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Diet, metabolism and lifespan in Drosophila
Experimental Gerontology 40 (2005) 857–862 www.elsevier.com/locate/expgero
Mini review
Diet, metabolism and lifespan in Drosophila Matthew D.W. Pipera, Danielle Skorupab, Linda Partridgea,* a
UCL Centre for Research on Ageing, Department of Biology, University College London, Gower Street, London WC1E 6BT, UK b Huffington Center on Aging, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA Received 7 June 2005; received in revised form 19 June 2005; accepted 19 June 2005 Available online 30 August 2005
Abstract Dietary restriction (DR) by dilution of the food medium can extend lifespan in Drosophila. DR results in a state that is characterized by reduced fecundity, increased starvation resistance and higher total lipid levels. In the past, each of these correlated phenotypes has been proposed to play a causal role in the lifespan-extending effects of food reduction. However, more recent data show that each phenotype can be uncoupled from the long-lived state to varying extents. In this mini-review, we summarize the principal findings of the effects of DR on Drosophila in order to address what these phenotypes can tell us about the physiological remodeling required for Drosophila to be long-lived. Current data indicate lifespan-extension by DR is likely to involve both enhancement of various defense and detoxification mechanisms and a complex range of metabolic alterations that make energy available for these processes. q 2005 Elsevier Inc. All rights reserved.
1. Background More than 80 years ago, the first systematic investigations into ageing using Drosophila melanogaster as a model organism were initiated in a study by Pearl and Parker (1921). Originally they had planned to experiment on mice, however, ‘.just as the colony was ready to start definitive experimentation with, an accident completely destroyed it’. Since, this unintended initiation into Drosophila lifespan studies, the many advantages of this model of ageing have meant it has been extensively employed to study the effects of environment, genetics and behavior on lifespan. The functional context for such studies is provided by evolutionary life history theory, which suggests that length of life is dictated by the way limiting resources are allocated between traits that determine lifetime fitness, particularly between reproduction, on one hand, and growth and somatic maintenance on the other: the reproductive effort model (Hamilton, 1966; Kirkwood and Shanley, 2005). Food availability, the nutritional status of the organism and their effects upon reproductive rate play
* Corresponding author. Tel.: C44 20 7679 2983. E-mail address: [email protected] (L. Partridge).
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a key role in lifespan-determination and are central to evolutionary life history theory. Adult Drosophila are largely composed of post-mitotic tissue, and their nutritional requirements are therefore mainly to meet the costs associated with reproduction, movement and general maintenance for the preservation of self. Increased nutrient availability triggers a rapid elevation in reproductive activity in Drosophila. Both remating frequency as well as the daily and lifetime number of eggs laid per female are elevated with increased nutrition (Chapman and Partridge, 1996; Chippindale et al., 1993). In both of these studies, the same nutritional increases that led to elevated reproduction also resulted in reduced lifespan. Thus, an inverse relationship between reproduction and lifespan as a result of altered nutrition can be achieved in Drosophila—a phenomenon that has also been recognized in other organisms (Partridge et al., 2005a). This has led to the view in evolutionary theory that in times of plenty (when offspring survival is likely to be good) energy investment into reproduction is prioritized while during times of nutrient scarcity, self preservation is enhanced to enable survival until favorable conditions return and reproduction can be resumed (Harrison and Archer, 1988; Shanley and Kirkwood, 2000). That both the short-lived/ high-fecund flies (at high levels of nutrition) and the long-lived/low-fecund flies (at low levels of nutrition) are evolutionarily advantageous indicates that the organism is not malnourished in a general sense. This is distinct
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from the evolutionarily disadvantageous consequences of over-feeding or under-feeding, both of which result in malnourishment that causes decreased lifespan and lowered rates of reproduction (Partridge et al., 2005b). Elucidating the mechanistic link between nutrition without malnourishment to lifespan alteration is the focus of studies referred to herein as dietary restriction (DR). Many studies have investigated the effects of nutrition on Drosophila lifespan. Much of the early work establishing the basic nutritional requirements of flies reported very short lifespans due to malnutrition. The lifespan of flies kept on a nutritionally adequate diet of yeast and cane sugar was first reported in 1930 (Alpatov, 1930) and now forms the basis for most laboratory food recipes. Despite the existence of appropriate food recipes, there are still only a handful of studies on Drosophila longevity that first demonstrate decreased reproductive output across a range of nutrition that also increases lifespan, to establish true DR conditions. Here, we summarize work that has revealed information about the mechanisms of DR in D. melanogaster using only information from those situations where appropriate conditions for DR were first established. The focus here is on the role of metabolic changes in the response to DR. For a more detailed discussion of fundamental aspects of using Drosophila as a model of DR, readers are referred to (Partridge et al., 2005b).
