Melon juice concentrate supplementation in an animal model of obesity: Involvement of relaxin and fatty acid pathways

Journal of Functional Foods 59 (2019) 92–100

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Journal of Functional Foods journal homepage: www.elsevier.com/locate/jff

Melon juice concentrate supplementation in an animal model of obesity: Involvement of relaxin and fatty acid pathways

T

Julie Carillona,b, Marion Sabya, Sandy Bariala, Anna Sansonec, Roberta Scanferlatoc, Nathalie Gayrarda, Anne-Dominique Lajoixa, Bernard Jovera, Chryssostomos Chatgilialogluc, ⁎ Carla Ferreric, a

EA7288 Université de Montpellier, 15 Avenue Charles Flahault, BP 14491, 34093 Montpellier Cedex 5, France Bionov Sarl, Site Agroparc, Batiment Orion, Chemin des Meinajaries, CS 80501, Cedex 9, Avignon 84908 France c ISOF, Consiglio Nazionale delle Ricerche, Via Piero Gobetti 101, 40129 Bologna, Italy b

A R T I C LE I N FO

A B S T R A C T

Keywords: Superoxide dismutase Adipose tissue Membrane lipidome Inflammatory mediators Fibrosis Lipid remodeling

Chronic low-grade inflammation and oxidative stress associated with obesity induce molecular changes in extracellular matrix connected to relaxin pathway and fibrosis, the lipidome of various tissues and the level of lipid mediators. Increase of desaturase enzymatic activity and activation of the inflammatory mediator cascades are known to be associated with obesity. Decrease of enzymatic antioxidant defenses is strictly involved. The effects of a 10 days supplementation of encapsulated melon juice concentrate rich in superoxide dismutase were studied in Zucker Fat rats compared with Zucker Lean rats, following relaxin pathways, lipid mediators and fatty acidbased lipidomic analysis in adipose tissue and erythrocytes. Significant ameliorations in obese rats concerned the restored relaxin levels, connected with fibrosis reduction, the reduction of inflammatory mediators and a favorable fatty acid remodeling, with the decrease of unsaturated fatty acids in the lipidome of adipose tissue and erythrocytes becoming closer to the lipidome of normal rats.

1. Introduction Obesity, excessive adipose tissue growth, is associated with several metabolism dysfunctions and is characterized as a state of chronic lowgrade inflammation and oxidative stress (Valdecantos, Pérez-Matute, & Martinez, 2009; Yyer, Fairlie, Prins, Hammock, & Brown, 2010). The direct use of subcutaneous abdominal adipose tissue (SAT), the major fat accumulation, is performed to study obesity and its disorders (Pou et al., 2007), as well as the examination of other biological compartments such as plasma, both in animal models and in humans (CollRisco et al., 2016; Guerville et al., 2017; Klein-Platat, Drai, Oujaa, Schlienger, & Simon, 2005). In particular, in obese adipose tissues a number of lipid inflammatory mediators such as prostaglandins (PGs), thromboxanes (TxB), hydroxyeicosatetraenoic acids (HETE) and others are found (Gómez-Zorita et al., 2013), principally derived from polyunsaturated

fatty acids (PUFA), such as arachidonic acid (AA), eicosapentaenoic (EPA) and docosahexaenoic acid (DHA) (Stables & Gilroy, 2011). Since the status of the PUFA pathways derives from the omega-6 and omega-3 precursors, which are essential elements from the diet, an important piece of information is given by the PUFA content in tissues, in order to establish the subsequent balance between pro- and anti-inflammatory lipid mediators (Serhan, Chiang, & Van Dyke, 2008). The lipidomic analysis provides information on the lipidome composition (PUFA together with saturated and monounsaturated fatty acids, SFA and MUFA) of different tissues and compartments of the body, in animals and humans. Fatty acid-based functional membrane lipidomics can be a valid tool to follow up the fatty acid remodeling occurring in membranes during nutritional and nutraceutical treatments in a personalized manner (Ferreri & Chatgilialoglu, 2012). Examining the quality and quantity of fatty acids in cell membrane lipidome can delineate molecular unbalance, also reflecting poor

Abbreviations: 6kPGF1α, 6-ketoprostaglandin F1α; AA, arachidonic acids; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; GAPDH, glyceraldehyde 3phosphate dehydrogenase; HDoHE, hydroxy-docosahexaenoic acids; HEPE, hydroxyeicosapentaenoic acid; HETE, hydroxyeicosapentaenoic acid; LTB4, leukotrienes B4; LxB4 or LxA4, lipoxin B4 or A4; MUFA, monounsaturated fatty acids; PD1, programmed cell death protein 1; PGE2, prostaglandin E2; PUFA, polyunsaturated fatty acids; RBC, red blood cells; RvD2 or RvD1, resolvins D2 or D1; RXFP1, insulin-like family peptide receptor 1/relaxin receptor 1; SAT, subcutaneous adipose tissue; SFA, saturated fatty acids; TxB2, thromboxane B2; ZF, Zucker Fatty; ZL, Zucker Lean ⁎ Corresponding author. E-mail address: [email protected] (C. Ferreri). https://doi.org/10.1016/j.jff.2019.05.027 Received 5 March 2019; Received in revised form 15 May 2019; Accepted 17 May 2019 Available online 24 May 2019 1756-4646/ © 2019 Elsevier Ltd. All rights reserved.

