Melissa Buscato

Morgane Davezac

Rana Zahreddine

Marine Adlanmerini

Raphaël Métivier

Marianne Fillet

Gael Cobraiville

Cedric Moro

Jean Michel

Jean-Michel M Foidart

Françoise Lenfant

Mélissa Buscato

Pierre Gourdy

Jean-François Arnal
email: jean-francois.arnal@inserm.fr

Coralie Fontaine
email: coralie.fontaine@inserm.fr

Estetrol prevents western diet-induced

Keywords: estrogen receptor, menopause, fatty liver, atherosclerosis, metabolic syndrome

INTRODUCTION

The risk for women to develop metabolic disorders increases significantly after menopause, especially in countries with unbalanced diet habits. Accordingly, postmenopausal women exhibit a higher risk of developing type 2 diabetes, non-alcoholic fatty liver disease (NAFLD) and atherosclerosis (32). Estrogens are well-recognized as key regulators of energy balance and glucose homeostasis. Indeed, cessation of the production of 17-β estradiol (E2) by ovaries at menopause promotes visceral adiposity and insulin resistance resulting in an increased risk of type 2 diabetes (7). In addition, hormonal therapy (HT) was found to reduce the incidence of type 2 diabetes in postmenopausal women (29). In randomized placebocontrolled studies, a significant 21-35% decrease in diabetes occurrence was observed in menopausal women receiving the association of conjugate equine estrogens (CEE) and medroxyprogesterone acetate (MPA) [START_REF] Rossouw | Writing Group for the Women's Health Initiative Investigators. Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results From the Women's Health Initiative randomized controlled trial[END_REF]. However, HT is associated with two major adverse effects: i) an increased risk of thromboembolic events, probably as a consequence, at least in part, of an increase in hepatic-derived coagulation factors and ii) an increased incidence of breast cancers [START_REF] Scarabin | Hormones and venous thromboembolism among postmenopausal women[END_REF]. The identification of safer compounds for HT is thus needed.

Estetrol (E4) is a natural estrogen produced only by the human fetal liver from the 9 th week gestation. It could be an interesting candidate in oral contraception (14) but also for HT (17). Indeed, E4 treatment did not appear to increase the level of hepatic-derived coagulation factors, and therefore might not increase the risk of thromboembolic events (17, 26). E4 used in clinical studies is synthesized from estrone contained in soy and is identical to the natural hormone with over 99% purity. E4 is currently evaluated in phase III clinical studies for HT and a recent multiple-rising-dose study showed that it had beneficial effects on vasomotor symptoms, bone metabolism and genito-urinary syndrome in menopausal women (10, 11).

However, the effect of E4 on metabolic disorders and associated diseases are poorly described.

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

E4 is a natural estrogen but its physiological role remains unknown. As E2, the main natural estrogen in adults, E4 activates the nuclear Estrogen Receptor alpha (ERα) to regulate genomic transcription [START_REF] Abot | The uterine and vascular actions of estetrol delineate a distinctive profile of estrogen receptor α modulation, uncoupling nuclear and membrane activation[END_REF]. But in contrast with E2, E4 does not activate membrane ERα and even inhibits these membrane-initiated steroid signals (MISS) effects of E2 [START_REF] Abot | The uterine and vascular actions of estetrol delineate a distinctive profile of estrogen receptor α modulation, uncoupling nuclear and membrane activation[END_REF]. Thus, while E2 elicits endothelial NO synthase activation and accelerates endothelial healing, E4 exerts no effect by itself, and even abrogates these E2-induced actions [START_REF] Abot | The uterine and vascular actions of estetrol delineate a distinctive profile of estrogen receptor α modulation, uncoupling nuclear and membrane activation[END_REF]. E4 is thus considered as a natural estrogen with selective activation of ERα depending on the tissues, meaning that E4 can act as an agonist or an antagonist of ERα in some target cells.

Selective estrogen receptor modulators (SERMs) belong to a family of drugs used in various estrogen-related diseases such as tamoxifen (TAM) for the treatment of breast cancer, raloxifene for the treatment of osteoporosis, or bazedoxifene, in association to estrogens, for the management of postmenopausal symptoms. As E2, SERMs confer protection from metabolic disorders observed in response to high-fat diets (9, [START_REF] Xu | Effect of selective estrogen receptor modulators on metabolic homeostasis[END_REF][START_REF] Xu | The effect of selective estrogen receptor modulators on type 2 diabetes onset in women: Basic and clinical insights[END_REF]. However, molecular and cellular targets accounting for their protective action against a metabolic stress appear to differ greatly from that of E2, although mediated by ERα in all cases (9). While E2 improves metabolic homeostasis, at least partially, by increasing hepatic production of fibroblast growth factor 21 (FGF21), bazedoxifene increases hepatic expression of Sirtuin1, PPARα and AMPK activity [START_REF] Kim | Tissue-selective estrogen complexes with bazedoxifene prevent metabolic dysfunction in female mice[END_REF]. In addition, we recently demonstrated that, whereas E2 still prevented obesity and glucose intolerance in liver-specific ERα knock out (LERKO) mice, similar beneficial metabolic actions of tamoxifen were totally abrogated in those mice. Furthermore, and by contrast to E2, the whole-body metabolic protection of tamoxifen is entirely dependent on the hepatokine growth differentiation factor 15 (GDF15) [START_REF] Guillaume | Selective Liver Estrogen Receptor α Modulation Prevents Steatosis, Diabetes, and Obesity Through the Anorectic Growth Differentiation Factor 15 Hepatokine in Mice[END_REF]. Interestingly, GDF15 is induced in response to chronic exposure to tamoxifen [START_REF] Guillaume | Selective Liver Estrogen Receptor α Modulation Prevents Steatosis, Diabetes, and Obesity Through the Anorectic Growth Differentiation Factor 15 Hepatokine in Mice[END_REF] and bazedoxifene [START_REF] Buscato | Estetrol (E4), the next generation hormone therapy (HT) for menopausal symptoms: phase 2b clinical trial results[END_REF] but not to E2.

Accordingly, ERα binding sites were found in Gdf15 regulatory region in liver from TAMtreated mice but not from E2-treated mice, due to specific epigenetic modifications [START_REF] Guillaume | Selective Liver Estrogen Receptor α Modulation Prevents Steatosis, Diabetes, and Obesity Through the Anorectic Growth Differentiation Factor 15 Hepatokine in Mice[END_REF].

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

The aim of this study was to evaluate the ability of E4 to prevent obesity and associated metabolic and vascular disorders in ovariectomized female mice fed a western diet.

We demonstrated that E4 protects against body weight gain and glucose intolerance in both C57Bl/6 and LDLR -/-mice and also prevents steatosis and atherosclerosis. We finally demonstrate that, whereas liver ERα is necessary to the prevention of steatosis in response to E4, it is fully dispensable for E4 protection against obesity and atheroma, highlighting dissociation between global metabolic effect of E4 and its liver impact.

MATERIAL & METHODS

Mice and surgical procedure

All procedures involving experimental animals were performed in accordance with the 2014-56). The investigation is consistent with the directive 2010/63/EU of the European parliament. ERαAF2 0 mouse line was generated by deletion of seven amino acids from helix 12 of the AF2 domain as described previously [START_REF] Billon-Galés | Activation function 2 (AF2) of estrogen receptor-alpha is required for the atheroprotective action of estradiol but not to accelerate endothelial healing[END_REF]. To generate the double-deficient mice, LDLR -/-female mice, purchased from Charles River (L'Arbresle, France), were crossed with AlbCreERα flox/flox mice (LERKO) [START_REF] Guillaume | Selective Liver Estrogen Receptor α Modulation Prevents Steatosis, Diabetes, and Obesity Through the Anorectic Growth Differentiation Factor 15 Hepatokine in Mice[END_REF]. WT mice are on a C57Bl/6 background and genetically manipulated mice are systematically compared to their wild type littermates control (n= 7 to 12 per group). Bilateral ovariectomy was performed on 4 weeks of age females, after intraperitoneal injection of ketamine (100 mg.kg -1 ) and xylazine (10 mg.kg -1 ).

Two weeks after surgery, mice were switched to a western diet (42% kcal fat, 0.2% cholesterol, Envigo) and concomitantly implanted with s.c. pellets releasing either vehicle or E4 at 6, 9 and 12 weeks of age to evaluate metabolic and vascular functions.

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

Pellet preparation and E4 Dosage

To deliver E4 over prolonged periods of time, E4 (Mithra) and cholesterol (C3045 Sigma-Aldrich) were thoroughly mixed in powder form and compacted to obtain pellets (weight/weight ratio of E4/cholesterol at 1/20). Mice were implanted subcutaneously every 3 weeks with E4 pellets (1 mg E4/pellet total) or a vehicle (cholesterol only) pellets as recently described (16). Blood E4 concentration was evaluated as previously described (34).

Preliminary experiments determined a 5 ng/ml plasma concentration of E4 after 3 weeks of this treatment, corresponding to the level observed 3h after a single oral administration of E4 at 1 mg/kg/day (19) (Supplementary Figure 1). This plasma concentration is clinically relevant, as it is comparable to those observed in women receiving chronically a daily administration of 15 mg E4 (2 to 5 fold higher) (Mithra communication study MIT ES 0001).

Metabolic tests

Intra-peritoneal glucose tolerance test (IPGTT) was performed by injecting D-glucose (1 g/kg, i.p) after overnight fasting in 13-week-old mice. Blood glucose concentrations were monitored with a glucose meter (Roche Diagnostic, Meylan, France) at the indicated time points: 30 min prior to (-30), just before (0) and at 30, 60, and 90 min after glucose injection.

Basal metabolism was evaluated by indirect calorimetry after 8 weeks of treatment. Mice were individually housed for 48 hours where the first 24 hours corresponded to acclimatization and the subsequent 24 hours to measurement of energy expenditure (Phenomaster; TSE-Systems) at 10-min intervals at constant temperature (22°C). Physical activity was monitored by an infrared photocell beam interruption method.

Liver and perigonadal adipose tissue histology

Liver and perigonadal adipose tissues were quickly excised, fixed in 10% buffered formalin and embedded in paraffin. Sections (3 µm) were stained with haematoxylin/eosin (H&E) and oil red O as previously described [START_REF] Guillaume | Selective Liver Estrogen Receptor α Modulation Prevents Steatosis, Diabetes, and Obesity Through the Anorectic Growth Differentiation Factor 15 Hepatokine in Mice[END_REF]. Images were obtained with nanozoomer scanner

Liver lipid content analysis

Hepatic levels of triglycerides and cholesterol esters were determined using Bligh & Dyer methodology and lipid extracts were analyzed by gas-liquid chromatography as previously described [START_REF] Guillaume | Selective Liver Estrogen Receptor α Modulation Prevents Steatosis, Diabetes, and Obesity Through the Anorectic Growth Differentiation Factor 15 Hepatokine in Mice[END_REF].

