Epigenetic regulation of hepatic endoplasmic reticulum stress pathways in the ethanol-fed cystathionine beta synthase-deficient mouse.

UNLABELLED: 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. Hepatic endoplasmic reticulum stress signals including glucose-regulated protein-78 (GRP78), activating transcription factor 4, growth arrest and DNA damage-inducible gene 153 (GADD153), caspase 12, and transcription factor sterol response element binding protein-1c (SREBP-1c) were up-regulated in ethanol-fed mice with genotype interactions and negative correlations with the SAM/SAH ratio. Immunohistochemical staining showed reduction in trimethylated histone H3 lysine-9 (3meH3K9) protein levels in centrilobular regions in both ethanol groups, with no changes in trimethylated histone H3 lysine-4 levels. The chromatin immunoprecipitation assay revealed a decrease in levels of suppressor chromatin marker 3meH3K9 in the promoter regions of GRP78, SREBP-1c, and GADD153 in ethanol-treated heterozygous cystathionine beta synthase mice. The messenger RNA expression of the histone H3K9 methyltransferase EHMT2 (G9a) was selectively decreased in ethanol-fed mice. CONCLUSION: The pathogenesis of alcoholic steatohepatitis is mediated in part through the effects of altered methionine metabolism on epigenetic regulation of pathways of endoplasmic reticulum stress relating to apoptosis and lipogenesis.

Quantitative lipid metabolomic changes in alcoholic micropigs with fatty liver disease.

BACKGROUND: Chronic ethanol consumption coupled with folate deficiency leads to rapid liver fat accumulation and progression to alcoholic steatohepatitis (ASH). However, the specific effects of alcohol on key liver lipid metabolic pathways involved in fat accumulation are unknown. It is unclear whether lipid synthesis, lipid export, or a combination of both is contributing to hepatic steatosis in ASH. METHODS: In this study we estimated the flux of fatty acids (FA) through the stearoyl-CoA desaturase (SCD), phosphatidylethanolamine-N-methyltransferase (PEMT), and FA elongation pathways in relation to liver triacylglycerol (TG) content in Yucatan micropigs fed a 40% ethanol folate-deficient diet with or without supplementation with S-adenosyl methionine (SAM) compared with controls. Flux through the SCD and PEMT pathways was used to assess the contribution of lipid synthesis and lipid export respectively on the accumulation of fat in the liver. Liver FA composition within TG, cholesterol ester (CE), phosphatidylethanolamine, and phosphatidylcholine classes was quantified by gas chromatography. RESULTS: Alcoholic pigs had increased liver TG content relative to controls, accompanied by increased flux through the SCD pathway as indicated by increases in the ratios of 16:1n7 to 16:0 and 18:1n9 to 18:0. Conversely, flux through the elongation and PEMT pathways was suppressed by alcohol, as indicated by multiple metabolite ratios. SAM supplementation attenuated the TG accumulation associated with alcohol. CONCLUSIONS: These data provide an in vivo examination of liver lipid metabolic pathways confirming that both increased de novo lipogenesis (e.g., lipid synthesis) and altered phospholipid metabolism (e.g., lipid export) contribute to the excessive accumulation of lipids in liver affected by ASH.

S-adenosylmethionine attenuates oxidative liver injury in micropigs fed ethanol with a folate-deficient diet.

BACKGROUND: To demonstrate a causative role for abnormal methionine metabolism in the pathogenesis of alcoholic steatohepatitis (ASH), we measured the preventive effects of supplementing folate deficient and ethanol containing diets in the micropig 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, or with ethanol supplemented with SAM at 0.4 g/1000 kcal for 14 weeks. Histopathology, markers of liver injury, and regulatory enzymes were measured in terminal liver samples. RESULTS: Among the ethanol group, livers showed hepatocellular necrosis together with increased levels of S-adenosylhomocysteine (SAH) and reduced levels of SAM and its ratio to SAH and glutathione (GSH), together with increased malondialdehyde plus hydroxynonenol (MDA + HNE) and nitrotyrosine (NT), transcripts and protein levels of cytochrome P4502E1 (CYP2E1), activity of NADPH oxidase, and activity and protein levels of inducible nitric oxide (iNOS). These findings were attenuated partially or completely to control levels by SAM supplementation of the ethanol diet. CONCLUSIONS: 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.

