Frédéric Nicolas Daussin
email: frederic.daussin@univ-lille.fr

Alexa Cuillerier

Julianne Touron
email: julianne.touron@gmail.com

Samir Bensaid
email: samir.bensaid@univ-lille.fr

Bruno Melo

Ali Al Rewashdy

Goutham Vasam
email: gvasam@uottawa.ca

Keir J Menzies
email: kmenzies@uottawa.ca

Mary-Ellen Harper
email: maryellen.harper@uottawa.ca

Elsa Heyman
email: elsa.heyman@univ-lille.fr

Yan Burelle
email: yburell2@uottawa.ca

Dietary Cocoa Flavanols Enhance Mitochondrial Function in Skeletal Muscle and Modify Whole-Body Metabolism in Healthy Mice

Keywords: cocoa flavanols, NAD metabolism, mitochondrial mass, glucose metabolism, skeletal muscle

Mitochondrial dysfunction is widely reported in various diseases and contributes to their pathogenesis. We assessed the effect of cocoa flavanols supplementation on mitochondrial function and whole metabolism, and we explored whether the mitochondrial deacetylase sirtuin-3 (Sirt3) is involved or not. We explored the effects of 15 days of CF supplementation in wild type and Sirt3 -/- mice. Whole-body metabolism was assessed by indirect calorimetry, and an oral glucose tolerance test was performed to assess glucose metabolism. Mitochondrial respiratory function was assessed in permeabilised fibres and the pyridine nucleotides content (NAD + and NADH) were quantified. In the wild type, CF supplementation significantly modified whole-body metabolism by promoting carbohydrate use and improved glucose tolerance. CF supplementation induced a significant increase of mitochondrial mass, while significant qualitative adaptation occurred to maintain H 2 O 2 production and cellular oxidative stress. CF supplementation induced a significant increase in NAD + and NADH content. All the effects mentioned above were blunted in Sirt3 -/-mice. Collectively, CF supplementation boosted the NAD metabolism that stimulates sirtuins metabolism and improved mitochondrial function, which likely contributed to the observed whole-body metabolism adaptation, with a greater ability to use carbohydrates, at least partially through Sirt3.

Introduction

Mitochondria are subcellular organelles that are involved in multiple cellular functions, such as energy transduction through mitochondrial oxidative phosphorylation and in mitochondrial hydrogen peroxide production or mitochondrial-mediated cell death activation [START_REF] Kuznetsov | Analysis of mitochondrial function in situ in permeabilized muscle fibers, tissues and cells[END_REF][START_REF] Sena | Physiological Roles of Mitochondrial Reactive Oxygen Species[END_REF][START_REF] Wacquier | Interplay Between Intracellular Ca2+ Oscillations and Ca2+-stimulated Mitochondrial Metabolism[END_REF]. Emerging evidence suggests that mitochondrial dysfunction is involved in many pathologies and plays a central role in their pathogenesis [START_REF] Suomalainen-Wartiovaara | Mitochondrial diseases: The contribution of organelle stress responses to pathology[END_REF]. Our group reported mitochondrial impairment in patients with type 1 diabetes long before clinical complications [START_REF] Heyman | Muscle Oxygen Supply and Use in Type 1 Diabetes, From Ambient Air to the Mitochondrial Respiratory Chain: Is There a Limiting Step?[END_REF]. Moreover, a decrease of mitochondrial oxidative capacity occurs with aging even in healthy people [START_REF] Daussin | From mitochondria to sarcopenia: Role of inflammaging and RAGE-ligand axis implication[END_REF]. Therefore, identifications of strategies to mitigate these dysfunctions or enhance mitochondrial mass are opportunities to prevent, or at least to reduce, the incidence of many disorders or increase aerobic capacity of healthy subjects.

In this context, the identification of safe and natural compounds that enhance mitochondrial function and whole-body metabolism with or without limited side effects is of interest. Among natural compounds, cocoa flavanols (CF) are considered promising molecules as their consumption was shown to positively affect cardiovascular health, insulin resistance, or immune function [START_REF] Katz | Cocoa and Chocolate in Human Health and Disease[END_REF]. Indeed, CF was shown to improve glucose metabolism, and a short term administration of CF is followed by an increase in insulin sensitivity, even in glucose-intolerant hypertensive patients [START_REF] Grassi | Short-term administration of dark chocolate is followed by a significant increase in insulin sensitivity and a decrease in blood pressure in healthy persons[END_REF][START_REF] Grassi | Blood Pressure Is Reduced and Insulin Sensitivity Increased in Glucose-Intolerant, Hypertensive Subjects after 15 Days of Consuming High-Polyphenol Dark Chocolate[END_REF].

Among the flavanols, (-)-epicatechin (EPI) is the most commonly found monomer in CF, and EPI is considered the major bioavailable and bioactive molecule of CF [START_REF] Ottaviani | The stereochemical configuration of flavanols influences the level and metabolism of flavanols in humans and their biological activity in vivo[END_REF]. A consistent body of evidence indicates that EPI supplementation improves mitochondrial function and/or content in skeletal muscle [START_REF] Daussin | Effects of (-)-epicatechin on mitochondria[END_REF]. EPI supplementation stimulates multiple pathways converging on peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) and major nuclear transcriptional complexes. EPI directly enhances nitric oxide generation, which stimulates mitochondrial biogenesis [START_REF] Daussin | Effects of (-)-epicatechin on mitochondria[END_REF]. Concomitantly, the sirtuins 1 and 3 are also stimulated by EPI supplementation [START_REF] Daussin | Effects of (-)-epicatechin on mitochondria[END_REF]. The use of proanthocyanidins, oligomeric flavonoids which contain EPI, was shown to increase the intracellular nicotinamide adenine dinucleotide (NAD + ) levels through the increase of several precursors for NAD biosynthesis and upregulate sirtuin-1 mRNA levels in rat liver [START_REF] Aragonès | Dietary proanthocyanidins boost hepatic NAD+ metabolism and SIRT1 expression and activity in a dose-dependent manner in healthy rats[END_REF]. The sirtuin family (Sirt), an NAD + -dependent deacetylase, comprises seven members that differ by their subcellular distribution, substrate specificity, and cellular functions [START_REF] Michan | Sirtuins in mammals: Insights into their biological function[END_REF]. Sirt1, an extensively studied member of this family, stimulate mitochondrial biogenesis by promoting deacetylation of PGC1α, thereby enhancing its transcriptional activity [START_REF] Cantó | PGC-1alpha, SIRT1 and AMPK, an energy sensing network that controls energy expenditure[END_REF]. Sirt1, a key metabolic sensor, was stimulated following EPI administration [START_REF] Aragonès | Dietary proanthocyanidins boost hepatic NAD+ metabolism and SIRT1 expression and activity in a dose-dependent manner in healthy rats[END_REF][START_REF] Taub | Alterations in Skeletal Muscle Indicators of Mitochondrial Structure and Biogenesis in Patients with Type 2 Diabetes and Heart Failure: Effects of Epicatechin Rich Cocoa[END_REF][START_REF] Moreno-Ulloa | Recovery of Indicators of Mitochondrial Biogenesis, Oxidative Stress, and Aging With (-)-Epicatechin in Senile Mice[END_REF][START_REF] Ramírez-Sánchez | Epicatechin-induced recovery of mitochondria from simulated diabetes: Potential role of endothelial nitric oxide synthase[END_REF]. However, the influence of EPI on other sirtuins have not been explored. The assessment of EPI effect will be particularly interesting on sirtuins located within the mitochondria (Sirt3, Sirt 4, and Sirt5), which are known to modulate the activity of the Krebs cycle and respiratory chain enzymes [START_REF] Lombard | Mitochondrial Sirtuins in the Regulation of Mitochondrial Activity and Metabolic Adaptation[END_REF]. Sirt3, which is highly expressed in tissues with high metabolic turnover and mitochondrial content, is of particular interest concerning its critical role in maintaining normal mitochondrial function through reversible protein lysine deacetylation [START_REF] Schwer | Reversible lysine acetylation controls the activity of the mitochondrial enzyme acetyl-CoA synthetase 2[END_REF][START_REF] Ahn | A role for the mitochondrial deacetylase Sirt3 in regulating energy homeostasis[END_REF][START_REF] Hirschey | SIRT3 regulates mitochondrial fatty-acid oxidation by reversible enzyme deacetylation[END_REF]. Moreover, Sirt3 plays an important role in the regulation of whole-body metabolism [START_REF] Ahn | A role for the mitochondrial deacetylase Sirt3 in regulating energy homeostasis[END_REF]. Sirt3 was shown to be altered in skeletal muscle of models of type 1 and type 2 diabetes and cardiovascular diseases [START_REF] Jing | Sirtuin-3 (Sirt3) regulates skeletal muscle metabolism and insulin signaling via altered mitochondrial oxidation and reactive oxygen species production[END_REF][START_REF] Wu | Emerging role of SIRT3 in mitochondrial dysfunction and cardiovascular diseases[END_REF]. Furthermore, it was suggested that activation of Sirt3 might represent a promising therapeutic strategy for the improving mitochondrial function and metabolism [START_REF] Wu | Emerging role of SIRT3 in mitochondrial dysfunction and cardiovascular diseases[END_REF][START_REF] Jing | Sirt3 Regulates Metabolic Flexibility of Skeletal Muscle Through Reversible Enzymatic Deacetylation[END_REF]. Therefore, determining whether Sirt3 underlies part of the beneficial effects of CF supplementation on whole-body metabolism and mitochondrial function is of interest.

In this study, we used an integrative approach to investigate, in mice, the effect of a 15-day CF supplementation. We hypothesised that the CF supplementation would: (i) modify whole-body metabolism and glucose metabolism, (ii) increase mitochondrial function in oxidative and glycolytic muscle in permeabilized fibers, and (iii) boost NAD metabolism. We also explored the putative involvement of Sirt3 in CF-induced mitochondrial biogenesis and whole-body metabolism. Our hypotheses were that CF's effects on whole-body metabolism and mitochondrial mass would be blunted in Sirt3 -/-mice.

Methods

Animals and Diet

For this study, 129S1/SvlmJ 10-week-old male mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA). To determine whether sirtuins are involved in CF administration-induced mitochondrial mass and function adaptation, we also used 10-week-old Sirt3 whole-body knock-out male mice on a same strain as wild-type [START_REF] Sebaa | SIRT3 controls brown fat thermogenesis by deacetylation regulation of pathways upstream of UCP1[END_REF]. The Sirt3 -/-mice were used to explore the involvement of Sirt3 in CF-induced mitochondrial biogenesis and whole-body metabolism adaptations. The mice were kept in a room at a regulated temperature of 23-25 • C and controlled lighting (12-h light and dark cycles). The animals had access to rodent chow and tap water ad libitum.

