Glycation of glutamate cysteine ligase by 2-deoxy-d-ribose and its potential impact on chemoresistance in glioblastoma.

The antioxidant glutathione (GSH) plays a critical role in maintaining intracellular redox homeostasis but in tumors the GSH biosynthetic pathway is often dysregulated, contributing to tumor resistance to radiation and chemotherapy. Glutamate-cysteine ligase (GCL) catalyzes the first and rate-limiting reaction in GSH synthesis, and enzyme function is controlled by GSH feedback inhibition or by transcriptional upregulation of the catalytic (GCLC) and modifier (GCLM) subunits. However, it has recently been reported that the activity of GCLC and the formation of GCL can be modified by reactive aldehyde products derived from lipid peroxidation. Due to the susceptibility of GCLC to posttranslational modifications by reactive aldehydes, we examined the potential for 2-deoxy-D-ribose (2dDR) to glycate GCLC and regulate enzyme activity and GCL formation. 2dDR was found to directly modify both GCLC and GCLM in vitro, resulting in a significant inhibition of GCLC and GCL enzyme activity without altering substrate affinity or feedback inhibition. 2dDR-mediated glycation also inhibited GCL subunit heterodimerization and formation of the GCL holoenzyme complex while not causing dissociation of pre-formed holoenzyme. This PTM could be of particular importance in glioblastoma (GBM) where intratumoral necrosis provides an abundance of thymidine, which can be metabolized by thymidine phosphorylase (TP) to form 2dDR. TP is expressed at high levels in human GBM tumors and shRNA knockdown of TP in U87 GBM cells results in a significant increase in cellular GCL enzymatic activity.

Rapid activation of glutamate cysteine ligase following oxidative stress.

Glutamate cysteine ligase (GCL) catalyzes the rate-limiting step in the formation of the cellular antioxidant glutathione (GSH). The GCL holoenzyme consists of two separately coded proteins, a catalytic subunit (GCLC) and a modifier subunit (GCLM). Both GCLC and GLCM are controlled transcriptionally by a variety of cellular stimuli, including oxidative stress. This study addresses post-translational control of GCL activity, which increased rapidly in human lymphocytes following oxidative stress. Activation of GCL occurred within minutes of treatment and without any change in GCL protein levels and coincided with an increase in the proportion of GCLC in the holoenzyme form. Likewise, GCLM shifted from the monomeric form to holoenzyme and higher molecular weight species. Normal rat tissues also showed a distribution of monomeric and higher molecular weight forms. Neither GCL activation, nor the formation of holoenzyme, required a covalent intermolecular disulfide bridge between GCLC and GCLM. However, in immunoprecipitation studies, a neutralizing epitope associated with enzymatic activity was protected following cellular oxidative stress. Thus, the N-terminal portion of GCLC may undergo a change that stabilizes the GCL holoenzyme. Our results suggest that a dynamic equilibrium exists between low and high activity forms of GCL and is altered by transient oxidative stress. This provides a mechanism for the rapid post-translational activation of GCL and maintenance of cellular GSH homeostasis.

Manipulation of cellular GSH biosynthetic capacity via TAT-mediated protein transduction of wild-type or a dominant-negative mutant of glutamate cysteine ligase alters cell sensitivity to oxidant-induced cytotoxicity.