Gene expression analysis

Tissues were homogenized using a Precellys tissue homogenizer (Bertin Technol., Cedex, France) and total RNA from tissues was extracted using TRIzol (Invitrogen, Carlsbad, CA). 1000 ng for liver and 500 ng for aorta of RNA was reverse transcribed (RT) at 25°C for 10 min and then at 37°C for 2 h using the High Capacity cDNA reverse transcriptase kit (Applied Biosystems). For gene expression in aorta and liver, qPCR were performed using SsoFast EvaGreen Supermix (Bio-Rad) with primers efficiency validated using standard curves method (95% < efficiency < 105%) (Supplemental Table 1). Gene expression was quantified using the comparative C t (threshold cycle) method. Tpt1 and Ppia were used as housekeeping gene to normalize the mRNA levels respectively in the aorta and the liver.

Analysis of atherosclerosis lesions

Overnight fasted mice were anesthetized, and blood was collected from the retro-orbital venous plexus before dissecting the heart. Lipid deposition was evaluated at the aortic sinus as previously described (4). Briefly, each heart was frozen on a cryostat mount with OCT compound. One hundred 10 µm thick sections were prepared from the top of the left ventricle, where the aortic valves were first visible, up to a position in the aorta where the valve cusps were just disappearing from the field. After drying for 2 hours, the sections were stained with oil red O and counterstained with Mayer's hematoxylin. Ten sections out of the 100, each separated by 90 µm, were used for specific morphometric evaluation of intimal lesions using A c c e p t e d M a n u s c r i p t a computerized Biocom morphometry system. The first and most proximal section to the heart was taken 90 µm distal to the point where the aorta first becomes rounded. The mean lesion size (expressed in µm 2 ) of these 10 sections was used for each animal.

Determination of plasma lipids

Plasma triglycerides were assayed using ABX Pentra Triglycerides CP (ABX Pentra, Horiba, Montpellier, France). The high density lipoprotein (HDL) and low density lipoprotein (LDL) fractions were isolated from 10 µl of serum and assayed using the "ABX Pentra HDL Direct CP" method (ABX Pentra, Horiba, Montpellier, France).

Statistics

Results are expressed as means ± SEM. Statistical analyses were performed using GraphPad Prism version 6.00 for Windows (GraphPad Software, San Diego, CA). T-test was used to analyze the effect of treatment in WT or LDLR -/-mice. ANOVA-2 factor were used to analyze the effects of treatment and genotype. Fischer LSD post-test were subsequently performed in case of interaction. P < 0.05 was considered statistically significant (*P<0.05, **P<0.01, ***P<0.001).

RESULTS

ERαAF2 is necessary to mediate E4 beneficial effect against western-diet induced obesity and steatosis.

To evaluate the metabolic impact of E4, we first determined whether E4 was able to confer protection against western diet-induced obesity as previously described in response to E2 and SERMs. To this aim, ovariectomized WT female mice were fed a western diet (42% kcal fat, 0.2% cholesterol) and concomitantly implanted with subcutaneous E4 pellets or a vehicle for A c c e p t e d M a n u s c r i p t 9 weeks (Figure 1A). Weekly body weight measurements revealed that E4-treated mice are protected against western diet-induced obesity from the first weeks of treatment (Figure 1B).

After 7 weeks of western diet feeding, E4 treatment improved glucose tolerance in overnightfasted mice, as illustrated by lower plasma glucose levels at 30, 60 and 90 min following glucose challenge, resulting in a 20% decrease in the Area Under the Curve (AUC) for glucose (Figure 1C). At the end of the protocol, E4-treated mice were remarkably leaner (Supplementary Figures 2A and2B). Fasting glucose levels were significantly lower in E4treated mice (Supplementary Figure 2C), while insulin levels tended to be lower, although this difference did not reach statistical significance (Supplementary Figure 2D).

Nevertheless, insulin sensitivity was preserved in E4-treated mice compared to the vehicle group, as attested by a lower HOMA-IR mean value (Figure 1D). Uterine impregnation was chosen as a positive control of estrogenic action and, as expected, ovariectomy led to uterine atrophy, whereas chronic E4 treatment increased uterine weight (Figure 1E). E4 treatment led to reduced accumulation of subcutaneous, perigonadal and mesenteric adipose tissues (Figure 1F). The mean area of adipocytes from perigonadal adipose tissue was also significantly lower in E4-treated mice compared to control mice (Figure 1G). Moreover, protection conferred by E4 included a lower lipid accumulation in the liver (Figure 1H). Accordingly, mRNA levels of genes involved in lipid metabolism, including cholesterol metabolism and lipoprotein assembly (i.e. Cd36, Pltp and Apoa4) were decreased in the liver from E4-treated mice compared to vehicle control mice (Supplementary Figure 3). E4 treatment also regulated expression of some hepatokines with an increase in Growth differentiation factor 15 (Gdf15) but a decrease in Fibroblast growth factor 21 (Fgf21) and Angiopoietin-like 6 (Angptl6) mRNA levels. (Supplementary Figure 3).

To further explore the beneficial effect of E4 on metabolism, we then analyzed basal metabolism by indirect calorimetry after 8 weeks of E4 treatment. As expected, both control A c c e p t e d M a n u s c r i p t and E4-treated mice elicited a marked increase in physical activity during the dark (active) phase (Figure 2A). More importantly, during this period the physical activity was significantly higher in E4-treated mice than in the control group. Accordingly, E4 treatment induced higher energy expenditure during the dark phase (Figure 2B). Altogether, these results suggest that E4 protects ovariectomized WT female mice from western diet-induced obesity at least in part by increasing locomotor activity and energy expenditure.

To evaluate the role of ERα and more particularly of its transcriptional activity in this E4 metabolic protection, we then used ERαAF2 0 mice. In this mouse model, nuclear action of ERα is disrupted due to the deletion of seven amino acids in the helix 12 while membrane initiated signalling are preserved, as previously reported (2) (Figure 3A). As expected, E4 led to an increase of uterine weight in ERαAF2 +/+ mice but not in ERαAF2 0 mice, reinforcing the demonstration of the crucial role of the nuclear function of ERα in uterine response to E4 (1) (Figure 3B). Likewise, although E4 prevented body weight gain (Figure 3C and3D) and accumulation of subcutaneous, perigonadal and mesenteric adipose tissues (Table 1) in the control ERαAF2 +/+ mice, this beneficial action of E4 was no longer observed in ERαAF2 0 mice. The protective effect of E4 against lipid accumulation in the liver was also abrogated in ERαAF2 0 mice compared to their ERαAF2 +/+ littermates (Figure 3E and Table 1), highlighting the role of ERα and its nuclear/AF2-dependent action in E4 metabolic protection.

Estetrol prevents obesity as well as associated vascular and hepatic metabolic disorders

induced by western diet in ovariectomized LDLR -/-mice.

We then evaluated the effect of E4 on lipid lesion deposits at the aortic sinus using LDLR -/- mice known to develop both atherosclerosis and steatosis in response to western diet (3, 24, 40, 42) (Figure 4A). As previously observed in WT C57Bl/6 mice, E4 also prevented dietinduced obesity (Figure 4B), improved glucose tolerance (Figure 4C) and protected the liver against triglycerides and cholesterol ester accumulation (Figures 4D and4E) in this LDLR -/- A c c e p t e d M a n u s c r i p t mouse model. Importantly, this metabolic protection was associated with less fatty streak at the aortic sinus (Figures 4F and4G) and a decreased expression of endothelial activation markers, such as Vascular Cell Adhesion Molecule 1 (Vcam1) and Intercellular Adhesion Molecule 1 (Icam1) (Figure 4H) in the aorta from E4-treated mice. Moreover, E4 treatment also decreased mRNA expression of pro-inflammatory cytokines such as interleukin (Il1β), macrophage inflammatory protein 1-alpha (Mip1α), and of the macrophage marker (Cd68) (Figure 4H). Altogether, these results demonstrate that, in addition to the protection against diet-induced obesity and steatosis, E4 also conferred atheroprotection.

Hepatic ERα is necessary for E4-mediated prevention of steatosis but dispensable for its protective action against obesity and atherosclerosis.

Hepatic ERα has been proposed to contribute to the protective effect of E2 on both atherosclerosis (49) and steatosis (13, 21, 23, 31), and is also necessary for the prevention of diet-induced obesity in response to some SERMs such as TAM [START_REF] Guillaume | Selective Liver Estrogen Receptor α Modulation Prevents Steatosis, Diabetes, and Obesity Through the Anorectic Growth Differentiation Factor 15 Hepatokine in Mice[END_REF]. Chronic E4 treatment did not regulate ERα protein level in liver from chow diet-fed mice (Supplementary Figure 4A). However, ERα protein level was decreased in liver from mice on western diet compared to mice on chow diet but this effect was prevented by E4 treatment (Supplementary Figure 4B). We then evaluated the transcriptional response induced by E4 on a set of ERα targetgenes previously identified in liver (35). To avoid the bias associated to the protective action of E4 against steatosis under western diet feeding, the transcriptional response induced by E4 was studied in liver from chow diet-fed mice. Most of the known ERα target genes tested were found to be regulated by E4 (Supplemental Table 2). The comparison of the transcriptional responses induced by E4, the full agonist E2 or the well-known SERM, TAM, identified subsets of genes specifically regulated by E4 or regulated by both E4 and TAM but not by E2 (Supplemental Table 2). Importantly, ChIP-qPCR analyses revealed the recruitment of ERα induced by E4 in the regulatory region of several of these genes, further Then, to directly evaluate the role of hepatic ERα, we analyzed the metabolic effects of chronic E4 treatment in mice harbouring a hepatocyte-specific ERα deletion (LERKO) bred with atheroprone LDLR -/-mice. Efficiency of ERα deletion in hepatocytes was confirmed by the 80 % decrease of ERα mRNA level measured by qPCR in LERKO-LDLR -/-mice compared to the control littermate mice (Supplementary Figure 6). Ovariectomized LERKO-LDLR -/-mice treated or not with E4 were fed a western diet for 9 weeks. The protection conferred by E4 against western diet-induced obesity was fully preserved in LERKO-LDLR -/-mice (Figure 5A and Supplementary Figure 7), demonstrating that hepatic ERα is not required to induce this beneficial action of E4. Hence, E4 reduced fat mass accumulation to a similar extent in WT-LDLR -/-and LERKO-LDLR -/-mice, at both subcutaneous and visceral sites (Figure 5C). In addition, E4 exposure protected similarly WT-LDLR -/-and LERKO-LDLR -/-female mice from glucose intolerance (Figure 5B) and atherosclerosis (Figure 5D) induced by western diet. Plasma HDL-and LDL-cholesterol profiles were also similar in WT-LDLR -/-and LERKO-LDLR -/-mice whereas the decrease in plasma triglycerides induced by E4 treatment in WT-LDLR -/-mice was totally abrogated in LERKO-LDLR -/-mice (Figure 5E). In addition, whereas E4 treatment prevented intrahepatic accumulation of triglycerides and cholesterol ester in WT-LDLR -/-control mice, this effect was abolished in LERKO-LDLR -/-mice (Figures 6A and6B). Accordingly, analysis of lipid droplets revealed that the liver protection conferred by E4 treatment was no longer observed in LERKO-LDLR -/-mice (Figure 6C), demonstrating the crucial role of hepatic ERα to the protection against steatosis in response to E4.

