Previously, we showed that feeding micropigs ethanol with a folate-deficient diet promoted the development of hepatic injury while increasing hepatic levels of homocysteine and S-adenosylhomocysteine (SAH) and reducing the level of S-adenosylmethionine (SAM) and the SAM-to-SAH ratio. Our present goals were to evaluate mechanisms for hepatic injury using liver specimens from the same micropigs. The effects of ethanol feeding or folate-deficient diets, singly or in combination, on cytochrome P-450 2E1 (CYP2E1) and signal pathways for apoptosis and steatosis were analyzed using microarray, real-time PCR, and immunoblotting techniques. Apoptosis was increased maximally by the combination of ethanol feeding and folate deficiency and was correlated positively to liver homocysteine and SAH. Liver CYP2E1 and the endoplasmic reticulum stress signals glucose-regulated protein 78 (GRP78), caspase 12, and sterol regulatory element binding protein-1c (SREBP-1c) were each activated in pigs fed folate-deficient or ethanol diets singly or in combination. Liver mRNA levels of CYP2E1, GRP78, and SREBP-1c, and protein levels of CYP2E1, GRP78, nuclear SREBP, and activated caspase 12 each correlated positively to liver levels of SAH and/or homocysteine and negatively to the SAM-to-SAH ratio. The transcripts of the lipogenic enzymes fatty acid synthase, acetyl-CoA carboxylase, and stearoyl-CoA desaturase were elevated in the ethanol-fed groups, and each was positively correlated to liver homocysteine levels. The induction of abnormal hepatic methionine metabolism through the combination of ethanol feeding with folate deficiency is associated with the activation of CYP2E1 and enhances endoplasmic reticulum stress signals that promote steatosis and apoptosis.

Background: To demonstrate a causative role of abnormal methionine metabolism in the pathogenesis of alcoholic steatosis, we measured the effects on hepatic lipid synthesis of supplementing ethanol and folate-deficient diets with S-adenosylmethionine (SAM), a metabolite that regulates methionine metabolism.Methods: Yucatan micropigs were fed folate-deficient diets as control, with ethanol at 40% of kcal, and with ethanol supplemented with SAM at 0.4 g/1,000 kcal for 14 weeks. Histopathology, triglyceride levels and transcripts, and protein levels of the regulatory signals of hepatic lipid synthesis were measured in terminal omental adipose and liver samples.Results: Feeding ethanol at 40% of kcal with folate-deficient diets for 14 weeks increased and supplemental SAM maintained control levels of liver and plasma triglyceride. Serum adiponectin, liver transcripts of adiponectin receptor-1 (AdipoR1), and phosphorylated adenosine monophosphate kinase-b (p-AMPKb) were each reduced by ethanol feeding and were sustained at normal levels by SAM supplementation of the ethanol diets. Ethanol feeding activated and SAM supplementation maintained control levels of ER stress-induced transcription factor sterol regulatory element-binding protein-1c (SREBP-1c) and its targeted transcripts of lipid synthesizing enzymes acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), and glycerol-3-phosphate acyltransferase (GPAT).Conclusions: Ethanol feeding with a folate-deficient diet stimulates hepatic lipid synthesis by down-regulating adiponectin-mediated pathways of p-AMPK to increase the expression of nSREBP1c and its targeted lipogenic enzymes. Preventing abnormal hepatic methionine metabolism by supplementing ethanol diets with SAM reduces liver triglyceride levels by up-regulation of adiponectin-mediated pathways to decrease fatty acid and triglyceride synthesis. This study demonstrates that ethanol-induced hepatic lipid synthesis is mediated in part by abnormal methionine metabolism, and strengthens the concept that altered methionine metabolism plays an integral role in the pathogenesis of steatosis.

We tested the hypothesis that the pathogenesis of alcoholic liver injury is mediated by epigenetic changes in regulatory genes that result from the induction of aberrant methionine metabolism by ethanol feeding. Five-month-old cystathionine beta synthase heterozygous and wild-type C57BL/6J littermate mice were fed liquid control or ethanol diets by intragastric infusion for 4 weeks. Both ethanol-fed groups showed typical histopathology of alcoholic steatohepatitis, with reduction in liver S-adenosylmethionine (SAM), elevation in liver S-adenosylhomocysteine (SAH), and reduction in the SAM/SAH ratio with interactions of ethanol and genotype effects. P revious studies established associations of abnormal hepatic methionine metabolism with the development and clinical expression of alcoholic steatohepatitis (ASH). 1,2 In transmethylation reactions, homocysteine is methylated to methionine and then Sadenosylmethionine (SAM), which is a substrate and principal methyl donor in methylation reactions, whereas S-adenosylhomocysteine (SAH) is both a product and

The present results indicate that SAM supplementation attenuates ethanol induced liver injury through its effects on the expressions and activities of oxidative stress pathways, and are consistent with the concept that the pathogenesis of oxidative liver injury is regulated in part through altered hepatic methionine metabolism.