Adaptive Regulation of Vitamin B1 Transport

说明(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的酶辅助因子，是维持所有细胞的生物能量和合成代谢需求所必需的。当缺陷出现时，硫胺转运体表达的适应性上调已被描述为试图恢复硫胺稳态。然而，长期饮酒期间的硫胺素缺乏与硫胺素转运体的下调有关。酒精和硫胺素对硫胺转运蛋白基因调控的拮抗作用尚不清楚，因为缺乏关于硫胺素转运蛋白如何适应性调节的基础知识。因此，这项应用致力于了解在硫胺素缺乏期间，负责调节适应性上调的顺式和跨式调节元件。在这种对适应性调节的机械性洞察被了解之前，了解酒精如何在相关的硫胺缺乏期间减弱硫胺转运体的基因表达是不可能的。我们对此应用程序的总体目标是确定 转录途径参与硫胺转运体在硫胺缺乏时的适应性调节。我们的中心假设是缺氧诱导因子-1(HIF-1)是一种中枢转录因子，通过反式激活硫胺转运体SLC19A3的表达来调节硫胺的可获得性。这一假设是根据申请人实验室提供的初步数据提出的。这项拟议研究的基本原理是，一旦知道了适应性硫胺转运体表达的转录途径，就可以获得关于酒精消费对硫胺转运体能力的潜在影响的新的见解和方法。在我们强大的初步数据的指导下，这一假说将通过追求两个具体目标来验证：1)建立硫胺素缺乏和假低氧对硫胺适应性运输表达的联系。我们认为，硫胺素缺乏的代谢后果可以导致丙酮酸的积累，进而激活HIF-1介导的基因调控。因此，我们将结合代谢谱和评估HIF-1靶基因的表达，特别是硫胺素缺乏期间的SLC19A3。我们还将确定酒精代谢的后果是否会减弱丙酮酸的水平，以抵消这种适应性调节。2)检测HIF-1对SLC19A3基因表达的反式激活作用。我们的工作假设是硫胺转运体SLC19A3的适应性上调是通过近端启动子区域的低氧反应元件实现的。结合启动子-荧光素酶分析和芯片技术，我们将确定HIF-1是SLC19A3表达所必需的反式激活剂。总体而言，这项拟议的研究具有重要意义，因为它有望导致对硫胺缺乏相关疾病中硫胺转运体调节的机械性理解。

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.