ArticleLiterature Review

Glutamate cysteine ligase and the age-related decline in cellular glutathione: The therapeutic potential of γ-glutamylcysteine

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Abstract

A consistent underlying index of aging is a decline in the cellular levels of the tripeptide glutathione (GSH). GSH is an essential thiol antioxidant produced in the cytosol of all cells and plays a key role in protecting against oxidative stress by neutralising free radicals and reactive oxygen species (ROS). The decline in GSH has been associated with changes in the expression and activity of the rate-limiting enzyme glutamate cysteine ligase (GCL), which produces the intermediate dipeptide γ-glutamylcysteine (γ-GC). The molecular mechanisms that affect these age-related changes remain unclear due to the complexity of GCL regulation. Impairment of the transcriptional activity of Nrf2 has been demonstrated to contribute to GCL dysregulation in aged rats. However, considering the complex nature of GCL regulation, relatively little research has been conducted to investigate the age-associated post-transcriptional controls of the enzyme. Defining these unknown mechanisms may inform our understanding of the aetiology of many age-related diseases and assist in formulating appropriate therapeutic strategies. This review focuses on the suitability of treatment with exogenous γ-GC to raise GSH levels by circumventing the age-related dysregulation of the rate-limiting step of GSH, providing promise for future research for the treatment of chronic oxidative stress-related diseases.

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... While the capacity to defend against free radicals is quite complex, one major cellular pathway, the glutathione (GSH) system, has been investigated for its known changes occurring with age (Jones et al., 2002;Liu, 2002;Ghosh et al., 2014;Ferguson and Bridge, 2016). Cellular GSH regulation utilizes a series of GSH peroxidases (Gpx) to sequester different types of free radicals. ...
... GSH is a re-usable tripeptide antioxidant that is used as a substrate by Gpx to reduce radicals and is recycled back into a usable form by GSH reductase at the expense of NADPH. Advanced age diminishes cellular levels of both GSH as well as the availability of its amino-acid constituent, cysteine, within the plasma (Jones et al., 2002;Liu, 2002;Ghosh et al., 2014;Ferguson and Bridge, 2016). The ability to produce and maintain adequate cellular GSH levels is believed to be bottlenecked by either the availability of its cysteine substrate or the availability of the enzyme responsible for the first of two ligation reactions: glutamate-cysteine ligase (GCL) (Griffith, 1999;Lu, 2009). ...
... The ability to produce and maintain adequate cellular GSH levels is believed to be bottlenecked by either the availability of its cysteine substrate or the availability of the enzyme responsible for the first of two ligation reactions: glutamate-cysteine ligase (GCL) (Griffith, 1999;Lu, 2009). Both GSH and GCL abundance decrease with age in other organ systems (Liu, 2002;Ghosh et al., 2014;Ferguson and Bridge, 2016). Further, both GSH and GCL activity diminishes following SCI. ...
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Pre-clinical and clinical spinal cord injury (SCI) studies differ in study design, particularly in the demographic characteristics of the chosen population. In clinical study design, criteria such as such as motor scores, neurological level, and severity of injury are often key determinants for participant inclusion. Further, demographic variables in clinical trials often include individuals from a wide age range and typically include both sexes, albeit historically most cases of SCI occur in males. In contrast, pre-clinical SCI models predominately utilize young adult rodents and typically use only females. While it is often not feasible to power SCI clinical trials to test multi-variable designs such as contrasting different ages, recent pre-clinical findings in SCI animal models have emphasized the importance of considering age as a biological variable prior to human experiments. Emerging pre-clinical data have identified case examples of treatments that diverge in efficacy across different demographic variables and have elucidated several age-dependent effects in SCI. The extent to which these differing or diverging treatment responses manifest clinically can not only complicate statistical findings and trial interpretations but also may be predictive of worse outcomes in select clinical populations. This review highlights recent literature including age as a biological variable in pre-clinical studies and articulates the results with respect to implications for clinical trials. Based on emerging unpredictable treatment outcomes in older rodents, we argue for the importance of including age as a biological variable in pre-clinical animal models prior to clinical testing. We believe that careful analyses of how age interacts with SCI treatments and pathophysiology will help guide clinical trial design and may improve both the safety and outcomes of such important efforts.
... Glutation występuje w kilku postaciach redoksowych, a najważniejsze z nich to glutation zredukowany (GSH) i glutation utleniony (GSSG). W większości komórek eukariotyczych dominuje postać zredukowana GSH (>98%), która w warunkach stresu oksydacyjnego utleniana jest do GSSG w reakcji katalizowanej przez peroksydazę glutationową (glutathione peroxidase, GPx) [3]. Potencjał oksydacyjno-redukcyjny układu GSH/GSSG jest odpowiedzialny za antyoksydacyjne działanie glutationu, czyli neutralizację reaktywnych form tlenu i azotu (reactive oxygen and nitrogen species, RONS), nadtlenku wodoru (H 2 O 2 ), nadtlenków organicznych i związków elektrofilnych (detoksykacja ksenobiotyków) oraz za chelatowanie jonów metali [4]. ...
... Dzięki aktywności γ-GT, GSH uczestniczy w magazynowaniu i transporcie cysteiny, która jest aminokwasem limitującym syntezę GSH. Pozostałe aminokwasy (glutaminian i glicyna) nie odgrywają już tak ważnej roli, gdyż pozyskiwane są w wielu szlakach metabolicznych -a przez to stale dostępne w komórce [3]. Natomiast cysteina wytwarzana jest w wyniku transsulfuracji z dostarczanej z pożywieniem metioniny. ...
... Wewnątrzkomórkowe stężenie GSH warunkuje dynamiczna równowaga między jego syntezą, metabolizmem i transportem [3]. Istnieje kilka mechanizmów przez które komórki utrzymują wewnętrzną homeostazę GSH, tj. ...
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Abstrakt Glutation (γ-glutamylocysteinyloglicyna, GSH) jest najbardziej rozpowszechnionym tiolowym antyoksydantem wytwarzanym w cytozolu wszystkich komórek ssaków, który pełni ważną rolę ochronną przed stresem oksydacyjnym. GSH jest syntetyzowany de novo przez sekwencyjne działanie dwóch enzymów: ligazy γ-glutamylocysteiny (GCL) i syntetazy glutationowej (GS). GCL katalizuje pierwszy etap biosyntezy GSH, którego produktem jest γ-glutamylocysteina (γ-GC). GCL jest heterodimerycznym enzymem zbudowanym z podjednostki katalitycznej (GCLc) i modulatorowej (GCLm), kodowanych przez dwa różne geny. Podjednostki GCL podlegają złożonej regulacji zarówno na poziomie przed-, jak i potranslacyjnym. Zmiany w ekspresji i aktywności GCL mogą zaburzać poziom GSH i homeostazy redoks. Przyczyną wielu przewlekłych schorzeń związanych ze stresem oksydacyjnym jest upośledzenie aktywności katalitycznej GCL oraz spadek stężenia GSH. Badania przedkliniczne sugerują, że podawanie egzogennej γ-GC podwyższa wewnątrzkomórkowe GSH przez dostarczenie brakującego substratu i może wykazywać potencjał jako terapia uzupełniająca w chorobach związanych z deplecją GSH.
