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.

项目总结/摘要 我们实验室的研究重点是研究的分子生理学，病理生理学和细胞生物学， 水溶性维生素在消化系统器官中的运输过程。在本建议中，我们的目标是 继续我们的研究生理学，病理生理学和细胞生物学的吸收过程， 饮食和微生物群产生的维生素B1形式[i.例如，游离硫胺素和焦磷酸硫胺素 （TPP）]在小肠和大肠中。维生素B1（以其生物活性形式，即例如，TPP）对正常 由于它在氧化能量代谢，ATP产生， 和减少细胞氧化应激。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，必须从 通过肠道吸收的外源性来源。宿主可获得两种维生素来源：饮食和 微生物产生的。我们以前的特点不同方面的吸收过程中的游离硫胺素 沿着肠道，并显示涉及两个运输系统：硫胺素转运蛋白-1和-2（THTR-1）。 1和-2;分别为SLC 19 A2和SLC 19 A3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 表明这两种形式在结肠中都是可吸收的;游离硫胺素的吸收发生在小的 肠通过载体介导的过程，涉及THTR-1和-2，而TPP通过不同的，高- 亲和力和特异性（即，不运输游离硫胺素）载体介导的过程。我们的后续研究 实验室已经从结肠克隆了一种特异性TPP转运蛋白（cTPPT; SLC 44 A4基因的产物） 并发现其沿着肠道的表达仅限于大肠， 仅在衬里上皮的顶膜区域。我们在这项建议中的目标是：1） 确定cTPPT对微生物群产生的TPP的总载体介导的摄取的贡献， 天然结肠，其在正常结肠生理学/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（LPS）和鞭毛蛋白] 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 Slc 44 a4敲除（KO）小鼠模型，其在初始表征时显示清楚的表型（具有受损的 结肠TPP摄取）。我们还确定了与cTPPT的假定相互作用， 结肠细胞，并显示其细胞质尾在顶端靶向的重要作用。此外，我们获得 有证据表明，结肠/小肠上皮细胞暴露于缺氧、促炎细胞因子、LPS 和鞭毛蛋白，导致维生素B1形式的摄取抑制。根据这些新的（和以前的）发现，我们的 该建议中的工作假设是cTPPT（SLC 44 A4）是涉及载体的主要系统， 在天然结肠中微生物群产生的TPP介导的摄取，这种转运蛋白对结肠 生理学和健康，cTPPT具有相互作用的配偶体，并且cTPPT的顶端靶向由以下决定： 在其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 上皮细胞缺氧，促炎细胞因子和细菌产物，导致抑制TPP和游离 硫胺素摄取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/细胞/分子方法。这些研究的结果应该提供新的信息， 维生素B1的吸收沿着肠道在健康和疾病。这将有助于设计有效的 优化整体（身体）和局部（肠道粘膜）维生素B1营养的策略，从而改善健康。