RÉSUMÉ
The infectivity of Leishmania is determined by its ability to invade and evade host and its thriving capacity within the macrophage. Our study revealed the role of Leishmania donovani mevalonate kinase (MVK), an enzyme of mevalonate pathway in visceral leishmaniasis pathogenesis. Peritoneal exudate cells (PEC)-derived macrophages from BALB/c mice were infected with wild type (WT), MVK over expressing (MVK OE) and knockdown (KD) parasites and MVK OE parasites were found to be more infective than WT and MVK KD parasites. Incubation of macrophages with MVK OE parasites declined inducible nitric oxide synthase (iNOS) expression as well as nitric oxide (NO) production, both by 2 times in comparison to WT parasites. Moreover, â¼3 fold increase in Arginase1 expression indicated that MVK might induce polarization of macrophage towards M2, favouring the survival of parasite within the macrophages. Post 24 h infection of the macrophages with mutant strains, the levels of different cytokines (TNF-α, IL-12, IL-10 and IFN-γ) were measured. Infection of macrophages with MVK OE parasites showed an increase in the level of anti-inflammatory cytokine: IL-10 while infection with MVK KD parasites exhibited an increase in the level of pro-inflammatory cytokines: TNF-α, IL-12, and IFN-γ. Hence, Leishmania donovani mevalonate kinase (LdMVK) modulates macrophage functions and has a significant role in pathogenesis.
Sujet(s)
Cytokines , Leishmania donovani , Leishmaniose viscérale , Macrophages péritonéaux , Souris de lignée BALB C , Nitric oxide synthase type II , Monoxyde d'azote , Phosphotransferases (Alcohol Group Acceptor) , Leishmania donovani/enzymologie , Leishmania donovani/pathogénicité , Leishmania donovani/génétique , Leishmania donovani/physiologie , Animaux , Souris , Leishmaniose viscérale/parasitologie , Leishmaniose viscérale/immunologie , Monoxyde d'azote/métabolisme , Macrophages péritonéaux/parasitologie , Macrophages péritonéaux/enzymologie , Cytokines/métabolisme , Phosphotransferases (Alcohol Group Acceptor)/métabolisme , Phosphotransferases (Alcohol Group Acceptor)/génétique , Nitric oxide synthase type II/métabolisme , Arginase/métabolisme , Arginase/génétique , Femelle , Techniques de knock-down de gènesRÉSUMÉ
BACKGROUND: Helicobacter pylori infection is one of the main causes of gastric cancer. thioredoxin-1 (Trx1) and arginase (RocF) expressed by H. pylori were found to be closely related to its pathogenicity. However, whether Trx1 and RocF can be used in clinical screening of highly pathogenic H. pylori and the pathogenesis of trx1 high expressing H. pylori remain still unknown. MATERIALS AND METHODS: We investigated the expression level of H. pylori trx1 and H. pylori rocF in human gastric antrum tissues using reverse transcription and quantitative real-time PCR (RT-qPCR) and clarified the clinical application value of trx1 and rocF for screening highly pathogenic H. pylori. The pathogenic mechanism of Trx1 were further explored by RNA-seq of GES-1 cells co-cultured with trx1 high or low expressing H. pylori. Differentially expressed genes and signaling pathways were validated by RT-qPCR, Enzyme-linked immunosorbent assay (ELISA), western blot, immunohistochemistry and immunofluorescence. We also assessed the adherence of trx1 high and low expressing H. pylori to GES-1 cells. RESULTS: We found that H. pylori trx1 and H. pylori rocF were more significantly expressed in the gastric cancer and peptic ulcer group than that in the gastritis group and the parallel diagnosis of H. pylori trx1 and H. pylori rocF had high sensitivity. The trx1 high expressing H. pylori had stronger adhesion ability to GES-1 cells and upregulated the interleukin (IL) 23A/nuclear factor κappaB (NF-κB)/IL17A, IL6, IL8 pathway. CONCLUSIONS: H. pylori trx1 and H. pylori rocF can be used in clinical screening of highly pathogenic H. pylori and predicting the outcome of H. pylori infection. The trx1 high expressing H. pylori has stronger adhesion capacity and promotes the development of gastric diseases by upregulating the activation of NF-κB signaling pathway.
