Endocrinology

Ovarian disorders, including anovulation, primary ovarian insufficiency (POI), and polyendocrine metabolic ovarian syndrome (PMOS), affect millions of reproductive-age women worldwide; however, mechanistic studies of ovarian biology and pathophysiology remain challenging because current experimental approaches often lack selectivity, tunability, or physiological relevance. Genetically modified animal models are labor-intensive and irreversible; small molecules frequently exhibit off-target effects; and conventional antibodies have limited tissue penetration and restricted temporal control. Designed ankyrin repeat proteins (DARPins) represent a highly modular protein engineering platform with advantages in specificity, size, stability, and extracellular targeting, but their utility in reproductive biology remains largely unexplored. Here, we used epidermal growth factor receptor (EGFR)-targeting DARPins as a proof-of-concept platform to interrogate ovarian signaling during ovulation. Screening of engineered anti-EGFR DARPins identified SX-006, a bispecific tetravalent construct with robust cross-species EGFR binding and potent biological activity. Using an ex vivo murine ovulation system, SX-006 inhibited follicle rupture in a dose-dependent manner with IC50 of 1.21 M without overt cytotoxicity. Lower concentrations of SX-006 preferentially perturbed follicle rupture while largely preserving oocyte meiotic maturation and luteinization, suggesting differential sensitivity of ovulatory processes to extracellular EGFR blockade. Comparative transcriptomic analyses further revealed that extracellular EGFR blockade and small molecule-based intracellular EGFR kinase inhibition produce overlapping but also distinct transcriptional responses, supporting biologically distinct modes of ovulatory signaling pathway perturbation. Together, these findings establish DARPins as a selective, tunable, and physiologically relevant platform for studying ovarian signaling and provide proof-of-concept for extracellular receptor targeting in ovarian biology, infertility research, and non-hormonal contraceptive development. Summary sentenceAn engineered EGFR-targeting DARPin selectively inhibits ovulation through extracellular receptor blockade and establishes a versatile platform for investigating ovarian signaling and reproductive disorders.

Prolactinomas, the most common secretory pituitary tumour subtype, frequently occur in patients with Multiple Endocrine Neoplasia type 1, caused by germline MEN1 mutations encoding menin. While menin loss is well established in MEN1-associated prolactinomas, its role in sporadic tumours remains unclear. We investigated menin expression, subcellular localization, and downstream signalling in two murine models of non-MEN1 prolactinomas, the dopamine D2-receptor knockout and the hCG{beta}-subunit-overexpressing mice, in which only females develop prolactinoma. Pituitary Men1 expression, analysed by qPCR, remained unchanged despite the genotype, in both sexes. However, in prolactinomas, lactotrophs exhibited a marked loss of nuclear MEN1 immunostained, with protein restricted to the cytoplasm. Male mice pituitaries retained nuclear MEN1 localization regardless their genotype. Loss of nuclear menin in prolactinomas was associated with reduced p27 and Pten expression, increased Ccnd1 expression, and enhanced pAKT. Moreover, by using in vivo pharmacological and surgical approaches we demonstrated that dopamine-agonist treatment preserved nuclear menin in lactotrophs, whereas dopamine blockade or estradiol induced its nuclear loss. Importantly, analysis of human pituitary biopsies confirmed nuclear and cytoplasmic menin localization in lactotrophs from normal pituitaries, and in prolactinomas from both genders following dopamine agonist therapy. However, in a prolactinoma from an untreated female, nuclear menin was partially lost. Therefore, our findings identify a state of functional MEN1-deficiency in sporadic prolactinomas (characterized by preserved MEN1 expression), but its exclusion from the nucleus (linked to activation of proliferative pathways, impaired tumour suppressor signalling, and tumour development) highlights the restoration of nuclear MEN1 localization as a potential therapeutic strategy.

Purpose: Gonadotropin releasing hormone (GnRH) agonists are clinically used to delay pubertal progression by suppressing the hypothalamic-pituitary-gonadal (HPG) axis. While GnRH agonists have long been used clinically, the developmental characterization of HPG axis suppression during puberty remains incompletely understood. Thus, we examined the effects of GnRH receptor agonism in juvenile rats. Hypothesis: Sustained GnRH receptor agonism will result in lower gonadal mass, blunt peripheral pubertal landmarks, and alter hormonal signaling dynamics within the HPG axis. Methods: Animals received a single injection of extended-release leuprolide acetate depot (LA) or vehicle control on postnatal day (PND) 23. Animals were assessed for body mass and peripheral markers of puberty. On PND 44, animals were euthanized and tissues were evaluated to assess additional markers of pubertal maturation, pituitary gene transcript levels, and hormone concentrations in serum and gonads. Results: In females, LA treatment resulted in a smaller gonad size, increased body mass, and less vaginal openings. In males, LA treatment resulted in smaller gonads but did not significantly alter body mass or preputial separation. In the pituitary, LA-treated rats had lower Gnrhr, Fshb, and Lhb transcript levels regardless of sex, while females exhibited higher Cga and Nr5a1. Serum FSH and ACTH were lower in LA-treated animals, and treated females also had lower progestins and androstenedione, and higher LH. Conclusions: LA treatment reduced aspects of pubertal maturation and HPG axis output, with sex specific outcomes. These findings highlight the need for integrated, multi-level approaches to understand how altered GnRH signaling impacts pubertal and long-term physiology.