The glutathione (GSH) antioxidant defense system plays a central role in protecting mammalian cells against oxidative injury. Glutamate cysteine ligase (GCL) is the rate-limiting enzyme in GSH biosynthesis and is a heterodimeric holoenzyme composed of catalytic (GCLC) and modifier (GCLM) subunits. As a means of assessing the cytoprotective effects of enhanced GSH biosynthetic capacity, we have developed a protein transduction approach whereby recombinant GCL protein can be rapidly and directly transferred into cells when coupled to the HIV TAT protein transduction domain. Bacterial expression vectors encoding TAT fusion proteins of both GCL subunits were generated and recombinant fusion proteins were synthesized and purified to near homogeneity. The TAT-GCL fusion proteins were capable of heterodimerization and formation of functional GCL holoenzyme in vitro. Exposure of Hepa-1c1c7 cells to the TAT-GCL fusion proteins resulted in the time- and dose-dependent transduction of both GCL subunits and increased cellular GCL activity and GSH levels. A heterodimerization-competent, enzymatically deficient GCLC-TAT mutant was also generated in an attempt to create a dominant-negative suppressor of GCL. Transduction of cells with a catalytically inactive GCLC(E103A)-TAT mutant decreased cellular GCL activity in a dose-dependent manner. TAT-mediated manipulation of cellular GCL activity was also functionally relevant as transduction with wild-type GCLC(WT)-TAT or mutant GCLC(E103A)-TAT conferred protection or enhanced sensitivity to H(2)O(2)-induced cell death, respectively. These findings demonstrate that TAT-mediated transduction of wild-type or dominant-inhibitory mutants of the GCL subunits is a viable means of manipulating cellular GCL activity to assess the effects of altered GSH biosynthetic capacity.

TGFbeta1-induced suppression of glutathione antioxidant defenses in hepatocytes: caspase-dependent post-translational and caspase-independent transcriptional regulatory mechanisms.

TGFbeta1-induced hepatocyte apoptosis involves the production of reactive oxygen species. An effective cellular defense mechanism against oxidative stress is the tripeptide glutathione (GSH), and the rate-limiting step in GSH biosynthesis is catalyzed by the heterodimeric holoenzyme glutamate cysteine ligase (GCL). Here, we demonstrate that TGFbeta1-induced apoptosis in the TAMH murine hepatocyte cell line is accompanied by both the cleavage and loss of the catalytic subunit of GCL (GCLC) and the down-regulation of GCLC gene expression resulting in a reduction in GCL activity and depletion of intracellular GSH. TGFbeta1-induced apoptosis is also accompanied by a reduction in Bcl-XL, an effect that may facilitate TGFbeta1-induced apoptosis as Bcl-XL overexpression inhibits TGFbeta1-induced caspase activation and cell death. Interestingly, Bcl-XL overexpression prevents TGFbeta1-induced cleavage of GCLC protein but not down-regulation of GCLC mRNA. Furthermore, TGFbeta1-induced down-regulation of GCLC mRNA is prevented by inhibition of histone deacetylase activity, suggesting that this is an active repression of GCLC gene transcription. These findings suggest that the suppression of GSH antioxidant defenses associated with the caspase-dependent cleavage of GCLC protein, caspase-independent suppression of GCLC gene expression, and depletion of intracellular GSH may play a role in enhancing TGFbeta1-induced oxidative stress and potentiating apoptotic cell death.

Glutamate-cysteine ligase attenuates TNF-induced mitochondrial injury and apoptosis.

Glutathione (GSH) is important in free radical scavenging, maintaining cellular redox status, and regulating cell survival in response to a wide variety of toxicants. The rate-limiting enzyme in GSH synthesis is glutamate-cysteine ligase (GCL), which is composed of catalytic (GCLC) and modifier (GCLM) subunits. To determine whether increased GSH biosynthetic capacity enhances cellular resistance to tumor necrosis factor-alpha- (TNF-alpha-) induced apoptotic cell death, we have established several mouse liver hepatoma (Hepa-1) cell lines overexpressing GCLC and/or GCLM. Cells overexpressing GCLC alone exhibit modest increases in GCL activity, while cells overexpressing both subunits have large increases in GCL activity. Importantly, cells overexpressing both GCL subunits exhibit increased resistance to TNF-induced apoptosis as judged by a loss of redox potential; mitochondrial membrane potential; translocation of cytochrome c to the cytoplasm; and activation of caspase-3, caspase-8, and caspase-9. Analysis of the effects of TNF on these parameters indicates that maintaining mitochondrial integrity mediates this protective effect in GCL-overexpressing cells.

Cell culture model for acetaminophen-induced hepatocyte death in vivo.

