Type II Interleukin-4 Receptor Activation in Basal Breast Cancer Cells Promotes Tumor Progression via Metabolic and Epigenetic Modulation.

Interleukin-4 (IL4) is a Th2 cytokine that can signal through two different receptors, one of which-the type II receptor-is overexpressed by various cancer cells. Previously, we have shown that type II IL4 receptor signaling increases proliferation and metastasis in mouse models of breast cancer, as well as increasing glucose and glutamine metabolism. Here, we expand on those findings to determine mechanistically how IL4 signaling links glucose metabolism and histone acetylation to drive proliferation in the context of triple-negative breast cancer (TNBC). We used a combination of cellular, biochemical, and genomics approaches to interrogate TNBC cell lines, which represent a cancer type where high expression of the type II IL4 receptor is linked to reduced survival. Our results indicate that type II IL4 receptor activation leads to increased glucose uptake, Akt and ACLY activation, and histone acetylation in TNBC cell lines. Inhibition of glucose uptake through the deletion of Glut1 ablates IL4-induced proliferation. Additionally, pharmacological inhibition of histone acetyltransferase P300 attenuates IL4-mediated gene expression and proliferation in vitro. Our work elucidates a role for type II IL4 receptor signaling in promoting TNBC progression, and highlights type II IL4 signaling, as well as histone acetylation, as possible targets for therapy.

Mixed-Lineage Leukaemia Gene Regulates Glucose-Sensitive Gene Expression and Insulin Secretion in Pancreatic Beta Cells.

The escalating prevalence of diabetes mellitus underscores the need for a comprehensive understanding of pancreatic beta cell function. Interest in glucose effectiveness has prompted the exploration of novel regulatory factors. The myeloid/lymphoid or mixed-lineage leukaemia gene (MLL) is widely recognised for its role in leukemogenesis and nuclear regulatory mechanisms through its histone methyltransferase activity in active chromatin. However, its function within pancreatic endocrine tissues remains elusive. Herein, we unveil a novel role of MLL in glucose metabolism and insulin secretion. MLL knockdown in ßHC-9 pancreatic beta cells diminished insulin secretion in response to glucose loading, paralleled by the downregulation of the glucose-sensitive genes SLC2a1 and SLC2a2. Similar observations were made in MLL heterozygous knockout mice (MLL+/-), which exhibited impaired glucose tolerance and reduced insulin secretion without morphological anomalies in pancreatic endocrine cells. The reduction in insulin secretion was independent of changes in beta cell mass or insulin granule morphology, suggesting the regulatory role of MLL in glucose-sensitive gene expression. The current results suggest that MLL interacts with circadian-related complexes to modulate the expression of glucose transporter genes, thereby regulating glucose sensing and insulin secretion. Our findings shed light on insulin secretion control, providing potential avenues for therapeutics against diabetes.

The protective role of endothelial GLUT1 in ischemic stroke.

OBJECTIVE: To provide thorough insight on the protective role of endothelial glucose transporter 1 (GLUT1) in ischemic stroke. METHODS: We comprehensively review the role of endothelial GLUT1 in ischemic stroke by narrating the findings concerning biological characteristics of GLUT1 in brain in depth, summarizing the changes of endothelial GLUT1 expression and activity during ischemic stroke, discussing how GLUT1 achieves its neuroprotective effect via maintaining endothelial function, and identifying some outstanding blind spots in current studies. RESULTS: Endothelial GLUT1 maintains persistent high glucose and energy requirements of the brain by transporting glucose through the blood-brain barrier, which preserves endothelial function and is beneficial to stroke prognosis. CONCLUSION: This review underscores the potential involvement of GLUT1 trafficking, activity modulation, and degradation, and we look forward to more clinical and animal studies to illuminate these mechanisms.

Cadmium contributes to cardiac metabolic disruption by activating endothelial HIF1A-GLUT1 axis.

