Atrazine (ATR) is one of the most widely used herbicides in global agricultural production, it enters the body mainly through drinking water and diet. Liver is the main organ of ATR metabolism. As the end product of tryptophan metabolism, 5-HIAA can regulate liver metabolism. To investigate the hepatotoxic mechanism of ATR and the rescue effect of 5-HIAA against ATR-induced hepatic lipid accumulation, Wistar rats were treated with ATR for 90 days, L02 cells were treated with ATR for 24 hours. Transcriptomic and metabolomic analyses revealed that differentially expressed genes and metabolites were enriched in non-alcoholic fatty liver disease and the tryptophan metabolic pathway, respectively. In this study, ATR suppressed the AMPK/p38 signaling pathway, inhibiting FAO and leading to hepatic lipid accumulation. Conversely, 5-HIAA indirectly modulated the activation of the AMPK/p38 signaling pathway to alleviate ATR-induced lipid accumulation and restore reduced FAO in the liver.
Fibroblast growth factor 23 (FGF23) maintains phosphate and vitamin D homeostasis. In the kidney, recognition of FGF23 is mediated by fibroblast growth factor receptors (FGFRs), α-Klotho, and heparan sulfate. Structural studies indicate that an FGF23-FGFR-α-Klotho recognition unit recruits a second FGFR with assistance from heparan sulfate to form an asymmetric 1:2:1:1 complex that activates Ras-mitogen-activated protein kinase (MAPK) signaling. Circulating intact FGF23 reflects transcription, post-translational processing, secretion, and clearance. In FGF23-producing cells, polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3)-mediated O-glycosylation at Thr178 protects the cleavage site, whereas phosphorylation of Ser180 by FAM20C, a Golgi-associated secretory pathway kinase, limits this protection. Furin and related proprotein convertases cleave FGF23 within the secretory pathway, although their in vivo contributions remain unresolved. In cell-free experiments, tissue-type and urokinase-type plasminogen activators directly cleave recombinant FGF23; mouse data also implicate the plasminogen activator inhibitor-1 axis, but its quantitative contribution to the human circulating pool is unknown. Receptor availability further shapes signaling. Although osteocytes and osteoblasts are the principal endocrine sources, human skin cells express and secrete FGF23 in vitro. Their response to 1,25-dihydroxyvitamin D3 depends partly on the vitamin D receptor, but whether skin contributes to circulating FGF23 remains undefined. Chronic kidney disease, autosomal dominant hypophosphatemic rickets, hyperphosphatemic familial tumoral calcinosis, X-linked hypophosphatemia, Raine syndrome, and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) deficiency illustrate the relationships among FGF23 processing, bone-matrix signaling, and pyrophosphate homeostasis. This review integrates these mechanisms and evaluates burosumab, selective FGFR inhibition, C-terminal peptides, small-molecule antagonists, and ENPP1 enzyme replacement according to current evidence.
Obesity is a recognized risk factor for breast cancer, the leading cause of cancer death worldwide. Vitamin D (vit D), a molecule with anti-inflammatory effects, can act on different cell types through the presence of its VDR receptor. Our aim was therefore to study the impact of vit D in a 3D co-culture model between PreAd (spheroids of pre-adipocytes from obese women) or Mat-Ad (spheroids of mature adipocytes from obese women), M1-like pro-inflammatory macrophages and mammary cancer spheroids, to mimic the inflammatory breast tumor microenvironment (TME) of a person with obesity. The results showed a decrease in the expression of adipocyte differentiation genes in PreAd, inflammatory cytokines genes in inflammatory macrophages and tumor proliferation genes in cancer spheroids in the coculture system with PreAd, M1-macrophages and cancer cells. In addition, there was a decrease in the secretion of certain key inflammatory cytokines involved in obesity thus decreasing the overall inflammation of the microenvironment. These results were not observed in the coculture with Mat-Ad, M1-macrophages and cancer cells, with little to no effect of vit D on gene expression and an increase of the secretion of key inflammatory cytokines involved in obesity in the microenvironment thus favoring inflammatory microenvironment. These results suggest that, at a physiological concentration, vit D has therefore a more pronounced action on PreAd. Our findings suggest that the effects of vit D on TME depend on adipocyte differentiation, with potential benefits in preadipocytes rich microenvironment but limited efficacy in mature adipocytes rich microenvironment. Further studies are needed to determine the therapeutic relevance of vit D supplementation.
