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Caloric restriction (CR) is the leading non-pharmacological intervention to delay induced and spontaneous tumors in pre-clinical models. These effects of CR are largely attributed to canonical inhibition of pro-growth pathways. However, our recent data suggest that CR impairs primary tumor growth and cancer progression in the murine 4T1 model of triple negative breast cancer (TNBC), at least in part, through reduced frequency of the myeloid-derived suppressor cells (MDSC). In the present study, we sought to determine whether injection of excess MDSCs could block regression in 4T1 tumor growth and metastatic spread in BALB/cJ female mice undergoing daily CR. Our findings show that MDSC injection impeded CR-mediated protection against tumor growth without increasing lung metastatic burden. Overall, these results reveal that CR can slow cancer progression by affecting immune suppressive cells. Impact statement: Inoculation of MDSCs from donor mice effectively impedes the ability of calorie restriction to protect against primary tumor growth without impacting lung metastatic burden in recipient animals. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11357-022-00635-y.

In population studies, dietary patterns clearly influence the development, progression, and therapeutic response of cancers. Nonetheless, interventional dietary trials have had relatively little impact on the prevention and treatment of malignant disease. Standardization of nutritional interventions combined with high-level mode-of-action studies holds the promise of identifying specific entities and pathways endowed with antineoplastic properties. Here, we critically review the effects of caloric restriction and more specific interventions on macro- and micronutrients in preclinical models as well as in clinical studies. We place special emphasis on the prospect of using defined nutrition-relevant molecules to enhance the efficacy of established anticancer treatments. Significance: The avoidance of intrinsically hypercaloric and toxic diets contributes to the prevention and cure of cancer. In addition, specific diet-induced molecules such as ketone bodies and micronutrients, including specific vitamins, have drug-like effects that are clearly demonstrable in preclinical models, mostly in the context of immunotherapies. Multiple trials are underway to determine the clinical utility of such molecules.

To define metrics of phenotypic aging, it is essential to identify biological and environmental factors that influence the pace of aging. Previous attempts to develop aging metrics were hampered by cross-sectional designs and/or focused on younger populations. In the Baltimore Longitudinal Study of Aging (BLSA), we collected longitudinally across the adult age range a comprehensive list of phenotypes within four domains (body composition, energetics, homeostatic mechanisms and neurodegeneration/neuroplasticity) and functional outcomes. We integrated individual deviations from population trajectories into a global longitudinal phenotypic metric of aging and demonstrate that accelerated longitudinal phenotypic aging is associated with faster physical and cognitive decline, faster accumulation of multimorbidity and shorter survival. These associations are more robust compared with the use of phenotypic and epigenetic measurements at a single time point. Estimation of these metrics required repeated measures of multiple phenotypes over time but may uniquely facilitate the identification of mechanisms driving phenotypic aging and subsequent age-related functional decline.

Supplementary Table from Science-Driven Nutritional Interventions for the Prevention and Treatment of Cancer

In population studies, dietary patterns clearly influence the development, progression, and therapeutic response of cancers. Nonetheless, interventional dietary trials have had relatively little impact on the prevention and treatment of malignant disease. Standardization of nutritional interventions combined with high-level mode-of-action studies holds the promise of identifying specific entities and pathways endowed with antineoplastic properties. Here, we critically review the effects of caloric restriction and more specific interventions on macro- and micronutrients in preclinical models as well as in clinical studies. We place special emphasis on the prospect of using defined nutrition-relevant molecules to enhance the efficacy of established anticancer treatments.Significance:The avoidance of intrinsically hypercaloric and toxic diets contributes to the prevention and cure of cancer. In addition, specific diet-induced molecules such as ketone bodies and micronutrients, including specific vitamins, have drug-like effects that are clearly demonstrable in preclinical models, mostly in the context of immunotherapies. Multiple trials are underway to determine the clinical utility of such molecules.

Sulfonylureas (SUs) are effective and affordable antidiabetic drugs. However, chronic use leads to secondary failure, limiting their utilization. Here, we identify cytochrome b5 reductase 3 (Cyb5r3) down-regulation as a mechanism of secondary SU failure and successfully reverse it. Chronic exposure to SU lowered Cyb5r3 abundance and reduced islet glucose utilization in mice in vivo and in ex vivo murine islets. Cyb5r3 β cell–specific knockout mice phenocopied SU failure. Cyb5r3 engaged in a glucose-dependent interaction that stabilizes glucokinase (Gck) to maintain glucose utilization. Hence, Gck activators can circumvent Cyb5r3-dependent SU failure. A Cyb5r3 activator rescued secondary SU failure in mice in vivo and restored insulin secretion in ex vivo human islets. We conclude that Cyb5r3 is a key factor in the secondary failure to SU and a potential target for its prevention, which might rehabilitate SU use in diabetes.

BackgroundAging is associated with increased levels of reactive oxygen species and inflammation that disrupt proteostasis and mitochondrial function and leads to organism-wide frailty later in life. ARA290 (cibinetide), an 11-aa non-hematopoietic peptide sequence within the cardioprotective domain of erythropoietin, mediates tissue protection by reducing inflammation and fibrosis. Age-associated cardiac inflammation is linked to structural and functional changes in the heart, including mitochondrial dysfunction, impaired proteostasis, hypertrophic cardiac remodeling, and contractile dysfunction. Can ARA290 ameliorate these age-associated cardiac changes and the severity of frailty in advanced age?MethodsWe conducted an integrated longitudinal (n = 48) and cross-sectional (n = 144) 15 months randomized controlled trial in which 18-month-old Fischer 344 x Brown Norway rats were randomly assigned to either receive chronic ARA290 treatment or saline. Serial echocardiography, tail blood pressure and body weight were evaluated repeatedly at 4-month intervals. A frailty index was calculated at the final timepoint (33 months of age). Tissues were harvested at 4-month intervals to define inflammatory markers and left ventricular tissue remodeling. Mitochondrial and myocardial cell health was assessed in isolated left ventricular myocytes. Kaplan–Meier survival curves were established. Mixed ANOVA tests and linear mixed regression analysis were employed to determine the effects of age, treatment, and age-treatment interactions.ResultsChronic ARA290 treatment mitigated age-related increases in the cardiac non-myocyte to myocyte ratio, infiltrating leukocytes and monocytes, pro-inflammatory cytokines, total NF-κB, and p-NF-κB. Additionally, ARA290 treatment enhanced cardiomyocyte autophagy flux and reduced cellular accumulation of lipofuscin. The cardiomyocyte mitochondrial permeability transition pore response to oxidant stress was desensitized following chronic ARA290 treatment. Concurrently, ARA290 significantly blunted the age-associated elevation in blood pressure and preserved the LV ejection fraction. Finally, ARA290 preserved body weight and significantly reduced other markers of organism-wide frailty at the end of life.ConclusionAdministration of ARA290 reduces cell and tissue inflammation, mitigates structural and functional changes within the cardiovascular system leading to amelioration of frailty and preserved healthspan.

