Functional characterization and immunolocalization of the transporter encoded by the life-extending gene Indy

Caloric restriction extends life span in a variety of species, highlighting the importance of energy balance in aging. A new longevity gene, Indy (for I'm not dead yet), which doubles the average life span of flies without a loss of fertility or physical activity, was postulated to extend life by affecting intermediary metabolism. We report that functional studies in Xenopus oocytes show INDY is a metabolite transporter that mediates the high-affinity, disulfonic stilbene-sensitive flux of dicarboxylates and citrate across the plasma membrane by a mechanism that is not coupled to Na(+), K(+), or Cl(-). Immunocytochemical studies localize INDY to the plasma membrane with most prominent expression in adult fat body, oenocytes, and the basolateral region of midgut cells and show that life-extending mutations in Indy reduce INDY expression. We conclude that INDY functions as a novel sodium-independent mechanism for transporting Krebs and citric acid cycle intermediates through the epithelium of the gut and across the plasma membranes of organs involved in intermediary metabolism and storage. The life-extending effect of mutations in Indy is likely caused by an alteration in energy balance caused by a decrease in INDY transport function.     

Knockdown of Indy/CeNac2 extends Caenorhabditis elegans life span by inducing AMPK/aak-2

Reducing the expression of the Indy (Acronym for ‘I''m Not Dead, Yet’) gene in lower organisms promotes longevity and leads to a phenotype that resembles various aspects of caloric restriction. In C. elegans, the available data on life span extension is controversial. Therefore, the aim of this study was to determine the role of the C. elegans INDY homolog CeNAC2 in life span regulation and to delineate possible molecular mechanisms. siRNA against Indy/CeNAC2 was used to reduce expression of Indy/CeNAC2. Mean life span was assessed in four independent experiments, as well as whole body fat content and AMPK activation. Moreover, the effect of Indy/CeNAC2 knockdown in C. elegans with inactivating variants of AMPK (TG38) was studied. Knockdown of Indy/CeNAC2 increased life span by 22 ± 3% compared to control siRNA treated C. elegans, together with a decrease in whole body fat content by ~50%. Indy/CeNAC2 reduction also increased the activation of the intracellular energy sensor AMPK/aak2. In worms without functional AMPK/aak2, life span was not extended when Indy/CeNAC2 was reduced. Inhibition of glycolysis with deoxyglucose, an intervention known to increase AMPK/aak2 activity and life span, did not promote longevity when Indy/CeNAC2 was knocked down. Together, these data indicate that reducing the expression of Indy/CeNAC2 increases life span in C. elegans, an effect mediated at least in part by AMPK/aak2.     

Patterns of metabolic activity during aging of the wild type and longevity mutants of Caenorhabditis elegans

At least three mechanisms determine life span in Caenorhabditis elegans. An insulin-like signaling pathway regulates dauer diapause, reproduction and longevity. Reduction-or loss-of-function mutations in this pathway can extend longevity substantially, suggesting that the wild-type alleles shorten life span. The mutations extend life span by activating components of a dauer longevity assurance program in adult life, resulting in altered metabolism and enhanced stress resistance. The Clock (Clk) genes regulate many temporal processes, including life span. Mutation in the Clk genes clk-1 and gro-1 mildly affect energy production, but repress energy consumption dramatically, thereby reducing the rate of anabolic metabolism and lengthening life span. Dietary restriction, either imposed by mutation or by the culture medium increases longevity and uncovers a third mechanism of life span determination. Dietary restriction likely elicits the longevity assurance program. There is still uncertainty as to whether these pathways converge on daf-16 to activate downstream longevity effector genes such as ctl-1 and sod-3. There is overwhelming evidence that the interplay between reactive oxygen species (ROS) and the capacity to resist oxidative stress controls the aging process and longevity. It is as yet not clear whether metabolic homeostasis collapses with age as a direct result of ROS-derived damage or is selectively repressed by longevity-determining genes. The dramatic decline of protein turnover during senescence results in the accumulation of altered enzymes and in a gradual decline of metabolic performance eventually followed by fatal failure of the system.     