Cell culture systems are indispensable tools for studying cellular metabolism and signaling. However, subtle variations in nutrient composition, serum content, culture duration, and handling can influence cellular physiology. Despite widespread use, conditions commonly regarded as "standard" are rarely systematically evaluated for their impact on metabolic and insulin signaling pathways. Here, we systematically examined the effects of glucose concentration, fetal bovine serum (FBS) levels, culture duration, and medium volume on metabolic dynamics, stress responses, and insulin signaling in the hypothalamic cell line CLU468. Continuous oxygen consumption monitoring was combined with acute mitochondrial function assays and molecular analyses of stress pathways, autophagy, apoptosis, and insulin-responsive signaling cascades, including AKT and ERK activation. Prolonged culture duration alone (72 h vs. 24 h) reduced insulin receptor phosphorylation by approximately 70% and AKT activation by 77%. Continuous oxygen monitoring revealed distinct metabolic profiles dependent on glucose and serum availability: serum deprivation caused early stagnation of oxygen consumption and robust activation of autophagy and apoptosis, whereas glucose restriction permitted a delayed but ultimately more severe energy crisis, coinciding with pronounced cell death. Acute insulin signaling was shaped by a significant interaction between culture time and media condition, such that prolonged culture shifted the balance of AKT and ERK activation towards ERK dominance in standard conditions, but towards AKT dominance under glucose or serum restriction. Increasing medium volume did not uniformly delay these signaling shifts, but altered their direction in a condition-specific manner, while supplementation with the fatty acid palmitate selectively extended mitochondrial function in conditions with sufficient serum support. Key aspects of this nutrient- and time-dependent regulation, including the time-dependent decline in insulin signaling and its shift in AKT/ERK balance, were conserved in the insulin-sensitive pre-adipocyte line 3T3-L1 and were robust across multiple FBS lots. These findings demonstrate that routine culture parameters are active determinants of cellular metabolic state and shape insulin-induced metabolic versus mitogenic signaling by altering the pAKT/pERK balance, and that this regulation extends beyond a single cell type. Careful consideration and reporting of culture conditions are therefore essential for the interpretation and reproducibility of neuroendocrine and metabolic studies.
Liver function is critical for maintenance of body homeostasis since it regulates several metabolic processes, including glucose and fatty acids metabolism. Metabolic and nutritional altered states such as obesity, insulin resistance and diabetes negatively impact the liver leading to a condition termed Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD). Connexins (Cxs) and pannexins (Panxs) are plasma membrane channels, central for correct liver function, including the coordination of metabolic functions. These channels allow direct communication between the cytoplasm of adjacent cells or via paracrine signaling. Overnutrition conditions that lead to MASLD impact Cxs and Panxs channel functions in hepatocytes. Changes in Cxs and Panxs expression during hepatic metabolic disturbances further contribute to the pathogenesis of MASLD. This situation appears to be a pathological condition, which involves alterations mainly in the expression of connexin 43 (Cx43), connexin 32 (Cx32) and pannexin 1 (Panx1) in the transmission of damage signals. In the liver, Panx1 activity is traditionally associated with inflammation and apoptosis. Nevertheless, recent findings challenge this view. Therefore, these channels and their associated pathways arise as potential therapeutic targets in liver metabolic conditions such as MASLD. In this narrative review we analyzed the role of Cxs and Panxs channels in the development of this pathology as well as possible targets for treatment.
