Adaptive Regulation of Vitamin B1 Transport

描述（由申请人提供）：维生素B1（硫胺素）是糖酵解代谢网络中的一种关键酶辅因子，是维持所有细胞生物能量和合成代谢需求所必需的。当缺陷出现时，已经描述了硫胺素转运蛋白表达的适应性上调，其试图恢复硫胺素稳态。然而，在慢性饮酒过程中硫胺素缺乏与硫胺素转运蛋白的下调有关.这种酒精和硫胺素之间的拮抗作用对硫胺素转运蛋白的基因调控尚不清楚，因为缺乏关于硫胺素转运蛋白如何适应性调节的基本知识。因此，本申请致力于理解负责在硫胺素缺乏期间介导适应性上调的顺式和反式调节元件。直到这种适应性调节机制的见解是已知的，了解酒精如何减弱硫胺素转运蛋白的基因表达在相关的硫胺素缺乏症将不会实现。我们在此应用程序中的总体目标是确定 转录途径参与硫胺素转运蛋白在硫胺素缺乏的适应性调节。我们的中心假设是缺氧诱导因子-1（HIF-1）是通过反式激活硫胺素转运蛋白SLC 19 A3的表达来调节硫胺素可用性的中心转录因子。这一假设是根据申请人实验室的初步数据提出的。这项研究的基本原理是，一旦已知自适应硫胺素转运蛋白表达的转录途径，就可以实现酒精消费对硫胺素转运能力的潜在影响的新见解和方法。在我们强有力的初步数据的指导下，这一假设将通过追求两个特定的目标进行测试：1）建立硫胺素缺乏和假性缺氧对适应性硫胺素转运表达之间的联系。我们的论点是，硫胺素缺乏的代谢后果可以导致丙酮酸的积累，这反过来又激活HIF-1介导的基因调控。因此，我们将使用代谢分析和评估HIF-1靶基因表达的组合，特别是在硫胺素缺乏症期间的SLC 19 A3。我们还将确定酒精代谢的后果是否会减弱丙酮酸水平，以抵消这种适应性调节。2)确定HIF-1介导的SLC 19 A3基因表达的反式激活。我们的工作假设是，硫胺素转运蛋白SLC 19 A3的适应性上调是通过近端启动子区的缺氧反应元件来实现的。使用启动子荧光素酶测定和ChIP的组合，我们将建立HIF-1作为SLC 19 A3表达的必需反式激活因子。总的来说，拟议的研究是重要的，因为它有望导致硫胺素缺乏症相关疾病的硫胺素转运蛋白调节的机制的理解。

DESCRIPTION (provided by applicant): Vitamin B1 (thiamine) is a critical enzyme cofactor within the glycolytic metabolism network that is fundamentally required to sustain the bioenergetic and anabolic needs of all cells. When deficiencies arise, an adaptive up-regulation in thiamine transporter expression has been described that attempts to restore thiamine homeostasis. However, thiamine deficiencies during chronic alcohol consumption are associated with a down- regulation of thiamine transporters. This antagonism between alcohol and thiamine availability on gene regulation of thiamine transporters is not clearly understood because there is a lack of fundamental knowledge on how thiamine transporters are adaptively regulated. Hence, this application endeavors to understand the cis- and trans-regulatory elements that are responsible for mediating an adaptive up-regulation during thiamine deficiency. Until this mechanistic insight into adaptive regulation is known, understanding how alcohol attenuates the gene expression of thiamine transporters during associated thiamine deficiency will not be realized. The overall objective we have for this application is to determine the transcriptional pathways involved in the adaptive regulation of thiamine transporters during thiamine deficiency. It is our central hypothesis that hypoxia inducible factor-1 (HIF-1 is a centrl transcription factor modulating thiamine availability by trans- activating expression of the thiamine transporter SLC19A3. This hypothesis has been formulated on the basis of preliminary data produced in the applicants' laboratory. The rationale for the proposed research is that once the transcriptional pathways for adaptive thiamine transporter expression are known, new insights and approaches into the underlying effects of alcohol consumption on thiamine transport capacity can be achieved. Guided by our strong preliminary data, this hypothesis will be tested by pursuing two specific aims: 1) Establishing the connection between thiamine deficiency and pseudo-hypoxia on adaptive thiamine transport expression. It is our contention that the metabolic consequences of thiamine deficiency can lead to an accumulation of pyruvate which in turn activates HIF-1 mediated gene regulation. Thus we will use a combination of metabolic profiling and assessment of HIF-1 target gene expression and in particular SLC19A3 during thiamine deficiency. We will also determine if the consequences of alcohol metabolism attenuates the level of pyruvate to counteract this adaptive regulation. 2) Determining HIF-1 mediated trans- activation of SLC19A3 gene expression. Our working hypothesis is that adaptive up-regulation of the thiamine transporter SLC19A3 is conferred through hypoxia response elements in the proximal promoter region. Using a combination of promoter-luciferase assays and ChIP we will establish HIF-1 as an essential trans-activator for SLC19A3 expression. Overall, the proposed research is significant because it is expected to lead to a mechanistic understanding of thiamine transporter regulation within thiamine deficiency related disorders.