Endonuclease G (EndoG) is one of the most abundant

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1 ORIGINAL RESEARCH EndoG Knockout Mice Show Increased Brown Adipocyte Recruitment in White Adipose Tissue and Improved Glucose Homeostasis Rosario Pardo, Natividad Blasco,* Maria Vilà,* Daniel Beiroa,* Rubén Nogueiras, Xavier Cañas, Rafael Simó, Daniel Sanchis, and Josep A. Villena Laboratory of Metabolism and Obesity (R.P., M.V., J.A.V.) and Group of Diabetes and Metabolism (R.S.), Vall d Hebron-Institut de Recerca, Universitat Autònoma de Barcelona, Barcelona, Spain; Cell Signaling and Apoptosis Group (N.B., D.S.), Biomedical Research Institute of Lleida, University of Lleida, Lleida, Spain; Department of Physiology (D.B., R.N.), Center for Research in Molecular Medicine and Chronic Diseases, Universidad de Santiago de Compostela, and Centro de Investigación Biomédica en Red on Physiopathology of Obesity and Nutrition (D.B., R.N.), Santiago de Compostela, Spain; Laboratory Animal Applied Research Platform (X.C.), Scientific Park of Barcelona, Barcelona, Spain; Centro de Investigación Biomédica en Red on Diabetes and Associated Metabolic Diseases (J.A.V. R.S.), Barcelona, Spain Brown adipose tissue (BAT) plays a central role in the regulation of whole-body energy and glucose homeostasis owing to its elevated capacity for lipid and glucose oxidation. The BAT thermogenic function, which is essential for the defense of body temperature against exposure to low environmental temperatures, relies on the expression in the inner membrane of brown adipocyte s mitochondria of uncoupling protein-1, a protein that uncouples substrate oxidation from oxidative phosphorylation and leads to the production of heat instead of ATP. BAT thermogenesis depends on proper mitochondrial biogenesis during the differentiation of brown adipocytes. Despite the data that support a role for Endonuclease G (EndoG) in the process of mitochondrial biogenesis, its function in BAT has not been explored. Here, using an EndoG knockout mouse model, we demonstrate that EndoG is not essential for the expression of mitochondrial genes involved in substrate oxidation or for the induction of thermogenic genes in BAT in response to cold exposure. We also show that a lack of EndoG is associated with an increased expression of thermogenic genes (ie, uncoupling protein-1, peroxisome proliferator-activated receptor- coactivator-1 ) in white adipose tissue (WAT) that correlates with the appearance of brown adipocyte-like cells interspersed among white adipocytes. Interestingly, the increased browning of WAT elicited by the lack of EndoG was associated with a better glucose tolerance and reduced fat mass. Our results suggest that the induction of browning in WAT by means of inhibiting EndoG activity appears as a potential therapeutic strategy to prevent obesity and ameliorate glucose intolerance. (Endocrinology 157: , 2016) Endonuclease G (EndoG) is one of the most abundant nucleases in eukaryotic cells. The nuclease activity of EndoG is highly unspecific, acting on RNA, single- and double-stranded DNA, or DNA-RNA duplexes (1, 2). Nevertheless, the cellular function of EndoG remains controversial. Several studies have implicated EndoG in the process of programmed cell death. These studies have reported that EndoG, during the process of cell death, is released from mitochondria and translocated to the nucleus (3, 4), in which it has been shown to contribute to the ISSN Print ISSN Online Printed in USA Copyright 2016 by the Endocrine Society Received April 14, Accepted August 16, First Published Online August 22, 2016 * N.B., M.V., and D.B. contributed equally to this work. Abbreviations: ANP, atrial natriuretic peptide; BAT, brown adipose tissue; COX, cytochrome oxidase; EndoG, endonuclease G; FBS, fetal bovine serum; KO, knockout; mtdna, mitochondrial DNA; PGC-1, peroxisome proliferator-activated receptor- coactivator 1; SVF, stromal vascular fraction; UCP1, uncoupling protein-1; WAT, white adipose tissue; Wt, wild type. doi: /en Endocrinology, October 2016, 157(10): press.endocrine.org/journal/endo 3873

2 3874 Pardo et al EndoG in Energy and Glucose Homeostasis Endocrinology, October 2016, 157(10): fragmentation of chromosomal DNA in a variety of cell types, such as hepatocytes (5), neurons (6), and cardiomyocytes (7). However, the function of EndoG as an effector of cell death remains dubious. Indeed, the independent generation of two EndoG knockout mouse models has unequivocally demonstrated that EndoG is dispensable for normal embryogenesis and development, two processes in which programmed cell death is essential during tissue remodeling (8, 9). This, together with the fact that fibroblasts from EndoG knockout mice retain their sensitivity to a wide variety of cell death-inducing stimuli (8), strongly advocates against a determinant role of EndoG in programmed cell death. Consistent with its localization in the mitochondria (1, 10), early studies also suggested that EndoG could play a fundamental role in mitochondrial biogenesis by regulating mitochondrial DNA (mtdna) replication. More specifically, EndoG was proposed to participate in the generation of the primers required for mtdna replication by cleaving the RNA from DNA-RNA duplexes, known as R loops, that are generated during mtdna transcription (1). Although the function of EndoG in the synthesis of mtdna has not been unambiguously clarified (11, 12), a recent study has provided additional data that support a central role for EndoG in the process of mitochondrial biogenesis (13). In this study, the authors showed that overexpression of EndoG in cultured cardiomyocytes resulted in an increase in mitochondrial mass, whereas a lack of EndoG was associated with reduced mitochondrial content, decreased mtdna copy number, impaired oxidative function, and increased reactive oxygen species in the hearts of EndoG null mice. These results highlight the importance of EndoG in the energetic homeostasis of cardiac cells. The exact mechanisms by which EndoG controls the process of mitochondrial biogenesis are unclear, but the finding that EndoG binds throughout the mtdna molecule, in an analogous manner as mitochondrial transcription factor A (14), suggests that EndoG participates in the regulation of mtdna replication and/or transcription. Brown adipose tissue (BAT) is a specialized type of fat tissue present in most