Expression Of Glutamine Synthase Research Articles

Peroxiredoxin 6 Regulates Glutathione Peroxidase 1-Medited Glutamine Synthase Preservation in the Hippocampus of Chronic Epilepsy Rats.

Clasmatodendrosis (an autophagic astroglial degeneration) plays an important role in the regulation of spontaneous seizure duration but not seizure frequency or behavioral seizure severity in chronic epilepsy rats. Recently, it has been reported that N-acetylcysteine (NAC), a precursor to glutathione (GSH), attenuates clasmatodendritic degeneration and shortens spontaneous seizure duration in chronic epilepsy rats, although the underlying mechanisms of its anti-convulsive effects are not fully understood. To elucidate this, the present study was designed to investigate whether NAC affects astroglial glutamine synthase (GS) expression mediated by GSH peroxidase 1 (GPx1) and/or peroxiredoxin 6 (Prdx6) in the epileptic hippocampus. As compared to control animals, GS and GPx1 expressions were upregulated in reactive CA1 astrocytes of chronic epilepsy rats, while their expressions were significantly decreased in clasmatodendritic CA1 astrocytes and reactive astrocytes within the molecular layer of the dentate gyrus. Prdx6 expression was increased in reactive CA1 astrocytes as well as clasmatodendritic CA1 astrocytes. In the molecular layer of the dentate gyrus, Prdx6 expression levels were similar to those in control animals. NAC ameliorated clasmatodendrosis through the increment of GS and GPx1 expressions, while it abolished Prdx6 upregulation. 1-hexadecyl-3-(trifluoroethgl)-sn-glycerol-2 phosphomethanol (MJ33, a selective inhibitor of aiPLA2 activity of Prdx6) alleviated clasmatodendrosis by enhancing GPx1 and GS expressions in clasmatodendritic CA1 astrocytes without changing the Prdx6 level. NAC or MJ33 did not affect GS, GPx1 and Prdx6 expression in astrocytes within the molecular layer of the dentate gyrus. These findings indicate that upregulated aiPLA2 activity of Prdx6 may abolish GPx1-mediated GS preservation and lead to clasmatodendrosis in CA1 astrocytes, which would extend spontaneous seizure duration due to impaired glutamate-glutamine conversion regulated by GS. Therefore, the present data suggest that aiPLA2 activity of Prdx6 in astrocytes may be one of the upstream effectors of seizure duration in the epileptic hippocampus.

Time-dependent changes in hypoxia- and gliosis-related factors in experimental diabetic retinopathy

Diabetes causes various biochemical changes in the retina; long-term changes in the factors associated with hypoxia and gliosis have rarely been reported. The present study was conducted to explore the changes in these factors in a time-dependent manner in experimental diabetic retinopathy (DR). Diabetes was induced in Sprague–Dawley rats by intraperitoneal injection of streptozotocin. The expression of the following factors was examined using immunofluorescence and western blot analysis at 0.5, 1, 2, 4 and 6 months after diabetes onset: hypoxia-inducible factor-1alpha (HIF-1alpha), vascular endothelial growth factor (VEGF), erythropoietin (EPO), erythropoietin receptor (EPOR), glial fibrillary acidic protein (GFAP), vimentin, glutamate-aspartate transporter (GLAST) and glutamine synthase (GS). The expression of factors such as HIF-1alpha, VEGF, EPO, EPOR, GFAP and vimentin, was up-regulated with the progression of diabetes in the diabetic rat retinas compared to the expression in normal control retinas, whereas the expression of GS and GLAST was down-regulated. Changes in EPO and EPOR appeared 2 weeks after diabetes onset. HIF-1alpha, VEGF and GFAP started to increase at 1 month and vimentin at 4 months after diabetes onset. GS and GLAST started to decrease at 1 month after diabetes onset. The expression of these factors, which are involved in the processes of hypoxia and gliosis, varied at different stages of DR. The time-course change may be helpful in the evaluation of the progression of DR, and it may indicate the optimal intervention time points for DR.

