Influence of Aromatase Inhibition on the Bone-Protective Effects of Testosterone

Abstract

The influence of the aromatase enzyme in androgen-induced bone maintenance after skeletal maturity remains somewhat unclear. Our purpose was to determine whether aromatase activity is essential to androgen-induced bone maintenance. Ten-month-old male Fisher 344 rats (n = 73) were randomly assigned to receive Sham surgery, orchiectomy (ORX), ORX + anastrozole (AN; aromatase inhibitor), ORX + testosterone-enanthate (TE, 7.0 mg/wk), ORX + TE + AN, ORX + trenbolone-enanthate (TREN; nonaromatizable, nonestrogenic testosterone analogue; 1.0 mg/wk), or ORX + TREN + AN. ORX animals exhibited histomorphometric indices of high-turnover osteopenia and reduced cancellous bone volume compared with Shams. Both TE and TREN administration suppressed cancellous bone turnover similarly and fully prevented ORX-induced cancellous bone loss. TE− and TREN-treated animals also exhibited greater femoral neck shear strength than ORX animals. AN co-administration slightly inhibited the suppression of bone resorption in TE-treated animals but did not alter TE-induced suppression of bone formation or the osteogenic effects of this androgen. In TREN-treated animals, AN co-administration produced no discernible effects on cancellous bone turnover or bone volume. ORX animals also exhibited reduced levator ani/bulbocavernosus (LABC) muscle mass and elevated visceral adiposity. In contrast, TE and TREN produced potent myotrophic effects in the LABC muscle and maintained fat mass at the level of Shams. AN co-administration did not alter androgen-induced effects on muscle or fat. In conclusion, androgens are able to induce direct effects on musculoskeletal and adipose tissue, independent of aromatase activity.

Introduction

Both androgens and estrogens influence skeletal development and maintenance in males.^(1,2) In older men, hypogonadism (ie, low circulating total/bioavailable testosterone [T]) is associated with reduced bone mineral density (BMD)^(3,4) and increased fracture risk,⁽⁵⁾ despite elderly men experiencing an elevated estradiol (E₂)/T ratio within the circulation,⁽⁶⁾ suggesting that bone loss in hypogonadal men results primarily from a T deficiency. Within this population, T administration dose-dependently improves BMD, with physiologic T-replacement therapy providing modest enhancement of bone mass and higher-than-replacement T required for more robust musculoskeletal and lipolytic results.^(7,8) However, it remains unclear whether the skeletal protection induced by T administration occurs directly, via androgen-mediated actions, or indirectly, via estrogen-mediated actions that occur after the gonadal and/or tissue-specific aromatization of T to E₂.⁽⁹⁾ For example, we previously demonstrated that supraphysiologic T administration dramatically increases circulating and intraskeletal androgen concentrations and completely prevents bone loss in orchiectomized (ORX) rats, without altering circulating or intraskeletal E₂ concentrations.^(10,11) In addition, several clinical studies have reported that elderly men experience very minimal to no bone loss after pharmacologic aromatase inhibition, despite reductions in circulating E₂ ranging from 30% to 50%.^(12–14) Interestingly, administration of either dihydrotestosterone (DHT, a potent nonaromatizable androgenic metabolite of T)⁽¹⁵⁾ or trenbolone (TREN, a synthetic nonestrogenic, nonaromatizable androgen)⁽¹⁶⁾ completely prevents ORX-induced bone loss in skeletally mature rodents, providing further evidence that elevated systemic and/or skeletal-specific E₂ is not required for androgen-induced bone maintenance or that only a very minimal threshold concentration of E₂ may be required for bone health in adult males.⁽⁵⁾

In contrast, evidence supporting the role of E₂ in mediating androgen-induced bone maintenance in adult men is derived from studies demonstrating that co-administration of physiologic T plus letrozole (an aromatase inhibitor) is unable to blunt the increase in bone resorption that occurs in elderly men after experimental sex-steroid hormone ablation (via leuprolide), whereas physiologic E₂ successfully inhibits the elevation in bone resorption⁽¹⁷⁾ and from several large cohort studies reporting that circulating total/bioavailable E₂ is associated with BMD maintenance in adult men, whereas total/bioavailable T is not.^(6,18,19) Additionally, at least one study of older intact male rats demonstrates that pharmacologic aromatase inhibition increases bone resorption and reduces BMD,⁽²⁰⁾ effects that are completely prevented by E₂ administration.⁽²¹⁾ As such, some controversy exists regarding the role of the aromatase enzyme in mediating the effects of androgens on bone maintenance in adult males.

Our primary purpose was to determine whether aromatase inhibition reduces the osteogenic effects of administered T or trenbolone in skeletally mature ORX rats. We chose the ORX rat model because aromatase inhibition produces profound elevations in circulating T in gonadally intact animals,⁽²⁰⁾ which confounds the interpretation of this treatment on the skeletal outcomes in animals with intact sex organs. Secondary purposes of this study were to determine whether aromatase inhibition influences the myotrophic or lipolytic effects of administered androgens.

