NAD-dependent Deacetylase SIRT1 Research Articles

Nicotinamide Mononucleotide Prevents Free Fatty Acid-Induced Reduction in Glucose Tolerance by Decreasing Insulin Clearance.

The NAD-dependent deacetylase SIRT1 improves β cell function. Accordingly, nicotinamide mononucleotide (NMN), the product of the rate-limiting step in NAD synthesis, prevents β cell dysfunction and glucose intolerance in mice fed a high-fat diet. The current study was performed to assess the effects of NMN on β cell dysfunction and glucose intolerance that are caused specifically by increased circulating free fatty acids (FFAs). NMN was intravenously infused, with or without oleate, in C57BL/6J mice over a 48-h-period to elevate intracellular NAD levels and consequently increase SIRT1 activity. Administration of NMN in the context of elevated plasma FFA levels considerably improved glucose tolerance. This was due not only to partial protection from FFA-induced β cell dysfunction but also, unexpectedly, to a significant decrease in insulin clearance. However, in conditions of normal FFA levels, NMN impaired glucose tolerance due to decreased β cell function. The presence of this dual action of NMN suggests caution in its proposed therapeutic use in humans.

The Role of Tryptophan-Nicotinamide (TRP-NAM) Pathway in Malnutrition Induced Liver Dysfunction

Abstract Objectives Malnutrition contributes to 45% of the deaths of children < 5. Mortality in severe malnutrition is often related to metabolic disturbances, including hypoglycemia, indicating hepatic metabolic dysfunction. Children with severe malnutrition have been found to have significantly lower circulating tryptophan (TRP) levels in their blood. Disturbances in TRP-NAM pathway have been implicated in liver and intestine diseases, potentially mediated by NAD-dependent deacetylase SIRT1. This study aim to evaluate the effect of modulating TRP-NAM pathway on hepatic metabolism dysfunction in a mouse model of severe malnutrition. Methods Weanling male C57BL/6J mice were randomized into different groups fed with a control diet (18% protein), or a malnourished diet (1% protein) with or without supplementation of modulators of the TRP-NAM pathway (NAM, NR and TRP). Supplementation therapies were given from days 7–14 after start of the dietary intervention. In addition, SIRT1 modulators treatment including resveratrol, EX-527 and vehicle were tested. Comprehensive metabolic, histological and molecular analyses were performed. Results After diet treatment, the mice in the 1% protein group had significantly lower body weight and body length compared to the 18% protein group, without any effect of modulators of the TRP-NAM pathway. Feeding mice a 1% protein diet led to severe hepatic steatosis, mitochondrial structural alterations, lower ATP levels, disrupted TRP-NAM pathway metabolites, and a decrease of phospholipids to triglycerides ratio. NAM, NR and TRP treatment all led to a partial reversal of the hepatic steatosis in the 1% protein-fed mice. ATP levels, mtDNA copy number, and PPARα target genes in the β-oxidation pathway were also recovered in treatment group. We postulate that the effect is mediated through changes in levels of NAD+, which is critical in redox reactions and is a substrate for SIRT1, important for mitochondrial function. Conclusions Modulating TRP-NAM pathway can partially improve liver metabolic function in a mouse model of severe malnutrition. This study improves our understanding of the cellular pathophysiology of severe malnutrition. The results of this project could lead to the development of new interventions which could then be taken to clinical trials. Funding Sources Bill & Melinda Gates Foundation.

NAD-dependent deacetylase sirtuin-1 mediates the mechanical stress-induced degeneration of articular cartilage in osteoarthritis: mechanical stress stimulates the expression of osteogenic transcription factor RUNX2 via the srituin-1 activation in chondrocytes