Sujet(s)
Infections à Helicobacter , Helicobacter pylori , Interleukine-8 , Facteur de transcription NF-kappa B , Thiorédoxines , Humains , Helicobacter pylori/génétique , Helicobacter pylori/physiologie , Helicobacter pylori/pathogénicité , Thiorédoxines/métabolisme , Thiorédoxines/génétique , Facteur de transcription NF-kappa B/métabolisme , Infections à Helicobacter/microbiologie , Infections à Helicobacter/métabolisme , Interleukine-8/métabolisme , Interleukine-8/génétique , Régulation positive , Transduction du signal , Arginase/métabolisme , Arginase/génétique , Lignée cellulaire , Maladies de l'estomac/microbiologie , Maladies de l'estomac/métabolisme , Tumeurs de l'estomac/microbiologie , Tumeurs de l'estomac/génétique , Tumeurs de l'estomac/métabolisme , Tumeurs de l'estomac/anatomopathologieRÉSUMÉ
Elevation of arginase enzyme activity in the lung contributes to the pathogenesis of various chronic inflammatory diseases and infections. Inhibition of arginase expression and activity is able to alleviate those effects. Here, we investigated the immunomodulatory effect of arginase inhibitor in C. neoformans infection. In the pulmonary cryptococcosis model that was shown to recapitulate human infection, we found arginase expression was excessively induced in the lung during the late stage of infection. To inhibit the activity of arginase, we administered a specific arginase inhibitor, nor-NOHA, during C. neoformans infection. Inhibition of arginase reduced eosinophil infiltration and level of IL-13 secretion in the lungs. Whole lung transcriptome RNA-sequencing analysis revealed that treatment with nor-NOHA resulted in shifting the Th2-type gene expression patterns induced by C. neoformans infection to the Th1-type immune profile, with higher expression of cytokines Ifng, Il6, Tnfa, Csf3, chemokines Cxcl9 and Cxcl10 and transcription factor Stat1. More importantly, mice treated with arginase inhibitor had more infiltrating brain leukocytes and enhanced gene expression of Th1-associated cytokines and chemokines that are known to be essential for protection against C. neoformans infection. Inhibition of arginase dramatically attenuated spleen and brain infection, with improved survival. Taken together, these studies demonstrated that inhibiting arginase activity induced by C. neoformans infection can modulate host immune response by enhancing protective type-1 immune response during C. neoformans infection. The inhibition of arginase activity could be an immunomodulatory target to enhance protective anti-cryptococcal immune responses.
Sujet(s)
Arginase , Arginine/analogues et dérivés , Cryptococcose , Cryptococcus neoformans , Souris de lignée C57BL , Animaux , Arginase/métabolisme , Arginase/antagonistes et inhibiteurs , Arginase/génétique , Cryptococcose/immunologie , Cryptococcose/traitement médicamenteux , Cryptococcus neoformans/immunologie , Cryptococcus neoformans/effets des médicaments et des substances chimiques , Souris , Poumon/immunologie , Poumon/anatomopathologie , Poumon/effets des médicaments et des substances chimiques , Cytokines/métabolisme , Cytokines/immunologie , Femelle , Modèles animaux de maladie humaine , Mycoses pulmonaires/immunologie , Mycoses pulmonaires/traitement médicamenteux , Humains , Lymphocytes auxiliaires Th2/immunologie , Lymphocytes auxiliaires Th2/effets des médicaments et des substances chimiques , Lymphocytes auxiliaires Th1/immunologie , Lymphocytes auxiliaires Th1/effets des médicaments et des substances chimiques , Encéphale/immunologie , Encéphale/effets des médicaments et des substances chimiques , Encéphale/anatomopathologie , Antienzymes/pharmacologie , Antienzymes/usage thérapeutiqueRÉSUMÉ
Bacteria-enhanced inducible nitric oxide synthase (iNOS) overproduces nitric oxide (NO) leading to mitochondrial and cellular damage. In mammals, arginase (ARG), the enzyme consuming the same substrate l-arginine with iNOS, was believed to inhibit iNOS activity by competing the substrate. But in fish, this conception has been widely challenged. In this study, the gene expression using real-time quantitative PCR (RT-qPCR) technology showed that when stimulated by Aeromonas hydrophila (A. hydrophila), grass carp (gc) iNOS was up-regulated in head kidney monocytes/macrophages (M0/MФ), and its changes were not detected in the whole tissue of liver or spleen, showing a high degree of cell-specific expression pattern. At the same time, gcARG2 had a high basal expression in tissues and was up-regulated by A. hydrophila stimulation. Next, phthalaldehyde-primaquine reaction was first used in the determination of intracellular urea in fish cells. It was found that the induced gcARG2 led to an increase in the intracellular urea content. Moreover, urea and NO production in M0/MФ were increased in a substrate dose-dependent manner from 30 to 100 µM of l-arginine and reached the highest yield at 300 and 3000 µM of l-arginine, respectively. Furthermore, head kidney M0/MФ was cultured in RPMI1640 medium containing physiological concentration (500 µM) of l-arginine to evaluate the effect of ARG. Under A. hydrophila stimulation, treatment with the arginase inhibitor S-(2-boronoethyl)-l-cysteine (BEC) showed that inhibition of arginase could further enhance the NO production stimulated by A. hydrophila. This in turn led to a cumulation in peroxynitrite (ONOO-) content and an injury of the mitochondrial membrane potential. Our study showed for the first time that fish ARG in head kidney M0/MФ can limit excessive production of NO and harmful products by iNOS to maintain mitochondrial and cellular homeostasis.
