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The discovery of the lifespan-extending effect of restricted food intake in rats by McCay et al. (1935) has promoted calorie restriction (CR) as a key research area in biogerontology. CR is currently the only experimental method that has been shown to extend the lifespan of a number of different organisms, including yeast, flies, worms, rodents and possibly primates (Imai, 2009). In addition, the onset and progression of numerous age-related disorders are postponed, including diabetes, cancer and atherosclerosis (Fontana, 2009). However, despite many years of research, the mechanisms underlying these effects and their applicability to humans remain unresolved.
The effects of calorie restriction
The CR model features a reduction in available calories of up to 50% compared to ad libitum (freely fed) intake, without restricting essential nutrients and vitamins. This has been shown to be responsible for increasing average and maximum lifespan in a range of organisms by up to 50%, retarding ageing and increasing the mortality rate doubling time in rodent populations (Masoro, 2000). A study of a long-lived strain (C3B10RF) of female mice indicated that those restricted to either 40 or 50 kcal/week exhibited mean and maximal lifespans 35-65% greater than those on an ad libitum diet, and 20-40% greater than those restricted to 85 kcal/week (Weindruch et al., 1986). Mice on the lowest calorie diet lived the longest of all groups (Figure 1). These results have been replicated in many other studies. Holehan and Merry (1985) also reported that CR slowed the age-associated increase in mortality rate in rats. Some mice studies, however, have shown that CR extends lifespan without slowing the increase in mortality rate (Turturro et al., 2002).
The delaying or prevention of age-associated diseases is one of the more striking characteristics of CR. Microarray analysis, which provides the most detailed description of the CR phenotype so far, has shown that CR prevents many of these progressive changes that occur during ageing (Lee et al., 1999). For example, CR has been shown to extend the relatively short lifespan of p53-/- mice, which die early from cancers due to loss of the tumour suppressor (Berrigan et al., 2002). Other changes characteristic of CR animals include lower body temperature, blood glucose and insulin levels, and reduced body weight and fat. Resistance to external stresses such as oxidative stress also appears to be higher (Sohal and Weindruch, 1996). Such physiological changes elicited by CR are thus believed to trigger increased longevity. However, consequences of CR in rodents include lower growth rates and maximum body size (McCay et al., 1935), a delay in sexual maturation and lower fertility (Merry and Holehan, 1979). This latter feature is analogous with the disposable soma theory of ageing, in which animals with increased longevity suffer from reduced fertility as a trade off (Kirkwood, 1977). Indeed, Holliday (1989) proposed that this diversion of energy to maintenance in CR is the basis of its anti-ageing action.
Whilst most studies indicating a link between CR on longevity have used rodent models, long term effects in primates are uncertain, although preliminary evidence appears to suggest a link (Roth et al., 2004). Studies in humans indicate that certain markers of ageing, including blood pressure, blood glucose and cholesterol levels, improve with CR diets (Walford et al., 2002). Indeed, inhabitants of the Japanese Okinawan islands, the longest-living people on earth, consume 40% fewer calories than Americans and live four years longer (Everett and Le Couteur, 2007). This increase in lifespan is far less than those found in animal studies, reflecting the complexity of comparing animal models to humans.
Fig 1 | Effect of calorie intake on A) body weight, B) percent survival, and C) lifespan in female C3B10F mice. Symbols in A) also apply to B).
Adapted from Sohal and Weindruch, 1996.
Many different hypotheses have been proposed regarding the mechanisms underlying CR-induced longevity. These are summarised in Figure 2. Early suggestions by McCay et al. (1935) considered an extension of lifespan due to growth retardation. This was later disregarded following the observation of lifespan extension in adult mice. Other mechanisms proposed include a reduction in body fat and metabolic rate. Findings regarding the latter theory show varying effects of CR on metabolic rate, however (Masoro, 2009). On the other hand, recent evidence points to roles for decreased oxidative damage and alterations in transcription in providing plausible explanations for the effect of CR on ageing.
Fig 2 | List of proposed mechanisms underlying calorie restriction-induced lifespan extension in rats and mice
From Masoro, 2009.