Cadmium (Cd) is an environmental risk factor of cardiovascular diseases. Researchers have found that Cd exposure causes energy metabolic disorders in the heart decades ago. However, the underlying molecular mechanisms are still elusive. In this study, male C57BL/6 J mice were exposed to cadmium chloride (CdCl2) through drinking water for 4 weeks. We found that exposure to CdCl2 increased glucose uptake and utilization, and disrupted normal metabolisms in the heart. In vitro studies showed that CdCl2 specifically increased endothelial glucose uptake without affecting cardiomyocytic glucose uptake and endothelial fatty acid uptake. The glucose transporter 1 (GLUT1) as well as its transcription factor HIF1A was significantly increased after CdCl2 treatment in endothelial cells. Further investigations found that CdCl2 treatment upregulated HIF1A expression by inhibiting its degradation through ubiquitin-proteasome pathway, thereby promoted its transcriptional activation of SLC2A1. Administration of HIF1A small molecule inhibitor echinomycin and A-485 reversed CdCl2-mediated increase of glucose uptake in endothelial cells. In accordance with this, intravenous injection of echinomycin effectively ameliorated CdCl2-mediated metabolic disruptions in the heart. Our study uncovered the molecular mechanisms of Cd in contributing cardiac metabolic disruption by inhibiting HIF1A degradation and increasing GLUT1 transcriptional expression. Inhibition of HIF1A could be a potential strategy to ameliorate Cd-mediated cardiac metabolic disorders and Cd-related cardiovascular diseases.

Design of Evodiamine-Glucose Conjugates with Improved In Vivo Antitumor Activity.

Natural product evodiamine is a multitargeting antitumor lead compound. However, clinical development of evodiamine derivatives was hampered by poor water solubility and limited in vivo antitumor potency. Herein, a series of evodiamine-glucose conjugates were designed by additional targeting glucose transporter-1 (GLUT1). Compared with the lead compound, conjugate 8 exhibited obvious enhancement in water solubility and in vivo antitumor efficacy. Furthermore, the effect of GLUT1 targeting also led to lower cytotoxicity to normal cells. Antitumor mechanism studies manifested that conjugate 8 acted by Top1/Top2 dual inhibition, apoptosis induction, and G2/M cell cycle arrest, which selectively targeted tumor cells with a high expression level of GLUT1. Thus, evodiamine-glucose conjugates showed promising features as potential antitumor agents.

Endocytic vesicles act as vehicles for glucose uptake in response to growth factor stimulation.

Glycolysis is a fundamental cellular process, yet its regulatory mechanisms remain incompletely understood. Here, we show that a subset of glucose transporter 1 (GLUT1/SLC2A1) co-endocytoses with platelet-derived growth factor (PDGF) receptor (PDGFR) upon PDGF-stimulation. Furthermore, multiple glycolytic enzymes localize to these endocytosed PDGFR/GLUT1-containing vesicles adjacent to mitochondria. Contrary to current models, which emphasize the importance of glucose transporters on the cell surface, we find that PDGF-stimulated glucose uptake depends on receptor/transporter endocytosis. Our results suggest that growth factors generate glucose-loaded endocytic vesicles that deliver glucose to the glycolytic machinery in proximity to mitochondria, and argue for a new layer of regulation for glycolytic control governed by cellular membrane dynamics.

Rapid Enrichment of a Native Multipass Transmembrane Protein via Cell Membrane Electrophoresis through Buffer pH and Ionic Strength Adjustment.

Supported membrane electrophoresis is a promising technique for collecting membrane proteins in native bilayer environments. However, the slow mobility of typical transmembrane proteins has impeded the technique's advancement. Here, we successfully applied cell membrane electrophoresis to rapidly enrich a 12-transmembrane helix protein, glucose transporter 1 with antibodies (GLUT1 complex), by tuning the buffer pH and ionic strength. The identified conditions allowed the separation of the GLUT1 complex and a lipid probe, Fast-DiO, within a native-like environment in a few minutes. A force model was developed to account for distinct electric and drag forces acting on the transmembrane and aqueous-exposed portion of a transmembrane protein as well as the electroosmotic force. This model not only elucidates the impact of size and charge properties of transmembrane proteins but also highlights the influence of pH and ionic strength on the driving forces and, consequently, electrophoretic mobility. Model predictions align well with experimentally measured electrophoretic mobilities of the GLUT1 complex and Fast-DiO at various pH and ionic strengths as well as with several lipid probes, lipid-anchored proteins, and reconstituted membrane proteins from previous studies. Force analyses revealed the substantial membrane drag of the GLUT1 complex, significantly slowing down electrophoretic mobility. Besides, the counterbalance of similar magnitudes of electroosmotic and electric forces results in a small net driving force and, consequently, reduced mobility under typical neutral pH conditions. Our results further highlight how the size and charge properties of transmembrane proteins influence the suitable range of operating conditions for effective movement, providing potential applications for concentrating and isolating membrane proteins within this platform.