2. Extension of lifespan by DR in Drosophila Lifespan-extension by DR in rodents is achieved by restricting the amount of food that the animal has access to. This technique appears to be unsuitable for Drosophila, both because direct observations have shown that flies visit the food to eat several times each hour (Piper, unpublished data) and because studies that have restricted access to food showed considerable deaths during periods of intermittent starvation (Oishi et al., 2004). This complication confounds any DR-related interpretation of studies on flies that have failed to report lifespan-extension after restricting access to food (Carey et al., 2002; Cooper et al., 2004; Kopec, 1928). It is therefore more practical and effective to dilute the concentration of nutrients in the food to which flies have free access. Both rates of egg-production (Chapman and Partridge, 1996) and of feeding behavior (Mair et al., 2005) indicate that flies do not regulate their feeding activity to compensate for the lowered nutritional value of DR food. Thus, lifespan-extension by DR in Drosophila appears to be a consequence of reduced nutrient ingestion, as it is for rodents. An important insight into the nature of the lifespanextension promoted by DR was recently made using demographic analyses of mortality rates when flies were switched between dietary treatments (Mair et al., 2003). In this study, a sub-population of flies maintained throughout adult life on control food (relatively high nutritional
content) was switched to DR food (relatively low nutritional content). A reciprocal switch was also performed by taking flies from DR to control food. Within 48 h of the change in diet, control flies adopted the low mortality of flies subject to lifelong DR. Likewise, the mortality of DR flies rapidly rose to the rate of flies maintained on control food for life. This is consistent with the hypothesis that irreversible deathcausing damage accumulates at the same rate in flies of both treatments and that a relatively high-nutrient diet causes an added risk of death. This added risk of dying is acquired rapidly and is completely reversible indicating that the physiological changes responsible for the action of DR in Drosophila are also likely to be rapid in effect and reversible.
3. Physiological features of Drosophila subjected to DR As mentioned above, reproductive activity responds rapidly to nutrition. In particular, the removal of yeast from a sugar/yeast diet has been shown to rapidly arrest egglaying of female Drosophila (Partridge et al., 1987; Good and Tatar, 2001). Recent work has shown that the yeast component of a sugar/yeast diet can elicit the majority of the effects of DR in Drosophila (Mair et al., 2005). These data suggest that the costs associated with egg-laying could be the direct cause of shortened life for flies maintained on high food. This possibility has been excluded, however, because female flies made sterile by X-irradiation or a mutation that stops oogenesis at stage 4 (before vitellogenesis of the eggs commences) still exhibit full lifespan-extension by DR that is rapidly reversible during nutrition switches (Mair et al., 2004). Furthermore, both post-reproductive females and males exhibit the characteristic mortality reduction and lifespan-extension in response to DR (Mair et al., 2003; Mair et al., 2004). Thus, if there is an obligatory trade-off between reduced reproduction and extension of lifespan by DR, it must operate both in males and prior to stage 4 of oogenesis in females, where it cannot be explained as a simple competition for limiting storage macromolecules. In addition to increased longevity and reduced fecundity, it has been noted that DR flies survive longer during starvation than do control-food fed flies (Chippindale et al., 1993). It has also been shown that Drosophila under DR have elevated relative lipid content compared with controlfood flies (Simmons and Bradley, 1997). Thus, the high lipid content of DR flies was proposed to account for their starvation resistance and increased longevity (Rauser et al., 2004). It is not known whether the higher lipid levels of DR flies is the result of increased fatty acid anabolism, decreased fatty acid catabolism or a combination of the two. In a recent study of a TAG-lipase mutant fly, it was shown that decreased rates of fatty acid catabolism result in higher relative body lipid content (Gro¨nke et al., 2005). These flies can still mobilize their fat reserves by the action of other TAG-lipases and exhibit higher starvation
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resistance than controls due to their increased lipid reserves. However, these mutant flies are shorter lived than controls, demonstrating that higher lipid content, elevated starvation resistance and reduced rates of lipid catabolism are not in themselves sufficient to extend Drosophila lifespan. While it is possible there is some unknown effect of the lipase mutation on lifespan, the data are also compatible with an explanation that fatty acid anabolism is enhanced under DR and that it is this aspect of the altered metabolic environment that can tell us something about how increased longevity can be achieved. These findings point to a similar conclusion to those from the effect of abolition of egglaying, namely that there is no direct competition for limiting storage macromolecules at the metabolic level. Rather, there is a diversion of resources upstream towards reproduction on the one hand, or starvation resistance and the processes required for lifespan-extension on the other. Aerobic metabolism is required for energy production but is also the source of numerous reactive oxygen species (ROS). These species can cause damage to major cellular constituents that is generally believed to underlie the aging process (Harman, 1956). Because the metabolic network of any organism is highly sensitive to nutrient supply, it has been proposed that dietary alterations could elicit changes in the rate of ROS (and therefore damage) production, by altering metabolic rate. Measurements by respirometry and calorimetry however show that there is no difference in mass-specific metabolic rate between DR and control flies (Hulbert et al., 2004). This observation is supported by studies that show DR flies have similar, if not elevated mitochondrial density when compared with flies fed high food (Magwere et al., in press) and mitochondria isolated from DR flies show no difference in ROS-production rates when compared with mitochondria from high food flies in vitro (Miwa et al., 2004). Therefore, under the conditions tested, ROS-generation seems not to account for the mortality differences observed between nutritional treatments, although in vivo measurements of the relevant variables are needed. Cellular damage can also be controlled by regulating the systems of defense and detoxification. It is still possible, therefore, that increased turnover of damaged macromolecules is the reduced risk factor associated with DR. This idea could be tested by studying markers of molecular damage during nutritional switches and how lifespan-extending interventions involved in defense (e.g. over-expression of the superoxide dismutases Sun et al., 2004) interact with DR.
