Prebiotic effects of wheat arabinoxylan related to the increase in bifidobacteria, Roseburia and Bacteroides/Prevotella in diet-induced obese mice.

Abstract

Alterations in the composition of gut microbiota--known as dysbiosis--has been proposed to contribute to the development of obesity, thereby supporting the potential interest of nutrients targeting the gut with beneficial effect for host adiposity. We test the ability of a specific concentrate of water-extractable high molecular weight arabinoxylans (AX) from wheat to modulate both the gut microbiota and lipid metabolism in high-fat (HF) diet-induced obese mice.
Mice were fed either a control diet (CT) or a HF diet, or a HF diet supplemented with AX (10% w/w) during 4 weeks. AX supplementation restored the number of bacteria that were decreased upon HF feeding, i.e. Bacteroides-Prevotella spp. and Roseburia spp. Importantly, AX treatment markedly increased caecal bifidobacteria content, in particular Bifidobacterium animalis lactis. This effect was accompanied by improvement of gut barrier function and by a lower circulating inflammatory marker. Interestingly, rumenic acid (C18:2 c9,t11) was increased in white adipose tissue due to AX treatment, suggesting the influence of gut bacterial metabolism on host tissue. In parallel, AX treatment decreased adipocyte size and HF diet-induced expression of genes mediating differentiation, fatty acid uptake, fatty acid oxidation and inflammation, and decreased a key lipogenic enzyme activity in the subcutaneous adipose tissue. Furthermore, AX treatment significantly decreased HF-induced adiposity, body weight gain, serum and hepatic cholesterol accumulation and insulin resistance. Correlation analysis reveals that Roseburia spp. and Bacteroides/Prevotella levels inversely correlate with these host metabolic parameters.
Supplementation of a concentrate of water-extractable high molecular weight AX in the diet counteracted HF-induced gut dysbiosis together with an improvement of obesity and lipid-lowering effects. We postulate that hypocholesterolemic, anti-inflammatory and anti-obesity effects are related to changes in gut microbiota. These data support a role for wheat AX as interesting nutrients with prebiotic properties related to obesity prevention.

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Prebiotic Effects of Wheat Arabinoxylan Related to the
Increase in Bifidobacteria, Roseburia and Bacteroides/
Prevotella in Diet-Induced Obese Mice
Audrey M. Neyrinck1, Sam Possemiers2, Ce ´line Druart1, Tom Van de Wiele2, Fabienne De Backer1,
Patrice D. Cani1, Yvan Larondelle3, Nathalie M. Delzenne1*
1Metabolism and Nutrition Research Group, Louvain Drug Research Institute, Universite ´ catholique de Louvain, Brussels, Belgium, 2Laboratory of Microbial Ecology and
Technology, Ghent University, Ghent, Belgium, 3Institut des Sciences de la Vie, Universite ´ catholique de Louvain, Louvain-la-Neuve, Belgium
Abstract
Background: Alterations in the composition of gut microbiota - known as dysbiosis - has been proposed to contribute to
the development of obesity, thereby supporting the potential interest of nutrients targeting the gut with beneficial effect
for host adiposity. We test the ability of a specific concentrate of water-extractable high molecular weight arabinoxylans
(AX) from wheat to modulate both the gut microbiota and lipid metabolism in high-fat (HF) diet-induced obese mice.
Methodology/Principal Findings: Mice were fed either a control diet (CT) or a HF diet, or a HF diet supplemented with AX
(10% w/w) during 4 weeks. AX supplementation restored the number of bacteria that were decreased upon HF feeding, i.e.
Bacteroides-Prevotella spp. and Roseburia spp. Importantly, AX treatment markedly increased caecal bifidobacteria content,
in particular Bifidobacterium animalis lactis. This effect was accompanied by improvement of gut barrier function and by a
lower circulating inflammatory marker. Interestingly, rumenic acid (C18:2 c9,t11) was increased in white adipose tissue due
to AX treatment, suggesting the influence of gut bacterial metabolism on host tissue. In parallel, AX treatment decreased
adipocyte size and HF diet-induced expression of genes mediating differentiation, fatty acid uptake, fatty acid oxidation and
inflammation, and decreased a key lipogenic enzyme activity in the subcutaneous adipose tissue. Furthermore, AX
treatment significantly decreased HF-induced adiposity, body weight gain, serum and hepatic cholesterol accumulation and
insulin resistance. Correlation analysis reveals that Roseburia spp. and Bacteroides/Prevotella levels inversely correlate with
these host metabolic parameters.
Conclusions/Significance: Supplementation of a concentrate of water-extractable high molecular weight AX in the diet
counteracted HF-induced gut dysbiosis together with an improvement of obesity and lipid-lowering effects. We postulate
that hypocholesterolemic, anti-inflammatory and anti-obesity effects are related to changes in gut microbiota. These data
support a role for wheat AX as interesting nutrients with prebiotic properties related to obesity prevention.
Citation: Neyrinck AM, Possemiers S, Druart C, Van de Wiele T, De Backer F, et al. (2011) Prebiotic Effects of Wheat Arabinoxylan Related to the Increase in
Bifidobacteria, Roseburia and Bacteroides/Prevotella in Diet-Induced Obese Mice. PLoS ONE 6(6): e20944. doi:10.1371/journal.pone.0020944
Editor: Lorraine Brennan, University College Dublin, Ireland
Received December 8, 2010; Accepted May 16, 2011; Published June 9, 2011
Copyright: ? 2011 Neyrinck et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work supported by a grant from the Walloon Region (General Directory of Agriculture, convention D31-1107, http://agriculture.wallonie.be). PDC is
a Research Associate from the Fonds de la Recherche Scientifique (FRS-FNRS) Belgium (http://www.frs-fnrs.be/). SP and TVDW are Postdoctoral Researchers from
the Research Foundation - Flanders (Fonds voor Wetenschappelijk Onderzoek [FWO] - Vlaanderen, http://www.fwo.be/). CD benefits from a Danone Institute
grant (http://www.danoneinstitute.be/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: nathalie.delzenne@uclouvain.be
Introduction
Recent studies demonstrated that diet-induced obesity was
linked to changes in the gut microbial ecology, resulting in an
increased capacity of the distal gut microbiota to promote host
adiposity [1,2]. We have previously shown that inulin-type
fructans, non-digestible carbohydrates obtained from chicory root,
restore the drop of bifidobacteria numbers occurring in the caeco-
colon of high fat/carbohydrate-free diet-fed mice and thereby
improves metabolic alterations associated with obesity, including
dyslipidemia, impaired gut permeability, endotoxemia, inflamma-
tion and diabetes [3–6]. Inulin-type fructans are typically studied
as they were the first compounds to respond to the prebiotic
concept, defined as the selective stimulation of growth and/or
activity of one or a limited number of microbial genus(era)/species
in the gut microbiota that confer(s) health benefits to the host [7].