Journal of Functional Foods 59 (2019) 92–100

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palm oil, by spray drying method, in order to preserve SOD activity from the digestive enzymes secreted above the small intestine. Detailed information about the antioxidant content of SODB has been published in a previous study (Carillon et al., 2012).

homeostasis and pro-inflammatory conditions, to be transferred for diagnostic and therapeutical applications (Ferreri & Chatgilialoglu, 2015). As a matter of facts, membrane lipid replacement therapy has been demonstrated to afford amelioration of illnesses and chronic fatigue (Nicolson & Ash, 2017). Together with the inflammatory conditions represented by PUFAs, another important metabolic aspect that occurs in obesity is the activation of desaturase enzymes, specifically in the transformation of saturated into monounsaturated fatty acids (Steffen et al., 2008; Warensjo et al., 2009). The activation of desaturase can be monitored by the levels of oleic and palmitoleic acids, and indeed especially the latter fatty acid is found to be connected with obesity without interferences from the diet (Gong et al., 2011). As far as the adipose tissue conditions are concerned, obesity is often accompanied by several modifications of extracellular matrix constituents (Henegar et al., 2008; Pasarica et al., 2009). In obese adipose tissue, collagens and other extracellular matrix constituents are overexpressed (Divoux & Clément, 2011; Kos et al., 2009). Obesity is therefore associated with adipose tissue fibrosis and particularly pericellular fibrosis that may slow down fat mass loss (Divoux et al., 2010). Inflammation as well as degenerative processes are involved in the development of fibrosis (Sun, Tordjman, Clément, & Scherer, 2013). Finally, Keaney et al reported that obesity is a strong independent predictor of systemic oxidative stress (Keaney et al., 2003). All the modifications of the adipose tissue in obesity, such as inflammation, fibrosis and alteration of lipid metabolism induce an overproduction of reactive oxygen species (ROS) and a diminution of antioxidant defense leading to an increased oxidative stress (Furukawa et al., 2004; Meigs et al., 2007). To increase antioxidant defenses antioxidant supplementations can be carried out. In this context, antioxidant properties of SODB, a gastroresistant encapsulated melon juice concentrate particularly rich in superoxide dismutase (SOD), have already been demonstrated in several models. Indeed, this specific product had previously revealed preventive effect on SAT fibrosis through the reduction of oxidative stress in a hamster model of cafeteria diet-induced obesity mimicking modern Western lifestyle (Carillon et al., 2014). Among the anti-fibrotic peptides, relaxin was reported to reduce collagen in many organs (Van der Westhuizen et al., 2008). The activation of relaxin/insulin-like family peptide receptor 1 (RXFP1) receptors by relaxin binding plays a role in extracellular matrix remodeling (Du, Bathgate, Samuel, Dart, & Summers, 2010; Scott et al., 2005). Interestingly, in left ventricular of hypertensive rats, it was shown in a previous study that relaxin pathway is implicated in the anti-fibrotic properties of the melon juice concentrate (Carillon et al., 2016). The present investigation was designed to characterize the effects of a supplementation with melon juice concentrate on SAT fibrosis in the Zucker Fatty rat (ZF) and its lean counterpart, the Zucker Lean rat (ZL). In particular, the relaxin pathway, connected with fibrosis reduction, was followed up together with the levels of lipid mediators in adipose tissue and plasma, and with the fatty acid-based lipidomic profiles of adipose tissue and red blood cell (RBC) membrane. We aimed at obtaining an integrated scenario of the beneficial influence of this supplementation on genetic obesity.

