Canagliflozin (Cana) is an FDA-approved diabetes drug that protects against cardiovascular and kidney diseases. It also inhibits the sodium glucose transporter 2 by blocking renal reuptake and intestinal absorption of glucose. In the context of the mouse Interventions Testing Program, genetically heterogeneous mice were given chow containing Cana at 180 ppm at 7 months of age until their death. Cana extended median survival of male mice by 14%. Cana also increased by 9% the age for 90th percentile survival, with parallel effects seen at each of 3 test sites. Neither the distribution of inferred cause of death nor incidental pathology findings at end-of-life necropsies were altered by Cana. Moreover, although no life span benefits were seen in female mice, Cana led to lower fasting glucose and improved glucose tolerance in both sexes, diminishing fat mass in females only. Therefore, the life span benefit of Cana is likely to reflect blunting of peak glucose levels, because similar longevity effects are seen in male mice given acarbose, a diabetes drug that blocks glucose surges through a distinct mechanism, i.e., slowing breakdown of carbohydrate in the intestine. Interventions that control daily peak glucose levels deserve attention as possible preventive medicines to protect from a wide range of late-life neoplastic and degenerative diseases.

To see if variations in timing of rapamycin (Rapa), administered to middle aged mice starting at 20 months, would lead to different survival outcomes, we compared three dosing regimens. Initiation of Rapa at 42 ppm increased survival significantly in both male and female mice. Exposure to Rapa for a 3-month period led to significant longevity benefit in males only. Protocols in which each month of Rapa treatment was followed by a month without Rapa exposure were also effective in both sexes, though this approach was less effective than continuous exposure in female mice. Interpretation of these results is made more complicated by unanticipated variation in patterns of weight gain, prior to the initiation of the Rapa treatment, presumably due to the use of drug-free food from two different suppliers. The experimental design included tests of four other drugs, minocycline, β-guanidinopropionic acid, MitoQ, and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), but none of these led to a change in survival in either sex.

Several previous lines of research have suggested, indirectly, that mouse lifespan is particularly susceptible to endocrine or nutritional signals in the first few weeks of life, as tested by manipulations of litter size, growth hormone levels, or mutations with effects specifically on early-life growth rate. The pace of early development in mice can also be influenced by exposure of nursing and weanling mice to olfactory cues. In particular, odors of same-sex adult mice can in some circumstances delay maturation. We hypothesized that olfactory information might also have a sex-specific effect on lifespan, and we show here that the lifespan of female mice can be increased significantly by odors from adult females administered transiently, that is from 3 days until 60 days of age. Female lifespan was not modified by male odors, nor was male lifespan susceptible to odors from adults of either sex. Conditional deletion of the G protein Gαo in the olfactory system, which leads to impaired accessory olfactory system function and blunted reproductive priming responses to male odors in females, did not modify the effect of female odors on female lifespan. Our data provide support for the idea that very young mice are susceptible to influences that can have long-lasting effects on health maintenance in later life, and provide a potential example of lifespan extension by olfactory cues in mice.

Longevity in mammals is influenced by sex, and lifespan extension in response to anti-aging interventions is often sex-specific, although the mechanisms underlying these sexual dimorphisms are largely unknown. Treatment of mice with 17-α estradiol (17aE2) results in sex-specific lifespan extension, with an increase in median survival in males of 19% and no survival effect in females. Given the links between lifespan extension and metabolism, we performed untargeted metabolomics analysis of liver, skeletal muscle and plasma from male and female mice treated with 17aE2 for eight months. We find that 17aE2 generates distinct sex-specific changes in the metabolomic profile of liver and plasma. In males, 17aE2 treatment raised the abundance of several amino acids in the liver, and this was further associated with elevations in metabolites involved in urea cycling, suggesting altered amino acid metabolism. In females, amino acids and urea cycling metabolites were unaffected by 17aE2. 17aE2 also results in male-specific elevations in a second estrogenic steroid-estriol-3-sulfate-suggesting different metabolism of this drug in males and females. To understand the underlying endocrine causes for these sexual dimorphisms, we castrated males and ovariectomized females prior to 17aE2 treatment, and found that virtually all the male-specific metabolite responses to 17aE2 are inhibited or reduced by male castration. These results suggest novel metabolic pathways linked to male-specific lifespan extension and show that the male-specific metabolomic response to 17aE2 depends on the production of testicular hormones in adult life.

