Marielle Margier

Xavier Collet

Cédric Le May

Charles Desmarchelier

François André

Chantal Lebrun

Catherine Defoort

Alice Bluteau

Patrick Borel

Anne Lespine
email: anne.lespine@inra.fr.

Cédric Le May

Dr Emmanuelle Reboul
email: emmanuelle.reboul@univ-amu.fr.

ABCB1 (P-glycoprotein) regulates vitamin D absorption and contributes to its transintestinal efflux

Keywords: Cholecalciferol, 25-hydroxycholecalciferol, bioavailability, enterocyte, MDR1

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Introduction

Vitamin D is essential for health and is involved in many functions including the optimal functioning of the musculoskeletal system. Indeed, it stimulates both dietary calcium absorption and osteoid mineralization, and has major regulating roles in bone turnover and muscle function [START_REF] Wimalawansa | Vitamin D: an essential component for skeletal health[END_REF]. Consequently, hypovitaminosis D is associated with defects in bone development in infants as well as with osteoporosis in postmenopausal women, and it can severely impact welfare and health in elderly populations in general. As vitamin D deficiency and insufficiency are extremely prevalent in the general population worldwide, this represents today a major public health concern [START_REF] Holick | The vitamin D deficiency pandemic: Approaches for diagnosis, treatment and prevention[END_REF].

Vitamin D is a generic term that includes several cholesterol-derived compounds such as vitamin D 3 (D 3, cholecalciferol), and its metabolites 25-hydroxycholecalciferol (25(OH)D 3 ) and 1,25dihydroxycholecalciferol (1,25(OH) 2 D 3 ). D 3 can be either synthetized in the skin from 7-dehydrocholesterol under the influence of UV light, or provided through the diet. After synthesis/absorption, D 3 is taken up by the liver where it is metabolized into 25(OH)D 3 and released to the blood stream associated with its specific binding protein. Vitamin D insufficiency and deficiency are defined as low 25(OH)D 3 circulating levels [START_REF] Holick | The vitamin D deficiency pandemic: Approaches for diagnosis, treatment and prevention[END_REF].

The vitamin D active hormonal form 1,25-dihydroxycholecalciferol is finally synthetized from 25(OH)D 3 in the kidneys or locally in target tissues [START_REF] Bikle | Vitamin D metabolism, mechanism of action, and clinical applications[END_REF]. Since sun exposure cannot provide sufficient vitamin D in most people, supply through dietary intake is usually required. Therefore, intestinal absorption of the dietary vitamin D is an essential process for maintaining optimal vitamin D status.

Vitamin D intestinal absorption has long been thought to occur through passive diffusion (4), but we have recently shown that membrane transporters of cholesterol such as Scavenger Receptor Class B type I (SR-BI), CD36 molecule (CD36) and to a lesser extend NPC1 like Transporter 1 (NPC1L1), facilitate D 3 intestinal absorption [START_REF] Reboul | Vitamin D intestinal absorption is not a simple passive diffusion: evidences for involvement of cholesterol transporters[END_REF], but not that of 25(OH)D 3 [START_REF] Desmarchelier | Comparison of the Micellar Incorporation and the Intestinal Cell Uptake of Cholecalciferol, 25-Hydroxycholecalciferol and 1-alpha-Hydroxycholecalciferol[END_REF]. In addition, the efflux of neo-absorbed D 3 [START_REF] Reboul | Vitamin D intestinal absorption is not a simple passive diffusion: evidences for involvement of cholesterol transporters[END_REF] and 25(OH)D 3 [START_REF] Desmarchelier | Comparison of the Micellar Incorporation and the Intestinal Cell Uptake of Cholecalciferol, 25-Hydroxycholecalciferol and 1-alpha-Hydroxycholecalciferol[END_REF] from Caco-2 cells into the apical medium in the presence of mixed micelles strongly suggests the existence of an active excretion of the vitamin D from the intestine into the lumen. Such luminal efflux is a key limiting process of the absorption of a large number and variety of lipids and xenobiotics, and its complete mechanisms is poorly understood. Several export pumps are likely to be involved in this phenomenon, among which multidrug resistance proteins of the ABCB subfamilies, such as ABCB, also known as P-glycoprotein (Pgp) [START_REF] Sharom | The P-glycoprotein multidrug transporter[END_REF].

ABCB1 is a 170 kDa transmembrane protein from the ATP-powered pump family, made of two ATPbinding domains and transmembrane hydrophobic domains that delineate a large multispecific binding pocket with a wide drug recognition capacity. This protein can thus actively efflux a broad range of structurally unrelated hydrophobic compounds, and plays a central role in protecting tissues from accumulation of xenobiotics and their endogenous metabolites [START_REF] Sharom | The P-glycoprotein multidrug transporter[END_REF]. It also affects the uptake and distribution of several endogenous lipids from the intestine, such as steroid hormones [START_REF] Uhr | Penetration of endogenous steroid hormones corticosterone, cortisol, aldosterone and progesterone into the brain is enhanced in mice deficient for both mdr1a and mdr1b P-glycoproteins[END_REF]. In particular, ABCB1 participates in cholesterol transintestinal excretion (TICE), contributing to cholesterol homeostasis [START_REF] May | Transintestinal cholesterol excretion is an active metabolic process modulated by PCSK9 and statin involving ABCB1[END_REF].

Since cholesterol and vitamin D are structurally related and share common uptake routes [START_REF] Reboul | Vitamin D intestinal absorption is not a simple passive diffusion: evidences for involvement of cholesterol transporters[END_REF], we hypothesized that they could also share common efflux pathways. We thus investigated whether ABCB1 could export this vitamin at the intestinal level by conducting cellular, animal, in silico and human experiments.

Material and methods

In vitro studies

Caco-2 cells. Caco-2 clone TC-7 cells were cultured, seeded and grown on transwells as previously described [START_REF] Goncalves | Fatty acids affect micellar properties and modulate vitamin D uptake and basolateral efflux in Caco-2 cells[END_REF][START_REF] Goncalves | Pinoresinol of olive oil decreases vitamin D intestinal absorption[END_REF]. Twelve hours prior to each experiment, cells received micellar vitamin D at the apical side (10 µM) and complete medium (DMEM -Dulbecco's Modified Eagle's medium -supplemented with 10% fetal bovine serum, 1% nonessential aminoacids and 1% penicillin/ streptomycin) at the basolateral side. Cells were rinsed once with PBS and once with mixed micelles without vitamin D. Cells then received mixed micelles without vitamin D supplemented or not with either valspodar 0.1-5 µM (PSC 833, MedChemExpress), Zosuquidar 1µM (Sigma Aldrich) or KO143 1µM (Sigma Aldrich) at the apical side, and complete media at the basolateral side for 4 h. The media were harvested, cells were rinsed with PBS and scraped into 0.5 ml ice-cold PBS.

Griptite cells. Griptite 293MSR cells were cultured and transfected with either ABCB1-pCMV

plasmid or empty pCMV plasmid as previously described [START_REF] Borel | CD36 and SR-BI Are Involved in Cellular Uptake of Provitamin A Carotenoids by Caco-2 and HEK Cells, and Some of Their Genetic Variants Are Associated with Plasma Concentrations of These Micronutrients in Humans[END_REF][START_REF] Goncalves | Intestinal scavenger receptors are involved in vitamin K1 absorption[END_REF]. Transfection efficiency was verified by western blotting [START_REF] Reboul | Lutein transport by Caco-2 TC-7 cells occurs partly by a facilitated process involving the scavenger receptor class B type I (SR-BI)[END_REF]. Cells then received complete medium enriched with 10 µM vitamin D in ethanol (final ethanol concentration <0.1 %) for 12 h. Cells were then rinsed twice with DMEM and received mixed micelles diluted in DMEM (1:4; vol:vol) as efflux medium. After 2 h incubation, 1 ml of medium was harvested and replaced by the same volume of efflux medium. After 4 h, the media were harvested, cells were rinsed with PBS and scraped into 0.5 ml ice-cold PBS.

MDCK cells. MDCK-II (Madin-Darby canine kidney strain II) epithelial cells and MDCK-II cells

transfected with human ABCB1 gene (MDCKII-ABCB1) were provided by The Netherland Cancer Institute (Amsterdam). Cells (passage 10 to 16) were cultured similarly to Griptite cells. For experiment, cells were seeded at a density of 5.0×10 6 cells/well on polycarbonate membrane inserts (6-well plate, 1 µm-pore-size Polyethylene Terephthalate membrane; Merck Millipore). Four to 5 days after seeding cells reached monolayers and they were washed in PBS. The basolateral side of the cell monolayers received HBSS (Hanks' Balanced Salt Solution) supplemented with 1% BSA and containing 10 µM D 3 or 25(OH)D 3 , whereas the apical side received HBSS supplemented with 1% BSA (efflux medium). At 2 h, 0.5 ml of the apical medium were harvested and replaced by the same volume of new efflux media. At 4 h, both apical and basolateral media were harvested. Cells were rinsed and scraped into 0.5 ml ice-cold PBS.

