ARTICLE
Received 27 Mar 2015 | Accepted 25 May 2015 | Published 2 Jul 2015
DOI: 10.1038/ncomms8629
Farnesoid X receptor inhibits glucagon-like
peptide-1 production by enteroendocrine L cells
Mohamed-Sami Trabelsi1,2,3,4, Mehdi Daoudi1,2,3,4, Janne Prawitt1,2,3,4, Sarah Ducastel1,2,3,4, Véronique
Touche1,2,3,4, Sama I. Sayin5,6, Alessia Perino7, Cheryl A. Brighton8, Yasmine Sebti1,2,3,4, Jérôme Kluza2,9,
Olivier Briand1,2,3,4, Hélène Dehondt1,2,3,4, Emmanuelle Vallez1,2,3,4, Emilie Dorchies1,2,3,4, Grégory Baud1,2,10,11,
Valeria Spinelli1,2,3,4, Nathalie Hennuyer1,2,3,4, Sandrine Caron1,2,3,4, Kadiombo Bantubungi1,2,3,4,
Robert Caiazzo1,2,10,11, Frank Reimann8, Philippe Marchetti2,9, Philippe Lefebvre1,2,3,4, Fredrik Bäckhed5,6,12,
Fiona M. Gribble8, Kristina Schoonjans7, Franc¸ois Pattou1,2,10,11, Anne Tailleux1,2,3,4, Bart Staels1,2,3,4
& Sophie Lestavel1,2,3,4
Bile acids are signalling molecules, which activate the transmembrane receptor TGR5 and the
nuclear receptor FXR. BA sequestrants (BAS) complex bile acids in the intestinal lumen and
decrease intestinal FXR activity. The BAS–BA complex also induces glucagon-like peptide-1
(GLP-1) production by L cells which potentiates b-cell glucose-induced insulin secretion.
Whether FXR is expressed in L cells and controls GLP-1 production is unknown. Here, we
show that FXR activation in L cells decreases proglucagon expression by interfering with the
glucose-responsive factor Carbohydrate-Responsive Element Binding Protein (ChREBP) and
GLP-1 secretion by inhibiting glycolysis. In vivo, FXR deficiency increases GLP-1 gene
expression and secretion in response to glucose hence improving glucose metabolism.
Moreover, treatment of ob/ob mice with the BAS colesevelam increases intestinal
proglucagon gene expression and improves glycaemia in a FXR-dependent manner. These
findings identify the FXR/GLP-1 pathway as a new mechanism of BA control of glucose
metabolism and a pharmacological target for type 2 diabetes.
1 European Genomic Institute for Diabetes (EGID), FR 3508, Lille F-59000, France. 2 Université de Lille, Lille F-59000, France. 3 INSERM UMR 1011, Lille
F-59000, France. 4 Institut Pasteur de Lille, Lille F-59000, France. 5 Wallenberg Laboratory/Sahlgrenska Center for Cardiovascular and Metabolic Research,
Sahlgrenska University Hospital, Gothenburg 413445, Sweden. 6 Department of Molecular and Clinical Medicine, University of Gothenburg, Gothenburg
41345, Sweden. 7 Institute of Bioengineering, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne CH-1015, Switzerland.
8 Cambridge Institute for Medical Research and Institute of Metabolic Sciences, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 0XY, UK. 9 INSERM
U 837, Lille Cedex F- 59045, France. 10 Centre de Bio-Pathologie, Plate-forme de Biothérapie, Banque de Tissus, Centre Hospitalier Régional Universitaire,
Lille F-59000, France. 11 INSERM UMR 859, Lille F-59000, France. 12 Section for Metabolic Receptology and Enteroendocrinology, Novo Nordisk Foundation
Center for Basic Metabolic Research, Faculty of Health Sciences, University of Copenhagen, Copenhagen 2200, Denmark. Correspondence and requests for
materials should be addressed to B.S. (email: bart.staels@pasteur-lille.fr).
NATURE COMMUNICATIONS | 6:7629 | DOI: 10.1038/ncomms8629 | www.nature.com/naturecommunications
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1
ARTICLE
B
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms8629
ile acids (BAs) are amphipathic molecules derived from
cholesterol, synthesized and conjugated in the liver, stored
in the gallbladder, expelled in the intestinal lumen after
meal ingestion and further metabolized by the gut microbiota
into secondary BAs. Initially considered to be dietary lipid
detergents, BAs are now recognized as signalling molecules
which, through binding and activation of the membrane receptor
TGR5, the nuclear receptors vitamin D receptor, Pregnane X
Receptor and Farnesoid X Receptor (FXR, NR1H4), play key roles
in the control of energy homeostasis1. Secondary bile acids
are more potent ligands for TGR5 (affinity for TGR5:
LCA4DCA4CDCA4CA) than for FXR (affinity for FXR:
CDCA4DCA ¼ LCA4CA) (for review, see refs 1,2).
TGR5, a G protein-coupled receptor, first described as a
regulator of cytokine production in a human monocyte cell line3,
was also shown to promote glucagon-like peptide-1 (GLP-1)
secretion in response to BA by intestinal enteroendocrine L cells4.
Although representing o1% of the intestinal epithelial cells,
enteroendocrine cells play key roles in the regulation of energy
metabolism through their capacity to secrete various bio-active
peptides. After food ingestion, the incretins GLP-1 and glucosedependent insulinotropic polypeptide are secreted by L and K
cells, respectively, and subsequently potentiate postprandial
insulin secretion by pancreatic b cells in response to glucose,
the so-called incretin effect5. Even though the insulinotropic
property of GLP-1 is maintained, its secretion in response to a
meal is decreased in type 2 diabetic patients6,7. This observation
has led to the development of DPP-IV inhibitors that increase
GLP-1 half-life and GLP-1 mimetics, which improve glucose
homeostasis with less risk for hypoglycemia.
Together with TGR5, FXR controls BA-induced signalling
pathways. FXR, predominantly expressed in the liver and
intestine, was first identified as a regulator of hepatic BA
metabolism through the induction of small heterodimer partner
(SHP) in the liver and fibroblast growth factor 15 (FGF15) in
the intestine, resulting in the subsequent inhibition of the
rate-limiting enzyme in hepatic BA synthesis, cholesterol
7a-hydroxylase (CYP7A1) and the regulation of BA transporters
(for review, see refs 8,9). FXR in the liver also plays a modulatory
role in the regulation of lipid and glucose homeostasis. FXR
activation with the synthetic agonist GW4064 decreases hepatic
gluconeogenic gene expression and increases glycogenesis10 thus
improving glucose homeostasis in diabetic mice. In the
postprandial state, FXR activation in hepatocytes also inhibits
the induction of glycolytic gene expression by glucose through
negative interference with the carbohydrate response element
binding protein ChREBP (also known as MlxIPL)11,12. Recent
studies have highlighted a metabolic role of non-hepatic and
specifically intestinal FXR in mice under pathophysiological
conditions such as obesity13–16. Indeed, oral treatment of
high-fat-diet-fed mice with GW4064 promotes hyperglycaemia
and obesity13, whereas an opposite effect is observed upon
intraperitoneal injection of the drug10. Moreover, in two different
models of obesity, whole-body FXR deficiency improved
metabolic parameters, an effect not observed in hepatocytespecific FXR-deficient animals14. Furthermore, intestinal specific
FXR-deficient mice are protected against diet-induced obesity and
non-alcoholic fatty liver disease15,16. These studies also identified
a crucial role of the intestinal microbiota in BA signalling by
regulating the ratio of BAs with agonist or antagonist activity on
FXR. Indeed, the primary BA tauro beta-muricholic acid
(TbMCA), whose levels are elevated in germ-free (GF) mice,
acts as an FXR antagonist to improve glucose metabolism via
intestinal FXR15,17. Finally, BA pool composition is modulated by
BA sequestrants (BAS) (for review, see ref. 18). BAS, such as
colesevelam, are anion exchange resins that trap BAs in the
2
intestinal lumen. Initially used for their cholesterol-lowering
effects, they have more recently been shown to act as antidiabetic
drugs2,14,19. In diabetic db/db mice, BAS administration
deactivates intestinal FXR and increases glucose clearance in
peripheral tissues19. Among the proposed action mechanism of
BAS is a TGR5-mediated increase of GLP-1 secretion in dietinduced obese mice20,21. In addition to their acute effects on
GLP-1 secretion, BAS-bound BAs enhance proglucagon gene
expression through TGR5, another mechanism via which this
transmembrane receptor regulates GLP-1 production20.
Whether FXR is expressed and plays a role in L cells has not
been reported yet. Using the murine GLUTag L cell line, human
intestinal biopsies and different mouse models, we demonstrate
that FXR is expressed and functional in enteroendocrine L cells.
In mice and in human ex vivo intestinal biopsies, activated
FXR downregulates proglucagon mRNA levels. In vitro, FXR
activation decreases both proglucagon mRNA and protein levels
by interfering with ChREBP-mediated glucose-induction of
proglucagon mRNA levels. FXR activation also inhibits the
glycolysis pathway translating into decreased intracellular ATP
levels, contributing to a decreased GLP-1 secretion in response to
glucose. In vivo, FXR deficiency increases proglucagon mRNA
levels and glucose-induced GLP-1 secretion in chow diet-fed
mice, whereas the protective effect of FXR deficiency on
glycaemic control in high-fat-fed mice is blunted by the GLP-1
receptor (GLP-1R) antagonist Exendin-4(9-39). Finally, treatment of ob/ob mice with colesevelam improves glycaemia at least
in part by an FXR-dependent increase of proglucagon mRNA
levels.