DISCUSSION

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

E4 is an estrogen currently considered for its highly promising clinical potential, in particular as a new HT for menopause. In this therapeutic indication, phase III development is ongoing but phase II clinical investigations already provided positive results [START_REF] Genazzani | Oral investigational drugs currently in phase I or phase II for the amelioration of menopausal symptoms[END_REF]. Indeed, in a multiple-rising-dose study involving postmenopausal women, E4 improved vasomotor symptoms and vaginal cytology, and a dose-dependent estrogenic effect was observed on endocrine parameters, bone turnover markers, lipids and lipoproteins, along with only a small effect on haemostatic parameters (10, 11). However, although this is an important issue to consider for HT, the ability of E4 to prevent postmenopausal metabolic disorders was still unknown. Indeed, the decline in endogenous estrogens at menopause leads to a redistribution of body fat, characterized by accumulation of visceral adiposity (7, 29, 41), which contributes to an increased risk of cardiovascular diseases, type 2 diabetes and steatosis (15). The major finding of this study is that a chronic E4 exposure prevents body weight gain, glucose intolerance and steatosis in ovariectomized C57Bl/6 mice submitted to a western diet. These findings were confirmed in the atheroprone LDLR -/-mouse model in which E4 treatment also prevented lipid accumulation at the aortic sinus highlighting the atheroprotective effect of E4 in such an obesity context.

We previously reported that E4 was able to activate the nuclear actions of ERα but not its membrane initiated signalling [START_REF] Abot | The uterine and vascular actions of estetrol delineate a distinctive profile of estrogen receptor α modulation, uncoupling nuclear and membrane activation[END_REF]. Here, we showed that ERαAF2 is necessary for the prevention of weight gain and associated disorders in response to E4, further demonstrating the importance of nuclear activation of ERα in E4-mediated action. However, to which extent this action is mediated through ERE-dependent pathway or involves other signalling pathways such as protein-protein interaction with other transcription factors as AP-1 or SP1 remains to be determined. Indeed, several studies highlighted the importance of the non-classical genomic ERα signalling pathways in the beneficial action of E2 against metabolic disturbances (36, 47).

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

Our observations in LERKO mice demonstrated that hepatic ERα is not necessary to the beneficial effects of E4 on body weight gain, glucose tolerance and atheroma. The increase in physical activity and energy expenditure conferred by E4 is reminiscent of the effects of a chronic E2 treatment that we and others previously reported [START_REF] Gourdy | Estrogen receptor subcellular localization and cardiometabolism[END_REF]28) and rather suggests a central effect of E4. Indeed, numerous studies have reported the crucial role of ERα in specific area of the central nervous system in mediating the regulation of energy balance in response to E2 (12,33,[START_REF] Xu | Central regulation of energy metabolism by estrogens[END_REF][START_REF] Xu | Distinct hypothalamic neurons mediate estrogenic effects on energy homeostasis and reproduction[END_REF]. ERα could also affects metabolism by controlling mitochondrial dynamics and function in both white and brown adipose tissues [START_REF] Zhou | Estrogen receptor α controls metabolism in white and brown adipocytes by regulating A c c e p t e d M a n u s c r i p t[END_REF] and the role of ERα in adipocytes could also contribute to the preventive effect of E4 against obesity and metabolic dysfunction.

Hepatic ERα is necessary for the prevention of steatosis conferred by E4. Nevertheless, we cannot exclude the contribution of other nuclear receptors since crosstalk of ERα with other nuclear receptor such as LXR and GR have been reported to mediate part of the protective effect of E2 against steatosis [START_REF] Han | Estrogen receptor ligands ameliorate fatty liver through a nonclassical estrogen receptor/Liver X receptor pathway in mice[END_REF]37). Interestingly, we found that E4 chronic treatment induces Gdf15 mRNA level in the liver, correlated with a direct binding of ERα on its promoter. We recently reported such a regulation in response to tamoxifen but not to E2 due to epigenetic modification [START_REF] Guillaume | Selective Liver Estrogen Receptor α Modulation Prevents Steatosis, Diabetes, and Obesity Through the Anorectic Growth Differentiation Factor 15 Hepatokine in Mice[END_REF]. Gdf15 induction by tamoxifen is sufficient to prevent dietinduced obesity, type 2 diabetes, and NAFLD [START_REF] Guillaume | Selective Liver Estrogen Receptor α Modulation Prevents Steatosis, Diabetes, and Obesity Through the Anorectic Growth Differentiation Factor 15 Hepatokine in Mice[END_REF]. However, by contrast to tamoxifen, selective deletion of hepatic ERα does not impact here the beneficial actions of E4 on body weight gain and glucose tolerance suggesting that GDF15 is not the main effector of E4 action. More generally, we reported that E4 displays a specific transcriptional program in the liver which differs from those of E2 and TAM. This underlines that each molecule displays a highly specific profile of gene regulation in the liver, consistently with their differential action on hepatic coagulation factors in humans [START_REF] Kluft | Reduced hemostatic effects with drospirenone-based oral contraceptives containing estetrol vs. ethinyl estradiol[END_REF]30). In addition to the nuclear activity of ERα, selective activation of membrane ERα using "pathway preferential estrogens" (PaPEs) was A c c e p t e d M a n u s c r i p t also reported to prevent steatosis (8, 27) and to regulate a specific pattern of genes that differs from E2, through activation of mTOR and MAPK signalling pathways independently of ERα DNA binding [START_REF] Madak-Erdogan | c r i p t[END_REF]. Altogether, these results demonstrate that different ERα subfunctions can be involved in a similar biological effect depending on the nature of the ligand through specific transcriptional program.

To conclude, this study demonstrates that E4 confers metabolic and vascular protection in mice. Together with the protection against osteoporosis and the limited impact on coagulation factors associated with E4 administration, our results complete the really promising clinical potential of this estrogen as a new hormonal treatment for menopause.

ACKNOWLEDGMENTS

The staff of the animal facilities and of the "Plateforme d'expérimentation fonctionnelle" is acknowledged for their skillful technical assistance. We acknowledge the skillful technical assistance of Anexplo-Genotoul, GeT-TQ Genopole and MetaToul-lipidomique. ERα accounts for sex differences in the ability to cope with an excess of dietary lipids.

GRANTS

Mol Metab 32: 97-108, 2020. doi: 10.1016/j.molmet.2019.12.009. 106-116, 2018. doi: 10.1016/j.molmet.2017.12.012. When a significant interaction between the 2 factors were revealed, a Fisher's LSD post-test was performed (Veh versus E4: ***P < 0.001, n=6 mice/group).

E.

Veh E4 C.

Intrahepatic lipid content

F.

Fatty streak

Veh E4

Surface (x1000) µm²

G.

Fatty streak quantification H.

C.

WT-LDLR -/-Veh WT-LDLR -/-E4 LERKO-LDLR -/-Veh LERKO-LDLR -/-E4 D.

Surface (x 1000) µm²

  principles and guidelines established by the National Institute of Medical Research (INSERM) and were approved by the local Animal Care and Use Committee (CEEA-122 :

A c c e

  p t e d M a n u s c r i p t (Hamamatsu Photonics, Hamamatsu, Japan) and analyzed with NDP view software (Hamamatsu Photonics, Hamamatsu, Japan).

A c c eFigure 5

 5 Figure 5).

  The work at INSERM U1048 was supported by INSERM, CHU and Université de Toulouse III, Faculté de Médecine Toulouse-Rangueil, ANR (NOSE4), Fondation de France (FDF, N°00086486), Fondation pour la Recherche Médicale (FRM ECO201806006691), Institut Universitaire de France and Mithra Pharmaceuticals. Work at the CNRS UMR6290 was supported by the CNRS and University of Rennes1. DISCLOSURES JM. Foidart is a consultant at Mithra, the company that develops estetrol-based women's healthcare products. JF Arnal at INSERM U 1048 / University of Toulouse 3 has received a research grant from Mithra. The remaining authors have no disclosures to report.

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  p t e d M a n u s c r i p t Polg1 and mitochondrial remodeling. Sci Transl Med 12, 2020. doi: 10.1126/scitranslmed.aax8096. 49. Zhu L, Shi J, Luu TN, Neuman JC, Trefts E, Yu S, Palmisano BT, Wasserman DH, Linton MF, Stafford JM. Hepatocyte estrogen receptor alpha mediates estrogen action to promote reverse cholesterol transport during Western-type diet feeding. Mol Metab 8:

Figure 1 :

 1 Figure 1: Estetrol (E4) prevents western diet (WD)-induced obesity and steatosis in wildtype (WT) female mice.

Figure 2 :

 2 Figure 2: Estetrol (E4) enhances energy expenditure in wild-type (WT) female mice fed a western diet.

Figure 3 :

 3 Figure 3: E4 prevents western diet-induced obesity and steatosis through ERαAF2 activation.

Figure 4 :

 4 Figure 4: Estetrol (E4) prevents atherosclerosis and hepatic steatosis in LDLR -/-female mice fed a western diet.

A c c e

  p t e d M a n u s c r i p t means ± SEM. t-test were used to analyze the effect of treatment, except for body weight gain evolution and glucose tolerance test for which two-way ANOVA of variance test with repeated measures and a Fisher's LSD post test was performed. (E4 exposure versus vehicle *P<0.05, **P < 0.01, ***P < 0.001, n=5/6 mice/group).

