The circadian clock plays a critical role in coordinating energy metabolism across tissues, including brown adipose tissue (BAT), a major site of nonshivering thermogenesis. This study aimed to elucidate the cell-autonomous role of the peripheral circadian clock in brown adipocyte thermogenesis using an in vitro model independent of extrinsic cues. Primary brown adipocytes were differentiated from the stromal vascular fraction of interscapular BAT isolated from C57BL/6J mice. An in vitro model of BAT clock disruption was established by siRNA-mediated knockdown of the core clock gene Bmal1. Thermogenic function was assessed via measurement of oxygen consumption rate (OCR) using an extracellular flux analyzer. To further assess the thermogenic process, protein expression levels of lipolytic enzymes and mitochondrial oxidative phosphorylation (OXPHOS) complexes were analyzed by Western blotting. Bmal1-knockdown markedly reduced both basal and β-adrenergic-stimulated OCR, indicating impaired thermogenic function, despite comparable cellular differentiation, preserved β-adrenergic responsiveness, and elevated uncoupling protein 1 (Ucp1) expression. Notably, Bmal1-deficient cells exhibited decreased protein expression of key lipolytic enzymes, adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL), as well as multiple mitochondrial oxidative phosphorylation (OXPHOS) subunits, suggesting decreased free fatty acid supply and reduced mitochondrial ability to generate the proton gradient required for UCP1-mediated thermogenesis. The peripheral circadian clock in brown adipocytes supports thermogenic function by regulating lipid mobilization and mitochondrial oxidative function, thus its disruption may lead to decreased energy expenditure and increased susceptibility to metabolic disorders.

The binding of human growth hormone (hGH) to the human growth hormone receptor (hGHR) is a key endocrinological process that controls critical aspects of cell growth, proliferation and differentiation. Mechanistically, this sequential, asymmetric binding event involves the interaction between a single hGH molecule and distinct sites (site 1 and site 2) on the extracellular domain of a preformed hGHR homodimer. Our group recently identified S1H, a rationally-designed peptide sequence mimetic of the hGH site 1-binding helix (residues 36-51) that disrupts the hGH-hGHR interaction and inhibits hGH-mediated phosphorylation of signal transducer and activator of transcription 5 (STAT5) in hGHR-positive cell lines. Structure-activity relationship studies revealed a positive correlation between helical propensity and inhibitory potency of the S1H peptide, prompting the design of structurally "stabilized" S1H variants (SS1H) with improved biological activity. In this study, we employed a chemical strategy, termed hydrocarbon stapling, to generate a series of SS1H peptides that proved to be more helical, proteolytically stable and biologically active compared to linear (unstructured) S1H. Notably, one SS1H derivative (SS1HB) inhibited hGH-induced STAT5 phosphorylation in hGHR-positive human bladder cancer cells more effectively than pegvisomant, the only hGHR antagonist currently approved by the FDA. Collectively, our results demonstrate that hydrocarbon stapling improves the antagonistic effects of S1H peptides and elevates their potential as chemical probes to study the molecular mechanisms of hGH signaling. It is also anticipated that SS1H peptides will serve as potent lead compounds for developing next-generation therapeutics designed to treat endocrine disorders that manifest along the hGH-hGHR signaling axis.

High ovarian cancer cell glucocorticoid receptor (GR) expression is associated with reduced progression-free survival (PFS) despite standard debulking surgery and adjuvant chemotherapy. Although not previously linked to tumor-cell GR expression, a "cold" (immune-suppressive) ovarian cancer tumor microenvironment (TME) is also associated with poor prognosis. In this study, analysis of The Cancer Genome Atlas (TCGA) ovarian cancer database revealed that NR3C1 (GR) mRNA was positively correlated with immunosuppressive cytokine gene expression. Higher tumor NR3C1 expression also associated with gene expression encoding cellular markers of immunosuppressive myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs). In vitro, GR activation in human and mouse ovarian cancer cell lines led to increased secretion of pro-tumorigenic cytokines, including G-CSF, M-CSF, TGFβ2 and CXCL2. Co-treatment with a GR agonist (mimicking endogenous cortisol) and a selective GR modulator (SGRM) significantly reduced cytokine secretion. In human xenograft mouse models, systemic administration of a SGRM decreased serum immunosuppressive cytokine concentrations and mouse MDSC tumor infiltration, suggesting that tumor-cell GR activity and cytokine secretion contribute to MDSC generation and recruitment. Additionally, in a syngeneic model of GR-positive ovarian cancer, both pharmacologic GR antagonism and tumor cell-specific GR knockdown reduced intratumoral and circulating MDSCs, as well as intratumoral Tregs. Collectively, these findings suggest that ovarian cancer-cell GR activity contributes to maintaining a highly immunosuppressive TME through secretion of tumor-derived cytokines. We conclude that ovarian cancer cell GR activity has a previously unrecognized role in inhibiting anti-tumor immunity and that systemic GR modulation could improve immunotherapy outcomes in ovarian cancer.

