Martine ; Auclair

Natacha ; Roblot

Emilie ; Capel

Bruno ; Fève

Dr Bénédicte Antoine
email: benedicte.antoine@inserm.fr

Pharmacological modulation of RORa controls the beiging of murine and human white adipose tissue

Keywords: white adipose tissue, browning, RORα, synthetic ligands, in vivo

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Introduction

Activating non-shivering thermogenesis in brown adipose tissue (BAT) and white adipose tissue (WAT) to enhance the rate of energy expenditure is a new pharmacological target to protect against obesity. BAT burns glucose and fatty acids, thanks to UCP1 which dissociates substrate oxidation from ATP production [START_REF] Ricquier | Mitochondrial protein alteration in active brown fat[END_REF], while WAT is specialized in energy storage and mobilization. Brown and white adipocytes share several transcription factors critical for the development and maintenance of mature fat cells [START_REF] Kuri-Harcuch | Cellular perspective of adipogenesis transcriptional regulation[END_REF][START_REF] Seale | Transcriptional regulatory circuits controlling brown fat development and activation[END_REF]. However, the ways used to generate the different adipocyte subtypes are still not fully understood. In brown adipocytes, PRDM16 and PGC1a interact with PPARg, the master regulator of adipocyte differentiation, to activate the thermogenic program [START_REF] Seale | Transcriptional control of brown fat determination by Prdm16[END_REF]. Nevertheless, in white adipocytes, specific transcriptional effectors of white adipocyte gene expression are scarce [START_REF] Lee | Transcriptional and epigenomic regulation of adipogenesis[END_REF].

Moreover, the ability of some white mature adipocytes to adopt a thermogenic phenotype in response to cold has led to the discovery of "beige adipocytes", molecularly distinct from the brown ones. These beige adipocytes can emerge either from the differentiation of few "beige" progenitors hidden amongst "white" precursors present within the WAT or, from the direct conversion of existing white adipocytes into beige fat cells (transdifferentiation) [START_REF] Barbatelli | The emergence of cold-induced brown adipocytes in mouse white fat depots is determined predominantly by white to brown adipocyte transdifferentiation[END_REF][START_REF] Lee | Cellular origins of cold-induced brown adipocytes in adult mice[END_REF]. This latter hypothesis suggests the presence of transcriptional suppressors of the thermogenic program in white adipocytes and such molecular brakes have been described, including Rb, RIP 140, TLE3, or ZFP423 (reviewed in [START_REF] Shao | Transcriptional brakes on the road to adipocyte thermogenenesis[END_REF].

Elucidating the molecular mechanisms that switch on the beige phenotype could identify new molecular targets to be addressed in the fight against obesity and its metabolic complications. Whereas many extracellular effectors able to modulate WAT browning have been already reported [START_REF] Forest | What induces watts in WAT?[END_REF], new gene candidates can still emerge from the phenotype of mutated mice. Given that the RORa-deficient (sg/sg) mice exhibited higher rate of energy expenditure both in chow diet [START_REF] Bertin | Effects of Cold Acclimation on the Energetic Metabolism of the Staggerer Mutant Mouse[END_REF] and high fat diet [START_REF] Kang | Transcriptional profiling reveals a role for RORa in regulating gene expression in obesity-associated inflammation and hepatic steatosis[END_REF], a relationship between RORa and thermogenesis has been suspected. Muscat 's group was the first to establish an inverse correlation between RORa and UCP1 expression and to suggest that enhanced UCP1 and thermogenic activity may be an important contributor to the lean, obesity-resistant phenotype of sg/sg mice [START_REF] Lau | RORa deficiency and decreased adiposity are associated with induction of thermogenic gene expression in subcutaneous white adipose and brown adipose tissue[END_REF][START_REF] Tuong | Transgenic adipose-specific expression of the nuclear receptor RORa drives a striking shift in fat distribution and impairs glycemic control[END_REF]. We further confirmed an increased "browning" of all the fat depots in these mice, thus conferring to RORa an inhibitory role on the thermogenic program that could be counteracted ex vivo by the use of a synthetic RORa inverse-agonist [START_REF] Monnier | The nuclear Retinoid-related Orphan Receptor RORa controls circadian thermogenic programming in white fat depots[END_REF]. Indeed, we showed that a short treatment with this specific RORa ligand, in the absence of other external stimulation, was sufficient to induce UCP1 protein appearance in WAT explants, within a timing compatible with a potential browning process by transdifferentiation or revealing the presence of pre-existing beige adipocytes.

RORa, b and g constitute a subfamily of nuclear receptors involved in many physiological processes. RORa is the major form expressed in WAT and is increased during adipogenesis [START_REF] Ohoka | The orphan nuclear receptor RORa restrains adipocyte differentiation through a reduction of C/EBPb activity and perilipin gene expression[END_REF]. The recent characterization of endogenous ligands for these former orphan receptors has stimulated the development of synthetic ligands by pharmaceutical companies since targeting these receptors could represent new opportunities to treat several diseases [START_REF] Kojetin | Reverb and ROR nuclear receptors as drug targets[END_REF]. Several oxysterols have been found to function as RORa inverse-agonists, inhibiting G6PC (Glucose-6-Phosphatase) and Bmal expression [START_REF] Wang | Modulation of RORa and RORg activity by 7-oxygenated sterol ligands[END_REF]. The Scripps Research Institute designed several RORa ligands with, for some of them, an in vivo efficiency: SR3335 is a selective RORa inverse-agonist and SR1078, a RORa agonist able to suppress or increase some hepatic target genes in vivo, respectively [START_REF] Kumar | Identification of SR3335 (ML-176): a synthetic RORa selective inverse agonist[END_REF][START_REF] Wang | Identification of SR1078, a synthetic agonist for the orphan nuclear receptors RORa and RORg[END_REF].

The aim of this work was 1) to investigate the possibility of a pharmacologically-induced inverse relationship between RORa activity and UCP1 expression in adipose tissue explants by using either a RORa inverse-agonist or a RORa agonist; 2) to more precisely delineate the time-course of the molecular events produced by these RORa ligands in murine adipocyte cell lines and in human adipocytes; 3) to test the ability of in vivo administration of these RORa ligands on non-shivering thermogenesis and body weight in mice.

Material and Methods

Animals and treatment. sg/sg mice (a spontaneous mutation in C57BL6/ strain) were obtained by crossing heterozygous sg/+ mice, given by Pr. J. Mariani' s laboratory (IBPS, Paris, France), and compared with +/+ littermates. All animals care and use procedures were in accordance with the guidelines of the Charles Darwin Ethics Committee (Ce5/26092 and 26094). Mice were housed in a specific pathogen-free environment in a temperature-controlled room maintained at 24°C, with a 12h/12h light-dark cycle (lights on from 8h to 20h). Water and food (A03, UAR, Epinay-sur-Orge, France) were provided ad libitum.

