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Calorie Restriction and Skeletal Mass in Rhesus Monkeys (Macaca mulatta)

Evidence for an Effect Mediated Through Changes in Body Size

^a Molecular and Nutritional Physiology Unit, Gerontology Research Center, National Institute on Aging, Baltimore, Maryland
^b Obesity Research Center, St. Luke's/Roosevelt Hospital, New York, New York
^c Department of Nutritional Sciences, Rutgers, The State University, New Brunswick, New Jersey

M.A. Lane, Gerontology Research Center, 5600 Nathan Shock Blvd., Baltimore, MD 21224 E-mail: MLANE{at}vms.grc.nia.nih.gov.

Decision Editor: John A. Faulkner, PhD

	Abstract

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Little is known regarding the effects of prolonged calorie restriction (CR) on skeletal health. We investigated long-term (11 years) and short-term (12 months) effects of moderate CR on bone mass and biochemical indices of bone metabolism in male rhesus monkeys across a range of ages. A lower bone mass in long-term CR monkeys was accounted for by adjusting for age and body weight differences. A further analysis indicated that lean mass, but not fat mass, was a strong predictor of bone mass in both CR and control monkeys. No effect of short-term CR on bone mass was observed in older monkeys (mean age, 19 years), although young monkeys (4 years) subjected to short-term CR exhibited slower gains in total body bone density and content than age-matched controls. Neither biochemical markers of bone turnover nor hormonal regulators of bone metabolism were affected by long-term CR. Although osteocalcin concentrations were significantly lower in young restricted males after 1 month on 30% CR in the short-term study, they were no longer different from control values by 6 months on 30% CR.

CALORIE restriction (CR), undernutrition without malnutrition, is the only intervention known to slow several physiologic indices of aging ^(1)(2)(3), increase life span, attenuate the prevalence and severity of tumor formation, and slow the progression of age-related disease in short-lived species, such as rodents ⁽¹⁾⁽³⁾. Currently, two long-term studies at the National Institute on Aging (NIA; 4) and the University of Wisconsin—Madison ⁽⁵⁾ are examining the effects of moderate, long-term CR in rhesus monkeys. Data from these studies suggest that the effects of CR in these long-lived species will, in most ways, parallel the extensive results reported in rodents; for a review see Lane and colleagues ⁽⁶⁾.

Although the effects of CR on several physiologic parameters have been closely examined, the effects of this nutritional intervention on the skeleton are not well defined. The majority of studies examining the effects of CR on bone health have been conducted in rodents ^{(7)(8)(9)(10)}, although a couple of studies have used nonhuman primates ^(5)(11). Variability in the age at which CR was initiated, the degree and duration of CR, and the methods used to assess skeletal changes make these studies difficult to compare. McCay and colleagues ⁽¹²⁾ reported extreme fragility of femurs removed from CR rats, such that the bones crumbled on dissection. However, it is likely that the extreme nature of the calorie restriction used in these early studies, as well as calcium deficiency, contributed to the increased bone frailty. Overall, results from recent studies using a more moderate level of CR and fortified rations indicate that long-term CR results in the delayed development of a smaller but otherwise normal skeleton when the CR is initiated in young rats ⁽⁷⁾⁽⁸⁾ and monkeys ⁽¹¹⁾. The prevention of hyperparathyroidism and the associated decline in bone mineral density (BMD) in Fischer 344 rats subjected to lifelong CR may reflect the retardation of renal disease in this rodent model ⁽⁷⁾⁽⁸⁾. However, because the Fischer 344 rat is prone to developing renal disease late in life, it cannot be assumed that CR would have this affect in other species. Although CR tends to reduce measures of skeletal mass, such as BMD, bone mineral content (BMC), calcium content, and bone strength, when initiated in adult animals ^{(7)(8)(9)(10)(13)(14)(15)}, these changes may be related to reductions in body weight and altered body composition that accompany CR ^(9)(10)(15).

In humans, a growing body of literature demonstrates a positive association between body weight and BMD ^(16)(17). Low body weight is associated with decreased BMD in humans ^{(18)(19)(20)(21)} and rats ^(22)(23), although it is unclear whether the effects of body weight on the skeleton are mediated by changes in lean mass or fat mass ^{(18)(24)(25)(26)(27)(28)}.

