Endocrinology最新文献

Growth hormone (GH) controls sexual dimorphism in hepatocyte gene expression programs governing lipid metabolism, bile acid synthesis and xenobiotic processing, which contribute to sex differences in metabolic dysfunction-associated steatotic liver disease (MASLD) risk. Although GH-regulated sex-specific transcription is well-studied, the functional cis-regulatory hepatocyte enhancers that orchestrate these sex-dependent metabolic programs remain largely unknown. Here, we integrated single-nucleus multiomic profiling of hepatocyte chromatin accessibility with in vivo functional enhancer assays to identify and validate GH-responsive, sex-biased hepatocyte enhancers in intact mouse liver. We constructed a tiled HDI-STARR-seq library of 23,912 reporters spanning 1,839 liver ATAC regions and delivered it to liver by hydrodynamic injection, enabling enhancer activity assessment across different biological conditions. Reporters representing 840 ATAC regions showed sex-biased and/or GH-regulated enhancer activity, in many cases mirroring regulation of their accessibility in hepatocyte chromatin, validating them as functional, physiologically regulated enhancers. The regulated enhancer sequences were enriched for activating histone marks (H3K27ac, H3K4me1), and for binding sites for the STAT5-dependent, sex-specific repressors BCL6 and CUX2; whereas, STAT5 binding was enriched at both regulated and non-regulated enhancers. Motifs for HNF4A and for several novel factors identified de novo were specifically enriched at the regulated enhancers. Sex-biased and GH-regulated enhancers were linked to both MASLD-enabling and MASLD-protective genes, suggesting that GH-dependent chromatin remodeling at these loci contributes to sex-differential metabolic disease susceptibility. This integrated in vivo approach defines a validated set of GH-regulated hepatocyte enhancers through which chromatin accessibility and transcription factor binding drive sexual dimorphism in hepatic metabolism and MASLD risk.

The clinical characteristics of type 2 diabetes (T2D) differ between the sexes. For example, the risk of T2D is higher in males than in premenopausal females, whereas the risk of T2D-associated cardiovascular disease is higher in females. However, the sex-dependent mechanisms of T2D pathogenesis remain incompletely understood. Publicly available human islet datasets, such as HPAP and Humanislets.com, offer a valuable tool for uncovering the impact of biological sex on islet structure, gene expression, and function at a scale that was not previously possible. We performed an integrated analysis of data from publicly available sources to identify sex differences in baseline islet characteristics in donors without diabetes and subsequently examined these features in donors who lived with T2D. Among donors without diabetes, female islets had a greater proportion of alpha-cells compared with male islets and showed enriched expression of ribosomal and mitochondrial pathways in both beta- and alpha-cells. Measurements of mitochondrial function in female islets revealed lower spare respiratory capacity compared to male islets. Male and female islets had distinct changes in gene and protein expression in the context of T2D with female islets having greater preservation of insulin content and fewer defects in islet function. Together, these data show female islets have fewer islet impairments in T2D. This highlights the need for detailed mechanistic studies in both sexes to support effective and sex-informed interventions for T2D.

Orthopedia (Otp) transcription factor is a critical determinant in the development of the neuroendocrine hypothalamus, and its embryonic deletion results in lethality. Although Otp expression is maintained throughout life, its physiological function in adulthood is not well understood. Here, we generated a forebrain-specific, tamoxifen-inducible, conditional knockout mouse model to investigate the roles of Otp beyond development. Conditional deletion of Otp in two-month-old mice resulted in impaired stress responses, characterized by increased depressive-like behavior and elevated stress-induced cortisol levels. It also led to various metabolic changes, including reduced thyroid hormone levels and body temperature, a higher percentage of fat mass, and diminished responsiveness to ghrelin without affecting food intake, energy expenditure, or body weight. This composite metabolic phenotype was associated with reduced expression of hypothalamic neuropeptides TRH, AgRP, and NPY. Our findings highlight the role of Otp in adult physiological functions as a key neuroendocrine integrator of adaptive stress response and energy balance.

Effector kinases of the lipid second messenger diacylglycerol (DAG), including protein kinase C (PKC) and protein kinase D (PKD) isozymes, have been widely implicated in the development and progression of prostate cancer. By acting as central hubs of growth factor-mediated signaling, these kinases integrate oncogenic signals with the androgen receptor (AR) pathway, contributing to prostate tumor growth. Distinct members of the DAG-regulated kinases contribute to the acquisition of castration-resistant prostate cancer (CRPC) and bypass AR dependence, promoting the proliferative, migratory, and invasive competencies of androgen-independent prostate cancer cells. As predicted from their coupling to signaling cascades that impact gene expression, PKC/PKD isozymes control the activation of transcription factors such as NF-κB, E2F, and STAT3, and additionally regulate epithelial-to-mesenchymal transition (EMT) transcription factors in prostate cancer cells, providing an additional layer of control in invasive signaling. The aberrant expression/activation of DAG-regulated kinases during prostate cancer progression results in pronounced deregulation and rewiring of transcriptional networks associated with cell cycle control, invasiveness, and cancer cell interactions with the tumor microenvironment (TME). The multifaceted regulation of nuclear functions by these pleiotropic kinases underscores their convoluted roles in prostate cancer development and progression, offering new opportunities for therapeutic targeting.

