Aging and Nutrition: Novel Approaches and Techniques

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Journal Articles

Azpurua J, Ke Z, Chen IX, et al. Naked mole-rat has increased translational fidelity compared with the mouse, as well as a unique 28S ribosomal RNA cleavage. Proc Natl Acad Sci USA. 2013 Oct 22;110(43):17350-5.

Baker DJ, Childs BG, Durik M, et al. Naturally occurring p16(Ink4a)-positive cells shorten healthy lifespan. Nature. 2016 Feb 11;530(7589):184-9.

Bitto A, Ito TK, Pineda VV, et al. Transient rapamycin treatment can increase lifespan and healthspan in middle-aged mice. Elife. 2016 Aug 23;5. pii: e16351.

Childs BG, Baker DJ, Wijshake T, et al. Senescent intimal foam cells are deleterious at all stages of atherosclerosis. Science. 2016 Oct 28;354(6311):472-477.

Colman RJ, Beasley TM, Kemnitz JW, et al. Caloric restriction reduces age-related and all-cause mortality in rhesus monkeys. Nat Commun. 2014 Apr 1;5:3557.

Gorbunova V, Seluanov A, Zhang Z, et al. Comparative genetics of longevity and cancer: insights from long-lived rodents. Nat Rev Genet. 2014 Aug;15(8):531-40.

Kaeberlein M, Creevy KE, Promislow DE. The dog aging project: translational geroscience in companion animals. Mamm Genome. 2016 Aug;27(7-8):279-88.

Kim EB, Fang X, Fushan AA, et al. Genome sequencing reveals insights into physiology and longevity of the naked mole rat. Nature. 2011 Oct 12;479(7372):223-7.

Ma S, Lee SG, Kim EB, et al. Organization of the mammalian ionome according to organ origin, lineage dpecialization, and longevity. Cell Rep. 2015 Nov 17;13(7):1319-26.

Ma S, Upneja A, Galecki A, et al. Cell culture-based profiling across mammals reveals DNA repair and metabolism as determinants of species longevity. Elife. 2016 Nov 22;5. pii: e19130.

Ma S, Yim SH, Lee SG, et al. Organization of the mammalian metabolome according to organ function, lineage specialization, and longevity. Cell Metab. 2015 Aug 4;22(2):332-43.

McCay CM, Crowell MF, Maynard LA. The effect of retarded growth upon the length of life span and upon the ultimate body size. 1935. Nutrition. 1989 May-Jun;5(3):155-71; discussion 172.

Seim I, Fang X, Xiong Z, et al. Genome analysis reveals insights into physiology and longevity of the Brandt's bat Myotis brandtii. Nat Commun. 2013;4:2212.

Seluanov A, Hine C, Azpurua J, et al. Hypersensitivity to contact inhibition provides a clue to cancer resistance of naked mole-rat. Proc Natl Acad Sci USA. 2009 Nov 17;106(46):19352-7.

Stroustrup N, Anthony WE, Nash ZM, et al. The temporal scaling of Caenorhabditis elegans ageing. Nature. 2016 Feb 4;530(7588):103-7.

Stroustrup N, Ulmschneider BE, Nash ZM, et al. The Caenorhabditis elegans lifespan machine. Nat Methods. 2013 Jul;10(7):665-70.

Swindell WR. Accelerated failure time models provide a useful statistical framework for aging research. Exp Gerontol. 2009 Mar;44(3):190-200.

Tian X, Azpurua J, Hine C, et al. High-molecular-mass hyaluronan mediates the cancer resistance of the naked mole rat. Nature. 2013 Jul 18;499(7458):346-9.

Vural DC, Morrison G, Mahadevan L. Aging in complex interdependency networks. Phys Rev E Stat Nonlin Soft Matter Phys. 2014 Feb;89(2):022811.

Yu BP, Masoro EJ, McMahan CA. Nutritional influences on aging of Fischer 344 rats: I. Physical, metabolic, and longevity characteristics. J Gerontol. 1985 Nov;40(6):657-70.

