Rayane A Ghandour

Cecilia Colson

Maude Giroud

Stefanie Maurer

Samah Rekima

Gérard Ailhaud

Martin Klingenspor

Dr Ez-Zoubir Amri
email: amri@unice.fr

Dr Didier F Pisani
email: pisani@unice.fr

Impact of dietary ω3 polyunsaturated fatty acid supplementation on brown and brite adipocyte function

Keywords: PUFA, oxylipins, prostaglandins, adipose tissue, UCP1

The recent characterization of functional brown adipose tissue (BAT) in adult humans has opened new perspectives for regulation of energy expenditure with respect to obesity and diabetes. Furthermore, dietary recommendations have taken into account the insufficient dietary intake of ω3 polyunsaturated fatty acids (PUFA) and the concomitant excessive intake of ω6 PUFA associated with the occurrence of overweight/obesity. We aimed to study whether ω3 PUFAs could play a role in the recruitment and function of energy-dissipating brown/brite adipocytes. We show that ω3 PUFA supplementation has beneficial effect on the thermogenic function of adipocytes. In vivo, a low dietary ω6/ω3 ratio improved the thermogenic response of brown and white adipose tissues to β3-adrenergic stimulation. This effect was recapitulated in vitro by PUFAs treatment of hMADS adipocytes. We pinpoint the ω6-derived eicosanoid PGF2α as the molecular origin since the effects were mimicked with a specific PGF2α receptor agonist. PGF2α level in hMADS adipocytes was reduced in response to ω3 PUFA supplementation. The recruitment of thermogenic adipocytes is influenced by the local quantity of individual oxylipins, which is controlled by the ω6/ω3 ratio of available lipids. In human nutrition, energy homeostasis may thus benefit from the implementation of a more balanced dietary ω6/ω3 ratio.

Introduction

Dietary fats are the source of essential polyunsaturated fatty acids (PUFAs), i.e. ω6 linoleic acid (LA), a precursor of ω6 arachidonic acid (ARA), and ω3 α-linolenic acid, a precursor of ω3 eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids. These long-chain PUFAs trigger a variety of biological responses, particularly in adipose tissue, and are required for healthy development [START_REF] Simopoulos | The importance of the ratio of omega-6/omega-3 essential fatty acids[END_REF][START_REF] Simopoulos | An Increase in the Omega-6/Omega-3 Fatty Acid Ratio Increases the Risk for Obesity[END_REF]. New dietary recommendations take into account the insufficient intake of ω3 PUFAs and the excess of ω6 PUFAs, which correlate with overweight/obesity [START_REF] Ailhaud | Temporal changes in dietary fats: role of n-6 polyunsaturated fatty acids in excessive adipose tissue development and relationship to obesity[END_REF][START_REF] Muhlhausler | Omega-6 polyunsaturated fatty acids and the early origins of obesity[END_REF][START_REF] Simopoulos | The importance of a balanced omega-6 to omega-3 ratio in the prevention and management of obesity[END_REF]. Indeed, high ω6/ω3 ratios are positively associated with adiposity of infants at 6 months, 3 and 4 years of age [START_REF] Donahue | Prenatal fatty acid status and child adiposity at age 3 y: results from a US pregnancy cohort[END_REF][START_REF] Moon | Maternal plasma polyunsaturated fatty acid status in late pregnancy is associated with offspring body composition in childhood[END_REF][START_REF] Rudolph | Early infant adipose deposition is positively associated with the n-6 to n-3 fatty acid ratio in human milk independent of maternal BMI[END_REF], and ARA intake correlates positively with the body mass index (BMI) and the associated metabolic syndrome [START_REF] Inoue | Low serum eicosapentaenoic acid / arachidonic acid ratio in male subjects with visceral obesity[END_REF][START_REF] Savva | Association of adipose tissue arachidonic acid content with BMI and overweight status in children from Cyprus and Crete[END_REF][START_REF] Williams | Adipose tissue arachidonic acid and the metabolic syndrome in Costa Rican adults[END_REF][START_REF] Claria | Diversity of lipid mediators in human adipose tissue depots[END_REF][START_REF] Garaulet | Site-specific differences in the fatty acid composition of abdominal adipose tissue in an obese population from a Mediterranean area: relation with dietary fatty acids, plasma lipid profile, serum insulin, and central obesity[END_REF]. In fact, diets exhibiting a high ω6/ω3 ratio result in a higher ARA bioavailability for the synthesis of ω6 derived oxylipins due to an insufficient compensatory effect of EPA and DHA [START_REF] Fischer | Dietary omega-3 fatty acids modulate the eicosanoid profile in man primarily via the CYPepoxygenase pathway[END_REF]. These ω6 oxygenated derivatives are known to favor inflammatory responses [START_REF] Ghoshal | Cyclooxygenase-2 deficiency attenuates adipose tissue differentiation and inflammation in mice[END_REF], as well as to promote energy storage [START_REF] Massiera | Arachidonic acid and prostacyclin signaling promote adipose tissue development: a human health concern?[END_REF] and to inhibit energy expenditure [START_REF] Pisani | The omega6-fatty acid, arachidonic acid, regulates the conversion of white to brite adipocyte through a prostaglandin/calcium mediated pathway[END_REF]. These effects are mainly triggered by oxylipins arising from the cyclooxygenase (COX) pathway.

In contrast to the white adipose tissue (WAT) involved in energy storage and release, brown adipose tissue (BAT) is endowed with a thermogenic activity and regulates body temperature by dissipating energy through non-shivering thermogenesis [START_REF] Cannon | Brown adipose tissue: function and physiological significance[END_REF]. This mechanism is mediated by the uncoupling protein 1 (UCP1), which uncouples mitochondrial oxygen consumption from energy production. Interestingly, a further population of UCP1-positive adipocytes is present in WAT and is termed brite for "brown in white" or beige adipocytes [START_REF] Petrovic | Chronic peroxisome proliferator-activated receptor gamma (PPARgamma) activation of epididymally derived white adipocyte cultures reveals a population of thermogenically competent, UCP1-containing adipocytes molecularly distinct from classic brown adipocytes[END_REF][START_REF] Ishibashi | Medicine. Beige can be slimming[END_REF][START_REF] Wu | Beige adipocytes are a distinct type of thermogenic fat cell in mouse and human[END_REF]. In vivo, brite adipocytes stem from progenitors or emerge by direct conversion of mature white adipocytes [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 | Adipose tissue plasticity from WAT to BAT and in between[END_REF][START_REF] Rosenwald | Bi-directional interconversion of brite and white adipocytes[END_REF]. Several studies have demonstrated the involvement of oxylipins in fat mass development through the modulation of white and brown/brite adipocyte differentiation and activity [START_REF] Barquissau | Control of adipogenesis by oxylipins, GPCRs and PPARs[END_REF]. Mice exposed to diets with high levels of ω6 PUFA during the perinatal period display progressive accumulation of body fat across generations [START_REF] Massiera | A Western-like fat diet is sufficient to induce a gradual enhancement in fat mass over generations[END_REF]. This observation is in agreement with the consumption of Westernized diets rich in ω6 PUFAs as overweight and obesity develop earlier in life and have increased within a given population during the last decades [START_REF] Massiera | A Western-like fat diet is sufficient to induce a gradual enhancement in fat mass over generations[END_REF]. Moreover, oxylipins derived from ω6 PUFA inhibit brite and brown adipocyte activity both in vitro and in vivo [START_REF] Pisani | The omega6-fatty acid, arachidonic acid, regulates the conversion of white to brite adipocyte through a prostaglandin/calcium mediated pathway[END_REF]. ARA lowers the expression and function of UCP1, thus affecting the dissipation of energy in these thermogenic adipocytes. These effects are mediated via COX activities that lead to increased synthesis and release of PGE2 and PGF2α, which act as inhibitors of Ucp1-mediated thermogenesis via a calcium-dependent pathway.

It has been shown that healthy adult humans exhibit BAT in the cervical and thoracic part of the body, which consists in islets of energy-dissipating thermogenic adipocytes [START_REF] Cypess | Identification and importance of brown adipose tissue in adult humans[END_REF][START_REF] Nedergaard | Unexpected evidence for active brown adipose tissue in adult humans[END_REF][START_REF] Saito | High incidence of metabolically active brown adipose tissue in healthy adult humans: effects of cold exposure and adiposity[END_REF][START_REF] Van Marken Lichtenbelt | Cold-activated brown adipose tissue in healthy men[END_REF][START_REF] Virtanen | Functional brown adipose tissue in healthy adults[END_REF]. These adipocytes display a gene expression signature comparable to either rodent brown or brite adipocytes depending on the localization and the depth of the analyzed tissue [START_REF] Wu | Beige adipocytes are a distinct type of thermogenic fat cell in mouse and human[END_REF][START_REF] Cypess | Anatomical localization, gene expression profiling and functional characterization of adult human neck brown fat[END_REF][START_REF] Jespersen | A classical brown adipose tissue mRNA signature partly overlaps with brite in the supraclavicular region of adult humans[END_REF][START_REF] Sharp | Human BAT possesses molecular signatures that resemble beige/brite cells[END_REF]. As brown and brite adipocytes represent important candidates for controlling body weight, investigations in humans on the regulation of brown/brite adipocyte recruitment and activation are in demand, particularly from a nutritional point of view since quantitative and qualitative issues of dietary lipids are relevant to increased body weight [START_REF] Ailhaud | Temporal changes in dietary fats: role of n-6 polyunsaturated fatty acids in excessive adipose tissue development and relationship to obesity[END_REF].

As in mammals oxylipins govern a large part of the biology of adipose tissue, any dysregulation in their levels may disrupt tissue homeostasis. Thus, it is tempting to assume that prevention of excessive consumption of ω6 fatty acids or re-establishment of a balanced ω6/ω3 PUFA ratio from 30 may contribute to reduce excessive adipose tissue development by controlling white adipocyte formation and enhancing brite adipocyte recruitment.

Herein, we aimed to study whether the inhibitory effect of ω6-PUFA linoleic acid (via local action of ARA) on brite adipocyte recruitment could be reversed by supplementation with the ω3-PUFA αlinolenic acid associated to its metabolites EPA and DHA. We show, in vivo in mice and in vitro in human cells, that adjustment of a ω6/ω3 PUFA ratio from 30 to 3.7 by supplementation of ω6 PUFAenriched diet with ω3 PUFAs rescues the inhibitory effect of ω6-derived oxylipins on the activity of brown and brite adipocytes. These effects are mediated through oxylipins via decreased level of PGF2α.