Because sg/sg mice are leaner than WT, the adult mice used in this study were chosen to be weight-matched rather than age-matched because body weight influence thermogenenesis [START_REF] Lau | RORa deficiency and decreased adiposity are associated with induction of thermogenic gene expression in subcutaneous white adipose and brown adipose tissue[END_REF], average age difference being about 6 months. For in vivo studies, mice were i.p. injected with SR3335 or SR1078 (15 mg/kg) or with the excipient (10% dimethylsulfoxide (Dmso), and 10% Tween 80 solution), singlecaged, then shifted to 6°C for 3h for cold experiment. Body temperature was measured with a rectal probe (Physitemp, RET3, Clifton, New-jersey, USA) and a reader (Physitemp, BAT-12, Clifton, New-Jersey, USA) every hour during the test. Mice were sacrificed by cervical dislocation. Perigonadal and inguinal (white), and inter-scapular (brown) adipose tissues were cautiously dissected, weighted, snapfrozen in liquid nitrogen and stored at -80°C before RNA extraction or protein homogenate preparation.

For ex vivo studies, adipose tissues were minced into small pieces (5-10 mg) and incubated in DMEM containing 10 mM glucose, 0.5% bovine serum albumin (BSA) and 10 µM RORa ligands (or the excipient Dmso) for the times indicated in the figure legends.

Metabolic rate was measured by indirect calorimetry in TSE Phenomaster cages (Systems, Chesterfield, Missouri).19 wk-old C57BL6/j mice were housed individually one week before the experiment under a 12h light/12h dark cycle at 22°C. Food (chow diet) and water were provided ad libitum. 6 mice were injected daily with SR3335 (15mg/kg) or the excipient at the end of the light period (ZT11) for four days. On the fourth day morning (ZT5), each mouse was challenged with the b3 adrenoreceptor agonist CL316,243 (1 mg/kg) to explore adaptive thermogenesis. Body composition was measured by nuclear magnetic resonance (NMR) at the beginning and the end of the experiment.

Measurements in the metabolic chambers were made from D1 to D5.

Cell culture. Murine 3T3-F442A adipocytes were differentiated as previously described [START_REF] Tordjman | Cytosolic aspartate aminotransferase; a new partner in adipocyte glyceroneogenesis and an atypical target of thiazolidinedone[END_REF]. Human adipocytes have been obtained from adipose-derived stem cell (hASC) isolated from the abdominal subcutaneous adipose tissue of 3 healthy and pre-menopausal women (BMI< 25 kg/m 2 ) in a context of plastic surgery and with the free and informed consent of the donors, in accordance with the ethical standards of the local ethical committee and the Declaration of Helsinki (1964). The study was approved by the French regulatory authorities (CPP Ile de France V, n° 14964). hASC were isolated according to Zhu et al [START_REF] Zhu | Manual isolation of adiposederived stem cells from human lipoaspirates[END_REF]. Briefly, tissue was digested with 0.2% collagenase (Sigma, Darmstadt, Germany). After centrifugation, the stroma vascular fraction was filtered on 100 µM pores, rinsed and seeded. Cells were cultured in aMEM supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, 1% penicillinstreptomycin and 145 nM basic fibroblast growth factor (Pepro Tech, Neuilly sur Seine, France). Subconfluent hASC cells were trypsinized and seeded at 10,000 cells/cm 2 . The white differentiation process used was: two days post-confluence, adipogenesis was initiated by culturing hASC in DMEM with 10% FCS, 1 µM rosiglitazone, 1 µ M dexamethasone, 500 µM IBMX (3-isobutyl-1methylxanthine), 1 µM insulin during 5 days, then with 1 µM insulin and 1 µM rosiglitazone for the following 9 days. The impact of repeated doses of RORa ligands during adipogenesis was tested twice with four points in each kind of adipocytes. Treatment with 10 µM RORa ligands in mature adipocytes was performed in DMEM containing 10 mM glucose, 0.5% BSA and for the indicated times in the figure legends; three different experiments including 6 points have been performed for each RORa ligand.

Confocal immunofluorescence microscopy.

Adipocytes were grown and differentiated on 13-mm glass coverslips. They were fixed and permeabilized in cold methanol for 10 min at -20°C. Primary and secondary antibodies were applied to coverslips in PBS with 1% BSA for 15-30 min at room temperature. Confocal microscopy was performed using a Leica TCS-SP microscope (Lasertechnik, Berlin, Germany) equipped with a 63× objective. Double fluorescence images were acquired in a sequential mode. Serial optical sections of 1 µm were taken. Selected paired sections were then processed to produce single composite color-merged overlay images using Adobe Photoshop software (version 5.5). The antibody against PLIN1 (ab3526) was from Abcam, Paris, France.

RNA extraction, cDNA synthesis and quantitative PCR. Adipocyte and adipose tissue mRNA were extracted using the RNeasy Lipid Tissue Mini Kit (Qiagen, Courtaboeuf, France), then reversetranscribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems Carlsbad, CA, USA). Quantitative PCR of the genes of interest was performed using SYBR Green and a Light Cycler 480 Real-Time PCR System (Roche Diagnostics, Meylan, France) and the specific primers listed as followed: for mUcp1 ACAGAAGGATTGCCGAAAC (fwd) and AGCTGATTTGCCTCTGAATG (rev); for mPRDM16 ATGTGCTTAATTCCACCTTA (fwd) and GGAGAGGAGTGTCTTCAGAG (rev); for mTLE3 TGGTGAGCTTTGGAGCTGTT (fwd) and CGGTTTCCCTCCAGGAAT (rev) and for mADRB3 ATCGTGTCCGCTGCCGT (fwd) and ATCTGCCCCTACACGCCAC (rev); for m36B4 GCTGATGGGCAAGAACACCA (fwd) and CCCAAAGCCTGGAAGAAGGA (rev); for mHPRT AGGACCTCTCGAAGTGT (fwd) and TCAAATCCCTGAAGTACTCAT (rev); for PGC1a CAACCGCAGTCGCAACAT (fwd) and TGGGAACCCTTGGGGTCA (rev); for mcox8b, GAACCATGAAGCCAACGACT (fwd) and GCGAAGTTCACAGTGGTTCC (rev); for mcox7a CAGCGTCATGGTCAGTCTGT (fwd) and AGAAAACCGTGTGGCAGAGA (rev); for mCPTIb ATCATGTATCGCCGCAAACT (fwd) and CCATCTGGTAGGAGCACATGG (rev); for mCS: GGACAATTTTCCAACCAATCTGC (fwd) and TCGGTTCATTCCCTCTGCATA (rev); for mERRa GCAGGGCAGTGGGAAGCTA (fwd) and CCTCTTGAAGAAGGCTTTGCA (rev); for RIP140 TCAAAAGCCACACCATACCC (fwd) and TCGTCTCCACTGCTGTCATC (rev); for hCS TAGTGCTTCCTCCACGAATTTG (fwd) and CCACCATACATCATGTCCACAG (rev); for hCOX2 TACGGCGGACTAATCTTCAA (fwd) and CCGGGAATTGCATCTGTTTT (rev).

