Effect of Alcohol on Hepatic Creatine Biosynthesis: Role of Defective Methylation

描述（由申请人提供）： 在这个建议中，乙醇诱导的肝细胞S-腺苷甲硫氨酸（SAM）的减少的影响：腺苷高半胱氨酸（SAH）的比例上的一个重要的甲基转移酶，胍乙酸甲基转移酶（GAMT）将被检查。这种甲基转移酶催化甲基从SAM转移到胍基乙酸（GAA）以形成肌酸。最近已经确定，GAA主要在肾脏中合成，并通过循环转运到肝脏，在那里它被甲基化形成肌酸。肝脏是肌酸循环水平的主要来源，用于由具有高但可变能量需求的“肌酸需求”组织（例如骨骼肌和心肌）摄取。因此，乙醇暴露后GAMT功能受损的影响可导致两种不同的有害后果：1）它可以降低肝外组织的肌酸利用率，包括骨骼肌和心肌，其依赖于完整的肝肌酸生物合成途径以实现最佳功能;以及2）由于毒性肌酸的积累， 前体，GAA。基于这些考虑，提出了以下假设：乙醇诱导的改变的SAM：SAH通过其对GAMT活性的影响损害肝脏肌酸合成，在肝脏以及肝外组织（如骨骼肌和心肌）中引起有害后果。此外，甜菜碱补充凭借纠正改变的SAM：SAH比率可以使肝肌酸生物合成正常化，从而防止这些有害影响。 检验这一假设的具体目的是： 1)进一步研究和阐明慢性乙醇给药导致肝脏肌酸生物合成受损的机制。 2)确定乙醇和甜菜碱对循环肌酸和GAA水平、肝脏肌酸外排和肾脏GAA产生的影响。 3)证明乙醇诱导的肌酸前体GAA肝脏蓄积的细胞毒性后果。 4)评估乙醇暴露后肌酸生物合成和利用率改变对骨骼肌和心肌的影响和功能后果，并检查甜菜碱和/或 补充肌酸可以逆转或防止酒精引起的变化。本提案的目的是首先确定乙醇诱导的肝脏SAM：SAH比率降低在调节肝脏肌酸合成中的作用。然后，将检查通过校正SAM：SAH比率的甜菜碱是否也逆转酒精诱导的对肝肌酸生物合成途径的影响。由于身体对肌酸的需求是通过从循环中摄取来满足的，接下来将确定乙醇和甜菜碱治疗对肌酸和GAA的血浆水平的影响。此外，将检查肾脏产生和释放GAA的能力是否被乙醇和/或甜菜碱改变。在下一个具体目标中，将确定肝脏中肌酸前体GAA积累的有害后果。最后，将检查消耗乙醇的大鼠骨骼肌和心肌中肌酸缺乏的生化和功能后果，以确定这些有害后果是否可以通过在饮食中补充甜菜碱和/或肌酸来逆转。总之，在肝损伤的乙醇模型中完成这些研究并使用GAMT敲除小鼠模型进行验证，将提供对维持肝脏中GAMT催化的基本甲基化反应相对于肝脏和肝外组织的重要性的见解。

DESCRIPTION (provided by applicant): In this proposal, the effect of ethanol-induced reduction in hepatocellular S-adenosylmethionine (SAM):Sadenosylhomocysteine (SAH) ratios on an important methyltransferase, guanidinoacetate methyltransferase (GAMT) will be examined. This methyltransferase catalyzes the transfer of a methyl group from SAM to guanidinoacetate (GAA) to form creatine. It has been recently established that GAA is primarily synthesized in the kidney and transported through the circulation to the liver where it is methylated to form creatine. Liver is the principa source of circulating levels of creatine for uptake by "creatine-requiring" tissues that have a hig, but variable energy demand such as skeletal and cardiac muscles Therefore, the impact of compromised GAMT function following ethanol exposure can lead to two distinct detrimental consequences: 1) It can decrease creatine availability for extra-hepatic tissues, including skeletal and cardiac muscles that rely on an intact hepatic creatine biosynthetic pathway for optimal functioning; and 2) it can cause liver injury due to the accumulation of the toxic creatine precursor, GAA. Based on these considerations, the following hypothesis has been formulated: Ethanol induced altered SAM:SAH impairs hepatic creatine synthesis via its effect on GAMT activity causing detrimental consequences in the liver as well as extra-hepatic tissues such as the skeletal and cardiac muscles. Furthermore, betaine supplementation by virtue of correcting the altered SAM:SAH ratio could normalize hepatic creatine biosynthesis and thereby prevent these detrimental effects. The specific aims to test this hypothesis are: 1) To further examine and delineate the mechanism of impaired hepatic creatine biosynthesis by chronic ethanol administration. 2) To determine the effects of ethanol and betaine on circulating creatine and GAA levels, liver creatine efflux and kidney GAA production. 3) To demonstrate the cytotoxic consequences of ethanol-induced hepatic accumulation of creatine precursor, GAA. 4) To evaluate the effects and functional consequences of altered creatine biosynthesis and availability following ethanol exposure on skeletal and cardiac muscles and to examine whether betaine and/or creatine supplementation can revert or prevent the alcohol-induced changes. The objectives of this proposal are to first determine the role of ethanol-induced reduced liver SAM: SAH ratios in regulating hepatic creatine synthesis. Then, whether betaine by correcting the SAM: SAH ratio also reverses alcohol-induced effects on the liver creatine biosynthetic pathway will be examined. Since the body's requirement for creatine is met via uptake from the circulation, the effect of ethanol and betaine treatment on plasma levels of creatine and GAA will be determined next. Further, whether kidney's ability to produce and release GAA is altered by ethanol and/or betaine will be examined. In the next specific aim the detrimental consequences of accumulation of creatine precursor, GAA in the liver will be determined. Finally, the biochemical and functional consequences of creatine shortage in the skeletal and cardiac muscles of rats consuming ethanol will be examined to determine whether these detrimental consequences could be reversed by supplementing betaine and/or creatine in the diet. Overall, completion of these studies in an ethanol model of liver injury and validated using a GAMT knockout mouse model will provide insight in to the importance of maintaining the essential methylation reaction catalyzed by GAMT in the liver in relation to hepatic and extra hepatic tissues.