Benoit Julien

Claudie Pinteur

Nathalie Vega

Hubert Vidal

Danielle Naville

Dr Brigitte Le Magueresse-Battistoni

Estrogen withdrawal and replacement

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Bullet points:

Estrogens differentially impacted hepatic glucose and lipid metabolism in female mice Estrogens differentially impacted visceral and subcutaneous fat depots Exposure to pollutants altered lipid deposition in liver and adiponectin levels Pollutants impacted E2-regulated gene mRNA levels in a tissue-dependent manner Mixture contained pollutants at the tolerable daily intake dose

Introduction

Obesity-related metabolic dysfunctions constitute a major problem in Public Health. Over 1.9 billion adults were overweight, of those 650 million were obese in 2016 placing them at high risk for metabolic diseases including type 2 diabetes [START_REF] Who | Obesity and overweight[END_REF]. Risk of obesity is not equal between males and females. Sex differences appeared as the population aged. In 2005, 16-17% of women vs 10-11% of men over 50 were obese [START_REF] Kelly | Global burden of obesity in 2005 and projections to 2030[END_REF]. Risk of obesity in females is associated with metabolic changes occurring during the menopausal transition characterized by progressive reduction of ovarian estrogen production. According to different studies, the prevalence of the metabolic syndrome which is a cluster of conditions of dyslipidemia, central adiposity, hypertension, abnormal glucose metabolism and insulin resistance is enhanced among postmenopausal women varying between 30 to 70% compared with 14-45% in younger women [START_REF] Stefanska | Metabolic Syndrome and Menopause: Pathophysiology, Clinical and Diagnostic Significance[END_REF] delineating the post-menopausal women as a vulnerable population.

Metabolic disorders are multifactorial diseases. Besides genetic predispositions and imbalance between energy intake and expenditure, environmental pollutants have been suggested to be etiologic factors as well, based on robust epidemiologic data and experimental studies demonstrating changes in body weight and/or insulin resistance [START_REF] Casals-Casas | Endocrine disruptors: from endocrine to metabolic disruption[END_REF][START_REF] Nadal | Endocrine-disrupting chemicals and the regulation of energy balance[END_REF][START_REF] Thayer | Role of environmental chemicals in diabetes and obesity: a National Toxicology Program workshop review[END_REF][START_REF] Magueresse-Battistoni | Endocrine disrupting chemicals in mixture and obesity, diabetes and related metabolic disorders[END_REF]. Environmental pollutants may include persistent chemicals which bioaccumulate in the food chain. They are especially present in fatty foods of animal origin (meat, milk, fish) and characterized by halflives of several years in humans, e.g. dioxins and polychlorobiphenyls [START_REF] Schafer | Persistent toxic chemicals in the US food supply[END_REF]. Other pollutants may have short half-lives e.g. phthalates and bisphenols. However, as these chemicals are massively used in plastic goods, the leaching from packaging causes contamination resulting in a chronic exposure [START_REF] Muncke | Exposure to endocrine disrupting compounds via the food chain: Is packaging a relevant source? The Science of the total environment[END_REF][START_REF] Vandenberg | Hormones and endocrine-disrupting chemicals: low-dose effects and nonmonotonic dose responses[END_REF]. Pollutants are under regulation by international risk assessment evaluation bodies (e.g., the European Food Safety Agency, EFSA). However, the cocktail effect resulting from the chronic exposure to a myriad of chemicals originating from different sources (foods and beverages, air breathing, skin absorption of cosmetics) and to which the human population is involuntary exposed is underappreciated [START_REF] Kortenkamp | Low dose mixture effects of endocrine disrupters and their implications for regulatory thresholds in chemical risk assessment[END_REF][START_REF] Svingen | The risk of chemical cocktail effects and how to deal with the issue[END_REF][START_REF] Magueresse-Battistoni | Sex-specific metabolic alterations induced by environmental pollutants[END_REF]. Of striking importance are chemicals identified for their ability to interfere with hormonal action, the so-called endocrine disruptors [START_REF] Zoeller | A path forward in the debate over health impacts of endocrine disrupting chemicals[END_REF]. Populations vulnerable because of hormonal deficits such as postmenopausal women may thus be at particular risk when exposed to these chemicals. It has consistently been found that among a population of obese and post-menopausal women, plasma levels of persistent organic pollutants (POPs) were associated with insulin resistance [START_REF] Gauthier | The metabolically healthy but obese phenotype is associated with lower plasma levels of persistent organic pollutants as compared to the metabolically abnormal obese phenotype[END_REF].

The estrogeno-mimetic properties of endocrine disruptors are of particular concern.

Characterizing their mechanisms of actions could help to explain their deleterious impact on the reproductive functions but also on metabolic disorders [START_REF] Vandenberg | Hormones and endocrine-disrupting chemicals: low-dose effects and nonmonotonic dose responses[END_REF]. Indeed, it has been well demonstrated that estrogens exert protective roles against metabolic diseases in addition to regulating reproductive functions. They are for instance known to positively regulate glucose homeostasis and lipid metabolism favoring insulin sensitivity [START_REF] Nadal | Endocrine-disrupting chemicals and the regulation of energy balance[END_REF][START_REF] Mauvais-Jarvis | The role of estrogens in control of energy balance and glucose homeostasis[END_REF][START_REF] Riant | Estrogens protect against high-fat dietinduced insulin resistance and glucose intolerance in mice[END_REF][START_REF] Zhu | Estrogen treatment after ovariectomy protects against fatty liver and may improve pathway-selective insulin resistance[END_REF]. All these beneficial effects progressively disappear with menopause. Estrogens act through specific receptors distributed centrally and peripherally including ERα, ERβ and the G-protein-coupled receptor GPR30 which differ in abundance and tissue distribution in a sex-dependent manner [START_REF] Mauvais-Jarvis | The role of estrogens in control of energy balance and glucose homeostasis[END_REF][START_REF] Barros | Estrogen receptors: new players in diabetes mellitus[END_REF][START_REF] Sharma | G-Protein-Coupled Estrogen Receptor (GPER) and Sex-Specific Metabolic Homeostasis[END_REF]. Non-genomic actions of estrogens as well as cross-talk with various nuclear receptors and/or transcription factors have also been evidenced [START_REF] Maggi | Sex, metabolism and health[END_REF][START_REF] Matthews | Estrogen receptor and aryl hydrocarbon receptor signaling pathways[END_REF]. All these signaling pathways result in pleiotropic actions of estrogens in the peripherally and metabolically active tissues including the liver and the adipose tissues which express the estrogen receptors. Thus, these signaling pathways are potential targets for endocrine disruptors in a manner dependent on the age, the sex and the tissue considered [START_REF] Casals-Casas | Endocrine disruptors: from endocrine to metabolic disruption[END_REF][START_REF] Diamanti-Kandarakis | Endocrine-disrupting chemicals: an Endocrine Society scientific statement[END_REF][START_REF] Swanson | Role of nuclear receptors in lipid dysfunction and obesity-related diseases. Drug metabolism and disposition: the biological fate of chemicals[END_REF].