Sujet(s)
Aeromonas hydrophila , Arginase , Carpes (poisson) , Maladies des poissons , Protéines de poisson , Infections bactériennes à Gram négatif , Mitochondries , Monoxyde d'azote , Animaux , Aeromonas hydrophila/physiologie , Arginase/génétique , Arginase/métabolisme , Maladies des poissons/immunologie , Infections bactériennes à Gram négatif/immunologie , Infections bactériennes à Gram négatif/médecine vétérinaire , Monoxyde d'azote/métabolisme , Carpes (poisson)/immunologie , Protéines de poisson/génétique , Protéines de poisson/immunologie , Nitric oxide synthase type II/génétique , Nitric oxide synthase type II/métabolisme , ArginineRÉSUMÉ
The mitochondrial enzyme arginase-2 (Arg-2) is implicated in the pathophysiology of contrast-induced acute kidney injury (CI-AKI). Therefore, Arg-2 represents a candid target for CI-AKI prevention. Here, layer-by-layer (LbL) assembled renal-targeting polymeric nanoparticles are developed to efficiently deliver small interfering RNA (siRNA), knockdown Arg-2 expression in renal tubules, and prevention of CI-AKI is evaluated. First, near-infrared dye-loaded poly(lactic-co-glycolic acid) (PLGA) anionic cores are electrostatically coated with cationic chitosan (CS) to facilitate the adsorption and stabilization of Arg-2 siRNA. Next, nanoparticles are coated with anionic hyaluronan (HA) to provide protection against siRNA leakage and shielding against early clearance. Sequential electrostatic layering of CS and HA improves loading capacity of Arg-2 siRNA and yields LbL-assembled nanoparticles. Renal targeting and accumulation is enhanced by modifying the outermost layer of HA with a kidney targeting peptide (HA-KTP). The resultant kidney-targeting and siRNA loaded nanoparticles (PLGA/CS/HA-KTP siRNA) exhibit proprietary accumulation in kidneys and proximal tubular cells at 24 h post-tail vein injection. In iohexol-induced in vitro and in vivo CI-AKI models, PLGA/CS/HA-KTP siRNA delivery alleviates oxidative and nitrification stress, and rescues mitochondrial dysfunction while reducing apoptosis, thereby demonstrating a robust and satisfactory therapeutic effect. Thus, PLGA/CS/HA-KTP siRNA nanoparticles offer a promising candidate therapy to protect against CI-AKI.
