February 1998 EDITORIALS 403 12. Lehmann PV, Forsthuber T, Miller A, Sercarz EE. Spreading of T-cell autoimmunity to cryptic determinants of an autoantigen. Nature 1992;358:155 157. 13. Manns M, Gerken G, Kyriatsoulis A, Staritz M, Meyer zum Büshenfelde KH. Characterization of a new subgroup of autoimmune chronic active hepatitis by autoantibodies against a soluble liver antigen. Lancet 1987;1:292 294. 14. Hayes JD, Pulford DJ. The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance. Crit Rev Biochem Mol Biol 1995;30:445 600. 15. Horsfall AC. Molecular mimicry and autoantigens in connective tissue diseases. Mol Biol Rep 1992;16:139 147. 16. Maul GG, Jiminez SA, Riggs E, Ziemnicka-Kotula D. Determination of an epitope of the diffuse systemic sclerosis marker antigen DNA topoisomerase I: sequence similarities with retroviral p30gag protein suggests a possible cause for autoimmunity in systemic sclerosis. Proc Natl Acad Sci USA 1989;86:8492 8496. 17. Query CC, Keene JD. A human autoimmune protein associated with U1 RNA containing a region of homology that is crossreactive with retroviral p30gag antigen. Cell 1987;51:211 220. 18. Scofield RH, Harley JB. Autoantigenicity of Ro/SSA antigen related to a nucleocapsid protein of vesicular stomatitis virus. Proc Natl Acad Sci USA 1991;88:3343 3347. 19. Kasturi KN, Hatakeyama A, Spiera H, Bonsa CA. Antifibrallin autoantibodies present in systemic sclerosis and other connective diseases interact with similar epitopes. J Exp Med 1995;181: 1027 1036. 20. McNeilage LJ, Macmillan EM, Whittingham SF. Mapping of epitopes on the La(SS-B) autoantigen of primary Sjögren s syndrome: identification of a cross-reactive epitope. J Immunol 1990;145:3829 3835. 21. Kohsaka H, Yamamoto K, Fuji H, Miura H, Nishioka K, Miyamoto T. Fine epitope mapping of the human SS-B(La) protein. Identification of a distinct autoepitope homologous to a viral gag polypeptide. J Clin Invest 1990;85:1566 1574. 22. Harris DP, Vordermeier HM, Singh M, Moreno C, Jurcevic S, Ivanyi J. Cross-recognition by T cells of an epitope shared by two unrelated mycobacterial antigens. Eur J Immunol 1995;25:3173 3179. Address correspondence to: Judy Van de Water, M.D., Division of Rheumatology, Allergy and Clinical Immunology, University of California Davis, School of Medicine, Davis, California 95616. 1998 by the American Gastroenterological Association 0016-5085/98/$3.00 Methionine Adenosyltransferase and Liver Disease: It s All About SAM See article on page 364. Methionine adenosyltransferase (MAT, also known as S-adenosylmethionine synthetase and AdoMet synthetase) is the enzyme responsible for the synthesis of S-adenosyl-L-methionine (SAM) using methionine and adenosine triphosphate (ATP). 1 SAM is the principal biological methyl donor, the precursor of aminopropyl groups used in polyamine biosynthesis; in the liver, SAM is also a precursor of glutathione through its conversion to cysteine via the transsulfuration pathway. 1 SAM contains a sulfonium ion that makes it a high-energy reagent and can easily transfer its methyl group to a large variety of acceptor substrates including nucleic acids, proteins, phospholipids, biological amines, and a long list of small molecules. 2 Given the critical role of methylation in determining various cellular processes ranging from gene expression to membrane fluidity, any alteration in the availability of SAM may have profound effects on cellular growth, differentiation, and function. In mammals, two different genes, MAT1A and MAT2A, encode for two homologous MAT catalytic subunits, 1 and 2 3 5 (see Kotb et al. 5 for a consensus nomenclature of the mammalian MAT genes and gene products). MAT1A is expressed only in the liver and encodes the 1 subunit found in two native MAT isozymes, which are either a dimer (MATIII) or tetramer (MATI) of this single subunit. 5 MAT2A encodes for a catalytic subunit ( 2) found in a native MAT isozyme (MATII), which is widely distributed. 3,5 MAT2A and its gene product also predominate in the fetal liver and is progressively replaced by MAT1A during development. 6,7 In hepatocarcinogenesis, there is a switch in the gene expression from MAT1A to MAT2A, suggesting that the expression of MAT2A in liver correlates with more rapid cell growth. 8,9 Different isoforms of MAT differ in kinetic and regulatory properties. Of the two liver-specific MAT isoforms, MATI, the tetramer, exhibits much lower Michaelis constant (K m ) for its substrates (the highaffinity K m for methionine, 36 mol/l; K m for ATP, 300 mol/l) than MATIII, the dimer (K m for methionine, 700 mol/l; K m for ATP, 1 mmol/l). 2,10 Because the hepatic concentration of methionine is approximately 50 80 mol/l, 1,11 the specific activity of MATI is likely to be tenfold higher than that of MATIII under physiological conditions. 11
