Calorie Restriction Mimetics: an Emerging Research Field

Aging Cell (2006) 5, pp97–108 Doi: 10.1111/j.1474-9726.2006.00202.x

Blackwell Publishing Ltd REVIEW Calorie restriction mimetics: an emerging research field

Donald K. Ingram,1 Min Zhu,1 Jacek Mamczarz,1 fully assess the potential of any CRM. Nonetheless, this Sige Zou,1 Mark A. Lane,1 George S. Roth2 and strategy clearly offers a very promising and expanding Rafael deCabo1 research endeavor. 1Laboratory of Experimental Gerontology, Intramural Research Key words: aging; cancer; glucose; insulin; metabolism; stress Program, National Institute on Aging, 5600 Nathan Shock Drive, 2 Baltimore, MD 21224, USA, and GeroScience, Inc., Pylesville, Introduction MD 21132, USA It is intriguing that one of the oldest paradigms in gerontology is providing the newest synthesis of findings regarding mech- Summary anisms of aging and suggesting directions for intervention that When considering all possible aging interventions evalu- could prove to be highly fruitful. The formal study of calorie ated to date, it is clear that calorie restriction (CR) remains restriction (CR), also known as food restriction or diet restriction, the most robust. Studies in numerous species have is most often traced to the pioneering nutritional research of demonstrated that reduction of calories 30–50% below ad Clive McCay and colleagues at Cornell University beginning in libitum levels of a nutritious diet can increase lifespan, the 1930s (McCay & Crowell, 1934; McCay et al., 1935). As reduce the incidence and delay the onset of age-related reviewed by Kritchesky (1993), however, reports of CR effects diseases, improve stress resistance, and decelerate func- on cancer growth appeared much earlier in the work of More- tional decline. A current major focus of this research area schi in 1909 and Rous in 1914. Results of many subsequent is whether this nutritional intervention is relevant to studies in different laboratories have demonstrated that sub- human aging. Evidence emerging from studies in rhesus stantial reductions of food intake to 30–60% below ad libitum monkeys suggests that their response to CR parallels that (AL) intake levels can increase lifespan (median and maximum) observed in rodents. To assess CR effects in humans, clin- in a wide variety of species (Weindruch & Walford, 1988; Yu, ical trials have been initiated. However, even if results 1994; Weindruch & Sohal, 1997; Masoro, 2000). Moreover, in from these studies could eventually substantiate CR as hundreds of rodent studies ranging over several decades, CR an effective pro-longevity strategy for humans, the utility has been shown to decrease the incidence and age of onset of this intervention would be hampered because of the of many age-related diseases, increase resistance to toxicity and degree and length of restriction required. As an alternative stress, and maintain function at more youthful-like states strategy, new research has focused on the development compared to controls eating at or near AL levels (Weindruch & of ‘CR mimetics’. The objective of this strategy is to iden- Walford, 1988; Yu, 1994). Compared to numerous attempts to tify compounds that mimic CR effects by targeting met- manipulate the rate of aging in animal models, CR remains the abolic and stress response pathways affected by CR, but most robust aging intervention studied to date. without actually restricting caloric intake. For example, Given its robust nature and continued research prominence, drugs that inhibit glycolysis (2-deoxyglucose), enhance the three most compelling questions driving recent CR research insulin action (metformin), or affect stress signaling are: (i) Is this intervention relevant to human aging? (ii) If so, pathways (resveratrol), are being assessed as CR mimetics can mechanisms of CR relevant to human aging be identified? (CRM). Promising results have emerged from initial stud- (iii) If so, can interventions be identified that operate through ies regarding physiological responses which resemble these mechanisms to mimic CR? those observed in CR (e.g. reduced body temperature and plasma insulin) as well as protection against neurotoxicity Relevance of calorie restriction to humans (e.g. enhanced dopamine action and up-regulated neurotrophic factors). Ultimately, lifespan analyses in addition Arguments have been presented that the marked increases in to expanded toxicity studies must be accomplished to lifespan observed in rodents on CR are not likely to be observed in humans on CR because humans have not evolved such mechanisms (Harrison & Archer, 1989; de Grey, 2005; Phelan & Rose, Correspondence Donald K. Ingram, Laboratory of Experimental Gerontology, Intramural 2005). CR may be more relevant to short-lived species in which Research Program, National Institute on Aging, 5600 Nathan Shock Drive, a major loss of food supply would be catastrophic, whereas Baltimore, MD 21224, USA. Tel.: 410-558-8180; fax: 410-558-8320; e-mail: humans have evolved cultural mechanisms for dealing with such [email protected] events and might not have to rely on such evolutionary adap- Accepted for publication 16 November 2005 tation. Using a mathematical model relating longevity to relative

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98 Calorie restriction mimetics, D. K. Ingram et al.

investment in reproductive effort, Phelan & Rose (2005) have restriction mimetics, that can provide the pro-longevity bene- estimated that CR should have 10 times greater effects on fits of CR without actually having to reduce caloric intake. longevity of rodents than of humans. Of course, many epidemiological studies have reported that Calorie restriction mimetics caloric intake is related to the incidence of several chronic diseases, including cardiovascular disease, cancer, diabetes, as The field of calorie restriction mimetics (CRM) is still in its infancy well as neurodegenerative disorders (Logroscino et al., 1996; but is attracting increasing attention (Lane et al., 2002; Ingram Mayeux et al., 1999; Willet, 2000; Astrup, 2001; Kritchesky, et al., 2004; Everitt et al., 2005; Roth et al., 2005). A recent 2001; Hursting et al., 2003). Studies of Okinawan centenarians search yielded 21 citations, the oldest citation dated 2001. support the view that a low-calorie diet can increase prospects Ironically, one of the citations that does not appear on PubMed for good health and longevity in humans (Suzuki et al., 2001). is the paper by Lane et al. (1998) that first introduced the con- Findings from the small group of volunteers confined to Bio- cept and the first candidate CRM drug. sphere 2 confirmed that CR (∼30%) could be imposed for Unfortunately since that initial report, the concept itself has 2 years and would produce many of the physiological, hormo- evolved to a current context of broad connotation. To many nal, and morphological effects expected (Walford et al., 2002). researchers, CRM applies to any intervention that can evoke A recent study of self-selecting volunteers on CR also noted similar effects on aging, health, and lifespan to those of CR. remarkably low values on risk factors for cardiovascular disease Such interventions can include antioxidants, hormonal replace- (Fontana et al., 2004). These studies are valuable and clearly ment, metabolic enhancers, metal chelators, and even exercise suggestive of the relationship between caloric intake and aging (Hadley et al., 2001; Poehlman et al., 2001). In addition, appetite but are no substitute for experimental evidence. suppressants and even gastric bypass surgery might also qualify Experimental proof that CR can significantly increase lifespan as CRM because they reduce caloric intake. in organisms that live longer than rodents has been limited to We would argue that the concept of CRM should have a one study in dogs (Kealy et al., 2002) and one in cows (Pinney much more focused meaning and application. Specifically, we et al., 1972). Studies in nonhuman primates have indicated that propose the following features for any candidate CRM: (i) it many physiological responses in monkeys parallel those mimics the metabolic, hormonal, and physiological effects of observed in rodents on CR (Mattison et al., 2003; Roth et al., CR; (ii) it does not significantly reduce long-term food intake; 2004). In addition, primate studies relating several of these (iii) it activates stress response pathways observed in CR and responses to markers of disease risk, such as blood lipids and provides protection against a variety of stressors; and (iv) it hormones, also indicate a reduced incidence of chronic diseases, produces CR-like effects on longevity, reduction of age-related such as heart disease and diabetes (Roth et al., 2001). disease and maintenance of function. To assess the feasibility and potential of CR intervention in Taking this approach, the major question is what would humans, recent clinical studies sponsored by the National be the appropriate mechanism/s to target for a CRM? Several Institute on Aging were initiated at three sites – Washington mechanisms underlying the pro-longevity effects of CR have University, Tufts University, and the Pennington Center at Lou- been proposed and include the following: (i) reduced oxidative isiana State University (Heilbronn & Ravussin, 2003). These pilot stress and damage (Sohal & Weindruch, 1996); (ii) reduced studies have now been completed and provide valuable infor- glycation of macromolecules (Sell et al., 1996); (iii) reduced DNA mation on physiological responses to CR over a short-term damage and increased repair (Raffoul et al., 1999); (iv) reduced (6–12 months) as well as greatly needed information on pro- inflammation and autoimmunity (Chung et al., 2001); (v) increased cedures to obtain and maintain compliance for such regimens. mitochondrial metabolic efficiency to protect the plasma Another study which imposed alternate-day fasting was also membrane (de Cabo et al., 2004); (vi) reduced damage to completed by investigators at the Pennington Center which cellular components, such as lysosomes and peroxisomes, that showed that such regimens could produce many CR-like effects negatively impact autophagic proteolysis (Bergamini et al., but was questionable as a long-term diet strategy because of 2004); (vii) enhanced maintenance of age-related patterns of negative reports of hunger (Heilbroon et al., 2005). gene expression (Weindruch et al., 2001); and (viii) enhanced Even if science could advance to a point where it was indeed protection against stress, or hormesis (Masoro, 1998; Rattan, clear that 30–40% CR imposed in humans could reduce mor- 2001). Certainly it is possible that interventions providing pro- bidity and mortality and maintain function to the same degree tection against many of the deleterious processes listed above as observed in rodents, questions would remain; that is, could could produce CR-like effects, but would such interventions we do it? and would we do it? When considering the difficulty produce the robust, multisystem effects associated with CR? that Westerners have in maintaining low-calorie diets for prolonged periods (Burke et al., 1997; Knauper et al., 2005), much Hormesis less for a lifetime, the prospects for successful application of CR to humans on a broad scale would appear dim due to the severity In a recent, insightful review of CR and CRM, Sinclair (2005) and length of restriction required. The realization of this challenge pointed out that hypotheses pertaining to mechanisms of CR has generated interest in developing alternatives to CR, or calorie have for the most part been based on the view that underlying

