Ivan Weinsanto

Jinane Mouheiche

Alexis Laux-Biehlmann

Francois Delalande

Arnaud Marquette

Virginie Chavant

Florian Gabel

Sarah Cianferani

Alexandre Charlet

Marie-Odile Parat

François Delalande

& Yannick Goumon
email: yannick.goumon@inserm.u-strasbg.fr

Dr Yannick Goumon

Morphine Binds Creatine Kinase B and Inhibits Its Activity

Keywords: morphine, complex, ligand-binding protein, creatine kinase, high affinity 1-

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers,

2-

Background Morphine, an alkaloid from Papaver somniferum, is used to relieve pain in multiple clinical settings. In addition to its analgesic properties, morphine decreases intestinal motility, suppresses cough and has vasodilatory effects {Andersen, 2003 #5023}. It influences many other physiological processes and notably decreases ATP availability in specific brain structures [START_REF] Nasello | Effect of morphine on the RNA and ATP concentration of brain structures of the rat[END_REF]. In addition, endogenous morphine has been characterized in numerous mammalian cells and tissues, and its structure is identical to that of morphine isolated from the poppy (for review [START_REF] Laux-Biehlmann | Endogenous morphine and its metabolites in mammals: history, synthesis, localization and perspectives[END_REF]).

Only a few unspecific morphine-binding proteins with low affinity have been identified so far and include serum albumin [4; 5]. In the past, our laboratory has demonstrated that the phosphatidylethanolamine-binding protein (PEBP) [START_REF] Goumon | The hippocampal cholinergic neurostimulating peptide, the N-terminal fragment of the secreted phosphatidylethanolamine-binding protein, possesses a new biological activity on cardiac physiology[END_REF] binds to morphine-6-glucuronide (M6G) and morphine-3-glucuronide (M3G) with an affinity equal to that of its reference ligand, phosphatidylethanolamine, but has no affinity for morphine [START_REF] Atmanene | Characterization of human and bovine phosphatidylethanolamine-binding protein (PEBP/RKIP) interactions with morphine and morphine-glucuronides determined by noncovalent mass spectrometry[END_REF]. In addition to these low affinity binding-proteins, covalent binding of morphine to proteins has been proposed to result in irreversible binding to insoluble tissue components after administration [8; 9; 10].

The implication of the imidazoline system, and particularly the I2-binding site (I2B) [START_REF] Li | Imidazoline I2 receptors: An update[END_REF], in the modulation of morphine-induced analgesia and morphine analgesic tolerance has been documented [12; 13]. Interestingly, creatine kinase B (CK-B) represents the main I2B site for imidazoline and its derivatives (clonidine, guanfacine…) [11; 14]. Molecular modelling using the crystal structure of chicken CK-B and the irreversible ligand BU990006-binding site has indicated the importance of Thr71, Val72, Val75, Leu201, Leu202, Cys283 and Ser285 in this interaction [START_REF] Kimura | Identification of an imidazoline binding protein: creatine kinase and an imidazoline-2 binding site[END_REF]. Dopamine, a morphine precursor in plants and mammals [START_REF] Laux-Biehlmann | Endogenous morphine and its metabolites in mammals: history, synthesis, localization and perspectives[END_REF], has been shown to covalently bind to CK-B [START_REF] Van Laar | Proteomic identification of dopamine-conjugated proteins from isolated rat brain mitochondria and SH-SY5Y cells[END_REF]. CK-B also forms highly stable complexes with ankyrin repeat and SOCS box protein 9 (ASB9) [START_REF] Balasubramaniam | How the ankyrin and SOCS box protein, ASB9, binds to creatine kinase[END_REF]. CK-B is a 42 kDa protein expressed in neurons, oligodendrocytes and astrocytes of the central nervous system (CNS) [START_REF] Manos | Cellular and Subcellular Compartmentation of Creatine-Kinase in Brain[END_REF]. Different CK isoenzymes exist in the cytoplasm as dimers or multimers in specific cells: CK-BB in the brain, CK-MM in muscles and CK-MB in the heart [START_REF] Wyss | Creatine and creatinine metabolism[END_REF]. CK catalyzes the reversible phosphorylation of creatine by ATP to produce phosphocreatine and ADP. Mitochondrial isoforms of CK form phosphocreatine from ATP, whereas cytoplasmic CK isoenzymes form ATP from phosphocreatine.

In the present study, we have investigated whether CK-B might represent a morphine-binding protein. Recombinant mouse CK-B (rCK-B) and mouse CK-B-derived synthetic peptides, together with biochemistry approaches, were used to characterize the affinity of potential morphine-binding sites. To assess the functional significance of CK-B/morphine interactions, the impact of morphine on rCK-B activity was studied in vitro and in vivo.

3-Material and Methods

3.1-Experimental design

Our animal study is reported in accordance with the ARRIVE Guidelines for reporting experiments involving animals [START_REF] Mcgrath | Guidelines for reporting experiments involving animals: the ARRIVE guidelines[END_REF]. Experiments were carried out in a randomised and blind manner, and statistical analyses were done prior to revealing treatment groups. At least three technical replicates were used for in vitro experiments. Mice were assigned an identity number and assigned to groups randomly so that the experimenter was blind to treatment.

3.2-Drugs

Morphine base was purchased from Euromedex (Souffelweyersheim, France). A stock solution of 35 mM morphine was prepared by dissolving morphine in H2O after adding an equimolar concentration of HCl. Codeine monohydrate, morphine-3-β-D-glucuronide and morphine-6-β-D-glucuronide dihydrate powders were purchased from Sigma-Aldrich (Lyon, France) and dissolved in H2O to prepare stock solutions. Working concentrations for in vitro and in vivo experiments were prepared from the stock on the day of experiment. When necessary, the pH of the working solutions was adjusted to 7.4 with NaOH before use.

3.3-Animals

All procedures were performed in accordance with European directives (86/609/EEC) and were approved by the regional ethics committee and the French Ministry of Agriculture (license No. 00456.02 to Y.G.). Experiments were performed with C57BL/6 mice (45 day-old adult male, 24 ± 3g; Charles River, L'Arbresle, France). Animals were given food and water ad libitum, and maintained in a 12h light-dark cycle at a room temperature of 22°C ± 2°C. Cage bedding was from Anibed (Pontvallain, France; reference AB3) and food from SAFE (Augy, France; reference A04). Mice were kept group-housed at 5 per cage (Type II cage, 370cm 2 , height 14cm).

3.4-Drug injections

Mice were weighed and then i.p. injected (light phase at 10AM) with a single dose of 7.5 mg/kg morphine or an equivalent volume of saline (NaCl 0.9% in H2O). The volume of injection was 240 ± 30 µL for mice weighing 24 ± 3g. After 90 min, mice were anaesthetized with a solution of ketamine and xylazine (17 mg/mL ketamine and 2.5 mg/mL xylazine, 4 mL/kg i.p.; Centravet, Taden, France). After 5 min, adequate anaesthesia was ensured by pinching the hind paws and observing no reflex response. Animals were then euthanized by decapitation. Brains were then collected and frozen at -80°C.

3.5-Preparation of brain extracts from morphine and saline-treated mice

Brains from morphine-and saline-treated mice were homogenised with an Ultra Turrax instrument (Ika, Staufen, Germany) in 1 ml of H2O containing protease inhibitors (cOmplete Mini, EDTA-free, Roche, Basel, Switzerland). The homogenates were then sonicated (2 times 10 s, 90W) with a Vibra Cell apparatus (Sonics, Newtown, U.S.A.) and centrifuged (14,000 x g, 30 min) at 4°C. Supernatants were recovered and protein concentration was determined using the Bradford method (Protein Assay, Bio-Rad, Marnes-la-Coquette, France). Samples were then frozen at -80°C until further use.

3.6-Production of the mouse rCK-B

The N-terminal His-tagged cDNA coding region for the mouse Ckb (residues 1-318, accession number NM_021273; ref. MR205953, Origene, Herford, Germany) was cloned into the vector pET15b (CIGEx, CEA, Fontenay aux Roses, France). The N-terminal hexahistidine-tagged fusion protein was expressed in Escherichia coli BL21 (DE3). Cells were grown in LB medium (Euromedex) at 37°C until an A600 of 0.4 and subsequently induced for 3 h at 37°C with 0.1 mM isopropyl 1-thio-Dgalactopyranoside (Euromedex). Harvested bacterial pellets were resuspended in buffer containing isopropyl thio-β-d-galactoside (IPTG; Euromedex). The cell pellets were resuspended in binding buffer (10 mM Tris-HCl, 300 mM NaCl, 10% glycerol, 2 mM CHAPS and 10 mM imidazole, pH 8.0; Euromedex), lysed by sonication on ice (8 x 30 s, 90 W) and then clarified by centrifugation (20,000 x g, 50 min, 4°C). The cleared supernatant was loaded on a nickel-Hitrap column (GE Healthcare; Aulnay Sous Bois, France). The protein was eluted using 50 and 250 mM imidazole (10 mM Tris-HCl, 300 mM NaCl, pH 8.0; Euromedex). The protein was then dialyzed 12 h at 4°C against a buffer containing 10 mM Tris, and 150 mM NaCl (pH 8). The purity and homogeneity of the protein were assessed by SDS-PAGE (see below).

3.7-Enzyme-Linked ImmunoSorbent Assay (ELISA)

ELISAs were performed to determine the affinity and specificity of the binding of mouse rCK-B and its derived peptides to opiate alkaloids. 96-well plates (NUNC, Roskilde, Denmark) were coated for 1 h at 37°C with 100 μl of a solution of CK-B-derived peptides or rCK-B (10 µg/ml) in carbonatebicarbonate buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6). After three washes with 100 mM phosphate buffer pH 7.4 (PT buffer; 5 min), the wells were incubated for 30 min with 200 μl of BSA diluted in PT buffer (5%, w/v; PT-BSA buffer) to saturate non-specific sites. After saturation, wells were incubated for 1 h with 100 μl of morphine, M6G, M3G or codeine, diluted in H2O at increasing concentrations. The plate was then washed three times with PT buffer, and 100 μl of the mouse primary anti-morphine antibody (which detects morphine, codeine, M3G and M6G according to the manufacturer's antibody datasheet) diluted in PT-BSA buffer (1:2,000, v/v; 3A6, ref AMM00033; Aviva System Biology, San Diego, USA) were added. After three more washes with PT buffer, 100 μl of the secondary antibody in PT-BSA buffer (HRP-conjugated donkey anti-mouse IgG, P.A.R.I.S. CliniSciences, Nanterre, France; 1:500, v/v) were added and incubated for 30 min at room temperature. After two washes with PT buffer, followed by two washes with a pH 7.5 phosphate-citrate-0.05% Tween 20 buffer (10 min), revelation was performed with 200 μl of a 1-Step Ultra TMB substrate solution (3,3',5,5' tetramethylbenzidine, Fischer Scientific). After 15 min of incubation at RT, the reaction was stopped by the addition of 50 µl of 2N hydrochloric acid. Optical density was determined at 450 nm with a Multiskan EX plate reader (Thermo Life Sciences, Cergy Pontoise, France). Each ELISA points were tested in triplicate in three independent experiments. All samples with a triplicate CV>10% were retested to obtain a CV below or equal to 10%.

