RESUMEN
OBJECTIVES: This study aimed to explore the involvement of phosphoenolpyruvate carboxykinase 2 (PCK2) in gefitinib-resistant non-small cell lung cancer (NSCLC) cells and assess its feasibility as a therapeutic target against gefitinib resistance. METHODS: Gefitinib-resistant cell lines, PC9GR and HCC827GR, were generated through progressive exposure of parental cells to escalating concentrations of gefitinib. Transcriptomic analysis encompassed the treatment of PC9 and PC9GR cells with gefitinib or vehicle, followed by RNA extraction, sequencing, and subsequent bioinformatic analysis. Cell viability was determined via CCK-8 assay, while clonogenic assays assessed colony formation. Apoptosis was detected utilizing the Annexin V-FITC/7AAD kit. Iron ion concentrations were quantified using FerroOrange. mRNA analysis was conducted through quantitative RT-PCR. Western blotting was employed for protein analysis. H&E and immunohistochemical staining were performed on tumor tissue sections. RESULTS: The results revealed that depletion or inhibition of PCK2 significantly enhanced gefitinib's efficacy in inducing cell growth arrest, apoptosis, and ferroptosis in resistant NSCLC. Moreover, PCK2 knockdown led to the downregulation of key ferroptosis-related proteins, GPX4 and SLC7A11, while upregulating ASCL4. Conversely, overexpression of PCK2 in gefitinib-sensitive cells rendered resistance to gefitinib. In vivo experiments using a gefitinib-resistant xenograft model demonstrated that PCK2 silencing not only reduced tumor growth but also considerably increased the anti-tumor effect of gefitinib. CONCLUSIONS: In conclusion, our study presents compelling evidence indicating that PCK2 plays a pivotal role in gefitinib resistance in NSCLC. The modulation of ferroptosis-related proteins and the involvement of Akt activation further elucidate the mechanisms underlying this resistance. Consequently, PCK2 emerges as a promising therapeutic target for overcoming gefitinib resistance in NSCLC, offering a new avenue for the development of more effective treatment strategies.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Resistencia a Antineoplásicos , Ferroptosis , Gefitinib , Neoplasias Pulmonares , Ferroptosis/efectos de los fármacos , Ferroptosis/genética , Gefitinib/farmacología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/genética , Humanos , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Línea Celular Tumoral , Animales , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismo , Fosfoenolpiruvato Carboxiquinasa (ATP)/genética , Antineoplásicos/farmacología , Ratones , Ratones Desnudos , Apoptosis/efectos de los fármacosRESUMEN
Mitochondrial phosphoenolpyruvate carboxykinase (PCK2), a mitochondrial isoenzyme, supports the growth of cancer cells under glucose deficiency conditions in vitro. This study investigated the role and potential mechanism of PCK2 in the occurrence and development of Hepatocellular carcinoma (HCC). The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and other databases distinguish the expression of PCK2 and verified by qRT-PCR and Western blotting. Kaplan-Meier was conducted to assess PCK2 survival in HCC. The potential biological function of PCK2 was verified by enrichment analysis and gene set enrichment analysis (GSEA). The correlation between PCK2 expression and immune invasion and checkpoint was found by utilizing Tumor Immune Estimation Resource (TIMER). Lastly, the effects of PCK2 on the proliferation and metastasis of hepatocellular carcinoma cells were evaluated by cell tests, and the expressions of Epithelial mesenchymal transformation (EMT) and apoptosis related proteins were detected. PCK2 is down-regulated in HCC, indicating a poor prognosis. PCK2 gene mutation accounted for 1.3% of HCC. Functional enrichment analysis indicated the potential of PCK2 as a metabolism-related therapeutic target. Subsequently, we identified several signaling pathways related to the biological function of PCK2. The involvement of PCK2 in immune regulation was verified and key immune checkpoints were predicted. Ultimately, after PCK2 knockdown, cell proliferation and migration were significantly increased, and N-cadherin and vimentin expression were increased. PCK2 has been implicated in immune regulation, proliferation, and metastasis of hepatocellular carcinoma, and is emerging as a novel predictive biomarker and metabolic-related clinical target.
