Pharmacologic AMPK Activation Extends Lifespan in C. elegans and Improves Aspects of Healthspan in Mice.

The population around the world is graying, and as many of these individuals will spend years suffering from the burdens of age associated diseases, understanding how to increase healthspan, defined as the period of life free from disease and disability, is an urgent priority of geroscience research. The lack of agreed-upon quantitative metrics for measuring healthspan in aging mice has slowed progress in identifying interventions that do not simply increase lifespan, but also healthspan. Here, we define FAMY (Frailty-Adjusted Mouse Years) and GRAIL (Gauging Robust Aging when Increasing Lifespan) as new summary statistics for quantifying healthspan in mice. FAMY integrates lifespan data with longitudinal measurements of a widely utilized clinical frailty index, while GRAIL incorporates these measures and also adds information from widely utilized healthspan assays and the hallmarks of aging. Both metrics are conceptually similar to quality-adjusted life years (QALY), a widely utilized measure of disease burden in humans, and can be readily calculated from data acquired during longitudinal and cross-sectional studies of mouse aging. We find that interventions generally thought to promote health, including calorie restriction, robustly improve healthspan as measured by FAMY and GRAIL. Finally, we show that the use of GRAIL provides new insights, and identify dietary restriction of protein or isoleucine as interventions that robustly promote healthspan but not longevity in female HET3 mice. We suggest that the routine integration of these measures into studies of aging in mice will allow the identification and development of interventions that promote healthy aging even in the absence of increased lifespan.

A Ketogenic Diet Extends Longevity and Healthspan in Adult Mice

A Ketogenic Diet Extends Longevity and Healthspan in Adult Mice

Natural products improve healthspan in aged mice and rats: A systematic review and meta-analysis

The epithelial Na(+) channel (ENaC) regulates epithelial salt and water reabsorption, processes that require significant expenditure of cellular energy. To test whether the ubiquitous metabolic sensor AMP-activated kinase (AMPK) regulates ENaC, we examined the effects of AMPK activation on amiloride-sensitive currents in Xenopus oocytes and polarized mouse collecting duct mpkCCD(c14) cells. Microinjection of oocytes expressing mouse ENaC (mENaC) with either active AMPK protein or an AMPK activator inhibited mENaC currents relative to controls as measured by two-electrode voltage-clamp studies. Similarly, pharmacological AMPK activation or overexpression of an activating AMPK mutant in mpkCCD(c14) cells inhibited amiloride-sensitive short circuit currents. Expression of a degenerin mutant beta-mENaC subunit (S518K) along with wild type alpha and gamma increased the channel open probability (P(o)) to approximately 1. However, AMPK activation inhibited currents similarly with expression of either degenerin mutant or wild type mENaC. Single channel recordings under these conditions demonstrated that neither P(o) nor channel conductance was affected by AMPK activation. Moreover, expression of a Liddle's syndrome-type beta-mENaC mutant (Y618A) greatly enhanced ENaC whole cell currents relative to wild type ENaC controls and prevented AMPK-dependent inhibition. These findings indicate that AMPK-dependent ENaC inhibition is mediated through a decrease in the number of active channels at the plasma membrane (N), presumably through enhanced Nedd4-2-dependent ENaC endocytosis. The AMPK-ENaC interaction appears to be indirect; AMPK did not bind ENaC in cells, as assessed by in vivo pull-down assays, nor did it phosphorylate ENaC in vitro. In summary, these results suggest a novel mechanism for coupling ENaC activity and renal Na(+) handling to cellular metabolic status through AMPK, which may help prevent cellular Na(+) loading under hypoxic or ischemic conditions.

