Loss of epigenetic information as a cause of mammalian aging.

All living things experience an increase in entropy, manifested as a loss of genetic and epigenetic information. In yeast, epigenetic information is lost over time due to the relocalization of chromatin-modifying proteins to DNA breaks, causing cells to lose their identity, a hallmark of yeast aging. Using a system called "ICE" (inducible changes to the epigenome), we find that the act of faithful DNA repair advances aging at physiological, cognitive, and molecular levels, including erosion of the epigenetic landscape, cellular exdifferentiation, senescence, and advancement of the DNA methylation clock, which can be reversed by OSK-mediated rejuvenation. These data are consistent with the information theory of aging, which states that a loss of epigenetic information is a reversible cause of aging.

Impairment of an Endothelial NAD+-H2S Signaling Network Is a Reversible Cause of Vascular Aging.

A decline in capillary density and blood flow with age is a major cause of mortality and morbidity. Understanding why this occurs is key to future gains in human health. NAD precursors reverse aspects of aging, in part, by activating sirtuin deacylases (SIRT1-SIRT7) that mediate the benefits of exercise and dietary restriction (DR). We show that SIRT1 in endothelial cells is a key mediator of pro-angiogenic signals secreted from myocytes. Treatment of mice with the NAD+ booster nicotinamide mononucleotide (NMN) improves blood flow and increases endurance in elderly mice by promoting SIRT1-dependent increases in capillary density, an effect augmented by exercise or increasing the levels of hydrogen sulfide (H2S), a DR mimetic and regulator of endothelial NAD+ levels. These findings have implications for improving blood flow to organs and tissues, increasing human performance, and reestablishing a virtuous cycle of mobility in the elderly.

Slowing ageing by design: the rise of NAD+ and sirtuin-activating compounds.

The sirtuins (SIRT1-7) are a family of nicotinamide adenine dinucleotide (NAD+)-dependent deacylases with remarkable abilities to prevent diseases and even reverse aspects of ageing. Mice engineered to express additional copies of SIRT1 or SIRT6, or treated with sirtuin-activating compounds (STACs) such as resveratrol and SRT2104 or with NAD+ precursors, have improved organ function, physical endurance, disease resistance and longevity. Trials in non-human primates and in humans have indicated that STACs may be safe and effective in treating inflammatory and metabolic disorders, among others. These advances have demonstrated that it is possible to rationally design molecules that can alleviate multiple diseases and possibly extend lifespan in humans.

Reprogramming to recover youthful epigenetic information and restore vision.

Ageing is a degenerative process that leads to tissue dysfunction and death. A proposed cause of ageing is the accumulation of epigenetic noise that disrupts gene expression patterns, leading to decreases in tissue function and regenerative capacity1-3. Changes to DNA methylation patterns over time form the basis of ageing clocks4, but whether older individuals retain the information needed to restore these patterns-and, if so, whether this could improve tissue function-is not known. Over time, the central nervous system (CNS) loses function and regenerative capacity5-7. Using the eye as a model CNS tissue, here we show that ectopic expression of Oct4 (also known as Pou5f1), Sox2 and Klf4 genes (OSK) in mouse retinal ganglion cells restores youthful DNA methylation patterns and transcriptomes, promotes axon regeneration after injury, and reverses vision loss in a mouse model of glaucoma and in aged mice. The beneficial effects of OSK-induced reprogramming in axon regeneration and vision require the DNA demethylases TET1 and TET2. These data indicate that mammalian tissues retain a record of youthful epigenetic information-encoded in part by DNA methylation-that can be accessed to improve tissue function and promote regeneration in vivo.

SIRT1 Limits Adipocyte Hyperplasia through c-Myc Inhibition.

The expansion of fat mass in the obese state is due to increased adipocyte hypertrophy and hyperplasia. The molecular mechanism that drives adipocyte hyperplasia remains unknown. The NAD(+)-dependent protein deacetylase sirtuin 1 (SIRT1), a key regulator of mammalian metabolism, maintains proper metabolic functions in many tissues, counteracting obesity. Here we report that differentiated adipocytes are hyperplastic when SIRT1 is knocked down stably in mouse 3T3-L1 preadipocytes. This phenotype is associated with dysregulated adipocyte metabolism and enhanced inflammation. We also demonstrate that SIRT1 is a key regulator of proliferation in preadipocytes. Quantitative proteomics reveal that the c-Myc pathway is altered to drive enhanced proliferation in SIRT1-silenced 3T3-L1 cells. Moreover, c-Myc is hyperacetylated, levels of p27 are reduced, and cyclin-dependent kinase 2 (CDK2) is activated upon SIRT1 reduction. Remarkably, differentiating SIRT1-silenced preadipocytes exhibit enhanced mitotic clonal expansion accompanied by reduced levels of p27 as well as elevated levels of CCAAT/enhancer-binding protein ß (C/EBPß) and c-Myc, which is also hyperacetylated. c-Myc activation and enhanced proliferation phenotype are also found to be SIRT1-dependent in proliferating mouse embryonic fibroblasts and differentiating human SW872 preadipocytes. Reducing both SIRT1 and c-Myc expression in 3T3-L1 cells simultaneously does not induce the adipocyte hyperplasia phenotype, confirming that SIRT1 controls adipocyte hyperplasia through c-Myc regulation. A better understanding of the molecular mechanisms of adipocyte hyperplasia will open new avenues toward understanding obesity.

