Perturbation of one-carbon metabolism in ALD via inactivation of B12-dependent MS Hyperhomocysteinemia and ALD. It is now well established that excessive alcohol consumption leads to the perturbation of one-carbon metabolism and hyperhomocysteinemia (elevated blood homocysteine) (1-8). Although hyperhomocysteinemia is a risk factor for cardiovascular disease (9) and cognitive dysfunction (10), the role that elevated homocysteine plays in ALD progression, mortality and morbidity is not at all clear. Is it possible that the hyperhomocysteinemia associated with alcohol abuse is a mediator of ALD? This project will initially address mechanisms of alcohol-induced hyperhomocysteinemia. In non-alcoholic patients with homocystinuria (and hyperhomocysteinemia), hepatosteatosis occurs with high prevalence (11,12). Thus, hyperhomocysteinemia without alcohol consumption results in liver pathology. In healthy middle-aged adults, plasma total homocysteine (tHcy) concentrations range from 3.3 to 13.4 nmol/L (13). In most alcoholics tHcy concentrations are moderately elevated (>13 to 30 umol/L) (14-16) but severe hyperhomocysteinemia (>100 umol/L) has also been observed (17). Will homocysteine-lowering protocols using B-complex vitamins and/or betaine benefit alcoholic patients? Kaplowitz and colleagues have shown that lowering homocysteine with betaine reduces ER stress and liver injury in animal models of ALD (18). Methionine metabolism. In the liver, methionine derived from dietary protein and/or catabolism of intracellular proteins, is converted to S-adenosylmethionine (AdoMet or SAM) by methionine adenosyltransferase (MAT) (Reaction 1, Fig. 1) (19). SAM serves as the principal methyl donor in the body in reactions catalyzed by up to 100 different SAM-dependent methyltransferases (Reaction 2) (20). The S-adenosylhomocysteine (AdoHcy or SAH) formed in these methyltransferase reactions is hydrolyzed to adenosine and homocysteine by SAH hydrolase (Reaction 3) (21). [Note: the equilibrium constant for this reaction favors the formation of AdoHcy, which is an end-product inhibitor of most methyltransferases]. The methionine cycle is completed by the remethylation of homocysteine back to methionine. In the liver and kidney, this reaction is catalyzed by B12-dependent MS using A/-5-methyltetrahydrofolate as the methyl donor (Reaction 4) (22) and by homocysteinebetaine methyltransferase using betaine as the methyl donor (Reaction 5) (23). Homocysteine, a branch-point metabolite, is catabolized to cystathionine and then cysteine through the transsulfuration pathway by Reactions 6 and 7, which are catalyzed by pyridoxal-51- phosphate (PLP)-dependent cystathionine p-synthase (CBS) and PLP-dependent y-cystathionase, respectively (24). Alterations in methionine metabolism have been associated with liver disease since the late 1940s when Kinsell et al. (25) showed that methionine clearance was significantly impaired in several patients with chronic liver disease after receiving a single intravenous injection of methionine. Duce et al. (26) found that MAT activity was significantly decreased in liver biopsies from patients with alcoholic cirrhosis and non-alcoholic cirrhosis. Avila et al. (27) found that the mRNA levels of several enzymes of the methionine cycle, namely MAT1 A, GNMT, MS, BHMT and CBS, were all reduced in both alcoholic and non-alcoholic human cirrhotic livers. Recently, two proteomic studies have examined the liver proteome of alcohol-preferring and alcoholavoiding rats and mice (28,29). In the mouse study, Park et al. (29) found that 26 proteins were differentially increased or decreased in the alcohol-preferring mice, and two enzymes of the methionine cycle were included in that group, namely MAT and SAHH, which were both decreased. MAT was also significantly decreased in the rat study (28). It is well known that SAM levels are decreased in ALD (30-33). In fact, in most forms of liver disease, both MAT (28,34,35) and SAM metabolites are reduced (30,31,36). Hyperhomocysteinemia is caused by the loss of function of enzymes that catalyze Reactions 4 (MS), 6 (CBS), or 9 (MTHFR) in Fig. 1 as a result of either gene mutation or coenzyme/substrate deficiency (i.e., 8,2, B6 orfolate). The focus of Aim 1 is to gain a mechanistic understanding of the cause of hyperhomocysteinemia in ALD, specifically the role that B12-dependent MS plays in this process. Inactivation of B12-dependent MS in ALD.
In Aim 1, we hypothesize that ethanol-induced ROS in the liver attacks and inactivates cob(l)alamin, an intermediate in the catalytic cycle of MS (Reaction 4). Is there evidence for loss of MS function in ALD? Chronic exposure to ethanol in cell and animal models of ALD results in the inactivation of Bi2-dependent MS as shown by several investigators (37-47). Although ethanol perse does not inhibit MS activity, in vitro studies have shown that acetaldehyde at supraphysiological concentrations does inhibit MS (47). Therefore, we believe that other mechanisms are responsible for MS inactivation in ALD. Formation of corrinoid analogs in ALD. Inactivation of the cob(l)alamin intermediate formed during the catalytic cycle of MS by ROS is likely to result in decreased production of methylcobalamin (MeCbl) and the formation of corrinoid analogues. Cob(l)alamin is also generated during adenosylcobalamin (Bi2 coenzyme; AdoCbl) biosynthesis and is also likely to be targeted by ROS in ALD. If AdoCbl biosynthesis is impaired in ALD, then the activity of mitochondrial methylmalonyl-CoA mutase could be compromised. In fact, Lambert et al. (48) found that alcoholic patients did indeed have higher levels of methylmalonic acid compared to controls. There is also evidence for increased production of corrinoid analogues in ALD (48,49).
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