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An increase in the Akkermansia spp. Population induced by metformin

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 Gut Online First, published on June 26, 2013 as 10.1136/gutjnl-2012-303839
▸ Additional material is
published online only. To view
please visit the journal online
(http://dx.doi.org/10.1136/
gutjnl-2012-303839).
1 physiology and metabolism of the host. Metformin is
Department of Life and
Nanopharmaceutical Sciences
and Department of Biology,
Kyung Hee University, Seoul,
Korea
2
Department of Medicine and
Department of Biotechnology
& Bioengineering, Samsung
Medical Center, Sungkyunkwan
University School of Medicine,
Seoul, Korea
Correspondence to
Dr Jin-Woo Bae,
Department of Life and
Nanopharmaceutical Sciences
and Department of Biology,
Kyung Hee University, Seoul
130-701, Korea; baejw@khu.
ac.kr
or Dr Myung-Shik Lee,
Department of Medicine,
Samsung Medical Center,
Sungkyunkwan University
School of Medicine, Seoul 135-
710, Korea; mslee0923@skku.
edu
N-RS, J-CL and H-YL
contributed equally to this
work.
Received 28 September 2012
Revised 15 May 2013
Accepted 30 May 2013
To cite: Shin N-R, Lee J-C,
Lee H-Y, et al. Gut
Published Online First:
[please include Day Month
Year] doi:10.1136/gutjnl-
2012-303839
Copyright
Shin N-R, et Article authordoi:10.1136/gutjnl-2012-303839
al. Gut 2013;0:1–9. (or their employer) 2013. Produced by BMJ Publishing Group Ltd (& BSG) under licence.
Gut microbiota
ORIGINAL ARTICLE
An increase in the Akkermansia spp. population
induced by metformin treatment improves glucose
homeostasis in diet-induced obese mice
Na-Ri Shin,1 June-Chul Lee,2 Hae-Youn Lee,2 Min-Soo Kim,1 Tae Woong Whon,1
Myung-Shik Lee,2 Jin-Woo Bae1
ABSTRACT
Background Recent evidence indicates that the
composition of the gut microbiota contributes to the
development of metabolic disorders by affecting the
one of the most widely prescribed type 2 diabetes (T2D)
therapeutic agents.
Objective To determine whether the antidiabetic effect
of metformin is related to alterations of intestinal
microbial composition.
Design C57BL/6 mice, fed either a normal-chow diet or a
high-fat diet (HFD), were treated with metformin for
6 weeks. The effect of metformin on the composition of
the gut microbiota was assessed by analysing 16S rRNA
gene sequences with 454 pyrosequencing. Adipose tissue
inflammation was examined by flow cytometric analysis of
the immune cells present in visceral adipose tissue (VAT).
Results Metformin treatment significantly improved the
glycaemic profile of HFD-fed mice. HFD-fed mice treated
with metformin showed a higher abundance of the mucin-
degrading bacterium Akkermansia than HFD-fed control
mice. In addition, the number of mucin-producing goblet
cells was significantly increased by metformin treatment
(p<0.0001). Oral administration of Akkermansia
muciniphila to HFD-fed mice without metformin
significantly enhanced glucose tolerance and attenuated
adipose tissue inflammation by inducing Foxp3 regulatory
T cells (Tregs) in the VAT.
Conclusions Modulation of the gut microbiota (by an
increase in the Akkermansia spp. population) may contribute
to the antidiabetic effects of metformin, thereby providing a
new mechanism for the therapeutic effect of metformin in
patients with T2D. This suggests that pharmacological
manipulation of the gut microbiota in favour of Akkermansia
may be a potential treatment for T2D.
INTRODUCTION
Worldwide prevalence of obesity has increased over
the past 30–40 years owing to changes in dietary pat-
terns and reduced physical activity.1 The condition
has now become a pandemic.2 3 Although the
primary reason for obesity is an imbalance between
dietary energy intake and expenditure, it is becoming
clear that the gut microbiota plays a role in control-
ling energy balance and immune homeostasis.4–11
The human gut microbiota comprises 10–100 trillion
microorganisms and more than 1000 different bac-
terial species.12 It also plays important roles in regu-
lating the host’s metabolism and extracting energy
from ingested food.4 8 10 13
Significance of this study
What is already known about this subject?
▸ Alterations in the gut microbiota composition
play a role in the development of adipose
tissue inflammation and in the pathogenesis of
obesity-induced diabetes.
