Changes in bone and mineral homeostasis after short-term androgen deprivation therapy with or without androgen receptor signalling inhibitor - substudy of a single-centre, double blind, randomised, placebo-controlled phase 2 trial.

BACKGROUND: Prostate cancer (PCa) patients treated with androgen deprivation therapy (ADT) have an increased fracture risk. Exploring biomarkers for early bone loss detection is of great interest. METHODS: Pre-planned substudy of the ARNEO-trial (NCT03080116): a double blind, randomised, placebo-controlled phase 2 trial performed in high-risk PCa patients without bone metastases between March 2019 and April 2021. Patients were 1:1 randomised to treatment with gonadotropin-releasing hormone antagonist (degarelix) + androgen receptor signalling inhibitor (ARSI; apalutamide) versus degarelix + matching placebo for 12 weeks prior to prostatectomy. Before and following ADT, serum and 24-h urinary samples were collected. Primary endpoints were changes in calcium-phosphate homeostasis and bone biomarkers. FINDINGS: Of the 89 randomised patients, 43 in the degarelix + apalutamide and 44 patients in the degarelix + placebo group were included in this substudy. Serum corrected calcium levels increased similarly in both treatment arms (mean difference +0.04 mmol/L, 95% confidence interval, 0.02; 0.06), and parathyroid hormone and 1,25-dihydroxyvitamin D3 levels decreased. Bone resorption markers increased, and stable calcium isotope ratios reflecting net bone mineral balance decreased in serum and urine similarly in both groups. INTERPRETATION: This exploratory substudy suggests that 12 weeks of ADT in non-metastatic PCa patients results in early bone loss. Additional treatment with ARSI does not seem to more negatively influence bone loss in the early phase. Future studies should address if these early biomarkers are able to predict fracture risk, and can be implemented in clinical practice for follow-up of bone health in PCa patients under ADT. FUNDING: Research Foundation Flanders; KU Leuven; University-Hospitals-Leuven.

Inactivation of AR or ERα in Extrahypothalamic Neurons Does not Affect Osteogenic Response to Loading in Male Mice.

Failure of bone mass maintenance in spite of functional loading is an important contributor to osteoporosis and related fractures. While the link between sex steroids and the osteogenic response to loading is well established, the underlying mechanisms are unknown, hampering clinical relevance. Androgens inhibit mechanoresponsiveness in male mice, but the cell type mediating this effect remains unidentified. To evaluate the role of neuronal sex steroid receptor signaling in the male bone's adaptive capacity, we subjected adult male mice with an extrahypothalamic neuron-specific knockout of the androgen receptor (N-ARKO) or the estrogen receptor alpha (N-ERαKO) to in vivo mechanical stimulation of the tibia. Loading increased cortical thickness in the control animals mainly through periosteal expansion, as total cross-sectional tissue area and cortical bone area but not medullary area were higher in the loaded than the unloaded tibia. Trabecular bone volume fraction also increased upon loading in the control group, mostly due to trabecular thickening. N-ARKO and N-ERαKO males displayed a loading response at both the cortical and trabecular bone compartments that was not different from their control littermates. In conclusion, we show that the presence of androgen receptor or estrogen receptor alpha in extrahypothalamic neurons is dispensable for the osteogenic response to mechanical loading in male mice.

Testosterone Reduces Body Fat in Male Mice by Stimulation of Physical Activity Via Extrahypothalamic ERα Signaling.

Testosterone (T) reduces male fat mass, but the underlying mechanisms remain elusive, limiting its clinical relevance in hypogonadism-associated obesity. Here, we subjected chemically castrated high-fat diet-induced adult obese male mice to supplementation with T or the nonaromatizable androgen dihydrotestosterone (DHT) for 20 weeks. Both hormones increased lean mass, thereby indirectly increasing oxygen consumption and energy expenditure. In addition, T but not DHT decreased fat mass and increased ambulatory activity, indicating a role for aromatization into estrogens. Investigation of the pattern of aromatase expression in various murine tissues revealed the absence of Cyp19a1 expression in adipose tissue while high levels were observed in brain and gonads. In obese hypogonadal male mice with extrahypothalamic neuronal estrogen receptor alpha deletion (N-ERαKO), T still increased lean mass but was unable to decrease fat mass. The stimulatory effect of T on ambulatory activity was also abolished in N-ERαKO males. In conclusion, our work demonstrates that the fat-burning action of T is dependent on aromatization into estrogens and is at least partially mediated by the stimulation of physical activity via extrahypothalamic ERα signaling. In contrast, the increase in lean mass upon T supplementation is mediated through the androgen receptor and indirectly leads to an increase in energy expenditure, which might also contribute to the fat-burning effects of T.

