is the only endogenous (natural) ligand for the GHS-R, ever discovered; (2) ghrelin shares similar structure with motilin, the so-long called "orphan peptide," based on its unique structure; (3) ghrelin is the first known case of a peptide hormone modified by a fatty acid, the n-octanoic acid; a step necessary for ghrelin to exert its effects [1]; (4) ghrelin is a hunger hormone that stimulates appetite, food intake and promotes weight gain, that is, ghrelin antagonist would serve as a potential treatment for obesity. In addition, ghrelin's poly-faceted properties include prokinetic and possible anti-inflammatory abilities. Although there has been several excellent reviews published recently on the prokinetic effects on ghrelin, some were brief [3,4], or combined with motilin [5,6], and some were focused on the effect of ghrelin on the interdigestive motility [7], gastrointestinal [GI] disorders [8,9], or a certain subset of disease [10]. In the current review, we offer a complete detailed review solely on the prokinetic effects of exogenous ghrelin on the stomach and intestines in health and in disease.

Methods
This review evaluated published data regarding the prokinetic properties of exogenous ghrelin using the PubMed database for English articles from 1999 to September 2009. Our strategy was to combine and distil all currently available studies and reviews in order to provide an overview on exogenous ghrelin and its prokinetic abilities along the gut in health and disease. The search was performed by combining the terms "ghrelin" with "prokinetic" or with "gastrointestinal motility." Clinical trials and review articles were specifically identified, and their reference citation lists were searched for additional publications not identified in the database searches. We classified the literature into two major groups, depending on whether studies were done in health or in disease. We sub-classified the studies into 2 International Journal of Peptides stomach, small intestinal and colon, and broke them down further into in vitro, in vivo (animals) and in humans. The reviewed studies were presented in a chronological order to guide the readers across the scientific advances in the field. We have combined all the available information from previous literature, in order to provide a complete review on the prokinetic properties of exogenous ghrelin and its potential applications for the treatment of various gastrointestinal (GI) dysmotility ailments.

In Vitro Gastric Tissues in Health.
In vitro studies demonstrated the prokinetic potential of ghrelin or its (receptor) agonists in enhancing gastric muscle contractility via activation of the growth hormone secretagogue receptor (GHS-R) and direct neural stimulation of the enteric nervous system (ENSs); an effect involving the cholinergic and tachykininergic pathways. Earliest studies were done in 2003 by Dass et al. on rodent gastric fundic circular muscle strips. Ghrelin concentration dependently increased the amplitude of cholinergic off-contractions at concentrations from 0.1 to 10 μM. Ghrelin did not affect the neurogenic electrical field stimulation (EFSs) induced contractions [11]. They confirmed their results in 2005 [12]. Only a year later, were they able to prove that ghrelin increased the EFSinduced contractions using a concentration of 1 μM [13]. Fukuda et al. confirmed this effect on the antrum and body longitudinal muscle strips using the same concentration of 1 μM [14]. Depoortere et al. showed that ghrelin and ghrelin receptor synthetic peptide agonist, growth hormone receptor peptide 6 (GHRP-6), enhanced off-contractions on both the fundus and the antrum [15]. They went further to report such effects in the presence of N ω -Nitro-L -arginine-methyl-ester-hydrochloride (L-NAME), to verify the involvement of nitric oxide (NO) in the EFS-induced relaxation on-response. Ghrelin or GHRP-6 also enhanced L-NAME-induced contractions; an effect that was reproducible in mice [15,16]. The same research group confirmed the involvement of GHS-R in ghrelin's excitatory effect on gastric muscle tissue either by direct identification of the GHS-R 1a transcripts in the muscle strips, or by testing the effect of treatment with the synthetic nopeptide GHS-R agonist (capromorelin) [15,16]. In the same year, Levin et al. [17], reported that ghrelin's excitatory effect on the gastric fundus was blocked by the pretreatment with atropine, suggesting the involvement of the cholinergic pathway. Such conclusion was confirmed a year later by Bassil et al. who showed the involvement of the tachykininergic pathway, as well [13].
Recently, ghrelin's excitatory effects have also been reported in the upper gut of birds [18,19].  [20]. In fact, ghrelin appears to be the endogenous signal for the MMC in rodents [21]. In 2008, Taniguchi et al. showed that ghrelin infusion increased the motility index (MI) of antral phase III-like contractions, dose dependently, in conscious freely moving rats [7]. A year later, they confirmed the same finding of increase MI in conscious freely moving mice [22]. Zheng et al. reported that ghrelin-induced phase III-like contractions in the antrum of freely moving mice are mediated via the vagal cholinergic pathway [23]. In dogs, however, ghrelin was reported to have no effect on MMCs, suggesting a possible species-related variation [24]. Such variation may be related to the fact that rodents do not express the motilin receptor, whereas in dog, expressing ghrelin and motilin receptors, it is possible that the endogenous signal for the MMC to be modulated by motilin, with no response to ghrelin. In vivo studies revealed opposite effects of ghrelin on the proximal and distal gastric tone in anaesthetized rats. Kobashi et al. reported that ICV ghrelin, or direct injection of ghrelin into the dorsal vagal complex (DVC), relaxed the proximal stomach, while ICV ghrelin at a higher dose contracted the distal stomach [25]. These results suggested the presence of GHS-R in the DVC, as well as the involvement of vagal preganglionic neurons in the action of ghrelin on gastric tone.

