Vitamin D deficiency is highly prevalent in alcohol-associated liver disease and may contribute to metabolic and inflammatory dysregulation in alcoholic steatohepatitis (ASH). To examine the hepato-metabolic actions of vitamin D receptor (VDR) activation in ASH, we evaluated calcipotriol, a VDR agonist, in male C57BL/6N mice fed a 5% ethanol-containing Lieber-DeCarli diet. Calcipotriol (20 µg/kg) reduced intra-hepatic triglyceride accumulation and serum alanine aminotransferase activity, indicating attenuation of alcohol-induced liver injury. Integrated transcriptomic, metabolomic, and biochemical analyses showed that VDR agonism suppressed hepatic lipogenic programs, including de novo lipogenesis, and improved alcohol-induced metabolic derangements. Calcipotriol also reduced oxidative injury and endoplasmic reticulum stress, as evidenced by lower hepatic protein carbonyl content and reduced p-eIF2α, XBP1s, CHOP, ATF4, and BiP expression, together with diminished inflammasome-associated inflammatory signalling. Metabolomic profiling further showed partial restoration of hepatic metabolic homeostasis by calcipotriol, as evidenced by increased choline, uridine monophosphate, and taurine levels. These findings identify calcipotriol as an endocrine-metabolic modulator of ASH and support VDR activation as a mechanistically relevant therapeutic strategy for alcohol-induced liver injury.
Intracellular signalling is commonly represented as linear pathways that connect receptors to downstream effectors. While such models have been instrumental in defining signalling cascades, they fail to capture the spatial organisation that underlies signalling specificity in living cells. The cyclic adenosine monophosphate (cAMP) pathway provides a well-established example of this principle, where signalling is compartmentalised into nanometre-scale domains that generate highly localised and functionally distinct responses. However, a comprehensive framework for defining the molecular composition, spatial organisation, and functional outputs of these signalling domains and how they adapt to perturbations remains lacking. Here, we discuss how integrative proteomics approaches can be used to reconstruct the subcellular cartography of compartmentalised signalling networks. By combining isoform-specific interactomics, quantitative phosphoproteomics, network analysis, and spatial annotation, individual signalling platforms can be mapped within their native intracellular context. We introduce phosphoproteome-interactome networks (pPINs), a systems-level framework that integrates molecular interactions, subcellular localisation, and phosphorylation responses to define phosphodiesterase (PDE)-centred signalling platforms and their associated signalling outputs. Using PDE3A isoforms as an example, we illustrate how pPINs uncover multiple spatially distinct cAMP signalling nanodomains in cardiac myocytes and reveal previously unrecognised biology, including a nuclear PDE3A2/SMAD4/HDAC-1 platform that locally constrains PKA activity and suppresses prohypertrophic gene expression. More broadly, pPINs provide a conceptual and computational framework for resolving the spatial architecture of intracellular signalling networks and establish a foundation for precision therapeutic strategies targeting discrete signalling microenvironments.
GRAPHICAL ABSTRACT: ABSTRACT:Adropin is a novel protein that regulates energy homeostasis. Serum and follicular fluid (FF) levels of adropin are decreased in women with polycystic ovarian syndrome (PCOS); however, its role in ovarian function remains unknown. The aims of this study were to determine the expression of adropin and its receptor G protein-coupled receptor 19 (GPR19), in human granulosa cells (GC), its immunolocalization, and its in vitro effects on GC function. Blood plasma, FF, and GC samples were obtained from normal-weight, obese, and women diagnosed with or without PCOS (n = 8). The in vitro effects of adropin on GC proliferation, apoptosis, cell cycle progression, and steroidogenesis were analyzed. The results revealed that adropin plasma concentration was decreased in obese patients, with a similar reduction observed in obese patients with PCOS, whereas GPR19 expression was decreased in the GC of obese and PCOS women, as well as in obese patients with PCOS. We noted that in all investigated patient groups, adropin reduced GC proliferation and cell cycle progression, negatively influenced steroidogenic enzyme levels, and promoted apoptosis. Such disruptions in GC function are likely to impair ovarian follicular maturation and contribute to the subfertility commonly observed in PCOS. These alterations may ultimately affect oocyte competence and ovarian responsiveness, parameters that are clinically relevant for in vitro fertilization outcomes. Our findings suggest that adropin may act as a novel regulator of ovarian function and could contribute to the pathophysiology of PCOS, highlighting its potential clinical value as a marker of altered ovarian follicular function in affected women.
