Induction Of Synthetase Research Articles

4‐Aminoethylamino‐emodin – a novel potent inhibitor of GSK‐3β– acts as an insulin‐sensitizer avoiding downstream effects of activated β‐catenin

Glycogen synthase kinase-3β (GSK-3β) is a key target and effector of downstream insulin signalling. Using comparative protein kinase assays and molecular docking studies we characterize the emodin-derivative 4-[N-2-(aminoethyl)-amino]-emodin (L4) as a sensitive and potent inhibitor of GSK-3β with peculiar features. Compound L4 shows a low cytotoxic potential compared to other GSK-3β inhibitors determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide assay and cellular ATP levels. Physiologically, L4 acts as an insulin-sensitizing agent that is able to enhance hepatocellular glycogen and fatty acid biosynthesis. These functions are particularly stimulated in the presence of elevated concentrations of glucose and in synergy with the hormone action at moderate but not high insulin levels. In contrast to other low molecular weight GSK-3β inhibitors (SB216763 and LiCl) or Wnt-3α-conditioned medium, however, L4 does not induce reporter and target genes of activated β-catenin such as TOPflash, Axin2 and glutamine synthetase. Moreover, when present together with SB216763 or LiCl, L4 counteracts expression of TOPflash or induction of glutamine synthetase by these inhibitors. Because L4 slightly activates β-catenin on its own, these results suggest that a downstream molecular step essential for activation of gene transcription by β-catenin is also inhibited by L4. It is concluded that L4 represents a potent insulin-sensitizing agent favouring physiological effects of insulin mediated by GSK-3β inhibition but avoiding hazardous effects such as activation of β-catenin-dependent gene expression which may lead to aberrant induction of cell proliferation and cancer.

Antioxidant Properties of Raisins (Vitis viniferaL.)

Currants and sultanas (Vitis vinifera L., Family Vitaceae) are dried vine products produced in Greece and used widely in the Mediterranean diet. We investigated the polar methanol extracts from the raisins for the antiradical activity, polyphenol content, cytotoxicity in peripheral blood mononuclear cells, total glutathione (GSH) levels, oxidized glutathione (GSSG) levels, and inhibition of low-density lipoprotein (LDL) oxidation. Extracts exhibited 1,1-diphenyl-2-picrylhydrazyl scavenging activity and inhibited tert-butylhydroperoxide (tBHP)-induced cytotoxicity, GSH decrease, and LDL oxidation. Fluorescence microscopy revealed the characteristic apoptotic patterns with tBHP and inhibition with the extracts. Elevated levels of total GSH and unaltered levels of GSSG with extract treatment demonstrated the induction of gamma-glutamylcysteine synthetase. The antioxidant activity was correlated to the polyphenolic content. Greek currants and sultanas are suggested as antioxidant components of the Mediterranean basin.

The Comparative Immunomodulatory Effects of β-Glucans from Yeast, Bacteria, and Mushroom on the Function of Macrophages

The comparative immunomodulatory effects of β-glucans isolated from mushroom fungi (Coriolus versicol), yeast (Saccharomyces cerevisiae) and bacteria (Agrobacterium) on the major functions of macrophages were evaluated. As parameters of macrophage functions, we examined tumoricidal activity, phagocytosis, nitric oxide (NO) production, and the induction of inducible NO synthetase (iNOS) in RAW264.7 cells, following treatments with β-glucans from the three different sources. The results indicated that all β-glucan treatments significantly induced tumoricidal activity in the RAW264.7 cells, with a remarkable effect shown by the beta-glucan from Agrobacterium at a concentration of 10 ㎍/mL. There was also a significant increase in iNOS-NO system activity in macrophages treated with β-glucans extracted from yeast; however, iNOS-NO system activity was not markedly changed by the treatment of β-glucans from C. versicolor mushroom fungi or Agrobacterium. Furthermore, the β-glucans from C. versicolor had a significant phagocytotic effect at concentrations of 1, 10, and 100 ㎍/mL. Taken together, the present data suggest that these β-glucans, isolated from three different sources, have different effects on macrophage function, and therefore, may have different clinical uses in different for various types of diseases.

