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.Copyright (c) 2023 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Patients suffering from chronic kidney disease (CKD) often experience bone loss and arterial calcifications. It is unclear if hypogonadism contributes to the development of these complications and whether androgen therapy might prevent them. Male adult rats were randomized into four groups. The first group received standard chow (control), while three other groups were fed a 0.25% adenine/low vitamin K diet (CKD). Two CKD groups were treated with testosterone or dihydrotestosterone (DHT), whereas the control group and one CKD group received vehicle (VEH). CKD animals had 10-fold higher serum creatinine and more than 15-fold higher parathyroid hormone levels compared to controls. Serum testosterone levels were more than two-fold lower in the CKDVEH group compared to control + VEH and CKD + testosterone groups. Seminal vesicle weight was reduced by 50% in CKDVEH animals and restored by testosterone and DHT. CKD animals showed a low bone mass phenotype with decreased trabecular bone volume fraction and increased cortical porosity, which was not rescued by androgen treatment. Aortic calcification was much more prominent in CKD animals and not unequivocally prevented by androgens. Messenger RNA expression of the androgen receptor-responsive genes Acta1 and Col1a1 was reduced by CKD and stimulated by androgen treatment in levator ani muscle but not in the bone or aortic tissue. We conclude that adenine-induced CKD results in the development of hypogonadism in male rats. Androgen therapy is effective in restoring serum testosterone levels and androgen-sensitive organ weights but does not prevent bone loss or arterial calcifications, at least not in the presence of severe hyperparathyroidism.

Abstract Disclosure: K. David: None. G. Devos: None. W. Devlies: None. N. Narinx: None. L. Antonio: None. L. Deboel: None. D. Schollaert: None. D.M. Vanderschueren: None. F.A. Claessens: None. S. Joniau: None. B. Decallonne: None. Next to the classical androgen deprivation therapy (ADT) with GnRH-(ant)agonists, androgen receptor signaling inhibitors (ARSIs) such as apalutamide (APA) have become available for the treatment of prostate cancer (PC) patients in the last years. Hypothyroidism was more frequent in APA-treated patients compared to placebo in the SPARTAN trial (NCT01946204) (8.1% vs 2%). We collected serum of PC patients included in a RCT (ARNEO; NCT03080116) before and after 12 weeks of neo-adjuvant ADT (GnRH-antagonist degarelix (DGX) with APA or placebo). We aimed to explore changes in thyroid homeostasis under deep androgen suppression with combined GnRH-antagonist and ARSI. Of the 88 patients ending the trial, 1 was excluded because of history of hypothyroidism for which he received levothyroxine. 43 and 44 patients were included in the DGX+placebo and DGX+APA group, respectively. Mean age was 66.3y. Sex steroid levels measured by LC-MS/MS decreased significantly to castrate range with median testosterone 10.5 ng/dL and estradiol 3.6 ng/L. TSH levels increased in both treatment groups, but more pronounced in the DGX+APA group (DGX+placebo before: 1.18 mIU/L and after: 1.33 mIU/L, p 0.0351 vs. DGX+APA before: 1.07 mIU/L and after: 1.87 mIU/L, p <0.0001; difference between groups after treatment: p <0.0001). Free T4 and total T4 levels decreased in the DGX+APA group, but remained unaltered in the DGX+placebo group (free T4 after: 16.53 vs 13.87 pmol/L, p<0.0001 and total T4 after: 8.77 vs. 7.78 µg/dL, p 0.0055). Total T3 and free T3 increased in the DGX+APA group, and again did not differ after treatment in the DGX+placebo group. (free T3 after: 5.03 vs 5.41 pmol/L, p 0.0066 and total T3 after: 132.30 vs 149.50 ng/dL, p 0.0003). As a result, the total and free T3/T4 ratio increased in the DGX+APA group with 32% and 35%, respectively. Reverse T3 decreased in both treatment arms, but greater in the DGX+APA group (19.55 vs 14.20 ng/dL, p 0.0157). Thyroxine binding globulin (TBG) increased in both treatment arms, with 6.9% and 8.4%, respectively. In conclusion, interference with androgen signaling alters thyroid homeostasis in PC patients, and is most pronounced under deep androgen suppression with combined GnRH-antagonist and ARSI therapy. The increase in T3 and decrease in rT3 may indicate additional effects of ARSI on deiodinase activity. Longer follow-up trials are needed to confirm these findings and assess the clinical implications of these changes in thyroid homeostasis. Presentation: Friday, June 16, 2023

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.

Abstract 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.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

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 (ER alpha) on the skeleton. Based on these considerations, we hypothesized that neuronal ER alpha-signaling may contribute to the skeletal growth during puberty. Here, we generated mice with tamoxifen-inducible Thy1-Cre mediated ER alpha inactivation during late puberty specifically in extrahypothalamic neurons (N-ER alpha KO). Inactivation of neuronal ER alpha did not alter the body weight in males, whereas N-ER alpha KO 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-ER alpha KO 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-ER alpha KO. In contrast, inactivation of neuronal ER alpha had no major effect on bone growth in males. In conclusion, we demonstrate that central ER alpha-signaling limits longitudinal bone growth and radial bone expansion specifically in females potentially by interacting with the GH/IGF-1 axis.

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.

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.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

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.

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.

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.

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.

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.