Figure 5 :

 5 Figure 5: Hepatic ERα is not necessary for the beneficial effect of E4 on metabolic disorders and atherosclerosis in LDLR -/-female mice fed a western diet.Four-week-old ovariectomized female LERKO-LDLR -/-mice and their littermates were implanted subcutaneously with E4 or Vehicle (Veh) pellets and concomitantly fed a western diet from the age of 6 to 15 weeks. Cumulative weekly body weight gain evolution (A), intraperitoneal glucose tolerance (B) and adipose tissue distribution (C) are presented.Quantification of lipid deposition at the aortic sinus (scale bars: 1mm) (D) and plasmatic HDL-cholesterol, LDL-cholesterol and triglycerides content (E) were quantified. Two-way ANOVA were performed to evaluate interaction between Genotype (Gen) and treatment (E4).

Figure 6 :

 6 Figure 6: Hepatic ERα is necessary for the beneficial effect of E4 on hepatic steatosis in LDLR -/-female mice fed a western diet Four-week-old ovariectomized female LERKO-LDLR -/-mice and their littermates were implanted subcutaneously with E4 or Vehicle (Veh) pellets and concomitantly fed a western diet for 9 weeks from the age of 6 to 15 weeks. Quantification of intrahepatic triglycerides (A) and cholesterol ester (B) content. Representative images of haematoxylin and eosin (H/E) (scale bars: 160µm) and Oil Red O staining (scale bars: 510µm) on liver sections (C). Two-way ANOVA were performed to evaluate interaction between Genotype (Gen) and treatment (E4). When a significant interaction between the 2 factors were revealed, a Fisher's LSD posttest was performed (Veh versus E4: ***P < 0.001 n=6 mice/group).

  Figure 1

  Figure4

  Interaction p =0.75 E4 p < 0.0001 Gen p = 0.05 Interaction p = 0.96 E4 p < 0.0001 Gen p = 0.08 Interaction p = 0.67 E4 p < 0.0001 Gen p = 0.62

  Figure5

Table 1 : Metabolic parameters of ERαAF2 +/+ vs ERαAF2 0 mice treated with E4 or vehicle and fed a western diet

 1

		ERαAF2	+/+	ERαAF2 0	Two-factor
							ANOVA
		Veh		E4	Veh	E4	P, Interaction
	Body weight (g)	38.3 ± 2	24.5 ± 0.9 ***	37.7 ± 0.7	37.1 ± 1.2	*** p<0.0001
	Uterine weight (mg)	8.5 ± 1.2	109.2 ± 21 ***	5.5 ± 0.7	5.1 ± 0.5	*** p<0.0001
	Subcutaneous Tissue	7.3 ± 0.3	2.5 ± 0.2 ***	8 ± 0.3	7.1 ± 0.2 *	*** p<0.0001
	(% body weight)
	Perigonadal Tissue	5 ± 0.1	1.2 ± 0.05 ***	4.8 ± 0.2	4.6 ± 0.4	*** p<0.0001
	(% body weight)
	Mesenteric Tissue	2.4 ± 0.3	1 ± 0.1 ***	2.9 ± 0.2	2.6 ± 0.1	** p=0.0015
	(% body weight)
	Intrahepatic Triglycerides	60.6 ± 3.6	25.1 ± 5.7 *	50.7 ± 18.2	74.3 ± 11.8	* p=0.015
	(µg/mg of liver)
	Four-week-old ovariectomized ERαAF2 0 female mice and their littermates were implanted
	subcutaneously with E4 or vehicle (Veh) pellets and concomitantly fed a western diet from the
	age of 6 to 15 weeks. Body weight, uterine weight, subcutaneous, perigonadal and mesenteric
	tissues weight and intrahepatic triglycerides were evaluated. Results are expressed as means ±
	SEM. Two-way ANOVA were performed to evaluate interaction between Genotype and
	treatment (E4). Fisher's LSD post-test were performed (Veh versus E4: *P < 0.05, **P<0.01,
	***P < 0.001 n=6/8 mice/group).

AUTHORS CONTRIBUTION

MB, MD, RZ, RM performed and analyzed experiments. MF and GC performed estetrol dosage. CM and JMF provided intellectual guidance in all phases of the study. FL, PG, MA, JFA and CF designed the studies, analyzed experiments, wrote the manuscript and oversaw the project.


Qing Wang

Didier Tichit

Frederic Meunier

Hazar Guesmi

Combined DRIFTS and DFT study of CO adsorption and segregation modes in Pt-Sn nanoalloys

This contribution aims at rationalizing the observations made by in situ IR spectroscopy during CO adsorption over 1.8 nm Pt-Sn nanoparticles by Density Functional Theory (DFT) calculations and describing the environment and electronic properties of surface Pt atoms modified by Sn. Pt surface enrichment upon CO exposure was observed by DRIFTS (diffuse reflectance FT-IR spectroscopy) and rationalized by the theoretical calculations, which also indicated that isolated Pt was favored over Pt pairing in a Sn-rich alloy. DFT frequency calculations allowing the fine assignment of vibrational CO spectra. The results show gradual decay of the linear carbonyl wavenumber from plain Pt (2075 cm -1 ), through a Sn-poor Pt-Sn (2054 cm -1 ) and down to a Sn-rich Pt-Sn nanoparticles (2039 cm -1 ). This decay is accompanied by a weakening of Pt-CO bonds, which confirm the ability of Sn to prevent CO poisoning of Pt when present in a nanoalloy. Moreover, electronic structure analyses evidence a preponderant charge transfer from Sn to Pt atoms, which explains the weakening of both the Pt-CO bond and the C-O bond through back-donation to antibonding molecular orbitals. A weaker C-O bond would favor CO dissociation and the formation of Pt and SnOx ensembles, which is consistent with DRIFTS data recorded at low temperatures.

I. Introduction

Multimetallic catalyst formulations offer clear opportunities in terms of reducing the reliance on noble or rare metals and the improvement of catalyst performances [START_REF] Zafeiratos | Alloys in catalysis: phase separation and surface segregation phenomena in response to the reactive environment[END_REF][START_REF] Dasgupta | Intermetallics in catalysis: An exciting subset of multimetallic catalysts[END_REF][START_REF] Juárez | Catalytic properties of Au electrodes modified by an underlayer of Pd[END_REF] . In this context, tin has frequently been used as a promoter to alter the properties of noble metal-based catalysts.

For instance, Sn alloyed with Pd, showed significant increase in the selectivity of numerous hydrogenation reactions [START_REF] Verdier | Pd-Sn/Al2O3 catalysts from colloidal oxide synthesis -II. Surface characterization and catalytic properties for buta-1,3-diene selective hydrogenation[END_REF][START_REF] Aduriz | Alumina-supported bimetallics Bof palladium alloyed with germanium, tin, lead or antimony, from organometallic precursors .2. Gas-phase hydrogenation of 2-methyl-1-buten-3-yne (valylene) and 2methyl-1,3-butadiene (isoprene)[END_REF][START_REF] Choi | XAFS study of tin modification of supported palladium catalyst for 1,3-butadiene hydrogenation in the presence of 1-butene[END_REF] . Choi and Lee reported modifications of the Pd electronic density (so-called ligand effect) and nanoparticle morphologies (so-called geometric or ensemble effect) induced by Sn addition [START_REF] Choi | XAFS study of tin modification of supported palladium catalyst for 1,3-butadiene hydrogenation in the presence of 1-butene[END_REF] . The reported Pd K-edge XANES data indicated that Sn-free Pd was in an electro-deficient hydride state, while Sn-modified Pd was in a zero valent state and Pd ensembles were replaced with Pd-Sn entities. Using CO adsorption and temperature programmed methods some of us observed that the geometric effect of Sn over Pd was very significant, by totally removing Pd-Pd pairs on which bridged CO was able to form [START_REF] Jbir | Individual Heat of Adsorption of Adsorbed CO Species on Palladium and Pd-Sn Nanoparticles Supported on Al2O3 by Using Temperature-Programmed Adsorption Equilibrium Methods[END_REF] . In contrast, the effect on the heat of adsorption of linear CO, which is controlled by the electronic properties of the adsorption site (or ensemble), was modest.

Bimetallic Pt-Sn supported catalysts have been investigated in a large range of reactions such as selective hydrogenation of unsaturated compounds [START_REF] Mäki-Arvela | Chemoselective hydrogenation of carbonyl compounds over heterogeneous catalysts[END_REF][START_REF] Santori | Hydrogenation of crotonaldehyde on Pt/SiO2 catalysts modified with tin added via surface organometallic chemistry on metals techniques[END_REF][START_REF] Margitfalvi | Characterization of Sn-Pt/SiO2 catalysts used in selective hydrogenation of crotonaldehyde by Mossbauer spectroscopy[END_REF][START_REF] Taniya | Preparation of Sn-modified silica-coated Pt catalysts: A new Pt-Sn bimetallic model catalyst for selective hydrogenation of crotonaldehyde[END_REF][START_REF] Llorca | Activation of carbon dioxide by a silica-supported platinum-tin bimetallic complex[END_REF][START_REF] Ruiz-Martínez | Effect of tin content and reduction temperature on the catalytic behaviour of PtSn/TiO2 catalysts in the vapour-phase hydrogenation of crotonaldehyde[END_REF] , dehydrogenation of light alkanes to alkenes where Sn is the most studied promoter of the platinum-based catalysts [START_REF] Sattler | Catalytic Dehydrogenation of Light Alkanes on Metals and Metal Oxides[END_REF][START_REF] Kumar | Dehydrogenation of propane over Pt-SBA-15 and Pt-Sn-SBA-15: Effect of Sn on the dispersion of Pt and catalytic behavior[END_REF][START_REF] Furukawa | Catalytic properties of Pt-based intermetallic compounds in dehydrogenation of cyclohexane and n-butane[END_REF][START_REF] Kaylor | Propane dehydrogenation over supported Pt-Sn nanoparticles[END_REF][START_REF] Pham | Role of Sn in the Regeneration of Pt/gamma-Al2O3 Light Alkane Dehydrogenation Catalysts[END_REF] , low temperature oxidation of carbon monoxide [START_REF] Moscu | CO PROX over Pt-Sn/Al2O3: A combined kinetic and in situ DRIFTS study[END_REF][START_REF] Michalak | CO oxidation on PtSn nanoparticle catalysts occurs at the interface of Pt and Sn oxide domains formed under reaction conditions[END_REF][START_REF] Schubert | Bimetallic PtSn catalyst for selective CO oxidation in H-2-rich gases at low temperatures[END_REF] . A wide variety of parameters, mainly the Pt/Sn atomic ratio, the preparation procedure (e. g. conventional coimpregnation, chemical vapor deposition, polyalcohol reduction process), the nature of the precursors and of the support influence the electronic and geometric structure of the bimetallic particles and the formation of PtxSny alloys which determine the performances of the catalysts. For example, Taniya et al. [START_REF] Taniya | Preparation of Sn-modified silica-coated Pt catalysts: A new Pt-Sn bimetallic model catalyst for selective hydrogenation of crotonaldehyde[END_REF] have prepared unsupported bimetallic PtSnnanoparticles using a polyalcohol reduction process in order to avoid the influence of the metal-support interaction. Their catalytic behavior was examined for the selective hydrogenation of crotonaldehyde to crotyl alcohol (UOL). Pt3Sn1, Pt1Sn1 and Pt1Sn2 alloys were successively formed as the Pt/Sn atomic ratio increases. The UOL selectivity depends on the composition of the major PtxSny alloy species present in the catalyst. Highest total hydrogenation was obtained with Pt3Sn1 enhancing both C=C and C=O bonds hydrogenation.