Asunto(s)
Carcinoma Hepatocelular , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/inmunología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/mortalidad , Humanos , Pronóstico , Línea Celular Tumoral , Fosfoenolpiruvato Carboxiquinasa (GTP)/genética , Fosfoenolpiruvato Carboxiquinasa (GTP)/metabolismo , Transición Epitelial-Mesenquimal/genética , Mitocondrias/metabolismo , Mitocondrias/genética , Masculino , Femenino , Apoptosis , Movimiento Celular/genética , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Persona de Mediana Edad , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismo , Fosfoenolpiruvato Carboxiquinasa (ATP)/genéticaRESUMEN
OBJECTIVE: The main objective of this study was to find if thiamine disulfide (TD) lowers blood glucose level and improves insulin resistance (IR) in liver and muscle in rats with chronic type 1 diabetes (T1DM) using euglycemic-hyperinsulinemic clamp technique. METHODS: A total of fifty male Wistar rats were assigned to five groups consisted of: non-diabetic control (NDC), diabetic control (DC), diabetic treated with thiamine disulfide (D-TD), diabetic treated with insulin (D-insulin), and diabetic treated with both TD and insulin (D-insulin+TD). Diabetes was induced by a 60â¯mg/kg dose of streptozotocin. Blood glucose levels, pyruvate tolerance test (PTT), intraperitoneal glucose tolerance test (IPGTT), levels of glycosylated hemoglobin (HbA1c), glucose infusion rate (GIR), liver and serum lipid profiles, liver glycogen stores, liver enzymes ([ALT], [AST]), and serum calcium and magnesium levels. were evaluated. Additionally, gene expression levels of phosphoenolpyruvate carboxykinase (Pepck), forkhead box O1 (Foxo1), and glucose transporter type 4 (Glut4) were assessed in liver and skeletal muscle tissues. RESULTS: Blood glucose level was reduced by TD treatment. In addition, TyG index, HOMA-IR, serum and liver lipid profiles, HbA1c levels, and expressions of Foxo1 and Pepck genes were decreased significantly (P<0.05) in all the treated groups. However, TD did not influence Glut4 gene expression, but GIR as a critical index of IR were 5.0±0.26, 0.29±0.002, 1.5±0.07, 0.9±0.1 and 1.3±0.1â¯mg.min-1Kg-1 in NDC, DC, D-TD, D-insulin and D-insulin+TD respectively. CONCLUSIONS: TD improved IR in the liver primarily by suppressing gluconeogenic pathways, implying the potential use of TD as a therapeutic agent in diabetes.
Asunto(s)
Glucemia , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Resistencia a la Insulina , Hígado , Ratas Wistar , Tiamina , Animales , Masculino , Hígado/metabolismo , Hígado/efectos de los fármacos , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/metabolismo , Glucemia/efectos de los fármacos , Glucemia/metabolismo , Ratas , Tiamina/farmacología , Tiamina/administración & dosificación , Transportador de Glucosa de Tipo 4/metabolismo , Insulina/sangre , Transducción de Señal/efectos de los fármacos , Fosfoenolpiruvato Carboxiquinasa (GTP)/metabolismo , Fosfoenolpiruvato Carboxiquinasa (GTP)/genética , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismo , Estreptozocina , Proteína Forkhead Box O1 , Péptidos y Proteínas de Señalización IntracelularRESUMEN
Protein succinylation modification is a common post-translational modification (PTM) that plays an important role in bacterial metabolic regulation. In this study, quantitative analysis was conducted on the succinylated proteome of wild-type and florfenicol-resistant Vibrio alginolyticus to investigate the mechanism of succinylation regulating antibiotic resistance. Bioinformatic analysis showed that the differentially succinylated proteins were mainly enriched in energy metabolism, and it was found that the succinylation level of phosphoenolpyruvate carboxyl kinase (PEPCK) was highly expressed in the florfenicol-resistant strain. Site-directed mutagenesis was used to mutate the lysine (K) at the succinylation site of PEPCK to glutamic acid (E) and arginine (R), respectively, to investigate the function of lysine succinylation of PEPCK in the florfenicol resistance of V. alginolyticus. The detection of site-directed mutagenesis strain viability under florfenicol revealed that the survival rate of the E mutant was significantly higher than that of the R mutant and wild type, indicating that succinylation modification of PEPCK protein may affect the resistance of V. alginolyticus to florfenicol. This study indicates the important role of PEPCK during V. alginolyticus antibiotic-resistance evolution and provides a theoretical basis for the prevention and control of vibriosis and the development of new antibiotics.