GRAPHICAL ABSTRACT: ABSTRACT:Endocrine-disrupting pesticides (EDPs) exert deleterious effects on the endocrine system, with documented evidence implicating specific pesticides in endocrine disruption, resulting in developmental delays during puberty and thyroid gland dysfunction, thereby increasing susceptibility to metabolic diseases. The disruption of intracellular insulin signaling, which is characterized by redox imbalance, toxicological effects, and pro-inflammatory activity, facilitates cellular adaptation to stress. However, this adaptive response can aberrantly induce a dysfunctional feedback loop characterized by diminished cellular insulin responsiveness, a prevalent feature of metabolic disorders. Despite significant advancements in the scientific understanding of EDPs, substantial knowledge gaps and uncertainties persist, impeding progress toward improved health outcomes. This review highlights current findings on the metabolic toxicity of pesticides in the context of obesity and diabetes, concentrating on crucial signaling pathways and a mechanistic perspective that offers insight into resistance channels as reviewed. These findings enhance our understanding of the potential impacts of EDPs on human health.
Myogenesis is a tightly regulated process by a cascade of well-coordinated events and pathways. In addition to the known myogenic regulatory factor (MRF) family, including Myf5, MyoD, myogenin, and MRF4, our previous study identified that BAMBI is involved in muscle regeneration and siRNA-mediated BAMBI knockdown impairs C2C12 myoblast differentiation. However, the potential role of BAMBI in determining cell lineage and fate specification in C2C12 myoblasts remains unexplored. In this study, we analyzed the RNA-seq data and discovered that BAMBI expression was dynamically regulated during the proliferation and differentiation period of C2C12 myoblasts. By establishing a cell line with the loss of function and gain of function of BAMBI in C2C12 myoblasts, we demonstrated that BAMBI deletion totally abrogated myogenic differentiation of C2C12 myoblast cells. In addition, loss of BAMBI converts C2C12 myoblasts to brown adipocytes through downregulation of MyoD and upregulation of Prdm16. Conversely, BAMBI overexpression strikingly enhanced myogenic differentiation of C2C12 myoblasts by upregulating MyoD expression. Taken together, our results establish that BAMBI is a vital myogenic factor involved in myoblast differentiation and a key regulator governing the balance between myogenic and adipogenic lineages in C2C12 myoblast cells.
Obesity is characterized by chronic inflammation and impaired glucose homeostasis. Regulatory T cells (Tregs) preserve immunometabolic balance, and their reduction contributes to metabolic disturbances. Although IL-2/anti-IL-2 complex (IL-2C) and hyperbaric oxygen (HBO) therapy expand Tregs, their combined effects on obesity remain unclear. Male C57BL/6 mice were fed a low-fat diet (LFD) or a high-fat diet (HFD) for 16 weeks and treated with IL-2C and/or HBO. Both interventions significantly reduced HFD-induced body weight (9-22%, P < 0.05) independent of caloric intake, with no significant changes in LFD groups. Glucose tolerance and insulin sensitivity improved, showing significant reductions in IPGTT and IPITT area-under-the-curve values (20-25% and 30-35%, respectively; P < 0.05) compared with the HFD control. Combination therapy produced the most consistent metabolic improvements. Histological analysis demonstrated reduced adipocyte hypertrophy and crown-like structures in epididymal adipose tissue (P < 0.05). Treatments restored CD4+FoxP3+ Tregs, suppressed pro-inflammatory M1 macrophages, and decreased hypoxia-inducible factor-1α expression (P < 0.05). Consistent with these findings, histological examination revealed tissue-specific structural remodeling, characterized by restored multilocular adipocytes in iBAT and reduced adipocyte hypertrophy in iWAT under HFD conditions. These findings suggest that IL-2C and HBO improve immunometabolic dysfunction associated with obesity, in parallel with favorable adipose tissue remodeling and metabolic adaptation. Thus, IL-2C and HBO may represent complementary therapeutic strategies for enhancing metabolic health.
