Mechanisms mediating human enteroendocrine cell differentiation and function

介导人肠内分泌细胞分化和功能的机制

• 批准号: 10739834
• 负责人: Daniel Richard Zeve
• 金额: $ 16.94万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2028-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10739834
• 关键词: ATAC-seq; Address; Agonist; Amino Acids; Appetite Regulation; Attenuated; Automobile Driving; Award; Bioinformatics; Boston; CNR1 gene; CRISPR/Cas technology; Cell Differentiation process; Cell physiology; Cells; Childhood; Co-Immunoprecipitations; Collaborations; Cytokine Signaling; Data; Desire for food; Development Plans; Developmental Biology; Disease; Doctor of Philosophy; Duodenum; Endocrinologist; Enhancers; Enteroendocrine Cell; Enzyme-Linked Immunosorbent Assay; Epigenetic Process; Exposure to; FFAR1 gene; FOXO1A gene; Flow Cytometry; Foundations; Future; GPR119 receptor; Gastrointestinal tract structure; Gene Expression; Genes; Genetic Transcription; Glucose; Goals; Health; Homeobox; Hormonal; Hormone secretion; Hormones; Human; Hydrocortisone; Immunofluorescence Immunologic; Inflammatory; Insulin; Interleukin-6; Intestines; MAPK8 gene; Mediating; Medical; Mentors; Metabolism; Methods; Modeling; Molecular; Non-Insulin-Dependent Diabetes Mellitus; Nutrient; Obesity; Obesity Epidemic; Organoids; Palmitates; Pediatric Hospitals; Persons; Physiological; Play; Production; Proteins; Public Health; Rectum; Regulation; Research; Resources; Role; SP600125; Signal Induction; Signal Transduction; System; Testing; Time; Transcriptional Regulation; Twin Multiple Birth; United States; Up-Regulation; Volatile Fatty Acids; career development; cell growth; cell type; comparison control; cytokine; design; directed differentiation; epigenetic regulation; experience; hormonal signals; improved; incretin hormone; inhibitor; innovation; insulin secretion; knockout gene; new therapeutic target; novel therapeutic intervention; obesity development; rectal; response; rimonabant; single-cell RNA sequencing; small molecule; stem cells; transcription factor; treatment strategy

PROJECT SUMMARY The twin epidemics of obesity and type 2 diabetes mellitus (T2DM) continue to worsen, highlighting a growing need to understand the dysregulation of appetite and insulin secretion that contribute to these diseases. Enteroendocrine cells (EECs) are key regulators of both appetite and insulin secretion. Thus, the long-term goal of this project is to understand the transcriptional and epigenetic regulation of EEC differentiation and function, and how this is perturbed in disease states. The overall objective of this application is to identify and evaluate factors that are necessary for EE differentiation and hormone production/secretion. The central hypothesis is that EEC growth and function are controlled by intrinsic and extrinsic factors. Intrinsically, we have shown that FOXO1 inhibition (AS) and, separately, CB1/JNK inhibition (RSP) induce human EE differentiation, but the mechanisms driving this are not known. Extrinsically, nutrients are known to regulate EE hormone production, but it is unclear how other factors, including disease-related cytokines and hormones, alter this response. The rationale for this proposal is that through a better understanding of EEC differentiation and function, unique therapies can be developed to regulate appetite and insulin secretion via EE hormones. The central hypothesis will be evaluated in two specific aims: 1) to identify the role of HES6 and LMX1B in human EE differentiation and to identify the epigenetic and transcriptional changes driven by AS and RSP during early EE differentiation; and 2) to evaluate the impact of cytokines and hormones on nutrient-stimulated EEC function. In Aim 1, we will study gene expression changes (using CRISPR/Cas9, SHARE-seq), epigenetic changes (using SHARE-seq, bulk ATAC-seq), and protein interactions (using co-immunoprecipitation) in human duodenal organoids to investigate the roles of HES6 and LMX1B and to identify new factors involved in early stages of EE differentiation. In Aim 2, we will assess the ability of EECs (derived from duodenal and rectal organoids) to recapitulate the response of native EECs to nutrient stimulation. We will assess hormone production and secretion (using ELISA, qPCR), and how these responses are dysregulated following exposure to obesity/T2DM-associated cytokines and hormones. The candidate for this K08 proposal is Daniel Zeve, MD, PhD, a pediatric endocrinologist with expertise in developmental biology and metabolism. With his mentor, Dr. David Breault, Dr. Zeve has designed a career development plan to achieve scientific independence. This plan will be performed at Boston Children’s Hospital, allowing the applicant access to a multitude of resources throughout the Harvard Medical system. During the award period, the applicant will gain additional experience in SHARE-seq, epigenetics, CRISPR/Cas9, and bioinformatics through multiple avenues, including coursework, seminars, and high-level collaborations. This innovative project, which seeks to unravel the regulation of EEC differentiation and function, will highlight potential novel therapeutic targets for obesity and T2DM, and provide a strong foundation for Dr. Zeve’s scientific independence and future R01 submissions.

项目摘要 肥胖和2型糖尿病（T2DM）的双胞胎发作继续担心，突出了一个增长 需要了解有助于这些疾病的食欲和胰岛素分泌的失调。 肠内分泌细胞（EEC）是食欲和胰岛素分泌的关键调节剂。那，长期 该项目的目标是了解EEC差异化的转录和表观遗传调节 功能，以及如何在疾病状态下扰动。该应用程序的总体目的是识别和 评估EE分化和马酮产量/分泌所必需的因素。中央 假设是EEC的生长和功能受到内在和外在因素的控制。本质上，我们 已经表明FOXO1抑制（AS）和分别cb1/jnk抑制（RSP）会影响人EE 差异化，但是驱动此驱动的机制尚不清楚。外部，已知营养素可以调节EE 马酮的产量，但尚不清楚其他因素如何，包括疾病相关的细胞因子和马， 改变此响应。该提议的理由是，通过更好地理解EEC差异化 和功能，可以开发出独特的疗法，以通过EE激素调节食欲和胰岛素分泌。 中心假设将以两个具体的目的进行评估：1）确定HES6和LMX1B在 人类EE的分化并确定由AS和RSP驱动的表观遗传和转录变化 在EE早期分化期间； 2）评估细胞因子和马对营养刺激的影响 EEC功能。在AIM 1中，我们将研究基因表达变化（使用CRISPR/CAS9，share-seq），表观遗传学 更改（使用共享序言，批量ATAC-SEQ）和蛋白质相互作用（使用共免疫沉淀） 人十二指肠器官研究HES6和LMX1B的作用，并确定涉及的新因素 EE分化的早期阶段。在AIM 2中，我们将评估EEC的能力（源自十二指肠和 直肠器官）概括了天然EEC对营养刺激的反应。我们将评估本人 生产和分泌（使用ELISA，QPCR），以及暴露后这些反应如何失调 肥胖/T2DM相关的细胞因子和马。该K08提案的候选人是医学博士Daniel Zeve， 博士学位，一名具有发育生物学和代谢专家的儿科内分泌学家。他的精神，博士 Zeve博士David Breault设计了一项职业发展计划，以实现科学独立性。这个计划 将在波士顿儿童医院进行，允许适用于大量资源 在整个哈佛医疗系统中。在颁奖期间，申请人将获得额外的经验 通过多种途径，包括 课程工作，半手和高级合作。这个创新的项目，试图揭开 EEC分化和功能的调节将突出肥胖和 T2DM，为Zeve博士的科学独立性和未来R01提交提供了坚实的基础。