In mammals, ovarian follicle development is a highly coordinated process that underlies female fertility. Granulosa cells expressing anti-Mullerian hormone (AMH) are widely used as a marker of growing follicles. However, the in vivo roles of granulosa cells in follicular development and female fertility remain unclear. Here, we analyzed AMH-toxin receptor-mediated cell knockout (AMH-TRECK) transgenic (Tg) mice on a NOG background, in which AMH-expressing granulosa cells are specifically depleted by diphtheria toxin (DT). We first found that, after a single DT injection into postnatal AMH-TRECK Tg females, AMH-expressing granulosa cells in primary and secondary follicles exhibited cleaved caspase-3 signals 1 day later and were depleted 4 days later. Second, after repeated DT injections weekly from 1 to 7 weeks of age in AMH-TRECK Tg females, antral follicles and corpora lutea were rarely observed, and the numbers of primordial, primary, and secondary follicles were decreased. Following PMSG and hCG stimulation, repeated DT-injected Tg females exhibited a reduced number of ovulated oocytes with a low proportion of mature oocytes, resulting in reduced IVF rates and fertility. Further, after a cessation of repeated DT treatment, ovarian weight and follicular development recovered: the numbers of primary, secondary, and antral follicles were recovered, whereas the primordial follicle pool remains reduced. We conclude that selective depletion of AMH-expressing granulosa cells in vivo impairs follicular development and fertility. Our model enables assessment of the in vivo effects of granulosa cell depletion and may provide a useful platform for future transplantation-based studies to understand complex follicular dynamics.

Pregnancy induces or represses hepatic drug metabolism. Whether pregnancy affects hepatic drug transport is unexplored. We previously showed that a cocktail of pregnancy-related hormones (PRHC) induces mRNA expression and activity of sodium/taurocholate cotransporting polypeptide (NTCP), organic anion transporter 2 (OAT2), and organic cation transporter 1 (OCT1, mRNA only) in differentiated HepaRG cells. Here, using HepaRG cells, we identified cortisol as the hormone primarily responsible for this induction and explored the underlying mechanisms. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated knockdown studies in HepaRG cells showed that the glucocorticoid receptor (GR) is the primary mediator of this response. GR knockdown markedly attenuated cortisol-induced NTCP, OAT2, and OCT1 mRNA expression and activity. Cortisol also induced the mRNA expression of regulatory factors, including pregnane X receptor (PXR), constitutive androstane receptor (CAR), and hepatocyte nuclear factor (HNF) 4 alpha (HNF4). HNF4 knockdown selectively attenuated OAT2 and OCT1 induction, whereas HNF1 knockdown enhanced NTCP induction, attenuated OCT1 induction, and reduced basal organic anion transporting polypeptide 1B1 (OATP1B1) expression. In contrast, knockdown of CAR or PXR did not significantly alter cortisol-mediated transporter regulation. These data identify cortisol as the principal PRH driving regulation of the hepatic OAT2, NTCP, and OCT1 in HepaRG cells and indicate that this response is mediated primarily by GR, with selective downstream contributions from HNF4 and HNF1. These findings provide mechanistic insights into pregnancy-associated changes in hepatic transporter-mediated drug disposition, including when antenatal corticosteroids are administered to pregnant women to prevent respiratory distress syndrome in their prematurely born infants. Significance StatementThe extent and mechanisms by which pregnancy-related hormones regulate hepatic uptake transporters remain poorly defined. This study identifies cortisol as the principal pregnancy-related hormone driving NTCP, OAT2, and OCT1 induction in HepaRG cells and shows that this response is mediated primarily through GR, with transporter-specific contributions from HNF4 and HNF1.

Targeted mass spectrometry (multiple-reaction monitoring, MRM) enables reproducible, multiplexed quantification of plasma proteins, but whether a fixed targeted panel can resolve endocrine disorders with overlapping systemic features is unknown. We analyzed a 256-protein targeted panel (1,894 peptides; 3,790 transitions) quantified in 57 participants spanning autoimmune thyroid disease (Hashimotos thyroiditis, n=7; Graves disease, n=5) and growth-hormone deficiency (GHD; partial, n=26; complete, n=19). Protein abundances were obtained by transition summation, log2 transformation, and per-sample median normalization. We applied unsupervised analysis (PCA, PERMANOVA), differential expression (limma), an ordered severity-trend test, and leave-one-out cross-validated classification with feature selection performed strictly inside each fold. All 256 proteins were quantified in every sample (median inter-sample r=0.875). PC1 (36% variance) separated autoimmune thyroid disease from GHD (p=0.016), whereas the global four-group structure was not significant (PERMANOVA p=0.17). No protein reached FDR<0.05, but the autoimmune-versus-GHD contrast was strongly enriched for low p-values (26 proteins at p<0.05; binomial p=5.4x10-4). The signal was biologically coherent: immunoglobulin/B-cell-receptor proteins, including CD79A, were lower, whereas proteasome subunits (PSMC5, PSMC3) and the NF-{kappa}B subunit RELA were higher in autoimmune disease. A cross-validated classifier separated the two classes (AUC 0.72; permutation p=0.05; eight proteins selected in all folds), whereas GHD severity was not predictable (AUC 0.31). A fixed 256-protein targeted panel reproducibly captures an immunoglobulin/B-cell-receptor and proteasome/NF-{kappa}B axis that distinguishes autoimmune thyroid disease from GHD but cannot resolve within-class severity.