We previously demonstrated that a mixture of low-dose pollutants made of dioxin, PCB, phthalate and bisphenol A (BPA) could interfere with estrogen signaling and trigger hepatic insulin resistance and aggravation of glucose intolerance in female mice fed a high-fat highsucrose diet [START_REF] Naville | Low-dose food contaminants trigger sex-specific, hepatic metabolic changes in the progeny of obese mice[END_REF]. In addition, mice fed a standard diet but exposed to the same mixture of pollutants exhibited enhancement of hepatic triglycerides content and insulin resistance [START_REF] Labaronne | Low-dose pollutant mixture triggers metabolic disturbances in female mice leading to common and specific features as compared to a high-fat diet[END_REF].

The impact of this pollutant mixture depended on the age of the mice, with differences occurring between mature and immature females [START_REF] Naville | Low-dose food contaminants trigger sex-specific, hepatic metabolic changes in the progeny of obese mice[END_REF][START_REF] Naville | Metabolic outcome of female mice exposed to a mixture of low-dose pollutants in a diet-induced obesity model[END_REF]. However, the metabolic impact of the mixture of pollutants in conditions of hormonal deficit after ovariectomy to mimic menopause and hormonal replacement using 17β estradiol (E2) pellets implanted subcutaneously, has not yet been explored. The purpose of the current study was to fill this gap. After characterizing the metabolic phenotypes of liver, subcutaneous and visceral adipose tissues in ovariectomized mice, we delineated the estrogenic component using hormonal replacement.

Materials and methods

Animals, diets and experimental design

A schematic view of the 3 experimental protocols is described in Supplemental Figure 1.

C57Bl/6 female mice (4-week-old) were purchased from Envigo laboratories (Gannat, France), housed by 2 in polypropylene cages (to avoid BPA leaching from polycarbonate cages) at 21°C with a normal light-dark cycle and free access to water (polypropylene bottles) and standard chow (LASvendi-16R; Soest, Germany). After one week of acclimatization, mice of the experimental protocol 1 were fed a high fat-high sucrose (HF) diet (from Envigo) containing (HFp) or not (HF0) a mixture of pollutants (see below for the details). At 7 weeks of age, mice were sham-operated (HF0 sham) or underwent a bilateral ovariectomy after general anesthesia, as previously described [START_REF] Julien | Evidence for estrogeno-mimetic effects of a mixture of low-dose pollutants in a model of ovariectomized mice[END_REF] followed by the implantation, under the neck skin, of Silastic implants filled either with vehicle (HF0 Ovx and HFp Ovx) or with 50µg of 17β-estradiol-3-benzoate, E2 (Sigma-Aldrich, Saint-Quentin Fallavier, France) in 30µl of sesame oil (HF0 Ovx+E2 and HFp Ovx+E2) as described [START_REF] Raskin | Conditional inactivation of androgen receptor gene in the nervous system: effects on male behavioral and neuroendocrine responses[END_REF]. The implants were renewed at 13 weeks of age (8 weeks of diet) as the release of E2 was effective only 6 weeks based on body weight follow-up performed in a pilot experiment (data not shown). In the course of the study, two other protocols were conducted to assess the individual effects of the HF diet and of the ovariectomy (Supplemental Figure 1). All procedures were performed with the approval of the Regional Committee of Ethics for Animal Experiments and the French Ministry for Higher Education and Research. Body weight (for all protocols) and food intake (for protocol 1) were recorded weekly throughout the protocol until mice were 20 weeks of age. Validation of the surgery was assessed at the time of mouse euthanasia with the observation of uterine atrophy in the Ovx females (measurement of uterus weight; Supplemental Figure 2). Food intake measured in Protocol 1 did not change between groups, regardless of treatments (data not shown).

The pollutant mixture was made of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, CAS n° 1746-01-6 from LGC-Promochem, Molsheim, France), polychlorinated biphenyl (PCB)

153 (CAS n°35065-27-1), bisphenol A (BPA, CAS n°80-05-7) and di-[2-ethylhexyl]phthalate (DEHP, CAS n°117-81-7) (all three from Sigma-Aldrich). Each pollutant was used at a dose close to its tolerable daily intake (TDI) reference dose of either the pollutant itself (DEHP, BPA) or representative congeners (TCDD, PCB153) [START_REF] Naville | Low-dose food contaminants trigger sex-specific, hepatic metabolic changes in the progeny of obese mice[END_REF].

Pollutants were dissolved in dimethylsulfoxide (DMSO) and diluted in corn oil to facilitate uniform homogenization in the food and the making of pellets. Pollutant-free diet contained identical volumes of DMSO and corn oil as the pollutant-containing food.

HF0 and HFp diets contained 63% fat, 23.6% carbohydrate and 13.4% protein in kcal, as detailed previously [START_REF] Labaronne | Low-dose pollutant mixture triggers metabolic disturbances in female mice leading to common and specific features as compared to a high-fat diet[END_REF]. To globally ensure the daily exposure to 2pg/kg bw/d of TCDD, 80 ng/kg bw/d of PCB153, 50μg/ kg bw/d of DEHP and 5 μg/ kg bw/d of BPA 1 g of contaminated food per 17 g of body weight was given per day to mice otherwise fed ad libitum as previously described [START_REF] Naville | Low-dose food contaminants trigger sex-specific, hepatic metabolic changes in the progeny of obese mice[END_REF][START_REF] Naville | Metabolic outcome of female mice exposed to a mixture of low-dose pollutants in a diet-induced obesity model[END_REF].

Metabolic tests

After 13 wks of diet, 16 h-fasted mice were injected intraperitoneally with glucose (1 mg/g of body weight) for the glucose tolerance test (GTT). Blood glucose was measured using the Accu-Chek Performa glucometer (Roche Diabetes Care France, Meylan).

