Sirtuins are NAD+-dependent protein deacetylases that are broadly conserved from bacteria to humans. Because sirtuins extend the lifespan of yeast, worms and flies, much attention has been paid to their mammalian homologues. Recent studies have revealed diverse physiological functions of sirtuins that are essentially similar to those of their yeast homologue, Sir2 (silent information regulator 2). Sirtuins are implicated in the pathology of many diseases, for which sirtuin activators such as resveratrol have great promise as potential treatments. In the present review, we describe the functions of sirtuins in cell survival, inflammation, energy metabolism, cancer and differentiation, and their impact on diseases. We also discuss the organ-specific functions of sirtuins, focusing on the brain and blood vessels.
- DNA repair
- stress resistance
A PROTOTYPE OF SIRTUINS
Saccharomyces cerevisiae is a useful eukaryote model organism. Sir2 (silent information regulator 2) is first identified and most extensively studied among five sirtuins of yeast cells. Sir2 represses transcription at the mating type loci HML and HMR , telomeres  and of rRNA genes . Sir2 suppresses recombination in rRNA genes and inhibits the accumulation of ERCs (extrachromosomal rDNA circles), an important cause of aging in S. cerevisiae . Loss-of-function Sir2 mutants exhibit both sterility and a shortened lifespan, while overexpression of Sir2 extends the lifespan by ~30% . An increase in Sir2 expression also prolongs the lifespan of Caenorhabditis elegans  and Drosophila melanogaster . CR (calorie restriction) extends the lifespan of rodents  and a variety of other species. The lifespan extension of yeast and fly by CR is Sir2-dependent [7,9]. Sir2 binds to a forkhead transcription factor to increase mitochondrial biogenesis and the stress resistance of the cell . Upon DNA damage, Sir2 is recruited from telomeres to the DNA break point , in which Sir2 co-operates with the DNA end-binding protein Ku to repair DNA .
Recently, Sir2 was found to contribute to the transfer of damaged and aggregated proteins from the daughter to the mother cell during cytokinesis . An early study showed that lysine residues of histones are less acetylated in the mating type loci and telomeric regions than in other regions and that the overexpression of Sir2 elicits histone deacetylation in vivo . Later, Sir2 was found to be an unusual histone deacetylase that uses NAD+ as a coenzyme . Sir2 transfers an acetyl moiety from the acetyl-lysine of histones to NAD+, which is catalysed to AADPR (O-acetyl-ADP-ribose)  and nicotinamide. AADPR is a newly identified molecule that has some biological functions , and nicotinamide is an inhibitor of sirtuins .
MAMMALIAN SIRTUINS CONSIST OF SEVEN MEMBERS
Seven human sirtuins, SIRT1–SIRT7, have been reported . SIRT1 and SIRT2 exist in the nucleus and cytoplasm [20,21]. SIRT3, SIRT4 and SIRT5 localize in the mitochondria; however, SIRT3 is also found in the nucleus and cytoplasm [22,23]. SIRT6 and SIRT7 are nuclear sirtuins . As mentioned above, sirtuins, which are class III HDACs (histone deacetylases), require NAD+ for their activity, which distinguishes them from class I and class II HDACs. The modulators of sirtuins are also different from those that modulate classes I and II HDACs. Some of the mammalian sirtuins have been reported to be ADP-ribosyltransferases, but a recent study indicated that the ADP-ribosyltransferase activity probably reflects inefficient side reactions of the deacetylase activity . Surprisingly, the essential functions of the mammalian sirtuins are quite similar to those of yeast Sir2, except that they also affect inflammation and differentiation (Figure 1). Among the seven sirtuins, SIRT1 is the most extensively studied.