Other non-digestible/fermented carbohydrates, which are gradu-
ally fermented throughout the colon and which can be applied in
different food matrices, may be valuable alternative substrates
to test for their health effects related to their influence on gut
microbiota composition.
AX are the most important non-digestible carbohydrates pre-
sent in wheat. They represent 50% of dietary fibers and are mostly
present in the bran and aleurone fractions [8]. AX are selectively
degraded in the colon by intestinal bacteria possessing AX-
degrading enzymes such as xylanases and arabinofuranosidases
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and represent a new class of potential prebiotics [9–11]. Existing in
different forms, ranging from soluble to insoluble fibers and high-
molecular weight to enzymatically modified short-chain fractions,
the physiological effects of AX are largely unknown [12]. How-
ever, several studies indicate that they behave like fermentable
fibers in the colon, with different fermentation profiles depending
on the physicochemical properties and degree of polymerization,
and with potential impact on lipid and glucose metabolism
[8,9,13–16]. Molecular weight has been shown to play an im-
portant role, with highest activity for high-molecular weight AX
[17]. Accordingly, the purpose of this study was to examine
the effects of an AX concentrate, containing long-chain water-
extractable AX, on the gut microbiota and lipid metabolism with
focus on the expression of genes relevant to energy homeostasis
and fat storage in a mice model of HF diet-induced obesity. We
have chosen high molecular weight AX concentrate because
previous in vitro studies have described an efficient fermentation of
the AX, leading to stimulation of specific stimulation of certain
bifidobacterial species and specific fermentation profiles (high
propionate production) using Simulator of Human Intestinal
Microbial Ecosystem (SHIME) [18,19].
Materials and Methods
Ethics Statement
The animal experiments were approved by the local ethic
committee; the agreement of the animal experiments performed in
this study was given by the ethical committee for animal care of
the Health Sector of the Universite ´ catholique de Louvain, under
the supervision of prof. P. Gianello P et JP Dehoux under the
specific number ULC/MD/2007/003. Housing conditions were
as specified by the Belgian Law of 14 November, 1993 on the
protection of laboratory animals (agreement nu LA 1230314).
Animals and diet intervention
Twenty four male C57bl6/J mice (9 weeks old at the beginning
of the experiment, Charles River Laboratories, France) were
housed in groups of 4 per cage in a controlled environment (12-
hour daylight cycle) with free access to food and water. After one
week of acclimatisation, the mice were divided into 3 groups
(n=8/group): a control group (CT), fed with a control diet (AO4,
SAFE, Villemoison-sur-Orge, France), a group fed a HF diet and a
group fed the same HF diet, supplemented with AX (90% HF (w/
w)+10%AX; HF-AX group). The full composition of both the HF
diet (D12492, Research Diets) and the A04 standard diet was
given in the Table S1. The energy content of the HF diet consisted
of fat for 60%, carbohydrate for 20% and protein for 20%.
BioActor (Ghent, Belgium) supplied AX (NaxusH, batch 07NX-
001) with a purity of 62%, degree of substitution of 0.7, and a
varying degree of polymerization, with an average above 60; the
composition of the batch used for the study was 67% non-starch
polysaccharides (62% AX), 18% protein, 0.5% lipids, 3.8% ash.
Food intake was recorded taking into account spillage twice a week
during 4 weeks. The total caloric intake was obtained by
multiplying total food intake (g) for 4 mice per cage (n=2) by
the caloric value of the diets, i.e. 3.10 Kcal/g, 5.24 Kcal/g and
4.83 Kcal/g for CT, HF and HF-AX, respectively. The caloric
value of the HF-AX diet was calculated taking in to account that
this diet was composed of 90% HF diet and 10% AX. After 4
weeks and a 6-hour period of fasting, mice were anesthetised
(ketamine/xylazine i.p., 100 and 10 mg/kg, respectively) and
blood samples were harvested for further analysis. Liver, three
types of white adipose tissues (visceral, epididymal and subcuta-
neous), vastus lateralis muscle, jejunum and caecum were carefully
dissected and weighted before immersion in liquid nitrogen before
storage at 280uC.
Oral glucose tolerance test (OGTT)
After 3 weeks of treatment, an oral glucose tolerance test was
performed on 6 h fasted-mice. Glucose was administered orally
(3 g/kg body weight, 66% glucose solution) and blood glucose
levels were determined using a glucose meter (Roche Diagnostics)
on 3.5 ml of blood collected from the tip of the tail vein both before
(230 min and 0 min) and after glucose administration (15, 30, 60,
90, 120 min). Twenty microliters of blood were sampled 30 min
before and 15 min after the glucose load to assess plasma insulin
concentrations.
Microbial analysis of the caecal contents
At the end of the experiment, the total caecum content was
collected and weighed before storage at 280uC. For analysis of
the microbial content, metagenomic DNA was extracted from the
caecal content of all mice, using the QIAamp DNA stool mini kit
(Qiagen, Venlo, The Netherlands) according to the manufactur-
er’s instructions. Denaturing Gradient Gel Electrophoresis
(DGGE) on total bacteria, bifidobacteria, lactobacilli and
Bacteroides-Prevotella spp. was performed to study the qualitative
effect of the treatment on the structure and composition of the
intestinal microbial community [20]. DGGE with a 45–60%
denaturing gradient was used to separate the polymerase chain
reaction (PCR) products obtained with a nested approach for the
16S rRNA genes of bifidobacteria (primers BIF164f-BIF662r),
lactobacilli (SGLAB0159f-SGLAB0667r) and the Bacteroides-Pre-
votella cluster (FD1-RBacPre) [21]. The first PCR round was
followed by a second amplification with primers 338F-GC and
518R. The latter primers were also used to amplify the 16S
rDNA of all bacteria on total extracted DNA. The DGGE
patterns obtained were subsequently analyzed using the Bionu-
merics software version 5.10 (Applied Maths, Sint-Martens-
Latem, Belgium). In brief, the calculation of the similarities was
based on the Pearson (product–moment) correlation coeffi-
cient. Clustering analysis was performed using the unweighted
pair group method with arithmetic mean clustering algorithm
(UPGMA) to calculate the dendrograms of each DGGE gel and a
combination of all gels. The latter was performed on a created
composite dataset. The composite dataset of the 3 DGGE pa-
tterns was also used to perform principal component analysis
(PCA). PCA ordinations were calculated using the Pearson
product-moment correlation coefficient. Within each character
set, this coefficient subtracts each character from the average
value, and divides it by the variance of the character set.
