CHARACTERIZATION OF INTESTINAL ABSORPTION OF VITAMIN B1

本建议涉及维生素B1（硫胺素）在人体肠道细胞和分子水平的吸收过程的表征。硫胺素是一种水溶性维生素，在不同的细胞代谢反应中起着重要作用。硫胺素对正常人类健康和福祉的重要性体现在硫胺素缺乏症中出现的严重临床异常，包括心血管和神经系统疾病。在发达国家，硫胺素缺乏症是一个严重的营养问题，发生在酗酒者、糖尿病患者、乳糜泻患者、肾病患者、老年人、先天性硫胺素代谢错误以及长期使用利尿剂的人群中。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从外源获取维生素。因此，肠道在决定和调节硫胺素正常体内平衡中起着至关重要的作用。人体肠道中的硫胺素有两种来源：饮食和大肠中正常菌群的细菌合成产物。人们对饮食中硫胺素在人体小肠中的吸收机制知之甚少，对于细菌合成的硫胺素在大肠中的吸收机制也没有任何信息。硫胺素通过功能极化的小肠和结肠上皮细胞的运输代表维生素通过两个结构和功能不同的膜域（即管腔和基底外侧膜域）的运输。我们建议使用纯化的管腔和基底外侧膜囊泡制剂来表征硫胺素跨单个膜运输的机制。近年来，多种营养物质的肠道转运过程受到特定的细胞内蛋白激酶介导途径和细胞外底物水平的调节。然而，对于人体肠道中硫胺素吸收过程的细胞调节机制，我们所知甚少。我们建议在这个应用程序中解决这个问题。肠道硫胺素转运过程的分子特征尚不清楚。我们提出了一种基于酵母s . cerevisiae NKC6（一种硫胺素运输活性缺陷的酵母突变体）的人类cDNA文库互补的方法来克隆和表征人类肠道硫胺素转运体。我们还建议克隆和表征人类肠道硫胺素转运蛋白基因的5'调控区。各种生理和分子生物学技术将在这些研究中使用。这些关于人体肠道中硫胺素吸收的细胞/分子机制和调控的研究有望提供有价值的信息。这将最终帮助我们设计有效的策略来优化硫胺素体内平衡，并帮助我们了解在某些病理生理条件下硫胺素营养发生畸变的原因。

This proposal deals with characterization of the absorption process of vitamin B1(thiamine) in the human intestine at the cellular and molecular levels. Thiamine, a water-soluble vitamin, plays an essential role in different cellular metabolic reactions. The importance of thiamine to normal human health and well being is manifested by the serious clinical abnormalities that occur in thiamine deficiency which include cardiovascular and neurological disorders. Thiamine deficiency represents a significant nutritional problem in developed countries and occur in alcoholics, diabetics, coeliac disease patients, renal disease patients, the elderly, in inborn errors of thiamine metabolism, and in long-term users of diuretics. Humans and other mammals can not synthesize thiamine, and thus, must obtain the vitamin from exogenous sources via intestinal absorption. Therefore, the intestine plays a critical role in determining and regulating thiamine normal body homeostasis. Thiamine is presented to the human intestine from two sources: the diet, and as a product of bacterial synthesis by the normal microflora in the large intestine. Very little is known about the absorption mechanism of dietary thiamine in the human small intestine, and no information is available regarding the mechanism of absorption of the bacterially synthesized thiamine in the large intestine. Transport of thiamine across the functionally polarized small intestinal and colonic epithelial cells represents transport of the vitamin across two structurally and functionally different membrane domains (i.e., the luminal and basolateral membrane domains). We propose to characterize the mechanism(s) of thiamine transport across the individual membrane using purified luminal and basolateral membrane vesicle preparations. Intestinal transport processes of variety of nutrients have been shown in recent years to be regulated by specific intracellular protein kinase-mediated pathways, and by extracellular substrate levels. Very little, however, is known about the cellular regulation of the thiamine absorption process in the human intestine. We propose to address this issue in this application. The molecular characteristics of the intestinal thiamine transport process is not known. We propose to clone and characterize the human intestinal thiamine transporters(s) using a method that is based on the strategy of complementation with a human cDNA library of the yeast S. cerevisiae NKC6, a yeast mutant defective in thiamine transport activity. We also propose to clone and characterize the 5'-regulatory region of the human intestinal thiamine transporter(s) gene. Variety of physiological and molecular biology techniques will be used in these studies. Valuable information are expected to emerge from these investigations regarding the cellular/molecular mechanisms and regulation of thiamine absorption in the human intestine. This should ultimately assist us in designing effective strategies to optimize thiamine body homeostasis, and help us understand the causes of aberrations that occur in thiamine nutrition under certain pathophysiological conditions.