Calorie Restriction and Dietary Restriction Mimetics: A ...

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Calorie Restriction and Dietary Restriction Mimetics: A Strategy for Improving Healthy Aging and Longevity Gabriella Testa, Fiorella Biasi, Giuseppe Poli and Elena Chiarpotto* Department of Clinical and Biological Sciences, University of Torino, Italy Abstract: Improvements in health care have increased human life expectancy in recent decades, and the elderly population is thus increasing in most developed countries. Unfortunately this still means increased years of poor health or disability. Since it is not yet possible to modify our genetic background, the best anti-aging strategy is currently to intervene on environmental factors, aiming to reduce the incidence of risk factors of poor health. Calorie restriction (CR) with adequate nutrition is the only non-genetic, and the most consistent non-pharmacological intervention that extends lifespan in model organisms from yeast to mammals, and protects against the deterioration of biological functions, delaying or reducing the risk of many age-related diseases. The biological mechanisms of CR’s beneficial effects include modifications in energy metabolism, oxidative stress, insulin sensitivity, inflammation, autophagy, neuroendocrine function and induction of hormesis/xenohormesis response. The molecular signalling pathways mediating the anti-aging effect of CR include sirtuins, peroxisome proliferator activated receptor G coactivator-1, AMP-activated protein kinase, insulin/insulin growth factor-1, and target of rapamycin, which form a pretty interacting network. However, most people would not comply with such a rigorous dietary program; research is thus increasingly aimed at determining the feasibility and efficacy of natural and/or pharmacological CR mimetic molecules/treatments without lowering food intake, particularly in mid- to late-life periods. Likely candidates act on the same signalling pathways as CR, and include resveratrol and other polyphenols, rapamycin, 2-deoxy-D-glucose and other glycolytic inhibitors, insulin pathway and AMP-activated protein kinase activators, autophagy stimulators, alpha-lipoic acid, and other antioxidants.

Keywords: Calorie restriction, calorie restriction mimetics, aging, longevity. INTRODUCTION Aging is a complex multifactorial process common to nearly all organisms which leads to loss of function, increased vulnerability to disease, and death. Both endogenous (developmental-genetic) [1, 2] and exogenous (stochastic-environmental) causes [3, 4], not mutually exclusive, and both important, have been claimed to be responsible for it. Reports are accumulating on both the genetic determinants of aging, and the major cellular mechanisms involved in the process (among others free-radical-induced damage, mitochondrial dysfunction and increase of oxidative stress, decreasing autophagy, alteration of insulin-like growth factor/growth hormone signalling, alteration of cholesterol and glucose metabolism, telomere shortening). Improvements in health care have increased human life expectancy since the middle of the twentieth century, and consequently the elderly population is increasing more rapidly than other age groups in most developed countries. It is expected that the percent increase of the population aged above 65 will be about 200% by 2050 and about 300% by 2100, but the most astonishing projection is the predicted increase of over-eighties, about 400%, compared to a 16% increase of people aged 15-64 and only a 5% increase of under 15s [5, 6]. Unfortunately, this extended lifespan still means an increase in the years of poor health or disability. Since for the moment it is not possible to modify our genetic background, at the present time the best anti-aging strategy is to intervene on environmental factors. Reducing the incidence of risk factors of poor health, such as dyslipidemia, diabetes, obesity, hypertension, or mental stress, would help to prolong not only life, but healthy life. This review will focus first on calorie restriction (CR), the only non-genetic and the most consistently successful non-pharmacological intervention, extending lifespan in model organisms from yeast to mammals [7, 8] and protecting against the deterioration *Address correspondence to this author at the Department of Clinical and Biological Sciences, University of Torino, Regione Gonzole 10, 10043 Orbassano, (TO), Italy; Tel: +39-0116705423; Fax: +39-0116705424; E-mail: [email protected] 1381-6128/14 $58.00+.00

of biological functions, thus delaying or reducing the onset of many age-related diseases in rodents and also in humans [9]. However, even if CR is beneficial to both lifespan and healthspan, for social, economic and medical reasons it is unlikely that most people would comply with such a rigorous dietary program, in particular in the long term. Therefore, recent research is increasingly aimed at determining the feasibility and efficacy of natural and/or pharmacological CR mimetic (CRM) (or dietary restriction mimetic: DRM) molecules/treatments, which would act by mimicking the positive aspects of CR without lowering food intake, particularly as might be applicable to mid- to late-life periods [10]. The second part of this review will concentrate on several CRM, discussing their emerging applications in prevention/therapy. 1. CALORIE RESTRICTION AND AGING Hippocrates: “Let Food be Your Medicine, and Medicine be Your Food”. The pursuit of the legendary spring of youth has always been the mankind’s dream. The concept that limiting food intake results in health benefits, and may thus extend lifespan, is not recent. Fasting is thought to have been part of the regimen of huntsmen and warriors to prepare for hunting or battle, and was later practiced by athletes and religious practitioners. Fasting, or CR, was part of the religious–medical notions instilled in ancient India [11] and fasting is still a common habit in several different religions, including that performed to different degrees during Lent in the Christian religion, during Ramadan in the Islamic religion, and in the framework of Hindu and Buddhist ascetism. Starting from the mid 1500s reports began to appear on the connection between lifestyle and longevity, starting from “Discorsi della vita sobria” by Luigi Cornaro, first published in 1558 [12], via Francis Bacon (1561–1626) [13] and William Temple (1628–1699) [13], to Benjamin Franklin’s (1706-1790) epigram “Wouldst thou enjoy a long life, a healthy body, and a vigorous mind, ….. labor in the first place to bring thy appetite to reason” [13]. Thus, the con© 2014 Bentham Science Publishers

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cept that limiting food intake is profitable for both health and lifespan seems to have been widespread down the centuries. First of all, however, one must clarify what CR is, so as not to fall into the distortion propagated by Sir William Temple, who wrote that ‘‘Health and long life are usually blessings of the poor, not of the rich.’’ [13]. The CR regimen, as intended in the majority of experimental studies on vertebrates, provides essential nutrients and vitamins, thus avoiding malnutrition, while limiting the total energy intake, usually set at between 10% and 40% less than “ad libitum”(AL)-fed controls, depending on the experimental protocol [14]. Even if research by McCay and colleagues in the 1930s [15, 16] may be considered “seminal” in the field of the relationship between food restriction and extension of lifespan, the first experimental report on CR’s positive effect on the duration of life was in 1917, by Osborne and colleagues in rats [17] . In the same year, Loeb and Northrop showed that it was possible to extend the lifespan of fruit flies by restricting their food intake during the larval stage [18]. Nevertheless, starting from McCay’s research, dietary restriction has been shown to extend both median and maximum lifespan in a wide variety of animal species tested thus far, from invertebrates to larger mammals [9, 19]. Among these, unicellular organisms such as yeast (Saccharomyces cerevisiae) [20], invertebrates

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such as nematodes (Caenorhabditis elegans) [21, 22], insects such as the fruit fly (Drosophila melanogaster) [23, 24], and higher vertebrates such as the mouse (Mus musculus) and the rat (Rattus norvegicus) [25] are the most widely studied “model species”, but similar beneficial effects have also been reported in dogs (Canis domesticus, Labrador Retriever) [26, 27] and in other non-model organisms such as the silkworm (Bombyx mori) [28], the bowl and doily spider (Frontinella pyramitela) [29], the Mediterranean fruit fly (Ceratitis capitata) [30, 31], several rotifer species (Asplanchna brightwelli, Elosa worallii, Brachionus plicatilis) [32-35] and some fishes (guppies, Lebistes reticulatus, Peters [36] and zebrafish, Danio rerio [37]) (Fig. 1). A more complete review on CR’s effects on the longevity of different animal species is given by Le Bourg [38]. The positive effects of CR on such a wide range of different species stimulated the extension of the studies into the effects of dietary restriction to primates, and three separate studies on aging and CR were initiated at the end of the 1980s, using Rhesus monkeys (Macaca mulatta). The National Institute on Aging (NIA) and the Wisconsin National Primate Research Center (WNPRC) studies have been planned to evaluate whether moderate CR (about 30%) could modify lifespan also in this species, while the third study, by the group led by Barbara Hansen at the University of Maryland

Fig. (1). Calorie restriction (CR) and lifespan extension. Starting from McCay’s research in the 1930s [15-16], dietary restriction has been shown to extend both median and maximum lifespan in a wide variety of animal species, from invertebrates to larger mammals, among them yeast, nematodes, spiders, flies, fishes, rodents and dogs. The results thus far obtained in three separate and ongoing studies into the effects of dietary restriction on primates do not yet clarify the degree of CR action on lifespan; however they do show its capability to delay the onset of age-related diseases. The same effect is reported for humans.

Calorie Restriction and Dietary Restriction Mimetics

(UMD), concentrated more on general physiology and health benefits [39]. Rhesus monkeys were selected because of their many affinities to humans (genetics, physiology, endocrinology, neuroanatomy and learning function, and aging flow) [40]. All these studies are still ongoing, so that the exact degree of CR’s action on the animal’s lifespan is not yet known. However, the results obtained thus far are positive. Provisional analyses of the Wisconsin study data show that the frequency of death from an age-related cause in control animals is three fold that of the CR animals. Moreover, while median age at death was approximately 27 years in controls, more than 50% of the CR monkeys were still alive at age 32 [41]. Similar analyses of the NIA data showed a halving of death from age-related causes in CR animals versus controls [42] and in the Maryland study the median survival for the eight CR animals was six years longer than that of controls [43]. However, since the median life expectancy and the maximum lifespan of Rhesus monkeys are 27 and 40 years respectively, it will take some years more to obtain data on CR effect, particularly on the maximal lifespan of these primates. Apart from the tentative prediction of a positive effect of CR also on primate longevity, importantly, actual proof of the effectiveness of CR in this and other species is its demonstrated capability to delay the onset of age-related diseases. In particular, in all three studies, CR reduced body weight, fat mass, and decreased indices of risk for cardiovascular disease and diabetes, such as blood pressure, blood lipids and C-reactive protein, while increasing glucose tolerance, and insulin sensitivity [41, 43-45]. The WNPRC study also showed that also cancer and age-associated muscle mass loss (sarcopenia) were delayed and/or attenuated by CR [41, 46, 47]. On these foundations, if CR does indeed ameliorate health and potentially increase lifespan in non-human primates, it is difficult to give a definitive answer to whether or not it will do the same in humans. For ethical and logistic reasons, it is unthinkable to propose long-term prospective studies whose main objective is to evaluate the effect of CR on human survival. Some useful information on such effects on human health, however, can be acquired from studies involving longer-than-normal-lived persons, including centenarians, and subjects who voluntarily choose to practice CR. Among these, a pionieristic report has suggested a reduction of mortality and morbidity among the residents of a Spanish rest-cure center subjected to a reduced caloric intake on alternate days [48, 49]. However, the most compelling epidemiological indication in favor of the role of CR in extending the human lifespan is represented by the population of Okinawa (Japan) where the mean number of centenarians is 4-5 times that of most industrialized countries. The Japanese Ministry of Health, Labor, and Welfare reports increased mean and maximum lifespan among today’s Okinawans [50, 51], and this enhanced longevity has been associated with a low calorie intake that began in childhood more than 40 years ago, and continued into adulthood (20% CR versus other Japanese people) [52, 53]. A more recent evaluation of the dietary intake and energy expenditure at younger ages, in Okinawan septuagenarians, demonstrated a 11% energy deficit [54]. What is particularly interesting, though, is that the Okinawan longevity benefit has vanished except in older people (aged 65-plus), in coincidence with the shift to “western type” and “standard Japanese” diet from the 1960s [55, 56]. Most corroborative data on the beneficial effects of CR in humans comes from performing dietary restrictions to achieve weight loss in overweight or obese people. For example, changes in the lipid blood pattern have been observed in obese persons subjected to a strict low-calorie diet, including increased HDL cholesterol levels [57]. One consequence is a reduced predisposition to cardiovascular incidents, which are the first cause of death in industrialized countries. There is, however, a group of human studies, reporting upon the anti-aging effect of CR. The first such study to our

