Hepatic Deletion of SIRT1 Decreases Hepatocyte Nuclear Factor 1?/
Farnesoid X Receptor Signaling and Induces Formation of Cholesterol
Gallstones in Mice
Aparna Purushotham,aQing Xu,aJing Lu,aJulie F. Foley,bXingjian Yan,cDong-Hyun Kim,dJongsook Kim Kemper,dand Xiaoling Lia
Laboratory of Signal Transductionaand Cellular & Molecular Pathology Branch,bNational Institute of Environmental Health Sciences, Research Triangle Park, North
Carolina, USA; Undergraduate Programs of Biology and Biostatistics, University of North Carolina, Chapel Hill, North Carolina, USAc; and Department of Molecular and
Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USAd
First identified in yeast as key components in gene silencing com-
plexes (18), sirtuins have been increasingly recognized as crucial
regulators for a variety of cellular processes, ranging from energy
metabolism and stress response to tumorigenesis and aging (6).
The mammalian genome encodes seven sirtuins, SIRT1 to SIRT7
(15). As the most conserved mammalian sirtuin, SIRT1 couples
including p53, E2F1, NF-?B, FOXO, peroxisome proliferator-ac-
liver X receptor (LXR), farnesoid X receptor (FXR), CLOCK and
59), to the hydrolysis of NAD?. Therefore, SIRT1 has been con-
sidered as a metabolic sensor that directly links cellular metabolic
status to gene expression regulation, playing an important role in
a number of prosurvival and metabolic activities (19).
In the liver, the central metabolic organ that controls key as-
pects of nutrient metabolism (48), SIRT1 has been shown to reg-
ulate metabolism of both glucose and lipids (45). For instance,
SIRT1 inhibits TORC2, a key mediator of early phase gluconeo-
genesis, leading to decreased gluconeogenesis during the short-
term fasting phase (28). Prolonged fasting, on the other hand,
increases SIRT1-mediated deacetylation and activation of PGC-
1?, an essential coactivator for a number of transcription factors,
resulting in increased fatty acid oxidation and improved glucose
homeostasis (41, 42). Consistently, adenoviral knockdown of
SIRT1 reduces expression of fatty acid ?-oxidation genes in the
liver of fasted mice (43). Specific deletion of the exon 4 of the
tional SIRT1 protein, impairs peroxisome proliferator-activated
receptor ? (PPAR?) activity and fatty acid ?-oxidation, thereby
increasing the susceptibility of mice to high-fat diet-induced he-
patic steatosis and hepatic inflammation (41). Furthermore, a
IRT1 is a mammalian member of the silent information regu-
lator 2 (Sir2) family of proteins, also known as sirtuins (7).
to the development of liver steatosis, hyperglycemia, oxidative
damage, and insulin resistance, even on a normal chow diet (53,
54). Conversely, hepatic overexpression of SIRT1 mediated by
adenovirus attenuates hepatic steatosis and endoplasmic reticu-
lum (ER) stress and restores glucose homeostasis in mice (27). In
addition to glucose and fatty acid metabolism, SIRT1 has also
been reported to regulate hepatic lipid homeostasis through a
number of nuclear receptors and transcription factors (21, 26,
In this report, we show that hepatic SIRT1 modulates bile acid
pression. FXR is an important nuclear receptor in the regulation
report by Kemper et al. has shown that SIRT1 modulates the FXR
signaling through direct deacetylation of this transcription factor
in a mouse model in which hepatic SIRT1 was knocked-down by
short hairpin RNA (shRNA) (21). Using a liver-specific SIRT1
knockout mouse model (SIRT1 LKO), we show here that perma-
nent deletion of hepatic SIRT1 with the flox/albumin-Cre system
decreases FXR signaling largely through reduced activity of hepa-
tocyte nuclear factor 1? (HNF1?), a homeodomain-containing
transcription factor that plays an important role in the transcrip-
tional regulation of FXR (46). We found that deficiency of SIRT1
in the liver decreases the HNF1? recruitment to the FXR pro-
Received 22 July 2011 Returned for modification 16 August 2011
Accepted 13 January 2012
Published ahead of print 30 January 2012
Address correspondence to Xiaoling Li, email@example.com.
Supplemental material for this article may be found at http://mcb.asm.org/.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
mcb.asm.org0270-7306/12/$12.00 Molecular and Cellular Biology p. 1226–1236
moter and reduces the expression of FXR, resulting in impaired
transport of biliary bile acids and phospholipids and increased
incidence of cholesterol gallstones.
MATERIALS AND METHODS
Animal experiments. Liver-specific SIRT1 knockout (SIRT1 LKO) mice
in a C57BL/6 background were generated as described previously (41).