Quantitative PCR (Q-PCR) was performed to study the
quantitative effect of the treatment on the composition of the
intestinal microbial community. The Q-PCR for total bacteria
(using primers PRBA338f and P518r) and specific for bifidobac-
teria were performed as reported by Possemiers et al. [22]. The Q-
PCR for Roseburia spp. was performed as described before [23],
using the primers Ros-F1 and Ros-R1, and the Power SYBR
Green PCR Master kit (Applied Biosystems, Foster City). The Q-
PCR for Bacteroides-Prevotella spp. was performed as described by
Rinttila ¨ et al. [24], using the Q-PCR Core kit for SYBR Green I
(Eurogentec, Seraing, Belgium) and primers Bacter140f and
Bacter140r. The Q-PCR for Bifidobacterium animalis lactis was
performed as described by Ventura et al. [25] using the primers
(Bflact2 and Bflact5). All Q-PCR were performed with an ABI
PRISM SDS 7000 Sequence Detection System (Applied Biosys-
tems, Nieuwerkerk a/d Ijssel, the Netherlands).
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Blood parameters
Plasma insulin concentrations were determined using an ELISA
kit (Mercodia, Uppsala, Sweden). The insulin resistance index was
calculated by multiplying the area under the curve for glucose, and
the area under the curve for insulin, calculated from 230 min
until 15 min after glucose challenge [5,26]. Concentrations of IL-6
and MCP-1 were determined in 15 ml of plasma using a multiplex
immunoassay kit (Bio-Plex Cytokine Assay, Bio-Rad, Belgium)
and measured using LuminexH technology (BioplexH, Bio-Rad,
Belgium). Adiponectin concentrations were determined using an
ELISA kit designed to measure full-length mouse adiponectin
levels in serum (QuantikineH Mouse adiponectin, R&DSystems).
Plasma triglycerides, cholesterol and non esterified fatty acid
concentrations were measured using kits coupling enzymatic
reaction and spectrophotometric detection of reaction end-
products (Diasys Diagnostic and Systems, Holzheim, Germany).
High density lipoprotein cholesterol (HDL-cholesterol) concentra-
tion was measured enzymatically after very low density lipoprotein
(VLDL), chylomicrons and low density lipoprotein cholesterol
(LDL-cholesterol) antibodies precipitation (Diasys Diagnostic and
Systems, Holzheim, Germany). LDL was estimated by the
Friedewald formula [27].
Lipid analysis in the liver
Triglycerides and cholesterol were measured in the liver tissue
after extraction with chloroform–methanol as described by
Neyrinck et al. [28].
Fatty acid synthase (FAS) activity
Homogenate of subcutaneous adipose tissue was performed in
phosphate buffer (100 mg tissue/500 ml buffer). Cytosolic fractions
were obtained after 2 successive centrifugations at 4uC (1000 g-
15 min and 20000 g 30 min) The procedure described by Linn
was used for the measurement of FAS activity in cytosolic fractions
[29]. Protein was determined by the Bradford method.
Adipose tissue morphometry
Number of adipocytes per microscopic field (density) was
estimatedonparaffin-embedded
counterstained sections of subcutaneous adipose tissue using the
image analyzer software (Motic Image Plus 2.0 ML), as previously
described [30].
hematoxylin-stained eosin-
Fatty acid profile analysis in adipose tissue
Fatty acid analysis, including conjugated linoleic acid (c9, t11
CLA), in subcutaneous adipose tissue was performed according to
the method described by Dewulf et al [30].
Expression of selected genes in tissues
Total RNA was isolated using the TriPure isolation reagent kit
(Roche Diagnostics Belgium, Vilvoorde). cDNA was prepared by
reverse transcription of 1 mg total RNA using the Kit Reverse
transcription System (Promega, Leiden, The Netherlands). Real-
time PCRs were performed with the StepOnePlusTMreal time
PCR system and software (Applied Biosystems, Den Ijssel, The
Netherlands) using Mesa Fast qPCRTM(Eurogentec, Seraing,
Belgium) for detection according to the manufacturer’s instruc-
tions. RPL19 RNA was chosen as housekeeping gene. Primer
sequences for the targeted mouse genes are available on request
(audrey.neyrinck@uclouvain.be). All samples were run in
duplicate in a single 96-well reaction plate and data were analysed
according to the 2-DCT method [31]. The identity and purity of
the amplified product was checked through analysis of the melting
curve carried out at the end of amplification.
Statistical analysis
Results are presented as mean 6 SEM. Statistical analysis was
performed by ANOVA followed by post hoc Tuckey’s multiple
comparison test (GraphPad Software, San Diego, CA, USA);
p,0.05 was considered as statistically significant. Correlations
between parameters were assessed by Pearson’s correlation test;
correlations were considered significant as follows: *p,0.01,
**p,0.001, ***p,0.0001 with the absolute value of Pearson
r.0.5.
Results
Supplementation with arabinoxylan modified the gut
microbiota composition
HF feeding decreased both the caecal content weight and
tissue weight as compared to the control condition (Figures 1A and
1B). Clustering of the DGGE fingerprints for total bacteria
(Figure 1C) indicated a separate cluster for the CT and HF groups.
PCA analysis of combined DGGE fingerprints of total bacteria,
bifidobacteria, lactobacilli and Bacteroides-Prevotella spp. shows
distinct clusters between the HF and CT groups (Figure 1D).
This was further confirmed following Q-PCR analyses of different
bacterial groups (Figure 2 and Figure S1). HF diet induced a drop
in Roseburia spp. and Bacteroides-Prevotella spp. numbers. Conversely,
the number of bifidobacteria per gram of caecum content was
significantly higher in the HF group, as compared to control
mice (Figure 2B).
AX supplementation induced caecal enlargement (Figures 1A
and 1B) and a profound shift in the microbial community in
comparison with the HF group, as shown by the distinct clustering
in the DGGE profile and PCA analysis (Figures 1C and 1D). AX
supplementation restored the HF diet-induced microbial commu-
nity changes as shown by the significant increase in Bacteroides–
Prevotella spp. and Roseburia spp. (Figures 2C and 2D). Moreover,
AX induced a specific increase in bifidobacteria (Figure 2B), in
particular bifidobacterium animalis ssp lactis (expressed as Log10
[DNA copies/g caecal content]: 5,6760,07, 6,0360,07*, and
6,3760,06*1 for CT, HF and HF-AX, respectively; ANOVA, *
p,0.05 versus CT and 1p,0.05 versus HF).