2.2. Experimental design The present animal experiments complied with the European and French laws (permit numbers B-3417226 and 34179) and conform to the Guide for the Care and Use of Laboratory Animals published by the NIH (Publication No. 85-23, revised 1996). Twenty Zucker Fat (ZF) rats of 7 weeks-old and twenty Zucker Lean (ZL) rats of 7 weeks-old (Charles River, Arbresle, France) were used in the present experiments. All male animals were used. They were housed at 22 ± 1 °C, subjected to a 12 h light/dark cycle with free access to both food (Rats Mice Hamsters maintenance diet A04, SAFE, Augy, France; details in the website: http://www.safe-diets.com/wp-content/ uploads/2018/01/DS-SAFE-A04.pdf) and tap water. After one week of an adaptation period, rats were randomly divided into 4 treatment groups. One group of ZF (n = 10) and one group of ZL (n = 10) received the treatment at the daily dose of 10U SOD mixed with food, during 10 days. Everyday Every day controls were made for the entire ingestion of the food by the rats. The dose and duration of treatment were chosen according to previous studies where these conditions were efficient on adipose tissue remodeling (Carillon et al., 2016). The two other groups of ZF and ZL (n = 10 in each) remained untreated, i.e., they did not receive a placebo but only food without the supplement, and served as control. Food intake and body weight were recorded daily. The body weight of untreated rats was significantly different at the start (ZF 65 ± 3 g; ZL 36 ± 2 g) and remained different at the end of treatment (ZF 62 ± 3 g; ZF 32 ± 1 g). At the end of the experimental period, rats were anesthetized (ketamine and xylazine, 75 and 25 mg/kg). Then, 4 mL of blood was sampled in all rats by cardiac puncture. This blood was centrifuged (2000 g, 10 min, 4 °C) and plasma was stored at −80 °C until analysis. The SAT was excised, sectioned and stored at −80 °C or in formalin 10% until analysis. The weight (g) of SAT was obtained as a comparison between treated and untreated rats. 2.3. Plasma immunoassay measurements Plasma relaxin level was assessed using enzyme immunoassay kits from R&D Systems (Lille, France). Results are expressed as picograms per milligrams of proteins. 2.4. Determination of fatty acid content in SAT and RBC Commercially available cis and trans fatty acid methyl esters (FAME) were purchased from Sigma Aldrich (San Louis, MO, USA); chloroform, methanol, diethyl ether and n-hexane (HPLC grade) were purchased from Baker (New Jersey, USA) and used without further purification. Analytical thin-layer chromatography (TLC) was performed on Merck silica gel 60 plates 0.25 mm thickness, and spots were detected by spraying the plate with cerium ammonium sulfate/ammonium molybdate reagent and revealed by heating the plate. FAME were analyzed by GC (Agilent 6850, Milan), using the split mode (50:1), equipped with a 60 m × 0.25 mm × 0.25 µm (50%-cyanopropyl)-methylpolysiloxane column (DB23, Agilent, USA), and a flame ionization detector with the following oven program; temperature started from 165 °C, held for 3 min, followed by an increase of 1 °C/ min up to 195 °C, held for 40 min, followed by a second increase of 10 °C/min up to 240 °C, and held for 10 min. A constant pressure mode (29 psi) was chosen with helium as the carrier gas. FAMEs were identified by comparison with authentic samples and chromatograms were examined as described previously (Ferreri et al., 2012; Ferreri, Faraone Mennella, Formisano, Landi, & Chatgilialoglu, 2002; Sansone,

2. Materials and methods 2.1. Preparation and characterization of SODB SODB (Bionov, Avignon, France) is a dried melon juice concentrate, particularly rich in SOD, resulting from a patented process. Around 625 kg of a specific proprietary and no-GMO melon variety Cucumis melo L. (equivalent to 15 kg of dried melon pulp) are needed to produce 1 kg of this dried melon juice concentrate. Briefly, the melon pulp is separated from skin and seeds and crushed before centrifugation. Then, the melon juice undergoes filtration and concentration steps. Finally, the obtained melon juice concentrate is freeze-dried. For nutraceutical applications, this freeze-dried melon juice concentrate is coated with 93

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Computing, Vienna, Austria). Lipid mediators quantities were transformed to z-scores and clustered based on one Pearson correlation coefficient as distance and the Ward algorithm as agglomeration criterion. Results are expressed as picograms per milligram of tissue.

Melchiorre, Chatgilialoglu, & Ferreri, 2013; Fouret et al., 2015). 2.4.1. Lipid extraction and fatty acid analysis of adipose tissue Homogenate tissue sample (ca 20 mg) was added with tri distilled H2O (2 mL) and 2:1 chloroform/methanol (5 × 4 mL) according to the Folch method (Folch, Lees, & Sloane Stanley, 1957) The organic layer was collected and dried over anhydrous Na2SO4 then evaporated under vacuum to dryness. The weight of lipid extracts of the samples was found to be around 6–10 mg. TLC monitoring (n-hexane/Et20 8:2) revealed that extracts were composed mainly by triglycerides, and only traces of diglycerides, phospholipids and cholesterol were detected. 1 mL of 0.5 M solution of KOH in methanol was added to each sample for transesterification of the fatty acid-containing esters (mainly triglycerides) that was performed by stirring for 30 min at room temperature. The methanolic reaction mixture was quenched with brine (1 mL), then the extraction was repeated with n-hexane (3 × 2 mL) and the organic phases were collected, dried over anhydrous Na2SO4 and evaporated to dryness. The FAME residue of each sample was dissolved in 50 μL of n–hexane and 1 μL was injected for GC analysis.

2.6. Histology One piece of SAT was paraffin-embedded and cut into 5 µm sections and mounted on Superfrost-Plus glass slides (Menzel, Braunschweig, Germany). For fibrosis determination, SAT sections were stained with 0.1% picrosirius red and mounted in Eukitt medium (Sigma-Aldrich) following published protocols (Divoux et al., 2010). Fibrosis was quantified in five to ten given fields per animals, and expressed as the percentage of fibrous tissue area stained with picrosirius red per density of adipocytes. In these same sections, adipocytes were counted and the average is expressed in adipocytes number per square millimeter of tissue (density). All analyses were performed using image analysis software (ImageJ, National Institutes of Health, Bethesda, Maryland).