Androgen biosynthesis is physiologically necessary for generating the principal stimulus for androgen receptor (AR) signaling and thus plays an essential role for development of the normal prostate, prostate cancer growth and the development of resistance to hormonal therapies. Testosterone and dihydrotestosterone are both potent endogenous androgens that stimulate AR signaling. While the role of gonadal androgens has been recognized in stimulating prostate cancer progression for over 80 years, the appreciation for non-gonadal precursor steroids in prostate cancer has been more limited in duration of time, attention and focus in the field. Nevertheless, the very clearly established role of non-gonadal androgens in enabling prostate cancer progression, especially in the absence of gonadal testosterone, frames the essentiality of androgen metabolic processes for dictating prostate cancer clinical behavior. Here, the role of androgen metabolism in prostate cancer is reviewed, particularly within the context of hormonal therapy, hormone therapy resistance and with emphasis on recent advances.

Activin-class ligands of the transforming growth factor β family induce follicle-stimulating hormone (FSH) production by pituitary gonadotrope cells in mice via the actions of the transcription factors SMAD3, SMAD4, and FOXL2, which bind to cis-elements in the FSHβ subunit (Fshb) promoter. An enhancer region for murine Fshb transcription was identified in vitro. However, deletion of the region using CRISPR-Cas9 did not affect FSH synthesis or secretion in mice. Using single-nucleus ATAC-seq of whole murine pituitaries, we identified three additional open chromatin regions upstream of Fshb exclusively in gonadotropes. These regions, as well as the Fshb gene, were fully or partially closed in gonadotropes of FSH-deficient mice with genetically or pharmacologically inactivated activin type II receptors. The initially characterized enhancer region did not significantly alter basal or activin-stimulated murine Fshb promoter-reporter activity in homologous LβT2 cells. In contrast, the other three open chromatin regions enhanced basal and activin A-stimulated Fshb promoter-reporter activity in LβT2 cells, with the two most distal showing the greatest effects. These two regions were open, exhibited enrichment of the enhancer mark H3K27ac, and were bound by SMAD2/3 and FOXL2 in response to activin A in LβT2 cells. The most distal enhancer exhibited strong FOXL2 and weak SMAD4 binding in gel shift assays. SMAD4, but not FOXL2, directly bound the other distal enhancer. Mutation of defined FOXL2 and SMAD4 cis-elements diminished enhancer activity in reporter assays in LβT2 cells. Collectively, the data indicate that there may be as many as four activin-sensitive enhancers upstream of murine Fshb.

Anti-Müllerian hormone (AMH) is produced by granulosa cells within growing ovarian follicles and limits the number of follicles reaching ovulation. AMH is synthesised as a precursor protein comprising N-terminal prodomains and C-terminal mature domains, separated by a furin-like cleavage motif (RXXR). Proteolytic maturation of AMH (140 kDa) is required to release the bioactive mature dimer (25 kDa), which potentiates signalling via AMH receptors (AMHR2 and ALK2/3). However, the abundance of unprocessed AMH in human follicular fluid suggests that cleavage within the ovary is inefficient. This study hypothesised that enhancing AMH maturation would increase AMH activity in vitro and in vivo. Using targeted mutagenesis we optimised the murine AMH cleavage site (from wild-type (WT) 443RTGR445 to 443RKKR445) and showed in vitro that this favoured production of bioactive AMH. We then introduced this mutation into the Amh gene in C57Bl6/J mice using CRISPR/Cas9 and assessed the consequences for female reproduction. Analyses of 12-week-old AmhRKKR/RKKR mice revealed that the ovaries were significantly lower in mass (-25%, p<0.05) relative to AmhWT/WT controls. Despite differences in ovarian masses, estrous cyclicity, and fertility were unaltered. Although maturing follicle numbers did not differ, ovaries from 12- and 24-week-old AmhRKKR/RKKR females contained a greater proportion of atretic secondary follicles (1.6-4-fold more, p<0.05), underscoring AMH's role in preantral follicle survival. Analyses of adult male AmhRKKR/RKKR mice indicated testis mass and morphology were unaltered. These findings support a physiological role for ovarian AMH in limiting preantral follicle survival and indicate that enhancing AMH maturation is otherwise non-disruptive to female reproduction.

