Guest guest Posted June 5, 2005 Report Share Posted June 5, 2005 Hi All, How long we will live? Is it described by: 'the most likely sign to hang in any hypothetical ageing headquarters at the moment would read: " It's the free radicals, stupid! " '? Or maybe, is it modified from what the Clinton staff had as 'a simple, and now infamous, sign to remind them to stay on message: " It's the economy, stupid! " ', to: " It's the [calorie] economy, stupid! " ? From Jeanne Calment to yeast and back to Jeanne Calment seems like a long distance. Please see the below. Nemoto S, Finkel T. Ageing and the mystery at Arles. Nature. 2004 May 13;429(6988):149-52. No abstract available. PMID: 15141200 What determines how long we will live? Studies of simple organisms, single cells and mammals hint that certain shared principles underlie ageing, and raise the possibility of devising ways to extend life — if we want to. " I don't want to achieve immortality through my work. I want to achieve it through not dying " — Woody (1935–?) In August 1997, Jeanne Calment was laid to rest in a simple ceremony in the south of France. Those who eulogized her life that day noted that it was unremarkable in all but one respect: she had been born on 21 February 1875. Five years before her birth, Napoleon III had been overthrown and the French Third Republic was formed. In her native Arles, Impressionism would take root and flourish. Years later, she would often tell visitors of her recollection of the painter Van Gogh, whom she regarded as " dirty and badly dressed " . When pressed for the secret of her longevity, Madame Calment would stress her consumption of large quantities of olive oil and port wine, and the fact that she quit smoking, albeit aged 119. Insurance companies study life expectancy all the time (see Box 1), but science long neglected the issue. Although the events that initiate life and the processes that determine its end were avidly examined, there was curiously little study of what determines the speed at which we travel between these two points. Ageing, unlike other biological processes, was not felt to be a subject that is readily dissected into its components. Several laboratories have, however, spent the past decade chipping away at this notion. Although their collective efforts have not yet produced a completed work of art, they at least provide the first discernible outline of the molecular path-ways that regulate lifespan. Box 1. How long will you live? The theoretical maximum lifespan in humans is still a subject of considerable debate. For most of us, however, our life expectancy is determined not by theoretical limits but by a confluence of factors we can't control (genetic) and factors we struggle to control (behavioural and environmental). For a hint on how long you might live — or at least how long an insurance company thinks you might live — simply fill out the questionnaire below. (From ref. 36.) Begin with the number 76 and add or subtract as follows: If you are age 30–50 (+2), if 51–70 (+4) _____ Male (-3), Female (+4) _____ Live in an urban area of population more than 2 million (-2), or a town under 10,000 (+2) _____ One grandparent lived to age 85 (+2), or all grandparents lived to 80 (+6) _____ A parent died of cardiovascular disease or a stroke before age 50 (-4) _____ Brother, sister or parent under 50 has cancer or heart disease, or diabetes since childhood (-3) _____ Earn the equivalent of over US$50,000 (-2) _____ Finished college (+1), or have a graduate/professional degree (+2) _____ 65 or over and still working (+3) _____ Live with a spouse or friend (+5) _____ Live alone (-3) and (-3) for every decade you lived alone since age 25 _____ Work behind a desk (-3), do work requiring strenuous physical labour (-3) _____ Exercise strenuously for 30 minutes 5 times a week (+4), 2–3 times a week (+2)____ Sleep more than ten hours a day (-4) _____ Personality is relaxed (+3) or intense (-3), happy (+1) or unhappy (-2) _____ Speeding ticket in the previous year (-1) _____ Drink the equivalent of 1 oz of liquor per day (-1) _____ Smoke more than 2 packs per day (-8), 1–2 packs/day (-6), or 1/2–1 pack/day (-3) _____ Overweight by 50 lbs or more (-8), 30–49 lbs (-4), 1–29 lbs (-2) _____ If over age 40, do you have a check-up or (if female) see a gynaecologist annually? (+2) _____ >>>Calculated life expectancy ______ http://www.nature.com.qe2a-proxy.mun.ca/nature/journal/v429/n6988/box/429149a_bx\ 1.html Simple organisms Yeast would seem an unlikely model system for learning about human ageing. A single-celled organism, essential perhaps for our daily bread and our favourite brew, it seems to lack the complexity that we associate with higher-order functions such as ageing. Nonetheless, yeast 'replicative lifespan' — a measure of the number of divisions a mother yeast cell can undergo — has become a useful surrogate for mammalian ageing. Early in life, a mother yeast cell can readily divide, asymmetrically, to produce a daughter cell. Later on, when the mother cell has divided many times, it begins to enlarge and its capacity to produce progeny diminishes. The fact that middle-aged yeast as well as middle-aged humans are