Supplementation with arabinoxylan modified the
expression of markers related to gut barrier function
The mRNA levels of both ZO-1 and occludin, which are tight-
junction proteins, were measured in the jejunum, as well as the
mRNA level of proglucagon, which is the precursor of the
intestinotrophic peptide GLP-2, known to reduce gut permeability
[4]. HF feeding did not modify the expression of these genes
(Figure S2). Interestingly, AX supplementation significantly
increased the mRNA levels for both tight-junction proteins and
proglucagon expression, as compared to the HF and/or CT
groups.
Supplementation with arabinoxylan decreased body
weight gain and fat mass development
The mice fed with HF-AX did not gain weight as rapidly as HF-
fed mice (Fig. 3A). In fact, AX supplementation decreased body
weight gain by about 40% as compared to HF (fig. 3B). Moreover,
AX treatment induced a lower fat mass development as shown by
the weight of epididymal, subcutaneous and visceral adipose
tissues (Figures 3C, 3D and 3E). This effect could not be explained
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by changes in energy intake since the total calorie intake (for 4
mice, n=2) was not different between the HF and HF-AX groups
(1579630 kcal and 1539615 kcal, respectively).
Supplementation with arabinoxylan improved
insulinoresistance index and cholesterol levels
HF feeding induced fasting hyperglycemia and hyperinsulin-
emia, and increased the insulin resistance index upon OGTT, as
compared to mice fed normal chow diet (Table 1). Surprisingly,
adiponectin level was not modified after HF treatment. HF diet
feeding induced hepatic triglyceride accumulation (by about 28%)
and increased serum total, LDL- and HDL-cholesterol levels
without affecting serum triglycerides and non-esterified fatty acids
(Table 1). The HF-induced hypercholesterolemia was associated to
a higher hepatic content of free cholesterol.
AX did not change significantly fasting glycemia or insulinemia.
Interestingly, AX feeding improved the insulin resistance index as
compared to the HF group. However, we observed a lower
concentration of adiponectin after AX supplementation (Table 1).
AX supplementation furthermore decreased hypercholesterolemia
(both LDL- and HDL-cholesterol) and also led to a shift
from hepatic free cholesterol towards esterified cholesterol,
without changing total cholesterol or triglyceride level in the
tissue (Table 1).
Supplementation with arabinoxylan increased the
proportion of rumenic acid in the subcutaneous adipose
tissue reflecting changes in gut bacterial metabolism
The fatty acid profile in adipose tissue is influenced by that of
the dietary lipids [32,33]. In addition, specific fatty acids, such as
vaccenic acid (C18:1 t11) and conjugated linoleic acids, the major
being the rumenic acid (C18:2 c9,t11), may be formed upon
bacterial metabolism (biohydrogenation) of linoleic acid in the
intestine [34]. Therefore, we analyzed the fatty acid patterns in
subcutaneous adipose tissue of mice (Table 2). HF feeding induced
drastic changes in the fatty acid composition of the adipose tissue
with a higher proportion of monounsaturated fatty acids (MUFA),
at the expence of both polyunsaturated fatty acids (PUFA) and
saturated fatty acids (SFA). Interestingly, HF feeding induced a
significant increase in both vaccenic acid and rumenic acid.
Figure 1. Caecum weight, DGGE fingerprints and PCA analysis in the caecal content. Caecal content (A) and caecal tissue (B) weight.
Denaturing gradient gel electrophoresis (DGGE) fingerprint patterns of the caecal microbial community; the DGGE profiles were constructed using
primers for total bacteria (C). Principal Coordinate Analysis (PCA) was used to explore the similarity within a composite data set consisting of DGGE
fingerprints of total bacteria, bifidobacteria, lactobacilli and the Bacteroides-Prevotella spp. cluster (D). Mice were fed a standard (CT, green symbols), a
high fat diet (HF, red symbols) or a high fat diet supplemented with 10% arabinoxylan (HF-AX, blue symbols) for 4 weeks. *p,0.05 versus CT and
1p,0.05 versus HF (ANOVA).
doi:10.1371/journal.pone.0020944.g001
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Supplementation with AX significantly decreased the proportion
of palmitic acid (C16:0) whereas it further increased the rumenic
acid content as compared to the HF treatment.
Supplementation with arabinoxylan reduced adipocyte
size and decreased HF-induced gene expression in the
subcutaneous adipose tissue
Histological analysis revealed that the adipocyte size in
subcutaneous adipose tissues were increased in the HF-fed mice
versus controls, whereas the AX treatment normalized this
parameter (Figure 4; adipocyte number per field: 339649,
71613* and 177621*1 for CT, HF and HF-AX, respectively;
ANOVA, * p,0.05 versus CT and 1p,0.05 versus HF). HF
feeding increased the expression of genes controlling inflammation
(F4/80, IL-6, MCP-1), PPAR-a dependent-fatty acid oxidation
(CPT-1, ACO), PPARc-dependent differentiation and/or fatty acid
uptake (C/EBPa, FAT/CD36, aP2, LPL), and lipolysis (MGL)
(Figure 5). Furthermore, the mRNA content of GPR43 -a receptor
activated by short-chain fatty acids and implicated in the regulation
of lipolysis and adipocyte differentiation- was significantly increased
upon HF feeding. Interestingly, the AX treatment hugely decreased
the expression of the most of these genes in the subcutaneous tissue.
Of particular interest, AX treatment decreased the serum
concentrations of 2 inflammatory markers that were downregulated
in the adipose tissue through AX treatment, namely IL6
(53.9611.5 pg/ml and 21.967.7 pg/ml for HF and HF-AX
groups, respectively; t test p,0.05) and MCP-1 (32.165.3 pg/ml
and 12.362.4 pg/ml for HF and HF-AX groups, respectively; t test
p,0.05). In addition, AX supplementation inhibited the expression
of fatty acid synthase (FAS), a lipogenic enzyme that was already
downregulated by the HF diet. In accordance with its expression,
we confirmed that FAS activity was downregulated through AX
supplementation in adipose tissue since its activity was significantly
lower as compared to HF group (32.567.1, 25.567.5 and
7.161.3*1 for CT, HF and HF-AX respectively; ANOVA:
*p,0.05 versus CT and1p,0.05 versus HF). By contrast, it did
not affect the expression of the uncoupling protein UCP-2.
