UCP1-independent signaling involving SERCA2bmediated calcium cycling regulates beige fat thermogenesis and systemic glucose homeostasis

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1 ARTICLES UCP-independent signaling involving SERCA2bmediated calcium cycling regulates beige fat thermogenesis and systemic glucose homeostasis 27 Nature America, Inc., part of Springer Nature. All rights reserved. Kenji Ikeda 3, Qianqian Kang 3, Takeshi Yoneshiro 3, Joao Paulo Camporez 4, Hiroko Maki 5, Mayu Homma 5, Kosaku Shinoda 3, Yong Chen 3, Xiaodan Lu 3, Pema Maretich 3, Kazuki Tajima 3, Kolapo M Ajuwon 6, Tomoyoshi Soga 5 & Shingo Kajimura 3 Uncoupling protein (UCP) plays a central role in nonshivering thermogenesis in brown fat; however, its role in beige fat remains unclear. Here we report a robust UCP-independent thermogenic mechanism in beige fat that involves enhanced ATP-dependent Ca 2+ cycling by sarco/endoplasmic reticulum Ca 2+ -ATPase 2b (SERCA2b) and ryanodine receptor 2 (RyR2). Inhibition of SERCA2b impairs UCP-independent beige fat thermogenesis in humans and mice as well as in pigs, a species that lacks a functional UCP protein. Conversely, enhanced Ca 2+ cycling by activation of - and/or 3-adrenergic receptors or the SERCA2b RyR2 pathway stimulates UCP-independent thermogenesis in beige adipocytes. In the absence of UCP, beige fat dynamically expends glucose through enhanced glycolysis, tricarboxylic acid metabolism and pyruvate dehydrogenase activity for ATP-dependent thermogenesis through the SERCA2b pathway; beige fat thereby functions as a glucose sink and improves glucose tolerance independently of body weight loss. Our study uncovers a noncanonical thermogenic mechanism through which beige fat controls whole-body energy homeostasis via Ca 2+ cycling. UCP is a mitochondrial protein specific to brown adipose tissue (BAT), and it uncouples cellular respiration and mitochondrial ATP synthesis to dissipate energy in the form of heat. As UCP has been considered the sole thermogenin responsible for BAT thermogenesis,2, the prevailing dogma is that primarily the action of UCP mediates the functions of brown and beige fat, which promote antiobesity and antidiabetic effects when activated. Beige adipocytes are the inducible form of thermogenic fat cells that emerge within white adipose tissue (WAT) following a variety of external stimuli, such as chronic cold exposure, long-term treatment with peroxisome proliferator-activated receptor (PPAR ) agonists, cancer cachexia and bariatric surgery 3. Beige adipocytes are similar to brown adipocytes in that they possess multilocular lipid droplets and the capacity for thermogenesis 4 8, but beige fat in subcutaneous WAT expresses lower levels of UCP when compared to BAT in mice. Accordingly, beige fat was thought to play a marginal role in the regulation of whole-body energy metabolism 9. However, several lines of evidence suggest important biological roles for beige fat. For instance, earlier works demonstrated that obesity-resistant mouse strains possess higher amounts of UCP-positive adipocytes in WAT than obesity-prone strains,. It has also been shown that selective induction of beige adipocyte biogenesis through genetic and pharmacological means increases whole-body energy expenditure and protects animals from diet-induced obesity and glucose intolerance 2 5. Conversely, depletion of beige adipocytes through adipocyte-specific deletion of Prdm6 or its cofactor Ehmt causes diet-induced obesity and insulin resistance in mice 6,7. Of note, the inguinal WAT of cold-acclimated Ucp / mice exhibits increased oxygen consumption in response to succinate supplementation 8. Furthermore, chronic treatment with a 3-adrenergic receptor (AR) agonist increases the metabolic rate of epididymal WAT in Ucp / mice 9. Recent studies have also reported nonshivering thermogenic mechanisms in skeletal muscle mediated by sarcolipin 2,2 and in brown and beige fat mediated by a creatine-driven substrate cycle 22. These results collectively indicate the existence of UCP-independent thermogenesis; however, its underlying mechanisms and the physiological significance of UCP-independent processes in beige fat remain poorly understood. Here we report Ca 2+ cycling as a UCP-independent thermogenic mechanism in beige fat that controls whole-body energy homeostasis. RESULTS UCP is dispensable for beige fat thermogenesis in vivo We previously reported that adipose tissue selective transgenic expression of Prdm6 driven by the Fabp4 promoter and enhancer potently promoted beige adipocyte biogenesis in subcutaneous WAT, whereas Diabetes Center, University of California, San Francisco, San Francisco, California, USA. 2 Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, California, USA. 3 Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, California, USA. 4 Departments of Medicine and of Cellular & Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut, USA. 5 Institute for Advanced Biosciences, Keio University, Yamagata, Japan. 6 Department of Animal Sciences, Purdue University, West Lafayette, Indiana, USA. Correspondence should be addressed to S.K. (shingo.kajimura@ucsf.edu). Received March; accepted 2 September; published online 3 November 27; doi:.38/nm.4429 NATURE MEDICI ADVANCE ONLI PUBLICATION

2 A RTICLES 27 Nature America, Inc., part of Springer Nature. All rights reserved. it caused no morphological or molecular changes in the interscapular BAT (ibat) and epididymal WAT depots 2. To examine whether UCP is required for beige adipocyte function in vivo, we crossed Fabp4-Prdm6 transgenic mice () with Ucp / mice on the Bl6 background ( Ucp / ). First, we confirmed that the Prdm6 transgene was selectively and equally expressed in the adipose tissues of mice and Ucp / mice (Supplementary Fig. a,b). mice expressed significantly higher levels of Ucp in inguinal WAT, but not in ibat and epididymal WAT, than their littermate controls (Supplementary Fig. c). Expression of brown- and beige-fat-selective genes and mitochondrial genes was also higher in the inguinal WAT of mice than in controls (Supplementary Fig. d). Similarly, the inguinal WAT of Ucp / mice exhibited higher mrna expression for all of the brown- and beige-fat-selective genes except Ucp than the inguinal WAT of littermate Ucp / mice (Supplementary Fig. d). No major difference was seen in brown- and beige-fat-selective gene expression in ibat and epididymal WAT between the genotypes (Supplementary Fig. e,f). The activation of brown- and beige-fatselective genes in mice and Ucp / mice was accompanied by an increased number of