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Department of Biology, University College London, United Kingdom.
Address correspondence to Linda Partridge, Department of Biology, University College London, Darwin Building, Gower Street, London, WC1E 6BT, UK. E-mail: l.partridge{at}ucl.ac.uk
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Abstract |
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 Top Abstract BackgroundD. melanogaster as a...Mechanisms by Which DR...References |
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Dietary restriction (DR) appears to be a truly "public" modulator of life span (10) because it has been shown to increase longevity in organisms ranging from yeast (11,12), various invertebrates (6,13), and rodents (14) to primates, where preliminary data indicate that it is having positive results (15–18). Although conservation of the longevity phenotype spans the evolutionary distance from single-celled organisms to mammals, little is known of the mechanisms that connect diet and life span in the different species. For progress to be made in this area, we believe it is important that two criteria are fulfilled in studies of DR: 1) protocols are demonstrated to operate within the evolutionary trade-offs described above so that at the food-intake level for increased longevity (restricted), fecundity and/or growth are coordinately reduced, demonstrating that the relatively short-lived animals (fully fed) are likely to have increased evolutionary fitness; and 2) the diets are well defined. These rules allow dietary manipulations to be related to specific physiological responses that should inform us about the mechanisms by which diet affects the risk of death.
A great deal of literature has been published concerning the effects of DR on rodent life span since the first report in 1935 by McCay and colleagues (14), and many good comprehensive reviews are available on this topic (19–22). However, 70 years of research on DR has yielded relatively few insights into the mechanisms by which this intervention works to extend life. One experimental approach to elucidating these mechanisms is the study of food components that are critical for the effects of DR. We therefore begin this review by considering the potential role that calories have been proposed to play in longevity. To address the role of many more dietary components on life span in a thorough manner, it is appropriate to use relatively short-lived and easily contained model organisms. Data from such model-organism studies can then be used to provide direction for the more laborious and expensive work of testing dietary manipulations on mammals. It is our aim, therefore, to highlight the research value of the model organism Drosophila melanogaster for detailed DR studies and to provide guidelines for their appropriate experimental design. Finally, we also provide a brief summary of several important mechanistic insights into DR that have already been made using D. melanogaster that currently await testing in mammalian systems.
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BACKGROUND |
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TopAbstractBackgroundD. melanogaster as a...Mechanisms by Which DR...References |
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Possibly the most striking feature of life-span extension by reduced food intake is the delay in the onset of a broad range of age-related pathologies. The delay in appearance of rodent diseases such as chronic nephropathy, cardiomyopathy, and neoplasia are thought to underlie the magnitude of the impact of DR on life span, which is typically increased in restricted animals by 40%–50% for both median and maximum (26,27). Thus, the hallmarks of DR-induced life-span extension are its effectiveness in preventing a broad range of diseases and its substantial effect on life span.
Caloric Restriction or DR?
The term "calorie restriction" (CR) has gained common usage to label the extension of life span by food reduction. However, the term is often used without clear reference to what it actually describes. We believe that making this reference clear highlights the need for greater use of the term ‘dietary restriction’ to describe food-reduction interventions that extend life span by the criteria described above.
There are two possible ways of defining CR that have distinct and noninterchangeable meanings. These are: 1) to describe the theoretical energy content of a diet; and 2) to describe the actual biologically useable energy in a diet. This distinction is important to make, because the latter term can yield insights into the physiological mechanisms by which diet affects life span, whereas the former term cannot. Thus, published reports describing the effects of calorie reduction using the first definition must be interpreted extremely conservatively when drawing mechanistic conclusions. This is apparent after further considering the meaning of the two definitions.
Theoretical energy content of a diet.-- The theoretical calorie content of food used in the literature is derived from bomb calorimetry studies, modified by empirically derived data (28). This enables a formulaic approach to food design that assigns carbohydrate and protein a value of 4 kcal/g and fat 9 kcal/g. It should be stressed that this definition is the basis for the assertion in most of the literature that animals subject to different treatments ingest equal or unequal daily and lifetime calories [for examples, see diet designs in (26,27,29)].