Body composition, blood and tissue collection

One day before euthanasia, weight and whole body composition were analyzed using nuclear magnetic resonance (Minispec TD-NMR, Bruker BioSpin). Following a 6 hour fasting, blood was collected by retro-orbital sampling. Mice were euthanized by cervical dislocation and liver, inguinal subcutaneous (SAT) and visceral (periovarian+parametrial, VAT) adipose tissue were quickly dissected, snap-frozen in nitrogen liquid and conserved at -80°C until experiments. Small pieces of SAT and VAT were fixed in 4% buffered formaldehyde and embedded in paraffin for histology studies.

Blood glucose concentrations (Accu-Chek Performa glucometer) as well as plasma levels of insulin (Mouse Ultrasensitive ELISA, Eurobio), triglycerides (Biolabo, Maizy, France), cholesterol, free fatty acids (FFA), alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) activities (Sigma-Aldrich), leptin and adiponectin (Crystal Chem, Zaandam, Netherlands) were measured. Total cholesterol and triglycerides were also measured in the liver after extraction of lipids from frozen liver samples using a lipid Extraction kit chloroform-free (Clinisciences, Nanterre, France).

Adipose tissue histology

Paraffin-embedded adipose tissues were cut and adipocyte area was assessed by immunostaining of the basal membrane realized on 4µm sections using the rabbit antilaminin antibody (1:100, Merck, L9393). Image acquisition was obtained with the AXIOVERT 200 M inversed fluorescent microscopes (Carl Zeiss S.A.S., Marly le Roi, France) equipped with the AxioVision 4.8.2 software. Analyses were performed using the ImageJ software.

Real time PCR analysis

Total RNA extracted from frozen tissue samples was reverse-transcribed and analyzed by real-time PCR as described [START_REF] Naville | Direct and indirect impact of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on adult mouse Leydig cells: an in vitro study[END_REF] using a set of specific primers (Supplemental Table 1). In each tissue data were normalized relatively to TBP (encoding TATA-Box Binding Protein).

Statistical analysis

All results are expressed as means ± SEM and differences between means were considered significant at p-value <0.05. To test for statistical significance, one-way ANOVA followed by Tukey post hoc test was used when comparing HF0 sham, HF0

Ovx and HF0 Ovx+E2 groups. To test for pollutant effects, two-way ANOVA for nonrepeated measures with E2 and pollutants as factors was used, followed by a Bonferroni post hoc test. When significant effects without interaction were obtained, differences were tested using Student's t-test between the pollutant-exposed group and the nonexposed group. GraphPad Prism 5.0 software was used for all statistical analysis.

Results

Metabolic characterization of sham and ovariectomized females fed a HF diet and effect of 17ß estradiol replacement

Sham-operated (sham) and ovariectomized (Ovx) females having received E2 or vehicle supplementation, all fed a HF diet were thoroughly characterized (Protocol 1). Non-operated females fed either a standard diet or a HF diet were monitored in parallel (Protocol 3). As expected, females fed the HF diet were significantly heavier than ST fed mice with high levels of blood glucose and plasma insulin levels resulting in a 3-fold increase of the HOMA-IR (Fig. 1A-B; Supplemental Figure 3) which is an index of insulin resistance. Ovariectomy induced an additional increase of body weight and fat mass, a hyperglycaemia after a 16h fasting (time 0 of the GTT) with a trend after a 6h fasting (blood sample at euthanasia), and elevated insulin plasma levels, all resulting in glucose intolerance (GTT) and insulin resistance (enhanced HOMA-IR) (Fig. 1). Conversely, triglycerides (TG) and cholesterol levels (both plasma and liver) were not significantly impacted by ovariectomy (Table 1).

These metabolic features were also observed in Protocol 2 in which ovariectomy was performed in mice fed a standard diet (Supplemental Figure 4). Neither ovariectomy nor E2 treatment induced liver toxicity as there was no increase in plasma AST or ALT in HF0 Ovx and HF0 Ovx+E2 mice (data not shown).

Estradiol supplementation to HF0 Ovx mice led to a restoration of insulin and FFA plasma levels as well as glucose tolerance to values measured in HF0-sham mice (Fig. 1; Table 1).

Other metabolic parameters including glycaemia, body weight, VAT and SAT weight, plasma cholesterol (total and cholesteryl esters) and leptin levels were significantly lower than those measured in HF0 sham mice (Fig. 1 and Table 1). For some parameters, the range of values was identical to those in standard-fed mice (Fig. 1). Besides, impedance analyses revealed enhanced lean mass and reduced fat mass in E2-replaced Ovx mice compared to HF-fed sham mice (Fig. 1D) which is probably associated to the reduced adipocyte diameter observed in both AT depots (Fig. 1D and Supplemental Figure 5) with E2 replacement. Moreover, adiponectin levels, not affected by ovariectomy, were reduced in E2-replaced females (Table 1) as reported elsewhere [START_REF] Bryzgalova | Mechanisms of antidiabetogenic and body weight-lowering effects of estrogen in high-fat diet-fed mice[END_REF].

Fat depots were differently affected in response to ovariectomy and 17ß estradiol supplementation

The metabolic explorations showed that the adipose tissue was highly responsive to ovariectomy and E2 replacement consistent with estrogens playing major roles in fat tissue deposition and expansion in females [START_REF] Mauvais-Jarvis | The role of estrogens in control of energy balance and glucose homeostasis[END_REF][START_REF] Jia | Estrogen receptor alpha and beta in health and disease. Best practice & research Clinical endocrinology & metabolism[END_REF]. To go further in-depth, we analyzed by RT-qPCR a panel of genes encoding the estrogen receptors and markers of adipogenesis or inflammation. We observed that the expression level of Esr1 and Esr2 genes encoding the nuclear estrogen receptor (ER) α and β, respectively and Gper1 encoding the G protein coupled estrogen receptor 1 (GPR30) responded differently to estrogen withdrawal and replacement. Esr1 mRNA levels were unchanged in Ovx females but significantly increased

(2-fold) upon E2 replacement in both SAT and VAT. Gper1 and Esr2 mRNA levels were significantly higher and lower, respectively, in Ovx than in sham females. E2 replacement corrected these effects in SAT but not in VAT (Fig. 2). The pattern of expression of Nr3c4

(encoding the androgen receptor) and Esr1 mRNA was quite similar in SAT and VAT (i.e., almost no change following ovariectomy and augmentation following E2 replacement relative to sham mice) (Fig 2). Additionally, ten-fold more Esr1 and 1.5-fold more Nr3c4 mRNA levels were found in VAT than in SAT, and 2-fold more Gper1 mRNA levels were found in SAT than in VAT. As for Esr2, mRNA levels were low in both fat pads with large fluctuations in the VAT of sham mice (Fig. 2).

