Nuclear SIRT1 Research Articles

Sirtuins mediate mammalian metabolic responses to nutrient availability

Metabolic diseases are an increasing threat in developed countries. Dysregulation of metabolic pathways, caused by imbalances in energy homeostasis, leads to obesity, diabetes and cardiovascular disease with devastating results for both individuals and societies. Sirtuins, a conserved family of NAD(+)-dependent deacetylase enzymes found in many species, regulate various metabolic pathways and have emerged as important sensors of energy status in mammals. The nuclear sirtuins, SIRT1, SIRT6 and SIRT7, regulate the activity of key transcription factors and cofactors of numerous metabolic pathways in almost all tissues by linking nutrient signals with the cellular responses to energy demands. The mitochondrial sirtuins, SIRT3, SIRT4 and SIRT5, regulate the activity of important mitochondrial enzymes and drive metabolic cycles in response to fasting and calorie restriction. Accumulating evidence indicates that sirtuins can be beneficial in the prevention of metabolic and age-related diseases and suggests that they can be pharmacologically activated to ameliorate such diseases. This Review describes the latest advances in the understanding of the function of sirtuins as regulators of mammalian metabolism and focuses on the role of these enzymes as mediators of nutrient availability.

Involvement of FOXO Transcription Factors, TRAIL-FasL/Fas, and Sirtuin Proteins Family in Canine Coronavirus Type II-Induced Apoptosis

n our previous study, we have shown that canine coronavirus type II (CCoV-II) activates both extrinsic and intrinsic apoptotic pathway in a canine fibrosarcoma cell line (A-72 cells). Herein we investigated the role of Sirtuin and Forkhead box O (FOXO) families in this experimental model using Nortern Blot and Western Blot analysis. Our results demonstrated that mitochondrial SIRT3 and SIRT4 protein expression increased from 12 and 24 h post infection (p.i.) onwards, respectively, whereas the nuclear SIRT1 expression increased during the first 12 h p.i. followed by a decrease after 36 h p.i., reaching the same level of control at 48 h p.i. Sirtuins interact with/and regulate the activity of FOXO family proteins, and we herein observed that FOXO3A and FOXO1 expression increased significantly and stably from 12 h p.i. onwards. In addition, CCoV-II induces a remarkable increase in the expression of TNF-related apoptosis-inducing ligand (TRAIL), while we observed a slight up-regulation of FasL/Fas at 36 p.i. with a decrease of both proteins at the end of infection. Furthermore, we found that virus infection increased both bax translocation into mitochondria and decreased bcl-2 expression in cytosol in a time-dependent manner.These data suggest that FOXO transcription factors mediate pro-apoptotic effects of CCoV-II, in part due to activation of extrinsic apoptosis pathway, while some Sirtuin family members (such as SIRT3 and SIRT4) may be involved in intrinsic apoptotic pathway. Moreover, these results propose that TRAIL is an important mediator of cell death induced by CCoV-II during in vitro infection.

Oxidative Damage Targets Complexes Containing DNA Methyltransferases, SIRT1, and Polycomb Members to Promoter CpG Islands

Cancer cells simultaneously harbor global losses and gains in DNA methylation. We demonstrate that inducing cellular oxidative stress by hydrogen peroxide treatment recruits DNA methyltransferase 1 (DNMT1) to damaged chromatin. DNMT1 becomes part of a complex(es) containing DNMT3B and members of the polycomb repressive complex 4. Hydrogen peroxide treatment causes relocalization of these proteins from non-GC-rich to GC-rich areas. Key components are similarly enriched at gene promoters in an invivo colitis model. Although high-expression genes enriched for members of the complex have histone mark and nascent transcription changes, CpG island-containing low-expression genes gain promoter DNA methylation. Thus, oxidative damage induces formation and relocalization of a silencing complex that may explain cancer-specific aberrant DNA methylation and transcriptional silencing.