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Calorie restriction mimetics, D. K. Ingram et al. 99 mechanisms were passive in their protective modes. In contrast, CRM have targeted energy pathways to mimic the physiological the hormesis hypothesis of CR posits an active role of stress responses of CR and to enhance stress responses. This strategy protection. Specifically, CR imposes a low level of stress on the emerged from the growing evidence that energy sensing pathways organism that in turn activates stress responses providing appear critical in regulating aging in a number of model systems. protection against a variety of aging processes. The concept of A widely cited example of the evolutionarily conserved and hormesis is derived from toxicology where the term implies that adaptive strategy of CR is the diapause found in the nematode, small doses of a toxin might have long-term beneficial consequences Caenorhabiditis elegans. When placed in low-energy environ- as a means of conditioning the organism toward enhanced ments, this small roundworm transforms to its dauer state in stress responses (Masoro & Austad, 1996; Rattan, 2001). which development and reproduction are arrested. Within the Such a concept would be in tune with an evolutionary per- dauer state, the nematode becomes more stress resistant and spective provided by the disposable soma theory of aging, which can manifest a lifespan exceeding that of its normal adult proposes that when faced with low energy availability, an form (Johnson et al., 2001; Lithgow & Walker, 2002). Careful organism must shift its energy investment away from growth research has helped to identify several key genes that regulate and reproductive processes to energy investment in somatic the conversion to the dauer form, among them age-1, daf-2, maintenance and repair (Shanley & Kirkwood, 2000). This com- daf-16, daf-18, akt-1, akt-2 (Vanfleteren & Braeckman, 1999). pelling hypothesis would predict that it is adaptive for the This signal transduction pathway is now considered to be organism under CR to use available energy to enhance its homologous to the mammalian insulin/insulin-like growth protection against stress (Masoro & Austad, 1996). It is well factor-1 (IGF-1) pathway (Gems & Partridge, 2001; Johnson established that CR induces stress, manifested as higher levels et al., 2001; Lithgow & Walker, 2002; Tatar et al., 2003). When of circulating glucocorticoids (Masoro & Austad, 1996). Evi- selected mutations of genes regulating this pathway are pro- dence of enhanced stress responses in rodents on CR has been duced, for example in daf-2, signaling through this pathway is produced in numerous paradigms (Masoro & Austad, 1996), for reduced to a point that the worm does not transform to the example, increased resistance to a variety of neurotoxins in rats dauer form but still exhibits increased stress protection and and mice on various regimens of CR (Bruce-Keller et al., 1999; extended lifespan (Gems & Partridge, 2001; Tatar et al., 2003). Zhu et al., 1999; Calinagasan & Gibson, 2000; Duan et al., These findings regarding reduced signaling through the 2001). Based on this perspective, we propose that it would be insulin/IGF-1 pathways have also been observed in mammalian productive to develop CRM that could target a broadly acting models with evidence of increased lifespan (Holzenberger mechanism, such as an enhanced stress protection. The objective et al., 2003). For example, knockout mice for the IGF-1 receptor should be to trick the organism into a CR state and thereby acti- show increased lifespan and increased insulin resistance to vate the protective mechanisms that are induced in CR. More- oxidative stress (Masternak et al., 2005). No other phenotypic over, the parallel objective should be to minimize any reduction effects are observed in this mouse as their energy metabolism, in actual caloric consumption. This latter objective is driven more nutrient uptake, physical activity, fertility and reproduction do by scientific interests than by practical considerations. CRM can not appear to differ from normal littermates. Similar results of best be evaluated when this objective of avoiding actual CR is lifespan extension have been reported from studies of transgenic achieved; otherwise, one is merely evaluating CR. rats (Shimokawa et al., 2002) and mutant dwarf mice (Bartke et al., 2001) in which signaling through growth hormone/IFG-1 pathways has been reduced, but these mutants differ markedly Downstream vs. upstream mechanisms in phenotypic characteristics from their normal controls. In his review, Sinclair (2005) agrees strongly with the hormesis Many investigators interpret findings from these studies of hypothesis and emphasizes the importance of identifying the invertebrates and vertebrates as reasons for attempting to iden- cellular and genetic mechanisms so that CRM could directly tar- tify single genes as the longevity regulators and, thus, the most get the ‘longevity regulators’. Such efforts have thus focused promising targets for driving the development of CRM (Sinclair, on downstream, nuclear positioned mechanisms of CR, such as 2005). However, an equally important conclusion to draw from the sirtuin class of genes (Sir2 and SIRT1, primarily) that act as these studies is that manipulations of energy-processing path- histone deacetylases regulating gene expression. Considerable ways can be made at various upstream and downstream points experimental support for the involvement of this pathway has with similar effects. When considering that downstream targets been provided in many invertebrate studies. For example in might be more effective than upstream targets for the produc- Drosophila, mutations that reduce the levels of the histone tion of CRM (Sinclair, 2005), it is important to remember that deacetylases, Rdp3 deacetylase (Rogina et al., 2002) and over- CR effects are produced by a very upstream manipulation – expression of Sir2 (Rogina & Helfand, 2004) increase lifespan. reducing the amount food available to the organism. Thus, as In addition, when CR is imposed in Rdp3 mutants or in mutants a screen for identifying candidate CRM, we would argue for a that reduce Sir2, then no increase in lifespan is observed. strategy that manipulates systems involved in energy sensing, In addition to downstream targets, we contend that CRM can regulation, and metabolism and then to look for physiological also be developed for specific upstream targets involved in energy hallmarks of CR and enhanced stress protection in relevant metabolism. Our initial efforts in developing the concept of model systems.