Longevity for free? Increased reproduction with limited trade-offs in Drosophila melanogaster selected for increased life span

Selection for increased life span in Drosophila melanogaster has been shown to correlate with decreased early fecundity and increased fecundity later in life. This phenomenon has been ascribed to the existence of trade-offs in which limited resources can be invested in either somatic maintenance or reproduction. In our longevity selection lines, we did not find such a trade-off. Rather, we find that females have similar or higher fecundity throughout life compared to non-selected controls. To determine whether increased longevity affects responses in other traits, we looked at several stress resistance traits (chill coma recovery, heat knockdown, desiccation and starvation), geotactic behaviour, egg-to-adult viability, body size, developmental time as well as metabolic rate. Longevity selected flies were more starvation resistant. However, in females longevity and fecundity were not negatively correlated with the other traits assayed. Males from longevity selected lines were slower at recovering from a chill induced coma and resting metabolic rate increased with age, but did not correlate with life span.     

Is life span extension in single gene long-lived Caenorhabditis elegans mutants due to hypometabolism?

The nematode C. elegans is widely used in aging research largely because of the identification of numerous gene mutations that significantly increase worm longevity. While model organisms such as C. elegans can provide important insights into aging it is also important to consider the limitations of these systems. For example, ectothermic (poikilothermic) organisms are able to tolerate a much larger metabolic depression than humans and considering only chronological longevity when assaying for long-lived mutants provides a limited perspective on the mechanisms by which longevity is increased. In order to provide true insight into the aging process additional physiological processes, such as metabolic rate, must also be assayed. Currently it is controversial when long-lived C. elegans mutants retain normal metabolic function. Resolving this issue requires accurately measuring the metabolic rate of C. elegans under conditions that minimize environmental stress. Comparisons of metabolic rate between long-lived and wild-type C. elegans under more optimized conditions indicate that the extended longevity of at least some long-lived C. elegans mutants may be due to a reduction in metabolic rate, rather than an alteration of a metabolically-independent genetic mechanism specific to aging. Consistent with this assertion are studies showing that the disruption of mitochondrial function in C. elegans can extend worm's longevity, but typically causes worms to grow and develop more slowly than wild-type animals.     

Genetic and environmental conditions that increase longevity in Caenorhabditis elegans decrease metabolic rate

Mutations that increase the longevity of the soil nematode Caenorhabditis elegans could define genes involved in a process specific for aging. Alternatively, these mutations could reduce animal metabolic rate and increase longevity as a consequence. In ectotherms, longevity is often negatively correlated with metabolic rate. Consistent with these observations, environmental conditions that reduce the metabolic rate of C. elegans also extend longevity. We found that the metabolic rate of long-lived C. elegans mutants is reduced compared with that of wild-type worms and that a genetic suppressor that restored normal longevity to long-lived mutants restored normal metabolic rate. Thus, the increased longevity of some long-lived C. elegans mutants may be a consequence of a reduction in their metabolic rate, rather than an alteration of a genetic pathway that leads to enhanced longevity while maintaining normal physiology. The actual mechanism responsible for the inverse correlation between metabolic rate and longevity remains unknown.     

Individual fecundity and senescence in Drosophila and medfly

Evolutionary theory postulates that there should be a robust relationship between fecundity and longevity. Prior work has generally supported this concept, but has not shed much light on the mechanisms at play. In preceding work, we have developed and verified a mathematical model of Drosophila melanogaster female fecundity based on the analysis of empirical studies independently done by several different laboratories. Then we applied this technique to Mediterranean fruit fly (medfly) populations. In this article we analyze associations between individual longevity and the parameters of individual fecundity pattern in Drosophila and medfly. We cluster both Drosophila and medfly individuals by life span and discuss the differences. It allows us to demonstrate that only one fecundity-related parameter is associated with longevity in Drosophila, whereas two such parameters can be found in medflies. This difference demonstrates different ways of aging in various Diptera species. Finally, we discuss the possible implications of this finding.     

