Intestinal Vitamin B2 Absorption: Molecular/Cellular Aspects and Effects of Alcoh

肠道维生素 B2 吸收：分子/细胞方面和酒精的影响

• 批准号: 8244942
• 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: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8244942
• 关键词: Actins; Acyl CoA Dehydrogenases; Affect; Alcoholism; Alcohols; Amino Acids; Anemia; Animals; Area; Binding; Biological; Biological Assay; Birth Place; Brush Border; C-terminal; Caco-2 Cells; Calories; Carbohydrates; Carcinogens; Cell Differentiation process; Cell physiology; Cells; Cellular biology; Chimeric Proteins; Chronic; Clinical; Computer Analysis; Diabetes Mellitus; Diet; Disease; Down-Regulation; Elderly; Elements; Endoplasmic Reticulum; Enterocytes; Epithelial; Epithelial Cells; Epithelium; Ethanol; Event; Fluorescence Microscopy; Folate; Functional disorder; Funding; Genes; Genetic Transcription; Green Fluorescent Proteins; Growth; Growth and Development function; Health; Heterogeneous Nuclear RNA; High Prevalence; Homeostasis; Human; Human Activities; Image; Impairment; Indium; Individual; Inflammatory Bowel Diseases; Injury; Intestinal Absorption; Intestines; Investigation; Kinetics; Knowledge; Laboratories; Large Intestine; Lead; Life; Lipids; Liquid substance; Mammals; Measures; Mediating; Membrane; Membrane Protein Traffic; Membrane Transport Proteins; Messenger RNA; Metabolic; Metabolism; Microfilaments; Microtubules; Modeling; Molecular; Molecular Biology; Movement; Mutate; N-terminal; Nervous system structure; Nuclear; Nuclear Trans-Acting Factor; Nucleic Acid Regulatory Sequences; Nutrient; Nutritional; Organ Donor; Oxidation-Reduction; Patients; Personal Satisfaction; Physiological; Physiology; Play; Positioning Attribute; Predisposition; Preparation; Process; Proteins; Rattus; Reaction; Recovery; Regulation; Regulatory Element; Retrieval; Riboflavin; Riboflavin Deficiency; Role; Site; Small Intestines; Source; Supplementation; Testing; Therapeutic Agents; Time; Tissues; Transcriptional Regulation; Transport Process; Tyrosine; Up-Regulation; Vertebral column; Vesicle; Villus; Vitamin B6; Vitamins; Western Blotting; Work; absorption; acyl-CoA dehydrogenase; alcohol effect; apical membrane; base; basolateral membrane; brush border membrane; cancer therapy; cell growth regulation; chronic alcohol ingestion; clinically relevant; design; extracellular; feeding; glycosylation; intestinal epithelium; novel; polyclonal antibody; polypeptide; problem drinker; promoter; protein folding; response; secretion process; skin lesion; trafficking; uptake

DESCRIPTION (provided by applicant): The long-term objectives of this application are to characterize the molecular mechanisms involved in the regulation of the intestinal absorption of vitamin B2 (riboflavin, RF), and those involved in intracellular trafficking and membrane targeting of the involved membrane transporter. We also aim at examining the effect of chronic alcohol use on intestinal RF absorption and determining the cellular and molecular mechanisms involved. RF is involved in critical cellular metabolic reactions, and thus, is essential for normal human health and well-being. Its deficiency, which occurs in a variety of conditions like chronic alcoholism, leads to serious clinical abnormalities that include degenerative changes in the nervous system, anemia, growth retardation, and skin lesions. Humans (and other mammals) cannot synthesize RF, and thus, must obtain the vitamin from exogenous sources via intestinal absorption. Thus, the human intestine plays a central role in maintaining and regulating normal RF body homeostasis. For these reasons understanding the physiology/pathophysiology as well as cell/molecular biology of the intestinal RF absorption process is of significant importance and is the aim of this proposal. We have been investigating the physiology of intestinal RF transport for over two decades, but more is needed at the molecular level to fully understand the process. With the recent molecular identification of mammalian RF transporters, we are now in an excellent position to carry out such investigations. Thus, in new preliminary studies we have shown that the up-regulation in intestinal RF uptake in RF deficiency shown by us previously is mediated (at least in part) via a transcriptional regulatory mechanism(s) affecting hRFT-2. We also showed that the intestinal RF uptake process undergoes differentiation - dependent regulation and that this regulation again appears to be mediated (at least in part) via a transcriptional mechanism(s). Using confocal imaging of living human intestinal epithelia cells and Western blot analysis of native human intestinal brush border and basolateral membrane preparations, our preliminary studies further showed exclusive expression of the human RF transporter -2 (hRFT-2; the most relevant intestinal RF transporter) at the apical membrane domain of polarized human intestinal epithelial cells. Furthermore, distinct trafficking vesicles appear to be involved in intracellular movement of hRFT2 in human intestinal epithelial cells. Finally, we showed for the first time that chronic alcohol feeding significantly inhibit RF transport across the jejunal BBM which is associated with a significant decrease in the level of expression of RFT-2. Our Specific Objectives in this proposal are: 1) To continue our investigations into the molecular mechanisms involved in the adaptive up-regulation in intestinal RF uptake in RF deficiency and during cell differentiation; 2) To study the mechanism(s) involved in the targeting of the hRFT-2 protein to the apical membrane domain of polarized human intestinal epithelial cells, and to determine the factor(s) involved in its intracellular trafficking; and 3) to extend our basic physiological/nutritional investigations on the intestinal RF uptake process into a clinically-relevant area and will examine the effect of chronic alcohol consumption on cell and molecular parameters of intestinal RF absorption process. Collectively, results of these studies should provide novel and valuable information regarding the physiology/pathophysiology and cell/molecular biology of intestinal RF uptake and of the factors that interfere with the process. This should ultimately assist us in the designing of effective strategies to optimize RF body homeostasis, especially in conditions of RF deficiency and sub- optimal levels. 1

