Beyond the rodent model: Calorie restriction in rhesus monkeys

Lifespan extension and reduction of age-related disease by calorie restriction (CR) are among the most consistent findings in gerontological research. The well known effects of CR have been demonstrated many times in rodents and other short-lived species. However, effects of CR on aging in longer-lived species, more closely related to humans, were unknown until recently. Studies of CR and aging using nonhuman primates (rhesus monkeys) were begun several years ago at the National Institute on Aging, the University of Wisconsin-Madison, and the University of Maryland. These studies are beginning to yield useful data regarding the effects of this nutritional intervention in primates. Several studies from these ongoing investigations have shown that rhesus monkeys on CR exhibit physiological responses to CR that parallel findings in rodents. In addition, several potential biomarkers of aging are being evaluated and preliminary findings suggest the possibility that CR in rhesus monkeys could slow the rate of aging and reduce age-related disease, specifically diabetes and cardiovascular disease. It will be several years before conclusive proof that CR slows aging and extends life span in primates is established, however, results from these exciting studies suggest the possibility that the anti-aging effects of CR reported in rodents also occur in longer-lived species such as nonhuman primates, strenghtening the possibility that this nutritional intervention will also prove beneficial in longer-lived species, including humans.     

Accommodative function in rhesus monkeys: effects of aging and calorie restriction

Numerous degenerative changes in the visual system occur with age, including a loss of accommodative function possibly related to hardening of the lens or loss of ciliary muscle mobility. The rhesus monkey is a reliable animal model for studying age-related changes in ocular function, including loss of accommodation. Calorie restriction (CR) is the only consistent intervention to slow aging and extend lifespan in rodents, and more recently the beneficial effects of CR have been reported in nonhuman primates. The goal of the present study was to evaluate age-related changes in ocular accommodation and the potential effect of long-term (>8 years) CR on accommodation in male and female rhesus monkeys. Refraction, accommodation (Hartinger coincidence refractometer), and lens thickness (A-scan ultrasound) were measured in 97 male and female rhesus monkeys age 8–36 years under Telazol/acepromazine anesthesia. Refraction and accommodation measurements were taken before and after 40% carbachol corneal iontophoresis to induce maximum accommodation. Half the animals were in the control (CON) group and were fed ad libitum. The CR group received 30% fewer calories than age- and weight-matched controls. Males were on CR for 12 years and females for eight years. With increasing age, accommodative ability declined in both CON and CR monkeys by 1.03 ± 0.12 (P = 0.001) and 1.18 ± 0.12 (P = 0.001) diopters/year, respectively. The age-related decline did not differ significantly between the groups (P = 0.374). Baseline lens thickness increased with age in both groups by 0.03 ± 0.005 mm/year (P = 0.001) and 0.02 ± 0.005 mm/year (P = 0.001) for the CON and CR groups, respectively. The tendency for the for the lens to thicken with age occurred at a slower rate in the CR group vs. the CON group but the difference was not statistically significant (P = 0.086). Baseline refraction was –2.8 ± 0.55 and –3.0 ± 0.62 diopters for CON and CR, respectively. Baseline refraction tended to become slightly more negative with age (P = 0.070), but this trend did not differ significantly between the groups (P = 0.587). In summary, there was no difference in the slope of the age-related changes in accommodation, lens thickness, or refraction in the carbachol-treated eyes due to diet. These data are consistent with previous findings of decreased accommodative ability in aging rhesus monkeys, comparable to the age-dependent decrease in accommodative ability in humans. This study is the first to indicate that the accommodative system may not benefit from calorie restriction.     

Dietary caloric restriction prevents the age-related decline in plasma melatonin levels of rhesus monkeys.

Rhesus monkeys exhibit an age-associated decrease in peak plasma melatonin levels analogous to that reported for humans. This decrease is essentially abolished in monkeys subjected to a 30% reduction in caloric intake over a 12-yr period. The caloric restriction (CR) effect does not seem to be a reversal, but rather a long-term prevention, of the age-related decline in hormone concentrations. The age effect does not seem to be due to a phase shift in the peak of melatonin secretions, as has been observed in some populations of aged humans. It is also extremely unlikely that the CR effect simply reflects a phase shift, since old monkeys on the diet have nocturnal melatonin levels equal to or greater than adult fully fed controls. Thus, if peak times (approximately 0200 h) were actually shifted, maximal levels in old CR monkeys would be even higher. These findings, coupled with previous observations in humans, suggest that peak plasma melatonin levels may represent a possible candidate "biomarker of aging" in primates. Moreover, this index of age-associated physiological decrement seems to be inhibited by dietary CR.     

