Simon Lecoutre

Charlène Pourpe

Laura Butruille

Lucie Marousez

Christine Laborie

Céline Guinez

Jean J Lesage

Didier D Vieau

Jerome Eeckhoute

Anne Gabory

Jérôme Eeckhoute

Frederik Oger

Delphine Eberlé

Professor Christophe Breton
email: christophe.breton@univ-lille1.fr

Reduced PPARγ2 expression in adipose tissue of male rat offspring from obese dams is associated with epigenetic modifications

Keywords: perinatal programming, Developmental Origin of Health and Disease, epigenetic mechanisms, gene expression, fat expansion

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Introduction

Epidemiological studies demonstrated that adult-onset metabolic disorders may originate from events taking place during fetal and postnatal development [START_REF] Oestreich | Developmental and Transmittable Origins of Obesity-Associated Health Disorders[END_REF]. These observations led to the Developmental Origin of Health and Disease (DOHaD) hypothesis also called "developmental programming" [START_REF] Oestreich | Developmental and Transmittable Origins of Obesity-Associated Health Disorders[END_REF]. In particular, maternal obesity and accelerated growth in neonates program obesity later in life. Numerous studies confirmed that maternal obesity is correlated with increased offspring's adiposity in rodents [START_REF] Lecoutre | Depot-and sexspecific effects of maternal obesity in offspring's adipose tissue[END_REF][START_REF] Liang | Maternal obesity epigenetically alters visceral fat progenitor cell properties in male offspring mice[END_REF][START_REF] Shankar | Maternal obesity at conception programs obesity in the offspring[END_REF][START_REF] Borengasser | Maternal obesity enhances white adipose tissue differentiation and alters genome-scale DNA methylation in male rat offspring[END_REF][START_REF] Zambrano | Dietary intervention prior to pregnancy reverses metabolic programming in male offspring of obese rats[END_REF][START_REF] Desai | Maternal obesity and high-fat diet program offspring metabolic syndrome[END_REF] and in human neonates [START_REF] Modi | The influence of maternal body mass index on infant adiposity and hepatic lipid content[END_REF]. Thus, white adipose tissue (WAT) has been the focus of developmental programming events in a sex-and depot-specific manner [START_REF] Lecoutre | Depot-and sexspecific effects of maternal obesity in offspring's adipose tissue[END_REF]. In rodents, adipose tissue development occurs mainly during lactation [START_REF] Greenwood | Postnatal development of adipocyte cellularity in the normal rat[END_REF][START_REF] Birsoy | Analysis of gene networks in white adipose tissue development reveals a role for ETS2 in adipogenesis[END_REF][START_REF] Han | The spatiotemporal development of adipose tissue[END_REF], although adipose tissue remains to some extent expandable throughout life [START_REF] Joe | Depotspecific differences in adipogenic progenitor abundance and proliferative response to high-fat diet[END_REF][START_REF] Spalding | Dynamics of fat cell turnover in humans[END_REF]. Adipocyte progenitors are highly sensitive to maternal factors. Suboptimal nutritional and hormonal milieu during the perinatal period may affect adipogenesis, resulting in modified growth and persistent changes in the structure and function of WAT [START_REF] Lecoutre | Maternal nutritional manipulations program adipose tissue dysfunction in offspring[END_REF][START_REF] Lecoutre | The cellularity of offspring's adipose tissue is programmed by maternal nutritional manipulations[END_REF][START_REF] Lukaszewski | Nutritional manipulations in the perinatal period program adipose tissue in offspring[END_REF].