ObjectiveThe aryl hydrocarbon receptor (AhR) pathway primarily mediates pollutant responses by activating xenobiotic metabolism enzymes like cytochrome P450 1A1 and 1A2 (CYP1A). Although AhR has also been implicated in systemic metabolic dysfunction and is inducible in pancreatic islets, its role in islet physiology remains unclear. MethodsWe analyzed a publicly available bulk human islet transcriptomic dataset to identify pathways associated with CYP1A1 expression. We also assessed islet responses to the pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and glucolipotoxicity (GLT) in vitro using two mouse models: a global Cyp1a1/1a2 double knockout (CypKO) model, which disrupts canonical AhR-CYP1A signaling in whole islets, and a {beta}-cell-specific Ahr knockout ({beta}AhrKO) model, which abolishes AhR signaling selectively in {beta}-cells. We then examined the role of {beta}-cell Ahr in early adaptation to high-fat diet (HFD) feeding in vivo. ResultsXenobiotic and nutrient metabolism pathways were enriched in donors with high CYP1A1 expression. Global Cyp1a1/1a2 deletion increased susceptibility of female mouse islets to TCDD-induced impairments in insulin secretion but had minimal effects on GLT responses in either sex. In contrast, {beta}-cell Ahr deletion did not affect islet responses to TCDD, but exacerbated GLT-induced islet dysfunction in male islets and increased baseline insulin secretion in both vehicle- and GLT-exposed female islets in vitro. Lastly, {beta}-cell Ahr deletion prevented adaptive HFD-induced hyperinsulinemia in both sexes in vivo. ConclusionIslet AhR signaling shapes responses to chemical and nutrient stressors in a context- and sex-dependent manner. While the canonical AhR-CYP1A axis supports female islet resilience to TCDD, {beta}-cell AhR signaling more broadly regulates nutrient stress responses in both sexes.

The RNA-binding proteins Musashi1 and Musashi2 (MSI1 and MSI2) regulate stem cell function and tissue plasticity by modulating mRNA translation. While typically known as translational repressors, the MSI1 and MSI2 proteins can also act as context-dependent activators of mRNA translation, although the mechanism of MSI-mediated translational activation are unknown. Here, we identify Embryonic Lethal Abnormal Vision-like (ELAVL) proteins as essential co-regulators of MSI1-dependent translational activation. In Xenopus laevis oocytes, antisense oligonucleotide knockdown of Elavl4 inhibited progesterone-stimulated maturation and blocked polyadenylation and translation of key MSI target mRNAs, including the Mos and Cyclin B5 mRNAs. Exogenous expression of ELAVL4 rescued these defects, confirming its necessity for maturation and cell cycle progression. Mechanistically, we determined that the ELAVL4 C-terminal domain interacts with the N-terminal RNA recognition motifs of MSI1 in an RNA-independent manner. Mass spectrometry and functional assays revealed this interaction is evolutionarily conserved: mouse ELAVL1 interacts with MSI1 in the pituitary, and human ELAVL1 rescues Elavl4-depleted Xenopus oocytes. Furthermore, knockdown of Elavl1 in a mammalian cell line abrogated MSI-dependent translational activation of a pituitary Prop1 3-UTR mRNA reporter. Our results establish a conserved mechanism where ELAVL family members interact with MSI to promote MSI-dependent mRNA translational activation.

Summary/AbstractThe estrous cycle transcriptional and chromatin dynamics in pituitary cell types have not been investigated. We report single-nucleus multiomics assays in 18 adult mouse pituitaries (102,069 post quality-control nuclei) across 6 cycle time points. Differential analysis revealed cycle stage-dependent epigenetic and transcriptional remodeling across the major pituitary cell populations. In gonadotropes and lactotropes, we identified temporal patterns of differential gene expression linked to various biological processes, notably neuronal and synaptic-related ontologies. Pseudotime trajectory analysis was consistent with a rapid transition of individual gonadotropes through different cellular states. In gonadotropes and lactotropes, we uncovered gene regulatory circuits whose activity varies between consecutive cycle time points. We experimentally validated a gonadotrope ETS2-driven circuit that differentially regulates Fshb gene expression between 2 and 9 am on estrus. Our data and analyses, available at https://rstudio-connect.hpc.mssm.edu/snpit_estrous_browser/, provide a window into gene regulatory mechanisms underlying estrous cycle stage transitions. HighlightsO_LIPituitary cells show chromatin and transcriptome plasticity across the estrous cycle C_LIO_LIWe identify stage-modulated gene regulatory circuits across pituitary cell types C_LIO_LIGonadotrope DEGs form distinct pathway-annotated temporal trajectory clusters C_LIO_LIWe validate a gonadotrope ETS2-driven circuit regulating the Fshb gene C_LI eTOC blurbZhang et al. conduct a single-nucleus multiomics analysis of mouse pituitaries in vivo across the estrous cycle. They reveal an epigenetic and transcriptomic plasticity in pituitary cell populations. In gonadotropes and lactotropes, they demonstrate that DEGs clustered by temporal trajectories are enriched for distinct biological processes and identify stage-modulated gene regulatory circuits. They experimentally validate a gonadotrope ETS2-driven circuit regulating Fshb expression. Their dynamic molecular atlas of the cycling pituitary captures key cis-regulatory mechanisms underlying estrous cycle stage transitions.

