The FASEB Journal

Regular exercise induces adaptations in skeletal muscle and other organ systems to improve physical performance and overall health. Exercise results in phosphorylation of 5 AMP-activated protein kinase (AMPK) at threonine 172 (T172) of the 2 subunit; however, the role of this activation in cellular and functional adaptations has not been elucidated. To this end, we subjected non-activatable Ampk2(T172A) knock-in (KI) adult mice and wild-type (WT) littermates to 4 weeks of voluntary wheel running (VWR). Exercise training led to significant improvements in endurance capacity, maximal oxygen consumption ([Formula]O2max), and glucose tolerance, as well as skeletal muscle IIb-to-IIa fiber type shift in both WT and KI mice. Contrastingly, VWR resulted in increased mitochondrial OxPhos protein expression, mitochondrial volume density, and capillary density in skeletal muscle of WT but not KI mice. Exercise-induced improvements of mitochondrial respiration and conductance revealed by high-resolution respirometry of isolated mitochondria were blunted in KI mice. Therefore, for the first time, we reveal that AMPK2 T172 activation is required for exercise training-induced mitochondrial biogenesis, improvement of mitochondrial respiratory function, and angiogenesis in skeletal muscle, but that these adaptations are not solely responsible for improved [Formula]O2max and exercise endurance capacity. Significance StatementExercise is the most effective lifestyle intervention for promoting health and preventing chronic diseases through adaptive changes in skeletal muscle and many other tissues/organs. AMPK is an energy sensor and signaling regulator for exercise-induced skeletal muscle adaptation, yet its functional role and the impact on exercise capacity have been studied in mouse genetic models wherein protein stoichiometry is disrupted. Using non-activatable Ampk2(T172A) knock-in mice, we ascertained that AMPK2 activation via T172 phosphorylation is required for endurance training-induced mitochondrial and angiogenic adaptations in skeletal muscle. Importantly, these adaptations are not required for improved exercise capacity, challenging the prevailing concept that increased mitochondrial content and function and microvasculature are the sole driving factors for the performance gains with endurance training.

IntroductionObesity is a major health issue associated with metabolic and psychological comorbidities, as well as an increased prevalence of disorders of gut-brain interaction (DGBI). Obesity and DGBI share common mechanisms such as inflammation, gut barrier dysfunction, and alterations of gut microbiota, which are all known to be regulated by stress. Glutamine (Gln), which is essential to maintain intestinal integrity and immune response, may counteract these alterations. This study aimed to evaluate the effects of oral Gln supplementation on stress-induced response in obese mice. MethodsSeven-week-old male leptin-deficient ob/ob mice were assigned to four groups: control, chronic restraint stress (CRS), Gln-supplemented, or both CRS and Gln-supplemented. Gln was administered in drinking water for two weeks, and CRS was performed during the final 4 days. Metabolic parameters, intestinal permeability, inflammatory markers, gene and protein expression, and gut microbiota composition were assessed. ResultsStress increased plasma corticosterone levels but had a limited effect on metabolic parameters. In obese mice without stress, Gln supplementation reduced body weight gain, improved body composition and reduced inflammation in the visceral adipose tissue. These effects were lost under stress conditions, with an increase in fasting glycaemia. Stress reduced occludin protein levels, while Gln exerted context-dependent effects, decreasing gene expression of Tjp3, Cldn15 and Ccl2 in unstressed mice but increasing gene expression of multiple tight junction (Tjp2, Tjp3, Cldn12, Cgn, F11r, Marveld2) and inflammatory markers (Tlr2, Myd88, Irf3) under stress. Interestingly, in unstressed obese mice, Gln altered the composition of the gut microbiota, with changes in key bacterial taxa (Thermodesulfobacteriota and Clostridiaceae). This was associated with decreased levels of cecal short-chain fatty acids and increased levels of branched-chain fatty acids. ConclusionIn conclusion, Gln improves metabolic and adipose inflammatory parameters in genetically obese mice. However, these benefits are no longer observed when mice are under stress conditions. Since, Gln has been found to increase fasting glycaemia and colonic inflammation, in association with alterations of gut microbiota.

