Soumaya Kouidhi

Isabelle Seugnet

Stéphanie Decherf

Hajer Guissouma

Amel Benammar Elgaaied

Barbara Demeneix
email: demeneix@mnhn.fr

Marie-Stéphanie Clerget-Froidevaux

Amel Benammar Elgaaied

Peroxisome Proliferator-activated Receptor-γ (PPARγ) modulates hypothalamic regulation

Keywords: peroxisome proliferator-activated receptor type  (PPAR), thyroid hormone receptors (TRs), mouse hypothalamus, thyrotropin-releasing hormone (TRH), transcriptional regulation, in vivo gene

come

INTRODUCTION

Maintenance of energy homeostasis relies on tight regulation linking peripheral metabolism to central controls in the brain through complex cellular and molecular mechanisms [START_REF] Havel | Peripheral signals conveying metabolic information to the brain: short-term and long-term regulation of food intake and energy homeostasis[END_REF]. Hypothalamic control of energy balance integrates body metabolic signals, allowing adaptation to environmental challenges [START_REF] Gao | Neuronal control of energy homeostasis[END_REF]. In the hypothalamus the thyrotropin-releasing hormone (TRH) neurons are crucial actors in these energy regulating, homeostatic processes [START_REF] Herwig | Hypothalamic Thyroid Hormone in Energy Balance Regulation[END_REF][START_REF] Hollenberg | The role of the thyrotropin-releasing hormone (TRH) neuron as a metabolic sensor[END_REF][START_REF] Lechan | The TRH neuron: a hypothalamic integrator of energy metabolism[END_REF]. The cellular integration of various metabolic signals relies on stringent control of transcriptional responses, mainly through the coordinated action of transcription factors and associated co-regulators [START_REF] Lonard | The expanding cosmos of nuclear receptor coactivators[END_REF]. Several members of the nuclear receptor (NR) superfamily play critical roles in central and peripheral energy balance control. Their actions as metabolic gene-expression regulators has become a major focus in the development of preventive and therapeutic strategies [START_REF] Francis | Nuclear receptors and the control of metabolism[END_REF].

In an era marked by the increasing prevalence of obesity, diabetes and cardiovascular diseases [START_REF] Lehrke | The many faces of PPARgamma[END_REF], the peroxisome proliferator-activated receptor  (PPAR ), a member of the NR superfamily, has emerged as a major transcriptional regulator of metabolism. PPAR is highly expressed in adipose tissue where it regulates adipocyte differentiation and plays key roles in adipose tissue function [START_REF] Sharma | Review: Peroxisome proliferator-activated receptor gamma and adipose tissue--understanding obesity-related changes in regulation of lipid and glucose metabolism[END_REF]. PPAR is also involved in glucose and lipid metabolism and energy balance in many other peripheral tissues [START_REF] Cho | Peroxisome Proliferators-Activated Receptor (PPAR) Modulators and Metabolic Disorders[END_REF].

All the PPAR subtypes (α, γ, and δ) are expressed in the central nervous system (CNS) [START_REF] Wada | Peroxisome proliferator-activated receptor gamma-mediated regulation of neural stem cell proliferation and differentiation[END_REF]. However, of the three, it is PPARγ signaling pathway that is best characterized in the brain. Indeed, PPARγ and its cognate agonists appears to be an attractive therapeutic target for various disorders of the central nervous system [START_REF] Heneka | PPARs in the brain[END_REF]. PPARγ agonists have shown promising results in animal models of Alzheimer's disease, stroke, multiple sclerosis, Parkinson's disease, amyotrophic lateral sclerosis and pituitary adenoma [START_REF] Sundararajan | PPARgamma as a therapeutic target in central nervous system diseases[END_REF]. Clinical trials of PPARγ agonists are currently underway in several CNS diseases [START_REF] Jiang | The role of peroxisome proliferator-activated receptor-gamma (PPARgamma) in Alzheimer's disease: therapeutic implications[END_REF][START_REF] Schintu | PPARgamma-mediated neuroprotection in a chronic mouse model of Parkinson's disease[END_REF]. More importantly, it was confirmed that the PPARγ isoform is expressed in key neuronal subsets regulating energy homeostasis (Moreno et al, 2004), particularly in the PVN neurons of the hypothalamus [START_REF] Sarruf | Expression of PPAR{gamma} in key neuronal subsets regulating glucose metabolism and energy homeostasis[END_REF]. However, there is as yet no indication of a role of PPAR signaling in central metabolic regulations.

The thyroid hormone (TH) signaling pathway is also a major regulator of metabolism. Early ''lossand gain-of-function'' experiments demonstrated the importance of TH in human metabolism and disease [START_REF] Hollenberg | The thyroid and metabolism: the action continues[END_REF]. The active form of TH is tri-idothyronine (T 3 ), mainly produced in peripheral tissues by deiodination of the precursor thyroxine (T 4 ). T 3 regulates both energy expenditure in the peripheral tissues [START_REF] Kim | Thyroid hormone as a determinant of energy expenditure and the basal metabolic rate[END_REF] and energy balance in the brain [START_REF] Herwig | Hypothalamic Thyroid Hormone in Energy Balance Regulation[END_REF]. Regulation of gene expression by T 3 is mediated via thyroid hormone receptors (TRs) [START_REF] Wu | Thyroid hormone response element sequence and the recruitment of retinoid X receptors for thyroid hormone responsiveness[END_REF]. Three major thyroid hormone receptor isoforms (TR1, TR1, and TR2) bind T 3 . TR distribution is fairly ubiquitous, whereas that of TR is more restricted and tissue-specific [START_REF] Bernal | Thyroid hormone receptors in brain development and function[END_REF]. Specifically, the TR isoforms play important roles in the central control of the hypothalamicpituitary-thyroid (HPT) axis, by regulating hypothalamic Trh gene expression and production in a classic negative feedback fashion [START_REF] Guissouma | Physiological regulation of hypothalamic TRH transcription in vivo is T3 receptor isoform specific[END_REF]. Both TRisoforms bind to the Trh promoter at an unusual TH responsive element (TRE) as a heterodimer with the 9-cis retinoic acid X receptor, RXR [START_REF] Shibusawa | Thyroid hormone receptor DNA binding is required for both positive and negative gene regulation[END_REF].