Because of the many potentially beneficial effects of long-term CR on health and longevity, it is important to investigate fully the effect of this intervention on skeletal health. The present study was designed to assess the effects of moderate long-term (11 years) CR on BMD and BMC at several skeletal sites, as well as body composition, biochemical markers of bone turnover, and hormonal regulators of bone metabolism in male rhesus monkeys of various ages. A second aspect of the study involved investigating the temporal relationships between changes in bone and body composition during the initiation of CR.

	Materials and Methods

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Animals and Husbandry
Long-term CR study.-- Fifty-one adult male rhesus monkeys (Macaca mulatta) ranging from 12 to 34 years of age were included in this study. Roughly half (n = 24) of the monkeys had consumed a 30% calorie-restricted diet for the previous 11 years, whereas the remaining 27 monkeys served as controls, eating at or near ad-libitum levels. Thus, monkeys ranged in age from prepubertal to fully mature at the initiation of CR. The ages of the monkeys were not evenly distributed over the range of ages, but they were clustered into two age groupings, adult (12.2–16.4 years) and old (25.2–34.5 years). All animals were research naïve with documented dates of birth, and they were housed continuously at the Primate Unit of the Veterinary Resources Program, Office of Research Services National Institutes of Health (ORS), in Poolesville, MD. The monkeys were singly housed in weight-appropriate sized caging, and they were fed twice daily (7 AM and 2 PM).

Control (CON) and CR monkeys consumed the same balanced, natural ingredient chow. Therefore, our experimental paradigm was a reduction in total caloric intake and not a manipulation of a specific dietary component. The basic diet composition was 3.8 kcal/g, 15.4% protein, 5.0% fat and 5.0% fiber; 1.2% calcium, 0.69% phosphorus, and 3.3 IU/g of vitamin D. For a detailed description of the diet formulation, see Ingram and colleagues ⁽⁴⁾ or Lane and colleagues ⁽⁶⁾. Food allotments offered to CR monkeys were 30% less than those offered to CON monkeys of a comparable age and body weight. Thus, to ensure an adequate intake of all essential nutrients by CR animals, the chow was fortified to exceed recommendations of the National Research Council (NRC) for vitamins, minerals, and trace elements by 30–40% ⁽²⁹⁾. CON monkeys were offered a specified amount based on NRC guidelines for their age and weight ⁽²⁹⁾ and were, therefore, not fed strictly ad libitum. However, 5-day food consumption studies conducted quarterly on all monkeys over the course of the study have shown that the specified allotments given to controls approximated ad- libitum feeding (M.A. Lane, unpublished data). Monkey rooms were environmentally controlled to provide stable temperature (22–28°C) and humidity (50–60%) and a 12 hour on, 12 hour off light cycle (6 AM–6 PM).

Short-term CR study.-- One group of young adult (YA) male (4–5 years of age; 6 CON, 6 CR) and one group of mature, older (O) male (mean age 19 ± 2 years; 6 CON, 6 CR) rhesus monkeys were studied during the first 16 months of caloric restriction. Diet and housing conditions were identical to those described above for the long-term study. A 30% reduction in intake (based on individual animal ad-libitum intake) was gradually instituted in 10% increments over 3 months. CR was then maintained at a 30% reduced intake for 12 months.

Data Collection
All procedures were performed with the monkeys anesthetized (Telazol 3–4 mg/kg IM) following an overnight fast. Isoflurane, administered by mask, was used when additional immobilization was needed for dual energy x-ray absorptiometry (DXA) scans. The mask was removed for total body scans.

Long-term CR study.-- Bone mineral density (grams per square centimeter), bone mineral content (grams), and total body composition (lean mass and fat mass) were assessed once, after the animals had been on study for 11 years, by DXA, using a Lunar DPX- bone densitometer (Lunar Corp., Madison, WI). DXA scans were not initiated when CR began 11 years earlier. Thus, it is not possible to present longitudinal changes in DXA values over the course of the study. Animals were individually weighed on an electronic scale (Sartorius, Edgewater, NY) just prior to DXA scanning.

