The SNPs of candidate genes
The SNPs of candidate genes may exert their effect on associated traits through changing of gene expression (Bonafe et al., 2003, Wyszynska-Koko et al., 2006). In agreement, GHSR SNPs were significantly associated with upregulation of this gene in the pituitary gland of GG/GG chickens. The GHRL/GHSR/GH system contributes to the control of a number of key pathways including energy metabolism, GH secretion, and glucose metabolism (Lim et al., 2011). Thus, it is possible that the association with the studied growth traits resulted from altered regulation of the growth axis related genes, GHRL and GH. Strikingly, the gene expression of the GHRL in the hypothalamus and proventriculus and that of the GH in the pituitary gland was also significantly up-regulated in chickens with GG/GG haplotype. Chickens with GG/GG haplotype also have increased serum level of GH and GHRL. Since GH secretion is regulated by hypothalamic neuropeptides, including GHRL (Ahmed and Harvey, 2002), it is possible that change of lysine to arginine, due to G244A SNP, may directly or indirectly change receptor function in a way that increases GHRL secretion from hypothalamus which in turn stimulates secretion of GH from pituitary. The G244A SNP involves a residue (lysine 328) totally conserved in all species studied so far. This high degree of conservation of lysine 328 indicates that amino IPI-504 substitutions at this position may affect receptor function. Although, G244A SNP is located in the linear intracellular domain remote from the specific binding site, it is possible that the conformation of the GHSR protein may be changed by this SNP which in turn can affect its function. As a member of the G protein-coupled receptor superfamily, GHSR signals via activation of the protein kinase C (PKC) system and hydrolysis of phosphatidylinositol (phospholipase–IP3 pathway) resulting in the elevation of intracellular calcium levels (Korbonits et al., 2004, Ueno et al., 2005). The linear intracellular domain of GHSR plays a crucial role in this signaling pathway through its interaction with, and binding to, the downstream signaling effectors. It is possible that changing of the highly conserved lysine 328 into arginine causes conformational changes in the GHSR protein in a way that the linear intracellular domain becomes unable to interact with/bind to the downstream signaling effectors (PKC and IP3). Further investigations using yeast two hybrid or co-immunoprecipitation experiments can confirm this possibility. Regulation of appetite and food intake in birds remain open research questions. Here, we found a mutation in ghrelin receptor (GHSR) which increases expression of this gene in addition to expression and serum level of its specific ligand, GHRL, and GH which surprisingly accompanied by increase food intake and body weight gain in chicken. In parallel, a recent study by Aghdam Shahryar and Lotfi (2016) reported that the hunger GHRL signal was decreased following injection of GHSR antagonist in chicken. In mammals, administration of GHSR antagonist or ablation of GHSR also resulted in decreased food intake and body weight (Lin et al., 2011). To best of our knowledge, this is the first study confirms the orexigenic effect of endogenous GHRL in chicken. In agreement, previous reports suggest (without experimental proof) that the increased level of endogenous GHRL as seen naturally after fasting acts as a hunger signal rather than a satiety signal in birds (Kaiya et al., 2013a, Kaiya et al., 2007, Shousha et al., 2005). We hypothesized that effect of exogenous GHRL is quietly different from endogenous GHRL regarding the appetite of birds. Endogenous GHRL in birds may act, similar to central effect of GHRL in mammals (Nakazato et al., 2001), via orexigenic hypothalamic neuropeptide Y (NPY) neurons which stimulate appetite. However, exogenous GHRL may target hypothalamic corticotropin-releasing factor (CRF)-secreting neurons instead of NPY neurons, resulting in increased serum corticosterone levels and reduced appetite (Saito et al., 2005). Additionally, administration of GHRL increases vocalization, an indicator of anxiety, in newly hatched chicks via the CRF system (Carvajal et al., 2009).