... The very important role played by endogenous glutathione (GSH), known as the 'master antioxidant', is well known and need not be further elaborated. However, not so well known is the observation that the first enzyme in the two-step biosynthesis pathway for GSH is compromised as we get older, especially beyond the age of 60 [1,2]. Taking supplemental GSH orally is not an option, since oral GSH is rapidly degraded by the enzyme, gamma-glutamyl transpeptidase [2]. ...
... However, not so well known is the observation that the first enzyme in the two-step biosynthesis pathway for GSH is compromised as we get older, especially beyond the age of 60 [1,2]. Taking supplemental GSH orally is not an option, since oral GSH is rapidly degraded by the enzyme, gamma-glutamyl transpeptidase [2]. An orally bioavailable GSH has not yet been described in the literature; therefore, we now present an orally biooavailable GSH in the form of its prodrug. ...
... While mice and rat data are generally transferable to humans, it is incumbent that studies be conducted with CySSG in humans as soon as possible by clinician investigators relative to its oral bioavailability. GSH taken orally by humans is degraded by the enzyme, gamma-glutamyl transpeptidase [2], and is known not to be bioavailable [7]. In contrast, the mechanism that releases GSH from CySSG intracellularly is via a thiol-disulfide exchange reaction [5], and was shown here to be bioavailable in mice. ...
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L-Cysteine-glutathione mixed disulfide (CySSG), a prodrug of glutathione (GSH), was found to be orally bioavailable in mice, and protected mice against a toxic dose of acetaminophen. If oral bioavailability can also be demonstrated in humans, a wide range of applicability for CySSG can be envisioned.
... Alternatively, GGC can be taken up by cells intact where it is converted to glutathione [16][17][18]. Furthermore, GGC itself is an antioxidant by acting as a cofactor for GPx where it is effectively comparable to GSH for hydrogen peroxide detoxification [19] Animal safety trials have demonstrated that GGC is safe at a repeated limit dosage of 1000 mg/kg/day over a 90-day period [20]. ...
... Furthermore, GGC itself is an antioxidant by acting as a cofactor for GPx where it is effectively comparable to GSH for hydrogen peroxide detoxification [19] Animal safety trials have demonstrated that GGC is safe at a repeated limit dosage of 1000 mg/kg/day over a 90-day period [20]. Exogenous supply of GGC, which is sold as a dietary supplement in the USA, has been reported to suppress oxidative injury and improve mitochondrial function both in in vitro and in vivo animal models of oxidative stress-induced tissue damage [18,[21][22][23][24]. In the context of lung disease, GGC has been tested in an LPS-induced mouse model of sepsis where it was demonstrated that GGC administration suppressed the production of LPS-induced inflammatory and oxidative mediators, which reduced lung tissue damage and sepsis lethality [24]. ...
... Despite multiple clinical trials with current glutathionebased therapies, improved clinical outcomes in CF have not been achieved [6,7]. As a precursor to glutathione, GGC has been consistently shown to increase intracellular levels of glutathione in both in vivo and in vitro models [16][17][18][21][22][23][24], and to reduce inflammation and oxidative stress [19,43,44]. Recently, it was shown that GGC exhibits anti-inflammatory effects in an in vivo and in vitro mouse sepsis model [24]. ...
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Systemic glutathione deficiency, inflammation, and oxidative stress are hallmarks of cystic fibrosis (CF), an inherited disease that causes persistent lung infections and severe damage to the respiratory system and many of the body organs. Improvements to current antioxidant therapeutic strategies are needed. The dietary supplement, γ-glutamylcysteine (GGC), which is the immediate precursor to glutathione, rapidly boosts cellular glutathione levels following a single dose in healthy individuals. Efficacy of GGC against oxidative stress induced by Pseudomonas aeruginosa, which is a common and chronic pathogen infecting lungs of CF patients, remains unassessed. Primary mucocilliary differentiated airway (bronchial and/or nasal) epithelial cells were created from four individuals with CF. Airway oxidative stress and inflammation was induced by P. aeruginosa lipopolysaccharide (LPS). Parameters including global proteomics alterations, cell redox state (glutathione, oxidative stress), pro-inflammatory mediators (IL-8, IDO-1), and cellular health (membrane integrity, stress granule formation, cell metabolic viability) were assayed under six experimental conditions: (1) Mock, (2) LPS-challenged (3) therapeutic, (4) prophylactic (5) therapeutic and prophylactic and (6) GGC alone. Proteomic analysis identified perturbation of several pathways related to cellular respiration and stress responses upon LPS challenge. Most of these were resolved when cells were treated with GGC. While GGC did not resolve LPS-induced IL-8 and IDO-1 activity, it effectively attenuated LPS-induced oxidative stress and stress granule formation, while significantly increasing total intracellular glutathione levels, metabolic viability and improving epithelial cell barrier integrity. Both therapeutic and prophylactic treatments were successful. Together, these findings indicate that GGC has therapeutic potential for treatment and prevention of oxidative stress-related damage to airways in cystic fibrosis.
... Age-related decline of GSH levels is reported in mammalian cells, which increases the risk of oxidative tissue damages in the elderly [51]. The main reason for the declined GSH levels is reported to be the reduction of de novo synthesis of GSH in aged cells [51]. ...
... Thus, the adult mice exhibited higher resistance to apoptosis than the resistance exhibited by the weaning mice. Age-related decline of GSH levels is reported in mammalian cells, which increases the risk of oxidative tissue damages in the elderly [51]. The main reason for the declined GSH levels is reported to be the reduction of de novo synthesis of GSH in aged cells [51]. ...
... Age-related decline of GSH levels is reported in mammalian cells, which increases the risk of oxidative tissue damages in the elderly [51]. The main reason for the declined GSH levels is reported to be the reduction of de novo synthesis of GSH in aged cells [51]. GCL, the rate-limiting enzyme for GSH biosynthesis, condenses cysteine and glutamic acid to produce γ-glutamylcysteine, which reacts with glycine in the presence of GSH synthase to form GSH [32]. ...
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Age is a risk factor for drug-induced liver injury (DILI). However, there is a limited understanding of pediatric DILI. Here, 2-week-old weaning and 8-week-old adult male ICR mice were intraperitoneally injected with CCl4 (0.1 mmol/kg equal to 15.4 mg/kg) to comparatively evaluate the time-dependent liver damage and cellular events. CCl4 significantly enhanced the serum alanine aminotransferase/aspartate aminotransferase levels and hepatic centrilobular necrosis in the weaning mice, whereas it induced mild liver injury in the adult mice. CCl4-treated weaning mice exhibited higher hepatic levels of pro-apoptotic proteins (Bax, cleaved caspase-3, -7, and -9), activated MAPKs (p-JNK and p-Erk), and endoplasmic reticulum stress indicators (ATF6 and CHOP) and lower hepatic anti-apoptotic Bcl-2 levels than the adult mice. The weaning mice exhibited enhanced basal hepatic glutathione (GSH) levels due to high glutamate cysteine ligase (GCL) and low anti-cysteine dioxygenase (CDO) enzyme levels. However, CCl4 markedly reduced the hepatic GSH levels only in the weaning mice. Furthermore, higher hepatic levels of oxidative stress-induced malondialdehyde, 4-hydroxynonenal, nitrotyrosine-protein adducts, and oxidized proteins were observed in CCl4-treated weaning mice than in CCl4-treated adult mice. The enhanced levels of hepatic cytochrome P450 (CYP) 2E1 and CYP3A, and decreased hepatic GSH S-transferase (GST)-π and GSH reductase (GR) levels in the weaning mice may contribute to their enhanced susceptibility to liver damage.