Sujet(s)
Atteinte rénale aigüe , Arginase , Produits de contraste , Nanoparticules , Copolymère d'acide poly(lactique-co-glycolique) , Petit ARN interférent , Nanoparticules/composition chimique , Atteinte rénale aigüe/induit chimiquement , Atteinte rénale aigüe/prévention et contrôle , Atteinte rénale aigüe/métabolisme , Animaux , Petit ARN interférent/composition chimique , Copolymère d'acide poly(lactique-co-glycolique)/composition chimique , Produits de contraste/composition chimique , Souris , Arginase/métabolisme , Arginase/génétique , Chitosane/composition chimique , Techniques de knock-down de gènes , Rein/métabolisme , Rein/effets des médicaments et des substances chimiques , Rein/anatomopathologie , Acide hyaluronique/composition chimique , Mâle , Humains , Acide lactique/composition chimique , Acide polyglycolique/composition chimique , Nanoparticules couche par coucheRÉSUMÉ
Neuregulin receptor degradation protein 1 (Nrdp1) is a ring finger E3 ubiquitin ligase involved in some inflammation through ubiquitination, including macrophage polarization following cerebral hemorrhage. However, there is limited understanding regarding the mechanisms through which Nrdp1 modulates macrophage polarization and the potential impact of this modulation on neurological function. Using stereotactic injection and adenoviral transfection techniques, the corresponding animal models were constructed through injecting adenovirus, saline, or blood into the mouse striatum at different periods of time in this research. The alteration in the ratio of various M1/M2 phenotype-associated markers (e.g., CD86, CD206, IL-6, IL-10, etc.) was evaluated through immunohistochemistry, immunofluorescence, western blotting, and elisa assays. Additionally, neurological function scores and behavioral tests were utilized to evaluate changes in neurological function in mice after cerebral hemorrhage. Our results show that overexpression of Nrdp1 promotes the expression of a variety of M2 macrophage-associated markers and enhance transcriptional activity of arginase-1 (Arg1) protein through ubiquitination for early regulation M2 macrophage polarization. Additionally, Nrdp1 promotes hematoma absorption, increases IL-10 expression, inhibits inducible nitric oxide synthase (iNOS), IL-6, and TNF-α production, alleviates neurological impairment and brain edema, and accelerates functional recovery. These findings suggest that modulating macrophage polarization through Nrdp1 could be a therapeutic strategy for neurofunctional impairment in cerebral hemorrhage.
Sujet(s)
Hémorragie cérébrale , Macrophages , Récupération fonctionnelle , Ubiquitin-protein ligases , Animaux , Hémorragie cérébrale/métabolisme , Hémorragie cérébrale/anatomopathologie , Macrophages/métabolisme , Mâle , Ubiquitin-protein ligases/métabolisme , Ubiquitin-protein ligases/génétique , Souris , Récupération fonctionnelle/physiologie , Souris de lignée C57BL , Arginase/métabolisme , Arginase/génétique , Phénotype , Modèles animaux de maladie humaine , Ubiquitination , Activation des macrophages/physiologieRÉSUMÉ
Cystic echinococcosis (CE) is a zoonotic disease, caused by Echinococcus granulosus sensu lato (E. granulosus s. l.), which posed significant public health concern globally. E. granulosus s. l. annexin B18 (EgANXB18) acts as a secretory protein, exerting a crucial influence in mediating host-parasite interactions. Recombinant annexin B18 (rEgANXB18) was expressed by Escherichia coli and the immunoreactivity was assessed by western blotting. The binding affinity between rEgANXB18 and total protein of RAW264.7 cells was assessed by ELISA. The impact of rEgANXB18 on the metabolic activity of RAW264.7 cells was assayed by Cell Counting Kit-8 assay. The mRNA levels of polarization markers (inducible nitrous oxide synthase (iNOS) and arginase 1 (Arg1)) and key cellular factors (IL-1ßï¼IL-6ï¼IL-10 and TNFα) were evaluated by qRT-PCR. rEgANXB18 was successfully expressed and recognized by E. granulosus s.l. infected canine sera, as well as could bind to the total protein of RAW264.7 cells. Additionally, rEgANXB18 could promote metabolic activity at 5, 10, 20, and 40 µg/mL while no significant impact on metabolic activity was observed at 80 µg/mL. Co-culture RAW264.7 cells with rEgANXB18 resulted in significantly upregulation of the transcript levels of polarization markers iNOS and Arg1. Moreover, rEgANXB18 significantly upregulated the transcript levels of IL-1ß, IL-6, TNFα, and IL-10, while dose-effect relationship was observed in IL-1ß, IL-6, and IL-10. Our results indicated that EgANXB18 showed the potential to regulate immune response of macrophages by shifting the cell polarization and cytokine profile, thereby promoting the parasitism of CE.