After at least a week of habituation in the animal facility, experimental animals received CF supplementation through oral gavage of a natural extract (302.1 mg/kg body weight twice a day for 15 days) resuspended in carboxymethylcellulose (Sigma-Aldrich, St. Louis, Mo, USA). The composition of the cocoa powder is described in Table 1 and has been obtained from Naturex (Quart de Poblet, Spain). The composition has been established by high performance liquid chromatography method and 100 mg of extract are equivalent to an average of 475 mg of dry cocoas seeds. The control mice received a vehicle composed of similar content of theobromine and caffeine dissolved in carboxymethylcellulose by oral gavage. The daily dose was determined according to the industry's guidance that converts the human dose to animal equivalent doses based on body surface area, and we multiplied the human dose by 12.3 [START_REF] Alert | Guidance for Industry Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers[END_REF]. The oral gavage was performed by an experienced technician.

Tissue Collection

Twenty-four hours after the last oral gavage, unfasted animals were anaesthetised using ketamine and xylazine (respectively: 100 and 10 mg/kg). Collection of tissues was performed immediately after anaesthesia was achieved. The soleus and the white portion of gastrocnemius were excised. Samples were either immediately frozen in liquid nitrogen and stored at -80 • C for subsequent analysis or used to prepare skinned muscle fibres.

Metabolic and Physical Activity Assessments

Indirect calorimetry and spontaneous physical activity were measured using the Comprehensive Laboratory Animal Monitoring System (CLAMS; Columbus Instruments, Columbus, OH, USA). Mice were housed individually in the chambers at 28 • C, with light from 07h00 to 19h00 and ad libitum access to rodent chow and tap water. Mice were acclimated to the metabolic cages for three days before day one of data collection. The data collection was performed on the fifteenth day of supplementation. Oxygen consumption (VO 2 ), carbon dioxide production (VCO 2 ), and respiratory exchange ratio (RER) were measured with an airflow of 0.5 L/min and a sample air of 0.4 L/min. Percent relative cumulative frequency (PCRF) of RER was determined over a 24-h period as previously described by [START_REF] Riachi | Percent relative cumulative frequency analysis in indirect calorimetry: Application to studies of transgenic mice[END_REF]. Briefly, PCRF was determined by the addition of the frequency of each data point to the previous increment sequence (from 0.65 to 1.20 with an increment of 0.01) [START_REF] Riachi | Percent relative cumulative frequency analysis in indirect calorimetry: Application to studies of transgenic mice[END_REF]. The metabolic flexibility was determined by the Hill slope (H value) and the 50th percentile (EC50) of the PRCF curve and by measuring the RER amplitude over 24 h [START_REF] Riachi | Percent relative cumulative frequency analysis in indirect calorimetry: Application to studies of transgenic mice[END_REF]). General physical activity was monitored using an infrared beam-operated sensor system that detected xand z-axis activity. Both total counts (every time a beam is broken) and ambulatory counts (every time a new beam is broken) were assessed. An X-total (X-TOT) count is registered when the animal moves horizontally more than 0.5 in and is representative of small-scale, repetitive activities, such as scratching and grooming. The X-ambulatory count (X-AMB) measures actual locomotion by registering a count only when the animal breaks a new beam. A Z-total (Z-TOT) count is registered when 1.5 in of vertical movement occurs, i.e., rearing (Abreu-Vieira 2016). For estimating carbohydrate and fat oxidation from VO 2 and VCO 2 , we used the nonprotein respiratory quotient table from Péronnet and Massicotte previously used in mice [START_REF] Péronnet | Table of nonprotein respiratory quotient: An update[END_REF][START_REF] Overmyer | Maximal Oxidative Capacity during Exercise Is Associated with Skeletal Muscle Fuel Selection and Dynamic Changes in Mitochondrial Protein Acetylation[END_REF].

Total Body and Fat Mass

Total fat mass and total lean mass were determined in unanaesthetised mice by quantitative resonance interference analysis using the EchoMRI whole-body magnetic resonance analyser (Echo Medical System, Houston, TX, USA). Total fat mass represented the sum of all fat in the body. Total lean mass included mainly muscle and inner organs. Skeletal muscle mass is known to account for the largest portion of total lean mass.

Oral Glucose Tolerance Test (OGTT)

Glycemia was measured using a drop of blood collected from the tail with a glucometer (Accu-Chek Performa, Roche, Argentina). OGTT was performed on mice that were fasted for 5 to 6 h. The measurement was performed on the last day of CF supplementation. After measurement of the basal glycemia (time 0), mice were then injected per os with a solution of 20% glucose in sterile water at a dose of 1.5 g glucose•kg body weight -1 . Glycemia was measured at 15, 30, 60, 90, and 120 min after injection of the glucose solution. The incremental area of the glucose curve was then calculated as a measure of glucose tolerance.

qRT-PCR Analysis

The forward (F) and reverse (R) primers of the selected genes used in this study are presented in Table 2. Total RNA was obtained from pulverised frozen white gastrocnemius muscle using Trizol reagent (Invitrogen Life Technologies, Rockville, MD, USA). The purity, integrity and quantity of RNA were evaluated using a microplate reader (Synergy H1 (Biotek Instruments, Winooski, Vermont). The sample was considered pure when the ratio OD260/OD280 was >1.8. cDNA was synthesised from total RNA (2 µL) using the iScript reverse transcription supermix for RT-qPCR (Biorad, Hercules, CA, USA). Total RT products were analysed by quantitative polymerase chain reaction (PCR) using the 2x Platinium SYBR Green qPCR SuperMix-UDG according to the manufacturer's specifications (Invitrogen Lige Technologies, Burlington, Canada). Cycling was achieved in a CFX96 cycler (Biorad, Hercules, California; conditions: 95 • C for 10 min and 40 cycles of 95 • C for 30 s, 55 • C for 45 s and 72 • C for 45 s). At the end of each run, the absence of primer-dimer formation and the presence of a unique amplicon were confirmed using the melting curve. The threshold cycle (Ct) values were used following the 2-∆∆Ct method to analyse qPCR data, with either ß-Actin or HPRT1 as a housekeeping gene [START_REF] Schmittgen | Analyzing real-time PCR data by the comparative C T method[END_REF]. Quantitative RT-PCR values were related to those of the control group, which were set to 1.

Enzyme Activities

The measurement of the specific activity of electron transport chain complexes and citrate synthase were performed spectrophotometrically as previously described [START_REF] Jarreta | Mitochondrial function in heart muscle from patients with idiopathic dilated cardiomyopathy[END_REF]. Pulverised frozen white gastrocnemius muscles (~30 mg) were homogenised with a vibrating microbead homogeniser in 500 µL of homogenisation buffer (120 mM KCl, 20 mM of HEPES, 2 mM of MgCl2, 1 mM of EGTA, and 5 mg•mL -1 of BSA, pH 7.4) followed by the addition of 500 µL of hypotonic media (25 mM potassium phosphate and 5 mM MgCl 2 , pH 7.2). The samples were then submitted to three rounds of freeze-thaw cycle in liquid nitrogen. The samples were centrifuged for 10 min at 600× g at 4 • C, and the supernatant was kept on ice until the analysis. The protein content was determined in the supernatant in triplicate using a Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA).

The activity of citrate synthase (CS) was determined spectrophotometrically at 412 nm following the reduction of 2 mM 5,5 -dithio-bis(2-nitrobenzoic acid) in the presence of 0.1 mM acetyl-CoA and 12 mM of oxaloacetic acid in 200 mM Tris buffer (pH 7.4). The rotenone-sensitive NADH-decylubiquinone oxidoreductase (complex I) assay was performed at 340 nm using the acceptor 2,3-dimethoxy-5-methyl-6-n-decyl-1,4-benzoquinone (80 µM) and NADH as electron donor (200 µM) in 10 mM Tris buffer (pH 8.0). The addition of 4µM of rotenone was used to quantify the rotenone sensitive activity. The activity of the cytochrome c oxidase (complex IV) was performed at 550 nm using 10 µM reduced cytochrome c as donor and 2.5 mM of n-dodecyl-ß-maltoside to permeabilise both mitochondrial membranes in 100 mM potassium phosphate buffer (pH 7.0).

All the enzyme assays were determined in triplicate, and results were related to those of the control group, which were set to 100.

Mitochondrial Assessment

Mitochondrial respiration and mitochondrial H 2 O 2 production were studied in situ in saponin permeabilised fibres using white gastrocnemius and soleus muscles [START_REF] Kuznetsov | Analysis of mitochondrial function in situ in permeabilized muscle fibers, tissues and cells[END_REF]. Briefly, fibres were separated under binocular microscope in solution A at 4 • C (in mM: 2.77 CaK 2 EGTA, 7.23 K 2 EGTA, 6.56 MgCl 2 , 20 taurine, 0.5 DTT, 50 K-methane sulfonate, 20 imidazole, 5.7 Na2 ATP, and 15 creatine-phosphate, pH 7.1) and permeabilised in solution A with 50 µg•mL -1 of saponin for 30 min at 4 • C. Mitochondrial respiratory function was determined in an oximeter equipped with a Clark-type electrode (Oxygraph, Hansatech Instruments, Glasgow). The chamber was filled with 1 mL of Solution B (in mM: 2.77 CaK 2 EGTA, 7.23 K 2 EGTA, 6.56 MgCl 2 , 20 taurine, 0.5 DTT, 50 K-methane sulfonate, and 20 imidazole, pH 7.1) and after recording baseline oxygen content in the chamber, one bundle of 1-2 mg dry weight of permeabilised myofibres was placed into the chamber, which was then sealed shut. After baseline readings, the following additions were sequentially made: palmitoyl carnitine and malate (160 µM:5 mM), glutamate (10 mM), succinate (25 mM), rotenone (0.5 µm), the uncoupler CCCP (1 µM), antimycin-A (8 µM), and N,N',N'-tetramethyl-pphenylenediamine dihydrochloride and ascorbate (0.9:9 mM). Respiration was measured at 23 • C under continuous stirring. At the end of each test, fibres were carefully removed from the oxygraphic cell, blotted and dried at least 24 h at ~80 • C for determination of fibre weight. Rates of O 2 consumption (JO 2 ) were expressed in nmol O 2 •min -1 •(mg dry weight) -1 . All measurements were performed at least in duplicate.

