| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Division of Endocrinology and Metabolism, Center for Osteoporosis and Metabolic Bone Diseases, Department of Internal Medicine, and the Central Arkansas Veterans Healthcare System, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205-7199
Address all correspondence and requests for reprints to: Robert S. Weinstein, M.D, University of Arkansas for Medical Sciences, Division of Endocrinology and Metabolism, 4301 West Markham Street, Slot 587, Little Rock, Arkansas 72205-7199. E-mail: weinsteinroberts{at}uams.edu.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Loss of gonadal function in either sex stimulates the production of osteoblasts and osteoclasts, resulting in an increase in cancellous osteoblasts and bone turnover—changes quite distinct from those found in glucocorticoid-induced osteoporosis (3, 6, 7, 8, 9). Like glucocorticoid excess, however, sex steroid deficiency causes an increase in the prevalence of osteoblast apoptosis and an extension of the life span of osteoclasts (1, 3, 4, 9). Thus, hypogonadism and glucocorticoid treatment exert distinct effects on bone cell production but similar effects on bone cell apoptosis (10). Nevertheless, a contribution of glucocorticoid-induced hypogonadism to the adverse effects of the steroid on bone has often been suggested (1, 6, 11, 12). The contribution of hypogonadism to glucocorticoid-induced osteoporosis is of clinical significance because hypogonadal individuals are thought to be at greater risk of osteoporosis after glucocorticoid exposure (5, 6). Furthermore, administration of sex steroids has been recommended as a therapeutic maneuver in patients receiving excess glucocorticoids (1, 11, 12). However, antifracture efficacy has not been demonstrated, and modern guidelines for the prevention and therapy of glucocorticoid-induced osteoporosis do not advocate that sex hormone replacement therapy be used as the sole treatment (13).
To investigate the interaction between hypogonadism and glucocorticoid excess, we used established murine models of androgen deficiency and of glucocorticoid-induced osteoporosis and studied the changes in bone mineral density (BMD), vertebral compression strength, bone marrow cell progenitor numbers, bone histomorphometry, and the prevalence of osteoblast apoptosis in orchidectomized mice receiving prednisolone. We found that hypogonadism does not occur in or contribute to glucocorticoid-induced osteoporosis and that the adverse effects of glucocorticoids override those of hypogonadism.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Glucocorticoid administration
Slow-release pellets (Innovative Research of America, Sarasota, FL) of placebo or 2.1 mg/kg/d of prednisolone were implanted for 28 d after sham operation or orchidectomy (3, 8). The prednisolone dose was based on previous dose response studies and is equivalent to a daily dose of about 20 mg to humans, consistent with the much higher metabolic clearance of glucocorticoids and other compounds in small laboratory animals than in humans. Smaller doses in mice may not be accompanied by significant loss of spinal BMD, and larger doses cause animal deaths (3). The groups consisted of sham-operated mice with placebo pellets (sham/placebo), sham-operated mice with prednisolone pellets (sham/pred), orchidectomized mice with placebo pellets (orch/placebo), and orchidectomized mice with prednisolone pellets (orch/pred). There were 11–16 animals per group, and the experiment was repeated three times with similar results. The BMD and vertebral compression strength findings from these experiments were pooled. For dynamic histomorphometric measurements, tetracycline HCl (30 mg/kg body weight) was given ip 6 and 2 d before the mice were killed. At the time the mice were killed, lumbar vertebrae (L1–L4) were prepared for histomorphometric analysis, L6 was used for the determination of compression strength and bone marrow aspirates were obtained from the femora for ex vivo marrow cell cultures. The weight of the seminal vesicles (mg/100 g body weight) was used as an index of the androgen status of the animals (3). In addition, serum specimens were taken for determination of unbound testosterone using a competitive binding, biotin immunoassay (Diagnostics Systems Laboratories, Inc., Webster, TX).
