Rate-limiting Enzyme In GSH Synthesis Research Articles

Dimethyl Fumarate Reduces Methylglyoxal-derived Carbonyl Stress Through Nrf2/GSH Activation in SH-SY5Y Cells.

Carbonyl stress refers to the excessive accumulation of advanced glycation end products (AGEs) in mammalian tissues. This phenomenon plays a significant role in the pathogenesis of various diseases, including diabetes, chronic renal failure, arteriosclerosis, and central nervous system (CNS) disorders. We have previously demonstrated that an increase in glutathione concentration, dependent on the nuclear factor erythroid 2-related factor 2 (Nrf2) system, provides a potent cytoprotective effect against Methylglyoxal (MGO)-induced carbonyl stress. Meanwhile, dimethyl fumarate (DMF), known for its Nrf2-activating effects, was recently approved as a treatment for multiple sclerosis (MS), a neurodegenerative disease. DMF is a first line therapy for relapsing-remitting MS and may also be effective for other neurodegenerative conditions. However, the detailed mechanisms by which DMF mitigates neurodegenerative pathologies remain unclear. This study investigates the impact of DMF on anticarbonyl activity and its underlying mechanism focusing on the accumulation of carbonyl protein in the cell. MGO, a glucose metabolite, was used to induce carbonylation in the neuronal cell line. MGO is a typical carbonyl compound that readily reacts with arginine and lysine residues to form AGE-modified proteins. Methylglyoxal-derived hydroimidazolone 1 (MG-H1) often forms uncharged, hydrophobic residues on the protein surface, which can affect protein distribution and lead to misfolding. Our findings indicate that DMF increases levels of glutathione (GSH), glutamate cysteine ligase modifier subunit (GCLM), and nuclear Nrf2 in SH-SY5Y cells. Importantly, DMF pretreatment significantly reduced the accumulation of MG-H1-modified proteins. Furthermore, this effect of DMF was diminished when Nrf2 expression was suppressed and when GCL, a rate-limiting enzyme in GSH synthesis, was inhibited. Thus, the increase in GSH levels, leading to the activation of the Nrf2 pathway, a key factor in DMF's ability to suppress the accumulation of MG-H1-modified proteins. This study is the first to demonstrate that DMF possesses strong anticarbonyl stress activity in neuronal cells. Therefore, future research may extend the application of DMF to other CNS diseases associated with carbonyl stress, such as Alzheimer's and Parkinson's disease.

The glutathione biosynthesis is involved in metamorphosis, antioxidant function, and insecticide resistance in Tribolium castaneum.

Reduced glutathione (GSH) synthesis is vital for redox homeostasis, cell-cycle regulation and apoptosis, and immune function. The glutamate-cysteine ligase catalytic subunit (Gclc) is the first and rate-limiting enzyme in GSH synthesis, suggesting the potential use of Gclc as a pesticide target. However, the functional characterization of Gclc, especially its contribution in metamorphosis, antioxidant status and insecticide resistance, is unclear in Tribolium castaneum. In this study, we identified and cloned Gclc from T. castaneum (TcGclc) and found that its expression began to increase significantly from the late larvae (LL) stage (3.491 ± 0.490-fold). Furthermore, RNA interference-mediated knockdown of TcGclc resulted in three types of aberration (100% total aberration rate) caused by the downregulation of genes related to the 20-hydroxyecdysone (20E) pathway. This deficiency was partially rescued by exogenous 20E treatment (53.1% ± 3.2%), but not by antioxidant. Moreover, in the TcGclc knockdown group, GSH content was decreased to 62.3%, and total antioxidant capacity, glutathione peroxidase and total superoxide dismutase activities were reduced by 14.6%, 83.6%, and 82.3%, respectively. In addition, treatment with different insecticides upregulated expression of TcGclc significantly compared with a control group during the late larval stage (P < 0.01). Our results indicate that TcGclc has an extensive role in metamorphosis, antioxidant function and insecticide resistance in T. castaneum, thereby expanding our understanding of GSH functions and providing a scientific basis for pest control. © 2024 Society of Chemical Industry.

