Combined exercise and insulin-like growth factor-1 supplementation induces neurogenesis in old rats, but do not attenuate age-associated DNA damage

We have investigated the effects of 2 weeks of insulin-like growth factor-1 (IGF-1) supplementation (5 μg/kg per day) and 6 weeks of exercise training (60% of the maximal oxygen consumption [VO? max]) on neurogenesis, DNA damage/repair, and sirtuin content in the hippocampus of young (3 months old) and old (26 months old) rats. Exercise improved the spatial memory of the old group, but IGF-1 supplementation eliminated this effect. An age-associated decrease in neurogenesis was attenuated by exercise and IGF-1 treatment. Aging increased the levels of 8-oxo-7,8-dihydroguanine (8-oxoG) and the protein Ku70, indicating the role of DNA damage in age-related neuropathology. Acetylation of 8-oxoguanine DNA glycosylase (OGG1) was detected in vivo, and this decreased with aging. However, in young animals, exercise and IGF-1 treatment increased acetylated (ac) OGG1 levels. Sirtuin 1 (SIRT1) and SIRT3, as DNA damage-associated lysine deacetylases, were measured, and SIRT1 decreased with aging, resulting in a large increase in acetylated lysine residues in the hippocampus. On the other hand, SIRT3 increased with aging. Exercise-induced neurogenesis might not be a causative factor of increased spatial memory, because IGF-1 plus exercise can induce neurogenesis in the hippocampus of older rats. Data revealed that the age-associated increase in 8-oxoG levels is due to decreased acetylation of OGG1. Age-associated decreases in SIRT1 and the associated increase in lysine acetylation, in the hippocampus, could have significant impact on function and thus, could suggest a therapeutic target.     

Reduced mitochondrial function in obesity-associated fatty liver: SIRT3 takes on the fat

Aging is associated with various metabolic disorders that may have their origin in the liver, including non-alcoholic fatty liver disease, obesity, type 2 diabetes mellitus, and atherosclerosis. Although well-characterized in models of caloric restriction, relatively little is known about the role of sirtuins and acetylation under conditions of caloric excess. Sirtuins are NAD (+)-dependent protein deacetylases that mediate adaptive responses to a variety of stresses, including calorie restriction and metabolic stress. Sirtuin 3 (SIRT3) is localized within the mitochondrial matrix, where it regulates acetylation levels of a diverse set of metabolic enzymes. When normal mice are fed a high fat diet they demonstrate reduced SIRT3 activity, impaired mitochondrial function, and hyperacetylation of a diverse set of proteins in their livers. Furthermore, SIRT3 knockout mice have signs of accelerated aging and cancer. Understanding SIRT3?s biochemical function and regulation in the liver under conditions of caloric excess may potentially increase our understanding of the normal aging process and diseases associated with aging, such as diabetes, fatty liver disease, or cancer.     

Exercise training promotes SIRT1 activity in aged rats

The objective of this study was to determine the effects of aging and exercise training on SIRT1 activity and to identify a pathway linking SIRT1 to antioxidant response and cell cycle regulation in rats. SIRT1 is a NAD(+)-dependent deacetylase involved in the oxidative stress response and aging. The effects of aging and of moderate and prolonged exercise training in rats are unknown. We measured SIRT1 activity in heart and adipose tissue of young (6 months old), sedentary old (24 months), and trained old (24 months) rats using an assay kit. Peroxidative damage was determined by measuring levels of thiobarbituric reactive substances (TBARS) and the protein-aldehyde adduct 4-hydroxynonenal (4-HNE). MnSOD, catalase, and FOXO3a levels were evaluated by Western blot, and GADD45a, cyclin D(2), and FOXO3a mRNA by RT-PCR. Aging significantly reduced SIRT1 activity in heart, but not in adipose tissue, increased TBARS and 4-HNE and decreased Mn-SOD and catalase expression in both heart and adipose tissue. Aging did not affect FOXO3a protein expression in the heart or FOXO3a mRNA in adipose tissue. Exercise training significantly increased FOXO3a protein in the heart and FOXO3a mRNA in adipose tissue of aged rats. It also significantly increased Mn-SOD and catalase levels in both heart and adipose tissue. The exercise-induced increase in SIRT1 activity in the heart caused a decrease in cyclin D(2) and an increase in GADD45a mRNA expression. There was a similar decrease in cyclin D(2), and no changes in GADD45a mRNA expression in adipose tissue. We concluded that exercise training, which significantly increases SIRT1 activity, could counteract age-related systems impairment.     