Purpose: Mechanical stress is the major pathologic factors associated with osteoarthritis (OA). It has been indicated that the NAD-dependent deacetylase sirtuin-1 (Sirt-1) has an important role in human aging. Recent report demonstrated that SIRT-1 insufficiency induced the shear (mechanical) stress-induced vascular calcification and artherosclerosis in cardiovascular tissues, suggesting that SIRT-1 may be of benefit in the protection against vascular calcification and maintenance of cardiovascular function against shear stress. In addition, it has been suggested that Sirt-1 promotes osteogenic and chondrogenic differentiation of mesenchymal stem cells. However, the impact of Sirt-1 in the osteoarthritic chondrocyte state still remains unclear. We postulated that Sirt-1 regulates a mechanical stress-induced hypertrophic chondrocyte lineage and osteophyte formation through the activation of osteogenic transcription factor Runx2 in osteoarthritic chondrocytes. Since it is well known that Runx2 is a promotor of the activation of cartilage matrix degrading enzyme, matrix metalloproteinase (MMP)-13, Sirt-1 may also influence the expression of MMP-13 in chondrocytes, consequently facilitating the deterioration of articular cartilage in OA. To verify the impact of Sirt-1 in the pathology of OA, we investigated the mechanical stress-induced expressions of Sirt-1, Runx2 and MMP-13, and their correlations in human chondrocytes in OA. Methods: i) The expressions of Sirt-1 and Runx2 were histologically analyzed in the in vitro OA model (mechanical stress-culture system). ii) Human chondrocytes were isolated from articular cartilage tissues from OA patients who underwent the knee joint replacement surgery. Levels of expressions of Runx2, Sirt-1 and MMP-13 were analyzed in the presence or absence of mechanical stress (25 gf/cm2, 2.5 kPa), in chondrocytes. The impact of Sirt-1 insufficiency in the chondrocyte activity was also examined in chondrocytes cultures that were treated with Sirt-1 inhibitor. Results: i) The expression of Sirt-1 was ubiquitously observed in chondrocytes, in contrast, Runx2 expressed in the only osteophyte region in the knee joint of OA model mice. ii) Mechanical stress up-regulated the expression of Runx2 in osteoarthritic chondrocytes. The mechanical stress-accelerated expression of Runx2 was inhibited by the pretreatment with Sirt1 inhibitor in osteoarthritic chondrocytes. Also, the mechanical stress-induced production of MMP-13 from chondrocytes was reduced by the pretreatment with Sirt1 inhibitor. Conclusions: Our study revealed that Sirt-1 inactivation trended to inhibit the mechanical stress-induced expressions of Runx2 and MMP-13 in chondrocytes. The Runx2 has been reported to have an important role as a promotor of activation of MMP-13 in chondrocytes (4). Thus, our findings suggest that the NAD-dependent deacetylase Sirt-1 may upregulate the osteophyte formation and the expression of cartilage degrading enzyme MMP-13 through the mechanism involving the acceleration of the osteogenic transcription factor Runx2 in OA cartilage tissues. Since it is well known that Sirt-1 activity is affected by several stresses as well as mechanical stress, Sirt-1 may be involved in the pathology of OA. Our study may provide a pathologic mechanism linking the NAD-dependent deacetylase Sirt-1 and its modulation of Runx2 in OA.

A small molecule ApoE4-targeted therapeutic candidate that normalizes sirtuin 1 levels and improves cognition in an Alzheimer\u2019s disease mouse model

We describe here the results from the testing of a small molecule first-in-class apolipoprotein E4 (ApoE4)-targeted sirtuin1 (SirT1) enhancer, A03, that increases the levels of the neuroprotective enzyme SirT1 while not affecting levels of neurotoxic sirtuin 2 (SirT2) in vitro in ApoE4-transfected cells. A03 was identified by high-throughput screening (HTS) and found to be orally bioavailable and brain penetrant. In vivo, A03 treatment increased SirT1 levels in the hippocampus of 5XFAD-ApoE4 (E4FAD) Alzheimer’s disease (AD) model mice and elicited cognitive improvement while inducing no observed toxicity. We were able to resolve the enantiomers of A03 and show using in vitro models that the L-enantiomer was more potent than the corresponding D-enantiomer in increasing SirT1 levels. ApoE4 expression has been shown to decrease the level of the NAD-dependent deacetylase and major longevity determinant SirT1 in brain tissue and serum of AD patients as compared to normal controls. A deficiency in SirT1 level has been recently implicated in increased tau acetylation, a dominant post-translational modification and key pathological event in AD and tauopathies. Therefore, as a novel approach to therapeutic development for AD, we targeted identification of compounds that enhance and normalize brain SirT1 levels.

TSPYL2 is a novel regulator of SIRT1 and p300 activity in response to DNA damage.

Protein acetylation and deacetylation events are finely regulated by lysine-acetyl-transferases and lysine-deacetylases and constitute an important tool for the activation or inhibition of specific cellular pathways. One of the most important lysine-acetyl-transferases is p300, which is involved in the regulation of gene expression, cell growth, DNA repair, differentiation, apoptosis, and tumorigenesis. A well-known target of p300 is constituted by the tumor suppressor protein p53, which plays a critical role in the maintenance of genomic stability and whose activity is known to be controlled by post-translational modifications, among which acetylation. p300 activity toward p53 is negatively regulated by the NAD-dependent deacetylase SIRT1, which deacetylates p53 preventing its transcriptional activation and the induction of p53-dependent apoptosis. However, the mechanisms responsible for p53 regulation by p300 and SIRT1 are still poorly understood. Here we identify the nucleosome assembly protein TSPY-Like 2 (TSPYL2, also known as TSPX, DENTT, and CDA1) as a novel regulator of SIRT1 and p300 function. We demonstrate that, upon DNA damage, TSPYL2 inhibits SIRT1, disrupting its association with target proteins, and promotes p300 acetylation and activation, finally stimulating p53 acetylation and p53-dependent cell death. Indeed, in response to DNA damage, cells silenced for TSPYL2 were found to be defective in p53 activation and apoptosis induction and these events were shown to be dependent on SIRT1 and p300 function. Collectively, our results shed new light on the regulation of p53 acetylation and activation and reveal a novel TSPYL2 function with important implications in cancerogenesis.