... GCLC, also known as gamma-glutamylcysteine synthetase, is the first rate-limiting enzyme in de novo of glutathione biosynthesis [56]. Changes in the expression and activity of this enzyme lead to a decline in glutathione levels [57]. We have previously shown that perinatal exposure to GBH decreases the levels of glutathione in the hippocampus of rat offspring at PND15 [15]. ...
... We have previously shown that perinatal exposure to GBH decreases the levels of glutathione in the hippocampus of rat offspring at PND15 [15]. Interestingly, GCLC is induced by the major product of lipid peroxidation, 4-hydroxynonenal (4-HNE) [57,58], which was found to be increased in the GBH-treated animals. Therefore, the increased protein levels of these important antioxidant enzymes may represent an adaptive response against the oxidative insult exerted by GBH. ...
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Concerns have been raised regarding the potential adverse health effects of the ubiquitous herbicide glyphosate. Here, we investigated long-term effects of developmental exposure to a glyphosate-based herbicide (GBH) by analyzing serum melatonin levels and cellular changes in the striatum of adult male rats (90 days old). Pregnant and lactating rats were exposed to 3% GBH (0.36% glyphosate) through drinking water from gestational day 5 to postnatal day 15. The offspring showed reduced serum melatonin levels (43%) at the adult age compared with the control group. The perinatal exposure to GBH also induced long-term oxidative stress-related changes in the striatum demonstrated by increased lipid peroxidation (45%) and DNA/RNA oxidation (39%) together with increased protein levels of the antioxidant enzymes, superoxide dismutase (SOD1, 24%), glutamate–cysteine ligase (GCLC, 58%), and glutathione peroxidase 1 (GPx1, 31%). Moreover, perinatal GBH exposure significantly increased the total number of neurons (20%) and tyrosine hydroxylase (TH)-positive neurons (38%) in the adult striatum. Mechanistic in vitro studies with primary rat pinealocytes exposed to 50 µM glyphosate demonstrated a decreased melatonin secretion partially through activation of metabotropic glutamate receptor 3 (mGluR3), while higher glyphosate levels (100 or 500 µM) also reduced the pinealocyte viability. Since decreased levels of the important antioxidant and neuroprotector melatonin have been associated with an increased risk of developing neurodegenerative disorders, this demonstrates the need to consider the melatonin hormone system as a central endocrine-related target of glyphosate and other environmental contaminants.
... A consistent underlying index of aging is a decline in cellular levels of the tripeptide glutathione. 42 Glutathione is an essential thiol antioxidant produced in the cytosol of all cells that plays a key role in protection against oxidative stress by neutralizing free radicals and reactive oxygen species. 42 The precursors of glutathione include cysteine, glutamate, and glycine. ...
... 42 Glutathione is an essential thiol antioxidant produced in the cytosol of all cells that plays a key role in protection against oxidative stress by neutralizing free radicals and reactive oxygen species. 42 The precursors of glutathione include cysteine, glutamate, and glycine. In the present study, glycine levels decreased with increasing age. ...
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Age-related gut microbes and urine metabolites were investigated in 568 healthy individuals using metataxonomics and metabolomics. The richness and evenness of the fecal microbiota significantly increased with age, and the abundance of 16 genera differed between the young and old groups. Additionally, 17 urine metabolites contributed to the differences between the young and old groups. Among the microbes that differed by age, Bacteroides and Prevotella 9 were confirmed to be correlated with some urine metabolites. The machine learning algorithm eXtreme gradient boosting (XGBoost) was shown to produce the best performing age predictors, with a mean absolute error of 5.48 years. The accuracy of the model improved to 4.93 years with the inclusion of urine metabolite data. This study shows that the gut microbiota and urine metabolic profiles can be used to predict the age of healthy individuals with relatively good accuracy.
... Glutathione (GSH), a tripeptide comprised of glutamate, cysteine, and glycine, is the main nonenzymatic antioxidant in the cytoplasm, present in high concentrations (1-10 × 10 −3 m). [57,58] It reduces oxidants directly and through facilitation by glutathione peroxidase. It is also used in detoxification, becoming irreversibly conjugated to electrophilic xenobiotics and to endogenous lipid peroxides and other reactive oxygen and nitrogen species (ROS and RNS) by the glutathione-S-transferases, facilitating their elimination. ...
... It is also used in detoxification, becoming irreversibly conjugated to electrophilic xenobiotics and to endogenous lipid peroxides and other reactive oxygen and nitrogen species (ROS and RNS) by the glutathione-S-transferases, facilitating their elimination. [59,60] GSH levels decrease with age, [57] correlate with better physical and mental health in the elderly, [61,62] and are inversely associated with numerous age-related diseases. [63] Although oxidized glutathione can be recycled to its reduced form, it is also exported by the cell into the bloodstream, catabolized in the kidneys, can bind to proteins and, as noted, its conjugation to electrophiles is irreversible. ...
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Certain metabolic interventions such as caloric restriction, fasting, exercise, and a ketogenic diet extend lifespan and/or health span. However, their benefits are limited and their connections to the underlying mechanisms of aging are not fully clear. Here, these connections are explored in terms of the tricarboxylic acid (TCA) cycle (Krebs cycle, citric acid cycle) to suggest reasons for the loss of effectiveness and ways of overcoming it. Specifically, the metabolic interventions deplete acetate and likely reduce the conversion of oxaloacetate to aspartate, thereby inhibiting the mammalian target of rapamycin (mTOR) and upregulating autophagy. Synthesis of glutathione may provide a high‐capacity sink for amine groups, facilitating autophagy, and prevent buildup of alpha‐ketoglutarate, supporting stem cell maintenance. Metabolic interventions also prevent the accumulation of succinate, thereby slowing DNA hypermethylation, facilitating the repair of DNA double‐strand breaks, reducing inflammatory and hypoxic signaling, and lowering reliance on glycolysis. In part through these mechanisms, metabolic interventions may decelerate aging, extending lifespan. Conversely, with overnutrition or oxidative stress, these processes function in reverse, accelerating aging and impairing longevity. Progressive damage to aconitase, inhibition of succinate dehydrogenase, and downregulation of hypoxia‐inducible factor‐1α, and phosphoenolpyruvate carboxykinase (PEPCK) emerge as potentially modifiable reasons for the loss of effectiveness of metabolic interventions.
... When GLC is impaired, this leads to decreased GSH biosynthesis, reduced cellular antioxidant capacity and the induction of oxidative stress. GSH concentration and γ-GC ligase activity in the central nervous system decline with age in association with increased oxidative stress (24) . ...
... Improvement was seen in five of the six mood parameters measured: anger, confusion, depression, fatigue, and tension; only vigour was unchanged. All the parameters in the untreated patients remained unchanged (24) . ...
... In the progression of oxidative stress during aging, an important role is played by the suppression of the antioxidant defense system. This is evidenced by a signifi cant decrease in the content and synthesis of glutathione in the tissues of the cardiovascular system [4][5][6]. The levels of glutathione correlate positively with the endothelial-depended vasodilatation, which is greatly depressed with aging and hypertensive patients [7][8][9]. ...