Mitochondrial biogenesis and destruction in skeletal muscle are coordinated by distinct signaling pathways that are influenced by internal and exogenous variables including, but not limited to, muscle phenotype, physical activity, dietary composition, or drug administration. Previously we found that long-term resveratrol administration (up to 480 mg/day) ameliorates the slow-to-fast phenotypic shift in soleus muscles and promotes the expression in slow myosin heavy chain in the mixed plantaris muscle of non-human primates consuming a high fat/sugar (HFS) diet. Here, we expand on these earlier findings by examining whether mitochondrial content and the markers that dictate their biogenesis and mitophagy/autophagy are similarly affected by HFS and/or influenced by resveratrol while consuming this diet (HFSR). Compared to controls (n=9), there was a ~20–25% decrease in mitochondrial content in HFS (n=8) muscles as reflected in the COX2- and CYTB-to-GAPDH ratios using PCR analysis, which was blunted by resveratrol in HFSR (n=7) soleus and, to a lesser degree, in plantaris muscles. A ~1.5 and 3-fold increase in Rev-erb-α protein was detected in HFSR soleus and plantaris muscles compared to controls, respectively. Unlike in HFSR animals, HFS soleus and plantaris muscles exhibited a ~2-fold elevation in phosphor-AMPKα (Thr172). HFS soleus muscles had elevated phosphorylated-to-total TANK binding protein-1 (TBK1) ratio suggesting an enhancement in mito/autophagic events. Taken together, resveratrol appears to blunt mitochondrial losses with a high fat / sugar diet by tempering mito/autophagy rather than promoting mitochondrial biogenesis, suggesting that the quantity of daily resveratrol supplement ingested and / or its long-term consumption are important considerations.

Age-dependent differences in methylation at specific cytosine–guanine (CpG) sites have been used in “epigenetic clock” formulas to predict age. Deviations of epigenetic age from chronological age are informative of health status and are associated with adverse health outcomes, including mortality. In most cases, epigenetic clocks are performed on methylation from DNA extracted from circulating blood cells. However, the effect of neoplastic cells in the circulation on estimation and interpretation of epigenetic clocks is not well understood. Here, we explored this using Fischer 344 (F344) rats, a strain that often develops large granular lymphocyte leukemia (LGLL). We found clear histological markers of LGLL pathology in the spleens and livers of 27 out of 61 rats aged 17–27 months. We assessed DNA methylation by reduced representation bisulfite sequencing with coverage of 3 million cytosine residues. Although LGLL broadly increased DNA methylation variability, it did not change epigenetic aging. Despite this, the inclusion of rats with LGLL in clock training sets significantly altered predictor selection probability at 83 of 121 commonly utilized CpG sites. Furthermore, models trained on rat samples that included individuals with LGLL had greater absolute age error than those trained exclusively rats free of LGLL (39% increase; p < .0001). We conclude that the epigenetic signals for aging and LGLL are distinct, such that LGLL assessment is not necessary for valid measures of epigenetic age in F344 rats. The precision and architecture of constructed epigenetic clock formulas, however, can be influenced by the presence of neoplastic hematopoietic cells in training set populations.

The discovery of drugs targeting the fundamental mechanisms of aging (herein called ‘aging drugs’) poses a far greater challenge than the discovery of drugs for individual diseases. There are multi...

Sublethal cell damage can trigger a complex adaptive program known as senescence, characterized by growth arrest, resistance to apoptosis, and a senescence-associated secretory phenotype (SASP). As senescent cells accumulating in aging organs are linked to many age-associated diseases, senotherapeutic strategies are actively sought to eliminate them. Here, a whole-genome CRISPR knockout screen revealed that proteins in the YAP-TEAD pathway influenced senescent cell viability. Accordingly, treating senescent cells with a drug that inhibited this pathway, Verteporfin (VPF), selectively triggered apoptotic cell death and derepressed DDIT4, in turn inhibiting mTOR. Reducing mTOR function in senescent cells diminished endoplasmic reticulum (ER) biogenesis, causing ER stress and apoptosis due to high demands on ER function by the SASP. Importantly, VPF treatment decreased senescent cell numbers in the organs of old mice and mice exhibiting doxorubicin-induced senescence. We present a novel senolytic strategy that eliminates senescent cells by hindering ER activity required for SASP production.

