Level Of Glycolysis Research Articles

3,5-diCQA suppresses colorectal cancer cell growth through ROS/AMPK/mTOR mediated mitochondrial dysfunction and ferroptosis

ABSTRACT 3,5-diCQA has been shown to have anti-tumor effect by decreasing cancer cell growth. However, the molecular mechanism by which 3,5-diCQA impacts colorectal cancer (CRC) cells is unknown. This study discovered that 3,5-diCQA had a suppressive effect on CRC cells, mainly in the inhibition of proliferation, migration, and the enhancement of apoptosis in HCT116 and SW480 cells. Additionally, 3,5-diCQA was found to cause cell cycle arrest in CRC cells. Meanwhile, we found that 3,5-diCQA activates the AMPK pathway through the generation of ROS, mediates mitochondrial damage, and reduces mitochondrial aerobic glycolysis and oxidative phosphorylation levels. 3,5-diCQA promoted oxidative damage and ferroptosis in CRC cells. Hence, we added ROS inhibitor NAC and found that the NAC reversed the effects of 3,5-diCQA on proliferation, apoptosis, ROS generation, and ferroptosis in CRC cells. Moreover, 3,5-diCQA was also shown to suppress the development of CRC tumor in a tumor-forming model of nude mice. In conclusion, we found that 3,5-diCQA enhances the oxidative damage and ferroptosis while reducing proliferation and migration of CRC cells, depending on mitochondrial dysfunction caused by the ROS/AMPK/mTOR pathway.

ENO2 as a Biomarker Regulating Energy Metabolism to Promote Tumor Progression in Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most common and metastatic type of renal cell carcinoma. Despite significant advancements, the current diagnostic biomarkers for ccRCC lack the desired specificity and sensitivity, necessitating the identification of novel biomarkers and elucidation of their underlying mechanisms. Three gene expression profile datasets were obtained from the GEO database, and differentially expressed genes (DEGs) were screened. Gene Ontology and KEGG pathway analysis were conducted in ccRCC. To clarify the diagnosis and prognostic role of ENO2, Kaplan-Meier analysis and Cox proportional hazards regression analysis were performed. Functional experiments were also carried out to verify the significant role of ENO2 in ccRCC. Finally, tumor mutational burden analysis was utilized to investigate the potential role of ENO2 in gene mutations in ccRCC. The study showed that ENO2 is a potential biomarker for the diagnosis of ccRCC and can independently predict the clinical prognosis of ccRCC. Furthermore, we found that ENO2 can promote the occurrence and progression of ccRCC by affecting the glycolysis level of cells through the "Warburg effect". These findings provide new theories for the occurrence and development of ccRCC and can help formulate new strategies for its diagnosis and treatment.

Ubiquitination of ASCL1 mediates CD47 transcriptional activation of the AKT signaling pathway, and glycolysis promotes osteogenic differentiation of hBMSCs

Bones are extremely dynamic organs that continually develop and remodel. This process involves changes in numerous gene expressions. hBMSC cells can promote osteogenic differentiation. The purpose of this study was to elucidate the mechanism by which ASCL1 promotes osteogenic differentiation in hBMSC cells while decreasing glycolysis. hBMSCs were induced to differentiate into osteoblasts. The ASCL1 expression level during hBMSC osteogenic differentiation was measured by RT‒qPCR, Western blotting, and immunofluorescence. The differentiation level of osteoblasts was observed after staining with ALP and alizarin red. ChIP-qPCR were used to determine the relationship between ASCL1 and CD47, and the expression of glycolysis-related proteins was detected. Overexpression of ASCL1 was used to determine its impact on osteogenic differentiation. si-USP8 was used to verify the ubiquitination of ASCL1-mediated CD47/AKT pathway’s impact on hBMSC glycolysis and osteogenic differentiation. The results showed that the expression of ASCL1 was upregulated after the induction of osteogenic differentiation in hBMSCs. From a functional perspective, knocking down USP8 can promote the ubiquitination of ASCL1, while the osteogenic differentiation ability of hBMSCs was improved after the overexpression of ASCL1, indicating that ASCL1 can promote the osteogenic differentiation of hBMSCs. In addition, USP8 regulates the ubiquitination level of ASCL1 and mediates CD47 transcriptional regulation of the AKT pathway to increase the glycolysis level of hBMSCs and cell osteogenic differentiation. USP8 ubiquitination regulates the level of ASCL1. In addition, ubiquitination of ASCL1 mediates CD47 transcription to activate the AKT signaling pathway and increase hBMSC glycolysis to promote osteogenic differentiation.