Given that AX treatment decreased genes involved in fatty acid
uptake, we decided to explore some markers of lipid metabolism in
the liver and the muscle (Table S2). In the vastus lateralis muscle,
there were no changes in mRNA expression of genes involved in
fatty acid oxidation (ACO, PPARa, PPARd, CPT1) between the
groups. In the liver, HF feeding induced the expression of the genes
encoding sterol regulatory element-binding protein-1c (SREBP-1c)
and its downstream regulated protein FAS. AX supplementation
did not modify significantly lipogenic gene expression but it lowered
HF-induced PPARa expression with no consequences on genes
involved in fatty acid oxidation (CPT1 or ACO). Of note, there was
no change in the expression of genes involved in cholesterol
metabolism, whatever the dietary treatments.
Figure 2. Bacterial quantification per gram of caecal content. Caecal bacterial content of total bacteria (A), Bifidobacterium spp. (B),
Bacteroides-Prevotella spp. (C), Roseburia spp. (D). Bacterial quantities are expressed as Log10 (bacterial cells/ g caecal content wet weight). Mice were
fed a standard (CT), a high fat diet (HF) or a high fat diet supplemented with 10% arabinoxylan (HF-AX) for 4 weeks. *p,0.05 versus CT and1p,0.05
versus HF (ANOVA).
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Figure 3. Body weight and fat mass. Body weight evolution (A), body weight gain (B), visceral (C), epididymal (D), and subcutaneous (E) adipose
tissue weight (% versus body weight) of mice fed a standard (CT), a high fat diet (HF) or a high fat diet supplemented with 10% arabinoxylan (HF-AX)
for 4 weeks. *p,0.05 versus CT and1p,0.05 versus HF (ANOVA).
doi:10.1371/journal.pone.0020944.g003
Table 1. Blood and hepatic parameters.
CTHF HF-AX
Serum
- fasting glycemia (mM) 7.4960.28 9.5860.41* 8.4460.41
- fasting insulinemia (mM)83.9612.6 226.3632.2* 156.1616.6*
- insulin resistance index300306963113500619440* 7199067423*1
- adiponectin (pg/ml) 9.3660.409.3860.467.7660.55 *1
- triglycerides (mM) 1.4760.11 1.7160.131.5460.11
- non esterified fatty acids (mM)0.2760.030.3160.030.2960.04
- total cholesterol (mM)1.7560.06 3.0060.03 *2.4160.06 *1
- LDL-cholesterol (mM)0.3160.051.2260.08 *0.9060.06 *1
- HDL-cholesterol (mM)0.7860.021.0060.03 *0.8260.02 1
Liver lipid content, nmol/mg protein
- triglycerides60.963.077.865.3 * 66.168.0
- total cholesterol41.164.1 74.2611.0 *73.2610.5 *
- free cholesterol27.062.240.364.6 *27.861.3 1
- esterified cholesterol 14.163.233.966.6 45.569.3 *
Mice were fed a standard diet (CT), a high fat diet (HF) or a high fat diet supplemented with 10% arabinoxylan (HF-AX) for 4 weeks * p,0.05 versus CT and1p,0.05
versus HF (ANOVA). LDL, low density liprotein; HDL, high density lipoprotein.
doi:10.1371/journal.pone.0020944.t001
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Improved metabolism following arabinoxylan
supplementation was correlated with the increase in
bifidobacteria or the restoration of Roseburia spp. and
Bacteroides/Prevotella spp
To determine whether modifications of the gut microbiota in
mice fed AX were associated with an improvement in obesity and
lipid metabolism, a correlation analysis was performed between
bacteria and host metabolic parameters that were significantly
affected through AX supplementation (Figure 6 and Table S3).
The analysis revealed negative correlations (p,0.001) between
Roseburia spp, and fat mass development, body weight gain,
cholesterolemia, insulinoresistance index, and expression of several
genes that mediate differentiation and/or fatty acid uptake
(PPARc, aP2, FAT/CD36, LPL, FIAF), fatty acid oxidation
(CPT-1, ACO), short-chain fatty acid response (GPR43), and
inflammation (IL6, F4/80) in the subcutaneous adipose tissue.
Some of these metabolic parameters were also inversely correlated
with the number of Bacteroides/Prevotella spp. (p,0.01) whereas
none of these markers was significantly correlated with the number
of total bifidobacteria. However, bifidobacteria levels were
positively correlated with rumenic acid in the adipose tissue and
with the mRNA levels of the tight junction proteins (ZO-1 and
occludine) in the gut. The analysis revealed other interesting
negative correlation with bifidobacteria such as the FAS activity/
expression in the adipose tissue and circulating inflammatory
markers (IL-6 and MCP-1).
Discussion
Over the past 5 years, animal and human studies have revealed
a remarkable microbial influence on host metabolism, energy
Table 2. Fatty acid pattern of the subcutaneous adipose tissue from mice fed a standard diet (CT), a high fat diet (HF) or a high fat
diet supplemented with 10% arabinoxylan (HF-AX) for 4 weeks.
CTHFHF-AX
SFA28.197 ±
0.614 25.998 ±
0.180*25.010 ±
0.322*1
C6:00.017
6
0.0020.007
6
0.001*0.006
6
0.001*
C8:00.009
6
0.0010.004
6
0.001*0.004
6
0.001*
C10:00.045
6
0.0030.039
6
0.0030.040
6
0.002
C12:00.119
6
0.0090.080
6
0.006*0.082
6
0.004*
C13:00.014
6
0.0010.010
6
0.001*0.011
6
0.001
C14:01.563
6
0.0571.010
6
0.017*0.997
6
0.024*
C15:0 iso0.013
6
0.0010.003
6
0.000*0.003
6
0.001*
C15:0 anteiso0.013
6
0.0010.003
6
0.001*0.004
6
0.001*
C15:00.169
6
0.0050.106
6
0.001*0.107
6
0.003*
C16:0 iso0.133
6
0.0030.067
6
0.003*0.069
6
0.003*
C16:022.422
6
0.45919.844
6
0.138* 18.631
6
0.338*1
C17:0 iso0.150
6
0.0030.143
6
0.003 0.138
6
0.003*
C17:0 anteiso 0.683
6
0.015 0.737
6
0.0140.798
6
0.032*
C17:00.181
6
0.0050.250
6
0.004*0.248
6
0.004*
C18:02.525
6
0.1363.631
6
0.088*3.791
6
0.177*
C20:00.140
6
0.0050.065
6
0.004*0.080
6
0.007*
MUFA 44.442 ±
0.260 48.981 ±
0.133* 49.778 ±
0.438*
C14:1 C90.153
6
0.008 0.062
6
0.010*0.052
6
0.003*
C16:1C98.631
6
0.2074.727
6
0.160*4.407
6
0.229*
C18:1T90.040
6
0.0030.224
6
0.008* 0.240
6
0.007*
C18:1T11 0.055
6
0.0040.192
6
0.007*0.194
6
0.005*
C18:1C9 32.341
6
0.21441.253
6
0.143*42.283
6
0.593*
C18:1C11 3.222
6
0.0492.520
6
0.019*2.602
6
0.023*1
PUFA 27.361 ±
0.58325.021 ±
0.164*25.211 ±
0.375*
C18:2C9C12 25.698
6
0.54423.115
6
0.146*23.436
6
0.294*
C18:3C9C12C150.859
6
0.032 1.081
6
0.026* 0.991
6
0.062
C18:2C9T110.090
6
0.0050.171
6
0.002* 0.181
6
0.003*1
C20:3C11C14C170.013
6
0.001 0.061
6
0.001*0.060
6
0.002*
C20:4C5C8C11C140.323
6
0.0110.362
6
0.007 0.332
6
0.017
C20:5C5C8C11C14C170.041
6
0.0050.020
6
0.001*0.013
6
0.003*
C22:5C7C10C13C16C19 0.063
6
0.002 0.065
6
0.002 0.060
6
0.003
C22:6C4C7C10C13C16C19 0.264
6
0.0120.141
6
0.003*0.132
6
0.008*
Values are means 6 SEM (g/100 g of identified fatty acid methyl esters). SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty
acids.