▸ The presence of the mucin-degrading
bacterium, Akkermansia spp., is associated
with healthy mucosa.
▸ Immune cells resident in the adipose tissue are
critical promoters of obesity and insulin
resistance.
What are the new findings?
▸ The microbial community in the intestine of
metformin-treated high-fat diet-fed (HFD-Met)
mice exhibited a unique microbial consortium
that was distinct from that in the HFD-fed
control mice (HFD-CT) or normal-chow diet
(NCD)-fed mice (NCD-CT and NCD-Met).
▸ Marked changes in the abundance of 29
genera, particularly mucin-degrading
Akkermansia, accounted for the observed
differences in the gut microbial communities
following a HFD or metformin treatment.
▸ The number of mucin-producing goblet cells
increased upon metformin treatment, regardless
of the dietary composition or glucose tolerance.
▸ Akkermansia-administered HFD-fed mice
(HFD-Akk) showed improved glucose tolerance
and an increase in the number of goblet cells
and adipose tissue-resident CD4 Foxp3
regulatory T cells (Tregs).
How might it impact on clinical practice in
the foreseeable future?
▸ This study provides circumstantial evidence that
modulation of the gut microbial community,
either by metformin treatment or by
Akkermansia administration, may result in an
improved metabolic profile in patients with
type 2 diabetes.
Apart from its beneficial functions for the host,
gut microbiota can potentially take part in patho-
physiological interactions with the host, particularly
1
 Gut microbiota
in the case of obesity and related metabolic disorders. Recent
studies have shown that changes in the gut microbiota may play
a role in the pathogenesis of the obese and diabetic phenotypes.
For example, germ-free mice are protected from high-fat diet
(HFD)-induced obesity and metabolic dysfunction, including
glucose intolerance.4 6 In addition, colonisation of germ-free
animals with gut microbiota isolated from conventionally raised
obese donors led to a significant increase in body fat content
and insulin resistance in recipient mice.10 14 Similarly, the trans-
fer of gut microbiota from Toll-like receptor 5-deficient mice to
wild-type germ-free mice also transferred the diabetic pheno-
type of the Toll-like receptor 5-deficient donor to the recipi-
ent.15 Taken together, these studies suggest that alterations in
the gut microbial community increase the capacity of the host to
extract energy from a given diet, thereby triggering the develop-
ment of obesity and diabetes. This indicates a link between the
gut microbiota and the diabetic phenotype of the host.
Obesity and type 2 diabetes (T2D) are also associated with
chronic low-grade tissue inflammation.16 17 Numerous studies
suggest that adipose tissue is not just a fat storage depot; it also
controls systemic metabolism by acting as an endocrine organ.18
Moreover, inflammation within adipose tissue disrupts triglycer-
ide storage, and the subsequent excess of free fatty acids induces
insulin resistance.19 At the cellular level, immune cells present
in adipose tissue, such as neutrophils and macrophages, partici-
pate in obesity-induced pathology.20 21 Of the different types of
immune cells that reside in adipose tissue, CD4 Foxp3 regula-
tory T cells (Tregs) control local inflammation by counteracting
the infiltration of M1 macrophages and CD8 T cells, thereby
preventing adipose tissue inflammation.22 The protective role
played by these Tregs has also been demonstrated in several
animal models of obesity and insulin resistance, such as leptin-
deficient mice and HFD-fed mice.23 Both the percentage and
number of Tregs in the adipose tissue of obesity-induced dia-
betic animals are considerably lower than those in lean controls.
Metformin (1,1-dimethylbiguanide hydrochloride) has been
widely used to treat T2D for the past 50 years.24–26 In T2D,
increased hepatic glucose production is an important cause of
hyperglycaemia. Although the precise mechanism underlying
the glucose-lowering effect of metformin is not fully under-
stood, the most commonly accepted mechanism involves the
suppressed transcription of genes involved in gluconeogenesis
via activation of AMP-activated protein kinase (AMPK), an
enzyme that senses cellular energy levels and regulates the avail-
ability of fuel.27–29 The intestinal microbiota may also control