TRIM32 promotes oral squamous cell carcinoma progression by enhancing FBP2 ubiquitination and degradation

The aberrant expression of TRIM32, an E3 ubiquitin ligase, has been identified in multiple malignant cancer types. Nevertheless, the functional roles and detailed mechanisms of TRIM32 in oral squamous cell carcinoma (OSCC) remain to be elucidated. Here, we investigated TRIM32 expression and its functional role in OSCC. TRIM32 expression was consistently elevated in OSCC tissues, particularly in samples from patients with advanced clinical grades. Functionally, silencing TRIM32 dampened OSCC cell growth, migration and invasion. Additionally, a xenograft tumor model suggested that TRIM32 knockdown suppressed in vivo OSCC tumor growth and lung metastasis formation. Mechanistically, we discovered that TRIM32 directly bound to the FBP2 protein via mass spectrometry and co-immunoprecipitation. TRIM32 could interact with FBP2 and accelerates its degradation, eventually enhancing glycolysis in OSCC cell lines. Importantly, rescue assays demonstrated that FBP2 silencing could at least partially offset the tumor-suppressive and aerobic glycolysis inhibition effect induced by TRIM32 knockdown. Thus, our findings demonstrate that TRIM32 plays a crucial role in promoting tumor growth and enhancing glycolysis through FBP2 inhibition. Given OSCC is associated with increased glycolysis levels, our study suggests potential therapeutic targets for OSCC treatment.

Iguratimod promotes functional recovery after SCI by repairing endothelial cell tight junctions

Destruction of the blood-spinal cord barrier (BSCB) after spinal cord injury (SCI) is an important factor promoting the progression of the injury. This study addressed how to repair the BSCB in order to promote the repair of injured spinal cords. Iguratimod (IGU), an anti-rheumatic drug, has been approved for clinical use. A spinal cord injury mouse model and TNF-α-stimulated bEnd.3 cells were used to investigate the effect and mechanism of IGU on injured BSCB. An intracerebroventricular osmotic pump was used to administer drugs to the SCI mouse model. The results showed that the SCI mice in the treatment group had better recovery of neurological function than the control group. Examination of the tissue revealed better repair of the BSCB in injured spinal cords after medication. According to the results from the cell model, IGU promoted the expression of tight junction proteins and reduced cell permeability. Further research found that IGU repaired the barrier function by regulating glycolysis levels in the injured endothelial cells. In studying the mechanism, IGU was found to regulate HIF-1α expression through the NF-κB pathway, thereby regulating the expression of the glycolytic enzymes related to endothelial injury. In summary, IGU promoted functional recovery in vivo by repairing the BSCB. In vitro, IGU regulated the level of glycolysis in the damaged endothelium through the NF-κB pathway, thereby repairing the tight junctions between the endothelium. Therefore, IGU may become a potential drug for treating spinal cord injury.

Unraveling the transcriptome-based network of tfh cells in primary sjogren syndrome: insights from a systems biology approach.

Primary Sjogren Syndrome (pSS) is an autoimmune disease characterized by immune cell infiltration. While the presence of follicular T helper (Tfh) cells in the glandular microenvironment has been observed, their biological functions and clinical significance remain poorly understood. We enrolled a total of 106 patients with pSS and 46 patients without pSS for this study. Clinical data and labial salivary gland (LSG) biopsies were collected from all participants. Histological staining was performed to assess the distribution of Tfh cells and B cells. Transcriptome analysis using RNA-sequencing (RNA-seq) was conducted on 56 patients with pSS and 26 patients without pSS to uncover the underlying molecular mechanisms of Tfh cells. To categorize patients, we employed the single-sample gene set enrichment analysis (ssGSEA) algorithm, dividing them into low- and high-Tfh groups. We then utilized gene set enrichment analysis (GSEA), weighted gene co-expression network analysis (WGCNA), and deconvolution tools to explore functional and immune infiltration differences between the low- and high-Tfh groups. Patients with pSS had a higher positive rate of the antinuclear antibody (ANA), anti-Ro52, anti-SSA, anti-SSB and hypergammaglobulinaemia and higher levels of serum IgG compared to the non-pSS. Histopathologic analyses revealed the presence of Tfh cells (CD4+CXCR5+ICOS+) in germinal centers (GC) within the labial glands of pSS patients. GSEA, WGCNA, and correlation analysis indicated that the high-Tfh group was associated with an immune response related to virus-mediated IFN response and metabolic processes, primarily characterized by hypoxia, elevated glycolysis, and oxidative phosphorylation levels. In pSS, most immune cell types exhibited significantly higher infiltration levels in the high-Tfh group compared to the low-Tfh group. Additionally, patients in the Tfh-high group demonstrated a higher positive rate of the ANA, rheumatoid factor (RF), and hypergammaglobulinaemia, as well as higher serum IgG levels. Our study suggests that Tfh cells may play a crucial role in the pathogenesis of pSS and could serve as potential therapeutic targets in pSS patients.