HsABCB1 ATPase activity measurements. ATPase activity was measured in membranes of Sf9 insect cells infected with a recombinant baculovirus containing the human ABCB1 cDNA (Tebu-Bio), without or with 5 µM of verapamil, in absence or in the presence of 40µM of either D 3 or 25(OH)D 3 , and using a modification of the endpoint inorganic phosphate (Pi) release assay described by Sarkadi et al. [START_REF] Sarkadi | Expression of the human multidrug resistance cDNA in insect cells generates a high activity drug-stimulated membrane ATPase[END_REF].

A duplicate reaction mixture also containing an ABCB1 ATPase inhibitor, i.e. sodium orthovanadate 100 µM, was assayed in parallel as a negative control. The amount of Pi released was determined at each time point in triplicate, and the mean initial hydrolysis rate was calculated in nmol Pi/min/mg of total membrane protein.

Mouse experiments

Ethics. Animals and standard operating procedures were conducted under European laws on the protection of animals (86/609/EEC) and adhered to the guidelines from the institutional animal care committee for good clinical practice (CVMP/VICH 59598). The protocols for vitamin D gavage and postprandial measurement of plasma vitamin D response in mice were approved by the local institutional animal care and ethics committee of the "Direction Départementale des Services Vétérinaires de Haute-Garonne" (specific approval number B31555-25). Transintestinal excretion measurement in mice were approved by the Ethic Committee for Animal Experimentation of Pays de la Loire (specific approval number 01953.01).

Animals. FVB control mice (Abcb1 +/+ ) and FVB deficient mice for the two murine ABCB1s encoded by Abcb1a and Abcb1b genes (GenBankTM Accession numbers NM011076 and NM011075, respectively) (Abcb1 -/-mice), were obtained from Taconic (Germantown, NY, USA) and kept as previously described [START_REF] Menez | Relative neurotoxicity of ivermectin and moxidectin in Mdr1ab (-/-) mice and effects on mammalian GABA(A) channel activity[END_REF]. Experiments were carried out on 8-15-week-old mice (20-30 g). Tissue collection. Animals (n = 3 to 9) were euthanized at fast by cervical dislocation. Brain, kidney and liver samples were then taken and snap-frozen into liquid nitrogen.

Postprandial experiment with D 3.

Postprandial experiment was conducted on male mice (n=4 to 5 in each group) as previously described [START_REF] Goncalves | Intestinal scavenger receptors are involved in vitamin K1 absorption[END_REF]. Plasma samples were taken at fast. Gavage emulsions were prepared as previously described [START_REF] Reboul | Vitamin D intestinal absorption is not a simple passive diffusion: evidences for involvement of cholesterol transporters[END_REF] to provide 250 µg of D 3 to each mouse.

Measure of transintestinal vitamin D efflux in Ussing chambers.

Median jejunum was removed from Abcb1 +/+ (n=3) and Abcb1 -/-mice (n=3), mounted on sliders and fixed into Ussing chambers (Physiological Instruments, San Diego, CA). Both chambers contained 1 mL of oxygenated (95% O 2 /5% CO 2 ) Krebs-Henseleit medium (Sigma) supplemented with 1% of BSA, and maintained at 37 °C. At the start of the experiment, vitamin D 10 µM was added in the basolateral chamber, while the apical chamber contained vitamin D-free mixed micelles. Apical medium (250 µL) was collected at 15 min and replaced by fresh medium for additional 15 min incubation.

Transintestinal Efflux in vivo measurement. Perfusion procedures was performed on male mice (n=7 for Abcb1 +/+ and n=9 for Abcb1 -/-mice) as previously described [START_REF] May | Transintestinal cholesterol excretion is an active metabolic process modulated by PCSK9 and statin involving ABCB1[END_REF] after intravenous injection of H 3 -25(OH)D 3 dissolved in intralipid® (0.25 µCi of H 3 -25(OH)D 3 in 100 µl). Intestinal perfusates (500 µl) were mixed with 5 ml of liquid scintillation (ECOLITE, Perkin Elmer) and radiolabeled vitamin levels were assessed by liquid scintillation counting.

Lipid analysis

Total lipids in mouse plasma samples were analyzed as previously described (17).

Vitamin D extraction, HPLC and LC-MS² analysis.

All the samples from experiments were stored at -80°C under nitrogen before vitamin D analysis. All solvents used were from Carlo-Erba.

Cell samples and mouse postprandial experiment samples. The internal standards were ergocalciferol and retinyl acetate for samples containing 25(OH)D 3 and D 3 , respectively. Samples were extracted and analyzed in a Shimadzu HPLC system as previously described [START_REF] Reboul | Vitamin D intestinal absorption is not a simple passive diffusion: evidences for involvement of cholesterol transporters[END_REF][START_REF] Goncalves | Fatty acids affect micellar properties and modulate vitamin D uptake and basolateral efflux in Caco-2 cells[END_REF]. The dried extracts were dissolved in 80 µl acetonitrile and a volume of 50 µl was used for HPLC analysis. D 3 and retinyl acetate, as well as 25(OH)D 3 and ergocalciferol, were separated using a 100×4.6mm Kinetex 5 µm C18 100 Å column (Phenomenex) with a C18 guard column (Phenomenex). The mobile phase involved a gradient of acetonitrile (A) and water (B). Flow rate was 1.2 ml/min. The gradient profile of the mobile phase (A:B) was set at 70:30 for 6.5 min and changed linearly to 90:10 in 8.5 min, and then the mobile phase was changed back to 70:30 from 18.5 to 21 min. The column was kept at a constant temperature (40°C). D 3 and 25(OH)D 3 quantification was performed using Chromeleon software (version 6.50 SP4 Build 1000, Dionex) comparing peak area with standard reference curves.

Mouse plasma and tissue samples. Samples were extracted and analyzed by LC-MS². Isotopically labelled d3-D 3 , d3-25(OH)D 3 and d3-1,25 (Cambridge Isotope Laboratories) were used as internal standards. Plasma samples were prepared as previously described [START_REF] Wang | Simultaneous measurement of plasma vitamin D(3) metabolites, including 4beta,25dihydroxyvitamin D(3), using liquid chromatography-tandem mass spectrometry[END_REF] and derivatized with Ampliflex Diene (Sciex) reagent (19). Tissue samples were prepared as previously described [START_REF] Xue | Simultaneous Quantification of 25-Hydroxyvitamin D3 and 24,25-Dihydroxyvitamin D3 in Rats Shows Strong Correlations between Serum and Brain Tissue Levels[END_REF] and derivatized with either Ampliflex (19) or 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD, Sigma) reagents [START_REF] Xue | Simultaneous Quantification of 25-Hydroxyvitamin D3 and 24,25-Dihydroxyvitamin D3 in Rats Shows Strong Correlations between Serum and Brain Tissue Levels[END_REF]. Accurate mass measurements were performed on the Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific) equipped with a Heated Electrospray Ionization (H-ESI II) probe. Thermo Xcalibur 3.0.63 software was used for both instrument setup and control, and for data treatment. Tune Q Exactive Plus 2.5 software was used for direct control of the mass spectrometer. The autosampler tray was kept at 4 °C and the column was kept 40 °C. Five µL of sample were injected onto a Hypersil GOLD C18 column (2.1 x 100 mm; Thermo Fisher Scientific). The solvents were : (A) water -formic acid 0.1% and (B) was 0.1% acetonitrile -formic acid 0.1%. Solvent flow rate was set at 400 µL/min. For Amplifex assays, the proportions of solvent B used were: 0-4 min : 30%, 4-10 min : 30-65%, 10-12 min: 65%, 12-16 min: 65-100%, 16-18 min : 100%-30%.

For PTAD assays, the proportions of solvent B used were: 0-1 min : 50%, 1-13 min : 50-100%, 13-15 min : 100%, 15-16 min : 100-50%, 16-18 min : 50%. LC-HRMS/MS analyses were performed in positive ionization mode. The H-ESI probe and the transfer capillary temperature were kept at 310°C and 320°C, respectively. Spray voltage was set at 3500 V and the S-lens radiofrequency level at 55 V. Sheath and auxiliary gas were maintained at 30 and 8 (arbitrary units). Parallel Reaction Monitoring method was set up as MS/MS acquisition mode and full higher-energy collision dissociation spectra (HCD) spectra were acquired. For each compound, the most abundant ion was selected as precursor ion and isolated in a 2 Uma window in a specified time segment and fragmented under optimized collision energies. The resolving power was set to 35000 full width at half maximum at m/z 200.

Sample preparation and Real-time PCR.

Total RNA was extracted from the duodenal mucosa of mice (n = 4 per group) using TRIzol reagent (Euromedex, France). Total RNA concentration and quality were confirmed with the use of the biodrop µLite (Sciences tec). cDNAs were synthesized from 1µg total RNA. Real time quantitative PCR assays were performed with Sybr green mixes using a thermocycler Light Cycler 480 (Roche). For each condition, expression was quantified in duplicate, and 18s rRNA was used as endogenous control in the comparative cycle threshold (CT) method. Primer sequences were 18s [F: CGCCGCTAGAGGTGAAATTCT and R: CATTCTTGGCAAATGCTTTCG], vdr [F: CTGTACCCCAGGTCGGTCT and R: AACCCCTCATAAAGTTCCAGGT].