SIRT1 Deacetylase in SF1 Neurons Protects against Metabolic Imbalance

Chronic feeding on high-calorie diets causes obesity and type 2 diabetes mellitus (T2DM), illnesses that affect hundreds of millions. Thus, understanding the pathways protecting against diet-induced metabolic imbalance is of paramount medical importance. Here, we show that mice lacking SIRT1 in steroidogenic factor 1 (SF1) neurons are hypersensitive to dietary obesity owing to maladaptive energy expenditure. Also, mutant mice have increased susceptibility to developing dietary T2DM due to insulin resistance in skeletal muscle. Mechanistically, these aberrations arise, in part, from impaired metabolic actions of the neuropeptide orexin-A and the hormone leptin. Conversely, mice overexpressing SIRT1 in SF1 neurons are more resistant to diet-induced obesity and insulin resistance due to increased energy expenditure and enhanced skeletal muscle insulin sensitivity. Our results unveil important protective roles of SIRT1 in SF1 neurons against dietary metabolic imbalance.

Nuclear SIRT1 activity, but not protein content, regulates mitochondrial biogenesis in rat and human skeletal muscle

Silent mating type information regulator 2 homolog 1 (SIRT1)-mediated peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) deacetylation is potentially key for activating mitochondrial biogenesis. Yet, at the whole muscle level, SIRT1 is not associated with mitochondrial biogenesis (Gurd, BJ, Yoshida Y, Lally J, Holloway GP, Bonen A. J Physiol 587: 1817-1828, 2009). Therefore, we examined nuclear SIRT1 protein and activity in muscle with varied mitochondrial content and in response to acute exercise. We also measured these parameters after stimulating mitochondrial biogenesis with chronic muscle contraction and 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) administration in rodents and exercise training in humans. In skeletal and heart muscles, nuclear SIRT1 protein was negatively correlated with indices of mitochondrial density (citrate synthase activity, CS; cytochrome oxidase IV, COX IV), but SIRT1 activity was positively correlated with these parameters (r > 0.98). Acute exercise did not alter nuclear SIRT1 protein but did induce a time-dependent increase in nuclear SIRT1 activity. This increase in SIRT1 activity was temporally related to increases in mRNA expression of genes activated by PGC-1α. Both chronic muscle stimulation and AICAR increased mitochondrial biogenesis and muscle PGC-1α, but not nuclear PGC-1α. Concomitantly, muscle and nuclear SIRT1 protein contents were reduced, but nuclear SIRT1 activity was increased. In human muscle, training-induced mitochondrial biogenesis did not alter muscle or nuclear SIRT1 protein content, but it did increase muscle and nuclear PGC-1α and SIRT1 activity. Thus, nuclear SIRT1 activity, but not muscle or nuclear SIRT1 protein content, is associated with contraction-stimulated mitochondrial biogenesis in rat and human muscle, possibly via AMPK activation.

Abstract 1624: SIRT1 overexpression in pancreatic adenocarcinomas correlates with shortened patient survival and small molecule inhibition as well as target knockdown of SIRT1 induces growth arrest and apoptosis in pancreatic cancer cells

Abstract Aims: Several lines of evidence indicate that the nuclear enzyme SIRT1 belonging to the class III histone deacetylases is implicated in the initiation and progression of various malignancies. SIRT1 exerts its function by regulating deacetylation of histone and non-histone substrates leading to transcriptional repression of tumor suppressor genes. Recently, SIRT1 gained attraction as druggable target. Since chemotherapeutic treatment options are still very limited in pancreatic cancer we aimed at investigating the potential of SIRT1 in this regard by evaluating the effects of SIRT1 inhibition in vitro and SIRT1 expression in vivo. Methods: Employing immunohistochemistry SIRT1 expression was analyzed in a large cohort of pancreatic ductal adenocarcinomas and subsequently correlated with clinicopathological data and patient survival. Furthermore, we investigated the impact of SIRT1-specific small molecule inhibition and target knock down as well as combinatorial regimens including conventional chemotherapy and small molecule inhibitors directed against the EGFR in pancreatic cancer cell culture models. Using the xCELLigence system, cellular events were measured quantitatively in real time and corroborated by secondary conventional readouts including FACS analysis. Results: We found nuclear SIRT1 expression in 36 (27.9%) of 129 pancreatic carcinomas. SIRT1 expression was significantly higher in poorly differentiated carcinomas (p=0.002). SIRT1 expression was positively correlated with the expression of conventional HDACs suggesting a shared regulation. Strong SIRT1 expression was a significant predictor of poor survival both in univariate (p=0.002) and multivariate (HR 1.65, p=0.045) analysis under inclusion of WHO stage and grade. Small molecule inhibition and target knockdown of SIRT1 lead to a rapid growth arrest and influenced cell viability. This effect was even more pronounced in combinatorial regimens with Gefitinib, but not in combination with Gemcitabine. Conclusions: SIRT1 is shown to be an independent prognostic biomarker for pancreatic adenocarcinomas. Moreover our data suggest that SIRT1 plays an important functional role in pancreatic cancer cell growth, which can be levered out by combinatory small molecule inhibition. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1624. doi:10.1158/1538-7445.AM2011-1624

