Physiological and Pathological Aspects of Intestinal Vitamin B2 Absorption

 描述（由申请人提供）： 本次更新申请的长期目标继续关注于研究肠道维生素B2（核黄素; RF）摄取过程的细胞/分子生理学和病理生理学以及影响和干扰事件的因素。RF对正常人类健康至关重要，因为它在涉及脂质、碳水化合物和氨基酸代谢的生物氧化还原反应中以及在维生素B6和叶酸转化为其活性形式中发挥关键作用。最近的研究发现RF在正常免疫功能中的其他作用，作为抗炎和抗氧化剂，以及维持正常的肠道内稳态。 人类（哺乳动物）不能合成RF，因此必须通过肠道吸收从外源来源获得维生素。来自我们实验室和其他人的研究已经表征了肠道RF吸收的不同方面，并表明该过程是特异性的和载体介导的;最近克隆的所有三种RFVT（RFVT-1，-2和-3;分别是SLC 52 A1，SLC 52 A2和SLC 52 A3基因的产物）也在肠道中表达。 在当前资助期内进行的研究中，我们使用体外基因沉默（siRNA）方法表明，顶端表达的RFVT-3在肠道RF摄取中起主要作用。在旨在确定RFVT-3在体内天然肠中肠RF吸收中的作用的新的初步研究中，我们产生了条件性（结肠特异性）SLC 52 A3敲除（KO）小鼠模型，并计划使用它来确认和扩展我们的体外发现。在其他新的初步研究中，我们已经确定了（通过同源建模/配体对接分析）RFVT-3蛋白的推定结构特征，可能是重要的，其功能，获得的证据表明微RNA（miRNA）在转录后调节肠道RF摄取，并确定了几个潜在的相互作用伙伴与RFVT-3在肠上皮细胞。我们还获得了证据表明，肠上皮细胞感染S。鼠伤寒沙门氏菌，并暴露于促炎细胞因子和细菌LPS显着导致抑制肠RF摄取。最后，我们获得了新的初步证据，表明表观遗传机制可能参与了我们在当前资助期间观察到的长期酒精喂养对肠道/结肠RF摄取的抑制作用。 基于我们已发表的研究和新的初步发现，我们在本提案中的工作假设是：1）RFVT-3在体内天然肠道中的RF吸收中起重要作用;该转运蛋白受microRNA的转录后调节;并且它具有可能影响其生理学/细胞生物学的相互作用伴侣; 2）沙门氏菌感染，以及暴露于促炎细胞因子和细菌LPS抑制肠RF摄取;和3）慢性酒精的抑制作用 在肠中暴露于SLC 52 A3转录至少部分地通过表观遗传/分子机制介导。提出了三个具体目标来测试这些假设，并将利用最先进的体内和体外生理，细胞和分子方法。 这些研究的结果应继续提供新的和有价值的信息，关于细胞/分子生理学和病理生理学的肠道RF摄取和外部因素的影响和干扰的过程。这将最终帮助我们设计有效的策略来优化RF体内平衡，特别是在与RF缺乏和次优水平相关的情况下。

 DESCRIPTION (provided by applicant): The long-term objectives of this renewal application continue to focus on investigating the cell/molecular physiology and pathophysiology of the intestinal vitamin B2 (riboflavin; RF) uptake process and of the factors that affect and interfere with the event. RF is essential for normal human health due to the key roles it plays in biological oxidation-reduction reactions involving lipid, carbohydrate and amino acid metabolism, and in the conversion of vitamin B6 and folate into their active forms. Recent findings have uncovered additional roles for RF in normal immune function, as an anti-inflammatory and anti-oxidant agent, and in the maintenance of normal intestinal homeostasis. Humans (mammals) cannot synthesize RF, and thus, must obtain the vitamin from exogenous sources via intestinal absorption. Studies from our laboratory and others have characterized different aspects of intestinal RF absorption and shown the process is specific and carrier-mediated; also all the three recently cloned RFVTs (RFVT-1, -2 & -3; products of the SLC52A1, SLC52A2 & SLC52A3 genes, respectively) are expressed in the intestine. In studies performed during the current funding period, we used an in vitro gene-silencing (siRNA) approach to show that the apically expressed RFVT-3 plays a major role in intestinal RF uptake. In new preliminary studies aimed at establishing the role of RFVT-3 in intestinal RF absorption in native intestine in vivo, we generated a conditional (intestinal-specific) SLC52A3 knockout (KO) mouse model and plan to use it to confirm and extend our in vitro findings. In other new preliminary studies, we have identified (via homology modeling/ligand docking analyses) putative structural features in the RFVT-3 protein that may be important for its function, obtained evidence implicating microRNAs (miRNAs) in post-transcriptional regulation of intestinal RF uptake, and have identified several potential interactig partners with RFVT-3 in intestinal epithelial cells. We also obtained evidence showing that infection of intestinal epithelial cells with S. Typhimurium, and exposure to pro-inflammatory cytokines and to bacterial LPS significantly lead to inhibition in intestinal RF uptake. Finally, w obtained new preliminary evidence suggesting possible involvement of epigenetic mechanism(s) in the inhibitory effect of chronic alcohol feeding on intestinal/colonic RF uptake that we observed during the current funding period. Based on our published studies and new preliminary findings, our working hypotheses in this proposal are that: 1) the RFVT-3 plays an important role in RF absorption in native intestine in vivo; that the transporter is post-transcriptionally regulated by microRNA; and that it has interacting partner(s) that may influence its physiology/cell biology; 2) Salmonella infection, and exposure to pro-inflammatory cytokines and to bacterial LPS inhibits intestinal RF uptake; and 3) the inhibitory effect of chronic alcohol exposure on SLC52A3 transcription in the intestine is mediated, at least in part, via epigenetic/molecular mechanism(s). Three specific aims are proposed to test these hypotheses and will utilize state-of-the-art in vivo and in vitro physiological, cellular, and molecular approaches. Results of these studies should continue to provide novel and valuable information regarding the cell/molecular physiology and pathophysiology of intestinal RF uptake and of external factors that affect and interfere with the process. This should ultimately assist us in th designing of effective strategies to optimize RF body homeostasis, especially in conditions associated with RF deficiency and sub-optimal levels.