Biomechanical testing
The load bearing properties of L6 were measured using a single column material testing machine and a calibrated tension/compression load cell (Model 5542, Instron Corp., Canton, MA) as previously described (9). The length, width, and depth of the bones were recorded with a digital caliper at a resolution of 0.01 mm (Mitutoyo no. 500–196, Ace Tools, Ft. Smith, AR). Load-bearing measurements were normalized for vertebral size and ultimate strength or stress (Newtons/square millimeter; in megapascals) was calculated from the compression measurements and vertebral dimensions.
Detection and quantification of osteoblast and osteoclast progenitors
Femoral marrow cells were obtained and the number of osteoblast progenitors (CFU-OB), osteoblast progenitors capable of forming mineralized colonies in ex vivo marrow cell cultures in the marrow isolate was determined by culturing cells at 2.5 x 106 cells per 10-cm2 well for 25–28 d with irradiated guinea pig feeder cells in phenol red-free 
MEM containing 15% preselected fetal bovine serum and 1 mM ascorbate-2-phosphate (14). One half of the medium was replaced every 5 d. Colonies containing osteoblasts were visualized by Von Kossa staining.
The number of osteoclast progenitors within the marrow isolate was determined by coculturing 75,000 marrow cells with 8,000 UAMS-32 stromal/osteoblastic cells for 8 d in a 2-cm2 well in the presence of 10% fetal bovine serum in phenol red-free MEM supplemented with 10 nM 1,25(OH)2D3 to stimulate osteoclast formation (3, 4). Replicate cultures (n = 3) were established from each animal. Osteoclastic cells were visualized by staining for tartrate-resistant acid phosphatase (TRAP); both mononucleated and multinucleated TRAP-positive cells were counted.
Bone histomorphometry
The lumbar vertebrae were fixed and embedded undecalcified in methyl methacrylate as previously described (3, 4, 8). The histomorphometric examination was done with a computer and digitizer tablet (OsteoMetrics, Decatur, GA) interfaced to a Zeiss Axioscope (Carl Zeiss, Thornwood, NY) with a drawing tube attachment. The identity of each specimen was concealed from the histomorphometry reader. All cancellous measurements were made at x400 magnification (plan-neofluar objective, numerical aperture 0.75). Trabecular width, trabecular spacing, and wall width were measured directly, whereas the rate of bone formation per cancellous perimeter and activation frequency were calculated (3, 8). The terminology used was that recommended by the Histomorphometry Nomenclature Committee of the American Society for Bone and Mineral Research (15).
Measurement of apoptosis in undecalcified bone sections
Apoptosis of osteoblasts was detected by in situ nick-end labeling (ISEL) using the Klenow DNA Fragmentation Detection Kit (Oncogene Research Products, Cambridge, MA) (4, 16, 17). Undecalcified, 5 µm-thick, longitudinal, vertebral sections were mounted on silane-coated glass slides (Scientific Device Lab, Inc., Des Plains, IL) and incubated in 10 mM citrate buffer (pH 6.0) in a microwave processor (Energy Beam Sciences, Inc., Agawam, MA) at 48 C for 4 min. Slides were then immediately covered with 0.05% pepsin in 0.1 N HCl for 30 min at 37 C before using the Klenow kit in a Dako Autostainer (Dako Corp., Carpinteria, CA). To further improve the sensitivity of the reaction, sections were subsequently incubated for 2 min in a filtered solution of 0.15% CuSO4 in 0.9% NaCl. A laboratory counter (Clay Adams, Parsippany, NJ) was used to count apoptotic cells in sections counterstained for 1 min with 2% methyl green. Morphological changes typical of apoptosis accompanied the ISEL-positive osteoblasts and included discretely condensed chromatin, nuclear fragmentation, and cell shrinkage.
