Gene expression modifications in the liver caused by binge drinking and S-adenosylmethionine feeding. The role of epigenetic changes.

Chronic ethanol ingestion, achieved by feeding ethanol at a constant rate using intragastric tube feeding, alters the expression of genes in the liver. This is done by epigenetic mechanisms, which depend on the blood alcohol levels at the time of killing. However, acute bolus feeding of ethanol changes gene expression without lasting epigenetic changes. This occurs with histone 3 methylation and acetylation modifications. The gene expression response to an acute bolus of ethanol might be modified by feeding S-adenosylmethionine (SAMe), a methyl donor. In the present study, rats were given a bolus of ethanol (6 g/kg body weight (bw), SAMe (1 g/kg bw), ethanol + SAMe, or isocaloric glucose. The group of rats (n = 3) were killed at 3 and 12 h post bolus, and gene microarray analysis was performed on their liver cells. SAMe reduced the 3 h blood ethanol levels and increased the ALT levels at 3 h. Venn diagrams showed that alcohol changed the expression of 646 genes at 3 h post bolus and 586 genes at 12 h. SAMe changed the expression of 1,012 genes when fed with ethanol 3 h post ethanol bolus and 554 genes at 12 h post ethanol bolus. SAMe alone changed the expression of 1,751 genes at 3 h and 1,398 at 12 h. There were more changes in gene expression at 3 h than at 12 h post ethanol when ethanol alone was compared to the dextrose control. The same was true when SAMe was compared to SAMe + ethanol. Ethanol up regulated gene expression in most functional pathways at 3 h. However, when SAMe was fed with ethanol at 3 h, most pathways were down regulated. At 12 h, however, when ethanol was fed, the pathways were half up regulated and half down regulated. The same was true when SAMe + ethanol was fed. The expression of epigenetically important genes, such as BHMT and Foxn3, was up regulated 3 h post alcohol bolus. At 3 h, SAMe down regulated the expression of genes, such as BHMT, Mat2a, Jun, Tnfrs9, Ahcy 1, Tgfbr1 and 2, and Pcaf. At 12 h, the insulin signaling pathways were half down regulated by ethanol, which was partly prevented by SAMe. The MAPK pathway was up regulated by ethanol, but SAMe did not prevent this. In conclusion, profound changes in gene expression evolved between 3 h and 12 post ethanol bolus. SAMe down regulated these changes in gene expression at 3 h, and less so at 12 h.

Epigenetics of proteasome inhibition in the liver of rats fed ethanol chronically.

AIM: To examine the effects of ethanol-induced proteasome inhibition, and the effects of proteasome inhibition in the regulation of epigenetic mechanisms. METHODS: Rats were fed ethanol for 1 mo using the Tsukamoto-French model and were compared to rats given the proteasome inhibitor PS-341 (Bortezomib, Velcade(TM)) by intraperitoneal injection. Microarray analysis and real time PCR were performed and proteasome activity assays and Western blot analysis were performed using isolated nuclei. RESULTS: Chronic ethanol feeding caused a significant inhibition of the ubiquitin proteasome pathway in the nucleus, which led to changes in the turnover of transcriptional factors, histone-modifying enzymes, and, therefore, affected epigenetic mechanisms. Chronic ethanol feeding was related to an increase in histone acetylation, and it is hypothesized that the proteasome proteolytic activity regulated histone modifications by controlling the stability of histone modifying enzymes, and, therefore, regulated the chromatin structure, allowing easy access to chromatin by RNA polymerase, and, thus, proper gene expression. Proteasome inhibition by PS-341 increased histone acetylation similar to chronic ethanol feeding. In addition, proteasome inhibition caused dramatic changes in hepatic remethylation reactions as there was a significant decrease in the enzymes responsible for the regeneration of S-adenosylmethionine, and, in particular, a significant decrease in the betaine-homocysteine methyltransferase enzyme. This suggested that hypomethylation was associated with proteasome inhibition, as indicated by the decrease in histone methylation. CONCLUSION: The role of proteasome inhibition in regulating epigenetic mechanisms, and its link to liver injury in alcoholic liver disease, is thus a promising approach to study liver injury due to chronic ethanol consumption.

Chronic ethanol feeding alters hepatocyte memory which is not altered by acute feeding.