Novel model to study the physiological effects of temporary or prolonged sex steroid deficiency in male mice.

Sex steroids are critical for skeletal development and maturation during puberty as well as for skeletal maintenance during adult life. However, the exact time during puberty when sex steroids have the highest impact as well as the ability of bone to recover from transient sex steroid deficiency is unclear. Surgical castration is a common technique to study sex steroid effects in rodents, but it is irreversible, invasive, and associated with metabolic and behavioral alterations. Here, we used a low dose (LD) or a high dose (HD) of gonadotropin-releasing hormone antagonist to either temporarily or persistently suppress sex steroid action in male mice, respectively. The LD group, a model for delayed puberty, did not show changes in linear growth or body composition, but displayed reduced trabecular bone volume during puberty, which fully caught up at adult age. In contrast, the HD group, representing complete pubertal suppression, showed a phenotype reminiscent of that observed in surgically castrated rodents. Indeed, HD animals exhibited severely impaired cortical and trabecular bone acquisition, decreased body weight and lean mass, and increased fat mass. In conclusion, we developed a rodent model of chemical castration that can be used as an alternative to surgical castration. Moreover, the transient nature of the intervention enables to study the effects of delayed puberty and reversibility of sex steroid deficiency.NEW & NOTEWORTHY We developed a rodent model of chemical castration, which can be used as an alternative to surgical castration. Moreover, the transient nature of the intervention enables to study the effects of delayed puberty and reversibility of sex steroid deficiency.

Androgen action on renal calcium and phosphate handling: Effects of bisphosphonate treatment and low calcium diet.

Renal calcium and phosphate handling is an important contributor to mineral homeostasis and bone health and the androgen receptor (AR) is highly expressed in the kidney. We investigated the short term effects of androgen deprivation on renal calcium and phosphate reabsorption, independent of their effects on bone. Two weeks following orchidectomy (ORX) of adult mice, bone loss occurred along with hypercalciuria, which was similarly prevented by testosterone and dihydrotestosterone supplementation. Treatment with bisphosphonates prior to ORX also inhibited hypercalciuria, indicating that the calcium flux originated from the bone. Renal calcium and phosphate transporter expression was increased post-ORX, independent of bisphosphonates. Furthermore, androgen deprivation appeared to stimulate local synthesis of 1,25(OH)2D3. When bisphosphonate-treated mice were fed a low calcium diet, bone resorption was no longer blocked and secondary hyperparathyroidism developed, which was more pronounced in ORX mice than sham-operated mice. In conclusion, this study shows that androgen deprivation increased renal calcium and phosphate transporter expression, independent of bone, and underlines the importance of adequate intestinal calcium supply in circumstances of androgen deprivation and bisphosphonate treatment.

Estrogen receptor alpha signaling in extrahypothalamic neurons during late puberty decreases bone size and strength in female but not in male mice.

Sexually dimorphic bone structure emerges largely during puberty. Sex steroids are critical for peak bone mass acquisition in both genders. In particular, the biphasic effects of estrogens mediate the skeletal sexual dimorphism. However, so far the stimulatory vs inhibitory actions of estrogens on bone mass are not fully explained by direct effects on bone cells. Recently, it has become evident that there is possible neuroendocrine action of estrogen receptor alpha (ERα) on the skeleton. Based on these considerations, we hypothesized that neuronal ERα-signaling may contribute to the skeletal growth during puberty. Here, we generated mice with tamoxifen-inducible Thy1-Cre mediated ERα inactivation during late puberty specifically in extrahypothalamic neurons (N-ERαKO). Inactivation of neuronal ERα did not alter the body weight in males, whereas N-ERαKO females exhibited a higher body weight and increased body and bone length compared to their control littermates at 16 weeks of age. Ex vivo microCT analysis showed increased radial bone expansion of the midshaft femur in female N-ERαKO along with higher serum levels of insulin-like growth factor (IGF)-1 as well as IGF-binding protein (IGFBP)-3. Furthermore, the 3-point bending test revealed increased bone strength in female N-ERαKO. In contrast, inactivation of neuronal ERα had no major effect on bone growth in males. In conclusion, we demonstrate that central ERα-signaling limits longitudinal bone growth and radial bone expansion specifically in females potentially by interacting with the GH/IGF-1 axis.