The vitamin D receptor (VDR) is a ligand-activated nuclear receptor that mediates the biological actions of vitamin D and is a critical regulator of mineral homeostasis, cellular differentiation, immune function, and metabolism. VDR is a high-affinity intracellular binding protein for the most active vitamin D metabolite, 1,25-dihydroxyvitamin D3 (1,25D). Early biochemical and molecular studies established VDR as a member of the nuclear receptor superfamily, functioning as a transcription factor that heterodimerizes with the retinoid X receptor and binds vitamin D response elements to regulate gene expression. Since the cloning of the VDR gene in the 1980s, characterization of its structural domains and identification of coregulators significantly advanced understanding of its genomic mechanisms of action. Over the past several decades, research has expanded the scope of VDR biology beyond classical calcium and phosphate metabolism. Genome-wide binding analyses and transcriptomic studies have revealed extensive VDR cistromes and context-dependent gene networks across diverse tissues. These advances have positioned VDR as a key factor linking vitamin D availability to tissue-specific outcomes. Despite substantial progress, fundamental questions remain, including mechanisms governing tissue-specific VDR actions, integration of genomic signaling pathways, and the role of VDR in complex diseases, such as cancer, autoimmune disorders, and aging. In addition, how VDR function is modulated by chromatin context, metabolic state, and the microbiome remains incompletely understood. Here, we summarize what is known about these actions of VDR and its history of discovery. Addressing these questions will be essential for translating mechanistic insights into improved therapeutic strategies targeting the vitamin D axis.
Diabetes mellitus (DM) is characterized by chronic metabolic stress that promotes oxidative damage, genomic instability, and premature cellular ageing, with adipose tissue senescence being a pivotal contributor to metabolic dysfunction. Yet, the impact on DNA damage repair (DDR) and telomere maintenance in adipose tissue remains poorly defined. This study investigated DDR capacity, telomere integrity, and the senescence-associated secretory phenotype (SASP) in adipose tissue and adipose tissue-derived stromal cells (ADSCs) under diabetic conditions. Using an obese diabetic (ob/ob) mouse model, we confirmed whole-blood telomere shortening, significant adipose tissue hypertrophy, metabolic dysregulation, and elevated DNA damage, evidenced by increased γH2AX-positive staining. In vitro, ADSCs exposed to a diabetic microenvironment (AGEs and TNFα) exhibited increased reactive oxygen species and DNA damage without a corresponding activation of DDR pathways, as indicated by unchanged PARP1 levels and broad downregulation of key DNA repair genes, including sensors (ATM, ABL1, RAD17) and effectors across the MMR, NER, HR, and NHEJ pathways. This impaired genomic surveillance was accompanied by premature cellular senescence and a significant repression of genes involved in telomere protection (shelterin complex), telomerase activity, and telomere maintenance, together with marked telomere shortening following prolonged exposure. Furthermore, diabetic conditions increased the secretion of pro-inflammatory cytokines, chemokines, and growth factors. Collectively, these findings demonstrate that the diabetic microenvironment is associated with maladaptive DDR responses, telomere dysfunction, cellular senescence, and a pro-inflammatory secretory phenotype. This study highlights compromised genomic maintenance as a potential key mechanism underpinning adipose tissue dysfunction in DM and emphasizes the need for future investigations into the mechanisms underlying dysregulated DDR and telomere biology.
