Frontiers in Endocrinology

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.

Delta-like protein 1 (DLK1) is a transmembrane protein involved in the regulation of cellular differentiation and stem cell maintenance in several tissues, including the pituitary gland. Although DLK1 expression has been reported in the adult pituitary, its spatiotemporal distribution during mouse pituitary development remains incompletely characterized. The aim of this study was to analyse the distribution of DLK1 during embryonic and postnatal development of the mouse pituitary gland and to characterize its relationship with hormone-producing cell populations. Immunohistochemistry was performed in Swiss albino mice from embryonic day 9.5 (e9.5) to postnatal day 15 (p15). Double immunofluorescence was used at e18.5 and p15 to examine the association of DLK1 immunoreactivity with ACTH-, TSH-, GH-, FSH- and PRL-producing cells. DLK1 immunoreactivity was detected from the earliest stages of pituitary development in Rathkes pouch and the ventral diencephalon. During embryonic development, DLK1-ir cells were widely distributed throughout adenohypophyseal and neurohypophyseal primordia and subsequently became progressively regionalized within the anterior, intermediate and tuberal lobes, as well as in the median eminence and posterior lobe. Cells displaying overlapping immunoreactivity for DLK1 and all hormone-producing cell populations analysed were observed at late embryonic and postnatal stages. Semiquantitative analysis at p15 indicated that approximately 32% of adenohypophyseal cells were DLK1-immunoreactive. These findings provide a detailed description of the spatiotemporal distribution of DLK1 during mouse pituitary ontogeny and reveal its association with differentiating endocrine cell populations throughout pituitary development.

PurposeEstrogen deficiency drives bone loss through interacting endocrine, oxidative, inflammatory and bone-remodeling disturbances. Ergothioneine (EGT) is a diet-derived thiol/thione antioxidant whose effects on the estrogen-deficient skeleton are unknown. We evaluated whether EGT, alone or combined with vitamin K2, vitamin D3 and magnesium L-threonate, attenuates the skeletal and systemic consequences of ovariectomy (OVX) in mice. MethodsForty-eight female C57BL/6J mice underwent sham surgery or OVX and received daily oral gavage for 12 weeks of vehicle, alendronate (1.53 mg/kg), EGT (30 mg/kg/day), EGT with vitamin K2 (40 {micro}g/kg/day) and vitamin D3 (500 IU/kg/day), or EGT with vitamin K2, magnesium L-threonate (350 mg/kg/day) and vitamin D3 (n = 5-6 analysed per group). Outcomes included the uterine index, tibial micro-computed tomography, distal-femoral histology, and serum bone turnover markers (CTX-I, PINP, osteocalcin), sex hormones, TNF-, IL-6, SOD and MDA. OVX lowered the uterine index and induced tibial trabecular deterioration, with increased CTX-I, decreased PINP and osteocalcin, elevated TNF- and IL-6, reduced SOD and increased MDA (all P < 0.01 vs sham). Alendronate restored tibial micro-CT bone-volume fraction (BV/TV) and trabecular number (P < 0.01 vs OVX). The EGT-based regimens did not significantly restore tibial micro-CT BV/TV, trabecular thickness or trabecular number (all P > 0.05 vs OVX), but significantly increased trabecular area on distal-femoral histology (OVX 7.6% vs 14.2-15.0% across regimens; P < 0.05 vs OVX) and lowered CTX-I, TNF-, IL-6 and MDA while raising SOD and partially restoring PINP and osteocalcin (P < 0.05-0.01 vs OVX). Because the histological and micro-CT endpoints were assessed at different skeletal sites, structural interpretation is cautious. Apparent increases in serum estradiol were assay-dependent and are regarded as exploratory. Ergothioneine-based nutritional regimens improved the systemic oxidative, inflammatory and bone-turnover environment of estrogen-deficient bone loss and preserved distal-femoral trabecular area on histology, although tibial three-dimensional microarchitecture by micro-CT was not restored. Because the histological and micro-CT endpoints were assessed at different skeletal sites, the structural interpretation is necessarily cautious. These findings support further evaluation of EGT as a dietary adjunct, with mechanistic and dose-optimization studies warranted.