A successful pregnancy hinges on a finely coordinated dialogue between the maternal endometrium and the implanting embryo. Following embryo attachment to the uterine epithelium, underlying stromal cells undergo a transformation into decidual cells that promote a vascularized maternal-fetal interface and direct trophoblast lineage decisions through paracrine cues. However, the metabolic adaptations that enable decidual cells to support these energetically demanding processes remain poorly understood. Here, using a uterine-specific knockout mouse model, we identify Glucose Transporter 1 (Glut1) as a critical metabolic regulator linking endometrial glucose uptake to reproductive success. We demonstrate that stromal Glut1, induced by hypoxia-inducible factor 2 (Hif2), sustains a Hif2-Rab27b feed-forward circuit that drives vesicular trafficking during pregnancy through the glucose-sensing transcription factor MAX-like protein X (Mlx). Mice lacking endometrial Glut1 are severely subfertile despite normal embryo attachment. Glut1-deficient uteri exhibit impaired stromal extracellular vesicle secretion, defective decidual angiogenesis, and marked dysregulation of trophoblast differentiation, including expansion of trophoblast progenitors, accumulation of glycogen trophoblast cells, and altered placental lactogen production. These placental defects culminate in mid-gestation fetal loss and maternal gestational diabetes mellitus (GDM). Collectively, our findings establish endometrial Glut1 as a metabolic gatekeeper of maternal glucose homeostasis and placentation and introduce a genetically tractable mouse model of spontaneously developing GDM, a disorder affecting nearly one in seven pregnancies worldwide. SIGNIFICANCEThis study uncovers endometrial Glut1 as a previously unrecognized metabolic regulator of placentation, demonstrating that maternal stromal glucose uptake dictates trophoblast fate and maternal glycemic control, and providing the field with its first genetically defined mouse model of spontaneously arising gestational diabetes.

Early-life exposures during critical periods of development significantly impact lifelong metabolic risk and likely contribute to the rising rates of obesity, type 2 diabetes, and metabolic dysfunction-associated steatotic liver disease (MASLD) in children. Here, we evaluated the safety and metabolic effects of semaglutide, a GLP-1 receptor agonist (GLP-1 RA), administered from preconception through lactation in dams fed a high-fat diet (HFD) or standard diet, and assessed metabolic outcomes in dams and their offspring. Offspring were weaned to a standard diet. We found that semaglutide improved body composition and glucose metabolism in HFD-fed dams during pregnancy. These maternal changes persisted 10 weeks after weaning despite discontinuation of semaglutide treatment. HFD exposure impaired glucose homeostasis and promoted hepatic steatosis in offspring at 18 weeks. These effects were ameliorated by maternal semaglutide treatment. Importantly, metabolic improvements in dams and offspring occurred without adverse effects on conception rate or fetal viability. These findings suggest that GLP-1 RA during the perinatal period can improve maternal and offspring metabolic health in a mouse model of obesity and support further investigation of GLP-1-based therapies to mitigate maternal metabolic dysfunction and improve metabolic risk in children. ARTICLE HIGHLIGHTS* Rates of obesity, type 2 diabetes, and fatty liver disease are rising in children, in part due to maternal obesity and insulin resistance that program offspring metabolic risk during the perinatal period. * We asked whether the GLP-1 receptor agonist (GLP-1 RA), semaglutide, administered during critical developmental windows could prevent adverse outcomes in offspring using a diet-induced mouse model of maternal obesity. * Semaglutide, given to dams from preconception through lactation, improved maternal metabolism and ameliorated metabolic dysfunction in offspring caused by maternal high-fat diet. * These findings highlight a potential role for perinatal GLP-1 receptor agonism to improve maternal metabolic health and reduce metabolic risk in offspring.

Bipolar androgen therapy (BAT) exploits the paradoxical vulnerability of castration-resistant prostate cancer (CRPC) cells to rapid cycling between castrate and supraphysiologic androgen concentrations, but clinical BAT uses testosterone, which can also activate wild-type androgen receptor (AR) in androgen-responsive tissues, causing systemic side effects. 5{beta}-dihydrotestosterone (5{beta}-DHT) is a naturally occurring testosterone metabolite generally considered androgenically inactive because it binds wild-type AR weakly, yet its activity against clinically relevant AR mutants has not been systematically evaluated. Here, we tested whether 5{beta}-DHT and related 5{beta}-reduced testosterone metabolites activate AR signaling and growth programs in prostate cancer models that carry AR mutations. In C4-2 cells, 5{beta}-DHT and 3{beta}-etiocholanediol (3{beta}-ecdiol) increased canonical AR target genes, including KLK3 and TMPRSS2, with weaker activity than testosterone, whereas other 5{beta} metabolites showed limited activity. In androgen-responsive LNCaP and C4-2 models, 5{beta}-DHT and 3{beta}-ecdiol promoted cell growth under androgen-depleted conditions, and this effect was suppressed by enzalutamide, supporting AR dependence. RNA-seq confirmed that 5{beta}-DHT and 3{beta}-ecdiol induced androgen-response gene sets substantially overlapping with testosterone, albeit at lower transcriptional magnitude. Further, we found that 5{beta}-DHT, but not 3{beta}-ecdiol, suppresses cell proliferation of LNCaP, C4-2, and PC-3 cells stably expressing the clinically relevant AR gain-of-function mutants W742C and H875Y through activating AR-induced senescence-like features after high-dose exposure, consistent with the therapeutic logic of BAT. These findings identify 5{beta}-DHT as an overlooked mutant-AR agonist capable of BAT-like tumor suppression and propose it as a testosterone surrogate in BAT with potentially reduced systemic androgenic side effects. HighlightsO_LI5{beta}-DHT and 3{beta}-ecdiol promote AR-dependent prostate cancer cell growth C_LIO_LIBoth are weaker AR agonists than testosterone by RNA-seq and qPCR C_LIO_LISupraphysiologic 5{beta}-DHT suppresses growth via AR-mediated senescence C_LIO_LIGrowth suppression extends to AR mutants W742C and H875Y C_LIO_LI5{beta}-DHT may be a lower-androgenicity testosterone surrogate for BAT C_LI