In
In vivo studies debated the effect of ghrelin on gastric myoelectrical activity (GMA). We have previously reported no effects of ghrelin on GMA in healthy dogs [26] and have confirmed it in rats (Sallam-unpublished). However, Tümer et al. reported an enhancement of GMA following ghrelin treatment using the same dose we used in rats [27]. We tend to disagree that the reported enhancement was substantial considering that it was within 5%-10%; moreover, the rats had an almost 90% of normal slow waves before the ghrelin treatment.
A number of in vivo studies preceded the in vitro studies and demonstrated prokinetic effects of ghrelin on gastric motility exerted via a vagally mediated mechanism. Masuda et al. were the first to report that IV ghrelin enhanced gastric contractions dose-dependently in anesthetized rats (from 0.8, 4, and 20 μg/kg) and the effect was blocked by the pretreatment with atropine or vagotomy [28]. Later, Trudel et al. however, could not reproduce the results in anaesthetized rats; instead they performed the study in conscious rats and reported that IV ghrelin dose-dependently (5 and 20 μg/kg) accelerated gastric emptying [29]. A similar effect was reported with subcutaneous ghrelin (100 nmol/Kg) in conscious mice [30]. Fukuda et al. reported that IV ghrelin (20 μg/kg) accelerated gastric emptying of both nutrient and non-nutrient meals in conscious rats. Pretreatment with capsaicin blocked the effect of ghrelin on only the nutrient meal [14]. Using ghrelin infusion of 500 pmol/Kg/min and a similar gastric emptying assessment method, Levin et al. could not reproduce Fukuda's results regarding ghrelin's acceleration of the nutrient meal [17]. However, a number of other studies confirmed the ability of intravenous (IV) or intraperitoneal (IP) ghrelin or ghrelin (receptor) agonists in International Journal of Peptides 3 Table 1: Prokinetic effects of exogenous ghrelin on gastric motility in healthy human volunteers (D-Lys). GHRP-6: GHRP receptor antagonist; GE: gastric emptying; GHRP-6: ghrelin secretagogue receptor 6 (non-synthetic ghrelin receptor agonist); GHS-R: growth hormone secretagogue receptor; IV: intravenous; MI; motility index; MMC: migrating motor complex; SPECT: single photon emission computed tomography; VAS: visual analogue scale. the acceleration of gastric emptying in rats [15,27,31,32] and mice [16,32,33], using a variety of methods for the assessment of gastric emptying. Such results were equally reproducible in ghrelin receptor knockout mice, suggesting a minor role of ghrelin in gastric emptying regulation [34]. In dogs, ghrelin was reported to show no effect on gastric emptying in one study, but excitatory effects on gastric contractions in another [24,26].