2.4.2. Lipid extraction and fatty acid analysis of RBC Whole blood was centrifuged in a 1.5 mL Eppendorf at 4000 g for 10 min at 6 °C for two consecutive times to obtain plasma separation, followed by cell washings with phosphate buffer to eliminate any residual plasma. Cell lysis was obtained by adding distilled water (1 mL) and two subsequent centrifugations led to isolating the erythrocyte membrane pellet. The pellet was composed by phospholipids and cholesterol (as evidenced by the TLC using n-hexane/Et20 8:2 as the eluent) and lipids were extracted by partitioning between water (1 mL) and 2:1 chloroform/methanol (2 × 4 mL). The organic layers were collected and dried over anhydrous Na2SO4, then evaporated under vacuum to dryness. 1 mL of 0.5 M solution of KOH in methanol was added to each sample for transesterification of the phospholipids into FAME by stirring at room temperature for 10 min. The reaction was quenched with brine (1 mL), and the addition of n-hexane (4 × 2 mL) afforded the separation of the organic phase containing the FAME. The FAME residue was dissolved in 10 μL of n–hexane and 1 μL was injected for GC analysis.

2.7. Determination of adipose tissue hydroxyproline content Homogenates of SAT were hydrolysed in the presence of concentrated HCl in teflon-capped vials at 120 °C for 3 h. Hydrolyzates were dried and the determination of hydroxyproline content was performed with a commercial assay kit from BioVision (Milpitas, CA, USA). Hydroxyproline content was expressed as milligrams per gram of protein. The protein determination was assessed in the same samples by Bicinchoninic Acid determination (Sigma-Aldrich). 2.8. Adipose tissue Western blot analysis The SAT protein extraction was carried out on ice in 20 mM of Tris buffer (pH 6.8) containing 150 mM NaCl, 1 mM EDTA, 1% Triton 20%, 0.1% SDS, 1% protease inhibitor cocktail (Sigma-Aldrich). After centrifugation (5500 g, 15 min at 4 °C), the supernatant was collected and extracted tissue proteins were then separated by SDS polyacrylamide gel electrophoresis. Equal amounts of proteins were loaded onto a 10% or a 15% acrylamide gel with a 4% stacking acrylamide gel. Migration was conducted in a Tris-glycine-SDS buffer (Sigma-Aldrich). After separation, proteins were transferred onto nitrocellulose membranes (Sigma-Aldrich). Relaxin receptor 1 (RXFP1) and relaxin were detected by Western Blot analysis. The primary antibodies against rat RXFP1, relaxin, and the control protein glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased at R&D Systems or Sigma-Aldrich. Expression of GAPDH was used for checking the equal protein load across gel tracks. Secondary antibodies (Santa Cruz Biotechnology, Heidelberg, Germany), coupled with horseradish peroxidase, were used for revealing the primary antibodies. Western blotting was performed according to the Amersham ECL select protocol (GE Healthcare, VelizyVillacoublay, France) and was acquired with a chemiluminescence detection system (Chemi-smart 5000, Vilber Lourmat, Marne-la-Vallée, France). Image analysis (ImageJ) was used for quantification after standardization within membranes by expressing the intensity of each band of interest relative to that of GAPDH in the same lane. Results are then expressed as percent of values obtained in untreated animals.

2.5. Determination of lipid mediators in adipose tissue 200 mg of SAT were crushed in 500 μL of Hank's balanced salt solution (Invitrogen, Cergy-Pontoise, France) and 5 μL of internal standard mixture (400 ng/mL in methanol), then 300 μL of cold methanol was added. After centrifugation (2000 g, 15 min at 4 °C), supernatants were collected, completed to 2 mL in water and submitted to solidphase extraction using HRX-50 mg 96-well (Macherey Nagel, Hoerd, France). Lipid mediators were eluted with methanol. Quantifications of 6-ketoprostaglandin F1α (6kPGF1α), prostaglandins E2 (PGE2), leukotrienes B4 (LTB4), thromboxane B2 (TxB2), hydroxyeicosatetraenoic acids (5-HETE, 12-HETE and 15-HETE), lipoxins B4 (LxB4), 18-hydroxyeicosapentaenoic acid (18-HEPE), 14 and 17-hydroxy-docosahexaenoic acids (14-HDoHE and 17-HDoHE), 7maresin (7-MaR1), D1 and D2 resolvins (RvD1 and RvD1) and programmed cell death protein 1 (PD1) were performed by LC-MS/MS (Le Faouder et al., 2013). This technique was performed on Agilent LC1290 Infinity coupled to Agilent 6460 triple quadrupole MS (Agilent Technologies, Les Ulis, France) equipped with electrospray ionization operating in negative mode. Reverse-phase UHPLC was performed using ZorBAX SB-C18 column (Agilent Technologies) with a gradient elution. The mobile phases consisted of water, acetonitrile and formic acid (75:25:0.1; v/v/v) and acetonitrile, formic acid (100:0.1, v/v). The flow rate was 0.35 mL/min, during 12 min. Peak detection, integration and quantitative analysis were performed using Mass Hunter Quantitative analysis software (Agilent Technologies). Hierarchical clustering and heat-map were obtained with R software (R Foundation for Statistical