Heart failure is the leading cause of death in individuals with diabetes mellitus (DM), and no effective treatments exist to treat or prevent hyperglycemia-driven cardiomyopathy. Here, we demonstrate that high glucose triggers regulator of G protein signaling 6 (RGS6) up-regulation in both human and murine cardiomyocytes, the hearts of hyperglycemic mice, and cardiac tissue samples from individuals with heart failure and a history of diabetes. Modulation of RGS6 expression in isolated cardiomyocytes resulted in corresponding changes in the expression of the transcription factor Krüppel-like factor 4 (KLF4), a novel RGS6-interacting protein. Further, RGS6-dependent, KLF4-mediated suppression of microRNA 30e (miR-30e) increased expression of the pro-apoptotic miR-30e target Ca2+/calmodulin-dependent kinase II δ isoform (CaMKIIδ). Importantly, inhibition of either KLF4 or CaMKII or overexpression of miR-30e mitigated the deleterious impact of RGS6 overexpression on myocyte viability. Indeed, cardiac-specific RGS6 knockdown provided marked protection against hyperglycemia-driven oxidative stress, mitochondrial dysfunction, and activation of the intrinsic mitochondrial apoptosis pathway in the murine myocardium. Similarly, inhibition of KLF4 decreased cardiotoxicity resulting from viral overexpression of RGS6 in mouse heart. Thus, RGS6 is both necessary and sufficient to drive cardiac damage resulting from chronic elevations in blood glucose. Together, these data point to RGS6/KLF4 as key sources of pathogenic cardiac damage in individuals with DM.
OBJECTIVE:This study aimed to investigate the therapeutic potential of compound 18, an N-salicyloyl tryptamine derivative with established anti-neuroinflammatory and neuroprotective properties, in ameliorating male reproductive dysfunction induced by high-fat diet (HFD) in obese mice and to elucidate the underlying mechanisms. METHODS:Male mice were fed a HFD to induce obesity and subsequently treated with compound 18 at doses of 25 or 50 mg/kg. Systemic metabolic parameters including body weight, blood glucose, insulin, and lipid profiles were measured. Histopathological assessments were conducted on liver, adipose tissue, testes, and epididymis. Molecular analyses were performed to evaluate the expression of markers related to proliferation (PCNA), apoptosis (BAX and Bcl-2), components of the insulin signaling pathway (IGF1, IGF1R), and key glycolytic enzymes (HK2, PKM2, LDHA). RESULTS:Administration of compound 18 led to significant and dose-dependent improvements in systemic metabolism, characterized by reductions in body weight, blood glucose, insulin levels, and lipid parameters. The compound also attenuated hepatic steatosis and adipocyte hypertrophy, while notably restoring testicular and epididymal tissue architecture. At the molecular level, compound 18 up-regulated the proliferative marker PCNA, modulated apoptosis-related proteins (down-regulating Bax and up-regulating Bcl-2), enhanced insulin sensitivity-as indicated by increased IGF1R and decreased IGF1 expression-and augmented glycolytic capacity in Sertoli cells through elevated expression of HK2, PKM2, and LDHA. CONCLUSION:These results demonstrate that compound 18 effectively alleviates HFD-induced spermatogenic dysfunction concomitantly with ameliorating testicular insulin resistance and promoting glycolytic flux in Sertoli cells.
Preeclampsia (PE) is a major cause of maternal and perinatal morbidity, driven by placental dysfunction, oxidative stress, and trophoblast apoptosis. This study investigates the therapeutic potential of transcutaneous auricular vagus nerve stimulation (taVNS) in mitigating PE-induced placental injury via modulation of endoplasmic reticulum (ER) stress and calcium homeostasis. Using a reduced uterine perfusion pressure (RUPP) rat model and HTR-8/SVneo trophoblast cells, we assessed taVNS effects on hypertension, placental proteome, apoptosis, and molecular pathways. taVNS treatment was associated with significant reductions in mean arterial pressure, placental apoptosis, and ER ultrastructural damage in PE rats, while proteomic analysis revealed downregulation of calcium/calmodulin-dependent protein kinase II (CAMK II). In vitro, TNFα induced ROS accumulation, ER stress (via PERK activation), IP3R1-associated calcium dysregulation, and apoptosis, which were attenuated by taVNS-enhanced acetylcholine (ACh) through M3AChR signaling. Genetic silencing of PERK or IP3R1, or treatment with the ROS scavenger N-acetylcysteine or ER stress inhibitor 4-phenylbutyric acid, attenuated this injury cascade, providing functional support for a ROS-ER stress-calcium dysregulation-apoptosis axis in trophoblast dysfunction. These findings provide preliminary mechanistic and proof-of-concept evidence supporting further preclinical investigation of taVNS as a potential non-pharmacological approach in PE, offering mechanistic insights that may inform the design of future preclinical safety and translational feasibility studies.