Materials and Methods

Animal care

Barrier-raised and specific pathogen-free male Fischer 344 rats aged 10 months were obtained from Charles River Laboratories (Wilmington, MA, USA). Animals were individually housed in a temperature- and light-controlled room on a 12-hour light, 12-hour dark cycle. Rats were fed Harlan rodent chow containing 3.1 kcal/g, distributed as 58% carbohydrate, 24% protein, and 18% fat with 1.0% calcium and 0.7% phosphorus (2018 Teklad Global 18% Protein Rodent Diet, Harlan Laboratories Inc., Indianapolis, IN, USA) and tap water ad libitum. All experimental procedures conformed to the ILAR Guide to the Care and Use of Experimental Animals and were approved by the Institutional Animal Care and Use Committee at the Gainesville VA Medical Center.

Experimental design

Rats (n = 10–11/group) were stratified by initial weight and divided into the following groups: 1) Sham operated (SHAM); 2) orchiectomized (ORX); 3) ORX + anastrozole (AN); 4) ORX + T-enanthate (TE); 5) ORX + TE + AN; 6) ORX + trenbolone-enanthate (TREN); 7) ORX + TREN + AN. Animals received aseptic bilateral closed ORX involving removal of testes, epididymis, and epididymal fat, or Sham surgery performed by Charles River Laboratories. After ORX/SHAM surgery, rats received no intervention for 2 weeks to ensure full recovery from surgery and that the influence of endogenous gonadally derived sex-steroid hormones had subsided. Subsequently, animals received AN (0.5 mg/d/animal, Sigma-Aldrich Corp., St. Louis, MO, USA) or placebo via sc-implanted computer-programmed iPrecio micro-infusion drug pumps (Primetech Corporation, Tokyo, Japan) and TE (7.0 mg/wk/animal, Savient Pharmaceutical, East Brunswick, NJ, USA), TREN (1.0 mg/wk/animal, Steraloids, Newport, RI, USA), or vehicle (sesame oil) once weekly via im injection under brief isoflurane administration.

Animals were also injected sc with declomycin and calcein (all chemicals obtained from Sigma-Aldrich, unless noted) at a dose of 15 mg/kg body weight 10 and 3 days before euthanization, respectively, to label actively forming bone surfaces with fluorochromes. Rats were euthanized at day 28, via ip pentobarbital (120 mg/kg), blood was collected via cardiac puncture, and the right and left femurs and tibias, retroperitoneal fat pad, and levator ani-bulbocanernosus (LABC) muscle complex were harvested and weighed. Serum aliquots were stored at −80°C. The femora were wrapped in saline-soaked gauze to prevent dehydration and stored at −20°C for microcomputed tomography (μCT) and bone mechanical testing. Tibias were stored according to the methods below. The remaining soft tissues were snap-frozen in liquid nitrogen and stored at −80°C.

Drug selection rationale

AN was administered in a dose exceeding that which has previously been shown to produce a marked reduction in circulating E₂ and to dramatically elevate circulating T (a hallmark characteristic of aromatase inhibition) in intact female rodents within 7 days of treatment⁽²²⁾ and infused at a 2 μL/h/animal. Pumps were refilled biweekly to ensure that sufficient drug was available throughout the study. The enanthate esters of T and TREN allow for elevated circulating T⁽¹¹⁾ and trenbolone⁽¹⁰⁾ for ~7 days and were administered in doses that have consistently been shown to fully prevent cancellous bone loss and to produce potent myotrophic effects in both young and skeletally mature rodents after ORX,^(10,11,16) whereas lower doses do not provide full musculoskeletal protection. The dose and route of TE administration also mimicked that of a recent clinical trial conducted in our lab⁽²³⁾ and increases the likelihood of systemic and/or intraskeletal aromatization.

Serum measurements

All samples were assayed in duplicate within the same run. T was determined by EIA (ALPCO Diagnostics, Salem, NH, USA). Trenbolone was determined by qualitative ELISA (Neogen, Lexington, KY, USA) and a standard curve was developed using trenbolone and methods previously described.^(10,16) Osteocalcin and C-telopeptide were determined by EIA (Immunodiagnostic Systems, Fountain Hills, AZ, USA).

