Caloric restriction and physical exercise have proven beneficial against age-associated changes in body composition and declining physical performance; however, little is known regarding what benefit these interventions might have when initiated late in life. The study of mimetics of diet and exercise and the combination thereof may provide additional treatments for a vulnerable elderly population; however, how and when to initiate such interventions requires consideration in developing the most safe and efficacious treatment strategies. In this review, we focus on preclinical late-life intervention studies, which assess the relationship between physical function, sarcopenia, and body composition. We provide a conceptual framework for the ever-changing definition of sarcopenia and a rationale for the use of an appropriate rodent model of this condition. We finish by providing our perspective regarding the implications of this body of work and future areas of research that may also contribute to the ultimate goal of extending healthspan.

In this study, we explored the mechanisms by which the angiotensin converting enzyme inhibitor (ACEI), enalapril, and the Ang II receptor blocker (ARB), losartan suppress oxidative stress and NF-κB activation-induced inflammatory responses in aged rat kidney. The experimentations were carried out utilizing aged (24-month-old) Brown Norway x Fischer 344 (F1) male rats which were randomized into 3 groups and administered enalapril (40 mg/kg), losartan (30 mg/kg) or placebo for 6 months (daily p.o.). The level of reactive species (RS), peroxynitrite (ONOO−), GSH/GSSG and lipid peroxidation were measured. The activity of the pro-inflammatory transcription factor NF-κB, and gene expression of proteins in upstream signaling cascades were measured by electro-mobility shift assay (EMSA) and Western blotting. Enalapril and losartan differentially attenuated redox imbalance and the redox-sensitive transcription factor, NF-κB pathway. Furthermore, stimulation of the NF-κB activation pathway by phosphorylation of p65 was attenuated by both compounds. Moreover, mediation of phosphorylation of p65 by phosphorylation of IκB kinase αβ (IKKαβ) and mitogen- and stress-activated protein kinase-1 (MSK1), were also inhibited by enalapril and losartan. Finally, both compounds also lowered expression of NF-κB-dependent inflammatory genes, such as cyclooxygenase-2 (COX-2),) and inducible NO synthase (iNOS). Only losartan lowered levels of 5-lipoxygenase (5-LOX). These findings indicate that enalapril and losartan differentially suppress inflammatory responses via inhibition of oxidative stress-induced NF-κB activation in aged rat kidney.

The primary purpose of the present set of studies was to provide a direct comparison of the effects of the angiotensin-converting enzyme inhibitor enalapril and the angiotensin receptor blocker losartan on body composition, physical performance, and muscle quality when administered late in life to aged rats. Overall, enalapril treatment consistently attenuated age-related increases in adiposity relative to both placebo and losartan. The maximal effect was achieved after 3 months of treatment (between 24 and 27 months of age), at a dose of 40 mg/kg and was observed in the absence of any changes in physical activity, body temperature, or food intake. In addition, the reduction in fat mass was not due to changes in pathology given that enalapril attenuated age-related increases in tumor development relative to placebo- and losartan-treated animals. Both enalapril and losartan attenuated age-related decreases in grip strength, suggesting that changes in body composition appear dissociated from improvements in physical function and may reflect a differential impact of enalapril and losartan on muscle quality. To link changes in adiposity to improvements in skeletal muscle quality, we performed gene array analyses to generate hypotheses regarding cell signaling pathways altered with enalapril treatment. Based on these results, our primary follow-up pathway was mitochondria-mediated apoptosis of myocytes. Relative to losartan- and placebo-treated rats, only enalapril decreased DNA fragmentation and caspase-dependent apoptotic signaling. These data suggest that attenuation of the severity of skeletal muscle apoptosis promoted by enalapril may represent a distinct mechanism through which this compound improves muscle strength/quality.