Fatty acids (FAs) are bioactive dietary and metabolic molecules that participate in membrane architecture, energy homeostasis, inflammatory signaling, gene regulation and immune function, all of which intersect with breast cancer (BC) risk, progression and treatment response. In this narrative review we integrate epidemiological, clinical, translational and mechanistic evidence on the role of FAs in BC. Saturated, monounsaturated, trans- and polyunsaturated FAs (PUFAs) are treated as distinct biological exposures rather than interchangeable measures of total fat intake. Similarly, evidence from dietary assessment, circulating biomarkers, erythrocyte membrane composition, adipose tissue stores and tumor lipid signatures is interpreted separately, because each captures exposure and biology at a different level. BC subtypes differ in FA synthesis, uptake, oxidation, storage and remodeling: luminal tumors are frequently linked to hormone-regulated lipogenesis, human epidermal growth factor receptor 2 (HER2)-positive tumors to growth-factor-driven lipid metabolism, and triple-negative tumors to exogenous FA uptake, inflammatory lipid mediators and ferroptosis-related vulnerabilities. FA-derived mediators also shape immune-cell polarization, cytokine signaling and the tumor microenvironment, and dietary FAs may reshape the gut microbiota; the fiber-derived short-chain FAs it produces, distinct from dietary FAs, likewise help regulate immune and inflammatory tone. Clinical data suggest possible roles for fat-quality modification and selected n-3 PUFA interventions, but findings are heterogeneous and not yet sufficient to support routine biomarker-guided precision onco-nutrition. Candidate biomarkers, such as erythrocyte n-6:n-3 composition, require prospective validation before clinical implementation. FA biology thus represents a modifiable but complex axis in BC prevention, tumor biology and supportive care.
Obesity promotes cardiac remodeling and dysfunction through oxidative stress, inflammation, and metabolic disturbances. Citrus bergamia (bergamot) has shown beneficial effects on obesity-related cardiometabolic risk factors, but its cardiac effects remain unclear. We evaluated bergamot supplementation in a rat model of diet-induced obesity using integrated assessments of cardiac structure and function, redox and inflammatory status, biogenic amines, fatty acids, and metabolites. Wistar rats (n = 54) were randomized to Control, Obese, or Obese + Bergamot groups for 20 weeks; bergamot was administered by gavage at 250 mg/kg/day. Animals in both obese groups had higher body weight (p < .01) and adiposity index (p < .0001) than controls, confirming diet-induced obesity. Untreated obese rats also exhibited higher systolic blood pressure, cardiac remodeling, oxidative damage, higher cardiac TNF-α and IL-10 concentrations, and reduced NRF2 expression (p < .05). Bergamot attenuated cardiac remodeling, preserved systolic and diastolic function (p < .05), reduced malondialdehyde (p < .05), protein carbonyls (p < .0001), and cardiac TNF-α and IL-10 concentrations (p < .05), and increased ferric reducing antioxidant power (p < .01). Exploratory cardiac metabolic profiling showed partial recovery of putrescine and spermidine, higher spermine and histamine concentrations (p < .05), lower serotonin monoforms (p < .05), higher arachidonic acid abundance (C20:4n6; p < .05), and higher inosine abundance (p < .05) following bergamot supplementation. Bergamot-associated cardiac preservation occurred despite persistent obesity and without restoration of NRF2 expression, the NRF2/KEAP1 ratio, or myocardial NAD+ and NADP+ concentrations. Changes in polyamine, fatty acid, and purine-related profiles, including higher spermine and inosine, identify candidate metabolic features associated with this response but do not establish these metabolites as causal mediators.
