Extends Lifespan In Mice Research Articles

Polr3b heterozygosity in mice induces both beneficial and deleterious effects on health during ageing with no effect on lifespan.

The genetic pathways that modulate ageing in multicellular organisms are typically highly conserved across wide evolutionary distances. Recently RNA polymerase III (Pol III) was shown to promote ageing in yeast, C. elegans and D. melanogaster. In this study we investigated the role of Pol III in mammalian ageing using C57BL/6N mice heterozygous for Pol III (Polr3b+/-). We identified sexually dimorphic, organ-specific beneficial as well as detrimental effects of the Polr3b+/- mutation on health. Female Polr3b+/- mice displayed improved bone health during ageing, but their ability to maintain an effective gut barrier function was compromised and they were susceptible to idiopathic dermatitis (ID). In contrast, male Polr3b+/- mice were lighter than wild-type (WT) males and had a significantly improved gut barrier function in old age. Several metabolic parameters were affected by both age and sex, but no genotype differences were detected. Neither male nor female Polr3b+/- mice were long-lived compared to WT controls. Overall, we find no evidence that a reduced Pol III activity extends mouse lifespan but we do find some potential organ- and sex-specific benefits for old-age health.

Autophagy compensates for Lkb1 loss to maintain adult mice homeostasis and survival.

Liver kinase B1 (LKB1), also known as serine/threonine kinase 11 (STK11) is the major energy sensor for cells to respond to metabolic stress. Autophagy degrades and recycles proteins, macromolecules, and organelles for cells to survive starvation. To assess the role and cross-talk between autophagy and Lkb1 in normal tissue homeostasis, we generated genetically engineered mouse models where we can conditionally delete Stk11 and autophagy essential gene, Atg7, respectively or simultaneously, throughout the adult mice. We found that Lkb1 was essential for the survival of adult mice, and autophagy activation could temporarily compensate for the acute loss of Lkb1 and extend mouse life span. We further found that acute deletion of Lkb1 in adult mice led to impaired intestinal barrier function, hypoglycemia, and abnormal serum metabolism, which was partly rescued by the Lkb1 loss-induced autophagy upregulation via inhibiting p53 induction. Taken together, we demonstrated that autophagy and Lkb1 work synergistically to maintain adult mouse homeostasis and survival.

Acarbose has sex-dependent and -independent effects on age-related physical function, cardiac health, and lipid biology.

With an expanding aging population burdened with comorbidities, there is considerable interest in treatments that optimize health in later life. Acarbose (ACA), a drug used clinically to treat type 2 diabetes mellitus (T2DM), can extend mouse life span with greater effect in males than in females. Using a genetically heterogeneous mouse model, we tested the ability of ACA to ameliorate functional, pathological, and biochemical changes that occur during aging, and we determined which of the effects of age and drug were sex dependent. In both sexes, ACA prevented age-dependent loss of body mass, in addition to improving balance/coordination on an accelerating rotarod, rotarod endurance, and grip strength test. Age-related cardiac hypertrophy was seen only in male mice, and this male-specific aging effect was attenuated by ACA. ACA-sensitive cardiac changes were associated with reduced activation of cardiac growth–promoting pathways and increased abundance of peroxisomal proteins involved in lipid metabolism. ACA further ameliorated age-associated changes in cardiac lipid species, particularly lysophospholipids — changes that have previously been associated with aging, cardiac dysfunction, and cardiovascular disease in humans. In the liver, ACA had pronounced effects on lipid handling in both sexes, reducing hepatic lipidosis during aging and shifting the liver lipidome in adulthood, particularly favoring reduced triglyceride (TAG) accumulation. Our results demonstrate that ACA, already in clinical use for T2DM, has broad-ranging antiaging effects in multiple tissues, and it may have the potential to increase physical function and alter lipid biology to preserve or improve health at older ages.

