GSK2245840

Sirtuin activators: Designing molecules to extend life span

Abstract

Resveratrol (RESV) exerts important pharmacological effects on human health: in addition to its beneficial effects on type 2 diabetes and cardiovascular diseases, it also modulates neuronal energy homeostasis and shows antiaging properties. Although it clearly has free radical scavenger properties, the mechanisms involved in these beneficial effects are not fully understood. In this regard, one area of major interest concerns the effects of RESV on the activity of sirtuin 1 (SIRT1), an NAD+-dependent histone deacetylase that has been implicated in aging. Indeed, the role of SIRT1 is currently the subject of intense research due to the antiaging properties of RESV, which increases life span in various organisms ranging from yeast to rodents. In addition, when RESV is administered in experimental animal models of neurological disorders, it has similar beneficial effects to caloric restriction. SIRT1 activation could thus constitute a potential strategic target in neurodegenerative diseases and in disorders involving disturbances in glucose homeostasis, as well as in dyslipidaemias or cardiovascular diseases. Therefore, small SIRT1 activators such as SRT501, SRT2104, and SRT2379, which are currently undergoing clinical trials, could be potential drugs for the treatment of type 2 diabetes, obesity, and metabolic syndrome, among other disorders. This review summarises current knowledge about the biological functions of SIRT1 in aging and aging-associated diseases and discusses its potential as a pharmacological target.

1. Introduction

Once a country’s population has achieved the goals of a good quality of life in which basic needs are met, and where this is combined with access to good health service and drugs to successfully treat most diseases, the next step is to increase life expectancy [1]. To this end, it becomes necessary to develop drugs that act on different organs and tissues of the body to preserve their functioning. One way of accomplishing this would be through the development of drugs with antiaging 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 find molecules with such a variety of antiaging properties. However, nature has the ability to reveal surprising antiaging tools. For instance, it was observed that the French population, through the consumption of mild to moderate amounts of red wine, showed reduced mortality from coronary heart disease [2–4]. Researchers subsequently realised that resveratrol (RESV; 3,5,4′-trihydroxystilbene), a naturally occurring polyphenolic compound found in red wine, was responsible for the beneficial effects on the cardiovascular system [3]. RESV is the main nonflavonoid polyphenol found in black grapes and is characterised as a phytoalexin [3]. It is produced by a variety of plants in response to stress as protection against fungal colonisation [4]. Initial attempts to ascertain the mechanisms involved in the cardioprotective effects of RESV suggested that the latter were mainly due to its strong antioxidant properties [5–7].

However, the antioxidant properties of RESV alone cannot explain the pharmacological effects of this compound, which, in recent years, has been shown to have anti-inflammatory, antitumour, cardioprotective,and antiaging properties (Fig. 1) [7]. Therefore, there must be additional pathways activated by RESV that could explain its antiaging and beneficial coronary effects. Interestingly, a protein called SIRT1, which is an NAD+-dependent protein deacetylase, may be the target for RESV and responsible for its physiological actions.

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 with antiaging properties. Besides an increase in NF transcription factor may explain, among others, the neuroprotective properties of SIRT1. SIRT1 protects pancreatic cells and muscle cells against stress-induced apoptosis by increasing activity of the forkhead protein FOXO1. In the liver, SIRT1 deacetylases the coactivator PGC-1α, thereby increasing the expression of genes for gluconeogenesis. In the muscles, the effect of SIRT1 on FOXO1 increases mitochondrial biogenesis and insulin secretion.

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

Seven mammalian sirtuins (SIRT1-7) have been characterised, and they have different localisations and functions inside the cell [4]. At present, the most widely studied and best known is SIRT1. The development of a transgenic mouse for SIRT1 has revealed that in mammalians 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, recent data demonstrate that CR extends life span and delays the onset of age-associated diseases such as cardiovascular disease, cancer, and diabetes, as well as muscle atrophy in nonhuman primates [20–29]. This research provides strong support for the importance of CR in aging and age-associated disease and highlights the need for a better understanding of the pathways involved in CR-improved health. Several studies have demonstrated that CR regulates mammalian SIRT1 in different tissues, specifically by increasing SIRT1 activity [20,27]. Since RESV treatment produces beneficial effects similar to CR in mice, it was hypothesised that SIRT1 activation could be responsible for the antiaging effects of RESV [17]. However, the use of caloric restriction as a therapeutic tool is not a suitable health strategy, hence the need for drugs that mimic the process of CR and therefore selectively activate SIRT1. The synthesis of selective and specific compounds acting on the SIRT1 such as SRT1720, has shown the connection between SIRT1 in CR responses and life span. This compound has several physiological effects through the inhibition of adipogenesis and induction of lipid oxidation which mimics CR.