INHIBITION OF CELL DEATH BY SIRTUINS
Sirtuins promote cell survival through various mechanisms (Figure 2). The tumour suppressor p53 induces apoptosis. The deacetylation of p53 by SIRT1 represses the transcriptional activity of p53 and inhibits apoptosis [25,26]. AROS (active regulator of SIRT1)  and Necdin  bind to SIRT1 and p53, to accelerate the deacetylation of p53 by SIRT1. SIRT7 associates with RNA polymerase I and increases rRNA transcription, while SIRT7 knockdown stops cell proliferation and triggers apoptosis . SIRT7 deacetylates and inactivates p53, which corresponds to an increased rate of apoptosis in SIRT7-knockout cells . SIRT1 and SIRT3 deacetylate Ku70, causing Ku70 to bind and sequester the pro-apoptotic factor Bax from mitochondria, thereby inhibiting stress-induced cell apoptosis [31,32]. PARP [poly(ADP-ribose) polymerase-1] consumes NAD+ and contributes to caspase-independent cell death, and SIRT1 deacetylates and inhibits PARP . HSF1 (heat-shock factor 1) is a transcription factor to protect cells from heat shock and misfolded proteins. Deacetylation of HSF1 by SIRT1 is essential for cell survival in response to heat shock . SIRT1 deacetylates HSF1 and increases the DNA-binding activity of HSF1. SIRT1 also deacetylates and regulates the FOXO (forkhead box O) transcription factors FOXO1, FOXO3a and FOXO4, thereby arresting the cell cycle and decreasing ROS (reactive oxygen species) by inducing p27kip1 and stress-resistant proteins such as mitochondrial MnSOD [manganese SOD (superoxide dismutase)] and GADD45 (growth-arrest and DNA-damage-inducible protein 45) [35–38].
In cases of infarction and cardiomyopathy, SIRT1 is translocated from the cytoplasm to the nucleus of cardiomyocytes, and it decreases ROS-induced cell death by inducing MnSOD . CR increases nuclear SIRT1 via a nitric oxide signal and promotes myocardial ischaemic tolerance , which may be further enhanced by activation of eNOS (endothelial nitric oxide synthase) by SIRT1 . SIRT1 also increases several key ROS-detoxifying enzymes by deacetylating and activating PGC-1α [PPAR (peroxisome-proliferator-activated receptor) γ co-activator-1α], a transcriptional co-activator of FOXO family [42–44]. Nuclear SIRT3 decreases ROS by inducing MnSOD and catalase, through FOXO3a activation, and prevents cardiac hypertrophy . In mesangial cells, SIRT1 reduces apoptosis by deacetylating Smad7, which inhibits activation of Smad7 by TGF-β1 (transforming growth factor-β1) . In vivo, the overexpression of SIRT1 in transgenic mouse models promotes the survival of pancreatic islet β-cells  and cardiomyocytes  and of neurons in a mouse model of AD (Alzheimer's disease) . Although sirtuins modulate various effectors to inhibit cell death, it is not known which one is more important in physiological and pathological conditions.
INHIBITION OF INFLAMMATION BY SIRTUINS
Sirtuins are involved in inflammation (Figure 3). NF-κB (nuclear factor κB) is a transcription factor involved in inflammation. SIRT1 deacetylates the RelA/p65 subunit of NF-κB at Lys310 and inhibits NF-κB signalling . Aβ (amyloid β-peptide), which plays a fundamental role in AD, increases the acetylation of RelA/p65 in microglia, thus activating NF-κB; in contrast, SIRT1 reduces the acetylation and protects neurons . The SIRT1 level is reduced in the lungs of smokers and patients with COPD (chronic obstructive pulmonary disease), leading to increases in RelA/p65 acetylation  and MMP-9 (matrix metalloproteinase-9) transcription , which further promote the disease. SIRT6 inhibits NF-κB activity by a different mechanism. By binding to RelA/p65, SIRT6 is recruited to NF-κB target-gene promoters, where it deacetylates histone H3 at Lys9 and attenuates NF-κB signalling .