Exploring the potential of pheophorbide A, a chlorophyll-derived compound in modulating GLUT for maintaining glucose homeostasis.

Introduction: Pheophorbide A, a chlorophyll-breakdown product, is primarily investigated for its anti-oxidant and anti-inflammatory activity. Recent reports on pheophorbide A have shown its potential in lowering blood glucose levels, thus leading to the exploration of its use in diabetes management. Literature has also shown its effect on enhanced insulin secretion, whereas its mechanism on glucose stimulated insulin secretion (GSIS) in pancreatic ß cells remains unexplored. Methods: In-silico and in-vitro investigations were used to explore the effect of pheophorbide A on class I glucose transporters (GLUTs). In-silico studies include - Molecular docking studies and stability assessment using GROMACS. In-vitro studies include - MTT assay, Glucose uptake assay, Live-cell imaging and tracking of GLUTs in presence of Pheophorbide A compared to control. Results: Molecular docking studies revealed better binding affinity of pheophorbide A with GLUT4 (-11.2 Kcal/mol) and GLUT1 (-10.7 Kcal/mol) when compared with metformin (-5.0 Kcal/mol and -4.9 Kcal/mol, respectively). Glucose levels are largely regulated by GLUTs where GLUT1 is one of the transporters that is ubiquitously present in human ß cells. Thus, we confirmed the stability of the complex, that is, pheophorbide A-GLUT1 using GROMACS for 100 ns. We further assessed its effect on a pancreatic ß cell line (INS-1) for its viability using an MTT assay. Pheophorbide A (0.1-1 µM) showed a dose-dependent response on cell viability and was comparable to standard metformin. To assess how pheophorbide A mechanistically acts on GLUT1 in pancreatic ß cell, we transfected INS-1 cells with GLUT1-enhanced green fluorescent protein and checked how the treatment of pheophorbide A (0.50 µM) modulates GLUT1 trafficking using live-cell imaging. We observed a significant increase in GLUT1 density when treated with pheophorbide A (0.442 ± 0.01 µm-2) at 20 mM glucose concentration when compared to GLUT1 control (0.234 ± 0.01 µm-2) and metformin (0.296 ± 0.02 µm-2). The average speed and distance travelled by GLUT1 puncta were observed to decrease when treated with pheophorbide A. The present study also demonstrated the potential of pheophorbide A to enhance glucose uptake in ß cells. Conclusion: The current study's findings were validated by in-silico and cellular analyses, suggesting that pheophorbide A may regulate GLUT1 and might be regarded as a potential lead for boosting the GSIS pathway, thus maintaining glucose homeostasis.

Curcumin Inhibits the PPARδ-p-Akt-GLUT1 Pathway and Ameliorates the Antiproliferative Effects of Doxorubicin in MDA-MB-231 Cells.

OBJECTIVE: The aim of the present study was to examine whether GLUT1 was involved in the antiproliferative activity of curcumin and doxorubicin by understanding mechanistically how curcumin regulated GLUT1. METHODS: Expression level of GLUT1 in MCF-7 and MDA-MB-231 cells were quantitated using quantitative real-time PCR and western blot. GLUT1 activity was inhibited in MDA-MB-231 cells with the pharmacological inhibitor WZB117 to assess the anti-proliferative effects of doxorubicin using MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide).  To examine cell proliferation, trypan blue assay was used in cells transfected with GLUT1 siRNA or plasmid overexpressing GLUT1 with doxorubicin and/or commercially available curcumin. The role of PPARδ and Akt on the regulation of GLUT1 by curcumin was examined by overexpressing these proteins and western blot was employed to examine their protein expression. RESULTS: The data revealed that there was a 1.5 fold increase in GLUT1 mRNA and protein levels in MDA-MB-231 compared to MCF-7.  By inhibiting GLUT1 in triple negative breast cancer cell line, MDA-MB-231 with either the pharmacological inhibitor WZB117 or with GLUT1 siRNA, we observed the enhanced antiproliferative effects of doxorubicin. Additional observations indicated these effects can be reversed by the overexpression of GLUT1. Treatment of MDA-MB-231 with curcumin also revealed downregulation of GLUT1, with further growth suppressive effects when combined with doxorubicin.  Overexpression of GLUT1 blocked the growth suppressive role of curcumin and doxorubicin (p< 0.05). Mechanistically, we also observed that the regulation of GLUT1 by curcumin was mediated by the Peroxisome proliferator-activated receptor (PPAR) δ/Akt pathway. CONCLUSION: Our study demonstrates that regulation of GLUT1 by curcumin via the PPARδ/Akt signaling improves the efficacy of doxorubicin by promoting its growth inhibitory effects in MDA-MB-231 cells.