Asunto(s)
Antibacterianos , Farmacorresistencia Bacteriana , Lisina , Procesamiento Proteico-Postraduccional , Tianfenicol , Vibrio alginolyticus , Tianfenicol/farmacología , Tianfenicol/análogos & derivados , Tianfenicol/metabolismo , Vibrio alginolyticus/genética , Vibrio alginolyticus/efectos de los fármacos , Vibrio alginolyticus/metabolismo , Farmacorresistencia Bacteriana/genética , Lisina/metabolismo , Antibacterianos/farmacología , Mutagénesis Sitio-Dirigida , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Ácido Succínico/metabolismo , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismo , Fosfoenolpiruvato Carboxiquinasa (ATP)/genéticaRESUMEN
BACKGROUND: Phosphoenolpyruvate carboxykinase (PEPCK) plays a crucial role in gluconeogenesis, glycolysis, and the tricarboxylic acid cycle by converting oxaloacetate into phosphoenolpyruvate. Two distinct isoforms of PEPCK, specifically cytosolic PCK1 and mitochondrial PCK2, have been identified. Nevertheless, the comprehensive understanding of their dysregulation in pan-cancer and their potential mechanism contributing to signaling transduction pathways remains elusive. METHODS: We conducted comprehensive analyses of PEPCK gene expression across 33 diverse cancer types using data from The Cancer Genome Atlas (TCGA). Multiple public databases such as HPA, TIMER 2.0, GEPIA2, cBioPortal, UALCAN, CancerSEA, and String were used to investigate protein levels, prognostic significance, clinical associations, genetic mutations, immune cell infiltration, single-cell sequencing, and functional enrichment analysis in patients with pan-cancer. PEPCK expression was analyzed about different clinical and genetic factors of patients using data from TCGA, GEO, and CGGA databases. Furthermore, the role of PCK2 in Glioma was examined using both in vitro and in vivo experiments. RESULTS: The analysis we conducted revealed that the expression of PEPCK is involved in both clinical outcomes and immune cell infiltration. Initially, we verified the high expression of PCK2 in GBM cells and its role in metabolic reprogramming and proliferation in GBM. CONCLUSION: Our study showed a correlation between PEPCK (PCK1 and PCK2) expression with clinical prognosis, gene mutation, and immune infiltrates. These findings identified two possible predictive biomarkers across different cancer types, as well as a comprehensive analysis of PCK2 expression in various tumors, with a focus on GBM.
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Neoplasias , Fosfoenolpiruvato Carboxiquinasa (GTP) , Humanos , Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/patología , Fosfoenolpiruvato Carboxiquinasa (GTP)/metabolismo , Fosfoenolpiruvato Carboxiquinasa (GTP)/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismo , Fosfoenolpiruvato Carboxiquinasa (ATP)/genética , Animales , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Pronóstico , Proliferación CelularRESUMEN
Endothelial cells (ECs) are highly glycolytic, but whether they generate glycolytic intermediates via gluconeogenesis (GNG) in glucose-deprived conditions remains unknown. Here, we report that glucose-deprived ECs upregulate the GNG enzyme PCK2 and rely on a PCK2-dependent truncated GNG, whereby lactate and glutamine are used for the synthesis of lower glycolytic intermediates that enter the serine and glycerophospholipid biosynthesis pathways, which can play key roles in redox homeostasis and phospholipid synthesis, respectively. Unexpectedly, however, even in normal glucose conditions, and independent of its enzymatic activity, PCK2 silencing perturbs proteostasis, beyond its traditional GNG role. Indeed, PCK2-silenced ECs have an impaired unfolded protein response, leading to accumulation of misfolded proteins, which due to defective proteasomes and impaired autophagy, results in the accumulation of protein aggregates in lysosomes and EC demise. Ultimately, loss of PCK2 in ECs impaired vessel sprouting. This study identifies a role for PCK2 in proteostasis beyond GNG.
