Divalent Metal-ion Transporter 1 as a Therapeutic Target to Optimize Intestinal Iron Transport

项目摘要 铁是人类必需的营养物质，但过量的铁是有毒的。因此，铁超载和缺铁 导致严重的体内平衡紊乱。铁超载最常与遗传性疾病有关 血色沉着症(HH)，它困扰着美国约1：250的成年人。HH患者的组织铁积累 导致关节炎、骨质疏松症、肝脏损伤和癌症、心肌病、糖尿病和阳萎。 HH是由于铁调节荷尔蒙海普西丁(HepC)的产生受损或由于 HAMP基因(编码HepC)的突变。丙型肝炎限制肠道铁的吸收。此外，减少了 丙型肝炎细胞的合成是铁负荷的基础，铁负荷是无效红细胞生成障碍的典型表现(例如，β- 中间型地中海贫血[βTI])。因此，HH和βTI患者的肠道铁吸收过度。这导致了 病理性铁超载，因为人类不能排泄过量的铁。肠道铁吸收的调节是 因此，适当控制身体的铁水平至关重要。膳食铁主要以无机(或非血红素)铁的形式存在。 铁(Fe3+)非血红素铁首先还原为Fe2+，通过二价金属离子输入十二指肠肠上皮细胞 转运蛋白1(DMT1)，由铁转运蛋白1(FPN1)输出，氧化后与转铁蛋白结合。DMT1是 基础条件下肠道铁的初级进口者，但DMT1对铁的相对贡献 HH和βTI中的累积是未知的。因此，在目标1中，我们将测试需要DMT1的假设 对于HH(Hepc KO)和βTI(Hbbd3th；具有突变的β主要珠蛋白[Hbb- B1])基因。因此，我们将产生也缺乏肠道DMT1的HepC和Hbb-b1KO小鼠。我们进一步 假设减少DMT1的表达将阻止HH和βTI的铁负荷。因此，我们有 已开发的生姜纳米颗粒衍生脂质载体(GNLV)可以将功能性DMT1 siRNA运送到 小鼠在活体十二指肠(DMT1表达减少约40%)。在AIM 2中，这个GNLV发射系统将是 在HH和βTI的啮齿动物模型中测试其防止铁负荷的能力。此外，缺铁(ID)是 在美国也很常见，大约800万年轻女性和70万婴儿受到影响。ID经常出现在以下情况下 膳食铁的吸收不能满足人体的需求。ID最常见的发生原因是 快速生长、妊娠、月经失血、吸收障碍、胃旁路手术和慢性 发炎。ID的症状包括贫血、认知障碍、免疫反应降低和疲劳。 在肠梗阻期间，DMT1增加了铜向十二指肠肠细胞的转运，新出现的数据表明 在缺铁状态下，铜是支持铁补充的关键。转换DMT1的机制 然而，变成铜运输机还是个未知数。因此，目标3将检验DMT1蛋白是 在缺铁期间翻译后修饰，允许铜的运输。似是而非的替代假设 也可能被考虑。总体而言，这项以DMT1为重点的调查可能会加强 调节高危个体肠道铁吸收的新的治疗和营养方法。

Project Summary Iron is an essential nutrient for humans, yet excess iron is toxic. As such, iron overload and iron deficiency result in severe homeostatic perturbations. Iron overload is most frequently associated with hereditary hemochromatosis (HH), which afflicts ~1:250 adults in the U.S. Tissue iron accumulation in patients with HH leads to arthritis, osteoporosis, liver damage and cancer, cardiomyopathy, diabetes mellitus, and impotence. HH results from impaired production of the iron-regulatory hormone hepcidin (HEPC) or as a result of mutations in the HAMP gene (encoding HEPC). HEPC limits intestinal iron absorption. Moreover, reduced HEPC synthesis underlies the iron loading that typifies disorders of ineffective erythropoiesis (e.g. β- thalassemia intermedia [βTI]). In HH and βTI, intestinal iron absorption is thus excessive. This leads to pathological iron overload since humans cannot excrete excess iron. Regulation of intestinal iron absorption is thus critical to properly control body iron levels. Dietary iron exists primarily as inorganic (or nonheme) iron. Ferric (Fe3+) nonheme iron is first reduced to Fe2+, imported into duodenal enterocytes by divalent metal-ion transporter 1 (DMT1), exported by ferroportin 1 (FPN1) and oxidized for binding to transferrin. DMT1 is the primary intestinal iron importer under basal conditions, but the relative contribution of DMT1 to iron accumulation in HH and βTI is unknown. In Aim 1, we will thus test the hypothesis that DMT1 is required for iron loading in mouse models of HH (HEPC KO) and βTI (Hbbd3th; with a mutated β major globin [Hbb- b1]) gene. We will thus generate HEPC and Hbb-b1 KO mice that are also lacking intestinal DMT1. We further hypothesize that decreasing DMT1 expression will prevent iron loading in HH and βTI. Accordingly, we have developed ginger nanoparticle-derived lipid vectors (GNLVs) which can deliver functional DMT1 siRNA to the mouse duodenum in vivo (~40% reduction in DMT1 expression). In Aim 2, this GNLV delivery system will be tested for its ability to prevent iron loading in rodent models of HH and βTI. Furthermore, iron deficiency (ID) is also common in the U.S., afflicting ~8 million young women, and 700,000 infants. ID frequently occurs when absorption of dietary iron does not meet the body’s demand. ID most commonly occurs as a consequence of rapid growth, pregnancy, menstrual blood loss, malabsorptive disorders, gastric bypass surgery and chronic inflammation. ID symptoms include anemia, impaired cognition, decreased immune response, and fatigue. During ID, DMT1 increases Cu transport into duodenal enterocytes and emerging data demonstrates that copper is critical to support iron repletion during states of deficiency. The mechanism that transforms DMT1 into a copper transporter is, however, unknown. Aim 3 will thus test the hypothesis that the DMT1 protein is post-translationally modified during iron deficiency, allowing Cu transport. Plausible alternative hypotheses may also be considered. Overall, this DMT1-focused investigation is likely to potentiate the development of novel therapeutic and nutritional approaches to modulate intestinal iron absorption in at-risk individuals.