However, the mechanisms underlying developmental programming are poorly understood. Because the consequences of the events occurring in early life are maintained throughout adult life, epigenetic modifications are likely to play an important role in this process [START_REF] Gluckman | Effect of in utero and early-life conditions on adult health and disease[END_REF]. Epigenetics is defined as heritable changes in gene expression that do not involve changes to the underlying DNA sequence. Epigenetics relate to chromatin modifications, including DNA methylation and histone modifications [START_REF] Kouzarides | Chromatin modifications and their function[END_REF]. DNA methylation results from the transfer by DNA methyltransferase (DNMT) of a methyl group to the cytosine residue within CpG dinucleotides to form 5-methylcytosine (5mC) and serves to establish gene silencing [START_REF] Jones | The role of DNA methylation in mammalian epigenetics[END_REF]. 5mC both positively and negatively modulates binding of numerous DNA binding proteins including methyl-binding domain (MBD) proteins (MBD2, MeCP2). MBD can recruit protein and chromatin remodeling complexes known to promote the formation of a condensed chromatin structure [START_REF] Medvedeva | Effects of cytosine methylation on transcription factor binding sites[END_REF]. 5-hydroxymethylcytosine (5hmC) is another important cytosine modification catalyzed by the enzymes of the Ten-eleven translocation methylcytosine dioxygenase (TET) family that serves as an intermediate for demethylation. It can also promote chromatin opening at transcriptional regulatory regions [START_REF] Tahiliani | Conversion of 5methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1[END_REF]. Chromatin structure and accessibility are also controlled by histone posttranslational modifications [START_REF] Kouzarides | Chromatin modifications and their function[END_REF]. For example, acetylation of histone H3 lysine residues (H3ac) and methylation of H3K4 (H3K4me3) are associated with active transcription while methylation of H3K9 (H3K9me3) generally indicates silenced chromatin [START_REF] Kouzarides | Chromatin modifications and their function[END_REF]. These histone posttranslational modifications are catalyzed by various enzymes including histone acetyltransferase (HAT) such as KAT2 and P300 as well as histone deacetylase (HDAC) such as SIRT1. Histone-modifying enzyme activity is dependent on intermediary metabolites and hormonal responses [START_REF] Öst | Epigenetic modulation of metabolic decisions[END_REF]. Editing of epigenetic marks during critical developmental windows in early life may serve as a memory of exposure to potential environment insults [START_REF] Oestreich | Developmental and Transmittable Origins of Obesity-Associated Health Disorders[END_REF]. The persistence of these marks throughout life may program permanent changes in gene expression [START_REF] Oestreich | Developmental and Transmittable Origins of Obesity-Associated Health Disorders[END_REF].

We previously showed that adult male rat offspring from high-fat diet-fed dams (called HF) displayed higher adiposity (2), hyperleptinemia and elevated leptin gene expression in WAT [START_REF] Samuelsson | Diet-induced obesity in female mice leads to offspring hyperphagia, adiposity, hypertension, and insulin resistance: a novel murine model of developmental programming[END_REF]. Persistent leptin upregulation occurs via epigenetic malprogramming during lactation in a depot-specific manner [START_REF] Lecoutre | Maternal obesity programs increased leptin gene expression in rat male offspring via epigenetic modifications in a depot-specific manner[END_REF]. We also showed that adult HF offspring exhibited a decrease in PPAR gene expression in perirenal (pWAT) fat pad [START_REF] Lecoutre | Depot-and sexspecific effects of maternal obesity in offspring's adipose tissue[END_REF]. PPARis considered the master regulator of adipose tissue promoting all processes involved in adipocyte function and WAT expansion. PPAR is present in two isoforms, PPAR1 and PPAR2, generated by alternative promoter usage [START_REF] Tontonoz | Fat and Beyond: The Diverse Biology of PPARγ[END_REF]. PPAR1 is expressed at low level in many cell types including cells from stromal vascular fraction (SVF) of adipose tissue, whereas the expression of PPAR2 is mainly restricted to adipocyte [START_REF] Tontonoz | Fat and Beyond: The Diverse Biology of PPARγ[END_REF]. Although several studies reported dysregulation of PPAR expression in offspring from obese dams (3-5, 7, 25, 26), scarce data is available about programming mechanisms.

Here, we hypothesized that maternal obesity programs persistent PPARγ downregulation in HF offspring via epigenetic mechanisms malprogramming which take places during WAT development. Thus, we examined epigenetic modifications (i.e., DNA and histone modifications) focusing on both PPARγ promoters as well as activity and expression of chromatin modifying enzymes in pWAT and inguinal (iWAT) fat pads of HF offspring at weaning and 9 months of age.

Materials and methods

Animals

Animal use accreditation by the French Ministry of Agriculture (No. 04860) has been granted to our laboratory for experimentation with rats. Experiments were conducted in accordance with the principles of laboratory animal care (European Communities Council Directive of 1986, 86/609/EEC) as previously described [START_REF] Lecoutre | Depot-and sexspecific effects of maternal obesity in offspring's adipose tissue[END_REF][START_REF] Samuelsson | Diet-induced obesity in female mice leads to offspring hyperphagia, adiposity, hypertension, and insulin resistance: a novel murine model of developmental programming[END_REF]. One-month-old virgin female Wistar rats were purchased from Charles River Laboratories (L'Arbresle, France) and were housed in individual cages in a humidity-controlled room with a 12:12-h light-dark cycle.