Materials and Methods

Animals and diets.

The experiments were conducted in accordance with the French and European regulations (directive 2010/63/EU) for the care and use of research animals and were approved by national experimentation committees (MESR 01947.03). Ten week-old C57BL/6J male mice from Janvier Laboratory (France) were maintained at thermoneutrality (28 ± 2°C) and 12:12-hour light-dark cycles, with ad libitum access to food and water and killed at 10-11 AM. Mice were fed for 12 weeks with isocaloric ω6-or ω3-supplemented diets (12% energy content as lipids). These experimental diets were prepared from standard chow diets (ref. 2016, Harlan Lab., WI, USA). The ω6-supplemented diet was enriched with 0.5% linoleate-ethyl-ester (LA) and 0.7% oleate-ethyl-ester (OA) (ratio ω6/ω3 = 30); the ω3-supplemented diet comprised 0.5% LA, 0.54% α-linolenate-ethyl-ester (LNA), 0.08% eicosapentaenoate-ethyl-ester and 0.08% docosahexaenoate-ethyl-ester (ratio ω6/ω3 = 3.7) (see supplemental Table S1 for details) in order to follow human nutritional recommendations. Safflower oil (0.5%) was added to favor dispersion of ethyl esters in the diet. Fatty acid ethyl esters were from NuChekPrep (MIN, USA) and diets were produced by Harlan (WI, USA). Chronic β 3 -adrenergic receptor stimulation was carried out during the last week of the diet treatment by daily intra-peritoneal injections of CL316,243 (1 mg/kg in saline solution). Control mice were injected with vehicle only.

Blood, interscapular BAT (iBAT), epididymal (eWAT) and inguinal subcutaneous WAT (scWAT) were sampled and used for different analyses. hMADS cell culture. The establishment and characterization of hMADS cells has been described [START_REF] Pisani | Differentiation of Human Adipose-Derived Stem Cells into "Brite" (Brownin-White) Adipocytes[END_REF][START_REF] Rodriguez | Adipocyte differentiation of multipotent cells established from human adipose tissue[END_REF][START_REF] Elabd | Human multipotent adipose-derived stem cells differentiate into functional brown adipocytes[END_REF]. In the experiments reported herein, hMADS-3 cells were used between passages 14 and 20. All experiments were performed at least 3 times using different cultures. Cells were cultured and differentiated as previously described [START_REF] Pisani | The omega6-fatty acid, arachidonic acid, regulates the conversion of white to brite adipocyte through a prostaglandin/calcium mediated pathway[END_REF][START_REF] Pisani | Differentiation of Human Adipose-Derived Stem Cells into "Brite" (Brownin-White) Adipocytes[END_REF]. Briefly, cells were induced to differentiate at day 2 postconfluence (designated as day 0) in DMEM/Ham's F12 media supplemented with 10 µg/ml transferrin, 10 nM insulin, 0.2 nM triiodothyronine, 1 µM dexamethasone and 500 µM isobutyl-methylxanthine (IBMX). Two days later, the medium was changed (dexamethasone and IBMX omitted) and 100 nM rosiglitazone was added. At day 9 rosiglitazone was withdrawn to enable white adipocyte differentiation but again included between days 14 and 17 to promote white to brite adipocyte conversion as previously described [START_REF] Pisani | The omega6-fatty acid, arachidonic acid, regulates the conversion of white to brite adipocyte through a prostaglandin/calcium mediated pathway[END_REF][START_REF] Pisani | Differentiation of Human Adipose-Derived Stem Cells into "Brite" (Brownin-White) Adipocytes[END_REF]. Fatty acids were bound to bovine serum albumin (BSA) (0.04% for 15 min at 37°C) prior to addition to culture media.

Oxylipin quantification.

In vitro analysis of secreted PGF2α was performed on differentiated cells at day 17 after incubation for 1 or 24 h in fresh culture media. PGF2α was quantified by Elisa Immuno Assay (EIA) following the manufacturer's instructions (Cayman, BertinPharma, Montigny le Bretonneux, France).

Quantification of oxylipins was performed at METATOUL platform (MetaboHUB, INSERM UMR 1048, I2MC, Toulouse, France) by mass spectrometry analysis. All tissues were snap-frozen with liquid nitrogen immediately after collection and stored at -80°C until extraction. Extraction and analysis were performed as previously described [START_REF] Pisani | The omega6-fatty acid, arachidonic acid, regulates the conversion of white to brite adipocyte through a prostaglandin/calcium mediated pathway[END_REF][START_REF] Faouder | LC-MS/MS method for rapid and concomitant quantification of pro-inflammatory and proresolving polyunsaturated fatty acid metabolites[END_REF].

Measurement of oxygen consumption.

Oxygen consumption rate (OCR) of hMADS adipocytes was determined at day 17 using an XF24 Extracellular Flux Analyzer (Seahorse Bioscience, Agilent). ATP synthase uncoupled OCR was determined by addition of 1.2 µM oligomycin A (ATP synthase inhibitor) and maximal OCR by addition of 1 µM FCCP (Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone as a mitochondrial oxidative phosphorylation uncoupling agent). Rotenone and Antimycin A (2 µM each) were used to inhibit Complex I-and Complex III-dependent respiration, respectively. Parameters were measured for each individual well using the different value of OCR as previously described [START_REF] Brand | Assessing mitochondrial dysfunction in cells[END_REF].

Histology. Histological analysis was performed as previously described [START_REF] Pisani | The omega6-fatty acid, arachidonic acid, regulates the conversion of white to brite adipocyte through a prostaglandin/calcium mediated pathway[END_REF]. Sections (4 µm) were dewaxed and treated in boiling citrate buffer (10 mM, pH 6.0) for 6 min. Cooled sections were rinsed and then permeabilized in PBS 0.2 % triton X-100 at room temperature for 20 min. Sections were saturated in the same buffer containing 3% BSA for 30 min, incubated with perilipin antibody (#RDI-PROGP29, Research Diagnostic Inc., Flanders, USA) for 1h, and TRITC-coupled anti-guinea pig antibody for 45 min. Nuclear staining was performed with DAPI.

UCP1 immunohistochemistry was performed following manufacturer's instructions (LSAB+ system-HRP, Dako, Les Ulis, France) and using goat anti-UCP1 (clone C-17, Santa Cruz, Tebu-bio, Le Perray-en-Yvelines, France).Visualization was performed with an Axiovert microscope and pictures were captured with AxioVision software (Carl Zeiss, Jena, Germany). Lipid droplet diameters (visualized by perilipin staining) were measured using Fiji software [START_REF] Schindelin | Fiji: an open-source platform for biological-image analysis[END_REF]. Displayed pictures are representative of the 4 mice per group analyzed.

Isolation and analysis of RNA. These procedures follow MIQE recommendations [START_REF] Bustin | The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments[END_REF]. Total RNA was extracted using TRI-Reagent kit (Euromedex, Souffelweyersheim, France) according to the manufacturer's instructions. For RNA isolation from organs, tissues were homogenized in TRI-Reagent using a dispersing instrument (ULTRA TURRAX T25, Ika, Germany). Reverse transcriptionpolymerase chain reaction (RT-PCR) was performed using M-MLV-RT (Promega). SYBR qPCR premix Ex TaqII from Takara (Ozyme, France) was used for quantitative PCR (qPCR), and assays were run on a StepOne Plus ABI real-time PCR machine (PerkinElmer Life and Analytical Sciences, Boston).

The expression of selected genes was normalized to that of the TATA-box binding protein (TBP) and 36B4 housekeeping genes for human genes, and 36B4 and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for mouse genes, and then quantified using the comparative-ΔCt method. Primer sequences are available upon request.

Statistical analysis. Data are expressed as mean values ± SEM and were analyzed using InStat software (GraphPad Software). Data were analyzed by one-way ANOVA followed by a Student-Newman-Keuls post-test, or Student's t-test to assess statistical differences between experimental groups. Differences were considered statistically significant with p<0.01.

Results

ω3 PUFA supplementation makes mice sensitive to β3-adrenergic receptor agonist treatment.

Ten-week old mice were fed for 12 weeks with an isocaloric standard diet enriched in ω6 PUFAs (ω6 diet, ω6/ω3 = 30), or supplemented with ω3 PUFAs (ω3 diet, ω6/ω3 = 3.7) (Supplemental Table S1).

There was no difference in body weight (Figure 1A) and food intake during 12 weeks (ω6 diet, 4.49 g/day vs ω3 diet, 4.46 g/day per mouse). During the last week, mice received daily injections of the agonist CL316,243 (1 mg/kg) to activate the β3-adrenergic receptor pathway. As expected, such treatment decreased body weight (Figure 1A) as well as interscapular BAT and epididymal WAT (eWAT) mass (Figure 1B). Interestingly, ω3 diet-fed mice showed a larger decrease in body mass and adipose tissue mass upon CL316,243-treatment compared to ω6 diet-fed mice (Figure 1A and1B). This observation is in line with higher plasma glycerol levels (Figure 1C). Altogether, these results indicate a higher sensitivity of ω3 diet-fed mice to CL316,243 treatment. ω3 diet-fed mice showed an improved response to thermogenic stimulation in BAT and scWAT.

After CL316,243 treatment, mice fed the ω3-diet displayed an increase in BAT mass loss and in the plasma glycerol level, suggesting a higher lipolysis capacity. As expected, histological analysis of BAT showed clear activation of brown adipocytes after CL316,243 treatment as indicated by adipocyte morphological changes in the two groups of mice (Figure 2A). This observation was confirmed by immunostaining for perilipin 1 (PLN1), a known lipid droplet surface protein, which allowed the measure of droplet diameters. The data showed smaller lipid droplets in BAT of mice treated with CL316,243 (Figure 2C). Interestingly, the droplet size was smaller in ω3 compared to ω6 diet-fed mice of both treatment groups (Figure 2C). These morphological modifications were in line with elevated expression of brown adipocyte marker genes in ω3 vs. ω6 diet-fed mice (Figure 2B andD).