描述（由申请人提供）： 该应用的长期目标是表征参与调节维生素 B2（核黄素，RF）肠道吸收的分子机制，以及参与细胞内运输和相关膜转运蛋白的膜靶向的分子机制。我们还旨在研究长期饮酒对肠道射频吸收的影响，并确定所涉及的细胞和分子机制。 射频参与关键的细胞代谢反应，因此对于人类的正常健康和福祉至关重要。它的缺乏会发生在慢性酒精中毒等多种疾病中，导致严重的临床异常，包括神经系统的退行性变化、贫血、生长迟缓和皮肤病变。人类（和其他哺乳动物）无法合成RF，因此必须通过肠道吸收从外源获取维生素。因此，人体肠道在维持和调节正常的射频体内稳态方面发挥着核心作用。由于这些原因，了解肠道射频吸收过程的生理学/病理生理学以及细胞/分子生物学具有重要意义，也是本提案的目的。二十多年来，我们一直在研究肠道射频传输的生理学，但需要在分子水平上进行更多研究才能充分理解这一过程。随着最近对哺乳动物射频转运蛋白的分子鉴定，我们现在处于开展此类研究的绝佳位置。因此，在新的初步研究中，我们已经表明，我们之前所显示的 RF 缺陷中肠道 RF 摄取的上调是通过影响 hRFT-2 的转录调节机制介导（至少部分）的。我们还表明，肠道 RF 摄取过程经历分化依赖性调节，并且这种调节似乎再次（至少部分）通过转录机制介导。利用活人肠上皮细胞的共聚焦成像以及对天然人肠刷状缘和基底外侧膜制剂的蛋白质印迹分析，我们的初步研究进一步显示了人RF转运蛋白-2（hRFT-2；最相关的肠道RF转运蛋白）的独家表达在极化的人肠上皮细胞的顶膜域。此外，不同的运输囊泡似乎参与了人肠上皮细胞中 hRFT2 的细胞内运动。最后，我们首次表明，长期饮酒会显着抑制 RF 穿过空肠 BBM 的转运，这与 RFT-2 表达水平的显着降低有关。我们在本提案中的具体目标是： 1) 继续研究 RF 缺乏和细胞分化过程中肠道 RF 摄取适应性上调所涉及的分子机制； 2) 研究hRFT-2蛋白靶向极化人肠上皮细胞顶膜结构域的机制，并确定参与其细胞内运输的因素； 3）将我们对肠道射频吸收过程的基本生理/营养研究扩展到临床相关领域，并将检查长期饮酒对肠道射频吸收过程的细胞和分子参数的影响。 总的来说，这些研究的结果应该提供有关肠道射频吸收的生理学/病理生理学和细胞/分子生物学以及干扰该过程的因素的新颖且有价值的信息。这最终将有助于我们设计有效的策略来优化 RF 身体稳态，特别是在 RF 缺乏和次优水平的情况下。 1