SIRT1-null mice exhibit an autoimmune-like condition [54,55]. SIRT1 inhibits AP-1 (activator protein-1) by deacetylating c-Jun and plays a major role in clonal T-cell anergy, a mechanism important for the suppression of self-reactive T-cells . However, at present, the role of SIRT1 in human autoimmune diseases is unknown. The deacetylation of c-Fos by SIRT1 also contributes to the inhibition of AP-1 and reduces COX (cyclo-oxygenase)-2 expression in macrophages . Activation of macrophages plays a central role in chronic low-grade tissue inflammation of obesity-related diseases. SIRT1 inhibition in macrophages broadly activates inflammatory pathways evoked by LPS (lipopolysaccharide), whereas resveratrol, a SIRT1 activator, inhibits c-Jun phosphorylation, IκB (inhibitor of NF-κB) degradation and NF-κB phosphorylation induced by LPS in a SIRT1-dependent manner . LPS and NEFAs (non-esterified ‘free’ fatty acids) also down-regulate AMPK (AMP-activated protein kinase), resulting in the decrease of NAD+ content and inactivation of SIRT1 (see below) . Myeloid-cell-specific SIRT1-knockout mice fed on a high-fat diet increase levels of pro-inflammatory cytokines and develop insulin resistance . SIRT1 also inhibits fibrosis, by deacetylating and inhibiting activation of Smad3 by TGF-β1 and suppressing the up-regulation of collagen IV and fibronectin in renal fibroblasts . Because fibrosis is involved in various diseases including cirrhosis, pulmonary fibrosis and myelofibrosis, activation of SIRT1 may have a benefit for these diseases. SIRT7-knockout mice develop inflammatory cardiomyopathy with extensive fibrosis , suggesting SIRT7 is also involved in inflammation. In addition, induction of ROS-detoxifying enzymes by SIRT1 and SIRT3, as mentioned above, also suppresses inflammation. Additionally, STAT3 (signal transducer and activator of transcription 3) is deacetylated and modulated by SIRT1 , but its significance in immunology is not determined. These experiments indicate that sirtuin activation has the advantage in inflammation control.
REGULATION OF CELLULAR METABOLIC STATUS BY SIRTUINS
Sirtuins regulate energy metabolism (Figure 4). Exercise, metformin and thiazolidinediones, which are all typical remedies for Type 2 diabetes, activate AMPK, which promotes oxidative phosphorylation to produce ATP and reduces ATP consumption by inhibiting anabolic pathways, such as protein synthesis pathways. AMPK is regulated by the [AMP]/[ATP] levels and LKB1 (liver kinase B1). CR activates AMPK because it increases the [AMP]/[ATP] ratio; CR also leads to an increased SIRT1 level [31,42], which activates LKB1 by deacetylating it at Lys48 [62,63]. LKB1 is also activated by SIRT3-dependent deacetylation .
SIRT1 is regulated by the NAD+ level [65,66] and acts as a redox sensor to monitor the [NAD+]/[NADH] ratio, which changes dynamically according to the metabolic conditions. AMPK enhances SIRT1 activity by increasing the NAD+ level, which is mediated by the activation of mitochondrial oxidative phosphorylation  or by the induction of NAMPT (nicotinamide phosphoribosyltransferase) . NAMPT is a rate-limiting enzyme for NAD+ synthesis in the NAD+ salvage pathway . NAMPT and NAD+ levels, like many metabolic processes, display circadian oscillations and, with SIRT1, are involved in the feedback regulation of the circadian machinery [69,70]. In the oxidation of nutrients, NAD+ and NADH mediate electron transfer from the citric acid cycle to the electron transport chain in order to generate a proton gradient, which is then used for oxidative phosphorylation to produce ATP. SIRT1 is directly regulated by the material (nutrients) and the intermediate product (NAD+/NADH) and is indirectly controlled by the end product (ATP/ADP). The discovery of SIRT1 clarified the feedback loop in the conversion of nutrients into ATP.
SIRT1 activates SUV39H1 (suppressor of variegation 3–9 homologue 1), a histone methyltransferase, by binding to and deacetylating SUV39H1 at Lys266, and SUV39H1 then increases the trimethylated histone H3 at Lys9 to form heterochromatin . Under energy-starvation conditions, SIRT1 and SUV39H1 are recruited to the rRNA locus with nucleomethylin and repress rRNA transcription to reduce energy consumption . In addition, CR promotes autophagy, a cellular response to limited nutrients, and SIRT1 activates autophagy by interacting with and deacetylating essential components for this process . Roles of autophagy are not limited to nutrient recycling but are also involved in selective degradation of damaged mitochondria, protein aggregates, intracellular bacteria and so on. Studies of the function of SIRT1 on autophagy may expand in the near future.
