Jérôme Chevalier

What future for zirconia as a biomaterial?

Keywords: Zirconia, Ageing, Explant analysis, Orthopedics

The failure events of Prozyr s femoral heads in 2001-2002 have opened a strong, controversial issue on the future of zirconia as a biomaterial. The aim of this paper is to review and analyze the current knowledge on ageing process and on its effect on the long term performanc e of implants in order to distinguish between sc ientific fac ts and spec ulation. Current state of the art shows the strong variability of zirc onia to in vivo degradation, as a c onsequenc e of the strong influenc e of proc essing on ageing proc ess. As different zirc onia from different vendors have different proc ess related mic rostruc ture, there is a need to assess their ageing sensitivity with advanc ed and ac c urate tec hniques, and ISO standards should be modified, espec ially to gain c onfidenc e from clinicians. There is a trend today to develop alumina-zirconia composites as an alternative to monolithic alumina and zirconia: the issue of ageing is also disc ussed for these composites.

Introduction

Among the materials for orthopedics, biomedical grade zirconia is maybe the one for which there is the largest controversy among scientists, industrials or clinicians. Biomedical grade zirconia was introduced 20 years ago to solve the problem of alumina brittleness and the consequent potential failure of implants [START_REF] Christel | Biomechanical compatibility and design of ceramic implants for orthopedic surgery. Bioceramics :material caracteristics versus in vivo behavior[END_REF]. Today, more than 600 000 zirconia femoral heads have been implanted worldwide, mainly in the US and in Europe. On the one hand, biomedical grade zirconia exhibits the best mechanical properties of oxide cera-mics: this is the consequence of phase transformation toughening, which increases its crack propagation resistance. The stress-induced phase transformation involves the transformation of metastable tetragonal grains to the monoclinic phase at the crack tip, which, accompanied by volume expansion, induces compressive stresses [START_REF] Garvie | Ceramic Steel?[END_REF]. On the other hand, due to this metastability, zirconia is prone to ageing in the presence of water [START_REF] Lawson | Environmental degradation of zirconia ceramics[END_REF]. Zirconia manufacturers claimed that this problem was limited under in vivo situation until year 2001 when roughly 400 femoral heads failed in a very short period [4]. The failure origin is now associated to an accelerated ageing in two particular batches of the leader Prozyr s product [5]. Even if limited in time and number, and clearly identified to be process controlled, these events have had a catastrophic impact for the use of zirconia, some surgeons going back to other solutions. Some still claim that zirconia alone allows the use of a larger range of designs (cf. Fig. 1), for example 22 mm heads that fail with alumina, more brittle. They also rightly claim that the failure rate before 2001 was exceptionally low and that ageing Tel.: +33 4 72 43 61 25; fax: +33 4 72 43 85 28. E-mail address: jerome.chevalier@insa-lyon.fr. sensitivity can be controlled and decreased by a careful control of process. Others, also at right, claim that it is unsatisfactory to implant a material in the body, which is not fully stable. There is a dearth of clinical retrieval studies to really assess this issue. Part of the answer should appear in the next years with the revision of thousands of implants, related or not to this aspect. Up to date clinical reports appear to be again somewhat opposite: some results show excellent behavior of some heads after several years in vivo [START_REF] Caton | Phase Transformation in Zirconia Heads after THA Myth or Reality[END_REF], while others show poor follow up results [START_REF] Allain | Poor eighth year survival of cemented zirconia-polyethylene total hip replacements[END_REF], with severe wear and osteolysis around the implant. Few case studies report surface degradation of zirconia implants, which could be related to ageing [START_REF] Haraguchi | Phase transformation of a zirconia cermaic head after total hip arthroplasty[END_REF][START_REF] Catledge | Surface crystalline phases and nanoindentation hardness of explanted zirconia femoral heads[END_REF]. However, there is a clear lack today of correlation between ageing and clinical failures. So, why did some zirconia heads behave well while other show problematic results?

The orthopedic community is thus confronted to a dilemma concerning zirconia, and the market sale has decreased of more than 90% between 2001 and 2002 (end of Prozyr s activity) with no evidence of a clear renew. At the same time, and quite surprisingly, the dental community is 'discovering' the aesthetical and mechanical benefits of using zirconia (see Fig. 1) and seems not to be so concerned by ageing problems. For these dental applications, zirconia market increases of more than 12% per year.

As underlined recently by Clarke et al. [START_REF] Clarke | Current status of zirconia used in total hip implants[END_REF], the history of zirconia has been the subject of misleading interpretations and confusions, mainly due to an absence of rigorous scientific clarifications on ageing. The purpose of the present paper is to bring recent highlights on the ageing mechanism to analyze deeper some recent retrieval studies. This could help the readers to distinguish between scientific facts and speculations. The 400 failures of 2001 have also shown that ISO standard [START_REF]Implants for surgery-Ceramic materials based on yttria-stabilized tetragonal zirconia (Y-TZP)[END_REF] should be modified, at least taking into account ageing, in order to avoid any new dramatic event of that kind. This paper aims at showing the main changes necessary to the ISO standards in the future.

At last, another source of confusion can be associated to the present tendency to develop zirconia-toughened alumina composite [START_REF] Rack | A new ceramic material for orthopaedics[END_REF]. Is it possible indeed that zirconia grains inside these composites could be affected by ageing and lead to potential problems as it was the case for monolithic zirconia? This paper tries to define some rules to be followed for future developments of zirconia-based products.

Ageing process and methods for assessing the ageing sensitivity of zirconia ceramics

The main issue concerning zirconia ceramics, not only in orthopedics, is their sensitivity to low temperature degradation (LTD). LTD has been associated to the roughening of the implants after steam sterilization [13] and more recently to the failure events of Prozyr s heads [4]. Giving a comprehensive review of the ageing mechanism would considerably extent the scope of the present paper. The readers not familiar with this field can refer to the excellent review of Lawson [START_REF] Lawson | Environmental degradation of zirconia ceramics[END_REF]. The monoclinic phase is the stable structure of zirconia ceramics at room temperature. When stabilized with yttria, zirconia ceramics can retain their high temperature tetragonal structure, which is metastable at room temperature. Ageing occurs by a slow surface transformation to the stable monoclinic phase in the presence of water or water vapor. Transformation starts first in isolated grains on the surface by a stress corrosion type mechanism. For a femoral head, surface means the polished wearing surface, but also the interior of the cone, in contact with the metallic taper. This surface was somewhat 'forgotten' before the failure events of Prozyr s heads. The initial transformation of specific grains can be related to their disequilibrium state, i.e. either to a larger size [START_REF] Lawson | Environmental degradation of zirconia ceramics[END_REF], a lower yttria content [START_REF] Lawson | Environmental degradation of zirconia ceramics[END_REF], a specific orientation from the surface [START_REF] Deville | Martensitic transformation in zirconia, part I: nanometer scale prediction and measurement of transformation induced relief[END_REF], the presence of residual stresses [START_REF] Deville | Influence of surface finish and residual stresses on the ageing sensitivity of biomedical grade zirconia[END_REF] or even the presence of cubic phase [START_REF] Chevalier | Critical effect of cubic phase on aging in 3 mol% yttria-stabilized zirconia ceramics for hip replacement prosthesis[END_REF], that has been underestimated in most of the existing literature. As schematically described in Fig. 2, this nucleation of the transformation leads then to a cascade of events occurring neighbor to neighbor: the transformation of one grain leads to a volume increase stressing up the neighboring grains and to microcraking. This offers a path for the water to penetrate down into the specimen. The transformation occurs therefore by a nucleation and growth process, well described by Mehl-Avrami-Johnson laws [START_REF] Chevalier | Low temperature aging of Y-TZP ceramics[END_REF] (Fig. 3). The growth stage again depends of several microstructure patterns: porosity, residual stresses, grain size, etc. It is quite clear at this stage that both nucleation and growth will be highly process related. Table 1 summarizes the potential effect of different process stages on microstructure of zirconia and consequently on ageing. During the short-20 years-story of zirconia, we have seen black/white zirconia, HIPed heads, non-HIPed heads, etc., all these differences affecting LTD resistance. As different zirconia from different vendors (or even from different processes for a given vendor) will have different microstructural characteristics, there is a need to assess the ageing sensitivity of each with advanced and accurate techniques. This can be done via accelerated tests in vitro and from the analysis of in vivo femoral heads explanted for any reason after a given duration (we will review some existing retrieval studies further in the text). Ageing being thermally activated [START_REF] Chevalier | Low temperature aging of Y-TZP ceramics[END_REF], accelerated tests can be performed at temperatures higher than 37 1C, in the framework of the classical timetemperature equivalence principle. The standard, now forbidden, steam sterilization procedure at 134 1C was proven to induce some degree of ageing. It was therefore a good basis to propose an accelerated test. It was thus calculated that 1 h of autoclave treatment at 134 1C had theoretically the same effect as 3-4 years in vivo [START_REF] Chevalier | Low temperature aging of Y-TZP ceramics[END_REF]. If proved to be accurate, this should avoid heavy and long experiments to assess the ageing sensitivity of a given zirconia prior to commercialization. A critical comparison of methods for the determination of the ageing sensitivity of biomedical grade zirconia can be found in Ref. [START_REF] Deville | A critical comparison of methods for the determination of the aging sensitivity in biomedical grade yttria-stabilized zirconi[END_REF]. X-ray diffraction (XRD) analysis was traditionally used to follow quantitatively the transformation. However, this technique suffers some limits such as a poor precision during the first stages of ageing (when the monoclinic content measured by XRD is typically less than 5%), and the absence of local information on ageing process. However, due to its simplicity, this technique can be considered as a first step to investigate the ageing sensitivity of a particular zirconia. Scanning Electron Microscopy (SEM) has been used to investigate the potential impact of ageing on the surface degradation. However, SEM suffers poor vertical resolution and can be also inadequate to investigate the first stages of ageing. It should be preferred to follow the transformation in the bulk or the effect of ageing on surface grain pull out. Moreover, SEM, especially on cross sections, requires specific preparation that can modify the observed surface. Some care should be taken on reports revealing an apparent poor initial density of damaged monoclinic zones with some SEM analyses. Indeed, microcraked zones can lead to extensive pull out during the preparation of the samples. More recent methods, such as optical interferometer (OI) and Atomic Force Microscopy (AFM) can provide valuable insights on the nucleation and growth process, especially during the very first stages of ageing, when SEM and XRD lack precision. Fig. 3 represents the relevance domains of the experimental techniques as a function of the transformation stage. The combination of each technique provides a wide range of characterization, that will inevitably validate or not a given batch of zirconia for future in vivo situation.

