The purpose of this study was to evaluate if long-term (6 months) treatment with lasofoxifene (LAS), a new selective estrogen receptor modulator (SERM), can protect against age-related changes in bone mass and bone strength in intact aged male rats. Sprague-Dawley male rats at 15 months of age were treated (daily oral gavage) with either vehicle (n = 12) or LAS at 0.01 mg/kg per day (n = 12) or 0.1 mg/kg per day (n = 11) for 6 months. A group of 15 rats was necropsied at 15 months of age and served as basal controls. No significant change was found in body weight between basal and vehicle controls. However, an age-related increase in fat body mass (+42%) and decrease in lean body mass (−8.5%) was observed in controls. Compared with vehicle controls, LAS at both doses significantly decreased body weight and fat body mass but did not affect lean body mass. No significant difference was found in prostate wet weight among all groups. Total serum cholesterol was significantly decreased in all LAS-treated rats compared with both the basal and the vehicle controls. Both doses of LAS treatment completely prevented the age-related increase in serum osteocalcin. Peripheral quantitative computerized tomography (pQCT) analysis at the distal femoral metaphysis indicated that the age-related decrease in total density, trabecular density, and cortical thickness was completely prevented by treatment with LAS at 0.01 mg/kg per day or 0.1 mg/kg per day. Histomorphometric analysis of proximal tibial cancellous bone showed an age-related decrease in trabecular bone volume (TBV; −46%), trabecular number (Tb.N), wall thickness (W.Th), mineral apposition rate, and bone formation rate-tissue area referent. Moreover, an age-related increase in trabecular separation (Tb.Sp) and eroded surface was observed. LAS at 0.01 mg/kg per day or 0.1 mg/kg per day completely prevented these age-related changes in bone mass, bone structure, and bone turnover. Similarly, the age-related decrease in TBV and trabecular thickness (Tb.Th) and the age-related increase in osteoclast number (Oc.N) and osteoclast surface (Oc.S) in the third lumbar vertebral cancellous bone were completely prevented by treatment with LAS at both doses. Further, LAS at both doses completely prevented the age-related decrease in ultimate strength (−47%) and stiffness (−37%) of the fifth lumbar vertebral body. These results show that treatment with LAS for 6 months in male rats completely prevents the age-related decreases in bone mass and bone strength by inhibiting the increased bone resorption and bone turnover associated with aging. Further, LAS reduced total serum cholesterol and did not affect the prostate weight in these rats. Our data support the potential use of a SERM for protecting against the age-related changes in bone and serum cholesterol in elderly men.
OSTEOPOROSIS AND associated fractures are a common skeletal disorder in aging men.(1,2) The risk factors for osteoporosis in men include smoking, alcohol abuse, long-term use of glucocorticoids, decreased physical activity, and androgen deficiency.(3,4) More recently, the importance of estrogen in skeletal growth, development, and maintenance in males has been recognized. Clinical investigations have indicated that bone loss in elderly men is more significantly correlated with declining estrogen levels than with declining androgen levels.(5–8) Moreover, the reports of a mutation in the estrogen receptor gene in a man causing osteoporosis(9) and a loss of in vivo estrogen receptor-α protein expression in osteoblast and osteocytes in male idiopathic osteoporosis(10) further support the importance of estrogen in the male skeleton.
It is well documented that selective estrogen receptor modulators (SERMs) prevent bone loss and preserve bone strength in ovariectomized (OVX) rats by inhibiting the increased bone resorption and bone turnover associated with estrogen deficiency.(11–15) In postmenopausal women, SERMs prevent bone loss, increase bone mineral density (BMD), decrease skeletal fractures, and decrease serum cholesterol.(16,17) More recent clinical investigations established that SERMs decreased the risk of invasive breast cancer in postmenopausal women and in women at higher risk for breast cancer.(18,19) These study results indicate that SERMs might have multiple therapeutic benefits in postmenopausal women.
