Mechanism Study On Folic Acid Regulating Lipid Metabolism In Broilers Based On Insulin/IGF Signal And PI3K/AKT Pathway

For a long period,the genetic breeding goal of meat-type chickens was to improve the growth rate,but excessive abdominal fat deposition has also been accompanied.Physiologically inessential fats have negative effects on the quality and yield of meat and eggs,and also reduce feed efficiency,and improve slaughter cost.In addition,some diseases related to lipid metabolism disorder are triggered.All these cause huge economic loss in the poultry industry.During the long time genetic breeding,could the old feeding standard and nutritional requirement meet the growing need of broilers at present? Whether it is the deviation between the old nutritional requirement and real amounts what birds need that leads to these problems mentioned above.Folic acid,as one of B vitamins,is involved in the process of one-carbon metabolism and was also reported to be closely associated with lipid metabolism.However,different animals have different regulation patterns about lipid metabolism,the results from one species are not completely applied directly into other ones.Hence,we firstly systematically assessed developmental changes in hepatic lipid metabolism of chicks during the embryonic periods and the first week of post-hatch.Accordingly,the regulation mechanism of folic acid on lipid metabolism was studied in primary chicken hepatocytes in vitro and broilers in vivo with the help of metabolomics and proteomics technology,aiming to provide the theoretical and scientific basis for the precaution of lipid metabolism diseases and dietary folic acid effective application in poultry.Experiment 1 Developmental changes in hepatic lipid metabolism of chicks during the embryonic periods and the first week of post-hatchThe main site of lipid metabolism is the liver in poultry,and hepatic lipid metabolism disorder will lead to excessive abdominal fat deposition or fatty liver disease,thereby causing huge economic loss.The present study was conducted to investigate developmental changes in hepatic lipid metabolism from embryonic periods to the first week after hatching.Liver samples were collected from embryonic day 11(E11)to the age of day 7 post-hatch(D7)for lipid metabolism analysis.Results showed that hepatic lipids increased gradually during the embryonic period and declined after hatching based on hematoxylin-eosin and Oil Red O staining;ELISA analysis also revealed that the sum of hepatic triglycerides(TG)and cholesterol(TC)reached the peak around the hatched day(P<0.05).Levels of Acetyl-Co A carboxylase(ACC)and fatty acid synthase(FAS)m RNA expression in the liver were higher from E17 to D1 with the peak at E19 as well as Acyl-Co A desaturase 1(SCD1)when compared with those at E13 and E15(P<0.05).Hepatic elongase of very-long-chain fatty acids 6(ELOVL6)and microsomal triglyceride transfer protein(MTTP)m RNA abundance was lower during embryonic periods but at a relatively higher level after hatching(P<0.05).On the contrary,hepatic carbohydrate response element binding protein(Ch REBP),carnitine palmitoyltransferase 1(CPT1)and peroxisome proliferators-activated receptor α(PPARα)expression were higher during embryonic periods but decreased post-hatch(P<0.05).The m RNA abundance of sterol-regulatory element binding protein 1c(SREBP-1c)was the lowest at E13 and E15,then increased gradually from E17 to D1,but decreased from D3 to D7 little by little(P<0.05).In summary,hepatic lipogenesis genes have different expression patterns during the embryonic periods and the first week of post-hatch,which might be activated and regulated by different transcription factors.The liver of newly hatched chick is full of lipid with higher de novo lipogenesis ability and weak fatty acid β-oxidation,and might be a good model for primary hepatocyte isolation to carry out studies about lipid metabolism disorder in vitro.Experiment 2 Culture of primary chicken hepatocytes and genes expression associated with lipid metabolism affected by of folic acidThe present study was conducted to establish the method for the