REGULATION OF GLUCOSE METABOLISM BY SIRTUINS
AMPK phosphorylates and activates PGC-1α, a key co-activator that regulates a number of genes involved in metabolic pathways by activating both nuclear receptors and FOXO transcription factors. The activation of PGC-1α promotes gluconeogenesis and represses glycolysis (Figure 4). PGC-1α is also activated by its deacetylation. Under high-glucose conditions, PGC-1α is highly acetylated on at least 13 lysine residues by the acetyltransferase GCN5, and PGC-1α is, in turn, deacetylated by SIRT1 in response to low glucose . In the fasted state, liver SIRT1 supplies blood glucose via PGC-1α activation . Furthermore, SIRT1 promotes nuclear localization of FOXO1, which may further enhance gluconeogenesis . SIRT1 deacetylates STAT3, a repressor of PGC-1α and inhibits STAT3 phosphorylation, thereby maintaining the PGC-1α activity . PGC-1β, a homologue of PGC-1α, is also activated by SIRT1 and induces the expression of the glucose transporter GLUT4 in skeletal muscle .
SIRT1 affects insulin signalling. It reduces the expression levels of PTP1B (tyrosine phosphatase 1B), which dephosphorylates and inhibits the insulin receptor, resulting in an increase in insulin sensitivity . SIRT1 is also involved in insulin secretion. The overexpression of SIRT1 in pancreatic islet β-cells increases ATP production by repressing the mito-chondrial UCP2 (uncoupler protein 2) expression, thereby leading to the closing of ATP-sensitive K+ channels and to insulin secretion . Mitochondria are a master generator of intracellular ROS, and UCP2 might affect ROS production; however, function of SIRT1 on mitochondrial ROS production is unknown. HIF-1 (hypoxia-inducible factor-1) induces glycolysis and inhibits mitochondrial respiration pathways under low-oxygen conditions. SIRT1 deacetylates HIF-1α at Lys674 to inhibit the HIF-1α-dependent transcription . During hypoxia, the decrease in NAD+ level suppresses SIRT1 and maintains the HIF-1α activity. SIRT6-knockout mice die before 1 month after birth with lethal hypoglycaemia because SIRT6 binds to HIF-1α and represses HIF-1α-dependent transcription by deacetylating histone H3 at Lys9 around the HIF-1α-target gene promoters . On the other hand, HIF-2α, which promotes MnSOD, VEGF-A (vascular endothelial growth factor-A) and EPO (erythropoietin) expression during hypoxia, is activated upon its deacetylation by SIRT1 .
MITOCHONDRIAL FUNCTION AND SIRTUINS
PGC-1α is the master regulator of mitochondrial biogenesis and function (Figure 4). PGC-1α that is deacetylated by SIRT1 induces proteins that promote mitochondrial oxidative phosphorylation in skeletal muscle . Adiponectin, an anti-diabetic adipokine, induces the PGC-1α deacetylation by SIRT1 via elevated [NAD+]/[NADH] levels, thereby increasing the mitochondrial function in skeletal muscle . FGF21 (fibroblast growth factor 21), an anti-hyperglycaemic and triacylglycerol (triglyceride)-lowering growth factor, increases mitochondrial activity through a SIRT1/AMPK/PGC-1α-dependent mechanism in adipocytes . SIRT3 is dominantly expressed in the mitochondria. SIRT3-knockout mice develop a fatty liver and show reduced ATP production. SIRT3 activates mitochondrial acetyl-CoA synthetase 2 by deacetylation, thereby increasing the synthesis of acetyl-CoA from acetate; acetyl CoA is used as an alternative energy source in fasting [84,85]. SIRT3 also augments the mitochondrial electron transport chain activities of Complex I  and Complex II  by deacetylating some of the components of the complexes, to maintain ATP production.