Plasma leptin levels of Ovx females (Table 1) which correlated with obesity were consistent with the increase of lep mRNA levels. They returned to sham levels in SAT and VAT of E2treated mice (Fig. 3). A similar pattern of mRNA expression was observed for Ccl2 which encodes the monocyte chemoattractant protein 1 (MCP1) along with estrogen loss and recovery challenges in both fat pads (Fig. 4). Conversely, fluctuations in level of adipogenesis, lipogenesis, lipolysis and inflammation markers were found to vary in fat depots according to body localization i.e., subcutaneous or visceral (Fig. 3,4). Specifically, we surveyed Nr1c3 encoding peroxisome proliferator-activated receptor PPAR, Fabp4 respectively. On the one hand, no variations were observed in the level of Nr1c3, Pnpla2, Tnfα and Il12a mRNA in SAT across the groups, while the same genes were up-regulated in VAT in the Ovx group. Specifically, VAT samples from E2-replaced females had higher mRNA levels of Nr1c3 and Pnpla2 than samples from Ovx females which was consistent with PPARγ being a direct transcriptional regulator of ATGL [START_REF] Kershaw | PPARgamma regulates adipose triglyceride lipase in adipocytes in vitro and in vivo[END_REF] while inflammatory marker mRNA levels increased in Ovx females and returned to sham levels in E2-replaced females (Fig. 3,4). On the other hand, Fabp4, Lipe and Lpl had a profile of gene expression consistent with estrogens negatively regulating these genes in SAT but not in VAT samples (Fig. 3,4).

Regarding Il1β, the profile was identical to the other inflammatory markers in VAT while the mRNA levels were enhanced following E2 replacement in SAT.

Estrogens differentially impact glucose and lipid metabolism in the liver

Estrogens act in liver to positively regulate insulin sensitivity and glucose tolerance [START_REF] Mauvais-Jarvis | The role of estrogens in control of energy balance and glucose homeostasis[END_REF]. ERα being the primary estrogen receptor expressed in mouse liver [START_REF] Gao | Genome-wide identification of estrogen receptor alpha-binding sites in mouse liver[END_REF], we explored the hepatic expression of the Esr1 gene in ovariectomized females. We observed a significant decrease of Esr1 mRNA levels (Fig. 5A). Interestingly, in E2-replaced Ovx mice a significant doubling in Esr1 mRNA levels was measured compared to those detected in sham mice. We next performed RT-qPCR analyses to discriminate, among genes regulating glucose and lipid metabolism, those under negative and positive estrogen regulation. Consistent with E2 positively regulating glucose tolerance, the expression levels of G6pc encoding the catalytic subunit of the gluconeogenic enzyme G6Pase (glucose-6 phosphatase) doubled in Ovx mice and was restored in E2-replaced females, relative to the level of sham mice (Fig. 5B).

Expression levels of Gck encoding Glucokinase, mirrored those of G6pc (Fig. 5B) consistent with an adaptive role of Gck in counteracting hyperinsulinemia and insulin resistance and with the reduction of glycaemia [START_REF] Winzell | Chronic glucokinase activation reduces glycaemia and improves glucose tolerance in high-fat diet fed mice[END_REF].

To complete the study and although we did not observe significant changes in hepatic TG and cholesterol levels in ovariectomized mice (Table 1), we undertook a comprehensive analysis of genes encoding proteins involved in hepatic lipid metabolism. Specifically, genes surveyed included Nr1h3 encoding Liver X receptor (LXR)α and its downstream target gene Srebf1 hydroxylase which is the rate-limiting enzyme in the synthesis of bile acids [START_REF] Wagner | Nuclear receptors in liver disease[END_REF].

On this panel of genes, Nr1h4 was unique in that the mRNA level was enhanced by estrogen withdrawal compared to sham mice. There was reduction in mRNA abundance of Srebf1, Dgat2 and Ldlr in the Ovx mice (Fig. 5). All returned to sham levels after E2 replacement.

Lower Nr1c1 and Cd36 mRNA levels were found in E2 replaced mice than in sham mice (Fig. 5); mRNA levels of Nr0b2 encoding small heterodimeric partner (SHP) and those of Cpt1α, Nr1h3, Scd1 remained the same between the different groups (data not shown). In addition, several genes belonging to the cholesterol metabolism pathway had their mRNA levels strongly increased following E2 supplementation. This included Srebf2 and the target genes Hmgcr, Pcsk9 and Cyp7a1 (Fig. 5E).

A low-dose mixture of pollutants differentially impacted the expression levels of E2regulated genes depending on the metabolic tissue explored

The expression levels of estrogen receptors and estrogen-regulated genes were analyzed in the liver and adipose tissues of mice exposed to a mixture of low-dose pollutants previously shown to exhibit estrogeno-mimetic activities [START_REF] Naville | Low-dose food contaminants trigger sex-specific, hepatic metabolic changes in the progeny of obese mice[END_REF][START_REF] Naville | Metabolic outcome of female mice exposed to a mixture of low-dose pollutants in a diet-induced obesity model[END_REF][START_REF] Julien | Evidence for estrogeno-mimetic effects of a mixture of low-dose pollutants in a model of ovariectomized mice[END_REF].

Metabolic exploration revealed that exposure to pollutants did not change food intake (not shown), body weight and fat pad weight of the exposed animals (Supplemental Figure 6). No effect of pollutants was observed on blood glucose and plasma insulin levels, HOMA-IR, glucose tolerance, as well as on TG (plasma and hepatic) and FFA levels (Table 1).

Notwithstanding, cholesterol (plasma and hepatic) and leptin levels were significantly increased in HFp-Ovx females compared to HF0 sham mice while these parameters were not significantly changed by ovariectomy itself arguing for a pollutant effect. Moreover, liver TG accumulation in HFp Ovx mice was 1.5-fold higher than in HF0 Ovx mice although it did not reach significance. In addition, its level significantly decreased following E2 replacement (HFp Ovx+E2 versus HFp Ovx) which was not observed in pollutant-free conditions (Table 1). Adiponectin levels were significantly different in HFp Ovx+E2 versus levels measured in HF0-Ovx+E2 mice which also indicated a pollutant effect (Table 1).

