Walter F. Ward

Impact of caloric restriction on health and survival in rhesus monkeys from the NIA study

Calorie restriction (CR), a reduction of 10–40% in intake of a nutritious diet, is often reported as the most robust non-genetic mechanism to extend lifespan and healthspan. CR is frequently used as a tool to understand mechanisms behind ageing and age-associated diseases. In addition to and independently of increasing lifespan, CR has been reported to delay or prevent the occurrence of many chronic diseases in a variety of animals. Beneficial effects of CR on outcomes such as immune function, motor coordination and resistance to sarcopenia in rhesus monkeys have recently been reported. We report here that a CR regimen implemented in young and older age rhesus monkeys at the National Institute on Aging (NIA) has not improved survival outcomes. Our findings contrast with an ongoing study at the Wisconsin National Primate Research Center (WNPRC), which reported improved survival associated with 30% CR initiated in adult rhesus monkeys (7–14 years) and a preliminary report with a small number of CR monkeys. Over the years, both NIA and WNPRC have extensively documented beneficial health effects of CR in these two apparently parallel studies. The implications of the WNPRC findings were important as they extended CR findings beyond the laboratory rodent and to a long-lived primate. Our study suggests a separation between health effects, morbidity and mortality, and similar to what has been shown in rodents, study design, husbandry and diet composition may strongly affect the life-prolonging effect of CR in a long-lived nonhuman primate.

Protein stability and resistance to oxidative stress are determinants of longevity in the longest-living rodent, the naked mole-rat

The widely accepted oxidative stress theory of aging postulates that aging results from accumulation of oxidative damage. Surprisingly, data from the longest-living rodent known, naked mole-rats [MRs; mass 35 g; maximum lifespan (MLSP) > 28.3 years], when compared with mice (MLSP 3.5 years) exhibit higher levels of lipid peroxidation, protein carbonylation, and DNA oxidative damage even at a young age. We hypothesize that age-related changes in protein structural stability, oxidation, and degradation are abrogated over the lifespan of the MR. We performed a comprehensive study of oxidation states of protein cysteines [both reversible (sulfenic, disulfide) and indirectly irreversible (sulfinic/sulfonic acids)] in liver from young and old C57BL/6 mice (6 and 28 months) and MRs (2 and >24 years). Furthermore, we compared interspecific differences in urea-induced protein unfolding and ubiquitination and proteasomal activity. Compared with data from young mice, young MRs have 1.6 times as much free protein thiol groups and similar amounts of reversible oxidative damage to cysteine. In addition, they show less urea-induced protein unfolding, less protein ubiquitination, and higher proteasome activity. Mice show a significant age-related increase in cysteine oxidation and higher levels of ubiquitination. In contrast, none of these parameters were significantly altered over 2 decades in MRs. Clearly MRs have markedly attenuated age-related accrual of oxidation damage to thiol groups and age-associated up-regulation of homeostatic proteolytic activity. These pivotal mechanistic interspecies differences may contribute to the divergent aging profiles and strongly implicate maintenance of protein stability and integrity in successful aging.

Rapamycin Extends Life and Health in C57BL/6 Mice

Target of rapamycin inhibition by rapamycin feeding has previously been shown to extend life in genetically heterogeneous mice. To examine whether it similarly affected mouse health, we fed encapsulated rapamycin or a control diet to C57BL/6Nia mice of both sexes starting at 19 months of age. We performed a range of health assessments 6 and 12 months later. Rapamycin feeding significantly reduced mTOR activity in most but not all tissues. It also reduced total and resting metabolic rate during the light (inactive) phase of the light:dark cycle in females only but had no effect on spontaneous activity or metabolism during the dark (active) phase of either sex. Males only had less fragmented sleep when fed rapamycin, whereas stride length and rotarod performance were improved in both sexes. Survival was also improved by this late-life rapamycin feeding, and some pathological lesions were delayed. We found no adverse health consequences associated with rapamycin treatment.

Detection of protein carbonyls in aging liver tissue: A fluorescence-based proteomic approach.