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reduce insulin and body temperature, with little or no effects Glycolytic inhibition on food intake and body weight. Therefore, 2DG appeared as In the first publication directly proposing the development of a promising CRM candidate. CRM, Lane et al. (1998) targeted glucose metabolism, specifi- However, why should effects on only two physiological mark- cally to reduce glycolysis without reducing food intake. For this ers evoke such confidence in the potential of this compound? initial study, the compound selected was 2-deoxy-D-glucose First, we would argue that reduced body temperature and (2DG), which inhibits the enzyme phosphohexose isomerase insulin levels are highly indicative of the altered metabolic state and thus reduces glycolytic processing. Results of previous that CR produces. Second, the validity of these biomarkers of rodent studies had shown that injections of 2DG could inhibit CR was further confirmed in an analysis of survival data in tumor growth (Gridley et al., 1985), produce torpor (Dark et al., human males derived from the Baltimore Longitudinal Study 1994) and increase glucocorticoids (Weidenfeld et al., 1994), all of Aging (BLSA). Analysis of the BLSA data revealed that the indicative of a CRM. In the initial evaluation of 2DG as a CRM, probability of survival in a healthy sample of men was greater young male Fischer-344 (F344) rats were fed diets supplemented in those with the lowest temperature and plasma insulin levels (by weight) with 0.2%, 0.4%, or 0.6% 2DG, approximating (Roth et al., 2001). − doses of 100–150, 250–300, or 400–450 mg kg 1, respectively. In addition to the in vivo data emerging from the initial studies Within the first few weeks, the high dose proved to be toxic, of 2DG, other in vitro studies confirmed the ability of the resulting in a number of deaths; thus, this group was then fed compound to enhance stress protection. For example, 2DG the 0.6% diet every other week which appeared to be well protected against glutamate excitoxicity in fetal hippocampal tolerated. Toxicity is predicted with a high 2DG dose because neurons (Lee et al., 1999). Confirmation of up-regulated stress of insufficient cellular energy due to an intolerably high degree responses observed in CR rats was produced in 2DG treated of glycolysis inhibition. After all, CR works only to a particular cultures showing, specifically, increased heat shock protein-70 degree of restriction, typically 60%. (HSP-70) and glutamate responsive protein-78 (GRP-78). After The 2DG diets produced physiological endpoints indicative 12 weeks of 2DG injections in rats, their cortical synaptosomes of CR. As presented in Figs 1 and 2, plasma insulin and body exhibited greater protection against iron and amyloid-β-peptide temperature were reduced in rats on the 0.4% and 0.6% con- (Guo & Mattson, 2000). In addition, HSP-70 and GRP-78 were centration 2DG diets, without significant reduction in plasma significantly elevated compared to control synaptosome pre- glucose. While all doses initially reduced food intake and body parations (Guo & Mattson, 2000). weight, the 0.2% and 0.4% groups caught up to controls within Other short-term in vivo studies have provided further con- a few weeks and by the end of the study at 6 months of age, firmation that 2DG can enhance stress protection. As examples, they did not differ significantly from controls, while the 0.6% group in a model of focal ischemia using middle cerebral artery occlusion- did maintain reduced body weights throughout the experiment. reperfusion, 2DG treatment attenuated damage similar to the The major findings from this initial study were that a 2DG degree observed in CR (Yu & Mattson, 1999). When mice are injected diet could affect two major biomarkers of CR, specifically to with the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

Fig. 1 Mean (SEM) plasma concentrations of glucose and insulin in male Fischer-344 rats fed control (CON) diet or diet supplemented with 2-deoxyglucose (2DG) in three concentrations. *Significantly different from CON group (data from Lane et al., 1998).

Calorie restriction mimetics, D. K. Ingram et al. 101

Fig. 2 Mean (SEM) body temperature recorded in male Fischer-344 rats fed control (CON) diet or diet supplemented with 2-deoxyglucose (2DG) in three concentrations. *Significantly different from CON group (data from Lane et al., 1998).

(MPTP), they show motor impairments related to depletion of enhanced locomotor response to amphetamine, and this dopamine resulting from neuronal loss in the substantia nigra; response is also observed in rats fed a 0.4% 2DG diet over the however, 2DG treatments provided greater protection against same period (Mamczarz et al., 2005). These results are depicted MPTP and faster behavioral recovery (Duan & Mattson, 1999). in Fig. 3, which presents locomotor responses first to placement Additionally, similar to in vitro results, 2DG treatment again within a novel environment followed by response to a saline increased HSP-70 and GRP-78 in the DA-rich brain regions of injection and then to the amphetamine injection. The increased these mice. locomotor response of the 40% CR group relative to controls Results from a recent long-term study in Sprague-Dawley rats following amphetamine treatment is readily evident. However, comparing 2DG feeding to CR also reported CRM effects of this the initial response of the 2DG group is equivalent to that of compound (Wan et al., 2003). Compared to AL controls, CR the CR group although the locomotor response dissipates more (fed every other day) and 2DG-fed groups alike had lower serum quickly in this group compared to CR animals. It was noteworthy glucose and insulin concentrations as well as reduced heart rate in this study that the 2DG-fed rats did not differ in body weight and blood pressure but not body temperature. However, food from AL fed controls. This behavioral paradigm might also prove intake and body weight of 2DG-fed rats were similar to controls highly useful for screening candidate CRM. Previously it has unlike the markedly reduced body weight of CR rats. In a follow- been noted that CR can attenuate the age-related loss of striatal up study, Wan et al. (2004) demonstrated that 2DG-fed rats dopamine D2 receptors (Roth et al., 1984). It would be inter- also exhibited increased recovery from stress measured as heart esting to determine if long-term 2DG feeding could produce rate, blood pressure, and body temperature following restraint similar results. and cold water stress. Beneficial effects of 2DG injections on tumor growth had Other 2DG studies have documented very interesting effects been reported previously, but a recent study involving 2DG on the brain to indicate additional parallels to CR. For example, feeding has shown that a dietary treatment at concentrations in studies of both CR and 2DG, the neurotrophic factor, brain- of the compound that do not affect food intake and body derived neurotrophic factor (BDNF), has been reported in several weight is also highly effective. Specifically, Zhu et al. (2005) studies (Mattson et al., 2003; Mattson, 2005) to be up-regulated reported that 2DG attenuated mammary tumor growth in in specific regions of the rat brain, although a recent study of female Sprague-Dawley rats produced by injection of 1-methyl- hippocampal BDNF in F344 × BN rats did not support this 1-nitrosourea. Moreover, these investigators used dietary con- finding (Newton et al., 2005). BDNF is involved in enhancing centrations of 0.02% and 0.03% 2DG after determining that dopamine neurotransmission and has been shown to be neuro- the concentrations used in the Lane et al. (1998) study produced protective (Hyman et al., 1991). Results of other studies have major body weight growth inhibition in these rats. Indeed, they demonstrated that short-term CR (e.g. 2 weeks) can increase found that 2DG concentrations of 0.06% disrupted normal dopamine-related locomotor responses in rats (Fuemayor & body weight growth. Even at the low concentrations used, 2DG Diaz, 1984). Specifically, rats exhibit hyperactivity when challenged still reduced serum insulin and raised serum corticosterone levels with the dopamine agonist, amphetamine, and this response is but had no significant effects on glucose, leptin or IGF-1-values. enhanced in CR rats. In a recent study from our laboratory, it It is clear that results have accumulated to suggest that 2DG has been shown that rats on CR for 4 months exhibit an presents a highly favorable profile as a CRM (Kang & Hwang,

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Fig. 3 Time course of locomotor activity of rats tested 4 months after initiating a diet regimen of ad libitum feeding (Control), 40% calorie restriction (CR), or 0.4% 2DG diet ad libitum. Mean locomotor response (SEM) accumulated over 10-min intervals consisted of (1) 120 min of spontaneous response; (2) 60 min of response following a saline injection; and (3) 180 min of response to an amphetamine − injection (0.75 mg kg 1 s.c.) (data from Mamczarz et al., 2005).