multilocular adipocytes in inguinal WAT (Supplementary Fig. g). In ibat, brown adipocytes from Ucp / mice and Ucp / mice contained lipid droplets larger than those in Ucp +/+ mice (Supplementary Fig. h). No noticeable difference was observed in the morphology of ibat and epididymal WAT as a result of transgenic expression of Prdm6 (Supplementary Fig. h,i). Accordingly, this mouse model provides valuable insights regarding the extent to which the action of UCP mediates the physiological function of beige fat in whole-body energy metabolism. Upon cold exposure at 6 C, mice and littermate controls maintained their core body temperature (Fig. a). Consistent with previous studies,23, Ucp / mice showed a severe impairment in cold tolerance; in contrast, body weight matched Ucp / mice were capable of maintaining their core body temperature under cold conditions (Fig. b). mice displayed a modestly but significantly higher whole-body oxygen consumption rate (VO 2 ) and heat production as compared to control mice following cold exposure at 6 C (Fig. c,d), although there was no difference in energy expenditure between these groups at 3 C (Supplementary Fig. 2a). Notably, VO 2 and heat generation in Ucp / mice were significantly higher than in littermate Ucp / mice at 6 C (Fig. e,f). During cold exposure, no significant difference was observed in either food intake or locomotor activity among the genotypes (Supplementary Fig. 2b,c). To determine the specific tissues responsible for thermogenesis in Ucp / mice, we next monitored tissue temperature in ibat, inguinal WAT and skeletal muscle following norepinephrine () treatment (Fig. g). The ibat temperature of mice and littermate control mice increased by. C or more following treatment, whereas such an increase was not observed in Ucp / mice and Ucp / mice (Fig. h). On the other hand, the inguinal WAT temperature in Ucp / mice underwent a significant increase, comparable to that in ibat, following treatment. In skeletal muscle, no increase in tissue temperature was seen in response to treatment. We also found that the oxygen consumption rate () of ibat was significantly increased by in Prdm6 Tg mice and control mice, but not in Ucp / mice and Ucp / mice (Supplementary Fig. 2d). In contrast, the in the inguinal WAT of and Ucp / mice was significantly higher than that in their respective controls following treatment (Supplementary Fig. 2e). In epididymal WAT, no significant difference in was seen among the genotypes (Supplementary Fig. 2f). Of note, the contribution of skeletal muscle shivering appeared to be negligible for heat generation in this mouse model, as no difference in muscle shivering was found by electromyography between Ucp / mice and Ucp / mice, either at 3 C or 6 C (Fig. i,j). We also observed no difference in the serum concentration of creatine kinase, a marker of muscle damage, between the two groups after cold exposure (Supplementary Fig. 2g). SERCA2b controls UCP-independent thermogenesis in beige adipocytes To investigate the mechanisms of UCP-independent thermogenesis in Ucp / mice, we performed RNA-seq analysis of inguinal WAT followed by Metascape pathway analysis 24. We identified 39 genes that were significantly (P <.5) upregulated in the inguinal WAT of mice and Ucp / mice (Fig. 2a). We also found 64 genes whose expression was selectively elevated in Ucp / mice relative to mice with the other genotypes. Metascape pathway analysis found that the upregulated pathways in both mice and Ucp / mice were linked to PPAR signaling, fatty acid metabolism and brown fat differentiation, which are involved in beige adipocyte biogenesis (Fig. 2b). Furthermore, several metabolic pathways, including tricarboxylic acid (TCA) metabolism, branched-chain amino acid (BCAA) oxidation and glycolysis, were solely upregulated in Ucp / mice as compared to mice with the other genotypes. Notably, the pathway linked to cardiac muscle contraction was only upregulated in Ucp / mice; mrna expression of several cardiac muscle related genes encoding proteins involved in Ca 2+ cycling, including Serca2 and Ryr2, was significantly higher in Ucp / mice than in the other groups (Fig. 2c). The changes in Ca 2+ -cycling-related genes caught our attention because mutations in the RYR gene cause malignant hyperthermia in humans and pigs 25,26 and sarcolipin, a SERCA regulator, is required for nonshivering thermogenesis in skeletal muscle 2,2. SERCA2b, one of the isoforms of SERCA2 encoded by the Atp2a2 gene (Serca2a, Serca2b), is the predominant form of SERCA in beige adipocytes; mrna expression of Serca2b was much higher than that of Atp2a (Serca) (a skeletal muscle selective form of SERCA), Serca2a and Atp2a3 (Serca3) (Fig. 2d). ATP2A2 (SERCA2b) also encodes the predominant form of SERCA in differentiated human beige adipocytes 27 (Fig. 2e). Of note, Serca2b mrna expression in inguinal WAT was increased by chronic cold exposure at 4 C for 7 d (Supplementary Fig. 3a). SERCA2 protein expression was also increased in inguinal WAT by cold exposure, whereas such a change was not observed in ibat (Supplementary Fig. 3b). Immunohistochemistry analysis of beige adipocytes showed that SERCA2 was localized to the endoplasmic reticulum (ER), on the basis of its colocalization with a marker of the ER and sarcoplasmic reticulum (SR), calnexin (Supplementary Fig. 3c). As from sympathetic neurons is known to activate 3-AR and trigger intracellular cyclic AMP (camp) signaling 28, we examined whether SERCA2 expression was regulated by the camp pathway. To do so, primary beige adipocytes were treated with forskolin, an adenylyl cyclase activator that generates camp. We found that forskolin treatment for 4 h significantly increased Serca2b and SERCA2b mrna expression in mouse and human beige adipocytes, respectively, but did not alter that of the other isoforms (Fig. 2d,e). 2 ADVANCE ONLI PUBLICATION NATURE MEDICI

