Physiological and Pathological Aspects of Intestinal Vitamin B2 Absorption

 描述(由申请人提供)： 这一更新应用的长期目标继续侧重于研究肠道维生素B2(核黄素；RF)摄取过程的细胞/分子生理学和病理生理学，以及影响和干预这一事件的因素。由于RF在涉及脂肪、碳水化合物和氨基酸代谢的生物氧化还原反应中起着关键作用，并且在维生素B6和叶酸转化为其活性形式的过程中发挥关键作用，因此对正常的人类健康是必不可少的。最近的发现揭示了RF在正常免疫功能中的其他作用，作为抗炎和抗氧化剂，以及在维持正常的肠道稳态方面。人类(哺乳动物)不能合成RF，因此必须通过肠道吸收从外源获得维生素。我们实验室和其他实验室的研究已经表征了肠道RF吸收的不同方面，并表明这一过程是特异的和载体介导的；最近克隆的三个RFVT(RFVT-1、-2和-3；分别是SLC52A1、SLC52A2和SLC52A3基因的产物)都在肠道表达。在当前资助期间进行的研究中，我们使用了体外基因沉默(SiRNA)方法来表明顶部表达的RFVT-3在肠道RF吸收中起主要作用。在旨在建立RFVT-3在活体天然肠道中对肠道RF吸收的作用的新的初步研究中，我们建立了条件(肠道特异性)SLC52A3基因敲除(KO)小鼠模型，并计划使用它来证实和扩展我们的体外研究结果。在其他新的初步研究中，我们已经(通过同源建模/配体对接分析)确定了RFVT-3蛋白中可能对其功能重要的假定结构特征，获得了暗示microRNAs(MiRNAs)参与肠道RF摄取的转录后调节的证据，并确定了几个潜在的与RFVT-3在肠上皮细胞中相互作用的伙伴。我们还获得了证据表明，肠上皮细胞感染鼠伤寒沙门氏菌，暴露在促炎细胞因子和细菌内毒素下，显著抑制肠道RF的摄取。最后，我们获得了新的初步证据，表明表观遗传学机制(S)可能参与了我们在本资金期间观察到的慢性酒精喂养对肠道/结肠RF摄取的抑制效应。基于我们已发表的研究和新的初步发现，我们在这项提议中的工作假设是：1)rfvt-3在活体的天然肠道中对rf的吸收起重要作用；rfvt-3是由microrna转录后调控的；它具有可能影响其生理/细胞生物学的相互作用的伙伴(S)；2)沙门氏菌感染，暴露于促炎细胞因子和细菌内毒素可以抑制肠道rf的吸收；以及3)慢性酒精的抑制作用。 肠道中SLC52A3转录的暴露至少部分是通过表观遗传/分子机制调节的(S)。提出了三个具体的目标来检验这些假说，并将利用体内和体外最先进的生理、细胞和分子方法。这些研究的结果应该继续提供有关肠道RF摄取的细胞/分子生理学和病理生理学以及影响和干扰这一过程的外部因素的新的和有价值的信息。这最终将有助于我们设计有效的策略，以优化射频身体的动态平衡，特别是在与射频缺乏和次优水平相关的条件下。

 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.