Biochimica et Biophysica Acta 1799 (2010) 740–749

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Review

Sirtuin activators: Designing molecules to extend life span

Antoni Camins a,⁎, Francesc X. Sureda b, Felix Junyent a,c, Ester Verdaguer a, Jaume Folch c, Carme Pelegri e,Jordi Vilaplana e, Carlos Beas-Zarate d, Mercè Pallàs a

a Unitat de Farmacologia i Farmacognòsia Facultat de Farmàcia, Institut de Biomedicina (IBUB), Centros de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CIBERNED),Universitat de Barcelona, Nucli Universitari de Pedralbes, 08028 Barcelona, Spainb Unitat de Farmacología, Centros de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CIBERNED), Facultat de Medicina i Ciències de la Salut,Universitat Rovira i Virgili, C./ St. Llorenç 21 43201 Reus, Tarragona, Spainc Unitat de Bioquimica, Facultat de Medicina i Ciències de la Salut, Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CIBERNED),Universitat Rovira i Virgili, C./ St. Llorenç 21 43201 Reus, Tarragona, Spaind Departamento de Biología Celular y Molecular, CUCBA, Universidad de Guadalajara, División de Neurociencias, Centro de Investigación Biomédica de Occidente,IMSS, Sierra Mojada 800, Col. Independencia, Guadalajara, Jalisco 44340, Mexicoe Departament de Fisiologia, Centro de Investigación de Biomedicina en Red de Enfermedades Neurodegenerativas (CIBERNED), Facultat de Farmàcia,Universitat de Barcelona, Av. Joan XXIII s/n, 08028 Barcelona, Spain

⁎ Corresponding author. Institut de Biomedicina (IBBiomédica en Red de Enfermedades NeurodegeneraFarmacologia i Farmacognòsia, Facultat de Farmàcia, UUniversitari de Pedralbes, 08028 Barcelona, Spain. Te934035982.

E-mail address: camins@ub.edu (A. Camins).

1874-9399/$ – see front matter © 2010 Elsevier B.V. Adoi:10.1016/j.bbagrm.2010.06.005

a b s t r a c t

a r t i c l e i n f o

Article history:Received 24 February 2010Received in revised form 31 May 2010Accepted 10 June 2010Available online 23 June 2010

Keywords:SirtuinsResveratrolNeurodegenerative diseasesDiabetesSirtuin activators

Resveratrol (RESV) exerts important pharmacological effects on human health: in addition to its beneficialeffects on type 2 diabetes and cardiovascular diseases, it also modulates neuronal energy homeostasis andshows antiaging properties. Although it clearly has free radical scavenger properties, the mechanismsinvolved in these beneficial effects are not fully understood. In this regard, one area of major interestconcerns the effects of RESV on the activity of sirtuin 1 (SIRT1), an NAD+-dependent histone deacetylase thathas been implicated in aging. Indeed, the role of SIRT1 is currently the subject of intense research due to theantiaging properties of RESV, which increases life span in various organisms ranging from yeast to rodents. Inaddition, when RESV is administered in experimental animal models of neurological disorders, it has similarbeneficial effects to caloric restriction. SIRT1 activation could thus constitute a potential strategic target inneurodegenerative diseases and in disorders involving disturbances in glucose homeostasis, as well as indyslipidaemias or cardiovascular diseases. Therefore, small SIRT1 activators such as SRT501, SRT2104, andSRT2379, which are currently undergoing clinical trials, could be potential drugs for the treatment of type 2diabetes, obesity, and metabolic syndrome, among other disorders. This review summarises currentknowledge about the biological functions of SIRT1 in aging and aging-associated diseases and discusses itspotential as a pharmacological target.

UB), Centros de Investigacióntivas (CIBERNED), Unitat deniversitat de Barcelona, Nuclil.: +34 934024531; fax: +34

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© 2010 Elsevier B.V. All rights reserved.

1. Introduction

Once a country's population has achieved the goals of a goodquality of life inwhich basic needs aremet, andwhere this is combinedwith access to good health service and drugs to successfully treat mostdiseases, the next step is to increase life expectancy [1]. To this end, itbecomes necessary to develop drugs that act on different organs andtissues of the body to preserve their functioning. One way ofaccomplishing this would be through the development of drugs withantiaging properties, which should help to prevent or treat aging-associated diseases [1,2]. Because of the complex and multifactor

nature of aging, it would appear to be almost impossible to findmoleculeswith such a variety of antiaging properties. However, naturehas the ability to reveal surprising antiaging tools. For instance, it wasobserved that the French population, through the consumption ofmildto moderate amounts of red wine, showed reduced mortality fromcoronary heart disease [2–4]. Researchers subsequently realised thatresveratrol (RESV; 3,5,4′-trihydroxystilbene), a naturally occurringpolyphenolic compound found in red wine, was responsible for thebeneficial effects on the cardiovascular system [3]. RESV is the mainnonflavonoid polyphenol found in black grapes and is characterised asa phytoalexin [3]. It is produced by a variety of plants in response tostress as protection against fungal colonisation [4]. Initial attempts toascertain the mechanisms involved in the cardioprotective effectsof RESV suggested that the latter were mainly due to its strongantioxidant properties [5–7].

However, the antioxidant properties of RESV alone cannot explainthe pharmacological effects of this compound,which, in recentyears, hasbeen shown to have anti-inflammatory, antitumour, cardioprotective,

Fig. 1. SIRT1 produces different outputs as a result of different stimuli. Activation of SIRT 1 to the brain causes an increase in the expression of the transcription factor FOXO 3A withantiaging properties. Besides an increase in NF transcription factor may explain, among others, the neuroprotective properties of SIRT1. SIRT1 protects pancreatic cells and musclecells against stress-induced apoptosis by increasing activity of the forkhead protein FOXO1. In the liver, SIRT1 deacetylases the coactivator PGC-1α, thereby increasing the expressionof genes for gluconeogenesis. In the muscles, the effect of SIRT1 on FOXO1 increases mitochondrial biogenesis and insulin secretion.