V = 4% Shear strain H 2 O H 2 O Surface uplift (a) (b) (c) H 2 O H 2 O H 2 O H 2 O
3. Current state of the art concerning retrieval studies: some different stories Some retrieval studies give positive clinical experiences with zirconia [START_REF] Caton | Phase Transformation in Zirconia Heads after THA Myth or Reality[END_REF], while others show surface degradation of zirconia implants and in some occasions severe wear and/or osteolysis around the implant [START_REF] Allain | Poor eighth year survival of cemented zirconia-polyethylene total hip replacements[END_REF].

The consequences of ageing process on the long term performance of zirconia implants can be twofold: ageing is associated (a) to roughening-this will lead to increased wear, (b) to microcracking-this will lead to grain pull-out and generation of particle debris and possibly premature failure when the microcracked, damaged zone reaches the critical size for slow crack growth to proceed. These two effects are poorly documented in the existing literature: despite 20 years of use and several thousands of implanted heads, only few reports have detailed retrieval analysis, often with no attention dedicated to microstructural features and structural modification. Recent studies give a first scenario of the ageing of zirconia in the body, and its effect on the biomechanical performance of the implants [START_REF] Haraguchi | Phase transformation of a zirconia cermaic head after total hip arthroplasty[END_REF][START_REF] Catledge | Surface crystalline phases and nanoindentation hardness of explanted zirconia femoral heads[END_REF][START_REF] Clarke | Current status of zirconia used in total hip implants[END_REF][START_REF] Maccauro | Fracture of a Y-TZP ceramic femoral head, analysis of a fault[END_REF].

At this stage, we must come back and separate between 'normal' and 'unusual' ageing in typical batches of Prozyr s heads. The two case reports from Maccauro et al. [START_REF] Maccauro | Fracture of a Y-TZP ceramic femoral head, analysis of a fault[END_REF] and from Varner et al. (in Ref. [START_REF] Clarke | Current status of zirconia used in total hip implants[END_REF]) deal with a batch in which fracture rates could be as high as 42%. In these series, fracture occurred after 21-46 months after surgery. It was said by the manufacturer that failure occurred via 'unexpected' and accelerated ageing in some batches [5]. It is clear today that these failures were indeed attributed to an accelerated ageing of the ceramic, in the cone region [START_REF] Clarke | Current status of zirconia used in total hip implants[END_REF][START_REF] Maccauro | Fracture of a Y-TZP ceramic femoral head, analysis of a fault[END_REF]. Due to a change in the processing technique (change from batch to tunnel furnace) the microstructure was varied so that the ageing resistance was poor in this region. We might suppose that such changes in process may have similar dramatic events in any materials. These two reports, and the crisis of 2001, emphasis the importance of accuracy in process control, but should not conduct to any conclusion on the normal behavior of zirconia in the body.

Maybe more critical are the reports which show, under 'normal' situation, surface degradation of zirconia implants, or strong osteolysis associated with the use of zirconia heads. Haraguchi et al. [START_REF] Haraguchi | Phase transformation of a zirconia cermaic head after total hip arthroplasty[END_REF] reported for the first time two cases of surface degradation (roughening and microcracking) caused by phase transformation. The zirconia used in this work, presumably processed by Kiocera followed ISO criteria. They measured monoclinic contents of 20% and 30% in both heads, after only 3 or 6 years respectively, associated with a strong increase of roughness (from 6 to 120 nm). From the photomicrographs provided by the authors, small surface domes, of some dozen of microns, were present on the pole of the heads. They were likely to be monoclinic 'spots', which were observed in previous works with accelerated, in vitro tests [START_REF] Chevalier | Low temperature aging of Y-TZP ceramics[END_REF]. Craters of the same size were visible at the equator (region of wear contact). They should correspond to the same monoclinic spots, but worn out, i.e. inducing large amount of pull out. This gives us the scenario of surface degradation, due to the combination of LTD and wear, as schematically described in Fig. 4. More recent paper from Shane et al. [START_REF] Catledge | Surface crystalline phases and nanoindentation hardness of explanted zirconia femoral heads[END_REF] confirms the degradation of surface properties for some zirconia heads, by means of nano-indentation hardness measurements on explanted heads that had undergone various degrees of phase transformation in service (ranging from 0 to E78% monoclinic content 1 ). The hardness dropped from 18 to 11 GPa for high transformation ratios, which indicates extensive microcracking on the transformation sites. Little information was given on the origin of the heads, their microstructural features and on their clinical history, making an extended analysis of the results difficult. However, it should be noted that one heads exhibited about 78% of monoclinic phase after 62 months of implantation.

Contrasting with these bad clinical experiences, some results still show good, at least middle term follow up [START_REF] Caton | Phase Transformation in Zirconia Heads after THA Myth or Reality[END_REF]. For instance, 22 mm diameter zirconia heads after 45 months showed less than 10% monoclinic content, with apparently neither increase in roughness nor grain pull out. Explanted heads from the same team should be regarded with special interest, since LTD occurs by nucleation and growth and may be at the nucleation stage (small but significant monoclinic content).

These different clinical experiences seem to confirm the strong variability of zirconia heads as regard to LTD resistance. Many processing factors may affect LTD resistance (Table 1) and it seems that, even in the framework of ISO standards, some heads behave well while other show catastrophic behavior. There is, thus, a great need of more advanced studies on zirconia implants, with a deeper correlation between microstructure and LTD resistance in vivo. We recently performed a retrieval analysis of two heads that supports the large variability of zirconia heads to LTD, and in consequence to in vivo situation. Two independent surgeons provided the retrieved heads. One was retrieved after 8 years, the origin of revision being other than related to failure or aseptic loosening. This head was processed by Saint Gobain Desmarquest within the year 1996 (Prozyr s laser marking). The second head was retrieved due to breakage of the zirconia femoral head after only 4.5 years in vivo. The femoral head was broken into four pieces. No information could be obtained on the fabrication, since no trade-name Laser marking was visible in the head. The retrieved heads were first analyzed at different locations on the surface via XRD in order to estimate the amount of phase transformation in vivo. SEM and AFM were conducted in order to investigate possible surface degradation induced by phase transformation. The explants were then cut in parts in order to obtain sections at the core of the heads free of any surface transformation, i.e. not in contact with body fluid. These sections were polished at a laboratory scale with diamond pastes down to 1 mm grain size, leading to surface roughness of about 2 nm. On one polished section of each head, ageing kinetics were performed at 134 1C, 2 bars in autoclave in order to get insight into the expected kinetics at 37 1C for each head, and to compare with results obtained experimentally at the surface in contact with body fluid. On another polished section of each head, thermal etching at 1300 1C, for 30 min was conducted so as to get information on grain size. Density and composition were also checked in agreement with the ISO standard.

Table 2 summarizes the grain size (intercept dimension), density and chemical composition of the two heads, in comparison to ISO standard. XRD monoclinic content at the surface (including measurements at different locations) and approximate size of monoclinic spots visible at the surface via AFM are included. Both heads follow the ISO recommendations. The major difference lies in the grain size, in the upper limit of the norm for the second head. This probably traduces differences in the sintering process (temperature, time, etc.), which may have a strong influence on ageing kinetics. This may be the major origin for the strong difference lying in monoclinic content: about 10% after 8 years for the first one, and 20% after only 4.5 years for the latter. This leads to large surface degradation on the second head, with the occurrence of large damaged zones appearing like 'craters' (Fig. 5a), which have to be related to monoclinic nuclei formed by ageing [START_REF] Chevalier | Low temperature aging of Y-TZP ceramics[END_REF]. In comparison the surface of the present Prozyr s head after 8 years exhibited minor topographic changes. Only AFM could reveal the presence of transformed zones (Fig. 5b). No craters were observed. Fig. 6 shows the ageing kinetics exhibited by the two materials, measured by XRD at 134 1C. The kinetics are given versus time at this reference temperature and expected at 37 1C from the time-temperature equivalence discussed above. Also included are the experimental data obtained at the surface of the retrieved heads in contact with body fluids. The results show an excellent agreement between prediction at 37 1C from the accelerated test and experimental measurements at the surface in contact with body fluid performed before. This shows that accelerated tests can accurately predict in vivo behavior of a given zirconia and should be used as a quality control test before any commercialization. This shows also how slight variations in the process of zirconia can lead to difference in LTD resistance. Among difference things it also shows that the hypothesis of significant elevation of the temperature of the surface during motion in vivo [START_REF] Haraguchi | Phase transformation of a zirconia cermaic head after total hip arthroplasty[END_REF] seems not valid and may be an artifact of hip joint simulators [START_REF] Lu | Frictional heating of bearing materials tested in a hip joint wear simulator[END_REF].

The future of zirconia?

A need for advanced specifications for ISO standards

The tendency of zirconia to ageing both in vitro and in vivo, and the differences observed from one zirconia to another lead us to come back to the crucial question of the title 'what future for zirconia as a biomaterial?'. Indeed, from their inception 20 years ago, the performance of zirconia ceramic heads has been controversial, mainly because of a lack of accurate standards and specifications giving emphasis on LTD. To gain the confidence of surgeons community, a revision of current specifications on zirconia is mandatory. How is it possible that no reference to ageing is made after 20 years experience in the orthopedic community, and much more for other fields? Why is it still possible to process 3Y-TZP with a real grain size approaching 1 mm (the ISO standard referring to a linear intersection distance of 0.6 mm), while a number of studies clearly show dramatic decrease of ageing resistance for a real grain size above 0.6 mm [START_REF] Munoz-Saldana | Mechanical properties and low temperature aging of tetragonal zirconia polycristals processed by hot isostatic pressing[END_REF] (i.e. linear intersect distance of 0.38 mm)? Table 3 summarizes actual ISO 13356 requirements for a given number of parameters (which clearly affect long term behavior of zirconia materials) and necessary changes to be adopted. In particular, accelerated tests in steam, for a given batch of products or after a change in the process or even in the development of new 'ageing free' zirconia ceramics are the key for gaining confidence in the future.