IL-27 enhances peripheral B cell glycolysis of rheumatoid arthritis patients via activating mTOR signaling.

Our previous study found that increased serum IL-27 could promote rheumatoid arthritis (RA) B cell dysfunction via activating mTOR signaling pathway. This study aimed to explore the effects of IL-27 on B cell metabolism and clarify the mechanisms via which IL-27 enhancing glycolysis to induce B cells hyperactivation. Peripheral CD19+ B cells were purified from healthy controls (HC) and RA patients and then cultured with or without anti-CD40/CpG and glycolysis inhibitor 2-deoxy-D-glucose (2-DG) or mTOR inhibitor rapamycin. Furthermore, the isolated CD19+ B cells were treated by HC serum or RA serum in the presence and absence of recombinant human IL-27 or anti-IL-27 neutralizing antibodies or 2-DG or rapamycin. The B cell glycolysis level, proliferation, differentiation and inflammatory actions were detected by qPCR, flow cytometry or ELISA. We found that the glycolysis in RA B cells was increased significantly compared with HC B cells. Glycolysis inhibition downregulated the proliferation, differentiation, and inflammatory actions of RA B cells. RA serum and IL-27 promoted B cell glycolysis, which could be obviously rescued by anti-IL-27 antibodies or mTOR inhibitor rapamycin. Our results suggest that the enhanced cellular glycolysis of RA B cells induced by IL-27 may contribute to B cells hyperactivation through activating the mTOR signaling pathway.

Metabolic reprogramming of proinflammatory macrophages by target delivered roburic acid effectively ameliorates rheumatoid arthritis symptoms

Rheumatoid arthritis (RA) is a common chronic inflammatory disorder that usually affects joints. It was found that roburic acid (RBA), an ingredient from anti-RA herb Gentiana macrophylla Pall., displayed strong anti-inflammatory activity. However, its medical application is limited by its hydrophobicity, lack of targeting capability and unclear functional mechanism. Here, we constructed a pH responsive dual-target drug delivery system hitchhiking RBA (RBA-NPs) that targeted both CD44 and folate receptors, and investigated its pharmacological mechanism. In rat RA model, the nanocarriers effectively delivered RBA to inflammatory sites and significantly enhanced the therapeutic outcomes compared with free RBA, as well as strongly reducing inflammatory cytokine levels and promoting tissue repair. Following analysis revealed that M1 macrophages in the joints were reprogrammed to M2 phenotype by RBA. Since the balance of pro- and anti-inflammatory macrophages play important roles in maintaining immune homeostasis and preventing excessive inflammation in RA, this reprogramming is likely responsible for the anti-RA effect. Furthermore, we revealed that RBA-NPs drove M1-to-M2 phenotypic switch by down-regulating the glycolysis level via blocking ERK/HIF-1α/GLUT1 pathway. Thus, our work not only developed a targeting delivery system that remarkably improved the anti-RA efficiency of RBA, but also identified a potential molecular target to reversely reprogram macrophages though energy metabolism regulation.

GLUCOSE LEVELS OF PLEURAL EFFUSION FLUID AND HER2 STATUS IN PLEURAL-METASTATIC BREAST CANCER

Highlights Patients with breast cancer who have distant metastases are frequently impacted by pleural effusion. HER2 status was found to be substantially correlated with glucose levels in pleural effusion fluid. Abstract Background: Patients diagnosed with breast cancer who have also been affected by distant metastases often suffer from pleural effusion. The prognosis of malignant pleural effusions worsens when glucose levels in the pleural fluid are low. Elevated levels of anaerobic glycolysis due to HER2 overexpression cause breast cancer cells to take in more glucose. Objective: This study aimed to identify any correlation between glucose levels in pleural fluid and HER2 status in breast cancer patients. Material and Method: In this research, 35 female patients with thoracocentesis pleural metastatic breast cancer participated in an analytical observational study using a cross-sectional design. Patients who had undergone thoracocentesis had their HER2 status determined based on the information included in their medical records in the Integrated Oncology Clinic (Poli Onkologi Satu Atap (POSA)) Dr. Soetomo General Academic Hospital, Surabaya, Indonesia. After obtaining data on glucose levels in pleural fluid and HER2 status, statistical analysis was carried out to determine the relationship between the two parameters. Results: The statistical test results with a 95% significance level obtained p=0.004 (p<0.05), so it could be statistically said that there was a significant relationship between the glucose level of pleural fluid effusion with HER2 status. The obtained OR values were 10.93 and CI=95% (1.87-63.97), so that it could be interpreted that the low-glucose levels of pleural effusion fluid increase the proportion of patients with positive HER2 compared to not low-glucose levels of pleural effusion fluid. The correlation between the glucose level of pleural effusion fluid and HER2 status was moderately positive (Chi-Square Contingency Coefficient = 0.44). Conclusion: Glucose levels in pleural effusion fluid were significantly correlated with HER2 status.