404 EDITORIALS GASTROENTEROLOGY Vol. 114, No. 2 Liver-specific MAT plays an essential role in methionine metabolism. Up to 48% of methionine is converted to 6 8 g of SAM in the liver daily. Under normal conditions, SAM can be decarboxylated to be used as a precursor for the synthesis of polyamines, but most of it is used in transmethylation reactions in which methyl groups are added to compounds and SAM is converted to S-adenosylhomocysteine (SAH). 2 SAH is a potent competitive inhibitor of transmethylation reactions; both an increase in SAH level and a decrease in the SAM to SAH ratio are known to inhibit transmethylation reactions. 1,2 For this reason, the removal of SAH is essential. The reaction that converts SAH to homocysteine and adenosine is reversible and catalyzed by SAH hydrolase. 1,2 In fact, the thermodynamics favor synthesis of SAH. 1 In vivo, the reaction proceeds in the direction of hydrolysis only if the products, adenosine and homocysteine, are rapidly removed. 1,2 In the liver, there are three pathways that metabolize homocysteine. One is the transsulfuration pathway that converts homocysteine to cysteine. Two other enzymatic reactions resynthesize methionine from homocysteine; one is catalyzed by methionine synthase and the other by betaine homocysteine methyltransferase. 1,2 If homocysteine metabolism is impaired in these pathways, SAH level may increase and the ratio of SAM to SAH may decrease, leading to inhibition of transmethylation reactions. It has long been realized that patients with cirrhosis of different causes often have hypermethioninemia and delayed plasma clearance of methionine after intravenous injection. 12,13 Subsequent studies showed that the hypermethioninemia in cirrhotic patients can be attributed to a 50% 60% decrease in the activity of hepatic MAT. 11,14 Interestingly, the decrease in MAT activity was restricted to the tetramer without affecting the dimer. 11 Because the intracellular reduced glutathione (GSH) ratio to oxidized glutathione (GSSG) is known to be an important modulator of the oligomeric equilibrium of the hepatic MAT isozymes, 10 it had been proposed that the selective loss of the tetramer was due to a reduction in the GSH/GSSG ratio. 2 More recent studies from Mato et al. suggest that oxidative stress may also play an important role in the selective loss of the tetramer (see below). Decreased hepatic MAT activity has also been found in different animal models of hepatic injury. 2,15 19 In most of these models, decreased hepatic SAM levels paralleled decreased MAT activities. Because SAM is an important precursor for GSH synthesis in the liver, a significant decrease in hepatic GSH was often noted. 2,19 A decrease in hepatic GSH may jeopardize antioxidant defense and lead to further reduction in MAT activity. In all of these models of hepatic injury except hypoxic injury, 17,18 decreased MAT activity occurred in the absence of any change in the steady-state MAT1A messenger RNA (mrna) levels. 2,3,16 Thus, depending on the model of hepatopathology, different mechanisms may be involved. In addition to abnormal MAT activity, decreased methionine synthase activity has also been reported in several animal models of alcoholic liver disease. 20,21 In the rat, decreased methionine synthase activity is accompanied by increased betaine homocysteine methyltransferase activity, thus conserving the methionine pool at the expense of betaine. 20 In this model, betaine supplementation greatly increased the hepatic SAM level and protected against fatty infiltration of the liver. In micropigs, long-term alcohol feeding led to decreased hepatic methionine synthase activity as in the rat. 21 Hepatic SAM level remained unchanged, SAH level increased (perhaps because of lack of compensatory increase in the betaine homocysteine methyltransferase activity), and the SAM to SAH ratios decreased. 21 MAT activity was not determined in this model. 21 Whether methionine synthase activity is also decreased in humans with alcoholic liver disease is unknown. This is important because human liver produces very little betaine and is not likely to have a compensatory increase in betaine homocysteine methyltransferase activity. 20 Thus, depending on the model of liver injury and the species involved, different pathways of methionine metabolism may be affected. Recent studies from Mato et al. have greatly advanced our understanding of liver-specific MAT both at the gene transcription and protein function levels. 22 25 The promoter of the rat MAT1A gene has been characterized, and the gene expression of MAT1A can be up-regulated by glucocorticoids and adenosine 3,5 -cyclic monophosphate. 