Fine-tuning of SIRT 1 expression is essential to protect the liver from cholestatic liver disease . Short title : SIRT 1 contributes to cholestasis

Cholestasis comprises aetiologically heterogeneous conditions characterized by accumulation of bile acids in the liver that actively contribute to liver damage. Sirtuin 1 (SIRT1) regulates liver regeneration and bile acid metabolism via modulating the farnesoid X receptor (FXR); we here investigate its role in cholestatic liver disease. We determined SIRT1 expression in livers from patients with cholestatic disease, in two experimental models of cholestasis, as well as in human and murine liver cells in response to bile acid loading. SIRT1 overexpressing (SIRT) and hepatocyte-specific SIRT1-KO mice (SIRT) were subjected to BDL and were fed with 0.1%DDC diet to determine the biological relevance of SIRT1 during cholestasis. The effect of NorUDCA was tested in BDL/SIRT mice. A cc ep te d A rt ic le This article is protected by copyright. All rights reserved. We found that SIRT1 was highly expressed in livers from cholestatic patients, mice after BDL and Mdr2 animals. The detrimental effects of SIRT1 during cholestasis were validated in vivo and in vitro. SIRT mice showed exacerbated parenchymal injury whereas SIRT mice evidenced a moderate improvement after BDL and 0.1%DDC feeding. Likewise, hepatocytes isolated from SIRT mice showed increased apoptosis in response to bile acids, while a significant reduction was observed in SIRT hepatocytes. Importantly, the decrease, but not complete inhibition of SIRT1 exerted by NorUDCA treatment correlated with pronounced improvement in liver parenchyma in BDL/SIRT mice. Interestingly, both SIRT1 overexpression and hepatocyte-specific SIRT1 depletion correlated with inhibition of the farnesoid X receptor (FXR), whereas modulation of SIRT1 by NorUDCA associated with restored FXR-signalling. Conclusion: SIRT1 expression is increased during human and murine cholestasis. Finetuning expression of SIRT1 is essential to protect the liver from cholestatic-liver damage. The term ‘cholestatic liver disease’ includes a broad spectrum of aetiologically heterogeneous hepatobiliary disorders, mainly comprising primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) in adults. These conditions are characterised by accumulation of bile acids in the liver, leading to hepatocellular necrosis and apoptosis, progressive fibrosis and end-stage liver disease(1-3). Current therapeutic approaches for treating cholestasis mainly rely on the use of Ursodeoxycholic acid (UDCA); however, this treatment has no proven efficacy for PSC and a proportion of patients with PBC(2, 3). The therapeutic options for such unresponsive patients are currently limited though there have been recent promising advances including the use of 24-norursodeoxycholic acid (NorUDCA)(4), which has shown to improve liver function in PSC patients in a recent clinical trial(5). Also, novel treatments using fibrates(6) and FXR agonists, such as obeticholic acid(7-9), have shown efficacy for PBC patients unresponsive to UDCA. Still, a better A cc ep te d A rt ic le This article is protected by copyright. All rights reserved. understanding of the molecular mechanism underpinning the pathogenesis of cholestasis will enable the development of efficient therapies for cholestatic patients. FXR is an orphan nuclear receptor that plays a key role in the regulation of bile acid metabolism, and in the pathogenesis of cholestasis (6, 10-13). The regulation of FXR involves a dynamic acetylation/deacetylation process mediated by p300 and Sirtuin 1 (SIRT1) respectively(14). SIRT1 deacetylates FXR, increasing its DNA binding and dependent gene transcription. Interestingly, SIRT1/FXR interaction must be finely tuned, as prolonged SIRT1-mediated FXR deacetylation leads to ubiquitination and proteasome degradation(14). SIRT1 is an evolutionarily conserved NAD-dependent histone III deacetylase that is activated in response to energy deprivation, controlling key metabolic functions including bile acid metabolism(15, 16). Initial work delineating the implication of SIRT1 in prolonging the lifespan in lower organisms(17) and in promoting healthy ageing in mammals(18) led to SIRT1 being hyped as a ‘magic bullet’ to preserve lifelong health. Nevertheless, the role of SIRT1 has revealed to be highly complex in a wide range of biological functions including tumorigenesis. We and others have described SIRT1 as being highly expressed in human liver tumours(16, 19, 20), pointing to the potential contribution of SIRT1 to liver disease. Supporting this, we demonstrated that SIRT1 overexpression leads to impaired liver regeneration after partial hepatectomy, which associated with disturbances in bile acid homeostasis, including reduced FXR signalling, increased synthesis and accumulation of toxic bile acids in the liver(16). Overall, these results led us to hypothesise that SIRT1 may play a role during cholestatic liver disease. In accordance, in this study, we provide evidence that SIRT1 is up-regulated in the liver during human cholestasis in PSC and PBC patients and in two murine models of cholestasis; after bile duct ligation (BDL) and in Mdr2 mice. We further demonstrate that SIRT1 contributes to liver parenchymal damage in the context of obstructive cholestasis, as A cc ep te d A rt ic le This article is protected by copyright. All rights reserved. the overexpression of SIRT1 aggravates liver injury, whereas hepatocyte-specific SIRT1 depletion exerts a moderate cell protection after BDL and feeding with a diet containing 0.1% of DDC. Importantly, the improvement in liver function observed in hepatocyte-specific SIRT1 KO mice is only transient, likely involving mechanisms including the attenuation of FXR signalling. Ultimately, we describe that the beneficial effect of NorUDCA treatment in reducing liver injury in cholestatic SIRT1 overexpressing mice, associates with the modulation, whilst not complete depletion, of SIRT1 expression. Overall, our results support the importance of maintaining SIRT1 fine-tuned expression to preserve liver function in the context of cholestatic disease. MATERIALS AND METHODS Human PBC and PSC samples SIRT1 gene expression was determined by qPCR analysis in mRNA isolated from cirrhotic livers of patients with PBC (n=10) and PSC (n=10) who underwent liver transplantation. Control liver tissues (n=5) were acquired from large margin liver resections from patients undergoing of colorectal metastases with no microscopic changes of liver disease identified by a pathologist, all collected in the Department of General, Transplant and Liver Surgery, Medical University of Warsaw, Poland as described elsewhere(21). Supplemental Table 1 includes detailed clinical and biochemical data of these patients. Protein expression of SIRT1 was assessed by immunohistochemistry (IHC) in paraffin embedded sections from livers obtained by percutaneous biopsy from n=9 PBC patients, n=5 PSC patients and in liver samples obtained from n=4 healthy individuals at the Norwich Norfolk University Hospital, UK. The diagnosis was established by pathological analysis of liver biopsies together with presence of anti-mitochondrial antibodies in the case of PBC. Clinical and biochemical data of these patients is included in Supplemental Table 2. The use A cc ep te d A rt ic le This article is protected by copyright. All rights reserved. of human tissue samples was approved by the Faculty of Medicine and Health Sciences Research Ethics committee (University of East Anglia, UK). Collection and handling of human samples used in this study conformed to the Declaration of Helsinki and the Human Tissue Act (UK) and Good Clinical Practice Guidelines (UK). More information in Supplemental Material and Methods.