Hyperpolarized Carbon-13 MRI in Breast Cancer.

One of the hallmarks of cancer is metabolic reprogramming, including high levels of aerobic glycolysis (the Warburg effect). Pyruvate is a product of glucose metabolism, and 13C-MR imaging of the metabolism of hyperpolarized (HP) [1-13C]pyruvate (HP 13C-MRI) has been shown to be a potentially versatile tool for the clinical evaluation of tumor metabolism. Hyperpolarization of the 13C nuclear spin can increase the sensitivity of detection by 4-5 orders of magnitude. Therefore, following intravenous injection, the location of hyperpolarized 13C-labeled pyruvate in the body and its subsequent metabolism can be tracked using 13C-MRI. Hyperpolarized [13C]urea and [1,4-13C2]fumarate are also likely to translate to the clinic in the near future as tools for imaging tissue perfusion and post-treatment tumor cell death, respectively. For clinical breast imaging, HP 13C-MRI can be combined with 1H-MRI to address the need for detailed anatomical imaging combined with improved functional tumor phenotyping and very early identification of patients not responding to standard and novel neoadjuvant treatments. If the technical complexity of the hyperpolarization process and the relatively high associated costs can be reduced, then hyperpolarized 13C-MRI has the potential to become more widely available for large-scale clinical trials.

Integrated application of transcriptomics and metabolomics provides insights into acute hepatopancreatic necrosis disease resistance of Pacific white shrimp Litopenaeus vannamei.

Acute hepatopancreatic necrosis disease (AHPND) has caused a huge economic loss to shrimp aquaculture. Vibrio parahaemolyticus (VpAHPND) is regarded as a major causative agent of AHPND in the Pacific white shrimp Litopenaeus vannamei. However, knowledge about how shrimp resist to AHPND is very limited. In order to learn the molecular mechanisms underlying AHPND resistance of shrimp, comparison between disease-resistant family and susceptible family of L. vannamei were performed at transcriptional and metabolic levels. Integrated analysis of transcriptomics and metabolomics on hepatopancreas of shrimp, the target tissue of VpAHPND, showed that significant differences existed between resistant family and susceptible family of shrimp. The susceptible family showed higher level of glycolysis, serine-glycine metabolism, purine and pyrimidine metabolism, but lower level of betaine-homocysteine metabolism in the hepatopancreas in comparison with the resistant family without VpAHPND infection. Curiously, VpAHPND infection induced up-regulation of glycolysis, serine-glycine metabolism, purine metabolism, pyrimidine metabolism, and pentose phosphate pathway, and down-regulation of betaine-homocysteine metabolism in resistant family. In addition, arachidonic acid metabolism and some immune pathways, like NF-κB and cAMP pathways, were up-regulated in the resistant family after VpAHPND infection. In contrast, amino acid catabolism boosted via PEPCK-mediated TCA cycle flux was activated in the susceptible family after VpAHPND infection. These differences in transcriptome and metabolome between resistant family and susceptible family might contribute to the resistance of shrimp to bacteria. IMPORTANCE Vibrio parahaemolyticus (VpAHPND) is a major aquatic pathogen causing acute hepatopancreatic necrosis disease (AHPND) and leads to a huge economic loss to shrimp aquaculture. Despite the recent development of controlling culture environment, disease resistant broodstock breeding is still a sustainable approach for aquatic disease control. Metabolic changes occurred during VpAHPND infection, but knowledge about the metabolism in resistance to AHPND is very limited. Integrated analysis of transcriptome and metabolome revealed the basal metabolic differences exhibited between disease-resistant and susceptible shrimp. Amino acid catabolism might contribute to the pathogenesis of VpAHPND and arachidonic acid metabolism might be responsible for the resistance phenotype. This study will help to enlighten the metabolic and molecular mechanisms underlying shrimp resistance to AHPND. Also, the key genes and metabolites of amino acid and arachidonic acid pathway identified in this study will be applied for disease resistance improvement in the shrimp culture industry.

