| Maternal nutrition during pregnancy and lactation which has been considered as critical windows of development,plays an important role in regulating growth and metabolic consequences in offspring via epigenetic regulation,such as DNA methylation.Betaine is a highly efficient methyl donor and contains three active methyl groups.Moreover,betaine involves in one-carbon metabolism acting as a substrate of the methionine cycle and donates methyl groups for most methylation reactions in the body.Previously we have reported that maternal betaine supplementation during pregnancy increased hepatic cholesterol content and reduced triglyceride level in neonatal piglets,which is accompanied by changes in DNA methylation and histone modifications.However,the effect of maternal betaine supplementation during pregnancy and lactation on the lipid metabolism in weaning stage is unclear.In addition,over-nutrition and long-term stress can lead to excessive deposition of fat and finally induces non-alcoholic fatty liver disease(NAFLD).In recent years,animal model studies have shown that betaine supplementation has been proven effective in attenuating high-fat or high-sugar induced NAFLD.However,the effect of maternal betaine supplementation during pregnancy and lactation on the glucocorticoid-induced intrahepatic lipid deposition in offsprings is unknown.Moreover,the role of adipose tissue in the development of NAFLD remains to be further studied.Finally,the mechanism of action of betaine to alleviate lipid deposition was explored in vitro model.1 Effects of maternal betaine supplementation on hepatic lipid metabolism in weaning offspring rats and its mechanismsIn order to explore the effect of maternal betaine on hepatic lipid metabolism in offspring rats.We used 3 months-old female Sprague-Dawley rats and then confirmed pregnancy by the presence of a vaginal smear plug.The pregnant rats were transferred to individual cages and randomly divided into control and experimental groups throughout gestation and lactation,fed with a basal diet or betaine-supplemented diet(1%),respectively.At 21 days of weaning,serum and liver samples were immediately collected,frozen in lipuid nitrogen and stored at-80℃ for further analysis.The results showed that the weaning body weight tended to decrease(P=0.085)in betaine-supplemented group compared with control group,but no significant changes were observed in liver weight and liver weight/body weight.Serum concentrations of total cholesterol and high-density lipoprotein cholesterol(HDLC)were significantly increased in the offspring rats by betaine-supplemented dams(P<0.05),while serum glucose,total triglyceride and low-density lipoprotein cholesterol(LDLC)were not affected.In addition,maternal betaine exposure significantly increased hepatic cholesterol contents(P<0.05),not triglyceride,in weaned offspring rats.Real-time PCR showed that maternal betaine supplementation during gestation and lactation significantly reduced hepatic 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase(HMGCR)and cholesterol 7 alpha-hydroxylase(CYP7A1)mRNA expression(P<0.05),not affect sterol regulatory element-binding protein 2(SREBP2),cholesterol 27 alpha-hydroxylase(CYP27A1)and low-density lipoprotein receptor(LDLR)mRNA expression.However,western blot results showed that the protein level of CYP7A1 and LDLR were significantly upregulated and downregulated(P<0.05),respectively,in betaine-supplemented dams,while the protein levels of fatty acid-related genes were not affected between two groups.Additionally,we also found that maternal betaine exposure markedly increased hepatic betaine-homocysteine methyltransferase(BHMT)mRNA and protein expression(P<0.05),not affected glycine N-methyltransferase(GNMT),s-adenosyl homocysteine hydrolase-like 1(AHCYL1)and DNA methyltransferase 1(DNMT1)mRNA and protein level.Furthermore,we used methylated DNA immunoprecipitation(MeDIP)to analyze the DNA methylation enrichment on the promoter of cholesterol-related genes,which demonstrated that maternal betaine supplementation resulted in the hepatic CYP7A1 hypermethylation in weaning offspring rats(P<0.05).Our results provide that maternal betaine supplementation significantly increases hepatic cholesterol contents without triglyceride,accompanied by the changes of cholesterol-related genes and DNA methylation enrichment in weaned offspring rats.2 Effects of maternal betaine supplementation on deamethasone-induced hepatic lipid metabolism and its mechanismsTo investigate the effect of maternal betaine on glucocorticoid-induced hepatic lipid metabolism in offspring rats.After weaning,both group rats were fed with basal diet.At 90 days,all of rats from two group were then divided into subgroups(Con-Con,Con-Dex,Bet-Con,Bet-Dex),being subject to vehicle or dexamethasone treatment with intraperitoneal injection of physiological saline or dexamethasone in a dose of 0.1mg/kg body mass every day for 3 weeks.After 3 weeks,all of rats were sacrificed.Serum,adipose and liver samples were immediately collected,frozen in lipuid nitrogen and stored at-80℃for further analysis.The results showed that dexamethasone-treated rats significantly down-regulated final body weight(P<0.05)and daily food intake(P=0.06)compared with Con-Con group,whereas maternal betaine exposure did not restore the effect.In addition,no marked differences were observed in liver weight,epididymal fat weight,liver weight/body weight and epididymal fat weight/body weight among groups.Compared with Con-Con group,dexamethasone administration markedly increased serum total triglyceride level(P<0.05)and decreased serum total cholesterol,HDLC and LDLC concentrations(P<0.05),while maternal betaine exposure did not improve