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and antiaging properties (Fig. 1) [7]. Therefore, theremust be additionalpathways activated by RESV that could explain its antiaging andbeneficial coronary effects. Interestingly, a protein called SIRT1, whichis an NAD+-dependent protein deacetylase, may be the target for RESVand responsible for its physiological actions.

SIRT1 was first identified as the human orthologue of yeast Sir2(silent information regulator 2), which belongs to a family of histonedeacetylases (HDACs) that have been divided into four groups [6–11].The major interest is in class III HDACs, which share common featureswith the yeast transcriptional repressor Sir2 and are referred to assirtuins [12]. Class III histone deacetylases were named after thefoundingmember, Saccharomyces cerevisiae silent information regulator2 (Sir2) proteins, and they are essential formaintaining silent chromatinduring histone deacetylation [10–13]. A feature of class III HDACs is thatthey are nicotinamide adenine dinucleotide (NAD+)-dependent, andthey are conserved from bacteria to humans [12]. Since the discovery ofthe involvement of SIRT 1 in apoptosis, cell survival, transcription,metabolism, and aging, these activities have been implicated as diseasemodifiers [3,9,14–16].

Seven mammalian sirtuins (SIRT1-7) have been characterised, andthey have different localisations and functions inside the cell [4]. Atpresent, the most widely studied and best known is SIRT1. Thedevelopment of a transgenic mouse for SIRT1 has revealed that inmammalians this protein exerts a key role in cell metabolism [17–19].

A very interesting discovery was the fact that caloric restriction(CR) has beneficial effects on mammalian health [20]. Indeed, recentdata demonstrate that CR extends life span and delays the onset ofage-associated diseases such as cardiovascular disease, cancer, anddiabetes, as well as muscle atrophy in nonhuman primates [20–29].This research provides strong support for the importance of CR inaging and age-associated disease and highlights the need for a betterunderstanding of the pathways involved in CR-improved health.Several studies have demonstrated that CR regulates mammalianSIRT1 in different tissues, specifically by increasing SIRT1 activity[20,27]. Since RESV treatment produces beneficial effects similar to CRin mice, it was hypothesised that SIRT1 activation could beresponsible for the antiaging effects of RESV [17]. However, the use

of caloric restriction as a therapeutic tool is not a suitable healthstrategy, hence the need for drugs that mimic the process of CR andtherefore selectively activate SIRT1. The synthesis of selective andspecific compounds acting on the SIRT1 such as SRT1720, has shownthe connection between SIRT1 in CR responses and life span. Thiscompound has several physiological effects through the inhibition ofadipogenesis and induction of lipid oxidation which mimics CR.

An in vitro screen for activators of SIRT1 identified RESV as themost potent of eighteen inducers of deacetylase activity [18].Accordingly, neuroprotective and antiaging effects of RESV might bemediated through SIRT1 activation [2,17,19]. Current research istherefore focused on understanding the mechanisms involved in theability of RESV to increase SIRT1 activity and on the intracellularpathways that are activated or regulated by SIRT1 [20].

There are several interesting findings in this regard. One was thediscovery that RESV extends the life span of S. cerevisiae, Caenorhab-ditis elegans, and Drosophila melanogaster, but only if the gene thatencodes SIR2 is present in these organisms [20,26]. RESV alsoincreased the life span of the fish Nothobranchius furzeri, althoughthe target involved in this effect is still unknown [25,27]. Given thesefindings, however, drugs that activate SIRT1might also have antiagingand other beneficial actions on metabolism. Indeed, it is hoped thatsuch compounds may be of benefit in the treatment of diabetes andneurodegenerative diseases, as well as in the prevention of cardio-vascular diseases.

Therefore, this article reviews current knowledge on the mecha-nism of action of RESV and related compounds, mainly specific SIRT1activators. The synthesis of these new specific compounds may have apotential application in aging associated diseases such as neurode-generative diseases, cardiovascular, and metabolic diseases.

2. Intracellular pathways regulated by SIRT1

SIRT1 proteins exert their effects through two different pathways:

a) histone modificationsb) nonhistone substrates

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a) Histone modificationsEpigenetic modulation involves changes in the activity andexpression of chromatin that include variations such as methyla-tions and histonemodifications [29]. Similarly, many aging-relatedeffects are caused by chromatin changes. Since SIRT1 is localisedmainly in the nucleus its physiological actions are partly mediatedthrough its ability to deacetylate nucleosomal histones at specificresidues [2]. For instance, aging produces a decrease in the repairof chromatin aberrations, as well as telomere shortening [29–31].The vastmajority of life span studies have been performed in yeast,where histone methylation is catalysed by histone lysine methyl-transferases, while histone acetyltransferase and histone deacety-lases are involved in regulating, respectively, the acetylation anddeacetylation of lysine residues [30]. Histone acetylation isessential for the control of chromatin structure and, hence, theregulation of gene expression. Thus, chromatin is a target for Sir2,which, in turn, regulates yeast life span [30]. SIRT1 (Sir2) isinvolved in the formation of two different forms of heterochro-matin: facultative and constitutive and both play a role in theexpression of genes. Facultative heterochromatin refers to chro-matin regions that become tightly packed during some processes,such as cell differentiation. On the other hand, the constitutiveheterochromatin describes those chromatin regions that, onceformed, always are condensed. Sir2 has been shown throughprocesses of deacetylation of histones, particularly H4K16 andH3K9 regulates the formation of facultative chromatin, this beingaccompanied by aging increase [30–34]. Moreover, one of thechromatin elements that has been implicated in aging is H1, sinceaging produces changes in the distribution of H1 subtypes [29,30],deamination of H1 molecules, and loss of acetylation of serine 1[30,35]. All of these chromatin changes may result in chromatinsilencing and repression of transcription.Interestingly, a recent study demonstrated that SIRT1, throughdeacetylation of HSF1, could promote chromatin silencing andrepression. Thus, SIRT1 favours HSF1 binding to the heat shockpromoter Hsp70 and may be a pathway in the regulation of lifespan [35].