mammals, including adult humans, whose primary function is the production of heat to maintain body temperature in response to cold. The process of heat production, known as nonshivering adaptive thermogenesis, relies on the exclusive presence in the mitochondria of brown adipocytes of uncoupling protein-1 (UCP1), a protein located in the inner mitochondrial membrane that acts as a proton channel and uncouples oxidative phosphorylation from the synthesis of ATP. Upon exposure to low temperatures, -adrenergic stimulation of brown adipocytes activates an intracellular signaling cascade that leads to the activation of lipolysis, which provides fatty acids that serve as substrate for -oxidation but also directly enable UCP1 thermogenic activity (reviewed in reference 15). Simultaneously, sympathetic stimulation leads to the transcriptional activation of the thermogenic program, enhancing the expression of UCP1 and mitochondrial oxidative genes. As a result, the electrochemical proton gradient generated by the oxidation of substrates through the complexes of the mitochondrial respiratory chain/oxidative phosphorylation (OxPhos) system is dissipated by UCP1, generating heat instead of ATP. Clusters of UCP1-positive adipocytes can be also found in white adipose tissue (WAT) depots, interspersed among white adipocytes (16, 17). These so-called brite (brown in white) adipocytes, beige adipocytes, or inducible brown adipocytes are endowed with all the machinery required to carry thermogenesis, including high mitochondrial content and UCP1 expression (18). Contrary to brown adipocytes from BAT, which originate from a myf5 precursor of the muscle cell lineage, brite adipocytes derive from a distinct, yet poorly characterized, precursor cell (19). Brite adipocytes in WAT are heavily recruited in response to cold exposure or 3 -adrenergic receptor agonist treatment (16), a phenomenon known as browning of WAT. In addition, other hormones or drugs have been shown to increase brite adipocyte recruitment, including thiazolidinediones (20), natriuretic peptides (21), or fibroblast growth factor-21 (22). In animal models, the recruitment of brite adipocytes in WAT, as well as the activation of BAT thermogenesis, has been associated with an improvement of metabolic diseases, like obesity or type 2 diabetes (23 26). Consequently, the finding of new pharmacological targets that increase the mass and thermogenic activity of brown and brite adipocytes has aroused a great deal of interest as a novel therapeutic approach for the treatment of human metabolic diseases. The thermogenic activity of both brown and brite adipocytes greatly depends on their high mitochondrial content and elevated oxidative capacity. Despite recent data that indicate a crucial role of EndoG in the regulation of mitochondrial biogenesis, the function of EndoG in the control of mitochondrial biogenesis and thermogenic function of BAT has not been explored. Here, using a mouse model devoid of EndoG, we show that lack of EndoG impairs neither the expression of OxPhos mitochondrial genes nor the induction of thermogenic genes in BAT in response to cold exposure. Interestingly, we found that lack of EndoG leads to an increase in the occurrence of brite adipocytes in WAT, even when animals are acclimated at thermoneutrality. We also show that the increased browning of WAT resulting from EndoG loss is

3 doi: /en press.endocrine.org/journal/endo 3875 associated with reduced fat mass and improved glucose homeostasis. Materials and Methods Animals For this study, 16-week-old male EndoG knockout (EndoG KO) mice and their wild-type (Wt) counterparts were used. The generation of EndoG KO mice has been described elsewhere (9). Unless otherwise stated, animals were housed in a room maintained at 21 C and 60% humidity with a 12-hour light, 12-hour dark cycle. Mice were given free access to water and fed a standard laboratory diet (2018 Teklad Global 18% Protein Rodent Diet; Harlan Laboratories). For experiments involving acute cold exposure, 8- and 16- week-old EndoG KO mice and Wt male mice were first acclimated to thermoneutrality (30 C) for 15 days. Then individually caged mice, with free access to water and food, were exposed to 4 C during a period of 5 hours. A group of EndoG KO and Wt mice was kept at thermoneutrality as a control. Body temperature was measured every hour by using a digital thermometer and a rectal probe, as previously described (27). After the exposure period, mice were euthanized by cervical dislocation and WAT and BAT dissected for subsequent analysis. All procedures involving animal experimentation were performed according to the institutional animal care guidelines of the Vall d Hebron-Institut de Recerca, the Scientific Park of Barcelona, and the Universitat de Lleida and were approved by the Animal Experimentation and Ethics Committee of each Institution (12/11 CEEA, 02/03 CEEA, and 05/13 CEEA). mrna isolation and gene expression analysis To analyze gene expression, total RNA was first isolated from BAT, WAT, and skeletal muscle or from white adipocyte primary cultures with TRIzol (Life Technologies). Then 400 ng of RNA was used to synthesize cdna with SuperScript II reverse transcriptase (Life Technologies) and oligo(dt), according to the manufacturer s instructions. Gene expression was assessed by real-time quantitative PCR using SYBR Green dye and genespecific primers in an ABI PRISM 7500 sequence detection system (Applied Biosystems Inc), as described previously (28). Relative gene expression was calculated according to the 2 - CT threshold method using cyclophilin as a reference gene (29). Western blot Protein extracts were prepared from one lobe or interscapular BAT or the dorsal half portion of the inguinal WAT depot in homogenization buffer (50 mm Tris-HCl, 150 mm NaCl, 1% Nonidet P-40, 5 mm EGTA, 5 mm EDTA, 1 mm phenylmethylsulfonyl fluoride) containing protease inhibitors. Thirty to forty micrograms of protein extract were subjected to electrophoresis in a 15% SDS-PAGE and transferred to a polyvinyl difluoride membrane. Immunodetection was performed with specific antibodies to detect UCP1, NDUFB9, atrial natriuretic peptide (ANP) (Abcam), EndoG (Cell Signaling), tyrosine hydroxylase, cytochrome oxidase (COX)-IV, and -tubulin (Merck Millipore). Histological analysis For histological analysis, interscapular BAT and sc inguinal WAT depots were removed