Gene expression was normalized to 36B4 and HPRT and data analysis was based on the D CCT method.

Protein extraction and western blot analysis. Frozen adipose tissue was homogenized on ice in Tissue

Protein Lysis Buffer (Euromedex, Souffelweyersheim, France) then centrifuged firstly at 10,000 g, 4°C for 8 min to remove lipids, and then at 20,000 g, 4°C for 10 min to remove insoluble material.

Supernatants were subjected to SDS-PAGE and western blotted with antibodies against RORa (sc-28612), PPARg (sc-7273), hUCP1 (sc-293418) (Santa-Cruz Biotechnology, Heidelberg, Germany) and against PRDM16 (ab 106410), mUCP1 (ab 10938) (Abcam, Paris, France, against TLE3 (Proteintech 11372-1-AP, Manchester, UK) and tubulin (T5168, Sigma, Darmstadt, Germany). Bands were measured on unsaturated exposition by densitometry using the software "Image J" (NIH, USA) and expressed vs tubulin used as loading control.

Mitochondrial bioenergetics analysis. Mitochondrial mass of the differentiated adipocytes were appreciated by the fluorescent labeling of the Mitotracker Red probe (MTR, M-7512, Molecular Probes, Eugene, OR). Measurement of oxygen consumption was performed using a Seahorse Bioscience XF96 Analyzer (Agilent, Santa Clara, CA, USA), according to the manufacturer's protocol. OCR measurement were obtained following sequential additions of oligomycin (10 µM), FCCP (6.3µM) and antimycin A (AA)/rotenone (10 µM).

ROR synthetic ligand. SR3335 and SR1001 were purchased from Cayman (Interchim, Montigny le Bretonneux, France) and SR1078 from Calbiochem (Merck Chemicals, Nottingham, UK).

Statistical analysis.

Values are presented as means +/-SEM. Statistical analysis was performed by oneway or two-way ANOVA or by unpaired Student 's t test as mentioned in the figures caption (Graph Pad Software, San Diego, CA, USA). P<0.05 was considered the limit for statistical significance.

Results

UCP1 expression is modulated by RORa inverse-agonist or agonist in scWAT and BAT explants

A 16h exposure of scWAT explants from WT mice to the RORa inverse-agonist SR3335 enabled a transient appearance of UCP1 protein (Fig 1A ); this effect was found to be specific of RORa because explants from RORa -/-(sg/sg) mice, physiologically expressing UCP1, were unsensitive to SR3335 addition (histogram Fig 1A). SR3335 was also able to increase the UCP1 amount in the BAT of WT mice, but with a lower magnitude than in scWAT (x1.8 and x3 in BAT and scWAT, respectively) (Fig 1B). In order to test if the reciprocity was true, BAT explants from both genotypes were treated with a RORa agonist, SR1078. Fig 1C confirms that this ligand-dependent increase of RORa activity led to a half-decrease of UCP1 amount in BAT. Expectedly, SR1078 had no effect on UCP1 expression in explants from sg/sg mice. Our data thus show that the amount of UCP1 in adipose tissue is ex vivo modulated by ligands of the nuclear receptor RORa, in the absence of other culture manipulations.

Beige cells can arise from 3T3-F442A adipocytes treated with RORa inverse-agonists

Loss of RORa in mice promotes adipose tissue browning and its decreased activity in adipose explants leads to an increase of UCP1 expression [START_REF] Lau | RORa deficiency and decreased adiposity are associated with induction of thermogenic gene expression in subcutaneous white adipose and brown adipose tissue[END_REF][START_REF] Monnier | The nuclear Retinoid-related Orphan Receptor RORa controls circadian thermogenic programming in white fat depots[END_REF]. This shows that RORa exerts a negative effect on UCP1 expression and suggests an indirect mechanism of action. Indeed, experiments of global run analysis have shown that, if the mechanism of gene induction by RORa was direct and transcriptional, the way used by RORa to repress brown fat genes expression was indirect [START_REF] Zhang | The hepatic circadian clock fine-tunes the lipogenic response to feeding through RORa/g[END_REF]. We previously observed that in WAT of sg/sg mice, the higher expression level of brown genes was concomitant with a decreased level of two transcriptional suppressors of the thermogenic program, TLE3 and RIP 140 [START_REF] Monnier | The nuclear Retinoid-related Orphan Receptor RORa controls circadian thermogenic programming in white fat depots[END_REF], making them putative direct target genes of RORa. We thus investigated whether and how RORa ligands were able to modulate the thermogenic pathway in a murine white adipocyte cell line, either in differentiating adipocytes or in mature adipocytes (Fig 2).

We first compared the effect of both kinds of RORa ligands (two inverse-agonists and one agonist) on the "white" or "brown" phenotype of 3T3-F442A cells treated during the course of adipose conversion from D4 to D14, and with isoproterenol for the last 6h. Figure 2A shows representative Western blots and histograms averaging the respective protein amount accumulated into the adipocytes from D7 to D14. We first verified if these ligands were able to exert an effect on the RORa protein amount; indeed, ROR gene promoters contained several ROREs suggesting that RORa could regulate its own transcription [START_REF] Jetten | Retinoid-related orphan receptors (RORs) : critical roles in development, immunity, circadian rhythm, and cellular metabolism[END_REF]. Consistently, the content of RORa that accumulated during adipogenesis was found to be increased by the treatment with the RORa agonist SR1078 and decreased with the RORa inverse-agonist SR3335, while the other inverse-agonist SR1001 tested had no significant effect (Fig 2A). The RORa inverse-agonist SR3335 was able to increase the UCP1 and PRDM16 protein amount and the mitochondrial mass of the adipocytes (Fig 2D ), whereas the number of multilocular lipid droplets into the adipocytes seemed also to be enhanced (sup Fig 1A, red arrows). At the opposite, the RORa agonist SR1078 decreased the UCP1 and PRDM16 protein amount, while increasing that of TLE3, a suppressor of the thermogenic program. Finally, the RORa inverse-agonist SR1001 was not found to exert any significant variation on these proteins. Thus, our data show that the browning of murine adipocytes is modulated by the ligands of RORa, suggesting a putative role for this nuclear receptor in the maintenance of a "white" phenotype.