4. Genetic pathways that interact with the response to DR Signaling pathways mediate the necessary flexibility of any organism’s metabolic network to changeable nutrition. The insulin/IGF-like signaling (IIS) and target of rapamycin (TOR) signaling pathways have been identified in flies and
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are known to couple growth to nutrition as well as playing a role in the control of adult lifespan (Tatar et al., 2001; Clancy et al., 2001; Hwangbo et al., 2004; Giannakou et al., 2004; Broughton et al., 2005; Kapahi et al., 2004; Leevers and Hafen, 2004). In particular, mutations that reduce signaling through these pathways slow growth, reduce fecundity and extend lifespan, thus conforming to the tradeoffs associated with reduced nutrition. It is also interesting that those long-lived IIS mutants tested possess increased lipid stores (Clancy et al., 2001; Tatar et al., 2001; Broughton et al., 2005). In light of these similarities, it is important to ask how the interactions of these mutations and DR affect lifespan. The interaction between DR and the lifespan-extension conferred by the IIS mutation chico has been reported (Clancy et al., 2002) as has the interaction between DR and reduced TOR-pathway activity resulting from overexpression of TSC2, which antagonizes TOR (Kapahi et al., 2004). Both of these studies show that the maximum lifespan achieved by DR cannot be further extended by the signaling pathway mutation at any of the food concentrations tested. Moreover, the peak lifespan of the mutant occurs at a higher food concentration than the peak lifespan of the control flies. This is consistent with the explanation that these flies are partially dietarily restricted by their genotype and therefore that IIS and TOR signaling pathways are involved in effecting altered lifespan in response to DR. However, neither mutation completely abolishes lifespan variation in response to altered nutrition, which would be expected if the intervention were solely responsible for mediating the effects of DR. This remaining lifespan variation could be mediated either by residual signaling activity in the mutated pathway (neither mutation completely abolishes signaling through its respective pathway) or due to the existence of alternative, parallel, nutrient sensing pathways that also affect lifespan variation. Two other genetic interventions that alter lifespan have also been proposed to operate in a manner similar to DR in Drosophila. First, heterozygous mutation of the dicarboxylate transporter INDY was reported to extend lifespan (Rogina et al., 2000). However, there are as yet no published data on the interaction between diet and lifespan for this intervention. The second intervention is through altered levels of either of two protein deacetylases Rpd3 or dSir2. These mutant flies (rpd3 heterozygotes and dSir2 over-expressors) had extended lifespan and were proposed to operate in the same genetic pathway to extend lifespan (Rogina et al., 2002; Rogina and Helfand, 2004). Wild-type dSir2 and rpd3 are apparently required for the lifespanextending effects of DR because both mutant genotypes were equally long-lived at both control and low food concentrations (Rogina et al., 2002; Rogina and Helfand, 2004), thus representing a complete block of lifespan variation in response to altered nutrition. The evidence, however, is not conclusive since only two food types were used. As food concentration is lowered, lifespan first
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increases, in response to DR, and then decreases, as a result of starvation-related malnutrition. Even with a normal response to DR, use of only two food types could show an increase, decrease or no effect on lifespan (Partridge et al., 2005b). In addition, the two food types used differed in their proportional nutrient composition. The flies therefore received qualitatively different foods, confounding interpretation of the effects of quantitatively altered nourishment. Further work should be performed using a dilution range of a single food type to establish the interaction between these genotypes and DR. Since, it is known that TOR signaling and IIS pathways can both be partial effectors of the lifespan response to DR (above), SIR2 would lie in the same pathway (either up or downstream of both) to block their effects.
5. Molecular insights into the effects of DR Comprehensive screens (e.g. transcriptomics, proteomics and metabolomics) are ideally suited to provide information on responses to interventions that alter physiology, such as changing nutrition. Of relevance to the field of Drosophila ageing are a number of microarray studies that have investigated the effects of nutrition, stress and age on gene expression (Pletcher et al., 2005). The only such study that has specifically examined the effects of DR in Drosophila reported a time-course of expression profiles from flies chronically exposed to DR or control-feeding conditions (Pletcher et al., 2002). By coupling individual gene transcript profiles to demographic data, these authors distinguished groups of genes whose expression profiles track with biological age or chronological age in both dietary treatments. Genes whose expression was similar between diets when scaled to biological age (determined from survivorship) were classified as health markers, while those with similar profiles between diets when scaled to chronological age were identified as ageing markers. In light of the demographic data on DR, the former category that are affected by both age and dietary treatment track with the nutrition-associated risk of dying, while the latter category track to death-causing irreversible damage that is unaffected by diet. From the identity of genes in the group that tracked with biological age, several functional categories were identified that potentially provide physiological information about the effects of DR on flies. Groups of genes whose transcript levels decreased with chronological age, but were delayed by DR, had functions related to reproduction, while groups of genes related to fatty acid synthesis, anti-microbial peptides, metalothionines and a subset of cytochrome P450s had increased expression with chronological age that was similarly delayed by DR (Pletcher et al., 2002; Pletcher et al., 2005). With the caveat that gene expression only represents an indirect measure of physiology, the changes in reproduction agree well with the physiological changes
that occur with DR (above). Furthermore, several categories contain genes involved in self-preservation (defense and detoxification), which is a popular candidate mechanism to delay the onset of death (above). This conforms to the proposal that DR-related signaling changes alter defenses to enhance survival at the same time as altering the metabolic network to reduce reproductive output and channel reserves to storage.