Sujet(s)
Annexines , Arginase , Échinococcose , Echinococcus granulosus , Macrophages , Nitric oxide synthase type II , Animaux , Echinococcus granulosus/génétique , Echinococcus granulosus/immunologie , Souris , Macrophages/parasitologie , Macrophages/métabolisme , Cellules RAW 264.7 , Arginase/métabolisme , Arginase/génétique , Échinococcose/parasitologie , Échinococcose/immunologie , Nitric oxide synthase type II/métabolisme , Nitric oxide synthase type II/génétique , Annexines/génétique , Annexines/métabolisme , Chiens , Protéines recombinantes/génétique , Protéines recombinantes/métabolisme , Cytokines/métabolisme , Cytokines/génétique , ARN messager/métabolisme , Test ELISA , Technique de Western , Interactions hôte-parasiteRÉSUMÉ
Glaciozyma antarctica PI12 is a psychrophilic yeast isolated from Antarctica. In this work, we describe the heterologous production, biochemical properties and in silico structure analysis of an arginase from this yeast (GaArg). GaArg is a metalloenzyme that catalyses the hydrolysis of L-arginine to L-ornithine and urea. The cDNA of GaArg was reversed transcribed, cloned, expressed and purified as a recombinant protein in Escherichia coli. The purified protein was active against L-arginine as its substrate in a reaction at 20 °C, pH 9. At 10-35 °C and pH 7-9, the catalytic activity of the protein was still present around 50%. Mn2+, Ni2+, Co2+ and K+ were able to enhance the enzyme activity more than two-fold, while GaArg is most sensitive to SDS, EDTA and DTT. The predicted structure model of GaArg showed a very similar overall fold with other known arginases. GaArg possesses predominantly smaller and uncharged amino acids, fewer salt bridges, hydrogen bonds and hydrophobic interactions compared to the other counterparts. GaArg is the first reported arginase that is cold-active, facilitated by unique structural characteristics for its adaptation of catalytic functions at low-temperature environments. The structure and function of cold-active GaArg provide insights into the potentiality of new applications in various biotechnology and pharmaceutical industries.
Sujet(s)
Basidiomycota , Saccharomyces cerevisiae , Arginase/génétique , Basidiomycota/génétique , Arginine , Escherichia coliRÉSUMÉ
Endometrial cancer (EC) is the most common gynecologic malignancy. While the majority of patients present with early-stage and low-grade EC and have an excellent prognosis, a subset has metastatic disease at presentation or develops distant recurrence after initial treatment of the primary. However, the lack of prognostic biomarkers for metastatic EC is a critical barrier. Arginase 1 (ARG1) regulates the last step of the urea cycle, and an increase in ARG1 has been correlated as a poor prognostic factor in a variety of cancers. In the present study, ARG1 expression was evaluated as a potential prognostic marker for metastatic EC in endometrial hyperplasia and cancer of mice with Pten mutation as well as Pten and Mig-6 double mutations. While Pten mutation in the uterus is not sufficient for distant metastasis, mice with concurrent ablation of Mig-6 and Pten develop distant metastasis. Our immunostaining and RT-qPCR analysis revealed that the expression of ARG1 in early stage of EC as well as endometrial hyperplasia from mice deficient in Mig-6 and Pten mutations significantly increased compared to Pten mutation in the uterus. The results suggest that a high level of ARG1 is associated with poor prognosis in association with EC of mouse.
Sujet(s)
Arginase , Marqueurs biologiques tumoraux , Tumeurs de l'endomètre , Phosphohydrolase PTEN , Femelle , Tumeurs de l'endomètre/anatomopathologie , Tumeurs de l'endomètre/génétique , Tumeurs de l'endomètre/métabolisme , Animaux , Arginase/génétique , Arginase/métabolisme , Phosphohydrolase PTEN/génétique , Phosphohydrolase PTEN/métabolisme , Marqueurs biologiques tumoraux/génétique , Marqueurs biologiques tumoraux/métabolisme , Pronostic , Souris , Humains , Mutation , Hyperplasie endométriale/génétique , Hyperplasie endométriale/métabolisme , Hyperplasie endométriale/anatomopathologie , Métastase tumoraleRÉSUMÉ
BACKGROUND: Arginase 1 (Arg1) encodes a key enzyme that catalyzes the metabolism of arginine to ornithine and urea. In our recent study, we found that knockdown of Arg1 in the lungs of fetal mice induces apoptosis of epithelial cells and dramatically delays initiation of labor. As the most abundant internal mRNA modification, N6 -methyladenosine (m6 A) has been found to play important roles in lung development and cellular differentiation. However, if the knockdown of Arg1 affects the RNA m6A modification in fetal lungs remains unknown. METHODS: In the current study, the RNA m6A levels and the expression of RNA m6A related enzymes were validated in 13.0 dpc fetal lungs that Arg1 was knocked down by adeno-associated virus carrying Arg1-shRNA, using western blot, immunofluorescence, and RT-qPCR. RESULTS: No statistical differences were found in the expression of methyltransferase, demethylases, and binding proteins in the fetal lungs between AAV-shArg1-injected mice and AAV-2/9-injected mice. Besides, there is no significant change of overall RNA m6A level in fetal lungs from AAV-shArg1-injected mice, compared with that from AAV-2/9-injected mice. CONCLUSIONS: These results indicate that arginase 1 does not affect RNA m6A methylation in mouse fetal lung, and the mechanisms other than RNA m6A modification underlying the effects of Arg1 knockdown on the fetal lung development and their interaction with labor initiation need to be further explored.
