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Frontiers in Immunology 2022Alcohol abuse, reported by 1/8 critically ill patients, is an independent risk factor for death in sepsis. Sepsis kills over 270,000 patients/year in the US. We reported...
Alcohol abuse, reported by 1/8 critically ill patients, is an independent risk factor for death in sepsis. Sepsis kills over 270,000 patients/year in the US. We reported that the ethanol-exposure suppresses innate-immune response, pathogen clearance, and decreases survival in sepsis-mice sirtuin 2 (SIRT2). SIRT2 is an NAD+-dependent histone-deacetylase with anti-inflammatory properties. We hypothesized that in ethanol-exposed macrophages, SIRT2 suppresses phagocytosis and pathogen clearance by regulating glycolysis. Immune cells use glycolysis to fuel increased metabolic and energy demand of phagocytosis. Using ethanol-exposed mouse bone marrow- and human blood monocyte-derived macrophages, we found that SIRT2 mutes glycolysis deacetylating key glycolysis regulating enzyme phosphofructokinase-platelet isoform (PFKP), at mouse lysine 394 (mK394, human: hK395). Acetylation of PFKP at mK394 (hK395) is crucial for PFKP function as a glycolysis regulating enzyme. The PFKP also facilitates phosphorylation and activation of autophagy related protein 4B (Atg4B). Atg4B activates microtubule associated protein 1 light chain-3B (LC3). LC3 is a driver of a subset of phagocytosis, the LC3-associated phagocytosis (LAP), which is crucial for segregation and enhanced clearance of pathogens, in sepsis. We found that in ethanol-exposed cells, the SIRT2-PFKP interaction leads to decreased Atg4B-phosphorylation, decreased LC3 activation, repressed phagocytosis and LAP. Genetic deficiency or pharmacological inhibition of SIRT2 reverse PFKP-deacetylation, suppressed LC3-activation and phagocytosis including LAP, in ethanol-exposed macrophages to improve bacterial clearance and survival in ethanol with sepsis mice.
Topics: Animals; Humans; Mice; Ethanol; Macrophages; Phagocytosis; Sirtuin 2; Phosphofructokinase-1, Type C
PubMed: 36865524
DOI: 10.3389/fimmu.2022.1079962 -
Cell Death & Disease Sep 2022Endometriosis (EM) is one of the vanquished wonted causes of chronic pelvic sting in women and is closely associated with infertility. The long-term, complex, systemic,...
Endometriosis (EM) is one of the vanquished wonted causes of chronic pelvic sting in women and is closely associated with infertility. The long-term, complex, systemic, and post-treatment recurrence of EM wreaks havoc on women's quality of life. Extensive metabolic reprogramming (aerobic glycolysis, glucose overweening intake, and high lactate production) and cancer-like changes have been found in EM, which bears striking similarities to tumorigenesis. The key glycolysis regulator PFKFB4 is overexpressed in EM. However, the mechanism of PFKFB4 in EM remains unknown. We found that PFKFB4 was upregulated and was closely related to the progression of EM. We identified focus PIM2 as a new pioneering adjoin protein of PFKFB4. Vigorous biochemical methods were used to confirm that PIM2 phosphorylated site Thr140 of PFKFB4. PIM2 also could enhance PFKFB4 protein expression through the ubiquitin-proteasome pathway. Moreover, PIM2 expression was really corresponding prevalent with PFKFB4 in endometriosis in vivo. Importantly, phosphorylation of PFKFB4 on Thr140 by PIM2 promoted EM glycolysis and cell growth. Our study demonstrates that PIM2 mediates PFKFB4 Thr140 phosphorylation thus regulating glycolysis and EM progression. We illustrated a new mechanism that PIM2 simulated a central upstream partnership in the regulation of PFKFB4, and reveal a novel means of PIM2-PFKFB4 setting EM growth. Our research provided new theoretical support for further clarifying the reprogramming of EM glucose metabolism, and provided new clues for exploring non-contraceptive treatments for EM.
Topics: Anaerobiosis; Cell Proliferation; Endometriosis; Female; Glucose; Glycolysis; Humans; Lactates; Phosphofructokinase-2; Phosphoric Monoester Hydrolases; Phosphorylation; Proteasome Endopeptidase Complex; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Quality of Life; Ubiquitins
PubMed: 36109523
DOI: 10.1038/s41419-022-05241-6 -
Cell Death & Disease May 2023F-box/LRR-repeat protein 7 (FBXL7) was predicted as a differentially expressed E3 ubiquitin ligase in non-small cell lung cancer (NSCLC), which has been suggested to...
