3 ± 4.1 U/L [WT], 266.3 ± 27 U/L [GNMT-KO], 61.8 ± 9.5 U/L [NAM-treated GNMT-KO]; alanine aminotransferase, 24.5 ± 3 U/L [WT], 177.8 ± 10.4 U/L [GNMT-KO], selleck compound 35.6 ± 1.4 U/L [NAM-treated GNMT-KO]; NAM-treated GNMT-KO versus untreated GNMT-KO for both aminotransferases [n = 5; P < 0.05]). Furthermore, histological examination revealed that the livers of 3-month-old GNMT-KO mice treated with NAM lacked signs of steatosis or fibrosis. As reported,6 3-month-old GNMT-KO mice exhibited extensive accumulation of liver fat (hematoxylin-eosin

staining) and fibrosis (Sirius Red staining and α-SMA immunohistochemical analysis) (Fig. 2). In contrast, NAM-treated GNMT-KO mice showed no signs of steatosis or fibrosis (Fig. 2). Liver histology was normal in NAM-treated WT animals. Consistent with the high SAM levels, the liver expression of methionine adenosyltransferase 2A (MAT2A), a gene whose expression is inhibited by SAM,16 was markedly reduced in GNMT-KO mice but was normal in NAM-treated KO animals (Fig. 3E). Similarly, the livers of 3-month-old GNMT-KO mice showed marked alterations in the expression of critical genes involved in lipid metabolism (fatty acid translocase CD36 [CD36], adipose differentiation-related protein [ADFP],

peroxisome proliferator-activated receptor-α [PPARα], and PPARγ), oxidative stress and inflammation (cytochrome P4502E1 [CYP2E1], cytochrome P45039A1 [CYP39A1], cytochrome P4504A10 [CYP4A10], cytochrome P4504A14 [CYP4A14], uncoupling protein-2 [UCP2], PPARγ, interleukin-6 [IL6], and inducible nitric oxide synthase [iNOS]), and extracellular click here matrix regulation (pro-α1-collagen type I [COL1A1], TIMP tissue inhibitor of metalloproteinase-1 [TIMP-1], α-SMA). Furthermore, the treatment of GNMT-KO mice with NAM prevented completely (CD36, ADFP, CYP4A10, CYP4A14, CYP39A1, UCP2, IL6, iNOS, COL1A1, α-SMA) or largely (PPARα, PPARγ, CYP2E1, TIMP-1) the abnormal expression of these genes in the liver (Fig. 3A-E). NAM administration had no significant effect on the expression of these genes in WT mice (Fig. 3A-E),

indicating that the effect of NAM on gene expression in GNMT-KO mice is mediated by its capacity to reduce hepatic SAM content. The hepatic expression of sirtuin-1 (SIRT1), a NAD+-dependent protein deacetylase PRKACG that is an important regulator of energy metabolism modulating many aspects of glucose and lipid homeostasis,17 was similar in WT and GNMT-KO mice and was not modified by NAM administration (Fig. 3A). Finally, the livers of 3-month-old GNMT-KO mice showed marked apoptosis as demonstrated by poly(ADP-ribose) polymerase (PARP) cleavage and TUNEL immunostaining, which was also prevented in NAM-treated GNMT-KO animals (Fig. 4A,B). We have reported the existence of global DNA hypermethylation in the livers of GNMT-KO mice.6 Global DNA methylation, assayed both by the quantification of 5mC groups (Fig. 5A) and by the measurement of the number of unmethylated CpG sites (Fig.