Aging decreases expression and activity of glutathione peroxidase-1 in human endothelial progenitor cells

The mechanisms underlying effects of aging on functions of pro-angiogenic endothelial progenitor cells (EPCs) are poorly understood. Previous studies demonstrated that human EPCs express high levels of antioxidant enzymes as compared to mature endothelial cells. Here, we hypothesized that aging impairs antioxidant capacity of EPCs. So called “early EPCs” derived from cultured blood mononuclear cells were obtained from healthy young (average = 24 years old) and old (average = 72 years old) subjects. In EPCs of old subjects, the levels of glutathione peroxidase-1 (GPX1) protein and enzymatic activity were significantly reduced. The serum selenium levels in young and old subjects were not significantly different. Increasing selenium concentration in the cell culture also did not affect the protein levels of GPX1, suggesting the reduced GPX1 in old subject's EPCs is selenium independent. Expressions of catalase, Mn-superoxide dismutase (MnSOD), and CuZnSOD were not affected by aging. EPCs of old subjects were more sensitive to oxidative stress induced by H₂O₂ as compared with EPCs of young subjects, suggesting that impairment of GPX1 during aging may contribute to low survival ability of EPCs in response to oxidative stress. The results indicate that GPX1 may represent a potential therapeutic target for enhancement of regenerative capacity of EPCs in old subjects.     

The role of SIRT3 in mitochondrial homeostasis and cardiac adaptation to hypertrophy and aging

Although acetyl-modification of protein lysine residues has been recognized for many decades, the appreciation that this post-translational modification is highly prevalent in mitochondria and plays a pivotal regulatory role in mitochondrial function has only become apparent since 2006. The classical biological stressors that modulate mitochondrial protein acetylation include alterations in caloric levels and redox signaling and the major enzyme orchestrating deacetylation is the mitochondrial enriched sirtuin SIRT3. Overall the action of SIRT3 modulates mitochondrial homeostasis and SIRT3 target proteins include mediators of energy metabolism and mitochondrial redox stress adaptive program proteins. Given these effects, it is not surprising that the role of SIRT3 has begun to be implicated in cardiac biology. This review gives a brief overview of sirtuin biology and then focuses on the role of the SIRT3 regulatory program in the control of cardiac hypertrophy and aging. This article is part of a Special Section entitled "Post-translational Modification."     

Exercise training enhanced SIRT1 longevity signaling replaces the IGF1 survival pathway to attenuate aging-induced rat heart apoptosis

Cardiovascular disease is the second leading cause of death (9.1 %) in Taiwan. Heart function deteriorates with age at a rate of 1 % per year. As society ages, we must study the serious problem of cardiovascular disease. SIRT1 regulates important cellular processes, including anti-apoptosis, neuronal protection, cellular senescence, aging, and longevity. In our previous studies, rats with obesity, high blood pressure, and diabetes exhibiting slowed myocardial performance and induced cell apoptosis were reversed via sports training through IGF1 survival signaling compensation. This study designed a set of experiments with rats, in aging and exercise groups, to identify changes in myocardial cell signaling transduction pathways. Three groups of three different aged rats, 3, 12, and 18 months old, were randomly divided into aging groups (C3, A12, and A18) and exercise groups (E3, AE12, and AE18). The exercise training consisted of swimming five times a week with gradual increases from the first week from 20 to 60 min for 12 weeks. After the sports training process was completed, tissue sections were taken to observe cell organization (hematoxylin and eosin (H&E) stain) and apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays) and to observe any changes in the myocardial tissues and proteins (Western blotting). The experimental results show that cardiomyocyte apoptotic pathway protein expression increased with age in the aging groups (C3, A12, and A18), with improvement in the exercise group (E3, AE12, and AE18). However, the expression of the pro-survival p-Akt protein decreased significantly with age and reduced performance. The IGF1R/PI3K/Akt survival pathway in the heart of young rats can indeed be increased through exercise training. As rats age, this pathway loses its original function, even with increasing upstream IGF1. However, levels of SIRT1 and its downstream target PGC-1α were found to increase with age and compensatory performance. Moreover, exercise training enhanced the SIRT longevity pathway compensation instead of IGF1 survival signaling to improve cardiomyocyte survival.     