Protein carbonyls are commonly used as a marker of protein oxidation in cells and tissues. Currently, 2,4-dinitrophenyl hydrazine (DNPH) is widely used (spectrophotometrically or immunologically) to quantify the global carbonyl levels in proteins and identify the specific proteins that are carbonylated. We have adapted a fluorescence-based approach using fluorescein-5-thiosemicarbazide (FTC), to quantify the global protein carbonyls as well as the carbonyl levels on individual proteins in the proteome. Protein carbonyls generated in vitro were quantified by labeling the oxidized proteins with FTC followed by separating the FTC-labeled protein from free probe by gel electrophoresis. The reaction of FTC with protein carbonyls was found to be specific for carbonyl groups. We measured protein carbonyl levels in the livers of young and old mice, and found a significant increase (two-fold) in the global protein carbonyl levels with age. Using 2-D gel electrophoresis, we used this assay to directly measure the changes in protein carbonyl levels in specific proteins. We identified 12 proteins showing a greater than two-fold increase in carbonyl content (pmoles of carbonyls/microg of protein) with age. Most of the 12 proteins contained transition metal binding sites, with Cu/Zn superoxide dismutase containing the highest molar ratio of carbonyls in old mice. Thus, the fluorescence-based assay gives investigators the ability to identify potential target proteins that become oxidized under different pathological and physiological conditions.

Neuronal mitochondrial amelioration by feeding acetyl-L-carnitine and lipoic acid to aged rats

Brain function declines with age and is associated with diminishing mitochondrial integrity. The neuronal mitochondrial ultrastructural changes of young (4 months) and old (21 months) F344 rats supplemented with two mitochondrial metabolites, acetyl‐L‐carnitine (ALCAR, 0.2%[wt/vol] in the drinking water) and R‐α‐lipoic acid (LA, 0.1%[wt/wt] in the chow), were analysed using qualitative and quantitative electron microscopy techniques. Two independent morphologists blinded to sample identity examined and scored all electron micrographs. Mitochondria were examined in each micrograph, and each structure was scored according to the degree of injury. Controls displayed an age‐associated significant decrease in the number of intact mitochondria (P = 0.026) as well as an increase in mitochondria with broken cristae (P < 0.001) in the hippocampus as demonstrated by electron microscopic observations. Neuronal mitochondrial damage was associated with damage in vessel wall cells, especially vascular endothelial cells. Dietary supplementation of young and aged animals increased the proliferation of intact mitochondria and reduced the density of mitochondria associated with vacuoles and lipofuscin. Feeding old rats ALCAR and LA significantly reduced the number of severely damaged mitochondria (P = 0.02) and increased the number of intact mitochondria (P < 0.001) in the hippocampus. These results suggest that feeding ALCAR with LA may ameliorate age‐associated mitochondrial ultrastructural decay and are consistent with previous studies showing improved brain function.

Caretaker or undertaker? The role of the proteasome in aging

Despite intensive studies, the molecular basis of the decline of protein degradation with age still remains unresolved. It is suspected that the proteasome is one of the key factors controlling the age-dependent turnover of intracellular proteins. This hypothesis is based on the observation that the proteasome is a part of the ubiquitin-proteasome pathway, which together with the lysosomal pathway constitute the major mechanisms of protein degradation. While there are alterations in proteasome structure and function with age, the observed changes do not provide a clear mechanism for explaining the decline of protein degradation. In addition, there are no consistent changes in the ubiquitination system to account for this decline. On the other hand, because of the essential role played by the proteasome in the maintenance of cellular homeostasis, the observation of age-related changes in structure and function will ultimately be demonstrated to contribute to the aging process. The fact that food restriction, the only currently available experimental paradigm that can alter the aging process, modulates the age-related changes in proteasome structure and function provides presumptive evidence that the proteasome is involved in the aging process.

Protein Degradation in the Aging Organism

It is now generally accepted that protein degradation declines with age but a mechanism of action for this decline has not yet been delineated. Although intracellular and extracellular proteins can enter multiple pathways of degradation, there primarily appears to be two final mediators of this degradation, the lysosome and the proteasome. Studies on the effects of age on lysosomal function suggest that, if anything, lysosomal enzyme activity increases with age (Ward 2000). The peptidase activities of the proteasome are altered with age, but not in a consistent manner. There is a significant age-related decline of the PGPH activity, but the rate-limiting peptidase activity, ChT-L activity, as well as T-L activity have both been reported either to increase, not change, or decrease (Table 1). In addition, proteasomal degradation of casein does not appear to be altered with age. As a result, it has not been possible to definitively implicate either of the two primary final mediators of protein degradation, the lysosome and the proteasome, as mechanisms of action for the decline in protein degradation observed in the aging organism. However, there are experimental observations suggesting that age may have strong effects on both macroautophagic and the chaperone-mediated autophagic processes. Therefore, it is important that more research activity be devoted to the investigation of the effects of age on these processes as this may be where mechanism(s) of action for the age-related decline in protein degradation lies.

