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Original Articles |
Gerontology Research Center (G.S.R., D.K.I., M.A.L.), National Institute on Aging, Baltimore, Maryland 21224; Fred Hutchinson Cancer Research Center (V.L., M.L.), Seattle, Washington 98109; and Jean Choay Institute for Biomedical Research (M.L.), CH-6826 Riva San Vitale, Switzerland
Address correspondence and requests for reprints to: George S. Roth, Ph.D., Gerontology Research Center, National Institute on Aging, 5600 Nathan Shock Drive, Baltimore, Maryland 21224.
Abstract
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.
DIETARY CALORIC RESTRICTION (CR) is the only intervention reproducibly shown to slow aging and reduce age-related diseases in mammals (1). Although most of the early work in this area used rodents, the applicability of CR to primates has been under investigation since 1987 (2). A number of parallels between the beneficial effects of this regimen in rodents and monkeys have now been established. These include reductions in body mass and fat, delayed maturation, reductions in plasma insulin and glucose with increased insulin sensitivity, lower body temperature and initially reduced metabolic rate, lower blood lipids with increased high-density lipoprotein, and delay or reduction in a number of other age-associated decrements (for a review see Ref. 3).
To determine conclusively whether CR exerts "anti-aging" effects in monkeys, it is necessary to demonstrate an extended life span or a slower rate of change in some indices of aging. Because the maximum life span of rhesus monkeys in captivity is on the order of 40 yr (3), we chose the latter strategy initially. This strategy entails establishing a battery of candidate "biomarkers of aging" that can be used to determine whether CR has altered aging rates. Valid biomarkers should exhibit both cross-sectional age differences and longitudinal age changes in the same direction (4). In addition, individual differences should be stable, such that over time the rank order of subjects is maintained (4). We have previously demonstrated the feasibility of this approach, using various routine blood chemistry and hematology measures (5, 6), as well as plasma levels of dehydroepiandrosterone sulfate (7).
One potential candidate biomarker of aging that has been the subject of much recent attention is plasma melatonin (8, 9, 10, 11). This hormone, secreted by the pineal gland and several tissues including the gastrointestinal tract, is released into circulation in a pulsatile fashion with the sharpest peaks in the early morning hours (12). A number of studies have reported that the amplitude of these peaks is progressively reduced during human aging (e.g. Refs. 8, 9, 10, 11). In addition, the phasing of these peaks may also be shifted (11). Therefore, it became of interest to examine the nocturnal peak of melatonin secretion in rhesus monkeys subjected to CR to determine (8) whether this hormonal parameter is affected by age, and (9) whether CR diminishes these age differences. To our knowledge, the present study is the first to demonstrate an age-related decrease in the nocturnal peak of melatonin secretion in monkeys and, more importantly, that long-term CR greatly reduces, or delays, this increase.
Materials and Methods
Subjects
The present study included a total of 96 (51 males and 45 females) rhesus monkeys (Macaca mulatta). Monkeys used in this study were part of an ongoing study of aging and CR in nonhuman primates at the National Institute on Aging (1). The monkeys ranged from 2–17 yr of age at the time CR was initiated. The data included here were collected from male monkeys that had been maintained on 30% CR for nearly 12 yr, whereas females were on 30% CR for approximately 7 yr.
Animal husbandry and diet composition have been described in detail elsewhere (1). Briefly, all monkeys were housed individually but had extensive auditory, visual, and olfactory interaction with other monkeys in the vivarium. The vivaria were maintained on a 12 h light (0600 h):12 h dark (1800 h) cycle, and all rooms were controlled for temperature (22-28 C) and humidity (50–60%).
Monkeys in both the control (CON) and CR groups were fed two meals a day (0700 and 1400 h). Food allotments for CON monkeys were based on National Research Council (see Ref. 1) requirements for monkeys of a given age and weight. Regular measurements of food consumption have determined that these allotments approximate ad libitum feeding. The daily food allotment offered to CR monkeys was 30% less than that given to age- and body weight-matched monkeys in the CON group. Diet composition (1) did not differ between the two groups. As such, the experimental manipulation was a reduction in total caloric intake.