Previous in vitro studies have underlined interactions between PPAR signaling and other pathways also involved in metabolic regulation, particularly TR-dependent pathways [START_REF] Hyyti | Molecular mechanisms of cross-talk between thyroid hormone and peroxisome proliferator activated receptors: focus on the heart[END_REF]. Crosstalk between TR and PPAR signals could involve competition for their common heterodimeric-partner, the 9-cis-retinoic acid X receptor (RXR) [START_REF] Juge-Aubry | Peroxisome proliferator-activated receptor mediates cross-talk with thyroid hormone receptor by competition for retinoid X receptor. Possible role of a leucine zipper-like heptad repeat[END_REF]. Indeed, both PPAR and TR heterodimerize with RXR on their respective responsive elements (RE) in target gene promoters. TR and PPAR can also compete for DNA-binding on their respective hormoneresponsive elements as TR has been shown to bind PPAR responsive elements (PPRE) [START_REF] Araki | Thyroid hormone receptor beta mutants: Dominant negative regulators of peroxisome proliferator-activated receptor gamma action[END_REF][START_REF] Miyamoto | Inhibition of peroxisome proliferator signaling pathways by thyroid hormone receptor. Competitive binding to the response element[END_REF][START_REF] Ying | Mutant thyroid hormone receptor beta represses the expression and transcriptional activity of peroxisome proliferator-activated receptor gamma during thyroid carcinogenesis[END_REF]. However, despite increasing data showing interactions between PPAR and TR in peripheral tissues, their potential interactions in the hypothalamus remain to be investigated. This absence of data is all the more surprising given that this brain region is the central integrator of whole body energy homeostasis [START_REF] Hollenberg | The role of the thyrotropin-releasing hormone (TRH) neuron as a metabolic sensor[END_REF].

A c c e p t e d M a n u s c r i p t

One previous report has shown a peripheric effect of PPAR activation by a seven days treatment of rosiglitazone on hypothalamic-pituitary-thyroid axis concentrated on the PPARγ isoform, demonstrating a significant effect on thyroid hormone release [START_REF] Festuccia | Peroxisome proliferator-activated receptor-gamma-mediated positive energy balance in the rat is associated with reduced sympathetic drive to adipose tissues and thyroid status[END_REF]. We thus investigated whether the hypothalamic PPARγ signaling pathway affected hypothalamic THdependent gene regulations. To this end, we focused on TH-dependent and independent regulations of Trh transcription, as Trh plays a key role in central metabolic homeostasis. The hypothesis tested was that hypothalamic PPAR could modify TR-dependent regulation of the Trh promoter. To this end, a well documented non-viral reporter gene approach was applied to the newborn mouse model [START_REF] Boussif | A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine[END_REF][START_REF] Guissouma | Physiological regulation of hypothalamic TRH transcription in vivo is T3 receptor isoform specific[END_REF]. This polyethylenimine based method allows for both overexpression and gene silencing approaches to be carried out in an in vivo context, in defined brain areas. The regulations documented in this in vivo, tissue specific, context therefore reflect integrated, physiological conditions [START_REF] Dupre | Both thyroid hormone receptor (TR)beta 1 and TR beta 2 isoforms contribute to the regulation of hypothalamic thyrotropin-releasing hormone[END_REF][START_REF] Guissouma | Physiological regulation of hypothalamic TRH transcription in vivo is T3 receptor isoform specific[END_REF].

The results reported here show first, that pharmacological activation of central, endogenous PPAR signaling leads to increased T 3 -independent Trh transcription and increased circulating T 4 levels. In contrast, inhibition of PPAR activity did not affect Trh transcriptional activity in the absence of T 3 , but interfered with T 3 -dependent Trh repression. Modifying local PPAR protein levels by overexpression or knock-down also modulated transcription from the Trh promoter. Use of small hairpin RNA (shRNA) against PPAR increased T 3 -independent Trh transcription, whilst PPAR overexpression abrogated T 3 -dependent Trh repression. The effect of PPAR overexpression was reversed by co-expression of either TRβ1 or RXR  suggesting that PPAR titrates the TR and PPAR common heterodimeric partner RXR.

Taken together the data demonstrate potential crosstalk between PPARand TRβ1 signaling pathways at the level of the TRH neuron. Hypothalamic PPAR signaling might thus play a critical but as yet unrecognized role in central control of metabolism through Trh transcription.