An in vitro screen for activators of SIRT1 identified RESV as the most potent of eighteen inducers of deacetylase activity [18]. Accordingly, neuroprotective and antiaging effects of RESV might be mediated through SIRT1 activation [2,17,19]. Current research is therefore focused on understanding the mechanisms involved in the ability of RESV to increase SIRT1 activity and on the intracellular pathways that are activated or regulated by SIRT1 [20].

There are several interesting findings in this regard. One was the discovery that RESV extends the life span of S. cerevisiae, Caenorhab- ditis elegans, and Drosophila melanogaster, but only if the gene that encodes SIR2 is present in these organisms [20,26]. RESV also increased the life span of the fish Nothobranchius furzeri, although the target involved in this effect is still unknown [25,27]. Given these findings, however, drugs that activate SIRT1 might also have antiaging and other beneficial actions on metabolism. Indeed, it is hoped that such compounds may be of benefit in the treatment of diabetes and neurodegenerative 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 SIRT1 activators. The synthesis of these new specific compounds may have a potential 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: Epigenetic modulation involves changes in the activity and expression of chromatin that include variations such as methyla- tions and histone modifications [29]. Similarly, many aging-related effects are caused by chromatin changes. Since SIRT1 is localised mainly in the nucleus its physiological actions are partly mediated through its ability to deacetylate nucleosomal histones at specific residues [2]. For instance, aging produces a decrease in the repair of chromatin aberrations, as well as telomere shortening [29–31]. The vast majority 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 and deacetylation of lysine residues [30]. Histone acetylation is essential for the control of chromatin structure and, hence, the regulation of gene expression. Thus, chromatin is a target for Sir2, which, in turn, regulates yeast life span [30]. SIRT1 (Sir2) is involved in the formation of two different forms of heterochro- matin: facultative and constitutive and both play a role in the expression 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 constitutive heterochromatin describes those chromatin regions that, once formed, always are condensed. Sir2 has been shown through processes of deacetylation of histones, particularly H4K16 and H3K9 regulates the formation of facultative chromatin, this being accompanied by aging increase [30–34]. Moreover, one of the chromatin elements that has been implicated in aging is H1, since aging 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 chromatin silencing and repression of transcription.

Interestingly, a recent study demonstrated that SIRT1, through deacetylation of HSF1, could promote chromatin silencing and repression. Thus, SIRT1 favours HSF1 binding to the heat shock promoter Hsp70 and may be a pathway in the regulation of life span [35].

b) Nonhistone substrates of SIRT1 regulation

Once SIRT1 is activated it mediates intracellular responses that promote cell survival, enhance the repair of damaged DNA, and reduce cell division. Moreover, analysis of SIRT1 enzymatic activity has demonstrated that it acts in a different way to other previously described histone deacetylases. Experimental data using purified SIRT1 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 possess two enzymatic activities: the deacetylation of a target protein and the metabolism of NAD+.