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knowledge is that by Vallejo, involving 120 volunteers, of whom 60 received 2300 Kcal/day (controls) and 60 received an average of 1500 Kcal/day, for 3 years (CR: 2300 Kcal or one liter of milk plus 500 g of fruit on alternate days), resulting in about 35% calorie restriction versus controls [48]. A later re-analysis of these results by Stunkard and colleagues [58] showed a decrease in hospital admissions of about 50% in the CR group compared to controls, and a decreased death rate, although it was not statistically significant. More recent and better controlled studies are the Biosphere2 experiment and the study by the TNO Institute of Toxicology and Nutrition, the Netherlands organization for Applied Scientific Research. Biosphere 2 involved 8 healthy subjects (4 men, among whom Dr. Roy Walford, and 4 women, aged from 22-67 years) who lived from 1991 to 1993 in a confined glass and steel ecological laboratory structure, 1.28 hectares in size, that reproduced different earth ecosystems (agro-forest, human/animal habitats, ocean, tropical rainforest, savannah, marsh and desert). It was hermetic versus the outside; the air and water were entirely recycled and the diet depended on the food grown inside the structure. Unexpectedly, food production in Biosphere 2 was lower than foreseen and the biospherian subjects underwent a 30% calorie restriction. Although the sample is too small to provide any firm conclusions, many of the same physiological, hematological, biochemical and metabolic modifications associated with the CR anti-aging effect in rodents and primates were also observed in these subjects, including a decrease of core temperature, blood pressure, insulin, fasting glycemia and metabolic rate [59, 60]. The TNO study was better controlled; it comprised 8 control subjects fed “ad libitum” and 16 subjects fed a 20% reduced caloric intake. As in the Biosphere 2 study, the CR subjects showed positive effects compared to controls, including decreased fat mass and systolic pressure [61]. To date, however, the best controlled and most complete study on the effect of CR on humans is the CALERIE (Comprehensive Assessment of Longterm Effects of Reducing Intake of Energy) study, funded by the NIA. This began about 6 years ago and is still running, in three different centers in the USA (Pennington Biomedical Research Center, Baton Rouge, LA, Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University Boston, MA and Washington University in St. Louis, School of Medicine St. Louis, Mo). It enrolled 225 healthy non-obese (BMI 22-27.9) men and women aged 21-50 years and its purpose is to find out what effect two years of 25% CR have on reducing risk of disease and on slowing the aging process [62, 63]. This multicenter controlled trial was designed on the basis of the results obtained in the three independent short-term phase I studies performed in the above clinical centers [64-66]. The results obtained thus far in the first phase studies show a general improvement of cardiovascular health indices and of glucose metabolism. In particular, CR has been shown to lower blood triacylglycerol, LDL-cholesterol, total cholesterol/HDL ratio and CRP concentrations [67, 68] and to ameloriate left ventricular diastolic function [69]. With regard to glucoregulation, circulating insulin and glucose levels are decreased by CR [65,70] while insulin sensitivity is increased [67,70]. Further insight into CR’s effects on humans has been provided by the possibility of studying long-term CR, thanks to the collaboration of 18 individuals (15 men, 3 women) enrolled through the Caloric Restriction Optimal Nutrition Society (aged 35-82 years) who have been practicing a strict self-imposed restricted diet (about 1800 kcal/day; 30% less than age-matched and sex-matched volunteers consuming a typical Western diet) for periods ranging from 3 to 15 years [71]. In these subjects, the protective effects against cardiovascular diseases (CVD) and diabetes exerted by short-term CR were confirmed, in terms of blood and cardiovascular parameters (lipid and lipoprotein profile, glucose metabolism, carotid artery intima media thickness) [72] and of cardiac function (systolic and diastolic pressure) [73]. In particular, left ventricular diastolic function was similar to that found in subjects approximately 16 years younger [71]. Moreover, plasma concentrations of some in-


flammatory markers (C reactive protein, soluble tumor necrosis factor- receptors, and interleukin-6) were reduced compared to age- and sex-matched controls eating a typical USA diet [72, 74]. This further confirms the cardiovascular protection offered by CR, in view of the known association of circulating inflammatory markers with CVD [75]. Additionally, other metabolic and hormonal indices, such as reduced body temperature, circulating triiodothyronine (T3), testosterone and estradiol, and increased adiponectin and steroid hormone binding protein concentrations, which are predictive of increased survival and have been reported in rodents and rhesus monkeys on CR [40,42,45,76], are also found in CR humans [74,77-79]. Scientific evidence obtained in animal models also supports the view that lower calorie diets in humans may decrease the incidence of other age-related diseases apart from CVD and diabetes, i.e. cancer, and neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases [80-84]. An alternative protocol to the classic CR described above is the so-called “Every Other Day” (EOD) feeding, also known as “intermittent feeding” or “intermittent fasting” (IF) or “Alternate-Day Fasting” (ADF), first introduced in the 1980s [85]. This regimen alternates 24 hours of ad libitum food consumption and 24 hours of total or partial food restriction; the calorie consumption as a whole may not be extremely reduced, since subjects may balance the reduced caloric intake during fasting periods by overeating on ALfeeding days. Recent studies measuring food intake have, indeed, shown that EOD induces mild (20%) CR [86]. For this reason, these regimens often do not reduce body weight to the same extent as CR [87]. Nevertheless, like CR, ADF has been shown in animals to decrease some risk factors for diseases often associated with aging, such as cancer, CVD, diabetes, kidney disease, and neurological diseases [87-92], thus favouring increased longevity. As regards humans, relatively few ADF trials have been attempted and the results are somewhat contrasting [93]; however, one such study, on a fairly large population, reported improvements in insulin sensitivity, asthma, seasonal allergies, autoimmune diseases, osteoarthritis, infectious disease of different origins, inflammatory central nervous system lesions, cardiac arrhythmias, and menopause-related hot flashes [94]. Be that as it may, even if no definitive answer yet exists as to whether CR has beneficial effects on intrinsic aging and maximal lifespan in humans, these early human studies are encouraging, and further stimulate the systematic search for the mechanism to slow aging in other species, as a means to understand human longevity.

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The mass of literature on this topic (a search done in March 2013 on PubMed, for publications between 1980 and 2013, using the search terms ‘‘caloric’’ or ‘‘dietary’’ and ‘‘restriction” retrieved almost 20,000 papers) makes it impossible to review the subject in its entirely in a single article. We thus refer readers to other comprehensive reviews for an in-depth examination of the implications of CR on healthy human aging [93, 95-98]. 2. BIOLOGICAL MECHANISMS OF THE ANTI-AGING EFFECT OF CR Despite the plethora of scientific studies on the anti-aging effect of CR, the underlying mechanism/s have not yet been fully clarified. Over the past 77 years, at least 10 different hypotheses have been proposed to elucidate how CR works; however, many of these have been found to be conflicting, and the remainders fail to explain the countless observations about CR and life-span extension (Fig. 2). A comprehensive review of studies, in favour of, as well as against, the numerous hypotheses on this topics, was attempted by Chiarpotto and colleagues [99]. Among such theories, considerable consensus still surrounds that of the protection that CR may provide against the ageassociated increase of oxidative stress and consequent cell damage [100]. Compatible with this theory, older animals present higher levels of oxidized lipids and proteins [101-103] as well as impaired/mutated DNA, especially the mitochondrial DNA [104, 105]. In parallel, CR reduces the age-associated accumulation of oxidatively damaged lipids, proteins, and DNA [101, 102, 106, 107]. Opposers of this theory sustain that the administration of a number of different antioxidants to laboratory mammals has failed to increase their lifespan [108, 109] and that rodents genetically modified to overexpress or partially lacking antioxidant enzymes (e.g. Sod2+/) show neither increased nor shortened lifespans [for a review see 110]. However, firstly, the lack of lifespan extension provided by antioxidant overexpression only implies that oxidative stress is not the only life-span-restricting agent; secondly, complete knockout mice for CuZnSOD, Sod1, Prxd1, and Sod2 actually do show reduced lifespans, whereas in partially knock-out animals, not all markers of oxidative stress are increased; and thirdly, the true life span of antioxidant knockout mice is not always certain [110]. Other outstanding hypotheses that have been put forth to explain lifespan extension by CR are: variation of the glucorticoid cascade [111]; decrease of body fat [112]; modulation of cell survival/apoptosis [113, 114]; and various neuroendocrinological

Fig. (2). Biological mechanisms that have been proposed to explain lifespan extension by calorie restriction (CR).


changes [115]. In this latter connection, the insulin/insulin-like growth factor 1 (IGF-1) receptor signal transduction system has been suggested to play a significant role in regulating the rate of aging, both in invertebrates [116, 117] and in mammals [118-120]. Moreover, it has been shown that single mutations of genes involved in growth hormone (GH)/IGF-1 signalling that cause dwarfism, also increase the lifespan in mice [121]. CR reduces serum IGF-1, insulin and glucose levels in rodents [122] and is known to suppress the GH-IGF-1 axis [123]. Moreover, it further extends the already enhanced lifespan of Ames dwarf mice [124]. Other important CR-induced endocrinological changes are: enhanced insulin sensitivity [74, 125]; decreased anabolic hormone panel (insulin, testosterone, estradiol and leptin) [126]; decreased levels of hormones regulating thermogenesis and basal metabolism (T3) [77, 126]; and increased anti-inflammatory hormone levels (adiponectin, corticosterone and ghrelin) [74, 127]. In connection with this last-named metabolic effect, another CR-mediated anti-aging mechanism is recognized to be the reduction of chronic systemic inflammation [128]. Chronic inflammation is often associated with tissue injury, increased fibrosis, and organ dysfunction, and is involved in the pathogenesis of many agerelated diseases and in the aging process itself [129]. For this reason, in 2000 Franceschi coined the term “inflammaging” to describe the phenomena of age-related immune function remodeling and derangement of inflammatory process regulation [130]. Indeed, aging is characterized by increased levels of inflammatory proteins (IL-6, IL-1 and TNF) both in tissues [102] and in plasma [131], and this phenomenon in the elderly has been associated with severe diseases (Parkinson’s, dementia, etc.) and in general with frailty, functional disability, and a higher mortality risk [132]. CR has been shown to decrease the inflammatory cytokine content in the plasma [133] and tissues [102, 134] of experimental animals, as well as in humans [127], and to decrease the production of other chemical mediators of inflammation (PGE2, PGI2 and TXA2) [135]. Moreover, in in vivo studies on rodents, CR modifies the expression of genes involved in inflammation, enhancing those with antiinflammatory properties (nuclear factor k B inhibitor alpha: NFkBi, tissue inhibitor of metalloproteinases-3:Timp3 and peroxisome proliferator-activated receptors: PPARs) [136, 137] and inhibiting pro-inflammatory ones (TNF, IL-6, COX-2, iNOS, VCAM-1 and ICAM-1) [138, 139]. It also decreases the expression and DNA binding activity of transcription factors known to activate genes involved in the inflammatory process, such as NFkB, forkhead box O-1 (FoxO1) transcription factor, and activator protein-1 (AP-1) [101, 102, 140]. The anti-inflammatory action of CR is associated with the protection it provides against the impairment of cardiac function often seen in aged subjects [73], and also ADF has been demonstrated beneficial against cardiac hypertrophy and fibrosis lowering inflammation and decreasing the transforming growth factor 1 (TGF1) and ERK-PI3K signalling pathways [101, 141]. This general CR-mediated anti-inflammatory effect is likely related to the above-cited increase in the blood levels of some anti-inflammatory hormones [74, 127], to the remodelling of white adipose tissue with consequent lowering of proinflammatory adipokine and cytokine levels [98, 142], and to the decrease of oxidative stress [97, 100-102]. Another proposed mechanism underlying the anti-aging action of CR is that fasting stimulates autophagy and leads to an increased turnover of cell proteins, membranes and organelles [143, 144]. When starved, cells can activate autophagy to stimulate protein degradation, in order to meet metabolic demands and to remove redundant or damaged cell membranes and organelles, including mitochondria, endoplasmic reticulum and peroxisomes [145]. This mechanism is particularly important in non-proliferating cells: intracellular aggregation of misfolded protein is a common peculiarity of many neurodegenerative diseases (Alzheimer’s, Parkinson’s, and Hungtington’s diseases).


