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.     

Anti‐aging drugs reduce hypothalamic inflammation in a sex‐specific manner

Aging leads to hypothalamic inflammation, but does so more slowly in mice whose lifespan has been extended by mutations that affect GH/IGF‐1 signals. Early‐life exposure to GH by injection, or to nutrient restriction in the first 3 weeks of life, also modulate both lifespan and the pace of hypothalamic inflammation. Three drugs extend lifespan of UM‐HET3 mice in a sex‐specific way: acarbose (ACA), 17‐α‐estradiol (17αE2), and nordihydroguaiaretic acid (NDGA), with more dramatic longevity increases in males in each case. In this study, we examined the effect of these anti‐aging drugs on neuro‐inflammation in hypothalamus and hippocampus. We found that age‐associated hypothalamic inflammation is reduced in males but not in females at 12 months of age by ACA and 17αE2 and at 22 months of age in NDGA‐treated mice. The three drugs blocked indices of hypothalamic reactive gliosis associated with aging, such as Iba‐1‐positive microglia and GFAP‐positive astrocytes, as well as age‐associated overproduction of TNF‐α. This effect was not observed in drug‐treated female mice or in the hippocampus of the drug‐treated animals. On the other hand, caloric restriction (CR; an intervention that extends the lifespan in both sexes) significantly reduced hypothalamic microglia and TNF‐α in both sexes at 12 months of age. Together, these results suggest that the extent of drug‐induced changes in hypothalamic inflammatory processes is sexually dimorphic in a pattern that parallels the effects of these agents on mouse longevity and that mimics the changes seen, in both sexes, of long‐lived nutrient restricted or mutant mice.     

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.     

lac‐1 and lag‐1 with ras‐1 affect aging and the biological clock in Neurospora crassa

Using an automated cell counting technique developed previously (Case et al., Ecology and Evolution 2014; 4: 3494), we explore the lifespan effects of lac‐1, a ceramide synthase gene paralogous to lag‐1 in Neurospora crassa in conjunction with the band bd (ras‐1) gene. We find that the replicative lifespan of a lac‐1^KO bd double mutants is short, about one race tube cycle, and this double mutant lacks a strong ~21‐hr clock cycle as shown by race tube and fluorometer analysis of fluorescent strains including lac‐1^KO. This short replicative lifespan phenotype is contrasted with a very long estimated chronological lifespan for lac‐1^KO bd double mutants from 247 to 462 days based on our regression analyses on log viability, and for the single mutant lac‐1^KO, 161 days. Both of these estimated lifespans are much higher than that of previously studied WT and bd single mutant strains. In a lac‐1 rescue and induction experiment, the expression of lac‐1⁺ as driven by a quinic acid‐dependent promoter actually decreases the median chronological lifespan of cells down to only 7 days, much lower than the 34‐day median lifespan found in control bd conidia also grown on quinic acid media, which we interpret as an effect of balancing selection acting on ceramide levels based on previous findings from the literature. Prior work has shown phytoceramides can act as a signal for apoptosis in stressed N. crassa cells. To test this hypothesis of balancing selection on phytoceramide levels, we examine the viability of WT, lag‐1^KO bd, and lac‐1^KO bd strains following the dual stresses of heat and glycolysis inhibition, along with phytoceramide treatments of different dosages. We find that the phytoceramide dosage–response curve is altered in the lag‐1^KO bd mutant, but not in the lac‐1^KO bd mutant. We conclude that phytoceramide production is responsible for the previously reported longevity effects in the lag‐1^KO bd mutant, but a different ceramide may be responsible for the longevity effect observed in the lac‐1^KO bd mutant.     

