Why is motilin active in some studies with mice, rats, and guinea pigs, but not in others? Implications for functional variability among rodents

Abstract The gastrointestinal (GI) hormone motilin helps control human stomach movements during hunger and promotes hunger. Although widely present among mammals, it is generally accepted that in rodents the genes for motilin and/or its receptor have undergone pseudonymization, so exogenous motilin cannot function. However, several publications describe functions of low concentrations of motilin, usually within the GI tract and CNS of mice, rats, and guinea pigs. These animals were from institute‐held stocks, simply described with stock names (e.g., “Sprague–Dawley”) or were inbred strains. It is speculated that variation in source/type of animal introduces genetic variations to promote motilin‐sensitive pathways. Perhaps, in some populations, motilin receptors exist, or a different functionally‐active receptor has a good affinity for motilin (indicating evolutionary pressures to retain motilin functions). The ghrelin receptor has the closest sequence homology, yet in non‐rodents the receptors have a poor affinity for each other's cognate ligand. In rodents, ghrelin may substitute for certain GI functions of motilin, but no good evidence suggests rodent ghrelin receptors are highly responsive to motilin. It remains unknown if motilin has functional relationships with additional bioactive molecules formed from the ghrelin and motilin genes, or if a 5‐TM motilin receptor has influence in rodents (e.g., to dimerize with GPCRs and create different pharmacological profiles). Is the absence/presence of responses to motilin in rodents’ characteristic for systems undergoing gene pseudonymization? What are the consequences of rodent supplier‐dependent variations in motilin sensitivity (or other ligands for receptors undergoing pseudonymization) on gross physiological functions? These are important questions for understanding animal variation.


| INTRODUC TI ON TO MOTILIN
In humans, the hormone motilin is found mostly within endocrine cells of the mucosa of the duodenum and jejunum, and to a lesser extent the gastric antrum. 1,2 Motilin is released from these cells during hunger to induce phase III activity of the gastric migrating motor complex, a wave of high amplitude and propulsive contractions which occurs during fasting every 90-120 min and moves from the stomach and into the small intestine. Its purpose is thought to help clear the stomach of any undigested material, prevent bacterial overgrowth, and stimulate a sensation of hunger. [3][4][5] Intravenous infusions of motilin to humans have also been shown to stimulate the motility of the gastric antrum, 6,7 increase gastric emptying of a solid meal, 8 and induce postprandial nausea. 7 Experiments with human isolated stomach showed that the gastric prokinetic activity of motilin occurs primarily because of an ability to act prejunctionally within the enteric nervous system to strongly facilitate cholinergic activity in a concentration-dependent manner, with the higher concentrations also directly contracting the muscle; the large magnitude of the excitatory nerve-muscle responses at the higher concentrations, perhaps in conjunction with vagal nerve activation, have been argued to help promote the ability of high doses of motilin and of motilin receptor agonists to cause nausea and vomiting. 9