Background: Prolonged unsupplemented spiritual fasting (USF), complete caloric abstinence motivated by spiritual practice, is undertaken by many communities worldwide, yet its physiological consequences remain poorly characterized. No prior study has documented continuous real-time monitoring during free-living fasting beyond 10 days. Methods: We conducted a self-controlled observational study of 23 experienced Jain practitioners undergoing USF. Seventeen completed at least 8 days of fasting (11 completed eight days, six continued to 30 days); six discontinued early. Continuous glucose monitoring (CGM) and blood biomarkers at baseline (Day-0), post-fasting (Day-9), and 60-day follow-up (Day-69) were obtained. Mood was assessed daily using PANAS. Within-participant comparisons used both parametric and non-parametric t-tests. Results: CGM revealed near-complete suppression of glycaemic variability within 24-48 hours of fasting onset, sustained throughout with no clinical hypoglycaemia in either cohort. Blood biomarkers showed transient perturbation during fasting -- including rises in hepatic enzymes, bilirubin, uric acid, creatinine, and lipid fractions -- broadly reversible by follow-up (Day-69). hsCRP rose during fasting then fell below baseline at follow-up (5.52{+/-}13.67 to 3.68{+/-}13.18 mg/L, p=0.034). HDL significantly rose above baseline (45.71{+/-}112.32 to 48.97{+/-}111.19, p=0.045) and LDL similarly declined below baseline (122.33{+/-}132.10 to 106.96{+/-}131.62 mg/dL, p=0.035); and then both significantly improved by follow-up. Thyroid axis suppression fully normalized by follow-up. Psychological wellbeing was maintained throughout. Conclusions: Extended USF produces a safely reversible pattern of acute physiological adaptation with net cardiometabolic benefit. Absence of clinical hypoglycaemia during 30-day water-only USF, documented here for the first time with CGM, provides empirical grounding for future controlled trials.

Introduction: Central regulation of energy homeostasis is essential for balancing lipid storage and reproductive investment; however, the hypothalamic factors governing this trade-off remain incompletely defined in birds. Neurosecretory protein GM (NPGM), an 83-amino acid hypothalamic factor, was investigated for its role in energy allocation during sexual maturation in Japanese quail (Coturnix japonica). Methods: Male and female quails at the onset of sexual maturation received chronic intracerebroventricular administration of NPGM for 13 days via osmotic pumps, during which their body mass, food intake, and water intake were monitored daily. At the endpoint, peripheral tissue and muscle masses, serum metabolite levels (glucose, fatty acids, triglycerides, testosterone, and 17{beta}-estradiol), hepatic triglyceride content, and gene expression profiles of hypothalamic feeding/reproductive genes and hepatic/adipose lipid metabolic genes were evaluated. Results: NPGM increased subcutaneous and abdominal fat in both sexes and was associated with suppressed gonadal maturation, as indicated by reduced testicular mass relative to body mass and lower testosterone levels in males, as well as a trend toward reduced ovarian mass and lower 17{beta}-estradiol levels in females. Sex-dependent metabolic phenotypes emerged: males exhibited increased body mass gain, hyperphagia, elevated water intake, enlarged liver, pancreas, and heart, higher serum and hepatic triglyceride levels, increased hepatic SCD1 expression, and reduced hepatic CGI-58, PPAR{gamma}, SLC2A2, and CD36. In contrast, females showed fat accumulation without hyperphagia or hepatic triglyceride elevation, accompanied by reduced hepatic VTG2 and APOV1 and decreased adipose ATGL, LPL, and FATP. Hypothalamic AGRP expression decreased in males, whereas both NPY and AGRP decreased in females. Discussion: These findings demonstrate that central NPGM shifts energy allocation from reproduction toward lipid storage through sex-dependent endocrine and metabolic mechanisms, identifying NPGM as a neuroendocrine regulator of energy allocation during sexual maturation in Japanese quails.

