Absorption of vitamin B1 along the intestinal tract: Physiology, pathophysiology and cell biology aspects

该提案的目标继续集中在研究肠道中维生素B1吸收过程的生理学/病理生理学和细胞生物学。维生素B1（以其生物活性形式，TPP）对所有细胞的正常生理和健康至关重要，因为它在氧化能量代谢，ATP产生和减少细胞氧化应激中起着关键作用。维生素B1缺乏症发生在各种情况下，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，因此必须通过肠道吸收从外源性来源获得维生素。宿主可获得两种维生素来源：饮食和微生物群产生的。我们先前已经表征了游离硫胺素沿着肠道的摄取过程的不同方面，并且显示了两个转运系统的参与：硫胺素转运蛋白-1和-2（分别为SLC 19 A2和SLC 19 A3）。至于微生物群产生的维生素B1，相当数量的这种来源是以TPP的形式提供的，我们已经确定了一种高亲和力和特异性的载体介导的过程，用于其被结肠细胞摄取。我们还从结肠克隆了一种特异性TPP转运蛋白（cTPPT;产物SLC 44 A4基因），显示其沿着胃肠道的表达仅限于大肠，并证明其仅定位于上皮细胞的顶膜结构域。我们在该提案中的目标是确定cTPPT对天然结肠组织中TPP的总载体介导的摄取的贡献，其在结肠生理学/健康中的作用，其是否具有影响其生理学/细胞生物学的相互作用物，以及决定其顶端靶向的分子决定因素。我们还旨在研究在某些疾病状态下肠道暴露于的条件/因素[即缺氧，促炎细胞因子和细菌脂多糖（LPS）和鞭毛蛋白]对TPP摄取的影响。在新的初步研究中，我们已经产生了纯合子Slc 44 a4 loxP+/+小鼠[产生大的丝氨酸特异性Slc 44 a4敲除（KO）小鼠的关键步骤]，鉴定了与cTPPT的假定相互作用，并发现其胞质尾在顶端靶向中的关键作用。我们还发现，暴露于结肠上皮细胞缺氧，促炎细胞因子，LPS和鞭毛蛋白，导致抑制维生素B1的摄取。基于这些新的（和以前的）发现，我们在该提案中的工作假设是cTPPT（SLC 44 A4）是天然结肠中参与载体介导的TPP摄取的主要系统，它对结肠生理学/健康很重要，它具有相互作用的伴侣，并且它的顶端靶向由其结构中的特定结构基序决定。我们还假设结肠上皮细胞暴露于缺氧、促炎细胞因子和细菌产物导致TPP摄取抑制。我们将通过实现两个具体目标来测试这些假设，并将利用最先进的生理/分子方法。这些研究的结果应该提供关于健康和疾病中肠道维生素B1吸收的新信息，这应该有助于设计有效的策略来优化其稳态/营养，从而改善健康。

The objectives of this proposal continue to focus on investigating the physiology/pathophysiology and cell biology of the absorption processes of vitamin B1 in the intestinal tract. Vitamin B1 (in its biologically active form, TPP) is essential for normal physiology and health of all cells due to its critical roles 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 (SLC19A2 and SLC19A3, respectively). As to the microbiota-generated vitamin B1, a considerable amount of this source is provided in the form of TPP, and we have identified a high-affinity and specific carrier-mediated process for its uptake by colonocytes. We have also cloned a specific TPP transporter from the colon (the cTPPT; product SLC44A4 gene), showed its expression along the GI tract to be restricted to the large intestine, and demonstrated its exclusive localization at the apical membrane domain of epithelia. Our objectives in this proposal are to determine the contribution of cTPPT toward total carrier-mediated uptake of TPP in native colonic tissue, its role in colon physiology/health, whether it has interactor(s) that influence its physiology/cell biology, and molecular determinant(s) that dictates its apical targeting. We also aim to investigate the effects of conditions/factors that the intestinal tract is exposed to under certain disease states [namely, hypoxia, pro-inflammatory cytokines, and the bacterial lipopolysaccharides (LPS) and flagellin] on TPP uptake. In new preliminary studies, we have generated homozygous Slc44a4 loxP+/+ mice [a critical step towards generating a large intestine-specific Slc44a4 knockout (KO) mice], identified putative interactors with cTPPT, and found a critical role for its cytoplasmic tail in apical targeting. We also found that exposure of colonic epithelia to hypoxia, pro-inflammatory cytokines, LPS and flagellin to lead to inhibition in vitamin B1 uptake. 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 TPP uptake in native colon, it is important for colon physiology/health, it has interacting partner(s), and that its apical targeting is dictated by specific structural motif(s) in its structure. We also hypothesize that exposure of colonic epithelia to hypoxia, pro- inflammatory cytokines, and to bacterial products to lead to inhibition in TPP uptake. We will test these hypotheses by accomplishing two specific aims and will utilize state-of the-art physiological/molecular approaches. Results of these studies should provide new information regarding intestinal vitamin B1 absorption in health and disease, which should assist in the designing effective strategies to optimize its homeostasis/nutrition, and thus, improve health.