22 They have also shown the importance of the oxidation/reduction state of the certain critical cysteine residues of the enzyme and the intracellular GSH/GSSG ratio on the oligomeric equilibrium of liver-specific MATs and the overall MAT activity. 2,10 Modifications of these critical cysteine residues can inactivate the enzyme by direct interference with the substrate binding site(s) or by causing dissociation of the oligomers. 2,10,23 25 One cysteine residue in particular, the cysteine at position 121, is conserved in rat and human liver-specific MAT and is a target of oxidative stress. 24,25 The model structure of the rat liver MAT shows that cysteine 121 is localized at a flexible loop over the active site cleft of MAT. 25 Although this cysteine is not essential for activity, because substitution of serine for this residue had no effect on MAT activity, when cysteine is modified either by oxidation or by the formation of a nitrosothiol, the enzyme is inactivated. 23,25 The inactivation could be reversed by GSH and other thiol-reducing agents. The
February 1998 EDITORIALS 405 amount of GSH required to reverse the inactivation was 3 mmol/l for the dimer and 25 mmol/l for the tetramer. 25 Because normal hepatic GSH concentration is 5 10 mmol/l, the difference in reversibility of inactivation by GSH may contribute to the selective loss of the tetramer in the liver injuries described above. In this issue of GASTROENTEROLOGY, Avila et al. 26 report results of their ongoing studies characterizing regulation of the liver-specific MAT by focusing on the effect of hypoxia. Because hypoxia and oxidative stress have been implicated in the pathogenesis of different hepatic injuries, their current work was aimed at elucidating the molecular mechanisms of hypoxia-induced decrease in MAT activity and MAT1A gene expression as originally described by Chawla et al. 17,18 In their original work, Chawla et al. found that levels of hepatic SAM, MAT activity, and MAT1A mrna were significantly lower in rats exposed to 10% oxygen for 9 10 days. 17,18 To elucidate the molecular mechanisms, Avila et al. studied primary cultures of rat hepatocytes subjected to 3% oxygen for up to 24 hours. This 3% oxygen was chosen to induce quick and measurable changes in MAT levels while preserving cellular viability. Although it can be argued that 3% oxygen may be nonphysiological, the physiological oxygen tension in the perivenous area is estimated to be approximately 8% and is believed to be lower in alcoholic liver disease. 27 Hypoxia led to decreased MAT activity by two mechanisms; one was exerted at the MAT protein and the other was at the gene expression of MAT1A. The rapid decrease (within the first 8 hours) in MAT activity after exposure of hepatocytes to 3% oxygen was clearly mediated at the protein level. Although the MAT1A mrna level decreased by 2 hours after hypoxia, the MAT protein level remained unchanged even after 8 hours of hypoxia, suggesting that the half-life of the liver-specific MAT protein is quite long. The inactivation of the liver-specific MAT in the first 6 hours was most likely mediated by nitric oxide, whose production was increased under hypoxia. In support of this, blocking nitric oxide synthesis by N G -nitro- L-arginine methyl ester (L-NAME) also blocked hypoxic inactivation of MAT. Although two other important factors for the decrease in MAT activity, namely, decreases in ATP and GSH levels, may also contribute, L-NAME protected against hypoxia-induced loss in MAT activity despite reduced ATP and GSH levels. N-Acetyl-Lcysteine also protected partially against hypoxia-induced MAT inactivation. However, this may have reduced the nitrosothiol bond formed between liver-specific MAT and nitric oxide. Within the first 8 hours of exposure to hypoxia, there was no induction in manganous superoxide dismutase to suggest increased generation of reactive oxygen species. However, if L-NAME treatment prevented the decrease in MAT activity, why was GSH level still lower? The degree of ATP depletion should not have affected the enzymatic activities of the GSH synthesis pathway. 28 Did GSH decrease because of increased efflux, oxidative stress, or increased utilization? This remains to be resolved. Hypoxia also exerted an effect on the gene expression of MAT1A. Although this was apparent early on, because of the long half-life of the liver-specific MAT, the effect on gene expression is not likely to be seen until much later, at least more than 8 hours later. Both decreased MAT1A gene transcription and mrna stability seemed to contribute to a decreased steady-state MAT1A mrna level. Fairly convincing evidence was then provided to show that an oxygen sensing heme protein was involved in mediating the effect of hypoxia on MAT1A gene expression. Lower ATP or increased intracellular hydrogen peroxide levels had no effect on MAT1A gene expression. What does this elegant study of the molecular mechanisms of hypoxia-induced decrease in MAT activity in cultured hepatocytes have to do with clinical liver disease? Theoretically, any liver injury resulting in hypoxic hepatocytes may result in inactivation of MAT. However, studies by Chawla et al. suggest that in vivo even at 10% oxygen, a decrease in hepatic MAT activity does not occur until 9 10 days later. 