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: 8139616
• 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/8139616
• 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 PUBLIC HEALTH RELEVANCE: Riboflavin (RF) is essential for normal human health and its deficiency leads to a variety of clinical abnormalities. Humans cannot synthesize RF and must obtain the vitamin from exogenous sources via intestinal absorption. Thus, the gut plays a central role in regulating normal RF body level. The objectives of this application are to characterize the molecular mechanisms involved in the regulation of the intestinal RF absorption and those involved in intracellular trafficking and membrane targeting of the involved transporter, as well as investigate the effect of chronic alcohol consumption on this process. Results of these studies are expected to provide important information on how the intestine regulates the absorption of vitamin B2, and how chronic alcohol use interferes with this process. Such knowledge should help in the designing of effective strategies to optimize RF homeostasis, especially in conditions associated with RF deficiency and sub-optimal level. 1

描述（由申请人提供）： 本申请的长期目标是表征参与调节维生素B2（核黄素，RF）的肠吸收的分子机制，以及参与所涉及的膜转运蛋白的细胞内运输和膜靶向的分子机制。我们还旨在研究长期饮酒对肠道RF吸收的影响，并确定所涉及的细胞和分子机制。 RF参与关键的细胞代谢反应，因此对正常的人类健康和福祉至关重要。它的缺乏，发生在各种条件下，如慢性酒精中毒，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）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。因此，人类肠道在维持和调节正常RF体稳态中起着核心作用。由于这些原因，了解肠道RF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，并且是本提案的目的。二十多年来，我们一直在研究肠道RF转运的生理学，但需要在分子水平上进行更多的研究以充分了解这一过程。随着最近哺乳动物RF转运蛋白的分子鉴定，我们现在处于一个很好的位置进行这样的调查。因此，在新的初步研究中，我们已经表明，我们先前所示的RF缺乏时肠RF摄取的上调是通过影响hRFT-2的转录调节机制介导的（至少部分）。我们还表明，肠RF摄取过程经历分化依赖性调节，并且这种调节再次似乎是通过转录机制介导的（至少部分地）。使用活的人肠上皮细胞的共聚焦成像和天然人肠刷状缘和基底侧膜制备物的Western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RF转运蛋白）在极化的人肠上皮细胞的顶端膜结构域的排他性表达。此外，不同的运输囊泡似乎参与人肠上皮细胞中hRFT 2的细胞内运动。最后，我们首次发现，长期酒精喂养显着抑制RF运输通过空肠BBM，这是与RFT-2的表达水平显着下降。我们的具体目标是：1）继续研究RF缺乏和细胞分化过程中肠RF摄取适应性上调的分子机制; 2）研究hRFT-2蛋白靶向极化的人肠上皮细胞顶端膜结构域的机制，并确定参与其细胞内运输的因子;和3）将我们对肠道RF摄取过程的基本生理/营养研究扩展到临床-本研究旨在研究长期饮酒对肠道RF吸收过程中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新的和有价值的信息，关于生理学/病理生理学和细胞/分子生物学的肠道RF摄取和干扰的因素的过程。这将最终帮助我们设计有效的策略来优化RF体内稳态，特别是在RF缺乏和次优水平的情况下。1 公共卫生相关性： 核黄素（RF）是人体正常健康所必需的，其缺乏会导致各种临床异常。人类不能合成RF，必须通过肠道吸收从外源性来源获得维生素。因此，肠道在调节正常RF体水平中起着核心作用。本申请的目的是表征参与调节肠RF吸收的分子机制以及参与细胞内运输和相关转运蛋白的膜靶向的分子机制，以及研究慢性饮酒对该过程的影响。这些研究的结果有望提供关于肠道如何调节维生素B2吸收以及长期饮酒如何干扰这一过程的重要信息。这些知识应该有助于设计有效的策略来优化RF稳态，特别是在与RF缺乏和次优水平相关的条件下。1