Asunto(s)
Células Endoteliales , Gluconeogénesis , Fosfoenolpiruvato Carboxiquinasa (GTP) , Proteostasis , Gluconeogénesis/genética , Humanos , Células Endoteliales/metabolismo , Fosfoenolpiruvato Carboxiquinasa (GTP)/metabolismo , Fosfoenolpiruvato Carboxiquinasa (GTP)/genética , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Glucosa/metabolismo , Autofagia , Respuesta de Proteína Desplegada , Fosfoenolpiruvato Carboxiquinasa (ATP)RESUMEN
In most actinomycetes, GlnR governs both nitrogen and non-nitrogen metabolisms (e.g., carbon, phosphate, and secondary metabolisms). Although GlnR has been recognized as a global regulator, its regulatory role in central carbon metabolism [e.g., glycolysis, gluconeogenesis, and the tricarboxylic acid (TCA) cycle] is largely unknown. In this study, we characterized GlnR as a direct transcriptional repressor of the pckA gene that encodes phosphoenolpyruvate carboxykinase, catalyzing the conversion of the TCA cycle intermediate oxaloacetate to phosphoenolpyruvate, a key step in gluconeogenesis. Through the transcriptomic and quantitative real-time PCR analyses, we first showed that the pckA transcription was upregulated in the glnR null mutant of Amycolatopsis mediterranei. Next, we proved that the pckA gene was essential for A. mediterranei gluconeogenesis when the TCA cycle intermediate was used as a sole carbon source. Furthermore, with the employment of the electrophoretic mobility shift assay and DNase I footprinting assay, we revealed that GlnR was able to specifically bind to the pckA promoter region from both A. mediterranei and two other representative actinomycetes (Streptomyces coelicolor and Mycobacterium smegmatis). Therefore, our data suggest that GlnR may repress pckA transcription in actinomycetes, which highlights the global regulatory role of GlnR in both nitrogen and central carbon metabolisms in response to environmental nutrient stresses. IMPORTANCE: The GlnR regulator of actinomycetes controls nitrogen metabolism genes and many other genes involved in carbon, phosphate, and secondary metabolisms. Currently, the known GlnR-regulated genes in carbon metabolism are involved in the transport of carbon sources, the assimilation of short-chain fatty acid, and the 2-methylcitrate cycle, although little is known about the relationship between GlnR and the TCA cycle and gluconeogenesis. Here, based on the biochemical and genetic results, we identified GlnR as a direct transcriptional repressor of pckA, the gene that encodes phosphoenolpyruvate carboxykinase, a key enzyme for gluconeogenesis, thus highlighting that GlnR plays a central and complex role for dynamic orchestration of cellular carbon, nitrogen, and phosphate fluxes and bioactive secondary metabolites in actinomycetes to adapt to changing surroundings.
Asunto(s)
Proteínas Bacterianas , Regulación Bacteriana de la Expresión Génica , Gluconeogénesis , Nitrógeno , Gluconeogénesis/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Nitrógeno/metabolismo , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Amycolatopsis/metabolismo , Amycolatopsis/genética , Regiones Promotoras Genéticas , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismo , Fosfoenolpiruvato Carboxiquinasa (ATP)/genética , Ciclo del Ácido Cítrico/genética , Actinobacteria/genética , Actinobacteria/metabolismoRESUMEN
The repeated emergence of NADP-malic enzyme (ME), NAD-ME and phosphoenolpyruvate carboxykinase (PEPCK) subtypes of C4 photosynthesis are iconic examples of convergent evolution, which suggests that these biochemistries do not randomly assemble, but are instead specific adaptations resulting from unknown evolutionary drivers. Theoretical studies that are based on the classic biochemical understanding have repeatedly proposed light-use efficiency as a possible benefit of the PEPCK subtype. However, quantum yield measurements do not support this idea. We explore this inconsistency here via an analytical model that features explicit descriptions across a seamless gradient between C4 biochemistries to analyse light harvesting and dark photosynthetic metabolism. Our simulations show that the NADP-ME subtype, operated by the most productive crops, is the most efficient. The NAD-ME subtype has lower efficiency, but has greater light harvesting plasticity (the capacity to assimilate CO2 in the broadest combination of light intensity and spectral qualities). In both NADP-ME and NAD-ME backgrounds, increasing PEPCK activity corresponds to greater light harvesting plasticity but likely imposed a reduction in photosynthetic efficiency. We draw the first mechanistic links between light harvesting and C4 subtypes, providing the theoretical basis for future investigation.