Sujet(s)
Arginase , , Souris , Animaux , Arginase/génétique , Arginase/métabolisme , Poumon/métabolisme , Methyltransferases/génétique , Methyltransferases/métabolisme , ARN/métabolismeRÉSUMÉ
BACKGROUND: L-arginase, is a powerful anticancer that hydrolyzes L-arginine to L-ornithine and urea. This enzyme is widely distributed and expressed in organisms like plants, fungi, however very scarce from bacteria. Our study is based on isolating, purifying, and screening the marine bacteria that can produce arginase. RESULTS: The highest arginase producing bacteria will be identified by using microbiological and molecular biology methods as Bacillus licheniformis OF2. Characterization of arginase is the objective of this study. The activity of enzyme was screened, and estimated beside partial sequencing of arginase gene was analyzed. In silico homology modeling was applied to generate the protein's 3D structure, and COACH and COFACTOR were applied to determine the protein's binding sites and biological annotations based on the I-TASSER structure prediction. The purified enzyme was undergone an in vitro anticancer test. CONCLUSIONS: L-arginase demonstrated more strong anti-cancer cells with an IC50 of 21.4 ug/ml in a dose-dependent manner. L-arginase underwent another investigation for its impact on the caspase 7 and BCL2 family of proteins (BCL2, Bax, and Bax/Bcl2). Through cell arrest in the G1/S phase, L-arginase signals the apoptotic cascade, which is supported by a flow cytometry analysis of cell cycle phases.
Sujet(s)
Arginase , Bacillus licheniformis , Arginase/génétique , Arginase/métabolisme , Bacillus licheniformis/génétique , Bacillus licheniformis/métabolisme , Protéine Bax/génétique , Arginine/métabolisme , Ornithine/métabolisme , Protéines proto-oncogènes c-bcl-2RÉSUMÉ
BACKGROUND & AIMS: Restoring hepatic and peripheral insulin sensitivity is critical to prevent or reverse metabolic syndrome and type 2 diabetes. Glucose homeostasis comprises in part the complex regulation of hepatic glucose production and insulin-mediated glucose uptake and oxidation in peripheral tissues. We previously identified hepatocyte arginase 2 (Arg2) as an inducible ureahydrolase that improves glucose homeostasis and enhances glucose oxidation in multiple obese, insulin-resistant models. We therefore examined structure-function determinants through which hepatocyte Arg2 governs systemic insulin action and glucose oxidation. METHODS: To do this, we generated mice expressing wild-type murine Arg2, enzymatically inactive Arg2 (Arg2H160F) and Arg2 lacking its putative mitochondrial targeting sequence (Arg2Δ1-22). We expressed these hepatocyte-specific constructs in obese, diabetic (db/db) mice and performed genetic complementation analyses in hepatocyte-specific Arg2-deficent (Arg2LKO) mice. RESULTS: We show that Arg2 attenuates hepatic steatosis, independent of mitochondrial localization or ureahydrolase activity, and that enzymatic arginase activity is dispensable for Arg2 to augment total body energy expenditure. In contrast, mitochondrial localization and ureahydrolase activity were required for Arg2-mediated reductions in fasting glucose and insulin resistance indices. Mechanistically, Arg2Δ1-22 and Arg2H160F failed to suppress glucose appearance during hyperinsulinemic-euglycemic clamping. Quantification of heavy-isotope-labeled glucose oxidation further revealed that mistargeting or ablating Arg2 enzymatic function abrogates Arg2-induced peripheral glucose oxidation. CONCLUSION: We conclude that the metabolic effects of Arg2 extend beyond its enzymatic activity, yet hepatocyte mitochondrial ureahydrolysis drives hepatic and peripheral oxidative metabolism. The data define a structure-based mechanism mediating hepatocyte Arg2 function and nominate hepatocyte mitochondrial ureahydrolysis as a key determinant of glucose oxidative capacity in mammals.