We next tested whether, in fully mature 3T3-F442A adipocytes, a single exposure to the RORa inverse-agonist SR3335 was also able to induce the onset of the thermogenic program. These data show that a single dose of SR3335 was able to promote the browning of mature adipocytes. The time-course of the events allows the hypothesis of an initial action of RORa on the level of TLE3 mRNA but does not seem to implicate RIP140 gene expression.

RORa inhibition induces the emergence of beige characteristics in human adipocytes whereas RORa activation counteracts this effect

In order to determine whether the regulation of the thermogenic program by RORa was also effective in human adipocytes, we analyzed the effect of RORa ligands on differentiating and mature adipocytes derived from human adipose stem cells (hASC). hASC were differentiated in white adipogenic medium supplemented with 10 µM As observed in the murine preadipocyte cell line, these data confirmed that the ligand-dependent RORa inhibition enables the emergence of beige characteristics in human adipocytes as well, whereas RORa activation counteracts it.

In vivo pharmacological modulation of UCP1 in scWAT by RORa ligands affects the mass of the fat depot, body weight and energy expenditure of the mice.

Because the two RORa ligands that we ex vivo used have been demonstrated to be active in vivo [START_REF] Kumar | Identification of SR3335 (ML-176): a synthetic RORa selective inverse agonist[END_REF][START_REF] Wang | Identification of SR1078, a synthetic agonist for the orphan nuclear receptors RORa and RORg[END_REF], we then tested their ability to control UCP1 amount in mice adipose tissues after a single I.P. injection of SR3335 or SR1078 (15 mg/kg). Given the average 16h period of time necessary for UCP1 to rise in WAT explants and the short half-life of the compounds, we chose to test the effect of the ligands 18-20h after injection.

The representative Western blot and the side-histograms (Fig 5A ) show that SR3335 induced a 4-fold increase in UCP1 amount in scWAT of WT mice, rising an average level similar to that detected in sg/sg mice. Under the same conditions, no significant effect of SR3335 on UCP1 protein amount was observed in the BAT (Fig 5B ). By contrast, in vivo exposure to the RORa agonist exerted the opposite effect: SR1078 injection reduced by 3-fold the UCP1 amount in the scWAT of WT mice, and by 1.3fold in the BAT, but without any effect on sg/sg mice (Fig 6 A,B). These data confirmed that RORa ligands could be useful tools to modulate UCP1 protein amount in adipose tissues in vivo.

We thus investigated whether these effects of RORa ligands on UCP1 expression have been associated with variations of whole body weight and/or adipose tissue depot mass of the mice. We found consumption and CO2 production, especially during the night period without affecting neither food and drink intake nor the locomotor activity of the mice. Finally, given that the respiratory exchange rate (RER) was nightly decreased by SR3335, data suggests an increased lipid oxidation in this period of time at the expense of the other nutrients present in the chow diet.

The decreased body weight and fat mass induced by the RORa inverse-agonist SR3335 is associated with increased body temperature and browning of scWAT in cold-stressed mice

To investigate if the SR3335-dependent increase of UCP1 in scWAT was functionally associated with a gain in adaptive thermogenesis, mice from both genotypes were treated or not for 24h with a single dose of SR3335, then challenged by a 6°C cold exposure for 3h. Rectal temperature was measured every hour. Figure 8A confirmed that sg/sg mice maintain higher rectal temperature than WT mice during cold exposure, as previously shown by Lau et al [START_REF] Lau | RORa deficiency and decreased adiposity are associated with induction of thermogenic gene expression in subcutaneous white adipose and brown adipose tissue[END_REF]. Indeed, while WT mice lost 3.5°C after 2h in the cold, sg/sg mice only lost 2°C. Strikingly, SR3335 injection partially prevented the decrease in the rectal temperature of WT mice that exhibited a rectal temperature pattern similar than sg/sg mice. The measurement of body weight before and after the cold challenge showed that the higher thermogenesis of sg/sg mice was associated with a higher weight loss (-7.5%) than in WT mice (-5%).

Remarkably, SR3335 injection allowed the WT mice to lose similar body weight than sg/sg mice (-8%) Because the weight-matched sg/sg mice used in our experiments were necessarily six-months older than WT mice, we investigated if the failure of sg/sg response to RORa ligands was not due to the fact that they are much older than WT mice. By examining the absolute weight loss in response to SR3335 injection in old age-matched (65-wk-old) mice from both genotypes after acute cold exposure (4h at 4°C), we could observe that only WT mice lost more weight in response to SR 3335+cold vs cold alone (WT: -0.77g vs sg/sg -0.06g). Cold-stressed sg/sg mice lost 1.22 +/-0.27 and 1.16 +/-0.24 gram with and without SR 3335 injection, respectively, while WT mice lost 2.18+/-0.34 and 1.41 +/-0.14 g with and without SR3335 pre-treatment, respectively (p<0.05), n=4 for sg/sg and 5 for WT mice). These data thus confirm that aged WT mice still respond to SR3335 whereas aged sg/sg mice do not.

Taken together, these data show that during cold exposure, a single SR3335 injection enables WT mice to acquire the same performance than untreated sg/sg mice, and suggests a more efficient adaptive thermogenesis associated with an increased UCP1 expression and a specific loss of scWAT.

The browning effect of RORa inverse-agonist SR3335 affects the non-shivering thermogenesis in WT mice

To unequivocally demonstrate that browning induced by pharmacological modulation of RORa affect non-shivering thermogenesis, we performed indirect calorimetry analysis in C57BL6/j mice in response to a b3-adrenoreceptor agonist. 6 mice having been pre-treated in vivo by SR3335 or not for 3 days were challenged by a CL316,243 injection on D4. Taken together, these data confirm that, as the loss of RORa function in sg/sg mice, pharmacological antagonism of RORa by an inverse-agonist allows the browning of the scWAT depot of WT mice and potentiates the decrease in the mass of this fat pad subsequently to the activation of non-shivering thermogenesis that improves lipid oxidation.