2006). The next major objective then would be to complete a receptor reported increased lifespan, and, additionally, CR was long-term study to evaluate the effects of the compound on ineffective in further lifespan extension (Bartke, 2005). Thus, mortality and morbidity. To meet this objective, a study of 2DG these results would support a major involvement of IGF-1 in the feeding (0.25% and 0.4%) was initiated in 6-month-old male pro-longevity effects of CR. F344 rats. After about 2 months on the diet, deaths were noted Similar effects on lifespan associated with manipulating the in the higher dose group, and these accelerated with time. insulin/IGF-like signaling (IIS) pathway has also been observed Based on preliminary pathological analysis, the primary cause in Drosophila, but the role of IIS in flies does not appear to be of death was congestive heart failure manifested as enlarged as robust as that observed in mice. The chico1 mutation in the hearts with marked vacuolation, which had been noted in the IIS pathway produces dwarf long-lived flies at normal nutrition, initial study in younger rats (Lane et al., 1998). We are currently and responds with increased lifespan under appropriate condi- completing this analysis in addition to assessing whether this tions of CR, thus leading to the conclusion that CR mechanisms cardiac pathology is observed in other rat strains. Notably Zhu of pro-longevity overlap with those involving IIS (Clancy et al., et al. (2005) did not report this pathology, but these investiga- 2002). Using mutations of the insulin/IGF-1-like (IIF) pathway tors used much lower doses of 2DG. in nematodes with various regimens of CR, Houthoofd et al. Many targets for inhibiting glycolysis exist; therefore, many (2003) concluded that the pro-longevity effects of CR were potential candidates could have potential efficacy as CRM. Targets independent of this pathway. In Drosophila, it appears that could include glucose transporters as well as other enzymatic other signaling pathways, particularly the target of rapamycin steps, such as hexokinase inhibitors. For example, another com- (TOR), appear to be critical for lifespan effects. For example, pound, iodoacetate acid, which inhibits glyceraldehyde-3- overexpression of TSC1 and TSC2 (tuberous sclerosis complex phosphate, has shown potential as a CRM based on in vitro genes 1 and 2) can inhibit TOR and increase lifespan substantially analysis. Pretreatment of fetal hippocampal neurons with this (Kapahi et al., 2004). compound provided protection against several stresses, includ- In their review of the literature, Hursting et al. (2003) also ing excessive glutamate, iron, and trophic factor withdrawal and argued for the primary role of IGF-1 in the antiaging and anti- also stimulated an up-regulation of heat shock proteins, HSP70 cancer effects of CR. A number of possible candidates for inhib- and HSP90 (Guo et al., 2001). iting the IGF-1 pathway were identified in this review including retinoids (e.g. fenretinide and all-trans retinoic acid), soy isofla- vones (e.g. genistein and daidzein), flavonoids (e.g. quercetin IGF-1 and kaempferol), somatostatin analogues (e.g. octreotide, and Evidence emerging from studies of dwarf mice has made a selective estrogen receptor modulators such as tamoxifen). strong case for the role of growth hormone and IGF-1 pathways Other strategies could include small molecule inhibitors of in aging and longevity (Bartke, 2005). However, the phenotypic IGF-1 as well as antisense IGF-1. Regarding 2DG in this context, complexity of these mutants made it difficult to specify exactly it is important to note in the Zhu et al. (2005) study described which pathways were involved. Moreover, CR was equally effec- previously, there was no effect of the 2DG supplemented diets tive in lifespan extension in these mice compared to controls. on plasma IGF-1 levels. Nonetheless, manipulations of IGF-1 A more recent study using a specific knockout of the IGF-1 remain a viable strategy for developing CRM.

2005). In addition, epidemiological studies have indicated that Insulin sensitizers resveratrol consumption can lower the risk of many age-related Compounds that increase sensitivity to insulin offer another diseases, including cancer (Sinclair, 2005). Presumably then, area for development of CRM. As described previously, lower resveratrol can meet the criteria presented above as a CRM circulating insulin is a key biomarker of CR and appears predic- although these pro-longevity effects have yet to be confirmed tive of longevity (Roth et al., 2001). Anisimov (2003) provided in mammalian systems. Nonetheless, the results are certainly an excellent review of possible interventions that affect insulin/ promising. In addition, research on a variety of other STACs, IGF-1 signaling pathways with a major emphasis on the bigua- with possibly even more robust effects than resveratrol, continue nides. Dilman (1971) was instrumental in bringing attention to to emerge, including particularly plant polyphenolics (Quideau, the class of compounds that are derived from the French lilac 2004; Sinclair, 2005). (Galega officinalis) as potential antidiabetic drugs. Compounds of interest include phenformin, buformin, and metformin, PPARs and thiazolidinediones which collectively have been shown to increase glucose utilization and reduce hyperglycemia, free fatty acid utilization, Other possible candidates as CRM likely exist among the large gluconeogenesis, intestinal glucose absorption, serum lipids, class of agonists for peroxisome proliferator activated receptors insulin, and somatomedin. In a number of rodent studies, phen- (PPARs). Many such compounds are under intense development formin has been shown to increase lifespan and reduce cancer for the treatment of obesity and diabetes. There are currently (Anisimov, 2003). Regarding stress protection, phenformin has two approved drugs in the class of thiazolidinediones – rosiglita- been reported to increase resistance to glutamate toxicity in zone and pioglitazone – that act on the PPARγ isoform of these primary neuronal cultures (Lee et al., 2002). Clinical use of receptors. phenformin in the treatment of type 2 diabetes was attempted, There is clear linkage between metabolic consequences of but the drug was removed from the market due to the severe CR and activation of the fasting-responsive transcriptional lactic acidosis observed in a number of patients. Metformin is coactivator, peroxisome proliferator-activated coactivator 1α now a widely prescribed medication for treating type 2 diabetes (PGC-1α); however, sorting out the specific beneficial effects that can be used alone or in combination with sulfonylurea of the different PPAR agonists as CRM will require considerable drugs (Kirpichnikov et al., 2002). In addition to improving the research investment. Using gene transcript profiling in mice, metabolic profile of diabetes, increased survival from all-cause Corton et al. (2004) made a strong case that manipulation of mortality has been associated with metformin treatment in both PPARα parallels that observed in CR. In vivo studies in nonhuman diabetic and cardiovascular disease patients (Scarpello, 2003; primate models of obesity and diabetes have also indicated the Eurich et al., 2005). therapeutic potential of a PPARα activator. Results included In an impressive study in mice evaluating the potential of reduced hyperinsulinemia and improvement of insulin-stimulated several candidate CRM using microarray profiling, Dhahbi et al. glucose uptake rate as well as lowered triglycerides and body (2005) reported that the 8 weeks’ treatment with metformin weight (Schafer et al., 2004). Use of a PPARδ activator also produced a gene expression pattern that was closely aligned markedly improved lipid and insulin profiles in this primate with long-term CR. Other candidate CRM were also evaluated model (Oliver et al., 2001). in this study including glipizide, rosiglitazone, and soy isofla- PPARγ activators can also increase sensitivity to insulin, but vone, but the metformin profile was far superior to these com- recent studies have begun to question the rationale for this pounds in reproducing the gene profile of CR. The authors strategy. Rather than activating this pathway, a more effective made a strong case for the utility of this technology in screening strategy for treating metabolic disorders might be to modulate candidate CRM. PPARγ activity to improve glucose homeostasis while preventing adipogenesis (Argmann et al., 2005). A connection between SIRT1 and PPAR has been made because SIRT1 represses PPARγ Sirtuins transactivation and inhibits lipid accumulation in adipocytes Growing interest in the sirtuins as possible downstream medi- (Picard & Guarente, 2005). It has been hypothesized that reduc- ators of longevity in a number of organisms has stimulated the ing adipose tissue could have beneficial effects on lifespan development of sirtuin-activating compounds, or STACs (Sinclair, related to the production of adipokines acting on target tissues, 2005). Howitz et al. (2003) described the results of a screen to due to the indirect prevention of age-related metabolic disorders identify activators of human SIRT1 and reported 18 small mol- including diabetes or atherosclerosis (Picard & Guarente, ecules, including butein, piceatannol, and resveratrol. Of these 2005). molecules, resveratrol has received the most attention. This polyphenol has been shown to increase lifespan in nematodes Lipid targets and fruit flies in a Sir2-dependent manner that appeared to mimic CR (Wood et al., 2004). In vitro studies have also dem- Pharmacological manipulation of lipids offers many additional onstrated its abilities to protect against a wide range of stressors, targets for candidate CRM; however, concepts for intervention including oxidative stress, radiation, and ischemia (Sinclair, are currently much less developed than for other targets. For