JAK1-mediated Sirt1 phosphorylation functions as a negative feedback of the JAK1-STAT3 pathway

The type III NAD-dependent histone deacetylase Sirt1 plays important roles in a variety of pathobiological functions through targeting either the acetylated histones or transcription factors. However, the molecular mechanisms underlying how the Sirt1 functions are regulated remain vague. Herein we identified that the Janus kinase 1 (JAK1) interacts with Sirt1 and catalyzes its phosphorylation at the tyrosine residues of 280 and 301, both of which are highly conserved and located in the histone deacetylase catalytic domain of Sirt1. IL-6 stimulation enhanced Sirt1 interaction with JAK1 and JAK1-mediated Sirt1 phosphorylation. Interestingly, JAK1-mediated Sirt1 phosphorylation did not alter Sirt1 deacetylase catalytic activity, but instead it is required for Sirt1 interaction with the downstream transcription factor STAT3. JAK1-mediated phosphorylation enhanced Sirt1 suppression of STAT3 acetylation and transcriptional activity. As a consequence, Sirt1 activation attenuates IL-6 activity in protecting cancer cells from chemotherapeutic drug-induced apoptosis. Our studies identify JAK1 as a previously unappreciated tyrosine kinase of Sirt1 and reveal a novel negative feedback of the JAK1-STAT3 pathway.

The Role of FoxOs in Bone Health and Disease.

Recent studies with murine models of cell-specific loss- or gain-of-function of FoxOs have provided novel insights into the function and signaling of these transcription factors on the skeleton. They have revealed that FoxO actions in chondrocytes are critical for normal skeletal development, and FoxO actions in cells of the osteoclast or osteoblast lineage greatly influence bone resorption and formation and, consequently, bone mass. FoxOs also act in osteoblast progenitors to inhibit Wnt signaling and bone formation. Additionally, FoxOs decrease bone resorption via direct antioxidant effects on osteoclasts and upregulation of the antiosteoclastogenic cytokine OPG in cells of the osteoblast lineage. Deacetylation of FoxOs by the NAD-dependent histone deacetylase Sirt1 in both osteoblasts and osteoclasts stimulates bone formation and inhibits bone resorption, making Sirt1 activators promising therapeutic agents for diseases of low bone mass. In this chapter, we review these advances and discuss their implications for the pathogenesis and treatment of estrogen deficiency-, Type 1 diabetes-, and age-related osteoporosis.

Functional Link Between Heat Shock Protein HSP90alpha and Sirtuin 1 (SIRT1) in the Pathogenesis of Diffuse Large B Cell Lymphoma