Abstract 4697: Metabolic regulation of caspase-2

Abstract Caspase-2 has been shown to act as an apical caspase, upstream of cytochrome c release, in a variety of cell death settings including chemotherapy-induced apoptosis. However, the mechanism governing caspase-2 activation in response to chemotherapeutics is poorly understood. We previously demonstrated that production of NADPH via the pentose phosphate pathway (PPP) stimulates caspase-2 suppression via a CaMKII-dependent phosphorylation on Ser135 (Nutt et al., 2005), and that as PPP activity declines, caspase-2 becomes dephosphorylated and activated to promote cell death. Our current efforts are focused on identifying the cellular factors that link metabolism to caspase-2 activation, with the hope that such factors could be exploited therapeutically in cancer. In this study we show that inhibition of Sirt1 deacetylase overrides the anti-apoptotic effect of high PPP activity (stimulated by G6P) to cause caspase-2 activation and cell death. In an effort to more precisely understand the role of Sirt1 in caspase-2 activation, we developed a novel biotin-switch-based proteomics approach to identify Sirt1 substrates in an unbiased manner. This approach yielded a variety of high-confidence Sirt1 substrates, many of which are cytoplasmic proteins that primarily group into the areas of glycolysis/metabolism, oxidative stress, cytoskeletal dynamics, and apoptosis; all areas where Sirt1 has been implicated. We had previously shown that one of these substrates, 14-3-3ζ, binds to caspase-2 in a phosphorylation-dependent manner and protects Ser135 from dephosphorylation (Nutt et al., 2009). We show here that acetylation of 14-3-3ζ occurs in response to a drop in PPP activity and triggers 14-3-3ζ release from caspase-2, leading to caspase-2 activation. Conversely, we show that high PPP levels promote Sirt1 activity, which, in turn, promotes 14-3-3ζ binding to caspase-2 by antagonizing the acetylation of 14-3-3ζ. We have extended these results to a panel of breast tumor cell lines, in which inhibition of Sirt1 induces the rapid dissociation of caspase-2 from 14-3-3ζ, and results in a marked enhancement of the cells’ sensitivity to paclitaxel. Interestingly, we observe cytoplasmic localization of Sirt1 at high levels in these cells with very little to none in the nucleus, in contrast to a more nuclear Sirt1 distribution in normal breast tissue. In conclusion, these data support a model in which Sirt1 promotes the pro-survival effect of high metabolic activity by directly regulating 14-3-3ζ-mediated inhibition of caspase-2, and suggest that as PPP activity declines, a consequent decrease in Sirt1 activity triggers caspase-2 activation. As Sirt1 is known to also modulate numerous transcription factors, its suppressive effect on caspase-2 may play a particularly key role under conditions in which transcription is not ongoing (e.g., paclitaxel-induced mitotic arrest) or when Sirt1 is excluded from the nucleus (breast and other tumor cell lines). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4697. doi:10.1158/1538-7445.AM2011-4697