Background Neuraminidase 1 (Neu1) plays a crucial role in the removal of sialic acid from glycoproteins and glycolipids, significantly influencing hepatic glycolipid metabolism. However, its precise contributions to liver gluconeogenesis and bile acid metabolism are not well defined. Although the sialylation of the hepatic glucagon receptor (GCGR) has been linked to the regulation of hepatic glucose homeostasis, the impact of Neu1 on hepatic gluconeogenesis through the desialylation of GCGR remains to be elucidated. Methods To explore the physiological function of Neu1 in liver glycolipid metabolism, we constructed liver-specific Neu1 knockout mice. Glucagon induced HepG2 cells were used to assess the role of Neu1 in gluconeogenesis. Results Phenotypic analysis indicated that these knockout mice exhibited a late-onset glycolipid metabolism disorder. Mechanistically, Neu1 deficiency caused the upregulation of key gluconeogenic genes, with cAMP-PKA signaling and the sialylation mediated activation of GCGR influencing the Akt-FoxO1 pathway, contributing to the disruption of hepatic glucose metabolism in 21-month-old Neu1 deficient mice. Transcriptomic analysis revealed an increase in lipogenesis and bile acid synthesis pathways in the livers of aged Neu1 knockout mice. Notably, there were significant alterations in steroid hormone biosynthesis, with elevated levels of lithocholic acid and allolithocholic acid detected in the Neu1 deficient mice. Additionally, the activation of inflammatory responses and reduced lipolysis seemed to correlate with the observed abnormalities in glycolipid metabolism. Conclusion These results indicate that Neu1 is essential for GCGR-dependent gluconeogenesis, suggesting that Neu1 could be a potential therapeutic target for addressing hepatic glycolipid metabolism disorders associated with aging.
Background Heart failure (HF) is the terminal stage of various cardiovascular diseases. Mitochondrial Ca2+ overload and dysfunction mediated by mitochondria-associated ER membranes (MAMs) are recognized contributors to HF pathogenesis; however, the underlying molecular mechanisms remain incompletely understood. Methods An in vitro model of cardiomyocyte injury was established using angiotensin II (Ang II)-stimulated H9c2 cells. IP3R was knocked down using specific siRNA. Cell viability (CCK-8), apoptosis (Annexin V/TUNEL), and hypertrophy (phalloidin staining) were assessed. IP3R expression and mitochondrial complex subunits were analyzed by western blotting. MAM formation was examined by transmission electron microscopy and immunofluorescence co-localization. Mitochondrial function was evaluated by measuring ATP levels, ROS (MitoSOX), mitochondrial membrane potential (TMRM), and Ca2+ uptake (Rhod-2). Results Ang II stimulation reduced cell viability, induced hypertrophy and apoptosis, and promoted MAM formation. Mechanistically, Ang II upregulated IP3R expression and enhanced MAM-mediated mitochondrial Ca2+ overload, leading to decreased mitochondrial membrane potential, elevated ROS, and reduced ATP production. Notably, genetic knockdown of IP3R attenuated these pathological changes, reducing MAM formation and mitochondrial Ca2+ overload while restoring mitochondrial function and cell viability. Conclusion Our findings suggest that IP3R may exacerbate cardiomyocyte injury by facilitating MAM-mediated Ca2+ transfer and mitochondrial dysfunction, indicating that targeting IP3R could represent a potential therapeutic strategy for HF. However, the upstream mechanisms of IP3R upregulation and the causal role of mitochondrial Ca2+ uptake require further investigation.