Age-related alterations of plasma glutathione and oxidation of redox potentials in chimpanzee (Pan troglodytes) and rhesus monkey (Macaca mulatta)

Chimpanzee (Pan troglodytes) and rhesus macaque (Macaca mulatta) and humans (Homo sapiens) share physiological and genetic characteristics, but have remarkably different life spans, with chimpanzees living 50–60 % and the rhesus living 35–40 % of maximum human survival. Since oxidative processes are associated with aging and longevity, we might expect to see species differences in age-related oxidative processes. Blood and extracellular fluid contain two major thiol redox nodes, glutathione (GSH)/glutathione-disulfide (GSSG) and cysteine (Cys)/cystine (CySS), which are subject to reversible oxidation–reduction reactions and are maintained in a dynamic non-equilibrium state. Disruption of these thiol redox nodes leads to oxidation of their redox potentials (E_hGSSG and E_hCySS) which affects cellular physiology and is associated with aging and the development of chronic diseases in humans. The purpose of this study was to measure age-related changes in these redox thiols and their corresponding redox potentials (E_h) in chimpanzees and rhesus monkeys. Our results show similar age-related decreases in the concentration of plasma GSH and Total GSH as well as oxidation of the E_hGSSG in male and female chimpanzees. Female chimpanzees and female rhesus monkeys also were similar in several outcome measures. For example, similar age-related decreases in the concentration of plasma GSH and Total GSH, as well as age-related oxidation of the E_hGSSG were observed. The data collected from chimpanzees and rhesus monkeys corroborates previous reports on oxidative changes in humans and confirms their value as a comparative reference for primate aging.

Graphical abstract

Open in a separate windowGSH declined while the GSH/GSSG redox potential (EhGSSG) increased with age indicating age-related increased oxidative stress.     

Nonhuman primate models in biogerontology

A variety of animal models are utilized in biogerontological studies including yeast, nematodes, fruit flies, hamsters, mice, rats, and nonhuman primates. Species selection for research is based on many factors including economic feasibility, husbandry, generalizability of findings, available background information, adaptability to experimentation, and often, relevance to human aging. Each model offers its own strengths and limitations; however, nonhuman primates offer the unique advantage of phylogenetic proximity to humans. Among others, costs to purchase and maintain research subjects represent major limitations of nonhuman primate models. Although several nonhuman primate species have been utilized in aging research, rhesus monkeys (Macaca mulatta) are the best characterized and most extensively studied in biomedical gerontology. Nonhuman primate models have been employed as models for human aging in many research areas including neurobiology, skeletal, and reproductive aging and age-related diseases such as cardiovascular disease and diabetes. Primate models are now also being utilized to study interventions into aging such as caloric restriction. It will be several more years until definitive conclusions regarding lifespan effects can be made. However, existing data strongly suggest that many of the beneficial effects reported in rodents on CR also occur in primate models thereby strengthening the possibility that this nutritional paradigm may also impact favorably upon human aging.     

Locomotor activity in female rhesus monkeys: assessment of age and calorie restriction effects

As a component of a long-term, longitudinal study of aging in this primate model, the objective of the current experiment was to assess age and diet effects on locomotor activity in a cross-sectional analysis. By attaching a motion detection device to the home cage, locomotor activity was monitored over a week in a group (N = 47) of female rhesus monkeys (Macaca mulatta) 6-26 yrs of age. About half these monkeys composed a control group fed a nutritionally fortified diet near ad libitum levels, whereas an experimental group had been fed the same diet at levels 30% less than comparable control levels for approximately 5 yrs prior to testing. Among control monkeys, a marked age-related decline in activity was noted when total activity was considered and also when diurnal and nocturnal periods of activity were analyzed separately. When comparing activity levels between control and experimental groups, only one significant diet effect was noted, which was in the youngest group of monkeys (6-8 yrs of age) during the diurnal period. Monkeys in the experimental group exhibited reduced activity compared to controls. Body weight was not consistently correlated to activity levels. In some older groups, heavier monkeys tended to show greater activity, but in younger groups the opposite pattern was observed.     