Regarding gene expression, exposure to pollutants resulted in the increase of Esr1 mRNA levels in SAT and in the ratio of Esr1 to Nr3c4 expression in SAT and VAT of Ovx mice (Fig. 6). In conditions of E2 supplementation, effects of pollutant exposure included the augmentation of Esr2 mRNA levels in both fat pads as well as Esr1 /Nr3c4 ratio in SAT (HFp Ovx+E2 versus HF0 Ovx+E2) (Fig. 6). Among genes involved in cholesterol metabolism, we observed a significant doubling of Nr1h3, Nr0b2 and Cd36 in pollutant-exposed Ovx mice compared to non-exposed Ovx mice. Interestingly, in E2-replaced females, pollutant exposure triggered significant changes in Cyp7a1 (half-decrease) and Nr1h3 (a doubling) mRNA levels (Fig. 6C). These pollutant-induced effects were different than those exerted by E2 replacement (Fig. 4).

To complete the study, we measured the expression levels of genes encoding xenobiotic receptors including the aryl hydrocarbon receptor (AhR), the constitutive androstane receptor CAR (Nr1i3) and the pregnane xenobiotic receptor PXR (Nr1i2) as well as CAR and PXR immediate target genes which encode the cytochrome P450 enzymes CYP2B10 and CYP3A11 [START_REF] Aleksunes | Coordinated regulation of hepatic phase I and II drug-metabolizing genes and transporters using AhR-, CAR-, PXR-, PPARalpha-, and Nrf2-null mice. Drug metabolism and disposition: the biological fate of chemicals[END_REF]. These genes were not found impacted by the pollutant mixture as compared to non-exposed mice except Cyp2b10 which mRNA abundance slightly increased (Supplemental figure 7).

Discussion

In the present study, we developed a model allowing the exploration of the metabolic consequences of ovarian withdrawal and E2 replacement on liver and adipose tissues along with investigating the impact of a low-dose mixture of pollutants previously reported to exhibit some estrogeno-mimetic properties in female mice [START_REF] Naville | Low-dose food contaminants trigger sex-specific, hepatic metabolic changes in the progeny of obese mice[END_REF][START_REF] Naville | Metabolic outcome of female mice exposed to a mixture of low-dose pollutants in a diet-induced obesity model[END_REF]. Because of differences in the literature regarding the extent of the ovariectomy-induced deleterious metabolic effects, the experimental model developed herein was first characterized. Accordingly [START_REF] Riant | Estrogens protect against high-fat dietinduced insulin resistance and glucose intolerance in mice[END_REF][START_REF] Barros | Estrogen receptors: new players in diabetes mellitus[END_REF][START_REF] Camporez | Cellular mechanism by which estradiol protects female ovariectomized mice from high-fat diet-induced hepatic and muscle insulin resistance[END_REF][START_REF] Lavoie | Dynamics of hepatic and intestinal cholesterol and bile acid pathways: The impact of the animal model of estrogen deficiency and exercise training[END_REF][START_REF] Mauvais-Jarvis | Menopausal Hormone Therapy and Type 2 Diabetes Prevention: Evidence, Mechanisms, and Clinical Implications[END_REF],

ovariectomy induced body weight increase, glucose intolerance and insulin resistance and tended toward an increase of leptin and insulin plasma levels. No changes in TG plasma levels and cholesterol or in hepatic lipid deposition could be measured in the ovariectomized mouse model.

Importantly, treatment of ovariectomized mice with E2 either reversed the ovariectomyinduced adverse effects (e.g., glucose tolerance) to sham levels or improved metabolic functions over the physiological levels (e.g., adipose tissue weight and leptin levels). It suggested that diet-induced obesity could impair estrogenic signaling in the adipose tissues in female mice and that part of the HF effects were counter-balanced by E2 supplementation, extending previous data [START_REF] Bryzgalova | Mechanisms of antidiabetogenic and body weight-lowering effects of estrogen in high-fat diet-fed mice[END_REF]. Importantly, while ovariectomy and E2 replacement similarly regulated the ERs and AR encoding mRNA expression in SAT and VAT depots, adipogenesis and inflammatory markers were differently regulated in response to estrogen withdrawal and E2 replacement. Specifically, our findings were consistent with estrogens negatively regulating adipogenesis in SAT but not in VAT, and inflammation in VAT but not in SAT. In addition, enhanced Nr1c3 and Atgl mRNA expression in VAT, two markers of adipocyte differentiation, suggested a positive effect of E2-treatment on this tissue in Ovx mice.

Extending the literature [START_REF] Maury | Adipokine dysregulation, adipose tissue inflammation and metabolic syndrome[END_REF][START_REF] Frank | Determinants of body fat distribution in humans may provide insight about obesity-related health risks[END_REF], the herein findings underlined distinct functions of SAT and VAT in the development of obesity-induced metabolic disorders in females, with estrogens regulating fat distribution and expansion preferably in subcutaneous depots and opposing inflammation particularly in VAT thus protecting from insulin resistance. Noticeably, the mRNA abundance changes of the estrogen receptors induced by ovariectomy and E2 replacement emphasized that, in physiological conditions, circulating estrogens could regulate

Gper1 and Esr2 at a transcriptional level. In addition, if considering the reported phenotypes of enhanced adiposity in Esr2-deficient female mice [START_REF] Foryst-Ludwig | Metabolic impact of estrogen signalling through ERalpha and ERbeta[END_REF] and the protection from diet-induced obesity in Gper1-deficient female mice [START_REF] Wang | GPR30 regulates diet-induced adiposity in female mice and adipogenesis in vitro[END_REF], the alterations (herein observed) of Gper1 and Esr2 expression in the adipose tissues could well be linked to the obesity phenotype of the Ovx mice. The E2 replacement data suggested that the up-regulation of Esr1 and Nr3c4 expression in both fat pads and the restored Esr2 and Gper1 mRNA abundance (at least in SAT) could be linked to the observed decreased in adiposity in line with previous data [START_REF] Barros | Estrogen receptors: new players in diabetes mellitus[END_REF][START_REF] Foryst-Ludwig | Metabolic impact of estrogen signalling through ERalpha and ERbeta[END_REF][START_REF] Fagman | The androgen receptor confers protection against diet-induced atherosclerosis, obesity, and dyslipidemia in female mice[END_REF]. Moreover, the data presented herein indicated that the described estrogen antiinflammatory properties [START_REF] Barros | Estrogen receptors: new players in diabetes mellitus[END_REF][START_REF] Foryst-Ludwig | Metabolic impact of estrogen signalling through ERalpha and ERbeta[END_REF] could be mediated through ERß in VAT extending previous reports in female mice [START_REF] Davis | The sexually dimorphic role of adipose and adipocyte estrogen receptors in modulating adipose tissue expansion, inflammation, and fibrosis[END_REF]. Interestingly, SAT and VAT share identically regulated genes including Ccl2 (in line with previous findings [START_REF] Julien | Evidence for estrogeno-mimetic effects of a mixture of low-dose pollutants in a model of ovariectomized mice[END_REF] ) and Lep which could be positively regulated by estradiol in adipose tissues via Gper1or negatively via ERß. Such regulations remain to be demonstrated.

