Nuclear receptor mediated bile acid alterations and coagulopathy in protein-energy undernutrition

项目总结/摘要 蛋白质-能量营养不良（PEU）与全球五岁以下儿童死亡人数的一半有关， 仍然是当今儿科最紧迫的挑战之一。PEU启动肝脏恶性循环 功能异常进一步损害健康。例如，肠内胆汁酸显著减少， 患有PEU的儿童，导致膳食脂肪吸收不良和体重增加受损。在严重的情况下，维生素K- 独立的凝血障碍可导致灾难性出血。PEU改变这两种机制的机制 尽管有证据表明营养感应核受体法尼醇X 受体（FXR）和过氧化物酶体增殖物激活受体（PPAR）α。FXR在进食状态下被胆汁激活 酸，而PPAR α在禁食状态下被脂解产物激活。这些受体调节胆汁酸 体内平衡和其他肝脏功能，竞争结合到许多相同的启动子区域， 转录效应编码FXR基因突变的儿童也具有维生素K非依赖性 伴有包括纤维蛋白原在内的多种凝血因子转录抑制的凝血病，涉及FXR PEU的凝血障碍中的信号传导。为了研究核受体在这两种肝功能中的作用， 我们检查了早期PEU的小鼠模型，每种模型都表现出胆汁酸的全面减少， 凝血病我们的年轻成年小鼠的基因表达模式表明Ppar α激活，Fxr信号丢失， 以及促进胆汁酸合成和凝血的基因的转录抑制。基于这些 结果，我们的假设是，Ppar α激活的脂肪分解产物产生的PEU 转录抑制胆汁酸合成中的关键基因，导致肠道胆汁酸减少 和体重增加受损。此外，我们假设激活的Ppar α取代了共享的Fxr， DNA启动子区，通过减少Fxr依赖性基因的转录介导凝血病。 这一假设将以两个具体目标进行检验。在目标1中，PEU和健康野生型小鼠用 Ppar α激动剂或拮抗剂，沿着PEU和健康Ppar α-/-小鼠，将用于确定Ppar α 通过qPCR驱动调节胆汁酸池大小的基因表达的变化， LC-MS/MS和随时间的生长损害。在目标2中，用Ppar α和Fxr激动剂处理的野生型小鼠和 拮抗剂将用于确定两个核受体之间的竞争性DNA结合 通过Fxr依赖性凝血因子的转录抑制导致凝血病;这些研究 将采用ChIP-seq、RNA-seq和血浆凝固测量。我们的预期结果是 表征一种新的分子连接，由核激素受体介导，之间的调节， 胆汁酸稳态和凝血，这两者在PEU中都发生病理改变。这些研究将提供 开发新的药理学和基于饮食的治疗策略的基础， 可推广到由于各种医学和社会经济原因而体重减轻的儿童。 Geoffrey a.医学博士，博士目前是儿科胃肠病学、肝病学和营养学研究员 在贝勒医学院他在宏基因组学、全球健康和营养方面有着出色的出版记录 儿童和PEU动物模型的研究。他的长期目标是成为一个独立的NIH资助的 一位医生兼科学家，研究生命早期营养缺乏导致的并发症。这些研究 旨在通过建立其儿科胃肠病学背景来支持PI的职业发展， 在宏基因组学的研究生工作，在核受体生物学和先进的应用， 通量测序和生物信息学分析。培训计划的其他关键要素包括： 由国际公认的、独立供资的、具有专门知识的调查人员组成的辅导和咨询小组 在发展计划的各个方面，包括大卫摩尔，博士。（核受体生物学），亚历山大 Milosavljevic博士（全基因组生物信息学）和Robert Shulman，M.D.（临床转化研究） 和儿科胃肠病学家学术职业发展）; 2）核受体高级课程 贝勒医学院生物医学科学研究生院的生物学和生物信息学， 春港实验室，FASEB科学研究会议和Keystone研讨会;和3）学术 旨在培养独立性的活动。PI的培训环境是一个首屈一指的学术研究 该机构与世界上最大的医疗中心，全国最大的儿童医院， 美国国立卫生研究院资助的德克萨斯医学中心消化疾病中心。这种环境将提供一种协作、 支持性和富有成效的气氛，以促进完成所有研究目标和发展目标， 拟议的时间轴。总之，这个培训计划将使PI直接通往成功的职业生涯 作为独立研究者，同时确定PEU患者肝功能损害的新途径， 发展，并最终可以预防和治疗。

PROJECT SUMMARY/ABSTRACT Protein-energy undernutrition (PEU) is implicated in half of all global deaths under five years of age and remains one of the most pressing challenges in pediatrics today. PEU sets into motion a vicious cycle of liver function abnormalities that further erode health. For example, intestinal bile acids are markedly reduced in children with PEU, resulting in poor dietary fat absorption and impaired weight gain. In severe cases, vitamin K- independent coagulopathy can lead to catastrophic bleeding. Mechanisms by which PEU alters these two processes are unknown, although evidence implicates the nutrient-sensing nuclear receptors, farnesoid X receptor (FXR) and peroxisome proliferator-activated receptor (PPAR)α. FXR is activated in the fed state by bile acids, while PPARα is activated in the fasted state by products of lipolysis. These receptors regulate bile acid homeostasis and other liver functions, competing for binding to many of the same promoter regions with opposite transcriptional effects. Children with mutations in the gene encoding FXR also have vitamin K-independent coagulopathy with transcriptional repression of multiple coagulation factors including fibrinogen, implicating FXR signaling in the coagulopathy of PEU. To investigate the role of nuclear receptors in these two liver functions, we examined mouse models of early-life PEU, each of which exhibits globally decreased bile acids and coagulopathy. Gene expression patterns in our young adult mice demonstrate Pparα activation, Fxr signal loss, and transcriptional repression of genes that promote bile acid synthesis and coagulation. Based on these findings, our hypothesis is that Pparα activation by products of lipolysis generated in PEU transcriptionally represses key genes in bile acid synthesis leading to decreased