from mice, washed in saline solution, and fixed overnight in formalin. The tissue was then dehydrated and embedded in paraffin for subsequent sectioning. Tissue sections (5 8 m) were stained with hematoxylin/eosin. Image J software (National Institutes of Health, Bethesda, Maryland) was used to measure white adipocyte size in inguinal WAT of EndoG KO mice and Wt counterparts (n 3 animals/group). At least 300 cells from each animal were measured from three independent tissue sections. Glycogen content Glycogen content in skeletal muscle of mice was determined with the glycogen colorimetric assay kit (Sigma-Aldrich) according to the manufacturer s instructions. Triglyceride content in feces Triglyceride content in feces was determined as previously described (30). Briefly, lipids from the feces collected over a 48- hour period were extracted in a mixture of chloroform/methanol (2:1 [vol/vol]) during 5 hours with constant shaking. MilliQ water was then added and samples were centrifuged to allow phase separation. The organic layer containing the neutral lipids was collected and the solvent was dried using a Speed Vac. Triglycerides were determined using an enzymatic-based colorimetric method (Spinreact). Oxygen consumption/locomotor activity Oxygen consumption and locomotor activity were assessed using a TSE LabMaster modular research platform (TSE Systems). Oxygen consumption after treatment with the 3 -adrenoreceptor agonist CL316,243 was determined to evaluate the function of BAT in Wt and EndoG KO mice. For this, mice were placed in the calorimetric system cages 48 hours prior to the experiment. On the day of the experiment, basal oxygen consumption was recorded for 3 hours, and then mice were injected ip with 1 mg/kg of CL316,243, and oxygen consumption was measured for an additional period of 2 hours. Glucose and insulin tolerance tests Glucose tolerance test was performed on 16-week-old Wt and EndoG KO mice that were fasted for 12 hours. After an ip injection of glucose (2 g/kg body weight), blood glucose was measured at 0, 15, 30, 60, 90, and 120 minutes. For the insulin tolerance test, mice were fasted for 5 hours, and then blood glucose was measured at 0, 15, 30, 60, 90, and 120 minutes after an ip injection of insulin (0.75 U/kg of body weight). Glucose levels were measured in blood using an ELITE glucometer (Bayer). Insulin was determined in serum with an ultrasensitive mouse insulin ELISA kit (Crystal Chem). Primary culture of white adipocytes Primary adipocytes from Wt and EndoG KO males or females were differentiated from precursor cells in culture following the protocol described previously for brown adipocytes with minor modifications (31). Briefly, the stromal vascular fraction (SVF), which contains adipocyte precursors, was first isolated from inguinal WAT by collagenase digestion. For this, inguinal WAT

4 3876 Pardo et al EndoG in Energy and Glucose Homeostasis Endocrinology, October 2016, 157(10): Figure 1. EndoG is highly expressed in BAT. EndoG protein levels were determined by Western blot in total protein extracts from different tissues of Wt mice. COXIV protein was also detected as a marker of the mitochondrial content in the tissues analyzed. Coomassie blue staining of SDS-PAGE is shown to verify equal protein loading, sk. muscle, skeletal muscle. was first washed in PBS, minced with scissors, and then subjected to collagenase digestion for approximately 30 minutes at 37 C with constant shaking in digestion buffer (100 mm HEPES, ph 7.4; 123 mm NaCl; 1 mm KCl; 1.3 mm CaCl 2 ; 5 mm glucose; 1.5% BSA; and 1 mg/ml collagenase A). Once digested, the tissue was filtered through a 100- m nylon cell strainer to remove undigested tissue fragments, and the cell suspension was centrifuged at 700 g for 10 minutes to collect SVF. Approximately 10 5 precursor cells/well were plated in a six-well plate and grown in DMEM medium supplemented with 10% fetal bovine serum (FBS) until cells reached confluence. Two days after confluence, cells were treated with induction media (DMEM, 10% FBS, 1 M dexamethasone, 0.5 mm isobutylmethylxantine, 100 nm insulin) for 2 more days. Then cells were cultured in differentiation media (DMEM, 10% FBS, 100 nm insulin) for 10 additional days until cells were fully differentiated. Cells were then harvested for RNA isolation and analysis of gene expression. In addition, to analyze their response to adrenergic stimulation, primary cultured adipocytes from WAT were treated with 1 M norepinephrine and incubated for 24 hours prior to their harvest for RNA isolation. Statistics All values are presented in figures as mean SEM. To determine the significance of differences between experimental groups, an unpaired Student s t test was used. Differences were considered significant at P.05. Results EndoG is highly expressed in BAT EndoG is ubiquitously expressed, but its mrna and protein levels have been found particularly elevated in tissues such as heart, skeletal muscle, and liver (32, 33). Analysis by Western blot of different mouse tissues show that EndoG is also highly expressed in BAT, in which EndoG protein content reaches levels similar to those found in heart and skeletal muscle (Figure 1). Consistent with the strong association between EndoG expression and tissue mitochondrial mass, the levels of EndoG protein tightly correlate with those of COXIV, a surrogate marker of mitochondrial content (Figure 1). Despite the high expression of EndoG, its role in BAT has not been previously explored. Mice lacking EndoG are intolerant to cold Recent studies have provided evidence supporting the contribution of EndoG to the control of mitochondrial biogenesis and oxidative capacity in the heart (13). To investigate the function of EndoG in BAT, we first determined how lack of EndoG affects BAT thermogenic function, a process that absolutely relies on proper mitochondrial activity. For this purpose, 16-week-old EndoG KO male mice and their Wt counterparts were first acclimated to thermoneutrality (30 C) for 2 weeks and then exposed acutely to 4 C for 5 hours. When housed at thermoneutrality, both EndoG KO and Wt mice exhibited a similar and constant core body temperature (Figure 2A). However, when exposed to cold, EndoG KO mice suffered a much larger drop in body temperature than Wt mice (Figure 2B), indicating that EndoG KO mice are more cold intolerant than the Wt mice. These results point toward a potential defect in adaptive thermogenesis as a consequence of the lack of EndoG in brown adipocytes. We then investigated whether the inability of EndoG KO mice to maintain their body temperature when exposed to cold was due to defects in the activation of the thermogenic program of BAT. Despite the impaired thermoregulatory capacity observed, the transcriptional activation of the thermogenic pathway was preserved in EndoG KO mice and the mrna levels of UCP1 as well as those of peroxisome proliferator-activated receptor- coactivator 1 (PGC-1)-, the coactivator that controls the transcriptional activation of the thermogenic program in BAT, were dramatically induced upon cold exposure in both EndoG KO and Wt mice (Figure 3A). The induction of genes of the thermogenic program appeared to be