Statistics
To evaluate changes in BMD measurements, vertebral compression, cell culture and histomorphometric data, we used one-way ANOVA (18). Comparisons of interest were specified a priori. The Shapiro-Wilk test was used to investigate within-group normality for a given parameter of interest. Levene’s test was conducted to determine the homogeneity of the variance assumption. When normality or equality of variance assumptions were not met, nonparametric analysis (the Kruskal-Wallis test) was used to evaluate drug administration and operation effects. P < 0.05 was considered significant after Bonferroni’s correction. Pearson correlation coefficients were calculated to test for an association between two independently measured variables (19).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). In orchidectomized animals, the serum testosterone was at or below the lower limit of detection of the assay (25–30 pg/ml). Concordant with these findings, seminal vesicle weight (which had less than half the variance of the testosterone measurements and indicated cumulative androgen status in mice better than a single serum sample) decreased by 61% in the orchidectomized mice receiving placebo (P < 0.001) but did not change in the group receiving prednisolone alone when compared with sham-operated animals receiving placebo. In the simultaneous orchidectomy and prednisolone group, seminal vesicle weight decreased by 68% (P < 0.001). Mean body weight in each group of mice at the beginning of the experiments was equivalent. Mean body weight after orchidectomy or prednisolone alone was not significantly different from sham-operated animals receiving placebo but was 13.3% lower in the simultaneous orchidectomy and prednisolone group when compared with sham-operated animals receiving placebo (P < 0.01). However, there were no significant differences in body weights among the three treatment groups.
|
, A and B). Remarkably, osteoblastogenesis and osteoclastogenesis were 37% and 47% lower in orchidectomized animals receiving prednisolone than in the group with orchidectomy alone (P < 0.02 and P < 0.001, respectively). When compared with sham-operated animals receiving placebo, there were no significant difference in the number of osteoblast or osteoclast progenitors when orchidectomy and prednisolone were combined.
|
). Consistent with these findings, vertebral compression strength was decreased in animals after orchidectomy, prednisolone treatment, or both (P < 0.05) (Fig. 2B). However, there were no significant differences in compression strength among the three treatment groups.
|
and Table 2; also see Fig. 5, A and B). Also in agreement with the effects on osteoclastogenesis, osteoclast perimeter increased by 47% with orchidectomy alone (P < 0.02), decreased by 40% with prednisolone alone (P < 0.05) but remained decreased when orchidectomy and prednisolone were combined (Fig. 3B). Increases with orchidectomy alone, decreases with prednisolone alone and values characteristic of prednisolone alone when orchidectomy and prednisolone were combined were also seen with the mineralizing perimeter, mineral appositional rate, bone formation rate, and activation frequency (Fig. 3, C and D, and Table 2). The imbalance between bone formation and bone resorption in the sham-operated group receiving prednisolone is accentuated when expressed as a ratio of the osteoclast perimeter to the osteoblast perimeter (Table 2). In addition, the strong relationship between the osteoblast perimeter and activation frequency (r = 0.67, P < 0.001) suggests that the orchidectomy-induced increase in bone turnover cannot occur without osteoblastic cells (Fig. 4).
|
|
|
|
and Table 2). |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Our study compared the effects of glucocorticoid excess and loss of gonadal function on peak adult murine bone mass, compression strength, marrow progenitor numbers, and histomorphometry. The data show that, despite orchidectomy, glucocorticoid excess suppresses osteoblastogenesis and osteoclastogenesis in the bone marrow, the number of mature osteoblasts and osteoclasts on cancellous bone and the rate of bone formation and frequency of activation of new bone remodeling units. The negative effect of glucocorticoid excess on osteoblastic cells may account for the absence of additional effects from hypogonadism on the loss of BMD and strength that occurs in glucocorticoid-induced osteoporosis. Although it is possible that if the experiment was longer than 28 d, a difference in BMD or bone strength between the group receiving prednisolone alone and the orchidectomized group receiving prednisolone may have become apparent, we think that this is unlikely. A 28-d experiment in mice is equivalent in duration to about 3–4 yr in humans when based on relative life spans (3, 22). Furthermore, the high bone turnover typical of orchidectomy begins to wane in longer experiments (20).