Blood samples
Blood samples for the determination of plasma melatonin were
collected in fasted, anesthetized (7–10 mg/kg, ketamine im) monkeys
from a single venipuncture of the femoral vein. All sampling was
conducted under red light to avoid artificial inhibition of melatonin
secretion during nighttime hours. We initially determined the time
course of rhesus melatonin secretion by collecting blood samples
every 4 h in two young adult monkeys over a 24-h period. The peak
melatonin concentration occurred between 2200 and 0200 h (Fig. 1
). To be certain that age changes in the
peak time do not occur, the sampling was repeated on four young adult
and four old monkeys using a similar schedule. Maximum melatonin levels
were observed to occur between 2400 and 0300 h and between 2315
and 0215 h (SE ± 1.5 h, P >
0.5) for young and old animals, respectively. All blood samples from
CON and CR monkeys were then collected within this time period. Whole
blood was centrifuged, and the resultant plasma was sorted at -80 C
until assayed.
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Statistical analyses
Melatonin data measured as pg/mL was subjected to a log transformation before statistical analysis because of marked interindividual variability. The effects of age on plasma melatonin were assessed by linear regression in CON and CR monkeys. Additional effects of age group and CR were analyzed using two-way (age x CR) ANOVA. When the age by diet interaction was significant, additional within group effects were determined by a test for simple main effects. Statistical significance was accepted as P less than 0.05.
Results
Characteristics of CON and CR rhesus monkeys under study
Table 1 lists the ages and
body weights of the monkeys in the present study. Both Adult and Old
males are slightly older than their female counterparts, due to the
demographics of the colony. Males in both CON and CR groups are heavier
than females, whereas CR animals are 20–25% lighter than controls
(except for the Old female group, which suffered some deaths of heavier
CON animals).
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Figure 1
shows that, as in humans, plasma melatonin in rhesus
monkeys varies with the light cycle, with a peak at approximately
0200 h when the light period is from 0600–1800 h. Despite
considerable interindividual variability in the magnitude of the peak,
the time course is quite consistent. Two Adult female monkeys, 12 yr of
age, with extremes in absolute plasma melatonin values are depicted to
illustrate this point. Also, as indicated in Materials and
Methods, peak melatonin times were assessed in animals of various
ages and did not significantly differ. Therefore, a sampling time of
0200 h was chosen for the full study.
Effect of age and diet on peak plasma levels of melatonin in rhesus monkeys
Because no gender differences were observed (gender effect,
P > 0.05), data from both males and females are
combined for analysis. Figure 3 shows the
effects of aging CON (left) and CR rhesus monkeys
(right) on the nocturnal peak plasma melatonin levels (log
transformed). A statistically nonsignificant trend toward a progressive
reduction with increasing age, as in humans, is observed in fully fed
CON animals (Fig. 3A, P < 0.06). In contrast, CR
counterparts do not exhibit any hint of an age-related decline.
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).
Data from this analysis are depicted in Fig. 3B. Two-way ANOVA
confirmed a significant age x diet interaction (P
< 0.04). It is apparent from the figure that a statistically
significant age-related reduction in melatonin concentrations was
apparent (left) (P < 0.01).
Surprisingly, many Old CR monkeys seem to have plasma melatonin values
even higher than many Adult CON (Fig. 3B). Because, unfortunately,
longitudinal data on melatonin levels in these same animals over the
entire 12-yr course of this ongoing study are not available, it became
important to determine whether CR was reducing the normal
age-associated decrease in hormone concentrations or actually reversing
(or more than reversing) the decrement. Data (not shown) from a
12-month CR exposure in both Adult and Old monkeys did not reveal any
change in peak melatonin levels. Thus, the CR effect seems to be a
chronic reduction or delay in the age-related hormonal decline rather
that an acute reversal in old monkeys. Alternatively, a 1-yr duration
of CR may not be long enough to affect plasma melatonin levels under
the present conditions.