A need for 'ageing free' zirconia materials

The differences observed under in vitro and in vivo situations have shown that zirconia products could behave well. It is difficult to talk about 'ageing free' zirconia since the transformation occurring upon ageing consists in a 'natural' return back to the monoclinic equilibrium state. However, the transformation kinetics can be much affected by microstructural issues. Some solutions were proposed in the literature, but were

Aging:

No reference to ageing An accelerated ageing test should be carried out in steam at 134 1C, 2 bars, for 5 h. After this period, the variation of monoclinic content should be lower than 10% (for every surface in contact with body fluids). No strength degradation should be accepted after the test.

Grain size distribution and microstructures: Intercept distanceo0.6 mm. Intercept distanceo0.4 mm, with a standard deviation less than 0.2 mm (large deviation possibly indicating the presence of cubic phase). No reference to initial monoclinic content Initial monoclinic content less than 20% for every surface in contact with body fluids.

hardly followed by industrial changes. Among them, the addition of small amounts of silica [START_REF] Gremillard | Improving the durability of a biomedical grade Zirconia ceramic by the addition of Silica[END_REF] or the use of Yttria coated instead of co-precipitated powders [START_REF] Piconi | Y-TZP for artificial joint replacements[END_REF] seem to have a clear benefit to the ageing resistance, while preserving good toughness and strength. These solutions were proposed in the Y-TZP system. It has to be said that the issue of ageing of zirconia medical devices stands to the use of yttria as a dopant. Yttrium, as a trivalent ion, creates oxygen vacancies that help hydroxyl group diffusion in the lattice, generating nucleation of the transformation by stress corrosion type mechanism. Ceria doped zirconia ceramics were almost never considered while they exhibit superior toughness (up to 20 MPaOm) and almost no ageing (i.e. non-significant during the lifetime of an implant). There is thus still a door open for zirconia ceramics with improved properties. However, the stop of Prozyr s activity and the new generation of zirconia-alumina composites promoted by major ceramic companies (i.e. Ceramtec or Metoxit) play against further development of such zirconia based products in orthopedics.

Zirconia toughened alumina composites: the ultimate choice?

Given the moderate toughness of alumina and the issue of ageing in zirconia, there is a trend today to develop alumina-zirconia composites. This may be the way to benefit from zirconia transformation toughening without the major drawback associated with its transformation under steam or body fluid condition. In the recent literature concerning alumina-zirconia composites for biomedical applications, different compositions have been tested, from the zirconia rich to the alumina rich side [START_REF] De Aza | Crack growth resistance of alumina, zirconia and zirconia toughened alumina ceramics for joint prostheses[END_REF][START_REF] Affatato | Mixed oxides prosthetic ceramic ball heads. Part 2: effect of the ZrO 2 fraction on the wear of ceramic on ceramic joints[END_REF]. Major ceramic companies are developing such materials. A composite material processed with 80% tetragonal zirconia polycrystals (ZrO 2 -TZP) and 20% alumina (Al 2 O 3 ) is reported to have outstanding mechanical and tribological properties. The alumina-toughened zirconia (ATZ) Bio-Hip s , developed by Metoxit AG (Thayngen, Switzerland), has a bending strength of up to 2000 MPa, indicating that it can withstand loads that are four times greater than conventional Al 2 O 3 implants. This product is still not in the market. At the same time, Ceramtec AG (Plochingen, Germany) recently developed BIOLOX s delta, which consists of approximately 75% aluminum oxide, the basis for hardness and wear resistance, and approximately 25% zirconium oxide, for improved mechanical properties. A strength higher than 1150 MPa is reported, associated with a toughness of 8.5 MPaOm. This product is now in the market.

The addition of alumina to zirconia clearly hinders ageing, or at least reduces drastically its kinetic. It is shown that strength of Biolox delta s for example does not decrease even when repeatedly steam sterilized. However, 'no decrease in strength' does not mean necessary 'no ageing', since other manifestations of ageing are grain pull out and roughening. Few studies have been devoted to ageing in alumina-zirconia systems, but they show that, even if limited and possibly reduced to zero, some degree of degradation can be observed, depending on microstructural features [START_REF] Pecharroman | Percolative mechanism of ageing in zirconia containing ceramics for medical applications[END_REF][START_REF] Deville | Low temperature ageing of zirconia toughened alumina composites[END_REF][START_REF] Deville | Torrecillas 'Microstructural investigation of the aging behavior of Y-TZP toughened alumina composites[END_REF]. As an example, we showed in a previous work [START_REF] Pecharroman | Percolative mechanism of ageing in zirconia containing ceramics for medical applications[END_REF] that ageing could be significant in a 3Y-TZPalumina composite above 16 vol% zirconia. This critical content was related to the percolation threshold above which a continuous path of zirconia grains allowed transformation to proceed. Any extrapolation to other laboratory scale or industrial composites could be hazardous, but it shows again how ageing must be checked carefully prior to clinical development of a given alumina-zirconia composite. Anyway, the presence of zirconia aggregates, especially if the zirconia is stabilized with yttria, should be avoided.

Conclusion

The bad story of Prozyr s femoral heads in 2001, even if dramatic in some aspects, have led the scientific and orthopedic community to deeply study the behavior of zirconia, especially in respect to ageing in vitro and in vivo. Biomedical grade zirconia is by far much deeper understood than ever and powerful tools can be used to assess its sensitivity against ageing. However, given the consequence of the Prozyr s event and some controversial retrieval studies, some of them clearly demonstrating ageing in vivo, further effort will be necessary to gain confidence from the orthopedic community. In this field, it seems that the future, at least for the short and middle term, stands on the combination of alumina and zirconia to obtain advanced composites. However, even if more limited, there is also for these materials a need for more detailed understanding of ageing related issue. The use of zirconia for dental implants is quite young and in development phase. The issue of ageing is still not discussed for these applications.

Fig. 1 .

 1 Fig. 1. Range of femoral heads dimensions allowed with zirconia (courtesy HTI, Decines, France) and example of a zirconia dental bridge (courtesy Diatomic, Louey, France).

Fig. 2 .Fig. 3 .

 23 Fig. 2. Scheme of the ageing process occurring in a cross section, showing the transformation neighbor to neighbor. (a) Nucleation on a particular grain at the surface, leading to microcracking and stresses to the neighbors. (b) Growth of the transformed zone, leading to extensive microcracking and surface roughening. Transformed grains are gray. Red path represents the penetration of water due to microcracking around the transformed grains.

Fig. 4 .

 4 Fig. 4. Scheme of the surface degradation of zirconia hip prostheses due to the combination of ageing and wear. (a) Surface roughening and microcracking on a surface not subjected to wear (i.e. pole of the head). (b) Grain pull-out induced by wear, leading to craters at the surface (i.e. equator of the head).

Fig. 5 .

 5 Fig. 5. (a) SEM picture of one retrieved head ('second' head in the text) after 4.5 years. Note the large crater at the surface, induced by ageing associated to wear. (b) AFM picture (height image) of the Prozyr s after 8 years. The transformed zone does not lead yet to grain pull out.

Fig. 6 .

 6 Fig.6. Kinetics measured on polished 'non-aged' (see text) sections of the two heads at 134 1C, calculated at 37 1C from apparent activation energy, and compared with experimental in vivo points.

Table 1

 1 Potential effect of the different process stages on the microstructure of zirconia ceramics

	Process stage	Potential effect on microstructure
	Initial powder	Yttria content and distribution, presence of additives (leading to secondary phases)
	Forming	Pore distribution of green compacts, and consequently porosity of final components
	Sintering temperature and duration	Density, grain size, amount of cubic phase
	Cooling rate after sintering	Phase partitioning, residual stresses
	Hot isostatic pressing	Density, phase assemblage, increase of oxygen vacancies content, residual stresses
	Whitening	Decrease of oxygen vacancies, modification of residual stresses
	Grinding and machining	Surface roughness, residual stresses, initial monoclinic content
	Cleaning, sterilization	Initial monoclinic content

Table 2

 2 Physico-chemical characteristics of the two retrieved heads of the present study, together with measured XRD monoclinic content and monoclinic spots mean diameter

		Prozyr s head after 8 years in vivo	Second head after 4.5 years in vivo	ISO 13356
	Density	6.0870.2 g/cm 3	6.0570.2 g/cm 3	4 6 g/cm 3
	Grain size (intercept dimension)	0.4270.04 mm	0.6770.07 mm	o 0.6 mm
	Chemical composition
	ZrO 2 +HfO 2	94.4%71	94%71	4 94%
	HfO 2	51% (non-detected)	51% (non-detected)	o5%
	Y 2 O 3	5.6%71	6.0%71	5 % 70.5
	Al 2 O 3	51% (non-detected)	51% (non-detected)	o0.5%
	XRD monoclinic content	7-10%	18-23%
	Monoclinic spots diameter	6 mm	15 mm

Table 3

 3

	Personal recommendations for modification of ISO 13356 standards
	Current ISO 13356 specifications	Personal recommendations for ISO modifications

Values re-calculated from intensity ratios given on the paper.

Acknowledgments

No benefit in any form has been received or will be received from a commercial party that can have modified the content and/or the conclusions of the present paper. Many thanks to Sylvain Deville and Laurent Gremillard for their strong implication to the work devoted to zirconia.


Eva Rial-Pensado

Laurence Canaple

Romain Guyot

Christoffer Clemmensen

Joëlle Wiersema

Shijia Wu

Sabine Richard

Anita Boelen

Timo D Müller

Miguel López

Eva Rial Pensado

Laurence Canaple B-C

Frédéric Flamant

Karine Gauthier
email: karine.gauthier@ens-lyon.fr

Neuronal Blockade of Thyroid Hormone Signaling Increases Sensitivity to Diet-Induced Obesity in Adult Male Mice

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Original data generated and analyzed during this study are included in this published article or in the data repository listed in the references (https://doi.org/10.6084/m9.figshare.22047992).