the activation status of AMPK. For example, germ-free mice
that are protected against diet-induced obesity show increased
levels of AMPK activity in the liver and in muscle tissue.6
Furthermore, the intestines themselves make an important con-
tribution to the glucose-lowering effect of metformin.30–32
Several studies report much higher concentrations of metformin
in the intestinal mucosa than in other tissues.31 32 These find-
ings raise the possibility that metformin may have both direct
and indirect effects on the gut microbiota, which may in turn
contribute to its antidiabetic effects.
However, no experimental or clinical studies have examined
the effects of antidiabetic drugs on the gut microbiota, although
gut microbial dysbiosis has been reported in patients with
T2D.33 34 Therefore, questions about whether metformin, as an
antidiabetic agent, regulates glucose metabolism by modulating
the gut microbiota remain unanswered. This study examined the
therapeutic effect of metformin on the progression of the dia-
betic phenotype in HFD-induced obese and diabetic mouse
models and studied the possible contribution of the gut
2 Shin N-R, et al. Gut 2013;0:1–9. doi:10.1136/gutjnl-2012-303839
microbiota. The results show that metformin induces a pro-
found shift in the composition of the gut microbiota community
and suggest a mechanism by which this drug exerts its thera-
peutic effect in patients with diabetes.
MATERIALS AND METHODS
Animals
C57BL/6 mice (4-week-old, n=24; Charles River Laboratories,
L’Arbresle, France) were maintained in groups of no more than
six mice per cage with free access to food and water. Mice were
fed either a normal-chow diet (NCD) or a HFD (60% fat, 20%
protein, 20% carbohydrate (kcal/100 g), #D12492; Research
Diets, New Brunswick, New Jersey, USA). After 8 weeks, the
mice were divided into four groups of six and fed the following
diets: (1) a NCD without metformin treatment (NCD-CT), (2)
a NCD with metformin treatment (NCD-Met), (3) a HFD
without metformin treatment (HFD-CT) or (4) a HFD with
metformin treatment (HFD-Met). The two groups of
metformin-treated mice (NCD-Met and HFD-Met) received
300 mg/kg/day of metformin by oral gavage for a period of
6 weeks. Body weight was monitored once a week. Akkermansia
muciniphila MucT (CIP107 961) were cultured anaerobically on
brain–heart infusion (BBL) agar plates containing porcine mucin
(Sigma) at 37°C. The bacteria were harvested at the late expo-
nential growth phase, suspended in thioglycolate–phosphate-
buffered saline (PBS) (4.0×108 cfu) and orally administered to
HFD-fed mice (HFD-Akk; n=6).
Biochemical analyses
Glucose homeostasis
Mice were fasted overnight and a glucose tolerance test was per-
formed after an intraperitoneal injection of glucose (1 g/kg body
weight). The homeostasis model assessment of insulin resistance
(HOMA-IR) index was calculated as previously described.35
Blood glucose concentrations were measured with an
Accu-Check glucometer (Roche) both before (0 min) and after
(15, 30, 60, 120 and 180 min) the glucose injections.
Histology
The distal ileum was removed from all the mice, flushed with
PBS, fixed in 4% formaldehyde overnight at room temperature
and paraffin-embedded. The specimens were then cut into 5 μm
sections and stained with Alcian blue periodic acid–Schiff (PAS)
(the goblet cells were stained blue). The results were expressed
as the number of goblet cells per intestinal villus.
Analysis of gut microbiota and quantification of
Akkermansia muciniphila
The methods used to analyse the gut microbiota and to quantify
A muciniphila are described in the online supplementary
methods (see online supplementary table S1).
Isolation of stromal vascular fraction cells and flow
cytometry
The visceral adipose tissue (VAT) was excised and minced into
small pieces (>2 mm) and incubated for 20 min in a collagenase
solution (1 mg/mL of collagenase type 2 (Worthington) in
Dulbecco’s modified Eagle’s medium). The digested tissue was
centrifuged at 1000g for 8 min. The pellet containing the
stromal vascular fraction (SVF) was resuspended in PBS and fil-
tered through a 70 μm mesh filter. The cells were then washed
with PBS, incubated in red blood cell (RBC) lysis buffer (Sigma)
for 3 min and resuspended in fluorescence-activated cell sorting
(FACS) buffer (2 mM EDTA, 2% fetal bovine serum in PBS).