b) Nonhistone substrates of SIRT1 regulationOnce SIRT1 is activated it mediates intracellular responses thatpromote cell survival, enhance the repair of damaged DNA, andreduce cell division. Moreover, analysis of SIRT1 enzymatic activityhas demonstrated that it acts in a different way to other previouslydescribed histone deacetylases. Experimental data using purifiedSIRT1 indicate that for every acetyl lysine group that is removed,one molecule of NAD+ is cleaved, and nicotinamide and O-acetyl-ADP-ribose are produced [18]. Therefore, SIRT1 appears to possesstwo enzymatic activities: the deacetylation of a target protein andthe metabolism of NAD+.Some beneficial effects of SIRT1 are therefore mediated throughthe regulation of nonhistone cellular substrates, specifically, bydeacetylating transcription factors such as the tumour suppressorp53, the FOXO family (also called FKHR, a member of the forkheadfamily of transcription factors FOXO1, FOXO3, and FOXO4, inwhich it prevents the nuclear translocation and activation of itstargets, such as BIM), and NF-κB. Moreover, deacetylating also thefactor Ku70 reduce their ability to trigger apoptosis or induce theexpression of their target genes involved in stress protection, cellcycle arrest, senescence, or apoptosis [13,15,36–38]. Thus, SIRT1-mediated deacetylation of p53 attenuates stress-induced apopto-sis by reducing the ability of p53 to induce transcriptionally theexpression of the proapoptotic factor Bax [14]. The tumoursuppressor protein p53 plays an essential role in the response toa multitude of cellular stresses such as oxidative stress, deregu-lated oncogene expression, and DNA damage [14,17]. However,the molecular and signalling cascades may differ from cell to cell.For example, p53 regulates the cell cycle in response to stress by

activating the cyclin inhibitor p21 and initiates the apoptoticprocess through the activation of BH3-only proteins such as PUMA,NOXA, and BAX [2,14,18]. The antiproliferative effects of RESV areassociated with inhibition of cell cycle proteins and induction ofp53 in tumour cells. However, in neuronal cells, neuroprotectiveeffects via SIRT1 activation may be mediated by p53 inhibition[14]. As mentioned earlier, another SIRT1 target involves deace-tylation of the FOXO family. Early studies in C. elegans demon-strated that FOXO (DAF-16) proteins interact with severalpathways that regulate cellular life span and thus increaselongevity and aging. In addition to the regulation of longevityprocesses, members of the FOXO family of transcription factors arealso involved in regulating other cellular processes such asmetabolism; for instance, FOXO1 modulates glucose tolerance inadipose tissue and in the liver and pancreas [36,37]. Similarly,FOXO3a is involved in the prevention of apoptosis activity via theregulation of Bim. Moreover, SIRT1 also deacetylates the nuclearreceptor peroxisome proliferator-activated receptor-γ (PPARγ)and its transcriptional coactivator PPARγ coactivator-α (PGC-α),which regulates a wide range of metabolic activities in muscle,adipose tissue, heart, and liver [37]. Through the regulation of NF-κB, SIRT1 could be involved in inhibiting the expression of genesimplicated in inflammation and aging [40,41]. This effect of SIRT1on cytokines could bring additional benefits in the context ofneurodegenerative and cardiovascular diseases.

3. Sirtuin 1 activation and prevention of age-related diseases

3.1. Antiaging effects

Aging is a natural process that produces deleterious changes in alltissues of the organism. One leading theory about the causes of agingsuggests that oxidative stress plays a major role in pathogenesis [40].Oxidative stress induces intracellular cell damage that affects allbiological components, including DNA, lipids, sugars, and proteins[41–44]. Therefore, the imbalance between intracellular ROS andantioxidant defence mechanisms results in harmful oxidative stress.One of themost widely considered strategies for preventing aging andfor treating age-related diseases is the use of natural antioxidantagents [42]. The development of specific antiaging treatments hasattracted considerable attention in recent years. Antiaging medicinesinclude ginkgo biloba extracts, RESV, melatonin, quercetin, catechin,curcumin, carotenoids, and flavonoids [1,2]. The molecular mechan-isms by which these compounds may act as antiaging drugs are notfully understood, and obviously, clinical trials demonstrating theeffects of these compounds on life span in humans have not beencarried out. RESV has such antioxidant effects, and indeed, this wasthe first mechanism described to explain its pharmacologicalproperties [2]. As already noted, its strong antioxidant propertieshave been associated with the beneficial effects of red wineconsumption in protecting against coronary heart disease [45,46]. Inaddition, RESV may target mitochondrial systems that are involved inenergy and free radical metabolism and could stabilise mitochondrialfunction under stress conditions [17,18]. In fact, RESV was found toattenuate mitochondrial ROS production in cultured human coronaryarterial endothelial cells. It also increased the expression of twoantioxidant enzymes: manganese superoxide dismutase (MnSOD)and glutathione (GSH) [18]. Moreover, RESV has been shown toincrease plasma antioxidant capacity and decrease lipid peroxidation.However, a study using low-dose RESV demonstrated that thebeneficial effects on aging are not mediated through its antioxidantproperties, since a diet with a low dose of RESV (4.9 mg/kg/day) wasused [47]. Taken together, therefore, these studies confirm that theantiaging effects of RESV are not due to its antioxidant properties.Thus, the life span extension by RESV could result from its putativesirtuin-activating properties.