FGF19 Promotes the Proliferation and Insulin Secretion from Human Pancreatic ß Cells Via the IRS1/GLUT4 Pathway.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is a commonly observed complication associated with obesity. The effect of fibroblast growth factor 19 (FGF19), a promising therapeutic agent for metabolic disorders, on pancreatic ß cells in obesity-associated T2DM remains poorly understood. METHODS: Human pancreatic ß cells were cultured with high glucose (HG) and palmitic acid (PA), followed by treatment with FGF19. The cell proliferation, apoptosis, and insulin secretion were evaluated by CCK-8, qRT-PCR, ELISA, flow cytometry, and western blotting. The expression of the insulin receptor substrate (IRS)/glucose transporter (GLUT) pathway was evaluated. The interaction between FGF19 and IRS1 was predicted using the STRING database and verified by co-immunoprecipitation and immunofluorescence. The regulatory effects of the IRS1/GLUT4 pathway on human pancreatic ß cells were assessed by overexpressing IRS1 and silencing IRS1 and GLUT4. RESULTS: HG+PA treatment reduced the human pancreatic ß cell proliferation and insulin secretion and promoted cell apoptosis. However, FGF19 treatment restored these alterations and significantly increased the expressions of IRS1, GLUT1, and GLUT4 in the IRS/GLUT pathway. Furthermore, FGF19 and IRS1 were found to interact. IRS1 overexpression partially promoted the proliferation of pancreatic ß cells and insulin secretion through GLUT4. Additionally, the silencing of IRS1 or GLUT4 attenuated the therapeutic effects of FGF19. CONCLUSION: In conclusion, FGF19 partly promoted the proliferation and insulin secretion of human pancreatic ß cells and inhibited apoptosis by upregulating the IRS1/GLUT4 pathway. These findings establish a theoretical framework for the clinical utilization of FGF19 in the treatment of obesity-associated T2DM.

Aberrantly Glycosylated GLUT1 as a Poor Prognosis Marker in Aggressive Bladder Cancer.

Muscle-invasive bladder cancer (MIBC) remains a pressing health concern due to conventional treatment failure and significant molecular heterogeneity, hampering the development of novel targeted therapeutics. In our quest for novel targetable markers, recent glycoproteomics and bioinformatics data have pinpointed (glucose transporter 1) GLUT1 as a potential biomarker due to its increased expression in tumours compared to healthy tissues. This study explores this hypothesis in more detail, with emphasis on GLUT1 glycosylation patterns and cancer specificity. Immunohistochemistry analysis across a diverse set of human bladder tumours representing all disease stages revealed increasing GLUT1 expression with lesion severity, extending to metastasis, while remaining undetectable in healthy urothelium. In line with this, GLUT1 emerged as a marker of reduced overall survival. Revisiting nanoLC-EThcD-MS/MS data targeting immature O-glycosylation on muscle-invasive tumours identified GLUT1 as a carrier of short glycosylation associated with invasive disease. Precise glycosite mapping uncovered significant heterogeneity between patient samples, but also common glycopatterns that could provide the molecular basis for targeted solutions. Immature O-glycosylation conferred cancer specificity to GLUT1, laying the molecular groundwork for enhanced targeted therapeutics in bladder cancer. Future studies should focus on a comprehensive mapping of GLUT1 glycosites for highly specific cancer-targeted therapy development for bladder cancer.

Identification and validation of ferroptosis-related gene SLC2A1 as a novel prognostic biomarker in AKI.