primary culture of chicken hepatocytes and investigate the effects of folic acid on gene expression associated with lipid metabolism in primary chicken hepatocytes,further providing a basis for exploring lipid metabolism of poultry in vitro.Primary chicken hepatocytes were isolated by enzymatic digestion,and PAS staining was used to evaluate the morphology and purity of cells.When the cell confluence reached about 80%,cells were divided into 6 groups and changed with the new treatment medium with different concentrations of folic acid(0,1,5,10,15 and 20 mg/L)for 12 h,and each group has 6 replicates.Hepatocytes proliferation and injury were detected by the MTT method and LDH activity of culture medium respectively.Genes expression was determined by RT-PCR.The results showed that hepatocytes were isolated well with high purity.At first,the cells were polygonal like epithelial cells,and then cell clusters contacted with each other and began to come to a fusion,and last primary hepatocytes were islands and fused into pieces.Folic acid with different doses didn’t affect cell proliferation and LDH activity(P>0.05).When compared with the control group(1 mg/L folic acid),10,15 and 20 mg/L folic acid significantly reduced FAS and ACC expression in chicken hepatocytes(P<0.05).However,folic acid had no effects on CPT-1 and PPARα expression(P>0.05).In addition,there existed linear and quadratic regression between FAS and ACC gene expression and folic acid concentration(P<0.05).In conclusion,folic acid could reduce gene expression associated with fatty acid synthesis in chicken hepatocytes,and regression analysis revealed that the optimum treatment dose of folic acid was 15 mg/L in this study.Experiment 3 Combined proteomics and metabolomics methods reveal the effects of folic acid on lipid metabolism in primary chicken hepatocytesBased on the folic acid addition dose identified in trial 2,the present study was conducted out to reveal the effects of folic acid on lipid metabolism by omics technology.New-born chicks were used for primary chicken hepatocytes isolation.There are two groups,one for the control(1 mg/L)and another one for folic acid addition(15 mg/L)with 6 replicates for each group.After appropriate treatments,cells were collected for proteomics and metabolomics analysis.In addition,biochemical parameters of lipid metabolism were also determined.Proteomics results found 81 differential proteins when compared with the control(P<0.05),26 down-regulation including FAS and PI3 K,55 up-regulation including ATGL and MTTP.Metabolomics analysis revealed that folic acid addition significantly reduced 20 metabolites and improved 25 metabolites levels(P<0.05).KEGG pathway also found that amino acid metabolism and lipid metabolism pathways were enriched significantly based on differential metabolites(P<0.05).When compared with the control,folic acid prominently decreased TG and increased NEFA and VLDL contents(P<0.05),while total cholesterol was not affected as well as HDL,LDL,HDL-c,LDL-c,apolipoprotein A,and apolipoprotein B(P>0.05).Meanwhile,oil red O staining also showed that lipid contents were lower in the folic acid group(P<0.05).In conclusion,our findings indicated that folic acid could remarkably inhibit de novo fatty acid synthesis and coordinately promotes triglyceride hydrolysis and exportation,thereby reducing lipid deposition in primary chicken hepatocytes.But the detailed regulation mechanism needs to be explored deeply in the future.Experiment 4 The mechanism study on lipid metabolism regulated by folic acid in primary chicken hepatocytesBased on the findings in trial 3,the present study is aiming to explore the regulation mechanism of folic acid on lipid metabolism.Firstly,experimental design was the same as trial 3,m RNA and protein level of pathway factors about lipid metabolism were detected.Then recombinant IGF2 protein,IGF2 inhibitor at the transcription level and LY294002,the inhibitor of the PI3K/AKT signal,were used to verify and evaluate the roles of IGF2 and PI3K/AKT signal in lipid metabolism regulation.When compared with the control,folic acid addition significantly