S-adenosylmethionine attenuates hepatic lipid synthesis in micropigs fed ethanol with a folate-deficient diet.

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-beta (p-AMPKbeta) 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 nSREBP-1c 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.

Abnormal transsulfuration and glutathione metabolism in the micropig model of alcoholic liver disease.

BACKGROUND: Alcoholic liver disease is associated with abnormalities of methionine metabolic enzymes that may contribute to glutathione depletion. Previously, we found that feeding micropigs a combination of ethanol with a folate-deficient diet resulted in the greatest decreases in S-adenosylmethionine and glutathione and increases in liver S-adenosylhomocysteine and oxidized disulfide glutathione. METHODS: To study the mechanisms of glutathione depletion, we analyzed the transcripts and activities of enzymes involved in its synthesis and metabolism in liver and plasma specimens that were available from the same micropigs that receive folate-sufficient or folate-depleted diets with or without 40% of energy as ethanol for 14 weeks. RESULTS: Ethanol feeding, folate deficiency, or their combination decreased liver and plasma glutathione and the activities of hepatic copper-zinc superoxide dismutase and glutathione peroxidase and increased the activity of manganese superoxide dismutase and glutathione reductase. Hepatic levels of cysteine and taurine were unchanged while plasma cysteine was increased in the combined diet group. Cystathionine beta-synthase transcripts and activity were unaffected by ethanol feeding, while the activities of other transsulfuration enzymes involved in glutathione synthesis were increased. Glutathione transferase transcripts were increased 4-fold and its mean activity was increased by 34% in the combined ethanol and folate-deficient diet group, similar in magnitude to the observed 36% reduction in hepatic glutathione. CONCLUSIONS: Chronic ethanol feeding and folate deficiency acted individually or synergistically to affect methionine metabolism in the micropig by depleting glutathione pools and altering transcript expressions and activities of enzymes involved in its synthesis, utilization, and regeneration. The data suggest that the observed decrease in hepatic glutathione during ethanol feeding reflects its increased utilization to meet increased antioxidant demands, rather than reduction in its synthesis.

Chronic ethanol feeding and folate deficiency activate hepatic endoplasmic reticulum stress pathway in micropigs.

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.

Methyl deficiency causes reduction of the methyl-CpG-binding protein, MeCP2, in rat liver.

MeCP2 is a member of a family of proteins [methyl- (cytosine-guanine)CpG-binding proteins] that bind specifically to methylated DNA and induce chromatin remodeling and gene silencing. Dietary deficiency of folate, choline and methionine causes decreased tissue S-adenosylmethionine concentrations (methyl deficiency), global DNA hypomethylation, hepatic steatosis, cirrhosis and ultimately hepatic tumorigenesis in rodents. We investigated the effects of this diet on expression of MeCP2 during pre-neoplastic transformation of liver tissue. After 9 weeks, MeCP2 mRNA level was slightly higher in methyl-deficient rats compared with replete controls, while after 36 weeks, a difference in MeCP2 mRNA level was no longer observed. In contrast, MeCP2 protein level was reduced almost 2-fold in the deficient rats compared with replete controls at both 9 and 36 weeks. Conversely, a second methyl-CpG-binding protein, MBD2, showed increased levels of both message and protein at the two time points. Low MeCP2 protein in the deficient rats was associated with a low level of the co-repressor protein, Sin3a, at 36 weeks. Moreover, a known gene target of MeCP2, the tumor suppressor gene metallothionein-I, was over-expressed in the deficient rat livers at both 9 and 36 weeks, suggesting that reduction in MeCP2 may have functional consequences. Methyl deficiency also caused an increase in the ratio of long to short variants of MeCP2 transcripts. This finding suggests that reduced MeCP2 protein level is the result of a reduced rate of translation. Reduction of MeCP2 protein expression may influence the initiation and/or progression of hepatic cancer induced by methyl deficiency and may provide a useful marker of pre-neoplastic change.