BACKGROUND: Emerging evidence reveals the key role of ferroptosis in the pathophysiological process of acute kidney injury (AKI). Our study aimed to investigate the potential ferroptosis-related gene in AKI through bioinformatics and experimental validation. METHODS: The AKI single-cell sequencing dataset was retrieved from the GEO database and ferroptosis-related genes were extracted from the GENECARD website. The potential differentially expressed ferroptosis-related genes of AKI were selected. Functional enrichment analysis was performed. Machine learning algorithms were used to identify key ferroptosis-related genes associated with AKI. A multi-factor Cox regression analysis was used to construct a risk score model. The accuracy of the risk score model was validated using receiver operating characteristic (ROC) curve analysis. We extensively explored the immune landscape of AKI using CIBERSORT tool. Finally, expressions of ferroptosis DEGs were validated in vivo and in vitro by Western blot, ICH and transfection experiments. RESULTS: Three hub genes (BAP1, MDM4, SLC2A1) were identified and validated by constructing drug regulatory network and subsequent screening using experimentally determined interactions. The risk mode showed the low-risk group had significantly better prognosis compared to high-risk group. The risk score was independently associated with overall survival. The ROC curve analysis showed that the prognosis model had good predictive ability. Additionally, CIBERSORT immune infiltration analysis suggest that the hub gene may influence cell recruitment and infiltration in AKI. Validation experiments revealed that SLC2A1 functions by regulating ferroptosis. CONCLUSIONS: In summary, our study not only identifies SLC2A1 as diagnostic biomarker for AKI, but also sheds light on the role of it in AKI progression, providing novel insights for the clinical diagnosis and treatment of AKI.

HNRNPC Regulates GLUT1/LDHA Pathway by Stabilizing FOXM1 mRNA to Promote the Progression and Aerobic Glycolysis of Multiple Myeloma.

OBJECTIVE: Multiple Myeloma (MM) is a malignant hematological disease. Heterogeneous nuclear ribonucleoprotein C1/C2 (HNRNPC) acts as an oncogene in a variety of cancers. However, the role of HNRNPC in MM has not been reported so far. METHODS: The mRNA and protein expressions of HNRN-PC and FOXM1 were detected by qRT-PCR and western blot. CCK8, EDU staining, flow cytometry and western blot were used to detect cell viability and cell cycle. The extracellular flux analyzer XF96 was used to detect the production of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Lactic acid and glucose levels in culture medium were detected by lactic acid assay kits and glucose assay kits, respectively. Then, the binding ability of HNRNPC with FOXM1 was detected by RIP and the stability of FOXM1 mRNA was appraised with qRT-PCR. With the application of qRT-PCR and western blot, the transfection efficacy of si-HNRNPC and Oe-FOXM1 was examined. Western blot was applied for the estimation of GLUT1/LDHA signaling pathway-related proteins. RESULTS: The expression of HNRNPC in MM cell line was abnormally elevated. HNRNPC silence significantly inhibited the proliferation, facilitated the apoptosis, induced cycle arrest, and suppressed aerobic glycolysis in MM cells, which were all reversed by FOXM1 overexpression. It was also found that the regulatory effect of HNRNPC is realized by stabilizing FOXM1 mRNA and regulating GLUT1/LDHA pathway. CONCLUSION: HNRNPC regulated GLUT1/LDHA pathway by stabilizing FOXM1 mRNA to promote the progression and aerobic glycolysis of MM.

Elucidating the role of the GC/GR/GLUT1 axis in steroid-induced osteonecrosis of the femoral head: A proteomic approach.

BACKGROUND: Steroid-induced osteonecrosis of the femoral head (SONFH) is a prevalent and incapacitating condition that affects the hip joint. Unfortunately, early diagnostic and treatment measures are limited. METHODS: Our study employed Tandem Mass Tag (TMT) labeling mass spectrometry (MS)-based quantitative proteome to compare the proteins of femoral head tissues in patients with SONFH with those of patients who sustained femoral neck fracture (FNF). We investigated the level and effects of glucose transporter member 1 (GLUT1) in SONFH patients and MC3T3-E1 cells and examined the function and molecular mechanism of GLUT1 in the context of SONFH using in vivo and in vitro approaches. RESULTS: The SONFH group exhibited significant changes in protein expression levels compared to the fracture group. Specifically, we observed the up-regulation of 86 proteins and the down-regulation of 138 proteins in the SONFH group. Among the differentially expressed proteins, GLUT1 was down-regulated and associated with glucose metabolic processes in the SONFH group. Further analysis using Parallel Reaction Monitoring (PRM), WB, and PCR confirmed that the protein was significantly down-regulated in both femoral head tissue samples from SONFH patients and dexamethasone-treated MC3T3-E1 cells. Moreover, overexpression of GLUT1 effectively reduced glucocorticoid (GC)-induced apoptosis and the suppression of osteoblast proliferation and osteogenic differentiation in MC3T3-E1 cells, as well as GC-induced femoral head destruction in GC-induced ONFH rat models. Additionally, our research demonstrated that GC down-regulated GLUT1 transcription via glucocorticoid receptors in MC3T3-E1 cells. CONCLUSIONS: GLUT1 was down-regulated in patients with SONFH; furthermore, down-regulated GLUT1 promoted apoptosis and inhibited osteoblast ossification in dexamethasone-induced MC3T3-E1 cells and contributed to GC-induced femoral head destruction in a SONFH rat model. Glucocorticoids inhibited the transcriptional activity of GLUT1, leading to a reduction in the amount and activity of GLUT1 in the cells and ultimately promoting apoptosis and inhibiting osteoblast ossification via the GC/GR/GLUT1 axis in SONFH.