Bone histomorphometry

The tibia was cut in half, cross-sectionally, and placed in 10% phosphate-buffered formalin for 48 hours for tissue fixation, dehydrated in ethanol, and embedded undecalcified in methyl methacrylate. The proximal tibias were sectioned longitudinally at 4- and 8-μm thicknesses with a Leica/Jung 2065 microtome. The 4-μm bone sections were stained by the Von Kossa method with a tetrachrome counterstain (Polysciences Inc., Warrington, PA, USA) and the 8-μm sections remained unstained to measure fluorochrome-based indices of bone formation. The sample area within the proximal tibial metaphysis began 1 mm distal to the growth plate to exclude the primary spongiosa and cancellous bone tissue within 0.25 mm of the endocortical surfaces. Osteoblast (Ob.S/BS) and osteoclast (Oc.S/BS) surfaces were measured as percentages of total cancellous perimeter. Fluorochrome-based indices of cancellous bone formation were measured under ultraviolet illumination. The percentages of cancellous bone surfaces with double fluorochrome labels were measured with the Osteomeasure System. Mineralizing surface (MS/BS), an index of active bone formation, was calculated as the percentage of cancellous bone surface with a double fluorochrome label. Mineral apposition rate (MAR), an index of osteoblast activity, was calculated by dividing the interlabel distance by the time interval between administration of fluorochrome labels. Bone formation rate (BFR/BS) was calculated by multiplying MS/BS by MAR. The terminology used was based on the recommendations by the Histomorphometry Nomenclature Committee of the American Society of Bone and Mineral Research.⁽²⁴⁾

μCT analysis of bone morphometry

The right distal femoral metaphysis, diaphysis, and femoral neck were scanned by μCT with a Bruker Skyscan 1172 (Kontich, Belgium). Images were acquired using the following parameters: 80 kVP, 12 0μA, 0.5 mm aluminum filter, 1 k camera resolution, 19.2 μm voxel size, 0.5° rotation step, and 180° tomographic rotation. The regions of interest (ROI) at the distal femoral metaphysis began 1.5 mm proximal to the growth plate and encompassed 4 mm. Cross-sectional images were reconstructed with a filtered back-projection algorithm (NRecon, Kontich, Belgium). 2D and 3D morphometric measurements were calculated with CTan software (Bruker Skyscan) and include cancellous bone volume (BV/TV), trabecular number (Tb.N), trabecular separation (Tb.Sp), and trabecular thickness (Tb.Th). Medullary volumetric (v)BMD (cancellous bone only) was evaluated using the previously defined distal femoral metaphysis ROI. In addition, medullary vBMD was assessed at the femoral neck ROI, which encompassed 0.35 mm surrounding the smallest diameter of the neck. The μCT nomenclature used is in accordance with that of the American Society of Bone and Mineral Research.⁽²⁵⁾

Bone mechanical evaluation

Subsequent to μCT, the femoral neck underwent a compression and bending test using a material testing system (Bose ElectroForce 3220, Eden Prairie, MN, USA) to assess bone mechanical characteristics. The femora were thawed to room temperature and remained wrapped in saline-soaked gauze except during measurements. The distal two-thirds of the shaft was fixed using bondo, and a custom-made fixture was used to align the femoral head with the actuator, which moved downward at 0.05 mm/s during loading. The applied force was measured with a 225 N load cell and the force/displacement was recorded at 2000 Hz. The ultimate strength (failure load) and displacement at failure were determined from the force-displacement curves. Stiffness (load per unit displacement) was calculated using least square fitting of the best-fit line from the force-displacement curves.

Statistical analysis

Results are reported as mean ± SEM and p ≤ 0.05 was defined as the threshold of significance. One-way ANOVAs (for normally distributed data) were used to separately analyze dependent variables, and the Tukey post hoc test was performed for multiple comparisons among groups when appropriate. The Kruskal-Wallis and Mann-Whitney U tests were performed when data were not normally distributed and the Holm-Bonferroni correction was utilized to correct for potential type I error that can occur when performing multiple comparisons. Hormone values that were below the lowest detectable standards were assigned a value equal to the sensitivity of the assay for the purposes of statistical analysis. Data were analyzed with the SPSS v15.0.0 statistical software package (IBM, Chicago, IL, USA).

Results

Body mass and bone mass/length

No differences in body mass were present among groups at baseline (data not shown). Briefly, body mass remained relatively stable in SHAM animals until day 7 and progressively increased thereafter (Supplemental Fig. S1). Improvements in body mass in ORX and ORX + AN animals were similar to that of SHAMs. Body mass gains in ORX + TE and ORX + TE + AN animals were greater than that of SHAMs on days 3 to 7, and the increases in ORX + TREN and ORX + TREN + AN animals were greater than that of SHAMs on day 7. By day 21, ORX + TE + AN animals gained more body mass than all other groups, with the exception of SHAMs (p < 0.05), and this difference was maintained throughout the remainder of the study. No differences in femoral or tibial length/mass were present among groups (data not shown).