A c c e p t e d M a n u s c r i p t

MATERIAL AND METHODS

Animals

All aspects of animal care and experimentation were in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of the Animal Protection and Health, Veterinary Services Direction, Paris, France. Swiss wild-type mice were purchased from Janvier (Le Genest St Isle, France), bred in our animal facility and maintained on a 12h light, 12h dark cycle, with food and water ad libitum. Hypothyroidism: Our experiments are generally realized on hypothyroid mice to reduce the variability between animals, being sure that they are all hypothyroid (that means, with homogenous, low level of T3). To induce fetal and neonatal hypothyroidism, pregnant female OF1 mice were given iodinedeficient food containing 0.15 % 6-n-propyl-2-thiouracil (PTU) (Harlan, Gannat, France) and drinking water with 0.5 g/l PTU (Sigma-Aldrich, St Quentin Fallavier, France) from day 14 of gestation through lactation. Hypothyroid 1-day-old pups were injected subcutaneously with 2.5 µg of T 3 /g of body weight (b.w) (in 0.9% saline). Controls received vehicle (saline 0.9%) injections.

Plasmids

The TRH reporter gene construct consisting of 631 bp of the 5' Trh gene regulatory region cloned upstream the Firefly luciferase coding sequence (TRH-luc) was a gift from W. Balkan (University of Miami School of Medicine, FL) [START_REF] Balkan | Expression of rat thyrotropinreleasing hormone (TRH) gene in TRH-producing tissues of transgenic mice requires sequences located in exon 1[END_REF]. The Glut4 reporter gene construct (Glut4-luc) was a gift from Hilde I. Nebb (University of Oslo, Norway) [START_REF] Dalen | Expression of the insulin-responsive glucose transporter GLUT4 in adipocytes is dependent on liver X receptor alpha[END_REF]. The pSG5/stop-mPPAR1 (pSG5-PPAR) expression vector construct was generously provided by Dr Liliane Mickalik (University of Lausanne, Switzerland). The pSG5-RXRα plasmid was a generous gift from Patrick Kastner [START_REF] Kastner | Genetic analysis of RXR alpha developmental function: convergence of RXR and RAR signaling pathways in heart and eye morphogenesis[END_REF] (IGBMC, Illkrich, France). The pSG5-TRβ1 has been described previously [START_REF] Dupre | Both thyroid hormone receptor (TR)beta 1 and TR beta 2 isoforms contribute to the regulation of hypothalamic thyrotropin-releasing hormone[END_REF].

Design and cloning of the small hairpin RNAs

The double-stranded small hairpin RNA (shRNA) coding sequence itself contains sense and antisense siRNA sequence against the target gene separated by a loop sequence and is inserted in the pCMV-H1 plasmid backbone [START_REF] Hassani | A hybrid CMV-H1 construct improves efficiency of PEI-delivered shRNA in the mouse brain[END_REF]. Plasmids coding for shRNA against PPAR (shPPAR were designed from previously published siRNA against PPAR analyzed by immunoblot for their efficiency and validated for their capacity to completely abolish the rosiglitazone induced differentiation of 3T3-L1 pre-adipocytes into adipocytes [START_REF] Liao | Suppression of PPAR-gamma attenuates insulin-stimulated glucose uptake by affecting both GLUT1 and GLUT4 in 3T3-L1 adipocytes[END_REF]. The shRNA coding sequence was purchased from Eurogentec (Angers, France). The following shRNA sequence against PPAR mRNA (shPPAR) 5'TCGAGTGAATTATCTGATTGAGGCTTATCAAGAGTAAGCTTCAATCGGATGGTTCTTT TTTGC3' was subcloned between XhoI and NotI sites in the pCMV-H1 backbone, downstream the hybrid promoter CMV-H1 following the Quick Ligation kit protocol (Invitrogen, Life Technologies Corporation, Carlsbad, CA, USA). All plasmid DNA preparations were prepared using commercial columns (Qiagen, Chatsworth, CA, USA), suspended in 10 mM Tris-HCl, 1 mM EDTA pH 8, sequenced (Eurofins MWG Operon, Germany) and stored as aliquots at -20°C.

Q-PCR

Hypothalami of pups were dissected under a binolocular microscope (limits for hypothalamic dissection: posterior to the optic chiasma, anterior to the mammillary bodies, along both lateral sulcus and 2 mm in depth) immediately froze in liquid nitrogen, and stored at -80°C until processing. Total RNA was extracted following the protocol provided with RNAble reagent (Eurobio, Les Ulis, France)and stored in Tris 10 mM /EDTA 0,1 mM (PH 7,4) at -80°C. To quantify mRNAs in hypothalami, 1µg of total RNA were reverse-transcribed using High capacity cDNA Reverse Transcription kit (Applied Biosystems, Courtaboeuf, France). Control reactions without reversetranscriptase were done in parallel. Taqman probes for the detection of, TRH, PPARRXRα, Glut4, Dio2 and Dio3 (Taqman Gene expression Assays) were purchased from Applied Biosystems (Courtaboeuf, France). Direct detection of the PCR product was monitored by measuring the increase in fluorescence generated by the TaqMan probe. Samples containing 2 µl of cDNA (diluted 20 fold), A c c e p t e d M a n u s c r i p t 6 1µl of specific probe and 10 µl of 2x TaqMan R universal PCR Master Mix (Applied Biosystems) were prepared in a final volume of 20 µl. The gene-specific PCR products were measured continuously using ABI PRISM 7300 Sequence Detection System (Applied Biosystems) during 40 cycles. The threshold cycle (CT) of each target product was determined and ΔCT between target and endogenous control was calculated. The difference in ΔCT values of two groups (ΔΔCT) was used to calculate the fold increase (F=2 -ΔΔCT ) and to determine the target gene expression.

Reagents

PPAR agonists (pioglitazone and rosiglitazone) and antagonist GW9662 (2-chloro-5nitrobenzanilide) were purchased from Alexis Biochemicals (Enzo Life Sciences Inc., Farmingdale, NY, USA) and dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich Corporation, St. Louis, MO, USA) as a 5 mM stock solution at -80°C.