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The mitochondria, the primary producers of reactive oxygen species (ROS) [146], are also highly susceptible to oxidant-induced damage, because of the proximity of ROS. This has led to the formulation of another prominent hypothesis of aging that suggests senescence is the result of damage caused by ROS to the mitochondrial genome in post-mitotic cells. Once mitochondria become injured, they become less efficient at producing energy and produce larger amounts of ROS [147]. In view of the fact that aging is associated with a decline in the so-called “mitophagy” [148], the reversal of this age-associated decline by CR may well be considered an anti-aging mechanism [143, 144, 149]. This hypothesis is corroborated by the fact that anti-lipolytic agents that stimulate autophagy protect against age-associated mitochondrial damage and proteolytic failure, both in vitro and in vivo [145, 150]. The latest hypothesis on the antiaging effect of CR is the “Hormesis hypothesis”, whereby CR represents a low-intensity biological stress on the organism, eliciting a defensive response that helps protect it against other, stronger, subsequent stressors, thus favouring enhanced longevity [151]. Indeed, both CR and ADF have been shown to enhance the expression of a number of heatshock proteins (Hsp) [152-154] as well as to increase the content of antoxidant enzymes and antioxidants in various tissues in aged animals [102, 154, 155]. This theory was subsequently expanded by Sinclair and colleagues, who formulated the “Xenohormesis hypothesis”, which proposes that small molecules with CR-mimetic action can regulate life-span [156]. 3. MOLECULAR MECHANISMS UNDERLYING THE ANTI-AGING EFFECT OF CR One of the most attractive theories put forth to explain how CR improves lifespan was the ‘‘rate of living theory’’ [157]. This hypothesis assumed that a decrease in the metabolic rate (rate of living) lowers the flow of energy, with a consequent decrease in ROS production and oxidative damage to living tissues [158]. CR is, indeed, correlated to a marked lowering in energy metabolism. [159]. However, during the last decade the concept that CR modulates lifespan by decreasing the metabolic rate, and thus reducing the accrual of macromolecule damage over time, has been replaced with the concept that CR influences the activity of several signalling pathways tha play roles in regulating metabolism, particularly energy metabolism, which have been maintained relatively unaltered during evolution from yeast to humans. The most widely studied of these are the sirtuins, particularly sirtuin 1 (SIRT-1, silent information regulator 2 Sir2 homolog), peroxisome proliferator activated receptor G coactivator-1 (PGC-1), AMP-activated protein kinase (AMPK), insulin growth factor-1 (IGF-1), and target of rapamycin (TOR) protein kinase signalling. 3.1. Sirtuins The sirtuin proteins comprise a highly conserved family of NAD+-dependent deacetylases and mono-ADP-ribosyl transferases, which are homologs of the yeast silent information regulator 2 (Sir2) [160]. They are present in organisms from prokaryotes to humans, and show a high degree of functional diversification that has led to two different enzymatic activities, a wide range of substrates, and a highly diversified pattern of cellular localization. In mammals, seven sirtuin genes (SIRTs-1-7) have been identified that are differentially located within the cellular compartments, with different biochemical activities. SIRT-1, SIRT-6 and SIRT-7 are predominantly in the nucleus [161]; SIRT-1 is also found in the cytoplasm, as is SIRT-2 [162], whereas SIRT-3, SIRT-4 and SIRT5 are in the mitochondria [163]. In terms of activity, SIRT-1 and SIRT-5 exhibit deacetylase activity [160, 164], SIRT-4 and SIRT-6 are ADP-ribosyl transferases [165, 166], while SIRT-2 and SIRT-3 have both deacetylase and ADP-ribosyl transferase activities [162, 163]. No certain catalytic activity has yet been found for SIRT-7.


Sirtuin proteins modulate the activity of a wide array of proteins associated with energy metabolism, stress resistance, and cell survival and longevity, including p53, Forkhead box O (FoxOs), PGC1 and NFkB [167-169], and also appear to be involved in mediating key effects of CR during aging [157]. In particular, overexpression of Sir2, orthologue of the mammalian SIRT-1, has been demonstrated to increase the yeast lifespan [170], and that of Caenorhabditis elegans [171] and Drosophila [172], and Sir2 has been shown to be essential in the mechanism of lifespan extension by CR in the same species [173-175]. In mammals, SIRT-1 is activated by CR in several tissues [176], and in the cerebral tissue of rats CR enhances the expression of both SIRT-1 and SIRT-5, which, together with SIRT-3 and SIRT-4, are involved in mitochondrial function [177]. Moreover, over-expression of SIRT-1 in mice is associated with positive modifications of important metabolic parameters, including glucose homeostasis and insulin sensitivity [178], and is also linked to providing protection from the development of metabolic disease, induced by high-fat diets [179], analogously to what happens in mice on a CR diet [180]. Several epidemiological studies have shown the impact of p53 on aging and longevity in humans [181, 182]. In Drosophila melanogaster, the fly orthologs of Sir2 and p53, respectively dSir2 and Dmp53, physically interact in vivo. Dmp53-derived peptides can be deacetylated by dSir2, and Dmp53 transcriptional activity is inhibited in vitro by dSir2 activation [167]. As regards NFkB, through its modulation SIRT-1 inhibits the expression of genes involved in inflammation [169]. Significantly, SIRT-1 over-expression can also increase longevity by stimulating autophagy [183]: SIRT-1 binds and activates some autophagy-related proteins (ATG), and autophagy is markedly decreased in cells from SIRT-1 knockout animals [183]; in the cells of these animals, mitochondria and organelles show alterations similar to those found in cells from ATG5 knockouts [183]. SIRT proteins can further stimulate autophagy by deacetylating and activating FoxO [184, 185]. By this same mechanism, SIRT-1 may further promote longevity through FoxO-dependent induction of stress response genes [186, 187]: FoxO transcription factors, or the invertebrate ortholog DAF-16, have been shown to interact with several pathways that regulate cell lifespan and increase longevity [188], and Sir2/SIRT-1 directly activate DAF16/FoxO [186, 189]. Very recently, Fusco and colleagues demonstrated that, in the brain of mice knock-out for the cAMP responsive-element binding (CREB)-1, the positive effects of calorie restriction on neuronal plasticity, memory and social behaviour are abolished, these negative effects being associated with a drastic reduction of SIRT-1 expression. The study demonstrated a complex interaction between CREB and SIRT-1, in that CREB directly regulates SIRT-1 transcription, whereas SIRT-1 stimulates CREB-dependent expression of target genes, including PGC1-. These target genes are, indeed, considerably down-regulated in the brain of SIRT-1 knockout mice, which are also insensitive to CR [190]. PGC-1 is another important substrate of SIRT-1 deacetylating activity. In the liver, in response to fasting signals, SIRT-1 deacetylates PGC-1 at a specific lysine residue, with consequent triggering of gluconeogenesis and of glucose output, and inhibition of glycolysis [191, 192]. Numerous indications point to SIRT-1’s tuning glucose and lipid metabolism through its deacetylase activity for many substrates, and playing a protective role against diseases related to insulin resistance, such as metabolic syndrome and diabetes mellitus, through its involvement in insulin signalling. Indeed, SIRT-1 induces a glucose-dependent insulin secretion from pancreatic beta cells, and directly triggers insulin signalling pathways in insulin-sensitive organs [193]. In addition, SIRT-1 reduces adipogenesis by inhibiting peroxisome proliferator-activated receptor gamma, and enhances lipid mobilization in white adipose tissue [194].

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Moreover, SIRT-1 is also implicated in mTOR signalling, in that SIRT-1 activation lowers TOR signalling whereas SIRT-1 inhibition enhances it [195]. Other components of the sirtuin group may also be important in the CR effect. The nuclear sirtuins SIRT-6 and SIRT-7, as well as the mitochondrial sirtuins SIRT-3, SIRT-4 and SIRT-5, which modulate several metabolic responses [196], are sensitive to fasting and to CR [196-198]. The cytoplasmic SIRT-2 has also recently been identified as a therapeutic target for treating age-related disorders, such as metabolic syndromes, cancer, and neurological disorders [199]. SIRT-3 is emerging as an important player in the metabolic adaptations to diet and lifestyle that may well influence mammalian lifespan. Several reports have established a role for SIRT-3 in the protective effects of CR; it mediates the induction of antioxidant defences and metabolic adaptations during CR [200, 201] and, being activated by CR, regulates mitochondrial function in brown adipose tissue [163] and activates AMPK and PGC-1 in skeletal muscle [198]. In addition, a recent study showed that SIRT6 can extend the lifespan in male mice [202]. Moreover, SIRT-6 overexpression lowered serum levels of IGF1 and, as was mentioned above, an attenuation of IGF1 signalling is associated with increased longevity in many animal models [121]. 3.2. PGC-1 PGC-1 is a member of the peroxisome-proliferatoractivated receptor coactivator-1(PGC-1) family of transcriptional coativators, and is a key regulator of genes involved in mitochondria metabolism and biogenesis [203, 204]. Although there is little experimental data about age-dependent changes in PGC-1 function and activity [205], severel indirect indications support its role in aging and anti-aging protection. In general, PGC-1 preserved activity is associated with protection against several effects: the decline in mitochondrial function that characterizes skeletal muscle aging; the age-induced metabolic shift in the heart, from lipid metabolism toward carbohydrate metabolism, also typical of heart disease; and the age-associated alteration of adipose tissue distribution and function [206]. Interestingly, CR opposes these ageinduced metabolic alterations [205, 206], induces the expression of genes involved in the mitochondrial electron transport system [207], and also increases mitochondrial biogenesis through activation of PGC-1 via SIRT-1 [176, 208]. Anderson and colleagues have identified another possible mechanism for the maintenance of PGC-1 function by CR: following stress glycogen synthase kinase 3 beta (GSK3) is activated and modifies PGC-1 protein, addressing it to proteasomal degradation inside the nucleus. Nuclear PGC1 thus decreases reducing gene transcription, whereas PGC-1 levels in the cytosol are restored to pre-stress levels [209]. Data obtained in vivo in mice demonstrate that GSK3 is decreased by CR, and suggest that this mechanism can play a role in the improvement of mitochondrial function by CR, by preserving elevated nuclear levels of PGC-1 [209]. However, quite recently the group of Holloszy reported contrasting data concerning the effect of CR on mitochondrial biogenesis. They showed that, in the heart, brain, liver, WAT, and skeletal muscle of rats on a 30% calorie restricted diet for 14 weeks, PGC-1 protein levels were not increased, and that in the heart, brain and skeletal muscle, other mitochondrial proteins showed no modifications [210]. Nevertheless, in the liver of mice on EOD feeding, a decrease of PGC-1 was demonstrated, but the maximum longevity was still increased; the study authors speculate that this might be due to a decrease in mitochondrial oxidative stress [211]. The behaviour of PGC-1 thus appears to differ depending on the type and duration of DR, but it is in all cases connected to increased longevity. 3.3. AMPK Another key regulator of mitochondrial biogenesis in the adaptive response to energy deprivation is AMPK [212]. It is a highly


sensitive sensor of the energy status of the cell, in that it is activated in response to an increase in the intracellular AMP/ATP ratio, and inhibited by a low AMP/ATP ratio [213]. AMPK also both fits to and governs food intake and systemic energy consumption, by reacting to hormonal signals in the central nervous system and peripheral tissues [214]; in particular, AMPK is sensitive to the adipokines leptin and adiponectin, and to ghrelin and tetraiodothyronine (T4) [215]. Its activation in response to several metabolic modifications induced by CR, such as reduced energy and glucose, suggests it may play a role in CR-induced longevity [212]. Moreover, increased AMPK signalling extends the lifespan of Caenorhabditis elegans and yeast [216, 217] while inhibition of Drosophila AMPK, especially in muscle, shortens the lifespan of Drosophila [218]. AMPK’s association with CR-induced increased longevity in these organisms is still debated, although in Caenorhabditis elegans its activation has been related to the extension of lifespan brought about by a new type of dietary restriction, through an increased resistance to oxidative stress [219] . In mammals, AMPK is markedly induced by fasting, but the effect of CR is again debated. Some studies report that long-term CR does not modify AMPK kinase activity in skeletal muscle, liver or heart [220] or that it reduces it [221], but conversely more recent studies report increased AMPK activity in the heart and skeletal muscle after CR [198, 222]. This latter report appears to be confirmed by a very recent paper, showing an increase in AMPK phosphorylation in heart and liver of lifelong CR male B6D2F1 mice [223]. The mechanism/s underlying AMPK-induced lifespan extension are still under study, but it has been shown that AMPK directly phosphorylates PGC-1 and FoxO transcription factors [224, 225], targeting them for deacetylation by SIRT-1 [226]. AMPK may thus regulate mitochondrial biogenesis and energy production. 3.4. Insulin/IGF Signalling As has already been said, it has been suggested that the IGF-1 signalling pathway is related to CR-induced regulation of the lifespan, in animal species ranging from invertebrates to mammals and humans [227]. It is known that the insulin/IGF-1 signalling pathway controls the downstream phosphatidylinositol 3-kinase (PI(3)K)/Akt/phosphoinositide-dependent kinase-1 (PDK-1) cascade, which results in the inhibition of FoxO transcriptional activity, through phosphorylation and consequent sequestration of FoxO in the cytoplasm [228]. Mutations that lead to decreased function of insulin/IGF-1 signalling, including those resulting in a decreased activity of this cascade components, have indeed been reported to increase lifespan, to retard the onset of age-related diseases, and to increase resistance to oxidative stress [229-232]. However, this finding is contrasted by a recent paper by Bokov and colleagues who show that, unlike worms with similar mutations, the longevity of Igf1r (+/-) mice was not increased, apart from a slight increase in the mean lifespan of Igf1r(+/-) females compared to female wild type mice [233]. Conversely, mice overexpressing the Klotho gene, whose product inhibits intracellular insulin signalling, showed an increased lifespan together with increased protection against oxidative stress [234]. In Ashkenazi Jews, a functional variant of Klotho, KL-VS, has been shown to be advantageous in heterozygosis, but disadvantageous in homozygosis, in terms of the bearer’s longevity [235]. Moreover, in the Ashkenazi population, mutations known to alter IGF-1 receptor function are overrepresented in a cohort of centenarians [236]; in a Japanese cohort, variants in the insulin receptor gene are linked to longevity [237]. In Caenorhabditis elegans it has been shown that mutations leading to decreased activity of daf-2, whose transcript is a hormone receptor similar to the insulin and IGF-1 receptors, more than double the animal’s lifespan [227]. Moreover, insulin/IGF-1 signalling down-regulates activity of the transcription factor Daf-16,