SIRT1 Regulation of Wakefulness and Senescence-Like Phenotype in Wake Neurons

Wake neurons in the basal forebrain and brainstem provide critical inputs to optimize alertness and attention. These neurons, however, evidence heightened vulnerability to a diverse array of metabolic challenges, including aging. SIRT1 is an nicotinamide adenine dinucleotide responsive deacetylase serving diverse adaptive responses to metabolic challenges, yet this metabolic rheostat may be downregulated under conditions of significant oxidative stress. We hypothesized that SIRT1 might serve as a critical neuroprotectant for wake neurons in young animals but that this protectant would be lost upon aging, rendering the neurons more vulnerable to metabolic insults. In this collection of studies, we first established the presence of nuclear SIRT1 in wake neurons throughout the forebrain and brainstem. Supporting functional and behavioral roles for SIRT1 in wake-active neurons, transgenic whole animal, and conditional loss of brain SIRT1 in the adult mouse impart selective impairments in wakefulness, without disrupting non-rapid eye movement or rapid eye movement sleep. Populations of wake neurons, including the orexinergic, locus ceruleus, mesopontine cholinergic, and dopaminergic wake neurons, evidence loss of dendrites and neurotransmitter synthesis enzymes and develop accelerated accumulation of lipofuscin, consistent with a senescence-like phenotype in wake neurons. Normal aging results in a progressive loss of SIRT1 in wake-active neurons, temporally coinciding with lipofuscin accumulation. SIRT1 is a critical age-sensitive neuroprotectant for wake neurons, and its deficiency results in impaired wakefulness.

Mammalian Sirtuins and Energy Metabolism

Sirtuins are highly conserved NAD+-dependent protein deacetylases and/or ADP-ribosyltransferases that can extend the lifespan of several lower model organisms including yeast, worms and flies. The seven mammalian sirtuins, SIRT1 to SIRT7, have emerged as key metabolic sensors that directly link environmental signals to mammalian metabolic homeostasis and stress response. Recent studies have shed light on the critical roles of sirtuins in mammalian energy metabolism in response to nutrient signals. This review focuses on the involvement of two nuclear sirtuins, SIRT1 and SIRT6, and three mitochondrial sirtuins, SIRT3, SIRT4, and SIRT5, in regulation of diverse metabolic processes.

Induction of Manganese Superoxide Dismutase by Nuclear Translocation and Activation of SIRT1 Promotes Cell Survival in Chronic Heart Failure

Oxidative stress plays a pivotal role in chronic heart failure. SIRT1, an NAD(+)-dependent histone/protein deacetylase, promotes cell survival under oxidative stress when it is expressed in the nucleus. However, adult cardiomyocytes predominantly express SIRT1 in the cytoplasm, and its function has not been elucidated. The purpose of this study was to investigate the functional role of SIRT1 in the heart and the potential use of SIRT1 in therapy for heart failure. We investigated the subcellular localization of SIRT1 in cardiomyocytes and its impact on cell survival. SIRT1 accumulated in the nucleus of cardiomyocytes in the failing hearts of TO-2 hamsters, postmyocardial infarction rats, and a dilated cardiomyopathy patient but not in control healthy hearts. Nuclear but not cytoplasmic SIRT1-induced manganese superoxide dismutase (Mn-SOD), which was further enhanced by resveratrol, and increased the resistance of C2C12 myoblasts to oxidative stress. Resveratrol's enhancement of Mn-SOD levels depended on the level of nuclear SIRT1, and it suppressed the cell death induced by antimycin A or angiotensin II. The cell-protective effects of nuclear SIRT1 or resveratrol were canceled by the Mn-SOD small interfering RNA or SIRT1 small interfering RNA. The oral administration of resveratrol to TO-2 hamsters increased Mn-SOD levels in cardiomyocytes, suppressed fibrosis, preserved cardiac function, and significantly improved survival. Thus, Mn-SOD induced by resveratrol via nuclear SIRT1 reduced oxidative stress and participated in cardiomyocyte protection. SIRT1 activators such as resveratrol could be novel therapeutic tools for the treatment of chronic heart failure.

Impact of 6-mo caloric restriction on myocardial ischemic tolerance: possible involvement of nitric oxide-dependent increase in nuclear Sirt1

