High circulating concentrations of estradiol are anabolic for bone mass and strength in an adult male to female transgender mouse model.

The effects of gender affirming hormone therapy (GAHT) on bone microarchitecture and fracture risk in adult transgender women is unclear. To investigate the concept that skeletal integrity and strength in trans women may be improved by treatment with a higher dose of GAHT than commonly prescribed, we treated adult male mice with a sustained, high dose of estradiol. Adult male mice at 16 weeks of age were administered ~1.3 mg estradiol by silastic implant, implanted intraperitoneally, for 12 weeks. Controls included vehicle treated intact females and males. High-dose estradiol treatment in males stimulated the endocortical deposition of bone at the femoral mid-diaphysis, increasing cortical thickness and bone area. This led to higher stiffness, maximum force, and the work required to fracture the bone compared to male controls, while post-yield displacement was unaffected. Assessment of the material properties of the bone showed an increase in both elastic modulus and ultimate stress in the estradiol treated males. Treatment of male mice with high dose estradiol was also anabolic for trabecular bone, markedly increasing trabecular bone volume, number and thickness in the distal metaphysis which was accompanied by an increase in the histomorphometric markers of bone remodelling, mineralizing surface/bone surface, bone formation rate and osteoclast number. In conclusion, a high dose of estradiol is anabolic for cortical and trabecular bone in a male to female transgender mouse model, increasing both stiffness and strength. These findings suggest that increasing the current dose of GAHT administered to trans women, while considering other potential adverse effects, may be beneficial to preserving their bone microstructure and strength.

Estradiol increases cortical and trabecular bone accrual and bone strength in an adolescent male-to-female mouse model of gender-affirming hormone therapy.

The effects of gender-affirming hormone therapy on the skeletal integrity and fracture risk in transitioning adolescent trans girls are unknown. To address this knowledge gap, we developed a mouse model to simulate male-to-female transition in human adolescents in whom puberty is first arrested by using gonadotrophin-releasing hormone analogs with subsequent estradiol treatment. Puberty was suppressed by orchidectomy in male mice at 5 weeks of age. At 3 weeks post-surgery, male-to-female mice were treated with a high dose of estradiol (~0.85 mg) by intraperitoneal silastic implantation for 12 weeks. Controls included intact and orchidectomized males at 3 weeks post-surgery, vehicle-treated intact males, intact females and orchidectomized males at 12 weeks post-treatment. Compared to male controls, orchidectomized males exhibited decreased peak bone mass accrual and a decreased maximal force the bone could withstand prior to fracture. Estradiol treatment in orchidectomized male-to-female mice compared to mice in all control groups was associated with an increased cortical thickness in the mid-diaphysis, while the periosteal circumference increased to a level that was intermediate between intact male and female controls, resulting in increased maximal force and stiffness. In trabecular bone, estradiol treatment increased newly formed trabeculae arising from the growth plate as well as mineralizing surface/bone surface and bone formation rate, consistent with the anabolic action of estradiol on osteoblast proliferation. These data support the concept that skeletal integrity can be preserved and that long-term fractures may be prevented in trans girls treated with GnRHa and a sufficiently high dose of GAHT. Further study is needed to identify an optimal dose of estradiol that protects the bone without adverse side effects.

The Utility of Preclinical Models in Understanding the Bone Health of Transgender Individuals Undergoing Gender-Affirming Hormone Therapy.

PURPOSE OF REVIEW: To summarise the evidence regarding the effects of gender-affirming hormone therapy (GAHT) on bone health in transgender people, to identify key knowledge gaps and how these gaps can be addressed using preclinical rodent models. RECENT FINDINGS: Sex hormones play a critical role in bone physiology, yet there is a paucity of research regarding the effects of GAHT on bone microstructure and fracture risk in transgender individuals. The controlled clinical studies required to yield fracture data are unethical to conduct making clinically translatable preclinical research of the utmost importance. Novel genetic and surgical preclinical models have yielded significant mechanistic insight into the roles of sex steroids on skeletal integrity. Preclinical models of GAHT have the potential inform clinical approaches to preserve skeletal integrity and prevent fractures in transgender people undergoing GAHT. This review highlights the key considerations required to ensure the information gained from preclinical models of GAHT are informative.

