Intrinsic differences in study design

Most of the early rodent CR studies involved very young onset life-long CR initiated post-weaning, usually in inbred genetic backgrounds. In the 1980s it became clear that adult onset CR (12-month-old mice) was also effective in delaying ageing and extending lifespan in rodents, albeit to a lesser extent than the young onset model22. Many rodent CR studies opt to feed control animals ad libitum amounts of food while others provide less than ad libitum amounts arguing that this strategy avoids the confounding effects of obesity and reduces variability in food intake among individuals. With the launch of the NIA rhesus monkey study in 1987, the implementation of CR was such that the control monkeys were not free-fed. Food allotments were determined in accordance with data published by the National Research Council to provide approximate ad libitum intake based on their age and bodyweight for the maturing control monkeys without overfeeding23. Rations were increased to maintain growth and development until full stature was attained. CR monkeys received 30% less food than height-, age- and sex-matched control monkeys. The intervention was initiated as young-onset and old-onset groups of males, and young, adult, and old-onset groups of females24 (Table 1). Launched in 1989, the UW study initiated the CR diet in adult animals only, after full stature was achieved (∼8 years of age for rhesus monkeys)25. Food was provided at levels approximating ad libitum to control animals. To accommodate heterogeneity in the feeding behaviours within the cohort, the ad libitum reference for each individual was established using baseline food intake measured over 3–6 months, and CR was implemented on a per-individual basis. The rationale for these design features at UW was to implement a study as it might have been conducted in humans.

Table 1 Study design. Full size table

The source of the monkeys in each cohort and the population type represented is also a point of difference for the two studies. The UW monkeys were born and raised at the Wisconsin National Primate Research Center and were all of Indian origin. The NIA monkeys were sourced from several locations and included monkeys of both Indian and Chinese origin. Chinese male rhesus monkeys are generally heavier and longer than their Indian counter parts with the reverse being the case for females, and Chinese rhesus monkeys are also thought to exhibit greater sexual dimorphism26. Monkeys of different origin are sufficiently genetically different that they can be distinguished using a panel of single nucleotide polymorphisms27. Apart from population differences, rhesus monkeys share a similar degree of inter-individual genetic variation as humans28. In this way, the contribution of population type to differences in outcomes of the two studies as opposed to the contribution of individual genetic heterogeneity is difficult to ascertain.

The diet compositions were another important difference between the two studies. First, the source of diet components was different. A naturally sourced diet was employed at the NIA facility to ensure that micronutrients such as phytochemicals and trace minerals were provided, acknowledging that there was potential for seasonal variation. In contrast, a semi-purified diet was employed at UW to ensure that intake could be fully defined and consistent throughout the course of the study. Second, although diets at both locations had a similar caloric density, the relative macronutrient composition of the diets was not equivalent (Table 2). Compared to the UW diet, the NIA diet was lower in fat, higher in protein and higher in fibre. Finally, the nutrient content of the diets was also different. At both locations diets contained∼60% carbohydrates by weight, but sucrose comprised less than 7% of total carbohydrates at NIA and 45% of total carbohydrates at UW. Diets at both locations were replete for vitamins that were provided at or above the recommended daily allowance.

Table 2 Diet composition at each location. Full size table

Feeding practices also differed between studies. At NIA, the monkeys were fed two meals at ∼6:30 and 13:00 each day. Any food remaining after the morning meal was removed after about 3 h, and a low calorie treat was provided, typically in the form of a small piece of fruit. The afternoon meal was not removed so that monkeys had access to food at night. At UW, all monkeys were fed in the morning at∼8:00 and any remaining food was removed at ∼16:00 when a treat of fresh fruit or vegetable, which was quickly and completely eaten, was provided. Food allotment for control animals was adjusted to ensure that there was always some uneaten food to be removed at the end of the day. In this way UW animals were ad libitum fed during the day but food deprived overnight. While there were considerable differences in study design as outlined above, it should be noted that animal housing and routine animal care were equivalent at NIA and UW primate facilities. This included identical housing conditions, temperature and humidity range, light cycles, and the use of tap water, which was continuously available. Both studies included animal monitoring several times per day, and a designated veterinary staff that inspected the animals routinely and provided outstanding care as needed.