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As discussed above, RESV has been reported to increase life spanin nonmammalian species such as worms and flies through Sir 2activation. Likewise, this effect was also observed in N. furzeri, a veryshort-lived seasonal fish. In fact, RESV supplementation extends themaximum life span of this fish species by up to 59% [48]. RESV alsoimproved locomotor activity and cognitive performance in thefish anddecreased aggregated proteins in elderly fish brains [48]. However,there are controversies about the effects of RESV in reference to theactivation of SIRT1 (Sir2). Regarding this aspect, Borra et al. [11]suggest that RESV activated SIRT1 but not others who have studiedsuch as Sir2 and SIRT2. Also, Kaeberlein et al. [13] questioned the roleof RESV on the activation of SIRT1 and suggest other potentialpathways at themitochondriamodulated by RESV. Furthermore, therearemajor problemswhen trying to extrapolate the results from in vitroto in vivo experiments [11,13].

In vitro data demonstrate that sirtuin activation by RESV involvesFOXO3A regulation, promoting its localisation in the nucleus andinitiating FOXO-dependent gene expression [49]. Although thismechanism has not been demonstrated in humans, different studiescarried out on specific isolated populations with a high prevalence oflongevity evidenced an association between increased longevity andFOXO3A gene expression [50–53]. The three independent studieswere conducted in an ethnic Japanese population in Hawaii, anisolated Italian population from a region to the southeast of Naples,and a population of German centenarians. The data from these studiescollectively demonstrate the association of this gene with the abilityto attain an exceptionally old age [50–53].

Current research is therefore focused on understanding themechanisms involved in the ability of RESV to increase the activityof SIRT1 and on the intracellular pathways that are activated orregulated by SIRT1 [2,17,18]. One theory is that RESV might alter thesubstrate specificity of SIRT1 in vivo [17,18]. At all events, the questionof whether enhanced SIRT1 activity and/or RESV treatment increasemammalian life span, and therefore their potential as antiagingtreatments, remains unresolved [2]. Indeed, antiaging effects arelikely to be more complicated to understand, since more than onebiochemical pathway may be involved.

In addition to the sirtuins, several other proteins are now known toinfluence longevity, energy use, and the response to caloric restriction[53]. These potential pathways, which are also involved in antiaging,include the receptors for insulin, for another hormone called IGF-1, andfor a protein of increasing interest that is known as TOR (“target ofrapamycin”) [54,55]. Rapamycin is an antimicrobial that was recentlyfound to extend life span significantly, even when given to mice at anadvanced age [54]. Since TOR is involved in the response to caloricrestriction, it has been hypothesised that rapamycin may extend life viathis pathway.Moreover, by acting on adenosinemonophosphate (AMP)-activated protein kinase (AMPK), RESV could inhibit mTOR activation[55]. Further studies are required todemonstrate thepotential interactionbetween RESV andmTOR activation [18]. Another interesting hypothesisis that aging is associated with inhibited autophagy. Suppression ofautophagy in neural cells is also responsible for the appearance ofneurodegenerative disease in mice [56,57]. Recent data indicate that theactivation of SIRT1 (by the pharmacological agent RESV) triggersautophagy in nematode cells [57]. Moreover, the effects of Sirtuin-1activators are lost in autophagy-deficient C. elegans [57]. Therefore,autophagy might be involved in the antiaging properties of RESV.

3.2. Neurodegenerative diseases

Neurodegenerative diseases are closely related with aging [58].Moreover, one of the most common changes in the aging brain ismemory loss. Therefore, the main purpose of antiaging drugs is toprevent the decline in learning and memory caused by aging. In thiscontext, it should be noted that brain SIRT1 is mainly localised in thehippocampus, cerebellum and cerebral cortex [2,58].

3.2.1. Alzheimer's disease (AD)A link between SIRT1 and AD is increasingly evident. Decreased

SIRT1 expression was found in patients with AD, and this decreasewas correlated with β-amyloid deposits [59]. Several lines of evidencesupport the involvement of SIRT1 in AD:

a) Sirt1 is activated by caloric restriction and plays a role in extendinglife span [60]. As such, it may have a beneficial effect on ADneuropathology and could be of use in the development oftherapeutic dietary strategies in AD and, possibly, other neurode-generative disorders. Moreover, experimental studies of caloricrestriction in AD mouse models showed improved conditioningmemory and reductions in both caspase-3 activation and astrogliosis(two markers of the apoptotic process) [60,61]. Likewise, tauhyperphosphorylation was reduced through the decrease in CDK5,a cyclin-dependent kinase that is involved in neurodegeneration.Therefore, it could be hypothesised that a reduction of caloric intakemay be a preventive measure in populations at high risk for AD, incombination with specific AD drugs. However, a hypocaloric diet isan unpopular strategy. Patel et al. [60] showed that short-term CRsubstantially decreased the accumulation of β-amyloid plaques intwo AD-prone APP/presenilin transgenic mice lines. Furthermore, itwas demonstrated that CR reduced the content of β-amyloid in thetemporal cortex of squirrel monkeys, this being inversely correlatedwith SIRT1 protein concentrations in the same brain region [62].