The gut microbiome is increasingly linked to endocrine physiology, but human evidence that individual microbial traits contribute to male reproductive and skeletal phenotypes remains largely observational. We integrated MiBioGen genus-level and Dutch Microbiome Project species-level microbiome genome-wide association studies with sex-stratified steroid traits, sex hormone-binding globulin (SHBG), and heel estimated bone mineral density (eBMD) in a two-sample Mendelian randomization (MR) screen of 1204 microbe-outcome tests. The leading male endocrine association linked genetically proxied abundance of the MiBioGen-defined Eubacterium rectale group to greater odds of detectable male estradiol (odds ratio = 1.36; 95% CI, 1.12-1.65; P = 0.00246; eight instruments). This binary endpoint denotes assay detectability above 175 pmol/L, not circulating estradiol concentration. Independent GTEx V10/SuSiE fine-mapping of CYP19A1 expression in subcutaneous adipose provided aromatase-relevant tissue context but did not localize or mediate the microbial association. Complementary associations linked Roseburia hominis with SHBG and Alistipes finegoldii with heel eBMD; the latter was the only study-wide Bonferroni-significant result (P = 6.55 × 10-8). A Bifidobacterium longum-female testosterone association attenuated after exclusion of the LCT/MCM6 region, indicating substantial host-diet genetic influence. Across 172 matched genus-species pairs, effect estimates showed little correlation, directional agreement was no better than chance, and no pair shared clumped instruments. These findings nominate the E. rectale group for targeted male endocrine follow-up and show that taxonomic resolution is part of the exposure definition in microbiome MR.
OBJECTIVE:Early detection of diabetic kidney disease (DKD) remains challenging because currently available clinical markers mainly reflect established renal injury rather than early pathogenic changes. Ferroptosis has been increasingly implicated in DKD development, we aimed to identify ferroptosis-related molecular signatures and candidate diagnostic biomarkers for DKD. METHODS:We integrated public kidney transcriptomic datasets from DKD and control samples and analyzed them together with curated ferroptosis-related genes. Differential expression and gene co-expression analyses were combined with protein-interaction and machine-learning approaches to prioritize candidate biomarkers. Single-cell transcriptomic analysis was used to explore cellular localization, and the leading candidate was further validated in an independent clinical transcriptomic database and a diabetic mouse model. RESULTS:We identified genes that were consistently dysregulated across DKD datasets and linked them to ferroptosis-related processes. Network-based analysis prioritized several hub genes, among which CD36 emerged as the most robust candidate biomarker. Independent database validation demonstrated that CD36 was significantly upregulated and associated with renal function indicators. Consistent with these findings, experimental validation confirmed increased renal CD36 protein expression and elevated serum CD36 levels in diabetic mice, further supporting its potential as a biomarker for DKD. CONCLUSIONS:Our ferroptosis-oriented screening strategy identified CD36 as a promising diagnostic biomarker for DKD. These findings support the potential value of ferroptosis-related signatures for biomarker discovery in DKD and warrant further mechanistic validation.
The melanocortin-4 receptor (MC4R) is a critical regulator of energy homeostasis, affecting both energy intake and expenditure. It is also involved in regulating reproduction. Recent clinical success with peptide agonists has validated MC4R as a therapeutically actionable target. However, orally active small-molecule ligands would offer important advantages over injectable peptides. Small-molecule agonists may provide therapeutic opportunities for obesity and sexual dysfunction, whereas small-molecule antagonists may have utility in cachexia, anorexia, and related wasting states. In addition, some hydrophobic small molecules can function as pharmacoperones, rescuing trafficking-defective naturally occurring MC4R mutations and thereby offering a precision medicine avenue for a subset of monogenic obesity. Moreover, some MC4R ligands display biased signaling properties, suggesting that pathway-selective modulation may further expand the pharmacological and therapeutic landscape of this receptor. In this review, we summarize the major classes of small-molecule ligands reported for MC4R, with emphasis on their pharmacology, biased signaling, and pharmacoperone activity.