Calorie-Restriction-Induced Insulin Sensitivity Is Mediated by Adipose mTORC2 and Not Required for Lifespan Extension

Calorie restriction (CR) extends the healthspan and lifespan of diverse species. In mammals, a broadly conserved metabolic effect of CR is improved insulin sensitivity, which may mediate the beneficial effects of a CR diet. This model has been challenged by the identification of interventions that extend lifespan and healthspan yet promote insulin resistance. These include rapamycin, which extends mouse lifespan yet induces insulin resistance by disrupting mTORC2 (mechanistic target of rapamycin complex 2). Here, we induce insulin resistance by genetically disrupting adipose mTORC2 via tissue-specific deletion of the mTORC2 component Rictor (AQ-RKO). Loss of adipose mTORC2 blunts the metabolic adaptation to CR and prevents whole-body sensitization to insulin. Despite this, AQ-RKO mice subject to CR experience the same increase in fitness and lifespan on a CR diet as wild-type mice. We conclude that the CR-induced improvement in insulin sensitivity is dispensable for the effects of CR on fitness and longevity.

17-α estradiol ameliorates age-associated sarcopenia and improves late-life physical function in male mice but not in females or castrated males.

Pharmacological treatments can extend mouse lifespan, but lifespan effects often differ between sexes. 17‐α estradiol (17aE2), a less feminizing structural isomer of 17‐β estradiol, produces lifespan extension only in male mice, suggesting a sexually dimorphic mechanism of lifespan regulation. We tested whether these anti‐aging effects extend to anatomical and functional aging—important in late‐life health—and whether gonadally derived hormones control aging responses to 17aE2 in either sex. While 17aE2 started at 4 months of age diminishes body weight in both sexes during adulthood, in late‐life 17aE2‐treated mice better maintain body weight. In 17aE2‐treated male mice, the higher body weight is associated with heavier skeletal muscles and larger muscle fibers compared with untreated mice during aging, while treated females have heavier subcutaneous fat. Maintenance of skeletal muscle in male mice is associated with improved grip strength and rotarod capacity at 25 months, in addition to higher levels of most amino acids in quadriceps muscle. We further show that sex‐specific responses to 17aE2—metabolomic, structural, and functional—are regulated by gonadal hormones in male mice. Castrated males have heavier quadriceps than intact males at 25 months, but do not respond to 17aE2, suggesting 17aE2 promotes an anti‐aging skeletal muscle phenotype similar to castration. Finally, 17aE2 treatment benefits can be recapitulated in mice when treatment is started at 16 months, suggesting that 17aE2 may be able to improve aspects of late‐life function even when started after middle age.

Long term rapamycin treatment improves mitochondrial DNA quality in aging mice

Age-induced mitochondrial DNA deletion mutations may underlie cell loss and tissue aging. Rapamycin extends mouse lifespan and modulates mitochondrial quality control. We hypothesized that reduced deletion mutation abundance may contribute to rapamycin's life extension effects. To test this hypothesis, genetically heterogeneous male and female mice were treated with rapamycin, compounded in chow at 14 or 42 ppm, from 9 months to 22 months of age. Mice under a 40% dietary restriction were included as a control known to protect mtDNA quality. To determine if chronic rapamycin treatment affects mitochondrial DNA quality, we assayed mtDNA deletion frequency and electron transport chain deficient fiber abundances in mouse quadriceps muscle.At 42 ppm rapamycin, we observed a 57% decrease in deletion frequency, a 2.8-fold decrease in ETC deficient fibers, and a 3.4-fold increase in the number of mice without electron transport chain deficient fibers. We observed a similar trend with the 14 ppm dose. DR significantly decreased ETC deficient fiber abundances with a trend toward lower mtDNA deletion frequency. The effects of rapamycin treatment on mitochondrial DNA quality were greatest in females at the highest dose. Rapamycin treatment at 14 ppm did not affect muscle mass or function. Dietary restriction also reduced deletion frequency and ETC deficient fibers. These data support the concept that the lifespan extending effects of rapamycin treatment result from enhanced mitochondrial DNA quality.

Sex differences in lifespan extension with acarbose and 17-α estradiol: gonadal hormones underlie male-specific improvements in glucose tolerance and mTORC2 signaling.