Exceptional longevity is associated with decreased reproduction

A number of leading theories of aging, namely The Antagonistic Pleiotropy Theory (Williams, 1957), The Disposable Soma Theory (Kirkwood, 1977) and most recently The Reproductive-Cell Cycle Theory (Bowen and Atwood, 2004, 2010) suggest a tradeoff between longevity and reproduction. While there has been an abundance of data linking longevity with reduced fertility in lower life forms, human data have been conflicting. We assessed this tradeoff in a cohort of genetically and socially homogenous Ashkenazi Jewish centenarians (average age ~100 years). As compared with an Ashkenazi cohort without exceptional longevity, our centenarians had fewer children (2.01 vs 2.53, p<0.0001), were older at first childbirth (28.0 vs 25.6, p<0.0001), and at last childbirth (32.4 vs 30.3, p<0.0001). The smaller number of children was observed for male and female centenarians alike. The lower number of children in both genders together with the pattern of delayed reproductive maturity is suggestive of constitutional factors that might enhance human life span at the expense of reduced reproductive ability.     

The endeavor of high maintenance homeostasis: resting metabolic rate and the legacy of longevity

Metabolism, the continuous conversion between structural molecules and energy, is life in essence. Size, metabolic rate, and maximum life span appear to be inextricably interconnected in all biological organisms and almost follow a "universal" law. The notion of metabolic rate as the natural "rate of living" filled most of the academic discussion on aging in the early 20th century to be later replaced by the free-radical theory of aging. We argue that the rate of living theory was discarded too quickly and that studying factors affecting resting metabolic rate during the aging process may provide great insight into the core mechanisms explaining differential longevity between individuals, and possibly the process leading to frailty. We predict that measures of resting metabolic rate will be introduced in geriatric clinical practice to gather information on the degree of multisystem dysregulation, exhaustion of energy reserve, and risk of irreversible frailty.     

Experimental evolution reveals antagonistic pleiotropy in reproductive timing but not life span in Caenorhabditis elegans

Many mutations that dramatically extend life span in model organisms come with substantial fitness costs. Although these genetic manipulations provide valuable insight into molecular modulators of life span, it is currently unclear whether life-span extension is unavoidably linked to fitness costs. To examine this relationship, we evolved a genetically heterogeneous population of Caenorhabditis elegans for 47 generations, selecting for early fecundity. We asked whether an increase in early fecundity would necessitate a decrease in longevity or late fecundity (antagonistic pleiotropy). Caenorhabditis elegans experimentally evolved for increased early reproduction and decreased late reproduction but suffered no total fitness or life-span costs. Given that antagonistic pleiotropy among these traits has been previously demonstrated in some cases, we conclude that the genetic constraint is not absolute, that is, it is possible to uncouple longevity from early fecundity using genetic variation segregating within and among natural populations.     

Oxidative stress: Its potential relevance to human disease and longevity determinants

Most evidence indicates that aging is a result of normal metabolic processes that are essential for life. Thus an important approach in biogerontology is to identify specific metabolic reactions necessary for life but which could also lead to aging. A unique characteristic of this approach is an explanation of what governs aging rate or longevity of a species or even individuals within a species. These would be mechanisms that would act to reduce the long-term toxic or aging effects of the normal metabolic and developmental reactions. The reactions involving oxygen metabolism clearly fit into this model for they are essential for life yet can potentially cause many of the dysfunctions associated with aging. Such a model can also account for differences in aging rate or longevity of different animal species by differences that may exist in their innate ability to reduce oxidative stress state. Our laboratory has been testing this oxidative stress state (OSS) hypothesis of aging and longevity by determining if a positive correlation exists between OSS of an animal and its aging rate. Much of our data has found such a positive correlation, yet there is some indication that separate causative mechanisms may exist in determining aging rate as opposed to those related to age-dependent specific diseases such as cancer or cardiovascular disease.     