Mitochondria and oxidative damage
Current evidence suggests that CR may delay ageing and extend lifespan by the reduction of oxidative damage (Sohal and Weindruch, 1996). This is consistent with the Free Radical/Oxidative Stress Theory of Ageing, which proposes that reactive oxygen species (ROS) produced during normal aerobic respiration cause cumulative oxidative cell damage, resulting in ageing and eventually death. Mitochondria are the main source of ROS in most cells, mediated by complexes I and II of the electron transport chain (ETC) (Raha and Robinson, 2000). Under normal conditions when ATP requirements are low or NADH levels are high, electrons are channelled through complexes I-IV of the ETC, enabling protons (H+) to be pumped from the mitochondrial matrix to the cytoplasm via uncoupling proteins, generating a proton gradient. Excess H+ ions dissipate as heat, and the inefficient transfer of electrons results in their leakage from complexes I and II, reacting with oxygen to generate numerous ROS. Studies of H2O2 formation in rat heart mitochondria indicate that an increase in the protonic potential increases H2O2 production, with a threshold value above which a very strong increase in H2O2 production takes place (Korshunov et al., 1997). This process is summarised in Figure 3.
Fig 3 | The formation of ROS. A) Leakage of electrons from complexes I and II lead to the formation of ROS. B) Electrons combine with oxygen to form superoxide ions. This can lead to the formation of other ROS, resulting in further oxidative damage.
Adapted from Bordone and Guarante, 2005.
The generation of ROS has wide-ranging effects in vivo, including damage to lipids, proteins and nucleic acids. Lipid peroxidation by free radicals leads to the formation of unsaturated aldehydes, which are highly reactive and can act as mutagens (Marnett et al., 1985). Oxidative damage to nucleic acids may also cause base alterations and induce breakages in strands, leading to mutations and cell senescence. Mitochondrial DNA damage is particularly apparent (Vermulst et al., 2007). Such cellular changes are characteristic of ageing, and as such, oxidative damage has been shown to increase with age in mitochondria, contributing to the ageing phenotype (Farmer and Sohal, 1989).
Data from numerous CR studies have reported a reduction in age-associated oxidative damage in different tissues in rodents on a CR diet. These include decreases in lipid peroxidation, DNA oxidation and lipofuscin levels (Bokov et al., 2004). Measurement of global protein carbonyl content in CR also indicates prevention of the age-related increase in protein oxidation in mouse skeletal muscle mitochondria (Figure 4; Lass et al., 1998).
Fig 4 | Carbonyl content of skeletal muscle mitochondria, determined as a function of age and dietary regime.
Adapted from Lass et al., 1998.
Based on these findings, Sohal and Weindruch (1996) proposed that CR delays ageing by decreasing oxidative stress. Indeed, there is evidence that CR decreases the rate of mitochondrial ROS generation (Sohal and Dubey, 1994). This has been supported by similar findings in mitochondria isolated from both rat heart and liver (Gredilla et al., 2001). These observations may be explained by increased mitochondrial biogenesis and lower mitochondrial NADH levels in CR due to lower glucose intake. This results in greater utilisation of H+ ions and reduced electron leakage, which direct to more efficient ATP production. As a result, there is likely to be a decrease in protonmotive force from increased proton leakage, and a concomitant fall in ROS generation. The reduced heat dissipation may explain the compromised thermogenesis observed in CR animals. Whilst most studies indicate supporting roles for this theory, others have revealed a number of inconsistencies. In a study observing rat liver DNA, a significant reduction was found in the concentration of 8-OHdg, an oxidative marker, after 6 weeks in CR rats (Chung et al., 1992). A later study using the same strain of rat failed to confirm this early decrease, with CR and control animals showing similar levels of damage (Kaneko et al., 1997). Another hypothesis implies that decreased methionine ingestion in CR leads to a higher cellular reduced/oxidised glutathione ratio, decreasing complex I ROS generation (Sanz et al., 2006).