Results
FXR decreases proglucagon mRNA levels in mice and humans.
Previous studies have reported high expression of FXR in
intestinal epithelial cells22,23. However, its expression in
enteroendocrine L cells has not yet been assessed. We analyse
Fxr expression in L cells sorted by fluorescence-activated cell
sorting (FACS) from transgenic proglucagon-VENUS mice24,25.
FACS-sorted L þ cells were separated from L cells with a purity
495% (ref. 24). As expected, the Fxr gene is more abundantly
expressed in ileal non-L cells (ileum L ) than in colonic non-L
cells (colon L ) (Fig. 1a). Surprisingly, compared with non-L
cells, Fxr expression is higher in L cells from the ileum (ileum
L þ ) and, albeit nonsignificantly, the colon (colon L þ ) (Fig. 1a).
Confocal microscopy analysis on human intestinal biopsies reveal
that FXR is expressed in GLP-1-positive cells from the jejunum
(Fig. 1b, Supplementary Movie 1) and colon (Supplementary
Fig. 1a).
To assess whether FXR activation controls the production of
GLP-1, one of the major bio-active peptides produced by L cells,
8-week-old C57Bl6-J wild-type mice were daily treated by gavage
with the FXR agonist GW4064 (30 mpk) for 5 days. As expected
Fgf15 mRNA levels increase after FXR agonist treatment
(Supplementary Fig. 1b), whereas proglucagon mRNA levels
decrease in both the ileum and colon (Fig. 1c). Since treatment
with GW4064 modulates the bile acid pool composition leading
to lower amount of TGR5 activators13, proglucagon mRNA levels
were measured in intestines of Tgr5 / mice treated during 5
days with GW4064 (30 mpk). FXR activation significantly
decreases proglucagon mRNA levels in the ileum of Tgr5 /
mice and to a lesser extent in the colon, suggesting a crosstalk
between FXR and TGR5 in the colon, but not the ileum (Fig. 1d).
This finding is consistent with elevated levels of secondary BA
that activate TGR5 in the colon. In addition, primary murine
intestinal epithelial cells treated ex vivo with GW4064
(5 mmol l 1) also exhibited decreased proglucagon mRNA levels
NATURE COMMUNICATIONS | 6:7629 | DOI: 10.1038/ncomms8629 | www.nature.com/naturecommunications
& 2015 Macmillan Publishers Limited. All rights reserved.
ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms8629
a
b
mRNA relative levels
3.0
Fxr
2.0
**
DAPI
FXR
GLP-1
FXR/GLP-1/DAPI
1.0
0.0
L–
L+
L–
Ileum
mRNA relative levels
2.0
d
Proglucagon
1.5
Vehicle
GW4064
1.5
***
**
1.0
0.5
mRNA relative levels
c
L+
Colon
Ileum
TGR5KO-Veh
TGR5KO-GW4064
1.0
0.5
Ileum
Colon
Proglucagon
DMSO
*
f
1.5
GW4064
1.0
0.5
0.0
mRNA relative levels
mRNA relative levels
1.5
***
0.0
0.0
e
Proglucagon
Colon
Proglucagon
DMSO
***
GW4064
1.0
0.5
0.0
Figure 1 | FXR decreases proglucagon mRNA levels in mice and in human. (a) Fxr expression by qPCR in FACS-sorted proglucagon-negative
and proglucagon-positive cells from the ileum (ileum L ; ileum L þ ) and colon (colon L ; colon L þ ) of GLU-VENUS mice (n ¼ 3).
(b) Twelve-micrometre-thick slices from human jejunal biopsies were incubated with antibodies against FXR (in green) and GLP-1 (in red). Nuclei
are in blue. Co-expression in GLP-1-positive cells (dotted line) was assessed on a confocal microscope. Representative of three different FXR/GLP-1
immunostaining experiments. Scale bar, 2 mm. Proglucagon qPCR on cDNA from ileum and colon of 8-week-old wild-type (c) or Tgr5 / (d) mice
treated by gavage for 5 days with GW4064 (30 mpk; n ¼ 5 mice per group. Data are represented as mean ±s.d. (e) Proglucagon qPCR on cDNA from
isolated primary intestinal epithelial cells from two wild-type mice ex vivo treated for 24 h with DMSO or with GW4064 (5 mmol l 1). (f) Proglucagon qPCR
on cDNA of human jejunal biopsies from four normoglycemic patients ex vivo treated for 16 h with DMSO or with GW4064 (5 mmol l 1). Data are
represented as mean ± s.e.m. Student’s t-test, *Pr0.05, **Pr0.01 and ***Pr0.001.
(Fig. 1e) showing that in addition to changes in bile acid pool
composition, FXR activation directly decreases proglucagon gene
expression. As FXR is also expressed in human intestinal L cells
(Fig. 1b), human jejunal biopsies were treated with GW4064
(5 mmol l 1). FXR activation results in the expected induction
of mRNA levels of FGF19, the human FGF15 orthologue
(Supplementary Fig. 1c), whereas proglucagon gene expression
decreases (Fig. 1f).
FXR activation decreases proglucagon expression in vitro. To
study the mechanisms underlying the regulation of proglucagon
gene expression by FXR, the well-characterized murine L-cell
model GLUTag was used26,27. FXR mRNA (Fxr Ct ¼ 28;
cyclophilin Ct ¼ 22) and protein are expressed and enriched in
the nuclear fraction (Fig. 2a). Moreover, incubation of GLUTag
cells with increasing concentrations of GW4064 results in the
induction of the FXR target genes Shp (Fig. 2b) and Fgf15
(Fig. 2c). Similar as in human and murine hepatocytes11,28,
GW4064 treatment (5 mmol l 1) increases FXR mRNA and
protein expression, whereas siRNA knockdown of FXR decreases
450% FXR mRNA (Fig. 2d) and protein levels (Fig. 2e).
The induction of Fgf15 by GW4064 decreases by 10-fold in
siFxr-transfected GLUTag cells (Fig. 2f). Altogether, these data
indicate the presence of functional FXR in GLUTag cells.
Incubation of GLUTag cells for 24 h with increasing concentrations of GW4064 or with GW4064 (5 mmol l 1) for different
times in standard culture glucose concentrations (5.6 mmol l 1)
decreases proglucagon mRNA levels in a dose- and timedependent manner with a maximum effect at 5 mmol l 1
(Fig. 3a), a concentration often used to study FXR activation12,28,
after 24 h of treatment (Fig. 3b). Moreover, a similar decrease is
observed with the natural FXR ligand chenodeoxycholic acid
(CDCA, 100 mmol l 1) (Supplementary Fig. 2a). Using primer
combination covering exon 2 to intron 2 of the proglucagon gene,
a similar decrease of proglucagon pre-mRNA levels is observed,
suggesting that FXR negatively regulates proglucagon gene
transcription (Supplementary Fig. 2b). GW4064 incubation
NATURE COMMUNICATIONS | 6:7629 | DOI: 10.1038/ncomms8629 | www.nature.com/naturecommunications
& 2015 Macmillan Publishers Limited. All rights reserved.
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ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms8629
mRNA relative levels
Nu
Cy
t.
To
t.
2
pG
He
MW
b
c.
a
GLUTag
55 kDa
4.0
Shp
*
3.0
*
*
2.0
1.0
d
Fgf15
25
***
**
20
2.0
***
mRNA relative levels
mRNA relative levels
c
15
10
5
0
)
μM
(1
0
40
64
(5
G
W
40
64
W
G
μM
)
)
μM
)
(1
μM
(0
.1
40
64
W
Fxr
DMSO
GW4064
**
1.5
1.0
§§§
§§§
0.5
)
)
0
μM
μM
(1
W
G
–
+
150
55 kDa
FXR
43 kDa
β-Actin
2.5 FXR
DMSO
*
2.0
GW4064
1.5
***
1.0
mRNA relative levels
+
siFxr
40
40
W
40
G
W
G
–
f
siFxr
siCtrl
GW4064
siCtrl
64
64
(1
64
(0
64
40
G
W
(5
μM
μM
.1
M
D
)
)
SO
0.0
e
FXR /β-actin protein
(arbitrary unit)
G
G
W
40
64
D
M
SO
0.0
DMSO
Fgf15
***
GW4064
100
50
§§§
0
siCtrl
siFxr
§§§
§§§
0.5
0.0
siCtrl
siFxr
Figure 2 | FXR is expressed and functional in GLUTag L cells. (a) Representative western blot of four experiments performed with fractioned protein
extracts from GLUTag cells. Total proteins from HepG2 are used as control. Shp (b) and Fgf15 (c) qPCRs on cDNA from GLUTag cells treated for 24 h with
GW4064 (0.1, 1, 5 and 10 mmol l 1). Data are represented as mean ± s.d. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test.
