Ghrelin, cholecystokinin, and peptide YY in Atlantic salmon (Salmo salar): Molecular cloning and tissue expression
Introduction
The gastrointestinal (GI) tract is the largest endocrine organ in vertebrates and produces around 30 different peptide hormones. These hormones act on several tissues including the GI tract itself, exocrine glands and the central nervous system (CNS) (Mendieta-Zerón et al., 2008). Almost all of the GI hormones are sensitive to gut nutrient content and some of them play an important role in control of appetite and food ingestion (Konturek et al., 2004, Murphy and Bloom, 2004).
Cholecystokinin (CCK), secreted by the proximal intestine, is one of the anorexigenic GI peptides (Raybould, 2007). The C-terminal octapeptide (CCK-8) is well conserved among vertebrates except for a single substitution of the sixth amino acid from the C-terminal position (Johnsen, 1998). The tyrosine sulfated form has the highest biological activity (Himick et al., 1993, Nielsen et al., 1998, Chandra and Liddle, 2007). The physiological role of CCK is not only in regulation of food intake and satiation but also in digestion where it has been described as playing key roles in the regulation of the intestinal phase. CCK stimulates the exocrine pancreas and discharge of bile from the gallbladder and also affects smooth muscle contraction in the jejunum and pyloric sphincter (Ramirez and Farrar, 1970, Shiratori et al., 1986). Peptide YY (PYY), which is produced in the distal intestine, is known to act as an antagonistic peptide for CCK in the control of pancreatic exocrine function (Walsh, 1994). PYY is a peptide hormone belonging to the neuropeptide Y (NPY) family that consists of 36 amino acids. Though NPY is well known to have a strong orexigenic function in the hypothalamus (Schwartz et al., 2000), PYY inhibits food intake, and reduces weight gain (Moran, 1955, Batterham et al., 2002, Challis et al., 2003, Moran et al., 2005). In contrast to CCK and PYY, ghrelin (GHRL), which is mainly expressed in the stomach, is the only orexigenic GI peptide that has been isolated to date. While the orexigenic function of GHRL has been well described in mammals, it is still uncertain whether it plays a similar role in teleosts (Jönsson et al., 2007). GHRL was first identified as the endogenous ligand for the growth hormone secretagogue receptor (GHS-R) in rat and human (Kojima et al., 1999). The first four amino acid residues (GSSF) with the n-octanoyl modification in the third serine residue in mature GHRL are considered to be the active core of the biological activity of GHRL (Bednarek et al., 2000).
We report here the cloning of full-length cDNAs that encode for the GHRL, CCK and PYY of Atlantic salmon (Salmo salar). Atlantic salmon is one of the most important aquaculture species in cold-water regions, and has been targeted for studies of physiology of growth, digestion, and energy homeostasis. While there is a high level of understanding of how GHRL, CCK and PYY affect these processes in mammals, knowledge of their roles in teleosts is still very fragmentary. The basic information regarding GI peptides in Atlantic salmon contributes to the elucidation of the physiological relationships of fish gut peptide hormones.
There is a growing use of vegetable ingredients in aquafeeds due to limitations in the availability of available marine protein sources. One of the main problems that arise when fish are fed high levels of plant proteins is reduced growth. This has been partially explained by a reduced appetite and lower voluntary feed intake level. For this reason, it is important to elucidate the physiological mechanisms involved in the regulation of appetite, feed intake, and satiety in important aquaculture species like Atlantic salmon.
Section snippets
Animals and samples
For cDNA cloning and analysis of tissue distribution, Atlantic salmon (S. salar), average body weight 44.7 g, were reared at the Bergen High-Technology Centre (Bergen, Norway) in indoor tanks supplied with a continuous flow of fresh water at 8 °C. The fish were fed a commercial pellet diet (EWOS innovation, Bergen, Norway). At sampling four males and four females were killed with an overdose of MS-222, and tissues were collected and stored in RNAlater (Ambion, Austin, TX, USA) at −20 °C until RNA
GHRL
From the RACE PCR for salmon GHRL, full-length cDNA sequences of two different GHRL isoforms were obtained. Atlantic salmon prepro-GHRL-1 (GenBank Accession No. AB443431) nucleotide sequence was 470 bp in length, and contained a 57 bp of 5′-untranslated region (5′-UTR), a 77 bp of 3′-untranslated region (3′-UTR) and a 336 bp of open reading frame (ORF) encoding a prepro-GHRL-1 with 111 amino acids (Fig. 1). Atlantic salmon prepro-GHRL-2 (461 bp, GenBank Accession No. AB443432) nucleotide encoded a
Discussion
This study identified full-length cDNA sequences for GHRL-1, GHRL-2, CCK-L, CCK-N, and PYY in Atlantic salmon. Comparative analysis of the sequences obtained clearly indicates that they are the respective GHRL, CCK, and PYY gene homologs in Atlantic salmon. In the rainbow trout GHRL gene, an alternative splicing site has been found at the second intron, which results in the production of two types of GHRL (rtGHRL and des-VRQ-rtGHRL) (Kaiya et al., 2003). The salmon GHRL-1 and GHRL-2 were quite
Acknowledgments
We thank Anne-Grethe Gamst Moen (BIO, UiB) and Dr. Ann-Elise Olderbakk Jordal (BIO, UiB) for assistance during sampling. We are grateful to the staff of EWOS Innovation (Lønningdal, Norway) for supplying fish. This work was supported by a Research Fellowship of the Japan Society for the Promotion of Science (JSPS) for Young Scientists to K.M., Research Council of Norway Grant No. 172548/S40, grants from Helse Vest and the University of Bergen (NettMettBAC) to I.R and in part by “the promotion
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