Interleukin-8 and interleukin-10, brain volume and microstructure, and the influence of calorie restriction in old rhesus macaques.

Higher systemic levels of the proinflammatory cytokine interleukin-6 (IL-6) were found to be associated with lower gray matter volume and tissue density in old rhesus macaques. This association between IL-6, and these brain indices were attenuated by long-term 30 % calorie restriction (CR). To extend these findings, the current analysis determined if a CR diet in 27 aged rhesus monkeys compared to 17 normally fed controls reduced circulating levels of another proinflammatory cytokine, interleukin-8 (IL-8), and raised levels of anti-inflammatory interleukin-10 (IL-10). Further, these cytokines were regressed onto imaged brain volume and microstructure using voxel-wise regression analyses. CR significantly lowered IL-8 and raised IL-10 levels. Across the two dietary conditions, higher IL-8 predicted smaller gray matter volumes in bilateral hippocampus. Higher IL-10 was associated with more white matter volume in visual areas and tracts. Consuming a CR diet reduced the association between systemic IL-8 and hippocampal volumes. Conversely, CR strengthened associations between IL-10 and microstructural tissue density in the prefrontal cortex and other areas, particularly in a region of dorsal prefrontal cortex, which concurred with our prior findings for IL-6. Consumption of a CR diet lowered proinflammatory and increased anti-inflammatory cytokine concentrations, which lessened the statistical association between systemic inflammation and the age-related alterations in important brain regions, including the hippocampus.

Effects of aging and calorie restriction on white matter in rhesus macaques.

Rhesus macaques on a calorie restricted diet (CR) develop less age-related disease, have virtually no indication of diabetes, are protected against sarcopenia, and potentially live longer. Beneficial effects of caloric restriction likely include reductions in age-related inflammation and oxidative damage. Oligodendrocytes are particularly susceptible to inflammation and oxidative stress, therefore, we hypothesized that CR would have a beneficial effect on brain white matter and would attenuate age-related decline in this tissue. CR monkeys and controls underwent diffusion tensor imaging (DTI). A beneficial effect of CR indexed by DTI was observed in superior longitudinal fasciculus, fronto-occipital fasciculus, external capsule, and brainstem. Aging effects were observed in several regions, although CR appeared to attenuate age-related alterations in superior longitudinal fasciculus, frontal white matter, external capsule, right parahippocampal white matter, and dorsal occipital bundle. The results, however, were regionally specific and also suggested that CR is not salutary across all white matter. Further evaluation of this unique cohort of elderly primates to mortality will shed light on the ultimate benefits of an adult-onset, moderate CR diet for deferring brain aging.

Age-related changes in neural volume and microstructure associated with interleukin-6 are ameliorated by a calorie-restricted diet in old rhesus monkeys.

Systemic levels of proinflammatory cytokines such as interleukin-6 (IL-6) increase in old age and may contribute to neural atrophy in humans. We investigated IL-6 associations with age in T1-weighted segments and microstructural diffusion indices using MRI in aged rhesus monkeys (Macaca mulatta). Further, we determined if long-term 30% calorie restriction (CR) reduced IL-6 and attenuated its association with lower tissue volume and density. Voxel-based morphometry (VBM) and diffusion-weighted voxelwise analyses were conducted. IL-6 was associated with less global gray and white matter (GM and WM), as well as smaller parietal and temporal GM volumes. Lower fractional anisotropy (FA) was associated with higher IL-6 levels along the corpus callosum and various cortical and subcortical tracts. Higher IL-6 concentrations across subjects were also associated with increased mean diffusivity (MD) throughout many brain regions, particularly in corpus callosum, cingulum, and parietal, frontal, and prefrontal areas. CR monkeys had significantly lower IL-6 and less associated atrophy. An IL-6xCR interaction across modalities also indicated that CR mitigated IL-6 related changes in several brain regions compared to controls. Peripheral IL-6 levels were correlated with atrophy in regions sensitive to aging, and this relationship was decreased by CR.

Short-term consumption of a resveratrol-containing nutraceutical mixture mimics gene expression of long-term caloric restriction in mouse heart.

