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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.

Previously, we showed that extracellular matrices (ECMs), produced ex vivo by various types of stromal cells, direct bone marrow mesenchymal stem cells (BM-MSCs) in a tissue-specific manner and recapitulate physiologic changes characteristic of the aging microenvironment. In particular, BM-MSCs obtained from elderly donors and cultured on ECM produced by young BM stromal cells showed improved quantity, quality and osteogenic differentiation. In the present study, we searched for matrix components that are required for a functional BM-MSC niche by comparing ECMs produced by BM stromal cells from "young" (≤25 y/o) versus "elderly" (≥60 y/o) donors. With increasing donor age, ECM fibrillar organization and mechanical integrity deteriorated, along with the ability to promote BM-MSC proliferation and responsiveness to growth factors. Proteomic analyses revealed that the matricellular protein, Cyr61/CCN1, was present in young, but undetectable in elderly, BM-ECM. To assess the role of Cyr61 in the BM-MSC niche, we used genetic methods to down-regulate the incorporation of Cyr61 during production of young ECM and up-regulate its incorporation in elderly ECM. The results showed that Cyr61-depleted young ECM lost the ability to promote BM-MSC proliferation and growth factor responsiveness. However, up-regulating the incorporation of Cyr61 during synthesis of elderly ECM restored its ability to support BM-MSC responsiveness to osteogenic factors such as BMP-2 and IGF-1. We next examined aging bone and compared bone mineral density and Cyr61 content of L4-L5 vertebral bodies in "young" (9-11 m/o) and "elderly" (21-33 m/o) mice. Our analyses showed that low bone mineral density was associated with decreased amounts of Cyr61 in osseous tissue of elderly versus young mice. Our results strongly demonstrate a novel role for ECM-bound Cyr61 in the BM-MSC niche, where it is responsible for retention of BM-MSC proliferation and growth factor responsiveness, while depletion of Cyr61 from the BM niche contributes to an aging-related dysregulation of BM-MSCs. Our results also suggest new potential therapeutic targets for treating age-related bone loss by restoring specific ECM components to the stem cell niche.

With evolving cores, enrichment and training programs, and supported research projects, the San Antonio (SA) Nathan Shock Center has for 26 years provided critical support to investigators locally, nationally, and abroad. With its existing and growing intellectual capital, the SA Nathan Shock Center provides to local and external investigators an enhanced platform to conduct horizontally integrated (lifespan, healthspan, pathology, pharmacology) transformative research in the biology of aging, and serves as a springboard for advanced educational and training activities in aging research. The SA Nathan Shock Center consists of six cores: Administrative/Program Enrichment Core, Research Development Core, Aging Animal Models and Longevity Assessment Core, Pathology Core, Analytical Pharmacology and Drug Evaluation Core, and Integrated Physiology of Aging Core. The overarching goal of the SA Nathan Shock Center is to advance knowledge in the basic biology of aging and to identify molecular and cellular mechanisms that will facilitate the development of pharmacologic interventions and other strategies to extend healthy lifespan. In pursuit of this goal, we provide an innovative “one-stop shop” venue to accelerate transformative research in the biology of aging through our integrated research cores. Moreover, we aim to foster and promote career development of early-stage investigators in aging biology through our research development programs, to serve as a resource and partner to investigators from other Shock Centers, and to disseminate scientific knowledge and enhanced awareness about aging research. Overall, the SA Nathan Shock Center aims to be a leader in research that advances our understanding of the biology of aging and development of approaches to improve longevity and healthy aging.

Mice bred in 2017 and entered into the C2017 cohort were tested for possible lifespan benefits of (R/S)-1,3-butanediol (BD), captopril (Capt), leucine (Leu), the Nrf2-activating botanical mixture PB125, sulindac, syringaresinol, or the combination of rapamycin and acarbose started at 9 or 16 months of age (RaAc9, RaAc16). In male mice, the combination of Rapa and Aca started at 9 months and led to a longer lifespan than in either of the two prior cohorts of mice treated with Rapa only, suggesting that this drug combination was more potent than either of its components used alone. In females, lifespan in mice receiving both drugs was neither higher nor lower than that seen previously in Rapa only, perhaps reflecting the limited survival benefits seen in prior cohorts of females receiving Aca alone. Capt led to a significant, though small (4% or 5%), increase in female lifespan. Capt also showed some possible benefits in male mice, but the interpretation was complicated by the unusually low survival of controls at one of the three test sites. BD seemed to produce a small (2%) increase in females, but only if the analysis included data from the site with unusually short-lived controls. None of the other 4 tested agents led to any lifespan benefit. The C2017 ITP dataset shows that combinations of anti-aging drugs may have effects that surpass the benefits produced by either drug used alone, and that additional studies of captopril, over a wider range of doses, are likely to be rewarding.

AMP-activated protein kinase (AMPK) is a central regulator of both lifespan and health across multiple model organisms. β-Guanidinopropionic acid (GPA) is an endogenous AMPK activator previously shown to improve metabolic function in young and obese mice. In this study, we tested whether age of administration significantly affects the physiological outcomes of GPA administration in mice. We report that intervention starting at 7–8 months (young) results in activation of AMPK signaling and a phenotype consisting of lower body mass, improved glucose control, enhanced exercise tolerance, and altered mitochondrial electron transport chain flux similar to previous reports. When GPA treatment is started at 18–19 months (old), the effect of GPA on AMPK signaling is blunted compared to younger mice despite similar accumulation of GPA in skeletal muscle. Even so, GPA administration in older animals delayed age-related declines in lean mass, improved measures of gait performance and circadian rhythm, and increased fat metabolism as measured by respiratory exchange ratio. These results are likely partially driven by the relative difference in basal function and metabolic plasticity between young and old mice. Our results suggest that age-related declines in AMPK sensitivity may limit potential strategies targeting AMPK signaling in older subjects and suggest that further research and development is required for AMPK activators to realize their full potential. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11357-021-00372-8.

We conducted a dose-response study of dexamethasone to investigate an optimal dexamethasone suppression test for common marmosets. Twelve marmosets received 0.1, 0.5, or 1.0 mg/kg dexamethasone. Doses of 0.5 and 1.0mg/kg both suppressed endogenous cortisol for at least 18 h with greater individual variability in the lower 0.5mg/kg dose.