generally slower, fatter and less interested in reproduction provides our first (albeit broad) clue that certain biological markers of ageing — and so, perhaps, the underlying mechanisms — might have been conserved during evolution. So what determines how many times a mother yeast cell can divide? Numerous environmental and genetic determinants have been identified. Among the environmental factors are several non-lethal stresses, for example reducing the glucose levels in the growth medium from 2% to 0.5%; such 'caloric restriction' can significantly increase the replicative lifespan of yeast. This concept of 'less is more' when it comes to calories and longevity is a theme that we see again and again, from yeast to mice. In yeast, low-glucose conditions extend lifespan through the action of a gene termed SIR2 (ref. 1). Strikingly, a similar lengthening of lifespan ensues when yeast are simply manipulated to produce too much of the protein product of this gene2. This product, Sir2, was first identified as a protein that modifies the physical state of DNA, causing a phenomenon known as genetic silencing ('Sir' stands for 'silent information regulator'). Most evidence now supports the idea that Sir2 extends lifespan in yeast by regulating gene expression or suppressing recombination (the exchange of chunks of DNA between chromosomes), although the relevant genetic targets of Sir2 are unknown. But what is the link between caloric restriction and Sir2? The protein's effects on DNA are achieved through its histone deacetylase activity — its ability to remove specific acetyl groups from histones and other proteins that wrap up DNA. This activity of Sir2 in turn depends on the cellular levels of nicotinamide adenine dinucleotide (NAD)2. NAD and its reduced form, NADH, represent a sort of basic energy currency in cells. Caloric restriction in yeast might increase Sir2 activity by altering either the NAD:NADH ratio or the levels of the NAD derivative nicotinamide. In other simple organisms, such as the fruitfly Drosophila melanogaster, caloric restriction might not only increase the activity of Sir2, but also directly regulate its levels3. Increased Sir2 levels and activity might then dampen gene expression and recombination, leading (somehow) to an extension in lifespan. Too much Sir2 also extends lifespan in the nematode worm Caenorhabditis elegans2. Like yeast, C. elegans has become a useful model in ageing research. Old and young worms look and act differently (Fig. 1), and the creature's lifespan is an experimentally manageable 20–25 days. Moreover, the worm genome has been completely sequenced, it is reasonably straightforward to mutate its genes and study the effects, and the expression of its genes can be easily manipulated using RNA interference (RNAi) methods — a technique for knocking out the middle step in the 'DNA to messenger RNA to protein' cascade that constitutes gene expression. Figure 1 Life and death of a worm. Over the past decade, studies of worms carrying mutations in one gene or another have revealed many genes besides SIR2 that can, individually, regulate C. elegans lifespan (reviewed in ref. 4). We don't have space to describe them all here, but many seem to regulate metabolic parameters. Some of the longer-lived mutant worms, for instance, have difficulty eating and thus are caloric-restricted through genetic hardwiring. Another long-lived mutant, named clk-1, cannot synthesize coenzyme Q, a molecule that is involved in metabolism in mitochondria (the cell's major energy-generating bodies). Finally, an interesting cluster of mutations affects the well-characterized insulin/insulin-like growth factor-1 (IGF-1) pathway. This signalling cascade is involved in nutritional sensing and metabolic regulation in a wide spectrum of organisms4. In long-lived animals with mutations in genes encoding components of this pathway, the activity of the DAF-16 protein (a member of the so-called Forkhead family, which regulates gene expression) is modestly increased (see Fig. 2 for why this happens). Two research groups5, 6 have begun to track down the transcriptional targets of DAF-16. Although their different approaches uncovered non-overlapping sets of targets, both groups identified genes that apparently regulate metabolism and the organism's response to 'oxidative stress' — caused by the imbalance between intracellular levels of reactive oxygen species (also termed free radicals or oxidants), generated in part as a by-product of metabolism, and the cellular antioxidant system. The mammalian counterparts of DAF-16 activate the transcription of similar stress-related genes7, 8. Figure 2 The DAF-16 signalling pathway and lifespan. The lifespan of the worm is regulated in part by a detailed biochemical pathway (simplified here) that influences the activity of the gene-transcription factor DAF-16. The binding of insulin-like molecules to their receptor (DAF-2) on the cell membrane activates a series of protein kinase enzymes: AAP-1–AGE-1 adds phosphate groups (P) to phosphatidylinositol-4,5-bisphosphate (PIP2), which activates AKT-1–AKT-2. This enzyme then phosphorylates DAF-16, keeping it in the cytoplasm. Some mutations that