Much research into oxidative damage has also focused on antioxidants, although findings are conflicting. Several groups have reported significant increases in liver antioxidant enzymes in CR mice compared to ad libitum mice, including Semsei et al. (1989), who showed increased catalase and Cu/Zn superoxide dismutase (SOD) activity and gene expression with CR in rat liver (Figure 5). Subsequent findings, however, failed to show a clear overall trend with CR, which may be attributed to the complex interactions between CR and antioxidant defences (Bokov et al., 2004). Strong evidence also suggests that CR may reduce oxidative damage through enhanced molecular repair and turnover. Repair of DNA damage in hepatocytes, for example, appears to be higher with CR (Weraarchakul et al., 1988). In summary, oxidative damage to molecules has been shown to be reduced by CR. This reduction is likely to arise from reduced ROS generation and enhanced macromolecule repair.
Fig 5 | Effect of calorie restriction on the expression of superoxide dismutase and catalase
From Semsei et al., 1989.
Sirtuins, PGC-1α and insulin
CR results in numerous metabolic adaptations that ultimately increase longevity in different organisms. Sirtuins, a group of proteins with histone deacetylase activity, have been implicated in enhancing survival by increasing gene expression in the nucleus. SIRT1 protein levels have been reported to increase in rodents and humans, with sir2 activity in non-mammals also increasing with CR (Chen et al., 2008). Glucose restriction in yeast has provided a useful model for identifying increases in longevity associated with sir2 activation (Lin et al., 2000). Following the production of NADH during CR, ATP is rapidly made, causing an increase in the NAD+/NADH ratio. This activates SIRT1, which in turn activates forkhead transcription factor (FOXO), which promotes increased gene expression in the nucleus, resulting in a number of pro-longevity transcriptional adaptations (Yu and Auwerx, 2009). These include increases in fatty acid oxidation, hepatic glucose production, insulin secretion and mitochondrial activity (Finkel et al., 2009). Additionally, low glucose levels lead to increased AMP levels due to limited ATP synthesis. This activates AMP kinase (AMPK), which also leads to SIRT1 activation. Glucose restriction in skeletal myoblasts has been shown to inhibit their differentiation by activating SIRT1 through AMPK-mediated regulation of Nampt, an NAD+ biosynthetic enzyme (Fulco et al., 2008). This elucidates the importance of this functional signalling pathway in response to restricted nutrient availability. Resveratrol, a natural phyoalexin, has also been shown to activate SIRT1, prolonging lifespan and retarding the onset of various age-related markers in a short-lived fish. These results are displayed in Figure 6 (Valenzano et al., 2006). However, CR does not induce SIRT1 activity in all tissues, with implications of reduced activity in the liver (Chen et al., 2008).
Fig 6 | Age-dependent survival of treated and untreated Nothobranchius furzeri.
A) Survival curves for controls and differently dosed resveratrol-treated fishes, B) Comparison between the age-dependent survival of males and females in controls and 120 mg/g resveratrol-treated fishes, C) Death trajectories in controls and 120 mg/g resveratrol-treated fishes
Adapted from Valenzano et al., 2006.
Mitochondria have also been shown to increase levels of the transcription factor PGC-1α. An increase in the ATP:ADP ratio, which indicates mitochondrial efficiency during CR, stimulates AMPK, thus activating PGC-1α in the nucleus. This induces mitochondrial biogenesis and respiration, increasing mitochondria production and complex activity. The conversion to highly oxidative mitochondria-rich type I fibres also occurs in skeletal muscle cells, and the interaction of PGC-1α with other transcription factors induces fatty acid oxidation genes. SIRT1 has also been shown to have a regulatory role, where it positively regulates mitochondrial and fatty acid oxidation genes by deacetylating and activating PGC-1α (Gerhart-Hines et al., 2007). This confirms the crucial role of SIRT1 in maintaining energy and nutrient homeostasis by increasing fatty acid oxidation in response to low glucose levels.