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

Context: Prader-Willi syndrome is a genetic neurodevelopmental disorder characterized by hyperphagia and early-onset obesity from hypothalamic dysfunction with endocrinopathies and learning disability. Management is challenging with strict control of the food environment needed. While newer glucagon-like peptide-1 receptor agonists, such as semaglutide, have efficacy in non-PWS obesity, there have been limited case reports in PWS. Objective/Design/Setting: Retrospective records review of 12 adults with PWS and overweight/obesity treated with semaglutide at a UK academic hospital centre specialist clinic. Patients: mean +/- SD age 28.3 +/- 10.1 years, 83% female, BMI 46.6 +/- 8.2kg/m2, 75% type 2 diabetes mellitus. Intervention: Median follow-up 17.2 months (range 8.7-36.1) with median semaglutide dose 2.4mg once weekly (1.0-2.4). Results: Although there was no significant weight loss on semaglutide, there was stabilisation of the weight gain prior to treatment over previous 12.4 months (7.6-23.0) (post -3.1 +/- 9.9% vs. pre +5.7 +/- 5.6%: d -0.72, P=0.037). There was a significant decrease in hyperphagia on semaglutide from hyperphagia questionnaire for clinical trials (n=11, -7.3 +/- 6.1 (max 36), d -1.19, P=0.003), having been stable before treatment. HbA1c improved in those with elevated baseline levels (n=6, -4.2 +/- 4.9%, d -0.74, P=0.13). Mild gastrointestinal side effects were seen in 25% but did not lead to discontinuation. Conclusions: In adults with PWS, semaglutide produced weight maintenance, reduced hyperphagia, and improved glycaemic control, with good tolerability. Larger placebo-controlled trials are needed to confirm these findings in adults and adolescents with PWS, especially in those without T2DM, where efficacy may be greater.

Introduction. Diabetes is a growing global health challenge, necessitating effective management strategies. Glycosylation, a highly regulated post-translational protein modification, has emerged as a pivotal factor in diabetes pathophysiology. However, the modulation of protein glycosylation by antidiabetic treatment is still largely unknown. This study explored the longitudinal effects of four distinct antidiabetic therapies - metformin, insulin, sodium-glucose cotransporter-2 (SGLT2) inhibitors, and glucagon-like peptide-1 receptor agonists (GLP-1RA) - on plasma protein and immunoglobulin G (IgG) glycosylation in patients with type 2 diabetes (T2D). Research Design and Methods. Plasma protein and IgG N-glycans were enzymatically released, purified and chromatographically profiled in a cohort of 124 patients, examined at four time points, to assess therapy-induced glycan alterations. Linear mixed models adjusting for covariates and multiple testing (FDR<0.05) were used to investigate the associations between plasma protein and IgG N-glycosylation and antidiabetic therapy. Results. Our findings reveal that metformin, SGLT2 inhibitors, and GLP-1RA induce significant alterations in IgG glycosylation, including the increased core fucosylation and galactosylation, features associated with a reduced inflammatory IgG potential. Notably, IgG monogalactosylation, previously linked to cardioprotective effects in women, was elevated in response to GLP-1RA and SGLT2 inhibitor treatments. Plasma protein glycosylation changes were more limited, with distinct alterations observed for each therapy. Metformin and GLP-1RA similarly reduced certain fucosylated and sialylated glycans, while SGLT2 inhibitors decreased a high-mannose glycan, previously positively associated with diabetes progression. Insulin therapy had a minimal effect on protein glycosylation, with only one plasma glycan significantly altered. Conclusions. Our findings emphasise the importance of protein glycosylation as a dynamic and responsive marker in T2D treatment. The distinct glycan alterations observed in response to metformin, SGLT2 inhibitors, and GLP-1 receptor agonists provide novel insights into the molecular effects of these therapies, potentially contributing to the development of glycan-based biomarkers for personalized diabetes management.

Background: Insulin resistance is a core pathophysiologic feature of metabolic disease, but its reference-standard assessment by steady-state plasma glucose (SSPG) testing is procedurally demanding and labor-intensive, limiting use in routine clinical care and large-scale research. Because OGTT glucose profiles are widely available, we aimed to develop a glucose-only metric to characterize dynamic glucose responses and estimate SSPG-measured insulin resistance. Methods: We developed the Width-Delay Index (WDI), a glucose-only OGTT metric integrating relative exposure width, delayed exposure timing, and glycemic floor. In a dataset of 32 subjects with 16-point venous OGTT profiles and paired SSPG measurements, WDI performance was assessed using leave-one-out cross-validation (LOOCV) for SSPG prediction, together with insulin-resistance discrimination and sparse-sampling robustness analyses. Results: The 15-120 min OGTT window yielded the strongest WDI performance. WDI15-120 predicted SSPG with LOOCV R2 = 0.57 (95% CI, 0.27-0.77), Pearson r = 0.77, and Spearman rho = 0.74. WDI15-120 showed higher predictive performance than standard OGTT glucose measures and insulin-derived indices, including HOMA-IR, Matsuda index, and disposition index. WDI15-120 also discriminated insulin-resistant from insulin-sensitive subjects with AUROC = 0.969. When recalculated from conventional 5-point OGTT sampling, WDI15-120 retained substantial performance, with LOOCV R2 = 0.41 and AUROC = 0.945. Conclusions: WDI provides a simple, glucose-only, physiologically interpretable approach for estimating SSPG-measured insulin resistance from OGTT glucose dynamics.