Vitamin D docking on ABCB1

The ABCB1 of the nematode C. elegans p-glycoprotein 1 (Cel-Pgp-1) X-ray structure, determined at a resolution of 3.4 Å (PDB code 4F4C) [START_REF] Jin | Crystal structure of the multidrug transporter P-glycoprotein from Caenorhabditis elegans[END_REF], was used in all docking calculations. Preparation of protein target structure and of ligands conformations, docking procedure, data analysis and identification of key residues are detailed in [START_REF] David | In silico analysis of the binding of anthelmintics to Caenorhabditis elegansP-glycoprotein 1[END_REF]. The molecular structures of ligands were extracted from Chemspider (CID 4444353 and 4446820 for D 3 and 25(OH)D 3 , respectively), and ten conformers generated by MarvinSketch in order to select several lowest energy ring conformers as starting point for flexible docking calculations.

Molecular docking experiments were performed using AutoDock 4 (release 4.2.6), with 100 poses in each docking run. The most representative poses of the docking experiments were chosen for data presentation, according to the score calculated by AutoDock, which can be considered as an estimated free energy of ligand binding, thus indicative of binding affinity. Other parameters and observables were used in the interpretation of docking data issued by AutoDock: histogram bars energy range, profile of the histogram, and location of calculated positions in the protein structure. For each lowest energy pose of selected clusters, the number and nature of interacting residues were analyzed within the protein. Among these, particular interest was given to residues belonging to the "hotspots" for drug binding [START_REF] David | In silico analysis of the binding of anthelmintics to Caenorhabditis elegansP-glycoprotein 1[END_REF].

Association between single-nucleotide polymorphisms (SNPs) in ABCB1 and fasting plasma 25(OH)D 3 concentrations in healthy male adults

Thirty-nine healthy male adults were recruited for the study (NCT02100774, ClinicalTrials.gov) and their fasting plasma 25(OH)D 3 concentration was measured as previously described [START_REF] Desmarchelier | A Combination of Single-Nucleotide Polymorphisms Is Associated with Interindividual Variability in Cholecalciferol Bioavailability in Healthy Men[END_REF]. They were genotyped using whole-genome microarrays [START_REF] Desmarchelier | The postprandial chylomicron triacylglycerol response to dietary fat in healthy male adults is significantly explained by a combination of single nucleotide polymorphisms in genes involved in triacylglycerol metabolism[END_REF]. One hundred and fourteen SNPs in ABCB1 were expressed on the arrays. SNPs for which the genotype call rate was <95%, or SNPs that presented a significant departure from the Hardy-Weinberg equilibrium (p<0.05; Chi-squared test), were excluded from all subsequent analyses. TagSNPs were selected using the LD TAG SNP Selection (TagSNP) tool from the SNPinfo Webserver (https://snpinfo.niehs.nih.gov/snpinfo/snptag.html) using genotype data from HapMap in the CEU population and with a linkage disequilibrium cut-off of 0.8. This resulted in the selection of 36 candidate tag SNPs. A dominant genetic model was assumed: for each SNP, subjects homozygous for the lesser frequent allele were grouped with heterozygous subjects. SNPs for which they were fewer than 5 subjects in a genotypic group were excluded from further statistical analysis.

Statistical analysis

Results are expressed as means ± SEM. Differences between more than two groups of unpaired data were analyzed using the nonparametric Kruskal-Wallis test while differences between two groups of unpaired For all tests, an adjusted p-value <0.05 was considered significant.

Results

ABCB1 is involved in vitamin D apical efflux in vitro in cells

We first measured vitamin D efflux in several epithelial cell models expressing different levels of ABCB1.

In Caco-2 TC7 cells, the ABCB1 inhibitor PSC833 (valspodar 1µM or 5 µM) induced a significant decrease of the apical efflux of both D 3 and 25(OH)D 3 (up to -28.1% and -21.8%, Figure 1A). Valspodar 0.1µM also significantly decreased D 3 efflux by 18.5%. Zosuquidar 1µM significantly reduced of both D 3 and 25(OH)D 3 efflux (-27.7% and -34.9%, Figure 1B). Conversely, KO143 1µM reduced D 3 (-30.4%) but not 25(OH)D 3 efflux (Figure 1C). The transitory transfection with ABCB1 of Griptite cells significantly increased the D 3 apical efflux, when compared with cells transfected with an empty plasmid (+32.2% at 2 h, +34.8% at 4 h; p<0.05; Figure 1D). This transitory transfection also significantly increased the 25(OH)D 3 apical efflux when compared with cells transfected with an empty plasmid (+21.2% at 2 h, +25.0% at 4 h; p<0.05; Figure 1D). Finally, when ABCB1 was overexpressed in stable ABCB1 gene-transfected MDCK-II cells, the trans-epithelial effluxes of both D 3 and 25(OH)D 3 were significantly increased compared with the parental MDCK-II (+167.1% at 2 h, +125.7% at 4 h for D 3 and +139.0% at 2 h, +54.1% at 4 h for 25(OH)D 3 ; p<0.05) (Figures 1E).

Vitamin D modulates ABCB1 ATPase activity

It is generally admitted that the ATPase activity of ABCB1 can be modulated by its own substrates.

Therefore, we measured this activity on inversed vesicles prepared from cells that overexpressed HsABCB1, in presence of D 3 or 25(OH)D 3 (Figure 1F). In our experimental conditions, D 3 at 40 µM activated moderately the ATPase activity while 25(OH)D 3 had no effect on the basal ABCB1-ATPase activity. Nevertheless, both compounds inhibited ABCB1-verapamil-stimulated ATPase, which confirms that D 3 and 25(OH)D 3 interacted directly with ABCB1.

ABCB1 deficiency in mice is associated with higher vitamin D status

In order to confirm our in vitro results, we assessed the impact of ABCB1 on vitamin D homeostasis in mice. The plasma concentrations of both D 3 and 25(OH)D 3 were respectively 58.8% and 42.6% higher in Abcb1 -/-mice compared with Abcb1 +/+ mice (p<0.01 and p<0.001, respectively), while plasma 1,25(OH) 2 D 3 remained unchanged (Figure 2A). Vitamin D concentrations were significantly higher in both fasting intestine (+115.4%; p<0.05 for D 3 and +43.0%; p<0.05 for 25(OH)D 3 , respectively, Figure 2B) and brain of Abcb1 -/-mice (+65.6%; p<0.005 for D 3 and +58.2%; p<0.05 for 25(OH)D 3 , respectively, Figure 2C). In addition, in Abcb1 -/-mice, 25(OH)D 3 concentration was significantly higher in kidney (+20.6%; p<0.02, Figure 2D) and liver (+59.2%; p<0.02, Figure 2C), while D 3 concentration did not change in both tissues, when compared with control mice. 1,25(OH) 2 D 3 was not delectable locally in tissues.

Intestinal vdr expression tended to be decreased by half in Abcb1 -/-mice compared to control mice (relative expression of 100.0 ± 20.3 % in control mice vs 51.2 ± 12.5% in Abcb1 -/-mice), but this difference remained nonsignificant.

Postprandial absorption of D 3 is increased by ABCB1 deficiency in mice

The postprandial plasma response in vitamin D was assessed with vitamin D dietary form, i.e. D 3 . D 3 postprandial plasma response was significantly increased in Abcb1 -/-mice compared with Abcb1 +/+ mice after 3 h of gavage (+196.2%, p<0.05), and after 6 h (+59.1%, p<0.05) (Figure 3A). Subsequently, the overall postprandial plasma D 3 response, expressed as area under the postprandial plasma concentration versus time curves at 0-6 h, tended to be higher in the Abcb1 -/-animals compared with control mice (Figure 3B). The increase in D 3 postprandial response in Abcb1 -/-mice was more pronounced and became significant after plasma total lipid (i.e. plasma triacylglycerols, phospholipids and cholesterol) adjustment: 3.43 and 6.71 µmol.h/ g of total lipids for Abcb1 -/-and Abcb1 +/+ mice, respectively (p<0.05; Figures 3C and3D). Finally, the D 3 content of the last intestinal fragment (i.e. ileum) of the Abcb1 -/-mice, was about two times higher, although non-significantly because of the high variability, than the D 3 content of the last fragment of Abcb1 +/+ mice (Figure 3E).

ABCB1 is involved in the transintestinal excretion of vitamin D in mice

In order to follow the transintestinal transport of vitamin D, we used 25(OH)D 3 , which is the main vitamin D form in the blood compartment. We first measured the apical transfer of 25(OH)D 3 through intestinal explants using Ussing chambers. After 30 min, the 25(OH)D 3 concentration in the apical compartment was twice more important for the parental explants than for the Abcb1 -/-mice jejunum (p<0.05, Figure 4A). A mice intestinal perfusion model was then used to investigate the 25(OH)D 3 transintestinal transport in vivo.

Results showed a significant decrease in the amounts of plasma H 3 -25(OH)D 3 measured in the Abcb1 -/- intestine perfusate after 30, 60 and 120 min, compared with Abcb1 +/+ mice after 30, 60 and 120 min (-33.7%; -26.1% and -25.3% respectively; p<0.005; p<0.05; p<0.005) (Figure 4B). Therefore, the global 25(OH)D 3 intestinal excretion after weight adjustment within the 2 hours of experiment was -36.6% lower in Abcb1 -/-mice, compared with Abcb1 +/+ mice (p<0.005; Figure 4C).