Serum sex-steroid concentrations and circulating markers of bone turnover

At death, serum T concentrations were 2.8 ± 0.4 ng/mL in SHAM animals (Table 1). ORX resulted in the near complete ablation of circulating T (p < 0.001), and neither TREN nor AN co-administration altered this effect. In contrast, TE administration produced peak T concentrations that were 4 to 5 times that of SHAMs (p < 0.001). Serum trenbolone concentrations were elevated similarly in both groups receiving TREN treatment (p < 0.001), whereas values in other groups were below the sensitivity of the assay (Table 1). Serum osteocalcin was 20% higher in ORX animals compared with SHAMs (p < 0.05) (Table 1). Conversely, serum osteocalcin was 40% to 65% lower in all androgen-treated animals compared with that of SHAM, ORX, and ORX + AN animals (p < 0.001). No differences were present among groups for serum C-telopeptide.

Table 1.

Serum Hormones and Biochemical Markers

	SHAM (a)	ORX (b)	ORX+AN (c)	ORX+TE (d)	ORX+TE+AN (e)	ORX+TREN (f)	ORX+TREN+AN (g)
Testosterone, ng/ml	2.8 ± 0.4b*,c*,d*,e*,f*,g*	0.1 ± 0.0a*,d*,e*	0.1 ± 0.00a*,d*,e*	13.6 ± 0.55a*,b*,c*,f*,g*	12.8 ± 1.5a*,b*,c*,f*,g*	0.1 ± 0.0a*,d*,e*	0.1 ± 0.0a*,d*,e*
Trenbolone, ng/ml	N/D	N/D	N/D	N/D	N/D	7.7 ± 0.8a*,b*,c*,d*,e*	8.0 ± 0.9a*,b*,c*,d*,e*
Osteocalcin, ng/ml	305 ± 14b,d*,e*,f*,g*	368 ± 20a,c,d*,e*,f*,g*	314 ± 18b,d*,e*,f*,g*	132 ± 8a*,b*,c*,e*	187 ± 14a*,b*,c*,d*,f*	134 ± 5a*,b*,c*,e*	151 ± 6a*,b*,c*
CTX, ng/ml	16.0 ± 1.2	17.3 ± 1.0	14.3 ± 1.3	18.9 ± 2.1	15.1 ± 1.0	16.3 ± 1.2	19.3 ± 2.0

Values are means ± SE of n = 8–11/group. Letter a–g indicate differences from respectively labeled groups at p<0.05 or * p<0.01 (a = vs. SHAM, b = vs. ORX, c = vs. ORX + AN, d = vs. ORX + TE, e = vs. ORX + TE + AN, f = vs. ORX + TREN, g = vs. ORX + TREN + AN). N/D = Not detectable, below assay sensitivity.

μCT analysis of bone structure

μCT analysis of the distal femur indicated that cancellous BV/TV was 25% lower in ORX animals compared with SHAMs (p < 0.001, Fig. 1) and AN co-administration did not alter this effect. This difference was characterized by a 20% reduction in Tb.N (p < 0.001) and a 20% increase in Tb.Sp (p < 0.01), with no difference in Tb.Th. Both TE and TREN completely prevented cancellous bone loss, with BV/TV being 23% to 35% higher than ORX animals (p < 0.01, Table 2). Ultimately, all cancellous bone variables in androgen-treated animals were maintained at the level of SHAMs, with no differences resulting from co-administration of AN.

Fig. 1.

μCT images of cancellous bone at the distal femoral metaphysis from animals receiving SHAM surgery or orchiectomy (ORX) in combination with anastrozole (AN), testosterone-enanthate (TE), or trenbolone-enanthate (TREN). Values represent mean ± SE of 8–11/group. Letters a–g indicate differences between respective groups at p < 0.05 or *p < 0.01 (a versus SHAM; b versus ORX; c versus ORX + AN; d versus ORX + TE; e versus ORX + TE + AN; f versus ORX + TREN; and g versus ORX + TREN + AN). VOI = volume of interest; BV/TV = cancellous bone volume.

Table 2.

Cancellous Structural Characteristics at the Distal Femoral Metaphysis Measured Via μCT

	SHAM (a)	ORX (b)	ORX+AN (c)	ORX+TE (d)	ORX+TE+AN (e)	ORX+TREN (f)	ORX+TREN+AN (g)
BV/TV, %	17.6 ± 0.6b*,c*	13.3 ± 0.3a*,d*,e*,f,g*	13.3 ± 0.5a*,d*,e*,f*,g*	17.6 ± 0.5b*,c*	17.2 ± 0.7b*,c*	16.4 ± 0.6b,c*	18.0 ± 0.6b*,c*
Tb.N, #/mm	1.63 ± 0.03b*,c*	1.31 ± 0.02a*,d*,e*,f*,g*	1.29 ± 0.03a*,d*,e*,f*,g*	1.66 ± 0.04b*,c*	1.63 ± 0.04b*,c*	1.60 ± 0.04b*,c*	1.68 ± 0.05b*,c*
Tb.Th, mm	0.11 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.11 ± 0.00	0.11 ± 0.00	0.10 ± 0.00	0.11 ± 0.00
Tb.Sp, mm	0.46 ± 0.01 b*,c*	0.55 ± 0.02a*,d*,e*,f,*,g*	0.55 ± 0.02a*,d*,e*,f*,g*	0.44 ± 0.02b*,c*	0.47 ± 0.02b*,c*	0.43 ± 0.02 b*,c*	0.43 ± 0.01 b*,c*