Positioning was standardized for all DXA scans. Total body and posterior-anterior lumbar spine scans DXA scans were performed with each animal in a supine position. Lumbar scans started just above the wing of the ileum and proceeded cranially until the 11th and 12th thoracic vertebrae were visible on the screen. A forearm positioning board (Lunar, Madison, WI) was used to correctly position each monkey's left arm palm down, fist open, on the scan table. Total body scans were not performed on five of the older monkeys as a result of severe curvature of the spine, which prevented proper positioning. The spinal curvature was such in three of these monkeys that it was not possible to perform lumbar spine scans. Thus, total body scans were performed on 46 monkeys, lumbar spine scans were performed on 48 monkeys, and forearm scans were performed on all 51 monkeys.

All scans were analyzed by one operator. Total body and lumbar spine scans were performed and analyzed by using Lunar Pediatric Software (Version 1.3g), whereas Lunar Small Animal Software (Version 1.0c) was used to perform and analyze forearm scans. A lumbar spine analysis was performed on lumbar vertebrae 3 through 5. The ends of the radius were defined on the forearm scans as the points where the ulna and radius meet distally and proximally. The scanned image of the radius was divided into three equal regions of interest (ROI), and bone mass in the middle one-third and distal one-third ROI was determined. The lower areal density of the distal third of the radius, relative to the mid-third, suggests a higher concentration of cancellous bone. Thus, these two regions of interest were chosen to represent appendicular sites of high (distal) and low (mid-) cancellous bone.

The stability of the scanner was determined by measuring an aluminum phantom (Lunar, Madison, WI) every day on which animals were scanned. The long-term reproducibility obtained with the phantom was <1% during the study period. For in vivo precision to be assessed, five consecutive DXA scans were performed, with repositioning, on five adult male monkeys at each of the skeletal sites of interest (Table 1 ).

Mid-morning voided urine samples were also collected and assayed for two markers of bone resorption, pyridinoline (PYD) and deoxypyridinoline (DPD). Total urinary pyridinium cross-links (PYD and DPD) were measured by high-performance liquid chromatography after hydrolyzed samples were submitted to a prefractionation procedure. Peaks were detected by fluorescence, quantified by external standards, and expressed per gram of urinary creatinine (#555, Sigma, St. Louis, MO). The interassay coefficients of variation (CV) for urinary PYD and DPD were 3.8% and 5.9%, respectively.

Short-term CR study.-- With the use of the same techniques as described above, total body and forearm (middle and distal radius only) BMD, BMC, and body composition were measured in YA and O males during the initiation of CR. Measurements were made at baseline (ad-libitum consumption) and after 1, 6, and 12 months at 30% CR. Individual body weight data were also collected at each intake level.

Femoral venous blood samples were drawn at each time point and analyzed as described above for plasma osteocalcin concentrations.

Statistical Analysis
Long-term CR.-- The relationship between age and BMC in CON monkeys was initially investigated by using simple linear regression. Student's nonpaired t-test analysis was used to examine the effects of CR on BMD, BMC, body weight, lean mass, and fat mass. An analysis of covariance (ANCOVA) was then used to determine the independent effects of CR, age, and body weight on BMD and BMC, as well as the effects of CR and age on body weight, lean mass, and fat mass. The normality of residuals was tested. Although assumptions of normality were generally met, when this was not the case, data were transformed by using a Box–Cox type of procedure ⁽³⁰⁾. Homogeneity of regression (parallelism of slopes) was tested across the two diet conditions to evaluate the validity of the ANCOVA-based inferences regarding the effects of CR. Parallelism was observed in all cases. The same statistical techniques were used to explore the relationships between bone measurements (dependent variables of interest: BMD and BMC) and body composition (independent variables of interest: lean mass and fat mass), as well as between indices of bone metabolism, and diet and age. In cases in which data were transformed to approximate normality, a linear transformation was applied to return them to their original scale (i.e., to have the same standard deviation as the untransformed variable) for ease of interpretation. All calculations were made by using NCSS 97 (NCSS Statistical Software, Kaysville, UT) and, statistical significance was accepted at p < .05.