In Human-Stomach of Healthy Subjects.
Studies on the prokinetic effects of exogenous ghrelin on the stomach of healthy human volunteers are summarized in Table 1.
Researchers have been able to reproduce some of the in vitro and in vivo results in healthy humans. The effects of ghrelin on the fasting, not fed, human stomach was similar to in vivo studies. In fasted volunteers, ghrelin induced phase III-like contractions [35]; an effect that was confirmed two years later by Bisschops [36]. However, both research groups, in addition to Cremonini et al. [37], reported that ghrelin increased the tone of the proximal stomach in the fasting state in healthy volunteers. Even postprandially, ghrelin increased the tone of the proximal stomach [38]. Such result is contradictory to an in vivo study in which ghrelin decreased proximal stomach tone in rats [25]. However, it is noteworthy that this particular in vivo study was performed in anesthetized, not conscious animals.
In 2006, researchers debated the prokinetic effect of ghrelin on gastric emptying [37,39]. It is possible that the use of different techniques for assessment of gastric emptying might have been responsible for this discrepancy.
Ghrelin was reported to inhibit gastric accommodation in healthy volunteers [38]. This raises the question of whether ghrelin would induce side effects, similar to erythromycin, the macrolide antibiotic and motilin receptor agonist that inhibits postprandial accommodation and induces postprandial symptoms. However, ghrelin has been shown not to affect meal related symptoms in healthy obese or lean subjects [37,38]. This is conceivable, as healthy volunteers would not be expected to have pronounced postprandial symptoms.

In Vitro Gastric Tissues in Disease.
Two recent studies by Qiu et al. reported that ghrelin or GHRP-6 increased the amplitude of carbachol-induced contractions of the gastric fundus of various diabetic rodent models [40,41]. These results were reproducible in vivo (see below).

In Vivo Gastric Motility in Animal Model of Diseases.
Studies on the prokinetic effects of exogenous ghrelin on diseased stomach in vivo are summarized in Table 2 [29]. A year later, they confirmed the same finding in dogs [42]. In 2005, the same group reported that RC-1139, a ghrelin receptor agonist, accelerated gastric emptying, dose-dependently, in postoperative ileus or in healthy morphine-treated rodent model. However, when postoperative ileus animals were also given a dose of morphine, a higher dose of RC-1139 was needed to accelerate gastric emptying [31].  [43]. reported that IP ghrelin improved gastric emptying in a mouse model of dyspepsia.

In Human-Patients with Gastric Motility Disorders.
Studies on the prokinetic effects of exogenous ghrelin in dyspeptic and/or gastroparetic patients are summarized in Table 3. From 2005 till present, most of the researchers seem to agree that ghrelin can accelerate gastric emptying in dyspeptic and/or gastroparetic patients. Unlike animal studies, the effects of ghrelin on gastric emptying in these patients were irrespective to vagal contribution [49,50], giving hope to neuropathy gastroparetic patients. In 2009, Ejskjaer et al. reported that TZP-101, the ghrelin receptor agonist, also accelerated gastric emptying in diabetic patients with gastroparesis [51]. Though the effect of ghrelin on patients' symptoms remained debatable [36,48,49], TZP-101 was reported to show no effect on the postprandial symptoms of diabetic gastroparesis patients and its safety profile has been determined in a phase I trial [51,52].
Despite the optimistic results of acute ghrelin administration on gastric motility in patients with gastroparesis, many researchers showed their concern regarding the side effects of the chronic use of ghrelin. Being the ligand of growth hormone secretagogue receptor, ghrelin has been shown to induce growth hormone secretion [53] and insulin resistance [54]; such effects would cause serious unfavorable side effects in particular subsets of patients. Modification of ghrelin (receptor) agonists might be necessary to avoid such problems. Long term studies will show whether the ghrelin receptor would be desensitized by chronic activation, similar to the motilin receptor. Treatment alteration between ghrelin and motilin receptor agonists has been suggested [3]. The new ghrelin receptor agonist, TZP-101, is promising as it did not induce growth hormone secretion following either peripheral or central administration [53]. TZP-101 has also been claimed to have a lower tendency to provoke ghrelin receptor desensitization [5,52,55].