3 ARTICLES 27 Nature America, Inc., part of Springer Nature. All rights reserved. a c d Core body temperature ( C) 4 8, 9,.2.2 7, 8,. 35 6, 7, 6, , 5, 4,.6.6 4, 25 3, 3,.4.4 2, 2, ,, 6 C 6 C Time (h) Time (h) Time (h) T ( C) VO 2 (ml/h per kg body weight) b e f Core body temperature ( C) g P = ibat lng WAT Skeletal muscle (back) Time (h) Temperature recording h VO 2 (ml/h per kg body weight) 7, 6, 5, 4, 3, 2,, VO 2 (ml/h per kg body weight) Heat (kcal) 7,... 6,.9.9 5, ,.6.6 3, , ,.. 6 C 6 C Ucp Time (h) / Time (h) T ( C) VO 2 (ml/h per kg body weight), 3 C 6 C 5 ibat lng WAT Skeletal muscle ,..., 5 3 C 6 C , 5 s j Time (min after ) Time (min after ) Time (min after ) T ( C) Heat (kcal) i EMG (µv) EMG (µv) EMG RMS (µv) Heat (kcal) Heat (kcal) 3 C 6 C Figure UCP is dispensable for beige fat thermogenesis. (a) Rectal core body temperature of mice and littermate controls () maintained at 6 C at the indicated time points. n = 7 for both genotypes. (b) Rectal core body temperature of Ucp / mice and littermate Ucp / mice maintained at 6 C. Ucp / mice, n = 3; Ucp / mice, n = 7. (c) Whole-body VO 2 (left) and averaged VO 2 s.e.m. (right) of mice and littermate controls following cold exposure at 6 C. n = 5 for both groups. (d) Whole-body heat generation (kcal) of the mice in c. (e) Whole-body VO 2 of Ucp / mice and littermate Ucp / mice following cold exposure at 6 C. n = 5 for both groups. (f) Whole-body heat generation (kcal) of the mice in e. (g) Schematic illustrating tissue temperature recording in ibat, inguinal WAT (ing WAT) and skeletal muscle. (h) Changes in tissue temperature ( T) in the indicated tissues following treatment (arrows) in the indicated groups of mice. rol, n = 6;, n = 4; Ucp /, n = 5; Ucp /, n = 4. (i) Representative electromyography (EMG) traces. n = 5 for both groups. (j) Quantification of the images in i converted to the root mean square (RMS) for Ucp / mice and Ucp / mice at 3 C and 6 C. n = 5 for all groups. Data in a f, h and j are expressed as means s.e.m. Data were analyzed by Student s t-test (a,c f,j), two-way ANOVA followed by Fisher s LSD test (b) or one-way ANOVA followed by Tukey s test (h). P <.5, P <., P <.., not significant. Because expression of Ucp2 and Ucp3 in inguinal WAT did not differ among the four genotypes (Supplementary Fig. 3d,e) and mitochondrial patch clamp recordings in a previous study showed that proton current in the mitochondrial membranes of brown and beige adipocytes is highly UCP dependent 29,3, UCP-independent thermogenesis in the inguinal WAT of Ucp / mice likely occurs through a mechanism independent of mitochondrial proton uncoupling. Next, we aimed to determine the requirement for SERCA2b-mediated Ca 2+ cycling in UCP-independent thermogenesis. To this end, we established Ucp / beige adipocytes through differentiation of immortalized stromal vascular fractions (SVFs) from the inguinal WAT of Ucp / mice. treatment significantly increased basal and oligomycin-resistant cellular s in Ucp / beige adipocytes by 7% and 78%, respectively (Fig. 2f). Acute inhibition of SERCA2 using a pharmacological SERCA inhibitor, thapsigargin, for h completely blunted the -induced increase in cellular respiration (Fig. 2f). Next, we used the CRISPR Cas9 system to introduce homozygous null mutations into Atp2a2 in Ucp / beige adipocytes (Fig. 2g). We confirmed with immunohistochemistry analysis that SERCA2 protein was abundantly expressed in beige adipocytes expressing a scrambled guide RNA, whereas it was not detected in Atp2a2-null cells (Fig. 2h). Genetic deletion of Atp2a2 in Ucp / beige adipocytes significantly reduced basal and -stimulated s as compared to control cells (Fig. 2i). Furthermore, acute inhibition of SERCA by thapsigargin for h diminished the -induced increase in in control cells, whereas this inhibitory effect was not observed in Atp2a2 / ; Ucp / cells (Fig. 2i). The requirement of SERCA2 for NATURE MEDICI ADVANCE ONLI PUBLICATION 3

4 A RTICLES beige fat thermogenesis was confirmed in two independent clonal cell lines (Supplementary Fig. 4a,b). As an alternative approach, we used lentiviruses expressing a scrambled control RNA or short hairpin RNAs (shrnas) targeting Atp2a2 to deplete endogenous SERCA2b in beige adipocytes (Supplementary Fig. 4c). We found that depletion of SERCA2b by two distinct shrnas significantly reduced the -stimulated in Ucp / beige adipocytes (Supplementary Fig. 4d). SERCA2b depletion also reduced 27 Nature America, Inc., part of Springer Nature. All rights reserved. a b log (P value) c d e Common pathway Serca2b Mouse primary Human beige adipocytes beige adipocytes PPAR signaling Fatty acid metabolism Upregulated genes in Brown fat differentiation Forskolin Forskolin Prdm 6 Tg and Connective tissue development.5 2. Prdm 6 Tg 2 Estrogen response.5 (39 genes). log. Upregulated genes only in (P value) Prdm 6 Tg Unique pathway (64 genes) Electron transport chain n.d. n.d... Coenzyme metabolism Z-score Cardiac muscle contraction Low High TCA cycle.7.7 Glycolysis BCAA oxidation (pmol/min per µg protein) Prdm 6 Tg Prdm 6 Tg Relative mrna expression Prdm 6 Tg Prdm 6 Tg beige adipocytes f g i Oligo FCCP AA rol Allele & Allele ( 2) 4 Atp2a2 / Allele 2 (+) Thapsi 2 + thapsi SERCA2/DAPI 8 h rol Atp2a2 / Relative mrna expression Serca Serca2a Serca2b Serca3 j beige adipocytes k Ing WAT l ibat m Ing WAT n ibat o (pmol/min per µg protein) Vector SERCA2b (pmol/min per mg) (pmol/min per µg protein) (pmol/min per mg) Veh Thapsi + thapsi 6 rol Adipo-Atp2a2 / rol Adipo-Atp2a2 / T ( C).5.5 Adipo-Atp2a2 / Time (min after ) T ( C).5.5 (pmol/min per µg protein) Time (min after ) Adipo-Atp2a2 / Relative mrna expression SERCA SERCA2a ; Atp2a2 / T ( C) Ing WAT SERCA2b SERCA3 Thapsi + thapsi Adipo-Atp2a2 / ibat Skeletal muscle Figure 2 SERCA2b controls UCP-independent thermogenesis in beige fat. (a) Hierarchical clustering and heat-map of RNA-seq data in the inguinal WAT. n = 3 for all groups. The color scale shows Z-scored fragments per kilobase of transcript per million mapped reads (FPKM) representing the mrna level of each gene in a blue (low expression) white red (high expression) scheme. (b) The commonly upregulated pathways in mice and Ucp / mice relative to the littermate controls (top) and the uniquely upregulated pathways in Ucp / mice relative to other genotypes (bottom). P values are shown on the top. n = 3 for all groups. (c) mrna expression of Serca2b in the inguinal WAT. rol, n = 9; Prdm6 Tg, n = 8; Ucp /, n = 9; Ucp /, n = 7. (d) mrna expression of the indicated genes in differentiated mouse primary beige adipocytes treated with forskolin or vehicle. n = 3 for both groups. (e) mrna expression of indicated genes in differentiated human beige adipocytes treated with forskolin or vehicle. n = 3 for both groups. n.d., not detected. (f) in Ucp / beige adipocytes treated with vehicle (veh),, thapsigargin (thapsi) or plus thapsigargin. Oligomycin (oligo), FCCP and antimycin (AA) were added at the time points indicated by dashed lines (left), and averaged basal s.e.m. is shown (right)., n = 4;, n = 3; thapsigargin, n = 3; + thapsigargin, n = 4. (g) Genomic sequences of a clonal Ucp / beige adipocyte line carrying the homozygous mutations in Atp2a2 introduced by the CRISPR Cas9 system (Atp2a2 / ). Mutations (red, insertion;, deletion) and wild-type