Ghrelin, an acylated brain and gut peptide, is primarily produced by endocrine cells of the gastric mucosa for secretion into the circulation. The major active form of ghrelin is a 28-amino-acid peptide containing an n-octanoyl modification at serine that is essential for activity. Studies have identified multiple physiological functions for ghrelin, including GH release, appetite stimulation, and metabolic fuel preference. Until now, there has not been any report detailing the mechanism of ghrelin acyl modification. Here we report that ingestion of either medium-chain fatty acids (MCFAs) or medium-chain triacylglycerols (MCTs) increased the stomach concentrations of acylated ghrelin without changing the total (acyl- and des-acyl-) ghrelin amounts. After ingestion of either MCFAs or MCTs, the carbon chain lengths of the acyl groups attached to nascent ghrelin molecules corresponded to that of the ingested MCFAs or MCTs. Ghrelin peptides modified with n-butyryl or n-palmitoyl groups, however, could not be detected after ingestion of the corresponding short-chain or long-chain fatty acids, respectively. Moreover, n-heptanoyl ghrelin, an unnatural form of ghrelin, could be detected in the stomach of mice after ingestion of either n-heptanoic acid or glyceryl triheptanoate. These findings indicate that ingested medium-chain fatty acids are directly used for the acylation of ghrelin.

Complementary deoxyribonucleic acid (cDNA) encoding goldfish preproghrelin was identified using rapid amplification of the cDNA ends (RACE) and reverse transcription (RT)-polymerase chain reaction (PCR). The 490 bp cDNA encodes a 103 amino acid preproghrelin which has a 26 amino acid signal region, 19 amino acid mature peptide and a 55 amino acid C-terminal peptide region. The mature peptide region of goldfish ghrelin has two putative cleavage sites and amidation signals (GRR); one after 12 amino acids and the other after 19 amino acids. The serine (S) in the second amino acid position in the "active core" of ghrelin is substituted with threonine (T). The goldfish ghrelin gene has four exons and three short introns and resembles the human ghrelin gene. Ghrelin messenger RNA (mRNA) expression was detected in the brain, pituitary, intestine, liver, spleen and gill by RT-PCR followed by Southern blot analysis, and in the intestine by Northern blot. Intracerebroventricular (ICV) injection of n-octanoylated goldfish ghrelin (1-19) stimulates food intake in goldfish.

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We have identified the amphibian ghrelin from the stomach of the bullfrog. We also examined growth hormone (GH)-releasing activity of this novel peptide in both the rat and bullfrog. The three forms of ghrelin identified, each comprised of 27 or 28 amino acids, possessed 29% sequence identity to the mammalian ghrelins. A unique threonine at amino acid position 3 (Thr 3 ) in bullfrog ghrelin differs from the serine present in the mammalian ghrelins; this Thr 3 is acylated by either n-octanoic or n-decanoic acid. The frog ghrelin-28 has a complete structure of GLT (O-n-octanoyl)FLSPAD-MQKIAERQSQNKLRHGNM; the structure of frog ghrelin-27 was determined to be GLT(O-n-octanoyl)FLSPAD-MQKIAERQSQNKLRHGN; frog ghelin-27-C10 possessed a structure of GLT(O-n-decanoyl)FLSPADMQKIAER-QSQNKLRHGN. Northern blot analysis demonstrated that ghrelin mRNA is predominantly expressed in the stomach. Low levels of gene expression were observed in the heart, lung, small intestine, gall bladder, pancreas, and testes, as revealed by reverse transcription polymerase chain reaction analysis. Bullfrog ghrelin stimulated the secretion of both GH and prolactin in dispersed bullfrog pituitary cells with potency 2-3 orders of magnitude greater than that of rat ghrelin. Bullfrog ghrelin, however, was only minimally effective in elevating plasma GH levels following intravenous injection into rats. These results indicate that although the regulatory mechanism of ghrelin to induce GH secretion is evolutionary conserved, the structural changes in the different ghrelins result in species-specific receptor binding. Growth hormone (GH)1 secretion from the pituitary gland is regulated by hypothalamic hormones; growth hormone-releasing hormone (GHRH) stimulates GH secretion, whereas somatostatin is inhibitory (1). Derivatives of Met-enkephalin stimulate GH release (2), the first demonstration that small synthetic peptides and nonpeptide molecules, dubbed growth hormone secretagogues (GHSs), can mediate GH release through a receptor distinct from that of GHRH (3-5). The G-protein-coupled GHS receptor (GHS-R) was subsequently identified in swine, rat, and human (6, 7), suggesting that one or more unknown ligands for this receptor are endogenously present.We recently discovered an endogenous ligand for GHS-R from the rat stomach, using an intracellular calcium influx assay in stable cell lines expressing rat GHS-R (8). This novel molecule, a 28-amino acid peptide named ghrelin (from "ghre," the Proto-Indo-European root of "grow"), possesses a unique serine residue at the third N-terminal position (Ser 3 ) that is n-octanoylated (8, 9). Acylation of Ser 3 is essential for ghrelin bioactivity. cDNA analysis revealed that the rat ghrelin sequence follows the 23-residue signal sequence within the 117-residue prepro-ghrelin. Ghrelin stimulates GH secretion both in vivo and in vitro. Accumulating evidence in mammals suggests that, in addition to regulating GH release, ghrelin also influences feeding behavior (10, 11), gastrointestinal function (12, 13), and ener...

In this study, we report the purification, cDNA cloning, and characterization of the novel growth hormone-releasing peptide, ghrelin, in the chicken (Gallus gallus). Chicken ghrelin is composed of 26 amino acids (GSSFLSPTYKNIQQQKDTRKPTARLH) and possesses 54% sequence identity with human ghrelin. The serine residue at position 3 (Ser(3)) is conserved between the chicken and mammalian species, as its acylation by either n-octanoic or n-decanoic acid. Chicken ghrelin mRNA is predominantly expressed in the stomach, where it is present in the proventriculus but absent in the gizzard. Using RT-PCR analysis, low levels of expression were also detectable in brain, lung, and intestine. Administration of chicken ghrelin increases plasma GH levels in both rats and chicks, with a potency similar to that of rat or human ghrelin. In addition, chicken ghrelin also increases plasma corticosterone levels in growing chicks at a lower dose than in mammals. The present results indicate that the stimulatory effect of ghrelin on GH secretion is evolutionarily conserved, whereas its effect on adrenal function seems to be unique in the chicken.