African American men experience a higher incidence and severity of prostate cancer relative to European American men, and there is a range of risk factors that may contribute to this disparity. Prostate adenocarcinoma originates from the epithelium, which is significantly influenced by signaling from the surrounding fibromuscular stroma. To identify ancestry-associated differences in the stroma, gene expression profiling was compared between laser-capture microdissected prostate cancer stroma from patients of African descent and those of European descent. Estrogen receptor signaling was the top differential pathway between the groups, with the steroid hormone dehydrogenase HSD17B7 identified as the most differentially expressed gene. In a separate cohort of patients, protein expression of HSD17B7 was higher in African American patients relative to European American patients in a radical prostatectomy tissue microarray, validating the transcriptional findings. African American patients also exhibited significantly increased levels of HSD17B7 protein in the stroma surrounding benign areas compared to the stroma near tumors. These studies provide important evidence of ancestry-associated differences in stromal estrogen signaling.

Leptin receptor positive (LepR+) cells are multipotent stromal cells and a source of osteogenic and adipogenic cells. Inactivation of Notch signaling in LepR+ cells increases bone mass in mature mice, but the target gene responsible was not identified. Because in LepR+ cells the expression of the Notch target gene Hes1 prevails over that of other genes, we explored the role of the Hes1 deletion in LepR+ cells. To this end, LepR-Cre;Hes1Δ/Δ mice were compared to Hes1loxP/loxP littermates. Male and female 5-month-old LepR-Cre;Hes1Δ/Δ mice exhibited an increase in femoral bone volume/total volume due to an increase in trabecular number; vertebral (L3) and cortical bone was not affected. Bone histomorphometry demonstrated decreased osteoclast number and eroded surface, decreased osteoblast number only in male mice, and no changes in bone formation. Neither osteogenesis nor adipogenesis was modified by the Hes1 deletion in bone marrow stromal cell cultures, although Tnfsf11 (encoding RANKL) was suppressed in osteogenic cultures of Hes1Δ/Δ cells. Single-cell RNA sequencing of femurs from 5-month-old LepR-Cre;Hes1Δ/Δ and control mice revealed the presence of 23 cell clusters including clusters composed of hematological cells (myeloid, B cells, and neutrophils), endothelial cells, and osteoblasts. There were no substantial differences in gene expression, cluster distribution, or trajectory finding between control and Hes1 inactivated cells. In conclusion, Hes1 inactivation in LepR+ cells results in an increase in bone mass secondary to a decrease in RANKL, osteoclast number, and bone resorption, but HES1 has little influence on osteogenesis or adipogenesis in bone.

Water movement across cell membranes through aquaporin water channels creates osmotic equilibrium between extracellular and intracellular fluid compartments. Plasma osmolality is tightly regulated by the kidneys and brain through the process of osmoregulation. The antidiuretic hormone, arginine vasopressin (AVP), is normally released from the posterior pituitary in response to increased osmolality or decreased intravascular volume. Defects in the synthesis or release of AVP result in AVP deficiency (AVP-D) and the syndrome of central diabetes insipidus, characterized by inappropriate aquaresis leading to hyperosmolality and insatiable thirst. While most cases of AVP-D are due to local mechanical, infiltrative, compressive, infectious or inflammatory processes, some recreational and pharmacological substances can cause AVP-D. In this review, we discuss the history and current knowledge about these substances, including cannabinoids, ethanol, kappa opioid receptor agonists, phenytoin, and anesthetic agents.

Patients with diabetes are disproportionately affected by cardiovascular and kidney disease. Mineralocorticoid receptor antagonists (MRA) show organ protection against cardiovascular and renal injury; however, major side effects including hyperkalaemia and reduced renal function limit their use in individuals with diabetic complications. The non-steroidal MR modulator, balcinrenone, may offer end-organ protection with fewer side effects. We compared responses to balcinrenone and eplerenone delivered from 8 weeks post-induction of streptozotocin (STZ)-induced type 1 diabetes in male mice. RNA-sequencing revealed diabetes induced modulation of immune function, and metabolic and vascular targets in the kidney, which were similarly attenuated by balcinrenone or eplerenone treatment. Urine K+ excretion was lower following eplerenone treatment, but not balcinrenone treatment, compared to diabetes without treatment. We identified a 5.90-fold increase in the expression of K+ transporter G protein-activated inward rectifier potassium channel 1 (GIRK-1) in eplerenone-, but not balcinrenone-treated diabetic mice. Balcinrenone and eplerenone similarly attenuated the diabetes-induced reduction in peak E-wave/A-wave velocity (E/A) compared to mice without treatment at 15 weeks post-STZ. Gene markers of cardiac injury, B-type natriuretic peptide (Bnp) and beta-myosin heavy chain protein (Myh7), were higher in diabetic versus non-diabetic left ventricles (LV). Conversely, gene expression of Ca2+ ion channel subunits, voltage-dependent L type, calcium channel subunit alpha 1C (Cav1.2) and ryanodine receptor 2 (Ryr2), in LV was lower in diabetic but not eplerenone- or balcinrenone-treated diabetic mice. Although balcinrenone and eplerenone similarly modified cardiac changes, potassium excretion was greater with balcinrenone, consistent with a reduced risk of hyperkalemia with the non-steroidal MR modulator.