Rapamycin fed late in life extends lifespan in genetically heterogeneous mice.

Inhibition of the TOR signalling pathway by genetic or pharmacological intervention extends lifespan in invertebrates, including yeast, nematodes and fruitflies; however, whether inhibition of mTOR signalling can extend lifespan in a mammalian species was unknown. Here we report that rapamycin, an inhibitor of the mTOR pathway, extends median and maximal lifespan of both male and female mice when fed beginning at 600 days of age. On the basis of age at 90% mortality, rapamycin led to an increase of 14% for females and 9% for males. The effect was seen at three independent test sites in genetically heterogeneous mice, chosen to avoid genotype-specific effects on disease susceptibility. Disease patterns of rapamycin-treated mice did not differ from those of control mice. In a separate study, rapamycin fed to mice beginning at 270 days of age also increased survival in both males and females, based on an interim analysis conducted near the median survival point. Rapamycin may extend lifespan by postponing death from cancer, by retarding mechanisms of ageing, or both. To our knowledge, these are the first results to demonstrate a role for mTOR signalling in the regulation of mammalian lifespan, as well as pharmacological extension of lifespan in both genders. These findings have implications for further development of interventions targeting mTOR for the treatment and prevention of age-related diseases.

Genetically diverse mice are novel and valuable models of age-associated susceptibility to Mycobacterium tuberculosis.

BACKGROUND: Tuberculosis, the disease due to Mycobacterium tuberculosis, is an important cause of morbidity and mortality in the elderly. Use of mouse models may accelerate insight into the disease and tests of therapies since mice age thirty times faster than humans. However, the majority of TB research relies on inbred mouse strains, and these results might not extrapolate well to the genetically diverse human population. We report here the first tests of M. tuberculosis infection in genetically heterogeneous aging mice, testing if old mice benefit from rapamycin. FINDINGS: We find that genetically diverse aging mice are much more susceptible than young mice to M. tuberculosis, as are aging human beings. We also find that rapamycin boosts immune responses during primary infection but fails to increase survival. CONCLUSIONS: Genetically diverse mouse models provide a valuable resource to study how age influences responses and susceptibility to pathogens and to test interventions. Additionally, surrogate markers such as immune measures may not predict whether interventions improve survival.

An Aging Interventions Testing Program: study design and interim report.

The National Institute on Aging's Interventions Testing Program (ITP) has developed a plan to evaluate agents that are considered plausible candidates for delaying rates of aging. Key features include: (i) use of genetically heterogeneous mice (a standardized four-way cross), (ii) replication at three test sites (the Jackson Laboratory, TJL; University of Michigan, UM; and University of Texas, UT), (iii) sufficient statistical power to detect 10% changes in lifespan, (iv) tests for age-dependent changes in T cell subsets and physical activity, and (v) an annual solicitation for collaborators who wish to suggest new interventions for evaluation. Mice in the first cohort were exposed to one of four agents: aspirin, nitroflurbiprofen (NFP), 4-OH-alpha-phenyl-N-tert-butyl nitrone (4-OH-PBN), or nordihydroguiaretic acid (NDGA). An interim analysis was conducted using survival data available on the date at which at least 50% of the male control mice had died at each test site. Survival of control males was significantly higher, at the interim time-point, at UM than at UT or TJL; all three sites had similar survival of control females. Males in the NDGA group had significantly improved survival (P = 0.0004), with significant effects noted at TJL (P < 0.01) and UT (P < 0.04). None of the other agents altered survival, although there was a suggestion (P = 0.07) of a beneficial effect of aspirin in males. More data will be needed to determine if any of these compounds can extend maximal lifespan, but the current data show that NDGA reduces early life mortality risks in genetically heterogeneous mice at multiple test sites.

Heterozygous kit mutants with little or no apparent anemia exhibit large defects in overall hematopoietic stem cell function.