Food and water were available ad libitum. After two weeks of acclimatization on a control (C) diet (3.85 kcal/g with 10% of total calories as fat consisting of soybean oil (5.6%) and lard (4.4%), 70% as carbohydrate and 20% as protein; D12450J, Research Diets, New Brunswick, NJ, USA), female rats were fed either a high-fat (HF) diet (5.24 kcal/g with 60% of total calories as fat consisting of soybean oil (5.6%) and lard (54.4%), 20% as carbohydrate and 20% as protein; D12492B, Research Diets, New Brunswick, NJ, USA) or a C diet for 16 weeks (n=30 per group). After mating with a male rat fed a C diet, 22-week-old pregnant females were transferred into individual cages with free access to water and to their respective diets (C or HF diet) throughout gestation and lactation. At parturition, pups were weighed and sexed. Litter size was adjusted to 8 pups per dam (four males and four females). Body weight of pups was assessed on postnatal days 21 (PND21). Only male offspring have been used for further analysis. After weaning, male offspring from C or HF dams were housed individually with free access to water and C diet. Body weight of animals was measured weekly until 9 months of age. Male offspring were sacrificed at 2 different stages: PND21 and 9 months of age (n=10 from C or HF dams). To obviate any litter effects, animals used for each experiment were randomly chosen in different litters and only a limited number of animals (n= 1 to 2) was used from each litter.

Plasma and tissue collections

21-day-old male neonates were separated from their dams and 9-month-old (9M) male rats were fasted 16 hours before they were sacrificed by decapitation (between 9 and 10 a.m.).

Trunk blood samples were collected into prechilled tubes containing EDTA (20 μl of a 5% solution) and centrifuged at 4000 g for 10 min at 4°C. Plasma was stored at -20°C. Fat pads were weighed, frozen in liquid nitrogen and stored at -80°C.

Quantification of endocrine parameters

Plasma hormone levels were evaluated in PND21 and 9M male offspring. Blood glucose was determined using a glucometer (Glucotrend 2; Roche Diagnostics, France).

Plasma corticosterone levels were determined by a competitive enzyme immunoassay (Immunodiagnostic Systems Ltd, Boldon, U.K). Plasma insulin concentrations were measured by ELISA (DRG, Internatlukaional, Inc. USA). The assay sensitivity was 0.07 ng/ml (insulin), 0.55 ng/ml (corticosterone) and the intra-and inter-assay coefficients of variation were 4% and 9.1% (insulin), 4.9% and 7.8% (corticosterone), respectively. Assay kits were applied to determine the contents of plasma triglycerides (61238 Triglyceride Enzymatique PAP100).

Western blot analysis

Immunoblotting analysis were carried out as described previously [START_REF] Lecoutre | Maternal obesity programs increased leptin gene expression in rat male offspring via epigenetic modifications in a depot-specific manner[END_REF]. Monoclonal mouse antibody against PPAR (catalogue no. sc-7273) was purchased from Santa Cruz Biotech (Dallas, TX, USA) and was diluted 1:200. Actin antibody (catalogue no. A2228) was purchased from Sigma Aldrich (Saint Quentin Fallavier, France) and was diluted 1:1000.

Anti-mouse IgG, HRP-linked Antibody (catalogue no. 7076) secondary antibodies were purchased from Cell Signalling (Danvers, MA, USA) and were diluted 1:10,000.

Pyrosequencing analysis

Genomic DNA was extracted from frozen WAT using a DNA extraction kit (DNeasy blood and tissue kit, Qiagen, Courtaboeuf, France) and modified with sodium bisulfite using the EpiTect Fast Bisulfite Conversion kit (Qiagen, Courtaboeuf, France) according to the manufacturer's protocol. The percentage of cytosine methylation was determined by pyrosequencing bisulfite converted DNA using Pyro-Mark Q24 (Qiagen, Courtaboeuf, France). Pyrosequencing primers were designed to amplify PPARand PPARCpG sites (Table 1). PCR was performed with 20 l final reaction volumes with 1.5 ml of bisulfite DNA (10 ng), 10 l of QuantiTect EurobioGreen PCR Mix Hi-ROX (Eurobio, Les Ulis, France), 7.7 l of H 2 O, and 0.4 l of each primer set (10 mM). Sequencing reactions used 10 l of PCR product and 20 l of 0.375 mM sequencing primer (Qiagen, Courtaboeuf, France).

Pyrosequencing assays were validated with a DNA methylation scale (0%, 5%, 25%, 50%, 75% and 100%) (EpigenDX, Hopkinton, USA). Each assay included a bisulfite conversion check to verify full conversion of the DNA.