3.8-Association assays

Association assays were performed with 0.5 µg of the mouse rCK-B in the presence of 1 or 5 µg of morphine, in a final volume of 10 µl incubated during 15 min at 37 °C. Samples were diluted 1:2 with the gel-loading buffer (see below).

3.9-Gel electrophoresis

Proteins were separated on SDS-PAGE gradient gels (4-12 % acrylamide; Novex, MES running buffer). Samples were suspended in 20 µl of loading buffer containing 60 mM Tris HCl pH 6.8, 2 % SDS (w/v), 4 M urea, 5 % glycerol (v/v), 5 mM EDTA, 1 % -mercaptoethanol (v/v) and 0.05 % bromophenol blue (w/v) and subjected to heat treatment (100°C, 5 min). Gels were run in duplicate. One gel was silver-stained, and the other one was used for Western blot analysis (see below).

3.10-Silver staining

Silver staining was done according to the manufacturer's (Proteabio Europe, Langlade, France) instructions. Silver-stained gels and Western blot analysis with an anti-CK antibody were used to assess the stability of CK after heat treatment. No difference in silver-stained band intensity or immunoreactivity was noticed in any experiment.

3.11-Western Blot

After electrophoretic separation, the gel was electrotransferred onto a polyvinyldifluorene (PVDF) membrane (Bio-Rad). PVDF membranes were treated for 30 min with a saturation solution containing PBS, 5 % BSA (w/v) and 0.05 % Tween 20 (v/v). Then, PVDF membranes were incubated with the 3A6 mouse monoclonal anti-morphine primary antibody for 1 h at room temperature (1:1,000). The mouse monoclonal 3A6 anti-morphine antibodies (ref AMM00033; Aviva System Biology) were raised against a ((5 alpha 6 alpha) 7 8 didehydro-4 5 epoxy-17methylmorphinan-3 5diol)-BSA conjugate. Binding of the primary antibody was detected using HRP-conjugated donkey anti-mouse antiserum (P.A.R.I.S.; 1:50,000 in PBS, 5 % of BSA (w/v), 0.05 % Tween 20 (v/v)). Luminata FORTE™ (Millipore, France) was used as the substrate. Non-specific binding of the secondary antibody was ruled out by omitting the primary antibody: non-specific labelling was absent with the secondary antibody alone (data not shown).

3.12-Peptide synthesis

CK-B-derived peptides (73 to 96 residues) were synthesized by Proteogenix (Schiltigheim, France): CK-B1-75, CK-B65-140, CK-B127-199, CK-B184-258, CK-B248-343, CK-B286-381, CK-B184-258, CK-B199-223, CK-B214-238, CK-B228-258.

3.13-Circular dichroism

Circular dichroism spectra were recorded from 260 to 190 nm using a J-810 spectropolarimeter (Jasco, Tokyo, Japan). Spectral resolution and data pitch were adjusted to 1 nm while the scanning speed was set up at 50 nm/min. Samples containing 0.2 mg/mL of CK-B1-75 in H2O (pH 6) and CK-B184-258 (in 2.5 mM of NaOH, pH 7) were transferred into a quartz cuvette of 1 mm path length and maintained at the temperature of 23°C. The secondary structures of the peptides were calculated from the spectra using a least squares fit procedure implemented in the DicroProt analysis software [START_REF] Deleage | An interactive graphic program for calculating the secondary structure content of proteins from circular dichroism spectrum[END_REF].

3.14-CK activity assay

We used a commercial creatine kinase activity assay kit (MAK116, Sigma-Aldrich) to measure the potential impact of morphine binding on CK-B enzymatic activity. The protocol followed the manufacturer's instructions at room temperature. Briefly, 1 µg of rCK-B or 75 µg of brain extract were added per well (96 well plates, NUNC) with increasing concentrations of morphine (0, 0.01, 0.1, 1, 10, 25, 50, 100 µM final). The reconstituted reagent (made of assay buffer, enzyme mix and substrate solution) was then added to start the reaction. Fluorescence was recorded using a Mithras LB940 fluorescence plate reader (Berthold, Bad Wildbad, Germany), with excitation and emission filters of 350 nm and 460 nm, respectively. Four replicates were used for each data point.

3.15-Statistics

Data were analyzed using Graphpad Prism Software 7.0. Non-parametric statistical tests were used due to low sample size and the non-Gaussian distribution of some groups. For in vitro rCK-B assays, data were expressed as percentage of the control (no morphine condition) to account for variability across replicates done on separate days. Statistical differences were tested with the Kruskal-Wallis test and Dunn's multiple comparisons as post-hoc. Brain extract assays were analyzed using the Mann-Whitney U test. Differences between groups were considered statistically significant at p < 0.05.

4-Results

4.1-Characterization of the CK-B affinity for morphine

The binding of mouse rCK-B, produced in E. coli to increasing concentrations (1.56 µM to 400 μM) of morphine, M3G or M6G was tested by ELISA. The results show that mouse rCK-B binds morphine (Figure 1A) with a calculated Kd of 10.8 µM. Our results further revealed that morphine-3-glucuronide (M3G), morphine-6-glucuronide (M6G) and codeine only weakly bind to rCK-B (Kd >100 µM) compared to morphine. As a non-specific binding control, a series of wells were coated with bovine serum albumin (BSA) instead of rCK-B and incubated with increasing concentrations of morphine, M3G or M6G. No binding of morphine, M3G or M6G to BSA was observed (Figure 1A,control).

Together, these results indicate that rCK-B binds morphine with a micromolar range affinity.

4.2-Characterization of a morphine-rCK-B complex resistant to SDS-PAGE

Mouse rCK-B (0.5 µg) was incubated 15 min in the presence of 1 or 5 µg of morphine. Samples were resuspended in loading buffer containing both SDS and urea. A heating step (100°C; 5 min) was performed before polyacrylamide gel electrophoresis (PAGE).

Western blot analysis using anti-morphine antibody showed that the mouse rCK-B alone is not labelled by the anti-morphine antibody (Figure 1B, left panel). Morphine immunolabelling was observed at 42 kDa (CK-B monomers) and 84 kDa (CK-B dimers) only when CK-B was incubated with 1 and 5 µg of morphine. As a control, an identical gel was run in parallel and silver-stained (Figure 1B, right panel). This control shows the presence of two bands at 42 kDa and 84 kDa in each condition. These two bands correspond to the monomers and dimers of mouse rCK-B, respectively.

These results indicate that morphine-mouse rCK-B complexes are resistant to SDS-PAGE.

4.3-Identification of the CK-B fragments displaying an affinity for morphine

To identify the morphine-binding motifs of CK, six overlapping peptides, namely CK-B1-75, CK-B65-140, CK-B127-199, CK-B184-258, CK-B248-343, and CK-B286-381 were tested for morphine, M3G, M6G and codeine binding via ELISA (Figure 2A). Increasing concentrations of opiates (0.1 µM to 324 μM) were added to wells pre-coated with the peptides (0.5 µg). CK-B1-75 and CK-B184-258 were able to bind morphine (Figure 2B-C) with a calculated Kd of 12.5 µM and 13.2 µM, respectively. However, ELISA analysis revealed that the six CK-derived peptides have no affinity for M3G, M6G or codeine (Figure 2B-C). Circular dichroism analysis revealed that in H2O, CK-B1-75 adopts a structure consisting of 3% alpha helix, 15% beta sheets and 82% random coils. Similarly, the predicted secondary structure of CK-B184-258 is 8% alpha helix, 19% beta sheets and 73% random coils. Focusing on the CK-B184-258 peptide, we tested the affinity of four smaller overlapping peptides (CK-B184-258, CK-B199-223, CK-B214-238, CK-B228-258). None of these four peptides displayed an affinity for morphine using an ELISA approach (data not shown).

Taken together, these results indicate that mouse CK-B1-75 and CK-B184-258 have a micromolar range affinity restricted to morphine, as observed for the mouse rCK-B.

4.4-Characterization of the effect of morphine binding on rCK-B activity in vitro

Next, we measured the potential impact of morphine binding to CK-B on the formation of ATP from phosphocreatine. Mouse rCK-B (1 µg) was incubated in the presence of increasing concentrations of morphine (0, 0.01, 0.1, 1, 10, 25, 50 and 100 µM; Figure 3A). The activity of rCK-B was monitored by measuring the production of ATP during 20 min. Morphine (1 µM) significantly decreased the formation of ATP by 27 % (36.4±1.8 nmol/min/mg) compared to the control condition (50 ±1.4 nmol/min/mg). Further increasing morphine concentrations dose-dependently inhibited CK-B activity until a maximal level of inhibition was reached (80% decrease in activity for morphine >50 µM). This experiment shows that 1 µM morphine, corresponding to a concentration close to the Kd of CK-B for morphine, significantly decreases CK-B activity in vitro.

4.4-Characterization of the effect of morphine on CK-B activity in vivo

In mice, the effect of morphine on CK-B activity in vivo was measured after a single injection of morphine (7.5 mg/kg, i.p.). Endogenous CK-B activity was tested on 75 µg of brain extract, by monitoring ATP production over 10 min. Results indicate that acute morphine treatment induces a statistically significant decrease of CK-B activity by 15.3% (1.60±0.09 µmole/min/mg of total brain protein) compared to saline-treated animals (1.89±0.06 µmole/min/mg of total brain protein; Figure 3B).

5-Discussion

The present study reports that recombinant mouse rCK-B displays a micromolar affinity for morphine but not for M3G, M6G or codeine. Two morphine-binding sites were identified, namely peptides CK-B1-75 and CK-B184-258, which displayed a similar micromolar affinity for morphine. In addition, we show that CK-B-morphine complexes are resistant to detergents (SDS), chaotropic agents (urea), reducing agents and heat treatment. Finally, our results show that at a concentration that results in complex formation, morphine significantly decreases the activity of the mouse rCK-B in vitro and endogenous mouse brain CK-B in vivo.

5.1-The morphine-CK-B complexes

CK-B is the main I2B site for imidazoline and derivatives (such as clonidine , guanfacine…) [11; 13; 14]. In addition, CK was described to covalently bind dopamine [START_REF] Van Laar | Proteomic identification of dopamine-conjugated proteins from isolated rat brain mitochondria and SH-SY5Y cells[END_REF], which is a morphine precursor in plants and animals [START_REF] Laux-Biehlmann | Endogenous morphine and its metabolites in mammals: history, synthesis, localization and perspectives[END_REF], and to form highly stable complexes with ASB9 [16; 21]. However, the potential binding of CK-B to morphine, as well as a potential modulatory role of morphine on CK-B activity, have never been studied.