Adult (3 month) mice with cardiac-specific overexpression of adenylyl cyclase (AC) type VIII (TGAC8) adapt to an increased cAMP-induced cardiac workload (~30% increases in heart rate, ejection fraction and cardiac output) for up to a year without signs of heart failure or excessive mortality. Here, we show classical cardiac hypertrophy markers were absent in TGAC8, and that total left ventricular (LV) mass was not increased: a reduced LV cavity volume in TGAC8 was encased by thicker LV walls harboring an increased number of small cardiac myocytes, and a network of small interstitial proliferative non-cardiac myocytes compared to wild type (WT) littermates; Protein synthesis, proteosome activity, and autophagy were enhanced in TGAC8 vs WT, and Nrf-2, Hsp90α, and ACC2 protein levels were increased. Despite increased energy demands in vivo LV ATP and phosphocreatine levels in TGAC8 did not differ from WT. Unbiased omics analyses identified more than 2,000 transcripts and proteins, comprising a broad array of biological processes across multiple cellular compartments, which differed by genotype; compared to WT, in TGAC8 there was a shift from fatty acid oxidation to aerobic glycolysis in the context of increased utilization of the pentose phosphate shunt and nucleotide synthesis. Thus, marked overexpression of AC8 engages complex, coordinate adaptation "circuity" that has evolved in mammalian cells to defend against stress that threatens health or life (elements of which have already been shown to be central to cardiac ischemic pre-conditioning and exercise endurance cardiac conditioning) that may be of biological significance to allow for proper healing in disease states such as infarction or failure of the heart.

Citrate is a critical metabolic substrate and key regulator of energy metabolism in mammalian cells. It has been known for decades that the skeleton contains most (>85%) of the body’s citrate, but the question of why and how this metabolite should be partitioned in bone has received singularly little attention. Here, we show that osteoblasts use a specialized metabolic pathway to regulate uptake, endogenous production, and the deposition of citrate into bone. Osteoblasts express high levels of the membranous Na + -dependent citrate transporter solute carrier family 13 member 5 ( Slc13a5 ) gene. Inhibition or genetic disruption of Slc13a5 reduced osteogenic citrate uptake and disrupted mineral nodule formation. Bones from mice lacking Slc13a5 globally, or selectively in osteoblasts, showed equivalent reductions in cortical thickness, with similarly compromised mechanical strength. Surprisingly, citrate content in mineral from Slc13a5 −/− osteoblasts was increased fourfold relative to controls, suggesting the engagement of compensatory mechanisms to augment endogenous citrate production. Indeed, through the coordinated functioning of the apical membrane citrate transporter SLC13A5 and a mitochondrial zinc transporter protein (ZIP1; encoded by Slc39a1 ), a mediator of citrate efflux from the tricarboxylic acid cycle, SLC13A5 mediates citrate entry from blood and its activity exerts homeostatic control of cytoplasmic citrate. Intriguingly, Slc13a5 -deficient mice also exhibited defective tooth enamel and dentin formation, a clinical feature, which we show is recapitulated in primary teeth from children with SLC13A5 mutations. Together, our results reveal the components of an osteoblast metabolic pathway, which affects bone strength by regulating citrate deposition into mineral hydroxyapatite.

Cellular senescence is characterized by cell cycle arrest, resistance to apoptosis, and a senescence-associated secretory phenotype (SASP) whereby cells secrete pro-inflammatory and tissue-remodeling factors. Given that the SASP exacerbates age-associated pathologies, some aging interventions aim at selectively eliminating senescent cells. In this study, a drug library screen uncovered TrkB (NTRK2) inhibitors capable of triggering apoptosis of several senescent, but not proliferating, human cells. Senescent cells expressed high levels of TrkB, which supported senescent cell viability, and secreted the TrkB ligand BDNF. The reduced viability of senescent cells after ablating BDNF signaling suggested an autocrine function for TrkB and BDNF, which activated ERK5 and elevated BCL2L2 levels, favoring senescent cell survival. Treatment with TrkB inhibitors reduced the accumulation of senescent cells in aged mouse organs. We propose that the activation of TrkB by SASP factor BDNF promotes cell survival and could be exploited therapeutically to reduce the senescent-cell burden.

Skeletal muscle adapts to different exercise training modalities with age; however, the impact of both variables at the systemic and tissue levels is not fully understood. Here, adult and old C57BL/6 male mice were assigned to one of three groups: sedentary, daily high-intensity intermittent training (HIIT), or moderate intensity continuous training (MICT) for 4 weeks, compatible with the older group’s exercise capacity. Improvements in body composition, fasting blood glucose, and muscle strength were mostly observed in the MICT old group, while effects of HIIT training in adult and old animals was less clear. Skeletal muscle exhibited structural and functional adaptations to exercise training, as revealed by electron microscopy, OXPHOS assays, respirometry, and muscle protein biomarkers. Transcriptomics analysis of gastrocnemius muscle combined with liver and serum metabolomics unveiled an age-dependent metabolic remodeling in response to exercise training. These results support a tailored exercise prescription approach aimed at improving health and ameliorating age-associated loss of muscle strength and function in the elderly., This work was supported by funding from the Intramural Research Program of the National Institute on Aging/NIH. Work in JMV laboratory was supported by the Spanish Ministerio de Economía y Competitividad (MINECO) grant BFU2015-64630-R, Ministerio de Ciencia, Innovación y Universidades (MICIU) grant RTI2018-100695-B-I00, Spanish Junta de Andalucía grants P18-RT-4264, 1263735-R and BIO-276, the FEDER Funding Program from the European Union, and Universidad de Córdoba. MCR was supported by a FPU fellowship from the Spanish Ministerio de Educación, Cultura y Deporte (reference FPU14/06308). SRL held a FPI predoctoral contract funded by MINECO (reference BES-2016-078229).

Nutrition profoundly influences the risk for many age-related diseases. Whether nutrition influences human aging biology directly is less clear. Studies in different animal species indicate that reducing food intake ("caloric restriction" [CR]) can increase lifespan and delay the onset of diseases and the biological hallmarks of aging. Obesity has been described as "accelerated aging" and therefore the lifespan and health benefits generated by CR in both aging and obesity may occur via similar mechanisms. Beyond calorie intake, studies based on nutritional geometry have shown that protein intake and the interaction between dietary protein and carbohydrates influence age-related health and lifespan. Studies where animals are calorically restricted by providing free access to diluted diets have had less impact on lifespan than those studies where animals are given a reduced aliquot of food each day and are fasting between meals. This has drawn attention to the role of fasting in health and aging, and exploration of the health effects of various fasting regimes. Although definitive human clinical trials of nutrition and aging would need to be unfeasibly long and unrealistically controlled, there is good evidence from animal experiments that some nutritional interventions based on CR, manipulating dietary macronutrients, and fasting can influence aging biology and lifespan.