Metabolic reprogramming regulates microglial polarization and its role in cerebral ischemia reperfusion.

The brain is quite sensitive to changes in energy supply because of its high energetic demand. Even small changes in energy metabolism may be the basis of impaired brain function, leading to the occurrence and development of cerebral ischemia/reperfusion (I/R) injury. Abundant evidence supports that metabolic defects of brain energy during the post-reperfusion period, especially low glucose oxidative metabolism and elevated glycolysis levels, which play a crucial role in cerebral I/R pathophysiology. Whereas research on brain energy metabolism dysfunction under the background of cerebral I/R mainly focuses on neurons, the research on the complexity of microglia energy metabolism in cerebral I/R is just emerging. As resident immune cells of the central nervous system, microglia activate rapidly and then transform into an M1 or M2 phenotype to correspond to changes in brain homeostasis during cerebral I/R injury. M1 microglia release proinflammatory factors to promote neuroinflammation, while M2 microglia play a neuroprotective role by secreting anti-inflammatory factors. The abnormal brain microenvironment promotes the metabolic reprogramming of microglia, which further affects the polarization state of microglia and disrupts the dynamic equilibrium of M1/M2, resulting in the aggravation of cerebral I/R injury. Increasing evidence suggests that metabolic reprogramming is a key driver of microglial inflammation. For example, M1 microglia preferentially produce energy through glycolysis, while M2 microglia provide energy primarily through oxidative phosphorylation. In this review, we highlight the emerging significance of regulating microglial energy metabolism in cerebral I/R injury.

#4645 HISTONE LACTYLATION ENHANCES PERITONEAL FIBROSIS BY INDUCING PERITONEAL MESOTHELIAL CELL SENESCENCE

Abstract Background and Aims Peritoneal fibrosis (PF) is a serious complication limiting the application of peritoneal dialysis (PD). Histone lactylation has recently been found to increase with glycolysis and intracellular lactate levels, thereby mediating chronic organ damage. This study investigated the role and mechanism of histone lactylation in high glucose peritoneal dialysate-induced peritoneal fibrosis in mice. Method The PF mouse model induced by high glucose peritoneal dialysate was constructed by daily intraperitoneal injection of 4.25% glucose peritoneal dialysate combined with 40 mM Methylglyoxal for 2 weeks. We also used a lactylation enhancer (rotenone) and a lactylation inhibitor (oxamate) to validate the effects of histone lactylation in vivo and in vitro. The parietal peritoneum samples and cultured peritoneal mesothelial cells in various groups were analyzed for evidence of senescence, fibrosis, glycolysis, and histone lactylation levels. In addition, we generated DcR2 knockout mice to further verify the effects of histone lactylation on peritoneal mesothelial cell (PMCs) senescence by clearing senescent cells. Results In the PF group, the results of Masson staining and immunohistochemistry revealed increased peritoneal thickness and expression of fibrosis markers (α-SMA, Collagen I, and Fibronectin) and senescence markers (P16, P21, and DcR2) compared to the control group. We found that lactate and glycolysis levels were significantly higher in the PF group compared to the control group, and further immunoprecipitation results also showed enhanced levels of histone lactylation modifications in the PF group. We further found that the level of lactylation modification of H3K18la site increased significantly, suggesting that H3K18la site may play an important role in promoting PF. Similarly, in vitro, we verified that peritoneal mesothelial cells (PMCs) exhibited enhanced levels of lactate and lactylation in response to stimulation by high glucose, while we found an increasing expression of cell senescence phenotype (P16, P21, and DcR2) and senescence-associated cytokines secretion (IL-1β, IL-6, and TNF-α). By enhancing or inhibiting lactylation, we found that PMCs senescence was significantly enhanced in the lactylation-enhanced group, while the lactylation-inhibited group alleviated PMCs senescence induced by high glucose peritoneal dialysate. In the PF model of DcR2 knockout mice, compared with the wild group, there was no significant difference in the level of histone lactylation, while peritoneal thickness and the expression of markers of fibrosis markers (α-SMA, Collagen I, and Fibronectin) were significantly reduced. Conclusion Histone lactylation leads to peritoneal fibrosis induced by high glucose peritoneal dialysate by promoting PMSCs senescence. Histone lactylation may be a vital mechanism of peritoneal dialysis-related peritoneal fibrosis.