OBJECTIVE: The evolutionarily conserved Kit receptor is vital for function of hematopoietic stem cells (HSC). Kit(W-41) (W-41) and Kit(W-42) (W-42) are single residue changes in the KIT intracellular phosphotransferase domain, while Kit(W-v) (W-v) is a single residue change in the ATP binding domain. This study tests how each mutation affects HSC function. METHODS: Cells in mutant and C57BL/6J(+/+) blood and marrow were compared. Overall HSC function was measured by competitive repopulation. Functions of specific progenitor populations were tested with stage-specific competitive repopulation and standard colony-forming unit assays. RESULTS: Bone marrow cells from these Kit mutants are severely defective at reconstituting peripheral blood lineages and bone marrow of irradiated recipients, when compared to +/+ control marrow. These defects increased with time. Marrow from W-41/+ and W-v/+ functions similarly but better than marrow from W-41/W-41 and W-42/+, to repopulate the erythroid and lymphoid lineages. Long-term (LT) and short-term (ST) HSC from W-v/+, W-41/W-41, and W-42/+ are more defective at reconstituting bone marrow than LT- and ST-HSC from W-41/+ and +/+. Common myeloid progenitor (CMP) cells from W-42/+ and W-41/W-41 are more defective at producing spleen colonies than CMP from W-v/+ and W-41/+. CONCLUSION: Heterozygous Kit mutants with little or no apparent anemia exhibit surprisingly large defects in overall HSC function. Multiplying the fractional defects in LT-HSC, ST-HSC, and CMP can account for overall effects of W-v/+, but does not completely account for the defects observed with W-41/+, W-42/+, and W-41/W-41.

Reduced in vivo hepatic proteome replacement rates but not cell proliferation rates predict maximum lifespan extension in mice.

Combating the social and economic consequences of a growing elderly population will require the identification of interventions that slow the development of age-related diseases. Preserved cellular homeostasis and delayed aging have been previously linked to reduced cell proliferation and protein synthesis rates. To determine whether changes in these processes may contribute to or predict delayed aging in mammals, we measured cell proliferation rates and the synthesis and replacement rates (RRs) of over a hundred hepatic proteins in vivo in three different mouse models of extended maximum lifespan (maxLS): Snell Dwarf, calorie-restricted (CR), and rapamycin (Rapa)-treated mice. Cell proliferation rates were not consistently reduced across the models. In contrast, reduced hepatic protein RRs (longer half-lives) were observed in all three models compared to controls. Intriguingly, the degree of mean hepatic protein RR reduction was significantly correlated with the degree of maxLS extension across the models and across different Rapa doses. Absolute rates of hepatic protein synthesis were reduced in Snell Dwarf and CR, but not Rapa-treated mice. Hepatic chaperone levels were unchanged or reduced and glutathione S-transferase synthesis was preserved or increased in all three models, suggesting a reduced demand for protein renewal, possibly due to reduced levels of unfolded or damaged proteins. These data demonstrate that maxLS extension in mammals is associated with improved hepatic proteome homeostasis, as reflected by a reduced demand for protein renewal, and that reduced hepatic protein RRs hold promise as an early biomarker and potential target for interventions that delay aging in mammals.

Longer lifespan in male mice treated with a weakly estrogenic agonist, an antioxidant, an α-glucosidase inhibitor or a Nrf2-inducer.

The National Institute on Aging Interventions Testing Program (ITP) evaluates agents hypothesized to increase healthy lifespan in genetically heterogeneous mice. Each compound is tested in parallel at three sites, and all results are published. We report the effects of lifelong treatment of mice with four agents not previously tested: Protandim, fish oil, ursodeoxycholic acid (UDCA) and metformin - the latter with and without rapamycin, and two drugs previously examined: 17-α-estradiol and nordihydroguaiaretic acid (NDGA), at doses greater and less than used previously. 17-α-estradiol at a threefold higher dose robustly extended both median and maximal lifespan, but still only in males. The male-specific extension of median lifespan by NDGA was replicated at the original dose, and using doses threefold lower and higher. The effects of NDGA were dose dependent and male specific but without an effect on maximal lifespan. Protandim, a mixture of botanical extracts that activate Nrf2, extended median lifespan in males only. Metformin alone, at a dose of 0.1% in the diet, did not significantly extend lifespan. Metformin (0.1%) combined with rapamycin (14 ppm) robustly extended lifespan, suggestive of an added benefit, based on historical comparison with earlier studies of rapamycin given alone. The α-glucosidase inhibitor, acarbose, at a concentration previously tested (1000 ppm), significantly increased median longevity in males and 90th percentile lifespan in both sexes, even when treatment was started at 16 months. Neither fish oil nor UDCA extended lifespan. These results underscore the reproducibility of ITP longevity studies and illustrate the importance of identifying optimal doses in lifespan studies.