Net H 2 O 2 release by respiring mitochondria was measured in permeabilised fibre bundles with the fluorescent probe Amplex Red (20 µM: excitation-emission: 563-587 nm), as described previously [START_REF] Picard | Mitochondrial Structure and Function Are Disrupted by Standard Isolation Methods[END_REF]. Following preparation of permeabilised fibres, samples destined for H 2 O 2 measurements were rinsed three times in buffer Z (in mM: 110 K-Mes, 35 KCl, 1 EGTA, 5 K 2 HPO 4 , 3 MgCl 2 6H 2 O, and 0.5 mg•mL -1 BSA, pH 7.3 at 4 • C). Fibre bundles (0.3-1.0 mg dry weight) were incubated at 37 • C in a quartz microcuvette with continuous magnetic stirring in 600 µL of buffer Z (pH 7.3 at 37 • C) supplemented with 1.2 U•mL -1 horseradish peroxidase. Baseline fluorescence readings were taken in the absence of any exogenous respiratory substrates. The following additions were then made sequentially: glutamate (5 mM), succinate (5 mM), rotenone (0.5 µM), ADP (10 mM), and antimycin-A (8 µM). At the end of each test, fibres were carefully removed from the cuvette, blotted, and dried to determine fibre weight. Rates of H 2 O 2 production were expressed in AU•min -1 •(mg dry weight) -1 and per oxphos capacity which corresponded to the maximal mitochondrial respiration under phosphorylation state. All measurements were performed at least in duplicate.

Mitochondrial calcium retention capacity (CRC) was assessed in white gastrocnemius muscle ghost fibres as previously described [START_REF] Picard | Resistance to Ca2+-induced opening of the permeability transition pore differs in mitochondria from glycolytic and oxidative muscles[END_REF]. Briefly, fibres were incubated in a quartz microcuvette under continuous stirring in 600 µL of CRC buffer (in mM: 250 sucrose, 10 MOPS, 0.005 EGTA, and 10 P i -Tris, pH 7.3). Fibres were then exposed to a single pulse of 20 nM of Ca 2+ . Changes in extramitochondrial calcium concentration were monitored fluorometrically using Calcium green 5N (1 mM: excitation-emission: 505-535 nm). Permeability transition pore susceptibility (PTP) was assessed by measuring the time required for PTP opening, and CRC was taken as the total amount of Ca 2+ accumulated by mitochondria before its release. At the end of each test, fibres were carefully removed from the cuvette, blotted, and dried to determine fibre weight. CRC values were expressed in nM of Ca 2+ per mg of dry weight.

Western Blot Analysis

Proteins from white gastrocnemius and soleus muscles were diluted in Laemmli buffer and separated on Mini-PROTEAN TGX Stain-Free 10% precast polyacrylamide gels (Biorad); an internal standard was loaded on each gel. Electrophoretic separation was done at 200 V for 35 min in migration buffer (25 mM TrisBase, 0.2 M glycine, and 1% SDS (p/v)). Stain-Free (SF) technology contains a proprietary trihalo compound that reacts with proteins, rendering them detectable through UV exposure. SF imaging was performed using ChemiDoc MP Imager and Image Lab 4.0.1 software (Biorad, Hercules, California, USA) with a 5-min stain activation time, and total protein patterns were therefore visualised. Proteins were then transferred on a 0.2 µm nitrocellulose sheet using the Trans-Blot Turbo Transfer System (Biorad). The quality of transfer was controlled by imaging membranes using the SF technology. Following the transfer step, the carbonyl proteins were derivatised with dinitrophenylhydrazine diluting in 2N HCL, and were finally extensively washed with methanol. The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline containing Tween-20 (TBST: 15 mM Tris/HCl, pH 7.6, 140 mM NaCl, and 0.05% Tween-20) for 1 h at room temperature. Membranes were then incubated at 4 • C overnight or 2 h at room temperature with the following primary antibodies: protein carbonyl primary antibody (Anti-DNP; # STA-308, Cell Biolabs) and SOD2 (#Ab13533, Abcam). After three 10 min washes in TBST, membranes were probed with secondary antibodies for protein carbonyl (HRP-conjugate; #STA-308, Cell Biolabs) and SOD2 (anti-rabbit IgG-HRP linked; #7074, Cell Signalling) in blocking solution for 2 h at room temperature and were finally extensively washed with TBST. The dilution of primary and secondary antibodies was optimised for each antibody. Chemiluminescence detection was carried out using ECL Clarity (Biorad), and images capture was done with ChemiDocMP. All the images were analysed using the Image Lab 4.0.1 software. Normalisation of protein signal intensities was carried out following the quantification of respective total protein levels on SF images, sample control, and internal standards.

NAD Measurement

Total NAD and NADH levels were measured in homogenised white gastrocnemius using a commercial kit (#K337-100, Biovision, USA) and following the manufacturer's instructions. Briefly, the total pool of NAD (NADt = NAD + and NADH) was extracted. For each of the extracted samples, half of the sample was heated to 60 • C for 30 min to decompose NAD + while keeping NADH intact. Both NADt and NADH samples were mixed with NAD cycling enzyme, and absorbance was measured at 450 nm. NADt and NADH were quantified by comparing with NADH standard curve and normalising to mg of protein. Finally, the ratio of NAD + (NADt-NADH) to NADH was calculated. All the assays were performed in triplicate.

Statistics

Data are reported as mean ± standard deviation (SD). Data normality was assessed using the D'Agostino-Pearson normality test. Data analysis was performed using the Student's t-test and two-way ANOVA (group and time) for repeated measurements followed by Bonferroni's post hoc test using Prism 8.4.1 (GraphPad Software, San Diego, CA, USA). A two-sided level of 5% for type I error was applied.

Results

Body Composition and Whole-Body Metabolism

Fifteen days of CF supplementation, in wild-type (WT) mice, did not modify body weight (p = 0.091), lean mass (p = 0.073) and fat mass assessed by EchoMRI (p = 0.89; Figure 1A). O 2 consumption rates expressed per lean mass was significantly higher in the dark cycle (8 ± 8%, p = 0.042), whereas it remained similar in the light cycle (p = 0.392; Figure 1B.). There were no changes in locomotor activity (data not shown).CF mice showed a significant increase of whole-body energy expenditure by 15 ± 15% (p = 0.042; Figure 1C) and in food intake (0.08 ± 0.01 vs. 0.11 ± 0.01 g per lean body weight, respectively CF group and WT, p = 0.013, Figure 1D). CF supplementation significantly modified whole-body metabolism: a main effect of higher RER in the CF group was observed over 24 h (p = 0.023, Figure 2A) and mean RER over 12 h was higher in the CF group in light and dark cycles (+5 ± 3%, p = 0.035 and +9 ± 4%, respectively, p = 0.026). Moreover, CF supplementation promoted carbohydrate (CHO) use (51 ± 13% of energy consumption depended on CHO oxidation in the CF group vs. 37 ± 12% in the control group during the light cycle, p = 0.108 and 71 ± 21% in CF group vs. 47 ± 24% in the control group during the dark cycle, p = 0.004; Figure 2B.). However, when expressed in kJ, there was no difference in CHO oxidation nor fatty acid oxidation (CHO oxidation: 3.7 ± 1.4 kcal•d -1 in control vs. 4.4 ± 1.7 kcal•lean body mass -1 •d -1 in CF, p = 0.33 and fatty acid oxidation: 2.3 ± 1.0 kcal•d -1 in control vs. 2.6 ± 0.87 kcal•lean body mass -1 •d -1 in CF, p = 0.608). Analysis of PRCF of RER revealed an increase of EC50 values in the CF group (p = 0,009; Figure 2D) meaning that CF induce a shift toward a greater use of carbohydrates. A decrease of the Hill slope was observed in the CF group (H values: 25.16 ± 4.44 in controls vs. 12.79 ± 3.96 in CF group, p < 0.001). Moreover, the RER amplitude over 24 h was increased by 31 ± 31% in the CF group (p = 0.035). CF supplementation improved glucose tolerance following oral glucose administration from unchanged baseline blood glucose levels after an overnight fast (Figure 2E, p = 0.003). The area under the capillary blood glucose curve was lower in the CF group (1018 ± 135 vs. 1181 ± 170 mM•120 min, p = 0.005; Figure 2F).

Mitochondrial Bioenergetics

We tested the effect of CF supplementation on the energetics of mitochondria in the white gastrocnemius and soleus muscle in WT mice. A higher mitochondrial respiration using complex IV substrates in the white gastrocnemius and soleus muscles was observed (p = 0.004 and p = 0.028, respectively; Figure 3A,B). No modification of the CII/CI and CIV/CI respiration ratios were observed (p = 0.899 and p = 0.701 for the white gastrocnemius and p = 0.693 and p = 0.912 for the soleus muscles, respectively, data not shown). Moreover, complex I, II, IV, and citrate synthase activities were significantly increased in the white gastrocnemius muscle (respectively: +31 ± 27%, p = 0.004; +28 ± 38%, p = 0.027; +35 ± 38%, p = 0.009; +14 ± 13%, p = 0.040; Figure 3C). The ability of mitochondria to oxidise palmitoyl-carnitine tends to increase in the white gastrocnemius (+24 ± 12%, p = 0.096; Figure 3D), whereas no difference was observed in the soleus (p = 0.363). The relative rates of palmitoyl-carnitine-stimulated respiration were unchanged when expressed in function of complex I respiration (62 ± 11 in the control group vs. 64 ± 20 in the CF group in the white gastrocnemius and 70 ± 18 vs. 72 ± 16 in the soleus). Next, we explored the sensitivity to Ca 2+ -induced PTP opening in the white gastrocnemius. As shown in Figure 3E, neither the time to PTP opening nor the Ca 2+ retention capacity was modified following CF supplementation (p = 0.608 and p = 0.943, respectively). mRNA assessment of genes involved in mitochondrial biogenesis showed a 70% upregulation of NRF1 mRNA, whereas no difference was reported for PGC1α, Tfam, CS, ND1, ND2, SDHa and Cox2 (Table 3). We explored, on a few samples, the effect of CF supplementation on mitochondrial supercomplexes (Figure S1, Supplementary Materials). The densitometric analysis suggests that CF supplementation increased overall supercomplexes content without qualitative adaptation or rearrangements. Indeed, respirasome density was improved by 58% using complex I and complex II probes and by 93% using complex III probe. Similarly, complex I and complex III content embedded in supercomplexes were higher in the CF group. Table 3. mRNA levels of genes involved in mitochondrial biogenesis, anti-oxidant defences and NAD metabolism. The mRNA levels were determined by real-time quantitative RT-PCR and expressed relative to the value of GAPDH, an internal control. Data are presented as the mean ± SD, n = 8. 4A,B, respectively), the rates were decreased when expressed per mitochondrial content (Figure 4C). A main positive effect of CF supplementation was observed in various conditions in the white gastrocnemius (p < 0.048; Figure 4C). The mitochondrial H 2 O 2 emission was also significantly reduced when expressed per oxidative phosphorylation capacity assessed by state 4 respiration using complex I+II substrates by 26 ± 13% in the white gastrocnemius (p = 0.044) and by 29 ± 12% in the soleus (p = 0.024; Figure 4D).