b) The potential beneficial effects of RESV in AD are also supported byevidence suggesting that moderate wine consumption is associ-ated with a lower incidence of AD and improved neuropathologyin a mouse model of the disease [63]. In vitro and in vivo studieshave investigated the neuroprotective molecular mechanismsassociated with RESV [64–67]. β-Amyloid peptide induces celldeath through apoptosis in many cell types via reactive oxygenspecies, but this effect was blocked by RESV [65,68]. In addition,RESV reduced the secretion of the β-amyloid peptide in twoAPP695-transfected cell lines (HEK293 and N2A) [77]. This effectcould be due to an increase in β-amyloid peptide degradation.Recently, it has been suggested that RESV is an inhibitor ofacetylcholinesterase, and this new pharmacological effect lendssupport to the potential application of RESV in AD [69].Interestingly, overexpression of SIRT1 and/or RESV treatmentmarkedly reduced the NF-κB signalling stimulated by β-amyloidand showed strong neuroprotective effects [68]. This finding isconsistent with the known role of SIRT1 in modulating NF-κBactivity in AD.

c) In a recent study using a transgenic mouse which overexpressesthe CDK5 activator p25, used as a model of AD, Kim et al. [70]demonstrated that up-regulation of SIRT1 showed neuroprotectiveeffects. Likewise, it has been reported that SIRT1 is upregulated inmouse models of AD. Moreover, in transgenic mouse, a model ofAD and tauopathies, RESV showed beneficial effects, reducingneurodegeneration in the hippocampus and preventing learningimpairment [71]. Accordingly, an increase in SIRT1 activationstrongly indicates that it may be a suitable target in the treatmentof AD.

3.2.2. Parkinson's disease (PD)PD is neuropathologically characterised by the selective and

progressive degeneration and loss of dopaminergic neurons in thesubstantia nigra pars compacta, as well as the frequent presence ofintraneuronal inclusions called Lewy bodies, which are mainlycomposed of α-synuclein [72]. Although the precise mechanismunderlying the neurodegeneration has yet to be determined, there is agrowing body of evidence that both genetic and environmentalfactors contribute to the disease. In particular, mitochondrialdysfunction has been considered one of the most important factorsin the pathogenesis of PD, and it is now more than twenty years since

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it was discovered that the administration of 1-methyl-4-phenyl-1,2,3,4-tetrahydropyridine (MPTP) causes parkinsonism in bothlaboratory animals and humans [73]. This activation occurs throughthe 1-methyl-4-phenyl pyridinium ion (MPP+), the active metaboliteof MPTP, which inhibits complex I in the chain of mitochondrialelectron transfer [74]. Moreover, complex I inhibition is known to be amajor source of free radicals [74,75].

RESV has shown beneficial effects in PD models [74]. For example,the administration to adult mice of a diet containing RESV preventedthe loss of dopaminergic neurons and protected against MPTPneurotoxicity [74]. However, as mentioned above the main mecha-nism involved in the neuroprotective effects of RESV could be its freeradical scavenging properties [75]. Consequently, the role of sirtuinsin RESV neuroprotection requires further clarification. In this context,Okawara et al. [76] used organotypic midbrain slice cultures toinvestigate the neuroprotective effects of RESV on dopaminergicneurons after treatment with the neurotoxin MPP+. They demon-strated that RESV and quercetin, another SIRT-activating compound,prevented the dopaminergic neuronal loss induced by MPP+,concluding that both antioxidant and SIRT-activating activities areinvolved in the neuroprotective effects of RESV in dopaminergicneurons. In another study performed in cerebellar granule cells,sirtinol (a SIRT-1 inhibitor) did not prevent neuroprotective effects ofRESV [74]. Hence, Alvira et al. [75] suggested that SIRT1 activation isnot involved in the neuroprotective effects of RESV against MPP+

cytotoxicity. Instead, they proposed that the antioxidant effects of thiscompound are responsible for the neuroprotection offered by RESVagainst MPP+ [76].

Similarly, Albani et al. [77] demonstrated in SK-N-BE neuroblastomacells that RESV showed neuroprotective effects against the toxicitytriggered by hydrogen peroxide or 6-hydroxydopamine (6-OHDA)through a SIRT1-independent mechanism. Neuroprotection was pre-vented bypharmacological inhibition using sirtinol and by siRNAdown-regulation of SIRT1 expression. In a model of PD using transgenicDrosophila, flies that had been treated with an extract prepared fromwhole grape (Vitis vinifera) exhibited a significant extension in averagelife span and protection from the enzyme activities of themitochondrialrespiratory electron transport chain (complexes I and II). The beneficialeffects of this extract could be mediated by its antioxidant properties[78].

Accordingly, RESV could be an interesting candidate in the treat-ment of PD, although probably only on the basis of its antioxidantproperties. At present, it remains to be clarified whether RESV mightoffer neuroprotection in PD through the activation of SIRT1.

3.2.3. Amyotrophic lateral sclerosisAmyotrophic lateral sclerosis (ALS) is an adult-onset neurodegen-

erative disease characterised by the selective vulnerability of motorneurons in the spinal cord, brainstem, and motor cortex [82,83]. Itcauses progressive muscle weakness, atrophy, paralysis, and bulbardysfunction, and in most cases, death occurs within 3–5 years ofdisease onset. The cause of the disease is unknown, being classified assporadic ALS in 90% of cases and genetic ALS in the remaining 10%.ALS-causingmutations have been identified in several genes, of whichthe best described is the mutation of Cu/Zn superoxide dismutase(SOD1), which is responsible for approximately 20% of familial cases[81]. Thus, oxidative stress is involved in the pathogenesis of ALS andexacerbates other mechanisms that contribute to the neurodegener-ative process in this disease.