A large "gene desert" located far upstream from Fshb and Kcna4 contains several gonadotrope-specific accessible chromatin sites which were seen in chromatin conformation capture (3C) to make distinct contacts with both genes. Expression of Fshb and Kcna4 was strongly inhibited by JQ-1, which represses super-enhancer activity, and the region displays super-enhancer characteristics. The sites of open chromatin were seen, in chromatin immunoprecipitation (ChIP), to bind Brd4 and Med1, most notably at a site -67 kb from the Fshb gene, as well as binding Ctcf further upstream (-123 kb), all of which were increased following activin exposure. The locus is transcribed to chromatin-associated lncRNAs whose levels correlate with Fshb and Kcna4 mRNA levels in vivo and in cultured gonadotrope cells, indicating coordinated regulation. CRISPR interference (CRISPRi) confirmed distinct functions for each element and, together with the 3C data, indicate that the -67 kb locus mediates basal and activin-stimulated Fshb expression, while the site at -59 kb contributes to activin-stimulation of both genes. Single-cell multiomics revealed that the -67 kb locus is accessible in pituitary stem cells and throughout gonadotrope differentiation, preceding opening of the Fshb promoter, although it is closed in other differentiated cell types, suggesting a gonadotrope-specific factor that keeps it open at this stage. Foxl2 was found to bind this element, contributes to maintaining its chromatin accessibility, and recruits Supt16h, a component of the Facilitates Active Chromatin Transcription (FACT) histone chaperone complex. These findings define a distal, Foxl2-bound super-enhancer that regulates Fshb transcription and shapes the gonadotrope regulatory landscape.

Background: Circulating estradiol is predominantly protein-bound, with human serum albumin (HSA) serving as its major carrier. While traditionally considered a carrier with low affinity and readily reversible binding at a single site, the molecular details and kinetics of estradiol-HSA interactions remain incompletely understood.

Methods: We employed equilibrium dialysis, steady-state and time-resolved fluorescence spectroscopy to characterize estradiol-HSA interactions. Surface plasmon resonance (SPR) was used to elucidate the kinetics of estradiol's association and dissociation with HSA. Structural and energetic features of binding were investigated using molecular docking and structure network analyses.

Results: Binding isotherms generated using equilibrium dialysis, steady-state and time-resolved fluorescence spectroscopy revealed non-linear asymmetric binding with apparent Kd that varied as a function of estradiol and HSA concentrations, inconsistent with canonical model of low-affinity, single-site interaction characterized by a fixed Kd. Kinetic analyses by SPR revealed initial rapid association dynamics followed by a slower second phase. Molecular modeling identified a high-affinity estradiol-binding pocket in Sudlow's Site I and two additional low-affinity sites within a highly interconnected hub of structural blocks. Spatially coordinated conformational rearrangements accompanying estradiol partitioning into the high-affinity pocket of Sudlow's Site I and two additional moderate-affinity sites suggest an allosterically coupled binding architecture that enables albumin to actively regulate estradiol bioavailability across a broad, physiologically relevant concentration range.

Conclusion: Estradiol's binding to HSA is a dynamic, multi-equilibrium process driven by ligand-induced conformational rearrangements within HSA; the binding data are inconsistent with canonical model of estradiol-HSA interaction with 1:1 stoichiometry and a fixed Kd.

Polycystic ovary syndrome (PCOS) is a systemic endocrine disorder characterized by perturbations in both androgen and insulin signaling pathways that result in anovulatory infertility and metabolic syndrome. This study aimed to elucidate insulin signaling in the PCOS ovary using a mouse model that develops both the metabolic and reproductive manifestations of PCOS due to chronic postnatal dihydrotestosterone exposure. PCOS mice developed anovulation, cystic follicles, systemic insulin resistance with compensatory hyperinsulinemia and mild excess adiposity, but not hepatic steatosis, adipose inflammation or frank obesity, suggesting that hyperandrogenism is the main driver of the metabolic perturbations in this model. Insulin signaling was then assessed in the ovary, liver, and skeletal muscle from hyperinsulinemic, fasting PCOS mice. Ovarian theca and granulosa cells showed upregulated markers of insulin signaling, while the liver and skeletal muscle from the same mice showed no changes compared to controls. However, cultured primary PCOS hepatocytes were profoundly insulin resistant in vitro, while primary theca cells (TCs) and granulosa cells (GCs) isolated from the same PCOS mice were insulin-sensitive. Both PCOS TCs and GCs produced significantly more steroid hormones than control cells when stimulated with insulin and gonadotropins. Our findings indicate that the PCOS ovary remains sensitive to insulin despite systemic insulin resistance, and that insulin works synergistically with gonadotropins to stimulate ovarian testosterone production in PCOS. We therefore suggest that insulin resistance is not merely a byproduct of hyperandrogenism but is a disease-driving factor in PCOS and should be treated as a clinical target in PCOS management.

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 in stimulating prostate cancer progression has been recognized for over 80 years, the appreciation for nongonadal 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 nongonadal 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 and hormone therapy resistance, and with emphasis on recent advances.

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 (sites 1 and 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.