Gene

Oxidative Stress Markers in Skeletal Muscle

The cellular protein oxidative stress level in WT mice was unaffected by CF supplementation as the cellular content of SOD2 was similar in the experimental and control groups in the white gastrocnemius and soleus (Figure 4E). Moreover, no difference was observed in protein carbonylation in the white gastrocnemius and soleus (Figure 4F). Additionally, no difference in catalase and MnSod mRNA expression was observed after CF supplementation (Table 3).

NAD Metabolism

The total pool of pyridine nucleotides increased following CF supplementation by 36 ± 33% (p = 0.012). Specifically, the NAD + and NADH contents increased in the white gastrocnemius (respectively by 69 ± 60%, p = 0.026 and 29 ± 42%, p = 0.017; Figure 5A,B) and the NAD + /NADH ratio tended to increase in the CF group (p = 0.084). No significant effect was observed for Sirt3 mRNA (p = 0.096), NMNAT mRNA (p = 0.094), and Sirt1 mRNA(p = 0.869; Table 3).

Whole-Body and Cellular Metabolism Response Following CF Supplementation in Sirt3 -/-Mice

We examined the effect of CF supplementation in Sirt3 -/-mice. Body composition as weight, lean mass, and fat mass were not different between the CF and control groups (p = 0.52, p = 0.66, p = 0.45, respectively; Figure 6A). Similarly, the daily energy expenditure was unaffected by CF supplementation (0.38 ± 0.01 Kcal•lean body mass -1 •day -1 in the controls vs. 0.39 ± 0.01 Kcal•lean body mass -1 •day -1 in CF, p = 0.502; Figure 6B). The RER was similar between both groups in the light and dark cycles (p = 0.301; Figure 6C), and substrate oxidation was not affected by CF supplementation (44 ± 17% of energy consumption depended on CHO oxidation in the control group vs. 50 ± 11% in the CF group during the light phase, p = 0.209 and 65 ± 19% in the control group vs. 77 ± 21% in the CF group during the dark phase, p = 0.395; Figure 6D). The metabolic flexibility assessed by RER amplitude over 24 h remained similar between the two groups (0.19 ± 0.05 in control vs. 0.20 ± 0.03 in CF, p = 0.604), whereas the PRCF slope was decreased in the CF group (H values: 17.77 ± 4.38 in control vs. 12.79 ± 3.93 in CF, p = 0.039). Next, we compared the effect of CF supplementation on mitochondrial enzyme activities. CF supplementation failed to increase the enzyme activity of complex I, II, IV, and CS in Sirt3 -/-mice as observed previously in WT (respectively: p = 0.623, p = 0.629, p = 0.283, and p = 0.791, Figure 6E). A greater enzyme activity of complex I and IV was observed in WT compared to Sirt3 -/-mice after CF supplementation (respectively: p = 0.027 and p = 0.045)

Discussion

In this study, we provide an integrative analysis of the effect of CF ingestion. We observed that chronic CF ingestion improved mitochondrial respiration and reduced H 2 O 2 production, assessed in their myofibre environment, whereas the susceptibility of PTP opening was unchanged. CF ingestion enhanced NAD metabolism, supporting the involvement of sirtuin pathways on the observed mitochondrial adaptations. We also observed wholebody metabolism modifications towards a greater ability to use carbohydrates as a main substrate suggesting that mitochondrial adaptations likely contributed to the observed whole-body metabolism adaptation.

Metabolism

Dietary administration of cocoa flavanols has been identified as an effective strategy for alleviating glucose intolerance, allowing opportunities to ameliorate metabolic diseases [START_REF] Strat | Mechanisms by which cocoa flavanols improve metabolic syndrome and related disorders[END_REF]. Our results are in accordance with previous studies that reported an increased glucose tolerance following flavanols intake [START_REF] Yamashita | Cacao liquor procyanidin extract improves glucose tolerance by enhancing GLUT4 translocation and glucose uptake in skeletal muscle[END_REF][START_REF] Watanabe | Flavan-3-ol fraction from cocoa powder promotes mitochondrial biogenesis in skeletal muscle in mice[END_REF]. The mechanisms proposed for this effect involve an increase in the translocation of GLUT4 towards the cell membrane, an increase in the phosphorylation of AMPK and the up-regulation of UCP-2 gene expression in the skeletal muscle [START_REF] Grassi | Short-term administration of dark chocolate is followed by a significant increase in insulin sensitivity and a decrease in blood pressure in healthy persons[END_REF][START_REF] Tomaru | Dietary supplementation with cacao liquor proanthocyanidins prevents elevation of blood glucose levels in diabetic obese mice[END_REF]. Moreover, CF ingestion was associated with insulin sensitivity improvement in humans [START_REF] Grassi | Short-term administration of dark chocolate is followed by a significant increase in insulin sensitivity and a decrease in blood pressure in healthy persons[END_REF]. After 15 days of dark chocolate consumption, a decrease of the homeostasis model assessment of insulin resistance index was observed in healthy subjects concomitantly with an increase of the quantitative insulin sensitivity check index and the insulin sensitivity index [START_REF] Grassi | Short-term administration of dark chocolate is followed by a significant increase in insulin sensitivity and a decrease in blood pressure in healthy persons[END_REF]. We also observed a concomitant increase in glucose tolerance and metabolic flexibility in WT mice, which refers to the the organism's capacity to adapt fuel oxidation to fuel availability [START_REF] Goodpaster | Metabolic Flexibility in Health and Disease[END_REF]. Interestingly, while impaired mitochondrial oxidative capacity and the related substrate oxidation was associated with insulin resistance [START_REF] Sergi | Mitochondrial (Dys)function and Insulin Resistance: From Pathophysiological Molecular Mechanisms to the Impact of Diet[END_REF], endurance exercise training increased mitochondrial content, insulin sensitivity, and metabolic flexibility [START_REF] Goodpaster | Metabolic Flexibility in Health and Disease[END_REF]. Therefore, the present results are specific to CF supplementation and are not influenced by spontaneous activity.

The positive effect of CF on metabolic flexibility, as well as the increase of mitochondrial complexes activity, were blunted in Sirt3 -/-mice, indicating that the positive effects of CF occur at least partially through Sirt3. These results support previous findings and a pivotal role of Sirt3 on metabolic flexibility regulation [START_REF] Jing | Sirt3 Regulates Metabolic Flexibility of Skeletal Muscle Through Reversible Enzymatic Deacetylation[END_REF]. Our results suggest that an increase in mitochondrial function may enhance metabolic flexibility. However, besides mitochondrial function, the basal respiratory quotient, glucose disposal rate, adipose tissue lipid storage capacity, and plasma free fatty acid concentration were also identified as determinant factors of metabolic flexibility and should be considered [START_REF] Galgani | Metabolic flexibility and insulin resistance[END_REF]. Future studies should assess the cellular determinants of metabolic flexibility more deeply to deepen our understanding of the underlying causes of enhancement of metabolic flexibility following CF supplementation.

The whole-body metabolism assessment revealed an increase in the relative CHO oxidation following CF supplementation. This result contrasts with a previous study that reported an increase of lipolysis following an acute flavanols ingestion or a two-week supplementation with flavan-3-ols in mice [START_REF] Watanabe | Flavan-3-ol fraction from cocoa powder promotes mitochondrial biogenesis in skeletal muscle in mice[END_REF]. Besides the extract composition differences, these contradictory findings may result from the pleiotropic effects of NO. It is well accepted that CF ingestion stimulates the production or the bioavailability of NO metabolism [START_REF] Katz | Cocoa and Chocolate in Human Health and Disease[END_REF]. On one side, NO stimulates glucose transport and uptake in skeletal muscle. Stimulation of NO production in cell line-derived myotubes by insulin or hydrogen peroxide resulted in an increase of GLUT4 translocation, which was reduced with nNOS inhibition [START_REF] Kellogg | Neuronal nitric oxide synthase mediates insulin-and oxidative stress-induced glucose uptake in skeletal muscle myotubes[END_REF]. Moreover, NO can increase glucose transport in an insulin-independent pathway leading to an increase in the levels of cyclic GMP and AMPK [START_REF] Deshmukh | Nitric oxide increases cyclic GMP levels, AMP-activated protein kinase (AMPK)Alpha1-specific activity and glucose transport in human skeletal muscle[END_REF], which promotes glucose oxidation in rat skeletal muscle independent of fatty acid availability and oxidation [START_REF] Smith | AMP kinase activation with AICAR simultaneously increases fatty acid and glucose oxidation in resting rat soleus muscle[END_REF]. On the other side, NO promotes fatty acid oxidation by reducing the level of malonyl-CoA via inhibition of acetyl-CoA carboxylase and activation of malonyl-CoA decarboxylase in skeletal muscle [START_REF] Jobgen | Regulatory role for the arginine-nitric oxide pathway in metabolism of energy substrates[END_REF]. While we observed a shift towards a lower relative proportion to use fatty acid as substrate with CF, there was no difference regarding the absolute values expressed in kJ. This suggests that the increase in energy expenditure observed with CF supplementation relies on an increase in glucose oxidation. Therefore, these results support the idea that CF consumption increases insulin sensitivity and promotes glucose oxidation without a concomitant effect on fatty acid oxidation capacity. These results raise the question of the relevance to use cocoa flavanols in the context of high fat diet. Future studies should evaluate a possible harmful effect of an association of a CF supplementation and a high-fat diet.

Mitochondrial Function

Dietary administration of cocoa flavanols has been shown to improve mitochondrial function, including (i) mitochondrial respiration and electron transport chain enzyme activities; (ii) mitochondrial H 2 O 2 release, which reflects mitochondrial reactive oxygen species (ROS) production; and (iii) mitochondrial calcium retention capacity which reflects the susceptibility to mitochondrial permeability transition pore (mPTP) opening (for review see [START_REF] Daussin | Effects of (-)-epicatechin on mitochondria[END_REF]. Our results are in accordance with previous studies; nonetheless, our study was the first to evaluate the effect of CF supplementation on mitochondrial function assessed in situ in permeabilised fibre and in different muscle types. Unlike the previous studies on isolated mitochondria, the assessment of mitochondrial function in permeabilised fibre preserves mitochondrial morphology, functional interactions with other intracellular components [START_REF] Kuznetsov | Analysis of mitochondrial function in situ in permeabilized muscle fibers, tissues and cells[END_REF] and avoids mitochondrial structure disruption during the mitochondrial isolation process [START_REF] Picard | Mitochondrial Structure and Function Are Disrupted by Standard Isolation Methods[END_REF]. Therefore, it is important to study mitochondrial function in tissue preparations where mitochondrial structure is preserved, and all of the mitochondrial pool is represented to better evaluate the response to a treatment.