F-box/LRR-repeat protein 7 (FBXL7) was predicted as a differentially expressed E3 ubiquitin ligase in non-small cell lung cancer (NSCLC), which has been suggested to influence cancer growth and metastasis. In this study, we aimed to decipher the function of FBXL7 in NSCLC and delineate the upstream and downstream mechanisms. FBXL7 expression was verified in NSCLC cell lines and GEPIA-based tissue samples, after which the upstream transcription factor of FBXL7 was bioinformatically identified. The substrate PFKFB4 of the FBXL7 was screened out by tandem affinity purification coupled with mass-spectrometry (TAP/MS). FBXL7 was downregulated in NSCLC cell lines and tissue samples. FBXL7 ubiquitinated and degraded PFKFB4, thus suppressing glucose metabolism and malignant phenotypes of NSCLC cells. Hypoxia-induced HIF-1α upregulation elevated EZH2 and inhibited FBXL7 transcription and reduced its expression, thus promoting PFKFB4 protein stability. By this mechanism, glucose metabolism and the malignant phenotype were enhanced. In addition, knockdown of EZH2 impeded tumor growth through the FBXL7/PFKFB4 axis. In conclusion, our work reveals that the EZH2/FBXL7/PFKFB4 axis plays a regulatory role in glucose metabolism and tumor growth of NSCLC, which is expected to be potential biomarkers for NSCLC.
Topics: Humans; Carcinoma, Non-Small-Cell Lung; Lung Neoplasms; Cell Line, Tumor; Hypoxia; Phosphoric Monoester Hydrolases; Glucose; Cell Proliferation; Gene Expression Regulation, Neoplastic; Enhancer of Zeste Homolog 2 Protein; Phosphofructokinase-2
PubMed: 37179372
DOI: 10.1038/s41419-023-05795-z -
JCI Insight Dec 2022Acute lung injury (ALI) is a severe form of lung inflammation causing acute respiratory distress syndrome in patients. ALI pathogenesis is closely linked to uncontrolled...
Acute lung injury (ALI) is a severe form of lung inflammation causing acute respiratory distress syndrome in patients. ALI pathogenesis is closely linked to uncontrolled alveolar inflammation. We hypothesize that specific enzymes of the glycolytic pathway could function as key regulators of alveolar inflammation. Therefore, we screened isolated alveolar epithelia from mice exposed to ALI induced by injurious ventilation to assess their metabolic responses. These studies pointed us toward a selective role for isoform 3 of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3). Pharmacologic inhibition or genetic deletion of Pfkfb3 in alveolar epithelia (Pfkfb3loxP/loxP SPC-ER-Cre+ mice) was associated with profound increases in ALI during injurious mechanical ventilation or acid instillation. Studies in genetic models linked Pfkfb3 expression and function to Hif1a. Not only did intratracheal pyruvate instillation reconstitute Pfkfb3loxP/loxP or Hif1aloxP/loxP SPC-ER-Cre+ mice, but pyruvate was also effective in ALI treatment of wild-type mice. Finally, proof-of-principle studies in human lung biopsies demonstrated increased PFKFB3 staining in injured lungs and colocalized PFKFB3 to alveolar epithelia. These studies reveal a specific role for PFKFB3 in counterbalancing alveolar inflammation and lay the groundwork for novel metabolic therapeutic approaches during ALI.
Topics: Humans; Animals; Mice; Lung; Acute Lung Injury; Pneumonia; Inflammation; Phosphofructokinase-2; Hypoxia-Inducible Factor 1, alpha Subunit
PubMed: 36326834
DOI: 10.1172/jci.insight.157855 -
Frontiers in Bioscience (Landmark... Nov 2023The dilation of lymphatic vessels plays a critical role in maintaining heart function, while a lack thereof could contribute to heart failure (HF), and subsequently to...
BACKGROUND
The dilation of lymphatic vessels plays a critical role in maintaining heart function, while a lack thereof could contribute to heart failure (HF), and subsequently to an acute myocardial infarction (AMI). Macrophages participate in the induction of lymphangiogenesis by secreting vascular endothelial cell growth factor C (VEGF-C), although the precise mechanism remains unclear.
METHODS
Intramyocardial injections of adeno-associated viruses (AAV9) to inhibit the expression of ( shRNA) or promote the expression of ( ORF) in the heart; Myh6-mCherry B6 D2-tg mice and flow cytometry were used to evaluate the number of myocellular debris in the mediastinal lymph nodes; fluorescence staining and qPCR were used to evaluate fluorescence analysis; seahorse experiment was used to evaluate the level of glycolysis of macrophages; , , and mice were used as a model to knock out the expression of and in macrophages.