Within the present study, ovariectomy clearly impacted glucose metabolism but not hepatic steatosis while E2 replacement resulted in altered cholesterol metabolism concurrently with marked changes in liver Esr1, Nr1c1 and Cd36 mRNA abundance linking estrogen signaling to these effects. It could indicate that impairment of glucose metabolism might be an early metabolic consequence of estrogen loss while alteration of lipid metabolism could be indirectly affected. This hypothesis is consistent with the phenotype of hepatosteatosis observed after a long-term ovariectomy [START_REF] Villa | Tetradian oscillation of estrogen receptor alpha is necessary to prevent liver lipid deposition[END_REF] but not in the present study. The finding that TG accumulation did not change with ovariectomy is in agreement with the observation that the mRNA levels of FXR, a protein known to oppose hepatic steatosis and hyperlipidemia [START_REF] Cave | Nuclear receptors and nonalcoholic fatty liver disease[END_REF],

was increased while the abundance of genes related to lipogenesis (Srebpf1, Dgat2) was reduced. Noticeably, the impairment of cholesterol metabolism observed following E2 supplementation extended previous data demonstrating that Esr1 overexpression could trigger a loss in the fine tuning of negative feedback regulation of cholesterol synthesis [START_REF] Wang | Overexpression of estrogen receptor alpha increases hepatic cholesterogenesis, leading to biliary hypersecretion in mice[END_REF].

Deregulation of lipid metabolism may as well result from the lack of cycles in E2-replaced females since it has been demonstrated that reproduction and lipid metabolism are tightly coupled in cycling females [START_REF] Villa | Tetradian oscillation of estrogen receptor alpha is necessary to prevent liver lipid deposition[END_REF]. Whether changes in the mRNA levels of Esr1, Nr1c1 and of Cd36 in the E2-replaced Ovx mice also resulted from a loss in feed-back regulatory mechanism will await further studies. Collectively, it could be concluded that regulation of glucose metabolism is tightly controlled by estradiol but that lipid metabolism escaped physiological regulatory feedback in E2-treated females. In addition, because ovaries produce progestogens as well as androgens, E2 replacement is not expected to normalize all disorders induced by ovariectomy.

The use of a model of estrogen withdrawal and replacement has allowed the identification of a set of estrogen-regulated genes. We expected these genes to exhibit changes in mRNA levels in response to chronic exposure to a mixture of archetypal endocrine disruptors previously identified as bearing some estrogeno-mimetic activities as well as metabolic disrupting effects [START_REF] Naville | Low-dose food contaminants trigger sex-specific, hepatic metabolic changes in the progeny of obese mice[END_REF][START_REF] Naville | Metabolic outcome of female mice exposed to a mixture of low-dose pollutants in a diet-induced obesity model[END_REF][START_REF] Naville | Chronic exposure to a pollutant mixture at low doses led to tissue-specific metabolic alterations in male mice fed standard and highfat high-sucrose diet[END_REF]. Specifically, the protocol used herein was adapted from the published previous model [START_REF] Naville | Low-dose food contaminants trigger sex-specific, hepatic metabolic changes in the progeny of obese mice[END_REF][START_REF] Labaronne | Low-dose pollutant mixture triggers metabolic disturbances in female mice leading to common and specific features as compared to a high-fat diet[END_REF] except that female mice were not exposed to the mixture of pollutants from fetal life onwards but continuously from week 5 to week 20 through diet. As reviewed previously [START_REF] Magueresse-Battistoni | Endocrine disrupting chemicals in mixture and obesity, diabetes and related metabolic disorders[END_REF][START_REF] Magueresse-Battistoni | Lifelong consumption of low-dosed food pollutants and metabolic health[END_REF][START_REF] Magueresse-Battistoni | Environmental Pollutants and Metabolic Disorders: The Multi-Exposure Scenario of Life[END_REF], the mixture of pollutants was designed to question the metabolic health consequences of a multi-exposure scenario to various pollutants at low dosage to approach a real life situation. Specifically, the mixture was made of shortly metabolized (BPA, DEHP) and persistent pollutants (TCDD, PCB153) contaminating food either through bioaccumulation along the food chain (TCDD, PCB153) or leaching from packaging (BPA, DEHP), activating a wide range of signaling molecules, not to mention the many cross-talks occurring between them and triggering estrogeno-mimetic and anti-androgen activities among others [START_REF] Casals-Casas | Endocrine disruptors: from endocrine to metabolic disruption[END_REF][START_REF] Thayer | Role of environmental chemicals in diabetes and obesity: a National Toxicology Program workshop review[END_REF][START_REF] Vandenberg | Hormones and endocrine-disrupting chemicals: low-dose effects and nonmonotonic dose responses[END_REF]. Pollutants constituent parts of the mixture were also chosen because of high concern for links with metabolic diseases in epidemiological and experimental studies [START_REF] Casals-Casas | Endocrine disruptors: from endocrine to metabolic disruption[END_REF][START_REF] Magueresse-Battistoni | Endocrine disrupting chemicals in mixture and obesity, diabetes and related metabolic disorders[END_REF][START_REF] Gore | EDC-2: The Endocrine Society's Second Scientific Statement on Endocrine-Disrupting Chemicals[END_REF][START_REF] Alonso-Magdalena | Endocrine disruptors in the etiology of type 2 diabetes mellitus[END_REF][START_REF] Heindel | Metabolism disrupting chemicals and metabolic disorders[END_REF]. Interestingly, we herein observed modest but significant effects in the set-up model of Ovx and E2 replacement in response to the mixture of pollutants. Phenotypically, and in agreement with our previous studies [START_REF] Naville | Low-dose food contaminants trigger sex-specific, hepatic metabolic changes in the progeny of obese mice[END_REF][START_REF] Naville | Metabolic outcome of female mice exposed to a mixture of low-dose pollutants in a diet-induced obesity model[END_REF][START_REF] Naville | Chronic exposure to a pollutant mixture at low doses led to tissue-specific metabolic alterations in male mice fed standard and highfat high-sucrose diet[END_REF], the mixture did not impact body weight. No changes were observed in glucose tolerance or insulin resistance in the mice exposed compared to those not exposed to the pollutant mixture.