allele sequences (control) are shown. (h) Immunohistochemistry analysis of SERCA2 in differentiated clonal Ucp / beige adipocytes with homozygous mutations in Atp2a2 / or control cells expressing a scrambled guide RNA. DAPI (blue) was used for nuclear staining. Scale bar, 25 m. (i) Basal in Ucp / beige adipocytes expressing a control guide RNA (Ucp / ) and Atp2a2 / ; Ucp / beige adipocytes. Ucp / with vehicle and, n = each; Ucp / with thapsigargin, n = ; Ucp / with + thapsigargin, n = 7; Atp2a2 / ; Ucp / with vehicle, n = ; Atp2a2 / ; Ucp / with, n = 2; Atp2a2 / ; Ucp / with thapsi, n = 8; Atp2a2 / ; Ucp / with + thapsi, n =. (j) Basal in Ucp / beige adipocytes expressing SERCA2b or an empty vector. Differentiated cells were treated with vehicle or for h. Vector with vehicle and with, n = 8 for both; SERCA2b with vehicle, n = 4; SERCA2b with, n = 5. (k) in the inguinal WAT of control and Adipo-Atp2a2 / mice after tissue isolation and treatment with or vehicle for h. rol with vehicle, n = ; control with, n = 8; Adipo-Atp2a2 / with vehicle, n = 6; Adipo-Atp2a2 / with, n = 8. (l) in the ibat of control and fat-specific Atp2a2 / mice. rol with vehicle, n = ; control with, n = 6; Adipo-Atp2a2 / with vehicle, n = ; Adipo-Atp2a2 / with, n = 8. (m) Real-time changes in tissue temperature in the inguinal WAT of control and Adipo-Atp2a2 / mice following treatment. n = 5 for both groups. (n) Real-time changes in tissue temperature in the ibat of mice in m. (o) Quantification in tissues temperature change in the indicated tissues of mice in m. Data in c f and i o are expressed as means s.e.m. Data were analyzed by Student s t-test (c e,m o) or one-way ANOVA followed by Tukey s test (f,i l). P <.5. P <., P <.. 4 ADVANCE ONLI PUBLICATION NATURE MEDICI

5 ARTICLES 27 Nature America, Inc., part of Springer Nature. All rights reserved. oligomycin-resistant respiration, but not carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP)-stimulated respiration, in Ucp / beige adipocytes (Supplementary Fig. 4d). SERCA2b depletion similarly blunted the -induced increase in in wild-type beige adipocytes (Supplementary Fig. 4e). These results indicate that SERCA2b is required for beige fat thermogenesis both in the presence and absence of UCP. The SERCA2b pathway also plays an important role in human beige adipocytes: treatment significantly increased the in UCP / human beige adipocytes, whereas this increase was completely blunted by thapsigargin (Supplementary Fig. 5a,b). Next, we employed a gain-of-function approach to determine the role of SERCA2b in UCP-independent thermogenesis. For this purpose, we overexpressed SERCA2b in Ucp / beige adipocytes by infecting the cells with a low titer of lentivirus expressing SERCA2b (Supplementary Fig. 5c,d). We found a modest elevation in Serca2b mrna expression of approximately.7-fold, a degree of increase equivalent to that found in Ucp / mice (Fig. 2c), which led to a significant increase in Ca 2+ release from the ER in response to treatment, likely as a result of enhanced Ca 2+ loading to the ER (Supplementary Fig. 5e). This increase was accompanied by enhanced thermogenesis following treatment (Fig. 2j). On the basis of the above results, we examined the genetic requirement for SERCA2b in beige fat thermogenesis in vivo. To this end, we crossed Atp2a2 flox/flox mice with Adipoq (Adiponectin)-Cre mice (Adipo-Atp2a2 / ) and found that Serca2b mrna was significantly lower in adipose tissues, but not in heart, of Adipo-Atp2a2 / mice than in littermate control mice (Supplementary Fig. 6a). The partial reduction in Serca2b expression in adipose tissues was likely due to the expression of Serca2b in other nonadipocytes within the tissues. We also found no difference in the expression of UCP and mitochondrial proteins between the Adipo-Atp2a2 / and control mice at 22 C (Supplementary Fig. 6b e). Histological examination of adipose tissues identified no obvious abnormality in cell and tissue structure or mitochondrial morphology in Adipo-Atp2a2 / mice (Supplementary Fig. 6f,g). We observed no difference in body weight, adipose tissue mass and food intake between Adipo-Atp2a2 / mice and controls on a regular diet at 22 C (Supplementary Fig. 6h j). However, the inguinal WAT of Adipo-Atp2a2 / mice had a significantly lower than that of littermate controls following treatment (Fig. 2k), suggesting that SERCA2b is required for beige fat thermogenesis. On the other hand, potently increased the to a similar degree in the ibat of Adipo-Atp2a2 / mice and controls (Fig. 2l), indicating that SERCA2 is dispensable for BAT thermogenesis in the presence of UCP. This is consistent with the results indicating that pharmacological inhibition of SERCA by thapsigargin did not attenuate the -induced increase in in wild-type brown adipocytes, whereas Ucp / brown adipocytes failed to respond to (Supplementary Fig. 6k). To further probe the requirement for SERCA2b in adipose thermogenesis in vivo, we recorded the adipose tissue temperature of Adipo-Atp2a2 / mice. To induce beige adipocyte biogenesis pharmacologically, we chronically treated the mice with CL36,243 for five consecutive days. We found that the inguinal WAT temperature of control mice rapidly increased by. C or more following treatment, indicating that beige fat thermogenesis occurred. However, this -induced beige fat thermogenesis was completely blunted in Adipo- Atp2a2 / mice (Fig. 2m). In contrast, the ibat temperature was significantly increased by injection both in Adipo-Atp2a2 / mice and controls (Fig. 2n). No difference in skeletal muscle temperature was seen between the two groups (Fig. 2o and Supplementary Fig. 6l). Enhanced Ca 2+ cycling stimulates UCP-independent thermogenesis in beige fat Given the established role of SERCA in intracellular Ca 2+ cycling, we examined the extent to which Ca 2+ flux controls UCP-independent thermogenesis. We observed a robust increase in intracellular Ca 2+ levels both in wild-type and Ucp / beige adipocytes within min of treatment (Fig. 3a). Of note, depletion of extracellular Ca 2+ did not affect -induced respiration in Ucp / beige adipocytes (Fig. 3b), whereas acute depletion of intracellular Ca 2+ by a cell-permeant Ca 2+ chelator, BAPTA, completely blocked the -induced increase in in both wild-type and Ucp / beige adipocytes (Fig. 3c and Supplementary Fig. 7a). In contrast, brown fat thermogenesis was highly dependent on UCP and not affected by BAPTA (Fig. 3d). is known to bind to -ARs and also to -adrenergic receptors ( -ARs), and it triggers intracellular Ca 2+ signaling 3. As our RNA-seq analysis found