The isolated cells were then incubated with labelled monoclonal
antibodies (eBioscience and BD Pharmingen) and analysed by
flow cytometry using a FACSAria flow cytometer (Becton
Dickinson). Data were analysed with FlowJo software (Tree Star,
Inc). We validated the flow cytometric identification of CD11c
F4/80 CD11b (M1) and CD206 F4/80 CD11b (M2) adipose
tissue macrophages. The percentage of Tregs was analysed by
three-colour flow cytometry after staining with antibodies to
CD3, CD4 and mouse/rat Foxp3 (FJK-16 s), as previously
described.36
Statistical analysis
Data were expressed as the mean±SEM. Significance of the dif-
ferences between the groups of mice was assessed using the
Student t test. For experiments comparing multiple groups, the
differences were analysed by one-way analysis of variance fol-
lowed by Duncan’s post-hoc test. p Values <0.05 were consid-
ered significant. The nearest shrunken centroid (NSC) method
was used to detect the bacterial genera that were specifically
over- or under-represented within each category (diet, treatment
or diet–treatment combinations).37 The amount of shrinkage
was chosen to minimise the overall misclassification error. These
analyses allowed the identification of bacterial genera whose
relative abundance was significantly different between the cat-
egories.38 The analysis was performed using the Predictive
Analysis for Microarrays package under R software.
RESULTS
Metformin improves glucose homeostasis under HFD
conditions
To assess the effect of metformin on obesity and glucose intoler-
ance, we used a glucose tolerance test to examine glucose
homeostasis in metformin-treated and non-treated mice fed
either a NCD or a HFD. As expected, in comparison with
NCD-CT mice, HDF-CT mice showed an increase in the area
under the curve (AUC), an increased HOMA-IR index and an
increased incidence of fasting hyperglycaemia (figure 1), all of
which suggest glucose intolerance. Total body weight and fat
pad weight also increased in mice fed a HFD (see online supple-
mentary figure S1). Compared with HFD-CT mice, HFD-Met
mice showed a significant improvement in glucose tolerance,
fasting blood glucose levels and the HOMA-IR index (p<0.01)
(figure 1A–D), demonstrating that metformin has an anti-
hyperglycaemic effect. Metformin treatment did not affect
fasting blood glucose levels, glucose tolerance or the HOMA-IR
index in NCD-fed mice. In contrast to its effects on glucose
metabolism, 6 weeks of metformin treatment had no effect on
body weight or fat pad weight in NCD- or HFD-fed mice (see
online supplementary figure S1), indicating that the improve-
ments in glucose tolerance seen in the HFD-Met mice are not
related to changes in body weight or fat mass.
Effect of metformin on the composition of the gut microbial
community
Since the composition of the gut microbiota is associated with
T2D,33 we next determined the effect of metformin on the
composition of the gut microbiota using metagenomic analysis
based on 454 pyrosequencing technology. A total of 121 051
raw reads were obtained from 20 fecal samples. After perform-
ing quality control procedures to remove low-quality sequences,
63 132 sequences (with an average length of 370±25 bases)
were subjected to further analysis (see online supplementary
table S2).
Shin N-R, et al. Gut 2013;0:1–9. doi:10.1136/gutjnl-2012-303839
Gut microbiota
Classification of the sequences was carried out by non-OTU
(operational taxonomic units)-based assessment using the
Ribosomal Database Project classifier. All the sequences were
assigned to eight bacterial phyla, comprising three phyla that
dominate the distal gut in both mice and humans: Firmicutes,
Bacteroidetes and Actinobacteria.8 Although all individuals are
born with a specific gut microbiome, dietary factors have a sig-
nificant influence on the development of the gut microbial com-
position.8 10 13 We observed that HFD-CT mice had a greater
abundance of Firmicutes (p<0.001) and a lower abundance of
Bacteroidetes (p<0.001) than NCD-CT mice (see online supple-
mentary figure S2A). In addition, HFD-CT mice showed a sig-
nificantly lower abundance of the phyla Verrucomicrobia
(p<0.001) and TM7 ( p<0.01) than NCD-CT mice.
Thus, we hypothesised that metformin may alter the compos-
ition of the gut microbiota in parallel with its antidiabetic effect
on host glucose metabolism. Metformin treatment resulted in a
profound shift in the fecal microbial community profiles of the
gut microbiota in diabetic mice (see online supplementary figure
S2B). The tendency toward phyla-wide changes after metformin
treatment was similar in NCD- and HFD-fed mice; however,
the changes were more marked in the HFD-fed group. There
were significant differences in the abundance of Firmicutes and
Bacteroidetes between HFD-CT mice and HFD-Met mice
(p<0.001), but there was no significant difference in the abun-
dance of these two phyla between NCD-Met mice and
NCD-CT mice. Importantly, the abundance of Verrucomicrobia
was significantly increased in the HFD group after metformin
treatment ( p<0.001), whereas no significant change was seen in
the NCD group. This suggests that metformin-induced modula-
tion of the gut microbiota is diet-dependent.
The overall composition of the bacterial community in the
different groups was characterised by quantifying and interpret-
ing similarities based on phylogenetic distances using UniFrac
metric. The principal coordinates analysis of UniFrac-based pair-
wise comparisons of community structures disclosed an unex-
pected distribution of the microbial community among the four
groups of mice. The main finding of the principal coordinates
analysis was that different diets promote the development of dif-
ferent gut microbial communities. Furthermore, HFD-Met mice
formed a cluster that was distinct from that of HFD-CT mice
and not even intermediate between those of the control
HFD-fed and NCD-fed mice (figure 2). The microbial commu-
nities of the NCD-fed groups were, however, closely clustered,
even in mice treated with metformin; this indicates that metfor-
min has a more marked effect on gut microbial community
composition in HFD-fed mice than it does in NCD-fed mice.
Taken together, these results indicate that metformin has differ-
ent effects on the gut microbial composition of NCD- and
HFD-fed mice.
Next, to determine which bacterial genera contributed to
these differences in microbiota composition between the four
groups, we performed a NSC analysis. The specifically defined
comparisons between community characteristics were tabulated
on a heat map representing the relative abundance of each
genus. By combining both statistical and NSC analyses, we
found that changes in the abundance of 29 genera, belonging to
six phyla, accounted for the differences in the gut microbial
communities seen in mice fed different diet or metformin treat-
ment (figure 3), which suggests the possibility that the antidia-
betic effect of metformin may be mediated by a specific subset
of bacterial taxa. The relative abundance of Anaerotruncus,
Lactococcus, Akkermansia, Parabacteroides, Odoribacter,
Alistipes, Lawsonia, Blautia and Lactonifactor was altered by a
3
 Gut microbiota