Asunto(s)
Malato Deshidrogenasa , Fotosíntesis , Malato Deshidrogenasa/metabolismo , Luz , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismo , Modelos BiológicosRESUMEN
BACKGRUOUND: Sodium-dependent glucose cotransporter 2 (SGLT2) mediates glucose reabsorption in the renal proximal tubules, and SGLT2 inhibitors are used as therapeutic agents for treating type 2 diabetes mellitus. This study aimed to elucidate the effects and mechanisms of SGLT2 inhibition on hepatic glucose metabolism in both serum deprivation and serum supplementation states. METHODS: Huh7 cells were treated with the SGLT2 inhibitors empagliflozin and dapagliflozin to examine the effect of SGLT2 on hepatic glucose uptake. To examine the modulation of glucose metabolism by SGLT2 inhibition under serum deprivation and serum supplementation conditions, HepG2 cells were transfected with SGLT2 small interfering RNA (siRNA), cultured in serum-free Dulbecco's modified Eagle's medium for 16 hours, and then cultured in media supplemented with or without 10% fetal bovine serum for 8 hours. RESULTS: SGLT2 inhibitors dose-dependently decreased hepatic glucose uptake. Serum deprivation increased the expression levels of the gluconeogenesis genes peroxisome proliferator-activated receptor gamma co-activator 1 alpha (PGC-1α), glucose 6-phosphatase (G6pase), and phosphoenolpyruvate carboxykinase (PEPCK), and their expression levels during serum deprivation were further increased in cells transfected with SGLT2 siRNA. SGLT2 inhibition by siRNA during serum deprivation induces nuclear localization of the transcription factor forkhead box class O 1 (FOXO1), decreases nuclear phosphorylated-AKT (p-AKT), and p-FOXO1 protein expression, and increases phosphorylated-adenosine monophosphate-activated protein kinase (p-AMPK) protein expression. However, treatment with the AMPK inhibitor, compound C, reversed the reduction in the protein expression levels of nuclear p- AKT and p-FOXO1 and decreased the protein expression levels of p-AMPK and PEPCK in cells transfected with SGLT2 siRNA during serum deprivation. CONCLUSION: These data show that SGLT2 mediates glucose uptake in hepatocytes and that SGLT2 inhibition during serum deprivation increases gluconeogenesis via the AMPK/AKT/FOXO1 signaling pathway.
Asunto(s)
Diabetes Mellitus Tipo 2 , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Humanos , Proteínas Quinasas Activadas por AMP/metabolismo , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Gluconeogénesis/genética , Glucosa , Fosfoenolpiruvato Carboxiquinasa (ATP)/genética , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-akt/farmacología , Proteínas Proto-Oncogénicas c-akt/uso terapéutico , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , ARN Interferente Pequeño/farmacología , Transducción de Señal , Sodio/metabolismo , Sodio/farmacología , Sodio/uso terapéutico , Transportador 2 de Sodio-Glucosa/metabolismo , Transportador 2 de Sodio-Glucosa/farmacología , Transportador 2 de Sodio-Glucosa/uso terapéutico , Inhibidores del Cotransportador de Sodio-Glucosa 2/farmacología , Inhibidores del Cotransportador de Sodio-Glucosa 2/uso terapéuticoRESUMEN
Aberrant PCK2 overexpression has been linked to an unfavorable prognosis and shorter survival, particularly in hepatocellular carcinoma (HCC). Thus, the inactivation of PCK2 provides a promising strategy for HCC treatment. In this study, we used a chemical genetic strategy to identify a natural-derived small-molecule cucurbitacin B (CuB) as a selective PCK2 inhibitor. CuB covalently bound to PCK2 at a unique Cys63 site, blocking the Ω-loop lid domain formation via a previously undisclosed allosteric mechanism. Additionally, targeted lipidomics analysis also revealed that CuB destroyed mitochondrial membrane integrity, leading to the disruption of mitochondrial fusion-fission dynamics. Taken together, this study highlights the discovery of a small-molecule CuB, which reprograms lipid metabolism for controlling mitochondrial dynamics via targeting PCK2 in cancer cells.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/patología , Dinámicas Mitocondriales , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Regulación Alostérica , Fosfoenolpiruvato Carboxiquinasa (ATP)RESUMEN
The fat body is responsible for a variety of functions related to energy metabolism in arthropods, by controlling the processes of de novo glucose production (gluconeogenesis) and glycogen metabolism. The rate-limiting factor of gluconeogenesis is the enzyme phosphoenolpyruvate carboxykinase (PEPCK), generally considered to be the first committed step in this pathway. Although the study of PEPCK and gluconeogenesis has been for decades restricted to mammalian models, especially focusing on muscle and liver tissue, current research has demonstrated particularities about the regulation of this enzyme in arthropods, and described new functions. This review will focus on arthropod PEPCK, discuss different aspects to PEPCK regulation and function, its general role in the regulation of gluconeogenesis and other pathways. The text also presents our views on potentially important new directions for research involving this enzyme in a variety of metabolic adaptations (e.g. diapause), discussing enzyme isoforms, roles during arthropod embryogenesis, as well as involvement in vector-pathogen interactions, contributing to a better understanding of insect vectors of diseases and their control.