extend lifespan appear to modestly increase the nuclear localization and hence activity of DAF-16 in adult worms. The targets of DAF-16 include genes involved in overall metabolism or adaptation to stress. A related pathway exists in fruitflies. The closest mammalian counterparts of the key proteins are shown in parentheses. Studies of mutations in single genes are an effective but narrowly focused way of investigating the genetic basis of ageing. In the hope of defining the full spectrum of genes that regulate ageing, one group9 has turned to RNAi — a higher-throughput approach — to selectively inactivate more than 5,000 worm genes in turn. A familiar theme emerged: the most frequent category of gene product that appeared to extend lifespan when knocked out comprised regulators of mitochondrial function9. RNAi that was directed towards several different elements of mitochondrial electron transport, a crucial step in metabolism, also increased lifespan10 (although, curiously, only when mitochondrial function was inhibited in young worms). From these studies, preliminary estimates suggest that, on average, inactivation of 1 out of every 50 random genes results in a reproducible lifespan extension9. In other words, these genes usually decrease lifespan. But why do worms — and presumably other species — hang on to so many life-reducing genes? One answer may be that, as our genes are (or so it is presumed) selected for only on the basis of their ability to promote reproduction and hence the continuance of the species, life-shortening genes must provide certain reproductive advantages to a young animal. In other words, as Darwin proposed, and as those of us with teenage children can readily verify, post-reproductive adults are of little to no utility. Single cells Researchers have also been studying ageing by looking at mammalian cells in culture dishes. Like yeast, normal, non-reproductive mammalian cells generally lose their ability to proliferate after a fixed number of doublings — a phenomenon termed the Hayflick limit. With each ensuing division, a greater proportion of the cells enter a state called senescence, characterized by a permanent withdrawal from the cell-division cycle. So what determines the Hayflick limit? One explanation for human cells is that every population doubling results in a shortening of the ends of each chromosome — protective caps called telomeres. Reduction of telomere length below a critical threshold can trigger senescence. Notably, normal non-reproductive cells can be 'immortalized' — meaning that they multiply indefinitely — when engineered to express telomerase, an enzyme that restores eroded telomeric DNA11. The immortal nature of cancer cells, and of normal stem cells in embryos and adults, might relate to their continuous telomerase activity. Given these observations, some have argued that the forced expression of telomerase could potentially reverse or delay the ravages of ageing. Sceptics have warned, however, that this strategy might lead to unacceptable levels of malignancies — a case of the ends, telomeric or otherwise, not justifying the means. Other manipulations of cells in culture influence lifespan, too. For instance, reduced levels of ambient oxygen delay senescence. The RAS gene, which regulates yeast replicative lifespan, can also contribute to mammalian cell senescence. Mutant Ras proteins are closely linked to the transformation of immortalized cells into tumour cells, but, paradoxically, the effect of Ras in a normal cell background is to block cell division irreversibly12. This has strengthened the concept that senescence, like apoptosis (programmed cell death), is a protective mechanism. Interestingly, in both yeast and mammalian cells, RAS induces the production of a high level of mitochondrial oxidants, and in human cells these oxidants are required to induce senescence13, 14. Mammals Although the progress made in simple organisms and single cells has been remarkable, much less is known about mammalian ageing. But numerous parallels are emerging. For instance, several genetically different strains of long-lived mice have been described that are characterized by a deficiency in secreted hormones such as growth hormone and IGF-1 (ref. 15). These mice are at least vaguely reminiscent of those long-lived worms that show reduced IGF-1 signalling. Strikingly, mice in which the insulin receptor is inactivated only in adipose (fat) cells also show a 20% increase in their lifespan16, as do mice in which the IGF-1 receptor is only partially inactivated17. The tissue-specific inactivation of the insulin receptor is reminiscent of studies in worms in which increased DAF-16 activity in just a few tissues increased the organism's lifespan18. Another regulator of mammalian lifespan is the p66shc protein; deletion of this signal-transduction intermediate in mice results in a roughly 30% increase in lifespan19. Like many of the long-lived mutant worms, these mice can survive oxidative stress better than their normal litter-mates. Moreover, cells from these animals have lower levels of oxidants7, 20. Notably, p66shc seems to regulate the activity of the mammalian Forkhead-family member that