Aims/hypothesisDisruption of the circadian glucocorticoid rhythm occurs in human settings including chronic stress, sleep restriction, circadian misalignment, ageing and autonomous cortisol secretion; in mild autonomous cortisol secretion (MACS) and Cushings syndrome, loss of the normal cortisol trough is clinically informative, and flatter diurnal cortisol profiles are associated with cardiometabolic disease. We previously showed that flattening of glucocorticoid rhythms in mice induces rapid and sustained hyperinsulinemia without hyper or hypo-glycaemia, implying that glucocorticoid rhythms may directly regulate the relationship between circulating glucose and systemic insulin output. Here we tested the hypothesis that beta cell glucocorticoid receptor (GR) signalling is required for the compensatory hyperinsulinaemia that maintains glucose homeostasis during glucocorticoid rhythm flattening, and that this reflects glucocorticoid-dependent reprogramming of beta cell stimulus-secretion coupling. MethodsGlucocorticoid rhythms were flattened in male C57BL/6J mice by subcutaneous corticosterone pellet implantation, which elevates trough levels and reduces peak amplitude while preserving the daily mean hormone concentration. Fasting plasma insulin and blood glucose were measured longitudinally and compared with placebo-implanted controls and high-fat diet-fed mice. Beta cell secretory function was assessed by static and dynamic glucose-stimulated insulin secretion in isolated islets, and beta cell excitability by GCaMP6f Ca{superscript 2} imaging in islets from Ins1-Cre;GCaMP6f mice. To test the requirement for beta cell GR in mature beta cells while avoiding developmental effects of constitutive GR deletion, we generated adult-inducible beta cell-specific GR knockout mice (MIP-CreERT;Nr3c1fl/fl; {beta}GRKO). Combined beta cell and hepatic GR knockout mice (double-GRKO) were used to examine an additional extra-pancreatic contribution to systemic insulin availability. Glucose tolerance and insulin sensitivity were assessed by intraperitoneal glucose and insulin tolerance tests. As a secondary question, a possible contribution of altered insulin clearance was examined from plasma C-peptide:insulin ratios and hepatic insulin-degrading enzyme (IDE) abundance. ResultsGlucocorticoid rhythm flattening produced sustained hyperinsulinaemia with maintained euglycaemia, distinct from the delayed hyperinsulinaemia and hyperglycaemia observed in high-fat diet-fed mice. Islets from glucocorticoid-flattened mice exhibited increased insulin secretion at subthreshold (3 mmol/l) glucose, enhanced secretory responses to stimulatory glucose and increased Ca{superscript 2} responses, indicating a lowered glucose threshold for beta cell activation that persisted ex vivo. Beta cell-specific deletion of GR markedly attenuated the hyperinsulinaemic response to glucocorticoid flattening (insulin AUC reduced [~]40% vs controls; p < 0.001) and produced progressive hyperglycaemia and impaired glucose tolerance, despite unchanged or improved insulin sensitivity. A reduced plasma C-peptide:insulin molar ratio (p = 0.007) and decreased hepatic IDE abundance (p = 0.032) indicated that reduced insulin clearance contributes additionally to the elevated circulating insulin, and combined beta cell and hepatic GR deletion lowered circulating insulin further than beta cell GR deletion alone. The absence of hypoglycaemia despite persistent hyperinsulinaemia is consistent with concurrent insulin resistance. Conclusions/interpretationBeta cell GR signalling is required for the compensatory hyperinsulinaemia that maintains glucose homeostasis when glucocorticoid rhythmicity is disrupted, acting through glucocorticoid-dependent lowering of the glucose threshold for insulin secretion; reduced insulin clearance contributes additionally to the rise in circulating insulin. These findings identify beta cell GR signalling as a key determinant of glucose homeostasis during disrupted glucocorticoid rhythmicity. Clinically, the work is most relevant not simply to nonspecific chronic stress, but to human states in which the cortisol rhythm is measurably flattened or the nocturnal trough is lost, including MACS, Cushings syndrome, sleep restriction, shift work/circadian misalignment and ageing. RESEARCH IN CONTEXTO_ST_ABSWhat is already known about this subject?C_ST_ABSO_LIFlattened or disrupted glucocorticoid rhythmicity in humans is observed most directly in MACS and Cushings syndrome, where loss of the late-night cortisol nadir is clinically informative, and more broadly as flatter salivary cortisol slopes or elevated evening cortisol in ageing, sleep restriction and circadian misalignment; these patterns are associated with type 2 diabetes, cardiovascular disease and mortality. C_LIO_LIFlattening of glucocorticoid rhythms in mice induces rapid and sustained hyperinsulinaemia without hypoglycaemia, indicating that circulating insulin can be elevated independently of glucose. C_LIO_LIHepatic insulin clearance, mediated in part by insulin-degrading enzyme and CEACAM1, is a major determinant of circulating insulin levels. C_LI What is the key question?O_LIHow does disruption of glucocorticoid rhythmicity increase circulating insulin while maintaining glycaemic control, and is beta cell glucocorticoid receptor signalling required for this adaptive response? C_LI What are the new findings?O_LIGlucocorticoid rhythm flattening lowers the glucose threshold for beta cell activation through enhanced Ca{superscript 2} excitability, an effect that persists in isolated islets and indicates in vivo reprogramming of beta cell function. C_LIO_LIBeta cell-specific deletion of the glucocorticoid receptor blunts the hyperinsulinaemic response to glucocorticoid flattening and produces hyperglycaemia and impaired glucose tolerance despite unchanged or improved insulin sensitivity. C_LIO_LIReduced insulin clearance, associated with decreased hepatic insulin-degrading enzyme abundance, contributes additionally to the elevated circulating insulin, but is not required for maintenance of glucose homeostasis. C_LI How might this impact on clinical practice in the foreseeable future?O_LIIdentifying beta cell glucocorticoid receptor signalling as a requirement for glucose homeostasis during disrupted glucocorticoid rhythmicity may inform strategies for understanding hyperinsulinaemia and steroid-associated metabolic dysfunction in human conditions marked by loss of the cortisol trough or flatter diurnal cortisol profiles, particularly MACS, Cushings syndrome, shift work/circadian misalignment and ageing. C_LI