Using this definition there are many studies that do demonstrate a correlation between reduced daily calorie intake and life-span extension (24,25,30–33). However, for the term CR to be valid in this sense, an obligate relationship must exist between the theoretical calorie content of the diet and the longevity phenotype. There are several studies that show that this is not the case (24,27,34–40). Despite the concern that in several of these cases the full criteria outlined above for the characteristic "CR" effect on life span were not met, others of these do meet those criteria and do so by the ingestion of diets where daily calorie intakes were not reduced. Thus, "dietary restriction" is a more appropriate term to describe the intervention by this definition.
Actual biologically useable energy in a diet.-- Quantifying the actual, biologically accessible nutrients in a diet is extremely difficult to do. This is because its determination involves complex experimental procedures, and the value itself depends on the nutritional balance of the food as well as the physiology of the animal (that is influenced by genotype, age, and various aspects of the environment). Thus, the actual biologically useable nutrients in a diet are different for each organism, and vary throughout an animal's lifetime. This point has been recognized previously and well made by Payne (41) concerning biologically useable protein content of diets and by Weindruch and Walford (19) in their book aptly entitled "The Retardation of Aging and Disease by Dietary Restriction." In light of the difficulties in ascertaining the actual biologically useable energy in a diet, we also believe the term "dietary restriction" should be used to label the literature in this field.
Methionine Restriction and Life-Span Extension in Rodents
Of the studies that show life-span extension without a reduction in theoretical daily calorie intake, three that are published do so by the specific reduction of the essential amino acid methionine in the diet of rats (37–39) and there is one as yet unpublished report on mice (Miller RA, personal communication). In the first of the published studies, the methionine level administered was shown to be growth limiting (an effect that could not be reversed by supplementation with extra calories), indicating that this intervention was not due to ingestion of a calorie-limited diet (37). Furthermore, equivalence of theoretical daily caloric intake between experimental cohorts throughout the life-span experiments was ensured by pair-feeding (39). Importantly, this intervention was shown to extend mean and maximum life span by up to 50% and 44%, respectively, while pathological data indicate a delay in all major ageing-related illnesses (Zimmerman JA, personal communication). Thus, it fulfils the criteria previously used for the full effects of DR on longevity and does so without altered caloric intake. Further preliminary data in (39) indicate that this effect is conserved for males of four different strains of rat commonly used in the laboratory. Thus a diet containing growth-restricting amounts of methionine is sufficient to elicit life-span extension irrespective of genetic background. It remains to be seen if the protective effects of this intervention can be acquired postdevelopmentally, as all work to date has initiated the restricted diet during development and is accompanied by dramatically slowed growth.
In the absence of information on the critical physiological changes required for longer life, it is possible that methionine restriction and whole-food reduction operate through the same or different mechanistic pathways to extend life. Thus, the intriguing possibility exists that food components can affect physiology in many ways to promote longevity. To elucidate these differences or similarities, a comprehensive analysis of the food components that can affect life span without malnutrition is required. This should lead to a better understanding of the interaction between physiology and life-span determination.
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D. MELANOGASTER AS A MODEL FOR STUDIES ON THE INTERACTION BETWEEN DR AND LIFE SPAN |
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TopAbstractBackgroundD. melanogaster as a...Mechanisms by Which DR...References |
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Studies with yeast have made a significant impact on the ageing field because DR protocols exist based both on reduction of nutrients in the medium and on mutations in single genes. Furthermore, mechanistic studies have been aided by the fact that the organism is particularly amenable to genome-wide molecular biology approaches. However, the relevance of single-cell replicative ageing to that in multicellular, differentiated organisms is questionable, and at least one of the genetic factors thought to be an essential determinant of life span (42) is apparently yeast specific. Furthermore, S. cerevisiae has atypical energy metabolism when compared with higher organisms because it will preferentially initiate anaerobic, fermentative sugar catabolism, instead of respiratory metabolism, in the presence of high glucose (43). This finding in S. cerevisiae complicates interpretations of the way diet affects physiology to alter life span.