An antioxidant feedforward cycle coordinated by linker histone variant H1.2 and NRF2 that drives nonsmall cell lung cancer progression

Nonsmall cell lung cancer (NSCLC) is highly malignant with limited treatment options, platinum-based chemotherapy is a standard treatment for NSCLC with resistance commonly seen. NSCLC cells exploit enhanced antioxidant defense system to counteract excessive reactive oxygen species (ROS), which contributes largely to tumor progression and resistance to chemotherapy, yet the mechanisms are not fully understood. Recent studies have suggested the involvement of histones in tumor progression and cellular antioxidant response; however, whether a major histone variant H1.2 (H1C) plays roles in the development of NSCLC remains unclear. Herein, we demonstrated that H1.2 was increasingly expressed in NSCLC tumors, and its expression was correlated with worse survival. When crossing the H1c knockout allele with a mouse NSCLC model (KrasLSL-G12D/+), H1.2 deletion suppressed NSCLC progression and enhanced oxidative stress and significantly decreased the levels of key antioxidant glutathione (GSH) and GCLC, the catalytic subunit of rate-limiting enzyme for GSH synthesis. Moreover, high H1.2 was correlated with the IC50 of multiple chemotherapeutic drugs and with worse prognosis in NSCLC patients receiving chemotherapy; H1.2-deficient NSCLC cells presented reduced survival and increased ROS levels upon cisplatin treatment, while ROS scavenger eliminated the survival inhibition. Mechanistically, H1.2 interacted with NRF2, a master regulator of antioxidative response; H1.2 enhanced the nuclear level and stability of NRF2 and, thus, promoted NRF2 binding to GCLC promoter and the consequent transcription; while NRF2 also transcriptionally up-regulated H1.2. Collectively, these results uncovered a tumor-driving role of H1.2 in NSCLC and indicate an "H1.2-NRF2" antioxidant feedforward cycle that promotes tumor progression and chemoresistance.

C-Jun-N Terminal Kinase-Mediated Degradation of γ-Glutamylcysteine Ligase Catalytic Subunit Inhibits GSH Recovery After Acetaminophen Treatment: Role in Sustaining JNK Activation and Liver Injury.

Aims: Acetaminophen (APAP) overdose is the most common cause of acute liver failure in the United States. Liver glutathione (GSH) depletion and sustained P-JNK (c-Jun-N-terminal kinase) activation are key modulators in the mechanism leading to hepatic necrosis. GSH depletion is directly related to the consumption of GSH by APAP metabolites N-acetyl-p-benzoquinone imine (NAPQI). We previously noticed that the glutamate-cysteine ligase catalytic subunit (GCLC), the rate-limiting enzyme in GSH synthesis, rapidly decreased at the same time P-JNK increased. Our aims were to determine if JNK was directly responsible for decreased GCLC causing impaired recovery of GSH and if this was an important factor in determining APAP hepatotoxicity. Results: Immunoprecipitation of JNK after APAP identified binding to GCLC. Expression of a site-directed mutated canonical JNK docking site in GCLC was resistant to degradation and led to rapid restoration of GSH and inhibited sustained JNK activation. The JNK-resistant GCLC markedly protected against necrosis and alanine aminotransferase (ALT) elevation. The proteolytic loss of GCLC was abrogated by inhibition of the proteasome, ubiquitination, or calpain. Innovation: Using mutated-GCLC resistant to JNK-induced degradation, the results allowed us to identify impaired GSH recovery as an important contributor to early progression of APAP toxicity after the metabolism of APAP and initial GSH depletion had occurred. Conclusion: Activated JNK interacts directly with GCLC and leads to proteolytic degradation of GCLC. Degradation of GCLC impairs GSH recovery after APAP allowing the continued activation of JNK. Conversely, rapid recovery of GSH inhibits the sustained activation of the mitogen-activated protein (MAP) kinase cascade and dampens APAP toxicity by suppressing the continued activation of JNK. Antioxid. Redox Signal. 38, 1071-1081.

Glutathione deficiency decreases lipid droplet stores and increases reactive oxygen species in mouse oocytes†.

Glutathione (GSH) is a tripeptide thiol antioxidant that has been shown to be important to overall reproductive health. Glutamate cysteine ligase, the rate-limiting enzyme in GSH synthesis consists of a catalytic and a modifier (GCLM) subunit. We previously showed that oxidative stress in the ovary and oocytes of Gclm-/- mice is associated with accelerated age-related decline in ovarian follicles and decreased female fertility due to preimplantation embryonic mortality. Mammalian preimplantation development is a highly regulated and energy-intensive process that primarily relies on coordination between lipid droplets (LDs) and mitochondria to maintain cellular homeostasis. In this study, we hypothesized that GSH deficiency in oocytes increases oxidative stress, leading to increased mitochondrial dysfunction and decreased LD consumption, thereby decreasing oocyte developmental competence. We observed that Gclm-/- oocytes have increased oxidative stress, primarily in the form of mitochondrial superoxide and decreased subcortical mitochondrial clusters. Further, Gclm-/- oocytes have decreased mitochondrial membrane potential (ΔΨm) compared with Gclm+/+. We surmise this is likely due to the decreased availability of LDs, as we observed a significant decrease in LD content in Gclm-/- oocytes compared with Gclm+/+. The decreased oocyte LD content is likely related to an altered serum lipidome, with Gclm-/- serum having relatively lower unsaturated fatty acids and triglycerides than that of Gclm+/+ and Gclm+/- females. Altogether these data support that decreased LDs and increased oxidative stress are primary drivers of decreased oocyte developmental competence in GSH-deficient oocytes.