Thyroid hormone, adaptive thermogenesis, cold exposure, diet induced obesity, neurons, brown adipose tissue, sympathetic nervous system (SNS)

Introduction

As obesity and associated metabolic disorders became pandemic [START_REF] Blüher | Obesity: global epidemiology and pathogenesis[END_REF], the metabolic activities of thyroid hormone (3,3′,5-triiodo-L-thyronine, or T3), the active metabolite of thyroxine (T4) gained renewed interest. Indeed, in humans, hyperthyroidism often results in weight loss, whereas hypothyroidism tends to favor weight gain. Most importantly, exogenous T3 increases energy expenditure (EE) both in humans and rodents [START_REF] Zekri | Central vs. Peripheral Action of Thyroid Hormone in Adaptive Thermogenesis: A burning topic[END_REF]. As a result, an excess of T3 induces weight loss despite an increase in food intake [START_REF] Cicatiello | Metabolic Effects of Intracellular Regulation of Thyroid Hormone: Old Players, New Concepts[END_REF].

T3 can increase EE either directly or indirectly. Its direct metabolic action takes place in BAT, white adipose tissue (WAT), muscle and liver, where it stimulates the mitochondrial metabolism. In BAT, T4 can be deiodinated to produce T3. This local production of T3 triggers the expression of UCP1, the protein responsible for the uncoupling between oxidative phosphorylation and ATP production, which has an important function in thermogenesis [START_REF] Chouchani | New Advances in Adaptive Thermogenesis: UCP1 and Beyond[END_REF][START_REF] Golozoubova | UCP1 is essential for adaptive adrenergic nonshivering thermogenesis Am[END_REF]. After hormonal stimulation, several enzymes involved in lipid synthesis and calories production are also induced in BAT [START_REF] Zekri | Brown adipocytes local response to thyroid hormone is required for adaptive thermogenesis in adult male mice[END_REF], and EE increases in muscle [START_REF] Obregon | Adipose tissues and thyroid hormones[END_REF][START_REF] Nicolaisen | Thyroid hormone receptor alpha in skeletal muscle is essential for T3-mediated increase in energy expenditure[END_REF]. T3 also triggers WAT browning, i.e. the conversion of some white adipocytes into thermogenic adipocytes producing UCP1 [START_REF] Martinez-Sánchez | Thyroid hormones induce the browning of white fat[END_REF].

In addition to its direct influence on peripheral tissues, T3 is also involved in the central control of adaptive thermogenesis [START_REF] López | Hypothalamic AMPK and fatty acid metabolism mediate thyroid regulation of energy balance[END_REF][START_REF] Capelli | Thyroid wars : the rise of central actions[END_REF], a process that is activated to face two types of external stress: cold exposure and excess of food intake [START_REF] Cannon | Brown adipose tissue: Function and Physiological Significance[END_REF]. Perception of cold exposure by skin receptors is sensed by the hypothalamus and results in an increased activity of the sympathetic nervous system (SNS). The subsequent stimulation of adrenergic synapses in adipose tissues triggers immediate BAT thermogenesis, and late browning of WAT [START_REF] Fliers | Novel neural pathways for metabolic effects of thyroid hormone[END_REF][START_REF] López | Regulation of energy balance by thyroid hormones at central level[END_REF]. SNS stimulation leads to both UCP1-dependent and UCP1-independent thermogenesis in BAT [15-16-17] and muscle [START_REF] Bal | The Role of Sarcolipin in Muscle Nonshivering Thermogenesis[END_REF]. How a high-fat diet (HFD) provokes a thermogenic response is less documented. While the involvement of the SNS in this response is well established, the contribution of BAT seems limited [START_REF] Saito | Brown adipose tissue, dietinduced thermogenesis, and Thermogenic Food Ingredients: From Mice to Men[END_REF][START_REF] Kozak | Brown fat and the myth of diet-induced thermogenesis[END_REF]. Importantly, stimulation of BAT via the SNS results in increased expression of the Dio2 gene and a concomitant increase in type-2 deiodinase (D2) activity [START_REF] De Jesus | The type 2 iodothyronine deiodinase is essential for adaptive thermogenesis in brown adipose tissue[END_REF]. The resulting local conversion of T4 into T3 contributes to an increased thermogenic activity. Dio2 knock-out mice are more sensitive to cold [START_REF] Kozak | Brown fat and the myth of diet-induced thermogenesis[END_REF] and more prone to diet-induced obesity (DIO) [START_REF] Castillo | Disruption of thyroid hormone activation in type 2 deiodinase knockout mice causes obesity with glucose intolerance and liver steatosis only at thermoneutrality[END_REF].

Hypothalamic glial cells including astrocytes and tanycytes contain D2, which allows a local conversion of T4 into T3 [START_REF] Guadaño-Ferraz | Type 2 iodothyronine deiodinase is expressed primarily in glial cells in the neonatal rat brain[END_REF]. The fact that this local conversion is involved in adaptative thermogenesis is supported by experiments that show that an intrahypothalamic excess of T3 is sufficient to stimulate BAT activity [START_REF] López | Hypothalamic AMPK and fatty acid metabolism mediate thyroid regulation of energy balance[END_REF]. As all cell types in the hypothalamus express the nuclear receptors of T3, either TRα1 encoded by the Thra gene, or TRβ1/2 encoded by Thrb [START_REF] Samarut | Thyroid Hormone Receptors: Several players for One Hormone and Multiple Functions[END_REF], they all have the capacity to respond to a local increase in T3. However, the link between the intrahypothalamic T3 concentration and the increase in sympathetic tone remains unclear. Elevation of intra-hypothalamic T3 concentration is characterized by rapid activation of the AMPK-ER stress-JNK1 pathway in neurons . Although their T3 response is not documented, neuronal populations located in other areas of the brain, notably the brainstem, also participate in EE [START_REF] Roh | Brain regulation of Energy Metabolism[END_REF].

We address here the importance of T3 signaling in neurons for the regulation of EE under thermogenic stresses. To this end, we used the Cre/loxP methodology to produce a mouse model in which the T3 response is selectively eliminated from a significant fraction of neurons, notably in 20 to 42% of the neurons in the hypothalamus depending of the nucleus. We evaluated the ability of these mice to face thermogenic stress. Mice were exposed either to an intense cold or fed for several months with an HFD. Mutant mice display an intact thermogenic response after exposure to cold at 4°C, but were more prone to DIO. The decrease in SNS tone in mutants mimics what was observed after virogenetic inhibition of TR signaling in the hypothalamus and mirrors the increase triggered by ICV T3 administration [START_REF] López | Hypothalamic AMPK and fatty acid metabolism mediate thyroid regulation of energy balance[END_REF][START_REF] Capelli | Thyroid wars : the rise of central actions[END_REF][START_REF] Cannon | Brown adipose tissue: Function and Physiological Significance[END_REF][START_REF] Fliers | Novel neural pathways for metabolic effects of thyroid hormone[END_REF][START_REF] López | Regulation of energy balance by thyroid hormones at central level[END_REF][START_REF] Kazak | A creatine-driven substrate cycle enhances energy expenditure and thermogenesis in beige fat[END_REF][START_REF] Rahbani | Creatine kinase B controls futile creatine cycling in thermogenic fat[END_REF][START_REF] Sun | Mitochondrial TNAP controls thermogenesis by hydrolysis of phosphocreatine[END_REF][START_REF] Bal | The Role of Sarcolipin in Muscle Nonshivering Thermogenesis[END_REF][START_REF] Saito | Brown adipose tissue, dietinduced thermogenesis, and Thermogenic Food Ingredients: From Mice to Men[END_REF][START_REF] Kozak | Brown fat and the myth of diet-induced thermogenesis[END_REF][START_REF] De Jesus | The type 2 iodothyronine deiodinase is essential for adaptive thermogenesis in brown adipose tissue[END_REF][START_REF] Castillo | Disruption of thyroid hormone activation in type 2 deiodinase knockout mice causes obesity with glucose intolerance and liver steatosis only at thermoneutrality[END_REF][START_REF] Guadaño-Ferraz | Type 2 iodothyronine deiodinase is expressed primarily in glial cells in the neonatal rat brain[END_REF][START_REF] Samarut | Thyroid Hormone Receptors: Several players for One Hormone and Multiple Functions[END_REF][START_REF] Martínez-Sánchez | Hypothalamic AMPK-ER Stress-JNK1 Axis Mediates the Central Actions of Thyroid Hormones on Energy Balance[END_REF]. Overall, our data show that endogenous T3 signaling exerts a significant influence on neurons involved in the regulation of energy metabolism.

2.Material and Methods

Chemicals:

Tri-iodothyronine (T3) and thyroxine (T4) were from Sigma-Aldrich (l'Isle D'Abeau, France).

Mice lines:

Mutant mice were obtained by crosses in the C57BL6/J genetic background to introduce different recombinant alleles. Thra AMI allows the production of the TRα1 L400R mutant, which has dominantnegative properties, after Cre/loxP-mediated excision of a stop cassette [START_REF] Quignodon | A point mutation in the 2 domain of thyroid hormone receptor alpha1 expressed after CRE-mediated recombination partially recapitulates hypothyroidism[END_REF]. Despite the persistence of an intact Thra allele in Cre3 Tg/+ xThra AMI/+ mice, the dominant negative action of TRα1 L400R eliminates the capacity of cells expressing Thra to respond to T3. Thrb lox has 2 tandem-arranged loxP sequences, allowing Cre-mediated excision of exon 3, which encodes the DNA binding domain of the TRβ1/TRβ2 receptor, resulting in a frameshift and a loss of function [START_REF] Winter | Deafness in TRbeta mutants is caused by malformation of the tectorial membrane[END_REF]. Cre3 Tg [START_REF] Banares | Novel pan neuronal Cre transgenic line for conditional ablation of genes in the nervous system[END_REF] is a randomly integrated transgene that drives the expression of the Cre recombinase expression in neurons of the central nervous system, but also the peripheral nervous system. The fraction of neurons with active Cre differs in the different areas and is markedly higher in hypothalamus as compared to other areas of the brain [START_REF] Richard | Neuronal expression of a thyroid hormone receptor α mutation alters mouse behavior[END_REF]. Cre activity was not detected in astrocytes or non-neuronal cell types [START_REF] Banares | Novel pan neuronal Cre transgenic line for conditional ablation of genes in the nervous system[END_REF][START_REF] Richard | Neuronal expression of a thyroid hormone receptor α mutation alters mouse behavior[END_REF]. We previously verified this specific neuronal expression using the rosa YFP reporter line [START_REF] Srinivas | Cre reporter strains produced by the targeted insertion of EYFP and ECFP into theROSA26 locus[END_REF].