Impact of CR on survival

The initial goal of both NIA and UW studies was to determine the impact of CR on the health of rhesus monkeys, as it was not a foregone conclusion that CR would be an appropriate intervention in long-lived species. The investigation of the impact of CR on longevity was not considered a primary outcome at either study location. Even though 121 monkeys were enrolled in the NIA study, the differences in age of onset (from 1 to 23 years) precluded the animals from being grouped together for data analyses. Although the age range for time of onset is smaller for the UW study (ages 7–15), with only 38 outbred genetically distinct monkeys per group (including both sexes), it seemed unlikely that the study would have the statistical power required to test CR’s effect on longevity. While neither study reports longevity data, both studies have yielded survival data. For rhesus monkeys in captivity, the previously reported median survival was ∼26 years of age, 10% survival was ∼35 years of age and maximal survival was ∼40 years of age29. Mortality curves were generated separately for UW and NIA (Fig. 1). Survival estimates for monkeys at both sites were calculated based on data captured up to July 2015 using the three most common statistical methods: Kaplan-Meier product-limit method; Cox proportional hazard regression and parametric survival analysis assuming a Weibull distribution (Table 3). Because the Weibull distribution is a special case of the generalized extreme value distribution, it can accommodate estimation of the upper quantiles of a survival distribution and maximal lifespan, especially when there are censored data due to animals that remain alive30.

Figure 1: Mortality curves for monkeys at UW and at NIA. These curves depict data for male and female monkeys on the UW study and on the NIA study. Animals are grouped by age where male J/A include juvenile and adolescent onset animals, female J/A include juvenile and adult onset animals, and old include the advanced age onset animals. Inset boxes indicate animals still alive, dashed line marks 50% mortality. Statistics related to this figure are provided in Supplementary Information, Supplementary Table 1. Full size image

Table 3 Survival estimates. Full size table

In the UW adult-onset study, the estimated survival of UW control animals was close to that of the average recorded for monkeys in captivity (∼26 years of age). Considering both males and females together, a statistically significant effect of CR in increasing survival was observed (Cox regression P=0.017; Supplementary Table 1). The hazard ratio (HR) of 1.865 (95% confidence interval (CI): 1.119–3.108) indicated that at any time-point the control monkeys had almost twice the rate of death when compared to CR animals. The effect of sex on the response to CR was not statistically significant. Kaplan-Meier analysis showed that median survival estimates were greater for CR animals for both males and females (Table 3). In the NIA study large differences in ages of monkeys at time of recruitment to the study (Table 1) prompted a separation of data from the early and late onset groups. Here and throughout this report, NIA male juveniles and adolescents (J/A) were grouped and female juveniles and adults were grouped (J/A). The Kaplan-Meier median estimated survival was not different between NIA control and CR animals for the J/A onset groups of males or females (Fig. 1). Although Cox proportional hazard regression indicated that the differences in survival between J/A control and CR were not statistically significant (Supplementary Table 1), CR monkeys reached 80% mortality before the controls for both sexes. With 38% of the NIA J/A cohort still alive, the survival curves are incomplete and the impact on survival remains to be determined; however, the early mortality suggests that for some individuals implementation of CR in the very young may confer a survival risk. For old-onset CR, Kaplan-Meier estimated survival was not different between control and CR groups for either males or females (Table 3), but survival estimates were higher than those of J/A monkeys and UW controls. For both males and females, survival estimates for the NIA old-onset cohort were comparable to or exceeded those for UW CR.