is a counterpart of C. elegans DAF-16 (ref. 7). Earlier this year, it was shown that mammalian Forkhead proteins also interact, physically and functionally, with NAD-dependent deacetylases from the Sir2 family21, 22. For all their evolutionary distance, mammals and nematodes seem to have kept the same bag of tricks. Microarray analyses of the genes that are expressed in young and old tissues provide another means of trying to understand ageing in mammals. These analyses look at the messenger RNAs (mRNAs) that are expressed, and initial studies revealed that, in this respect, young and old tissues are remarkably similar23, 24. Indeed, comparative analysis of the brain and skeletal muscle from old and young mice showed that only some 1–2% of all mRNAs differ by more than twofold in their expression levels. Those of us who wish to resist a mandatory retirement could argue that, at the cellular level, we are at roughly 98% of our youthful effectiveness. Nonetheless, although only a handful of genes may differ, those genes are clustered into functional classes that regulate the response to oxidative stress, inflammation and overall metabolism. Strikingly, these age-related changes in gene expression did not occur when the animals' calorie intake was restricted23, 24. Whether or not human ageing and longevity could be influenced by caloric restriction is unknown, and understandably there are few people willing to participate in the lifelong experiment that would be necessary to find out. But further insight into human ageing has come from the study of the rare accelerated-ageing syndromes called progerias. The most widely known of these inherited disorders is Werner's syndrome, which is characterized by the early appearance of grey and thinning hair, osteoporosis, atherosclerosis and cancer25. The gene that is altered in Werner's syndrome encodes a DNA helicase; other members of this enzyme family have been implicated in inherited predispositions to cancer. Such enzymes seem to be involved in normal DNA replication, repair and recombination. These accelerated-ageing syndromes re- mind us of an important distinction between our understanding of ageing in people and that in lower organisms. For the most part, we die from specific diseases and events; the cause of death in lower organisms is less well understood. Can our understanding of the pathways that regulate ageing teach us anything about the mechanisms that underlie age-related diseases? If, for instance, we had a magic potion that slowed human ageing, would we also decrease the incidence of diseases such as atherosclerosis and cancer, which rises so dramatically as we get older? For the case of malignancies, some insight may come from a study in yeast showing that daughters derived from older mother yeast cells had a roughly 100-fold greater genetic instability than cells from younger mothers26. If the same occurs in human cells, then cancer and ageing mechanisms may be inextricably intertwined. In another example, circulating endothelial progenitor cells — which repair damaged blood vessels — decline as a function of human age27. Similarly, in mice, the ability of resident skeletal-muscle progenitor cells to proliferate and hence repair damage appears to decline markedly as a function of age28. These results hint that different tissues age at different rates, and that the exhaustion, depletion or senescence of progenitor cells (including stem cells) might contribute to overall ageing as well as age-related diseases. In this context, atherosclerosis, neurodegeneration and other specific disease states could be viewed as a form of tissue-specific progeria. A unified theory? During the 1992 US presidential campaign between Bill Clinton and Bush Sr, out of a whole array of issues it was the economy that most exercised the electorate. In their campaign room the Clinton staff had a simple, and now infamous, sign to remind them to stay on message: " It's the economy, stupid! " In our view, the most likely sign to hang in any hypothetical ageing headquarters at the moment would read: " It's the free radicals, stupid! " Indeed, the cellular effects of free radicals represent the most likely contender to explain the ageing process across a wide range of species. In a nutshell, the idea is that when mitochondrial metabolism — fuelled by food and oxygen — increases, more reactive oxygen species are produced as a by-product, with greater adverse effects on cells. Although such a theory was proposed close to 50 years ago29, its current incarnation has nuances (Fig. 3). For instance, there is a growing appreciation that the production of reactive oxygen species in cells is intricately regulated, and that the effects of these species include both random damage to cellular components and the regulation of specific pathways30. Similarly, although metabolic rate and the production of reactive oxygen species are generally correlated, this relationship can be complex. For instance, a group of mitochondrial proteins known as uncoupling proteins is activated by superoxide, one type of reactive oxygen species, and can induce a metabolic state in which oxygen consumption increases but overall