Covalent binding of morphine to proteins has been first proposed by Misra and colleagues [8; 22]. This group was able to demonstrate that a single subcutaneous injection of 10 mg/kg of [ 14 C]morphine-N-methyl led to morphine-related radioactivity lasting in the rat CNS for at least 3 weeks. Later, [C1-3 H]-morphine and [N-14 CH3]-morphine were found to irreversibly bind insoluble tissue components after administration [9; 10]. In addition, S-glutathionylmorphine complexes have been characterized [23; 24] and are likely formed first by the action of morphine-6-deshydrogenase (EC 1.1.1.218) leading to the formation of morphinone [START_REF] Endo | Characterization of rabbit morphine 6-dehydrogenase and two NAD(+)-dependent 3alpha(17beta)-hydroxysteroid dehydrogenases[END_REF], followed by a spontaneous step allowing conjugation to glutathione (GSH) or proteins displaying cysteine residues. Besides, the binding of morphine to 35-50 kDa proteins in mice has also been reported [10; 26; 27]. Morphine binding to these unknown proteins was described as "covalent" because morphine-protein complexes were resistant to both methanol precipitation and SDS-PAGE denaturing conditions [START_REF] Nagamatsu | Covalent binding of morphine to isolated rat hepatocytes[END_REF].

In this article, we similarly unveil the formation of mouse rCK-B-morphine complexes that are resistant to SDS-PAGE treatment. Such a strong binding seems incompatible with the micromolar range affinity measured by ELISA. However, ELISA only reflects a biased affinity (i) resulting from conditions that do not especially promote interactions of morphine with CK-B or its derived fragments and (ii) dependent on the primary and secondary antibodies. This point is strengthened by the fact that both CK-B1-75 and CK-B184-258 display an affinity (µM range) similar to that of the full-length mouse rCK-B. In addition, the control condition performed with a nonspecific morphine-binding protein (i.e., BSA) did not result in any binding and therefore highlights the stringency of our ELISA [4; 5]. It is likely that morphine-binding sites are present in a well-defined structure of the peptides rather than in unstructured parts. Smaller CK-B184-258-derived peptides did not show any affinity for morphine, therefore we could not determine a specific morphine-binding site. This suggests that multiple structural parts all along the sequence of CK-B184-258 are required for the binding of morphine. The formation of strong morphine-CK-B complexes can be explained by two hypotheses: (i) non-covalent interactions involving ionic, hydrophobic and/or van der Waals interactions lead to a highly stable complex or (ii) a covalent binding of morphine to cysteine residues present in the CK-B occurs. However, our in vitro experimental conditions rule out a possible cross-linking involving cysteine residues (experiments performed in reducing conditions) and the action of morphine-6-deshydrogenase (reaction media do not contain this enzyme). Thus, it is likely that non-covalent complexes are formed with morphine and involve specific interactions as shown for the CK-ASB9 complexes [START_REF] Balasubramaniam | How the ankyrin and SOCS box protein, ASB9, binds to creatine kinase[END_REF].

5.2-CK-B activity modulation by morphine

As CK-B represents a crucial enzyme that regulates ATP bioavailability in the brain, the ability of morphine to alter CK-B activity may dramatically affect CNS metabolism. Notably, the binding of the irreversible I2B ligand BU99006 (20 µM) to CK-B was shown to decrease the apparent Vmax of CK-B by up to 16% [START_REF] Kimura | Identification of an imidazoline binding protein: creatine kinase and an imidazoline-2 binding site[END_REF]. However, other I2B ligands (2-BFI, BU224, agmatine) did not affect CK-B activity. In addition, it was shown that a chronic administration of cannabidiol increases CK activity by up to 20% in the rat brain [START_REF] Valvassori | Acute and chronic administration of cannabidiol increases mitochondrial complex and creatine kinase activity in the rat brain[END_REF].

The implication of I2B sites in the modulation of morphine-induced analgesia has been well documented [12; 13; 29; 30] and the involvement of I2B sites in the modulation of morphine withdrawal and tolerance has been reported in rodents [31; 32; 33]. For example, the I2 ligand agmatine increases morphine-induced analgesia and inhibits tolerance and dependence to opioids [START_REF] Dardonville | Synthesis and pharmacological studies of new hybrid derivatives of fentanyl active at the mu-opioid receptor and I2-imidazoline binding sites[END_REF].

In the brain, changes in the expression of energy metabolism enzymes contribute to behavioural abnormalities during chronic morphine treatment [START_REF] Chen | Expression changes of hippocampal energy metabolism enzymes contribute to behavioural abnormalities during chronic morphine treatment[END_REF] but can also deeply impact gliotransmission through the modulation of ATP release [START_REF] Harada | Gliotransmitter Release from Astrocytes: Functional, Developmental, and Pathological Implications in the Brain[END_REF]. Accordingly, morphine-binding significantly decreases CK-B enzymatic activity by to 27% when morphine is present at a concentration close to the Kd value. In addition, we have shown that in vivo, CK-B activity in brain tissue is decreased by 15 % after injection of an analgesic dose of morphine. Interestingly, our results are in agreement with the effect of morphine on ATP levels in the rat neocortex and thalamus, where respective decreases of 14% and 26% have been observed in vivo [START_REF] Nasello | Effect of morphine on the RNA and ATP concentration of brain structures of the rat[END_REF].

Our results reveal two potential morphine-CK-B binding sites, namely CK-B1-75 and CK-B184-258. The former is not involved in CK-B activity. Conversely, CK-B184-258 contains a conserved, negatively charged amino acid cluster, Glu226, Glu227, Asp228 (EED) located in the active site of CK isoenzymes [START_REF] Eder | A conserved negatively charged cluster in the active site of creatine kinase is critical for enzymatic activity[END_REF]. This particular motif has been described as essential for CK enzymatic activity. Thus, we can hypothesize that morphine alters CK-B function through binding to the CK-B184-258 sequence. A limitation of our study is that we only evaluated a fixed time point after euthanizing the animals, and a different pattern of CK-B responses might occur at different time points. Further clarification of the impact of morphine on CK-B function in vivo is needed, e.g. through brain microdialysis and ATP quantification. Another open question is the impact of chronic morphine treatment in patients on CK-B activity, which is likely to differ from our acute morphine-exposure treatment. Indeed, a chronic morphine exposure might inhibit CK-B during a long period leading to important physiological changes and a measurable phenotype.

5.3-In vivo complex formation

After administration, morphine rapidly reaches the CNS where it binds to mu opioid receptors (MORs) to produce analgesia. Then, morphine is taken up by glial cells including microglial cells and astrocytes through the action of the organic cation transporter OCT1 [START_REF] Tzvetkov | Morphine is a substrate of the organic cation transporter OCT1 and polymorphisms in OCT1 gene affect morphine pharmacokinetics after codeine administration[END_REF]. After conversion by UDPglucuronosyl-transferase (UGT) enzymes [39; 40], glucuronides are released through the efflux MRP3 transporter (multidrug resistance protein 3) [START_REF] Zelcer | Mice lacking multidrug resistance protein 3 show altered morphine pharmacokinetics and morphine-6-glucuronide antinociception[END_REF]. In this context, it is plausible that after administration and cellular uptake, morphine can form complexes with cytoplasmic CK-B and therefore negatively modulate its activity. Interestingly, injection (i.v., i.p. or s.c.) of an analgesic dose of morphine (10 to 20 mg/kg) to mice results in serum concentrations ranging from 2 to 35 µM. Similarly, morphine content in the brain reaches nmol/g of tissue levels (corresponding to a µM range), indicating that our findings are relevant in vivo [42; 43; 44; 45].

Endogenous morphine has been detected in numerous cerebral areas and brain cells of different species including the mouse [1; 46; 47]. Specifically, endogenous morphine is detected in GABAbasket cells, astrocytes and microglial cells [46; 47; 48; 49]. In addition, an increase of endogenous morphine and M6G blood levels has been linked to stress [50; 51; 52] and pathologies such as sepsis, with concentrations reaching up to 0.25 µM [53; 54]. In contrast to opioidergic peptides (e.g., enkephalins), the functional role of endogenous morphine in the CNS remains to be elucidated. Endogenous morphine is found in the cytoplasm of astrocytes expressing both OCT1 [START_REF] Schreiber | Astrocytes and glioblastoma cells express novel octamer-DNA binding proteins distinct from the ubiquitous Oct-1 and B cell type Oct-2 proteins[END_REF] and CK-B [START_REF] Tachikawa | Distinct cellular expressions of creatine synthetic enzyme GAMT and creatine kinases uCK-Mi and CK-B suggest a novel neuron-glial relationship for brain energy homeostasis[END_REF]. Although endogenous morphine concentrations are unlikely to reach 1 μM, uptake by astrocytic processes could theoretically bring endogenous morphine in contact with cytoplasmic CK-B. Whether endogenous morphine-CK-B complexes exist and might influence bioavailability of ATP in astrocytes and impact ATP-mediated gliotransmission through CK-B inhibition remains to be investigated in further studies.

5.4-Clinical relevance of morphine-CK-B interactions

The in vitro and in vivo impact of morphine on CK-B activity in the mouse raises the question of a potential relevance of morphine-CK-B interactions in the case of clinical or recreational use of morphine or its derivatives in humans.

First, doses administered to morphine-naive humans are usually two orders of magnitude lower than those needed to elicit analgesia in rodents. Thus, peak morphine blood concentrations (and presumably tissue concentrations) after oral therapeutic dosing typically only reach 100 nM [START_REF] Klimas | Morphine-6-glucuronide is responsible for the analgesic effect after morphine administration: a quantitative review of morphine, morphine-6glucuronide, and morphine-3-glucuronide[END_REF], a concentration far below the 1 µM of morphine needed for CK-B modulation. However, it is possible that intracellular morphine could reach micromolar concentrations in chronically treated, tolerant patients. Indeed, dose escalation following long-term treatment can lead to patients receiving 500 mg of morphine or more per day. Thus, in the case of chronic use of morphine or codeine, whether in clinical (e.g. cancer patients) or drug abuse settings, morphine concentrations rise to low µM levels in both plasma and brain [58; 59]. In addition, morphine concentrations in the cerebrospinal fluid (CSF) and brain extracellular fluid (ECF) have been reported to reach nM to µM values in different conditions, including epidural injection during chronic intractable pain due to cancer and codeine overdoses [60; 61; 62]. It is also important to mention the fact that M3G concentrations in these cases are far higher than those of morphine. CK-B is an intracellular protein highly expressed in astrocytes which are known to metabolize morphine into M3G [63; 64; 65; 66]. The active uptake of morphine by astrocytes might allow intracellular morphine to reach µM levels. Unfortunately, intracellular morphine concentrations have never been measured in the human brain, although it has been shown in rodents that morphine preferentially accumulates inside cells, especially astrocytes rather than in the ECF or CSF [66; 67].

