Mechanism/Regulation of Intestinal Thiamin Uptake

肠道硫胺素摄取的机制/调节

• 批准号: 7905077
• 负责人: HAMID M SAID
• 金额: $ 36.44万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7905077
• 关键词: Address; Affect; Amino Acids; Animals; Apical; Apoptosis; Binding; Biological; Biological Assay; Biopsy; Biopsy Specimen; Biotinylation; Caco-2 Cells; Cardiovascular Diseases; Cardiovascular system; Celiac Disease; Cell Line; Cell membrane; Cell physiology; Cells; Cellular biology; Chemicals; Chronic; Clinical; Co-Immunoprecipitations; Coenzymes; Computer Analysis; Country; Decarboxylation; Developed Countries; Developing Countries; Development; Diabetes Mellitus; Diabetic Retinopathy; Diarrhea; Diet; Disease; E-Cadherin; Elderly; Energy Metabolism; Enterocytes; Epithelial; Epithelial Cells; Escherichia coli Infections; Event; Exogenous Factors; Firefly Luciferases; Funding; Genes; Goals; Green Fluorescent Proteins; Growth; Half-Life; Health; Health Status; Homeostasis; Human; Human body; Hyperglycemia; Image; Impairment; In Vitro; Incidence; Indium; Individual; Infection; Inflammatory Bowel Diseases; Integral Membrane Protein; Intestinal Absorption; Intestines; Investigation; Kidney; Kinetics; Knock-out; Knockout Mice; Knowledge; Laboratories; Laboratory Finding; Lead; Length; Life; Maintenance; Mammals; Mediating; Megaloblastic Anemia; Membrane; Membrane Transport Proteins; Messenger RNA; Metabolic Pathway; Metabolism; Methods; Micronutrients; Microtubules; Modeling; Molecular; Mus; Mutate; Mutation; Nature; Neurologic; New York; Normal Cell; Nuclear; Nuclear Trans-Acting Factor; Nucleic Acid Regulatory Sequences; Nutrient; Nutritional; Oxidative Stress; Patients; Pentosephosphate Pathway; Pentosephosphates; Pentoses; Perfusion; Pharmacology; Physiological; Physiology; Play; Post-Translational Regulation; Preparation; Process; Production; Proteins; Publishing; Pyruvic Acid; RNA; Reaction; Regulation; Regulatory Element; Reporter Genes; Risk; Role; Screening procedure; Series; Signal Transduction; Site; Small Interfering RNA; Source; Specificity; Structure of beta Cell of islet; Structure of retinal pigment epithelium; Subfamily lentivirinae; Symptoms; Syndrome; System; Techniques; Testing; Thiamine; Thiamine Deficiency; Thiamine Pyrophosphate; Time; Tissues; Trans-Activators; Transgenic Mice; Transmembrane Transport; Transport Process; Type III Secretion System Pathway; Up-Regulation; Vesicle; Vitamins; Water-Soluble Vitamin; Western Blotting; Wild Type Mouse; Work; absorption; alpha-ketoglutamic acid; apical membrane; base; basolateral membrane; brush border membrane; cDNA Library; design; enteropathogenic Escherichia coli; foodborne pathogen; human PHEMX protein; in vivo; killings; knock-down; medical schools; monolayer; mouse model; mutant; nervous system disorder; novel; nutrition; pathogen; pathogenic Escherichia coli; prevent; problem drinker; promoter; protein protein interaction; public health relevance; red fluorescent protein; response; small hairpin RNA; trafficking; uptake; yeast two hybrid system