Effects of Ghrelin on Intestinal
Motility in Health In the colonic tissues, studies have shown that the prokinetic effects of ghrelin were species-specific. While it induced colonic contractions in fish or birds [19,57], ghrelin had no effect on the colon of rodents or humans [11,12].

In Vivo-Intestinal Motility in Healthy Animals.
In vivo studies showed that ghrelin induced intestinal MMCs in fed rats involving the activation of the cholinergic pathway, and the NO, NPY or 5-hydroxytryptamine 4 (5-HT 4 ) receptors. In 2003, Fujino et al. reported that ghrelin induced MMCs in fed and/or vagotomized rats; this effect was blocked by immunoneutralization of the NPY receptor [20]. In 2004, Edholm reported that ghrelin dose-dependently shortened the intestinal MMC cycles. Pretreatment with atropine blocked the ghrelin effect [56]. In 2007, Wang et al. reproduced the exact same results in rats and showed that  the NO and 5-HT pathways might be involved in the action of ghrelin on intestinal interdigestive motility [58]. One year later, Taniguchi et al. pinpointed the involvement of the 5-HT 4 receptor in the ghrelin's action on intestinal MMCs [7]. Recently, using a manometric method for simultaneous assessment of gastro-duodenal motility, Tanaka et al. showed that ghrelin induced MMCs in fed conscious freely moving mice [22]. In dogs, however, ghrelin was shown to have no effect on intestinal MMCs [24]. Using a variety of techniques for the assessment of intestinal transit, researchers proved that ghrelin or its receptor agonist accelerated intestinal transit in rodents. In 2002, Trudel et al. showed that IV ghrelin 20 μg/kg accelerated intestinal transit in rats [29]. This prokinetic effect of ghrelin on intestinal transit has been confirmed by several researchers using IV or IP ghrelin using the same, or different dose in rats [14,15] or mice [32,46]. Ghrelin receptor agonists GHRP-6 or EX-1314 has also been shown to have similar prokinetic results. EX-1314 did not accelerate the intestinal transit in ghrelin receptor knockout mice, confirming the necessity of GHS-R activation for the prokinetic action of ghrelin, in accordance to what has been shown in the in vitro studies [15,32,36].
As for the colon, studies showed that to induce colon propulsion in conscious, not anesthetized, rats, central administration of ghrelin was needed. Although, ghrelin may exert an indirect effect on the colon merely by triggering upper GI MMCs, such possibility was not evidenced in the literature. The lack of direct effect of ghrelin on the colon may possibly be due to the lack of ghrelin immunoreactive cells and/or ghrelin receptors in the colon [1,59,60]. In 2002, Trudel et al. reported no effect of IV ghrelin on the colon of conscious rats [29]. In 2005, Tebbe et al. showed that ghrelin, injected in the paraventricular nucleus (PVN), stimulated colonic motility, dose dependently, in freely moving conscious rats. This effect involved central activation of the PVN via the corticotrophin releasing factor 1 (CRF 1 ) and the NPY 1 receptors [61,62]. In 2006, Shimizu et al. reported that intrathecal ghrelin increased the colonic propulsive function in anesthetized rats. Shimizu et al. reported similar effects with intrathecal, or IV, or IV infusion of CP464709, a synthetic ghrelin receptor agonist which exerted defecation in conscious rats that was dependent on intact pelvic nerves [63]. In 2009, Charoenthongtrakul et al. reported that oral EX-1314 increased fecal output in conscious mice [32]. Shafton et al. tested a centrally acting ghrelin receptor agonist, GSK894281 that was administrated orally in conscious rats. They reported a dose-dependent increase in the fecal output both acutely and after 8 days of treatment [64].

In Human-Intestines of Healthy Subjects. Tack et al.
reported premature intestinal phase III contractions following IV ghrelin in healthy volunteers. These contractions were of gastric origin [35]. Although ghrelin, like motilin, triggered intestinal MMCs in humans, but unlike motilin, plasma ghrelin has not been reported to fluctuate with phase III, suggesting that motilin remains the main hormone dominating interdigestive motility in man.
No studies were found on the effects of ghrelin on colon motility in healthy subjects.