17,18 The one human liver disease in which chronic hypoxia, especially in the perivenous area, and decreased hepatic MAT activity are well recognized is alcoholic liver disease. However, the steady-state MAT1A mrna level was unchanged in patients with alcoholic cirrhosis. 2,4 One possible explanation may be that there is zonal heterogeneity in the steady-state levels of MAT1A mrna and liver-specific MATs so that a selective decrease in the pericentral zone may not be reflected by the whole liver. This remains to be examined. The other explanation may be that in cirrhotic patients and various models of liver injury, oxidative stress and GSH depletion, which decrease MAT activity at the posttranslational level, play more dominant roles than hypoxia. Is the decrease in MAT activity by hypoxia or oxidative stress good or bad for the hepatocyte? Sánchez-Góngora et al. suggested that acute inactivation may be a protective mechanism to conserve the cellular ATP and nicotinamide adenine dinucleotide stores. 28 In support of this, overexpression of liver-specific MAT in Chinese hamster ovary cells resulted in 50% depletion of ATP and nicotinamide adenine dinucleotide levels and increased sensitivity to oxidative stress. 29 However, prolonged inactivation is likely to lead to detrimental effects because decreased SAM levels can affect various transmethylation
406 EDITORIALS GASTROENTEROLOGY Vol. 114, No. 2 reactions, polyamine synthesis, and GSH synthesis. In support of this, in animal models of alcoholic liver disease and carbon tetrachloride hepatotoxicity, exogenous administration of SAM prevented the depletion of SAM and GSH levels and significantly ameliorated liver injury, including fibrosis. 2,16,19,30 Furthermore, decreased hepatic SAM levels and hypomethylation of certain oncogenes have correlated with development of experimental hepatocarcinogenesis, which were completely prevented by exogenous administration of SAM. 31 Clinical trials using SAM have also begun in patients with alcoholic liver disease, and improvement in 2-year survival has been reported in those with less advanced liver disease. 32 However, in contrast to the animal models in which decreased hepatic SAM levels paralleled MAT activities, despite a 50% 60% decrease in hepatic MAT activity, hepatic SAM levels were unchanged in cirrhotic patients. 2,11 SAH levels were not measured in these samples. Thus, animal models of hepatotoxicity may not reflect liver disease in humans. This also raises the question of the exact mechanisms of SAM s protective effect. Colell et al. showed recently that SAM administration prevented impaired GSH uptake into mitochondria in long-term ethanol-fed rats by preventing changes in mitochondrial membrane fluidity. 33 Preliminary results from Chawla et al. showed that SAM down-regulated tumor necrosis factor (TNF- ) mrna and protein synthesis by murine macrophage cells on stimulation by lipopolysaccharide and resulted in lower serum TNF- levels. 34 From these studies it is clear that SAM exerts numerous effects, from increasing membrane fluidity to altering gene expression. Where do we go from here? We have certainly come a long way since mammalian MAT was first discovered almost one-half century ago. Studies using animal models will continue to provide fresh insights about alterations of methionine metabolism in liver disease. Now that the promoter of rat MAT1A has been characterized, studies at the molecular level will be instrumental in furthering our understanding of how MAT1A gene expression is altered. Comparative studies using human hepatocytes and patients with liver disease will also be essential before extrapolating findings from animal studies to humans. Finally, as the use of SAM as a therapeutic agent in liver disease increases, more studies examining the molecular mechanisms of SAM s protective effect are clearly needed. SHELLY C. LU Center for Liver Disease Research Division of Gastrointestinal and Liver Diseases Department of Medicine University of Southern California School of Medicine Los Angeles, California References 1. Finkelstein JD. Methionine metabolism in mammals. J Nutr Biochem 1990;1:228 237. 2. Mato JM, Alvarez L, Corrales FJ, Pajares MA. S-Adenosylmethionine and the liver. In: Arias IM, Boyer JL, Fausto N, Jakoby WB, Schachter DA, Shafritz DA, eds. 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