Ethylene and salicylic acid control glutathione biosynthesis in ozone‐exposed Arabidopsis thaliana

Ozone produces reactive oxygen species and induces the synthesis of phytohormones, including ethylene and salicylic acid. These phytohormones act as signal molecules that enhance cell death in response to ozone exposure. However, some studies have shown that ethylene and salicylic acid can instead decrease the magnitude of ozone-induced cell death. Therefore, we studied the defensive roles of ethylene and salicylic acid against ozone. Unlike the wild-type, Col-0, Arabidopsis mutants deficient in ethylene signaling (ein2) or salicylic acid biosynthesis (sid2) generated high levels of superoxide and exhibited visible leaf injury, indicating that ethylene and salicylic acid can reduce ozone damage. Macroarray analysis suggested that the ethylene and salicylic acid defects influenced glutathione (GSH) metabolism. Increases in the reduced form of GSH occurred in Col-0 6 h after ozone exposure, but little GSH was detected in ein2 and sid2 mutants, suggesting that GSH levels were affected by ethylene or salicylic acid signaling. We performed gene expression analysis by real-time polymerase chain reaction using genes involved in GSH metabolism. Induction of gamma-glutamylcysteine synthetase (GSH1), glutathione synthetase (GSH2), and glutathione reductase 1 (GR1) expression occurred normally in Col-0, but at much lower levels in ein2 and sid2. Enzymatic activities of GSH1 and GSH2 in ein2 and sid2 were significantly lower than in Col-0. Moreover, ozone-induced leaf damage observed in ein2 and sid2 was mitigated by artificial elevation of GSH content. Our results suggest that ethylene and salicylic acid protect against ozone-induced leaf injury by increasing de novo biosynthesis of GSH.

S1852 Induction of Glutamine Synthetase By Exercise in Healthy Volunteers: Exploring An Alternative Proposal for Nonhepatic Ammonia Metabolism

S1852 Induction of Glutamine Synthetase By Exercise in Healthy Volunteers: Exploring An Alternative Proposal for Nonhepatic Ammonia Metabolism

C/EBP Homology Protein (CHOP) Interacts with Activating Transcription Factor 4 (ATF4) and Negatively Regulates the Stress-dependent Induction of the Asparagine Synthetase Gene

C/EBP homology protein (CHOP), a stress-induced transcription factor, is involved in transcriptional regulation, cell cycle, and apoptosis. The present studies identified CHOP as an interacting partner of activating transcription factor (ATF) 4 in a yeast two-hybrid screen and confirmed their interaction in HEK293T cells. CHOP protein levels rose modestly and transiently during amino acid deprivation, whereas endoplasmic reticulum stress caused a much higher and sustained expression of CHOP protein. Exogenous CHOP expression enhanced the TRB3 gene induction by amino acid deprivation. Conversely, CHOP suppressed the induction of the endogenous asparagine synthetase (ASNS) gene and inhibited transcription from a reporter gene driven by the ASNS promoter following activation by ATF4 or amino acid deprivation. Short interfering RNA-mediated knockdown of CHOP further enhanced the induction of ASNS by either amino acid deprivation or endoplasmic reticulum stress. The CHOP-dependent repression of the ASNS gene required the entire CHOP protein, arguing against the possibility of simple sequestration of ATF4 by the CHOP leucine zipper domain, and chromatin immunoprecipitation analysis showed association of CHOP with the ASNS and TRB3 promoters. Interestingly, chromatin immunoprecipitation also showed that CHOP was associated with the C/EBP-ATF composite site regions of the SNAT2, VEGF, and CAT-1 genes, despite no significant effect on their expression after exogenous CHOP overexpression. Collectively, the results document that CHOP is a member of the transcription factor network that controls the stress-induced regulation of specific C/EBP-ATF-containing genes, such as ASNS.

Antioxidant therapy in critical care—Is the microcirculation the primary target?

This review presents the rationale for the therapeutic use of antioxidants in treating critically ill patients; it is not a systematic review of the clinical evidence that has been assessed recently by others. Clinical and nonclinical evidence is presented to support the notion that natural antioxidants are of therapeutic value in treating cardiovascular shock. Oxidative stress is a major promoter and mediator of the systemic inflammatory response. The microcirculation is particularly susceptible to oxidative stress that causes hemodynamic instability, leading to multiple organ failure due to systemic inflammatory response syndrome. Vitamin C is the antioxidant used experimentally to demonstrate oxidative stress as a key pathophysiologic factor in septic shock. Pharmacologic studies reveal that vitamin C (as ascorbate), at supraphysiologic doses, significantly affects the bioavailability of nitric oxide during acute inflammation, including inhibiting nitric oxide synthetase induction. Parenteral high-dose vitamin C inhibits endotoxin-induced endothelial dysfunction and vasohyporeactivity in humans and reverses sepsis-induced suppression of microcirculatory control in rodents. In severe burn injury, in both animals and patients, parenteral high-dose vitamin C significantly reduces resuscitation fluid volumes. Therefore, a significant body of pharmacologic evidence and sound preliminary clinical evidence supports the biological feasibility of using the exemplary antioxidant, vitamin C, in the treatment of the critically ill.