Furthermore, in the present study, using the ROSA tdTomato reporter transgene [START_REF] Madisen | A robust and high-throughput Cre reporting and characterization system for the whole mouse brain[END_REF], we verified the absence of recombination activity outside the nervous system, except for a small fraction of cardiomyocytes (Fig. S1) [START_REF] Rial Pensado | ERP Supplemental data file[END_REF]. Cre3 Tg/+ x ROSAtdTomato mice were also used to highlight with red fluorescence the hypothalamic cells in which Cre-mediated recombination occurred and compare the pourcentage of neurons that present Cre activity in the different part of the hypothalamus (Fig. S2 [33]). Mice with the Cre3 Tg/+ xThra AMI/+ Thrb lox/lox genotype are called Neuron TRKO (NTRKO).

Thra AMI/+

Cold exposure and thermography

The cold response was assessed in mice implanted with IPTT-300 transponders (Plexx BV, The Netherlands) by exposing them to 4°C for 58 hours. Infrared thermography was performed at different time points in awake and free running animals. To capture static dorsal thermographic images, thermal videos of 2 min duration each were recorded using an infrared camera (FLiR Systems, Inc.). Thermal images were analyzed using the FLiR Research IR program. Regions of interest (ROI) were drawn so that each type of ROI was overlaid on all thermal images at once. This allowed for identical sizing of each type of ROI across all mice. An ROI on the basis of the tail served as the temperature control, while an ROI surrounding the interscapular region served to evaluate BAT thermogenesis. The temperature difference between these two ROI was calculated and used as a proxy of BAT activation. For each time point, four images per mouse were analyzed. Four mice per genotype were included in the experiment.

When mentioned, the experiments were performed at thermoneutrality in thermoregulated chambers.

Induction of hypo/hyperthyroidism

T3 deficiency in adult animals was induced as previously described with a diet containing PTU (Propylthiouracil) (Harlan Teklad TD95125, Madison, WI) (25.3% protein, 10% fat, 64.8% carbohydrates and 0,15% PTU) and followed or not by thyroid hormones (TH) (mix of T4 and T3) daily injections [START_REF] Weiss | Thyroid hormone action on liver, heart, and energy expenditure in thyroid hormone receptor beta-deficient mice[END_REF] for five consecutive days. Mice ate between 3.75 and 5.25 mg/day of PTU (2.5 and 3.5 g of diet per day). Serum concentrations of FT3 and FT4 were measured as described in paragraph 2.6, and were around 1 pm/L for both FT3 and FT4 in all genotypes in the PTU treated groups and respectively above 50pm/L and 100mg/L (the maximum concentrations detectable by the assay) in all genotypes in the PTU/TH treated groups. As expected PTU leads to hypothyroidism whereas TH injection leads to hyperthyroidism.

HFD feeding

The response to HFD (Research Diets, New Brunswick, USA, D12331) (16.4 % proteins; 58% fat and 25.5% carbohydrates) was evaluated after acute feeding periods (3.5 days) or prolonged feeding periods (3 to 7 months).

Indirect calorimetry

A calorimetric system (LabMaster; TSE Systems; Bad Homburg, Germany) was used to assess EE and locomotor activity. Animals were placed one per cage with controlled conditions of air and temperature (24°C). Mice were in adaptation for a week before starting the measurements. After calibrating the system with the reference gases (20.9% O2, 0.05% CO2 and 79.05% N2), the metabolic rate was measured for 48 hours as previously described [START_REF] Imbernon | Central melanin-concentrating hormone influences liver and adipose metabolism through specific hypothalamic nuclei and efferent autonomic/JNK1 pathways[END_REF]. EE, RQ (VCO2/VO2) and LA were recorded every 30 min. Mice were injected subcutaneously with 1 mg / kg of norepinephrine bitartrate salt monohydrate (Sigma, Germany) to analyse the response to norepinephrine (NE). The O2 consumption was then measured for another 120-160 min every 10 min.

Tissue sampling

At the end of the experiments, mice were anesthetized at 2pm (intraperitoneal injection of a mixture of xylazine and ketamine) to collect blood from the inferior vena cava in heparin coated tubes.

Plasma was obtained after centrifugation at 3500 rpm for 15mn. Tissues were dissected and snapped frozen in liquid nitrogen and stored at -80°C for later RNA preparation or fixed in zinc formal Fixx (ThermoFisher Scientific, Waltham, MA, USA) for histology and immunohistochemistry.

For neuroanatomy experiments, the thorax was opened and each mouse was perfused with 4% paraformaldehyde in 0.1M phosphate buffer at room temperature and brain processed as previously described [START_REF] Richard | Neuronal expression of a thyroid hormone receptor α mutation alters mouse behavior[END_REF] 2.4 RNA extraction and expression analyses by relative RT-qPCR Total RNA was prepared using the TRI Reagent (ThermoFisher Scientific) protocol followed by a RNAse free DNAse treatment (Qiagen, Hilden, Germany). Concentration and purity were quantified with a Nanodrop. 1µg of RNA was used for reverse transcription with M-MLV reverse transcriptase (Promega, Madison, Wi, USA).

For qPCR, Protocols were described before [START_REF] Chatonnet | Genome-wide analysis of thyroid hormone receptors shared and specific functions in neural cells[END_REF] Duplicates were run for each sample. The results were analyzed according to the ∆∆Cq method [START_REF] Bookout | Quantitative real-time PCR protocol for analysis of nuclear receptor signaling pathways[END_REF]. Hprt was used as the reference gene, and the control group was CHOW fed, HFD fed or PTU CTRL groups. The numbers of mice used for each experiment are reported in each figure legend. All the primer sequences are listed in Table S1 [START_REF] Rial Pensado | ERP Supplemental data file[END_REF].

More details on the procedure is provided in supplemental data file SI1 [START_REF] Rial Pensado | ERP Supplemental data file[END_REF].

Western blot

The proteins were extracted from the hypothalamus in lysis buffer (50mM Tris-HCl, 10 mM EGTA, 1mM EDTA, 16mM Triton X-100, 1 mM sodium orthovanadate, 50mM sodium fluoride, 10 mM sodium pyrophosphate and 250 mM sucrose). Western blots were transferred to PVDF membranes (PVDF; Millipore; Billerica, MA, USA) and probed with antibodies against pACCα (Ser79) (Cell Signaling Cat# 3661; RRID: AB_330337; Danvers; MA, USA), pAMPKα (Thr172) (Cell Signaling Cat# 2535S; RRID: AB_331250) or β-actin (Sigma-Aldrich Cat# A5316; RRID: AB_476743; St. Louis,MO, USA) as described [START_REF] Nicolaisen | Thyroid hormone receptor alpha in skeletal muscle is essential for T3-mediated increase in energy expenditure[END_REF][START_REF] Martinez-Sánchez | Thyroid hormones induce the browning of white fat[END_REF]. Adequate secondary antibody: anti-mouse (Agilent Cat# P0260, RRID: AB_2636929; Santa Clara, CA, USA) or anti-rabbit (Agilent Cat# P0450, RRID: AB_2630354) was added. Signal of autoradiographic films was quantified by densitometry using ImageJ-1.33 software (NIH; Bethesda, MD, USA). Values were expressed in relation to β-actin. Data are expressed as a percentage of CTRL vehicle and NTRKO vehicle. Representative images for all proteins are shown with all bands for each picture derived from the same gel, although they may be spliced for clarity (represented by vertical black lines). pACCα and pAMPKα were assayed in the same membranes, therefore, some of the β-actin bands are common in their representative panels; this has been specified in the appropriate figure legend.

Plasma biochemistry

Plasma was assayed on a Cobas 6000 automats for free T3 and free T4 with the Cobas e601module (Roche, ECL analyzers),

D2 activity

Hypothalamic block or 50 mg BAT tissue was homogenized in lysis buffer (100 mM sodium phosphate, 2mM EDTA, 50mM DTT). D2 activity was measured in fresh homogenates. D2 activity was measured in duplicate using 50 µl undiluted homogenate (50 -100 μg protein) that was incubated for 3h (hypothalamus) or 2h (BAT) at 37°C in a final volume of 100 µl in the presence of 0.25 M sucrose, 1 mM PTU (to block D1 activity in the hypothalamus), 500 nM of T3 (to block D3 activity in the hypothalamus), 1 nM T4, and approximately 1.10 5 cpm 125 I-T4 (in-house, single labeled tracer according to [START_REF] Wiersinga | Radioimmunoassay of thyroxine (T4), 3,5,3'-triiodothyronine (T3), 3,3',5'-triiodothyronine (reverse T3, rT3) and 3,3'-diiodothyronine (T2)[END_REF] Wiersinga et al in Phospate-EDTA buffer (PE buffer, 100mM sodium phosphate, 2nM EDTA, pH7.2). D2 activity was expressed as 125 I-fmol released per minute per gram of protein.

The assay is based on the protocol previously described by Werneck-de-Castro et al. [START_REF] Werneck-De-Castro | Thyroid hormone signaling in Male Mouse Skeletal Muscle Is Largely Independent of D2 in myocytes[END_REF].

Histology, immunohistochemistry 2.8.1 Adipose tissues

Fixed adipose tissues were embedded in paraffin. Tissue sections (4 µm for BAT; 8 µm inguinal WAT (iWAT) were stained with hematoxylin and eosin (H&E). Rabbit poly-clonal antibodies directed against UCP1 (Abcam Cat# ab10983, RRID: AB_2241462, Cambridge, UK) and Tyrosine Hydroxylase (Abcam Cat# ab112, RRID: AB_297840) were diluted in PBS/2.5% goat serum for immunohistochemistry, (1:400 and 1:750 dilution respectively). Sections were deparaffinized and incubated overnight at 4°C with a primary antibody. Sections were then incubated with an HRP-labeled antirabbit (1:300) (Promega Cat# W4011, RRID: AB_430833) for 1h at room temperature. Peroxidase activity was visualized with diaminobenzidine staining (DAB, D5905 Sigma-Aldrich) and counterstained with hematoxylin. The images were acquired using an AxioObserver Zeiss microscope at a 16x magnification. Data were collected and the average intensity of the DAB signal was quantified after color deconvolution was performed to separate the relative contributions of DAB and hematoxylin, using ImageJ software. Background correction was performed using a negative control slide, in which the primary antibody was omitted. Three slides per animal were processed.