example, similar to the effects of CR, use of hypolipidemic drugs nematodes or fruit flies, to assess response to in vivo stressors, has shown efficacy in removal of damaged cellular components such as heat or hydrogen peroxide, and also to quantitate survival through enhanced autophagy (Donati et al., 2004). On the as an endpoint. other hand, higher circulating levels of certain lipids, such as The recent development in our laboratory of an in vitro model long-chain fatty acids, during CR may serve as an important of CR can also help accelerate development of CRM (de Cabo signal for generating effects on tumor suppression, and enhanced et al., 2003). A candidate CRM can first be administered to rats immune responses as well as protection against oxidative stress and mice over a short duration, e.g. 4 weeks. Then blood can (Yu, 1994). Higher levels of ketone bodies have been shown to be withdrawn and serum prepared and added to an in vitro provide protection against neurotoxic insults (Veech, 2004); cell system that is then subjected to a stress, such as heat or however, this response appears to occur under regimens of CR hydrogen peroxide. Using this assay, it has been shown that cells that involve intermittent feeding rather than a sustained reduc- treated with serum derived from CR rats and monkeys exhibit tion in caloric intake (Anson et al., 2003). enhanced survival after heat or oxidative stress (de Cabo et al., Manipulation of adipokines can also be considered a target 2003). Importantly, serum from CR animals was also capable for CRM. Following the discovery of leptin, there was great of inducing Sir2 expression in in vitro cell systems (Cohen et al., optimism that manipulation of this adipokine would be a highly 2004). Thus, the rationale would be that serum derived from effective target that could regulate appetite and energy utiliza- rats or mice treated with a candidate CRM should provide cells tion (Wolf, 1996). This potential has not been fulfilled because grown in vitro with greater protection against stress. of the apparent development of leptin resistance with long-term Beyond the in vitro screens, additional evaluation of in vivo treatment. Nonetheless, a reduction in the level of circulating effects will be needed. As we have argued, reduced body leptin observed in CR has been suggested to be the critical temperature and plasma insulin levels remain two important neuroendocrine modulator of the antiaging effects induced by biomarkers of CR. Other hormonal responses should also be this intervention (Shimokawa & Higami, 1999). To overcome the examined, including glucocorticoids, thyroid hormones, and issue of leptin resistance, leptin gene transfer directly into the adipokines, such as leptin and adiponectin. Another important hypothalamus has been considered as a strategy (Kalra & Kalra, in vivo screen that should be considered is effects of a candidate 2005). A single injection of a recombinant adeno-associated CRM on tumor growth in experimental models. virus encoding the leptin gene can result in long-term reductions For both in vitro and in vivo screens, DNA microarrays can in fat, insulin, tryglycerides and free fatty acids (Kalra & Kalra, also be a highly useful strategy for developing CRM. The objec- 2005). tive would be to determine if a candidate CR mimetic produced Manipulation of adiponectin as a CRM offers another poten- in a particular tissue produced a gene expression profile that was tially important strategy. Levels of this adipokine are elevated similar to that produced by CR (Dhahbi et al., 2005; Weindruch in CR rats (Zhu et al., 2004). Circulating levels of adiponectin et al., 2001). appear to be predictive of insulin responses to different diets Ultimately, any candidate CRM would still need to be validated in humans. Specifically, low levels of adiponectin correlate with by demonstrating its beneficial effects on mortality, morbidity, reduced insulin sensitivity when nonobese subjects are placed and function in a relevant animal model, most likely a rodent on a short-term high-fat diet (Thamer et al., 2004). Injections model. Of course, this stage of development would be the most of adiponectin in mice produces weight loss and reduced levels time-consuming and expensive component in the process. To of serum glucose and lipids (Qi et al., 2004). Production of assist in the discovery process, the National Institute on Aging higher levels of adiponectin through gene therapy techniques has organized the Interventions Testing Program (Warner et al., has been shown to be effective in achieving reduced body 2000; National Institute on Aging Website, 2005). Acceptance weight and higher peripheral insulin sensitivity (Shklyave et al., into this program would allow an investigator to move a can- 2003). didate CRM into a mammalian model and would assure quality control of the assessments. Development of screens Conclusions and future directions It is clear that the search for effective CRM can include a wide variety of targets. This search will be aided greatly by the develop- Further evidence of the increased interest in the development ment of a screening process to identify potential candidates. of CRM can be found in Table 1. The websites of the biotech Screening processes can utilize simple cell systems or possibly companies presented there now list CRM as one their primary simple invertebrate model systems, such as yeast. Targeting a development projects. High throughput in vitro screens estab- cellular response, such as sirtuin activation, has already proven lished by such commercial enterprises will clearly accelerate the effective (Sinclair, 2005). Other targets, such as enhanced stress identification of candidate CRM. responses or even survival from stress, would likely also be As outlined in this review, many logical targets exist for useful. In these systems, the candidate CRM would be added assessing the potential effectiveness of these candidates. Use directly to the model systems. Candidates emerging from such of different in vivo model systems will be applied to move these screens could then be evaluated in higher organisms, such as candidates forward. As candidates undergo different validation