SummaryInterventions that extend lifespan in mice can show substantial sexual dimorphism. Here, we show that male‐specific lifespan extension with two pharmacological treatments, acarbose (ACA) and 17‐α estradiol (17aE2), is associated, in males only, with increased insulin sensitivity and improved glucose tolerance. Females, which show either smaller (ACA) or no lifespan extension (17aE2), do not derive these metabolic benefits from drug treatment. We find that these male‐specific metabolic improvements are associated with enhanced hepatic mTORC2 signaling, increased Akt activity, and phosphorylation of FOXO1a – changes that might promote metabolic health and survival in males. By manipulating sex hormone levels through gonadectomy, we show that sex‐specific changes in these metabolic pathways are modulated, in opposite directions, by both male and female gonadal hormones: Castrated males show fewer metabolic responses to drug treatment than intact males, and only those that are also observed in intact females, while ovariectomized females show some responses similar to those seen in intact males. Our results demonstrate that sex‐specific metabolic benefits occur concordantly with sexual dimorphism in lifespan extension. These sex‐specific effects can be influenced by the presence of both male and female gonadal hormones, suggesting that gonadally derived hormones from both sexes may contribute to sexual dimorphism in responses to interventions that extend mouse lifespan.

Diverse interventions that extend mouse lifespan suppress shared age-associated epigenetic changes at critical gene regulatory regions

BackgroundAge-associated epigenetic changes are implicated in aging. Notably, age-associated DNA methylation changes comprise a so-called aging “clock”, a robust biomarker of aging. However, while genetic, dietary and drug interventions can extend lifespan, their impact on the epigenome is uncharacterised. To fill this knowledge gap, we defined age-associated DNA methylation changes at the whole-genome, single-nucleotide level in mouse liver and tested the impact of longevity-promoting interventions, specifically the Ames dwarf Prop1df/df mutation, calorie restriction and rapamycin.ResultsIn wild-type mice fed an unsupplemented ad libitum diet, age-associated hypomethylation was enriched at super-enhancers in highly expressed genes critical for liver function. Genes harbouring hypomethylated enhancers were enriched for genes that change expression with age. Hypermethylation was enriched at CpG islands marked with bivalent activating and repressing histone modifications and resembled hypermethylation in liver cancer. Age-associated methylation changes are suppressed in Ames dwarf and calorie restricted mice and more selectively and less specifically in rapamycin treated mice.ConclusionsAge-associated hypo- and hypermethylation events occur at distinct regulatory features of the genome. Distinct longevity-promoting interventions, specifically genetic, dietary and drug interventions, suppress some age-associated methylation changes, consistent with the idea that these interventions exert their beneficial effects, in part, by modulation of the epigenome. This study is a foundation to understand the epigenetic contribution to healthy aging and longevity and the molecular basis of the DNA methylation clock.

A New Preclinical Paradigm for Testing Anti-Aging Therapeutics.

Testing drugs for anti-aging effects has historically been conducted in mouse life-span studies, but are costly and time consuming, and more importantly, difficult to recapitulate in humans. In addition, life-span studies in mice are not well suited to testing drug combinations that target multiple factors involved in aging. Additional paradigms for testing therapeutics aimed at slowing aging are needed. A new paradigm, designated as the Geropathology Grading Platform (GGP), is based on a standardized set of guidelines developed to detect the presence or absence of low-impact histopathological lesions and to determine the level of severity of high-impact lesions in organs from aged mice. The GGP generates a numerical score for each age-related lesion in an organ, summed for total lesions, and averaged over multiple mice to obtain a composite lesion score (CLS). Preliminary studies show that the platform generates CLSs that increase with the age of mice in an organ-dependent manner. The CLSs are sensitive enough to detect changes elicited by interventions that extend mouse life span, and thus help validate the GGP as a novel tool to measure biological aging. While currently optimized for mice, the GGP could be adapted to any preclinical animal model.

Rapamycin Attenuates Age-associated Changes in Tibialis Anterior Tendon Viscoelastic Properties.

