Matthias Van Hul

Lucie Geurts

Hubert Plovier

Céline Druart

Amandine Everard

Marcus Ståhlman

Moez Rhimi

Kleopatra Chira

Pierre Louis Teissedre

Nathalie M Delzenne

Emmanuelle Maguin

Angèle Guilbot

Amandine Brochot

Philippe Gérard #

Fredrik Bäckhed #

Patrice D Cani
email: patrice.cani@uclouvain.be23

Prof D Patrice

Cani

Van Hul

Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with changes in gut microbiota and markers of gut barrier

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

With more than one third of the adult population affected worldwide, obesityassociated metabolic disorders have become a major global health challenge that extends well beyond the developed world (1). Obesity is caused by a disparity between energy intake and energy expenditure, although genetic and environmental factors also influence this balance and modify metabolism [START_REF] Bogardus | Missing heritability and GWAS utility[END_REF].

Obesity is associated with an excess of (white) adipose tissue mass, insulin resistance, liver fat accumulation, chronic pro-inflammatory state and major health issues that include a plethora of comorbidities, such as type 2 diabetes, cardiovascular diseases, hypertension, stroke, and certain types of cancer [START_REF] Pi-Sunyer | The medical risks of obesity[END_REF].

Weight-reducing programs that recommend switching to a healthier lifestyle with reduced caloric intake and increased physical activity, although efficient, are difficult to maintain in the long term and therefore often remain unsuccessful [START_REF] Christian | Interpreting weight losses from lifestyle modification trials: using categorical data[END_REF]. Unfortunately, available drugs are of limited efficacy and are associated with side effects [START_REF] Patel | Pharmacotherapy for the management of obesity[END_REF]. Plant extracts may represent an additional option in the support of weight management strategies [START_REF] Thaiss | Persistent microbiome alterations modulate the rate of postdieting weight regain[END_REF]. Among these, polyphenols, a large family of compounds identified in plants, have attracted great interest because of their beneficial health effects [START_REF] Del Rio | Dietary (poly)phenolics in human health: structures, bioavailability, and evidence of protective effects against chronic diseases[END_REF]. These effects are often attributed to their ability to relieve oxidative stress-induced tissue damage associated with chronic diseases via their antioxidant activity and free radical scavenging capacities. Interestingly, some polyphenols were shown to have anti-inflammatory and antimicrobial properties, thereby influencing the host gut microbiota, and eventually the inflammatory and metabolic status [START_REF] Chuang | Potential mechanisms by which polyphenol-rich grapes prevent obesity-mediated inflammation and metabolic diseases[END_REF][START_REF] Selma | Interaction between phenolics and gut microbiota: role in human health[END_REF][START_REF] Nohynek | Berry phenolics: antimicrobial properties and mechanisms of action against severe human pathogens[END_REF][START_REF] Puupponen-Pimia | Antimicrobial properties of phenolic compounds from berries[END_REF][START_REF] Anhe | A polyphenol-rich cranberry extract protects from diet-induced obesity, insulin resistance and intestinal inflammation in association with increased Akkermansia spp. population in the gut microbiota of mice[END_REF]. In addition, we and others have demonstrated that the gut microbiota Comment citer ce document : Van Hul, M., Geurts, Plovier, Druart, Everard, A., Ståhlman, M., Rhimi, M., Chira, K., Teissedre, P. L., Delzenne, Maguin, E., Guilbot, A., Brochot, A. (Co-dernier auteur), Gérard, P. (Co-dernier auteur), Bäckhed, F. (Co-dernier auteur), Cani, P. D. (Auteur de correspondance) (2018).

Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with 4 contributes to the development of the metabolic disorders associated with obesity by modulating appetite [START_REF] Cani | Inulin-type fructans modulate gastrointestinal peptides involved in appetite regulation (glucagon-like peptide-1 and ghrelin) in rats[END_REF][START_REF] Backhed | The gut microbiota as an environmental factor that regulates fat storage[END_REF], energy harvest and absorption (14-16), gut motility, intestinal barrier function, inflammation (17, 18), glucose and lipid metabolism, as well as hepatic and adipose tissue fat storage (19). It was also reported that diets containing high fat levels diminish intestinal microbial diversity, often at the expense of more beneficial bacteria (20).

The aim of the present study was to determine the effects of two extracts of plants, cinnamon and grape pomace, known to be rich in polyphenols, in a mouse model of diet-induced obesity. Anti-diabetic and anti-inflammatory properties of cinnamon were reported in experimental studies (21-26) and the beneficial effects of grapes and grape by-products were documented in different aspects of the metabolic syndrome including dyslipidaemia, diabetes, hypertension, and obesity (27-29). However, only few studies have explored the impact of dietary polyphenols on the gut microbiota and intestinal barrier functions (28, [START_REF] Baldwin | Table grape consumption reduces adiposity and markers of hepatic lipogenesis and alters gut microbiota in butter fat-fed mice[END_REF][START_REF] Roopchand | Dietary Polyphenols Promote Growth of the Gut Bacterium Akkermansia muciniphila and Attenuate High-Fat Diet-Induced Metabolic Syndrome[END_REF][START_REF] Wang | Dietary grape seed extract ameliorates symptoms of inflammatory bowel disease in IL10-deficient mice[END_REF], and many aspects of this interaction remain largely unknown. This assessment was therefore one of the main objectives of this study.

Materials and Methods

Extracts

The cinnamon bark extract (CBE), ChalCinn ® , (3inature, Saint-Bonnet-de-Rochefort, France) was extracted from Cinnamomum cassia, rich in polyphenol type-A polymers (oligomeric proanthocyanidins) (21,[START_REF] Anderson | Isolation and characterization of polyphenol type-A polymers from cinnamon with insulin-like biological activity[END_REF].

The grape (Vitis vinifera L.) pomace extract (GPE) was supplied by 3iNature and sourced from Alicante from red wine cultivar in the Rhône valley. Grape pomace is a wine by-product that is characterized by high contents of phenolic

Comment citer ce document : Van Hul, M., Geurts, Plovier, Druart, Everard, A., Ståhlman, M., Rhimi, M., Chira, K., Teissedre, P. L., Delzenne, Maguin, E., Guilbot, A., Brochot, A. (Co-dernier auteur), Gérard, P. (Co-dernier auteur), Bäckhed, F. (Co-dernier auteur), Cani, P. D. (Auteur de correspondance) (2018).

Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with 5 compounds due to an incomplete extraction during the winemaking process. It is a mixture of grape skins and seeds in near-equivalent amounts together with a small amount of stems (<6 %). The main polyphenols of grape pomace are anthocyanins, hydroxycinnamic acids, flavanols and flavanol glycosides [START_REF] Kammerer | Polyphenol screening of pomace from red and white grape varieties (Vitis vinifera L.) by HPLC-DAD-MS/MS[END_REF].

Extraction was performed with ethanol/water (30:70 v/v) at 85 °C and the extract was concentrated and vacuum-dried on non-extracted grape pomace.

The final product thus consisted of Alicante grape pomace enriched with its own polyphenols.

Reagents and Standards

Deionized water was purified with a Milli-Q water system (Millipore, Bedford, MA, USA). HPLC grade acetonitrile, ethyl acetate, chloroform, methanol, ethanol and acetone purchased from Scharlau (Sentmenat, Barcelona, Spain). The

Grape pomace extract analysis

Comment citer ce document : Van Hul, M., Geurts, Plovier, Druart, Everard, A., Ståhlman, M., Rhimi, M., Chira, K., Teissedre, P. L., Delzenne, Maguin, E., Guilbot, A., Brochot, A. (Co-dernier auteur), Gérard, P. (Co-dernier auteur), Bäckhed, F. (Co-dernier auteur), Cani, P. D. (Auteur de correspondance) (2018).

Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with 6

Tannins Extraction

GPE (1 gr of dry matter) was solubilized in water/ethanol (250 mL, 95:5, v/v) and partitioned three times with chloroform (250 mL) to remove lipophilic material. The aqueous phase was then extracted three times with ethyl acetate (250 mL) to obtain two distinctive fractions: a low molecular weight procyanidin fraction (monomeric/oligomeric tannins) in the organic phase and a high weight procyanidin fraction (polymeric tannins) in the aqueous phase. These two fractions were concentrated and lyophilized.

Anthocyanins Extraction

Anthocyanin extraction was adapted from the method of Sriram et al. (1999).

GPE (1 g) was extracted four times with acidified methanol (40 mL, 0.1% HCl 12N) successively for 4 h, 12 h, 4 h and 12 h. The centrifugal supernatants were combined and evaporated in vacuo at 30 °C to remove methanol; the residue was dissolved in water and lyophilized to obtain an anthocyanin-rich powder.

Total Phenolics and Tannins, Anthocyanins

Total polyphenol contents (TPC) were determined in GPE and cinnamon bark extract (CBE). Tannin and anthocyanin contents were determined only in GPE.

HPLC Analysis of Anthocyanins in GPE

GPE was dissolved in water/methanol solution (50:50, v/v) at a concentration of 10 mg/mL prior to UPLC-UV analyses using a Thermo-Accela HPLC system (Thermo-Fisher, San Jose, CA, USA) composed of a PDA detector, an autosampler and a quaternary 600 series pump system controlled by an Xcalibur data system.

Separation was performed on a C18 Kinetex column (100 mm × 2.1 mm, 1.7 µm).

The injected volume was 2 µL. The mobile phase pumped at 200 µL/min comprised a 20 min, 7%-26% gradient of acetonitrile in water with both solvents containing 5% formic acid. Eluting peaks were monitored at 520 nm.

Identification of mean peaks

Cinnamon bark extract analysis

Total proanthocyanidins

In CBE, total proanthocyanidin content (PAC) was determined in EP using the BL-DMAC method as previously described (Lee et al., 2009) and quantified as Atype proanthocyanidin equivalents.

Determination of essential oil in CBE

CBE was ground and submitted to steam distillation (20,0 g of dried material) for 8 h, using a Clevenger-type apparatus without hexane at a rate of 3-4 ml/min, according European Pharmacopoeia. The volatile distillates were analyzed by GC-MS. The ratio of CBE and water (acidified by 2 ml HCL 37% m/m) was 1:10.

The essential oils were stored in amber vials at 4°C until analysis. The yield oil were kept frozen at temperature -20°C up to their utilization. respectively. The essential oil is diluted in acetone: 1/100.

Components were identified by both GC retention times and by comparison of their mass with those present in the computer data bank and published spectra.

Quantification was performed by area percent, FID-response factor = 1.