Distinct and shared pathways of health and lifespan can be untangled following a concerted approach led by experimental design and a rigorous analytical strategy where the confounding effects of diet and feeding regimens can be dissected. In this chapter, we use integrated analysis of multiomics (transcriptomics-metabolomics) data in liver from mice to gain insight into pathways associated with improved health and survival. We identify a unique metabolic hub involving glycine-serine-threonine metabolism at the core of lifespan, and a pattern of shared pathways related to improved health.

Increased age is blamed for a wide range of bone physiological changes, and although the underlying mechanisms affecting the decreased capacity for fracture healing are not fully understood, they are clearly linked to changes at the cellular level. Recent evidence suggests potential roles of senescent cells in response to most tissue injuries, including bone fractures. In this issue of the JCI, Liu, Zhang, and co-authors showed that a senolytic drug cocktail cleared senescent cells from the callus and improved bone fracture repair in aged mice. Understanding how senescent cells emerge at fracture sites and how their timely removal improves fracture healing should provide insights for effective therapeutic approaches in old age.

Males and females may respond differently to medications, yet knowledge about sexual dimorphisms in the effects of polypharmacy remains limited, particularly in aging. This study aimed to assess the effect of high Drug Burden Index (DBI) polypharmacy treatment compared to control on physical function and behavior in young and old, male and female mice. We studied whether age and sex play a role in physical function and behavior following polypharmacy treatment and whether they are paralleled by differences in serum drug levels. Young (2.5 months) and old (21.5 months), C57BL/6 mice were randomized to control or high DBI polypharmacy treatment (simvastatin, metoprolol, oxybutynin, oxycodone, and citalopram; n = 6–8/group) for 4–6 weeks. Compared to control, polypharmacy reduced physical function (grip strength, rotarod latency, gait speed, and total distance), middle zone distance (increased anxiety), and nesting score (reduced activities of daily living) in mice of both ages and sexes (p < .001). Old animals had a greater decline in nesting score (p < .05) and midzone distance (p < .001) than young animals. Grip strength declined more in males than females (p < .05). Drug levels at steady state were not significantly different between polypharmacy-treated animals of both ages and sexes. We observed polypharmacy-induced functional impairment in both age and sex groups, with age and sex interactions in the degree of impairment, which were not explained by serum drug levels. Studies of the pathogenesis of functional impairment from polypharmacy may improve management strategies in both sexes.

Aging is one of the greatest risk factors for cancers, and epigenetic changes involving DNA methylation show close similarities during age-related cellular processes and tumor development. We have shown orthogonal relationships between the accrual of aberrant DNA methylation changes associated with cell division cycles during senescence and transformation. Consequently, we have hypothesized that while both senescent and proliferative aging cells coexist in the same tissues, the risk for cancer most likely comes from, and due to, epigenetic changes occurring in the latter, long-term, self-renewing cells. Using colon organoid models, we have studied the importance of genes undergoing DNA methylation mediated silencing during aging and their role in predisposing to tumorigenesis. Analyses of the in vivo and ex vivo aging-associated epigenetic/expression changes indicate the importance of age-related aberrant methylation in tumor promoting states. Citation Format: Daniel A. Petkovich, Rachael Powers, Yong Tao, Byunghak Kang Nibedita Patel, Kathy Gabrielson, Rafael de Cabo, Stephen B. Baylin, Hariharan P. Easwaran. Aberrant age-related methylation leads to tumor predisposition in colorectal organoids [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr B016.

Aging is associated with functional and metabolic decline and is a risk factor for all noncommunicable diseases. Even though mice are routinely used for modeling human aging and aging-related conditions, no comprehensive assessment to date has been conducted on normative mouse aging. To address this gap, the Study of Longitudinal Aging in Mice (SLAM) was designed and implemented by the National Institute on Aging (NIA/NIH) as the mouse counterpart to the Baltimore Longitudinal Study of Aging (BLSA). In this manuscript, we describe the premise, study design, methodologies, and technologies currently employed in SLAM. We also discuss current and future study directions. In this large population mouse study, inbred C57BL/6J and outbred UM-HET3 mice of both sexes are longitudinally evaluated for functional, phenotypic, and biological health, and collection of biospecimens is conducted throughout their life span. Within the longitudinal cohorts, a cross-sectional arm of the study has also been implemented for the well-controlled collection of tissues to generate a biorepository. SLAM and studies stemming from SLAM seek to identify and characterize phenotypic and biological predictors of mouse aging and age-associated conditions, examine the degrees of functional and biomolecular variability that occur within inbred and genetically heterogeneous mouse populations with age, and assess whether these changes are consistent with alterations observed in human aging in BLSA. The findings from these studies will be critical for evaluating the utility of mouse models for studying different aspects of aging, both in terms of interpreting prior findings and designing and implementing future studies.

SummarySulfonylureas (SU) are effective and affordable anti-diabetic drugs. But chronic use leads to secondary failure, limiting their utilization. The mechanism of secondary failure is unknown. Here we identify Cyb5r3 downregulation as a mechanism of SU failure and successfully reverse it. Chronic exposure to SU impairs Cyb5r3 levels and reduces islet glucose utilization with a metabolomics signature characterized by low acetyl-CoA and amino acid levels. Cyb5r3 engages in a glucose-dependent interaction that stabilizes glucokinase (Gck) to maintain glucose utilization. Accordingly, activating Gck mutations in patients with hyperinsulinemia reduce Cyb5r3 binding, whereas inactivating MODY mutations increase it, providing evidence for a role of Cyb5r3 in determining flux through Gck. The Cyb5r3 activator tetrahydroindenoindole (THII) rescues secondary failure to SU in an animal model of chronic SU treatment and restores insulin secretion from ex vivo islets. We conclude that Cyb5r3 loss-of-function is a key factor in the secondary failure to SU and a potential target for its prevention, which may lead to a rehabilitation of SU use in diabetes.