Maternal obesity and viral infection induce placental inflammation, but how their co-exposure influence fetoplacental development remains unclear. We hypothesized that maternal high fat (HF) diet and viral infection would independently induce placental inflammation and lipid peroxidation, reduce antioxidant defence, and cellular turnover. Further, HF diet would compromise placental capacity to adapt to infection. Female C57BL/6J mice were fed a control (CON) or 62% HF diet six weeks before and throughout pregnancy and injected with poly(I:C) (viral mimic) or vehicle (VEH) 24h before sacrifice at gestational days (GD) 12.5, 15.5, and 18.5 (n=5-8/group/GD). Placental inflammasome (NLRP3), oxidative stress (4-HNE), antioxidant defence (GPx-4), and cellular proliferation-to-death ratio (Ki-67, Caspase-3) were assessed by immunohistochemistry, and mRNA expression of Tlr3, Irf3, Tlr4, Tirap, and Il-1{beta} were measured by qPCR. Data were analysed by linear mixed models (p[≤]0.05). At GD12.5, infection was associated with increased Tlr3 mRNA and immunoreactive (ir)-4-HNE, and reduced ir-GPx-4 expression in the placental labyrinth zone (LZ). By GD15.5, HF diet was associated with increased ir-NLRP3 in both LZ and junctional zones (JZ). Exposure to infection alone and co-exposure to HF diet and infection further increased LZ ir-NLRP3. At GD18.5, HF diet was associated with increased Tirap and Il-1{beta} mRNA expression, ir-4-HNE in the JZ and ir-Caspase-3 in the LZ. Maternal HF diet and infection exert distinct effects on the placenta across gestation, suggesting that maternal overnutrition might reduce the placentas capacity to handle adverse exposures, which may increase susceptibility to poor fetal outcomes.

Angiotensin-converting enzyme 2 (ACE2) functions as the receptor for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The virus utilizes the cellular endocytic machinery for entry by binding to defined residues on ACE2 with its spike protein (S protein), whose activation requires a priming process by another transmembrane protease, the transmembrane protease serine 2 (TMPRSS2). In addition, ACE2 itself is cleaved by TMPRSS2, which has been shown to be critical for viral pathology. This study aimed to elucidate the relationship between ACE2 and TMPRSS2 and the mechanism of ACE2 processing under normal cellular conditions. It is shown that interaction of ACE2 with TMPRSS2 results in altered processing, modification and cellular localization. Glycosylation of ACE2 has a major impact on TMPRSS2 interaction, trafficking and shedding of the enzyme. Studies in newly generated TMPRSS2-knockout rats reveal increased ACE2 levels in tissues supporting an important role of TMPRSS2 in ACE2 shedding also in vivo.

Skeletal muscle homeostasis depends on the coordinated regulation of protein turnover and mitochondrial quality control; however, the molecular mechanisms linking these processes remain unclear. In this study, we examined the physiological role of leucine aminopeptidase 3 (LAP3), a post-proteolytic aminopeptidase, using constitutive LAP3-deficient mice. LAP3 deficiency preferentially affected skeletal muscle, causing reduced muscle mass and mitochondrial enlargement in both sexes. Female LAP3-deficient mice also showed reduced myofiber size, impaired endurance capacity, increased energy expenditure, elevated lipid oxidation, and lipid droplet accumulation adjacent to the mitochondria. Proteomic analyses revealed remodeling of pathways related to lipid metabolism and protein homeostasis. Consistent with these findings, LAP3 deficiency increased the expression of Pink1 and Tax1bp1 and promoted the accumulation of ubiquitinated proteins, suggesting alterations in mitochondrial quality control and proteostatic regulation. In cultured myogenic cells, LAP3 localized to mitochondrial fractions, and both LAP3 knockdown and overexpression altered mitochondrial morphology. Taken together, these results identify LAP3 as a regulator of skeletal muscle homeostasis and support a role for LAP3 in linking intracellular peptide turnover to mitochondrial homeostasis, with female skeletal muscle showing greater susceptibility to LAP3 deficiency.