Glutamine, insulin and glucocorticoids regulate glutamine synthetase expression in C2C12 myotubes, Hep G2 hepatoma cells and 3T3 L1 adipocytes

The cell-specific regulation of glutamine synthetase expression was studied in three cell lines. In C2C12 myotubes, glucocorticoids increased the abundance of both glutamine synthetase protein and mRNA. Culture in the absence of glutamine also resulted in very high glutamine synthetase protein abundance but mRNA levels were unchanged. Glucocorticoids also increased the abundance of glutamine synthetase mRNA in Hep G2 hepatoma cells but this was not reflected in changes in protein abundance. Culture of Hep G2 cells without glutamine resulted in very high levels of protein, again with no change in mRNA abundance. Insulin was without effect in both C2C12 and Hep G2 cells. In 3T3 L1 adipocytes glucocorticoids increased the abundance of both glutamine synthetase mRNA and protein, insulin added alone had no effect but in the presence of glucocorticoids resulted in lower mRNA levels than seen with glucocorticoids alone, although protein levels remained high under such conditions. In contrast to the other cell lines glutamine synthetase protein levels were relatively unchanged by culture in the absence of glutamine. The results support the hypothesis that in myocytes, and hepatomas, but not in adipocytes, glutamine acts to moderate glutamine synthetase induction by glucocorticoids.

Role of Glutamine Depletion in Directing Tissue-specific Nutrient Stress Responses to L-Asparaginase

Role of Glutamine Depletion in Directing Tissue-specific Nutrient Stress Responses to L-Asparaginase

Electroconvulsive seizure increases adult hippocampal angiogenesis in rats

Electroconvulsive seizure has a proven therapeutic application in the treatment of severe depression and treatment-resistant depression. Despite the efficacy of electroconvulsive seizure as a non-chemical antidepressant treatment, the mechanism of action is unclear. Elevation in hippocampal trophic factor expression and concomitant cellular proliferation are thought to play a role in its action. We examined whether the reported induction of angiogenic factors and endothelial cell proliferation leads to an increase in vascular density. Two hippocampal regions, the dentate gyrus and the stratum lacunosum moleculare (SLM), were examined employing a combination of vascular density quantification, angiogenic gene expression analysis and immunohistochemistry. A 6% increase in vascular density was observed in the dentate gyrus but this did not achieve statistical significance. The SLM of the hippocampus exhibited a robust 20-30% increase in vascular density and was accompanied by an increase in expression of inhibitor of differentiation-3. There was also an induction of the angiogenesis markers alphaVbeta3 integrin and Del1. Increases in the vascular density of the SLM could be in response to enhanced metabolic activity in this region. This is supported by the induction of glutamine synthetase and the glutamate transporter GLT1.

Cytoskeletal and cell contact control of the glucocorticoid pathway

The cytoskeleton is a dynamic network that undergoes restructuring during a variety of cellular events including cell contact formation, cell invasion and the mitotic phase of the cell cycle. Here, we review the contribution of the cytoskeletal network to the inductive activity of glucocorticoids by focusing on the hormonal control of glutamine synthetase in the chick neural retina. Depolymerization of the cytoskeleton in cells of the intact retinal tissue inhibits the hormonal induction of glutamine synthetase, but does not alter the cellular amount of the glucocorticoid-receptor protein or the ability of the receptor molecules to translocate into the nucleus. Inhibition of glutamine synthetase induction occurs via a mechanism that involves elevation of c-Jun protein accumulation and repression of glucocorticoid-receptor transcriptional activity. Unlike growth factors and other c-Jun inducing stimuli that control the transcription of the c-Jun gene, depolymerization of the cytoskeleton elevates c-Jun accumulation by upregulating the translation of the c-Jun transcript. We postulate that the cytoskeletal-dependent increase in c-Jun accumulation is involved in cell contact control of both cell proliferation and transcriptional activity of the glucocorticoid-receptor protein.