Glucose metabolism and function of CD4+ Tregs are regulated by the TLR8/mTOR signal in an environment of SKOV3 cell growth.

To investigate the role of mammalian target of rapamycin (mTOR) signal in Toll-like receptor (TLR) 8-mediated regulation of glucose metabolism and its effect on reversing immunosuppression in CD4+ regulatory T-cells (Tregs) in ovarian cancer (OC). Fluorescence-activated cell sorting was used to detect the expression levels of mTOR+ and 4E-BP1+ cells in CD4+ Tregs. The prognosis and immune infiltration analysis of mTOR mRNA in OC were performed using the TIMER and Kaplan-Meier plotter database. Furthermore, real-time polymerase chain reaction (RT-PCR) and western blot (WB) were used to detect expression levels of glucose metabolism-related genes and proteins in CD4+ Tregs. Glucose uptake and glycolysis levels were detected by colorimetry, while the effects of CD4+ Tregs on the proliferation of CD4+ T-effector cells (Teffs) were evaluated by carboxyfluorescein diacetate succinimidyl ester (CFSE). mTOR expression in CD4+ Tregs was significantly higher in patients with OC compared with controls and in CD4+ Tregs than in CD4+ Teffs in OC. Additionally, the expression level of mTOR mRNA was related to prognosis and immune infiltration levels in patients with OC. Blocking the mTOR signal resulted in downregulation of glucose metabolism in CD4+ Tregs. Simultaneous inhibition of the mTOR signal while activation of the TLR8 signal had a coordinated inhibitory effect on glucose metabolism and the immunosuppressive function of CD4+ Tregs. Furthermore, the mTOR signal played an essential role in TLR8-mediated reversal of immunosuppressive function in CD4+ Tregs. These findings imply that activation of the TLR8 signal inhibits glucose metabolism in CD4+ Tregs by downregulating mTOR signaling, thereby reversing the immunosuppressive function of these cells in an OC cell growth environment.

Exosomes from PYCR1 knockdown bone marrow mesenchymal stem inhibits aerobic glycolysis and the growth of bladder cancer cells via regulation of the EGFR/PI3K/AKT pathway.

Bladder cancer (BC) is a heterogeneous disease, and pyrroline‑5‑carboxylate reductase 1 (PYCR1) can promote the proliferation and invasion of BC cells and accelerate BC progression. In the present study, si‑PYCR1 was loaded into bone marrow mesenchymal stem cell (BMSC)‑derived exosomes (Exos) in BC. First, PYCR1 levels in BC tissues/cells were assessed, and cell proliferation, invasion, and migration were evaluated. Aerobic glycolysis levels (glucose uptake, lactate production, ATP production, and the expression of relevant enzymes) and the EGFR/PI3K/AKT pathway phosphorylation levels were determined. PYCR1‑EGFR interactions were examined by co‑immunoprecipitation experiments. RT4 cells transfected with oe‑PYCR1 were treated with EGFR inhibitor CL‑387785. Exos were loaded with si‑PYCR1 and identified, followed by an assessment of their effects on aerobic glycolysis and malignant cell behaviors. Nude mouse models of xenograft tumors were established by injecting mice with Exo‑si‑PYCR1 and Exo‑si‑PYCR1. PYCR1 was upregulated in BC cells, with the highest expression observed in T24 cells and the lowest expression in RT4 cells. Following PYCR1 knockdown, the malignant behaviors of T24 cells and aerobic glycolysis were decreased, while PYCR1 overexpression in RT4 cells averted these trends. PYCR1 interacted with EGFR, and CL‑387785 inhibited the EGFR/PI3K/AKT pathway and attenuated the effects of PYCR1 overexpression on RT4 cells but had no effect on PYCR1 expression. Exo‑si‑PYCR1 showed stronger inhibitory effects on aerobic glycolysis and on the malignant behaviors of T24 cells than si‑PYCR1. Exo‑si‑PYCR1 blocked xenograft tumor growth and had good biocompatibility. Briefly, PYCR1 knocking loaded by BMSC‑derived Exos suppressed aerobic glycolysis and BC growth via the PI3K/AKT pathway by binding to EGFR.

