Effects And Mechanism Analysis Of Arachidonic Acid (ARA) On Lipid Metabolism Of Grass Carp(Ctenopharygodon Idellus)

As the main n-6 highly-unsaturated fatty acid(HUFA),arachidonic acid(ARA;20:4n-6)serves as a precursor of one of the most important groups of bioactive lipid mediators,the eicosanoids,which play important roles in regulating lipid metabolism,biological processes and immune response.Recent studies about ARA nutrition in fish focused on its effects on growth performance,survival,stress resistance,immune response,reproductive performance,pigmentation and skeletogenesis.However,rare studies have focused on its effects on lipid metabolism since ARA and its metabolites have been demonstrated to play a vital role in regulating adipogenesis and other lipid metabolism processes in mammals.Grass carp is easily to retain excess fat in the abdominal cavity,and the regulation of lipid accumulation in grass carp in the nutritional approach is always one of the important themes in aquaculture industry.Hence,we designed five experiments to explore the effects and mechanism of ARA on the lipid metabolism of grass carp,aiming to provide reference for the health aquaculture and development of functional feeds.The main results obtained as follows:1.Dietary 0.3% ARA decreases lipid accumulation and improves the health status in grass carp.A 60-day feeding(initial weight 6.87 ± 1.18 g)experiment was conducted using three isonitrogenous and isoenergetic semi-purified diets containing 0.03%(control group),0.30%,0.60% ARA.Results showed that the intraperitoneal fat(IPF)ratio in the 0.30% group was significantly lower compared with that in the control group,dietary ARA also decreased the fat content in hepatopancreas.The mRNA expression of peroxisome proliferator activated receptor γ(PPARγ)and fatty acid synthase(FAS)showed significantly lower levels in the hepatopancreas and IPF of ARA groups.The lowest serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)activities were found in the 0.30% group,and showed significantly difference compared with the 0.60% group.The serum tumor necrosis factor α(TNFα)and interleukin-6(IL-6)concentrations decreased with the increased dietary ARA content(P<0.05).The mRNA expression of toll-like receptor 22(TLR22)and myeloid differentiation factor 88(MyD88)in kidney showed the highest levels in the 0.30% group and the lowest levels in the control group.Overall,the appropriate dietary ARA inclusion(0.30%)effectively suppressed the lipid accumulation and altered the key genes expression of lipidmetabolism,improved the health status by decreasing the aminotransferase activities and pro-inflammatory cytokines concentrations in serum and increased the expression of immune-related genes expression in kidney of juvenile grass carp.2.Cyclooxygenase(COX)-mediated metabolites participate in the in lipid accumulation in juvenile grass carp fed arachidonic acid.Three purified diets,namely ARA-free(control),ARA(0.30%),and ARA + acetylsalicylic acid(ASA;a COX inhibitor)were designed,and used them to feed grass carp(27.65 ± 3.05 g)for 8 weeks.The results showed that ARA decreased the lipid content in the hepatopancreas,whereas ASA recovered lipid content to a significant level.ARA significantly decreased the mRNA expression levels of FAS and stearoyl-CoA desaturase(SCD)in the hepatopancreas.However,ASA did not rescue the mRNA expression of these genes.Interestingly,ARA significantly enhanced the level of PPARα gene expression,and this increase was attenuated by ASA.Overall,our findings suggest that COX metabolites play important roles in the inhibition of lipid accumulation in the hepatopancreas of grass carp fed with ARA,and that regulation of gene expression promotes lipid catabolism rather than lipogenic activities.3.Dual function of ARA on the adipogenesis in grass carp.Based on the feeding experiment above using three isonitrogenous and isoenergetic purified diets: ARA-free,ARA,and ARA+ASA.Fish were sampled after 4 and 8 weeks of feeding.Results showed that ARA-fed fish had a significantly lower IPFI and smaller adipocytes;these decreases were reversed by ASA after 8 weeks of feeding.Nevertheless,at week 4,the IPFI and adipocyte size were higher in the ARA group,and were comparable to those of fish fed ARA+ASA.To further investigate the