Cathepsin B is a differentiation-resistant target for nitroxyl (HNO) in THP-1 monocyte/macrophages

We previously showed that the one-electron reduction product of nitric oxide (NO), nitroxyl (HNO), irreversibly inhibits the proteolytic activity of the model cysteine protease papain. This result led us to investigate the differential effects of the nitrogen oxides, such as nitroxyl (HNO), NO, and in situ-generated peroxynitrite on cysteine modification-sensitive cellular proteolytic enzymes. We used Angeli's salt, diethylaminenonoate (DEA/NO), and 3-morpholinosydnoniminehydrochloride (SIN-1), as donors of HNO, NO, and peroxynitrite, respectively. In this study we evaluated their inhibitory activities on the lysosomal mammalian papain homologue cathepsin B and on the cytosolic 26S proteasome in THP-1 monocyte/macrophages after LPS activation or TPA differentiation. HNO-generating Angeli's salt caused a concentration-dependent (62 ± 4% at 316 μM) inhibition of the 26S proteasome activity, resulting in accumulation of protein-bound polyubiquitinylated proteins in LPS-activated cells, whereas neither DEA/NO nor SIN-1 showed any effect. Angeli's salt, but not DEA/NO or SIN-1, also caused (94 ± 2% at 316 μM) inhibition of lysosomal cathepsin B activity in LPS-activated cells. Induction of macrophage differentiation did not significantly alter the inhibitory effect of HNO on lysosomal cathepsin B activity, but protected the proteasome from HNO-induced inhibition. The protection awarded by macrophage differentiation was associated with induction of the GSH synthesis rate-limiting enzyme γ-glutamylcysteine synthetase, as well as with increased intracellular GSH. In conclusion, HNO abrogates both lysosomal and cytosolic proteolysis in THP-1 cells. Macrophage differentiation, associated with upregulation of antioxidant defenses such as increased cellular GSH, does not protect the lysosomal cysteine protease cathepsin B from inhibition.

Exercise interrupts ongoing glucocorticoid-induced muscle atrophy and glutamine synthetase induction.

This study was undertaken to determine whether regular endurance exercise is a deterrent to a developing state of muscle atrophy from glucocorticoids and to evaluate whether the contractile activity antagonizes the hormonal actions on glutamine synthetase, alanine aminotransferase, and cytosolic aspartate aminotransferase (cAspAT). Adult female rats were administered cortisol acetate (CA, 100 mg/kg body wt) or an equal volume of the vehicle solution for up to 15 days. Exercise (treadmill running at 31 m/min, 10% grade, 90 min/day) was introduced after 4 days of CA treatment, at which time plantaris and quadriceps muscle mass had been reduced to 90% of control levels. Running for 11 consecutive days prevented 40 mg of the 90-mg loss and 227 mg of the 808-mg loss that were subsequently observed in plantaris and quadriceps muscles, respectively, in the sedentary animals. Glutamine synthetase mRNA and enzyme activity were elevated threefold by glucocorticoid treatment in the deep quadriceps (fast-twitch red) muscles after 4 days. Initiating exercise completely interfered with the further hormonal induction (to approximately 5-fold) of this enzyme and, after 11 consecutive days of the exercise regimen, glutamine synthetase mRNA and enzyme activity were 58 and 68% of values from CA-treated sedentary animals. In vehicle-treated groups, basal levels of glutamine synthetase expression were also diminished by exercise to approximately 40% of the values in sedentary controls. Hormone treatment did not alter either aminotransferase enzyme activity but reduced cAspAT mRNA in fast-twitch red muscles by 50%. Exercise abolished the glucocorticoid effect on cAspAT mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Direct molecular and spectroscopic evidence for increased ammonia removal capacity of skeletal muscle in acute liver failure

It has been proposed that, in acute liver failure, skeletal muscle adapts to become the principle organ responsible for removal of blood-borne ammonia by increasing glutamine synthesis, a reaction that is catalyzed by the cytosolic ATP-dependent enzyme glutamine synthetase. To address this issue, glutamine synthetase expression and activities were measured in skeletal muscle of rats with acute liver failure resulting from hepatic devascularization. Glutamine synthetase protein and gene expression were investigated using immunoblotting and semi-quantitative RT-PCR analysis. Glutamine synthetase activity and glutamine de novo synthesis were measured using, respectively, a standard enzymatic assay and [13C]-nuclear magnetic resonance spectroscopy. Glutamine synthetase protein (but not gene) expression and enzyme activities were significantly up-regulated leading to increased de novo synthesis of glutamine and increased skeletal muscle capacity for ammonia removal in acute liver failure. In contrast to skeletal muscle, expression and activities of glutamine synthetase in the brain were significantly decreased. These findings demonstrate that skeletal muscle adapts, through a rapid induction of glutamine synthetase, to increase its capacity for removal of blood-borne ammonia in acute liver failure. Maintenance of muscle mass together with the development of agents with the capacity to stimulate muscle glutamine synthetase could provide effective ammonia-lowering strategies in this disorder.

