Increased Glutamate Cysteine Ligase Activity Research Articles

Pharmacological elevation of glutathione inhibits status epilepticus-induced neuroinflammation and oxidative injury

Glutathione (GSH) is a major endogenous antioxidant, and its depletion has been observed in several brain diseases including epilepsy. Previous studies in our laboratory have shown that dimercaprol (DMP) can elevate GSH via post-translational activation of glutamate cysteine ligase (GCL), the rate limiting GSH biosynthetic enzyme and inhibit neuroinflammation in vitro. Here we determined 1) the role of cysteamine as a new mechanism by which DMP increases GSH biosynthesis and 2) its ability to inhibit neuroinflammation and neuronal injury in the rat kainate model of epilepsy. DMP depleted cysteamine in a time- and concentration-dependent manner in a cell free system. To guide the in vivo administration of DMP, its pharmacokinetic profile was determined in the plasma, liver, and brain. The results confirmed DMP's ability to cross the blood-brain-barrier. Treatment of rats with DMP (30 mg/kg) depleted cysteamine in the liver and hippocampus that was associated with increased GCL activity in these tissues. GSH levels were significantly increased (20 %) in the hippocampus 1 h after 30 mg/kg DMP administration. Following DMP (30 mg/kg) administration once daily, a marked attenuation of GSH depletion was seen in the SE model. SE-induced inflammatory markers including cytokine release, microglial activation, and neuronal death were significantly attenuated in the hippocampus with DMP treatment. Taken together, these results highlight the importance of restoring redox status with rescue of GSH depletion by DMP in post epileptogenic insults.

Post-translational Activation of Glutamate Cysteine Ligase with Dimercaprol: A NOVEL MECHANISM OF INHIBITING NEUROINFLAMMATION IN VITRO

Neuroinflammation and oxidative stress are hallmarks of various neurological diseases. However, whether and how the redox processes control neuroinflammation is incompletely understood. We hypothesized that increasing cellular glutathione (GSH) levels would inhibit neuroinflammation. A series of thiol compounds were identified to elevate cellular GSH levels by a novel approach (i.e. post-translational activation of glutamate cysteine ligase (GCL), the rate-limiting enzyme in GSH biosynthesis). These small thiol-containing compounds were examined for their ability to increase intracellular GSH levels in a murine microglial cell line (BV2), of which dimercaprol (2,3-dimercapto-1-propanol (DMP)) was found to be the most effective compound. DMP increased GCL activity and decreased LPS-induced production of pro-inflammatory cytokines and inducible nitric-oxide synthase induction in BV2 cells in a concentration-dependent manner. The ability of DMP to elevate GSH levels and attenuate LPS-induced pro-inflammatory cytokine production was inhibited by buthionine sulfoximine, an inhibitor of GCL. DMP increased the expression of GCL holoenzyme without altering the expression of its subunits or Nrf2 target proteins (NQO1 and HO-1), suggesting a post-translational mechanism. DMP attenuated LPS-induced MAPK activation in BV2 cells, suggesting the MAPK pathway as the signaling mechanism underlying the effect of DMP. Finally, the ability of DMP to increase GSH via GCL activation was observed in mixed cerebrocortical cultures and N27 dopaminergic cells. Together, the data demonstrate a novel mechanism of GSH elevation by post-translational activation of GCL. Post-translational activation of GCL offers a novel targeted approach to control inflammation in chronic neuronal disorders associated with impaired adaptive responses.

Curcumin, quercetin, and tBHQ modulate glutathione levels in astrocytes and neurons: importance of the glutamate cysteine ligase modifier subunit

A decrease in GSH levels, the main redox regulator, can be observed in neurodegenerative diseases as well as in schizophrenia. In search for substances able to increase GSH, we evaluated the ability of curcumin (polyphenol), quercetin (flavonoid), and tert-butylhydroquinone (tBHQ) to up-regulate GSH-synthesizing enzymes. The gene expression, activity, and product levels of these enzymes were measured in cultured neurons and astrocytes. In astrocytes, all substances increased GSH levels and the activity of the rate-limiting synthesizing enzyme, glutamate cysteine ligase (GCL). In neurons, curcumin and to a lesser extent tBHQ increased GCL activity and GSH levels, while quercetin decreased GSH and led to cell death. In the two cell types, the gene that showed the greatest increase in its expression was the one coding for the modifier subunit of GCL (GCLM). The increase in mRNA levels of GCLM was 3 to 7-fold higher than that of the catalytic subunit. In astrocytes from GCLM-knock-out mice showing low GSH (-80%) and low GCL activity (-50%), none of the substances succeeded in increasing GSH synthesis. Our results indicate that GCLM is essential for the up-regulation of GCL activity induced by curcumin, quercetin and tBHQ.