| LOSS OF A FUN C TI ONAL MOTILIN SYS TEM AMONG RODENTS
Motilin is found within the mammalian kingdom, with orthologues identified in birds, reptiles, amphibians, and fishes; the receptor for motilin, a seven-transmembrane (TM), G-protein-coupled structure (first identified in human 10 ), has a matching presence. [11][12][13] However, examinations of genomic databases, including those assembled by Ensembl, found that among the mammals, the genes in rodents (mouse, rat, kangaroo rat, guinea pig, squirrel, a strain of pika) for the motilin receptor and often for motilin itself, have become pseudogenes (e.g., 80%-90% identity to the human motilin receptor, but with an in-frame stop-codon). This indicates that in rodents the functions of motilin have been lost 11,12,14,15 For the mouse and rat, this is thought to have occurred via mutations in the genes encoding the motilin receptor and motilin, and not by a disruptive chromosomal rearrangement that potentially could have removed both genes. 11 Interestingly, among the amphibians, the reverse may be true. Certain frogs (e.g., the tropical clawed frog may have retained a motilin receptor but lost the presence of motilin; the authors suggest the possibility of a different endogenous agonist acting at the motilin receptor). 13 Among rodents, the evolutionary pressures that led to the elimination of the presence and functions of motilin are unclear.
However, it has been speculated that because rodents have also lost the ability to vomit (with marked and associated changes in the presence of other genetic markers and in neuronal, hormonal, and structural functions regulating upper gastrointestinal (GI) functions) all, or many of these events may have been somehow driven by an environmental pressure for water conservation in arid or semi-arid regions. 12 Early pseudonymization of the motilin receptor was followed by loss of the motilin peptide, complete in some rodent species but not in others. For example, although a potentially functional form of motilin was not identified in the guinea pig by He et al, 11  qPCR and by Southern blot hybridization), which when synthesized were inactive when applied to guinea pig GI muscle strips but were effective stimulants when applied in similar experiments using rabbit duodenum (the evoked activity was reduced by the motilin receptor antagonist GM-109 and by human motilin desensitization). Interestingly the later RT-PCR using various primer sets failed to amplify the mRNA for one of the putative motilin structures. 16 Nevertheless, the differences in data obtained by different investigators suggested that the gene for motilin in guinea pigs is undergoing pseudogenization but highly divergent alleles of the gene exist within the cDNA and genomic sequences of the guinea pig population. 18 Another study into the North American kangaroo mouse and rat (members of the Dipodomyinae subfamily of rodents) identified potentially functional forms of motilin but since the motilin receptor pseudogene was formed well before the radiation of this subfamily, the retention of a potentially functional motilin was suggested to represent a lineage-specific physiological adaptation to a new function. 18

| THE RODENT ANOMALIE S
In contrast with the failure to identify a functional motilin gene within genomic databases of rats and mice (see above), or identify the presence of motilin, 19,20 there are several publications in which motilin is reported to be present within these animals (recent examples include [21][22][23]. Furthermore, although the application of motilin has been found to be without activity in several experiments with stomach and intestinal preparations from rats, mice, and guinea pigs (examples include 16,[24][25][26][27][28], several other publications report an ability of motilin to exert functional activity in the stomach, brain and other tissues of rats, mice, and guinea pigs (briefly noted previously 11,29 ). Table 1 lists these studies and when provided by the authors, gives the sources of rodents and ligands used within each investigation.
Examination of Table 1 reveals several features:

| SOURCE-AND S TR AIN -DEPENDENT DIFFEREN CE S AMONG RODENTS
In the experiments in which a function of motilin was identified, the sources of animals were not always provided (   Could a non-motilin receptor, sensitive to motilin, exist within the cDNA and genomic sequences of the rodent population? The receptor with the closest sequence homology is the ghrelin receptor.