Background: Endothelial progenitor cells (EPCs) contribute to endothelial repair and neovascularization, and EPC dysfunction is closely associated with oxidative stress-related vascular injury. Forkhead box O3a (FoxO3a) regulates cellular stress responses, whereas miR-34a has been implicated in endothelial dysfunction, senescence, and apoptosis. However, the relationship between FoxO3a and miR-34a-3p in oxidatively injured EPCs remains incompletely defined. Objective: This study investigated the role of FoxO3a in H2O2-induced EPC dysfunction and examined whether miR-34a-3p directly interacts with the FoxO3a 3' untranslated region (3'UTR). Methods: Human umbilical cord blood-derived EPCs were identified by DiI-ac-LDL uptake, FITC-UEA-1 binding, and the expression of EPC-related markers. Oxidative stress was induced by H2O2. Cell viability, apoptosis, and angiogenic capacity were evaluated using CCK-8 assay, Annexin V/7-AAD flow cytometry, and Matrigel tube formation assay, respectively. FoxO3a expression was modulated using adenoviral overexpression or knockdown vectors, and miR-34a was modulated using mimics or antagomir. FoxO3a and miR-34a expression levels were detected by Western blot and qPCR. A dual-luciferase reporter assay was used to verify the interaction between hsa-miR-34a-3p and the FoxO3a 3'UTR. Results: H2O2 reduced EPC viability, increased apoptosis, and impaired tube formation in a concentration-dependent manner. H2O2 increased FoxO3a protein abundance and miR-34a expression, whereas FoxO3a mRNA did not change markedly. FoxO3a overexpression aggravated, whereas FoxO3a knockdown partially alleviated, H2O2-induced EPC dysfunction. Similarly, miR-34a mimics further suppressed EPC viability and tube formation, while miR-34a antagomir exerted a protective effect. Dual-luciferase reporter analysis showed that hsa-miR-34a-3p significantly reduced the activity of the wild-type FoxO3a 3'UTR reporter, while mutation of the predicted binding site abolished this suppression. Conclusion: FoxO3a and miR-34a participate in oxidative stress-induced EPC dysfunction. The dual-luciferase data demonstrate that hsa-miR-34a-3p directly targets the FoxO3a 3'UTR, suggesting the presence of miR-34a-3p-mediated post-transcriptional feedback within the FoxO3a-related stress-response network in EPCs.

Sedentary behavior promotes chronic low-grade inflammation in adipose tissue, contributing to metabolic dysfunction and insulin resistance. High-intensity interval training (HIIT) is a time-efficient exercise strategy with potent anti-inflammatory and metabolic benefits; however, its effects on adipose tissue inflammatory signaling and microRNA (miRNA) regulation remain incompletely understood. This study investigated the effects of eight weeks of HIIT on inflammatory and epigenetic markers in interscapular white adipose tissue (iWAT) of male Wistar rats. Fourteen rats were randomly assigned to either a sedentary (SED; n = 7) or HIIT (n = 7) group. The HIIT protocol consisted of treadmill running five days per week for eight weeks. Body weight and iWAT mass were assessed, and molecular adaptations were evaluated at multiple regulatory levels using RT-qPCR for mRNA targets (NLRP3, TNF-, PPAR-{gamma}, and IL-10) and miRNAs (miR-21 and miR-30d-5p), while protein levels of NLRP3 and PPAR-{gamma} were assessed using Western blotting. Compared with the SED group, HIIT significantly reduced body weight (p < 0.001) and iWAT mass (p = 0.002). Furthermore, HIIT downregulated the expression of pro-inflammatory mediators, including NLRP3 (gene: p = 0.001; protein: p < 0.001) and TNF- (p = 0.025), while upregulating anti-inflammatory regulators PPAR-{gamma} (gene: p = 0.026; protein: p = 0.020) and IL-10 (p = 0.010). In parallel, inflammation-associated miRNAs, including miR-21 (p = 0.004) and miR-30d-5p (p = 0.002), were markedly downregulated. These coordinated transcriptional, post-transcriptional, and translational adaptations suggest that HIIT attenuates adipose tissue inflammation and promotes a favorable immunometabolic phenotype through integrated molecular and epigenetic mechanisms.

