Effect of Pathophysiological Conditions on Intestinal Absorption of Free Thiamin

病理生理条件对游离硫胺素肠道吸收的影响

• 批准号: 10651601
• 负责人: HAMID M SAID
• 金额: --
• 依托单位: VETERANS HEALTH ADMINISTRATION
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10651601
• 关键词: Affect; Affinity; Alcoholism; Apical; C-terminal; Cell physiology; Cells; Cellular biology; Chronic; Colon; Cytoplasm; Cytoplasmic Tail; Diet; Diphosphates; Diphosphotransferases; Disease; Energy Metabolism; Enzymes; Epithelial Cells; Epithelium; Exposure to; Flagellin; Functional disorder; General Population; Generations; Genes; Genomics; Gut Mucosa; Health; Homeostasis; Human; Hypoxia; Impairment; Inflammatory; Inflammatory Bowel Diseases; Intestinal Absorption; Intestines; Investigation; Knockout Mice; Laboratories; Large Intestine; Lipopolysaccharides; Mammals; Mediating; Mitochondria; Molecular; Normal Cell; Nutritional; Organ; Oxidative Stress; Phenotype; Phosphorylation; Physiological; Physiology; Play; Process; Production; Proteins; Regulation; Research; Role; Sepsis; Small Intestines; Source; Susceptibility Gene; System; Testing; Thiamine; Transport Process; Ulcerative Colitis; Veterans; Vitamin Deficiency; Vitamins; Water-Soluble Vitamin; absorption; apical membrane; cytokine; design; dietary; gastrointestinal system; gut microbiota; improved; intestinal epithelium; microbiota; mouse model; novel; nutrition; uptake

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.

项目摘要/摘要 我们实验室的研究重点是研究研究分子生理，病理生理学和细胞生物学 消化系统器官器官中水溶性维生素的运输过程。在此提案中，我们的目标是 继续我们对吸收过程的生理，病理生理学和细胞生物学的研究 维生素B1的饮食和微生物群生成形式[i。例如，硫胺素和硫胺素的硫酸硫酸盐 （TPP）]在小肠和大肠中。维生素B1（以其生物学活性形式，即e。，TPP）对于正常 由于其在氧化能量代谢中起着关键作用，ATP产生，所有细胞的生理和健康 并减少细胞氧化应激。维生素B1的缺乏发生在各种情况下 炎症性肠病，败血症和慢性酒精中毒。 人/哺乳动物不能内源性地合成维生素B1，因此必须从中获得维生素 通过肠吸收来源。宿主可以使用两种维生素来源：饮食和 微生物群生成。我们以前已经表征了自由硫胺素吸收过程的不同方面 沿着肠道，显示两个传输系统的参与：硫胺素转运蛋白1＆-2（thtr- 1＆-2; SLC19A2和SLC19A3基因的产物）。至于微生物群生成的维生素B1， 该来源以自由和磷酸化（TPP）形式提供硫胺素。我们实验室的研究 表明这两种形式在结肠中都是可吸收的。自由硫胺素的吸收如小 通过涉及Thtr-1和-2的载体介导的过程，TPP的肠道介导的过程是通过独特的高 - 亲和力和特定（即不传输硫胺素）载体介导的过程。随后的研究 实验室已将特定的TPP转运蛋白从结肠（CTPPT； SLC44A4基因的乘积）克隆 并发现其沿着肠道的表达仅限于大肠，并且发生 仅在衬里上皮的顶端膜结构域。我们在此提案中的目标是：1） 确定CTPPT对微生物群生成的TPP的总载体介导的摄取的贡献 本地结肠，其在正常结肠生理/健康中的作用以及研究其细胞生物学方面；和2）到 检查肠道暴露于某些疾病状态下的条件/因素的影响 [即：缺氧，促炎细胞因子和细菌脂多糖（LPS）和鞭毛蛋白] 结肠/小肠道TPP和免费的硫胺素吸收。因此，在新的初步研究中，我们产生了 SLC44A4敲除（KO）小鼠模型，最初表征显示清晰的表型（受损 与野生型同窝室相比，结肠TPP的吸收）。我们还确定了与CTPPT的推定互动 结肠细胞，并在顶端靶向中显示其细胞质尾巴的重要作用。此外，我们获得了 证据表明，结肠/小肠上皮暴露于缺氧，促炎细胞因子，LPS 并导致抑制维生素B1形式的抑制作用。基于这些新的（和以前）的发现，我们的 该提案中的工作假设是CTPPT（SLC44A4）是载体的主要系统 在天然结肠中介导的菌群生成的TPP的摄取，该转运蛋白对结肠很重要 生理学和健康，CTPPT具有相互作用的伴侣，并且CTPPT的顶端靶向由 其C末端结构域中的特定结构基序。我们还假设结肠/小肠暴露 对缺氧，促炎性细胞因子和细菌产物的上皮症，导致TPP抑制和游离 硫胺素的吸收。我们将通过实现两个具体目标来检验这些假设，并将利用最先进 生理/细胞/分子方法。这些研究的结果应提供有关有关的新信息 沿着健康和疾病的肠道吸收维生素B1。这应该有助于设计有效 优化全球（身体）和局部（肠粘膜）维生素B1营养的策略，从而改善健康。