The ability to generate in vitro cultures of neuronal cells has been instrumental in advancing our understanding of the nervous system. Rodent models have been the principal source of brain cells used in primary cultures for over a century, providing insights that are widely applicable to human diseases. However, therapeutic agents that showed benefit in rodent models, particularly those pertaining to aging and age-associated dementias, have frequently failed in clinical trials. This discrepancy established a potential “translational gap” between human and rodent studies that may at least partially be explained by the phylogenetic distance between rodent and primate species. Several non-human primate (NHP) species, including the common marmoset (Callithrix jacchus), have been used extensively in neuroscience research, but in contrast to rodent models, practical approaches to the generation of primary cell culture systems amenable to molecular studies that can inform in vivo studies are lacking. Marmosets are a powerful model in biomedical research and particularly in studies of aging and age-associated diseases because they exhibit an aging phenotype similar to humans. Here, we report a practical method to culture primary marmoset neurons and astrocytes from brains of medically euthanized postnatal day 0 (P0) marmoset newborns that yield highly pure primary neuron and astrocyte cultures. Primary marmoset neuron and astrocyte cultures can be generated reliably to provide a powerful NHP in vitro model in neuroscience research that may enable mechanistic studies of nervous system aging and of age-related neurodegenerative disorders. Because neuron and astrocyte cultures can be used in combination with in vivo approaches in marmosets, primary marmoset neuron and astrocyte cultures may help bridge the current translational gap between basic and clinical studies in nervous system aging and age-associated neurological diseases. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s11357-020-00284-z) contains supplementary material, which is available to authorized users.

Methionine restriction (MR) extends lifespan and improves several markers of health in rodents. However, the proximate mechanisms of MR on these physiological benefits have not been fully elucidated. The essential amino acid methionine plays numerous biological roles and limiting its availability in the diet directly modulates methionine metabolism. There is growing evidence that redox regulation of methionine has regulatory control on some aspects of cellular function but interactions with MR remain largely unexplored. We tested the functional role of the ubiquitously expressed methionine repair enzyme methionine sulfoxide reductase A (MsrA) on the metabolic benefits of MR in mice. MsrA catalytically reduces both free and protein-bound oxidized methionine, thus playing a key role in its redox state. We tested the extent to which MsrA is required for metabolic effects of MR in adult mice using mice lacking MsrA. As expected, MR in control mice reduced body weight, altered body composition, and improved glucose metabolism. Interestingly, lack of MsrA did not impair the metabolic effects of MR on these outcomes. Moreover, females had blunted MR responses regardless of MsrA status compared to males. Overall, our data suggests that MsrA is not required for the metabolic benefits of MR in adult mice.

Objective: Mitochondrial dysfunction comprises part of the etiology of myriad health issues, particularly those that occur with advancing age. Methionine sulfoxide reductase A (MsrA) is a ubiquitous protein oxidation repair enzyme that specifically and catalytically reduces a specific epimer of oxidized methionine: methionine sulfoxide. In this study, we tested the ways in which mitochondrial bioenergetic functions are affected by increasing MsrA expression in different cellular compartments. Methods: In this study, we tested the function of isolated mitochondria, including free radical generation, ATP production, and respiration, from the skeletal muscle of two lines of transgenic mice with increased MsrA expression: mitochondria-targeted MsrA overexpression or cytosol-targeted MsrA overexpression. Results: Surprisingly, in the samples from mice with mitochondrial-targeted MsrA overexpression, we found dramatically increased free radical production though no specific defect in respiration, ATP production, or membrane potential. Among the electron transport chain complexes, we found the activity of complex I was specifically reduced in mitochondrial MsrA transgenic mice. In mice with cytosolic-targeted MsrA overexpression, we found no significant alteration made to any of these parameters of mitochondrial energetics. Conclusions: There is also a growing amount of evidence that MsrA is a functional requirement for sustaining optimal mitochondrial respiration and free radical generation. MsrA is also known to play a partial role in maintaining normal protein homeostasis by specifically repairing oxidized proteins. Our studies highlight a potential novel role for MsrA in regulating the activity of mitochondrial function through its interaction with the mitochondrial proteome. Keywords: Superoxide, oxidative stress, mitochondria, protein homeostasis, electron transport chain

Pharmaceutical intervention of aging requires targeting multiple pathways, thus there is rationale to test combinations of drugs targeting different but overlapping processes. In order to determine if combining drugs shown to extend lifespan and healthy aging in mice would have greater impact than any individual drug, a cocktail diet containing 14 ppm rapamycin, 1000 ppm acarbose, and 1000 ppm phenylbutyrate was fed to 20-month-old C57BL/6 and HET3 4-way cross mice of both sexes for three months. Mice treated with the cocktail showed a sex and strain-dependent phenotype consistent with healthy aging including decreased body fat, improved cognition, increased strength and endurance, and decreased age-related pathology compared to mice treated with individual drugs or control. The severity of age-related lesions in heart, lungs, liver, and kidney was consistently decreased in mice treated with the cocktail compared to mice treated with individual drugs or control, suggesting an interactive advantage of the three drugs. This study shows that a combination of three drugs, each previously shown to enhance lifespan and health span in mice, is able to delay aging phenotypes in middle-aged mice more effectively than any individual drug in the cocktail over a 3-month treatment period.

Pharmaceutical intervention of aging requires targeting multiple pathways, thus there is rationale to test combinations of drugs each targeting different but overlapping processes. In order to determine if combining drugs previously shown to improve lifespan would have greater impact than any individuyal drug, a diet containing rapamycin at 14 ppm, acarbose at 1000 ppm, and phenylbutyrate at 1000 ppm was fed to 20-month-old C57BL/6 and HET3 4-way cross mice of both sexes for three months. Mice fed the cocktail diet showed a strain and gender-dependent phenotype consistent with healthy aging including decreased body fat and blood glucose, improved cognition, and increased grip strength and walking ability compared to mice fed individual drug or control diets. A cocktail diet containing ½ dosing of each compound was overall less effective than the full dose. The composite age-related lesion score of heart, lungs, liver and kidney was decreased in mice fed the cocktail diet compared to mice fed individual drug or control diets suggesting an interactive advantage of the three drugs. Senescence and inflammatory cytokine levels in kidneys from mice fed the cocktail diet were lower than in kidneys from mice fed control diet, and consistent with low expression levels in kidneys from young untreated mice, suggesting the cocktail diet delayed aging partly by senolytic and anti-inflammatory effects.