Determination of coumarin content in CBE

About 3.0 g (accurately weighed to 0,0001 g) of powdered whole plant was extracted using 150 ml methanol R as solvent under reflux during 30 min. After filtration, the filtrate was adjusted to 250,0 ml with the same solvent. The sample were filtered using 0,45 µm membrane filters (Millipore). Quantification of the constituent coumarin was carried out from the calibration curves of the HPLC chromatograms using authentic compounds. Coumarin (> 98%, HPLC) purchased from Sigma (St. Louis, MO, USA) was used as an external standard.

Insulin resistance index

Plasma insulin concentration was determined using an ELISA kit (Mercodia, Uppsala, Sweden) according to the manufacturer's instructions. Insulin resistance index was determined by multiplying the area under the curve of both blood glucose (-30 to 120 min) and plasma insulin (-30 and 15 min) obtained following the oral glucose tolerance test [START_REF] Geurts | Adipose tissue NAPE-PLD controls fat mass development by altering the browning process and gut microbiota[END_REF]. Glucose-induced insulin secretion was calculated as the difference between plasma insulin levels 30 min before and 15 min after oral glucose load.

Tissue sampling

At the end of the treatment period (week 8), 9 animals from each group were anesthetized with isoflurane (Forene, Abbott, Queenborough, Kent, England) and blood was sampled from the portal and cava veins. After exsanguination, mice were killed by cervical dislocation. Subcutaneous adipose tissue depots, intestines, muscles and liver were precisely dissected, weighed and immediately immersed in liquid nitrogen followed by storage at -80°C for further analysis.

Comment citer ce document : Van Hul, M., Geurts, Plovier, Druart, Everard, A., Ståhlman, M., Rhimi, M., Chira, K., Teissedre, P. L., Delzenne, Maguin, E., Guilbot, A., Brochot, A. (Co-dernier auteur), Gérard, P. (Co-dernier auteur), Bäckhed, F. (Co-dernier auteur), Cani, P. D. (Auteur de correspondance) (2018).

Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with 13

Indirect calorimetry experiments

The remaining mice (n = 5 per group) were housed individually in specialized metabolic chambers (Phenomaster, TSE Systems GmbH, Bad Homburg, Germany) to measure whole energy expenditure, oxygen (O 2 ) consumption and carbon dioxide (CO 2 ) production, respiratory exchange ratio (RER, calculated as vCO 2 /vO 2 ), food intake and spontaneous locomotor activity [START_REF] Geurts | Adipose tissue NAPE-PLD controls fat mass development by altering the browning process and gut microbiota[END_REF][START_REF] Everard | Intestinal epithelial MyD88 is a sensor switching host metabolism towards obesity according to nutritional status[END_REF]. Activity was recorded using an infrared light beam-based locomotion monitoring system (expressed as counts per hour). Mice were allowed 48 h acclimatization before experimental measurements. After six days of measurements, mice were killed and tissues were sampled as described above.

Histological analyses

Subcutaneous adipose tissue depots were fixed in 4% paraformaldehyde for 24 hours at room temperature. Samples were then immersed in ethanol 100 % for 24 hours prior to processing for paraffin embedding.

To determine the adipocyte tissue diameter, paraffin sections of 8 µm were stained with hematoxylin and eosin.

Macrophage infiltration in the adipose tissue was assessed by staining them with a MAC-2/galectin-3 antibody (CL8942AP, Cedarlane Laboratories, Burlington, Ontario, Canada) diluted 1:500 in blocking buffer overnight, and was detected with an anti-rat igG antibody (AI-4001, Vector Laboratories, Inc., Burlingame, California, USA) (10 mg/ml). Immune complexes were detected by the Dako Envision kit (Agilent Technologies, Santa Clara, California, USA) according to the manufacturer's instructions, and briefly counterstained with haematoxylin.

Hepatic lipid content was visualized by using Oil red O staining.

Comment citer ce document : Van Hul, M., Geurts, Plovier, Druart, Everard, A., Ståhlman, M., Rhimi, M., Chira, K., Teissedre, P. L., Delzenne, Maguin, E., Guilbot, A., Brochot, A. (Co-dernier auteur), Gérard, P. (Co-dernier auteur), Bäckhed, F. (Co-dernier auteur), Cani, P. D. (Auteur de correspondance) (2018).

Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with 14

All analyses were performed in a blinded manner by the investigator and quantified using ImageJ software (Version 1.50a, National Institutes of Health, Bethesda, Maryland, USA). At least five high-magnification fields were selected at random for each mouse. Images were obtained using a SCN400 slide scanner and Digital Image Hub software (Leica Biosystems, Wetzlar, Germany).

RNA preparation and Real-time qPCR analysis

Total RNA was prepared from tissues using TriPure reagent (Roche).

Quantification and integrity analysis of total RNA were performed by analyzing 1 μl of each sample in an Agilent 2100 Bioanalyzer (Agilent RNA 6000 Nano Kit, Agilent, Santa Clara, California, USA). cDNA was prepared by reverse transcription of 1 μg total RNA using a Reverse Transcription System kit (Promega, Madison, Wisconsin, USA). Real-time PCR was performed with the CFX96 real-time PCR system and CFX Manager 3.1 software (Bio-Rad, Hercules, California, USA) using Mesa Fast qPCR (Eurogentec, Liège, Belgium) for detection according to the manufacturer's instructions. RPL19 was chosen as the housekeeping gene. All samples were performed in duplicate, and data were analyzed according to the 2 -ΔΔCT method. The identity and purity of the amplified product were assessed by melting curve analysis at the end of amplification. The primer sequences for the targeted mouse genes are presented in Table 1.

Biochemical analyses

Plasma adipokines (leptin, resistin) and inflammatory markers (IL1b, IFNg, MCP1, MIP1a, PAI1) were detected by using a Bio-Plex Milliplex kit (Millipore, Billerica, Massachusetts, USA) and their concentrations were measured by using Plasma non-esterified fatty acids, cholesterol and triglyceride concentrations were measured using kits coupling an enzymatic reaction with spectrophotometric detection of the reaction end-products (Diasys Diagnostic and Systems, Holzheim, Germany) according to the manufacturer's instructions.

Total lipids were measured in the liver tissue after extraction in CHCl3:MeOH according to Folch et al. [START_REF] Folch | A simple method for the isolation and purification of total lipides from animal tissues[END_REF] and adapted as follows: Briefly, 100 mg of liver tissue was homogenized in 2 ml of CHCl 3 :MeOH (2:1) using a Tissue Lyser followed by an ultrasonic homogenizer. 400 µl of 0.9% NaCl solution was added and lipids were then extracted by vigorous shaking. After centrifugation, the lipidic phase was recovered in glass tubes and dried under a stream of N 2 . Glass tubes were weighed before and after lipid extraction to quantify total lipid content. The dried residue was solubilized in 1.5 to 3 ml isopropanol depending on the lipid content.

Short chain fatty acid (SCFA) measurements

Short-chain fatty acids in cecal samples were analyzed by gas-liquid chromatography as described previously [START_REF] Lan | Increased induction of apoptosis by Propionibacterium freudenreichii TL133 in colonic mucosal crypts of human microbiota-associated rats treated with 1,2dimethylhydrazine[END_REF].

Bile Acids (BA) measurements

The extraction and analysis of bile acids were performed according to a previous work [START_REF] Tremaroli | Roux-en-Y Gastric Bypass and Vertical Banded Gastroplasty Induce Long-Term Changes on the Human Gut Microbiome Contributing to Fat Mass Regulation[END_REF]. Briefly, bile acids from portal vein plasma were extracted by protein precipitation with 10 volumes of IS-containing methanol. After the samples were vortexed and centrifuged, the supernatant was diluted 50 times in

Comment citer ce document : Van Hul, M., Geurts, Plovier, Druart, Everard, A., Ståhlman, M., Rhimi, M., Chira, K., Teissedre, P. L., Delzenne, Maguin, E., Guilbot, A., Brochot, A. (Co-dernier auteur), Gérard, P. (Co-dernier auteur), Bäckhed, F. (Co-dernier auteur), Cani, P. D. (Auteur de correspondance) (2018).

Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with 16 methanol:water (1:1). Bile acids from cecum (15-100 mg in 2 ml polypropylene tubes filled with ceramic beads) were extracted in 500µl IS-containing methanol using a TissueLyser II instrument (Retsch, Haan, Germany). The supernatant was diluted 50 times in methanol:water (1:1). Separation was performed using water and acetonitrile on a Kinetex C18 column (2.1 ×100 mm with 1.7 µm particles;

Phenomenex, Torrance, California, USA). Detection was performed using a QTRAP 5500 instrument (AB Sciex, Toronto, Canada) with MRM in negative mode.

Gut microbiota analysis

The V3-V4 region was amplified from purified DNA with the primers F343

(CTTTCCCTACACGACGCTCTTCCGATCTACGGRAGGCAGCAG) and R784

(GGAGTTCAGACGTGTGCTCTTCCGATCTTACCAGGGTATCTAATCCT) using 30 amplification cycles with an annealing temperature of 65°C. The amplicon lengths were about 510 bp (the exact length varies depending on the species).

Because MiSeq sequencing enables paired 250-bp reads, the ends of each read overlap and can be stitched together to generate extremely high-quality, fulllength reads covering the entire V3-V4 region. Single multiplexing was performed using a home-made 6 bp index, which was added to the R784 primer during a second PCR with 12 cycles using the forward primer Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with 17 further analysis, each pair-end sequence was assigned to its sample using the previously integrated index.

Bioinformatics analysis

Sequences were trimmed for adaptors and assembled with Flash1.6.2 [START_REF] Magoc | FLASH: fast length adjustment of short reads to improve genome assemblies[END_REF]. PCR primers were removed and sequences with sequencing errors in the primers were excluded (Mothur) [START_REF] Schloss | Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities[END_REF]. For each sample, 12000 reads were randomly selected for each sample. Chimera were removed with UCHIME [START_REF] Edgar | UCHIME improves sensitivity and speed of chimera detection[END_REF] and Mothur (42) softwares. Reads were clustered into Operational Taxonomic Units (OTUs)

at the 97% identity level using Esprit-tree [START_REF] Sun | A large-scale benchmark study of existing algorithms for taxonomy-independent microbial community analysis[END_REF]. A reference sequence was picked for each OTU and assigned it at different taxonomic levels (from phylum to species) using the Greengenes database (release 13-5) (45) and the RDP classifier [START_REF] Wang | Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy[END_REF].