influence of ARA on adipocyte differentiation,confluent pre-adipocytes of grass carp were incubated with ARA for 3 days.This in vitro experiment demonstrated that ARA promoted adipogenesis in a dose-dependent manner.Pre-treatment with the lipoxygenase(LOX)inhibitor nordihydroguaiaretic acid(NDGA)and PPARγinhibitor GW9662 attenuated the pro-adipogenic function of ARA.However,after treatment with ARA for 8 days,adipocytes had a lower lipid content than cells treated with oleic acid(OA),and ASA could suppress this effect.We thus revealed the potential dual function of ARA in adipogenesis in grass carp.The LOX pathway may play a key role in pro-adipogenesis after short-term treatment with ARA,whereas the COX pathway is possibly responsible for the inhibition of adipogenesis after long-term treatment.4.Dietary 0.3% ARA decrease adipocyte development and adipose tissue inflammation in grass carp after long-term feeding.A 10 weeks feeding experiment was performed by using two isonitrogenous and isoenergetic purified diets,namely ARA free(control group)and 0.30% ARA(ARA group),then the transcriptome and DNA methylometechniques were used.RNA-seq data showed that 611 genes were up-regulated and 973 genes were down-regulated.Differentially expressed genes(DEGs)results demonstrate that dietary ARA had different effects on the ARA metabolism-related enzymes,ARA decreased the adipocyte proliferation,adipogenesis and apoptosis-related genes,increased lipid catabolism and bile acid synthesis related genes;moreover,dietary ARA decreased the mRNA level of chemokines,cytokines and related receptors,as well as down-regulated oxidative stress and angiogenesis.By integrating analysis the transcriptome and DNA methylome,153 differentially methylated and expressed genes(DMEGs)were obtained,including 4 related to ARA metabolism,5 related to cell proliferation,4 related to cell apoptosis,11 related to adipogenesis,2 related to fatty acid metabolism and 14 related to inflammation.Overall,these data will provide basic references for the influence of ARA on the adipose tissue in transcriptional and epigenetic levels.5.Dietary ARA has more potent ability in regulating lipid accumulation then EPA at same concentration in juvenile grass carp.Four isonitrogenous and isoenergetic purified diets containing free HUFA(control group),0.30% ARA,0.30% EPA and 0.30% ARA+EPA(equivalent)were designed to feed juvenile grass carp(10.21 ± 0.10 g)for 10-weeks.The results showed that both ARA and EPA decreased the hepatopancreatic triglyceride(TG)levels,accompanied by the decrease of lipoprotein lipase(LPL)activity in the ARA group and hepatic lipase(HL)activity in the ARA and EPA groups(P<0.05).ARA and EPA also decreased the FAS,diacylgycerol acyltransferase(DGAT)and apolipoprotein E(apoE)gene expression in the hepatopancreas,but only ARA increased the mRNA level of adipose triglyceride lipase(ATGL).As to the IPF,ARA significantly decreased IPFI and adipocyte sizes,but EPA had no obvious effect.Meanwhile,ARA decreased the adipogenic genes CCAAT enhancer binding protein α(C/EBPα),LPL and FAS,increased the lipid catabolic genes PPARα,ATGL,hormone-sensitive lipase(HSL)and carnitine palmitoyltransferase 1(CPT-1),whereas EPA showed less efficiency in regulating these genes.Overall,the present study demonstrates that ARA is more potent in regulating lipid deposition in grass carp,especially in IPF.In conclusion,1)diet supplemented with 0.30% could effectively decrease the lipid accumulation of grass carp mainly through decreasing the lipid synthesis and increasing lipid catabolism,among them,COX mediated metabolites play important roles in regulating lipid catabolism.2)Dietary ARA had a time-differential role in the adipocyte development,ARA might promote adipogenesis through LOX pathway at the initial stage of cell differentiaion,whereas COX pathway might be the key in the inhibition of adipogenesis at the final stage.3)Dietary ARA decreased the adipocyte development,as well as the inflammatorycytokines and chemokines,and DNA methylation play important roles in these process.4)At the same concentration,ARA exhibited more potent of regulating lipid metabolism related genes than EPA.Overall,these results provided references and new approach for the regulation of ARA on the lipid accumulation in grass carp.