The cyclic pattern of blood alcohol levels during continuous ethanol feeding in rats: The effect of feeding S-adenosylmethionine

S-adenosylmethionine (SAMe), the major methyl donor for DNA and histone methylation was fed with ethanol for 1 month in order to modify the effects of ethanol on rat liver. The following parameters were studied to determine the effects of SAMe; liver histology, the blood alcohol cycle (BAL), changes in gene expression mined from microarray analysis, changes in histone methylation, changes in liver SAMe levels and its metabolites and ADH. SAMe changed the type of fatty liver, reduced liver ALT levels and prevented the BAL cycle caused by intragastric ethanol feeding. Microarray analysis showed that SAMe feeding prevented most of the changes in gene expression induced by ethanol feeding, presumably by inducing H3K27me3 and gene silencing. H3K27me3 was significantly increased by SAMe with or without ethanol feeding. It is concluded that SAMe feeding stabilized global gene expression so that the changes in gene expression involved in the blood alcohol cycle were prevented.     

SAMe prevents the up regulation of toll-like receptor signaling in Mallory–Denk body forming hepatocytes

Mallory–Denk body (MDB) formation is a component of alcoholic and non alcoholic hepatitis. In the present study, the role of the toll-like receptor (TLR) signaling pathway was investigated in the mechanism of MDB formation in the DDC-fed mouse model. Microarray analysis data mining, performed on the livers of drug-primed mice refed DDC, showed that TLR2/4 gene expression was significantly up regulated by DDC refeeding. SAMe supplementation prevented this up regulation and prevented the formation of MDBs. qRT-PCR analysis confirmed these results. TLR2/4 activates the adapter protein MyD88. The levels of MyD88 were increased by DDC refeeding. The increase of MyD88 was also prevented by SAMe supplementation. Results showed that MyD88-independent TLR3/4-TRIF-IRF3 pathway was not up regulated in the liver of DDC refed mice. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is the downstream protein recruited by the MyD88/IRAK protein complex, and is involved in the regulation of innate immune responses. Results showed a significant increase in the levels of TRAF-6. TRAF-6 activation leads to activation of NFkB and the mitogen-activated protein kinase (MAPK) cascade. The TRAF-6 increase was ameliorated by SAMe supplementation. These results suggest that DDC induces MDB formation through the TLR2/4 and MyD88-dependent signaling pathway. In conclusion, SAMe blocked the over-expression of TLR2/4, and their downstream signaling components MyD88 and TRAF-6. SAMe prevented the DDC-induced up regulation of the TLR signaling pathways, probably by preventing the up regulation of INF-γ receptors by DDC feeding. INFγ stimulates the up regulation of TLR2. The ability of SAMe feeding to prevent TLR signaling up regulation has not been previously described.     

Betaine feeding prevents the blood alcohol cycle in rats fed alcohol continuously for 1 month using the rat intragastric tube feeding model

Background

Blood alcohol levels (BAL) cycle up and down over a 7–8 day period when ethanol is fed continuously for one month in the intragastric tube feeding rat model (ITFRM) of alcoholic liver disease. The cycling phenomenon is due to an alternating increase and decrease in the metabolic rate. Recently, we found that S-adenosyl-methionine (SAMe) fed with alcohol prevented the BAL cycle.

Method

Using the ITFRM we fed rats betaine (2 g/kg/day) with ethanol for 1 month and recorded the daily 24 h urine ethanol level (UAL) to measure the BAL cycle. UAL is equivalent to BAL because of the constant ethanol infusion. Liver histology, steatosis and BAL were measured terminally after 1 month of treatment. Microarray analysis was done on the mRNA extracted from the liver to determine the effects of betaine and alcohol on changes in gene expression.