Statistical analysis

Mouse data are expressed as the mean ± SEM. Differences between groups were assessed using non-parametric Kruskal-Wallis one-way analysis of variance Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with 18 OTUs assigned to those families. For each such family, Mann-Whitney test with 424 BH correction [START_REF] Hochberg | More powerful procedures for multiple significance testing[END_REF] were performed to detect the combinations (treatment) that 425 were significantly different in terms of abundance. The same procedure was 426 applied for each genus and for each OTUs. All analyses were done using R (R 427 Core Team, 2015, R Foundation for Statistical Computing, Vienna, Austria) 428 A two-way ANOVA analysis with a Bonferonni post-hoc test on repeated 429 measurements was performed for the evolution of glycaemia and insulinemia 430 during the OGTT. For all analyses and for each group, any exclusion decision was 431 supported by the use of the Grubbs test for outlier detection. 2).

The mean degree of polymerization of the proanthocyanidins in the GPE fraction was 4.2 ± 0.025 (data not shown).

CBE contained 79 mg/g of total phenolics as GAE. Proanthocyanidin A content was 90 mg/g. Coumarin and cinnamaldehyde represented 9 mg/g and 1.8 mg/g respectively.

Effects on body weight, body composition, adipose tissues and adipokines

Body weight gain and fat mass gain were both significantly greater in all high-fat diet treated groups (HFD, HFD-CBE, HFD-GPE) than in mice fed the control diet

Effects on energy homeostasis

The reduced fat mass gain observed in HFD-GPE mice could not be explained by any difference in energy intake (Fig 3A). On the contrary, there was a trend

Effects on nutrients absorption

To investigate the mechanism associated with reduced energy harvest, we measured different nutrient transporters in the proximal part of the intestines (jejunum) (Fig3K). Glucose transporters (Slc5a1/SGLT1, Slc2a2/GLUT2) were slightly lower in the high-fat fed group, whereas FABP1/LFABP and CD36, fatty acid binding proteins, are higher, confirming the switch from glucose consumption to lipid oxidation. Both CBE and GPE slightly increased SGLT1, but only GPE increased GLUT2 as well. LFABP was somewhat decreased by the GPE, as was CD36 by CBE. However, these changes were too subtle to achieve statistical significance.

Effects on whole-body and hepatic lipid metabolism

The management of dyslipidemia is a key element in the prevention of cardiovascular diseases in obese and diabetic patients. Therefore, we performed an analysis of circulating and liver lipids. Interestingly, circulating non-esterified fatty acids levels (NEFAs) were higher in the HFD group than in the control To evaluate systemic inflammation, we measured circulating inflammatory markers in plasma (Fig 5D). We did not find a marked HFD effect for any of the markers, indicating that although there is a tissue inflammatory tone, they have not yet reached systemic inflammation. However, the GPE tended to be systematically lower than the other groups, especially for IFNg (p = 0,02 versus HFD, Mann-Whitney test).

Effects on gut microbiota

We and others have previously linked the gut microbiota with low-grade inflammation and metabolic disorders associated with HFD feeding (17, 50-52).

The composition of the gut microbiota of mice that received HFD was significantly changed compared to those fed with CT diet, with an enrichment in

Effects on bile acids

Primary bile acids (BAs) are synthesized by the liver and may be converted into secondary BAs as a result of biotransformation by the intestinal microbiota [START_REF] Gerard | Metabolism of cholesterol and bile acids by the gut microbiota[END_REF].

They serve many important physiological functions, including glucose and lipid metabolism [START_REF] Sayin | Gut microbiota regulates bile acid metabolism by reducing the levels of tauro-beta-muricholic acid, a naturally occurring FXR antagonist[END_REF]. In portal vein plasma, total BA concentrations tended to be reduced in untreated HFD mice as compared to CT mice, but were higher in the HFD-CBE and HFD-GPE groups than in the HFD group, reaching levels comparable to that of the CT Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with 28

Discussion

Although polyphenols are not strictly required for vital body functions in humans, there is compelling clinical and epidemiological evidence that they significantly reduce the risk of chronic diseases and promote health [START_REF] Del Rio | Dietary (poly)phenolics in human health: structures, bioavailability, and evidence of protective effects against chronic diseases[END_REF]56,[START_REF] Zhang | Polyphenols in Health and Disease[END_REF].

However, a significant proportion of the population is not consuming sufficient quantities of dietary polyphenols as a result of inadequate vegetable and fruit intake. Therefore, concentrated polyphenol extracts might be valuable dietary supplements offering an interesting additional strategy for metabolic disorders management [START_REF] Anhe | A polyphenol-rich cranberry extract protects from diet-induced obesity, insulin resistance and intestinal inflammation in association with increased Akkermansia spp. population in the gut microbiota of mice[END_REF][START_REF] Anhe | Triggering Akkermansia with dietary polyphenols: A new weapon to combat the metabolic syndrome?[END_REF][START_REF] Anhe | Gut Microbiota Dysbiosis in Obesity-Linked Metabolic Diseases and Prebiotic Potential of Polyphenol-Rich Extracts[END_REF].

In this study, we demonstrated that both cinnamon bark and grape pomace extracts are able to ameliorate the overall metabolic profile in a model of dietinduced obesity. This is evidenced by a decrease in fat mass gain and adipose tissue inflammation and by reduced hepatic lipid content, especially in the grape pomace-treated mice, which was not compensated by elevated plasma lipid concentrations. Our data are consistent with previous reports that showed moderate but significant beneficial effect of table grape extracts on adiposity, hepatic steatosis, insulin resistance and adipose tissue inflammation (28, 30, 60-

63).

We also found a clear improvement of glucose homeostasis by both extracts, as evidenced by an improved glucose tolerance and lower insulin resistance index.

This was associated with a marked reduction of non-esterified fatty acids (NEFAs, free fatty acids), which have previously been found to be modulators of insulin sensitivity [START_REF] Kahn | Mechanisms linking obesity to insulin resistance and type 2 diabetes[END_REF]. Interestingly, although improvement of insulin resistance index was achieved by both grape and cinnamon extracts, the mechanisms A large fraction of dietary polyphenols reaches the colon and can be metabolized by the intestinal microbiota. Moreover, polyphenols are well known to affect intestinal bacteria [START_REF] Nohynek | Berry phenolics: antimicrobial properties and mechanisms of action against severe human pathogens[END_REF][START_REF] Puupponen-Pimia | Antimicrobial properties of phenolic compounds from berries[END_REF]. Here, we report a significant impact of our extracts on the microbial composition, which was more profound for the GPE than for the CBE. One of the genera significantly increased by the GPE is Roseburia. These are bacteria that were previously found to be at a low abundance in patients with type 2 diabetes and proposed to play an important role in gut health as they have anti-inflammatory effects in the gut [START_REF] Aminov | Molecular diversity, cultivation, and improved detection by fluorescent in situ hybridization of a dominant group of human gut bacteria related to Roseburia spp. or Eubacterium rectale[END_REF][START_REF] Karlsson | Gut metagenome in European women with normal, impaired and diabetic glucose control[END_REF][START_REF] Qin | Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with 41 composition of the gut microbiota shapes the colon mucus barrier[END_REF]. Interestingly, Roseburia are increased by prebiotics and associated with improvements in metabolic disorders (71, 72). We also found a higher abundance of Allobaculum Indeed, H 2 S has been shown to disrupt energy metabolism in the gut epithelium [START_REF] Babidge | Sulfides impair short chain fatty acid beta-oxidation at acyl-CoA dehydrogenase level in colonocytes: implications for ulcerative colitis[END_REF]. This leads to cell death and ultimately results in intestinal inflammation [START_REF] Hond | In vivo butyrate metabolism and colonic permeability in extensive ulcerative colitis[END_REF]. As for the HFD-CBE mice, we found similar trends for Roseburia, Desulfovibrio and Lactococcus genera as in the HFD-GPE mice, although they did not reach statistical significance. While the genus Peptococcus was not consistently increased by HFD, we observed that CBE strongly decreased its level.

Comment citer ce document : Van Hul, M., Geurts, Plovier, Druart, Everard, A., Ståhlman, M., Rhimi, M., Chira, K., Teissedre, P. L., Delzenne, Maguin, E., Guilbot, A., Brochot, A. (Co-dernier auteur), Gérard, P. (Co-dernier auteur), Bäckhed, F. (Co-dernier auteur), Cani, P. D. (Auteur de correspondance) (2018).

Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with

31

Previous studies have shown a strong association between the ingestion of polyphenol extracts and the species Akkermansia municiphila, a bacterium known to improve metabolic disorders [START_REF] Anhe | A polyphenol-rich cranberry extract protects from diet-induced obesity, insulin resistance and intestinal inflammation in association with increased Akkermansia spp. population in the gut microbiota of mice[END_REF][START_REF] Anhe | Triggering Akkermansia with dietary polyphenols: A new weapon to combat the metabolic syndrome?[END_REF]. However, none of the tested treatments were associated with a modulation of A. municiphila (data not shown). This was unexpected, as previous studies have shown an increase in abundance of A. muciniphila following polyphenol treatment (reviewed in ( 58)).

In contrast, a recent study using a grape seed extract showed no changes in A.

muciniphila [START_REF] Liu | Grape seed proanthocyanidin extract ameliorates inflammation and adiposity by modulating gut microbiota in high-fat diet mice[END_REF] and it was also reported that Resveratrol, a polyphenol mainly found in grapes, berries and a wide range of fruits, decreases A. muciniphila in mice [START_REF] Sung | Improved Glucose Homeostasis in Obese Mice Treated With Resveratrol Is Associated With Alterations in the Gut Microbiome[END_REF]. In vitro, a pomegranate extract significantly inhibited the growth of A.

muciniphila [START_REF] Henning | Pomegranate ellagitannins stimulate the growth of Akkermansia muciniphila in vivo[END_REF]. This may suggests that polyphenols have varied prebiotic potential on A. muciniphila. However, there are several design differences between the studies that may have contributed to the divergent results concerning the gut microbiota composition. First, depending on the origin of the polyphenols (for example the grape terroir) and the extraction procedure the composition of the final extracts may vary significantly. Secondly, it has been shown that gut microbiota composition is affected by diet (type and amount of fat/sugar in the diet), mouse strain and age, and mouse provider [START_REF] Laukens | Heterogeneity of the gut microbiome in mice: guidelines for optimizing experimental design[END_REF]. Thirdly, the increase of a certain bacterial species, such as A. muciniphila, may depend on its baseline intestinal abundance.