Background and AimsPregnancy requires dynamic, stage-specific adaptations in maternal liver metabolism and growth to sustain fetal development while preserving systemic homeostasis. Estrogen signaling, which significantly increases during pregnancy, is primarily mediated in hepatocytes by estrogen receptor (ER). Although hepatic ER regulates female liver metabolism under non-pregnant conditions, its role in pregnancy-induced hepatic remodeling remains unclear. MethodsWe studied non-pregnant and pregnant control and liver-specific ER knockout (LERKO) mice across gestational stages using longitudinal physiological measurements, liver transcriptomics, targeted metabolomics, histological assessment of cell proliferation, and metabolic phenotyping. ResultsIn control mice, pregnancy elicited sequential hepatic remodeling characterized by early induction of cell-cycle programs, a mid-gestational peak in hepatocyte proliferation with transient suppression of selected metabolic pathways, and late reactivation of specific metabolic programs. Chronic hepatic ER deficiency alters this temporal pattern. LERKO livers showed premature activation of proliferative and anabolic transcriptional programs, changes in amino acid- and fatty acid-related metabolic pathways, and altered temporal regulation of AKT-mTORC1-related signaling. At mid-gestation, LERKO mice displayed reduced hepatocyte proliferation, altered expression of metabolic and insulin-related genes, blunted gestational glucose adaptation without overt evidence of systemic insulin resistance, and changes in the light/dark-phase metabolic patterns. ConclusionsThese findings suggest that hepatic ER is required for the appropriate stage-specific coupling of liver growth, metabolic remodeling, and insulin-responsive signaling during pregnancy. Its loss is associated with gestational hepatic maladaptation and systemic metabolic phenotypes, providing a framework for investigating estrogen-dependent mechanisms underlying pregnancy-associated metabolic and liver disorders. HighlightsHepatic ER is required for stage-specific liver remodeling during pregnancy. Loss of hepatic ER alters temporal coupling of liver growth and metabolism. LERKO mice show early changes in amino acid- and fatty acid-related pathways. Hepatic ER loss reduces proliferation and alters gestational glucose adaptation. Hepatic ER loss is associated with altered light/dark-phase metabolic organization. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/743939v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@d52bborg.highwire.dtl.DTLVardef@b27511org.highwire.dtl.DTLVardef@23b286org.highwire.dtl.DTLVardef@19d9314_HPS_FORMAT_FIGEXP M_FIG C_FIG

Background: Biological sex and endocrine signaling influence cancer biology, immune response, and therapeutic outcomes. Recent evidence suggests that testosterone signaling may exert brain context dependent protective effects in glioblastoma through the hypothalamic-pituitary-adrenal axis, reduced glucocorticoid-mediated immune suppression, and altered tumor-immune interactions. We assessed whether testosterone replacement therapy (TRT) exposure was associated with survival in solid tumor central nervous system (CNS) metastases and glioblastoma (GBM, IDHwildtype, WHO grade 4), settings in which post-diagnosis survival and TRT timing can be clinically defined. Methods: We performed a retrospective Mayo Clinic cohort study of adult patients with molecularly confirmed glioblastoma and solid tumor CNS metastases confirmed from neuroimaging reports using large language model-assisted adjudication. TRT exposure was defined by testosterone-specific prescription evidence within prespecified peri-diagnostic windows. Overall survival was evaluated using propensity score-matched Cox models, 24 month administratively censored Cox models, time-dependent Cox sensitivity analyses, and 24 month restricted mean survival time. Results: In the pooled solid tumor CNS metastasis cohort, TRT exposure was associated with improved overall survival after propensity score matching (HR 0.80, 95% CI 0.65 to 0.98, p=0.029) and a 3.22-month improvement in 24 month restricted mean survival time. In glioblastoma, TRT exposure was similarly associated with improved overall survival after propensity score matching (HR 0.56, 95% CI 0.38 to 0.82, p=0.003) and a 5.81-month improvement in 24 month restricted mean survival time. Conclusions: TRT exposure was associated with improved survival in CNS metastases and glioblastoma. These hypothesis-generating findings support prospective studies incorporating TRT timing, hormone levels, corticosteroid exposure, immune correlates, and tumor-specific stratification.