ADCK2 haploinsufficiency-mediated mitochondrial coenzyme Q deficiency in skeletal muscle causes mitochondrial myopathy associated with defects in beta-oxidation of fatty acids, aged-matched metabolic reprogramming, and defective physical performance. Calorie restriction has proven to increase lifespan and delay the onset of chronic diseases associated to aging. To study the possible treatment by food deprivation, heterozygous Adck2 knockout mice were fed under 40% calorie restriction (CR) and the phenotype was followed for 7 months. The overall glucose and fatty acids metabolism in muscle was restored in mutant mice to WT levels after CR. CR modulated the skeletal muscle metabolic profile of mutant mice, partially rescuing the profile of WT animals. The analysis of mitochondria isolated from skeletal muscle demonstrated that CR increased both CoQ levels and oxygen consumption rate (OCR) based on both glucose and fatty acids substrates, along with mitochondrial mass. The elevated aerobic metabolism fits with an increase of type IIa fibers, and a reduction of type IIx in mutant muscles, reaching WT levels. To further explore the effect of CR over muscle stem cells, satellite cells were isolated and induced to differentiate in culture media containing serum from animals in either ad libitum or CR diets for 72 h. Mutant cells showed slower differentiation alongside with decreased oxygen consumption. In vitro differentiation of mutant cells was increased under CR serum reaching levels of WT isolated cells, recovering respiration measured by OCR and partially beta-oxidation of fatty acids. The overall increase of skeletal muscle bioenergetics following CR intervention is paralleled with a physical activity improvement, with some increases in two and four limbs strength tests, and weights strength test. Running wheel activity was also partially improved in mutant mice under CR. These results demonstrate that CR intervention, which has been shown to improve age-associated physical and metabolic decline in WT mice, also recovers the defective aerobic metabolism and differentiation of skeletal muscle in mice caused by ADCK2 haploinsufficiency., This work was supported by Junta de Andalucía grant BIO-177, the Instituto de Salud Carlos III FIS grant FIS PI20/00541, CIBERER (U729)-ISCIII, the FEDER Funding Program from the European Union and the Spanish Ministry of Science, Innovation and Universities grant RED2018-102576-T. This work was supported by the Spanish Ministry of Education, Culture and Sports through fellowship FPU16/03264 to JH-C, and the Association Française contre les Myopathies (AFM) through fellowship grant #22450 to CV-G. This work was funded in part by the Intramural Research Program of the National Institute on Aging, NIH. This research was also supported by the Instituto de Salud Carlos III (PI19/01310) (Co-funded by the European Union) and by the Agència de Gestió d'Ajuts Universitaris i de Recerca (AGAUR) (2017: SGR 1428) and the CERCA.

SUMMARYThere is currently no established intervention for Cockayne syndrome, a disease characterized by progressive early onset neurodegeneration with features of premature aging. Here, we tested if acetyl-CoA precursors, citrate and beta-hydroxybutyrate, could reduce features of Cockayne syndrome in three model systems. We identified the gene Helicase 89B as a homologue of CSB in drosophila and found that the ketone beta-hydroxybutyrate rescued features of premature aging in Hel89B deficient flies. In mammals, loss of the citrate carrier Indy exacerbated the phenotype of Csbm/m mice which was rescued by a ketogenic diet. The rescue effect appeared to be mediated through ketone stimulated histone acetylation and facilitation of transcriptional readthrough of secondary DNA structures. These findings link a ketogenic diet with transcriptional resolution of secondary structures and DNA repair.

The combined influences of sinoatrial nodal (SAN) pacemaker cell automaticity and its response to autonomic input determine the heart’s beating interval variability and mean beating rate. To determine the intrinsic SAN and autonomic signatures buried within EKG RR interval time series change in advanced age, we measured RR interval variability before and during double autonomic blockade at 3-month intervals from 6 months of age until the end of life in long-lived (those that achieved the total cohort median life span of 24 months and beyond) C57/BL6 mice. Prior to 21 months of age, time-dependent changes in intrinsic RR interval variability and mean RR interval were relatively minor. Between 21 and 30 months of age, however, marked changes emerged in intrinsic SAN RR interval variability signatures, pointing to a reduction in the kinetics of pacemaker clock mechanisms, leading to reduced synchronization of molecular functions within and among SAN cells. This loss of high-frequency signal processing within intrinsic SAN signatures resulted in a marked increase in the mean intrinsic RR interval. The impact of autonomic signatures on RR interval variability were net sympathetic and partially compensated for the reduced kinetics of the intrinsic SAN RR interval variability signatures, and partially, but not completely, shifted the EKG RR time series intervals to a more youthful pattern. Cross-sectional analyses of other subsets of C57/BL6 ages indicated that at or beyond the median life span of our longitudinal cohort, noncardiac, constitutional, whole-body frailty was increased, energetic efficiency was reduced, and the respiratory exchange ratio increased. We interpret the progressive reduction in kinetics in intrinsic SAN RR interval variability signatures in this context of whole-body frailty beyond 21 months of age to be a manifestation of “heartbeat frailty.”

Cellular senescence is characterized by cell cycle arrest, resistance to apoptosis, and a senescence-associated secretory phenotype (SASP) whereby cells secrete pro-inflammatory and tissue-remodeling factors. Given that the SASP exacerbates age-associated pathologies, some aging interventions aim at selectively eliminating senescent cells. In this study, a drug library screen uncovered TrkB (NTRK2) inhibitors selectively capable of triggering apoptosis of senescent, but not proliferating, human fibroblasts. Senescent cells expressed high levels of TrkB, which supported senescent cell viability, and secreted the TrkB ligand BDNF. The reduced viability of senescent cells after ablating BDNF signaling suggested an autocrine function for TrkB and BDNF, including increased expression of BCL2L2 downstream of BDNF-TrkB, favoring senescent cell survival. Strikingly, treatment with TrkB inhibitors reduced the accumulation of senescent cells in aged mouse organs. We propose that the activation of TrkB by SASP factor BDNF promotes cell survival and could be exploited therapeutically to reduce the senescent-cell burden.