Pt1Sn1 alloy leads to the highest UOL selectivity which then decreases at the expense of butyraldehyde when Pt1Sn2 alloy is formed due to preferential C=C bond hydrogenation. Kaylor et al. prepared Pt-Sn/SiO2 and Pt-Sn/Al2O3 bimetallic supported catalysts with different Sn loading via incipient wetness impregnation or polyol method for propane dehydrogenation to propene [START_REF] Kaylor | Propane dehydrogenation over supported Pt-Sn nanoparticles[END_REF] . Pt3Sn and Pt1Sn1 alloys were detected by XRD whatever the preparation method and the support. Catalytic performances, particularly stability and selectivity to propene, depended on the strength of interaction of Sn with Pt relative to the support.

Pt-Sn alloy is also promising materials for Proton-Exchange Membrane Fuel Cells (PEM-FC), as Pt3Sn1 supported on carbon showed much improved resistance to CO poisoning as compared to Pt and many other alloys [START_REF] Leng | Assessment of CO-tolerance for different Pt-alloy anode catalysts in a polymer electrolyte fuel cell using ac impedance spectroscopy[END_REF] . The improved resistance to CO poisoning was later attributed in part to lower CO adsorption coverage on the Pt sites [START_REF] Wang | Kinetics investigation of H-2/CO electro-oxidation on carbon supported Pt and its alloys using impedance based models[END_REF] . As reported on our previous study [START_REF] Moscu | Direct evidence by in situ IR CO monitoring of the formation and the surface segregation of a Pt-Sn alloy[END_REF] , the heat of adsorption of CO on Pt in alumina-supported Pt-Sn alloy nanoparticles was measured to be 95 ± 5 kJ mol -1 , which is about half the value measured on plain Pt (180 ± 5 kJ mol -1 ). This large difference makes the bonding of CO on the Pt of this Pt-Sn alloy similar to that measured on Cu°/alumina (85 ± 5 kJ mol -1 ) [START_REF] Dulaurent | Heats of adsorption of CO on a Cu/Al2O3 catalyst using FTIR spectroscopy at high temperatures and under adsorption equilibrium conditions[END_REF] and can explain the better resistance of Sn-modified Pt against CO poisoning. The sample above-mentioned was prepared from colloid-stabilized nanoparticles that exhibited the stoichiometry Pt3Sn [START_REF] Boualleg | Unexpected, spontaneous and selective formation of colloidal Pt3Sn nanoparticles using organometallic Pt and Sn complexes[END_REF] . The exact stoichiometry of the derived alumina-supported Pt-Sn nanoparticles could not be determined accurately because a fraction of tin was converted to SnOx species interacting with the alumina during the deposition/calcination process, while some of the Sn-containing ligand could have also decomposed and further reacted with the nanoparticles upon the final reduction [START_REF] Moscu | CO dissociation on Pt-Sn nanoparticles triggers Sn oxidation and alloy segregation[END_REF] . This Pt3Sn-like sample is therefore forthwith noted "Sn-poor" Pt-Sn/alumina. The colloidal procedure was used to prepare a Sn-rich sample (vide infra), except that the ratio of the Sn/Pt precursors was increased 12-fold as compared to the case of the Sn-poor material, aiming at a PtSn4-like structure. Note that Pt3Sn and PtSn4 are the two extreme compositions of Pt-Sn intermetallic compounds, which also include Pt1Sn1, Pt2Sn3 and PtSn2.

All the previous results emphasize the main role of the environment and electronic properties of surface Pt atoms modified by Sn. This was an incitement to focus in the present work on the use of CO adsorption monitored by in situ diffuse reflectance FT-IR spectroscopy (DRIFTS) as a means to characterize these properties. The IR experimental data are then compared to DFT models of CO adsorption applied to idealized surfaces and nanoparticles.

The main aim is to highlight any possible difference between the Sn-rich and Sn-poor Pt-Sn nanoparticles in terms of electronic density probed by CO adsorption. To this end, IR monitoring of CO adsorption is a powerful technique as it probes only the surface atoms, the most relevant for catalysis, and provides a statistical average over a large sample volume.

However, CO adsorption can yet modify alloy surfaces, even at room temperature. Surface segregation was observed on Au-Pd nanoparticles as Pd, which forms a much stronger bond with CO than Au does, was gradually pulled towards the surface [START_REF] Creuze | Surface segregation in AuPd alloys: Ab initio analysis of the driving forces[END_REF][START_REF] Zhu | Evidence of Pd segregation and stabilization at edges of AuPd nano-clusters in the presence of CO: A combined DFT and DRIFTS study[END_REF][START_REF] Zhu | CO Adsorption-Induced Surface Segregation and Formation of Pd Chains on AuPd(100) Alloy: Density Functional Theory Based Ising Model and Monte Carlo Simulations[END_REF] . More, phase segregation induced by CO dissociation was also reported on Pt-Sn nanoparticles, as the resulting oxygen reacted with Sn forming tin oxide domains intimately mixed with Pt [START_REF] Tsuda | Ab initio study of alloying and straining effects on CO interaction with Pt[END_REF] . These phenomena are yet kinetically slow (timescale of tens of minutes) and can be mitigated by carrying out rapid analyses. The effect of CO dissociation can also be mitigated by carrying out CO adsorption in the presence of H2, which probably removes any oxygen formed preventing Sn oxidation [START_REF] Tsuda | Ab initio study of alloying and straining effects on CO interaction with Pt[END_REF] . Such approach was fruitfully used in the present work. In addition, DFT calculations were performed to provide insights into the atomistic-level processes of CO adsorbate-induced segregation in Pt-Sn alloy surfaces with models representing Sn-poor and Sn-rich Pt-Sn nanoalloys.

II. Experimental and Theoretical methods

II.1. Experimental methods

Materials and gases

The preparations of the Pt/alumina and Sn-poor Pt-Sn/alumina materials have been reported elsewhere [START_REF] Boualleg | Unexpected, spontaneous and selective formation of colloidal Pt3Sn nanoparticles using organometallic Pt and Sn complexes[END_REF] . In brief, the method consisted in a two-step synthesis using a colloidal approach.

A mixture of a Pt complex [Pt(dba)2 (dba=dibenzylidene acetone)] and octylsilane, which plays the role of ligand, was used for the synthesis of Sn-free Pt monometallic nanoparticles. The Sn-poor Pt-Sn nanoparticles were obtained by contacting equimolar amounts of Pt(dba)2 dried under vacuum and tributyltin hydride [(n-Bu)3SnH] without the addition of any other ligand.

The precursors were dissolved in dry tetrahydrofuran (THF) and pressurized with H2 (3 bar) during 10 h at room temperature. A fraction of the Sn-precursors reduced forming Pt-Sn nanoparticles, while another fraction remained as ligands stabilizing the nanoparticles. These colloidal suspensions were then used for the second step of the catalyst preparation. Following the evaporation of the solvent, a small volume of the suspension was added to the γ-Al2O3 support (specific BET surface area 106 m 2 /g) under continuous stirring and non-oxidative atmosphere and dried overnight under Ar. The dry crude catalysts were subsequently calcined under flowing dry air at 320 °C in order to remove most of the alkyl fragments. Note that a fraction of tin was converted to SnOx species interacting with the alumina during this process [START_REF] Tsuda | Ab initio study of alloying and straining effects on CO interaction with Pt[END_REF] . An identical procedure was used for the preparation of the Snrich sample, except that the ratio of the Sn/Pt precursors was increased 12-fold as compared to the case of the Sn-poor material. The particle size distribution of this sample determined by TEM was narrow and centred around 1.8 nm (data not shown), that is essentially identical to those of the plain Pt and Sn-poor samples that were reported in our previous work [START_REF] Moscu | Direct evidence by in situ IR CO monitoring of the formation and the surface segregation of a Pt-Sn alloy[END_REF] .

High purity He and H2 from Air Liquide (Alphagaz, 99.999% purity) further purified with using VICI® traps (models P-100-2 and P-200-2 for He and H2, respectively) were used, which should have brought down O2 level below 1 ppm. High purity CO (>99%, main impurity N2 ~ 0.4 mol. % and H2 ~ 0.5 mol. %) was used without further purification.

Diffuse Reflectance FT-IR spectroscopy analysis.

The DRIFTS experiments were performed at ambient pressure with a high temperature DRIFT cell (from Spectra-Tech®) fitted with KBr windows, using a collector assembly. A description and properties of the cell can be found elsewhere [START_REF] Li | Effective bulk and surface temperatures of the catalyst bed of FT-IR cells used for in situ and operando studies[END_REF] . The spectrophotometer used was a Nicolet 6700 (ThermoFischer Scientific) fitted with a liquid-N2 cooled MCT detector. The DRIFT spectra were recorded at a resolution of 4 cm -1 and 8 or 32 scans accumulation depending on the time-resolution desired.