Human DNA methylation data have previously been used to develop highly accurate biomarkers of aging (“epigenetic clocks”). Subsequent studies demonstrate that similar epigenetic clocks can also be developed for mice and many other mammals. Here, we describe epigenetic clocks for common marmosets (Callithrix jacchus) based on novel DNA methylation data generated from highly conserved mammalian CpGs that were profiled using a custom Infinium array (HorvathMammalMethylChip40). From these, we developed and present here two epigenetic clocks for marmosets that are applicable to whole blood samples. We find that the human-marmoset clock for relative age exhibits moderately high age correlations in two other non-human primate species: vervet monkeys and rhesus macaques. In a separate cohort of marmosets, we tested whether intervention with rapamycin, a drug shown to extend lifespan in mice, would alter the epigenetic age of marmosets, as measured by the marmoset epigenetic clocks. These clocks did not detect significant effects of rapamycin on the epigenetic age of marmoset blood. The common marmoset stands out from other mammals in that it is not possible to build accurate estimators of sex based on DNA methylation data: the accuracy of a random forest predictor of sex (66%) was substantially lower than that observed for other mammals (which is close to 100%). Overall, the epigenetic clocks developed here for the common marmoset are expected to be useful for age estimation of wild-born animals and for anti-aging studies in this species. Supplementary Information The online version contains supplementary material available at 10.1007/s11357-021-00438-7.

Funder: Calico Life Sciences, LLC, The naked mole‐rat (Heterocephalus glaber) has fascinated zoologists for at least half a century. It has also generated considerable biomedical interest not only because of its extraordinary longevity, but also because of unusual protective features (e.g. its tolerance of variable oxygen availability), which may be pertinent to several human disease states, including ischemia/reperfusion injury and neurodegeneration. A recent article entitled ‘Surprisingly long survival of premature conclusions about naked mole‐rat biology’ described 28 ‘myths’ which, those authors claimed, are a ‘perpetuation of beautiful, but falsified, hypotheses’ and impede our understanding of this enigmatic mammal. Here, we re‐examine each of these ‘myths’ based on evidence published in the scientific literature. Following Braude et al., we argue that these ‘myths’ fall into four main categories: (i) ‘myths’ that would be better described as oversimplifications, some of which persist solely in the popular press; (ii) ‘myths’ that are based on incomplete understanding, where more evidence is clearly needed; (iii) ‘myths’ where the accumulation of evidence over the years has led to a revision in interpretation, but where there is no significant disagreement among scientists currently working in the field; ( iv ) ‘myths’ where there is a genuine difference in opinion among active researchers, based on alternative interpretations of the available evidence. The term ‘myth’ is particularly inappropriate when applied to competing, evidence‐based hypotheses, which form part of the normal evolution of scientific knowledge. Here, we provide a comprehensive critical review of naked mole‐rat biology and attempt to clarify some of these misconceptions.

Methionine restriction (MR) extends lifespan and improves several markers of health in rodents. However, the proximate mechanisms of MR on these physiological benefits have not been fully elucidated. The essential amino acid methionine plays numerous biological roles and limiting its availability in the diet directly modulates methionine metabolism. There is growing evidence that redox regulation of methionine has regulatory control on some aspects of cellular function but interactions with MR remain largely unexplored. We tested the functional role of the ubiquitously expressed methionine repair enzyme methionine sulfoxide reductase A (MsrA) on the metabolic benefits of MR in mice. MsrA catalytically reduces both free and protein-bound oxidized methionine, thus playing a key role in its redox state. We tested the extent to which MsrA is required for metabolic effects of MR in adult mice using mice lacking MsrA. As expected, MR in control mice reduced body weight, altered body composition, and improved glucose metabolism. Interestingly, lack of MsrA did not impair the metabolic effects of MR on these outcomes. Moreover, females had blunted MR responses regardless of MsrA status compared to males. Overall, our data suggests that MsrA is not required for the metabolic benefits of MR in adult mice.

The mechanism of kidney injury in aging are not well understood. In order to identify hitherto unknown pathways of aging‐related kidney injury, we performed RNA‐Seq on kidney extracts of young and aged mice. Expression of chloride (Cl) channel accessory 1 (CLCA1) mRNA and protein was increased in the kidneys of aged mice. Immunostaining showed a marked increase in CLCLA1 expression in the proximal tubules of the kidney from aged mice. Increased kidney CLCA1 gene expression also correlated with aging in marmosets and in a human cohort. In aging mice, increased renal cortical CLCA1 content was associated with hydrogen sulfide (H2S) deficiency, which was ameliorated by administering sodium hydrosulfide (NaHS), a source of H2S. In order to study whether increased CLCA1 expression leads to injury phenotype and the mechanisms involved, stable transfection of proximal tubule epithelial cells overexpressing human CLCA1 (hCLCA1) was performed. Overexpression of hCLCA1 augmented Cl− current via the Ca++‐dependent Cl− channel TMEM16A (anoctamin‐1) by patch‐clamp studies. hCLCA1 overexpression also increased the expression of fibronectin, a matrix protein, and induced the senescence‐associated secretory phenotype (SASP). Mechanistic studies underlying these changes showed that hCLCA1 overexpression leads to inhibition of AMPK activity and stimulation of mTORC1 as cellular signaling determinants of injury. Both TMEM16A inhibitor and NaHS reversed these signaling events and prevented changes in fibronectin and SASP. We conclude that CLCA1‐TMEM16A‐Cl− current pathway is a novel mediator of kidney injury in aging that is regulated by endogenous H2S., Chloride channel accessory 1 expression is increased in the tubular epithelial cells of aged kidneys. In vitro experiments show that CLCA1 augments the activity of TMEM16A, a Ca++‐dependent Chloride Channel, activates mTORC1, augments the synthesis of matrix proteins, and induces SASP, whcih contribute to aging associated kidney injury.