[18F]FDG PET/CT Signal Correlates with Neoangiogenesis Markers in Patients with Fibrotic Interstitial Lung Disease Who Underwent Lung Biopsy: Implication for the Use of PET/CT in Diffuse Lung Diseases.

The use of [18F]FDG PET/CT as a biomarker in diffuse lung diseases is increasingly recognized. We investigated the correlation between [18F]FDG uptake with histologic markers on lung biopsy of patients with fibrotic interstitial lung disease (fILD). Methods: We recruited 18 patients with fILD awaiting lung biopsy for [18F]FDG PET/CT. We derived a target-to-background ratio (TBR) of maximum pulmonary uptake of [18F]FDG (SUVmax) divided by the lung background (SUVmin). Consecutive paraffin-embedded lung biopsy sections were immunostained for alveolar and interstitial macrophages (CD68), microvessel density (MVD) (CD31 and CD105/endoglin), and glucose transporter 1. MVD was expressed as vessel area percentage per high-power field (Va%/hpf). Differences in imaging and angiogenesis markers between histologic usual interstitial pneumonia (UIP) and non-UIP were assessed using a nonparametric Mann-Whitney test. Correlation of imaging with angiogenesis markers was assessed using the nonparametric Spearman rank correlation. Univariate Kaplan-Meier survival analysis assessed the difference in the survival curves for each of the angiogenesis markers (separated by their respective optimal cutoff) using the log-rank test. Statistical analysis was performed using SPSS. Results: In total, 18 patients were followed for an average of 41.36 mo (range, 5.69-132.46 mo; median, 30.07 mo). Only CD105 MVD showed a significantly positive correlation with [18F]FDG TBR (Spearman rank correlation, 0.556; P < 0.05, n = 13). There was no correlation between [18F]FDG uptake and macrophage expression of glucose transporter 1. CD105 and CD31 were higher for UIP than for non-UIP, with CD105 reaching statistical significance (P = 0.011). In all patients, MVD assessed with either CD105 or CD31 quantification on biopsy predicted overall survival. Patients with CD105 MVD of less than 12 Va%/hpf or CD31 MVD of less than 35 Va%/hpf had a significantly better prognosis (no deaths during follow-up in the case of CD105) than did patients with higher scores of CD105 MVD (median survival, 35 mo; P = 0.041, n = 13) or CD31 MVD (median survival, 28 mo; P = 0.014, n = 13). Conclusion: Previous work has used [18F]FDG uptake in PET/CT as a biomarker in fILD. Here, we highlight a correlation between angiogenesis and [18F]FDG TBR. We show that MVD is higher for UIP than for non-UIP and is associated with mortality in patients with fILD. These data set the scene to investigate the potential role of vasculature and angiogenesis in fibrosis.

Therapeutic Effect of a Novel M1 Macrophage-Targeted Nanodrug in Chronic Periodontitis Mice.

Chronic periodontitis is a chronic, progressive, and destructive disease. Especially, the large accumulation of advanced glycation end products (AGEs) in a diseased body will aggravate the periodontal tissue damage, and AGEs induce M1 macrophages. In this project, the novel nanodrugs, glucose-PEG-PLGA@MCC950 (GLU@MCC), are designed to achieve active targeting with the help of glucose transporter 1 (GLUT1) which is highly expressed in M1 macrophages induced by AGEs. Then, the nanodrugs release MCC950, which is a kind of NLRP3 inhibitor. These nanodrugs not only can improve the water solubility of MCC950 but also exhibit superior characteristics, such as small size, stability, innocuity, etc. In vivo experiments showed that GLU@MCC could reduce periodontal tissue damage and inhibit cell apoptosis in periodontitis model mice. In vitro experiments verified that its mechanism of action might be closely related to the inhibition of the NLRP3 inflammatory factor in M1 macrophages. GLU@MCC could effectively reduce the damage to H400 cells caused by AGEs, decrease the expression of NLRP3, and also obviously reduce the M1-type macrophage pro-inflammatory factors such as IL-18, IL-1ß, caspase-1, and TNF-α. Meanwhile, the expression of anti-inflammatory factor Arg-1 in the M2 macrophage was increased. In brief, GLU@MCC would inhibit the expression of inflammatory factor NLRP3 and exert antiperiodontal tissue damage in chronic periodontitis via GLUT1 in the M1 macrophage as the gating target. This study provides a novel nanodrug for chronic periodontitis treatment.