levels of reactive oxygen species decline31. Conversely, expression of an activated RAS gene in yeast leads to decreased oxygen consumption but increased levels of reactive oxygen species13. Figure 3 A current version of the free-radical theory of ageing. Nutrients (detected by sensors such as the insulin receptor at the cell membrane) and oxygen fuel metabolism in the mitochondria, generating reactive oxygen species (ROS). The levels of ROS might determine the rate of ageing by inducing random damage in proteins and DNA, and by activating specific pathways that depend on the reduction–oxidation status of particular proteins. DNA damage or recombination might be decreased by proteins that influence lifespan, including the Werner helicase or members of the Sir2 family. Although ROS levels and oxygen consumption are related, the relationship is complex; proteins such as uncoupling proteins and Ras may alter the levels of ROS produced or released per molecule of oxygen consumed. It also remains unclear how the cell partitions substrates to the inefficient but ROS-sparing non-mitochondrial pathways, or the highly efficient but ROS-generating mitochondrial pathways. Nonetheless, many of the known regulators of longevity can be mapped to a scheme resembling Fig. 3. For instance, inhibiting mitochondrial function — as many of the genes uncovered in the C. elegans RNAi screens9, 10 seem to do — might cause the organism to rely on alternative, non-mitochondrial pathways for energy production. These pathways are inherently less efficient, but produce fewer reactive oxygen species, perhaps leading to the lifespan extension observed. Mutations that activate DAF-16, meanwhile, would induce the expression of genes that encode antioxidants, and might also shift the organism to a different overall metabolic state. Caloric restriction could lead to many changes, including Sir2 activation and a consequent reduction in gene expression and recombination, as well as reduced activation of the insulin/IGF-1 pathways. The net effect might be to lower oxidant production and increase the removal of free radicals. But although this theory is the best candidate to explain the wealth of experimental data, disquieting observations persist. Take two examples. First, if ageing represents the legacy of the combustible mixture of food and oxygen in our mitochondria, then oxidant-related damage should be, to some degree, cumulative. But when fruitflies were caloric-restricted for just two days, their mortality rate became equivalent to that of flies that spent their whole life hungry32. Second, at least in yeast, glucose withdrawal extends life but results in a tripling in the rate of oxygen consumption33. Oxygen consumption and the generation of reactive oxygen species are not necessarily equivalent. Still, one would expect such a large change in oxygen consumption to be more likely to increase than decrease the levels of such species. So the free-radical theory remains a useful, but still unproven, framework within which to understand how we age, and questions remain as to whether levels of oxygen radicals cause, or are merely correlated with, ageing34. Conclusions The discovery of genes that are associated with the regulation of lifespan in simple organisms, the apparent clustering of these genes into a handful of discrete categories and pathways, and the evolutionary conservation of such genes and pathways have led to the hope that treatments can be developed to slow ageing and increase lifespan — if we want to. The idea has obvious social implications, with ageing populations already becoming a problem in many countries. Nonetheless, as caloric restriction seems to be such a robust way to extend life, there is considerable enthusiasm for the idea of molecules that mimic its effects. At least in yeast, such a strategy may hold water: last year, researchers identified numerous structurally related small molecules that increase Sir2 activity and so mimic the effects of caloric restriction on lifespan35. For those of us who enjoy life's desserts, the fact that yeast exposed to these compounds lived longer despite being cultured in a food-rich environment provides some cause for optimism. Other potentially promising compounds could include partial inhibitors of the insulin/IGF-1 pathways, small molecules that block p66shc, or compounds that modestly activate the Forkhead-family proteins. Will such approaches be successful? Can we produce drugs that preserve the quality of life and yet extend its length? Some would argue that the proof that such strategies will work is already discernible from the menagerie of long-lived worms, fruitflies and mice in existence. Others would say that for evidence of how much remains a mystery, one need only visit a small graveyard in Arles and find a stone that bears the simple inscription: Jeanne Calment, born 1875, died 1997. Al Pater, PhD; email: old542000@... __________________________________ Discover Find restaurants, movies, travel and more fun for the weekend. Check it out! http://discover./weekend.html Quote Link to comment Share on other sites More sharing options...
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