5 doi: /en press.endocrine.org/journal/endo 3877 Figure 2. EndoG KO mice exhibit cold intolerance. A, 16-week-old EndoG KO mice and Wt counterparts were acclimated at thermoneutrality (30 C) for 15 days, and body temperature was measured during a period of 5 hours. B, After the acclimation period at thermoneutrality, mice were exposed to 4 C and body temperature was measured every hour over a period of 5 hours. Data represent mean SEM of five to six animals/group. *, P.05. greater in EndoG KO mice, perhaps indicating the requirement of a higher, although clearly insufficient, thermogenic effort to maintain body temperature in response to cold temperature. Unexpectedly, increased UCP1 and PGC-1 mrna levels, as well as elevated UCP1 protein content, were also observed at thermoneutrality in EndoG KO mice (Figure 3, A and B). These results indicate that increased thermal sensitivity of EndoG KO mice is not due to their incapacity to activate the thermogenic program in response to cold. Proper mitochondrial biogenesis and function is required to sustain heat production by BAT (27). Therefore, we analyzed whether the expression of genes involved in the mitochondrial oxidative function was altered in BAT of EndoG KO mice. As shown in Figure 3C, independently of whether they were acclimated at 30 C or exposed at 4 C, the expression of mitochondrial genes encoding for proteins of the OxPhos system was similar in BAT of EndoG KO and Wt mice. Moreover, the expression of some OxPhos genes (Cycs, Cox7a1, Cox8b) was even increased in mice lacking EndoG. Similarly, the expression of genes related to fatty acid metabolism, like Cpt1b or Cidea, was also modestly increased in BAT of EndoG KO mice (Figure 3C). Histological analysis of BAT revealed notable morphological differences between Wt and EndoG KO mice. When housed at thermoneutrality, brown adipocytes of Wt mice adopt a white adipocyte-like morphology, with increased triglyceride accumulation in single vacuoles (Figure 3D, left panel), that results from the inactivation of thermogenesis in the absence of thermogenic stimuli (eg, low environmental temperatures, -adrenergic stimulation). Intriguingly, at thermoneutrality, brown adipocytes from EndoG KO mice exhibit the typical morphology of a fully active BAT, with brown adipocytes accumulating lipid in numerous small droplets (Figure 3D, right panel). This, together with the increased expression of UCP1 and PGC-1, suggests that lack of EndoG results in a hyperactivated BAT, an effect that is more evident when animals are housed at thermoneutrality. To unequivocally exclude the possibility that the cold sensitivity exhibited by EndoG KO mice is the result of a dysfunctional BAT, we measured oxygen consumption in mice upon acute stimulation with the 3 -adrenoreceptorspecific agonist CL316,243. As seen in Figure 3E, both Wt and EndoG KO mice responded similarly to pharmacological adrenergic stimulation by increasing oxygen consumption, indicating that the inability of mice lacking EndoG to mount a proper thermogenic response is not the reason of their thermal sensitivity. Although nonshivering thermogenesis in BAT is a crucial process to maintain body temperature during exposure to low temperatures, other processes, such as muscle shivering, contribute to core body temperature maintenance during the initial phase of cold adaptation. Therefore, we analyzed whether muscle dysfunction could contribute to the impaired cold tolerance observed in EndoG KO mice. For this, we first measured the expression of mitochondrial genes of the OxPhos system in skeletal muscle of Wt and EndoG KO mice housed at thermoneutrality or exposed to cold. As shown in Supplemental Figure 1A, the expression of mitochondrial genes was similar in Wt and EndoG KO mice, independently of the environmental temperature. Moreover, the mrna expression of PGC-1 and UCP3, which are known to be rapidly induced by muscle contraction, was similarly increased in Wt and EndoG KO mice exposed at 4 C (Supplemental Figure 1A), indicating higher muscle contractile activity as a result of cold exposure. Glycogen, which is rapidly used by muscle as a substrate to sustain contraction, was similarly decreased in Wt and EndoG KO mice during cold exposure (Supplemental Figure 1B). The similar mobilization of glycogen and the increase in UCP3 and PGC-1 expression

6 3878 Pardo et al EndoG in Energy and Glucose Homeostasis Endocrinology, October 2016, 157(10): Figure 3. BAT of EndoG KO mice is not dysfunctional. A, mrna expression of the thermogenic genes encoding for UCP1 and PGC-1 was analyzed by quantitative PCR in BAT of 16-week-old Wt and EndoG KO mice at the end of the acclimation period to 30 C or after 5 hours of exposure to 4 C. B, UCP1 protein levels were analyzed in BAT by Western blot. Asterisk indicates a nonspecific band that was used as a loading control. C, mrna expression of representative genes encoding for proteins of mitochondrial OxPhos complexes I-V or genes involved in lipid oxidation (Cpt1b and Cidea) was determined by quantitative PCR in BAT of Wt and EndoG KO mice that have been housed at 30 C for 15 days (upper panel) or exposed to cold (4 C) for 5 hours (lower panel). D, Morphological appearance of BAT from EndoG KO and Wt mice acclimated to thermoneutrality was analyzed in histological sections stained with hematoxylin/eosin. E, Oxygen consumption was determined in Wt and EndoG KO mice treated with 1 mg/kg of the 3 -adrenoreceptor agonist CL316,243 (CL). Data are expressed as mean SEM of four to six mice/group. *, P.05, **, P.01, Wt vs KO; #, P.05, ##, P.01, 30 C vs 4 C.