*Pr0.05, **Pr0.01 and ***Pr0.001 versus DMSO (n ¼ 3; performed three times). (d) Fxr qPCR on cDNA from GLUTag cells electroporated with siCtrl or
siFxr and treated for 24 h with GW4064 (5 mmol l 1; n ¼ 3; performed three times). (e) Representative western blot of four experiments performed on
protein extracts from GLUTag cells electroporated with a siCtrl or with a siFxr and treated for 24 h with GW4064 (5 mmol l 1; upper panel) and
quantification (lower panel) of FXR from four western blots (n ¼ 3). (f) Fgf15 qPCR on cDNA from GLUTag cells electroporated with siCtrl or siFxr and
treated for 24 h with GW4064 (5 mmol l 1; n ¼ 3; performed three times). Data are represented as mean ± s.d. Two-way ANOVA analysis followed by
Bonferronni’s post hoc test. *Pr0.05, **Pr0.01 and ***Pr0.001 versus DMSO of transfection-matched condition and yyyPr0.001 versus siCtrl of
treatment-matched condition.
decreases proglucagon mRNA and protein levels in siCtrl cells,
whereas such a decrease is not observed in siFxr cells, indicating
that FXR is required for the decrease of proglucagon gene
expression upon GW4064 treatment (Fig. 3c,d).
FXR inhibits glucose-induced proglucagon expression. It has
previously been shown that glucose induces proglucagon gene
expression in GLUTag L cells26. Moreover, in hepatocytes, FXR
activation inhibits the induction of glycolytic and lipogenic genes
4
by glucose12. To determine whether FXR activation also inhibits
the glucose response in L cells, GLUTag cells were cultured for
12 h in glucose-free medium and subsequently incubated in
medium containing lactate (10 mmol l 1), glucose (5.6 mmol l 1)
or 2-deoxyglucose (5.6 mmol l 1; Fig. 4a). As expected26, glucose
increases proglucagon gene expression (Fig. 4a). 2-Deoxyglucose,
a non-metabolised glucose analogue, does not increase
proglucagon mRNA levels, highlighting that glucose metabolism
is mandatory for the glucose-induced proglucagon gene
expression. Moreover, FXR activation for 24 h with either
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& 2015 Macmillan Publishers Limited. All rights reserved.
ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms8629
b 1.5
1.0
**
***
0.5
**
0.5
0.0
Time (h)
0
6
12
24
0
μM
1.0
64
(1
(5
64
GW4064
W
40
siFxr
siCtrl
GW4064
–
+
–
+
22 kDa
c
DMSO
1.5
mRNA relative levels
Proglucagon
d
G
W
40
G
)
)
μM
)
)
μM
(1
μM
64
W
40
G
G
W
40
64
(0
.1
D
M
SO
0.0
mRNA relative levels
Proglucagon
1.5
Proglucagon
GW4064
Proglucagon
NS
43 kDa
**
1.0
β-Actin
0.5
0.0
siCtrl
siFxr
Proglucagon/β-actin protein
(arbitrary unit)
mRNA relative levels
a
NS
2.5
2.0
1.5
1.0
**
0.5
0.0
siCtrl
siFxr
Figure 3 | FXR activation decreases proglucagon mRNA in GLUTag cells. (a) Proglucagon qPCR on cDNA from GLUTag cells treated for 24 h with
GW4064 (0.1, 1, 5 or 10 mmol l 1; n ¼ 3; performed three times). Data are represented as mean ± s.d. One-way analysis of variance (ANOVA) followed by
Tukey’s post hoc test. **Pr0.01 and ***Pr0.001 versus DMSO. (b) Proglucagon qPCR on cDNA from GLUTag cells treated for 0, 6, 12 or 24 h with
GW4064 (5 mmol l 1; n ¼ 3; performed three times). Data are represented as mean ± s.d. One-way ANOVA followed by Tukey’s post hoc test. **Pr0.01
versus t0. (c) Proglucagon qPCR on cDNA from GLUTag cells electroporated with siCtrl or siFxr and treated for 24 h with GW4064 (5 mmol l 1).
(d) Representative western blot of four experiments performed on protein extracts from GLUTag cells electroporated with a siCtrl or with a siFxr and treated
for 24 h with GW4064 (5 mmol l 1; upper panel) and quantification (lower panel) of proglucagon from four western blots (n ¼ 3). Data are represented as
mean ±s.d. Two-way ANOVA analysis followed by Bonferronni’s post hoc test. **Pr0.01: effect of GW4064 in each transfection condition.
GW4064 or CDCA inhibits proglucagon gene expression only in
L cells incubated in standard (5.6 mmol l 1), but not in low
glucose concentrations (Fig. 4a).
As ChREBP is a glucose-sensitive transcription factor activated
by glucose metabolites29 and since FXR activation interferes with
the ChREBP-mediated induction of glycolytic enzyme gene
expression by glucose in hepatocytes12, we next assessed
whether this regulatory mechanism is operational also in
L cells. Using L cells isolated from GLU-VENUS mice, ChREBP
is found to be expressed at higher levels in L cells than in
non-L cells (Fig. 4b). Moreover, ChREBP is also expressed in
GLUTag L-cells (Fig. 4b insert). FXR immunoprecipitation from
cytoplasmic and nuclear protein fractions demonstrates that
ChREBP and FXR physically interact or are in the same
complexes in high glucose conditions (Fig. 4c), similar as in
hepatocytes12. Finally, siRNA knockdown of ChREBP, resulting
in Z50% decrease of Chrebp mRNA levels (Supplementary
Fig. 3), prevents the induction of proglucagon mRNA levels by
glucose (Fig. 4d). FXR activation by GW4064 inhibits the increase
of proglucagon mRNA levels by glucose in siCtrl transfected cells,
whereas this effect is not observed in ChREBP knockdown cells.
Taken together, these data demonstrate that glucose
metabolism through the glycolysis pathway is necessary for the
glucose-dependent ChREBP-mediated increase of proglucagon
gene expression, which is inhibited upon FXR activation.
FXR decreases glycolysis and glucose-induced GLP-1 secretion.
GLP-1 secretion in response to glucose occurs, at least in
part, through glucose metabolism by glycolysis24,30,31. DNA
microarray followed by Gene Ontology analysis was performed
on GW4064-treated GLUTag cells. Interestingly, GW4064
treatment downregulates the expression of several glycolytic
genes leading to a significant inhibition of this pathway
(P ¼ 4.33 10 6; Fig. 5a). This decrease translates into lower
basal and glucose-enhanced intracellular ATP levels (Fig. 5b).
GW4064 treatment does not decrease mitochondrial mass as
assessed by the Mitotracker Green assay (Fig. 5c). However,
glycolytic capacity assessed by measurement of the extracellular
acidification rate (ECAR) after oligomycin treatment is lower in
GW4064 pre-treated cells (Fig. 5d). In parallel, FXR activation
decreases basal- and ATP-dependent oxygen consumption rates
(OCR) in GW4064-treated cells (Supplementary Fig. 4) likely
reflecting the decrease in activity of the global glycolysis pathway
as also suggested by the microarray results. Finally, FXR
activation results in lower basal and glucose-induced GLP-1
secretion (Fig. 5e). As GW4064 does not modulate KCl-induced
GLP-1 secretion (Fig. 5e), it is unlikely that FXR modulates
electrogenic events and that its action occurs rather upstream of
membrane depolarization. Even though initial reports suggested
that the SGLT-1/electrogenic pathway is the main mechanism
by which glucose induces GLP-1 secretion in primary intestinal
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ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms8629
CDCA
1.5
*** ***
1.0
0.5
.
.
Chrebp
C yt
4
GW4064
mRNA relative levels
mRNA relative levels
DMSO
§§§
Nuc
Proglucagon
2.0
GLUTag
WB ChREBP
b
§§§
MW
a
**
3
-95 kDa
2
*
1
0
0.0
Lactate
Glucose
L–
2-Deoxyglucose
L+
L–
Ileum
d
IP-FXR
Glc
GW4064 5 μM
–
–
+
–
+
+
–
–
+
–
+
+
95 kDaWB ChREBP
55 kDa-
Proglucagon
2.0
mRNA relative levels
c
Cyt.
DMSO
GW4064
**
1.5
§§§
1.0
0.5
0.0
Glc
WB FXR
*
–
+
+
siCtrl
Nuc.
L+
Colon
–
+
+
siChrebp
Figure 4 | FXR inhibits glucose-induced proglucagon expression. (a) Proglucagon qPCR on cDNA from GLUTag cells starved for 12 h with lactate
(10 mmol l 1) and then incubated for 24 h in lactate (10 mmol l 1), glucose (5.6 mmol l 1) or 2-deoxyglucose (5.6 mmol l 1) media containing DMSO,
GW4064 (5 mmol l 1) or CDCA (100 mmol l 1; n ¼ 3; performed three times). Data are represented as mean ±s.d. Two-way analysis of variance
(ANOVA) followed by Bonferronni’s post hoc test. ***Pr0.001: effect of GW4064 and CDCA on proglucagon mRNA levels in each medium conditions.
yyyPr0.001: effect of glucose on proglucagon mRNA levels in DMSO, GW4064 and CDCA conditions. (b) Chrebp qPCR on cDNA from FACS-sorted
proglucagon-negative and proglucagon-positive cells from the ileum (ileum L ; ileum L þ ) and colon (colon L ; colon L þ ) of GLU-VENUS mice (lower
panel; n ¼ 3) and ChREBP protein expression from cytoplasm and nucleus extract from GLUTag cells (upper panel; performed three times). Data are
represented as mean ±s.d. Student’s t-test. *Pr0.05 and **Pr0.01 (c) ChREBP and FXR western blots after FXR immunoprecipitation on lysates from
cytoplasm and nucleus of GLUTag cells treated or not with GW4064 (5 mmol l 1) in the presence or not of glucose (5.6 mmol l 1; performed two times).