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Highlights

Dietary restriction is the most well-studied aging intervention. Some forms of restriction are definitively shown to extend lifespan, although the mechanisms of this effect are not fully understood.

Dietary restriction can induce changes in gene expression and DNA methylation, and many of these changes persist when a normal diet is resumed.

mTOR inhibitors such as rapamycin extend lifespan in a wide range of species including yeast, worms, flies, and mice. Efforts to translate such results to higher-order animals are underway.

Short-term treatment with rapamycin improves multiple measures of cardiac function in middle-aged companion dogs.

Some especially long-lived animals, such as the naked mole rat, have evolved biological processes that naturally inhibit the mTOR pathway, mimicking the effects of rapamycin and contributing to longevity.

Effects of dietary restriction on DNA methylation

Eighty years of research data show definitively that caloric restriction in the 30–40 percent range extends lifespan in species both simple and complex, from yeast and worms to flies, rodents, dogs, and non-human primates, explained Arlan Richardson of University of Oklahoma Health Sciences Center. This phenomenon is evidenced when such restriction begins early in life, i.e. shortly after weaning, as well as when initiated in adulthood.

Dietary restriction (DR) is the most well-studied aging intervention, yet the complete mechanisms of its impact on longevity are unknown. Increased stress resistance, reduced inflammation and oxidative damage, as well as a reduction in mTOR signaling, have been shown to contribute to the longevity effects of DR, but recently, researchers have begun probing the possibility that DR may also induce epigenetic changes that promote longevity.

Richardson noted that environmental and physical stresses on the body are known to induce changes in DNA methylation, and that these modifications can persist long after the stress abates. If DNA methylation plays a role in the anti-aging effects of dietary restriction, these changes should be evidenced soon after DR is implemented, and persist upon resumption of a normal diet. "This would be an example of a memory effect, which is a hallmark characteristic of epigenetics," Richardson said.

Mouse studies published dating from the 1980s to 2009 confirm that dietary restriction for as few as 20 days and up to four months, implemented as early as the first days of life and as late as six months of age, universally extended lifespan, offering evidence of both early impact and a memory effect. Additional studies show that DR has an early and long-lasting impact on physiological parameters such as insulin resistance: after just ten days of DR, mice show a significant improvement in glucose utilization, and these benefits persist long after DR is discontinued.

Such phenomena neither confirm nor illuminate the precise impact of dietary restriction on the transcriptome—the dynamic set of RNA transcripts produced by the genome. Richardson and his collaborators have investigated more than 30,000 genes in tissues from the liver, colon, fat, hypothalamus, and hippocampus in search of changes in DNA methylation following a period of dietary restriction—a key step in determining whether such changes exist, what the precise changes are, and if they persist after DR is removed. Mice exposed to one month of DR showed significant changes in gene expression (both up-and downregulation), and 20–30 percent of those changes persisted once the animals were fed ad libitum (AL).

Short-term dietary restriction changes gene expression, and many of these changes persist once a normal diet is resumed.

Richardson analyzed a select group of genes that showed significant expression changes following DR to determine if and how such restriction affects methylation. While the results varied considerably between the genes discussed—some genes remained consistently up- or downregulated following resumption of an AL diet with no changes in methylation—one gene, NTSR1, displayed changes in methylation indicative of a memory effect following a period of dietary restriction. Specifically, NTSR1 is significantly upregulated during dietary restriction, and decreases only slightly upon discontinuation. Methylation is decreased overall, and each of the three CpG sites of the NTSR1 promoter region shows decreased methylation at the onset of DR, with even greater decreases after DR is discontinued. This is the first evidence that dietary restriction can alter DNA methylation in the promoter region of a gene, and that such changes can be long-lasting.