To test the hypothesis that exercise increases central leptin signaling, and thus reduces dietary weight gain in an aged obese model, we assessed the effects of voluntary wheel running (WR) in 23-month-old F344×BN rats fed a 60% high-fat (HF) diet for 3 months. After 2 months on the HF diet, half of the rats were provided access to running wheels for 2 weeks while the other half remained sedentary. Following the removal of the wheels, physical performance was evaluated, and 4 weeks later leptin signaling was assessed in hypothalamus and VTA after an acute bout of WR. Introduction of a HF diet led to prolonged hyperphagia (63.9 ± 7.8 kcal/day on chow diet vs. 88.1 ± 8.2 kcal/day on high-fat diet (when food intake stabilized), p < 0.001). As little as 9 (ranging to 135) wheel revolutions per day significantly reduced caloric consumption of HF food (46.8 ± 11.2 kcal/day) to a level below that on chow diet (63.9 ± 7.8 kcal/day, p < 0.001). After 2 weeks of WR, body weight was significantly reduced (7.9 ± 2.1% compared with prerunning weight, p < 0.001), and physical performance (latency to fall from an incline plane) was significantly improved (p = 0.04). WR significantly increased both basal (p = 0.04) and leptin-stimulated (p = 0.001) STAT3 phosphorylation in the ventral tegmental area (VTA), but not in the hypothalamus. Thus, in aged dietary obese rats, the act but not the extent of voluntary WR is highly effective in reversing HF consumption, decreasing body weight, and improving physical performance. It appears to trigger a response that substitutes for the reward of highly palatable food that may be mediated by increased leptin signaling in the VTA.

Pro-opiomelanocortin (POMC) neurons are identified in two brain sites, the arcuate nucleus (ARC) of the hypothalamus and nucleus of the solitary tract (NTS) in brainstem. Earlier pharmacological and POMC gene transfer studies demonstrate melanocortin activation in either site alone improves insulin sensitivity and reduces obesity. The present study, for the first time, investigated the long-term efficacy of POMC gene transfer concurrently into both sites in the regulation of energy metabolism in aged F344xBN rats bearing adult-onset obesity. Pair feeding was included to reveal food-independent POMC impact on energy expenditure. We introduced adeno-associated virus encoding either POMC or green fluorescence protein to the two brain areas in 22-month-old rats, then recorded food intake and body weight, assessed oxygen consumption, serum leptin, insulin and glucose, tested voluntary wheel running, analyzed POMC expression, and examined fat metabolism in brown and white adipose tissues. POMC mRNA was significantly increased in both the hypothalamus and NTS region at termination. Relative to pair feeding, POMC caused sustained weight reduction and additional fat loss, lowered fasting insulin and glucose, and augmented white fat hormone-sensitive lipase activity and brown fat uncoupling protein 1 level. By wheel running assessment, the POMC-animals ran twice the distance as the control or pair-fed rats. Thus, the dual-site POMC treatment ameliorated adult-onset obesity effectively, involving a moderate hypophagia lasting ~ 60 days, enhanced lipolysis and thermogenesis, and increased physical activity in the form of voluntary wheel running. The latter finding provides a clue for countering age-related decline in physical activity.

There is growing concern over the increasing use of opioids to treat chronic pain in the elderly primarily because of the potential increased sensitivity to the adverse side effects. Here, we use a preclinical model (male Brown Norway X F344 rats aged 12, 18, 24, and 30 months) to describe the outcome of chronic fentanyl administration (1.0 mg/kg/day) on various physiological and behavioral measures. Continuous fentanyl administration resulted in an initial decrease in food consumption, followed by the development of tolerance to this effect over a 4-week period and a subsequent increase in food consumption during withdrawal. This change in food consumption was associated with decreases in body weight (predominantly due to a loss of fat mass) that was maintained through early withdrawal. After one month of withdrawal, only the 12-month old animals had fully regained body weight. Fentanyl administration resulted in a decrease in grip strength and an increase in locomotor activity that did not differ across age groups. There was no effect of fentanyl administration on rotarod performance. These results demonstrate that while there is a delayed recovery of body mass with age, the observed changes in behavioral responses are uniform across ages.