Figure 1 Metformin improves glucose homeostasis in HFD-fed (diabetic) mice. The glucose tolerance test (GTT) (A), the area under the curve
(AUC) for the GTT curves (B), the fasting glucose levels (C) and the homeostasis model assessment of insulin resistance (HOMA-IR) index (D) are
shown for both control (CT) and metformin-treated (Met) mice fed a normal-chow diet (NCD) or a high-fat diet (HFD). All mice were fasted
overnight (16 h) before the GTT. Data are expressed as the mean±SEM (n=6 mice/group). The letters denote the level of statistical significance
( p<0.05) using one-way analysis of variance followed by Duncan’s post-hoc test.

HFD; however, metformin rescued these HFD-induced
changes, restoring the levels to those seen in NCD-fed mice.
Despite the low relative abundance (0.3–2.9%) of sequences
assigned to Akkermansia, this genus made a large contribution
to the observed differences in the composition of the gut micro-
bial communities in HFD-CT and HFD-Met mice. Moreover,
Akkermansia was responsible for the greater abundance of
Verrucomicrobia seen in HFD-Met mice compared with
HFD-CT mice. Thus, metformin restored the relative abun-
dance of specific bacterial genera in HFD-Met mice to that seen
in NCD-fed mice, an action that may play a role in its glucose-
lowering effects.
The unexpected changes in the composition of the gut micro-
biota in HFD-Met mice prompted us to examine whether deplet-
ing the microbiota using broad-spectrum antibiotics would
abrogate the antidiabetic effects of metformin. To examine this,
HFD-fed mice were treated with a combination of antibiotics
(carbenicillin, metronidazole, neomycin and vancomycin) before
metformin treatment. It is noteworthy that HFD-fed mice
treated with antibiotics, but not treated with metformin
(HFD-Abx), showed a significant improvement in glucose toler-
ance in comparison with HFD-CT mice ( p<0.05) (see online
supplementary figure S3). However, the reduction in the AUC
seen for metformin- and antibiotic-treated HFD-fed mice
(HFD-Abx+Met) was not significantly different from that seen
for HFD-Abx mice. This suggests that antidiabetic effect of met-
formin can be abrogated in the absence of the gut microbiota.
Metformin treatment increases the number of goblet cells
Because metformin significantly increased the relative abun-
dance of Akkermansia in HFD-Met mice and Akkermansia uses
mucus as a nutrient source,39 we next examined the ileum in
mice from the different treatment groups and counted the
number of mucin-producing goblet cells. PAS–Alcian blue stain-
ing showed that metformin treatment increased the number of
PAS-positive goblet cells (figure 4A–D). To evaluate these
changes further, we counted the number of PAS-positive goblet
cells per villus. An average of 6.6±0.3 goblet cells was seen in
HFD-CT mice, which was lower than that in NCD-CT mice
(7.8±0.2) (figure 4E). Interestingly, metformin treatment

Figure 2 Cluster analysis of control (CT) or metformin-treated (Met) mice fed a normal-chow diet (NCD) or a high-fat diet (HFD). Bacterial
communities were clustered using the principal coordinates analysis (PCoA) of unweighted (A) and weighted (B) UniFrac distance matrices. The first
two principal coordinates (PC1 and PC2) from the PCoA of unweighted and weighted UniFrac are plotted for each sample. The percentage variation
in the plotted principal coordinates is indicated on the axes. Each spot represents one sample and each group of mice is denoted by a different
colour.

4 Shin N-R, et al. Gut 2013;0:1–9. doi:10.1136/gutjnl-2012-303839

 Gut microbiota

Figure 3 Striking differences in the

abundance of the different bacterial
genera that comprise the gut microbial
communities in NCD-fed and HFD-fed
mice in CT and Met mice. (A) Nearest
shrunken centroids of the 29 genera
that account for the differences in the
composition of the microbial
communities between the four groups
of mice. For the genera listed in the
centre, those that are over-represented
are designated by rightward-extending
bars. The bars denoting
under-represented genera extend to
the left. The length of the bar indicates
the strength of the effect. (B) Heat
map showing the relative abundance
of each classified genus. Each column
on the heat map represents a sample
of 20 mice (n=5 mice/group) and each
row represents the genus listed in the
centre. A range of colours, from yellow
to black, indicates the prevalence of
each genus. All the genera listed in the
centre showed significant differences
( p<0.05; one-way analysis of variance
followed by Duncan’s post-hoc test).
NCD-CT, normal-chow diet (NCD)-fed
control mice; NCD-Met, NCD-fed
metformin-treated mice; HFD-CT,
high-fat diet (HFD)-fed control mice;
HFD-Met, HFD-fed metformin-treated
mice.

significantly increased the number of goblet cells in both NCD-
and HFD-fed mice (NCD-Met, 9.1±0.2; HFD-Met, 9.5±0.5;
p<0.001), indicating that metformin alters the goblet cell popu-
lation regardless of metabolic profile or dietary composition. In
addition, Pearson correlation analysis showed that the number
of goblet cells was positively correlated with the abundance of
Akkermansia (figure 4F).
Administration of A muciniphila improves insulin signalling
by inducing VAT-resident CD4 Foxp3 Tregs
Since we observed both a greater abundance of Akkermansia and
an increase in the number of goblet cells in HFD-Met mice, we
next investigated whether a selective increase in the Akkermansia
population had any effect on the metabolic profile or the number
of goblet cells in HFD-fed mice. Mice were fed a HFD for

Figure 4 The goblet cell population

is affected by metformin treatment.
(A–D) Representative light
photomicrographs showing ileal
sections stained with periodic acid–
Schiff (PAS)/Alcian blue. (A) NCD-CT
mice. (B) NCD-Met mice. (C) HFD-CT
mice. (D) HFD-Met mice. (E) Changes
in goblet cells (blue in A–D) are
expressed as means±SEM of the
number of PAS-positive goblet cells per
villus. The letters above the bars
denote statistically significant
differences ( p<0.05) between the
means (one-way analysis of variance
followed by Duncan’s post-hoc test).
(F) The relationship between the
relative abundance of Akkermansia
and the number of goblet cells
(Pearson’s correlation test; r=0.4996,
p=0.02). NCD-CT, normal-chow diet
(NCD)-fed control mice; NCD-Met,
NCD-fed metformin-treated mice;
HFD-CT, high-fat diet (HFD)-fed control
mice; HFD-Met, HFD-fed
metformin-treated mice.

