Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology
Sequence, genomic organization and expression of ghrelin receptor in grass carp, Ctenopharyngodon idellus
Introduction
Growth hormone secretagogue receptor (GHS-R) belongs to the family of G-protein-coupled receptors containing seven transmembrane domains (TMD). Mammalian GHS-R gene is composed of two exons, and two types of GHS-R mRNAs, GHS-R1a and 1b, are generated by alternative transcription process of the gene (Howard et al., 1996). So far, GHS-Rs have been identified in mammals (Howard et al., 1996, Katayama et al., 2000, Kitazawa et al., 2011, Suzuki et al., 2012), avians (Geelissen et al., 2003, Tanaka et al., 2003) and several fish species (Palyha et al., 2000, Chan and Cheng, 2004, Olsson et al., 2008, Kaiya et al., 2009a, Kaiya et al., 2009b, Small et al., 2009, Eom et al., 2014). In fish, GHS-R1a was first identified by Palyha et al. (2000) in pufferfish Takifugu rubripes. After that, Chan and Cheng (2004) identified two isoforms of GHS-R in black seabream Acanthopagrus schlegeli, sbGHSR-1a and sbGHSR-1b. It was the first report on the identification of a GHSR-1b transcript from species other than mammals and the demonstration that receptor interaction might provide a possible explanation for the existence and biological significance of the sbGHSR-1b transcript. Since then GHS-R has also been identified in the zebrafish Danio rerio (Olsson et al., 2008), orange-spotted grouper Epinephelus coioides (Chen et al., 2008), channel catfish Ictalurus punctatus (Small et al., 2009), rainbow trout Oncorhynchus mykiss (Kaiya et al., 2009a), Mozambique tilapia Oreochromis mossambicus (Kaiya et al., 2009b), goldfish Carassius auratus (Kaiya et al., 2010) and Atlantic salmon Salmo salar (Hevrøy et al., 2011).
Ghrelin was identified as the endogenous ligand of GHS-R and named for its potent growth hormone (GH)-secreting properties (Kojima et al., 1999). Several studies have also examined ghrelin's role in body weight homeostasis and as an important indicator of energy insufficiency (Kamegai et al., 2000, Tschop et al., 2000, Wisse et al., 2001, Wang et al., 2002, Asakawa et al., 2003, Yasuda et al., 2003). The central role of ghrelin in body weight homeostasis and the critical importance of GHS-R in transmitting ghrelin's energy balance and GH secretory messages are highlighted by recent studies on a GHS-R knockout mouse model (Sun et al., 2004, Sun et al., 2008). In teleost, a goldfish that has four GHS-Ra that is divided into two types, 1a and 2a, and three of these four receptors (except 2a-2 receptor), were activated by goldfish ghrelin or GHS (Kaiya et al., 2010). Hevrøy et al. (2012) have reported that lower circulating ghrelin during negative energy homeostasis induces down-regulation of GHSR1a-LR, neuropeptide Y, and anorexigenic factors at transcriptional levels in the hypothalamus, which over time lead to a voluntary anorexia development in adult salmon held at 19 °C. The expression of the GH secretagogue receptors (sbGHSR-1a and sbGHSR-1b) was significantly increased in the hypothalamus of the food-deprived seabream (Zhang et al., 2008). However, the information about the physiological function on GHS-R of fish is still lacking.
Although accumulating evidence have demonstrated that ghrelin is a brain-gut peptide with multiple functions in fish like in mammals, the mechanism of its functions is still not well defined. Rainbow trout ghrelin stimulates GH release from the pituitary of juvenile rainbow trout in vivo and in vitro (Kaiya et al., 2003). Jönsson et al. (2007) have reported that rainbow trout ghrelin does not stimulate food intake when injected intraperitoneally. For further understanding how these physiological actions occur, it is necessary to identify and characterize ghrelin receptor in fish. The grass carp is one of the four major domesticated cyprinid fish species in China. This species has become the principal species for freshwater aquaculture and has been widely favored in China (Wang et al., 2008). Detailed study into the regulation of ghrelin and its receptors expression in different physiological states is anticipated to provide meaningful insight into grass carp ghrelin physiology. Therefore, the purpose of this study was to identify and characterize GHS-R1a in grass carp. We found that GHS-R1a was mainly expressed in pituitary. Tissue expression distribution of the receptor mRNA was determined by using quantitative real-time PCR, and investigated the effects of fasting and intraperitoneally injected with ghrelin on grass carp ghrelin and GHS-R1a mRNAs expression in the pituitary.
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Fish and experimental conditions and samples collection
The experimental grass carp (initial body mass: 43.91 ± 0.46 g) were provided by Wuhan Academy of Agricultural Science & Technology and kept in 1000-L tanks with a constant flow of filtered water. Prior to the initiation of the experiment, grass carp were fed twice a day at 8:00 h and 17:00 h for 2 weeks to acclimate to feeding conditions for 3 weeks. After acclimation, six grass carp were deeply anesthetized with MS-222 (200 mg L− 1), and killed by immediate spinal destroying for measure and dissection.
