FOLATE DEFICIENCY AND ONE-CARBON METABOLISM

叶酸缺乏和一碳代谢

• 批准号: 3248135
• 负责人: CONRAD WAGNER
• 金额: $ 12.44万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 1998-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3248135
• 关键词: DNA methylation; S adenosylmethionine; cell type; creatine; folate deficiency; gene expression; homocysteine; immunocytochemistry; laboratory rat; methyl group; methylation; methyltransferase; nutrition related tag; pancreas; pathogenic diet; phospholipids; tissue /cell culture

The broad objective of this proposal is to determine whether a deficiency of folate, which is needed for the de novo synthesis of methyl groups, causes similar metabolic consequences as a deficiency of preformed methyl groups. Diets that contain no methionine and choline cause spontaneous liver cancer. Ethionine, an analogue of methionine that interferes with methylation, is a carcinogen, but when injected to choline deficient mice, ethionine rapidly causes acute pancreatitis. One explanation for the effects of methyl deficiency has been the decreased ratio of S- adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH) that results. A wide variety of cellular methylation reactions are inhibited when the ratio of SAM/SAH is decreased, including the methylation of DNA which controls gene expression in eukaryotes. It has been shown by the P.I. that folate deficiency produces a decrease in the SAM/SAH ratio, in liver and pancreas, which is as great as that caused by methyl group deficiency. This SAM/SAH ratio is regulated by the enzyme, glycine N- methyltransferase (GNMT), which contains tightly bound folate. Immunohistochemical studies have shown that GNMT is concentrated in the exocrine pancreas. A high concentration of GNMT implies that methylation and folate are particularly important for certain cell types. These aspects are investigated in the specific aims which are: 1. To investigate whether folate deficiency affects pancreatic secretion in vivo, in situ and in tissue culture using the AR42J cell line. 2. To investigate whether folate deficiency affects the methylation of small molecules (creatine, phospholipids) and macromolecules (DNA). 3. To extend the immunohistochemical localization of GNMT to a variety of tissues. 4. To develop an immunohistochemical method for detecting folate within tissues. Immunohistochemical localization of folate will be modeled after the method used by the neurochemists for the localization of brain neurotransmitters. Rapid folate deficiency will be produced in rats by feeding the amino defined diet of Walzem and Clifford. Undermethylation of DNA will be evaluated by the ability to enzymatically incorporate additional methyl groups, by an altered expression of poly A+ RNA, and by an altered response of DNA to restriction endonucleases. Undermethylation of DNA in folate deficiency can affect gene expression. The ability to estimate folate levels of specific cells within tissues may provide a means of looking for localized folate deficiency as a cause of pre-neoplastic lesions in smokers and oral-contraceptive users.

该提案的总体目标是确定是否存在缺陷 叶酸，这是甲基从头合成所需的， 导致与预制甲基缺乏类似的代谢后果 组。 不含蛋氨酸和胆碱的饮食会导致自发性 肝癌。 乙硫氨酸是蛋氨酸的类似物，可干扰 甲基化，是一种致癌物质，但注射到胆碱缺乏时 乙硫氨酸迅速引起小鼠急性胰腺炎。 一种解释是 甲基缺乏的影响是S-比例下降 腺苷甲硫氨酸 (SAM) 转化为 S-腺苷高半胱氨酸 (SAH)。 当多种细胞甲基化反应被抑制时 SAM/SAH 的比率降低，包括 DNA 甲基化 控制真核生物中的基因表达。 P.I. 已经证明了这一点。 叶酸缺乏会导致肝脏中 SAM/SAH 比率降低 和胰腺，与甲基引起的一样大 不足。 该 SAM/SAH 比例由甘氨酸 N- 酶调节 甲基转移酶（GNMT），含有紧密结合的叶酸。 免疫组织化学研究表明，GNMT 集中在 外分泌胰腺。 高浓度的 GNMT 意味着甲基化 和叶酸对于某些细胞类型特别重要。 这些 在具体目标中对各个方面进行了调查，这些目标是： 1. 探讨叶酸缺乏是否影响胰腺分泌 使用 AR42J 细胞系进行体内、原位和组织培养。 2. 探讨叶酸缺乏是否影响甲基化 小分子（肌酸、磷脂）和大分子（DNA）。 3. 将GNMT的免疫组化定位扩展到多种 组织。 4. 建立检测叶酸的免疫组化方法 组织。 叶酸的免疫组织化学定位将按照 神经化学家用于大脑定位的方法 神经递质。 大鼠体内会出现快速叶酸缺乏症 喂养 Walzem 和 Clifford 的氨基限定饮食。 甲基化不足 DNA 的含量将通过酶促掺入的能力来评估 通过改变 Poly A+ RNA 的表达来增加额外的甲基，以及 DNA 对限制性核酸内切酶反应的改变。 叶酸缺乏时 DNA 甲基化不足会影响基因表达。 估计组织内特定细胞的叶酸水平的能力 可能提供一种寻找局部叶酸缺乏原因的方法 吸烟者和口服避孕药使用者的肿瘤前病变。