Adipose tissue transplantation protects ob/ob mice from obesity, normalizes insulin sensitivity and restores fertility.

Adipose tissue affects metabolism by secreting various adipokines. Lipodystropic mice benefit both from leptin replacement therapy and from transplantation of normal fat. Leptin-deficient Lep(ob)/Lep(ob) (ob/ob) mice can also be treated with leptin. Surprisingly, there have been no reports of successful treatment of obese ob/ob mice by transplantation of normal white adipose tissue (WAT). If WAT transplantation is ineffective in treating insulin resistance and diabetes in obese individuals, its applicability may be limited in humans as such abnormalities are usually associated with obesity. In the current study, we tested whether WAT transplantation might prevent, and even reverse, abnormalities characteristic of ob/ob mice. To assess the preventive potential, 6-week-old ob/ob mice were transplanted, subcutaneously, with gonadal fat pads from normal mice. Profound effects on multiple physiological phenotypes were achieved despite leptin levels below 25% of those in control mice. WAT from one donor reduced body weight gain, and WAT from 4 or 8 donors prevented obesity in ob/ob mice. Nonfasting insulin levels and insulin tolerance test were normalized. Corticosterone elevation was also prevented. Finally, WAT from 4 donors restored fertility in ob/ob females. The effects of WAT transplantation were long-lasting, with body weight gain suppressed for at least 40 weeks. To assess the therapeutic potential, obese 13-month-old ob/ob mice with a long history of leptin deficiency were used. Their body weight decreased by approximately 50% when transplanted with WAT from 8 donors. As in young recipients, transplantation greatly reduced nonfasting insulin, suggesting normalized insulin sensitivity. Thus, WAT transplantation was effective for both prevention and therapy. In the future, WAT transplantation may become a useful alternative to hormone replacement in treating not only lipodystropy, but also certain types of obesity.

Hematopoietic senescence is postponed and hematopoietic stem cell function is enhanced by dietary restriction.

OBJECTIVE: The aim of this study was to test dietary restriction (DR) as an intervention to alleviate senescence-associated functional defects in hematopoietic stem cells (HSCs). MATERIALS AND METHODS: BALB/cByJ (BALB) mice were fed ad libitum (AL) or were diet restricted (DR) to 75% of the AL food intake after 1 month of age. Peripheral blood and bone marrow cell compositions were compared in 3- and 25-month-old AL (AL-3, AL-25) mice and in 25-month-old DR (DR-25) mice using fluorescence-activated cell staining. Relative HSC functions in vivo were compared using competitive repopulation, and were also tested in 6-month-old BALB mice to measure the effects of short-term DR. RESULTS: Compared to AL-3, AL-25 blood had significantly lower levels of red blood cells and hemoglobin. AL-25 marrow contained less than half the concentration of Lin(-)CD34(-)Sca1(+)CD117(+) HSCs and showed only half the in vivo functional ability of AL-3 marrow. In vivo, AL-25 HSCs failed to produce the strong correlations over time that demonstrate clonal stability during competitive repopulation. These correlations were shown in AL-3 HSCs. DR for 24 months alleviated hematopoietic deficiencies in the blood, increased concentrations of bone marrow Lin(-)CD34(-)Sca1(+)CD117(+) HSCs and improved HSC functional abilities in DR-25 mice to values far greater than those in normally aged mice. Surprisingly, HSC function in 25-month-old DR mice was better than that in young adults. Degrees of recipient repopulation by HSCs from DR-25 mice also correlated well over time, demonstrating clonal stability. Short-term DR for 5 months also improved HSC function, but to a much smaller degree. CONCLUSIONS: Aged BALB mice show hematopoietic and HSC senescence and clonal succession. Lifelong DR slows hematopoietic senescence and prevents HSC aging.