Cloning and sequence analysis of grass carp GHE-R1a coding domain sequence
The complete coding domain sequence of GHS-R1a was 1803 bp in length, encoding a 360 aa protein, the start codon was ATG and stop codon was TGA. The nucleotide sequence showed high similarity to GHS-R1a in other species, especially in teleost. Seven TMD were predicated to be in grass carp GHS-R1a, and they were highly conserved in the Cyprinidae fish (Fig. 1). Multiple amino acid sequences alignment showed that amino acid sequences of GHS-R1a were highly conserved (Fig. 1). The phylogenetic
Discussion
In this study, for the first time, we identified the complete coding domain sequence and tissue distribution of GHS-R1a from grass carp. Furthermore, we gained an evolutionary prospective on the function of the gene. Two types of GHS-R have been identified, GHS-R1a and GHS-R1b. GHS-R1a mediateed biological actions of GHS and ghrelin (Unniappan and Peter, 2005). In the present study, the grass carp GHS-R1a complete coding domain sequence was successfully obtained. Grass carp GHS-R1a shared the
Acknowledgments
This work was financially supported by the National Nature Science Foundation of China (NSFC) Grant (31302203), the Research Fund for the Doctoral Program of Higher Education of China (20120146120010), the Fundamental Research Funds for State Key Laboratory of Freshwater Ecology and Biotechnology of China (Grant No. 2013FB11), the National Innovation Experiment Program for University Students (201210504099), and supported by the Natural Science Foundation of Hubei Province of China (2012FFC08101
References (50)
- et al.
Fasting induces preproghrelin mRNA expression in the brain and gut of zebrafish, Danio rerio
Gen. Comp. Endocrinol.
(2009) - et al.
Identification and functional characterization of two alternatively spliced growth hormone secretagogue receptor transcripts from the pituitary of black seabream Acanthopagrus schlegeli
Mol. Cell. Endocrinol.
(2004) - et al.
Ghrelin ligand-receptor mRNA expression in hypothalamus, proventriculus and liver of chicken (Gallus gallus domesticus): studies on ontogeny and feeding condition
(2007) - et al.
Growth performance and adiposity in gilthead seabream (Spaurus aurata): risks and benefits of high energy diets
Aquaculture
(1999) - et al.
Molecular cloning, regulation, and functional analysis of two GHS-R genes in zebrafish
Exp. Cell Res.
(2014) - et al.
Distribution and regulation of chicken growth hormone secretagogue receptor isoforms
Gen. Comp. Endocrinol.
(2003) - et al.
Ghrelin is involved in voluntary anorexia in Atlantic salmon raised at elevated sea temperatures
Gen. Comp. Endocrinol.
(2012) - et al.
Dominance, nutritional state, and growth hormone levels in rainbow trout (Oncorhynchus mykiss)
Horm. Behav.
(1996) - et al.
Plasma ghrelin levels in rainbow trout in response to fasting, feeding and food composition, and effects of ghrelin on voluntary food intake
Comp. Biochem. Physiol. A
(2007) - et al.
Ghrelin: a multifunctional hormone in non-mammalian vertebrates
Comp. Biochem. Physiol. A
(2008)
Ghrelin receptor (GHS-R)-like receptor and its genomic organisation in rainbow trout, Oncorhynchus mykiss
Comp. Biochem. Physiol. A
Two functional growth hormone secretagogue receptor (ghrelin receptor) type 1a and 2a in goldfish, Carassius auratus
Mol. Cell. Endocrinol.
Molecular identification of ghrelin receptor (GHS-R1a) and its functional role in the gastrointestinal tract of the guinea-pig
Peptides
Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method
Methods
Ghrelin, cholecystokinin, and peptide YY in Atlantic salmon (Salmosalar): molecular cloning and tissue expression
Gen. Comp. Endocrinol.
Identification of genes for the ghrelin and motilin receptors and a novel related gene in fish, and stimulation of intestinal motility in zebrafish (Danio rerio) by ghrelin and motilin
Gen. Comp. Endocrinol.
Time course of the GH/IGF-I axis response to fasting and increased ration in chinook salmon (Oncorhynchus tshawytscha)
Gen. Comp. Endocrinol.
Food restriction, ghrelin, its antagonist and obestatin control expression of ghrelin and its receptor in chicken hypothalamus and ovary
Comp. Biochem. Physiol. A
Sequence, genomic organization and expression of two channel catfish, Ictalurus punctatus, ghrelin receptors
Comp. Biochem. Physiol. A
The effect of starvation on growth and plasma growth hormone concentrations of rainbow trout, Oncorhynchus mykiss
Gen. Comp. Endocrinol.
Molecular identification of GHS-R and GPR38 in Suncus murinus
Peptides
Molecular characterization of chicken growth hormone secretagogue receptor gene
Gen. Comp. Endocrinol.
Structure, distribution and physiological functions of ghrelin in fish
Comp. Biochem. Physiol. A
Peripheral ghrelin selectively increases Fos expression in neuropeptide Y-synthesizing neurons in mouse hypothalamic arcuate nucleus
Neurosci. Lett.
Extraction of pepsin-soluble collagen from grass carp (Ctenopharyngodon idella) skin using an artificial neural network
Food Chem.
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These authors contributed equally to the study.