Discovery of ghrelin, the motilin related peptide

The discovery of ghrelin is yet another surprising finding which developed from studies aimed at understanding the regulation of growth hormone (GH) secretion from the pituitary gland. When it became clear around the middle of the 20th century that the hypothalamus produced releasing factors which caused the anterior pituitary to secrete tropic hormones, the search for the factor causing the release of somatotropin, i.e. GH, led instead to the discovery of a peptide with an inhibitory effect. This peptide, originally designated as “somatotropin release inhibiting factor (SRIF), is now known with the less cumbersome name somatostatin. In the 1970s several reports appeared proposing peptides with “growth hormone releasing hormone (GHRH)” activity, but all had to be discarded. The observation that excess growth hormone secretion was often associated with pancreatic tumors led finally to the isolation of GHRH from such a tumor in 1982 and later its identity with the hypothalamic factor was demonstrated (1).

Meanwhile as part of the search for GHRH, studies had been made of analogues of the enkephalins, small peptides (five amino acid residues) present in the brain with opiate activity, because it was thought that these peptides could be related to GHRH. Indeed opiates were known to stimulate GH release be it with low potency. From these studies several molecules were derived which had lost opiate activity but were potent GH releasers and which were therefore called growth hormone secretagogues (GHSs). A potent member of this group of molecules is growth hormone releasing peptide-6 (GHRP-6) (2) a hexapeptide with sequence HisDTrpAlaTrpDPheLysNH2. For a recent review see (3). Non-peptide analogues of GHRP-6, for example MK-067, were also developed (4).

From studies of the effects of GHSs in vivo and in vitro it became clear that these molecules acted through another receptor than GHRH. The concept was finally proven by the cloning of the GHS-receptor (GHS-R) (5). The GHS-R is a classical seven transmembrane G protein coupled receptor present, as expected in the hypothalamus-pituitary area, but surprisingly also found in peripheral, endocrine and non-endocrine tissues (6) suggesting broader functions of the GHS-R beyond the control of GH secretion.

Increasing evidence that GHSs had cardiovascular activity, led a Japanese group active in the cardiovascular field, to a search for the endogenous ligand of the GHS-R. They used a CHO cell-line expressing the GHS-R and measured the ability of tissue extracts to induce Ca2+ release. To their surprise they found the highest GHS-R activation with extracts from the stomach and went on to purify the peptide which they named ghrelin, from “ghre” the Proto-Indo-European root of the word “growth” (7). Ghrelin is a peptide of 28 amino acids in which the serine 3 residue is octanoylated (Fig. 1).

Fig.1. Amino acid sequences reported for motilin homolog, motilin related peptide (MTLRP) and ghrelin and alignment with the human motilin sequence. Identification of the octanoylation of ser3 is indicated with an asterisk. Homologous residues in bold, conserved residues underlined. Also shown is the alignment of human motilin and human and des-Gln14-ghrelin.

Almost simultaneously another group identified in mouse a peptide with the same amino acid sequence as rat ghrelin. They gave it the name “motilin related peptide” because its precursor structure and amino acid sequence were similar to motilin's (8). It is of interest that while the papers of Kojima and Tomasetto were published with 6 months difference, Kojima submitted his sequence to the EMBL/genbank database just one day before Tomasetto did the same with her sequence (9). However, both were also unaware of the fact that the same sequence had already been submitted as part of a US patent application for “motilin homologs” filed in 1997 by the company Zymogenetics (10). Therefore the ghrelin sequence has been independently discovered three times. However, it is now known that the unique posttranslational modification present in ghrelin is essential for bio-activity (11). Because Tomasetto (8) and the patent for motilin homologs (10) derived the amino acid sequence from the nucleotide sequence, they were unaware of this, and it seems therefore indicated to use only the name ghrelin and to consider Kojima et al., (7) as the true discoverers of the endogenous ligand for the GHS-R.

The amino acid sequence of ghrelin, along with the amino acid sequence of motilin is given in Fig. 1. Recently a splice variant of ghrelin has been described in which residue 14 is missing (12). It is interesting that this splice variant, des-Gln14-ghrelin, shows an even better alignment with motilin. While motilin was until now not a member of a group of related peptides, the discovery of ghrelin has changed this. Moreover, in vitro data to be discussed below, suggest that ghrelin and motilin may be part of a larger family of peptides (13).