SIRT4 is highly expressed in pancreatic islet β-cells. SIRT4-knockout mice exhibit higher blood insulin level than control mice, and SIRT4 has been shown to suppress insulin secretion by ADP-ribosylating and -inhibiting glutamate dehydrogenase, which supplies α-ketoglutarate for ATP production . Although SIRT1 is a positive regulator of insulin secretion, SIRT4 has an opposing role. Because SIRT1 and SIRT4 are controlled by the NAD+ level, how these two enzymes participate in insulin secretion remains to be elucidated. SIRT5 binds to and deacetylates carbamoyl phosphate synthetase 1, which catalyses the initial step of urea cycle to detoxify ammonia . During fasting, amino acids are used as energy sources that increase ammonia production. Because fasting increases the NAD+ level, activation of SIRT5 by NAD+ promotes detoxification of ammonia. Accordingly, SIRT5-knockout mice show hyperammonaemia during fasting .
REGULATION OF LIPID METABOLISM BY SIRTUINS
Sirtuins regulate adipogenesis and lipid metabolism (Figure 5). PPARγ, also known as the glitazone receptor, is a nuclear receptor that regulates adipogenesis. SIRT1, with N-CoR (nuclear receptor co-repressor), represses the transcriptional activity of PPARγ, which leads to the inhibition of adipogenesis . Under fasting conditions, PGC-1α activation by SIRT1 promotes fatty acid oxidation and ketogenesis . Enzymes for fatty acid oxidation such as MCAD (medium-chain acyl-CoA dehydrogenase) and CPT1b (carnitine palmitoyltransferase 1b) and PDK-4 (pyruvate dehydrogenase kinase-4), a key regulator of metabolic shift to fatty acid oxidation under nutrient deprivation, are induced by PGC-1α. SIRT1 also binds to PPARα and enhances the transcriptional activity of PPARα with its co-activator PGC-1α and promotes fatty acid oxidation . Because NEFAs are the main energy source of cardiomyocytes, SIRT1 may contribute to the heart function by the lipid catabolism.
LXRs (liver X receptors) and FXRs (farnesoid X receptors) are nuclear receptors that regulate lipid metabolism. LXRs regulate cholesterol and fat metabolism and enhance cholesterol transport from peripheral tissues to the liver, whereas FXR reduces serum glucose and lipid levels by regulating bile acid, lipid and glucose metabolism. SIRT1 deacetylates and activates these nuclear receptors and improves metabolic outcomes [92,93]. Deacetylation of LXRs and FXR by SIRT1 also promotes their ubiquitination and degradation [92,93], but how the quick turnover activates these nuclear receptors is not known. SIRT1 also deacetylates SREBP (sterol-regulatory-element-binding protein)-1 and SREBP-2, transcription factors that promote the expression of lipogenic and cholesterogenic genes for fat storage and are active in the fed state. Their deacetylation makes them targets for ubiquitination and results in their down-regulation . SIRT3 promotes fatty acid oxidation during fasting by deacetylating and thereby activating LCAD (long-chain acyl CoA dehydrogenase), a key enzyme in the fatty acid oxidation pathway . Thus therapeutics that activate sirtuins, especially SIRT1, have potential for treating metabolic disorders including obesity and atherosclerosis.
SIRTUINS IN DNA REPAIR AND CANCER
Sirtuins are involved in DNA damage responses that are closely related to aging and carcinogenesis (Figure 6). Werner syndrome, a disorder characterized by premature aging, is caused by a mutation of WRN (Werner syndrome, RecQ helicase-like), a member of the RecQ DNA helicase family. The acetylation of WRN by p300 and CBP [CREB (cAMP-response-element-binding protein)-binding protein] decreases its activity, while deacetylation by SIRT1 activates it . SIRT6-knockout mice show genomic instability and aging-like phenotype . SIRT6 associates with WRN and suppresses end-to-end chromosomal fusions and premature cellular senescence at telomeres. SIRT6 depletion leads to telomere dysfunction with end-to-end chromosomal fusions and premature senescence . NBS (Nijmegen breakage syndrome) 1 is a sensor of DNA double-strand breaks, and its mutation causes NBS, a rare disorder involving chromosomal instability. The deacetylation of NBS1 by SIRT1 is required for the ionizing-radiation-induced phosphorylation of NBS1 by ATM kinase, which enables an efficient DNA repair response . SIRT6 deacetylates CtIP (C-terminal-binding protein-interacting protein) to improve genome stability and promote DNA end resection, a crucial step in DNA repair by homologous recombination .