An analysis of the relationship between metabolism, developmental schedules, and longevity using phylogenetic independent contrasts

Comparative studies of aging are often difficult to interpret because of the different factors that tend to correlate with longevity. We used the AnAge database to study these factors, particularly metabolism and developmental schedules, previously associated with longevity in vertebrate species. Our results show that, after correcting for body mass and phylogeny, basal metabolic rate does not correlate with longevity in eutherians or birds, although it negatively correlates with marsupial longevity and time to maturity. We confirm the idea that age at maturity is typically proportional to adult life span, and show that mammals that live longer for their body size, such as bats and primates, also tend to have a longer developmental time for their body size. Lastly, postnatal growth rates were negatively correlated with adult life span in mammals but not in birds. Our work provides a detailed view of factors related to species longevity with implications for how comparative studies of aging are interpreted.     

The dietary restriction effect in C. elegans and humans: is the worm a one-millimeter human?

Dietary restriction (DR) lengthens life span in wide range of vertebrate and invertebrate species. The molecular mechanism by which DR increases life span and the universality of its effects (and hence its applicability to humans) are currently debated in gerontology. This article addresses these two problems from both an experimental perspective, using the nematode C. elegans as a model system, and a theoretical viewpoint, by appealing to recent mechanistic and evolutionary models of aging. Molecular mechanisms of aging are analysed by contrasting the rate of living/oxidative stress hypothesis with the metabolic stability/longevity hypothesis, a new model of aging which postulates that the robustness of metabolic networks, rather than metabolic rate per se, is the major determinant of aging. Studies of food-restricted worms are shown to be consistent with the metabolic stability/longevity hypothesis. The universality of the effects of DR is addressed in terms of directionality theory, an evolutionary model, which is based on the analytical fact that the robustness or the stability of demographic networks determines Darwinian fitness. Directionality theory, in conjunction with the metabolic stability hypothesis, predicts that DR will have negligible effects on equilibrium species (late age of sexual maturity, small size of progeny sets and broad reproductive span) and large effects on opportunistic species (early age of maturity, large size of progeny sets, narrow reproductive span). Empirical studies using C. elegans (an opportunistic species) and computational studies on human populations (an equilibrium species) are shown to be consistent with these predictions.     

Lamotrigine extends lifespan but compromises health span in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

The discovery of life extension in Caenorhabditis elegans treated with anticonvulsant medications has raised the question whether these drugs are prospective anti-aging candidate compounds. The impact of these compounds on neural modulation suggests that they might influence the chronic diseases of aging as well. Lamotrigine is a commonly used anticonvulsant with a relatively good adverse-effects profile. In this study, we evaluated the interaction between the impacts of lamotrigine on mortality rate, lifespan, metabolic rate and locomotion. It has been proposed in a wide range of animal models that there is an inverse relationship between longevity, metabolic rate, and locomotion. We hypothesized that the survival benefits displayed by this compound would be associated with deleterious effects on health span, such as depression of locomotion. Using Drosophila as our model system, we found that lamotrigine decreased mortality and increased lifespan in parallel with a reduction in locomotor activity and a trend towards metabolic rate depression. Our findings underscore the view that assessing health span is critical in the pursuit of useful anti-aging compounds.     

Biological Versus Chronological Aging: JACC Focus Seminar

Aging is the main risk factor for vascular disease and ensuing cardiovascular and cerebrovascular events, the leading causes of death worldwide. In a progressively aging population, it is essential to develop early-life biomarkers that efficiently identify individuals who are at high risk of developing accelerated vascular damage, with the ultimate goal of improving primary prevention and reducing the health care and socioeconomic impact of age-related cardiovascular disease. Studies in experimental models and humans have identified 9 highly interconnected hallmark processes driving mammalian aging. However, strategies to extend health span and life span require understanding of interindividual differences in age-dependent functional decline, known as biological aging. This review summarizes the current knowledge on biological age biomarkers, factors influencing biological aging, and antiaging interventions, with a focus on vascular aspects of the aging process and its cardiovascular disease related manifestations.     