A20 controls RANK-dependent osteoclast formation and bone physiology.

The anti-inflammatory protein A20 serves as a critical brake on NF-κB signaling and NF-κB-dependent inflammation. In humans, polymorphisms in or near the TNFAIP3/A20 gene have been associated with several inflammatory disorders, including rheumatoid arthritis (RA), and experimental studies in mice have demonstrated that myeloid-specific A20 deficiency causes the development of a severe polyarthritis resembling human RA. Myeloid A20 deficiency also promotes osteoclastogenesis in mice, suggesting a role for A20 in the regulation of osteoclast differentiation and bone formation. We show here that osteoclast-specific A20 knockout mice develop severe osteoporosis, but not inflammatory arthritis. In vitro, osteoclast precursor cells from A20 deficient mice are hyper-responsive to RANKL-induced osteoclastogenesis. Mechanistically, we show that A20 is recruited to the RANK receptor complex within minutes of ligand binding, where it restrains NF-κB activation independently of its deubiquitinating activity but through its zinc finger (ZnF) 4 and 7 ubiquitin-binding functions. Together, these data demonstrate that A20 acts as a regulator of RANK-induced NF-κB signaling to control osteoclast differentiation, assuring proper bone development and turnover.

The AR in bone marrow progenitor cells protects against short-term high-caloric diet-induced weight gain in male mice.

We previously identified a novel pathway of testosterone action via the androgen receptor (AR) in bone marrow mesenchymal precursor cells (BM-PCs) to negatively regulate fat mass and improve metabolic function in male mice. This was achieved using our PC-AR Gene Replacement mouse model in which the AR is only expressed in BM-PCs and deleted in all other tissues. We hypothesise that the markedly reduced fat mass and increased insulin sensitivity of PC-AR Gene Replacements will confer protection from diet-induced overweight and obesity. To test this, 6-week-old male PC-AR Gene Replacements and controls (WT, global-AR knockouts (KOs)) were fed a chow or high-caloric diet (HCD) for 8 or 18 weeks. Following 8 weeks (short-term) of HCD, WT and Global-ARKOs had markedly increased subcutaneous white adipose tissue (WAT) and retroperitoneal visceral adipose tissue (VAT) mass compared to chow-fed controls. In contrast, PC-AR Gene Replacements were resistant to WAT and VAT accumulation following short-term HCD feeding accompanied by fewer large adipocytes and upregulation of expression of the metabolic genes Acaca and Pnlpa2. Following long-term HCD feeding for 18 weeks, the PC-AR Gene Replacements were no longer resistant to increased WAT and VAT adiposity, however, maintained their improved whole-body insulin sensitivity with an increased rate of glucose disappearance and increased glucose uptake into subcutaneous WAT. In conclusion, the action of testosterone via the AR in BM-PCs to negatively regulate fat mass and improve metabolism confers resistance from short-term diet-induced weight gain and partial protection from long-term diet-induced obesity in male mice.

The role of the androgen receptor in the pathogenesis of obesity and its utility as a target for obesity treatments.

Obesity is associated with hypothalamic-pituitary-testicular axis dysregulation in males. Here, we summarize recent evidence derived from clinical trials and studies in preclinical animal models regarding the role of androgen receptor (AR) signaling in the pathophysiology of males with obesity. We also discuss therapeutic strategies targeting the AR for the treatment of obesity and their limitations and provide insight into the future research necessary to advance this field.

Distinct roles of androgen receptor, estrogen receptor alpha, and BCL6 in the establishment of sex-biased DNA methylation in mouse liver.

