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: 10246647
• 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/10246647
• 关键词: Affect; Affinity; Alcoholism; Apical; C-terminal; Cell physiology; Cells; Cellular biology; Chronic; Colon; Cytoplasmic Tail; Diet; Diphosphates; Diphosphotransferases; Disease; Energy Metabolism; Enzymes; Epithelial; Epithelial Cells; 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; Lead; Lipopolysaccharides; Mammals; Mediating; Mitochondria; Molecular; Normal Cell; Nutritional; Organ; Oxidative Stress; Phenotype; 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; base; 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.

项目概要/摘要 我们实验室的研究重点是研究分子生理学、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。在本提案中，我们的目标是 继续我们对吸收过程的生理学、病理生理学和细胞生物学的研究 膳食和微生物产生的维生素 B1 [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（其生物活性形式，即 TPP）对于正常的生命活动至关重要 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/哺乳动物不能内源合成维生素 B1，因此必须从体内获取维生素 外源性通过肠道吸收。宿主有两种维生素来源：饮食和 微生物群产生的。我们之前已经描述了游离硫胺素吸收过程的不同方面 沿着肠道，并显示出两个运输系统的参与：硫胺素转运蛋白-1和-2（THTR- 1 & -2;分别是SLC19A2和SLC19A3基因的产物）。至于微生物产生的维生素 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 表明这两种形式均可在结肠中吸收；游离硫胺素的吸收发生在小 肠道通过涉及 THTR-1 和 -2 的载体介导过程进行，而 TPP 则通过独特的、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 实验室从结肠中克隆了一种特定的 TPP 转运蛋白（cTPPT；SLC44A4 基因的产物） 并发现其沿着肠道的表达仅限于大肠，并且发生 仅位于衬里上皮的顶膜区域。我们在此提案中的目标是： 1) 确定 cTPPT 对载体介导的微生物群产生的 TPP 的总吸收的贡献 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的各个方面； 2) 至 检查肠道在某些疾病状态下所暴露的条件/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 Slc44a4 敲除 (KO) 小鼠模型，在初步表征后显示出清晰的表型（受损 结肠 TPP 摄取）与野生型同窝小鼠相比。我们还确定了与 cTPPT 的假定相互作用者 结肠细胞，并显示其细胞质尾部在顶端靶向中发挥重要作用。此外，我们还获得了 有证据表明结肠/小肠上皮暴露于缺氧、促炎细胞因子、LPS 和鞭毛蛋白导致维生素 B1 形式的摄取受到抑制。根据这些新的（和之前的）发现，我们 该提案中的工作假设是 cTPPT (SLC44A4) 是涉及运营商的主要系统 介导天然结肠中微生物群产生的 TPP 的摄取，表明该转运蛋白对于结肠很重要 生理学和健康，cTPPT 具有相互作用的伙伴，并且 cTPPT 的顶端靶向取决于 其 C 端结构域中的特定结构基序。我们还假设结肠/小肠的暴露 上皮细胞对缺氧、促炎细胞因子和细菌产物的影响导致 TPP 和游离的抑制 硫胺素吸收。我们将通过实现两个具体目标来测试这些假设，并将利用最先进的技术 生理/细胞/分子方法。这些研究的结果应该提供有关以下方面的新信息： 维生素 B1 在健康和疾病中沿着肠道吸收。这应该有助于设计有效的 优化整体（身体）和局部（肠粘膜）维生素 B1 营养的策略，从而改善健康。