Nine- to 10-month-old SIRT1 LKO mice and their age-matched litter-
mate Lox controls (albumin-Cre negative, SIRT1flox/flox) were fed ad libi-
tum either a standard laboratory chow diet or a lithogenic diet (D12383;
Research Diets) for 6 weeks. All animal experiments were conducted in
accordance with guidelines of the National Institute of Environmental
Health Sciences (NIEHS)/NIH Animal Care and Use Committee.
Histological and biochemical analysis. Paraffin-embedded liver sec-
tions were stained with hematoxylin and eosin for morphology. Serum
enzyme-linked immunosorbent assay (ELISA) (Meso scale discovery).
Serum alanine transaminase (ALT) activities were measured using the
ALT kit from Catechem.
To examine the biliary lipid profiles of control and SIRT1 LKO mice,
bile was collected from the gallbladder, and then total bile acids were
measured with the total bile acid kit based on 3?-hydroxysteroid dehy-
drogenase (Diazyme Laboratories). Biliary phospholipids and total cho-
lesterol were determined using commercial kits from Wako. The choles-
terol saturation indices (CSI) were then calculated from the critical tables
in reference 10.
To determine the fecal bile acid outputs, feces were collected from
individually housed mice over 24 h and bile acids from feces were ex-
tracted with 75% ethanol at 50°C for 2 h, followed by centrifugation at
1,500 ? g for 10 min. Bile acids were then measured in the resulting
Cell culture. HEK293T cells stably infected by pSuper or pSuper-
SIRT1 RNA interference (RNAi) were described previously (26). Mouse
collagenase perfusion, seeded on collagen-coated plates in seeding me-
fetal bovine serum [FBS], 100 nM insulin, 1 ?M dexamethasone), and
maintained in maintenance medium (high-glucose DMEM, 0.1% bovine
serum albumin). To induce the expression of FXR target genes, primary
hepatocytes were treated with dimethyl sulfoxide (DMSO) or 1 ?M
GW4064 in high-glucose medium for 24 h.
munoprecipitation (ChIP) analysis. Liver total-cell homogenates were
prepared in SDS buffer (50 mM Tris-HCL [pH 6.8], 4% SDS), incubated
at 100°C for 10 min, and then immunoblotted using antibodies against
SIRT1 (Sigma), HNF1? (Santa Cruz Biotechnology), FXR (Santa Cruz
Biotechnology), and actin.
For immunoprecipitation between SIRT1 and HA-HNF1?, HEK293T
48 h later, cell lysates were prepared in NP-40 buffer (50 mM Tris-HCl [pH
phatase inhibitors (Roche) were immunoprecipitated with antihemaggluti-
To determine the acetylation levels of FXR protein in liver, 1 mg of
protein of liver extracts from control or SIRT1 LKO mice was incubated
for 3 h with 1 ?g of FXR antibody (goat polyclonal, sc-1204; Santa Cruz
Biotechnology) under stringent conditions with SDS-containing radio-
immunoprecipitation assay (RIPA) buffer. Acetylation levels of endoge-
nous FXR in the immunoprecipitates were detected with anti-acetyl-Lys
anti-FXR antibodies (mouse monoclonal, sc-25309; Santa Cruz Biotech-
Chromatin immunoprecipitation (ChIP) analysis was performed es-
sentially as described by Upstate Biotechnology with modifications.
Briefly, cells were cross-linked and harvested in IP buffer (10 mM Tris-
HCl [pH 8.0], 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate,
and Complete protease inhibitor mixture). Chromatins were then soni-
lipore), or normal rabbit IgG. DNA fragments were subjected to quanti-
tative PCR (qPCR) using primers flanking FXRE on the small het-
erodimer partner (SHP) promoter or different regions on the FXR
zol (Invitrogen) and the Qiagen RNeasy minikit (Qiagen). For real-time
qPCR, cDNA was synthesized with the ABI reverse transcriptase kit and
normalized to lamin A expression.
Luciferase assay. For transactivation experiments, the mouse FXR
promoter (fragment from ?2644 to ?149) was cloned into the pGL3
basic vector (Promega). Hepa1-6 cells were then transfected with FXR
firefly luciferase reporter and control pRL-CMV (cytomegalovirus) re-
porter (Renilla luciferase; Promega), together with the indicated con-
the dual-luciferase reporter assay system (Promega). The final firefly lu-
ciferase activity was normalized to the coexpressed Renilla luciferase ac-
Statistical analysis. Values are expressed as means ? standard errors
by two-tailed, unpaired Student’s t test, and differences were considered
significant at P ? 0.05.
Hepatic deletion of SIRT1 reduces the expression of FXR. To
examine the function of SIRT1 in the liver, we previously gener-
ated liver-specific SIRT1 knockout (SIRT1 LKO) mice and ana-
lyzed the hepatic transcriptional expression profiles of these mice
on a chow diet by microarray analyses (41). Interestingly, two
pathways, their products Abcb4 (?1.967), Abcc3 (?1.734),
Abcg8 (?1.153), and Slc10a2 (?2.010) are transporters that me-
S1 and S2 in the supplemental material), and Abcb4 is a direct
transcriptional target of FXR.
