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Articles

Energy Restriction Reduces Bone Density and Biomechanical Properties in Aged Female Rats^{1 ,2}

Department of Nutritional Sciences, Rutgers University, New Brunswick, NJ and ^* Department of Surgery (Division of Orthopaedics), Robert Wood Johnson Medical School, New Brunswick, NJ

³To whom correspondence should be addressed. E-mail: Shapses{at}aesop.rutgers.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS Laboratory determinations RESULTS DISCUSSION LITERATURE CITED

 
Bone mineral density (BMD) is highly correlated with body weight, and weight loss is associated with reduced BMD. Whether such losses of BMD increase skeletal fragility is unclear. We examined the effect of 9 wk of energy restriction (ER) on bone density, mineral and matrix protein composition and biomechanical properties in mature (20 wk old, n = 12) and aged (48 wk old, n = 16) female rats. Energy-restricted rats were fed 40% less energy than controls that consumed food ad libitum. Bone content of mineral (ash and calcium content) and matrix proteins (hydroxyproline, pyridinium crosslinks and proteoglycans), serum hormones, site-specific bone density and biomechanical properties (peak load, peak torque, shear stiffness and bending stiffness) were measured at the conclusion of the study. In both age groups, ER reduced body weight by 15 ± 10% (P < 0.001) and dramatically decreased femoral bone density by 32–35% (P < 0.01) compared with controls. Energy restriction resulted in a small reduction in tibia and humerus density, as well as biomechanical properties in the aged but not mature rats (P < 0.05). Reduced serum levels of insulin and estradiol due to ER in aged rats (P < 0.05) may play a role in altering bone quality. These data show that although weight loss due to ER is detrimental to some bone parameters in mature rats, only aged rats show consistent reductions in bone density and biomechanical properties.

KEY WORDS: • bone • biomechanical • energy restriction • diet • rats

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS Laboratory determinations RESULTS DISCUSSION LITERATURE CITED

 
Body weight loss has been associated with reductions in bone mass in both humans (1 2 3) and animals (4 5 6 7 8 9) . Epidemiologic studies suggest that weight loss in older women increases the risk of fracture (10 11 12 13) . It is unclear, however, in these population studies whether fracture risk is increased due to secondary causes of weight loss (i.e., undiagnosed disease states). The change in structural and material biomechanical properties and composition of the bone, as an index of skeletal fragility has not been examined after adult-onset weight loss. Inadequate energy intake may influence bone mass and strength via several mechanisms including alterations in hormonal profiles (14) , reductions in bone content of minerals (5) and matrix proteins (15) as well as changes in bone geometry (16 17) . In addition, although bone strength changes with age (18) , it is not known whether the response of bone biomechanical properties to energy restriction (ER)⁴ is age sensitive as well. This study was conducted to determine the influence of ER on bone density and biomechanical properties in mature and aged female rats.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS Laboratory determinations RESULTS DISCUSSION LITERATURE CITED

 
Animals.

Two age groups of female Sprague-Dawley rats (Taconic Farms, Germantown, NY) were examined in this study. At the start of the study, rats were 20 wk (mature; n = 12) and 48 wk of age (aged; n = 16). Rats were housed in hanging wire-bottomed cages and maintained on a 12-h light:dark cycle with constant room temperature. Rats were weighed using an XT top-loading balance scale (Fisher Scientific, Pittsburgh, PA), matched for body weight within age groups and assigned to one of two diet groups, representing two levels of energy intake. One ER mature rat was excluded from the study after 2 wk of treatment due to the presence of a tumor, leaving 11 rats in the mature group. At the conclusion of the study (9 wk of ER or ad libitum consumption), mature and aged rats (29 and 57 wk of age, respectively) were anesthetized by CO₂ exposure and killed by decapitation. Blood samples were collected and serum was separated by centrifugation (16,000 x g for 10 min) and frozen at -70°C until determination of hormones. Long bones (femur, tibia and humerus) of each rat were removed, cleaned of soft tissue, wrapped in saline-soaked gauze and stored at -70°C until analyzed for bone density, biomechanical testing and chemical composition.

All studies were approved by the Institutional Review Board of Rutgers University and conformed to procedures set forth for the Care and Use of Laboratory Animals.

Diets.

Diets (Research Diets, New Brunswick, NJ) were formulated to provide two levels of energy intake, i.e., control and 40% ER (Table 1 ). Daily intakes of protein, fat, fiber, vitamins and other minerals were the same in each energy group. Energy restriction was accomplished by reducing the carbohydrate content (sucrose and cornstarch) and feeding a reduced quantity of the ER diets. ER rats were pair-fed to the mean daily intake of control rats in the same age group.