ELISA assay

Euthyroid newborn mice were injected bilaterally in the hypothalamus with 2 µl of a 5% glucose solution containing Rosiglitazone at a final concentration of 1 µM, or DMSO in control animals. Blood samples were collected and pooled 20h and 36h post-treatment. Total T 4 blood concentrations were quantified using ELISA kits (AbCys, Paris, France) following the manufacturer's instructions.

In vivo gene transfer

Transfection of plasmid/polyethylenimine (PEI) complexes was previously described [START_REF] Guissouma | Physiological regulation of hypothalamic TRH transcription in vivo is T3 receptor isoform specific[END_REF]. Briefly, pups were anaesthetized by hypothermia on ice and transfected on postnatal day one with 2x2 l of a 125 ng/l solution of TRH-luc complexed with PEI (500 ng/pup). To assess PPAR overexpression effect, we added either pSG5-PPAR  (5-25-50-100 ng/pup), pSG5-TR1 (100 ng/pup), pSG5-RXR (100 ng/pup) or empty pSG5 expression vector (Promega, Madison, WI, USA) in the mixture. Luciferase activity was assayed 20h after transfection. In shRNA experiments, we added small hairpin expression vector in the complexes at 2x2 l of a 100 ng/µl concentration (400 ng/pup) and luciferase activity was measured 48h later to allow for shRNA expression. To assess the pharmacological activation or inhibition of endogenous PPAR, pioglitazone, rosiglitazone or GW9662 diluted stock solutions were added to the TRH-luc /PEI complexes solution to reach final concentrations (0.1; 0.5; 1 µM). In control animals, diluted DMSO was added to the plasmid/PEI complex solution.

Statistical analysis

Q-PCR results boxes represent 5 to 95 percentiles around the median with whiskers for minimum and maximum values. Statistical analysis for Q-PCR data compared the medians of fold increase, using a Student's t-test to compare control and treated group. Independent experiments (5≤n≤8) were repeated twice providing similar results, and data were pooled. In vivo transfection results are presented as means ± SEM per group. ANOVA was used to compare groups followed by multiple comparisons Bonferroni's post-test. Each experiment was carried out with n≥10, repeated at least three times providing the same results. Statistical analysis for Elisa data compared the means of T 4 values ± SEM, using Student's t-test to compare control and treated group at each time (4≤n≤6). In all cases, representative experiments are shown. Differences were considered significant at p<0.05. *, p<0.05; **, p<0.01; ***, p<0.001.

A c c e p t e d M a n u s c r i p t 7

RESULTS

Hypothalamic expression of PPAR and Glut4 in the newborn mouse

The main objective of this study was to test the hypothesis that crosstalk between PPAR and TH signaling could occur in the mouse hypothalamus and play a physiological role in regulation of genes involved in central control of metabolism, such as Trh.

Q-PCR showed that mRNA encoding PPAR and a key PPAR target gene, Glut4, were expressed in euthyroid (Fig. 1A) and hypothyroid (Fig. 1B) newborn mouse hypothalamus. Measurable levels of both PPAR and Glut4 mRNAs were found. Their expression levels were independent of thyroid status.

Pharmacological modulation of endogenous PPAR activity at the central level modifies HPT function

As PPAR affects peripheral TR signaling, we studied whether pharmacological activation of endogenous PPAR receptors modulated expression of Trh, PPAR and certain genes known to be implicated in Trh regulation in the mouse hypothalamus. The well-characterized PPAR agonist rosiglitazone was administered (1 µM) to euthyroid newborn mice by hypothalamic injection. QPCR was used to assess expression of Trh, PPARRXRα, Dio2 and Dio3 gene levels after 20h (Fig. 2A) or 36h (Fig. 2B) exposure to PPAR agonist. Rosiglitazone administration was without significant effect on the global level of expression of these genes in the hypothalamus at the time points used.

To test more directly whether PPAR activity affected the highly neuron-specific transcription an in vivo PEI-based Trh reporter paradigm [START_REF] Guissouma | Physiological regulation of hypothalamic TRH transcription in vivo is T3 receptor isoform specific[END_REF][START_REF] Guissouma | Transcriptional repression of TRH promoter function by T3: analysis by in vivo gene transfer[END_REF] was exploited. The PPAR agonists rosiglitazone and pioglitazone, were administered to euthyroid newborn mice by intracerebral hypothalamic injection simultaneous with a TRH promoter-luciferase reporter gene (TRH-luc) construct. Injecting increasing amounts (0.1, 0.5 and 1 µM) of either rosiglitazone or pioglitazone, induced dose-dependent activation of TRH-luc expression by 68 % (p<0.01) and 50 % (p<0.05) respectively at 1µM (Fig. 3A).

The effects of these agonists were then tested on hypothyroid mice, as the hypothyroid state reduces variability in endogenous thyroid hormone levels and therefore in the transcriptional responses between animals. Both pioglitazone and rosiglitazone (each at 1µM) increased T 3independent TRH-luc transcription by 35% (p≤0.05) and 50% (p≤0.05) respectively. Moreover at this concentration, both pioglitazone (Fig. 3B) and rosiglitazone (Fig. 3C) increased levels of both T 3independent TRH-luc activation and T 3 -dependent TRH-luc repression. Thus, endogenous PPAR activation by its agonists in the hypothalamus raised the set-point of Trh transcription, maintaining T 3dependent negative feedback on Trh transcription but at a higher transcriptional level than in the absence of PPAR ligands.