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Artrópodos , Animales , Artrópodos/metabolismo , Fosfoenolpiruvato Carboxiquinasa (GTP)/metabolismo , Fosfoenolpiruvato Carboxiquinasa (ATP)/genética , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismo , Glucosa/metabolismo , Homeostasis , Mamíferos/metabolismoRESUMEN
Deoxynivalenol (DON) is one of the most plentiful trichothecenes occurring in food and feed, which brings severe health hazards to both animals and humans. This study aims to investigate whether sodium butyrate (NaB) can protect the porcine intestinal barrier from DON exposure through promoting mitochondrial homeostasis. In a 4-week feeding experiment, 28 male piglets were allocated according to a 2 by 2 factorial arrangement of treatments with the main factors including supplementation of DON (< 0.8 vs. 4.0 mg/kg) and NaB (0.0 vs. 2 g/kg) in a corn/soybean-based diet. Dietary NaB supplementation mitigated the damaged mitochondrial morphology within the jejunal mucosa and the disrupted gut epithelial tight junctions irritated by DON. In IPEC-J2 cells, we found efficient recovery of the intestinal epithelial barrier occurred following NaB administration. This intestinal barrier reparation was facilitated by NaB-induced PCK2-mediated glyceroneogenesis and restoration of mitochondrial structure and function. In conclusion, we elucidated a mechanism of PCK2-mediated improvement of mitochondrial function by NaB to repair porcine intestinal barrier disruption during chronic DON exposure. Our findings highlight the promise of NaB for use in protecting against DON-induced gut epithelial tight junction disruption in piglets.
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Tricotecenos , Humanos , Porcinos , Animales , Masculino , Ácido Butírico/farmacología , Ácido Butírico/metabolismo , Tricotecenos/toxicidad , Mucosa Intestinal/metabolismo , Mitocondrias , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismoRESUMEN
Phosphoenolpyruvate carboxykinases (PEPCK) are a well-studied family of enzymes responsible for the regulation of TCA cycle flux, where they catalyze the interconversion of oxaloacetic acid (OAA) and phosphoenolpyruvate (PEP) using a phosphoryl donor/acceptor. These enzymes have typically been divided into two nucleotide-dependent classes, those that use ATP and those that use GTP. In the 1960's and early 1970's, a group of papers detailed biochemical properties of an enzyme named phosphoenolpyruvate carboxytransphosphorylase (later identified as a third PEPCK) from Propionibacterium freudenreichii (PPi -PfPEPCK), which instead of using a nucleotide, utilized PPi to catalyze the same interconversion of OAA and PEP. The presented work expands upon the initial biochemical experiments for PPi -PfPEPCK and interprets these data considering both the current understanding of nucleotide-dependent PEPCKs and is supplemented with a new crystal structure of PPi -PfPEPCK in complex with malate at a putative allosteric site. Most interesting, the data are consistent with PPi -PfPEPCK being a Fe2+ activated enzyme in contrast with the Mn2+ activated nucleotide-dependent enzymes which in part results in some unique kinetic properties for the enzyme when compared to the more widely distributed GTP- and ATP-dependent enzymes.
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Propionibacterium freudenreichii , Fosfoenolpiruvato , Propionibacterium freudenreichii/metabolismo , Fosfoenolpiruvato Carboxiquinasa (ATP)/química , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismo , Ácido Oxaloacético/química , Guanosina Trifosfato , Nucleótidos , Adenosina Trifosfato , CinéticaRESUMEN
Phosphoenolpyruvate carboxykinase (PEPCK) is a well-known lyase involved in gluconeogenesis, while their evolution and function differentiation have not been fully understood. In this study, by constructing a phylogenetic tree to examine PEPCKs throughout the evolution from poriferans to vertebrates, Mollusk was highlighted as the only phylum to exhibit two distinct lineages, Mollusca_PEPCK-1 and Mollusca_PEPCK-2. Further study of two representative members from Crassostrea gigas (CgPEPCK-1 and CgPEPCK-2) showed that they both shared conserved sequences and structural characteristics of the catalytic enzyme, while CgPEPCK-2 displayed a higher expression level than CgPEPCK-1 in all tested tissues, and CgPEPCK-1 was specifically implicated in the immune defense against LPS stimulation and Vibrio splendidus infection. Functional analysis revealed that CgPEPCK-2 had stronger enzymatic activity than CgPEPCK-1, while CgPEPCK-1 exhibited stronger binding activity with various PAMPs, and only the protein of CgPEPCK-1 increased significantly in hemolymph during immune stimulation. All results supported that distinct sequence and function differentiations of the PEPCK gene family should have occurred since Mollusk. The more advanced evolutionary branch Mollusca_PEPCK-2 should preserve its essential function as a catalytic enzyme to be more specialized and efficient, while the ancient branch Mollusca_PEPCK-1 probably contained some members, such as CgPEPCK-1, that should be integrated into the immune system as an extracellular immune recognition receptor.