An active area of aging research is focused on identifying compounds having the ability to mimic the effects of caloric restriction (CR). From 2 to 5 months of age, we fed male B6C3F(1) mice either a 40% CR diet, a control diet supplemented with a commercially available nutraceutical mixture (NCM) containing resveratrol, quercetin and inositol hexaphosphate, or a diet supplemented with an equivalent dose of chemical-grade resveratrol (RES; 1.25 mg resveratrol kg(-1) day(-1)) from 2 to 5 months of age. Cardiac gene expression profiles were generated for the three groups of treated mice and compared to age-matched control (CO) mice. All three treatments were associated with changes in several cytoskeletal maintenance pathways, suggesting that RES and NCM are able to mimic short-term CR. CR uniquely affected several immune function pathways while RES uniquely affected multiple stress response pathways. Pathway analysis revealed that NCM (but not CR or RES) regulated multiple metabolic pathways that were also changed by long-term CR, including glucose and lipid metabolism, oxidative phosphorylation and chromatin assembly. Examination of key genes and pathways affected by NCM suggests that Foxo1 is a critical upstream mediator of its actions.

Mitochondrial DNA mutations, oxidative stress, and apoptosis in mammalian aging.

Mutations in mitochondrial DNA (mtDNA) accumulate in tissues of mammalian species and have been hypothesized to contribute to aging. We show that mice expressing a proofreading-deficient version of the mitochondrial DNA polymerase g (POLG) accumulate mtDNA mutations and display features of accelerated aging. Accumulation of mtDNA mutations was not associated with increased markers of oxidative stress or a defect in cellular proliferation, but was correlated with the induction of apoptotic markers, particularly in tissues characterized by rapid cellular turnover. The levels of apoptotic markers were also found to increase during aging in normal mice. Thus, accumulation of mtDNA mutations that promote apoptosis may be a central mechanism driving mammalian aging.

Ageing, oxidative stress, and mitochondrial uncoupling.

Mitochondria are a cell's single greatest source of reactive oxygen species. Reactive oxygen species are important for many life sustaining processes of cells and tissues, but they can also induce cell damage and death. If their production and levels within cells is not effectively controlled, then the detrimental effects of oxidative stress can accumulate. Oxidative stress is widely thought to underpin many ageing processes, and the oxidative stress theory of ageing is one of the most widely acknowledged theories of ageing. As well as being the major source of reactive oxygen species, mitochondria are also a major site of oxidative damage. The purpose of this review is a concise and current review of the effects of oxidative stress and ageing on mitochondrial function. Emphasis is placed upon the roles of mitochondrial proton leak, the uncoupling proteins, and the anti-ageing effects of caloric restriction.

Caloric restriction and body weight independently affect longevity in Wistar rats.

OBJECTIVE: To evaluate the independent effects of caloric restriction (CR) and body weight (BW) on mortality rate (MR) and the extent to which BW may mediate the effect of CR on MR. DESIGN AND SUBJECTS: Data were from the Biosure Study, a randomized, controlled, prospective intervention study of diet regimens in 1200 Wistar rats. Animals were followed until they died spontaneously, were euthanized because of illness, or reached age 30 months. STATISTICAL ANALYSIS: Cox regression was performed to evaluate the effects of CR and BW on MR. Bootstrap procedures were used to test the contribution of BW to the effect of CR on MR. RESULTS: CR initiated after age 13 weeks decreased the rate of subsequent mortality. The MR increased with higher BW in early adulthood (21 weeks) and this effect persisted even after adjustment for CR. After adjustment for BW in early adulthood, we did not find a similar relation between mortality and BW in late adulthood (105 weeks). Mediation analysis indicated that low BW associated with CR appeared to mediate some of the mortality-reducing effects of CR, but CR clearly had effects independent of BW. The reductions in BW appeared to account for approximately 11% of the effect of CR. CONCLUSION: CR and BW have independent effects on MR in Wistar rats. BW may mediate a small part of the CR effects on MR.

Lifelong voluntary exercise in the mouse prevents age-related alterations in gene expression in the heart.

We present the first quantitative gene expression analysis of cardiac aging under conditions of sedentary and active lifestyles using high-density oligonucleotide arrays representing 11,904 cDNAs and expressed sequence tags (ESTs). With these data, we test the hypothesis that exercise attenuates the gene expression changes that normally occur in the aging heart. Male mice (Mus domesticus) were sampled from the 16th generation of selective breeding for high voluntary exercise. For the selective breeding protocol, breeders were chosen based on the maximum number of wheel revolutions run on days 5 and 6 of a test at 8 wk of age. For the colony sampled herein, mice were housed individually over their entire lifetimes (from weaning) either with or without access to running wheels. The hearts of these two treatment groups (active and sedentary) were assayed at middle age (20 mo) and old age (33 mo). Genes significantly affected by age in the hearts of the sedentary population by at least a 50% expression change (n = 137) were distributed across several major categories, including inflammatory response, stress response, signal transduction, and energy metabolism. Genes significantly affected by age in the active population were fewer (n = 62). Of the 42 changes in gene expression that were common to both treatment groups, 32 (72%) displayed smaller fold changes as a result of exercise. Thus exercise offset many age-related gene expression changes observed in the hearts of the sedentary animals. These results suggest that adaptive physiological mechanisms that are induced by exercise can retard many effects of aging on heart muscle at the transcriptional level.