Age-dependent renal damage is influenced by genetic background and the Fisher344xBrown Norway (F344xBN) rat is resistant to glomerular injury. In vulnerable strains, a fall in renal nitric oxide synthase (NOS) contributes to age-dependent renal damage. Here, we investigated renal NOS in young (3 months) and old (30 months) male F344xBN to test the hypothesis that renal NOS is maintained in “protected” strains. We also examined if 6 months of renin-angiotensin system (RAS) blockade using angiotensin converting enzyme inhibition (ACEI) and angiotensin receptor blockade (ARB) provides further benefit in these “protected” old rats. Aging increased tubulointerstitial injury but glomerular sclerosis was minimal and NOS and superoxide dismutase abundance increased. There was no change in the NOS inhibitor, ADMA (asymmetric dimethylarginine) or its regulatory enzymes. RAS blockade with ARB protected against tubulointerstitial injury and increased nNOSα, but ACEI, which also increased nNOSα, had no protective effect on the tubulointerstitium. We conclude that the glomerular sclerosis-resistant aged male F344xBN rat maintains renal NOS, thus reinforcing our hypothesis that progressive glomerular injury is related to renal NOS deficiency. The tubulointerstitial injury seen with aging is reversed with 6 months of ARB but not ACEI and is not associated with renal NOS.

Synopsis

Evidence from animal models and preliminary studies in humans indicate that calorie restriction (CR) delays cardiac aging and can prevent cardiovascular disease. These effects are mediated by a wide spectrum of biochemical and cellular adaptations, including redox homeostasis, mitochondrial function, inflammation, apoptosis and autophagy. Despite the beneficial effects of CR, its large-scale implementation is challenged by applicability issues as well as health concerns. However, preclinical studies indicate that specific compounds, such as resveratrol, may mimic many of the effects of CR, thus potentially obviating the need for drastic food intake reductions. Results from ongoing clinical trials will reveal whether the intriguing alternative of CR mimetics represents a safe and effective strategy to promote cardiovascular health and delay cardiac aging in humans.

Controversy exists as to whether lifelong 40% calorie restriction (CR) enhances, has no effect on, or disrupts cognitive function during aging. Here, we report the effects of CR versus ad-lib feeding on cognitive function in male Brown Norway × Fisher344 rats across a range of ages (8–38 months), using two tasks that are differentially sensitive to age-related cognitive decline: object recognition and Morris water maze (MWM). All ages performed equally in object recognition, whereas, as a group, CR rats were impaired. In contrast, there was an age-related impairment in the MWM that was attenuated by CR as measured by time in proximity with and latency to reach the platform. Distance to the platform, a more sensitive measure, was not affected by CR. Finally, CR resulted in an overall increase in physical activity, one of several behavioral confounders to consider in the interpretation of cognitive outcomes in both tasks.

Background

Mitochondrial dysfunction and oxidative stress are central mechanisms underlying the aging process and the pathogenesis of many age-related diseases. Selected antioxidants and specific combinations of nutritional compounds could target many biochemical pathways that affect both oxidative stress and mitochondrial function and, thereby, preserve or enhance physical performance.

Methodology/Principal Findings

In this study, we evaluated the potential anti-aging benefits of a Q-ter® based nutritional mixture (commercially known as Eufortyn®) mainly containing the following compounds: terclatrated coenzyme Q10 (Q-ter®), creatine and a standardized ginseng extract. We found that Eufortyn® supplementation significantly ameliorated the age-associated decreases in grip strength and gastrocnemius subsarcolemmal mitochondria Ca2+ retention capacity when initiated in male Fischer344 x Brown Norway rats at 21 months, but not 29 months, of age. Moreover, the increases in muscle RNA oxidation and subsarcolemmal mitochondrial protein carbonyl levels, as well as the decline of total urine antioxidant power, which develop late in life, were mitigated by Eufortyn® supplementation in rats at 29 months of age.

Conclusions/Significance

These data imply that Eufortyn® is efficacious in reducing oxidative damage, improving the age-related mitochondrial functional decline, and preserving physical performance when initiated in animals at early midlife (21 months). The efficacy varied, however, according to the age at which the supplementation was provided, as initiation in late middle age (29 months) was incapable of restoring grip strength and mitochondrial function. Therefore, the Eufortyn® supplementation may be particularly beneficial when initiated prior to major biological and functional declines that appear to occur with advancing age.