6. Self-preservation and metabolic network alterations under DR DR in Drosophila acts rapidly to lower mortality rate, suggesting that shifts in the whole metabolic network are efficiently coordinated with changes in defense capabilities. Recent reports on the wider functions of the IIS transcription factor FOXO and the TOR signaling pathway can perhaps shed light on how this is achieved, since they reveal that these pathways can act as molecular switches between growth promotion when nutrients are abundant versus enhancement of general defense and detoxification systems when nutrients are scarce (Wang et al., 2005; Scott et al., 2004). In particular, it has been shown that the stress responsive Jun-N-terminal kinase (JNK) signaling pathway signals through FOXO to extend lifespan and increase stress defenses (Wang et al., 2005). This signal is associated with nuclear localization of FOXO, which occurs during lowered IIS signaling, the circumstance under which IIS modulation extends lifespan. Lowered signaling through TOR is associated with an increase in autophagy (Scott et al., 2004) that functions to enhance nutrient recovery by degrading cellular structures. Detoxification of endogenous damage occurs as a byproduct of this process and has large implications for cellular survival—especially in postmitotic tissues (Cuervo, 2004). These molecular switches thus enable up-regulation of the machinery required for improved defense and detoxification and remodeling of the metabolic network to withdraw resources from reproduction and make energy available for repair. The fact that these adjustments are made simultaneously is important because this indicates that they are interconnected branches of the same upstream pathway that is activated by DR. Drosophila exposed to DR contain higher relative lipid and carbohydrate levels than control-fed flies (Simmons and Bradley, 1997), which is in agreement with a recent report that long-lived flies under DR are not limited for calories (Mair et al., 2005). The prerequisites for fatty acid anabolism are the availability of activated carbon as malonyl CoA and acetyl CoA as well as reducing energy in the form of NADPH. Availability of NADPH is also critical for the biosynthetic reactions that accompany egg production and in stress defense and detoxification to maintain a reduced cellular environment. The pentose phosphate pathway is critical for the reduction of NADPC to NADPH and is controlled at the committed step catalyzed
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by glucose 6-phosphate dehydrogenase (encoded by Zw in Drosophila). During increasing biological age, this gene is dramatically up-regulated under DR conditions, but not in control food fed flies (Pletcher et al., 2002) indicating a greater potential for NADPC reduction in DR individuals. Activation of this gene is consistent with the rise in lipid levels in DR flies, but is counter-intuitive in light of the removal of the heavy anabolic demand associated with reproduction. Thus, the increase in expression of this gene under DR may reflect greater availability of reducing power to defense systems such as the glutathione/thioredoxin system (Missirlis et al., 2001) and aspects of xenobiotic detoxification, which have both been implicated in controlling lifespan (Gems and McElwee, 2005). It is interesting to note that in an experiment to select long- and short-lived Drosophila, glucose 6-phosphate dehydrogenase activity was positively correlated with longevity (Luckinbill et al., 1990) and that this metabolic enzyme is also critical for defense responses in human cells (Salvemini et al., 1999) and yeast (Slekar et al., 1996). Furthermore, recent array data on chico-mutant flies that are long-lived, stress resistant and possess high lipid levels show increased Zw expression (Piper, unpublished data). This is consistent with chico playing a role in mediating the effects of DR (Clancy et al., 2002). A similar case has been made for the interrelationship of energy metabolism (in terms of the NAD:NADH redox couple) and the mode of action of Sir2 to extend lifespan under calorie restriction (Koubova and Guarente, 2003). In view of the complex network of reactions that comprise metabolism, it is conceivable (and indeed likely) that both the NAD:NADH and NADP:NADPH couples play a role in lifespandetermination under DR. These cofactors are present in only catalytic amounts in cells and the ratio of their redox couples is known to be critical in determining metabolic fluxes. By coupling the activation of defense pathways to nutrientsensitive signaling (such as the TOR and IIS pathways) enhancement of defense systems occurs only in the presence of the appropriate metabolic alterations. In fact, the requirement for general physiological change may explain why the most successful lifespan-enhancing interventions identified to date (DR and the nutrient signaling pathway mutants) intervene high in the hierarchy of physiologyaltering signaling events. This argument is supported by a study in worms that found many genes whose expression was changed by mutations in the IIS pathway could elicit only small changes in lifespan individually, arguing it is the sum of all these changes together that is required to alter lifespan (Murphy et al., 2003).
7. Conclusion DR has been shown to extended lifespan in a wide variety of organisms from yeast to mammals. This offers the potential to study the mechanisms of DR in short-lived
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model organisms like Drosophila and use any insights gained to direct research in longer-lived higher organisms. At the present time it is still unknown whether the precise mechanisms that mediate the longevity effects of DR in different organisms are conserved or whether it is an example of convergent evolution. Such interspecies comparisons are important to answer this question. Further research on the mechanisms of DR in Drosophila should focus on the interaction between diet and nutrientsensing signaling pathways across acute nutritional switches. An additional level of information can be gained from these studies if the nutritional switches are performed with defined diets rather than simple dilution of the yeast component. However, this first requires development of a defined diet that is suitable for Drosophila lifespan studies. Within the relatively narrow time window required for mortality changes across the switches, analyses of the transcriptome, proteome and metabolome will help build information about how physiology is altered to ensure longevity. Since these individual changes are likely to be many and complex, involving both up-regulation of protective mechanisms as well as remodeling metabolism, reverse engineering DR-type lifespan-extension is likely to be most successful by manipulating nutrient-sensitive signaling pathways.
Acknowledgements We thank Will Mair for helpful comments on the manuscript, as well as the BBSRC, Wellcome Trust and National Institutes of Health for funding. We apologize to authors for the omission of several references to primary research due to space limitations.