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Fosfoenolpiruvato Carboxiquinasa (ATP) , Vibriosis , Animales , Filogenia , Fosfoenolpiruvato Carboxiquinasa (ATP)/genética , Gluconeogénesis , Secuencia ConservadaRESUMEN
Phosphoenolpyruvate carboxykinase (PCK) plays a critical role in cytosolic gluconeogenesis, and defects in PCK1 cause a fasting-aggravated metabolic disease with hypoglycemia and lactic acidosis. However, there are two genes encoding PCK, and the role of the mitochondrial resident PCK (encoded by PCK2) is unclear, since gluconeogenesis is cytosolic. We identified three patients in two families with biallelic variants in PCK2. One has compound heterozygous variants (p.Ser23Ter/p.Pro170Leu), and the other two (siblings) have homozygous p.Arg193Ter variation. All three patients have weakness and abnormal gait, an absence of PCK2 protein, and profound reduction in PCK2 activity in fibroblasts, but no obvious metabolic phenotype. Nerve conduction studies showed reduced conduction velocities with temporal dispersion and conduction block compatible with a demyelinating peripheral neuropathy. To validate the association between PCK2 variants and clinical disease, we generated a mouse knockout model of PCK2 deficiency. The animals present abnormal nerve conduction studies and peripheral nerve pathology, corroborating the human phenotype. In total, we conclude that biallelic variants in PCK2 cause a neurogenetic disorder featuring abnormal gait and peripheral neuropathy.
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Enfermedades del Sistema Nervioso Periférico , Fosfoenolpiruvato Carboxiquinasa (ATP) , Ratones , Animales , Humanos , Fosfoenolpiruvato , Fosfoenolpiruvato Carboxiquinasa (ATP)/genética , Fosfoenolpiruvato Carboxiquinasa (GTP)/genética , Gluconeogénesis/genética , Fosfoenolpiruvato Carboxilasa/metabolismo , Enfermedades del Sistema Nervioso Periférico/genéticaRESUMEN
Phosphoenolpyruvate carboxykinase (PEPCK) catalyses the reversible reaction of decarboxylation and phosphorylation of oxaloacetate (OAA) to generate phosphoenolpyruvate (PEP) and CO2 playing mainly a gluconeogenic role in green algae. We found two PEPCK isoforms in Chlamydomonas reinhardtii and we cloned, purified and characterised both enzymes. ChlrePEPCK1 is more active as decarboxylase than ChlrePEPCK2. ChlrePEPCK1 is hexameric and its activity is affected by citrate, phenylalanine and malate, while ChlrePEPCK2 is monomeric and it is regulated by citrate, phenylalanine and glutamine. We postulate that the two PEPCK isoforms found originate from alternative splicing of the gene or regulated proteolysis of the enzyme. The presence of these two isoforms would be part of a mechanism to finely regulate the biological activity of PEPCKs.
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Chlamydomonas reinhardtii , Fosfoenolpiruvato , Chlamydomonas reinhardtii/genética , Fosfoenolpiruvato Carboxiquinasa (ATP)/genética , Isoformas de Proteínas , Fenilalanina , CitratosRESUMEN
BACKGROUND: Tumor cells may aberrantly express metabolic enzymes to adapt to their environment for survival and growth. Targeting cancer-specific metabolic enzymes is a potential therapeutic strategy. Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the conversion of oxaloacetate to phosphoenolpyruvate and links the tricarboxylic acid cycle and glycolysis/gluconeogenesis. Mitochondrial PEPCK (PEPCK-M), encoded by PCK2, is an isozyme of PEPCK and is distributed in mitochondria. Overexpression of PCK2 has been identified in many human cancers and demonstrated to be important for the survival program initiated upon metabolic stress in cancer cells. We evaluated the expression status of PEPCK-M and investigated the function of PEPCK-M in breast cancer. METHODS: We checked the expression status of PEPCK-M in breast cancer samples by immunohistochemical staining. We knocked down or overexpressed PCK2 in breast cancer cell lines to investigate the function of PEPCK-M in breast cancer. RESULTS: PEPCK-M was highly expressed in estrogen receptor-positive (ER+ ) breast cancers. Decreased cell proliferation and G0 /G1 arrest were induced in ER+ breast cancer cell lines by knockdown of PCK2. PEPCK-M promoted the activation of mTORC1 downstream signaling molecules and the E2F1 pathways in ER+ breast cancer. In addition, glucose uptake, intracellular glutamine levels, and mTORC1 pathways activation by glucose and glutamine in ER+ breast cancer were attenuated by PCK2 knockdown. CONCLUSION: PEPCK-M promotes proliferation and cell cycle progression in ER+ breast cancer via upregulation of the mTORC1 and E2F1 pathways. PCK2 also regulates nutrient status-dependent mTORC1 pathway activation in ER+ breast cancer. Further studies are warranted to understand whether PEPCK-M is a potential therapeutic target for ER+ breast cancer.