Androgen Receptor in Neurons Slows Age-Related Cortical Thinning in Male Mice.

Androgens via the androgen receptor (AR) are required for optimal male bone health. The target cell(s) for the effects of androgens on cortical bone remain(s) incompletely understood. In females, estrogen receptor alpha in neurons is a negative regulator of cortical and trabecular bone. Whether neuronal AR regulates bone mass in males remains unexplored. Here, we inactivated AR in neurons using a tamoxifen-inducible CreERT2 under the control of the neuronal promoter Thy1. Tamoxifen induced a 70% to 80% reduction of AR mRNA levels in Thy1-CreERT2-positive brain regions cerebral cortex and brainstem as well as in the peripheral nervous tissue of male neuronal AR knockout (N-ARKO) mice. Hypothalamic AR mRNA levels were only marginally reduced and the hypothalamic-pituitary-gonadal axis remained unaffected, as determined by normal levels of serum testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH). In contrast to orchidectomy, deletion of neuronal AR did not alter body weight, body composition, hindlimb muscle mass, grip strength, or wheel running. MicroCT analysis of the femur revealed no changes in bone accrual during growth in N-ARKO mice. However, 36- and 46-week-old N-ARKO mice displayed an accelerated age-related cortical involution, namely a more pronounced loss of cortical thickness and strength, which occurred in the setting of androgen sufficiency. Neuronal AR inactivation decreased the cancellous bone volume fraction in L5 vertebra but not in the appendicular skeleton of aging mice. MicroCT findings were corroborated in the tibia and after normalization of hormonal levels. Serum markers of bone turnover and histomorphometry parameters were comparable between genotypes, except for a 30% increase in osteoclast surface in the trabecular compartment of 36-week-old N-ARKO mice. Cortical bone loss in N-ARKO mice was associated with an upregulation of Ucp1 expression in brown adipose tissue, a widely used readout for sympathetic tone. We conclude that androgens preserve cortical integrity in aging male mice via AR in neurons. © 2018 American Society for Bone and Mineral Research.

Testosterone boosts physical activity in male mice via dopaminergic pathways.

Low testosterone (T) in men, especially its free fraction, has been associated with loss of energy. In accordance, orchidectomy (ORX) in rodents results in decreased physical activity. Still, the mechanisms through which T stimulates activity remain mostly obscure. Here, we studied voluntary wheel running behavior in three different mouse models of androgen deficiency: ORX, androgen receptor (AR) knock-out (ARKO) and sex hormone binding globulin (SHBG)-transgenic mice, a novel mouse model of "low free T". Our results clearly show a fast and dramatic action of T stimulating wheel running, which is not explained by its action on muscle, as evidenced by neuromuscular studies and in a muscle-specific conditional ARKO mouse model. The action of T occurs via its free fraction, as shown by the results in SHBG-transgenic mice, and it implies both androgenic and estrogenic pathways. Both gene expression and functional studies indicate that T modulates the in vivo sensitivity to dopamine (DA) agonists. Furthermore, the restoration of wheel running by T is inhibited by treatment with DA antagonists. These findings reveal that the free fraction of T, both via AR and indirectly through aromatization into estrogens, stimulates physical activity behavior in male mice by acting on central DA pathways.

A shortened tamoxifen induction scheme to induce CreER recombinase without side effects on the male mouse skeleton.

The selective estrogen receptor modulator tamoxifen exerts estrogen agonistic or antagonistic actions on several tissues, including bone. The off-target effects of tamoxifen are one of the most widely recognized pitfalls of tamoxifen-inducible Cre recombinases (CreERs), potentially confounding the phenotypic findings. Still, the validation of tamoxifen induction schemes that minimize the side effects of the drug has not been addressed. Here, we compared the side effects on the skeleton and other androgen-responsive targets of a shortened tamoxifen regimen (2 doses of 190 mg/kg body weight by oral gavage) to a standard protocol (4 doses) and determined their efficiency in inducing CreER-mediated gene deletion. In addition, both a vehicle- and a 10-dose group, which served as a positive control for tamoxifen side effects, were also included. For this purpose, we generated male mice with a floxed androgen receptor (AR) and a neuron-specifically expressed CreER. Treatment with two doses of tamoxifen was the only regimen that did not diminish androgenic bioactivity, as assessed by both seminal vesicles and levator ani/bulbocavernosus muscle weights and serum testosterone concentrations. Similarly, trabecular and cortical femoral bone structure were dramatically altered by both the standard and high-dose protocols but not by the shortened version. Serum osteocalcin and bone-gene expression analyses confirmed the absence of effects on bone by 2 doses of tamoxifen. This protocol decreased AR mRNA levels efficiently and specifically in the nervous system. Thus, we optimized a protocol for tamoxifen-induced CreER gene deletion in mice without off-target effects on bone and male reproductive organs.