Discussion
Results from the present study of rhesus monkeys confirm and
extend observations in humans, which show a progressive age-related
decrease in the nocturnal peak of melatonin secretion
(8, 9, 10, 11). Not only do monkeys exhibit a similar
age-associated reduction in peak hormone levels, but animals that have
been maintained on 30% CR for a 12-yr period, do not show the
decrease. Indeed, the night peak melatonin levels are considerably
higher in Old CR monkeys compared with age-matched controls (Fig. 3B).
The CR effect seems to be still another example of how CR can reduce or
delay an age-dependent physiological change (for a review see Ref.
1), because short-term restriction (12 months) does not
alter melatonin secretion. Age effects do not seem to reflect phase
shifts in the melatonin peak as has been reported for some elderly
humans (11). Moreover, a phase shift would be a very
unlikely explanation for the CR effect since Old monkeys in this group
already have melatonin levels equal to, or greater than, Adult control
counterparts.
Several investigators have implicated changes in melatonin concentrations in age-related physiological deterioration (14, 15, 16). Although this point has been somewhat controversial (17, 18, 19), reasonable evidence suggests that decreases in plasma levels of melatonin may represent a possible candidate biomarker of aging. Unfortunately, the lack of serial measurements throughout the 12 yr of this ongoing study precludes any conclusions regarding longitudinal changes or stability of individual differences over time. Substantial animal-to-animal variability may present another problem. However, it will now be possible to follow potential age changes over the remainder of the project to determine whether these criteria for valid biomarkers are satisfied. In addition, it will be important to determine whether Old CR monkeys eventually exhibit some decrease in peak plasma melatonin secretion, because most CR effects are retardation or partial amelioration, rather than complete prevention, of age changes (1).
In any case, the present findings are consistent with data obtained by others in rats (20) and further support the idea that primate aging may be slowed by CR. Several mechanisms have been proposed to explain the anti-aging and "anti-disease" effects of restriction (1). These include protection against oxidative stress and other stressors, reduced glycation of macromolecules, as well as neuroendocrine regulation of genes involved in energy metabolism. These proposed mechanisms are not mutually exclusive and more than one may be involved in the phenomenology described here. Apparent beneficial effects of this intervention in rhesus monkeys have also been reported with respect to the normal age-associated decrease in plasma dehydrepiandrosterone sulfate concentrations (7) and several blood chemistry parameters (5, 6) as well as plasma glucose (21) and high-density lipoprotein levels (22). Ultimately, it will be necessary to combine a number of such candidate biomarkers of aging to obtain a global assessment of organismic aging rate (5, 6). Whether or not CR monkeys will actually outlive fully fed counterparts, as has been repeatedly demonstrated in rodents and invertebrates (1), remains to be determined, although preliminary results on morbidity and mortality from the current study suggest that this may, indeed, eventually be the case (23). Moreover, antagonism of any physiological decline by this intervention bodes well for the possibility that it may indeed exert at least some beneficial effects in primates, and in theory could be applicable to humans.
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We thank Drs. Walter Pierpaoli and Douglas Powell, Mr. Edward Tilmont, and Ms. April Handy for advice, veterinary care, and technical assistance, respectively.
Received February 29, 2000.
Revised September 1, 2000.
Revised February 7, 2001.
Accepted March 20, 2001.
References
This article has been cited by other articles:
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  G. S. Roth, J. A. Mattison, M. A. Ottinger, M. E. Chachich, M. A. Lane, and D. K. Ingram Aging in Rhesus Monkeys: Relevance to Human Health Interventions Science, September 3, 2004; 305(5689): 1423 - 1426. [Abstract] [Full Text] [PDF]
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G. S. Roth, M. A. Lane, D. K. Ingram, J. A. Mattison, D. Elahi, J. D. Tobin, D. Muller, and E. J. Metter Biomarkers of Caloric Restriction May Predict Longevity in Humans Science, August 2, 2002; 297(5582): 811 - 811. [Full Text] [PDF]
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,
Analysis of the simple main effect of diet indicated a significant
effect in the Old group (P < 0.01), but not in the
young group.