Brain

For neuroanatomy experiments, immunohistochemistry was performed on free-floating brain sections as previously described [START_REF] Richard | Neuronal expression of a thyroid hormone receptor α mutation alters mouse behavior[END_REF]. Brain Coronal sections (50 μm) were cut with a vibrating microtome (Integraslice 7550 SPDS, Campden Instruments, Loughborough, UK), and stored at -20°C in cryoprotectant (30% ethylene glycol and 20% glycerol in 10 mM PBS) prior to immunohistochemistry.

The following primary antibodies were used: mouse anti-NeuN (1:500) (Merck Millipore Cat# MAB377, RRID: AB_2298772, Burlington, MA, USA), and rabbit anti-GFAP (1:2000) (Agilent Cat# Z0334, RRID: AB_10013382). Secondary antibodies were made in donkey (ThermoFisher Scientific, anti-rabbit DyLight 488 Cat# SA5_10038, RRID: AB_2556618, anti-mouse DyLight 650 Cat# SA5_10169, RRID:AB_25556749) and used at a 1:1000 dilution. Sections were imaged using an inverted confocal microscope (Zeiss LSM 780). ImageJ software was used to estimate the proportion of NeuN+ cells that were also tdTomato+ at different levels of the rostrocaudal hypothalamus.

Quantification and statistical analyzes

For mouse experiments, the data presented represent the average values of different animals from the same genotype given the same treatment. The number of animals (n) used is indicated in the figure legends. For RT-qPCR, each sample was run in duplicate. The error bars represent SEM, since it is more appropriate than SD when n≥6. Statistical relevance was determined using the one-way Anova or the T-Test method as indicated in the figure legends. * or $ or £ stands for p ≤0,05, ** or $$ for 0,005≤ p≤ 0,05, *** or $$$ for 0,0005≤ p≤ 0,005, **** or $$$$ for p≤ 0,0005. This method is appropriate since the size of the compared groups is similar and the distribution of the samples normal. Given the variability of the phenotype that we observed, we used n≥ 6 to obtained statistically significant results. For only two groups, n = 5, because the animals died during the experimentation procedures. ns stands for non-significant.

3.Results

Generation of mice with selective elimination of T3 signaling in neurons

Recombination with the Cre / LoxP system was used to generate somatic mutations in the Thra and Thrb genes. The Cre3 Tg , Thra AMI and Thrb lox alleles were combined to obtain Cre3 Tg/+ Thra AMI/+ Thrb lox/lox mice, called NTRKO mice (see methods 2.2). These mice are heterozygous for the Thra AMI mutation and express the TRα1 L400R in neurons as a result of Cre/loxP mediated recombination. This mutant receptor exerts a dominant-negative activity toward the remaining intact TRα1 receptor. As mice are also homozygous for a Thrb floxed allele, Cre/loxP recombination eliminates the receptors encoded by Thrb [START_REF] Quignodon | A point mutation in the 2 domain of thyroid hormone receptor alpha1 expressed after CRE-mediated recombination partially recapitulates hypothyroidism[END_REF][START_REF] Winter | Deafness in TRbeta mutants is caused by malformation of the tectorial membrane[END_REF]. Therefore, the T3 response is selectively eliminated from all Cre expressing cells.

The efficacy of Cre-dependent recombination in the Cre3 Tg transgenic mice was first described to be pan-neuronal in both the central and peripheral nervous systems [START_REF] Banares | Novel pan neuronal Cre transgenic line for conditional ablation of genes in the nervous system[END_REF]. Later studies from our lab showed that the efficiency of recombination was the highest in hypothalamus as compared to other brain area [START_REF] Richard | Neuronal expression of a thyroid hormone receptor α mutation alters mouse behavior[END_REF]. Here we used a Rosa-tdTomato lox reporter [START_REF] Madisen | A robust and high-throughput Cre reporting and characterization system for the whole mouse brain[END_REF] to show that Cre activity is merely absent in non-nervous tissues (Fig. S1 [START_REF] Rial Pensado | ERP Supplemental data file[END_REF]) and to better evaluate the recombination pattern in the hypothalamus (Fig. S2 [33], Fig. 1). The red fluorescence systematically colocalized with the neuronal NeuN marker and did not overlap with the glial marker GFAP (Fig. 1) as previously described in other area of the brain [START_REF] Richard | Neuronal expression of a thyroid hormone receptor α mutation alters mouse behavior[END_REF]. The density of red fluorescent neurons was high in all hypothalamic nuclei (Fig. 1B, Fig. S2 [START_REF] Rial Pensado | ERP Supplemental data file[END_REF]), except the suprachiasmatic nucleus (not shown), representing 20 to 42% of all neurons in the counted area (Fig. S2 [START_REF] Rial Pensado | ERP Supplemental data file[END_REF]). Therefore, mutations take place in a large fraction of neurons but not in glial cells or other cell types of the brain. We also used TRβ1 mRNA level as a marker for Cre-mediated recombination in NTRKO mice and found that it was only decreased in the hypothalamus but not in the peripheral tissues (Fig. 2A). As TRH is produced by the hypothalamic neurons and involved in the feed-back control of TH production by the hypothalamus-pituitary axis, we measured the expression of TRH. In NTRKO TRH levels were unchanged under either CHOW or HFD (Fig. 2B left panel) and repression of TRH by TH in hypothyroid animals was also normal (Fig. 2B right panel). In agreement free T3 and free T4 concentrations in serum were normal in the mutants under all physiological conditions tested in the paper (Fig. S3A [START_REF] Rial Pensado | ERP Supplemental data file[END_REF]).

In order to address the capacity of neurons to respond to T3, we treated NTRKO with PTU to make them hypothyroid. In response to TH the expression of the Shh and Klf9 genes, previously described to be targets of T3 in neurons [START_REF] Chatonnet | Genome-wide analysis of thyroid hormone receptors shared and specific functions in neural cells[END_REF][START_REF] Bookout | Quantitative real-time PCR protocol for analysis of nuclear receptor signaling pathways[END_REF][START_REF] Wiersinga | Radioimmunoassay of thyroxine (T4), 3,5,3'-triiodothyronine (T3), 3,3',5'-triiodothyronine (reverse T3, rT3) and 3,3'-diiodothyronine (T2)[END_REF][START_REF] Werneck-De-Castro | Thyroid hormone signaling in Male Mouse Skeletal Muscle Is Largely Independent of D2 in myocytes[END_REF][START_REF] Desouza | Thyroid hormone regulates the expression of the sonic hedgehog signaling pathway in the embryonic and adult Mammalian brain[END_REF] was blunted in the hypothalamus of the mutants (Fig. 2C).

Moreover, whereas administration of TH increased food intake in CTRL mice [START_REF] Kong | Triiodothyronine Stimulates Food Intake via the Hypothalamic Ventromedial Nucleus Independent of Changes in Energy Expenditure[END_REF], this response was reduced in the NTRKO mice (Fig. 2D), supporting an inactivation of TH signaling in hypothalamic neurons. In contrast, a normal TH response was observed in all peripheral tissues that we tested. This was ascertained by measuring the mRNA levels for Klf9 and Hr in inguinal WAT (iWAT), BAT and heart, or Hr and Thrsp in liver (Fig. S3B [START_REF] Rial Pensado | ERP Supplemental data file[END_REF]).

In summary, NTRKO mice are euthyroid with functional T3 signaling in all cell types except for a fraction of neurons.

T3 signaling in neurons regulates EE

The NTRKO phenotype was first analyzed under standard conditions of food and temperature. Their body weight (Fig. 3A) and temperature (Fig. 3B) were normal. Their EE (Fig. 3C) measured by indirect calorimetry was lower than that of CTRL mice. Their locomotor activity (Fig. 3D) was similar but NTRKO food intake (Fig. 3E) was slightly lower. RT-qPCR analyzes did not reveal any major alteration of neither the thermogenic program in BAT (Dio2, Ucp1, mCKB and mAlpl mRNA) (Fig. S4A [33]) nor the browning program in iWAT (Ppargc1a, Ucp1, Cidea, Cox7c and Prdm6) (Fig. S4B [33]). However, UCP1 protein level was lower in BAT of NTRKO mice (Fig. 3F). We also assessed UCP1-independent non-shivering thermogenesis in skeletal muscle, by measuring the expression of the genes involved in Ca 2+ cycling (Serca2A, Serca2B, Sln, Ryr1, Ryr2) [START_REF] Bal | The Role of Sarcolipin in Muscle Nonshivering Thermogenesis[END_REF]. We found an increase in Ryr2 expression in NTRKO muscles (Fig. 3G). Thermogenic mechanisms might be turned on in muscle to compensate defective BAT activity.

T3 signaling in neurons is not involved in severe cold-induced thermogenesis

As increased D2 activity and local production of T3 in the BAT are critical to the cold response in wildtype mice (WT) [START_REF] De Jesus | The type 2 iodothyronine deiodinase is essential for adaptive thermogenesis in brown adipose tissue[END_REF], the activity of D2 was checked in the hypothalamus of WT mice. It was not modified during cold exposure in the hypothalamus but, as expected, was strongly induced in the BAT (Fig. 4A).

To test whether despite the unchanged D2 activity in hypothalamus, impaired T3 signaling in neurons prevents the cold-induced process of adaptive thermogenesis, mice were exposed to 4°C. Their body weight (Fig 4B ) and body temperature (Fig. 4C) were monitored for 2.5 days. Both NTRKO and CTRL mice managed to maintain their body temperature by increasing their food intake (Fig. 4D).

Thermogenesis of BAT was observed by infrared thermography after 24 hours and 48 hours at 4°C. The temperature of this tissue was similar in the two genotypes at both time points (Fig. S5A [33]).

Accordingly, the UCP1 protein was expressed at the same level in NTRKO and CTRL in both BAT (Fig. 4E) and in iWAT (Fig. S5B [33]) at 4°C. Moreover RT-qPCR analyzes did not reveal any major alteration of either the thermogenic program in BAT (Dio2, Ucp1, mCKB and mAlpl mRNA) (Fig. 4E) or the browning program in iWAT (Ppargc1a, Ucp1, Cidea, Cox7c and Prdm6) at this temperature (Fig. S5B [33]).

The general response of BAT tissue to SNS stimulation, estimated by the level of expression of Dio2, Ucp1, and Elovl6, is similar in the NTRKO BAT at 4°C compared to CTRL (Fig. 4F). However, the expression of the Adrb3 gene encoding the adrenergic receptor beta 3 the main sensor in BAT for cold-triggered SNS activation, and of one of the genes up-regulated after this activation (Ppargc1a) were higher in the NTRKO BAT under the same condition. A possible interpretation of these results would be that a slight increase in BAT sensitivity to SNS stimulation allows NTRKO mice to face a cold challenge.