Skeletal effects of long-term caloric restriction in rhesus monkeys

Age-related bone loss is well established in humans and is known to occur in nonhuman primates. There is little information, however, on the effect of dietary interventions, such as caloric restriction (CR), on age-related bone loss. This study examined the effects of long-term, moderate CR on skeletal parameters in rhesus monkeys. Thirty adult male rhesus monkeys were subjected to either a restricted (R, n = 15) or control (C, n = 15) diet for 20 years and examined throughout for body composition and biochemical markers of bone turnover. Total body, spine, and radius bone mass and density were assessed by dual-energy X-ray absorptiometry. Assessment of biochemical markers of bone turnover included circulating serum levels of osteocalcin, carboxyterminal telopeptide of type I collagen, cross-linked aminoterminal telopeptide of type I collagen, parathyroid hormone, and 25(OH)vitamin D. Overall, we found that bone mass and density declined over time with generally higher levels in C compared to R animals. Circulating serum markers of bone turnover were not different between C and R with nonsignficant diet-by-time interactions. We believe the lower bone mass in R animals reflects the smaller body size and not pathological osteopenia.     

Calorie restriction in rhesus monkeys

Calorie restriction (CR) extends lifespan and reduces the incidence and age of onset of age-related disease in several animal models. To determine if this nutritional intervention has similar actions in a long-lived primate species, the National Institute on Aging (NIA) initiated a study in 1987 to investigate the effects of a 30% CR in male and female rhesus macaques (Macaca mulatta) of a broad age range. We have observed physiological effects of CR that parallel rodent studies and may be predictive of an increased lifespan. Specifically, results from the NIA study have demonstrated that CR decreases body weight and fat mass, improves glucoregulatory function, decreases blood pressure and blood lipids, and decreases body temperature. Juvenile males exhibited delayed skeletal and sexual maturation. Adult bone mass was not affected by CR in females nor were several reproductive hormones or menstrual cycling. CR attenuated the age-associated decline in both dehydroepiandrosterone (DHEA) and melatonin in males. Although 81% of the monkeys in the study are still alive, preliminary evidence suggests that CR will have beneficial effects on morbidity and mortality. We are now preparing a battery of measures to provide a thorough and relevant analysis of the effectiveness of CR at delaying the onset of age-related disease and maintaining function later into life.     

Strategy for identifying biomarkers of aging in long-lived species

If effective anti-aging interventions are to be identified for human application, then the development of reliable and valid biomarkers of aging are essential for this progress. Despite the apparent demand for such gerotechnology, biomarker research has become a controversial pursuit. Much of the controversy has emerged from a lack of consensus on terminology and standards for evaluating the reliability and validity of candidate biomarkers. The initiation of longitudinal studies of aging in long-lived non-human primates has provided an opportunity for establishing the reliability and validity of biomarkers of aging potentially suitable for human studies. From the primate study initiated in 1987 at the National Institute on Aging (NIA), the following criteria for defining a biomarker of aging have been offered: (1) significant cross-sectional correlation with age; (2) significant longitudinal change in the same direction as the cross-sectional correlation; (3) significant stability of individual differences over time. These criteria relate to both reliability and validity. However, the process of validating a candidate biomarker requires a greater standard of proof. Ideally, the rate of change in a biomarker of aging should be predictive of lifespan. In short-lived species, such as rodents, populations differing in lifespan can be identified, such as different strains of rodents or groups on different diets, such as those subjected to calorie restriction (CR), which live markedly longer. However, in the NIA primate study, the objective is to demonstrate that CR retards the rate of aging and increases lifespan. In the absence of lifespan data associated with CR in primates, validation of biomarkers of aging must rely on other strategies of proof. With this challenge, we have offered the following strategy: If a candidate biomarker is a valid measure of the rate of aging, then the rate of age-related change in the biomarker should be proportional to differences in lifespan among related species. Thus, for example, the rate of change in a candidate biomarker of aging in chimpanzees should be twice that of humans (60 vs 120 years maximum lifespan); in rhesus monkeys three times that of humans (40 vs 120 years maximum lifespan). The realization of this strategy will be aided by developing a primate aging database, a project that was recently launched in cooperation with the NIA, the National Center for Research Resources, and the University of Wisconsin Regional Primate Research Center.     