Indole-3-butyric acid synthesis in ecotypes and mutants of Arabidopsis thaliana under different growth conditions

Although IBA is a naturally occurring auxin, its role in plant development is still under debate. In this study a set of Arabidopsis mutants was used to analyze the biosynthesis of IBA in vitro. The mutants chosen for this study can be classified as: (1) involvement in auxin metabolism, transport or synthesis (amt1, aux1, ilr1, nit1, rib1, sur1, trp1-100); (2) other hormones possibly involved in the regulation of IBA synthesis (aba1, aba3, eto2, fae1, hls1, jar1); (3) photomorphogenesis (det1, det2, det3); and (4) root architecture (cob1, cob2, scr1). In addition, two transgenic lines overexpressing the IAA glucose synthase (iaglu) gene from maize were analyzed. The ecotypes No-0 and Wassilewskija showed the highest IBA synthetase activity under control conditions, followed by Columbia, Enkheim and Landsberg erecta. In the mutant lines IBA synthetase activity differed in most cases from the wild type, however no particular pattern of up- or down-regulation, which could be correlated to their possible function, was found. For rib1 mutant seedlings it was tested whether reduced IBA synthetase activity correlates with the endogenous IBA levels. Free IBA differed only depending on the culture conditions, but gave no clear correlation with IBA synthetase activity compared to the wild type. Since drought and osmotic stress as well as abscisic acid (ABA) application enhanced IBA synthesis in maize, it was tested whether IBA synthetase from Arabidopsis is also inducible by drought stress conditions. This was confirmed for the two ecotypes Col and Ler which showed different IBA synthetase activity when cultivated with various degrees of drought stress. IBA synthetase was also determined in photomorphogenic mutants under different light regimes. Induction of IBA synthetase in det1 and det3 plants was found under short day plus a red light pulse or in the dark, respectively. The results are discussed with respect to the functions of the mutated genes.

Ornithine α-ketoglutarate metabolism in the healthy rat in the postabsorptive state

To gain further insight into the ability of ornithine α-ketoglutarate (OKG) to generate key metabolites, the aim of this work was to study the short-term metabolism, that is, 1 hour after administration, of OKG in plasma and tissues. Particular attention was paid to keto acids ( α-ketoglutarate and branched-chain keto acids). Young (3 weeks old) male Wistar rats in the postabsorptive state received either 1.5 g/kg of monohydrated OKG (OKG group, n = 8) diluted in distilled water or an equivalent volume of saline solution at 0.9% (control group, n = 8) by gavage and were killed 1 hour later. Plasma, liver, jejunal and ileal mucosa, and the extensor digitorum longus muscle were removed to analyze amino and keto acid contents. Major metabolites detected after OKG ingestion (ornithine [ORN], α-ketoglutarate, proline and glutamate; OKG vs control, P < .05) and the absence of increased arginine (and even a decrease in jejunum and muscle) and citrulline levels suggested that ORN was mainly metabolized by the ORN aminotransferase pathway. In addition, significantly decreased plasma branched-chain keto acids and increased hepatic branched-chain amino acids (OKG vs control, P < .05) were observed upon OKG ingestion. Finally, glutamine accumulation restricted to the intestine, as evidenced in this short-term study, suggests that the effects of OKG on glutamine pools in other tissues in various pathological states after several days of treatment, as observed in previous studies, may be related to a long-term induction of glutamine synthetase.