Translational geroscience: from mice to dogs to humans

Currently, the most promising research on mitigating the impacts of aging and extending longevity is limited to rodents living in laboratories, not humans living in the real world. "We are on the verge of actually having interventions that we may be able to apply to improve the quality of life of people ... but how do we move out of the laboratory?" asked University of Washington researcher Matt Kaeberlein. "How do we get from mice to people?" Kaeberlein's work in translational geroscience seeks to close the gap between promising lab findings and possible applications for humans by using a familiar stepping stone: man's best friend.

Dogs age similarly to humans and suffer from many of the same age-related diseases, but their lives are shorter, and thus they age much faster. Unlike a clinical trial using human subjects, where results take decades and are prone to ambiguities of correlation and causality, dog studies on aging can yield answers in as little as 3–5 years. Companion dogs, which are the subject of Kaeberlein's work, are especially relevant models for learning about human aging and healthy longevity, as they share the human environment.

The Dog Aging Project is the first national, large-scale longitudinal study of the genetic and environmental factors that influence healthy aging in dogs. Kaeberlein will enroll up to 10,000 dogs living different lifestyles in various environments—urban, rural, active, sedentary, and stray—using veterinary records, owner surveys, and biological samples to build a rich database of factors that influence canine aging and health. "We can actually do this for dogs in a decade, where it would take 50 years to do it with humans," Kaeberlein said.

Among the enrollees are several experimental cohorts, the first of which is a group of 40 middle-aged companion dogs treated with a 10-week course of the drug rapamycin. Most often used to prevent organ transplant rejection, rapamycin—an mTOR inhibitor—has the remarkable and well-documented side effects of delaying age-related diseases and increasing healthy longevity in every species in which it has been tested, including worms, mice, flies, and yeast. The drug is typically well-tolerated, and experiments in mice show that even a transient course of rapamycin in middle age produces functional improvements in cardiac and immune function. "This is exactly the kind of intervention we're interested in from a translational perspective—something you can start in middle-age and still see benefits," said Kaeberlein, adding that among the 24 dogs that completed his first rapamycin trial, those who received the drug showed improvements in multiple measurements of cardiac function. The dogs showed improved systolic function, as measured by ejection fraction and fractional shortening—both indicators of cardiac output and the heart's pumping capabilities—and improved relaxation, or diastolic function, as measured by E/A ratio. Further, dogs with weaker cardiac output at the beginning of the trial showed the greatest improvement after 10 weeks of treatment.

Middle-aged mice and dogs treated with a 10-week course of rapamycin experience similar gains in cardiac function.

While Kaeberlein cautioned that broad conclusions about the benefits of a short course of rapamycin cannot be drawn from a single trial in a small cohort of dogs, he believes the initial results are promising enough to expand the protocol to include 300 dogs, with long-term follow up of cardiac and cognitive functioning.

Mechanisms of longevity in long-lived mammals

The naked mole rat is a hairless, nearly blind, underground-dwelling rodent that more than compensates in intrigue for what it lacks in beauty, according to Vera Gorbunova of University of Rochester in her introduction to the longest living member of the rodent family. With a lifespan that can top 30 years and an innate resistance to cancer and age-related degeneration, the naked mole rat is considered perhaps the ultimate animal model for longevity and oncology research.

When grown in culture, naked mole rat cells do not grow to confluence. Unlike mouse cells, which easily proliferate to fill a dish, naked mole rat cells are highly sensitive to contact inhibition—a critical trait in preventing tumor formation, as malignant cells lose the ability to interpret cellular cues to cease division. Gorbunova's team discovered that naked mole rat cells secrete hyaluronic acid (HA), a viscous substance present in connective tissues throughout the body, as a signal to halt cell division. This finding led to several additional implications for the role of hyaluronic acid in both longevity and cancer resistance.

The biological properties of HA depend on the molecule's length—long molecules are anti-proliferative and anti-inflammatory, and short chains, which are found at wound sites and in metastases, have the opposite properties. The naked mole rat genome has an unusual sequence of hyaluronan synthase 2 (HAS2) which produces extremely high molecular weight HA—up to five times the length of human or mouse HA. Further, while other mammals regularly degrade and replace HA in cells, naked mole rats recycle very little HA, allowing it to accumulate in tissues throughout the body, including in the bone marrow.