Like yeast, C. elegans has been used successfully to study the mechanisms of ageing. In light of its short life span, the accessibility of single-gene knockdowns and a published protocol for DR (13), worms are an attractive and useful model species in which to study the mechanisms of DR. Furthermore, a genetic model of DR has been reported in the eat mutants that have slower pharyngeal pumping and are long lived (44). However, eat-mutant worms have recently been shown to be longer lived than wild-type worms under dietary conditions that maximize wild-type life span (Walker G, Keaney M, Gems D, unpublished observations) (45). This finding indicates that the mutation must alter longevity by mechanisms in addition to those for DR-induced life-span extension, making eat mutants an unsuitable model for DR. Unfortunately, a significant hurdle has been encountered when using worms to elucidate the effects of individual dietary components on life span. In defined, axenic media C. elegans is very long-lived with very low fecundity in a manner that is insensitive to nutrient concentration. It is odd that these phenotypes can only be reversed by the addition of a living bacterial food source and not by the addition of organic extracts or killed bacteria (46) (Walker G, Keaney M, Gems D, unpublished observations).
DR has been shown to extend life span in D. melanogaster in a manner that conforms to the evolutionary trade-off hypothesis (6). Furthermore, large populations of individuals can be screened relatively easily, which aids demographic analyses, and precisely defined media recipes have been developed (47–49). Unlike yeast and worms, generating gene knockdowns is not a trivial matter, although large stocks of publicly available mutants exist. These make it possible to study the mechanisms of DR using D. melanogaster at both the level of food components critical for its effects and through interaction between dietary components and the genotype of the fly. These attributes thus make D. melanogaster the best suited of the short-lived model organisms in which to study the effects of dietary components on life span.
Determining the Limits for DR in D. melanogaster
Food consumption is difficult to control directly in small insects, and DR is commonly imposed by whole-food dilutions. In several studies (50–52), researchers have attempted to implement DR in flies by restricting the amount of food they eat, but in no studies is an extension of life span reported for the limitations imposed. These studies, however, do not test whether this is because DR does not work in flies or because the cycles of feeding and starvation introduced by this technique effectively cause malnourishment that is detrimental to life span. This raises the problem of how to subject D. melanogaster to DR without malnourishment. The solution to this problem is outlined by the Chapman and Partridge study (6), in which the appropriate range of food dilutions was identified as the range over which life span is extended and daily and lifetime fecundity coordinately reduced (Figure 1). Thus, the relatively short-lived flies on high concentrations of food leave more progeny than do the relatively long-lived animals on DR. This makes it distinct from high food concentrations that result in shortened life span due to a pathological effect of increasing the amount of food. An example of this is found in a study by Klass (13) for C. elegans, where at the highest food concentration there is a concomitant reduction of life span and fecundity. This observation is consistent with an explanation that worms suffocate at high food concentrations in liquid medium and that the shortened life span is therefore nonaging-related. Furthermore, if the food is too dilute, both life span and fecundity decline, probably due to starvation (see food dilutions in Figure 1 that are less than half that of standard laboratory food).
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Just as it is important that food concentration is optimized as a preliminary step to studies of DR, studies of food quality should be designed so that malnourishment is avoided. This point is illustrated by work performed in (61), where the effects of dietary components such as lard (pig fat), dripping (beef fat), or butter were studied for their effects as calorie sources on D. melanogaster life span. The life-shortening effects of these meat-derived additives to the diet of fruit flies is likely to reflect the fact that they are not biologically useable for D. melanogaster, meaning that the animals were effectively starved or poisoned when placed on these media. However, without reference to a physiological indicator of fitness such as egg laying, this distinction cannot be made.
Diets to Study Interaction of Dietary Components With Life Span
Most laboratories use a semidefined, killed-yeast diet for D. melanogaster cultivation that supports vigorous growth and development and is adequate for adult maintenance (62,63). Based on this knowledge, invaluable work in the laboratory of Sang and colleagues (47–49,64) has described a set of defined media that provide all the physiological requirements for D. melanogaster growth and egg-laying. This diet offers the opportunity to dissect the effects of different food components on the life span of D. melanogaster.
One study on the effects of sugar, protein, pH, and vitamins on longevity in D. melanogaster adults kept on defined media (65) serves as an example of where to begin when approaching this topic. Life-span optimization experiments using various combinations and concentrations of food components were performed, but, most interestingly, life span was optimized when sucrose and casein were covaried in an otherwise complete dietary background. A life-span peak was found for both components. Unfortunately, this study did not examine fecundity, and so lacks the reference for improved lifetime reproductive success of the relatively short-lived animals in higher nutrient concentrations. This study does, however, illustrate that, as in mammals, both the carbohydrate and protein components of the diet may be important for life-span determination in D. melanogaster.