(d) Proglucagon qPCR on cDNA from GLUTag cells electroporated with a siCtrl or siChrebp, starved for 12 h with lactate (10 mmol l 1) and then incubated
for 24 h in lactate 10 mmol l 1 (Glc ) or glucose 5.6 mmol l 1 (Glc þ ) media supplemented with DMSO or GW4064 (5 mmol l 1; n ¼ 3; performed three
times). Data are represented as mean ±s.d. Two-way ANOVA analysis followed by Bonferronni’s post hoc test. *Pr0.05 and **Pr0.01: effect of
treatments on each transfection condition. yyyPr0.001: effect of siChrebp in each treatment condition.
epithelial cells24,27, recent observations on perfused rat ileums
indicate that, in addition to the SGLT-1 pathway, the glucose
metabolism-mediated pathway contributes to the full GLP-1
secretion response to glucose30,31. Therefore, the response to
glucose (5.6 mmol l 1) with or without the GLUTs inhibitor
phloretin on GLP-1 secretion in murine ileal biopsies was tested.
Glucose alone induces GLP-1 secretion by tissue explants from
vehicle-treated mice, whereas co-incubation with phloretin
completely blocked the glucose response (Fig. 5f). Moreover,
in vivo GW4064 treatment (5 days, 30 mpk) prevents the increase
in glucose-induced GLP-1 secretion by the ileum ex vivo (Fig. 5f),
but is without effect in the presence of phloretin (Fig. 5f),
indicating that glucose transport by the GLUT-transporter
appears mandatory for the inhibitory effect of FXR activation
on the GLP-1 response to glucose.
Taken together, these data show that FXR activation decreases
the glycolysis pathway leading to decreased ATP levels and a
lower GLP-1 secretion in response to glucose.
Fxr deficiency increases proglucagon mRNA and GLP-1 levels.
FXR-deficient mice display elevated proglucagon mRNA levels in
the ileum and colon (Fig. 6a) and enhanced active GLP-1 plasma
concentrations 15 min after oral glucose administration (Fig. 6b).
Since conventionally raised (CONV-R) and GF mice display
6
different FXR antagonist:agonist ratios leading to FXR inhibition
in GF mice17, proglucagon mRNA levels were compared in
CONV-R and GF WT and FXR-deficient mice. As expected17,
Fgf15 gene expression is repressed in GF compared with
CONV-R mice, similar to that in Fxr / mice (Fig. 6c).
Moreover, CONV-R Fxr / mice display higher proglucagon
mRNA levels compared with CONV-R Fxr þ / þ mice (Fig. 6d),
whereas in GF mice, FXR deficiency no further modulates
proglucagon mRNA levels (Fig. 6d). In vitro incubation of
GLUTag cells with TbMCA (100 mmol l 1) decreases Fgf15 gene
expression (Fig. 6e) and increases proglucagon gene expression
(Fig. 6f), suggesting a crosstalk between BA, FXR and microbiota
in the regulation of proglucagon gene expression in L cells.
Thus, modulation of endogenous FXR ligands by the
microbiota may influence proglucagon gene expression in an
FXR-dependent manner.
FXR deficiency improves glycaemia through the GLP-1R pathway.
It is well known that FXR deficiency protects mice against
diet-induced obesity and improves glucose homeostasis14,32,33.
To test the metabolic impact of the FXR/GLP-1 pathway in a
pathophysiological context, Fxr þ / þ and Fxr / mice were
fed a high-fat diet (HFD) during 6 weeks before metabolic
tests with or without the GLP-1R antagonist Exendin-4(9-39).
NATURE COMMUNICATIONS | 6:7629 | DOI: 10.1038/ncomms8629 | www.nature.com/naturecommunications
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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms8629
a
Glc
DMSO
: –1.11 to –1.30
: –1.31 to –1.40
: < –1.50
Gpi1
§§
Fru6P
Pfkp,m,l
Fru1,6biP
DHAP
Cacna1g
Aldo a Aldo c
Glyceraldehyde-3-P
Tpi1
2+
Ca
Ca
2+
GW4064
3
Relative ATP levels
Hk2
Glc6P
Kir6.2
Sur1
b
Microarray fold change
between
DMSO- and GW4064treated cells
Gapdh
1,3-biP-glycerate
Pgk1
2
NS
**
1
**
3-P-glycerate
↑ATP
Pgam1
0
Glucose
2-P-glycerate
Eno1-4
–
+
P-enolpyruvate
Ldha
Pyr
Lactate
Mitochondria
d
DMSO
c
Mitotracker green
100
CTRL
DMSO
% of max
80
GW4064
60
40
ECAR
(mpH min–1 per10,000 cells)
GLP-1
100
101
102
FL1-H
103
e
DMSO
5
0
104
§§§
§§§
2
**
***
–
Glc
KCl
C
D
**
§§§
15
30
45
60
75
Time (min)
A Glucose
C 2-Deoxyglucose
B Oligomycin D Rotenone/antimycin
Vehicle
pmol l–1 per microgram
protein
Relative
GLP-1 secretion
§§§
***
0
f
GW4064
3
0
Secretagogue
§§§
GLP-1
4
1
B
10
20
0
GW4064 5 µM
A
15
15
GW4064
§§§
§§§
10
10
**
9
9
9
0
Glucose 5.6 mM –
Phloretin 0.5 mM –
–
–
12
12
5
+
–
+
–
+
+
+
+
Figure 5 | FXR inhibits GLP-1 secretion by decreasing glycolysis. (a) DNA microarrays on 24 h DMSO- and GW4064(5 mmol l 1)-treated GLUTag cells
were performed using Agilent Technology. Genes whose expression is downregulated by 10%, 10% 30% and upregulated by 50% after GW4064
treatment are written with black letters in rectangles filled in grey, with white letters in rectangles filled in grey and with white letters in black filled
rectangles, respectively. P value of the glycolysis biological process: P ¼ 4.33 10 6. (b) ATP measurements on GLUTag cells treated for 24 h with
GW4064(5 mmol l 1) and stimulated or not for 1 h with glucose(5.6 mmol l 1). yyPr0.01: effect of glucose on ATP levels. **Pr0.01: effect of GW4064 on
ATP levels. (c) Fluorescence measurements by Mitotracker Green in GLUTag cells incubated with DMSO or GW4064 (n ¼ 3; performed three times).
(d) Extracellular acidification rate (ECAR) after successive injection of glucose(10 mmol l 1), oligomycin(1 mmol l 1), 2-deoxyglucose(100 mmol l 1) and
rotenone(1 mmol l 1)/antimycin A(1 mmol l 1) on GLUTag cells incubated 24 h with DMSO or GW4064. yyyPr0.001: effect of GW4064 on ECAR between
t ¼ 15 min and t ¼ 40 min. **Pr0.01 and ***Pr0.001: effect of GW4064 on ECAR between t ¼ 40 min and t ¼ 55 min. Representative results of four
independent experiments. (e) GLP-1 measurements in supernatants of GLUTag cells treated for 24 h with GW4064(5 mmol l 1) and stimulated or not for
1 h with 5.6 mmol l 1 of glucose- or 30 mmol l 1 of KCl-containing buffer (n ¼ 3; performed four times). **Pr0.01, ***Pr0.001: effect of GW4064
treatment in each secretion condition. yyy Pr0.001: effect of secretagogue in each treatment condition. (f) GLP-1 measurements in supernatants of
intestinal biopsies from WT mice treated for 5 days with vehicle or with GW4064(30 mpk) and then stimulated with medium alone, medium plus
glucose(5.6 mM) or medium plus glucose(5.6 mol l 1) plus phloretin(0.5 mmol l 1). **Pr0.01: effect of GW4064 treatment on GLP-1 secretion in each
secretion condition. yyyPr0.001: effect of secretagogues on each treatment condition. On the bars, number of biopsies used from three vehicle- or
GW4064 (30 mpk)-treated mice. Data are represented as mean ±s.d. (b, d and e) or mean±s.e.m. (f). Statistical analysis were performed using two-way
analysis of variance followed by Bonferronni’s post hoc test.
NATURE COMMUNICATIONS | 6:7629 | DOI: 10.1038/ncomms8629 | www.nature.com/naturecommunications
& 2015 Macmillan Publishers Limited. All rights reserved.
