Molecular Cloning And Appetite Regulation Of Leptin And Cholecystokinin In Trachinotus Blochii

Leptin and cholecystokinin(CCK)have been well-established as a class of cytokines that inhibit appetite in mammals,but many of their biological functions in teleosts are still unclear.In the present study,in order to elucidate the physiological mechanisms involved in the regulation of appetite of leptin and cholecystokinin,leptin-B,leptin receptor(LeptinR)and CCK genes were identified and isolated in Snubnose pompano(Trachinotus blochii).The alignment of the genes amino acid(aa)sequences among various species was performed and phylogenetic analysis was constructed with biological software.At the same time,we also characterized the tissue expression profiles of leptin and CCK in Snubnose pompano by real-time quantitative PCR,evaluated the effects of a single meal and fasting on the genes expression,and finally achieved the following experimental results.1.In the present study,full-length cDNA sequences of Leptin-B,LeptinR and CCK were cloned and analyzed from Snubnose pompano.The nucleotide sequences of Leptin-B and CCK were 1223 bp and 822 bp in length,encoding the peptides of 158 aa and 137 aa residues,respectively.The full-length cDNA of Snubnose pompano LeptinR was 3809 bp,encoding a protein of 1158 aa.Further analyzed the deduced amino acid sequence,the three-dimensional(3D)structure models showed strong conservation of tertiary structure of the Snubnose pompano leptin-A and leptin-B with other vertebrates leptin,comprised of four a-helices,in spite of considerable divergence in the primary structures.The Snubnose pompano leptinR protein possessed a 26 aa signal peptide,and includes all functionally important domains that are conserved among vertebrate leptin receptors:three fibronectin type Ⅲ(FN Ⅲ)domains,the immunoglobulin(Ig)C2-like domain,a pair of repeated tryptophan/serine motifs(WSXWS)at an extracellular segment,two JAK2-binding motif boxes,and a STAT-binding domain at an intracellular segment.The predicted prepro-CCK peptide contained a putative signal peptide of 20 amino acids and a 117 amino acid mature peptide that included a C-terminal octapeptide,DYLGWMDF(the sixth amino acid from the C-terminal end was L:leucine).A identity comparison of aa sequences showed that leptin-B shared only 24.7%aa identity with leptin-A,and leptin-A and leptin-B was grouped with the fish Acanthoptergii-A and fish Acanthopterygii-B branch,respectively.LeptinR and CCK have high homology with the amino acids of the same species,and are clustered together in the phylogenetic analysis.2.The tissue expression pattern of Snubnose pompano leptin-A,leptin-B,leptinR and CCK mRNA was analyzed with quantitative RT-PCR normalized by β-actin.In all fish tested,leptin-A and leptin-B were widely observed in various tissues,and leptin-A was expressed primarily in the live,adipose and all parts of the brain,with minimal expression in the kidney,while leptin-B was expressed relatively highly in the adipose,heart and goand and relatively weakly in the kidney and muscle.LeptinR mRNA was mainly expressed in the pituitary,muscle and live,but weakly expressed in the hypothalamus,cerebellum and stomach tissue.Conversely,the relative expression level of CCK was the highest in the telencephalon,followed by the pituitary and then muscle tissue,and relatively lowly in adipose,gill,kidney.3.Both the hepatic expression of leptin-A and the hypothalamic expression of leptin-B was gradually decreased in preprandial groups(-3,-1,and 0 h),the expression.of leptin-A/-B showed a rapid increase after 1 h feeding,then reduced back to normal level at 3 h.the hypothalamic expression of leptin-A was slightly higher than that in the postprandialgroup at 3h and Oh before meals.The expression of Leptin-A in the postprandial group was slightly lower than that in the control group at 1h and 3h,respectively.The expression levels of Leptin-B and LeptinR in the liver were higher than those in the postprandial group at 3h,1h and Oh before the meal.The expression level of Leptin-B and LeptinR was slightly lower than that of the control group at 1h after the meal.The expression level of Leptin-B and LeptinR was similar to that of the control group at 3h after the meal.And there was no significant difference in the expression of LeptinR in the hypothalamus between preprandial groups and postprandial groups(+1 and +3h).For CCK in the hypothalamic,the mRNA expression level was gradually increased and was maximum at 0 h,then also decreased to normal level at 3 h.These results indicated that leptin might be a postprandial satiety signal in Snubnose pompano,but not CCK.4.During the fasting and re-feeding period,within the liver and hypothalamic,leptin-A and leptin-B expression were significantly higher in fasting groups than in fed groups,and decreased back to normal level after re-fed in the 7 th day.The expression level of leptinR in the hypothalamus was significantly lower in fasting groups than in fed groups during the fasting period.The hepatic expression of leptinR was slightly lower in fasting groups than in fed groups,but increased to normal level after re-fed in the 7 th day.In contrast,the hypothalamus CCK mRNA levels of fasting groups shown gradually decreased and appeared significant differences in the 5th day,then increased return to normal level after re-fed with 5 days.The above results showed that the expression levels of Leptin/LeptinR/CCK were significantly changed by starvation,suggesting that Leptin and CCK may be involved in the appetite regulation in Snubnose pompano.