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Peptide Purification, Complementary Deoxyribonucleic Acid (DNA) and Genomic DNA Cloning, and Functional Characterization of Ghrelin in Rainbow Trout

Department of Biochemistry (H.Kai., H.H., K.K.), National Cardiovascular Center Research Institute, Osaka 565-8565, Japan; Division of Molecular Genetics (M.K.), Institute of Life Science, Kurume University, Fukuoka 839-0861, Japan; and School of Fisheries Sciences (S.M., A.T., H.Kaw.), Kitasato University, Iwate 022-0101, Japan

Address all correspondence and requests for reprints to: Hiroyuki Kaiya, Ph.D., Department of Biochemistry, National Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan. E-mail: kaiya{at}ri.ncvc.go.jp.

We have identified ghrelin from the stomach of rainbow trout. Four isoforms of ghrelin peptide were isolated: the C-terminal amidated type of rainbow trout ghrelin (rt ghrelin) composed of 24 amino acids (GSSFLSPSQKPQVRQGKGKPPRV-amide) is a basic form; des-VRQ-rt ghrelin, which deleted three amino acids (V¹³R¹⁴Q¹⁵) from rt ghrelin; and further two types of rt ghrelin that retained the glycine residue at the C terminus, rt ghrelin-Gly, and des-VRQ-rt ghrelin-Gly. The third serine residue was modified by octanoic acid, decanoic acid, or the unsaturated form of those fatty acids. In agreement with the isolated peptides, two cDNAs of different lengths were isolated. The rt ghrelin gene has five exons and four introns, and two different mRNA molecules are predicted to be produced by alternative splicing of the gene. A high level of ghrelin mRNA expression was detected in the stomach, and moderate levels were detected in the brain, hypothalamus, and intestinal tracts. Des-VRQ-rt ghrelin stimulated the release of GH in the rat in vivo. Furthermore, des-VRQ-rt ghrelin stimulated the release of GH, but not the release of prolactin and somatolactin in rainbow trout in vivo and in vitro. These results indicate that ghrelin is a novel GH secretagogue in rainbow trout that may affect somatic growth or osmoregulation through GH. Because ghrelin is expressed in various tissues other than stomach, it may play important role(s) in cellular function as a local regulator.

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