Maximum lifespan of a species is the oldest that individuals can survive, reflecting the genetic limit of longevity in an ideal environment. Here we report methylation-based models that accurately predict maximum lifespan (r=0.89), gestational time (r=0.96), and age at sexual maturity (r=0.87), using cytosine methylation patterns collected from over 12,000 samples derived from 192 mammalian species. Our epigenetic maximum lifespan predictor corroborated the extended lifespan in growth hormone receptor knockout mice and rapamycin treated mice. Across dog breeds, epigenetic maximum lifespan correlates positively with breed lifespan but negatively with breed size. Lifespan-related cytosines are located in transcriptional regulatory regions, such as bivalent chromatin promoters and polycomb-repressed regions, which were hypomethylated in long-lived species. The epigenetic estimators of maximum lifespan and other life history traits will be useful for characterizing understudied species and for identifying interventions that extend lifespan.

Traditional animal models have been used to make seminal discoveries in biomedical research including a better understanding of the biology of the aging process. However, translation of these findings from laboratory to clinical populations has likely been hindered due to fundamental biological and physiological differences between common laboratory animals and humans. Non-human primates (NHP) may serve as an effective bridge towards translation, and short-lived NHP like the common marmoset offer many advantages as models for aging research. Here, we address these advantages and discuss what is currently understood about the changes in physiology and pathology that occur with age in the marmoset. In addition, we discuss how aging research might best utilize this model resource, and outline an ongoing study to address whether pharmaceutical intervention can slow aging in the marmoset. With this manuscript, we clarify how common marmosets might assist researchers in geroscience as a potential model for pre-clinical translation.

Since the initial suggestion that rapamycin, an inhibitor of target of rapamycin (TOR) nutrient signaling, increased lifespan comparable to dietary restriction, investigators have viewed rapamycin as a potential dietary restriction mimetic. Both dietary restriction and rapamycin increase lifespan across a wide range of evolutionarily diverse species (including yeast, Caenorhabditis elegans, Drosophila, and mice) as well as reducing pathology and improving physiological functions that decline with age in mice. The purpose of this article is to review the research comparing the effect of dietary restriction and rapamycin in mice. The current data show that dietary restriction and rapamycin have different effects on many pathways and molecular processes. In addition, these interventions affect the lifespan of many genetically manipulated mouse models differently. In other words, while dietary restriction and rapamycin may have similar effects on some pathways and processes; overall, they affect many pathways/processes quite differently. Therefore, rapamycin is likely not a true dietary restriction mimetic. Rather dietary restriction and rapamycin appear to be increasing lifespan and retarding aging largely through different mechanisms/pathways, suggesting that a combination of dietary restriction and rapamycin will have a greater effect on lifespan than either manipulation alone.

Aging is associated with progressive loss of cellular homeostasis resulting from intrinsic and extrinsic challenges. Lack of a carefully designed, well-characterized, precise, translational experimental model is a major limitation to understanding the cellular perturbations that characterize aging. Here, we tested the feasibility of primary fibroblasts isolated from nonhuman primates (baboons) as a model of cellular resilience in response to homeostatic challenge. Using a real-time live-cell imaging system, we precisely defined a protocol for testing effects of prooxidant compounds (e.g., hydrogen peroxide (H(2)O(2)), paraquat), thapsigargin, dexamethasone, and a low glucose environment on cell proliferation in fibroblasts derived from baboons across the life course (n = 11/sex). Linear regression analysis indicated that donor age significantly reduced the ability of cells to proliferate following exposure to H(2)O(2) (50 and 100 µM) and paraquat (100 and 200 µM) challenges in cells from males (6.4–21.3 years; average lifespan 21 years) but not cells from females (4.3–15.9 years). Inhibitory effects of thapsigargin on cell proliferation were dependent on challenge duration (2 vs 24 h) and concentration (0.1 and 1 µM). Cells from older females (14.4–15.9 years) exhibited greater resilience to thapsigargin (1 µM; 24 h) and dexamethasone (500 µM) challenges than did those from younger females (4.3–6.7 years). The cell proliferation response to low glucose (1 mM) was reduced with age in both sexes. These data indicate that donor’s chronological age and sex are important variables in determining fibroblast responses to metabolite and other challenges. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11357-021-00395-1.

DNA methylation data have been successfully used to develop highly accurate estimators of age (“epigenetic clocks”) in many mammalian species. With a view of extending such epigenetic clocks to all primate species, we analyzed DNA methylation profiles of 2400 tissues derived from 37 primate species including 11 haplorhine species (baboons, marmosets, vervets, rhesus macaque, chimpanzees, gorillas, orangutan, humans) and 26 strepsirrhine species (suborders Lemuriformes and Lorisiformes). From these we present here, pan-primate epigenetic clocks which are highly accurate for all primates including humans (age correlation R=0.98). We also carried out in-depth analysis of baboon DNA methylation profiles and generated five epigenetic clocks for baboons (Olive-yellow baboon hybrid), one of which, the pan-tissue epigenetic clock, was trained on seven tissue types (fetal cerebral cortex, adult cerebral cortex, cerebellum, adipose, heart, liver, and skeletal muscle) with ages ranging from late fetal life to 22.8 years of age. To facilitate translation of findings in baboons to humans, we further constructed two dual-species, human-baboon clocks. We also identified and present here, epigenetic predictors of sex that apply to all primate species. Low overlap can be observed between age- and sex-related CpGs. Overall, this study advances our understanding of conserved age- and sex-related epigenetic changes in primates, and provides biomarkers to study the aging of all primate species with the facility to readily translate any findings between primate species.