The purpose of this review is to describe how recent pharmacological and genetic studies have contributed to our understanding of the role of the renin–angiotensin system (RAS) in age-related sarcopenia and diastolic dysfunction. Treatment strategies are limited in the context of both of these conditions, although interventions, which include blockade of the RAS (using angiotensin-converting enzyme inhibitors and angiotensin-receptor blockers) are successful and lead to improvements in functional outcomes that are not necessarily mediated by hemodynamic effects of the drugs. Studies in animal models of sarcopenia and diastolic dysfunction point to ubiquitous effects of RAS blockade on multiple biological mechanisms, including inflammation, oxidative damage and metabolic dysregulation. Therefore, a re-evaluation of the use of these drugs in other conditions should be considered for maintaining functional independence in older individuals.

TNF-α-mediated apoptosis is enhanced in aged rodent muscles, suggesting that this pathway may be involved in sarcopenia. Interleukin-15 (IL-15), a muscle-derived anabolic cytokine, mitigates muscle wasting and apoptosis in cachectic rats. This effect is thought to occur through inhibition of TNF-α-triggered apoptosis. We investigated IL-15 signaling and the TNF-α-mediated pathway of apoptosis in the gastrocnemius muscle of Fischer344×Brown Norway rats across the ages of 8, 18, 29 and 37 months, in relation to life-long calorie restriction (CR, 40% calorie intake reduction). Aging caused loss of muscle mass and increased apoptotic DNA fragmentation, which were mitigated by CR. Protein levels of IL-15 and mRNA abundance of IL-15 receptor α-chain decreased in senescent ad libitum (AL) fed rats, but were maintained in CR rodents. Elevations of TNF-α, TNF-receptor 1, cleaved caspase-8 and -3 were observed at advanced age in AL rats. These changes were prevented or mitigated by CR. Our results indicate that aging is associated with decreased IL-15 signaling in rat gastrocnemius muscle, which may contribute to sarcopenia partly through enhanced TNF-α-mediated apoptosis. Preservation of IL-15 signaling by CR may therefore represent a further mechanism contributing to the anti-aging effect of this dietary intervention in skeletal muscle.

Accelerated apoptosis in skeletal muscle is increasingly recognized as a potential mechanism contributing to the development of sarcopenia of aging and disuse muscle atrophy. Given their central role in the regulation of apoptosis, mitochondria are regarded as key players in the pathogenesis of myocyte loss during aging and other atrophying conditions. Oxidative damage to mitochondrial constituents, impaired respiration and altered mitochondrial turnover have been proposed as potential triggering events for mitochondrial apoptotic signaling. In addition, iron accumulation within mitochondria may enhance the susceptibility to apoptosis during the development of sarcopenia and possibly acute muscle atrophy, likely through exacerbation of oxidative stress. Mitochondria can induce myocyte apoptosis via both caspase-dependent and independent pathways, although the apoptogenic mediators involved may be different depending on age, muscle type and specific atrophying conditions. Despite the considerable advances made, additional research is necessary to establish a definite causal link between apoptotic signaling and the development of sarcopenia and acute atrophy. Furthermore, a translational effort is required to determine the role played by apoptosis in the pathogenesis of sarcopenia and disuse-induced muscle loss in human subjects.

Inhibition of the TOR signalling pathway by genetic or pharmacological intervention extends lifespan in invertebrates, including yeast, nematodes and fruit flies1–5. However, whether inhibition of mTOR signalling can extend life in a mammalian species was unknown. We report here that rapamycin, an inhibitor of the mTOR pathway, extends median and maximal lifespan of both male and female mice when fed beginning at 600 days of age. Based on age at 90% mortality, rapamycin led to an increase of 14% for females and 9% for males. The effect was seen at three independent test sites in genetically heterogeneous mice, chosen to avoid genotype-specific effects on disease susceptibility. Disease patterns of rapamycin-treated mice did not differ from those of control mice. In a separate study, rapamycin fed to mice beginning at 270 days of age also increased survival in both males and females, based on an interim analysis conducted near the median survival point. Rapamycin may extend lifespan by postponing death from cancer, by retarding mechanisms of ageing, or both. These are the first results to demonstrate a role for mTOR signalling in the regulation of mammalian lifespan, as well as pharmacological extension of lifespan in both genders. These findings have implications for further development of interventions targeting mTOR for the treatment and prevention of age-related diseases.