Ion fluxes, regulated by membrane potential (Vm), are vital to white fat adipocyte (WFA) function and adipogenesis. These processes present a novel target to intervene in obesity. To explore this possibility, we applied transcriptomics, immunohistochemistry, electrophysiology, and adipogenesis assays alongside siRNA and pharmacological tools to identify the chloride-permeable channel(s) that determine the Vm of WFA and to test whether those channels - specifically SWELL1 (Lrrc8 family), TTYH2/3, and EAAT1 - regulate adipocyte differentiation and adipogenesis. RNA sequencing and qPCR confirmed the existence of Lrrc8a, Lrrc8b, Lrrc8c, Lrrc8d, Ttyh2, Ttyh3, Slc1a1, and Ano1 transcripts across different primary fat depots of rats and mice, as well as a subset within 3T3-L1 adipocytes. The corresponding protein products, SWELL1 (Lrrc8x), TTYH2/3 (Ttyh2/3), and EAAT1 (Slc1a1), were found in both the plasma membrane and cytoplasm of primary and differentiated 3T3-L1 adipocytes, whereas TMEM16A protein was only in primary adipocytes and undifferentiated 3T3-L1 pre-adipocytes. Functional studies showed that although siRNA knockdown or pharmacological blockade of SWELL1, TTYH2/3, or EAAT1 proteins did not affect adipocyte Vm or electrophysiological characteristics, SWELL1 and TTYH2/3 knockdown significantly suppressed adipocyte differentiation and adipogenesis. Together, these findings reveal a role for SWELL1, TTYH2/3, and EAAT1 in adipogenesis and highlight their potential as targets for therapeutic strategies in obesity management. Although the precise chloride permeability responsible for adipocyte Vm remains to be defined, this work advances our understanding of adipocyte biology.
This study aims to provide a comprehensive overview of age- and Tanner stage-specific reference intervals (RIs) for testosterone, estradiol, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and sex hormone-binding globulin (SHBG) in children and adolescents and to highlight the importance of age- and Tanner stage-specific RIs for clinical practice. A literature review was performed to identify studies reporting RIs on testosterone and estradiol quantified using GC-MS/MS or LC-MS/MS and LH, FSH, and SHBG quantified using immunoassays in healthy individuals aged 0-18 years, categorized by age and/or Tanner stage. Sample sizes were evaluated according to the Clinical Laboratory Standards Institute (CLSI) guideline CLSI EP28-A3c, with ≥120 samples per group being acceptable, 39-119 samples being poor, and ≤38 samples being unacceptable. Analytical methods and statistical approaches were carefully documented. Fourteen studies were included for testosterone, seven for estradiol, thirteen for LH and FSH, and nine for SHBG. The results revealed considerable variability in the availability and quality of RIs, with many studies lacking adequate sample size or appropriate partitioning. Testosterone, estradiol, LH, and FSH RIs demonstrated significant changes during mini-puberty and puberty, with clear sex-specific patterns. SHBG RIs fluctuated throughout childhood and adolescence. This literature review demonstrates that RIs for HPG axis hormones show significant changes during mini-puberty and puberty, although data using appropriate analytical methods, using sufficient sample sizes, and reporting on age categories, Tanner stage, and menstrual cycle phase are scarce. Robust age- and Tanner stage-specific RIs are essential to improve diagnostic accuracy and individualized care in children and adolescents.