β-Catenin is the effector of the canonical Wnt signal and functions in development and carcinogenesis. The acetylation of β-catenin by p300 and CBP increases its transcriptional activity and oncogenicity; SIRT1 deacetylates β-catenin and inhibits the development of colon cancer in a mouse model . BRCA1 (breast cancer early-onset 1) is a tumour suppressor that is involved in DNA repair by homologous recombination; mutations in BRCA1 predispose women to breast and ovarian cancers. BRCA1-mutant mice have low SIRT1 levels and high levels of survivin, a negative regulator of apoptosis. Normally, BRCA1 transactivates SIRT1, which in turn inhibits survivin expression by changing the epigenetic modification of histone H3 . These results indicate that sirtuins inhibit cancer.
On the other hand, there are conflicting data in vitro that SIRT1 is up-regulated in cancer cells and has a tumour-promoting activity. SIRT1 silencing induces growth arrest and/or apoptosis in epithelial cancer cells, while cell growth of non-cancer epithelial cells is not affected by SIRT1 siRNA (small interfering RNA) . SIRT1 inhibitor sirtinol induces senescence-like growth arrest of breast cancer MCF-7 and lung cancer H1299 cells. Activation of Ras and MAPK (mitogen-activated protein kinase) pathways in response to EGF (epidermal growth factor) and IGF1 (insulin-like growth factor 1) is impaired by sirtinol . HIC1 (hypermethylated in cancer 1), a tumour suppressor and an inhibitor of SIRT1 transcription, is down-regulated with age and may increase SIRT1 expression in elderly individuals . The tumour suppressor DBC1 (deleted in breast cancer 1) binds to and inhibits SIRT1 activity . Angiogenesis is necessary during tumour progression, and SIRT1 has angiogenic activity (see below), which is supposed to help development of cancer. As shown in Figure 2, an increase in sirtuin level promotes survival of DNA-damaged cells via suppression of apoptosis-inducing factors such as p53 and may raise the risk of cancer.
Although these cell-based experiments indicate the cancer-promoting activity of SIRT1 and more than 50% of human tumours have abnormality of p53, in vivo experiments using transgenic mice have not shown the oncogenic feature of SIRT1. Because sirtuins could affect a cell at multiple levels by multiple mechanisms, either of cancer-promoting or cancer-inhibiting activities may be found in a cell type-specific manner. Alternatively, the effect of sirtuins may be different in stages of cancer development. In vivo studies are important to elucidate the role of sirtuins in cancer.
REGULATION OF DIFFERENTIATION BY SIRT1
Among the seven sirtuins, SIRT1 affects development. SIRT1-knockout mice are small and most die during the early postnatal period [107,108]. Eyes exhibit abnormal closure of the optic fissure, and retinal layers are thin with disorganized structures in SIRT1-knockout mice. Septal defects or valve abnormalities are often found in the heart of SIRT1-knockout mice. In an outbred background, SIRT1-null mice often survive to adulthood, but both sexes are sterile . Thus, SIRT1 is required for developmental processes and reproduction. SIRT1 inhibits muscle and adipocyte differentiation by MyoD (myogenic differentiation 1) deacetylation and inhibition of PPARγ activity respectively [65,90]. SIRT1 is highly expressed in the embryonic brain . It is located in the cytoplasm of neuronal precursor cells of mice at late embryonic stages and is transiently translocated into the nucleus by differentiation stimuli, where it binds to the co-repressor N-CoR and inhibits Notch signalling, thereby inducing neuronal differentiation . Interestingly, abnormalities found in SIRT1-null mice could occur in the disturbance of Notch signalling . The regulating mechanism of SIRT1 translocation in differentiation is unknown. In the postnatal period, SIRT1 binds to Hes1 and promotes gliogenesis . Because neural precursor cells preferentially differentiate into glial cells after birth, the epigenetic modulation of chromatin DNA, such as methylation, might elicit the opposite function of SIRT1. Further studies are necessary to elucidate molecular function of SIRT1 in the development.