Histone modifications change with age, dietary restriction and rapamycin treatment in mouse brain.

The risk of developing neurodegenerative disorders such as Alzheimer's disease (AD) increases dramatically with age. Understanding the underlying mechanisms of brain aging is crucial for developing preventative and/or therapeutic approaches for age-associated neurological diseases. Recently, it has been suggested that epigenetic factors, such as histone modifications, maybe be involved in brain aging and age-related neurodegenerations. In this study, we investigated 14 histone modifications in brains of a cohort of young (3 months), old (22 months), and old age-matched dietary restricted (DR) and rapamycin treated BALB/c mice. Results showed that 7 out of all measured histone markers were changed drastically with age. Intriguingly, histone methylations in brain tissues, including H3K27me3, H3R2me2, H3K79me3 and H4K20me2 tend to disappear with age but can be partially restored by both DR and rapamycin treatment. However, both DR and rapamycin treatment also have a significant impact on several other histone modifications such as H3K27ac, H4K16ac, H4R3me2, and H3K56ac, which do not change as animal ages. This study provides the first evidence that a broad spectrum of histone modifications may be involved in brain aging. Besides, this study suggests that both DR and rapamycin may slow aging process in mouse brain via these underlying epigenetic mechanisms.

Rapamycin-mediated lifespan increase in mice is dose and sex dependent and metabolically distinct from dietary restriction.

Rapamycin, an inhibitor of mTOR kinase, increased median lifespan of genetically heterogeneous mice by 23% (males) to 26% (females) when tested at a dose threefold higher than that used in our previous studies; maximal longevity was also increased in both sexes. Rapamycin increased lifespan more in females than in males at each dose evaluated, perhaps reflecting sexual dimorphism in blood levels of this drug. Some of the endocrine and metabolic changes seen in diet-restricted mice are not seen in mice exposed to rapamycin, and the pattern of expression of hepatic genes involved in xenobiotic metabolism is also quite distinct in rapamycin-treated and diet-restricted mice, suggesting that these two interventions for extending mouse lifespan differ in many respects.

Acarbose, 17-α-estradiol, and nordihydroguaiaretic acid extend mouse lifespan preferentially in males.

Four agents--acarbose (ACA), 17-α-estradiol (EST), nordihydroguaiaretic acid (NDGA), and methylene blue (MB)--were evaluated for lifespan effects in genetically heterogeneous mice tested at three sites. Acarbose increased male median lifespan by 22% (P < 0.0001), but increased female median lifespan by only 5% (P = 0.01). This sexual dimorphism in ACA lifespan effect could not be explained by differences in effects on weight. Maximum lifespan (90th percentile) increased 11% (P < 0.001) in males and 9% (P = 0.001) in females. EST increased male median lifespan by 12% (P = 0.002), but did not lead to a significant effect on maximum lifespan. The benefits of EST were much stronger at one test site than at the other two and were not explained by effects on body weight. EST did not alter female lifespan. NDGA increased male median lifespan by 8-10% at three different doses, with P-values ranging from 0.04 to 0.005. Females did not show a lifespan benefit from NDGA, even at a dose that produced blood levels similar to those in males, which did show a strong lifespan benefit. MB did not alter median lifespan of males or females, but did produce a small, statistically significant (6%, P = 0.004) increase in female maximum lifespan. These results provide new pharmacological models for exploring processes that regulate the timing of aging and late-life diseases, and in particular for testing hypotheses about sexual dimorphism in aging and health.

Young and old genetically heterogeneous HET3 mice on a rapamycin diet are glucose intolerant but insulin sensitive.