Discussion

The regulation of adipose tissue browning was not fully deciphered and required further studies explaining how the regulation of this process may be of interest for tackling obesity and related metabolic disorders. Our data confirmed the involvement of the transcription factor RORa in the regulation of non-shivering thermogenesis, and importantly, revealed the possibility to in vivo modulate its activity by synthetic ligands with beneficial consequences on fat mass and body weight of the mice.

The first evidence for the association between RORa signaling and UCP1 mRNA expression was provided by the Muscat 's team. They observed a decrease in the expression of UCP1 mRNA in the BAT of an adipocyte-specific RORa gain of function mouse model [START_REF] Tuong | Transgenic adipose-specific expression of the nuclear receptor RORa drives a striking shift in fat distribution and impairs glycemic control[END_REF] whereas, on the opposite, an increased UCP1 expression was found in the BAT and WAT of the RORa-deficient sg/sg model [START_REF] Lau | RORa deficiency and decreased adiposity are associated with induction of thermogenic gene expression in subcutaneous white adipose and brown adipose tissue[END_REF] that we further confirmed [START_REF] Monnier | The nuclear Retinoid-related Orphan Receptor RORa controls circadian thermogenic programming in white fat depots[END_REF]. These findings pointed to RORa as a new transcription factor negatively involved in UCP1 expression. We previously showed that a synthetic RORa inverse-agonist was a useful tool for increasing UCP1 protein amount and oxygen consumption in scWAT explants of WT mice, thus pharmacologically ex vivo mimicking the effect of the in vivo RORa deficiency in WAT [START_REF] Monnier | The nuclear Retinoid-related Orphan Receptor RORa controls circadian thermogenic programming in white fat depots[END_REF]. We confirmed here a similar regulation by the RORa inverse-agonist into BAT explants and also demonstrated that reciprocally, a RORa agonist was able to down-regulate their UCP1 protein amount. Moreover, we could observe an increase of the COX4 protein in BAT explants treated by SR3335 (by 2,4fold, not shown). From these experiments, emerged the possibility to ex vivo modulate UCP1 expression (and perhaps mitochondrial function) into adipose tissues by the use of synthetic ligands of the nuclear factor RORa. Moreover, the action of these ligands appeared to be specific of RORa, since no effect was detectable on adipose explants isolated from sg/sg mice.

We then investigated if this "up and down" browning effect of RORa ligands was reproducible in cultured adipocytes. We observed for the first time that the ligand-dependent RORa modulation can control the emergence of beige characteristics, not only in murine but also in human adipocytes; the RORa inverse agonist increasing thermogenic genes and mitochondrial biogenesis and function, and the RORa agonist exerting the opposite effect.

The first protocol we used was several repeated doses added every 2-3 days along the differentiation process in a way to "optimize" the expected answer. It took into account the kinetics of expression of RORa that appears only significant from D2 or D4 of differentiation (1,7 respectively). Thus, this approach was performed in a way to prevent an interference with the initial differentiation cocktail used for hASC, for example. Our goal was not to decipher the origin of the beige cells, i.e.

whether the RORa ligands interfered with the differentiation of specific precursors, but rather to determine whether modulating RORa activity allowed their switch in an active or inactive thermogenic state [START_REF] Kajimura | Brown fat: physiological roles beyond heat generation[END_REF]. Our demonstration of a browning effect obtained only few hours after the addition of a RORa inverse-agonist on mature adipocytes would fit with a transdifferentiating effect [START_REF] Barbatelli | The emergence of cold-induced brown adipocytes in mouse white fat depots is determined predominantly by white to brown adipocyte transdifferentiation[END_REF][START_REF] Lee | Cellular origins of cold-induced brown adipocytes in adult mice[END_REF]. At the opposite, the fact that RORa activation by agonists was still able to rapidly decrease some residual brown gene expression in the human adipocytes was compatible with such hypothesis. However, one cannot exclude the possibility that RORa ligands acted on pre-existing beige adipocytes into the F442A cell line or hASC. In that field, molecules reported as affecting beige activation and differentiation have been most often tested ex vivo on cell systems of differentiation (and by addition as soon as the beginning of the differentiation process) [START_REF] Forest | What induces watts in WAT?[END_REF]. Few studies have been performed on mature adipocytes, [START_REF] Lone | Curcumin induces brown fat-like phenotype in 3T3-L1 and primary white adipocytes[END_REF][START_REF] Rodriguez | Peripherally admisnistered melanocortins induce mice fat browning and prevent obesity[END_REF][START_REF] Zhao | Linifanib exerts dual anti-obesity effect by regulating adipocyte browning and formation[END_REF], or on explants [START_REF] Joffin | Acute induction of uncoupling protein 1 by citrulline in cultured explants of white adipose tissue from lean and high-fat-died-fed rats[END_REF].

Our data revealed that, in both murine and human mature adipocytes, treatment with the RORa inverse-agonist SR3335 resulted in a rapid increase in PRDM16 and UCP1 protein amount reaching a peak after about 16h before decreasing at 24h. The appearance of the UCP1 protein as soon as 6-7h after the treatment corroborated the time course observed in primary mouse adipocytes exposed to 31°C [START_REF] Ye | Fat cells directly sense temperature to activate thermogenesis[END_REF].

The maximal increase in their respective mRNA, peaking at 9h before decreasing at 16h, fits with the pattern of UCP1 transcript induction by trans-retinoids [START_REF] Mercader | Induction of UCP1 in mouse embryonic fibroblasts-derived adipocytes by retinoic acid[END_REF]. The transient effect of SR3335 on UCP1 expression is coherent with the reported half-lives of UCP1 transcript and protein (5h and 10h, respectively), in brown adipocytes treated with the b3-adrenoceptor agonist CL316,243 [START_REF] Mao | Visualization and quantification of browning using a UCP1-2A-luciferase knock-in mouse model[END_REF]. Our results suggest transcriptional events activated by SR3335 or repressed by SR1078. Given that RORa activation exerts a negative effect on UCP1, and since it has been reported that negative gene regulation by RORa tends to be indirect [START_REF] Zhang | The hepatic circadian clock fine-tunes the lipogenic response to feeding through RORa/g[END_REF], we focused on the pattern of expression of two different suppressors of the thermogenic program [START_REF] Debevec | Receptor interacting protein 140 regulates expression of uncoupling protein 1 in adipocytes through specific peroxisome proliferator activated receptor isoforms and estrogen-related receptor alpha[END_REF][START_REF] Villanueva | Adipose subtype-selective recruitment of TLE3 or Prdm16 by PPARs specifies lipid storage versus thermogenic gene programs[END_REF]. We previously found these suppressors to be decreased in sg/sg mice [START_REF] Monnier | The nuclear Retinoid-related Orphan Receptor RORa controls circadian thermogenic programming in white fat depots[END_REF], suggesting they could be putative targets for RORa. RIP140 mRNA levels were not changed by the RORa ligands in murine F442A adipocytes and decreased by the RORa agonist SR1078 in hASC, whereas an increase would have been expected for a repressor. At the opposite, TLE3 mRNA levels were reciprocally regulated by the RORa agonist or inverse-agonist. TLE3 mRNA level peaked as soon as 3h after the addition of the RORa agonist SR1078 in the medium, but decreased maximally 6h after the addition of the RORa inverse-agonist SR3335. These data suggest that the suppressor of the thermogenic program TLE3 could better fit as a candidate target of RORa transcriptional activity than RIP140.