Results

Betaine fed with ethanol completely prevented the BAL cycle similar to SAMe. Betaine also significantly reduced the BAL compared to ethanol fed rats without betaine. This was also observed when SAMe was fed with ethanol. The mechanism involved in both cases is that SAMe is required for the conversion of epinephrine from norepinephrine by phenylethanolamine methyltransferase (PNMT). Epinephrine is 5 to 10 fold more potent than norepinephrine in increasing the metabolic rate. The increase in the metabolic rate generates NAD, permitting ADH to increase the oxidation of alcohol. NAD is the rate limiting factor in oxidation of alcohol by alcohol dehydrogenase (ADH). This explains how SAMe and betaine prevented the cycle. Microarray analysis showed that betaine feeding prevented the up regulation of a large number of genes including TLR2/4, Il-1b, Jax3, Sirt3, Fas, Ifngr1, Tgfgr2, Tnfrsf21, Lbp and Stat 3 which could explain how betaine prevented fatty liver.

Conclusion

Betaine feeding lowers the BAL and prevents the BAL cycle by increasing the metabolic rate. This increases the rate of ethanol elimination by generating NAD.     

S-adenosylmethionine decreases the peak blood alcohol levels 3 h after an acute bolus of ethanol by inducing alcohol metabolizing enzymes in the liver

Introduction

An alcohol bolus causes the blood alcohol level (BAL) to peak at 1–2 h post ingestion. The ethanol elimination rate is regulated by alcohol metabolizing enzymes, primarily alcohol dehydrogenase (ADH1), acetaldehyde dehydrogenase (ALDH), and cytochrome P450 (CYP2E1). Recently, S-adenosylmethionine (SAMe) was found to reduce acute BALs 3 h after an alcohol bolus. The question, then, was: what is the mechanism involved in this reduction of BAL by feeding SAMe? To answer this question, we investigated the changes in ethanol metabolizing enzymes and the epigenetic changes that regulate the expression of these enzymes during acute binge drinking and chronic drinking.

Methods

Rats were fed a bolus of ethanol with or without SAMe, and were sacrificed at 3 h or 12 h after the bolus.

Results

RT-PCR and Western blot analyses showed that SAMe significantly induced ADH1 levels in the 3 h liver samples. However, SAMe did not affect the changes in ADH1 protein levels 12 h post bolus. Since SAMe is a methyl donor, it was postulated that the ADH1 gene expression up regulation at 3 h was due to a histone modification induced by methylation from methyl transferases. Dimethylated histone 3 lysine 4 (H3K4me2), a modification responsible for gene expression activation, was found to be significantly increased by SAMe at 3 h post bolus.

Conclusion

These results correlated with the low BAL found at 3 h post bolus, and support the concept that SAMe increased the gene expression to increase the elimination rate of ethanol in binge drinking by increasing H3K4me2.     

Sirt1 is involved in energy metabolism: the role of chronic ethanol feeding and resveratrol

Sirt1, a deacetylase involved in regulating energy metabolism in response to calorie restriction, is up regulated after chronic ethanol feeding using the intragastric feeding model of alcohol liver disease. PGC1α is also up regulated in response to ethanol. These changes are consistent with activation of the Sirt1/PGC1α pathway of metabolism and aging, involved in alcohol liver disease including steatosis, necrosis and fibrosis of the liver. To test this hypothesis, male rats fed ethanol intragastrically for 1 month were compared with rats fed ethanol plus resveratrol or naringin. Liver histology showed macrovesicular steatosis caused by ethanol and this change was unchanged by resveratrol or naringin treatment. Necrosis occurred with ethanol alone but was accentuated by resveratrol treatment, as was fibrosis. The expression of Sirt1 and PGC1α was increased by ethanol but not when naringin or resveratrol was fed with ethanol. Sirt3 was also up regulated by ethanol but not when resveratrol was fed with ethanol. These results support the concept that ethanol induces the Sirt1/PGC1α pathway of gene regulation and both naringin and resveratrol prevent the activation of this pathway by ethanol. However, resveratrol did not reduce the liver pathology caused by chronic ethanol feeding.     

