PROJECT SUMMARY In our continued efforts to probe pituitary hormone action on body composition [Nature, 2017, PMID: 28538730; PNAS, 2019, PMID: 31843930]1,2 and given that weight gain in post–menopausal women is accompanied by variable increases in FSH and LH3-7, we examined the action of LH on body fat and energy homeostasis. We found that LH receptor (LHCGR) activation resulted in leanness—opposite to the pro– obesity effects of FSH1. This is particularly telling as a subset of women with polycystic ovarian syndrome, with high LH levels, display a lean phenotype8-10. We made three sets of key observations. First, we found that LHCGR transcript and protein was expressed abundantly in fat tissue, with gonadal white adipose tissue levels in female mice approaching that of the ovaries themselves. Second, LHCGR activation by LH, hCG and a small molecule agonist, ORG43553, which has undergone clinical testing for infertility11-14, resulted in less fat accrual in mice on a high–fat diet independently of testosterone. Third and importantly, ORG43553 reduced adipocyte differentiation in organoid cultures and induced thermogenesis both in vitro (Seahorse) and in vivo (indirect calorimetry). These responses mimicked those of FSH blockade1—raising an important clinical question whether ORG43553 and MS-Hu6 can be used in combination for therapeutic synergy in obesity—a crippling disease that affects 600 million men and women worldwide, with an increasing prevalence in older adults. It is thus imperative that we understand the cellular and molecular underpinnings of LH action on body fat, and the interactions between LH and FSH. In Specific Aim 1, we will determine how LH induces leanness given that, not only do we find abundant receptors on adipocytes, but that LHCGRs are expressed in hypothalamic neurons that regulate adipose tissue sympathetic tone [eLife, 2022, PMID: 36052994]15. We will therefore delete Lhcgr selectively in adipocytes by crossing Lhcgrfl/fl mice with doxycycline–inducible Adipoq- CrertTA,tetO mice, and use UCP1–reporter ThermoMice to study LH–induced beiging. In parallel, we will inject siLhcgr stereotactically into LHCGR–rich hypothalamic nuclei15. Mutant mice will be put on a high–diet and undergo detailed body composition and metabolic phenotyping. In Specific Aim 2, to determine whether FSH and LH intersect, we will probe downstream thermogenesis and cell differentiation signals using UCP1– reporter ThermoCells and 3T3.L1 organoids, respectively. This will be complemented by an unbiased search for early and late events using phosphoproteomics and RNA sequencing. We will also determine which gene has a dominant role by studying genetic epistasis between Fshr+/- and Lhcgr+/- crosses. This, in turn, will inform us whether LHCGR agonism and FSHR blockade could be combined for treating obesity. To determine if there is synergy, we will first perform dosing studies to determine the minimum effective dose for ORG43553, and then combine it...