Investigation Of Mechanism Of TGR5 And FXR Mediated Bile Acid Regulation Of Hepatic Lipid Deposition In Pearl Gentian Grouper

There is an increasing trend towards higher levels of fat in fish diets due to its protein sparing ability,but high level of fat in the diet can lead to excessive lipid deposition in fish.The addition of bile acids（BAs）to diets can promote growth and reduce body fat in fish.Studies in terrestrial animals have shown that BAs regulate host lipid metabolism and BAs metabolism mainly through the BAs receptors TGR5（G protein-coupled bile acid receptor 1）and FXR（farnesol X receptor）,but fewer relevant studies have been conducted in fish.Therefore,this study used 16S r RNA sequencing,proteomics and BAs omics to investigate the mechanisms by which BAs alleviate the high-fat diet（HD）-induced excessive hepatic lipid accumulation in pearl gentian grouper（Epinephelus fuscoguttatus♀×E.lanceolatus♂）.The main results of this study include:1)The full-length sequence of fxr and tgr5 from pearl gentian grouper was cloned and characterized.The results showed that the full length of fxr gene was2242bp and contained a 1458bp ORF（open reading frame）which encoding 485amino acids.fxr gene contained a Zn F＿C4（c4 zinc finger in the nuclear hormone receptors）domain and a HOLI（ligand-binding domain of the hormone receptors）domain.fxr gene was highly expressed in hindgut,liver,spleen and body kidney.The full tgr5 gene in hybrid grouper was 2525bp in length.The ORF of tgr5 gene was1029 bp and encoded 342 amino acids,which contained 7TM（seven transmembrane）structural domains.tgr5 gene was highly expressed in the spleen,midgut and brain of grouper.The FXR and TGR5 of pearl gentian grouper were highly conserved with those of all fish species in Perciformes.2)Seven diets were formulated:control diet（CD,8.27%of lipid）,HD（14.92%）,HD supplemented with BAs at 300（B1D）,600（B2D）,900（B3D）,1200（B4D）,and1500（B5D）mg kg^-1to fed pearl gentian grouper for 8 weeks.After feeding,compared to the CD group,the HD treatment did not significantly affect the growth performance of fish,but it promoted lipid deposition,as revealed by a significantly higher crude lipid content of the whole body,muscle,and liver.Among the HD supplemented with BAs groups,and compared to the HD,fish fed B3D exhibited the best growth performance and lowest hepatic lipid deposition.In most HD supplemented with BAs groups,the content of total cholesterol（T-CHO）,low-density lipoprotein cholesterol（LDL）,and triglycerides（TG）in serum,as well as the content of T-CHO in the liver,were decreased,whereas the content of high-density lipoprotein cholesterol（HDL）in serum was increased.In addition,the most strongly influenced pathways between the control,HD,and B3D groups were fatty acid biosynthesis,insulin signaling pathway,and AMPK signaling pathway.The supplementation of BAs decreased the expression of lipogenesis genes,proteins and enzymes,while increased the expression of lipolysis genes,proteins and enzymes.HD treatment altered the gut microbiota structure,suppressing microbes associated with bile-salt hydrolase（BSH）activity,thus decreasing the levels of unconjugated primary BAs[CA（cholic acid）and CDCA（chenodeoxycholic acid）].B3D treatment increased the BSH-producing microbes level,thus increasing the level of unconjugated BAs.The BAs pool size was significantly decreased in the HD group,whereas the BAs pool increased in all HD supplemented with BAs groups.The HD group had a significantly increased expression of BAs synthesis and BAs recycling genes;while exhibiting a significantly decreased expression of BAs transport,BAs reabsorption and BAs receptors genes.After BAs intervention,the expression of BAs synthesis,BAs transport,BAs reabsorption and BAs receptors genes was increased in most HD supplemented with BAs groups,whereas the expression of BAs recycling genes was decreased in all HD supplemented with BAs groups.High-fat diets disrupted fat metabolism and BAs metabolism,and promoted fat deposition in grouper pearl gentian.Addition of exogenous BAs improved fat metabolism and BAs metabolism in the fish,and alleviated fat deposition.The appropriate level of BAs addition to high-fat diets for pearl gentian grouper was 900mg kg^-1.3)The primary hepatocytes of pearl gentian grouper were treated as follows:V-CN（without any treatment）,V-HL（0.5 ml/L lipid emulsion）,V-TCA（V-HL+TCA）,V-T747（V-TCA+obeticholic acid）,V-TGU（V-TCA+guggulsterone）,V-T777（V-TCA+INT-777）and V-TSBI（V-TCA+SBI-115）.The results showed that fatty degenerated hepatocytes induced by V-HL accumulated the intracellular TG,the enhanced expression of sterol responsive element binding protein 1（SREBP1）protein,and the repressed expression of peroxisome proliferator-activated receptor alpha（PPARA）and phosphorylated PPARA（P-PPARA）proteins.V-TCA treatment reduced the content of TG,while increased the expression of PPARA,FXR and TGR5 proteins.V-T747 