Diffuse large B-cell lymphomas (DLBCL) are heterogenous tumors. A large subset of DLBCL is reliant on B-cell receptor activity ('BCR'-DLBCLs). Additional subset of tumors that does not exhibit addiction to BCR signaling features transcriptional program is associated with increased oxidative phosphorylation ('OxPhos'-DLBCLs, Blood 2005; 105: 1851-61). These OxPhos tumros are resistant to BCR inhibition and remain functionally poorly defined. We and others found that OxPhos-DLBCLs, compared to BCR subtype, exhibit overexpression of heat shock protein HSP90alpha (Br J Haematol 2009; 144: 358-66). Expression of multiple heat shock proteins is regulated by the activity of a stress-responsive, acetylation-dependent transcription factor HSF1. Acetylation blocks, whereas deacetylation by sirtuins increases HSF1 activity. However, molecular mechanisms responsible for increased HSP90alpha expression in OxPhos-DLBCLs remain unknown.We investigated the abundance of NAD-dependent deacetylase SIRT1 in a panel of DLBCL cell lines and found that SIRT1 protein level was higher in OxPhos- than BCR-DLBCLs and correlated positively with HSP90alpha gene expression (p=0.02, r=0.7857). To assess the role of SIRT1 in transcriptional regulation of HSP90alpha, we used a small molecule SIRT1 inhibitor tenovin-6 or interfering RNA, which dramatically reduced HSP90alpha induction after heat shock. We next characterized the mechanisms of SIRT1 overexpression in OxPhos cells. SIRT1 transcript abundance was similar in OxPhos and BCR cells, suggesting that changes in SIRT1 expression result from different protein half-life/stability. To test this assumption, OxPhos and BCR cells were incubated with a protein synthesis inhibitor, cycloheximide, and assessed for SIRT1 protein decays over time. Our experiments revealed increased stability of SIRT1 protein in OxPhos-DLBCLs, compared to BCR-DLBCLs. Addition of an HSP90 inhibitor 17-AAG accelerated SIRT1 protein degradation, suggesting that HSP90 might chaperone and thus protect SIRT1 against degradation. We further found that SIRT1 coimmunoprecipitated with HSP90alpha, confirming interaction between these proteins and highlighting a self-reinforcing feedback loop linking HSP90alpha and SIRT1 in OxPhos-DLBCLs.We next assessed the therapeutic potential of SIRT1 inhibition in DLBCLs. SIRT1 knockdown with small interfering RNA or chemical SIRT1 inhibition (tenovin-6) decreased proliferation of OxPhos-DLBCL cells, but also of BCR-DLBCLs, indicating that SIRT1 inhibition exhibits broader, subtype-independent activity in DLBCL cell lines. Surprisingly, BCR-type DLBCLs were significantly more sensitive to SIRT1 inhibitor, tenovin-6. We thus sought to identify a mechanism responsible for SIRT1 inhibition in BCR-DLBCLs. Since SIRT1 deacetylase modulates the transcriptional activity of BCL6 oncogene, critical for survival of BCR-type DLBCLs (PNAS 2007; 104: 3207-12), we assessed the expression of BCL6-regulated genes in these cells. SIRT1 inhibition upregulated the abundance of multiple BCL6-dependent transcripts (including FCER2, CCL3, BLIMP1 and CD74), suggesting that toxicity of SIRT1 inhibition in BCL6-dependent DLBCLs is, at least partially, mediated by decreased BCL6 repressor activity.Although OxPhos-DLBCLs were less sensitive to tenovin-6 than BCR-type DLBCLs, we hypothesized that, given the feedback loop between SIRT1 and HSP90, simultaneous inhibition of HSP90 would increase the cellular sensitivity to tenovin-6. Consistent with this, we found that combination of these two compounds exhibited synergistic activity in OxPhos-DLBCL. Taken together, our data demonstrate that SIRT1 and HSP90alpha are mutually linked and involved in the pathogenesis of OxPhos-DLBCL. Targeting SIRT1-HSP90alpha loop with combinatorial use of SIRT1 and HSP90 inhibitors might be a promising treatment strategy in OxPhos-DLBCLs. DisclosuresWarzocha:BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Juszczynski:Selvita S.A.: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Acetylation of FOXM1 is essential for its transactivation and tumor growth stimulation

Forkhead box transcription factor M1 (FOXM1) plays crucial roles in a wide array of biological processes, including cell proliferation and differentiation, the cell cycle, and tumorigenesis by regulating the expression of its target genes. Elevated expression of FOXM1 is frequently observed in a multitude of malignancies. Here we show that FOXM1 can be acetylated by p300/CBP at lysines K63, K422, K440, K603 and K614 in vivo. This modification is essential for its transactivation on the target genes. Acetylation of FOXM1 increases during the S phase and remains high throughout the G2 and M phases, when FOXM1 transcriptional activity is required. We find that the acetylation-deficient FOXM1 mutant is less active and exhibits significantly weaker tumorigenic activities compared to wild-type FOXM1. Mechanistically, the acetylation of FOXM1 enhances its transcriptional activity by increasing its DNA binding affinity, protein stability, and phosphorylation sensitivity. In addition, we demonstrate that NAD-dependent histone deacetylase SIRT1 physically binds to and deacetylates FOXM1 in vivo. The deacetylation of FOXM1 by SIRT1 attenuates its transcriptional activity and decreases its protein stability. Together, our findings demonstrate that the reversible acetylation of FOXM1 by p300/CBP and SIRT1 modulates its transactivation function.

AU-1 from Agavaceae plants causes transient increase in p21/Cip1 expression in renal adenocarcinoma ACHN cells in an miR-34-dependent manner.

Here, we show that AU-1, spirostanol saponin isolated from Agavaceae plants, causes a transient increase in cyclin-dependent kinase inhibitor (CDKI) p21/Cip1 through the upregulation of miRNAs, miR-34 and miR-21. AU-1 stimulated p21/Cip1 expression without exerting cytotoxicity against different types of carcinoma cell lines. In renal adenocarcinoma ACHN cells, AU-1 transiently elevated the expression level of p21/Cip1 protein without marked increases in p21/Cip1 mRNA levels. Rapid and transient increases in miR-34 and miR-21, both of which are known to upregulate p21/Cip1, were observed in AU-1-treated cells. Inhibitor for miR-34 and for miR-21 significantly blocked the AU-1-caused increase in p21/Cip1, indicating that elevation of p21/Cip1 protein by AU-1 is dependent on these microRNAs. We further clarified that NAD-dependent deacetylase SIRT1, a direct target of miR-34, is decreased by the treatment with AU-1. Furthermore, we found that SIRT1-knockdown increases p21/Cip1 protein levels in an miR-21-dependent manner. On the other hand, ectopic expression of p21/Cip1 resulted in the lowered expression of miR-34 and miR-21, suggesting that reciprocal regulation exists between p21/Cip1 and these miRNAs. We propose that the following feedback network composed of miR-34/SIRT1/miR-21/p21 is triggered by the treatment with AU-1: in cells treated with AU-1, transient elevation of miR-34 leads to the downregulation of SIRT1, thereby miR-21 is freed from SIRT1-dependent suppression. Then, elevated miR-21 upregulates p21/Cip1 protein, followed by the suppression of miR-34 expression.