Sex‐ and tissue‐specific changes in mTOR signaling with age in C57BL/6J mice

Inhibition of the mTOR (mechanistic Target Of Rapamycin) signaling pathway robustly extends the lifespan of model organisms including mice. The precise molecular mechanisms and physiological effects that underlie the beneficial effects of rapamycin are an exciting area of research. Surprisingly, while some data suggest that mTOR signaling normally increases with age in mice, the effect of age on mTOR signaling has never been comprehensively assessed. Here, we determine the age‐associated changes in mTORC1 (mTOR complex 1) and mTORC2 (mTOR complex 2) signaling in the liver, muscle, adipose, and heart of C57BL/6J.Nia mice, the lifespan of which can be extended by rapamycin treatment. We find that the effect of age on several different readouts of mTORC1 and mTORC2 activity varies by tissue and sex in C57BL/6J.Nia mice. Intriguingly, we observed increased mTORC1 activity in the liver and heart tissue of young female mice compared to male mice of the same age. Tissue and substrate‐specific results were observed in the livers of HET3 and DBA/2 mouse strains, and in liver, muscle and adipose tissue of F344 rats. Our results demonstrate that aging does not result in increased mTOR signaling in most tissues and suggest that rapamycin does not promote lifespan by reversing or blunting such an effect.     

Rapamycin inhibits the secretory phenotype of senescent cells by a Nrf2‐independent mechanism

Senescent cells contribute to age‐related pathology and loss of function, and their selective removal improves physiological function and extends longevity. Rapamycin, an inhibitor of mTOR, inhibits cell senescence in vitro and increases longevity in several species. Nrf2 levels have been shown to decrease with aging and silencing Nrf2 gene induces premature senescence. Therefore, we explored whether Nrf2 is involved in the mechanism by which rapamycin delays cell senescence. In wild‐type (WT) mouse fibroblasts, rapamycin increased the levels of Nrf2, and this correlates with the activation of autophagy and a reduction in the induction of cell senescence, as measured by SA‐β‐galactosidase (β‐gal) staining, senescence‐associated secretory phenotype (SASP), and p16 and p21 molecular markers. In Nrf2KO fibroblasts, however, rapamycin still decreased β‐gal staining and the SASP, but rapamycin did not activate the autophagy pathway or decrease p16 and p21 levels. These observations were further confirmed in vivo using Nrf2KO mice, where rapamycin treatment led to a decrease in β‐gal staining and pro‐inflammatory cytokines in serum and fat tissue; however, p16 levels were not significantly decreased in fat tissue. Consistent with literature demonstrating that the Stat3 pathway is linked to the production of SASP, we found that rapamycin decreased activation of the Stat3 pathway in cells or tissue samples from both WT and Nrf2KO mice. Our data thus suggest that cell senescence is a complex process that involves at least two arms, and rapamycin uses Nrf2 to regulate cell cycle arrest, but not the production of SASP.     

Diet restriction‐induced healthy aging is mediated through the immune signaling component ZIP‐2 in Caenorhabditis elegans

Dietary restriction (DR) robustly delays the aging process in all animals tested so far. DR slows aging by negatively regulating the target of rapamycin (TOR) and S6 kinase (S6K) signaling pathway and thus inhibiting translation. Translation inhibition in C. elegans is known to activate the innate immune signal ZIP‐2. Here, we show that ZIP‐2 is activated in response to DR and in feeding‐defective eat‐2 mutants. Importantly, ZIP‐2 contributes to the improvements in longevity and healthy aging, including mitochondrial integrity and physical ability, mediated by DR in C. elegans. We further show that ZIP‐2 is activated upon inhibition of TOR/S6K signaling. However, DR‐mediated activation of ZIP‐2 does not require the TOR/S6K effector PHA‐4/FOXA. Furthermore, zip‐2 was not activated or required for longevity in daf‐2 mutants, which mimic a low nutrition status. Thus, DR appears to activate ZIP‐2 independently of PHA‐4/FOXA and DAF‐2. The link between DR, aging, and immune activation provides practical insight into the DR‐induced benefits on health span and longevity.     