	Laboratory determinations

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS Laboratory determinations RESULTS DISCUSSION LITERATURE CITED

Ash weight and calcium content were determined on right tibiae. Bones were ashed for 24 h at 550°C in a muffle furnace, and ash weight was determined using a Mettler balance (accuracy to 0.0001 g). Calcium content of bones was measured by atomic absorption spectrophotometry (Thermo Jarrell Ash, Franklin, MA).

Total pyridinium crosslinks of collagen [pyridinoline (PYD) and deoxypyridinoline (DPD)] of the left tibia were measured by HPLC. A modified method (19) was used in which a 0.5-mL aliquot of solubilized bone powder was added to an equal volume of 6 mol/L hydrochloric acid and hydrolyzed at 108°C for 18 h. Pyridinium crosslink values are expressed as nmol/mg bone.

Hydroxyproline content of the left tibia was analyzed after hydrolysis in 6 mol/L HCl at 110°C for 16 h, drying in a desiccator and diluting with assay buffer. The solution was centrifuged (15 min at 16,000 x g) and the supernatant mixed thoroughly in a ratio of 1:2:1 with Chloramine-T and dimethylaminebenzaldehyde and then incubated at 60°C for 15 min. Hydroxyproline concentration was measured by spectrophotometry at 550 nm (19) and expressed as pmol/mg bone.

Proteoglycan content of the left tibia was analyzed by quantitative determination of total sulfated glycosaminoglycans (GAG). Briefly, bone samples were demineralized in 1.7 mol/L acetic acid for 3 d. The demineralized bone cartilage was solubilized by papain digestion at 60°C for 16 h and GAG were determined using a dye binding assay with 1,9-dimethylmethylene blue and spectrophotometry at a dual wavelength of 540 and 595 nm.

Biomechanical properties.

Biomechanical testing was performed on left femurs and left humeri. Bone samples were thawed to room temperature before biomechanical tests and kept moist during all handling and testing procedures. Left femurs were tested in three-point bending using an Instron model 1312 (Camden, MA) at a displacement rate of 2.0 mm/min. During testing, femurs were immersed in a saline bath at 37°C with load applied midway between two supports positioned 18 mm apart. Peak load (N) and bending stiffness (N · mm) were determined for each sample. Left humeri were tested in torsion using an Instron model 4204 materials tester. Humeri length was measured with laser micrometer (Laser Mike, Middletown, CT). Bones were then mounted on a torsional test fixture specially designed to convert axial tensile deformation to torsional load. Testing was performed at an axial deformation rate of 100 mm/min corresponding to an angular displacement rate of 7.85 rad/min. Torque and angle of deformation were recorded on an X-Y recorder. After failure, the location of the failure was recorded and peak torque (N · mm) and shear stiffness were calculated. Inner and outer cortical diameters were assessed histologically. After testing, bone samples with a clean break (n = 14) were fixed in 10% buffered formalin and embedded in paraffin. Sections (10 µm thick) were cut as close to the fracture site as possible and stained with hematoxylin and eosin for measurement of inner and outer cortical diameter and calculation of cortical area and moment of inertia.

Bone density.

Radiography (standard single beam X-ray) was performed on right tibiae, right humeri and right femurs using an aluminum alloy step standard (Faxitron radiograph at 50 kVp for 4.5 min). Radiographs were digitized and density measurements were taken using Sigma Scan software (Jandel Scientific, San Rafael, CA). Each individual image was calibrated using the steps as the density standards, and values of bone density are expressed as a percentage of rats consuming food ad libitum.

Serum hormones.

Serum concentrations of parathyroid hormone (PTH), estradiol and insulin were measured by RIA after 9 wk of control or ER diets. Assay kits of PTH (DSL-8000), estradiol (DSL-39100) and insulin (DSL-1600) were purchased from Diagnostic Systems Laboratories (Webster, TX) with intra- and interassay CV <8.3 and 12.2%, respectively.

Data analysis.

Data were analyzed by two-way ANOVA using dietary energy (control and ER) and age (mature and aged) as independent factors and measurements (bone density, hormones, mechanical properties and bone composition parameters) as dependent variables. When significant, results were further analyzed by Scheffé’s post-hoc comparisons test to determine differences among groups (SuperANOVA, version 1.1, Abacus Concepts) (20) . Variables with unequal variances were analyzed using log-transformed numbers. The percentage difference in bone density measurements between the weight-matched ER and rats that consumed food ad libitum was tested by Student’s t test. Pearson’s correlation coefficients were calculated to determine which variables (body weight, bone density, composition) best predicted the skeletal fragility of bone after ER. Values are expressed as means ± SD and differences were considered significant at P < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS Laboratory determinations RESULTS DISCUSSION LITERATURE CITED

 
Food intake, body weight and bone density.

