The Effect And DNA Methylation Mechanism Of Betaine Intake During Pregnancy On The Mice Offspring Of Fetal Non-alcoholic Fatty Liver Disease

BackgroundNon-alcoholic fatty liver disease(NAFLD),as a common chronic liver disease,not only has a higher incidence in adults,but also gradually becomes a common chronic disease in children and adolescents.The pathogenesis of NAFLD is currently unclear.Early thought that the incidence of NAFLD was related to the "second strike hypothesis" and "multiple strike model".In recent years,with the focus on nutrition during pregnancy,it has been revealed that the hypothesis of embryonic origin of human diseases is related to the occurrence of NAFLD.The hypothesis of embryonic origin of human diseases is that nutrition and diseases during pregnancy or lactation will have certain effects on the offspring’s organ structure,physiological function and disease development in adulthood.If the female mouse is obese or eats a high-fat diet during pregnancy and lactation,it will increase the incidence of NAFLD in the offspring and cause various metabolic dysfunctions;Obesity and diabetes in pregnant women increase the risk of metabolic system disease in their offspring as adults.Therefore,maternal obesity and diabetes are important risk factors for fetal NAFLD.Betaine,also called trimethylglycine,as a pure natural substance in plant food,is an important methyl donor in our body.Insufficient intake of betaine in the daily diet can promote fatty liver disease.Our previous research found that the disease level of the NAFLD population increased with the decrease of betaine content,and betaine supplementation could improve liver fatty degeneration in NAFLD mice.Whether betaine intervention during pregnancy can prevent fetal-derived NAFLD has not been reported.The liver is the central organ of lipid metabolism.The levels of triglyceride(TG)in hepatocytes mainly depend on the synthesis,oxidative decomposition and transport of fatty acids,and the metabolic enzymes in each pathway are regulated by their genes.Abnormal gene expression of enzymes will inevitably lead to the accumulation of TG in the liver.Peroxisome proliferator-activated receptor γ coactivator 1α(PPARGC1A,PGC1α),which is an activator of peroxisome proliferator-activated receptor α(PPARα),is an important factor that affects insulin resistance and oxidative phosphorylation during NAFLD.PPARα can activate the expression of related enzymes,enhance the fatty acid scavenging ability in liver cells and further reduce fat accumulation.The whole-genome DNA methylation chip found that the liver PGC1α of patients with NAFLD showed hypermethylation and decreased gene expression compared with normal people.At present,there are few reports on the methylation of NAFLD-related genes and related mechanisms.Whether fetal NAFLD is related to abnormal methylation of lipid metabolism-related genes has not been reported.Based on the above background,this study aims to verify the improvement effect of betaine intake on fetal NAFLD through animal experiments.At the same time,expression of lipid metabolism genes,methylation level and methyl donor metabolites were measured to elucidate the mechanism of betaine to improve the methylation of fetal NAFLD.Furthermore,it provides a reference for the targeted dietary guidance for the pregnant and lying-in population and prevents NAFLD from the perspective of nutrition in early life.Objects and Methods 1.Animal modeling and interventionEighty four-week-old SPF C57BL/6J female mice were divided into 4 groups according to fasting weight with a random number table: control group,gestational diabetes mellitus(GDM)group,gestational diabetes mellitus + 1% betaine group and gestational diabetes mellitus + 2% betaine group.The control group had normal feed and drinking water,and the other three groups had high-fat feed and regular drinking water within 4 weeks.Feeding to 8 weeks of age,and then put the cage together in a 1:2 male-female ratio,and then give the pregnant mice intraperitoneal injection of streptozotocin(STZ)to construct a model of GDM,while giving 1% and 2% betaine(wt/vol)in drinking water.The experiment has been fed until the weaning of the offsprings(the offsprings are about 3 weeks old).After that,the offsprings were killed to 8 weeks of age with normal feed and drinking water,and whole blood,heart,liver,spleen,lung,and other organs were removed.2.Experimental technology and detection method(1)The serum biochemical parameters were determined using commercially available kits on automatic biochemistry analyzer;(2)According to the instructions of the tissue triglyceride enzyme kit,50 mg of liver sample were took to lyse,then 500 μl of lysate was measured using a tissue triglyceride enzyme assay kit;(3)After pretreatment of liver tissue,histological analysis was evaluated by hematoxylin and eosin and oil red O stains;(4)Total RNA was isolated from the liver with the TRIZOL reagent,then