PROJECT SUMMARY/ABSTRACT Obesity is a global pandemic with accelerating trends in morbidity, mortality, and medical expenditures. Treatments have been limited in efficacy or burdened with safety concerns. Leptin is a potent anorexigenic hormone and compelling candidate to treat diet-induced obesity (DIO). However, DIO induces leptin resistance, in part, by downregulating leptin receptors (LepRs) on the surface of hypothalamic neurons, rendering leptin therapy ineffective. Overcoming leptin resistance has emerged as a promising goal for obesity therapy. Recently, we discovered that the guanylyl cyclase C (GUCY2C) receptor is specifically expressed in hypothalamic LepR(+) neurons. Like LepR, hypothalamic GUCY2C induces satiety through binding of its hormone, uroguanylin, produced in intestine. In DIO, uroguanylin expression is lost, silencing hypothalamic GUCY2C. Importantly, reconstitution of GUCY2C signaling restores leptin sensitivity and produces weight loss in DIO mice. These data suggest that loss of intestinal uroguanylin in obesity produces leptin resistance by silencing GUCY2C signaling in hypothalamus, decreasing cell-surface LepR. The ultimate long-term goal of this project is to elucidate mechanisms by which GUCY2C signaling in hypothalamus regulates satiety and resists weight gain, to inform the utility of GUCY2C ligand therapy in the treatment and prevention of obesity. To achieve this goal, three specific aims are proposed. Aim 1, the pathophysiologic aim, will demonstrate that the uroguanylin-GUCY2C axis regulates leptin sensitivity. Leveraging unique tissue-specific conditional mouse models developed by our laboratory, we will measure leptin sensitivity of mice before and after eliminating the uroguanylin-GUCY2C signaling axis. Leptin sensitivity in mice with altered GUCY2C signaling will be assessed through phenotypic analysis of satiety and metabolism, and biochemical analysis of LepR signaling. Aim 2, the mechanistic aim, will establish that GUCY2C signaling controls the availability of neuronal surface LepRs. Preliminary data suggest that LepR signaling is reduced in GUCY2C-/- mice. We will use LepR(+) neurons enriched from LepR reporter mice to quantify the effect of GUCY2C signaling on LepR translocation to the cell surface and signaling. Finally, Aim 3, the therapeutic aim, will define the ability of GUCY2C ligands to overcome leptin resistance in DIO mice. Here, we will use clinically available GUCY2C ligands, and mice with a conditional uroguanylin transgene, to reconstitute hypothalamic GUCY2C signaling in DIO mice. Using methods described in Aim 1, we will explore the effects of exogenous or transgenic GUCY2C ligand supplementation on hypothalamic leptin signaling in DIO animals. These studies will reveal new mechanisms contributing to obesity, and novel solutions to overcome canonical leptin resistance and alleviate dysregulation of appetite and satiety in obesity. The potential to translate these results into patients ...