Caloric restriction lowers plasma lipoprotein (a) in male but not female rhesus monkeys.

Many age-associated pathophysiological changes are retarded by caloric restriction (CR). The present study has investigated the effect of CR on plasma lipoprotein (a) [Lp(a)], an independent risk factor for the age-associated process of atherosclerosis. Rhesus monkeys were fed a control diet (n=19 males, 12 females) or subjected to CR (n=20 males, 11 females fed 30% less calories) for >2 years. All female animals were premenopausal. Plasma Lp(a) levels in control animals were almost two fold higher for males than females (47+/-9 vs 25+/-5mg/dl mean+/-SEM, p=0.05). CR resulted in a reduction in circulating Lp(a) in males to levels similar to those measured in calorie-restricted females, (27+/-5 vs 24+/-4 mg/dl mean+/-SEM). For all animals, plasma Lp(a) was correlated with total cholesterol (r=0.27, p=0.03) and LDL cholesterol (r=0.50, p=0.0001) whether unadjusted or after adjustment for treatment, gender or group. These studies introduce a new mechanism whereby CR may have a beneficial effect on risk factors for the development of atherosclerosis in primates.

Influences of aging and caloric restriction on the transcriptional profile of skeletal muscle from rhesus monkeys.

In laboratory rodents, caloric restriction (CR) retards several age-dependent physiological and biochemical changes in skeletal muscle, including increased steady-state levels of oxidative damage to lipids, DNA, and proteins. We have previously used high-density oligonucleotide arrays to show that CR can prevent or delay most of the major age-related transcriptional alterations in the gastrocnemius muscle of C57BL/6 mice. Here we report the effects of aging and adult-onset CR on the gene expression profile of 7,070 genes in the vastus lateralis muscle from rhesus monkeys. Gene expression analysis of aged rhesus monkeys (mean age of 26 years) was compared with that of young animals (mean age of 8 years). Aging resulted in a selective up-regulation of transcripts involved in inflammation and oxidative stress, and a down-regulation of genes involved in mitochondrial electron transport and oxidative phosphorylation. Middle-aged monkeys (mean age of 20 years) subjected to CR since early adulthood (mean age of 11 years) were studied to determine the gene expression profile induced by CR. CR resulted in an up-regulation of cytoskeletal protein-encoding genes, and also a decrease in the expression of genes involved in mitochondrial bioenergetics. Surprisingly, we did not observe any evidence for an inhibitory effect of adult-onset CR on age-related changes in gene expression. These results indicate that the induction of an oxidative stress-induced transcriptional response may be a common feature of aging in skeletal muscle of rodents and primates, but the extent to which CR modifies these responses may be species-specific.

Effects of caloric restriction on skeletal muscle mitochondrial proton leak in aging rats.

Long-term caloric restriction (CR) retards aging processes and increases maximum life span. We investigated the influence of CR on mitochondrial proton leaks in rat skeletal muscle. Because CR lowers oxidative damage to mitochondrial membrane lipids and proteins, we hypothesized that leak would be lower in mitochondria from old CR rats than in age-matched controls. Three groups (n = 12) were studied: 4-month-old "young" control rats (body weight: 404 g +/- 7 SEM), 33-month-old CR rats (body weight: 262 g +/- 3), and 33-month-old control rats (body weight: 446 g +/- 5). CR rats received 67% of the energy intake of old control rats, with adequate intakes of all essential nutrients. Maximum leak-dependent O2 consumption (State 4) was 23% lower in CR rats than in age-matched controls, whereas protonmotive force values were similar, supporting our hypothesis. The overall kinetics of leak were similar between the two groups of old rats; in the young, kinetics indicated higher protonmotive force values. The latter indication is consistent with aging-induced alterations in proton leak kinetics that are independent of dietary intervention. There was no influence of age or diet on serum T4 level, whereas T3 was lower in young than in old control rats. These results support and extend the oxidative stress hypothesis of aging.

Microarray profiling of gene expression in aging and its alteration by caloric restriction in mice.