The insulin-like growth factor (IGF)/IGF-binding protein (IGFBP) axis has been implicated in diabetes mellitus and the associated burden of cardiovascular complications. Higher circulating levels of IGFBP-1 and IGFBP-2 have been established as markers of protection from incident type 2 diabetes, yet their associations with cardiovascular disease remain unclear. Utilising the UK Biobank (UKB) resource to integrate disease outcomes, plasma proteomics and MRI data, we examined associations of IGFBP-1 and IGFBP-2 with incident diabetes and cardiovascular disease. Approximately 50,000 UKB participants with plasma proteomic measurements for IGFBP-1 and IGFBP-2 were included. Multivariate Cox regression models revealed that participants in the highest quartiles of IGFBP-1 and IGFBP-2 had a substantially lower risk of incident diabetes (hazard ratio (HR) = 0.31 and 0.32 respectively), but, paradoxically, had increased risks of incident macrovascular disease, all-cause and cardiovascular-related mortality (HR = 1.81 and 2.39). Both proteins were negatively associated with HbA1c levels, triglyceride/HDL ratio and abdominal adiposity, yet positively associated with NT-proBNP, troponin I, cardiac chamber size and aortic dimensions. In summary, negative associations of IGFBP-1 and IGFBP-2 with incident diabetes mellitus did not translate to a reduced cardiovascular risk, suggesting potentially complex actions of IGFBP-1 and IGFBP-2 in the pathophysiology of cardiometabolic disease.

Glucagon is a key metabolic hormone regulating blood glucose and appetite, yet little is known about its actions within the brain. Here, we investigated its receptor (GCGR) localization in periventricular brain barrier interfaces in young rats using immunohistochemical and immunofluorescence approaches. GCGR was enriched in the proximal region of motile ependymal cilia lining the ventricles, as well as in tanycytic primary cilia and cytoplasmic extensions within the hypothalamus. Additional immunostaining was observed in ciliated cells of the subcommissural organ and, more heterogeneously, in choroid plexus epithelium and associated primary cilia, while other circumventricular organs lacked detectable GCGR. These findings identify brain cilia and tanycytes as previously unrecognized sites of glucagon receptor localization and suggest that glucagon signaling at brain barrier interfaces may contribute to integrating peripheral metabolic cues with central homeostatic circuits.

Aims: Sarcopenia and sarcopenic obesity are associated with increased risks of cardiovascular (CV) disease and mortality. This study examined the associations of body composition and daily physical activity with mortality, CV events and cancer in patients with diabetes. Methods: This prospective cohort study included patients with diabetes treated at a specialised clinic in Japan between January 2018 and March 2023. Body composition, including visceral adipose tissue (VAT), was assessed by bioelectrical impedance analysis. Daily physical activity was evaluated using the non-exercise activity thermogenesis (NEAT) questionnaire, and handgrip strength (HGS) was measured by dynamometry. Cox proportional hazards models were used to assess associations with mortality, CV events, and cancer. Results: Among 2,024 patients (mean age 63.0 years, BMI 24.6 kg/m^2, HbA1c 7.8%), NEAT, HGS, and VAT were not independently associated with all-cause mortality. Higher VAT was associated with increased cancer risk (HR 1.485; 95% CI 1.101-2.003; p = 0.009). Higher HGS was inversely associated with CV event risk (HR 0.951; 95% CI 0.919-0.984; p = 0.004). NEAT was not associated with any outcome. Conclusions: Higher VAT was associated with increased cancer risk, whereas higher HGS was protective against CV events. Incorporating body composition and HGS assessments into clinical practice may improve risk stratification and management in patients with diabetes.