WNT-inhibitory factor 1-mediated glycolysis protects photoreceptor cells in diabetic retinopathy.

BACKGROUND: In diabetic retinopathy (DR), hypoxia-inducible factor (HIF-1α) induces oxidative stress by upregulating glycolysis. This process leads to neurodegeneration, particularly photoreceptor cell damage, which further contributes to retinal microvascular deterioration. Further, the regulation of Wnt-inhibitory factor 1 (WIF1), a secreted Wnt signaling antagonist, has not been fully characterized in neurodegenerative eye diseases. We aimed to explore the impact of WIF1 on photoreceptor function within the context of DR. METHOD: Twelve-week-old C57BL/KsJ-db/db mice were intravitreally injected with WIF1 overexpression lentivirus. After 4 weeks, optical coherence tomography (OCT), transmission electron microscopy (TEM), H&E staining, and electroretinography (ERG) were used to assess the retinal tissue and function. The potential mechanism of action of WIF1 in photoreceptor cells was explored using single-cell RNA sequencing. Under high-glucose conditions, 661 W cells were used as an in vitro DR model. WIF1-mediated signaling pathway components were assessed using quantitative real-time PCR, immunostaining, and western blotting. RESULT: Typical diabetic manifestations were observed in db/db mice. Notably, the expression of WIF1 was decreased at the mRNA and protein levels. These pathological manifestations and visual function improved after WIF1 overexpression in db/db mice. TEM demonstrated that WIF1 restored damaged mitochondria, the Golgi apparatus, and photoreceptor outer segments. Moreover, ERG indicated the recovery of a-wave potential amplitude. Single-cell RNA sequencing and in vitro experiments suggested that WIF1 overexpression prevented the expression of glycolytic enzymes and lactate production by inhibiting the canonical Wnt signaling pathway, HIF-1α, and Glut1, thereby reducing retinal and cellular reactive oxygen species levels and maintaining 661 W cell viability. CONCLUSIONS: WIF1 exerts an inhibitory effect on the Wnt/ß-catenin-HIF-1α-Glut1 glycolytic pathway, thereby alleviating oxidative stress levels and mitigating pathological structural characteristics in retinal photoreceptor cells. This mechanism helps preserve the function of photoreceptor cells in DR and indicates that WIF1 holds promise as a potential therapeutic candidate for DR and other neurodegenerative ocular disorders.

Identification of SLC2A1 as a predictive biomarker for survival and response to immunotherapy in lung squamous cell carcinoma.

BACKGROUND: As one of the common subtypes of non-small lung cancer, lung squamous cell carcinoma (LUSC) patients with advanced stage have few choices of treatment strategies. Therefore, it is urgent to discover genes that are associated with the survival and efficacy of immunotherapies. METHOD: Differential gene expression analyses were conducted using TCGA LUSC bulk-sequencing and single-cell RNA-sequencing data. Prognostic genes were identified from the TCGA LUSC cohort. Protein expression validation and survival analyses were performed. Experiments were conducted to explore the underlying mechanisms. In addition, the correlation between gene expression and pathological response to adjuvant immunochemotherapy was also investigated. RESULTS: After a series of bioinformatic analyses, solute carrier family 2 member 1(SLC2A1), encoding glucose transporter-1 (GLUT1), was found to be differentially expressed between tumor and normal tissues. GLUT1 was subsequently identified as an independent prognostic factor for LUSC. GSEA analysis revealed the glycolysis metabolism pathway of KEGG enriched in SLC2A1high tumor tissues. LASSO analyses revealed that tumor tissues with high expression of SLC2A1 were associated with high levels of protein lactylation. We found that SLC2A1 was preferentially expressed by SPP1+ macrophages in the tumor microenvironment, and the expression of SLC2A1 was associated with the abundance of SPP1+ macrophages. Immunofluorescence demonstrated GLUT1 and HIF1α colocalization in tumor-infiltrating macrophages. In vitro experiments showed HIF-1α-induced macrophage polarization under hypoxia, and GLUT1 inhibition blocked this polarization. In addition, SLC2A1 was negatively associated with the common immune checkpoint molecules, such as programmed cell death 1(PD-1), T cell immunoreceptor with Ig and ITIM domains (TIGIT), cytotoxic T-lymphocyte associated protein 4 (CTLA4) and lymphocyte activating 3 (LAG3), while showed a positive association with CD44. Finally, we observed that there was a significant correlation between pre-adjuvant-treatment GLUT1 expression and the pathological response. CONCLUSION: SLC2A1 expression was differentially upregulated in tumor tissues, and elevated GLUT1 expression was associated with worse survival and poor pathological response to adjuvant immunochemotherapy. Upregulation of GLUT1 promoted macrophage polarization into the M2 phenotype. The findings will contribute to guiding the treatment selection for LUSC patients and providing personalized immunotherapy strategies.

