Sirtuin1 (SIRT1) regulates central metabolic functions such as lipogenesis, protein synthesis, gluconeogenesis, and bile acid (BA) homeostasis through deacetylation. Here we describe that SIRT1 tightly controls the regenerative response of the liver. We performed partial hepatectomy (PH) to transgenic mice that overexpress SIRT1 (SIRT). SIRT mice showed increased mortality, impaired hepatocyte proliferation, BA accumulation, and profuse liver injury after surgery. The damaging phenotype in SIRT mice correlated with impaired farnesoid X receptor (FXR) activity due to persistent deacetylation and lower protein expression that led to decreased FXR-target gene expression; small heterodimer partner (SHP), bile salt export pump (BSEP), and increased Cyp7A1. Next, we show that 24-norUrsodeoxycholic acid (NorUDCA) attenuates SIRT protein expression, increases the acetylation of FXR and neighboring histones, restores trimethylation of H3K4 and H3K9, and increases miR34a expression, thus reestablishing BA homeostasis. Consequently, NorUDCA restored liver regeneration in SIRT mice, which showed increased survival and hepatocyte proliferation. Furthermore, a leucine-enriched diet restored mammalian target of rapamycin (mTOR) activation, acetylation of FXR and histones, leading to an overall lower BA production through SHP-inhibition of Cyp7A1 and higher transport (BSEP) and detoxification (Sult2a1) leading to an improved liver regeneration. Finally, we found that human hepatocellular carcinoma (HCC) samples have increased presence of SIRT1, which correlated with the absence of FXR, suggesting its oncogenic potential. Conclusion: We define SIRT1 as a key regulator of the regenerative response in the liver through posttranscriptional modifications that regulate the activity of FXR, histones, and mTOR. Moreover, our data suggest that SIRT1 contributes to liver tumorigenesis through dysregulation of BA homeostasis by persistent FXR deacetylation. (Hepatology 2014;59:1972–1983)
Sirtuin 1 (SIRT1) is a class III NAD+-dependent histone deacetylase that tightly regulates lipid, glucose, and bile acid (BA) metabolism.[1, 2] SIRT1 is activated in situations of low energy availability and links nutritional status with metabolic homeostasis. SIRT1 regulates adenosine monophosphate activated protein kinase (AMPK) by deacetylation of LKB1, which are involved in tissue repair processes. Contrary to SIRT1, mammalian target of rapamycin (mTOR) is activated in high-energy conditions and controls cell growth and proliferation. mTORC1 promotes protein synthesis by activating the S6 ribosomal protein and this axis is essential to regulate the cell cycle during liver regeneration after partial hepatectomy (PH). BA is also essential for the regeneration of the liver after PH, although, when present in excess, BA can be toxic and promote hepatocyte death. Therefore, a fine regulation of BA metabolism is essential to preserve liver homeostasis and a proper response to injury. The orphan nuclear receptor (NR) farnesoid X receptor (FXR; NR1H4) is the master regulator of BA, lipid, and glucose metabolism. SIRT1 directly modulates FXR activity by deacetylation of this NR and neighboring histones that strictly control target gene transcription.[9, 10] In the present work we describe that modulation of SIRT1 is essential for the regenerative response in the liver through mechanisms involving the regulation of (1) FXR and (2) mTOR signaling pathways by dynamic acetylation/deacetylation, which overall maintain BA homeostasis.
BA-mediated toxicity is also involved in the pathogenesis of cirrhosis from diverse etiologies.[11, 12] The role of SIRT during tumorigenesis remains controversial, as both pro- and antioncogenic properties have been described.[13, 14] Here we show that increased SIRT1 expression correlated with a low presence of FXR in human HCC samples, supporting the significance of SIRT1 deacetylase activity in regulating BA homeostasis and the response to liver injury.
Overall, our data defines SIRT1 as a key regulator of the regenerative response of the liver, controlling BA homeostasis, protein synthesis, and cell proliferation through deacetylation of FXR and histones and regulation of mTOR. Importantly, our results underscore the implication of mTOR in regulating BA metabolism as a negative regulator of SIRT1.
SIRT1 is essential for cell metabolism control as, in situations of low energy availability (low adenosine triphosphate [ATP]), it promotes catalytic processes to restore cell-energy homeostasis.[1, 2] Here we describe that SIRT1 overexpression has a detrimental impact on liver regeneration, as poor survival and impaired proliferation were found after PH. In apparent contradiction, recent work showed that reduced SIRT1 expression in older mice correlated with impaired liver regeneration, which was restored after normalization of SIRT levels in these aged mice. Interestingly, both Jin et al. and our work emphasize the critical relevance of the fine-tuned regulation of SIRT deacetylase activity for the regeneration of the liver, as either deficiency or excess have a detrimental impact on this process. Here we show that SIRT-overexpressing mice have persistent deacetylase activity that profoundly alters key metabolic responses such as (1) BA metabolism and (2) protein synthesis, highlighting the role of SIRT1 in controlling the regenerative response of the liver.