Loss of NDG‐4 extends lifespan and stress resistance in Caenorhabditis elegans

NDG‐4 is a predicted transmembrane acyltransferase protein that acts in the distribution of lipophilic factors. Consequently, ndg‐4 mutants lay eggs with a pale appearance due to lack of yolk, and they are resistant to sterility caused by dietary supplementation with the long‐chain omega‐6 polyunsaturated fatty acid dihommogamma‐linolenic acid (DGLA). Two other proteins, NRF‐5 and NRF‐6, a homolog of a mammalian secreted lipid binding protein and a NDG‐4 homolog, respectively, have previously been shown to function in the same lipid transport pathway. Here, we report that mutation of the NDG‐4 protein results in increased organismal stress resistance and lifespan. When NDG‐4 function and insulin/IGF‐1 signaling are reduced simultaneously, maximum lifespan is increased almost fivefold. Thus, longevity conferred by mutation of ndg‐4 is partially overlapping with insulin signaling. The nuclear hormone receptor NHR‐80 (HNF4 homolog) is required for longevity in germline less animals. We find that NHR‐80 is also required for longevity of ndg‐4 mutants. Moreover, we find that nrf‐5 and nrf‐6 mutants also have extended lifespan and increased stress resistance, suggesting that altered lipid transport and metabolism play key roles in determining lifespan.     

β‐Guanidinopropionic acid extends the lifespan of Drosophila melanogaster via an AMP‐activated protein kinase‐dependent increase in autophagy

Previous studies have demonstrated that AMP‐activated protein kinase (AMPK) controls autophagy through the mammalian target of rapamycin (mTOR) and Unc‐51 like kinase 1 (ULK1/Atg1) signaling, which augments the quality of cellular housekeeping, and that β‐guanidinopropionic acid (β‐GPA), a creatine analog, leads to a chronic activation of AMPK. However, the relationship between β‐GPA and aging remains elusive. In this study, we hypothesized that feeding β‐GPA to adult Drosophila produces the lifespan extension via activation of AMPK‐dependent autophagy. It was found that dietary administration of β‐GPA at a concentration higher than 900 mm induced a significant extension of the lifespan of Drosophila melanogaster in repeated experiments. Furthermore, we found that Atg8 protein, the homolog of microtubule‐associated protein 1A/1B‐light chain 3 (LC3) and a biomarker of autophagy in Drosophila, was significantly upregulated by β‐GPA treatment, indicating that autophagic activity plays a role in the effect of β‐GPA. On the other hand, when the expression of Atg5 protein, an essential protein for autophagy, was reduced by RNA interference (RNAi), the effect of β‐GPA on lifespan extension was abolished. Moreover, we found that AMPK was also involved in this process. β‐GPA treatment significantly elevated the expression of phospho‐T172‐AMPK levels, while inhibition of AMPK by either AMPK‐RNAi or compound C significantly attenuated the expression of autophagy‐related proteins and lifespan extension in Drosophila. Taken together, our results suggest that β‐GPA can induce an extension of the lifespan of Drosophila via AMPK‐Atg1‐autophagy signaling pathway.     

Reducing sphingolipid synthesis orchestrates global changes to extend yeast lifespan

Studies of aging and longevity are revealing how diseases that shorten life can be controlled to improve the quality of life and lifespan itself. Two strategies under intense study to accomplish these goals are rapamycin treatment and calorie restriction. New strategies are being discovered including one that uses low‐dose myriocin treatment. Myriocin inhibits the first enzyme in sphingolipid synthesis in all eukaryotes, and we showed recently that low‐dose myriocin treatment increases yeast lifespan at least in part by down‐regulating the sphingolipid‐controlled Pkh1/2‐Sch9 (ortholog of mammalian S6 kinase) signaling pathway. Here we show that myriocin treatment induces global effects and changes expression of approximately forty percent of the yeast genome with 1252 genes up‐regulated and 1497 down‐regulated (P < 0.05) compared with untreated cells. These changes are due to modulation of evolutionarily conserved signaling pathways including activation of the Snf1/AMPK pathway and down‐regulation of the protein kinase A (PKA) and target of rapamycin complex 1 (TORC1) pathways. Many processes that enhance lifespan are regulated by these pathways in response to myriocin treatment including respiration, carbon metabolism, stress resistance, protein synthesis, and autophagy. These extensive effects of myriocin match those of rapamycin and calorie restriction. Our studies in yeast together with other studies in mammals reveal the potential of myriocin or related compounds to lower the incidence of age‐related diseases in humans and improve health span.     