The goal of the current study was to determine the role of maternal diet in the perinatal period on the health and survival of the offspring. AKR/J mice, a model described to be susceptible to leukemia development, was used where females were maintained on either standard diet (SD), high sucrose diet, Western diet, or calorie restriction (CR) as they were mated with SD-fed males. Body weights, pregnancy rates, litter size, and litter survival were used as markers of successful pregnancy and pup health. Data indicated that maternal diet had significant effects on litter size, early pup survival, and early pup body weights. As pups matured, the makeup of their respective maternal diet was a predictor of adult metabolic health and survival. Overall, these results suggest that perinatal maternal diet is an important determinant of the health and survival of the offspring and that these effects continue well into adulthood, strongly correlating with lifespan.

Effects of Intermittent Fasting on Health and Aging Evidence is accumulating that eating in a 6-hour period and fasting for 18 hours can trigger a metabolic switch from glucose-based to ketone-base...

ATP synthase (F1Fo) synthesizes daily our body's weight in ATP, whose production-rate can be transiently increased several-fold to meet changes in energy utilization. Using purified mammalian F1Fo-reconstituted proteoliposomes and isolated mitochondria, we show F1Fo can utilize both ΔΨm-driven H+- and K+-transport to synthesize ATP under physiological pH = 7.2 and K+ = 140 mEq/L conditions. Purely K+-driven ATP synthesis from single F1Fo molecules measured by bioluminescence photon detection could be directly demonstrated along with simultaneous measurements of unitary K+ currents by voltage clamp, both blocked by specific Fo inhibitors. In the presence of K+, compared to osmotically-matched conditions in which this cation is absent, isolated mitochondria display 3.5-fold higher rates of ATP synthesis, at the expense of 2.6-fold higher rates of oxygen consumption, these fluxes being driven by a 2.7:1 K+: H+ stoichiometry. The excellent agreement between the functional data obtained from purified F1Fo single molecule experiments and ATP synthase studied in the intact mitochondrion under unaltered OxPhos coupling by K+ presence, is entirely consistent with K+ transport through the ATP synthase driving the observed increase in ATP synthesis. Thus, both K+ (harnessing ΔΨm) and H+ (harnessing its chemical potential energy, ΔμH) drive ATP generation during normal physiology.

Purpose Age-related cataracts affect the majority of older adults and are a leading cause of blindness worldwide. Treatments that delay cataract onset or severity have the potential to delay cataract surgery, but require relevant animal models that recapitulate the major types of cataracts for their development. Unfortunately, few such models are available. Here, we report the lens phenotypes of aged mice lacking the critical antioxidant transcription factor Nfe2l2 (designated as Nrf2 −/−). Methods Three independent cohorts of Nrf2 −/− and wild-type C57BL/6J mice were evaluated for cataracts using combinations of slit lamp imaging, photography of freshly dissected lenses, and histology. Mice were fed high glycemic diets, low glycemic diets, regular chow ad libitum, or regular chow with 30% caloric restriction. Results Nrf2 −/− mice developed significant opacities between 11 and 15 months and developed advanced cortical, posterior subcapsular, anterior subcapsular, and nuclear cataracts. Cataracts occurred similarly in male mice fed high or low glycemic diets, and were also observed in 21-month male and female Nrf2 −/− mice fed ad libitum or 30% caloric restriction. Histological observation of 18-month cataractous lenses revealed significant disruption to fiber cell architecture and the retention of nuclei throughout the cortical region of the lens. However, fiber cell denucleation and initiation of lens differentiation was normal at birth, with the first abnormalities observed at 3 months. Conclusions Nrf2 −/− mice offer a tool to understand how defective antioxidant signaling causes multiple forms of cataract and may be useful for screening drugs to prevent or delay cataractogenesis in susceptible adults.

Mild uncoupling of oxidative phosphorylation is an intrinsic property of all mitochondria and may have evolved to protect cells against the production of damaging reactive oxygen species. Therefore, compounds that enhance mitochondrial uncoupling are potentially attractive anti-aging therapies; however, chronic ingestion is associated with a number of unwanted side effects. We have previously developed a controlled-release mitochondrial protonophore (CRMP) that is functionally liver-directed and promotes oxidation of hepatic triglycerides by causing a subtle sustained increase in hepatic mitochondrial inefficiency. Here, we sought to leverage the higher therapeutic index of CRMP to test whether mild mitochondrial uncoupling in a liver-directed fashion could reduce oxidative damage and improve age-related metabolic disease and lifespan in diet-induced obese mice. Oral administration of CRMP (20 mg/[kg-day] × 4 weeks) reduced hepatic lipid content, protein kinase C epsilon activation, and hepatic insulin resistance in aged (74-week-old) high-fat diet (HFD)-fed C57BL/6J male mice, independently of changes in body weight, whole-body energy expenditure, food intake, or markers of hepatic mitochondrial biogenesis. CRMP treatment was also associated with a significant reduction in hepatic lipid peroxidation, protein carbonylation, and inflammation. Importantly, long-term (49 weeks) hepatic mitochondrial uncoupling initiated late in life (94-104 weeks), in conjugation with HFD feeding, protected mice against neoplastic disorders, including hepatocellular carcinoma (HCC), in a strain and sex-specific manner. Taken together, these studies illustrate the complex variation of aging and provide important proof-of-concept data to support further studies investigating the use of liver-directed mitochondrial uncouplers to promote healthy aging in humans.