Nonetheless, adiponectin levels were found to be lower in E2-replaced and pollutant-exposed mice than in E2-replaced and non-exposed females. This finding is indicative of an adverse hormonal and metabolic effect triggered by pollutant exposure since adiponectin is an adipokine with positive metabolic properties [START_REF] Turer | Adiponectin: mechanistic insights and clinical implications[END_REF]. In addition, TG accumulation in liver of HFp Ovx mice was trending upward compared to non-exposed Ovx mice. Some genes involved in lipid metabolism in the liver and which mRNA levels were not modified by ovariectomy, were found impacted in response to the mixture of pollutants in Ovx mice (i.e., HFp Ovx versus HF0 Ovx mice). All these findings indicated that the mixture of pollutants had some metabolic impact but did not exert estrogeno-mimetic activities at least on the set of hepatic genes studied. These data do not fully reproduce previous findings in which the same mixture of pollutants was found to enhance Esr1 gene expression in the liver of ovariectomized females and alleviate glucose intolerance [START_REF] Julien | Evidence for estrogeno-mimetic effects of a mixture of low-dose pollutants in a model of ovariectomized mice[END_REF]. One possible explanation resides in the timing of exposure to the mixture encompassing gestation and lactation in the previous protocol [START_REF] Julien | Evidence for estrogeno-mimetic effects of a mixture of low-dose pollutants in a model of ovariectomized mice[END_REF] but not in the present study. Indeed, the maternal period has been found to be a highly vulnerable period [START_REF] Barker | Fetal origins of adult disease: strength of effects and biological basis[END_REF][START_REF] Barouki | Developmental origins of noncommunicable disease: implications for research and public health[END_REF]. For example, it was demonstrated that hepatic metabolic gene expression in adult mice including Esr1 was strongly impacted in mice exposed in utero to BPA as compared to control mice [START_REF] Ilagan | Bisphenol-A exposure in utero programs a sexually dimorphic estrogenic state of hepatic metabolic gene expression[END_REF]. In addition, the vulnerability of the fetal period towards exposure to the presently used mixture of pollutants has been evidenced in male mice [START_REF] Naville | Chronic exposure to a pollutant mixture at low doses led to tissue-specific metabolic alterations in male mice fed standard and highfat high-sucrose diet[END_REF].

Importantly, we observed that the response to the mixture of pollutants was highly tissue-dependent extending our previous data [START_REF] Labaronne | Low-dose pollutant mixture triggers metabolic disturbances in female mice leading to common and specific features as compared to a high-fat diet[END_REF][START_REF] Naville | Chronic exposure to a pollutant mixture at low doses led to tissue-specific metabolic alterations in male mice fed standard and highfat high-sucrose diet[END_REF]. While we did not identify estrogenomimetic activities of the pollutant mixture in the liver, the situation was clearly distinct in SAT. Indeed, the estrogen receptors Esr1 and Esr2 were found regulated the same way by either the pollutant mixture or E2 supplementation. Conversely, the mixture of pollutants tended to oppose E2 effects in VAT with an up-regulation of Esr2 and Il12a mRNA abundance in E2-replaced mice. The meaning of these distinct effects depending on fat depots, remains to be investigated but may be linked to the distinct functions of the fat depots regarding adipogenesis and inflammation [START_REF] Maury | Adipokine dysregulation, adipose tissue inflammation and metabolic syndrome[END_REF][START_REF] Frank | Determinants of body fat distribution in humans may provide insight about obesity-related health risks[END_REF]. In addition, it should be reminded that the dosage of the mixture was based on the tolerable daily intake (TDI) doses defined empirically by risk assessment agencies, e.g., the European Food Safety Authority (EFSA) or the U.S.

Food and Drug administration (FDA) [START_REF] Dorne | Metabolism, variability and risk assessment[END_REF], thus not expected individually to generate a metabolic effect. Therefore, the herein demonstration that several genes were impacted (at least at the mRNA level) by a mixture of low-dose pollutants together with previous data on the adverse metabolic effects of low-dosed mixture [START_REF] Magueresse-Battistoni | Environmental Pollutants and Metabolic Disorders: The Multi-Exposure Scenario of Life[END_REF], emphasizes that current legislation on chemical risk assessment may underestimate the risk and should be updated.

In summary, analysis of female mice in conditions of withdrawal of estrogen, E2 replacement or exposure to a mixture of pollutants with reported estrogeno-mimetic activities, extended knowledge on the estrogenic regulation of glucose and lipid metabolism in the liver and of adipogenesis and inflammatory markers in the fat depots. Importantly, this model highlighted that the disrupting effect of the mixture of low-dosed pollutants which did not affect body weight, glycaemia, plasma insulin and lipidemia was different in the liver and adipose tissues.

Noticeably, alterations concerned adiponectin plasma levels, estrogen receptors in the adipose tissue and lipid metabolism in the liver. The present findings may have important implications for the understanding of the estrogen-dependent metabolic alterations with regards to situations of loss of estrogens as observed with menopause, especially if considering the worldwide context of exposure to chemicals. A significant overall effect was obtained using a Two-way ANOVA followed by the Bonferroni test. Standard: standard diet; HF0: pollutant-free high-fat high-sucrose diet; Sham: sham-operated mice; Ovx: ovariectomized mice; Ovx+E2: ovariectomized mice with E2 replacement. (γ) significant pollutant effect (using a one-way ANOVA followed by Tukey post hoc test).

HF0: pollutant-free high-fat high-sucrose diet; HFp: high-fat high-sucrose diet containing the mixture of pollutants; Sham: sham-operated mice; Ovx: ovariectomized mice; Ovx+E2: ovariectomized mice with E2 replacement.

  encoding fatty-acid binding protein4, Lipe encoding hormone-sensitive lipase (HSL), Lpl encoding lipoprotein lipase (LPL), Pnpla2 encoding Patatin Like Phospholipase Domain Containing 2 also known as Adipose triglyceride lipase (ATGL) and inflammatory cytokines, Tnfα, Il1β and Il12a encoding tumor necrosis factor (TNF)α and the interleukins 1ß and 12a,

  encoding sterol regulatory element binding protein 1c (SREBP1c), a master regulator of de novo lipogenesis; Scd1 encoding the stearoyl-CoA desaturase-1 (SCD1), a rate-limiting enzyme of lipogenesis; Dgat2 encoding diacylglycerol O-acyltransferase 2 which catalyzes the final reaction in the synthesis of triglycerides; Cd36 encoding Cluster of differentiation 36 involved in fatty acid uptake; Nr1c1encoding peroxisome proliferator-activated receptor (PPAR)α which regulates fatty acid oxidation and its target gene Cpt1a encoding carnitine palmitoyltransferase 1α; Srebf2 which encodes the transcription factor Sterol Regulatory Element Binding Transcription Factor 2 and its downstream target genes Hmgcr and Pcsk9 encoding 3-Hydroxy-3-Methylglutaryl-CoA Reductase, the rate limiting enzyme of cholesterol metabolism and Proprotein convertase subtilisin/kexin type 9 which regulates lipoprotein metabolism, respectively. We surveyed Ldlr encoding the low density lipoprotein receptor which mediates the endocytosis of cholesterol-rich LDL; Nr1h4 encoding the bile acid receptor Farnesoid X receptor (FXR), and Cyp7a1 encoding the cholesterol 7α