Lasofoxifene (LAS), a newly discovered SERM,(20,21) is currently under clinical investigation for the prevention and treatment of postmenopausal osteoporosis. We have previously reported that LAS binds selectively (greater than 100-fold selectivity against all other steroid receptors) and with high affinity to the human estrogen receptor α and human estrogen receptor β with half inhibition concentration (IC50) of 1.5 nM and 1.2 nM, respectively, which are similar to those seen with estradiol.(20,21) Further, LAS completely prevented OVX-induced bone loss and inhibited the increased bone turnover associated with estrogen deficiency in female rats.(20) More recently, we reported that the loss of bone mass and bone strength in adult male rats induced by orchidectomy (ORX) was prevented completely by treatment with LAS at 0.01 mg/kg per day or 0.1 mg/kg per day.(21) These results suggest that SERMs such as LAS might be useful agents for the prevention of osteoporosis in men with hypogonadism. In this study, we examine the long-term (6 months) effects of LAS on intact, aged (15 months of age) male rats to test whether LAS can protect against the age-related changes in bone mass, bone strength, total serum cholesterol, and body composition.
MATERIALS AND METHODS
Animals and study protocol
Fifty Sprague-Dawley male rats at 15 months of age (Charles River Inc., Wilmington, MA, USA), weighing approximately 725 g, were used for this study. The rats were obtained at 3 months of age and they were housed singly in 20 × 32 × 20-cm cages at local vivarium conditions (24°C and 12/12 h light-dark cycle) for 12 months before the study. All rats were allowed free access to water and a pelleted commercial diet (Agway ProLab 3000; Agway County Food, Inc., Syracuse, NY, USA), containing 0.97% calcium, 0.85% phosphorus, and 1.05 IU/g of vitamin D3 before 12 months of age. Thereafter, each rat was given 30 g of rat diet per day until the end of the study. During the study period, we found that rats consumed all 30 g of the diet each day. The experiments were conducted according to Pfizer animal care approved protocols and animals were maintained in accordance with the National Institutes of Health (NIH) guide for the care and use of laboratory animals.
A group of 15 rats was necropsied at day 0 as basal controls. The remaining rats were treated by daily oral gavage with vehicle (0.5% methylcellulose; 1 ml/rat; n = 12) or LAS at doses of 0.01 mg/kg per day (n = 12) or 0.1 mg/kg per day (n = 11) for 6 months. All rats were given subcutaneous injections of demeclocycline (Sigma Chemical Co., St. Louis, MO, USA) at 20 mg/kg 14 days and 13 days before death and calcein (Sigma Chemical Co.) at 10 mg/kg 4 days and 3 days before death to determine dynamic changes in bone tissues.(22)
After 6 months of treatment, the body weight was measured. Fat and lean body mass was determined using dual-energy X-ray absorptiometry (Hologic QDR-4500; Hologic, Inc., Waltham, MA, USA) equipped with whole body scan software. The rats were then necropsied under anesthesia by intraperitoneal (ip) injection of a mixture of ketamine/xylazine (57 mg per 0.86 mg/ml solution per kilogram body weight). The prostate wet weight was determined immediately at necropsy. Blood was obtained by cardiac puncture. Total serum cholesterol was determined using a high-performance cholesterol colorimetric assay (Boehringer Mannheim Biochemicals, Indianapolis, IN, USA). Serum osteocalcin was determined using a rat osteocalcin immunoradiometric assay (IRMA) kit (Immutopics, Inc., San Clemente, CA, USA).
Excised femurs were scanned by a peripheral quantitative computerized tomography (pQCT) X-ray machine (Stratec XCT Research M; Norland Medical Systems, Fort Atkinson, WI, USA) with software version 5.40. A 1-mm-thick cross-section of the femur metaphysis was taken 5.0 mm proximal from the distal end with a voxel size of 0.10 mm. Cortical bone was defined and analyzed using contour mode 2 and cortical mode 4. An outer threshold setting of 340 mg/cm3 was used to distinguish the cortical shell from soft tissue and an inner threshold of 529 mg/cm3 was used to distinguish cortical bone along the endocortical surface. Trabecular bone was determined using peel mode 4 with a threshold setting of 655 mg/cm3 to distinguish (sub)cortical from cancellous bone. An additional concentric peel of 1% of the defined cancellous bone was used to ensure (sub)cortical bone was eliminated from the analysis. Volumetric content, density, and area were determined for both trabecular and cortical bone.(23,24) Using the 340-, 529- and 655-mg/cm3 threshold settings, we determined that the ex vivo precision of volumetric content, density, and area of total bone, trabecular, and cortical regions ranged from 0.99% to 3.49% with repositioning.