ORGAN-SPECIFIC FUNCTIONS OF SIRTUINS
Specific functions of sirtuins in various organs have been reported. In the present review, we will discuss their specific roles in brain and blood vessels.
SIRT1 is widely expressed in the brain . SIRT1 is shown to protect neurons expressing mutant SOD1, a model of ALS (amyotrophic lateral sclerosis) . In a mouse model of AD, SIRT1 protects neurons and reduces the Aβ levels [48,114,115]. AD mice develop Aβ plaques in the brain at 3–5 months after birth. AD mice crossed with SIRT1 transgenic mice show a marked reduction of plaques in the brain, while AD mice with inactivated brain SIRT1 increase Aβ plaques and all die between 3 and 5 months . The sequential cleavage of APP (amyloid precursor protein) by the β and γ secretases produces Aβ, and this process is inhibited by an alternate cleavage of APP by the α and γ secretases. SIRT1 increases the α secretase levels by deacetylating and activating retinoic acid receptor β  and by reducing the expression level of ROCK1 (Rho-associated kinase 1), a downstream serine/threonine kinase of Rho .
In addition, the brains of SIRT1-knockout mice exhibit decreases in dendritic branching, branch length and complexity of dendritic arbours, and these mice show impaired cognitive abilities . Consistent with this, cytoplasmic SIRT1 promotes neurite extension in pheochromocytoma PC12 cells . Conditional SIRT1-knockout mice generated with Nestin-Cre transgenic mice exhibit increased expression of a brain-specific microRNA, miR-134, resulting in the down-regulation of CREB and brain-derived neurotrophic factor . SIRT1 is involved in the brain-based regulation of feeding and energy expenditure in the arcuate nucleus of the hypothalamus. Fasting increases hypothalamic SIRT1 levels and promotes deacetylation and activation of FOXO1, which inhibits anorexigenic POMC (pro-opiomelanocortin) transcription, whereas SIRT1 inhibitor EX-527 increases food intake via increasing the POMC level and decreasing orexigenic Agrp (agouti-related protein) level . The number of inhibitory synapses of Agrp neurons onto POMC neurons reduces 4 h after administration of EX-527. Moreover, SIRT1-knockout in Agrp neurons reduces the activity of Agrp neurons and decreases food intake . Thus SIRT1 affects synaptic plasticity and firing rate of neurons and controls feeding in the brain. Because spine density changes shortly after the inhibition of SIRT1 activity, SIRT1 may have another function than the microRNA regulation.
SIRT2, a tubulin deacetylase, is also associated with mitotic structures . SIRT2 is phosphorylated and inactivated by CDKs (cyclin-dependent kinases) including CDK5, which regulates neurite outgrowth and cell migration . Although SIRT2 inhibits neurite outgrowth and impairs growth cone collapse induced by ephrin-A , in vivo studies are needed.
In blood vessels, SIRT1 promotes endothelium-dependent vasodilatation by deacetylating and activating eNOS . SIRT1 promotes the sprouting and branching angiogenesis of endothelial cells, in a process involving FOXO1 modulation . Endothelial-restricted SIRT1-knockout mice show an impaired ability to form new vessels in the ischaemic tissue . SIRT1 may also promote angiogenesis by enhancing VEGF-A expression via HIF-2α activation in hypoxia . In addition, SIRT1 deacetylates and activates cortactin, an actin-binding protein that is involved in actin–cytoskeleton rearrangement, lamellipodia formation, endocytosis and cell migration; its deacetylation by SIRT1 promotes the migration of fibroblasts and some cancer cells . Because cortactin is present in all types of cells including endothelial cells and is concentrated in dendritic spines, deacetylation of cortactin by SIRT1 may modulate angiogenesis, dendrite formation, neurite extension and synaptic plasticity via actin reorganization. Yeast Sir2 also links to the actin network by interacting with polarisome that is a protein complex to determine cell polarity . It will be important to elucidate the interaction of actin cytoskeleton and sirtuins.
