Alcoholic liver disease (ALD) is one of the most serious medical consequences of chronic ethanol use. Alcohol can affect most organs of the body including brain, gastrointestinal tract, immune system, kidney, and pancreas, but its effect on the liver is better characterized than other organs. In the liver, alcohol consumption induces a spectrum of liver diseases ranging from steatosis, steatohepatitis, fibrosis, cirrhosis, and cancer. Multiple factors and cofactors have been implicated in the pathogenesis of alcohol-induced liver injury (recently reviewed in ).
METHIONINE METABOLISM IN THE LIVER
Ongoing investigations in many laboratories including ours have established that alterations in hepatic methionine metabolism are important factors contributing to the development of alcoholic liver injury (reviewed in [2–4]). The importance of the methionine metabolic cycle is primarily to conserve methionine so that S-adenosylmethionine (SAM), a universal methyl donor, can be generated. This reaction is catalyzed by methionine adenosyltransferase (MAT I and III), which are liver-specific enzymes. The generated SAM is then utilized for more than 85% of all transmethylation reactions that occur in this organ. In the SAM-dependent methylation reactions, a methyl group is transferred from SAM to a variety of acceptors to form methylated acceptors that play a vital role in maintaining important biological functions in the liver. The other product of the methylation reaction is S-adenosylhomocysteine (SAH), which is hydrolyzed to adenosine and homocysteine by S-adenosylhomocysteine hydrolase (SAHH). Although the equilibrium dynamics of the SAHH-catalyzed reaction strongly favor SAH synthesis over homocysteine synthesis, the efficient removal of homocysteine and adenosine by multiple pathways (indicated in Fig. 1) allows homocysteine synthesis to proceed so that it can be converted to methionine or to glutathione (GSH).
There are two pathways in the liver that participate equally in converting homocysteine to methionine. One pathway is folate-dependent and utilizes N5-methyltetrahydrofolate (MTHF), methionine synthase, and vitamin B12. MTHF is derived from endogenous 5,10, methylenetetrahydrofolate (CH2-THF) by way of its reductase, 5,10 methylenetetrahydrofolate reductase (MTHFR). Through the action of methionine synthase, a methyl group is transferred from MTHF to vitamin B12 to form methylcobalamine. The methylcobalamine in turn transfers the methyl group to homocysteine to produce methionine. The other pathway is folate independent and utilizes betaine (derived from the oxidation of choline) in a reaction catalyzed by betaine-homocysteine methyltransferase (BHMT) to form methionine and dimethylglycine. Key enzymes of the methionine cycle (except MAT III) share several properties including low Kms for sulphur-containing substrates, downregulation by dietary methionine, and inhibition by methionine and/or SAM.
Homocysteine can also be irreversibly catabolized through the transsulfuration pathway by the action of cystathionine β-synthase (CBS). The tight regulation of homocysteine metabolism depends on the different affinities of methionine synthase, BHMT, and CBS for homocysteine that favor methionine conservation at low and the transsulfuration pathway at high homocysteine concentrations. Through the action of vitamin B6-dependent CBS, homocysteine is converted to cystathionine, which is hydrolyzed via B6-dependent cystathionine γ-lyase to cysteine that is then incorporated into GSH. GSH, because of its thiol group, is very active in protecting the cell from metabolic oxidants that could cause cellular injury. Regulatory mechanisms favor transsulfuration when the supply of SAM is high. A redox sensitive regulation of CBS ensures increased production of cysteine under conditions of increased requirement for GSH . Given the critical roles of transmethylation reactions and GSH levels in determining various cellular processes and antioxidant status, respectively, changes in methionine metabolism could have far reaching effects.
EFFECT OF ETHANOL ON THE METHIONINE METABOLIC PATHWAY
Ethanol exposure impairs several of the multiple steps in methionine metabolism in the liver (reviewed in [2–4]). Briefly, ethanol consumption has been reported to inhibit the activity of methionine synthase, which is involved in remethylating homocysteine, causing increases in homocysteine and SAH levels and the lowering of the hepatocellular SAM : SAH ratio . By way of compensation in some species such as the Wistar rat, ethanol can also increase the activity of BHMT  that help maintain SAM levels. However, during extended periods of ethanol feeding (more than 2 months of feeding), this alternate pathway cannot be maintained due to the depletion of hepatic betaine stores resulting eventually in decreased hepatocellular SAM levels .