RESULTS
The escalation of in cardiac tissue can facilitate the drainage of myocardial debris to the mediastinal lymph nodes, thereby improving cardiac function and reducing fibrosis after reperfusion injury. Conversely, myeloid deficiency displayed an increase in macrophage counts and inflammation levels following reperfusion injury. The inhibition of the critical enzyme in macrophage glycolysis can stimulate the manifestation of in macrophages. A deficiency in myeloid is associated with induced lymphangiogenesis following reperfusion injury.
CONCLUSIONS
Our initial investigations suggest that the suppression of expression in macrophages could potentially stimulate the production of in these immune cells, which in turn may facilitate lymphangiogenesis and mitigate the inflammatory effects of I/R injury.
Topics: Animals; Mice; Lymphangiogenesis; Macrophages; Myocardial Infarction; Reperfusion Injury; Vascular Endothelial Growth Factor C; Phosphofructokinase-2
PubMed: 38062830
DOI: 10.31083/j.fbl2811277 -
Cellular and Molecular Gastroenterology... 2023Metabolic reprogramming, in particular, glycolytic regulation, supports abnormal survival and growth of hepatocellular carcinoma (HCC) and could serve as a therapeutic...
BACKGROUND & AIMS
Metabolic reprogramming, in particular, glycolytic regulation, supports abnormal survival and growth of hepatocellular carcinoma (HCC) and could serve as a therapeutic target. In this study, we sought to identify glycolytic regulators in HCC that could be inhibited to prevent tumor progression and could also be monitored in vivo, with the goal of providing a theragnostic alternative to existing therapies.
METHODS
An orthotopic HCC rat model was used. Tumors were stimulated into a high-proliferation state by use of off-target liver ablation and were compared with lower-proliferating controls. We measured in vivo metabolic alteration in tumors before and after stimulation, and between stimulated tumors and control tumors using hyperpolarized C magnetic resonance imaging (MRI) (hC MRI). We compared metabolic alterations detected by hC MRI to metabolite levels from ex vivo mass spectrometry, mRNA levels of key glycolytic regulators, and histopathology.
RESULTS
Glycolytic lactate flux increased within HCC tumors 3 days after tumor stimulation, correlating positively with tumor proliferation as measured with Ki67. This was associated with a shift towards aerobic glycolysis and downregulation of the pentose phosphate pathway detected by mass spectrometry. MRI-measured lactate flux was most closely coupled with PFKFB3 expression and was suppressed with direct inhibition using PFK15.
CONCLUSIONS
Inhibition of PFKFB3 prevents glycolytic-mediated HCC proliferation, trackable by in vivo hC MRI.
Topics: Rats; Animals; Carcinoma, Hepatocellular; Phosphofructokinase-2; Liver Neoplasms; Cell Proliferation; Glycolysis; Lactic Acid
PubMed: 36162723
DOI: 10.1016/j.jcmgh.2022.09.009 -
The Journal of Clinical Endocrinology... Oct 2021
Topics: Diabetes Mellitus; Diabetic Retinopathy; Humans; Phosphofructokinase-2; Signal Transduction
PubMed: 34171101
DOI: 10.1210/clinem/dgab469 -
FASEB Journal : Official Publication of... Jul 2021Proliferation and differentiation of preadipocytes, and other cell types, is accompanied by an increase in glucose uptake. Previous work showed that a pulse of high...
Proliferation and differentiation of preadipocytes, and other cell types, is accompanied by an increase in glucose uptake. Previous work showed that a pulse of high glucose was required during the first 3 days of differentiation in vitro, but was not required after that. The specific glucose metabolism pathways required for adipocyte differentiation are unknown. Herein, we used 3T3-L1 adipocytes as a model system to study glucose metabolism and expansion of the adipocyte metabolome during the first 3 days of differentiation. Our primary outcome measures were GLUT4 and adiponectin, key proteins associated with healthy adipocytes. Using complete media with 0 or 5 mM glucose, we distinguished between developmental features that were dependent on the differentiation cocktail of dexamethasone, insulin, and isobutylmethylxanthine alone or the cocktail plus glucose. Cocktail alone was sufficient to activate the capacity for 2-deoxglucose uptake and glycolysis, but was unable to support the expression of GLUT4 and adiponectin in mature adipocytes. In contrast, 5 mM glucose in the media promoted a transient increase in glucose uptake and glycolysis as well as a significant expansion of the adipocyte metabolome and proteome. Using genetic and pharmacologic approaches, we found that the positive effects of 5 mM glucose on adipocyte differentiation were specifically due to increased expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), a key regulator of glycolysis and the ancillary glucose metabolic pathways. Our data reveal a critical role for PFKFB3 activity in regulating the cellular metabolic remodeling required for adipocyte differentiation and maturation.