Sujet(s)
Arginase , Diabète de type 2 , Souris , Animaux , Arginase/génétique , Arginase/métabolisme , Glucose , Hépatocytes/métabolisme , Obésité/métabolisme , Insuline , Mammifères/métabolismeRÉSUMÉ
Objective. The study was performed to elucidate whether nicotinamide (NAm) can attenuate the diabetes-induced liver damage by correction of ammonia detoxifying function and disbalance of NAD-dependent processes in diabetic rats. Methods. After four weeks of streptozotocin-induced diabetes, Wistar male rats were treated for two weeks with or without NAm. Urea concentration, arginase, and glutamine synthetase activities, NAD+ levels, and NAD+/NADH ratio were measured in cytosolic liver extracts. Expression of parp-1 gene in the liver was estimated by quantitative polymerase chain reaction and PARP-1 cleavage evaluated by Western blotting. Results. Despite the blood plasma lipid peroxidation products in diabetic rats were increased by 60%, the activity of superoxide dismutase (SOD) was reduced. NAm attenuated the oxidative stress, but did not affect the enzyme activity in diabetic rats. In liver of the diabetic rats, urea concentration and arginase activity were significantly higher than in the controls. The glutamine synthetase activity was decreased. Decline in NAD+ level and cytosolic NAD+/NADH ratio in the liver of diabetic rats was observed. Western blot analysis demonstrated a significant up-regulation of PARP-1 expression accompanied by the enzyme cleavage in the diabetic rat liver. However, no correlation was seen between mRNA expression of parp-1 gene and PARP-1 protein in the liver of diabetic rats. NAm markedly attenuated PARP-1 cleavage induced by diabetes, but did not affect the parp-1 gene expression. Conclusions. NAm counteracts diabetes-induced impairments in the rat liver through improvement of its detoxifying function, partial restoration of oxidative stress, NAD+ level, normalization of redox state of free cytosolic NAD+/NADH-couples, and prevention of PARP-1 cleavage.
Sujet(s)
Diabète expérimental , Nicotinamide , Rats , Mâle , Animaux , Nicotinamide/pharmacologie , Nicotinamide/métabolisme , NAD/métabolisme , NAD/pharmacologie , Diabète expérimental/traitement médicamenteux , Diabète expérimental/métabolisme , Rat Wistar , Inhibiteurs de poly(ADP-ribose) polymérases/métabolisme , Inhibiteurs de poly(ADP-ribose) polymérases/pharmacologie , Arginase/génétique , Arginase/métabolisme , Arginase/pharmacologie , Glutamate-ammonia ligase/génétique , Glutamate-ammonia ligase/métabolisme , Glutamate-ammonia ligase/pharmacologie , Stress oxydatif , Foie/métabolisme , Urée/métabolisme , Urée/pharmacologieRÉSUMÉ
Obesity is a risk factor for vascular endothelial cell dysfunction characterized by low-grade, chronic inflammation. Increased levels of arginase I and concomitant decreases in l-arginine bioavailability are known to play a role in the pathogenesis of vascular endothelial cell dysfunction. In the present study, we focused on changes in the systemic expression of arginase I as well as l-arginine metabolism in the pre-disease state of early obesity prior to the onset of atherosclerosis. C57BL/6 mice were fed a control diet (CD; 10% fat) or high-fat diet (HFD; 60% fat) for 8 weeks. The mRNA expression of arginase I in the liver, adipose tissue, aorta, and muscle; protein expression of arginase I in the liver and plasma; and systemic levels of l-arginine bioavailability and NO2 − were assessed. HFD-fed mice showed early obesity without severe disease symptoms. Arginase I mRNA and protein expression levels in the liver were significantly higher in HFD-fed obese mice than in CD-fed mice. Arginase I levels were slightly increased, whereas l-arginine levels were significantly reduced, and these changes were followed by reductions in NO2 − levels. Furthermore, hepatic arginase I levels positively correlated with plasma arginase I levels and negatively correlated with l-arginine bioavailability in plasma. These results suggested that increases in the expression of hepatic arginase I and reductions in plasma l-arginine and NO2 − levels might lead to vascular endothelial dysfunction in the pre-disease state of early obesity
Sujet(s)
Animaux , Mâle , Souris , Arginase/métabolisme , Arginine/sang , Endothélium vasculaire/métabolisme , Foie/métabolisme , Monoxyde d'azote/sang , Obésité/métabolisme , Vascularite systémique/métabolisme , Aorte/enzymologie , Aorte/métabolisme , Arginase/sang , Arginase/génétique , Athérosclérose/étiologie , Marqueurs biologiques , Endothélium vasculaire/immunologie , Endothélium vasculaire/anatomopathologie , Vascularite systémique/physiopathologie , Indice de gravité de la maladieRÉSUMÉ
The incidence of cardiovascular disease is predicted to increase as the population ages. There is accumulating evidence that arginase upregulation is associated with impaired endothelial function. Here, we demonstrate that arginase II (ArgII) is upregulated in aortic vessels of aged mice and contributes to decreased nitric oxide (NO) generation and increased reactive oxygen species (ROS) production via endothelial nitric oxide synthase (eNOS) uncoupling. Inhibiting ArgII with small interfering RNA technique restored eNOS coupling to that observed in young mice and increased NO generation and decreased ROS production. Furthermore, enhanced vasoconstrictor responses to U46619 and attenuated vasorelaxation responses to acetylcholine in aged vasculature were markedly improved following siRNA treatment against ArgII. These results might be associated with increased L-arginine bioavailability. Collectively, these results suggest that ArgII may be a valuable target in age-dependent vascular diseases.