Although there were slight discrepancies in the estimated median survival between the non-parametric Kaplan-Meier and parametric Weibull estimation methods, the survival comparisons between study sites using either analysis were consistent. A certain degree of sexual dimorphism was observed in survival outcomes where incidence of early death appeared to be greater for females. This observation might be explained in part by endometriosis, which is the proliferation of endometrial tissue outside of the uterus. Endometriosis can occur at relative high incidence in monkeys in captivity (∼25%), and risk is considerably greater for nulliparous females31,32. Incidence of endometriosis was equivalent for control and CR groups. For the J/A cohorts in the NIA study, 12 of the 44 females died of complications due to endometriosis, and of these the juvenile onset females were confirmed nulliparous. Females recruited to the UW study, in contrast, had at least one but no more than three healthy infants33, and only 2 of 30 females died of complications due to endometriosis. A further contributing factor relates to the policy on treatment of clinical conditions. At UW the policy to treat clinical conditions was implemented from the outset. At NIA, although acute pain and suffering were always treated, chronic medical conditions, including endometriosis, were monitored but not medically treated. A policy change was implemented in 2010 due to the high incidence of endometriosis. The power to assess the impact of CR on survival for NIA J/A females has been compromised somewhat by this one condition.

Biometric and food intake measures from both studies

For over a quarter of a century during these studies, bodyweight, body composition and food intake were measured for all 197 monkeys. Bodyweight was determined in fasted and anesthetized monkeys 2–4 times per year during routine procedures. Longitudinal data for all monkeys were averaged by age of the animal (Fig. 2a). As is the case for humans, monkeys often experience cachexia or end-of-life rapid weight loss. To avoid confounding effects of weight change that is not related to food intake or diet, data from the last year of life for each monkey were excluded. To facilitate comparisons among the cohorts, data were grouped into three age categories representing young adult (11–13 years of age), late mid-age (18–20 years of age) and advanced age (25–27 years of age) (Supplementary Tables 2 and 3).

Figure 2: Bodyweight data for monkeys at NIA and UW. (a) Bodyweight (kg) for male and female monkeys at UW and at NIA grouped by age where male J/A include juvenile and adolescent onset animals, female J/A include juvenile and adult onset animals, and old include the advanced age onset animals. Digits shown in white within the boxes are the numbers of individual animals contributing to each data point, data are shown as mean±s.e. of the mean. (b) Comparison of bodyweight averages for monkeys from UW and NIA studies with records of the internet Primate Aging Database (iPAD). Average bodyweight for control and CR monkeys at both study locations were determined by age category including adult (11–13 years of age), late mid-age (18–20 years of age) and advanced age (25–27 years of age). Data are expressed as percent deviation from the iPAD average for females and males from each age category. Statistics related to this figure are provided in Supplementary Information, Supplementary Tables 2 and 3. Full size image

Considering first the female monkeys, bodyweight for the NIA J/A was not significantly different between control and CR monkeys for any age categories. UW CR females weighed significantly less (17–26%) than controls throughout the study period, and UW female controls weighed significantly more than NIA J/A female controls throughout (Supplementary Table 2). For NIA old-onset females, bodyweight was not significantly different between controls and CR, and was significantly lower than bodyweight of UW female controls. In summary, for NIA J/A and old-onset female cohorts, bodyweight for control and CR monkeys was not different from each other and all were significantly lower than the UW controls. Considering next the male monkeys, NIA J/A CR males weighed significantly less (19–22%) than their control counterparts throughout the study. The difference between UW control and CR was slightly greater (24–35%), with CR males weighing significantly less than controls. The average peak weight for NIA J/A control males was ∼15% lower than that of UW control males, but differences in bodyweight were significant for the young age category only (Supplementary Table 3). Bodyweight of the old-onset NIA control and CR males were not significantly different at either mid-age or advanced ages, and old-onset NIA male controls weighed significantly less than UW controls. In summary, NIA J/A and UW male cohorts showed a clear bodyweight response to CR, but old-onset NIA control and CR males were not different from each other and were significantly lower than the UW controls.