reverse transcribed it into c DNA by reverse transcription kit;A quantitative analysis of the gene expression of liver FAS,ACC,PPARα,PGC1α,ACOX,MTTP,AGPAT2,Apo B48 were performed by real-time polymerase chain reaction;(5)DNA was isolated from the livers of mice using the DNA Kit,and DNA bisulfite conversion was performed.The product of target gene promoters were amplified by PCR,then the product were directly sequenced to measure ACC and PGC1α methylation levels by the dideoxy chain termination method;(6)The genomic DNA methylation levels were measured using liver DNA by Methyl Flash Methylated DNA Quantification Kit;(7)Serum betaine,choline,liver homocysteine(Hcy),hepatic S-adenosyl methionine(SAM)and S-adenosyl homocysteine(SAH)were detected by high performance liquid chromatography.Results 1 The effect of betaine on the liver lipid accumulation of dams and offspringsIn dams,the weight,visceral fat,serum alanine aminotransferase(ALT),aspartate aminotransferase(AST)and glucose(GLU)levels in the GDM group were significantly higher than those in the control group(P<0.05).Liver pathological slices showed steatosis in the GDM group,and the TG level(0.025±0.004 μmol/mg vs.0.017±0.003 μmol/mg,P=0.05)increased significantly,indicating successful fetalderived NAFLD modeling.Compared with the GDM group,the 1% betaine group and the 2% betaine group were able to reduce the weight of visceral fat(0.47±0.17 g,0.47±0.13 g vs.0.65±0.17 g),and the serum ALT(103.30±7.61U/ L,76.83±2.67U/L vs.116.13±1.1U/L),GLU(6.40±0.18 mmol/L,7.08±0.52 mmol/L vs.11.01±0.17 mmol/L).Betaine intervention can significantly reduce the degree of hepatic steatosis,and the liver TG level is also significantly reduced(0.0180±0.002 μmol/mg,0.019±0.002 μmol/mg vs.0.025±0.004 μmol/mg).Among the pups,the serum ALT in the GDM group was higher than that in the control group(72.48±3.32 U/L vs.47.75±2.92 U/L,P<0.001),and liver TG also increased significantly(0.036±0.004 μmol/mg vs.0.017±0.008 μmol/mg,P=0.008).Compared with the GDM group,the serum ALT level(51.71±2.36 U/L,55.35±1.60 U/L vs.72.48±3.32 U/L)and liver TG content(0.018±0.002 μmol/mg,0.020±0.003 μmol/mg vs.0.036±0.004 μmol/mg)also decreased significantly.2 The effect of betaine on the expression of liver lipid metabolism genes in fetal NAFLDCompared with the control group,the expression of FAS(0.538±0.060 vs.1.097±0.164,P=0.003)and ACC(0.525±0.080 vs.1.031±0.086,P=0.011)decreased in the progeny liver GDM group,the expression levels of FAS(0.627 ± 0.083,0.923±0.099 vs.0.538±0.060)and ACC(0.593±0.098,1.001±0.138 vs.0.525±0.080)rebounded after 1% and 2% betaine intervention.The expression of genes related to fatty acid oxidation PGC1α(0.215±0.036 vs.1.107±0.170,P<0.001),PPARα(0.286±0.063 vs.1.108±0.171,P=0.005)and ACOX(0.037±0.006 vs.0.963±0.335,P=1.25)in GDM group were lower than those of the control group,and the corresponding gene expression increased significantly after 1% and 2% betaine intervention(0.326±0.052,0.558±0.090 vs.0.215±0.036;0.487±0.041,0.656±0.103 vs.0.286±0.063;0.066±0.007,1.539±0.276 vs.0.037±0.006).Compared with the GDM group,the expression level of MTTP in fat-related genes decreased after 1% and 2% betaine intervention(0.025±0.008,0.034±0.002 vs.0.682±0.073).3 The effect of betaine on the methylation level of PGC1α gene promoter in fetal NAFLD liverCompared with the control group,the degree of methylation at the-3685 Cp G site of the PGC1α gene promoter region in the liver of GDM piglets was significantly increased(50.82±1.40% vs.42.65±2.36%,P=0.016),Compared with GDM group,1% and 2% betaine intervention can reduce PGC1α methylation level(45.56±1.74%,44.84±1.25% vs.50.82±1.40%).The methylation level of PGC1α promoter was negatively correlated with its corresponding mRNA expression level(r=-0.500,P=0.021).4.The effect of betaine on fetal NAFLD liver methyl donor metabolitesThere was no statistically significant difference in the level of DNA methylation of the liver genome among the groups(P>0.05).Compared with the GDM group,the homocysteine level in the 1% betaine group was significantly reduced(44.36±6.35 ng/mg vs.76.10±6.84 ng/mg,P=0.004),level of SAH in the 1% and 2% betaine group decreased significantly(11.09±0.667 ng/mg,8.87±1.098 ng/mg vs.14.70±0.912 ng/mg).Conclusion 1.Gestational diabetes mellitus female mice will cause the occurrence of fetal nonalcoholic fatty liver disease,and betaine intake during pregnancy has a certain relief effect on liver steatosis and liver damage in the dams and offsprings.2.The expression of FAS,ACC,PGC1α,PPARα and ACOX decreased in the gestational diabetes mellitus offspring group,and betaine intake during pregnancy could correct the expression disorder of these lipid metabolism genes.3.Betaine intake during pregnancy reduces the levels of liver homocysteine and SAH,and at the same time increases the expression of PGC1α mRNA by reducing the methylation level of the PGC1α promoter in the liver of the offspring.