Age- and pregnancy-related changes in serum total cholesterol and triglyceride levels in the Cayo Santiago rhesus macaques

Ninety-six free-ranging rhesus monkeys were evaluated for age-, sex-, and pregnancy-related changes in total serum cholesterol and triglyceride levels and compared with previous studies. Our findings indicate that pregnancy depressed total cholesterol in females and that cholesterol levels tend to increase in males with age. Triglycerides decreased significantly with advancing age in males.The Cayo Santiago rhesus monkeys represent a unique opportunity to study the effects of age on a population of nearly 1200 nonhuman primates on which there is accurate data onbirth date, lineage,behavior, reproduction and post-mortem morphology (skeletons). Further gerontological studies are necessary to take full advantage of this resource and to increase the presently-scant body of information on aging in monkeys for comparative studies on human and for the development of animal models of gerontological diseases of humans.     

Recent advances in calorie restriction research on aging

The extension of both median and maximum lifespan and the suppression of age-related diseases in laboratory animals by reduced food intake, i.e., calorie restriction (CR) are regarded as hallmarks of CR's anti-aging action. The diverse efficacy of CR to counteract aging effects and its experimental reproducibility has made it the gold standard of many aging intervention studies of recent years. Although CR originally was used as a tool to perturb the aging process of laboratory animals as to uncover clues of underlying mechanisms of aging processes, current CR research interests have shifted to the retardation of aging-related functional decline and the prevention of age-related diseases. Advances in CR research on non-human primates and recent endeavors using human subjects offer a promising outlook for CR's beneficial effects in healthy human aging.     

Age-related pathology and biosenescent markers in captive rhesus macaques

During the past 15 years, our aging colony of rhesus monkeys, consisting of animals from 20 to 37 years of age, had an annual average population of 88.2 live monkeys and, of this population, an annual average of 13.9 monkeys died spontaneously or were terminated due to severe illness. From 1980 to 1994, a total of 175 autopsies of rhesus macaques, from 20 to 37 years of age, were performed. By cumulative autopsy data, the incidence of age-related pathology in various organs was surveyed. Major geriatric diseases such as coronary sclerosis, emphysema, degenerative joint disorders, cancer, and cerebral amyloid plaque began to develop in 10 to 40% of macaques after 20 years and the incidence significantly increased after 26 years of age. Approximately 12% of aged macaques from 20 to 30 years of age died annually due to such geriatric diseases with severe complications. The average survival rate indicated that half the population at 20 years of age died by 25 years and 73% died by 30 years of age. Less than 10% of macaques survived over 30 years. Using these aged macaques as well as other juvenile to adult monkeys in our Center, clinical opththalmological and reproductive endocrinological studies, and magnetic resonance imaging (MRI) of the brain were conducted to define bioaging markers of captive rhesus monkeys. Cataracts began to develop in 20% of rhesus monkeys at 20 to 22 years of age and the rate significantly increased after 26 years of age. Menopause occurred at 26 to 27 years of age. Multiple cerebral infarctions and iron deposits in the globus pallidus and substantia nigra were detected by MRI in the aged brains. These geriatric disorders in captive aged macaques appear to be natural aging outcomes, since the simple lifestyle of these captive animals offers minimal exposure to environmental factors. Our data will offer useful paradigms for preventive or experimental studies on age-related diseases.     