2.9. Statistical analyses Values are presented as means ± SEM. Statistical analysis of the data was carried out Using GraphPad Prism software (La Jolla, California, USA) by one-way ANOVA followed by Mann & Whitney’s tests. P-values less than 0.05 were considered to be significant. 94

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65 ± 3 g; ZL 36 ± 2 g) and remained different at the end of treatment (ZF 62 ± 3 g; ZL 32 ± 1 g). The SAT sample weights were compared at the end of the treatment. This parameter differed between ZF and ZL animals at start (ZF 1.35 ± 0.05 g; ZL 0.43 ± 0.04 g), as expected, however it was also found significantly reduced between untreated and SODB-treated ZF (1.35 ± 0.05 g vs 1.16 ± 0.04 g; p = 0.013). The evaluation of SAT collagens using the staining by Picrosirius red (Divoux et al., 2010) is shown in Fig. 1A, where at start the staining was twice higher in ZF, compared to ZL group (0.046 ± 0.003% vs 0.022 ± 0.002%, p < 0.001). ZF-associated collagen increase was corrected by melon concentrate supplementation (0.029 ± 0.003%), resulting significantly different from the ZF at start and similar to ZL (p = 0.007) (Fig. 1A). Hydroxyproline quantification confirmed this result with significant higher content of collagens in ZF SAT, compared to ZL group (96 ± 11 vs 204 ± 32 mg/g proteins) (Fig. 1B). No difference was observed in ZL SAT whatever the group and the method (Fig. 1). 3.2. Melon concentrate supplementation modulated relaxin pathway As shown in Fig. 2A, plasma relaxin concentration was decreased by 40% in ZF animals, compared to ZL. Melon concentrate treatment reduced this alteration by increasing circulating relaxin in ZF-treated group. No difference was observed in plasma relaxin concentration in the two group of ZL. As depicted in Fig. 2B, SAT relaxin measured by western blot analysis was two-fold increased in ZF, compared to ZL. Melon concentrate supplementation fully corrected the level of SAT relaxin in ZF, whereas no difference was observed in ZL groups. As presented in Fig. 2C, SAT level of RXFP1 protein expression was significantly impaired in ZF, i.e. 38% lower when compared to ZL rats. Melon concentrate treatment induced an increase level of this receptor in ZF-treated animals. Finally, no difference was observed in SAT RXFP1 level between ZL (fixed to 100%) and ZL-treated groups.

Fig. 1. Collagen quantification in SAT. A: Picrosirius red staining of adipose tissue sections from a representative rats from each group. The values found in the ZF and ZL rat groups at start were: 0.046 ± 0.003% vs 0.022 ± 0.002%, p < 0.001, respectively. After supplementation, the value of 0.029 ± 0.003% was found significantly reduced in ZF rats, p = 0.007. B: Hydroxyproline content determination. Results (means ± SEM) are expressed as milligrams per gram of protein. ** p < 0.01 compared to untreated ZL; § p < 0.05 effect of melon concentrate, compared to untreated group.

3.3. Melon concentrate supplementation modulated lipid pathways and fatty acid composition in tissues As presented in Fig. 3A, SAT 6kPGF1α, TxB2, PGE2, 12-HETE, and 5-HETE concentrations were significantly increased in ZF animals, compared to ZL group. All these markers were reduced after melon concentrate supplementation, by respectively 59%, 65%, 53%, 53% and 23%, compared to untreated ZF. No difference was observed in ZL groups. As depicted in Fig. 3B, LxB4 and 7-MaR1 were not found in SAT of the two groups of ZL and untreated ZF group, whereas these markers are present (30 ± 17 and 5 ± 2 pg/g of SAT respectively) in SAT of treated ZF. No difference was observed in SAT LTB4, 18-HEPE, 15-HETE, 17HDoHE, 14-HDoHE concentrations between groups (Table 1). Finally,

3. Results 3.1. Melon concentrate supplementation reduced obesity-induced fibrosis in SAT During the 10 days treatment of melon juice concentrate supplementation, the weight of the rats was monitored to compare ZF and ZL animals. The body weight gain was not significantly different among animals, but obviously different among the two rat groups ((ZF

Fig. 2. Expression of relaxin pathway. A: Plasma relaxin level. Results (means ± SEM) are expressed as picograms per milligrams of protein. B. Adipose tissue protein expression of relaxin. C: Adipose tissue protein expression of relaxin receptor 1 (RXFP1). Quantification was made after standardization within membrane by expressing the intensity of the band of relaxin/RXFP1 relative to that of GAPDH in the same lane. Results (means ± SEM) are then expressed as relative change from untreated ZL group. * p < 0.05 and ** p < 0.01 compared to untreated ZL; § p < 0.05 and §§ p < 0.01 effect of melon concentrate, compared to untreated group. 95