... Although the values of prothrombin index before and after therapy were within the range of reference values, this suggests the possibility of garlic usage as a mild antithrombotic agent in patients with hypertension and hypercholesterolemia. Our data correlate with those obtained earlier in hypertensive patients of younger age (20-39 years old) who showed H 2 S content in blood plasma of healthy donors(1), hypertensive patients at the initial examination (2), hypertensive patients after 10 days of standard treatment (3), hypertensive patients after 10 days of standard treatment and 14 days of garlic supplement consumption (4), hypertensive patients after 10 days of standard treatment and 30 days of garlic supplement consumption(5). The comparison between healthy donors and hypertensive patients was made by unpaired t test. The comparison in group of hypertensive patients was made by paired t test. ...
... Supplementation with GSH is generally ineffective at increasing cellular glutathione levels because most human cells cannot uptake extracellular GSH due to the lack of the membrane-bound ectoenzyme γ-glutamyltranspeptidase (γ-GT) or due to an unfavorable concentration gradient across the cell membrane 10,16 . In contrast, γ-EC can be readily absorbed by many cell types without thermodynamic limitations on transmembrane transport 19,24,48 . With cytosolic concentrations of γ-EC around 7 μM 49 , any passive influx of exogenous γ-EC, unlike GSH, would be directed into the cell 50 . ...
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Durian (Durio zibethinus L.) fruit pulp is a rich source of γ-glutamylcysteine (γ-EC), a direct precursor to the antioxidant glutathione (GSH). This study elucidated the in vitro neuroprotective potential of unripe durian fruit pulp extract (UDE) against H2O2-induced neurotoxicity in SH-SY5Y cells and neuroinflammation in lipopolysaccharide (LPS)-stimulated BV-2 cells. Treatments with γ-EC, GSH standards, or UDE exhibited no cytotoxicity in SH-SY5Y and BV-2 cells, except at high concentrations. A 4-h pretreatment with 100 µM γ-EC or UDE containing 100 µM γ-EC significantly increased SH-SY5Y cell viability post H2O2 induction. Moreover, a similar pretreatment reduced LPS-stimulated production of proinflammatory cytokines in BV-2 cells. The neuroprotective effect of UDE is primarily attributed to γ-EC provision and the promotion of GSH synthesis, which in turn elevates intracellular GSH levels and reduces proinflammatory cytokines. This study identifies γ-EC in UDE as a potential neuroprotective biomarker boosting intracellular GSH levels, providing insights into UDE's therapeutic potential.
... GSTA4 is particularly interesting as it detoxifies 4-hydroxynonenal, a product of arachidonic acid peroxidation and a biomarker of oxidative tissue damage [66]. Similarly to the case of glucuronidation, activity of GST enzymes was supported by upregulation of both the catalytic and regulatory subunits of gamma-glutamate-cysteine ligase, a rate-limiting enzyme in biosynthesis of GSH, and of glutathione synthetase [67]. Furthermore, the most upregulated protein in liver of I278T mice was asparagine synthetase (ASNS), which catalyzes conversion of aspartate and glutamine into asparagine and glutamate [68], likely to provide glutamate for GSH biosynthesis. ...
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Background Cystathionine β-synthase (CBS)-deficient homocystinuria (HCU) is an inherited disorder of sulfur amino acid metabolism with varying severity and organ complications, and a limited knowledge about underlying pathophysiological processes. Here we aimed at getting an in-depth insight into disease mechanisms using a transgenic mouse model of HCU (I278T). Methods We assessed metabolic, proteomic and sphingolipidomic changes, and mitochondrial function in tissues and body fluids of I278T mice and WT controls. Furthermore, we evaluated the efficacy of methionine-restricted diet (MRD) in I278T mice. Results In WT mice, we observed a distinct tissue/body fluid compartmentalization of metabolites with up to six-orders of magnitude differences in concentrations among various organs. The I278T mice exhibited the anticipated metabolic imbalance with signs of an increased production of hydrogen sulfide and disturbed persulfidation of free aminothiols. HCU resulted in a significant dysregulation of liver proteome affecting biological oxidations, conjugation of compounds, and metabolism of amino acids, vitamins, cofactors and lipids. Liver sphingolipidomics indicated upregulation of the pro-proliferative sphingosine-1-phosphate signaling pathway. Liver mitochondrial function of HCU mice did not seem to be impaired compared to controls. MRD in I278T mice improved metabolic balance in all tissues and substantially reduced dysregulation of liver proteome. Conclusion The study highlights distinct tissue compartmentalization of sulfur-related metabolites in normal mice, extensive metabolome, proteome and sphingolipidome disruptions in I278T mice, and the efficacy of MRD to alleviate some of the HCU-related biochemical abnormalities.
... This may reflect the fact that all of the animals in the cross-sectional study were healthy and offered appropriate amounts of complete and balanced diets. As the plasma levels of GLY and CYS were within normal range for both age groups, the lower levels of glutathione in the senior cats may be due to age related changes in the activity of enzymes involved in glutathione synthesis (17) . Reports in humans suggest that older individuals have lower intracellular levels of GLY and CYS due to slower body protein turnover or decreased de novo synthesis (21,47) . ...
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Intracellular levels of glutathione, the major mammalian antioxidant, are reported to decline with age in several species. To understand whether ageing affects circulating glutathione levels in cats, blood was sampled from two age groups, < 3 years and > 9 years. Further, to determine whether dietary supplementation with glutathione precursor glycine (GLY) affects glutathione concentrations in senior cats (> 9 years), a series of free GLY inclusion level dry diets were fed. Subsequently, a 16-week GLY feeding study was conducted in senior cats (age), measuring glutathione, and markers of oxidative stress. Whole blood (WB) and red blood cell (RBC) total, oxidised and reduced glutathione levels were significantly decreased in senior cats, compared to their younger counterparts (P ≤ 0.02). The inclusion level study identified 1.5% free GLY for the subsequent dry diet feeding study. Significant increases in RBC total and reduced glutathione were observed between senior cats fed supplemented and control diets at 4 weeks (P £ 0.03; maximum difference of 1.23 mM). Oxidative stress markers were also significantly different between groups at 8 (P = 0.004; difference of 0.68 nG/mL in 8-OHdG) and 12 weeks (P ≤ 0.049; maximum difference of 0.62 nG/mG Cr in F2-isoprostane PGF2α). Senior cats have lower circulating glutathione levels compared to younger cats. Feeding senior cats a complete and balanced dry diet supplemented with 1.5% free GLY for 12 weeks elevated initial RBC glutathione, and altered markers of oxidative stress. Dietary supplementation with free GLY provides a potential opportunity to restore age-associated reduction in glutathione in cats.
... The interconversion of the 2 forms maintains the redox balance in cells [93]. The abnormal metabolism of cysteine leads to the inhibition of GSH synthesis, causing disrupted intracellular oxidative homeostasis and tumor cell ferroptosis [94,95]. ...