ABCB1 shows in silico high affinity binding sites for D 3 and 25(OH)D 3

We then studied the putative molecular interactions between the protein and D 3 or 25(OH)D 3 using in silico docking on well-resolved crystal structure of C. elegans Pgp-1 in an open-inward configuration. The most favored docked poses for D 3 and 25(OH)D 3 on Cel-Pgp-1 exhibited both low binding energies scores (-8.9 and -8.8 kcal/mol, respectively), consistent with ligand-protein interaction of high affinity. The binding sites of the two compounds were proved to be very close to each other and located in the inner chamber delineated by the transmembrane domains of the protein (Figure 5A). The molecular poses revealed favored interactions of vitamin D with several residues on TM1, TM11 and TM12. Of particular interest, the 3hydroxy substituent present on both forms of vitamin D could engage in 3 hydrogen bonds with T1028, G1032, and to a weaker extent L1031, all located on TM12 (Figure 5B and 5C), while the 25-hydroxy group present only in the 25(OH)D 3 proved to form 2 additional H-bonds with the Q98 residue (TM1) (Figure 5C). A number of other contact residues such as M1021 and T1025 on TM12 and L990, L993, N994 and Y998 on TM11 engaged hydrophobic interactions with the two forms of vitamin D (Figure 5D).

A SNP in ABCB1 is associated with the fasting plasma 25(OH)D 3 concentration in a group of healthy male adults

Subjects carrying the lesser frequent allele at rs17064, localized in the le 3'-Untranslated Region of ABCB1 gene, displayed a 35.9% decrease in their 25(OH)D 3 status compared with subjects homozygous for the most frequent allele (respectively 35.94 ± 4.50 nmol/L, n=9 vs 56.07 ± 3.00 nmol/L, n=30; adjusted p=0.041, t-test with Benjamini-Hochberg correction). Of note, 3 other SNPs (rs1045642, rs17149810, rs2235015) exhibited a significant p-value but they did not reach significance following multiple comparison correction. A complete list of the SNPs tested and their respective p-value and adjusted p-value are presented in Table 1.

Discussion

Given the prominent role of ABCB1 in effluxing a large spectrum of lipophilic compounds, including cholesterol, we considered this transporter as a good candidate that could participate in the turn-over of vitamin D. We thus explored the ABCB1-mediated transport and intestinal export of vitamin D, and we detailed the molecular interactions supporting this process.

We first used the popular Caco-2 TC-7 intestinal cell model, which constitutionally expresses ABCB1 [START_REF] Collett | Predicting P-glycoprotein effects on oral absorption: correlation of transport in Caco-2 with drug pharmacokinetics in wild-type and mdr1a(-/-) mice in vivo[END_REF].

We confirmed that these cells were able to efflux both neo-absorbed D 3 and 25(OH)D 3 to the apical medium, in line with our previous data [START_REF] Reboul | Vitamin D intestinal absorption is not a simple passive diffusion: evidences for involvement of cholesterol transporters[END_REF][START_REF] Desmarchelier | Comparison of the Micellar Incorporation and the Intestinal Cell Uptake of Cholecalciferol, 25-Hydroxycholecalciferol and 1-alpha-Hydroxycholecalciferol[END_REF]. The fact that the efflux of both vitamin D forms could be impaired by PSC833, the reference inhibitor of ABCB1 activity, is taken as primary evidence that ABCB1 is involved in this process. Zosuquidar, a third generation ABCB1 specific inhibitor, led to similar results. We then explored vitamin D status in vivo in an Abcb1 -/-mouse model lacking both Abcb1a and Abcb1b genes encoding the two forms of ABCB1 (ABCB1a and ABCB1b) expressed in rodents [START_REF] Schinkel | Disruption of the mouse mdr1a P-glycoprotein gene leads to a deficiency in the blood-brain barrier and to increased sensitivity to drugs[END_REF]. We observed that fasting plasma 25(OH)D 3 and D 3 concentrations were significantly increased in Abcb1 -/-mice compared with control animals, supporting a role of ABCB1 in the elimination of vitamin D. Plasma 1,25(OH) 2 D 3 was not modified, which is likely due to its tight regulation process [START_REF] Anderson | Determinants of circulating 1, 25-dihydroxyvitamin D3 levels: the role of renal synthesis and catabolism of vitamin D[END_REF]. Interestingly, Abcb1 -/-mice displayed increased D 3 and 25(OH)D 3 levels in the brain as well, which is consistent with the important role of ABCB1 at the blood-brain-barrier level [START_REF] Cui | Tissue distribution, gender-divergent expression, ontogeny, and chemical induction of multidrug resistance transporter genes (Mdr1a, Mdr1b, Mdr2) in mice[END_REF]. Increased 25(OH)D 3 levels were also measured in Abcb1 -/- liver and kidneys compared with organs from control mice where ABCB1 is physiologically expressed [START_REF] Cui | Tissue distribution, gender-divergent expression, ontogeny, and chemical induction of multidrug resistance transporter genes (Mdr1a, Mdr1b, Mdr2) in mice[END_REF].

ABCB1 is largely expressed in the gut where it mediates the luminal efflux of its substrates, thereby regulating the intestinal absorption of a broad range of dietary components. At fast, a significant accumulation of both D 3 and 25(OH)D 3 was observed in Abcb1 -/-mouse intestines compared with control intestines. After a D 3 gavage in oil-in-water emulsion, the lipid-adjusted postprandial plasma D 3 response was significantly increased in Abcb1 -/-mice compared with control animals. This strongly supports that ABCB1 impacts on vitamin D absorption process by secreting back to the intestinal lumen a fraction of neoabsorbed vitamin D. Furthermore, this effect was, at least partly, specific to vitamin D, as it was not driven by a concomitant increase in dietary lipid absorption. Finally, D 3 content of the distal intestinal mucosa, where ABCB1 is predominant in control mice [START_REF] Maclean | Closing the gaps: a full scan of the intestinal expression of p-glycoprotein, breast cancer resistance protein, and multidrug resistance-associated protein 2 in male and female rats[END_REF], tended to be increased in ABCB1-deficient animals, in accordance with a defect in the efflux of vitamin D.

As ABCB1 is involved in cholesterol transintestinal excretion [START_REF] May | Transintestinal cholesterol excretion is an active metabolic process modulated by PCSK9 and statin involving ABCB1[END_REF], we further explored its capacity to participate in vitamin D transintestinal secretion as well using ex vivo intestinal explants. The basolateral to apical transfer of 25(OH)D 3 was significantly lower when intestinal explants were lacking ABCB1. The existence of an active intestinal excretion pathway for vitamin D was further reinforced by data obtained in vivo in in situ perfused mice. Indeed, we clearly showed for the first time that when administered by intravenous injection, 25(OH)D 3 can be excreted from the plasma compartment to the intestinal lumen. We also showed that this process is mediated, at least partly, by ABCB1. By using in silico docking approach, we provide computational evidence that the two forms of vitamin D can bind efficiently a close ortholog of human ABCB1. ABCB protein structures and functions are well conserved within all living kingdoms, including nematodes and mammals [START_REF] Higgins | ABC transporters: from microorganisms to man[END_REF], and the nematode ABCB1, i.e. Cel-Pgp-1, has been previously described to share similar structural features with the mammalian ABCB1 in ligand binding [START_REF] David | In silico analysis of the binding of anthelmintics to Caenorhabditis elegansP-glycoprotein 1[END_REF][START_REF] David | Structural model, functional modulation by ivermectin and tissue localization of Haemonchus contortus P-glycoprotein-13[END_REF]. We therefore confidently used Cel-Pgp-1 3D structure as relevant model for human ABCB1 in an open-inward configuration, which is supposed to be the first conformational state recognized by a substrate [START_REF] Jin | Crystal structure of the multidrug transporter P-glycoprotein from Caenorhabditis elegans[END_REF]. Both vitamin D forms specifically bound in a region deeply embedded in the transmembrane hydrophobic domains of the transporter. This positioning is consistent with subsequent triggering of the cascade of events that leads to the translocation of the compound out of the cells. Interestingly, similar positioning was found for D 3 and 25(OH)D 3 on an area corresponding to the multispecific drug binding domain, previously described as the upper binding site in Cel-Pgp-1 [START_REF] David | In silico analysis of the binding of anthelmintics to Caenorhabditis elegansP-glycoprotein 1[END_REF] and in mammalian ABCB1 (33). The 3 and 4 hydrogen bonds engaged respectively by D 3 and 25(OH)D 3 with residues on TM1 and on TM12 were consistent with the high-affinity binding of vitamin D on Cel-Pgp-1.

Importantly, most of the interacting residues are pointed out as hotspots for drug binding in mammalian ABCB1, further validating the relevance of the homology modeling. Interestingly, the hydrophobic TMD11 appeared as a "Velcro®" strap that fixed the molecules on the protein between residues 98 and 1028, further stabilizing the vitamin D in their binding sites, giving high confidence in these poses. Remarkably, the vitamin D binding modes were very similar, in terms of location and interacting residues, to that of the ABCB1 substrate progesterone (unpublished data), strengthening the idea that, beside transporting many drugs, ABCB1 can also transport steroid derivatives, including vitamin D. Overall, the binding modes as described herein for vitamin D on Cel-Pgp-1, were proven to be compatible with a transport by ABCB1 [START_REF] Jin | Crystal structure of the multidrug transporter P-glycoprotein from Caenorhabditis elegans[END_REF][START_REF] David | In silico analysis of the binding of anthelmintics to Caenorhabditis elegansP-glycoprotein 1[END_REF].

