The communication between adipose immune cells and neighboring adipocytes has become increasingly appreciated over the past two decades, and its relevance to metabolic disorders such as obesity, insulin resistance and diabetes is now well-recognized. We have recently discovered that acetylcholine-producing immune cells within subcutaneous adipose tissue influence thermogenic beige fat function through paracrine mechanisms via CHRNA2 (nicotinic acetylcholine receptor, alpha2 subunit). Here we propose to thoroughly investigate how this immune-beige fat acetylcholine signaling, particularly via ChAT+(choline acetyltransferase) macrophages, influences adipose tissue function and whole body metabolic homeostasis under physiological and pathological conditions. Our preliminary studies reveal that cold exposure significantly increased percentage of macrophages that express ChAT in subcutaneous fat. We have generated multiple mouse lines with genetic deletion of Chat in various immune cell subsets and only macrophage deletion of Chat ablated induction of acetylcholine production and rendered thermogenic defects in inguinal fat upon cold exposure. Preliminary studies with pharmacological activation using agonists in genetic models including single knockouts of subtype of the b-ARs (adrenergic receptors), suggested that these ChAT+ macrophages are mediated through β2-AR. Aim 1. We will thoroughly investigate how these cholinergic macrophages are activated using both cultured macrophages (BMDM) and adipose resident macrophages in a double reporter mouse line (ChAT-Cre;tdTomato;ChATBAC-eGFP). Spatial distribution of ChAT+ cells in vivo will be visualized in Adipo-Clear prepared subcutaneous fat and single cell RNA-seq will be carried out to characterize the transcriptome landscape of these cells. Aim 2. We will investigate how Ca2+ influx influences CHRNA2 downstream signaling in activated beige adipocytes. Preliminary results indicated that newly generated Chrna2HA-Cre knockin mice may provide a functional beige selective Cre system for the field. We also propose to characterize the composition of the CHRNA2-containing ion channel complex in beige fat using this mouse model. Aim 3. We will investigate how acetylcholine-CHRNA2 signaling is affected throughout the development of obesity. We propose to study how CHRNA2 mediated beige fat activation contributes to the adaptive response to streptozotocin-induced loss of glycemic control. All required tools and genetic models are at hand and have been validated. Conditions for key experiments have been optimized. A team of collaborators have been recruited to carry out proposed studies with interdisciplinary approaches. Ultimately, understanding the mechanisms underlying this circuitry will lead us to new molecular and cellular candidates counteracting human metabolic disorders.