Values are means ± SE of n = 8–11/group. Letter a–g indicate differences from respectively labeled groups at p < 0.05 or *p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs. ORX + AN, d = vs. ORX + TE, e = vs. ORX + TE + AN, f = vs. ORX + TREN, g = vs. ORX + TREN + AN). BV/TV = Bone volume fraction, Tb. Th = Trabecular thickness, Tb.N = Trabecular number, Tb.Sp = Trabecular separation.

Cancellous (medullary) vBMD was ~20% lower at the distal femoral metaphysis and at the femoral neck in ORX animals compared with SHAMs (p < 0.001, Table 3); AN co-administration did not alter this effect. Both TE and TREN administration completely prevented these reductions, with cancellous vBMD values being 21% to 26% higher at the distal femoral metaphysis (p < 0.01) and 22% to 30% higher at the femoral neck compared with ORX animals (p < 0.01) and not different than SHAMs (Table 3). AN co-administration did not alter these androgen-induced effects.

Table 3.

Medullary Volumetric Bone Mineral Density (vBMD) and Mechanical Characteristics of the Femoral Neck

	SHAM (a)	ORX (b)	ORX+AN (c)	ORX+TE (d)	ORX+TE+AN (e)	ORX+TREN (f)	ORX+TREN+AN (g)
Medullary vBMD, g/cm2	0.51 ± 0.01b*,c*	0.40 ± 0.02a*,d*,e*,f*,g*	0.42 ± 0.01a*,d*,e*,g*	0.52 ± 0.01b*,c*	0.52 ± 0.02b*,c*	0.49 ± 0.02b*	0.50 ± 0.02b*,c*
Failure Load, N	148 ± 4	127 ± 9d	124 ± 7d,f	158 ± 7b,c	144 ± 8	156 ± 7c	144 ± 4
Displacement, mm	0.73 ± 0.08	0.59 ± 0.05	0.64 ± 0.06	0.82 ± 0.07	0.71 ± 0.08	0.86 ± 0.17	0.81 ± 0.07
Stiffness, N/mm	212 ± 18	220 ± 25	206 ± 25	197 ± 15	212 ± 23	203 ± 24	184 ± 17

Values are means SE of n = 8–11/group. Stiffness was calculated using least mean square fitting of the best fit line from the force-displacement curves. Letter a–g indicate differences from respectively labeled groups at p < 0.05 or *p < 0.01 (a = vs. SHAM, b = vs. ORX, c = vs. ORX + AN, d = vs. ORX + TE, e = vs. ORX + TE + AN, f = vs. ORX + TREN, g = vs. ORX + TREN + AN).

Cancellous bone histomorphometry

Cancellous Oc.S/BS was ~fivefold higher in ORX animals compared with SHAMs (p < 0.001, Table 4). AN partially blunted this increase, resulting in Oc.S/BS values that were 40% lower than ORX animals (p < 0.05) but which remained ~threefold above SHAMs (p < 0.001, Table 4). Both TE and TREN administration prevented the ORX-induced increase in Oc.S/BS (p < 0.01), resulting in values that were not different than SHAMs (Table 4). AN co-administration partially inhibited the TE-induced reduction in Oc.S/BS (p < 0.05), although no difference in Oc.S/BS was present between ORX + TE + AN and SHAM animals (Table 4). Conversely, AN co-administration did not inhibit TREN-induced reductions in Oc.S/BS.

Table 4.