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which has been shown to be essential for longevity of daf-2 mutants: the presence of the double mutation daf-2 (-) and daf-16 (-) completely suppressed the lifespan increase found in animals with the daf-2 mutation alone [227], but in the same animals expression of daf-16 in only one tissue can increase longevity of the whole animal [227] indicating that Daf-16 may also influence other longevity signals independent of daf-16 in other tissues. Similarly, activation of dFOXO, the Drosophila orthologue of Daf-16, whether by inhibition of insulin/IGF-1 signalling or by gene overexpression, is linked to increased lifespan in Drosophila [227]. The mammalian daf-16 homologues are FoxO proteins, including FoxO1, FoxO3a, FoxO4, and FoxO6 [238], and a genetic variation of the FoxO3A gene has been reported in long-lived men, together with several signs of healthy aging, e.g lower prevalence of cancer and cardiovascular disease, and greater insulin sensitivity [239]. As has already been said, FoxO can induce a number of stress response genes [186, 187, 238], further favouring longevity. 3.5. TOR Other well-known targets of the IGF-1 signalling pathways are members of the target of rapamycin (TOR) family; these are a conserved family of kinases that are responsive to stress, nutrients and growth factors. In particular, TOR signalling stimulates cell growth in the case of food wealth [240]. The name derives from the presence of a binding domain for the macrocyclic lactone antibiotic, rapamycin (sirolimus) [240]. In mammalian cells, the corresponding protein is the mammalian target of rapamycin, mTOR, which is part of two complexes: mTOR complex 1 (mTORC1), which contains Raptor (rapamycin-sensitive adaptor protein of mTOR ) and is rapamycin sensitive, and mTORC2, which contains Rictor and is rapamycin insensitive [240]. mTORC1 plays an important role in inhibiting autophagy and in stimulating protein synthesis and cell proliferation [240]. The signalling pathway of mTORC1 activation by growth factors, including insulin/IGF-1, is the same pathway described above, involving PI3K, Akt and PDK-1. Once phosphorylated by PDK-1, Akt in turn phosphorylates the Tuberous sclerosis complex (TSC) 2, with consequent inactivation of the TSC1/TSC2 complex, which becomes unable to break the bond between GTP and Rheb [241]. This causes an increase in Rheb-GTP, which binds to and activates mTORC1 kinase [241]. Again, the implication of TOR in aging was first demonstrated in Caenorhabditis elegans and Drosophila, in which its downregulation led to a significant lifespan expansion [for a review see 242]. An increased lifespan was also observed in mice, associated with decreased mTOR signalling [243], and through mTOR inhibition by rapamycin treatment [244]. A primary role of this kinase in lifespan extension by CR has been demonstrated in yeast and fruit flies [245, 246]. To our knowledge, no direct data are yet available about the implications of this important regulatory molecule in primate aging and CR, but very recently, in a Dutch cohort of families with extended longevity, it was shown that the mTOR signalling gene set is associated with old age [247]. Significantly, it has been demonstrated that some factors downstream of mTORC1 also modulate the aging process: decreased ribosomal protein S6 kinase (S6K) activity extends lifespan in yeast and in Caenorhabditis elegans and Drosophila [245, 248, 249], and female mice lacking S6K1 showed an increased lifespan and retarded progression of age-related diseases [243], together with a significant overlap of gene expression profiles with mice under long-term CR, including AMPK and PGC-1 [242, 243]. Another typical mTORC1 substrate that has been shown to modulate the aging process is eukaryotic initiation factor 4E-binding protein 1 (4E-BP 1). In Drosophila it is upregulated upon CR, and is thought to mediate CR-dependent changes in mitochondrial activity and lifespan extension. Accordingly, its total absence completely cancels these CR-dependent changes [250].


TOR may also regulate lifespan through other mechanisms, e.g. by modulating autophagy. In fact, TOR normally inhibits autophagy [251], which has been shown to be one of the biological mechanisms whereby CR extends the lifespan [143, 144]; amino acid or glucose deprivation, as well as the removal of growth factors including insulin, also inhibit TOR activity and induce autophagy [251]. In this respect, autophagy has been shown to be essential for lifespan expansion in TOR knockout yeast [252], and in Caenorhabditis elegans the inhibition of autophagy abolishes the lifespan expansion induced by TOR inhibition [253]. Moreover, within the cells, amino acid deprivation decreases Rheb binding to mTORC1, decreasing its activity and thus inducing autophagy [254]. Finally, mTOR may be inhibited by AMPK, probably by acting on the TSC2 protein and the mTORC1 binding subunit raptor [255], as well as trough SIRT-1[195]. In conclusion, by down-regulating the IGF-1 pathway [121], CR induces activation of FoxO transcription factors, through SIRT1 activation and the resulting FoxO deacetylation [157]. Once activated, FoxO can induce the transcription of antioxidant genes in the nucleus. Another consequence of SIRT-1 activation by CR is PGC1 modulation [176, 208] with improved mitochondrial function and increased mitochondrial biogenesis, and with consequent further decrease in oxidative stress. Significantly, PGC-1 as well as the FoxOs can be directly activated through AMPK-dependent phosphorylation [224, 225]. Moreover, nutrient deprivation and removal of growth factors can stimulate autophagy, directly through

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SIRT-1, or via SIRT-1-dependent FoxO induction [184, 185], or by inhibiting TOR activity [195, 251, 255] (Fig. 3). In this context, it is of considerable importance to improve the caloric restriction approach, in view of its positive interference with the most important mechanisms that lead to aging of the organism. 4. CALORIE RESTRICTION MIMETICS (CRM) Even if the CR regimens generally applied to man are not in fact particularly restrictive (about 1800 Kcal/day) [62, 71] it is difficult for most individuals to respect them, particularly in the long term. Moreover, some disagreable side effects have been reported, such as a lowering of the body temperature [ 256] and reduced circulating sex hormones [257] possibly causing a decreased libido, together with some changes that may be health threatening, such as decreased bone mineral density (although this has been reported not to reduce bone quality) [258], or slowed wound healing [259]. For these reasons, increasing interest is now being shown in the search for organic or inorganic compounds able to elicit the same beneficial effects on aging, health and lifespan as those of CR, by activating the same metabolic- and stress-responses as CR does without decreasing food consumption, in particular in the mid- to late- life periods. In other words, to find compounds that can mime the biological effects of CR, often called CR mimetics (CRM) or dietary restriction mimetics (DRM). An ideal CRM, as first proposed by Ingram and colleagues [260], should: 1) reproduce the metabolic, hormonal and physiological effects of CR, 2) activate stress response pathways similar to

Fig. (3). Molecular mechanisms involved in the anti-aging effect of calorie restriction (CR). CR, by down-regulating the IGF-1 pathway and through SIRT-1 activation, induces activation of FoxO transcription factors. Activated FoxOs can induce the transcription of antioxidant genes in the nucleus. SIRT-1 activation by CR also leads to PGC-1 modulation, with improved mitochondrial function and increased mitochondrial biogenesis, leading to a further decrease in oxidative stress. PGC-1 as well as FoxOs can be directly activated through AMPK-dependent phosphorylation. Moreover, nutrient deprivation and removal of growth factors can stimulate autophagy directly, through SIRT-1, or via SIRT-1-dependent FoxO induction, or by inhibiting TOR activity through SIRT-1 and AMPK activation and IGF-1 signaling inhibition. The nuclear sirtuins SIRT-6 and SIRT-7, and the mitochondrial sirtuins SIRT-3, SIRT-4 and SIRT-5, which modulate several metabolic responses, are also sensitive to CR.


CR, increasing the protection against stress, 3) produce CR-like beneficial effects on mortality and age-related disease , and 4) induce no significant reduction in long-term food consumption. This latter point is not always respected, since some CRM may induce significant modifications of body weight/fat content, with a consequent reduction of food consumption in the long term. Increasingly, studies are targeting this area of research, aimed at determining the feasibility and efficacy of natural and/or pharmacological CRM molecules. The NIA and the NIH are both running the Interventions Testing Program, to test a number of these CRM candidate molecules, with assorted results [for a review, see 261]. Thus far, the most likely candidate CRMs are SIRT-1 activators, especially resveratrol [262, 263], inhibitors of TOR, especially rapamycin [264, 265], 2-deoxy-D-glucose (2DG) and other glycolytic inhibitors [for a review see 266], insulin pathway and AMPK activators [267, 268], autophagy stimulators [140], and -lipoic acid (LA) [269]; dietary antioxidants may also delay specific aspects of aging [270] (Fig. 4). As far as mannan oligosaccharides (MOS) are concerned, their potential use as CRM have been recently disputed [271]. 4.1. Sirtuin 1 Activating Compunds (STACS) In recent years, the sirtuin protein family has attracted considerable attention for its critical regulatory role of physiological responses to CR; at the same time, chemical compounds that can activate the mammalian Sir2 ortholog SIRT-1, known as STACs (sirtuin activating coumpounds), have also stimulated broad interest in CRM [272-274]. 4.1.1. Resveratrol and other Polyphenolic STACS The first STACs identified were polyphenolic plant metabolites [275, 276], a vast group of compounds having aromatic ring(s), characterized by the presence of one or more hydroxyl groups with varying structural complexities [277]. Among polyphenols, resveratrol (RSV), commonly found in the grape Vitis vinifera, was identified as the most potent SIRT-1activator [263, 278]. RSV is highly concentrated in red wine (5


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mg/L on average, ranging from 0.5 to 10 ppm) [279], which is considered the principal source of this compound for humans. RSV has repeatedly been indicated as the compound responsible for the results of the many epidemiological studies correlating moderate redwine consumption with reduced risk of a series of diseases [280, 281]. The wide spectrum of biological effects is surprising, in view of the fact that the bioavailability of orally-ingested RSV is very low. Once ingested, it is very quickly absorbed [282] and 75% of it is lost by excretion via the faeces. The remainder is mostly metabolized to glucuronide- and sulfate-resveratrol conjugates [283], and less than 2% remains in the aglycone form. This raises the doubt that the glucuronide- and sulfate-resveratrol conjugates might be the forms responsible for at least some of RSV’s biological effects in vivo [284]. Thus, RSV is a molecule that is well known in the field of medical nutrition, with numerous possible positive effects on human health [285], but its precise mechanisms of action in vertebrates await clarification, especially in the light of its very low bioavailability. RSV (3,5,4’-trihydroxystilbene), belonging to the stilbene class, consists of two phenol rings connected by a 2-carbon methylene bridge [277]. It exists in both isomeric trans and cis forms, but most of its health benefits are attributed to the trans-isomer. The structure-activity relationship of RSV has been explored for SIRT-1 activation, and the hydroxyl substituents at the 3 and 5 positions of the A ring have been identified as crucial, while modification at the 4’ position of the B ring can be tolerated. RSV is hypothesized to bind the non-catalytic N-terminus of SIRT-1, inducing a conformational change that lowers the Michaelis-Menten constant (Km) for the substrate, promoting a more productive conformation that enhances deacetylating activity [275]. This model is supported by studies showing that the mutations SIRT-1-E230K and Sir2D223K, within the N-terminal sequences, close to the catalytic cores of SIRT-1/Sir2, prevent activation by RSV without affecting basal activity [286]. However, the nature of RSV’s action on SIRT1 in vivo remains controversial: some studies have reported that it extends lifespan in a Sir2-dependent manner, whereas others report that RSV does not affect lifespan.

Fig. (4). Molecular targets for calorie restriction mimetics (CRM). SIRT-1, known to modulate the activity of many proteins associated with energy metabolism, stress resistance, cell survival and longevity, including FoxO and PGC-1, is activated by 2-DG, resveratrol and the thiazolidinediones. These latter two agents, together with metformin, activate AMPK, which in turn inhibits TOR. Metformin and rapamycin have the same effect on TOR directly, and resveratrol indirectly, via PI3K inhibition. The negative regulation of TOR by resveratrol, metformin and rapamycin stimulates autophagy, which may also be achieved with Acipimox, an anti-lipolytic drug. Finally, lipoic acid, and several nutraceuticals with antioxidant and metabolic modulating effects, reduce oxidative damage due to reactive oxygen species overproduction.