Sexual dimorphism in gene regulation, including DNA methylation, is the main driver of sexual dimorphism in phenotypes. However, the questions of how and when sex shapes DNA methylation remain unresolved. Recently, using mice with different combinations of genetic and phenotypic sex, we identified sex-associated differentially methylated regions (sDMRs) that depended on the sex phenotype. Focusing on a panel of validated sex-phenotype dependent male- and female-biased sDMRs, we tested the developmental dynamics of sex bias in liver methylation and the impacts of mutations in the androgen receptor, estrogen receptor alpha, or the transcriptional repressor Bcl6 gene. True hermaphrodites that carry both unilateral ovaries and contralateral testes were also tested. Our data show that sex bias in methylation either coincides with or follows sex bias in the expression of sDMR-proximal genes, suggesting that sex bias in gene expression may be required for demethylation at certain sDMRs. Global ablation of AR, ESR1, or a liver-specific loss of BCL6, all alter sDMR methylation, whereas presence of both an ovary and a testis delays the establishment of male-type methylation levels in hermaphrodites. Moreover, the Bcl6-LKO shows dissociation between expression and methylation, suggesting a distinct role of BCL6 in demethylation of intragenic sDMRs.

Genetic Depletion of Amylin/Calcitonin Receptors Improves Memory and Learning in Transgenic Alzheimer's Disease Mouse Models.

Based upon its interactions with amyloid ß peptide (Aß), the amylin receptor, a class B G protein-coupled receptor (GPCR), is a potential modulator of Alzheimer's disease (AD) pathogenesis. However, past pharmacological approaches have failed to resolve whether activation or blockade of this receptor would have greater therapeutic benefit. To address this issue, we generated compound mice expressing a human amyloid precursor protein gene with familial AD mutations in combination with deficiency of amylin receptors produced by hemizygosity for the critical calcitonin receptor subunit of this heterodimeric GPCR. These compound transgenic AD mice demonstrated attenuated responses to human amylin- and Aß-induced depression of hippocampal long-term potentiation (LTP) in keeping with the genetic depletion of amylin receptors. Both the LTP responses and spatial memory (as measured with Morris water maze) in these mice were improved compared to AD mouse controls and, importantly, a reduction in both the amyloid plaque burden and markers of neuroinflammation was observed. Our data support the notion of further development of antagonists of the amylin receptor as AD-modifying therapies.

Neuronal androgen receptor is required for activity dependent enhancement of peripheral nerve regeneration.

Neuronal activity after nerve injury can enhance axon regeneration and the restoration of function. The mechanism for this enhancement relies in part on hormone receptors, and we previously demonstrated that systemic androgen receptor antagonism blocked the effect of exercise or electrical stimulation on enhancing axon regeneration after nerve injury in both sexes. Here, we tested the hypothesis that the site of this androgen receptor signaling is both neuronal and involves the classical, genomic signaling pathway. In vivo, dorsal root ganglion neurons successfully regenerate in response to activity-dependent neuronal activation, and conditional deletion of the DNA-binding domain of the androgen receptor in adults blocks this effect in males and females. Motoneurons in males and females also respond in this manner, but we also observed a sex difference. In vitro, cultured sensory dorsal root ganglion neurons respond to androgens via traditional androgen receptor signaling mechanisms leading to enhanced neurite growth and did not respond to a testosterone conjugate that is unable to cross the cell membrane. Given our previous observation of a requirement for activity-dependent androgen receptor signaling to promote regeneration in both sexes, we interpret our results to indicate that genomic neuronal androgen receptor signaling is required for activity-dependent axon regeneration in both sexes.

Changes in white adipose tissue gene expression in a randomized control trial of dieting obese men with lowered serum testosterone alone or in combination with testosterone treatment.