The upward resetting of Trh promoter set-point by PPAR agonists should lead to downstream repercussions on circulating thyroid hormone levels, endpoint of the HPT axis regulatory system. To examine whether this was the case, newborn mice were given a single intra-hypothalamic 1 µM injection of rosiglitazone or vehicle, and total plasma T 4 levels were measured 20h and 36h later by ELISA assay. No changes in circulating T 4 were seen at 20h, whereas at 36h post-injection, rosiglitazone treatment significantly increased plasma total T 4 by nearly two-fold (see Fig. 4,4.3 ng/ml ± 0.7 vs 6.9 ng/ml ± 0.93, p≤0.05, control vs 36h group). Thus, the upward shift in the Trh transcriptional set-point induced by the PPAR agonist produces a coherent, physiological stimulation of the HPT axis.

Next, the effects of the potent PPAR selective antagonist GW9662 were investigated. GW9662 (0.1, 0.5 or 1 µM) had no effect on T 3 -independent TRH-luc transcription (data not shown). However, GW9662 (1 µM) completely abolished T 3 -dependent TRH-luc repression (Fig. 5, far right column) increasing mean levels by 33% (p ≤ 0.05) compared to controls (compare white columns in Fig. 5).

Hypothalamic PPAR knockdown affects basal Trh promoter activity

Having demonstrated that pharmacological modulation of PPAR activity modulates the set point of Trh transcription, the effects of modulating hypothalamic PPAR expression level on Trh A c c e p t e d M a n u s c r i p t regulation were examined. First, the effect of hypothalamic PPAR expression knockdown on Trh promoter activity was assessed using a plasmid-based RNA interference (shPPAR) approach (see Materials and Methods).

The shPPAR strategy was validated exploiting the previously published effect of PPAR ligands on Glut4 regulation, i.e. rosiglitazone induction of Glut4 expression [START_REF] Armoni | Peroxisome proliferator-activated receptor-gamma represses GLUT4 promoter activity in primary adipocytes, and rosiglitazone alleviates this effect[END_REF]. shPPAR plasmids were transfected into hypothyroid mice hypothalami together with a Glut4-luc construct. Effects of shPPAR on Glut4-luc expression were tested in the absence or presence of rosiglitazone. Rosiglitazone significantly increased Glut4-luc transcription by 53 % (p<0.001) in control animals (Fig. 6A, left pair of columns). This potent rosiglitazone induction of Glut4-luc was completely abolished in presence of shPPAR (Fig. 6A, right pair of columns). However, shPPAR plasmids had no effect on basal Glut4 promoter activity. Our results thus confirmed the specific role of liganded-PPAR in activating Glut4 gene transcription (Fig. 6A), and demonstrated the efficiency of the shPPAR plasmid used.

Having validated that shPPAR abolished rosigliatzone-dependent induction of Glut4 promoter activity, we examined the effect of shPPAR on T 3 -independent Trh transcription. As shown in Figure 6B, expressing shPPAR in newborn mice hypothalami induced a somewhat unexpected, but highly significant, increase (circa 50%, p < 0.001) of T 3 -independent TRH-luc transcription, showing that PPAR knockdown enhanced T 3 -independent Trh promoter activity.

Hypothalamic PPAR overexpression interferes with T 3 -dependent Trh regulation

We next evaluated the effect of PPAR over-expression on T 3 -dependent regulation of hypothalamic Trh transcription. Hypothyroid newborn mice were transfected in the hypothalamic region with a PPAR expression vector (pSG5-PPAR) or a control pSG5 vector, and the TRH-luc construct (see Materials and Methods). Following transfection, mice were treated or not with T 3 (2.5 µg/g of bw).

As shown in Figure 7A (left pair of columns), T 3 treatment significantly reduced TRH-luc expression (p<0.001) by more than 55% in control mice. In contrast, PPAR overexpression (100 ng) totally abolished T 3 -dependent TRH-luc repression without affecting TRH-luc transcription levels in the absence of T 3 (right pair of columns fig. 7A). The effect of PPAR on T 3 -dependent TRH-luc repression was dose-dependent, increasing amounts of PPAR (5, 25, 50 and 100 ng) progressively reversing the T 3 -dependent inhibition of TRH-luc (Fig. 7B).

Co-transfection of RXRα or TRβ1 reverses PPAR effects on Trh promoter activity

The demonstration that overexpressed PPAR significantly (p<0.01) inhibited T 3 -dependent TRH-luc repression led to the hypothesis that this abrogation could be due to a PPAR-dependent titration of RXRand/or TRβ1-required partners to achieve T 3 -dependent TRH-luc repression. To test this hypothesis, TRH-luc was co-transfected with expression vectors coding for PPAR and RXRα or TRβ1 into newborn mice hypothalami. As shown in Figure 8, co-transfection of RXR (100 ng) with PPAR (50 ng) completely restored the T 3 feedback on TRH-luc (P<0.001) (Fig. 8, 4 th column). A similar reversal of the effect of PPAR was seen when TRβ1 (100 ng) was overexpressed (Fig. 8, far right column), confirming the hypothesis that PPAR abrogation of T 3 -dependent TRH-luc repression was due to an imbalance and a competition between transcription factors. The data presented here show that hypothalamic PPAR and its cognate ligands interfere with T 3 -independent and T 3 -dependent Trh transcription, demonstrating that hypothalamic Trh regulation of metabolism can integrate information from PPAR signaling pathways.

