Store-operated Orai3 calcium channels in metabolism and obesity

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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.