Short-term study.-- Although monkeys included in the short-term study were randomly assigned to dietary groups, there were significant differences between the CON and CR groups in several baseline measurements. Thus, short-term data are presented as the percent change from baseline values. BMD, BMC, body composition, and osteocalcin data were analyzed by a repeated measures analysis of variance (NCSS 97) to determine the effect of CR on these parameters over time.

	Results

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BMD and BMC
Long-term CR study.-- Older CON monkeys had lower forearm BMC than younger CON monkeys (p values = .03; Fig. 1). Initial analyses indicated that CR monkeys had lower BMD (lumbar spine, middle and distal radius) and BMC (all sites) than CON monkeys (p values < .05; Table 2 ). However, when body weight and age were added as covariates in the ANCOVA analysis, there were no significant dietary effects on bone parameters (p values > .4; Table 3 ). Heavier monkeys had higher total body and forearm BMD and BMC values, whereas there was a negative relationship between total body and forearm BMD and BMC values and age (p values < .04; Table 3 ). Although age did not significantly affect lumbar spine BMD or BMC measurements, a positive relationship was observed between these parameters and body weight (p values < .02; Table 3 ).

View larger version (17K): [in this window] [in a new window]   Figure 1. Total body, A, lumbar spine, B, mid-radius, C, and distal radius, D, bone mineral content (grams) as a function of age in control (CON) and calorie restricted (CR: 30%) male rhesus monkeys after 11 years on study. Points represent data from individual animals; results of a linear regression analysis on CON monkey data are shown; n = 26 CON and 20 CR (total body), 26 CON and 22 CR (lumbar spine; L2–L4), 27 CON and 24 CR (middle and distal radius) monkeys.

View larger version (23K): [in this window] [in a new window]   Figure 2. Effects of short-term calorie restriction (CR) on total body, A, mid-radius, B, and distal radius, C, bone mineral content in young (all monkeys 4 years of age at initiation of study) male rhesus monkeys. Bars represent mean (± SE) percentage change from baseline values for six control (CON) and six CR monkeys. Measurements were made at baseline (ad libitum), and again after 1, 6, and 12 months on 30% CR. Notations above graphs represent significant results of an analysis of variance for repeated measures.

View larger version (24K): [in this window] [in a new window]   Figure 3. Effects of short-term calorie restriction (CR) on total body, A, mid-radius, B, and distal radius, C, bone mineral content in old (mean age = 19 ± 2 years at initiation of study) male rhesus monkeys. Bars represent mean (± SE) percentage change from baseline values for six control (CON) and six CR monkeys. Measurements were made at baseline (ad libitum), and again after 1, 6, and 12 months on 30% CR. Notations above graphs represent significant results of an analysis of variance for repeated measures.

View larger version (23K): [in this window] [in a new window]   Figure 4. Effects of short-term calorie restriction (CR) on body weight, A, lean mass, B, and fat mass, C, in young (all monkeys 4 years of age at initiation of study) male rhesus monkeys. Bars represent mean (± SE) percentage change from baseline values for six control (CON) and six CR monkeys. Measurements were made at baseline (ad libitum), and again after 1, 6, and 12 months on 30% CR. Notations above graphs represent significant results of analysis of variance for repeated measures.

View larger version (22K): [in this window] [in a new window]   Figure 5. Effects of short-term calorie restriction (CR) on body weight, A, lean mass, B, and fat mass, C, in old (mean age = 19 ± 2 years at initiation of study) male rhesus monkeys. Bars represent mean (± SE) percentage change from baseline values for six control (CON) and six CR monkeys. Measurements were made at baseline (ad libitum), and again after 1, 6, and 12 months on 30% CR. Notations above graphs represent significant results of an analysis of variance for repeated measures.

View larger version (9K): [in this window] [in a new window]   Figure 6. Serum osteocalcin concentration as a function of age in control (CON) and calorie restricted (CR: 30%) male rhesus monkeys after 11 years on study. Points represent data from individual animals; n = 27 CON and 24 CR male monkeys.

View larger version (13K): [in this window] [in a new window]   Figure 7. Urinary excretion of the pyridinium cross-links, pyridinoline, A, and deoxypyridinoline, B, as a function of age in control (CON) and calorie restricted (CR: 30%) male rhesus monkeys after 11 years on study. Points represent data from individual animals; n = 27 CON and 24 CR male monkeys.