Circadian rhythm disruption is associated with metabolic and inflammatory disorders; however, the mechanisms linking circadian dysfunction to endocannabinoidome (eCBome) signaling and mitochondrial metabolism remain unclear. In our previous in vivo study, constant light exposure altered hepatic eCBome profiles, reduced N-acylethanolamines (NAEs), increased monoacylglycerols (MAGs), and elevated inflammatory cytokines. Here, we investigated the underlying mechanisms using CRISPR/Cas9-generated BMAL1 knockout (KO) HepG2 cells as an in vitro model of circadian alteration. The BMAL1 KO model showed broad lipid remodeling characterized by increased fatty acids, prostaglandins, and MAGs together with reduced NAEs and enhanced lipid accumulation. These changes were accompanied by increased inflammatory signaling and cytokine production. Among the assessed genes, GPR110 was significantly altered in mice exposed to constant light (in vivo study) and BMAL1 KO model and emerged as a potential mediator linking circadian signaling to mitochondrial function. BMAL1 KO cells also exhibited significantly increased calcium (Ca{superscript 2}+) levels in mitochondria and the endoplasmic reticulum (ER), along with attenuation of mitochondrial and glycolytic ATP production. BMAL1KO did not abolish the rhythmicity of NAEs level over 24 hours from medium deprivation and read ministration except for N-docosahexaenoyl-ethanolamide (DHEA). Further, experiments showed that DHEA acts through GPR110 and suppress inflammatory lipid-associated pathways, enhances ATP production, and increases mitochondrial and ER Ca{superscript 2}+ accumulation and inflammatory signaling. Together, these mitochondrial Ca{superscript 2}+ signaling, and inflammation in hepatocytes, highlighting DHEA-GPR110 signaling as a potential regulator of hepatic metabolic homeostasis. HighlightsCircadian disruption increases hepatic monoacylglycerols and decreases N-acylethanolamines. Circadian disruption decreases ATP production and enhances mitochondrial and endoplasmic reticulum Ca{superscript 2}+ levels in hepatocytes DHEA-GPR110 signaling regulates hepatocytes mitochondrial Ca{superscript 2}+ dynamics and ATP production GPR110-mediated Ca{superscript 2}+ signaling significantly alters hepatocytes glycolysis and glycolytic ATP production

Defective intestinal epithelial tight junction (TJ) barrier function and endoplasmic reticulum (ER) stress are central pathological features of inflammatory bowel disease (IBD), yet the molecular mechanisms ER stress to TJ disruption remains poorly understood. Here, we investigated the role of autophagy in regulating intestinal TJ homeostasis during ER stress. ER stress was elevated in inflamed Crohns disease tissue and chronic dextran sulfate sodium (DSS) colitis. In human intestinal epithelial Caco-2 monolayers, murine colon, and human colonic explants, induction of ER stress with tunicamycin, thapsigargin, or brefeldin A disrupted TJ barrier integrity, as demonstrated by reduced transepithelial electrical resistance and increased paracellular permeability. ER stress selectively increased the pore-forming TJ protein claudin-2 and altered occludin localization without significantly affecting other claudins. Pharmacologic activation of autophagy with rapamycin attenuated ER stress, restored TJ barrier function, reduced claudin-2 accumulation, and preserved occludin localization. Conversely, CRISPR-Cas9-mediated deletion of autophagy gene ATG7 exacerbated ER stress, apoptosis, and TJ barrier dysfunction in vitro, while intestinal epithelial-specific Atg7 knockout mice exhibited enhanced ER stress-induced intestinal permeability in-vivo. Mechanistically, prolonged ER stress impaired autophagic flux through IRE1 kinase signaling, resulting in accumulation of p62 and claudin-2. Inhibition of IRE1 kinase activity restored autophagy, reduced claudin-2 levels, and preserved TJ barrier function. We further identified adaptor-associated kinase 1 (AAK1) as a downstream mediator of IRE1 signaling during ER stress, with increased AP2M1 phosphorylation and altered claudin-2 trafficking. Claudin-2 overexpression alone induced ER stress and lysosomal damage, suggesting a feed-forward mechanism amplifying epithelial injury. Finally, enteric rapamycin administration reduced ER stress and restored autophagy in murine DSS colitis. Collectively, these findings identify an IRE1-AAK1-autophagy axis as a critical regulator of intestinal TJ barrier integrity during ER stress.