Proximal tibial metaphyseal cancellous bone histomorphometry
At necropsy, the proximal third of the right tibia from each rat was removed, dissected free of soft tissue, fixed in 70% ethanol, stained in Villanueva bone stain, dehydrated in graded concentrations of ethanol, defatted in acetone, and embedded in methyl methacrylate. Longitudinal sections of the proximal tibial metaphysis (PTM) at a 220-μm thickness were cut using a low-speed metallurgical saw and then ground to 20 μm for histomorphometric analysis(25) using an Image Analysis System (Osteomeasure, Inc., Atlanta, GA, USA). Histomorphometric measurements were performed in cancellous bone tissue of the PTM between 0.5 and 3.5 mm distal to the growth plate-epiphyseal junction and extended to the endocortical surface in the lateral dimension.
Measurements and calculations related bone mass and structure included trabecular bone volume (TBV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb.Sp), and wall thickness (W.Th; average distance between reversal line and quiescent trabecular surface), while measurements and calculations related to bone resorption included eroded surface and percent eroded surface. The parameters related to bone formation included percent mineralizing surface [(double labeling surface + ½ single labeling surface)/total trabecular surface × 100], percent osteoid surface, mineral apposition rate, bone formation rate/tissue volume (TV). Further, the parameters related to bone turnover included bone formation rate/bone volume (BV), bone formation rate/bone surface (BS), formation period, resorption period, quiescent period, and activation frequency. The definitions and formulae for calculations of these parameters were described previously by Parfitt et al.(26) and Jee et al.(27)
Cancellous bone histomorphometry of third lumbar vertebral body
Undecalcified, methyl methacrylate embedded sagittal sections of the third lumbar vertebral body (LV3) at 4 μm thickness were stained with modified Masson's trichrome stain.(26,27) Using an Image Analysis System (Osteomeasure, Inc.), TBV, Tb.Th, Tb.N, Tb.Sp, osteoclast surface/BS (Oc.S/BS), and osteoclast number/BS (Oc.N/BS) were determined as described previously.(26,27) These measurements were performed in the cancellous bone tissue of LV3 at distance greater than 0.5 mm from the cranial and caudal growth plates.
Mechanical testing of fifth lumbar vertebral body
Using a Material Testing System (model 810; MTS Systems Corp., Minneapolis, MN, USA), a compression test was used to determine the mechanical properties of the fifth lumbar vertebral body (LV5).(21,28,29) The load and displacement curve was obtained from each test. The maximal load is the force that results in mechanical failure of the LV5. Stiffness was calculated from the linear portion of the load and displacement curve. The ultimate strength, toughness, and elastic modulus also were obtained.(30)
Data are expressed as mean ± SEM. Statistics were calculated using StatView 4.0 packages (Abacus Concepts, Inc., Berkeley, CA, USA). The analysis of variance (ANOVA) test was used for all four group comparisons and Fisher's protected least significant difference (PLSD) was used to compare the differences between each group.(31) A value of p < 0.05 was considered a significant difference.
Body weight and body composition
There was no significant difference in body weight at necropsy between basal controls (15 months of age) and vehicle-treated controls (21 months of age). However, there was a significant increase in fat body mass and percent fat body mass and a significant decrease in lean body mass and percent lean body mass in vehicle-treated controls as compared with basal controls (Table 1).