In accordance with our previous findings and the changes in the gut microbiota composition, the expression of several antimicrobial peptides, including Ang4 (effective against Gram-positive and Gram-negative bacteria), Reg3γ (effective against Gram-positive bacteria), and Lyz1 (mostly effective against Gram-positive bacteria) was found to be increased in response to both

Comment citer ce document : Van Hul, M., Geurts, Plovier, Druart, Everard, A., Ståhlman, M., Rhimi, M., Chira, K., Teissedre, P. L., Delzenne, Maguin, E., Guilbot, A., Brochot, A. (Co-dernier auteur), Gérard, P. (Co-dernier auteur), Bäckhed, F. (Co-dernier auteur), Cani, P. D. (Auteur de correspondance) (2018).

Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with 32 CBE and GPE supplementation [START_REF] Gallo | Epithelial antimicrobial defence of the skin and intestine[END_REF]. In the CBE-treated group, this was accompanied by a putative increase in intestinal mucosal turnover and barrier integrity in the colon, as evidenced by an increase of intectin and claudin3.

Gut microbiota may affect metabolic parameters by influencing the bile acid pool composition. Bile acids facilitate the digestion and absorption of lipids, but they also act as signaling molecules by binding to FXR, contributing to the regulation of various metabolic processes [START_REF] Wang | FXR: a metabolic regulator and cell protector[END_REF]. Bile acid content tended to be increased with CBE, solely due to an increase in conjugated bile acids, suggesting a decrease in bile salt hydrolase (BSH) activity within the microbial community.

This seems to be supported by the fact that we did not find any effect on the biological markers associated with the synthesis of bile acids in this group. In addition, evidence has revealed that bile acids are also able to alter the gut microbiota via direct and indirect antimicrobial effects [START_REF] Begley | The interaction between bacteria and bile[END_REF], and promote the survival of some bile acid-tolerant bacteria such as some Lactobacillus and Bifidobacterium species [START_REF] Devkota | Dietary-fat-induced taurocholic acid promotes pathobiont expansion and colitis in Il10-/-mice[END_REF]. In contrast, we found evidence for an increase bile acid production in GPE treated mice. However, this was not translated to higher bile acid contents in caecum of plasma.

Taken together, our data demonstrate that polyphenols derived from grapes or cinnamon can partially counter the deleterious effects of HFD and ameliorate overall metabolic parameters related to adiposity, glucose homeostasis and gut barrier integrity. These changes are associated with a modulation of the microbiota composition and a reduction in inflammation. Interestingly, all these beneficial effects resemble that of prebiotics, even though the doses used were much lower than those generally required for classical prebiotics [START_REF] Anhe | Gut Microbiota Dysbiosis in Obesity-Linked Metabolic Diseases and Prebiotic Potential of Polyphenol-Rich Extracts[END_REF][START_REF] Bindels | Towards a more comprehensive concept for prebiotics[END_REF].

Comment citer ce document : Van Hul, M., Geurts, Plovier, Druart, Everard, A., Ståhlman, M., Rhimi, M., Chira, K., Teissedre, P. L., Delzenne, Maguin, E., Guilbot, A., Brochot, A. (Co-dernier auteur), Gérard, P. (Co-dernier auteur), Bäckhed, F. (Co-dernier auteur), Cani, P. D. (Auteur de correspondance) (2018).

Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with

33

Modes of action of both compounds were found to be different, indicating that polyphenols have a broad range of targets that require further investigations.

Thus, although both studied extracts positively improve glucose and lipid metabolism and reinforce the gut barrier together with changes in the gut microbiota, it is currently unknown how this beneficial effects occur. The health benefits on the host may be mediated by the microbial production of bioactive polyphenol-derived metabolites and/or by the modulation of the gut microbial community itself. Phenolic analysis indicated that the most abundant anthocyanins found in our grape pomace extract were Malvidin-3-O-glucoside, and Peonidin-3-O-glucoside (Table 2). While the antibesity and antidiabetic Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with

  following chemicals were obtained from Sigma Aldrich (St. Louis, MO, USA): (+)catechin, ()-epicatechin, B1 [()-epicatechin-(4β-8)-(+)-catechin], procyanidin dimer B2 [()-epicatechin-(4β-8)-()-epicatechin], cyanidin-3-O-glucoside chloride, delphinidin-3-Oglucoside chloride, malvidin-3-O-glucoside chloride, peonidin-3-O-glucoside chloride, gallic acid. The Laboratory of Organic Chemistry and Organometallic (Université Bordeaux 1) synthesized procyanidins dimers B3 [(+)-catechin-(4α-8)-(+)-catechin], B4 [(+)-catechin-(4α-8)-()-epicatechin] and a trimer (C1) [(+)-catechin-(4β-8)-(+)catechin-(4β-8)-()-epicatechin] (Tarascou et al., 2006).

  Crude extracts were solubilized in water/ethanol (90:10, v/v; pH 3.5 with tartaric acid) at appropriate concentrations. TPC was determined by the Folin-Ciocalteu assay [Singleton et al., 1965] and the data expressed as mg of gallic acid equivalents (GAE) per g dry weight. Total tannin content was measured by acidic hydrolysis using the method of Ribereau-Gayon and Stonestreet [Ribéreau et al., Comment citer ce document : Van Hul, M., Geurts, Plovier, Druart, Everard, A., Ståhlman, M., Rhimi, M., Chira, K., Teissedre, P. L., Delzenne, Maguin, E., Guilbot, A., Brochot, A. (Co-dernier auteur), Gérard, P. (Co-dernier auteur), Bäckhed, F. (Co-dernier auteur), Cani, P. D. (Auteur de correspondance) (2018). Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with 7 1966]. Anthocyanin content was determined by the SO2 bleaching procedure [Ribéreau et al., 1965].

  was performed by comparison to external standards (Anthocyans monoglucosides (cyanidin-3-O-glucoside, delphinidin-3-O-glucoside, paeonidin-3-O-glucoside, malvidin-3-O-glucoside, petunidin-3-Oglucoside), acylated anthocyans (paeonidin-3-O-glucoside, malvidin-3-Oglucoside) and coumaroylated (paeonidin-3-O-glucoside, malvidin-3-Oglucoside.The data was expressed as Malvidine3-O-glucoside equivalent/g dry weight GPE. (Ky et al. ,2014). Determination of Individual Tannins in GPE by HPLC Analysis GPE extract was solubilized in a methanol/water solution (50:50, v/v) at appropriate concentrations and analyses of monomeric/oligomeric tannins (catechin, epicatechin, dimers B1, B2,B3,B4 ; Trimer C2) were carried out Comment citer ce document : Van Hul, M., Geurts, Plovier, Druart, Everard, A., Ståhlman, M., Rhimi, M., Chira, K., Teissedre, P. L., Delzenne, Maguin, E., Guilbot, A., Brochot, A. (Co-dernier auteur), Gérard, P. (Co-dernier auteur), Bäckhed, F. (Co-dernier auteur), Cani, P. D. (Auteur de correspondance) (2018). Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with 8 according to the method of Silva et al. [Silva et al, 2012]. The data was expressed as catechin equivalent/g dry weight GPE. Determination of Mean Degree of Polymerization (mDP) in GPE The proanthocyanidin mean degree of polymerization (mDP) was determined for GPE in monomeric/oligomeric and polymeric tannin fractions by the means of phloroglucinolysis [Drinkine et al., 2007]. Analyses were carried out using the same method as described by Lorrain et al. [Lorrain et al., 2011]. The oligomeric and polymeric proanthocyanidins were depolymerised in the presence of a nucleophilic agent phloroglucinol in an acidic medium. Reversedphase HPLC analysis of the products formed allows determination of the structural composition of proanthocyanidins, which are characterised by the nature of their constitutive extension units (released as flavan-3-ols phloroglucinol adducts) and terminal units (released as flavan-3-ols). To calculate the apparent mDP, the sum of all subunits (flavan-3-ol monomer and phloroglucinol adducts, in mols) was divided by the sum of all flavan-3-ol monomers (in mols). GPE sample was analysed with a Surveyor series instrument (Thermo-Finnigan, Les Ullis, France) equipped with a 100 x 4.6 mm i.d., 3.5 µm X-Terra reversed-phase C18 column (Waters) thermostated at 25 C. Detection was carried out at 280 nm using a Finnigan Surveyor PDA Plus detector. The mass detection was carried out using a Finnigan LCQ DECA XP MAX mass spectrometer with an ESI interface, performed in positive mode with the following parameters: capillary temperature 325 _C, capillary voltage 4 V, nebulizer gas flow 1.75 L/min, desolvation gas flow 1 L/min, and spray voltage 5 kV. The solvents used were solvent A: H2O/AcOH (99:1 v/v), and B: MeOH. The Comment citer ce document : Van Hul, M., Geurts, Plovier, Druart, Everard, A., Ståhlman, M., Rhimi, M., Chira, K., Teissedre, P. L., Delzenne, Maguin, E., Guilbot, A., Brochot, A. (Co-dernier auteur), Gérard, P. (Co-dernier auteur), Bäckhed, F. (Co-dernier auteur), Cani, P. D. (Auteur de correspondance) (2018). Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with 9 gradient consisted of 5% B during 25 min, linear gradient 5%-20% B in 20 min, then 20%-32% B in 15 min, finally 32%-100% B in 2 min. The column was washed with 100% B for 5 min and then stabilized with the initial conditions for 10 min. The injection volume was 20 µL. The flow rate was 1 mL/min.

  Comment citer ce document : Van Hul, M., Geurts, Plovier, Druart, Everard, A., Ståhlman, M., Rhimi, M., Chira, K., Teissedre, P. L., Delzenne, Maguin, E., Guilbot, A., Brochot, A. (Co-dernier auteur), Gérard, P. (Co-dernier auteur), Bäckhed, F. (Co-dernier auteur), Cani, P. D. (Auteur de correspondance) (2018). Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with 15 Luminex technology (Bio-Rad Bioplex; Bio-Rad) following the manufacturer's instructions.

  The resulting PCR products were purified and loaded onto the Illumina MiSeq cartridge according to the manufacturer instructions. The quality of the run was checked internally using PhiX, and for Comment citer ce document : Van Hul, M., Geurts, Plovier, Druart, Everard, A., Ståhlman, M., Rhimi, M., Chira, K., Teissedre, P. L., Delzenne, Maguin, E., Guilbot, A., Brochot, A. (Co-dernier auteur), Gérard, P. (Co-dernier auteur), Bäckhed, F. (Co-dernier auteur), Cani, P. D. (Auteur de correspondance) (2018).

