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

Calorie restriction (CR), a reduction of 10­40% 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 (7­14 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.

Maintaining PGC-1α expression following pressure overload-induced cardiac hypertrophy preserves angiogenesis but not contractile or mitochondrial function.

During pathological hypertrophy, peroxisome proliferator-activated receptor coactivator 1α (PGC-1α) is repressed in concert with reduced mitochondrial oxidative capacity and fatty acid oxidation (FAO). We therefore sought to determine if maintaining or increasing PGC-1α levels in the context of pressure overload hypertrophy (POH) would preserve mitochondrial function and prevent contractile dysfunction. Pathological cardiac hypertrophy was induced using 4 wk of transverse aortic constriction (TAC) in mice overexpressing the human PGC-1α genomic locus via a bacterial artificial chromosome (TG) and nontransgenic controls (Cont). PGC-1α levels were increased by 40% in TG mice and were sustained following TAC. Although TAC-induced repression of FAO genes and oxidative phosphorylation (oxphos) genes was prevented in TG mice, mitochondrial function and ATP synthesis were equivalently impaired in Cont and TG mice after TAC. Contractile function was also equally impaired in Cont and TG mice following TAC, as demonstrated by decreased +dP/dt and ejection fraction and increased left ventricular developed pressure and end diastolic pressure. Conversely, capillary density was preserved, in concert with increased VEGF expression, while apoptosis and fibrosis were reduced in TG relative to Cont mice after TAC. Hence, sustaining physiological levels of PGC-1α expression following POH, while preserving myocardial vascularity, does not prevent mitochondrial and contractile dysfunction.

Calorie restriction alleviates the age-related decrease in neural progenitor cell division in the aging brain.

Production of new neurons from stem cells is important for cognitive function, and the reduction of neurogenesis in the aging brain may contribute to the accumulation of age-related cognitive deficits. Restriction of calorie intake and prolonged treatment with rapamycin have been shown to extend the lifespan of animals and delay the onset of the age-related decline in tissue and organ function. Using a reporter line in which neural stem and progenitor cells are marked by the expression of green fluorescent protein (GFP), we examined the effect of prolonged exposure to calorie restriction (CR) or rapamycin on hippocampal neural stem and progenitor cell proliferation in aging mice. We showed that CR increased the number of dividing cells in the dentate gyrus of female mice. The majority of these cells corresponded to nestin-GFP-expressing neural stem or progenitor cells; however, this increased proliferative activity of stem and progenitor cells did not result in a significant increase in the number of doublecortin-positive newborn neurons. Our results suggest that restricted calorie intake may increase the number of divisions that neural stem and progenitor cells undergo in the aging brain of females.

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.

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-alpha-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.

PGC-1alpha-induced mitochondrial alterations in 3T3 fibroblast cells.

Peroxisome proliferation activator receptor (PPAR) gamma-coactivator 1alpha (PGC-1alpha), a transcription coactivator, functions as a master regulator of a wide array of metabolic and physiological processes and is an essential factor in the process of mitochondrial biogenesis. Transfection of NIH 3T3 fibroblasts with a mouse cDNA for PGC-1alpha led to the induction of markers of mitochondrial biogenesis, that is, mitochondrial transcription factor A (mtTFA), cytochrome c, and mitochondrial DNA (mtDNA). Mitochondrial biogenesis-associated net protein synthesis appears to be accomplished by a reduction in the rate of mitochondrial protein degradation with little or no change in the rate of protein synthesis. Overexpression of PGC-1alpha did not adversely affect cellular proliferation. Cellular ATP levels were increased in the transfected cells and they were more resistant to oxidative stress than the control nontransfected 3T3 cells. This resistance to oxidative stress was manifested by both an improved viability and the maintenance of mitochondrial membrane potential in the transfected cells when exposed to t-butyl hydroperoxide (t-BOOH). It therefore appears that PGC-1alpha overexpression stimulates mitochondrial biogenesis in 3T3 cells making them more resistant to oxidative stressors.

Effect of aging and caloric restriction on the mitochondrial proteome.