The β3-adrenergic agonist CL316,243 is a potent inducer of brite adipocytes and therefore we hypothesized a contribution of these thermogenic cells to CL316,243-induced plasma glycerol levels and body weight loss. No morphological or gene expression differences were found in scWAT between the 2 groups of mice in response to vehicle treatment (Figure 3A andB). However, after CL316,243 treatment, visual examination of sections suggested increased Ucp1 positive adipocytes in scWAT of ω3 diet-fed mice compared to ω6 diet-fed mice (Figure 3A). This observation was supported by the analysis of Ucp1 mRNA expression, which tended to be increased in the ω3 diet-fed group after CL316,243 treatment. Gene expression of other markers in response to CL316,243 was not affected by the diet (Figure 3B). As shown in Supplemental Figure S1, eWAT did not display multiloculated adipocytes which are characteristic of UCP1+ adipocytes. However, a decrease of adipocyte mean diameter after CL316,243 treatment was found in both groups in agreement with, eWAT weight decrease (Figure 1B).

Altogether, these data indicate that the dietary ω6/ω3 ratio affects the induction/activation of thermogenic adipocytes in BAT and scWAT upon stimulation with a β-adrenergic agonist. As the dietary fatty acid composition can considerably influence the quality and quantity of fatty acid metabolites [START_REF] Simopoulos | An Increase in the Omega-6/Omega-3 Fatty Acid Ratio Increases the Risk for Obesity[END_REF][START_REF] Simopoulos | The importance of a balanced omega-6 to omega-3 ratio in the prevention and management of obesity[END_REF] this differential response to thermogenic stimulation may be the consequence of a modulated eicosanoid level in adipose tissues.

Dietary ω3 PUFA supplementation controls the level of ω6-derived oxylipins

To investigate diet-induced differences in the quantity of ω3-and ω6-derived oxylipins, we quantified the levels of more than 30 selected metabolites in scWAT and iBAT from ω3 and ω6 diet-fed mice.

Depending on their origin, oxygenated metabolites derived from ω6 (ARA) or ω3 (EPA and/or DHA) PUFAs, due to COX, LOX and CYP450 activities (Supplemental Figure S2A andB, left panels, oxylipins) or to COX activity only (right panels, eicosanoids: a group of oxylipins) were analyzed (analyzed oxylipins detailed in Supplemental Table S2).

As expected, ω3 PUFAs supplementation allowed higher quantity of their metabolites in BAT and scWAT (Supplemental Figure S2A andB, black columns). In BAT, where the molecular effect of a ω3diet was the most important, we did not find any significant impact of the diet on ω6-derived oxylipin levels (Supplemental Figure S2A, grey columns). In scWAT, similar data were obtained in untreated mice, however when the mice were treated with the β3-adrenergic receptor agonist, the decrease in the levels of ω6-derived oxylipins was higher in ω3 diet-fed mice than in ω6-diet fed mice (Supplemental Figure S2).

Analysis of COX derived eicosanoids showed an increase in EPA derived eicosanoids in the ω3 dietfed group and no significant decrease of ω6-derived eicosanoids, except after CL316,243 treatment (Supplemental Figure S2A and B, right panels). Of note, the treatment with CL316,243, a situation where the UCP1 activity is increased via the release of fatty acids, led to a decrease in the levels of the various oxygenated metabolites, possibly reflecting the preferential use of PUFAs as fuel for thermogenesis, thus limiting their availability for oxylipin production (Figure 4 and Supplemental Figure S2).

Previous work from our laboratory has demonstrated that COX-derived oxylipins are crucial for the formation and activation of thermogenic adipocytes [START_REF] Pisani | The omega6-fatty acid, arachidonic acid, regulates the conversion of white to brite adipocyte through a prostaglandin/calcium mediated pathway[END_REF][START_REF] Ghandour | IP-receptor and PPARs trigger the conversion of human white to brite adipocyte induced by carbaprostacyclin[END_REF]. Accordingly, further analyses focused on the levels of individual COX-derived oxylipins (i.e. 6kPGF1α (representative of PGI2), PGF2α, PGE2, TXB2, PGD2 and 15dPGJ2) in BAT and scWAT. The levels of most individual metabolites were similar in iBAT and scWAT of vehicle-treated ω6 diet-and ω3 diet-fed mice, except for a decrease in TXB2 levels in BAT and in PGE2 levels in scWAT (Figure 4A andB). Treatment with CL316,243 resulted in decreased quantity of these oxylipins in both diet groups in BAT and scWAT, although down regulation of TXB2 and PGE2 was more pronounced in scWAT than in iBAT. Interestingly, PGF2α was exempt from this mode of regulation: CL316,243-treatment resulted in a significant decrease in PGF2α level in ω3 diet-fed but not in ω6 diet-fed mice. Thus, the level of this eicosanoid in response to CL316,243treatment is inversely correlated with Ucp1 expression in BAT and scWAT.

Our previous results demonstrated (17) that PGF2α is a negative regulator of thermogenic adipocyte recruitment. Indeed, the ω6-enriched diet PGF2α level was not affected by CL316,243 treatment (Figure 4A andB) and correlated with inhibition of brown and brite adipocyte recruitment and activation (Figures 2 and3). In contrast, for a ω3-enriched diet, after CL316,243 treatment, no alteration in the brown and brite adipocyte recruitment and activation was found though a striking decrease in the PGF2α level was observed (Figure 4A andB).

Taken together these results demonstrated that diet supplementation with ω3 PUFAs reversed the inhibitory effect of a ω6-enriched diet. This effect could be due to competition between ω6 and ω3 PUFAs at the COX activity level leading in turn to a decrease in PGF2α synthesis. To further investigate the effect of PUFAs and eicosanoids on adipocyte function, we used brite adipocytes derived from hMADS cells as a model system.

EPA reversed the ARA-inhibitory effect in vitro.

hMADS cells are a human stem cell model that is able to differentiate into white adipocytes and to convert into functional brite adipocytes upon rosiglitazone treatment (Figure 5A, [START_REF] Pisani | The omega6-fatty acid, arachidonic acid, regulates the conversion of white to brite adipocyte through a prostaglandin/calcium mediated pathway[END_REF][START_REF] Pisani | Differentiation of Human Adipose-Derived Stem Cells into "Brite" (Brownin-White) Adipocytes[END_REF]). As described previously [START_REF] Pisani | The omega6-fatty acid, arachidonic acid, regulates the conversion of white to brite adipocyte through a prostaglandin/calcium mediated pathway[END_REF], this process can be modified by ω6-PUFA ARA: treatment of hMADS adipocytes during the conversion to brite adipocytes with ARA inhibited the expression of UCP1 and other brite adipocyte markers (CIDEA, CPT1M, PLN5) (Figure 5A). Such treatment did not affect adipogenesis per se, as the expression of PLN1 and ADIPQ was not affected (Figure 5A). Treatment of hMADS adipocytes with the ω3-PUFA EPA (molar ARA/EPA ratio of 3) reversed the inhibitory effect of ARA on brite adipocyte marker expression (Figure 5A). This effect was further investigated at the functional level. Oxygen consumption analysis of hMADS brite adipocytes revealed that ARA inhibited all mitochondrial respiration parameters (Figure 5B), i.e. basal, uncoupled and maximal respiration as well as the spare respiratory capacity (SRC). EPA partially reversed this inhibitory effect of ARA on mitochondrial oxygen consumption, thus affecting the overall thermogenic capacity of these cells (Figure 5B). No change in adipogenic marker expression was observed when DHA was added as an alternative ω3 PUFA (Supplemental Figure S3) indicating EPA to specifically represent the effective compound within this class of molecules.

Taken together these results indicated the thermogenic function of adipocytes to be positively and negatively affected by EPA (ω3) and ARA (ω6), respectively.

EPA reduced PGF2α synthesis and secretion.

To identify the pathway involved in the EPA effect, we used fluprostenol, an agonist of the FP receptor (PGF2α receptor), instead of the precursor ARA during the conversion of white hMADS adipocytes into brite adipocytes. In agreement with our previous work [START_REF] Pisani | The omega6-fatty acid, arachidonic acid, regulates the conversion of white to brite adipocyte through a prostaglandin/calcium mediated pathway[END_REF], fluprostenol mimicked the effect of ARA by inhibiting the expression of UCP1 mRNA (Figure 6A). Co-treatment with EPA did not reverse this effect (Figure 6A), indicating that the UCP1 expression of brite hMADS adipocytes was directly affected by an interaction between the FP receptor and its ω6-derived, endogenous ligand PGF2α. Thus, we hypothesized that EPA reversed the ARA-induced effects in brite hMADS adipocytes by modulating the availability of PGF2α as ligand for its receptor. Accordingly, we measured PGF2α secretion after ARA treatment of brite hMADS adipocytes in the absence or presence of EPA. PGF2α secretion, was not altered by EPA during the first hour of treatment. However, a striking decrease in PGF2α levels was observed after 24 h (Figure 6B). This decrease in quantity was likely not due to a lower expression of the major enzymes involved in PGF2α synthesis. Indeed, the expression of COX-1, COX-2 (allowing the metabolization of ARA to PGG2), AKR1B1 and AKR1C3 (allowing the metabolization of PGG2 to PGF2α) mRNAs was not altered by EPA treatment (Figure 6C). As both ARA and EPA can be recognized and metabolized by COX-1 and COX-2, EPA most likely blocks PGF2α synthesis by competing with ARA.