In initial screening, RSV was found to be the most potent SIRT1 activator. When tested on Saccaromyces cerevisiae, RSV increased lifespan by 70% in a Sir2 dependent manner. The ability of Sir2 to extend lifespan in this yeast is thought to be due to a role it may play in stabilizing repetitive DNA sequences. Dose–response experiments show that RSV doubles the rate of deacetylation by human recombinant SIRT-1, whereas at a saturated concentration, RSV activates SIRT-1 8-fold [275]. Another study on three different yeast strains showed that RSV had no detectable effect on Sir2 activity in vivo, as measured by rDNA recombination, transcriptional silencing near telomeres, and lifespan. In vitro analyses have shown that RSV enhances binding and deacetylation of peptide substrates that contain Fluor de Lys, a non-physiological fluorescent moiety, but has no effect on binding or deacetylation of acetylated peptides lacking the fluorophore [287]. Wood and colleagues have evaluated the effect of RSV in invertebrate model systems, including the nematode worm Caenorhabditis elegans and the fruit fly Drosophila melanogaster. Supplementation with RSV appears to extend the lifespan of C. elegans and D. melanogaster in a dosedependent manner. The study also found that RSV does not further extend the lifespan of flies under a restricted diet with reduced amount of macronutrients compared to that of flies on a full diet, and nor does it extend the lifespan of D. melanogaster with mutations in the dSir2 gene [172]. Other studies have confirmed the prolongevity effect of RSV in metazoans: it extends the lifespan of the short-lived D. melanogaster flies [288]. In C. elegans, RSV increased both mean and maximum lifespan, by delaying the onset of the exponential increase in mortality characterizing the so-called “dying phase”, but it did not affect the dying phase itself, suggesting that it does not act by directly affecting metabolism [289]. Microarray analysis of C. elegans treated with RSV has shown that the pro-longevity effect induced by this compound occurs through a Sir2.1-dependent endoplasmic reticulum stress pathway [290]. However, another study found no significant effects of RSV on the lifespan of D. melanogaster and C. elegans in independent trials [291]. The reason for the mixed findings in yeast, worms, and fruit flies is not fully clear, but a study on tephritid fruit flies of the species Anastrepha ludens suggests that other factors might influence the response to RSV. The study found RSV to have a modest effect on lifespan in females, but no effect in males; the effect on females only occurred when the diet composition was within a very narrow range of sugar contents, suggesting that the prolongevity effect of RSV in this species of fruit fly was both gender- and diet-dependent [292]. In a recent report, two RSV doses (30 and 130 μM) were shown to lengthen the average lifespan of wild-type honey bees under normal oxygen conditions, by 38% and by 33%, respectively. Both RSV doses also lengthened maximum and median lifespan. In contrast, hyperoxic stress abolished the RSV life-extension response [293]. However, thus far studies on RSV in invertebrates appear to indicate that it may be a potential CRM. One of the first studies showing an effect of RSV on lifespan in vertebrates was on a very-short-lived seasonal fish, Nothobranchius furzeri. Food supplementation with RSV starting in early adulthood caused a dose-dependent increase of both median and maximum lifespan. Moreover, RSV delayed the age-dependent decay of locomotor activity and cognitive performances, and reduced the expression of neurofibrillary degeneration in the brain [294]. In addition to the effects in model organisms, RSV has been consistently shown to produce SIRT-1-dependent effects in mammalian cell culture models. It increases p53-mediated cell survival in human embryonic kidney 293 cell line [275], reduces fat storage and triglyceride release in differentiated 3T3-L1 adipocytes [194], inhibits NFkB-dependent transcription in non-small-cell lung cancer cell lines [295], stimulates glucose uptake in L6 skeletal muscle cells [296] and induces mitochondrial biogenesis in cultured human coronary artery endothelial cells [297]. These RSV effects were abolished in the corresponding SIRT-1 knockdown model. How-

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ever, in vitro effects of RSV on SIRT-1 activity have been found to be highly substrate-dependent and questionable, suggesting they might be an artifact derived from the use of a fluorescent substrate [287, 298-300]. Regardless of the controversy about its mode of action, RSV has been confirmed to have numerous health benefits in various species. Several reports indicate that RSV treatment produces beneficial effects similar to those of CR in mice fed a high-fat diet. Baur and colleagues showed that RSV shifts the physiology of middleaged mice on a high-calorie diet towards that of mice on a standard diet, and significantly increases survival, by protecting against insulin resistance [301]. Moreover, RSV inhibits insulin secretion by the pancreatic islets in normal rats [302] and, similarly to CR, it lowers blood insulin in rodents [301, 303]. It has also been shown that, in high-fat-fed mice, RSV significantly increases mitochondrial oxidative phosphorylation and aerobic capacity, which are associated with increased longevity [304]. RSV is reported to increase the metabolic rate and to reduce fat mass in wild-type mice, but not in AMPK-deficient mice, in which it failed to increase insulin sensitivity and glucose tolerance. In addition, RSV increased the NAD+-to-NADH ratio in an AMPK-dependent manner, which may explain how RSV may activate SIRT-1 indirectly [305]. A recent paper reports that RSV increases the activity of SIRT-1 by inhibiting cAMP-specific phosphodiesterases (PDE) and identifies the cAMP effector protein Epac1 as a key mediator of RSV’s effects, including prevention of diet-induced obesity and glucose intolerance in mice [306]. This study also shows that RSV causes an increase in mitochondrial biogenesis, in PGC-1, and AMPK activities. Among CR benefits are its positive effects on the brain and cognition in rodent models. In a mouse model of Huntington's disease, SIRT-1 induction by RSV protected neurons against mutant polyglutamines toxicity [307]. The neuro-protective effect of RSV was also demonstrated in an in vitro model of Parkinson's disease, using rat cerebellar granule neurons (CGNs), in which the loss of cell viability and apoptosis were prevented by RSV (1-100 μM) treatment. However, in this case RSV’s neuroprotective effects were not mediated by the activation of SIRT-1, since sirtinol, an inhibitor of this enzyme, did not attenuate them [308]. Further, two studies in mice show that long-term administration of RSV induces gene expression patterns similar to those induced by CR and delays aging-related deterioration, but does not extend lifespan when mice are fed a regular diet [309, 310]. Conversely, RSV does not mimic other aspects of CR in mice, such as slowing heart rate and decreasing core body temperature [311]. It has recently been shown, that dietary supplemention with RSV increases mean life expectancy and maximal life span in SAMP8 mice, a model of age-related Alzheimer’s disease. Furthermore, long-term dietary RSV activated AMPK pathways and pro-survival routes, including SIRT-1 [312]. As has already been said, despite the numerous studies on CR and aging in short-lived species (yeast, flies, nematodes, mice and rats), only a few are ongoing in long-lived species, and the full effects of CR and CRM have not yet been compared in primates. Recent studies report the beneficial effects of RSV in a non-human primate model, the grey mouse lemur Microcebus murinus. Dietary supplementation with RSV, as well as chronic CR, enhanced cognitive function by ameliorating executive function and spatial memory [313], and also increased insulin sensitivity by improving glucose tolerance at early–middle age, without altering baseline insulin secretion [314]. However, after one year’s treatment, CR and RSV induced differential metabolic responses in M. Murinus; in particular, CR induced a decrease in daily energy expenditure (DEE) without causing changes in the resting metabolic rate (RMR), whereas RSV induced a concomitant increase in DEE and RMR [315]. Although RSV has been widely studied for its potential health benefits, little is known about its metabolic effects in humans. Several clinical trials have been conducted to this end: the results ob-


tained to date suggest that RSV may improve insulin sensitivity and mimic some aspects of CR [316-318]. At least some of the beneficial effect of RSV on human health stem from its capacity to promote autophagy by activating the NAD-dependent deacetylase SIRT-1 [319, 320]. Other studies have raised the possibility that RSV may modulate lifespan through alternative pathways, independent of SIRT-1 activation: RNA interference experiments have shown that the inhibitory effects of RSV on insulin signaling pathways are not reduced in cells with reduced expression of SIRT-1 [321]. Other STACs belonging to the group of polyphenols, while not as potent sirtuin activators as RSV, have also been studied as candidate CRMs. The flavonol quercetin, present in many fruits and vegetables, has been found to extend lifespan [322, 323] and to increase stress resistance in the nematode C. elegans. [324, 325]. These pro-longevity effects do not appear to be dependent on sirtuins, but rather to be related to quercetin’s influence on the expression of other genes in this species. Three different flavonoids were isolated from onion, i.e. quercetin and quercetin 3’-O--Dglucopyranoside (Q3’G), and one novel compound, quercetin 3-O-D-glucopyranoside-(41)--D-glucopyranoside (Q3M). Among these, in C. elegans quercetin showed the highest antioxidant activity, whereas Q3M showed the strongest anti-aging activity, which might be related to its high hydrophilicity. On the contrary, rutin, structurally similar to Q3M, did not show any lifespan extension effect. Also in this case, the observed pro-longevity action was independent of the sir-2.1 gene, but dependent on old-1, a gene responsible for stress resistance and adult longevity [326]. Quercetin increases SIRT-1 in skeletal muscle and brain in mice [327] and in skeletal muscle in humans [328]; in both cases, the quercetin-induced increase in SIRT-1 is associated with improved exercise performance [327, 328]. Quercetin and its O-methylated metabolites, isorhamnetin and tamarixetin, significantly prolonged the lifespan of C. elegans providing an increase in mean lifespan ranging from 11% to 16% versus controls. However, only quercetin significantly increased the reproductive capacity of the nematode, and enlarged its body size [329]. In contrast, a very old study reported that dietary supplementation with 0.1% quercetin significantly reduced lifespan in mice [330], possibly because of SIRT-1 inhibition by the quercetin metabolite quercetin-3-O-glucuronide in vivo [331]. Quercetin and its derivative quercetin caprylate have been shown to be potent proteasome activators with antioxidant properties in vitro, consequently influencing lifespan, survival and viability of primary human fibroblasts. Moreover, when these compounds were supplemented to already senescent fibroblasts, a rejuvenating effect was observed [332]. Quercetin also possesses antioxidant properties [333-335] reducing lipid peroxidation products and increasing glutathione levels in the mouse brain [336]. Rat lung epithelial cells treated with quercetin are protected against hydrogen peroxide-induced DNA damage, probably thanks to the flavonol’s OH. radical scavenging action, but paradoxically in these cells quercetin’s reactivity towards thiol lowers glutathione levels [337]. In addition to its potential antioxidant effect, quercetin is also a significant anti-inflammatory and anti-cancer agent [338-341], and protects against several chronic diseases [342]. A recent study reports that, in C. elegans, myricetin increased the mean adult lifespan by 18%, with quercetin being less effective (5.8%) followed by kaempferol (5.6%). The natural flavonols also increased maximum lifespan, with myricetin showing the strongest effect (21.7%), followed by quercetin (18.4%) and kaempferol (6.7%). The lifespan-extending effect depends on the structural properties of the C-ring of the flavonol, which increases proportionally with the number of hydroxyl substituents in the B-ring. [343]. Fisetin, present in many fruits and vegetables, and butein, present in extracts of Rhus verniciflua Stokes (Anarcadiaceae family) and those of the heartwood of Dalbergia odorifera (Fabaceae fam-


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ily), respectively caused a 33% and a 5% increase in the lifespan of the yeast S. cerevisiae [275]. Fisetin has also been shown to stimulate signalling pathways that enhance long-term memory [344], whereas butein has also been shown to have blood-pressurelowering [345], anti-cancer [346, 347], and anti-inflammatory effects [348]. Very recently the flavanone hesperidin and the aglycon hesperetin, both derived from the Citrus genus, have been shown to exhibit opposing effects upon aging in S. Cerevisiae, in that the former extended the lifespan via activation of SIRT-1 activity, while the latter showed no anti-aging effect [349]. Piceatannol, a hydroxylated analog of resveratrol that is an important component of rhubarb extract, has been shown to have a pro-longevity effect likely due to its anti-cancer [350, 351] and antioxidant [352] properties. As regards the polyphenols present in green tea, the flavanol catechin has been reported to modulate an energy-intensive stress response and repair system in C. elegans, that results in reduced body length and enhanced lifespan. Lifespan tests upon various stress-relevant and lifespan-relevant mutant strains of C. elegans, revealed that the lifespan extending phenotype was absent in mev-1, daf-2, akt-2 and nhr-8 mutants, suggesting the involvement of these genes, and of the molecular systems they participate in, in catechinmediated longevity [353]. Epicatechin, also abundant in cocoa, promoted the survival of obese diabetic mice and Drosophila melanogaster, also increasing the mean lifespan in this latter experimental model. In diabetic mice it also induced modifications related to a healthier and longer lifespan, including reducing circulating systemic inflammation markers, LDL cholesterol and IGF-1, increasing antioxidant defences, and improving AMP-activated protein kinase- activity in the liver and skeletal muscle [354]. Daily administration of epigallocatechin gallate (EGCG), the most abundant catechin in green tea, increased the mean lifespan in both the wildtype N2 and the transgenic MEV-1 (KN1) strain of C. elegans, the latter being hypersensitive to oxidative stress and prone to premature aging. This pro-longevity effect was also evident under lethal oxidative stress conditions [355]. Epicatechin and catechin procyanidins are the main polyphenols present in apples. A quite recent study in fruit flies showed that the mean lifespan was extended by 10% by apple polyphenols, and that this was accompanied by upregulation of CuZnSOD, MnSOD, catalase and Rpn11 genes, and down-regulation of methuselah (Mth), suggesting that the antiaging activity of procyanidins was, at least in part, mediated by its interaction with these genes [356]. In particular, down-regulation of the mRNA Mth gene is known to be associated with a long lifespan [357] and Rpn11 overexpression could delay the age-related reduction of 26 S proteasome activity, suppressing the progression of age-associated neurodegenerative diseases [358]. A similar study has been conducted on C. elegans. Treatment with procyanidins from apple extended the mean lifespan of wild-type N2 and fem-1 nematodes. Moreover, treatment with procyanidins had no effect on the longevity of sir-2.1 nematodes, which lack Sir-2 activity, suggesting that procyanidins have sir-2.1-dependent anti-aging effects in C. elegans [359]. A recent study demonstrated that black rice extracts, rich in anthocyanins, could prolong the mean lifespan of fruit flies by 14%, by acting on the above genes involved in the antioxidant system and in aging, up-regulating CuZnSOD, MnSOD, catalase and Rpn11, and down-regulating methuselah (Mth) at the transcriptional level [360]. The same prolongevity effect has been reported for blueberry extracts, based on the same molecular mechanism [361]. Moreover, proanthocyanidin components of blueberry increased lifespan and slowed aging-related decline in C. elegans, likely modulating osmotic stress resistance, since treatment with this fraction of blueberry extract did not prolong lifespan of osr-1 (rm1), sek-1(ag1) or unc-43 (n1186) animals, carrying mutations in the osmotic stress resistance pathway, while it extended the lifespan of sir-2.1(ok434)