Introduction: Physical exercise is well-recognized for its beneficial effects on health. Acute or short-term effects of exercise have been extensively studied; however, it remains unclear whether exercise in juvenile has sustained beneficial effects until old age. Hypothesis: We hypothesized that juvenile exercise would delay frailty and promote metabolic health in aged mice. Methods: Male and female mice were divided into sedentary and exercise groups, and each group contains 50 mice. Mice in exercise group started swimming at 1 month of age and stopped at 4 month of age (1.5 hour per day and 5 days per week). No exercise intervention was conducted in other time. The general health, cardiac function, metabolism and other parameters were examined in aged mice. Transcriptional profiling of various tissues and organs was performed to obtain insight into mechanisms that how juvenile exercise affects aged mice. Results: Juvenile exercise mice showed significant reduction in frailty and improvement in metabolic health compared with sedentary controls. Both male and female juvenile exercise mice were scored lower in frailty at the age of 24 months. Juvenile exercise improved the integument and physical/musculoskeletal status of male mice. For females, juvenile exercise reduced frailty in all aspects. Female juvenile exercise mice weighted more than sedentary mice at 24 months old but male groups showed no difference. The cardiac function, food consumption, grip strength and rotarod running time between two groups also presented no difference at 24 months. Juvenile exercise mice showed improved flexibility in substrate usage at the age of 24 months under fasting condition. Aged mice in exercise group showed significant higher energy expenditure, fat and carbohydrate oxidation than their controls. KEGG overrepresentation analysis for the differentially expressed genes demonstrated that pathways altered by juvenile exercise were most related to metabolism, especially lipid metabolism. Conclusions: These results demonstrate that regular and moderate physical exercise in juvenile age extends health span in mice, highlighting the sustained beneficial effects of juvenile exercise.

Ageing is associated with progressive metabolic dysregulation. Rutin is a metabolic regulator with a poor solubility. Using soluble sodium rutin we investigating the effect and mechanisms of rutin in ageing process. Wild type male mice were treated with or without sodium rutin ( 0.2mg·ml-1 in drinking water from 8-month-old until end of life. Kaplan-Meier survival curve was used for lifespan assay, ageing-related histopathology analysis and metabolic analysis were performed to determine the effects of chronic sodium rutin on the longevity. Serological test, liver tissue metabolomics and transcriptomics were used for liver function assay. SiRNA knockdown Angptl8 and autophagy flux assay in HepG2 cell lines explored the mechanism through which sodium rutin might impact the function of hepatocyte. Sodium rutin treatment extends the lifespan of mice by 10%. Sodium rutin supplementation alleviates ageing-related pathological changes and promotes behaviour performance in ageing mice. Sodium rutin supplementation altered the whole-body metabolism in mice, which exhibited increased energy expenditure and lower respiratory quotient. Transcriptomics analysis showed that Sodium rutin affected the expression of metabolic genes. Metabolomics analysis showed that Sodium rutin reduced liver steatosis through increased lipid β-oxidation. Sodium rutin treatment increased the autophagy level both in vivo and in vitro. The inhibition of autophagy partially abolished the sodium rutin-mediated effect on lipolysis in HepG2 cells. Sodium rutin treatment extends the lifespan and health span of mice with beneficial effects on metabolism, which were achieved by enhancing the autophagy activity in hepatocytes. This article is part of a themed issue on Inflammation, Repair and Ageing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.9/issuetoc.