that -AR, -AR and 3-AR were highly expressed in the inguinal WAT of Ucp / mice (Supplementary Fig. 7b), we asked which forms of AR mediate the stimulatory effect of on UCP-independent thermogenesis. We found that -AR and 3-AR mediate the action of to activate UCP-independent thermogenesis, as pharmacological stimulation of -AR and 3-AR by phenylephrine and CL36,243, respectively, significantly increased the and intracellular Ca 2+ levels in Ucp / beige adipocytes (Fig. 3e and Supplementary Fig. 7c). On the other hand, the -AR agonist (denopamine) and 2-AR agonist (clonidine) did not alter the. This lack of effect from the 2-AR agonist is likely because 2-AR is expressed at an undetectable level in beige adipocytes (Supplementary Fig. 7b). Conversely, pharmacological inhibition of -AR and 3-AR by phenoxybenzamine and SR5923A, respectively, partially but significantly blunted the -stimulated increase in in Ucp / beige adipocytes (Fig. 3f). We next employed the following gain-of-function approaches to test whether enhanced Ca 2+ cycling through the SERCA2b RyR2 pathway stimulates UCP-independent thermogenesis. First, we examined the extent to which enhanced Ca 2+ flux through RyR2 stimulates beige fat thermogenesis. The rationale for this approach is based on our RNA-seq data showing that Ryr2 expression was significantly higher in the inguinal WAT of Ucp / mice relative to that of mice with the other genotypes (Supplementary Fig. 8a). As RyR2 is the predominant form among the ryanodine receptor family members in beige adipocytes (Supplementary Fig. 8b), we overexpressed RyR2 or an empty vector in Ucp / beige adipocytes (Supplementary Fig. 8c,d). Although RyR2 overexpression did not affect beige adipocyte differentiation or mitochondrial contents (Supplementary Fig. 8e), Ucp / beige adipocytes expressing RyR2 displayed a higher than vector-expressing cells (Fig. 3g). Furthermore, overexpression of SERCA2b and RyR2 additively increased the -stimulated in Ucp / beige adipocytes (Supplementary Fig. 8f). Additionally, pharmacological inhibition of RyR2 by ryanodine at a high dose ( M) or ruthenium red partially but significantly blunted the -stimulated increase in (Supplementary Fig. 8g,h). Second, we overexpressed calstabin2 (encoded by Fkbpb), a key subunit of the RyR2 complex, in Ucp / beige adipocytes (Supplementary Fig. 8i). Previous studies have demonstrated that Calstabin2 overexpression in cardiomyocytes reduces Ca 2+ leak and increases Ca 2+ loading in the SR, thereby enhancing Ca 2+ release and muscle contractility in response to caffeine stimulation Of note, Calstabin2 is abundantly expressed in adipose tissue (Supplementary Fig. 8j), although its mrna expression was unchanged by Prdm6 transgene expression (Supplementary Fig. 8k). We found that Calstabin2 overexpression NATURE MEDICI ADVANCE ONLI PUBLICATION 5

6 A RTICLES in Ucp / beige adipocytes potentiated Ca 2+ release from the ER following treatment (Fig. 3h). The enhanced Ca 2+ release in Calstabin2-expressing cells was accompanied by a significant increase in -induced relative to vector-expressing cells (Fig. 3i). Third, we tested whether acute enhancement of RyR2 function by a pharmacological stabilizer of RyR2 (S7) activates UCP-independent thermogenesis in vivo. S7 treatment is known to prevent Ca 2+ leak and enhance Ca 2+ loading in the SR by stabilizing the interaction between Calstabin2 and RyR2, leading to an improvement in cardiac contractility in vivo 35. S7 treatment also enhances caffeine-induced 27 Nature America, Inc., part of Springer Nature. All rights reserved. a b c Normalized fluorescence intensity (F/F) (pmol/min per g protein) WT Time (s) 25 (pmol/min per g protein) (pmol/min per g protein) Basal (% in vehicle-treated cells) Total Uncoupled EGTA Thapsi + d # e f g BAPTA WT + + -AR 2-AR -AR 2-AR (% in -treated cells) (pmol/min per g protein) Vector Vector, veh Calstabin2 Vector, beige adipocytes Calstabin2, veh Calstabin2, Time (s) Selective agonists h i j F/F WT ## ## BAPTA BAPTA Core body temperature ( C) -AR 3-AR -AR + 3-AR Selective inhibitors mice (pmol/min per g protein) (pmol/min per g protein) RyR stabilizer (S7) Time (h) after cold exposure Oligo FCCP AA Vector RyR EMG ( V) EMG ( V), 5 5,, 5 5, EMG RMS ( V) Figure 3 Enhanced Ca 2+ cycling stimulates UCP-independent thermogenesis in beige fat. (a) Intracellular Ca 2+ levels in differentiated beige adipocytes from wild-type (WT) and Ucp / mice following treatment (arrow). n = 6 for all groups. P <., pretreatment versus treatment. (b) Total and oligomycin-resistant (uncoupled) in Ucp / beige adipocytes in a Ca 2+ -depleted medium containing egtazic acid (EGTA) in the presence of or thapsigargin. n = 6 for all groups. P <.. (c) Basal in WT and Ucp / beige adipocytes treated with BAPTA or vehicle. WT cells, n = 5 for all the treatment; Ucp / with vehicle or, n = 2; Ucp / with BAPTA or BAPTA +, n = 5. P <. between vehicle and, ##P <. between vehicle and BAPTA. (d) Basal in WT and Ucp / brown adipocytes incubated in a medium containing BAPTA or vehicle. WT with vehicle, n = 8; WT with, n = 6; WT with BAPTA, n = 8; WT with BAPTA +, n = 6; Ucp / with vehicle n = 8; Ucp / with, n = 6; Ucp / with BAPTA, n = 8; Ucp / with BAPTA +, n = 6. P <., P <. between vehicle and, #P <.5 between WT and Ucp /. (e) Basal in Ucp / beige adipocytes treated with or specific agonists for -AR (phenylephrine), 2-AR (clonidine), -AR (denopamine) and 3-AR (CL36243). and, n = 4; phenylephrine, n = 5; clonidine, n = 5; denopamine, n = 5; CL36243, n = 5. P <.5, P <. relative to vehicle. (f) in Ucp / beige adipocytes treated with specific inhibitors for -AR (phenoxybenzamine) and 3-AR (SR5923A) in the presence of., n = 8; phenoxybenzamine, n = 8; SR5923A, n = 9; phenoxybenzamine + SR5923A, n = 8. P <.. (g) in Ucp / beige adipocytes expressing RyR2 or an empty vector. Oligomycin, FCCP and antimycin (AA) were added at indicated time points. n = 5 for all groups. P <.. (h) -induced Ca 2+ release in Ucp / beige adipocytes expressing Calbstabin2 or an empty vector in a Ca 2+ -depleted medium. n = 5 for all groups. P <.. (i) Basal in Ucp / beige adipocytes expressing Calbstabin2 or an empty vector. or vehicle was added at the indicated time point (arrow). Vector with vehicle n = 8; vector with, n = 5; Calstabin2 with vehicle or, n = for both. P <.5, P <., P <.. (j) Rectal core body temperature of Ucp / mice treated with the RyR2 stabilizer S7 or vehicle under conditions of 6 C at the indicated time points., n = 9; S7, n = 8. P <.5. (k) Representative EMG traces of Ucp / mice treated with vehicle or S7 under conditions at 6 C. n = 4 for both groups. Data are expressed as means s.e.m. Data were analyzed by Student s t-test (a,g,h,j,k) or one-way ANOVA followed by Tukey s test (b f,i). k S7 5 s S7 6 ADVANCE ONLI PUBLICATION NATURE MEDICI