Resveratrol enhanced FOXO3 phosphorylation via synergetic activation of SIRT1 and PI3K/Akt signaling to improve the effects of exercise in elderly rat hearts

Exercise training is considered a benefit to heart function, but the benefit in aging hearts remains unknown. Activation of the PI3K-Akt survival pathway and suppression of Fas/FADD/caspase-8 apoptotic signaling by exercise training in hearts from young subjects have been described in our previous studies. However, the mechanisms are still unclear and need to be explored in aging hearts. Thus, 18-month-old rats were used as a model and underwent swimming exercise training, resveratrol treatment (15 mg/kg/day), or exercise training with resveratrol treatment for 1 month. The results showed that heart function in each group improved. However, the 18-month-old rats in the exercise-only group experienced the slightly inevitable impact of increased TNF-α, cell apoptosis, and fibrosis. In the protein analysis, the PI3K-Akt pathway was slightly increased with exercise training and resveratrol treatment, but Sirtuin 1 (SIRT1) was only highly activated with resveratrol treatment in the aged rat hearts. Moreover, the exercise training plus resveratrol group benefited from SIRT1 and PI3K-Akt dual pathways and blocked FOXO3 accumulation. Our experimental results strongly suggest that resveratrol treatment improves the beneficial effects of exercise training in aging rat hearts.     

Effect of exercise training on antioxidant and metabolic functions in senescent rat skeletal muscle.

Aging is known to be associated with alterations of both oxidative capacity and antioxidant status in skeletal muscle. In the present investigation we compared the activity of enzymes involved in both metabolic functions and antioxidant defense capacity in young adult (5 months old) and senescent (27.5 months old) Fischer 344 rats. In addition, we studied the effects of chronic exercise training on these enzymes in the senescent skeletal muscle. Old sedentary rats had significantly lower glutathione peroxidase (GPX) activity (-22%, p less than 0.05) in the deep portion of vastus lateralis muscle (DVL) than young sedentary rats, but after a progressive 10-week treadmill training program GPX activity in DVL was significantly increased in old rats to a level higher than that seen in young sedentary rats. Superoxide dismutase and catalase activities in the DVL were not altered significantly with aging or by training. Glutathione S-transferase activity in the same muscle was elevated (p less than 0.05) with aging but unaffected by training. Citrate synthase and malate dehydrogenase activities in the DVL muscle were significantly decreased in senescence, whereas training increased these two enzyme activities by 71 and 48%, respectively (both p less than 0.05). Lactate dehydrogenase activity in the same muscle decreased with age but increased 23% (p less than 0.05) in old rats. These data indicate that while aging may significantly affect antioxidant and metabolic capacities in skeletal muscle, regular exercise can preserve functions of these enzyme systems at old age.     

Endurance exercise increases the SIRT1 and peroxisome proliferator-activated receptor gamma coactivator-1alpha protein expressions in rat skeletal muscle

Peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) is considered to play a pivotal role in the exercise-induced metabolic adaptation of skeletal muscle. Although the oxidized form of nicotinamide adenine dinucloetide (NAD(+))-dependent histone deacetylase SIRT1 has been shown to mediate PGC-1alpha-induced metabolic adaptation, the effect of endurance exercise on the SIRT1 protein remains to be elucidated. The purposes of this study were (1) to investigate the distribution of SIRT1 and PGC-1alpha proteins in skeletal muscle and (2) to examine the effects of acute endurance exercise and low- or high-intensity exercise training on SIRT1 and PGC-1alpha protein expressions and on the metabolic components in rat skeletal muscle. Both the SIRT1 and PGC-1alpha proteins preferentially accumulate in red oxidative muscles. Acute endurance exercise on a motor-driven treadmill (20 m/min, 18.5% incline, 45 minutes) increases the PGC-1alpha protein expression at 18 hours after exercise and the SIRT1 protein expression at 2 hours after exercise in the soleus muscle. In the training experiment, the rats were divided into control, low-intensity (20 m/min, 18.5% incline, 90 min/d), and high-intensity (30 m/min, 18.5% incline, 60 min/d) training groups. After 14 days of training, the SIRT1 and PGC-1alpha proteins, hexokinase activity, mitochondrial proteins and enzyme activities, and glucose transporter 4 protein in the soleus muscle were increased by both trainings. In the plantaris muscle, SIRT1, hexokinase activity, mitochondrial proteins and enzyme activities, and glucose transporter 4 were increased by high-intensity training whereas the PGC-1alpha was not. These results suggest that endurance exercise increases the skeletal muscle SIRT1 protein content. In addition, the findings also raise the possibility that the SIRT1 protein expression may play a potentially important role in such adaptations, whereas an increase in the PGC-1alpha protein expression is not necessary for such adaptations.     