Continuous glucose monitoring (CGM) is increasingly used for inpatient glycemic surveillance, but evidence in non-critical care wards remains limited, particularly in real-world public healthcare settings. Intermittent capillary glucose testing may fail to detect transient, nocturnal, or asymptomatic dysglycemia. We sought to evaluate whether CGM improves detection of clinically significant dysglycemia compared with seven-point capillary glucose monitoring in hospitalized patients with type 2 diabetes mellitus or hyperglycemia. This is a prospective, observational, non-randomized, real-world study performed in a tertiary referral center in Mexico. 56 hospitalized patients were included: 28 underwent flash CGM and 28 underwent seven-point capillary glucose monitoring. Patients were followed for up to 6 hospitalization days. The main analytical focus was detection of clinically significant dysglycemia, including hypoglycemia <70 mg/dL, clinically significant hypoglycemia <54 mg/dL, and severe hyperglycemia >250 mg/dL. Secondary outcomes included time in range, mean daily glucose, insulin requirements, infectious complications, length of stay, and mortality. CGM detected more hypoglycemia <70 mg/dL than capillary monitoring (71.4% vs 35.7%, p=0.005), more clinically significant hypoglycemia <54 mg/dL (median 3 [IQR 0-6.5] vs 0, p=0.030), and more severe hyperglycemia >250 mg/dL (median 8.5 [IQR 0.5-17] vs 0 [IQR 0-9.52], p=0.030). Time in range was not significantly different between groups (59.86 +/- 23.46% vs 69.28 +/- 24.99%, p=0.151). After adjustment for age, diabetes duration, and admission hyperglycemia, CGM remained associated with hypoglycemia detection (OR 4.7, 95% CI 1.2-19.0, p=0.027). We concluded that CGM improved detection of clinically significant dysglycemia during up to 6 hospitalization days. Although CGM did not improve time in range or short-term clinical outcomes, it provided superior glycemic surveillance compared with intermittent capillary glucose testing.

Introduction: Lipids are considered both drivers and biomarkers of cardiometabolic diseases. As glucagon-like peptide-1 receptor agonists (GLP-1RAs) are widely used for diabetes and obesity management, it is crucial to understand how they affect the related comorbidities through the circulating lipidome. This study investigated the lipidomic changes induced by liraglutide treatment when compared to placebo in people with type 2 diabetes (T2D) to characterise lipid remodelling and its association with clinical outcomes. Research design and methods: This post-hoc study analysed plasma samples using liquid chromatography-mass spectrometry (LC-MS/MS) from LIRAFLAME, a randomised, double-blind, placebo-controlled, parallel-group trial. A hundred people with T2D received up to 1.8 mg of liraglutide or placebo once daily for 26 weeks. Plasma samples were collected at baseline, week 13 and week 26. Results: Liraglutide treatment resulted in a statistically significant increase in multiple lysophospholipid subclasses, including LPCs, LPC(O)s, LPC(P)s, LPEs, and LPE(P)s, observed at 13 weeks and sustained at 26 weeks compared to placebo. These increases were not mediated by the change in BMI. Triglyceride concentrations decreased at 13 weeks, while fatty acid levels declined at 26 weeks, consistent with enhanced lipid remodelling. The increase in LPC(O)s was associated with favourable decreases in ALAT, MCP-1, and UACR, suggesting anti-inflammatory effects with hepatic, renal, and cardiovascular benefits. Conclusions: Compared to placebo, 26 weeks of liraglutide treatment resulted in a favourable lipidomic shift from a triglyceride-rich profile towards one enriched in lysophospholipids. This lipid remodelling was associated with improvements in hepatic, renal, and inflammatory markers.

