Mechanism/Regulation of Intestinal Thiamin Uptake

项目摘要： 本次更新申请的长期目标是继续调查 水的肠道吸收过程中所涉及的细胞和分子机制- 可溶性维生素B1（硫胺素）及其调节。我们还旨在研究 肠致病性E.大肠杆菌（EPEC），一种常见的肠道病原体，对肠道 硫胺素吸收过程 硫胺素是正常细胞功能所必需的，它的缺乏（这代表了一种免疫缺陷）。 严重营养问题）导致多种临床异常，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，必须通过肠道吸收获得维生素。本期研究 资金期间的特点是肠道硫胺素吸收过程的许多方面。这些 包括人硫胺素基因的5 ′-调控区的表征 转运蛋白1和2（hTHTR-1和hTHTR-2），证明在体外和体内， 肠道硫胺素摄取过程在硫胺素缺乏时通过以下途径适应性上调： 转录介导的机制，并且该过程也经历分化-并且 发育依赖性调节。我们还描述了 上皮细胞中硫胺素转运蛋白的膜靶向和细胞内运输。 使用基因特异性siRNA方法，我们实验室最近的研究表明， hTHTR 1和2都参与人肠上皮细胞Caco-2对硫胺素的摄取， 2细胞系。关于这些硫胺素转运蛋白在硫胺素代谢中的作用， 在天然肠道中的吸收。因此，在新的初步研究中，我们开发了Slc 19 a3-/- 敲除小鼠模型，并显示肠道硫胺素摄取显著受损 与野生型小鼠相比，我们还建立了Slc 19 a2-/-敲除小鼠的群体， 我们的实验室此外，我们还利用细菌双杂交系统筛选了一个人 肠cDNA文库，并鉴定了两个推定的可以与hTHTR-1相互作用的蛋白质 （四跨膜蛋白和E-钙粘蛋白-1）。此外，我们还研究了 肠致病性大肠杆菌（EPEC），并发现显着抑制硫胺素的摄取。 根据我们以前和新的初步研究结果，我们提出这一建议的目的是：1）进一步 使用Slc 19 a2-/-和Slc 19 a3-/-敲除表征肠道硫胺素吸收过程 小鼠模型，并确定THTR-1和THTR-2在跨上皮和跨- 天然肠中的膜转运事件，2）为了鉴定顺式调节元件， 反式作用核因子参与硫胺素摄取的适应性上调 3）在人肠上皮细胞中鉴定与hTHTR-1和hTHTR-2相互作用的蛋白， 上皮细胞，并了解其生物学/生理学作用，以及4）确定 EPEC抑制小肠硫胺素摄取的细胞和分子机制 过程 这些研究的结果应继续提供新的和有价值的信息 关于肠道吸收硫胺素的细胞和分子机制， 这一过程及其监管以及对这一过程产生负面影响的因素。这 应该最终帮助我们设计有效的策略，以优化硫胺素体 与维生素B1缺乏和次优水平相关的疾病中的体内平衡，以及 使可能对该营养参数产生负面影响的外源因素的影响最小化。

Project Summary: 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.