7 ARTICLES 27 Nature America, Inc., part of Springer Nature. All rights reserved. Ca 2+ release from the ER and promotes insulin secretion by pancreatic beta cells 36. Accordingly, we implanted osmotic pumps, each containing S7 at a dose of 2 mg per kg body weight or vehicle, in Ucp / mice for 7 d and afterward transferred the mice to an environment at 6 C. Although vehicle-treated Ucp / mice developed hypothermia following cold exposure, S7-treated Ucp / mice were capable of maintaining their core body temperature (Fig. 3j). As muscle shivering in Ucp / mice was already active under cold conditions, we did not observe any further enhancement in muscle shivering due to S7 treatment (Fig. 3k). A recent study reported a futile cycle of creatine-driven thermogenesis in brown and beige fat 22. We examined the extent to which Ca 2+ cycling and creatine cycling converge to control UCP-independent thermogenesis because both of these processes involve ATP-dependent thermogenesis. To this end, we depleted SERCA2b in Ucp / beige adipocytes using the lentiviral shrna targeting Atp2a2 and inhibited the creatine cycle by treating the cells with -guanidinopropionic acid ( -GPA), a creatine analog that potently inhibits creatine transport. We found that SERCA2b depletion significantly reduced the in Ucp / beige adipocytes even in the presence of -GPA, whereas inhibition of the by -GPA was not observed in SERCA2b-depleted cells (Supplementary Fig. 8l). The antiobesity and antidiabetic effects of beige fat are independent of UCP The distinct function of the SERCA2b RyR2 pathway in UCP- independent thermogenesis led us to hypothesize that the activation of beige adipocyte biogenesis could prevent diet-induced obesity and glucose intolerance independently of UCP. To test this hypothesis, Ucp / mice and controls were subjected to a high-fat diet (HFD) for up to 24 weeks. We found that mice gained significantly less body weight at 9 weeks on a HFD and thereafter under ambient temperature at 22 C (Fig. 4a). Ucp / mice also gained significantly less body weight than littermate Ucp / mice at weeks of a HFD and thereafter (Fig. 4b). The difference in body weight was due to reduced fat mass but not to reduced lean mass (Supplementary Fig. 9a). Notably, Ucp / mice fully normalized the obese phenotype to a similar degree as mice did (Fig. 4c). We did not observe any difference in food intake or locomotor activity among the genotypes (Supplementary Fig. 9b,c). We next investigated the requirement for UCP in systemic glucose homeostasis. A glucose tolerance test (GTT) showed that mice exhibited significantly improved glucose tolerance relative to littermate controls at weeks of a HFD even when there was no difference in body weight between the two groups (Fig. 4d). Ucp / mice similarly displayed a marked increase in glucose tolerance at weeks on a HFD relative to littermate Ucp / mice (Fig. 4d). Glucose tolerance of Ucp / mice was improved to a comparable degree to that of mice when normalized to their respective controls (Supplementary Fig. 9d). Likewise, an insulin tolerance test (ITT) found that Prdm6 mice and Ucp / mice were both more insulin sensitive than their respective littermate controls at weeks of a HFD (Fig. 4e and Supplementary Fig. 9e). The improvements shown in the GTT and ITT were not observed under a regular chow diet (Supplementary Fig. 9f,g). When animals were fed a HFD under thermoneutrality at 3 C, mice gained slightly less body weight than littermate controls (Fig. 4f). The reduced body weight gain in mice was associated with reduced fat mass but not with reduced lean mass (Supplementary Fig. 9h). On the other hand, Ucp / mice and Ucp / mice gained a similar degree of body weight and fat mass at 3 C (Fig. 4f and Supplementary Fig. 9i); however, Prdm6 Tg mice and Ucp / mice exhibited improved glucose tolerance relative to their respective littermate controls (Fig. 4g). Enhanced glucose utilization in UCP-null beige fat To investigate the molecular mechanisms through which beige fat improves systemic glucose tolerance in a UCP-independent manner, we performed metabolomics in inguinal WAT of mice (Supplementary Table ). The metabolomics data combined with the RNA-seq data suggest that glycolysis and the TCA cycle were substantially activated in the inguinal WAT of Ucp / mice (Figs. 2b and 5a). For instance, mrna expression of a large number of genes encoding products involved in glycolysis and TCA metabolism was significantly higher in Ucp / mice relative to mice from the other groups (Supplementary Fig. a). This alteration was selective in inguinal WAT because no major difference between groups was seen in ibat and epididymal WAT (Supplementary Fig. b,c). The results lead to the hypothesis that Ucp / beige adipocytes actively utilize glucose through enhanced glycolysis and TCA metabolism for ATP-dependent thermogenesis by SERCA2b. In fact, glucose uptake in the inguinal WAT of Ucp / mice was significantly higher than that in the other genotypes (Fig. 5b). In contrast, glucose uptake in ibat and epididymal WAT was not different between Ucp / mice and controls. Furthermore, enzymatic activity of the pyruvate dehydrogenase (PDH) complex, a key regulator that converts pyruvate into acetyl-coa, and expression of the genes encoding it were significantly higher in the inguinal WAT of Ucp / mice relative to the other groups (Fig. 5c and Supplementary Fig. d). PDH activity in ibat and epididymal WAT was not different between Ucp / mice and Ucp / mice (Fig. 5c). Of note, the enhanced glucose oxidation in Ucp / beige fat significantly affected whole-body metabolism; Ucp / mice displayed a higher respiratory exchange ratio (RER; VO 2 /VCO 2 ) than littermate Ucp / mice, indicating that Ucp / mice utilize carbohydrate (glucose) as their primary fuel source rather than fat (Fig. 5d). The SERCA2 RyR2 pathway controls glucose oxidation in beige fat The above results led us to examine the extent to which the SERCA2b RyR2 pathway controls glucose utilization in beige fat. Accordingly, we measured the extracellular acidification rate (ECAR), a cellular index of glycolysis, in differentiated Ucp / beige adipocytes under a culture condition with low or high glucose concentrations. We found that treatment potently increased the ECAR in Ucp / beige adipocytes both under low- and high-glucose conditions (Fig. 6a). Oligomycin treatment further increased the ECAR in Ucp / beige adipocytes, whereas blockage of glycolysis by 2-deoxy-D-glucose (2-DG) potently reduced basal and -induced ECAR to a similar level. When SERCA2b was depleted by the lentiviral shrna targeting Atp2a2, we found a significant reduction in basal and -stimulated ECAR (Fig. 6b). Acute depletion of intracellular Ca 2+ by BAPTA completely blocked -induced ECAR (Supplementary Fig. a), indicating that -stimulated Ca 2+ flux is required for glycolytic ATP production in beige adipocytes. In accordance with this observation, depletion of SERCA2b led to a significant reduction in glucose oxidation in Ucp / beige adipocytes (Fig. 6c). The decreases in ECAR and NATURE MEDICI ADVANCE ONLI PUBLICATION 7