Gorbunova reported the results of experiments to determine whether this unusually long-chain, well-preserved HA plays a role in the naked mole rat's cancer resistance. Typically, the perturbations in signaling pathways that trigger malignancy in mouse fibroblasts fail to produce cancer in naked mole rat cells. However, inhibiting HAS2 or overexpressing an HA-degrading enzyme makes naked mole rat cells susceptible to malignancy. "If you remove the ability to make hyaluronan from a naked mole rat, it becomes as susceptible [to cancer] as a mouse," she said. "But what happens if you add the ability to produce high-molecular weight hyaluronan to a mouse? Can we make them, or maybe even humans, more resistant to cancer?"

Interfering with hyaluronan production in naked mole rats makes typically cancer-resistant cells susceptible to malignancies.

Gorbunova and her collaborators have yet to answer the latter question, but by creating a transgenic mouse that overexpresses the naked mole rat variant of HAS2, they discovered that long chain hyaluronan confers several benefits likely associated with longevity.

Hyaluronan is a key part of the stem cell niche, and naked mole rats have higher quantities of HA in the niche than mice and other mammals, as well as more hematopoietic stem cells. These cells are quiescent compared to mouse stem cells, multiplying less frequently and displaying increased stress resistance. The bone marrow of transgenic mice that overexpress HAS2 shows a threefold increase in fully functional, long-term hematopoietic stem cells versus control mice, with stress resistance similar to that of naked mole rat stem cells. The evidence indicates that long-chain HA serves both to protect quiescent stem cells and bolster stress resistance, allowing for long-term maintenance of the hematopoietic system and likely staving off age-related decline.

Naked mole rats also show a natural downregulation of mTOR, a factor associated with longevity when induced by drugs like rapamycin. Gorbunova explained that this suppression of the cell's main nutrient sensing pathway in the naked mole rat is directly related to the synthesis of high molecular weight hyaluronan. Producing such long-chain polymers is energy intensive, forcing cells to deplete their glucose stores and activating AMPK, which mediates inhibition of mTOR. In experiments with transgenic HAS2 mice, inhibiting the AMPK pathway diminishes mTOR suppression.

Highlights

Wide variations in lifespan exist between individual of the same species and even individuals with identical genes; the factors that influence this distribution of lifespan are not well understood.

Lifespan varies greatly among mammals, as do the genetic, epigenetic, and metabolic factors that confer longevity benefits on the longest-lived animals.

Technological advances in collecting lifespan data have allowed researchers to conduct large-scale studies of factors that influence longevity in model animals, including C. elegans.

Studies of senescent cells have shown that the unique, pro-inflammatory secretome of such cells is a key driver of atherosclerosis in mice.

Removing senescent cells significantly extend life and healthspan in mice, and can both reduce arterial plaque formations in early-stage atherosclerosis and stabilize mature plaques.

Linking molecular interventions to systemic outcomes in aging

Nicholas Stroustrup delivered the first of three presentations on new approaches to studying aging and lifespan. Referencing a graph depicting the range of human lifespan from birth to age 100, he explained that while individuals of the same species show wide variation in lifespan, little is known about the factors that influence this variation. Genetics, environmental factors, and chance all play some role in determining longevity, but even when genetic and environmental diversity is eliminated, as is the case in cloned laboratory animals raised in isolation, variations in lifespan between seemingly identical individuals persist.

Stroustrup seeks to understand lifespan diversity by placing aging in the context of a multiscale systems biology problem. Thus, variations in the makeup and performance of individual organs, tissues, cells, and molecules interact in parallel to determine a person's healthspan and longevity, and these differ from person to person. To quantify the impact of these variations on the lifespan of a model animal, he devised novel experiments and measurement techniques in C. elegans populations.