Some beneficial effects of SIRT1 are therefore mediated through the regulation of nonhistone cellular substrates, specifically, by deacetylating transcription factors such as the tumour suppressor p53, the FOXO family (also called FKHR, a member of the forkhead family of transcription factors FOXO1, FOXO3, and FOXO4, in which it prevents the nuclear translocation and activation of its targets, such as BIM), and NF-κB. Moreover, deacetylating also the factor Ku70 reduce their ability to trigger apoptosis or induce the expression of their target genes involved in stress protection, cell cycle 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 the expression of the proapoptotic factor Bax [14]. The tumour suppressor protein p53 plays an essential role in the response to a 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 apoptotic process through the activation of BH3-only proteins such as PUMA, NOXA, and BAX [2,14,18]. The antiproliferative effects of RESV are associated with inhibition of cell cycle proteins and induction of p53 in tumour cells. However, in neuronal cells, neuroprotective effects 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 several pathways that regulate cellular life span and thus increase longevity and aging. In addition to the regulation of longevity processes, members of the FOXO family of transcription factors are also involved in regulating other cellular processes such as metabolism; for instance, FOXO1 modulates glucose tolerance in adipose tissue and in the liver and pancreas [36,37]. Similarly, FOXO3a is involved in the prevention of apoptosis activity via the regulation of Bim. Moreover, SIRT1 also deacetylates the nuclear receptor 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 genes implicated in inflammation and aging [40,41]. This effect of SIRT1 on cytokines could bring additional benefits in the context of neurodegenerative 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 all tissues of the organism. One leading theory about the causes of aging suggests that oxidative stress plays a major role in pathogenesis [40]. Oxidative stress induces intracellular cell damage that affects all biological components, including DNA, lipids, sugars, and proteins [41–44]. Therefore, the imbalance between intracellular ROS and antioxidant defence mechanisms results in harmful oxidative stress. One of the most widely considered strategies for preventing aging and for treating age-related diseases is the use of natural antioxidant agents [42]. The development of specific antiaging treatments has attracted considerable attention in recent years. Antiaging medicines include 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 not fully understood, and obviously, clinical trials demonstrating the effects of these compounds on life span in humans have not been carried out. RESV has such antioxidant effects, and indeed, this was the first mechanism described to explain its pharmacological properties [2]. As already noted, its strong antioxidant properties have been associated with the beneficial effects of red wine consumption in protecting against coronary heart disease [45,46]. In addition, RESV may target mitochondrial systems that are involved in energy and free radical metabolism and could stabilise mitochondrial function under stress conditions [17,18]. In fact, RESV was found to attenuate mitochondrial ROS production in cultured human coronary arterial endothelial cells. It also increased the expression of two antioxidant enzymes: manganese superoxide dismutase (MnSOD) and glutathione (GSH) [18]. Moreover, RESV has been shown to increase plasma antioxidant capacity and decrease lipid peroxidation. However, a study using low-dose RESV demonstrated that the beneficial effects on aging are not mediated through its antioxidant properties, since a diet with a low dose of RESV (4.9 mg/kg/day) was used [47]. Taken together, therefore, these studies confirm that the antiaging effects of RESV are not due to its antioxidant properties. Thus, the life span extension by RESV could result from its putative sirtuin-activating properties.

As discussed above, RESV has been reported to increase life span in nonmammalian species such as worms and flies through Sir 2 activation. Likewise, this effect was also observed in N. furzeri, a very short-lived seasonal fish. In fact, RESV supplementation extends the maximum life span of this fish species by up to 59% [48]. RESV also improved locomotor activity and cognitive performance in the fish and decreased aggregated proteins in elderly fish brains [48]. However, there are controversies about the effects of RESV in reference to the activation of SIRT1 (Sir2). Regarding this aspect, Borra et al. [11] suggest that RESV activated SIRT1 but not others who have studied such as Sir2 and SIRT2. Also, Kaeberlein et al. [13] questioned the role of RESV on the activation of SIRT1 and suggest other potential pathways at the mitochondria modulated by RESV. Furthermore, there are major problems when trying to extrapolate the results from in vitro to in vivo experiments [11,13].

In vitro data demonstrate that sirtuin activation by RESV involves FOXO3A regulation, promoting its localisation in the nucleus and initiating FOXO-dependent gene expression [49]. Although this mechanism has not been demonstrated in humans, different studies carried out on specific isolated populations with a high prevalence of longevity evidenced an association between increased longevity and FOXO3A gene expression [50–53]. The three independent studies were conducted in an ethnic Japanese population in Hawaii, an isolated Italian population from a region to the southeast of Naples, and a population of German centenarians. The data from these studies collectively demonstrate the association of this gene with the ability to attain an exceptionally old age [50–53].

Current research is therefore focused on understanding the mechanisms involved in the ability of RESV to increase the activity of SIRT1 and on the intracellular pathways that are activated or regulated by SIRT1 [2,17,18]. One theory is that RESV might alter the substrate specificity of SIRT1 in vivo [17,18]. At all events, the question of whether enhanced SIRT1 activity and/or RESV treatment increase mammalian life span, and therefore their potential as antiaging treatments, remains unresolved [2]. Indeed, antiaging effects are likely to be more complicated to understand, since more than one biochemical pathway may be involved.

