1

Hepatic deletion of SIRT1 decreases HNF1α/FXR signaling and 1

induces formation of cholesterol gallstones in mice 2

3

Aparna Purushotham1, Qing Xu1, Jing Lu1, Julie F. Foley2, Xingjian Yan3, 4

Dong-Hyun Kim4, Jongsook Kim Kemper4, and Xiaoling Li1, * 5

1 Laboratory of Signal Transduction, 2 Cellular & Molecular Pathology Branch, 6

National Institute of Environmental Health Sciences, Research Triangle Park, NC 7

27709, 3 Undergraduate Programs of Biology and Biostatistics, University of 8

North Carolina, Chapel Hill, NC 27514, 4 Department of Molecular and Integrative 9

Physiology, University of Illinois at Urbana-Champaign, Urbana, IL 61801 10

* Correspondence: lix3@niehs.nih.gov 11

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Running title: SIRT1 regulates HNF1α 15

Key words: SIRT1, HNF1α, FXR, cholesterol gallstones 16

17

Word count for the Materials and Methods: 740 18

Word count for the Introduction, Results, and Discussion: 3803 19

20

Copyright © 2012, American Society for Microbiology. All Rights Reserved.Mol. Cell. Biol. doi:10.1128/MCB.05988-11 MCB Accepts, published online ahead of print on 30 January 2012

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Abstract 21

SIRT1, a highly conserved NAD+-dependent protein deacetylase, is a key 22

metabolic sensor that directly links nutrient signals to animal metabolic 23

homeostasis. Although SIRT1 has been implicated in a number of hepatic 24

metabolic processes, the mechanisms by which hepatic SIRT1 modulates bile 25

acid metabolism are still not well understood. Here we report that deletion of 26

hepatic SIRT1 reduces the expression of farnesoid X receptor (FXR), a nuclear 27

receptor that regulates bile acid homeostasis. We provide evidence that SIRT1 28

regulates the expression of FXR through hepatocyte nuclear factor 1α (HNF1α). 29

SIRT1 deficiency in hepatocytes leads to decreased binding of HNF1α to the 30

FXR promoter. Furthermore, we show that hepatocyte-specific deletion of SIRT1 31

leads to derangements in bile acid metabolism, predisposing the mice to 32

development of cholesterol gallstones on a lithogenic diet. Taken together, our 33

findings indicate that SIRT1 plays a vital role in the regulation of hepatic bile acid 34

homeostasis through the HNF1α/FXR signaling pathway. 35

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Introduction 44

SIRT1 is a mammalian member of the Silent Information Regulator 2 45

(Sir2) family of proteins, also known as sirtuins (7). First identified in yeast as key 46

components in gene silencing complexes (18), sirtuins have been increasingly 47

recognized as crucial regulators for a variety of cellular processes, ranging from 48

energy metabolism and stress response to tumorigenesis and aging (6). The 49

mammalian genome encodes seven sirtuins, SIRT1 to SIRT7 (15). As the most 50

conserved mammalian sirtuin, SIRT1 couples the deacetylation of numerous 51

transcription factors and co-factors, including p53, E2F1, NFκB, FOXO, PGC-1α, 52

c-myc, HIF-1, HIF-2α, HSF1, LXR, FXR, CLOCK and PER2, and TORC2 (2, 9, 53

13, 21, 26, 28, 29, 32, 34, 35, 42, 49, 55, 58, 59), to the hydrolysis of NAD+. 54

Therefore, SIRT1 has been considered as a metabolic sensor that directly links 55

cellular metabolic status to gene expression regulation, playing an important role 56

in a number of pro-survival and metabolic activities (19). 57

In the liver, the central metabolic organ that controls key aspects of 58

nutrient metabolism (48), SIRT1 has been shown to regulate both glucose and 59

lipid metabolism (45). For instance, SIRT1 inhibits TORC2, a key mediator of 60

early phase gluconeogenesis, leading to decreased gluconeogenesis during 61

short-term fasting phase (28). Prolonged fasting, on the other hand, leads to 62

increased SIRT1 deacetylation and activation of PGC-1α, an essential co-63

activator for a number of transcription factors, resulting in increased fatty acid 64

oxidation and improved glucose homeostasis (41, 42). Consistently, adenoviral 65

knockdown of SIRT1 reduces expression of fatty acid β-oxidation genes in the 66

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liver of fasted mice (43). Specific deletion of the exon 4 of the hepatic mouse 67

SIRT1 gene, which results in a truncated non-functional SIRT1 protein, impairs 68

peroxisome proliferator-activated receptor (PPARα) activity and fatty acid β-69

oxidation, thereby increasing the susceptibility of mice to high-fat diet induced 70

hepatic steatosis and hepatic inflammation (41). Furthermore, a complete 71

deletion of hepatic SIRT1 by floxing exons 5 and 6 leads to the development of 72

liver steatosis, hyperglycemia, oxidative damage, and insulin resistance even 73

under a normal chow diet (53, 54). Conversely, hepatic overexpression of SIRT1 74

mediated by adenovirus attenuates hepatic steatosis and ER stress, and restores 75

glucose homeostasis in mice (27). In addition to glucose and fatty acid 76

metabolism, SIRT1 has also been reported to regulate hepatic lipid homeostasis 77

through a number of nuclear receptors and transcription factors (21, 26, 40, 51). 78