The regulation of non-coding RNA expression in the liver of mice fed DDC

Mallory-Denk bodies (MDBs) are found in the liver of patients with alcoholic and chronic nonalcoholic liver disease, and hepatocellular carcinoma (HCC). Diethyl 1,4-dihydro-2,4,6,-trimethyl-3,5-pyridinedicarboxylate (DDC) is used as a model to induce the formation of MDBs in mouse liver. Previous studies in this laboratory showed that DDC induced epigenetic modifications in DNA and histones. The combination of these modifications changes the phenotype of the MDB forming hepatocytes, as indicated by the marker FAT10. These epigenetic modifications are partially prevented by adding to the diet S-adenosylmethionine (SAMe) or betaine, both methyl donors. The expression of three imprinted ncRNA genes was found to change in MDB forming hepatocytes, which is the subject of this report. NcRNA expression was quantitated by real-time PCR and RNA FISH in liver sections. Microarray analysis showed that the expression of three ncRNAs was regulated by DDC: up regulation of H19, antisense Igf2r (AIR), and down regulation of GTL2 (also called MEG3). S-adenosylmethionine (SAMe) feeding prevented these changes. Betaine, another methyl group donor, prevented only H19 and AIR up regulation induced by DDC, on microarrays. The results of the SAMe and betaine groups were confirmed by real-time PCR, except for AIR expression. After 1 month of drug withdrawal, the expression of the three ncRNAs tended toward control levels of expression. Liver tumors that developed also showed up regulation of H19 and AIR. The RNA FISH approach showed that the MDB forming cells' phenotype changed the level of expression of AIR, H19 and GTL2, compared to the surrounding cells. Furthermore, over expression of H19 and AIR was demonstrated in tumors formed in mice withdrawn for 9 months. The dysregulation of ncRNA in MDB forming liver cells has been observed for the first time in drug-primed mice associated with liver preneoplastic foci and tumors.     

Changes in IL12A methylation pattern in livers from mice fed DDC

Mallory-Denk body (MDB) formation is a component of alcoholic and non alcoholic hepatitis. Proteins of the TLR pathway were shown to be involved in the formation of MDBs, in mice fed DDC. TLR genes are upregulated and SAMe supplementation prevents this up regulation and prevented the formation of MDBs. DNA of livers from control mice, from mice fed DDC 10weeks, refed 1week with DDC and with DDC+SAMe were extracted and used to study the methylation pattern of genes involves in the TLR pathway. A PCR array was used to analyze it. Using PCR arrays for the mouse TLR pathway,24 genes were found whose expression of IL12A was regulated by the methylation of its gene. DDC fed for 10weeks reduced the methylation of the IL12A gene expression. This expression was also reduced when DDC was refed. However, when SAMe was fed, the intermediate level methylation of IL12A was up regulated to the intermediate level and the methylation of the promoter decreased compared to DDC refeeding or DDC 10weeks. IL12A is known to induce the production of IFNg by NK and L(T). We showed in a previous publication that IFNg is one of the major cytokines involved in the induction of MDB formation. The low expression of IL12A associated with the intermediate methylation of its promoter could explain one step in the mechanism which leads to the formation of MDBs.     

SAMe treatment prevents the ethanol-induced epigenetic alterations of genes in the Toll-like receptor pathway