Ischemic tolerance decreases with aging, and the cardioprotective effect of ischemic preconditioning (IPC) is impaired in middle-aged animals. We have demonstrated that short-term caloric restriction (CR) improves myocardial ischemic tolerance in young and old animals via the activation of adiponectin-AMP-activated protein kinase (AMPK)-mediated signaling. However, it is unknown whether prolonged CR confers cardioprotection in a similar manner. Furthermore, little is known regarding the myocardial expression of silent information regulator 1 (Sirt1; which reportedly mediates various aspects of the CR response) with prolonged CR. Thus, 6-mo-old male Fischer-344 rats were randomly divided into ad libitum (AL) and CR groups. Six months later, isolated perfused hearts were subjected to 25 min of global ischemia followed by 120 min of reperfusion with or without IPC. CR improved the recovery of left ventricular function and reduced infarct size after ischemia-reperfusion and restored the IPC effect. Serum adiponectin levels increased, but myocardial levels of total and phosphorylated AMPK did not change with prolonged CR. Total levels of Sirt1 did not change with CR; however, in the nuclear fraction, CR significantly increased Sirt1 and decreased acetyl-histone H3. Eleven rats from each group were given N-nitro-l-arginine methyl ester in their drinking water for 4 wk before death. In these hearts, chronic inhibition of nitric oxide synthase prevented the increase in nuclear Sirt1 content by CR and abrogated CR-induced cardioprotection. These results demonstrate that 1) prolonged CR improves myocardial ischemic tolerance and restores the IPC effect in middle-aged rats and 2) CR-induced cardioprotection is associated with a nitric oxide-dependent increase in nuclear Sirt1 content.

Inflammation and endothelial dysfunction during aging: role of NF-κB

One of the major conceptual advances in our understanding of the pathogenesis of age-associated cardiovascular diseases has been the insight that age-related oxidative stress may promote vascular inflammation even in the absence of traditional risk factors associated with atherogenesis (e.g., hypertension or metabolic diseases). In the present review we summarize recent experimental data suggesting that mitochondrial production of reactive oxygen species, innate immunity, the local TNF-alpha-converting enzyme (TACE)-TNF-alpha, and the renin-angiotensin system may underlie NF-kappaB induction and endothelial activation in aged arteries. The theme that emerges from this review is that multiple proinflammatory pathways converge on NF-kappaB in the aged arterial wall, and that the transcriptional activity of NF-kappaB is regulated by multiple nuclear factors during aging, including nuclear enzymes poly(ADP-ribose) polymerase (PARP-1) and SIRT-1. We also discuss the possibility that nucleophosmin (NPM or nuclear phosphoprotein B23), a known modulator of the cellular oxidative stress response, may also regulate NF-kappaB activity in endothelial cells.

The SIRT1 deacetylase suppresses intestinal tumorigenesis and colon cancer growth.

Numerous longevity genes have been discovered in model organisms and altering their function results in prolonged lifespan. In mammals, some have speculated that any health benefits derived from manipulating these same pathways might be offset by increased cancer risk on account of their propensity to boost cell survival. The Sir2/SIRT1 family of NAD+-dependent deacetylases is proposed to underlie the health benefits of calorie restriction (CR), a diet that broadly suppresses cancer in mammals. Here we show that CR induces a two-fold increase SIRT1 expression in the intestine of rodents and that ectopic induction of SIRT1 in a β-catenin-driven mouse model of colon cancer significantly reduces tumor formation, proliferation, and animal morbidity in the absence of CR. We show that SIRT1 deacetylates β-catenin and suppresses its ability to activate transcription and drive cell proliferation. Moreover, SIRT1 promotes cytoplasmic localization of the otherwise nuclear-localized oncogenic form of β-catenin. Consistent with this, a significant inverse correlation was found between the presence of nuclear SIRT1 and the oncogenic form of β−catenin in 81 human colon tumor specimens analyzed. Taken together, these observations show that SIRT1 suppresses intestinal tumor formation in vivo and raise the prospect that therapies targeting SIRT1 may be of clinical use in β−catenin-driven malignancies.

Sirtuin, an oxidant sensitive deacetylase, is post‐translationally modified and degraded by the proteasome in response to cigarette smoke in lung epithelial cells