reduced m RNA levels of IGF2,IR,IGF1 R,AKT2,SREBP-1c,ACC and FAS genes(P<0.05),but did not affect Ch REBP expression(P>0.05).Consistently,protein abundances of IGF2,PI3K85,PI3K110,p-AKT,SREBP,and FAS were all lower in the folic acid group(P<0.05).IGF2 inhibitor significantly reduced TG content in hepatocytes(P<0.05),while IGF2 addition did not increase TG deposition.However,IGF2 addition indeed increased gene expression about de novo lipogenesis such as FAS,ACC,SCD,and SREBP and activated the PI3K/AKT/ SREBP pathway with higher PI3K85,PI3K110,p-AKT,SREBP,and FAS protein levels when compared with the control(P<0.05).In the IGF2 inhibitor group,gene expression of lipogenesis and protein abundance of the pathway were all lower(P<0.05).On the other hand,lipid contents were significantly reduced when LY294002 existed(P<0.05).IGF2 did not develop its activation function for the PI3K/AKT/SREBP pathway when LY294002 was pretreated and added together with IGF2.Also,folic acid addition increased free folic acid and 5-Me-THF levels in hepatocytes(P<0.05).The total methylation level of the IGF2 promoter and genomic 5m C contents were also higher in the folic acid group(P<0.05).DNMT3 A and DNMT3 B m RNA expression were down-regulated by folic acid addition(P<0.05).In conclusion,folic acid could down-regulate IGF2 expression by improving DNA methylation of gene promoter,then weaken insulin/IGF signal,finally inhibit de novo fatty acid synthesis in primary chicken hepatocytes through PI3K/AKT/SREBP pathway.Experiment 5 The mechanism study on lipid metabolism regulated folic acid perfusion administration in starter broilersBased on the results in vitro,the current study was to investigate the role of folic acid in lipid metabolism regulation in broilers.A total of 105 one-day-old healthy Arbor Acres broilers were randomly distributed into 3 treatments,including the control(Con),saline-perfusion group(NS)and folic acid perfusion group(FA).The growth performance,biochemical characteristics in serum,lipid metabolism in the liver and abdominal fat tissues were evaluated.Results have shown that folic acid significantly reduced abdominal fat percentage(P<0.05)and had no effects on BW,ADFI,ADG and FCR(P>0.05).Folic acid perfusion reduced RFC and increased FR m RNA expression in duodenum and jejunum(P<0.05).Serum triglycerides(TG),total cholesterol(TC)and alanine aminotransferase(ALT)levels were lower in the FA group but albumin concentration was higher(P<0.05).Hepatic ACC,SCD,ELOVL6,PI3 K,LDLR,HMGCR and ABCA1 m RNA abundance were all down-regulated in the FA group(P<0.05)when compared with the Con and NS groups,while CPT1 and PPARα were not affected.In addition,MTTP m RNA abundance was higher in the liver of birds subjected to folic acid(P<0.05).There was no difference in TG deposition in the liver among all groups based on hematoxylin-eosin(HE)and Oil Red O staining.On the other hand,ELOVL6,PPARγ,IGF1 and TGFβ2 expression were notably decreased in the abdominal fat in the FA group(P<0.05).In conclusion,our data demonstrated that folic acid has reduced abdominal fat percentage by decreasing hepatic lipogenesis and suppressing adipocytes proliferation and differentiation.And the inhibiting effect of adipocytes might be mediated by IGF1 and TGFβ2 down-regulation.To sum up,hepatic lipogenesis genes have different expression patterns during the embryonic periods and the first week of post-hatch,and hepatic lipid metabolism features of newly hatched chicken were similar with those in early fatty liver to some extent,which might be a good model for primary hepatocyte isolation to carry out studies about lipid metabolism disorder in vitro.Folic acid could inhibit de novo lipogenesis through PI3K/AKT/SREBP pathway mediated by insulin/IGF signal,and coordinately promotes triglyceride hydrolysis and exportation,thereby reducing lipid deposition in primary chicken hepatocytes.Moreover,folic acid perfusion could improve serum lipid metabolism and hepatic function,meanwhile decrease hepatic de novo lipidgenesis,adipocyte proliferation and differention,thereby reducing abdominal fat deposition in broilers.