Previous studies performed in cell culture or in vivo in mice have shown that supplementation with EPI, which is considered the main bioactive molecule in CF, increases maximal ADP-stimulated respiration (e.g., state 3 respiration) when mitochondria are energised with a combination of energy substrates feeding various sites along the respiratory chain [START_REF] Panneerselvam | Epicatechin regulation of mitochondrial structure and function is opioid receptor dependent[END_REF][START_REF] Elbaz | Epicatechin Stimulates Mitochondrial Activity and Selectively Sensitizes Cancer Cells to Radiation[END_REF][START_REF] Rowley | Monomeric cocoa catechins enhance β-cell function by increasing mitochondrial respiration[END_REF][START_REF] Bitner | Common gut microbial metabolites of dietary flavonoids exert potent protective activities in β-cells and skeletal muscle cells[END_REF][START_REF] Kener | High-resolution Respirometry to Measure Mitochondrial Function of Intact Beta Cells in the Presence of Natural Compounds[END_REF]. Consistent with these results, we observed a main effect of CF supplementation on mitochondrial respiration in oxidative and glycolytic muscles. To determine whether this increase is related to a mitochondrial mass increase or the remodelling of mitochondrial respiratory complexes, we assessed mitochondrial supercomplexes from the quadriceps muscle. These supramolecular structures are thought to decrease ROS production, stabilise or assist in the assembly of individual complexes, regulate respiratory chain activity, and prevent protein aggregation in the protein-rich inner mitochondrial membrane [START_REF] Lapuente-Brun | Supercomplex Assembly Determines Electron Flux in the Mitochondrial Electron Transport Chain[END_REF][START_REF] Acin-Perez | The function of the respiratory supercomplexes: The plasticity model[END_REF][52]. Despite a limited number of measures, the visual analysis of the results (Supplementary Materials) suggests that CF supplementation induces an increase of mitochondrial mass, as reflected by an increase in all supercomplex species, rather than specific respiratory chain complex reorganisations. Collectively, these results suggest that CF promotes a mitochondrial mass increase without qualitative adaptation of respiratory chain complex arrangements.

During mitochondrial respiration, variable amounts of superoxide are formed and can be metabolised to form other types of ROS [START_REF] Muller | The nature and mechanism of superoxide production by the electron transport chain: Its relevance to aging[END_REF]. Although excessive ROS production from mitochondria is involved in a broad spectrum of pathologies, low physiological concentrations of ROS can have beneficial effects, such as protecting against infectious agents and participating in cellular signalling [START_REF] Sena | Physiological Roles of Mitochondrial Reactive Oxygen Species[END_REF][START_REF] Powers | Reactive oxygen and nitrogen species as intracellular signals in skeletal muscle[END_REF]. Previous studies performed in vivo in rodents and humans and cell culture models reported beneficial CF supplementation effects on ROS production and various antioxidant systems [START_REF] Daussin | Effects of (-)-epicatechin on mitochondria[END_REF]. For instance, oral gavage with EPI during 15 days increases SOD2 and catalase activity in mice quadriceps muscles [START_REF] Ramírez-Sánchez | Epicatechin-induced recovery of mitochondria from simulated diabetes: Potential role of endothelial nitric oxide synthase[END_REF]. While mitochondrial ROS production has not been widely assessed, we observed that ROS production levels were similar when values were expressed per muscle weight, whereas the production was lowered when expressed per mitochondrial mass, suggesting that qualitative adaptations occur to maintain the physiological concentrations of ROS. Moreover, we did not observe any effect of CF on SOD2 nor ROS-induced damages assessed by protein carbonylation. While CF enhances antioxidant protein pools, including widely reported SOD2 and catalase levels [START_REF] Moreno-Ulloa | Recovery of Indicators of Mitochondrial Biogenesis, Oxidative Stress, and Aging With (-)-Epicatechin in Senile Mice[END_REF][START_REF] Ramírez-Sánchez | Epicatechin-induced recovery of mitochondria from simulated diabetes: Potential role of endothelial nitric oxide synthase[END_REF][START_REF] Ramirez-Sanchez | (-)-Epicatechin improves mitochondrial-related protein levels and ameliorates oxidative stress in dystrophic δ-sarcoglycan null mouse striated muscle[END_REF], this adaptation was not always associated with a decrease in ROS-induced damages [START_REF] Wiegand | Effects of Quercetin and Catechin on Hepatic Glutathione-S Transferase (GST), NAD(P)H Quinone Oxidoreductase 1 (NQO1), and Antioxidant Enzyme Activity Levels in Rats[END_REF][START_REF] Taub | Beneficial effects of dark chocolate on exercise capacity in sedentary subjects: Underlying mechanisms. A double blind, randomized, placebo controlled trial[END_REF][START_REF] Decroix | Acute cocoa Flavanols intake has minimal effects on exercise-induced oxidative stress and nitric oxide production in healthy cyclists: A randomized controlled trial[END_REF][START_REF] Santos | Catechin and epicatechin reduce mitochondrial dysfunction and oxidative stress induced by amiodarone in human lung fibroblasts[END_REF]. These data support the theory that low levels of ROS production are required for normal cell homeostasis [START_REF] Sena | Physiological Roles of Mitochondrial Reactive Oxygen Species[END_REF]. Collectively, these results suggest that, concomitantly to an increase of mitochondrial mass, CF promotes qualitative mitochondrial adaptations to enhance its antioxidant capacity and maintain low levels of ROS production.

Mitochondrial Biogenesis

Mitochondrial mass is the result of the interplay between mitochondrial biogenesis and mitophagy. Our study focused on mitochondrial biogenesis activated by polyphenols consumption [START_REF] Davinelli | Polyphenols as Caloric Restriction Mimetics Regulating Mitochondrial Biogenesis and Mitophagy[END_REF]. Mitochondrial biogenesis is the cellular process that improve mitochondrial mass. This complex and highly regulated process involve several transcription factors, nuclear hormone receptors, and transcription coactivators that act collectively to coordinate changes in the expression of nuclear and mitochondrial DNA encoded genes [START_REF] Dominy | Mitochondrial Biogenesis through Activation of Nuclear Signaling Proteins[END_REF]. Previous studies have attempted to identify which pathway(s) may underlie CF supplementationinduced mitochondrial biogenesis. Stimulation of NO-dependent signalling has emerged as a central pathway involved in mitochondrial biogenesis. CF supplementation enhances the generation of NO by inhibiting the arginine degrading enzyme arginase, which increases the availability of L-arginine for NO biosynthesis [START_REF] Schnorr | Cocoa flavanols lower vascular arginase activity in human endothelial cells in vitro and in erythrocytes in vivo[END_REF][START_REF] Lee | Epicatechin Attenuates Degradation of Mouse Oxidative Muscle Following Hindlimb Suspension[END_REF]. Moreover, using an eNOS inhibitor, the stimulatory effect of epicatechin on mitochondrial biogenesis was partially blunted [START_REF] Ramírez-Sánchez | Epicatechin-induced recovery of mitochondria from simulated diabetes: Potential role of endothelial nitric oxide synthase[END_REF]. These results suggest the involvement of other signalling pathways to stimulate mitochondrial biogenesis.

Recently, Aragones et al. (2016) reported that dietary proanthocyanidins, members of the polyphenols family, boost hepatic NAD + metabolism by enhancing the de novo NAD + biosynthesis pathway and Sirt1 activity in a dose-dependent manner in rats [START_REF] Aragonès | Dietary proanthocyanidins boost hepatic NAD+ metabolism and SIRT1 expression and activity in a dose-dependent manner in healthy rats[END_REF]. Furthermore, the liver exports NAD + precursors to other organs, such as skeletal muscle, which have a lower capacity to synthetise NAD, raising the possibility of extending the effects observed in the liver to the whole organism [START_REF] Imai | Dissecting systemic control of metabolism and aging in the NAD World: The importance of SIRT1 and NAMPT-mediated NAD biosynthesis[END_REF]. While some studies reported an effect of CF supplementation on Sirt1 protein content, activity, or mRNA [START_REF] Aragonès | Dietary proanthocyanidins boost hepatic NAD+ metabolism and SIRT1 expression and activity in a dose-dependent manner in healthy rats[END_REF][START_REF] Taub | Alterations in Skeletal Muscle Indicators of Mitochondrial Structure and Biogenesis in Patients with Type 2 Diabetes and Heart Failure: Effects of Epicatechin Rich Cocoa[END_REF][START_REF] Moreno-Ulloa | Recovery of Indicators of Mitochondrial Biogenesis, Oxidative Stress, and Aging With (-)-Epicatechin in Senile Mice[END_REF][START_REF] Ramírez-Sánchez | Epicatechin-induced recovery of mitochondria from simulated diabetes: Potential role of endothelial nitric oxide synthase[END_REF][START_REF] Ramirez-Sanchez | Epicatechin rich cocoa mediated modulation of oxidative stress regulators in skeletal muscle of heart failure and type 2 diabetes patients[END_REF][START_REF] Ramirez-Sanchez | Epicatechin induced reversal of endothelial cell aging and improved vascular function: Underlying mechanisms[END_REF], the involvement of NAD metabolism in mitochondrial biogenesis following CF supplementation using loss or gain of function has never been tested. While a muscle-specific knock-out would have been preferable over the whole-body Sirt3 KO mice used in our study, our results suggest that CF improves NAD metabolism, and Sirt3 is involved in this supplementation-induced mitochondrial electron transport chain activity improvement. The positive effects observed on mitochondrial complexes activity following CF supplementation were not observed using Sirt3 -/-mice and a greater activity of complex I and IV activities were observed after CF supplementation only in WT. These observations extend previous works showing that Sirt3 plays a pivotal role in mitochondrial function [START_REF] Giralt | SIRT3, a pivotal actor in mitochondrial functions: Metabolism, cell death and aging[END_REF]. Collectively, these findings suggest that sirtuins, through the modulation of NAD metabolism, are involved in mitochondrial mass improvement observed following CF supplementation.