Chromatin immunoprecipitation

ChIP assays were performed as previously described [START_REF] Oger | Peroxisome proliferatoractivated receptor α regulates genes involved in insulin/insulin-like growth factor signaling and lipid metabolism during adipogenesis through functionally distinct enhancer classes[END_REF] with antibodies against H3K4me3, H3ac and H3K9me3 (Abcam, Cambridge, UK, ab8580, ab47915 and ab1773) and IgG (Cell Signaling Technology, Danvers, USA, #2729) as a negative control. H3ac, H3K4me3 and H3K9me3 levels were analyzed using immunoprecipitated and input DNA as control.

RNA isolation and real-time PCR

PPARγ isoforms (PPARγ1, PPARγ2), PPARγ targets (C/EBP, SREBP1, FAS, DGAT2, LPL, CD36, FABP4, GLUT4, ACOX1, AdipoQ, Resistin), enzymes of the epigenetic machinery (DNMT1, MBD2, MECP2, TET2, TET3, KAT2, P300, HDAC1, SIRT1, LSD1, SUV39) mRNA levels were determined by RT-qPCR using SyberGreen-based chemistry as previously described [START_REF] Lecoutre | Depot-and sexspecific effects of maternal obesity in offspring's adipose tissue[END_REF]. Cyclophilin A (PpiA) and Ribosomal Protein Lateral Stalk Subunit P1 (RPLP1) were used as reference genes for RT-qPCR. Primer sequences are listed in Table 1.

Global DNA methylation and hydroxymethylation

Relative levels of global DNA methylation were assessed by me-CpG ELISA using the MethylFlashTM Methylated DNA Quantification Kit (Cat # P-1034; Epigentek, Mundolsheim, France). Global DNA hydroxymethylation were measured using the Quest 5-hmC™ DNA ELISA Kit (Zymo Research, Irvine, CA, USA) according to manufacturer's instructions. The amount of DNA methylation and DNA hydroxymethylation was calculated based on a standard curve generated using the kit controls.

Enzyme activity

DNMT, TET, HAT and HDAC activities were studied using the EpiQuick DNMT Assay kit (Cat # P-3001-2; Epigentek, Mundolsheim, France), TET Hydroxylase Activity Quantification kit (Cat # ab156912; Abcam, Cambridge, UK), EpiQuick HAT Assay kit (Cat # P-4003-96; Epigentek, Mundolsheim, France) and EpiQuick HDAT Assay kit (Cat # P-4002-96; Epigentek, Mundolsheim, France). These assays were performed according to manufacturer's instructions.

Statistical analysis

All data are expressed as means ± standard error of the mean (SEM). Statistical analysis was carried out using GraphPad Prism6. A direct comparison between a pair of groups was made using an unpaired Student's t test. P values < 0.05 was considered statistically significant.

Results

Effects of maternal obesity on HF offspring metabolic parameters

Given that metabolic signals including hormonal signaling pathways play critical roles in determining chromatin structure [START_REF] Lu | Metabolic regulation of epigenetics[END_REF], we assessed the plasma metabolic profile in HF offspring. We previously reported that weaned and adult HF offspring exhibited hyperleptinemia [START_REF] Lecoutre | Depot-and sexspecific effects of maternal obesity in offspring's adipose tissue[END_REF][START_REF] Lecoutre | Maternal obesity programs increased leptin gene expression in rat male offspring via epigenetic modifications in a depot-specific manner[END_REF]. Here, we show that serum triacylglycerol and insulin levels were elevated in HF male offspring at PND21 (Table 2). At 9 months of age, HF male offspring still displayed hyperinsulinemia and had hypercorticosteronemia [START_REF] Lecoutre | Depot-and sexspecific effects of maternal obesity in offspring's adipose tissue[END_REF]. No difference in plasma glucose concentration was observed at both stages (Table 2,

Discussion

Here, we show, for the first time, that persistent decreased mRNA PPAR2 levels in adult HF offspring's WAT may occur via epigenomic remodeling in the PPARγ2 promoter which take place during lactation.

We extended our previous work [START_REF] Lecoutre | Depot-and sexspecific effects of maternal obesity in offspring's adipose tissue[END_REF] reporting that obesity-prone 9-month-old HF offspring had lower PPAR2 mRNA expression levels in both pWAT and iWAT fat pads.