Discussion

The obesogenic effect of ω6 PUFAs, particularly ARA, is thought to originate from their metabolization to oxylipins thus promoting fat storage and a reduction in energy expenditure [START_REF] Massiera | Arachidonic acid and prostacyclin signaling promote adipose tissue development: a human health concern?[END_REF][START_REF] Pisani | The omega6-fatty acid, arachidonic acid, regulates the conversion of white to brite adipocyte through a prostaglandin/calcium mediated pathway[END_REF][START_REF] Fjaere | Indomethacin Treatment Prevents High Fat Diet-induced Obesity and Insulin Resistance but Not Glucose Intolerance in C57BL/6J Mice[END_REF][START_REF] Javadi | The effect of six different C18 fatty acids on body fat and energy metabolism in mice[END_REF]. However, this system and its regulation appear to underlie an unanticipated complexity. In fact, previous studies demonstrated the ω6 eicosanoid prostacyclin (PGI2, which is derived from ARA via COX-dependent metabolization) as a positive modulator of UCP1 expression and brite adipogenesis in human and murine cellular model systems [START_REF] Ghandour | IP-receptor and PPARs trigger the conversion of human white to brite adipocyte induced by carbaprostacyclin[END_REF][START_REF] Mossenbock | Browning of white adipose tissue uncouples glucose uptake from insulin signaling[END_REF][START_REF] Vegiopoulos | Cyclooxygenase-2 controls energy homeostasis in mice by de novo recruitment of brown adipocytes[END_REF][START_REF] Babaei | Immuno-Magnetic Isolation and Thermogenic Differentiation of White Adipose Tissue Progenitor Cells[END_REF]. In line with these findings, transgenic mice with constitutive overexpression of COX-2 (the inducible isoform of COX) show enhanced browning of WAT and resistance to diet-induced obesity [START_REF] Vegiopoulos | Cyclooxygenase-2 controls energy homeostasis in mice by de novo recruitment of brown adipocytes[END_REF]. The complexity of this system becomes apparent in IP receptor knockout mice, which are protected from ω6 PUFA-induced body and fat mass gain [START_REF] Massiera | Arachidonic acid and prostacyclin signaling promote adipose tissue development: a human health concern?[END_REF]. In a similar way, when mice were fed a high fat diet, the inhibition of COX activities with indomethacin prevented body weight gain, due to decreased fat storage and enhanced recruitment of brite adipocytes in the scWAT [START_REF] Ghoshal | Cyclooxygenase-2 deficiency attenuates adipose tissue differentiation and inflammation in mice[END_REF][START_REF] Fjaere | Indomethacin Treatment Prevents High Fat Diet-induced Obesity and Insulin Resistance but Not Glucose Intolerance in C57BL/6J Mice[END_REF]. These examples emphasize the need to increase our current understanding of the proand anti-adipogenic properties of PUFAs on the level of oxylipin metabolism to modulate energy balance regulation.

In our previous work, we elucidated in more detail the relationship between ARA-derived eicosanoid level, browning of WAT and energy expenditure. Mice were fed an ARA-supplemented standard diet and showed impaired brown adipocyte activation and brite adipocyte recruitment in response to CL316,243 treatment, an effect that was attributed to the abundance of PGF2α [START_REF] Pisani | The omega6-fatty acid, arachidonic acid, regulates the conversion of white to brite adipocyte through a prostaglandin/calcium mediated pathway[END_REF]. Using hMADS adipocytes as an in vitro cell model, we demonstrated that this eicosanoid translates its inhibitory effect on the thermogenic capacity of adipocytes via an interaction with its natural cell surface receptor, the FP receptor, upon its synthesis from ARA via COX activity [START_REF] Pisani | The omega6-fatty acid, arachidonic acid, regulates the conversion of white to brite adipocyte through a prostaglandin/calcium mediated pathway[END_REF]. Herein we show that this ARAdependent production of PGF2α is attenuated in the presence of ω3-PUFAs both in vitro in brite hMADS adipocytes and in vivo in thermogenically activated brown and brite adipocytes. We conclude that Ucp1 expression in these models is less affected by the elevated quantity of ω3-derived metabolites but rather depends on the reduced level of individual ω6-derived metabolites (such as PGF2α) with EPAtreatment.

Both ω6 and ω3 PUFAs are transported in the bloodstream between tissues and are incorporated in plasma membrane under the form of phospholipids or as triglycerides within adipocytes [START_REF] Fickova | Dietary (n-3) and (n-6) polyunsaturated fatty acids rapidly modify fatty acid composition and insulin effects in rat adipocytes[END_REF][START_REF] Herzberg | Differential accumulation and release of long-chain n-3 fatty acids from liver, muscle, and adipose tissue triacylglycerols[END_REF][START_REF] Luo | Dietary (n-3) polyunsaturated fatty acids improve adipocyte insulin action and glucose metabolism in insulin-resistant rats: relation to membrane fatty acids[END_REF]. PUFAs are released into the cell by lipases and metabolized into oxylipins using similar pathways. ω6 and ω3

PUFAs are known to compete at different steps that modulate the availability of their respective metabolites. LA and LNA use the same Δ-desaturases and elongases to generate PUFA metabolites such as ARA, DGLA (Dihomo-γ-linolenic acid), EPA and DHA [START_REF] D'andrea | The same rat Delta6-desaturase not only acts on 18-but also on 24carbon fatty acids in very-long-chain polyunsaturated fatty acid biosynthesis[END_REF]. They can also be used as substrates via β-oxidation, ω3 PUFAs being more rapidly oxidized compared to ω6 PUFAs and mono-unsaturated fatty acids [START_REF] Cunnane | Problems with essential fatty acids: time for a new paradigm?[END_REF].

Although our data support a model in which dietary PUFAs act on the level of oxylipins, this mechanism is not exclusive in the context of thermogenic adipocyte recruitment since ω3-PUFAs per se are associated with browning properties [START_REF] Laiglesia | Eicosapentaenoic acid promotes mitochondrial biogenesis and beige-like features in subcutaneous adipocytes from overweight subjects[END_REF][START_REF] Zhao | Eicosapentaenoic acid promotes thermogenic and fatty acid storage capacity in mouse subcutaneous adipocytes[END_REF]. For example, when mice or rats are fed a LA-enriched diet, the increase in fat mass can be prevented by LNA supplementation under isolipidic and isocaloric conditions [START_REF] Massiera | Arachidonic acid and prostacyclin signaling promote adipose tissue development: a human health concern?[END_REF][START_REF] Muhlhausler | Opposing effects of omega-3 and omega-6 long chain polyunsaturated Fatty acids on the expression of lipogenic genes in omental and retroperitoneal adipose depots in the rat[END_REF][START_REF] Martinez-Fernandez | Omega-3 fatty acids and adipose tissue function in obesity and metabolic syndrome[END_REF]. Using this strategy, we demonstrated herein that ω3 PUFA diet supplementation ameliorates brown adipocyte function in response to β-adrenergic stimulation, by promoting a more oxidative phenotype and to a lesser extent brite adipocyte recruitment. Analysis of PUFA metabolites associated to this improvement showed a decrease in the n-2 series prostaglandins levels, especially PGF2α. Altogether, in vivo and in vitro data show that the competition between ω6 and ω3 PUFAs takes place at the level of COX activities, favoring the production of EPA-derived metabolites instead of ARA-derived metabolites. DHA treatment was inefficient. The lack of effect could be due to the fact that DHA does not compete with ARA for metabolite production but was described to inhibit activity and expression of COX-2 (57).

Interestingly, this mechanism does not appear to contribute to the regulation of body weight when mice are fed under thermoneutral conditions but rapidly initiates the mobilization of fat as an energy substrate in response to thermogenic stimulation. This observation may be of particular relevance to the treatment of overweight and obesity in human subjects as humans constantly live in a thermoneutral environment. Thus, adjusting the ω6/ω3-ratio of human nutrition according to dietary recommendations may benefit the recruitment of brown and brite adipocytes associated with other therapeutic strategies in order to promote a negative energy balance.

Figure 1 .

 1 Figure 1. A low dietary ω6/ω3 ratio enhances body weight loss in response to a β3-adrenergic receptor agonist. Mice were fed with diets supplemented with ω6 PUFAs or ω3 PUFAs, respectively, at 28°C during 12 weeks. CL316,243 or vehicle (NaCl) treatment was performed daily during the last week of feeding. A) Left: Body weight development of mice; Right: body weight loss of mice after CL316,243 treatment relative to vehicle treatment. B) Interscapular brown adipose tissue (iBAT) and epidydimal white adipose tissue (eWAT) weight loss after CL316,243 treatment. C) Plasma glycerol levels after NaCl or CL316,243 treatment. Data are displayed as the mean ± SEM, n = 12 mice per group. a = p<0.01 ω6 vs ω3 and b = p<0.01 NaCl vs CL316,243.

Figure 2 .

 2 Figure 2. Morphological and molecular analysis of brown adipose tissue. A) 2 and (B) expression of Ucp1 mRNA. C) PLN1 immunostaining of iBAT sections from mice fed ω6 or ω3-diet. Lipid droplet (white arrows) diameters were evaluated using PLN1 staining, data are displayed as mean ± sem, n = 200 lipid droplets/mice, 4 mice per group. D) Expression of brown adipocyte marker mRNAs was determined by RT-qPCR. mRNA expressions are expressed as fold increase relative to "NaCl-ω6" values and are represented as a mean ± SEM, n = 12 mice per group. a = p<0.01 ω6 vs ω3 and b = p<0.01 NaCl vs CL316,243.

Figure 3 .

 3 Figure 3. Morphological and molecular analysis of sub-cutaneous white adipose tissue. A) UCP1 immunohistological analysis of sub-cutaneous WAT (scWAT) sections from mice fed ω6 or ω3-diet. Slides were counter-stained with hematoxylin and eosin. B) Expression of Ucp1 and brite/white adipocyte marker mRNAs was determined by RT-qPCR. mRNA expressions are expressed as fold increase relative to "NaCl-ω6" values and are presented as mean ± SEM, n = 12 mice per group. a = p<0.01 ω6 vs. ω3 and b = p<0.01 NaCl vs CL316,243.

Figure 4 .

 4 Figure 4. Abundance of ω6 PUFA-derived eicosanoids in BAT and WAT. Eicosanoid levels were measured by LC-MS/MS in (A) iBAT and (B) scWAT of vehicle and CL316,243-treated mice fed with ω6 and ω3 diet. Data are presented as mean ± SEM of 8 mice per group. a = p<0.01 ω6 vs ω3 and b = p<0.01 NaCl vs CL316,243.

29

 29

Figure 5 .

 5 Figure 5. EPA reversed the effect of ARA on adipocyte browning in vitro. hMADS cells were differentiated into white or brite adipocytes. Brite hMADS adipocytes were treated during the last 3 days of differentiation with 10 µM ARA in the presence or absence of 3.3 µM EPA. A) Expression of adipocyte markers was determined by RT-qPCR and is expressed as fold increase relative to "brite" group values. B) Basal, ATP synthase uncoupled respiration (= % of residual respiration after addition of ATP synthase inhibitor oligomycin A) and maximal respiration (obtained after addition of the chemical uncoupling agent FCCP) were assessed at the end of treatment to determine the oxygen consumption rate (OCR) of mitochondria. The spare respiratory capacity represents a measure for the full respiratory potential of mitochondria and is calculated as the difference between maximal and basal respiration. Data are presented as mean ± SEM of 3 (A) or 6 (B) independent experiments. a = p<0.01 vs brite and b = p<0.01 vs brite + ARA.