Human DNA methylation data have previously been used to develop highly accurate biomarkers of aging (“epigenetic clocks”). Subsequent studies demonstrate that similar epigenetic clocks can also be developed for mice and many other mammals. Here, we describe epigenetic clocks for common marmosets (Callithrix jacchus) based on novel DNA methylation data generated from highly conserved mammalian CpGs that were profiled using a custom Infinium array (HorvathMammalMethylChip40). From these, we developed and present here, two epigenetic clocks for marmosets that are applicable to whole blood samples. We find that the human-marmoset clock for relative age exhibits moderately high age correlations in two other non-human primate species: vervet monkeys and rhesus macaques. In a separate cohort of marmosets, we tested whether intervention with rapamycin, a drug shown to extend lifespan in mice, would alter the epigenetic age of marmosets, as measured by the marmoset epigenetic clocks. These clocks did not detect significant effects of rapamycin on the epigenetic age of marmoset blood. The common marmoset stands out from other mammals in that it is not possible to build accurate estimators of sex based on DNA methylation data: the accuracy of a random forest predictor of sex (66%) was substantially lower than that observed for other mammals (which is close to 100%). Overall, the epigenetic clocks developed here for the common marmoset are expected to be useful for age estimation of wild-born animals and for anti-aging studies in this species.

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., Intermittent (1 month on/1 month off) or a limited 3‐month exposure to rapamycin, beginning at 20 months, was as effective as continuous exposure in increasing survival in males. In females, only intermittent exposure increased survival but the increase was not as great as continuous exposure.

The objective of the proposed study is to investigate the feasibility of the marmoset as an animal model to study age-associated nonalcoholic fatty liver disease (NAFLD). This chronic liver disease includes a spectrum of disorders ranging from increased triglyceride accumulation in the liver or hepatic steatosis to the more severe inflammatory form nonalcoholic steatohepatitis that can lead to cirrhosis and even hepatocellular carcinoma in individuals who do not have a history of alcohol abuse. Aging increases the prevalence of NAFLD and is strongly associated with the progression and severity of this disease. End-stage hepatic failure and liver cancer resulting from advanced NAFLD are leading indications for liver transplantation enhancing the burden on our healthcare systems. Accumulating clinical evidence also suggests that patients with NAFLD have a higher prevalence of cardiovascular disease. Pathogenetic mechanisms involved in the development and progression of NAFLD are poorly understood and as such, there is a lack of effective therapies. The common marmoset is a relatively short-lived non-human primate that recapitulates many of the physiological changes that occur in human aging. We hypothesized an age-associated increase in hepatic steatosis and alterations in serum lipid profile in the marmoset model. An increase in triglyceride levels and oil red o staining in liver tissues of old marmosets compared to young animals was observed suggesting an age-associated increase in hepatic steatosis in marmosets as observed in humans. Lipidomic studies were also performed using blood samples from male and female marmosets to investigate age-associated changes in specific lipid species, which are characteristic of aberrant lipid metabolism. Analysis of the results revealed significant decreases in several phosphatidylethanolamine, phosphatidylcholine, and sphingomyelin species in the plasma of old marmosets compared to young marmosets. We are now performing studies to determine whether the observed changes in different lipid species could influence the development of cardiovascular disease and provide new insights underlying the mechanisms of NAFLD development with aging.

Canagliflozin (Cana) is an inhibitor of the sodium glucose transporter 2 (SGLT2), and is thought to act by blocking renal reuptake and intestinal absorption of glucose. Cana is FDA-approved for treatment of diabetes, and affords protection from cardiovascular and kidney diseases. In the context of the mouse Interventions Testing Program, genetically heterogeneous mice were given chow containing 180 ppm Cana at 7 months of age until their death. Cana extended median survival of male mice by 14%, with p < 0.001 by log-rank test. Cana also increased by 9% the age for 90th percentile survival (p < 0.001 by Wang/Allison test), with parallel effects seen at each of three test sites. Cana did not alter the distribution of inferred cause of death, nor of incidental pathology findings at end-of-life necropsies. No benefits were seen in female mice. The lifespan benefit of Cana is likely to reflect blunting of peak glucose levels, because similar longevity effects are seen in 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.

Understanding masseter muscle (MM) innervation is critical for the study of cell-specific mechanisms of pain induced by temporomandibular disorder or after facial surgery. Here, we identified trigeminal (TG) sensory neuronal subtypes (MM TG neurons) innervating masseter muscle fibers, masseteric fascia, tendons, and adjusted tissues. A combination of patch clamp electrophysiology and immunohistochemistry on TG neurons backtraced from reporter mouse MM found 9 distinct subtypes of MM TG neurons. Of these neurons, 24% belonged to non-peptidergic IB-4+/TRPA1- or IB-4+/TRPA1+ groups, while two TRPV1+ small-sized neuronal groups were classified as peptidergic/CGRP+ One small-sized CGRP+ neuronal group had a unique electrophysiological profile and were recorded from Nav1.8- or trkC+ neurons. The remaining CGRP+ neurons were medium-sized, could be divided into Nav1.8-/trkC- and Nav1.8low/trkC+ clusters, and showed large 5HT-induced current. The final two MM TG neuronal groups were trkC+ and had no Nav1.8 and CGRP. Among MM TG neurons, TRPV1+/CGRP- (somatostatin+), tyrosine hydroxylase+ (C-LTMR), TRPM8+, MrgprA3+ or trkB+ (Aδ-LTMR) subtypes have not been detected. Masseteric muscle fibers, tendons and masseteric fascia in mice and the common marmoset, a new world monkey, were exclusively innervated by either CGRP+/NFH+ or CGRP-/NFH+ medium-to-large neurons, which we found using a Nav1.8-YFP reporter, and labeling with CGRP, TRPV1, neurofilament heavy chain (NFH) and pgp9.5 antibodies. These nerves were mainly distributed in tendon and at junctions of deep-middle-superficial parts of MM. Overall, the data presented here demonstrates that MM is innervated by a distinct subset of TG neurons, which have unique characteristics and innervation patterns.Significance StatementIdentification of sensory neuron subtypes innervating masseter muscle (MM) will enable the study of cell-specific mechanisms of masticatory myofascial pain, including temporomandibular disorder and after restorative surgeries involving MM. Combining back tracing from MM, patch-clamp electrophysiology, and immunohistochemistry with sensory neuronal markers on mouse and nonhuman primate tissues, we identified trigeminal neuronal groups innervating MM (MM TG neurons). MM and adjacent tissues are innervated by 9 distinct types of TG neurons, some of which are substantially different from L3-L5 DRG neurons. However, MM fibers, tendon, muscle-tendon junction, and fascia in mice and common marmosets are exclusively innervated by medium-to-large neurons. These neurofilament heavy chain-positive sensory nerve fibers are mainly distributed in tendon and at junctions between deep, middle, and superficial parts of MM.