Alteration in mitochondrial function and glutamate metabolism affected by 2-chloroethanol in primary cultured astrocytes

The aim of this study was to explore the mechanisms that contribute to 1,2-dichloroethane (1,2-DCE) induced brain edema by focusing on alteration of mitochondrial function and glutamate metabolism in primary cultured astrocytes induced by 2-chloroethanol (2-CE), a metabolite of 1,2-DCE in vivo. The cells were exposed to different levels of 2-CE in the media for 24h. Mitochondrial function was evaluated by its membrane potential and intracellular contents of ATP, lactic acid and reactive oxygen species (ROS). Glutamate metabolism was indicated by expression of glutamine synthase (GS), glutamate–aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1) at both protein and gene levels. Compared to the control group, exposure to 2-CE could cause a dose dependent damage in astrocytes, indicated by decreased cell viability and morphological changes, and supported by decreased levels of nonprotein sulfhydryl (NPSH) and inhibited activities of Na+/K+-ATPase and Ca2+-ATPase in the cells. The present study also revealed both mitochondrial function and glutamate metabolism in astrocytes were significantly disturbed by 2-CE. Of which, mitochondrial function was much vulnerable to the effects of 2-CE. In conclusion, our findings suggested that mitochondrial dysfunction and glutamate metabolism disorder could contribute to 2-CE-induced cytotoxicity in astrocytes, which might be related to 1,2-DCE-induced brain edema.

Abstract 5151: Yap reprograms glutamine metabolism and supports growth during liver development and tumorigenesis

Abstract Hepatocellular carcinoma is a global health problem with poor prognosis and limited therapeutic options. Recently, the Hippo pathway has emerged as a master regulator of organ size control and tumorigenesis. However, the metabolic impact of the pathway is poorly understood. Using a transgenic zebrafish model with liver-specific activation of the Hippo pathway effector Yap, we have shown that Yap promotes hepatomegaly and liver dysplasia. In addition, we demonstrate that the Yap transgenics are highly susceptible to chemically-induced hepatocarcinogenesis. Transcriptomic and metabolomic profiling reveals that Yap enhances glutamine synthase (GS) expression and elevates steady-state levels of glutamine, respectively. Intervention studies with the GS inhibitor methionine sulfoximine established that elevated GS activity contributes to the rapid liver growth observed during Yap-driven hepatomegaly. Finally, studies in cultured human cancer cells identify GS as a bone-fide Yap target gene, confirming that the GS regulation by Yap is evolutionarily conserved. We conclude that Yap regulates GS expression and reprograms nitrogen metabolism, which contributes to liver growth during development and tumorigenesis. We hypothesize that Yap integrates the anabolic demands of rapid cell proliferation by increasing the flux of glutamine into nucleotide biosynthesis. Citation Format: Andrew G. Cox, Katie L. Hwang, Kimberley Evason, Kristin K. Brown, Sebastian Beltz, Keelin O'Connor, Giorgio G. Galli, Dean Yimlamai, Sagar Chhangawala, Evan Lien, Fernando D. Camargo, John Asara, Yariv Houvras, Didier Y. Stainier, Wolfram Goessling. Yap reprograms glutamine metabolism and supports growth during liver development and tumorigenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5151. doi:10.1158/1538-7445.AM2015-5151

Pigment epithelium-derived factor protects the morphological structure of retinal Müller cells in diabetic rats.

To investigate if pigment epithelium-derived factor (PEDF) has any protective effect on the retinal Müller cells of Sprague-Dawley rats suffering from diabetes mellitus. Sixty Sprague-Dawley rats were randomly divided into a negative control group, a group receiving 0.1 µg/µL PEDF, another group receiving 0.2 µg/µL PEDF, and a group receiving balanced salt solution (BSS). Rats in both the PEDF and BSS groups were treated intravitreally based on previously established diabetic models. After 4wk of treatment, morphological alterations of Müller cells and protein expression of glutamine synthase (GS) and glial fibrillary acidic protein (GFAP) were analyzed. PEDF at either 0.1 µg/µL or 0.2 µg/µL significantly improved the structures of both nuclei and organelles of Müller cells compared to the BSS-treated group. Expression of GS was significantly higher in the 0.2 µg/µL PEDF group than that in the BSS group (P=0.012), but expression of GFAP was significantly lower in the 0.2 µg/µL PEDF group than that in the BSS group (P=0.000); however, there were no significant differences in expression of these proteins between the 0.1 µg/µL PEDF group and the BSS group (P=0.608, P=0.152). PEDF protects the morphological ultrastructure of Müller cells, improves the expression of glutamate synthase and prevents cell gliosis.