Given that PPARγ2 promotes adipocyte differentiation and triglyceride storage in WAT [START_REF] Tontonoz | Fat and Beyond: The Diverse Biology of PPARγ[END_REF], it may appear paradoxical that maternal obesity reduces PPARγ2 expression in WAT of HF offspring. However, others have also shown that obesity is associated with a decline in the PPARγ2 activity and expression [START_REF] Guilherme | Adipocyte dysfunctions linking obesity to insulin resistance and type 2 diabetes[END_REF][START_REF] Soccio | Targeting PPARγ in the epigenome rescues genetic metabolic defects in mice[END_REF][START_REF] Zhang | Negative regulation of peroxisome proliferator-activated receptor-gamma gene expression contributes to the antiadipogenic effects of tumor necrosis factor-alpha[END_REF][START_REF] Fujiki | Expression of the peroxisome proliferator activated receptor gamma gene is repressed by DNA methylation in visceral adipose tissue of mouse models of diabetes[END_REF]. These modifications appear to be strongly associated with the pathogenesis of metabolic syndrome [START_REF] Soccio | Targeting PPARγ in the epigenome rescues genetic metabolic defects in mice[END_REF][START_REF] Zhang | Negative regulation of peroxisome proliferator-activated receptor-gamma gene expression contributes to the antiadipogenic effects of tumor necrosis factor-alpha[END_REF][START_REF] Fujiki | Expression of the peroxisome proliferator activated receptor gamma gene is repressed by DNA methylation in visceral adipose tissue of mouse models of diabetes[END_REF]. The decrease in PPARγ2 gene expression might be seen as an adaptive mechanism to prevent further fat accumulation in WAT [START_REF] Ortega | The gene expression of the main lipogenic enzymes is downregulated in visceral adipose tissue of obese subjects[END_REF]. It may, in turn, durably change the expression of PPARγ target genes.

Consistent with this hypothesis, recent findings have demonstrated that PPARγ downregulation in obese mice is associated with a decrease in genome-wide association occupancy of PPARγ [START_REF] Zhang | Negative regulation of peroxisome proliferator-activated receptor-gamma gene expression contributes to the antiadipogenic effects of tumor necrosis factor-alpha[END_REF].

We previously reported that HF neonates exhibited a rapid weight gain during lactation. At PND21, HF male offspring displayed higher iWAT and pWAT deposit weights with increased leptin mRNA levels, marked adipocyte hypertrophy and hyperplasia. HF 9-monthold male rats showed persistent increased pWAT mass with elevated leptin mRNA levels whereas this "expandable" phenotype was no longer observed in iWAT [START_REF] Lecoutre | Depot-and sexspecific effects of maternal obesity in offspring's adipose tissue[END_REF][START_REF] Lecoutre | Maternal obesity programs increased leptin gene expression in rat male offspring via epigenetic modifications in a depot-specific manner[END_REF]. By contrast, both fat pads had persistent lower PPAR expression. These varying outcomes may reflect difference in postnatal and depot-specific programming. Depot-specific adipogenesis is regulated by the anatomic location, the unique signature of developmental gene expression and the hormonal microenvironment of WAT [START_REF] Yan | Prenatal Polycyclic Aromatic Hydrocarbon, Adiposity, Peroxisome Proliferator-Activated Receptor (PPAR) γ Methylation in Offspring, Grand-Offspring Mice[END_REF]. PPAR governs biological processes by modifying the expression of a large number of target genes. As already reported [START_REF] Lecoutre | Depot-and sexspecific effects of maternal obesity in offspring's adipose tissue[END_REF][START_REF] Samuelsson | Diet-induced obesity in female mice leads to offspring hyperphagia, adiposity, hypertension, and insulin resistance: a novel murine model of developmental programming[END_REF], gene expression profiling revealed differences in adipogenic and lipogenic activities between iWAT and pWAT of adult HF offspring, suggesting a depot-specific programming. It may appear paradoxical that persistent reduced PPARγ2 expression was correlated with an increase in expression of genes associated with fat accumulation (i.e., FAS, DGAT2 and LPL) in pWAT. One possible explanation is that regulation of lipogenic genes in adult HF offspring's WAT may occur, at least in part, via additional PPAR-independent signaling pathways. Consistent with this hypothesis, HF adult male offspring exhibited hyperinsulinemia with elevated SREBP-1c mRNA levels within pWAT (2) that might be due for activation of insulin signaling. Indeed, offspring from obese dams showed greater AKT phosphorylation in response to insulin challenge associated with up-regulation of lipogenic pathways in WAT [START_REF] Borengasser | Maternal obesity enhances white adipose tissue differentiation and alters genome-scale DNA methylation in male rat offspring[END_REF].