Effects of ghrelin on gastrointestinal motility

Ghrelin is predominantly present in gastric endocrine cells and secreted into the bloodstream, suggesting that it may have an effect on gastrointestinal function. Because of the structural similarity with motilin, the effect of ghrelin on gastrointestinal motility has been examined. in vivo the most typical effect of motilin is the induction of the migrating motor complex in the fasted state, while in the fed state motilin accelerates gastric emptying. These two effects have now also been observed with ghrelin.

Indeed there is ample evidence that ghrelin, and the ghrelin agonist GHRP-6, accelerate gastric emptying in rats (14, 15) and in mice (16, 17). In rats it has also been shown that ghrelin may reverse the delay of gastric emptying in a post-operative ileus model (15). No studies have been performed in humans or dogs, but it has been noted that gastric emptying half-time was correlated with fasting plasma ghrelin levels (18).

Ghrelin also induces the migrating motor complex. In fed rats ghrelin, given iv or icv, increases motor in the stomach, and induces the MMC pattern in the duodenum (19). In a recent study it was shown that infusion of 40 µg ghrelin iv in healthy volunteers, twenty minutes after the occurrence of phase III of the MMC, induced a premature phase III originating in the stomach after 14 ± 4 min in all subjects studied (6/6) (20).

It was also found in rats that ghrelin accelerates the transit of the small intestine (14, 15) but had no effect in the colon (15).

Involvement of the motilin receptor

The motor effects of ghrelin are therefore comparable with those observed for motilin, although motilin induces phase 3 of the migrating motor complex at a lower dose than ghrelin (21). On the other hand, high doses of motilin (22) and low doses of ghrelin (7) stimulate growth hormone secretion. These data, illustrated in Table 1, suggest that both peptides may cross-react with their receptors. It is of interest in this respect that not only the peptides show structural similarity, but their receptors also have a marked sequence homology with an overall identity of 44 %, rising to 87% in the transmembrane regions. In fact the discovery of the motilin receptor is the inverse story of the discovery of ghrelin, as an orphan receptor structurally related to the GHS-R was shown to have motilin as its ligand (23).

Table.1. Doses (in mg) required for biological effect in vivo1

1 Comparable models were selected. The data are for growth hormone release from Kojima et al., (7) and Samson et al., (22) and for the induction of phase 3 of the MMC from Vantrappen et al., (21) and Tack et al. (20). 2 To calculate the ratio, the molar mass was taken into account (motilin 2700, ghrelin 3315.

On the other hand the pharmacophore of both peptides is quite different. An Ala-scan of the 1-14 fragment of motilin led to the conclusion that the side chains of residues Phe1, Phe4 and Tyr7 are involved in receptor contact (24). Ghrelin does not have a residue corresponding to Phe1 in motilin, and the only alanine substitution which has an effect on ghrelin, Phe4, (25) is not matched by a similar effect in motilin if the corresponding Phe5 is replaced by alanine. Of course the value of such arguments may be limited. Thus, judging form the amino acid sequence it is hard to predict that GHRP-6 would interact with the same receptor as ghrelin and the final answer must therefore be obtained from in vitro experiments.

The rabbit antrum is the classical in vitro model for motilin. The affinity of ghrelin [1-28] for the rabbit receptor, as determined from binding studies is almost 10,000 times smaller (pKd 4.23) than the affinity of motilin (pKd 9.13) (13). In agreement with these findings ghrelin did not induce contractions, nor did it enhance the response to electrical field stimulation in rabbit antral strips. Interestingly, GHRP-6 showed some affinity in binding experiments (IC50: 3 µM), and at 10-5 M enhanced the response to electrical field stimulation. Pharmacological analysis revealed that this effect is partially mediated via a motilin receptor on non-cholinergic nerves with tachykinins as mediator, partially via another receptor, that may be a GHS-R subtype, on cholinergic nerves that corelease tachykinins (13).

It may also be noted that we have found, in collaboration with the laboratory of prof. P. Robberecht (ULB, Brussels), that in CHO-K1 cells expressing the human motilin receptor, and in CHO-K1 cells expressing the ghrelin receptor, there is only very weak cross-reactivity of ghrelin analogues, motilin analogues and motilides (unpublished data).

Activation of central or peripheral pathways

The myenteric plexus. The results mentioned in the previous section suggest that ghrelin, in contrast to motilin, is not able to activate neural pathways in the myenteric plexus. However, while the rabbit is almost the only species, apart from man, responding to motilin in vitro, the opposite may be true for ghrelin. Thus in rats, ghrelin and GHRP-6, but not motilin, enhance neurally induced contractions in rat fundic and antral strips via cholinergic pathways (3, 14). In this species the GHS-R is present on nerves associated with the muscle layers (3).

