PROJECT SUMMARY/ABSTRACT The gastrointestinal (GI) epithelium directly senses components of foods we eat, which contain a variety of nutritive and non-nutritive stimuli. These stimuli come from foods with a range of osmolality from zero (water) to >1,500 mmol/kg-H2O for some of the saltiest foods. GI epithelial cells are not only first to sense luminal osmoles, but these cells, along with the kidney tubular cells, encounter the largest osmole swings of all cells in the body. In functional and motility GI disorders, such as irritable bowel syndrome (IBS), which affect 10 - 20% of the US population, non-nutritive aspects of diets such as osmolality are often first line therapies. For example, FODMAPs (Fermentable Oligo-, Di-, Monosaccharides and Polyols) are targeted for elimination in IBS diets. FODMAPs are also highly osmotically active – they are known to drive luminal volume shifts which may lead to symptoms. Both hypo- and hyper-osmotic stimuli invoke downstream signaling that regulates GI motility and systemic physiologic responses. The enteroendocrine cells (EECs) are specialized sensory epithelial cells that interact with luminal stimuli, both nutritive and non-nutritive, and these cells are capable of regulating both local and systemic physiology including GI motility. While EECs are traditionally considered to be nutrient sensors, our lab discovered an EEC subpopulation that transduces mechanical signals, thereby opening the door to EECs being sensors of non-nutritive stimuli. Literature has shown that EEC receptor activation drives two main signal transduction pathways: calcium (Ca2+) and cAMP which lead to release of a range of signaling molecules, including serotonin (5HT). To investigate how EECs sense hyper and hypoosmotic stimuli respectively, our lab is manipulating EEC receptors: V1aR, VRAC, and Piezo2. In VRAC proteins, Ca2+ signaling drives Cl- currents. Understanding osmo-transduction will provide mechanistic insights into commonly used clinical therapies. The overall goal of this proposal is to uncover the mechanisms by which osmolality is sensed by EECs and how osmotic stimuli may engage EEC signal transduction to alter release of signaling molecules and systemic GI physiology. The hypothesis is that EECs transduce osmotic stimuli in location and subtype-specific ways - via cytoplasmic Ca2+, Cl- and cAMP, through osmoregulatory proteins, and secrete signaling molecules to modulate GI motility. Aim 1 investigates the cellular pathways by which EECs transduce osmotic stimuli. Aim 2 investigates the osmotically induced extracellular release of signaling molecules by EECs and subsequent changes in GI motility. The results of this work are poised to bridge knowledge gaps in EEC osmo-transduction, as well as inform broader osmosensing mechanisms in sensory epithelia. The proposed work will be carried out in an environment that provides expert knowledge towards achieving the specified goal, including collaborations with experts in visceral signal transduction, organoid work, and osmolality. This proposal includes a comprehensive training plan by which the PI will gain valuable skills in the study of molecular osmo-transduction on clinically relevant questions. Along with research activities, the plan also includes clinical training and shadowing activities to prepare the PI for her transition to the next stage of training as a future surgeon-scientist.

PROJECT NARRATIVE Disorders of gut-brain interaction (DGBIs) like irritable bowel syndrome (IBS) collectively affect around 15% of US adults. Patients suffering from DGBIs suffer significant disability and have few successful therapeutic options. Dietary modifications are the first line therapies and have been most successful at alleviating symptoms. One such dietary modification is the elimination of FODMAPs. FODMAPs are highly osmotically active, driving luminal volume shifts in the small bowel, but mechanistic understanding of osmotic signal transduction remains unclear, and how it affects cellular, multicellular, and systemic processes to alter GI physiology remains unknown. Our lab discovered a population of enteroendocrine cells (EECs) that sense non-nutritive stimuli such as mechanical force, thereby opening the door for EECs to be sensors of other non- nutritive stimuli, such as osmoles. In this proposal, we seek to uncover the role of EECs in sensing osmotic stimuli and how these pathways alter signal molecule release and GI motility. This knowledge may pinpoint targets for the treatment of DGBIs.