BACKGROUND: Gene expression changes in the liver after acute binge drinking may differ from the changes seen in chronic ethanol feeding in the rat. The changes in gene expression after chronic ethanol feeding may sensitize the liver to alcohol-induced liver damage, which is not seen after acute binge drinking. METHODS: To test this hypothesis, gene microarray analysis was performed on the livers of rats (n = 3) fed an acute binge dose of ethanol (6 g/kg body wt) and killed at 3 and 12 hours after ethanol by gavage. The gene microarrays were compared with those made on the liver of rats from a previous study, in which the rats were fed ethanol by intragastric tube for 1 month (36% of calories derived from ethanol). RESULTS: Microarray analysis data varied between the acute and chronic models in several important respects. Growth factors increased mainly in the chronic alcohol fed rat. Changes in enzymes involved in oxidative stress were noted only with chronic ethanol feeding. Gene expression of fat metabolism was increased only with chronic ethanol feeding. Most importantly, epigenetic related enzymes and acetylation and methylation of histones changed only after chronic ethanol feeding. CONCLUSIONS: The results support the concept that chronic ethanol ingestion induces altered gene expression as a result of changes in epigenetic mechanisms, where acetylation and methylation of histones were altered.

Epigenetic mechanisms regulate Mallory Denk body formation in the livers of drug-primed mice.

The mechanism of Mallory Denk body formation is still not fully understood, but growing evidence implicates epigenetic mechanisms in MDB formation. In a previous study the epigenetic memory of MDB formation remained intact for at least 4 months after withdrawal from the DDC diet. In the present study, mice were fed a diet containing DDC or a diet containing DDC and S-adenosylmethionine (SAMe) to investigate the epigenetic memory of MDB formation. DDC feeding caused an increase in histone 3 acetylation, a decrease in histone 3 trimethylation, and an increase in histone ubiquitinylation. The addition of SAMe to the DDC diet prevented the DDC induced decrease of H3K4 and H3K9 trimethylation and the increase in histone ubiquitinylation. Changes in histone modifying enzymes (HATs and HDACs), were also found in the liver nuclear extracts of the DDC/SAMe fed mice. Data mining of microarray analysis confirmed that gene expression changed with DDC refeeding, particularly the SAMe metabolizing enzymes, Mat2a, AMD, AHCY and Mthfr. SAMe supplementation prevented the decrease of AHCY and GNMT, and prevented the increase in Mthfr, which provides a mechanism to explain how DDC inhibits methylation of histones. The results indicate that SAMe prevented the epigenetic cellular memory involved in the MDB formation.

S-adenosylmethionine prevents Mallory Denk body formation in drug-primed mice by inhibiting the epigenetic memory.

UNLABELLED: In previous studies, microarray analysis of livers from mice fed diethyl-1,4-dihydro-2,4,6-trimethyl-3,5-pyridine decarboxylate (DDC) for 10 weeks followed by 1 month of drug withdrawal (drug-primed mice) and then 7 days of drug refeeding showed an increase in the expression of numerous genes referred to here as the molecular cellular memory. This memory predisposes the liver to Mallory Denk body formation in response to drug refeeding. In the current study, drug-primed mice were refed DDC with or without a daily dose of S-adenosylmethionine (SAMe; 4 g/kg of body weight). The livers were studied for evidence of oxidative stress and changes in gene expression with microarray analysis. SAMe prevented Mallory Denk body formation in vivo. The molecular cellular memory induced by DDC refeeding lasted for 4 months after drug withdrawal and was not manifest when SAMe was added to the diet in the in vivo experiment. Liver cells from drug-primed mice spontaneously formed Mallory Denk bodies in primary tissue cultures. SAMe prevented Mallory Denk bodies when it was added to the culture medium. CONCLUSION: SAMe treatment prevented Mallory Denk body formation in vivo and in vitro by preventing the expression of a molecular cellular memory induced by prior DDC feeding. No evidence for the involvement of oxidative stress in induction of the memory was found. The molecular memory included the up-regulation of the expression of genes associated with the development of liver cell preneoplasia.

Mallory body formation is associated with epigenetic phenotypic change in hepatocytes in vivo.

Microarrays were done on the livers of mice fed DDC for 10 weeks, withdrawn 1 month (DDC primed livers) and refed 6 days, and compared with mice fed the control diet. The expression of a large number of genes changed when DDC was fed or refed. A Venn diagram analysis identified 649 genes where gene expression was changed in the same direction. The epigenetic memory of the DDC primed liver involved an increase in the expression of ubiquitin D, alpha fetoprotein, connective tissue growth factor, integrin beta 2, DNA methyl transferase 3a and DNA damage-inducible 45 gamma. DNA methyl transferase 3b was down-regulated as was Cbp/p300. When DDC was refed, DNA methyltransferase and histone deacetylase were up-regulated as shown by microarray analysis. Histone3 lysine9 acetylation was increased by DDC and DDC refeeding and DNA methyltransferases were not changed as shown by Western blot analysis. The data suggest the concept that the epigenetic memory that explains why DDC primed hepatocytes form MBs in 7 days of DDC refeeding is primarily the result of epigenetic modifications of gene expression through changes in histone acetylation and methylation, as well as DNA methylation.