Shin N-R, et al. Gut 2013;0:1–9. doi:10.1136/gutjnl-2012-303839 5

 Gut microbiota
Figure 5 Akkermansia administration
improves glucose tolerance and
modulates the number of goblet cells
in HFD-fed mice. (A) Changes in goblet
cells are expressed as the number of
periodic acid–Schiff (PAS)-positive
goblet cells per villus. (B)
Representative light photomicrographs
showing ileal sections stained with
PAS/Alcian blue. (C) Area under the
curve (AUC) obtained after the glucose
tolerance test (GTT). The results are
representative of three independent
experiments. All data are expressed as
the mean±SEM (n=6 mice/group). p
Values were determined using a
one-tailed unpaired t test. *p<0.05;
**p<0.01; ***p<0.001. NCD-CT,
normal-chow diet (NCD)-fed control
mice; HFD-CT, high-fat diet (HFD)-fed
control mice; HFD-Met, HFD-fed
metformin-treated mice; HFD-Akk,
HFD-fed Akkermansia-administered
mice.
4 weeks, after which they were given 4.0×108 cfu of A mucini-
phila orally every day for a further 6 weeks. Colonisation of
Akkermansia was confirmed by calculating the relative fractional
abundance of Akkermansia by quantitative PCR. Despite differ-
ences in the initial abundance of Akkermansia between HFD-CT
and HFD-Akk mice, Akkermansia treatment prevented any
decrease in abundance in HFD-fed mice as they aged (see online
supplementary figure S4). Consistent with the positive correl-
ation between the number of goblet cells and the abundance of
Akkermansia, supplementation with Akkermansia restored both
the number ( per villus) and density ( per unit surface area) of
goblet cells in HFD-fed mice (figure 5A–B and online supple-
mentary figure S5).
Furthermore, HFD-Akk mice showed a significant improve-
ment in glucose tolerance ( p<0.01) (figure 5C), similar to that
seen in HFD-Met mice. This indicates that Akkermansia has
the potential to ameliorate the glucose tolerance induced by a
HFD. However, treatment with a lower dose (4.0×106 cfu) of
live Akkermansia or with the same dose of autoclaved (dead)
cells did not ameliorate the impaired glucose tolerance (see
online supplementary figure S6), suggesting that the bacteria
must be metabolically active and used at a dose > 4.0×107 cfu
to exert their beneficial effect. We next studied the effect of
Akkermansia on metabolic hormones. As expected, metformin
significantly reduced the increases in serum insulin and leptin
levels induced by a HFD ( p<0.001 for insulin; p<0.05 for
leptin) (see online supplementary figure S7A and B), an effect
that is probably secondary to improvements in glucose homeo-
stasis. Similarly, HFD-Akk mice showed reduced serum concen-
trations of insulin and leptin compared with HFD-CT mice,
although the differences were not statistically significant (see
online supplementary figure S7A and B). The HFD-induced
increases in serum lipopolysaccharide (LPS) levels reduced by
metformin or Akkermansia, suggesting that the decreased endo-
toxaemia seen in HFD-Akk mice might be related to improved
glucose tolerance. However, the differences were not statistic-
ally significant compared with HFD-CT mice (see online sup-
plementary figure S7C). Furthermore, neither metformin nor
Akkermansia affected intestinal permeability in HFD-fed mice
(see online supplementary figure S7D).
6 Shin N-R, et al. Gut 2013;0:1–9. doi:10.1136/gutjnl-2012-303839
Because changes in intestinal permeability cannot explain the
improved metabolic profile seen after Akkermansia administra-
tion, we next examined the possibility that Akkermansia might
exert an antidiabetic effect by reducing SVF inflammation in the
VAT, which has been implicated in the aetiology of insulin resist-
ance.21 40 FACS analysis showed a significant increase in the
proportion of M1 macrophages and a decrease in the propor-
tion of M2 macrophages in the VAT of HFD-CT mice compared
with that in NCD-CT mice. However, M1/M2 macrophage
polarisation in the SVF of HFD-fed mice was not affected by
Akkermansia or metformin (see online supplementary figure
S8). In addition, we also observed a significant difference in the
CD4/CD8 T cell ratio in the SVF of NCD-CT and HFD-CT
mice; however, the CD4/CD8 T cell ratio in the SVF of
HFD-fed mice was not affected by Akkermansia or metformin
(see online supplementary figure S9).
We next examined the number of Tregs, which are critical
regulators of immune or inflammatory responses in the adipose
tissues of obese animals. We observed a significant reduction in
the proportion of Tregs in the SVF from the VAT of HFD-CT
mice compared with that in NCD-CT mice (p<0.05) (figure 6A
and C), consistent with previous reports.23 Interestingly, admin-
istration of Akkermansia to HFD-fed mice restored the Treg
proportion to a level comparable to that in NCD-CT mice
(NCD-CT, 49.5±2.3; HFD-CT, 32.5±5.7; HFD-Akk, 52.47
±2.176; p<0.01; figure 6A and C). There was also a significant
increase in the overall number of Tregs within the SVF in
HFD-Met and HFD-Akk mice compared with that in HFD-CT
mice (figure 6B). When we examined the expression of proin-
flammatory cytokines that play a direct role in tissue inflamma-
tion and insulin resistance, we found that the expression of
interleukin (IL)-6 and IL-1β mRNA was significantly increased
in HFD-CT mice compared with that in NCD-CT mice;
however, after treatment with Akkermansia, the expression of
IL-6 and IL-1β was similar to that in NCD-CT mice (figure 6D
and E). Thus, a single bacterial species, A muciniphila, can
attenuate VAT inflammation by inducing the generation of Tregs
and suppressing the production of proinflammatory cytokines in
the SVF, thereby leading to increased insulin signalling in
HFD-fed mice.
 Gut microbiota