An active research area in biological gerontology concerns the mechanisms by which caloric restriction (CR) retards the aging process in laboratory rodents. We used high density oligonucleotide arrays representing 6347 genes to determine the gene expression profile of the aging process in gastrocnemius muscle of male C57BL/6 mice. Aging resulted in a differential gene expression pattern indicative of a marked stress response and lower expression of metabolic and biosynthetic genes. Most alterations were completely or partially prevented by CR. Transcriptional patterns of muscle from calorie-restricted animals suggest that CR retards the aging process by causing a metabolic shift toward increased protein turnover and decreased macromolecular damage. The use of high density oligonucleotide microarrays provides a new tool to measure biological age on a tissue-specific basis and to evaluate at the molecular level the efficacy of nutritional interventions designed to retard the aging process.

Caloric restriction mimetics: metabolic interventions.

Caloric restriction (CR) retards diseases and aging in laboratory rodents and is now being tested in nonhuman primates. One way to apply these findings to human health is to identify and test agents that may mimic critical actions of CR. Panel 2 focused on two outcomes of CR, reduction of oxidative stress and improved glucoregulation, for which candidate metabolic mimics exist. It was recommended that studies on oxidative stress should emphasize mitochondrial function and to test the efficacy of nitrone and other antioxidants in mimicking CR's effects. Studies should also focus on the long-term effects of compounds known to lower circulating glucose and insulin concentrations or to increase insulin sensitivity. Also, four other developing areas were identified: intermediary metabolism, response to infection, stress responses, and source of dietary fat. These areas are important because either they hold promise for the discovery of new mimetics or they need to be explored prior to initiation of CR trials in humans. Other recommendations were that transgenic approaches and adult-onset CR should be emphasized in future studies.

Dietary restriction and aging in rhesus monkeys: the University of Wisconsin study.

Dietary restriction (DR) retards aging and extends the maximum lifespan of laboratory mice and rats. To determine whether DR has similar actions in a primate species, we initiated a study in 1989 to investigate the effects of a 30% DR in 30 adult male rhesus monkeys. In 1994, an additional 30 females and 16 males were added to the study. Although the animals are still middle-aged, a few differences have developed between the control and DR animals suggesting that DR may induce physiologic changes in the rhesus monkey similar to those observed in rodents. Fasting basal insulin and glucose concentrations are lower in DR compared to control animals while insulin sensitivity is higher in the restricted animals. DR has also altered circulating LDL in a manner that may inhibit atherogenesis. These results suggest that DR may be slowing some age-related physiologic changes. In addition to measures of glucose and lipid metabolism, the animals are evaluated annually for body composition, energy expenditure, physical activity, hematologic indices, and blood or urinary hormone concentrations. In the next few years, the first animals will reach the average lifespan ( approximately 26 years) of captive rhesus monkeys and it will become possible to determine if DR retards the aging process and extends the lifespan in a primate species.

Restriction of energy intake, energy expenditure, and aging.

Energy restriction (ER), without malnutrition, increases maximum life span and retards the development of a broad array of pathophysiological changes in laboratory rodents. The mechanism responsible for the retardation of aging by ER is, however, unknown. One proposed explanation is a reduction in energy expenditure (EE). Reduced EE may increase life span by decreasing the number of oxygen molecules interacting with mitochondria, thereby lowering reactive oxygen species (ROS) production. As a step toward testing this hypothesis, it is important to determine the effect of ER on EE. Several whole-body, organ, and cellular studies have measured the influence of ER on EE. In general, whole-body studies have reported an acute decrease in mass-adjusted EE that disappears with long-term ER. Organ-specific studies have shown that decreases in EE of liver and gastrointestinal tract are primarily responsible for initial reductions in EE with ER. These data, however, do not determine whether cellular EE is altered with ER. Three major processes contributing to resting EE at the cellular level are mitochondrial proton leak, Na(+)-K(+)-ATPase activity, and protein turnover. Studies suggest that proton leak and Na(+)-K(+)-ATPase activity are decreased with ER, whereas protein turnover is either unchanged or slightly increased with ER. Thus, two of the three major processes contributing to resting EE at the cellular level may be decreased with ER. Although additional cellular measurements are needed, the current results suggest that a lowering of EE could be a mechanism for the action of ER.

Caloric restriction of rhesus monkeys lowers oxidative damage in skeletal muscle.