A key mechanism of SR3335 action could be to release the brake of RORa on the thermogenic pathway. TLE3 has been described as a white-selective cofactor that acts reciprocally with the brownselective co-factor PRDM16 to specify thermogenic gene program. Indeed, TLE3 is able to disrupt the physical interaction between PRDM16 and PPARg [START_REF] Villanueva | Adipose subtype-selective recruitment of TLE3 or Prdm16 by PPARs specifies lipid storage versus thermogenic gene programs[END_REF]. Consistently, TLE3 is less abundant in BAT than in WAT (Fig 2A and 37). The same distribution between fat depots was observed for RORa expression (Fig 2A ), allowing to hypothesize that these two proteins, whose expression rises during white adipogenesis [START_REF] Austin | Induction of the nuclear orphan receptor RORgamma during adipocyte differentiation of D1 and 3T3-L1 cells[END_REF][START_REF] Duez | Inhibition of adipocyte differentiation by RORa[END_REF][START_REF] Villanueva | TLE3 is a dual-function transcriptional coregulatory of adipogenesis[END_REF], could act in concert to maintain a "white" adipocyte phenotype. Finally, the RORa ligands did not affect the expression of the master adipogenic gene PPARg (Fig 3A ), and thus did not interfere with the global adipogenic program, but rather act downstream to switch the fat cell towards a white or beige phenotype.

There are limitations in the interpretation of our data: i) we have not tested the expression of other thermogenic brakes than TLE3 and RIP140, ii) TLE3 transcript could not be strictly RORa-dependent, because it is not entirely absent from scWAT of sg/sg mice [START_REF] Monnier | The nuclear Retinoid-related Orphan Receptor RORa controls circadian thermogenic programming in white fat depots[END_REF]; however, comparing the circadian rhythms of the thermogenic program in the scWAT of sg/sg compared to WT mice [START_REF] Monnier | The nuclear Retinoid-related Orphan Receptor RORa controls circadian thermogenic programming in white fat depots[END_REF], we observed that the highest reciprocal variation of Prdm16 and TLE3 mRNA levels took place at the same period of the day (at the beginning of the dark period), suggesting a higher disproportion in the abundance of these reciprocal actors of thermogenesis in the absence of RORa than in its presence. This suggested that the stoichiometric equilibrium between these two factors was modified in sg/sg mice during the circadian cycle of gene expression.

However, only experiments of TLE3 gain and function in the presence of RORa inverse agonist would demonstrate the involvement of TLE3 in the regulation of UCP1 by RORa.

iii) the specificity of synthetic ligands is always questionable. While these RORa ligands were shown to have no activity at other nuclear receptors as defined in a Gal4-chimeric receptor assay [START_REF] Kojetin | Reverb and ROR nuclear receptors as drug targets[END_REF], some potential interaction within the ROR subfamily existed for some of them. Indeed, while SR3335 is a RORa-specific inverse agonist [START_REF] Kumar | Identification of SR3335 (ML-176): a synthetic RORa selective inverse agonist[END_REF], SR1001 and SR1078 are able to bind both RORa and RORg [START_REF] Kojetin | Reverb and ROR nuclear receptors as drug targets[END_REF][START_REF] Wang | Identification of SR1078, a synthetic agonist for the orphan nuclear receptors RORa and RORg[END_REF]. This is the reason why we previously verified the reciprocal effects of the inverse agonist SR3335 and of the agonist SR1078 on the RORa protein amount in 3T3-F442A adipocytes and then excluded SR1001 because it was not found to decrease the amount of RORa (Fig 2A). In the case of SR1078, its inhibitory effect on UCP1 expression in scWAT and BAT (Fig6) could be due to the inhibition of both RORa and RORg; this is an option because a specific RORg inverse agonist (SR1555) has been reported to increase UCP1 expression into the BAT [START_REF] Chang | Antiobesity effect of a small molecule repressor of RORa[END_REF].

Another important point to discuss was whether the link between thermogenesis and RORa activity involved b3-adrenoceptor signaling. Cold being the natural stimulus for the activation of thermogenesis, the prevailing model was that it exerts its effect on adipocytes indirectly i.e. via the central nervous system, relayed by the b3-adrenoceptor signaling. However, it was reported that the induction of thermogenic genes in the scWAT from b-adrenoceptor-deficient mice was largely preserved on cold exposure, suggesting that there is a b-adrenoceptor-independent pathway that controls thermogenic gene expression in scWAT [START_REF] Ye | Fat cells directly sense temperature to activate thermogenesis[END_REF]. In sg/sg mice, there was no increase of the b3adrenoceptor expression (ADBR3) in BAT and WAT vs WT mice (Fig 8 and17). Whereas increased levels of norepinephrine have been reported in the BAT of sg/sg mice (4), these mice displayed similar increase of oxygen consumption after i.p. injection of the b3-adrenoceptor agonist CL316,243 than WT mice [START_REF] Lau | RORa deficiency and decreased adiposity are associated with induction of thermogenic gene expression in subcutaneous white adipose and brown adipose tissue[END_REF]. The group of Lau et al [START_REF] Lau | RORa deficiency and decreased adiposity are associated with induction of thermogenic gene expression in subcutaneous white adipose and brown adipose tissue[END_REF] concluded that the increased thermogenic activity of sg/sg mice suggested that RORa deficiency results in a cell-autonomous effect that is not driven by the central nervous system. The ligand-dependent RORa modulation of thermogenesis we observed here confirmed a cell-autonomous process. Indeed, it was obtained ex vivo in the absence of other stimuli.