In conclusion, CK-B/morphine interactions have the potential to be of clinical relevance and this warrants further investigation. While morphine concentrations may not reach sufficient levels to significantly impact CK-B activity in normal dosing regimens, chronic administration and high doses administered to tolerant patients might affect ATP bioavailability in humans.

6-Conclusion

Taken together, our results unveil a strong interaction between CK-B and morphine, occurring in vitro and in vivo. This interaction negatively impacts CK-B activity and thus has the potential to affect energy metabolism and glial transmission. Whether or not morphine is able to bind to and modulate CK-M or mitochondrial CK remains to be studied, but this seems likely given a high sequence homology between these enzymes and CK-B. Additional experiments will allow to further elucidate the binding site of morphine and study, in detail, the consequences of this interaction. The impact of CK-B and its derived peptides on morphine analgesia and metabolism will be investigated in the future. Finally, a detailed study has to focus on endogenous morphine in order to determine its potential implication in energy bioavailability in both physiological and pathological processes.

FOOTNOTES

Figure 1 -

 1 Figure 1-Characterization of the CK-B affinity for morphine. A, Affinity of mouse rCK-B for morphine, M3G and M6G using an immunoenzymatic assay (ELISA). ELISAs were performed with the 3A6 morphine antibody which detects morphine, codeine, M3G and M6G with the same affinity. Optical density increases with formation of peptide-alkaloid complexes. Data are expressed as mean ± SEM of triplicates (representative result of n=3 independent experiments). B, Characterization of CK-B-morphine complexes resistant to SDS-PAGE. Left panel, Western blot analysis showing morphine-immunoreactivity after incubation of rCK-B with morphine (representative result of n=3 independent experiments). Right panel, silver staining of a duplicate gel performed in parallel.

Figure 2 -

 2 Figure 2-Affinity of synthetic peptides generated from mouse CK-B sequence for morphine and its derivatives. A, Map of the six CK-B-derived peptides. B, Immunoenzymatic (ELISA) determination of the binding properties of peptides CK-B1-75 and CK-B184-258 to morphine, M6G, M3G and codeine. Data are expressed as mean ± SEM of triplicates (representative result of n=3 independent experiments). ELISAs were performed with the 3A6 anti-morphine antibody which binds morphine, codeine, M3G and M6G. Optical density increases with formation of peptide-alkaloid complexes.

Figure 3 -

 3 Figure 3-Effect of morphine on CK-B activity A-In vitro effect of increasing concentrations of morphine on CK-B activity. Data are expressed as mean ± SEM. Kruskal-Wallis test with Dunn's multiple comparison; n=8; *, p<0.05;***, p<0.001. B-In vivo effect of an injection of saline or 7.5 mg/kg of morphine on brain CK-B activity. Data are expressed as mean ± SEM. Mann-Whitney test; n=8 animals per group. *, p<0.05.

ACKNOWLEDGMENTS

GRANT SPONSOR -This work was funded by Inserm, CNRS (Postdoctoral fellowship to A.L.), University of Strasbourg, French Ministère Délégué à la Recherche et à l'Enseignement Supérieur (Ph.D. fellowship to A.L., and I.W.), Alsace contre le Cancer, National Council for Scientific Research of Lebanon (CNRS-Lebanon to J.M.) and SATT-Conectus Alsace. We thank the following research programs of excellence for their support: FHU Neurogenycs, French National Research Agency (ANR) through the Programme d'Investissement d'Avenir (contract ANR-17-EURE-0022, EURIDOL graduate school of pain). We acknowledge Morgane Razafimahatratra for her technical help and Tando Maduna for her advice on the manuscript.

COMPETING INTERESTS -

The authors declare that they have no competing interests. ABBREVIATIONS ASB9, ankyrin repeat and SOCS box protein 9; CK-B, brain creatine kinase; CK-M, muscular creatine kinase; CNS, central nervous system; I2B, I2-binding; M3G, morphine-3-glucuronide; M6G, morphine-6-glucuronide; PEBP, phosphatidylethanolamine-binding protein; SEM, standard error of the mean.


Melha Benlebna

Laurence Balas

Béatrice Bonafos

Laurence Pessemesse

Claire Vigor

Jacques Grober

Florence Bernex

Gilles Fouret

Veronika Paluchova

Sylvie Gaillet

Jean François Landrier

Ondrej Kuda

Thierry Durand

Charles Coudray

François Casas

Christine Feillet-Coudray

Long-term high intake of 9-PAHPA or 9-OAHPA increases basal metabolism and insulin sensitivity but disrupts liver homeostasis in healthy mice

Keywords: FAHFA, Insulin sensitivity, Liver steatosis, Liver fibrosis, Inflammation, Mice

est destinée au dépôt

Introduction

Recently, a new class of endogenous bioactive lipids, branched fatty acid esters of hydroxy fatty acids (FAHFAs), has been discovered [START_REF] Yore | Discovery of a class of endogenous mammalian lipids with anti-diabetic and antiinflammatory effects[END_REF]. FAHFAs are a combination of fatty acid (FA) and hydroxylated fatty acid (HFA), and thus, a great diversity of FAHFAs structure exists regarding the numerous types of naturally occurring FA and HFA. Major FAHFAs are a combination of palmitic acid (PA), stearic acid (SA), oleic acid (OA) or palmitoleic acid (PO) with their corresponding HFA providing, for example, PAHPA, OAHPA, PAHOA, OAHOA, PAHSA and OAHSA [START_REF] Balas | Branched fatty acyl esters of hydroxyl fatty acids (FAHFAs), appealing beneficial endogenous fat against obesity and type-2 diabetes[END_REF]. It was reported that palmitic acid esters of hydroxystearic acid (PAHSAs) exert antidiabetic and anti-inflammatory effects [START_REF] Yore | Discovery of a class of endogenous mammalian lipids with anti-diabetic and antiinflammatory effects[END_REF][START_REF] Moraes-Vieira | GLUT4 expression in adipocytes regulates de novo lipogenesis and levels of a novel class of lipids with antidiabetic and anti-inflammatory effects[END_REF], suggesting that it could have a high therapeutic potential to prevent and/or treat type 2 diabetes. However, little if any is known regarding biological effects of other types of FAHFAs. Recently, 9-PAHPA and 9-OAHPA, two FAHFAs shown by Yore et al. [START_REF] Yore | Discovery of a class of endogenous mammalian lipids with anti-diabetic and antiinflammatory effects[END_REF] to be highly up-regulated in the adipose-specific Glut4 overexpressing mice (AG4OX), were synthesized by Balas et al. [START_REF] Balas | Regiocontrolled syntheses of FAHFAs and LC-MS/MS differentiation of regioisomers[END_REF], allowing to study both the biological activities of FAHFAs other than PAHSAs and the influence of FA and HFA chains inside the FAHFA. Regarding human dietary intake of FAHFAs, both PAHPA and OAHPA are present in the diet, in yolk and meat but also in numerous plant foods, with the highest abundancy in strawberry, radish, pineapple, apple and oat [START_REF] Liberati-Cizmek | Analysis of fatty acid esters of hydroxyl fatty acid in selected plant food[END_REF].

The liver is a key metabolic organ which governs body energy metabolism; it acts as a hub to metabolically connect to various tissues, including skeletal muscle and white adipose tissue (WAT) [START_REF] Rui | Energy metabolism in the liver[END_REF]. In addition, the liver metabolic activity, particularly glucose production, is tightly controlled by insulin [START_REF] Liberati-Cizmek | Analysis of fatty acid esters of hydroxyl fatty acid in selected plant food[END_REF]. Moreover, mitochondria play a central role in cellular energy metabolism and in metabolic regulation of liver pathophysiology [START_REF] Sato | Central role of mitochondria in metabolic regulation of liver pathophysiology[END_REF]. Indeed, previous work from our team demonstrated that various aspects of mitochondrial activity were modified by changes in diet lipid composition or by bioactive molecules such as polyphenols and ubiquinone [START_REF] Aoun | Rat liver mitochondrial membrane characteristics and mitochondrial functions are more profoundly altered by dietary lipid quantity than by dietary lipid quality: effect of different nutritional lipid patterns[END_REF][START_REF] Fouret | The mitochondrialtargeted antioxidant, MitoQ, increases liver mitochondrial cardiolipin content in obesogenic diet-fed rats[END_REF][START_REF] Hokayem | Grape polyphenols prevent fructose-induced oxidative stress and insulin resistance in first-degree relatives of type 2 diabetic patients[END_REF][START_REF] Feillet-Coudray | Impact of high dietary lipid intake and related metabolic disorders on the abundance and acyl composition of the unique mitochondrial phospholipid, cardiolipin[END_REF].

Therefore, the aim of this work was to determine whether dietary intake of 9-PAHPA or 9-OAHPA may influence insulin sensitivity and glucose metabolism in vivo and to study whether the potential beneficial effects of these FAHFAs are sustained with long-term dietary intake. In addition, the liver being a key metabolic organ tightly controlled by insulin, this study also aimed to determine if 9-PAHPA or 9-OAHPA might affect liver metabolic activity. To address these questions, healthy C57Bl/6J mice were fed a control diet supplemented or not with 9-PAHPA or 9-OAHPA for 12 weeks.

Our results show that the long-term high intake of 9-PAHPA or 9-OAHPA increased basal metabolism and enhanced insulin sensitivity in healthy mice. However, 9-PAHPA or 9-OAHPA intake promoted liver steatosis and fibrosis in some mice, underlining the fact that a likely beneficial effect of these FAHFAs in pathological situations can be harmful in a situation of good health at least at this high dietary intake.

Materials and methods

FAHFAs synthesis

The synthesis of 9-PAHPA and 9-OAHPA was performed using our previously reported procedure [START_REF] Balas | Regiocontrolled syntheses of FAHFAs and LC-MS/MS differentiation of regioisomers[END_REF]. The molecular weight of 9-PAHPA is 510.5 g/mol, and the molecular weight of 9-OAHPA is 537 g/mol.

Animals and diets

Thirty 6-week-old male C57BL/6J mice (Charles River, L'Arbresle, France), weighing about 22 g, were housed (5 per cage) under conditions of constant temperature (20°C-22°C), humidity (45%-50%) and a standard dark cycle (20:00-08:00 h). The mice were randomized, according to their initial weight, into 3 groups of 10 animals and fed for 12 weeks one of the three following semipurified diets: (1) control diet, (2) control diet+9-PAHPA and (3) control diet+9-OAHPA.

