| Among the nutrients required by fish,such as protein,lipids,and carbohydrates,protein is the most expensive ingredient in aquatic feeds.Therefore,it is common to increase the level of lipids in aquatic feeds to maintain the growth of fish and to save protein for cost saving purposes.However,excessive lipid levels can easily cause lipidty liver in fish,reduce production performance and induce related metabolic diseases.Studies in terrestrial animals have found that glycerol monolaurate(GML)addition can affect lipid metabolism and reduce lipid accumulation,but few studies have been conducted in fish,and the specific metabolic mechanisms are not yet known.The aim of this experiment was to evaluate the effect and regulatory mechanism of GML on liver lipid metabolism in hybrid grouper(Epinephelus fuscoguttatus♀×E.lanceolatus♂)and the effect of lipid level on the regulation of lipid metabolism by GML.In the present study,a total of five experiments were conducted,and the results were as follows:1.Seven isonitrogenous and isolipid feeds were prepared by adding 0(control),600,1200,1800,2400,3000 and 3600 mg/kg GML to the basal diets(the protein level is about49% and the lipid level is about 9%)and were recorded as G1-G7.A total of 840 juvenile grouper of uniform size and healthy color(9.10 ± 0.03)g were selected and randomly divided into seven groups with four replicates each for the experiment.The experimental results showed that the final body weight(FBW),weight gain rate(WGR)and specific growth rate(SGR)of group G4 were significantly higher than those of the control group,while the feed conversion rate(FCR)and hepatopancreas somatic indices(HSI)were significantly lower than those of the control group(P < 0.05).The whole fish crude protein(CP)content was significantly increased in groups G4 and G5(P < 0.05),but there was no significant effect on whole fish crude lipid(CL)and moisture(P < 0.05).The G4 group significantly reduced serum cholesterol(TCHOL),triglyceride(TG)and low density lipoprotein(LDL)levels in all experimental groups(P < 0.05),while high density lipoprotein(HDL)levels were significantly increased in the G5 and G6 groups(P < 0.05).The G4 group increased the activity of lipolytic enzymes(LPS(lipase)and MDH(malate dehydrogenase)),inhibited the activity of lipidty acid synthase(FAS),and regulated the expression of genes related to lipid metabolism,increasing the expression of lipolytic genes(HMGCR,FAS,ELOVL5 and SREBP1)and inhibiting the expression of lipolytic genes(CPT-1 and LPS)expression(P < 0.05).Compared with the control group,the G4 group significantly increased lauric acid(C12:0),n-3 polyunsaturated lipidty acids(n-3 PUFA),n-6 PUFA and n-3/n-6 PUFA ratio,while saturated lipidty acids(SFA)content was significantly lower(P < 0.05).In the intestinal flora results,the addition of GML didn’t change the dominant phylum of grouper intestinal flora,but significantly increased the abundance of beneficial bacteria Bacillus(P < 0.05).According to the regression equation model fitted by WGR,the optimal level of GML was 1700 mg/kg.The addition of GML to the feed significantly promoted lipolysis,inhibited lipid synthesis,reduced liver lipid deposition,improved liver health.2.To investigate the regulatory effect of GML on lipid metabolism in grouper at high lipid levels,three experimental diets were formulated for this study: control(CN,9% lipid level),high lipid control(HC,15%)and high lipid experimental(HG,15% + 1700 mg/kg GML).The results showed that the high-lipid diet HC group inhibited the growth performance(FBW,WGR and SGR)and promoted liver lipid accumulation in grouper compared to the CN group(P < 0.05),while the supplementation with GML significantly improved the growth performance and reduced liver lipid accumulation in grouper(P <0.05).The HG group significantly increased the activity of lipolytic metabolizing enzymes,decreased the lipid synthesis-related enzyme activity,inhibited lipid accumulation while promoting β-oxidation of lipid,and improved the status of hepatic lipidty liver(P < 0.05).The HC group increased the abundance of harmful bacteria,while the addition of GML regulated the composition of the intestinal flora,increased the abundance of beneficial bacteria,and promoted intestinal health(P < 0.05).By transcriptomic analysis,GML reduced the expression of PPARγand RXR through the core pathway(PPAR signaling pathway)and decreased lipid synthesis by inhibiting the expression of downstream FADS2,SCD,FABP family(2/3/4)and CD36;while increasing the expression of CYP7A1 and CYP27A1 expression to improve bile acid metabolism and promote lipid digestion and catabolism,and to increase the expression of CPT-1,CPT-2 