SIRT1 Is Necessary for Proficient Telomere Elongation and Genomic Stability of Induced Pluripotent Stem Cells

SummaryThe NAD-dependent deacetylase SIRT1 is involved in chromatin silencing and genome stability. Elevated SIRT1 levels in embryonic stem cells also suggest a role for SIRT1 in pluripotency. Murine SIRT1 attenuates telomere attrition in vivo and is recruited at telomeres in induced pluripotent stem cells (iPSCs). Because telomere elongation is an iPSC hallmark, we set out to study the role of SIRT1 in pluripotency in the setting of murine embryonic fibroblasts reprogramming into iPSCs. We find that SIRT1 is required for efficient postreprogramming telomere elongation, and that this effect is mediated by a c-MYC-dependent regulation of the mTert gene. We further demonstrate that SIRT1-deficient iPSCs accumulate chromosomal aberrations and show a derepression of telomeric heterochromatin. Finally, SIRT1-deficient iPSCs form larger teratomas that are poorly differentiated, highlighting a role for SIRT1 in exit from pluripotency. In summary, this work demonstrates a role for SIRT1 in the maintenance of pluripotency and modulation of differentiation.

SIRT1 modulates miRNA processing defects in p53-mutated human keratinocytes

BackgroundTogether with p53, the NAD-dependent lysine deacetylase SIRT1 and the microRNA miR-34a form a feedback loop which self-regulates SIRT1 expression and modulates p53-dependent responses. In addition to its well-described role in mediating transcriptional responses to genotoxic stress, p53 may also regulate microRNA processing and maturation. ObjectiveThis study explored the functional relationship among p53, SIRT1 and miR-34a, and the influence of p53 and SIRT1 on microRNA biogenesis and maturation in primary (NHEK) and p53-mutated (HaCaT) keratinocyte cell lines. MethodsRNAi, miRNA target site blocking oligonucleotides and small molecule inhibitors were used to modulate activity and expression of SIRT1 and p53. Changes in microRNA and mRNA were analysed by qRT-PCR and protein expression was determined by immunoblotting. ResultsMature miR-34a decreased in p53-suppressed NHEK cells, whereas ablation of SIRT1 reduced the primary transcript (pri-miR-34a). When either SIRT1 expression or activity was inhibited in combination with p53 ablation, pri-miR-34a levels increased and mature miR-34a levels decreased. Under these same conditions, additional p53-regulated microRNAs (miRs 16-1/15, 145 and 107) also failed to mature. In HaCaT cells, primary microRNA transcripts for miR-16-1/15, miR-145 miR200c/141 and miRNA-107, but not miR-34a, were approximately 8-fold higher than in NHEK cells. However, the levels of mature microRNA sequences in HaCaT cells were only 1.5–2 fold higher (miR-16-1, miR-145), unchanged (miR-107) or decreased (miR-200c/141, miR-34a) compared to NHEK cells. ConclusionsOur results suggest that p53 mutations interfere with efficient microRNA biogenesis in keratinocytes, and that SIRT1 functions in combination with p53 in this process.

Transcription factor NFκB regulates the expression of the histone deacetylase SIRT1

BackgroundThe NAD-dependent protein deacetylase SIRT1 has a wide range of different targets, which may be regulated either directly through deacetylation and thus potentially altering their activity or localization or indirectly by deacetylation of histones, which in turn alters their transcription rate and availability. SIRT1 is therefore involved in the regulation of many different and fundamental cellular processes such as apoptosis, metabolism, differentiation and cell cycle arrest. It is also involved in the regulation of resistance of cells against oxidative stress and longevity under conditions of caloric restriction. Even though the targets and role of SIRT1 have been studied quite intensively, only little is known about the mechanisms affecting SIRT1 transcriptional regulation. The nuclear factor NFκB is a well-studied and widely known transcription factor, which is involved in the regulation of many important cellular activities. The regulation of NFκB by SIRT1 has been reported recently, but it is, however, still unknown whether a feedback mechanism affects the regulation of SIRT1 too, particularly in view of the fact that putative NFκB binding sites within the SIRT1 promoter suggest just that.ResultsIn the study presented herein we show that there is activation of the SIRT1 promoter by overexpression of different NFκB subunits. Direct binding of NFκB to the SIRT1 promoter can be demonstrated by an electrophoretic mobility shift assay. Further investigations indicated enhanced expression of SIRT1 on the mRNA levels in cells overexpressing NFκB. A functional assay showed that acetylation of one of the main target proteins of SIRT1 is reduced in these cells.ConclusionsThese finding together indicate SIRT1 expression to be regulated in a positive feedback loop by NFκB. The putative binding sites for NFκB found within the SIRT1 promoter appears to be functional and several NFκB subunits are able to enhance the expression of SIRT1 if they are overexpressed.