In this report, we show that hepatic SIRT1 modulates bile acid metabolism 79

through regulation of farnesoid X receptor (FXR) expression. FXR is an important 80

nuclear receptor in the regulation of systemic cholesterol and bile acid 81

metabolism (12, 20). A recent report by Kemper et al. has shown that SIRT1 82

modulates the FXR signaling through direct deacetylation of this transcription 83

factor in a mouse model where hepatic SIRT1 was knocked-down by shRNA 84

(21). Using a liver-specific SIRT1 knockout mouse model (SIRT1 LKO), we show 85

here that permanent deletion of hepatic SIRT1 with flox/Albumin-cre system 86

decreases FXR signaling largely through reduced activity of hepatocyte nuclear 87

factor 1α (HNF1α), a homeodomain-containing transcription factor that plays an 88

important role in the transcriptional regulation of FXR (46). We found that 89

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deficiency of SIRT1 in the liver decreases the HNF1α recruitment to the FXR 90

promoter and reduced the expression of FXR, resulting in impaired transport of 91

biliary bile acids and phospholipids and increased incidence of cholesterol 92

gallstones. 93

94

Material and methods 95

Animal experiments 96

Liver-specific SIRT1 knockout mice (SIRT1 LKO) in C57BL/6 background 97

were generated as described (41). 9-10-month-old SIRT1 LKO mice and their 98

age-matched littermate Lox controls (Albumin-Cre negative, SIRT1 flox/flox) were 99

fed ad libitum either a standard laboratory chow diet or a lithogenic diet (D12383, 100

Research Diets) for 6 weeks. All animal experiments were conducted in 101

accordance with guidelines of NIEHS/NIH Animal Care and Use Committee. 102

103

Histological and biochemical analysis 104

Paraffin-embedded liver sections were stained with hematoxylin and eosin 105

for morphology. Serum LDL, HDL and total cholesterol and triglycerides were 106

measured using commercially available kits (Wako and Sigma). Serum insulin 107

and leptin levels were measured by ELISA (Meso scale discovery). Serum 108

alanine transaminase (ALT) activities were measured using the ALT kit from 109

Catechem. 110

To examine the biliary lipid profiles of control and SIRT1 LKO mice, bile 111

was collected from the gallbladder, then total bile acids were measured with the 112

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total bile acid kit based on 3α-hydroxysteroid dehydrogenase (Diazyme 113

Laboratories). Biliary phospholipids and total cholesterol were determined using 114

commercial kits form Wako. The Cholesterol Saturation Indices (CSI) were then 115

calculated from the critical tables in reference 10 (10). 116

To determine the fecal bile acid outputs, feces were collected from 117

individually housed mice over 24 h and bile acids from feces were extracted with 118

75% ethanol at 50 °C for 2 h, followed by centrifugation at 1500 g for 10 min. Bile 119

acids were then measured in the resulting supernatants. 120

121

Cell culture 122

HEK293T cells stably infected with pSuper or pSuper-SIRT1 RNAi have 123

been described (26). Mouse primary hepatocytes were isolated from control or 124

SIRT1 LKO mice using collagenase perfusion, seeded on collagen-coated plates 125

in seeding medium (high glucose DMEM, 10% FBS, 100 nM insulin, 1 µM 126

dexamethasone), and maintained in maintenance medium (high glucose DMEM, 127

0.1% bovine serum albumin). To induce the expression of FXR target genes, 128

primary hepatocytes were treated with DMSO or 1 µM of GW4064 in high 129

glucose medium for 24 h. 130

131

Western blot analysis, co-immunoprecipitation and chromatin 132

immunoprecipitation (ChIP) analysis 133

Liver total-cell homogenates were prepared in the SDS buffer (50mM Tris-134

HCL, pH 6.8, 4% SDS), incubated at 100 °C for 10 min, and then immunoblotted 135

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using antibodies against SIRT1 (Sigma), HNF1α (Santa Cruz biotechnology), 136

FXR (Santa Cruz biotechnology), and actin. 137

For immuno-precipitation between SIRT1 and HA-HNF1α, HEK293T cells 138

were transfected with indicating constructs. Cells were then harvested 48 h later 139

and cell lysates prepared in NP40 buffer (50mM Tris-HCL, pH 8.0, 150mM NaCl, 140

0.5% NP40) containing Complete TM protease and phosphatase inhibitors 141

(Roche) were immunoprecipitated with anti-HA antibodies (Santa Cruz 142

biotechnology). 143

To determine the acetylation levels of FXR protein in liver, 1 mg protein of 144

liver extracts from control or SIRT1 LKO were incubated for 3 h with 1 μg of FXR 145

antibody (goat polyclonal, sc-1204, Santa Cruz biotechnology) under stringent 146

conditions with SDS-containing RIPA buffer. Acetylation levels of endogenous 147

FXR in the immunoprecipitates were detected with anti-acetyl-Lys antibody (Cell 148

Signaling). The membrane was stripped and re-probed with anti-FXR antibodies 149

(mouse monoclonal, sc-25309, Santa Cruz biotechnology). 150

Chromatin immunoprecipitation (ChIP) analysis was performed essentially 151

as described by the Upstate Biotechnology with modifications. Briefly, cells were 152

crosslinked and harvested in IP buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1% 153