Finally, to evaluate whether ABCB1 could play a significant role in vitamin D status in humans, we explored the associations between polymorphisms in ABCB1 and the interindividual variability in 25(OH)D 3 concentration in a group of healthy male adults. The observed association between a SNP in the 3'-untranslated region of ABCB1 (rs17064) and decreased fasting 25(OH)D status strengthens our data obtained with both in vitro and animal models. The untranslated regions at the 3' end of mRNA transcripts are known to exhibit important sequences regulating the fate of mRNA and thus proteosynthesis. A previous study showed that rs17064 did not alter ABCB1 mRNA stability [START_REF] Gow | The effects of ABCB1 3'-untranslated region variants on mRNA stability[END_REF], but further research is needed to evaluate whether this variant can influence ABCB1 protein function or expression levels through other mechanisms, e.g. microRNA-mediated gene regulation [START_REF] Moszyńska | SNPs in microRNA target sites and their potential role in human disease[END_REF]. Indeed, miRNAs have been involved in the regulation of ABCB1 mRNA degradation [START_REF] Zhu | Role of MicroRNA miR-27a and miR-451 in the regulation of MDR1/P-glycoprotein expression in human cancer cells[END_REF]. Based on these data, we can speculate that rs17064 affects ABCB1 expression, which may lead to altered vitamin D transport and status. Since SNPs on the microarrays are tag SNPs, i.e. they are highly correlated to neighboring SNPs and therefore represent many unmeasured SNPs located in the same genomic region, it cannot be excluded that rs17064 is in linkage disequilibrium with the unidentified causal variant [START_REF] Schaid | From genome-wide associations to candidate causal variants by statistical fine-mapping[END_REF]. However, together with the observation that 2 SNPs in ABCB1 were previously associated with D 3 bioavailability in the same group of subjects [START_REF] Desmarchelier | A Combination of Single-Nucleotide Polymorphisms Is Associated with Interindividual Variability in Cholecalciferol Bioavailability in Healthy Men[END_REF], this result highly suggests that ABCB1 is involved in vitamin D homeostasis in humans by affecting both the net absorption of dietary vitamin D and the net efflux of circulating vitamin D. As gender differences in clearance have been reported for ABCB1 [START_REF] Ellen | Faster clearance of sustained release verapamil in men versus women: Continuing observations on sex-specific differences after oral administration of verapamil[END_REF] and because estrogens can influence ABCB1 activity [START_REF] Kim | P-glycoprotein (P-gp/MDR1)-Mediated Efflux of Sex-Steroid Hormones and Modulation of P-gp Expression In Vitro[END_REF], the specific effect of ABCB1 on vitamin D status in women remains to be evaluated.

Interestingly, 1,25-dihydroxyvitamin D 3 was previously shown to modulate ABCB1 expression via the Vitamin D Receptor VDR in human intestine [START_REF] Tachibana | Involvement of Vitamin D receptor in the intestinal induction of human ABCB1[END_REF], which further emphasizes the close link between ABCB1 and vitamin D turnover. Our data showed that vdr intestinal expression tended to be downregulated by in Abcb1 -/-mice compared with Abcb1 +/+ mice. The regulation of vitamin D intestinal efflux by intestinal VDR is thus another point of interest that would require specific research.

Our data highlight for the first time a new ABCB1-mediated pathway for vitamin D cellular efflux, as well as for vitamin D export from the bloodstream to the intestinal lumen. These data reinforce the idea that ABCB1 can control the transport of important dietary lipid components such as vitamin D. The large variability of intestinal ABCB1 expression levels (41) may thus participate in the variability also observed regarding vitamin D status. Since ABCB1 activity can be simultaneously regulated by many dietary components, drugs or polymorphisms, deciphering further the contribution of ABCB1 to vitamin D excretion balance will help to better apprehend vitamin D metabolism.

The new metabolic excretion pathway highlighted here can be described as a "TransIntestinal fat-soluble Micronutrient Excretion" (TIME), which may have a significant impact on fat-soluble vitamin homeostasis. This novel finding raises several questions. Firstly, we cannot exclude that transporters other than ABCB1 are involved in neo-absorbed vitamin D efflux and in TIME. Since TICE is more affected by the deletion of other cholesterol transporters than by ABCB1 deficiency in mice [START_REF] May | Transintestinal cholesterol excretion is an active metabolic process modulated by PCSK9 and statin involving ABCB1[END_REF][START_REF] Van Der Veen | Activation of the liver X receptor stimulates trans-intestinal excretion of plasma cholesterol[END_REF][START_REF] Jakulj | Transintestinal Cholesterol Transport Is Active in Mice and Humans and Controls Ezetimibe-Induced Fecal Neutral Sterol Excretion[END_REF], it would be interesting to determine the contribution of additional membrane proteins to TIME. Secondly, the physiological relevance of such excretion pathway remains to be evaluated. Given that several ABCB1 endogenous ligands (cholesterol, steroids, xenobiotics) are present in the organism at rather high concentrations compared with vitamin D, neo-absorbed vitamin efflux or TIME may be active in situation of oral vitamin D supply only.

The capacity of vitamin D to interact at physiological concentrations with ABCB1 thus remains to be fully characterized. Finally, TICE is currently largely investigated because it might be an interesting therapeutic target for increasing cholesterol disposal with the feces [START_REF] Reeskamp | Transintestinal cholesterol excretion in humans[END_REF]. It would consequently be of primary interest to assess whether TICE activation can lead to a concomitant increase in TIME that would potentially be deleterious for patients.

  data were tested by the Mann-Whitney U test, using Statview software, version 5.0 (SAS Institute, Cary, NC). The associations between the 21 selected SNPs in ABCB1 and the 25(OH)D 3 concentration were analyzed by Student's t-test with the Benjamini-Hochberg correction for multiple testing, using QVALUE (version 1.0, designed by researcher) (25) and R softwares (version 3.0.2, R foundation of statistical computing).

Finally

  , KO143, a relative specific inhibitor of ABCG2, impaired D 3 efflux but not that of 25(OH)D 3 , suggestion that D 3 efflux may be mediated by other transporters. The significant increase in D 3 and 25(OH)D 3 efflux and transepithelial secretion in two other cell models overexpressing ABCB1 provides additional evidence on the specific involvement of ABCB1 in vitamin D transport. Finally, direct interaction of D 3 and 25(OH)D 3 with HsABCB1 was further confirmed by in vitro experiments showing that both compounds inhibited the ABCB1-verapamil-stimulated ATPase activity.

Figure 1 :

 1 Figure 1: ABCB1 involvement in vitamin D apical efflux in epithelial cell models. (A-C) Apical efflux of D 3 and 25(OH)D 3 by Caco-2 TC7 differentiated cell monolayers. Cells were charged overnight with vitamin D before efflux to mixed micelles was measured in the absence (white bar) or presence (black bar) of valspodar 0.1-5µM (A), zosuquidar 1µM (B) or KO143 1µM (C). Vitamin D efflux was estimated by the amount of vitamin found in the apical medium and expressed as a percentage of the amount vitamin D taken up by the cells (i.e vitamin D found in the medium plus vitamin D remaining in the cells at the end of the experiment). (D) Apical efflux of D 3 and 25(OH)D 3 by Griptite cells transfected or not with ABCB1. Cells were transfected with either an empty pCMV plasmid (○) or with a pCMV plasmid containing ABCB1 (•). The cell monolayers received overnight vitamin D 10 µM before efflux was measured at 2 h and 4 h. Vitamin D efflux was estimated by the amount of vitamin D found in the efflux medium and expressed as a percentage of the amount of vitamin D taken up by the cells (i.e. vitamin D found in the medium at 2 h plus vitamin D found in the medium at 4 h plus vitamin D found in the cells at the end of the experiment). (E) Transcellular efflux of D 3 and 25(OH)D 3 by MDCK-II cells overexpressing ABCB1 (•) compared with control cells (○). The basolateral side of the cell monolayers received media enriched with 10 µM vitamin D. Two sequential samples were taken after 2 h and 4 h from the apical side. Vitamin D efflux was estimated by the amount of vitamin found in the apical and expressed as a percentage of the amount vitamin D initially delivered at the basolateral side. (F) Modulation of basal and verapamil-stimulated ABCB1 ATPase activity by D 3 and 25(OH)D 3 . Left panel: ABCB1 ATPase activity was measured in membranes of Sf9 insect cells expression human ABCB1, in absence (control) or in the presence of 40 µM D 3 or 25(OH)D 3 . Right panel : The verapamil-stimulated-ATPase activity of ABCB1 was determined in the presence of verapamil 5 µM (concentration giving the maximal stimulation), in absence (control) or in the presence of 40 µM D 3 or 25(OH)D 3 . Data are mean ± SEM of 3 assays. * p<0.05 when compared with the control cells.

