Park E I, Garrow T A
Department of Food Science and Human Nutrition and the Division of Nutritional Sciences, University of Illinois, Urbana, Illinois 61801, USA.
J Biol Chem. 1999 Mar 19;274(12):7816-24. doi: 10.1074/jbc.274.12.7816.
We previously showed that rat liver betaine-homocysteine methyltransferase (BHMT) mRNA content and activity increased 4-fold when rats were fed a methionine-deficient diet containing adequate choline, compared with rats fed the same diet with control levels of methionine (Park, E. I., Renduchintala, M. S., and Garrow, T. A. (1997) J. Nutr. Biochem. 8, 541-545). A further 2-fold increase was observed in rats fed the methionine-deficient diet with supplemental betaine. The nutrition studies reported here were designed to determine whether other methyl donors would induce rat liver BHMT gene expression when added to a methionine-deficient diet and to define the relationship between the degree of methionine restriction and level of methyl donor intake on BHMT expression. Therefore, rats were fed amino acid-defined diets varying in methionine and methyl donor composition. The effect of diet on BHMT expression was evaluated using Northern, Western, and enzyme activity analyses. Similar to when betaine was added to a methionine-deficient diet, choline or sulfonium analogs of betaine induced BHMT expression. The diet-induced induction of hepatic BHMT activity was mediated by increases in the steady-state level of its mRNA and immunodetectable protein. Using methyl donor-free diets, we found that methionine restriction was required but alone not sufficient for the high induction of BHMT expression. Concomitant with methionine restriction, dietary methyl groups were required for high levels of BHMT induction, and a dose-dependent relationship was observed between methyl donor intake and BHMT induction. Furthermore, the severity of methionine restriction influenced the magnitude of BHMT induction. To study the molecular mechanisms that regulate the expression of BHMT, we have cloned the human BHMT gene. This gene spans about 20 kilobases of DNA and contains 8 exons and 7 introns. Using RNA isolated from human liver and hepatoma cells, a major transcriptional start site has been mapped using the 5' rapid amplification of cDNA ends technique, and this start site is 26 nucleotides downstream from a putative TATA box.
我们之前的研究表明，与喂食含对照水平蛋氨酸的相同日粮的大鼠相比，当给大鼠喂食含充足胆碱但蛋氨酸缺乏的日粮时，大鼠肝脏甜菜碱 - 高半胱氨酸甲基转移酶（BHMT）的mRNA含量和活性增加了4倍（Park, E. I., Renduchintala, M. S., and Garrow, T. A. (1997) J. Nutr. Biochem. 8, 541 - 545）。在喂食含补充甜菜碱的蛋氨酸缺乏日粮的大鼠中，观察到BHMT又增加了2倍。本文报道的营养研究旨在确定当添加到蛋氨酸缺乏的日粮中时，其他甲基供体是否会诱导大鼠肝脏BHMT基因表达，并确定蛋氨酸限制程度与甲基供体摄入量水平对BHMT表达的关系。因此，给大鼠喂食蛋氨酸和甲基供体组成不同的氨基酸定义日粮。使用Northern、Western和酶活性分析评估日粮对BHMT表达的影响。与向蛋氨酸缺乏的日粮中添加甜菜碱时类似，胆碱或甜菜碱的锍类似物诱导了BHMT表达。日粮诱导的肝脏BHMT活性是由其mRNA和免疫可检测蛋白的稳态水平增加介导的。使用无甲基供体的日粮，我们发现需要蛋氨酸限制，但仅蛋氨酸限制不足以高度诱导BHMT表达。与蛋氨酸限制同时，高水平的BHMT诱导需要日粮甲基基团，并且在甲基供体摄入量与BHMT诱导之间观察到剂量依赖性关系。此外，蛋氨酸限制的严重程度影响BHMT诱导的幅度。为了研究调节BHMT表达的分子机制，我们克隆了人BHMT基因。该基因跨越约20千碱基的DNA，包含8个外显子和7个内含子。使用从人肝脏和肝癌细胞中分离的RNA，使用5' cDNA末端快速扩增技术定位了一个主要转录起始位点，该起始位点位于假定的TATA框下游26个核苷酸处。