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.

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.

Interventions 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.

The female survival advantage is a robust characteristic of human longevity. However, underlying mechanisms are not understood, and rodent models exhibiting a female advantage are lacking. Here, we report that the genetically heterogeneous (UM-HET3) mice used by the National Institute on Aging Interventions Testing Program (ITP) are such a model. Analysis of age-specific survival of 3,690 control ITP mice revealed a female survival advantage paralleling that of humans. As in humans, the female advantage in mice was greatest in early adulthood, peaking around 350 days of age and diminishing progressively thereafter. This persistent finding was observed at three geographically distinct sites and in six separate cohorts over a 10-year period. Because males weigh more than females and bodyweight is often inversely related to lifespan, we examined sex differences in the relationship between bodyweight and survival. Although present in both sexes, the inverse relationship between bodyweight and longevity was much stronger in males, indicating that male mortality is more influenced by bodyweight than is female mortality. In addition, male survival varied more across site and cohort than female survival, suggesting greater resistance of females to environmental modulators of survival. Notably, at 24 months the relationship between bodyweight and longevity shifted from negative to positive in both sexes, similar to the human condition in advanced age. These results indicate that the UM-HET3 mouse models the human female survival advantage and provide evidence for greater resilience of females to modulators of survival.

Chaperone-mediated autophagy (CMA) is the most selective form of lysosomal proteolysis, where individual peptides, recognized by a consensus motif, are translocated directly across the lysosomal membrane. CMA regulates the abundance of many disease-related proteins, with causative roles in neoplasia, neurodegeneration, hepatosteatosis, and other pathologies relevant to human health and aging. At the lysosomal membrane, CMA is inhibited by Akt-dependent phosphorylation of the CMA regulator GFAP. The INS-PI3K-PDPK1 pathway regulates Akt, but its role in CMA is unclear. Here, we report that inhibition of class I PI3K or PDPK1 activates CMA. In contrast, selective inhibition of class III PI3Ks does not activate CMA. Isolated liver lysosomes from mice treated with either of two orally bioavailable class I PI3K inhibitors, pictilisib or buparlisib, display elevated CMA activity, and decreased phosphorylation of lysosomal GFAP, with no change in macroautophagy. The findings of this study represent an important first step in repurposing class I PI3K inhibitors to modulate CMA in vivo.

PTEN is a crucial negative regulator of the INS/PI3K/AKT pathway and is one of the most commonly mutated tumor suppressors in cancer. Global overexpression (OE) of PTEN in mice shifts metabolism to favor oxidative phosphorylation over glycolysis, reduces fat mass, and extends the lifespan of both sexes. We demonstrate that PTEN regulates chaperone-mediated autophagy (CMA). Using cultured cells and mouse models, we show that PTEN OE enhances CMA, dependent upon PTEN's lipid phosphatase activity and AKT inactivation. Reciprocally, PTEN knockdown reduces CMA, which can be rescued by inhibiting class I PI3K or AKT. Both PTEN and CMA are negative regulators of glycolysis and lipid droplet formation. We show that suppression of glycolysis and lipid droplet formation downstream of PTEN OE depends on CMA activity. Finally, we show that PTEN protein levels are sensitive to CMA and that PTEN accumulates in lysosomes with elevated CMA. Collectively, these data suggest that CMA is both an effector and a regulator of PTEN.