Figure 2 :

 2 Figure 2: Plasma vitamin D concentration and postprandial plasma vitamin D response to vitamin D gavage in Abcb1 +/+ and Abcb1 -/-mice. (A) Fasting plasma D 3 , 25(OH)D 3 and 1,25(OH) 2 D 3 concentrations in Abcb1 +/+ (+/+; n=9) and Abcb1 -/-(-/-; n=9) mice. (B) Fasting intestinal D 3 and 25(OH)D 3 concentrations in Abcb1 +/+ (+/+) and Abcb1 -/-(-/-) mice (n=3 for D 3 and n=4 for 25(OH)D 3 ).

( C )

 C Brain D 3 and 25(OH)D 3 concentrations in Abcb1 +/+ (+/+) and Abcb1 -/-(-/-) mice (n=9 for D 3 and n=5 for 25(OH)D 3 ). (D) Kidney D 3 and 25(OH)D 3 concentration in Abcb1 +/+ (+/+) and Abcb1 -/-(-/-) mice (n= 4 per group). (E) Liver D 3 and 25(OH)D 3 concentration in Abcb1 +/+ (+/+) and Abcb1 -/-(-/-) mice (n=4 per group). Data are mean ± SEM. * p<0.05; ** p<0.01; ***p<0.001 when compared with the Abcb1 +/+ mice.

Figure 3 :

 3 Figure 3: Postprandial plasma vitamin D response to vitamin D gavage and vitamin D intestinal content in Abcb1 +/+ and Abcb1 -/-mice. (A) The mice were force-fed with D 3 -rich emulsion, and plasma samples were collected every 1.5 h during a period of 6 h. Postprandial plasma D 3 responses in Abcb1 +/+ (○; n=5) and Abcb1 -/-(•; n=4) mice. (C) Postprandial plasma D 3 responses adjusted for plasma total lipids (cholesterol plus phospholipids plus triacylglycerols) in Abcb1 +/+ (○; n=4) and Abcb1 -/-(•; n=4) mice. (B-D) Corresponding areas under the curves at 0-6 h.(E) D 3 content of Abcb1 +/+ and Abcb1 -/-mice intestinal fragments after force-feeding with a D 3 -enriched emulsion. 6 h after force-feeding with a D 3 -enriched emulsion, intestines from Abcb1 +/+ and Abcb1 -/-mice were harvested and cut into fragments before their D 3 content was assessed. Data are means ± SEM. * p<0.05 when compared with the Abcb1 +/+ mice.

Figure 4 :

 4 Figure 4: Vitamin D transintestinal excretion by Abcb1 +/+ and Abcb1 -/-mouse intestine. (A) Transintestinal 25(OH)D 3 excretion measured by using Ussing chambers. Abcb1 +/+ (○; n=3) and Abcb1 - /-(•; n=3) mice jejunum explants were mounted on Ussing Chambers. 25(OH)D 3 10 µM was added to Krebs/glucose buffer with 1% of BSA free fatty in the basolateral chamber. The transintestinal 25(OH)D 3 excretion was determined after 15 min and 30 min by measuring the appearance of 25(OH)D 3 in the apical chamber filled with Krebs buffer enriched with 25(OH)D 3 acceptors (micelles). (B) Transintestinal 3 H-25(OH)D 3 excretion using in situ perfusion. Mouse duodenum was perfused with Krebs supplemented with taurocholate-phosphatidylcholine micelles. 3 H-25(OH)D 3 was injected to Abcb1 +/+ mice (○; n=7) and Abcb1 -/-mice (•; n=9) by the penis vein. Samples of the perfusate were taken and the 3 H-25(OH)D 3 content was measured at 30, 60 and 120 min.(C) Transintestinal 3 H-25(OH)D 3 excretion of Abcb1 +/+ mice (white bar) and Abcb1 -/-mice (black bar) after weight adjustment after 2 h. Data are means ± SEM. * p<0.05; ** p<0.01 when compared with Abcb1 +/+ mice.

Figure 5 :

 5 Figure 5: D 3 and 25(OH)D 3 docking positions on the ABCB1 Cel-Pgp-1.

TableTable 1 . List of the SNPs in ABCB1 whose association with fasting plasma 25(OH)D 3 concentration was tested in a group of 39 healthy male adults. SNP rs number P-value Adjusted p-value

 1

	rs17064	0.002	0.042
	rs1045642	0.011	0.105
	rs17149810	0.015	0.105
	rs2235015	0.034	0.179
	rs2235023	0.065	0.237
	rs12539936	0.079	0.237
	rs4148807	0.079	0.237
	rs2178658	0.103	0.270
	rs11772987	0.213	0.497
	rs4148737	0.239	0.502
	rs2235033	0.294	0.561
	rs4148732	0.541	0.855
	rs1202179	0.562	0.855
	rs1211152	0.605	0.855
	rs17327624	0.659	0.855
	rs1922240	0.679	0.855
	rs9282564	0.692	0.855
	rs10264990	0.743	0.867
	rs868755	0.849	0.916
	rs4148733	0.872	0.916
	rs2188526	0.934	0.934

Acknowledgements:

We acknowledge Marion Nowicki and Lea Lasvaux for technical contributions, as well as Marion David for sharing knowledge on docking experiments.

Marielle Margier received a doctoral fellowship (2015-2018) from Region PACA (France) in partnership with the Centre Technique de Conservation des Produits Alimentaires (CTCPA Avignon, France). This work was partly supported by a Research Price from the French Nutrition Society (SFN).

Marielle Margier received a doctoral fellowship (2015-2018) from Region PACA (France) in partnership with the Centre Technique de Conservation des Produits Alimentaires (CTCPA Avignon, France). This work was partly supported by a Research Price from the French Nutrition Society (SFN).

Author contributions:

All authors have declared that no conflict of interest exists. ER and AL designed the research project with the participation of XC; MM and CL performed in vitro experiments; MM and CLM performed in vivo experiments; MM and CD performed LC-MS analyses; ChD and PB performed SNP analysis; FA performed docking experiments; all authors participated in analysis and interpretation of data; MM and ChD did the statistical analyses; ER and AL drafted the manuscript; XC did a critical revision of the manuscript; all authors have read and approved the final manuscript; ER and AL have primary responsibility for the final content of the manuscript.


I< Brousse

S Nguyen

A Gillet

S Pinaud

R Tan

A Meffre

I< Soulantica

B Chaudret

P L Tabema

M Respaud

P Simon

Laser-scribed Ru organometallic complex for the preparation of Ru0 2 micro-supercapacitor electrodes on flexible substrate

Molecular Ru(COD)(COT) Ruthenium oxide thin films Laser-writing Flexible micro

The current technological development towards miniaturized and flexible electronics for wearable applications requires new designs for energy storage devices [START_REF] Kyeremateng | Microsupercapacitors as miniaturized energy-storage components for on-chip electronics[END_REF]. While energy delivery is mainly ensured by batteries and supercapacitors, these are actually assembled in a sandwich configuration with metallic current col lectors, thus making impossible their integration in roll-up dis plays, electronic papers, smart cloth, sensors and biomedical devices. To tackle the need for flexible, lightweight, high power and long cycle life systems, carbon-based micro-supercapacitors (MSCs) have been integrated onto flexible substrates. Therefore, carbon nanotubes (CNT) [START_REF] Hsia | Highly flexible, ail solid-state micro supercapacitors from vertically aligned carbon nanotubes[END_REF], graphene [START_REF] El-Kady | Scalable fabrication of high-power graphene micro supercapacitors for flexible and on-chip energy storage[END_REF][START_REF] Wen | Enhanced laser scribed flexible graphene-based micro-supercapacitor performance with reduction of carbon nanotubes diameter[END_REF][START_REF] Wu | Graphene-based in-plane micro supercapacitors with high power and energy densities[END_REF][START_REF] Peng | Flexible and stackable laser induced graphene supercapacitors[END_REF], reduced graphite oxide (r GO) [START_REF] Gao | Direct laser writing of micro-supercapacitors on hydrated graphite oxide films[END_REF] and porous carbon [START_REF] Jost | Carbon coated textiles for flexible energy storage[END_REF] have been explored in the past five years. Another strategy to improve the electrochemical perfor mance of flexible devices consists in the use of pseudocapacitive materials, which store energy via fast and reversible redox re actions occurring mainly at their surface. Meta! oxides, such as Mn0 2 [9.10], Ru0 2 [START_REF] Sie Ben | 6n, D. Cazorla-Amor6s, Flexible ruthenium oxide activated carbon cloth composites prepared by simple electrodeposition methods[END_REF], or MXenes [START_REF] Peng | All-MXene (2D titanium carbide) solid-state microsupercapacitors for on-chip energy storage[END_REF], as well as conductive polymers [START_REF] Meng | Ultrasmall integrated 3D micro supercapacitors solve energy storage for miniature devices[END_REF], have been integrated on flexible substrates via many fabrication processes. E-mail addresses: respaud@insa-toulouse.fr (M. Respaud), simon@chimie.ups tlse.fr (P. Simon). https: / /doi.org/10.1016/j.electacta.2018. 05.198 Laser-writing has attracted particular research interest as it provides a facile and scalable one-step process under ambient conditions to manufacture bendable MSCs [START_REF] Arnold | Direct-write planar microultracapacitors by laser engineering[END_REF][START_REF] El-Kady | Laser scribing of high performance and flexible graphene-based electrochemical capacitors[END_REF][START_REF] Peng | Flexible boron-doped laser induced graphene microsupercapacitors[END_REF]. Based on the pioneer work from Arnold 's and Kaner's groups [START_REF] Arnold | Direct-write planar microultracapacitors by laser engineering[END_REF][START_REF] El-Kady | Laser scribing of high performance and flexible graphene-based electrochemical capacitors[END_REF], direct laser writing was used to embed pseudocapacitive materials in poly imide [START_REF] Ye | In situ formation of metal oxide nanocrystals embedded in laser induced graphene[END_REF]. Ruthenium oxide is a good candidate for the preparation of MSCs thanks to its extremely high theoretical pseudocapacitance (> 1300 F g-1 ), its acceptable electronic conductivity and long-term cyclability [START_REF] V. Augustyn | Pseudocapacitive oxide materials for high-rate electrochemical energy storage[END_REF]. Besides, the low RuO 2 loadings used in micro supercapacitor electrodes do not make the cost of RuO 2 pre cursors a major issue [START_REF] Dinh | Hydrous RuO2/carbon nanowalls hierarchical structures for all-solid state ultrahigh-energy-density micro-supercapacitors[END_REF]. Recently, Kaner's group prepared laser scribed graphene (LSG)/RuO 2 based MSCs from a graphene oxide (GO)/RuCh mixture drop-casted on PET [START_REF] Hwang | Direct preparation and processing of graphene/RuO2 nano composite electrodes for high-performance capacitive energy storage[END_REF]. Laser irradiation allowed both GO conversion into graphene and RuCl 3 oxidation into RuO 2 . However, RuCIJ•3H 2 O, used as a soluble precursor, is an ill-defined compound displaying Ru centers adopting the +II and +IV oxidation states, which can further leave chloride con taminants in the material. Aside, GO is needed to ensure a good adherence of the laser-scribed RuO 2 . In search for precursors able at providing a proof of concept of the deposition of thin RuO 2 layers on a flexible substrate by laser writing technology, we turned to ruthenium 1,5-cyclooctadiene 1,3,5-cyclooctatriene (Ru(COD)( COT)) known to decompose in mild conditions without leaving any residues [START_REF] Duteil | Preparation of organic solutions or solid films of small particles of ruthenium, palladium, and platinum from organometallic precursors in the presence of cellulose derivatives[END_REF].