Recent initiatives by the U.S. Food and Drug Administration and the National Institutes of Health to reduce animal testing in drug development have highlighted the need for in vitro platforms that better recapitulate human biology for preclinical safety assessment. Drug-induced nephrotoxicity remains a major cause of drug attrition, underscoring the need for human-relevant kidney models. To address this, a pump-free human patient-derived proximal tubule microphysiological system was developed by integrating human renal proximal tubular epithelial cells (hRPTECs), isolated from non-tumorous nephrectomy cortex, with a porous membrane-based microfluidic device. Expanded hRPTECs were cultured for 10 days under static conditions or rocker-driven shear stress approximating physiological proximal tubular flow. Shear stress increased epithelial density, enhanced proximal tubule marker expression (Na+/K+-ATPase and aquaporin-1), and improved Zonula occludens-1 and occludin localization. Bulk RNA sequencing demonstrated transcriptomic changes associated with enhanced apical maturation and epithelial signature. In cisplatin-induced injury assays, shear-conditioned epithelia exhibited reduced cell density and increased {gamma}H2AX staining, indicating greater sensitivity to nephrotoxicity. These findings demonstrate that rocker-driven shear stress promotes epithelial maturation in patient-derived hRPTECs. The pump-free human patient-derived proximal tubule microphysiological system offers a practical, scalable, and physiologically relevant platform for modeling flow-dependent proximal tubule biology and assessing human-relevant nephrotoxicity.

Under homeostatic conditions, the cornea is avascular and contains few immune cells, but this changes rapidly following injury. Although the long-term consequences of corneal damage are well characterized, the earliest vascular and immune responses remain poorly understood. Here, we used a murine corneal alkali-burn model to examine limbal vascular activation and leukocyte recruitment immediately and at 2, 6, and 24 hours after injury. Limbal blood vessels underwent immediate dilation; however, vascular leakage into the corneal stroma occurred only in males. Lymphatic capillaries rapidly formed directed extensions toward the injury without significantly increasing their total vascular area, with males exhibiting longer extensions than females. Fluorescent dextran uptake provided evidence that these lymphatic vessels were functionally engaged in early tracer drainage. Despite pronounced vascular activation, early recruitment of neutrophils, monocytes, dendritic cells, macrophages, T cells, B cells, and natural killer cells remained limited. Thus, limbal blood and lymphatic vessels initiate the earliest response to corneal alkali injury before substantial leukocyte infiltration. These findings reveal sex-dependent differences in vascular permeability and lymphatic remodeling and identify the limbal vasculature as an early regulator of corneal inflammation and tissue repair.