Although much data document how PPAR signaling contributes to energy balance in the periphery by regulating glucose and lipid metabolism [START_REF] Ferre | The biology of peroxisome proliferator-activated receptors: relationship with lipid metabolism and insulin sensitivity[END_REF][START_REF] Sharma | Review: Peroxisome proliferator-activated receptor gamma and adipose tissue--understanding obesity-related changes in regulation of lipid and glucose metabolism[END_REF], little is known about the potential role of PPAR in central metabolic regulations. The hypothalamus controls energy homeostasis at the central level. In particular, paraventricular TRH neurons exert a dominant role on the HPT axis regulation as key metabolic sensors [START_REF] Lechan | The TRH neuron: a hypothalamic integrator of energy metabolism[END_REF] and notably as transducers of leptin and melanocortin signals that modulate the HPT axis [START_REF] Garza | Adeno-associated virus-mediated knockdown of melanocortin-4 receptor in the paraventricular nucleus of the hypothalamus promotes high-fat diet-induced hyperphagia and obesity[END_REF]. Here, the working hypothesis was that as PPAR is expressed in the adult hypothalamus [START_REF] Sarruf | Expression of PPAR{gamma} in key neuronal subsets regulating glucose metabolism and energy homeostasis[END_REF], it might contribute to central control of metabolic homeostasis by modulating TR-dependent Trh transcription.

This hypothesis was tested in newborn mice. Q-PCR confirmed that PPAR is expressed in the newborn hypothalamus. Next, effects of two synthetic PPAR agonists, rosiglitazone and pioglitazone, were assessed on Trh transcriptional activity. These thiazolidinediones (TZD) drugs are commonly used for the treatment of metabolic disease such as insulin resistance or cardiovascular dismetabolic syndrome [START_REF] Murphy | PPAR-gamma agonists: therapeutic role in diabetes, inflammation and cancer[END_REF]. Because of their poor blood-brain-barrier permeability [START_REF] Pedersen | Insulin resistance contributes to aberrant stress responses in the Tg2576 mouse model of Alzheimer's disease[END_REF][START_REF] Risner | Efficacy of rosiglitazone in a genetically defined population with mild-to-moderate Alzheimer's disease[END_REF], they were administrated directly into the hypothalamus. Both agonists significantly increased T 3 -independent Trh transcription. Interestingly, a parallel activation of hypothalamic Glut4 transcription was seen within the same time-scale as for Trh transcription. Liganded-PPAR induction of Glut4 transcription is well documented in peripheral tissues [START_REF] Armoni | Peroxisome proliferator-activated receptor-gamma represses GLUT4 promoter activity in primary adipocytes, and rosiglitazone alleviates this effect[END_REF] and so served as a positive control in these experiments on the effects of TZDs in the brain. PPAR activates the Glut 4 promoter through direct binding of liganded PPAR on the PPAR responsive element (PPRE) at -66/+163 bp from the transcription start site [START_REF] Armoni | Peroxisome proliferator-activated receptor-gamma represses GLUT4 promoter activity in primary adipocytes, and rosiglitazone alleviates this effect[END_REF]. No such PPRE is present on the TRH-luc construct used for the reporter gene experiments, nor on the first 630 bp of the 5' region for the mouse Trh gene. Thus, the transcriptional activation observed is most probably due to an indirect effect of liganded PPAR. Such a regulatory cascade could implicate liganded PPAR-dependent gene pathways in other hypothalamic or extrahypothalamic nuclei connected with TRH expressing neurons. One potential pathway could be the leptin pathway which provides information on energy stores to brain centers regulating appetite and energy expenditure, notably the HPT axis [START_REF] Guo | Leptin signaling targets the thyrotropin-releasing hormone gene promoter in vivo[END_REF].

Here we show that PPARagonists increase not only T 3 -independent hypothalamic Trh transcription, but also importantly, T 4 production. Thus, TZDs or endogenous PPAR-agonists activate hypophysiotropic Trh, which stimulates pituitary TSH, consequently stimulating TH production and release. In turn, TH affects peripheral metabolism at multiple levels including lowering lipids and atherogenic lipoproteins and increasing energy expenditure [START_REF] Erion | Targeting thyroid hormone receptor-beta agonists to the liver reduces cholesterol and triglycerides and improves the therapeutic index[END_REF]. These effects contrast with the peripheral effects of systemic TZDs treatment, which induces positive energy balance and weight gain. These differences are probably due to the fact that with systemic TZDs delivery only small amount of TZDs reach the brain [START_REF] Festuccia | Peroxisome proliferator-activated receptor-gamma-mediated positive energy balance in the rat is associated with reduced sympathetic drive to adipose tissues and thyroid status[END_REF][START_REF] Risner | Efficacy of rosiglitazone in a genetically defined population with mild-to-moderate Alzheimer's disease[END_REF].

Overexpression or knock-down of PPAR in the hypothalamus differentially affects T 3dependent and T 3 -independent Trh transcription

Using a previously published shRNA sequence to knockdown hypothalamic PPAR we observed a loss of Glut 4 regulation by PPAR agonists and significant enhancement of T 3independent TRH transcription. Previous in vitro experiments have already described this phenomenon, referred as de-repression. Notably, in adipocytes, PPAR deletion increases basal transcriptional activity of positively regulated PPAR target genes (Guan et al., 2005;[START_REF] Bensinger | Integration of metabolism and inflammation by lipidactivated nuclear receptors[END_REF].

