Page 1 of 63 Diabetes Inhibition of mitochondrial calcium overload by SIRT3 prevents obesity or age- related whitening of brown adipose tissue Peng Gao1,#, Yanli Jiang1,2,#, Hao Wu1, Fang Sun1, Yaohong Li2, Hongbo He1, Bin Wang1, Zongshi Lu1, Yingru Hu1, Xiao Wei1, Yuanting Cui1, Chengkang He1, Lijuan Wang1, Hongting Zheng3, Gangyi Yang4, Daoyan Liu1, Zhencheng Yan1,*, Zhiming Zhu1,* 1Department of Hypertension and Endocrinology, Center for Hypertension and Metabolic Diseases, Daping Hospital, Third Military Medical University, Chongqing Institute of Hypertension, Chongqing 400042, China 2Department of Endocrinology, Menghai County People's Hospital, Xishuangbanna, Yunnan 666200, China. 3Department of Endocrinology, Translational Research Key Laboratory for Diabetes, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, China 4Department of Endocrinology, The Second Affiliated Hospital, Chongqing Medical University and Chongqing Clinical Research Center for Geriatrics, Chongqing 400010, China #These authors contributed equally to this work *Correspondence to Zhencheng Yan, MD, PhD and Zhiming Zhu, MD, PhD, Department of Hypertension and Endocrinology, Center for Hypertension and Metabolic Diseases, Daping Hospital, Third Military Medical University, Chongqing Institute of Hypertension, 10 Chang Jiang Zhi Lu, Yuzhong District, Chongqing Diabetes Publish Ahead of Print, published online November 11, 2019 Diabetes Page 2 of 63 400042, China. Tel. +86-23-6876-7849. Email address: [email protected] (Z.Y.); [email protected] (Z.Z.). Word Count: 5207 Running title: Capsaicin inhibits BAT whitening by activating AMPK/SIRT3 pathway Key words: capsaicin, SIRT3, AMPK, brown adipose tissue, mitochondrial calcium overload Abstract The whitening and loss of brown adipose tissue (BAT) during obesity and aging promote metabolic disorders and related diseases. The imbalance of Ca2+ homeostasis accounts for the dysfunction and clearance of mitochondria during BAT whitening. Capsaicin, a dietary factor activating TRPV1, can inhibit obesity induced by high-fat diet (HFD), but whether capsaicin inhibits BAT loss and the underlying mechanism remain unclear. In this study, we determined that the inhibitory effects capsaicin on HFD-induced obesity and BAT whitening were dependent on the participation of SIRT3, a critical mitochondrial deacetylase. SIRT3 also mediated all the beneficial effects of capsaicin on alleviating ROS generation, elevating mitochondrial activity and restricting mitochondrial calcium overload induced by HFD. Mechanistically, SIRT3 inhibits mitochondrial calcium uniporter (MCU)-mediated mitochondrial calcium overload by reducing the H3K27ac level on MCU promoter in an AMPK-dependent manner. In addition, HFD also inhibits AMPK activity to reduce SIRT3 expression, which could be reversed by capsaicin. Capsaicin intervention also inhibited aging- induced BAT whitening through this mechanism. In conclusion, this study emphasizes Page 3 of 63 Diabetes a critical role of AMPK/SIRT3 pathway in the maintenance of BAT morphology and function, and suggests that intervention in this pathway may be an effective target for preventing obesity or age-related metabolic diseases. Introduction The prevalence of obesity has doubled in more than 70 countries and has continuously increased, leading to 4.0 million deaths globally (1). Obesity also promotes alterations in other intermediate risk factors such as hypertension, dyslipidemia, and glucose intolerance, etc. (2). Previous studies have shown that obesity promotes the accumulation of white adipose tissue (WAT) to stimulate immune cell infiltration, contributing to systemic metabolic dysfunction (3). Compared to WAT, the distribution of brown adipose tissue (BAT) is more specific, mainly located in cervical, supraclavicular, paravertebral, mediastinal and perirenal regions in humans (4, 5). As the amount and function of BAT in humans is decreased with aging and obesity, the decline in BAT function may also facilitate energy storage and metabolic dysfunction under these conditions (5, 6). However, the molecular mechanisms that account for the reduced BAT function in obesity and its physiological implications remain elusive. During obesity, loss of normal structure and function of BAT, also referred as “BAT whitening”, involves mitochondrial dysfunction as featured by altered oxidative function, ultrastructure abnormalities and increased oxidative stress (7, 8). As mitochondrial Ca2+ plays a critical role in regulating mitochondrial activity (9), an aberrant modulation of mitochondrial calcium level, especially continuous calcium Diabetes Page 4 of 63 overload, increases mitochondrial reactive oxygen species (ROS) production, which is directly implicated in mitophagy, a process