Exploratory Project 2 "Homocysteine and Alcoholic Liver Disease"

探索性项目2“同型半胱氨酸与酒精性肝病”

基本信息

批准号：
8137306
负责人：
Donald Weldon Jacobsen
金额：
$ 7.55万
依托单位：
CLEVELAND CLINIC LERNER COM-CWRU
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8137306
关键词：
Acetaldehyde Address Adenosine Adenosylhomocysteinase Adult Alcohol abuse Alcohol consumption Alcoholic Liver Cirrhosis Alcoholic Liver Diseases Alcoholism Alcohols Anabolism Animal Model Antioxidants Apoptotic Back Betaine Biological Assay Blood Carbon Cardiovascular Diseases Catabolism Cell Death Cells Chronic Cirrhosis Clinical Cobalamin Coenzymes Corrinoids Cystathionine Cysteine Cysteine Desulfhydrase Cytochrome P-450 CYP2E1 Dietary Proteins Disease Progression Endoplasmic Reticulum Enzyme-Linked Immunosorbent Assay Enzymes Equilibrium Ethanol Exposure to Figs - dietary Functional disorder Future Gene Mutation Generations Heavy Drinking High Prevalence Homocysteine Homocystine Homocystinuria Human Hydrolysis Hyperhomocysteinemia Immunoprecipitation Impaired cognition Individual Injury Kidney Lead Liquid Chromatography Liver Liver diseases MTHFR gene Mediator of activation protein Messenger RNA Metabolism Metallothionein Methionine Methionine Metabolism Pathway Methylmalonic Acid Methylmalonyl-CoA Mutase Methyltransferase Mitochondria Modeling Molecular Molecular Chaperones Molecular Target Morbidity - disease rate Mus Oxidative Stress Pathology Pathway interactions Patients Pilot Projects Plasma Play Process Production Proteins Proteome Proteomics Protocols documentation Pyridoxal Rattus Reaction Reactive Oxygen Species Research Research Personnel Risk Factors Role S-Adenosylmethionine Spectrometry, Mass, Electrospray Ionization Staging Steatohepatitis Subarachnoid Hemorrhage Testing Therapeutic Time Tissues Vitamin B Complex Western Blotting alcohol preferring mice analog cobamamide design endoplasmic reticulum stress feeding in vitro activity in vivo inhibitor/antagonist inorganic phosphate intravenous injection liver biopsy loss of function mRNA Expression methionine adenosyltransferase middle age mortality non-alcoholic novel problem drinker

项目摘要

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).