treatment reduced the content of TG,while down-regulated the expression of SREBP1 and PPARA,and up-regulated the expression of P-PPARA,FXR,small heterodimer partner（SHP）and TGR5.V-TGU treatment increased the level of TG,while decreased the expression of TGR5,increased the expression of SHP.V-T777 treatment reduced the content of TG,while down-regulated the expression of SREBP1 and SHP,up-regulated the expression of TGR5.V-TSBI treatment elevated the level of TG,while reduced the expression of P-PPARA and TGR5.Lipid metabolism is disturbed in a pearl grouper model of hyperlipidemic hepatocytes,as evidenced by enhanced lipogenesis and reduced lipolysis.Incubation with BAs may have improved lipid metabolism and fat accumulation in hepatocytes by inhibiting lipogenesis and promoting lipolysis through activation of FXR and/or TGR5 signalling pathways.4)Fish were intraperitoneally injected with taurocholic acid（I-TCA）,obeticholic acid（FXR agonist,I-T747）,guggulsterone（FXR antagonist,I-TGU）,SBI-115（TGR5 antagonist,I-TSBI）,and INT-777（TGR5 agonist,I-T777）,respectively.The results showed that the fish injected with 50 mg kg^-1TCA three times during a 144 h period exhibited significantly lower hepatic lipid accumulation,compared to other concentrations and periods.Compared to the I-TCA group,the I-T747 treatment significantly increased the activities of hepatic adipose triglyceride lipase（ATGL）and carnitine palmitoyltransferase 1（CPT1）,as well as the expression of lipolysis and fatty acid uptake genes,while significantly decreased the content of hepatic crude lipid,the activity of CPT1,and the expression of lipogenesis genes.Meanwhile,the I-TGU treatment significantly increased hepatic lipid deposition and the expression of lipogenesis genes.Compared to the I-TCA group,the I-T777treatment did not significantly affect the parameters related to lipid deposition,but it significantly decreased the activities of acetyl-Co A carboxylase（ACC）,CPT1,and fatty acid synthase（FAS）,while significantly increasing the activity of ATGL and the expression of lipolysis and fatty acid uptake-related genes.Meanwhile,the I-TSBI treatment significantly increased the hepatic lipid accumulation,the activities of ACC,CPT1 and FAS,and the expression of lipogenesis and lipogenic transcriptional factors genes.I-T747 treatment increased the transport and reabsorption of BAs,whereas the I-TGU treatment reduced the above processes.I-TSBI treatment decreased the transport and recycling of BAs;whereas I-T777treatment increased the transport and reabsorption of BAs.Injection of BAs at appropriate concentrations(50 mg kg^-1)reduced lipid accumulation in the liver of pearl gentian grouper;inhibition of FXR exacerbated hepatic lipid deposition,disrupted lipid metabolism and inhibited the transport and reabsorption of BAs;inhibition of TGR5 exacerbated hepatic lipid deposition,disrupted lipid metabolism and inhibited the transport of BAs;the effect of BAs on lipid metabolism may be dependent on the activation of FXR or TGR5 signalling pathways.5)The pearl gentian groupers were fed B3D diet（SBD）or B3D diet supplemented with antibiotics mixture（ASBD）for a short-term period.The results showed that ASBD treatment significantly decreased the level of hepatic lipid droplets,the content of hepatic TG and T-CHO,and serum LDL,while significantly increasing the content of HDL.ASBD treatment enhanced activities of lipolytic enzymes,expression of lipolytic proteins and transcriptional factors,whereas the expression of lipogenic transcriptional factor genes was significantly reduced.The ASBD group decreased the size of the BAs pool,as well as the expression of BAs reabsorption and recycling genes;while increasing the expression of BAs synthesis,transport,and receptor genes.ASBD group significantly increased the abundance of Bacteroidetes.No significant shifts were observed in theα-diversity indices in SBD and ASBD groups,although the structure and potential functions of the intestinal microbiota were clearly separated.The presence of intestinal flora inhibited lipolysis,promoted lipogenesis,increased lipid deposition,enhanced BAs reabsorption and maintained BAs pool size in pearl gentian grouper.In conclusion,the potential mechanism by which the high-lipid dietary supplementation of BAs reduced the lipid accumulation in pearl gentian grouper involves the following:BAs remodeling the structure of gut microbiota and increasing the relative abundance of BSH-producing microbes,increasing the levels of unconjugated BAs and the size of the BAs pool,restoring the enterohepatic BAs metabolism by enhancing the BAs transport and reabsorption,activating the hepatic TGR5 and FXR signaling pathway,improving the lipid metabolism by increasing lipolysis and decreasing lipogenesis,promoting the growth performance of fish.The appropriate level of BAs addition to high-fat diets for pearl gentian grouper was 900mg kg^-1.