Table Table 1.. Changes in Body Weight, Body Composition, Prostate Weight, Total Serum Cholesterol, and Serum Osteocalcin
Compared with vehicle-treated controls, body weight was significantly decreased in rats treated with LAS at 0.01 mg/kg per day or 0.1 mg/kg per day after 6 months of treatment (Table 1). Fat body mass and percent fat body mass decreased significantly, whereas lean body mass showed no difference and percent lean body mass increased significantly in rats treated with LAS at 0.01 mg/kg per day as compared with vehicle controls. In rats treated with LAS at 0.1 mg/kg per day, a significant decrease in fat body mass and nonsignificant change in percent fat body mass, lean body mass, and percent lean body mass was observed as compared with rats treated with vehicle (Table 1).
Prostate weight, total serum cholesterol, and serum osteocalcin
There was no significant difference among all four groups in prostate weight (Table 1).
Total serum cholesterol significantly increased by 34% in vehicle-treated rats as compared with basal controls (Table 1). LAS at 0.01 mg/kg per day or 0.1 mg/kg per day significantly decreased total serum cholesterol as compared with both basal and vehicle controls.
A significant increase in serum osteocalcin (+40%) was found in vehicle controls as compared with basal controls. Serum osteocalcin was significantly lower in both doses of LAS-treated rats as compared with vehicle controls. There was no significant difference in serum osteocalcin among basal controls and rats treated with LAS at both doses (Table 1).
Femoral bone mineral measurements by pQCT
Cortical content and cortical density did not differ among the groups (data not shown). Cortical thickness decreased significantly by 11% in vehicle controls (0.78 ± 0.09 mm) as compared with basal controls (0.70 ± 0.09 mm), and this decrease was prevented completely by LAS at both doses.
A significant decrease in distal femoral metaphyseal total density and trabecular density and a nonsignificant decrease in total content and trabecular content was found in vehicle controls as compared with basal controls (Fig. 1). At doses of 0.01 mg/kg per day or 0.1 mg/kg per day, LAS significantly prevented the decrease in total content, total density, trabecular content, and trabecular density (Fig. 1). These parameters did not differ between rats treated with LAS at 0.01 mg/kg per day and 0.1 mg/kg per day.
Effects of aging:
Compared with basal controls, there was a significant decrease in TBV (−46%), Tb.N, W.Th, mineral apposition rate, and bone formation rate/TV in vehicle controls (Table 2 and Fig. 2). Furthermore, Tb.Sp and percent eroded surface (+13%) increased significantly in vehicle controls compared with basal controls.
Table Table 2.. Histomorphometric Analysis of PTM Cancellous Bone
Effects of LAS:
Rats treated with LAS at 0.01 mg/kg per day or 0.1 mg/kg per day had significantly higher TBV (+68% and +78%, respectively) and W.Th (Table 2 and Fig. 2) than those treated with vehicle (Table 2 and Fig. 2). Further, a significant decrease in Tb.Sp, percent eroded surface, percent osteoid surface, percent mineralizing surface, bone formation rate/BV, bone formation rate/BS, and activation frequency was found in rats treated with LAS at 0.01 mg/kg per day or 0.1 mg/kg per day as compared with those treated with vehicle. Formation, resorption, and quiescent periods increased significantly in rats treated with LAS as compared with vehicle controls. In addition, Tb.N was significantly higher in rats treated with LAS at 0.1 mg/kg per day than in those treated with vehicle (Table 2). Compared with basal controls, there was no significant difference in TBV, Tb.N, Tb.Th, Tb.Sp, percent eroded surface, and percent osteoid surface in rats treated with LAS at 0.01 mg/kg per day or 0.1 mg/kg per day. W.Th in rats treated with LAS at 0.1 mg/kg per day increased significantly compared with basal controls. Furthermore, rats treated with LAS at 0.01 mg/kg per day or 0.1 mg/kg per day had significantly lower mineral apposition rate, bone formation rate/BV, bone formation rate/BS, and activation frequency and significantly higher formation, resorption, and quiescent periods than basal controls (Table 2 and Fig. 2).
Effects of aging:
Compared with basal controls, vehicle-treated rats had significantly lower TBV (−16%) and thickness and significantly higher Oc.S (+62%) and Oc.N (+82%; Table 3).