Figure 6 The induction of Tregs and

a reduction in the levels of
proinflammatory cytokines attenuate
visceral adipose tissue (VAT)
inflammation in Akkermansia
treatment to HFD-fed mice. (A)
Percentage of Foxp3 Tregs within the
CD3 CD4 T cell population in the
stromal vascular fraction (SVF) of the
VAT. (B) Absolute number of CD3 CD4
Foxp3 Tregs per million cells in the SVF
of the VAT. (C) Representative
fluorescence activated cell sorting plots
showing the gated Foxp3 Tregs within
the total SVF CD4 T cell population in
the VAT of each mouse. (D and E)
Relative expression of interleukin (IL)-6
and IL-1β mRNA in the VAT. All data
are expressed as the mean±SEM (n=6
mice/group). p Values were determined
using a one-tailed unpaired t test.
*p<0.05; **p<0.01; ***p<0.001.
NCD-CT, normal-chow diet (NCD)-fed
control mice; HFD-CT, high-fat diet
(HFD)-fed control mice; HFD-Met,
HFD-fed metformin-treated mice;
HFD-Akk, HFD-fed
Akkermansia-administered mice.

DISCUSSION reduction in the AUC over that seen in HFD-Abx mice.

The gut microbiota is highly associated with obesity and T2D.8–10 33
Metformin is one of the most common drugs used to treat patients
with T2D.25 41 Here, we used a metagenomic approach to demon-
strate a role for metformin in modulating the composition of the gut
microbiota in diet-induced obese and diabetic C57BL/6 mice.
Consistent with previous findings,7 8 10 42 a HFD led to dysbiosis,
increased fasting glucose levels and glucose intolerance. Metformin
treatment significantly improved the hyperglycaemia in HFD-CT
mice; however, the gut microbiota of HFD-Met mice showed an
altered microbial composition, which was distinct from that in
HFD-CT or NCD-CT mice. Based on these results, we suggest that
the gut microbiota might be a contributing factor to the glucose-
lowering effect of metformin.
Depending on the diet fed to the mice, metformin had differ-
ent effects on microbial composition. The gut microbial com-
munity in HFD-Met mice was distinct from that in NCD-CT
and NCD-Met mice (figure 2). Specifically, NSC analysis identi-
fied several genera that contributed to the observed differences
in the distribution of bacterial communities between the four
groups (figure 3). Metformin offset the changes in the propor-
tions of several genera that were induced by a HFD. For
example, the significant reductions in the proportions of
Akkermansia and Alistipes and the increases in the proportions
of Anaerotruncus, Lactococcus, Parabacteroides, Odoribacter,
Lawsonia, Blautia and Lactonifactor, seen in HFD-CT mice,
were reversed by metformin. Consequently, the proportions of
these genera were similar in HFD-Met and NCD-CT mice (the
non-diabetic phenotype). Depleting the gut microbiota using a
combination of antibiotics before metformin treatment abro-
gated the antidiabetic effects of the drug, as indicated by the
finding that HFD-Abx+Met mice did not show any additional
However, both HFD-Abx and HFD-Abx+Met mice showed a
significant improvement in glucose tolerance compared with
HFD-CT mice. Accordingly, recent studies suggest that antibio-
tics improve glucose homeostasis of HFD-fed mice.43 44
Furthermore, it is not possible to test whether the antidiabetic
effects of metformin are abrogated in germ-free animals because
HFD-induced dysbiosis is likely to be responsible for impaired
glucose homeostasis.4 6 Despite this, our study supports the
hypothesis that changes in the composition of the gut micro-
biota induced by antidiabetic drugs contribute to the improved
glucose homeostasis seen in diabetic mice.
Recently, several studies have highlighted the effects of mucus
degradation on host physiology.45 46 The mucin-degrading bac-
terium, A muciniphila, which belongs to the phylum
Verrucomicrobia, is more abundant in the mucosa of healthy
subjects than in that of diabetic patients or animals.47–50 Also,
the colonisation of germ-free mice with A muciniphila led to
altered host mucosal transcriptome profiles, which showed
balanced immune responses indicative of tolerance to com-
mensal bacteria.46 We observed that a HFD reduced the abun-
dance of Akkermansia, suggesting an unhealthy state in these
mice. The abundance of Akkermansia in HFD-Met mice was,
however, significantly higher than that in HFD-CT mice, sug-
gesting increased caecal mucin levels.45 In this regard, we
observed a greater number of mucin-producing goblet cells in
both NCD- and HFD-fed mice after metformin treatment. We
also noted a positive correlation between the abundance of
Akkermansia and the number of PAS-positive goblet cells (figure
4). Additionally, the amelioration of glucose intolerance and an
increase in the goblet cell number were seen in HFD-Akk mice
(figure 5). Akkermansia is a mucin-degrading bacterium and the