In laboratory rodents, caloric restriction (CR) retards several age-dependent physiological and biochemical changes in skeletal muscle, including increased steady-state levels of oxidative damage to lipids, DNA, and proteins. We used immunogold electron microscopic (EM) techniques with antibodies raised against 4-hydroxy-2-nonenal (HNE) -modified proteins, dinitrophenol, and nitrotyrosine to quantify and localize the age-dependent accrual of oxidative damage in rhesus monkey vastus lateralis skeletal muscle. Using immunogold EM analysis of muscle from rhesus monkeys ranging in age from 2 to 34 years old, a fourfold maximal increase in levels of HNE-modified proteins was observed. Likewise, carbonyl levels increased approximately twofold with aging. Comparing 17- to 23-year-old normally fed to age-matched monkeys subjected to CR for 10 years, levels of HNE-modified proteins, carbonyls, and nitrotyrosine in skeletal muscle from the CR group were significantly less than control group values. Oxidative damage largely localized to myofibrils, with lesser labeling in other subcellular compartments. Accumulation of lipid peroxidation-derived aldehydes, such as malondialdehyde and 4-hydroxy-2-alkenals, and protein carbonyls were measured biochemically and confirmed the morphological data. Our study is the first to quantify morphologically and localize the age-dependent accrual of oxidative damage in mammalian skeletal muscle and to demonstrate that oxidative damage in primates is lowered by CR.

Cellular phenotypes of age-associated skeletal muscle mitochondrial abnormalities in rhesus monkeys.

Rhesus monkey vastus lateralis muscle was examined histologically for age-associated electron transport system (ETS) abnormalities: fibers lacking cytochrome c oxidase activity (COX(-)) and/or exhibiting succinate dehydrogenase hyperreactivity (SDH(++)). Two hundred serial cross-sections (spanning 1600 microm) were obtained and analyzed for ETS abnormalities at regular intervals. The abundance and length of ETS abnormal regions increased with age. Extrapolating the data to the entire length of the fiber, up to 60% of the fibers were estimated to display ETS abnormalities in the oldest animal studied (34 years) compared to 4% in a young adult animal (11 years). ETS abnormal phenotypes varied with age and fiber type. Middle-aged animals primarily exhibited the COX(-) phenotype, while COX(-)/SDH(++) abnormalities were more common in old animals. Transition region phenotype was affected by fiber type with type 2 fibers first displaying COX(-) and then COX(-)/SDH(++) while type 1 fibers progressed from normal to SDH(++) and then to COX(-)/SDH(++). In situ hybridizations studies revealed an association of ETS abnormalities with deletions of the mitochondrial genome. By measuring cross-sectional area along the length of ETS abnormal fibers, we demonstrated that some of these fibers exhibit atrophy. Our data suggest mitochondrial (mtDNA) deletions and associated ETS abnormalities are contributors to age-associated fiber atrophy.

In vitro oxidation of low-density lipoprotein in two species of nonhuman primates subjected to caloric restriction.

Caloric restriction (CR), which increases longevity and retards age-associated diseases in laboratory rodents, is being evaluated in nonhuman primate trials. CR reduces oxidative stress in rodents and appears to improve risk factors for cardiovascular disease in nonhuman primates. We tested the hypothesis that low-density lipoprotein (LDL) oxidizability is reduced in two monkey species (rhesus and cynomolgus) subjected to chronic CR. In both species, no significant differences occurred between CR and control animals on total, LDL, or high-density lipoprotein (HDL) cholesterol. In rhesus monkeys, triglycerides were higher in controls than CR (139 +/- 23 vs 66 +/- 8 mg/dl,p < .01, respectively). LDL from CR rhesus monkeys was reduced in triglyceride content and molecular weight compared to controls, whereas LDL composition in cynomolgus monkeys was similar in CR and control animals. In keeping with minor deviations in lipids, antioxidants, and LDL composition, no consistent differences in in vitro LDL oxidizability were apparent between CR and controls in either species.

Gene-expression profile of the ageing brain in mice.

Ageing of the brain leads to impairments in cognitive and motor skills, and is the major risk factor for several common neurological disorders such as Alzheimer disease (AD) and Parkinson disease (PD). Recent studies suggest that normal brain ageing is associated with subtle morphological and functional alterations in specific neuronal circuits, as opposed to large-scale neuronal loss. In fact, ageing of the central nervous system in diverse mammalian species shares many features, such as atrophy of pyramidal neurons, synaptic atrophy, decrease of striatal dopamine receptors, accumulation of fluorescent pigments, cytoskeletal abnormalities, and reactive astrocytes and microglia. To provide the first global analysis of brain ageing at the molecular level, we used oligonucleotide arrays representing 6,347 genes to determine the gene-expression profile of the ageing neocortex and cerebellum in mice. Ageing resulted in a gene-expression profile indicative of an inflammatory response, oxidative stress and reduced neurotrophic support in both brain regions. At the transcriptional level, brain ageing in mice displays parallels with human neurodegenerative disorders. Caloric restriction, which retards the ageing process in mammals, selectively attenuated the age-associated induction of genes encoding inflammatory and stress responses.