animals, which lack sir-2.1 gene activity, suggesting that this pathway is not involved in the pro-longevity effect of this proanthocyanidin molecule [362]. Proanthocyanidins contained in persimmon skin have also been shown to exercise pro-longevity activity, being able to extend the lifespan of human fibroblast cells in vitro [363] and in vivo in the senescence-accelerated mouse P8 [364]. In both cases, the effect on lifespan was related to increased SIRT-1 expression. The anti-aging effects of cyanidin, a particular type of anthocyanidin, were investigated under stress-induced premature senescence (SIPS) using WI-38 human diploid fibroblasts. Cyanidin treatment attenuated oxidative stress induced by H2O2 treatment, increasing cell viability and inhibiting lipid peroxidation. In particular, cyanidin treatment significantly limited the H2O2induced increase in mRNA and protein expression of NFkB, COX2, and iNOS [365]. Other plant polyphenols investigated for their action on longevity, i.e. tannic acid (TA), gallic acid (GA) and ellagic acid (EA), have been found to extend the lifespan of C. elegans, but whereas for TA and EA CR mimetism and hormetic properties appear to be the main pro-longevity mechanism of action, for GA (and in part also for EA) antimicrobial effects appear more likely, and in general for all three compounds their antioxidant activity does not appear to be correlated with lifespan extension [366]. Another polyphenol that might delay cellular senescence is curcumin. The action of curcumin on TOR pathway genes has pointed to possible implications in the treatment of cancer, as well as for the reversal of aging [367, 368]. Feeding D. melanogaster with curcumin induced an extended lifespan, with significantly increased median and maximum longevities in the adult fly. Gene expression data from curcumin-fed fruit-flies show that the TOR pathway is inhibited in the larvae and the young to midlife adults, although several other genes involved in extending longevity are also affected [369]. Lifespan extension by curcumin in Drosophila is associated with up-regulation of the Mn-SOD and CuZn-SOD genes, and down-regulation of the dInR, ATTD, Def, CecB, and DptB genes, suggesting that curcumin increases the mean lifespan of Drosophila primarily through antioxidant activity, regulating SOD gene expression and thus reducing lipid peroxidation and MDA accumulation [370]. Likewise in C. elegans, curcumin increased lifespan and reduced intracellular ROS and lipofuscin content during aging. Lifespan tests with selected stress- and lifespanrelevant mutant strains of C. elegans revealed that the lifespanextending phenotype was absent in the osr-1, sek-1, mek-1, skn-1, unc-43, age-1, and sir-2.1 mutants, whereas curcumin treatment prolonged the lifespan of mev-1 and daf-16 mutants, suggesting that curcumin may improve survival by modulating osmotic stress resistance, through its antioxidative properties, acting on the Sir.2 pathway and independently of DAF 16 [371]. In addition, Kitani and colleagues showed that tetrahydrocurcumin had a differential effect on mouse longevity, depending on the animal’s age at start of treatment: the effect was positive if treatment began at 13 months of age, wheras there was no effect if treatment began at 19 months [372]. Curcumin and other structurally similar polyphenols have also recently been demonstrated to antagonize age-related cognitive decline [373, 374]. Conversely, Strong and colleagues recently reported that lifelong treatment of mice, beginning at 4 months of age, with one of five agents (curcumin, green tea catechins, oxaloacetic acid, medium-chain triglyceride oil, or resveratrol) had a statistically significant effect on the lifespan of male and female genetically heterogeneous mice, as determined by the log-rank test, although a secondary analysis suggested that green tea extract might have decreased the risk of midlife death, but only in females [375]. To our knowledge, there is only one experimental study on the effects on longevity of the isoflavone genistein, the most abundant phytoestrogen in soya. This study used D. melanogaster as experimental model, and found that genistein induced a concentration-

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dependent decrease in both maximum and mean lifespan of female and male animals [376]. However, Viña and colleagues demonstrated that, both in the healthy aging human population and in Alzheimer's disease, genistein reproduces the antioxidant effect of estradiol at nutritionally-relevant concentrations, by activating estrogen receptors and the MAPK and NFB pathways, with subsequent activation of the expression of Mn-SOD and glutathione peroxidase [377]. 4.1.2. Non-polyphenolic Natural STACS Several studies have shown that omega-3 fatty acids from fish oils improve cardiovascular and autoimmune disorders. A foodrestricted/omega-3-fatty acid-enriched diet was noted to significantly increase lifespan in mice [378]. Dietary supplementation of omega-3 fatty acids is effective in reversing the reduction of SIRT1 levels in rats with mild traumatic brain injury [379]. SIRT-1 mRNA levels have also been shown to increase in the adipose tissue of non-diabetic human subjects after treatment with a combination of ephedrine, caffeine, and pioglitazone, an antidiabetic drug [380]. Melatonin has been found to improve pro-survival signals and reduce pro-death signals in age-related impairment of neural processes in mice. Melatonin supplementation between the first and the tenth month of life increased SIRT-1 and resulted in improved protein deacetylation [381]. Poeggeler and colleagues have also reported the action of a novel endogenous indole derivative, indolepropionamide, similar in structure to melatonin; it reverses the agedependent decline of mitochondrial energy capacity and increases rotifer lifespan, by binding to the rate-limiting component of oxidative phosphorylation in complex I of the respiratory chain, and acting as a stabilizer of energy metabolism. It thereby reduces ROS production, and may potentially represent a novel endogenous antiaging substance of physiological importance [382]. -Lapachone, an o-naphthoquinone extracted from bark of the lapacho tree, has been shown to stimulate NADH oxidation, leading to SIRT-1 activation: the pharmacological administration of lapachone increased SIRT-1 mRNA expression in muscle and white adipose tissues in mice [383]. 4.1.3. Resveratrol-containing Nutraceuticals and Pharmaceuticals Since RSV is a natural compound characterized by low bioavailability in vivo and non-patentability, nutraceutical compositions, such as RSV-containing foods or beverages, and pharmaceutical formulations for the sustained release of RSV have been developed; this has encouraged the development of dietary supplements or foods that contain RSV. LifeGen Technologies, in collaboration with the Swiss-based DSM Nutritional Products, have created ResVidaTM, a high-purity form of RSV for use in many types of dietary supplements and in food and beverage formulations. Consumption of ResVidaTM for 30 days has been found to induce changes in the metabolic profile in healthy obese humans, mimicking the effects of CR [318]. Another nutraceutical mixture that comprises RSV supplemented with 5% quercetin and 5% rice bran phytate, commercially known as Longevinex®, has been marketed by Resveratrol Partners Llc. Longevinex®-treated rats showed better cardiac performance, reduced infarct size, and induction of survival signals in the heart. Moreover, prolonged feeding with Longevinex® increased autophagy markers, such as beclin and LC3-II, and phosphorylation and nuclear translocation of FoxO1, FoxO3a, and FoxO4, indicating involvement of FoxOs with autophagy. Since Sirts and FoxOs are reliable markers of longevity, it seems conceivable that Longevinex might induce longevity after prolonged feeding, via induction of autophagy [384]. In rabbits fed with a high-cholesterol diet, Longevinex® was also shown to exert cholesterol-lowering ability, thus protecting hypercholesterolemic hearts from ischemic reperfusion


injury [385]. In humans, it has recently been reported that Longevinex® administration improved endothelial function in adults with metabolic syndrome who were receiving standard therapy for lifestyle-related diseases (diabetes mellitus, dyslipidemia, or hypertension), but it did not significantly affect blood pressure, insulin resistance, the lipid profile, or inflammatory markers [386]. A commercial micronized RSV formulation called SRT501 has been developed by Sirtris Pharmaceuticals. Micronization allows increased drug absorption, thus increasing bioavailability. SRT501 administration to mice fed a high-fat diet induced many of the molecular events triggered by CR, improving glucose homeostasis and insulin sensitivity, increasing mitochondrial biogenesis, and decreasing inflammatory signaling both via SIRT-1 activation and through transcriptional activation of Ppargc1 and Ppar family members [387]. SRT501 has been tested in a Phase I clinical trial in patients with hepatic metastases, to assess its safety, pharmacokinetics, and pharmacodynamics. It was found to be well tolerated and a single dose produced mean plasma RSV levels 3.6 times higher than those achieved by equivalent doses of non-micronized RSV; moreover, following SRT501 administration, RSV was detected in hepatic tissue, where it exerted a significant pro-apoptotic effect (39% increase versus placebo) [388]. 4.1.4. Non-resveratrol-related Synthetic STACS In recent years, chemically distinct molecules mimicking RSV’s effects have been developed, which activate SIRT-1 at much lower doses. The pharmaceutical company S*BIO PTE LTD has formulated three quinoxaline-based compounds that are as potent activators as RSV, increasing SIRT-1 activity more than double. These compounds are potent lipolytic agents, and also show anti-inflammatory properties in vitro in mouse adipocytes, as well as in human THP-1 monocytes, markedly reducing tumor necrosis factor alpha (TNF-) [389]. SIRT-1 activation may also be achieved by using agents that increase NAD+ concentrations: Qin and colleagues demonstrated that CR-induced increased intracellular NAD+ concentration activates SIRT-1 in the brain of Tg2576 transgenic mice, a model of Alzheimer’s-disease-type amyloid neuropathology. The study authors claim that SIRT-1 activation down-regulates ROCK1 expression, thus promoting -secretase activity and favoring the nonamyloidogenic processing of amyloid precursor protein; this suggests that NAD+-dependent induction of SIRT-1 may be a mechanism whereby CR influences Alzheimer’s disease [390]. Moreover, nicotinamide riboside, nicotinic acid riboside, O-ethylnicotinate riboside, O-methylnicotinate riboside, and several N-alkyl derivatives have also been developed that markedly increase NAD+ concentrations (from 120% to 270%) in several mammalian cell lines [391]. Sirtris Pharmaceuticals has identified and characterized a number of other small molecules that are activators of SIRT-1, i.e. SRT1720, SRT1460, and SRT2183 [392, 262]. These compounds are structurally unrelated to any of the previously identified SIRT-1 activators, significantly more potent, orally bioavailable, and active in vivo. After about 4 weeks’ treatment with the most potent one, SRT1720, a significant improvement in the response to insulin, lower plasma glucose and increased mitochondrial capacity were evident in both diet-induced obese and genetically-obese mice. SRT1460 and SRT2183 had similar, but less pronounced effects. All these compounds are reported to increase SIRT-1 activity by binding to the SIRT-1 enzyme-peptide substrate complex, thus lowering the Michaelis-Menten constant for acetylated substrates [392]. In mice, SRT1720 administration markedly enhanced endurance running performance, and protected against diet-induced obesity and insulin resistance, by enhancing oxidative metabolism in skeletal muscle, liver, and brown adipose tissue. It also induced chronic metabolic adaptations that involve the indirect stimulation