In this communication, we describe, as a proof of concept, the preparation of adherent pseudocapacitive electrodes for micro supercapacitor applications starting from the above mentioned organometallic complex in which ruthenium is zerovalent (Ru(0)), using a simple laser-writing process. Thus, RuO 2 -based electrodes were obtained from direct laser-writing of a Ru(COD)(COT)/cellu lose acetate mixture, supported on a flexible Kapton ™ /Ti/Au sub strate, without using any additional capacitive material or nanostructuration step. Different from Atomic Layer Deposit (ALD) or sputtering techniques, the laser-writing of this original ruthe nium metallic precursor provided a facile and scalable fabrication of thin-films RuO 2 -based electrodes for designing flexible MSCs.

Experimental

Electrodes preparation

The Ru(COD)(COT)/cellulose acetate mixture was obtained ac cording to previous work [START_REF] Duteil | Preparation of organic solutions or solid films of small particles of ruthenium, palladium, and platinum from organometallic precursors in the presence of cellulose derivatives[END_REF]. Briefly, in a glove box ( 0 2 and H 2 O contents lower than 0.1 ppm), 67.4 mg of cellulose acetate were dissolved in 3 ml of tetrahydrofuran (THF, Sigma Aldrich). In a different recipient, 125 mg of Ru(COD)(COT) (NanoMePS, France) were dissolved in 1 ml of THF. Then, the Ru(COD)(COT) solution was added into the cellulose acetate solution, resulting in a yellow solution (Ru/polymer 60% wt).

Prior to Ru(COD)(COT)/cellulose acetate mixture deposition, Ti/ Au(S0 nm/200 nm) was evaporated onto a Kapton ™ foi! (75 µm, RadioSpare, UK) to insure the current collection during the elec trochemical characterizations. Polyimide was chosen for its ther mal stability [START_REF] Park | Flexible molecular-scale electronic devices[END_REF]. The as-prepared Ru(COD)(COT)/cellulose acetate mixture was then spin-coated on the flexible substrate (500 rpm, 30 s) to achieve a homogeneous deposit of the ruthenium precursor (thickness of 1.50 ± 0.25 µm). The use of cellulose acetate allows the smooth and homogeneous deposition of the Ru(COD)(COT) pre cursor during spin-coating, which is not the case when spin coating is performed without cellulose acetate. Finally, the Kap ton ™ /Ti/Au/Ru( COD)( COT)/cellulose acetate mixture was irradi ated with a UV laser ( 405 nm, power up to 285 mW on the sample, DILASE 250, KLOÉ, France, designed for direct laser-writing), con verting the Ru precursor into RuO 2 through combustion of the organic parts of the precursor and the cellulose acetate, and oxidation of the Ru(0). The best results were obtained with the irradiation parameters: 40% of the full power (116 mW), a spot size of 1 µm, a distance between two adjacent lines of 1 µm and a writing speed of 2 mm ç 1. The area that was not irradiated by the laser was easily removed by washing the non-laser-scribed Ru(COD)(COT)/cellulose acetate with acetone and ethanol. The procedure could be repeated several times to increase the amount of coated RuO 2 , with a short 20 min annealing at 250 °C after each deposition step.

Structural and electrochemical characterizations

The chemical composition and morphology of the electro chemically active deposit were investigated by X-ray photoelectron spectroscopy (XPS). The analyses were carried out with a Kalpha XPS Thermoscientific (Thermo fisher scientific, USA) using a monochromatic AlKa source (1486.6 eV). The pressure in the analysis chamber was close to 10-9 mbar. Constant passed energy was kept at 30 eV with 0.1 eV energy steps for the high resolution scans. Ali the binding energies refer to a C (ls) peak binding energy of 284.5 eV.

The structure of the laser-scribed RuO 2 was observed through Scanning Electron Microscopy (SEM) with a JSM 7100 F üEOL, Japan), and the thickness of the coating was estimated from the cross-sectional view of the layered Kapton ™ /Ti/Au/laser-scribed Ru(COD)(COT).

The electrochemical characterizations of the as-prepared elec trodes were carried out using a Biologie VMP3 Potentiostat in 1 M H 2 SO 4 in a three-electrode configuration. A Hg/Hg 2 SO 4 elec trode was used as reference electrode and a 0.64 cm 2 Pt foi! as counter electrode. Cyclic voltammetry was performed at several scan rates and the areal capacitance (C, in F cm-2 ) was calculated from equation (1 ):

j IdV C=- A.v . ..:::JV (1)
where I stands for the discharge current (A), v the scan rate (V ç 1 ), /1 V the potential window (V) and A the footprint area of the active material (cm 2 ).

Results and discussion

Laser-scribed ruthenium oxide

An optical view of a 0.25 cm 2 laser-scribed Ru(COD)(COT) film (center) deposited onto a Kapton ™ foi! covered with a conducting Ti/Au thin layer (border) is presented in Fig. 1A. The RuO 2 layer grown on the gold layer looks homogeneous and exhibits a gran ular structure (magnification Fig. 1A). In addition, the material resulting from Ru(COD)(COT) after laser-writing remained onto the flexible substrate even after a scotch scratch test. A thickness of 100 nm was first estimated with an optical profiler (SENSOFAR, Spain) (Fig. Sl ).

The conversion of the Ru(COD)(COT) precursor into RuO 2 was monitored by XPS, since the diffraction peaks related to RuO 2 were not clearly visible from X-Ray diffraction analyses (Figs. S2 andS3). As important information can be missed on the Ru(3p) spectra, the Ru(3d) core level was first studied. A deconvolution was necessary as the Ru(3d) core level overlaps the C(ls) core level. The presence of carbon ( detailed in Supplementary Materials) is assumed to be a surface contamination layer formed on the electrode surface during storage, since the C-bindings are no longer visible after a 30s Ar + erosion of the surface (Figs. S4A andS4B). Also, the possible pres ence of carbon coming from the laser-writing of cellulose acetate was definitely excluded by performing cyclic voltammetry on laser scribed cellulose acetate, where no additional capacitive current was evidenced (Fig. SS). Fig. 1B (left) shows the Ru(3d) core level recorded for the laser-scribed Ru(COD)(COT). The Ru(3d) peaks are resolved as two 3d 5 1 2 and 3d 3 1 2 doublets, with a spin-orbit splitting of 4.2 eV. The Ru 3ds1 2 binding energy is determined as 280.9 eV, which is characteristic for Ru(IV) [START_REF] Morgan | Resolving ruthenium: XPS studies of common ruthenium mate rials[END_REF]. The presence of a second lower binding energy spin-orbit doublet, with a Ru 3d 5 1 2 compo nent located at 279.7 eV, shows that the electrode contains zer ovalent Ru [START_REF] Mun | XPS investigations of ruthenium deposited onto representative inner surfaces of nuclear reactor containment buildings[END_REF]. The corresponding satellites were added accordingly. Hence, the laser-writing of Ru(COD)(COT) leads to a mixture of metallic ruthenium and ruthenium oxide, arising either from a spontaneous oxidation or induced by the temperature reached upon laser irradiation. This supports the assumption that the laser irradiation removes the organic chains of the Ru precursor under ambient atmosphere. Then, the Ru(3p) core level spectrum shown Fig. 1B (right) was deconvoluted accordingly [START_REF] Morgan | Resolving ruthenium: XPS studies of common ruthenium mate rials[END_REF]. The atomic ratio between metallic Ru(0) and Ru(IV) is calculated to be 1:2 from both Ru(3d) and Ru(3p) core level analysis.