It is not surprising that the bone area per tissue area measurements using histomorphometry did not show significant differences between the groups. Spinal BMD is a more sensitive indicator of bone mass than the histomorphometric analysis of bone area. Bone area per tissue area is obtained from one to three longitudinal sections of L1–L4, whereas the murine spinal BMD analysis is an integral measurement of all the vertebrae from below the skull to the base of the tail, a much larger skeletal sample. Another reason for the greater sensitivity of BMD over the histomorphometric measurement of cancellous bone area is probably related to the variance of the determinations. Cancellous bone area has a coefficient of variation of 13.2% when quantifying multiple sections taken from the same specimen (23), whereas the coefficient of variation of repeated measurements of a plastic-embedded murine BMD phantom is only 1.8% (4).
Either glucocorticoid excess or loss of gonadal function reduce bone mass and increase the risk of fracture, but it remains unclear whether compromised gonadal function is an obligatory contributor to the adverse effects of glucocorticoid excess on bone (1, 6, 11, 12). Postmenopausal women receiving glucocorticoid treatment are indeed at greater risk of fracture than premenopausal women or men, but it is unknown whether this is because they have already experienced bone loss or because they are hypogonadal per se. In contrast to the osteoporosis caused by loss of sex steroids where increased bone resorption and turnover are the hallmarks, osteoporosis caused by long-term glucocorticoid excess is characterized by depressed bone formation and turnover (3). The purported evidence for reduced secretion of gonadal hormones in the pathogenesis of the deleterious skeletal effects of glucocorticoids is based primarily on observations in small groups of patients receiving long-term glucocorticoid treatment for severe disease (24, 25, 26, 27). When present, the hypogonadism could have been the result of the underlying disease rather than the consequence of glucocorticoid therapy (26). Compelling evidence against a role for sex steroid deficiency in the pathogenesis of glucocorticoid-induced osteoporosis is supplied by Pearce et al. (28), who found a 4.6% decrease in spinal BMD in men after 6 months of treatment with 50 mg/d of prednisolone despite maintenance of a normal testosterone/sex hormone binding globulin ratio. Additional strong evidence against a universal role of hypogonadism in glucocorticoid-treated patients is provided by registries containing the records of thousands of successful pregnancies in woman receiving prednisone to prevent rejection of renal transplants (29, 30). In support of the contention that loss of sex steroids may not contribute to the adverse effects of glucocorticoid excess on bone, we show here that murine serum unbound testosterone concentration and seminal vesicle weight were not diminished when prednisolone was administered alone. These results are fully consistent with our previous work on the effects of androgen deficiency on bone remodeling in senescence-accelerated mice (SAMP6), an animal model of defective osteoblastogenesis. Indeed, in the SAMP6 mice, the expected changes in bone cell progenitor numbers, histomorphometry and mineral density that occur after orchidectomy either were absent or greatly attenuated (8, 14). These findings, along with those of the present experiments, demonstrate that the adverse effects of sex steroid withdrawal on bone are mediated by osteoblastic cells and are obviated by constraints on osteoblastogenesis whether genetically defective, as in SAMP6 mice, or acquired because of glucocorticoid excess. Likewise, in young rats, glucocorticoid administration induced osteopenia of a similar magnitude in the presence or absence of ovarian function, suggesting that estrogen sufficiency or deficiency does not influence the osteopenic action of glucocorticoid excess (31, 32). However, paradoxically and in contrast to glucocorticoid action in humans and mice, glucocorticoids have been reported to inhibit bone resorption and promote apoptosis of osteoclasts in the rat, thus complicating interpretation of the findings in that particular animal model (33).
Finally, our findings are relevant to the decreased skeletal mass found in patients with anorexia nervosa (34). Despite amenorrhea and estrogen deficiency, biochemical markers of bone turnover are normal or reduced in anorectic women—in striking contrast to the increases in these markers found in postmenopausal women (35, 36). Elevated plasma cortisol levels and urinary free cortisol excretion in women with anorexia nervosa (37, 38) may be part of the explanation for the suppressed bone turnover with hypogonadism, although weight loss and malnutrition may also contribute to bone loss in these women (34, 39).