GSH and Zinc Supplementation Attenuate Cadmium-Induced Cellular Stress and Stimulation of Choline Uptake in Cultured Neonatal Rat Choroid Plexus Epithelia.

Choroid plexus (CP) sequesters cadmium and other metals, protecting the brain from these neurotoxins. These metals can induce cellular stress and modulate homeostatic functions of CP, such as solute transport. We previously showed in primary cultured neonatal rat CP epithelial cells (CPECs) that cadmium induced cellular stress and stimulated choline uptake at the apical membrane, which interfaces with cerebrospinal fluid in situ. Here, in CPECs, we characterized the roles of glutathione (GSH) and Zinc supplementation in the adaptive stress response to cadmium. Cadmium increased GSH and decreased the reduced GSH-to-oxidized GSH (GSSG) ratio. Heat shock protein-70 (Hsp70), heme oxygenase (HO-1), and metallothionein (Mt-1) were induced along with the catalytic and modifier subunits of glutamate cysteine ligase (GCL), the rate-limiting enzyme in GSH synthesis. Inhibition of GCL by l-buthionine sulfoximine (BSO) enhanced stress protein induction and stimulation of choline uptake by cadmium. Zinc alone did not induce Hsp70, HO-1, or GCL subunits, or modulate choline uptake. Zinc supplementation during cadmium exposure attenuated stress protein induction and stimulation of choline uptake; this effect persisted despite inhibition of GSH synthesis. These data indicated up-regulation of GSH synthesis promotes adaptation to cadmium-induced cellular stress in CP, but Zinc may confer cytoprotection independent of GSH.

Antioxidant supplementation partially rescues accelerated ovarian follicle loss, but not oocyte quality, of glutathione-deficient mice†.

The tripeptide thiol antioxidant glutathione (GSH) has multiple physiological functions. Female mice lacking the modifier subunit of glutamate cysteine ligase (GCLM), the rate-limiting enzyme in GSH synthesis, have decreased GSH concentrations, ovarian oxidative stress, preimplantation embryonic mortality, and accelerated age-related decline in ovarian follicles. We hypothesized that supplementation with thiol antioxidants, N-acetyl cysteine (NAC), or α-lipoic acid (ALA) will rescue this phenotype. Gclm-/- and Gclm+/+ females received 0 or 80mM NAC in drinking water from postnatal day (PND) 21-30; follicle growth was induced with equine chorionic gonadotropin (eCG) on PND 27, followed by an ovulatory dose of human CG and mating with a wild type male on PND 29 and zygote harvest 20h after hCG. N-acetyl cysteine supplementation failed to rescue the low rate of second pronucleus formation in zygotes from Gclm-/- versus Gclm+/+ females. In the second study, Gclm-/- and Gclm+/+ females received diet containing 0, 150, or 600mg/kg ALA beginning at weaning and were mated with wild type males from 8 to 20 weeks of age. α-Lipoic acid failed to rescue the decreased offspring production of Gclm-/- females. However, 150mg/kg diet ALA partially rescued the accelerated decline in primordial follicles, as well as the increased recruitment of follicles into the growing pool and the increased percentages of follicles with γH2AX positive oocytes or granulosa cells of Gclm-/- females. We conclude that ovarian oxidative stress is the cause of accelerated primordial follicle decline, while GSH deficiency per se may be responsible for preimplantation embryonic mortality in Gclm-/- females.

Targeting the Vulnerability of Glutathione Metabolism in ARID1A-Deficient Cancers

ARID1A encodes an SWI/SNF chromatin-remodeling factor and is frequently mutated in various cancers. This study demonstrates that ARID1A-deficient cancer cells are specifically vulnerable to inhibition of the antioxidant glutathione (GSH) and the glutamate-cysteine ligase synthetase catalytic subunit (GCLC), a rate-limiting enzyme for GSH synthesis. Inhibition of GCLC markedly decreased GSH in ARID1A-deficient cancer cells, leading to apoptotic cell death triggered by excessive amounts of reactive oxygen species. The vulnerability of ARID1A-deficient cancer cells results from low basal levels of GSH due to impaired expression of SLC7A11. The SLC7A11-encoded cystine transporter supplies cells with cysteine, a key source of GSH, and its expression is enhanced by ARID1A-mediated chromatin remodeling. Thus, ARID1A-deficient cancers are susceptible to synthetic lethal targeting of GCLC.