The corticotropin-releasing factor (CRF) system bidirectionally interacts with cytokines and other immune-related components in mammals. However, the nature of these interactions remains poorly characterized in other vertebrates, including teleost fishes. To gain insight into the relationship between immune responses and the CRF system in teleosts, we explored how CRF system components were regulated in immune organs of rainbow trout (Oncorhynchus mykiss). We first characterized the CRF system in the spleen and head kidney—two primary immune organs in teleosts—and found that many CRF system components were present in both tissues, but splenic expression was consistently greater. Changes in the abundance of splenic CRF system transcripts following vaccination (which transiently stimulated inflammatory responses and cytokine production) indicated that this inflammatory challenge had contrasting and time-dependent effects on transcripts related to CRF receptor 1 (CRFR1; suppression) versus CRFR2 (stimulation). Using spleen explant cultures, we evaluated whether these effects were mediated by either of nuclear factor kappa B (NF-κB; a pro-inflammatory transcription factor) or cortisol (an anti-inflammatory hormone). At baseline, cultured spleens increased cytokine production and exhibited changes in mRNA levels of CRF system components comparable to those observed following vaccination. Cortisol treatment and NF-κB inhibition both attenuated the rise in cytokine mRNA levels; however, cortisol treatment generally affected transcripts related to CRFR1, while NF-κB inhibition reduced levels of transcripts related to CRFR2. Overall, our data provide novel insight into CRF system regulation in the spleen and suggest that cortisol and inflammatory cytokines differentially regulate CRFR1 and CRFR2 signalling within this organ.
Inducible nitric oxide synthase (iNOS) is activated in obesity and is involved in the regulation of cellular pathways, including the unfolded protein response and insulin signaling. We previously showed that iNOS deficiency is associated with reduced obesity-induced insulin resistance (IR), particularly in skeletal muscle, in iNOS knockout (iNOS KO) mice fed a high-fat diet (HFD). However, the mechanisms underlying protection against HFD-induced IR in iNOS KO mice remain incompletely understood. Several studies have reported associations between gut microbiota composition and metabolic outcomes, including body weight regulation and insulin resistance, with differences observed between lean and obese subjects. However, the potential contribution of the gut microbiota to the metabolic phenotype observed in iNOS KO mice has not been fully investigated. To address this question, we assessed glucose homeostasis, adipose tissue inflammation, gut microbiota composition, and intestinal barrier integrity. Fecal microbiota transfer (FMT) experiments were also performed between HFD-fed iNOS KO and C57BL/6J mice in both directions. HFD-fed iNOS KO mice exhibited reduced adipose tissue inflammation, characterized by decreased numbers of mast cells and pro-inflammatory M1 macrophages, together with increased numbers of anti-inflammatory M2 macrophages, compared with HFD-fed C57BL/6J mice. The gut microbiota profile of HFD-fed iNOS KO mice was associated with increased expression of genes related to intestinal tight junction integrity. Fecal microbiota transfer from HFD-fed iNOS KO mice was associated with improved glucose tolerance, enhanced insulin sensitivity, and increased expression of genes related to intestinal tight junction integrity in recipient mice. These findings suggest that gut microbiota contributes, at least in part, to the protection against HFD-induced IR in iNOS KO mice and support the potential of targeting gut microbiota as a therapeutic strategy to improve metabolic disturbances.
BACKGROUND:β-cell dysfunction is a key factor in the progression of diabetes. It was reported that lipotoxicity impaired β-cell mitochondrial function. However, the molecular mechanisms regulating lipotoxicity-induced β-cell mitochondrial dysfunction remain unclear. METHODS:Islet cells were isolated from diabetic db/db and control db/m mice. Palmitic acid (PA) was used to treat MIN6 cells to construct a lipotoxicity-induced β-cell model. Co-IP was employed to verify the interaction between Sirt1 and Trim67. Enrichment of Klf5, Klf2, Klf6, and Ahr on the Trim67 promoter was analyzed using DNA pull-downs. Binding of Klf6 to the Trim67 promoter region was detected using ChIP and dual-luciferase reporter assays. Cell proliferative capacity and mitochondrial function were measured by CCK-8, EdU, JC-1, Seahorse XF-96 and ATP detection kits. Protein expression was tested using RT-qPCR and western blotting. Sirt1 deacetylase activity was measured fluorometrically. Ubiquitination sites were mapped using site-directed mutagenesis. In vivo experiments were performed using AAV8-mediated Trim67 overexpression in the db/db mice. RESULTS:Trim67 was downregulated in pancreatic islet of diabetic mice and in PA-induced β-cells. AAV8-mediated Trim67 overexpression in db/db mice significantly improved glucose tolerance, enhanced insulin secretion, and preserved islet integrity. Overexpression of Trim67 alleviated PA-induced mitochondrial dysfunction in β-cells. Moreover, Trim67 was transcriptionally activated by Klf6 in PA-induced β-cells. Trim67 increased Sirt1 stability through K63-linked ubiquitination at lysines 491 and 591 to mediate the Sirt1/Pgc-1α pathway, which in turn alleviates PA-induced mitochondrial dysfunction in β-cells. CONCLUSION:Klf6 transcriptionally activated Trim67 to regulate the Sirt1/Pgc-1α axis, thereby alleviating PA-induced mitochondrial dysfunction in β-cells. This study offers new insights into the treatment of diabetes.