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Fig. 3. Lipid mediators in adipose tissue. A: Inflammatory markers concentration measured by LC-MS/MS. Data (means ± SEM) are expressed in nanograms per milligram of protein. B: Pro-resolving mediators concentration measured by LC-MS/MS. Data (means ± SEM) are expressed in picograms per milligram of protein. * p < 0.05 and ** p < 0.01 compared to untreated ZL; § p < 0.05 and §§ p < 0.01 effect of melon concentrate, compared to untreated group.

decrease in PUFA, remaining lower the omega-6/omega-3 ratio, but did not change the increased levels of SFA and MUFA in SAT. In RBC membranes, SFA were higher (by 17%), whereas PUFA were lower (by 21%) in ZF, compared to ZL group. Melon concentrate supplementation significantly restored the SFA level and increased PUFA levels, both becoming similar to untreated ZL rats. As shown in Table 3, in RBC membranes at start the relative percentage of stearic acid (18:0) was higher (by 58%) in ZF group,

SAT LxA4, RvD2, RvD1 and PD1 weren't detected in the present study whatever the groups. Regarding the fatty acid-based lipidomic analyses at initial time, as presented in Table 2, in SAT, SFA and MUFA were higher (respectively by 17% and 19%), whereas PUFA were lower (by 35%) in ZF, compared to ZL group. Interestingly, in obese rats the significant changes of PUFA concerned the omega-6/omega-3 ratio, which was decreased compared to lean rats. Melon concentrate supplementation induced a further

Table 1 Concentration of metabolites and bioactive lipid mediators in SAT showing no differences. LTB4 (ng/mg of protein)

18-HEPE (ng/mg of protein)

15-HETE (ng/mg of protein)

17-HDoHE (ng/mg of protein)

14-HDoHE (ng/mg of protein)

ZL treated ZL

0.97 ± 0.70 0.89 ± 0.62

5.62 ± 1.26 5.47 ± 1.42

99.79 ± 15.04 98.18 ± 12.23

56.98 ± 12.33 52.84 ± 19.56

35.25 ± 6.82 32.63 ± 8.69

ZF treated ZF

1.70 ± 1.04 1.45 ± 0.67

4.81 ± 1.21 4.78 ± 0.81

88.71 ± 15.67 97.40 ± 15.35

65.48 ± 14.57 58.37 ± 22.37

44.13 ± 12.99 36.96 ± 14.94

See Materials and methods for the details. Values are means ± SEM. LTB4: leukotrienes B4, 18-HEPE: 18-hydroxyeicosapentaenoic acid, 15-HETE: 15-hydroxyeicosatetraenoic acids, 17-HDoHE and 14-HDoHE: 17 and 14-hydroxydocosahexaenoic acids. 96

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Table 2 Relative percentages (% rel) of the fatty acid families and two relevant indexes in SAT and RBC membrane phospholipids of melon concentrate-treated and untreated Zucker Lean (ZL) and Zucker Fat (ZF) rats. SFA (%rel) n = 10

MUFA (%rel) n = 10

PUFA (%rel) n = 10

SFA/MUFA

ω6/ω3

in SAT

ZL treated ZL ZF treated ZF

33.56 32.93 39.11 39.16

± ± ± ±

0.80 0.54 0.23* 0.27*

33.48 33.70 39.77 40.70

± ± ± ±

1.21 0.88 0.38* 0.49*

33.06 33.68 21.43 20.28

± ± ± ±

1.87 1.24 0.26* 0.44*,§

1.01 0.98 0.98 0.96

± ± ± ±

0.26 0.00 0.01 0.02

14.17 ± 0.71 14.16 ± 0.75 8.70 ± 0.72*** 9.13 ± 0.66***

in RBC

ZL treated ZL ZF treated ZF

48.97 50.39 57.42 48.76

± ± ± ±

0.95 1.15 1.66* 1.65§

21.92 17.81 19.68 23.63

± ± ± ±

1.94 0.57 0.67 2.10

28.96 31.80 22.86 27.61

± ± ± ±

2.01 1.06 1.62* 1.75§

2.84 2.36 2.95 2.19

± ± ± ±

0.12 0.22 0.13 0.18§§

7.00 ± 0.24 9.42 ± 0.33§§§ 11.18 ± 0.91** 7.76 ± 0.44§§

Values are means ± SEM and are obtained from the fatty acid peak areas of the gas chromatographic (GC) analysis, recognized and calibrated by the appropriate standards (see Experimental). Peak area recognition > 98% of the total GC peaks. SFA: saturated fatty acids. MUFA: monounsaturated fatty acids. PUFA: polyunsaturated fatty acids. * p < 0.05. ** p ≤ 0.01. *** p ≤ 0.001 compared to untreated ZL. § p < 0.05. §§ p ≤ 0.01. §§§ p ≤ 0.001 compared to the untreated group.