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Cellular metabolism is the fundamental process by which cells maintain growth and self‐renewal. It produces energy, furnishes raw materials, and intermediates for biomolecule synthesis, and modulates enzyme activity to sustain normal cellular functions. Cellular metabolism is the foundation of cellular life processes and plays a regulatory role in various biological functions, including programmed cell death. Ferroptosis is a recently discovered form of iron‐dependent programmed cell death. The inhibition of ferroptosis plays a crucial role in tumorigenesis and tumor progression. However, the role of cellular metabolism, particularly glucose and amino acid metabolism, in cancer ferroptosis is not well understood. Here, we reviewed glucose, lipid, amino acid, iron and selenium metabolism involvement in cancer cell ferroptosis to elucidate the impact of different metabolic pathways on this process. Additionally, we provided a detailed overview of agents used to induce cancer ferroptosis. We explained that the metabolism of tumor cells plays a crucial role in maintaining intracellular redox homeostasis and that disrupting the normal metabolic processes in these cells renders them more susceptible to iron‐induced cell death, resulting in enhanced tumor cell killing. The combination of ferroptosis inducers and cellular metabolism inhibitors may be a novel approach to future cancer therapy and an important strategy to advance the development of treatments.
... In our study, group I had significantly lower GSH concentration compared with groups II group III; the effect size of physical-activity effect on GSH turned out to be considerable (η 2 H ≥ 0.14). These results are aligned with the findings of other researchers, who found a relationship between the concentration of GSH and the level of oxidative stress and age [36]. ...
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Background Given a lack of studies precisely indicating how many steps elderly people should take daily for their antioxidant defence, bone metabolism, and cognitive abilities to improve, our study set out to compare the selected antioxidant, prooxidant, bone turnover, and BDNF indicators between elderly women differing in physical activity (PA) measured by the daily number of steps. Methods The PA levels of 62 women aged 72.1 ± 5.4 years were assessed based on their daily number of steps and then were used to allocate the participants to three groups: group I (n = 18; <5,000 steps a day); group II (n = 22; from 5,000 to 9,999 steps a day); and group III (n = 22; ≥10,000 steps a day). Blood samples were collected from the participants in early morning hours and subjected to biochemical analysis for prooxidant-antioxidant balance indicators (SOD, CAT, GPx, GR, GSH, UA, MDA and TOS/TOC), bone metabolism indicators (Ca, 25-OH vitamin D, osteocalcin, CTX-I, and PTH), and BDNF levels. Results The groups were not statistically significantly different in the activity of SOD, CAT, GPx, and GR, but their concentrations of GSH (H = 22.10, p < 0.001) and UA (H = 12.20, p = 0.002) proved to be significantly associated with the groups’ daily PA. The between-group differences in the concentrations of MDA and TOS/TOC were not significant, with both these indicators tending to take higher values in group I than in groups II and III. Significant differences between the groups were established for the concentrations of 25-OH vitamin D (H = 24.21, p < 0.001), osteocalcin (H = 7.88, p = 0.019), CTX-I (H = 12.91, p = 0.002), and BDNF (H = 14.47, p = 0.001), but not for Ca and PTH. Conclusions Significantly higher concentrations of GSH, slightly lower oxidative stress indicators, significantly higher BDNF levels, and moderately better bone turnover indicators and resorption markers in the group taking more than 5,000 steps a day suggest that this level of PA can promote successful aging. More research is, however, needed to confirm this finding.
... MSC also has the ability to improve cognitive deficits due to Alzheimer's disease (39). An important indicator of cellular senescence evaluation is the decrease of glutathione content (40), which has the role of scavenging free radicals and maintains intracellular oxidative homeostasis (41), and its content is regulated by the intermediate dipeptide gamma-glutamylcysteine (γ-GC) (42). Some scholars believe that GSH cannot be synthesized ab initio in mitochondria and it is not the primary compound that exerts antioxidant homeostasis. ...
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Sanghuangporus vaninii is a profitable traditional and medicinal edible fungus with uncommon therapeutic properties and medicinal value. The accumulation of active ingredients in this fungus that is used in traditional Chinese medicine is affected by its years of growth, and their pharmacological activities are also affected. However, the effects of age on the medicinal value of fruiting bodies of S. vaninii cultivated on cut log substrate remain unclear. In this study, an untargeted liquid chromatography mass spectrometry (LC-MS)-based metabolomics approach was performed to characterize the profiles of metabolites from 1-, 2- and 3-year-old fruiting bodies of S. vaninii. A total of, 156 differentially accumulated metabolites (DAMs) were screened based on the criterion of a variable importance projection greater than 1.0 and p < 0.01, including 75% up regulated and 25% down regulated. The results of enrichment of metabolic pathways showed that the metabolites involved the biosynthesis of plant secondary metabolites, biosynthesis of amino acids, central carbon metabolism in cancer, steroid hormone biosynthesis, linoleic acid metabolism, prolactin signaling pathway, and arginine biosynthesis, and so on. The biosynthesis of plant secondary metabolites pathway was significantly activated. Five metabolites were significantly elevated within the growth of fruiting bodies, including 15-keto-prostaglandin F2a, (4S, 5R)-4,5,6-trihydroxy-2-iminohexanoate, adenylsuccinic acid, piplartine, and chenodeoxycholic acid. 15-keto-prostaglandin F2a is related to the pathway of arachidonic acid metabolism and was significantly increased up to 1,320- and 535-fold in the 2- and 3-year-old fruiting bodies, respectively, compared with those in the 1-year-old group. The presence of these bioactive natural products in S. vaninii is consistent with the traditional use of Sanghuang, which prompted an exploration of its use as a source of natural prostaglandin in the form of foods and nutraceuticals. These findings may provide insight into the functional components of S. vaninii to develop therapeutic strategies.
... Our findings reveal PNS provision up-regulated GCLc gene expression. GCLc is the rate limiting enzyme in GSH synthesis that directly repress ROS [46,47]. Interestingly, the down-regulation of miR-139-5p and miR-24-3p under PNS exposure both positively affect GCLc expression as GCLc targets is a target of both miRNAs (Fig. 9). ...
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Background: Herbal medicine Sanqi (SQ), the dried root or stem of Panax notoginseng (PNS), has been reported to have anti-diabetic and anti-obesity effects and is usually administered as a decoction for Chinese medicine. Alternative to utilizing PNS pure compound for treatment, we are motivated to propose an unconventional scheme to investigate the functions of PNS mixture. However, studies providing a detailed overview of the transcriptomics-based signaling network in response to PNS are seldom available. Methods: To explore the reasoning of PNS in treating metabolic disorders such as insulin resistance, we implemented a systems biology-based approach with RNA sequencing (RNA-seq) and miRNA sequencing data to elucidate key pathways, genes and miRNAs involved. Results: Functional enrichment analysis revealed PNS up-regulating oxidative stress-related pathways and down-regulating insulin and fatty acid metabolism. Superoxide dismutase 1 (SOD1), peroxiredoxin 1 (PRDX1), heme oxygenase-1 (Hmox1) and glutamate cysteine ligase (GCLc) mRNA and protein levels, as well as related miRNA levels, were measured in PNS treated rat pancreatic β cells (INS-1). PNS treatment up-regulated Hmox1, SOD1 and GCLc expression while down-regulating miR-24-3p and miR-139-5p to suppress oxidative stress. Furthermore, we verified the novel interactions between miR-139-5p and miR-24-3p with GCLc and SOD1. Conclusion: This work has demonstrated the mechanism of how PNS regulates cellular molecules in metabolic disorders. Therefore, combining omics data with a systems biology strategy could be a practical means to explore the potential function and molecular mechanisms of Chinese herbal medicine in the treatment of metabolic disorders.