The underlying molecular mechanisms regulating PPAR gene expression remain unclear. However, decreased PPARγ expression via modifications of epigenetic mechanisms may play a pivotal role in the development of obesity and diabetes [START_REF] Guilherme | Adipocyte dysfunctions linking obesity to insulin resistance and type 2 diabetes[END_REF][START_REF] Soccio | Targeting PPARγ in the epigenome rescues genetic metabolic defects in mice[END_REF][START_REF] Fujiki | Expression of the peroxisome proliferator activated receptor gamma gene is repressed by DNA methylation in visceral adipose tissue of mouse models of diabetes[END_REF][START_REF] Yan | Prenatal Polycyclic Aromatic Hydrocarbon, Adiposity, Peroxisome Proliferator-Activated Receptor (PPAR) γ Methylation in Offspring, Grand-Offspring Mice[END_REF][START_REF] Zwamborn | Prolonged high-fat diet induces gradual and fat depotspecific DNA methylation changes in adult mice[END_REF][START_REF] Zhang | Negative regulation of peroxisome proliferator-activated receptor-gamma gene expression contributes to the antiadipogenic effects of tumor necrosis factor-alpha[END_REF]. Few studies have shown that DNA methylation and histone modifications regulate the expression of PPARγ in WAT [START_REF] Fujiki | Expression of the peroxisome proliferator activated receptor gamma gene is repressed by DNA methylation in visceral adipose tissue of mouse models of diabetes[END_REF][START_REF] Yan | Prenatal Polycyclic Aromatic Hydrocarbon, Adiposity, Peroxisome Proliferator-Activated Receptor (PPAR) γ Methylation in Offspring, Grand-Offspring Mice[END_REF][START_REF] Zwamborn | Prolonged high-fat diet induces gradual and fat depotspecific DNA methylation changes in adult mice[END_REF][START_REF] Jiao | Protective effects of maternal methyl donor supplementation on adult offspring of high fat diet-fed dams[END_REF][START_REF] Kosaka | Human adipocytes from the subcutaneous superficial layer have greater adipogenic potential and lower PPAR-γ DNA methylation levels than deep layer adipocytes[END_REF][START_REF] Sun | Epigenetic DNA methylation in the promoters of Peroxisome Proliferator-Activated Receptor in chicken lines divergently selected for fatness[END_REF]. First, during adipogenesis, DNA demethylation [START_REF] Ortega | The gene expression of the main lipogenic enzymes is downregulated in visceral adipose tissue of obese subjects[END_REF] as well as enrichment of active marks H3ac [START_REF] Salma | Temporal Recruitment of Transcription Factors and SWI / SNF Chromatin-Remodeling Enzymes during Adipogenic Induction of the Peroxisome Proliferator-Activated Receptor ␥ Nuclear Hormone Receptor[END_REF] and H3K4me3 [START_REF] Cho | Histone methylation regulator PTIP is required for PPARgamma and C/EBPalpha expression and adipogenesis[END_REF][START_REF] Mikkelsen | Comparative epigenomic analysis of murine and human adipogenesis[END_REF] were observed in the PPARγ promoter region that positively regulates gene expression. Second, luciferase reporter assays demonstrated that PPARγ promoter methylation may repress reporter gene expression [START_REF] Ortega | The gene expression of the main lipogenic enzymes is downregulated in visceral adipose tissue of obese subjects[END_REF]. Third, hypermethylation of the PPARγ2 promoter, which was negatively associated with PPARγ2 expression, was evidenced in visceral WAT of obese animals [START_REF] Fujiki | Expression of the peroxisome proliferator activated receptor gamma gene is repressed by DNA methylation in visceral adipose tissue of mouse models of diabetes[END_REF][START_REF] Zwamborn | Prolonged high-fat diet induces gradual and fat depotspecific DNA methylation changes in adult mice[END_REF][START_REF] Jiao | Protective effects of maternal methyl donor supplementation on adult offspring of high fat diet-fed dams[END_REF][START_REF] Sun | Epigenetic DNA methylation in the promoters of Peroxisome Proliferator-Activated Receptor in chicken lines divergently selected for fatness[END_REF]. However, to our knowledge, no data are available regarding PPARγ programming mechanisms in offspring from obese dams.

We showed that PND21 is a period characterized by active epigenomic remodeling (i.e., higher DNA methylation and lower active histone modification H3ac and H3K4me3) of the PPARγ2 promoter in pWAT, consistent with decreased PPARγ2 mRNA expression levels.

