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...