Prior studies showed that Toll-like receptor (TLR) signaling pathway genes were upregulated in the liver of rats fed ethanol, but not in rats fed ethanol plus S-adenosylmethionine (SAMe). These results were obtained using a PCR microplate array analysis for TLRs and associated proteins such as proinflammatory cytokines and chemokine mRNA levels. A large number of genes were upregulated by the ethanol diet, but not the ethanol plus SAMe diet. In the present study, using the same experimental rat livers, DNA methylation analysis was done by using an Epitect Methyl DNA Restriction Kit (Qiagen, 335451) (24 genes). The results of all the genes combined show a highly significant increase in methylation in the ethanol-fed group of rats, but not in the dextrose-fed, SAMe-fed or ethanol plus SAMe-fed groups of rats. There was also an increase in DNA methylation in rats with high blood alcohol levels compared to a rat with a low blood alcohol level. The individual genes that were upregulated as indicated by the increased mRNA measured by qPCR correlated positively with the increased methylation of the DNA of the corresponding genes as follows: Cd14, Hspa1a, Irf1, Irak1, Irak2, Map3k7, Myd88, Pparα, Ripk2, Tollip and Traf6.     

Betaine prevents Mallory-Denk body formation in drug-primed mice by epigenetic mechanisms

Previous studies showed that S-Adenosylmethionine (SAMe) prevented MDB formation and the hypomethylation of histones induced by DDC feeding. These results suggest that formation of MDBs is an epigenetic phenomenon. To further test this theory, drug-primed mice were fed the methyl donor, betaine, together with DDC, which was refed for 7 days. Betaine significantly reduced MDB formation, decreased the liver/body weight ratio and decreased the number of FAT10 positive liver cells when they proliferate in response to DDC refeeding. Betaine also significantly prevented the decreased expression of BHMT, AHCY, MAT1a and GNMT and the increased expression of MTHFR, caused by DDC refeeding. S-Adenosylhomocysteine (SAH) levels were reduced by DDC refeeding and this was prevented by betaine. The results support the concept that betaine donates methyl groups, increasing methionine available in the cell. SAMe metabolism was reduced by the decrease in GNMT expression, which prevented the conversion of SAMe to SAH. As a consequence, betaine prevented MDB formation and FAT10 positive cell proliferation by blocking the epigenetic memory expressed by hepatocytes. The results further support the concept that MDB formation is the result of an epigenetic phenomenon, where a change in methionine metabolism causes global gene expression changes in hepatocytes.     

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.     

Experimental rat model for alcohol‐induced osteonecrosis of the femoral head

Alcohol‐induced osteonecrosis of the femoral head (ONFH) is observed in alcohol abusers and patients with alcoholic fatty liver disease. It has been reported that Toll‐like receptor 4 (TLR4) signalling plays a crucial role in the pathogenesis of alcoholic fatty liver disease. We previously reported a corticosteroid‐induced ONFH rat model, and suggested that TLR4 signalling contributes to the pathogenesis of ONFH. Thus, it is thought that the pathogenesis of alcohol‐induced ONFH is probably similar to that of corticosteroid‐induced ONFH. The aim of this study was to develop a new animal model for alcohol‐induced ONFH and to evaluate the relationship between the pro‐inflammatory response via TLRs and the development of ONFH in rats. Male Wistar rats were fed a Lieber–DeCarli liquid diet containing 5% ethanol (experimental group) or dextran (control group) for 1–24 weeks. Histopathological and biochemical analyses were performed. Feeding the ethanol‐containing liquid diet resulted in the development of ONFH with hepatic steatosis, hepatic dysfunction and hyperlipidaemia, whereas feeding the dextran‐containing diet did not cause ONFH. However, we could not recognize any relationship between the pro‐inflammatory response via TLR4 and the development of alcohol‐induced ONFH. Thus in this study we have developed a new rat model for alcohol‐induced ONFH based on the feeding of an ethanol liquid diet. ONFH was observed within seven days from the start of feeding with 5% ethanol‐containing liquid diet. Although this was linked to hepatic steatosis, a TLR4 association was not a feature of this model.     