Table 1 Websites of biotech companies engaged in research on calorie Bartke A, Brown-Borg H, Mattison J, Kinney B, Hauck S, Wright C (2001) restriction mimetics Prolonged longevity of hypopituitary dwarf mice. Exp. Gerontol. 36, 21–28. Company name Website Bartke A (2005) Minireview: role of the growth hormone/insulin-like growth factor system in mammalian aging. Endocrinology 146, 3718– Biomarker Pharmaceuticals www.biomarkerinc.com/html/home.htm 3723. Chronogen www.chronogen-inc.com/en/index.php Bergamini E, Cavallini G, Donati A, Gori Z (2004) The role of macro- Elixer Pharmaceucticals www.elixirpharm.com/ autophagy in the ageing process, anti-ageing intervention and age- GeroNova Research www.geronova.com/ associated diseases. Int. J. Biochem. Cell Biol. 36, 2392–2404. Irazu Biodiscovery www.irazubiodiscovery.net/ Bruce-Keller AJ, Umberger G, McFall R, Mattson MP (1999) Food restric- Juvenon www.juvenon.com/ tion reduces brain damage and improves behavioral outcome follow- Longenity www.longenity.com/index.html ing excitotoxic and metabolic insults. Ann. Neurol. 45, 8–15. Phoenix Biomolecular www.phoenixbiomolecular.com/index.html Burke LE, Dunbar-Jacob JM, Hill MN (1997) Compliance with cardio- Corporation Sirtris Pharmaceuticals www.sirtrispharma.com/ vascular disease prevention strategies: a review of the research. Ann. Behav. Med. 19, 239–263. de Cabo R, Cabellob R, Riosa M, Lo’pezLluchb G, Ingram DK, Lane MA, Navas P (2004) Calorie restriction attenuates age-related alterations assessments, the ultimate objective would be to evaluate a CRM in the plasma membrane antioxidant system in rat liver. Exp. Gerontol. in human trials. Because the acceptance of biomarkers of aging 39, 297–304. de Cabo R, Fürer-Galban SR, Anson RM, Gilman C, Gorospe M, Lane MA has still not been realized within the scientific community and, (2003) An in vitro model of caloric restriction. Exp. Gerontol. 38, 631–639. more importantly, within the regulatory arena, it is likely that Calingasan NY, Gibson GE (2000) Dietary restriction attenuates the neu- disease endpoints will be used to assess the effectiveness of the ronal loss, induction of heme oxygenase-1 and blood-brain barrier candidates. Endpoints could include assessment of risk factors breakdown induced by impaired oxidative metabolism. Brain Res. for heart disease and diabetes and for treatment or prevention 885(1), 62–69. of cancer, arthritis, stroke, and neurodegenerative disorders, Chung HY, Kim HJ, Kim JW, Yu BP (2001) The inflammation hypothesis such as Alzheimer and Parkinson diseases. of aging: molecular modulation by calorie restriction. Ann. N. Y. Acad. Sci. 928, 327–335. Another concept that will likely guide further development Clancy DJ, Gems D, Hafen E, Leevers SJ, Partridge L (2002) Dietary restric- of CRM is that combinations of compounds could be more tion in long-lived dwarf flies. Science 296, 319. effective than single compounds (Everitt et al., 2005; Roth et al., Cohen HY, Miller C, Bitterman KJ, Wall NR, Hekking B, Kessler B, Howitz KT, 2005). This ‘cocktail’ approach may emerge in recognition of Gorospe M, de Cabo R, Sinclair DA (2004) Calorie restriction promotes two key issues. First, because energy regulation is so key to an mammalian cell survival by inducing the SIRT1 deacetylase. Science organism’s survival, there are likely to be compensatory mech- 305, 390–392. anisms activated in response to manipulating a single target; Corton JC, Apte U, Anderson SP, Limaye P, Yoon L, Latendresse J, Dunn C, Everitt JI, Voss KA, Swanson C, Kimbrough C, Wong JS, Gill SS, thus, an intervention aimed at multiple targets could offset Chandraratna RAS, Kwak M, Kensler TW, Stulnig TM, Steffensen KR, these compensatory responses. Second, effects of aging and the Gustafsson J, Mehendale HM (2004) Mimetics of caloric restriction antiaging effects of CR involve multiple pathways; thus, inter- include agonists of lipid-activated nuclear receptors. J. Biol. Chem. ventions intended to provide protection to multiple systems and 279, 46204–46212. multiple tissues should be inherently more beneficial. Dark J, Miller DR, Zucker I (1994) Reduced glucose availability induces In conclusion, this emerging field appears poised for major torpor in Siberian hamsters. Am. J. Physiol. 267, 496–501. advances. There will be many new concepts developed, many Dhahbi JM, Mote PL, Fahy GM, Spindler SR (2005) Identification of potential caloric restriction mimetics by microarray profiling. Physiol. new approaches suggested, and many new candidate CRM Genomics 23, 343–350. identified as interest in this concept grows and matures. Dilman VM (1971) Age-associated elevation of hypothalamic threshold to feedback control and its role in development, aging and disease. Lancet 1, 1211–1219. References Donati A, Cavallini G, Carresi C, Gori Z, Parentini I, Bergamini E (2004) Anti-aging effects of anti-lipolytic drugs. Exp. Gerontol. 39, 1061–1067. Anisimov VN (2003) Insulin/IGF-1 signaling pathway driving aging and Duan W, Mattson MP (1999) Dietary restriction and 2-deoxyglucose cancer as a target for pharmacological intervention. Exp. Gerontol. administration improve behavioral outcome and reduce degeneration 38, 1041–1049. of dopaminergic neurons in models of Parkinson's disease. J. Neurosci. Anson A, Guo Z, de Cabo R, Iyun T, Rios M, Hagepanos A, Ingram DK, Res. 57, 195–206. Lane MA, Mattson MP (2003) Periodic fasting dissociates beneficial Duan W, Lee J, Guo Z, Mattson MP (2001) Dietary restriction stimulates effects of dietary restriction from calorie intake. Proc. Natl Acad. Sci. BDNF production in the brain and thereby protects neurons against USA 100, 6216–6220. excitotoxic injury. J. Mol. Neurosci. 16, 1–12. Argmann CA, Cock TA, Auwerx J (2005) Peroxisome proliferator- Eurich DT, Majumdar SR, McAlister FA, Tsuyuki RT, Johnson JA (2005) activated receptor gamma: the more the merrier? Eur. J. Clin. Invest. Improved clinical outcomes associated with metformin in patients 35, 82–92. with diabetes and heart failure. Diabetes Care 28, 2345–2351. Astrup A (2001) Healthy lifestyles in Europe: prevention of obesity and Everitt AV, Roth GS, Le Couteur DG, Hilmer SN (2005) Caloric restriction type II diabetes by diet and physical activity. Public Health Nutr. 4, versus drug therapy to delay the onset of aging diseases and extend 499–515. life. Age 27, 39–48.