Gene expression patterns of the liver in response to alcohol: in vivo and in vitro models compared.

Two basic models of alcoholic liver disease pathogenesis exist, one in vivo and one in vitro. To justify the in vitro model, evidence is needed to show that it stimulates the in vivo model. Therefore, changes in gene expression caused by high ethanol level were compared using the two models. Many functional pathways were upregulated in both models. These included the insulin signaling pathway, TGFbeta signaling pathway, apoptosis, MAPK signaling pathway, wnt signaling pathway and apoptosis. Differences were found in the fatty acids synthesis pathway, which was upregulated in vivo; and glycosylation enzymes which were downregulated in vivo. Also, downregulated in vitro were beta oxidation by mitochondria and translation factors. Catalase and superoxide dismutase in mitochondria were upregulated in vitro. These two enzymes have antioxidant effects. In summary, remarkably similar responses to high alcohol levels in the form of changes in gene expression pathways were found in the in vivo and in vitro models tested.

Mallory body (cytokeratin aggresomes) formation is prevented in vitro by p38 inhibitor.

Microarray analysis of livers from mice fed diethyl-1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate (DDC) to induce Mallory body (MB) cytokeratin aggresome formation showed that gene expression for cellular adhesion molecules, cytokeratins, kinases and aggresome forming proteins were upregulated, when MBs were formed in vivo. This response was enhanced when the DDC was refed (mice fed DDC for 10 weeks followed by DDC withdrawal for 1 month, then refed DDC for 7 days). Immunofluorescent antibody staining of the MBs that formed showed that MAPK p38 was colocalized with ubiquitin and p62 in the MBs. To investigate further the mechanisms of MB formation, primary cultures derived from DDC primed mice and their controls were incubated for 6 days. Liver cells cultured for 3 h and 6 days were used for microarray analysis. At 3 h, there were no MBs formed, but MBs were numerous after 6 days of culture. At 3 h, the expression of a large number of genes was different when the control, and the DDC primed hepatocytes were compared, which indicates that the primed hepatocytes were phenotypically changed. The gene expression of many kinases including p38 was upregulated after 6 days where the gene expression of cytokeratins, adhesion molecules and aggresome forming proteins were upregulated when MBs formed. An inhibitor of p38 phosphorylation (SB202190) completely prevented MB formation. Western blot showed that phosphorylated p38 MAPK and total p38 were absent in vitro after the p38 inhibitor treatment. Immunostaining of 6-day DDC-primed hepatocyte cultures stained with antibodies to p62 and phospho-p38 MAPK showed that phosphorylated p38 MAPK was concentrated within the MBs. Antibodies to specific serine phosphorylated sites 73 and 431, located in cytokeratin 8, localized to Mallory bodies in vivo, indicating that cytokeratin 8 was hyperphosphorylated. The data supported the concept that MBs form as the result of hyperphosphorylation of cytokeratin 8 by p38.

The role of laminin-integrin signaling in triggering MB formation. An in vivo and in vitro study.

It is still unclear as to how hepatocytes perceive external factors and transduce the signals which initiate MB formation. To investigate this phenomenon, the model of MB formation in liver in vivo and in primary culture of hepatocytes derived from drug-primed mice was used. Control mice were fed the control diet (group 1). MBs were induced in the livers of mice fed diethyl-1, 4-dihydro-2, 4, 6-trimethyl-3, 5-pyridinedicarboxylate (DDC) for 10 weeks (group 2). The induced MBs completely disappeared after the withdrawal of DDC for 4 weeks (group 3). Newly formed MBs were numerous after DDC was refed for 1 week (group 4). Relative mRNA abundance was determined by quantitative real-time RT-PCR in the liver from the mice. The expression of integrin alpha(6) and beta(2) was significantly increased in the livers of DDC-treated (group 2) and drug refed mice (group 4), when compared with the livers from controls (group 1) and DDC-withdrawn (group 3) mice. The increased mRNA of these two integrin genes was associated with the increased expression of laminin (a ligand for integrin alpha(6)beta(1) and alpha(6)beta(4)), Icam1 (a ligand of alphaLbeta2), Src, MEKK1, and ERK1. Primary cultures of isolated DDC-primed hepatocytes (group 4 mice were withdrawn from DDC-CMZ for 4-6 weeks) produced significantly more MBs on laminin-coated coverslips compared with plastic uncoated, fibronectin-, collagen-, or fibrinogen-coated coverslips. U0126, an inhibitor of MEK1 protein, significantly reduced the phosphorylated forms of ERK1/2 and MB formation in vitro. In conclusion, the current study revealed an association between MB formation and integrin-mediated signaling in vivo. The data indicate that laminin-integrin signaling which activates ERK, triggered MB formation in vitro, and an inhibitor of the signaling cascade reduced MB formation.