The detailed composition of the control diet is given in the Supplementary Table 1. The control diet contains 5% lipids as a mixture of rapeseed oil, high oleic sunflower oil, sunflower oil and linseed oil (oil mixture of Carrefour). The lipid fraction of the control diet was composed of 12.2% saturated fatty acids, 60.6% monounsaturated fatty acids and 27.3% polyunsaturated fatty acids (Supplementary Table 2). The 9-PAHPA or the 9-OAHPA was incorporated into the diet after dissolution in the oil mixture of Carrefour, and the final content of each FAHFA was set at 300 μmol/kg diet. For a mouse of 25 g that eats 2.5 g diet per day, this corresponds to a FAHFA intake of about 30 μmol/day/kg b.w. of mice (or about 15 mg/day/kg). FAHFAs content was quantified in the three experimental diets (Supplementary Table 3). No 9-PAHPA and 9-OAHPA was found in the control diet. The administered dosage of the studied FAHFAs was based on the work of [START_REF] Yore | Discovery of a class of endogenous mammalian lipids with anti-diabetic and antiinflammatory effects[END_REF] [START_REF] Yore | Discovery of a class of endogenous mammalian lipids with anti-diabetic and antiinflammatory effects[END_REF]. It is also in good agreement with the conditions recently published by [START_REF] Paluchova | Lipokine 5-PAHSA is regulated by adipose triglyceride lipase and primes adipocytes for de novo lipogenesis in mice[END_REF] [START_REF] Paluchova | Lipokine 5-PAHSA is regulated by adipose triglyceride lipase and primes adipocytes for de novo lipogenesis in mice[END_REF]. Throughout the study, mice were given free access to food and tap water. Mice body weight was followed weekly, and food consumption was determined every 2 days (week) or 3 days (weekend). Our institution guidelines for the care and use of laboratory animals were observed, and all experimental procedures were approved by the local ethical committee in Montpellier, France (Reference APAFIS#12759-2017121912214385). A detailed scheme of the study design is provided in Fig. 1.

Body composition analysis

Mice whole-body composition (fat and lean masses) was measured every 2 weeks throughout the study by an EchoMRI-700 wholebody composition analyzer (Echo Medical Systems, Houston, TX, USA) according to the manufacturer's instructions.

Metabolic analyses

Mice oxygen consumption and carbon dioxide production were measured using a Comprehensive Lab Animal Monitoring System (Columbus Instruments, Columbus, OH, USA). Mice were housed in individual cage inside a controlled cabinet. The environmental enclosure allows precise control over the temperature and light/dark cycle. Male mice were acclimatized individually in metabolic cages with ad libitum access to standard control and water for 24 h, prior to a 24-h period of automated recordings. Sample air from individual cages was passed through sensors to determine O 2 and CO 2 content. Sensors were calibrated before each experiment against commercial gas mixtures of accurately determined composition (20.5% O 2 , 0.5% CO 2 and 79% N 2 ). For each mouse, volume of oxygen (VO 2 ) and volume of carbon dioxide (VCO 2 ) were measured 111 times in about 24 h. The respiratory exchange ratio (RER) was calculated as the VCO2/VO 2 ratio [START_REF] Marvyn | Data on oxygen consumption rate, respiratory exchange ratio, and movement in C57BL/6J female mice on the third day of consuming a high-fat diet[END_REF].

Fig. 1. Design of the study.Thirty 6-week-old male C57BL/6 were fed for 12 weeks a control diet, a control diet+9-PAHPA or a control diet+9-OAHPA. Whole-body composition was measured every 2 weeks using an EchoMRI-700 analyzer. Mice oxygen consumption and carbon dioxide production were measured using a Comprehensive Lab Animal Monitoring System the last weeks of the study. Oral glucose tolerance test and insulin tolerance test were performed the last weeks of the study. At the end of the study, blood was collected from retro-orbital sinus. After cervical dislocation, the liver, WAT and pancreas were removed, rinsed and frozen into liquid nitrogen until analyses.

Oral glucose tolerance test and insulin tolerance test

Following an overnight fasting, mice were administrated glucose (2 g/kg) by oral gavage, and blood samples were collected from the tail vein at the indicated times for glucose and insulin determination. Insulin tolerance was also assessed after 2-h fasting by administration of human insulin (0.75 U/kg) and blood glucose monitoring. Glycemia was measured using an OneTouch Verio glucometer (Lifescan). Insulin was measured using an ultrasensitive mouse insulin enzyme-linked immunosorbent assay (ELISA) kit (Crystal Chem).

Sampling and routine biochemical analyses

Four to 5 days after the OGTT or the ITT, blood from 12-h fasted mice was collected from the retro-orbital sinus and distributed into heparinized tubes. Blood tubes were centrifuged at 1000g for 10 min at 4°C; plasma was collected and stored at -80°C until analysis. Erythrocytes were washed with saline solution, hemolyzed and stored at -80°C until analysis. After cervical dislocation of mice, liver, WAT and pancreas were removed, rinsed with 0.9% NaCl, weighed and kept at -80°C until analysis.

Plasma levels of glucose, total cholesterol, triglycerides and free fatty acids as well as enzymatic activity of alanine aminotransferase (ALT) were measured at the ANEXPLO/CREFRE analysis platform (CHU RANGUEIL-BP 84225, France). Plasma levels of insulin, leptin and IL-6 were quantified with ELISA kits (Merck Millipore, Darmstadt, Germany; Crystal Chem, Zaandam, Netherlands; Abcam, Paris, France, respectively). Total glucagon-like peptide-1 (GLP-1) plasma concentration was measured by chemiluminescence using commercially available ELISA kit (Mercodia AB, Sweden).

Liver neutral lipids and histology

To assess liver neutral lipids, liver samples were homogenized in NaCl (9 g/L) and Triton X-100 (0.1%), and free fatty acids, triglycerides and total cholesterol levels were quantified on the tissue homogenate by enzymatic methods (Wako-NEFA-C kit, Oxoid, Dardilly, France; Cholesterol CHOD-PAP SOBIODA kit and triglycerides LQ SOBIODA kit, Sobioda 38330 Montbonnot-Saint-Martin, France) [START_REF] Coudray | A mitochondrial-targeted ubiquinone modulates muscle lipid profile and improves mitochondrial respiration in obesogenic diet-fed rats[END_REF].

After euthanasia by cervical dislocation, liver samples were collected, freshly frozen in Tissue-Tek (Microm Microtech) and then stored at -80°C, whereas pancreas were collected, fixed in 4% formaldehyde and then paraffin embedded. For liver, 10-μm sections were stained with Oil Red O or Sirius Red. For Oil Red O staining, the sections were fixed in PBS, 4% PFA at room temperature (RT) 5 min, washed in H 2 O, incubated with 60% isopropanol 5 min and then incubated in oil Red O solution (0.6%) 7 min. Then, sections were briefly washed with 60% isopropanol, incubated with Harris' hematoxylin for 30 s, washed in H 2 O 3 min and mounted. For Sirius Red staining, the sections were fixed in PBS, 4% PFA at RT 5 min, washed in H 2 O and incubated in 0.01% Sirius Red F3B 1 h. Then, sections were washed twice in acidified water, dehydrated thrice in 100% ethanol, then cleared in xylene and mounted. For pancreas, 5-μm sections were stained by hematoxylin and eosin. For the morphometric analysis, liver and pancreatic sections were scanned using a NanoZoomer (Hamamatsu Photonics, Japan) with a 20× objective. The surface of islets was assessed using NDP.View 2 software (Hamamatsu Photonics). The degree of liver fibrosis was evaluated and scored using Image J software as described [START_REF] Standish | An appraisal of the histopathological assessment of liver fibrosis[END_REF].

Liver oxidative stress status

Long-established oxidative stress parameters

A part of liver was homogenized in phosphate buffer (50 mM, pH 7) 1 g for 9 ml buffer using a Polytron homogenizer. The liver thiobarbituric acid reactive substances (TBARS) and total glutathione (GSH) levels were measured in homogenate according to the methods of Sunderman [START_REF] Jr | Increased lipid peroxidation in tissues of nickel chloride-treated rats[END_REF] and Griffith [START_REF] Griffith | Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine[END_REF], respectively. The remaining homogenate was centrifuged at 1000g for 10 min at 4°C, and the supernatant was used for the other analyses of oxidative stress. Protein oxidation was assessed by measurement of thiol groups [START_REF] Faure | Measurement of plasma sulfhydryl and carbonyl groups as a possible indicator of protein oxidation[END_REF]. Catalase activity was measured according to the method of Beers and Sizer [START_REF] Beers | A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase[END_REF]. Glutathione peroxidase (GPx) was measured according to the method of Flohe and Gunzler [START_REF] Flohe | Assays of glutathione peroxidase[END_REF]. Total superoxide dismutase (SOD) was measured according to the method of Marklund [START_REF] Marklund | Spectrophotometric study of spontaneous disproportionation of superoxide anion radical and sensitive direct assay for superoxide dismutase[END_REF].

Isoprostanoid metabolites measurement

To measure oxidative damage to lipids, the liver isoprostanoids levels were measured based on micro-LC-MS/MS technique [START_REF] Dupuy | Simultaneous quantitative profiling of 20 isoprostanoids from omega-3 and omega-6 polyunsaturated fatty acids by LC-MS/MS in various biological samples[END_REF]. Briefly, after lipid extraction with Folch mixture, the extracts were mixed with a cocktail of internal standards, and an alkaline hydrolysis was performed. The metabolites were concentrated thanks to a solid phase extraction step conduced on weak-anion exchange materials. The metabolites were then analyzed by micro-LC-MS/MS. Mass spectrometry analysis was performed in an AB Sciex QTRAP5500 (Sciex Applied Biosystems). The ionization source was electrospray in negative mode. Detection of the fragmentation ion products from each deprotonated molecule was performed in the multiple reaction monitoring modes. Metabolites quantification was done, using Multi-Quant 3.0 software, by measuring the ratio of area under the specific metabolite peak/area under the internal standard peak and compared to the ratio of area under the metabolite calibration peak/area under the internal standard peak.

Liver mitochondrial enzymatic activities

The different mitochondrial respiratory complex activities were determined as previously described [START_REF] Feillet-Coudray | Oxidative stress in rats fed a high-fat high-sucrose diet and preventive effect of polyphenols: involvement of mitochondrial and NAD(P)H oxidase systems[END_REF]. Complex I activity was measured spectrophotometrically at 600 nm during 45 s by following the reduction of 2,6-dichloroindophenol by electrons accepted from decylubiquinol, itself reduced after oxidation of NADH by complex I [START_REF] Janssen | Spectrophotometric assay for complex I of the respiratory chain in tissue samples and cultured fibroblasts[END_REF]. Complex II (CII) activity was measured spectrophotometrically at 600 nm by following the reduction of 2,6-dichloroindophenol by the succinate during 120 s [START_REF] Feillet-Coudray | Oxidative stress in rats fed a high-fat high-sucrose diet and preventive effect of polyphenols: involvement of mitochondrial and NAD(P)H oxidase systems[END_REF]. Complex II + III (CII + CIII) activities were measured spectrophotometrically by following the oxidation of oxidation of cytochrome c at 550 nm during 90 s [START_REF] Rustin | Biochemical and molecular investigations in respiratory chain deficiencies[END_REF]. Cytochrome c oxidase (COX) activity was measured spectrophotometrically by following the oxidation of reduced cytochrome c at 550 nm during 30 s [START_REF] Wharton | Cytochrome oxidase from beef heart mitochondria[END_REF]. In addition, mitochondrial β-hydroxyacyl-CoA dehydrogenase (β-HAD), a marker of last step of mitochondrial β-oxidation activity, was determined spectrophotometrically according to the procedure described by Clayton et al. [START_REF] Clayton | Hyperinsulinism in short-chain L-3-hydroxyacyl-CoA dehydrogenase deficiency reveals the importance of beta-oxidation in insulin secretion[END_REF].