FXR is the master regulator of BA metabolism. FXR activates SHP that represses the expression of Cyp7A1, the BA synthesis rate-limiting enzyme. Additionally, essential BA transporters like BSEP are positively regulated by FXR. Little is known regarding the mechanisms by which FXR regulates gene expression, although posttranscriptional modifications (PTM) seem to play a key role. PTM occur at two levels: modifying the NR and its cofactors, and/or modifying histones at the promoters of the NR-target genes (reviewed). FXR is tightly regulated by p300- and SIRT1-mediated acetylation/deacetylation. Whereas acetylation is generally related to gene activation, Kemper et al. showed that acetylation of FXR by p300 leads to lower transactivation potential. Accordingly, deacetylation of FXR by SIRT1 allows interaction with RXR, leading to DNA binding or to its degradation by ubiquitination. Interestingly, the FXR-target gene SHP is differently regulated, as histone deacetylation, by SIRT1, of its promoter contributes to silence transcription. This underlines that the complex mechanism by which SIRT1 controls BA metabolism through deacetylation must be a dynamic process. In addition to the ostensible deacetylation of FXR and low transcriptional capacity, we found aberrant methylation of histones in SIRT mice, as both H3K4 and H3K9 were tri-methylated. The histone methyltransferase Set9 competes with histone deacetylases to methylate H3 on the lysine 4. H3K4me3 inhibits the methyltransferase Suv39H1, which (tri-)methylates H3K9. This regulatory mechanism avoids simultaneous methylation of H3K4me3 and H3K9me3, as they have generally opposing activities: activation and repression of gene transcription, respectively. Sir2, the SIRT1 analog in yeast, promotes trimethylation of H3K4 and H3K9.[26, 27] H3K4m3 relates with FXR activation of target gene expression such as BSEP, whereas acetylation of H3K9 by p300 is essential for SHP-mediated activation of its transcriptional activity.
Overall, we propose that SIRT1 modulates BA metabolism through two mechanisms: (1) PTM of NR (FXR) and its cofactors (SHP) that repress target gene transcription (CYp7a1, BSEP), and (2) PTM of histones permitting methylation through deacetylation. Both of these mechanisms are altered when SIRT1 is overexpressed, leading to dysregulated FXR activity and aberrant methylation of H3K4 and H3K9.
SIRT mice show no signs of liver injury at basal conditions and have comparable levels of liver BA than WT. However, a closer analysis shows that misregulation of FXR-related BA metabolism found basally correlates with a shift towards the enhanced presence of secondary BA in SIRT livers. These data suggest that the role of the intestine in BA homeostasis might be affected in SIRT mice. Although it is out of the scope of this work, the unbalance towards secondary BA in SIRT mice, despite having a similar BA pool size to WT animals, may indicate enhanced intestine elimination rate due to reduced reuptake by the ileum. Also, consistent with enhanced intestinal elimination, our data points to a role of intestinal bacteria in changing the BA pool composition in SIRT mice.
Previous work showed that BA homeostasis is essential for liver regeneration, as FXR−/− mice show impaired response after PH. Accordingly, our data suggest that persistent deacetylation of FXR by SIRT1 contributes to defective liver regeneration through misregulation of BA synthesis, transport, and detoxification. Consequently, accumulation of BA in regenerating livers of SIRT mice may cause severe necrosis and tissue damage, supporting the toxicity of BA when present in excess. We show that BA-mediated liver injury was attenuated and liver regeneration was fully restored in NorUDCA/SIRT animals. Our data not only confirms what was previously described regarding the therapeutic role of NorUDCA during liver damage,[15, 16] but also provides new insights in the mechanisms by which NorUDCA exerts its beneficial effects. Our results suggest that NorUDCA controls the acetylation status of the cell by regulating the expression of SIRT1. Thus, in agreement with the known regulation of SIRT1 by miR34a, we found that NorUDCA recovered the expression of this microRNA attenuating SIRT1. Overall, our data suggest that regulation of FXR by NorUDCA undergoes through transcriptional and translational mechanisms, involving restoration of mRNA and protein expression, and by regulating the acetylation status in the cell through SIRT1.