Limits

Cocoa beans are composed by a mixture of monomeric, oligomeric, and polymeric flavanols. Among the flavanols, EPI appears to be the most abundant polyphenolic monomer found in cacao products, representing up to 35% of the polyphenol content [START_REF] Wollgast | Review on polyphenols in Theobroma cacao: Changes in composition during the manufacture of chocolate and methodology for identification and quantification[END_REF]. EPI was established as the main bioactive molecule underlying benefits associated with cocoa and chocolate supplementation [START_REF] Rowley | Monomeric cocoa catechins enhance β-cell function by increasing mitochondrial respiration[END_REF][START_REF] Schroeter | Epicatechin mediates beneficial effects of flavanol-rich cocoa on vascular function in humans[END_REF]. However, EPI metabolites (EPIm) may be involved in the EPI-induced biological effects observed in in vivo studies. EPIm can reach higher plasma concentration than EPI [START_REF] Ottaviani | The metabolome of [2-14C](-)epicatechin in humans: Implications for the assessment of efficacy, safety and mechanisms of action of polyphenolic bioactives[END_REF]. Moreover, major differences in EPIm were observed across species. Indeed, 80% of EPIm present in humans were not detected in rats, while the similarity between mice and humans was found to be slightly more favourable as the two major human metabolites were detected [START_REF] Ottaviani | The metabolome of [2-14C](-)epicatechin in humans: Implications for the assessment of efficacy, safety and mechanisms of action of polyphenolic bioactives[END_REF]. We used enriched CF extract containing up to five times more EPI than unfermented cocoa beans [START_REF] Payne | Impact of Fermentation, Drying, Roasting, and Dutch Processing on Epicatechin and Catechin Content of Cacao Beans and Cocoa Ingredients[END_REF]. Moreover, EPI content was reduced by 10-20% by fermentation and decrease in a temperature-dependent manner during roasting [START_REF] Payne | Impact of Fermentation, Drying, Roasting, and Dutch Processing on Epicatechin and Catechin Content of Cacao Beans and Cocoa Ingredients[END_REF][START_REF] Kofink | Catechin in Cocoa and Chocolate: Occurence and Analysis of an Atypical Flavan-3-ol Enantiomer[END_REF]. Collectively, these data suggest that attention should only be paid on the EPI content when assessing the benefits of EPI supplementation on human health, and the present results should be considered with caution before extrapolating the observed underlying mechanisms in humans.

Conclusions

Daily CF supplementation for 15 days leads to mitochondrial function improvements in oxidative and glycolytic muscles. The present results suggest that the increased mitochondrial respiration results in an increase in mitochondrial mass while the cellular level of ROS production was maintained, suggesting qualitative adaptations within the mitochondria. Concomitant to the cellular effect on NO metabolism previously described [START_REF] Moreno-Ulloa | Effects of (-)-epicatechin and derivatives on nitric oxide mediated induction of mitochondrial proteins[END_REF], CF modulates NAD metabolism, which stimulates sirtuins activity. Sirt3 plays a central role in mitochondrial adaptations and metabolic flexibility following CF supplementation. As sirtuins are recent targets in various disease pathogenesis, such as diabetes or asthma [START_REF] Turkmen | Sirtuins as novel players in the pathogenesis of diabetes mellitus[END_REF][START_REF] Ma | Sirtuins as novel targets in the pathogenesis of airway inflammation in bronchial asthma[END_REF], further studies will explore the potential interest of using CF as a natural activator of sirtuins.

Figure 1 .

 1 Figure 1. Fifteen days of CF supplementation influences body composition and increases energy expenditure in 129S1/SvlmJ mice. (A) Body weight, lean mass, and fat mass. (B) Measurement of whole-body oxygen consumption during light and dark phases. (C) Daily energy expenditure. (D) Daily food intake. n = 10, means ± SD. * p < 0.05 vs. the control group.

Figure 2 .

 2 Figure 2. Fifteen days of CF supplementation modified whole-body metabolism in 129S1/SvlmJ mice. (A) Respiratory exchange ratio (RER) over the 24 h cycle. (B) Carbohydrate and fat oxidation in light and dark cycles. (C) Percent relative cumulative frequency (PCRF) of RER. (D) Fiftieth percentile values (EC50) of PCRF of RER (E) OGTT after an overnight fasting. (F) Area under the curve of the OGTT. n = 10-14, means ± SD. * p < 0.05 vs. the control group, ** p < 0.01 vs. the control group.

Figure 3 .

 3 Figure 3. Fifteen days of supplementation improved mitochondrial respiration and enzyme activities but did not modify mitochondrial sensitivity to Ca 2+ in 129S1/SvlmJ mice. (A) Respiration rates (JO 2 ) expressed per unit of dry weight from the white gastrocnemius muscle. (B) Enzyme activities of respiratory chain complexes and citrate synthase in the white gastrocnemius muscle. (C) Respiration rates (JO 2 ) using palmitoyl-carnitine as substrate in the white gastrocnemius and soleus muscles. (D) Effects on the time to PTP opening and the Ca 2+ retention capacity in the white gastrocnemius muscle. (E) Respiration rates (JO 2 ) expressed per unit of dry weight from the soleus muscle. n = 10, means ± SD. * p < 0.05 vs. the control group, ** p < 0.01 vs. the control group, *** p > 0.001 vs. the control group.

Figure 4 .

 4 Figure 4. Fifteen days of supplementation modified ROS metabolism in 129S1/SvlmJ mice. (A) Rates of H 2 O 2 production expressed per unit of dry weight from the white gastrocnemius muscle. (B) Rates of H 2 O 2 production expressed per unit of the marker enzyme citrate synthase (CS) from the white gastrocnemius muscle. (C) Rates of H 2 O 2 production expressed per unit of dry weight from soleus muscle. (D) Maximal rate of H 2 O 2 production expressed per Oxphos capacity from the white gastrocnemius and soleus muscle. (E) SOD2 content in the white gastrocnemius and soleus muscles, and representative examples of SOD2 and stain free total protein. (F) Total protein carbonylation content per total protein content and representative examples of protein carbonylation (detected as ubiquitin-conjugated proteins) in the white gastrocnemius and soleus muscles. n = 8-10, means ± SD. * p < 0.05 vs. the control group, ** p < 0.01 vs. the control group.

Figure 5 .

 5 Figure 5. Fifteen days of supplementation modified NAD + metabolism in the white gastrocnemius in 129S1/SvlmJ mice. (A) NAD + content. (B) NADH content. (C) NAD + /NADH ratio. n = 10, means ± SD. * p < 0.05 vs. the control group.

Figure 6 .

 6 Figure 6. Impact of 15 days of CF supplementation on whole-body and mitochondrial metabolism of Sirt3 -/-mice. (A) Body weight, lean mass, and fat mass. (B) Daily energy expenditure. (C) Respiratory exchange ratio (RER) over a 24 h cycle. (D) Carbohydrate and fat oxidation in light and dark cycles. (E) Enzyme activities of respiratory chain complexes and citrate synthase in the white gastrocnemius muscle. n = 10 in the control group and n = 9 in the Sirt3 -/-group, means ± SD, * p < 0.05, ** p < 0.01.

Table 1 .

 1 Composition of the cocoa powder.

	Compound	Content
	Total flavanols (%)	31.62
	(-)-epicatechin (%)	5.93
	(-)-catechin (%)	1.21
	Théobromin (%)	7.12
	Caffeine (%)	0.92

Table 2 .

 2 Primer sequences used in real-time PCR analysis.

	Target Gene	Official Full Name	GenBank Accession Number	Forward Primer (5 -3 ) Reverse Primer (3 -5 )
	PGC1α	Peroxisome proliferative activated receptor, gamma, coactivator 1 alpha	NM_008904.2	AAACTTGCTAGCGGTCCTCA TGGCTGGTGCCAGTAAGAG
	NRF1	Mus musculus nuclear respiratory factor 1	NM_001164226	GCACCTTTGGAGAATGTGGT GGGTCATTTTGTCCACAGAGA
	TFAM	Mus musculus transcription factor A, mitochondrial	NM_009360	CCTTCGATTTTCCACAGAACA GCTCACAGCTTCTTTGTATGCTT
	CS	Citrate synthase	NM_026444.4	GGAGCCAAGAACTCATCCTG TCTGGCCTGCTCCTTAGGTA
	ND1	NADH dehydrogenase subunit 1	NC_005089.1	ACACTTATTACAACCCAAGAACACAT TCATATTATGGCTATGGGTCAGG
	ND2	NADH dehydrogenase subunit 2	NC_005089.1	CCATCAACTCAATCTCACTTCTATG GAATCCTGTTAGTGGTGGAAGG
	SDHa	Succinate dehydrogenase complex, subunit A	NM_023281.1	GGAACACTCCAAAAACAGACCT CCACCACTGGGTATTGAGTAGAA
	Cox2	Cytochrome c oxidase subunit II	NC_005089.1	CATCTGAAGACGTCCTCCACTCAT TGCTTGATTTAGTCGGCCTGGGAT
	Cat	Catalase	NM_009804.2	TGAGAAGCCTAAGAACGCAATTC CCCTTCGCAGCCATGTG
	MnSOD	Superoxide dismutase [Mn]	NM_013671.3	TTAACGCGCAGATCATGCA GGTGGCGTTGAGATTGTTCA
	Sirt1	Sirtuin 1	NM_019812	AAAGGAATTGGTTCATTTATCAGAG TTGTGGTTTTTCTTCCACACA
	Sirt3	Sirtuin 3	NM_022433.2	AGGTGGAGGAAGCAGTGAGA GCTTGGGGTTGTGAAAGAAA
	NMNAT1	Nicotinamide nucleotide adenyltransferase 1	NM_133435.2	TGTGCCCAAGGTGAAATTGCT CCACGATTTGCGTGATGTCC
	GAPDH	glyceraldehyde-3-phosphate dehydrogenase	NM_008084.3	ACTCCACTCACGGCAAATTC GTTAGTGGGGTCTCGCTCCT

Nutrients 2021, 13, 3466. https://doi.org/10.3390/nu13103466 https://www.mdpi.com/journal/nutrients

Acknowledgments:

The authors are grateful for the technical expertise and assistance of technicians of the animal core facility of the university of Ottawa. The authors also thank Linde Homecare France for their kind donation which helped to pay for publication costs.

Data Availability Statement:

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Supplementary Materials:

The following are available online at https://www.mdpi.com/article/ 10.3390/nu13103466/s1, Figure S1 Informed Consent Statement: Not applicable.

Conflicts of Interest:

The authors have no conflict of interests that are relevant to the content of this article.


Dr David Goyard

Dr Benoît Roubinet

Federica Vena

Dr Ludovic Landemarre

Prof Olivier Renaudet

Homo-and Heterovalent Neoglycoproteins as Ligands for Bacterial Lectins

Keywords: bacterial lectin, click chemistry, glycodendrimer, multivalency, neoglycoprotein

) allows the interaction with both lectins (IC 50 of 10 nM) despite the presence of unspecific moieties, and even with similar efficiency for LecB. These results demonstrate the potential of multivalent and multispecific neoGPs as a promising strategy to fight against resistant pathogens.

Table of Contents

Extending molecular diversity of multivalent systems: the utilization of click chemistry allowed the preparation of homo-and heterovalent neoglycoproteins from a carrier protein and glycodendrons. Binding assays with LecA and LecB lectins from P. aeruginosa highlighted the impact of the multivalent presentation of glycodendrons by the carrier protein and demonstrated that the heterovalent display of glycans allows the interaction with both lectins.