Canagliflozin (Cana), a clinically important anti-diabetes drug, leads to a 14% increase in median lifespan and a 9% increase in the 90th percentile age when given to genetically heterogeneous male mice from 7 months of age, but does not increase lifespan in female mice. A histopathological study was conducted on 22-month-old mice to see if Cana retarded diverse forms of age-dependent pathology. This agent was found to diminish incidence or severity, in male mice only, of cardiomyopathy, glomerulonephropathy, arteriosclerosis, hepatic microvesicular cytoplasmic vacuolation (lipidosis), and adrenal cortical neoplasms. Protection against atrophy of the exocrine pancreas was seen in both males and females. Thus, the extension of lifespan in Cana-treated male mice, which is likely to reflect host- or tumor-mediated delay in lethal neoplasms, is accompanied by parallel retardation of lesions, in multiple tissues, that seldom if ever lead to death in these mice. Canagliflozin thus can be considered a drug that acts to slow the aging process and should be evaluated for potential protective effects against many other late-life conditions.

Diets low in methionine extend lifespan of rodents, though through unknown mechanisms. Glycine can mitigate methionine toxicity, and a small prior study has suggested that supplemental glycine could extend lifespan of Fischer 344 rats. We therefore evaluated the effects of an 8% glycine diet on lifespan and pathology of genetically heterogeneous mice in the context of the Interventions Testing Program. Elevated glycine led to a small (4%-6%) but statistically significant lifespan increase, as well as an increase in maximum lifespan, in both males (p = 0.002) and females (p < 0.001). Pooling across sex, glycine increased lifespan at each of the three independent sites, with significance at p = 0.01, 0.053, and 0.03, respectively. Glycine-supplemented females were lighter than controls, but there was no effect on weight in males. End-of-life necropsies suggested that glycine-treated mice were less likely than controls to die of pulmonary adenocarcinoma (p = 0.03). Of the 40 varieties of incidental pathology evaluated in these mice, none were increased to a significant degree by the glycine-supplemented diet. In parallel analyses of the same cohort, we found no benefits from TM5441 (an inhibitor of PAI-1, the primary inhibitor of tissue and urokinase plasminogen activators), inulin (a source of soluble fiber), or aspirin at either of two doses. Our glycine results strengthen the idea that modulation of dietary amino acid levels can increase healthy lifespan in mice, and provide a foundation for further investigation of dietary effects on aging and late-life diseases.

Chaperone-mediated autophagy (CMA) selectively degrades proteins that are crucial for glycolysis, fatty acid metabolism, and the progression of several age-associated diseases. Several previous studies, each of which evaluated males of a single inbred mouse or rat strain, have reported that CMA declines with age in many tissues, attributed to an age-related loss of LAMP2A, the primary and indispensable component of the CMA translocation complex. This has led to a paradigm in the field of CMA research, stating that the age-associated decline in LAMP2A in turn decreases CMA, contributing to the pathogenesis of late-life disease. We assessed LAMP2A levels and CMA substrate uptake in both sexes of the genetically heterogeneous UM-HET3 mouse stock, which is the current global standard for the evaluation of anti-aging interventions. We found no evidence for age-related changes in LAMP2A levels, CMA substrate uptake, or whole liver levels of CMA degradation targets, despite identifying sex differences in CMA.

Rapamycin inhibits mechanistic (or mammalian) target of rapamycin (mTOR) that promotes protein production in cells by facilitating ribosome biogenesis (RiBi) and eIF4E-mediated 5'cap mRNA translation. Chronic treatment with encapsulated rapamycin (eRapa) extended health and life span for wild-type and cancer-prone mice. Yet, the long-term consequences of chronic eRapa treatment are not known at the organ level. Here, we report our observations of chronic eRapa treatment on mTORC1 signaling and RiBi in mouse colon and visceral adipose. As expected, chronic eRapa treatment decreased detection of phosphorylated mTORC1/S6K substrate, ribosomal protein (rpS6) in colon and fat. However, in colon, contrary to expectations, there was an upregulation of 18S rRNA and some ribosomal protein genes (RPGs) suggesting increased RiBi. Among RPGs, eRapa increases rpl22l1 mRNA but not its paralog rpl22. Furthermore, there was an increase in the cap-binding protein, eIF4E relative to its repressor 4E-BP1 suggesting increased translation. By comparison, in fat, there was a decrease in the level of 18S rRNA (opposite to colon), while overall mRNAs encoding ribosomal protein genes appeared to increase, including rpl22, but not rpl22l1 (opposite to colon). In fat, there was a decrease in eIF4E relative to actin (opposite to colon) but also an increase in the eIF4E/4E-BP1 ratio likely due to reductions in 4E-BP1 at our lower eRapa dose (similar to colon). Thus, in contrast to predictions of decreased protein production seen in cell-based studies, we provide evidence that colon from chronically treated mice exhibited an adaptive 'pseudo-anabolic' state, which is only partially present in fat, which might relate to differing tissue levels of rapamycin, cell-type-specific responses, and/or strain differences.