Context: Polycystic ovary syndrome (PCOS) is a heterogeneous endocrine disorder involving complex interactions among endocrine, metabolic, inflammatory, and thyroid pathways. However, the systems-level organization of these interactions remains poorly understood. Objective: To reconstruct the endocrine-metabolic biomarker network in women with PCOS and identify bridge biomarkers integrating distinct physiological domains. Design: Retrospective cross-sectional study. Setting: Single tertiary referral center. Participants: A total of 1,286 women diagnosed with PCOS according to the revised Rotterdam criteria. Methods: Twenty-nine routinely measured laboratory biomarkers representing endocrine, metabolic, hematological/inflammatory, and thyroid domains were analyzed. Sparse Gaussian graphical models were estimated using Graphical LASSO with Extended Bayesian Information Criterion model selection. Network topology, node centrality, bridge centrality, bootstrap resampling, and predefined sensitivity analyses were performed. Results: The reconstructed network comprised 29 biomarkers connected by 73 conditional dependency edges (network density, 0.18), demonstrating a modular but highly integrated endocrine-metabolic architecture. Conventional centrality analysis primarily identified biomarkers organizing local physiological modules, whereas bridge-centrality analysis revealed biomarkers coordinating communication between biological domains. Sex hormone-binding globulin exhibited the highest bridge strength, followed by fasting insulin, triglycerides, and high-density lipoprotein cholesterol. Additional reproducible bridge biomarkers included free thyroxine, white blood cell count, 2-hour plasma glucose, absolute neutrophil count, androstenedione, and anti-thyroglobulin antibodies. The leading bridge biomarkers remained stable across bootstrap resampling, complete-case reconstruction, and alternative network specifications. Conclusions: PCOS is characterized by an integrated endocrine-metabolic network organized around a limited number of reproducible bridge biomarkers linking multiple physiological systems. Network analysis provides complementary systems-level information beyond conventional biomarker evaluation and may facilitate future biological phenotyping and precision medicine approaches in PCOS.

The coexistence of obesity and insulin resistance is associated with elevated plasma amino acid concentrations. However, it remains unclear whether adiposity or insulin resistance is the stronger determinant of plasma amino acid dysregulation in this setting. Twenty-two adults (10 women, 12 men) spanning a broad range of body mass index (BMI) and insulin resistance underwent a 75-g oral glucose tolerance test (OGTT) after an overnight fast. Plasma glucose, insulin, and amino acid concentrations were measured serially, and insulin resistance/sensitivity was estimated from OGTT-derived glucose and insulin responses, using the homeostasis model assessment of insulin resistance (HOMA-IR) and the Matsuda insulin sensitivity index (Matsuda-ISI). Principal component analysis (PCA) of fasting plasma amino acid concentrations showed no clear separation by obesity or insulin resistance classifications. In contrast, PCA of OGTT-stimulated plasma amino acid concentrations revealed clearer clustering by BMI, fat mass, and waist circumference, whereas separation by HOMA-IR and Matsuda-ISI was less distinct. Importantly, regression analyses showed that BMI, fat mass, and waist circumference were significant predictors of OGTT-stimulated, but not fasting, amino acid responses, with waist circumference accounting for the greatest proportion of the variance in branched-chain amino acid responses during the OGTT (R2 = 0.54). In conclusion, measures of adiposity, particularly total fat mass and waist circumference, accounted for a greater proportion of the variance in plasma amino acid responses under physiologically stimulated conditions than indices of insulin resistance. These findings support the view that plasma amino acid concentrations reflect adiposity-related metabolic alterations more strongly than insulin resistance.

Fructooligosaccharides (FOS) are prebiotic fibers that influence gut microbiota and host metabolic function. In a diet-induced obesity (DIO) mouse study, FOS supplementation was compared with PBS-treated obese controls. Blood glucose was markedly lower at Day 42 (221.9 {+/-} 7.8 vs 138.3 {+/-} 9.0 mg/dL), and remained lower at Day 56. FOS reduced body-weight gain from 8.4 {+/-} 0.9 g in PBS controls to 2.6 {+/-} 0.2 g, corresponding to an approximate 69.5% reduction in gain over Days 1-70. Cumulative feed consumption was not significantly different between PBS and FOS cages, suggesting that the observed metabolic effects were not explained simply by reduced food intake. These data support our thesis that FOS works as an active metabolic ingredient acting through the gut-liver-metabolic axis. Thus, in the present study, dietary FOS supplementation produced marked improvements in glucose homeostasis in a severe DIO model characterized by diabetic-range hyperglycemia that more closely resembles poorly controlled human type 2 diabetes. HIGHLIGHTSO_LIFructooligosaccharide (FOS) normalized glucose levels in a severe DIO model that mimics poorly controlled human type 2 diabetes. C_LIO_LIDay-42 blood glucose was reduced by [~]37.7% in FOS-treated DIO mice. C_LIO_LIFOS reduced body-weight gain by [~]69.5% versus controls over 70 days. C_LIO_LIMetabolic benefits occurred without a statistically significant reduction in feed intake. C_LIO_LIFindings support a gut-liver-metabolic mechanism rather than simple caloric restriction. C_LIO_LIData position FOS as an active metabolic ingredient with potential utility in diabetes and metabolic health. C_LI

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.