The mechanisms involved in motor neuron degeneration aremultifactorial and complex. Nonetheless, mitochondrial dysfunctionand neuroinflammation have been implicated in ALS pathogenesis.Recent studies suggest that RESV acting on SIRT1 activation offers invitro protection against the cell line SOD1G93A, which has a mutantsuperoxide dismutase that causes familial ALS [70]. Moreover, RESVwas very effective at rescuing NSC34motor neuron cells expressing an

ALS-associated mutation of superoxide dismutase 1 from cell death[81].

3.3. Cardiovascular diseases

Red wine consumption is associated with a lower incidence ofcardiovascular diseases, and it has been hypothesised that theprotective effects of red wine in heart diseases are mediated by RESV[82]. Possible explanations for the cardioprotective effect of RESVinclude the prevention of platelet aggregation, COX-1 inhibition,activation of nitric oxide (NO)/cyclic guanosine, or the antioxidantproperties of this compound, among others. In addition, RESV preventsoxidation of LDL by scavenging reactive oxygen species [83–85].However, recent studies suggest that SIRT1 activation induces theregulation of eNOS and that the nitric oxide which is generatedincreases SIRT1 expression [83–87]. Consequently, there is positivefeedback between the two enzymes and this contributes to vasodila-tation and other beneficial effects of RESV in the heart. Overexpressionof SIRT1 also induces eNOS in cultured rat aortas [85]. Moreover, SIRT1exerts a protective effect via FOXO1 deacetylation that regulatesvasodilatation and promotes vascular health [83]. Studies in culturedhuman coronary arterial endothelial cells have demonstrated thatRESV shows beneficial effects in terms of increasing mitochondrialmass, upregulating the protein expression of electron transport chaincomponents and mitochondrial biogenesis factors.

3.4. Type 2 diabetes

This disease is also associated with aging. Published experimentaldata demonstrate that RESV can protect mice against diabetes anddiet-induced obesity [87–90]. As a result, it was hypothesised thatSIRT1 may act as a regulator of energy andmetabolic homeostasis andmight even regulate metabolic diseases such as insulin resistance.Thus, through SIRT1 activation, RESV ameliorated glucose homeostasisand insulin sensitivity in tissues such as liver, muscle, and fat [88,89].The mechanisms by which SIRT1 decreases insulin resistance andimproves glucose and lipid homeostasis are mainly associated withPGC-1γ, PPARγ, and FOXO-1 [87]. Glucose production in the liver isregulated through the activation of PGC-1α. Consequently, SIRT1 actsas a modulator of PGC-1α in regulating glucose homeostasis,gluconeogenic genes, andhepatic glucose output throughPGC-1γ [91].

FOXO-1 regulates hepatic glucose production and stimulation ofβ-cell proliferation in insulin-resistant mice. Furthermore, FOXO-1and PGC-1γ interact in insulin-regulated gluconeogenesis. SIRT1binds FOXO-1, decreases its acetylation, and inhibits its transcriptionalactivity [89]. Thus, increasing SIRT1 activity in pancreatic β cells canlead to enhanced β-cell function and provide beneficial effects onglucose homeostasis in the process of aging.

Furthermore, the SIRT1 protein may, through regulation of theacetylation level of insulin receptor substrate 2 (IRS-2) proteins, directlyregulate insulin-induced IRS-2 tyrosine phosphorylation [88]. Afterphosphorylation, IRS proteins further transmit insulin signalling todownstream events, mainly through two kinase cascades, themitogen-activated protein kinase cascade and the phosphatidylinositol 3-kinase-Akt cascade [90].

Also in this context, adiponectin is known to be secreted byadipose tissue in response to metabolic effectors to sensitise the liverand muscle to insulin. Insulin resistance could therefore be due to areduction in the circulating levels of adiponectin that is usuallyassociated with obesity. Interestingly, adiponectin secretion isregulated by SIRT1 [88–91].

3.5. Osteoporosis

Osteoporosis mainly affects elderly women, and age-relateddeficiency of osteoblast differentiation is one well-known pathogenic

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mechanism [92]. The treatment of this disease aims to promoteosteoblast differentiation to enhance bone formation. PPARγ is animportant regulator of osteoblast differentiation and SIRT1 activationis involved in the regulation of PPARγ [93]. In mesenchymal stemcells, RESV increases osteoblast differentiation and, consequently,could be considered as a means of treating osteoporosis through theactivation of SIRT1 [93,94].

4. SIRT1 activators

Based on the observations that SIRT1 activation by RESV has awidespectrum of beneficial effects in cardiovascular, metabolic andneurodegenerative diseases, there has been increasing interest indeveloping more potent SIRT1 activators for the treatment of theseaging-associated diseases [95–100] (Figs. 2 and 3). Although RESV isan interesting molecule because of its low toxicity in humans, as adrug it lacks specificity for SIRT1. To solve this problem, more specificand selective SIRT1 activators have been developed with the aim oftreating diseases or conditions that are regulated by this protein [96–99]. Compounds that activate sirtuin 1 can be classified into twogroups: those of natural origin, which are phytochemical compounds(polyphenols) such as RESV, buteine, quercetine, and myricetin; andnonrelated synthetic compounds. Natural compounds only activateSIRT1 at high concentrations because of their lower potency [97].Experiments with polyphenols have mainly been performed in HT29cells and HeLa cells [17]. In studies carried out using S. cerevisiae, thecompounds cerebutein, fisetin, and RESV significantly increasedaverage life span by 31%, 55%, and 70%, respectively, and this effect

Fig. 2. Chemical structures of resveratrol and n

was mediated by sir2 [97]. However, although quercetin andpiceatannol have a marked effect on SIRT1 activity, they did notproduce significant effects on life span. Interestingly, RESV effectswere abolished in the analogous SIRT1 knockdown model, indicatingthat the antiaging properties of RESV are mediated through SIRT1activation. With respect to the polyphenolic compounds, it has beenreported that quercetin induced apoptosis and inhibits cell prolifer-ation in tumor cells [3,6,7].