Triton X-100, 0.1% Na-Deoxycholate, and Complete™ protease inhibitor 154

mixture). Chromatins were then sonicated to an average length of 200-500 bp 155

and immunoprecipitated with antibodies against FXR, HNF1α (Santa Cruz 156

biotechnology), SIRT1 (Millipore), or normal rabbit IgG. DNA fragments were 157

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subjected to qPCR using primers flanking FXRE on the SHP promoter or 158

different regions on the FXR promoter. 159

160

RNA analysis 161

Total RNA was isolated from cells or tissues using Trizol (Invitrogen) and 162

Qiagen RNeasy mini-kit (Qiagen). For quantitative real-time PCR (qPCR), cDNA 163

was synthesized with the ABI reverse transcriptase kit, and analyzed using 164

SYBR Green Supermix (Applied Biosystems). All data were normalized to 165

Lamin A expression. 166

167

Luciferase assay 168

For trans-activation experiments, the mouse FXR promoter (-2644 to +149 169

fragment) was cloned into the pGL3 basic vector (Promega). Hepa1-6 cells were 170

then transfected with FXR fire-fly luciferase reporter and control pRL-CMV 171

reporter (Renilla Luciferase, Promega) together with indicated constructs, and 172

were cultured for 24 h. Luciferase activity was measured using the Dual-173

Luciferase Reporter Assay System (Promega). The final firefly luciferase activity 174

was normalized to the co-expressed renilla luciferase activity. 175

176

Statistical analysis 177

Values are expressed as mean + standard error of mean (SEM). 178

Significant differences between the means was analyzed by two-tailed, unpaired 179

Student’s t test and differences were considered significant at p< 0.05. 180

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181

Results 182

Hepatic deletion of SIRT1 reduces the expression of FXR 183

To examine the function of SIRT1 in the liver, we have previously 184

generated liver-specific SIRT1 knockout mice (SIRT1 LKO mice) and analyzed 185

the hepatic transcriptional expression profiles of these mice under a chow diet by 186

microarray analyses (41). Interestingly, two related pathways that were 187

significantly affected in the SIRT1 LKO mice were hepatic cholestasis and 188

FXR/RXR activation (Figure S1). Among genes involved in these two pathways, 189

Abcb4 (-1.967), Abcc3 (-1.734), Abcg8 (-1.153), and Slc10a2 (-2.010) are 190

transporters that mediate the uptake and efflux of bile acids and cholesterol 191

(Table S1 and S2), and Abcb4 is a direct transcriptional target of FXR. 192

To confirm our microarray data that SIRT1 deficiency in the liver results in 193

decreased FXR signaling, we isolated primary hepatocytes from Lox control or 194

SIRT1 LKO mice and treated them with a FXR agonist GW4064 (GW). As shown 195

in Figure 1A, the induction of a number of FXR target genes, including Small 196

heterodimer partner (SHP), Abcb11, and Abcb4, was significantly blunted in the 197

SIRT1 deficient hepatocytes. Moreover, lentivirus mediated overexpression of 198

SIRT1 in LKO hepatocytes completely restored the mRNA levels of these genes 199

(Figure 1B). These data indicate that SIRT1 positively regulates FXR signaling 200

directly in hepatocytes. To dissect the molecular mechanism underlying this 201

regulation, we analyzed the expression levels of FXR in hepatocytes and livers. 202

As shown in Figure 2A and 2B, both the mRNA and proteins levels of FXR were 203

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significantly decreased in SIRT1 deficient livers and primary hepatocytes. 204

Furthermore, the chromatin associated FXR levels at the FXRE of SHP were 205

significantly decreased in the liver of SIRT1 LKO mice as well as in SIRT1 206

deficient primary hepatocytes (Figure 2C). These observations suggest that 207

SIRT1 may positively regulate the transcription of FXR, and that the reduced 208

FXR signaling in the SIRT1 LKO mice may be in part due to decreased 209

expression of FXR. In support of our hypothesis, overexpression of SIRT1 in the 210

SIRT1 deficient hepatocytes resulted in a dose-dependent increase of FXR 211

mRNA levels, and further stimulated the expression of FXR in the control 212

hepatocytes (Figure 2D). 213

FXR has recently been reported as an acetylated transcription factor (21). 214

It has been shown that acetylation of FXR by the acetyltransferase p300 leads to 215

decreased heterodimerization with its partner, RXRα, resulting in reduced DNA 216

binding and transactivation activity. SIRT1, on the other hand, is able to 217

deacetylate FXR thereby activating its activity (21). To test the importance of the 218

observed transcriptional regulation of FXR by SIRT1 (Figure 2) relative to the 219

previously defined role of SIRT1 in enhancing the transactivation activity of FXR 220

through deacetylation (21), we analyzed the transactivation activity of exogenous 221

murine FXR protein in control and SIRT1 deficient primary hepatocytes. As 222

shown in Figure 3A, lentiviral expression of FXR in the SIRT1 KO hepatocytes 223

completely rescued the deficient expression of SHP, and almost completely 224

recovered the levels of Abcb4, indicating that the transactivation activity of 225

exogenous wild-type (WT) FXR protein is almost normal in the SIRT1 deficient 226

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hepatocytes. To further assess the transactivation activities of acetylated-FXR 227

and deacetylated-FXR proteins in primary hepatocytes, we generated 228

lentiviruses expressing mutant FXR proteins in which the previously identified 229

lysine acetylation sites were mutated either to arginine (K168R/K228R, KR) to 230

mimic the deacetylation protein or to glutamine (K168Q/K228Q, KQ) to mimic the 231

acetylated FXR protein. The transactivation activities of these mutants were then 232