Neuronal SIRT1 regulates endocrine and behavioral responses to calorie restriction.

Mammalian life span can be extended by both calorie restriction (CR) and mutations that diminish somatotropic signaling. Sirt1 is a mediator of many effects of CR in mammals, but any role in controlling somatotropic signaling has not been shown. Since the somatotropic axis is controlled by the brain, we created mice lacking Sirt1 specifically in the brain and examined the impacts of this manipulation on somatotropic signaling and the CR response. These mutant mice displayed defects in somatotropic signaling when fed ad libitum, and defects in the endocrine and behavioral responses to CR. We conclude that Sirt1 in the brain is a link between somatotropic signaling and CR in mammals.

Age and life expectancy clocks based on machine learning analysis of mouse frailty.

The identification of genes and interventions that slow or reverse aging is hampered by the lack of non-invasive metrics that can predict the life expectancy of pre-clinical models. Frailty Indices (FIs) in mice are composite measures of health that are cost-effective and non-invasive, but whether they can accurately predict health and lifespan is not known. Here, mouse FIs are scored longitudinally until death and machine learning is employed to develop two clocks. A random forest regression is trained on FI components for chronological age to generate the FRIGHT (Frailty Inferred Geriatric Health Timeline) clock, a strong predictor of chronological age. A second model is trained on remaining lifespan to generate the AFRAID (Analysis of Frailty and Death) clock, which accurately predicts life expectancy and the efficacy of a lifespan-extending intervention up to a year in advance. Adoption of these clocks should accelerate the identification of longevity genes and aging interventions.

Publisher Correction: Age and life expectancy clocks based on machine learning analysis of mouse frailty.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Insulin signaling cascade in the hearts of long-lived growth hormone receptor knockout mice: effects of calorie restriction.

Calorie restriction (CR) improves insulin sensitivity and increases life span in normal but not in long-lived growth hormone-resistant knockout (GHRKO) mice. In this study, we examined interactive effects of GH resistance and long-term CR on cardiac insulin action. GHRKO mice exhibited marked increases in the insulin-induced phosphorylation of the insulin receptor (IR), insulin receptor substrate-1 (IRS-1), Akt, and ERK1/2 along with elevated insulin-stimulated IRS-1-associated regulatory subunit of phosphatidylinositol 3-kinase in the heart. These changes were associated with elevated protein levels of IR, IRS-1, and Akt and with a down-regulation of cardiac glucose transporter 4 (GLUT4). In normal mice, CR induced an important increase in the phosphorylation of cardiac Akt without elevation of Akt protein, reaching activation levels similar to those seen in GHRKO mice. This change may be cardioprotective and thus contribute to increased longevity in response to CR. Interestingly, the insulin signaling cascade in the heart of GHRKO mice was unaffected by CR.

Effects of mild calorie restriction on reproduction, plasma parameters and hepatic gene expression in mice with altered GH/IGF-I axis.

The somatotropic axis, the hypothalamic-pituitary-gonadal axis and the nutritional status are deeply interrelated in mammals. Calorie restriction (CR) prolongs lifespan, but usually at some cost to reproduction. Interestingly, many of the physiological characteristics of animals with interruption in the somatotropic axis are shared by CR animals. The level of CR in most studies is 30-60%. We tested if a milder (20%) CR would promote health benefits without inhibiting reproduction in four types of mice with altered somatotropic axis: Ames dwarfs, GHR-KO, and PEPCK-bGH and MT-bGH transgenics. Fertility was not altered by CR in any of the examined groups. Mild CR did not affect final body weights or relative reproductive organ weights; did not alter plasma levels of glucose, insulin, IGF-I, testosterone, progesterone or estradiol; and did not influence hepatic expression of genes related to longevity. Altered activity of the GH/IGF-I axis in the different mice models studied had a major impact on the parameters analyzed. This preliminary study encourages speculation that mild regimens of CR can produce health and longevity benefits without the "costs" of impaired reproductive potential.

