OGhrelin human

Fig. 5. Amino acid sequence of human ghrelin

O 40 20

O 40 20

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Mg/kg iv

0.2 ghrelin

1.0 GHRH

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0.2 ghrelin

1.0 GHRH

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Time (min)

Fig. 6. Ghrelin and GHRP each synergise similarly with GHRH to stimulate GH release. Low-dose sensitisation of GHRP-2 (leftpanel) and ghrelin (rightpanel) in normal young men.Values are means ± SEM. (Data from [8])

logical isolation of the natural hormone ghrelin did not alter the interesting attributes of the GHRP and GHS compounds; rather, it stimulated further new biochemical analysis to understand the novel functionality of this uniquely octanoy-lated peptide.

Because the chemistry of the ghrelin molecule and its bioactivity on the ghrelin GHS receptor type 1a are only achievable after the post-transla-tional octanoyl addition, it is apparent that a mixture of special strategies, approaches and techniques was required for the isolation and identification of ghrelin. This included the unnatural GHRPs, the cloned GHRP/GHS receptor, the classical purification of ghrelin from stomach extracts and finally mass spectrometry. Even a seemingly simple decision based on conceptualisation and strategy guiding the approaches may profoundly influence the success of the isolation of a natural hormone. For example, because of the bioactive dependency of the ghrelin octanoylation step and because octanoylation is a post-translational step, a singular recombinant molecular cDNA approach would have been unsuccessful. Additionally, our studies of unnatural GHRPs encouraged us to believe that the putative natural GHRP hormone would be another hypothalamic hypophysiotropic hormone and for that reason the ideal tissue from which to isolate the natural hormone would be the hypothalamus. Thus for several years we pursued the same classical isolation procedure and approaches from thousands of porcine hypothala-mi as utilised for TRH and LHRH, but this was unsuccessful. Several years later, Kojima et al. showed that the primary anatomical origin of ghrelin is the stomach and not the hypothalamus, which again emphasised the role and importance of conceptualisation and strategy in the isolation process of a new hormone. Furthermore, before the isolation of ghrelin was accomplished, the focus of the GHRPs was on increasing GH secretion, although limited studies in rats and mice had already demonstrated that GHRPs also increased food intake. However, once ghrelin was isolated and its origin was found to be primarily from the stomach, the focus on the biological role of ghre-lin and GHRP increasingly has been on food intake.

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