In addition to the sirtuins, several other proteins are now known to influence 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, and for a protein of increasing interest that is known as TOR (“target of rapamycin”) [54,55]. Rapamycin is an antimicrobial that was recently found to extend life span significantly, even when given to mice at an advanced age [54]. Since TOR is involved in the response to caloric restriction, it has been hypothesised that rapamycin may extend life via this pathway. Moreover, by acting on adenosine monophosphate (AMP)- activated protein kinase (AMPK), RESV could inhibit mTOR activation [55]. Further studies are required to demonstrate the potential interaction between RESV and mTOR activation [18]. Another interesting hypothesis is that aging is associated with inhibited autophagy. Suppression of autophagy in neural cells is also responsible for the appearance of neurodegenerative disease in mice [56,57]. Recent data indicate that the activation of SIRT1 (by the pharmacological agent RESV) triggers autophagy in nematode cells [57]. Moreover, the effects of Sirtuin-1 activators 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 is memory loss. Therefore, the main purpose of antiaging drugs is to prevent the decline in learning and memory caused by aging. In this context, it should be noted that brain SIRT1 is mainly localised in the hippocampus, 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 decrease was correlated with β-amyloid deposits [59]. Several lines of evidence support the involvement of SIRT1 in AD: a) Sirt1 is activated by caloric restriction and plays a role in extending life span [60]. As such, it may have a beneficial effect on AD neuropathology and could be of use in the development of therapeutic dietary strategies in AD and, possibly, other neurode- generative disorders. Moreover, experimental studies of caloric restriction in AD mouse models showed improved conditioning memory and reductions in both caspase-3 activation and astrogliosis (two markers of the apoptotic process) [60,61]. Likewise, tau hyperphosphorylation 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 intake may be a preventive measure in populations at high risk for AD, in combination with specific AD drugs. However, a hypocaloric diet is an unpopular strategy. Patel et al. [60] showed that short-term CR substantially decreased the accumulation of β-amyloid plaques in two AD-prone APP/presenilin transgenic mice lines. Furthermore, it was demonstrated that CR reduced the content of β-amyloid in the temporal cortex of squirrel monkeys, this being inversely correlated with SIRT1 protein concentrations in the same brain region [62].

b) The potential beneficial effects of RESV in AD are also supported by evidence suggesting that moderate wine consumption is associ- ated with a lower incidence of AD and improved neuropathology in a mouse model of the disease [63]. In vitro and in vivo studies have investigated the neuroprotective molecular mechanisms associated with RESV [64–67]. β-Amyloid peptide induces cell death through apoptosis in many cell types via reactive oxygen species, but this effect was blocked by RESV [65,68]. In addition, RESV reduced the secretion of the β-amyloid peptide in two APP695-transfected cell lines (HEK293 and N2A) [77]. This effect could be due to an increase in β-amyloid peptide degradation. Recently, it has been suggested that RESV is an inhibitor of acetylcholinesterase, and this new pharmacological effect lends support to the potential application of RESV in AD [69]. Interestingly, overexpression of SIRT1 and/or RESV treatment markedly reduced the NF-κB signalling stimulated by β-amyloid and showed strong neuroprotective effects [68]. This finding is consistent with the known role of SIRT1 in modulating NF-κB activity in AD.

c) In a recent study using a transgenic mouse which overexpresses the CDK5 activator p25, used as a model of AD, Kim et al. [70] demonstrated that up-regulation of SIRT1 showed neuroprotective effects. Likewise, it has been reported that SIRT1 is upregulated in mouse models of AD. Moreover, in transgenic mouse, a model of AD and tauopathies, RESV showed beneficial effects, reducing neurodegeneration in the hippocampus and preventing learning impairment [71]. Accordingly, an increase in SIRT1 activation strongly indicates that it may be a suitable target in the treatment of AD.