Aqueous humor oxidative stress proteomic levels in primary open angle glaucoma

The purpose of this work was to investigate the expression of glutamine synthase (GS), nitric oxide synthase (NOS) superoxide dismutase (SOD) and glutathione transferase (GST) in the aqueous humor of patients with primary open angle glaucoma and controls. Aqueous humor proteome was analyzed by antibody microarray. The expression of tested proteins was detected by protein Cy3/Cy5 labeling, column purification and hybridization on antibody-spotted glass microarray. Fluorescent signals were detected by fluorescence laser scanning. Aqueous humor levels of SOD as well as of GST were significantly lower (2.0- and 2.2-fold, p < 0.01) among patients than controls; both NOS and GS expression were significantly higher (2.2- and 2.6 fold, p < 0.01) among patients than controls. Our data showed substantial differences of GS, NOS2, SOD and GST aqueous humor levels between glaucomatous patients and controls as measured by antibody microarray technology. The overproduction of NO through inducible NOS can form toxic products and change the metabolic conditions of the TM. The GS over-expression might be related to neuronal injury or to the potential role of glutamate as a modulator in the ciliary body signaling. The reduced expression of the antioxidant enzymes SOD and GST could aggravate the unbalance between both oxygen- and nitrogen-derived free radicals production and detoxification. Based on our results, GS, NOS2, SOD and GST as measured by antibody microarray technology may be useful oxidative markers in aqueous humor of glaucomatous patients.

Alterations in the Mitochondrial Proteome in Broilers fed a Lysine‐Deficient Diet

In liver, the primary site of lysine degradation is the mitochondrial matrix, but the protein(s) responsible for lysine uptake to the matrix is(are) unknown. Changes in inner mitochondrial membrane protein abundances were observed in broilers fed lysine‐adequate (LA) or lysine‐deficient (LD) diets for 14 days. Two transport proteins were affected by the LD diet. Broilers consuming the LD diet expressed only 50% of a voltage‐gated anion‐selective and a voltage‐gated potassium channel compared to those consuming a LA diet, although presumably lysine is not transported via either of these proteins. The consumption of the LD diet impacted many aspects of metabolism. Glutamine synthase expression was decreased over 95% in the chickens consuming the LD diet. Electron transport chain (ETC) components were also altered. Broilers consuming the LD diet had over a 30% reduction in NADH dehydrogenase expression and almost a 90% decrease in the expression of ATP sythase. Proteins involved in fatty acid oxidation were also affected by the LD diet, as Acyl CoA dehydrogenase was reduced about 75%. These data indicate that a LD diet affects many aspects of metabolism that may contribute to the reduction of growth typically seen with a lysine deficiency. (Support USDA‐NRI 2004‐35206‐14160)

N-acetylcysteine prevents HIV gp 120-related damage of human cultured astrocytes: correlation with glutamine synthase dysfunction