and a decrease in SAT relaxin, while the treatment was devoid of effect in ZL. These observations favor an induction by the melon concentrate of relaxin secretion from the adipose tissue of ZF. This result confirms first data achieved in SHR with a decrease in cardiac fibrosis associated to an induction of relaxin secretion after the melon concentrate administration (Van der Westhuizen et al., 2008). Concomitantly, the SAT content of RXFP1, which was lower in ZF, markedly increased after melon concentrate administration. Altogether, these results suggest that the beneficial effect of the melon concentrate in the SAT of ZF could be linked to the normalization of relaxin secretion and upregulation of its receptor, RXFP1, that very likely improve the anti-fibrotic effect of the peptide (Du et al., 2010; Scott et al., 2005). Another mechanism that may have an important role in the SAT inflammation, and thus fibrosis, is the modulation of fatty acid content. Membrane lipidomics offers a crucial point of view of fatty acid content in phospholipids, which are modulators of the cell homeostatic adaptation, with an important application to the estimation of the membrane natural balance and its changes due to stress consequences, including those caused by free radical reactivity. This effect has been demonstrated by some of us in different types of cells and animal models (Audette-Stuart, Ferreri, Festarini, & Carr, 2012; Bolognesi, Chatgilialoglu, Polito, & Ferreri, 2013; Cohen et al., 2011; Coudray et al., 2016; Fouret et al., 2015; Marini, Abruzzo, Bolotta, Veicsteinas, & Ferreri, 2011). It is useful to recall, using Scheme 1, that de novo fatty acid synthesis provides palmitic acid (C16:0) that can be elongated into stearic acid (C18:0), the saturated fatty acid with eighteen carbon atoms, and subsequently transformed into oleic acid (C18:1) with the double bond in the C9-C10 positions. The desaturase enzyme (delta-9 desaturase) can also work directly on palmitic acid and provides palmitoleic acid (C16:1), with the double bond between C9-C10 of a C16

compared to ZL animals. Melon concentrate supplementation fully corrected stearic acid level in ZF. Palmitic acid (16:0) also diminished in RBC phospholipids of treated ZF, compared to untreated ZF. No difference was observed in the other MUFA and SFA levels whatever the groups. 4. Discussion As shown by several authors, SAT remodeling in ZF animals was associated with enhanced fibrosis, evidenced by an increase in SAT collagen content (Divoux et al., 2010; Sun, Kusminski, & Scherer, 2011). While melon concentrate treatment did not modify SAT in ZL rats, it decreased fibrosis in ZF after 10 days of treatment. It is worth noting that the quantity of the adipose tissue in ZF rats was also diminished significantly of ca. 15% (1.35 ± 0.05 g vs 1.16 ± 0.04 g ; p = 0.013). The present results confirm and extend previous report demonstrating in hamsters that the specific melon concentrate could reverse abdominal adipose tissue fibrosis, equated here with picrosirius red staining and hydroxyproline measurement (Carillon et al., 2014). The reduction of SAT fibrosis in the present study could be explained by a modulation in relaxin pathway. In the present experiments, at start we observed an increase in SAT relaxin and a decrease in circulating (plasma) relaxin in ZF, compared to ZL (cfr., Fig. 2) together with the lower concentration of RXFP1 in ZF SAT, all conditions that can facilitate SAT fibrosis. Indeed, relaxin is known to have anti-fibrotic properties and to stimulate remodeling action in extracellular matrix in many tissues: liver, heart, lungs (Du et al., 2010; Van der Westhuizen et al., 2008). Interestingly, the reduction of SAT fibrosis by the melon concentrate in ZF was accompanied by an increase in plasma relaxin

Table 3 Relative percentages (% rel) of saturated (SFA) and monounsaturated fatty acids (MUFA) detected in RBC membrane phospholipids of melon concentrate-treated and untreated Zucker Lean (ZL) and Zucker Fat (ZF) rats. See the transformations of Scheme 1 describing the SFA-MUFA pathway.

in RBC

ZL treated ZL ZF treated ZF

16:0 (n = 10)

16:1 9c (n = 10)

18:0 (n = 10)

18:1 9c (n = 10)

34.83 34.37 35.91 32.36

3.13 2.27 2.51 3.59

13.32 15.40 21.10 15.95

13.08 10.64 13.22 16.10

± ± ± ±

0.67 0.69 0.87 0.96§

± ± ± ±

0.43 0.22 0.13 0.69

± ± ± ±

0.73 0.51 0.85*** 1.68§

± ± ± ±

1.36 0.34 0.63 1.64

Values are means ± SEM and are obtained from the fatty acid peak areas of the gas chromatographic (GC) analysis, recognized and calibrated by the appropriate standards (see Experimental). Peak area recognition > 98% of the total GC peaks. *** p ≤ 0.001 compared to untreated ZL. § p < 0.05, significant effect of melon concentrate compared to untreated group. 97

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the omega-6 arachidonic acid from membrane phospholipids is the first step for the production of prostaglandin of series 2, with PGE2 levels connected with visceral adipose tissue in obese subjects (Madan, Tichansky, Coday, & Fain, 2006). In RBC membranes SFA content after treatment was significantly lower and PUFA content was significantly higher (see Table 2) in the ZF treated group compared to ZF untreated rats. Interestingly, at the end of treatment the SFA and PUFA levels of ZF treated group resembled those of ZL (Table 2). On the other hand, AA metabolism is not only directed toward proinflammatory mediators, such as PGE2, but also can produce lipoxin, which is an anti-inflammatory mediator (Anderson & Ma, 2009; Yyer et al., 2010). After supplementation, AA metabolism seems to be directed toward less inflammatory effects, that can be also obtained by omega-3-PUFA transformations (Anderson & Ma, 2009; Ariel & Timor, 2013; Calder, 2006). LxB4 and Mar-1, which are omega-3 derived mediators of the resolution of inflammation, were produced in SAT of ZF treated rats. Lipoxins are potent inhibitors of fibrosis and attenuate adipocyte inflammation (Brennan et al., 2011; Maderna & Godson, 2009; Serhan et al., 2008; Stables & Gilroy, 2011). It could have beneficial effects in inflammation context because it is known to be implicated in homeostasis, inflammation resolution and wound healing (Maderna & Godson, 2009; Serhan et al., 2009).