Activation of Sterol Regulatory Element Binding Protein and NLRP3 Inflammasome in Atherosclerotic Lesion Development in Diabetic Pigs

BackgroundAberrantly elevated sterol regulatory element binding protein (SREBP), the lipogenic transcription factor, contributes to the development of fatty liver and insulin resistance in animals. Our recent studies have discovered that AMP-activated protein kinase (AMPK) phosphorylates SREBP at Ser-327 and inhibits its activity, represses SREBP-dependent lipogenesis, and thereby ameliorates hepatic steatosis and atherosclerosis in insulin-resistant LDLR−/− mice. Chronic inflammation and activation of NLRP3 inflammasome have been implicated in atherosclerosis and fatty liver disease. However, whether SREBP is involved in vascular lipid accumulation and inflammation in atherosclerosis remains largely unknown.Principal FindingsThe preclinical study with aortic pouch biopsy specimens from humans with atherosclerosis and diabetes shows intense immunostaining for SREBP-1 and the inflammatory marker VCAM-1 in atherosclerotic plaques. The cleavage processing of SREBP-1 and -2 and expression of their target genes are increased in the well-established porcine model of diabetes and atherosclerosis, which develops human-like, complex atherosclerotic plaques. Immunostaining analysis indicates an elevation in SREBP-1 that is primarily localized in endothelial cells and in infiltrated macrophages within fatty streaks, fibrous caps with necrotic cores, and cholesterol crystals in advanced lesions. Moreover, concomitant suppression of NAD-dependent deacetylase SIRT1 and AMPK is observed in atherosclerotic pigs, which leads to the proteolytic activation of SREBP-1 by diminishing the deacetylation and Ser-372 phosphorylation of SREBP-1. Aberrantly elevated NLRP3 inflammasome markers are evidenced by increased expression of inflammasome components including NLPR3, ASC, and IL-1β. The increase in SREBP-1 activity and IL-1β production in lesions is associated with vascular inflammation and endothelial dysfunction in atherosclerotic pig aorta, as demonstrated by the induction of NF-κB, VCAM-1, iNOS, and COX-2, as well as by the repression of eNOS.ConclusionsThese translational studies provide in vivo evidence that the dysregulation of SIRT1-AMPK-SREBP and stimulation of NLRP3 inflammasome may contribute to vascular lipid deposition and inflammation in atherosclerosis.

FLT3-ITD+ AML Stem/Progenitor Cells Demonstrate Increased SIRT1 Expression and Enhanced Sensitivity to SIRT1 Inhibition