The decrease in hepatic levels of SAM can also occur because of the effect of prolonged alcohol feeding on MAT, the only enzyme responsible for synthesizing SAM from methionine. Effects ranging from diminished, normal, or increased MAT 1A expression have been reported (reviewed in ). Most authors, however, agree that the decreased activity of MAT observed after alcohol exposure is caused by alcohol-induced oxidative stress and reactive aldehydes that inactivate the liver-specific MAT (reviewed in ). Species such as C57Bl mice, that instead of an adaptive increase in BHMT, display reduced or no change in BHMT expression, exhibit more profound changes in ethanol-induced increases in homocysteine and decreases in SAM levels and SAM:SAH ratio , implying that BHMT plays an important role in maintaining liver homeostasis. A recent study proposed that reductive stress mediates the effects of ethanol on liver methionine metabolism as induction of the reductive stress achieved by increasing the NADH/NAD(+) ratio was associated with a marked decrease in SAM, an increase in SAH, and a lowering of hepatocellular SAM : SAH ratio [8▪].
CONSEQUENCES OF LOWERING THE HEPATOCELLULAR S-ADENOSYLMETHIONINE: S-ADENOSYLHOMOCYSTEINE RATIO
Changes in the SAM:SAH ratio affect the hepatic methylation capacity by affecting the activity of those SAM-dependent methyltransferases that have a lower Ki for SAH compared to the Km for SAM . As all methyltransferases have a broad range of cellular functions , there is a myriad of possible detrimental consequences of inhibiting the activity of many critical SAM-dependent methyltransferase in this organ as shown in Figure 2. The methyltransferases that we have studied in detail are phosphatidylethanolamine methyltransferase (PEMT), isoprenylcysteine carboxyl methyltransferase (ICMT), protein-L-isoaspartate methyltransferase (PIMT), and protein arginine methyltransferase (PRMT).
PEMT catalyzes three sequential transfers of methyl groups to phosphatidylethanolamine to generate phosphatidylcholine, an important constituent of very low-density lipoproteins (VLDLs) and cellular membranes (reviewed in ). ICMT is involved in the carboxyl methylation of isoprenylated proteins, a step that is crucial for activation of these proteins to enable their participation in antiapoptotic signaling pathways (reviewed in ). ICMT also plays a role in decreasing the expression of acyl-CoA:diacylglycerol acyltransferase 2, an enzyme that catalyzes the last step for triglyceride synthesis . PIMT is required for catalyzing the repair of isoaspartyl sites of spontaneously damaged proteins, thereby preventing the consequences of their accumulation as these atypical aspartyl residues compromise the biological function and modify the immunogenicity of proteins [11–13]. PRMT catalyzes the addition of monomethyl or dimethyl groups to the guanidine nitrogen atom of arginine. These methylated arginine proteins have been shown to regulate protein–protein interactions and signaling events . Studies in our laboratory have shown that decreased activity of these enzymes results in increased fat deposition , increased apoptosis [15,16], increased accumulation of damaged proteins [17,18], and altered signaling , respectively – all of which are hallmark features of alcoholic liver injury .
Studies recently conducted in our laboratory have shown that the lowered SAM:SAH ratio is also associated with impaired proteasome activity  and changes in mitochondrial respiratory chain proteome and function [22▪▪]. Both lysine methyltransferase and PRMT have been reported to methylate residues on proteasome subunits that appear to regulate proteasome function . It is not known yet whether an ethanol-induced alteration in mitochondrial proteome and function is linked to the disruption in the activity of specific mitochondrial SAM-dependent methyltransferase(s). Ethanol effects on specific DNA methyltransferases, histone methyltransferases, acetylases, and deacetylases also appear to be crucial for the epigenetic control of the expression of genes relevant to liver injury. (reviewed in ).
