| Liver is known to be a central metabolic organ,in particular at the stage of newborn.It is the predominant metabolic site of glucose,lipid and protein,and thus its normal development plays a vital role in maintaining metabolic homeostasis in vivo.It is reported that maternal nutrition modulates neonatal hepatic metabolism via the epigenetic regulation,which involves DNA methylation,histone modification and miRNA-mediated post-transcriptional mechanism.Moreover,methyl donors participate in epigenetic regulation by controlling the methylation status,suggesting their potential crucial values in the studies of maternal nutritional programming.Betaine is one substate of methionine metabolism and it donates methyl donors to almost methylation reactions in vivo.Moreover,betaine plays critical roles in fetal development and metabolism of glucose and lipid,yet the mechanism is still unknown.Furthermore,the function and mechanism of maternal betaine-supplemented diet on the neonatal hepatic glucose and lipid metabolism has not been reported.Neonatal metabolic disorders always result in chronic metabolis diseases at adulthood which impair animal health.Especially in animal husbandry,it not only impairs the quanlity of animal products,but causes other lethal diseases to increase mortality rate.Therefore,the present study aims to investigate the effects of feeding pregnant dams with betaine-supplemented diet on hepatic gluscose and lipid metabolism as well as the underlying mechanisms in neonatal piglets.Based on our research,it will benefit animal healthy growth and diseases prevention in animal husbandry.1 Effects of maternal betaine supplementation during pregnancy on hepatic betaine and methionine metabolism in newborn pigletsSixteen second parity Landrace×Yorkshire crossbred sows were artificially inseminated,at the observation of estrus,using the mixed Duroc semen samples obtained from two littermate boars.Sows were divided randomly into control and betaine groups(8 per group)one week after the artificial insemination.Sows in control group were fed basal diet,while those in betaine group received betaine-supplemented(3g/kg)diet throughout the pregnancy.All sows were fed three times per day at 05:00,10:00 and 17:00 h,respectively,with free access to water.Newborn piglets were individually weighed immediately after birth and the piglets from the same litter were kept together in the warm creep area.One male piglet of the mean body weight were selected per litter and sacrificed before suckling.Serum and liver samples were collected immediately,snap-frozen in liquid nitrogen and stored at-80℃.Neonatal piglets born to betaine-supplemented sows had significantly higher serum betaine(P<0.05),methionine(P<0.05),s-adenosyl methionine(SAM,P<0.05),and lower serum homocysteine level(P<0.05),as well as higher hepatic content of betaine(P<0.05),SAM(P<0.05)and SAM/SAH ratio(P<0.05).Moreover,maternal betaine supplementation demonstrated significantly greater expression of methionine metabolic enzymes including adenosylhomocysteine hydrolase-like 1(AHCYL1,P<0.05),betaine-homocysteine methyltransferase(BHMT,P<0.05),methionine adenosyltransferaseⅡbeta(MAT2B,P<0.05),and glycine N-methyltransferase(GNMT,P<0.05)in the liver.To summary,maternal betaine supplementation enhanced hepatic methionine metabolism in neonatal piglets.2 Effects of maternal betaine supplementation during pregnancy on hepatic gluconeogenic genes in newborn pigletsSignificantly higher serum concentrations of lactic acid(P<0.05)and glucogenic amino acids,including serine(P<0.05),glutamate(P<0.05),methionine(P<0.05)and histidine(P<0.05)were detected in the piglets born to betaine-supplemented sows,which were coincident with higher hepatic glycogen content and PEPCK1 enzyme activity(P<0.05),as well as greater protein expression of gluconeogenic enzymes(P<0.05),pyruvate carboxylase(PC),cytoplasmic phosphoenolpyruvate carboxykinase(PEPCK1),mitochondrional phosphoenolpyruvate carboxykinase(PEPCK2)and fructose-1,6-bisphosphatase(FBP1).Moreover,maternal betaine supplementation significantly changed