Table Table 3.. Histomorphometric Analysis of LV3 Cancellous Bone
Effects of LAS:
Compared with vehicle controls, rats treated with LAS at both doses had significantly higher TBV and significantly lower Oc.S and Oc.N (Table 3). In addition, rats treated with LAS at 0.01 mg/kg per day had significantly higher Tb.Th, whereas rat treated with LAS at 0.1 mg/kg per day had significantly lower Tb.Sp compared with vehicle-treated rats. All parameters listed in Table 3 did not differ between basal controls and rats treated with LAS at both doses.
Mechanical strength of LV5
An age-related decrease in ultimate strength (−50%), stiffness, toughness, and elastic modulus was found in vehicle controls as compared with basal controls (Fig. 3). LAS at 0.01 mg/kg per or 0.1 mg/kg per day completely prevented these age-related changes. Ultimate strength, stiffness, toughness, and elastic modulus in LAS-treated rats were significantly higher than vehicle controls and did not differ from basal controls (Fig. 3).
Bone loss in elderly males and resulting skeletal fractures are of growing medical importance as the number of males reaching >65 years of age increases. Men account for about one-third of hip fractures, and vertebral fractures may be as common in men as in women.(32) Agents that would prevent bone loss and osteoporotic fractures in males are of significant importance. Currently, there is no approved agent for the prevention and treatment of osteoporosis in males.
The results from this study show that treatment with LAS, a newly discovered SERM, for 6 months was efficacious in preventing age-related bone loss and preserving bone strength in aged male rats. Prevention of the bone loss recorded between 15 and 21 months of age in male rats was achieved by the inhibition of bone resorption and bone turnover associated with aging as measured by serum bone marker and bone histomorphometric methods. pQCT analysis of distal femoral metaphyses and compression mechanical testing of LV5 confirmed that the loss in bone mass and strength induced by aging was completely prevented by 6 months of treatment with LAS. The ability of LAS to prevent bone loss in aged male rats was similar to that observed in OVX female rats(20) and ORX adult male rats.(21) Doses of LAS given orally at 0.01 mg/kg per day and 0.1 mg/kg per day were both maximally efficacious in bone loss prevention that is in concert with findings in OVX and ORX rats. Because there was no dose-dependent effect between 0.01 and 0.1 mg/kg per day, the minimal maximal effective dose of LAS in preventing bone loss in these aged male rats is not clear. These data suggest that LAS is a potent SERM with significant potential to inhibit bone loss in both males and females. In addition to its benefits in bone protection, LAS significantly decreased body weight and total serum cholesterol while not affecting the prostate weight in these aged male rats compared with vehicle controls.
Although the detailed mechanistic insights into the weight loss in LAS-treated rats as compared with vehicle-treated rats currently are unknown, this study clearly showed that the weight loss induced by LAS treatment was resulting from a reduction in fat body mass. Lean body mass was not affected by LAS treatment as compared with vehicle controls. Because the rats' diet was restricted to approximately 30 g/day for each rat during the study period, food intake is not one of the factors responsible for the reduction in fat body mass and body weight in this study. Nevertheless, the mechanism responsible for the effects of LAS on fat metabolism in rodents requires further investigation.
In this study, we found that LAS not only prevented the age-related increase in total serum cholesterol but also significantly reduced total serum cholesterol in male rats compared with both the vehicle and the basal controls. These findings are consistent with those in OVX female rats and ORX male rats.(20,21) Decreased total serum cholesterol in rats and decreased total serum cholesterol and low-density lipoprotein in postmenopausal women have been found with other SERMs such as tamoxifen and raloxifene.(11,12,16,17) Further, it also has been reported that oral estrogen treatment improves serum lipid levels in elderly men.(33) Thus, one would predict that LAS has beneficial effects on the lipid profile in postmenopausal women as well as in elderly men, although the rat is not an ideal model of human lipid modulation.