通过 B12 依赖性 MS 失活扰动 ALD 中的一碳代谢高同型半胱氨酸血症和酒精性肝病。现在已经证实，过量饮酒会导致一碳代谢紊乱和高同型半胱氨酸血症（血液同型半胱氨酸升高）(1-8)。尽管高同型半胱氨酸血症是心血管疾病 (9) 和认知功能障碍 (10) 的危险因素，同型半胱氨酸升高在 ALD 进展、死亡率和发病率中所起的作用尚不清楚。是吗与酗酒相关的高同型半胱氨酸血症是否可能是 ALD 的介质？该项目将最初解决酒精引起的高同型半胱氨酸血症的机制。在非酒精患者中同型半胱氨酸尿症（和高同型半胱氨酸血症）、肝脂肪变性的发生率很高 (11,12)。因此，不饮酒的高同型半胱氨酸血症会导致肝脏病变。对于健康的中年人来说，血浆总同型半胱氨酸 (tHcy) 浓度范围为 3.3 至 13.4 nmol/L (13)。在大多数酗酒者中浓度中度升高 (>13 至 30 umol/L) (14-16)，但严重高同型半胱氨酸血症 (>100 umol/L) 也已被观察到 (17)。使用 B 族复合维生素和/或甜菜碱对酒精患者有益吗？ Kaplowitz 和他的同事已经证明，降低同型半胱氨酸甜菜碱可减少 ALD 动物模型中的 ER 应激和肝损伤 (18)。蛋氨酸代谢。在肝脏中，蛋氨酸来源于膳食蛋白质和/或细胞内的分解代谢蛋白质，通过甲硫氨酸腺苷转移酶 (MAT) 转化为 S-腺苷甲硫氨酸（AdoMet 或 SAM）（反应1，图1）（19）。 SAM 在体内催化的反应中充当主要的甲基供体 100 种不同的 SAM 依赖性甲基转移酶（反应 2）(20)。 S-腺苷同型半胱氨酸（AdoHcy 或 SAH）形成于这些甲基转移酶反应是水解为腺苷和 SAH 水解酶检测同型半胱氨酸（反应3）（21）。 [注：平衡常数反应有利于形成 AdoHcy，这是一种最终产品大多数甲基转移酶的抑制剂]。蛋氨酸循环完成由同型半胱氨酸的再甲基化回到蛋氨酸。在肝脏中和肾脏，这个反应是由 B12 依赖性 MS 催化使用A/-5-甲基四氢叶酸作为甲基供体（反应 4） (22) 和同型半胱氨酸甜菜碱甲基转移酶使用甜菜碱作为甲基供体（反应5）（23）。同型半胱氨酸，分支点代谢物是分解代谢为胱硫醚和然后半胱氨酸通过反应 6 和 7 的转硫途径，由吡哆醛-51-催化磷酸盐 (PLP) 依赖性胱硫醚 p-合酶 (CBS) 和 PLP 依赖性 y-胱硫醚酶，分别 (24)。自 20 世纪 40 年代末以来，蛋氨酸代谢的改变一直与肝脏疾病有关。金塞尔等人。（25）表明，几位患有慢性疾病的患者的蛋氨酸清除率显着受损。接受单次静脉注射蛋氨酸后出现肝脏疾病。杜斯等人。 (26) 发现 MAT 酒精性肝硬化和非酒精性肝硬化患者的肝活检中的活性显着降低肝硬化。阿维拉等人。 (27)发现蛋氨酸循环的几种酶的mRNA水平，即 MAT1 A、GNMT、MS、BHMT 和 CBS 在酒精性和非酒精性人类肝硬化中均降低肝脏。最近，两项蛋白质组学研究检查了饮酒者和避免饮酒者的肝脏蛋白质组大鼠和小鼠 (28,29)。在小鼠研究中，Park 等人。 (29)发现26种蛋白质存在差异在喜欢酒精的小鼠中增加或减少，并且包括蛋氨酸循环的两种酶在该组中，即 MAT 和 SAHH 均有所下降。 MAT 也显着下降大鼠研究 (28)。众所周知，ALD (30-33) 中 SAM 水平会降低。事实上，在大多数形式的肝脏中疾病时，MAT (28,34,35) 和 SAM 代谢物均减少 (30,31,36)。高同型半胱氨酸血症是由催化反应 4 (MS)、6 的酶功能丧失引起的 (CBS) 或图 1 中的 9 (MTHFR) 由于基因突变或辅酶/底物缺乏（即， 8,2, B6 叶酸）。目标 1 的重点是从机制上了解高同型半胱氨酸血症的原因在 ALD 中，特别是 B12 依赖性 MS 在此过程中发挥的作用。 ALD 中 B12 依赖性 MS 失活。在目标 1 中，我们假设乙醇诱导肝脏中的 ROS 攻击并使 cob(l)alamin 失活，cob(l)alamin 是 MS 催化循环中的中间体（反应 4）。有没有 ALD 患者 MS 功能丧失的证据？细胞和动物模型中长期接触乙醇的 ALD 结果几位研究人员表明，Bi2 依赖性 MS 失活 (37-47)。虽然乙醇本身不抑制 MS 活性，体外研究表明，超生理浓度的乙醛确实抑制 MS (47)。因此，我们认为其他机制是 ALD 中 MS 失活的原因。 ALD 中类咕啉类似物的形成。 cob(l)alamin 中间体的失活 ROS 的 MS 催化循环可能会导致甲钴胺 (MeCbl) 的产生减少，并且类咕啉类似物的形成。 Cob(l)alamin 也在腺苷钴胺素（Bi2 辅酶；Bi2 辅酶； AdoCbl) 生物合成，也可能是 ALD 中 ROS 的目标。如果 AdoCbl 生物合成受损 ALD，则线粒体甲基丙二酰辅酶A变位酶的活性可能会受到损害。事实上，兰伯特等人等人。 (48) 发现，与对照组相比，酗酒患者的甲基丙二酸水平确实较高。还有证据表明 ALD 中类咕啉类似物的产生增加 (48,49)。