PURPOSE: The aim of this study was to determine early weight loss-associated changes in subcutaneous abdominal white adipose tissue (WAT) gene expression in obese men with lowered serum testosterone by RNA next-generation sequencing. METHODS: Fourteen men, mean age (IQR) 51.6 years (43.4-54.5), BMI 38.3 kg/m2 (34.6-40.8) and total testosterone 8.4 nmol/L (7.5-9.5) provided subcutaneous WAT samples at baseline and after 2 weeks of a very low energy diet. RESULTS: Body weight loss was similar in participants receiving testosterone (n = 6), -5.27 kg [95% CI -6.17; -4.26], and placebo (n = 8), -4.57 kg [95% CI -6.10; -3.55], p = 0.86. In placebo-treated men, of the 14,410 genes expressed in subcutaneous WAT, four genes, Angiopoietin-like 4, Semaphorin 3 G, Neuropilin 2 and Angiopoietin 4, were upregulated (adjusted false discovery rate P < 0.05). In an exploratory analysis comparing men receiving testosterone and placebo, the most-upregulated gene in the testosterone group (exploratory p < 0.0005) was the neuropeptide y receptor 2. CONCLUSIONS: In obese men, dieting is associated with upregulation of WAT-expressed Angiopoietin-like 4, a secreted protein that regulates lipid metabolism, Semaphorin 3 G, a proposed adipocyte differentiation factor and secreted adipokine, and its receptor Neuropilin 2, as well as Angiopoietin 4, a vascular integrity factor. In an exploratory analysis, testosterone was associated with the upregulation of neuropeptide y receptor 2, a receptor involved in appetite regulation. Further studies are needed to confirm these observations and their potential biological implications. TRIAL REGISTRATION: clinicaltrials.gov, Identifier NCT01616732, Registration date: June 8, 2012.

Vitamin D receptor expression in mature osteoclasts reduces bone loss due to low dietary calcium intake in male mice.

Mature osteoclasts express the vitamin D receptor (VDR) and are able to respond to active vitamin D (1α, 25-dihydroxyvitamin D3; 1,25(OH)2D3) by regulating cell maturation and activity. However, the in vivo consequences of vitamin D signalling directly within functionally mature osteoclasts is only partially understood. To investigate the in vivo role of VDR in mature osteoclasts, conditional deletion of the VDR under control of the cathepsin K promoter (CtskCre/Vdr-/-), was assessed in 6 and 12-week-old mice, either under normal dietary conditions (NormCaP) or when fed a low calcium (0.03 %), low phosphorous (0.08 %) diet (LowCaP). Splenocytes from CtskCre/Vdr-/- mice were co-cultured with MLO-Y4 osteocyte-like cells to assess the effect on osteoclastogenesis. Six-week-old CtskCre/Vdr-/- mice demonstrated a 10 % decrease in vertebral bone volume (p < 0.05), which was associated with increased osteoclast size (p < 0.05) when compared to Vdrfl/fl control mice. Control mice fed a LowCaP diet exhibited extensive trabecular bone loss associated with increased osteoclast surface, number and size (p < 0.0001). Interestingly, CtskCre/Vdr-/- mice fed a LowCaP diet showed exacerbated loss of bone volume fraction (BV/TV%) and trabecular number (Tb.N), by a further 22 % and 21 %, respectively (p < 0.05), suggesting increased osteoclastic bone resorption activity with the loss of VDR in mature osteoclasts under these conditions. Co-culture of CtskCre/Vdr-/- splenocytes with MLO-Y4 cells increased resulting osteoclast numbers 2.5-fold, which were greater in nuclei density and exhibited increased resorption of dentine compared to osteoclasts derived from Vdrfl/fl splenocyte cultures. These data suggest that in addition to RANKL-mediated osteoclastogenesis, intact VDR signalling is required for the direct regulation of the differentiation and activity of osteoclasts in both in vivo and ex vivo settings.

The calcitonin receptor regulates osteocyte lacunae acidity during lactation in mice.