Effects of caloric restriction and growth hormone resistance on insulin-related intermediates in the skeletal muscle.

Growth hormone receptor-deficient (GHRKO) mice are long-lived and have reduced insulin-like growth factor (IGF)-1 and insulin levels and enhanced insulin sensitivity thus resembling the phenotype of animals subjected to calorie restriction (CR). In contrast to its effects in normal mice, CR does not improve insulin sensitivity or increase longevity in GHRKO males. In an attempt to identify mechanisms underlying this differential response to CR, effects of CR on the expression of insulin-related genes were compared in GHRKO and normal mice. In addition to changes detected in both genotypes, and responses unique to GHRKO mice, the levels of Akt2 and peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC1alpha) were increased and levels of phosphorylated c-Jun N-terminal kinase (JNK)1 were reduced in response to CR only in normal mice. These changes may be related to mechanisms of improving insulin sensitivity and life expectancy.

Mild calorie restriction does not affect testosterone levels and testicular gene expression in mutant mice.

The hypothalamic-pituitary-gonadal (HPG) axis and the somatotropic axis are influenced by nutritional factors. Calorie restriction (CR) extends lifespan but suppresses both the HPG and the somatotropic axes. Since most CR studies use a fairly severe (40%-60%) reduction of calorie intake, we hypothesized that a milder CR (20%) might not be deleterious to reproduction in male mice. To test this hypothesis, we evaluated the effects of 20% CR on testicular testosterone content and on testicular expression of genes that are relevant to testicular function and reproductive competence, including insulin-like growth factor-I, cytochrome P450 aromatase (Cyp19a1), androgen receptor, luteinizing hormone receptor, follicle-stimulating hormone receptor, cytochrome P450c17 and 3-beta-hydroxysteroid dehydrogenase/isomerase. To relate CR effects to the activity of the somatotropic axis, we have used growth hormone-resistant GHR knockout mice as well as transgenic mice overexpressing GH. Mild CR did not affect testosterone levels in testis homogenates and had little effect on expression of the examined genes in the reproductive organs. Altered activity of the GH/insulin-like growth factor-1 axis had a major impact on the parameters analyzed. The results also suggest that expression of several key genes involved in the control of testicular function is preserved under conditions of mild CR and encourage speculation that mild regimens of CR can produce longevity benefits without impairing reproduction.

A conserved NAD+ binding pocket that regulates protein-protein interactions during aging.

DNA repair is essential for life, yet its efficiency declines with age for reasons that are unclear. Numerous proteins possess Nudix homology domains (NHDs) that have no known function. We show that NHDs are NAD+ (oxidized form of nicotinamide adenine dinucleotide) binding domains that regulate protein-protein interactions. The binding of NAD+ to the NHD domain of DBC1 (deleted in breast cancer 1) prevents it from inhibiting PARP1 [poly(adenosine diphosphate-ribose) polymerase], a critical DNA repair protein. As mice age and NAD+ concentrations decline, DBC1 is increasingly bound to PARP1, causing DNA damage to accumulate, a process rapidly reversed by restoring the abundance of NAD+ Thus, NAD+ directly regulates protein-protein interactions, the modulation of which may protect against cancer, radiation, and aging.

Resveratrol Improves Vascular Function and Mitochondrial Number but Not Glucose Metabolism in Older Adults.

BACKGROUND: Resveratrol, a plant-derived polyphenol, has been reported to improve glucose metabolism and vascular function and to extend life span in animal models, but studies in humans have been inconclusive. METHODS: In a randomized, double-blind crossover study, we treated older glucose-intolerant adults (n = 30) with resveratrol (2-3 g/daily) or placebo, each for 6 weeks. A standard mixed-meal test was used to assess insulin sensitivity (Matsuda index) and secretion (C-peptide deconvolution) and vascular function by reactive hyperemia peripheral arterial tonometry. Skeletal muscle samples were obtained for gene expression using RNA-Seq analysis and to assess mitochondrial morphology. RESULTS: There were no changes in glucose tolerance, insulin sensitivity, weight, blood pressure, or lipid profile following resveratrol treatment. Fasting reactive hyperemia index improved with resveratrol (2.02 ± 0.2 vs 1.76 ± 0.02, p = .002). RNA-Seq analysis yielded 140 differentially expressed transcripts (corrected p-value ≤ .05), predominantly associated with mitochondrial genes and noncoding RNA. Ingenuity Pathway Analysis confirmed that mitochondrial dysfunction (p = 2.77 × 10-12) and oxidative phosphorylation (p = 1.41 × 10-11) were the most significantly perturbed pathways. Mitochondrial number, but not size, was increased. CONCLUSIONS: Resveratrol treatment of older adults with impaired glucose regulation may have beneficial effects on vascular function, but not glucose metabolism or insulin sensitivity. Changes in gene expression suggest effects similar to those observed with caloric restriction, which has been shown to increase life and health span in animal models, although its significance for humans is uncertain. Future human studies should address the appropriate dose range and low bioavailability of resveratrol.