Protein isolation and Western blotting analysis

Frozen liver samples were homogenized using an Ultra Turax homogenizer in an ice-cold extraction buffer containing 20 mM Tris-HCL, 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 0.1% SDS, 1 mM PMSF, 10 μM leupeptin and 1 μM pepstatin. Proteins (50 μg) were separated with 6%-15% SDS-PAGE and then transferred to a nitrocellulose membrane (120 min, 100 V). Membranes were blocked in 5% fat-free milk for 1 h at room temperature. Then, membranes were incubated overnight with primary antibody against ChREBP, GLK, GLUT2, IL-6, PEPCK and TNF-α in blocking buffer (Supplementary Table 4). After washes in TBS/Tween under gentle agitation, membranes were incubated for 1 h with horseradish-peroxidaselabeled antibody. After further washes, blots were treated with enhanced chemiluminescence detection reagents (ECL, ThermoScientific, F67403 Illkirch cedex, France). β-Actin or α-tubulin was used as loading references, and blot intensities were measured using Image Lab Software 5.2.1 (Bio-Rad Laboratories, Inc., France).

Liver real-time quantitative real-time quantitative polymerase chain reaction (RT-qPCR) analysis

RT-qPCR was used to measure target genes mRNA expression in liver. Total RNA was extracted with Trizol reagent (Invitrogen Life Technologies, Cergy Pontoise, France). Reverse transcription reaction was performed with 2 μg total RNA. cDNA was synthesized with the use of SuperScript II Reverse Transcriptase for first-strand cDNA synthesis (Invitrogen Life Technologies, Cergy Pontoise, France) and Oligo (dT) primers. The mRNA expressions of target genes were determined by RT-qPCR using IQTM SYBR Green Supermix (Biorad, Hercules, CA, USA) with a MiniOpticon detection system (Biorad, Hercules, CA, USA). Results were normalized with the gene encoding RPS9 used as the reference. The primer sequences used for RT-PCR are given in the Supplementary Table 5.

WAT RT-qPCR analysis

RT-qPCR was applied to measure key inflammation genes mRNA expression in the adipose tissue. RNA from WAT was extracted with Trizol reagent (Invitrogen Life Technologies, Cergy Pontoise, France). Reverse transcription reaction was performed with 1 μg total RNA. cDNA was synthesized with the use of moloney murine leukemia virus reverse transcriptase for first-strand cDNA synthesis and random primers. The mRNA expressions of target genes were determined by RT-qPCR. RT-qPCR analysis was performed using SYBR Green Mastermix (Eurogentec, liege, Belgium) with an Mx3005P Real-Time PCR System (Stratagene, La Jolla, CA, USA). Results were normalized with the gene encoding 18S. The primer sequences used for RT-PCR are given in the Supplementary Table 5.

WAT lipidomic analysis

Sample extraction

Extraction of WAT metabolites was carried out using a biphasic solvent system of cold methanol, methyl tert-butyl ether (MTBE) and water [START_REF] Cajka | Validating quantitative untargeted lipidomics across nine liquid chromatography-high-resolution mass spectrometry platforms[END_REF] with some modifications. WAT samples (20 mg) were homogenized with 275 μl MeOH and 275 μl 10% MeOH both containing internal standards for 1.5 min using a grinder (MM400, Retsch, Germany). Then, 1 ml of MTBE with internal standard was added, and the tubes were shaken for 1 min and centrifuged at 16,000 rpm for 5 min.

For profiling of high-abundant TAG, 10 μl of upper organic phase was collected, resuspended using a chloroform/MeOH/IPA (1:2:4) mixture, shaken for 30 s and centrifuged at 16,000 rpm for 2 min, and extract was further 100-times diluted with methanol containing CUDA internal standard. For profiling of minor-lipid species in positive and negative ion mode, 100 μl of upper organic phase was collected, resuspended using 80% MeOH with CUDA internal standard, shaken for 30 s, centrifuged at 16,000 rpm for 2 min and used for LC-MS analysis. The LC-MS systems consisted of a Vanquish UHPLC System (Thermo Fisher Scientific, Bremen, Germany) coupled to a QExactive Plus mass spectrometer (Thermo Fisher Scientific, Bremen, Germany).

Lipidomics

Lipids were separated on an Acquity UPLC BEH C18 column (50×2.1 mm; 1.7 μm) coupled to an Acquity UPLC BEH C18 VanGuard precolumn (5×2.1 mm; 1.7 μm) (Waters, Milford, MA, USA). The column was maintained at 65°C at a flow-rate of 0.6 ml/min. For LC-ESI(+)-MS analysis, the mobile phase consisted of (A) 60:40 (v/v) acetonitrile:water with ammonium formate (10 mM) and formic acid (0.1%) and (B) 90:10:0.1 (v/v/v) isopropanol:acetonitrile:water with ammonium formate (10 mM) and formic acid (0.1%). For LC-ESI(-)-MS analysis, the composition of the solvent mixtures was the same with the exception of the addition of ammonium acetate (10 mM) and acetic acid (0.1%) as mobile-phase modifier. Separation was conducted under the following gradient for LC-ESI(+)-MS: 0 min 15% (B); 0-1 min 30% (B); 1-1.3 min from 30% to 48% (B); 1.3-5.5 min from 48% to 82% (B); 5.5-5.8 min from 82% to 99% (B); 5.8-6 min 99% (B); 6-6.1 min from 99% to 15% (B); 6.1-7.5 min 15% (B). For LC-ESI(-)-MS, the following gradient was used: 0 min 15% (B); 0-1 min 30% (B); 1-1.3 min from 30% to 48% (B); 1.3-4.8 min from 48% to 76% (B); 4.8-4.9 min from 76% to 99% (B); 4.9-5.3 min 99% (B); 5.3-5.4 min from 99% to 15% (B); 5.4-6.8 min 15% (B). A sample volume of 0.5-5 μl was used for injection based on extract type. Sample temperature was maintained at 4°C. The source and MS parameters were sheath gas pressure, 60 arbitrary units; aux gas flow, 25 arbitrary units; sweep gas flow, 2 arbitrary units; capillary temperature, 300°C; aux gas heater temperature, 370 °C. For general lipidomics profiling, the mass spectrometer was operated under following conditions: MS1 mass range, m/z 200-1700; MS1 resolving power, 35,000 FWHM (m/z 200); number of data-dependent scans per cycle, 3; MS/MS resolving power, 17,500 FWHM (m/z 200). For ESI(+), spray voltage of 3.6 kV and normalized collision energy of 20% were used, while for ESI(-), spray voltage of -3.0 kV and normalized collision energy of 10%, 20% and 30% were set up. LC-MS and LC-MS/MS data were processed through the software MS-DIAL v. 2.52 [START_REF] Tsugawa | MS-DIAL: dataindependent MS/MS deconvolution for comprehensive metabolome analysis[END_REF]. Metabolites were annotated using an in-house retention time-m/z library and using MS/MS libraries available from public sources (MassBank, MoNA).

Statistical analysis

Results were expressed as means±S.D. All the groups were tested for the effects of FAHFAs intake by a one-way analysis of variance (ANOVA) test followed up by a Fisher's least significant difference test. In some case, Student's t test was also performed. Correlations were performed with the Kendall method. The limit of statistical significance was set at Pb.05. The means with different letters were significantly different. Statistical analyses were performed using the StatView program (SAS Institute, Cary, NC, USA).

Results

3.1. 9-PAHPA and 9-OAHPA had no effect on food intake, body weight and circulating lipids but increased basal metabolism Taking into account a mice food intake of about 2.5 g/day, the daily intake of 9-PAHPA or 9-OAHPA correspond to about 0.37 mg FAHFA/ day/mouse. The long-term high intake of 9-PAHPA or 9-OAHPA had no effect on body weight, fat and lean mass, food intake and plasma leptin levels (Fig. 2A). Moreover, plasma triglycerides, cholesterol and free fatty acids levels were not affected (Fig. 2B). Serum IL-6 level, a blood marker of inflammation, tended to increase (P=.0854) in the 9-PAHPA mice compared to the control and 9-OAHPA mice (Supplementary fig. 1).

In order to investigate the basal metabolism, oxygen consumption and carbon dioxide production were measured using CLAM system. We found that oxygen consumption reflecting the basal metabolism was increased with both 9-PAHPA and 9-OAHPA (respectively, +8% with 9-PAHPA and +17% with 9-OAHPA, Pb.001) (Fig. 2C). Moreover, analysis of RER revealed that 9-OAHPA but not 9-PAHPA favored the oxidation of carbohydrates, which was reflected by a higher RER value (Fig. 2D).

9-PAHPA and 9-OAHPA had no effect on glucose tolerance but improved insulin sensitivity

In both fasting and fed conditions, 9-PAHPA and 9-OAHPA intake had no impact on blood glycemia (Fig. 3A) or on glucose tolerance (Fig. 3B). However, insulin sensitivity was markedly increased by the intake of both 9-PAHPA and 9-OAHPA, and it was even necessary to stop the insulin tolerance test and reinject glucose 30 min after glucose gavage to the 9-PAHPA-or 9-OAHPA-supplemented mice. Indeed, their glycemia levels became so low that these were under the limit of detection of glucometer (Fig. 3C).

The more surprising observation was that plasma insulin levels did not respond to feeding in the 9-PAHPA-supplemented mice, whereas, as expected, they were significantly increased in the control and in 9-OAHPA mice (Fig. 3D). However, insulin levels were significantly higher in 9-PAHPA mice during fasting compared to control and 9-OAHPA mice (ANOVA, P=.0314) (Fig. 3D). Fasting level of plasma total GLP-1, an incretin with proven effects in promoting insulin secretion, was not significantly increased in 9-PAHPA or 9-OAHPA mice by comparison to control mice (Fig. 3E). However, plasma total GLP-1 was positively correlated to fasted plasma insulin level (P= .0394, r=0.308) (Fig. 3F).