intestinal bile acids and impaired weight gain. Furthermore, we hypothesize that activated Pparα displaces Fxr from shared DNA promoter regions, mediating coagulopathy by decreasing transcription of Fxr-dependent genes. This hypothesis will be tested with two specific aims. In Aim 1, PEU and healthy wild type mice treated with Pparα agonist or antagonist, along with PEU and healthy Pparα-/- mice, will be used to determine how Pparα drives changes in the expression of genes that regulate bile acid pool size by qPCR, bile acid concentrations by LC-MS/MS, and growth impairment over time. In Aim 2, wild type mice treated with Pparα and Fxr agonists and antagonists will be used to determine whether competitive DNA binding between the two nuclear receptors results in coagulopathy through transcriptional repression of Fxr-dependent coagulation factors; these studies will employ ChIP-seq, RNA-seq, and plasma coagulation measurements. Our expected outcomes are characterization of a novel molecular link, mediated by nuclear hormone receptors, between the regulation of bile acid homeostasis and coagulation, both of which are pathologically altered in PEU. These studies will provide the foundation for developing new pharmacologic and diet-based therapeutic strategies that could ultimately be generalizable to children with weight loss due to a variety of medical and socioeconomic causes. Geoffrey A. Preidis, M.D., Ph.D. is currently a fellow in Pediatric Gastroenterology, Hepatology, and Nutrition at Baylor College of Medicine. He has a strong publication record in metagenomics, global health, and nutrition research with children and animal models of PEU. His long-term goal is to become an independent NIH-funded physician-scientist investigating co-morbidities that result from nutrient deprivation early in life. These research aims support the PI’s career development by building upon his background in pediatric gastroenterology and graduate work in metagenomics, with new training in nuclear receptor biology and advanced applications of high- throughput sequencing and bioinformatic analysis. Additional key elements of the training plan include: 1) A mentorship and advisory team of internationally recognized, independently funded investigators with expertise in all aspects of the development plan, including David Moore, Ph.D. (nuclear receptor biology), Aleksandar Milosavljevic, Ph.D. (genome-wide bioinformatics), and Robert Shulman, M.D. (clinical translational research and pediatric gastroenterologist academic career development); 2) Advanced coursework in nuclear receptor biology and bioinformatics from the Baylor College of Medicine Graduate School of Biomedical Sciences, Cold Spring Harbor Laboratory, FASEB Science Research Conferences, and Keystone Symposia; and 3) Scholarly activities designed to foster independence. The PI’s training environment is a premiere academic research institution closely allied with the world’s largest medical center, the nation’s largest children’s hospital, and the NIH-funded Texas Medical Center Digestive Disease Center. This environment will provide a collaborative, supportive, and productive atmosphere to facilitate completion of all research aims and development goals in the proposed timeline. In summary, this training plan will place the PI on a direct route to a successful career as an independent investigator while identifying novel pathways by which liver function impairments in PEU develop and may ultimately be prevented and treated.