Lifespan experiments have long been hampered in scale by the arduous process of data collection— in the case of C. elegans, a researcher must manually assess the vitality of each individual worm daily for several weeks. The tedium of this task dictates the trend toward relatively small population studies. Stroustrup developed an automated scanning and image analysis system consisting of fleets of consumer flatbed scanners retrofitted to house dishes of C. elegans and capable of producing daily vitality information as well as high-resolution images and video for thousands of individual worms.

Thus equipped with robust imaging and assessment tools for collecting lifespan data at unprecedented scale and resolution, the researchers began testing the impact of various interventions on C. elegans populations. These included environmental and dietary interventions as well as genetic mutations.

Some interventions, such as raising environmental temperature, are known to shorten lifespan in C. elegans. Others, such as inducing a mutation in the IGF receptor gene DAF-2, are known to extend it. Stroustrup and his collaborators were surprised to observe that all interventions altered the lifespan distribution curve in C. elegans populations through an apparent stretching or shrinking of time, where individuals experienced the same set of expected outcomes, only at a proportionally earlier or later time. Stroustrup explained that in the high temperature experiments, "the proportion of animals that would have lived a shorter time at a normal temperature [based on controls] had their lives shortened by the same fold factor as the animals who would have been living longer. Regardless of the intervention, it's as if all the physical processes in the worm that are dependent on time are being sped up or slowed down by a constant amount."

Dietary and genetic interventions all produced a temporal scaling in the lifespan distribution of C. elegans, as if each member of the population had its lifespan shortened or extended by the exact same proportion.

While an intervention like temperature could feasibly affect many molecular mechanisms that impact lifespan, it is unlikely that the single genetic mutations tested has an equally global impact on these factors. Perplexed by the universality of temporal scaling in all tested lifespan interventions, Stroustrup and his collaborators hypothesized that the phenomenon may be a result of a relationship between the factors that influence lifespan. "Maybe if you influence a subset of these mechanisms, you can have a global effect on all such mechanisms," he said.

While a single intervention is unlikely to universally hobble all molecular mechanisms that influence longevity, researchers hypothesize that these mechanisms are interdependent.

The mechanisms that influence lifespan may more closely resemble a signaling network, where interdependent nodes rely on the strength of their neighboring nodes, and failure of a single node propagates additional failed dependencies throughout the network. Conversely, if a subset of molecular mechanisms or nodes are strengthened, it may increase the resilience of the entire network. As Stroustrup commented in closing, "Perhaps there's an aspect of aging that is inherently systemic, where different intervention targets are interacting in complex ways that systematize the consequences of these interventions."

Comparative genomics of lifespan control

Vadim Gladyshev of Brigham and Women's Hospital at Harvard Medical School steered the conversation to the wide range of maximum lifespan among mammals, which can top four years in a mouse and more than 200 for a bowhead whale. As mammalian species diverged from a common ancestor, their lifespan, as well as morphology, has been shaped through evolution, indicating that there are many routes for a species to achieve longevity. Gladyshev questions whether there are common mechanisms and patterns among the longest-lived animals, and if so, whether they may form the basis for interventions capable of shifting an organism from a short-lived state to a longer one.

The genetic determinants of longevity exist on multiple levels and in a range of mammalian systems. Even within a single organism, variations in lifespan arise at the cellular level: neurons can function for a century, whereas monocytes die within days. Identifying the mechanisms that drive longer-living cells, as well as commonalities in gene expression and metabolite profiles in long-lived species, is key to understanding the possibilities for boosting longevity.

"There are various ways to extend lifespan from an evolutionary standpoint," said Gladyshev, explaining that genomic data from long-lived animals indicates that there are both private mechanisms of longevity as well as common ones. For example, the naked mole rat and Brandt's bat—two different species with exceptional longevity—show distinct gene alterations that confer an advantage in longevity. However, many long-lived animals also display common gene expression patterns associated with longevity, such as upregulation in genes involved in DNA repair and glucose metabolism, and downregulation in genes involved with proteolysis.

Mouse studies show that different types of interventions enhance longevity, including pharmacological treatments like rapamycin and acarbose, nutritional approaches such as dietary and methionine restriction, and genetic alterations. "The question is whether these things extend lifespan using similar mechanisms or not, and if there is any overlap," said Gladyshev.

