ABSTRACT Sympathetic outflow to adipose tissue regulates whole body energy homeostasis by stimulating lipolysis in white adipocytes and thermogenesis in brown or beige adipocytes. Using a novel approach to study human adipose tissue in mice [1, 2], we have found that, during development, human adipocytes express a high level of monoamine oxidase A (MAOA), a major mechanism for degradation of norepinephrine, and a known target of antidepressant drugs [3]. Notably, expression of Maoa is virtually undetectable in mouse adipocytes, indicating that this control mechanism may have evolved to meet the metabolic features of larger species. We find that human adipocyte MAOA is decreased during beige adipose tissue development, potentially increasing norepinephrine bioavailability, adrenergic tone and thermogenic capacity. Thus, MAOA expression in human adipocytes is a previously underappreciated, key mechanism controlling adipose tissue functions, potentially underlying susceptibility to obesity and metabolic disease. In this proposal, we will further test the hypothesis that adipocyte MAOA controls systemic energy homeostasis through its effects on adipose tissue sympathetic responsiveness. We have developed methodologies to obtain large numbers of human multipotent mesenchymal progenitor cells that can differentiate into multiple adipocyte subtypes, and can generate functional adipose tissue upon implantation in vivo. We have also successfully deleted MAOA from these cells using a novel nanoparticle-based, CRISPR-Cas9 protein delivery technique, resulting in a >90% depletion of MAOA protein while avoiding non-specific effects of expressed Cas9. Leveraging these technologies, we shall: Aim 1. Test the hypothesis that human adipocyte MAOA limits lipolytic and thermogenic responses to norepinephrine. We will measure lipolysis and induction of thermogenesis in control and MAOA-deleted human adipocytes exposed to norepinephrine, as well as steady-state norepinephrine levels and dependency on the monoamine transporter Oct3. Aim 2. We will test the hypothesis that MAOA in human adipocytes regulates the development and responsiveness of thermogenic adipose tissue in vivo. We will measure the rate and extent of vascularization and innervation, and thermogenic responsiveness to environmental stimuli of tissue developed in NSG mice from control or MAOA deleted human adipocytes. Aim 3. We will test the hypothesis that expression of MAOA in adipocytes will determine susceptibility to obesity, systemic insulin sensitivity and systemic glucose homeostasis. We will analyze weight gain, adipocyte size, insulin sensitivity, glucose disposal under hyper insulinemic, hyperglycemic clamps, and basal metabolic rate in mice harboring adipose depots formed from control or MAOA deleted human adipocytes, and in mice overexpressing MaoA in subcutaneous adipose tissue. These aims will provide the basis for further development of tissue specific MAOA-targeting strategies as novel t...