Overdose of the popular, and relatively safe, analgesic acetaminophen (N-acetyl-p-aminophenol, APAP, paracetamol) can produce a fatal centrilobular liver injury. APAP-induced cell death was investigated in a differentiated, transforming growth factor alpha (TGFalpha)-overexpressing, hepatocyte cell line and found to occur at concentrations, and over time frames, relevant to clinical overdose situations. Coordinated multiorganellar collapse was evident during APAP-induced cytotoxicity with widespread, yet selective, protein degradation events in vitro. Cellular proteasomal activity was inhibited with APAP treatment but not with the comparatively nonhepatotoxic APAP regioisomer, N-acetyl-m-aminophenol (AMAP). Low concentrations of the proteasome-directed inhibitor MG132 (N-carbobenzoxyl-Leu-Leu-Leucinal) increased chromatin condensation and cellular stress responses preferentially in AMAP-treated cultures, suggesting a contribution of the proteasome in APAP- but not AMAP-mediated cell death. APAP-specific alterations to mitochondria were observed morphologically with evidence of mitochondrial proliferation in vitro. Biochemical alterations to cellular proteolytic events were also found in vivo, including APAP- or AMAP-mediated inhibition of caspase-3 processing. These results indicate that, although retaining some attributes of apoptosis, both APAP- and AMAP-mediated cell death have additional distinctive features consistent with longer term necrosis.

Methods to detect protein glutathionylation.

Glutathionylation is a posttranslational modification that results in the formation of a mixed disulfide between glutathione and the thiol group of a protein cysteine residue. Glutathionylation of proteins occurs via both nonenzymatic mechanisms involving thiol/disulfide exchange and enzyme-mediated reactions. Protein glutathionylation is observed in response to oxidative or nitrosative stress and is redox-dependent, being readily reversible under reducing conditions. Such findings suggest that glutathionylation plays an important role in mediating redox-sensitive signaling. Indeed, glutathionylation can affect protein function by altering activity, protein-protein interactions, and ligand binding. Glutathionylation may also serve to prevent cysteine residues from undergoing irreversible oxidative modification. Thus, determining the ability of a given protein to become glutathionylated can provide insight into its redox regulation and putative role in dictating cellular response to oxidative and nitrosative stress. Methods to measure protein glutathionylation using immunoblotting and mass spectrometry are described.

The role of glutathione in brain tumor drug resistance.

Chemotherapy is central to the current treatment modality for primary human brain tumors, but despite high-dose and intensive treatment regimens there has been little improvement in patient outcome. The development of tumor chemoresistance has been proposed as a major contributor to this lack of response. While there have been some improvements in our understanding of the molecular mechanisms underlying brain tumor drug resistance over the past decade, the contribution of glutathione (GSH) and the GSH-related enzymes to drug resistance in brain tumors have been largely overlooked. GSH constitutes a major antioxidant defense system in the brain and together with the GSH-related enzymes plays an important role in protecting cells against free radical damage and dictating tumor cell response to adjuvant cancer therapies, including irradiation and chemotherapy. Glutamate cysteine ligase (GCL), glutathione synthetase (GS), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-S-transferases (GST), and GSH complex export transporters (GS-X pumps) are major components of the GSH-dependent enzyme system that function in a dynamic cascade to maintain redox homeostasis. In many tumors, the GSH system is often dysregulated, resulting in a more drug resistant phenotype. This is commonly associated with GST-mediated GSH conjugation of various anticancer agents leading to the formation of less toxic GSH-drug complexes, which can be readily exported from the cell. Advances in our understanding of the mechanisms of drug resistance and patient selection based on biomarker profiles will be crucial to adapt therapeutic strategies and improve outcomes for patients with primary malignant brain tumors.

A synthetic chalcone as a potent inducer of glutathione biosynthesis.

