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

在过去的二十年里，脂肪免疫细胞和邻近脂肪细胞之间的沟通越来越受到重视，它与肥胖、胰岛素抵抗和糖尿病等代谢疾病的相关性现在得到了很好的认识。我们最近发现，皮下脂肪组织中产生乙酰胆碱的免疫细胞通过CHRNA2(烟碱型乙酰胆碱受体，α2亚基)旁分泌机制影响生热米色脂肪的功能。在这里，我们建议深入研究这种免疫米色脂肪乙酰胆碱信号，特别是通过胆碱乙酰转移酶(ChAT)巨噬细胞，在生理和病理条件下如何影响脂肪组织功能和全身代谢动态平衡。我们的初步研究表明，寒冷暴露显著增加了皮下脂肪中表达ChAT的巨噬细胞的百分比。我们已经在不同的免疫细胞亚群中产生了多个ChAT基因缺失的小鼠系，并且只有巨噬细胞缺失ChAT，消融诱导了乙酰胆碱的产生，并在冷暴露时呈现腹股沟脂肪的生热缺陷。在包括b-AR(肾上腺素能受体)亚型的单一敲除的遗传模型中使用激动剂进行药理激活的初步研究表明，这些ChAT巨噬细胞通过β2-AR介导。目的1.在双报告小鼠系(ChAT-CRE；tdTomato；ChATBAC-EGFP)中，我们将深入研究培养巨噬细胞(BMDM)和脂肪驻留巨噬细胞(ChAT-CRE；tdTomato；ChATBAC-EGFP)如何激活这些胆碱能巨噬细胞。体内ChAT细胞的空间分布将在脂肪-Clear皮下脂肪中可视化，并将进行单细胞RNA-SEQ来表征这些细胞的转录组情况。目的2.我们将研究钙离子内流如何影响活化的米色脂肪细胞的CHRNA2下游信号。初步结果表明，新建立的基因敲击小鼠可能为该领域提供了一个具有功能的米色选择性Cre系统。我们还建议使用这个小鼠模型来表征米色脂肪中含有CHRNA2的离子通道复合体的组成。目的3.我们将研究乙酰胆碱-CHRNA2信号如何在肥胖的整个发展过程中受到影响。我们建议研究CHRNA2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，而且已经 已验证。优化了重点实验条件。已经招募了一个合作者小组，以跨学科的方法开展拟议的研究。最终，了解这一电路背后的机制将引导我们找到新的分子和细胞候选药物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.