It is well established that males, like females, lose bone mass during the aging process, and that this bone loss accelerates after 50 years of age. The precise mechanisms responsible for age-related bone loss in males are not completely understood. Along with many other factors associated with aging in males, declining androgen as well as estrogen levels might play an important role in the process of age-related bone loss.(5–7) The effects of androgens on the male skeleton may be direct through an interaction with the androgen receptor.(34) However, it is also possible that the effects of androgens on the male skeleton may be indirect through aromatization of androgens into estrogens and thereafter, through an interaction with estrogen receptors.(34) It has been reported that men with estrogen deficiency resulting from aromatase deficiency have profound osteoporosis(35) and estrogen treatment in a man with aromatase deficiency resulted in an increase in bone mass.(36) These clinical results indicate the importance of aromatization of androgens into estrogens, which then interacts with estrogen receptors in maintaining bone mass in men. In rodents, Vidal and colleagues(37) reported that estrogen receptor α knockout in male mice resulted in a decrease in longitudinal growth as well as radial skeletal growth. Further, it has been reported that estrogen can prevent bone loss induced by ORX in the male rat.(34) More recently, we reported that LAS, a SERM, completely prevented bone loss induced by ORX in adult male rats.(21) In this study, we reported that LAS completely protects against the age-related bone loss in intact aged male rats. Taken together, these data show the important role of estrogen and/or stimulation of estrogen receptors, not only in growth and development, but also in the maintenance of male skeletal mass.
Between 15 and 21 months of age, male rats underwent age-related increases in bone resorption and bone turnover and age-related decreases in bone mass and bone strength as determined by serum bone turnover marker, histomorphometric methods, pQCT, and bone biomechanical testing. In LV3, the age-related decrease in TBV was accompanied by a decrease in Tb.Th and an increase in Oc.S and Oc.N. In the PTM, the age-related decrease in TBV was accompanied with an increase in eroded surface and a decrease in Tb.N, W.Th, mineral apposition rate, and bone formation rate-tissue referent. These data indicate that bone loss associated with aging was a result of increased bone resorption and decreased bone formation at 21 months of age as compared with 15 months of age. These histomorphometric findings are in agreement with other reports of a significant decrease in bone formation associated with aging in both humans and rodents.(38–40) In this study, we found that the serum osteocalcin level increased significantly by 40% whereas mineral apposition rate decreased significantly by 16% and bone formation rate-tissue referent decreased significantly by 41% at 21 months of age as compared with 15 months of age. These results indicate that serum osteocalcin may not be a good marker of bone formation in these aged male rats.
The results from the current study show that LAS protected against age-related bone loss by inhibition of bone resorption and bone turnover. Bone mass and bone structural indices in proximal tibiae, LV3, and femur did not differ between basal controls and all LAS-treated rats, indicating the maintenance of bone mass and structure by LAS. The inhibition of bone resorption by LAS was evident by lower eroded surface in the proximal tibial cancellous bone and lower Oc.N and Oc.S in lumbar vertebral cancellous bone in LAS-treated rats than in vehicle controls. Increased formation, resorption, and quiescent periods and decreased activation frequency in the proximal tibial cancellous bone and decreased serum osteocalcin in LAS-treated rats compared with vehicle controls show the inhibition of bone turnover by LAS. Although mineral apposition rate in proximal tibial cancellous bone did not differ between rats treated with LAS and vehicle, a longer formation period in rats treated with LAS may lead to an increase in W.Th compared with rats treated with vehicle.
In summary, we found that LAS treatment for 6 months protected against age-related loss in bone mass and bone strength by inhibition of bone resorption and bone turnover in intact aged male rats. In addition, LAS decreased total serum cholesterol while not affecting prostate weight. These results suggest that SERMs such as LAS might be useful agents for the prevention of osteoporosis not only in postmenopausal women but also in elderly men. Furthermore, we found that the 15-month-old intact male rat model is a useful model for studying age-related changes in bone in elderly men.
The authors thank the LAS Development Team at Pfizer for their support of this study. We thank Dr. Wei Yao, Dr. Chaoyang Li, and Dr. Webster Jee of the University of Utah for help with the histomorphometric measurements and Dr. Victor Shen of Skeletech, Inc. for help with the mechanical tests.