T24. REDOX REGULATORS AND OXIDATIVE STRESS IN SCHIZOPHRENIA

BackgroundElevated levels of oxidative stress have been reported in schizophrenia and may play a role in the underlying psychopathology. The antioxidant defense system may be disrupted in schizophrenia. We have earlier shown how levels of membrane polyunsaturated fatty acids (PUFA) change during the course of schizophrenia, increasing from a low level in a subgroup, remaining stable in another. Here, we aimed at comparing levels of redox regulators, oxidative stress, and related genotypes in schizophrenia patients and healthy controls, and at identifying how these biomarkers are related to membrane fatty acids and clinical characteristics in acute and chronic phase of schizophrenia.MethodsPatients with schizophrenia spectrum disorders (n=55) examined during an acute phase and five years later during a chronic phase, and healthy controls (n=51) were included. We assessed blood levels of redox regulators [(alpha-tocopherol, bilirubin, uric acid, glutathione (GSH), glutathione peroxidase (GPx)], glutathione reductase (GR), markers of oxidative stress [F2-isoprostane, reactive oxygen metabolites (D-ROMs)] and PUFA. We examined genotypes and gene expression related to glutamate cysteine ligase (GCL), the rate-limiting enzyme of GSH synthesis, and its catalytic (GCLC) and modulator (GCLM) subunits. Links between redox measures and Positive and Negative Syndrome Scale (PANSS) were studied.ResultsIn the chronic phase, levels of alpha-tocopherol (p=0.03) and bilirubin (p<0.001) were lower, while uric acid (p=0.02) was higher in patients than in controls. In patients, the levels of alpha-tocopherol were higher in the acute phase than in the chronic phase (p=0.001). However, the changes depended on the PUFA levels in the acute phase. In the low PUFA group, alpha-tocopherol levels remained stable, whereas in the high PUFA group, they decreased to those of the low PUFA group. Levels of D-ROMs were linked to PUFA (r=0.39, p=0.007) and long-chain PUFA (r=0.42, p=0.003) in controls but not in patients.There was no significant difference in the distribution of GCLC genotypes between patients and controls. Compared to other GAG trinucleotide-repeat (TNR) genotypes, 7/9 GAG genotype was linked to higher gene expression of GCL (mRNA GCLC, p=0.049; mRNA GCLM, p=0.02), higher levels of GSH in blood (p=0.02) and higher GR activity in blood (p=0.007). Only when combined with C-129T high risk genotype, the 7/7 GAG genotype induced a strong reduction of CGLC expression (p=0.045, bootstrapping 10000 samples). Gene expression related to glutathione synthesis was non-significantly (p=0.10–0.19) higher among patients. Levels of long-chain PUFA were significantly positively linked to gene expression of glutathione related enzymes.DiscussionThe findings of abnormal levels of alpha-tocopherol, bilirubin, uric acid and glutathione synthesis in the chronic phase of schizophrenia indicate oxidative stress. Dysregulation of antioxidant defenses may be involved in the pathophysiology of schizophrenia and warrants further experimental studies.

