Nt staining in the ethanol group that was normalized by betaine

Nt staining in the ethanol group that was normalized by betaine supplementation (Fig. 4). On the other hand, protein levels of Ppara did not follow the same trend as their gene expressions, since its mean level was increased by ethanol feeding and there was no difference after betaine treatment. Pyrosequencing Samples from CS heterozygote mice were selected for pyrosequencing analyses of the same three genes with L 663536 biological activity differential gene expressions according to diet, and DNA regions were chosen according to anticipated changes according to prior MethylC-seq analyses. Since there were no significant methylation differences among groups in promoter region CpG sites in any of the three genes, representative sites were selected according to CpG rich sites in gene bodies. As shown in Table 4, DNA methylation of CpG rich regions in Dnmt1 and Ppar were not significantly different in liver samples from the three diet groups, except for increased methylation of Dnmt1 in the betaine supplement group at a region over 1 kb upstream of the transcription start site in chromosome 9. On the other hand, the percent DNA methylation in a region spanning 7 CpG sites in the second 11-Deoxojervine site intron of Nos2 was significantly reduced by ethanol feeding and was sustained at control levels by betaine supplementation. Among all three diet groups in this CS heterozygote cohort, the percent DNA methylation of Nos2 in this region was correlated negatively with its relative expression (r=-0.5104; p<0.03) and positively with liver SAM (r=0.7538, p<0.0001) and the SAM:SAH ratio (r=0.79850, p<0.0001). Whereas a previous study found that the transcription of Nos2 in mouse mesangial cells was inhibited by hypermethylation of a specific promoter region in its DNA (Yu and Kone, 2004), we were unable to find methylation differences in the same Nos2 promoter region in the heterozygote livers of control, ethanol-fed, and betaine supplemented mice.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptDiscussionEpigenetic regulation of genes relevant to liver injury in ASH may be closely related to underlying ethanol-induced changes in methionine metabolism. Our initial studies in micropigs fed ethanol-containing diets that were deficient in the folate methyl donorAlcohol Clin Exp Res. Author manuscript; available in PMC 2015 June 01.Medici et al.Pagedemonstrated accelerated onset of the histopathology of ASH with significant decreases in hepatic SAM and the SAM:SAH methylation ratio compared to those fed folate replete diet (Halsted et al., 2002) together with increased transcript levels of genes involved in endoplasmic reticulum stress and lipogenesis (Esfandiari et al., 2005). Subsequent intervention studies in the same micropig model showed that the histopathology of ASH, abnormal SAM:SAH ratio, and the activation of the same liver injury genes could be prevented by concurrent supplementation of ethanol diets with the methyl donor SAM (Esfandiari et al., 2007; Villanueva et al., 2007). More recently, we demonstrated that 4 wk intragastric feeding of ethanol to CS-heterozygous mice reduced the SAM:SAH ratio while elevating the expressions of selected endoplasmic reticulum genes, each of which were associated with decreased H3K9me3 histone methylation (Esfandiari et al., 2010). The main findings of the present study are, first, that 4- wk of ethanol feeding resulted in increased steatosis and total hepatic histopathology scores typical of early ASH in both genotypes, toget.Nt staining in the ethanol group that was normalized by betaine supplementation (Fig. 4). On the other hand, protein levels of Ppara did not follow the same trend as their gene expressions, since its mean level was increased by ethanol feeding and there was no difference after betaine treatment. Pyrosequencing Samples from CS heterozygote mice were selected for pyrosequencing analyses of the same three genes with differential gene expressions according to diet, and DNA regions were chosen according to anticipated changes according to prior MethylC-seq analyses. Since there were no significant methylation differences among groups in promoter region CpG sites in any of the three genes, representative sites were selected according to CpG rich sites in gene bodies. As shown in Table 4, DNA methylation of CpG rich regions in Dnmt1 and Ppar were not significantly different in liver samples from the three diet groups, except for increased methylation of Dnmt1 in the betaine supplement group at a region over 1 kb upstream of the transcription start site in chromosome 9. On the other hand, the percent DNA methylation in a region spanning 7 CpG sites in the second intron of Nos2 was significantly reduced by ethanol feeding and was sustained at control levels by betaine supplementation. Among all three diet groups in this CS heterozygote cohort, the percent DNA methylation of Nos2 in this region was correlated negatively with its relative expression (r=-0.5104; p<0.03) and positively with liver SAM (r=0.7538, p<0.0001) and the SAM:SAH ratio (r=0.79850, p<0.0001). Whereas a previous study found that the transcription of Nos2 in mouse mesangial cells was inhibited by hypermethylation of a specific promoter region in its DNA (Yu and Kone, 2004), we were unable to find methylation differences in the same Nos2 promoter region in the heterozygote livers of control, ethanol-fed, and betaine supplemented mice.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptDiscussionEpigenetic regulation of genes relevant to liver injury in ASH may be closely related to underlying ethanol-induced changes in methionine metabolism. Our initial studies in micropigs fed ethanol-containing diets that were deficient in the folate methyl donorAlcohol Clin Exp Res. Author manuscript; available in PMC 2015 June 01.Medici et al.Pagedemonstrated accelerated onset of the histopathology of ASH with significant decreases in hepatic SAM and the SAM:SAH methylation ratio compared to those fed folate replete diet (Halsted et al., 2002) together with increased transcript levels of genes involved in endoplasmic reticulum stress and lipogenesis (Esfandiari et al., 2005). Subsequent intervention studies in the same micropig model showed that the histopathology of ASH, abnormal SAM:SAH ratio, and the activation of the same liver injury genes could be prevented by concurrent supplementation of ethanol diets with the methyl donor SAM (Esfandiari et al., 2007; Villanueva et al., 2007). More recently, we demonstrated that 4 wk intragastric feeding of ethanol to CS-heterozygous mice reduced the SAM:SAH ratio while elevating the expressions of selected endoplasmic reticulum genes, each of which were associated with decreased H3K9me3 histone methylation (Esfandiari et al., 2010). The main findings of the present study are, first, that 4- wk of ethanol feeding resulted in increased steatosis and total hepatic histopathology scores typical of early ASH in both genotypes, toget.