View larger version (24K): [in this window] [in a new window]   Figure 8. Effects of short-term calorie restriction (CR) on serum osteocalcin concentrations in young, A, and old, B, male rhesus monkeys. Young and old monkeys were 4 years and 19 ± 2 years at initiation of study, respectively. Bars represent mean (± SE) percent change from baseline values for six control (CON) and six CR monkeys in each age group. Measurements were made at baseline (ad libitum), and again after 1, 6, and 12 months on 30% CR. Notations above graphs represent significant results of an analysis of variance for repeated measures; the asterisk denotes significant differences between CON and CR monkeys (p < .05) at specific time points.

	Discussion

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This study is the first to describe the effects of CR, a nutritional intervention that extends life span and retards aging in rodents, on skeletal mass in a long-lived species. Data from our long-term (11 years) study suggest that lower BMC and BMD values in CR male rhesus monkeys result from lower body mass, especially lean tissue, and not from a disruption of bone turnover in these animals. These findings agree with at least one study in rodents in which Sanderson and colleagues ⁽¹⁰⁾ reported that the reduced femoral BMC observed in CR rats was accounted for by the accompanying reduction in body weight.

Male rhesus monkeys reach skeletal maturity at approximately 10 years of age ⁽³¹⁾. Thus, the present long-term study included monkeys that were both skeletally mature and immature at the initiation of CR, and all animals were mature at the time of this cross-sectional study. Results of this study previously showed slower skeletal growth and reduced total body BMC, but not areal density, in skeletally immature (1.5–5 years of age at initiation of CR) male rhesus monkeys after 7 years of 30% CR ⁽¹¹⁾. Similarly, Kalu and colleagues ⁽⁷⁾⁽⁸⁾ reported reduced femoral length, calcium content, and delayed closure of the epiphyseal plates in male rats subjected to lifelong CR initiated at 28 days of age. Most CR studies initiated in adult rats also report significant skeletal effects, including lower total body BMD ⁽⁷⁾; lower femoral ash, calcium, phosphorus and magnesium content ⁽¹⁴⁾; reduced femoral bone area and three-point-bending strength; and decreased bone stress values ⁽¹³⁾. The absence of significant age-by-diet interactions in the statistical analysis of the bone parameters in the present study suggests that the overall long-term effects of CR on bone did not differ by age at initiation of CR.

Results of our 12-month study in YA male monkeys indicate that CR slowed the growth-associated increase in total BMD and BMC. Our findings in O adult monkeys are consistent with those of Kemnitz and colleagues ⁽⁵⁾ and indicate that bone mass was not affected after 12 months on CR, although Colman and colleagues ⁽¹⁵⁾ reported reduced total body BMC in adult monkeys after 18 months of CR. Interestingly, this reduction in BMC was accompanied by a concurrent reduction in lean tissue ⁽¹⁵⁾.

In light of these results and those from our 11-year study, the skeletal effects of CR in nonhuman primates appear to develop slowly, most likely as a result of changes in body weight and composition. This is supported by the observation in our long-term study that body weight and lean mass were consistent significant predictors of BMD and BMC at each of the skeletal sites examined. It has been suggested that changes in BMD with weight loss may be the result of a lack of instrument sensitivity when body weight and composition change ⁽³²⁾. These claims were made based on the "artificial" reduction in BMD during the course of a weight-loss study, created by insignificant but opposite changes in BMC and bone area. This was not the case in the present studies; that is, reductions in BMD were not present without concurrent reductions in BMC, and bone area was not affected by CR. Also, similar changes in BMC were observed at forearm sites, which do not experience significant changes in tissue cover with weight loss, as do total body and lumbar spine sites.

