Effect of Pathophysiological Conditions on Intestinal Absorption of Free Thiamin

项目摘要/摘要 我们实验室的研究主要集中在分子生理学、病理生理学和细胞生物学方面。 消化系统器官中水溶性维生素的运输过程。在这项建议中，我们的目标是 继续我们对阿司匹林吸收过程的生理学、病理生理学和细胞生物学的研究 饮食和微生物产生的维生素B1[即游离硫胺素和焦磷硫胺素] (TPP)]在小肠和大肠。维生素B1(具有生物活性的形式，即TPP)对于正常人体是必不可少的。 所有细胞的生理和健康，因为它在氧化能量代谢，ATP产生， 和减少细胞的氧化应激。维生素B1缺乏可在多种情况下发生，包括 炎症性肠病、败血症和慢性酒精中毒。 人类/哺乳动物不能内源性合成维生素B1，因此必须从 通过肠道吸收的外源物质。寄主可获得两种维生素来源：膳食和 微生物区系产生的。我们之前已经描述了游离硫胺素摄取过程的不同方面 发现有两个转运系统参与其中：硫胺素转运体-1和-2(THTR-2)。 分别是SLC19A2和SLC19A3基因的产物)。至于微生物群产生的维生素B1， 该来源提供游离和磷酸化(TPP)形式的硫胺素。我们实验室的研究表明 表明这两种形式的硫胺素在结肠中都是可吸收的；游离硫胺素的吸收与小的 肠道通过载体介导的过程，涉及Thtr-1和-2，而TPP的过程通过不同的，高水平的 亲和力和特异性(即不运输游离硫胺素)载体介导的过程。来自我们的后续研究 实验室已经从结肠中克隆出一种特殊的TPP转运蛋白(cTPPT；SLC44A4基因的产物) 并发现其在肠道中的表达仅限于大肠，并发生 仅位于衬里上皮细胞的顶膜区。我们在这项建议中的目标是：1) 确定cTPPT对载体介导的微生物群产生的TPP的总摄取的贡献 天然结肠，其在正常结肠生理/健康中的作用，并研究其细胞生物学方面；以及2) 检查肠道在某些疾病状态下暴露的条件/因素的影响 [即：缺氧、促炎细胞因子、细菌脂多糖和鞭毛] 结肠/小肠TPP和游离硫胺素摄取。因此，在新的初步研究中，我们产生了一个 Slc44a4基因敲除(KO)小鼠模型，初步特征显示明显的表型(伴有受损 结肠型TPP摄取)与野生型产仔鼠进行比较。我们还确定了与cTPPT可能的相互作用。 其胞浆尾部在根尖靶向中起重要作用。此外，我们还获得了 有证据表明，结肠/小肠上皮细胞暴露于低氧、促炎细胞因子、内毒素 和鞭毛导致维生素B1形式的吸收受到抑制。基于这些新的(和以前的)发现，我们的 本方案中的工作假设是cTPPT(SLC44A4)是涉及载波- 微生物区系产生的TPP在天然结肠中的介导性摄取，即该转运蛋白对结肠重要 生理和健康，cTPPT有互动伙伴(S)，ctpt顶端靶点由 C-末端结构域中的特定结构基序(S)。我们还假设暴露在结肠/小肠中 上皮细胞对低氧、促炎细胞因子和细菌产物的抑制作用导致TPP和游离 硫胺素摄取。我们将通过实现两个特定目标来检验这些假设，并将利用最先进的技术 生理/细胞/分子方法。这些研究的结果应该提供关于以下方面的新信息 维生素B1在健康和疾病中的肠道吸收。这应该有助于有效地设计 优化全球(身体)和局部(肠道粘膜)维生素B1营养的战略，从而改善健康。

PROJECT SUMMARY/ABSTRACT Research in our laboratory focuses on studying the molecular physiology, pathophysiology and cell biology of the transport processes of water-soluble vitamins in organs of the digestive system. In this proposal, we aim to continue our investigations into the physiology, pathophysiology and cell biology of the absorption processes of both dietary and microbiota-generated forms of vitamin B1 [i. e., free thiamin and that of thiamin pyrophosphate (TPP)] in the small and large intestine. Vitamin B1 (in its biologically active form, i. e., TPP) is essential for normal physiology and health of all cells due to the critical roles it plays in oxidative energy metabolism, ATP production, and reduction of cellular oxidative stress. Deficiency of vitamin B1 occurs in a variety of conditions including inflammatory bowel diseases, sepsis and chronic alcoholism. Humans/mammals cannot synthesize vitamin B1 endogenously, and thus, must obtain the vitamin from exogenous sources via intestinal absorption. Two sources of the vitamin are available to the host: dietary and microbiota-generated. We have previously characterized different aspects of the uptake process of free thiamin along the intestinal tract, and showed involvement of two transport systems: thiamin transporter-1 & -2 (THTR- 1 & -2; products of the SLC19A2 & SLC19A3 genes, respectively). As to the microbiota-generated vitamin B1, this source provides thiamin in both free and phosphorylated (TPP) forms. Studies from our laboratory have shown that both of these forms are absorbable in the colon; absorption of free thiamin occurs as in the small intestine via a carrier-mediated process that involves THTR-1 & -2, while that of TPP occurs via a distinct, high- affinity and specific (i.e., does not transport free thiamin) carrier-mediated process. Subsequent studies from our laboratory have cloned a specific TPP transporter from the colon (the cTPPT; product of the SLC44A4 gene) and found its expression along the intestinal tract to be restricted to the large intestine only, and occurs exclusively at the apical membrane domain of the lining epithelia. Our objectives in this proposal are: 1) To determine the contribution of cTPPT toward total carrier-mediated uptake of the microbiota-generated TPP in the native colon, its role in normal colon physiology/health, and to study aspects of its cell biology; and 2) To examine the effect of conditions/factors that the intestinal tract is exposed to under certain disease states [namely: hypoxia, pro-inflammatory cytokines, and bacterial lipopolysaccharides (LPS) and flagellin] on colonic/small intestinal TPP and free thiamin uptake. Thus, in new preliminary studies we have generated a Slc44a4 knockout (KO) mouse model, which upon initial characterization showed clear phenotype (with impaired colonic TPP uptake) compared to wild-type littermates. We also identified putative interactors with cTPPT in colonocytes, and showed an essential role for its cytoplasmic tail in apical targeting. Furthermore, we obtained evidence to show that exposure of colonic/small intestinal epithelia to hypoxia, pro-inflammatory cytokines, LPS and flagellin to lead to inhibition in uptake of vitamin B1 forms. Based on these new (and previous) findings, our working hypotheses in this proposal are that the cTPPT (SLC44A4) is the predominant system involved in carrier- mediated uptake of the microbiota-generated TPP in native colon, that this transporter is important for colon physiology and health, that cTPPT has interacting partner(s), and that apical targeting of cTPPT is dictated by specific structural motif(s) in its c-terminal domain. We also hypothesize that exposure of colonic/small intestinal epithelia to hypoxia, pro-inflammatory cytokines, and to bacterial products to lead to inhibition in TPP and free thiamin uptake. We will test these hypotheses by accomplishing two specific aims and will utilize state-of the-art physiological/cellular/molecular approaches. Results of these studies should provide new information regarding vitamin B1 absorption along the intestinal tract in health and disease. This should assist in the designing effective strategies to optimize global (body) and local (gut mucosal) vitamin B1 nutrition, and thus, improve health.