3.2.2. Parkinson’s disease (PD)

PD is neuropathologically characterised by the selective and progressive degeneration and loss of dopaminergic neurons in the substantia nigra pars compacta, as well as the frequent presence of intraneuronal inclusions called Lewy bodies, which are mainly composed of α-synuclein [72]. Although the precise mechanism underlying the neurodegeneration has yet to be determined, there is a growing body of evidence that both genetic and environmental factors contribute to the disease. In particular, mitochondrial dysfunction has been considered one of the most important factors in the pathogenesis of PD, and it is now more than twenty years since it was discovered that the administration of 1-methyl-4-phenyl- 1,2,3,4-tetrahydropyridine (MPTP) causes parkinsonism in both laboratory animals and humans [73]. This activation occurs through the 1-methyl-4-phenyl pyridinium ion (MPP+), the active metabolite of MPTP, which inhibits complex I in the chain of mitochondrial electron transfer [74]. Moreover, complex I inhibition is known to be a major 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 prevented the loss of dopaminergic neurons and protected against MPTP neurotoxicity [74]. However, as mentioned above the main mecha- nism involved in the neuroprotective effects of RESV could be its free radical scavenging properties [75]. Consequently, the role of sirtuins in RESV neuroprotection requires further clarification. In this context, Okawara et al. [76] used organotypic midbrain slice cultures to investigate the neuroprotective effects of RESV on dopaminergic neurons 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 are involved in the neuroprotective effects of RESV in dopaminergic neurons. In another study performed in cerebellar granule cells, sirtinol (a SIRT-1 inhibitor) did not prevent neuroprotective effects of RESV [74]. Hence, Alvira et al. [75] suggested that SIRT1 activation is not involved in the neuroprotective effects of RESV against MPP+ cytotoxicity. Instead, they proposed that the antioxidant effects of this compound are responsible for the neuroprotection offered by RESV against MPP+ [76].

Similarly, Albani et al. [77] demonstrated in SK-N-BE neuroblastoma cells that RESV showed neuroprotective effects against the toxicity triggered by hydrogen peroxide or 6-hydroxydopamine (6-OHDA) through a SIRT1-independent mechanism. Neuroprotection was pre- vented by pharmacological inhibition using sirtinol and by siRNA down- regulation of SIRT1 expression. In a model of PD using transgenic Drosophila, flies that had been treated with an extract prepared from whole grape (Vitis vinifera) exhibited a significant extension in average life span and protection from the enzyme activities of the mitochondrial respiratory electron transport chain (complexes I and II). The beneficial effects 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 antioxidant properties. At present, it remains to be clarified whether RESV might offer neuroprotection in PD through the activation of SIRT1.

3.2.3. Amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegen- erative disease characterised by the selective vulnerability of motor neurons in the spinal cord, brainstem, and motor cortex [82,83]. It causes progressive muscle weakness, atrophy, paralysis, and bulbar dysfunction, and in most cases, death occurs within 3–5 years of disease onset. The cause of the disease is unknown, being classified as sporadic ALS in 90% of cases and genetic ALS in the remaining 10%. ALS-causing mutations have been identified in several genes, of which the 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 and exacerbates other mechanisms that contribute to the neurodegener- ative process in this disease.

The mechanisms involved in motor neuron degeneration are multifactorial and complex. Nonetheless, mitochondrial dysfunction and neuroinflammation have been implicated in ALS pathogenesis. Recent studies suggest that RESV acting on SIRT1 activation offers in vitro protection against the cell line SOD1G93A, which has a mutant superoxide dismutase that causes familial ALS [70]. Moreover, RESV was very effective at rescuing NSC34 motor 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 of cardiovascular diseases, and it has been hypothesised that the protective effects of red wine in heart diseases are mediated by RESV [82]. Possible explanations for the cardioprotective effect of RESV include the prevention of platelet aggregation, COX-1 inhibition, activation of nitric oxide (NO)/cyclic guanosine, or the antioxidant properties of this compound, among others. In addition, RESV prevents oxidation of LDL by scavenging reactive oxygen species [83–85]. However, recent studies suggest that SIRT1 activation induces the regulation of eNOS and that the nitric oxide which is generated increases SIRT1 expression [83–87]. Consequently, there is positive feedback between the two enzymes and this contributes to vasodila- tation and other beneficial effects of RESV in the heart. Overexpression of SIRT1 also induces eNOS in cultured rat aortas [85]. Moreover, SIRT1 exerts a protective effect via FOXO1 deacetylation that regulates vasodilatation and promotes vascular health [83]. Studies in cultured human coronary arterial endothelial cells have demonstrated that RESV shows beneficial effects in terms of increasing mitochondrial mass, upregulating the protein expression of electron transport chain components and mitochondrial biogenesis factors.

3.4. Type 2 diabetes

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

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

Furthermore, the SIRT1 protein may, through regulation of the acetylation level of insulin receptor substrate 2 (IRS-2) proteins, directly regulate insulin-induced IRS-2 tyrosine phosphorylation [88]. After phosphorylation, IRS proteins further transmit insulin signalling to downstream events, mainly through two kinase cascades, the mitogen- activated protein kinase cascade and the phosphatidylinositol 3-kinase- Akt cascade [90].