Fontana L, Meyer TE, Klein S, Holloszy JO (2004) Long-term calorie therapeutic, but not any more a low glucose mimetic. Life Sci. 78, restriction is highly effective in reducing the risk for atherosclerosis in 1392–1399. humans. Proc. Natl Acad. Sci. USA 101, 6659–6663. Kapahi P, Zid BM, Harper T, Koslover D, Sapin V, Benzer S (2004) Regu- Fuemayor LD, Diaz S (1984) The effect of feeding on the stereotyped lation of lifespan in Drosophila by modulation of genes in the TOR behaviour induced by amphetamine and by apomorphine in the signaling pathway. Curr. Biol. 14, 885–890. albino rat. Eur. J. Pharmacol. 99, 153–158. Kealy RD, Lawler DE, Ballam JM, Mantz SL, Biery DN, Greeley EH, Lust G, Gems D, Partridge L (2001) Insulin/IGF signaling and ageing: seeing the Segre M, Smith GK, Stowe HD (2002) Effects of diet restriction on bigger picture. Curr. Opin. Genet. Dev. 122, 673–693. lifespan and age-related changes in dogs. J. Am. Vet Med. Assoc. 220, de Grey AD (2005) The unfortunate influence of the weather on the 1315–1320. rate of ageing: why human caloric restriction or its emulation may Kirpichnikov D, McFarlane SI, Sowers JR (2002) Metformin: an update. only extend life expectancy by 2–3 years. Gerontology 51, 73–82. Ann. Intern. Med. 137, 25–33. Gridley DS, Nutter RL, Kettering JD, Mantik DW, Slater JM (1985) Mouse Knauper B, Cheema S, Rabiau M, Borten O (2005) Self-set dieting rules: neoplasia and immunity: effects of radiation, hyperthermia, 2-deoxy- adherence and prediction of weight loss success. Appetite 44, 283– D-glucose, and Corynebacterium parvum. Oncology 42, 391–398. 288. Guarente L, Picard F (2005) Calorie restriction – the SIR2 connection. Kritchesky D (1993) Undernutrition and chronic disease: cancer. Proc. Cell 120(4), 473–482. Nutr Soc. 52, 39–47. Guo Z, Mattson MP (2000) In vivo 2-deoxyglucose administration pre- Kritchesky D (2001) Caloric restriction and cancer. J. Nutr. Sci. Vitaminol. serves glucose and glutamate transport and mitochondrial function 47, 13–19. in cortical synaptic terminals after exposure to amyloid β-peptide Lane MA, Ingram DK, Roth GS (1998) 2-Deoxy-D-glucose feeding in rats and iron: evidence for a stress response. Exp. Neurol. 166, 173– mimics physiological effects of calorie restriction. J. Anti Aging Med. 179. 1, 327–337. Guo Z, Lee J, Lane M, Mattson M (2001) Iodoacetate protects hippo- Lane MA, Ingram DK, Roth GS (2002) Easy to swallow. The serious search campal neurons against excitotoxic and oxidative injury: involvement for an anti-aging pill. Sci. Am. 287, 24–29. of heat-shock proteins and Bcl-2. J. Neurochem. 79, 361–370. Lee J, Bruce-Keller AJ, Kruman Y, Chan SL, Mattson MP (1999) 2-deoxy- Hadley EC, Dutta C, Finkelstein J, Harris TB, Lane MA, Roth GS, D-glucose protects hippocampal neurons against excitotoxic and oxi- Sherman SS, Starke-Reed PE (2001) Human implications of caloric dative injury: evidence for the involvement of stress proteins. J. Neu- restriction’s effects on aging in laboratory animals: an overview of rosci. Res. 57, 48–61. opportunities for research. J. Gerontol. A Biol. Sci. Med. Sci. 56, 5–6. Lee J, Chan SL, Mattson MP (2002) Phenformin protects hippocampal Harrison DE, Archer JR (1989) Natural selection for extended longevity neurons against excitotoxicity by suppressing NMDA receptor- from food restriction. Growth Dev. Aging 53, 3. mediated calcium influx. Exp. Neurol. 175, 161–167. Heilbronn LK, Ravussin E (2003) Calorie restriction and aging: reviews Lithgow GJ, Walker GA (2002) Stress resistance as a determinate of C. of the literature and implications for studies in humans. Am. J. Clin. elegans lifespan. Mech. Ageing Dev. 123, 765–771. Nutr. 78, 361–369. Logroscino G, Marder K, Cote L, Tang MX, Shea S, Mayeux R (1996) Heilbroon LK, Smith SR, Martin CK, Anton SD, Ravussin E (2005) Alternate- Dietary lipids and antioxidants in Parkinson’s disease: a population- day fasting in nonobese subjects: effects on body weight, body com- based, case-control study. Ann. Neurol. 39, 89–94. position, and energy metabolism. Am. J. Clin. Nutr. 81, 69–73. Mamczarz J, Duffy K, Bowker J, Zhu M, Hagepanos A, Ingram D (2005) Holzenberger M, Dupont J, Ducos B, Leneuve P, Geloen A, Even PC, Enhancement of amphetamine-induced locomotor response in rats on Cervera P, Le Bouc Y (2003) IGF-1 receptor regulates lifespan and different regimens of diet restriction and 2-deoxyglucose treatment. resistance to oxidative stress in mice. Nature 421, 182–187. Neurosci. 131, 451–461. Houthoofd K, Johnson TE, Vanfleteren JR (2003) Life extension via Masoro EJ (1998) Hormesis and the antiaging action of dietary restric- dietary restriction is independent of the Ins/IGF-1 signalling pathway tion. Exp. Gerontol. 33, 61–66. in Caenorhabditis elegans. Exp. Gerontol. 38, 947–954. Masoro EJ (2000) Caloric restriction and aging: an update. Exp. Gerontol. Howitz KT, Bitterman KJ, Cohen HY, Lamming DW, Lavu S, Wood JG, 35, 299–305. Zipkin RE, Chung P, Kisielewski A, Zhang LL, Scherer B, Sinclair DA Masoro EJ, Austad SN (1996) The evolution of the antiaging action of (2003) Small molecule activators of sirtuins extend Saccharomyces dietary restriction: a hypothesis. J. Gerontol. A Biol. Sci. Med. Sci. 51, cerevisiae lifespan. Nature 425, 191–196. B387–B391. Hursting SD, Lavigne JA, Berrigan D, Perkins SN, Barrett JC (2003) Calorie Masternak MM, Al-Regaiey KA, Del Rosario Lim MM, Jimenez-Ortega V, restriction, aging, and cancer prevention: Mechanisms of action and Panici JA, Bonkowski MS, Bartke A (2005) Effects of caloric restriction applicability in humans. Annu. Rev. Med. 54, 131–152. on insulin pathway gene expression in the skeletal muscle and liver Hyman C, Hofer M, Barde Y-A, Jahasz M, Yancopoulos GD, Squinto SP, of normal and long-lived GHR-KO mice. Exp. Gerontol. 40, 679– Lindsay RM (1991) BDNF is a neurotrophic factor for dopaminergic 684. neurons of the substantia nigra. Nature 350, 230–232. Mattison JA, Lane MA, Roth GS, Ingram DK (2003) Calorie restriction Ingram DK, Anson RM, Decabo R, Mamczarz J, Zhu M, Mattison J, in rhesus monkeys. Exp. Gerontol. 38, 35–46. Lane MA, Roth GS (2004) Development of calorie restriction mimetics Mattson MP (2005) Energy intake, meal frequency, and health: a neuro- as a prolongevity strategy. Ann. N. Y. Acad. Sci. 1019, 412–423. biological perspective. Annu. Rev. Nutr. 25, 237–260. Johnson TE, de Castro E, Hegi de Castro S, Cypser J, Henderson S, Mattson MP, Duan W, Guo Z (2003) Meal size and frequency affect Tedesco P (2001) Relationship between increased longevity and stress neuronal plasticity and vulnerability to disease: cellular and molecular resistance as assessed through gerontogene mutations in Caenorhab- mechanisms. J. Neurochem. 84, 417–431. ditis elegans. Exp. Gerontol. 36, 1609–1617. Mayeux R, Costa R, Bell K, Merchant C, Tung MX, Jacobs D (1999) Kalra SP, Kalra PS (2005) Gene-transfer technology: a preventive neuro- Reduced risk of Alzheimer’s disease among individuals with low therapy to curb obesity, ameliorate metabolic syndrome and extend calorie intake. Neurology 59, S296–S297. life expectancy. Trends Pharmacol. Sci. 26, 488–495. McCay CM, Crowell MF (1934) Prolonging the life span. Sci. Mon. 39, Kang HT, Hwang ES (2006) 2-Deoxyglucose: an anticancer and antiviral 405–414.