Figure 6 .

 6 Figure 6. EPA competes with ARA at the cyclooxygenase level. hMADS cells were differentiated into white or brite adipocytes. A) Brite hMADS adipocytes were treated with 10 nM fluprostenol (agonist of PGF2α receptor) in the presence or absence of 3.3 µM EPA during the last 3 days of adipogenic differentiation. UCP1 and PLN1 were evaluated by RT-qPCR. B) Brite hMADS were exposed to 10 µM ARA in the presence or absence of 3.3 µM EPA for 1 or 24 h. PGF2α was quantified in culture media by EIA. C) Brite hMADS adipocytes were treated with 10 µM ARA in the presence or absence of 3.3 µM EPA during the last 3 days of differentiation. Expression of enzymes involved in the metabolization of ARA to PGF2α was evaluated by RT-qPCR. mRNA expressions are expressed as fold increase relative to "brite" values and are presented as mean ± SEM of 3 independent experiments. a = p<0.01 vs brite and b = p<0.01 vs brite + ARA.

Acknowledgments/grant support. The authors greatly acknowledge the IRCAN Animal core facilitiy and the Cytomed platform as well as the IBV histology platform. We thank Pauline Le Faouder and Justine Bertrand-Michel from the METATOUL platform (MetaboHUB, INSERM UMR 1048, I2MC, Toulouse, France) for oxylipins analysis. We thank Amanda Balzo and Clémence Auverdin for their technical suport. This work was supported by CNRS, French Agence Nationale de la Recherche and Deutsche Forschungsgemeinschaft (ANR/DFG-15-CE14-0033 "Nutribrite") and Nutricia Research Foundation ("2015-26"). The manuscript has been corrected by Dr. Brahimi-Horn (EditDocSci, http://cbrahimihorn.free.fr), a native English speaking scientific editor.

Supporting the physiological relevance of ω3 PUFA-induced effects, fish oil supplementation has been reported to induce UCP1 expression via the sympathetic nervous system, although this mechanism is restricted to BAT [START_REF] Kim | Fish oil intake induces UCP1 upregulation in brown and white adipose tissue via the sympathetic nervous system[END_REF][START_REF] Pahlavani | Eicosapentaenoic acid regulates brown adipose[END_REF].

In conclusion, herein we demonstrate that ω3 PUFA supplementation compensates for the inhibitory effect of ω6 PUFAs on the thermogenic function of adipocytes. We identified the eicosanoid PGF2α as a causal effector, the level of which is influenced by the availability of ω6 and ω3 PUFAs competing at the level of their metabolization. Our data indicate that the dietary ω6/ω3 ratio is a major regulator of adaptive thermogenesis consequently affecting energy homeostasis. These findings are of particular importance in a human nutritional context as the dietary ω6/ω3 ratio is associated with the development of obesity, cardiovascular and inflammatory diseases. tissue metabolism in high-fat-fed mice and in clonal brown adipocytes. J Nutr Biochem 39: 101-109.


Didier F Pisani

Anne-Sophie Coldefy

Christian Elabd

Candice Cabane

Jerome Salles

Martine Le Cunff

Benoit Derijard

Ez-Zoubir Amri

Christian Dani

Jean J Leger

Claude A Dechesne
email: dechesne@unice.fr.

INVOLVEMENT OF BTBD1 IN MESENCHYMAL DIFFERENTIATION

Keywords: BTB domain, DNA topoisomerase 1, myogenesis, adipogenesis, osteogenesis

BTBD1 is a recently cloned BTB-domain containing protein particularly expressed in skeletal muscle and interacting with DNA topoisomerase 1 (Topo1), a key enzyme of cell survival. We have previously demonstrated that stable overexpression of a N-terminal truncated BTBD1 inhibited ex vivo myogenesis but not adipogenesis of pluripotent C2C12 cells. Here, BTBD1 expression was studied in three models of cellular differentiation: myogenesis (C2C12 cells), adipogenesis (3T3-L1 cells) and osteogenesis (hMADS cells). BTBD1 mRNA was found to be upregulated during myogenesis. At the opposite, we have not observed BTBD1 upregulation in an altered myogenesis cellular model and we observed a downregulation of BTBD1 mRNA expression in adipogenesis. Interestingly, amounts of Topo1 protein, but not Topo1 mRNA, were found to be modulated at the opposite of BTBD1 mRNA. No variation of BTBD1 expression was measured during osteogenesis. Taken together, these results indicate that BTBD1 mRNA is specifically regulated during myogenic and adipogenic differentiation, in relation with Topo1 expression. Moreover, they corroborate observations made previously with truncated BTBD1 and show that BTBD1 is a key protein of balance between adipogenesis and myogenesis. Finally, a transcriptome analysis gave molecular clues to decipher BTBD1 role, with an emphasis on the involvement in ubiquitin/proteasome degradation pathway.

INTRODUCTION

BTBD1 is a recently characterized protein expressed in the whole animal kingdom from C. elegans to human, with an unknown function. This protein was detected first by Carrim-Todd et al. in 2001 along with its homologue BTBD2 [START_REF] Carim-Todd | Identification and characterization of BTBD1, a novel BTB domain containing gene on human chromosome 15q24[END_REF]. BTBD1 gene encodes a 482 amino acid protein with a N-terminal end containing a proline-rich region and a BTB domain, which are both involved in protein-protein interactions [START_REF] Zollman | The BTB domain, found primarily in zinc finger proteins, defines an evolutionarily conserved family that includes several developmentally regulated genes in Drosophila[END_REF]. In 2002, Xu et al. demonstrated that BTBD1 and BTBD2 interact with DNA topoisomerase 1 (Topo1) by their C-terminal region (317-482 positions for BTBD1) [START_REF] Xu | Characterization of BTBD1 and BTBD2, two similar BTB-domain-containing Kelch-like proteins that interact with Topoisomerase I[END_REF]. BTBD1 function has not been characterized, but its colocalization in cytoplasmic bodies with TRIM5δ [START_REF] Xu | BTBD1 and BTBD2 colocalize to cytoplasmic bodies with the RBCC/tripartite motif protein, TRIM5delta[END_REF], an E3 ubiquitin ligase, suggests its involvement in protein degradation. Moreover, BTBD1 can be classed in a novel functional family of BTB-domain containing proteins: the family of "Cullin-3 (Cul3) associated proteins" [START_REF] Geyer | BTB/POZ domain proteins are putative substrate adaptors for cullin 3 ubiquitin ligases[END_REF][START_REF] Pintard | Cullin-based ubiquitin ligases: Cul3-BTB complexes join the family[END_REF][START_REF] Furukawa | Targeting of protein ubiquitination by BTB-Cullin 3-Roc1 ubiquitin ligases[END_REF]. Cul3 is a subclass of Cullin-dependent ubiquitin ligases, a multi-subunit enzyme class of E3s involved in specific degradation of various proteins by the ubiquitin/proteasome pathway [START_REF] Kipreos | cul-1 is required for cell cycle exit in C. elegans and identifies a novel gene family[END_REF]. In this model, BTB proteins, like BTBD1, would determine selectivity of the degradation complex by linking specific substrates to Cul3 [START_REF] Furukawa | Targeting of protein ubiquitination by BTB-Cullin 3-Roc1 ubiquitin ligases[END_REF][START_REF] Van Den | Protein degradation: CUL-3 and BTB--partners in proteolysis[END_REF][START_REF] Xu | BTB proteins are substrate-specific adaptors in an SCF-like modular ubiquitin ligase containing CUL-3[END_REF]. For instance, since Topo1 degradation can be performed by the ubiquitin/proteasome pathway [START_REF] Desai | Ubiquitin-dependent destruction of topoisomerase I is stimulated by the antitumor drug camptothecin[END_REF][START_REF] Desai | Transcription-dependent degradation of topoisomerase I-DNA covalent complexes[END_REF], BTBD1 could link Cul3 by its N-terminal region and Topo1 by its C-terminal region, to promote Topo1 degradation. Topo1 is the key enzyme of DNA molecule relaxation, a paramount event in the cell life for all species. It is a determining factor for DNA replication, recombination and repair, chromatin condensation and remodelling, and finally RNA transcription [START_REF] Wang | Action at a distance along a DNA[END_REF][START_REF] Wang | Cellular roles of DNA topoisomerases: a molecular perspective[END_REF][START_REF] Kimura | Phosphorylation and activation of 13S condensin by Cdc2 in vitro[END_REF]. The crucial activity of Topo1 for cell survival and differentiation requests a strict regulation of its expression, which is principally determined by the balance between its synthesis and degradation [START_REF] Morham | Targeted disruption of the mouse topoisomerase I gene by camptothecin selection[END_REF][START_REF] Onishi | Topoisomerase inhibitors induce apoptosis in thymocytes[END_REF][START_REF] Pommier | Repair of and checkpoint response to topoisomerase I-mediated DNA damage[END_REF].

Our studies are focused on BTBD1 function in skeletal muscle. We have previously demonstrated that BTBD1 is essential for myoblast growth and differentiation [START_REF] Pisani | The topoisomerase 1-interacting protein BTBD1 is essential for muscle cell differentiation[END_REF]. Analysis of stable altered BTBD1 protein expression (BTBD1 truncated of its BTB and proline-rich Nterminal domains, designated as ∆-BTBD1) in C2C12 cells (a murine cell line of myoblasts) has shown a defect of these cells to accomplish myogenesis. The mechanism of BTBD1 implication in myogenic differentiation is unknown, but several characteristics of these modified cells are in favour of Topo1 deregulation. Thus, ∆-BTBD1 overexpression was found to be associated with deregulation of the nuclear Topo1 level and localization [START_REF] Pisani | The topoisomerase 1-interacting protein BTBD1 is essential for muscle cell differentiation[END_REF].