Previous studies have demonstrated that mechanistic target of rapamycin complex 2 (mTORC2) deficiency provides complete protection against steatotic liver disease driven by constitutive activation of the phosphoinositide 3-kinase (PI3K)-Akt signaling pathway and de novo lipogenesis, and partial protection against disease induced by a high-fat diet. We investigated herein whether mTORC2 deficiency in hepatocytes and myeloid cells, including Kupffer cells and recruited macrophages, influences the development of liver disease induced by intake of a choline-deficient, amino acid-defined high-fat diet (CDAHFD), a model in which liver disease is induced by impaired hepatic secretion of very low-density lipoprotein (VLDL) triacylglycerol. For this, mice with either hepatocyte- or myeloid cells-specific deletion of mTORC2 essential component rapamycin-insensitive companion of mTOR (Rictor) and their respective littermate controls were fed with either chow or CDAHFD for 10 weeks and evaluated for hepatic steatosis, inflammation and fibrosis. Our main findings indicate that hepatocyte Rictor/mTORC2 deficiency slightly attenuated the CDAHFD-induced increases in liver mass, macrovesicular steatosis and triacylglycerol accumulation, without affecting though liver cholesterol, serum markers of liver injury (AST and ALT), as well as the upregulation in proinflammatory cytokine IL-1{beta} and expression of fibrosis-related genes. Myeloid cells-Rictor deletion had no detectable impact on liver steatosis, inflammatory, or fibrosis induced by CDAHFD. In conclusion, mTORC2 deficiency show modest beneficial effects in counteracting liver disease induced by CDAHFD intake.

The mitochondrial fatty acid synthesis (mtFAS) pathway is a highly conserved process in mitochondria implicated in metabolic state sensing. Aberrant functioning of this pathway leads to neurodegenerative diseases in humans. Animal experiments indicates that the mtFAS pathway is essential in mammals, and mtFAS inactivation leads to neuronal cell death. Nuclear encoded mitochondrial 3-ketoacyl-acyl carrier protein reductase (KAR) is a heterotetrameric enzyme in this process, consisting of two CBR4 and two HSD17B8 polypeptides. CBR4 works as the catalytic subunit of the enzyme. Here, we provide evidence that CBR4 function is essential in mammals. In contrast, a skeletal muscle-specific Cbr4 KO in mice did not result in any measurable defects in muscle strength and endurance, and the overall structure of the muscle remained unchanged. The Cbr4 KO did not affect the lipoylation process in quadriceps muscle samples, and high-resolution respirometry analysis of soleus muscle samples showed no defects in mitochondrial respiration capacity. The lack of a phenotype of a muscle-specific Cbr4 KO is consistent with previous reports on a lack of effects of mtFAS inactivation in muscle and re-iterates the question about the existence of bypass mechanisms that can alleviate mtFAS deficiencies in non-neuronal cell types.

Statins, widely used cholesterol-lowering drugs, inhibit the mevalonate pathway and reduce coenzyme Q (CoQ) biosynthesis, potentially impairing mitochondrial function. Because astrocytes are essential for maintaining brain redox homeostasis, statin-induced mitochondrial dysfunction in these cells may contribute to CNS pathology. We examined the effects of a six-day statin exposure on mitochondrial bioenergetics in rat astrocytes, focusing on mitochondrial CoQ (mtCoQ) deficiency. Treatment with 200 nM atorvastatin or simvastatin decreased the total mtCoQ pool (mtCoQ9 + mtCoQ10) by 30-35% and decreased the antioxidant pool mtCoQH2 by 40%, whereas the levels of mitochondrial antioxidant proteins, including superoxide dismutase 2 and uncoupling proteins, remained unchanged. Mitochondria of statin-treated astrocytes showed decreased respiratory activity, membrane potential, and ATP synthesis, and increased mtCoQ reduction leading to increased H2O2 production during the oxidation of complex I (CI) and CII substrates. Statin treatment also altered the organization of the respiratory chain, leading to a downregulation of the CI+CIII2+CIV and CIII2+CIV supercomplexes and decreased protein levels and activity of all respiratory chain complexes. Furthermore, a decrease in cytochrome a + a3 content was accompanied by a reduction in the maximum activity of CIV. CoQ10 supplementation elevated mtCoQ levels, restored respiratory function, and decreased H2O2 production in the mitochondria of statin-treated astrocytes. Prolonged statin exposure alters mtCoQ redox homeostasis and impairs mitochondrial bioenergetic function in astrocytes. CoQ10 supplementation attenuates these changes, supporting its potential role in protecting astrocyte mitochondria from statin-induced dysfunction.