Rapamycin extends mouse life span, but the extent to which rapamycin prevents aging-associated changes in specific tissues remains unclear. Stiffness increases and collagen turnover decreases in mouse tendon with aging; thus, our aim was to determine the effect of long-term rapamycin treatment on the mechanical and structural properties of tendons from old mice. Tendons were harvested from female UM-HET3 mice maintained on a standard chow diet for 4 (adult) or 22 (old) months or fed chow containing polymer-encapsulated rapamycin (eRAPA) from 9 to 22 months of age (old RAPA). Stiffness was twofold higher for tendons of old compared with adult mice, but in old RAPA mice, tendon stiffness was maintained at a value not different from that of adults. Additionally, expression of collagen decreased, expression of matrix metalloproteinase-8 increased, and total hydroxyproline content trended toward decreased levels in tendons of old eRAPA-fed mice compared with controls. Finally, age-associated calcification of Achilles tendons and accompanying elevations in expression of chondrocyte and osteoblast markers were all lower in old eRAPA-fed mice. These results suggest that long-term administration of rapamycin alters the molecular pathways responsible for aging of tendon extracellular matrix, resulting in tissue that is structurally and mechanically similar to tendons in adult mice.

P53 and rapamycin are additive.

Mechanistic target of rapamycin (mTOR) is a kinase found in a complex (mTORC1) that enables macromolecular synthesis and cell growth and is implicated in cancer etiology. The rapamycin-FK506 binding protein 12 (FKBP12) complex allosterically inhibits mTORC1. In response to stress, p53 inhibits mTORC1 through a separate pathway involving cell signaling and amino acid sensing. Thus, these different mechanisms could be additive. Here we show that p53 improved the ability of rapamycin to: 1) extend mouse life span, 2) suppress ionizing radiation (IR)-induced senescence-associated secretory phenotype (SASP) and 3) increase the levels of amino acids and citric acid in mouse embryonic stem (ES) cells. This additive effect could have implications for cancer treatment since rapamycin and p53 are anti-oncogenic.

Tetrahydroxystilbene glucoside extends mouse life span via upregulating neural klotho and downregulating neural insulin or insulin-like growth factor 1

A Chinese herb, Polygonatum multiflorum, has been reported to prolong animal life span, but the relevant molecular mechanism remains unclear. Tetrahydroxystilbene glucoside (TSG) is one main component of P. multiflorum and may contribute to extending life span of mammals. On the other hand, neuronal insulin signaling mediates the life span of mammals. Therefore, we investigated the effects of TSG on memory ability, life span, and the neural insulin signaling in the senescence-accelerated prone mouse (SAMP8). TSG improved the memory ability significantly (p < 0.01, compared with a control group). TSG prolonged the life span of SAMP8 by 17% at the most (p < 0.01, compared with a control group). TSG increased the protein level of neural klotho and reduced the levels of neural insulin, insulin-receptor, insulin-like growth factor-1, and insulin-like growth factor-1 receptor in the brain of SAMP8 (p < 0.01, compared with a control group). All these proteins are key factors of the pathways related to neural insulin/IGF-1 signaling. These findings suggest that TSG has anti-aging effects on mammals. From these results, TSG from P. multiflorum should be developed as a potential anti-age drug.

