Cloning, Tissue Distribution And Appetite Regulation Of Appetite-related Neuropeptides In Schizothorax Prenanti

To study appetite-related neuropeptides on their regulation appetite in fish. From the RT-PCR and RACE PCR, Schizothorax prenanti NPY nucleotide sequence was 789 bp in length, and contained a 74 bp of 5’-UTR, a 424 bp of 3’-UTR and a 288 bp of open reading frame (ORF) encoding a prepro-NPY with 96 amino acids. Sequenee comparison showed that the partial NPY amino acids residues had the highest identity with Danio rerio (84%). In this study, we have cloned the full-length cDNA sequence for AgRP in Schizothorax prenanti. The AgRP gene, encoding 126-amino acids, was strongly expressed in the brain and testis. Homologous alignment analysis showed AgRP gene had Cyprinus carpio, Carassius auratus and Danio rerio identity to that of 96%,93% and 84%, resppectively. Schizothorax prenanti POMC nucleotide sequence was 1031 base pair (bp) in length, and included a 666 bp of open reading frame (ORF) encoding a precursor POMC with 222 amino acids. Homologous alignment analysis showed POMC gene had Carassius auratus, Gobiocypris raru,Ctenopharyngodon idella and Danio rerio identity to that of 92%、 91%、90% and 82%, resppectively. From the RT-PCR, the cDNA sequence of MC4R was obtained. Schizothorax prenanti MC4R nucleotide sequence was 1004 base pair (bp) in length, and included a 978 bp of open reading frame (ORF) encoding a precursor MC4R with 326 amino acids (aa). Homologous alignment analysis showed MC4R gene had Cyprinus carpio^ Carassius auratus and Danio rerio identity to that of 97%、96% and 96%, resppectively.Using real-time RT-PCR, NPY was mainly expressed in the brain and eye, and weakly expressed in the pituitary, heart, gill, gut, skin, testis, ovary, white muscle and red muscle. AgRP was mainly expressed in the brain and testis, and less expression in the ovary, pituitary, eye, heart and liver, suggesting that the gene could be involved in a wide range of physiological functions in various organs. Our study reveals that POMC was mainly expressed in the pituitary and head kidney. MC4R was mainly expressed in the brain and ovary, moderate in the heart, liver, pituitary, eye, spleen, skin, red muscle and testis, low in gill, cholecyst, gut and white muscle.To evaluate the effect of the post-fasting and feeding status, fed and fasted group of S. prenanti (average body weight 25.4 ± 4.1 g, n= 40×4) were sampled in four glass aquariums. In the course of a meal deprivation, fasting did not affect pituitary POMC expression levels but induced an increase in brain NPY and hypothalamus AgRP expression levels (P<0.05), which is consistent with the orexigenic roles for NPY and AgRP, respectively. Our results suggest that NPY, AgRP and MC4R have a major role in the regulation of feeding in S. prenanti and might contribute to the circadian feeding rhythm fluctuations in appetite in this species. Our results show that NPY, MC4R, but not AgRP, show significant differences in expression with higher NPY, MC4R and lower POMC expression levels after 14 days fasting (P<0.05), which revealed some direct and/or indirect relations of the expression to long-term food deprivation in S. prenant.Our result showed that NPY mRNA expression was observed from blastulation to hatch stage of the S. prenanti embryo. AgRP mRNA was detected in embryos at all developmental stages, its mRNA was also detectable in unfertilized eggs, suggesting that AgRP could be classified as maternal mRNA. Further, POMC is expressed in early embryo. Our study reveals that MC4R is expressed in early embryo and increased gradually until postnatal life, and that the level is influenced by proliferative conditions.Conclusion:1、The full-length NPY、AgRP、POMC and partial MC4R cDNA were first cloned from the brain of S. prenanti2、Many tissues that expressed appetite-related key neuropeptide could result in multiple physiological processes in fish.3、Our results suggest that NPY, AgRP and MC4R have a major role in the regulation of feeding in S. prenanti and might contribute to the circadian feeding rhythm fluctuations in appetite in this species. Expression levels of NPY, MC4R and POMC after 14 days fasting, which revealed some direct and/or indirect relations of the expression to long-term food deprivation in S. prenant.4、The presence of ppetite-related neuropeptides mRNA indicated that these genes may be involved in the development of the Schizothorax prenanti.