doi:10.1371/journal.pone.0020944.t002
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utilization and storage, and metabolic diseases [1,35–37]. A shift in
the composition the murine gut microbiota, such as a decrease
in Bacteroidetes or an increase in Firmicutes, was reported to
be induced by dietary interventions in favour of a HF diet
[3,28,38]. The microbial-related response of mice towards a HF
diet, in terms of fat mass development and metabolic diseases, is
dependent on the diet composition [39]. Here, we show that
feeding mice with a HF diet decreases the dominant members of
the mouse intestinal microbiota - Bacteroides-Prevotella ssp. from the
Bacteroidetes phylum together with the specific populations of
clostridial cluster XIVa, i.e. Roseburia spp from the Firmicutes
phylum. However, it slightly increased the number of total
bifidobacteria. The composition of the HF diet as compared to the
standard diet -in term of dietary fiber (concentration and nature)-
is probably responsible for the important changes in the
parameters related to the gut fermentation (caecal content weight
and microbial composition). Indeed, according to the manufac-
turer, the control diet contains 84% of cereal products that brings
4% of cellulose as dietary fibre without taking into account other
non digestible/fermentable carbohydrates such as arabinoxylan,
resistant starch, fructooligosaccharides present in cereal in
particular in bran fractions [8]. In contrast, the dietary fibre of
the HF diet is originating from cellulose BW200 (6.5%) which is
poorly fermentable. Moreover, the HF diet has only maltodextrin
and sucrose as digestible carbohydrate sources whereas the control
diet has native starch coming from cereals, bran and remilling; a
part of this starch being resistant to digestion and consequently
entering in the caeco-colon as fermentable fraction (resistant
starch). Interestingly, AX added to the HF diet led to an increase
of bifidobacteria and in particular of Bifidobacterium animalis subsp.
Lactis. AX also restored to control level, the bacterial populations
in the caecal content which were decreased upon HF feeding,
namely the gram negative Bacteroides-Prevotella spp. and the gram
positive bacteria Roseburia spp.
In previous studies, we demonstrated that feeding rats with the
inulin-type prebiotic (oligofructose) protected against liver triglyc-
eride accumulation induced by fructose and that the lower
lipogenic capacity of the liver could be the key event in this
protection; indeed FAS activity remained significantly lower in
oligofructose-fed rats [40–42]. More recently, we found that a
selective change in gut microbiota composition through inulin-
type prebiotic treatment improves gut barrier functions through a
GLP-2-dependent mechanism during obesity and diabetes [4]. As
shown for oligofructose, the bifidogenic effect of AX demonstrated
here, were accompanied by a decrease in FAS activity in the
adipose tissue. Moreover, we observed an increase in both tight
junction proteins and proglucagon expression after AX supple-
mentation, suggesting an increase in gut barrier-related functions
as a physiological consequence of the changes in the composition
of gut microbiota. Of particular interest, we observed lower levels
of inflammatory markers in the serum due to AX supplementation
that could be the result of improvement of gut barrier functions as
suggested by Cani et al. [3,5]. Interestingly, those metabolic
changes were correlated with the number of bifidobacteria in the
caecal content confirming prebiotic properties of AX.
Here, we demonstrate for the first time that a prebiotic
approach is also able to change the occurrence of PUFA meta-
bolites in the white adipose tissue. Such an effect was previously
obtained with a probiotic strategy, leading to modifications of fatty
acid pattern in the adipose tissue (including higher concentrations
of the n23 fatty acids eicosapentaenoic acid and docosahexaenoic
acid) [43]. AX increases the amount of a linoleic acid metabolite,
namely rumenic acid which belong to the conjugated linoleic acid
(CLA) family. CLAs comprise a set of positional (eg, 9,11; 10,12;
11,13) and geometric (cis or trans) isomers of linoleic acid with
conjugated double bonds. CLA isomers have been shown to exert
a variety of biological activities and some of them have been
shown to exert anti-obesity effects [44]. Commensal bifidobacteria
from the mammalian gut have been shown to generate CLA,
predominantly rumenic acid -the c9,t11 isomer from free linoleic
acid- whereas Roseburia spp. formed either vaccenic acid (18:1 t11)
or a 10-hydroxy-18:1, two precursors of rumenic acid [34,45,46].
We observed a higher proportion of rumenic acid upon HF
feeding alone, probably due to a higher concentration of its
substrate (linoleic acid) in the HF diet versus control diet. AX
supplementation further increased the rumenic acid proportion in
host adipose tissue, suggesting a specific linoleic acid metabolism
Figure 4. Histological pictures of subcutaneous adipose tissue.
Mice were fed a standard diet (CT), a high fat diet (HF) or a high fat diet
supplemented with 10% arabinoxylan (HF-AX) for 4 weeks.
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by CLA-producing bacteria such as bifidobacteria and indirectly
Roseburia spp, which are both promoted upon AX feeding.