Erratum: Epigenetic changes in the repression and induction of asparagine synthetase in human leukemic cell lines

Erratum: Epigenetic changes in the repression and induction of asparagine synthetase in human leukemic cell lines

2′,5′-Oligoadenylate size is critical to protect RNase L against proteolytic cleavage in chronic fatigue syndrome

A dysregulation in the 2′,5′-oligoadenylate (2-5A)-dependent RNase L antiviral pathway has been detected in peripheral blood mononuclear cells (PBMC) of chronic fatigue syndrome (CFS) patients, which is characterized by upregulated 2-5A synthetase and RNase L activities, as well as by the presence of a low molecular weight (LMW) 2-5A-binding protein of 37-kDa related to RNase L. This truncated protein has been shown to originate from proteolytic cleavage of the native 83-kDa RNase L by m-calpain and human leukocyte elastase (HLE). We investigated the possible role of 2-5A oligomers in the proteolytic action toward the endonuclease and show that incubation of CFS PBMC extracts with 2-5A trimer and tetramer, but not with the dimer, results in a significant protection of the native 83-kDa RNase L against cleavage by endogenous and purified proteases. Similar results are obtained with a purified recombinant RNase L. An analysis of the size of 2-5A oligomers produced by the catalytic activity of the 2-5A synthetase present in PBMC extracts further shows that samples containing the 37-kDa RNase L preferentially produce 2-5A dimers instead of higher oligomers. Taken together, our results indicate that homodimerization of RNase L by 2-5A oligomers higher than the dimer prevents its cleavage by proteolytic enzymes. The presence of the truncated 37-kDa RNase L in PBMC extracts is therefore likely to result, not only from the abnormal activation of inflammatory proteases, but also from a dysregulation in 2-5A synthetase induction or activation towards the preferential production of 2-5A dimers.

Adipocytic Differentiation and Liver X Receptor Pathways Regulate the Accumulation of Triacylglycerols in Human Vascular Smooth Muscle Cells

Lipid accumulation by vascular smooth muscle cells (VSMC) is a feature of atherosclerotic plaques. In this study we describe two mechanisms whereby human VSMC foam cell formation is driven by de novo synthesis of fatty acids leading to triacylglycerol accumulation in intracellular vacuoles, a process distinct from serum lipoprotein uptake. VSMC cultured in adipogenic differentiation medium accumulated lipids and were induced to express the adipocyte marker genes adipsin, adipocyte fatty acid-binding protein, C/EBPalpha, PPARgamma, and leptin. However, complete adipocyte differentiation was not observed as numerous genes present in mature adipocytes were not detected, and the phenotype was reversible. The rate of lipid accumulation was not affected by PPARgamma agonists, but screening for the effects of other nuclear receptor agonists showed that activation of the liver X receptors (LXR) dramatically promoted lipid accumulation in VSMC. Both LXRalpha and LXRbeta were present in VSMC, and their activation with TO901317 resulted in induction of the lipogenic genes fatty acid synthetase, sterol regulatory element binding protein (SREBP1c), and stearoyl-CoA desaturase. 27-Hydroxycholesterol, an abundant oxysterol synthesized by VSMC acted as an LXR antagonist and, therefore, may have a protective role in preventing foam cell formation. Immunohistochemistry showed that VSMC within atherosclerotic plaques express adipogenic and lipogenic markers, suggesting these pathways are present in vivo. Moreover, the development of an adipogenic phenotype in VSMC is consistent with their known phenotypic plasticity and may contribute to their dysfunction in atherosclerotic plaques and, thus, impinge on plaque growth and stability.

Critical Role of Mitochondrial Glutathione in the Survival of Hepatocytes during Hypoxia

Hypoxia is known to stimulate reactive oxygen species (ROS) generation. Because reduced glutathione (GSH) is compartmentalized in cytosol and mitochondria, we examined the specific role of mitochondrial GSH (mGSH) in the survival of hepatocytes during hypoxia (5% O2). 5% O2 stimulated ROS in HepG2 cells and cultured rat hepatocytes. Mitochondrial complex I and II inhibitors prevented this effect, whereas inhibition of nitric oxide synthesis with Nomega-nitro-L-arginine methyl ester hydrochloride or the peroxynitrite scavenger uric acid did not. Depletion of GSH stores in both cytosol and mitochondria enhanced the susceptibility of HepG2 cells or primary rat hepatocytes to 5% O2 exposure. However, this sensitization was abrogated by preventing mitochondrial ROS generation by complex I and II inhibition. Moreover, selective mGSH depletion by (R,S)-3-hydroxy-4-pentenoate that spared cytosol GSH levels sensitized rat hepatocytes to hypoxia because of enhanced ROS generation. GSH restoration by GSH ethyl ester or by blocking mitochondrial electron flow at complex I and II rescued (R,S)-3-hydroxy-4-pentenoate-treated hepatocytes to hypoxia-induced cell death. Thus, mGSH controls the survival of hepatocytes during hypoxia through the regulation of mitochondrial generation of oxidative stress.