Chalcones continue to attract considerable interest due to their anti-inflammatory and antiangiogenic properties. We recently reported the ability of 2',5'-dihydroxychalcone (2',5'-DHC) to induce both breast cancer resistance protein-mediated export of glutathione (GSH) and c-Jun N-terminal kinase-mediated increased intracellular GSH levels. Herein, we report a structure-activity relationship study of a series of 30 synthetic chalcone derivatives with hydroxyl, methoxyl, and halogen (F and Cl) substituents and their ability to increase intracellular GSH levels. This effect was drastically improved with one or two electrowithdrawing groups on phenyl ring B and up to three methoxyl and/or hydroxyl groups on phenyl ring A. The optimal structure, 2-chloro-4',6'-dimethoxy-2'-hydroxychalcone, induced both a potent NF-E2-related factor 2-mediated transcriptional response and an increased formation of glutamate cysteine ligase holoenzyme, as shown using a human breast cancer cell line stably expressing a luciferase reporter gene driven by antioxidant response elements.

Oxidative Stress and the ER Stress Response in a Murine Model for Early-Stage Alcoholic Liver Disease.

Alcoholic liver disease (ALD) is a primary cause of morbidity and mortality in the United States and constitutes a significant socioeconomic burden. Previous work has implicated oxidative stress and endoplasmic reticulum (ER) stress in the etiology of ALD; however, the complex and interrelated nature of these cellular responses presently confounds our understanding of ethanol-induced hepatopathy. In this paper, we assessed the pathological contribution of oxidative stress and ER stress in a time-course mouse model of early-stage ALD. Ethanol-treated mice exhibited significant hepatic panlobular steatosis and elevated plasma ALT values compared to isocaloric controls. Oxidative stress was observed in the ethanol-treated animals through a significant increase in hepatic TBARS and immunohistochemical staining of 4-HNE-modified proteins. Hepatic glutathione (GSH) levels were significantly decreased as a consequence of decreased CBS activity, increased GSH utilization, and increased protein glutathionylation. At the same time, immunoblot analysis of the PERK, IRE1α, ATF6, and SREBP pathways reveals no significant role for these UPR pathways in the etiology of hepatic steatosis associated with early-stage ALD. Collectively, our results indicate a primary pathogenic role for oxidative stress in the early initiating stages of ALD that precedes the involvement of the ER stress response.

Posttranslational modification and regulation of glutamate-cysteine ligase by the α,ß-unsaturated aldehyde 4-hydroxy-2-nonenal.

4-Hydroxy-2-nonenal (4-HNE) is a lipid peroxidation product formed during oxidative stress that can alter protein function via adduction of nucleophilic amino acid residues. 4-HNE detoxification occurs mainly via glutathione (GSH) conjugation and transporter-mediated efflux. This results in a net loss of cellular GSH, and restoration of GSH homeostasis requires de novo GSH biosynthesis. The rate-limiting step in GSH biosynthesis is catalyzed by glutamate-cysteine ligase (GCL), a heterodimeric holoenzyme composed of a catalytic (GCLC) and a modulatory (GCLM) subunit. The relative levels of the GCL subunits are a major determinant of cellular GSH biosynthetic capacity and 4-HNE induces the expression of both GCL subunits. In this study, we demonstrate that 4-HNE can alter GCL holoenzyme formation and activity via direct posttranslational modification of the GCL subunits in vitro. 4-HNE directly modified Cys553 of GCLC and Cys35 of GCLM in vitro, which significantly increased monomeric GCLC enzymatic activity, but reduced GCL holoenzyme activity and formation of the GCL holoenzyme complex. In silico molecular modeling studies also indicate these residues are likely to be functionally relevant. Within a cellular context, this novel posttranslational regulation of GCL activity could significantly affect cellular GSH homeostasis and GSH-dependent detoxification during periods of oxidative stress.

2',5'-Dihydroxychalcone-induced glutathione is mediated by oxidative stress and kinase signaling pathways.