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Mini review
Diet, metabolism and lifespan in Drosophila Matthew D.W. Pipera, Danielle Skorupab, Linda Partridgea,* a
UCL Centre for Research on Ageing, Department of Biology, University College London, Gower Street, London WC1E 6BT, UK b Huffington Center on Aging, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA Received 7 June 2005; received in revised form 19 June 2005; accepted 19 June 2005 Available online 30 August 2005
Abstract Dietary restriction (DR) by dilution of the food medium can extend lifespan in Drosophila. DR results in a state that is characterized by reduced fecundity, increased starvation resistance and higher total lipid levels. In the past, each of these correlated phenotypes has been proposed to play a causal role in the lifespan-extending effects of food reduction. However, more recent data show that each phenotype can be uncoupled from the long-lived state to varying extents. In this mini-review, we summarize the principal findings of the effects of DR on Drosophila in order to address what these phenotypes can tell us about the physiological remodeling required for Drosophila to be long-lived. Current data indicate lifespan-extension by DR is likely to involve both enhancement of various defense and detoxification mechanisms and a complex range of metabolic alterations that make energy available for these processes. q 2005 Elsevier Inc. All rights reserved.
1. Background More than 80 years ago, the first systematic investigations into ageing using Drosophila melanogaster as a model organism were initiated in a study by Pearl and Parker (1921). Originally they had planned to experiment on mice, however, ‘.just as the colony was ready to start definitive experimentation with, an accident completely destroyed it’. Since, this unintended initiation into Drosophila lifespan studies, the many advantages of this model of ageing have meant it has been extensively employed to study the effects of environment, genetics and behavior on lifespan. The functional context for such studies is provided by evolutionary life history theory, which suggests that length of life is dictated by the way limiting resources are allocated between traits that determine lifetime fitness, particularly between reproduction, on one hand, and growth and somatic maintenance on the other: the reproductive effort model (Hamilton, 1966; Kirkwood and Shanley, 2005). Food availability, the nutritional status of the organism and their effects upon reproductive rate play
* Corresponding author. Tel.: C44 20 7679 2983. E-mail address: [email protected] (L. Partridge).
0531-5565/$ - see front matter q 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2005.06.013
a key role in lifespan-determination and are central to evolutionary life history theory. Adult Drosophila are largely composed of post-mitotic tissue, and their nutritional requirements are therefore mainly to meet the costs associated with reproduction, movement and general maintenance for the preservation of self. Increased nutrient availability triggers a rapid elevation in reproductive activity in Drosophila. Both remating frequency as well as the daily and lifetime number of eggs laid per female are elevated with increased nutrition (Chapman and Partridge, 1996; Chippindale et al., 1993). In both of these studies, the same nutritional increases that led to elevated reproduction also resulted in reduced lifespan. Thus, an inverse relationship between reproduction and lifespan as a result of altered nutrition can be achieved in Drosophila—a phenomenon that has also been recognized in other organisms (Partridge et al., 2005a). This has led to the view in evolutionary theory that in times of plenty (when offspring survival is likely to be good) energy investment into reproduction is prioritized while during times of nutrient scarcity, self preservation is enhanced to enable survival until favorable conditions return and reproduction can be resumed (Harrison and Archer, 1988; Shanley and Kirkwood, 2000). That both the short-lived/ high-fecund flies (at high levels of nutrition) and the long-lived/low-fecund flies (at low levels of nutrition) are evolutionarily advantageous indicates that the organism is not malnourished in a general sense. This is distinct
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from the evolutionarily disadvantageous consequences of over-feeding or under-feeding, both of which result in malnourishment that causes decreased lifespan and lowered rates of reproduction (Partridge et al., 2005b). Elucidating the mechanistic link between nutrition without malnourishment to lifespan alteration is the focus of studies referred to herein as dietary restriction (DR). Many studies have investigated the effects of nutrition on Drosophila lifespan. Much of the early work establishing the basic nutritional requirements of flies reported very short lifespans due to malnutrition. The lifespan of flies kept on a nutritionally adequate diet of yeast and cane sugar was first reported in 1930 (Alpatov, 1930) and now forms the basis for most laboratory food recipes. Despite the existence of appropriate food recipes, there are still only a handful of studies on Drosophila longevity that first demonstrate decreased reproductive output across a range of nutrition that also increases lifespan, to establish true DR conditions. Here, we summarize work that has revealed information about the mechanisms of DR in D. melanogaster using only information from those situations where appropriate conditions for DR were first established. The focus here is on the role of metabolic changes in the response to DR. For a more detailed discussion of fundamental aspects of using Drosophila as a model of DR, readers are referred to (Partridge et al., 2005b).