In this study, we assess the involvement of BTBD1 in mesenchymal differentiation and we attempt to clarify its specificity of action. First, by ∆-BTBD1 C2C12 cell differentiation assays based on C2C12 pluripotentiality, we confirmed the involvement of BTBD1 in myogenesis and adipogenesis, but not in osteogenesis. Then, we analyzed BTBD1 basal expression along myoblast, adipocyte and osteoblast differentiations, as well as in a myoblast altered differentiation model. We found close correlations between BTBD1 mRNA expression and progression of adipocytic and myogenic differentiations, and we correlated those with Topo1 expression. A transcriptome analysis of gene deregulations induced by the overexpression of ∆-BTBD1 in C2C12 cells submitted to myogenic differentiation conditions gave some molecular basis to decipher BTBD1 function. Finally, our study demonstrates that BTBD1 is a key protein involved in adipogenic/myogenic differentiation balance, potentially via Topo1 regulation through its degradation by the ubiquitin/proteasome pathway.

MATERIALS AND METHODS

Cell culture. The C2C12 mouse skeletal muscle cell line (CRL 1772) was obtained from the American Type Culture Collection. Wild type (WT) and transfected cell lines were grown at 37°C in a 5% CO2 atmosphere in a growth medium (GM) composed of Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Myotube C2C12 differentiation was induced by withdrawing fetal calf serum from confluent cells and adding 10 µg/mL insulin, 5 µg/mL transferrin and 2% horse serum; this medium was named differentiation medium (DM).

C2C12 adipogenic differentiation was induced by adding rosiglitazone (5 µM final concentration) in GM to confluent cells. Rosiglitazone is a member of the glitazone family known to induce adipogenesis in various cell types [START_REF] Grimaldi | Trans-differentiation of myoblasts to adipoblasts: triggering effects of fatty acids and thiazolidinediones[END_REF]. C2C12 osteogenic differentiation was induced by adding 0.1 μM dexamethasone, 10 mM β-glycerophosphate and 50 μg/mL ascorbic acid 2-phosphate in GM to confluent cells [START_REF] Halvorsen | Extracellular matrix mineralization and osteoblast gene expression by human adipose tissue-derived stromal cells[END_REF]. The C2C12 DN MKK3 cell line has been previously described [START_REF] Cabane | Regulation of C2C12 myogenic terminal differentiation by MKK3/p38alpha pathway[END_REF].

For adipogenesis experiments, 3T3-L1 cells (ATCC-CL 173) were grown in GM until confluence and stimulated to differentiate by addition of 0.5μM dexamethasone, 0.5 mM 1methyl-3-isobutylmethyl-xanthine, and 170 nM insulin [START_REF] Green | An established preadipose cell line and its differentiation in culture. II. Factors affecting the adipose conversion[END_REF]. Two days later, cells were replaced in GM complemented with 170 nM insulin. For osteogenesis analysis, hMADS cells were grown in GM supplemented with 2,5 ng/mL FGF2 and differentiated in osteoblasts at confluence by adding 0.1 µM dexamethasone, 10 mM -glycerophosphate, and 50 µg/ml ascorbic acid 2phosphate [START_REF] Rodriguez | Transplantation of a multipotent cell population from human adipose tissue induces dystrophin expression in the immunocompetent mdx mouse[END_REF].

For WT C2C12, DN MKK3 C2C12, 3T3-L1 and hMADS cells, experiments were performed with a pool of clones routinely used. For ∆-BTBD1 C2C12 cells, only experiments reproducible on three independent modified cell lines were considered because of the clonal heterogeneity of derived C2C12 cells.

Cells staining and visualization. Oil red O and Alizarin red staining were performed as previously described [START_REF] Grimaldi | Trans-differentiation of myoblasts to adipoblasts: triggering effects of fatty acids and thiazolidinediones[END_REF][START_REF] Halvorsen | Extracellular matrix mineralization and osteoblast gene expression by human adipose tissue-derived stromal cells[END_REF]. Cells were mounted and visualized on a Leitz Wetzlar light microscope. Photographs were taken with a Nikon Coolpix 990 digital camera. Mineralization areas were quantified after numerization with Multi Gauge V 2.3 (Fuji) software.

Total RNA extraction and quantitative RT-PCR analysis. Total RNAs were extracted using TRI-REAGENT (Euromedex) according to the manufacturer's instructions. RNAs were treated with DNase I for 30 min (Promega) before reverse transcription. First strand cDNA was generated with Superscript II reverse transcriptase (Invitrogen) in presence of 12.5 ng/l random primers (Promega), following the manufacturer's instructions. Real-time PCRs were performed on an ABI Prism 7000 real-time PCR machine (Applied Biosystem). For each PCR run, a master mix was prepared and composed of "qPCR MasterMix Plus for SYBR green I" (Eurogentec) and 300 nM of each primer. Primers were designed using Primer Express software (Applied Biosystem) and validated by testing PCR efficiency using standard curves (efficiency >85%).

Gene expression was quantified using the comparative Ct (threshold cycle) method, mouse 36B4 and human TATA box binding protein (TBP) RNAs were used as reference. Forward and reverse primers are as follows : mouse 36B4 5'-TCCAGGCTTTGGGCATCA-3' and 5'-CTTTATCAGCTGCACATCACTCAGA-3' ; mouse aP2 5'-CTTCAAACTGGGCGTGGAA-3' Western blot analysis. Cell protein extracts and Western blots analysis were performed as previously described [START_REF] Pisani | The topoisomerase 1-interacting protein BTBD1 is essential for muscle cell differentiation[END_REF]. Anti-Topo1 rabbit polyclonal (H-300) and anti--actin (AC-15) mouse monoclonal antibodies were purchased respectively from Santa Cruz and Sigma-Aldrich.

The anti-aP2 rabbit polyclonal antibody was a gift of Dr. David Bernlohr (University of Minnesota, St. Paul, MN, USA).

Microarrays analysis. 50mers oligonucleotide-microarrays manufacturing, labelling, hybridizations and imaging, array data analysis and expression statistical analysis are described previously [START_REF] Rouger | Global/temporal gene expression in diaphragm and hindlimb muscles of dystrophin-deficient (mdx) mice[END_REF]. Briefly, total RNAs for each conditions under comparison were prepared, using the RNeasy Midi Kit (Qiagen) with DNAse treatment, from three independent culture of WT C2C12 cells pool or one culture of three independent ∆-BTBD1 C2C12 cells clones. [START_REF] Kimura | Phosphorylation and activation of 13S condensin by Cdc2 in vitro[END_REF]

RESULTS

Multiple differentiation assays with ∆-BTBD1 C2C12 cells. In a previous work [START_REF] Pisani | The topoisomerase 1-interacting protein BTBD1 is essential for muscle cell differentiation[END_REF], we have constructed a plasmid expressing ∆-BTBD1 protein, a partially truncated form of BTBD1 that lacks the 108 N-terminal amino acid residues, which correspond to the complete proline-rich domain and a large part of the BTB domain, basis of the N-terminal protein/protein interaction domain. ∆-BTBD1 stable expression in pluripotent C2C12 cells, i.e. a murine skeletal muscle cell line that is able to differentiate ex vivo, prevented their myogenesis capacity, but not their adipogenesis capacity. To precise these observations, and to analyze the general effect of ∆-BTBD1 expression on C2C12 cell pluripotency, we performed in parallel myogenesis, osteogenesis and adipogenesis assays with ∆-BTBD1 C2C12 (3 clones) and wild-type cells (WT)

as control (Fig. 1). Confluent cells were cultivated with the adequate differentiation medium.

Then, cells differentiation potential was evaluated on the basis of specific terminal morphologic modification. Myogenesis was assessed by myotube apparition within 2-4 days; adipogenesis was assessed by lipid droplets Oil Red O specific staining after 1 week; osteogenesis was assessed by calcification Alizarin red specific staining after 2 weeks. As expected, WT C2C12 cells were able to differentiate into myotubes, osteoblasts and adipocytes (Fig. 1 A-F), while ∆-BTBD1 C2C12 cells were able to differentiate into osteoblasts and adipocytes, but not in myotubes (Fig. 1 G-L).

Moreover, their capacity to differentiate in adipocytes was found to be increased in comparison with WT C2C12 cells (Fig. 1: compare E-F with K-L). This was confirmed by real-time PCR quantification of an adipocyte marker, the fatty acid binding protein (aP2) (Fig 1M). Conversely, ∆-BTBD1 C2C12 cells capacity to differentiate in osteoblasts was not found different than WT C2C12 cells capacity, as assessed by quantification of mineralization area (Fig. 1N). These results establish that ∆-BTBD1 expression affects C2C12 cell pluripotency: myogenesis capacity is inhibited, adipogenesis is enhanced and osteogenesis seems unaffected.

Moreover, confluent ∆-BTBD1 C2C12 cells are able to differentiate spontaneously in adipocytes, but not in osteoblasts, without adequate differentiation medium (data not shown).

This result is in favour of a specific implication of BTBD1 in adipocyte differentiation.

BTBD1 expression is positively correlated with C2C12 myogenic differentiation. In order to investigate BTBD1 function in skeletal muscle and to clarify the relation between ∆-BTBD1 expression and defect in ex vivo myogenic differentiation, we studied BTBD1 basal expression in differentiation of C2C12 cells. In growth medium (GM), C2C12 cells are mononucleated and undifferentiated (myoblasts). At confluence, if these cells are switched to differentiation medium (DM), they differentiate into myocytes which fuse after 2-3 days to form syncitial structures (myotubes) (Fig. 1B). The kinetic of BTBD1 mRNA variations was studied by quantitative RT-PCR during C2C12 cell differentiation, from confluence (day 0) to day 5 in DM, in comparison with mRNA variations of myogenin, a key myogenic regulator crucial for myocyte fusion.

Quantification of RNA content indicated a continuous increase of BTBD1 mRNA amounts throughout the differentiation process with an approximate seven-fold maximum reached 4 days after differentiation induction (Fig. 2A). This pattern was very well correlated with the important up-regulation of myogenin.

To further assess BTBD1 involvement in C2C12 cell myogenic differentiation, we investigated BTBD1 mRNA expression in a C2C12 cell model in which differentiation is altered.

This model is a stable C2C12 cell line expressing a dominant-negative form of the p38 activator MAP kinase kinase 3 and designated DN MKK3 C2C12 cells [START_REF] Cabane | Regulation of C2C12 myogenic terminal differentiation by MKK3/p38alpha pathway[END_REF]. The cells are characterized by their inability to complete differentiation due to inhibition of p38 activity. They are blocked at the myocyte step and are unable to fuse into myotubes (similar morphology than Fig. 1G). BTBD1

and myogenin expressions were compared by quantitative RT-PCR throughout 5 days in DM, the treatment that induces myogenic differentiation in WT C2C12 cells (Fig. 2B). A basal and constant expression, without any upregulation was found for both genes. Thus, in our model, alteration of C2C12 differentiation aborts BTBD1 and myogenin expression regulation.