Also in this context, adiponectin is known to be secreted by adipose tissue in response to metabolic effectors to sensitise the liver and muscle to insulin. Insulin resistance could therefore be due to a reduction in the circulating levels of adiponectin that is usually associated with obesity. Interestingly, adiponectin secretion is regulated by SIRT1 [88–91].

3.5. Osteoporosis

Osteoporosis mainly affects elderly women, and age-related deficiency of osteoblast differentiation is one well-known pathogenic mechanism [92]. The treatment of this disease aims to promote osteoblast differentiation to enhance bone formation. PPARγ is an important regulator of osteoblast differentiation and SIRT1 activation is involved in the regulation of PPARγ [93]. In mesenchymal stem cells, RESV increases osteoblast differentiation and, consequently, could be considered as a means of treating osteoporosis through the activation of SIRT1 [93,94].

4. SIRT1 activators

Based on the observations that SIRT1 activation by RESV has a wide spectrum of beneficial effects in cardiovascular, metabolic and neurodegenerative diseases, there has been increasing interest in developing more potent SIRT1 activators for the treatment of these aging-associated diseases [95–100] (Figs. 2 and 3). Although RESV is an interesting molecule because of its low toxicity in humans, as a drug it lacks specificity for SIRT1. To solve this problem, more specific and selective SIRT1 activators have been developed with the aim of treating diseases or conditions that are regulated by this protein [96– 99]. Compounds that activate sirtuin 1 can be classified into two groups: those of natural origin, which are phytochemical compounds (polyphenols) such as RESV, buteine, quercetine, and myricetin; and nonrelated synthetic compounds. Natural compounds only activate SIRT1 at high concentrations because of their lower potency [97].

Experiments with polyphenols have mainly been performed in HT29 cells and HeLa cells [17]. In studies carried out using S. cerevisiae, the compounds cerebutein, fisetin, and RESV significantly increased average life span by 31%, 55%, and 70%, respectively, and this effect was mediated by sir2 [97]. However, although quercetin and piceatannol have a marked effect on SIRT1 activity, they did not produce significant effects on life span. Interestingly, RESV effects were abolished in the analogous SIRT1 knockdown model, indicating that the antiaging properties of RESV are mediated through SIRT1 activation. With respect to the polyphenolic compounds, it has been reported 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 for SIRT1 have been synthesised. Yang et al. [97] developed stilbene derivatives, with certain modifications to their chemical structure, which showed higher activity in comparison with RESV. These compounds were found to prolong yeast life span to the same or a greater extent than RESV. Nayagam et al. [96] described the identification and in vitro characterisation of quinoxaline derivative compounds, which are SIRT1 activators. In a human leukaemia cell line, 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 potent than RESV at inhibiting TNFα. They also inhibit lipid accumulation in adipocytes and thus have a potential therapeutic application in the treatment of obesity and type 2 diabetes [96].

The most comprehensive studies conducted to develop new activators of SIRT1 are those by a pharmaceutical biotechnology company called Sirtris Pharmaceuticals [95,100,102]. Using a high-throughput screening methodology, they discovered novel selective SIRT1 activators. These newly synthesised compounds are potent small-molecule activators of SIRT1 that are structurally unrelated to natural polyphenols. SRT2183, SRT1460, SRT1720, and SRT501 are the most representative compounds from this series. To evaluate the activity of these compounds, two different parameters were used: the concentration of compound required to increase enzyme activity by 50% (EC1.5) and the maximum percentage activation achieved at the highest dose tested. The SIRT1 activators exhibited nanomolar- to low-micromolar potency in vitro, yielding a range of maximum activation (MA) above 250% when measuring functional activity in a SIRT1 cell-based deacetylation assay (RESV EC1.5=46.2 μM and MA =201%; SRT1460 EC1.5=2.9 μM and MA= 447%; SRT2183 EC1.5 = 0.36 μM and MA = 296%; SRT1720 EC1.5=0.16 μM and MA=781%) [95,102]. In addition to the evaluation of biological activity, research has also shown these or related compounds to have several beneficial effects. For instance, the effects of SRT647 and SRT501 were evaluated in retinal ganglion cells. In mice, the intravitreal administration of both compounds prevented optic neuritis [101]. Since RESV demonstrated beneficial effects on insulin resistance in diet-induced obesity in mice, the therapeutic efficacy of SRT1720 was evaluated with respect to the treatment of type 2 diabetes and other metabolic disorders [102]. This was examined using different models, in both diet-induced obesity and genetically determined strains (the obese mice Leo ob/ob and in Zucker fa/fa rats). In all these models, SRT1720 improved the metabolic parameters related to glucose metabolism, and this led to several clinical trials.