Over the course of the 9-wk study, mature rats consumed 8 and 13 g/d in the ER (131 kJ/d) and control (214 kJ/d) groups, respectively. Aged rats consumed 11 and 18 g/d in the ER (180 kJ/d) and control (286 kJ/d) groups, respectively. Energy restriction reduced body weight to 244 ± 34 in mature (-11.3 ± 6.5%; n = 5) and to 237 ± 19 g in aged (-17.7 ± 11.4%; n = 8) rats (P < 0.0001), and was not significantly different between age groups. Control rats consuming food ad libitum increased body weight by 22.4 ± 7.8% in the mature and 12.0 ± 6.0% in aged groups to 328 ± 26 and 356 ± 68 g, respectively. Mature control rats gained more weight than the aged controls (P < 0.05).

Bone densities of the femur, humerus, and tibia were greater in aged than in mature rats (P < 0.05; data not shown). In ER rats, femur density was less than that of controls in both age groups (P < 0.01, Fig. 1 ). In aged rats, ER also reduced bone density of the humerus and tibia (P < 0.05, Fig. 1 ).

View larger version (22K): [in this window] [in a new window]   Figure 1. Effect of 9 wk of energy restriction on densities of the femur, humerus and tibia in mature (n = 10) and aged (n = 16) rats. Each bar represents the mean percentage difference ± SEM from aged-matched controls that consumed food ad libitum. Values differ from controls; *P < 0.05; **P < 0.01 (two-tailed t test).

Serum PTH levels were more than threefold higher in aged rats than in mature rats (Table 2 ; P < 0.05). Serum PTH was not affected by ER in aged or mature rats. Serum estradiol was higher in aged than in mature rats (P < 0.05) and was lower due to ER (P < 0.01). Although there was no interaction between energy and age, post-hoc analysis indicated that ER reduced serum estradiol in aged (P < 0.05) but not mature rats compared with their age-matched controls. Serum insulin concentrations did not differ between age groups, but ER resulted in a lower insulin concentration in both mature and aged rats (P < 0.05).

Moment of inertia of both humerus and femur, and cortical area of the humerus were lower in mature than aged control rats (Table 3 , P < 0.05). Cortical area of the humerus and moment of inertia of the humerus and femur did not differ significantly between ER and control rats. Femoral cortical area was decreased by ER in mature rats (Table 3 , P < 0.05), however, there was no interaction effect for energy and age. Humerus length was not significantly affected by age or energy intake (Table 3) .

Tibia composition.

Fat-free dry weight (FFDW) was 15% greater in aged rats than in mature rats (Table 4 ; P < 0.05), and was decreased by ER only in aged rats (P < 0.01). Hydroxyproline content of the tibia was not affected by age and was reduced 14–19% by ER in both age groups (Table 4 ; P < 0.05); post-hoc analysis indicated a significant reduction in aged rats. The concentrations of the collagen crosslinks (PYD and DPD), and proteoglycan (as estimated by glycosaminoglycan content) were not affected by age or energy intake.

In regression analyses of all rats after treatment (both age and energy groups included), humerus and femur bone mineral density (BMD) correlated with FFDW (r = 0.55, P = 0.002; and r = 0.53, P < 0.005, respectively) and ash of the bones (r = 0.57, P < 0.002; r = 0.50, P < 0.02, respectively). Bone density of the femur and humerus were positively correlated with calcium content of the bones (r = 0.52, P 0.01; and r = 0.40, P < 0.05, respectively). Femur density was positively correlated with peak torque (r = 0.57, P < 0.01) and shear stiffness (r = 0.40, P < 0.05) (Fig. 2 ). Body weight was correlated with the dry weight of the bone (FFDW, r = 0.46, P < 0.05), femur density (r = 0.43, P 0.05) and peak torque (r = 0.45, P < 0.05) (Fig. 2) .

View larger version (26K): [in this window] [in a new window]   Figure 2. Relationship between body weight and (A) femoral bone mineral density (BMD) and (B) peak torque; and between femoral BMD and (C) shear stiffness and (D) peak torque after 9 wk of consuming food ad libitum (control) or 40% restricted in energy. Each point represents individual rats; both age (mature and aged) and energy (controls and energy-restricted) groups are included.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS Laboratory determinations RESULTS DISCUSSION LITERATURE CITED

Food deprivation studies (reductions in both energy and nutrients) have been shown to reduce osteoblastic activity (21) and bone formation (15 ,22) , reduce overall bone turnover (23 24) and bone mass (25) , and attenuate the normal age-associated increase in serum calcitonin (26) . Chronic food deprivation can impair linear bone growth and maturation (27) , collagen metabolism (28) , bone mineralization and biomechanics (29 30 31) . One study, however, showed no effect on bone composition of collagen, calcium and magnesium (32) . In aged rats, most studies agree that weight loss results in reduced bone mass (6 ,9 ,25 ,33 34) . The current study results are consistent with these and show reduced bone density at the femur, humerus and tibia after ER.