the methylation status of both CpGs and histones on the promoter of gluconeogenic genes(P<0.05).The lower PEPCK1 mRNA was associated with DNA hypermethylation(P<0.05)and more enriched repression histone mark H3K27me3(P<0.05),while the up-regulated PEPCK2(P<0.05)and FBP1 mRNA was associated with DNA hypomethylation(P<0.05)and more enriched activation histone mark H3K4me3(P<0.05).Furthermore,piglets born to betaine-supplemented sows demonstrated a significant down-regulation in the hepatic expression of miRNA-184(P<0.01)and miRNA-196b(P<0.01),which are predicted to target PC,and miRNA-1403p(P<0.01),miRNA-424-3p(P<0.01),miRNA-196b(P<0.01),miRNA-370(P<0.01),miRNA-30b-3p(P<0.05)and miRNA-92b-5p(P<0.05),which are predicted to target PEPCK1.Our results provide the first evidence that maternal betaine supplementation affects hepatic gluconeogenic genes expression in newborn piglets through epigenetic regulations which involve DNA and histone methylations,and possibly miRNAs-mediated post-transcriptional mechanism.3 Effects of maternal betaine supplementation during pregnancy on hepatic lipogenic genes in newborn pigletsBetaine-exposed piglets demonstrated significantly lower liver triglyceride content(P<0.05)associated with down-regulated hepatic expression of lipogenic genes acetyl-CoA Carboxylase(ACC,P<0.05),fatty acid synthase(FAS,P<0.05),stearoyl-CoA desaturase(SCD,P<0.05)and sterol regulatory element-binding protein-1c(P<0.05).This was accompanied by DNA hypermethylation(P<0.05)on FAS and SCD gene promoters and more enriched repression histone mark H3K27me 3(P<0.05)on the SCD genes promoter.Furthermore,glucocorticoid receptor(GR)binding to SCD gene promoter was diminished(P<0.05)along with reduced serum cortisol(P<0.05)and liver GR protein content(P<0.05)in betaine-exposed piglets.GR-mediated SCD gene regulation was confirmed in HepG2 cells in vitro.Dexamethasone(Dex)drastically increased the luciferase activity of porcine SCD promoter(P<0.05),while the deletion of GR response element on SCD promoter blocked the Dex-mediated SCD transactivation.In addition,hepatic expression of both miR-let-7e and miR-1285,which respectively target porcine SCD and ACC,was significantly increased(P<0.05),being in accordance with decreased protein content of these two genes.Taken together,our results suggest that maternal dietary betaine supplementation during gestation attenuates hepatic lipogenesis in neonatal piglets via epigenetic and GR-mediated mechanisms.4 Effects of maternal betaine supplementation during pregnancy on cholesterol metabolic genes in neonatal pigletsHepatic 3-hydroxy-3-methylglutary1 coenzyme A reductase(HMGCR)mRNA expression was down-regulated(P<0.05)in betaine-exposed piglets as well as its predominant regulatory factor sterol regulator element-binding protein 2(SREBP2,P<0.05).This was in accordance with DNA hypermethylation(P<0.05)and increased repression histone mark H3K27me3(P<0.05)on the promoter of HMGCR gene.However the HMGCR protein expression was reversely up-regulated(P<0.05),which may due to significantly decreased miR-497(P<0.05)which targets HMGCR.Furthermore,mRNA and protein levels of hepatic low density lipoprotein receptor(LDLR)were significantly down-regulated(P<0.05)in the betaine-exposed piglets,paralleled to the hypermethylation(P<0.05)of CpGs on the promoter of LDLR gene promoter.In addition,cholesterol-7alpha-hydroxylase(CYP7a1)was increased(P<0.05)at mRNA level,but was unchanged at protein levels,accompanied by the dramatically(P<0.05)activated miR-181 targeting CYP7a1 3’UTR.However,cholesterol-27alpha-hydroxylase(CYP27al)was up-regulated both at mRNA and protein level in the liver of betaine-exposed piglets.Collectively,our results indicate that maternal betaine supplementation modifies the expression of genes in cholesterol biosynthesis,transportation and transformation,through epigenetic regulations including DNA and histone methylation,as well as miRNAs expression and consequently leading to elevated hepatic cholesterol content in neonatal piglets. |
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