Increased levels of PD1 and glycolysis in CD4+ T cells are positively associated with lymph node metastasis in OSCC

BackgroundCervical lymph node metastasis is one of the poorest prognostic factors in oral squamous cell carcinoma (OSCC). Activated immune cells generally have metabolic abnormalities in the tumour microenvironment. However, it is unknown whether abnormal glycolysis in T cells could facilitate metastatic lymph nodes in OSCC patients. The aim of this study was to investigate the effects of immune checkpoints in metastatic lymph nodes and determine the correlation between glycolysis and immune checkpoint expression in CD4+ T cells.MethodsFlow cytometry and immunofluorescence staining were used to analyse the differences in CD4+ PD1+ T cells between metastatic lymph nodes (LN+) and negative lymph nodes (LN−). RT‒PCR was performed to detail the expression of immune checkpoints and glycolysis-related enzymes in LN+ and LN−.ResultsThe frequency of CD4+ T cells decreased in LN+ patients (p = 0.0019). The PD1 expression of LN+ increased markedly compared to that of LN− (p = 0.0205). Similarly, the PD1 of CD4+ T cells in LN+ increased significantly compared to that of LN−. Additionally, glycolysis-related enzyme levels in CD4+ T cells from LN+ patients were dramatically higher than those in LN− patients. PD1 and Hk2 expression in CD4+ T cells was also increased in LN+ OSCC patients with prior surgical treatment compared to those without.ConclusionsThese findings suggest that lymph node metastasis and recurrence in OSCC are associated with increases in PD1 and glycolysis in CD4+ T cells; this response may serve as a potential regulator of OSCC progression.

Lactate-induced lactylation circularly regulates glucose metabolism enzymes in breast cancer.

e13092 Background: Breast cancer is the most common malignant tumor in the female population worldwide and has become the disease with the highest cancer-related mortality. Among the molecular subtypes of breast cancer, triple-negative breast cancer accounts for only 15% of primary malignancies, but due to its increasing recurrence, metastasis, and mortality, we need to find new therapeutic targets to address this phenomenon. Analysis from the field of metabolism found that the level of glycolysis in triple-negative breast cancer was significantly higher than that in other cancers. Glycolysis is a common malignant phenotype of cancer, namely the famous Warburg effect. LDHA is the last metabolic enzyme in the glycolytic pathway, and its expression is increased in triple-negative breast cancer. Lactate is the final product of glycolysis, which converts pyruvate to lactate and NADH to NAD+ under the catalysis of LDHA. The acidic tumor microenvironment formed by the accumulation of lactate will not only affect the progression of tumors and the therapeutic effect of anticancer drugs, but also inhibit the function of immune cells. Lactic acid groups can bind to histones and non-histone proteins to produce a new post-translational modification, lactylation. Methods: Breast cancer cells were infected with lentivirus vectors overexpressing or knocking down LDHA, and cell proliferation and cytotoxicity assay, transwell assay and Lactic Acid Content Assay were performed. To further explore the presence of lactylation of LDHA, immunoprecipitation experiments were performed and the modification sites were mutated to examine the enzymatic activity and function of LDHA. Results: In triple negative breast cancer with strong glycolytic metabolic phenotype, altering LDHA level can affect the biological function of breast cancer cells. Secondly, RNA-seq sequencing results showed that LDHA can affect the protein and transcription levels of MEST and CSTA. Finally, the lactylation of histones was also significantly changed after LDHA dysregulated. Reduced levels of LDHA were accompanied by reduced levels of MEST, CSTA, and histone lactylation. In addition, LDHA itself also has lactic acid modification, and mutations in the modification sites can change the enzymatic activity and function of LDHA. Conclusions: The regulatory axis of LDHA-lactate-lactylation has important value and function in the occurrence and development of breast cancer. LDHA regulates the function of some genes by lactate-induced lactylation of histones. In addition, the lactylation of LDHA can alter the enzyme activity, which may be a new therapeutic target for breast cancer.

Molecular Beacon Imaging System to Discriminate the Differentiation State of Cells from Energy Metabolic Pathways.

Metabolic pathways of energy production play an essential role as a function of cells. It is well recognized that the differentiation state of stem cells is highly associated with their metabolic profile. Therefore, visualization of the energy metabolic pathway makes it possible to discriminate the differentiation state of cells and predict the cell potential for reprogramming and differentiation. However, at present, it is technically difficult to directly assess the metabolic profile of individual living cells. In this study, we developed an imaging system of cationized gelatin nanospheres (cGNS) incorporating molecular beacons (MB) (cGNSMB) to detect intracellular pyruvate dehydrogenase kinase 1 (PDK1) and peroxisome proliferator-activated receptor γ, coactivator-1α (PGC-1α) mRNA of key regulators in the energy metabolism. The prepared cGNSMB was readily internalized into mouse embryonic stem cells, while their pluripotency was maintained. The high level of glycolysis in the undifferentiated state, the increased oxidative phosphorylation over the spontaneous early differentiation, and the lineage-specific neural differentiation were visualized based on the MB fluorescence. The fluorescence intensity corresponded well to the change of extracellular acidification rate and the oxygen consumption rate of representative metabolic indicators. These findings indicate that the cGNSMB imaging system is a promising tool to visually discriminate the differentiation state of cells from energy metabolic pathways.