Sertoli cells (SCs) are crucial for testicular development, transitioning from a proliferative to a metabolically active state during puberty to facilitate germ-cell differentiation. However, the molecular switches governing this maturation remain unclear. Here, we identified a previously uncharacterized circular RNA, circLDHC, as a key regulator of SCs fate. Transcriptomic analysis and RT-qPCR validation showed that circLDHC expression increased during pubertal testicular development and was higher in mature than in immature SCs. In immature SCs, circLDHC overexpression reduced proliferative activity, accompanied by decreased expression of proliferation and cellcycle related genes and reduced S-phase entry. In peripubertal SCs, circLDHC increased the expression of AR and HSD17B4 and promoted lactate production and lipid droplet accumulation. Mechanistically, circLDHC acted as a competing endogenous RNA that sponged miR-370, thereby releasing androgen receptor (AR) from miRNA mediated repression. Dual-luciferase assays confirmed that miR-370 directly targets AR, and functional rescue assays supported the circLDHC/miR-370/AR axis. These findings reveal a circLDHC/miR-370/AR regulatory axis that coordinates SC proliferation arrest and metabolic maturation. This work provides new insights into the noncoding RNA mediated control of puberty onset in boars. It offers potential molecular targets for improving reproductive performance and understanding Sertoli cell dysfunction in mammals.
Background Micro- and nanoplastics (MNPs) have been detected in human placenta, though their impact on steroidogenesis during pregnancy remains unknown. Existing in vitro placental models are limited in capturing both barrier function and maternal-fetal steroidogenic interactions. Method A Transwell tri-culture model was developed, consisting of BeWo b30 (apical) and HUVEC cells on the insert and H295R cells at the bottom (basolateral). Fluorescently-labelled (F) polymers were added apically: commercially PS (50 nm, 200 nm and 1000 nm) and custom-made PMMA (340-740 nm), PVC (2140-5700 nm), PA6,6 (280-640 nm) and PET (1460-5200 nm). Translocation from apical to basolateral side was measured after 72 h, and uptake assessed by confocal microscopy. Mitochondrial activity, steroid hormone levels and gene expression of steroidogenic enzymes were also evaluated. Results F-PS translocation was size-dependent, with the highest observed for F-PS50 nm (4.4%). F-PMMA showed the highest translocation (10.7%), followed by F-PVC (6.5%) and F-PET (4.2%), while F-PA could not be reliably quantified. In the tri-culture, 19 steroid hormones were detected apically and 18 basolaterally. PS1000 nm and PS50 nm (10 μg/mL) decreased 17-hydroxy-dihydroprogesterone (17-OH-DHP) by 36% and 29%, respectively. PET, PMMA, PA6,6, and PVC (10 μg/mL) reduced apical etiocholanolone by 23-25%. PET (1 μg/mL) reduced it by 22%. PS1000 nm (1 μg/mL) lowered apical 17β-estradiol by 14%. CYP17 expression decreased after PS200 nm and PS1000 nm exposure; other polymers showed no effect. Conclusions This in vitro model demonstrates that MNPs cross the placental barrier and alter steroidogenesis, raising concerns about their potential endocrine-disrupting effects during pregnancy.