Perineural differentiation in neurotized nevi.

BACKGROUND: Perineuriomatous melanocytic nevi are rare and this may indicate the similar embryological source of melanocytes and peripheral nerves in the neural crest. Neurotized melanocytic nevi may resemble nerve sheath tumors histologically, and show schwannian differentiation. However, literature on whether neurotized nevi differentiate into perineural cells is controversial. We examined our cases of neurotized nevi for evidence of perineural differentiation. MATERIALS AND METHODS: A total of 100 benign nevi with large neurotized component (microscopically involved a low power field 4.2 mm in diameter) were prospectively evaluated in excisional biopsy samples. Immunohistochemical stainings for EMA, Claudin1, Glut1 and neurofilament were performed. RESULTS: Perineural differentiation was immunohistochemically detected in the neurotized component of the nevi in 61% of the cases with EMA and in all the cases with Glut1 and Claudin1. Axonal differentiation was not detected with neurofilament. The expression pattern, especially with Glut1, was usually in form of partial or complete staining surrounding the Meissner's corpuscle-like structure (MCLS). Also, a linear/curvilinear staining pattern was observed particularly with Claudin1. A diffuse staining pattern with EMA, Glut1 and Claudin1 was detected in a case with a microscopically distinct whorl structure, and in which spindle cells are separated from the superficial epithelioid melanocytes with an abrupt transition histologically. These findings of the case are compatible with previous reports of perineuromatous nevus. CONCLUSION: Perineural differentiation is not uncommon and immunohistochemically observed in all nevi with a relatively large component of neurotization. To prevent misdiagnosing desmoplastic melanoma and overtreating patients, it is crucial to be aware of perineuromatous nevi.

The role of adhesion molecules in osteocalcin-induced effects on glucose and lipid metabolism in adipocytes.

Recent findings suggest that uncarboxylated osteocalcin (GluOC) promotes glucose and lipid metabolism via its putative receptor GPRC6A; however, its direct effect on adipocytes remains elusive. In this study, we elucidated the effects of GluOC on adipocytes, with an emphasis on the role of cell adhesion molecules. We determined that GluOC promoted the expression of adipocyte adhesion molecule (ACAM) and its transcription factor Krüppel-like factor 4 and enhanced the cortical actin filament assembly, which ameliorated lipid droplet hypertrophy. Additionally, GluOC upregulated the expression of integrin αVß3 and activation of focal adhesion kinase (FAK) and prevented insulin receptor substrate 1 (IRS1) degradation by inhibiting the ubiquitin-proteasome system via the FAK-PLC-PKC axis, which activated IRS1-Akt-mediated glucose transporter 4 (GLUT4) transport. Furthermore, we showed that GluOC elevated the expression of the insulin-independent glucose transporters GLUT1 and GLUT8, which facilitated insulin stimulation-independent glucose transport. The GluOC-induced activation of integrin αVß3 signaling promoted microtubule assembly, which improved glucose and lipid metabolism via its involvement in intracellular vesicular transport. GluOC treatment also suppressed collagen type 1 formation, which might prevent adipose tissue fibrosis in obese individuals. Overall, our results imply that GluOC promotes glucose and lipid metabolism via ACAM, integrin αVß3, and GLUT1 and 8 expression, directly affecting adipocytes.