Role of intestinal luminal and epigenetic factors to mediate intestinal metabolic remodeling after gastric bypass surgery

项目总结 2型糖尿病(T2D)发病率的上升突显出越来越需要了解其背后的机制 高效的治疗方法，如Roux-en-Y胃旁路手术(RYGB)，以发展更好、更多 广泛适用的治疗方法。我们的长期目标是了解流明因素在调节 肠道改道对能量代谢的影响，包括T2D。这个应用程序的总体目标是 检测RYGB术后肠道组织水平的表观遗传学变化，作为Roux肢体的潜在机制 (RL)重塑，我们已经证明与人类T2D的缓解有关。中心假设是RL 代谢重编程是由片段特异性的表观遗传修饰驱动的，这种修饰是可塑性的，需要 持续的管腔刺激。这个项目的基本原理是更好地了解肠道基因 RYGB之后的表达调控网络将揭示以更小的侵入性模仿RYGB的效果的方法 另类疗法。中心假设将在两个具体目标中进行检验：(1)检验一个 意外的管腔营养负荷影响关键基因的RL表观遗传和转录签名 与新陈代谢显著的RL重塑有关；以及(2)测试RL表观遗传学的依赖性 利用肠道上皮有机物(IEO)对体内因子的特征和代谢底物的利用。 在目标1中，基因表达(rna-seq)和染色质重塑(atac-seq)将在RL与 搭桥肠肢(胆胰肢)或与袖状胃切除术相比，这些方法也将 用来测试热量限制和再喂养过程中RL重塑的可塑性。在目标2中，IEO将是 用于询问表观遗传学(ATAC-SEQ)和代谢重编程(RNA-SEQ；海马XF分析) 在没有体内因素的情况下发生变化。此次职业发展奖的候选人玛格丽特博士 Stefater是一位儿科内分泌学家，拥有医学博士学位和能量综合生理学专业知识 平衡，尤其是在减肥手术后。为了实现候选人的科学独立性，一个 已经制定了全面的职业发展计划，以获得以下领域的更多经验：(1) 表观遗传学和基因调控网络，(2)生物信息学，和(3)使用肠道器官作为模型 肠道生物学。这将通过课程作业和研讨会来实现，在 减肥外科(Nicholas Stylopoulos)和肠道生物学(David Breault)领域的专家。这个 在申请人看来，本申请中提议的研究是创新的，因为它代表了 通过使用最先进的测序和生物信息学技术大幅偏离现状 阐述RYGB的核心外科要素(例如，胆道排斥、加速营养输送)如何影响 基因表达的表观遗传调控。最终，这个项目将阐明鲁米纳连接的机制 已知的RYGB后肠道重塑的因素，从而强调潜在的治疗RYGB的新药靶点 开发创新的T2D疗法，并为申请者的第一份R01申请铺平道路。

PROJECT SUMMARY The rising incidence of type 2 diabetes (T2D) highlights a growing need to understand mechanisms behind highly-effective therapies such as Roux-en-Y gastric bypass surgery (RYGB) in order to develop better, more widely-applicable treatments. The long-term goal is to understand the role of luminal factors to mediate the effect of intestinal re-routing upon energy metabolism, including T2D. The overall objective of this application is to test for tissue-level epigenetic changes in the intestine after RYGB, as a potential mechanism for Roux limb (RL) remodeling that we have shown to relate to T2D remission in humans. The central hypothesis is that RL metabolic reprogramming is driven by segment-specific epigenetic modifications which are plastic and require continuous luminal stimulation. The rationale for this project is that a greater understanding of intestinal gene expression regulatory networks after RYGB will shed light on ways to mimic RYGB’s effects with less invasive alternative therapies. The central hypothesis will be tested in two specific aims: (1) to test the role of an unexpected luminal nutrient load to influence RL epigenetic and transcriptomic signatures for key genes associated with metabolically-significant RL remodeling; and (2) to test the dependency of RL epigenetic signatures and metabolic substrate utilization upon in vivo factors using intestinal epithelial organoids (IEOs). In aim 1, gene expression (RNA-seq) and chromatin remodeling (ATAC-seq) will be studied in RL versus bypassed intestinal limb (biliopancreatic limb) or versus sleeve gastrectomy, and these methods will also be employed to test the plasticity of RL remodeling during caloric restriction and refeeding. In aim 2, IEOs will be used to ask whether epigenetic (ATAC-seq) and metabolic reprogramming (RNA-seq; Seahorse XF Analysis) change in the absence of in vivo factors. The candidate for this career development award, Dr. Margaret Stefater, is a pediatric endocrinologist with an MD/PhD and expertise in the integrated physiology of energy balance, especially after bariatric surgery. In order to achieve scientific independence for the candidate, a comprehensive career development plan has been developed to gain additional experience in the areas of (1) epigenetics and gene regulatory networks, (2) bioinformatics, and (3) use of intestinal organoids as a model of intestinal biology. This will be accomplished through coursework and seminars, under the mentorship of experts in the fields of bariatric surgery (Nicholas Stylopoulos) and intestinal biology (David Breault). The proposed research in this application is innovative, in the applicant’s opinion, because it represents a substantial departure from the status quo by using state-of-the-art sequencing and bioinformatics techniques to address how RYGB’s core surgical elements (e.g., biliary exclusion, accelerated nutrient delivery) influence epigenetic regulation of gene expression. Ultimately, this project will shed light on mechanisms linking luminal factors to known intestinal remodeling after RYGB, thus highlighting potential novel drug targets for the development of innovative T2D therapies, and paving the way for the applicant’s first R01 submission.