DESCRIPTION (provided by applicant): The long-term objective of this renewal application is to continue our investigations into the cellular and molecular mechanisms involved in the intestinal absorption process of the water- soluble vitamin B1 (thiamin) and their regulation. We also aim at examining the effect of infection with enteropathogenic E. coli (EPEC), a common intestinal pathogen, on the intestinal thiamin absorption process. Thiamin is essential for normal cellular functions and its deficiency (which represents a significant nutritional problem) leads to a variety of clinical abnormalities including cardiovascular and neurological disorders. Humans (and other mammals) cannot synthesize thiamin, and thus, must obtain the vitamin via intestinal absorption. Studies during the current funding period have characterized many aspects of the intestinal thiamin uptake process. These include characterization of the 5' -regulatory regions of the genes of the human thiamin transporters 1 & 2 (hTHTR-1 & hTHTR-2) both in vitro and in vivo, demonstration that the intestinal thiamin uptake process is adaptively up-regulated in thiamin deficiency via transcriptionally-mediated mechanism(s) and that the process also undergoes differentiation- and developmental- dependent regulation. We have also characterized the mechanisms involved in membrane targeting and intracellular trafficking of the thiamin transporters in epithelial cells. Using gene specific siRNA approaches, recent studies from our laboratory have shown that both the hTHTR 1 & 2 are involved in thiamin uptake by a human intestinal epithelial Caco- 2 cell line in vitro. Nothing is known about the role of these thiamin transporters in thiamin uptake in the native intestine. Thus, in new preliminary studies we have developed an Slc19a3-/- knockout mouse model and showed significant impairment in intestinal thiamin uptake compared to wild-type mice; we have also established a colony of Slc19a2-/- knockout mice in our laboratory. In addition, we have used the bacterial two-hybrid system to screen a human intestinal cDNA library and have identified two putative proteins that can interact with hTHTR-1 (tetraspanin and E-cadherin-1). Furthermore, we have examined the effect of the enteropathogenic Escherichia coli (EPEC) and found significant inhibition in thiamin uptake. Based on our previous and new preliminary findings, our aims in this proposal are: 1) To further characterize the intestinal thiamin absorption process using Slc19a2 -/-and Slc19a3 -/- knockout mouse models and to determine the role of THTR-1 and THTR-2 in trans-epithelial and trans- membrane transport events in the native intestine, 2) To identify the cis-regulatory elements and trans-acting nuclear factors involved in the adaptive up-regulation of thiamin uptake in thiamin deficiency, 3) To identify proteins that interact with hTHTR-1 and hTHTR-2 in human intestinal epithelial cells and to understand their biological/physiological roles, and 4) to determine the cellular and molecular mechanisms involved in EPEC inhibition of the intestinal thiamin uptake process. Results of these studies should continue to provide novel and valuable information regarding the cellular and molecular mechanisms involved in the intestinal thiamin uptake process and their regulation as well as of the factors that negatively impact the process. This should ultimately assist us in the designing of effective strategies to optimize thiamin body homeostasis in conditions associated with thiamin deficiency and sub-optimal levels, and in minimizing the effect of exogenous factors that may negatively impact this nutritional parameter. PUBLIC HEALTH RELEVANCE: Humans and other mammals cannot synthesize vitamin B1 (an essential micronutrient for normal health), and thus, must obtain it from exogenous sources via intestinal absorption. The aims of this proposal since its inception ten years ago were (and continue to be) the delineation of the mechanisms involved in intestinal thiamin absorption, how the process is regulated, and what factors affect its function. Results of these investigations should help in the designing of effective strategies to optimize thiamin body levels, especially in conditions associated with thiamin deficiency and sub-optimal levels.

描述（由申请人提供）：本延续申请的长期目标是继续研究水溶性维生素B1（硫胺素）肠道吸收过程中涉及的细胞和分子机制及其调节。我们的目的也是研究感染肠致病性大肠杆菌的影响。大肠杆菌（EPEC），一种常见的肠道致病菌，对肠道硫胺素吸收过程。硫胺素对于正常的细胞功能是必不可少的，其缺乏（这代表了一个重要的营养问题）导致各种临床异常，包括心血管和神经系统疾病。人类（和其他哺乳动物）不能合成硫胺素，因此必须通过肠道吸收获得维生素。在目前的资助期间进行的研究已经描述了肠道硫胺素吸收过程的许多方面。这些包括在体外和体内对人硫胺素转运蛋白1和2（hTHTR-1和hTHTR-2）的基因的5 '-调节区的表征，证明了在硫胺素缺乏时肠硫胺素摄取过程通过转录介导的机制适应性上调，并且该过程也经历分化和发育依赖性调节。我们还表征了上皮细胞中硫胺素转运蛋白的膜靶向和细胞内运输机制。使用基因特异性siRNA方法，我们实验室最近的研究表明，hTHTR 1和2都参与体外人肠上皮Caco-2细胞系对硫胺素的摄取。目前还不清楚这些硫胺素转运蛋白在天然肠道中硫胺素摄取中的作用。因此，在新的初步研究中，我们开发了一种Slc19a3-/-敲除小鼠模型，与野生型小鼠相比，肠道硫胺素摄取明显受损;我们还在实验室建立了一个Slc19a2-/-敲除小鼠群体。此外，我们还利用细菌双杂交系统筛选了人肠道cDNA文库，并鉴定了两个可能与hTHTR-1相互作用的蛋白质（tetraspanin和E-cadherin-1）。此外，我们还研究了肠道致病性大肠杆菌（EPEC）的影响，并发现显着抑制硫胺素的摄取。根据我们以前和新的初步调查结果，我们在本提案中的目标是：1）为了使用Slc19a2-/-和Slc19a3-/-敲除小鼠模型进一步表征肠硫胺素吸收过程，并确定THTR-1和THTR-2在天然肠中的跨上皮和跨膜转运事件中的作用，2）鉴定在硫胺素缺乏时参与硫胺素摄取适应性上调的顺式调节元件和反式作用核因子，3）鉴定人肠上皮细胞中与hTHTR-1和hTHTR-2相互作用的蛋白，并了解其生物学特性。生理作用，以及4）确定EPEC抑制肠道硫胺素摄取过程中涉及的细胞和分子机制。这些研究的结果应继续提供新的和有价值的信息，有关的细胞和分子机制参与肠道硫胺素的摄取过程和他们的监管，以及对该过程产生负面影响的因素。这最终将有助于我们设计有效的策略，以优化硫胺素体内稳态的条件下与硫胺素缺乏和次优水平，并在尽量减少外源性因素的影响，可能会产生负面影响这一营养参数。公共卫生相关性：人类和其他哺乳动物不能合成维生素B1（正常健康所必需的微量营养素），因此必须通过肠道吸收从外源来源获得。自10年前提出以来，该提案的目的是（并继续是）描述肠道硫胺素吸收的机制，该过程如何调节，以及哪些因素影响其功能。这些调查的结果应有助于设计有效的策略，以优化硫胺素的身体水平，特别是在与硫胺素缺乏和次优水平相关的条件下。