Abstract 1368In acute myeloid leukemia (AML) small populations of leukemia stem and progenitor cells are resistant to available therapies and represent an important barrier to response and cure. There is pressing need to develop new treatment approaches to target AML stem/progenitor cells. The NAD-dependent deacetylase SIRT1 plays an important role in protection of stem cells from stress. We have recently shown that SIRT1 inhibition enhances elimination of CML LSC via acetylation and activation of p53 (Cancer Cell 2012, 21:266). Here we evaluated the role of SIRT1 in growth and maintenance of AML stem/progenitor cells. We observed higher levels of SIRT1 expression in AML compared to normal CD34+CD38− and CD34+CD38+ cell, with highest expression levels in samples from patients with poor-risk cytogenetic abnormalities. We tested the effects of the SIRT1 inhibitor Tenovin-6 (TV-6) on CD34+ cells from high-risk AML patients (n=12) and healthy controls (n=4). AML CD34+ cells demonstrated varying degrees of sensitivity to TV-6, but TV-6 treatment did not significantly reduce viability of AML CD34+ cells taken as a group, compared to normal cells (p=0.8). However, we observed significantly reduced viability of a subset of samples bearing the FLT3-ITD mutation following TV-6 treatment (p=0.01). We therefore tested the effects of TV-6 on a larger set of poor risk AML samples with the FLT3-ITD mutation (n=11), or wild-type FLT3 (FLT3-WT, n=11). The FLT3-ITD mutation was associated with significantly enhanced sensitivity of AML CD34+ cells to TV-6 induced apoptosis (LD50 for FLT3-ITD=1.9μM; FLT3-WT=3.9μM, p=0.009), and inhibition of cell growth measured using the CellTitre Glo luminescent cell viability assay (FLT3-ITD IC50=0.73μM; versus FLT3-WT IC50=1.4μM, p=0.01). Therefore the presence of the FLT3-ITD mutation identifies a subset of AML samples that are very sensitive to SIRT1 inhibition. Tenovin-6 treatment increased acetylated as well as total p53 levels in FLT3-ITD+ AML CD34+ cells. Tenovin-6 also induced apoptosis and inhibited growth of the FLT3-ITD+ and p53 wild type cell line MV4-11. SIRT1 knock-down reduced TV-6-induced apoptosis and growth inhibition in MV4-11 cells confirming that TV-6 effects are related to SIRT1 inhibition. The growth inhibitory and pro-apoptotic effects of TV-6 in MV4-11 cells were also significantly reduced by p53 knock-down (TV-6 1μM, p53 shRNA 39±8% inhibition, control ShRNA 66±2% inhibition, p=0.04, n=3). Taken together these results support a potential role for p53 activation in mediating the effects of TV-6 treatment in FLT3-ITD+ AML cells. Sequencing studies indicated that only one sample in the FLT3-WT group harbored a p53 mutation, suggesting that their resistance to SIRT1 inhibitors is related to mechanisms other than p53 mutation. Flow cytometry analysis demonstrated increased SIRT1 levels in FLT3-ITD (n=7) compared to FLT3-WT (n=9) AML CD34+ cells (p=0.01). We used lentivirus vectors to ectopically express a FLT3-ITD construct in human cord blood CD34+ cells. Expression of FLT3-ITD resulted in significantly increased SIRT1 expression levels compared to FLT3-WT or vector controls. Consistent with results obtained with samples from AML patients, FLT3-ITD transformed CB CD34+ cells demonstrated enhanced sensitivity to TV-6 mediated apoptosis (TV6 1μM, FLT3-ITD 44±3% survival, n=5; FLT3-WT 59±2% survival, n=5, p=0.04; empty vector 64±4% survival, p=0.02, n=5) and cell growth inhibition. These results indicate that FLT3-ITD-mediated SIRT1 overexpression may contribute to the increased sensitivity of FLT3-ITD AML CD34+ cells to SIRT1 inhibition. In conclusion, our studies indicate that AML stem/progenitor cells demonstrate varying degrees of sensitivity to SIRT1 inhibition reflecting underlying genetic lesions. Importantly the poor prognosis FLT3-ITD mutation is associated with increased SIRT1 expression and enhanced sensitivity to SIRT1 inhibition. In view of the limited success of small molecule FLT3 inhibitors so far, the use of SIRT1 inhibitors may offer an attractive approach to target FLT3-ITD+ AML stem/progenitor cells. Disclosures:No relevant conflicts of interest to declare.

Brown Remodeling of White Adipose Tissue by SirT1-Dependent Deacetylation of Pparγ

Brown adipose tissue (BAT) can disperse stored energy as heat. Promoting BAT-like features in white adipose (WAT) is an attractive, if elusive, therapeutic approach to staunch the current obesity epidemic. Here we report that gain of function of the NAD-dependent deacetylase SirT1 or loss of function of its endogenous inhibitor Deleted in breast cancer-1 (Dbc1) promote "browning" of WAT by deacetylating peroxisome proliferator-activated receptor (Ppar)-γ on Lys268 and Lys293. SirT1-dependent deacetylation of Lys268 and Lys293 is required to recruit the BAT program coactivator Prdm16 to Pparγ, leading to selective induction of BAT genes and repression of visceral WAT genes associated with insulin resistance. An acetylation-defective Pparγ mutant induces a brown phenotype in white adipocytes, whereas an acetylated mimetic fails to induce "brown" genes but retains the ability to activate "white" genes. We propose that SirT1-dependent Pparγ deacetylation is a form of selective Pparγ modulation of potential therapeutic import.

USP22 Antagonizes p53 Transcriptional Activation by Deubiquitinating Sirt1 to Suppress Cell Apoptosis and Is Required for Mouse Embryonic Development

The NAD-dependent histone deacetylase Sirt1 antagonizes p53 transcriptional activity to regulate cell-cycle progression and apoptosis. We have identified a ubiquitin-specific peptidase, USP22, one of the 11 death-from-cancer signature genes that are critical in controlling cell growth and death, as a positive regulator of Sirt1. USP22 interacts with and stabilizes Sirt1 by removing polyubiquitin chains conjugated onto Sirt1. The USP22-mediated stabilization of Sirt1 leads to decreasing levels of p53 acetylation and suppression of p53-mediated functions. In contrast, depletion of endogenous USP22 by RNA interference destabilizes Sirt1, inhibits Sirt1-mediated deacetylation of p53 and elevates p53-dependent apoptosis. Genetic deletion of the usp22 gene results in Sirt1 instability, elevated p53 transcriptional activity and early embryonic lethality in mice. Our study elucidates a molecular mechanism in suppression of cell apoptosis by stabilizing Sirt1 in response to DNA damage and reveals a critical physiological function of USP22 in mouse embryonic development.