Mechanisms that preserve and maintain the cellular proteome are associated with long life and healthy aging. Oxidative damage is a significant contributor to perturbation of proteostasis and is dealt with by the cell through regulation of antioxidants, protein degradation, and repair of oxidized amino acids. Methionine sulfoxide reductase A (MsrA) repairs oxidation of free- and protein-bound methionine residues through enzymatic reduction and is found in both the cytosol and the mitochondria. Previous studies in Drosophila have shown that increasing expression of MsrA can extend longevity. Here we test the effects of increasing MsrA on longevity and healthy aging in two transgenic mouse models. We show that elevated expression of MsrA targeted specifically to the cytosol reduces the rate of age-related death in female mice when assessed by Gompertz analysis. However, neither cytosolic nor mitochondrial MsrA overexpression extends lifespan when measured by log-rank analysis. In mice with MsrA overexpression targeted to the mitochondria, we see evidence for improved insulin sensitivity in aged female mice. With these and our previous data, we conclude that the increasing MsrA expression in mice has differential effects on aging and healthy aging that are dependent on the target of its subcellular localization., Graphical abstract fx1

While caloric restriction (CR) provides highly robust improvements to longevity and health, dietary restriction of the essential amino acid methionine can provide similar benefits including improved metabolic function and increased longevity. Despite these similarities between CR and methionine restriction (MR), there is growing evidence to suggest they may be mediated by different mechanisms that require further elucidation. The sulfur side-chain of methionine is highly prone to oxidation, even in vivo, with redox changes of these residues potentially altering protein function and interfering with its use as a substrate. An entire family of enzymes, methionine sulfoxide reductases, have evolved in aerobic organisms to regulate the redox status of methionine. We tested the role of methionine sulfoxide reductase A (MsrA) in the physiological and metabolic benefits of MR. After three months of MR, mice lacking MsrA (MsrA KO) showed significant loss of weight, including both fat and lean mass, in comparison to wild-type mice under MR. Both MsrA KO and wild-type mice responded to MR with improvements to both glucose and insulin tolerance. However, MR MsrA KO mice showed lower HbA1c and reduced leptin compared to MR wild-type mice. Overall, our results show mice lacking MsrA have a stronger response to MR suggesting that methionine redox may play an important role in some of the mechanisms responsible for these metabolic outcomes. Further studies clarify whether MsrA could also be a potential regulator of the longevity benefits of MR.

Metformin has beneficial effects on several age-related diseases (e.g., diabetes, obesity, cancer) and extends lifespan in nematodes and mice. Acarbose, an FDA-approved agent for treating type 2 diabetes, prevents breakdown of complex carbohydrates. Both compounds have been suggested as potential anti-aging interventions and acarbose has been shown to extend mouse longevity by the Intervention Testing Program (ITP). One potential next step is to assess the effect of these interventions on healthspan and lifespan in non-human primates. The common marmoset (Callithrix jacchus) is a small new world monkey with a relatively short life span and small size, both valuable for the translation potential of this nonhuman primate species for the study of aging and chronic disease. However, the dosing and assessment of potential side effects of either metformin or acarbose in this species have yet to be assessed. This study evaluated the pharmacokinetics of two dosage levels each of metformin or acarbose (given separately) in two small groups of young marmosets (n = 5/group) treated for 24 h to define the pharmacokinetics of each drug. The ability to rapidly and reliably dose socially housed marmosets with an oral form of acarbose or metformin that is well tolerated indicates that this species is a reliable model for testing acarbose and metformin in a safe and efficient way in a long-term intervention.

Aging is associated with progressive loss of cellular homeostasis which results from intrinsic and extrinsic challenges. Testing how such challenges relate to the aging process is often limited by the available model systems. We use primary cells (fibroblasts) isolated from baboons as a non-human primate cellular model to address how stressful challenges affect resilience. Using a real-time live-cell imaging system, we characterized a protocol for testing the effects of pro-oxidant compounds (e.g hydrogen peroxide (H2O2), paraquat and thapsigargin), dexamethasone and low glucose environment on cellular proliferation in fibroblasts derived from baboons across the life-course. The inhibitory effect of H2O2 (50 and 100µM), an oxidative stress, on cell proliferation was dose-dependent, with a higher impact in old males (age 14.52-14.80 years; average life span 21 years) compared to young males (6.35-6.39 years). Exposure to a different oxidative stress, paraquat (100 and 200µM) tended to reduce cell proliferation rate with age in males but not females. Inhibitory effects of thapsigargin, an endoplasmic reticulum stress inducer, on cell proliferation were dependent on challenge duration (2 vs 24h), concentration (0.1 and 1µM) and donor age, with greater resilience in young males than young females (4.33-6.70 years). Dexamethasone (100 and 500µM), a glucocorticoid, reduced cell proliferation dose-dependently, with older males exhibiting more resilience than females. In response to low glucose (1mM), cell proliferation reduced with age. Donor’s chronological age and sex are important variables in cellular response to challenge compounds faced during aging, which will guide our on-going studies on the cellular transcriptome and proteome.

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-alpha-estradiol and nordihydroguaiaretic acid (NDGA), at doses greater and less than used previously. 17-alpha-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 alpha-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.