McCay CM, Crowell MF, Maynard LA (1935) The effect of retarded Shimokawa I, Higami Y, Utsuyama M, Tuchiya T, Komatsu T, Chiba T, growth upon the length of life span and upon the ultimate body size. Yamaza H (2002) Lifespan extension by reduction in growth hormone- J. Nutr. 10, 63–79. insulin-like growth factor-1 axis in a transgenic rat model. Am. J. National Institute on Aging website: http://www.nia.nih.gov/ResearchIn- Pathol. 160, 2259–2265. formation/ScientificResources/InterventionsTestingProgram. Accessed: Shklyave S, Aslanidi G, Tennant M, Prima V, Kohlbrenner E, Kroutov V, 8 October 2005. Campbell-Thompson M, Crawford J, Shek EW, Scarpace PJ, Zolo- Newton IG, Forbes ME, Legault C, Johnson JE, Brunso-Bechtold JK, tukhin S (2003) Sustained peripheral expression transgene adiponec- Riddle DR (2005) Caloric restriction does not reverse aging-related tin offsets the development of diet-induced obesity in rats. Proc. Natl changes in hippocampal BDNF. Neurobiol. Aging 26, 683–688. Acad. Sci. USA 100, 14217–14222. Oliver WR Jr, Shenk JL, Snaith MR, Russell CS, Plunket KD, Bodkin NL, Sinclair DA (2005) Toward a unified theory of caloric restriction and lon- Lewis MC, Winegar DA, Sznaidman ML, Lambert MH, Xu HE, gevity regulation. Mech. Ageing Devel. 126, 987–1002. Sternbach DD, Kliewer SA, Hansen BC, Willson TM (2001) A selective Sohal RS, Weindruch R (1996) Oxidative stress, caloric restriction, and peroxisome proliferator-activated receptor δ agonist promotes reverse aging. Science 273, 59–63. cholesterol transport. Proc. Natl Acad. Sci. USA 98, 5306–5311. Suzuki M, Wilcox BJ, Wilcox CD (2001) Implications from and for food Phelan JP, Rose MR (2005) Why dietary restriction substantially increases cultures for cardiovascular disease: longevity. Asia Pac. J. Clin. Nutr. longevity in animal models but won’t in humans. Ageing Res. Rev. 10, 165–171. 4, 339–350. Tatar M, Bartke A, Antebi A (2003) The endocrine regulation of aging Pinney DO, Stephens DF, Pope LS (1972) Lifetime effects of winter sup- by insulin-like signals. Science 299, 1346-1351. plemental feed level and age at first parturition on range beef cows. Thamer C, Haap M, Bachmann O, Zur Nieden T, Tschritter O, Stefan N, J. Anim Sci. 34, 1067–1074. Fritsche A, Jacob S, Stumvoll M, Haring H (2004) Serum adiponectin Poehlman ET, Turturro A, Bodkin N, Cefalu W, Heymsfield S, Holloszy levels predict the effect of short-term dietary interventions on insulin J, Kemintz J (2001) Caloric restriction mimetics: physical activity and sensitivity in humans. Diabetologia. 47, 1303–1305. body composition changes. J. Gerontol. A Biol. Sci. Med. Sci. 56, 45–54. Vanfleteren JR, Braeckman BP (1999) Mechanisms of life span detere- Qi Y, Takahashi N, Hileman SM, Patel Hr, Berg AH, Pajvani UB, Scherer mination in Caenorhabditis elegans. Neurobiol. Aging 20, 487–502. PE, Ahima RS (2004) Adiponectin acts in the brain to decrease body Veech RL (2004) The therapeutic implications of ketone bodies: the weight. Nat. Med. 10, 524–529. effects of ketone bodies in pathological conditions: ketosis, ketogenic Quideau S (2004) Plant ‘polyphenolic’ small molecules can induce a diet, redox states, insulin resistance, and mitochondrial metabolism. calorie restriction-mimetic life-span extension by activating sirtuins: Prostaglandins Leukot. Essent. Fatty Acids 70, 309–319. will ‘polyphenols’ some-day be used as chemotherapeutic drugs in Walford RL, Mock D, Verdery R, MacCallum T (2002) Calorie restriction Western medicine? Chembiochem. 4, 427–430. in biosphere 2: alterations in physiologic, hematologic, hormonal, and Raffoul JJ, Guo Z, Soofi A, Heydari AR (1999) Caloric restriction and biochemical parameters in humans restricted for a 2-year period. J. genomic stability. J. Nutr. Health Aging 3, 102–110. Gerontol. A Biol. Sci. Med. Sci. 57, B211–B224. Rattan SI (2001) Applying hormesis in aging research and therapy. Hum. Wan R, Camandola S, Mattson MP (2003) Intermittent fasting and Exp. Toxicol. 20, 281–285. dietary supplementation with 2-deoxy-D-glucose improve functional Rogina B, Helfand SL (2004) Sir2 mediates longevity in the fly through and metabolic cardiovascular risk factors in rats. FASEB J. 17, 1133– a pathway related to calorie restriction. Proc. Natl Acad. Sci. USA 101, 1134. 15998–16003. Wan R, Camandola S, Mattson MP (2004) Dietary supplementation with Rogina B, Helfand SL, Frankel S (2002) Longevity regulation by Dro- 2-deoxy-D-glucose improves cardiovascular and neuroendocrine sophila Rpd3 deacetylase and caloric restriction. Science 298, 1745. stress adaptation in rats. Am. J. Physiol. Heart Circ Physiol. 287, Roth GS, Ingram DK, Joseph JA (1984) Delayed loss of striatal dopamine H1186–H1193. receptors during aging of dietarily restricted rats. Brain Res. 300, 27–32. Warner HR, Ingram D, Miller RA, Nadon NL, Richardson AG (2000) Pro- Roth GS, Ingram DK, Lane MA (2001) Caloric restriction in primates and gram for testing biological interventions to promote healthy aging. relevance to humans. Ann. N. Y. Acad. Sci. 928, 305–315. Mech. Ageing Dev. 115, 199–207. Roth GS, Lane MA, Ingram DK (2005) Caloric restriction mimetics: The Weidenfeld J, Corcos AP, Wohlman A, Feldman S (1994) Characteriza- next phase. Ann. N Y Acad. Sci. in press. tion of the 2-deoxyglucose effect on the adrenocortical axis. Endo- Roth GS, Mattison JA, Ottinger MA, Chachich M, Lane MA, Ingram DK crinol. 134, 1924–1931. (2004) Aging in rhesus monkeys: Relevance to human health inter- Weindruch R, Walford RL (1988) The retardation of aging and disease ventions. Science 305, 1423–1426. by dietary restriction. Springfield, IL: Charles C. Thomas. Scarpello JH (2003) Improving survival with metformin: the evidence Weindruch R, Sohal RS (1997) Seminars in medicine of the Beth Israel base today. Diabetes Metab. 29, 6S36–6S43. Deaconess Medical Center. Caloric intake and aging. N. Engl. J. Med. Schafer SA, Hansen BC, Volkl A, Fahimi HD, Pill J (2004) Biochemical 337, 986–994. and morphological effects of K-111, a peroxisome proliferator-activated Weindruch R, Kayo T, Lee CK, Prolla TA (2001) Microarray profiling of receptor (PPAR)α activator, in non-human primates. Biochem. Pharmacol. gene expression in aging and its alteration by calorie restriction in 68, 239–251. mice. J. Nutr. 131, 918S–923S. Sell DR, Lane MA, Johnson WA, Masoro EJ, Mock OB, Reiser KM, Fozarty JF, Weindruch R, Keenan KP, Carney JM, Fernandes G, Feuers RJ, Floyd RA, Cutler RG, Ingram DK, Roth GS, Monnier VM (1996) Longevity and Halter JB, Ramsey JJ, Richardson A, Roth GS, Spindler SR (2001) the genetic determination of glycooxidation kinetics in mammalian Caloric restriction mimetics: metabolic interventions. J. Gerontol. A senescence. Proc. Natl Acad. Sci. USA 93, 485–490. Biol. Sci. Med. Sci. 56, 20–33. Shanley DP, Kirkwood TB (2000) Calorie restriction and aging: a life- Willet WC (2000) Nutritional epidemiology issues in chronic disease at history analysis. Evolution Int J Org Evol 54, 740–750. the turn of the century. Epidemiol. Rev. 22, 82–86. Shimokawa I, Higami Y (1999) A role for leptin in the antiaging action Wolf G (1996) Leptin: the weight-reducing plasma protein encoded by of dietary restriction: a hypothesis. Aging 11, 380–382. the obese gene. Nutr. Rev. 54, 91–93.

Wood JG, Rogina B, Lavu S, Howitz K, Helfand SL, Tatar M, Sinclair D campal neurons against the death-promoting action of a presenilin-1 (2004) Sirtuin activators mimic caloric restriction and delay ageing in mutation. Brain Res. 842, 224–229. metazoans. Nature 430, 686–689. Zhu M, Miura J, Lu LX, Bernier M, de Cabo R, Lane MA, Roth GS, Yu BP (1994) Modulation of Aging Processes by Dietary Restriction. Boca Ingram DK (2004) Elevated circulation adiponectin levels are associ- Raton, FL: CRC Press. ated with peripheral insulin sensitivity in rats on caloric restriction. Exp. Yu ZF, Mattson MP (1999) Dietary restriction and 2-deoxyglucose admin- Gerontol. 39, 1049–1059. istration reduce focal ischemic brain damage and improve behavioral Zhu Z, Jiang W, McGinley JN, Thompson HJ (2005) 2-deoxyglucose as outcome: evidence for a preconditioning mechanism. J. Neurosci. Res. an energy restriction mimetic agent: effects on mammary carcino- 57, 830–839. genesis and on mammary tumor cell growth in vitro. Cancer Res. 65, Zhu H, Guo Q, Mattson MP (1999) Dietary restriction protects hippo- 7023–7030.