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of AMPK signaling [393]. In a mechanism operating via SIRT-1 activation and through the transcriptional activation of Ppargc1 and Ppar family members, SRT1720, like SRT501, increased mitochondrial biogenesis, insulin sensitivity and glucose homeostasis in multiple tissues and decreased inflammatory signaling, abrogating the diabetic phenotype in several different animal models of type 2 diabetes [387]. SRT1720 treatment also reduced liver lipid accumulation, by directly reducing the expressions of lipogenic genes in a mouse model of non-alcoholic fatty liver disease [394]; it also induced mitochondrial biogenesis through SIRT-1 activation and PGC-1 deacetylation, but not involving AMPK, in primary cultures of renal proximal tubule cells after acute oxidant injury [395]. SRT1720 has also been reported to extend both mean and maximum lifespan of adult mice fed a high-fat diet; the lifespan extension was accompanied by health benefits including reduced liver steatosis, increased insulin sensitivity, and enhanced locomotor activity [396]. In addition, Chauhan and colleagues have examined the anti-cancer activity of SRT1720. Treatment with SRT1720 inhibited growth and induced apoptosis in multiple myeloma cells resistant to conventional and bortezomib therapies, without significantly affecting the viability of normal cells [397]. Other novel small molecule activators of SIRT-1 have been synthesized and identified by the same company, Sirtris Pharmaceuticals, including oxazolo [4,5-b] pyridine derivatives, structurally unrelated to and more potent than RSV. In particular, the most potent analog within the benzimidazole series, compound 17, showed 8-fold activation of SIRT-1 [398]. Other SIRT-1 activating compounds developed by this company are the imidazo[1,2-b] thiazole derivatives, potential new therapeutic agents for various metabolic disorders. The most potent analogue in this series, compound 29, showed oral anti-diabetic activity in three different rodent models of type 2 diabetes [399]. Moreover, 1,4-dihydropyridine derivative compounds have also been developed and shown to be potent, dose-dependent, SIRT-1, -2 and -3 activatiors [400]. Finally, Sirtris Pharmaceuticals has recently started clinical studies with novel highly-selective non-resveratrol-related SIRT-1 activators. One of them, namely SRT2104, was tested on male and female volunteers in a series of Phase I clinical studies, to elucidate its tolerability and pharmacokinetics. SRT2104 was well tolerated in all of these studies. Although there were no substantial differences resulting from gender or formulation, a notable food-related effect was observed, in that levels of circulating unmodified drug in individuals receiving SRT2104 immediately after a meal were approximately 4-fold those measured in fasted individuals. Exploratory clinical trials in patient populations are not yet ongoing, but preclinical testing in murine models [unpublished data, as reported in 401] have shown SRT2104 to be effective against inflammation [lipopolysaccharide (LPS)-induced tumour necrosis factor- (TNF-) production, dextran sulphate sodium (DSS) and trinitrobenzesulphonic (TNBS) acid-induced colitis, caecal ligation and puncture-induced sepsis, experimental autoimmune encephalomyelitis (EAE)] and diabetes (improved glucose and insulin homeostasis in DIO mice and ob/ob mice) [401]. 5.1. Rapamycin and Rapalogs As reported in section 4.5, TOR is implicated in aging since its down-regulation is associated with a significant lifespan expansion in C. elegans, Drosophila, and mice [242, 243] and mTOR signalling has been associated with old age in man [247]. Therefore rapamycin, from which TOR (target of rapamycin) derives its name, may have potential applications as anti-aging molecule and CRM. Rapamycin, also known as sirolimus, was first discovered in 1975 as a macrolide antibiotic with strong antifungal activity, produced from the bacteria Streptomyces hygroscopicus, isolated from soil on the island Rapa Nui (hence its name) [402]. It binds to FRAP⁄FKBP512 and forms a ternary inhibitory complex with TOR kinase (mTOR in mammals) in TORC1, thus impeding downstream


phosphorylation events that would normally stimulate protein synthesis and cell growth [for a review see 240, 403]. TORC2, unlike TORC1, is considered to be rapamycin-insensitive [404] although, in certain cell types, long-term rapamycin treatment in cultures has been found to induce inhibition of TORC2 assembly and to reduce TORC2 activity [405]. Today rapamycin and its derivatives are used clinically as immunosuppressants to prevent rejection after organ transplantation, in particular in kidney transplant patients [406]; a rapidly expanding literature addresses their anti-proliferative and anti-cancer actions [407]. In this connection, rapamycin was found not only to prevent tumors in kidney transplant patients [407], but also to induce regression in pre-existing tumors [408-410]. Moreover, quite recently rapamycin has been shown to delay spontaneous cancer in 129/Sv mice, which have normal lifespan and cancer incidence [411] as well as in cancer-prone (heterozygous p53+/ [412] and HER-2/neu transgenic [413]) mice. As regards aging, rapamycin has been shown to increase lifespan in yeast [414], fruit flies [415] and nematodes [416] and to slow down senescence in normal [417] and progeric [418] human cells. Moreover, rapamycin extends both mean and maximum lifespan in normal [411], genetically-heterogenous [419] and cancer-prone mice [412, 413]. In particular, the maximum lifespan was increased by 9.3%, 28% and 12.4% in 129/Sv, heterozygous p53+/ and HER-2/neu transgenic mice, respectively [411-413]. Interestingly, while inbred 129/Sv mice and HER-2/neu transgenic were given rapamycin intermittently (three times a week for 2 weeks, followed by a 2-week break) lifelong, starting from 2 months of age [411], the genetically-heterogeneous mice were fed rapamycin starting relatively late, from 9 or 20 months of age (corresponding roughly to 50-60 years in humans [420]), and this nevertheless afforded an increased maximum lifespan in both male and female mice, extending it by 9% and 14%, respectively [419, 421]. Harrison was awarded the Methuselah mouse prize in 2010 for this large-scale study; it is part of the NIA Intervention Testing Program, which evaluates several substances that may extend lifespan in genetically-heterogeneous mice. The program is organized at three different sites in the USA: the Jackson Laboratory, the University of Michigan and the University of Texas Health Science Center (http://www.nia.nih.gov/ ResearchInformation/ ScientificResources/ InterventionsTestingProgram.htm) [422]. Apart from its non-competitive inhibitory effect on TOR/mTOR kinase, other mechanisms claimed to underlie rapamycin’s pro-longevity effect may depend on its action on factors downstream of mTORC1. In this connection, rapamycin-fed mice showed a significant decrease of phosphorylated ribosomal protein subunit S6 (rpS6, a substrate of S6K1 in the mTOR signaling pathway [403]) in the adipose tissue, compared to control animals [419], suggesting a decrease in S6K1 activity, shown to extend the lifespan in yeast, C. elegans, and Drosophila [245, 248, 249] as well as in mice [243]. In support of this possibility, rapamycin did not extend the lifespan of flies overexpressing S6K [415]. Moreover, rapamycin inactivates eukaryotic initiation factor eIF4E, leading to decreased cap-dependent translation [423]; the downregulation of various components of the eIF4E cap-binding complex has been shown to extend the lifespan of C. elegans [424]. An alternative possibility is that rapamycin may favour longevity by enhancing autophagy: it has been shown that mTORC1 suppresses the activity of the serine/threonine kinase unc-51-like kinase 1 (ULK1 or Atg1), an initiator of autophagy, while rapamycin upregulates Atg1 thus promoting autophagy [425]. Besides its clinical use as an immunosuppressant to prevent rejection of kidney transplants, rapamycin has shown promise in pre-clinical trials that it may be used to treat age-related diseases, since it delays atherosclerosis, osteoarthritis, type II diabetes, metabolic syndrome, neurodegeneration, organ fibrosis, age-related

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macular degeneration, and cancer [407, 426, 427]. Rapamycin has thus been proposed for use to extend the healthy lifespan, by slowing down the aging process [428]. The main drawback to such pro-longevity/therapeutic use might be that rapamycin may show unwanted side effects. It has been reported that rodents treated with rapamycin or rapalogues become diabetic and hyperlipidemic [429, 430], with hyperglycemia, glucose intolerance, higher levels of free fatty acids in skeletal muscle, hypertriglyceridemia and hypercholesterolemia [429]. Rapamycin treatment induces a size decrease in the pancreatic islets, with a decrease in glucose-stimulated insulin synthesis and secretion, and a 90% decrease in circulating insulin levels [430]. As has been said, rapamycin and rapalogues are also immunosuppressants [406, 407] and some kidney transplant recipients under chronic treatment with rapamycin presented oral ulcers, skin lesions (e.g., acne) and various forms of edema, together with impaired wound healing, lymphoceles, delayed graft function, anemia, pneumonia, hypercholesterolemia and proteinuria [407, 431]. However, quite recently Blagosklonny reveiwed some of these adverse side effects, particularly those related to insulin resistance [432]: he believes that rapamycin is a starvation-mimetic, causing metabolic alterations typical of starvation diabetes, a reversible condition described 160 years ago [for references see 433], including gluconeogenesis, ketogenesis, insulin resistance, low insulin levels, and increased lipolysis, thus inducing a starvation-like diabetes, which, unlike true diabetes, prevents diabetic complications [432]. Several studies, indeed, have shown that rapamycin prevents nephropathy [434-436], nutrient-induced insulin resistance in humans [437] and recently also age-related macular degeneration in an animal model [438]. Moreover, diabetes incidence in renal transplant patients was only slightly increased by chronic rapamycin administration, and showed a late symptomatology [432]. Further, rapamycin prevents atherosclerosis in rodents [439-441], coronary re-stenosis in humans [442, 443], and inhibits cellular proatherogenic effects [444-446]. Moreover, rapamycin exerts antiosteoporotic action, by blocking bone resorption through inhibition of osteoclast formation and activity [447], and anti-fibrotic action, by modulating collagen and matrix metalloproteinase gene expression [448]. It has been suggested that rapamycin’s adverse side effects might be prevented by its intermittent, rather than chronic, administration [426, 432]. As already said, rapamycin given intermittently extended the lifespan in mice [411, 413], may rejuvenate stem cells and improve wound healing [449]; as a single dose it reverses insulin resistance and does not influence mTORC2 [404]. Rapamycin could be administered in association with other drugs that control its adverse effects, for example an association with resveratrol might be particularly interesting. Be that as it may, quite recently rapamycin was proposed as a therapeutic anti-aging agent for treating Hutchinson-Gilford progeria [450], since increased autophagy induced by rapamycin reduces the accumulation of progerin, an alternate spliced form of lamin A/C that forms insoluble toxic aggregates leading to growth inhibition, epigenetic dysregulation, and genomic instability. Rapamycin has also shown beneficial effects in a mouse model of Duchenne muscolar distrophy, with significant decreased muscle fiber necrosis, probably thanks to decreased immune-cell infiltration [451]; however, several studies show that rapamycin has detrimental effects on healthy muscle, inhibiting protein synthesis after exercise [452-454]. It has been suggested that rapamycin might be a good tool for cancer prevention in patients with Li-Fraumeni syndrome [412], an autosomal dominant disorder with a germline p53 mutation, which increases the incidence of cancer by 50% at the age of 40 and by 90% at the age of 60 [455]. In the past 10 years, several pharmacological rapamycin analogs (or rapalogs) have been designed, e.g. CCI-779/temsirolimus


(Wyeth/Pfizer), RAD001/everolimus (Novartis) and AP23573/ ridaforolimus (Ariad). They have the same mechanism of action as rapamycin, but different pharmacokinetic properties due to different substitutions at the C40 position [456]. They have been approved for cancer therapy, and several clinical trials are ongoing for the treatment of metastatic renal cell carcinoma and metastatic nonclear cell carcinoma [457, 458], subependimal giant cell astrocytoma, different hematological malignancies, sarcoma, lung, gastric, endometrial and prostate cancers [457]. More recently, everolimus has also been approved for treatment of neuroendocrine tumours of the pancreas [457, 459] and temsirolimus and everolimus, alone or in association with other drugs, have been proposed for breast cancer treatment [457, 460]. Since rapamycin is very little hydrosoluble, and is not very stable in water, different oral preparations such as microemulsions [461], liposomes [462, 463], nanoparticles [464] and solid dispersions [465, 466] have been formulated to increase its bioavailability and effectiveness; these have been tested in pre-clinical and clinical studies. Recently, a novel formulation of rapamycin, called Rapatar, based on Pluronic block copolymers as nanocarriers, has been shown to have significantly higher bioavailability after oral administration than classic rapamycin, to inhibit mTOR in mouse tissues, and to increase lifespan and delay carcinogenesis in highly-tumourprone p53-deficient mice if given life-long [467]. Importantly, the biological effects of Rapatar were prominent at low doses (0.5 mg/kg) and intermittent schedules [467]. On the basis of its efficiency in delaying age-related diseases and in increasing lifespan, Blagosklonny has proposed re-labelling rapamycin, from immunosuppressant to aging-suppressant (gerosuppressant) [468]. Neverthless, more studies are necessary to clarify the benefits and side-effects of rapamycin and rapalogs, not only in mice but also in higher mammals, as possible cancer-preventive and anti-aging agents. In this connection, positive findings about the efficiency of intermittent rapamycin treatment, which might decrease the side effects linked to its chronic administration, are very encouraging [411, 413, 426, 432]. 5.2. 2-Deoxy-d-Glucose and Other Glycolysis Inhibitors The hypothesis that glycolysis inhibition may induce cellular reactions similar to those induced by CR was first proposed by Lane and colleagues [469]: lowering cellular energy production might reproduce a situation similar to that caused by lowering the calorie intake. The glucose analog 2DG is a promising candidate, since it is taken up by cells and, being not further metabolizable, competitively inhibits glucose utilization [469], thus resulting in decreased energy production. 2DG has been shown to increase the lifespan of Caenorhabditis elegans [470] and short-term 2DG treatment induced some characteristics of the CR phenotype in rats, namely decreased insulin levels and body temperature [266], and increased levels of glucocorticoids [471] and heat shock proteins [472]. However, long-term treatment proved to be cardiotoxic, and to increase the incidence of pheochromocytoma in the adrenal medulla [473]. It has recently been suggested that 2DG, like CR, might influence the lifespan by increasing SIRT-1 [474] and AMPK activities [475]. It appears probable that other glycolysis inhibitors, such as glucose transporter blockers or various glycolytic enzyme inhibitors, might prove to be good CRMs. In this connection, iodoacetate has been shown to protect fetal rat hippocampal neurons against stress, mainly through up-regulation of HSPs [476]; however, more recently it was shown to be neurotoxic [477, 478]. Some years ago, Roth and Ingram’s group proposed mannoheptulose, a sevencarbon sugar contained in avocado and an inhibitor of hexokinase II, as candidate CRM [479, 480], but no reports have yet emerged concerning studies in progress.