Since natural compounds did not show high activity on SIRT1,more potent compounds with a greater substrate-binding affinity forSIRT1 have been synthesised. Yang et al. [97] developed stilbenederivatives, with certain modifications to their chemical structure,which showed higher activity in comparison with RESV. Thesecompounds were found to prolong yeast life span to the same or agreater extent than RESV. Nayagam et al. [96] described theidentification and in vitro characterisation of quinoxaline derivativecompounds, which are SIRT1 activators. In a human leukaemia cellline, these compounds showed anti-inflammatory activity, as mea-sured by TNFα release after stimulation with lipopolysaccharide.Moreover, some of these new compounds are ten times more potentthan RESV at inhibiting TNFα. They also inhibit lipid accumulation inadipocytes and thus have a potential therapeutic application in thetreatment of obesity and type 2 diabetes [96].

The most comprehensive studies conducted to develop newactivators of SIRT1 are thosebyapharmaceutical biotechnology companycalled Sirtris Pharmaceuticals [95,100,102]. Using a high-throughputscreeningmethodology, they discovered novel selective SIRT1 activators.These newly synthesised compounds are potent small-molecule

atural compounds that are sirtuin agonist.

Fig. 3. Chemical structures of synthetic compounds that are more selective and potent sirtuin agonist.

746 A. Camins et al. / Biochimica et Biophysica Acta 1799 (2010) 740–749

activators of SIRT1 that are structurally unrelated to natural polyphenols.SRT2183, SRT1460, SRT1720, and SRT501 are the most representativecompounds from this series. To evaluate the activity of these compounds,two different parameters were used: the concentration of compoundrequired to increase enzyme activity by 50% (EC1.5) and the maximumpercentage activation achieved at the highest dose tested. The SIRT1activators exhibited nanomolar- to low-micromolar potency in vitro,yielding a range of maximum activation (MA) above 250% whenmeasuring functional activity in a SIRT1 cell-based deacetylation assay(RESV EC1.5=46.2 μM and MA=201%; SRT1460 EC1.5=2.9 μM andMA=447%; SRT2183 EC1.5=0.36 μM and MA=296%; SRT1720EC1.5=0.16 μM and MA=781%) [95,102]. In addition to the evaluationof biological activity, researchhas also showntheseor related compoundsto have several beneficial effects. For instance, the effects of SRT647 andSRT501 were evaluated in retinal ganglion cells. In mice, the intravitrealadministration of both compounds prevented optic neuritis [101]. SinceRESV demonstrated beneficial effects on insulin resistance in diet-

induced obesity in mice, the therapeutic efficacy of SRT1720 wasevaluated with respect to the treatment of type 2 diabetes and othermetabolic disorders [102]. This was examined using different models, inboth diet-induced obesity and genetically determined strains (the obesemice Leo ob/ob and in Zucker fa/fa rats). In all these models, SRT1720improved the metabolic parameters related to glucose metabolism, andthis led to several clinical trials.

The efficacy of SRT501 has been evaluated in patients with type 2diabetes. Phase II clinical trials indicate that this drug is well toleratedand safe for humans at oral doses of 1.25 or 2.5 g twice daily during a28-day treatment. At the higher dose, there is a significant decrease inglucose levels compared to placebo. Moreover, a phase II study incancer patients has been started (see Table 1 for details of clinicaltrials using SIRT1 activators).

SRT2104 has also been evaluated in phase I trials in healthyvolunteers and has demonstrated both safety and tolerability. In onestudy, the effects of oral doses of SRT2104 on several interleukins

Table 1Clinical trials with drugs that act by activating the SIRT1.

Disease Drug ClinicalTrials.govIdentifier

Status Start date tocompletion date

Sponsor/Collaborators Phase Observations

Healthy adults Resveratrol NCT00721877 Completed Aug 2008 University of Arizona/Natl. Cancer Institute

I Effect of resveratrol on CYPenzymes, phase II metabolism.Safety assessment of resveratrol

SRT2104 NCT00920660 Completed Apr 2009 to Jun 2009 GlaxoSmithKline I Effects on several biomarkersNCT00933062 Completed Mar 2009 to May 2009 GlaxoSmithKline INCT00933530 Completed May 2008 to Nov 2008 GlaxoSmithKline I Dose-escalation clinical studyNCT00937872 Completed Nov 2008 to Dec 2008 GlaxoSmithKline INCT00938275 Completed Jan 2009 to Mar 2009 GlaxoSmithKline I Effect of food and gender o

pharmacokineticsNCT00964340 Recruiting Oct 2009 to Jan 2010 GlaxoSmithKline I Evaluation of exercise tolerance

SRT2379 NCT01018628 Recruiting Dec 2009 to Jul 2010 GlaxoSmithKline I Dose-escalation clinical studyAlzheimer's disease Resveratrol NCT00678431 Recruiting Jan 2008 to Jun 2011 Dept. Veterans Affairs/

Alzheimer's AssociationIII Coadministered with glucose and

malate as a dietary supplementNCT00743743 Not yet open Sept 2008 to Dec 2010 Medical College of

WisconsinIII

Cancer (solid tumor) Resveratrol NCT00098969 Completed Sept 2004 University of Michigan/Natl. Cancer Institute

I Fast elimination of resveratroland its metabolites (1)

Cognitive function andcerebral blood flow

Resveratrol NCT01010009 Completed Jun 2008 to Mar 2009 Northumbria University – Resveratrol as a dietarysupplement

Colon cancer Resveratrol NCT00256334 Recruiting Jul 2005 to Dec 2009 University of California I-II Testing for Wnt signalingNCT00433576 Completed Dec 2006 University of Michigan/

