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 Biologyp. 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 7mcb.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.org Molecular 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 7 mcb.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
by these two factors.
To examine the pathophysiological effects of blunted HNF1?
decreased fatty acid oxidation, leading to a mild body weight gain
and the development of hepatic steatosis and inflammation on a
Western-style high-fat diet (41). When challenged with the litho-
genic diet, SIRT1 LKO mice showed no obvious signs of body
weight abnormality (data not shown) and had normal levels of
serum insulin and leptin (Fig. 6A). However, they displayed mild
but significant hypercholesterolemia, primarily through an in-
crease of the LDL fraction in serum (Fig. 6B). They also displayed
modest but significant hepatomegaly and had greater lipid accu-
mulation in the liver (Fig. 6C and D). In line with these observa-
tions, SIRT1 LKO mice showed significantly altered expression of
a couple of genes involved in cholesterol efflux and triglyceride
biogenesis (Fig. 6E). Additionally, 6 weeks of lithogenic diet feed-
LKO mice, as revealed by elevated serum alanine transaminase
(ALT) activities (Fig. 6F).
Hepatic deletion of SIRT1 impairs bile acid metabolism and
induces formation of cholesterol gallstones on the lithogenic
diet. Disruption of FXR in mice has also been associated with the
development of cholesterol gallstone disease upon lithogenic diet
formation of gallstones, which were visible at the macroscopic
level (Fig. 7B) in 73% of SIRT1 LKO mice (Fig. 7A). In contrast,
only 27% of the control mice acquired gallstones under the same
conditions (Fig. 7A).
Since the formation of gallstones is primarily determined by
the relative biliary concentrations of bile salts/phospholipids and
respectively (31), we analyzed the biliary lipid profile in control
and SIRT1 LKO mice. As shown in Fig. 7C, SIRT1 LKO mice
showed reduced levels of biliary bile acids and phospholipids
compared to control mice, whereas their biliary cholesterol con-
centrations were normal. As a result, their biliary cholesterol was
SIRT1 LKO mice have deficient FXR signaling on the lithogenic
FXR targets located at the outer leaflet of the hepatocyte canalic-
salts and phospholipids (31), Abcb11 and Abcb4, were signifi-
cantly lower in SIRT1 LKO mice compared to control mice (Fig.
7E). In contrast, the levels of two cholesterol transporters that are
under the control of LXR, Abcg5 and Abcg8, were not changed
SIRT1 leads to defective FXR signaling, resulting in reduced bili-
ary bile salt and phospholipid concentrations and increasing the
risk of cholesterol gallstones while on the lithogenic diet.
synthesis, primarily through SHP, an odd member of the nuclear
receptor superfamily (16, 20). Upon activation by bile acids, FXR
induces the expression of SHP, which in turn binds to LXR, liver
receptor homolog 1 (LRH-1), and possibly other nuclear recep-
tors to attenuate further bile acid synthesis (44). Consistently,
*, P ? 0.05. MOI ? 2.5 and 5, respectively. a-Fetoprotein, ?-fetoprotein; a-Fibrinogen, ?-fibrinogen.
SIRT1 Regulates HNF1?
April 2012 Volume 32 Number 7 mcb.asm.org 1231
deletion of FXR in mice decreases the expression of SHP while
increasing the mRNA levels of bile acid synthesis genes, such as
Cyp7a1, Cyp8b1, and Cyp27a1 (31). Loss of SHP, on the other
hand, partially impairs negative feedback regulation of bile acid
SIRT1 LKO mice may also suffer from an abnormally high rate of
bile acid synthesis. However, unexpectedly, the expression of a
number of bile acid synthesis genes was lower in these mice (Fig.
7F). In line with this observation, their bile acid synthesis rates,
based on the steady-state fecal bile acid output rate, were de-
genic conditions (Fig. 7G). However, there were no significant
alterations in the total bile acid pool size levels (Fig. 7H) and liver
the FXR-SHP-bile acid synthesis feedback loop was impaired in
the SIRT1 LKO mice.