7 doi: /en press.endocrine.org/journal/endo 3879 upon exposure to low temperature indicates that muscle shivering is as functional in EndoG KO mice as in Wt. Consistent with a normal muscle function, lack of EndoG did not affect the locomotor activity of mice (Supplemental Figure 1C). Altogether these results suggest that the impaired capacity of EndoG KO mice to maintain body temperature when exposed to cold is due neither to a defect in mitochondrial biogenesis or the inability to activate the transcription of the thermogenic program in BAT nor to a defect in muscle shivering. EndoG KO mice exhibit decreased adiposity Because insulation exerts a protective function against cold-induced stress, we next analyzed fat mass of EndoG KO mice. Although Wt and EndoG KO mice show similar food intake (Figure 4A) and intestinal lipid uptake (Figure 4B), mice lacking EndoG exhibit a significant decrease in the mass of major WAT depots, such as inguinal, gonadal, and retroperitoneal, as well as a reduction in the mass of interscapular BAT (Figure 4E). This reduction in fat mass correlates with a significant reduction in body weight (Figure 4, C and D). Conversely, no differences were observed in the weight of other major organs, such as the liver, heart, or gastrocnemious/soleus skeletal muscles (Figure 4E). The decreased weight of WAT depots in EndoG KO mice was associated with a reduction in the size of white adipocytes (Figure 4F) and therefore with a limited capacity for fat storage. Indeed, a detailed morphometric analysis revealed that inguinal the WAT depots of mice lacking EndoG contain smaller adipocytes than their Wt counterparts, in which big adipocytes appear with a higher frequency (Figure 4G). The average size of the white adipocytes was significantly smaller in EndoG KO mice (Wt m 2 vs EndoG KO m 2 ; P.009; n 6 mice/group). To provide evidence that impaired insulation derived from a reduced fat mass could contribute to the cold sensitivity exhibited by EndoG KO mice, we performed a cold exposure test in 8-week-old mice, an age at which the EndoG KO mice exhibit a similar body weight and fat mass as their Wt counterparts (Supplemental Figure 2, A and B). Interestingly, at this age, EndoG KO mice do not show signs of cold intolerance compared with Wt mice when exposed to 4 C (Supplemental Figure 2C), supporting our notion that cold sensitivity in the EndoG KO mice could be due to a defect in body insulation as a result of a reduced adipose mass. Increased brite adipocyte recruitment in WAT and improved glucose homeostasis in EndoG KO mice Similar to what we observed in BAT, lack of EndoG did not negatively affect the mrna expression of most mitochondrial genes analyzed in WAT, independently of whether they were encoded by the nuclear (eg, CoxIV, Atp5o) or the mitochondrial (eg, CoII, CoIII, Atp6) genomes (Figure 5A). Increased thermogenic demand, as it occurs when mice are exposed to cold, is known to increase the occurrence of brown adipocytes within WAT depots. Acute exposure to cold dramatically increased the expression of UCP1 and PGC-1 mrna in both Wt and EndoG KO mice (Figure 5B). However, the level of expression achieved by both genes was greater in the EndoG KO mice than in the Wt mice. Interestingly, at thermoneutrality, EndoG KO mice exhibited a 10- and 1.5-fold increase in the expression of UCP1 and PGC-1, respectively, when compared with Wt mice (Figure 5B). Western blot demonstrated that increased UCP1 mrna levels correlated with increased levels of UCP1 protein in EndoG KO mice, irrespective of the environmental temperature (Figure 5C). Higher levels of mitochondrial proteins COXIV and NDUFB9 were also observed in the WAT of the EndoG KO mice (Figure 5C). Moreover, histological analysis of inguinal WAT from mice acclimated to 30 C revealed the appearance of numerous cells with the characteristic multilocular morphology of brown adipocytes, interspersed within white adipocytes of EndoG KO mice (Figure 5D). This suggests that lack of EndoG favors the recruitment of brite adipocytes and promotes browning of WAT. Brown adipose tissue activity and increased occurrence of brite adipocytes in WAT is often associated with a healthy metabolic profile and improved glucose homeostasis. To test whether hyperactivation of BAT and increased recruitment of brite adipocytes in WAT of EndoG KO mice have any effect on glucose homeostasis, we first subjected mice to a glucose tolerance test. As shown in Figure 6A, EndoG KO mice were more glucose tolerant than their Wt counterparts. Then we analyzed wholebody insulin sensitivity in mice fasted for 5 hours by performing an insulin tolerance test. As shown in Figure 6B, no statistically significant differences were found between EndoG KO and Wt mice when their capacity to lower blood glucose concentration was assessed after insulin administration. Interestingly, we found that basal insulin levels were decreased by 40% in EndoG KO mice (Wt ng/ml vs EndoG KO ng/ml; P.05). To rule out a potential defect in insulin secretion, serum insulin levels were determined during the course of a glucose tolerance test. As shown in Figure 6C, no differences in insulin secretion were observed between Wt and EndoG KO mice, although EndoG KO mice were more glucose tolerant than Wt. Altogether these results indicate that EndoG KO mice exhibit improved glucose homeostasis.