The rat mitochondrial proteome was analyzed using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), and proteins altered by age or caloric restriction (CR) were identified using mass spectrometry. Of 2061 mitochondrial proteins analyzed in the three tissues, a significant change with age occurred in 25 liver proteins (19 increased, 6 decreased), 3 heart proteins (1 increased, 2 decreased), and 5 skeletal muscle proteins (all increased). CR prevented the age-related change in the level of one liver mitochondrial protein, altered the levels of four proteins (one increased, three decreased) from heart, and one protein (decreased) from skeletal muscle. Identification of the proteins that changed with age or CR revealed that they were varied among the three tissues, that is, not one mitochondrial protein was changed, in common, by age or CR in any tissue studied. Thus, the effect of age on the mitochondrial proteome appears to be tissue-specific, and CR has a minor effect on age-related protein changes.

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.

PGC-1alpha: a key regulator of energy metabolism.

Peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1alpha is a member of a family of transcription coactivators that plays a central role in the regulation of cellular energy metabolism. It is strongly induced by cold exposure, linking this environmental stimulus to adaptive thermogenesis. PGC-1alpha stimulates mitochondrial biogenesis and promotes the remodeling of muscle tissue to a fiber-type composition that is metabolically more oxidative and less glycolytic in nature, and it participates in the regulation of both carbohydrate and lipid metabolism. It is highly likely that PGC-1alpha is intimately involved in disorders such as obesity, diabetes, and cardiomyopathy. In particular, its regulatory function in lipid metabolism makes it an inviting target for pharmacological intervention in the treatment of obesity and Type 2 diabetes.

Effects of age and caloric restriction on lipid peroxidation: measurement of oxidative stress by F2-isoprostane levels.

The free radical theory of aging proposes that the accumulation of oxidative damage is a key component of the aging process. The discovery of F2-isoprostanes (F2-isoPs) and their establishment as a sensitive and accurate biomarker of lipid peroxidation represents a major advance for measuring the oxidative stress status of an organism. We have shown that plasma free and total (free plus esterified) F2-isoPs increase with age (185% and 66%, respectively), and that these increases are reduced by life-extending caloric restriction (50% and 23%, respectively). In addition, we found that levels of esterified F2-isoPs increase 68% with age in liver, and 76% with age in kidney. Caloric restriction modulated the age-related increase, reducing the esterified F2-isoPs levels 27% in liver and 35% in kidney. These age-related increases in esterified F2-isoPs levels correlate well with DNA oxidation, as measured by 8-oxodeoxyguanosine production demonstrating that F2-isoPs are an excellent biomarker for age-related changes in oxidative damage to membranes.

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.

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.

Detection and quantification of protein disulfides in biological tissues a fluorescence-based proteomic approach.

While most of the amino acids in proteins are potential targets for oxidation, the thiol group in cysteine is one of the most reactive amino acid side chains. The thiol group can be oxidized to several states, including the disulfide bond. Despite the known sensitivity of cysteine to oxidation and the physiological importance of the thiol group to protein structure and function, little information is available on the oxidative modification of cysteine residues in proteins because of the lack of reproducible and sensitive assays to measure cysteine oxidation in the proteome. We have developed a fluorescence-based assay that allows one to quantify both the global level of protein disulfides in the cellular proteome as well as the disulfide content of individual proteins. This fluorescence-based assay is able to detect an increase in global protein disulfide levels after oxidative stress in vitro or in vivo. Using this assay, we show that the global protein disulfide levels increase significantly with age in liver cytosolic proteins, and we identified 11 proteins that show a more than twofold increase in disulfide content with age. Thus, the fluorescence-based assay we have developed allows one to quantify changes in the oxidation of cysteine residues to disulfides in the proteome of a cell or tissue.

Whole body overexpression of PGC-1alpha 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.

Processing of data generated by 2-dimensional gel electrophoresis for statistical analysis: missing data, normalization, and statistics.

Several high-throughput statistical methods were evaluated for processing data generated by two-dimensional polyacrylamide gel electrophoresis, including how to handle missing data, normalization, and statistical analysis of data obtained from 2-D gels. Quantile normalization combined with a nonparametric permutation test based on minimizing false discover rates gave the highest yield of proteins that changed with genotype and detected the anticipated 50% decrease in Mn-superoxide dismutase (MnSOD) protein levels in mitochondrial extracts obtained from MnSOD-deficient mice.