Changes in S-adenosylmethionine and GSH homeostasis during endotoxemia in mice

Endotoxemia participates in the pathogenesis of many liver injuries. Lipopolysaccharide (LPS) was shown to inactivate hepatic methionine adenosyltransferase (MAT), the enzyme responsible for S-adenosylmethionine (SAMe) biosynthesis. SAMe treatment was shown to prevent the LPS-induced increase in tumor necrosis factor-alpha, which may be one of its beneficial effects. SAMe is also an important precursor of glutathione (GSH) and GSH was shown to ameliorate LPS-induced hepatotoxicity. The aims of this work were to examine changes in SAMe and GSH homeostasis during endotoxemia and the effect of SAMe. Mice received SAMe or vehicle pretreatment followed by LPS and were killed up to 18 h afterward. Unexpectedly, we found hepatic SAMe level increased 67% following LPS treatment while S-adenosylhomocysteine level fell by 26%, suggesting an increase in SAMe biosynthesis and/or block in transmethylation. The mRNA and protein levels of MAT1A and MAT2A were increased following LPS. However, despite increased MAT1A expression, MAT activity remained inhibited 18 h after LPS. The major methyltransferase that catabolizes hepatic SAMe is glycine N-methyltransferase, whose expression fell by 65% following LPS. Hepatic GSH level fell more than 50% following LPS, coinciding with a comparable fall in the mRNA and protein levels of glutamate-cysteine ligase (GCL) catalytic (GCLC) and modifier subunits (GCLM). SAMe pretreatment prevented the fall in GCLC and attenuated the fall in GCLM expression and GSH level. SAMe pretreatment prevented the LPS-induced increase in plasma alanine transaminases levels but not the LPS-induced increase in hepatic mRNA levels of proinflammatory cytokines. It further enhanced LPS-induced increase in interleukin-10 mRNA level. Taken together, the hepatic response to LPS is to upregulate MAT expression and inhibit SAMe utilization. GSH is markedly depleted largely due to lower expression of GCL. Interestingly, SAMe treatment prevented the fall in GCL and helped to preserve the GSH store and prevent liver injury.     

Toll-like receptor 2 mediates inflammatory cytokine induction but not sensitization for liver injury by Propioni- bacterium acnes

Recognition of Gram-positive bacteria by Toll-like receptor 2 (TLR2) induces activation of proinflammatory pathways. In mice, sensitization with the Gram-positive Propionibacterium acnes followed by a challenge with the TLR4 ligand, lipopolysaccharide (LPS), results in fulminant hepatic failure. Here, we investigated the role of TLR2 in liver sensitization to LPS-induced injury. Stimulation of Chinese hamster ovary cells and peritoneal macrophages with heat-killed P. acnes required expression of TLR2 but not of TLR4, suggesting that P. acnes was a TLR2 ligand. Cell activation by P. acnes was myeloid differentiation primary-response protein 88 (MyD88)-dependent, and it was augmented by coexpression of CD14 in mouse peritoneal macrophages. In vitro, P. acnes behaved as a TLR2 ligand and induced TLR4 hetero- and TLR2 homotolerance in peritoneal macrophages. In vivo priming of wild-type mice with P. acnes, but not with the selective TLR2 ligands peptidoglycan and lipotheicoic acid, resulted in hepatocyte necrosis, hyperelevated serum levels of tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-6, interferon-gamma (IFN-gamma), and IL-12 (p40/p70), and increased RNA expression of proinflammatory cytokines (IL-12p40, IL-1alpha, IL-6, IL-1beta, IL-18, IFN-gamma) in the liver after a LPS challenge. Furthermore, P. acnes priming sensitized TLR2-deficient (TLR2-/-) but not MyD88-/- mice to LPS-induced injury, evidenced by hepatocyte necrosis, increased levels of serum TNF-alpha, IFN-gamma, IL-6, and liver proinflammatory cytokine mRNA expression. IFN-gamma, a cytokine sensitizing to endotoxin, was induced by P. acnes in splenocytes of TLR2-/- and TLR9-/- but not MyD88-/- mice. These results suggest that although P. acnes triggers TLR2-mediated cell activation, TLR2-independent but MyD88-dependent mechanisms mediate in vivo sensitization by P. acnes for LPS-induced liver injury.     