8 3880 Pardo et al EndoG in Energy and Glucose Homeostasis Endocrinology, October 2016, 157(10): Figure 4. EndoG KO mice exhibit reduced WAT mass and white adipocyte size. A, Food intake was measured in individually caged mice by measuring the amount of food consumed over a 48-hour period. B, Triglyceride content in feces was determined as an index of intestinal nutrient absorption. C, Body weight of Wt and EndoG KO mice housed at 21 C was measured every 2 weeks over a period of 9 months, starting at 3 weeks of age (n animals/group). D, Body weight of Wt and EndoG KO mice 16 weeks of age at the end of the acclimation period to thermoneutrality. E, Weight of the major WAT fat pads (ie, gonadal, inguinal, and retroperitoneal), interscapular BAT, liver, skeletal muscles (gastrocnemius/soleus), and heart was determined in Wt and EndoG KO mice that had been acclimated to 30 C for 15 days (n 5 mice/group). F, Morphology of inguinal WAT depots from EndoG KO and Wt mice acclimated at thermoneutrality was analyzed in histological sections stained with hematoxylin/eosin. G, Distribution of white adipocyte size in inguinal WAT of EndoG KO mice and Wt counterparts (n 3 animals/group). At least 300 cells from each animal were measured from three independent tissue sections. *, P.05, **, P.01, Wt vs KO.

9 doi: /en press.endocrine.org/journal/endo 3881 Figure 5. EndoG KO mice exhibit increased recruitment of brite adipocytes. A, mrna expression of genes encoding for proteins of mitochondrial OxPhos complexes I-V or genes involved in lipid oxidation (CPT1b and CIDEA) was determined by quantitative PCR in inguinal WAT from 16- week-old Wt and EndoG KO mice that have been housed at 30 C for 15 days (upper panel) or exposed to cold (4 C) for 5 hours (lower panel). B, mrna expression of the thermogenic genes UCP1 and PGC-1 was analyzed by quantitative PCR in inguinal WAT of Wt and EndoG KO mice at the end of the acclimation period to 30 C or after 5 hours of exposure to 4 C. Data are expressed as mean SEM of five to six mice/group. *, P.05, **, P.01, Wt vs KO; #, P.05, ##, P.01, 30 C vs 4 C. C, Western blot of UCP1, COXIV, and NDUFB9 in inguinal WAT of Wt and EndoG KO mice acclimated to thermoneutrality or exposed to 4 C for 5 hours. D, Morphology of inguinal WAT depots from EndoG KO and Wt mice acclimated at thermoneutrality was analyzed in histological sections stained with hematoxylin/eosin. Arrowheads indicate clusters of brite adipocytes. Increased brite adipocyte differentiation in WAT of EndoG KO mice is a noncellautonomous process We next investigated to which extent the increased appearance of brite adipocytes within WAT depots of EndoG KO mice is associated with an increased capacity of adipocyte precursor cells to differentiate into brite adipocytes as a direct consequence of the lack of EndoG. For this purpose, the SVF containing adipose precursor cells was isolated from inguinal WAT of Wt and EndoG KO mice and differentiated in vitro. No gross morphological differences were observed between Wt and EndoG KO primary adipocytes, which differentiate well in culture, as judged by the triglyceride accumulation in typical small lipid droplets (Figure 7A) and the similar expression levels of mature adipocyte markers, such as CCAAT/ enhancer-binding protein-, peroxisome proliferator-activated receptor-, adipose triglyceride lipase, or leptin (Figure 7, B and C). In culture, fully differentiated brown and white adipocytes exhibit a similar morphology and lipid distribution in small droplets that makes them morphologically indistinguishable from each other (data not shown). Therefore, we determined the expression of brown/brite adipocyte-specific markers in primary cultures of adipocytes from WAT, as an index of the presence of brite adipocytes in the culture. No differences were found in the expression of UCP1, PGC-1, or PR domain-containing protein 16 in adipocyte cultures obtained from WAT of Wt and EndoG KO mice (Figure 7D), indicating that the occurrence of brite adipocytes was similar. Furthermore, treatment with norepinephrine similarly induced the expression of brown adipocyte-specific genes (UCP1 and PGC-1 ) in primary white adipocyte cultures of Wt and EndoG KO mice (Figure 7E). Together these results strongly advocate

10 3882 Pardo et al EndoG in Energy and Glucose Homeostasis Endocrinology, October 2016, 157(10): Figure 6. Glucose homeostasis in EndoG KO mice. A, A glucose tolerance test was performed in 12-hour-fasted mice. Blood glucose levels were measured over time after an ip injection of glucose (2 g/kg). B, For insulin tolerance tests, mice were fasted for 5 hours and then injected ip with a single dose of insulin (0.75 U/kg). C, Insulin secretion (right panel) was determined over the course of a glucose tolerance test (left panel) in serum of Wt and EndoG KO mice. Results are expressed as mean SEM of five to seven mice/group. *, P.05; **, P.01. for a noncell-autonomous defect of the lack of EndoG in the enhanced recruitment of brite adipocytes in WAT. Because -adrenergic stimulation is the strongest and best-characterized stimulus involved in brite adipocyte recruitment, we sought to investigate whether mice lacking EndoG could be subjected to a continuous adrenergic stimulation that could explain the increase in WAT browning. To check this hypothesis, we analyzed the expression of tyrosine hydroxylase, the enzyme that catalyzes the rate-limiting step in the synthesis of catecholamines, as a marker of sympathetic innervation in WAT. As observed in Figure 8, we found that EndoG KO mice exhibited higher levels of tyrosine hydroxylase protein, independently of whether animals were kept at 30 C or exposed to cold. These results suggest that WAT from EndoG KO mice is more innervated with sympathetic adrenergic fibers than Wt mice, suggesting that adipose depots of EndoG KO mice have a higher adrenergic tone. Discussion Proper mitochondrial biogenesis is required for the full thermogenic activity of BAT in response to cold exposure. This is paradigmatically exemplified by mice devoid of estrogen-related receptor-, which exhibit a severe decrease in the mitochondrial mass of brown adipocytes that results in impaired oxidative capacity of BAT and cold intolerance, even if the mice normally respond to low environmental temperature by activating the thermogenic program and increasing UCP1 expression (27). Interestingly, a recent study has demonstrated that estrogen-related receptor-, in partnership with PGC-1, regulates the expression of EndoG, which in turn appears essential for the maintenance of mitochondrial mass and oxidative function in heart (13). Based on these data, we would have predicted a relevant role for EndoG in the regulation of mitochondrial biogenesis in brown adipocytes, in which it is highly expressed and therefore in the thermogenic activity of BAT in response to cold exposure. However, although EndoG KO mice appeared more sensitive to cold than their Wt counterparts, the inability to maintain their body temperature to the same extent as Wt mice when acutely exposed to 4 C is not related to a defect in mitochondria biogenesis in BAT or to the inability to mount a proper thermogenic response. Indeed, neither the expression of OxPhos genes, independently of whether they are encoded by the nuclear or the mtdna genome, nor the expression of the thermogenic program (ie, PGC-1, UCP1) are decreased in EndoG KO mice. In fact, a statistically significant increase in