7
ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms8629
a
b
Proglucagon
10
Fxr +/+
*
Fxr –/–
2
*
1
Fxr –/–
**
6
4
2
0
0
Ileum
1.5
Colon
d
§§
Fgf15
2.5
**
1.0
mRNA relative levels
c
mRNA relative levels
Fxr +/+
GLP-1 secretion
8
pmol l–1
mRNA relative levels
3
Fxr +/+
Fxr –/–
0.5
0.0
2.0
Fxr +/+
Fxr –/–
Proglucagon
**
1.5
1.0
0.5
0.0
CONV-R
CONV-R
GF
e
GF
f
25
20
15
10
5
1.5
Fgf15
***
DMSO
GW4064
TβMCA
**
1.0
0.5
0.0
2.0
mRNA relative levels
mRNA relative levels
DMSO
GW4064
Proglucagon
**
1.5
TβMCA
**
1.0
0.5
0.0
Figure 6 | Fxr / mice exhibit higher proglucagon mRNA and GLP-1 levels. (a) Proglucagon qPCR on cDNA from ileum and colon of 8-week-old
Fxr þ / þ or Fxr / mice. n ¼ 5–6 mice per group. (b) GLP-1 secretion in 8-week-old Fxr þ / þ or Fxr / mice 15 min after an oral challenge with
glucose 2 g kg 1. n ¼ 5–6 mice per group. Data are represented as mean ±s.e.m. Student’s t-test, *Pr0.05 and **Pr0.01. Fgf15 (c) and Proglucagon
(d) qPCR on cDNA from ileum of 8-week-old GF or CONV-R mice on a Fxr þ / þ or a Fxr / background (n ¼ 11–12 mice per group). Data are
represented as mean ±s.e.m. Two-way analysis of variance (ANOVA) followed by Bonferronni’s post hoc test. **Pr0.01: effect of FXR deficiency on gene
expression in each raised condition. yy Pr0.01: effect of gut microbiota on gene expression in each genotype. Fgf15 (e) and Proglucagon (f) qPCR on cDNA
from GLUTag cells treated for 24 h with GW4064 (5 mmol l 1) or with TbMCA (100 mmol l 1; n ¼ 3; performed three times). Data are represented as
mean ±s.d. One-way ANOVA followed by Tukey’s post hoc test. **Pr0.01, ***Pr0.001 versus DMSO.
After 6 weeks of diet, Fxr þ / þ mice gain 44% of their initial
body mass, whereas, as expected14,32,33, high-fat-fed Fxr /
mice only gain 23%. Intraperitoneal GTT (2 g kg 1) reveals only
a minor, nonsignificant decrease in glucose excursion curve in
Fxr / versus Fxr þ / þ mice (Fig. 7a). However, when
challenged with an oral glucose bolus (2 g kg 1), Fxr /
mice display an improved glucose tolerance compared with
Fxr þ / þ mice (45% decrease in integrated area under the curve
(iAUC), Pr0.05, Fig. 7b) reflecting a role of the intestine in
the regulation of glucose homeostasis by FXR. To assess the
involvement of the GLP-1 pathway in this improvement, the
GLP-1R antagonist Exendin-4(9-39) was administered before
the oral glucose gavage test. Glucose tolerance worsens in
Exendin-4(9-39)-treated mice of both genotypes (Fig. 7b–d)
with a more pronounced effect in Fxr / mice (Fig. 7b, þ 30%
in iAUC in Fxr þ / þ versus þ 80% in iAUC in Fxr / mice),
hence abolishing the beneficial effect on oral glucose tolerance of
FXR deficiency.
These results show that the GLP-1/GLP-1R pathway
contributes to the improved glucose homeostasis upon FXR
deficiency.
8
BAS improve glycaemia and GLP-1 production by FXR. To test
the response to pharmacological targeting of the FXR/GLP-1
pathway, ob/ob Fxr þ / þ and ob/ob Fxr / mice were treated
for 2 weeks with colesevelam, a BAS known to deactivate
intestinal FXR23. As expected, BAS administration inhibits FXR
activity in the ileum and colon as reflected by repression of Fgf15
gene expression (Supplementary Fig. 5a,b). BAS improves glucose
metabolism after an OGTT in ob/ob Fxr þ / þ mice, an effect not
observed in ob/ob Fxr / mice (Fig. 8a–c). Moreover, BAS
administration increases proglucagon gene expression in the
ileum (Fig. 8d) and colon (Supplementary Fig. 5c) only in ob/ob
Fxr þ / þ mice, an effect which may contribute to the improved
glucose control.
Discussion
Using transgenic VENUS mice expressing the reporter gene only
in proglucagon-positive cells and human biopsies, we show
that FXR is not only expressed in the enterocyte but also in
L cells, where its activity was demonstrated by agonist-induced
regulation of two bona fide FXR target genes, Fgf15 and Shp.
NATURE COMMUNICATIONS | 6:7629 | DOI: 10.1038/ncomms8629 | www.nature.com/naturecommunications
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ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms8629
a
Blood glucose (mg dl–1)
b
IPGTT
600
30
*
NaCl
**
iAUC (×103)
Ex-9
400
200
10
0
0
30
90
60
Time (min)
120
Fxr+/+
d
600 OGTT
*
400
200
Fxr+/+ (NaCl)
Fxr+/+ (Ex9)
0
Fxr–/–
OGTT
600
Blood glucose (mg dl–1)
Blood glucose (mg dl–1)
§
Fxr–/–
Fxr+/+
0
c
20
*
400
***
**
200
Fxr–/–(NaCl)
Fxr–/–(Ex9)
0
0
30
60
90
120
0
30
60
90
120
Time (min)
Time (min)
Figure 7 | FXR deficiency improves glucose metabolism via the GLP-1 pathway. (a) Intraperitoneal glucose tolerance test in 12-week-old Fxr þ / þ and
Fxr / mice fed for 6 weeks with a 60% HFD (n ¼ 6 mice per group). Data are represented as mean ±s.e.m. (b) Integrated area under the curve (iAUC)
of glucose excursion curves after 0.9% NaCl or Exendin-4(9-39) (0.5 mpk) injection 45 min before an oral glucose tolerance test (OGTT, 2 g kg 1) in
Fxr þ / þ and Fxr / mice fed for 6 weeks with a 60% HFD (n ¼ 6 mice per group). Data are represented as mean ±s.e.m. Two-way analysis of variance
(ANOVA) followed by Bonferronni’s post hoc test. *Pr0.05 and **Pr0.01: effect of Exendin-4(9-39) on iAUC in each genotype. yPr0.05: effect of
genotype in each treatment condition. OGTT after 0.9% NaCl or Exendin-4(9-39) (0.5 mpk) in 12-week-old Fxr þ/ þ (c) and Fxr / (d) mice fed for
6 weeks with a HFD 60% (n ¼ 6 mice per group). Data are represented as mean ±s.e.m. Two-way ANOVA analysis followed by Bonferronni’s post hoc
test. *Pr0.05, **Pr0.01 and ***Pr0.001: effect of Exendin-4(9-39) treatment on glucose excursion in each genotype.
*
**
*
ob/ob Fxr+/+ control
ob/ob Fxr+/+ colesevelam
0
15
30
60
Time (min)
c
AUC (×103)
80
60
§
800
700
600
500
400
300
200
100
0
90
Control
Colesevelam
*
40
20
0
ob/ob
Fxr+/+
Blood glucose (mg dl–1)
b
800
700
600
500
400
300
200
100
0
ob/ob
Fxr –/–
ob/ob Fxr–/– control
ob/ob Fxr–/– colesevelam
0
15
30
60
Time (min)
d
Ileal mRNA relative levels
Blood glucose (mg dl –1)
a
90
Control
2.0 Proglucagon
1.5
§§
Colesevelam
**
1.0
0.5
0.0
ob/ob
Fxr+/+
ob/ob
Fxr–/–
Figure 8 | BA sequestration improves glycaemia and GLP-1 production through FXR. OGTT after 3 weeks of vehicle or colesevelam treatment in ob/ob
Fxr þ / þ (a) and ob/ob Fxr / mice (b) (n ¼ 6–7 mice per group). (c) Corresponding area under the curve (AUC). (d) Proglucagon qPCR on cDNA from
ileum of these mice (n ¼ 6–7 mice per group). Data are represented as mean ±s.e.m. Two-way analysis of variance (ANOVA) followed by Bonferronni’s
post hoc test. *Pr0.05 and **Pr0.01: effect of Colesevelam treatment on glucose excursion curve during an OGTT (a and b), on AUC (c) or on
proglucagon gene expression (d) in each genotype. y Pr0.05, yy Pr0.01: effect of FXR deficiency on AUC (c) or on proglucagon gene expression
(d) in each treatment condition.
NATURE COMMUNICATIONS | 6:7629 | DOI: 10.1038/ncomms8629 | www.nature.com/naturecommunications
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9
ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms8629
Colesevelam
L-cell
activated FXR
Glycolysis
pathway
Intracellular
ATP
Proglucagon
Pr
od
uc
tio
n
n
tio
cre
Se
GLP-1
Figure 9 | Proposed mechanism by which L-cell FXR decreases GLP-1
production and secretion. Glucose induces proglucagon gene expression in
L-cells via the glycolysis pathway and ChREBP, and subsequently promotes
GLP-1 production. Moreover, glycolysis increases intracellular ATP
concentrations and induces GLP-1 secretion. FXR activation, by inhibiting
glucose metabolism, decreases both GLP-1 production and secretion.
Colesevelam, by inhibiting FXR transcriptional activity in L-cells, promotes
GLP-1 production and secretion.
Both in vitro, ex vivo and in vivo, in mice as well as in human
intestines, activated FXR decreases proglucagon mRNA levels.
This decrease is the result of FXR activation in L cells rather than
an indirect effect of bile acid pool modification after FXR
activation.
ChREBP is a transcription factor activated by glucose
metabolites. As previously evoked by microarray data25, we
demonstrate by quantitative PCR (qPCR) analysis that ChREBP
is expressed to a higher extent in murine intestinal L cells than in
non-L cells. Moreover, glucose induction of proglucagon mRNA
levels26 is inhibited by FXR activation. We identify here that
glucose-induced proglucagon gene expression is dependent on
glucose metabolism and mediated by ChREBP, identifying a role
for ChREBP in enteroendocrine L cells in the control of
proglucagon gene expression. FXR physically interacts or is
complexed to ChREBP in L cells, a result also observed in human
hepatocytes where FXR activation inhibits the glucose-induced
glycolytic gene expression by ChREBP12. These observations are
in accordance with an overall decrease of the glycolysis pathway
after FXR activation in GLUTag cells similar to that in human
hepatocytes.