Relationship between enzymatic activity loss and post-translational protein modification in aging

During aging, there is a decrease in the activity of many enzymes. The mechanism causing the loss of activity is still not well understood in most cases. We have studied the decrease in the activities of the malic, 6-phosphogluconate dehydrogenase and superoxide dismutase enzymes. The old malic enzyme is about 36% less active than the young enzyme and the old 6-phosphogluconic dehydrogenase enzyme is about 26% less active than the young enzyme. In this paper, some chemical properties of these enzymes are studied. Diethyl pyrocarbonate measurements indicate that the old malic enzyme has 1 histidine residue less than the young malic enzyme. Moreover, the treatment of the young malic enzyme with ascorbate for 15 min produces the loss of 36% of enzymatic activity and the loss of 1.2 histidine residues. 2,4,6-trinitrobenzenesulfonic acid measurements indicate that the old 6-phosphogluconate dehydrogenase enzyme has 11 lysine residues less than the young 6-phosphogluconate dehydrogenase enzyme. The proteolysis with trypsin produces more peptides in the young 6-phosphogluconate dehydrogenase enzyme than in the old one. However, similar numbers of peptides were produced when endoproteinase Arg-C was used in both enzymes, young and old 6-phosphogluconate dehydrogenase. Moreover, the treatment of young 6-phosphogluconate dehydrogenase enzyme with ascorbate for 15 min produces the loss of 8 lysine residues. These results suggest that during aging the modification of histidine residue could be involved in the loss of malic enzyme activity, and the modification of lysine residues could be involved in the loss of 6-phosphogluconate dehydrogenase activity. These results could also suggest that the modification of histidine and lysine residues during aging could be produced by oxidation. This could be a general process in aging, with an increase in the oxidation of many proteins. The relevance of this process in the aging effects must be related to the kind of proteins that are susceptible of oxidation and that this oxidation affects their enzymatic or biological function. We have also studied other enzymes one of which is the superoxide dismutase enzyme involved in the protection against oxidative damage. Our results are similar to those described for malic enzyme. In the latter case, the failure to measure one of the histidines in the Cu/Zn SOD is due to a chemical modification, probably caused by oxidation of the residue.     

The loss of cardiac SIRT3 decreases metabolic flexibility and proteostasis in an age-dependent manner

SIRT3 is a longevity factor that acts as the primary deacetylase in mitochondria. Although ubiquitously expressed, previous global SIRT3 knockout studies have shown primarily a cardiac-specific phenotype. Here, we sought to determine how specifically knocking out SIRT3 in cardiomyocytes (SIRTcKO mice) temporally affects cardiac function and metabolism. Mice displayed an age-dependent increase in cardiac pathology, with 10-month-old mice exhibiting significant loss of systolic function, hypertrophy, and fibrosis. While mitochondrial function was maintained at 10 months, proteomics and metabolic phenotyping indicated SIRT3 hearts had increased reliance on glucose as an energy substrate. Additionally, there was a significant increase in branched-chain amino acids in SIRT3cKO hearts without concurrent increases in mTOR activity. Heavy water labeling experiments demonstrated that, by 3 months of age, there was an increase in protein synthesis that promoted hypertrophic growth with a potential loss of proteostasis in SIRT3cKO hearts. Cumulatively, these data show that the cardiomyocyte-specific loss of SIRT3 results in severe pathology with an accelerated aging phenotype.

     

From the Cover: Structure of the ��-tubulin acetyltransferase, ��TAT1, and implications for tubulin-specific acetylation

Protein acetylation is an important posttranslational modification with the recent identification of new substrates and enzymes, new links to disease, and modulators of protein acetylation for therapy. α-tubulin acetyltransferase (αTAT1) is the major α-tubulin lysine-40 (K40) acetyltransferase in mammals, nematodes, and protozoa, and its activity plays a conserved role in several microtubule-based processes. Here, we present the X-ray crystal structure of the human αTAT1/acetyl-CoA complex. Together with structure-based mutagenesis, enzymatic analysis, and functional studies in cells, we elucidate the catalytic mechanism and mode of tubulin-specific acetylation. We find that αTAT1 has an overall fold similar to the Gcn5 histone acetyltransferase but contains a relatively wide substrate binding groove and unique structural elements that play important roles in α-tubulin–specific acetylation. Conserved aspartic acid and cysteine residues play important catalytic roles through a ternary complex mechanism. αTAT1 mutations have analogous effects on tubulin acetylation in vitro and in cells, demonstrating that it is the central determining factor of α-tubulin K40 acetylation levels in vivo. Together, these studies provide general insights into distinguishing features between histone and tubulin acetyltransferases, and they have specific implications for understanding the molecular basis of tubulin acetylation and for developing small molecule modulators of microtubule acetylation for therapy.     