Rapamycin has been shown to extend lifespan in rodent models, but the effects on metabolic health and function have been widely debated in both clinical and translational trials. Prior to rapamycin being used as a treatment to extend both lifespan and healthspan in the human population, it is vital to assess the side effects of the treatment on metabolic pathways in animal model systems, including a closely related non-human primate model. In this study, we found that long-term treatment of marmoset monkeys with orally-administered encapsulated rapamycin resulted in no overall effects on body weight and only a small decrease in fat mass over the first few months of treatment. Rapamycin treated subjects showed no overall changes in daily activity counts, blood lipids, or significant changes in glucose metabolism including oral glucose tolerance. Adipose tissue displayed no differences in gene expression of metabolic markers following treatment, while liver tissue exhibited suppressed G6Pase activity with increased PCK and GPI activity. Overall, the marmosets revealed only minor metabolic consequences of chronic treatment with rapamycin and this adds to the growing body of literature that suggests that chronic and/or intermittent rapamycin treatment results in improved health span and metabolic functioning. The marmosets offer an interesting alternative animal model for future intervention testing and translational modeling.

The ability of an organism to respond to physical stresses and return to homeostasis (i.e. resilience) has been suggested to correlate with longevity. Here, we investigated whether this extends to resilience at a cellular level using primary fibroblasts isolated from tail skin of genetically heterogeneous young adult UM-HET3 mice. Cells isolated from each individual mouse (cell line) were tested in their response to concentrations of agents or conditions predicted to induce a cellular challenge, including paraquat, hydrogen peroxide, antimycin a, cadmium chloride, mdivi-1, thapsigargin, and nutrient starvation. Cell viability was monitored in real-time using an incucyte S3 live cell analysis system and we addressed the response following challenge as a marker of resilience. Cellular uptake of ethidium homodimer-1 was used to determine the loss of viability. Cellular bioenergetics were assessed using a seahorse XF24. We found that cell lines that were resistant to paraquat were also resistant to antimycin a, and hydrogen peroxide. Cell lines that were resistant to nutrient starvation were also resistant to mdivi-1. Indices of cellular bioenergetics status including ATP production rate and cell respiratory control ratio, revealed potential relationships with resiliency. Taken together, our data indicate that skin fibroblasts retain individual physiological programs that may in part explain the patterns of resiliency or sensitivity to a stressor at the organismal level. Since the cell lines tested in this study were obtained from living mice, future work will investigate whether these patterns of resiliency change with age and elucidate their utility in predicting future health outcome.

β-Guanidinopropionic acid (β-GPA) is a naturally occurring compound reported to activate AMP Activated Protein Kinase (AMPK) signaling in vivo. Acute administration of β-GPA in young animals has been reported to improve multiple functional measures including a switch to oxidative fatigue-resistant muscle fibers, improved glucose uptake, and increased mitochondrial biogenesis. However, it is unknown if β-GPA may promote healthy aging or prevent late-life functional decline. To address this knowledge gap, we tested the effects of β-GPA on mitochondrial energetics and cellular function in young and old genetically heterogenous mice (HET3). Both age groups were fed either 1% β-GPA or control chow for ~5 months and basic functional parameters including metabolism were assessed. β-GPA treatment decreased lean and fat mass in young males, but prevented late-life losses in these parameters in old animals. Notably, glycated hemoglobin (HbA1c) levels were lower in treated young and old males suggesting improved glucose homeostasis. Citrate synthase activity was also higher in old males fed β-GPA suggesting increased mitochondrial biogenesis. At the molecular level, mitochondrial Complex I expression decreased with β-GPA treatment in old males versus controls. High resolution respirometry revealed generally decreased respiration in old animals compared to young and decreased Complex 1 coupled respiration in soleus of β-GPA treated young but not old males. These findings indicate that the mitochondrial effects attributed to β-GPA may be mediated by its action on Complex I. While treatment outcomes varied in young and old males these results suggest β-GPA may prove beneficial in combating age-related declines in function.

Age is a primary risk factor for osteoarthritis (OA). The mechanisms that contribute to OA are poorly understood and disease modifying treatments have not been identified. A critical shortcoming in developing therapies is the limited number of translational models available to identify the causes of naturally occurring OA. Our goal is to use the common marmoset as a non-human primate (NHP) model of age-related OA. NHP are the closest evolutionary relative to humans and share many characteristics of human aging. The marmoset has advantages over other NHP for aging research because of their relatively short maximal lifespan and small size. Micro-computed tomography (uCT) was performed on whole-knee joints obtained from young (10 yrs, n=3) marmosets at necropsy. OA was evaluated using a clinical uCT scoring system and quantitative assessments of subchondral bone structure and ossified meniscal volume. Advancing age was positively correlated to increased uCT OA score (p

Interventions to extend lifespan and improve health with increasing age will have significant impact on a growing aged population. Several pharmaceutical interventions extend lifespan in laboratory rodent models with rapamycin, an inhibitor of mechanistic target of rapamycin (mTOR) being the most well studied. Bridging towards translation, we have an ongoing long-term study testing whether rapamycin treatment can extend lifespan and delay the progression of age-related disease in a short-lived non-human primate species, the common marmoset (Callithrix jacchus). We show that daily oral dosing of slow-releasing, encapsulated rapamycin will result in clinically effective concentrations of rapamycin in the blood and inhibit mTOR signaling. This treatment is well tolerated and does not dramatically promote known side effects of this drug, including altering clinical hematology, immune cell subsets, or promoting metabolic dysfunction including glucose intolerance in comparison to control aging marmosets. Unlike previous reports in rodents, rapamycin does not have clear effects on aging cardiovascular function in marmosets. However, in our oldest cohorts daily rapamycin treatment tends to prevent age-associated changes in body mass and composition and prevent decline in kidney function. Now more than three years after beginning treatment, we are now starting to assess the effects of rapamycin on marmoset longevity. When complete, this study will describe for the first time the potential for pharmaceutical intervention to extend longevity of a primate species with the ultimate goal of significant translational impact to human aging.