Effects of Ghrelin on Intestinal
Motility in Disease

In Vitro-Intestinal Tissues in Disease.
No studies were found on the effects of ghrelin on small intestinal tissues obtained from diseased animal models. However, ghrelin effects on colitis rodent model were reported. Recently, De Smet et al. showed that ghrelin decreased the colonic inhibitory responses in healthy mice and aggravated colitis in a dextran sodium sulfate (DDS)induced colitis mice model [65]. This is contradictory to several studies in which ghrelin exerted an anti-inflammatory Table 3: Prokinetic effects of exogenous ghrelin in dyspeptic and/or gastroparetic patients. IDDM: insulin-dependent diabetes mellitus; GE: gastric emptying; GHRP-6: ghrelin secretagogue receptor 6 (non-synthetic ghrelin receptor agonist); GHS-R: growth hormone secretagogue receptor; h: hour; IV: intravenous; VAS: visual analogue scale.  [66,67]. Further investigation is needed to explore the role of ghrelin in colitis-induced dysmotility.

In Vivo-Intestinal Motility in Animal Model of Diseases.
Studies on the prokinetic effects of exogenous ghrelin on diseases intestines in vivo are summarized in Table 4. Similar to its effects on gastric emptying, ghrelin or its receptor agonists have been reported to accelerate or normalize intestinal transit in a variety of diseased animal models; these include diabetic, postoperative, or morphine, or septic, or burn induced ileus, and opiate-induced bowel disorder models.

In Human-Patients with Intestinal Motility Disorders.
No studies were found regarding the effects of ghrelin in patients with intestinal motility disorders.

Summary and Conclusion
In conscious animals, exogenous ghrelin was reported to (1) induce gastric and intestinal MMCs in fed rodents, but not in the canine; (2) exert controversial effects on gastric myoelectrical activity in rodents; (3) induce antral contractions in dogs; (4) accelerate gastric emptying in healthy, diabetic, postoperative, or morphine, or septic, or burn-induced ileus, and cisplatin-induced dyspepsia animal models; (5) accelerate intestinal transit in healthy, diabetic, postoperative, or morphine, or septic, or burn induced ileus, and opiate-induced bowel disorder rodent models; (6) accelerate colonic transit in healthy rodents, when centrally administrated. Clinically, exogenous ghrelin was reported to (1) induce gastric and intestinal MMCs in fasted healthy subjects; (2) increase fundic tone in both fasted and fed healthy subjects; (3) exert controversial effects on gastric emptying and have no effect on postprandial symptoms in healthy subjects; (4) accelerate gastric emptying in dyspeptic and/or gastroparetic patients and have debatable effects on postprandial symptoms in these patients. Luckily, the prokinetic effects of ghrelin in gastroparesis and/or dyspepsia patients were independent of vagal involvement.
The prokinetic effects of ghrelin on GI motility involve the exclusive activation of the GHS-R 1a receptor, not the motilin receptor, the enteric nervous system (specifically the myenteric plexus), excitatory neurons involving 5-HT4 and NO, capsaicin-sensitive afferent neurons, tachykininergic motor neurons, as well as intact vagal cholinergic neurons.
Oral ghrelin use is limited due to its instant inhibition by the gastric acidic milieu; however, other routes for its administration are possible. The emergence of IV ghrelin agonists (e.g., synthetic peptide GHRP-6; synthetic nonpeptide capromorelin or ipamorelin) or ghrelin receptor agonists (e.g., GSK894281, EX-1314, EX-1315, RC-1139, TZP-101) are paving the way for possible uses in patient treatment. Oral TZP-102 soon followed and has been tested in healthy volunteers [70]. Other oral agonists include TZP-102, EX-1314 and RC-1141.
In conclusion, the prokinetic face of ghrelin enables it to serve as a strong tool in the clinical practice for the treatment of various GI dysmotility ailments. The prokinetic properties of ghrelin or its (receptor) agonists have the potential to serve in the treatment of diabetic, neurogenic or idiopathic gastroparesis and possibly, chemotherapy-associated dyspepsia, postoperative, septic or post-burn ileus, opiate-induced bowel dysfunction and chronic idiopathic constipation. Further research is necessary to close the gap in knowledge about the effect of ghrelin on the human intestines in health and disease.