Gene expression studies reveal that while global gene expression in mice following life-extending interventions varies considerably, certain interventions do produce similar changes. A map of these changes illustrates that genetic alterations tend to trigger correlated changes in gene expression, and pharmacological interventions including rapamycin and metformin induce completely different, unrelated changes.

Lifespan interventions impact gene expression in different ways. Uncovering commonalities in affected pathways may lead to new therapies that can produce similar effects.

Organ-specific gene regulation also governs some degree of longevity in mammals, as a study of brain, kidney, and liver tissue from 41 mammals reveals. A common cohort of genes in each organ is differentially up-or down-regulated in each organ over time. Notably, genes that regulate metabolic processes are significantly downregulated in the liver and kidney as mammals age, yet expression of these same genes remains largely unchanged in the brain. "What this means is that living a long time is gene-specific, mechanism-specific, and organ-specific," Gladyshev said. "We don't know the best way to extend lifespan, because it's clear there are multiple ways."

Identifying gene expression patterns associated with longer life, which Gladyshev terms "longevity signatures," has allowed him and his collaborators to test small molecules in cell culture to identify those that produce similar effects. Rapamycin, as well as several of its analogs, have emerged as top compounds.

Slowing aging and disease through senescent cell elimination

In the meeting's final presentation, Jan van Deursen of the Mayo Clinic discussed the role of senescent cells as potent drivers of age-related disease and degeneration, particularly in the case of atherosclerosis. Cellular senescence occurs when the cumulative impact of stressors and DNA damage causes replicating cells to cease dividing—a normal, irreversible arrest process that is important for tumor prevention. However, senescent cells possess a unique secretory phenotype associated with aging, and senescent cells accumulate in the body over time.

The secretome of senescent cells is rich in pro-inflammatory cytokines, growth factors, and proteases, which can disrupt neighboring cell function and tissue architecture, and, paradoxically, may even contribute to tumor progression. Senescent cells are abundant in atherosclerotic and Alzheimer's-associated plaques, as well as at sites of osteoarthritis.

Van Deursen reports that several different transgenes have been used in concert with small molecules to induce apoptosis in senescent mouse cells, effectively clearing them from the animal. Mice treated weekly with this protocol experienced a 25–30 percent extension in lifespan and attenuation of age-related deterioration in several organ systems with few or no side effects. Video footage of an experimental mouse next to a mouse given only the transgene (without the small molecule to trigger apoptosis) showed dramatic differences in ease of mobility and external measures of wellness and vitality between the two animals.

Additional mouse studies show that senescent cells are not merely present in atherosclerotic plaques, but are in fact key drivers of atherogenesis. Van Deursen tracked the process in both wild-type and LDL-receptor knockout mice, revealing the role of senescent cells from the earliest stages of atheroma formation through eventual plaque rupture resulting in myocardial infarction or stroke.

Arterial plaques, even in the earliest stages, are highly SABG-positive, a primary biomarker for senescent cells. SABG staining and transmission electron microscopy analysis of arterial deposits show that foamy macrophages possess these senescence markers well before a mature plaque forms. Further, the pro-inflammatory secretome of senescent cells advances atherosclerosis through production of VCAM1 and MCP1, recruitment factors that further draw monocytes from the circulation into the subendothelial space. In late-stage plaques, senescent cells produce enzymes that erode the fibrous cap that serves to stabilize plaques, rendering them vulnerable to rupture.

Van Deursen explained that frequent removal of senescent cells in LDL-receptor knockout mice, either through a transgene/small molecule approach or by using senolytic drugs, shows positive results during all stages of plaque development. Thus, removal of senescent cells can inhibit plaque initiation and growth in the early stages of atherosclerosis, and can stabilize mature plaques in late-stage disease.

Removing senescent cells from LDL-receptor knockout mice results in smaller, fewer arterial plaques and lower lipid levels, even among mice fed a high-fat diet.