The sperm surface glycocalyx undergoes extensive remodeling during epididymal maturation, required for sperm to reach and bind the oviductal epithelium. N-acetylglucosamine (GlcNAc)-containing glycans are candidate mediators of these events, however, how these residues are regulated across the reproductive tract, and whether their changes depend on specific epididymal enzymes or functionally contribute to sperm-oviduct epithelial adhesion, remains poorly defined. Here, we addressed this gap by examining how surface GlcNAc changes as sperm mature and become functionally competent, from epididymal maturation through capacitation and the acrosome reaction. We further asked whether these changes relate to the ability of spermatozoa to bind the oviductal epithelium. Surface GlcNAc, assessed by WGA reactivity, increased progressively from caput to cauda epididymal spermatozoa, with a corresponding shift in GlcNAc-bearing protein profiles, while remaining predominantly localized to the acrosomal region throughout maturation. Incubation of caput spermatozoa with cauda epididymal fluid reduced WGA labeling, an effect blocked by the selective {beta}-N-acetylglucosaminidase ({beta}-NAG) thiourea derived hydroxy pyrrolidine inhibitor VP150, identifying luminal {beta}-NAG as an active contributor to GlcNAc remodeling in the epididymis. In ejaculated spermatozoa, capacitation induced minor changes in surface GlcNAc, whereas the calcium ionophore-induced acrosome reaction produced a marked reduction in WGA reactivity and acrosomal labeling, consistent with glycoprotein loss during acrosomal exocytosis. Functionally, spermatozoa that bound to BOEC monolayers were preferentially WGA-positive, and pre-incubation of BOECs with WGA significantly reduced sperm adhesion, implicating surface GlcNAc in sperm-oviduct epithelial recognition. Together, these findings define surface GlcNAc as a dynamically regulated glycan that is progressively established during epididymal transit, partly through luminal {beta}-NAG activity, redistributed during capacitation and acrosomal exocytosis, and functionally engaged during sperm-BOEC adhesion, providing a mechanistic framework for glycocalyx-mediated sperm selection in cattle.

Lymphedema is a chronic disease characterized by impaired lymph drainage and accumulation of protein-rich interstitial fluid, which progresses to develop irreversible fibrosis. Importantly, effective therapies and treatments are lacking to alleviate and mitigate the disease. The pathological inflammation and fibrogenesis underlying lymphedema prompted us to evaluate a preclinical medicine IVA337, a pan-peroxisome proliferator-activated receptor (PPAR) agonist that improves liver fibrosis in patients with metabolic dysfunction-associated steatohepatitis (MASH) by activating three PPAR isoforms (, {beta}/{delta}, {lambda}), which play critical roles in lipid metabolism, anti-inflammation responses, and anti-fibrogenesis. Here, we investigate the therapeutic effects of IVA337 during the early stage of surgery-induced secondary lymphedema in mice and explored the underlying mechanisms. IVA337 administration alleviated lymphedema progression, improved lymphatic drainage, reduced dermal thickness, and resolved lymphatic vessel dilation. Mechanistically, IVA337 suppressed the TGF{beta}/SMAD2/3 signaling pathway, reduced immune cells infiltration, and improves lymphatic vessels integrity. In human dermal lymphatic endothelial cells (HDLECs), IVA337 attenuated TGF{beta} induced SMAD2/3 phosphorylation and preserved the expression of cell junction Claudin5, reduced VE-Cadherin-stained cell-cell gaps. Collectively, our findings demonstrate that IVA337 protects against early stage lymphedema by inhibiting TGF{beta}/SMAD2/3-mediated inflammatory and fibrotic responses. This study provides a potential therapeutic strategy to improve lymphatic function during the early phase of lymphedema and prevent progressive fibrosis in patients with lymphedema and related disorders.