Rapamycin, an inhibitor of the mechanistic target of rapamycin (mTOR) signaling pathway, extends the life span of yeast, worms, flies, and mice. Interventions that promote longevity are often correlated with increased insulin sensitivity, and it therefore is surprising that chronic rapamycin treatment of mice, rats, and humans is associated with insulin resistance (J Am Soc Nephrol., 19, 2008, 1411; Diabetes, 00, 2010, 00; Science, 335, 2012, 1638). We examined the effect of dietary rapamycin treatment on glucose homeostasis and insulin resistance in the genetically heterogeneous HET3 mouse strain, a strain in which dietary rapamycin robustly extends mean and maximum life span. We find that rapamycin treatment leads to glucose intolerance in both young and old HET3 mice, but in contrast to the previously reported effect of injected rapamycin in C57BL/6 mice, HET3 mice treated with dietary rapamycin responded normally in an insulin tolerance test. To gauge the overall consequences of rapamycin treatment on average blood glucose levels, we measured HBA1c. Dietary rapamycin increased HBA1c over the first 3 weeks of treatment in young animals, but the effect was lost by 3 months, and no effect was detected in older animals. Our results demonstrate that the extended life span of HET3 mice on a rapamycin diet occurs in the absence of major changes in insulin sensitivity and highlight the importance of strain background and delivery method in testing effects of longevity interventions.

Evaluation of resveratrol, green tea extract, curcumin, oxaloacetic acid, and medium-chain triglyceride oil on life span of genetically heterogeneous mice.

The National Institute on Aging Interventions Testing Program (ITP) was established to evaluate agents that are hypothesized to increase life span and/or health span in genetically heterogeneous mice. Each compound is tested in parallel at three test sites. It is the goal of the ITP to publish all results, negative or positive. We report here on the results of lifelong treatment of mice, beginning at 4 months of age, with each of five agents, that is, green tea extract (GTE), curcumin, oxaloacetic acid, medium-chain triglyceride oil, and resveratrol, on the life span of genetically heterogeneous mice. Each agent was administered beginning at 4 months of age. None of these five agents had a statistically significant effect on life span of male or female mice, by log-rank test, at the concentrations tested, although a secondary analysis suggested that GTE might diminish the risk of midlife deaths in females only.

Aging Kit mutant mice develop cardiomyopathy.

Both bone marrow (BM) and myocardium contain progenitor cells expressing the c-Kit tyrosine kinase. The aims of this study were to determine the effects of c-Kit mutations on: i. myocardial c-Kit(+) cells counts and ii. the stability of left ventricular (LV) contractile function and structure during aging. LV structure and contractile function were evaluated (echocardiography) in two groups of Kit mutant (W/Wv and W41/W42) and in wild type (WT) mice at 4 and 12 months of age and the effects of the mutations on LV mass, vascular density and the numbers of proliferating cells were also determined. In 4 month old Kit mutant and WT mice, LV ejection fractions (EF) and LV fractional shortening rates (FS) were comparable. At 12 months of age EF and FS were significantly decreased and LV mass was significantly increased only in W41/W42 mice. Myocardial vascular densities and c-Kit(+) cell numbers were significantly reduced in both mutant groups when compared to WT hearts. Replacement of mutant BM with WT BM at 4 months of age did not prevent these abnormalities in either mutant group although they were somewhat attenuated in the W/Wv group. Notably BM transplantation did not prevent the development of cardiomyopathy in 12 month W41/W42 mice. The data suggest that decreased numbers and functional capacities of c-Kit(+) cardiac resident progenitor cells may be the basis of the cardiomyopathy in W41/W42 mice and although defects in mutant BM progenitor cells may prove to be contributory, they are not causal.

Life extension by diet restriction and N-acetyl-L-cysteine in genetically heterogeneous mice.

We used a heterogeneous stock of mice-UM-HET3, the first generation offspring of CByB6F1/J and C3D2F1/J parents-to test effects of six antiaging treatments on life span. In the first report of diet restriction in a structured, segregating heterogeneous population, we observed essentially the same increases in mean and maximum life span as found in CByB6F1/J hybrid positive controls. We also report results of treatment with N-acetyl-L-cysteine started at 7 months, and aspirin, nitroflurbiprofen, 4-hydroxy phenyl N-tert-butyl nitrone, and nordihydroguaiaretic acid, all started at 16-18 months. Only male UM-HET3 mice receiving N-acetyl-L-cysteine had significantly increased life span, and this may have been due to treatment-related inadvertent diet restriction. The other agents had no significant effects on life span. The use of UM-HET3 mice helps assure that these results are not the result of unresponsiveness of a single genotype but that they more broadly represent laboratory mice.