Scheme 1. Transformation of palmitic acid in the fatty acid biosynthesis with the interplay of delta-9 desaturase and elongase enzymes.

5. Conclusions carbon atom chain. Our results, shown in Table 2, indicate that at start SFA were found significantly higher in RBC membranes and SAT of ZF rats compared to ZL rats. In Table 3, stearic acid (18:0) is the significantly increased saturated fat in ZF rats showing the involvement of the palmitic acid transformation by elongase enzyme. On the other hand, the MUFA components were found higher only in the adipose tissue and not in RBC membranes of ZF rats compared to ZL. Another interesting insight came from the PUFA status, that has lower levels in ZF rats than in ZL rats (cfr., Table 2). These data indicated that the obese rat model has similarity with lipidome profiling reported for overweight and obese people (Steffen et al., 2008; Warensjo et al., 2009), where the increase of palmitic acid (16:0) can activate the desaturase enzymatic transformation (Scheme 1). Moreover, the diminution of membrane PUFA lipids can be considered a consequence of an increased oxidative injury (Cazzola, Rondanelli, Russo-Volpe, Ferrari, & Cestaro, 2004). The marked decrease of PUFA in obese subjects is an indication of oxidative stress level associated to the disease, and also that the lipid remodeling is impaired and unable to compensate stress level (Pietiläinen et al., 2011; Sansone et al., 2016). As far as lipid mediators are concerned, they are biosynthesized from membrane fatty acids and involved in signal transduction participating to several steps of inflammatory response (Eyster, 2007; Serhan et al., 2009). Omega-6 PUFA are known precursors of pro-inflammatory markers, such as PGs, TxB, and HETE that are often increased in obese experimental animals (Chakrabarti et al., 2011; García-Alonso et al., 2016; Yyer et al., 2010). Inversely, the reduction of TxB could attenuate adipose tissue fibrosis (Lei et al., 2015). The results of fatty acid levels and pro-inflammatory markers (6kPGF1α, TxB2, PGE2, 12-HETE, and 5-HETE) found at higher levels in SAT of ZF compared to ZL, pointed to the activation of inflammation in obese rats, which is probably linked to the fibrosis observed in adipose tissue of ZF. The effects of supplementation was to bring the fatty acid levels in adipose tissue of ZF rats closer to the ZL rat condition. This is evident in the SFA-MUFA pathway (see Scheme 1 and Table 3) with a significant amelioration of 16:0 (palmitic acid) and 18:0 (stearic acid) levels, as well as in Table 2 with the fluidity index, i.e., the SFA/MUFA ratio, which is improved even better than the ZL rats, and the pro-inflammatory index, i.e., omega-6/omega-3 ratio, which is significantly diminished, becoming even lower than in ZL rats. It is also interesting to observe that the melon concentrate supplementation decreased ZF-induced pro-inflammatory markers in SAT such as PGE2. The release of

In conclusion, beneficial effects of 10 days supplementation of the melon juice concentrate rich in SOD were observed in ZF-induced fibrosis with the restoration of relaxin pathway and the reduction of lipid-derived mediators, that can produce inflammatory resolution in SAT. Additionally, without any change of dietary conditions, a more favorable membrane and tissue fatty acid profiles were obtained, that account for a positive remodeling and restoration of homeostatic control. The effects herein described certainly needs further work to assess the exact role of molecular and enzymatic components of the melon extract, as well as its comprehensive physiological roles, but we believe that our approach using an omic-integrated monitoring showed its usefulness in the evaluation of anti-obesity treatments. 6. Ethics statement The present animal experiments complied with the European and French laws (permit numbers B-3417226 and 34179) and conform to the Guide for the Care and Use of Laboratory Animals published by the NIH (Publication No. 85-23, revised 1996). Declaration of Competing Interest JC is employee of Bionov, the producer of the melon supplement. The other authors do not have any conflicts of interest. Acknowledgment The authors acknowledge the management support of Mr. Joseph Lorang throughout the Lipidomel project duration. Funding The authors acknowledge the support given by the program Eurostars Eureka with the funding of Lipidomel project (E!7348). RS acknowledges a one-year grant given by this project. References Anderson, B. M., & Ma, D. W. (2009). Are all n-3 polyunsaturated fatty acids created equal? Lipids in Health and Disease, 33, 1–20. https://doi.org/10.1186/1476-511X8-33.

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