The satiety cascade includes post-prandial increases in plasma glucose and insulin, and changes in appetitive gut hormones, including decreases in orexigenic stomach-derived ghrelin, and increases in liver-foregut-derived LEAP2 hormone, a ghrelin antagonist. However, which contribute to post-prandial attenuation of eating behaviour with increasing preload meal size is unclear. In a randomised, single-blinded study, adults without obesity attended four visits, consuming 750mL liquid preloads 0, 600, 900, 1200 kcal, with assay of plasma glucose, acyl ghrelin (AG), LEAP2 and serum insulin over 0-3h with appetite ratings (n=17, 65 visits). At 2h a virtual portion size creation task measured desired food intake (n=15, 58 visits, after data exclusion), and at 3h an ad libitum meal measured actual food intake (n=11, 43 visits). The greater the pre-load meal size, the greater the post-prandial increase in plasma glucose and serum insulin, and greater the decrease in plasma AG and AG/LEAP2 ratio, associated with greater attenuation of eating behaviour (appetite ratings, desired/actual food intake). The strongest correlations of eating behaviour with blood measures were for serum insulin, plasma AG and AG/LEAP2 ratio, with weaker results for plasma glucose. Although there was a weak correlation of post-prandial plasma LEAP2 with pre-load meal size but not with eating behaviour. Post-prandial increases in serum insulin and decreases in plasma AG and AG/LEAP2 ratio appear part of the satiety cascade related to preload meal size in adults without obesity. Future studies should investigate potential mediation of satiety in this context via other appetitive gut hormones, and in obesity.

BackgroundExercise is an effective way to improve metabolic health, and the modulation of adipose tissue (AT) secretory functions may play a significant role in this process. AT produces various lipokines, including fatty acid esters of hydroxy fatty acids (FAHFA), which increase insulin sensitivity and have anti-inflammatory effects. While factors such as sex, age, obesity, and genetics influence FAHFA levels, their impact on exercise-induced FAHFA regulation remains unclear. MethodsFirst, sex-specific responses to an acute bout of exercise were assessed in wild-type (WT) and ADTRP-deficient (ADTRP KO) mice. Fasted mice underwent acute treadmill exercise until exhaustion, followed by analysis of non-esterified fatty acids in plasma, ex vivo lipolysis in the presence or absence of a hormone-sensitive lipase (HSL) inhibitor, and FAHFA release from AT (measured by LC-MS). Second, obese male WT and ADTRP KO mice fed a high-fat diet underwent 7 weeks of regular treadmill exercise (5 days/week), after which parameters of glucose homeostasis, plasma and AT FAHFA levels, and AT lipid profiles were analyzed. ResultsAcute exercise-induced increases in plasma non-esterified fatty acid levels, AT lipolysis, and FAHFA release from AT explants were more pronounced in male mice of both genotypes. Conversely, pharmacological inhibition of HSL using BAY 59-9435 increased FAHFA release from AT explants only in females. In obese sedentary ADTRP KO mice, insulin sensitivity was improved compared with their WT counterparts. Although regular exercise suppressed weight gain in obese animals of both genotypes, insulin sensitivity improved only in WT mice. Chronic exercise generally had no effect on plasma FAHFA levels in mice fed ad libitum; however, in WT mice, it increased the levels of FAHFA-containing triacylglycerol estolides, which were associated with improved insulin sensitivity. ConclusionsAcute exercise revealed sex-specific differences in AT lipolysis and FAHFA metabolism, with HSL playing an important role in FAHFA hydrolysis. Chronic exercise in obesity increases insulin sensitivity and FAHFA storage in AT; however, this effect is absent in ADTRP KO mice, which exhibit elevated FAHFA levels in AT, a condition associated with improved insulin sensitivity even in non-exercising animals.