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

描述(由申请人提供)： 在这项研究中，我们将研究乙醇诱导的肝细胞S-腺苷甲硫氨酸：腺苷同型半胱氨酸比值降低对一种重要的甲基转移酶--乙酸胍甲基转移酶的影响。这种甲基转移酶催化一个甲基从SAM转移到乙酸胍(GAA)，形成肌酸。最近已经证实，GAA主要在肾脏合成，并通过循环运输到肝脏，在那里它被甲基化形成肌酸。肝脏是“需要肌酸的”组织摄取肌酸的主要来源，但能量需求变化很大，因此，酒精暴露后GAMT功能受损的影响可能导致两种不同的有害后果：1)它可以减少肝外组织的肌酸可获得性，包括依赖完整的肝脏肌酸生物合成途径来实现最佳功能的骨骼和心肌；2)它可以由于有毒肌酸的积累而导致肝脏损伤。 前身，GAA。基于这些考虑，提出了以下假设：乙醇引起的SAM改变：SAH通过影响GAMT活性而损害肝脏肌酸的合成，导致肝脏以及肝外组织(如骨骼肌和心肌)的不利后果。此外，通过纠正改变的SAM：SAH比率补充甜菜碱可以使肝脏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标是： 1)进一步研究和阐明慢性乙醇致大鼠肝肌酸生物合成受损的机制。2)研究乙醇和甜菜碱对大鼠循环肌酸和GAA水平、肝脏肌酸外流和肾脏GAA生成的影响。3)研究乙醇诱导肝脏肌酸前体GAA蓄积的细胞毒性效应。4)评估酒精暴露后骨骼肌和心肌肌酸生物合成和可获得性改变的影响和功能后果，并检查甜菜碱和/或 补充肌酸可以逆转或阻止酒精引起的改变。这项建议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，通过校正SAM：SAH比率的甜菜碱是否也逆转了酒精对肝脏肌酸生物合成途径的影响。由于人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血浆肌酸和GAA水平的影响。此外，还将检查肾脏产生和释放GAA的能力是否会被乙醇和/或甜菜碱改变。在下一个具体目标中，将确定肌酸前体GAA在肝脏中积聚的有害后果。最后，将检查饮用酒精的大鼠骨骼肌和心肌肌酸缺乏的生化和功能后果，以确定是否可以通过在饮食中补充甜菜碱和/或肌酸来逆转这些有害后果。总体而言，在酒精肝损伤模型中完成这些研究，并使用GAMT基因敲除小鼠模型进行验证，将有助于深入了解在肝脏中维持由GAMT催化的与肝和肝外组织相关的基本甲基化反应的重要性。

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.