Hydroxychalcones are naturally occurring compounds that continue to attract considerable interest because of their anti-inflammatory and antiangiogenic properties. They have been reported to inhibit the synthesis of the inducible nitric oxide synthase and to induce the expression of heme oxygenase-1. This study examines the mechanisms by which 2',5'-dihydroxychalcone (2',5'-DHC) induces an increase in cellular glutathione (GSH) levels using a cell line stably expressing a luciferase reporter gene driven by antioxidant-response elements (MCF-7/AREc32). The 2',5'-DHC-induced increase in cellular GSH levels was partially inhibited by the catalytic antioxidant MnTDE-1,3-IP(5+), suggesting that reactive oxygen species (ROS) mediate the antioxidant adaptive response. 2',5'-DHC treatment induced phosphorylation of the c-Jun N-terminal kinase (JNK) pathway, which was also inhibited by MnTDE-1,3-IP(5+). These findings suggest a ROS-dependent activation of the AP-1 transcriptional response. However, whereas 2',5'-DHC triggered the NF-E2-related factor 2 (Nrf2) transcriptional response, cotreatment with MnTDE-1,3-IP(5+) did not decrease 2',5'-DHC-induced Nrf2/ARE activity, showing that this pathway is not dependent on ROS. Moreover, pharmacological inhibitors of mitogen-activated protein kinase (MAPK) pathways showed a role for JNK and p38MAPK in mediating the 2',5'-DHC-induced Nrf2 response. These findings suggest that the 2',5'-DHC-induced increase in GSH levels results from a combination of ROS-dependent and ROS-independent pathways.

Enhanced glutathione biosynthetic capacity promotes resistance to As3+-induced apoptosis.

Trivalent arsenite (As(3+)) is a known human carcinogen capable of inducing both cellular transformation and apoptotic cell death by mechanisms involving the production of reactive oxygen species. The tripeptide antioxidant glutathione (GSH) constitutes a vital cellular defense mechanism against oxidative stress. While intracellular levels of GSH are an important determinant of cellular susceptibility to undergo apoptotic cell death, it is not known whether cellular GSH biosynthetic capacity per se regulates As(3+)-induced apoptosis. The rate-limiting enzyme in GSH biosynthesis is glutamate cysteine ligase (GCL), a heterodimeric holoenzyme composed of a catalytic (GCLC) and a modifier (GCLM) subunit. To determine whether increased GSH biosynthetic capacity enhanced cellular resistance to As(3+)-induced apoptotic cell death, we utilized a mouse liver hepatoma (Hepa-1c1c7) cell line stably overexpressing both GCLC and GCLM. Overexpression of the GCL subunits increased GCL holoenzyme formation and activity and inhibited As(3+)-induced apoptosis. This cytoprotective effect was associated with a decrease in As(3+)-induced caspase activation, cleavage of caspase substrates and translocation of cytochrome c to the cytoplasm. In aggregate, these findings demonstrate that enhanced GSH biosynthetic capacity promotes resistance to As(3+)-induced apoptosis by preventing mitochondrial dysfunction and cytochrome c release and highlight the role of the GSH antioxidant defense system in dictating hepatocyte sensitivity to As(3+)-induced apoptotic cell death.

Structure, function, and post-translational regulation of the catalytic and modifier subunits of glutamate cysteine ligase.

Glutathione (GSH) is a tripeptide composed of glutamate, cysteine, and glycine. The first and rate-limiting step in GSH synthesis is catalyzed by glutamate cysteine ligase (GCL, previously known as gamma-glutamylcysteine synthetase). GCL is a heterodimeric protein composed of catalytic (GCLC) and modifier (GCLM) subunits that are expressed from different genes. GCLC catalyzes a unique gamma-carboxyl linkage from glutamate to cysteine and requires ATP and Mg(++) as cofactors in this reaction. GCLM increases the V(max) and K(cat) of GCLC, decreases the K(m) for glutamate and ATP, and increases the K(i) for GSH-mediated feedback inhibition of GCL. While post-translational modifications of GCLC (e.g. phosphorylation, myristoylation, caspase-mediated cleavage) have modest effects on GCL activity, oxidative stress dramatically affects GCL holoenzyme formation and activity. Pyridine nucleotides can also modulate GCL activity in some species. Variability in GCL expression is associated with several disease phenotypes and transgenic mouse and rat models promise to be highly useful for investigating the relationships between GCL activity, GSH synthesis, and disease in humans.

Distinct Nrf1/2-independent mechanisms mediate As 3+-induced glutamate-cysteine ligase subunit gene expression in murine hepatocytes.