To date, only a few studies have investigated whether the
consumption of wheat-derived AX could have potential beneficial
effects for health. We show here that, in parallel to changes in gut
bacterial population, the specific concentrate of water-extractable
high molecular weight AX significantly decreased body weight gain
and fat mass development in a model of HF diet-induced obesity. In
addition, AX supplementation counteracted both the HF-induced
hypercholesterolemia (total, LDL- and HDL-cholesterol) and the
higher content of free cholesterol in the liver upon HF feeding. The
hypocholesterolemic effect of AX has previously been observed in
vivo in rats, an effect attributed to a decreased dietary cholesterol
absorption and an increased fecal excretion of cholesterol and bile
acids, leading -as a metabolic signature- to an increased expression
of HMG-CoA reductase [13]. We may not exclude that the higher
production of propionate upon AX fermentation could be
implicated in the decrease in hepatic cholesterol synthesis and
hypocholesterolemic effect [19]. However, the fact that we were
unable to see any changes in hepatic HMG-CoA reductase
expression is not in favor of this mechanism in our study.
Thehistologicalanalysisofthesubcutaneousadiposetissueshowed
that the adipocytes were larger upon the HF treatment and became
smaller when the HF diet was combined with AX. PPARs are vital
for adipogenesis (adipocyte differentiation), fatty acid uptake,
lipogenesis and fatty acid oxidation (see fig. 5 and [47,48]). PPARc
Figure 5. mRNA levels of key factors and metabolic network in the subcutaneous adipose tissue. Expression of genes involved in
subcutaneous adipose tissue metabolism (A). Mice were fed a standard (CT), a high fat diet (HF) or a high fat diet supplemented with 10%
arabinoxylan (HF-AX) for 4 weeks. Values are expressed relative to CT group (set at 1). *p,0.05 versus CT and1p,0.05 versus HF (ANOVA). Genes that
regulate metabolic processes in white adipose tissue (B); some of them are dependent on PPARa (blue) or PPARc (orange) activation by an
endogenous ligand. PPARc, peroxisome proliferator-activated receptor c; aP2, adipocyte fatty acid binding protein; C/EBPa, CCAAT enhancer binding
protein a; GPR43, G protein-coupled receptor 43; LPL, lipoprotein lipase; CD-36, cluster of differenciation 36; FAS, Fatty acid synthase; ACC, AcylCoa
carboxylase; PPARa, peroxisome proliferator-activated receptor-alpha ; CPT-1, carnitine palmitoyl transferase-1 ; ACO, AcylCoA oxydase; MGL,
monoacylglycerol lipase; UCP-2, uncoupling protein-2; VLDL, very low density lipoprotein; CM, chylomicron; FA, fatty acids; TG, triglycerides.
doi:10.1371/journal.pone.0020944.g005
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isabundantlyexpressedinadiposetissue,whereitisakeyregulatorof
adipocyte differentiation, whereas PPARa governs fatty acid
oxidation [47]. Importantly, antagonism of PPARc using a synthetic
ligand suppresses the increased adiposity observed in HF induced
obesity [47]. Recently, we have shown that inulin-type fructans with
prebiotic properties counteract PPARc-related adipogenesis in the
white adipose tissue of HF fed mice [30]. Here, we demonstrate that
the supplementation of the specific AX concentrate is also able to
decrease the expression ofmostoftheHF-inducedmetabolicgenesin
subcutaneous adipose tissue, in particular genes depending on
PPARs-activation. Indeed, AX down-regulated genes involved in
adipocyte differentiation, fatty acid uptake, fatty acid oxidation,
lipolysiswhileitdecreasedfattyacidsynthesis(expressionandactivity)
that was already downregulated upon HF-feeding. Although we can
attribute the antiobesity effect of AX (lower fat mass development) to
a lower fatty acid synthesis (FAS expression and activity) or fatty acid
uptake in white adipose tissue (downregulation of lipoprotein lipase,
fatty acid translocase/CD36 and fatty acid binding protein (aP2)), we
maynotexclude aneffectofAXon lipid absorptionthrough eitherits
intrinsic capacity of fat binding or modulation of pancreatic lipase
activity [49]. Chitosan –coming from chitin- has the capacity to bind
fatty acids and to increase the lipid content in the caecum [50,51].
This mechanism seems also to be involved in the improvement of
HF-induced metabolic alterations by AX. Indeed, the proportion of
lipid accumulated in the caecum versus the lipid ingested during a
12 h-feeding period was increased due to AX or chitosan
supplementation in the HF diet (figure S3A). Furthermore, in this
experiment, AX or chitosan supplementation increased both the fatty
acid content and the cholesterol content in the caecum of mice fed a
HF diet (figure S3B and S3C). Although the host adiposity changes
and hypocholesterolemic effect of AX could be the result of its direct
fat binding capacity leading to fat leakage in faecal matter, we may
not exclude other potential mechanisms. The study of VO2 and
VCO2 by indirect calorimetry at rest and during physical activity
could constitute one interesting perspective to investigate further. In
fact, we postulate that the modulation of gut microbiota induced by
AX supplementation observed in the present study, was involved in
its anti-obesity action as well as in its cholesterol-lowering effects. This
hypothesis is supported by the fact that the number of bacteria from
the clostridial cluster XIVa, such as Roseburia spp. -and Bacteroides-
Prevotella spp. to a lesser extent- showed inverse correlations with
important markers of obesity and host lipid metabolism (fat mass
development, body weight gain, cholesterolemia, and expression of
several genes mediating differentiation and/or fatty acid uptake, fatty
acid oxidation, short chain fatty acid response and inflammation in
the subcutaneous adipose tissue). Recently, Martinez and coworkers
[52] provided evidence that modulation of the gut microbiota-host
metabolic interrelationship by dietary intervention has the poten-
tial to improve mammalian cholesterol homeostasis, which has
relevance for cardiovascular health. Several mechanisms could
explain how the gut microbiota may affect host lipid metabolism
since gut bacteria are able 1) to regulate chylomicron formation and
lipid uptake by affecting gut transit time and bile salt metabolism
(modulation of the enterohepatic circulation of bile acids) [1,53]; 2) to
fermentcomplexpolysaccharidesintoshortchainfattyacidsthatmay
act either as lipogenic substrates in the liver or an inhibitor of
cholesterologenesis and lipogenesis from acetate [54]; 3) to suppress
the expression of fasting-induced adipose factor (FIAF) in the
intestinal mucosa, a factor able to increase lipoprotein lipase (LPL)
dependent triglyceride storage in adipose tissue and to reduce serum
triglyceride level [1,55,56]. FIAF is indeed an important regulator of
lipidmetabolismandhasbeenshowntoincreasetotalcholesteroland
high-density lipoprotein (HDL) cholesterol levels when over-
expressed in transgenic mice [57]. This last mechanism could be
Figure 6. Interrelationship between gut microbiota composition and host metabolic parameters significantly modified by
arabinoxylan supplementation. Green connections indicate a positive correlation (Pearson r.0.5), while red connections show correlations that
are inverse (Pearson r,0.5). Solid lines represent significance with p,0.001 and shared lines represent significance with p,0.01. aP2, adipocyte fatty
acid binding protein; GPR43, G protein-coupled receptor 43; IL6, interleukin 6; LPL, lipoprotein lipase; FAS, Fatty acid synthase; CPT-1, carnitine
palmitoyl transferase-1 ; MCP-1, monocyte chemoattractant protein-1; MGL, monoacylglycerol lipase; SCFA, short chain fatty acid.
doi:10.1371/journal.pone.0020944.g006
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involved in the hypocholesterolemic effect of AX since FIAF
expression in white adipose tissue was decreased after AX
supplementation in the HF diet.