Polycystic ovary syndrome (PCOS), a common endocrine-metabolic disorder, lacks effective therapeutic options. Granulosa cell (GC) apoptosis and mitochondrial dysfunction are critical drivers of ovarian dysfunction in PCOS, yet targeted therapies are scarce. Scutellarin, a bioactive flavonoid, is a promising but unexplored candidate for treating PCOS. We investigated scutellarin's effects in a dehydroepiandrosterone (DHEA)-induced PCOS mouse model and in DHEA-treated human granulosa-like KGN cells. We assessed metabolic and reproductive parameters, ovarian histology, and fertility and examined molecular mechanisms using transcriptomics, qRT-PCR, and western blotting. Apoptosis and mitochondrial function were evaluated via TUNEL staining, flow cytometry, and real-time mitochondrial assays. We found that scutellarin treatment was associated with improved metabolic phenotypes in PCOS mice, including glucose intolerance and insulin resistance, a normalized estrous cycle, lower serum testosterone and luteinizing hormone levels, better ovarian morphology, and enhanced fertility. Mechanistically, scutellarin correlated with reduced ovarian GC apoptosis and modulation of BCL2, BAX, and cleaved caspase-3. Transcriptomic analysis identified the PI3K/Akt signaling pathway as a key mediator, and scutellarin dampened its abnormal activation in DHEA-induced PCOS in vivo and in vitro. Furthermore, scutellarin was associated with improved mitochondrial function in DHEA-treated KGN cells, evidenced by reduced ROS production and restored membrane potential. We conclude that scutellarin is associated with the amelioration of metabolic and reproductive abnormalities in a PCOS mouse model, correlating with reduced GC apoptosis and improved mitochondrial function. By modulating these cellular defects, scutellarin offers potential dual benefits on ovarian and systemic dysfunctions, highlighting its value for clinical investigation and providing novel mechanistic insights.
Metabolic dysfunction-associated steatotic liver disease (MASLD), previously known as non-alcoholic fatty liver disease (NAFLD), remains a major health concern worldwide. Hepatic steatosis manifests by the aberrant accumulation of lipids in hepatocytes. We have previously shown that pharmacological inhibition of mTOR complex 1 (mTORC1) by rapamycin, a widely utilized potent immunosuppressant, induces MASLD under normal conditions. Notably, this phenotype was found exacerbated in mice with genetic or pharmacological inhibition of the master transcriptional regulator of energy metabolism, nuclear receptor ERRα. In this study, we show that combining antimalarial drug chloroquine with rapamycin attenuates the severity of hepatic lipid deposition observed with rapamycin monotherapy. Bulk mRNA-seq profiling showed that chloroquine co-injection reverses the upregulation of a large proportion of genes linked to lipid metabolism homeostasis found induced by rapamycin alone. Interrogation of these genes for direct transcriptional regulators identified ERRα among top candidates. Using a mouse model with genetic ERRα ablation, we demonstrate a crucial dependency on ERRα activity for the observed amelioration of rapamycin-induced hepatic steatosis by chloroquine addition. In ERRα-null liver, chloroquine failed to reverse and, in some instances, aggravated the upregulation of lipid metabolism genes by rapamycin, with evidence linking the impaired management of hepatic lipid overload to the underlying mitochondrial dysfunction. Together, these findings underscore a critical role of ERRα in reversing MASLD.
1,25-Dihydroxyvitamin D3 (1,25(OH)2D3), parathyroid hormone (PTH), and fibroblast growth factor 23 (FGF23) are endocrine regulators of calcium homeostasis. To assess the contribution of 1,25(OH)2D3-mediated renal calcium reabsorption to systemic calcium balance and bone homeostasis, we generated mice with a targeted deletion of the vitamin D receptor (VDR) in renal tubules (VdrRen- mice), weaned them onto diets containing 1% or 0.2% calcium, and examined their phenotype at 8 weeks of age. Despite higher urinary calcium excretion, VdrRen- mice maintained normocalcemia, suggesting compensatory adaptations in the intestine and bone. Transcript levels of intestinal calcium transporters were higher in male VdrRen- mice, particularly under dietary calcium restriction, whereas genotype-dependent changes were minimal in females. Bone mass was lower in VdrRen- mice of both sexes than in diet-matched wild-type littermates. On the 1% calcium diet, changes in calcium absorption and bone remodeling occurred without alterations in serum PTH and 1,25(OH)2D3, while FGF23 was elevated. Calcium restriction increased circulating PTH, 1,25(OH)2D3, and FGF23, with additional genotype- and sex-dependent effects on 1,25(OH)2D3 and FGF23. Together, these findings demonstrate that renal VDR signaling contributes to serum calcium conservation, and its loss triggers sexually dimorphic compensatory mechanisms in intestinal calcium absorption, especially under conditions of dietary calcium restriction.