Inhibition of T3 signaling in neurons alters the response to DIO

When fed an HFD, NTRKO mice gained more weight than CTRL (Fig. 5A). This resulted from higher body fat accumulation (Fig. 5B) without any significant changes in food intake (Fig. 5C) or locomotor activity (Fig. 5D). Circulating levels of total cholesterol and triglycerides were regulated on HFD to a similar extent in both genotypes (Fig. S3C [START_REF] Rial Pensado | ERP Supplemental data file[END_REF]). In WT animals, D2 enzymatic activity in the hypothalamus increased slightly but significantly after 3.5 days of HFD (Fig. 5E). To characterize the altered response of mutant mice to DIO, we measured gene expression in the BAT of NTRKO and CTRL mice after an HFD. The expression of genes involved in the thermogenic program or the response to SNS stimulation and the level of UCP1 protein were not affected (Fig. S6A [START_REF] Rial Pensado | ERP Supplemental data file[END_REF]). The gene expression analysis in muscle did not provide an indication of a defect in non-shivering thermogenesis in NTRKO mice (Fig. S6B [START_REF] Rial Pensado | ERP Supplemental data file[END_REF]). By contrast, quantification of Ucp1 mRNA and UCP1 protein (Fig. 5F) in iWAT showed that browning was less efficient in the mutants under HFD.

Therefore, an impairment in iWAT browning might explain the observed sensitivity to DIO (Fig. 5A).

Neuronal T3 signaling modulates SNS signaling to regulate EE

Inefficient browning of iWAT under HFD, increased expression of the Adrb3 gene in BAT at 4°C, and decreased levels of UCP1 protein in both BAT and iWAT under different conditions, all point to possible inadequate SNS signaling in NTRKO mutants. We housed mice at 30°C, to address the sensitivity of mutant mice to DIO in the absence of thermogenic SNS stimulation [START_REF] Ganeshan | Warming the mouse to model human diseases[END_REF]. In this condition, obesity was more pronounced in CTRL mice than at 23°C (∆body weight after 12 weeks of HFD, 9 g versus 5 g at 23°C). Most importantly, the difference between mutant and control mice disappeared at 30°C (Fig. S7A [33]). The level of UCP1 protein in BAT was similar in both genotypes under the CHOW diet (Fig. S7B [START_REF] Rial Pensado | ERP Supplemental data file[END_REF]) in contrast to what was observed at 23°C. Normalization of the NTRKO phenotype at thermoneutrality provides a strong argument suggesting that the sensitivity of NTRKO to DIO at 23°C originates from a defect in the tone of the SNS. To address the alternative possibility, which would be that mutant thermogenic organs are less sensitive to SNS stimulation, we treated NTRKO and CTRL mice with NE to activate β adrenergic receptor in BAT and measured the induced change in EE (Fig. 6A). Mutant and control mice responded equally well to this stimulation, reinforcing our initial interpretation. This was further reinforced by performing tyrosine hydroxylase (TH) immunostaining in BAT and iWAT, which allows us to indirectly assess the autonomic nervous input and adrenergic signaling on both CHOW (Fig. 6B) and HFD (Fig. 6C) [START_REF] De Matteis | Immunoreactive nerves of TH, NPY, SP, and CGRP in interscapular brown adipose tissue of adult rats acclimated to different temperatures: an immunohistochemical study[END_REF]. This revealed a slight alteration in the capacity of NTRKO mice to produce catecholamines on HFD (Fig. 6C).

We then assessed more directly the capacity of the hypothalamus to respond to a local T3 stimulation by performing intracerebroventricular (ICV) injections of T3 (Fig. 6 D, E andF). This treatment reduced AMPK and acetyl-coA carboxylase (ACC) phosphorylation levels in the hypothalamus of CTRL mice, as previously described [9-10-44] (Fig. 6D). Notably, the steady-state levels of pAMPKα and pACCα was higher in NTRKO VMH than in the VMH of CTRL mice (Fig. 6E) and was not decreased after local T3 injection (Fig. 6F). This supports the inhibitory effect of T3 on AMPK signaling and the requirement for functional TR in neuron to mediate this response.

Discussion

Data published in recent years provided distinct but complementary pieces of information on the mechanisms by which exogenous T3 influences energy homeostasis [9-10-11-44-45]. ICV or intra VMH injection of T3 for short term or up to 28 days increases SNS tone, BAT activity and EE [9-10-44].

Despite increased UCP1 expression in BAT and iWAT, systemic hyperthyroidism increases EE also via the activation of thermogenic mechanisms in the muscle . Our study provides strong genetic support to previous hypotheses drawn from these experiments and sheds light on the importance of endogenous T3 signaling in neurons in facing thermogenic stresses.

Here we generated NTRKO, a new mouse model in which Cre-mediated mutations of TR in neurons induce a selective blockade of T3 signaling in these neurons. In a previous study, using the same Cre (Cre3 Tg/+ Thra AMI/+ ) Richard et al [START_REF] Richard | Neuronal expression of a thyroid hormone receptor α mutation alters mouse behavior[END_REF]. described that the expression of hairless (Hr), a T3 target gene, was deregulated in hypothalamus but not in other cerebral areas such as the hippocampus, the cortex and the cerebellum. The hypothalamus must be one of the brain areas that is the most impacted by the mutations. Here we show that the decrease of Thrb expression was only observed in the hypothalamus and not in peripheral tissues. This decrease was only partial, since Thrb is also expressed in glia and only 20 to 42% of neurons express Cre. However, it was sufficient to observe a significant decrease of Shh and Klf9 expression, two well described T3 target genes, in response to T3. Importantly, the circulating levels of TH in NTRKO mice are similar to CTRL in all physiological conditions tested and so is the regulation of TRH expression by TH. This suggests that TR are still functional in TRH-expressing neurons to repress TRH expression in response to TH and that regulation of the hypothalamo-pituitary-thyroid axis by TH remains unaltered in NTRKO mice.

Altogether these mice are euthyroid with functional T3 signaling in all cell types except for a fraction of neurons mainly in hypothalamus.

Selective blockade of T3 signaling in NTRKO neurons triggers a decrease in EE that mirrors the increase of EE previously described after T3 injection in the hypothalamus. Our data suggest that EE is altered in NTRKO as a result of decreased uncoupling of catabolism and ATP production in BAT.

Calcium futile cycling in myotubes might produce heat at 23°C to compensate for the BAT defect and to maintain normal body temperature in NTRKO mice under this mild cold stress. Importantly, our data show that endogenous T3 signaling in neurons is mainly relevant for an adequate response to HFD, as NTRKO manage to maintain body temperature after a cold challenge.

D2 activity is increased in the hypothalamus in response to HFD but not cold. That suggests that the local production of T3 in the hypothalamus does not change during cold exposure and that the neuronal response to T3 is not recruited for cold-induced adaptive thermogenesis. In contrast local production of T3 in the hypothalamus would increase under HFD and the inability of NTRK0 to properly detect intrahypothalamic production of T3 would be responsible for their higher sensitivity to DIO.

Interaction with the SNS is involved in the NTRKO sensitivity to DIO, as it is normalized at thermoneutrality. Modulation of the hypothalamic AMPK pathway plays an important role in the central action of T3 on both BAT and liver metabolism [9-10-44]. Our data show that while central T3 decreases pAMPKα and pACCα protein levels, the absence of T3 signaling in neurons increases the basal level of these two phosphoproteins and blunts their regulation by T3. The expected result is a reduced SNS tone to BAT in NTRKO mice at 23°C on CHOW diet. Consequently, under these conditions the level of UCP1 protein in BAT is reduced. The associated decreased in BAT activity might be responsible for the decrease in EE and could be compensated by alternative thermogenic mechanisms, which include the activation of the Ca 2+ futile cycle in the skeletal muscle to maintain body temperature of NTRKO at 23°C. In any case, these results confirm that T3-induced thermogenesis in the brain mostly relies on stimulation of the BAT, while peripheral T3 administration also stimulates not only BAT [START_REF] Zekri | Brown adipocytes local response to thyroid hormone is required for adaptive thermogenesis in adult male mice[END_REF] but also muscle thermogenesis . Thus, T3 signaling in neurons is an important player in the regulation of EE. The interference with the AMPK regulation for SNS activation, previously described after central administration of T3, appears to be also relevant when a physiological thermogenic stress stimulates the hypothalamic production of T3. Importantly NTRKO mice efficiently maintained their body temperature when exposed to an intense cold, displaying normal BAT and WAT response, despite a suboptimal SNS tone. We propose a model for the role played by T3 in neurons for the regulation of EE in response to cold and HFD in Figure 7.

This contrasts with data obtained in a mouse model that ubiquitously expressed TRα1 R384C another dominant negative TRα1 [START_REF] Tinnikov | Retardation of post-natal development caused by a negatively acting thyroid hormone receptor alpha1[END_REF]. Unlike NTRKO, these mice are hypermetabolic and hypothermic, with a theoretical defended temperature of 38°C [START_REF] Sjögren | Hypermetabolism in mice caused by the central action of an unliganded thyroid hormone receptor alpha1[END_REF]. In depth phenotyping led to the conclusions that this phenotype is primarily due to a chronic vasodilatation, which increases heat dissipation. The defect in vasoconstriction was found to reflect a reduced sensitivity of blood vessels to SNS stimulation [START_REF] Warner | Inappropriate heat dissipation ignites brown fat thermogenesis in mice with a mutant thyroid hormone receptor α1[END_REF] that should not take place in NTRKO mice.

Conclusions

We show that T3 does control EE under physiological stresses via its action in neurons. Moreover, although selective inhibition of T3 signaling in neurons leads to a less severe metabolic phenotype than hypothyroidism, modulation of T3 concentration and signaling in neurons definitively represents a significant fraction of metabolic regulation by T3. Targeting T3 to neurons in adult might help fight obesity.