8 A RTICLES a Body weight (g) d C High fat diet Week 5 b Body weight (g) C High fat diet Week e 25 c Fat mass/body weight Nature America, Inc., part of Springer Nature. All rights reserved. Blood glucose (mg/dl) f Body weight (g) Blood glucose (mg/dl) Body weight (g) C 22 C C 3 C High-fat diet P = Week 3 6 P =. High-fat diet Week Blood glucose (mg/dl) Blood glucose (mg/dl) Blood glucose (mg/dl) Blood glucose (mg/dl) 2 5 P =.474 P = C 22 C P =.9 P = Figure 4 UCP-independent mechanisms in beige fat are involved in the regulation of body weight and glucose metabolism in vivo. (a) Body weight of mice and the littermate controls under a HFD at 22 C. n = 7 for both groups. (b) Body weight of Ucp / and the littermate Ucp / mice under a HFD at 22 C. Ucp /, n = 8; Ucp /, n = 2. (c) Adiposity (ratio of fat mass to body weight) of mice at 8 weeks of HFD. rol, n = 7;, n = 7; Ucp /, n = 8; Ucp /, n = 2. (d) Results from a GTT in and the littermate controls (left) and Ucp / and the littermate Ucp / mice (right) at weeks of a HFD at 22 C. rol, n = 9;, n = 8; Ucp /, n = ; Ucp /, n = 7. (e) Results from an ITT in the mice in d at weeks of a HFD. (f) Body weight of and the littermate controls (left) and Ucp / and the littermate Ucp / mice (right) on a HFD under conditions of 3 C. rol, n = 9; Prdm6 Tg, n = 7; Ucp /, n = 7; Ucp /, n = 8. (g) Results from a GTT in and the littermate controls (left) and Ucp / and the littermate Ucp / mice (right) at weeks of a HFD under conditions of 3 C. rol, n = 9;, n = 7; Ucp /, n = 7; Ucp /, n = 5. Data are expressed as means s.e.m. Data were analyzed by Student s t-test (c) or two-way ANOVA followed by Fisher s LSD test (a,b,d g). P <.5, P <., P <.. g glucose oxidation due to SERCA2b depletion were accompanied by reduced glucose uptake (Fig. 6d). On the other hand, fatty acid oxidation was not affected by SERCA2b depletion (Fig. 6e). Conversely, overexpression of RyR2 significantly increased ECAR and glucose uptake in Ucp / beige adipocytes (Fig. 6f and Supplementary Fig. b). Glycolytic ATP production profoundly contributes to ATP-dependent thermogenesis in Ucp / beige fat because blockade of the glycolysis pathway by 2-DG reduced in Ucp / beige adipocytes and potently and quickly increased in vehicletreated cells (Fig. 6g). Ca 2+ cycling thermogenesis is an evolutionarily conserved mechanism Lastly, we asked whether Ca 2+ cycling mediated thermogenesis is evolutionarily relevant in other mammalian species. To do this, we chose pig adipocytes as a model because pigs lack a functional UCP protein owing to deletion of exons 3 5 in the UCP gene 37. Pig SVFs isolated from the subcutaneous WAT of neonatal piglets were infected with retroviruses expressing PRDM6 or an empty vector and differentiated into mature adipocytes under adipogenic conditions (Fig. 6h). We found that PRDM6-expressing pig adipocytes had significantly 8 ADVANCE ONLI PUBLICATION NATURE MEDICI

9 ARTICLES 27 Nature America, Inc., part of Springer Nature. All rights reserved. a HK2 Glycolysis Glucose Glucose 6-phosphate Gpi Fructose 6-phosphate Pfk Glyceraldehyde 3P Pgk Fructose-,6-BP,3BPG 3P-glycerate 2P-glycerate P-enolpyruvate Pyruvate Fbp2 No change d RER (VO 2 /VCO 2 ) Night Pdha Pdhb Dld Pdhx Pyruvate Malate Fh 3 C Oxaloacetate Mdh2 Fumarate Sdhd Sdhc Sdhb Sdha Succinate higher mrna expression of the beige-fat-selective genes, including CIDEA, DIO2, ELOVL3 and TMEM26, than the vector-expressing adipocytes (Fig. 6i). The pig beige adipocytes also expressed higher amounts of mitochondrial proteins than the vector-expressing white adipocytes (Fig. 6j). When pig SERCA2b was depleted by the lentiviral shrna targeting Atp2a2 (Fig. 6k), we observed a significant reduction in basal and -stimulated, whereas potently increased in beige adipocytes expressing a scrambled RNA (Fig. 6l). treatment also increased ECAR in pig beige adipocytes, whereas SERCA2b depletion reduced basal and -stimulated ECAR (Fig. 6m). Conversely, RyR2-expressing pig adipocytes displayed higher than vector-expressing cells (Fig. 6n). DISCUSSION Our results suggest the following model (Fig. 6o). In response to cold exposure, binds to -AR and 3-AR to increase intracellular Ca 2+ flux by activating SERCA2b and RyR2 in beige adipocytes. Ca 2+ is transported back to its storage in the ER by the action of SERCA2b and likely to the mitochondria through a voltage-dependent anion channel and mitochondrial calcium uniporter. Increased Ca 2+ in the mitochondria, in turn, activates the PDH phosphatase PDPc 38, thereby activating the PDH enzyme and ATP synthesis. In the absence -oxidation Acetyl CoA TCA cycle Electron transport chain Day Night Day Night Day Night 22 C Suclg Suclg2 Increased in PRDM6 Tg Fatty acid synthesis Cs Citrate Aco2 Isocitrate Idh3a Idh3b Idh3g -ketoglutarate Ogdh Dlst Dld Succinyl CoA b Glucose uptake (% ID/g kg body weight) c PDH activity (OD/protein per min) Ing WAT ibat 2, RER (VO 2 /VCO 2 ).8 Ing WAT 3 C Glucose uptake (% ID/g kg body weight),2, PDH activity (OD/protein per min) C Day ibat Glucose uptake (% ID/g kg body weight) Epi WAT Epi WAT of UCP, beige adipocytes utilize glucose as the primary fuel source for ATP synthesis through enhanced glycolysis, TCA metabolism and the mitochondrial electron transport chain (ETC). Noncanonical thermogenesis occurs when calcium transport is uncoupled from ATP hydrolysis by SERCA2b, although the regulators of SERCA2 activity in beige adipocytes remain undetermined. Because the inhibition of RyR2 partly, but not completely, blocks -induced thermogenesis, it is conceivable that inositol,4,5-trisphosphate receptors (IP 3 Rs), such as IP 3 R and IP 3 R2, are also involved in the regulation of Ca 2+ release and noncanonical thermogenesis in beige adipocytes. Additionally, protein kinase A (PKA) signaling is known to phosphorylate RyR2, leading to dissociation between Calstabin2 