To further characterize the as-prepared electrode material, SEM observations were carried out. Fig. 2A is a top view SEM image of the RuO 2 coating (left) deposited onto a Kapton ™ /Ti/Au substrate (right). While focusing on the laser-scribed area (Fig. S6A), it is seen that the deposited material grown on the Kapton ™ /Ti/Au (Fig. 2B) consists in 5 µm-length Ru containing elongated islands ( evidenced by EDX as shown Fig. S6B) presenting a flake-like open porous structure for the coating. Cross-sectional observations of the layered Kapton ™ /Ti/Au/RuO 2 electrode (Fig. 2C) were used to es timate an average thickness of 210 ± 50 nm for the RuO 2 deposit (Fig. 2D).

Electrochemical characterization

To investigate the electrochemical performance of the as prepared RuO 2 , laser writing was performed on both Si/Ti/Au and Kapton ™ /Ti/Au. Electrochemical characterizations were carried out in 1 M H 2 SO 4 from cyclic voltammetry at different scan rates. Fig. 3A shows the cyclic voltammogram recorded at 100 mV ç 1 for a Si/Ti/ Au/RuO 2 electrode. The current was normalized to the RuO 2 area. The electrode exhibits the electrochemical signature expected for RuO 2 [START_REF] Zheng | Hydrous ruthenium oxide as an electrode material for electrochemical capacitors[END_REF] and delivers 24.5 mF cm-2 , which is comparable to the values reported for planar design [START_REF] Dinh | High-resolution on-chip supercapacitors with ultra-high scan rate ability[END_REF][START_REF] Warren | Highly active ruthenium oxide coating via ALD and electrochemical activation in super capacitor applications[END_REF]; however, a deviation from the ideal response is observed at low potentials. This devia tion has already been observed for hydrated RuO 2 . xH 2 O [START_REF] Zheng | Hydrous ruthenium oxide as an electrode material for electrochemical capacitors[END_REF]. The current arising from reversible ion adsorption on the bare Si/Ti/Au substrate is negligible (dashed line).

A pseudocapacitive signature is still observed with the flexible Kapton ™ /Ti/Au/RuO 2 electrode, although only 6.5 mF cm-2 are delivered. This difference is assumed to originate from the heat diffusion, which depends on the nature of the substrate used, thus influencing the growth of the deposit during laser-writing. Since such a low areal capacitance is likely due to a low mass of active material, five successive Ru(COD)(COT)/cellulose acetate spin coating and laser writing cycles were performed on Kapton ™ /Ti/ Au, leading to a labeled 5-Kapton ™ /Ti/Au/RuO 2 electrode. The Pôten.tial {VS: Hg/ ti�SQi /V D 20-1"""""1�-l"""""l""""""-1"""""1,..,,..,_r--l l'lf. • E corresponding CV curve recorded at 100 mV ç 1 in 1 M H 2 SO 4 is presented in Fig. 3C (solid circles). Again, the electrochemical signature of pseudocapacitive RuO 2 is visible, delivering an areal capacitance value of 16 mF cm-2 , which is about three times higher than that of the RuO 2 active layer previously obtained. The areal capacitance does not increase linearly with the number of layers; this was ascribed to a change of the hydration number of the RuO 2 during the annealing steps, as confirmed by the shape of the cyclic voltammogram obtained such as previously reported by Zheng et al. [START_REF] Zheng | Hydrous ruthenium oxide as an electrode material for electrochemical capacitors[END_REF]. This moderate areal capacitance value is similar to most of the capacitance reported for interdigitated micro-supercapacitor electrodes [START_REF] Hsia | Highly flexible, ail solid-state micro supercapacitors from vertically aligned carbon nanotubes[END_REF][START_REF] El-Kady | Scalable fabrication of high-power graphene micro supercapacitors for flexible and on-chip energy storage[END_REF][START_REF] Peng | Flexible boron-doped laser induced graphene microsupercapacitors[END_REF], although hybrid LSG/RuO 2 electrodes deliver higher capacitance of 171 mF cm-2 [START_REF] Hwang | Direct preparation and processing of graphene/RuO2 nano composite electrodes for high-performance capacitive energy storage[END_REF]. However, this method al lows to prepare thin layers of active materials using a wet synthesis route that avoids the use of complex Atomic Layer Deposition (ALD) or sputtering techniques [START_REF] Warren | Highly active ruthenium oxide coating via ALD and electrochemical activation in super capacitor applications[END_REF]. Besides, a loading of 25 µg cm-2 was calculated from !CP-MS measurements, thus corresponding to a gravimetric capacitance of 640 F g-1, which is consistent with the values reported in the literature [START_REF] Zheng | Hydrous ruthenium oxide as an electrode material for electrochemical capacitors[END_REF].

The rate performances of the laser-scribed RuO 2 electrodes were assessed from the CVs performed at different scan rates. Fig. 3D shows the change of the areal capacitance, calculated from the integration of the CVs during discharge, with the scan rate. At a high scan rate of 10 V ç 1 , the Kapton ™ /Ti/Au/RuO 2 electrode still delivers 11 mF cm-2 , which is more than 60% of the initial capaci tance at the low scan rate of 10 mV ç 1 • Thus, the thin RuO 2 coating ensures high rate performances, as well as its binder-free structure.

Conclusion

Ruthenium oxide was successfully deposited as a thin layer on both gold-coated silicon and Kapton ™ substrates with good adherence by UV laser irradiation of an organometallic Ru(COD)( COT) precursor, dispersed in a cellulose acetate film. Laser-writing led to the release of the Ru(0) atoms from the organometallic complex, which were partially oxidized under air. Although a modest capacitance value of 6.5 mF cm-2 was recorded in sulfuric acid, the I<apton ™ /Ti/Au/RuO 2 electrodes exhibited a pseudocapa citive ruthenium oxide signature, despite the fact that no additional material was used to improve the adherence and the electronic/ protonic conductivity of the laser-scribed RuO 2 . The amount of active material could be further increased by repeating the fabri cation process several times. Doing so, an areal capacitance of 16 mF cm-2 was obtained at 100 mV ç 1 with a flexible RuO 2 elec trode prepared from five repetitions of spin coating/laser scribing cycles. In addition, more than 60% of the initial capacitance was still delivered at a high scan rate of 10 V ç 1. Spin-coating and laser writing constitute a facile and scalable route to design thin films RuO 2 electrodes under ambient conditions.

Fig. 1 .

 1 Fig. 1. Chemical composition of the as-prepared electrode material. (A) Optical view of the as-prepared Kapton ™ {Ti/Au/laser-scribed Ru(COD)(COT) electrode (right) and mi croscope observation of the laser-scribed Ru(COD)(COT) (left). (B) Corresponding high resolution XPS spectra of the Ru(3d) and Ru(3p) regions.

Fig. 2 .

 2 Fig. 2. SEM observation of the Kapton ™ /fi/Au/Ru0 2 electrode. (A) Tilted view of the Ru0 2 /substrate interface. (B) Observation offlake-like Ru0 2 (inset) present within the 5 µm long islands. (C) Cross-sectional view of the flexible electrode with (D) a 210 nm-thick Ru02 deposit.

A 4 - 4 F

 44 -0;6 -;o,4 -0.2 o�o 0,2 o�• 0:6 P<>ter : ittal (vs 1-:1g/1-:1giso,)/V c3 ___________ _ 0.'tlll'I :i;il _) / \/ B l.0 .... , .... _. __ ,. , .... -;Q.6 -0.4 --0.2 o.o o;;z 0.4 o,e

Fig. 3 .

 3 Fig. 3. Electrochemical characterization of the laser-scribed Ru0 2 . (A) Cyclic voltammograms of a Si{fi/Au/Ru0 2 electrode and (B) Kapton ™ {fi/Au/Ru0 2 electrode recorded at 100 mV ç 1 in 1 M H 2S04• The current represented as dashed line is attributed to the substrate. (C) lmprovement of the areal capacitance after five successive spin-coating/laser writing cycles performed on the flexible substrate and (D) performances of the 5-Kapton ™ /Ti/Au/Ru02 electrode.

•

  Corresponding author. CIRIMAT, Université de Toulouse, UMR CNRS 5085, INPT, UPS, 118 Route de Narbonne, 31062 Toulouse Cedex 09, France. •• Corresponding author. Université de Toulouse, LPCNO, UMR 5215 INSA-UPS CNRS, Institut National des Sciences Appliquées, 135 Avenue de Rangueil, 31077 Toulouse, France.

Acknowledgements

K.B. was supported by the Chair of Excellence from the Airbus Group. P.S. acknowledges support from ANR Labex Store Ex pro gram. We are grateful to CNRS, INSA and Université Paul Sabatier (Toulouse III) for their support. We thank J. Esvan for the XPS characterization and A. Moreau for the !CP-MS measurements.

Appendix A. Supplementary data

Supplementary data related to this article can be found at https: // doi.org/10.1016/j.electacta.2018.05.198.