Insulin has also emerged as a key molecule affecting mitochondrial efficiency and gene expression. CR is known to decrease plasma glucose and insulin concentrations, whilst loss of the insulin receptor appears to increase longevity (Kalant et al., 1988). Masoro (1996) proposed that long-term reductions in plasma glucose and insulin levels are responsible for the lifespan-extending effects of CR. Decreased insulin/IGF-1 signalling is believed to increase mitochondrial efficiency and promote the expression of mitochondrial antioxidants. This is mediated by the translocation of FOXO3a to the nucleus, where mitochondrial gene expression is induced, stimulating MnSOD and glutathione peroxidizes (Page et al., 2010). Enhanced MnSOD activity lowers superoxide and peroxynitrite levels, and has been associated with reduced apoptosis, whereas increased glutathione peroxidize levels are protective against hydrogen peroxide. In contrast, high insulin levels normally downregulate FOXO and gene transcription, suppressing antioxidant production and preventing pro-longevity adaptations. Significantly, Lambert and Merry (2004) demonstrated the anti-ageing properties of insulin by showing that the ROS-reducing effect of CR was abolished with the administration of insulin, which increased hydrogen peroxide production in mitochondria from liver tissue of male Brown Norway rats (Figure 7).
Fig 7 | The effect of calorie restriction (CR) and CR with insulin on hydrogen peroxide production by mitochondria.
Adapted from Lambert and Merry, 2004
Advanced glycosylation end products (AGEs) are products of spontaneous chemical reactions between sugars and proteins, and are implicated in causing random protein aggregation and dysfunction, affecting the biochemical and physical properties of proteins and the extracellular matrix. Figure 8 illustrates the multiple pathways involved in the generation of AGEs. Some AGEs are additional sources of oxidative stress, enabling an acceleration of oxidative damage to proteins during ageing. In addition, the damaging effects of glucose have been linked to glycation and glycoxidation, particularly in diabetes (Baynes, 2001). Whilst it is known that AGEs increase in tissue with age, excessive AGEs are also characteristic of numerous chronic age-related diseases, including diabetes, atherosclerosis, Alzheimer's and other neurodegenerative diseases. Hence, methylglyoxal has been implicated in playing a causal role in limitation of lifespan in the nematode. Glucose has also been shown to shorten the lifespan of this worm by downregulating FOXO activity and aquaporin gene expression (Morcos et al., 2008). CR in rats, however, has shown a decrease in the age-associated accumulation of glycation and glycoxidation products in skin collagen (Cefalu et al., 1995). A decreased dietary intake of glycated proteins has also yielded increased lifespans in mice (Morcos et al., 2008). Despite the lack of concrete evidence supporting a role for AGEs as a primary determinant of ageing, it is however, likely that AGEs are one of many chemical mechanisms contributing to molecular damage with age.
Fig 8 | Multiple pathways leading to the formation of the AGE, CML.
Adapted from Baynes, 2001.
CR has long been recognised as a useful model for studying ageing theories, and has consistently been shown to extend mammalian lifespan, prolong functional competence and delay the onset of many age-related diseases. Whilst the CR and oxidative damage findings are consistent with the Free Radical/Oxidative Stress Theory of Ageing, they do not prove that reduced oxidative damage is the mechanism causing the lifespan-extending effect of CR. Numerous other biological and chemical pathways are affected by CR, indicating that many mechanisms are likely to at least contribute to the retarding of ageing. Hormesis, an increasingly popular theory, has also been proposed as a likely mechanism. This involves beneficial actions resulting from the response of an organism to a low intensity stressor, including the activation of stress response genes and heat shock proteins (Masoro, 2000).
Future studies may look into the effect of time between meals on lifespan, as intermittently fed mice have showed similar metabolic changes to those on a CR diet (Bordone and Guarente, 2005). Considering maximum lifespan as a main indicator compared to average lifespan may be more meaningful, as the latter may be influenced by optimising environmental conditions rather than a delay in ageing. Whilst significant knowledge has been gained from CR studies, much remains unanswered, particularly with regards to effects on humans. It is clear that high chronic calorie intake results in an increased risk of many disorders in humans. However, no studies have yet observed a reduction in risk when comparing CR to lean ad libitum individuals. Research in non-human primates may provide a more valuable tool for understanding the causes of ageing processes, and these may open new corridors for treatment of age-related disorders and ageing.