The study of ghrelin secretion and acyl-modification using mice and ghrelinoma cell lines.

Ghrelin is a peptide hormone with a unique structure comprising a medium chain fatty acid modification. Ghrelin cells are known to be abundantly localized in the gastric mucosa and are released into the blood stream to exert their multifunctional physiological effects. To elucidate the regulatory mechanisms of ghrelin secretion and acyl-modification, we developed novel ghrelin-producing cell lines. Using ghrelinoma cell lines, we focused on the mechanisms of ghrelin secretion and found that several GPCRs were highly expressed in ghrelin cells. Then, we showed that noradrenaline treatment stimulated ghrelin secretion via ß1-adrenergic receptor, and fasting-induced ghrelin elevation was completely inhibited by the ß1-adrenergic receptor antagonist in mice. In addition, we demonstrated that long chain fatty acids, glucose, and L-glutamate significantly inhibited ghrelin secretion. Furthermore, we recently revealed that the genes involved in fatty acid synthesis and long chain fatty acid metabolism were expressed in ghrelin cells, and that CPT-1 inhibitor treatment dramatically decreased the levels of acyl-modified ghrelin. Here, we introduce the current knowledge of the mechanisms involving ghrelin secretion and its acyl-modification.

Acyl modifications in bovine, porcine, and equine ghrelins.

Ghrelin is a peptide hormone with various important physiological functions. The unique feature of ghrelin is its serine 3 acyl-modification, which is essential for ghrelin activity. The major form of ghrelin is modified with n-octanoic acid (C8:0) by ghrelin O-acyltransferase. Various acyl modifications have been reported in different species. However, the underlying mechanism by which ghrelin is modified with various fatty acids remains to be elucidated. Herein, we report the purification of bovine, porcine, and equine ghrelins. The major active form of bovine ghrelin was a 27-amino acid peptide with an n-octanoyl (C8:0) modification at Ser3. The major active form of porcine and equine ghrelin was a 28-amino acid peptide. However, porcine ghrelin was modified with n-octanol (C8:0), whereas equine ghrelin was modified with n-butanol (C4:0) at Ser3. This study indicates the existence of structural divergence in ghrelin and suggests that it is necessary to measure the minor and major forms of ghrelin to fully understand its physiology.

Determination of Ghrelin Structure in the Barfin Flounder (Verasper moseri) and Involvement of Ingested Fatty Acids in Ghrelin Acylation.

Ghrelin is a peptide hormone that is acylated with a fatty acid, usually n-octanoic acid, at the third amino acid (aa) residue (usually a serine or threonine), and this acylation is known to be essential for ghrelin activity not only in mammals but also in non-mammals, such as fish. However, the modification mechanisms of ghrelin modification in fish are not known. In this study, we elucidated the structure of ghrelin in a teleost, the barfin flounder (Verasper moseri), and determined whether ingested free fatty acids of various chain lengths participated in ghrelin acylation. Complementary DNA cloning revealed the barfin flounder prepro-ghrelin to be a 106-aa peptide and the mature ghrelin to be a 20-aa peptide (GSSFLSPSHKPPNKGKPPRA). However, purification of ghrelin peptides from stomach extracts demonstrated that the major form of the hormone was a 19-aa decanoylated peptide [GSS(C10:0)FLSPSHKPPNKGKPPR] missing the last alanine of the 20-aa peptide. Ingestion of feed enriched with n-heptanoic acid (C7), n-octanoic acid (C8), or n-non-anoic acid (C9) changed the modification status of the peptide: ingestion of C8 or C9 increased the amount of C8:0 or C9:0 19-aa ghrelin, respectively, but no C7:0 ghrelin was isolated after ingestion of C7. These results indicate that ingested free fatty acids are substrates for ghrelin acylation in the barfin flounder, but the types of free fatty acids utilized as substrates may be limited.