Calorie restriction (CR) and CR mimetics for the prevention and treatment of age-related eye disorders

The morbidity of ocular diseases, including macular degeneration, diabetic retinopathy, and dry eye disease, has been gradually increasing worldwide. Because these diseases develop from age-associated ocular dysfunctions, interventions against the aging process itself may be a promising strategy for their management. Among the several approaches to interrupt aging processes, calorie restriction (CR) has been shown to recover and/or slow age-related functional declines in various organs, including the eye. Here, we review interventions against the aging process as potential therapeutic approaches to age-related ocular diseases. The effects of CR and CR mimetics in animal models of age-related eye diseases are explored. Furthermore, we discuss the possibilities of expanding this research to prospective studies to elucidate the molecular mechanisms by which CR and/or CR mimetics preserve ocular functions.     

Influences of aging and dietary restriction on red blood cell density profiles and antioxidant enzyme activities in rhesus monkeys

Dietary restriction (DR) retards aging processes in rodents and other animals but its influence on aging in primates is unknown. In rats, the average density of red blood cells (RBCs) reportedly increases with RBC age and decreases with host age and RBC antioxidant enzyme activities fall with both types of aging. We determined RBC density profiles and antioxidant enzyme activities in four groups (n = 5) of male rhesus monkeys. The “Control” group (11–14 years) was fed a purified diet ad lib and the “DR” group (11–16 years) were fed 70% of the ad lib level for two years. “Young” (6–10 years) and “Old” (27–36 years) monkeys were fed a nonpurified diet ad lib. The average RBC size was least in the most dense fraction (F4) and internal structural complexity increased with RBC density based on flow cytometry analysis but these were not influenced by host age or DR. Catalase activity decreased with increasing density. In contrast to findings in rats, age and fraction differences in glutathione peroxidase activities were insignificant. DR did not influence enzyme activities. These data suggest that aging in rhesus monkeys influences RBC density profiles and antioxidant enzyme activities far less strikingly than has been reported in rats.     

Caloric restriction <Emphasis Type="Italic">versus</Emphasis> drug therapy to delay the onset of aging diseases and extend life

There are two firmly established methods of prolonging life. Calorie restriction (CR) using nutrient-rich diets to prolong life in lower animals, and life saving medications in humans to delay the development of the major diseases of middle and old age. These two approaches have different mechanisms of action. In rats, CR at 40% below ad libitum intake begun soon after weaning and continued until death, reduces body weight by about 40% and increases lifespan. There have been no lifelong CR studies performed on humans. However, in healthy adult human subjects about 20% CR over a period of 2–15 years, lowers body weight by about 20% and decreases body mass index (BMI) to about 19. This CR treatment in humans reduces blood pressure and blood cholesterol to a similar extent as the specific drugs used to delay the onset of vascular disease and so extend human life. These same drugs may act by mechanisms that overlap with some of the mechanisms of CR in retarding these pathologies and thus may have similar antiaging and life prolonging actions. Such drugs may be regarded as CR mimetics which inhibit the development of certain life shortening diseases, without the need to lower calorie intake. In developed countries, better medical care, drug therapy, vaccinations, and other public health measures have extended human life by about 30 years during the 20th century without recourse to CR, which is so effective in the rat. The percentage gain in human life expectancy during the 20th century is twice that achieved by CR in rat survival. However, rat longevity studies now use specific pathogen-free animals and start CR after weaning or later, thereby excluding deaths from infectious diseases and those associated with birth and early life. There is a need to develop CR mimetics which can delay the development of life-threatening diseases in humans. In the 21st century due to the human epidemic of overeating with a sedentary lifestyle, it may necessary to utilize CR to counter the aging effects of overweight. Since the greatest life-extending effects of CR in the rodent occur when started early in life, long-term antiaging therapy in humans should be initiated soon after maturity, when physiological systems have developed optimally.     

Dehydroepiandrosterone sulfate (DHEAS) as an endocrine marker of aging in calorie restriction studies

The adrenal steroid, dehydroepiandrosterone sulfate (DHEAS), is generally regarded as being a reliable endocrine marker of aging, because in humans and nonhuman primates its circulating concentrations are very high during young adulthood, and the concentrations then decline markedly during aging. Despite promising results from early studies, we were recently surprised to find that caloric restriction (CR) did little to prevent or delay the decline of DHEAS concentrations in old rhesus macaques. Here we summarize the use of circulating DHEAS concentrations as a biomarker of aging in CR studies and suggest reasons for its limited value. Although DHEAS can reliably predict aging in animals maintained on a standard diet, dietary manipulations may affect liver enzymes involved in the metabolism of steroid hormones. Consequently, in CR studies the reliability of using DHEAS as a biomarker of aging may be compromised.     