Caloric restriction and growth hormone receptor knockout: effects on expression of genes involved in insulin action in the heart.

Blockade of growth hormone (GH), decreased insulin-like growth factor-1 (IGF1) action and increased insulin sensitivity are associated with life extension and an apparent slowing of the aging process. We examined expression of genes involved in insulin action, IR, IRS1, IRS2, IGF1, IGF1R, GLUT4, PPARs and RXRs in the hearts of normal and GHR-/- (KO) mice fed ad libitum or subjected to 30% caloric restriction (CR). CR increased the cardiac expression of IR, IRS1, IGF1, IGF1R and GLUT4 in normal mice and IRS1, GLUT4, PPARalpha and PPARbeta/delta in GHR-KO animals. Expression of IR, IRS1, IRS2, IGF1, GLUT4, PPARgamma and PPARalpha did not differ between GHR-KO and normal mice. These unexpected results suggest that CR may lead to major modifications of insulin action in the heart, but high insulin sensitivity of GHR-KO mice is not associated with alterations in the levels of most of the examined molecules related to intracellular insulin signaling.

Long-lived growth hormone receptor knockout mice show a delay in age-related changes of body composition and bone characteristics.

There is conflicting information on the physiological role of growth hormone (GH) in the control of aging. This study reports dual-energy x-ray absorptiometry (DXA) measurements of body composition and bone characteristics in young, adult, and aged long-lived GH receptor knockout (GHR-KO) and normal mice to determine the effects of GH resistance during aging. Compared to controls, GHR-KO mice showed an increased percentage of body fat. GHR-KO mice have reduced total-body bone mineral density (BMD), bone mineral content, and bone area, but these parameters increased with age. In addition, GHR-KO mice have decreased femur length, femur BMD, and lower lumbar BMD compared to controls in all age groups. These parameters also continued to increase with age. Our results indicate that GH resistance alters body composition, bone growth, and bone maintenance during aging in GHR-KO mice.

Measuring aging rates of mice subjected to caloric restriction and genetic disruption of growth hormone signaling.

Caloric restriction and genetic disruption of growth hormone signaling have been shown to counteract aging in mice. The effects of these interventions on aging are examined through age-dependent survival or through the increase in age-dependent mortality rates on a logarithmic scale fitted to the Gompertz model. However, these methods have limitations that impede a fully comprehensive disclosure of these effects. Here we examine the effects of these interventions on murine aging through the increase in age-dependent mortality rates on a linear scale without fitting them to a model like the Gompertz model. Whereas these interventions negligibly and non-consistently affected the aging rates when examined through the age-dependent mortality rates on a logarithmic scale, they caused the aging rates to increase at higher ages and to higher levels when examined through the age-dependent mortality rates on a linear scale. These results add to the debate whether these interventions postpone or slow aging and to the understanding of the mechanisms by which they affect aging. Since different methods yield different results, it is worthwhile to compare their results in future research to obtain further insights into the effects of dietary, genetic, and other interventions on the aging of mice and other species.

Restoration of normal embryogenesis by mitochondrial supplementation in pig oocytes exhibiting mitochondrial DNA deficiency.

An increasing number of women fail to achieve pregnancy due to either failed fertilization or embryo arrest during preimplantation development. This often results from decreased oocyte quality. Indeed, reduced mitochondrial DNA copy number (mitochondrial DNA deficiency) may disrupt oocyte quality in some women. To overcome mitochondrial DNA deficiency, whilst maintaining genetic identity, we supplemented pig oocytes selected for mitochondrial DNA deficiency, reduced cytoplasmic maturation and lower developmental competence, with autologous populations of mitochondrial isolate at fertilization. Supplementation increased development to blastocyst, the final stage of preimplantation development, and promoted mitochondrial DNA replication prior to embryonic genome activation in mitochondrial DNA deficient oocytes but not in oocytes with normal levels of mitochondrial DNA. Blastocysts exhibited transcriptome profiles more closely resembling those of blastocysts from developmentally competent oocytes. Furthermore, mitochondrial supplementation reduced gene expression patterns associated with metabolic disorders that were identified in blastocysts from mitochondrial DNA deficient oocytes. These results demonstrate the importance of the oocyte's mitochondrial DNA investment in fertilization outcome and subsequent embryo development to mitochondrial DNA deficient oocytes.