Glucose sensing by L cells and subsequent secretion of GLP-1
involves both ATP-independent and -dependent pathways24,30,31.
The ATP-independent mechanism is mediated by the
co-transport of Na þ and glucose through the membrane
transporter SGLT-1 (refs 27,31,34,35) and glucose binding to
the taste receptors Tas1R2 and Tas1R3 (ref. 36). The electrogenic
current generated by the Na þ entrance is sufficient to depolarize
L-cell membranes leading to GLP-1 secretion. This mechanism
has been shown to be involved in glucose-induced GLP-1
secretion in GLUTag cells, in perfused rat ileum and also in
primary intestinal epithelial cells24,27,31,34. The ATP-dependent
pathway involves glucose metabolism by glycolysis, thus
increasing intracellular ATP levels, closure of the voltagedependent KATP-dependent channel, membrane depolarization,
intracellular Ca2 þ increase and GLP-1 secretion. Such a
mechanism is involved in glucose-induced GLP-1 secretion in
GLUTag cells27, in perfused rat ileum31 but not in primary
intestinal epithelial cells27. We show that FXR activation does not
decrease the expression of Sglt1, Tas1r2, Tas1r3 in GLUTag cells.
Moreover, FXR activation did not change KCl-induced GLP-1
10
secretion, but lowered GLP-1 secretion in response to both low
and high glucose concentrations. Glucose transport by GLUT
transporters is required for intracellular ATP production in L
cells24. Indeed, incubation of murine intestinal biopsies with the
GLUTs inhibitor phloretin prevented glucose-induced GLP-1
secretion. FXR activation prevented glucose-induced GLP-1
secretion in isolated murine ileal biopsies to a similar extent as
phloretin. To delineate the mechanisms underlying the decrease
of ATP-dependent GLP-1 secretion by FXR, we assessed the
activity of the glycolysis pathway and the functionality of
mitochondria in GLUTag cells. FXR activation decreased
intracellular ATP levels, independent of a decrease in
mitochondrial mass or defective mitochondria, as seen with
Mitotracker Green and OCR experiments. ECAR measurements
after ATP-synthase inhibition with oligomycin showed that
GW4064-treated cells have a lower capacity to metabolize
glucose, which translates into lower ATP-dependent oxygen
consumption. Altogether, these results show that FXR activation
decreases the transcription of glycolytic enzymes not only in
human hepatocytes12, but also in enteroendocrine L cells leading
to a decrease in intracellular ATP levels and ATP-dependent
GLP-1 secretion in response to glucose.
An increasing body of evidence shows that BA pool size and
composition control glucose homeostasis through BA receptors.
Although their action through the transmembrane receptor
TGR5 is likely to occur rapidly after food ingestion, activation
of the nuclear receptor FXR induces a more delayed response thus
leading to a shift between early postprandial positive effects of
TGR5 activation and delayed effects through FXR activation. FXR
activation in the intestine increases FGF15/19 secretion which, in
addition to its role in the regulation of BA synthesis, reduces
adiposity, increases brown adipose tissue energy expenditure
and improves the metabolic rate in different obese murine
models37,38. In contrast, recent studies in obese mice highlight
that intestinal FXR antagonism results in improved energy
homeostasis15,16,39. Indeed, recently identified as endogenous BA
FXR antagonists in GF mice17, TbMCA and TaMCA inactivate
intestinal FXR15,39 and protect mice against diet-induced obesity
and improve glucose metabolism15,39. Treatment of GLUTag cells
with CDCA, a natural FXR agonist, decreased, whereas TbMCA
increased proglucagon gene expression. In agreement with these
findings, GF mice express higher intestinal levels of proglucagon
than CONV-R mice40. In addition, we show here that the increase
of intestinal proglucagon mRNA upon FXR deficiency requires
the gut microbiota since FXR deficiency in GF mice does not
further enhance proglucagon mRNA levels. Assessing the
dialogue between bile acids, gut microbiota and FXR in L cells
could be an interesting way to increase GLP-1 production.
Whole-body and intestinal FXR-deficient mice are protected
against obesity and have an improved glucose metabolism14,15. By
using the GLP-1R antagonist Exendin-4(9-39), we demonstrate
that the improved glucose metabolism in Fxr / mice fed a
HFD implies the GLP-1 pathway. BASs are resins which complex
BA, preventing ileal BA re-absorption and driving BA to the
colon thus facilitating their elimination41,42. Initially used for
their cholesterol-lowering effect, BAS treatment has been shown
to lower blood glucose in T2D patients (for review, see ref. 43).
Among the proposed mechanisms, FXR deactivation increases
energy expenditure in obese mice2,14,41. Another mechanism may
rely on the TGR5-mediated increase in GLP-1 production and
secretion. In the colon, which contains the highest density of
GLP-1 expressing cells, BAS-bound BA increase proglucagon
mRNA levels and meal-induced GLP-1 secretion thus improving
glucose metabolism in high-fat-fed mice20,21. In this study, we
demonstrate a role also for FXR in the response to colesevelam
resulting in an increase of proglucagon mRNA levels in ileum and
NATURE COMMUNICATIONS | 6:7629 | DOI: 10.1038/ncomms8629 | www.nature.com/naturecommunications
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ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms8629
colon translated to an improved response to oral glucose
suggesting that compounds with TGR5 agonist–FXR antagonist
activity may be most optimal to enhance incretin production.
Taken together, our results demonstrate that FXR activation
decreases glycolysis and ATP production, which, in turn,
decreases proglucagon transcription and GLP-1 secretion in
response to glucose. In pathophysiological conditions, the
beneficial effect of FXR deficiency is, at least in part, related to
this newly identified FXR/GLP-1 pathway. Our study further
provides a novel molecular mechanism contributing to the
beneficial effects of BAS on glucose control in T2DM through
increased GLP-1 production upon FXR deactivation in intestinal
L cells (Fig. 9). Thus, inhibiting FXR in L cells through a change
in BA pool composition or through combination TGR5-agonist
and FXR-antagonist treatment could be a promising approach to
treat T2DM.
Methods
Chemicals and reagents. The DPP-4 inhibitors diprotin A, FFA-free bovine
serum albumin (BSA), CDCA, TbMCA, exendin-4(9-39), phloretin, DMSO
and CMC were purchased from Sigma-Aldrich (St Quentin-Fallavier, France).
Sitagliptin was purchased from MSD. The synthetic FXR agonist GW4064 was
purchased from Tocris (R&D Systems, Lille, France). For in vitro or ex vivo
experiments, CDCA, TbMCA and GW4064 were dissolved in dimethylsulfoxide
(DMSO) at 0.1% final. For in vivo experiments, GW4064 was dissolved in 1%
carboxymethylcellulose (CMC) and Exendin-4(9-39) was dissolved in NaCl 0.9%.
Chow and high fat diets were purchased from UAR (A04, Villemoison/Orge,
France). Colesevelam–HCl was a kind gift of Daiichi Sankyo.
Animal models and experimental protocols. Eight-week-old C57Bl6/J male
Fxr þ / þ , Fxr / , ob/ob Fxr þ / þ , ob/ob Fxr / littermates (INSERM
U1011), 12-week-old C57Bl6/J male Tgr5 / (CNRS/INSERM/ULP, Illkirch)
and WT mice (Charles River Laboratories, Wilmington, MA), fed a chow diet, were
housed in a temperature-controlled room (22 °C) on a 12-h light–dark cycle.
GLU-VENUS, germ-free (GF) and conventionally raised (CONV-R) mice were
housed as previously stated17,40. Wild-type (eight mice per group) and Tgr5 /
(five mice per group) mice were gavaged for 5 days with 1% CMC containing or
not GW4064 (30 mpk). After randomization in four groups based on age and body
weight, C57Bl6/J male mice deficient (Fxr / ) or not (Fxr þ / þ ) for FXR were
fed a standard chow diet (five to six mice per genotype) or a high-fat diet (12 mice
Table 1 | Mouse small interfering RNA sequences used in
siRNA experiments.
Targeted gene
Fxr
Dharmacon Smartpool sequences (50 -30 )
GAAACUUCCUGCCGGACAU
GUGUAAAUCUAAACGGCUA
GAUUUGUGCCGGACGGGAU
UGCCAGGAGUGCCGGCUAA
Mlxipl
CAUCCGACCUUUAUUUGAA
AAGAGGCGGUUCAAUAUUA
GCAGCUGCGGGAUGAAAUA
UCAUGGAGAUAUCAGAUUU
per genotype). Age-matched C57Bl6/J male mice germ-free (GF) or conventional
raised (CONV-R), on Fxr þ / þ or Fxr / backgrounds (11–12 mice per group)
were fed a standard chow diet (Labdiet). Eight-week-old C57Bl6/J male Fxr þ / þ
and Fxr / mice on a leptin-deficient (ob/ob) background (six to seven mice per
group) were fed ad libitum during 3 weeks with a standard diet (UAR A04,
Villemoison/Orge, France) supplemented or not with 2% of colesevelam–HCl.