Introduction

In the past decades, glycosylated compounds based on multivalent synthetic scaffolds revealed essential tools for studying carbohydrate-protein (lectin) interactions and modulating biological events. [1], [2], [3] To design high affinity molecules, computational methods and structural data of the target protein allow the choice of sugar units, valency and linking arm, however predicting the influence of the scaffold itself remains difficult.

Besides being easy to synthesize, it should ideally be chosen to be both soluble and stable in vivo, to bring the sugar units at the closest proximity of the protein binding pockets and to have low flexibility to limit entropic cost of the interaction. If simple scaffolds fulfil these criteria, the development of chemical ligation has significantly extended the geometrical limits of multivalent systems to optimize their properties. [4], [5], [6] By combining scaffolds having either identical or different characteristics, a diversity of glycosylated architectures are now accessible and some of them are currently investigated as pathogen inhibitors, immunomodulators, delivery or theranostic tools. [7], [8], [9], [10], [11] In the present study, we report the conjugation of glycodendrons to a carrier protein using a convergent approach based on the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), [12] thus providing a new series of neoglycoproteins (neoGP) with clusters of glycans distributed at different positions (Fig. 1). Given the dense and expanded expression of glycoconjugates at the surface of the cell membrane, we reasoned that the combination of synthetic glycodendrons within a protein carrier could easily provide a diversity of relevant neoglycoprotein-based reagents for studying and/or modulating carbohydrate-protein interactions. In particular, the utilization of glycans involved in bacterial infections might have the potential to inhibit the infection process and to provide alternative to antibiotic treatments against resistant pathogens. [13] To this aim, we used Bovine Serum Albumin (BSA), a naturally unglycosylated protein as the carrier model.

BSA was first functionalized with alkyne linkers, then tetravalent αand β-galactosylated (αGal and βGal) or α-fucosylated (αFuc) dendrons functionalized with an azido group were conjugated to provide homovalent neoGPs. The resulting structures may thus bind selectively to the LecA and LecB lectins of Pseudomonas aeruginosa. [14], [15] These two tetrameric calcium-dependent lectins are involved in the bacterial adhesion to the host cells, the biofilm formation and the lethal effect for immunocompromised patients, thus making them ideal biological targets for this study. In addition, recent studies have highlighted the importance of heterovalent structures as high affinity and selective ligands for carbohydrate-binding proteins, often leading to original properties. [16], [17], [18], [19], [20], [21], [22] To this aim and to extend the interest of the present approach, another neoGP combining randomized grafting of the αGal and αFuc dendrons was prepared to provide a heterovalent structure. The binding ability of these homo-and heterovalent neoGPs was next evaluated with these two bacterial lectins.

Results and discussion

Synthesis of neoGPs

We previously reported several series of fully synthetic multivalent structures decorated with βGal or αFuc. [23], [24] Binding studies revealed that their affinity with LecA and LecB was closely dependent on either the valency or the ligand geometry. For example, only fucosylated structures of higher valency (i.e. 16 copies of αFuc) were found as excellent ligands for LecB with a significant impact of the sugar display on the affinity. [23] By contrast, tetravalent galactosylated structures have shown nanomolar affinity for LecA, while the increase of valency to sixteen βGal had only a limited effect on the binding. [24] Here we first synthesized tetravalent dendrons displaying both glycans to be further conjugated to BSA as the carrier protein. A polylysine structure functionalized with azido groups (1) was synthesized by solid-phase peptide synthesis using the Fmoc/tBu strategy (Scheme 1). Propargylated αFuc and βGal [24] , [25] were next conjugated by CuAAC under standard condition using copper sulfate, tris(3-hydroxypropyl-triazolylmethyl)amine (THPTA) and sodium ascorbate in a mixture of DMF and PBS (pH 7.4). After 2 hours, semi-preparative RP-HPLC purification provided tetravalent compounds that were subsequently functionalized on the free side chain of the C-terminal lysine with azido N-hydroxysuccinimidyl ester of azidoacetic acid (Fig. S1-9).

The neoGPs were synthesized using a similar protocol. BSA was functionalized at the lysine side chain with a commercial propargyl linker by amide coupling (Scheme 2). After 5h in PBS, the crude mixture was purified by size-exclusion chromatography on Sephadex G25 and MALDI-TOF analysis of BSA-alkyne 3 indicated that among the 60 lysines present in BSA, approximately 33 were functionalized with an alkyne group (Fig. S10). Finally, glycodendrons 2a-c (25 equiv. over the 33 reactive groups present in 3) were conjugated to 3 by CuAAC, with tris(benzyltriazolylmethyl)amine (TBTA), L-sodium ascorbate and CuSO 4 in PBS similarly to previous reports. [26], [27], [28] After 24h at room temperature, the resulting homovalent neoGPs 4a-c were purified on Sephadex G25. The purity was confirmed by SDS-PAGE and MALDI-TOF experiments enabled to estimate that between 12 to 19 tetravalent glycodendrons were coupled to BSA, which corresponds to 49-75 glycans per BSA and molecular weights from 92 to 105 kDa for homovalent neoGPs 4a-c (Table 1, Fig. S11 andS14). Of note, when the coupling reaction was performed with an excess of the fucosylated glycodendron 2c over BSA-alkyne 3 (i.e. 50 equiv.), the quantity of αFuc only increased to 73 (versus 53 using 25 equiv.), thus suggesting that the remaining alkyne groups are less accessible in the protein to be fully functionalized under these experimental conditions (Fig. S12).

Table 1. MALDI-TOF analysis of the functionalization of BSA-alkyne 3 with glycodendrons 2a-c by CuAAC.

Compound MW calc. [Da] MW found [Da] nDendron/BSA nGlycan/BSA

neoGP 4a (αGal, 25 eq.) 114578 [a] 103371 19 [d] 75 [e] neoGP 4b (βGal, 25 eq.) 114578 [a] 91894 12 [d] 49 [e] neoGP 4c (αFuc, 25 eq.) 112978 [a] 93322 13 [d] 53 [e] neoGP 4c (αFuc, 50 eq.) 126664 [b] 101321 18 [d] 73 [e] neoGP 5(αFuc/αGal, 50/50) [f] 113778 [c] 103303 19 [d] 76 [e] The value presented in the table are rounded values; In addition, with respect to the role of LecA and LecB in the infectious and lethal processes mediated by P. aeruginosa, heteroclusters combining βGal and αFuc could simultaneously bind these two lectins to strengthen inhibitory effect as demonstrated in previous studies based on heterovalent peptide glycodendrimers, [29] pillar [5]arenecontaining [2]rotaxane [30] or hexadecavalent cyclopeptide-based glycodendrimers. [24] To this aim, we performed the CuAAC reaction between BSA-alkyne 3 and a mixture of 2a and 2c to afford the heterovalent neoGP 5 with randomized distribution of glycodendrons exposed by BSA (Scheme 3, Table 1, Fig. S13 and Fig. S15).

Scheme 3. Synthesis of heterovalent neoGP 5 with randomized distribution of glycodendrons. Reagents and conditions: i) 2a (12.5 eq.) and 2c (12.5 eq.), CuSO 4 •5H 2 O, TBTA, sodium ascorbate, PBS (pH 7.5), r.t., 24h.

Direct binding assays

The binding ability of the homovalent neoGPs 4a-c was studied with LecA and LecB using the GLYcoPROFILE ® technology (GLYcoDiag, France). [26], [27], [28], [31] NeoGPs 4a-c were first labelled with biotin using the protocol described previously then were incubated in microtitration plates coated with lectins. Streptavidin labelled with 5-(4,6-dichlorotriazinyl)aminofluorescein (DTAF) was finally incubated in each well and the interaction was directly detected by reading the fluorescence intensity (FI) provided by the neoGP bound to the lectin. While no interaction was observed with the unspecific Wheat Germ Agglutinin (WGA), this experiment indicated that the galactosylated 4a-b and fucosylated 4c neoGPs were able to bind LecA and LecB, showing a FI proportional to the neoGP concentration (Fig. S16-18). In addition, at lower concentration, we observed higher FI for 4a than for 4b

which could be attributed to the higher valency in 4a rather than a clear preference of LecA for the alpha anomer of galactose. Altogether, these results confirm the benefit of the multivalent presentation of glycans.

Finally, we evaluated the interaction of the heterovalent neoGP 5, which statistically displays identical content of αGal and αFuc units, with LecA and LecB (See Fig. S19-20).

As expected, the fluorescent signal observed with LecA is higher when BSA only displays αGal due to the higher valency (75 in 4a vs 38 in 5). Conversely, when the grafting ratio of αFuc increases (4c) compared to 5, the fluorescent signal observed with LecB is of higher intensity. More interestingly, while containing half less specific glycan for a given lectin, the heterovalent neoGP 5 shows slightly lower binding ability with LecA and LecB than the parent homovalent neoGPs 4a-c. This result suggests that the heterovalent display of glycans in BSA allows the simultaneous interaction with both lectins without significant loss of specificity and efficiency.

Inhibition assays

To confirm these observations, we next performed inhibition assays with LecA and LecB using biotinylated neoGPs as tracers (respectively neoGP 4a for LecA and neoGP 4c for LecB). [31], [26], [27], [28] The neoGPs 4 a-c and 5, the two commercial neoglycoproteins NeoGa and NeoF functionalized with monovalent αGal or αFuc (grafting ratio of 13 and 14 units, respectively), and the corresponding monosaccharides have been tested as inhibitors (Table 2).

Table 2. Inhibition of the interaction of biotinylated neoGPs to LecA and LecB-coated plates with homo-and heterovalent neoGPs 4a-c and 5.

Compound n [a] IC 50 [µM] rp [b] rp/n [c] Gal (LecA)

1 13.2 ± 1.2 1 1
NeoGa [d] In this assay, lectin is immobilized in microplate wells and simultaneously incubated with a solution containing the biotinylated tracer and various concentration of the inhibitors.

After a final incubation with streptavidin-DTAF, the intensity of fluorescence enables to determine the inhibition potency of the tested compounds (Fig. S21 ). This highlights that the inhibition potency is closely related to the number of αGal and more importantly, that the presence of fucose units does not interfere with the inhibition process.