leading to progressive mitochondria loss (10, 11). The most critical channel mediating Ca2+ uptake is the mitochondrial Ca2+ uniporter (MCU) (12). It has been recently reported that the expression levels of MCU complex members were increased during obesity in adipose tissues of mice and humans (13). These findings all suggest a critical pathophysiological role of enhanced MCU- dependent mitochondrial Ca2+ uptake in the development of obesity, however, the underlying mechanism is still largely unknown. Nowadays, some active natural ingredients from food, especially the condiments, such as capsaicin, cinnamaldehyde and menthol, have been received much attention to be an effective anti-obesity lifestyle intervention approach (14). Since we first reported that capsaicin prevented obesity in mice in a transient receptor potential vanilloid 1 (TRPV1)-dependent manner (15), emerging evidence from laboratory and clinical studies support a role of capsaicin as an anti-obesity agent (16). Activation of TRPV1 by capsaicin increased intracellular Ca2+ level in adipocytes, which enhances the activity of Sirtuin 1 (SIRT1) by activating cytosolic AMP kinase (AMPK). Subsequently, the enhanced SIRT1 activity promoted browning of WAT by increasing the binding activity of PR domain containing 16 (PRDM16), a critical transcription factors promoting the differentiation and maintenance of BAT (17). However, whether capsaicin also exerts a protective role in mitochondrial calcium overload in brown adipocytes to counteract loss of BAT remains unclear. Page 5 of 63 Diabetes Unlike SIRT1 that is mainly enriched in nuclear, another member of the Sirtuin family, SIRT3, is preferentially located in mitochondria and highly expressed in BAT (18). Knockout of SIRT3 in mice on HFD resulted in accelerated obesity and metabolic syndrome by increasing mitochondrial oxidative stress (19, 20). Both activity and stability of SIRT3 were decreased by hyperacetylation during obesity and aging (21), thus the reduction of SIRT3 expression and activity might result in the loss of BAT in these conditions. Nevertheless, whether SIRT3 is also involved in the regulation of mitochondrial calcium homeostasis and the effect of capsaicin on obesity need to be confirmed. In this study, we aimed to determine the role of SIRT3 in the protective effect of capsaicin on obesity and aging-induced whitening of BAT and clarify the possible mechanism. Our results show that SIRT3 plays a crucial role in the maintenance of BAT and mediates the protective effect of capsaicin by repressing MCU-dependent mitochondrial Ca2+ overload in brown adipocytes, which might shed light on the mechanism of loss of BAT during obesity and aging. RESEARCH DESIGN AND METHODS Animals and treatment The SIRT3 knockout (SIRT3-KO) mice (22) were kindly provided by Professor De-Pei Liu from Chinese Academy of Medical Sciences and Peking Union Medical College (Beijing, China). The TRPV1 knockout (TRPV1-KO) mice (003770) were purchased from the Jackson laboratory. All mice were in C57BL/6 background and housed in Diabetes Page 6 of 63 cages at a controlled temperature (22 ± 1°C) and relative humidity (55 ± 5%) in a 12-h light/12-h dark cycle with standard laboratory chow and tap water ad libitum. At the age of 6 weeks, wild-type (WT), SIRT3-KO, or TRPV1-KO male mice were randomized into three groups and were fed standard chow (ND, 10% kcal% fat, 70% kcal% carbohydrate, 20% kcal% protein), a high-fat-diet (HFD, 45% kcal% fat, 35% kcal% carbohydrate, 20% kcal% protein), or a high-fat-diet plus 0.01% capsaicin (HFCD) for 32 weeks. In another experiment, we used 2-month-old male WT or SIRT3- KO mice as young and 18- to 20-month-old male mice as aged mice. And they were fed ND or a normal diet plus 0.01% capsaicin (CD) for 12 weeks. We also fed a small number of young WT and SIRT3-KO mice with CD for western blot. The approximate capsaicin intake in the CD or HFCD group was 1.64 μmol/day per mouse. Given of drug dosage between mouse and human is 12.3:1 (23), the converted human dose would be almost equal to that reported in a clinical trial (24). At the end of experiment, the mice were performed physiological tests before they were sacrificed. Tissues were stored in liquid nitrogen or fixed in 10% formalin for H&E staining and ROS detection. All experimental procedures were performed in accordance with protocols approved by the institutional animal care and research advisory committee at Daping hospital, Third Military Medical University. Indirect calorimetry The Comprehensive Laboratory Animal Monitoring System (CLAMS; Columbus Instrument) was used to measure oxygen consumption, carbon dioxide generation, Page 7 of 63 Diabetes energy expenditure (EE), respiratory exchange ratio (RER) and physical