ber of age-associated diseases. While it has been reported that
SIRT1 is a vital regulator in many aspects of hepatic lipid and
glucose metabolism in response to different nutrient signals (19,
45) and that SIRT1 regulates the FXR signaling by direct deacety-
lation of this transcription factor (21), we show in the present
study that hepatic SIRT1 modulates the message RNA levels of
FXR through HNF1?. As a result, deletion of SIRT1 in the liver
reduced expression of FXR, leading to decreased transport of bil-
iary bile acids and phospholipids and increased incidence of cho-
lesterol gallstones. These observations uncover a previously un-
known link between SIRT1, HNF1?, and transcriptional
regulation of FXR expression, suggesting that hepatic SIRT1 may
also be an important therapeutic target for cholesterol gallstone
the FXR signaling pathway predominantly at the transcriptional
of FXR is decreased not only in the SIRT1-deficient liver, but also
normal serum hormonal levels (A) and a mild increase of serum total cholesterol and LDL levels (B) (n ? 11). *, P ? 0.05. (C and D) SIRT1 LKO mice display
levels of SCD1 (n ? 11). *, P ? 0.05. (F) SIRT1 deficiency in the liver increases liver damage (n ? 11). *, P ? 0.05.
Purushotham et al.
mcb.asm.orgMolecular and Cellular Biology
in the SIRT1-deficient hepatocytes (Fig. 2A), indicating that
SIRT1 directly regulates FXR expression in a cell autonomous
fashion. Moreover, overexpression of SIRT1 in primary hepato-
cytes induces the expression of FXR (Fig. 2D). More importantly,
it appears that the transactivation activity of exogenous FXR is
normal in the SIRT1-deficient hepatocytes, as lentiviral expres-
sion of FXR almost completely rescues the defective FXR activity
in these cells (Fig. 3A). On the other hand, our results also point
toward the involvement of additional mechanisms. As shown in
Fig. 3A, putting back FXR did not completely restore the expres-
sion of all tested FXR downstream targets in the SIRT1-deficient
hepatocytes. It appears that the acetylation status of the FXR pro-
teins indeed affects their transactivation activities on some of the
Furthermore, the FXR proteins are markedly hyperacetylated in
the SIRT1 LKO mice (Fig. 8A). These observations indicate that
SIRT1 also plays a role in the posttranslational activation of FXR
through direct deacetylation of the receptor, thereby improving
its DNA binding ability (21). In addition to HNF1?, the expres-
sion and transactivation activity of FXR are also regulated by a
deletion of SIRT1 leads to decreased coactivation activity of
PGC-1? (41). Although our preliminary data indicate that
SIRT1-deficient hepatocytes (data not shown), the decreased ac-
tivity of PGC-1? may partially contribute to the reduction of the
FXR signaling in these cells. Interestingly, recent studies have
57). It has been shown that SHP, one of the direct FXR targets,
3= untranscribed region (3= UTR) of SIRT1 mRNA, inhibiting
the translation of SIRT1 protein (24, 57). Therefore, SIRT1 and
the FXR signaling pathway mutually interact at multiple levels,
coordinately regulating hepatic bile acid and cholesterol homeo-
stasis (Fig. 8B).
of liver and pancreas and that human HNF1? is commonly mu-
tated in patients with maturity onset diabetes of the young
LKO mice display increased incidence of cholesterol gallstones (n ? 11). The 9- to 10-month-old control and SIRT1 LKO mice were fed a lithogenic diet for 6
display decreased biliary concentrations of bile acids and phospholipids (n ? 8). *, P ? 0.05; #, P ? 0.068. (D) SIRT1 LKO mice have increased cholesterol
saturation indices (CSI) in gallbladder bile (n ? 8). *, P ? 0.05. (E) SIRT1 deficiency reduces expression of bile acid and phospholipid transporters at the
hepatocyte canalicular membrane (n ? 11). *, P ? 0.05. (F) Decreased expression of bile acid synthesis genes in the SIRT1 LKO mice (n ? 11). *, P ? 0.05. (G
to I) SIRT1 LKO mice show decreased fecal bile acid output (G) but normal total bile acid pool size (H) and hepatic bile acids (I) (n ? 5 to 6) *, P ? 0.05.
SIRT1 Regulates HNF1?