In contrast, overexpression of PPAR in the hypothalamus of newborn mice hypothalamus did not modify T 3 -independent Trh activation, but abolished T 3 -dependent Trh repression. This effect of A c c e p t e d M a n u s c r i p t 10 PPAR overexpression was dose-dependent. Previous in vitro studies have shown PPAR to counteract T 3 signaling [START_REF] Wang | Peroxisome proliferator activated receptor-gamma (PPARgamma) represses thyroid hormone signaling in growth plate chondrocytes[END_REF]. Our data suggest that unliganded PPAR acts as a dominant negative on hypothalamic TH signaling, preventing T 3 -induced Trh regulation. This inhibition could occur through at least two mechanisms. First, as PPAR and TRβ recruit similar transcriptional machineries, there would be competition for common cofactors (co-activators and corepressors) as described in the heart [START_REF] Hyyti | Molecular mechanisms of cross-talk between thyroid hormone and peroxisome proliferator activated receptors: focus on the heart[END_REF]. The other possibility is competition for the common heterodimer partner RXR, as demonstrated in in vitro studies [START_REF] Araki | Thyroid hormone receptor beta mutants: Dominant negative regulators of peroxisome proliferator-activated receptor gamma action[END_REF][START_REF] Hunter | Crosstalk between the thyroid hormone and peroxisome proliferator-activated receptors in regulating peroxisome proliferator-responsive genes[END_REF][START_REF] Juge-Aubry | Peroxisome proliferator-activated receptor mediates cross-talk with thyroid hormone receptor by competition for retinoid X receptor. Possible role of a leucine zipper-like heptad repeat[END_REF]. This explanation is all the more probable given that RXR enhances T 3 -dependent repression of Trh transcription [START_REF] Hollenberg | The human thyrotropin-releasing hormone gene is regulated by thyroid hormone through two distinct classes of negative thyroid hormone response elements[END_REF]. The present data consolidate this hypothesis, as the inhibitory effect of PPAR on T 3 -dependent Trh repression was abrogated by co-expression of either TRβ1 or RXR in a ligand-dependent manner. The formation of inactive PPAR:RXR heterodimers might be involved in the PPAR-induced modification of Trh  transcription regulation. In coherence with the roles of both TR1 and RXRα in regulating Trh transcription [START_REF] Dupre | Both thyroid hormone receptor (TR)beta 1 and TR beta 2 isoforms contribute to the regulation of hypothalamic thyrotropin-releasing hormone[END_REF][START_REF] Guissouma | Physiological regulation of hypothalamic TRH transcription in vivo is T3 receptor isoform specific[END_REF][START_REF] Hollenberg | The human thyrotropin-releasing hormone gene is regulated by thyroid hormone through two distinct classes of negative thyroid hormone response elements[END_REF], increasing TR1 would titrate PPAR:RXR heterodimer formation in favour of formation of active TR1:RXR heterodimers that mediate T 3 -dependent Trh repression. The effects of PPAR reported here could also been explained by direct interaction between hypothalamic PPAR (expressed in PVN, [START_REF] Sarruf | Expression of PPAR{gamma} in key neuronal subsets regulating glucose metabolism and energy homeostasis[END_REF] and TRβ1 (TR-PPAR heterodimers), as previously described in peripheral regulations on both TR and PPAR target genes [START_REF] Araki | Thyroid hormone receptor beta mutants: Dominant negative regulators of peroxisome proliferator-activated receptor gamma action[END_REF][START_REF] Bogazzi | A novel heterodimerization partner for thyroid hormone receptor. Peroxisome proliferator-activated receptor[END_REF]. The negative effect of overexpressed PPAR on T 3 -dependent Trh repression could arise from sequestration of TR1 in an inactive complex, through heterodimerisation with PPAR that would modulate T 3 action on Trh promoter. These possibilities are not mutually exclusive and in each case excess TR1 would reestablish PPAR-disrupted Trh regulation.

Together the data provide a basis for a model for PPAR interference with Trh transcription. Liganded-PPAR (holo-PPAR) would affect Trh transcription indirectly, via a third effector (hypothetically through leptin signaling, as PPAR is expressed in the center of leptin signaling, the arcuate nucleus [START_REF] Sarruf | Expression of PPAR{gamma} in key neuronal subsets regulating glucose metabolism and energy homeostasis[END_REF] Real-time PCR quantification of TRH, PPAR, RXRα, Dio2 and Dio3 mRNA was done on mRNA extracted from hypothalami of euthyroid newborn mice. Animals were given intracerebral injections of rosiglitazone and sacrificed 20h (A) or 36h (B) later. Boxes represent 5 to 95 percentiles around the median from pools of two independent experiments, with whiskers for minimum and maximum values, n between 5 and 8 samples per group, derived from pools of two independent experiments. Gene mRNA levels are expressed as relative to TRα2 mRNA levels. Hypothyroid newborn mice were transfected with TRH-luc reporter vector (500 ng) and animals treated either with DMSO as vehicle or with GW9662 (1 µM). Test substances were added to the transfection mixture, in the presence or absence of T 3 (2.5 μg/g bw). The data are expressed in RLU as means ± SEM; n≥ 10 per point. The whole experiment was repeated three times providing similar results. *, p<0.05; vs control mice.

  whereas unliganded-PPAR (apo-PPAR) would disrupt T 3induced Trh regulation more directly, competing with common partners as RXR. It is likely that the two mechanisms are independent.In conclusion, this study represents the first in vivo report of interplay between PPAR and TRin the hypothalamus, depicting a functional role of PPAR in regulating Trh transcription. Thus, PPAR, besides being implicated in regulating peripheral metabolism, can also contribute to central control of metabolism. Furthering the understanding of the mechanisms by which TR and PPAR crosstalk in TRH neurons will shed light on how this central group of neurons integrates a wide range of metabolic signals which in turn influence metabolism by setting TH levels. Such knowledge could open up therapeutic perspectives for pathologies implicating metabolic disorders.