analyzed in control and SIRT1 KO primary hepatocytes. As shown in Figure 3B, 233

the WT and mutant FXR proteins had comparable activities in both control and 234

SIRT1 deficient hepatocytes on two of FXR target genes, SHP and Abcb4. 235

However, the deacetylation mimetic, KR mutant protein, displayed significantly 236

increased activity on Abcb11. These observations suggest that acetylation status 237

of FXR impacts its transcriptional activity only on some target genes. Taken 238

together, our data demonstrate that permanent deletion of SIRT1 in hepatocytes 239

affects the activity of FXR largely through the transcriptional regulation of its 240

expression. 241

242

SIRT1 regulates the expression of FXR through HNF1α 243

As an important bile acid sensor that is critical for lipid and glucose 244

metabolism, the expression of FXR is tightly controlled by an intricate regulatory 245

network at multiple levels in response to various environmental stimuli. For 246

example, the expression of FXR is under control of HNF1α (46), a 247

homeodomain-containing transcription factor that is essential for diverse 248

metabolic processes in the pancreatic islets, liver, intestine and kidney (25, 38, 249

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39). The expression and transactivation activity of FXR is also regulated by an 250

important coactivator for a number of transcription factors, peroxisome 251

proliferator-activated receptor-gamma coactivator 1α (PGC-1α) (61), which is a 252

direct deacetylation target of SIRT1 in the regulation of gluconeogenesis and 253

fatty acid oxidation (41-43). FXR has also been reported to self-regulate its own 254

expression (reviewed by Eloranta & Kullak-Ublick (14)). 255

To dissect the molecular mechanisms by which loss of SIRT1 leads to the 256

reduction of FXR expression, we analyzed the association of SIRT1 on the 257

mouse FXR promoter. As shown in Figure 4A, SIRT1 was relatively concentrated 258

approximately 300 bp upstream of the transcription start site (TSS), where 259

multiple binding sites of HNF1α were identified by the Genomatix MatInspector 260

analyses (data not shown). Consistently, HNF1α was highly enriched near the 261

TSS of mouse FXR promoter (Figure 4A). This observation suggests that SIRT1 262

may regulate the expression of FXR through modulation of HNF1α. 263

HNF1α is an important transcription factor that is involved in the 264

differentiation program in several organs including the liver, kidney, intestine and 265

pancreas. Haploinsufficiency of HNF1α in humans (56) and knockout of HNF1α 266

in mice lead to the development of diabetes, renal Fanconi syndrome, hepatic 267

dysfunction, and hypercholesterolemia (25, 38, 39). Since both mRNA and 268

protein levels of HNF1α were normal in SIRT1 deficient mice and primary 269

hepatocytes (Figure 4B-4D), we speculated that SIRT1 might regulate the 270

activity of this transcription factor at the post-transcriptional level, which then 271

indirectly affects the expression of FXR. Consistent with this possibility, both WT 272

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and catalytic inactive (HY) SIRT1 were co-immunoprecipitated with HA-HNF1α in 273

HEK293T cells (Figure 4E). Moreover, the chromatin-associated HNF1α levels 274

were significantly reduced in the SIRT1 deficient hepatocytes compared to the 275

control hepatocytes in a chromatin-immunoprecipitation assay (Figure 4F), 276

suggesting that deletion of SIRT1 in hepatocytes decreases the DNA binding 277

affinity of HNF1α. To further confirm that SIRT1 regulates the expression of FXR 278

through HNF1α, we electroporated negative control siRNA or siRNA against 279

HNF1α into mouse hepatocyte Hepa1-6 cell line. We then transfected them with 280

vector (V) or constructs expressing WT or HY SIRT1 together with mouse FXR 281

promoter luciferase reporter (Figure 4G). As shown in Figure 4H, in Hepa1-6 282

cells transfected with control siRNA, overexpression of WT SIRT1 but not HY 283

mutant significantly induced luciferase reporter of FXR. However, this induction 284

was decreased in HNF1α RNAi cells, suggesting that SIRT1 induces the 285

expression of FXR in part through HNF1α. 286

In line with the observation that deletion of hepatic SIRT1 leads to reduced 287

expression of HNF1α target gene FXR, SIRT1 deficiency in hepatocytes also 288

reduced the expression of a number of other HNF1α target genes in both liver 289

and primary hepatocytes (Figure 5A and 5B). Furthermore, overexpression of 290

SIRT1 in the KO hepatocytes led to a dose-dependent increase of the mRNA 291

levels of HNF1α target genes and further stimulated the expression of these 292

targets in the control hepatocytes (Figure 5C). Collectively, these findings 293

demonstrate that SIRT1 deficiency in hepatocytes impairs the expression of FXR 294

through modulation of HNF1α transcriptional activity. 295

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296

Hepatic deletion of SIRT1 leads to impaired hepatic lipid metabolism on the 297

lithogenic diet 298

Decreased activity of HNF1α in humans and mice has been associated 299

with development of diabetes, hepatic dysfunction, and hypercholesterolemia 300

(25, 38, 39, 56). Disruption of FXR in mice is also associated with the 301

development of metabolic diseases, including diabetes and hypercholesterolemia 302