2. Extension of lifespan by DR in Drosophila Lifespan-extension by DR in rodents is achieved by restricting the amount of food that the animal has access to. This technique appears to be unsuitable for Drosophila, both because direct observations have shown that flies visit the food to eat several times each hour (Piper, unpublished data) and because studies that have restricted access to food showed considerable deaths during periods of intermittent starvation (Oishi et al., 2004). This complication confounds any DR-related interpretation of studies on flies that have failed to report lifespan-extension after restricting access to food (Carey et al., 2002; Cooper et al., 2004; Kopec, 1928). It is therefore more practical and effective to dilute the concentration of nutrients in the food to which flies have free access. Both rates of egg-production (Chapman and Partridge, 1996) and of feeding behavior (Mair et al., 2005) indicate that flies do not regulate their feeding activity to compensate for the lowered nutritional value of DR food. Thus, lifespan-extension by DR in Drosophila appears to be a consequence of reduced nutrient ingestion, as it is for rodents. An important insight into the nature of the lifespanextension promoted by DR was recently made using demographic analyses of mortality rates when flies were switched between dietary treatments (Mair et al., 2003). In this study, a sub-population of flies maintained throughout adult life on control food (relatively high nutritional
content) was switched to DR food (relatively low nutritional content). A reciprocal switch was also performed by taking flies from DR to control food. Within 48 h of the change in diet, control flies adopted the low mortality of flies subject to lifelong DR. Likewise, the mortality of DR flies rapidly rose to the rate of flies maintained on control food for life. This is consistent with the hypothesis that irreversible deathcausing damage accumulates at the same rate in flies of both treatments and that a relatively high-nutrient diet causes an added risk of death. This added risk of dying is acquired rapidly and is completely reversible indicating that the physiological changes responsible for the action of DR in Drosophila are also likely to be rapid in effect and reversible.
3. Physiological features of Drosophila subjected to DR As mentioned above, reproductive activity responds rapidly to nutrition. In particular, the removal of yeast from a sugar/yeast diet has been shown to rapidly arrest egglaying of female Drosophila (Partridge et al., 1987; Good and Tatar, 2001). Recent work has shown that the yeast component of a sugar/yeast diet can elicit the majority of the effects of DR in Drosophila (Mair et al., 2005). These data suggest that the costs associated with egg-laying could be the direct cause of shortened life for flies maintained on high food. This possibility has been excluded, however, because female flies made sterile by X-irradiation or a mutation that stops oogenesis at stage 4 (before vitellogenesis of the eggs commences) still exhibit full lifespan-extension by DR that is rapidly reversible during nutrition switches (Mair et al., 2004). Furthermore, both post-reproductive females and males exhibit the characteristic mortality reduction and lifespan-extension in response to DR (Mair et al., 2003; Mair et al., 2004). Thus, if there is an obligatory trade-off between reduced reproduction and extension of lifespan by DR, it must operate both in males and prior to stage 4 of oogenesis in females, where it cannot be explained as a simple competition for limiting storage macromolecules. In addition to increased longevity and reduced fecundity, it has been noted that DR flies survive longer during starvation than do control-food fed flies (Chippindale et al., 1993). It has also been shown that Drosophila under DR have elevated relative lipid content compared with controlfood flies (Simmons and Bradley, 1997). Thus, the high lipid content of DR flies was proposed to account for their starvation resistance and increased longevity (Rauser et al., 2004). It is not known whether the higher lipid levels of DR flies is the result of increased fatty acid anabolism, decreased fatty acid catabolism or a combination of the two. In a recent study of a TAG-lipase mutant fly, it was shown that decreased rates of fatty acid catabolism result in higher relative body lipid content (Gro¨nke et al., 2005). These flies can still mobilize their fat reserves by the action of other TAG-lipases and exhibit higher starvation
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resistance than controls due to their increased lipid reserves. However, these mutant flies are shorter lived than controls, demonstrating that higher lipid content, elevated starvation resistance and reduced rates of lipid catabolism are not in themselves sufficient to extend Drosophila lifespan. While it is possible there is some unknown effect of the lipase mutation on lifespan, the data are also compatible with an explanation that fatty acid anabolism is enhanced under DR and that it is this aspect of the altered metabolic environment that can tell us something about how increased longevity can be achieved. These findings point to a similar conclusion to those from the effect of abolition of egglaying, namely that there is no direct competition for limiting storage macromolecules at the metabolic level. Rather, there is a diversion of resources upstream towards reproduction on the one hand, or starvation resistance and the processes required for lifespan-extension on the other. Aerobic metabolism is required for energy production but is also the source of numerous reactive oxygen species (ROS). These species can cause damage to major cellular constituents that is generally believed to underlie the aging process (Harman, 1956). Because the metabolic network of any organism is highly sensitive to nutrient supply, it has been proposed that dietary alterations could elicit changes in the rate of ROS (and therefore damage) production, by altering metabolic rate. Measurements by respirometry and calorimetry however show that there is no difference in mass-specific metabolic rate between DR and control flies (Hulbert et al., 2004). This observation is supported by studies that show DR flies have similar, if not elevated mitochondrial density when compared with flies fed high food (Magwere et al., in press) and mitochondria isolated from DR flies show no difference in ROS-production rates when compared with mitochondria from high food flies in vitro (Miwa et al., 2004). Therefore, under the conditions tested, ROS-generation seems not to account for the mortality differences observed between nutritional treatments, although in vivo measurements of the relevant variables are needed. Cellular damage can also be controlled by regulating the systems of defense and detoxification. It is still possible, therefore, that increased turnover of damaged macromolecules is the reduced risk factor associated with DR. This idea could be tested by studying markers of molecular damage during nutritional switches and how lifespan-extending interventions involved in defense (e.g. over-expression of the superoxide dismutases Sun et al., 2004) interact with DR.