Cancellous Bone Histomorphometry in the Proximal Tibial Metaphysis

	SHAM (a)	ORX (b)	ORX+AN (c)	ORX+TE (d)	ORX+TE+AN (e)	ORX+TREN (f)	ORX+TREN+AN (g)
Oc.S/BS, %	0.7 ± 0.1b*,c*,g*	3.4 ± 0.4a*,c,d*,e*,f*,g*	2.0 ± 0.3a*,b,d*,e*,f*,g*	0.4 ± 0.0b*,c*,e*	0.8 ± 0.1b*,c*,d,g*	0.4 ± 0.1b*,c*	0.4 ± 0.0a*,b*,c*,e*
Ob.S/BS, %	8.1 ± 1.3b*,c*,d*,e*,f,g*	23.1 ± 3.0a*,d*,e*,f*,g*	22.5 ± 1.9a*,d*,e*,f*,g*	1.0 ± 0.2a*,b*,c*,e*	2.6 ± 0.3a*,b*,c*,d*,g	3.2 ± 1.2a,b,c*	1.4 ± 0.3a*,b*,c*,e
MS/BS, %	2.75 ± 0.60b*,c*,d*,e,f,g*	16.0 ± 2.59a*,d*,e*,f*,g*	13.5 ± 2.32a*,d*,e*,f*,g*	0.24 ± 0.10a*,b*,c*	0.84 ± 0.29a,b*,c*	0.55 ± 0.18a,b*,c*	0.42 ± 0.15a*,b*,c*
MAR, μm/day	0.60 ± 0.03f*	0.93 ± 0.13f*	0.64 ± 0.05f*	0.48 ± 0.15	0.47 ± 0.10	0.38 ± 0.06a*,b*,c*	0.80 ± 0.20
BFR/BS (dL), μm3/μm2/day	1.77 ± 0.38b*,c*,d*,e*,f*,g*	16.3 ± 3.74a*,d*,e*,f*,g*	8.96 ± 1.67a*,d*,e*,f*,g*	0.16 ± 0.05a*,b*,c*	0.41 ± 0.14a*,b*,c*	0.22 ± 0.06a*,b*,c*	0.31 ± 0.14a*,b*,c*

Values are means ± SE of n = 6–10/group. Letter a–g indicate differences from respectively labeled groups at p<0.05 or * p<0.01 (a = vs. SHAM, b = vs. ORX, c = vs. ORX + AN, d = vs. ORX + TE, e = vs. ORX + TE + AN, f = vs. ORX + TREN, g = vs. ORX + TREN + AN). Oc.S/BS = Osteoclast surface, Ob.S/BS = Osteoblast surface, MS/BS = Mineralizing surface, MAR = mineral apposition rate, BFR/BS = bone formation rate/bone surface.

ORX also produced a ~threefold increase in Ob.S/BS compared with SHAMs (p < 0.001), which was not inhibited by AN (Table 4). In contrast, TE and TREN administration reduced Ob. S/BS to values that were 60% to 88% below SHAMs (p < 0.01). AN co-administration partially inhibited the TE-induced reduction in OB.S/BS (p < 0.01) but did not alter the TREN-induced reductions (Table 4).

MS/BS was nearly fivefold higher in ORX animals compared with SHAMs (p < 0.001). Conversely, androgen treatments reduced MS/BS to values that were 70% to 90% below that of SHAM animals (p < 0.05). Similarly, BFR/BS was elevated >ninefold in ORX animals compared with SHAMs (p < 0.001), and androgen administration reduced BFR/BS to values that were 77% to 91% below SHAM animals (p < 0.01). Co-administration of AN did not alter any of these effects. MAR was also lower in ORX + TREN animals compared with SHAM, ORX, and ORX + AN animals (p < 0.01), with no other differences between groups.

Bone mechanical characteristics

The maximal (breaking) loads of the femoral neck were 148 ± 4 N (SHAM), 127 ± 9 N (ORX), 124 ± 4 N (ORX + AN), 158 ± 7 N (ORX + TE), 144 ± 8 N (ORX + TE + AN), 156 ± 7 (ORX + TREN), and 144 ± 4 N (ORX + TREN + AN) (Table 3). Maximal load in ORX + TE animals was 24% to 27% greater than ORX and ORX + AN animals (p < 0.05), and maximal load of ORX + TREN animals was 26% greater than ORX + AN animals (p < 0.05), with no other differences in bone mechanical characteristics present between groups.

Androgen-sensitive peripheral tissue mass

LABC mass was 45% lower in ORX and ORX + AN animals compared with SHAMs (p < 0.001, Supplemental Table S1). Conversely, LABC mass in TE and TREN animals was 38% to 42% greater than SHAMs (p < 0.001) and >2.5 times larger than ORX animals (p < 0.001), a result that was not inhibited by AN co-administration. Retroperitoneal fat mass was 22% to 24% higher in ORX and ORX + AN animals compared with SHAMs (p < 0.05, Supplemental Table S1). All androgen treatments prevented the ORX-induced increase in retroperitoneal fat mass, with fat mass being 33% lower in ORX + TE (p < 0.01), 23% lower in ORX + TE + AN (p < 0.05), 19% lower in ORX + TREN (nonsignificant), and 31% lower in ORX + TREN + AN animals (p < 0.01).