(47). The decreased activity of HNF1α and reduced expression of FXR in the 303

liver of the SIRT1 LKO mice suggest that these animals may display impaired 304

metabolic functions mediated by these two factors. 305

To examine the pathophysiological effects of blunted HNF1α and FXR 306

signaling in vivo, we challenged the SIRT1 LKO and their Lox control littermates 307

with a lithogenic diet containing high-fat high-cholesterol plus additional 0.5% 308

cholic acid. We have previously shown that deletion of SIRT1 in the liver results 309

in decreased fatty acid oxidation, leading to a mild body weight gain and the 310

development of hepatic steatosis and inflammation on a western-style high-fat 311

diet (41). When challenged with the lithogenic diet, SIRT1 LKO mice showed no 312

obvious signs of body weight abnormality (data not shown) and had normal 313

levels of serum insulin and leptin (Figure 6A). However, they displayed mild but 314

significant hypercholesterolemia, primarily through an increase of the LDL 315

fraction in serum (Figure 6B). They also displayed modest but significant 316

hepatomegaly and had greater lipid accumulation in the liver (Figure 6C and 6D). 317

In line with these observations, SIRT1 LKO mice showed significantly altered 318

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expression of a couple of genes involved in cholesterol efflux and triglycerides 319

biogenesis (Figure 6E). Additionally, six-week of lithogenic diet feeding also 320

resulted in significantly greater liver damage in the SIRT1 LKO mice, as revealed 321

by elevated serum alanine transaminase (ALT) activities (Figure 6F). 322

323

Hepatic deletion of SIRT1 impairs bile acid metabolism and induces 324

formation of cholesterol gallstones on the lithogenic diet 325

Disruption of FXR in mice has also been associated with the development 326

of cholesterol gallstone disease upon lithogenic diet feeding (31). Strikingly, six-327

week of lithogenic diet feeding induced formation of gallstones, which were 328

visible at the macroscopic level (Figure 7B) in 73% of SIRT1 LKO mice (Figure 329

7A). In contrast, only 27% of the control mice acquired gallstones under the 330

same conditions (Figure 7A). 331

Since the formation of gallstones is primarily determined by the relative 332

biliary concentrations of bile salts/phospholipids and cholesterol (10, 11), which 333

are under control of FXR and LXR respectively (31), we analyzed the biliary lipid 334

profile in control and SIRT1 LKO mice. As shown in Figure 7C, SIRT1 LKO mice 335

showed reduced levels of biliary bile acids and phospholipids compared to 336

control mice, whereas their biliary cholesterol concentrations were normal. As a 337

result, their biliary cholesterol was significantly more saturated (Figure 7D). 338

These data suggest that the SIRT1 LKO mice have deficient FXR signaling under 339

the lithogenic diet. In support of these observations, the expression levels of two 340

FXR targets located at the outer leaflet of the hepatocyte canalicular membrane 341

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that are responsible for the biliary transport of bile salts and phospholipids (31), 342

Abcb11 and Abcb4, were significantly lower in SIRT1 LKO mice compared to 343

control mice (Figure 7E). In contrast, the levels of two cholesterol transporters 344

that are under control of LXR, Abcg5 and Abcg8, were not changed (Figure 7E). 345

Together, these findings confirm that hepatic deletion of SIRT1 leads to defective 346

FXR signaling, resulting in reduced biliary bile salt and phospholipid 347

concentrations and increasing the risk of cholesterol gallstones under the 348

lithogenic diet. 349

In addition to regulating bile salts and phospholipid transport, HNF1α/FXR 350

also plays an important role in the regulation of bile acid synthesis, primarily 351

through SHP, an odd member of nuclear receptor superfamily (16, 20). Upon 352

activation by bile acids, FXR induces the expression of SHP, which in turn binds 353

to LXR, liver receptor homolog 1 (LRH-1) and possibly other nuclear receptors to 354

attenuate further bile acid synthesis (44). Consistently, deletion of FXR in mice 355

decreases the expression of SHP while increasing the mRNA levels of bile acid 356

synthesis genes, such as Cyp7a1, Cyp8b1 and Cyp27a1 (31). Loss of SHP, on 357

the other hand, partially impairs negative feedback regulation of bile acid 358

synthesis (22, 52). The reduced levels of FXR and SHP suggest that SIRT1 LKO 359

mice may also suffer from an abnormally high rate of bile acid synthesis. 360

However, unexpectedly, the expression of a number of bile acid synthesis genes 361

was lower in these mice (Figure 7F). In line with this observation, their bile acid 362

synthesis rates, based on the steady-state fecal bile acid output rate, were 363

decreased by 30% and 43% respectively in both basal and lithogenic conditions 364

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(Figure 7G). However, there were no significant alterations in the total bile acid 365

pool size levels (Figure 7H) and liver bile acid concentrations (Figure 7H). These 366

observations suggest that the FXR-SHP-bile acid synthesis feedback loop was 367

impaired in the SIRT1 LKO mice. 368

369

Discussion 370

As the best-studied member of the sirtuins family of anti-aging proteins, 371

SIRT1 is rising as an important therapeutic target for a number of age-associated 372

diseases. While it has been reported that SIRT1 is a vital regulator in many 373

aspects of hepatic lipid and glucose metabolism in response to different nutrient 374

signals (19, 45), and that SIRT1 regulates the FXR signaling by direct 375

deacetylation of this transcription factor (21), we show in the present study that 376

hepatic SIRT1 modulates the message RNA levels of FXR through HNF1α. As a 377

result, deletion of SIRT1 in the liver results in decreased HNF1α recruitment to 378

the FXR promoter and reduced expression of FXR, leading to decreased 379

transport of biliary bile acids and phospholipids and increased incidence of 380

cholesterol gallstones. These observations uncover a previously unknown link 381

between SIRT1, HNF1α, and transcriptional regulation of FXR expression, 382

suggesting that hepatic SIRT1 may also be an important therapeutic target for 383

cholesterol gallstone disease. 384

Several lines of evidence suggest that hepatic SIRT1 modulates the FXR 385

signaling pathway predominantly at the transcriptional level in the SIRT1 LKO 386

mouse model. For instance, the expression of FXR is decreased not only in the 387