Trivalent arsenite (As(3+)) is a known human carcinogen that is also capable of inducing apoptotic cell death. Increased production of reactive oxygen species is thought to contribute to both the carcinogenic and the cytotoxic effects of As(3+). Glutathione (GSH) constitutes a vital cellular defense mechanism against oxidative stress. The rate-limiting enzyme in GSH biosynthesis is glutamate-cysteine ligase (GCL), a heterodimeric holoenzyme composed of a catalytic (GCLC) and a modifier (GCLM) subunit. In this study, we demonstrate that As(3+) coordinately upregulates Gclc and Gclm mRNA levels in a murine hepatocyte cell line resulting in increased GCL subunit protein expression, holoenzyme formation, and activity. As(3+) increased the rate of transcription of both the Gclm and the Gclc genes and induced the posttranscriptional stabilization of Gclm mRNA. The antioxidant N-acetylcysteine abolished As(3+)-induced Gclc expression and attenuated induction of Gclm. As(3+) induction of Gclc and Gclm was also differentially regulated by the MAPK signaling pathways and occurred independent of the Nrf1/2 transcription factors. These findings demonstrate that distinct transcriptional and posttranscriptional mechanisms mediate the coordinate induction of the Gclc and Gclm subunits of GCL in response to As(3+) and highlight the potential importance of the GSH antioxidant defense system in regulating As(3+)-induced responses in hepatocytes.

Cigarette smoke extract-induced suppression of caspase-3-like activity impairs human neutrophil phagocytosis.

Neutrophils are the primary inflammatory cell in smokers' lungs, but little is known about the ability of cigarette smoke to modulate neutrophil function. Neutrophils undergo caspase-3-dependent spontaneous, as well as phagocytosis-induced, apoptosis. This study investigated the ability of cigarette smoke extract (CSE) to alter neutrophil caspase-3 activity, apoptosis, and phagocytosis. CSE treatment resulted in a dramatic suppression of neutrophil caspase-3-like activity, which correlated with reduced cleavage of glutamate-L-cysteine ligase catalytic subunit, a known target of active caspase-3. CSE did not affect procaspase-3 processing to its active fragment, suggesting a direct effect of CSE on active caspase-3. Consistent with this, CSE inhibited active recombinant caspase-3 activity, which was abolished by dithiothreitol, suggesting a redox-sensitive mechanism. CSE-induced suppression of caspase-3 activity did not alter spontaneous apoptosis but did impair phagocytic activity. Since CSE treatment resulted in profound suppression of caspase-3 activity but did not alter apoptosis, the possibility of a threshold level of caspase-3 activity was investigated. CSE reduced caspase-3 activity in a concentration-dependent manner. Despite near complete suppression of caspase-3 activity, spontaneous apoptosis was not altered. Conversely, treatment with the pan-caspase inhibitor, Z-Val-Ala-Asp-fluoromethylketone, reduced spontaneous apoptosis. These data demonstrate that CSE does not suppress caspase-3 activity below a threshold level to prevent spontaneous apoptosis, but the level of inhibition is sufficient to impair neutrophil phagocytic activity. These divergent functions of caspase-3 may contribute to the persistence of neutrophils in the lungs of smokers, as well as be a factor in their higher incidence of community-acquired pneumonia.

Caspase-3-Dependent Cleavage of the Glutamate-L-Cysteine Ligase Catalytic Subunit during Apoptotic Cell Death.

Apoptotic cell death is usually accompanied by activation of a family of cysteine proteases termed caspases. Caspases mediate the selective proteolysis of multiple cellular targets often resulting in the disruption of survival pathways. Intracellular levels of the antioxidant glutathione (GSH) are an important determinant of cellular susceptibility to apoptosis. The rate-limiting step in GSH biosynthesis is mediated by glutamate-L-cysteine ligase (GCL), a heterodimeric enzyme consisting of a catalytic (GCLC) and a modifier (GCLM) subunit. In this report we demonstrate that GCLC is a direct target for caspase-mediated cleavage in multiple models of apoptotic cell death. Mutational analysis revealed that caspase-mediated cleavage of GCLC occurs at Asp(499) within the sequence AVVD(499)G. GCLC cleavage occurs upstream of Cys(553), which is thought to be important for association with GCLM. GCLC cleavage is accompanied by a rapid loss of intracellular GSH due to caspase-mediated extrusion of GSH from the cell. However, while GCLC cleavage is dependent on caspase-3, GSH extrusion occurs by a caspase-3-independent mechanism. Our identification of GCLC as a target for caspase-3-dependent cleavage during apoptotic cell death suggests that this post-translational modification may represent a novel mechanism for regulating GSH biosynthesis during apoptosis.