9-PAHPA or 9-OAHPA intake induced liver alterations in some mice

During mice sacrifice and organs collection, we observed in 3 out of 10 mice fed with 9-PAHPA-supplemented diet and in 1 out of 10 mice fed with 9-OAHPA-supplemented diet a strong alteration of liver appearance (Fig. 4A). However, 9-PAHPA and 9-OAHPA intake had no effect on liver weight compared to control mice (Fig. 4B). In addition, liver content of triglycerides, cholesterol and total lipids was not modified compared to controls, whereas that of free fatty acid was slightly increased only in 9-PAHPA mice (Fig. 4C). Therefore, Oil Red O staining was performed to compare fat accumulation in mice liver. If overall there was no difference among the groups in agreement with previous results of liver lipid content, the livers that were visually strongly altered with 9-PAHPA and 9-OAHPA intake showed an overt accumulation of Oil Red O, suggesting hepatic steatosis in the concerned mice (Fig. 4D). In attempt to explain this high liver lipid content in some mice, we have investigated the gene and/or protein expression of major players in hepatic de novo lipogenesis, namely, PPARα, PPARγ, ACC and FAS. Although the obtained results (Supplementary Fig. 2) did not reveal any significant difference among the three studied groups, we however observed a significant correlation between some of these parameters, in particular p-ACC protein abundance, and the hepatic fibrosis index (Supplementary Fig. 3).

Fibrosis was then investigated using Sirius Red staining (Fig. 5A). Fibrosis percentage in liver was increased almost fourfold with 9-PAHPA and about twofold with 9-OAHPA compared to the control mice (Fig. 5B). As observed for Oil Red O staining, the livers that were visually strongly altered with 9-PAHPA and 9-OAHPA intakes presented more important fibrosis. Regarding liver alteration, we showed that plasma ALT activity, a biochemical marker of liver damage, was increased almost threefold with 9-PAHPA and twofold with 9-OAHPA (Fig. 5C). We then studied direct markers of fibrosis using Q-PCR. The expression of MMP2, a metalloproteinase implicated in complex extracellular matrix degradation [START_REF] Duarte | Matrix metalloproteinases in liver injury, repair and fibrosis[END_REF], was significantly increased with 9-PAHPA intake (threefold) but not with 9-OAHPA intake, while gene expression of CoL1A [START_REF] Hayashi | Identification of the collagen type 1 alpha 1 gene (COL1A1) as a candidate survival-related factor associated with hepatocellular carcinoma[END_REF], characteristic of liver fibrosis, was nonsignificantly increased (Fig. 5D). As liver fibrosis is often associated to inflammation, we studied specific inflammation markers such as CD68 and MCP-1. However, their expression was not modified by 9-PAHPA or 9-OAHPA intake (Fig. 5E). This result prompted us to study the expression of two major proinflammatory cytokines, TNFα and IL-6, at the protein levels. While liver IL-6 protein expression was unchanged whatever the diet (Fig. 5F), the high intake of both 9-PAHPA and 9-OAHPA significantly decreased liver TNF-α protein expression (Fig. 5G). Interestingly, TNF-α protein expression was inversely correlated to CoL1A (r=-0.394, P=.0048) (Fig. 5H).

3.4. 9-PAHPA and 9-OAHPA intake had no effect on liver mitochondrial activity and oxidative stress Because mitochondrial dysfunction and oxidative stress are interconnected and generally known to be involved in the 1A andB). The RER was calculated as the VCO 2 /VO 2 ratio (Fig. 1B). Mean VO 2 and RER values are mean of eight mice for each group considering all the cycles (Fig. 1C andD). Results were expressed as means±S.D., n=111. All the groups were tested for the effects of FAHFAs intake by a one-way ANOVA test followed up by a Fisher's least significant difference test. The limit of statistical significance was set at Pb.05. The means with different letters were significantly different. development of steatohepatitis and fibrosis [START_REF] Manne | Pathophysiology of nonalcoholic fatty liver disease/nonalcoholic steatohepatitis[END_REF], we investigated these parameters in the mice liver. The enzymatic activities of mitochondrial respiratory chain complexes were not modified by 9-PAHPA and 9-OAHPA, as well as the β-oxidation (Fig. 6A-B). Moreover, the activities of antioxidant enzymes (SOD, MnSOD, catalase and GPx) were not changed either (Fig. 6C). In line with these observations, 9-PAHPA and 9-OAHPA intake had no effect on biochemical markers of oxidized proteins (SH groups and GSSG/GSH) or oxidized lipids (TBARS, phytoprostanes, isoprostanes and neuroprostanes) (Fig. 6D-E).

3.5. High intake of 9-PAHPA slightly altered lipid composition of WAT Although the fat content of the mice was not changed regardless of the diet (Fig. 2A), the lipidomic analysis of WAT revealed that WAT from 9-PAHPA mice was redesigned and mildly enriched in saturated fatty acids compared to control and 9-OAHPA mice. However, this was observed only in half the mice of this group (Fig. 7A). The WAT mRNA expression of major proinflammatory cytokines (TNFα and IL-6) and chemokines (MCP-1 and RANTES) were not modified whatever the diet (Fig. 7B).

Discussion

In the present study, we have investigated for the first time the effect of 9-PAHPA and 9-OAHPA on basal metabolism, insulin sensitivity and glucose tolerance and liver metabolism in controldiet-fed mice. This study is a long-term high intake of 9-PAHPA and 9-OAHPA that lasted 12 weeks. The administered dosage of our FAHFAs was based on the work of Yore et al. ( 2014) [START_REF] Yore | Discovery of a class of endogenous mammalian lipids with anti-diabetic and antiinflammatory effects[END_REF]. It is also in good agreement with the conditions recently published by Paluchova et al. (2019) [START_REF] Paluchova | Lipokine 5-PAHSA is regulated by adipose triglyceride lipase and primes adipocytes for de novo lipogenesis in mice[END_REF]. We have to outline that these high dosages of FAHFAs applied in the present work cannot be taken up through normal dietary. We studied such high dosages in healthy mice because these Fig. 3. Glucose and insulin parameters.Glycemia in fast and fed state (A), OGTT (B), ITT (C), plasma insulin (D) level in fast and fed state, plasma total GLP-1 level (E) and correlation between plasma total GLP-1 and circulating insulin (F). Results were expressed as means±S.D., n=8-10 animals per group. Student's t test for comparison between fasted or fed state (30-min glucose injection) inside the same diet. The limit of statistical significance was set at *Pb.05 inside of fasted and fed states separately among control and FAHFAs. ANOVA between groups inside fasted state or fed state (30-min glucose injection). For plasma total GLP-1, all the groups were tested for the effects of FAHFAs intake by a one-way ANOVA test followed up by a Fisher's least significant difference test. The limit of statistical significance was set at Pb.05. The means with different letters were significantly different. dosages were investigated in different pathological situations; therefore, it seemed relevant to study their effects at these dosages in healthy mice as well [START_REF] Yore | Discovery of a class of endogenous mammalian lipids with anti-diabetic and antiinflammatory effects[END_REF][START_REF] Balas | Branched fatty acyl esters of hydroxyl fatty acids (FAHFAs), appealing beneficial endogenous fat against obesity and type-2 diabetes[END_REF][START_REF] Paluchova | Lipokine 5-PAHSA is regulated by adipose triglyceride lipase and primes adipocytes for de novo lipogenesis in mice[END_REF][START_REF] Smith | Adipose tissue regulates insulin sensitivity: role of adipogenesis, de novo lipogenesis and novel lipids[END_REF].

9-PAHPA and 9-OAHPA increased basal metabolic rate in healthy mice

Our data show for the first time that basal metabolic rate, measured by the rate of oxygen consumption, was increased with both 9-PAHPA and 9-OAHPA intakes but without impact on body weight. No comparison can be made with other types of FAHFAs, as this parameter has never been previously measured following the intake of other FAHFAs.

Our results also show that food intake and adiposity were not altered by 12-week intake of 9-PAHPA or 9-OAHPA. In fact, their dietary intake had no effect on food consumption, body weight or fat mass of mice. This is rather a surprising result because given the effect of 9-PAHPA and 9-OAHPA on basal metabolism, we would have expected a reduction in the weight of mice that eat less. This may suggest that 9-PAHPA and 9-OAHPA could modulate nutrient intestinal absorption. In addition, plasma leptin levels, an anorexigenic hormone mediator of long-term regulation of energy balance [START_REF] Klok | The role of leptin and ghrelin in the regulation of food intake and body weight in humans: a review[END_REF], was not modified whatever the diet. Circulating lipid levels also remained unchanged. In accordance, Syed et al. [START_REF] Syed | Palmitic acid hydroxystearic acids activate GPR40, which is involved in their beneficial effects on glucose homeostasis[END_REF] observed no effect on all these parameters in mice gavaged for 18 weeks with two structurally different FAHFAs: the 5-PAHSA and 9-PAHSA. These observations show that 9-PAHPA, 9-OAHPA, 5-PAHSA and 9-PAHSA have no effect on food consumption and body fat mass accumulation in control-dietfed mice. Regarding the numerous different types of FAHFAs that may exist, depending of the FA and HFA structure and the esterification position, this cannot be extrapolated to all FAHFAs and needs to be investigated for each individual FAHFA.

9-PAHPA and 9-OAHPA increased insulin sensitivity in healthy mice

The glucose and insulin tolerance tests are useful tools to investigate glucose metabolism. Our study demonstrates that longterm high intake of 9-PAHPA or 9-OAHPA markedly increased insulin sensitivity. In accordance with this observation, experiments per-formed using metabolic cages showed that, compared to control animals, mice supplemented with 9-OAHPA used glucose preferentially, as evidenced by the increase in the RER. In addition, the lack of increased RER in animals supplemented with 9-PAHPA could probably be explained by the fact that these mice no longer secrete insulin in response to glucose. However, since we did not find any alterations in the morphology and density of the islets of Langerhans in pancreas suggesting an attack of this tissue (Supplementary Fig. 4), we hypothesize that this loss of insulin secretion in response to glucose is probably an adaptation at the β-cell level that has been implemented in these mice to counteract the negative effects of insulin in the liver due to their hypersensitivity to the hormone.

Despite increased insulin sensitivity in 9-PAHPA and 9-OAHPA mice, glucose tolerance was not changed by any diet probably because glucose tolerance is already satisfactory in healthy mice. Regarding molecular glucose metabolism in the liver, the protein expression of Glut-2 (involved in glucose transport), GLK (a major component of the hepatic glucose-sensing system), PEPCK (a key molecule of gluconeogenesis) and ChREBP (a key player in the induction of genes of de novo fatty acid synthesis in response to glucose) remained unchanged after 9-PAHPA or 9-OAHPA intake (Supplementary Fig. 5), which indicates that glucose metabolism itself was not affected by the intake of these FAHFAs.