Leydig cells (LCs) are essential for male reproductive function due to their role in testosterone synthesis, a process critically dependent on mitochondrial cholesterol transport mediated by the Steroidogenic Acute Regulatory protein (StAR). Despite their importance, LCs are highly sensitive to metabolic and exogenous stressors. Ferroptosis, an iron-dependent form of regulated cell death driven by lipid peroxidation, has emerged as a key link between cellular metabolism and cell fate; however, its role in LCs and steroidogenesis remains poorly understood. In this study, we investigated the induction of ferroptosis in LCs and its impact on their steroidogenic capacity. We evaluated cellular responses to canonical ferroptosis inducers (Erastin and RSL3) alongside the transcriptional regulation of key genes. Our results demonstrate that LCs are vulnerable to ferroptotic stress, which significantly downregulates Star expression. Notably, we uncovered a novel endocrine-metabolic crosstalk: hormonal stimulation via hCG effectively rescues LCs from Erastin-induced toxicity and fully sustains maximal steroidogenesis. However, this hormone-driven cytoprotection fails against direct GPX4 inhibition by RSL3, indicating an absolute reliance on functional GPX4. These mechanistic findings highlight the paradoxical dual role of ACSL4 in Leydig cell biology and are further supported by bioinformatic analysis of public transcriptomic profiles from infertile patients, which reveal a detrimental imbalance in the ACSL4/GPX4 axis. Together, our data position ferroptosis as a critical disruptor of male endocrine function and reveal a hormone-mediated metabolic adaptation that could inform novel therapeutic strategies against oxidative stress in the testis. Highlights-Leydig cells exhibit a strong vulnerability to ferroptotic cell death. -Ferroptosis disrupts StAR expression and halts Leydig cell steroidogenesis. -hCG signaling promotes metabolic adaptation against Erastin-induced ferroptosis.

Aims/hypothesisHeterogeneity in the pathophysiology of type 2 diabetes is increasingly being realised. The currently available rodent models of type 2 diabetes all have limitations and do not accurately reflect all human type 2 diabetes subtypes. NOD.BR-H2k /Wicker mice (NODk), derived from the non-obese diabetic (NOD) mouse, are type 1 diabetes resistant. However, transgene induced beta-cell stress in male NODk mice induces hyperinsulinaemia followed by diabetes. Here we have investigated the propensity of NODk mice to develop a Western-diet (WD) induced hyperinsulinaemic subtype of type 2 diabetes. Comparator mouse strains used were BALB/c and B10.BR-H2k /SgSnJ mice (B10k). MethodsIn the longer-term studies (14-24 weeks), NODk, B10k and BALB/c mice were randomised to receive Chow or WD from 4 weeks of age, followed by serial measurement of body weight and fed-state blood glucose. IPGTT and IPITT tests were conducted at 13 weeks of age. Blood and pancreas were harvested for further analyses at 14 and 24 weeks of age, or sooner if diabetes developed (blood glucose concentrations [&ge;]20 mmol/l on two consecutive days). In the acute studies, metabolic characteristics of the three strains at 8 weeks of age, continued on Chow or after a 5-day WD challenge (WDC) were assessed, along with harvesting pancreas on day 5 for ex vivo islet insulin secretion, electron microscopy, and bulk islet transcriptomics analyses. ResultsMale WD-fed NODk mice became markedly hyperinsulinaemic, gained excess weight and developed a severe type 2 diabetes phenotype. Emergence of diabetes was associated with islet endocrine cell apoptosis and loss of beta-cell mass, without evidence of insulitis. Insulin resistance on IPITT testing, however, was not evident in Chow-fed NODk mice. In contrast, male B10k mice already had poor glucose tolerance on Chow diet and, despite having a hypoinsulinaemic phenotype, were resistant to WD-induced diabetes. BALB/c mice developed very mild glucose intolerance and hyperinsulinaemia in response to the WD. Female NODk mice were diabetes resistant. At 8 weeks of age, male Chow-fed NODk mice were mildly hyperinsulinaemic despite relative hypoglycaemia compared to the other strains. The acute 5-day WDC markedly increased hyperinsulinaemia in NODk mice. Transcriptomics analyses identified robust strain-specific differences, including altered islet cell differentiation, energy metabolism, endoplasmic reticulum to golgi vesicle transport and insulin processing. Conclusions/interpretationNODk mice, which exhibit mild hyperinsulinaemic hypoglycaemia on Chow diet and rapidly develop marked hyperinsulinaemia on WD, are type 2 diabetes prone. In contrast, B10k mice have poor glucose tolerance on Chow diet and no or limited capacity to increase insulinaemia in response to WD, are diabetes resistant. These findings support the hypothesis that hyperinsulinaemia is upstream to insulin resistance in the pathogenesis of severe insulin resistant subset of type 2 diabetes for which the WD-fed NODk mouse is a suitable new mouse model. Research in ContextO_ST_ABSWhat is already known about this subject?C_ST_ABSO_LIWhich of insulin hypersecretion and insulin resistance are upstream in the pathogenesis of the severe insulin resistant subtype of type 2 diabetes continues to be debated C_LIO_LIRodent models of type 2 diabetes do not accurately reflect all human subtypes of type 2 diabetes C_LIO_LINODk mice, derived from the non-obese diabetic (NOD) mouse, are type 1 diabetes resistant, but with transgene induction of islet beta-cell stress develop hyperinsulinaemia, followed by type 2 diabetes C_LI What is the key question?O_LICould Western-diet fed NODk mice be developed as a model of severe insulin resistant type 2 diabetes and shed light on its upstream pathogenesis? C_LI What are the new findings?O_LIMale NODk mice tend to hyperinsulinaemic hypoglycaemia on Chow diet, rapidly develop marked hyperinsulinaemia on Western-diet feeding, and then develop type 2 diabetes C_LIO_LIMale B10k mice (one of two comparator strains (B10k and BALB/c)) have poor glucose tolerance on Chow diet, limited capacity to increase insulinaemia in response to Western-diet feeding, but are resistant to develop Western-diet induced type 2 diabetes C_LIO_LIIsolated islet findings show strain differences that favour intrinsic hyper-responsiveness and hypo-responsiveness of islet beta-cells of NODk and B10k mice, underpinning their respective metabolic phenotypes C_LI How might this impact on clinical practice in the foreseeable future? O_LIThe findings are in support of the insulin hypersecretion hypothesis for severe insulin resistant type 2 diabetes, such that therapies to limit islet beta-cell hyperresponsiveness to prevent and treat this subtype of diabetes warrant investigation C_LI

