Cloning, Tissue Distribution And Appetite Regulation Of Leptin, CCK, PYY And CART In Schizothorax Prenanti

To study leptin, Cholecystokinin (CCK), Pepide YY (PYY) and cocaine- and amphetamine-regulated transcript (CART) on their regulation appetite in the Schizothorax prenanti, we employed RACE to obtained the full-length cDNA sequence of leptin, CCK, PYY and CART. Futhermore, we employed real-time quantitative PCR to characterize the tissue distribution, and appetite regulatory effects of leptin, CCK, PYY and CART in S. prenanti.The S. prenanti leptin full-length nucleotide sequence was 1121bp in length, and contained a 109bp of 5’-untranslated region (5’-UTR), a 496bp of 3’-untranslated region (3’-UTR) and a 516bp of open reading frame (ORF). The deduced leptin protein is composed of 171 amino acid residues. The S. prenanti leptin displayed a high degree of homology (85%) with common carp(cyprinus carpio) leptin-a-Ⅱ followed by goldfish (Carassius auratus auratus) leptin-a-Ⅰ (82%), common carp (cyprinus carpio) leptin a-Ⅰ (80%) and common carp(cyprinus carpio) leptin-a-Ⅱ (79%).For RACE PCR, a 776bp full-length CCK was obtained, including an 86bp of 5’-UTR, a 318bp of 3’-UTR and a 372bp of ORF for a 123 amino acid protein. The prepro-CCK contained a 21 amino acid signal peptide and a 102 amino acid mature peptide that included the C-terminal octapeptide CCK-8. The S. prenanti CCK showed high identity with the other species, such as goldfish(Carassius auratus) CCK (95%), grass carp(Ctenopharyngodon idellus) CCK (91%) and zebrafish(Danio rerio) CCK (82%).The S. prenanti PYY nucleotide sequence was 783 bp in length, and contained 152 bp of 5’-UTR,337 bp of 3’-UTR and 294bp of ORF. The deduced PYY protein is composed of 97 amino acid residues. The amino acid sequence of the S. prenanti PYY showed high identity with the other species, such as goldfish(Carassius auratus) PYY (97%), grass carp(Ctenopharyngodon idellus) PYY (96%) and zebrafish(Danio rerio) PPY (91%).The S. prenanti CART-1 nucleotide sequence was 597bp in length and contained a 42bp sequence of the 5’-UTR, a 201bp sequence of the 3’-UTR and a 354bp sequence of the ORF. The S. prenanti CART-2 nucleotide sequence was 694bp in length and contained a 109bp sequence of the 5’-UTR, a 222bp sequence of the 3’-UTR and a 363bp sequence of the ORF. The S. prenanti CART-3 nucleotide sequence was 749bp in length and contained a 128bp sequence of the 5’-UTR, a 306bp sequence of the 3’-UTR and a 315bp sequence of the ORF. The deduced CART-1, CART-2 and CART-3 protein is composed of 125,120 and 104 amino acids, respectively.The S. prenanti leptin, CCK, PYY and CART mRNA were widely distributed among the tissues. The S. prenanti leptin mRNA was expressed notably in liver. For CCK, the highest mRNA levels were observed in the hypothalamus, followed by myelencephalon, telencephalon and foregut. The PYY mRNA was distributed the highest levels in telencephalon, medulla oblongata, pituitary and hypothalamus. The CART-1 mRNA was mainly expressed in telencephalon and eye, followed in hypothalamus, myelencephalon, mesencephalon. The CART-2 mRNA was mainly expressed in mesencephalon, hypothalamus, telencephalon and myelencephalon. The S. prenanti CART-3 mRNA was widely distributed among the tissues, with the high levels in the foregut, hypothalamus, midgut, telencephalon and hindgut.The CCK, PYY and CART mRNA expression were highly elevated after feeding, whereas the leptin mRNA expression was not effected by single meal. In the fasting experiment, leptin, CCK, PYY and CART gene expression were both decreased after fasting and increased after refeeding.Our study suggested that CCK, PYY and CART are postprandial satiety signals in S. prenanti, but leptin might not be. In addition, leptin, CCK, PYY and CART might be involved in regulation of appetite in S. prenanti. This study provides an essential groundwork to further elucidate the appetite regulatory systems of leptin, CCK, PYY and CART in teleosts.