4. Genetic pathways that interact with the response to DR Signaling pathways mediate the necessary flexibility of any organism’s metabolic network to changeable nutrition. The insulin/IGF-like signaling (IIS) and target of rapamycin (TOR) signaling pathways have been identified in flies and
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are known to couple growth to nutrition as well as playing a role in the control of adult lifespan (Tatar et al., 2001; Clancy et al., 2001; Hwangbo et al., 2004; Giannakou et al., 2004; Broughton et al., 2005; Kapahi et al., 2004; Leevers and Hafen, 2004). In particular, mutations that reduce signaling through these pathways slow growth, reduce fecundity and extend lifespan, thus conforming to the tradeoffs associated with reduced nutrition. It is also interesting that those long-lived IIS mutants tested possess increased lipid stores (Clancy et al., 2001; Tatar et al., 2001; Broughton et al., 2005). In light of these similarities, it is important to ask how the interactions of these mutations and DR affect lifespan. The interaction between DR and the lifespan-extension conferred by the IIS mutation chico has been reported (Clancy et al., 2002) as has the interaction between DR and reduced TOR-pathway activity resulting from overexpression of TSC2, which antagonizes TOR (Kapahi et al., 2004). Both of these studies show that the maximum lifespan achieved by DR cannot be further extended by the signaling pathway mutation at any of the food concentrations tested. Moreover, the peak lifespan of the mutant occurs at a higher food concentration than the peak lifespan of the control flies. This is consistent with the explanation that these flies are partially dietarily restricted by their genotype and therefore that IIS and TOR signaling pathways are involved in effecting altered lifespan in response to DR. However, neither mutation completely abolishes lifespan variation in response to altered nutrition, which would be expected if the intervention were solely responsible for mediating the effects of DR. This remaining lifespan variation could be mediated either by residual signaling activity in the mutated pathway (neither mutation completely abolishes signaling through its respective pathway) or due to the existence of alternative, parallel, nutrient sensing pathways that also affect lifespan variation. Two other genetic interventions that alter lifespan have also been proposed to operate in a manner similar to DR in Drosophila. First, heterozygous mutation of the dicarboxylate transporter INDY was reported to extend lifespan (Rogina et al., 2000). However, there are as yet no published data on the interaction between diet and lifespan for this intervention. The second intervention is through altered levels of either of two protein deacetylases Rpd3 or dSir2. These mutant flies (rpd3 heterozygotes and dSir2 over-expressors) had extended lifespan and were proposed to operate in the same genetic pathway to extend lifespan (Rogina et al., 2002; Rogina and Helfand, 2004). Wild-type dSir2 and rpd3 are apparently required for the lifespanextending effects of DR because both mutant genotypes were equally long-lived at both control and low food concentrations (Rogina et al., 2002; Rogina and Helfand, 2004), thus representing a complete block of lifespan variation in response to altered nutrition. The evidence, however, is not conclusive since only two food types were used. As food concentration is lowered, lifespan first
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increases, in response to DR, and then decreases, as a result of starvation-related malnutrition. Even with a normal response to DR, use of only two food types could show an increase, decrease or no effect on lifespan (Partridge et al., 2005b). In addition, the two food types used differed in their proportional nutrient composition. The flies therefore received qualitatively different foods, confounding interpretation of the effects of quantitatively altered nourishment. Further work should be performed using a dilution range of a single food type to establish the interaction between these genotypes and DR. Since, it is known that TOR signaling and IIS pathways can both be partial effectors of the lifespan response to DR (above), SIR2 would lie in the same pathway (either up or downstream of both) to block their effects.
5. Molecular insights into the effects of DR Comprehensive screens (e.g. transcriptomics, proteomics and metabolomics) are ideally suited to provide information on responses to interventions that alter physiology, such as changing nutrition. Of relevance to the field of Drosophila ageing are a number of microarray studies that have investigated the effects of nutrition, stress and age on gene expression (Pletcher et al., 2005). The only such study that has specifically examined the effects of DR in Drosophila reported a time-course of expression profiles from flies chronically exposed to DR or control-feeding conditions (Pletcher et al., 2002). By coupling individual gene transcript profiles to demographic data, these authors distinguished groups of genes whose expression profiles track with biological age or chronological age in both dietary treatments. Genes whose expression was similar between diets when scaled to biological age (determined from survivorship) were classified as health markers, while those with similar profiles between diets when scaled to chronological age were identified as ageing markers. In light of the demographic data on DR, the former category that are affected by both age and dietary treatment track with the nutrition-associated risk of dying, while the latter category track to death-causing irreversible damage that is unaffected by diet. From the identity of genes in the group that tracked with biological age, several functional categories were identified that potentially provide physiological information about the effects of DR on flies. Groups of genes whose transcript levels decreased with chronological age, but were delayed by DR, had functions related to reproduction, while groups of genes related to fatty acid synthesis, anti-microbial peptides, metalothionines and a subset of cytochrome P450s had increased expression with chronological age that was similarly delayed by DR (Pletcher et al., 2002; Pletcher et al., 2005). With the caveat that gene expression only represents an indirect measure of physiology, the changes in reproduction agree well with the physiological changes
that occur with DR (above). Furthermore, several categories contain genes involved in self-preservation (defense and detoxification), which is a popular candidate mechanism to delay the onset of death (above). This conforms to the proposal that DR-related signaling changes alter defenses to enhance survival at the same time as altering the metabolic network to reduce reproductive output and channel reserves to storage.