and ACAA1 to improve lipidβ-oxidation and reduce liver lipid deposition.Through the analysis of liver metabolomics,we concluded that long-term intake of diets containing GML significantly increased the content of bile acids(taurine and goose deoxycholic acid),choline and acetyl L-carnitine,as a way to promote lipid absorption and utilization,inhibit lipid synthesis,and improve liver fat metabolism in grouper.3.In this study,three basal diets with 9%,12% and 15% lipid levels were formulated with 1700 mg/kg GML to prepare six experimental diets,respectively,and noted as: CN,CG,MC,MG,HC and HG.The 1020 juvenile grouper with an initial body weight of(10.65± 0.06)g,were randomly divided into 6 groups with 4 replicates in each group,for a total of 24 buckets.The experimental results showed that FBW,WGR and SGR in the CN,MC and HC groups rose and then fell with increasing lipid levels,and FCR decreased significantly(P < 0.05).This indicates that the appropriate level of lipid promoted the growth of grouper while the high lipid level inhibited the growth of grouper.In contrast,the CG,MG and HG groups with the addition of GML at each of the three lipid levels significantly promoted the growth performance of grouper(P < 0.05).And with the increase of lipid,the lipid accumulation of grouper increased,as shown by the significant increase of CL,liver lipid(LL),mesenteric lipid(ML),condition factor(CF),HSI and VSI(P <0.05);the supplementation of GML significantly improved the lipid accumulation and lipolysis of grouper,and promoted the healthy growth of grouper(P < 0.05).The relative abundance of Firmicutes was significantly increased with the increase of lipid level,although the dominant phylum species in grouper remained unchanged,and the addition of GML significantly reduced the abundance of Firmicutes and increased the ratio of Firmicutes and Bacteroidetes(P < 0.05).Transcriptomic results showed that GML can regulate lipid metabolism at different adiposity levels,the metabolic pathways it regulates are different.4.Three experimental diets were prepared by adding compound antibiotics and compound antibiotics + 1700 mg/kg GML(HAG)to the basal high-lipid diet(HCC,lipid level 15.14).The culture experiment lasted for 8 W.The experimental results showed that GML improved the growth performance of grouper(P < 0.05),while compensating for the shortcomings of reduced SR.In the body composition results,GML supplementation significantly reduced CL,LL and ML content and significantly reduced grouper lipid deposition(P < 0.05).Further analysis found that antibiotics had no significant effect on lipid synthase(FAS and ACC1)activity and lipid synthesis genes(FASN,ACC1,PPARγ,SREBP-1 and CD36)and only increased enzyme activity and gene expression of LPS(P <0.05);the addition of GML significantly inhibited lipid synthase(FAS and ACC1)activity and expression of lipid synthesis genes(FASN,ACC1,PPARγ,and CD36),and increased enzyme activity and gene expression of CPT-1 and LPS,significantly inhibited lipid synthesis,promoted β-oxidation of lipidty acids,and mediated lipid metabolism(P < 0.05).Intake of antibiotics significantly reduced grouper intestinal flora alpha diversity while increasing the abundance of harmful bacteria,causing intestinal disorders,but addition of GML significantly increased intestinal flora alpha diversity,increased bile salt hydrolase(BSH)producing bacteria(Lactobacillus and Bacillus)and promoted bile acid levels(P <0.05);inhibited the abundance of harmful bacteria and regulated intestinal flora disorders(P < 0.05);increased the abundance of Faecalibacterium and promoted the secretion of butyric acid,which favors intestinal mucosal integrity and promoted lipid digestion and absorption(P < 0.05).5.The PPARγ activator and inhibitor were injected into grouper to verify the regulatory effect of PPAR pathway on lipid metabolism in grouper.The injection experiments were divided into five groups,noted as DMSO(control group,injected with DMSO),GML(injected with 50 mg/kg GML),ROS(injected with 5 mg/kg ROS),GW9662(injected with 1 mg/kg GW9662)and GML-ROS(injected with a mixture of GML and ROS),with an injection dose of 100 μL,and the experimental period was 48 h.The experimental results showed that in vitro injection of GML reduced the LL content and lowered serum lipid levels(TG and TCHOL)(P < 0.05);inhibited lipid synthesis-related enzyme(FAS and ACC1)activity and increased lipolysis-related enzyme(LPS and CPT-1)activity(P < 0.05);inhibited the expression of PPARγ,while regulating the downstream genes(CD36,FASN,ACC1 and SREBP1)expression(P < 0.05),in order to regulate lipid metabolism. |
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