Caloric restriction (CR) is a daily reduction of total caloric intake without a decrease in micronutrients or disproportionate reduction of any one dietary component. CR can increase lifespan reliably in a wide range of species and appears to counteract some aspects of the aging process throughout the body. The effects on the brain are less clear, but moderate CR seems to attenuate age-related cognitive decline. Thus, we determined the effects of age and CR on key synaptic proteins in the CA3 region of the hippocampus and whether these changes were correlated with differences in behavior on a hippocampal-dependent learning and memory task. We observed an overall, age-related decline in the NR1, N2A and N2B subunits of the N-methyl-D-aspartate (NMDA)-type and the GluR1 and GluR2 subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA)-type ionotropic glutamate receptors. Interestingly, we found that CR initially lowers the glutamate receptor subunit levels as compared to young AL animals, and then stabilizes the levels across lifespan. Synaptophysin, a presynaptic vesicle protein, showed a similar pattern. We also found that both CR and ad libitum (AL) fed animals exhibited age-related cognitive decline on the Morris water maze task. However, AL animals declined between young and middle-age, and between middle-age and old, whereas CR rats only declined between young and middle-age. Thus, the decrease in key synaptic proteins in CA3 and cognitive decline occurring across lifespan are stabilized by CR. This age-related decrease and CR-induced stabilization are likely to affect CA3 synaptic plasticity and, as a result, hippocampal function.

Summary

Peripheral nerves from aged animals exhibit features of degeneration, including marked fiber loss, morphological irregularities in myelinated axons and notable reduction in the expression of myelin proteins. To investigate how protein homeostatic mechanisms change with age within the peripheral nervous system, we isolated Schwann cells from the sciatic nerves of young and old rats. The responsiveness of cells from aged nerves to stress stimuli is weakened, which in part may account for the observed age-associated alterations in glial and axonal proteins in vivo. While calorie restriction (CR) is known to slow the aging process in the central nervous system, its influence on peripheral nerves has not been investigated in detail. To determine if dietary restriction is beneficial for peripheral nerve health and glial function, we studied sciatic nerves from rats of four distinct ages (8-, 18-, 29- and 38-months) kept on an ad libitum (AL) or a 40% CR diet. Age-associated reduction in the expression of the major myelin proteins and widening of the nodes of Ranvier are attenuated by the dietary intervention, which is paralleled with the maintenance of a differentiated Schwann cell phenotype. The improvements in nerve architecture with diet restriction, in part, are underlined by sustained expression of protein chaperones and markers of the autophagy-lysosomal pathway. Together, the in vitro and in vivo results suggest that there might be an age-limit by which dietary intervention needs to be initiated to elicit a beneficial response on peripheral nerve health.

Identification of biological mediators in sarcopenia is pertinent to the development of targeted interventions to alleviate this condition. Iron is recognized as a potent pro-oxidant and a catalyst for the formation of reactive oxygen species in biological systems. It is well accepted that iron accumulates with senescence in several organs, but little is known about iron accumulation in muscle and how it may affect muscle function. In addition, it is unclear if interventions which reduced age-related loss of muscle quality, such as calorie restriction, impact iron accumulation. We investigated non-heme iron concentration, oxidative stress to nucleic acids in gastrocnemius muscle and key indices of sarcopenia (muscle mass and grip strength) in male Fischer 344 X Brown Norway rats fed ad libitum (AL) or a calorie restricted diet (60% of ad libitum food intake starting at 4 months of age) at 8, 18, 29 and 37 months of age. Total non-heme iron levels in the gastrocnemius muscle of AL rats increased progressively with age. Between 29 and 37 months of age, the non-heme iron concentration increased by approximately 200% in AL-fed rats. Most importantly, the levels of oxidized RNA in gastrocnemius muscle of AL rats were significantly increased as well. The striking age-associated increase in non-heme iron and oxidized RNA levels and decrease in sarcopenia indices were all attenuated in the calorie restriction (CR) rats. These findings strongly suggest that the age-related iron accumulation in muscle contributes to increased oxidative damage and sarcopenia, and that CR effectively attenuates these negative effects.