The Melanocortin System in Atlantic Salmon (Salmo salar L.) and Its Role in Appetite Control

Abstract

The melanocortin system is a key neuroendocrine network involved in the control of food intake and energy homeostasis in vertebrates. Within the hypothalamus, the system comprises two main distinct neuronal cell populations that express the neuropeptides proopiomelanocortin (POMC; anorexigenic) or agouti-related protein (AGRP; orexigenic). Both bind to the melanocortin-4 receptor (MC4R) in higher order neurons that control both food intake and energy expenditure. This system is relatively well-conserved among vertebrates. However, in Atlantic salmon (Salmo salar L.), the salmonid-specific fourth round whole-genome duplication led to the presence of several paralog genes which might result in divergent functions of the duplicated genes. In the current study, we report the first comprehensive comparative identification and characterization of Mc4r and extend the knowledge of Pomc and Agrp in appetite control in Atlantic salmon. In silico analysis revealed multiple paralogs for mc4r (a1, a2, b1, and b2) in the Atlantic salmon genome and confirmed the paralogs previously described for pomc (a1, a2, and b) and agrp (1 and 2). All Mc4r paralogs are relatively well-conserved with the human homolog, sharing at least 63% amino acid sequence identity. We analyzed the mRNA expression of mc4r, pomc, and agrp genes in eight brain regions of Atlantic salmon post-smolt under two feeding states: normally fed and fasted for 4 days. The mc4ra2 and b1 mRNAs were predominantly and equally abundant in the hypothalamus and telencephalon, the mc4rb2 in the hypothalamus, and a1 in the telencephalon. All pomc genes were highly expressed in the pituitary, followed by the hypothalamus and saccus vasculosus. The agrp genes showed a completely different expression pattern from each other, with prevalent expression of the agrp1 in the hypothalamus and agrp2 in the telencephalon. Fasting did not induce any significant changes in the mRNA level of mc4r, agrp, or pomc paralogs in the hypothalamus or in other highly expressed regions between fed and fasted states. The identification and wide distribution of multiple paralogs of mc4r, pomc, and agrp in Atlantic salmon brain provide new insights and give rise to new questions of the melanocortin system in the appetite regulation in Atlantic salmon.

Keywords: Atlantic salmon; agouti-related protein (agrp); appetite control centers; brain; food intake; melanocortin system; melanocortin-4 receptor (mc4r); proopiomelanocortin (pomc).

FIGURE 1

Phylogenetic relationship of melanocortin-4 receptor (Mc4r) in Salmonidae family. The phylogenetic tree was constructed based on the predicted full-length peptide sequences using the maximum likelihood (ML) method, 1,000 bootstraps replicates, and JTT + G matrix-based model in MEGA X. The tree with the highest log likelihood (–3598.40) is shown. Protein ID accession numbers are shown after the species name. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Phylogenetic tree is rooted to the human MC4R sequence. For additional information related to the protein sequence alignment, please refer to Supplementary Figure 2.

FIGURE 2

Primary protein sequence alignment of the human melanocortin-4 receptor (hMC4R) and the Atlantic salmon paralogs (ssMc4ra1, a2, b1, and b2). The transmembrane domains for hMC4R (as reviewed in UniProt) and ssMc4r (as predicted in UniProt) are marked in blue. The N-terminal, extracellular loops (ECLs) 1–3, intracellular loops (ICLs) 1 to 3, and C-terminus (with helix VIII) are also shown. The N-terminal glycosylated amino acid residues and the important conserved motifs of GPCRs are marked in red (Rodrigues et al., 2013). C-terminal palmitoylation Cys is shown in yellow. The Cys involved in the disulfide bonds in hMC4R and those conserved in ssMc4r are in green.

FIGURE 3

Tertiary structure of human melanocortin-4 receptor (MC4R) and Atlantic salmon Mc4r paralogs. Three-dimensional (3D) protein structures were obtained from IntFOLD and edited in the PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC. (A) Human MC4R 3D structure with agouti-related protein (AGRP) (PDB entry 2IQV). (B) Atlantic salmon Mc4r paralogs 3D structure. N-terminal domains are colored in dark blue, C-terminal domains in red, the seven transmembrane helices (TMH) in gray. The extracellular (ECL) and intracellular (ICL) loops are represented by different colors. The boundary amino acids of TMH are labeled according to the protein sequence alignment.

FIGURE 4

(A) Schematic representation of Atlantic salmon post smolt brain, showing dissection of the eight brain regions for gene expression analysis: olfactory bulb (OB), telencephalon (TEL), midbrain (MB), hypothalamus (HYP), cerebellum (CE), saccus vasculosus (SV), pituitary (PT), and brain stem (BS). Dashed line (i) represents the dissected area of the CE inside the MB, and P represents the pineal gland. (B) Effects of 4 days of fasting on the mRNA expression levels of melanocortin-4 receptor (mc4r) paralog genes in eight regions of Atlantic salmon brain. Black and gray columns represent fed (n = 6) and fasted (n = 6) fish, respectively. Values are expressed as copy number per total RNA used in the reaction. The dots represent the individual fish, and bars represent mean ± SEM. Two-tailed t-test was performed to assess the statistically significant differences between the two groups. Interaction between the brain region response and the treatment was analyzed with two-way ANOVA (Supplementary Table 1 for detailed information).

FIGURE 5

Effects of 4 days of fasting on the mRNA expression levels of proopiomelanocortin (pomc) and agouti-related protein (agrp) paralog genes in eight regions of Atlantic salmon brain. Black and gray columns represent fed (n = 6) and fasted (n = 6) fish, respectively. Values are expressed as copy number per total RNA used in the reaction. The dots represent individual fish, bars represent mean ± SEM, and asterisks show the significant degree (*p < 0.05, **p < 0.01). Two-tailed t-test was performed to assess the statistically significant differences between the two groups. Interaction between the brain region response and the treatment was analyzed with two-way ANOVA, followed by Sidak posttest (refer to Supplementary Table 1 for detailed information).