The G-protein-coupled melanocortin receptors (MCRs) play an important role in a variety of essential functions such as the regulation of pigmentation, energy homeostasis, and steroid production. We performed a comprehensive characterization of the MC system in Fugu (Takifugu rubripes). We show that Fugu has an AGRP gene with high degree of conservation in the C-terminal region in addition to a POMC gene lacking gamma-MSH. The Fugu genome contains single copies of four MCRs, whereas the MC3R is missing. The MC2R and MC5R are found in tandem and remarkably contain one and two introns, respectively. We suggest that these introns were inserted through a reverse splicing mechanism into the DRY motif that is widely conserved through GPCRs. We were able to assemble large blocks around the MCRs in Fugu, showing remarkable synteny with human chromosomes 16 and 18. Detailed pharmacological characterization showed that ACTH had surprisingly high affinity for the Fugu MC1R and MC4R, whereas alpha-MSH had lower affinity. We also showed that the MC2R gene in Fugu codes for an ACTH receptor, which did not respond to alpha-MSH. All the Fugu receptors were able to couple functionally to cAMP production in line with the mammalian orthologs. The anatomical characterization shows that the MC2R is expressed in the brain in addition to the head-kidney, whereas the MC4R and MC5R are found in both brain regions and peripheral tissues. This is the first comprehensive genomic and functional characterization of a GPCR family within the Fugu genome. The study shows that some parts of the MC system are highly conserved through vertebrate evolution, such as regions in POMC coding for ACTH, alpha-MSH, and beta-MSH, the C-terminal region of AGRP, key binding units within the MC1R, MC2R, MC4R, and MC5R, synteny blocks around the MCRs, pharmacological properties of the MC2R, whereas other parts in the system are either missing, such as the MC3R and gamma-MSH, or different as compared to mammals, such as the affinity of ACTH and MSH peptides to MC1R and MC4R and the anatomical expression pattern of the MCRs.

Background:The global prevalence of obesity and overweight is increasing rapidly among adults as well as among children and adolescents. Recent genome-wide association studies have provided strong support for association between variants in the FTO gene and obesity. We sequenced regions of the FTO gene to identify novel variants that are associated with obesity and related metabolic traits. Results: We screened exons 3 and 4 including exon-intron boundaries in FTO in 48 obese children and adolescents and identified three novel single nucleotide polymorphism in the fourth intronic region, (c.896 þ 37A4G, c.896 þ 117C4G and c.896 þ 223A4G). We further genotyped c.896 þ 223A4G in 962 subjects, 450 well-characterized obese children and adolescents and 512 adolescents with normal weight. Evidence for differences in genotype frequencies were not detected for the c.896 þ 223A4G variant between extremely obese children and adolescents and normal weight adolescents (P ¼ 0.406, OR ¼ 1.154 (0.768-1.736)). Obese subjects with the GG genotype, however, had 30% increased fasting serum insulin levels (P ¼ 0.017) and increased degree of insulin resistance (P ¼ 0.025). There were in addition no differences in body mass index (BMI) or BMI standard deviation score (SDS) levels among the obese subjects according to genotype and the associations with insulin levels and insulin resistance remained significant when adjusting for BMI SDS. Conclusion: These findings suggest that this novel variant in FTO is affecting metabolic phenotypes such as insulin resistance, which are not mediated through differences in BMI levels.

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