However, during the treatment of differentiating 3T3-F442A adipocytes with the RORa agonist SR1078, we observed a higher inhibition of UCP1 in presence of the b-adrenoceptor agonist isoproterenol (Fig 2A) than in its absence (not shown). This suggests a potential interference between RORa activation and b3-adrenoceptor signaling. Nevertheless, our investigations on the possibility for the RORa ligands to directly impact the cAMP/protein Kinase A signaling, thus downstream the activation of b-adrenoceptors, have not been conclusive (not shown). Interestingly, it was observed that TLE3 expression inhibited the ability of isoproterenol to induce UCP1 expression in PRDM16expressing cells [START_REF] Villanueva | Adipose subtype-selective recruitment of TLE3 or Prdm16 by PPARs specifies lipid storage versus thermogenic gene programs[END_REF]. Given that SR1078 was able to increase TLE3 protein amount in 3T3-F442A adipocytes that also expressed PRDM16 (Fig 2A ), our data confirmed a similar scenario, but the underlying molecular mechanisms remain to be explored. Finally, we observed that SR3335 was able to increase ADBR3 gene expression into the BAT of WT mice after acute cold challenge (Fig 8 ); thus perhaps improving the local adrenergic tone that was reduced, but still upgradable by a b3-specific agonist in 13-months aged mice [START_REF] Rogers | Aging leads to a programmed loss of brown adipocytes in murine subcutaneous white adipose tissue[END_REF].

On a physiological point of view, we show that RORa ligands could be also useful tools to modulate in vivo UCP1 expression in scWAT of WT mice with associated consequences on the mass of this fat depot. We found that SR3335 and SR1078 exert opposite effect on the WT mice body weight, and without any effect on sg/sg mice. Remarkably, a single injection of SR3335 was able to rapidly decrease their body weight (Fig 5C ), while that of SR1078 to increase it (Fig 6C ), by about 3% of their initial weight at the same period of the day. Indirect calorimetry allowed to demonstrate that SR3335 was in vivo able to promote an increase of energy expenditure associated with a decrease of RER, confirming an enhanced lipid oxidation in C57BL6/j mice. We then show that, during cold exposure, a single SR3335 injection enabled WT mice to acquire the same performance than untreated sg/sg mice, suggesting a more efficient adaptive thermogenesis associated with an increased UCP1 expression and a specific loss of scWAT. However, acute cold exposure was not sufficient to assess UCP1-dependent non-shivering thermogenesis since other thermogenic processes, including shivering, participate in the maintenance of body temperature during the first hours following exposure to low temperatures. Thus, to unequivocally demonstrate that browning induced by pharmacological modulation of RORa affects non-shivering thermogenesis, we performed indirect calorimetry in response to b3-specific agonist.

Data confirmed that SR3335 increased CL316,243-induced energy expenditure, lipid oxidation and fat mass loss in C57BL6/j mice.

We thus demonstrate that, similarly to the loss of RORa function in sg/sg mice, pharmacological antagonism of RORa by an inverse-agonist improves the browning of the scWAT depot and potentiates the decrease in the mass of this fat pad in cold-stressed WT mice and in global fat mass in response to a b3-specific agonist. It was previously reported that sg/sg mice did not lose more body weight than WT mice after cold exposure (10°C), whereas exhibiting a higher body temperature [START_REF] Lau | RORa deficiency and decreased adiposity are associated with induction of thermogenic gene expression in subcutaneous white adipose and brown adipose tissue[END_REF]. We thought that this discrepancy with our own data in sg/sg mice could be explained by the fact that the authors expressed their results in gram of weight lost (absolute value), but not as the percentage of variation of the initial body weight. Indeed, Lau et al [START_REF] Lau | RORa deficiency and decreased adiposity are associated with induction of thermogenic gene expression in subcutaneous white adipose and brown adipose tissue[END_REF] mentioned that the differences in weight between sg/sg and WT mice were <15%. In our case, the significant difference was 14%, but we rather chose weightrelated mice than age-related ones (about 10 and 18 months-old for WT and sg/sg mice, respectively). Indeed, sg/sg mice are leaner than their WT counterparts, and we performed our experiments on aged mice in order to minimize their body weight differences, and also to obtain enough adipose tissue explants to work with. Nevertheless, because age is known to have a deep impact on UC1 expression that declines precipitously between 3 and 12 months of age in C57BL6/j mice [START_REF] Rogers | Aging leads to a programmed loss of brown adipocytes in murine subcutaneous white adipose tissue[END_REF], we previously checked the potential effect of age in the differential response to RORa ligands between the two genotypes. We actually noticed a declined response towards in vivo SR3335 treatment in aged WT mice but, that was nevertheless conserved at the age of 52-65 wk; in 26-wk-old WT mice, the UCP1 protein amount in scWAT being increased by about 4-fold (fig 5), then by 1.82 +/-0.52 (n=5) in 52-65 wk-old mice. In parallel, in 52-65 wk-old-matched sg/sg mice, UCP1 failed to response to SR3335 (x 0.91+/-0.3) (n=5) and thus the difference between the two genotypes remained significant.

Given that aged mice have been shown to be defective in cold-induced beige adipocyte formation due to a cellular aging senescence-like phenotype of beige progenitors (3), this notion strengthens the therapeutic interest of our findings, with the new proposal to activate in vivo the white to beige switch in aged individuals, with beneficial effect on body weight. This effect mainly concerned the scWAT in mice and was only tested in adipocytes differentiated from precursors isolated from human abdominal scWAT. However, it was reported that in Human, visceral adipocytes were more prone to beiging [START_REF] Zuriaga | Human and mice display opposing patterns of « browning » gene expression in visceral and subcutaneous white adipose tissue depots[END_REF], thus it would be interesting to test the effect of RORa ligands on human deep fat depots. Finally, our in vivo study did not allow to distinguish in which proportion the energy balance of the mice was affected by the SR3335-induced beiging of scWAT and by the SR3335-activation of BAT itself. Nevertheless, we only observed a decrease of WAT fat pads and, not of interscapular BAT, by the RORa agonist, perhaps in line with the activation of FFA oxidation in these fat depots. In humans, it is difficult to delineate if BAT activity decline with age or obesity. Anyway, it was believed there would be good reason to attempt (pharmacologically) to oppose this development and thus decrease the risk for middle-age obesity [START_REF] Nedergaard | Three years with adult human brown adipose tissue[END_REF].

In conclusion, our data highlight insight into i) a new potential mechanism of WAT beiging that will require more investigations for a better knowledge in the specific biology of white and brown adipose tissues and ii) a potential therapeutic target to stimulate adipose tissue beiging in the absence of cold exposure. The fact that RORa inverse-agonist was also able to decrease neoglucogenesis in the liver ex vivo [START_REF] Kadiri | The nuclear retinoid-related orphan receptor-α regulates adipose tissue glyceroneogenesis in addition to hepatic gluconeogenesis[END_REF] and blood glucose in diet-induced rodent models of obesity (15) confirmed the therapeutic potential of antagonizing RORa to lower body fat percentage and improve glucose homeostasis. However, it should be kept in mind that RORa is an ubiquitous nuclear factor, the inhibition of which probably leading to adverse effects in other tissues. Further long-term investigations are mandatory to assess their efficiency and safety in animal models of metabolic diseases.

