■黑皮质素-3和-4受体(MC3R和MC4R),G蛋白偶联受体,在能量稳态的调节中起着至关重要的作用。目的了解mc3r和mc4r在红cru鱼(Carassiusauratusredvar)能量稳态中的作用。,RCC),我们克隆了mc3r和mc4r,分析了基因的组织表达和定位,并研究了mc3r(mc3r+/-)和mc4r(mc4r+/-)基因敲除在RCC中的作用。
■RCCmc3r和mc4r的全长cDNA分别为1459个碱基对(bp)和1894bp,分别。qRT-PCR表明mc3r和mc4r在大脑中大量表达,但在外周组织中表达较低。ISH透露mc3r和mc4r位于NPP中,NPO,NAPv,NSC,NAT,NRL,NLTl,和大脑的NLTp,提示mc3r和mc4r可能调节RCC的许多生理和行为方面。为了进一步验证MC3r和MC4r在能量稳态中的作用,通过CRISPR/Cas9系统获得mc3r/-和mc4r/-鱼。平均体重,总长度,身体深度,mc4r/-鱼的摄食量明显高于mc3r/-和正常野生型(WT)鱼,但是MC3r+/-和WT鱼没有区别,说明RCC表型和食物摄入量主要受mc4r影响,而不受mc3r影响。有趣的是,mc4r+/-鱼表现出比mc3r+/-和WT鱼更多的内脏脂肪量,与WT相比,mc3r/-鱼的内脏脂肪量也稍多。肝脏和肌肉的RNA-seq表明,WT与WT中的大量差异表达基因(DEGs)有所不同。mc3r+/-,WTvs.mc4r+/-,和mc3r+/-vs.mc4r+/-,主要与脂质有关,葡萄糖,和能量代谢。KEGG富集分析显示,DEGs主要富集在类固醇生物合成等途径中,脂肪酸代谢,脂肪酸生物合成,糖酵解/糖异生,Wnt信号通路,PPAR信号通路,和MAPK信号通路,从而影响脂质积累和生长。
■总而言之,这些结果将有助于进一步研究MC3R和MC4R参与鱼类能量稳态调节的分子机制。
Melanocortin-3 and -4 receptors (MC3R and MC4R), G protein-coupled receptors, play vital roles in the regulation of energy homeostasis. To understand the functions of mc3r and mc4r in the energy homeostasis of red crucian carp (Carassius auratus red var., RCC), we cloned mc3r and mc4r, analyzed the tissue expression and localization of the genes, and investigated the effects of knockout of mc3r (mc3r +/-) and mc4r (mc4r +/-) in RCC.
The full-length cDNAs of RCC mc3r and mc4r were 1459 base pairs (bp) and 1894 bp, respectively. qRT-PCR indicated that mc3r and mc4r were profusely expressed in the brain, but lower expressed in the periphery tissues. ISH revealed that mc3r and mc4r were located in NPP, NPO, NAPv, NSC, NAT, NRL, NLTl, and NLTp of the brain, suggesting that mc3r and mc4r might regulate many physiological and behavioral aspects in RCC. To further verify the roles of mc3r and mc4r in energy homeostasis, the mc3r+/- and mc4r+/- fish were obtained by the CRISPR/Cas9 system. The average body weights, total lengths, body depths, and food intake of mc4r+/- fish were significantly higher than those of mc3r+/- and the normal wild-type (WT) fish, but there was no difference between the mc3r+/- and WT fish, indicating that the RCC phenotype and food intake were mainly influenced by mc4r but not mc3r. Interestingly, mc4r+/- fish displayed more visceral fat mass than mc3r+/- and WT fish, and mc3r+/- fish also exhibited slightly more visceral fat mass compared to WT. RNA-seq of the liver and muscle revealed that a large number of differentially expressed genes (DEGs) differed in WT vs. mc3r+/-, WT vs. mc4r+/-, and mc3r+/- vs. mc4r+/-, mainly related to lipid, glucose, and energy metabolism. The KEGG enrichment analysis revealed that DEGs were mainly enriched in pathways such as steroid biosynthesis, fatty acid metabolism, fatty acid biosynthesis, glycolysis/gluconeogenesis, wnt signaling pathway, PPAR signaling pathway, and MAPK signaling pathway, thereby affecting lipid accumulation and growth.
In conclusion, these results will assist in the further investigation of the molecular mechanisms in which MC3R and MC4R were involved in the regulation of energy homeostasis in fish.