6. Self-preservation and metabolic network alterations under DR DR in Drosophila acts rapidly to lower mortality rate, suggesting that shifts in the whole metabolic network are efficiently coordinated with changes in defense capabilities. Recent reports on the wider functions of the IIS transcription factor FOXO and the TOR signaling pathway can perhaps shed light on how this is achieved, since they reveal that these pathways can act as molecular switches between growth promotion when nutrients are abundant versus enhancement of general defense and detoxification systems when nutrients are scarce (Wang et al., 2005; Scott et al., 2004). In particular, it has been shown that the stress responsive Jun-N-terminal kinase (JNK) signaling pathway signals through FOXO to extend lifespan and increase stress defenses (Wang et al., 2005). This signal is associated with nuclear localization of FOXO, which occurs during lowered IIS signaling, the circumstance under which IIS modulation extends lifespan. Lowered signaling through TOR is associated with an increase in autophagy (Scott et al., 2004) that functions to enhance nutrient recovery by degrading cellular structures. Detoxification of endogenous damage occurs as a byproduct of this process and has large implications for cellular survival—especially in postmitotic tissues (Cuervo, 2004). These molecular switches thus enable up-regulation of the machinery required for improved defense and detoxification and remodeling of the metabolic network to withdraw resources from reproduction and make energy available for repair. The fact that these adjustments are made simultaneously is important because this indicates that they are interconnected branches of the same upstream pathway that is activated by DR. Drosophila exposed to DR contain higher relative lipid and carbohydrate levels than control-fed flies (Simmons and Bradley, 1997), which is in agreement with a recent report that long-lived flies under DR are not limited for calories (Mair et al., 2005). The prerequisites for fatty acid anabolism are the availability of activated carbon as malonyl CoA and acetyl CoA as well as reducing energy in the form of NADPH. Availability of NADPH is also critical for the biosynthetic reactions that accompany egg production and in stress defense and detoxification to maintain a reduced cellular environment. The pentose phosphate pathway is critical for the reduction of NADPC to NADPH and is controlled at the committed step catalyzed
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by glucose 6-phosphate dehydrogenase (encoded by Zw in Drosophila). During increasing biological age, this gene is dramatically up-regulated under DR conditions, but not in control food fed flies (Pletcher et al., 2002) indicating a greater potential for NADPC reduction in DR individuals. Activation of this gene is consistent with the rise in lipid levels in DR flies, but is counter-intuitive in light of the removal of the heavy anabolic demand associated with reproduction. Thus, the increase in expression of this gene under DR may reflect greater availability of reducing power to defense systems such as the glutathione/thioredoxin system (Missirlis et al., 2001) and aspects of xenobiotic detoxification, which have both been implicated in controlling lifespan (Gems and McElwee, 2005). It is interesting to note that in an experiment to select long- and short-lived Drosophila, glucose 6-phosphate dehydrogenase activity was positively correlated with longevity (Luckinbill et al., 1990) and that this metabolic enzyme is also critical for defense responses in human cells (Salvemini et al., 1999) and yeast (Slekar et al., 1996). Furthermore, recent array data on chico-mutant flies that are long-lived, stress resistant and possess high lipid levels show increased Zw expression (Piper, unpublished data). This is consistent with chico playing a role in mediating the effects of DR (Clancy et al., 2002). A similar case has been made for the interrelationship of energy metabolism (in terms of the NAD:NADH redox couple) and the mode of action of Sir2 to extend lifespan under calorie restriction (Koubova and Guarente, 2003). In view of the complex network of reactions that comprise metabolism, it is conceivable (and indeed likely) that both the NAD:NADH and NADP:NADPH couples play a role in lifespandetermination under DR. These cofactors are present in only catalytic amounts in cells and the ratio of their redox couples is known to be critical in determining metabolic fluxes. By coupling the activation of defense pathways to nutrientsensitive signaling (such as the TOR and IIS pathways) enhancement of defense systems occurs only in the presence of the appropriate metabolic alterations. In fact, the requirement for general physiological change may explain why the most successful lifespan-enhancing interventions identified to date (DR and the nutrient signaling pathway mutants) intervene high in the hierarchy of physiologyaltering signaling events. This argument is supported by a study in worms that found many genes whose expression was changed by mutations in the IIS pathway could elicit only small changes in lifespan individually, arguing it is the sum of all these changes together that is required to alter lifespan (Murphy et al., 2003).
7. Conclusion DR has been shown to extended lifespan in a wide variety of organisms from yeast to mammals. This offers the potential to study the mechanisms of DR in short-lived
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model organisms like Drosophila and use any insights gained to direct research in longer-lived higher organisms. At the present time it is still unknown whether the precise mechanisms that mediate the longevity effects of DR in different organisms are conserved or whether it is an example of convergent evolution. Such interspecies comparisons are important to answer this question. Further research on the mechanisms of DR in Drosophila should focus on the interaction between diet and nutrientsensing signaling pathways across acute nutritional switches. An additional level of information can be gained from these studies if the nutritional switches are performed with defined diets rather than simple dilution of the yeast component. However, this first requires development of a defined diet that is suitable for Drosophila lifespan studies. Within the relatively narrow time window required for mortality changes across the switches, analyses of the transcriptome, proteome and metabolome will help build information about how physiology is altered to ensure longevity. Since these individual changes are likely to be many and complex, involving both up-regulation of protective mechanisms as well as remodeling metabolism, reverse engineering DR-type lifespan-extension is likely to be most successful by manipulating nutrient-sensitive signaling pathways.
Acknowledgements We thank Will Mair for helpful comments on the manuscript, as well as the BBSRC, Wellcome Trust and National Institutes of Health for funding. We apologize to authors for the omission of several references to primary research due to space limitations.
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