Taken together, these results demonstrate a strong positive correlation between BTBD1 expression and C2C12 cell myogenic differentiation.

BTBD1 gene is differently modulated during ex vivo mesenchymal differentiations. Firstly, to elucidate further the previous observation that C2C12 adipogenic differentiation was increased by ∆-BTBD1 stable expression (Fig. 1), we studied BTBD1 mRNA basal expression in a widely used adipogenesis model, the mouse pre-adipocyte 3T3-L1 cell line which is able to differentiate into mature adipocytes with more than 90% efficiency after a 6-day exposure to adipogenic signals [START_REF] Green | An established preadipose cell line and its differentiation in culture. II. Factors affecting the adipose conversion[END_REF]. Analysis by quantitative RT-PCR of 3T3-L1 cells mRNAs at different time-points during differentiation showed a significant gradual decrease of BTBD1 mRNA expression from day 0 to day 9 (Fig. 3A). More than 80% of decrease was observed at day 9, which is a time-point corresponding to 90% of 3T3-L1 cells adipogenic differentiation. Induction of the fatty acid binding protein aP2, a specific adipocyte marker, was quantified in parallel as a positive control of adipogenic differentiation.

Secondly, in the same manner, we studied BTBD1 expression during osteogenic differentiation. We used hMADS cells described to differentiate in osteoblasts under specific culture conditions [START_REF] Rodriguez | Transplantation of a multipotent cell population from human adipose tissue induces dystrophin expression in the immunocompetent mdx mouse[END_REF]. We detected no significant variation of BTBD1 mRNA level from day 0 to day 24 of differentiation (Fig. 3B), despite a clear expected extracellular matrix mineralization.

At the opposite, the mRNA level of phosphatase alkaline, an osteogenic differentiation marker, was found to be gradually increased between days 8 and 24.

Together, these data are consistent with the results obtained with ∆-BTBD1 and suggest that BTBD1 is differently implicated in mesenchymal differentiations.

Different correlations between BTBD1 and Topo1 expressions.

We have previously demonstrated that BTBD1 ─ which binds to Topo1 ─ is essential for normal Topo1 activity and speculated that BTBD1 could negatively contribute to Topo1 regulation, via its ubiquitination and degradation by the ubiquitin/proteasome pathway [START_REF] Pisani | The topoisomerase 1-interacting protein BTBD1 is essential for muscle cell differentiation[END_REF]. The upregulation of BTBD1 in C2C12 myogenic differentiation and its downregulation in C2C12 adipogenic differentiation prompted us to analyse Topo1 expression in the same cellular differentiations. Results are shown in Fig. 4. Topo1 protein levels were assessed on Western blots, but no information could be obtained on BTBD1 protein since no anti-BTBD1 antibodies are available. Topo1 mRNA were quantified with quantitative RT-PCR performed on the same C2C12 RNAs than mentioned above for BTBD1 expressions. Very interestingly, C2C12 myogenic differentiation, which is associated to an upregulation of BTBD1 mRNA (Fig. 2A), was found to be characterized by a significant approximately two-fold downregulation of Topo1 protein between days 0 and 4 (Fig. 4A).

However, no significant modification of Topo1 mRNA was seen during myogenic differentiation.

A converse situation was observed with C2C12 adipogenic differentiation. The downregulation of BTBD1 mRNA (Fig. 3A) contrasted with an approximately twofold upregulation of Topo1 protein between days 0 and 9 (Fig. 4B). As for myogenic differentiation, no variation in Topo1 mRNA amounts was detected during adipogenic differentiation. These data indicate opposite post-traductional regulations of Topo1 during C2C12 myogenic and adipogenic differentiations and corroborate our hypothesis of a negative regulation of Topo1 by BTBD1.

Transcriptome analysis of ∆-BTBD1 C2C12 cells myogenic differentiation.

To start to decipher BTBD1 function we have analyzed transcriptome modifications induced by ∆-BTBD1 overexpression in C2C12 cells at the onset of myogenic differentiation. Transcriptomes of ∆-BTBD1 and WT C2C12 cells were compared along a three time-points kinetic, i.e. day -2 (proliferating stage), day 0 (confluence) and day +1 (differentiating stage: myogenic differentiation is induced at day 0). The comparison was stopped at this latter stage before myotube apparition in WT C2C12 cells since ∆-BTBD1 C2C12 cells do not fuse into myotubes [START_REF] Pisani | The topoisomerase 1-interacting protein BTBD1 is essential for muscle cell differentiation[END_REF]. mRNAs were hybridized to murine cDNA microarrays developed in a previous work [START_REF] Rouger | Global/temporal gene expression in diaphragm and hindlimb muscles of dystrophin-deficient (mdx) mice[END_REF].

Figure 5 presents meaningful variations which were found in four gene families. A large defect of expression for many myogenic markers (troponin, tropomyosin, myosin…) including key muscle regulatory factors (MEF2C and MEF2D) have been found during ∆-BTBD1 C2C12 cells differentiation compared to WT cells. This is in accordance with the failure of ∆-BTBD1 C2C12 cells to differentiate into myotubes. Some of these genes are not upregulated at all, but some of them are induced in a much weaker manner than in WT C2C12 (i.e. troponin T3). The second striking result was the almost opposite expression profiles found for extracellular matrix genes, such as many collagen subtypes, between ∆-BTBD1 and WT C2C12 cells. In most of the cases, the genes were specifically induced in ∆-BTBD1 C2C12 cells. In fact only one gene clearly related to the extracellular matrix, i.e. fibronectin 1, was found to share a common expression profile between the two types of C2C12 cells (not shown). These opposite expression profiles were also observed for the four integrin genes found differentially expressed on the microarrays. This is important to note since integrins are involved in muscle differentiation, especially at the membrane fusion stage [START_REF] Schwander | Beta1 integrins regulate myoblast fusion and sarcomere assembly[END_REF]. All these results sustain important differences between ∆-BTBD1 and WT C2C12 cells and support myogenesis defect in ∆-BTBD1 C2C12 cells.

Finally, the last gene group of interest concerns genes involved in the ubiquitin/proteasome degradation pathway. This pathway is clearly affected in the ∆-BTBD1 showed that BTBD1 and BTBD2 were able to bind Topo1 and that BTBD2 has a weak Topo1 inhibitor activity in vitro. BTBD1 is preferentially expressed in skeletal muscle and heart, which suggests that BTBD1 has a more specific function in striated muscles [START_REF] Carim-Todd | Identification and characterization of BTBD1, a novel BTB domain containing gene on human chromosome 15q24[END_REF][START_REF] Xu | Characterization of BTBD1 and BTBD2, two similar BTB-domain-containing Kelch-like proteins that interact with Topoisomerase I[END_REF]. In a previous study, we have investigated BTBD1 function in modified murine C2C12 skeletal muscle cells expressing a truncated form of BTBD1 (Δ-BTBD1) which is expected to act as a dominantnegative form. The most important findings were the incapacity of Δ-BTBD1 C2C12 cells to differentiate in myotubes and alterations in Topo1 protein level and localization. These results suggested that BTBD1 may be involved in myogenic differentiation ex vivo [START_REF] Pisani | The topoisomerase 1-interacting protein BTBD1 is essential for muscle cell differentiation[END_REF].

In the present study, we investigated the effect of Δ-BTBD1 expression on C2C12 cells pluripotency by performing myogenesis, adipogenesis and osteogenesis assays. Then, to clarify BTBD1 implication in different lineage differentiations, we studied BTBD1 mRNA expression in cellular models for ex vivo myogenesis, adipogenesis and osteogenesis. In parallel, Topo1

expression was assessed at the RNA and protein levels. Finally, to get insights into the molecular mechanism involving BTBD1, we analyzed by cDNA microarrays gene expression modifications induced by overexpression of Δ-BTBD1 in C2C12 cells cultured under myogenic differentiation conditions.

Opposite involvement of BTBD1 in myogenic and adipogenic differentiations. To precise the general effect of Δ-BTBD1 expression on C2C12 cell pluripotency, Δ-BTBD1 C2C12 cells were treated to induce myogenesis, adipogenesis and osteogenesis. Our results indicated that expression of Δ-BTBD1 in C2C12 cells inhibits myogenesis, induces adipogenesis and does not affect osteogenesis. Two hypotheses can be raised to explain the opposite effect of Δ-BTBD1 expression on myogenic and adipogenic differentiations. (i) Inhibition of the process of myogenesis facilitates the differentiation of C2C12 cells into adipocytes as previously described with DN MKK3 C2C12 cells [START_REF] Yeow | Inhibition of myogenesis enables adipogenic trans-differentiation in the C2C12 myogenic cell line[END_REF]. (ii) BTBD1 has opposite functions in myogenic and adipogenic differentiations. To discriminate between these two hypotheses, we analyzed endogenous BTBD1 mRNA expression in models for ex vivo myogenesis and adipogenesis.

In vitro myogenic C2C12 differentiation is considered to mimic most of the in vivo differentiation steps. Under low serum culture conditions, confluent myoblasts complete a last cell cycle and then differentiate into myocytes, which align contiguously to each other and finally fuse into myotubes. This is the last structure before in vivo terminal myofiber formation. We found a close correlation between BTBD1 expression and differentiation from myoblasts to myotubes in C2C12 cells. A continuous increase in BTBD1 expression matched the progression of differentiation in WT C2C12 cells. Conversely, a low and constant BTBD1 expression was observed for several days under differentiation culture conditions in the DN MKK3 C2C12 cell line which stayed blocked at the myocyte stage for several days [START_REF] Cabane | Regulation of C2C12 myogenic terminal differentiation by MKK3/p38alpha pathway[END_REF]. In conclusion, a link between BTBD1 and myogenesis is underlined by our previous and present observations: (i) overexpression of altered BTBD1 protein blocks C2C12 cell myogenic differentiation, (ii) myogenic differentiation and progressive increase of BTBD1 expression are positively correlated and (iii) inhibition of myotube formation, leaded by inhibition of p38 activity in C2C12 cells, prevents normal upregulation of BTBD1 expression. An obvious hypothesis to explain these results is that upregulation of functional BTBD1 protein is necessary for myogenesis progress.