Extracellular matrix (ECM) remodeling and angiogenesis are essential processes underlying endometrial decidualization during embryo implantation. Fibrillar collagens, the major structural components of the ECM, form the architectural framework of the decidua and undergo dynamic reorganization during this process. However, the functional role of type V collagen--a key regulator of collagen fibrillogenesis-- remains undefined. Here, we demonstrate that Col5a1 is highly expressed in decidual stromal and endothelial cells of the mouse uterus. Conditional deletion of Col5a1 leads to progressive uterine hemorrhage beginning at gestation day 8, culminating in complete embryo resorption and pregnancy loss by day 12. Col5a1-deficient decidua exhibits severe structural distortion, marked disorganization of fibrillar collagen, shallow and misdirected embryo invasion, and profound disruption of decidual angiogenesis and vascular network formation. Transcriptomic profiling further reveals distinct gene expression signatures and signaling pathways regulated by COL5A1 in the decidua. Collectively, these findings identify type V collagen as a critical ECM regulator required for maintaining decidual integrity, supporting angiogenesis, and ensuring successful pregnancy.

CCCTC-binding factor (CTCF) is frequently mutated in endometrial cancer, resulting in genetic haploinsufficiency that contributes to tumour progression. We previously showed that depletion of CTCF disrupted cell polarity in KLE endometrial cancer spheroids; however, the implications for gene dysregulation and endometrial cancer pathophysiology remains poorly understood. ZNF185, an actin-associated and LIM domain-containing protein involved in cytoskeletal remodelling, was identified as a dysregulated target following CTCF haploinsufficiency. In this study, shRNA-mediated knockdown of CTCF was used to model haploinsufficiency in endometrial cancer cells, leading to the identification of a previously unrecognised isoform of ZNF185, named ZNF185B. Unlike the full-length protein, ZNF185B lacked co-localisation with F-actin and exhibited a diffuse cytoplasmic distribution, and ZNF185B was significantly upregulated in CTCF-depleted endometrial cancer cells and in an auxin-inducible degron model in a dose-dependent manner. Functional studies demonstrated that depletion of ZNF185 expression reduced endometrial cancer cell proliferation and clonogenic potential. Together, these findings identify ZNF185B as a novel isoform negatively regulated by CTCF protein dosage and establish ZNF185 as a requirement for endometrial cancer cell proliferation. Our results suggest that dysregulated ZNF185 expression is a crucial downstream consequence of CTCF haploinsufficiency and may contribute to tumour progression in endometrial cancer.

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.

Colorectal cancer cells increase glycolysis to help meet the metabolic demands required for cell growth. Many factors, both endogenous and exogenous, likely drive cellular metabolism and enhance glycolytic flux in colorectal cells. Interleukin-1 beta (IL-1{beta}) is a pro-inflammatory cytokine that is elevated in colorectal cancer. In this study, we investigated the effect of IL-1{beta} toward driving the cancer cell to increase glycolysis, while also suppressing the oxidation of the fiber-derived nutrient butyrate. The results presented here demonstrate that IL-1{beta} stimulated glycolysis and inhibited maximal mitochondrial respiration. IL-1{beta} also increased the phosphorylation of AKT and hypoxia-inducible factor 1 alpha (HIF1) levels. Utilizing colorectal cancer cells with AKT1/2 or HIF1 knocked out showed the requirement of these proteins in mediating the increase in glycolysis following IL-1{beta} treatment. Importantly, AKT1/2 was identified as upstream of HIF1, as IL-1{beta} still increased phosphorylation of AKT even in the absence of HIF1. However, loss of AKT1/2 completely abolished the ability of IL-1{beta} to increase HIF1 protein levels. Tumor necrosis factor alpha (TNF), another cytokine found to be elevated in colorectal cancer, also increased glycolysis in an AKT and HIF1-dependent manner. Our data point to a common pathway through AKT activation and HIF1 upregulation, by which pro-inflammatory cytokines increase glycolysis in colorectal cancer cells to help promote cancer progression.