PHARMACOLOGICAL ACTIVATORS OF SIRTUINS
As mentioned above, the activation of SIRT1 is beneficial for metabolic disorders except glucose metabolism in the liver. Resveratrol, a naturally occurring polyphenol found in grapes and red wine, has been proposed to be responsible for the cardioprotective effects of red wine, invoked by the so-called ‘French Paradox’ . Resveratrol, an antioxidant, is known as an inhibitor of tumour cell growth . Although resveratrol has multiple targets such as COXs [125,126], SIRT1 is found to be activated by resveratrol . The oral administration of resveratrol reduces plasma glucose and the triacylglycerol concentration in streptozotocin-induced diabetic rats by 25% and 50% compared with vehicle-treated rats respectively . Resveratrol promotes the deacetylation of PGC-1α by SIRT1, thereby reducing body weight and insulin resistance and increasing the aerobic capacity, motor function and survival of mice with high-fat-diet-induced obesity [128–130]. Resveratrol mimics the transcriptional gene expression profiles of CR in mice, but does not extend their lifespan . SIRT1 activation by resveratrol improves animal models for AD , BRCA1-associated breast cancer , chronic heart failure , renal fibrosis  and retinal degeneration . Administration of resveratrol to hamsters reduces the levels of acetyl-histone H3 significantly . It is not known whether the histone H3 deacetylation by resveratrol occurs in a specific gene and directly up-regulates the gene expression.
Synthetic SIRT1 activators have been developed and also show anti-obesity and anti-diabetic effects, but there is a debate about whether SIRT1 activators interact with SIRT1 directly or indirectly [126,134]. Although biological functions of SIRT1 activators are convincing, SIRT1 assay using an acetylated peptide with a fluorophore has a problem associated with fluorescently labelled non-native substrates. To this end, it is necessary to elucidate mechanisms of SIRT1 activation by resveratrol and also to develop a method to detect SIRT1 activity accurately.
Sirtuins are sensors of poor nutrient conditions and modulate metabolic pathways to a low-energy status. Intervention therapy by SIRT1 activators will soon be tested as a treatment for obesity and metabolic diseases. Sirtuins modulate physiological signals, in addition to their metabolic functions, and sirtuin activation provides benefits in many disease models. In the near future, sirtuin modulation will become a new tool for helping to overcome various diseases, although it might not be useful for generally extending the lifespan.
Our work was supported by a Grant-in-Aid for Scientific Research [grant number 22590245] and a National Project, “Knowledge Cluster Initiative,” (2nd stage, “Sapporo Biocluster Bio-S”) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and the Akiyama Lifescience Foundation.
Abbreviations: AADPR, O-acetyl-ADP-ribose; Aβ, amyloid β-peptide; AD, Alzheimer's disease; Agrp, agouti-related protein; AMPK, AMP-activated protein kinase; AP-1, activator protein-1; APP, amyloid precursor protein; BRCA1, breast cancer early-onset 1; CDK, cyclin-dependent kinase; COX, cyclo-oxygenase; CR, calorie restriction; CREB, cAMP-response-element-binding protein; CBP, CREB-binding protein; eNOS, endothelial NO synthase; LKB1, liver kinase B1; FOXO, forkhead box O; FXR, farnesoid X receptor; HDAC, histone deacetylase; HIF, hypoxia-inducible factor; HSF1, heat-shock factor 1; LCAD, long-chain acyl CoA dehydrogenase; LPS, lipopolysaccharide; LXR, liver X receptor; NAMPT, nicotinamide phosphoribosyltransferase; NBS, Nijmegen breakage syndrome; N-CoR, nuclear receptor co-repressor; NF-κB, nuclear factor κB; PARP, poly(ADP-ribose) polymerase-1; POMC, pro-opiomelanocortin; PPAR, peroxisome-proliferator-activated receptor; PGC-1α, PPARγ co-activator-1α; ROS, reactive oxygen species; Sir2, silent information regulator 2; SOD, superoxide dismutase; MnSOD, manganese SOD; SREBP, sterol-regulatory-element-binding protein; STAT3, signal transducer and activator of transcription 3; SUV39H1, suppressor of variegation 3–9 homologue 1; TGF-β1, transforming growth factor-β1; UCP2, uncoupler protein 2; VEGF-A, vascular endothelial growth factor-A; WRN, Werner syndrome, RecQ helicase-like
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