The drug acarbose is used to treat diabetes and, by inhibiting α-amylase in the small intestine, increases the amount of starch entering the lower digestive tract. This results in changes to the composition of the microbiota and their fermentation products. Acarbose also increases longevity in mice, an effect that has been correlated with increased production of the short-chain fatty acids propionate and butyrate. In experiments replicated across three study sites, two distantly related species in the bacterial family Muribaculaceae were dramatically more abundant in acarbose-treated mice, distinguishing these responders from other members of the family. Bacteria in the family Muribaculaceae are predicted to produce propionate as a fermentation end product and are abundant and diverse in the guts of mice, although few isolates are available. We reconstructed genomes from metagenomes (MAGs) for nine populations of Muribaculaceae to examine factors that distinguish species that respond positively to acarbose. We found two closely related MAGs (B1A and B1B) from one responsive species that both contain a polysaccharide utilization locus with a predicted extracellular α-amylase. These genomes also shared a periplasmic neopullulanase with another, distantly related MAG (B2) representative of the only other responsive species. This gene differentiated these three MAGs from MAGs representative of nonresponding species. Differential gene content in B1A and B1B may be associated with the inconsistent response of this species to acarbose across study sites. This work demonstrates the utility of culture-free genomics for inferring the ecological roles of gut bacteria, including their response to pharmaceutical perturbations. IMPORTANCE The drug acarbose is used to treat diabetes by preventing the breakdown of starch in the small intestine, resulting in dramatic changes in the abundance of some members of the gut microbiome and its fermentation products. In mice, several of the bacteria that respond most positively are classified in the family Muribaculaceae, members of which produce propionate as a primary fermentation product. Propionate has been associated with gut health and increased longevity in mice. We found that genomes of the most responsive Muribaculaceae showed signs of specialization for starch fermentation, presumably providing them a competitive advantage in the large intestine of animals consuming acarbose. Comparisons among genomes enhance existing models for the ecological niches occupied by members of this family. In addition, genes encoding one type of enzyme known to participate in starch breakdown were found in all three genomes from responding species but none of the other genomes.

Studies of the mTOR pathway have prompted speculation that diminished mTOR complex-1 (mTORC1) function may be involved in controlling the aging process. Our previous studies have shown diminished mTORC1 activity in tissues of three long-lived mutant mice: Snell dwarf mice, growth hormone receptor gene disrupted mice (GHRKO), and in this article, mice deficient in the pregnancy-associated protein-A (PAPPA-KO). The ways in which lower mTOR signals slow aging and age-related diseases are, however, not well characterized. Here, we show that Snell, GHKRO, and PAPPA-KO mice express high levels of two proteins involved in DNA repair, O-6-methylguanine-DNA methyltransferase (MGMT) and N-myc downstream-regulated gene 1 (NDRG1). Furthermore, we report that lowering mTOR enhances MGMT and NDRG1 protein expression via post-transcriptional mechanisms. We show that the CCR4-NOT complex, a post-transcriptional regulator of gene expression, is downstream of the mTORC1 pathway and may be responsible for the upregulation of MGMT and NDRG1 in all three varieties of long-lived mice. Our data thus suggest a novel link between DNA repair and mTOR signaling via post-transcriptional regulation involving specific alteration in the CCR4-NOT complex, whose modulation could control multiple aspects of the aging process.

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.