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SIRT1 deficient liver, but also in the SIRT1 deficient hepatocytes (Figure 2A), 388

indicating that SIRT1 directly regulates FXR expression in a cell autonomous 389

fashion. Moreover, overexpression of SIRT1 in primary hepatocytes induces the 390

expression of FXR (Figure 2D). More importantly, it appears that the 391

transactivation activity of exogenous FXR is normal in the SIRT1 deficient 392

hepatocytes, as lentiviral expression of FXR almost completely rescues the 393

defective FXR activity in these cells (Figure 3A). On the other hand, our results 394

also point toward the involvement of additional mechanisms. As shown in Figure 395

3A, putting back FXR did not completely restore the expression of all tested FXR 396

downstream targets in the SIRT1 deficient hepatocytes. It appears that the 397

acetylation status of the FXR proteins indeed affects their transactivation 398

activities on some of FXR targets, particularly in SIRT1 deficient hepatocytes 399

(Figure 3B). Furthermore, the FXR proteins are markedly hyperacetylated in the 400

SIRT1 LKO mice (Figure 8A). These observations indicate that SIRT1 also plays 401

a role in the post-translational activation of FXR through direct deacetylation of 402

the receptor, thereby improving its DNA binding ability (21). In addition to HNF1α, 403

the expression and transactivation activity of FXR is also regulated by a PGC-1α 404

(61). We have previously shown that hepatocyte-specific deletion of SIRT1 leads 405

to decreased co-activation activity of PGC-1α (41). Although our preliminary data 406

indicate that PGC-1α is still capable of promoting the expression of FXR in the 407

SIRT1 deficient hepatocytes (data not shown), the decreased activity of PGC-1α 408

may partially contribute to the reduction of the FXR signaling in these cells. 409

Interestingly, recent studies have demonstrated that the FXR-SHP pathway also 410

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positively regulates the translation of SIRT1 protein via the p53/miR-34a pathway 411

(24, 57). It has been shown that SHP, one of the direct FXR targets, inhibits 412

transactivation of transcription factor p53 and the expression of its downstream 413

target miR-34a, which in turn binds to the 3’ UTR of SIRT1 mRNA, inhibiting of 414

the translation of SIRT1 protein (24, 57). Therefore, SIRT1 and the FXR signaling 415

pathway mutually interact at multiple levels, coordinately regulating hepatic bile 416

acid and cholesterol homeostasis (Figure 8B). 417

Given the facts that HNF1α is paramount to normal functions of liver and 418

pancreas and that human HNF1α is commonly mutated in patients with maturity 419

onset diabetes of the young (MODY) which is characterized by severe insulin 420

secretory defects (1, 56), the positive forward link between SIRT1, HNF1α, and 421

FXR revealed in the present study is not altogether surprising. For example, it 422

has been shown that increased dosage of SIRT1 in pancreatic β cells improves 423

glucose tolerance and enhances insulin secretion in response to glucose (33), 424

whereas deletion of SIRT1 impairs glucose-stimulated insulin secretion (8). 425

Resveratrol, a polyphenol activator of SIRT1, potentiates glucose-stimulated 426

insulin secretion in β cells (50). In addition, activation of SIRT1 by its activators in 427

animals protects against high-fat induced obesity and insulin resistance (4, 23, 428

30), and modest overexpression of SIRT1 resulted in a protective effect against 429

high-fat induced hepatic steatosis and glucose intolerance (3, 37). Our data 430

suggest that activation of the HNF1α signaling pathway may partially underlie the 431

anti-diabetes action of SIRT1. Further exploring this possibility may provide novel 432

insights into SIRT1’s function in whole-body glucose homeostasis. Our data, 433

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however, are in contrast to those reported in a recent study (17). Grimm et al. 434

has shown that SIRT1 and HNF1α form a nutrient-sensitive complex, and this 435

interaction suppresses the transcriptional activity of HNF1α on its target genes, 436

particularly C-reactive protein (CRP), in hepatocytes. In addition, their data show 437

that SIRT1 inhibits HNF1α activity on the CRP promoter through deacetylation of 438

H4K16 instead of HNF1α itself (17). One possible factor contributing to the 439

discrepancy between our observations and those of Grimm et al. may be the 440

difference of environmental challenges in two studies. SIRT1 appears to 441

suppress HNF1α and the production of CRP only under conditions of nutrient 442

restriction (17), whereas SIRT1 activates the HNF1α/FXR pathway in 443

hepatocytes regardless of nutrient status. In addition, the HNF1α-mediated gene 444

expression involves formation of multi-unit transcriptional complexes with other 445

transcription factors. For instance, cytokine-driven expression of CRP gene 446

requires formation of c-Fos, STAT3, and HNF1α transcriptional complex (36). 447

Among which c-Fos can be deacetylated and inhibited by SIRT1 (60). Therefore, 448

it is possible that the net effect of SIRT1 on the expression of different HNF1α 449

target genes is determined by the combination of different transcriptional 450

partners. 451

How SIRT1 regulates the activity of HNF1α is still an on-going study. 452

Since the chromatin-associated HNF1α levels were significantly reduced in the 453

liver of SIRT1 LKO mice (Figure 4F), loss of SIRT1 may directly or indirectly 454

decrease the DNA binding affinity of HNF1α. Additional experiments are needed 455