Sirtuin1 (Sirt1) is a class III histone/protein deacetylase that has anti‐aging and anti‐inflammatory properties. We have previously shown that cigarette smoke (CS) decreased Sirt1 protein level in rat lung and human monocyte/macrophages leading to increased NF‐κB dependent pro‐inflammatory cytokine release. As CS contains greater than 1015–17 free radicals, we hypothesized that the cell's redox status influences Sirt1 protein level in response to CS, leading to post‐translational modifications and degradation. Human bronchial epithelial cells (BEAS.2B) were exposed for 24 hrs to different concentrations of cigarette smoke extract (CSE 0.5–1%) in the presence or absence of buthionine sulfoximine (BSO) or N‐acetyl‐L‐cysteine (NAC), an inhibitor and precursor for glutathione biosynthesis respectively. CSE decreased nuclear Sirt1 levels; co‐treatment with BSO further decreased Sirt1, whereas treatment with NAC rescued CSE depletion. Furthermore, CSE caused 4‐hydroxy‐2‐nonenol adduct formation on Sirt1, which was increased by BSO treatment and decreased by NAC. Decreased Sirt1 level was also found to be due to proteasomal degradation as proteasomal inhibitors (ALLN and MG132) attenuated CSE‐mediated degradation of Sirt1. These data suggest that oxidants in CSE modify Sirt1 post‐translationally, marking the protein for proteasomal degradation in lung epithelial cells, which may contribute to the pro‐inflammatory effect of cigarette smoke.Supported by the NIEHS Center Grant ES01247 and Toxicology Training Program Grant ES07026.

SIRT1, an Antiinflammatory and Antiaging Protein, Is Decreased in Lungs of Patients with Chronic Obstructive Pulmonary Disease

Abnormal inflammation and accelerated decline in lung function occur in patients with chronic obstructive pulmonary disease (COPD). Human sirtuin (SIRT1), an antiaging and antiinflammatory protein, is a metabolic NAD(+)-dependent protein/histone deacetylase that regulates proinflammatory mediators by deacetylating histone and nonhistone proteins. To determine the expression of SIRT1 in lungs of smokers and patients with COPD, and to elucidate the regulation of SIRT1 in response to cigarette smoke in macrophages, and its impact on nuclear factor (NF)-kappaB regulation. SIRT1 and NF-kappaB levels were assessed in lung samples of nonsmokers, smokers, and patients with COPD. Human monocyte-macrophage cells (MonoMac6) were treated with cigarette smoke extract (CSE) to determine the mechanism of CSE-mediated regulation of SIRT1 and its involvement in RelA/p65 regulation and IL-8 release. Peripheral lungs of smokers and patients with COPD showed decreased levels of nuclear SIRT1, as compared with nonsmokers, associated with its post-translational modifications (formation of nitrotyrosine and aldehyde carbonyl adducts). Treatment of MonoMac6 cells with CSE showed decreased levels of SIRT1 associated with increased acetylation of RelA/p65 NF-kappaB. Mutation or knockdown of SIRT1 resulted in increased acetylation of nuclear RelA/p65 and IL-8 release, whereas overexpression of SIRT1 decreased IL-8 release in response to CSE treatment in MonoMac6 cells. SIRT1 levels were reduced in macrophages and lungs of smokers and patients with COPD due to its post-translational modifications by cigarette smoke-derived reactive components, leading to increased acetylation of RelA/p65. Thus, SIRT1 plays a pivotal role in regulation of NF-kappaB-dependent proinflammatory mediators in lungs of smokers and patients with COPD.

Abstract 862: Ser517:-phosphorylation Of Sirt1 Is Required For Its Pi3k/akt-mediated Nuclear Translocation And Cardiomyocyte Protection Against Oxidant Stress In Failing Hearts.