GENETIC INFLUENCE ON ALCOHOLIC LIVER INJURY PROGRESSION
As only 10–20% of chronic alcohol abusers develop cirrhosis, there is a strong consideration that genetics may contribute to disease development. Individuals with commonly known inherited genetic defects in enzymes of the methionine metabolic pathway such as MTHFR or CBS and mice generated with targeted disruption of these genes spontaneously develop hepatic abnormalities [23–26]. There are also reports of significant interactions of ethanol and genotype effects in the context of liver disease [26,27].
The relationship between genetics, alterations in the methionine metabolic pathway, and indices of liver injury was further recently explored in 14 different strains of mice following 4 weeks of intragastric alcohol feeding. Despite a high alcohol dose delivered to all inbred strains of mice, minimal injury developed in some strains. Of all the genes examined, the different expression pattern of methionine metabolic pathway genes correlated with the interstrain differences in susceptibility to alcohol-induced liver injury. Specifically, the resistant strains were characterized by an upregulation of MAT 1A, whereas the most susceptible strains exhibited reduced methionine synthase expression, elevated liver SAH and homocysteine levels, and lowered SAM : SAH ratio that were significantly and inversely correlated with liver injury scores [28▪▪]. These results indicate that genetics influences the pathogenesis of alcoholic liver injury and polymorphism in genes affecting the methionine metabolic and related pathways could be a significant factor in modulating disease development.
The importance of preserving SAM levels and maintaining SAM : SAH ratio for normal liver function is further reinforced by observations on the various knockouts of the methionine metabolic pathway that have been generated within the last decade. Mice lacking MAT 1A have reduced hepatic SAM content and spontaneously developed steatohepatitis and hepatocellular carcinoma (HCC) . However, mice lacking in glycine N-methyltransferase (GNMT) that have elevated SAM levels also developed liver steatosis, fibrosis, and HCC . GNMT is a methyltransferase that normally maintains SAM : SAH ratio by utilizing excess SAM to methylate glycine to sarcosine and is one of the few liver methyltransferases that are less sensitive to inhibition by lower SAM : SAH ratio . Recently, a BHMT knockout mouse model was generated that at 5 weeks of age exhibited severe hyperhomocystenemia, reduced hepatic SAM levels, elevated SAH content, lowered SAM : SAH ratio, reduced methylation potential, reduced phosphatidylcholine, decreased VLDL secretion, and increased hepatic triglyceride accumulation [31▪▪]. These observed results were not surprising in light of the studies conducted in our laboratory over the last decade on these investigated parameters. We have shown that chronic ethanol consumption by lowering hepatocellular SAM : SAH ratio reduces phosphatidylcholine synthesis, decreases VLDL secretion, and causes increased hepatic triglyceride accumulation (reviewed in [2,3]). BHMT knockout mice, at 1 year of age, developed HCC or carcinoma precursors [31▪▪] similar to what had been reported for MAT 1 and GNMT knockouts [29,30].
Collectively, these findings in MAT 1, GNMT, and BHMT knockout mice, along with the observations in mice deficient in CBS or MTHFR (which also exhibit altered levels of hepatic SAM levels) emphasize the crucial role of methionine and folate metabolism in maintaining normal liver function. These studies also indicate that either too much or too little hepatic SAM, in concert with abnormal SAM : SAH ratio and decreased methylation potential, could lead to the development of liver injury. It has also been suggested that aberrant methylation of DNA and histones could cause epigenetic modulation of critical metabolic and carcinogenic pathways. Thus, acquiring the knowledge of the functional polymorphisms in the various enzymes of the methionine metabolic and related pathways (e.g., PEMT, PNPLA3 ) in humans could be helpful in assessing how these genotypes interact with ethanol to exacerbate liver disease progression.
Studies with these knockouts have further revealed that the various enzymes and metabolites of the methionine pathway could have additional roles separate from their known characteristic function [33–36]. For example, GNMT has recently been shown to improve intracellular folate retention and bioavailability in the liver [37▪] in addition to its well characterized role in catabolizing excess SAM.