ATP synthase (F(1)F(o)) synthesizes daily our body's weight in ATP, whose production-rate can be transiently increased several-fold to meet changes in energy utilization. Using purified mammalian F(1)F(o)-reconstituted proteoliposomes and isolated mitochondria, we show F(1)F(o) can utilize both ΔΨ(m)-driven H(+)- and K(+)-transport to synthesize ATP under physiological pH = 7.2 and K(+) = 140 mEq/L conditions. Purely K(+)-driven ATP synthesis from single F(1)F(o) molecules measured by bioluminescence photon detection could be directly demonstrated along with simultaneous measurements of unitary K(+) currents by voltage clamp, both blocked by specific F(o) inhibitors. In the presence of K(+), compared to osmotically-matched conditions in which this cation is absent, isolated mitochondria display 3.5-fold higher rates of ATP synthesis, at the expense of 2.6-fold higher rates of oxygen consumption, these fluxes being driven by a 2.7:1 K(+): H(+) stoichiometry. The excellent agreement between the functional data obtained from purified F(1)F(o) single molecule experiments and ATP synthase studied in the intact mitochondrion under unaltered OxPhos coupling by K(+) presence, is entirely consistent with K(+) transport through the ATP synthase driving the observed increase in ATP synthesis. Thus, both K(+) (harnessing ΔΨ(m)) and H(+) (harnessing its chemical potential energy, Δμ(H)) drive ATP generation during normal physiology.

A female UM-HET3 mouse from the Study of Longitudinal Aging in Mice (SLAM) was euthanized at 164 weeks of age due to hind limb weakness. Necropsy and histological analysis revealed that the most probable cause of the clinical finding was the compression of the thoracolumbar segment of the spinal cord by herniated intervertebral disks. In addition, a spontaneous chordoma was incidentally found in the coccygeal bones. Given the rarity of this type of tumor, bio-clinical annotations acquired throughout lifespan, detailed histopathological assessment, and comparative clinical-pathological correlations for this mouse are presented and discussed. Keywords: Geropathology, geroscience, study of longitudinal aging in mice (SLAM), spontaneous neoplasm, tumor burden, chordoma

According to a commonly held view, the obesity pandemic is caused by overconsumption of modern, highly palatable, energy-dense processed foods, exacerbated by a sedentary lifestyle. However, obesity rates remain at historic highs, despite a persistent focus on eating less and moving more, as guided by the energy balance model (EBM). This public health failure may arise from a fundamental limitation of the EBM itself. Conceptualizing obesity as a disorder of energy balance restates a principle of physics without considering the biological mechanisms that promote weight gain. An alternative paradigm, the carbohydrate-insulin model (CIM), proposes a reversal of causal direction. According to the CIM, increasing fat deposition in the body—resulting from the hormonal responses to a high-glycemic-load diet—drives positive energy balance. The CIM provides a conceptual framework with testable hypotheses for how various modifiable factors influence energy balance and fat storage. Rigorous research is needed to compare the validity of these 2 models, which have substantially different implications for obesity management, and to generate new models that best encompass the evidence.

Aging is associated with distinct phenotypical, physiological, and functional changes, leading to disease and death. The progression of aging-related traits varies widely among individuals, influenced by their environment, lifestyle, and genetics. In this study, we conducted physiologic and functional tests cross-sectionally throughout the entire lifespan of male C57BL/6N mice. In parallel, metabolomics analyses in serum, brain, liver, heart, and skeletal muscle were also performed to identify signatures associated with frailty and age-dependent functional decline. Our findings indicate that declines in gait speed as a function of age and frailty are associated with a dramatic increase in the energetic cost of physical activity and decreases in working capacity. Aging and functional decline prompt organs to rewire their metabolism and substrate selection and toward redox-related pathways, mainly in liver and heart. Collectively, the data provide a framework to further understand and characterize processes of aging at the individual organism and organ levels., eLife, 10, ISSN:2050-084X

Conserved mechanisms of aging often illuminate the development of novel therapeutic interventions to delay aging and the onset of age-related diseases and functional decline. Recently in Cell Metabolism, Zivanovic et al. (2019) demonstrated a decline during aging of the protective H2S-dependent protein persulfidation across species and its reversal by targeted strategies.

The American Geriatrics Society convened a conference in Bethesda, Maryland, to explore models and studies of aging. This was the second of three conferences, supported by a U13 grant from the National Institute on Aging, to aid recipients of Grants for Early Medical/Surgical Specialists Transition to Aging Research (GEMSSTAR) in integrating geriatrics into their specialties. Recognizing that aging is the largest risk factor for multiple chronic diseases and age-related loss of resilience, the conference organizers focused scientific sessions on how targeting age-related mechanisms can delay, prevent, or reverse geriatric syndromes, age-related chronic diseases, and loss of resilience. The rationale for studying models of aging as well as study designs, strategies, and challenges of studying human aging were reviewed. This article provides a summary of the full conference report, Models and Studies of Aging: Report from the U13 Conference Series, and summarizes key take-home messages that were designed to support GEMSSTAR awardees in developing their research careers focused on aging research (see supplementary text for the full report). J Am Geriatr Soc 67:428-433, 2019.