11 doi: /en press.endocrine.org/journal/endo 3883 Figure 7. Differentiation of adipocyte primary cultures from WAT is not impaired by the lack of EndoG. A, Stromal vascular fraction from inguinal WAT was isolated and differentiated in culture. Adipocytes from Wt and EndoG KO mice differentiate well in culture and do not exhibit significant morphological differences. B, EndoG expression in adipocyte primary culture from Wt and EndoG KO was assessed by real-time quantitative PCR. C, mrna expression of gene markers of adipocyte differentiation. D, mrna expression of brown/brite adipocyte markers. E, mrna expression of UCP1 and PGC-1 in adipocyte primary cultures obtained from WAT and treated with vehicle or 1 M norepinephrine for 24 hours. Data are expressed as mean SEM of two independent experiments with triplicates in which inguinal WAT from two Wt and two EndoG KO mice was pulled to obtain precursor cells. **, P.01; #, P.05; ##, P.01. the expression of some OxPhos and lipid oxidation-related genes was observed in the BAT of mice lacking EndoG, suggesting that the process of mitochondrial biogenesis would be actually enhanced in the absence of EndoG. Moreover, the capacity of EndoG KO mice to respond to the treatment with a 3 -adrenoreceptor-specific agonist by increasing oxygen consumption to the same extent as Wt mice clearly indicates that BAT function is not impaired despite the lack of EndoG. The elevated expression of genes related to the thermogenic program and other mitochondrial genes in BAT, together with the increase in the occurrence of brite adipocytes observed in WAT, could be viewed as a compensatory measure of EndoG KO mice to maintain their body temperature when exposed to cold. This would suggest that EndoG KO mice are subjected to a higher degree of thermal stress than Wt mice. An exacerbated heat loss, despite the hyperactivated thermogenic response observed, is compatible with a

12 3884 Pardo et al EndoG in Energy and Glucose Homeostasis Endocrinology, October 2016, 157(10): Figure 8. EndoG KO mice show increased sympathetic innervation of WAT. A, Tyrosine hydroxylase, an indicator of sympathetic innervation, was detected by Western blot in inguinal WAT of 16-week-old Wt and EndoG KO mice acclimated to thermoneutrality or exposed to cold. B, Data from the densitometric quantification of Western blots are expressed relative to levels of Wt at each environmental temperature, which were given an arbitrary value of 1. *, P.05. decrease in body insulation resulting from the diminished fat mass exhibited by EndoG KO mice. The importance of body insulation in conditioning the thermogenic response to maintain body temperature has been shown in different mouse models. For example, mice devoid of stearoyl-coa dehydrogenase-1 are characterized by a reduction in WAT mass that is accompanied by an increase in energy expenditure and the expression of thermogenic and other oxidative metabolismrelated genes in WAT and BAT (34). Similarly, mice deficient for syndecan-1, a proteoglycan involved in cell division and extracellular matrix metabolism, show a chronically activated BAT (35). Moreover, these animals show BAT hyperactivation when exposed to cold as an attempt to overcome poor insulation and excessive heat loss that results from a severe depletion of intradermal fat (35). Also, mice devoid of nardilysin (N-arginine dibasic convertase) exhibit signs of BAT hyperactivation and increased energy expenditure when kept under standard housing conditions (21 C-22 C) (36). When exposed to cold, nardilysin null mice show severe cold intolerance, in part as a consequence of poor body insulation (36). These models highlight the importance of proper insulation for the maintenance of body temperature and also show how altered insulation influences BAT activity and promotes brite adipocyte recruitment (37). In this regard, young EndoG KO mice, which show similar adipose tissue mass than Wt mice, do not exhibit cold intolerance when acutely exposed to 4 C. These data strongly advocates for a major contribution of impaired body insulation to the cold intolerance exhibited by 16-week-old EndoG KO mice. Nevertheless, the up-regulation of thermogenic genes in BAT and the increased browning of WAT observed in EndoG KO mice do not appear to be the direct result of poor body insulation and the consequent need for higher heat production to maintain body temperature. Indeed, at thermoneutrality, a condition at which adaptive thermogenesis is not required to maintain body temperature, BAT from EndoG KO mice shows the characteristic appearance of highly active BAT, with brown adipocytes accumulating triglycerides in numerous small lipid droplets. Such appearance contrasts with that of Wt mice acclimated at 30 C, in which brown adipocytes adopt a white adipocyte-like morphology characteristic of inactive BAT, accumulating lipids in a single or few large vacuoles (38, 39). In addition, a higher expression of thermogenic and other mitochondrial genes still persists in the BAT of EndoG KO mice at thermoneutrality. This is even more evident in WAT, in which mrna levels of UCP1 and PGC-1 are increased by more than 25- and 8-fold, respectively, in EndoG KO mice kept at thermoneutrality compared with Wt animals, concomitantly with the appearance of brite adipocytes. Therefore, in the absence of an objective thermal stress (ie, decreased environmental temperature), EndoG KO mice have a hyperactivated BAT and show increased WAT browning. The fact that primary cultures of adipocyte precursors isolated from WAT of Wt and EndoG KO mice differentiate normally, express comparable levels of UCP1 and PGC-1, and respond to pharmacological stimulation of 3 -adrenoreceptors, clearly indicates that lack of EndoG does not favor the differentiation of precursors cells into brite adipocytes in vitro and points to a noncell-autonomous effect as the main motive of the higher occurrence of brite adipocytes in WAT of EndoG KO mice. Our findings that tyrosine hydroxylase protein content is increased in WAT of EndoG KO mice rather suggests that increased browning could be the result of higher sympathetic activity in WAT depots, with the consequent sustained adrenergic tone. Such increased adrenergic stimulation is also compatible with the microscopic appearance of BAT and the higher expression of UCP1 and PGC-1 in this tissue. In this regard, the phenotype of EndoG KO mice is reminiscent of that of mice devoid of thyroid hormone receptor- and -, which are also cold sensitive, but show numerous signs of hyperactive BAT at thermoneutrality, including decreased lipid accumulation, increased mitochondrial gene expression and higher levels of UCP1 and PGC-1 mrna, as a result of chronic sympathetic tone (40). Although cold tolerance was not assessed, mice lacking interleukin-1 receptor also showed a similar phenotype, with hyperactive BAT and increased WAT browning as a result of enhanced adrenergic activity (41).