... The pharmacological benefit of supplementation of γ-Glu-Cys has recently been proposed under pathological conditions, such as strokes [237], inflammation [238][239][240][241], ALS [242], and ischemia/reperfusion injury [243]. The pharmacological action of γ-Glu-Cys appears to be largely attributable to an increased production of GSH [244][245][246]. Although the direct donation of an electron from γ-Glu-Cys to GPX1 has been also demonstrated [247], the presence of GSS mostly converts γ-Glu-Cys to GSH. ...
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γ-Glutamyl moiety that is attached to the cysteine (Cys) residue in glutathione (GSH) protects it from peptidase-mediated degradation. The sulfhydryl group of the Cys residue represents most of the functions of GSH, which include electron donation to peroxidases, protection of reactive sulfhydryl in proteins via glutaredoxin, and glutathione conjugation of xenobiotics, whereas Cys-derived sulfur is also a pivotal component of some redox-responsive molecules. The amount of Cys that is available tends to restrict the capacity of GSH synthesis. In in vitro systems, cystine is the major form in the extracellular milieu, and a specific cystine transporter, xCT, is essential for survival in most lines of cells and in many primary cultivated cells as well. A reduction in the supply of Cys causes GPX4 to be inhibited due to insufficient GSH synthesis, which leads to iron-dependent necrotic cell death, ferroptosis. Cells generally cannot take up GSH without the removal of γ-glutamyl moiety by γ-glutamyl transferase (GGT) on the cell surface. Meanwhile, the Cys–GSH axis is essentially common to certain types of cells; primarily, neuronal cells that contain a unique metabolic system for intercellular communication concerning γ-glutamyl peptides. After a general description of metabolic processes concerning the Cys–GSH axis, we provide an overview and discuss the significance of GSH-related compounds in the nervous system.
... Nevertheless, uptake of extracellular GSH is difficult because of lacking of a membrane-bound ectoenzyme γ-glutamyltranspeptidase (γ-GT) and an unfavorable concentration gradient [11]. γ-GC acts as an immediate precursor of GSH which not only is limited to transmembrane transport, but also exhibits the same antioxidant properties [12,13]. Recently, numerous studies have shown some new functions of γ-GC except for anti-oxidative including anti-inflammatory and anti-aging effects [14,15]. ...
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Objective: To explore the molecular mechanism of γ-glutamylcysteine (γ-GC) in response to inflammation in vivo and in vitro on regulating the polarization of macrophages. Methods: The expressions of gene or protein were assessed by qPCR and Western blot assays, respectively. Cell viability was investigated by CCK-8 assay. Eight-week-old male BALB/c mice were established to examine the therapeutic effects of γ-GC in vivo. The release of TNF-α and IL-4 was determined by ELISA assay. Macrophages polarization was identified by flow cytometry assay. Results: Our data showed that γ-GC treatment significantly improved the survival, weight loss, and colon tissue damage of IBD mice. Furthermore, we established M1- and M2-polarized macrophages, respectively, and our findings provided evidence that γ-GC switched M1/M2-polarized macrophages through activating AMPK/SIRT1 axis and inhibiting inflammation-related signaling pathway. Conclusion: Collectively, both in vivo and in vitro experiments suggested that γ-GC has the potential to become a promising novel therapeutic dipeptide for the treatment of IBD, which provide new ideas for the treatment of inflammatory diseases in the future.
... Among these amino acids, cysteine is the rate-limiting substrate for enzyme activity for GSH synthesis, because the other precursor amino acids, glutamate and glycine, have much higher intracellular concentrations than cysteine [90,94]. In agreement with these findings, it has been shown that GSH concentrations can only be elevated by increasing cytosolic cysteine availability [95]. The biosynthesis of GSH occurs via a two-step ATP-requiring enzymatic process that is catalyzed by glutamate-cysteine ligase (GCL; also known as γ-glutamylcysteine synthetase) and glutathione synthetase (GSS) [96,97]. ...
Article
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Multiple system atrophy (MSA) is a rare neurodegenerative disease characterized by parkinsonism, cerebellar impairment, and autonomic failure. Although the causes of MSA onset and progression remain uncertain, its pathogenesis may involve oxidative stress via the generation of excess reactive oxygen species and/or destruction of the antioxidant system. One of the most powerful antioxidants is glutathione, which plays essential roles as an antioxidant enzyme cofactor, cysteine-storage molecule, major redox buffer, and neuromodulator, in addition to being a key antioxidant in the central nervous system. Glutathione levels are known to be reduced in neurodegenerative diseases. In addition, genes regulating redox states have been shown to be post-transcriptionally modified by microRNA (miRNA), one of the most important types of non-coding RNA. miRNAs have been reported to be dysregulated in several diseases, including MSA. In this review, we focused on the relation between glutathione deficiency, miRNA dysregulation and oxidative stress and their close relation with MSA pathology.
... In addition, consuming pre-formed glutathione is an inefficient way to raise cellular levels of glutathione since published clinical studies demonstrate that it may take weeks or months to impact cellular levels when elevated levels are most needed acutely. [31][32][33][34][35][36][37][38][39][40][41][42][43] concept should be considered and tested using this approach in conjunction with measuring blood biomarkers that reflect individual and pathway proteins that correspond to inflammation and oxidative stress. ...
... 5) For many disorders, diseases, and aging, it is suggested that GSH depletion is associated with the onset of dysfunctional GCL regulation with insufficient γ-EC synthesis to supply GSH and maintain appropriate homeostasis. [6][7][8] However, exogenous GSH administration could not help alleviate those disorders and disease states, as the ability to ensure homeostatic regulation of GSH levels is decreased in those cases. 9) On the other hand, recent studies have revealed that the administration of γ-EC increases intracellular GSH levels in a human trial pilot study. ...
Article
Gamma-glutamylcysteine (γ-EC) is an intermediate generated in the de novo synthesis of glutathione (GSH). Recent studies have revealed that the administration of γ-EC shows neuroprotective effects against oxidative stress in age-related disorders and chronic diseases like Alzhiemer’s disease in model animals, which is not expected function in GSH. A phytochelatin synthase-like enzyme derived from Nostoc sp. (NsPCS) mediates γ-EC synthesis from GSH. To achieve low-cost and stable commercial level supply, the availability of immobilized NsPCS for γ-EC production was investigated in this study. Among the tested immobilization techniques, covalent binding to the cellulose carrier was most effective, and could convert GSH completely to γ-EC without decreasing the yield. The stable conversion of γ-EC from 100 mM GSH was achieved by both batch repeated and continuous reactions using the immobilized NsPCS on cellulose sheet and column shape monolith, respectively. The immobilization of NsPCS on those carriers is promising alternative technique for high-yielding and cost-effective production of γ-EC on its commercial applications. Fullsize Image
... γ-glutamylcysteine is the dipeptide and precursor of glutathione (Anderson & Meister, 1983). Unlike GSH, as a direct substrate for the production of GSH, supplemental γ-GC can be utilized by cells (Ferguson & Bridge, 2016). It is reported that oral administration of γ-GC could increase intracellular glutathione levels (Zarka & Bridge, 2017). ...