HIF1 Induced Glutathione Dysregulation in Platelets Associated with Increased Risk of Vascular Disease in Chuvash Polycythemia.

The von Hippel-Lindau protein-hypoxia-inducible factor pathway is a transcriptional system controlling cellular responses to hypoxia. Hypoxia-inducible factor-1 (HIF-1) is a heterodimer of α and β subunits. Under normoxia, the prolyl residues of the α subunit are hydroxylated allowing the von Hippel-Lindau protein (pVHL) to bind, which targets HIFα for proteasomal degradation. During hypoxia, HIFα does not bind to pVHL and instead forms a transcriptional complex with HIFβ leading to increased expression of a broad range of hypoxia-regulated genes. Patients with Chuvash polycythemia (CP) are homozygous for a 598 C->T mutation in the VHL gene, resulting in a pVHL that causes ineffective degradation of HIFα. Clinically, CP patients suffer from premature mortality related to vascular thrombotic events - an increased risk that is not related to their elevated hematocrit, blood pressure, or known cardio or cerebrovascular risk factors. Lab abnormalities seen in CP include erythrocytosis and an elevated plasma glutathione level (Sergueeva et al, Haematologica. Feb 2008). Glutathione (GSH) plays an essential role in cellular antioxidant protection, and its levels are controlled by two mechanisms. GSH is oxidized to glutathione disulfide (GSSG) but is replenished by reduction of GSSG via glutathione reductase (GSR). GSH is also increased by de novo synthesis, which is regulated at several levels, including GSH feedback inhibition, and glutathione synthetase (GSS) and glutamate cysteine ligase (GCL) activity. To establish the molecular basis of elevated GSH, we examined expression of GSS, GSR, and GCL in the platelets of 11 CP patients and 8 Chuvash controls using qRT-PCR. Analysis revealed a 2-fold increased expression of GCL in CP. GSR and GSS were not statistically different. This data suggests that increased GCL activity might be the mechanism by which GSH is elevated in CP, but whether HIF directly regulates GCL or whether these differences reflect a more global process are presently unknown. Since the promoter of GCL contains an oxidative stressresponse element, transcriptional up regulation of GCL by increased oxidative stress secondary to HIF dysregulation might drive increased GSH synthesis. To examine this question, we measured GSSG in our samples. Although CP patients had elevated GSH (8.00 uM vs. 4.32 uM, p 0.002), the GSH/GSSG ratio (a widely used marker of redox state) showed no differences between CP and controls. Thus, overexpression of GCL and elevated GSH appear not to be compensatory responses to increased oxidative stress in CP. We also found increased GCL expression in VHL mutant mice. To determine if HIF1 might regulate this expression, we next measured GCL expression in HIF1 deficient embryos at embryonic day 9.5 and found decreased expression of GCL. In the homozygote knock-out embryos where HIF1 is absent, GCL expression is decreased, in contrast to CP patients, where HIF1 and GCL expression are both increased. Collectively, our data suggests that HIF1 dysregulates cellular redox homeostasis by upregulating GCL and increasing GSH synthesis in an oxidative-stress-independent manner. The significance of elevated GSH in CP and its possible relationship to increased thromboses remains to be defined. Reactive oxygen species mediate the vascular inflammation seen in the development of atherosclerotic disease, and GSH is an important intracellular scavenger that protects cells against oxidative damage. Yet CP patients have increased GSH as well as increased thrombosis, which is especially provoking since recently published evidence suggests that increased redox potential may be harmful to the cardiovascular system (Rajasekaran, et al Cell 2007). Perhaps the GSH elevation may contribute to the increased vascular disease that constitutes the major cause of mortality in this disorder of hypoxia sensing. The cellular antioxidant defense system is intimately linked to oxidative stress, hypoxia regulation, and vascular homeostasis. Our proposed future studies employing HIF2 knock out mice, model cell systems for HIF1 and HIF2, and the role of GSH in platelet function will be used to further explore the molecular mechanisms that regulate these complex pathways.

