The role of immune-adipocyte cholinergic signaling during metabolic adaptation and dysfunction

在过去的二十年中，脂肪免疫细胞和邻近脂肪细胞之间的通信越来越受到重视，并且其与代谢紊乱如肥胖症，胰岛素抵抗和糖尿病的相关性现在得到了充分的认可。我们最近发现，皮下脂肪组织内产生乙酰胆碱的免疫细胞通过CHRNA 2（烟碱乙酰胆碱受体，α 2亚基）的旁分泌机制影响产热米色脂肪功能。在这里，我们建议彻底调查这种免疫米色脂肪乙酰胆碱信号，特别是通过ChAT+（胆碱乙酰转移酶）巨噬细胞，影响脂肪组织功能和全身代谢稳态的生理和病理条件下。我们的初步研究表明，冷暴露显着增加的百分比，表达ChAT的巨噬细胞在皮下脂肪。我们已经产生了多种小鼠品系，其在各种免疫细胞亚群中具有Chat的遗传缺失，并且仅巨噬细胞缺失Chat消除了乙酰胆碱产生的诱导，并且在冷暴露时在腹股沟脂肪中呈现产热缺陷。在遗传模型中使用激动剂进行药理学激活的初步研究，包括b-AR（肾上腺素能受体）亚型的单敲除，表明这些ChAT+巨噬细胞通过β2-AR介导。目标1。我们将彻底研究如何使用培养的巨噬细胞（BMDM）和脂肪驻留巨噬细胞在双报告小鼠系（ChAT-Cre;tdTomato; ChATBAC-eGFP）激活这些胆碱能巨噬细胞。体内ChAT+细胞的空间分布将在Adipo-Clear制备的皮下脂肪中可视化，并将进行单细胞RNA-seq以表征这些细胞的转录组景观。目标二。我们将研究钙离子内流如何影响激活的米色脂肪细胞中CHRNA 2下游信号传导。初步结果表明，新产生的Chrna 2 HA-Cre敲入小鼠可以提供一个功能性米色选择性Cre系统的领域。我们还建议使用这种小鼠模型来表征米色脂肪中含有CHRNA 2的离子通道复合物的组成。目标3。我们将研究乙酰胆碱-CHRNA 2信号是如何在肥胖的发展过程中受到影响的。我们建议研究CHRNA 2介导的米色脂肪激活如何有助于对链脲佐菌素诱导的血糖控制丧失的适应性反应。所有必要的工具和遗传模型都在手边， 验证.对关键实验的条件进行了优化。已经招募了一组合作者，以跨学科方法开展拟议的研究。最终，了解这种电路的机制将使我们找到新的分子和细胞候选人来对抗人类代谢紊乱。

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.