Higher DNMT activity parallels the increase in global DNA methylation and CpGs methylation in the PPARγ2 promoter. During lactation, adipocyte stem cells are plastic and highly sensitive to maternal factors [START_REF] Tang | Adipogenesis: from stem cell to adipocyte[END_REF]. Thus, maternal obesity and modified milk composition may influence offspring's nutritional and hormonal status [START_REF] Sun | Maternal high-fat diet during gestation or suckling differentially affects offspring leptin sensitivity and obesity[END_REF][START_REF] Vogt | Neonatal insulin action impairs hypothalamic neurocircuit formation in response to maternal high-fat feeding[END_REF]. Indeed, epigenetic enzyme activities are particularly sensitive to intracellular fluctuation in energy as well as metabolic intermediates [START_REF] Öst | Epigenetic modulation of metabolic decisions[END_REF]. In line with this notion, weaned HF offspring exhibited higher plasma leptin levels (2) as well as insulin and triglycerides concentrations. Consistent with epigenomic remodeling, global enzyme activity modifications were observed at PND21. However, the increase in DNMT and DNA methylation activities were restricted to pWAT in weaned HF offspring. It may rely on spatiotemporal developmental differences that occur between depots. The DNMT activity is maximal during adipogenesis [START_REF] Londoño Gentile | DNMT1 is regulated by ATP-citrate lyase and maintains methylation patterns during adipocyte differentiation[END_REF]. In rodents, iWAT depots begin to develop during embryogenesis and the progenitor compartment is established before the first few days of life whereas pWAT depots mainly develop postnatally during lactation which coincides with the period of maximum adipogenesis [START_REF] Han | The spatiotemporal development of adipose tissue[END_REF][START_REF] Berry | The developmental origins of adipose tissue[END_REF]. As already suggested (2, 25), offspring's pWAT seems to be much more sensitive to maternal obesity. Some of these epigenetic modifications were still observable in the PPARγ2 promoter in adult HF offspring. Sustained epigenetic modifications (i.e., higher DNA methylation and lower active histone modification H3ac) were observed in both fat pads of adult HF offspring, consistent with persistent decreased PPARγ gene expression. 5mC is the only known modification that targets the DNA itself. In most cases, the extent of 5mC of a promoter is inversely correlated with the activity of the respective gene [START_REF] Jones | The role of DNA methylation in mammalian epigenetics[END_REF]. We also showed that the global 5mC level of the PPARγ2 promoter in adult HF offspring was lower than weaned HF pups, suggesting that a demethylation process occurs during development of WAT. The enrichment of H3ac is characteristic of promoter activation [START_REF] Mikkelsen | Comparative epigenomic analysis of murine and human adipogenesis[END_REF]. A limitation of our work lies in the fact that DNA analysis was performed on entire tissue without distinguishing adipocyte and SVF fractions. Extracts may contain different cell type depending on the stage of development, tissue expansion and physiological states. However, gene expression profiling using several cell markers suggests that the composition of HF offspring's WAT is not modified regardless of the stage of development.

Our results provide compelling evidence that downregulation of PPARγ expression may have developmental origins through epigenetic mechanisms. Thus, editing of early-life epigenetic markings that occurs during development of WAT may persist throughout life.

Consistent with the DOHaD hypothesis, retained marks may provide a mechanistic basis for the cellular "memory" linking maternal obesity to a predisposition for later adiposity.