The physiological role of calcitonin, and its receptor, the CTR (or Calcr), has long been debated. We previously provided the first evidence for a physiological role of the CTR to limit maternal bone loss during lactation in mice by a direct action on osteocytes to inhibit osteocytic osteolysis. We now extend these findings to show that CTR gene expression is upregulated two- to three-fold in whole bone of control mice at the end of pregnancy (E18) and lactation (P21) compared to virgin controls. This was associated with an increase in osteoclast activity evidenced by increases in osteoclast surface/bone surface and Dcstamp gene expression. To investigate the mechanism by which the CTR inhibits osteocytic osteolysis, in vivo acidification of the osteocyte lacunae during lactation (P14 days) was assessed using a pH indicator dye. A lower pH was observed in the osteocyte lacunae of lactating Global-CTRKOs compared to controls and was associated with an increase in the gene expression of ATPase H+ transporting V0 subunit D2 (Atp6v0d2) in whole bone of Global-CTRKOs at the end of lacation (P21). To determine whether the CTR is required for the replacement of mineral within the lacunae post-lactation, lacunar area was determined 3 weeks post-weaning. Comparison of the largest 20% of lacunae by area did not differ between Global-CTRKOs and controls post-lactation. These results provide evidence for CTR activation to inhibit osteocytic osteolysis during lactation being mediated by regulating the acidity of the lacunae microenvironment, whilst the CTR is dispensable for replacement of bone mineral within lacunae by osteocytes post-lactation.

Paracrine signalling by cardiac calcitonin controls atrial fibrogenesis and arrhythmia.

Atrial fibrillation, the most common cardiac arrhythmia, is an important contributor to mortality and morbidity, and particularly to the risk of stroke in humans1. Atrial-tissue fibrosis is a central pathophysiological feature of atrial fibrillation that also hampers its treatment; the underlying molecular mechanisms are poorly understood and warrant investigation given the inadequacy of present therapies2. Here we show that calcitonin, a hormone product of the thyroid gland involved in bone metabolism3, is also produced by atrial cardiomyocytes in substantial quantities and acts as a paracrine signal that affects neighbouring collagen-producing fibroblasts to control their proliferation and secretion of extracellular matrix proteins. Global disruption of calcitonin receptor signalling in mice causes atrial fibrosis and increases susceptibility to atrial fibrillation. In mice in which liver kinase B1 is knocked down specifically in the atria, atrial-specific knockdown of calcitonin promotes atrial fibrosis and increases and prolongs spontaneous episodes of atrial fibrillation, whereas atrial-specific overexpression of calcitonin prevents both atrial fibrosis and fibrillation. Human patients with persistent atrial fibrillation show sixfold lower levels of myocardial calcitonin compared to control individuals with normal heart rhythm, with loss of calcitonin receptors in the fibroblast membrane. Although transcriptome analysis of human atrial fibroblasts reveals little change after exposure to calcitonin, proteomic analysis shows extensive alterations in extracellular matrix proteins and pathways related to fibrogenesis, infection and immune responses, and transcriptional regulation. Strategies to restore disrupted myocardial calcitonin signalling thus may offer therapeutic avenues for patients with atrial fibrillation.

Androgens stimulate erythropoiesis through the DNA-binding activity of the androgen receptor in non-hematopoietic cells.

BACKGROUND: Androgens function through DNA and non-DNA binding-dependent signalling of the androgen receptor (AR). How androgens promote erythropoiesis is not fully understood. DESIGN AND METHODS: To identify the androgen signalling pathway, we treated male mice lacking the second zinc finger of the DNA-binding domain of the AR (ARΔZF2 ) with non-aromatizable 5α-dihydrotestosterone (5α-DHT) or aromatizable testosterone. To distinguish direct hematopoietic and non-hematopoietic mechanisms, we performed bone marrow reconstitution experiments. RESULTS: In wild-type mice, 5α-DHT had greater erythroid activity than testosterone, which can be aromatized to estradiol. The erythroid response in wild-type mice following 5α-DHT treatment was associated with increased serum erythropoietin (EPO) and its downstream target erythroferrone, and hepcidin suppression. 5α-DHT had no erythroid activity in ARΔZF2 mice, proving the importance of DNA binding by the AR. Paradoxically, testosterone, but not 5α-DHT, suppressed EPO levels in ARΔZF2 mice, suggesting testosterone following aromatization may oppose the erythroid-stimulating effects of androgens. Female wild-type mice reconstituted with ARΔZF2 bone marrow cells remained responsive to 5α-DHT. In contrast, ARΔZF2 mice reconstituted with female wild-type bone marrow cells showed no response to 5α-DHT. CONCLUSION: Erythroid promoting effects of androgens are mediated through DNA binding-dependent actions of the AR in non-hematopoietic cells, including stimulating EPO expression.