MOLECULAR MECHANISMS OF β-AMYLOID-INDUCED INHIBITION OF GLUTATHIONE SYNTHESIS IN RATS

Oxidative stress is one of the main pathogenic mechanisms of Alzheimer's disease (AD). Glutathione (GSH) is an endogenous antioxidant protecting cells from oxidative injury. However, it is reported that the content of GSH is lower both in the brain of AD patients and the AD animal models. The reason why GSH content is lower in such an oxidative environment of AD brain is not clear. Glutamatecysteine ligase (GCL), composed of a catalytic (GCLC) and a modifier (GCLM) subunit, is the rate-limiting enzyme in GSH synthesis. The transcription of both GCLC and GCLM are regulated by Keap1-Nrf2-ARE signal pathway. Meanwhile, as a chaperone protein of Nrf2, small Maf proteins form heterodimers with Nrf2 leading to activation or repression of ARE-mediated gene expression. The current study aimed at investigating the molecular mechanisms of regulation on GCL transcription by amyloid-β (Aβ) involving Keap1-Nrf2-ARE signal pathway. Aβ intrahippocampal injection rat model and human neuroblastoma SH-SY5Y cells treated with Aβ were used. The expressions of Nrf2, GCLC, GCLM and small Maf proteins as well as GSH level under Aβ insult at different time point were tested. Overexpression and knockdown of Maf proteins were used to explore the roles of Maf proteins on GCL expression. The banding of Keap1 and Nrf2 was examined by CoIP. The expressions of GCLC, GCLM and GSH level were increased in acute stage of Aβ treatment (within 6h), while the expressions of GCLC, GCLM and GSH level were decreased accompanying with activation of Keap1-Nrf2-ARE signal pathway after Aβ treatment for 6–24h. The nuclear level of Nrf2 restored to normal level accompanying with down regulation of GCL and low GSH in the late stage of Aβ treatment. The nuclear expressions of MafG and MafK were largely induced and displacement of Nrf2 nuclear binding to ARE in the late stage of Aβ treatment, while, MafG or MafK knockdown restored GCLC expression and GSH level. Meanwhile, the banding of Keap1 and Nrf2 were enhanced with decreased expression of p-Nrf2 Ser40. Aβ-mediated inhibition of Keap1-Nrf2-ARE signal pathway and induction of nuclear Maf proteins contributed to lowering GSH content due to inhibition of GCL expression.

MYC-driven inhibition of the glutamate-cysteine ligase promotes glutathione depletion in liver cancer.

How MYC reprograms metabolism in primary tumors remains poorly understood. Using integrated gene expression and metabolite profiling, we identify six pathways that are coordinately deregulated in primary MYC-driven liver tumors: glutathione metabolism; glycine, serine, and threonine metabolism; aminoacyl-tRNA biosynthesis; cysteine and methionine metabolism; ABC transporters; and mineral absorption. We then focus our attention on glutathione (GSH) and glutathione disulfide (GSSG), as they are markedly decreased in MYC-driven tumors. We find that fewer glutamine-derived carbons are incorporated into GSH in tumor tissue relative to non-tumor tissue. Expression of GCLC, the rate-limiting enzyme of GSH synthesis, is attenuated by the MYC-induced microRNA miR-18a. Inhibition of miR-18a in vivo leads to increased GCLC protein expression and GSH abundance in tumor tissue. Finally, MYC-driven liver tumors exhibit increased sensitivity to acute oxidative stress. In summary, MYC-dependent attenuation of GCLC by miR-18a contributes to GSH depletion in vivo, and low GSH corresponds with increased sensitivity to oxidative stress in tumors. Our results identify new metabolic pathways deregulated in primary MYC tumors and implicate a role for MYC in regulating a major antioxidant pathway downstream of glutamine.

Anti-oxidative Activity of Hydrolysate from Rice Bran Protein in HepG2 Cells.

Glutathione (GSH) is an ubiquitous thiol-containing tripeptide, which plays important roles in cellular protection from oxidative stress. In our search for a dietary source that can increase GSH levels, we discovered that a 24 h treatment of HepG2 cells with rice bran protein hydrolysate (RBPH), prepared by Umamizyme G-catalyzed hydrolysis, increased the GSH content in a dose-dependent manner. RBPH elevated the expression levels of γ-glutamylcysteine synthetase (γ-GCS), which constitutes the rate-limiting enzyme of GSH synthesis, and of another two enzymes, hemeoxygenase-1 (HO-1) and reduced nicotinamide adenine dinucleotide (phosphate): quinone oxidoreductase 1 (NQO1). This induction was preceded by the accumulation of nuclear factor erythroid 2-related factor 2 (Nrf2) inside the nucleus, which is a key transcription factor for the expression of the γ-GCS, HO-1, and NQO1. Pre-treatment of cells with RBPH produced a significant protective effect against cytotoxicity caused by H2O2 or ethanol. These results indicate that RBPH exerts a protective effect against oxidative stress by modulating GSH levels and anti-oxidative enzyme expression via the Nrf2 pathway.

Effects of S-Adenosylmethionine and Its Combinations With Taurine and/or Betaine on Glutathione Homeostasis in Ethanol-induced Acute Hepatotoxicity.