Eight-week-old Fxr þ / þ and Fxr / mice (12 mice per genotype) received a
HFD (D12492; Research Diets; 60% kcal fat) and controls received a chow diet for
6 weeks. Body weight was monitored weekly and glucose tolerance tests were
measured as previously described14. Briefly, after 6 h fasting, mice were injected
with NaCl 0.9% (n ¼ 6 mice per group) containing or not Exendin-4(9-39)
(0.5 mpk, n ¼ 6 mice per group) 45 min before intragastric glucose gavage
(2 g kg 1). One week later, the same mice were subjected to intraperitoneal glucose
injection (2 g kg 1). Glycaemia were measured at 0, 15, 30, 60, 90 and 120 min
after either glucose gavage or intraperitoneal injection. For the in vivo GLP-1
experiments, mice were fasted 6 h, gavaged with sitagliptin (25 mpk) 45 min before
a glucose bolus (2 g kg 1). Fifteen minutes after glucose gavage, blood (250 ml) was
sampled by retro-orbital venipuncture under isoflurane anaesthesia and plasma
GLP-1 was measured as described below.
After 6 h fasting, mice were killed by cervical dislocation. Ileum (corresponding
to the five terminal centimetres of the small intestine) and colon were washed once
with phosphate-buffered saline (PBS), opened longitudinally on ice and the
intestinal mucosa was scrapped and snap-frozen in liquid nitrogen. The experiment
on GLU-VENUS mice was approved by local ethics committees and conformed to
United Kingdom Home Office regulations, the experiment on Tgr5 / mice
were approved by the local animal experimentation committee of the Canton de
Vaud (license no. 2614) and the experiment on GF/CONV-R mice was performed
with protocols approved by the University of Gothenburg Animal Studies
Committee. All the other experimental protocols were approved by the Lille
Pasteur Institute Ethical committee and carried out in agreement with European
Union (EEC no. 07430).
Ex vivo studies. GLP-1 secretion on murine intestinal biopsies. Murine ileal
biopsies from WT mice treated for 5 days with CMC or GW4064 (30 mpk) were
isolated as previously described4. Briefly, after 6 h fasting, mice were killed by
cervical dislocation. The last 8 cm of the small intestine, corresponding to the
ileum, was placed in cold Hank’s Balance Salt Solution (HBSS, Lonza) containing
2% horse serum (Life Technologies). Peyer’s patches were removed and the ileum
was opened longitudinally and cut into 5-mm-long pieces. Pieces were washed
5 in cold HBSS plus 2% horse serum and then incubated for 10 min at 4 °C
in HBSS containing 2% horse serum and 1,4-dithiothreitol (DTT, 1 mmol l 1).
After additional washing in cold HBSS plus 2% horse serum, ileum pieces were
distributed in 48-well plates and stabilized for 3 h at 37 °C in Iscove’s Modified
Dulbecco Medium (Life Technologies) containing 10% fetal calf serum (Life
Technologies). After 30 min glucose deprivation in DMEM No Glc medium,
a 1 h-GLP-1 secretion test in response to DMEM without glucose, to DMEM with
glucose (5.5 mmol l 1) or in response to DMEM with glucose (5.6 mmol l 1)
plus phloretin (0.5 mmol l 1) was performed at 37 °C in DMEM plus 1% DPP-4
inhibitor (Ile-Pro-Ile, Sigma-Aldrich). Finally, medium was removed, centrifuged
for 5 min at 4 °C at 13,000g and immediately frozen at 80 °C. GLP-1 content in
culture medium was measured as described below. The remaining tissue was
washed in cold PBS and frozen at 80 °C in NaOH 2 N. Protein content was
assessed according to BCA’s method (Thermoscientific).
Culture and immunostaining on human intestinal biopsies. Fresh human
jejunum biopsies from normoglycemic subjects were obtained with informed
consent as part of the A Biological Atlas of Severe Obesity (ABOS) study
(ClinicalTrials.gov; NCT01129297). Mucosa were dissociated from musculosa and
cut into small pieces of 1 cm2 before treatment for 16 h with DMSO or GW4064
(5 mmol l 1) in RPMI medium þ GlutaMAX-1 (catalogue (Cat.) No. 61870-010,
Life Technologies) containing glucose (11 mmol l 1), sodium pyruvate
(1 mmol l 1) and supplemented with 10% FBS (Life Technologies) and
penicillin/streptomycin (10,000 U l 1 per 10 mg l 1) in a 37 °C, 5% CO2
controlled atmosphere. RNA extraction and qPCR analysis were performed as
described below.
Table 2 | qPCR primer sequences.
Species
Mouse
Gene
Fgf15
Shp
Proglucagon
Exon/Intron proglucagon
Fxr
Forward (5’-3’)
GAGGACCAAAACGAACGAAATT
AGGAACCTGCCGTCCTTCTG
GATCATTCCCAGCTTCCCAG
CACTTCCACTCACAGATCATTCC
CCTGAGAACCCACAGCATTT
Reverse (5’-3’)
ACGTCCTTGATGGCAATCG
CTCAGCCACCTCGAAGGTCA
CTGGTAAAGGTCCCTTCAGC
CTTCAGACTCTTACCGGTTCCTC
GTGTCCATCACTGCACATCC
Human
Proglucagon
FGF19
GTTCCCAAAGAGGGCTTGCT
GGAGGAAGACTGTGCTTTCGA
GTTGCCAGCTGCCTTGTACC
GAAGAGCCTAGCCATGTGTAAC
NATURE COMMUNICATIONS | 6:7629 | DOI: 10.1038/ncomms8629 | www.nature.com/naturecommunications
& 2015 Macmillan Publishers Limited. All rights reserved.
11
ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms8629
For immunohistochemical analysis, human jejunal biopsies were overnight
(O/N) fixed with paraformaldehyde 4% (Sigma) before incubation with 20%
sucrose during 20 h and further inclusion in Jung Tissue Freezing Medium (Jung)
and storage at 80 °C before processing. Twelve-micrometre-thick slices were
post-fixed in methanol/acetone 50/50 (v/v) for 10 min at 20 °C. After two washes
in Tris/NaCl buffer (TRIZMA-Base (20 mmol l 1, Sigma)/NaCl (150 mmol l 1,
VWR), pH 7.6), an antigen retrieval step was performed (520 W, 5 min; 160 W,
10 min) in citrate buffer (ThermoScientific). After permeabilization during 10 min
(Tris/NaCl/0.1% TRITON X100), unspecific protein binding sites were masked by
Dako Protein Block (DAKO) during 2 h at room temperature. Then, the samples
were incubated O/N with mouse anti-human GLP-1 (SC-73508, Santa Cruz
Biotechnology) and rabbit anti-human FXR (Ab28676, Abcam) antibodies (1/100)
at 4 °C. The next day, immunoreactive cells were revealed after incubation with
goat anti-mouse IgG Alexa 568 (for GLP-1) and goat anti-rabbit IgG Alexa 488
(for FXR; dilutions: 1/200, Molecular Probes) and photographies were taken with a
confocal microscope (LSM 710 Zeiss). Image analysis was performed using the
ImageJ software (version 1.46c; WS Rasband, National Institutes of Health,
Bethesda, MD, USA, http://rsb.info.nih.gov/ij/).
In vitro studies. Cell culture and treatment. The mouse enteroendocrine L cell line
GLUTag was kindly provided by D.J. Drucker (University of Toronto, Toronto,
Canada) and grown in DMEM þ GlutaMAX-1 medium (Cat. No. 21885-025,
Invitrogen) containing glucose (5.6 mmol l 1), sodium pyruvate (1 mmol l 1) and
supplemented with 10% FBS. For treatments, 42 h cultured cells were incubated for
24 h in DMEM þ GlutaMAX-1 medium (Cat. No. 21885-025, Invitrogen) with
glucose (5.6 mmol l 1), sodium pyruvate (1 mmol l 1) and supplemented with
0.2% BSA containing TbMCA (100 mmol l 1), CDCA (100 mmol l 1) or GW4064
(5 mmol l 1) during 24 h unless specified. In some experiments, cells were deprived
of glucose by a 12 h incubation in glucose-free medium (DMEM GLUTamax,
Cat. No. 11966-025), supplemented with 1% glutamine, sodium pyruvate
(1 mmol l 1), 0.2% BSA and sodium lactate (10 mmol l 1) before FXR activation
in either lactate (10 mmol l 1)-, glucose (5.6 mmol l 1)- or 2-deoxyglucose
(5.6 mmol l 1)-containing medium.
Transient transfection assays. Cells were electroporated using the Neon
Transfection System (Life Technologies) with small interference RNA against
random (siCtrl), Fxr (siFxr) or Mlxipl (siChrebp) sequences (smart pool sequences
obtained from Dharmacon (Thermoscientific, Illkirch, France); see Table 1).
A total 140,000 electroporated cells cm 2 seeded into 24-well plates during 42 h
were treated as described above.