Sujet(s)
Animaux , Souris , Acide 15-hydroxy-11alpha,9alpha-(époxyméthano)prosta-5,13-diénoïque/pharmacologie , Vieillissement , Aorte/enzymologie , Arginase/génétique , Endothélium vasculaire/enzymologie , Induction enzymatique , Techniques de knock-down de gènes , Souris de lignée C57BL , Monoxyde d'azote/métabolisme , Nitric oxide synthase type III/métabolisme , Petit ARN interférent/génétique , Espèces réactives de l'oxygène/métabolisme , Régulation positive , Vasoconstriction/effets des médicaments et des substances chimiquesRÉSUMÉ
Introducción: Variaciones moleculares en el gen de la arginasa I, ARGl, podrían provocar un aumento de la expresión de la enzima o de su actividad, lo que puede llevar a una disfunción endotelial al disminuir la producción de óxido nítrico por parte de la eNOS. Así, el objetivo del presente estudio fue investigar la posible asociación del polimorfismo rs2781666 G>T y la presencia de enfermedad coronaria (confirmada con angiografía) en individuos de la región de La Araucanía. Material y Métodos: En el presente estudio, de tipo casos y controles, fueron evaluados 247 sujetos, con edades entre 30 y 74 años; 124 individuos pacientes con enfermedad arterial coronaria (casos) y 123 controles. La genotipificación del polimorfismo rs2781666 del gen ARGl fue realizada mediante PCR- RFLR Resultados: La frecuencia del genotipo homocigoto TT para el polimorfismo rs2781666 del gen ARGl fue de 6.5 por ciento en el grupo casos y 8 por ciento en el grupo control, difiriendo significativamente (p=0.032). La frecuencia relativa del alelo T también presentó diferencias significativas entre casos y controles (0.230 vs. 0.325, p=0.031). La OR relacionada al alelo mutado T fue de 0.63 (I.C. 95 por ciento, 0.43 - 0.94), confirmando la presencia de la asociación observada. Conclusión: Los resultados obtenidos sugieren que el polimorfismo rs2781666 G>T del gen ARGl confiere protección contra enfermedad coronaria en la población analizada. Sin embargo, este resultado debe ser replicado en otros grupos poblacionales de nuestro país.
Background: Recent data support a role for arginase 1 (ARG1) in the initiation, development, and complications of coronary artery disease. Thus, in the present study we investigated the possible association between the rs2781666 G>T variant of ARG1 and the presence of CAD confirmed by angiography in Chilean subjects. Methods: A total of 124 unrelated patients with diagnosis of CAD confirmed by angiography (stenosis > 70 percent) and 123 healthy controls (30 - 74 years old) were included in this study. The rs2781666 G>T variant of the ARG1 gene was evaluated by PCR-RFLR Results: The frequency of TT homozygous genotype in CAD patients was lower when compared to control group (6.5 percent vs. 8.0 percent, p=0.032). Similarly, the T allele frequency was different between CAD and control groups (0.230 vs. 0.325, p=0.031). The OR for CAD related to T allele was 0.64 (95 percent CI. = 0.43-0.94), con-firming the association founded. Conclusion: These findings suggest that the T allele for rs2781666 polymorphism of the ARG1 gene constitutes an inherited protective factor against CAD in Southern Chilean subjects.