Fig. 2. Chemical structures of resveratrol and natural compounds that are sirtuin agonist.

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

The efficacy of SRT501 has been evaluated in patients with type 2 diabetes. Phase II clinical trials indicate that this drug is well tolerated and safe for humans at oral doses of 1.25 or 2.5 g twice daily during a 28-day treatment. At the higher dose, there is a significant decrease in glucose levels compared to placebo. Moreover, a phase II study in cancer patients has been started (see Table 1 for details of clinical trials using SIRT1 activators). SRT2104 has also been evaluated in phase I trials in healthy volunteers and has demonstrated both safety and tolerability. In one study, the effects of oral doses of SRT2104 on several interleukins were compared to prednisolone in whole blood of healthy adult subjects. Moreover, a phase II clinical trial in type 2 diabetic human subjects will be finished in 2010. This compound is also under study in a phase I trial in elderly patients. SRT2379 is another compound that is in phase I to evaluate safety and pharmacokinetics at different doses in healthy male volunteers.

Recently, Bemis et al. [96] synthesised oxazolo [4,5-b]pyridine derivatives, which were identified as novel activators of SIRT1 using a high-throughput screening approach. Some compounds, specifically benzimidazoles, were found to be potent activators of SIRT1, with an eight-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, which are SIRT1 activators. Some of these new compounds had a C1.5 of 1 μM and 35 μM. They also showed a protective effect against senescence in human mesenchymal cells and improved mitochondrial function in murine C2C12 myoblasts, these effects being mediated through PGC-1α. Vu et al. [95] recently evaluated novel SIRT1 activators, specifically imidazol-[1,2-b]thiazole derivatives, some of which had an MA of 781% and an EC1.5 of 0.16 μM. Moreover, the most potent activator was also tested in mice and rats to determine its pharmacokinetic profile. This compound was not only the most potent but was also effective when administered orally in the same models of type 2 diabetes, the genetic obese mice (Leo ob/ob) and Zucker fa/fa rat, where it reduced glucose levels.

Note that a recent study the direct activation of SIRT1 by RESV and specific SIRT1 activators was questioned [104]. In this work, these
authors suggest that SIRT1720 did not have any beneficial metabolic effect for the treatment of type 2 diabetes.

5. Conclusions and future perspectives

In the last decade, SIRT1 has become an interesting and promising target in terms of its influence on both life span and age-related diseases. Research on the modulation of SIRT1 expression or its activity by using sirtuin-activating compounds has particularly focused on RESV, which has shown different beneficial effects in these diseases. SIRT1 activation plays a key role as a modulator of metabolism and might provide important new targets for the treatment of obesity and type 2 diabetes. The evidence that SIRT1 is decreased in AD patients and that this is accompanied by an accumulation of tau protein suggests that SIRT1 also plays a key role in AD pathology. The role of SIRT1 in neurodegenerative diseases is thus a further area of interest. Interestingly, in mice, low doses of RESV exert the same beneficial effects as caloric restriction. Although dietary restriction is not an appropriate strategy for the treatment of age-related diseases, its beneficial effects could be obtained via SIRT1 activators. Pharmacological development of active small molecule ligands is therefore essential to validate sirtuins as drug targets. It remains to be investigated whether new activators of SIRT1 could have additional beneficial effects on mitochondria, which could explain the protection against the decreased activity of this organelle in age-related diseases. Obviously, further studies in humans are needed to define the exact role of sirtuins in the pathophysiology of human diseases.

Acknowledgments

This study was supported by grants from the Spanish Ministry of Education and Science SAF-2009-13093, the Fondo de Investigación Sanitaria, 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 la Marató TV3 (063230). 610RT0405 was from Programa Iberoamericano de Ciencia y Tecnologia para el Desarrollo (CYTED). Ester Verdaguer holds a “Beatriu de Pinós” postdoctoral contract, awarded by the Generalitat. We thank the University of Barcelona Language Services for revising the manuscript.

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