The Role Of ABHD5 In Lipid Metabolism And Its Mechanism In Grass Carp(Ctenopharyngodon Idella)

Lipids are one of the important nutrients in the growth and development of fish and the main source of energy and essential fatty acids for fish.However,excess fat in feed tends to lead to excess fat deposition in different tissues,such as liver,muscle and kidney,and eventually lead to nutritional metabolic disease.The kidney in fish is equivalent to the bone marrow of vertebrates,and is an important immune organ.Therefore,it is of great significance to understand the lipid metabolism balance in response to different types of fatty acids in kidney,and eventually lead to occurrence and development of metabolic diseases in fish.ABHD5(α/β hydrolase domain containing 5,ABHD5)is an important lipid droplet-associated protein that belongs to the α/β hydrolase folding family.ABHD5 plays a very important role in the stability and metabolic process of lipid droplets by interaction with ATGL or other lipid droplet proteins(PLIN5 or PLIN1).It has been also shown that ABHD5 regulates autophagy and inflammation.However,the biochemical role of ABHD5 in Ctenopharyngodon idella has yet to be determined.In this study,by using Oil red O staining,Immunofluorescence staining,RT-q PCR,Western Blot,Co IP and RNA-seq and other technologies.We cloning and analyzing the sequence of ABHD5 gene,and studying the effects of different fatty acid on ABHD5 expression,and investigated the signaling pathways involved in that palmitic acid regulated ABHD5 expression.Then we mainly elucidate the role of ABHD5 in lipid metabolism induced by fatty acids.The specific results are as follows:1.Cloning and bioinformatics analysis of ABHD4 and ABHD5 in Ctenopharyngodon idella.Sequence analysis showed that the full-length c DNA of grass carp ABHD4(Gen Bank number: MK645600)was 1662 bp long,encoding 366 amino acids;the full-length c DNA of grass carp ABHD5(Gen Bank number: KP325482)was 1035 bp long,encoding 344 amino acids.The protein domain prediction showed that ABHD4 and ABHD5 contained the the α/β hydrolase domain.According to the phylogenetic tree,mammals and fish can be separated into two branches.Moreover,multiple sequence alignment and amino acid identity comparisons indicated that ABHD4 and ABHD5 share sequence identity with other species,exhibiting 58.61-98.09% and 60.97-93.31% amino acids sequence identities,respectively,and ABHD4 and ABHD5 are closely related.ABHD4 and ABHD5 were expressed in all the detected tissues.2.Screening and validation of target genes regulated by ABHD5 based on RNA-seq.The p GFP-ABHD5 vector was constructed and transfected into CIK cells,and the effect of overexpression of ABHD5 on CIK cells was analyzed by RNA-seq sequencing.GO analysis revealed that differentially expressed genes were involved in a variety of biological processes,such as metabolic process,localization,cellular process,development process,biological process regulation and so on.KEGG analysis revealed that differentially expressed genes were involved in protein processing in endoplasmic reticulum,sphingolipid metabolism,apoptosis and TNF signaling pathways.RNA-Seq data showed that 1105 differentially expressed genes were identified,of which 623 genes were up-regulated and 482 genes were down-regulated.Among them,upregulated genes include ATGL,AGPAT9,ATP5G3,ANXA2,and Smad7.The results of RT-q PCR verified the expression of these differential genes.The STRING database predicts that there was an interaction between ABHD5 with AGPAT9.These results suggested that ABHD5 can regulate ATGL and AGPAT9 expression.3.ABHD5 regulates ATGL expression and induces lipolysis.The results showed that ABHD5 protein was expressed in CIK and COS7 cells by Immunofluorescence.Overexpression of ABHD5 increased expression of ATGL m RNA and protein.Co IP assay showed that ABHD5 did not directly interact with ATGL in the basal state,but could rely on the expression of ATGL to reduce TAG accumulation induced by FAs.These results demonstrated that ABHD5 can induce the expression of ATGL to promote lipolysis.4.The effect of different fatty acids on the expression of ABHD5 in CIK cells.The results showed that compared with PA,OA and DHA can significantly induce the biogenesis of lipid droplets in CIK cells.At the same time,PA significantly induced the expression of ABHD5 and ER stress-related genes(GRP78,CHOP,ATF6,IRE1α,and IRE1β).Moreover,a certain concentration of OA or DHA decreased the expression of ABHD5 and increased the accumulation of lipid droplets.While,alleviated PA-induced the expression of ER stress-related and autophagy-related genes.These results suggested that the expression of ABHD5 was closely related to ER stress induced by PA.5.IRE1α-XBP1 signaling pathway involved in PA-induced ABHD5 expression.The results showed that the full-length c DNA of IRE1α(Gen Bank number: MH370854)and IRE1β(Gen Bank number: MK616212)gene were cloned,encoding 1031 and 944 amino acids,respectively.And they all contain signal peptide,Serine/Threonine protein kinase domain,and RNase domain.Different concentrations of thapsigargin(ER stress inducer)can significantly induced the expression of ER stress-related(IRE1α,IRE1β,GRP78,CHOP,and ATF6),and upregulated the expression of ABHD5.After adding 4-PBA or 4μ8c treatment,alleviated the expression ABHD5,GRP78,CHOP,and ATF6 induced by PA.Overexpression of XBP1 s significantly enhanced PA-induced ABHD5 expression.These results suggested that the IRE1α-XBP1 pathway mediates PA-induced ABHD5 expression.6.Overexpression of ABHD5 on AGPAT9 expression.The results show that the ABHD5 lentiviral vector was constructed,and the COS7 cell line stably expressing ABHD5 were generated by lentiviral technology.The results showed that the full-length c DNA of AGPAT9(Gen Bank number: OK044146)and AGPAT9L(Gen Bank number:OK044147)gene were cloned,encoding 450 and 444 amino acids,respectively.And they all contain three transmembrane and a PIs C domains.RT-q PCR results found that OA and DHA can significantly induce the expression of AGPAT9 and AGPAT9 L,but PA can down-regulate their expression in a dose-dependent manner.In addition,overexpression of ABHD5 enhanced the expression of AGPAT9.These results suggested that ABHD5 can regulate the expression of AGPAT9 and then affect lipid metabolism.In conclusion,the c DNA of grass carp ABHD4 and ABHD5 were successfully cloned.We also demonstrated that different FAs(OA,PA,and DHA)have different effects on ER stress,autophagy and ABHD5 expression in CIK cells,and it was determined that PA regulate the expression of ABHD5 by activating the IRE1α-XBP1 s signaling pathway.Overexpression of ABHD5 increases ATGL expression and then promotes lipolysis.While,it may regulate lipid metabolism by increased AGPAT9 expression.These studies have laid the foundation for a better understanding of the regulatory role of ABHD5 in lipid metabolism induced by fatty acids.