Announcement

Aging CellVolume 13, Issue 6 p. 1086-1086 AnnouncementOpen Access Announcement First published: 24 November 2014 https://doi.org/10.1111/acel.12292AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat The NIA Interventions Testing Program Announces the 2015 Solicitation of Proposals The National Institute on Aging (NIA) Interventions Testing Program (ITP) investigates dietary supplements purported to extend lifespan and/or delay the onset of disease and disability. The NIA ITP tests such compounds in mice, using a variety of measured endpoints to assess the efficacy of interventions. The NIA ITP is not a mechanism for funding sponsors’ laboratories to perform the work, but rather it is a collaborative effort between the three NIA-funded testing sites and the sponsors who propose interventions for study. The sponsor's role is to provide the rationale for investigating the intervention, make recommendations on the dose, route, and timing for administration of the intervention, and propose assays and measurements to document the efficacy of the intervention. The sponsors have access to all data developed from the treated mice, assist in analysis of the data, and co-author on resulting publications. One of the important aspects of the ITP protocol is that all compounds are tested on both male and female mice. The NIH has recently begun actively promoting the recognition of sex as a biological variable in animal studies (Clayton & Collins, 2014). The ITP has seen sex differences in the response to several interventions tested, such as the recent report on three interventions, acarbose, 17-α-estradiol, and nordihydroguaiaretic acid, that have far greater effects in males than in female mice (Harrison et al., 2014). These findings highlight the importance of testing both sexes in intervention studies. The NIA ITP is soliciting proposals for compounds to enter the study in 2016. The deadline for receipt of proposals is February 26, 2015. Information on the NIA ITP and guidelines for proposal development are posted at: http://www.nia.nih.gov/research/dab/interventions-testing-program-itp. Questions should be directed to Dr. Nancy Nadon (nadonn@nia.nih.gov). References Clayton JA, Collins FS (2014) Policy: NIH to balance sex in cell and animal studies. Nature 509, 282– 283. CrossrefPubMedWeb of Science®Google Scholar Harrison DE, Strong R, Allison DB, Ames BN, Astle CM, Atamna H, Fernandez E, Flurkey K, Javors MA, Nadon NL, Nelson JF, Simpkins JW, Smith D, Wilkinson JE, Miller RA (2014) Acarbose 17-α-estradiol, and nordihydroguaiaretic acid extend Mouse lifespan preferentially in males. Aging Cell 13, 273– 282. Wiley Online LibraryCASWeb of Science®Google Scholar Volume13, Issue6December 2014Pages 1086-1086 ReferencesRelatedInformation

MiR-17 extends mouse lifespan by inhibiting senescence signaling mediated by MKP7.

Here we show that transgenic expression of miR-17 extends lifespan and inhibits cellular senescence. We propose that miR-17 acts as a critical regulator of cellular senescence and tumorigenesis. We demonstrate that miR-17 targets both ADCY5 and IRS1, upregulating the downstream signals MKP7, FoxO3, LC3B, and HIF1α, and downregulating mTOR, c-myc, cyclin D1, and JNK. Silencing either ADCY5 or IRS1 promoted autophagy and repressed cellular senescence and apoptosis. Repression of ADCY5 by miR-17 translocated membrane-bound RGS2 into the nucleus, promoting interactions of RGS2 with HIF1α and the MKP7 promoter, enhancing MKP7 transcription. ADCY5 repression by miR-17 also facilitated the translocation of EGFR and MKP7 from membrane into cytoplasmic and mitochondrial fractions. Importantly, we found that MKP7 inhibited senescence by dephosphorylating PRAS40 at Thr246 and mTOR at Ser2248, facilitating the interaction and loss of function of both molecules. Thus, the oncogenic miR-17 also acts pleiotropically to inhibit cellular senescence and extend longevity.

Elevated ATF4 Function in Fibroblasts and Liver of Slow-Aging Mutant Mice

Work in yeast has shown that longevity extension induced by nutrient deprivation, altered ribosomal function, or diminished target of rapamycin action requires the activity of GCN4. We hypothesized that increased activity of ATF4, the mammalian equivalent of yeast GCN4, might be characteristic of mutations that extend mouse life span. Fibroblasts from the skin of two such mutants (Snell dwarf and PAPP-A knockout) were found to have higher levels of ATF4 protein and expression of several ATF4 target genes in responses to amino acid withdrawal, cadmium, hydrogen peroxide, and tunicamycin. ATF4 pathways were also elevated in liver of both kinds of long-lived mutant mice. Thus, a connection between ATF4 pathways and longevity may have deep evolutionary roots, and further studies of ATF4 mechanisms may provide insights into the links between cellular stress resistance, protein translation control, and aging.