Discussion

The aromatization of endogenous T to E₂ influences bone maintenance.⁽⁵⁾ However, some controversy exists as to the role of the aromatase enzyme in mediating the musculoskeletal and lipolytic effects of androgens. Herein, we report that TE (an aromatizable androgen) and TREN (a nonaromatizable, nonestrogenic T analogue) provide equal and complete protection against ORX-induced bone loss in skeletally mature adult male rats. In addition, co-administration of AN (a potent aromatase inhibitor) did not alter these effects, suggesting that the bone maintenance we observed was androgen-mediated and occurred independent of the aromatase enzyme. Interestingly, we observed similar myotrophic and lipolytic effects from TE and TREN, and aromatase inhibition did not alter these effects. These findings provide evidence that androgens exert direct effects on musculoskeletal and adipose tissue, and that aromatase activity is not essential for these effects.

Results from the current study corroborate previous findings from our laboratory indicating that TE and TREN completely prevent ORX-induced cancellous bone loss in skeletally mature male rodents⁽¹⁶⁾ and support work from others demonstrating that nonaromatizable androgens are able to fully prevent cancellous bone loss in adult male rats subsequent to ORX.⁽²⁶⁾ However, neither of the aforementioned studies accounted for the possibility that adrenal-derived E₂ influences skeletal outcomes in the absence of gonadal steroid production, which is an important concept given that only 15% of total E₂ is derived from the gonadal aromatization of T in males with the remaining 85% originating from peripheral aromatization.⁽⁹⁾ In this regard, androstendione (derived from dehydroepiandrosterone or directly produced in the adrenals) can be aromatized to estrone (E₁) and subsequently converted to E₂ by actions of 17β-hydroxysteroid dehydrogenase (17β-HSD)⁽²⁷⁾ in any target tissue expressing the necessary enzymatic machinery, including bone.⁽²⁸⁾

In the current study, animals receiving ORX exhibited skeletal characteristics consistent with high-turnover cancellous osteopenia, including elevated histomorphometric indices of bone resorption (ie, osteoclast surface) and bone formation (ie, osteoblast surface, MS/BS, and BFR/BS), along with elevated biochemical markers of bone formation (ie, osteocalcin) that resulted in reduced cancellous bone volume and bone strength.⁽²⁹⁾ Unexpectedly, we did not observe increased serum CTX (a marker of whole-body bone resorption), which is surprising given the known effects of ORX in elevating this marker.⁽²⁹⁾ Regardless, the histomorphometric measurements that we report clearly demonstrate elevated bone turnover. Interestingly, AN administration partially blunted the ORX-induced increase in osteoclast surface, an effect that is difficult to explain, although values remained well above SHAMs. In contrast, other bone parameters were unaltered by AN administration and a similar reduction in cancellous bone volume was present between ORX and ORX + AN animals, indicating that aromatase activity has little influence on cancellous bone maintenance in males in the absence of gonadal steroid production. In contrast, both androgen treatments reduced bone turnover, providing full preservation of cancellous bone volume and bone strength, likely via direct androgen-mediated effects.⁽³⁰⁾ Interestingly, the TE-induced reductions in osteoclast and osteoblast surfaces were partially ameliorated by AN co-administration, suggesting that the aromatase enzyme played a minor role in suppressing bone turnover subsequent to TE administration. In contrast, co-administration of AN did not alter bone turnover in TREN-treated animals, likely because TREN is nonaromatizable.⁽³¹⁾ Regardless, full preservation of cancellous bone occurred in both androgen-treated groups, and AN co-administration did not alter this or fluorochrome-based, dynamic indices of bone formation. As such, our results support the contention that aromatase activity is not essential for androgen-induced bone maintenance in skeletally mature males, at least in the presence of supraphysiologic androgens.

These results are somewhat surprising, given the plethora of direct and indirect evidence supporting the influence of E₂ on the adult male skeleton.⁽⁵⁾ For example, elevated bone resorption and reduced bone formation occur in adult men undergoing GnRH-induced sex-steroid deficiency.⁽¹⁷⁾ In this model, administration of either physiologic T or physiologic E₂ prevents the deleterious alterations in bone turnover. However, the effects of administered T appear to be primarily mediated by aromatase, given that elevated bone turnover persists subsequent to co-administration of a physiologic dose of T plus letrozole (an aromatase inhibitor).⁽¹⁷⁾ Similarly, pharmacologic aromatase inhibition (via vorozole) increased bone resorption and reduced BMD in aged (12-month) intact male rats,⁽²⁰⁾ effects that were prevented by E₂ administration.⁽²¹⁾ Interestingly, our evidence indicates that androgens are also capable of directly regulating bone turnover. This unique finding certainly does not undermine the known effects of E₂ on bone maintenance in males, especially considering that older hypogonadal men rarely exhibit estrogen deficiency and under most circumstances have higher circulating E₂ concentrations than postmenopausal females. Instead, our results appear to demonstrate that aromatase activity is not essential for bone maintenance in the presence of supraphysiologic androgens, which may explain why higher-than-replacement T enhances BMD in older hypogonadal men, whereas physiologic T-replacement therapy has a much reduced effect.^(7,8)