[Purpose] We recently found that SIRT1, a protein deacetylase, shuttles between the nucleus and cytoplasm. In this study, we examined the role of nuclear SIRT1 in cardiomyocyte protection against oxidant stress and involvement of PI3K/Akt in the nuclear translocation. [Methods and Results] First, the critical intracellular location of SIRT1 for its anti-apoptotic function was examined. C2C12 cells were transfected with wild-type SIRT1 (WT) or SIRT1 with site-directed mutations in the nuclear localizing signal (mtNLS) and exposed to antimycin A (AA), an oxidative stressor. AA-induced apoptosis was suppressed in WT-transfected cells expressing SIRT1 in the nuclei compared with that in mtNLS-transfected cells expressing SIRT1 in the cytoplasm (TUNEL-positive cells = 4.4±0.7% vs. 34.6±8.0%). AA-induced apoptosis and also angiotensin II-(angII)-induced apoptosis in neonatal rat cardiomyocytes (NRCM) were suppressed by resveratrol, a SIRT1 activator. This protective effect of resveratrol was attenuated by transfection of SIRT1-siRNA but not by transfection of control siRNA. Next, we assessed the role of PI3K/Akt in nuclear translocation of SIRT1. SIRT1 in NRCM was localized in both the nucleus and cytoplasm under baseline conditions, and IGF-1 induced its nuclear translocation. This effect of IGF-1 was suppressed by LY294002 (LY), a PI3K inhibitor. Deletion mutagenesis study showed that LY-induced nuclear exclusion was observed for SIRT1[223–540] but not for SIRT1[223– 489]. Replacement of serine517 with alanine (S517A) increased cytoplasmic SIRT1, and S517A showed attenuated nuclear translocation in response to IGF-1, indicating that serine517 is the target site of PI3K/Akt. Finally, to confirm heart failure-associated SIRT1 translocation in vivo, myocardial infarction was induced in WKY rats. The number of ventricular cardiomyocytes with nuclear SIRT1 at 4 weeks after infarction was significantly larger than that in sham-operated hearts (10.2±2.9% vs. 0.7±0.2%). [Conclusion] The results suggest that phosphorylation of SIRT1 at Ser517 by PI3K/Akt is involved in nuclear translocation of SIRT1, which contributes to cardiomyocyte protection from oxidant stress-mediated injury in failing hearts.

Nucleocytoplasmic Shuttling of the NAD+-dependent Histone Deacetylase SIRT1

Sir2 (silent information regulator 2) is an NAD(+)-dependent histone deacetylase that contributes to longevity in yeast. SIRT1, a mammalian Sir2 ortholog, deacetylates histones and various transcription factors, including p53, FOXO proteins, and peroxisome proliferator-activated receptor-gamma. We found that its subcellular localization varied in different tissues of the adult mouse. Some subsets of neurons predominantly expressed SIRT1 in the cytoplasm, but ependymal cells expressed it in both the nucleus and cytoplasm. On the other hand, spermatocytes expressed SIRT1 only in the nucleus. Cardiomyocytes in the day 12.5 mouse embryo expressed SIRT1 exclusively in the nucleus, but in the adult heart, they expressed it in both the cytoplasm and nucleus. C2C12 myoblast cells expressed SIRT1 in the nucleus, but it localized to the cytoplasm after differentiation. LY294002, an inhibitor of phosphoinositide 3-hydroxykinase, strongly inhibited the nuclear localization of SIRT1 in undifferentiated C2C12 cells. In a heterokaryon assay, SIRT1 shuttled between the nucleus and cytoplasm, and leptomycin B, an inhibitor of CRM1-mediated nuclear exportation, inhibited this shuttling. Two nuclear localization signals and two nuclear export signals were identified by deletion and site-directed mutation analyses. Overexpressed nuclear (but not cytoplasmic or dominant-negative) SIRT1 enhanced the deacetylation of histone H3 in C2C12 cells. Moreover, only the nuclear form suppressed the apoptosis of C2C12 cells induced by antimycin A, an oxidative stressor. These findings indicate that nucleocytoplasmic shuttling is a novel regulatory mechanism of SIRT1, which may participate in differentiation and in inhibition of cell death.

Activation of Sirt1 Decreases Adipocyte Formation During Osteoblast Differentiation of Mesenchymal Stem Cells