Controlled ovarian hyperstimulation (COH) is essential in assisted reproductive technologies, although some studies have associated it with reduced reproductive success. We have recently demonstrated that COH increases circulating ghrelin; however, its role in COH-induced alterations and the potential therapeutic impact of its inhibition remain unclear. Using a murine model, this study evaluated the effects of COH on preimplantation embryo development and implantation and examined whether the COH-induced alterations in these parameters are associated with hyperghrelinemia. Here, we evaluated hormonal profiles, gamete and embryo quality, implantation, and decidual characteristics in female mice, either in natural estrous cycles (controls) or following hyperstimulation, with or without treatment with ghrelin receptor antagonists ((D-Lys3)-GHRP-6 or PF-5190457). We found that COH increased ovulation rate and plasma progesterone and ghrelin levels compared with controls, without altering oocyte quality or early estradiol concentrations, although estradiol rose later (gestational day (GD) 7.5). COH also induced a delay in embryo development (on GD 3.5), which was reversed by the administration of (D-Lys3)-GHRP-6 or PF-5190457. COH showed a non-significant trend toward lower implantation, unrelated to hyperghrelinemia. COH did not affect decidual histology, embryotropic or embryotoxic factors, or uterine leukocyte infiltration; however, it reduced uterine IL-6 expression, which was restored by ghrelin receptor blockade, suggesting a mechanism for the delayed embryo development. Thus, COH-induced hyperghrelinemia negatively affects embryo development, potentially altering the uterine immune microenvironment.
Mitochondrial dysfunction driven by chronic hyperglycemia is a hallmark of diabetes, yet how this metabolic stress communicates pathological signals beyond individual cells remains poorly understood. In this study, we identified a novel mechanism linking chronic hyperglycemia to systemic metabolic impairment through ROS-mediated extracellular release of structurally intact mitochondria and mitochondrial DNA (mtDNA). In HepG2 cells exposed to high glucose (HG), extracellular release of structurally intact mitochondria was visualized by co-staining of mitochondria and the plasma membrane, together with electron microscopy. Mitochondria-enriched fractions isolated from culture supernatants were further quantified using flow cytometry and qPCR. Cell-free mtDNA (cf-mtDNA) was visualized with co-staining of mitochondria and double-stranded DNA, isolated through differential centrifugation and ultrafiltration, and quantified by qPCR. We demonstrate that HG stimulates the release of exosome-enclosed mtDNA and fragmented cf-mtDNA. Concurrently, HG induces mitochondrial dysfunction and markedly increases mitochondrial ROS (mtROS). Treatment with MitoTEMPO, a mitochondria-targeted ROS scavenger, significantly reduced HG-induced extracellular release of mitochondria and mtDNA, supporting the ROS dependence of this process. In diabetic mice, we detected elevated circulating mtDNA copy number and pronounced mitochondrial dysfunction in liver and muscle, including reduced ATP production, mitochondrial swelling, cristae disruption, and elevated MDA levels. Resting metabolic rate was markedly decreased, indicating impaired systemic respiratory metabolism. Serum analyses revealed increased 8-OHdG, pyruvic acid, GDF-15, and FGF-21, along with reduced FT3, reflecting severe oxidative stress and mtDNA damage. These findings uncover a novel mechanism in which hyperglycemia-induced ROS drive mitochondrial extrusion, potentially linking metabolic stress to systemic metabolic deterioration.