Regulation of selenoproteins and methionine sulfoxide reductases A and B1 by age, calorie restriction, and dietary selenium in mice.

Methionine residues are susceptible to oxidation, but this damage may be reversed by methionine sulfoxide reductases MsrA and MsrB. Mammals contain one MsrA and three MsrBs, including a selenoprotein MsrB1. Here, we show that MsrB1 is the major methionine sulfoxide reductase in liver of mice and it is among the proteins that are most easily regulated by dietary selenium. MsrB1, but not MsrA activities, were reduced with age, and the selenium regulation of MsrB1 was preserved in the aging liver, suggesting that MsrB1 could account for the impaired methionine sulfoxide reduction in aging animals. We also examined regulation of Msr and selenoprotein expression by a combination of dietary selenium and calorie restriction and found that, under calorie restriction conditions, selenium regulation was preserved. In addition, mice overexpressing a mutant form of selenocysteine tRNA reduced MsrB1 activity to the level observed in selenium deficiency, whereas MsrA activity was elevated in these animals. Finally, we show that selenium regulation in inbred mouse strains is preserved in an outbred aging model. Taken together, these findings better define dietary regulation of methionine sulfoxide reduction and selenoprotein expression in mice with regard to age, calorie restriction, dietary Se, and a combination of these factors.

Effects of dietary restriction on hematopoietic stem-cell aging are genetically regulated.

Diminished stem-cell functions with age may be a major cause of anemias and other defects. Unfortunately, treatments that increase stem-cell function can also increase the incidence of cancers. Lifelong dietary restriction (DR) is known to decrease spontaneous cancers and lengthen lifespan. This study examines the effect of DR on the ability of bone marrow cells to repopulate irradiated recipients and produce erythrocytes and lymphocytes. In BALB/cByJ (BALB) mice, repopulating abilities decline with age; DR ameliorates this trend. In C57BL/6J (B6) and (BALB x B6) F1 hybrid (F1) mice, repopulating abilities increase with age; DR maintains this increase. Hematopoietic stem cell (HSC) numbers are highly variable in aged BALB mice; however, the observed loss of marrow function results from a major loss in repopulating ability per HSC. DR greatly ameliorates this loss of function with age. In contrast, function per HSC in B6 mice is affected neither by age nor by DR. Thus, DR increases or maintains increased marrow repopulating ability with age in the 3 different genotypes tested, but effects on function per HSC depend on genotype. That DR increases or maintains stem-cell function with age, while decreasing cancer, has far-reaching health implications.

Nordihydroguaiaretic acid and aspirin increase lifespan of genetically heterogeneous male mice.

The National Institute on Aging's Interventions Testing Program was established to evaluate agents that are purported to increase lifespan and delay the appearance of age-related disease in genetically heterogeneous mice. Up to five compounds are added to the study each year and each compound is tested at three test sites (The Jackson Laboratory, University of Michigan, and University of Texas Health Science Center at San Antonio). Mice in the first cohort were exposed to one of four agents: aspirin, nitroflurbiprofen, 4-OH-alpha-phenyl-N-tert-butyl nitrone, or nordihydroguaiaretic acid (NDGA). Sample size was sufficient to detect a 10% difference in lifespan in either sex,with 80% power, using data from two of the three sites. Pooling data from all three sites, a log-rank test showed that both NDGA (p=0.0006) and aspirin (p=0.01) led to increased lifespan of male mice. Comparison of the proportion of live mice at the age of 90% mortality was used as a surrogate for measurement of maximum lifespan;neither NDGA (p=0.12) nor aspirin (p=0.16) had a significant effect in this test. Measures of blood levels of NDGA or aspirin and its salicylic acid metabolite suggest that the observed lack of effects of NDGA or aspirin on life span in females could be related to gender differences in drug disposition or metabolism. Further studies are warranted to find whether NDGA or aspirin, over a range of doses,might prove to postpone death and various age-related outcomes reproducibly in mice.