LegendsFigure 1 :

 1 Figure 1: (A) Body weight and weight of visceral and subcutaneous adipose tissues (VAT and SAT, respectively) measured at the end of the protocols (after 6 h of fasting). (B) Biochemical parameters: Blood glucose, plasma insulin and HOMA-IR. (C) Glucose Tolerance test performed after 13 wks of diet and corresponding AUC. (D) Measurement of whole body composition by NMR and of the adipocyte area. Values are means ± SEM with n=7-8. ***P<0.001; ** P<0.01; *P<0.05 (one-way ANOVA followed by Tukey post hoc test). For the adipocyte area, values are means ± SEM with n=3.

Figure 2 :

 2 Figure 2: Effect of ovariectomy alone (Ovx) or with 17β-estradiol (E2) supplementation (Ovx+E2) on the expression of genes encoding the estrogen receptors ERα, ERβ, GPR30 and the androgen receptor AR in subcutaneous (A) and visceral (B) adipose tissues (SAT and VAT, respectively). Values are means ± SEM with n=6-8. ***P<0.001; ** P<0.01; *P<0.05. Sham: sham -operated mice.

Figure 3 :

 3 Figure 3: Effect of ovariectomy alone (Ovx) or with 17β-estradiol (E2) supplementation (Ovx+E2) on the expression of genes related to adipogenesis and lipogenesis (A, A'), lipolysis (B, B') in subcutaneous (SAT, A, B) and visceral (VAT A', B') adipose tissues. Values are means ± SEM with n=6-8. ***P<0.001; ** P<0.01; *P<0.0; Sham: sham-operated mice.

Figure 4 :

 4 Figure 4: Effect of ovariectomy alone (Ovx) or with 17β-estradiol (E2) supplementation (Ovx+E2) on the expression of genes encoding inflammatory markers in subcutaneous (SAT) and visceral (VAT) adipose tissues.Values are means ± SEM with n=6-8. ***P<0.001; ** P<0.01; *P<0.0. Sham: sham-operated mice.

Figure 5 :

 5 Figure 5: Effect of ovariectomy alone (Ovx) or with 17β-estradiol (E2) supplementation (Ovx+E2) on the expression of genes encoding the nuclear estrogen receptor ERα (A), genes related to glucose metabolism (B), fatty acid metabolism (C), triglyceride metabolism (D), cholesterol metabolism (E) in liver. Values are means ± SEM with n=6-8. ***P<0.001; ** P<0.01; *P<0.05. Sham: sham-operated mice.

Figure 6 :

 6 Figure 6: Effect of the the mixture of pollutants on the expression of genes expressed in subcutaneous (SAT, A), visceral (VAT, B) adipose tissues, or liver (C). Values (expressed as fold-change relative to the HF0 group) are means ± SEM with n=6-8. ***P<0.001; ** P<0.01; *P<0.05. Ovx: ovariectomized mice; Ovx+E2: ovariectomized mice with E2 replacement. HF0: pollutant-free high-fat high-sucrose diet; HFp: high-fat high-sucrose diet containing the mixture of pollutants.

Figure 6 Supplemental Figure 2 :Supplemental Figure 3 :Supplemental Figure 4 :Supplemental Figure 5 :Supplemental Figure 6 : 1 :

 6234561 Figure 1

Table 1 :

 1 Biochemical parameters

		HF0 sham	HF0 Ovx	HF0 Ovx + E2	HFp Ovx	HFp Ovx + E2
	Plasma
	Total cholesterol (mg/mL)	1.54 ± 0.06	1.72 ± 0.08	1.32 ± 0.09 	1.84 ± 0.08	1.16 ± 0.17 
	Cholesteryl esters (mg/mL)	1.17 ± 0.04	1.31 ± 0.06	1.02 ± 0.07 	1.38 ± 0.07	0.87 ± 0.12 
	Triglycerides (mM)	0.70 ± 0.04	0.63 ± 0.03	0.62 ± 0.10	0.73 ± 0.06	0.46 ± 0.05 
	Free Fatty Acids (µM)	325 ± 36	500 ± 43	α	352 ± 44 	468 ± 50	281 ± 39 
	Leptin (ng/mL)	23.2 ± 3.8	32.3 ± 4.5	11.4 ± 1.2 	41.3 ± 4.0	12.1 ± 1.9 
	Adiponectin (µg/mL)	14.7 ± 0.4	15.1 ± 0.4	12.6 ± 0.6  , 	15.0 ± 0.8	9.82 ± 0.63 
	Liver
	Cholesterol (µg/mg)	3.19 ± 0.13	3.59 ± 0.40	3.70 ± 0.21	3.67 ± 0.15	3.27 ± 0.16
	Triglycerides (nmol/mg)	18.6 ± 3.6	25.8 ± 4.8	15.6 ± 1.4	38.6 ± 8.1	14.8 ± 1.7



Values are means ± SEM with n=6-8. (α): significant effect vs HF0 sham; (β) significant E2 effect;

Acknowledgments

We gratefully acknowledge Emmanuelle Loizon for technical help with PCR primers, Patrick Manas for animal care, Véronique Coxam and Patrice Lebecque for ovariectomy training and Sakina Mhaouty-Kodja for providing us with the E2 pellets. The authors wish to thank Dr Alexandrine Derrien-Colemyn for critically reading the manuscript.

This research project was supported by INSERM to InsermU1060. Benoit

Julien is supported by a research fellowship from the French "Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche". The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Conflict of interest

The authors have declared that no competing interests exist.

Supplemental Figures

Supplemental Figure 1: Experimental protocols. ST: standard chow diet; HF: high-fat highsucrose diet; HF0: pollutant-free high-fat high-sucrose diet; HFp: high-fat high-sucrose diet containing the mixture of pollutants. Sham: sham-operated mice; Ovx: ovariectomized mice; Ovx+E2: ovariectomized mice with E2 replacement.