Effects of Starvation and Refeeding on Glucose Metabolism and Immune Responses in Macrobrachium rosenbergii.

Starvation is a common challenge for aquatic animals in both natural and cultured environments. To investigate the effects of starvation and refeeding on glucose metabolism and immunity in Macrobrachium rosenbergii, prawns were starved for 14days and then refed for 7days. Results showed that both glucose and trehalose levels decreased significantly at the beginning of starvation, followed by a significant decrease in glycogen content in the hepatopancreas and muscle. Triglyceride and total protein reserves were also mobilized under starvation, with a slightly quicker response from triglycerides. The mRNA levels of glycolysis (glucokinase) and anabolism-related enzymes (glycogen branching enzyme, diacylglycerol acyltransferase, and transpeptidase) decreased during starvation, while gluconeogenic potential was induced, as indicated by up-regulated transcriptional levels of gluconeogenic enzymes (phosphoenolpyruvate carboxykinase) and catabolism-related enzymes (glycogen debranching enzyme, adipose triglyceride lipase, and cathepsin B). Starvation also stimulated the expression of the crustacean hyperglycemic hormone and inhibited insulin-like peptide expression, indicating their potential role in glucose metabolism regulation. In addition, starvation increased the mRNA levels of superoxide dismutase and prophenoloxidase, indicating an influence on the immune system. After bacterial infection, starved prawns showed enhanced activity of non-specific immunological parameters and reduced mortality. Refeeding for 7days led to a recovery of physiological and biochemical indices and transcriptional levels of metabolism/immune-related genes. Our findings provide a better understanding of the mechanisms underlying energy utilization, metabolic adaptation, and immune response to starvation in M. rosenbergii.

20-Hydroxyecdysone counteracts insulin to promote programmed cell death by modifying phosphoglycerate kinase 1

BackgroundThe regulation of glycolysis and autophagy during feeding and metamorphosis in holometabolous insects is a complex process that is not yet fully understood. Insulin regulates glycolysis during the larval feeding stage, allowing the insects to grow and live. However, during metamorphosis, 20-hydroxyecdysone (20E) takes over and regulates programmed cell death (PCD) in larval tissues, leading to degradation and ultimately enabling the insects to transform into adults. The precise mechanism through which these seemingly contradictory processes are coordinated remains unclear and requires further research. To understand the coordination of glycolysis and autophagy during development, we focused our investigation on the role of 20E and insulin in the regulation of phosphoglycerate kinase 1 (PGK1). We examined the glycolytic substrates and products, PGK1 glycolytic activity, and the posttranslational modification of PGK1 during the development of Helicoverpa armigera from feeding to metamorphosis.ResultsOur findings suggest that the coordination of glycolysis and autophagy during holometabolous insect development is regulated by a balance between 20E and insulin signaling pathways. Glycolysis and PGK1 expression levels were decreased during metamorphosis under the regulation of 20E. Insulin promoted glycolysis and cell proliferation via PGK1 phosphorylation, while 20E dephosphorylated PGK1 via phosphatase and tensin homolog (PTEN) to repress glycolysis. The phosphorylation of PGK1 at Y194 by insulin and its subsequent promotion of glycolysis and cell proliferation were important for tissue growth and differentiation during the feeding stage. However, during metamorphosis, the acetylation of PGK1 by 20E was key in initiating PCD. Knockdown of phosphorylated PGK1 by RNA interference (RNAi) at the feeding stage led to glycolysis suppression and small pupae. Insulin via histone deacetylase 3 (HDAC3) deacetylated PGK1, whereas 20E via acetyltransferase arrest-defective protein 1 (ARD1) induced PGK1 acetylation at K386 to stimulate PCD. Knockdown of acetylated-PGK1 by RNAi at the metamorphic stages led to PCD repression and delayed pupation.ConclusionsThe posttranslational modification of PGK1 determines its functions in cell proliferation and PCD. Insulin and 20E counteractively regulate PGK1 phosphorylation and acetylation to give it dual functions in cell proliferation and PCD.