The DRIFTS spectra are reported as log (1/R), where R is the sample reflectance. This pseudo-absorbance gives a better linear representation of the band intensity against surface coverage than that given by the Kubelka-Munk function for strongly absorbing media such as those based on metals supported on oxides [START_REF] Sirita | Quantitative analysis of adsorbate concentrations by diffuse reflectance FT-IR[END_REF] . The contribution of gas-phase CO was subtracted using a CO(g) spectrum collected at the reaction temperature over a SiC powder [START_REF] Paredes-Nunez | Spectrum baseline artefacts and correction of gas-phase species signal during diffuse reflectance FT-IR analyses of catalysts at variable temperatures[END_REF] .

The temperature of the sample surface was determined according to a calibration curve obtained using an optical pyrometer as described elsewhere [START_REF] Li | Effective bulk and surface temperatures of the catalyst bed of FT-IR cells used for in situ and operando studies[END_REF] .

The sample was reduced in situ under a flow of 50 mL min -1 of pure H2 at 400 °C for 30 min before going to the analysis temperature and introducing a flow of 2% CO/H2. The production of methane was monitored using an IR gas-cell connected to the DRIFTS cell vent and was found to be negligible.

II.2. Theoretical details

Assessment of the energetics and electronic structures relevant for adsorbate-induced segregation phenomena are provided by periodic self-consistent DFT analysis using the Vienna ab initio simulation package (VASP) [START_REF] Kresse | Abinitio molecular dynamics for liquid-metals[END_REF] . The exchange-correlation energy was calculated within the Perdew, Burke and Ernzerhof formulation of the generalized-gradient approximation (GGA-PBE) [START_REF] Perdew | Accurate and simple analytic representation of the electrongas correlation energy[END_REF] . The valence electrons were treated explicitly, and their interactions with the ionic cores were described by the projector augmented-wave (PAW) method [START_REF] Blöchl | Improved tetrehedron method for Brillouinzone integrations[END_REF][START_REF] Kresse | From ultrasoft pseudopotentials to the projector augmentedwave method[END_REF] , which allowed the use of a low energy cut off equal to 420 eV for the planewave basis. Super cell atomic positions were relaxed until the total energy differences decrease below 10 -4 eV. In order to accurately capture weak interactions such as Van der Waals forces, DFT-D3 calculations including dispersion correction proposed by Grimme [START_REF] Grimme | A consistent and accurate ab initio parametrization of density functional dispersion correction (DFT-D) for the 94 elements H-Pu[END_REF] were performed. The calculations of vibrational spectra of selected surface species were performed within harmonic approximation where only the CO center and its first and second neighbors (platinum and tin atoms) were considered in the Hessian matrix. This matrix was computed by the finite difference method followed by a diagonalization procedure. The eigenvalues of the resulting matrix led to the frequency values.

To calculate the segregation energies in presence and absence of CO gas, highly diluted systems formed by single Sn (Pt) atom in Pt (Sn) semi-infinite surfaces were considered. For Pt, three low index Pt surfaces (111), ( 100) and (110) were built from optimized FCC bulk structure. According to the work of Zhu et al. [START_REF] Zhu | Equilibrium Shape of Metal Nanoparticles under Reactive Gas Conditions[END_REF] these orientations are predicted to form the main facets of stable Pt nanoparticles under CO pressure. The bulk lattice constant was found to be of 3.96 Å which is in good agreement with experimental measured value of 3.92 Å [START_REF] Kittel | Introduction to Solid State Physics[END_REF] .

Concerning Sn, experiments indicate that bulk Sn has both diamond (-phase), and tetragonal (-phase) structures [START_REF] Busch | Semiconducting Properties of Gray Tin[END_REF] . Hörmann et al. [START_REF] Hörmann | Semiconductor-metal transition induced by nanoscale stabilization[END_REF] have reported that, independent of temperature, Sn nanoparticles smaller than 8 nm diameter always crystallize in the -phase structure in thermodynamic equilibrium. In addition, based on DFT calculations and thermodynamic considerations these authors have shown that the low-index (100) surfaces are the most stable. Based on these results, the low-index (100) surface was built from the optimized -Sn (body-centered tetragonal, BCT) bulk structure. The calculated equilibrium lattice constants (a, b = 5.94 Å, c = 3.21 Å) were found to be slightly overestimated compared to the experimental values (a = b = 5.83 Å, c = 3.18 Å at 298.15 K) [START_REF] Jette | Precision determination of lattice constants[END_REF] , but in good agreement with reported theoretical results [START_REF] Moscu | CO dissociation on Pt-Sn nanoparticles triggers Sn oxidation and alloy segregation[END_REF][START_REF] Tsuda | Ab initio study of alloying and straining effects on CO interaction with Pt[END_REF][START_REF] Paredes-Nunez | Spectrum baseline artefacts and correction of gas-phase species signal during diffuse reflectance FT-IR analyses of catalysts at variable temperatures[END_REF] . The surface slabs consist of 3 x 3 unit cells separated by 15 Å of vacuum space for Pt surfaces and 2 x 3 unit cells with 15 Å of vacuum space for Sn surface.

The Brillouin zone integrations were performed on a Monkhorst-Pack (3 x 3 x 1) and (2 x 3 x 1) k-point mesh for Pt and Sn surfaces, respectively. Total relaxation of all metallic atoms in the top four layers of the slab and the adsorbed CO molecule was allowed. The bottom two layers were constrained at the bulk geometries. Once the surfaces were optimized, Sn and Pt single impurities were substituted in different layers of the host Pt and Sn surfaces, respectively, and total energies were calculated. The segregation energy (Eseg) of Sn (Pt) in Pt (Sn) matrix was defined by equation 1.

Eseg. = [EPt-Sn(x-layer) -EPt-Sn (4th-layer)]

(Eq.1)

where EPt-Sn (x-layer) represents the total energy of the Pt-Sn alloy system with Sn(Pt) atoms located in the upper x Pt(Sn) layers (x= 1, 2 or 3), and EPt-Sn (4th-layer) represents the total energy of the Pt-Sn alloy system with the Sn(Pt) atom located in the 4 th Pt(Sn) layer, which corresponds to the center of the slab, i.e. to the bulk-like position. In the presence of adsorbed CO molecule, the segregation energies were calculated by considering the energetically most stable configuration.

To study the CO probe adsorption mechanisms and its vibrational frequencies on Pt-Sn nanoalloyed system, pure Pt and Pt-Sn clusters of different Sn ratio were modeled. Cubic cell with the side of 2 nm was chosen to provide enough spacing between metal clusters in the neighboring repeated cells. Truncated-cubo-octahedral (TOH) shape of 38 atoms was cut from bulk fcc metal presenting (111) and (100) facets. According to our previous theoretical studies [START_REF] Ismail | Investigation of the structures and chemical ordering of small Pd-Au clusters as a function of composition and potential parameterisation[END_REF][START_REF] Pittaway | Theoretical Studies of Palladium-Gold Nanoclusters: Pd-Au Clusters with up to 50 Atoms[END_REF] , this selected size and structure offers a good compromise between time calculation and theoretical level of accuracy. To model Sn-rich (PtSn4) and Sn-poor (Pt3Sn) Pt-Sn nanoparticles, TOH_Pt7Sn31 and TOH_Pt29Sn9 clusters were considered, respectively. As will be argued bellow, the choice of the selected configurations was based on the study of the energetics of the segregation and mixing of Pt-Sn system. Brillouin zone integrations were performed at the Gamma point and all atoms were relaxed during geometry optimizations.

According to their coordination numbers, the surface atoms may have two coordination numbers: 6 for edge position and 9 for center face position. Hereafter, we define all possible CO adsorption sites as follows: two top sites, over surface atom located at an edge position (top-ed) or at a center face position (top-fc); two bridge sites, between two atoms located at the edge position (BDG-ed-ed) and between edge and facet atoms (BDG-ed-fc); three-fold sites between three adjacent atoms on the (111) facet and four-fold sites between four adjacent atoms on the (100) facet. The adsorption energy of CO molecule was calculated by equation 2:

Eads=Ecluster+CO -ECO -Ecluster (Eq.2)
where Ecluster+CO is the total energy of the CO adsorbed on the metallic cluster, ECO and Ecluster are the total energies of free CO and metal cluster, respectively.

III. Results and discussions

III.1. Experimental results

DRIFTS spectra obtained between 200 °C down to 50 °C over the Sn-rich Pt-Sn/alumina under a flow of 2% CO/H2 are shown in Figure 1. H2 was kept in the feed to limit the phase segregation of the Pt-Sn nanoparticles due to minute CO dissociation and O reaction with Sn [START_REF] Moscu | CO dissociation on Pt-Sn nanoparticles triggers Sn oxidation and alloy segregation[END_REF][START_REF] Vandichel | A dimer path for CO dissociation on PtSn[END_REF] . The spectra recorded at 200 °C exhibited a main band at 2032 cm -1 , which shifted towards 2040 cm -1 at 100 °C. This band is assigned to linear CO adsorbed on Pt in a Pt-Sn phase [START_REF] Moscu | CO dissociation on Pt-Sn nanoparticles triggers Sn oxidation and alloy segregation[END_REF][START_REF] Virnovskaia | Characterization of Pt,Sn/Mg(Al)O catalysts for light alkane dehydrogenation by FT-IR Spectroscopy and catalytic measurements[END_REF] .

From 200 °C down to 100 °C the band remained mostly symmetric, indicating a homogeneity in the nature of the Pt sites present. However, the band developed a shoulder on the high wavenumber side below 100°C that was clearly visible at 50 °C. (Red spectrum in Fig. 1). This shoulder and change of the band shape are especially visible when comparing the spectra at 150 °C and 50 °C (Inset in Fig. 1). This observation is indicative of the presence of plain Pt nanoparticles, likely developing around SnOx domains, following the dissociation of CO leading to the oxidation of tin and carbon deposition, as was already reported for the Snpoor Pt-Sn/alumina sample [START_REF] Moscu | CO dissociation on Pt-Sn nanoparticles triggers Sn oxidation and alloy segregation[END_REF] . The spectra below 100 °C are thus associated with a mixture of Pt/SnOx and Pt-Sn phases and do not represent only the intermetallic compound. In fact, bridging CO becomes visible at 1885 cm -1 for the spectrum collected at 50 °C, confirming the formation of Pt-Pt pairs and the aggregation of Pt atoms at this temperature [START_REF] Meunier | Unravelling Platinum-Zirconia Interfacial Sites Using CO Adsorption[END_REF][START_REF] Hayden | The adsorption of CO on Pt(111) studied by infrared reflection-absorption spectroscopy[END_REF][START_REF] Aleksandrov | Can the state of platinum species be unambiguously determined by the stretching frequency of an adsorbed CO probe molecule?[END_REF] . The generalized Temkin model used to determine the heats of adsorption at zero and full CO coverage has been described in details elsewhere [START_REF] Jbir | Individual Heat of Adsorption of Adsorbed CO Species on Palladium and Pd-Sn Nanoparticles Supported on Al2O3 by Using Temperature-Programmed Adsorption Equilibrium Methods[END_REF][START_REF] Dulaurent | Adsorption isobars for CO on a Pt/Al2O3 catalyst at high temperatures using FTIR spectroscopy: isosteric heat of adsorption and adsorption model[END_REF] . The model assumes that the heat of adsorption linearly increases with decreasing coverage. The method consists in optimizing the values of adsorption heats at zero and full coverage to obtain the best agreement between the experimental and model curves representing the surface coverage as a function of temperature (Figure 2). According to Figures 1 and2 the full coverage by CO was assumed to be achieved at 100 °C, in view of the evolution of the band areas (Figure 1 and2).