The internet Primate Aging Database (iPAD; http://ipad.primate.wisc.edu) is a repository of clinical and biometric data from healthy, non-experimental, captive nonhuman primates housed at research facilities across the USA. Using data from over 1,200 individual rhesus monkeys of Indian origin, mean bodyweights were calculated for the above age categories for males (11.6, 12.1, 11.5 kg respectively) and females (7.4, 8.4, 7.8 kg respectively). UW control and CR monkeys fell on either side of these averages; control monkeys were heavier than the iPAD average (∼18% for males; ∼19% for females), and CR monkeys had lower bodyweight than the iPAD average (∼12% for males; ∼11% for females) (Fig. 2b). For NIA J/A, control males were the same to slightly heavier (5–10%) than the iPAD average and CR weighed less than the iPAD average (∼20%), while control and CR female monkeys both weighed less than the iPAD average throughout the study (∼10% and ∼20% respectively). All NIA old-onset monkeys weighed less than the iPAD average for both control (∼15% for females; ∼10% for males) and CR (∼22% for females; ∼21% for males) monkeys. In summary, bodyweights of UW and NIA control monkeys were not equivalent to each other, and apart from J/A males, were respectively higher and lower of the iPAD average.

To gain insight into differences in the effect of age and diet on body composition, dual X-ray absorptiometry measures were conducted at intervals throughout the course of the two studies (Fig. 3). Since each animal had multiple measures taken over time, estimates of the average percent adiposity (fat/bodyweight expressed as percent) were adjusted for age (Supplementary Fig. 1). Within groups a main effect of age on adiposity was detected for NIA J/A and UW cohorts. A main effect of diet was detected for NIA J/A males and for both males and females from the UW study, where CR was associated with significantly lower adiposity. The NIA J/A control and CR females did not differ from each other in adiposity and neither of the NIA old-onset monkey groups had a main effect of CR on adiposity. Combining the data from NIA J/A and UW, a difference in adiposity was detected between controls on the two studies for both males and females, where NIA monkeys had significantly lower percent body fat. Control monkeys from NIA J/A were not statistically different from UW CR in percent body fat for both sexes. These data show an impact of age on adiposity in all three groups and reveal that the impact of CR on adiposity was observed for both groups of UW monkeys and at NIA for J/A males only.

Figure 3: Adiposity data for female and male monkeys at NIA and UW. Percent adiposity (fat (g)/total bodyweight (g)) calculated from DXA (dual energy X-ray absorptiometry) measures conducted during the course of the studies for male and female monkeys at UW and at NIA grouped by age where male J/A include juvenile and adolescent onset animals, female J/A include juvenile and adult onset animals, and old include the advanced age onset animals. Digits shown in white within the boxes are the numbers of individual animals contributing to each data point, data are shown as mean±s.e. of the mean. Full size image

Food intake was monitored daily at both sites. At UW daily measures of food intake were used to calculate means. At NIA food intake means were calculated based on measures conducted during a single week per year as representative of typical intake. Longitudinal data for all monkeys were averaged by age of the animal (Fig. 4). Data from the last year of life of each monkey were excluded to avoid confounding effects of end-of-life feeding behaviours that usually include loss of appetite. Considering first the females and using the age categories defined above for both UW and NIA J/A, the controls consumed significantly more calories than CR at both young and mid-age, but the difference persisted only for UW female monkeys at advanced age. For the old-onset NIA, caloric intake was not different between control and CR. Among control monkeys, UW females consumed significantly more calories than NIA J/A at mid-age and advanced age and more than old-onset at advanced age. Considering next the males, the NIA J/A controls consumed significantly more calories than CR at young and mid-age and the difference between control and CR was significant for UW at mid-age only. Old-onset males at NIA differed significantly in their caloric intake between control and CR only at advanced age. Among controls, caloric intake was not different for NIA J/A and UW males at any point in the study, but old-onset males consumed significantly less than UW males and NIA J/A males at mid-age. In summary, significant differences in caloric intake were identified between control and CR monkeys for male and female NIA J/A and UW cohorts, but not for old-onset cohorts until advanced age and then for males only. Comparing between sites, caloric intake for NIA female controls of both J/A and old-onset was lower than that of UW controls, and for males, caloric intake of NIA J/A and UW controls were not different from each other but old-onset NIA controls were lower than both.

