Enteroendocrine cells (EECs) coordinate a wide variety of signaling networks to maintain metabolic homeostasis. As a rare secretory cell lineage of the gut epithelium, EECs sense and respond to luminal stimuli by releasing a diverse array of hormones that control nutrient sensing, appetite, glycemic regulation, and energy balance. Dietinduced obesity and bariatric surgery have been associated with the dysregulation and restoration of these hormonal pathways, respectively. Moreover, a growing number of pharmacological strategies have emerged that target key EEC signaling pathways to treat metabolic disease. However, despite these advances the molecular mechanisms regulating EEC biology remain incompletely defined. To address this knowledge gap, this proposal aims to determine the role of an EEC-enriched microRNA (miRNA), miR-375, in regulating the effects of dietary and surgical interventions on EEC biology. MiRNAs are short, non-coding RNA molecules that respond to changing environmental contexts and modulate gene expression at the post-transcriptional level. As such, miRNAs are critical regulators of a myriad of biological pathways, including intestinal epithelial development and function. Our lab has previously demonstrated that miR-375 is highly enriched both in intestinal stem cells (ISCs) and along the EEC lineage, and its expression is dramatically reduced by chronic high-fat diet. In addition, our preliminary data demonstrate significant rescue of miR-375 expression in ISCs following bariatric surgery, coinciding with increases in EEC abundance and circulating gut hormone levels. Therefore, I hypothesize that miR-375 exerts context-specific effects on EEC biology during the pathogenesis and amelioration of diet-induced obesity. The proposed studies will test this hypothesis through an interdisciplinary approach using our lab’s established colony of miR-375 knockout (375-KO) mice together with cutting-edge genomic and bioinformatic techniques. In Aim 1, I will assess how the loss of miR-375 exerts diet-specific effects on the distribution of different EEC subtypes by performing high-resolution single-cell RNA-sequencing (scRNA-seq) of small intestinal crypts and villi from wildtype (WT) and 375-KO animals fed either a chronic chow or high-fat diet. In Aim 2, I will determine how miR-375 contributes to surgically-induced EEC adaptations and metabolic improvements through scRNA-seq analyses of crypt and villus samples from diet-induced obese WT and 375- KO mice following bariatric surgery or a control procedure. With these single-cell datasets, I will bioinformatically determine context-dependent changes in overall EEC abundance, subtype distribution (correlated with circulating gut hormone levels), and gene expression (including identification of candidate miR-375 targets). I will also validate these molecular findings in vivo through immunohistochemical assays and metabolic parameters such as body weight and glucose tolerance. Altogether, these findings will further our understanding of EEC regulation and may provide novel therapeutic targets for the treatment of obesity and its comorbidities.