GLP-1 secretion assays, ATP and mitotracker-FACS measurement. After
treatment, GLUTag cells were starved for 30 min in glucose-free Krebs/phosphate
buffer (NaCl (120 mmol l 1), KCl (5 mmol l 1), MgCl2 (0.25 mmol l 1), CaCl2
(0.5 mmol l 1) and NaHCO3 (2.2 mmol l 1), pH 7.2) supplemented with diprotin
A (100 mmol l 1) and 0.2% BSA. Cells were subsequently stimulated for 1 h with
Krebs buffer with or without glucose (5.6 mmol l 1) or with a Krebs buffer
enriched with KCl (30 mmol l 1). The cell supernatants were then transferred into
ice-cold microtubes containing an equal volume of diprotin A (100 mmol l 1) in
Krebs buffer and centrifuged at 1,500g, 4 °C for 5 min. Cells were lysed in NaOH
(0.8 mol l 1) under agitation and total protein content was determined using the
BCA Protein Assay Kit (Pierce). Active 7-37 and 7-36 amide GLP-1 were measured
with an enzyme-linked immunosorbent assay kit (EGLP-35K; Merck-Millipore)
using Mithras Technology (Berthold) and normalized to the total quantity of
cellular proteins. ATP measurement (Cell Titre Glow, Promega) on GLUTag cells
in response to glucose (5.6 mmol l 1) was performed in the same condition as
GLP-1 secretion assays according to the manufacturer’s protocol and luciferase
activity was measured using Viktor apparatus (PerkinElmer). To estimate
mitochondrial quantity, 24 h DMSO- and GW4064-treated GLUTag cells were
washed with PBS, trypsinized and incubated at 37 °C for 20 min with 100 nmol l 1
MitoTracker Green FM (Molecular Probes). Samples were washed 3 in PBS and
subjected to flow cytometric analysis on a FACSCalibur apparatus (Becton
Dickinson, San Jose, CA).
Analysis of oxygen consumption and glycolytic rates. Measurements of OCR and
ECAR in GLUTag cells were performed using the XF24 analyser (Seahorse
Bioscience). A total 4 104 GLUTag cells per well were seeded in XF24 V7
microplates for 42 h before GW4064 treatment for 24 h. OCR and ECAR were
measured in Seahorse assay buffer containing basic glucose-free DMEM medium
(pH 7.4). The following compounds and concentrations were added successively:
glucose (10 mmol l 1); oligomycin (1 mmol l 1); 2-deoxyglucose (100 mmol l 1);
rotenone (1 mmol l 1) and antimycin A (1 mmol l 1).
Co-immunoprecipitation and western blot analysis. GLUTag cells were
glucose-deprived for 12 h and then treated 24 h with DMSO or with GW4064
(5 mmol l 1) in the presence of lactate (10 mmol l 1) or glucose (5.6 mmol l 1).
After three washes in ice-cold PBS, cells were scrapped in ice-cold PBS containing
protease inhibitors (1 , Roche). Cells were centrifuged for 5 min at 3,500g at 4 °C.
One volume of the cell lysis buffer (KCl (10 mmol l 1), TRIS HCl pH 7.9
(10 mmol l 1), DTT (0.5 mmol l 1), MgCl2 (1.5 mmol l 1), PIC 0.1%),
corresponding to the cell volume was added and cells were incubated on ice during
15 min. Cells were then centrifuged for 1 min at 11,500g at 4 °C. Supernatants were
collected (that is, cytoplasmic fraction) and pellets were lysed in modified RIPA
buffer (TRIS HCl pH 7.4 (50 mmol l 1), NaCl (150 mmol l 1), 0.25% DOC, 0.5%
12
NP40, EDTA (1 mmol l 1), 0.1% PIC) 30 min on ice. After centrifugation
(5 min at 13,000g at 4 °C) supernatants corresponding to the nuclear fraction were
collected. After a pre-clearing step in which protein fractions were incubated with
protein A agarose beads (1 h 30 min at 4 °C), cells were centrifuged (5 min at 1,000g
at 4 °C) and 200 mg of protein were immunoprecipitated (O/N at 4 °C) with
antibody against FXR (2 mg). Then immunocomplexes were captured with 100 ml
protein A agarose beads 3 h at 4 °C, centrifuged 1 min at 1,000g and washed
three times with 800 ml of RIPA modified buffer. Beads were resuspended in
2 migration buffer and heated for 3 min at 100 °C.
Western blots were generated as previously described29 and incubated with
proglucagon (SC-80730, Santa Cruz), FXR (PP-9033A, R&D), ChREBP (Novus
Biological, NB400-135) or b-actin (SC-1616, Santa Cruz) antibodies (diluted:
1/500). After 1 h incubation with horseradish peroxydase-conjugated secondary
antibodies (1/3,000, Sigma), protein revelation was performed using the enhanced
chemiluminescence FEMTO Plus reagents (ECL FEMTO, Thermofischer) by
autoradiography (Camera Gbox, SynGene) and band intensity was measured
(GeneTools software, SynGene).
Microarray analysis. GLUTag cells were treated or not with GW4064
(5 mmol l 1) for 24 h and four RNA samples of each treatment condition were
hybridized on mouse GEP 8 60 K arrays. Scanning clusters and data acquisition
were carried out following the manufacturer’s instructions (Agilent, One-colour
microarray Gene Expression Analysis). Data processing and analysis were
performed using the Genespring Software. Biological Processes Analysis was
performed using Gene Ontology Biological Processes on the Genomatix Software
(Genomatix, Deutschland). Data are deposited at EBI under the E-MTAB-2199
number.
RNA extraction and quantification by qPCR. Total RNAs from FACS-isolated
cells were isolated using a micro-scale RNA isolation kit (RNAeasy, Qiagen,
Crawley, UK) and were reverse transcribed according to standard protocols using a
Peltier Thermal Cycler-225 (MJ Research, Waltham, MA, USA). Quantitative PCR
with reverse transcription was performed with 7900 HT Fast Real-Time PCR
system (Applied Biosystems, Foster City, CA, USA). PCR reactions mix consisted
of first-strand cDNA template, primers (TaqMan gene expression assays, Applied
Biosystems) and PCR Master mix (Applied Biosystems). Fxr gene expression was
compared with that of b-actin measured on the same sample, in parallel, on the
same plate, giving a cycle threshold difference (DCT) for Fxr gene minus b-actin.
Total RNAs from GLUTag cells, mouse and human epithelial cells were
extracted using Extract-All Reagent (Eurobio, Courteboeuf, France) according to
the manufacturer’s protocol. After DNAse treatment (Fermentas, St Rémy Les
Chevreuse, France), total RNA (0.5–1 mg) was reverse transcribed using HighCapacity Multiscribe Reverse Transcriptase (Applied Biosystems, St Aubin, France)
according to the manufacturer’s protocol. qPCR with reverse transcription was
performed using the Master MIX SYBR Green Brillant Fast III (Agilent) on a
MX4000 apparatus (Stratagene) using specific oligonucleotides (see Table 2). The
results are presented using the DDCt method normalized to a reference gene
(Cyclophilin for in vitro and ex vivo experiments and TFIIB for in vivo
experiments). Controls were set at 1 and all conditions were expressed
comparatively to control.
Data analysis. In vitro experiments were performed in triplicates and repeated at
least three times. The in vitro and ex vivo data are presented as mean±s.d. The
in vivo data are presented as mean±s.e.m. All statistical analyses were performed
using two-tailed Student’s t-test or one-way analysis of variance followed by
Tukey’s post hoc test or two-way analysis of variance followed by Bonferronni’s
post hoc test and stated in the figure legends. P values r0.05 were considered as
significant.
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Acknowledgements
We wish to thank Meryem Tardivel and Anne-Sophie Drucbert of BiCeL-IFR114 Facility
for access to systems and technical advice. B.S. is a member of the Institut Universitaire
de France. M.-S.T. was supported by a grant from the French Ministry for Education and
Research. V.S. was supported by grant from the ‘‘Fondation pour la Recherche’’ (FRM).
This work was supported by grants from Région Nord-Pas de Calais, INSERM, A.N.R.
(FXREn), Université Lille 2, Université Lille Nord de France and European Genomic
Institute for Diabetes (EGID, ANR-10-LABX-46). Y.S. was supported by grant from the
Lille Métropole Communauté Urbaine (LMCU). F.M.G, F.R. and C.A.B. are funded
by the Wellcome Trust (WT088357/Z/09/Z, WT084210/Z/07/Z and the WT PhD
programme in Metabolic and Cardiovascular Disease).
Author contributions
M.-S.T., A.T., B.S. and S.L. conceived and designed the experiments; M.-S.T., J.P., V.T.,
E.D., E.V., S.D., Y.S., V.S., J.K., P.M., C.A.B., A.P. and S.I.S. acquired the data; M.-S.T.,
M.D., O.B., S.I.S., Y.S., J.K., B.S. and S.L. analysed and interpreted the data; M.-S.T.,
B.S. and S.L. drafted the manuscript; M.-S.T., F.M.G., A.T., B.S. and S.L. revised the
manuscript for important intellectual content; M.-S.T., S.S., S.C., K.B., P.L., C.A.B. and
V.T. participated in the statistical analysis; S.L. and B.S. obtained the funding; M.-S.T.,
V.T., H.D., E.D., E.V., N.H., R.C., G.B., S.S., A.P., F.R., F.M.G., A.T., K.S., F.B. and
F.P. gave technical and material supports; B.S. and S.L. supervised the study.
Additional information
Supplementary Information accompanies this paper at http://www.nature.com/
naturecommunications
Competing financial interests: The authors declare no competing financial interests.
Reprints and permission information is available online at http://npg.nature.com/
reprintsandpermissions/
How to cite this article: Trabelsi, M.S. et al. Farnesoid X receptor inhibits glucagon-like
peptide-1 production by enteroendocrine L cells. Nat. Commun. 6:7629
doi: 10.1038/ncomms8629 (2015).
NATURE COMMUNICATIONS | 6:7629 | DOI: 10.1038/ncomms8629 | www.nature.com/naturecommunications
& 2015 Macmillan Publishers Limited. All rights reserved.
13