Variable regulation of glutamate cysteine ligase subunit proteins affects glutathione biosynthesis in response to oxidative stress.

Glutamate cysteine ligase (GCL), composed of a catalytic (GCLC) and modulatory (GCLM) subunit, catalyzes the first step of glutathione (GSH) biosynthesis. Using 4-hydroxy-2-nonenal (4HNE), 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), and tertiary-butylhydroquinone (tBHQ) as models of oxidative stress which are known to work through different mechanisms, we measured changes in cellular GSH, GCL mRNA, and GCL protein. 4HNE and tBHQ treatments increased cellular GSH levels, while DMNQ exposure depleted GSH. Furthermore, changes in the two GCL mRNAs largely paralleled changes in the GCL proteins; however, the magnitudes differed, suggesting some form of translational control. The molar ratio of GCLC:GCLM ranged from 3:1 to 17:1 in control human bronchial epithelial (HBE1) cells and all treatments further increased this ratio. Data from several mouse tissues show molar ratios of GCLC:GCLM that range from 1:1 to 10:1 in support of these findings. These data demonstrate that alterations in cellular GSH are clearly correlated with GCLC to a greater extent than GCLM. Surprisingly, both control HBE1 cells and some mouse tissues have more GCLC than GCLM and GCLM increases to a much lesser extent than GCLC, suggesting that the regulatory role of GCLM is minimal under physiologically relevant conditions of oxidative stress.

Adenoviral overexpression of the glutamylcysteine ligase catalytic subunit protects pancreatic islets against oxidative stress.

The catalytic subunit of glutamylcysteine ligase (GCLC) primarily regulates de novo synthesis of glutathione (GSH) in mammalian cells and is central to the antioxidant capacity of the cell. However, GCLC expression in pancreatic islets has not been previously examined. We designed experiments to ascertain whether GCLC is normally expressed in islets and whether it is up-regulated by interleukin-1 beta (IL-1 beta). GCLC expression levels were intermediate compared with other metabolic tissues (kidney, liver, muscle, fat, and lung). IL-1 beta up-regulated GCLC expression (10 ng/ml IL-1 beta, 3.76 +/- 0.86; 100 ng/ml IL-1 beta, 4.22 +/- 0.68-fold control) via the p38 form of mitogen-activated protein kinase and NF kappa B and also increased reactive oxygen species levels (10 ng/ml IL-1 beta, 5.41 +/- 1.8-fold control). This was accompanied by an increase in intraislet GSH/GSSG ratio (control, 7.1 +/- 0.1; 10 ng/ml IL-1 beta, 8.0 +/- 0.5; 100 ng/ml IL-1 beta, 8.2 +/- 0.5-fold control; p < 0.05). To determine whether overexpression of GCLC increases the antioxidant capacity of the islet and prevents the adverse effects of IL-1 beta on glucose-induced insulin secretion, islets were infected with an adenovirus encoding GCLC. IL-1 beta significantly decreased glucose-stimulated insulin secretion (control, 123.8 +/- 17.7; IL-1 beta, 40.2 +/- 3.9 microunits/ml insulin/islet). GCLC overexpression increased intraislet GSH levels and partially prevented the decrease in glucose-stimulated insulin secretion caused by IL-1 beta. These data provide the first report of GCLC expression in the islet and demonstrate that adenoviral overexpression of GCLC increases intracellular GSH levels and protects the beta cell from the adverse effects of IL-1 beta.