In vitro, mesenchymal stem cells differentiate to osteoblasts when exposed to bone-inducing medium. However, adipocytes are also formed. We showed that activation of the nuclear protein deacetylase Sirt1 reduces adipocyte formation and promotes osteoblast differentiation. Mesenchymal stem cells (MSCs) can differentiate into osteoblasts, adipocytes, chondrocytes, and myoblasts. It has been suggested that a reciprocal relationship exists between the differentiation of MSCs into osteoblasts and adipocytes. Peroxisome proliferator-activated receptor gamma2 (PPARgamma2) is a key element for the differentiation into adipocytes. Activation of Sirt1 has recently been shown to decrease adipocyte development from preadipocytes through inhibition of PPARgamma2. We used the mouse mesenchymal cell line C3H10T1/2 and primary rat bone marrow cells cultured in osteoblast differentiation medium with or without reagents affecting Sirt1 activity. Adipocyte levels were analyzed by light microscopy and flow cytometry (FACS) after staining with Oil red O and Nile red, respectively. Osteoblast and adipocyte markers were studied with quantitative real-time PCR. Mineralization in cultures of primary rat bone marrow stromal cells was studied by von Kossa and alizarin red staining. We found that Sirt1 is expressed in the mesenchymal cell line C3H10T1/2. Treatment with the plant polyphenol resveratrol as well as isonicotinamide, both of which activate Sirt1, blocked adipocyte development and increased the expression of osteoblast markers. Nicotinamide, which inhibits Sirt1, increased adipocyte number and increased expression of adipocyte markers. Furthermore, activation of Sirt1 prevented the increase in adipocytes caused by the PPARgamma-agonist troglitazone. Finally, activation of Sirt1 in rat primary bone marrow stromal cells increased expression of osteoblast markers and also mineralization. In this study, we targeted Sirt1 to control adipocyte development during differentiation of MSCs into osteoblasts. The finding that resveratrol and isonicotinamide markedly inhibited adipocyte and promoted osteoblast differentiation may be relevant in the search for new treatment regimens of osteoporosis but also important for the evolving field of cell-based tissue engineering.

Tumor Suppressor HIC1 Directly Regulates SIRT1 to Modulate p53-Dependent DNA-Damage Responses

Hypermethylated in cancer 1 (HIC1) is an epigenetically regulated transcriptional repressor that functionally cooperates with p53 to suppress age-dependent development of cancer in mice. Here we show that the mechanism by which the loss of HIC1 function promotes tumorigenesis is via activating the stress-controlling protein SIRT1 and thereby attenuating p53 function. HIC1 forms a transcriptional repression complex with SIRT1 deacetylase, and this complex directly binds the SIRT1 promoter and represses its transcription. Inactivation of HIC1 results in upregulated SIRT1 expression in normal or cancer cells; this deacetylates and inactivates p53, allowing cells to bypass apoptosis and survive DNA damage. Inhibition of SIRT1 function in cells without HIC1 abolishes the resistance to apoptosis. Since aging increases promoter hypermethylation and epigenetic silencing of HIC1, we speculate that the resultant upregulation of SIRT1 may be a double-edged sword that both promotes survival of aging cells and increases cancer risk in mammals.

Increased nuclear proteins in muscle satellite cells in aged animals as compared to young growing animals

Evidence implies that satellite cells could play some limiting role in aged muscle undergoing repair or maintenance of mass, which is of potential clinical concern as this could contribute to sarcopenia. Further, insufficient information is available concerning the cellular mechanisms responsible for the lower rat satellite cell proliferation in old animals. Thus, it was hypothesized that the following proteins would be increased in nuclei of satellite cells from old rat skeletal muscle: the cyclin-dependent kinase (CDK) inhibitors p21 WAF1/CIP1 and p27 Kip1 as well as the transcription factors p53 and Forkhead box, subgroup O1 (FOXO1). In addition, the NAD +-dependent histone deacetylase SIRT1, the mammalian ortholog of the yeast SIR2 (silence information regulator 2) and a member of the Sirtuin family, was hypothesized to decrease in satellite cell nuclei of old rats. Old satellite cells (30-months old) exhibited a lesser number of BrdU-positive cells as compared to satellite cells (3-months old) from young growing animals. Western blot analysis demonstrated that nuclei of old satellite cells accumulated the cell cycle inhibitors p21 WAF1/CIP1 and p27 Kip1 . In addition, nuclear p53 and FOXO1 proteins were also higher in old satellite cells than in cells from young growing animals. These data indicated both p53/p21 WAF1/CIP1 - and FOXO1/p27 Kip1 -dependent pathways might contribute to the age-associated decrease in satellite cell proliferation. Cytoplasmic manganese superoxide dismutase (MnSOD), a gene driven by FOXO1, was higher in old satellite cells. Unexpectedly, nuclear SIRT1 was also increased in old satellite cells compared with satellite cells from young growing animals. The physiological significance of enhanced nuclear SIRT1 expression in old satellite cells remains elusive at this time. In summary, satellite cells in old rats have nuclear accumulation of proteins inhibiting the cell cycle as compared to young, growing animals.