Effects Of Dietary Betaine Supplementation On Hepatic Synthesis Of Yolk Precursors In Hens And Its Mechanism

In the egg-laying animals,the yolk is the most important source of nutrients needed for embryonic growth and development.The liver of oviparous vertebrates synthesizes yolk precursors under the control of estrogen(E2).Studies have shown that egg yolk precursors,vitellogenin(VTG)and yolk-targeted very low-density lipoprotein(VLDLy),are positively correlated with egg production.Betaine(BET),also known as trimethyl glycine,is synthesized by the oxidation of choline and can be obtained directly from many kinds of dietary sources.BET is an essential methyl donor in the methionine cycle,which plays an important role in the epigenetic regulation of gene expression.Many studies have reported that dietary betaine can increase egg production in hens.However,the effect and mechanism of betaine on yolk precursor synthesis in the liver are rarely reported.The present study found that dietary betaine significantly increased egg production rate in laying hens,which is accompanied by significantly upregulated hepatic expression of VTGII and lipid synthesis-and transport-related genes.Further studies show that betaine regulates glucocorticoid receptor(GR)and lipid metabolic genes through decreasing the DNA methylation in promoter regions and enhancing the interaction between GR and estrogen receptor(ERa),thereby activates the expression of genes involved in liver VTGII and lipid metabolism and promotes the synthesis of yolk precursors.1.Effects of Dietary Betaine Supplementation on Hepatic Expression of VTGII in Hens and Its MechanismThe aim of this study was to investigate the effect of BET on the expression of hepatic VTGII in hens and its mechanism.Thirty eight weeks old Rugao yellow feather laying hens were divided into two groups.One group was fed a basal diet and the other group was fed the basal diet containing 0.5%betaine,for 4 weeks.The changes in body weight and egg production rate were recorded throughout the experiment,and liver samples were taken at the end of the experiment.The results showed that dietary betaine did not affect body weight,but significantly(P<0.05)increased egg laying rate that was accompanied by a significant(P<0.05)increase in hepatic content and plasma level of VTGII.Plasmas E2 level did not change,but the hepatic expression of estrogen receptor a(ERa)mRNA was significantly(P<0.05)up-regulated.BET did not affect the protein content of ERa,but significantly(P<0.05)increased hepatic expression of GR at both mRNA and protein levels.Co-immunoprecipitation analysis revealed a relative increase in GR/ER? interaction in the liver nuclear lysates of BET-supplemented hens.Furthermore,dietary BET supplementation significantly increased(P<0.05)the hepatic expression of methyl-transfer enzymes,such as betaine homocysteine S-methyltransferase(BHMT),glycine N-methyltransferase(GNMT),and DNA methyltransferase 1(DNMT1).At the same time,hepatic content of S-adenosylmethionine(SAM)as well as SAM/S-adenosyl homocysteine(SAH)ratio were significantly(P<0.05)increased.These changes were accompanied by hypomethylation of GR gene promoter regions.In conclusion,dietary BET activates hepatic VTGII expression in association with modified GR gene promoter DNA methylation,GR expression and GR/ERa interaction.2.Effect of Dietary Betaine Supplementation on Hepatic Expression of Lipid Synthesis and Transport-related Genes and Its MechanismFeeding betaine-supplemented diet for 4 weeks significantly(P<0.05)increased liver and plasma triacylglycerol(TG)concentrations in laying hens.Simultaneously,the mRNA levels of the key genes involved in liver lipid synthesis,including sterol regulatory element binding protein 1(SREBP-1),fatty acid synthase(FAS),acetyl-CoA carboxylase(ACC)and stearoyl-CoA desaturase 1(SCD1),were significantly(P<0.05)upregulated.Western blot results showed that SREBP-1 and SCD1 protein levels were also increased significantly(P<0.05).In addition,mRNA expression of main apolipoprotein components of VLDLy,including apolipoprotein B(ApoB)and apolipoprotein-VLDL II(Apo-VLDLII),and microsomal triglyceride transfer proteins(MTTP)that is closely related to the synthesis and release of VLDL,was also significantly(P<0.05)up-regulated.Methylated DNA immunoprecipitation combined with PCR(MeDIP-PCR)detected a reduction(P<0.05)of methylation level in some promoter regions of the above genes.Chromatin immunoprecipitation(ChIP)PCR assays showed increased(P<0.05)binding of GR to SREBP1 and ApoB gene promoters.These results suggest that betaine supplementation in the diet can modify the expression of liver lipid synthesis-and transport-related genes by altering the methylation status and GR binding to their promoter regions,thereby promotes the liver synthesis and release of yolk precursors.3.Effect of Betaine Treatment on Expression of VTGII and Apo-VLDLII in Primary Embryonic Chicken Hepatocytes and Its MechanismTo explore whether betaine can induce the expression of VTG? and Apo-VLDL?in vitro,we used a model of primary embryonic hepatocytes of chicken.Cells were divided into 4 treatment groups,including Control(CON),10 mM BET,100 nM E2 and the combination of both(BET+E2).After 24 hours of treatment,cells were harvested for analysis.The results showed that VTGII and Apo-VLDLII were only expressed under E2 induction(P<0.05).Treatment with BET alone did not induce expression of either VTGII or Apo-VLDLII.However,BET+E2 co-treatment significantly promoted the expression of both genes.E2 treatment significantly(P<0.05)increased ERa mRNA and protein levels,while BET treatment significantly(P<0.05)increased mRNA and protein expression of GR and the methyltransferases genes,such as BHMT,GNMT and DNMT1.Co-immunoprecipitation analysis showed an increase in the relative content of GR co-precipitated with ERa in the nuclear protein extract of E2+BET co-treated cells,indicating increased interaction between these two proteins.In summary,betaine potentiates E2-induced expression of VTGII and Apo-VLDLII in hepatocytes through GR/ERa interaction.In conclusion,this study demonstrated possible mechanisms underlying the effects of dietary betaine supplementation on synthesis of egg yolk precursors in the liver of hens that contributes to improved egg production,and expanded the understanding of the biological effects of betaine,and provided an experimental basis for the application of betaine in the industry of laying hens.