(

  ANOVA), followed by the Dunn's multiple comparison test. Variance was compared using a Bartlett's test. If variances were significantly different between groups, values were normalized by Log-transformation before proceeding to the analysis. When only two groups were compared, a non-parametric Mann-Whitney test was used. Regimen and treatment effects on community compositions were assessed using permutational multivariate analysis of variances (PERMANOVA) after rarefaction of all communities to even sampling depths. The abundances of all families were computed by agglomerating the Comment citer ce document : Van Hul, M., Geurts, Plovier, Druart, Everard, A., Ståhlman, M., Rhimi, M., Chira, K., Teissedre, P. L., Delzenne, Maguin, E., Guilbot, A., Brochot, A. (Co-dernier auteur), Gérard, P. (Co-dernier auteur), Bäckhed, F. (Co-dernier auteur), Cani, P. D. (Auteur de correspondance) (2018).

(

  CT) (Fig 1A, B). Compared to the HFD-group, HFD-GPE (p = 0.03, 2-way repeated measurements ANOVA) and -CBE groups (trend, p = 0.1) had a lower fat mass gain during the last 4 week of follow-up (Fig 1B, E, F) but no significant difference in body weight and lean mass gain were observed in the extracttreated groups (Fig 1A-F). Comment citer ce document : Van Hul, M., Geurts, Plovier, Druart, Everard, A., Ståhlman, M., Rhimi, M., Chira, K., Teissedre, P. L., Delzenne, Maguin, E., Guilbot, A., Brochot, A. (Co-dernier auteur), Gérard, P. (Co-dernier auteur), Bäckhed, F. (Co-dernier auteur), Cani, P. D. (Auteur de correspondance) (2018). Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with 20 Brown and White adipose tissue depots were consistently smaller in the extracttreated groups compared to the HFD group, but there was no statistical difference (Fig 1G-I). These findings were mirrored by a trend in reduced leptin plasma levels in the HFD-GPE group (p = 0.08 versus HFD, Mann-Whitney test) (Fig 1J). The weights of liver, spleen and different muscles were not affected by the different diets (data not shown). Effects on glucose homeostasis Both CBE and GPE treatments significantly improved glucose tolerance, as evidenced by a lower blood glucose profile compared to the untreated HFD-fed group (Fig 2A). The effect was stronger for the HFD-CBE group as evidenced by the significantly reduced area under the curve (Fig 2B). Mice fed a HFD were hyperinsulinemic in the fasted state, as they exhibited more than two-fold higher levels of plasma insulin as compared to control mice (Fig 2C). Mice from the HFD-GPE group produced somewhat less insulin in response to oral glucose administration compared to the HFD and HFD-CBE mice without reaching statistical significance (Fig 2C) (p = 0.07 versus HFD, Mann-Whitney test). This result was corroborated by a decreasing trend for glucose-induced insulin secretion compared to the HFD and HFD-CBE groups (p = 0.08 versus HFD, non-parametric Mann-Whitney test)(Fig 2D) and a significant improvement of the insulin resistance index (Fig 2E) in the HFD-GPE treated group. This was in accordance with a smaller adipocyte size (Fig 2F-G) and a trend in lower circulating resistin levels (Fig 2H), factors that have previously been associated with insulin resistance (37). For the HFD-CBE group no difference in glucose-Comment citer ce document : Van Hul, M., Geurts, Plovier, Druart, Everard, A., Ståhlman, M., Rhimi, M., Chira, K., Teissedre, P. L., Delzenne, Maguin, E., Guilbot, A., Brochot, A. (Co-dernier auteur), Gérard, P. (Co-dernier auteur), Bäckhed, F. (Co-dernier auteur), Cani, P. D. (Auteur de correspondance) (2018). Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with 21 induced insulin secretion could be observed but there was a trend towards an improvement of the insulin resistance index compared to the HFD group (p = 0.06 versus HFD, Mann-Whitney test)(Fig 2E).

  towards a higher mean calorie intake in HFD-GPE mice (p = 0.08 versus HFD, Mann-Whitney test). HFD-GPE mice also had a significantly increased amount of feces excreted compared to the other HFD groups (Fig3B). In addition, bomb calorimetric analysis of the different groups revealed a higher energy content in the fecal material of mice supplemented with cinnamon or grape extract (Fig 3C), resulting in higher daily energy excretion in both groups, as compared to HFD (Fig3D). There was no difference when expressing this as percentage of the food intake (Fig3E).The basal energy expenditure, which can also affect energy balance, was calculated by measuring the O 2 consumption and the CO 2 production for each mouse (not shown). After correction for the individual lean masses, the analysis revealed a mean increase in energy expenditure for the HFD, HFD-CBE and HFD-GPE groups, as compared to the CT group, without any effect of dietary supplementation (Fig3F-G). In addition, body temperatures were not different between all four groups (Fig3H). The respiratory exchange rate (RER) showed a clear metabolic shift from carbohydrate to lipid oxidation in the three HFD treated groups as compared to CT mice (Fig 3I). Comment citer ce document : Van Hul, M., Geurts, Plovier, Druart, Everard, A., Ståhlman, M., Rhimi, M., Chira, K., Teissedre, P. L., Delzenne, Maguin, E., Guilbot, A., Brochot, A. (Co-dernier auteur), Gérard, P. (Co-dernier auteur), Bäckhed, F. (Co-dernier auteur), Cani, P. D. (Auteur de correspondance) (2018). Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with 22 Spontaneous physical activity (monitored by continuously counting the number of times a mouse crossed the different light beams in the metabolic cages) was increased in HFD-GPE mice in comparison with HFD mice (p = 0.02 versus HFD, 2-way repeated measurement ANOVA) (Fig 3J), whereas this effect was less pronounced in HFD-CBE mice (p = 0.18).

  group and were normalized in the CBE and GPE treated groups although without reaching significance (p = 0.06 and 0,08 versus HFD respectively, Mann-Whitney Comment citer ce document : Van Hul, M., Geurts, Plovier, Druart, Everard, A., Ståhlman, M., Rhimi, M., Chira, K., Teissedre, P. L., Delzenne, Maguin, E., Guilbot, A., Brochot, A. (Co-dernier auteur), Gérard, P. (Co-dernier auteur), Bäckhed, F. (Co-dernier auteur), Cani, P. D. (Auteur de correspondance) (2018). Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with 23 test). Cholesterol plasma levels were increased in all HFD-treated groups, whereas circulating triglycerides were similar between groups (Fig 4A). In the liver, HFD increased the total lipid content by about 40%. Interestingly, treatment with GPE completely blunted this effect (Fig 4B). This was reflected by a significant normalization of liver triglyceride levels and a similar trend for cholesterol. In the HFD-CBE group, a trend for normalization was also observed for total lipid content and triglycerides, while cholesterol levels remained unaffected (p = 0.06 versus CT, p = 0.6 versus HFD, Mann-Whitney test). This finding was confirmed by the histological analysis that revealed significantly increased hepatic lipid depots in HFD mice, and smaller lipid droplets in the HFD-CBE and HFD-GPE mice (Fig 4C). Effects on adipose tissue and systemic inflammation Diabetes and insulin resistance being frequently associated with adipose tissue inflammation (17, 48, 49), we measured various macrophage infiltration markers in the subcutaneous (SAT) and visceral adipose tissue (VAT) using qPCR analysis (Fig 5A and 5B, respectively). Integrin alpha X (ITGAX/CD11c), lipopolysaccharide binding protein (LBP) and monocyte chemoattractant protein-1 (MCP1) were upregulated by HFD and were reduced by GPE in both adipose tissues. CBE supplementation decreased CD11c and LBP, but not MCP1. Two other macrophage markers, F4/80 and CD68, were not differently expressed in any group (Fig 5A). Histological analysis of the SAT stained with MAC2/Galectin-3, a marker of activated macrophages, showed a 2.5-fold higher number of macrophages in the HFD mice than in CT mice (Fig 5B), whereas this accumulation of macrophages Comment citer ce document : Van Hul, M., Geurts, Plovier, Druart, Everard, A., Ståhlman, M., Rhimi, M., Chira, K., Teissedre, P. L., Delzenne, Maguin, E., Guilbot, A., Brochot, A. (Co-dernier auteur), Gérard, P. (Co-dernier auteur), Bäckhed, F. (Co-dernier auteur), Cani, P. D. (Auteur de correspondance) (2018). Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with 24 was markedly decreased in the HFD-CBE and HFD-GPE groups compared to the HFD group, although without reaching significance (Fig 5B).

Firmicutes and a decrease

  in Bacteroidetes (Fig 6A). At the phylum level, no clear differences were observed in HFD-CBE mice when compared to HFD. GPE treatment, however, increased the abundance of Bacteroidetes at the expense of the Proteobacteria (Fig 6A). As observed in the principal coordinates analysis (PCoA), HFD feeding caused a shift in microbiota composition along the axis 1, explaining more that 57% of the difference observed (Fig 6C). Conversely, most mice from the HFD-CBE and HFD-GPE groups were separated from the untreated HFD-fed mice according to the axis 2. At the operational taxonomic units (OTUs) level, this shift was modest in the HFD-CBE group but more profound in the HFD-GPE group (Fig 6B). More specifically, the abundance of 11 OTUs was significantly different in HFD-CBE Comment citer ce document : Van Hul, M., Geurts, Plovier, Druart, Everard, A., Ståhlman, M., Rhimi, M., Chira, K., Teissedre, P. L., Delzenne, Maguin, E., Guilbot, A., Brochot, A. (Co-dernier auteur), Gérard, P. (Co-dernier auteur), Bäckhed, F. (Co-dernier auteur), Cani, P. D. (Auteur de correspondance) (2018). Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with 25 mice compared with HFD mice. In HFD-GPE mice, 53 OTUs were significantly modified (Fig 6B). Interestingly, the gut microbiota from extract-treated mice differed from that of the HFD mice but also from that of the CT mice, suggesting that polyphenols may have specific effects on the gut microbiota (Fig 6D). At the family level, CBE supplementation significantly reduced the levels of Peptococcaceae (classified within the Firmicutes phylum) when compared to the HFD mice (Fig 6D, E). Supplementation with GPE reduced the levels of Desulfovibrionaceae and Streptococcaceae, while increasing the levels of Prevotellaceae and Erysipelotrichaceae (Fig 6D, E). At the genus level, Peptococcus were decreased in the CBE group (Fig 6F). In the GPE treated group, we observed a decrease of Desulfovibrio, Clostridium sensu stricto and Lactococcus, whereas Allobaculum and Roseburia were increased (Fig 6F). Effects on intestinal barrier HFD feeding and concomitant changes in the gut microbiota are linked to alterations in the intestinal gut barrier function and in the production of antimicrobial peptides. Here we found that HFD lowered the gene expression of the antimicrobial peptide Reg3γ all along the intestinal tract (Fig 7A and data not shown for jejunum and ileum) and lowered the expression of intectin, encoding a protein involved in the turnover of intestinal mucosa, in the colon (Fig 7B) and jejunum (data not shown). In the colon, CBE tended to increase levels of intectin (Fig 7B) (p = 0.008 versus HFD, Mann-Whitney test), of the antimicrobial peptides Lyz1 (p = 0.03) (Fig 7F) and of the tight-junction protein claudin3 (p = 0.03) (Fig 7E) compared to the Comment citer ce document : Van Hul, M., Geurts, Plovier, Druart, Everard, A., Ståhlman, M., Rhimi, M., Chira, K., Teissedre, P. L., Delzenne, Maguin, E., Guilbot, A., Brochot, A. (Co-dernier auteur), Gérard, P. (Co-dernier auteur), Bäckhed, F. (Co-dernier auteur), Cani, P. D. (Auteur de correspondance) (2018). Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with 26 HFD group. Levels of the microbicidal protein Ang4 were higher in the HFD-CBE group as compared to CT mice (Fig 7G). HFD-GPE treatment normalized the levels of Reg3γ (p = 0.02 versus HFD, Mann-Whitney test) (Fig 7A) in the colon and significantly increased the levels of Lyz1 (Fig 7F). The tight junction protein Occludin was somewhat higher in the treated groups when compared to CT and HFD mice, but this did not reach significance (Fig 7D). ZO-1 remained unaffected along the gastro-intestinal tract for all the HFD-treated groups (Fig 7C and data not shown).