Expression of klotho mRNA and protein in rat brain parenchyma from early postnatal development into adulthood

Without the age-regulating protein klotho, mouse lifespan is shortened and the rapid onset of age-related disorders occurs. Conversely, overexpression of klotho extends mouse lifespan. Klotho is most abundant in kidney and expressed in a limited number of other organs, including the brain, where klotho levels are highest in choroid plexus. Reports vary on where klotho is expressed within the brain parenchyma, and no data is available as to whether klotho levels change across postnatal development. We used in situ hybridization to map klotho mRNA expression in the developing and adult rat brain and report moderate, widespread expression across grey matter regions. mRNA expression levels in cortex, hippocampus, caudate putamen, and amygdala decreased during the second week of life and then gradually rose to adult levels by postnatal day 21. Immunohistochemistry revealed a protein expression pattern similar to the mRNA results, with klotho protein expressed widely throughout the brain. Klotho protein co-localized with both the neuronal marker NeuN, as well as, oligodendrocyte marker olig2. These results provide the first anatomical localization of klotho mRNA and protein in rat brain parenchyma and demonstrate that klotho levels vary during early postnatal development.

Rapamycin induces glucose intolerance in mice by reducing islet mass, insulin content, and insulin sensitivity

Rapamycin, a specific inhibitor for mTOR complex 1, is an FDA-approved immunosuppressant for organ transplant. Recent developments have raised the prospect of using rapamycin to treat cancer or diabetes and to delay aging. It is therefore important to assess how rapamycin treatment affects glucose homeostasis. Here, we show that the same rapamycin treatment reported to extend mouse life span significantly impaired glucose homeostasis of aged mice. Moreover, rapamycin treatment of lean C57B/L6 mice reduced glucose-stimulated insulin secretion in vivo and ex vivo as well as the insulin content and beta cell mass of pancreatic islets. Confounding the diminished capacity for insulin release, rapamycin decreased insulin sensitivity. The multitude of rapamycin effects thus all lead to glucose intolerance. As our findings reveal that chronic rapamycin treatment could be diabetogenic, monitoring glucose homeostasis is crucial when using rapamycin as a therapeutic as well as experimental reagent.

Longevity on the Brain

Several studies show that loss-of-function mutations in the insulin-like signaling cascade extend the life span of worms and flies; however, equivalent mutations are associated with metabolic disease and fatal diabetes in mice. In contrast, calorie restriction or genetic strategies in mice that enhance insulin sensitivity lower the risk of age-related disease and extend life span. Taguchi et al . resolve these conflicting results by pointing to the brain as the site where reduced insulin-like signaling can extend mouse life span. A. Taguchi, L. M. Wartschow, M. F. White, Brain IRS2 signaling coordinates life span and nutrient homeostasis. Science 317 , 369-372 (2007). [Abstract] [Full Text]

Oxidative Damage and Aging: Spotlight on Mitochondria

Whereas free radical damage has been proposed as a key component in the tissue degeneration associated with aging, there has been little evidence that free radical damage limits life span in mammals. The current research shows that overexpression of the antioxidant enzyme catalase in mitochondria can extend mouse life span. These results highlight the importance of mitochondrial damage in aging and suggest that when targeted appropriately, boosting antioxidant defenses can increase mammalian life span.

Methionine-deficient diet extends mouse lifespan, slows immune and lens aging, alters glucose, T4, IGF-I and insulin levels, and increases hepatocyte MIF levels and stress resistance.

SummaryA diet deficient in the amino acid methionine has previously been shown to extend lifespan in several stocks of inbred rats. We report here that a methionine‐deficient (Meth‐R) diet also increases maximal lifespan in (BALB/cJ × C57BL/6 J)F1 mice. Compared with controls, Meth‐R mice have significantly lower levels of serum IGF‐I, insulin, glucose and thyroid hormone. Meth‐R mice also have higher levels of liver mRNA for MIF (macrophage migration inhibition factor), known to be higher in several other mouse models of extended longevity. Meth‐R mice are significantly slower to develop lens turbidity and to show age‐related changes in T‐cell subsets. They are also dramatically more resistant to oxidative liver cell injury induced by injection of toxic doses of acetaminophen. The spectrum of terminal illnesses in the Meth‐R group is similar to that seen in control mice. Studies of the cellular and molecular biology of methionine‐deprived mice may, in parallel to studies of calorie‐restricted mice, provide insights into the way in which nutritional factors modulate longevity and late‐life illnesses.