Effect of long-term caloric restriction on GLUT4, phosphatidylinositol-3 kinase p85 subunit, and insulin receptor substrate-1 protein levels in rhesus monkey skeletal muscle

Caloric restriction (CR), a reduction in calorie intake without malnutrition, improves insulin sensitivity in various species, including mice, rats, rhesus and cynomolgus monkeys, and humans. Skeletal muscle is quantitatively the most important tissue for blood glucose clearance. Therefore, we assessed the effect of 6 years of CR (30% reduction in calorie intake) in male rhesus monkeys (14-20 years old) on muscle expression of several proteins involved in insulin action. Whole body insulin sensitivity (assessed by Modified Minimal Model) was significantly increased in CR relative to Control monkeys. CR did not alter the expression of GLUT4 glucose transporter or phosphatidylinositol-3 kinase p85 subunit (PI3K). Insulin receptor substrate-1(IRS-1) abundance tended to be greater for CR compared to Control monkeys (p = .051), but correlational analysis revealed no association between IRS-1 and insulin sensitivity (r2 = .075, p = .271). These findings indicate that the CR-induced increase in insulin sensitivity in rhesus monkeys is unrelated to alterations in GLUT4, P13K, and IRS-1 abundance.     

Long-term calorie restriction, but not endurance exercise, lowers core body temperature in humans

Reduction of body temperature has been proposed to contribute to the increased lifespan in calorie restricted animals and mice overexpressing the uncoupling protein-2 in hypocretin neurons. However, nothing is known regarding the long-term effects of calorie restriction (CR) with adequate nutrition on body temperature in humans. In this study, 24-hour core body temperature was measured every minute by using ingested telemetric capsules in 24 men and women (mean age 53.7 ± 9.4 yrs) consuming a CR diet for an average of 6 years, 24 age- and sex-matched sedentary (WD) and 24 body fat-matched exercise-trained (EX) volunteers, who were eating Western diets. The CR and EX groups were significantly leaner than the WD group. Energy intake was lower in the CR group (1769 ± 348 kcal/d) than in the WD (2302 ± 668 kcal/d) and EX (2798 ± 760 kcal/d) groups (P < 0.0001). Mean 24-hour, day-time and night-time core body temperatures were all significantly lower in the CR group than in the WD and EX groups (P ≤ 0.01). Long-term CR with adequate nutrition in lean and weight-stable healthy humans is associated with a sustained reduction in core body temperature, similar to that found in CR rodents and monkeys. This adaptation is likely due to CR itself, rather than to leanness, and may be involved in slowing the rate of aging.     

Caloric restriction delays aging-induced cellular phenotypes in rhesus monkey skeletal muscle

Sarcopenia is the age-related loss of skeletal muscle mass and function and is characterized by a reduction in muscle mass and fiber cross-sectional area, alterations in muscle fiber type and mitochondrial functional changes. In rhesus monkeys, calorie restriction (CR) without malnutrition improves survival and delays the onset of age-associated diseases and disorders including sarcopenia. We present a longitudinal study on the impact of CR on early stage sarcopenia in the upper leg of monkeys from ~ 16 years to ~ 22 years of age. Using dual-energy X-ray absorptiometry we show that CR delayed the development of maximum muscle mass and, unlike Control animals, muscle mass of the upper leg was preserved in CR animals during early phase sarcopenia. Histochemical analyses of vastus lateralis muscle biopsies revealed that CR opposed age-related changes in the proportion of Type II muscle fibers and fiber cross-sectional area. In contrast the number of muscle fibers with mitochondrial electron transport system enzyme abnormalities (ETS^ab) was not significantly affected by CR. Laser capture microdissection of ETS^ab fibers and subsequent PCR analysis of the mitochondrial DNA revealed large deletion mutations in fibers with abnormal mitochondrial enzyme activities. CR did not prevent stochastic mitochondrial deletion mutations in muscle fibers but CR may have contributed to the maintenance of affected fibers.