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to dissect the molecular mechanisms underlying this important modulation in vitro 456

and in vivo. 457

The impaired FXR-SHP-bile acid synthesis feedback loop in the SIRT1 458

LKO mice suggests that hepatic deletion of SIRT1 may disrupt bile acid synthesis 459

through FXR-independent mechanisms. For instance, increased liver damage in 460

the SIRT1 LKO mice under the lithogenic diet may activate c-Jun N-terminal 461

kinase (JNK), thereby inhibiting bile acid synthesis (22, 44, 52). Deletion of 462

SIRT1 in the hepatocytes may also result in hepatic insulin resistance, a 463

condition known to decrease the expression of the first committed enzyme in the 464

acidic pathway of bile acid synthesis, Cyp7b1 (5). However, comparison of levels 465

of phospho-JNK, a marker of the activated JNK pathway, in the liver extracts of 466

control and SIRT1 LKO mice failed to reveal significant alterations of this 467

signaling pathway (data not shown). Ser473 phosphorylation of Akt, a key 468

molecule in the insulin signaling pathway, was also normal in the livers of SIRT1 469

LKO mice (data not shown). Therefore, additional studies are required to dissect 470

the mechanism underlying this intriguing phenotype. 471

In summary, we have shown that hepatic SIRT1 plays an important role in 472

the regulation of hepatic bile acid metabolism. Hepatic deletion of SIRT1 leads to 473

an increased susceptibility to cholesterol gallstone disease through the 474

decreased HNF1α/FXR signaling. Our findings provide a direct link between 475

SIRT1 and cholesterol gallstone disease, and suggest that new therapeutic 476

strategies designed to modulate SIRT1 activity may be beneficial for preventing 477

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formation of cholesterol gallstones as well as for other metabolic diseases 478

associated with type-2 diabetes. 479

480

481

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692

Acknowledgements 693

We thank Drs. Anton Jetten, Stavros Garantziotis, Darryl Zeldin, and members of 694

Li laboratory for critical reading of the manuscript; Dr. Frederic Alt at Harvard 695

Medical School for providing SIRT1 exon 4 floxed allele. We also thank NIEHS 696

Laboratory of Experimental Pathology for histological staining and serum 697

hormone ELISA and NIEHS viral core facility for lentiviruses. This research was 698

supported by the Intramural Research Program of the NIH, National Institute of 699

Environmental Health Sciences to X.L. (Z01 ES102205). 700

701

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702

Figure legends 703

704

Figure 1. Loss of SIRT1 reduces the activity of FXR signaling pathway in primary 705

hepatocytes. (A) SIRT1 deficiency in primary hepatocytes reduces the induction 706

of FXR target genes by GW4064 (*p<0.05). (B) Overexpression of SIRT1 in 707

primary hepatocytes restores the expression of FXR targets. Primary 708

hepatocytes from control and SIRT1 LKO mice were infected with lentiviruses 709

expressing GFP or SIRT1. The mRNA levels of FXR target genes were analyzed 710

by qPCR (*p<0.05). 711

712

Figure 2. Loss of hepatic SIRT1 decreases the expression of FXR. (A) SIRT1 713

deficiency leads to reduced mRNA levels of FXR in the liver (n=11) and primary 714

hepatocytes (n=3), *p<0.05. (B) SIRT1 deficiency results in reduced FXR protein 715

levels in the liver (n=4, *p<0.05). (C) Reduced recruitment of FXR to the FXRE 716

on the promoter of SHP gene in the SIRT1 deficient livers and primary 717

hepatocytes (n=3, *p<0.05). (D) Overexpression of SIRT1 in SIRT1 deficient 718

primary hepatocytes restores the expression of FXR. Primary hepatocytes from 719

control and SIRT1 LKO mice were infected with lentiviruses expressing GFP or 720

increased doses of SIRT1 (MOI=2.5 and 5, respectively). The expression levels 721

of SIRT1 and FXR were determined by qPCR (*p<0.05). 722

723

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Figure 3. Loss of hepatic SIRT1 reduces the FXR signaling pathway primarily 724

through transcriptional regulation. (A) Lentivirus-mediated expression of FXR in 725

the SIRT1 KO hepatocytes rescues the deficient expression of FXR targets. 726

Primary hepatocytes from control and SIRT1 LKO mice were infected with 727

lentiviruses expressing GFP or FXR. The expression levels of FXR and FXR 728

target genes were determined by qPCR (*p<0.05). (B) Transactivation activities 729

of WT, KR, and KQ FXR proteins in control and SIRT1 LKO hepatocytes 730

(*p<0.05). Primary hepatocytes from control and SIRT1 LKO mice were infected 731

with lentiviruses expressing WT FXR, FXR KR or FXR KQ mutant proteins. The 732

expression levels of FXR and FXR target genes were determined by qPCR 733

(*p<0.05). 734

735

Figure 4. SIRT1 regulates the expression of FXR through HNF1α. (A) SIRT1 is 736

enriched on the HNF1α binding sites on the mouse FXR promoter. Primary 737

hepatocytes from control and SIRT1 LKO mice were ChIPed with SIRT1 or 738

HNF1α antibodies. DNA fragments were then subjected to qPCR using primers 739

flanking indicated regions on the FXR promoter. (B-D) SIRT1 deficiency does not 740

cause defective expression of HNF1α in the liver (B and C) and primary 741

hepatocytes (D). (E) SIRT1 interacts with HNF1α in HEK293T cells. Cell lysates 742

from HEK293T cells expressing HA-HNF1α with wild type (WT) or catalytically 743

inactive (HY) SIRT1 were immunoprecipitated (IP) with anti-HA antibodies. (F) 744

