As the best-studied member of the sirtuin family of antiaging proteins, SIRT1 is rising as an important therapeutic target for a number 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
) and that SIRT1 regulates the FXR signaling by direct deacetylation of this transcription factor (21
), we show in the present study that hepatic SIRT1 modulates the message RNA levels of FXR through HNF1α. As a result, deletion of SIRT1 in the liver results in decreased HNF1α recruitment to the FXR promoter and reduced expression of FXR, leading to decreased transport of biliary bile acids and phospholipids and increased incidence of cholesterol gallstones. These observations uncover a previously unknown link between SIRT1, HNF1α, and transcriptional regulation of FXR expression, suggesting that hepatic SIRT1 may also be an important therapeutic target for cholesterol gallstone disease.
Several lines of evidence suggest that hepatic SIRT1 modulates the FXR signaling pathway predominantly at the transcriptional level in the SIRT1 LKO mouse model. For instance, the expression of FXR is decreased not only in the SIRT1-deficient liver, but also in the SIRT1-deficient hepatocytes (A), indicating that SIRT1 directly regulates FXR expression in a cell autonomous fashion. Moreover, overexpression of SIRT1 in primary hepatocytes induces the expression of FXR (D). More importantly, it appears that the transactivation activity of exogenous FXR is normal in the SIRT1-deficient hepatocytes, as lentiviral expression of FXR almost completely rescues the defective FXR activity in these cells (A). On the other hand, our results also point toward the involvement of additional mechanisms. As shown in A, putting back FXR did not completely restore the expression of all tested FXR downstream targets in the SIRT1-deficient hepatocytes. It appears that the acetylation status of the FXR proteins indeed affects their transactivation activities on some of the FXR targets, particularly in SIRT1-deficient hepatocytes (B). Furthermore, the FXR proteins are markedly hyperacetylated in the SIRT1 LKO mice (A). 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 HNF1α, the expression and transactivation activity of FXR are also regulated by a PGC-1α (61
). We have previously shown that hepatocyte-specific deletion of SIRT1 leads to decreased coactivation activity of PGC-1α (41
). Although our preliminary data indicate that PGC-1α is still capable of promoting the expression of FXR in the SIRT1-deficient hepatocytes (data not shown), the decreased activity of PGC-1α may partially contribute to the reduction of the FXR signaling in these cells. Interestingly, recent studies have demonstrated that the FXR-SHP pathway also positively regulates the translation of SIRT1 protein via the p53/miR-34a pathway (24
). It has been shown that SHP, one of the direct FXR targets, inhibits transactivation of transcription factor p53 and the expression of its downstream target, miR-34a, which in turn binds to the 3′ untranscribed region (3′ UTR) of SIRT1 mRNA, inhibiting the translation of SIRT1 protein (24
). Therefore, SIRT1 and the FXR signaling pathway mutually interact at multiple levels, coordinately regulating hepatic bile acid and cholesterol homeostasis (B).
Fig 8 SIRT1 regulates FXR signaling at multiple levels. (A) Acetyl-FXR levels are significantly elevated in SIRT1 LKO mice. Total liver extracts from control and SIRT1 LKO mice were immunoprecipitated with goat anti-FXR antibodies and then immunoblotted with (more ...)
Given the facts that HNF1α is paramount to normal functions of liver and pancreas and that human HNF1α is commonly mutated in patients with maturity onset diabetes of the young (MODY), which is characterized by severe insulin secretory defects (1
), the positive forward link between SIRT1, HNF1α, and FXR revealed in the present study is not altogether surprising. For example, it has been shown that increased dosage of SIRT1 in pancreatic β cells improves glucose tolerance and enhances insulin secretion in response to glucose (33
), whereas deletion of SIRT1 impairs glucose-stimulated insulin secretion (8
). Resveratrol, a polyphenol activator of SIRT1, potentiates glucose-stimulated insulin secretion in β cells (50
). In addition, activation of SIRT1 by its activators in animals protects against high-fat-induced obesity and insulin resistance (4
), and modest overexpression of SIRT1 resulted in a protective effect against high-fat-induced hepatic steatosis and glucose intolerance (3
). Our data suggest that activation of the HNF1α signaling pathway may partially underlie the antidiabetes action of SIRT1. Further exploration 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 HNF1α form a nutrient-sensitive complex, and this interaction suppresses the transcriptional activity of HNF1α on its target genes, particularly C-reactive protein (CRP), in hepatocytes. In addition, their data show that SIRT1 inhibits HNF1α activity on the CRP promoter through deacetylation of H4K16 instead of HNF1α 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 HNF1α and the production of CRP only under conditions of nutrient restriction (17
), whereas SIRT1 activates the HNF1α/FXR pathway in hepatocytes regardless of nutrient status. In addition, the HNF1α-mediated gene expression involves formation of multiunit transcriptional complexes with other transcription factors. For instance, cytokine-driven expression of the CRP gene requires formation of c-Fos, STAT3, and the HNF1α transcriptional complex (36
), among which c-Fos can be deacetylated and inhibited by SIRT1 (60
). Therefore, it is possible that the net effect of SIRT1 on the expression of different HNF1α target genes is determined by the combination of different transcriptional partners.
How SIRT1 regulates the activity of HNF1α is still an ongoing study. Since the chromatin-associated HNF1α levels were significantly reduced in the livers of SIRT1 LKO mice (F), loss of SIRT1 may directly or indirectly decrease the DNA binding affinity of HNF1α. 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 mechanisms. For instance, increased liver damage in the SIRT1 LKO mice under the lithogenic diet may activate c-Jun N-terminal kinase (JNK), thereby inhibiting bile acid synthesis (22
). Deletion of SIRT1 in the hepatocytes may also result in hepatic insulin resistance, a condition known to decrease the expression of the first committed enzyme in the acidic pathway of bile acid synthesis, Cyp7b1 (5
). However, comparison of levels of phospho-JNK, a marker of the activated JNK pathway, in the liver extracts of control and SIRT1 LKO mice failed to reveal significant 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 important role in the regulation of hepatic bile acid metabolism. Hepatic deletion of SIRT1 leads to an increased susceptibility to cholesterol gallstone disease through decreased HNF1α/FXR signaling. Our findings provide a direct link between SIRT1 and cholesterol gallstone disease and suggest that new therapeutic strategies 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.