The ketogenic diet increases mitochondrial glutathione levels

The ketogenic diet (KD) is a high-fat, low carbohydrate diet that is used as a therapy for intractable epilepsy. However, the mechanism(s) by which the KD achieves neuroprotection and/or seizure control are not yet known. We sought to determine whether the KD improves mitochondrial redox status. Adolescent Sprague-Dawley rats (P28) were fed a KD or control diet for 3 weeks and ketosis was confirmed by plasma levels of beta-hydroxybutyrate (BHB). KD-fed rats showed a twofold increase in hippocampal mitochondrial GSH and GSH/GSSG ratios compared with control diet-fed rats. To determine whether elevated mitochondrial GSH was associated with increased de novo synthesis, the enzymatic activity of glutamate cysteine ligase (GCL) (the rate-limiting enzyme in GSH biosynthesis) and protein levels of the catalytic (GCLC) and modulatory (GCLM) subunits of GCL were analyzed. Increased GCL activity was observed in KD-fed rats, as well as up-regulated protein levels of GCL subunits. Reduced CoA (CoASH), an indicator of mitochondrial redox status, and lipoic acid, a thiol antioxidant, were also significantly increased in the hippocampus of KD-fed rats compared with controls. As GSH is a major mitochondrial antioxidant that protects mitochondrial DNA (mtDNA) against oxidative damage, we measured mitochondrial H2O2 production and H2O2-induced mtDNA damage. Isolated hippocampal mitochondria from KD-fed rats showed functional consequences consistent with the improvement of mitochondrial redox status i.e. decreased H2O2 production and mtDNA damage. Together, the results demonstrate that the KD up-regulates GSH biosynthesis, enhances mitochondrial antioxidant status, and protects mtDNA from oxidant-induced damage.

Optimization and application of glutamate cysteine ligase measurement in wildlife species

Glutamate cysteine ligase (GCL), synthesizing gamma-glutamylcysteine from glutamate and cysteine, is the rate-limiting enzyme in glutathione (GSH) biosynthesis. GCL activity measurement was optimized in tissues from deer mice, Sprague Dawley rats, and mallard ducks. Varying glutamic acid concentrations from 5 to 80 mM did not affect GCL activities markedly, whereas cysteine concentrations from 2.5 to 40 mM influenced GCL activities substantially. Optimal cysteine concentrations for deer mouse, Sprague Dawley rat, and mallard duck (respectively) were 30, 30, and 20 mM in liver, 10, 10, and 20 mM in kidney, 20, 20, and 30 mM in brain, and 30 mM in heart for all three species. Responses of mallard duck GCL activity to acid metalliferous water were evaluated. After subacute exposure, low doses increased GCL activity and GSH content in liver by 48.3% and 54.4%, respectively. High doses reduced GCL activities significantly in liver and kidney to 31.2% and 43.0% of the control, respectively.

Thiol-Based Regulation of Redox-Active Glutamate-Cysteine Ligase from Arabidopsis thaliana

Glutathione biosynthesis is a key component in the network of plant stress responses that counteract oxidative damage and maintain intracellular redox environment. Using a combination of mass spectrometry and site-directed mutagenesis, we examined the response of Arabidopsis thaliana glutamate-cysteine ligase (GCL) to changes in redox environment. Mass spectrometry identified two disulfide bonds (Cys186-Cys406 and Cys349-Cys364) in GCL. Mutation of either Cys-349 or Cys-364 to a Ser reduced reaction rate by twofold, but substitution of a Ser for either Cys-186 or Cys-406 decreased activity by 20-fold and abrogated the response to changes in redox environment. Redox titrations show that the regulatory disulfide bond has a midpoint potential comparable with other known redox-responsive plant proteins. Mutation of Cys-102, Cys-251, Cys-349, or Cys-364 did not alter the response to redox environment, indicating that modulation of activity depends on the Cys186-Cys406 disulfide bond. In vivo analysis of GCL in Arabidopsis root extracts revealed that multiple oxidative stresses altered the distribution of oxidized (active) and reduced (inactive) enzyme and that this change correlated with increased GCL activity. The thiol-based regulation of GCL provides a posttranslational mechanism for modulating enzyme activity in response to in vivo redox environment and suggests a role for oxidative signaling in the maintenance of glutathione homeostasis in plants.