Adipogenesis was simulated ex vivo using 3T3-L1 cells able to form mature adipocytes after 6 days in appropriate differentiation medium. Using this model, we have demonstrated a continuous decrease of BTBD1 mRNA expression during adipogenesis. While BTBD1 expression is upregulated during myogenesis, it is downregulated during adipogenesis. Since overexpression of Δ-BTBD1 increases C2C12 cell adipogenic differentiation [ref], we can speculate that BTBD1 is directly involved in adipogenesis by preventing this process. Thus, a defect in BTBD1 function, as caused by overexpression of Δ-BTBD1, could explain in the same time an inhibition in myogenic differentiation and an induction of adipogenic differentiation.

Therefore we propose that BTBD1 supports opposite functions in myogenic and adipogenic differentiations.

To precise a specific or a wide involvement of BTBD1 in mesenchymal differentiation, we also studied BTBD1 expression during osteoblastic differentiation. No significant variation in BTBD1 expression was detected during osteoblastic differentiation, in accordance with the lack of effect of Δ-BTBD1 expression in C2C12 cell osteoblastic differentiation. BTBD1 function in mesenchymal differentiation seems thus restricted to myogenic and adipogenic lineages. Maybe, BTBD1 could be another important protein of this adoptive potential of satellite cells and more generally of the differentiation engagement regulation of mesenchymal committed progenitors (myo-adipogenic or myo-adipo-chondro-osteogenic lineages).

Molecular events correlated to BTBD1 mRNA expression during myogenesis. Transcriptome modifications induced by Δ-BTBD1 in C2C12 cells grown under myogenic differentiation conditions, clearly indicate that the lack of differentiation in myotubes is associated with a wide and drastic impairment of myogenic markers RNA accumulation. This underlines the crucial role of BTBD1 in myogenic differentiation. Modifications in expression of extracellular matrix and integrin genes are likely related to the faint myogenic differentiation. Integrins are important for myoblasts membrane fusion in connection with extracellular matrix proteins. However it cannot be ruled out that expression modification of extracellular matrix genes can also be related to other cellular mechanisms since these genes are involved in many aspects of cell behaviour, as chondrogenic differentiation for collagen IIa1. The clearest hypothesis on BTBD1 role suggested by the microarray study is the implication in the ubiquitin/proteasome degradation pathway. It seems that Δ-BTBD1 expression profoundly modifies composition of multiple ubiquitin/proteasome complexes. This is based on the observation that 13 genes of this pathway showed opposite expression profiles between Δ-BTBD1 and WT C2C12 cells. They consist of ubiquitin activating enzymes, ubiquitin conjugating enzymes or ubiquitin ligases (Siah2), as well as proteasome subunits. The extent of these changes at mRNA level is surprising but of special interest, and could be a consequence of a large disruption in ubiquitin/proteasome system for protein degradation due to ∆-BTBD1 expression. These data reinforce our hypothesis for an implication of BTBD1 in this pathway, but with a more important involvement that we previously hypothesized.

Thus BTBD1 involvement in differentiation process is likely to consist in targeting key proteins to the ubiquitin/proteasome pathway. More precisely, BTBD1 could link specific substrates to E3 ubiquitin ligases, such as Cul3. In this context, expression of the truncated Nterminal form of BTBD1 which disrupts myogenesis and promotes adipogenesis, could prevent degradation of key substrates linked to its C-terminal region and not recruited to the E3 complex normally linked to the N-terminal containing proline-rich and BTB domains of BTBD1. Topo1 is likely to be one of the proteins targeted to degradation by BTBD1 during myogenesis and adipogenesis since i) BTBD1 is a Topo1 interacting protein [START_REF] Xu | Characterization of BTBD1 and BTBD2, two similar BTB-domain-containing Kelch-like proteins that interact with Topoisomerase I[END_REF], ii) its expression is negatively correlated with Topo1 protein expression and activity ─ without variations in Topo1 mRNA quantities ─during myogenesis [START_REF] Pisani | The topoisomerase 1-interacting protein BTBD1 is essential for muscle cell differentiation[END_REF] as well as adipogenesis (Fig. 4), iii) although no clear molecular evidence demonstrate the role of Topo1 in mesenchymal differentiation, links between Topo1 and myogenic differentiation are enlightened by the following point. A strategy to treat rhabdomyosarcoma, a malignant pathology characterized by uncontrolled skeletal muscle cell differentiation, is the use of Topo1 inhibitors such as topotecan or irinotecan [START_REF] Breitfeld | Rhabdomyosarcoma: new windows of opportunity[END_REF]. Thus, an abnormal upregulation of Topo1 activity can be linked to anarchic muscle cell differentiation, which suggests a direct link between Topo1 activity and myogenic differentiation. Taken together, these and our previous results clearly suggest an original function of Topo1 in mesenchymal differentiation with a main regulation at the protein level .

Finally, it also should be mentioned that Topo1 is a key enzyme of cell cycle progress in regard to its function in DNA replication. Another point to consider is that BTBD1 can be involved in cell cycle regulation, through Topo1 degradation via BTBD1 activity in the Cul3 complex. In this sense, mice invalidated for Cul3 die at day 7.5 post-coïtum with aberrations in several cyclin levels, which control cell cycle checkpoint [START_REF] Singer | Cullin-3 targets cyclin E for ubiquitination and controls S phase in mammalian cells[END_REF]. This lethality could be explained by the potential involvement of Cul3 complex in degradation of a multiplicity of substrates [START_REF] Guardavaccaro | Oncogenic aberrations of cullin-dependent ubiquitin ligases[END_REF].

More precisely, Cul3 disruption could involve Topo1 level aberrations leading to cell cycle perturbations. Because of the multiple functions of Topo1, it is difficult at this point to link or not the roles of Topo1 in cellular differentiation and cell cycle.

In conclusion, we clearly describe a primordial regulation of BTBD1 expression in myogenic differentiation, which could be related to its high expression in this tissue. Indeed, alteration of normal BTBD1 expression impairs myogenesis. Moreover, our results indicate a potential key function of BTBD1 in mesenchymal differentiation, regarding adipogenesis/myogenesis balance. The current and previous findings suggest that the dual and opposite involvement of BTBD1 in this two different differentiation pathways could be explained by its capacity to regulate Topo1 content and/or activity. We propose that BTBD1 is important for Topo1 regulation through its ubiquitin/proteasome pathway degradation during mesenchymal differentiation.

  '; mouse Topo1 5'-TCGAGCTGTTGCAATTCTTTGTA-3' and 5'-TGCAAGTTCATCATTGACTTCTCA-3' ; human alkaline phosphatase (ALP) 5'-GCGCAAGAGACACTGAAATATGC-3' and 5'-TGGTGGAGCTGACCCTTGAG-3' ; human BTBD1 5'-GACCAAGGCGTCGCTGAA-3' and 5'-CCCAGTACGAAGCGCACAT-3' ; human TBP 5'-CACGAACCACGGCACTGATT-3' and 5'-TTTTCTTGCTGCCAGTCTGGAC-3'. The data presented are means ± sem of triplicates from two independent experiments. Statistically significant changes (*, p< 0.01) were detected using unpaired Student's t -test.

C2C12 cells since 13

 13 out of 14 genes with differential microarray signals harboured opposite expression profiles (essentially down-regulated in ∆-BTBD1 cells) between ∆-BTBD1 and WT C2C12 cells (see Fig. 5 Proteasome panel). DISCUSSION BTBD1 is a new protein that contains several protein-protein interaction motifs. It was discovered at the same time as its homologue BTBD2 by Carrim-Todd et al. in 2001. Xu et al.

  Taken together, these results indicate that BTBD1 could be involved in myogenic/adipogenic differentiation balance. Recent experimental works have shown that satellite cells, the main myogenic precursors, can differentiate in several lineages and in particular give rise to adipocytes. [Asakura et al., Differentiation, 2001, 68, 245-253; De Coppi et al., Diabetologia, 2006]. Environmental cellular interactions are important cues for pluripotent-cell differentiation fates. Thus, Shefer et al. have shown that satellite cells, maintain in fact their myogenic potential when they are close to myofibers, but they can adopt an adipogenic potential when they are dissociated from myofibers [28]. Underlying molecular mechanisms need still to be clarify. An essential role has been found for Wnt signalling [Vertino et al., Mol Cell Biol, 2005, 16, 2039-2048].

Figure 1 .

 1 Figure 1. Analysis of Δ-BTBD1 C2C12 cells pluripotency potential. Control (A-F) and Δ-

Figure 2 .

 2 Figure 2. Analysis of BTBD1 mRNA expression during ex vivo myogenesis of C2C12 cells.

Figure 3 .

 3 Figure 3. Analysis of BTBD1 mRNA expression level during ex vivo adipogenesis of 3T3-L1

Figure 4 .

 4 Figure 4. Analysis of Topo1 expression level along myogenesis and adipogenesis. Total proteins

Figure 5 .

 5 Figure 5. Microarrays analysis of genes expression alteration during ∆-BTBD1 C2C12 cells

Figure 1 .

 1 Figure 1.

Figure 5 .

 5 Figure 5.

µg of RNA were reverse transcribed using the CyScribe cDNA Post labelling kit (Amersham

	filtered	and	normalized	with	Madscan	software	available	at

Biosciences) from oligo dT and random nonamers with dUTP-aminoallyl incorporation. cDNAs were purified on GFX columns (Amersham Biosciences) then coupled to Cy3 and Cy5. The microarrays contained 16254 spots representing 5418 genes in triplicates. Hybridization signals were red on ScanArray 3000 (Perkin Elmer) and analysed with Geneprix Pro software. Data were http://cardioserve.nantes.inserm.fr/mad/madscan/login.php. Statistical analyses were made with the Significance Analysis of Microarrays software.

ACKNOWLEDGMENTS

D.F. Pisani is a recipient of grants from the Ligue contre le cancer and Christian Elabd from the Fondation de la Recherche Médicale. This work was supported by the Association Française contre les Myopathies and the Centre National de la Recherche Scientifique. We are grateful to Dr. Nathalie Billon for critical review of the manuscript.