and RyR2 and enhancement of Ca 2+ release from the SR 39. -induced posttranslational modifications of RyR2 and SERCA2b in beige fat await future investigations. Although brown adipocytes are highly enriched in mitochondria, they express very low amounts of ATP synthase and thus possess low ATP synthesis capacity 4. As such, ATP-dependent thermogenesis is likely incapable of fully compensating for UCP loss in vivo. On the other hand, beige adipocytes express high levels of ATP synthase and generate ATP through enhanced glycolysis, the TCA cycle and the mitochondrial ETC; thus, the ATP-dependent thermogenesis by 2 4 Figure 5 Active glucose utilization occurs in Ucp / beige fat through enhanced glycolysis and TCA metabolism. (a) Enhanced metabolic pathways in the inguinal WAT of Ucp / mice. The genes and metabolites determined to be upregulated in the inguinal WAT of Ucp / mice based on RNA-seq and metabolomic data are highlighted in red. n = 3 for all groups. (b) 8 F-fluorodeoxyglucose ( 8 F-FDG) uptake in the indicated tissues and groups of mice. rol, n = 6;, n = 6; Ucp /, n = 5; Ucp /, n = 4. ID/g, injected dose per gram of tissue. (c) PDH enzymatic activity in the indicated tissues and groups of mice. rol, n = 8;, n = 7; Ucp /, n = ; Ucp /, n = 7. (d) RER in mice under conditions of 3 C and 22 C. rol, n = 6;, n = 5; Ucp /, n = 6; Ucp /, n = 6. Data in b d are expressed as means s.e.m. Data were analyzed by Student s t-test (a), one-way ANOVA followed by Tukey s test (b,c) or two-way ANOVA followed by Fisher s LSD test (d). P <.5, P <., P <.. PDH activity (OD/protein per min) 3 2 NATURE MEDICI ADVANCE ONLI PUBLICATION 9

10 A RTICLES 27 Nature America, Inc., part of Springer Nature. All rights reserved. a ECAR (mph/min per g protein) f ECAR (mph/min per g protein) SERCA2 -actin Glucose Oligo 2-DG Glucose Oligomycin Glucose oxidation Glucose uptake Fatty acid oxidation (a) (b) rol Scr RyR2 shatp2a2 (mph/min per g protein) DG ECAR (mph/min per g protein) 2-DG (mph/min per g protein) b c d e Scr shatp2a2 ECAR (mph/min per g protein).5..5 ECAR (mph/min per g protein) Pig white adipocytes. Scr shatp2a2 Scr shatp2a2 rol RyR2 (mph/min per g protein) Relative glucose oxidation (%) Relative glucose uptake (%) Scr shatp2a2 Scr shatp2a2 Scr shatp2a2 Scr shatp2a2 g h i j Pig beige adipocytes Relative mrna expression Pig white adipocytes Pig beige adipocytes CIDEA DIO2 k l m n o ELOVL3 TMEM26 Cold Norepinephrine -AR Complex V Complex IV Complex III Complex II Complex I -tubulin VDAC MCU camp Relative fatty acid uptake (%) Ca 2+ Cal2 Ca 2+ Heat ADP+Pi Ca 2+ ETC PDH Pig white adipocytes TCA cycle 3-AR RyR2 SERCA2b ATP Pig beige adipocytes Figure 6 The SERCA2 RyR2 pathway controls glucose utilization and thermogenesis in Ucp / beige fat. (a) ECAR in Ucp / beige adipocytes in culture medium with low or high glucose concentrations. Glucose, oligomycin and 2-DG were added at the time points indicated by dashed lines., n = 7;, n = 8. (b) ECAR in Ucp / beige adipocytes expressing a scrambled control RNA (Scr) or shrna targeting Atp2a2 (shatp2a2). Differentiated cells were treated with or vehicle in the culture medium with a high glucose concentration (left) and oligomycin (right). Scr with vehicle, n = 7; Scr with, n = 8; shatp2a2 with vehicle, n = 9; shatp2a2 with, n = 9. (c) Glucose oxidation in Ucp / beige adipocytes expressing a scrambled control RNA or shatp2a2. n = 6 for both groups. (d) Glucose uptake in Ucp / beige adipocytes expressing a scrambled control RNA or shatp2a2. n = 3 for both groups. (e) Fatty acid oxidation in Ucp / beige adipocytes expressing a scrambled control RNA or shatp2a2. n = 6 for both groups. (f) ECAR in Ucp / beige adipocytes expressing RyR2 or an empty vector (control). n = for both groups. (g) in Ucp / beige adipocytes treated with 2-DG, or oligomycin at indicated time points., n = 9; 2-DG, n =. (h) Oil Red O staining of differentiated pig adipocytes expressing PRDM6 (beige) and vector (white) at low magnification (top) and high magnification (bottom). Scale bars, 5 m. (i) mrna expression of the indicated beige-fat-selective genes in differentiated pig adipocytes expressing PRDM6 (beige) and an empty vector (white). n = 3 for both groups. (j) Mitochondrial oxidative phosphorylation (OXPHOS) proteins in differentiated pig adipocytes expressing PRDM6 (beige) or an empty vector (white). Tissue lysates from the rat heart were used as a positive control (p.c.). -tubulin antibody was used as a loading control. (k) SERCA2 protein in differentiated pig beige adipocytes expressing a scrambled control RNA or shatp2a2. -actin was shown as a loading control. Molecular weight (kda) is shown on the right in j and k. (l) Basal in pig beige adipocytes expressing a scrambled control RNA or shatp2a2. Scr with vehicle, n = 2; Scr with, n = 7; shatp2a2 with vehicle, n = 5; shatp2a2 with, n = 5. (m) ECAR in pig beige adipocytes expressing a scrambled control RNA or shatp2a2. Scr with vehicle, n = 5; Scr with, n = 6; shatp2a2 with vehicle, n = 5; shatp2a2 with, n = 6. (n) Basal in pig beige adipocytes expressing RyR2 or an empty vector (control). rol with vehicle, n = 2; control with, RyR2 with vehicle and RyR2 with, n =. (o) A proposed model of noncanonical thermogenesis in beige fat. Calstabin2, Cal2; voltage-dependent anion channel, VDAC; mitochondrial calcium uniporter, MCU. Data in a g, i and l n are expressed as means s.e.m. Data were analyzed by Student s t-test (a,c g,i) or one-way ANOVA followed by Tukey s test (b,l n). P <.5, P <., P <.. IP 3 RS Pyruvate Glycolysis Rat heart mitocondria 5 Glucose (p.c.) SERCA2 can compensate for UCP loss. These data suggest a distinct functional requirement for UCP-mediated canonical thermogenesis and the SERCA2b-mediated thermogenesis in brown fat and beige fat, respectively. Our study demonstrates the biological importance of noncanonical beige fat thermogenesis in whole-body energy metabolism: i.e., activation of beige fat biogenesis by fat-selective PRDM6 expression protects animals from cold-induced hypothermia and diet-induced obesity and diabetes independently of UCP. In skeletal muscle, nonshivering thermogenesis occurs through a UCP-independent mechanism in which sarcolipin controls the ATP hydrolysis activity of SERCA and Ca 2+ transport 2. Tangentially, Ca 2+ cycling by SERCA and RyR is suggested to be a mechanism of thermogenesis in the heat organ located in the extraocular muscle beneath the brain of endothermic fish, such as swordfish and blue marlin 4. These results together with those from the present study in mice, humans ADVANCE ONLI PUBLICATION NATURE MEDICI