  HFD increased total BAs concentration in cecal content (p = 0.03 versus CT, Mann-Whitney test) and supplementation with CBE further increased cecal content in BAs compared to the HFD group (Fig 8A), although this did not reach statistical significance. Interestingly, this effect was due solely to an increase in conjugated BAs (Fig 8B), since unconjugated BAs levels did not differ between HFD groups (Fig 8C). At the level of individual BAs, we could not pinpoint one specific BA responsible for this increase; it was rather an accumulation of small changes throughout the BA spectrum that contributed to this overall increase (Fig 8D).

  Comment citer ce document : Van Hul, M., Geurts, Plovier, Druart, Everard, A., Ståhlman, M., Rhimi, M., Chira, K., Teissedre, P. L., Delzenne, Maguin, E., Guilbot, A.,Brochot, A. (Co-dernier auteur), Gérard, P. (Co-dernier auteur), Bäckhed, F. (Co-dernier auteur), Cani, P. D. (Auteur de correspondance) (2018). Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with 27 group (Fig 8E). The percentage of conjugated BA tended to be increased in the HFD-GPE (p = 0.04 versus HFD, Mann-Whitney test)(Fig 8F), whereas unconjugated BAs tended to be decreased (p = 0.04) (Fig 8G). Similar to the cecum, no specific BA changed in concentration in the plasma (Fig 8H). Bile acids are synthesized via the classical pathway under control of cholesterol 7 alpha-hydroxylase (CYP7a1) and cholesterol 8 alpha-hydroxylase (CYP8a1), or via alternate pathways, such as the one under control of cholesterol 27hydroxylase (CYP27a1) and cholesterol 7 beta-hydroxylase (CYP7b1). To determine whether our extracts could affect bile acid production, we measured the mRNA levels of the main factors controlling these pathways in the liver (Fig 9 A-F) and ileum (Fig 9 G-H). We found a clear upregulation of CYP7a1 (Fig 9A) and a modest increase of CYP27a1 (Fig 9D) for the HFD-GPE mice, suggesting an increase in bile acid production in this group. In the liver, bile acids can activate FXR, which has been shown to activate the expression of FGF15 in the intestine (55). FGF15 functions as a metabolic hormone, but also signals through FGFR4 in hepatocytes to inhibit expression of CYP7a1 gene, thereby acting as a negative feedback loop. Interestingly FGF15 was upregulated in ileaum of the HFD and HFD-GPE groups, but not in the HFD-CBE group (Fig 9H). Suggesting an enlarged bile acid pool in these mice. Why this is not reflected in the bile acid content remains to be determined. Comment citer ce document : Van Hul, M., Geurts, Plovier, Druart, Everard, A., Ståhlman, M., Rhimi, M., Chira, K., Teissedre, P. L., Delzenne, Maguin, E., Guilbot, A., Brochot, A. (Co-dernier auteur), Gérard, P. (Co-dernier auteur), Bäckhed, F. (Co-dernier auteur), Cani, P. D. (Auteur de correspondance) (2018).

  Comment citer ce document : Van Hul, M., Geurts, Plovier, Druart, Everard, A., Ståhlman, M., Rhimi, M., Chira, K., Teissedre, P. L., Delzenne, Maguin, E., Guilbot, A., Brochot, A. (Co-dernier auteur), Gérard, P. (Co-dernier auteur), Bäckhed, F. (Co-dernier auteur), Cani, P. D. (Auteur de correspondance) (2018).Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with 29 behind this seem to be different. Indeed, the HFD-GPE mice needed less insulin to achieve the same overall glucose profile, while the HFD-CBE mice had similar insulin secretion as HFD treated mice, but achieved faster glucose uptake. It has been proposed that cinnamon facilitates glucose entrance into cells by inducing glucose transporter 4 (GLUT4) translocation to the plasma membrane mediated by the LKB1-AMPK signalling pathway[START_REF] Absalan | Hydro-Alcoholic Cinnamon Extract, Enhances Glucose Transporter Isotype-4 Translocation from Intracellular Compartments into the Cytoplasmic Membrane of C2C12 Myotubes[END_REF][START_REF] Shen | Cinnamon extract enhances glucose uptake in 3T3-L1 adipocytes and C2C12 myocytes by inducing LKB1-AMP-activated protein kinase signaling[END_REF], whereas grapes might activate the PI3K pathway and promote insulin action by reducing serine kinase activation and cytokine signaling[START_REF] Yogalakshmi | Grape seed proanthocyanidins and metformin act by different mechanisms to promote insulin signaling in rats fed high calorie diet[END_REF]. Our data thus suggest that both extracts might be useful additives in the management of glucose homeostasis in diabetic patients, as has been proposed previously (21, 23-25, 27).

(

  Erysipelotrichaceae). This genus has also been shown to be increased by prebiotics (73, 74) and grape extracts (28), and has been associated with improved intestinal integrity, increased Reg3γ levels in the colon and with resistance to NAFLD development[START_REF] Roy | Intestinal microbiota determines development of non-alcoholic fatty liver disease in mice[END_REF]. Moreover, Metformin and Berberine, two clinically effective drugs for the treatment of diabetes, are associated with Comment citer ce document : Van Hul, M., Geurts, Plovier, Druart, Everard, A., Ståhlman, M., Rhimi, M., Chira, K., Teissedre, P. L., Delzenne, Maguin, E., Guilbot, A., Brochot, A. (Co-dernier auteur), Gérard, P. (Co-dernier auteur), Bäckhed, F. (Co-dernier auteur), Cani, P. D. (Auteur de correspondance) (2018). Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with 30 increases in Allobaculum abundance (75). As for Roseburia, the major end product of Allobaculum fermentation is butyrate. This SCFA is of particular relevance in the gut because it is rapidly taken up by enterocytes where it serves as energy source (76). Conflicting data exist about the modulation of SCFA by polyphenols. Some studies reported an increase in SCFA after supplementation of the diet with extracts or phenolic compounds, whereas other studies showed no differences (77), but cecal SCFA content was not affected by treatments in our study (Fig 10A, B, D). GPE contains about 420 mg/g of fibers, which could be insufficient to induce a significant change in the microbial fermentation to markedly affect SCFA production. Alternatively, utilization of short-chain fatty acids by the colonocytes may be more important in this group. This is supported by the drastic increase of SLC5a8, a butyrate transporter, in the colon (Fig 10C). As previously described, HFD feeding increased the abundance of Desulfovibrionaceae (73, 78) and Lactococcus (79). This was completely reversed with GPE. Several genera belonging to the Desulfovibrionaceae family are considered opportunistic pathogens and have been linked to some inflammatory diseases (80, 81). They produce endotoxins and have the capacity to reduce sulphate to H 2 S (82), thereby damaging the intestinal barrier (83).

42 Figure Legends Figure 1 .

 42Legends1 Figure Legends

Figure 2 .

 2 Figure 1. Effects on body composition and adipose tissue. 8 week follow up of (A) body weight (g) with corresponding (B) fat mass (g) and (C) lean mass (g) measured by TD-NMR. (D) Body weight gain (g), (E) fat mass (g), (F) fat mass and lean mass (% of total body weight) at the end of the follow up. (G) Weight of brown adipose tissue (mg). (H) Weights of subcutaneous, epididymal and visceral adipose tissues (mg) and corresponding (I) adiposity index (%white adipose tissue/body weight). (J) Leptin plasma levels (ng/ml). Data are presented as the mean±s.e.m. '*' '**' and ' # ' indicate a significant difference versus HFD (P<0.05, P<0.01, P<0.001 respectively) as determined by a two-way ANOVA (A-C). Data with different superscript letters are significantly different (P<0.05) according to post-hoc one-way ANOVA (D-J).Figure 2. Effects on Glucose homeostasis. (A) Plasma glucose (mg/dl) profile and (B) the mean area under the curve (AUC) measured between 0 and 120 min after glucose loading (mg/dl/min). (C) Plasma insulin levels at 30 min before and 15 min after glucose loading (µg/l). (D) Glucose-induced insulin secretion, calculated as the difference between the fasting insulinemia and the insulinemia 15 min after an oral glucose load. (E) Insulin resistance index determined by multiplying the AUC of blood glucose by the AUC of insulin between 30 min before and 15 min after glucose loading. (F) Adipocyte distribution and frequency with respect to the mean diameter measured by histological analysis. (G) Mean adipocyte size (µm). (H) Resistin plasma levels measured in the vena cava (ng/ml). Data are presented as the mean±s.e.m. '*' '**' and ' # ' indicate a significant difference versus HFD (P<0.05, P<0.01, P<0.001 respectively) as determined by a two-way ANOVA (A-B). Data with different superscript letters are significantly different (P<0.05) according to post-hoc one-way ANOVA (B-E).Figure 3. Effects on energy metabolism. Energy intake: (A) mean food intake per week per mouse (kcal). Energy excretion: (B) mean amount of feces excreted per mouse in 24h (mg/24h), (C) mean energy content in feces (kcal/g) and (D) daily energy excretion as calculated using the previous values (kcal/mouse) and (E) as percentage of the food intake. (F) Energy expenditure per night per mouse (kcal/h/kg). (G) Daily energy expenditure (kCal/h/kg), (H) body temperature (°C) and (I) respiratory exchange ratio (RER). (J) Cumulative mean number of beam breaks recorded per mouse during six days. (J) qPCR analysis of glucose transporters SGLT1 and GLUT2, and of fatty acid transporters LFABP and CD36. Data are presented as the mean±s.e.m. Data with different superscript letters are significantly different (P<0.05) according to post-hoc one-way ANOVA (A-E). '*' and '**' indicate a

Figure 3 .