CD300a and CD300f differentially regulate the MyD88 and TRIF-mediated TLR signalling pathways through activation of SHP-1 and/or SHP-2 in human monocytic cell lines

CD300a, a membrane protein expressed on myeloid lineages and specific subsets of CD4(+) T cells, has been reported to have inhibitory activities in cellular activation. However, the role of CD300a in Toll-like receptor (TLR) -mediated macrophage activation has not been investigated. The human monocytic cell lines THP-1 and U937 were stimulated with various TLR ligands after triggering of CD300a with specific monoclonal antibody. Interestingly, CD300a blocked TLR4-mediated and TLR9-mediated expression of pro-inflammatory mediators without affecting TLR3-mediated events. In contrast, CD300f, another member of the CD300 family, blocked the activation of cells induced by all TLR ligands. A transient transfection assay using luciferase reporter gene under the regulation of nuclear factor-κB binding sites indicated that co-transfection of CD300f blocked reporter expression induced by over-expression of both myeloid differentiation factor 88 (MyD88) and toll-interleukin 1 receptor-domain-containing adapter-inducing interferon-β (TRIF), whereas CD300a blocked only MyD88-induced events. Synthetic peptides representing immunoreceptor tyrosine-based inhibitory motifs of CD300a or CD300f mimicked the differential inhibition patterns of their original molecules. The use of various signalling inhibitors and Western blotting analysis revealed that TLR9/MyD88-mediated signalling was regulated mainly by SH2-containing tyrosine phosphatase 1 (SHP-1), which could be activated by CD300a or CD300f. In contrast, regulation of the TLR3/TRIF-mediated pathway required the combined action of SHP-1 and SHP-2, which could be accomplished by CD300f but not CD300a. These data indicate that CD300a and CD300f regulate the MyD88 and TRIF-mediated TLR signalling pathways through differential activation of SHP-1 and SHP-2.     

TRAM is specifically involved in the Toll-like receptor 4-mediated MyD88-independent signaling pathway

Recognition of pathogens by Toll-like receptors (TLRs) triggers innate immune responses through signaling pathways mediated by Toll-interleukin 1 receptor (TIR) domain-containing adaptors such as MyD88, TIRAP and TRIF. MyD88 is a common adaptor that is essential for proinflammatory cytokine production, whereas TRIF mediates the MyD88-independent pathway from TLR3 and TLR4. Here we have identified a fourth TIR domain-containing adaptor, TRIF-related adaptor molecule (TRAM), and analyzed its physiological function by gene targeting. TRAM-deficient mice showed defects in cytokine production in response to the TLR4 ligand, but not to other TLR ligands. TLR4- but not TLR3-mediated MyD88-independent interferon-beta production and activation of signaling cascades were abolished in TRAM-deficient cells. Thus, TRAM provides specificity for the MyD88-independent component of TLR4 signaling.     

The effect of propranolol on gene expression during the blood alcohol cycle of rats fed ethanol intragastrically

Propranolol, a beta adrenergic blocker prevents the blood alcohol (BAL) cycle in rats fed ethanol intragastrically at a constant rate by preventing the cyclic changes in the metabolic rate caused by fluctuating levels of norepinephrine released into the blood. The change in the rate of metabolism changes the rate of alcohol elimination in the blood which causes the BAL to cycle. Microarray analysis of the livers from the rats fed ethanol and propranolol showed similar changes in clusters of functionally related gene expressions. The controls and the trough of the cycle differed dramatically from the cluster pattern seen in the rats at the peaks of the blood alcohol cycle. The changes in gene expression induced by ethanol were similar when propranolol was fed without ethanol especially with the changes in the kinases and phosphatases, Toll-like receptor signaling and cytokine-cytokine receptor interaction were also changed.The changes in gene expression caused by ethanol and propranolol feeding are alike probably because both drugs induce β adrenergic receptor desensitization.