Androgens have antiresorptive effects on trabecular disuse osteopenia independent from muscle atrophy.

Aging hypogonadal men are at increased risk of osteoporosis and sarcopenia. Testosterone is a potentially appealing strategy to prevent simultaneous bone and muscle loss. The androgen receptor (AR) mediates antiresorptive effects on trabecular bone via osteoblast-lineage cells, as well as muscle-anabolic actions. Sex steroids also modify the skeletal response to mechanical loading. However, it is unclear whether the effects of androgens on bone remain effective independent of mechanical stimulation or rather require indirect androgen effects via muscle. This study aims to characterize the effects and underlying mechanisms of androgens on disuse osteosarcopenia. Adult male mice received a unilateral botulinum toxin (BTx) injection, and underwent sham surgery or orchidectomy (ORX) without or with testosterone (ORX+T) or dihydrotestosterone (ORX+DHT) replacement. Compared to the contralateral internal control hindlimb, acute trabecular number and bone volume loss was increased by ORX and partially prevented DHT. T was more efficient and increased BV/TV in both hindlimbs over sham values, although it did not reduce the detrimental effect of BTx. Both androgens and BTx regulated trabecular osteoclast surface as well as tartrate-resistant acid phosphatase expression. Androgens also prevented BTx-induced body weight loss but did not significantly influence paralysis or muscle atrophy. BTx and ORX both reduced cortical thickness via endosteal expansion, which was prevented by T but not DHT. In long-term follow-up, the residual trabecular bone volume deficit in sham-BTx hindlimbs was prevented by DHT but T restored it more efficiently to pre-treatment levels. Conditional AR deletion in late osteoblasts and osteocytes or in the satellite cell lineage increased age-related trabecular bone loss in both hindlimbs without influencing the effect of BTx on trabecular osteopenia. We conclude that androgens have antiresorptive effects on trabecular disuse osteopenia which do not require AR actions on bone via muscle or via osteocytes.

Effects of sex hormone-binding globulin (SHBG) on androgen bioactivity in vitro.

Biochemical assessments of androgen status (hyper- or hypoandrogenism) are usually based on serum testosterone concentrations. According to the free hormone hypothesis, sex hormone-binding globulin (SHBG) determines free and bioavailable testosterone concentrations. Previous studies have suggested that in vitro androgen bioassay results may also be influenced by SHBG and correlate with free or bioavailable testosterone concentrations. To test this hypothesis, we established a stable HEK293 cell line with high expression of the human androgen receptor (AR) and a luciferase reporter downstream of a classical androgen response element. Importantly, we demonstrate that bioassay results are sensitive to dilution effects which increase apparent bioactivity in an SHBG-dependent manner. We therefore adopted a method using undiluted serum, which reduced cell proliferation but did not significantly affect the luciferase signal, cell viability or cytotoxicity. To correct for serum matrix effects, we applied signal correction based on internal controls with AR agonists or antagonists. Using this method, we provide direct evidence that in vitro androgen bioactivity reflects the inhibitory effects of SHBG, and correlates with free or bioavailable testosterone concentrations in adult hypogonadal men receiving androgen replacement therapy. In men receiving anti-androgens, serum bioactivity decreased tenfold while serum testosterone concentrations decreased only four-fold. Further pilot results in prostate cancer patients showed that androgen synthesis inhibitors result in more complete inhibition of androgen bioactivity than gonadorelin-based androgen deprivation therapy, even in patients whose testosterone concentrations were undetectable by mass spectrometry. We conclude that in vitro androgen reporter bioassays are useful tools to study how androgen bioactivity in serum is determined by androgens, anti-androgens as well as SHBG, provided that dilution and matrix effects are accounted for.