13 doi: /en press.endocrine.org/journal/endo Adrenergic stimulation is the most potent activation signal of BAT thermogenesis but also of brite adipocyte recruitment in WAT (42, 43). How lack on EndoG leads to an increased sympathetic activity is uncertain. It has recently been reported that EndoG KO mice show normal neurological functions, including learning capacities and motor coordination, but as a distinct trait they exhibit decreased anxiety levels (44). In line with these findings, it is known that stress, either negative or positive, does activate sympathetic nervous system and can induce browning and BAT activation. For example, keeping mice in an enriched environment has been recently shown to increase energy expenditure by mechanisms that involve BAT activation and browning of WAT mediated by increased adrenergic stimulation (45). Although alterations in the activity of the central nervous system of EndoG KO mice are plausible (44), further research is required to clarify whether the phenotype observed in EndoG KO mice is indeed the result of increased adrenergic stimulation of BAT and WAT due to altered hypothalamic activity. In this sense, beyond adrenergic stimulation, several paracrine and endocrine signals have been identified as activators of BAT and browning of WAT (46), and therefore, their potential contribution to the phenotype of EndoG KO mice cannot be totally ruled out. Furthermore, increasing evidence shows that cardiac-born signaling regulates systemic energy homeostasis and adipose tissue metabolism (47). Among these signaling molecules, natriuretic peptides, which are produced by the heart in response to cardiac dysfunction, have been shown to drive the recruitment of brite adipocytes in WAT and enhance thermogenesis in BAT through the activation of p38mapk signaling pathway (21). Interestingly, the major phenotypic characteristic of EndoG KO mice described to date is a cardiac dysfunction associated with cardiomyocyte hypertrophy (13). It has been previously reported that in vitro knockdown of EndoG expression in rat primary cardiomyocytes leads to increased ANP mrna expression, in close association with an augmentation of the size of cardiomyocytes (13). This is consistent with our results showing increased protein levels of ANP in hearts of EndoG KO mice (Supplemental Figure 3). Therefore, it is possible that the exacerbated thermogenic response and the changes in WAT of EndoG KO mice could be mediated by an increase in the secretion of natriuretic peptides as a result of the effects that lack of EndoG has on heart, an aspect that will deserve further investigation. Another interesting finding of our study is that the lack of EndoG has a remarkable beneficial effect on wholebody glucose homeostasis, significantly improving glucose tolerance and reducing fasting insulin levels. The improved metabolic profile of EndoG KO mice is in line with numerous studies in genetically engineered mouse models that report an amelioration of metabolic diseases, such as insulin resistance, type 2 diabetes, or obesity, associated with BAT and/or brite adipocyte activation. For example, mice deficient in the transcriptional corepressor receptor interacting protein 140 (24) or proteins of the retinoblastoma family like p107 (25) and retinoblastoma (48) show signs of increased BAT activation and/or WAT browning associated with a resistance to develop diet-induced obesity and insulin resistance. A similar phenotype has been described in transgenic mice overexpressing the transcription factors forkhead box C2 or PR domain-containing protein 16 (23, 26). Moreover, the higher capacity of certain mouse strains, like A/J and SWR/J, to recruit brite adipocytes in WAT or to activate BAT is also associated with their resistance to develop diet-induced obesity (49 51). In some of these models, calorimetric studies have revealed that increased WAT browning and improved metabolic profile is associated with increased oxygen consumption (24, 25), an effect that we have not been able to detect in EndoG KO mice under basal or adrenergic-stimulated conditions. Compared with mice devoid of receptor interacting protein 140 or p107, which exhibit an 8% 25% increase in oxygen consumption associated with a massive presence of brite adipocytes and more than a 100-fold increase in UCP1 mrna expression in WAT (24, 25, 52), EndoG KO mice show a rather modest browning of WAT. Therefore, even if enhanced WAT browning elicits noticeable, although modest, changes on body weight and WAT mass in EndoG KO mice in the long term, it might not be sufficient to bring about detectable changes in oxygen consumption or energy expenditure measured by indirect calorimetry over a short period of time. In this regard, recent studies have revealed that indirect calorimetry underestimates energy expenditure in mice by 7% 10% (53, 54). The magnitude of underestimation would be sufficient to explain the changes in body weight and fat mass observed in adult EndoG KO mice in the absence of detectable changes in oxygen consumption determined by indirect calorimetry (54). Furthermore, we cannot rule out the possibility that the metabolic changes observed in EndoG KO mice are not directly related to increased WAT brite adipocyte recruitment and increase energy expenditure. Indeed, it has been recently demonstrated that cardiac-specific modification of members of the Mediator coactivator complex induces systemic changes on insulin sensitivity and glucose tolerance (55, 56), supporting the notion that the glucose tolerance observed in EndoG KO mice could be derived from their cardiac phenotype. In summary, we have shown that a lack of EndoG leads to constitutive BAT activity and increased browning of WAT, independently of the environmental temperature. In addition, we have found that EndoG deficiency de-