Having a significant amount of excess body fat is a major
problem that increases the risk of developing insulin resistance and
type 2 diabetes. We have shown that the specific AX concentrate
decreased HF-induced hyperglycemia and hyperinsulinemia,
whereas its supplementation improved glucose tolerance after a
glucose load (data not shown) but these effects were not significant.
However, it is clear that the insulin resistance index was improved
due to AX treatment as compared to the HF group, suggesting
that the anti-obesity effect of AX was accompanied by a beneficial
effect on insulin sensitivity. This result is in accordance with a
clinical study demonstrating that consumption of the same AX
concentrate for 6 weeks improved postprandial glycemia and
insulinemia in overweight subjects with impaired glucose tolerance
[16].
In conclusion, this study has shown in a 4-week study in mice,
that supplementation of a concentrate of water-extractable high
molecular weight AX in the diet counteracted HF-induced gut
dysbiosis with a major effect on bacteria from clostridial cluster
XIVa (restoration of Roseburia spp.), gram negative Bacteroides-
Prevotella spp. and bifidobacteria (in particular, higher caecal
content in Bifidobacterium animalis subsp. Lactis). This prebiotic
effect was accompanied by a lower circulating inflammatory
markers and by an increase in both tight junction markers and
rumenic acid (18:2 c9,t11) in white adipose tissue. This last
phenomenon reflects the influence of gut bacterial metabolism
on host tissue. The gut microbiota changes due to the AX
treatment were accompanied by an improvement of obesity and
lipid-lowering effects together with an improvement of insulin
resistance markers in HF diet-induced obesity. In addition, AX
supplementation was able to decrease a number of PPARs-
dependent genes within white adipose tissue, without inducing
fat accumulation in the liver. Those metabolic effects may be
related to the intrinsic fat binding capacity of AX. However,
since it is known that gut bacteria could influence host lipid
metabolism, we postulate that both hypocholesterolemic and
anti-obesity effects conferred by AX are related, at least in part,
to changes in gut microbiota.
Even if a direct extrapolation of the present study to human is
still questionable due to differences in digestive tract structure and
in gut microbiota, our results suggest that water extractable high
molecular weight AX that is predominantly found in wheat
endosperm can confer positive health impacts through gut
microbiota modulation and may be a natural alternative in the
prevention of obesity and cardiovascular diseases.
Supporting Information
Figure S1
bacterial content of total bacteria (A), Bifidobacterium spp. (B),
Bacteroides-Prevotella spp. (C) and Roseburia spp. (D). Bacterial
quantities are expressed as Log10 (bacterial cells/ total caecal
content wet weight). Mice were fed a standard diet (CT), a high fat
diet (HF) or a high fat diet supplemented with 10% arabinoxylan
(HF-AX) for 4 weeks.*p,0.05 versus CT and1p,0.05 versus HF
(ANOVA).
(TIF)
Bacterial quantification in the caecum. Caecal
Figure S2
related to gut barrier function. Mice were fed a standard
(CT), a high fat diet (HF) or a high fat diet supplemented with 10%
arabinoxylan (HF-AX) for 4 weeks. Values are expressed relative
to CT group (set at 1). *p,0.05 versus CT and1p,0.05 versus HF
(ANOVA). ZO-1, zonula occludens-1.
(TIF)
mRNA levels of key markers in jejunum
Figure S3
ylan (AX) in vivo. Eighteen male C57bl6/J mice (10 week old)
were housed in groups of 3 per cage in a controlled environment
with free access to HF diet. After 3 days for acclimatisation, the
mice were divided into 3 groups (n=6/group): a group fed with a
HF diet, a group fed the same HF diet supplemented with 10%
AX (HF-AX) and a group fed with the HF diet supplemented with
10% chitosan (KiOnutrime-CsTMfrom KitoZyme sa, Belgium,
HF-Cs). Food intake was recorded and mice were killed 12 h after
access to the diets. Lipid content, fatty acids and cholesterol
concentration in the caecal content were determined as previously
described [50]. Proportion of caecal lipids versus ingested lipids
(A), caecal pool of fatty acids (B) and caecal pool of cholesterol (C);
*p,0.05 versus HF (ANOVA).
(TIFF)
Analysis of fat binding capacity of arabinox-
Table S1
the high fat diet.
(DOC)
The full composition of the control diet and
Table S2
of mice fed a standard diet (CT), a high fat diet (HF) or a
high fat diet supplemented with 10% arabinoxylan (HF-
AX) for 4 weeks.
(DOC)
Gene expression in the liver and in the muscle
Table S3
bacteria in the caecal content (expressed as Log10
(bacterial cells/ total caecal content wet weight) with
metabolic parameters that were significantly affected by
AX treatment.
(DOC)
Analysis of correlation between the number of
Acknowledgments
We would like to thank Christine Turu and Eric Mignolet from the Institut
des Sciences de la Vie (UCL) for their excellent technical assistance in the
fatty acid profile analysis.
Author Contributions
Conceived and designed the experiments: AMN NMD. Analyzed the data:
AMN CD NMD. Wrote the paper: AMN NMD. Performed the in vivo
experiments and biochemical analysis: AMN CD FDB. Performed RNA
extraction in tissues and measured mRNA levels by Q-PCR: CD FDB.
Participated to the in vivo follow-up of animal study, OGTT and sampling
at the end of the experiment: PDC. Performed and interpreted gut
microbiota analysis (DGGE and PCA, Q-PCR): SP TVDW. Analyzed the
data of fatty acid composition in the adipose tissue: YL. Provided
intellectual input on the paper and reviewed the paper: PDC SP TVDW
NMD YL. Planned and supervised all experiments and manuscript
preparation: NMD.
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Prebiotic Properties of Arabinoxylan in Obese Mice
PLoS ONE | www.plosone.org12 June 2011 | Volume 6 | Issue 6 | e20944