  Fig 1A, red arrows). Time-course analysis of the effect of SR 3335 on Ucp1 and Prdm16 mRNA levels

  RORa ligands from D5 to D13 before being harvested at D14. Protein expression was monitored along the differentiation process and illustrated by a representative Western blot (Fig 3A) and protein amount accumulated from D7 to D14 compared with control cells in side-histograms. At D14, their mitochondrial mass was measured with the MTK dye by fluorimetry and by immunocytochemistry (Fig 3B). The perilipin antibody was used to visualize the size of the lipid droplets (sup Fig 1B). Their oxygen consumption rate (OCR) was analyzed by the Seahorse technology. Data confirmed the results obtained in the murine 3T3-F442A cell line. When added during adipocyte differentiation, SR3335 enabled the emergence of a beige phenotype of human adipocytes with an increase of PRDM16 and UCP1 protein levels (Fig 3A) and with the appearance of smaller multilocular lipid droplets as delineated by the perilipin immunostaining (sup Fig 1B). The mitochondrial mass was enhanced in parallel, as visualized by the Mitotracker staining (Fig 3B), together with an increase of the uncoupled OCR (proton leak) and no variation of coupled respiration (ATP production) (Fig 3C). In addition, SR3335 decreased the amount of the TLE3 protein. Noteworthy, the RORa agonist SR1078 had the opposite effect on the TL3 protein (Fig 3A) and on the mitochondrial mass (Fig 3B). Finally, the amount of PPARg was not affected by the RORa ligands (Fig 3A). Thereafter, we performed the time-course analysis of a single dose of each RORa ligand on protein expression in differentiated human adipocytes (Fig 4). Western blots from Figure 4A illustrate representative data obtained with SR3335 or with SR1078, whereas kinetics shown in Figure 4A averaged the data obtained by protein density analysis. RORa inverse-agonist SR3335 increased PRDM16 (from 3h to 9h) and UCP1 (peaking at 16h), while decreasing TLE3 (lowest level after a 9h exposure) (Fig 4A). On the opposite, the TLE3 protein amount increased as soon as 3h after the RORa agonist SR1078 addition, while those of PRDM16 and UCP1 progressively decreased (Fig 4A). Some other candidates were analyzed by gene expression (Fig 4B). Concerning the other repressor of thermogenic genes we tested, RIP140 mRNA was decreased by the RORa agonist SR1078, whereas an increase would be expected for a repressor. Finally, we observed an increase of PGC1a, COX2 and citrate synthase mRNAs amount by SR3335 and not by SR1078, suggesting an enhanced b-oxidation capacity by SR3335.

  that SR3335 and SR1078 exerted opposite effect on the mice body weight. A single injection of SR3335 was able to decrease their body weight (Fig 5C), while that of SR1078 to increase it (Fig 6C), by about 2.8% of their initial weight compared to control animals. Likewise, SR3335 decreased the scWAT relative mass (Fig 5D) of the mice, while SR1078 induced a non-significant increase in the mass of this depot (Fig 6D). The relative BAT and liver mass were not affected by the RORa ligands (Fig 5D and 6D). Body weight and tissues depot mass being unsensitive to both treatments in sg/sg mice, this confirms a RORa-dependent effect of the synthetic ligands (Fig 5 and 6). To further functionally investigate the origin of the weight loss exerted by SR3335 on WT mice, we tested their energy expenditure by indirect calorimetry in response to the RORa agonist (Fig7). C57BL6/j mice were daily injected by SR3335 or the excipient for 3 days. Fig 7A illustrated the pattern of data acquisition on day 3 and night 3 while Fig 7B showed the averaged data upon 48h (days and nights 2 and 3). Data clearly showed that SR3335 was able to increase energy expenditure, O2

(

  Fig 8B). As expected, SR3335 did not induce an additional weight loss in sg/sg mice (Fig 8B). Mice were sacrificed immediately after cold exposure and their fat depots collected. SR3335 injection in WT mice decreased scWAT relative mass by 30%, at a level comparable to the subcutaneous fat depot of the untreated sg/sg mice (Fig 8C). The scWAT mass of sg/sg mice was unsensitive to SR3335 injection. Of note, the BAT and liver masses of the cold-stressed mice were not influenced by the genotype, or by the SR3335 treatment. The level of UCP1 amount in the scWAT depots of these mice fitted with their respective mass losses; it was increased by 1.7 +/-0.3 fold in the scWAT of all SR3335-treated vs vehicule-treated cold-stressed WT mice (Fig 8D). Finally, the BAT of these mice were compared at the level of gene expression (Fig 8E): UCP1 and ADBR3 mRNA levels were not different according to the genotype, and only enhanced in WT mice in response to SR3335 and acute cold challenge.

  Fig 9A and B compared the data 6h after the injection vs the same period of time on D3 and clearly showed that CL316,243 increased the energy expenditure and decreased the RER more importantly in mice pre-treated with SR3335 than in untreated mice. In parallel, their body temperature was increased by 0.3°C (Fig 9C) and, at the end of the experiment, these mice have lost the double of the fat mass loss due to the CL316,243 injection alone (Fig 9D).

Fig 1 :

 1 Fig 1: RORa inverse-agonist SR3335 and agonist SR1078 reciprocally modulated the amount of

Fig 2 :

 2 Fig 2: Beige cells can arise from 3T3-F442A adipocytes treated with RORa inverse-agonists:

Fig 3 :

 3 Fig 3: Effect of RORa inverse-agonist and agonist along the adipogenic process of human

Fig 4 :

 4 Fig 4: RORa inhibition enables the emergence of beige characteristics in human differentiated

Fig 5 :

 5 Fig 5: Injection of the RORa inverse-agonist (SR3335) enables the appearance of UCP1 in

Fig 6 :

 6 Fig 6: Injection of the RORa agonist (SR1078) decreases UCP1 in the BAT and scWAT and

Fig 9 :

 9 Fig 9: RORa inverse-agonist (SR3335) increases non-shivering thermogenesis in C57BL6/j mice

Disclosure Statement

The authors have no conflict of interest to declare. C: Analysis of body weight variation of the mice expressed in % of initial weight (n=6). D: Analysis of adipose tissue and liver masses expressed in % of body weight at the same period of the day (n=6).