Shin N-R, et al. Gut 2013;0:1–9. doi:10.1136/gutjnl-2012-303839 7

 Gut microbiota
proportion of Akkermansia correlates with an increase in the
number of goblet cells; thus, an increase in the number of
goblet cells may underlie the improved glucose profiles seen
after Akkermansia administration.
In addition to producing components of the mucus layer,
goblet cells also produce molecules that are associated with
innate defence mechanisms, such as intestinal trefoil factor and
resistin-like molecule β. Because these molecules augment barrier
integrity,51 52 an increase in the number of goblet cells after
Akkermansia administration may attenuate glucose intolerance by
reducing LPS translocation across the intestinal barrier. However,
serum LPS levels and gut permeability were not significantly dif-
ferent in HFD-Akk and HFD-CT mice, suggesting that factors
other than changes in barrier function are responsible for the
improved metabolic profile found in HFD-Akk mice.
Of the various T cell subsets, Tregs play a key role in control-
ling the classic adaptive immune responses and also the innate
immune responses associated with obesity-induced chronic
inflammation.53 In this context, we observed a significant
decrease in the Treg population in HFD-CT mice compared with
NCD-CT mice. However, oral administration of Akkermansia to
HFD-fed mice restored both the percentage and absolute
number of Tregs within the total CD4 T cell population in the
VAT, which was similar to the effect of metformin treatment
(figure 6A–C). These results provide evidence that a selective
increase in the Akkermansia population improves the metabolic
profile of individuals with diet-induced obesity via a mechanism
that is mediated by the anti-inflammatory activity of VAT-resident
Tregs. In addition, the increases in IL-6 and IL-1β mRNA expres-
sion seen in the VAT after a HFD were abolished by Akkermansia
treatment (figure 6D and E). This is consistent with the findings
of previous studies suggesting that Akkermansia has anti-
inflammatory activity.48 54 Recent papers report that goblet cells
affect intestinal immune homeostasis by delivering antigens from
the lumen to tolerogenic dendritic cells, which then induce
Tregs.55 Thus, the increase in the number of goblet cells after
Akkermansia treatment might also induce Tregs; however, the
increased goblet cell population does not appear to modulate sys-
temic LPS or intestinal permeability. Also, the increased goblet
cell population might expedite tolerogenic dendritic cell
responses to Akkermansia under HFD conditions and dendritic
cell-augmented Treg activity might suppress inflammatory
changes in adipose tissues, leading to increased glucose tolerance.
In summary, our results show that metformin treatment
induced an unexpected change in the composition of the gut
microbiota in mice receiving a HFD, which may constitute a new
mechanism for the antidiabetic activity of metformin. Moreover,
these findings reinforce the concept that changes in the gut
microbial community, aimed at increasing the Akkermansia popu-
lation, can rescue the glucose intolerance associated with T2D.
Although it is still unclear which of the molecular mechanisms or
interactions associated with Akkermansia (and which govern
glucose homeostasis) are involved in increasing the goblet cell
and Treg populations, our pharmacological intervention study
provides circumstantial evidence that modulation of the gut
microbial community improves the health of patients with T2D.
It also supports the suggestion that Akkermansia has a potential
role as a probiotic with antidiabetic effects. It would be interest-
ing to undertake a follow-up study to identify the immunomodu-
latory molecule(s) and/or soluble factors produced by
Akkermansia and their related interactions.
Contributors N-RS, J-CL and H-YL contributed equally to this work. N-RS, J-CL
and H-YL performed the experiments and data analysis. N-RS, J-CL, M-SL and J-WB
8 Shin N-R, et al. Gut 2013;0:1–9. doi:10.1136/gutjnl-2012-303839
planned the experiments. N-RS, J-CL, M-SK, TWW, M-SL and J-WB wrote the
manuscript. M-SL and J-WB conceived and supervised the study.
Funding This work was supported by a grant from the Mid-Career Researcher
Program (2011–0028854 to J-WB) through the National Research Foundation of
Korea (NRF), funded by the Ministry of Education, Science and Technology (MEST)
and by the Bio R&D Program (2008–04090 to M-SL). M-SL is the recipient of a
global research laboratory grant from the National Research Foundation of Korea
(K21004000003-12A0500-00310).
Competing interests None.
Provenance and peer review Not commissioned; externally peer reviewed.
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