Whole body overexpression of PGC-1α has opposite effects on hepatic and muscle insulin sensitivity

Type 2 diabetes is characterized by fasting hyperglycemia, secondary to hepatic insulin resistance and increased glucose production. Peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) is a transcriptional coactivator that is thought to control adaptive responses to physiological stimuli. In liver, PGC-1alpha expression is induced by fasting, and this effect promotes gluconeogenesis. To examine whether PGC-1alpha is involved in the pathogenesis of hepatic insulin resistance, we generated transgenic (TG) mice with whole body overexpression of human PGC-1alpha and evaluated glucose homeostasis with a euglycemic-hyperinsulinemic clamp. PGC-1alpha was moderately (approximately 2-fold) overexpressed in liver, skeletal muscle, brain, and heart of TG mice. In liver, PGC-1alpha overexpression resulted in increased expression of hepatocyte nuclear factor-4alpha and the gluconeogenic enzymes phosphoenolpyruvate carboxykinase and glucose-6-phosphatase. PGC-1alpha overexpression caused hepatic insulin resistance, manifested by higher glucose production and diminished insulin suppression of gluconeogenesis. Paradoxically, PGC-1alpha overexpression improved muscle insulin sensitivity, as evidenced by elevated insulin-stimulated Akt phosphorylation and peripheral glucose disposal. Content of myoglobin and troponin I slow protein was increased in muscle of TG mice, indicating fiber-type switching. PGC-1alpha overexpression also led to lower reactive oxygen species production by mitochondria and reduced IKK/IkappaB signaling in muscle. Feeding a high-fat diet to TG mice eliminated the increased muscle insulin sensitivity. The dichotomous effect of PGC-1alpha overexpression in liver and muscle suggests that PGC-1alpha is a fuel gauge that couples energy demands (muscle) with the corresponding fuel supply (liver). Thus, under conditions of physiological stress (i.e., prolonged fast and exercise training), increased hepatic glucose production may help sustain glucose utilization in peripheral tissues.

PGC-1α protects neurons and alters disease progression in an amyotrophic lateral sclerosis mouse model

Introduction: Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease. We sought to determine whether peroxisome proliferator–activated receptor γ coactivator 1α (PGC‐1α) would have a beneficial effect on this disease. Methods: PGC‐1α transgenic mice were crossed with SOD1 mutant G93A DL mice. Results: We observed a moderate but non‐significant increase in average lifespan in PGC‐1α/G93A DL mice, as compared with G93A DL mice (292 ± 3 days vs. 274 ± 7 days). Although the onset of ALS was not altered, progression of the disease was significantly slower (∽34% increase in duration) in the PGC‐1α/G93A DL mice. These mice also exhibited markedly improved performance on the rotarod test, and the improved motor activity was associated with a decreased loss of motor neurons and less degeneration of neuromuscular junctions. Conclusion: A sustained level of excitatory amino acid transporter protein 2 (EAAT2) in astrocytes of the PGC‐1α/G93A DL mice may contribute to neuronal protection. Muscle Nerve 2011

Comparative proteomics: characterization of a two-dimensional gel electrophoresis system to study the effect of aging on mitochondrial proteins.

To study the effect of aging and anti-aging strategies on mitochondria, we have characterized a two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) system to analyze the profile of mitochondrial proteins. We have optimized the separation of proteins by 2-D PAGE and established the linearity and reproducibility of the system with mitochondria isolated from skeletal muscle of mice. Using total mitochondria protein ranging from 10 to 200 microg, we found that 74% of the proteins resolved by 2-D PAGE had coefficient of determination (R2) values greater than 0.8, showing a linear increase in fluorescence with increasing protein concentration. The coefficient of variation (CV) was less than 50% for at least 93% of the 424 spots analyzed for both gel-to-gel variance and animal-to-animal variance. Using mitochondrial protein fractions prepared from skeletal muscle of 18-month-old mice, we show that 10 animals will be sufficient to detect a 100% difference in the 97% (i.e. 505) of the proteins resolved by 2-D PAGE. Thus, 2-D PAGE provides a sensitive and reliable technique for analysis of protein expression in mitochondria.