Preliminarily To Study The Feeding Regulation Mechanism Of Appetite Regulatory Genes And Mechanistic Target Of Rapamycin Signaling Genes In Schizothorax Prenanti

This research takes Schizothorax prenanti(S.prenanti)as an experimental fish investigate the role and mechanism that appetite regulatory genes and mechanistic target of rapamycin(mTOR)signaling played in feeding regulation in fish.Firstly,appetite regulatory genes(apelin,APJ,proglucagon,NUCB2)and mTOR signaling genes(mTOR,S6K1,S6,4E-BP1,4E-BP2,4E-BP3)were cloned.Subsequently,we analysed the genes expression profiles in various tissues and during different feeding status of S.prenanti by quantitative real-time polymerase chain reaction.Finally,we investigated the effects of L-leucine and rapamycin intraperitoneal injection on food intake and mRNA expressions of mTOR signaling and appetite regulatory genes in S.prenanti.1.Molecular cloning of appetite regulatory genes in S.prenanti:Characterization of tissue distribution and mRNA quantification during different feeding status This study cloned the cDNA sequences of appetite regulatory genes:The full length cDNA of apelin was 1001 bp,which encoded 77 amino acids including a 22 amino acids signal peptide.The cDNA fragment of APJ was 1080 bp,which encoded 359 amino acids with 7 transmembrane domains.The cDNA fragment of proglucagon was 504 bp,which encoded 121 amino acids including a 21 amino acids signal peptide.The full length cDNA of NUCB2 was 2140 bp,which encoded 487 amino acids including a 23 amino acids signal peptide.Apelin and APJ mRNAs were ubiquitously expressed in various tissues,relatively high expression levels were detected in the heart,spleen and brain.Proglucagon mRNA was abundantly expressed in intestine and brain,low expressed in other tissues.NUCB2 mRNA was ubiquitously expressed in various tissues,and expressed primarily in hepatopancreas.At 1 and 3 h post-feeding,hypothalamic apelin and APJ mRNA expressions significantly decreased(P<0.05);proglucagon and NUCB2 mRNA levels significantly elevated in hypothalamus and intestine(P<0.05,P<0.01).During the 7-day food deprivation,hypothalamic apelin mRNA expression significantly increased(P<0.05);hypothalamic proglucagon and NUCB2,and intestinal proglucagon mRNA levels significantly decreased(P<0.05,P<0.01);while NUCB2 mRNA levels dramatically elevated in hepatopancreas(P<0.01).APJ mRNA expression significantly increased at 3-day food deprivation(P<0.05),but no significant difference between fasted fish and fed control on 5-and 7-day(P>0.05).When 7-day fasted fish were re-fed,the mRNA expressions of apelin and APJ observably lower than the levels of 7-day fed control(P<0.05),and then returned to the normal levels on 9-day;there was no significant difference in proglucagon and NUCB2 mRNA levels between re-fed fish and fed control.These results provide that apelin and APJ act as orexigenic factors,proglucagon and NUCB2 act as satiety factors involved in the regulation of food intake in S.prenanti.2.Molecular cloning,tissue expression and role in feeding regulation of mTOR signaling genes in S.prenantiThe mTOR signaling genes were firstly cloned from S.prenanti:The full length cDNA of mTOR was 8046 bp,which encoded 2515 amino acids.The cDNA fragment of S6K1 was 1084 bp,which encoded 310 amino acids.The full length cDNA of S6 was 860 bp,which encoded 249 amino acids.The lengh of 4E-BP1,4E-BP2 and 4E-BP3 was 363 bp,342 bp and 336 bp,which encoded 120,113 and 111 amino acids,respectively.Multiple sequence alignments among amino acid sequences of mTOR signaling genes in S.prenanti and other vertebrates suggested that all of these gene sequences were highly conserved in vertebrate.The results of tissue distribution experiment showed that mTOR signaling genes were highly and ubiquitously expressed in all test tissues.It suggested that mTOR signaling may play pleiotropic biological functions in different tissues of S.prenanti.Rapamycin(1 mg/kg BW)significantly increased cumulative food intake at 4 h post intraperitoneal injection(P<0.05),and L-leucine(10 mg/kg BW)decreased slightly in food intake and there is no significant difference compare to control group at 4 h post intraperitoneal injection(P>0.05).L-leucine and rapamycin had few influence in the mRNA expressions of mTOR signaling genes.At 4 h after single intraperitoneal injection,L-leucine down-regulated the ghrelin mRNA level to about 1/8 and 2/5 of control levels in hepatopancreas and intestine,respectively(P<0.05).About 1.4-fold,5-fold and 16-fold up-regulated in ghrelin mRNA expression at 4 h after rapamycin injection was detected in hypothalamus,hepatopancreas and intestine compared to controls,respectively(P<0.05).Furthermore,L-leucine injection significantly up-regulated hypothalamic POMC and NPY mRNA levels,intestinal NUCB2 mRNA level,and NUCB2,leptin and CCK mRNA levels in hepatopancreas(P<0.05);and it down-regulated hypothalamic AgRP mRNA level(P<0.05).Rapamycin injection significantly down-regulated hypothalamic POMC and AgRP mRNA levels(P<0.05);and it up-regulated intestinal proglucagon and CCK mRNA levels(P<0.05).Both L-leucine and rapamycin injection had no effects on hypothalamic apelin,APJ,NUCB2 and CRH mRNA levels.These results suggested that L-leucine(10 mg/kg BW)and rapamycin(1 mg/kg BW)at 4 h after intraperitoneal injection control food intake in S.prenanti might through regulating both ghrelin and POMC expression and might be unrelated to the transcriptional level of mTOR signaling genes at 4 h post intraperitoneal injection.Collectively,apelin and APJ act as orexigenic factors,proglucagon and NUCB2 act as satiety factors involved in the regulation of food intake in S.prenanti.At 4 h after single intraperitoneal injection,L-leucine(10 mg/kg BW)and rapamycin(1 mg/kg BW)regulated both ghrelin and POMC expression to control food intake in S.prenanti.