Figure 4: Food intake data for monkeys at NIA and UW. Food intake (daily values in Kcalories) for male and female monkeys at UW and at NIA grouped by age where male J/A include juvenile and adolescent onset animals, female J/A include juvenile and adult onset animals, and old include the advanced age onset animals. Digits shown in white within the boxes are the numbers of individual animals contributing to each data point, data are shown as mean±s.e. of the mean. Full size image

Impact of CR on incidence of disease

The concept of healthspan is a fairly recent development in ageing research, where a distinction is drawn between chronological age and health status34. Traditionally, an increase in both median and maximum lifespan was considered the hallmark of delayed ageing, and improvements in health were deemed to be a necessary and obvious component of longevity. The perspective has shifted somewhat towards greater emphasis on health and morbidity, so an intervention that imparts improved health even in the absence of increased longevity, is viewed as a highly favourable and legitimate example of an ageing intervention. With advancing age, rhesus monkeys are vulnerable to many of the same conditions observed in humans. Among the most prevalent are cancer, cardiac disease, and conditions related to immune dysfunction and inflammation, and examples of each were identified in monkeys on the ageing and CR studies at both NIA and UW (Supplementary Table 4).

Fasting glucose measures were common to both studies and the longitudinal data are shown (Fig. 5). In healthy adult rhesus monkeys fasting glucose levels are 64–68 mg dl−1 (refs 18, 35). For NIA J/A, fasting glucose levels were equivalent for controls and CR up to ∼23 years of age, after which the control and CR males, but not females, began to diverge. Both control and CR females showed an age-related increase in fasting glucose levels after ∼21 years of age. For UW monkeys, the control males had higher fasting glucose levels than CR from 15 years of age with a further divergence of the curves after ∼23 years of age, while a noticeable difference between control and CR females emerged after only ∼21 years. For the NIA old-onset cohorts, fasting glucose was consistently low for the duration of the study period. These data point to an age-related increase in fasting glucose for rhesus monkeys and single out the UW control males as being predisposed to elevated circulating glucose in the fasted state. Using multilevel modelling to investigate the relationship between adiposity and fasting glucose levels a significant relationship was identified for UW males only (P=0.005). A significant age by diet interaction was also detected (P=0.014), suggesting that the impact of age on the relationship between adiposity and glucoregulatory parameters is distinct for control and CR monkeys.

Figure 5: Fasting glucose values for monkeys at NIA and UW. Circulating levels of glucose (mg dl−1) are shown for male and female monkeys at UW and at NIA grouped by age where male J/A include juvenile and adolescent onset animals, female J/A include juvenile and adult onset animals, and old include the advanced age onset animals. Digits shown in white within the boxes are the numbers of observations contributing to each data point, data are shown as mean±s.e. of the mean. Full size image

Veterinarians documented body condition and overall health of monkeys biannually at both study locations and indicators of diseases or disorders identified. The age at which a monkey was first diagnosed with an age-related condition was used to generate morbidity curves (Fig. 6). Age-related conditions included sarcopenia, osteoporosis, arthritis, diverticulosis, cataracts and persistent heart murmurs, in addition to age-related diseases including cancer, diabetes and cardiovascular disease. Cox proportional hazard regression modelling indicated that age-related conditions occurred at ∼2.7 times the rate in control animals compared to CR for UW monkeys (HR: 2.665; CI: 1.527–4.653; P=0.0006). In the NIA J/A cohort, age-related conditions occurred at twice the rate in control monkeys compared to CR (HR: 2.091; CI: 1.169–3.641; P=0.0125) (Supplementary Table 5; Supplementary Fig. 2). The advanced age of the old-onset NIA monkeys precluded detection of the first occurrence of an age-related condition.

Figure 6: Morbidity curves for monkeys at NIA and UW shown. (a) Graphs represent the first occurrence of any age-related disease, disorder or condition for combined males and females from UW (top) and NIA J/A (bottom). Statistics related to this figure are provided in Supplementary Information, Supplementary Table 4. (b) Incidence of prevalent age-related conditions in nonhuman primates for control and CR animals from UW and NIA (J/A and old-onset combined). To compare studies, cancer and cardiovascular disorders are reported as incidence upon necropsy and are expressed as a percentage of the animals that are deceased. Full size image