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Neoplasias de la Mama , Receptores de Estrógenos , Humanos , Femenino , Fosfoenolpiruvato/metabolismo , Receptores de Estrógenos/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Glutamina/metabolismo , Fosfoenolpiruvato Carboxiquinasa (ATP)/genética , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismoRESUMEN
Type 2 diabetes mellitus (T2DM) has become a worldwide public health problem. Epimedin C is considered one of the most important flavonoids in Epimedium, a famous edible herb in China and Southeast Asia that is traditionally used in herbal medicine to treat diabetes. In the present study, the therapeutic potential of epimedin C against T2DM was ascertained using a mouse model, and the mechanism underlying the hypoglycemic activity of epimedin C was explored using a label-free proteomic technique for the first time. Levels of fasting blood glucose (FBG), homeostasis model assessment of insulin resistance (HOMA-IR), and oral glucose tolerance, as well as contents of malondialdehyde (MDA) and low-density lipoprotein cholesterol (LDL-C) in the 30 mg·kg-1 epimedin C group (EC30 group), were significantly lower than those in the model control group (MC group) (p < 0.05), while the contents of hepatic glycogen, insulin, and high-density lipoprotein cholesterol (HDL-C), as well as activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the EC30 group were notably higher than those in the MC group (p < 0.05). The structures of liver cells and tissues were greatly destroyed in the MC group, whereas the structures of cells and tissues were basically complete in the EC30 group, which were similar to those in the normal control group (NC group). A total of 92 differentially expressed proteins (DEPs) were enriched in the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. In the EC30 vs. MC groups, the expression level of cytosolic phosphoenolpyruvate carboxykinase (Pck1) was down-regulated, while the expression levels of group XIIB secretory phospholipase A2-like protein (Pla2g12b), apolipoprotein B-100 (Apob), and cytochrome P450 4A14 (Cyp4a14) were up-regulated. According to the KEGG pathway assay, Pck1 participated in the gluconeogenesis and insulin signaling pathways, and Pla2g12b, Apob, and Cyp4a14 were the key proteins in the fat digestion and fatty acid degradation pathways. Pck1, Pla2g12b, Apob, and Cyp4a14 seemed to play important roles in the prevention and treatment of T2DM. In summary, epimedin C inhibited Pck1 expression to maintain FBG at a relatively stable level, promoted Pla2g12b, Apob, and Cyp4a14 expressions to alleviate liver lipotoxicity, and protected liver tissues and cells from oxidant stress possibly by its phenolic hydroxyl groups.
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Diabetes Mellitus Tipo 2 , Hipoglucemiantes , Humanos , Hipoglucemiantes/farmacología , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Proteómica , Flavonoides/farmacología , Insulina , Fosfoenolpiruvato Carboxiquinasa (ATP) , ColesterolRESUMEN
As an important enzyme for gluconeogenesis, mitochondrial phosphoenolpyruvate carboxykinase (PCK2) has further complex functions beyond regulation of glucose metabolism. Here, we report that conditional knockout of Pck2 in osteoblasts results in a pathological phenotype manifested as craniofacial malformation, long bone loss, and marrow adipocyte accumulation. Ablation of Pck2 alters the metabolic pathways of developing bone, particularly fatty acid metabolism. However, metformin treatment can mitigate skeletal dysplasia of embryonic and postnatal heterozygous knockout mice, at least partly via the AMPK signaling pathway. Collectively, these data illustrate that PCK2 is pivotal for bone development and metabolic homeostasis, and suggest that regulation of metformin-mediated signaling could provide a novel and practical strategy for treating metabolic skeletal dysfunction.