Furthermore, we found that persistent deacetylation by SIRT1 promotes a “fasting-like” status in the body characterized by increased BA synthesis through Cyp7a1. During fasting, the interaction of SIRT1 and FXR increases, whereas it is reduced upon refeeding and in the presence of BA, the natural FXR ligands. During fasting, SIRT1 regulates FXR through various mechanisms: repressing gene transcription by deacetylating histones or direct deacetylation of FXR and further ubiquitination and proteosomal degradation, which may explain the lower protein levels found in SIRT mice.
Fasting promotes activation of PGC1α by SIRT1-mediated deacetylation in order to increase gluconeogenesis and blood glucose levels. In SIRT mice, we found that low acetylation of PGC1α correlated with impaired gluconeogenesis, which may be explained by persistent deacetylation by SIRT1 and further degradation by ubiquitination. Overall, our data regarding the regulation of FXR and PGC1α further underlines the importance of SIRT1 as a regulator of the fine-tuning of the acetylation/deacetylation process, which seems essential to orchestrate a proper response to tissue injury.
SIRT1 regulates the energy status of the cell through activation of AMPK/LKB1.[1, 4, 19] Accordingly, SIRT mice showed phosphorylation of AMPK at basal conditions that was not further activated after PH, contrary to what we observed in WT animals. These results suggest that persistent phosphorylation of AMPK renders it nonfunctional in SIRT mice, as we found down-regulation of AMPK-target molecules like HuR, MAT2A, and NOS2. These data support the key role of LKB1/AMPK to mediate cell growth during liver regeneration.
We describe that mTOR signaling was attenuated in SIRT mice throughout the regenerative response, supporting the role of SIRT1 as a negative regulator of mTOR. Interestingly, we provide evidence of a feedback regulation of SIRT1 by mTOR, as Leu attenuated SIRT1 expression in SIRT mice. Our data links the previously described regulation of SIRT1-deacetylase activity by mTOR-mediated phosphorylation with the degradation of SIRT by phosphorylation. Leu restored mTORC1 signaling that correlated with reduced BA-accumulation and liver injury, pointing to a link between mTOR and BA metabolism. Accordingly, mTOR activation by Leu restored FXR acetylation and its target gene expression in SIRT mice and attenuated methylation of H3K4 and H3K9 reaching comparable WT liver levels. We propose that the direct regulation of SIRT1 by mTORC1 influences histone and protein acetylation, which may explain the potential of mTOR to regulate histone acetylation.
It is worth noting that the beneficial effects of Leu and NorUDCA seem directly related to counteracting the detrimental effects of SIRT overexpression. Thus, WT animals fed with Leu showed attenuated hepatocyte proliferation (Supporting Fig. 9A), whereas NorUDCA/WT mice showed a comparable number of proliferating hepatocytes than WT mice after PH (Supporting Fig. 9B).
PTM, such as chromatin alterations by acetylation/deacetylation, are involved in carcinogenesis. The role of SIRT1 during tumorigenesis remains controversial, as it has both pro- and anticarcinogenic effects. Here we confirm that SIRT1 is highly expressed in human HCC samples, regardless of etiology: HCV or ASH. Based on this fact, we propose that the tumorigenic characteristic of SIRT1 relies on its role as a regulator of BA metabolism, as both HCV- and ASH-cirrhosis are characterized by dysregulated BA metabolism.[11, 12] This might explain the apparent contradiction with a previous study where the protection against inflammation and ROS exerted by SIRT1 overexpression seemed sufficient to circumvent tumor development. Moreover, we convincingly show that SIRT1 overexpression leads to dysregulation of FXR, a well-known feature of metabolic disease and tumor development, as deletion of FXR leads to HCC development. Previous work related hyperacetylation of FXR with cancer, metabolic-, and aging-related diseases. Importantly, we propose that overexpression of SIRT1 correlates with an absence of FXR expression in tumors potentially due to hypoacetylation and further degradation of FXR. These compelling data evidence the inverse correlation between SIRT1 and FXR during tumorigenesis and points to the oncogenic role of SIRT1.
Overall, our data underscore the significance of maintaining the deacetylation activity of SIRT1 as a dynamic process in the liver during the regenerative response after injury. Also, our work highlights the need to propose the cautious use of SIRT1-activating drugs, as persistent SIRT1 activity may have detrimental effects on the liver in response to injury. Importantly, our findings underline the potential use of NorUDCA- and mTORC1-activators as therapeutic tools in the context of an aberrant overexpression of SIRT1, to counteract liver metabolic diseases involving dysregulated BA homeostasis and/or the induction of a regenerative response after injury.