Topics: 3T3-L1 Cells; Adipocytes; Adiponectin; Animals; Cell Differentiation; Cell Line; Dexamethasone; Glucose; Glucose Transporter Type 4; Glycolysis; Insulin; Male; Mice; Mice, Inbred C57BL; Phosphofructokinase-2; Xanthines
PubMed: 34110658
DOI: 10.1096/fj.202100381RR -
Archives of Biochemistry and Biophysics Jul 2023The phosphofructokinase (Pfk) reaction represents one of the key regulatory points in glycolysis. While most organisms encode for Pfks that use ATP as phosphoryl donor,...
The phosphofructokinase (Pfk) reaction represents one of the key regulatory points in glycolysis. While most organisms encode for Pfks that use ATP as phosphoryl donor, some organisms also encode for PP-dependent Pfks. Despite this central role, the biochemical characteristics as well as the physiological role of both Pfks is often not known. Clostridium thermocellum is an example of a microorganism that encodes for both Pfks, however, only PP-Pfk activity has been detected in cell-free extracts and little is known about the regulation and function of both enzymes. In this study, the ATP- and PP-Pfk of C. thermocellum were purified and biochemically characterized. No allosteric regulators were found for PP-Pfk amongst common effectors. With fructose-6-P, PP, fructose-1,6-bisP, and P PP-Pfk showed high specificity (K < 0.62 mM) and maximum activity (V > 156 U mg). In contrast, ATP-Pfk showed much lower affinity (K of 9.26 mM) and maximum activity (14.5 U mg) with fructose-6-P. In addition to ATP, also GTP, UTP and ITP could be used as phosphoryl donors. The catalytic efficiency with GTP was 7-fold higher than with ATP, suggesting that GTP is the preferred substrate. The enzyme was activated by NH, and pronounced inhibition was observed with GDP, FBP, PEP, and especially with PP (K of 0.007 mM). Characterization of purified ATP-Pfks originating from eleven different bacteria, encoding for only ATP-Pfk or for both ATP- and PP-Pfk, identified that PP inhibition of ATP-Pfks could be a common phenomenon for organisms with a PP-dependent glycolysis.
Topics: Phosphofructokinases; Clostridium thermocellum; Diphosphates; Amino Acid Sequence; Phosphofructokinase-1; Bacteria; Adenosine Triphosphate; Guanosine Triphosphate; Kinetics
PubMed: 37380119
DOI: 10.1016/j.abb.2023.109676 -
American Journal of Physiology. Cell... May 2022Although great effort has been expended to understand cancer's origins, less attention has been given to the primary cause of cancer deaths-cancer recurrences and their... (Review)
Review
Although great effort has been expended to understand cancer's origins, less attention has been given to the primary cause of cancer deaths-cancer recurrences and their sequelae. This interdisciplinary review addresses mechanistic features of aggressive cancer by studying metabolic enzyme patterns within ductal carcinoma in situ (DCIS) of the breast lesions. DCIS lesions from patients who did or did not experience a breast cancer recurrence were compared. Several proteins, including phospho-Ser226-glucose transporter type 1, phosphofructokinase type L and phosphofructokinase/fructose 2,6-bisphosphatase type 4 are found in nucleoli of ductal epithelial cells in samples from patients who will not subsequently recur, but traffic to the cell periphery in samples from patients who will experience a cancer recurrence. Large coclusters of enzymes near plasmalemmata will enhance product formation because enzyme concentrations in clusters are very high while solvent molecules and solutes diffuse through small channels. These structural changes will accelerate aerobic glycolysis. Agglomerations of pentose phosphate pathway and glutathione synthesis enzymes enhance GSH formation. As aggressive cancer lesions are incomplete at early stages, they may be unrecognizable. We have found that machine learning provides superior analyses of tissue images and may be used to identify biomarker patterns associated with recurrent and nonrecurrent patients with high accuracy. This suggests a new prognostic test to predict patients with DCIS who are likely to recur and those who are at low risk for recurrence. Mechanistic interpretations provide a deeper understanding of anticancer drug action and suggest that aggressive metastatic cancer cells are sensitive to reductive chemotherapy.
Topics: Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Epithelial Cells; Female; Humans; Neoplasm Recurrence, Local; Phosphofructokinase-2
PubMed: 35385324
DOI: 10.1152/ajpcell.00042.2022