Des-acyl ghrelin protects microvascular endothelial cells from oxidative stress-induced apoptosis through sirtuin 1 signaling pathway

ObjectiveGhrelin is a stomach-derived hormone. Acylation of ghrelin has been essential for its biological activities such as stimulating appetite. On the other hand, the function of des-acyl ghrelin (Des-G) has not been fully elucidated. The aim of the present study is to examine the anti-apoptotic effect of Des-G on endothelial cells.Materials/MethodsAfter human retinal microvascular endothelial cells (RMECs) were pretreated with or without 100 nmol/L Des-G, apoptosis was induced with 0.1 mmol/L hydrogen peroxide (H₂O₂). For pharmacological inhibition of surtuin 1 (SIRT1) catalytic activity, the cells were treated with 10 μmol/L Ex-527. Inhibition of SIRT1 with siRNA was also performed. The quantitative estimation of DNA fragmentation was used as a marker of apoptosis. Furthermore, total SIRT activity in nuclear extracts, mRNA and protein levels of SIRT1, manganese superoxide dismutase (MnSOD) and catalase were determined.ResultsDes-G pretreatment protected RMECs from oxidative stress-induced apoptosis and increased SIRTs deacetylase activity in nuclear extracts. On the other hand, both pharmacological and siRNA mediated inhibition of SIRT1 attenuated the anti-apoptotic effect of Des-G. Moreover, Des-G increased mRNA and protein levels of SIRT1 and antioxidant enzymes such as MnSOD and CAT, which are downstream targets of SIRT1. Although the treatment of Ex-527 did not alter mRNA expression levels of SIRT1, it decreased mRNA expression levels of antioxidant enzymes in the cells with Des-G pretreatment.ConclusionsOur results suggest that SIRT1 signaling pathway contributes to protective effect of Des-G against oxidative stress-induced apoptosis.     

Expression and Activity Patterns of Nitric Oxide Synthases and Antioxidant Enzymes Reveal a Substantial Heterogeneity Between Cardiac and Vascular Aging in the Rat

We investigated the effects of aging and ischemia–reperfusion (I/R) injury on the expression and activity of nitric oxide (^•NO) synthases and superoxide dismutase (SOD) isoforms. To this end we perfused excised hearts from young (6 months old) and old (31–34 months old) rats according to the Langendorff technique. The isolated hearts were, after baseline perfusion for 30 min, either subjected to 20 min of global no-flow ischemia followed by 40 min of reperfusion or were control-perfused (60 min normoxic perfusion). Both MnSOD and Cu,ZnSOD expression remained unchanged with increasing age and remained unaltered by I/R. However, SOD activity decreased from 7.55 ± 0.1 U/mg protein in young hearts to 5.94 ± 0.44 in old hearts (P<0.05). Furthermore, I/R led to a further decrease in enzyme activity (to 6.35 ± 0.41 U/mg protein; P<0.05) in myocardium of young, but not in that of old animals. No changes in myocardial protein-bound 3-nitrotyrosine levels could be detected. Endothelial NOS (eNOS) expression and activity remained unchanged in aged left ventricles, irrespective of I/R injury. This was in steep contrast to peripheral (renal and femoral) arteries obtained from the same animals where a marked age-associated increase of eNOS protein expression could be demonstrated. Inducible NOS expression was undetectable either in the peripheral arteries or in the left ventricle, irrespective of age. In particular when associated with an acute pathology, which is furthermore limited to a certain time frame, changes in the aged myocardium with respect to enzymes crucially involved in maintaining the redox homeostasis, seem to be much less pronounced or even absent compared to the vascular aging process. This may point to heterogeneity in the molecular regulation of the cardiovascular aging process.     