Despite the aforementioned findings, aromatase inhibition produces somewhat inconsistent outcomes in older men, with some studies reporting no change in serum bone turnover markers⁽¹³⁾ and no reduction in BMD⁽¹⁴⁾ after AN administration (even with a 30% to 50% reduction in circulating E₂). In contrast, others report increased bone resorption and reduced formation⁽³²⁾ that results in minor BMD deficits.⁽¹²⁾ The inconsistency in these findings may result from the fact that aromatase inhibition elevates serum T by >50% via stimulation of gonadal steroid production, as evidenced by elevated luteinizing hormone and follicle-stimulating hormone.^(14,32) This effect underlies our rationale for evaluating the effects of AN in ORX rodents but certainly does not reduce the significance of our findings given that ~85% of endogenous E₂ in males is derived from peripheral tissue-specific aromatization.⁽⁹⁾

We have previously reported that TE administration fully prevents ORX-induced cancellous bone loss in young (3-month-old) rodents.^(10,11) However, in contrast to our current results, TREN has been shown to only partially ameliorate ORX-induced cancellous bone loss when administered before skeletal maturity, even when given in doses up to 7 times that of the current study.⁽¹⁰⁾ The above results suggest that the aromatization of T remains essential for male adolescent bone development, likely because bone formation is much more rapid before skeletal maturity and osteoblasts express aromatase and locally synthesize E₂ as a means of regulating bone formation.⁽³³⁾ In support of this contention, MAR (an indicator of osteoblast activity) was maintained in TE animals, whereas it was reduced below SHAM values by TREN. Additional evidence supporting the role of aromatase in bone development comes from men with congenital aromatase deficiency who exhibit undetectable serum E₂ that results in a number of skeletal abnormalities, including osteoporosis, despite displaying normal circulating T.⁽³⁴⁾ For men with this condition, E₂ administration normalizes bone turnover and improves BMD, whereas T administration provides little skeletal benefit.⁽³⁵⁾ Male aromatase-knockout (ArKO) mice also exhibit osteopenia⁽³⁶⁾ that is reversible with E₂ administration.⁽³⁷⁾ In contrast to the above results, DHT (a nonaromatizable T metabolite) fully prevents ORX-induced bone loss when administered to young (7-week-old) ORX male rodents, although the potential influence of adrenal-derived E₂ was not accounted for in this study.⁽³⁸⁾ Regardless, neither T nor DHT administration restores bone mass in male androgen receptor-knockout mice,⁽¹⁵⁾ and ORX worsens bone loss in male ArKO mice,⁽³⁹⁾ demonstrating the complementary influence of androgens and estrogens in the developing male skeleton.

Androgens also influence skeletal muscle and adipose tissue. We observed equal myotrophic effects after TE and TREN administration, which corroborates previous findings from our laboratory^(10,16) and from a number of studies in ruminants demonstrating that aromatizable and nonaromatizable androgens produce similar improvements in lean mass.⁽³¹⁾ Interestingly, these myotrophic effects were not influenced by AN, which supports the results of a recent clinical trial reporting that aromatase inhibition does not alter T-mediated myotrophic effects in adult men.⁽⁴⁰⁾ We also observed that TE and TREN produced similar reductions in adiposity and that aromatase inhibition did not influence these androgen-induced lipolytic effects. It is likely that these results were direct androgen-mediated effects given that human preadipocytes and mature adipocytes express ARs⁽⁴¹⁾ and that other nonaromatizable androgens inhibit adipogenesis.⁽⁴²⁾ However, our results appear to contrast with that of Finkelstein and colleagues,⁽⁴⁰⁾ who reported that the aromatase enzyme mediates the lipolytic effects of T in adult men, at least when T is present in physiologic concentrations. Importantly, our paradigm differs slightly from that of the aforementioned trial, given that we administered androgens in supraphysiologic doses that produce robust myotrophic and lipolytic effects, whereas Finkelstein and colleagues⁽⁴⁰⁾ administered T in graded doses that produced T concentrations in the subphysiologic to physiologic range.

In summary, we report that TE and TREN (a nonaromatizable, nonestrogenic androgen) fully protect against ORX-induced cancellous bone loss and that these effects are not influenced by aromatase inhibition. Similarly, TE and TREN appeared to produce direct androgen-mediated myotrophic and lipolytic effects that did not require aromatase activity. These results indicate that androgens are able to produce potent effects in musculoskeletal and adipose tissue without influence of the aromatase enzyme, at least when androgens are present in supraphysiologic concentrations. This biologic concept is important given that supraphysiologic T administration is required for significant musculoskeletal and lipolytic effects in elderly hypogonadal men.^(7,8) Future clinical studies evaluating the health risks associated with aromatase inhibition concomitant to T-replacement therapy appear warranted, considering that this enzyme converts T to potent estrogenic metabolites with systemic effects.