LA SEGURIDAD SOCIAL

The cloning of the novel GHS Ghrelin mainly from the rat and human stomach has raised many new questions in GHS physiology. The nature of Ghrelin, its size of 28aa and the somewhat ‘bulky’ octanoyl group essential for Ghrelin’s functional activity, is surprising, considering that the artificial GHSs are comparatively small compounds. The process of Ghrelin’s octanoylation of the serineS needs future attention as to what the posttranslational mechanism for acyl modifications of proteins is.

Chapter 8: Ghrelin - a novel growth hormone secretagogue

In this study P. Le Tissier cloned the mouse Ghrelin homologue and established the genomic structure of stomach Ghrelin. The existence of two alternative splice forms of Ghrelin - Ghrelin 27 and 28 - were demonstrated. I measured their relative expression in stomach as 1:1.7, while the protein levels of Ghrelin 27:28 were previously shown to be 1:4 (Hosoda et a l, 2000).

Of interest is the Ghrelin expression in the heart, found by 5’RACE and in situ hybridisation. An alternative exon 1 suggests possible altered transcriptional and translational regulation for the heart Ghrelin. Hexarelin, a GHRP-6 analogue, has previously been shown to bind to rat cardiac membranes and Ong et al. suggested the existence of a different GHS-R subtype (Ong et al. 1998a,b). Hexarelin binding can be displaced by GHRP-6 but not MK-0677 (Bodart et al., 1999) and there is no GHS-R type la detectable in the heart (Guan et a l, 1997; Howard et a l, 1996), suggesting a different type of Hexarelin receptor in the heart than the GHS-R expressed in the hypothalamus and pituitary. There are now several studies of the cardiac and hemodynamic effects of GHSs (Bisi et a l, 1999; De Gennaro Colonna et a l, 1997; Locatelli et a l, 1999; Tivesten et a l, 2000). Therefore alternatively regulated Ghrelin expression in combination with a different GHS-R subtype or even another receptor could be of clinical interest in the heart. However, Ghrelin expression is extremely low in the heart, as was shown by in situ hybridisation and by it not being detectable in RPA. A specific signal for Ghrelin expression was absent in the hypothalamus as shown by in situ hybridisation and RNAse protection analysis. Although Ghrelin can be amplified from hypothalamic RNA extracts and many other tissues by RT-PCR, the physiological relevance of these very low levels of expression not only in the hypothalamus, but in all these tissues is questionable. Immunocytochemistry assays demonstrated by Kojima et al. had located Ghrelin in this area and a RIA for Ghrelin showed that

117.2±37.2fmol/ml circulated in the blood (Kojima et a l, 2000a). In Chapter 4 the GHS-R peptide was shown to be present in fibres of the ME. Since hypothalamic Ghrelin mRNA expression is low, but Ghrelin protein was located to this area, one could speculate that Ghrelin protein might be taken up into the hypothalamic area from the hypohpyseal blood system, since this area lies outside the blood brain barrier and by this means the rather ‘bulky’ Ghrelin would not need to cross it. Hewson et al. reported c-fos

expression in the ARC after Ghrelin injection, suggesting a direct central effect for Ghrelin (Hewson and Dickson, 2000). The very low levels of Ghrelin expression in the hypothalamus will make the analysis of Ghrelin’s possible involvement in the GH axis difficult. Whether Ghrelin is the or the only endogenous GH regulating ligand for the GHS-R remains to be proven.

Cloning of Ghrelin and its GH secreting action in vitro and in vivo suggests that our current understanding of the regulation of GH pulsatility may need modification. Ghrelin’s presence in the blood strengthens the notion of Ghrelin as a third GH regulator, but measurements of Ghrelin in portal blood and their relationship to GH pulses are needed to clarify its relevance to GH physiology.

Since the main expression of Ghrelin is in the stomach it also makes sense to look at its actions in the gastric area. The expression of Ghrelin in endocrine cells close to the nerve plexus suggests that these might be cells that are sympathetically innervated, thereby regulating stomach motility. Solution hybridisation experiments by D.F. Carmignac showed that Ghrelin expression in the stomach was down-regulated in starved mice, thereby implicating involvement of Ghrelin in feeding and maybe gastric motility (personal communication D.F. Carmignac). Interestingly, other family members of the GHS-R were identified as the motilin receptor (MTL-R) (Feighner et at., 1999; McKee et a i, 1997) and the NMU receptor (Fujii et a l, 2000; Howard et al.y 2000; Kojima et al.y 2000; Szekeres et al.y 2000). Both Motilin and NMU themselves are expressed throughout the gastrointestinal (GI) tract, being involved in GI tract motility (Howard et al. y 2000). It is thus feasible that Ghrelin might play a similar role in the GI system and food metabolism. Indeed, recently Masuda et al. confirmed that Ghrelin acts on the GI tract possibly by sympathetic innervation (Masuda et al. y 2000). I.v. injections of Ghrelin in rats dose-dependently stimulated gastric acid secretion and motility, and these stimulatory actions on gastric function were almost completely abolished by blocking vagal nerve activity (Masuda et al.y 2000). Whether Ghrelin’s actions on the GI tract via the vagal nerve pathway are mediated through the GHS-R remains to be proven. It was further shown that once daily s.c. Ghrelin injections for two weeks induced body weight gain in mice, but not food intake (Tschop et al.y 2000). However when Ghrelin was given i.c.v. food intake was enhanced. The body weight gain was shown to result from reduced fat utilisation, which was independent of GH and NPY (Tschop et al. y

Chapter 8: Ghrelin - a novel growth hormone secretagogue

2000). As shown previously in c-fos studies (Hewson and Dickson, 2000) and Ghrelin stomach expression studies (unpublished data D.F. Carmignac), Tschop et al. also indicated Ghrelin to be regulated by feeding state (Tschop et a l, 2000). Kamegai et al had previously demonstrated that Ghrelin upregulated hypothalamic NPY expression (Kamegai et a l, 2000) but Tschop et a l showed that presence of NPY was not necessary for Ghrelin to exert its effects on energy metabolism (Tschop et a l, 2000). Whether the effects of Ghrelin on GH secretion and metabolism are both mediated by the GHS-R remains to be proven. In preliminary RNAse protection assays I have shown that Ghrelin expression in the stomach was not affected by hypothalamic GHS-R over-expression in GHRH neurones, so the metabolic effects of Ghrelin might not be regulated by hypothalamic GHS-R, at least not those on GHRH neurones. Furthermore, by in situ hybridisation I have been unable to detect the GHS-R in the stomach or gut, suggesting that the effects of Ghrelin on the GI tract might not be mediated through local GHS-R.

In summary, our understanding of the physiological role of Ghrelin is still very much at the beginning. Its in vitro and in vivo actions suggest involvement in the regulation of GH secretion. In addition Ghrelin’s expression pattern and regulation in the stomach had implied a further action of Ghrelin in food metabolism and possibly gastric motility, which was recently confirmed (Masuda et a l, 2000; Tschop et a l, 2000). It seems that the GH secreting and metabolic effects of Ghrelin are similar to those of the artificial GHSs, although sympathetic innervation of the GI tract has never been reported for GHSs. Whether both the GH secreting and the metabolic effects of Ghrelin and the GHSs are mediated through the GHS-R and what the mechanism for Ghrelin’s influence on the GI tract is, remains unclear.

9

GHS treatment studies in transgenic mice and rats