Store-operated Orai3 calcium channels in metabolism and obesity

项目摘要/摘要 这项K99/R00提案旨在描述肥胖和肥胖的新分子机制。 开发治疗肥胖和肥胖相关心血管疾病的非侵入性方法。这个 该提案解决了知识上的一个根本性差距，并对治疗 肥胖。肥胖是几种疾病的主要诱因，包括2型糖尿病、心血管疾病 疾病和癌症。近年来的研究表明，钙信号在脂质中起着至关重要的作用 新陈代谢。Orai蛋白(Orai1-3)是一种具有高度钙选择性的通道，它对普遍存在的 受调控的钙进入所有细胞的进化保守途径，并在无数 细胞和生理功能，包括脂肪分解。在这里，我建议研究钙离子的功能 通过Orai3，一种哺乳动物特有的钙选择性Orai通道蛋白，进入代谢，神经元 神经支配、神经元激活及其在调节产热和肥胖中的作用。我们有 产生了Orai3全身基因敲除和Orai3flx/flx组织特异性基因敲除小鼠。我的初选 数据显示，饲养在15-18摄氏度的Orai3基因敲除小鼠和正常饮食的小鼠很快就会肥胖， 他们的体重增加是由脂肪积累引起的。这表明Orai3起着至关重要的作用。 在脂肪代谢方面。这些Orai3KO小鼠也减少了产热，交感神经减少 对棕色脂肪组织(BAT)的神经支配，表明肥胖是由于BAT减少所致 产生交感神经的去甲肾上腺素(NE)的产热和/或改变的神经支配或激活 神经元，它调节蝙蝠的产热。在本申请书中，我建议解决上述问题- 描述了Orai3KO小鼠肥胖的原因，有以下三个目的和关键实验：1) 通过Orai3进入CA2对蝙蝠线粒体产生热量以控制肥胖至关重要，而Orai3是控制肥胖的关键因素 将通过测量线粒体新陈代谢参数来确定。2)Orai3活动对 依赖于CA2的UCP1基因转录，是蝙蝠产热所必需的，这将被评估 通过研究调节UCP1表达的转录因子和拯救UCP1表达 在Orai3KO小鼠中，并测定Orai3KO和UCP1拯救小鼠的心脏代谢。3)Orai3 在中枢和外周神经系统(CNS和PNS)中需要调节BAT的活动，这是 将通过使用RNA范围分析CNS和PNS中的Orai3水平来确定，删除 CNS和测量BAT活动，删除CNS中的Orai3和测量寒冷和高脂肪饮食- 诱发性高血压和肥胖症。这项研究将有助于破译Orai3在BAT和 包括中枢神经元和交感神经元。它将弥合钙动态平衡和 线粒体功能，揭示了Orai3钙信号在脂肪代谢和肥胖中的新作用， 将Orai3确立为肥胖和肥胖相关心血管疾病的靶点。

Project Summary/Abstract This K99/R00 proposal aims to characterize novel molecular mechanisms of obesity and develop non-invasive methods for treating obesity and obesity-related cardiovascular diseases. The proposal addresses a fundamental gap in knowledge with a significant impact on the treatment of obesity. Obesity is a major contributor to several pathologies, including type 2 diabetes, cardiovascular disease, and cancer. In recent years, studies have shown that Ca2+ signals play a crucial role in lipid metabolism. Orai proteins (Orai1-3) are highly Ca2+ selective channels that contribute ubiquitous and evolutionary conserved routes of regulated Ca2+ entry into all cells and play a major role in a myriad of cellular and physiological functions, including lipolysis. Here, I propose to study the function of Ca2+ entry through Orai3, a mammalian specific Ca2+ selective Orai channel protein, in metabolism, neuronal innervation, neuronal activation, and its role in regulating thermogenesis and obesity. We have generated Orai3 whole-body knockout and Orai3flx/flx mice for tissue-specific knockouts. My preliminary data show that Orai3 knockout mice housed at 15-18°C and on regular diets quickly become obese, and their weight gain is derived from fat accumulation. This suggests that Orai3 plays an essential role in fat metabolism. These Orai3 KO mice also have reduced heat generation, reduced sympathetic innervation to brown adipose tissue (BAT), suggesting that adiposity is due to reduced BAT thermogenesis and/or altered innervation or activation of norepinephrine (NE) producing sympathetic neurons, which regulate BAT thermogenesis. In this application, I propose to address the above- described causes of adiposity in Orai3 KO mice with the following three aims and key experiments, 1) Ca2+ entry through Orai3 is crucial for BAT mitochondrial heat generation to control adiposity, which will be determined by measuring parameters of mitochondrial metabolism. 2) Orai3 activity is critical for Ca2+-dependent gene transcription of UCP1, required for BAT thermogenesis, which will be assessed by studying the transcription factors that regulate UCP1 expression and by rescuing UCP1 expression in Orai3 KO mice and determining cardio-metabolism on Orai3 KO and UCP1-rescued mice. 3) Orai3 is required in the central and peripheral nervous system (CNS and PNS) to regulate BAT activity, which will be determined by analyzing Orai3 levels in both CNS and PNS using RNA scope, deleting Orai3 in CNS and measuring BAT activity, and deleting Orai3 in CNS and measuring cold and high fat diet- induced hypertension and obesity. This study will help decipher the unique role of Orai3 in BAT and in both central and sympathetic neurons. It will bridge the gap between Ca2+ homeostasis and mitochondrial function and unveil a novel role for Orai3 Ca2+ signaling in lipid metabolism and obesity, establishing Orai3 as a target in obesity and obesity-related cardiovascular disease.