BackgroundHIV envelope gp 120 glycoprotein is released during active HIV infection of brain macrophages thereby generating inflammation and oxidative stress which contribute to the development of the AIDS-Dementia Complex (ADC). Gp120 has also been found capable to generate excitotoxic effect on brain tissue via enhancement of glutamatergic neurotransmission, leading to neuronal and astroglial damage, though the mechanism is still to be better understood.Here we investigated on the effect of N-acetylcysteine (NAC), on gp120-induced damage in human cultured astroglial cells and the possible contribution of gp120-related reacting oxygen species (ROS) in the imbalanced activity of glutamine synthase (GS), the enzyme that metabolizes glutamate into glutamine within astroglial cells playing a neuroprotective role in brain disorders.ResultsIncubation of Lipari human cultured astroglial cells with gp 120 (0.1–10 nM) produced a significant reduction of astroglial cell viability and apoptosis as evaluated by TUNEL reaction and flow cytometric analysis (FACS). This effect was accompanied by lipid peroxidation as detected by means of malondialdehyde assay (MDA). In addition, gp 120 reduced both glutamine concentration in astroglial cell supernatants and GS expression as detected by immunocytochemistry and western blotting analysis. Pre-treatment of cells with NAC (0.5–5 mM), dose-dependently antagonised astroglial apoptotic cell death induced by gp 120, an effect accompanied by significant attenuation of MDA accumulation. Furthermore, both effects were closely associated with a significant recovery of glutamine levels in cell supernatants and by GS expression, thus suggesting that overproduction of free radicals might contribute in gp 120-related dysfunction of GS in astroglial cells.ConclusionIn conclusion, the present experiments demonstrate that gp 120 is toxic to astroglial cells, an effect accompanied by lipid peroxidation and by altered glutamine release. All the effects of gp120 on astroglial cells were counteracted by NAC thus suggesting a novel and potentially useful approach in the treatment of glutammatergic disorders found in HAD patients.

The effect of inflammatory stimuli on NMDA-related activation of glutamine synthase in human cultured astroglial cells

Removal of glutamate from the synaptic cleft by astroglial glutamine synthase (GS) is a crucial step in the regulation of glutamate turnover and metabolism, thus participating in endogenous neuroprotective processes occurring within brain tissues. Here we investigated on the effect of inflammatory cytokines on GS activity in astroglial cells undergoing NMDA receptors stimulation. Incubation of human cultured astroglial cells with NMDA (100 μM) enhanced GS expression, an effect driven by the generation of nitric oxide (NO) since l-NAME (500 μM), an inhibitor of NO synthase, reversed this effect. NMDA-related increase of GS activity and glutamine concentration was antagonised by previous incubation of astroglial cells with a mixture of LPS plus γIFN, an effect counteracted by dexamethasone, the latter effect being accompanied by inhibition of inducible NO synthase. These results show that LPS plus γIFN inhibit elevation of GS activity subsequent to NMDA receptor stimulation in astroglial cells via enhancement of inducible NO synthase, and this may represent the site of interaction between pro-inflammatory and excitotoxic stimuli in the brain.

Müller cell response to laser-induced increase in intraocular pressure in rats.

The goal of this study was to investigate the reaction of the Müller cells to elevated intraocular pressure (IOP). Elevated IOP is one of the risk factors in glaucomatous retinal ganglion cell (RGC) degeneration. Müller cells play an important role in retinal homeostasis. The reaction of Müller cells was examined by evaluating temporal changes in glutamate aspartate transporter (GLAST), glutamine synthase (GS), glial fibrillary acidic protein (GFAP), and the B-cell lymphoma (Bcl-2) using immunoblotting and immunohistochemical techniques. After IOP was elevated for 4-60 days, there was a time-related decrease in RGC ranging from 6% to 44%. There was also a time-related increase in GLAST protein reaching maximum after 3 weeks of elevated IOP. On the other hand, there was very little change in the expression of GS during the first 2 weeks followed by some increase between 21 and 60 days. An increase in Bcl-2 was biphasic with maximum increase after 4 days followed by decline after 15 and 21 days. GFAP, which is usually not expressed in normal Müller cells, was present at all time points. In all cases, the increase was most intense in the vicinity of the ganglion cells where the astrocytes and endfeet of the Müller cells are located. These results indicate that Müller cells react to the insult of elevated IOP by expressing GFAP and Bcl-2, proteins that are expressed in reactive gliosis and other pathological conditions. The increase in GLAST along with minimum change in GS indicates a disturbance in glutamate homeostasis.