With LecB, the neoF with 14 sugar units showed a 200-fold better inhibition over fucose (rp/n = 14), whereas neoGP 4c displaying 53 glycans showed a remarkable improvement factor of 4200 times over fucose to inhibit the interaction, which corresponds to a rp/n of 79. Additionally, with the heterovalent neoGP 5 displaying only 38 αFuc (combined with 38 αGal units), the rp value reached similar efficiency albeit with lower rp/n. This unexpected result suggests that the presence of αGal in proximity of αFuc in 5 could induce a favourable orientation of the fucose units to inhibit the interaction of the tracer with LecB. This observation demonstrates the importance of spatial presentation of glycans rather than the valency for inhibition as already observed in other studies. [3]

Conclusions

In this paper, we report the synthesis of multivalent neoglycoproteins by click chemistry. The functionalization of lysine residues of BSA with alkyne linkers provided approximately 33 anchoring groups to the protein that were subsequently conjugated with tetravalent glycodendrons presenting αGal, βGal and/or αFuc by CuAAC. Homo-and heterovalent neoGPs (4a-c and 5) functionalized with up to 19 randomly distributed dendrons were thus prepared and their binding ability were studied with the LecA and LecB lectins from Pseudomonas aeruginosa using the GLYcoPROFILE ® technology. In a direct assay, we first showed the potential of the homovalent neoGPs 4a-c for recognition with the specific lectin and we observed that the presence of randomized fucosylated and galactosylated glycodendrons in BSA ( 5) allows the interaction with both LecA and LecB without any loss of efficiency. Moreover, inhibition assays have highlighted that the presence of βGal (4b) ensures a stronger inhibition for LecA, whereas αGal (4a) at higher valency is required to promote similar effect. Unexpectedly, the inhibitory potency of the heterovalent neoGP 5 with LecB proved similar to the fucosylated neoGP (4c), thus suggesting that a more favorable orientation of the fucose units in 5. Overall the simplicity of the synthetic strategy and the promising biological potency of carrier protein grafted with glycodendrimers opens the route towards the development of new therapeutic agents against resistant pathogens. In particular, due to their structural feature, such neoGP could efficiently mimic the cell surface and prevent the infection of host cells from pathogens. A large diversity of neoGPs is currently developed to explore this perspective.

Experimental section

General methods

All chemical reagents were purchased from Aldrich (Saint Quentin Fallavier, France) or For the neoGPs, Sephadex-G25 was obtained from GE Healthcare. Chemicals and solvents were used as received from commercial sources without further purification.

Fluorescence was recorded with a Fluostar Optima spectrometer (BMG labtech, Offenburg, Germany). The MALDI-TOF analysis were carried out with an UltrafleXtreme from Bruker.

General procedure for the preparation of glycodendrons by CuAAC (2a-c)

Propargyl glycoside (4.4 eq.) and azide-functionalized scaffold 1 (1 eq.) were dissolved in 1 mL of a 1:1 mixture of DMF and PBS buffer (pH 7.5). A solution of CuSO 4 .5H 2 O (0.5 eq.)

and THPTA (1 eq.) in PBS was added to a solution of sodium ascorbate (3 eq.) in PBS.

This mixture was added to the solution containing the azide and alkyne which was degassed with argon and stirred at r.t. for 2 hours after which RP-HPLC showed completion of the reaction. Chelex ® resin was then added to the reaction mixture which was stirred for 45 minutes. The resin was filtered off, rinsed with water and the filtrate was concentrated under reduced pressure. The crude product was used directly in the next step. The resulting tetravalent compound 2 (1 eq.) was dissolved in dry DMF (1 mL), DIPEA was added to reach pH ~ 9-10 (c.a. 20 µL) then N-hydroxy-succinimidyl ester of azidoacetic acid (1.5 eq.) was added. The reaction mixture was stirred at r.

Functionalization of BSA with an alkyne chain (3)

A solution of 3-(2-Propyn-1-yloxy)propanoic acid 2,5-dioxo-1-pyrrolidinyl ester (2 mg, 60 eq.) was added in a solution of BSA (10 mg) in PBS and the reaction mixture was stirred for 5 h at room temperature. BSA-alkyne (3) was purified on a Sephadex G25 and its purity was controlled by SDS-PAGE (Fig. S14-15). 90% yield. The number of alkyne group per BSA was determined by MALDI-TOF (Fig. S10).

Conjugation of BSA-alkyne 3 with glycodendrons 2a-c by CuAAC

TBTA (5 mg/mL), L-ascorbic acid (3 mg/mL), CuSO 4 .5H 2 O (3 mg/mL) and glycodendron 2a, 2b or 2c (10 mg/mL, 25 eq. or 50 eq.) was added to a solution of BSA-alkyne 3 (2 mg/mL) in PBS. The solution was stirred 24 h at room temperature then the crude mixture was purified by size-exclusion chromatography on a Sephadex G25 gel. The purity of each neoglycoproteins was controlled by SDS-PAGE (Fig. S14-15) and the ratio of glycodendrimers by BSA was determined by MALDI-TOF (Fig. S11-13).

Biotinylated of NeoGPs

The labelling of NeoGPs was carried out by amide coupling with the EZ-link sulfo-NHS-LCbiotin (10 equivalents) in PBS at pH 7.2 for 30 min at room temperature. The excess of biotin reagent was removed by dialysis process. The number of biotin coupled on the neoglycoprotein is determined according to the standard HABA/avidine quantification method.

Direct binding assays

The assays were performed according GlycoDiag's protocol already described. [21,22,23,24] For direct binding mode, each neoglycoproteins were preliminary labeled with biotin according to standard protocol already described. [23] Briefly, the different compounds (range of concentrations) prepared in PBS supplemented with 1 mM CaCl 2 and 0.5 mM MgCl 2 were deposited in each well of lectin (50 μL each) in triplicate and incubated for two hours at room temperature. After washing with PBS buffer, the streptavidin-DTAF conjugate was added (50 μL) and incubated for 30 min. The plate was then washed again with PBS. Finally, 100 μL of PBS were added for reading the plate using a fluorescence reader (λ ex = 485 nm, λ em = 530 nm). The intensity of the signal was directly correlated with the ability of the compound to be recognized by the lectin.

Inhibition assays

The interaction profiles of each compound were determined through an indirect method based on the inhibition by the compound of the interaction between a specific couple lectin-glycan (a neoglycoprotein labeled with biotin and used as a tracer). Briefly, a mix of tracer (fixed concentration) and the corresponding compounds (range of concentrations) prepared in PBS supplemented with 1 mM CaCl 2 and 0.5 mM MgCl 2 is deposed in each well (50 µL each) in triplicates and incubated two hours at room temperature. After washing with PBS buffer, the conjugate streptavidin-DTAF was added (50 µL) and incubated 30 min more. The plate was washed again with PBS. Finally, 100 µL of PBS was added for the readout of fluorescent plate performed with a fluorescence reader (λ ex = 485 nm, λ em = 530 nm). The signal intensity was inversely correlated with the capacity of the compound to be recognized by the lectin and expressed as inhibition percentage with comparison with the corresponding tracer alone.

Figure 1 .

 1 Figure 1. Assembly of homo-and heterovalent neoGPs from BSA and glycodendrons

Scheme 1 .

 1 Scheme 1. Synthesis of the polylysine-based glycodendrons 2a-c. Reagents and

Scheme 2 .

 2 Scheme 2. Synthesis of homovalent neoGPs 4a-c. Reagents and conditions: i) 3-(2-

  [a] MW of neoGPs 4a-c was calculated on the basis of the number of eq. of glycodendrons 2a-c: MW calc. = MW of BSA-alkyne 3 + 25 x MW of 2a-c; [b] MW of neoGP4c was calculated on the basis of the number of eq. of glycodendron 2c, considering that BSA-alkyne 3 displays a maximum of 33 alkyne groups: MW calc. = MW of BSA-alkyne 3 + 33 × MW of 2c; [c] MW of neoGP5 was calculated on the basis of the number of eq. of mixed glycodendrons 2a-c: MW calc. = MW of BSA-alkyne 3 + 25 × (50% MW of 2a + 50% MW of 2c); [d] nDendron/BSA = (MW found for 4a-c -MW of BSA-alkyne 3) / MW of 2a-c; [e] n Glycan/BSA = n Dendron / BSA × 4; [f] The ratio of 50% of αGal and 50% of αFuc residues was arbitrarily set to determine the number of dendrons and glycans per BSA and nGlycan/BSA.

  Acros (Noisy-Le-Grand, France) and were used without further purification. Commercial neoglycoproteins NeoGa and NeoF (i.e. BSA functionalized with αGal and αFuc,

	respectively), LecA and LecB 96-well plates (LEctPROFILE plates) were obtained from
	GLYcoDiag (France). All protected amino acids and Fmoc-Gly-Sasrin ® resin was obtained
	from Advanced ChemTech Europe (Brussels, Belgium), Bachem Biochimie SARL
	(Voisins-Les-Bretonneux, France) and France Biochem S.A. (Meudon, France). The EZ-
	link sulfo-NHS-LC-biotin was purchased from ThermoFisher Scientific (Ref: 21335) and
	the number of conjugated biotin quantified by the HABA/avidine method (ThermoFisher
	Scientific_UserGuide: HABA). For glycopeptides, analytical RP-HPLC was performed on a
	Waters alliance 2695 separation module, equipped with a Waters 2489 UV/visible
	detector. Analyses were carried out at 1.23 mL min -1 (Waters XBridge Shield RP18 3.5
	µM, C 18 , 100 × 4.6 mm) with UV monitoring at 214 nm and 250 nm using a linear A-B
	gradient (buffer A: 0.09% CF 3 CO 2 H in water; buffer B: 0.09% CF 3 CO 2 H in 90%
	acetonitrile). Preparative HPLC was performed on Waters equipment consisting of a
	Waters 2545 controller and a Waters 2487 dual absorbance detector. Purifications were
	carried out at 22 mL min-1 (VP 250 × 21 mm nucleosil 100-7 C 18 ) with UV monitoring at
	214 nm and 250 nm using a linear A-B gradient. 1 H NMR spectra were recorded on
	Bruker Avance III 500 MHz spectrometers and chemical shifts (δ) were reported in parts
	per million (ppm). Spectra were referenced to the residual proton solvent peaks relative to
	the signal of D 2 O (4.79 ppm). All mass spectrometry characterizations were performed at
	Mass Spectrometry facility, PCN-ICMG, Grenoble. ESI high resolution mass spectra of
	glycopeptides were measured on a LTQ Orbitrap XL spectrometer from Thermo Scientific.

Acknowledgments

This work was supported by CNRS, Université Grenoble Alpes, ICMG FR 2607, the French ANR projects Glyco@Alps (ANR-15-IDEX-02), LectArray (ANR-19-CE18-0019-03),

Labex ARCANE and CBH-EUR-GS (ANR-17-EURE-0003). O.R. acknowledges the European Research Council Consolidator Grant "LEGO" (647938) for D.G and the Proof of Concept Grant "THERA-LEGO" (963862). The European Union's Horizon 2020 research and innovation program (Marie Sklodowska-Curie grant agreement No. 814029 ITN SynBIOcarb) is acknowledged for F.V.