Nitric oxide-induced resistance to hydrogen peroxide stress is a glutamate cysteine ligase activity-dependent process

Nitric oxide ( NO) is a reactive nitrogen species known to be involved in cytotoxic processes. Cells respond to cytotoxic injury by stress response induction leading to the development of cellular resistance. This report describes an NO-induced stress response in Chinese hamster fibroblasts (HA1), which leads to glutathione synthesis-dependent resistance to H 2O 2-mediated oxidative stress. The development of resistance to H 2O 2 was completely abolished by the inhibition of glutamate cysteine ligase (GCL) during the first 8 h of recovery after NO exposure. Altered thiol metabolism was observed immediately after NO exposure as demonstrated by up to 75% decrease in intracellular thiol pools (glutathione, γ-glutamylcysteine, and cysteine), which then reaccumulated during the NO-mediated development of resistance. Immunoreactive protein and activity associated with GCL decreased immediately after exposure to NO and then reaccumulated during the development of resistance to H 2O 2 challenge. Moreover, compared to N 2 controls the activity levels of GCL in NO-exposed cells increased approximately twofold 24 h after H 2O 2 challenge. These results demonstrate that NO exposure is capable of inducing an adaptive response to H 2O 2-mediated oxidative stress in mammalian cells, which involves alterations in thiol metabolism and is dependent upon glutathione synthesis and increased GCL activity.

Cysteine Is the Metabolic Signal Responsible for Dietary Regulation of Hepatic Cysteine Dioxygenase and Glutamate Cysteine Ligase in Intact Rats

Cysteine, rather than a precursor or metabolite of cysteine, appears to mediate the upregulation of cysteine dioxygenase (CDO) and the downregulation of glutamate cysteine ligase (GCL) in cultured primary rat hepatocytes. However, similar experiments in intact rats have not been performed to confirm in vivo that changes in hepatic cysteine levels are associated with the regulation of CDO or GCL activity. Therefore, rats were fed a low protein basal diet (100 g casein/kg diet) with or without supplemental sulfur amino acids (8 g cystine, 9 g homocystine or 10 g methionine/kg diet) and with or without propargylglycine (PPG, 1 mmol/kg), an irreversible inhibitor of cystathionine gamma-lyase. Rats were fed the assigned diet for 2 full days and up until the mid-point of the dark cycle on d 3, at which time they were killed for collection of liver. Rats fed the PPG-containing diets had hepatic cystathionine gamma-lyase activities that were approximately 16% of the uninhibited level. PPG treatment reduced CDO activity by 50 and 54%, increased GCL activity by 41 and 61% and lowered total cysteine concentration by 33 and 64% in liver of the homocystine and methionine-supplemented groups, respectively, but not in the cystine-supplemented groups or unsupplemented groups. Glutathione levels were not affected by PPG treatment in any groups. These experiments are consistent with a role for cysteine, rather than a precursor or metabolite of cysteine, in the metabolic signaling responsible for diet-induced regulation of CDO and GCL.

Differential effect of nitric oxide on glutathione metabolism and mitochondrial function in astrocytes and neurones: implications for neuroprotection/neurodegeneration?

Primary culture rat astrocytes exposed to the long acting nitric oxide donor (Z)-1-[2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO) for 24 h approximately double their concentration of glutathione (GSH) and show no sign of cell death. In contrast, GSH was depleted by 48%, and significant loss of mitochondrial respiratory chain complex activity and cell death were observed in primary culture rat neurones subjected to DETA-NO for 18 h. Northern blot analysis suggested that mRNA amounts of both subunits of glutamate-cysteine ligase (GCL), the rate-limiting enzyme in GSH synthesis, were elevated in astrocytes following nitric oxide (NO) exposure. This correlated with an increase in astrocytic GCL activity. Neurones on the other hand did not exhibit increased GCL activity when exposed to NO. In addition, the rate of GSH efflux was doubled and gamma-glutamyltranspeptidase (gamma-GT) activity was increased by 42% in astrocytes treated with NO for 24 h. These results suggest that astrocytes, but not neurones, up-regulate GSH synthesis as a defence mechanism against excess NO. It is possible that the increased rate of GSH release and activity of gamma-GT in astrocytes may have important implications for neuroprotection in vivo by optimizing the supply of GSH precursors to neurones in close proximity.