In conclusion, the results of this study demonstrate that, in the murine model, the increases in osteoblast and osteoclast precursors, incidence of new remodeling cycles at sites on cancellous bone and rate of bone remodeling that occur after the loss of androgens are abrogated by glucocorticoid excess. This evidence strongly supports the concept that hypogonadism is not an inevitable accompaniment, nor does it contribute to the loss of BMD and strength that is caused by glucocorticoid excess.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Abbreviations: BMD, Bone mineral density; CFU-OB, osteoblast progenitors capable of forming mineralized colonies in ex vivomarrow cell cultures; ISEL, in situ nick end labeling; ORCH/placebo, orchidectomized mice with placebo pellets; orch/pred, orchidectomized mice with prednisolone pellets; sham/placebo, sham-operated mice with placebo pellets; sham/pred, sham-operated mice with prednisolone pellets; SAMP6, senescence-accelerated mice; TRAP, tartrate-resistant acid phosphatase.
Received August 28, 2003.
Accepted for publication December 22, 2003.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  M. Almeida, L. Han, M. Martin-Millan, L. I. Plotkin, S. A. Stewart, P. K. Roberson, S. Kousteni, C. A. O'Brien, T. Bellido, A. M. Parfitt, et al. Skeletal Involution by Age-associated Oxidative Stress and Its Acceleration by Loss of Sex Steroids J. Biol. Chem., September 14, 2007; 282(37): 27285 - 27297. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. S. Perrien, N. S. Akel, P. K. Edwards, A. A. Carver, M. S. Bendre, F. L. Swain, R. A. Skinner, W. R. Hogue, K. M. Nicks, T. M. Pierson, et al. Inhibin A Is an Endocrine Stimulator of Bone Mass and Strength Endocrinology, April 1, 2007; 148(4): 1654 - 1665. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Jia, C. A. O'Brien, S. A. Stewart, S. C. Manolagas, and R. S. Weinstein Glucocorticoids Act Directly on Osteoclasts to Increase Their Life Span and Reduce Bone Density Endocrinology, December 1, 2006; 147(12): 5592 - 5599. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Yirmiya, I. Goshen, A. Bajayo, T. Kreisel, S. Feldman, J. Tam, V. Trembovler, V. Csernus, E. Shohami, and I. Bab From the Cover: Depression induces bone loss through stimulation of the sympathetic nervous system PNAS, November 7, 2006; 103(45): 16876 - 16881. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Kristo, R. Jemtland, T. Ueland, K. Godang, and J. Bollerslev Restoration of the coupling process and normalization of bone mass following successful treatment of endogenous Cushing's syndrome: A prospective, long-term study Eur. J. Endocrinol., January 1, 2006; 154(1): 109 - 118. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F.-S. Wang, C.-L. Lin, Y.-J. Chen, C.-J. Wang, K. D. Yang, Y.-T. Huang, Y.-C. Sun, and H.-C. Huang Secreted Frizzled-Related Protein 1 Modulates Glucocorticoid Attenuation of Osteogenic Activities and Bone Mass Endocrinology, May 1, 2005; 146(5): 2415 - 2423. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. A. Ali, R. S. Weinstein, S. A. Stewart, A. M. Parfitt, S. C. Manolagas, and R. L. Jilka Rosiglitazone Causes Bone Loss in Mice by Suppressing Osteoblast Differentiation and Bone Formation Endocrinology, March 1, 2005; 146(3): 1226 - 1235. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. O'Brien, D. Jia, L. I. Plotkin, T. Bellido, C. C. Powers, S. A. Stewart, S. C. Manolagas, and R. S. Weinstein Glucocorticoids Act Directly on Osteoblasts and Osteocytes to Induce Their Apoptosis and Reduce Bone Formation and Strength Endocrinology, April 1, 2004; 145(4): 1835 - 1841. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