The adsorption heats at zero and full coverage thus determined for the Sn-rich Pt-Sn/alumina were 85 ± 5 and 75 ± 5 kJ mol -1 , respectively. These values are reported in Table 1 and compared to those measured earlier on alumina-supported Pt and Sn-poor Pt-Sn. a see reference [START_REF] Moscu | Direct evidence by in situ IR CO monitoring of the formation and the surface segregation of a Pt-Sn alloy[END_REF] No alloy segregation into Pt + SnOx can be observed above 100 °C in the presence of both CO and H2. However, a rather slow but gradual signal increase at essentially constant wavenumber was observed following the introduction of CO in the H2 feed on the pre-reduced Sn-rich Pt-Sn sample at higher temperatures. A set of spectra collected at 420 °C is presented in Figure 3 to exemplify this observation. This high temperature was chosen because it allowed observing the phenomenon over a reasonably short period of time of about 8 minutes. The observed signal increase was not due to the time required to flush the gas cell dead volume from pure H2 to the 2 % CO/H2 mixture, which requires less than 15 s.

Over the 8-min period the band hardly shifted from 2029 to 2032 cm -1 , which is within the experimental error. However, the band intensity grew by about 50 %, indicating a large increase of the concentration of Pt atoms at the surface of the Sn-rich Pt-Sn/alumina. This observation indicates that Pt was pulled to the surface from the bulk due to the formation of Pt-CO bond. A similar surface segregation phenomenon has been reported for Pd-Au alloys upon CO gas exposure [START_REF] Creuze | Surface segregation in AuPd alloys: Ab initio analysis of the driving forces[END_REF][START_REF] Zhu | Evidence of Pd segregation and stabilization at edges of AuPd nano-clusters in the presence of CO: A combined DFT and DRIFTS study[END_REF][START_REF] Zhu | CO Adsorption-Induced Surface Segregation and Formation of Pd Chains on AuPd(100) Alloy: Density Functional Theory Based Ising Model and Monte Carlo Simulations[END_REF] . After 8 min the signal was stable, indicating that a new equilibrium between surface and bulk Pt atoms had been reached. It is important to note that this surface segregation (implying variations of the Pt and Sn surface and bulk concentrations of the Pt-Sn nanoparticles) is different from the alloy segregation due to CO dissociation observed in Figure 1 (implying the formation of separate Pt and SnOx domains).

III.2. Theoretical results

Segregation phenomenon

In order to get an insight into the CO induced segregation of Sn and Pt in Pt-Sn, we performed DFT calculations of the segregation energies of Sn atom on Pt (111), ( 100) and ( 110)

surfaces and of Pt atom on Sn(100) surface (calculated from Eq. ( 1)). The analyzed systems could represent both highly diluted Sn-poor and Sn-rich Pt-Sn systems, respectively. Under vacuum conditions, the single Sn presents a similar behavior on the three facets;

it is highly stable in the first layer of the three considered surfaces (Eseg = -0.8 eV/atom, -1.01 eV/atom and -1.18 eV/atom for (111), (100) and (110) surface, respectively). Interestingly, the more open the surface is, the more stable the Sn in the first layer of Pt. This stability gets worse when Sn is located on the sub-surface layer (second layer position of the slab) and in

Layer position the Pt bulk (third and fourth layers). These results can be explained by two facts. On one hand, the lower surface energy of Sn compared to that of Pt favors the segregation of Sn toward the upper surface layer; experimental values are 0.709 J m -2 for Sn and 2.489 J m -2 for Pt [START_REF] Vitos | The surface energy of metals[END_REF] . On the other hand, because of the higher atomic radius of Sn compared to Pt (1.55 Å vs. 1.39 Å) [START_REF] Kittel | Introduction to Solid State Physics[END_REF] , the isolated Sn atoms prefer occupying under-coordinated surface sites. The segregation energy of Sn was also calculated in presence of CO adsorbed on the surfaces of Pt. As expected, CO does not directly interact with Sn atom and mostly prefers the top site of Pt atoms. As shown by the dashed lines in Figure 4a. the presence of CO slightly disturbs the stability of the system but do not change the chemical atomic ordering on the surface.

E seg (eV) 1 2 3 4 • • • • 1 2 3 4 • • • • 1 2 3 4 1 2 3
Considering Pt in Sn(100) surface, the Figure 4b shows the evolution of segregation energy in presence and in absence of CO. Under vacuum, the system shows better stability of Pt in the second layer then in the surface and the bulk. In the subsurface layer (2 nd layer) of Sn surface, the system is predicted to be slightly more stable (-0.06 eV). The energetically favorable location of the Pt atom at the second layer of Sn matrix can be explained by the fact that the Pt has higher surface energy and higher cohesive energy than Sn and that its presence in the second layer allows releasing the elastic energy due to the atomic size mismatch.

Interestingly, under gas, the segregation behavior drastically changes. Indeed, in the presence of one CO adsorbed on top of isolated Pt, this latter becomes highly stable on the surface while subsurface Pt become highly instable (Eseg= -0.76 eV/atom and +0.27 eV/atom when located on the first and second layers Sn surface, respectively). So, one can expect that for Pt-Sn nanoparticles exposed to CO gas, Pt atoms are either on the surface directly interacting with CO or in the bulk. This result corroborates well with the evolution of our experimental Pt-CO DRIFTS signal on the Sn-rich Pt-Sn/alumina (Figure 3) indicating that Pt was pulled to the surface from the bulk due to the formation of Pt-CO bond.

To go further and to learn more about the mixing tendency of Pt in Sn surface, we compared the stability of one Pt dimer and two isolated Pt atoms in the presence and in the absence of interacting CO. The calculated total energies predict that for diluted systems (Snrich Pt-Sn) the formation of isolated Pt surrounded by Sn atoms is more favorable than the formation of pure Pt ensembles.

Electronic structure analysis

In order to examine the local effect of CO-induced segregation of Pt atom on the topmost surface layer of Sn-rich and Sn-poor Pt-Sn alloys, the local density of states (DOS) were calculated, in presence and in absence of adsorbed CO. For the following analyses, the pure Pt(100) and Sn(100) surfaces as well as the Sn(100) with one diluted Pt on the surface (representing Sn-rich Pt-Sn alloy) are selected. Figure 5 intensity of occupied states of s-band can be distinguishable for the energy range between -8 and -5 eV.

As expected, in presence of CO on the top of the Pt atom surrounded by Sn, significant changes are recorded. In Figure 6 are reported the changes in DOSs of Pt and interacting CO molecule. In order to examine the charge transfer between CO and Pt, the charge density differences before and after the carbon monoxide adsorption were calculated by equation 3:

∆𝜌 = 𝜌 (𝑠𝑙𝑎𝑏+𝐶𝑂) -𝜌 (𝑠𝑙𝑎𝑏) -𝜌 (𝐶𝑂) Eq.3
where 𝜌 (𝑠𝑙𝑎𝑏+𝐶𝑂) represents the charge density of the CO adsorbed surface, 𝜌 (𝑠𝑙𝑎𝑏) and 𝜌 (𝐶𝑂)

are the charge density of the clean surface and of the absorbate CO molecular with geometries fixed from the optimized CO-adsorbed surface, respectively.

In Figure 7 are represented the charge density difference for CO adsorbed on pure Pt(100) and on a diluted Pt on Sn(100) surface. In the later surface ∆𝜌 was found to be mostly located on the Pt atom and its interacting CO; the neighboring Sn atoms not seem to be affected by the presence of CO. This corroborates well with the small perturbation found on the DOSs of s and p orbitals reported in Figure 5b. However, compared to Pt(100) surface, the ∆𝜌 of PtSn(100)

shows important electron transfer mainly from surface Sn atoms to single Pt (more than 0.8 é) which induces a strong electron accumulation around Pt. This charge transfer is expected as the electronegativity of Pt is much higher than that of Sn (2.28 for Pt vs. 1.96 for Sn). The charge transfer from Pt to CO is found to be small; about 0.12 é with more important charge depletion in the Pt-CO bond. This depletion is accompanied by a significant prolongation of Pt-C bond distance (from 1.842 Å in CO-Pt(100) to 1.900 Å in CO-PtSn(100) surface) which has also an incidence on the C-O bond length (elongation of 0.007 Å). The weakening of C-O bond is in line with the experimental prediction of CO dissociation over Pt-Sn nanoalloys inducing the formation of SnOx domains.

CO on Pt-Sn nano-clusters: Adsorption and vibrational frequencies

The above energetic and electronic studies of Pt and Sn in Pt-Sn alloys using semi-infinite surface models evidenced characteristics that could be extrapolated to nanosized system:

i) The interaction of CO with Sn-Pt induces the surface segregation of Pt from the bulk.

ii) In the presence as in the absence of CO, Sn prefers to occupy low coordinated sites To go further in the comparison between calculated and experimental features of CO adsorbed on Pt-Sn system, vibrational frequencies were calculated and reported in Table 2.

The calculated harmonic frequencies at the GGA/PBE level of adsorbed CO are below those observed in the DRIFTS experiments. At least, this is partially due to the considered low CO coverage case where dipole-dipole interaction is minimized (red-shift spectra) and for which the molecules are more strongly bonded to the surface (increased back-donation resulting in a red shift of the stretching frequency). To facilitate the comparison, a scaling factor of 1.025 was applied which corresponds to the adjustment of the calculated frequency of CO on pure Pt nanocluster (TOH_Pt38 (CO-Pt(top _ed)) to the value of CO of 2075 cm -1 , experimentally measured for CO linearly adsorbed on pure Pt nanoparticles (see Table 1). Interestingly, for

Figure 1 .