BackgroundExposure to ethanol abuse and severe oxidative stress are risk factors for hepatocarcinoma. The aim of this study was to evaluate the effects of S-adenosylmethionine (SAMe) and its combinations with taurine and/or betaine on the level of glutathione (GSH), a powerful antioxidant in the liver, in acute hepatotoxicity induced by ethanol.MethodsTo examine the effects of SAMe and its combinations with taurine and/or betaine on ethanol-induced hepatotoxicity, AML12 cells and C57BL/6 mice were pretreated with SAMe, taurine, and/or betaine, followed by ethanol challenge. Cell viability was detected with an MTT assay. GSH concentration and mRNA levels of GSH synthetic enzymes were measured using GSH reductase and quantitative real-time reverse transcriptase-PCR. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured with commercially available kits.ResultsPretreatment of SAMe, with or without taurine and/or betaine, attenuated decreases in GSH levels and mRNA expression of the catalytic subunit of glutamate-cysteine ligase (GCL), the rate-limiting enzyme for GSH synthesis, in ethanol-treated cells and mice. mRNA levels of the modifier subunit of GCL and glutathione synthetase were increased in mice treated with SAMe combinations. SAMe, taurine, and/or betaine pretreatment restored serum ALT and AST levels to control levels in the ethanol-treated group.ConclusionsCombinations of SAMe with taurine and/or betaine have a hepatoprotective effect against ethanol-induced liver injury by maintaining GSH homeostasis.

Nrf2-dysregulation correlates with reduced synthesis and low glutathione levels in experimental autoimmune encephalomyelitis.

This study investigates the possible mechanism(s) underlying glutathione (GSH) deficiency in the mouse spinal cord during the course of myelin oligodendrocyte glycoprotein35-55 peptide-induced experimental autoimmune encephalomyelitis (EAE), a commonly used animal model of multiple sclerosis. Using the classical enzymatic recycling method and a newly developed immunodot assay, we first demonstrated that total GSH levels (i.e. free GSH plus all its adducts) are reduced in EAE, suggesting an impaired synthesis. The decline in the levels of this essential antioxidant tripeptide in EAE coincides temporally and in magnitude with a reduction in the amount of γ-glutamylcysteine ligase, the rate-limiting enzyme in GSH synthesis. Other enzymes involved in GSH biosynthesis, whose genes also contain antioxidant-response elements, including glutathione synthetase, cystine/glutamate antiporter, and γ-glutamyl transpeptidase (γ-GT) are diminished in EAE as well. Low levels of γ-glutamylcysteine ligase, glutathione synthetase, and γ-GT are the consequence of reduced mRNA expression, which correlates with diminished expression of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in both the cytosol and nucleus. Interestingly, the low Nrf2 expression does not seem to be caused by increased degradation via Kelch-like ECH-associated protein 1-dependent or Kelch-like ECH-associated protein 1-independent mechanisms (such as glycogen synthetase kinase-3β activation), or by reduced levels of Nrf2 mRNA. This suggests that translation of this important transcription factor and/or other still unidentified post-translational processes are altered in EAE. These novel findings are central toward understanding how critical antioxidant and protective responses are lost in inflammatory demyelinating disorders.

The pulmonary inflammatory response to multiwalled carbon nanotubes is influenced by gender and glutathione synthesis

Inhalation of multiwalled carbon nanotubes (MWCNTs) during their manufacture or incorporation into various commercial products may cause lung inflammation, fibrosis, and oxidative stress in exposed workers. Some workers may be more susceptible to these effects because of differences in their ability to synthesize the major antioxidant and immune system modulator glutathione (GSH). Accordingly, in this study we examined the influence of GSH synthesis and gender on MWCNT-induced lung inflammation in C57BL/6 mice. GSH synthesis was impaired through genetic manipulation of Gclm, the modifier subunit of glutamate cysteine ligase, the rate-limiting enzyme in GSH synthesis. Twenty-four hours after aspirating 25 µg of MWCNTs, all male mice developed neutrophilia in their lungs, regardless of Gclm genotype. However, female mice with moderate (Gclm heterozygous) and severe (Gclm null) GSH deficiencies developed significantly less neutrophilia. We found no indications of MWCNT-induced oxidative stress as reflected in the GSH content of lung tissue and epithelial lining fluid, 3-nitrotyrosine formation, or altered mRNA or protein expression of several redox-responsive enzymes. Our results indicate that GSH-deficient female mice are rendered uniquely susceptible to an attenuated neutrophil response. If the same effects occur in humans, GSH-deficient women manufacturing MWCNTs may be at greater risk for impaired neutrophil-dependent clearance of MWCNTs from the lung. In contrast, men may have effective neutrophil-dependent clearance, but may be at risk for lung neutrophilia regardless of their GSH levels.