To confirm our microarray data that SIRT1 deficiency in the
tocytes from Lox control or SIRT1 LKO mice and treated them
tion of a number of FXR target genes, including the small het-
erodimer partner (SHP), Abcb11, and Abcb4 genes, was signifi-
cantly blunted in the SIRT1-deficient hepatocytes. Moreover,
lentivirus-mediated overexpression of SIRT1 in LKO hepatocytes
completely restored the mRNA levels of these genes (Fig. 1B).
These data indicate that SIRT1 positively regulates FXR signaling
directly in hepatocytes. To dissect the molecular mechanism un-
derlying this regulation, we analyzed the expression levels of FXR
in hepatocytes and livers. As shown in Fig. 2A and B, both the
mRNA and protein levels of FXR were significantly decreased in
chromatin-associated FXR levels at the FXRE of SHP were signif-
icantly decreased in the livers of SIRT1 LKO mice as well as in
SIRT1-deficient primary hepatocytes (Fig. 2C). These observa-
tions suggest that SIRT1 may positively regulate the transcription
SIRT1 Regulates HNF1?
April 2012 Volume 32 Number 7 mcb.asm.org 1227
may be in part due to decreased expression of FXR. In support of
our hypothesis, overexpression of SIRT1 in the SIRT1-deficient
hepatocytes resulted in a dose-dependent increase of FXR mRNA
levels and further stimulated the expression of FXR in the control
hepatocytes (Fig. 2D).
FXR has recently been reported as an acetylated transcription
factor (21). It has been shown that acetylation of FXR by the
its partner, RXR?, resulting in reduced DNA binding and trans-
the observed transcriptional regulation of FXR by SIRT1 (Fig. 2)
relative to the previously defined role of SIRT1 in enhancing the
alyzed the transactivation activity of exogenous murine FXR pro-
tein in control and SIRT1-deficient primary hepatocytes. As
shown in Fig. 3A, lentiviral expression of FXR in the SIRT1 KO
hepatocytes completely rescued the deficient expression of SHP
and almost completely recovered the levels of Abcb4, indicating
protein is almost normal in the SIRT1-deficient hepatocytes. To
further assess the transactivation activities of acetylated-FXR and
deacetylated-FXR proteins in primary hepatocytes, we generated
lentiviruses expressing mutant FXR proteins in which the previ-
ously identified lysine acetylation sites were mutated either to ar-
hepatocytes. (A) SIRT1 deficiency in primary hepatocytes reduces the induc-
in primary hepatocytes restores the expression of FXR targets. Primary hepa-
tocytes from control and SIRT1 LKO mice were infected with lentiviruses
by qPCR. *, P ? 0.05.
FIG 2 Loss of hepatic SIRT1 decreases the expression of FXR. (A) SIRT1 deficiency leads to reduced mRNA levels of FXR in the liver (n ? 11) and primary
hepatocytes (n ? 3). *, P ? 0.05. (B) SIRT1 deficiency results in reduced FXR protein levels in the liver (n ? 4). *, P ? 0.05. (C) Reduced recruitment of FXR to
the FXRE on the promoter of SHP gene in the SIRT1-deficient livers and primary hepatocytes (n ? 3). *, P ? 0.05. (D) Overexpression of SIRT1 in SIRT1-
*, P ? 0.05.
Purushotham et al.
mcb.asm.orgMolecular and Cellular Biology
ginine (K168R/K228R [KR]) to mimic the deacetylation protein
or to glutamine (K168Q/K228Q [KQ]) to mimic the acetylated
FXR protein. The transactivation activities of these mutants were
then analyzed in control and SIRT1 KO primary hepatocytes. As
shown in Fig. 3B, the WT and mutant FXR proteins had compa-
rable activities in both control and SIRT1-deficient hepatocytes
on two of FXR target genes, the SHP and Abcb4 genes. However,
the deacetylation-mimetic, KR mutant protein displayed signifi-
cantly increased activity on Abcb11. These observations suggest
that acetylation status of FXR impacts its transcriptional activity
only on some target genes. Taken together, our data demonstrate
ity of FXR largely through the transcriptional regulation of its
SIRT1 regulates the expression of FXR through HNF1?. As
an important bile acid sensor that is critical for lipid and glucose
metabolism, the expression of FXR is tightly controlled by an in-
scription factor that is essential for diverse metabolic processes in
the pancreatic islets, liver, intestine, and kidney (25, 38, 39). The
expression and transactivation activity of FXR are also regulated
peroxisome proliferator-activated receptor-gamma coactivator
1? (PGC-1?) (61), which is a direct deacetylation target of SIRT1
43). FXR has also been reported to self-regulate its expression
(reviewed by Eloranta and Kullak-Ublick ).