4.3. 9-PAHPA and 9-OAHPA intake induced liver steatosis and fibrosis in some healthy mice Visual observation of liver after euthanasia of mice revealed severe alterations of some livers in the groups supplemented with 9-PAHPA or 9-OAHPA. Histological analysis revealed steatosis and fibrosis in these same livers. Increased hepatic de novo lipogenesis is a significant pathway contributing to nonalcoholic fatty liver disease [START_REF] Chiu | Dietary carbohydrates and fatty liver disease: de novo lipogenesis[END_REF], and it is likely that both FAHFAs have insulin-sensitized the liver of some mice so much that de novo lipogenesis promoted steatosis/fibrosis. Although the gene/protein expression analysis of some major players in hepatic de novo lipogenesis, namely, PPARα, PPARγ, ACC and FAS, did not reveal significant differences among the three studied groups, it is not excluded that posttranslation protein modifications may be involved in the alteration of de novo lipogenesis. Nevertheless, histological observations were supported by the gene expression analysis of some liver fibrosis markers linked to matrix degradation [START_REF] Mak | Basement membrane type IV collagen and laminin: an overview of their biology and value as fibrosis biomarkers of liver disease[END_REF]. Plasma ALT levels, a biochemical marker of liver damage, confirmed also liver alterations with 9-PAHPA and 9-OAHPA as ALT levels were increased almost threefold with 9-PAHPA and twofold with 9-OAHPA.

In attempt to understand the origin of the liver fibrosis observed in some mice fed with the FAHFA-supplemented diet, we then explored various markers of inflammation. However, neither protein expression of IL-6 nor gene expression of IL-6, TNF-α, MCP-1 and CD-68 was increased whatever the diet. Even more surprisingly, protein expression of TNF-α was significantly decreased in mice liver fed the 9-PAHPA or 9-OAHPA and was inversely correlated to gene expression of one liver fibrosis marker. TNF-α is a multifunctional cytokine that plays important roles in diverse cellular events such as cell survival, proliferation, differentiation and cell death [START_REF] Wang | Tumor necrosis factor and cancer, buddies or foes?[END_REF], and anti-TNF-α may exert some undesirable effects on liver [START_REF] French | Hepatotoxicity associated with the use of anti-TNF-alpha agents[END_REF][START_REF] Ghabril | Liver injury from tumor necrosis factor-alpha antagonists: analysis of thirty-four cases[END_REF][START_REF] Borman | Anti-TNF-induced autoimmune hepatitis[END_REF].

Liver free fatty acid accumulation, inflammation, oxidative stress and mitochondrial dysfunction generally lead to steatosis and progressively switch it to steatohepatitis [START_REF] Manne | Pathophysiology of nonalcoholic fatty liver disease/nonalcoholic steatohepatitis[END_REF]. However, free fatty acids rather than triglycerides are responsible for liver injury via increased oxidative stress [START_REF] Liu | Free fatty acids, not triglycerides, are associated with nonalcoholic liver injury progression in high fat diet induced obese rats[END_REF]. To explore whether the accumulation of free fatty acids induced oxidative stress, various markers of oxidative stress were investigated as oxidized lipids (isoprostanoids and TBARs), oxidized protein (thiols and glutathione) and antioxidant enzyme activity. Surprisingly, none of these parameters was significantly modified regardless of the diet. Mitochondria are known to be one of the main ROS generation sites within the cell, and mitochondrial ROS production is closely associated with the respiratory chain complexes activity [START_REF] Feillet-Coudray | Oxidative stress in rats fed a high-fat high-sucrose diet and preventive effect of polyphenols: involvement of mitochondrial and NAD(P)H oxidase systems[END_REF]. Moreover, mitochondria are postulated to play a central role in the progression of liver diseases [START_REF] Mantena | Mitochondrial dysfunction and oxidative stress in the pathogenesis of alcohol-and obesityinduced fatty liver diseases[END_REF]. However, the enzymatic activity of mitochondrial respiratory chain complexes was not modified whatever the diet.

4.4. 9-PAHPA and 9-OAHPA had slight effect on WAT

The WAT lipidomic analysis revealed that 9-PAHPA intake induced a mild enrichment in saturated fats in the WAT of some mice. The mild enrichment in saturated fats of the WAT may be resulting either from the absorption of palmitate as a nutrient fatty acid or from the higher bioavailability of PAHPA. While hepatic de novo lipogenesis is thought to contribute to metabolic disease [START_REF] Eissing | De novo lipogenesis in human fat and liver is linked to ChREBP-beta and metabolic health[END_REF], WAT de novo lipogenesis is good, promoting "healthy" adipocyte [START_REF] Kuda | Omega-3 fatty acids and adipose tissue biology[END_REF]. In accordance, there was no increase of proinflammatory markers in this tissue.

Conclusion

This work demonstrates that long-term high intake of 9-PAHPA or 9-OAHPA, incorporated into a control diet, has some positive effects in healthy mice, namely, an increase in basal metabolic rate and in insulin sensitivity. However, 9-PAHPA intake and to a lesser extent 9-OAHPA intake induced liver steatosis and fibrosis in some mice. It is likely that both FAHFAs have insulin-sensitized the healthy liver so much that de novo lipogenesis promoted steatosis/fibrosis. This work highlights the fact that long-term high intake of some FAHFAs, because of their high insulin-sensitizing effect, can induce unexpected adverse metabolic effects in healthy mice. The results obtained in the present study are encouraging concerning the effects of these FAHFAs in pathological situations as diabetes. It should be emphasized that the studied FAHFAs cannot be taken up in the concentrations used in this study through normal diet. In vivo doseresponse studies should be considered to determine the safe intake of 9-PAHPA and 9-OAHPA. Fig. 6. Mitochondrial activity and hepatic oxidative stress parameters.Results were expressed as means±S.D., n=9-10 animals per group. All the groups were tested for the effects of FAHFAs intake by a one-way ANOVA test followed up by a Fisher's least significant difference test. The limit of statistical significance was set at Pb.05. The means with different letters were significantly different. CI, complex I; CII, complex II; CII + III, complexes II + III; COX, cytochrome c oxidase; β-HAD, β-hydroxyacyl-CoA dehydrogenase; SOD, total superoxide dismutase; MnSOD, manganese superoxide dismutase; GPx, glutathione peroxidase; -SH, thiols; GSSG/GSH, oxidized glutathione/reduced glutathione; TBARS, thiobarbituric acid reactive substances. Phytoprostanes: ent-16B1t-phytoprostane, ent-9L1t-phytoprostane, ent-16(RS)-13-epi-ST-d14-9-phytofuran; isoprostanes: 15(S)-F2t-isoprostane, 15(R)-F2tisoprostane, 5(RS)-5-F2t-isoprostane, 5(RS)-5-F2c-isoprostane; neuroprostanes: 10(S)-F4t-neuroprostane, 10(R)-F4t-neuroprostane, 4(RS)-4-F4t-neuroprostane. Fig. 7. WAT lipidomic and inflammation.Lipidomic analysis of WAT (A) according to Cajka et al. [START_REF] Cajka | Validating quantitative untargeted lipidomics across nine liquid chromatography-high-resolution mass spectrometry platforms[END_REF]. Gene expression of WAT inflammation markers (B) by qPCR. Results were expressed as means±S.D., n=9-10 animals per group. All the groups were tested for the effects of FAHFAs intake by a one-way ANOVA test followed up by a Fisher's least significant difference test. The limit of statistical significance was set at Pb.05. The means with different letters were significantly different. Mcp1, monocyte chemoattractant protein 1; IL-6, interleukin 6; TNFα, tumor necrosis factor α; RANTES, regulated upon activation, normal T cell expressed, and secreted.

Fig. 2 .

 2 Fig.2. Mice characteristics.Body weight, fat and lean mass and food intake (A), circulating lipids (B), VO 2 (C) and RER (D) in mice. For each mouse, VO 2 was measured 111 times in about 24 h (Fig.1A and B). The RER was calculated as the VCO 2 /VO 2 ratio (Fig.1B). Mean VO 2 and RER values are mean of eight mice for each group considering all the cycles (Fig.1C and D). Results were expressed as means±S.D., n=111. All the groups were tested for the effects of FAHFAs intake by a one-way ANOVA test followed up by a Fisher's least significant difference test. The limit of statistical significance was set at Pb.05. The means with different letters were significantly different.

Fig. 4 .

 4 Fig.4. Liver morphology and steatosis.Visual observation of liver after euthanasia of mice (A), liver weight after 12 weeks of diet (B), hepatic lipid content measured on tissue homogenate by enzymatic methods (C). Results were expressed as means±S.D., n=9-10 animals per group. All the groups were tested for the effects of FAHFAs intake by a one-way ANOVA test followed up by a Fisher's least significant difference test. The limit of statistical significance was set at Pb.05. The means with different letters were significantly different. Representative Oil Red O (D) of livers from all the mice (magnification ×10), n=10 animals per group.

Fig. 5 .

 5 Fig. 5. Liver fibrosis.Sirius Red stains of livers. Pictures are representative from each group (magnification ×10) (A), % of fibrosis (B) and plasma ALT level (C). Gene expression of liver fibrosis markers (D) and liver inflammation markers (E) by qPCR. Protein expression of IL-6 (F) and TNF-α (G) by Western blot and representative pictures from each group. (H) Correlation between TNF-α protein expression and Col1A1 gene expression. Results were expressed as means±S.D., n=9-10 animals per group. All the groups were tested for the effects of FAHFAs intake by a one-way ANOVA test followed up by a Fisher's least significant difference test. The limit of statistical significance was set at Pb.05. The means with different letters were significantly different. ALT, alanine aminotransferase; MMP2, matrix metalloproteinase-2; Col1A, collagen type α1; CD68, cluster of differentiation 68; Mcp1, monocyte chemoattractant protein 1; TNFα, tumor necrosis factor α; IL-6, interleukin 6.

Acknowledgments

The authors wish also to thank the animal staff from Metamus DMEM facility which belongs to Montpellier animal facilities network (RAM, Biocampus), Biocampus for technical support and expertise for metabolism phenotyping, and Metamontp platform supported by the

Funding

Melha Benlebna acknowledges the financial support from Ph.D. program of the Algerian Ministry of Higher Education and Scientific Research. The authors acknowledge the financial support of the French Lipid Nutrition Group, the National Institute for Agronomic Research, in particular the Human Nutrition Department (Alim-H department) and Czech Science Foundation (17-10088Y).

Authors' declaration

European fund for the regional development of Occitanie (FEDER). The authors wish to express their deep acknowledgements to Guillaume Reversat, Amandine Rocher and Camille Oger for their technical aid in the isoprostanoid analysis, Annabelle Tavernier for her technical aid in the GLP-1 analysis and Tomas Cajka (Institute of Physiology CAS) for the lipidomic analysis.

Conflict of interest

The authors confirm that this article content has no conflict of interest.

Appendix A. Supplementary data

Supplementary data to this article can be found online at .