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MECHANISMS BY WHICH DR EXTENDS LIFE SPAN—A NOTE ON RECENT DEVELOPMENTS AND CONSIDERATIONS |
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TopAbstractBackgroundD. melanogaster as a...Mechanisms by Which DR...References |
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Selection experiments using D. melanogaster have shown that the plastic response of life span to dietary manipulation has no intergenerational effects (66). Recent work has shown that DR in D. melanogaster is the product of an acute effect that causes fully-fed flies to adopt the mortality profile of lifelong DR flies within 48 hours of initiation of the treatment; this finding indicates that DR has no effect on the accumulation of irreversible, ageing-related damage (67) (Figure 2). This is in sharp contrast to the effects of different temperatures on life span and mortality; switches between thermal regimes demonstrated that life at higher temperature leads to greater accumulation of irreversible damage that causes death. The acute effect of DR on mortality rate in flies is critical for defining its mechanism of action, because it demonstrates that there is a completely reversible short-term risk of death associated with the nature of the animal's diet. Thus, the mortality switch can be used as an additional assay for the successful implementation of DR in D. melanogaster. It will be of great interest to see if this acute reversal is conserved in mammals, as has been suggested by the reversal of transcript profiles after short-term dietary changes in (68,69). Considering the apparently irreversible pathologies such as cancer and nephropathy that are delayed by DR in rodents, the data suggest that any acute effect of DR would operate in the first instance by raising the threshold for individuals entering the diseased state prior to its onset, rather than by reversing these illnesses after initiation. When defined, as in D. melanogaster, the rapid switch in mortality rate in response to newly imposed DR provides a useful paradigm for study of the initiation of events that set up the physiological changes that result in extension of life span by DR and of the downstream lesions that they ameliorate.
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In light of data from Colombani and colleagues (85), an interesting model has emerged for interaction between insulin and TOR signalling at the level of control of ligand availability (85). These authors found that, during larval growth, modulation of an amino acid transporter in the D. melanogaster fat body could alter expression of the D. melanogaster acid-labile subunit (ALS) protein, whose mammalian orthologue is responsible for chaperoning the IGF-1 ligand in circulation and increasing its half-life (86). Thus, a diffusible signal from the fat body (ALS) could modulate activity through the IIS pathway in Drosophila melanogaster. If flux through these signalling pathways forms a critical first step in the response to DR, these two parallel, but interacting pathways would function to modulate mortality rate by coordinating the response to energy in the diet as well as to other nutritional components such as protein. Recent work in C. elegans has also found regulatory interactions between TOR signalling and IIS, indicating that such coordination is not unique to D. melanogaster (84). Undoubtedly, the full response to DR involves coordination of even more signals required for metabolic homeostasis, and it will be important to investigate their roles in effecting the response of life span to DR. As the potential mechanisms by which DR has its effects increase in complexity, there is an increasing need to perform further investigations using interventions that involve specific dietary components important for life-span extension.
Conclusion
DR has been shown to extend life span in virtually all organisms tested. It has been traditionally considered, on the basis of work with rodents, that calories are critical for the response. However, recent work has illustrated that restriction of calories may be neither sufficient nor necessary for life-span extension by DR. Therefore, to investigate the mechanisms through which dietary interventions operate to extend life span, there is a need to identify the critical dietary components. The invertebrate model organism D. melanogaster is ideally suited to such investigations because of its relatively short life span, ease of handling, and the availability of a minimal, defined medium. However, investigations into DR using D. melanogaster have often omitted the essential preliminary step of defining the range of diets over which DR should be conducted. This is necessary to demonstrate that extension of life span is not caused by rescuing the flies from malnutrition. It is critical that each new study be accompanied by an empirical determination of food concentrations required for optimum life span and fecundity. Using these rules, work can progress to investigate the effects of specific dietary components on life span. In particular, it is of great interest to better characterize the acute mortality response to dietary change, as well as the interaction between dietary components and the activity of nutrient-related signalling pathways that are known to affect life span. This work in D. melanogaster will serve as a starting point for studies seeking to understand the mechanisms of DR in mammalian systems.
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Acknowledgments |
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Footnotes |
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Received September 10, 2004
Accepted January 19, 2005
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References |
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TopAbstractBackgroundD. melanogaster as a...Mechanisms by Which DR...References |
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