April 2012 Volume 32 Number 7 mcb.asm.org 1233
(MODY), which is characterized by severe insulin secretory de-
fects (1, 56), the positive forward link between SIRT1, HNF1?,
For example, it has been shown that increased dosage of SIRT1 in
pancreatic ? cells improves glucose tolerance and enhances insu-
lin secretion in response to glucose (33), whereas deletion of
SIRT1 impairs glucose-stimulated insulin secretion (8). Resvera-
trol, a polyphenol activator of SIRT1, potentiates glucose-stimu-
lated insulin secretion in ? cells (50). In addition, activation of
SIRT1 by its activators in animals protects against high-fat-in-
duced obesity and insulin resistance (4, 23, 30), and modest over-
expression of SIRT1 resulted in a protective effect against high-
fat-induced hepatic steatosis and glucose intolerance (3, 37). Our
data suggest that activation of the HNF1? signaling pathway may
ration of this possibility may provide novel insights into SIRT1’s
function in whole-body glucose homeostasis. Our data, however,
are in contrast to those reported in a recent study (17). Grimm et
al. have shown that SIRT1 and HNF1? form a nutrient-sensitive
ity of HNF1? on its target genes, particularly C-reactive protein
(CRP), in hepatocytes. In addition, their data show that SIRT1
tion of H4K16 instead of HNF1? itself (17). One possible factor
contributing to the discrepancy between our observations and
those of Grimm et al. may be the difference in environmental
challenges in two studies. SIRT1 appears to suppress HNF1? and
the production of CRP only under conditions of nutrient restric-
tion (17), whereas SIRT1 activates the HNF1?/FXR pathway in
mediated gene expression involves formation of multiunit tran-
scriptional complexes with other transcription factors. For in-
stance, cytokine-driven expression of the CRP gene requires
formation of c-Fos, STAT3, and the HNF1? transcriptional com-
the expression of different HNF1? target genes is determined by
the combination of different transcriptional partners.
How SIRT1 regulates the activity of HNF1? is still an ongoing
study. Since the chromatin-associated HNF1? levels were signifi-
cantly reduced in the livers of SIRT1 LKO mice (Fig. 4F), loss of
SIRT1 may directly or indirectly decrease the DNA binding affin-
ity of HNF1?. Additional experiments are needed to dissect the
molecular mechanisms underlying this important modulation in
vitro and in vivo.
The impaired FXR-SHP-bile acid synthesis feedback loop in
the SIRT1 LKO mice suggests that hepatic deletion of SIRT1 may
disrupt bile acid synthesis through FXR-independent mecha-
nisms. For instance, increased liver damage in the SIRT1 LKO
mice under the lithogenic diet may activate c-Jun N-terminal ki-
nase (JNK), thereby inhibiting bile acid synthesis (22, 44, 52).
Deletion of SIRT1 in the hepatocytes may also result in hepatic
the first committed enzyme in the acidic pathway of bile acid
synthesis, Cyp7b1 (5). However, comparison of levels of phos-
pho-JNK, a marker of the activated JNK pathway, in the liver
alterations of this signaling pathway (data not shown). Ser473
phosphorylation of Akt, a key molecule in the insulin signaling
pathway, was also normal in the livers of SIRT1 LKO mice (data
not shown). Therefore, additional studies are required to dissect
the mechanism underlying this intriguing phenotype.
In summary, we have shown that hepatic SIRT1 plays an im-
portant role in the regulation of hepatic bile acid metabolism.
SIRT1 LKO mice were immunoprecipitated with goat anti-FXR antibodies and then immunoblotted with rabbit anti-acetyl-K antibodies or mouse anti-FXR
antibodies (n ? 3). *, P ? 0.05. (B) The interaction network between SIRT1 and the FXR signaling pathway. SIRT1 modulates the activity of FXR signaling at
multiple levels (red lines). First, SIRT1 regulates the expression of FXR through interaction with HNF1?, which may also involve coactivator PGC-1?. Second,
SIRT1 deacetylates FXR, increasing its DNA binding affinity. Third, SIRT1 deacetylates PGC-1?, thereby activating the transactivation activity of FXR. Finally,
of SIRT1 protein via SHP-mediated inhibition of p53 transactivation and thereby miR34a expression (blue lines). This positive feedback network plays an
resulting in development of cholesterol gallstones.
Purushotham et al.
mcb.asm.orgMolecular and Cellular Biology
Hepatic deletion of SIRT1 leads to an increased susceptibility to
cholesterol gallstone disease through decreased HNF1?/FXR sig-
lesterol gallstone disease and suggest that new therapeutic strate-
gies designed to modulate SIRT1 activity may be beneficial for
preventing formation of cholesterol gallstones as well as for other
metabolic diseases associated with type 2 diabetes.
eric Alt at Harvard Medical School for providing the SIRT1 exon 4 floxed
allele. We also thank the NIEHS Laboratory of Experimental Pathology
for histological staining and serum hormone ELISA and the NIEHS viral
core facility for lentiviruses.
This research was supported by the Intramural Research Program of
the NIH, National Institute of Environmental Health Sciences, to X.L.
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