Body weight and composition changes in the short-term study suggest that CR affects these parameters differently in young, growing versus older adult monkeys. Previous studies of adult-onset CR in monkeys ^(5)(15) and rats ^(33)(34)(35) indicate that fat mass is more strongly affected than lean mass. Similarly, percentage reductions after 12 months on CR were greater in fat mass (45%) than in lean mass (8.5%) in old CR male monkeys (Fig. 5). Although CR significantly reduced lean and fat mass in old monkeys, these parameters were not affected by CR in young monkeys in the 12-month short-term study (Fig. 4). However, after long-term CR, which included animals that were immature and mature at the onset of CR, both lean and fat mass were reduced in all CR monkeys, regardless of age (Table 4 and Table 5 ). Thus, it must be concluded that significant differences in body composition in young CR monkeys develop over a longer period of time than the 12-month duration of this short-term study.

Measurements of serum concentrations of PTH and 1,25 dihydroxyvitamin D in the long-term study suggest that prolonged CR, with an adequate intake of essential nutrients, did not alter calcium homeostasis in these animals. The absence of a difference in 1,25 (OH)₂D concentrations between CON and CR monkeys suggests that CR monkeys received an adequate intake of this vitamin despite the overall higher intake in the CON monkeys.

The results of our 1-year study indicate that CR initially reduces the rate of bone turnover, as estimated by the serum osteocalcin concentrations, in YA but not O male monkeys. Similarly, short-term (4 week) calorie restriction decreases markers of bone turnover in young rats ⁽³⁶⁾, suggesting an inhibition of skeletal growth. However, the return of osteocalcin values to near baseline by 12 months on CR suggests that the reduction in the rate of bone turnover is not sustained. In contrast, bone turnover has been shown to increase after 9 weeks of CR in adult rats ⁽⁹⁾ and in response to weight loss in obese humans over 8 weeks ⁽¹⁹⁾ and 6 months ⁽³⁷⁾. Comparing these reports to the present short-term (12 month) study is problematic for several reasons. Data were not available that documented the initial response of bone turnover in rats to CR during the initiation of CR ⁽⁹⁾. Also, human studies focused on changes in bone turnover in obese subjects undergoing weight reduction ⁽¹⁹⁾, which is not necessarily representative of CR in healthy, lean subjects. Finally, it is difficult to compare studies because of differences in the duration of CR relative to species life span. Data from our 12-month study, suggesting that initial declines in the rate of bone turnover in response to CR in young animals are transitory, support the results of our long-term (11 year) study, in which no CR effects were observed in either osteocalcin (bone formation) concentrations or urinary pyridinium cross-link excretion (bone resorption).

Although long-term CR did not affect markers of bone turnover, significant age effects were observed (Fig. 6 and Fig. 7 and Table 7 ). Older monkeys had lower serum osteocalcin concentrations and higher urinary pyridinium cross-link (PYD and DPD) concentrations than younger monkeys. Coleman and colleagues ⁽³¹⁾ also reported lower osteocalcin values in older male monkeys, suggesting a decline in bone formation in older individuals. Although markers of bone turnover generally move in concert ⁽³⁸⁾, our findings suggest that bone formation and resorption may have been uncoupled in our older monkeys. A similar uncoupling has been observed, with increased urinary cross-link excretion without a concurrent rise in osteocalcin in osteoarthritis ^(39)(40), a condition known to increase with age in rhesus monkeys ^(31)(41). Future studies will examine the effect of age and CR on osteoarthritis in our colony.

In conclusion, long-term CR appears to reduce bone mass by means of a physiologic normalization to a smaller body size, specifically, a reduction in lean mass. Smaller body size is associated with lower BMD and an increased risk of fragility fracture in humans ^(42)(43). Thus, we cannot assume that lower BMD in CR monkeys does not carry a similar risk, although no obvious detrimental effects (e.g., fragility fractures) of lower BMC and BMD values were observed in these studies. Therefore, future studies attempting to maintain lean mass or biomechanical stresses on bone and to prevent or attenuate bone loss during CR may prove to be advantageous. DXA data were not available since the onset of the study. Future studies will report longitudinal effects of CR on bone mass.

	Acknowledgments

 
Osteocalcin and PTH assays were performed at the Wisconsin Regional Primate Center; the 1,25(OH)₂D analysis was performed at the Yerkes Regional Primate Research Center; and external standards for the pyridinoline analysis were provided courtesy of S. Robins, Bowett Research Institute.

Received December 21, 1999

Accepted September 7, 2000

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