To dissect the molecular mechanisms by which loss of SIRT1
leads to the reduction of FXR expression, we analyzed the associ-
ation of SIRT1 with the mouse FXR promoter. As shown in Fig.
4A, SIRT1 was relatively concentrated approximately 300 bp up-
stream of the transcription start site (TSS), where multiple bind-
ing sites of HNF1? were identified by the Genomatix MatInspec-
tor analyses (data not shown). Consistently, HNF1? was highly
observation suggests that SIRT1 may regulate the expression of
FXR through modulation of HNF1?.
the differentiation program in several organs, including the liver,
kidney, intestine, and pancreas. Haploinsufficiency of HNF1? in
ment of diabetes, renal Fanconi syndrome, hepatic dysfunction,
and hypercholesterolemia (25, 38, 39). Since both mRNA and
primary hepatocytes (Fig. 4B to D), we speculated that SIRT1
might regulate the activity of this transcription factor at the
posttranscriptional level, which then indirectly affects the ex-
pression of FXR. Consistent with this possibility, both WT and
SIRT1 KO hepatocytes rescues the deficient expression of FXR targets. Primary hepatocytes from control and SIRT1 LKO mice were infected with lentiviruses
KQ FXR proteins in control and SIRT1 LKO hepatocytes. *, P ? 0.05. Primary hepatocytes from control and SIRT1 LKO mice were infected with lentiviruses
expressing WT FXR, FXR KR, or FXR KQ mutant proteins. The expression levels of FXR and FXR target genes were determined by qPCR. *, P ? 0.05.
SIRT1 Regulates HNF1?
April 2012 Volume 32 Number 7mcb.asm.org 1229
catalytically inactive (HY) SIRT1 were coimmunoprecipitated
with HA-HNF1? in HEK293T cells (Fig. 4E). Moreover, the
chromatin-associated HNF1? levels were significantly reduced
in the SIRT1-deficient hepatocytes compared to the control
hepatocytes in a chromatin immunoprecipitation assay (Fig.
4F), suggesting that deletion of SIRT1 in hepatocytes decreases
the DNA binding affinity of HNF1?. To further confirm that
SIRT1 regulates the expression of FXR through HNF1?, we
(siRNA) or siRNA against HNF1? into the mouse hepatocyte
Hepa1-6 cell line. We then transfected these siRNAs with
vector (V) or constructs expressing WT or HY SIRT1 together
with mouse FXR promoter luciferase reporter (Fig. 4G). As
shown in Fig. 4H, in Hepa1-6 cells transfected with control
siRNA, overexpression of WT SIRT1 but not the HY mutant
significantly induced the luciferase reporter of FXR. However,
this induction was decreased in HNF1? RNAi cells, suggesting
that SIRT1 induces the expression of FXR in part through
In line with the observation that deletion of hepatic SIRT1
leads to reduced expression of HNF1? target gene FXR, SIRT1
of other HNF1? target genes in both liver and primary hepato-
cytes (Fig. 5A and B). Furthermore, overexpression of SIRT1 in
levels of HNF1? target genes and further stimulated the expres-
sion of these targets in the control hepatocytes (Fig. 5C). Collec-
tively, these findings demonstrate that SIRT1 deficiency in hepa-
tocytes impairs the expression of FXR through modulation of
HNF1? transcriptional activity.
tabolism on the lithogenic diet. Decreased activity of HNF1? in
tes, hepatic dysfunction, and hypercholesterolemia (25, 38, 39,
56). Disruption of FXR in mice is also associated with the devel-
opment of metabolic diseases, including diabetes and hypercho-
lesterolemia (47). The decreased activity of HNF1? and reduced
FIG 4 SIRT1 regulates the expression of FXR through HNF1?. (A) SIRT1 is enriched on the HNF1? binding sites on the mouse FXR promoter. Primary
hepatocytes from control and SIRT1 LKO mice were ChIPed with SIRT1 or HNF1? antibodies. DNA fragments were then subjected to qPCR using primers
inactive (HY) SIRT1 were immunoprecipitated (IP) with anti-HA antibodies. (F) SIRT1 deficiency leads to decreased association of HNF1? with the HNF1?
binding sites on the FXR promoter. Primary hepatocytes from control and SIRT1 LKO mice were ChIPed with IgG or HNF1? antibodies. (G and H) SIRT1
induces the expression of FXR through HNF1? in Hepa1-6 cells. Mouse hepatocyte Hepa1-6 cells were electroporated with negative control siRNA (control
promoter luciferase reporter. The expression levels of HNF1? and SIRT1 (G) were determined by qPCR, and the luciferase activity of FXR reporter was
determined as described in Materials and Methods.
Purushotham et al.
mcb.asm.org Molecular and Cellular Biology