Artificial selection,pre‐release diet,and gut symbiont inoculation effects on sterile male longevity for area‐wide fruit‐fly management

Longevity is an important life‐history trait for successful and cost‐effective application of the sterile insect technique. Furthermore, it has been shown that females of some species – e.g., Anastrepha ludens (Loew) (Diptera: Tephritidae) – preferentially copulate with ‘old’, sexually experienced males, rather than younger and inexperienced males. Long‐lived sterile males may therefore have greater opportunity to find and mate with wild females than short‐lived males, and be more effective in inducing sterility into wild populations. We explored the feasibility of increasing sterile male lifespan through selection of long‐lived strains and provision of pre‐release diets with added protein, and inoculated with bacterial symbionts recovered from cultures of the gut of wild Anastrepha obliqua (Macquart). Artificial selection for long‐lived A. ludens resulted in a sharp drop of fecundity levels for F1 females. Nevertheless, the cross of long‐lived males with laboratory females produced a female F1 progeny with fecundity levels comparable to those of females in the established colony. However, the male progeny of long‐lived males*laboratory females did not survive in higher proportions than laboratory males. Provision of sugar to A. obliqua adults resulted in increased survival in comparison to adults provided only with water, whereas the addition of protein to sugar‐only diets had no additional effect on longevity. Non‐irradiated males lived longer than irradiated males, and supplying a generic probiotic diet produced no noticeable effect in restoring irradiated male longevity of A. obliqua. We discuss the need to evaluate the time to reach sexual maturity and survival under stress for long‐lived strains, and the inclusion of low amounts of protein and specific beneficial bacteria in pre‐release diets to increase sterile male performance and longevity in the field.     

The role of lipid metabolism in aging,lifespan regulation,and age‐related disease

An emerging body of data suggests that lipid metabolism has an important role to play in the aging process. Indeed, a plethora of dietary, pharmacological, genetic, and surgical lipid‐related interventions extend lifespan in nematodes, fruit flies, mice, and rats. For example, the impairment of genes involved in ceramide and sphingolipid synthesis extends lifespan in both worms and flies. The overexpression of fatty acid amide hydrolase or lysosomal lipase prolongs life in Caenorhabditis elegans, while the overexpression of diacylglycerol lipase enhances longevity in both C. elegans and Drosophila melanogaster. The surgical removal of adipose tissue extends lifespan in rats, and increased expression of apolipoprotein D enhances survival in both flies and mice. Mouse lifespan can be additionally extended by the genetic deletion of diacylglycerol acyltransferase 1, treatment with the steroid 17‐α‐estradiol, or a ketogenic diet. Moreover, deletion of the phospholipase A2 receptor improves various healthspan parameters in a progeria mouse model. Genome‐wide association studies have found several lipid‐related variants to be associated with human aging. For example, the epsilon 2 and epsilon 4 alleles of apolipoprotein E are associated with extreme longevity and late‐onset neurodegenerative disease, respectively. In humans, blood triglyceride levels tend to increase, while blood lysophosphatidylcholine levels tend to decrease with age. Specific sphingolipid and phospholipid blood profiles have also been shown to change with age and are associated with exceptional human longevity. These data suggest that lipid‐related interventions may improve human healthspan and that blood lipids likely represent a rich source of human aging biomarkers.