| Ghrelin receptor
The motilin and ghrelin receptors belong to the same sub-family of 7-TM GPCRs, sharing significant amino acid identities in different species (e.g., the human motilin and ghrelin receptors and the receptors in the insectivore Suncus murinus each share, respectively, 52% and 42% overall amino acid identity and 86% and 62% in the seven-transmembrane region [81][82][83] ). Both hormones are released from endocrine cells of the upper GI tract at different times during fasting and both stimulate gastric motility and have roles in the feeding cycle in humans and other mammalian species; unlike motilin, ghrelin is also found outside the GI tract where it can exert significant additional non-GI functions. 84,85 Might the ghrelin receptor substitute for the absence of a functional motilin system? This seems to be a possibility in terms of the control of gastric functions, but good evidence to suggest that the rodent ghrelin receptor is highly responsive to exogenous (or endogenous) motilin is lacking.
In rodents, it has been suggested that the absence of a functional motilin system is compensated for by the actions of ghrelin. 29,86 This may be illustrated by the species-dependent roles of motilin and ghrelin in the mechanisms of the migrating motor complex (MMC). In humans, the release and subsequent actions of motilin during fasting mediate the propulsive phase III contractile activity of the gastric MMC, also associated with hunger (see Introduction). Although ghrelin is released during fasting in humans, this is not in association with phase III MMC activity, its purpose being to increase appetite. 4 In the insectivore Suncus murinus (house musk shrew), also possessing both motilin and ghrelin functional systems, the ability of motilin to induce phase III of the gastric MMC may involve the release of ghrelin. 87,88 In rats and mice, however, in which gastric MMCs are less well defined and more frequent, it is the release of ghrelin which evokes the phase-III-like contractions. [89][90][91] Notably, ghrelin can directly stimulate gastric enteric nerve functions in rat and mouse, but not in human. 27,92,93 Curiously, in rats with a mutant, non-functional ghrelin receptor, spontaneous gastric phase III-like contractions were still observed, suggesting the development of a different compensatory mechanism to maintain these contractions. 94 In species possessing both motilin and ghrelin, the receptors have a poor affinity for each other's cognate ligand (e.g., the human and rabbit receptors 29,95 ). In mice, it has been suggested that the ghrelin receptor is responsive to motilin at high concentrations.
Thus, using a whole-cell patch-clamp configuration, motilin 1-5 µM depolarized the pacemaker potentials of the interstitial cells of Cajal within the small intestine, in a concentration-dependent manner; this activity was inhibited by the ghrelin receptor antagonist [D-Lys] GHRP-6. 38 However, it is important to note that in most other in vitro studies in which motilin has been shown to exert activity in rodents, the efficacy is reported at nM concentrations (Table 1) Finally, a 5-TM motilin receptor has been identified, 10 with no known function. Similarly, a 5-TM ghrelin receptor, without sensitivity to ghrelin, is able to dimerize with the ghrelin receptor, changing its function and ability to form oligomeric complexes with the dopamine D 1 receptor, to create different pharmacological profiles. 104

| CON CLUS IONS AND QUE S TIONS
The absence of genes generating motilin and/ or its receptor, and the absence of a functional response to motilin in laboratory rodents has become the accepted status for motilin. Nevertheless, confusion remains over numerous reports, which demonstrate an ability of low concentrations of motilin to exert functional activity in some laboratory rodents, particularly within the GI tract and the CNS. There is no accepted explanation for this anomaly, but the very existence of such differences raises concerns, particularly in terms of the need to understand animal research reproducibility. 105 It is difficult to refute the suggestion that the variation in response to motilin is dependent on the source of rat, mouse, or guinea pig used. This includes outbred animals and genetically stable inbred strains of mice. The cause of the variation remains unknown, but it can be speculated that molecular differences in the receptors for motilin, ghrelin and perhaps for associated peptides might be involved. If correct, several questions need to be asked.  for MMCs originating in stomach and duodenum) and stimulate motilin release to sustain phase III activity. 110 By contrast, in rodents the 5-HT 3 receptor is not involved in regulating MMC activity 111 but 5-HT 3 receptor antagonists increase gastric emptying in rats and guinea pigs. 112 Thus, if different populations of rodents have lost or still retain an ability to respond to motilin, it seems reasonable to suggest that similar variability will be found among the actions of other endogenous ligands involved with the same physiological functions as motilin.
3. Are motilin-responsive rodents useful "knock-in" laboratory animals for studying the functions of motilin? This would avoid having to rely on other non-rodent species or rodents in which the human motilin receptor gene has been knocked-in [29].

ACKNOWLEDG EMENT
The author would like to thank everyone who generated the data discussed in this manuscript and in particular, his laboratory colleagues and friends in other institutions who have participated in trying to understand why the pharmacology and physiology of mice and other rodents are not always the same as humans.

CO N FLI C T O F I NTE R E S T
The author has no conflict of interest with respect to this study.

D I SCLOS U R E
None.

AUTH O R CO NTR I B UTI O N
GJS wrote this manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
This article contains no new data (other than a summation of the work of others).