SERUM MARKERS OF ALTERED METHIONINE METABOLIC PATHWAY AND SEVERITY OF ALCOHOLIC LIVER DISEASE
As abnormal hepatic methionine metabolism plays a central role in the pathogenesis of experimental ALD, could serum levels of methionine metabolites in chronic alcohol abusers predict the risk and pathological severity of ALD? Medici et al. conducted a clinical study to examine this premise by determining serum levels of methione metabolites (vitamin B6, vitamin B12, folate, homocysteine, methionine, SAM, SAH, cystathionine, cysteine, α-aminobutyrate, glycine, serine, and dimethylglycine) and biochemical markers of liver function in 40 ALD patients (of which 24 had liver biopsies), 26 active drinkers without liver disease, and 28 healthy individuals. They observed that whereas serum homocysteine was elevated in all alcohol abusers, ALD patients had low vitamin B6 with elevated cystathionine and decreased α-aminobutyrate/cystathionine ratio, consistent with decreased activity of vitamin B6-dependent cystathionine γ-lyase. The α-aminobutyrate/cystathionine ratio predicted the presence of ALD, whereas cystathionine levels correlated with the stage of fibrosis in all ALD patients indicating that the homocysteine transsulfuration pathway in ALD may have important diagnostic and therapeutic implications.
Over the years, there have been studies focusing on treatment modalities utilizing metabolites of the methionine metabolic pathway. Both betaine and SAM treatment have been shown to attenuate ethanol-induced liver injury in a variety of experimental models of liver disease. However, many clinical trials have failed to demonstrate a definitive effect of SAM in the treatment of established ALD. A 2006 meta-analysis of results from 434 patients in nine studies found insufficient evidence to support or refute the clinical use of SAM in the treatment for ALD . As noted, none of these studies evaluated the effect of SAM treatment on liver histopathology, and the largest clinical trial did not exclude patients with coexistent chronic viral hepatitis [40▪▪]. Recently, Medici et al.[40▪▪] undertook a double-blinded, placebo-controlled, 24-week clinical trial to determine the efficacy of SAM in the treatment for ALD and to define its mechanistic effects on hepatic methionine metabolism. They reported that 24-week treatment with SAM at the same dose that was used in prior studies is not effective in patients with moderately severe ALD according to Model End Stage Liver Disease scores. An increase in fasting serum SAM levels over timed intervals in the SAM treatment group and an overall improvement of aspartate transaminase, alanine transaminase, and bilirubin levels after 24 weeks of treatment were seen in the entire cohort. However, there were no differences between the treatment groups in any clinical or biochemical parameters nor any intragroup or intergroup differences or changes in liver histopathology scores for steatosis, inflammation, fibrosis, and Mallory-Denk hyaline bodies. They concluded that whereas abstinence improved liver function, 24 weeks of therapy with SAM was no more effective than placebo in the treatment for ALD [40▪▪]. The authors noted that a limitation of their study was that there were relatively small numbers of patients, which resulted from the combination of stringent inclusion criteria and the high, 30%, dropout rate secondary to alcoholic recidivism, which is similar to other treatment trials in outpatients with ALD [40▪▪].
Future treatment modalities for ALD should consider loss-of-function polymorphisms in the enzymes of the methionine metabolic and related pathways to account for significant interactions these gene variants could have with chronic ethanol abuse to generate more severe liver injury. Further, future new treatment modalities for ALD should consider supplementation with betaine, which may prove to be a promising protective agent. Further, any therapeutic regimen should include a larger number of compliant patients and longer periods of treatment for a successful outcome.
This study is the result of work supported with resources and the use of facilities through the VA Nebraska-Western Iowa Healthcare System. Research by the author cited in this study has been supported by a Merit Review Grant from the Biomedical Laboratory Research and Development, Office of Research and Development, Department of Veterans Affairs, Veterans Health Administration.
Conflicts of interest
There are no conflicts of interest.
REFERENCES AND RECOMMENDED READING
Papers of particular interest, published within the annual period of review, have been highlighted as:
▪ of special interest
▪▪ of outstanding interest
Additional references related to this topic can also be found in the Current World Literature section in this issue (p. 117).
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GNMT expression in hepatocytes could improve folate status and that normal GNMT function should be considered as a factor during antifolate chemotherapy or immunosuppressive treatments.
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Oral SAM was ineffective in treatment for ALD in this clinical study, which suggests that subsequent studies with a larger number of compliant patients and longer periods of treatment with SAM and betaine might produce a different outcome.