淫羊藿苷提高SIRT6酶活性及抑制小鼠NF-kB炎症信号通路的实验研究

目的观察淫羊藿苷（Icrrin,ICA）对组蛋白去乙酰化酶SIRT6的激活作用及对老年小鼠体内NF-kB（p65）、SIRT6蛋白表达及NF—kB信号通路下游炎症细胞因子表达的影响。方法以体外酶学实验观察ICA对SIRT6的激活作用并绘制不同剂量浓度激活效率曲线。选取24月龄老年组（N=11）、24月龄＋ICA干预组（N=12）和3月龄青年组小鼠（N=10）的心、肝、肌肉组织,用Westernblot检测NF-kB（p65）和SIRT6蛋白表达水平;采用ELISA法柃测小鼠血清中肿瘤坏死网子α（TNF—α）、细胞间粘附分子-1（ICAM-1）、白细胞介素-2（IL-2）和白细胞介素-6（IL-6）的含量。结果体外酶学实验显尔ICA随着药物浓度的下降,对SIRT6逐渐呈现激活效应,在10。mol／L浓度时达到最大激活效应为142％。小鼠实验显示ICA干预后,心脏、肝脏与肌肉组织中NF—kB（p65）蛋白表达与老年小鼠比较呈现下调趋势,而SIRT6蛋白表达较老年小鼠则呈现上调趋势;与青年小鼠比较,老年小鼠血清中炎症细胞TNF-α、ICAM-1、IL-2、IL-6的含量均明显上调（P〈0．05、P〈0．01）;与老年小鼠比较,ICA干预后小鼠血清中炎症因子TNF-α、ICAM-1、IL-2、IL-6的含量均明显下降（P〈0．05、P〈0．01）。结论ICA对SIRT6的酶活性具有显著的激活效应,且在极低的药物浓度下仍对SIRT6有激活作用;ICA能上调老年小鼠组织中SIRT6蛋白表达,抑制NF—kB（p65）蛋白表达及下调下游炎症细胞因子的产生,提示ICA延缓炎性衰老的作用机制及干预新靶点与NF—kB信号通路和组蛋白去乙酰化酶SIRT6密切相关。     

Reduced recognition memory is correlated with decrease in DNA methyltransferase1 and increase in histone deacetylase2 protein expression in old male mice

Chromatin modifying enzymes DNA methyltransferases (DNMTs), histone deacetylase (HDAC) 2 and CREB binding protein (CBP) play a crucial role in memory, particularly during consolidation process which declines with advancing age. However, the expression of these enzymes and their effect on memory consolidation during aging are not clearly understood. In the present study, novel object recognition test was used to assess the memory consolidation followed by expression analysis of DNMTs, HDAC2 and CBP in the cerebral cortex and hippocampus of young, adult and old male mice. Object recognition memory was reduced in old as compared to young and adult. DNMT1 protein expression was high in the cerebral cortex and hippocampus of young male mice, but declined gradually with age. On the other hand, HDAC2 mRNA and protein expression increased in the hippocampus of old male mice as compared to young and adult. Alteration in the expression of these enzymes is correlated with reduced recognition memory in old.     

Oxidative stress and the mitochondrial theory of aging in human skeletal muscle

According to the mitochondrial theory of aging, an age-related increase in oxidative stress is responsible for cellular damage and ultimately cell death. Despite compelling evidence that supports the mitochondrial theory of aging in some tissues, data regarding aging skeletal muscle are inconsistent. We collected resting muscle biopsies from the vastus lateralis, and 24 h urine samples from, young (N = 12, approximately 22 yr), and older (N = 12 approximately 72 yr) men. Urinary 8-OHdG was significantly higher in older as compared to younger men (Old: 7714 +/- 1402, Young: 5333 +/- 1191 ng g(-1) creatinine: p = 0.005), as were levels of protein carbonyls (Old: 0.72 +/- 0.42, Young: 0.26 +/- 0.14 nmol mg(-1) protein: p = 0.007). MnSOD activity (Old: 7.1 +/- 0.8, Young: 5.2 +/- 1.8 U mg(-1) protein: p = 0.04) and catalase activity (Old: 8.5 +/- 2.0, Young: 6.2 +/- 2.4 micro mol min(-1) mg(-1) protein: p = 0.03) were significantly higher in old as compared to young men, respectively, with no differences observed for total or CuZnSOD. Full-length mtDNA appeared lower in old as compared to young men, and mtDNA deletions were present in 6/8 old and 0/6 young men (p = 0.003). The maximal activities of citrate synthase, and complex II+III, and IV were not different between young and old men, however, complex I+III activity was marginally higher in older as compared to younger men (Old: 2.5 +/- 0.5, Young: 1.9 +/- 0.5 micromol min(-1) g(-1) w.w: p = 0.03) respectively. In conclusion, healthy aging is associated with oxidative damage to proteins and DNA, a compensatory up-regulation of antioxidant enzymes, and aberrations of mtDNA, with no reduction in electron transport chain maximal enzyme activity.