 1 Figure 1. DRIFTS in situ spectra during CO adsorption over Sn-rich Pt-Sn/alumina at various temperatures: (a) 200 °C, (b) 175 °C, (c) 150 °C, (d) 125 °C, (e) 100 °C and (f) 50 °C. The inset is a zoom in on the spectra at (c) 150°C and (f) 50°C. Feed: 2 % CO in H2. The spectrum of the sample reduced under H2 at 400 °C was used as background

Figure 2 .

 2 Figure 2. CO relative coverage as a function of temperature on the Sn-rich Pt-Sn/alumina (o) and corresponding Temkin model (dotted line). Full coverage was assumed to be achieved at 100 °C. The spectra at 75 and 50 °C also contained a non-negligible fraction of plain Pt domains alongside Pt-Sn.

Figure 3 .

 3 Figure 3. Evolution of the Pt-CO DRIFTS signal on the Sn-rich Pt-Sn/alumina at 420 °C with time following the introduction of CO in the H2 feed at 0 min. Feed: 2 % CO in H2. The spectrum of the sample reduced under H2 at 420 °C was used as background

Figure 4 aFigure 4 :

 44 Figure 4: Segregation energies in absence (full lines) and presence (dashed lines) of CO a): Sn in Pt(111), (100) and (110). b) Pt in Sn(100).

4 -

 4 (100) in presence of CO

Figure 5 :

 5 Figure 5: Density of states (DOS) calculated under vacuum. (a) 5d-band of Pt atom located in the first layer of Pt(100) surface (full line) and diluted in the topmost layer of the Sn(100) surface (dashed line). (b) 5s (cyan lines) and 5p (purple lines) band DOSs for Sn atom located in the topmost layer of pure Sn(100) surface (full lines) and in the first neighboring site of diluted Pt (dashed lines) in Sn(100) surface.

Fig.6a showsFig. 6 .

 6 Fig.6a shows a strong shift of the d-band center of Pt away from the Fermi level induced by the presence of CO. In addition, a significant hybridization is evidenced between p-band of CO and d-band of Pt in the conduction band -7.5 eV and -8.5 eV and valance band 2.5 eV regions. This corresponds to the bonding and antibonding states between p orbital of CO and d orbital of Pt. The additional peak appearing at the higher edge of the d-band DOS of the platinum surface atom (around 2.5 eV) (Figure 6a) could be the signature of a covalent CO-Pt bond where charge transfer may occur. To analyze the effect of Sn ensemble on the electronic structure of adsorbed CO, the Figure 6b. reports the superposition of the 2p-band orbitals of CO adsorbed on Pt atom surrounded by 1 Sn atom (Sn-poor Pt-Sn) and by 7 Sn atoms (Sn-rich Pt-Sn). The increase of Sn environment induces a significant shift toward the Fermi level of the peak located around 3.5 eV (see insert). The shift of this peak, corresponding to the 2* orbital of CO, indicates that Sn ensemble decreases the Pt-CO bonding which is in line with the measured heat of adsorption found to decrease when Sn content increase. Thus, the introduction of Sn improves the resistance against CO-poisoning of Pt surfaces.

Figure 7 .

 7 Figure 7. : Charge density difference plots for carbon monoxide adsorption on top site of Pt atom in (left) Pt(100) pure metal surface, (right) PtSn(100) alloy surface. The numerical values indicate calculated Bader charges. Blue (yellow) areas represent the electron accumulation (depletion) regions. dc-o is the distance between carbon and oxygen atom; dc-Pt is the distance between the carbon atom and the bonded Pt atom. Pt and Sn atoms are displayed as grey and indigo spheres, respectively.

Figure 8 :

 8 of the top most surface layer of Sn-poor Pt-Sn nanoalloys.Based on these results, truncated cuboctahedral nano-clusters of Pt-Sn were modeled to monitor the CO interactions on Pt-Sn nanoalloys as a function of surrounding chemical environment and to extract information of the structural arrangement under reacting gas.Pure Pt (TOH_Pt38), Sn-rich (TOH_Pt7Sn31) and Sn-poor (TOH_Pt29Sn9) Pt-Sn clusters were considered to study the adsorption mechanisms of probe CO molecule and to calculate the variation of adsorption energies and vibrational frequencies as a function of surface composition. TOH_Pt7Sn31 is a core-shell-like structure where 6 Pt atoms are located in the bulk and 1 Pt surface atom interacts with CO. All 31 Sn atoms are located in the surface. For TOH_Pt29Sn9 9 Sn atoms occupy the edge sites of the TOH surface structure and all the rest of atoms are Pt. In Figure8are illustrated the geometry optimized configurations of CO for which energetic and vibrational parameters are gathered in Table2. As CO does not adsorb on Sn sites, the unique selected configuration for Sn-rich Pt-Sn (TOH_Pt7Sn31) corresponds to CO adsorbed on top site of Pt atom. Over pure Pt (TOH_Pt38) and Sn-poor Pt-Sn (TOH_Pt29Sn9), three Pt adsorbed sites of CO are found to be favorable: top_ed, top-fc and BDG-ed-fc sites. The 3-fold and 4-fold sites were found to be unfavorable for the adsorption of CO. Illustration of the different CO adsorption sites on various Pt-Sn cluster configurations. Pt, Sn, C and O atoms are displayed as grey, indigo, brown and red spheres, respectively.

  TOH_Pt29Sn9 and TOH_Pt7Sn31 nanoclusters, the results of frequency calculations of CO adsorbed on top Pt edge atom show the same red shift as observed experimentally. The values of 2049 cm -1 and 2035 cm -1 , depicted in Table2, correspond very well with the experimental observed IR bands of CO in Sn-poor Pt-Sn (2054 cm -1 ) and Sn-rich Pt-Sn alloys (2039 cm -1 ), respectively. These results show that from DFT calculations we can reasonably assigned the various bands observed in DRIFTS spectra of Pt-Sn nanoparticles and describe their evolution induced by surface segregation phenomena. The concordance between experimental data and experimental results provides a clear picture of the surface chemical ordering of Pt-Sn nanoalloys and their evolution under reactive CO gas.IV. ConclusionCO adsorption on Sn-rich Pt-Sn nanoparticles supported on alumina allowed observing two modes of segregation depending on the experimental conditions. The first mode, observed at temperatures above 100 °C upon the introduction of CO in the H2 feed, is a surface segregation related to an enrichment of the surface of the Pt-Sn nanoparticles with Pt. This phenomenon, which does not lead to any significant band shift or shape change, is triggered by the formation of Pt-CO bond increasing the stability of Pt at the surface of the Pt-Sn nanoparticles. This Pt surface segregation was confirmed by DFT calculations on modeled Sn-rich and Sn-poor Pt-Sn alloy surfaces where geometric and electronic structure analyses highlighted the observed affinity of Pt to the CO gas. The second segregation phenomenon was observed experimentally at temperatures lower than 100 °C CO + H2 feed and led to a significant change of band shape and shift was due to the formation of Pt and SnOx domains triggered by CO dissociation. To learn more about the first segregation phenomenon, the heats of CO adsorption was determined by DRIFTS over various Pt-Sn surface. The DRIFTS data showed a gradual decay of the linear wavenumber from plain Pt (2075 cm -1 ), through a Sn-poor Pt-Sn (2054 cm -1 ) and to a Sn-rich Pt-Sn (2039 cm -1 ). The heat of adsorption at zero coverage also decayed in a parallel manner, from plain Pt (185 kJ mol -1 ), through Sn-poor Pt-Sn (95 kJ mol -1 ) and to Sn-rich Pt-Sn (85 kJ mol -1 ). DFT calculated adsorption energies as well as frequency calculations are fully consistent with the experimental results and thus provide an accurate model at the atomic level of Pt-Sn surface nanoparticles. Moreover, electronic structure analyses show significant charge transfer from Sn to Pt accompanied by a shift away (toward) the Fermi level of the bonding (antibonding) orbitals of the adsorbed CO. This result indicates that Sn decreases the Pt-CO bonding which is in line with the measured heat of adsorption found to decrease when increasing Sn composition, thereby improving the CO-poisoning resistance of Pt surface atoms.

Table 1 .

 1 Adsorption heats for linear CO on Pt in various samples and the corresponding wavenumber of CO at full coverage.

		Adsorption heat at	Adsorption heat at	CO stretch at
		zero coverage	full coverage	full coverage
		± 5 kJ/mol	± 5 kJ/mol	± 2 cm -1
	Pt a	180	85	2075
	Sn-poor Pt-Sn a	95	75	2054
	Sn-rich Pt-Sn	85	75	2039

Table 2 :

 2 DFT-D3 calculated adsorption energies and frequencies of CO adsorbed on PtSn nanoclusters. A scaling factor of 1.025 was applied to the calculated frequency values which corresponds to the adjustment of the calculated frequency of CO on pure Pt nanocluster with the observed experimental value. that on pure Pt nanocluster the CO prefers to adsorb on the top site rather than on the bridge site. The top of Pt atom located at the edge position is found to be the most favorable site with adsorption energy value of -2.20 eV. The adsorption energy on top of Pt located at the center face is predicted to be lower by 0.18 eV. For Pt-Sn, the preference of CO to the top site of under-coordinated Pt atoms remains true but alloying Pt with Sn induces a decrease in the adsorption energies. On TOH_Pt29Sn9 the adsorption energy of CO is found to decrease by about 0.02 eV on top Pt site by 0.5 eV on bridge one in comparison to pure Pt nanoclusters. The energy decrease is found to be more drastic on Sn-

	Clusters	CO adsorption site	Eads (eV)	DFT calculated	Experimental
				CO (cm -1 )	CO (cm-1)
	TOH_Pt38	Top Pt (edge site)	-2.20	2075	2075
		Top Pt (Fc site)	-2.02	2096	----
		Bridge Pt	-2.10	1895	1885

Acknowledgements:

A. Moscu and C. Theodoridi are acknowledged for sample preparation and help in carrying out the IR experiments, respectively. L. Veyre is acknowledged for the TEM analysis. This work was granted access to the HPC resources of [CCRT/CINES/IDRIS] under the allocation 2019 [x2019087369] made by GENCI (Grand Equipement National de Calcul Intensif).