JAK2 Mediates The Regualtion Of PepT1 By Leptin In Grass Carp(Ctenopharyngodon Idellus)Intestinal

The oligopeptide transporter 1(PepT1)is a transporter located in the brush-border membrane of intestinal epithelial cells.PepT1 is responsible for transporting dipeptides and tripeptides from extracellular to intracellular,and plays a key role in the small peptides absorption of the small intestinal epithelium.In this study,we cloned the Janus Kinase 2(JAK2)gene from Ctenopharyngodon idellus,and studied the effect of JAK2 on leptin(LP)regulating PepT1 gene expression in grass carp intestine in vivo and in vitro,which will preliminarily reveal the regulatory mechanism of leptin on PepT1 gene expression in grass carp intestine.1)Cloning,expression pattern of grass carp JAK2 gene and it's regulatory effect on the transcriptional activity of Pep T1 geneUsing molecular cloning technique,the Janus kinase JAK2 gene was cloned from the intestinal tissue of grass carp.The length of the JAK2 gene was 3676 bp which encoded 1126 amino acids with molecular weight of 129590.26 Da and theoretical pI of 6.37.Phylogenetic analysis showed that JAK2 gene was conservative during evolution and shared close evolutionary relationship with homologs from other cyprinidae fish.The expression of JAK2 gene in grass carp tissues was detected by RT-qPCR.The results showed that JAK2 gene was broadly expressed in kidney,hypophysis,muscle,heart,liver,spleen and intestine.The highest expression level was observed in intestine,which was significantly different from that in other tissues(P < 0.05).During embryonic development,JAK2 was expressed in all selected stages of the development process,with the highest expression level in gastrointestinal stage which was significantly higher than that in other stages(P <0.05).Dual luciferase reporter assay showed that JAK2 over expression could significantly up-regulate the activity of PepT1 promoter(P < 0.05),which preliminarily revealed that JAK2 might play a regulatory role in PepT1 gene expression in grass carp.2)Effects of leptin on the expression of JAK2 and PepT1 gene in grass carpIn vivo(injection)experiment: In this study,grass carps were injectedintraperitoneally with different concentrations of recombinant leptin(0.016 ?g/g,0.16?g/g,1.6 ?g/g 16 ?g/g and 160 ?g/g).After 8 hours,the expression of JAK2 and PepT1 genes in grass carp intestine was detected by RT-qPCR.The results showed that the expression of JAK2 and PepT1 genes increased first and then decreased with the increase of leptin concentration,and reached the highest level in 0.16 ?g/g leptin group(P < 0.05).The injection experimentwith AG490,a specific inhibitor of JAK2,showed that the expression level of intestinal PepT1 gene was significantly upregulated by leptin compared to the control group(P < 0.05),but this stimulating effect was blocked by the JAK2 inhibitor AG490,indicating that JAK2 may be involved in leptin regulating PepT1 gene expression in grass carp intestine.In vitro(cell)experiment: The primary intestinal cells of grass carp were cultured by tissue culture method in this study.The grass carp intestinal cells were identified by immunofluorescence using three kinds of rabbit epithelial cell specific antibodies:cytokeratin 8,E-cadherin and tight junction protein ZO-1.The grass carp intestinal epithelial cells were inoculated with different concentrations of recombinant leptin(0,50,100,250,500 and 1000 ng/mL),respectively.After 8 hours of continuous culture,the expression of JAK2 gene in cells was significanly increased by 50 and 100 ng/m L of leptin compared to the control,and the highest expression of PepT1 gene was found in the 100 ng/mL leptin group(P < 0.05).Then,according to the above experimental scheme,JAK2 specific inhibitor AG490 were added to the complete culture medium of intestinal cells of grass carp.After 8 hours of continuous culture,the collected cells were detected by RT-qPCR.Compared with the control group,the mRNA expression level of PepT1 gene was significantly upregualted in the leptin group(P < 0.05)which also has higher expression levels that that in the leptin+AG490 group(P < 0.05).These results further demonstrated that JAK2 may play an important role in leptin regulating PepT1 gene expression in intestine of grass carp.