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Alcohol abuse is a major cause of alcoholic liver disease (ALD) and can result in fibrosis and cirrhosis. γ‐glutamylcysteine (γ‐GC) is a precursor of glutathione (GSH) with antioxidant and anti‐inflammatory properties. Our research aimed to explore the protective impact of γ‐GC on ALD and its potential mechanisms of efficiency in vitro and in vivo. L02 cells were pretreated with γ‐GC (20, 40, and 80 μM) for 2 h and exposed to ethanol for 24 h. Cell viability, apoptosis, oxidative stress, and inflammatory levels were measured. The expression of protein cleaved caspase‐3 and cleaved PARP and flow cytometry results indicated that γ‐GC decreases apoptosis on L02 cells after ethanol treatment. Moreover, γ‐GC also attenuated oxidative stress and mitochondrial damage in hepatocytes caused by ethanol via increasing cellular GSH, SOD activity, and mitochondrial membrane potential. In vivo experiments, γ‐GC effectively reduced the AST, ALT, and TG levels in mice. The inflammation of ALD was alleviated by γ‐GC both in vivo and in vitro. Additionally, histopathological examination demonstrated that γ‐GC treatment lessened lipid droplet formation and inflammatory damage. In conclusion, these results showed that γ‐GC has anti‐inflammatory and anti‐apoptotic effects on ALD because it could help hepatocytes maintain sufficient GSH levels to combat the excess reactive oxygen species (ROS) generated during ethanol metabolism. Practical applications Alcohol intake is the fifth highest risk factor for premature death and disability among all risk variables. However, few medicines are both safe and effective for the treatment of ALD. As a direct precursor of GSH, γ‐GC has a broad variety of potential antioxidant and anti‐inflammatory applications for the treatment of numerous medical conditions. In conclusion, these results showed that γ‐GC could protect cells from ALD via suppressing oxidative stress, alleviating inflammation, and preventing apoptosis.
... The enzyme GCL that catalyzes the first and rate-limiting step plays a decisive role in GSH synthesis. 76 Thus, selectively blocking the rate-limiting step of GSH synthesis using specific drugs is a potential method for reducing GSH content in cancer cells. While several effective inhibitors of GSH synthesis are now utilized, the most anticipated inhibitor has been L-buthionine sulfoximine (BSO), which is commercially available. ...
Article
Glutathione (GSH), the main redox buffer, has long been recognized as a pivotal modulator of tumor initiation, progression and metastasis. It is also implicated in the resistance of platinum-based chemotherapy and radiation therapy. Therefore, depleting intracellular GSH was considered a potent solution to combating cancer. However, reducing GSH within cancer cells alone always failed to yield desirable therapeutic effects. In this regard, the convergence of GSH-scavenging agents with therapeutic drugs has thus been pursued in clinical practice. Unfortunately, the therapeutic outcomes are still unsatisfactory due to untargeted drug delivery. Advanced nanomedicine of synergistic GSH depletion and cancer treatment has attracted tremendous interest because they promise to deliver superior therapeutic benefits while alleviating life-threatening side effects. In the past five years, the authors and others have demonstrated that numerous nanomedicines, by simultaneously delivering GSH-depleting agents and therapeutic components, boost not only traditional chemotherapy and radiotherapy but also multifarious emerging treatment modalities, including photodynamic therapy, sonodynamic therapy, chemodynamic therapy, ferroptosis, and immunotherapy, to name a few, and achieved decent treatment outcomes in a large number of rodent tumor models. In this review, we summarize the most recent progress in engineering nanomedicine for GSH depletion-enhanced cancer therapies. Biosynthesis of GSH and various types of GSH-consuming strategies will be briefly introduced. The challenges and perspectives of leveraging nanomedicine for GSH consumption-augmented cancer therapies will be discussed at the end.
... A possible mechanism may be medicated through the induction of Nrf2 and its downtown genes by BDC to suppress oxidative stress, just like curcumin. g-GCL is a key rate-limiting enzyme in de novo synthesis cascade of GSH (Ferguson and Bridge, 2016). g-GCL consists of a catalytic subunit g-GCLc, and a modulatory subunit g-GCLm. ...
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This study was conducted to investigate the beneficial effects of bisdemethoxycurcumin (BDC) on growth performance, glutathione (GSH) redox potential, antioxidant enzyme defense, and gene expression in lipopolysaccharide (LPS)-challenged broilers. A total of 320, male, 1-day-old broilers were randomly assigned to 4 treatment groups including 8 replicates with 10 birds per cage in a 2 × 2 factorial arrangement: BDC supplementation (a basal diet with 0 or 150 mg/kg BDC) and LPS challenge (intraperitoneal injection of 1 mg/kg body weight saline or LPS at 16, 18, and 20 d of age). Results showed that dietary BDC supplementation prevented the LPS-induced decrease in ADG of broilers (P
... For example, GCLC was generally not found at the cell periphery. As the serum concentration of its product, cGC, is higher at 7 μM than its cytosolic level (42), cGC enters cells to participate in GSH synthesis (4,21,42,53,63). Moreover, cell-surface c-glutamyl transpeptidase (GGT), in the presence of a c-glutamyl donor such as glutamine, synthesizes cGC ( Fig. 1) (53,63). ...
Article
Some patients treated for ductal carcinoma in situ (DCIS) of the breast will experience cancer recurrences, whereas other patients will not. Unfortunately, current techniques cannot identify which pre-invasive lesions will lead to recurrent cancer. Because the mechanism of cancer recurrence is unknown, it is difficult to design a test that detects its activity. We propose that certain pentose phosphate pathway enzymes, glutathione synthesis enzymes, and RhoA cluster at the epithelial cell periphery during cancer recurrences. Enzyme clustering enhances metabolic flux. Using fluorescence microscopy we show that phosphophorylated glucose transporter type-1, transketolase-like protein-1, glutathione synthetase, GTP-loaded RhoA, and RhoA accumulate at the epithelial cell periphery in biopsies of women who will suffer recurrences, but not in samples from women who will not experience recurrences as judged using 2x2 contingency tables. A machine study of individual cribriform, papillary, micropapillary, and comedo forms of DCIS demonstrated 97% precision and 95% recall in the detection of samples from women will not experience a recurrence and a 90% precision and 95% recall in the detection of lesions that become recurrent. A holdout study of these patients showed 73% true negatives, 18% true positives, 4% false positives, and 4% false negatives at a 50% threshold. This work suggests mechanistic features of recurrences that may contribute to a new clinical test distinguishing high from low recurrence risk DCIS patients.
... A decreased concentration of gluta thione has been reported in erythrocytes and lymphocytes of healthy older people, and potentially could also occur in hepatocytes. 41 This decrease in glutathione concen tration suggests that older patients could be more prone to elevated oxidative stress than younger patients, which in conjunction with drug treatment might initiate DILI development. As cellular stress reaches a critical hepatocyte injury threshold, cell death signalling pathways are induced (eg, activation of the mitogen-activated protein kinase JNK), eventually causing apoptosis and necrosis through mitochondrial permea bility transition and outer membrane permea bilisation. ...