dexamethasone-induced these effects.HE and Oil Red staining on the liver sections showed that dexamethasone administration significantly increased hepatic fat vacuolation and lipid droplet accumulation,while prenatal betaine exposure could alleviate the liver injury(P<0.05).Biochemical analysis further demonstrated that maternal betaine supplementation reduced the dexamethasone-induced up-regulation of hepatic triglyceride contents(P<0.05).RT-PCR and WB results showed that dexamethasone administration significantly increased hepatic lipogenic genes ACC1,FAS and SCD1 mRNA and protein expression,which were alleviated by betaine-supplemented dams(P<0.05).However,other genes including ATGL,peroxisome proliferator-activated receptor alpha(PPARα),Carnitine palmitoyltransferase 1α(CPT1α),fatty acid binding protein(FABP)and Fatty acid translocase(CD36)were not observed markedly difference.We further used MeDIP method to detect the DNA methylation status on the promoter of lipogenic genes.MeDIP results showed that hepatic hypomethylation of ACC1,FAS and SCD1 in dexamethasone-induced offspring rats were reversed by prenatal betaine supplementation(P<0.05).Next,we predicted that there were GR and SP1 binding sites on the promoter of lipogenic genes.ChIP assay revealed that maternal betaine exposure significantly alleviated dexamethasone-induced increase of GR or SP1 binding to ACC1,FAS and SCD1 genes promoter,which were in line with the nucleus protein levels of GR and SP1(P<0.05).These results indicate that maternal betaine supplementation attenuates dexamethasone-induced fatty liver in adult offspring rats,which may be attributed to epigenetic and GR or SP1-mediated mechanism.3 Effects of maternal betaine supplementation on dexamethasone-induced lipid metabolism of adipose tissue and its mechanismsIn order to explore the role of adipose tissue in the alleviation of maternal betaine on glucocorticoid-induced NAFLD.we collected the epididymal adipose tissue of offspring rats.Our results showed that there was no significant change in epididymal fat weight and epididymal fat weight/body weight,whether prenatal betaine supplementation or dexamethasone injection.In addition,we found that maternal betaine supplementation during gestation and lactation attenuates dexamethasone-induced the up-regulation of serum insulin,IL6 and TNFa levels(P<0.05).Moreover,there were no significant effect on serum NEFA among four groups.RT-PCR and WB results showed that the increase of dexamethasone-induced adipose triglyceride lipase(ATGL)and hormone sensitive lipase(HSL)mRNA expressions were markedly decreased by maternal betaine exposure(P<0.05).Similar results were also observed in protein contents of ATGL and p-HSL(S855)(P<0.05).However,the protein expression of acetyl-CoA carboxylase(ACC)and fatty acid synthetase(FAS)were not affected among four groups.Additionally,we found that maternal betaine supplementation could significantly alleviate dexamethasone-induced down-regulation of serum endogenous corticosterone level and up-regulation of GR protein expression in adipose tissue(P<0.05).However,the protein levels of AMPK and AKT were not changed among four groups.We further used MeDIP method to detect DNA methylation enrichment on the promoter of ATGL and HSL genes.Our results showed that maternal betaine exposure alleviated dexamethasone-induced hypomethylation on the promoter of ATGL(P<0.05).In addition,chronic dexamethasone administration resulted in hypomethylation on the promoter of HSL(P<0.05),while maternal betaine supplementation did not restore the hypomethylation.These results suggest that maternal betaine supplementation regulates the expression of lipolytic genes through DNA methylation,thereby improving dexamethasone-induced adipose tissue dysfunction in offspring rats.4 The effects of betaine on lipid accumulation in HepG2 cells and its mechanismsTo explore the role of GR and DNA methylation in non-alcoholic fatty liver disease.We used oleic acid or dexamethasone in combination with oleic acid to stimulate HepG2 cell to establish a in vitro model to analyze the effect of betaine.Oil red O staining was used to observe accumulation of lipid droplet while the intracellular TG content was detected by enzymatic method.WB and MeDIP methods were also used to detect the changes of lipid metabolism related genes.The results showed that betaine significantly decreased the increase of oleic acid-induced,not dexamethasone in combination with oleic acid-induced,TG contents.Western blot demonstrated that neither oleic acid,betaine nor combined treatment affected the protein expression of GR,p-GR and p-GR/GR.Moreover,betaine treatment had no effect on the increase of dexamethasone in combination with oleic acid-induced p-GR/GR.MeDIP results showed that betaine treatment significantly alleviated the oleic acid-induced hypomethylation of ACC1,FAS and PPARγ genes(P<0.05).Additionally,Intracellular TG results showed that betaine treatment was able to reverse the lipid deposition induced by dexamethasone combined with oleic acid after treatment with the GR inhibitor RU486.Finally,the methylation inhibitor 5AZA suppressed the effect of betaine on TG content.The above results indicated that betaine treatment alleviated oleic acid-induced lipid accumulation in cells through DNA methylation.In conclusion,maternal betaine supplementation increases hepatic cholesterol contents without triglyceride level in weaning offspring rats.After dexamethasone administration,maternal betaine exposure was able to alleviate dexamethasone-induced NAFLD through regulating hepatic and adipose tissue lipid metabolism.This may be due to betaine altering DNA methylation and then affecting lipid metabolism. |