Alterations in glutamate cysteine ligase content in the retina of two retinitis pigmentosa animal models

Retinitis Pigmentosa (RP) comprises a group of rare genetic retinal disorders in which one of several different mutations induces photoreceptor death. Oxidative stress and glutathione (GSH) alterations may be related to the pathogenesis of RP. GSH has been shown to be present in high concentrations in the retina. In addition, the retina has the capability to synthesize GSH. In this study, we tested whether the two subunits of glutamate cysteine ligase, the rate-limiting enzyme in GSH synthesis, and the concentrations of retinal GSH, oxidized glutathione (GSSG), cysteine (Cys) and glutamate are altered in the retina of two different RP mice models. Retinas from C3H and rd1 mice at different postnatal days (P7, P11, P15, P19, P21 and P28) and from C57BL/6 and rd10 mice at P21 were obtained. Western blot analysis was performed to determine the protein content of catalytic and modulatory subunits from glutamate cysteine ligase (GCLC and GCLM, respectively). In another set of experiments, control and rd1 mice were administered buthinine sulfoximine, a glutathione synthase inhibitor, or paraquat. GSH, GSSG, glutamate and Cys concentrations were determined, by HPLC. A decrease in retinal GCLC content was observed in C3H and rd1 mice with age, nevertheless, there was an increase in retinal GCLC in rd1 mice compared to control retinas at P19. No modifications in GCLM content with age and no difference between GCLM content in rd1 and control retinas were observed. The GSH concentration decreased in the rd1 retinas compared with control ones at P15, it increased at P19, and was again similar at P21 and P28. No changes in GSSG concentration in control retinas with age were observed; the GSSG levels in rd1 retinas were similar from P7 to P19 and then increased significantly at P21 and P28. Glutamate concentration was increased in the rd1 retinas compared to control mice from P7 to P15 and were comparable at P21 and P28. The Cys concentrations was measured in control and rd1 retinas, but no significant changes were observed between them. BSO administration decreases GSH retinal concentration in control and rd1 mice, while paraquat administration induced an increase in GSH retinal concentration in control mice and a decrease in GSH in rd1 mice retina. Retinal GCLC was significantly increased in rd10 mice at P21 as well as GSSG. Our results suggest alterations in retinal GCLC content and GSH and/or its precursors in these two RP animal models. Regulation of the enzymes related to GSH metabolism and the retinal concentration of glutamate may be a possible target to delay especially cone death in RP.

Glutamate Cysteine Ligase-Modulatory Subunit Knockout Mouse Shows Normal Insulin Sensitivity but Reduced Liver Glycogen Storage.

Glutathione (GSH) deficits have been observed in several mental or degenerative illness, and so has the metabolic syndrome. The impact of a decreased glucose metabolism on the GSH system is well-known, but the effect of decreased GSH levels on the energy metabolism is unclear. The aim of the present study was to investigate the sensitivity to insulin in the mouse knockout (KO) for the modulatory subunit of the glutamate cysteine ligase (GCLM), the rate-limiting enzyme of GSH synthesis. Compared to wildtype (WT) mice, GCLM-KO mice presented with reduced basal plasma glucose and insulin levels. During an insulin tolerance test, GCLM-KO mice showed a normal fall in glycemia, indicating normal insulin secretion. However, during the recovery phase, plasma glucose levels remained lower for longer in KO mice despite normal plasma glucagon levels. This is consistent with a normal counterregulatory hormonal response but impaired mobilization of glucose from endogenous stores. Following a resident-intruder stress, during which stress hormones mobilize glucose from hepatic glycogen stores, KO mice showed a lower hyperglycemic level despite higher plasma cortisol levels when compared to WT mice. The lower hepatic glycogen levels observed in GCLM-KO mice could explain the impaired glycogen mobilization following induced hypoglycemia. Altogether, our results indicate that reduced liver glycogen availability, as observed in GCLM-KO mice, could be at the origin of their lower basal and challenged glycemia. Further studies will be necessary to understand how a GSH deficit, typically observed in GCLM-KO mice, leads to a deficit in liver glycogen storage.

Nuclear localization of glutamate-cysteine ligase is associated with proliferation in head and neck squamous cell carcinoma.

Glutathione (GSH) is the keystone of the cellular response toward oxidative stress. Elevated GSH content correlates with increased resistance to chemotherapy and radiotherapy of head and neck (HN) tumors. The purpose of the present cross-sectional study was to evaluate whether the expression of glutamate-cysteine ligase (GCL) accounts for the increased GSH availability observed in HN squamous cell carcinoma (SCC). For that purpose, the messenger (m)RNA levels of the modifier (M) and catalytic (C) subunits of GCL and its putative regulators (namely, nuclear factor erythroid 2-related factor 2, heme oxygenase-1 and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha) were monitored in 35 surgical resections of untreated HNSCC. The localization of GCLM was evaluated using in situ hybridization and immunohistochemistry. GCLM expression was significantly increased in tumor samples, compared with normal mucosa, both at the mRNA and protein level (P=0.029), but the pathway of GCLM activation remains to be elucidated. Protein expression of GCLM was detected in the cytoplasm and nucleus. GCLM and the proliferation marker Ki-67 displayed a similar distribution, being both mainly expressed at the periphery of tumor lobules. The present study reported increased expression of GCL and the rate-limiting enzyme of GSH synthesis, within HNSCC. The nuclear localization of GCLM and the concomitant expression of Ki-67 suggested that the localization of GSH synthesis contributes to the protection against oxidative stress within hotspots of cell proliferation.