 3 Figure 1. Effects on body composition and adipose tissue. 8 week follow up of (A) body weight (g) with corresponding (B) fat mass (g) and (C) lean mass (g) measured by TD-NMR. (D) Body weight gain (g), (E) fat mass (g), (F) fat mass and lean mass (% of total body weight) at the end of the follow up. (G) Weight of brown adipose tissue (mg). (H) Weights of subcutaneous, epididymal and visceral adipose tissues (mg) and corresponding (I) adiposity index (%white adipose tissue/body weight). (J) Leptin plasma levels (ng/ml). Data are presented as the mean±s.e.m. '*' '**' and ' # ' indicate a significant difference versus HFD (P<0.05, P<0.01, P<0.001 respectively) as determined by a two-way ANOVA (A-C). Data with different superscript letters are significantly different (P<0.05) according to post-hoc one-way ANOVA (D-J).Figure 2. Effects on Glucose homeostasis. (A) Plasma glucose (mg/dl) profile and (B) the mean area under the curve (AUC) measured between 0 and 120 min after glucose loading (mg/dl/min). (C) Plasma insulin levels at 30 min before and 15 min after glucose loading (µg/l). (D) Glucose-induced insulin secretion, calculated as the difference between the fasting insulinemia and the insulinemia 15 min after an oral glucose load. (E) Insulin resistance index determined by multiplying the AUC of blood glucose by the AUC of insulin between 30 min before and 15 min after glucose loading. (F) Adipocyte distribution and frequency with respect to the mean diameter measured by histological analysis. (G) Mean adipocyte size (µm). (H) Resistin plasma levels measured in the vena cava (ng/ml). Data are presented as the mean±s.e.m. '*' '**' and ' # ' indicate a significant difference versus HFD (P<0.05, P<0.01, P<0.001 respectively) as determined by a two-way ANOVA (A-B). Data with different superscript letters are significantly different (P<0.05) according to post-hoc one-way ANOVA (B-E).Figure 3. Effects on energy metabolism. Energy intake: (A) mean food intake per week per mouse (kcal). Energy excretion: (B) mean amount of feces excreted per mouse in 24h (mg/24h), (C) mean energy content in feces (kcal/g) and (D) daily energy excretion as calculated using the previous values (kcal/mouse) and (E) as percentage of the food intake. (F) Energy expenditure per night per mouse (kcal/h/kg). (G) Daily energy expenditure (kCal/h/kg), (H) body temperature (°C) and (I) respiratory exchange ratio (RER). (J) Cumulative mean number of beam breaks recorded per mouse during six days. (J) qPCR analysis of glucose transporters SGLT1 and GLUT2, and of fatty acid transporters LFABP and CD36. Data are presented as the mean±s.e.m. Data with different superscript letters are significantly different (P<0.05) according to post-hoc one-way ANOVA (A-E). '*' and '**' indicate a

  Comment citer ce document : Van Hul, M., Geurts, Plovier, Druart, Everard, A., Ståhlman, M., Rhimi, M., Chira, K., Teissedre, P. L., Delzenne, Maguin, E., Guilbot, A., Brochot, A. (Co-dernier auteur), Gérard, P. (Co-dernier auteur), Bäckhed, F. (Co-dernier auteur), Cani, P. D. (Auteur de correspondance) (2018). Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with Comment citer ce document : Van Hul, M., Geurts, Plovier, Druart, Everard, A., Ståhlman, M., Rhimi, M., Chira, K., Teissedre, P. L., Delzenne, Maguin, E., Guilbot, A., Brochot, A. (Co-dernier auteur), Gérard, P. (Co-dernier auteur), Bäckhed, F. (Co-dernier auteur), Cani, P. D. (Auteur de correspondance) (2018). Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with Comment citer ce document : Van Hul, M., Geurts, Plovier, Druart, Everard, A., Ståhlman, M., Rhimi, M., Chira, K., Teissedre, P. L., Delzenne, Maguin, E., Guilbot, A., Brochot, A. (Co-dernier auteur), Gérard, P. (Co-dernier auteur), Bäckhed, F. (Co-dernier auteur), Cani, P. D. (Auteur de correspondance) (2018). Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with Comment citer ce document : Van Hul, M., Geurts, Plovier, Druart, Everard, A., Ståhlman, M., Rhimi, M., Chira, K., Teissedre, P. L., Delzenne, Maguin, E., Guilbot, A., Brochot, A. (Co-dernier auteur), Gérard, P. (Co-dernier auteur), Bäckhed, F. (Co-dernier auteur), Cani, P. D. (Auteur de correspondance) (2018). Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with Comment citer ce document : Van Hul, M., Geurts, Plovier, Druart, Everard, A., Ståhlman, M., Rhimi, M., Chira, K., Teissedre, P. L., Delzenne, Maguin, E., Guilbot, A., Brochot, A. (Co-dernier auteur), Gérard, P. (Co-dernier auteur), Bäckhed, F. (Co-dernier auteur), Cani, P. D. (Auteur de correspondance) (2018). Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with Comment citer ce document : Van Hul, M., Geurts, Plovier, Druart, Everard, A., Ståhlman, M., Rhimi, M., Chira, K., Teissedre, P. L., Delzenne, Maguin, E., Guilbot, A., Brochot, A. (Co-dernier auteur), Gérard, P. (Co-dernier auteur), Bäckhed, F. (Co-dernier auteur), Cani, P. D. (Auteur de correspondance) (2018). Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with Comment citer ce document : Van Hul, M., Geurts, Plovier, Druart, Everard, A., Ståhlman, M., Rhimi, M., Chira, K., Teissedre, P. L., Delzenne, Maguin, E., Guilbot, A., Brochot, A. (Co-dernier auteur), Gérard, P. (Co-dernier auteur), Bäckhed, F. (Co-dernier auteur), Cani, P. D. (Auteur de correspondance) (2018). Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with Comment citer ce document : Van Hul, M., Geurts, Plovier, Druart, Everard, A., Ståhlman, M., Rhimi, M., Chira, K., Teissedre, P. L., Delzenne, Maguin, E., Guilbot, A., Brochot, A. (Co-dernier auteur), Gérard, P. (Co-dernier auteur), Bäckhed, F. (Co-dernier auteur), Cani, P. D. (Auteur de correspondance) (2018). Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with Comment citer ce document : Van Hul, M., Geurts, Plovier, Druart, Everard, A., Ståhlman, M., Rhimi, M., Chira, K., Teissedre, P. L., Delzenne, Maguin, E., Guilbot, A., Brochot, A. (Co-dernier auteur), Gérard, P. (Co-dernier auteur), Bäckhed, F. (Co-dernier auteur), Cani, P. D. (Auteur de correspondance) (2018). Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with Comment citer ce document : Van Hul, M., Geurts, Plovier, Druart, Everard, A., Ståhlman, M., Rhimi, M., Chira, K., Teissedre, P. L., Delzenne, Maguin, E., Guilbot, A., Brochot, A. (Co-dernier auteur), Gérard, P. (Co-dernier auteur), Bäckhed, F. (Co-dernier auteur), Cani, P. D. (Auteur de correspondance) (2018). Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with Comment citer ce document : Van Hul, M., Geurts, Plovier, Druart, Everard, A., Ståhlman, M., Rhimi, M., Chira, K., Teissedre, P. L., Delzenne, Maguin, E., Guilbot, A., Brochot, A. (Co-dernier auteur), Gérard, P. (Co-dernier auteur), Bäckhed, F. (Co-dernier auteur), Cani, P. D. (Auteur de correspondance) (2018).Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with

12 Oral glucose tolerance test (OGTT)

	(22:78 V/V). Coumarin content was quantified at 275 nm against the respective
	external standard. After 7 weeks of treatment, an oral glucose tolerance test (OGTT) was performed
	as previously described (35). Briefly, 6h-fasted mice were given an oral glucose
	Mice load (2 g glucose per kg body weight) and blood glucose levels were measured at
	Nine-week-old male C57BL/6J mice (Janvier, Le Genest-Saint-Isle, France) were different time points: 30 min before and 15, 30, 60, 90 and 120 min after oral
	housed in pairs in specific pathogen free conditions and in controlled glucose load. Blood glucose was measured with a standard glucose meter (Accu
	environment (room temperature of 23  2 °C, 12 h daylight cycle) with free Check, Roche, Basel, Switzerland) on blood samples collected from the tip of the
	access to food and water. After an acclimatization period of one week, mice were tail vein.
	randomly assigned to one of four dietary conditions (n = 14 per group). The
	different diets were as follow: control diet (CT) (10 kcal% fat, D12450Ji,
	Research Diet, New Brunswick, NJ, USA), high-fat diet (HFD) (60 kcal% fat,
	D12492i Research Diet), high-fat diet supplemented with 2 g cinnamon bark
	extract/kg (HFD-CBE) or a high-fat diet supplemented with 8.2 g grape pomace
	extract/kg (HFD-GPE) during 8 weeks. Body weight, food and water intake were
	recorded weekly. Body composition (lean and fat mass) was assessed by using
	7.5MHz time domain-nuclear magnetic resonance (TD-NMR) (LF50 Minispec,
	Bruker, Rheinstetten, Germany). In the final week of the experiment, feces were
	collected for each mouse and energy content was measured by calorimetric
	The coumarin content was determined according to reported methods [1] by an bomb analysis (Mouse Clinical Institute, Illkirch, France).
	HPLC apparatus (Hitachi) consisting of a quaternary pump, a autosampler, and a All mouse experiments were approved by and performed in accordance with the
	Photodiode Array Detector (PDA). Separation was carried with a C18 column guidelines of the local ethics committee. Housing conditions were specified by
	(4.6 mm x 250 mm) Purospher Star VWR. The column temperature was the Belgian Law of May 29, 2013, regarding the protection of laboratory animals
	maintained at 25°C and the mobile phase flow rate at 1.5 ml/min. The mobile (agreement number LA1230314).
	phase consisted of HPLC grade solvent Acetonitrile R and 5 g/L Phosphoric acid
	Comment citer ce document : Comment citer ce document :	11
	Van Hul, M., Geurts, Plovier, Druart, Everard, A., Ståhlman, M., Rhimi, M., Chira, K., Van Hul, M., Geurts, Plovier, Druart, Everard, A., Ståhlman, M., Rhimi, M., Chira, K.,
	Teissedre, P. L., Delzenne, Maguin, E., Guilbot, A., Brochot, A. (Co-dernier auteur), Gérard, P. Teissedre, P. L., Delzenne, Maguin, E., Guilbot, A., Brochot, A. (Co-dernier auteur), Gérard, P.
	(Co-dernier auteur), Bäckhed, F. (Co-dernier auteur), Cani, P. D. (Auteur de correspondance) (2018). (Co-dernier auteur), Bäckhed, F. (Co-dernier auteur), Cani, P. D. (Auteur de correspondance) (2018).
	Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with