Short-term administration of the rapamycin analogue RAD001 (everolimus), already approved for clinical use for different human disorders, has been found to counter-regulate age-related transcriptomic changes and kidney pathology in rats. The study offers a step forward in establishing a safer rapalog dosing regimen for the treatment of age-related diseases. The quest for therapies that slow aging and increase healthy life span is rapidly expanding, with a few compounds showing early promise in reaching this goal. The discovery that rapamycin, an inhibitor of mechanistic target of rapamycin (mTOR), can extend the life span of several model organisms, including yeasts, worms, flies, and mice, has elicited major interest in aging research by providing a molecular target for a potential pharmacological antiaging intervention in humans. Rapamycin-based therapy has shown benefits for patients in a range of clinical applications by acting as an immunosuppressant to prevent rejection of kidney and liver transplants, and in the treatment of autoimmune disorders, certain types of cancers and lymphangioleiomyomatosis (a rare, progressive lung disease). However, the use of rapamycin as an antiaging molecule is unlikely to be approved for healthy individuals due to considerable side effects related to its ability to suppress the immune system (1). Moreover, rapamycin has been found to promote glucose intolerance, insulin resistance, and hyperlipidemia in mice and rats (2,3), and predispose transplant patients to new-onset diabetes mellitus (4). A number of other adverse events associated with rapamycin-based therapy has also been reported (5). mTOR is a protein serine/threonine kinase that is found in two distinct protein complexes, mTOR complex 1 (mTORC1) and mTORC2. It is now accepted that the beneficial effects of rapamycin are largely mediated by the inhibition of mTORC1, which is acutely sensitive to rapamycin, while many of the negative side effects of the drug stem from the inhibition of mTORC2 after long-term exposure to rapamycin in a cell- and tissue-specific manner. Several derivatives of rapamycin (known as rapalogs) with improved pharmacokinetics have been developed (6); some of these have been shown to have reduced metabolic side effects in mice, likely due to decreased disruption of mTORC2 due to their pharmacokinetics (7). Different dosing schedules have been tested that minimize side effects while providing a safer strategy that may enable the translation of rapamycin-based therapies to the clinic (7). The development of a pro-longevity drug is hampered by difficulties in defining physiological parameters that are true indicators of aging and by the amount of time required to evaluate the drugs’s ability to extend human life. One of the approaches used to overcome the limitation of lengthy clinical trials has been to test whether short-time intervention with putative pro-longevity drugs can slow down phenotypic changes associated with the aging process. A recent clinical trial demonstrated that treatment for 6 weeks with the rapamycin analogue RAD001 (everolimus) ameliorated immuno-senescence and improved the response of elderly humans to influenza vaccination (8). In this issue, Shavlakadze et al. (9) evaluated the outcome of an intermittent administration of RAD001, initiated at 22.5 months of age in rats (roughly equivalent to a 60-year-old person), at doses and treatment duration similar to those previously used in humans (8). The authors compared the transcriptional profiles in liver, skeletal muscle, and kidney between young and old rats on RAD001, and found a striking (37%) reversal in the expression of age-regulated genes in the kidney, many of which linked to inflammation and fibrosis, together with a reduction in the severity of nephropathy lesions in aged rats (9). Under these conditions, the expression of age-regulated genes in liver and gastrocnemius of RAD001-treated rats was minimally altered despite lower activating phosphorylation of two surrogate markers of mTORC1 activity. Additional studies are needed to establish the presence of alternative RAD001-dependent mechanisms in the control of the liver and gastrocnemius transcriptome. RAD001 (at a dose of 1 mg/kg once a week for 6 weeks) was well-tolerated by old rats, with no significant changes in fasting blood glucose and body weight trajectories compared to controls. The authors attribute the safe metabolic profile to a “fairly selective effect” of this low-dose and short-term treatment paradigm on gene expression in the kidney, a tissue in which mTORC2 signaling is strongly resistant to rapamycin (10). These findings reinforce the idea that selective mTORC1 inhibition might be a safe and effective strategy to counteract age-related kidney pathology. How does inhibition of TOR activity influence aging? The authors report the role of c-Myc as a possible target of mTORC1 and point at a mechanism whereby RAD001 may suppress the age-dependent increase in the expression of c-Myc downstream target genes. The c-Myc proto-oncogene functions as a transcriptional regulator modulated by the mTORC1-CREB2 pathway. A recent study reports on the fact that Myc haploinsufficiency (Myc+/–) confers extended life span and health span in mice resulting from changes in multiple cellular processes such as those implicated in nutrient and energy sensing pathways (eg, AKT, TOR, and S6K) (11). It is unclear whether administration of RAD001 will counteract age-related kidney pathology in this longevity mouse model. In this issue, Shavlakadze and coworkers further demonstrate in HEK293 cells that RAD001 may affect c-Myc protein turnover by promoting its degradation; however, the fact that c-Myc protein was below detectable levels in the kidney puts into question the relevance of this observation in vivo. Nonetheless, this study helps define a therapeutic window in which rapalogs confer beneficial antiaging effects and guard against age-related diseases through mTORC1 inhibition while minimizing mTORC2-related side effects. Are rapalogues suitable antiaging drugs for people? Further research is needed to determine whether intermittent mTOR inhibition will promote human longevity and protect against age-related diseases.

The inflammaging clock strikes IL-11!