The development of type 2 diabetes mellitus is closely associated with mitochondrial dysfunction of pancreatic β-cells, but the mechanisms by which glucagon-like peptide-1 receptor activation preserves mitochondrial homeostasis under glucotoxic conditions remain incompletely understood. Herein, we investigated whether Exendin-4 protects β-cells against chronic high glucose (HG)-induced mitochondrial injury by regulating the cAMP/PKA/Drp1 signaling pathway. INS-1 β-cells, pancreatic tissues from db/db mice, and isolated primary islets were used to assess oxidative stress, apoptosis, mitochondrial function and morphology, insulin secretion, and cAMP/PKA/Drp1 signaling. Prolonged HG exposure increased oxidative stress and apoptosis, impaired mitochondrial membrane potential, elevated mitochondrial ROS accumulation, reduced ATP content, and promoted mitochondrial fragmentation in INS-1 β-cells. These changes were accompanied by increased Drp1 expression, reduced cAMP levels and PKA activity, decreased inhibitory phosphorylation of Drp1 at Ser637, and increased Ser616 phosphorylation. Exendin-4 attenuated HG-induced oxidative stress and apoptosis, restored mitochondrial function, improved mitochondrial morphology, and partially restored Drp1 Ser637 phosphorylation, whereas it did not significantly affect HG-induced Ser616 phosphorylation. In db/db mice, Exendin-4 improved metabolic parameters and alleviated β-cell apoptosis, with partial recovery of Drp1 Ser637 phosphorylation in pancreatic islets. Furthermore, glucose-stimulated insulin secretion assays in isolated primary islets showed that Exendin-4 improved β-cell secretory function in islets isolated from db/db mice. Pharmacological inhibition of PKA with H89 attenuated Exendin-4-induced Drp1 Ser637 phosphorylation and mitochondrial protection. Collectively, these results suggest that Exendin-4 protects pancreatic β-cells against HG-induced mitochondrial dysfunction and β-cell injury by restoring PKA-associated inhibitory phosphorylation of Drp1 at Ser637 and improving mitochondrial dynamics.
Gonadotropin-releasing hormone (GnRH) neurons regulate the hypothalamic-pituitary-gonadal (HPG) axis and are required for puberty onset and reproductive competence. However, the transcriptional regulators governing GnRH neuron specification and migration remain poorly defined. The homeodomain transcription factor DLX5 is expressed in fetal human GnRH neurons, its expression precedes that of GNRH1 in human pluripotent stem cell (hPSC)-derived GnRH neurons, and in mice, it serves as a guidance cue for GnRH neuron migration. We hypothesized that DLX5 may act as an upstream regulator of human GnRH neuron fate specification and migratory capacity. Using CRISPR activation, we upregulated DLX5 during FGF8b-directed differentiation of hPSCs to GnRH neurons via dual SMAD inhibition and Notch inhibition, as previously described. DLX5 activation increased neural progenitor motility (P < 0.001), upregulated FGF8 (P < 0.05), and induced GABAergic markers, including GAD1 and GAD2. Notably, DLX5 activation induced GNRH1 in the absence of exogenous FGF8b (P < 0.05), suggesting that in GnRH neurons, DLX5 regulates FGF8. When combined with exogenous FGF8b, DLX5 activation produced distinct neuronal patterning accompanied by upregulation of extracellular matrix genes, such as SPARC, which has been implicated in neurite outgrowth. Collectively, these data indicate that activation of DLX5 promotes GnRH neurogenesis from hPSCs, by driving GABAergic fate, inducing FGF8, and remodeling the extracellular matrix.