Natl. Cancer InstituteI

NCT00578396 Ongoing Jan 2008 to Jun 2010 University of California/Gateway for Cancer Research

I Dietary supplement, grape-derived

SRT501 NCT00920803 Completed Aug 2008 to Nov 2009 GlaxoSmithKline IDiabetes mellitus, type 2 Resveratrol NCT01038089 Not yet open Jan 2010 to Dec 2010 Boston University – Dietary supplement

SRT2104 NCT00937326 Recruiting Jul 2009 to Mar 2010 GlaxoSmithKline IINCT01018017 Not yet open Feb 2010 to Oct 2010 GlaxoSmithKline IINCT01031108 Not yet open Feb 2010 to Jan 2010 GlaxoSmithKline I

Metabolic syndrome Resveratrol NCT00654667 Recruiting May 2007 University of California IIMultiple myeloma SRT501 NCT00920556 Recruiting Mar 2009 to Dec 2010 GlaxoSmithKline II Alone or in combination with

bortezomibObesity Resveratrol NCT00998504 Recruiting Oct 2009 to Jul 2010 Maastricht University/DSM

Nutritional Products, Ltd– Dietary supplement. Effects on

fat oxidationObesity, metabolic syndrome,diabetes, aging

Resveratrol NCT00823381 Recruiting Jan 2009 to Jan 2010 Washington University/DSMNutritional Products, Inc.

– Dietary supplement. Effects onskeletal muscle gene expression

Sepsis SRT2104 NCT01014117 Recruiting Dec 2009 to Jun 2010 GlaxoSmithKline ISkeletal muscle atrophy SRT2104 NCT01039909 Not yet open Jan 2010 to Sept 2010 GlaxoSmithKline I

747A. Camins et al. / Biochimica et Biophysica Acta 1799 (2010) 740–749

were compared to prednisolone in whole blood of healthy adultsubjects. Moreover, a phase II clinical trial in type 2 diabetic humansubjects will be finished in 2010. This compound is also under study ina phase I trial in elderly patients. SRT2379 is another compound that isin phase I to evaluate safety and pharmacokinetics at different dosesin healthy male volunteers.

Recently, Bemis et al. [96] synthesised oxazolo [4,5-b]pyridinederivatives, which were identified as novel activators of SIRT1 using ahigh-throughput screening approach. Some compounds, specificallybenzimidazoles, were found to be potent activators of SIRT1, with aneight-fold increased activity (EC1.5=0.4 μM). However, no physio-logical studies with these compounds have yet been reported.

Mai et al. [103] have synthesised dihydropyridine derivatives, whichare SIRT1 activators. Some of these new compounds had a C1.5 of 1 μMand 35 μM. They also showed a protective effect against senescence inhuman mesenchymal cells and improved mitochondrial function inmurineC2C12myoblasts, these effects beingmediated throughPGC-1α.

Vu et al. [95] recently evaluated novel SIRT1 activators, specificallyimidazol-[1,2-b]thiazole derivatives, some of which had anMA of 781%and an EC1.5 of 0.16 μM. Moreover, the most potent activator was alsotested in mice and rats to determine its pharmacokinetic profile. Thiscompound was not only the most potent but was also effective whenadministered orally in the same models of type 2 diabetes, the geneticobese mice (Leo ob/ob) and Zucker fa/fa rat, where it reduced glucoselevels.

Note that a recent study the direct activation of SIRT1 by RESV andspecific SIRT1 activators was questioned [104]. In this work, these

authors suggest that SIRT1720 did not have any beneficial metaboliceffect for the treatment of type 2 diabetes.

5. Conclusions and future perspectives

In the last decade, SIRT1 has become an interesting and promisingtarget in terms of its influence on both life span and age-relateddiseases. Research on the modulation of SIRT1 expression or itsactivity by using sirtuin-activating compounds has particularlyfocused on RESV, which has shown different beneficial effects inthese diseases. SIRT1 activation plays a key role as a modulator ofmetabolism and might provide important new targets for thetreatment of obesity and type 2 diabetes. The evidence that SIRT1 isdecreased in AD patients and that this is accompanied by anaccumulation of tau protein suggests that SIRT1 also plays a key rolein AD pathology. The role of SIRT1 in neurodegenerative diseases isthus a further area of interest. Interestingly, inmice, low doses of RESVexert the same beneficial effects as caloric restriction. Althoughdietary restriction is not an appropriate strategy for the treatment ofage-related diseases, its beneficial effects could be obtained via SIRT1activators. Pharmacological development of active small moleculeligands is therefore essential to validate sirtuins as drug targets. Itremains to be investigated whether new activators of SIRT1 couldhave additional beneficial effects on mitochondria, which couldexplain the protection against the decreased activity of this organellein age-related diseases. Obviously, further studies in humans are

748 A. Camins et al. / Biochimica et Biophysica Acta 1799 (2010) 740–749

needed to define the exact role of sirtuins in the pathophysiology ofhuman diseases.

Acknowledgments

This study was supported by grants from the Spanish Ministry ofEducation and Science SAF-2009-13093, the Fondo de InvestigaciónSanitaria, and the Instituto de Salud Carlos III (PI080400 and PS09/01789).We thank the Catalan Government (Generalitat de Catalunya)for supporting the research groups (2009/SGR00853) and Fundació laMarató TV3 (063230). 610RT0405was fromPrograma Iberoamericanode Ciencia y Tecnologia para el Desarrollo (CYTED). Ester Verdaguerholds a “Beatriu de Pinós” postdoctoral contract, awarded by theGeneralitat. We thank the University of Barcelona Language Servicesfor revising the manuscript.

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