Use of different objective frailty assessment tools may improve understanding of the biology of frailty and allow evaluation of effects of interventions on frailty. Polypharmacy is associated with increased risk of frailty in epidemiologic studies, regardless of frailty definition, but the pathophysiology of the association is not well understood. This study aims to (1) assess and compare the prevalence of frailty from middle to old age following control, chronic polypharmacy or monotherapy treatment, when measured using the clinical frailty index assessment and the mouse frailty phenotype tools; and (2) to evaluate and compare the effects of chronic polypharmacy regimens with zero, low and high Drug Burden Index (DBI) and monotherapies from middle to old age on the rate of deficit accumulation on the frailty index, mean number of phenotype criteria, odds of being frail assessed by the frailty index or phenotype, and the time to onset of frailty assessed by the frailty index or phenotype. In a longitudinal study, middle-aged (12 months) male C57BL/6J(B6) mice were administered non medicated control feed and water, or therapeutic doses of different polypharmacy combinations or monotherapies in feed and/or water. Frailty assessments were performed at 12, 15, 18, 21 and 24 months. There was limited overlap between animals identified as frail using different frailty assessments. Polypharmacy has measurable and different effects on each frailty assessment. Long-term chronic administration of some polypharmacy and monotherapy therapeutic drug regimens increased the number of frailty deficits (clinical frailty index: low DBI polypharmacy (15 and 21 months), high DBI polypharmacy (15-21 months), oxycodone (15-18 months), oxybutynin (15-18 months), citalopram (15-21 months) and metoprolol monotherapy (15 months) and modified frailty phenotype assessment (over the whole duration of treatment, low DBI polypharmacy (adjusted Risk Ratio(aRR) = 1.97, 95% confidence interval (CI) 1.43-2.72), high DBI polypharmacy (aRR = 1.88; 95% CI 1.36-2.60), oxybutynin (aRR = 1.48; 95% CI 1.01-2.16) and citalopram monotherapy (aRR = 1.96; 95% CI 1.41-2.74), p 0.05) . The odds of developing frailty measured with the clinical frailty index increased with high DBI polypharmacy (adjusted odds ratio (aOR) = 3.13; 95% CI 1.01-9.66) and when measured with the frailty phenotype assessment increased with low DBI polypharmacy (aOR = 4.38, 95% CI 1.40-13.74), high DBI polypharmacy (aOR = 3.43; 95% CI 1.12-10.50) and citalopram monotherapy (aOR = 4.63; 95% CI 1.39-15.54)). No treatment affected time to frailty using either frailty assessment. Analysis of the number of deficits on the frailty index or number of positive criteria on the frailty phenotype allows analysis of rate of change and provides greater sensitivity, while the odds of being frail analysis provided a clinically relevant indicator of whether mice had greater chance of reaching a cut-off for becoming frail with medication exposure than without. Our results are consistent with clinical studies, demonstrating that certain polypharmacy regimens induce frailty, with different relationships observed when using different frailty assessments and analyses.

Nutrient composition and caloric intake have traditionally been used to devise optimized diets for various phases of life. Adjustment of meal size and frequency have emerged as powerful tools to ameliorate and postpone the onset of disease and delay aging, whereas periods of fasting, with or without reduced energy intake, can have profound health benefits. The underlying physiological processes involve periodic shifts of metabolic fuel sources, promotion of repair mechanisms, and the optimization of energy utilization for cellular and organismal health. Future research endeavors should be directed to the integration of a balanced nutritious diet with controlled meal size and patterns and periods of fasting to develop better strategies to prevent, postpone, and treat the socioeconomical burden of chronic diseases associated with aging.

The global human population has recently experienced an increase in life expectancy with a mounting concern about the steady rise in the incidence of age-associated chronic diseases and socio-economic burden. Calorie restriction (CR), the reduction of energy intake without malnutrition, is a dietary manipulation that can increase health and longevity in most model organisms. However, the practice of CR in day-to-day life is a challenging long-term goal for human intervention. Recently, daily fasting length and periodicity have emerged as potential drivers behind CR's beneficial health effects. Numerous strategies and eating patterns have been successfully developed to recapitulate many of CR's benefits without its austerity. These novel feeding protocols range from shortened meal timing designed to interact with our circadian system (e.g., daily time-restricted feeding) to more extended fasting regimens known as intermittent fasting. Here, we provide a glimpse of the current status of knowledge on different strategies to reap the benefits of CR on metabolic health in murine models and in humans, without the rigor of continuous reduction in caloric intake as presented at the USU State of the Science Symposium.

Cadmium exposure is ubiquitous and has been linked to diseases including cancers and reproductive defects. Since cadmium is nonmutagenic, it is thought to exert its gene dysregulatory effects through epigenetic reprogramming. Several studies have implicated germline exposure to cadmium in developmental reprogramming. However, most of these studies have focused on maternal exposure, while the impact on sperm fertility and disease susceptibility has received less attention. In this study, we used reduced representation bisulfite sequencing to comprehensively investigate the impact of chronic cadmium exposure on mouse spermatozoa DNA methylation. Adult male C57BL/J6 mice were provided water with or without cadmium chloride for 9 weeks. Sperm, testes, liver, and kidney tissues were collected at the end of the treatment period. Cadmium exposure was confirmed through gene expression analysis of metallothionein-1 and 2, 2 well-known cadmium-induced genes. Analysis of sperm DNA methylation changes revealed 1788 differentially methylated sites present at regulatory regions in sperm of mice exposed to cadmium compared with vehicle (control) mice. Furthermore, most of these differential methylation changes positively correlated with changes in gene expression at both the transcription initiation stage as well as the splicing levels. Interestingly, the genes targeted by cadmium exposure are involved in several critical developmental processes. Our results present a comprehensive analysis of the sperm methylome in response to chronic cadmium exposure. These data, therefore, highlight a foundational framework to study gene expression patterns that may affect fertility in the exposed individual as well as their offspring, through paternal inheritance.

Cancer incidence increases with age and is a leading cause of death. Caloric restriction (CR) confers benefits on health and survival and delays cancer. However, due to CR’s stringency, dietary alternatives offering the same cancer protection have become increasingly attractive. Short cycles of a plant-based diet designed to mimic fasting (FMD) are protective against tumorigenesis without the chronic restriction of calories. Yet, it is unclear whether the fasting time, level of dietary restriction, or nutrient composition is the primary driver behind cancer protection. Using a breast cancer model in mice, we compare the potency of daily CR to that of periodic caloric cycling on FMD or an isocaloric standard laboratory chow against primary tumor growth and metastatic burden. Here, we report that daily CR provides greater protection against tumor growth and metastasis to the lung, which may be in part due to the unique immune signature observed with daily CR., Caloric restriction (CR) has been shown as an effective intervention to reduce tumorigenesis, but alternative less stringent dietary interventions have also been considered. Here, the authors show that in a murine model of breast cancer CR has a larger effect on preventing tumorigenesis and metastasis compared to periodic caloric cycling.

A feeding schedule of prolonged overnight fasting periods extends healthy lifespan in fruit flies by promoting night-time autophagy, a process in which material in cells is degraded and recycled. Autophagy helps flies on an intermittent fasting schedule live longer.