Primary aldosteronism (PA) is commonly caused by somatic and germline mutations in various genes. Patients with unilateral PA may display classical or nonclassical histopathologic features in adrenal tumors. Somatic and germline mutations are linked to classical unilateral and familial PA, respectively. In this study, we aim to search for novel gene mutations causing nonclassical lateralizing PA. Surgically resected adrenal tumor tissues from patients enrolled in a PA cohort were subjected to histopathological evaluation. Genetic analyses were performed for genomic DNA isolated from peripheral blood leukocytes and CYP11B2 immunohistochemistry-guided macrodissected adrenal tissues to identify novel variants. A PA patient with a 1.8 cm mass in the right adrenal gland underwent laparoscopic right adrenalectomy and achieved complete biochemical success at 12 months, but partial clinical improvement. Post-adrenalectomy tissues displayed the nonclassical histopathologic feature with multiple aldosterone-producing micronodules (mAPMs), but no solitary aldosterone-producing adenoma. Genetic analyses identified a heterozygous CLCN2 germline variant, c.1087C>T (p.R363C), that was likely pathogenic based on bioinformatic evaluations. In vitro biochemical, electrophysiological, and hormone assays revealed that the human ClC-2 R363C Cl- channel displayed significant gain-of-function phenotypes, including enhanced protein stability and cell surface expression, higher channel open probability at physiological membrane potentials, and increased aldosterone production in human adrenocortical cells. The dominant gain-of-function effect of ClC-2 R363C was also observed when we co-expressed the variant with its wild-type counterpart. Altogether, we report the first association of gain-of-function CLCN2 germline mutation with nonclassical mAPMs, providing new insights into the pathogenic mechanisms of PA.
Prediabetes and Type 2 Diabetes represent major global health challenges and have escalated to pandemic levels. Adipose tissue functions as a critical endocrine organ, playing a central role in maintaining glucose homeostasis during fasting, feeding, and stress responses. In this study, we demonstrated that prolonged chronic hyperinsulinemic stress increases the burden of senescent adipocytes, accompanied by activation of the cGAS-STING signalling pathway. Chronic hyperinsulinemia-induced insulin-resistant 3T3-L1 and human mesenchymal stem cell-derived adipocytes exhibited elevated senescence-associated phenotypes, mitochondrial dysfunction and impaired cellular energetics. Notably, we found that mitochondrial DNA leakage triggered the cGAS-STING pathway in insulin-resistant adipocytes and mouse models. Temporal analysis revealed that mitochondrial dysfunction was detectable at earlier stages of chronic insulin exposure, preceding activation of the cGAS-STING pathway and senescence-associated markers, supporting a progressive model of cellular dysfunction. This phenomenon was also observed in adipose depots of individuals with Type 2 diabetes, underscoring the translational relevance of our findings. Targeting cGAS or STING, either pharmacologically or through genetic silencing, significantly reduced inflammatory and senescence-related features in hyperinsulinemia-induced insulin-resistant 3T3-L1 adipocytes. Furthermore, attenuation of senescence treatment with the combination of Dasatinib and Quercetin alleviated mitochondrial stress and associated adipose dysfunction. Collectively, our findings support a model in which prolonged hyperinsulinemic stress induces early mitochondrial dysfunction, followed by activation of cGAS-STING signalling and the subsequent emergence of adipocyte senescence-associated phenotypes, contributing to adipose tissue dysfunction in insulin resistance and Type 2 Diabetes.
In the era of personalized and precision medicine, sex is an important biological variable that needs to be taken into account in biomedical research. However, the role of the menstrual cycle has long been neglected because it requires clinical studies that are large, time-consuming, and expensive. A computational systems biology approach is a fundamentally different but powerful framework that explains how architectural features of the hormonal system, including feedback loops and cross-talk mechanisms, give rise to emergent behavior on the system level. Nevertheless, systems biology models that are centered around the menstrual cycle are still scarce. A major challenge both in medical research and from a modeling point of view is the large intra- and inter-individual variability in cycle length. In fact, the hypothalamic-pituitary-ovarian (HPO) axis, which is responsible for the regulation of the menstrual cycle, interacts with many of the other hormonal systems that are involved in, for example, the regulation of stress, energy balance, and glucose homeostasis. These interactions and their age-related changes need to be considered when exploring normal and pathological conditions since many endocrine diseases go along with disruptions in multiple hormonal axes. This paper focuses on the HPO axis and its interactions with the hypothalamic-pituitary-adrenal axis and glucose-insulin metabolism in both health and disease. It briefly summarizes the biomedical knowledge about the individual sub-systems and their cross-talk mechanisms and provides an overview of mathematical modeling approaches and opportunities in this field.