Energy restriction may adversely influence bone metabolism through a number of mechanisms, including effects on serum levels of PTH, insulin, insulin-like growth factors, calcium-regulating hormones and sex steroids. Serum PTH increases with moderate ER (35) , decreases with severe ER (36) and has been shown to increase with age (14) . Serum PTH levels, however, were not significantly lower in ER than in control rats. Acute and chronic periods of food deprivation (e.g., fasting and anorexia nervosa) have been shown to decrease levels of insulin and insulin-like growth factor-I (37) , both of which have anabolic effects on bone (38) . In addition, the decline in estrogen levels due to weight loss and its detrimental effects on bone are well established (7) . In the present study, serum insulin and estradiol were reduced in both age groups by ER; however, estradiol decreased significantly only in the aged rats (Table 2) . Low levels of both estrogen and insulin in aged rats due to ER may act together to reduce osteoblastic activity relative to osteoclastic activity, thereby contributing to the lower bone density and strength. Although there was no detrimental effect of ER on bone calcium content, cortical area or moment of inertia in these older rats, the reduction in both estradiol and insulin levels may have altered the pattern of bone mineral formation (39) . Such changes in mineral crystalline structure, which were not assessed as part of this study, would reduce bone strength (40) . A limitation of these data is that blood samples were obtained only at the end of the study; therefore, the response of hormones over time within each group is not known.

In aged, but not mature rats, 9 wk of ER reduced biomechanical properties (Table 3) compared with control rats. We hypothesized that a reduction in collagen content after ER in aged rats may partially explain the reduced bone strength (34) . Bone content of hydroxyproline (an index of collagen content), however, was reduced in both age groups after ER, but further post-hoc analysis indicated that the reduction may occur more consistently in aged than in mature rats (Table 4) . Another potential mechanism for reduced bone strength in older rats may be the absence of the modeling effects on bone geometry, which are present in younger rats (41) , but should be minimal in mature rats (16) . Our findings suggest that although bone length was not different between age groups at the end of the study, other geometric parameters were greater in the aged than mature rats (Table 3) . Also, although weight loss due to ER did not differ between the two age groups, the mature control rats gained more weight (22%) than the aged rats (12%). Together, these data suggest that the mature group may have experienced additional modeling of bone during this period of growth. Although our data showed that the influence of ER on bone parameters (density, biomechanical properties and composition) in adult rats is dependent on age when weight loss (15%) is similar, a future study could clarify these results by examining ex vivo bone properties before ER.

Regression analyses suggested that there is a direct relationship between bone density and bone strength. These data are consistent with observations of bone density and strength in human cadavers (42) and with density and fracture risk in clinical trials (43 44) . In addition, the finding of a direct relationship between body weight and femoral bone density and certain biomechanical properties suggests that the rat bone responds to changes in body weight in a manner similar to humans (12 ,45) . More specifically, the reduction in bone biomechanical properties due to ER is supported by epidemiologic studies showing that older women who lose weight have an increased risk of fracture (10 11 12 13) . It is interesting that in the present study, mature rats showed a decrease in femur density that was not associated with a reduction in any biomechanical properties. To our knowledge, there are no clinical data confirming the hypothesis that a decrease in bone density in young adult bone corresponds to the same rise in fracture risk as would be expected in older bone. A recent report, however, suggests that age as well as bone density should be considered in the estimation of fracture risk (46) . The question whether bone quality can be determined by BMD (i.e., that is independent of age) is raised by the current study results that showed different responses in the two age groups.

In summary, bone density and strength were reduced by ER in aged rats, with a smaller effect in younger mature rats. This suggests that adequate energy intake and maintenance of body weight are important factors in determining bone strength in aged rats.

	ACKNOWLEDGMENTS

 
We thank N. Camacho for her advice and help on the analysis of these data and C. Abjornsen for her technical support with the biomechanical testing; both associated with the Laboratory for Ultrastructural Biology in the Hospital for Special Surgery, New York, NY.

	FOOTNOTES

 
¹ Presented in part at the meeting of the American Society Bone and Mineral Research/International Bone Society [Talbott, S. M., Dunn, M. G. & Shapses, S. A. (1998) Energy restriction reduces bone density and biomechanical properties in older, but not younger female rats. Bone 12: S491 (abs.)].

² Supported by National Institutes of Health Grant (AG-12161) to S.A.S. and an American Institute of Nutrition predoctoral fellowship to S.M.T.

⁴ Abbreviations used: BMD, bone mineral density; DPD, deoxypyridinoline; ER, energy restriction/energy restricted; FFDW, fat-free dry weight; GAG, glycosaminoglycan; PTH, parathyroid hormone; PYD, pyridinoline.

Manuscript received January 12, 2001. Initial review completed March 8, 2001. Revision accepted June 19, 2001.

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