15

Since the glycolytic pathway is particularly active in many tumour types, glycolysis inhibitors are currently widely studied as possible anti-tumour agents [266]. 5.3. Metformin, Thiazolidinediones and Other Insulin/AMPK Activators As reported in section 4.4, the insulin/IGF-1 signalling pathway has been suggested to play a role in the CR-induced regulation of lifespan, in different animal species [227]. Indeed, some characteristic effects of CR are decreased circulating insulin and glucose levels, and increased insulin sensitivity [65, 67, 70, 72]. Thus molecules reducing insulin and glucose might be considered as potential CRMs [260], and promising candidates are metformin (N,Ndimethylbiguanide) and its biguanide correlates, phenformin (1phenylethylbiguanide) and buformin (1-butylbiguanide hydrochloride) [481]. They decrease glucose absorption in the gastrointestinal tract, and reduce hepatic gluconeogenesis, while increasing insulin sensitivity and glucose uptake, resulting in a reduction of glycemia; they are thus important anti-diabetic treatments [482]. However, phenformin and buformin are no longer in use in many countries, due to a high risk of causing lactic acidosis [483]. However, these drugs have been shown to extend the lifespan in Caenorhabditis elegans, mice and rats [484, 485]. As regards metformin, it, too, has been reported to exert a positive effect on the lifespan of Caenorhabditis elegans [486], mice, and rats [for a review, see 481], although this prolonged lifespan is in many cases in cancer-prone animals, and is associated with a delay in the appearance of tumors [481, 487]. Metformin may be associated with a reduced risk of cancer in humans and animals [488]; it has also been shown to improve clinical outcomes in type II diabetes and cardiovascular disease (CVD) patients [489] and to decrease the occurrence of some age-related diseases, such as CVD [490] and chronic kidney disease [491]. Although metformin ameliorates the health span in case of disease, to our knowledge there is no record to date showing that metformin can induce CR-like effects in healthy mammalian models. In this connection, Smith and colleagues have recently reported contrasting results: they found no increase in lifespan in healthy rats treated with metformin [492]; gender differences in the positive effects of metformin have also been shown, in that female rats seem to be more sensitive than males [493]. Moreover, in many studies metformin has been reported to decrease food intake [494, 495] suggesting that at least in part its lifespan extending effect could be due to CR. Indeed, in the liver of elderly mice, 8 weeks treatment with metformin reproduced the specific changes in gene expression profiles produced by longterm calorie restriction more closely than did 8 weeks CR [267, 496]. Even if research is increasingly addressing the physiologically positive effects of metformin, its precise molecular target/s are still unknown. It was initially thought to inhibit the mitochondrial electron transporting enzyme complex I of the respiratory chain [497], whereas it has subsequently been shown to activate AMPK [498, 499] and SIRT-1 [500] and to inhibit mTOR [501], thus mimicking CR’s action. Interestingly, AMPK activity is also regulated by liver kinase B1(LKB1) [502], which is required for metformin to lower glycemia [503]. Moreover, activation of AMPK by metformin is reportedly mediated by mitochondria-derived RNS, and activation of the c-Src/PI3K pathway might generate a metabolite or other molecule inside the cell to promote AMPK activation by the LKB1 complex [504]. In favour of LKB1 and AMPK involvement in metformin’s lifespan-extending action, in Caenorhabditis elegans mutants for AMPK (aak-2) and for LKB1 (par-4), this pro-longevity effect is abolished [486]. Thus, experimental data in general point to a key role of AMPK in the biological action of metformin, but it has not yet been clarified whether AMPK is activated by metformin directly or indirectly, through the modification of cellular energy status and AMP/ATP ratio and/or by upstream LKB1. Possibly metformin may activate different targets in different tissues, with


different timing. Metformin is thus a feasible CRM, deserving of continued research on the basis of its broad clinical use. Quite recently, a novel class of CRM was identified: the thiazolidinedione family of PPAR agonists (troglitazone, ciglitazone and others) [505, 506]. These compounds are widely used in the oral therapy of type II diabetes, since they enhance insulin sensitivity, by activating the transcription of insulin-sensitive genes coupled with glucose homeostasis, fatty acid metabolism and triglyceride accumulation in adipocytes [507]. Four different thiazolidinediones (troglitazone, ciglitazone, STG28 and OSU-CG12) have been shown to evoke a CR-like response in LNCaP prostate-cancer and MCF-7 breast-cancer cells, including activating AMPK, reducing the glycolytic rate, and causing transient induction of SIRT-1 [268]. In particular, OSU-CG12 has been shown to induce a higher response compared to those of two classical CRMs, resveratrol and 2-DG [268]. Recently, novel thiazolidinedione agents have been developed that, at low μM concentrations, exhibit the ability to activate AMPK independently of PPAR in human THP-1 macrophages [508]. Another putative target for developing CRMs is a novel insulin signalling molecule, WD-repeat protein 6 (WDR6), which interacts with LKB1 and might thus be implicated in the glucose and lipid metabolic changes in CR [509]. 5.4. Other Molecules As reported in section 3, aging is associated with a decline in autophagy, which is reversed by CR [143, 144, 149]. Bergamini’s group has shown that treatment with antilipolytic drugs can stimulate autophagy, also decreasing plasma free fatty acids, glucose and insulin, similarly to CR [150]; they may protect from age-related hypercholesterolemia [510]. In hepatocytes from old rats that had been given an antilipolytic synthetic analogue of nicotinic acid (Acipimox, 5-methylpyrazine-2-carboxylic acid-4-oxide) intragastrically, once-a-week, lifelong the age-induced accumulation of dolichol and decline in autophagic proteolysis were both prevented [511]. Moreover, as far as “mitophagy” is concerned, the same treatment prevented the age-related accumulation of oxidatively damaged mitochondrial DNA [512]. Acipimox has also been shown to decrease insulin and to enhance insulin sensitivity in metabolic syndrome patients [513]. Obviously, significant anti-aging effects might be obtained using drugs that stimulate autophagy by inhibiting mTOR. In this connection, Shinmura and coworkers quite recently showed that the positive effects of CR on age-related cardiac dysfunction may be a consequence of mTOR inhibition and autophagy stimulation [514]. However, it must be kept in mind that these drugs also exert immunosuppressive and cytostatic effects [515, 516] and that their continuous usage increases the risk of diabetes and cause hyperlipidemia [517, 518]. Another molecule proposed as CRM is -lipoic acid (LA) [269]. As reported in section 3, older animals present higher levels of oxidatively-damaged macromolecules, including mtDNA [105]. This may cause a decline in mitochondrial function with a further enhanced production of ROS. LA, a potent thiol antioxidant and mitochondrial metabolite, appears to increase low-molecularweight antioxidant status, and thereby decrease age-associated oxidative damage and mitochondrial dysfunction, in the rat heart and brain [519, 520]. This protective effect has been claimed to be associated with an improvement of age-associated cognitive dysfunction of the brain [521]. Microarray studies show that, similarly to CR, LA may inhibit some age-induced alterations of gene expression, thus reducing oxidative stress in the heart, although it had no effect on longevity in mice [109]. However, more recently dietary supplementation with LA hes been found to exert differing effects on rat survival, based on the dietary history of the animals: LA appears to set the survival curve to that determined by the initial feeding regime, i.e. it maintains the extended lifespan in animals that

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change from CR to AL feeding supplemented with LA, whereas in animals undergoing the reverse sequence, from AL feeding supplemented with LA to CR, it prevents any CR-related pro-longevity effect [269]. Thus, the administration of substances that protect the mitochondria from oxidative damage, and that consequently improve mitochondrial function, may be a good approach to mimic at least some of CR’s beneficial effects. LA is an antioxidant, and in general dietary antioxidants may delay specific aspects of aging [270]. In this connection, high levels of antioxidants are present in some plants, which in addition may regulate the activity of different enzymes and genes involved in modulating lifespan and age-related diseases [522-524]. A number of these so-called “nutraceuticals” have been demonstrated to act positively on lifespan and healthspan in different animal models. For example, nectarine extract, rich in various antioxidant compounds [525], promotes longevity and healthspan of Drosophila melanogaster, partly by modulating glucose metabolism and reducing oxidative damage [526]. Apple procyanidins extend the mean lifespan of Caenorhabditis elegans [527] and Drosophila melanogaster [356] and show neuroprotective action, suppressing amyloid -protein aggregation in PC-12 cells [528], in part by modulating oxidative stress response genes. The same pro-longevity effects, occurring through modulation of the expression of several agingrelated genes, have been demonstrated in the same animal models for curcumin, an extract from the rhizome of the plant Curcuma longa (turmeric) [529, 530]. Daily consumption of catechins derived from green tea, even starting in adulthood, protects against oxidative damage and suppresses age-related brain dysfunction in mice [531]. Finally, a freeze dried preparation of the pulp of the fruit of the açaí palm (Euterpe oleracea Mart.), native to South America and abundant in the Amazon River region, was shown to contain high levels of phytochemicals [532, 533] and to extend the lifespan in wild-type flies on a high-fat diet, as well as in sod1 knockdown flies on a standard diet [534]; moreover, its administration even in later life is sufficient to promote survival in sod1 knockdown flies, by reducing oxidative damage [535]. As regards humans, a pilot study showed that açai pulp decreases several risk markers of metabolic disease in overweight subjects, including fasting glucose and insulin levels, total and LDL cholesterol levels, the ratio of total cholesterol to HDL cholesterol and post-prandial glycemia [536]. On these foundations, it is conceivable that nutraceuticals may be considered as viable and cost-effective interventions to promote healthy aging in humans. 6. SCREENING FOR CR MIMETICS In the light of this huge bulk of data, it is important to assess screening systems that might be employed to identify possible CRM candidates. Testing the action of candidate CRM substances directly on mammalian lifespans takes time, is expensive, and may raise methodological queries [537]. Consequently, only a few compounds have entered the NIA and the NIH Interventions Testing Program [for a review see 261]. An option now emerging for direct drug testing on lifespan is known as expression-based screening for CRM [496]; in this procedure laboratory animals are treated with the candidate CRM, and gene expression modifications measured in tissue samples by microarrays are compared to those produced by CR. An evolution of these screening processes is based on the secreted alkaline phosphatase (SEAP) reporter system, commonly used to study the activity of known or putative enhancer/promoter elements [538]. This system has been adapted to detect the regulatory elements of supposed pro-longevity genes that are up-regulated by CR (CRISP—the CR-Imitating agent Screening Platform). DFCR-RE2 and -RE4 were identified as being activated by CR, and DFCR-RE reporter constructs have also been shown to be upregulated by CR, thus molecules that activate this reporter may be promising CRM candidates [539, 540]. Very recently, Fortney and colleagues developed an “in silico” version of expression-based CRM screening, which allows many drugs to be tested at the same


time. They made a cross-analysis of transcriptional effects of CR in mouse liver, and a public genome-scale data on the human cell line response to more than 1000 drugs, followed by a meta-analysis that pointed to 14 substances as being fair CRMs [541]. Since many data on CR’s transcriptional effects are available for the mouse and rat, but also some for primates and a few for humans, this method could be applied to speed up the identification and development of “longevity” drugs. CONCLUSIONS The pharmacological or nutritional manipulation of endogenous cellular mechanisms leading to the activation of cytoprotective genes of the cell life program, and to the down-regulation of inflammatory and oxidative processes, is potentially a novel therapeutic strategy for age-associated diseases, which might help to achieve healthy prolonged aging. In the light of subjects’ poor adherence to to continuous low calorie restricted diets, studies aimed at determining the feasibility and efficacy of natural and/or pharmacological CR mimetic molecules are ever-more-numerous. However, in the words of the Italian novelist Alessandro Manzoni, "Ai posteri l'ardua sentenza": only posterity will be know whether these prospects prove true or illusory. CONFLICT OF INTERESTS The authors confirm that this article content give rose to no conflict of interests. ACKNOWLEDGEMENTS The authors thank the Italian Ministry of the University (Prin 2009), the CRT Foundation, Turin, and the University of Turin, Italy, for supporting this work.

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Received: June 13, 2013

Accepted: September 24, 2013

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