Polyphenol content and profile of grape and cinnamon extracts

	GPE contained 82.663 ± 2.534 mg/g total phenolics as gallic acid equivalents
	(GAE). Total anthocyanin and tanin contents of the GPE fraction were 43.969 ±
	3.497 (as cyanidin-3-O-glucoside equivalent) and 26.006 ± 1.066 (as procyanidin
	B2 equivalent), respectively, (Table 2). Individual anthocyanin analysis revealed
	that the most abundant anthocyanins present in GPE were malvidin-3-O-
	glucoside (21.594 ± 0.213 mg/g), peonidin-3-O-glucoside (8.687 ± 0.258 mg/g)

Comment citer ce document : Van Hul, M., Geurts, Plovier, Druart, Everard, A., Ståhlman, M., Rhimi, M., Chira, K., Teissedre, P. L., Delzenne, Maguin, E., Guilbot, A., Brochot, A. (Co-dernier auteur), Gérard, P. (Co-dernier auteur), Bäckhed, F. (Co-dernier auteur), Cani, P. D. (Auteur de correspondance) (2018). Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with 19 Results and malvidin-3-O-(6''-p-coumaryl-glucoside) (4.624 ± 0.012 mg/g) (Table

Table 2

 2 Concentrations of the main components of grape pomace extract and cinnamon extract.

	43

Figure 7. Effects on intestinal barrier. (A-J) qPCR analysis of various markers of the intestinal barrier integrity and anti-microbial peptides in the colon (fold change versus CT group). Data are Comment citer ce document : Van Hul, M., Geurts, Plovier, Druart, Everard, A., Ståhlman, M., Rhimi, M., Chira, K., Teissedre, P. L., Delzenne, Maguin, E., Guilbot, A., Brochot, A. (Co-dernier auteur), Gérard, P. (Co-dernier auteur), Bäckhed, F. (Co-dernier auteur), Cani, P. D. (Auteur de correspondance) (2018).

Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with

  Comment citer ce document : Van Hul, M., Geurts, Plovier, Druart, Everard, A., Ståhlman, M., Rhimi, M., Chira, K., Teissedre, P. L., Delzenne, Maguin, E., Guilbot, A., Brochot, A. (Co-dernier auteur), Gérard, P. (Co-dernier auteur), Bäckhed, F. (Co-dernier auteur), Cani, P. D. (Auteur de correspondance) (2018).Reduced obesity, diabetes and steatosis upon cinnamon and grape pomace are associated with

	Primers	Forward Sequence	Reverse Sequence
	Rpl19	GAAGGTCAAAGGGAATGTGTTCA	CCTGTTGCTCACTTGT
	Cd11c	ACGTCAGTACAAGGAGATGTTGGA	ATCCTATTGCAGAATGCTTCTTTACC
	Mcp1	GCAGTTAACGCCCCACTCA	CCCAGCCTACTCATTGGGATCA
	Lbp	GTCCTGGGAATCTGTCCTTG	CCGGTAACCTTGCTGTTGTT
	Cd68	CTTCCCACAGGCAGCACAG	AATGATGAGAGGCAGCAAGAGG
	F4/80	TGACAACCAGACGGCTTGTG	GCAGGCGAGGAAAAGATAGTGT
	Reg3g	TTCCTGTCCTCCATGATCAAA	CATCCACCTCTGTTGGGTTC
	Intectin	GTTGCCCCTGATTCTGCTGG	GCACTATTGCAGAGGTCC-GT
	ZO-1	TTTTTGACAGGGGGAGTGG	TGCTGCAGAGGTCAAAGTTCAAG
	Occludin	ATGTCCGGCCGATGCTCTC	TTTGGCTGCTCTTGGGTCTGTAT
	Claudin3	TCATCGGCAGCAGCATCATCAC	ACGATGGTGATCTTGGCCTTGG
	Lyz1	GCCAAGGTCTACAATCGTTGTGAGTTG	CAGTCAGCCAGCTTGACACCACG
	Ang 4	CTCTGGCTCAGAATGTAAGGTACGA	GAAATCTTTAAAGGCTCGGTACCC
	Muc2	ATGCCCACCTCCTCAAAGAC	GTAGTTTCCGTTGGAACAGTGAA
	Klf4	AGAGGAGCCCAAGCCAAAGAGG	CCACAGCCGTCCCAGTCACAGT
	Proglucagon	TGGCAGCACGCCCTTC	GCGCTTCTGTCTGGGA
	CYP7a1	GGGATTGCTGTGGTAGTGAGC	GGTATGGAATCAACCCGTTGTC
	CYP7b1	TAGGCATGACGATCCTGAAA	TCTCTGGTGAAGTGGACTGAAA
	CYP8a1	GATCCGTCGCGGAGATAAGG	CGGGTTGAGGAACCGATCAT
	CYP27a1	TCTGGCTACCTGCACTTCCT	GTGTGTTGGATGTCGTGTCC
	FXR	TGGGTACCAGGGAGAGACTG	GTGAGCGCGTTGTAGTGGTA
	FGFR4	CTCGATCCGCTTTGGGAATTC	CAGGTCTGCCAAATCCTTGTC
	FGF15	GAGGACCAAAACGAACGAAATT	ACGTCCTTGATGGCAATCG
	SGLT1	TCTGTAGTGGCAAGGGGAAG	ACAGGGCTTCTGTGTCTTGG
	GLUT2	CTGGGTCTGCAATTTTGTCA	TGTAAACAGGGTGAAGACCA
	LFABP	ACCTCATCCAGAAAGGGAAGG	ACAATGTCGCCCAATGTCATG
	CD36	GCCAAGCTATTGCGACATGA	ATCTCAATGTCCGAGACTTTTCAAC
	SLC5a8	GCATATTCGGCATGGTTGGT	GGGCTCCAATTCCTACCCAT

Table 2 .

 2 Concentrations of the main components of grape pomace extract and cinnamon extract. Total phenolics, mg/g as gallic acid equivalent (Folin Ciocaleau assay)(2) Total anthocyanins, mg/g as cyanidin-3-O-glucoside equivalent (3) Total tanins, mg/g as procyanidin B2 equivalent (Bate-Smith assay)

	Main tanin concentrations in GPE (mg/g of the extract)
		mg/g of the extract	Daily dose (ng/d)
	Catechin	1.732  0.089	48,40 ± 3,23
	Procyanidin B1 dimer	0.100 ± 0.011	2,79 ± 0,18
	Procyanidin B2 dimer	0.311 ± 0.032	8,69 ± 0,58
	Procyanidin B3 dimer	0.350 ± 0.022	9,78 ± 0,65
	Procyanidin B4 dimer	0.090 ± 0.001	2,51 ± 0,16
	Procyanidin C2 trimer	0.071 ± 0.001	1,98 ± 0,13
	Epicatechin	1.011 ± 0.045	28,25 ± 1,88
	Total tanins	3.66 ± 0.625	102,27 ± 6,83
	Main anthocyanins concentrations in GPE
		mg/g of the extract	Daily dose (ng/d)
	Delphinidin-3-O-glucoside	1.635 ± 0.016	45,69 ± 3,05
	Cyanidin-3-O-glucoside	0.513 ± 0.007	14,33 ± 0,95
	Petunidin-3-O-glucoside	2.744 ± 0.027	76,67 ± 5,12
	Peonidin-3-O-glucoside	8.687 ± 0.258	242,74 ± 16,21
	Malvidin-3-O-glucoside	21.594 ± 0.213	603,39 ± 40,3
	Peonidin-3-O-(6" acetyl-glucoside)	0.546 ± 0.003	15,26 ± 1,02
	Malvidin-3-O-(6" acetyl-glucoside)	1.476 ± 0.026	41,24 ± 2,75
	Peonidin-3-O-(6''-p-coumaryl-glucoside)	1.596 ± 0.018	44,6 ± 2,98
	Malvidin-3-O-(6''-p-coumaryl-glucoside)	4.624 ± 0.012	129,21 ± 8,63
	Total anthocyanins	43.416 ± 6.793	1213,15 ± 81,03
	Phenolic composition of GPE
		mg/g of the extract	Daily dose (ng/d)
	Total phenolics (1)	82.663 ± 2.534	2309,81 ± 154,28
	Total anthocyanins (2)	43.969 ± 3.497	1228,6 ± 82,06
	Total tanins (3)	26.006 ± 1.066	726,67 ± 48,54
	(1) Main components in CBE
		mg/g of the extract	Daily dose (ng/d)
	Total polyphenols	79	1174,45 ± 40,46
	Proanthocyanidin A	90	1337,99 ± 46,09
	Coumarin	9	133,8 ± 4,609
	Cinnamaldehyde	1,8	26,76 ± 0,92
	Eucalyptol	not detected

Acknowledgments

We would like to thank A. Barrois, H. Danthinne, T. Pringels, M. Monnoye, C. Philippe and S. Boudebbouze for excellent technical assistance. We thank R-M. Goebbels for the processing of the samples prior to histological analysis. We thank E. Bourny from the Laboratoire Provençal des Plantes Aromatiques (LPPAM), Buis les Baronnies, France, for the analysis of cinnamon extract.

Conflict of interests

PileJe (Saint-Laurent-des-Autels, France) provided funding for this study to PDC, PG and EM. AB, AG are employed by PiLeJe. There are no patents or products in development to declare.

Comment citer ce document : Van Hul, M., Geurts, Plovier, Druart, Everard, A., Ståhlman, M., Rhimi, M., Chira, K., Teissedre, P. L., Delzenne, Maguin, E., Guilbot, A., Brochot, A. (Co-dernier auteur), Gérard, P. (Co-dernier auteur), Bäckhed, F. (Co-dernier auteur), Cani