Research On Mechanism Of Visfatin Regulating Feed Intake In Chicks By The Transcriptomics And Metabonomics Technology

Feed intake of poultry is one of the important factors that restrict its production efficiency.Studies have shown that the hypothalamus is an important central nervous tissue in the complicated and precise regulation network,which can collect,integrate and release signals associated with appetite regulation.Visfatin is a novel adipocytokines,which is preferentially produced by human abdominal visceral fat.In recent years,with the deepening of the research,reports have proved that Visfatin can significantly increase the feed intake of chicken and can be used as a orexigenicpotential factor.However,there has been not reported that the specific functionaltheoryand mechanism of the effects of Visfatin on increasing food intake by far.In order to explore the scientific problem furtherly,in this study,we observed the variation of feed intake after intracerebroventricular injection of visfatin in chicks,and conducted Illumia sequencing and metabonomics detection with the aid of transcriptome sequencing and GC-MS detection technology in hypothalamus.We hope to find the marker genes and metabolites during Visfatin increasing food intake in the level of transcriptional and molecular metabolism,explore the molecular mechanism of Visfatin regulation poultry feeding and lay the foundation for improving poultry appetite regulation and energy balance theory.Experiment 1 Transcriptomics studies on effects of Visfatin on feed intake in chicksIn this study,72 Roman brown cockerels of 1 day-old were randomly divided into three treatments,which consisted of 3 repeating group that including 8 chicks.They were intraventricular injected with 0ng(control,C),40ng(low dosage,LT)and 400ng(high dosage,HT)human recombinant Visfatin.The cumulative feed intake was recorded and the hypothalamus organization was obtained at 60 min after injection.The total RNA extracted from the hypothalamus was used tocreate library for RNA-Seq,and transcriptome sequencing was carried out on an Illumina Hi Seq2500 platform that generated about 125 bp paired-end(PE).After filtering and quality assessment,the original sequence obtained from RNA-Seq was mapped to the reference genome of Gallus gallus.Differential expression analysis of three treatment was performed based on the estimating levels of gene expression.To validate the repeatability and reproducibility of gene expression data obtained by RNA sequencing,q RT-PCR was carried out on 15 randomly selected differentially expressed genes(DEGs)using the total RNA used for RNA-seq.Then some of DEGs correlated with the regulation of feed intake were screened out though the Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis.Results showed that the feed intake of chicks in treatment group increased significantly compared with the control(P< 0.05),but there was no significant change between the 40 ng and 400 ng Visfatin(P> 0.05).According to the result of agarose gel electrophoresis and Agilent,the quality of hypothalamus RNA meet the requirement of building library and RNA-Seq.540174404 clean reads were obtained from RNA-Seq,and the total amount of data was 67.54 Gb.The quality evaluation results showed that the overall quality of RNA-Seq is good and the accuracy is high,which can meet the requirements of further analysis.With the analysis of differentially expressed genes(DEGs),406 DEGs were found in the treatment group contrast with the control group,and the LT treatment could cause the change of more gene transcriptions in the chicken hypothalamus.It was proved that the result of the transcriptome sequencing is reliable.We found out 20 DEGs closely related to the regulation of food intake.The expression of appetiteinhibitory factor POMC,GHRH,GHR,CCKand GLP-1R were increased significantly,and the expression of potential orexigenic factor HCRT,VIP,ABAT and NPFFR2 were reduced significantly.Most of the DEGs were related to the term of molecular function in analysis of GO enrichment and the pathway of information processing in analysis of KEGG.The pathway of neuroactive ligand-receptor interaction is the key pathway that the most DEGs enrichmented.The expression of thirteen genes,as examined by q RT-PCR,behaved similarly between q RT-PCR and RNA-Seq in fifteendifferential genes selected from RNA-Seq.Experiment 2 Metabonomics studies on effects of Visfatin on feed intake in chicksFollowing the design and treatment of experiment 1,experiment 2 included C(0 ng),L(40 ng),M(200 ng),H(400 ng)four treatment groupsconsisted of 5 repetitions.The cumulative feed intake was recorded and the hypothalamus organization was obtained at 60 min after injection.We extracted the metabolites from the hypothalamus sample with derivatization.Metabolomics instrumental analysis was performed on an Agilent 7890 A gas chromatography system coupled to an Agilent 5975 C inert MSD system.The data was normalized before performing univariate and multivariate statistics.Then we build the reliable model of PLS-DA and OPLS-DA.The differential metabolites were determined by the combination of the Variable importance in the projection(VIP)value(>1)of PLS-DA model and the p values(<0.05)from two-tailed Student’s T test on the normalized peak intensities.Then we analyzed the differential metabolites by correlation matrix,heatmap and pathway analysis.In this experiment,the feed intake of chicks were increased significantly in M/H vs C(P < 0.05).With the analysis of GC-MS,74 differential metabolites and 51 pathway were obtained.These metabolites covered to the metabolism of carbohydrate,lipid and amino acid,and oleamide,GABA and lysine maybe the marker metabolites of Visfatin in increasing feed intake.The differential metabolites in hypothalamus were related to nutrient metabolism closely,and the pathway of fatty acid biosynthesis was enriched significantly.The reduction of metabolites,such as carbohydrate,amino acid and free fatty acid,is the main change in hypothalamus in the level of metabolism after intracerebroventricular injection of Visfatin which increased feed intake of chicks.Above all,we can come to the conclusion that Visfatin can significantly increase the feed intake of the egg-type cockerels without dose-dependentthough intracerebroventricular injection.Visfatin may play a role though reducing the expression of anorexigenic genes POMC,GHRH,GHR,CCK,GLP-1R and increasing the expression of orexigenic genes HCRT,VIP,ABAT,NPFFR2.Neuroactive ligand-receptor interaction pathway maybe the key in the effect of Visfatin.At the metabolic level,the hypothalamus nutrient metabolic product content is lower,change the steady-state energy balance inside the body,so as to make the increase of feed intake in chicks.It was the main way for Visfatin to play the role of promoting appetite.And oleamide,GABA and lysine maybe the potential marker metabolites of Visfatin in increasing feed intake.