Figure 1 :

 1 FIGURE LEGENDS

Figure 2 :

 2 Figure 2: Endogenous TRH expression and its regulatory genes are not modulated by the PPAR agonist Rosiglitazone in the newborn mouse hypothalamus.

Figure 3 :

 3 Figure 3: The PPAR agonists Pioglitazone and Rosiglitazone activate T 3 -independent Trh promoter activity. A: Euthyroid newborn mice were transfected with TRH-luc reporter vector (500 ng per pup) and treated either with DMSO (vehicle) or with increasing concentrations of PPAR ligands Pioglitazone and Rosiglitazone (0.1, 0.5 or 1 µM). To ensure delivery to the hypothalamus, PPAR ligands were added to the transfection mixture together with TRH-Luc. B-C: To further investigate the effect of activation of endogenous PPAR on T 3 -dependent Trh regulation, hypothyroid newborn mice were transfected with TRH-luc reporter vector (500 ng). In each case, animals were treated either with DMSO (vehicle) or Pioglitazone (1 µM) (B) or Rosiglitazone (1 µM) (C) in the presence or absence of T 3 (2.5 μg/g bw), as mentioned. Data are expressed in RLU as means ± SEM; 10≤ n ≤33. The whole experiment was repeated three times. *, p<0.05; **, p<0.01; ***, p<0.001 vs control mice.

Figure 4 :

 4 Figure 4: Intracerebral injection of Rosiglitazone increases peripheral T 4 levels. Euthyroid one day-old newborn mice were acutely treated by a single intra-hypothalamic injection of either Rosiglitazone (1 µM) or DMSO (controls). Sixteen treated animals and sixteen controls were killed at 20h or 36h after treatment. Blood samples were collected and pooled (four individuals per pool) and T 4 assayed by Elisa. Data are expressed in ng/ml as means ± SEM of pooled samples; n≥ 4 for each point. *, p<0.05 vs control mice.

Figure 5 :

 5 Figure 5: The PPAR antagonist, GW9662, inhibits T 3 -dependent TRH-luc repression.Hypothyroid newborn mice were transfected with TRH-luc reporter vector (500 ng) and animals treated either with DMSO as vehicle or with GW9662 (1 µM). Test substances were added to the transfection mixture, in the presence or absence of T 3 (2.5 μg/g bw). The data are expressed in RLU as means ± SEM; n≥ 10 per point. The whole experiment was repeated three times providing similar results. *, p<0.05; vs control mice.

Figure 6 :

 6 Figure 6: PPAR mRNA knock-down upregulates T 3 -independent TRH-Luc transcription. A: intra-hypothalamic expression of shPPAR abolishes specific rosiglitazone-dependent Glut 4 promoter activity. B: shPPAR stimulates TRH-luc expression. Pups received intra-hypothalamic injection of a mix containing either DMSO (vehicle) or Rosiglitazone (1 µM) together with Glut4-luc (500 ng/pup) (A) or TRH-luc (500 ng/pup) (B) and a shRNA expression vector (either pCMV H1-shPPAR or the empty pCMVH1 (400 ng/pup), as specified). Luciferase activity was measured 48h later. Data are

Figure 7 :

 7 Figure 7: PPAR overexpression abrogates T 3 -dependent repression of TRH-luc transcription in a dose-dependent manner. Hypothyroid one-day-old mice were transfected in the hypothalamus with TRH-luc as reporter plasmid (500 ng/pup) and 100 ng (A) or increasing amounts (5-25-50-100 ng/pup) (B) of PPAR expression vector (pSG5/stop-mPPAR). Empty expression vector, pSG5, was used to keep DNA concentration constant (ct vector). Animals were treated with T 3 (2.5 µg/g of b.w.) or saline solution as vehicle (-T 3 ). Luciferase activity was measured 20h post-transfection. Data are expressed in RLU as means ± SEM; n≥ 10 per point. The whole experiment was repeated three times with similar results. *, p<0.05; **, p<0.01; ***, p<0.001 vs control mice.

Figure 8 :

 8 Figure8: Overexpression of RXR or TR1 reverses PPAR induced abrogation of T 3dependent TRH-luc regulation. Hypothyroid one-day-old mice received intrahypothalamic injections of TRH-luc reporter vector (500 ng/pup) and PPAR expression vector (pSG5/stop-mPPAR, 50 ng/pup). RXR or TR1 overexpression was obtained when 100 ng of respectively RXR or TR1 expression vectors were cotransfected with PPAR expression vector. Empty expression vector pSG5 was used to keep DNA concentration constant. Animals were treated with T 3 (2.5 µg/g of b.w.) or saline solution as vehicle (-T 3 ). The experiment was repeated three times providing similar results. Data are expressed in RLU as means ± SEM of triplicates; n≥ 10 per point. *, p<0.05; **, p<0.01; ***, p<0.001 vs control mice.

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ACKNOWLEDGEMENTS

We thank Z. Hassani for help with shRNA in vivo optimization. The excellent animal care provided by S. Sosinsky, P. Durand and C. Forgeron is gratefully acknowledged. This work was supported by a PHC-Utique program /CMCU grant n°06G0910 for S. Kouidhi and EU contract n°018652 "CRESCENDO". The authors declare there is no conflict of interest that would prejudice the impartiality of this work.