Neurons and astrocytes work in close metabolic collaboration, linking neurotransmission to brain energy and neurotransmitter metabolism. Dysregulated energy metabolism is a hallmark of the aging brain and may underlie the progressive age-dependent cognitive decline. However, astrocyte and neurotransmitter metabolism remains understudied in aging brain research. In particular, how aging affects metabolism of glutamate, being the primary excitatory neurotransmitter, is still poorly understood. Here we investigated critical aspects of cellular energy metabolism in the aging male mouse hippocampus using stable isotope tracing in vitro. Metabolism of [U–13C]glucose demonstrated an elevated glycolytic capacity of aged hippocampal slices, whereas oxidative [U–13C]glucose metabolism in the TCA cycle was significantly reduced with aging. In addition, metabolism of [1,2–13C]acetate, reflecting astrocyte energy metabolism, was likewise reduced in the hippocampal slices of old mice. In contrast, uptake and subsequent metabolism of [U–13C]glutamate was elevated, suggesting increased capacity for cellular glutamate handling with aging. Finally, metabolism of [15N]glutamate was maintained in the aged slices, demonstrating sustained glutamate nitrogen metabolism. Collectively, this study reveals fundamental alterations in cellular energy and neurotransmitter metabolism in the aging brain, which may contribute to age-related hippocampal deficits.

Dysregulated energy metabolism is a hallmark of aging, including brain aging; thus, strategies to restore normal metabolic regulation are at the forefront of aging research. Intermittent fasting, particularly time-restricted eating (TRE), is one of these strategies. Despite its well-established effectiveness in improving metabolic outcomes in older adults, the effect of TRE on preserving or improving cerebrovascular health during aging remains underexplored. We explored how aging itself affects energy metabolism and contextualized these age-related changes to cerebrovascular health. We also conducted a literature search on PubMed and Scopus to identify and summarize current studies on TRE in older adults. Finally, we provided preliminary data from our proof-of-concept pilot trial on the effect of 6-month TRE on cerebrovascular health in older adults. Current evidence shows the potential of TRE to improve energy metabolism and physiological outcomes in older adults. TRE may improve cerebrovascular function indirectly due to its effect on glucose homeostasis. However, to date, direct evidence of the effect of TRE on cerebrovascular parameters is lacking. TRE is a well-tolerated and promising dietary intervention for promoting and maintaining cerebrovascular health in older adults. Further studies on TRE in older adults must be better controlled for energy balance to elucidate its independent effects from those of caloric restriction.

Aging is associated with systemic, non-resolving inflammation and the accumulation of senescent cells. The resolution of inflammation (or inflammation-resolution) is in part mediated by the balance between specialized pro-resolving mediators (SPMs) and pro-inflammatory leukotrienes (LTs). Aged mice (i.e. 2 years of age) exhibit a significant decrease in the SPM:LT ratio in specific organs including the spleen, which suggests that this organ may exhibit heightened inflammation and may be particularly amenable to SPM therapy. Previous studies have shown that resolvin D1 (RvD1) is decreased in spleens of aged mice compared with young controls. Therefore, we asked whether treatment of RvD1 in aged mice would impact markers of cellular senescence in splenic macrophages, and downstream effects on splenic fibrosis, a hallmark of splenic aging. We found that in aged mice, both zymosan-elicited and splenic macrophages showed an increase in mRNA expression of inflammatory and eicosanoid biosynthesis genes and a dysregulation of genes involved in the cell cycle. Injections with RvD1 reversed these changes. Importantly, RvD1 also decreased splenic fibrosis, a hallmark of splenic aging. Our findings suggest that RvD1 treatment may limit several features of aging, including senescence and fibrosis in spleens from aged mice.SummaryAging is associated with systemic, low grade, non-resolving inflammation. The resolution of inflammation is in part mediated by the balance between specialized pro-resolving mediators (SPMs) and pro-inflammatory lipid mediators, like leukotrienes (LTs). A hallmark of aging is the accumulation of senescent cells that promote low grade inflammation by secreting pro-inflammatory cytokines and lipid mediators. Splenic macrophages contribute to systemic aging, and spleens of aged mice demonstrate decreased levels of the SPM called resolvin D1 (RvD1). Whether addition of RvD1 is protective in spleens of aged mice is unknown and is focus of this study. RvD1 treatment to aged mice led to decreased mRNA expression of markers of cellular senescence and inflammation in splenic macrophages compared with age-matched vehicle controls. Moreover, RvD1 decreased splenic fibrosis, which occurs due to persistent low-grade inflammation in aging. Promoting inflammation resolution with RvD1 thus limits macrophage senescence, pro-inflammatory signals and established splenic fibrosis in aging.