Genetic Polymorphism Associated Prefrontal Glutathione and Its Coupling With Brain Glutamate and Peripheral Redox Status in Early Psychosis.

Oxidative stress and glutathione (GSH) metabolism dysregulation has been implicated in the pathophysiology of schizophrenia. GAG-trinucleotide repeat (TNR) polymorphisms in the glutamate-cysteine ligase catalytic gene (GCLC), the rate-limiting enzyme for GSH synthesis, are associated with schizophrenia. In addition, GSH may serve as a reserve pool for neuronal glutamate (Glu) through the γ-glutamyl cycle. The aim of this study is to investigate brain [GSH] and its association with GCLC polymorphism, peripheral redox indices and brain Glu. Magnetic resonance spectroscopy was used to measure [GSH] and [Glu] in the medial prefrontal cortex (mPFC) of 25 early-psychosis patients and 33 controls. GCLC polymorphism was genotyped, glutathione peroxidases (GPx) and glutathione reductase (GR) activities were determined in blood cells. Significantly lower [GSHmPFC] in GCLC high-risk genotype subjects were revealed as compared to low-risk genotype subjects independent of disease status. In male subjects, [GSHmPFC] and blood GPx activities correlate positively in controls (P = .021), but negatively in patients (P = .039). In GCLC low-risk genotypes, [GlumPFC] are lower in patients, while it is not the case for high-risk genotypes. GCLC high-risk genotypes are associated with low [GSHmPFC], highlighting that GCLC polymorphisms should be considered in pathology studies of cerebral GSH. Low brain GSH levels are related to low peripheral oxidation status in controls but with high oxidation status in patients, pointing to a dysregulated GSH homeostasis in early psychosis patients. GCLC polymorphisms and disease associated correlations between brain GSH and Glu levels may allow patients stratification.

1,25-Dihydroxyvitamin D induces the glutamate transporter SLC1A1 and alters glutamate handling in non-transformed mammary cells

Genomic profiling of immortalized human mammary epithelial (hTERT-HME1) cells identified several metabolic genes, including the membrane glutamate transporter, SLC1A1, as 1,25-dihydroxyvitamin D3 (1,25D) regulated. In these studies we have surveyed the effects of 1,25D on known glutamate transporters and evaluated its impact on cellular glutamate handling. We confirm that expression of SLC1A1 and all of its known transcript variants are significantly upregulated in hTERT-HME1 cells following 1,25D treatment. Expression of the full-length cognate protein, EAAT3, is correspondingly increased in 1,25D treated hTERT-HME1 cells. Under the same conditions, the expression of two other glutamate transporters--SLC1A6 (EAAT4) and SLC1A2 (EAAT2 or GLT-1)--is enhanced by 1,25D while that of SLC1A3 (EAAT1 or GLAST) and SLC7A11 (xCT) is decreased. Glutamate is not essential for growth of hTERT-HME1 cells, and supplemental glutamate (up to 0.5 mM) does not abrogate the growth inhibitory effects of 1,25D. These data suggest that extracellular glutamate is not a major contributor to cellular energy metabolism in hTERT-HME1 cells under basal conditions and that the growth inhibitory effects of 1,25D are not secondary to its effects on glutamate handling. Instead, the effects of 1,25D on glutamate transporters translated to a decrease in cellular glutamate concentration and an increase in media glutamate concentration, suggesting that one or more of these transporters functions to export glutamate in response to 1,25D exposure. The reduced cellular glutamate concentration may also reflect its incorporation into the cellular glutathione (GSH) pool, which is increased upon 1,25D treatment. In support of this concept, the expression of GCLC (which codes for the rate-limiting enzyme in GSH synthesis) and genes which generate reducing equivalents in the form of NADPH (ie, G6PD, PGD, IDH2) are elevated in 1,25D-treated cells. Taken together, these data identify 1,25D as a physiological regulator of multiple membrane glutamate transporters that impacts on overall cellular glutamate handling.