SIRT1 deficiency leads to decreased association of HNF1α with the HNF1α 745

binding sites on the FXR promoter. Primary hepatocytes from control and SIRT1 746

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LKO mice were ChIPed with IgG or HNF1α antibodies. (G and H) SIRT1 induces 747

the expression of FXR through HNF1α in Hepa1-6 cells. Mouse hepatocyte 748

Hepa1-6 cell line were electroporated with negative control siRNA (control 749

siRNA) or siRNA against HNF1α (HNF1α siRNA), were then transfected with 750

vector (V) or constructs expressing WT or HY SIRT1 together with mouse FXR 751

promoter luciferase reporter. The expression levels of HNF1α and SIRT1 (G) 752

were determined by qPCR, and the luciferase activity of FXR reporter was 753

determined as described in Materials and Methods. 754

755

Figure 5. Loss of SIRT1 in hepatocytes results in decreased expression of other 756

HNF1α target genes. (A) SIRT1 deficiency in the liver leads to decreased 757

expression of other HNF1α target genes (n=11, *p<0.05). (B) SIRT1 deficiency 758

leads to decreased expression of other HNF1α target genes in primary 759

hepatocytes (n=3, *p<0.05). (C) Overexpression of SIRT1 in SIRT1 deficient 760

primary hepatocytes restores the expression of other HNF1α target genes in 761

primary hepatocytes (*p<0.05). MOI=2.5 and 5, respectively. 762

763

Figure 6. Hepatic loss of SIRT1 function impairs lipid homeostasis upon 764

lithogenic diet feeding. After six weeks of lithogenic diet feeding, SIRT1LKO 765

mice displayed normal serum hormonal levels (A) and a mild increase of serum 766

total cholesterol and LDL levels (B) (n=11, *p<0.05). (C and D) SIRT1 LKO mice 767

display modest hepatomegaly (C) and hepatic steatosis (D). (n=11, *p<0.05). 768

Bar, 50 μm. (E) SIRT1 LKO mice display decreased expression of Abca1 and 769

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32

increased levels of SCD1 (n=11, *p<0.05). (F) SIRT1 deficiency in the liver 770

increases liver damage (n=11, *p<0.05). 771

772

Figure 7. Hepatic deletion of SIRT1 induces formation of cholesterol gallstones 773

and impaired bile acid metabolism in mice upon a lithogenic diet feeding. (A) 774

SIRT1 LKO mice display increased incidence of cholesterol gallstones (n=11). 775

The 9-10-month old control and SIRT1 LKO mice were fed a lithogenic diet for 6 776

weeks. (B) Macroscopic appearance of gallbladders from control and SIRT1 LKO 777

mice fed with the lithogenic diet for 6 weeks. Bar, 1 mm. (C) SIRT1 LKO mice 778

display decreased biliary concentrations of bile acids and phospholipids (n=8, 779

*p<0.05, # p=0.068). (D) SIRT1 LKO mice have increased Cholesterol saturation 780

indices (CSI) in gallbladder bile (n=8, *p<0.05). (E) SIRT1 deficiency reduces 781

expression of bile acid and phospholipid transporters at the hepatocyte 782

canalicular membrane (n=11, *p<0.05). (F) Decreased expression of bile acid 783

synthesis genes in the SIRT1 LKO mice (n=11, *p<0.05). (G-I) SIRT1 LKO mice 784

show decreased fecal bile acid output (G) but normal total bile acid pool size (H) 785

and hepatic bile acids (I) (n=5-6, *p<0.05). 786

787

Figure 8. SIRT1 regulates the FXR signaling at multiple levels. (A) Acetyl-FXR 788

levels are significantly elevated in SIRT1 LKO mice. Total liver extracts from 789

control and SIRT1 LKO mice were immunoprecipitated with goat anti-FXR 790

antibodies, then immunoblotted with rabbit anti-acetyl-K antibodies or mouse 791

anti-FXR antibodies (n=3, *p<0.05). (B) The interaction network between SIRT1 792

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and FXR signaling pathway. SIRT1 modulates the activity of FXR signaling at 793

multiple levels (red lines). First, SIRT1 regulates the expression of FXR through 794

interaction with HNF1α, which may also involve co-activator PGC-1α. Second, 795

SIRT1 deacetylates FXR, increasing its DNA binding affinity. Third, SIRT1 796

deacetylates PGC-1α, thereby activating the transactivation activity of FXR. 797

Finally, SIRT1 stimulates the biosynthesis of bile acids, which in turn bind to FXR 798

and induce its transcriptional activity. FXR, on the other hand, increases the 799

translation of SIRT1 protein via SHP-mediated inhibition of p53 transactivation 800

thereby miR34a expression (blue lines). This positive feedback network plays an 801

essential role in the regulation of bile acid metabolism. Alterations of these loops 802

in the SIRT1 LKO mice lead to impairments in bile acid synthesis and transport, 803

resulting in development of cholesterol gallstones. 804

805

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mR

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Primary hepatocytes

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HNF1α

SIRT1PGC-1αTranscription

A B

Control SIRT1 LKO

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FXR SIRT1SIRT1

DNA binding

Ac-FXR

FXR

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