Mechanism/Regulation of Intestinal Thiamin Uptake

描述（由申请人提供）：本次更新申请的长期目标是继续我们对水溶性维生素B1（硫胺素）肠道吸收过程及其调控的细胞和分子机制的研究。我们还旨在研究肠致病性大肠杆菌（EPEC）感染对肠道维生素吸收过程的影响，这是一种常见的肠道病原体。硫胺素对正常细胞功能至关重要，缺乏硫胺素（这是一个严重的营养问题）会导致各种临床异常，包括心血管和神经系统疾病。人类（和其他哺乳动物）不能合成硫胺素，因此必须通过肠道吸收来获得维生素。在目前的资助期内的研究已经描述了肠道维生素摄取过程的许多方面。其中包括体外和体内人类硫胺素转运蛋白1和2 （hTHTR-1和hTHTR-2）基因的5'调控区域的表征，证明在硫胺素缺乏时，肠道硫胺素摄取过程通过转录介导的机制适应性上调，并且该过程也经历分化和发育依赖的调节。我们还描述了上皮细胞中硫胺素转运体的膜靶向和细胞内运输的机制。利用基因特异性siRNA方法，我们实验室最近的研究表明，hTHTR 1和2都参与了体外人肠上皮Caco- 2细胞系对硫胺素的摄取。对于这些硫胺素转运蛋白在天然肠道中摄取硫胺素的作用，我们一无所知。因此，在新的初步研究中，我们开发了Slc19a3-/-敲除小鼠模型，并显示与野生型小鼠相比，肠道硫胺素摄取显著受损；我们也在实验室建立了Slc19a2-/-基因敲除小鼠群体。此外，我们使用细菌双杂交系统筛选人类肠道cDNA文库，并鉴定出两种可能与hTHTR-1相互作用的蛋白（tetraspanin和E-cadherin-1）。此外，我们已经检查了肠致病性大肠杆菌（EPEC）的作用，并发现对硫胺素摄取的显著抑制。根据我们以前和新的初步调查结果，我们的建议目标是：1)利用Slc19a2 -/和Slc19a3 -/-敲除小鼠模型进一步表征肠内硫胺素吸收过程，并确定THTR-1和THTR-2在天然肠内跨上皮和跨膜转运事件中的作用。2)确定在硫胺素缺乏症中参与适应性上调硫胺素摄取的顺式调控元件和反式作用核因子。3)鉴定人肠上皮细胞中与hTHTR-1和hTHTR-2相互作用的蛋白并了解其生物学/生理学作用；4)确定EPEC抑制肠道硫胺素摄取过程的细胞和分子机制。这些研究的结果将继续提供有关肠道维生素摄取过程及其调控的细胞和分子机制以及对该过程产生负面影响的因素的新颖和有价值的信息。这将最终帮助我们设计有效的策略来优化与维生素缺乏和亚最佳水平相关的维生素体内稳态，并最大限度地减少可能对这一营养参数产生负面影响的外源因素的影响。公共卫生相关性：人类和其他哺乳动物不能合成维生素B1（正常健康所必需的微量营养素），因此必须通过肠道吸收从外源性来源获得。自十年前提出该建议以来，其目的一直是（并将继续是）描述肠道维生素吸收的机制，该过程是如何调节的，以及影响其功能的因素。这些研究结果将有助于设计有效的策略来优化体内的硫胺素水平，特别是在与硫胺素缺乏和亚理想水平相关的情况下。

DESCRIPTION (provided by applicant): The long-term objective of this renewal application is to continue our investigations into the cellular and molecular mechanisms involved in the intestinal absorption process of the water- soluble vitamin B1 (thiamin) and their regulation. We also aim at examining the effect of infection with enteropathogenic E. coli (EPEC), a common intestinal pathogen, on the intestinal thiamin absorption process. Thiamin is essential for normal cellular functions and its deficiency (which represents a significant nutritional problem) leads to a variety of clinical abnormalities including cardiovascular and neurological disorders. Humans (and other mammals) cannot synthesize thiamin, and thus, must obtain the vitamin via intestinal absorption. Studies during the current funding period have characterized many aspects of the intestinal thiamin uptake process. These include characterization of the 5' -regulatory regions of the genes of the human thiamin transporters 1 & 2 (hTHTR-1 & hTHTR-2) both in vitro and in vivo, demonstration that the intestinal thiamin uptake process is adaptively up-regulated in thiamin deficiency via transcriptionally-mediated mechanism(s) and that the process also undergoes differentiation- and developmental- dependent regulation. We have also characterized the mechanisms involved in membrane targeting and intracellular trafficking of the thiamin transporters in epithelial cells. Using gene specific siRNA approaches, recent studies from our laboratory have shown that both the hTHTR 1 & 2 are involved in thiamin uptake by a human intestinal epithelial Caco- 2 cell line in vitro. Nothing is known about the role of these thiamin transporters in thiamin uptake in the native intestine. Thus, in new preliminary studies we have developed an Slc19a3-/- knockout mouse model and showed significant impairment in intestinal thiamin uptake compared to wild-type mice; we have also established a colony of Slc19a2-/- knockout mice in our laboratory. In addition, we have used the bacterial two-hybrid system to screen a human intestinal cDNA library and have identified two putative proteins that can interact with hTHTR-1 (tetraspanin and E-cadherin-1). Furthermore, we have examined the effect of the enteropathogenic Escherichia coli (EPEC) and found significant inhibition in thiamin uptake. Based on our previous and new preliminary findings, our aims in this proposal are: 1) To further characterize the intestinal thiamin absorption process using Slc19a2 -/-and Slc19a3 -/- knockout mouse models and to determine the role of THTR-1 and THTR-2 in trans-epithelial and trans- membrane transport events in the native intestine, 2) To identify the cis-regulatory elements and trans-acting nuclear factors involved in the adaptive up-regulation of thiamin uptake in thiamin deficiency, 3) To identify proteins that interact with hTHTR-1 and hTHTR-2 in human intestinal epithelial cells and to understand their biological/physiological roles, and 4) to determine the cellular and molecular mechanisms involved in EPEC inhibition of the intestinal thiamin uptake process. Results of these studies should continue to provide novel and valuable information regarding the cellular and molecular mechanisms involved in the intestinal thiamin uptake process and their regulation as well as of the factors that negatively impact the process. This should ultimately assist us in the designing of effective strategies to optimize thiamin body homeostasis in conditions associated with thiamin deficiency and sub-optimal levels, and in minimizing the effect of exogenous factors that may negatively impact this nutritional parameter. PUBLIC HEALTH RELEVANCE: Humans and other mammals cannot synthesize vitamin B1 (an essential micronutrient for normal health), and thus, must obtain it from exogenous sources via intestinal absorption. The aims of this proposal since its inception ten years ago were (and continue to be) the delineation of the mechanisms involved in intestinal thiamin absorption, how the process is regulated, and what factors affect its function. Results of these investigations should help in the designing of effective strategies to optimize thiamin body levels, especially in conditions associated with thiamin deficiency and sub-optimal levels.