Alcoholic liver disease is associated with abnormal hepatic methionine metabolism and folate deficiency. Because folate is integral to the methionine cycle, its deficiency could promote alcoholic liver disease by enhancing ethanol-induced perturbations of hepatic methionine metabolism and DNA damage. We grouped 24 juvenile micropigs to receive folate-sufficient (FS) or folate-depleted (FD) diets or the same diets containing 40% of energy as ethanol (FSE and FDE) for 14 wk, and the significance of differences among the groups was determined by ANOVA. Plasma homocysteine levels were increased in all experimental groups from 6 wk onward and were greatest in FDE. Ethanol feeding reduced liver methionine synthase activity, S-adenosylmethionine (SAM), and glutathione, and elevated plasma malondialdehyde (MDA) and alanine transaminase. Folate deficiency decreased liver folate levels and increased global DNA hypomethylation. Ethanol feeding and folate deficiency acted together to decrease the liver SAM͞S-adenosylhomocysteine (SAH) ratio and to increase liver SAH, DNA strand breaks, urinary 8-oxo-2 -deoxyguanosine [oxo(8)dG]͞mg of creatinine, plasma homocysteine, and aspartate transaminase by more than 8-fold. Liver SAM correlated positively with glutathione, which correlated negatively with plasma MDA and urinary oxo(8)dG. Liver SAM͞SAH correlated negatively with DNA strand breaks, which correlated with urinary oxo(8)dG. Livers from ethanol-fed animals showed increased centrilobular CYP2E1 and protein adducts with acetaldehyde and MDA. Steatohepatitis occurred in five of six pigs in FDE but not in the other groups. In summary, folate deficiency enhances perturbations in hepatic methionine metabolism and DNA damage while promoting alcoholic liver injury. F olate deficiency is among the most common nutritional abnormalities in chronic alcoholic patients, especially in those who have developed alcoholic liver injury (1-5). In addition to poor diet, folate deficiency in chronic alcoholism can be ascribed to decreased intestinal absorption and hepatic uptake, increased renal excretion, and increased oxidative cleavage of the folate molecule (6-12). Folate in its 5-methyltetrahydrofolate (5-MTHF) form is integral to methionine metabolism. Folate deficiency perturbs hepatic methionine metabolism (13,14), which is associated with DNA nucleotide imbalance and increased hepatocellular apoptosis in experimental animals fed folate-deficient (FD) diets or exposed to chronic ethanol (15,16).Hepatic methionine metabolism is regulated by the availability of dietary and endogenous folate that appears in the circulation as 5-MTHF and is the substrate with cofactor vitamin B 12 for the methionine synthase (MS) reaction that generates methionine from homocysteine (Hcy) (see supporting information, which is published on the PNAS web site, www.pnas.org). In the alternate salvage pathway for methionine synthesis, choline is the precursor of betaine, which is the substrate for betaine homocysteine methyltransferase (BHMT). The methionine adeno...

The goals and objectives of these studies, conducted over the past 30 y, were to determine: a) how chronic alcoholism leads to folate deficiency and b) how folate deficiency contributes to the pathogenesis of alcoholic liver disease (ALD). The intestinal absorption of folic acid was decreased in binge drinking alcoholics and, prospectively, in volunteers fed alcohol with low folate diets. Monkeys fed alcohol for 2 y developed decreased hepatic folate stores, folic acid malabsorption and decreased hepatic uptake but increased urinary excretion of labeled folic acid. Micropigs fed alcohol for 1 y developed features of ALD in association with decreased translation and activity of intestinal reduced folate carrier. Another study in ethanol-fed micropigs demonstrated abnormal hepatic methionine and DNA nucleotide imbalance and increased hepatocellular apoptosis. When alcohol feeding was combined with folate deficiency, micropigs developed typical histological features of ALD in 14 wk, together with elevated plasma homocysteine levels, reduced liver S-adenosylmethionine and glutathione and increased markers for DNA and lipid oxidation. In summary, chronic alcohol exposure impairs folate absorption by inhibiting expression of the reduced folate carrier and decreasing the hepatic uptake and renal conservation of circulating folate. At the same time, folate deficiency accelerates alcohol-induced changes in hepatic methionine metabolism while promoting enhanced oxidative liver injury and the histopathology of ALD.

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.

Alcoholic liver disease is associated with abnormal hepatic methionine metabolism, including increased levels of homocysteine and S-adenosylhomocysteine (SAH) and reduced levels of Sadenosylmethionine (SAM) and glutathione (GSH). The concept that abnormal methionine metabolism is involved in the pathogenesis of alcoholic liver disease was strengthened by our previous findings in a micropig model where combining dietary folate deficiency with chronic ethanol feeding produced maximal changes in these metabolites together with early onset of microscopic steatohepatitis and an eightfold increase in plasma aspartate aminotransferase. The goal of the present study was to determine potential mechanisms for abnormal levels of these methionine metabolites by analyzing the transcripts and activities of transmethylation enzymes in the livers of the same micropigs. Ethanol feeding or folate deficiency, separately or in combination, decreased transcript levels of methylenetetrahydrofolate reductase (MTHFR), methionine adenosyltransferase (MAT1A), glycine-N-methyltransferase (GNMT) and S-adenosylhomocysteine hydrolase (SAHH). Ethanol feeding alone reduced the activities of methionine synthase (MS) and MATIII and increased the activity of GNMT. Each diet, separately or in combination, decreased the activities of MTHFR and SAHH. In conclusion, the observed abnormal levels of methionine metabolites in this animal model of accelerated alcoholic liver injury can be ascribed to specific effects of ethanol with or without folate deficiency on the expressions and activities of hepatic enzymes that regulate transmethylation reactions. These novel effects on transmethylation reactions may be implicated in the pathogenesis of alcoholic liver disease. ( A bnormal hepatic methionine metabolism can result from chronic exposure to ethanol or from folate deficiency. Hyperhomocysteinemia has been described in chronic alcoholics, 1,2 and decreased liver S-adenosylmethionine (SAM) levels were found in experimental animals and cultured hepatocytes exposed to ethanol. [3][4][5][6] Folate deficiency also results in abnormal methionine metabolism, because 5-methyltetrahydrofolate (5-MTHF) is substrate for the methionine synthase (MS) reaction that converts homocysteine to methionine, the substrate for SAM (Fig. 1). Furthermore, folate deficiency is frequently associated with alcoholic liver disease, in part, because chronic exposure to ethanol reduces the intestinal transport of folic acid and increases folate excretion in the urine. [7][8][9] Previous data support the concept that changes in methionine metabolism are involved in the development of alcoholic liver disease. Studies in rats and micropigs have demonstrated MS activity is reduced during chronic ethanol exposure. 10 -13 Rats that were fed ethanol by gastric tube demonstrated decreased methionine adenosyltransferase MAT1-III activity with decreased SAM and SAM/ S-adenosylhomocysteine (SAH) ratio in association with increased DNA strand breaks. 5 A MAT1A knockout mouse model developed he...