AbstractAdrenocortical carcinoma (ACC) is a rare and highly aggressive endocrine malignancy originating from the adrenal cortex with limited effective treatment options. The underlying pathophysiology of ACC is uniquely characterized by abnormal steroid production and increased metabolic activity, highlighting the critical role of mitochondria in adrenal steroid hormone biosynthesis and tumor metabolism. In this study, we investigated the therapeutic potential of TR-107, a novel and highly selective small-molecule agonist targeting the mitochondrial protease ClpP. Pharmacologic hyperactivation of ClpP disrupts mitochondrial proteostasis and bioenergetics and has shown promising antitumor activity in various preclinical models. Our results demonstrated that TR-107 induces potent dose-dependent cytotoxic effects at nanomolar concentrations in ACC cell lines NCI-H295R and mACC3 as well as short-term ACC patient-derived organoid (PDO) models, markedly reducing cell viability and confluency in vitro. Metabolic analyses revealed that TR-107 significantly impaired oxygen consumption, indicating a disruption of oxidative phosphorylation and substantial attenuation of basal cellular respiration. Mechanistic studies showed dose-dependent increases in reactive oxygen species (ROS) levels and upregulation of proteins involved in mediating the ferroptotic rheostat. Pharmacokinetic assessment uncovered that TR-107 was not a substrate of the ABCB1 (MDR1/P-glycoprotein) efflux transporter, suggesting potential to overcome common multidrug resistance mechanisms. Given the importance of IGF-2 signaling in ACC, we further explored the combinatorial effects of TR-107 with IGF-1 receptor (IGF-1R) inhibitors and discovered that co-treatment produced synergistic reductions in cell viability across NCI-H295R, mACC3, and ACC PDOs. Collectively, these findings support the potential of mitochondrial ClpP hyperactivation as a promising therapeutic strategy for ACC and demonstrate that TR-107 exhibits significant antitumor activity as a monotherapy or in combination with IGF-1R inhibitors. These findings provide a strong rationale for advancing ClpP agonists into clinical development for the management of ACC.

Metabolic dysfunction-Associated Steatotic Liver Disease (MASLD) and associated dyslipidemia is a growing health issue that gives rise to cardiovascular risk. Men are more prone to development of MASLD than women. Understanding mechanisms underlying sex differences in MASLD may lead to improved prevention and treatment approaches. Cholesteryl ester transfer protein (CETP) is a lipid transfer protein that shuttles triglycerides and cholesteryl esters between blood lipoproteins and tissues. In this study investigate the impact of hepatic CETP expression on MASLD. Hepatic CETP expression (L-HuCETP) was achieved by injecting liver-targeted CETP-expressing adeno-associated virus into C57BL/6J mice. In females, L-HuCETP improved glucose tolerance, consistent with our prior clamp results in global human CETP transgenic mice. Whereas in males, L-HuCETP worsened glucose metabolism and impaired insulin signaling. Correspondingly, L-HuCETP expression reduced the expression of gluconeogenic pathway genes in females but upregulated these genes in males. In males, L-HuCETP mice exhibited increased hepatic lipid droplet accumulation, lipogenesis proteins and these changes were not observed in females. L-HuCETP expression resulted in sex-specific hepatic responses, with increased expression of inflammation and fibrosis related genes in male, but decreased expression of these genes in females. Mechanistic studies indicate that L-HuCETP had sex specific effects on transcription factors ChREBP and HNF4, which are important for glucose and lipid metabolism. Our studies suggest that sex-specific roles of L-HuCETP with regard to liver metabolic adaptation and MASLD risk in obesity, highlighting CETP-mediated pathways as potential targets for sex-specific precision medicine approaches to improve MASLD.

IntroductionPrenatal dibutyl phthalate (DBP) exposure is associated with increased risks of adverse fetal outcomes as well as metabolic and cardiovascular diseases in the offspring in a fetal sex-specific manner. However, mechanisms underlying these prenatal DBP exposure-associated adverse fetal/offspring outcomes are unclear. We hypothesize that environmentally relevant low-dose prenatal DBP exposure dysregulates fetal-placental vascular function and lipid metabolism in a fetal sex-specific manner, thereby impairing placental efficiency and programming adverse offspring metabolic outcomes. MethodsFemale CD-1 adult mice (8-10wks) were orally dosed with vehicle or an environmentally relevant low-dose DBP (0.1 g/kg/day) daily from 30 days pre-pregnancy through gestational day (GD) 18.5. Fetal-placental vascular hemodynamics of these dams were examined using high-frequency ultrasound at multiple timepoints. The effect of prenatal environmentally relevant low-dose DBP exposure on placental efficiency, spatial transcriptomic profiles, lipid homeostasis, and placental vascular endothelial cells function in male and female fetuses were evaluated at gestational day (GD) 18.5. ResultsThe prenatal low-dose DBP exposure dysregulated the fetal-placental vascular hemodynamic indices from mid-to late gestation. DBP exposure impairs placental efficiency in male, but not female placenta at GD18.5. Further, female placentas exhibited fetal labyrinth vasculature-specific transcriptomic adaptations that preserves placental efficiency and endothelial function. In contrast, male placentas exhibited minimum transcriptomic adaptation, together with compromised placental efficiency and endothelial function associated with lipotoxic lipid profile. ConclusionsIn conclusion, prenatal low-dose DBP exposure dysregulates placental vascular function and lipid homeostasis in a fetal sex-specific manner, with male fetuses being more susceptible to DBP exposure.