  [START_REF] Lecoutre | Depot-and sexspecific effects of maternal obesity in offspring's adipose tissue[END_REF]).Offspring from obese dams show persistent decreased PPAR expression levels in adipose tissueAt PND21, HF offspring showed a decrease in PPAR mRNA and protein expression in pWAT and iWAT, consistent with global reduction in PPAR1 and PPAR2 expression (Fig 1A-D). At 9M, among PPAR1 and PPAR2, only the latter showed a persistent dowregulation in both fat pads. The impact of maternal obesity on HF offspring's PPAR target genes was more pronounced in pWAT at both stages, suggesting a depot-specific programming of adipogenesis and lipogenesis (Fig1E, F).Persistent lower PPAR expression is associated withCpG hypermethylation in the PPAR2 promoter of HF offspring We first assessed whether maternal obesity affects CpG methylation status of the PPAR1 and PPAR2 proximal promoter regions (Fig 2A) in offspring. To do this, we analyzed single CpG sites by bisulfite pyrosequencing. For the PPAR1 promoter, at PND21, higher 5mC levels with hypermethylation at two out of seven CpG sites analyzed (-364/-346) were observed in pWAT of HF offspring (Fig 2B). Only hypermethylation of CpG -346 persisted in HF offspring at 9 months of age (Fig 2B). No variation in DNA methylation levels were detected in iWAT (Fig 2C). For the PPAR2 promoter, higher 5mC levels were measured in both fat pads and stages in HF offspring. Two out of four CpG dinucleotides (-215/-199) were similarly affected by maternal obesity. Both CpG were hypermethylated at PND21 whereas only the CpG -199 remained hypermethylated at 9M (Fig 2D and E). Consistently, CpG hypermethylation were correlated with PPAR mRNA levels at PND21 and 9M in both deposits. Weaned offspring from obese dams display higher DNMT activity and global DNA methylation in pWAT We then assessed whether maternal obesity affects the epigenetic machinery activity in offspring. At PND21, DNMT activity (Fig 2F) and global DNA methylation (Fig 2G) were higher in pWAT, consistent with the CpG hypermethylation observed in the PPAR promoters of HF offspring. No variations in TET activity (Fig 2J) and global hydroxymethylation (Fig 2K) were observed. No changes of DNMT1, MBD2, MECP2, TET2 and TET3 mRNA levels were detected (Fig S1). No gross modifications were observed in iWAT at PND21 and in both fat pads at 9M for all parameters (Fig S1). Persistent lower PPAR expression is associated with depletion in H3ac/H3K4me3 in the PPAR2 promoter of HF offspring Finally, we assessed whether maternal obesity affects histone modifications in both promoters of offspring. For the PPAR1 promoter, at PND21, ChIP-qPCR revealed less enrichment of H3ac and H3K4me3 (active marks) in both fat pads of HF offspring (Fig 3A-F). These differences were no longer observable at 9 months of age (Fig 3A-F). For the PPAR2 promoter, at PND21, a reduction of active marks H3ac and H3K4me3 and a tendency towards enrichment of inactive mark H3K9me3 were evidenced only in pWAT of HF offspring (Fig 3A-F). At 9 months of age, persistent reduction of active mark H3ac was measured in both fat pads of HF offspring (Fig 3 A-F). No changes in global histone acetyl transferase and histone deacetylase activities were measured (Fig G and J). No variations of KAT2, P300, HDAC1, SIRT1, LSD1 and SUV39 mRNA levels were evidenced (Fig S1).

  Author contributions: S.L., F.O., D.E., A.G., J.E., C.B. designed the experiments, analyzed data and contributed to discussion and manuscript. C.P., L.B., L.M. performed the experiments. C.L., C.G., J.L., D.V. contributed to discussion.

Figure 1 :

 1 Figure 1: Maternal obesity induces persistent lower PPAR expression in adipose tissue

Figure 2 :

 2 Figure 2: Persistent lower PPAR expression is associated with CpG hypermethylation

Figure 3 :Figure 1 Figure 2 Figure 3

 3123 Figure 3: Persistent lower PPAR expression is associated with depletion in

Table 1

 1 Primers used for RT-qPCR and pyrosequencing. Primer sequences are indicated (see text for definitions).

	Targeted mRNA

Table 2

 2 Plasma parameters of weaned male offspring from obese dams fed either a control (C) or a

	Variable	Male Offspring PND21
				Maternal
		C	HF	diet effect
				p value
	Glucose (dg/ml)	168.22 ± 5.49 171.14 ± 2.54	ns
	Triacyglycerol(mg/dl)	210.9 ± 11.28 281.9 ± 13.10	*
	Insulin (µU/ml)	34.35 ± 5.94	60.99 ± 5.49	*
	Corticosterone (ng/ml)	221.21 ± 16.87 216.63 ± 20.84	ns

high-fat (HF) diet

All data are presented as means +/-SEM. Data were analyzed using Student's t test. Effects of maternal obesity at PND21 (*P  0.05). n = 20 per group.

Acknowledgements:

The authors thank Capucine Besenguez, Anne Dickes-Coopman and Valérie Montel for technical assistance during sacrifice.

Funding: This study was supported by grants from the French "Heart and Arteries" Foundation and grants of the French Ministry of Education. Work at INSERM U1011 was supported by grants from the Fondation pour la Recherche Médicale (Equipe labellisée, DEQ20150331724) and "European Genomic Institute for Diabetes" (E.G.I.D., ANR-10-LABX-46).

Conflicts of interest:

The author declares that he has no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.