Limitations to our study

First, the Cre-induced mutations occur in juveniles and are not restricted to adult life. Therefore, we cannot rule out that the post-natal maturation of neuronal circuits is altered in mutant mice even so we did not observe any obvious behavioral abnormality. Similarly, while the hypothalamus is the area of the brain that displays the highest percentage of Cre-mediated recombination and that is known to control EE, the intervention of other brain areas in the determination of the metabolic phenotype of NTRKO is possible. Finally, only male mice were included in the study. Since sex specificity can occur in metabolic responses, similar experiments should be run on females.

Further analyzes using models with selective inhibition of T3 signaling in other metabolic tissues, specific neuronal populations, and other brain cell types would clarify the mechanism by which T3 regulates energy balance. measured D2 activity. ER-P, KG, RG and SR performed in vitro experiments. KG, FF and ML wrote the paper. All authors helped analyze the data, reviewed and commented on the final manuscript.

Declaration of competing interest

We declare no competing interest. Statistical ANOVA tests were used for pairwise comparisons of CTRL and NTRKO mice. Stars indicated a significant difference between CTRL and mutants in a given condition, dollars a significant difference in a given genotype between two conditions. ns stands for non-significant. Representative images for all proteins are shown; all the bands for each picture are derived from the same gel, although they may be spliced for clarity (represented by vertical black lines). pAMPKα and pACCα were assayed in the same membranes, therefore, some of the β-actin bands are common in their representative panels, this has been specified in Figures 6D-F

Figure titles and legends

Fig. 1 :

 1 Fig. 1: Neuron-specific Cre-mediated recombination in the hypothalamus in mice that carry the Cre3

Fig. 2 :

 2 Fig. 2: Characterization of the NTRKO mice: a model of selective blockade of T3 signaling in neurons.

Fig. 3 :

 3 Fig. 3: T3 signaling in neurons regulates EE

Fig. 4 :Fig. 5 :

 45 Fig. 4: Blocking T3 signaling in neurons does not affect cold-induced thermogenesis

Fig. 6 :

 6 Fig. 6: Neuron T3 signaling interferes with SNS signaling to regulate EE

  . (A, D, E and F) Statistical T-tests or (B,C ) Statistical ANOVA tests were used for pairwise comparisons in (B, C, E) of CTRL and NTRKO mice , in (A) of CTRL NE and CTRL ( §) and of NTRKO NE and NTRKO , in (D) of CTRL T3 and CTRL VEH and in (F) of NTRKO T3 and NTRKO VEH. ns stands for non-significant.

Fig. 7 :

 7 Fig. 7: Proposed model for the role of T3 in neurone for regulation of energy expenditure in response

Fig. 1

 1 Fig. 1: color

Fig. S6 :

 S6 Fig.S6: Blocking T3 signaling in neurons did not modify the expression of thermogenic markers,

Figure. S7 :

 S7 Figure. S7: Blocking T3 signaling in neurons in NTRKO mice did not alter their response to Diet Induced

Fig

  Fig. S1: color

  Thrb lox/lox littermates were used as controls (CTRL). All experiments were carried out in accordance with the European Community Council Directive of September 22, 2010 (2010/63/EU) regarding the protection of animals used for experimental and other scientific purposes. The research project was approved in the different institutions. In Lyon, it was approved by a local animal care and use committee (C2EA015) and subsequently authorized by the French Ministry of Research, in Santiago by the USC Ethics Committee (Project ID 15010/14/006) and in München by the Animal Ethics Committee of the government of Upper Bavaria, Germany. A total of 180 mice have been used for this study.

	2.3 Animal procedures and preparation of tissue samples:
	2.3.1 Housing
	Mice were fed an ad libitum LASQC Rod16 R diet (Altromin, Germany) (16.9% proteins; 4.3% fat and
	79,2% carbohydrates, hereafter called CHOW), housed 2 per cage when possible, or otherwise
	mentioned at 23°, under a 7:30 am: 8:30 pm light/dark cycle. 3-to 5-month-old male mice were
	used for experiments.

Acknowledgements

We thank Andres del Valle who contributed with RT-qPCR analyzes; Nadine Aguilera, Marie Teixeira, and the ANIRA-PBES facility for help in transgenesis and mouse breeding; Catherine Cerrutti and the members of the Flamant Lab for fruitful discussions and Marja van Veen for her technical help. and HFD). The Blue arrows the regulation observed only at 4°C, the red the regulation observed only under HFD. The lightening sign stands for activation.

On the right panel are summarized three situation in the NTRKO. In green it shows, the difference of energy regulation pathways in the NTRKO as compared to CTRL on regular conditions (CHOW and 23°C). Black arrows picture the regulation observed in NTRK0 under either COLD or HFD as compared respectively to 23°C and CHOW diet. Red arrows picture the regulation observed only under HFD as compared to CHOW in the NTRKO and the Blue arrows the regulation only observed at 4°C as compared to 23°C in NTRKO as well.

This work was supported by grants from the French ANR (FF Thyromut2 program; ANR-15-CE14-0011-01); from ANSES (Thyrogenox); from Ministerio de Ciencia e Innovación co-funded by the EU FEDER Program (ML: PID2021-128145NB-I00) and 'la Caixa' Foundation (ID 100010434), under the agreement LCF/PR/HR19/52160022. ER-P was recipient of a fellowship from MINECO (BES-2015-072743). CC is supported by the Lundbeck Foundation (Fellowship R238-2016-2859) and the Novo Nordisk Foundation (grant number NNF17OC0026114). TDM received funding from the German Research Foundation (DFG TRR296, TRR152, SFB1123 and GRK 2816/1), the German Center for Diabetes Research (DZD e.V.) and the European Research Council ERC-CoG Trusted no.101044445.

Source of funding

This work was supported by grants from the French ANR (FF Thyromut2 program; ANR-15-CE14-0011-01); from ANSES (Thyrogenox); from Ministerio de Ciencia e Innovación co-funded by the EU FEDER Program (ML: PID2021-128145NB-I00) and 'la Caixa' Foundation (ID 100010434), under the agreement LCF/PR/HR19/52160022. ER-P was recipient of a fellowship from MINECO (BES-2015-072743). CC is supported by the Lundbeck Foundation (Fellowship R238-2016-2859) and the Novo Nordisk Foundation (grant number NNF17OC0026114). TDM received funding from the German Research Foundation (DFG TRR296, TRR152, SFB1123 and GRK 2816/1), the German Center for Diabetes Research (DZD e.V.) and the European Research Council ERC-CoG Trusted no.101044445.

Data analysis.

We are using the Software provided with the CFX, id BioRad CFX Maestro, use CFX Manager. Cq is automatically determined by the software. We consider a Cq of 35 as the upper limit of sensitivity.

Melting curve is provided for each sample, and amplification efficiency can be measured.

The 2 -∆∆Cq method is used for standardization using HPRT as the reference gene and control group was CHOW fed, HFD fed or PTU CTRL groups. We verify duplicate reproducibility and use the average value.

Statistics (average, standard deviation, etc…) are made with biological repeats, not technical repetitions. The numbers of mice used for each experiment are reported in each figure legend, but never lower than 5.

Author contributions

KG, FF and ML conceived the study. KG, ER-P, CC ran the in vivo experiments. TDM participated in study design and interpretation of data. LC ran the thermography experiments, and performed the histology/immune histology studies, SW performed some immune histology studies, JW and AB

Supplemental information SI1

RNA extraction and expression analyzes by relative RT-qPCR: detailed procedure RNA extraction Total RNA was prepared using the TRI Reagent (ThermoFisher Scientific) protocol followed by a RNAse free DNAse treatment (Qiagen). Reaction was stopped by EDTA. Concentration and purity was quantified with a Nanodrop.

Reverse transcription:

-Heat 1 µg RNA in 9,5 µl H20 + 1 µl Random Hexamer Primer 100ng/µl, 70°C 5 min. Put on ice after the 5mn to avoid re-hybridization of RNAs.

-Add 9,5µl of MIX (4µl 5X MLV buffer Promega, 2 µl dNTP (5mM each), 0,5 µl RNasin Promega,1 µl RT Promega (MMLV), 2 µl H20) Reaction is performed for 1h at 37°C and RT is inactivated by 10 min at 70°C on a BiORAD T100 thermal cycler.

cDNAs are diluted 1:10, aliquote by 10µl and stored at -20°C As we design our primers to span different exons, we do not use « no RT » control for each sample.

qPCR :

Triplicates are always better than duplicates, but more expensive. As qPCR is very reproducible in our hands we use duplicates. Should the CV of a sample >4%, this sample is discarded and should be analyzed again . It is however much more important to have biological repeats. cDNA are diluted 1 :10 and 2µl is used for each qPCR. Add 8µl of the reaction mix (5µl of 2X syber IQ supermix (Biorad), 330nM final concentration of each Forward and Reverse primers).

For each gene tested we use one "no template" control (no cDNA in QPCR) to check for contaminants in mixes and that is important for the melting curve (detection of primer dimers). Primers have been verified for efficiency on a comparable sample (same tissue, cell type...) → see primer design.

We use HPRT as our reference gene that display a similar aboundance to our genes of interest (usually HPRT for Cq around 24).

Samples are run on a BioRad CFX96 qPCR machin. The program used is universal, and the same for all the primers. The program end with a « melting curve » step to verify that only one amplicon is produced with neither water contamination nor primer dimers.

Program : 95°C 3'essential for release of the Taq chemical inhibition, (95°C 10'' denaturation, 64°C 30'' annealing / elongation, plate read out) x40, from 65 to 95°C, 5'' every 0,5°C, read-out to determine the melting curve.

Primer design and validation :

We use primer blast (primer3 + blast) on http://www.ncbi.nlm.nih.gov/tools/primerblast/index.cgi?LINK_LOC=BlastHome. The most important criteria are :

-primers must span an exon-exon junction" or "Primer pair must be separated by at least one intron on the corresponding genomic DNA" (to avoid amplification of contaminating genomic DNA).

-Amplified fragment should be between 80bp and 150bp. Tm around 60-62-64°C.

-%GC between 40% and 60%.

To test the efficiency of each primer pair, we performedva standard curve on cDNA dilutions spanning at least 4 or 5 log, and also a negative control. cDNA should be from the same tissue as our samples.

Efficiency is calculated, by the following formula : Eff = 10 (-1/slope) -1. It should be as close as possible to 100%, we qualify primers between 90 and 110% (3.6 > slope > 3.1) This experiment also checks for the unicity and size of the amplicon and the absence of primer dimerization.

Supplemental information titles and legends