Sex-specific adipose tissue imprinting of regulatory T cells.

Adipose tissue is an energy store and a dynamic endocrine organ1,2. In particular, visceral adipose tissue (VAT) is critical for the regulation of systemic metabolism3,4. Impaired VAT function-for example, in obesity-is associated with insulin resistance and type 2 diabetes5,6. Regulatory T (Treg) cells that express the transcription factor FOXP3 are critical for limiting immune responses and suppressing tissue inflammation, including in the VAT7-9. Here we uncover pronounced sexual dimorphism in Treg cells in the VAT. Male VAT was enriched for Treg cells compared with female VAT, and Treg cells from male VAT were markedly different from their female counterparts in phenotype, transcriptional landscape and chromatin accessibility. Heightened inflammation in the male VAT facilitated the recruitment of Treg cells via the CCL2-CCR2 axis. Androgen regulated the differentiation of a unique IL-33-producing stromal cell population specific to the male VAT, which paralleled the local expansion of Treg cells. Sex hormones also regulated VAT inflammation, which shaped the transcriptional landscape of VAT-resident Treg cells in a BLIMP1 transcription factor-dependent manner. Overall, we find that sex-specific differences in Treg cells from VAT are determined by the tissue niche in a sex-hormone-dependent manner to limit adipose tissue inflammation.

The androgen receptor in the hypothalamus positively regulates hind-limb muscle mass and voluntary physical activity in adult male mice.

We have previously shown that expression of the androgen receptor (AR) in neurons within the brain positively regulates hind-limb muscle mass and physical activity in male mice. To further investigate the region of the brain responsible for mediating these effects of testosterone and to determine whether they are only important for muscle mass accrual during development or whether they are also important for the maintenance of muscle mass in the adult, we deleted the AR specifically in the hypothalamus of adult male mice (Hyp-ARKOs). Hyp-ARKO mice were generated by bilateral stereotaxic microinjection of an adeno-associated virus (AAV) expressing GFP and iCre recombinase under the control of the e-synapsin promoter into the hypothalamus of 10-week-old exon 3-AR floxed male mice. AR mRNA was deleted by 45% in the hypothalamus of Hyp-ARKOs at 5 weeks post-AAV-eSyn-iCre injection. This led to an increase in the mass of the androgen-dependent organs, seminal vesicles and kidneys, by 30% (P < 0.01) and 10% (P < 0.05) respectively, and an increase in serum luteinizing hormone (LH) by 2 fold (P < 0.05). Whilst the mean value for serum testosterone was higher in the Hyp-ARKOs, this did not reach statistical significance. Despite a phenotype consistent with increased androgen bioactivity in Hyp-ARKOs, which would be expected to increase muscle mass, the mass of the hind-limb muscles, gastrocnemius (Gast) (P = 0.001), extensor digitorum longus (EDL) (P < 0.001) and soleus (Sol) (P < 0.01) were paradoxically decreased by 12-19% compared to controls. Voluntary physical activity was reduced by 65% (P < 0.05) in Hyp-ARKO male mice and was associated with a reduction in gene expression of Drd1a and Maob (P ≤ 0.05) in the hypothalamus, suggesting involvement of the brain dopaminergic system. These data provide compelling evidence that androgen signalling via the AR in the hypothalamus acts to positively regulate the maintenance of hind-limb muscle mass and voluntary activity in adult male mice, independent of AR signalling in peripheral tissues.