4-Phenylbutyrate (PBA) is a FDA approved drug for treating patients with urea cycle disorders. Additionally, PBA acts upon several pathways thought of as important modifiers of aging including: histone deacetylation, proteostasis as a chemical chaperone, and stress resistance by regulating expression of oxidative stress response proteins. PBA has also been shown to extend lifespan and improve markers of age-related health in Drosophila. Due to its wide range of effects PBA has been investigated for use in numerous age-related disorders including neurodegenerative and cardiovascular diseases. To better understand the effects of PBA on the molecular level, we used both in cellulo and in vivo studies. Treatment of primary mouse fibroblasts, C2C12 mouse muscle cells, and NCTC 1469 mouse liver cells with PBA demonstrated differential responses among cell lines to upregulation of oxidative stress response and histone acetylation. Specifically, upregulation of the oxidative stress response protein DJ-1 by PBA was found to have a corresponding dose response curve to histone H3 acetylation in primary fibroblasts. To study effects of PBA in vivo, four cohorts of HET3 mice were treated with PBA at different doses in drinking water for 4 weeks. PBA was well tolerated and led to different effects on body composition dependent on the sex of mice. We are currently investigating the molecular effects of PBA treatment in multiple tissues samples from these mice. The potential of PBA to alter many fundamental pathways, and specifically those related to stress responses, make it an attractive prospect for treatment of many age-related disorders.

Compelling data exist for programming of chronic later-life diseases and longevity by perinatal developmental programming challenges. Understanding mechanisms by which life course health trajectory and longevity are set is fundamental to understanding aging. Appropriate approaches are needed to determine programming effects on cellular function. We have developed a baboon model in which control mothers eat ad libitum while a second group eat 70% of the global diet fed controls, leading to male and female offspring intrauterine growth restriction (IUGR). We have shown that IUGR suffer from acceleration of several age-related physiological declines. Here, we report on a skin-derived fibroblast model with potential relevance for mechanistic studies on how IUGR impacts aging. Fibroblasts were cultured from the skin biopsies taken from adult baboons from control and IUGR cohorts. IUGR-derived fibroblasts grew in culture less well than controls and those derived from male, but not female, IUGR baboons had a significant reduction in maximum respiration rate compared to control-derived fibroblasts. We also show that relative levels of several mitochondrial protein subunits, including NDUFB8 and cytochrome c oxidase subunit IV, were reduced in IUGR-derived fibroblasts even after serial passaging in culture. The lower levels of electron transport system components provide potential mechanisms for accelerated life course aging in the setting of programmed IUGR. This observation fits with the greater sensitivity of males compared with females to many, but not all, outcomes in response to programming challenges. These approaches will be powerful in the determination of programming-aging interactions.

Aging is associated with replacement of normal kidney parenchyma by fibrosis. Because hydrogen sulfide (H2S) ameliorates kidney fibrosis in disease models, we examined its status in the aging kidney. In the first study, we examined kidney cortical H2S metabolism and signaling pathways related to synthesis of proteins including matrix proteins in young and old male C57BL/6 mice. In old mice, increase in renal cortical content of matrix protein involved in fibrosis was associated with decreased H2S generation and AMPK activity, and activation of insulin receptor (IR)/IRS-2-Akt-mTORC1-mRNA translation signaling axis that can lead to increase in protein synthesis. In the second study, we randomized 18–19 month-old male C57BL/6 mice to receive 30 μmol/L sodium hydrosulfide (NaHS) in drinking water vs. water alone (control) for 5 months. Administration of NaHS increased plasma free sulfide levels. NaHS inhibited the increase in kidney cortical content of matrix proteins involved in fibrosis and ameliorated glomerulosclerosis. NaHS restored AMPK activity and inhibited activation of IR/IRS-2-Akt-mTORC1-mRNA translation axis. NaHS inhibited age-related increase in kidney cortical content of p21, IL-1β, and IL-6, components of the senescence-associated secretory phenotype. NaHS abolished increase in urinary albumin excretion seen in control mice and reduced serum cystatin C levels suggesting improved glomerular clearance function. We conclude that aging-induced changes in the kidney are associated with H2S deficiency. Administration of H2S ameliorates aging-induced kidney changes probably by inhibiting signaling pathways leading to matrix protein synthesis.

Inflammation and oxidative stress through the production of reactive oxygen species (ROS) are consistently associated with metabolic syndrome/type 2 diabetes. Although the role of Nox2, a major ROS-generating enzyme, is well described in host defense and inflammation, little is known about its potential role in insulin resistance in skeletal muscle. Insulin resistance induced by a high fat diet was mitigated in Nox2-null mice compared with wild-type mice after 3 or 9 months on the diet. High fat feeding increased Nox2 expression, superoxide production, and impaired insulin signaling in skeletal muscle tissue of wild-type mice but not in Nox2-null mice. Exposure of C2C12 cultured myotubes to either high glucose concentration, palmitate, or H2O2 decreases insulin-induced Akt phosphorylation and glucose uptake. Pretreatment with catalase abrogated these effects, indicating a key role for H2O2 in mediating insulin resistance. Down-regulation of Nox2 in C2C12 cells by shRNA prevented insulin resistance induced by high glucose or palmitate but not H2O2. These data indicate that increased production of ROS in insulin resistance induced by high glucose in skeletal muscle cells is a consequence of Nox2 activation. This is the first report to show that Nox2 is a key mediator of insulin resistance in skeletal muscle.

The extension of lifespan due to reduced insulin-like growth factor 1 (IGF-I) signaling in mice has been proposed to be mediated through alterations in metabolism. Previously, we showed that mice homozygous for an insertion in the Igf1 allele have reduced levels of IGF-I, are smaller, and have an extension of maximum lifespan. Here, we tested whether this specific reduction of IGF-I alters glucose metabolism both on normal rodent chow and in response to high-fat feeding. We found that female IGF-I-deficient mice were lean on a standard rodent diet but paradoxically displayed an insulin-resistant phenotype. However, these mice gained significantly less weight than normal controls when placed on a high-fat diet. In control animals, insulin response was significantly impaired by high-fat feeding, whereas IGF-I-deficient mice showed a much smaller shift in insulin response after high-fat feeding. Gluconeogenesis was also elevated in the IGF-I-deficient mice relative to controls on both normal and high-fat diet. An analysis of metabolism and respiratory quotient over 24 h indicated that the IGF-I-deficient mice preferentially utilized fatty acids as an energy source when placed on a high-fat diet. These results indicate that reduction in the circulating and tissue IGF-I levels can produce a metabolic phenotype in female mice that increases peripheral insulin resistance but renders animals resistant to the deleterious effects of high-fat feeding.