This article investigates the effect on the mouse frailty index (FI), of factors known to influence lifespan and healthspan in mice: strain (short-lived DBA/2J mice vs long-lived C57BL/6J mice), calorie restriction (CR), and resveratrol treatment. The mouse FI, based on deficit accumulation, was recently validated in C57BL/6J mice by Whitehead JC, Hildebrand BA, Sun M, et al. (A clinical frailty index in aging mice: comparisons with frailty index data in humans. J Gerontol A Biol Sci Med Sci. 2014;69:621-632) and shares many characteristics of the human FI. FI scores were measured in male and female aged (18 months) ad-libitum fed and CR DBA/2J and C57BL/6J mice, as well as male aged (24 months) C57BL/6J mice ad-libitum fed with or without resveratrol (100 mg/kg/day) in the diet for 6 months. Mean scores of two raters were used, and the raters had excellent inter-rater reliability (ICC = 0.88, 95% CI [0.80, 0.92]). Furthermore, the interventions of CR and resveratrol were associated with a significant reduction in FI scores in C57BL/6J mice, compared to age-matched controls. The short-lived DBA/2J mice also had slightly higher FI scores than the C57BL/6J mice, for the male calorie-restricted groups (DBA/2J FI = 0.16±0.03, C57BL/6J FI = 0.11±0.03, p = .01). This study uses the mouse FI developed by Whitehead JC, Hildebrand BA, Sun M, et al. (A clinical frailty index in aging mice: comparisons with frailty index data in humans. J Gerontol A Biol Sci Med Sci. 2014;69:621-632) in a different mouse colony and shows that this tool can be applied to quantify the effect of dietary and pharmaceutical interventions on frailty.

Activation of adenosine monophosphate-activated kinase (AMPK) has been associated with the inhibition of inflammatory nociception and the attenuation of morphine antinociceptive tolerance. In this study, the authors investigated the impact of AMPK activation through resveratrol treatment on bone cancer pain. The nociception was assessed by measuring the incidence of foot withdrawal in response to mechanical indentation in rats (n = 8). Cytokine expression was measured using quantitative polymerase chain reaction (n = 8). Cell signalings were assayed by western blot (n = 4) and immunohistochemistry (n = 5). The microglial cell line BV-2, primary astrocytes, and neuron-like SH-SY5Y cells were cultured to investigate the in vitro effects. Resveratrol and 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide, the AMPK activators, significantly attenuated bone cancer pain in rats with tumor cell implantation (TCI; threshold of mechanical withdrawal, resveratrol vs. vehicle: 10.1 ± 0.56 vs. 4.1 ± 0.37; 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide vs. vehicle: 8.2 ± 0.17 vs. 4.1 ± 0.37, mean ± SEM); these effects were reversed by the AMPK inhibitor compound C (compound C vs. resveratrol: 6.2 ± 1.35 vs. 10.1 ± 0.56, mean ± SEM). Resveratrol has an AMPK-dependent inhibitory effect on TCI-evoked astrocyte and microglial activation. The antinociceptive effects of resveratrol were partially mediated by the reduced phosphorylation of mitogen-activated protein kinases and decreased production of proinflammatory cytokines in an AMPK-dependent manner. Furthermore, resveratrol potently inhibited inflammatory factors-mediated protein kinase B/mammalian target of rapamycin signaling in neurons. Acute pain evoked by proinflammatory cytokines in the spinal cord was significantly attenuated by resveratrol. AMPK activation in the spinal glia by resveratrol may have utility in the treatment of TCI-induced neuroinflammation, and our results further implicate AMPK as a novel target for the attenuation of bone cancer pain.

Neuropeptide Y Attenuates Aging-Induced Bone Marrow Niche Defects and Rejuvenate Aged Hematopoietic Stem/Progenitor Cells