MicroRNA Profiling Reveals Distinct Mechanisms Governing Cardiac and Neural Lineage-Specification of Pluripotent Human Embryonic Stem Cells

Realizing the potential of human embryonic stem cells (hESCs) has been hindered by the inefficiency and instability of generating desired cell types from pluripotent cells through multi-lineage differentiation. We recently reported that pluripotent hESCs maintained under a defined platform can be uniformly converted into a cardiac or neural lineage by small molecule induction, which enables lineage-specific differentiation direct from the pluripotent state of hESCs and opens the door to investigate human embryonic development using in vitro cellular model systems. To identify mechanisms of small molecule induced lineage-specification of pluripotent hESCs, in this study, we compared the expression and intracellular distribution patterns of a set of cardinal chromatin modifiers in pluripotent hESCs, nicotinamide (NAM)-induced cardiomesodermal cells, and retinoic acid (RA)-induced neuroectodermal cells. Further, genome-scale profiling of microRNA (miRNA) differential expression patterns was used to monitor the regulatory networks of the entire genome and identify the development-initiating miRNAs in hESC cardiac and neural lineage-specification. We found that NAM induced nuclear translocation of NAD-dependent histone deacetylase SIRT1 and global chromatin silencing, while RA induced silencing of pluripotence-associated hsa-miR-302 family and drastic up-regulation of neuroectodermal Hox miRNA hsa-miR-10 family to high levels. Genome-scale miRNA profiling indentified that a unique set of pluripotence-associated miRNAs was down-regulated, while novel sets of distinct cardiac- and neural-driving miRNAs were up-regulated upon the induction of lineage-specification direct from the pluripotent state of hESCs. These findings suggest that a predominant epigenetic mechanism via SIRT1-mediated global chromatin silencing governs NAM-induced hESC cardiac fate determination, while a predominant genetic mechanism via silencing of pluripotence-associated hsa-miR-302 family and drastic up-regulation of neuroectodermal Hox miRNA hsa-miR-10 family governs RA-induced hESC neural fate determination. This study provides critical insight into the earliest events in human embryogenesis as well as offers means for small molecule-mediated direct control and modulation of hESC pluripotent fate when deriving clinically-relevant lineages for regenerative therapies.

SIRT1 is required for long-term growth of human mesenchymal stem cells

Human mesenchymal stem cells (MSCs) have therapeutic potential because of their ability to self-renew and differentiate into multiple tissues. However, senescence often occurs in MSCs when they are cultured in vitro and the molecular mechanisms underlying this effect remain unclear. In this study, we found that NAD-dependent protein deacetylase SIRT1 is differentially expressed in both human bone marrow-derived MSCs (B-MSCs) and adipose tissue-derived MSCs after increasing passages of cell culture. Using lentiviral shRNA we demonstrated that selective knockdown of SIRT1 in human MSCs at early passage slows down cell growth and accelerates cellular senescence. Conversely, overexpression of SIRT1 delays senescence in B-MSCs that have undergone prolonged in vitro culturing and the cells do not lose adipogenic and osteogenic potential. In addition, we found that the delayed accumulation of the protein p16 is involved in the effect of SIRT1. However, resveratrol, which has been used as an activator of SIRT1 deacetylase activity, only transiently promotes proliferation of B-MSCs. Our findings will help us understand the role of SIRT1 in the aging of normal diploid cells and may contribute to the prevention of human MSCs senescence thus benefiting MSCs-based tissue engineering and therapies.

Regulation of Proliferation and Autophagy and Its Possible Mechanisms of Resveratrol on HepG2 Cells in Hypoxia

Resveratrol as a polyphenolic compound is naturally enriched in grapes, red wine and peanuts. Resveratrol has been reported to have potent anticancer and antioxidant properties. Most studies about the anti-cancer effect of resveratrol have been described previously when cells on the normoxia. The effect of resveratrol in hypoxia, the environment which mimics the state of tumor in vivo remains unclear. In this study, human hepatocellular carcinoma HepG2 cells were used to understand the effects and the possible mechanisms by which resveratrol acts as an anticancer agent in hypoxic conditions (1% O2). Our data show that resveratrol significantly inhibits HepG2 cell viability in hypoxia, and promotes hypoxia stress induced autophagy as well. We also found that the NAD-dependent deacetylase (Sirt1) and sphingosine kinase 1 (SPK1) might be involved in the effects of resveratrol in hypoxia. Collectively, our studies reveal the possible mechanisms of insensitive effect of the resveratrol in hypoxia, and further clarify the anticancer effect of resveratrol on solid tumor cells, providing a reference for drug development of resveratrol.