Dietary restriction studies in humans: focusing on obesity, forgetting longevity

Dietary restriction (DR: food restriction without malnutrition) is often considered as a nearly universal means to extend longevity in animal species and we could make the hypothesis that DR could increase longevity in humans. Some authors support the opinion that DR has already increased longevity in Okinawa inhabitants, and thus that DR can increase longevity in humans. The purpose of this article is to stress that no data on humans with a normal body mass index (neither overweight nor obese) indicate that DR can increase life span and health span, particularly because the results observed in Okinawa inhabitants can probably be considered as showing mainly deleterious effects of malnutrition rather than positive effects of DR. Since DR does not appear to increase human life span, studies testing for the effect of DR in humans should focus on the health effects of a mild DR in overweight and obese people, rather than in normal-weight people.     

From the stress theory of aging to energetic and evolutionary expectations for longevity

Stress targets energy carriers. Genes for stress resistance are selected that convey high metabolic efficiency enabling adaptation to the energetically restrictive and hence stressful environments of natural populations. Data from experimental organisms and from humans are consistent with a primary role for stress resistance underlying life span, which provides a hitherto neglected procedure for assaying longevity in natural populations. Taking into account the metabolic consequences of stressful environments, the free-radical theory of aging becomes a general stress theory of aging. A recent derivative, the deprivation-syndrome theory of aging, highlights resource and hence energy shortages. Energy balances under the stress theory of aging are primary for an understanding of the evolutionary limits of longevity of organisms in their habitats. In contrast, well-nourished humans of the modern era, and laboratory, domesticated and island populations are exposed to more benign conditions which appear to provide the background for other evolutionary theories of aging, especially the mutation accumulation and antagonistic pleiotropy theories. In modern human populations where selection for stress resistance is relaxed compared with earlier harsher conditions, substantial future evolutionary extensions to maximum life span may be difficult to attain because of the mutation accumulation process. However there is an urgent need for comparative empirical studies of life-history traits including longevity under benign and harsh environments. This revised version was published online in July 2006 with corrections to the Cover Date.     

Caloric restriction, metabolic rate, and entropy

Caloric restriction increases life span in many types of animals. This article proposes a mechanism for this effect based on the hypothesis that metabolic stability, the capacity of an organism to maintain steady state values of redox couples, is a prime determinant of longevity. We integrate the stability-longevity hypothesis with a molecular model of metabolic activity (quantum metabolism), and an entropic theory of evolutionary change (directionality theory), to propose a proximate mechanism and an evolutionary rationale for aging. The mechanistic features of the new theory of aging are invoked to predict that caloric restriction extends life span by increasing metabolic stability. The evolutionary model is exploited to predict that the large increases in life span under caloric restriction observed in rats, a species with early sexual maturity, narrow reproductive span and large litter size, and hence low entropy, will not hold for primates. We affirm that in the case of humans, a species with late sexual maturity, broad reproductive span and small litter size, and hence high entropy, the response of life span to caloric restriction will be negligible.     

Nutritional control of aging

For more than 60 years the only dietary manipulation known to retard aging was caloric restriction, in which a variety of species respond to a reduction in energy intake by demonstrating extended median and maximum life span. More recently, two alternative dietary manipulations have been reported to also extend survival in rodents. Reducing the tryptophan content of the diet extends maximum life span, while lowering the content of sulfhydryl-containing amino acids in the diet by removing cysteine and restricting the concentration of methionine has been shown to extend all parameters of survival, and to maintain blood levels of the important anti-oxidant glutathione. To control for the possible reduction in energy intake in methionine-restricted rats, animals were offered the control diet in the quantity consumed by rats fed the low methionine diet. Such pair-fed animals experienced life span extension, indicating that methionine restriction-related life span extension is not a consequence of reduced energy intake. By feeding the methionine restricted diet to a variety of rat strains we determined that lowered methionine in the diet prolonged life in strains that have differing pathological profiles in aging, indicating that this intervention acts by altering the rate of aging, not by correcting some single defect in a single strain.