In guinea pig, another species with poor response to motilin, ghrelin and GHS-R mRNA transcripts are present in the myenteric plexus and the GHS-R is colocalized with ChAT as demonstrated by immunocytochemistry (26). Also in guinea pig, there is an increase of the intracellular Ca2+ concentration in a subset of myenteric plexus neurons upon superfusion with ghrelin and GHRP-6. The neurons were loaded with a fluorescent indicator and observed under confocal microscopy (27). Using classical electrophysiology, A. Bugajski (Krakow, Poland), working in our laboratory, could show that ghrelin depolarizes neurons in the guinea pig myenteric plexus (unpublished data).

The species differences offer an interesting perspective. Motilin has never been isolated from rat or mouse, and recent attempts to identify using bio-informatics have failed. It is therefore tempting to speculate that ghrelin is the functional correlate of motilin in rodents.

Involvement of the vagus

The effect of ghrelin on the frequency and the amplitude of gastric contractions in urethane-anesthetized rats could be blocked by atropine and vagotomy indicating that peripheral ghrelin can also activate a vago-vagal reflex (28). Similarly, vagotomy blocked the acceleration of gastric emptying observed after ip administration of ghrelin in mice (16). The most detailed data were obtained in the recent study by Fujino et al. which demonstrated that ghrelin induces the migrating motor complex in fed rats and increases the frequency of the migrating motor complex in fasted rats (19). The effects could be obtained by icv or iv infusion, and were only blocked by a GHS-R antagonist when given by the same route. Of particular interest was the finding that the GHS-R antagonist when given to normal rats had no effect on fasted motor activity, but when given to vagotomized rats completely blocked the migrating motor complex. These data demonstrate that local and central pathways exist, but that in normal rats only central pathways are operational. However, when these pathways are eliminated by vagotomy, ghrelin is able to exert its effects via the myenteric plexus. This suggests that vagotomy may induce an upregulation of ghrelin receptors in the myenteric plexus.

In this respect studies should also be mentioned, not related to the effects of ghrelin on gastric motility, but supporting the concept that ghrelin released in the wall of the stomach, may affect vagal afferents in close proximity. Thus GHS-R mRNA has been detected in the rat nodose ganglion, and GHS-R is synthesized in vagal afferent cell bodies and transported to the periphery and ghrelin also suppresses firing of the vagal afferent nerve (29).

Central effects

As was already mentioned, in rats central administration of ghrelin induces the fasted motor pattern in fed animals and increases the frequency of the migrating motor complex in fasted animals (19). In mice, central administration of ghrelin accelerates gastric emptying (16). In both cases the effect depends upon vagal efferent pathways as it is abolished after vagotomy. The GHS-R receptor is present in the hypothalamus (30, 31) and it was recently shown that ghrelin changes the excitability of neurons in the paraventricular nucleus of the hypothalamus. The neurons responding to ghrelin were identified as receiving ascending afferent signals from mechanoreceptors in the stomach (32). It is however unclear whether under physiological conditions ghrelin released from the stomach or locally produced ghrelin activates these pathways. It has also been shown that centrally administered ghrelin increases food intake and body weight (33, 34) and stimulates gastric acid secretion (35).


CONCLUSIONS

Ghrelin accelerates gastric emptying and induces phase 3 of the migrating motor complex. Ghrelin's effect is not mediated via motilin receptors, although some ghrelin agonists may cross-react with motilin receptors and may also interact with related yet uncharacterized receptors. Ghrelin may act on motor neurons in the myenteric plexus, may activate a vago-vagal reflex or may stimulate central pathways. Presently the doses required to do so are rather pharmacological, and a physiological role for ghrelin in the regulation of gastrointestinal motility seems unlikely, except perhaps in rodents where ghrelin may be the functional correlate of motilin. Ghrelin agonists may find application as a prokinetics.

Acknowledgements: The research of the author on ghrelin is supported by grants from the Flemish Foundation for Scientific Research (FWO grant number G.0144.04), from the Ministry of the Flemish Community (International Scientific and Technological Cooperation with the P.R.China grant BIL 01/13) and from the Belgian Ministry of Science (GOA 03/11 and IAP P5/20 ).


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