Article
Drug-induced liver injury (DILI) is a rare, unpredictable, and potentially serious adverse reaction. It is induced by many drugs, herbs, and dietary supplements and represents a diagnostic challenge to clinicians. Older people (aged 65 years and older) are often polymedicated, and their declining physiological function affects drug pharmacokinetics. There is no consistent evidence that age is a general risk factor for DILI; however, age might be a risk factor with specific medications, with antimicrobials and cardiovascular drugs being the most likely medications to cause DILI in older people. Ageing influences DILI phenotypes, making cholestatic damage and chronic DILI more likely. In older people with DILI, comorbidities act as confounding causes and account for higher mortality unrelated to the liver. There are no specific therapies for DILI and supportive measures are still the mainstay of management. This Review highlights current advances and gaps in DILI epidemiology, mechanisms, and diagnosis that are pertinent to older individuals.
Article
Glutathione (GSH) is the most important low-molecular intracellular antioxidant. In addition to its antioxidant properties, it ensures many other biological functions, including the elimination of xenobiotics. Glutathione and glutathione disulfide (GSSG) levels can be used as biomarkers of oxidative stress associated with many physiological and pathological conditions at which intracellular GSH levels decrease. Thus enhancing intracellular GSH levels has a wide therapeutic potential. A promising candidate for effective elevation of intracellular GSH levels by simple supplementation is gamma-glutamylcysteine. The present review is focused on the description of available methods for characterization of GSH metabolism. Especially, methods utilizing liquid chromatography and liquid chromatography coupled with mass spectrometry for the determination of GSH, GSSG, and other thiols and related compounds are reviewed and critically discussed.
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The cellular defense system against exogenous substances makes therapeutics inefficient as intracellular glutathione (GSH) exhibits an astounding antioxidant activity in scavenging reactive oxygen species (ROS) or reactive nitrogen species (RNS) or other free radicals produced by the therapeutics. In the cancer cell microenvironment, the intracellular GSH level becomes exceptionally high to fight against oxidative stress created by the production of ROS/RNS or any free radicals, which are the byproducts of intracellular redox reactions or cellular respiration processes. Thus, in order to maintain redox homeostasis for survival of cancer cells and their rapid proliferation, the GSH level starts to escalate. In this circumstance, the administration of anticancer therapeutics is in vain, as the elevated GSH level reduces their potential by reduction or by scavenging the ROS/RNS they produce. Therefore, in order to augment the therapeutic potential of anticancer agents against elevated GSH condition, the GSH level must be depleted by hook or by crook. Hence, this Review aims to compile precisely the role of GSH in cancer cells, the importance of its depletion for cancer therapy and examples of anticancer activity of a few selected metal complexes which are able to trigger cancer cell death by depleting the GSH level.
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The dipeptide γ‐glutamylcysteine (γ‐GC), the first intermediate of glutathione (GSH) synthesis, is considered as a promising drug to reduce or prevent plethora of age‐related disorders such as Alzheimer and Parkinson diseases. The unusual γ‐linkage between the two constitutive amino acids, namely cysteine and glutamate, renders its chemical synthesis particularly challenging. Herein, we report on the metabolic engineering of the non‐conventional yeast Yarrowia lipolytica for efficient γ‐GC synthesis. The yeast was first converted into a γ‐GC producer by disruption of gene GSH2 encoding GSH synthase and by constitutive expression of GSH1 encoding glutamylcysteine ligase. Subsequently genes involved in cysteine and glutamate anabolism, namely MET4 , CYSE , CYSF , and GDH1 were overexpressed with the aim to increase their intracellular availability. With such a strategy, a γ‐GC titer of 464 nmol mg ⁻¹ protein (93 mg gDCW ⁻¹ ) was obtained within 24 h of cell growth.
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L-γ-Glutamyl-L-cysteinyl-glycine is commonly referred to as glutathione (GSH); this ubiquitous thiol plays essential roles in animal life. Conjugation and electron donation to enzymes such as glutathione peroxidase (GPX) are prominent functions of GSH. Cellular glutathione balance is robustly maintained via regulated synthesis, which is catalyzed via the coordination of γ-glutamyl-cysteine synthetase (γ-GCS) and glutathione synthetase, as well as by reductive recycling by glutathione reductase. A prevailing short supply of L-cysteine (Cys) tends to limit glutathione synthesis, which leads to the production of various other γ-glutamyl peptides due to the unique enzymatic properties of γ-GCS. Extracellular degradation of glutathione by γ-glutamyltransferase (GGT) is a dominant source of Cys for some cells. GGT catalyzes the hydrolytic removal of the γ-glutamyl group of glutathione or transfers it to amino acids or to dipeptides outside cells. Such processes depend on an abundance of acceptor substrates. However, the physiological roles of extracellularly preserved γ-glutamyl peptides have long been unclear. The identification of γ-glutamyl peptides, such as glutathione, as allosteric modulators of calcium-sensing receptors (CaSRs) could provide insights into the significance of the preservation of γ-glutamyl peptides. It is conceivable that GGT could generate a new class of intercellular messaging molecules in response to extracellular microenvironments.
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The present study aimed to evaluate the anti-aging effect of the leaves of Scutellaria baicalensis Georgi (LSBG) and investigate its mechanisms. For this purpose, SD rats were received D-galactose (D-gal) subcutaneously (0.3 g/kg) and LSBG intragastrically (0.4 g/kg or 0.8 g/kg) for 7 weeks. Behavior tests were conducted to evaluate the cognitive function of all rats. Results showed that memory impairment was reversed by LSBG. Then, metabolomics of the cortex and hippocampus were used to investigate the potential mechanisms. 21 metabolites in the cortex and 22 metabolites in the hippocampus of aging rats were altered, respectively. Additionally, results showed that the content of key metabolites and activities of enzymes in glutamate metabolism and its downstream metabolism (glutathione metabolism) could be regulated by the LSBG. Additionally, proteins in the Nrf2 signaling pathway were analyzed by western blot. And the protein expression levels of Nrf2, GCLC, HO-1, NQO-1 were significantly regulated by the LSBG in the cortex and hippocampus. Above all, the anti-aging effects of the LSBG were involved in regulating the glutamate metabolism and Nrf2 signaling pathway.
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Background Acute lung injury (ALI) represents a serious heterogenous pulmonary disorder with high mortality. Bone marrow mesenchymal stem cells (BMSCs) have a good therapeutic effect on ALI, but their survival rate in vivo is not high. GCLc has all the activities of Glutamate cysteine ligase (GCL) and can reduce reactive oxygen species, antioxidant stress response and improve cell survival. Therefore, in our study, overexpressing GCLc BMSCs were constructed by lentiviral transduction and intratracheally transplanted into ALI mice to evaluate their therapeutic effects, and we explored the mechanism of anti-apoptosis of GCLc in BMSCs. Methods Overexpressing GCLc hBMSCs were constructed using lentiviral vectors. The cell viability of MSCs was detected by CCK-8 assay. GSH, MDA, SOD and ROS were detected by the manufacturer's kit. Western blot and RT-qPCR were used to detect the expression of GCLc, bax, bcl2, cleaved-caspase 3, caspase 3, cleaved-caspase 9, caspase 9 and Foxo1 in BMSCs stimulated by H2O2. Apoptosis of BMSCs was analyzed by flow cytometry, JC-1 and TUNEL method. Confocal microscopy was to observe the nuclear extracellular migration of Foxo1. We then examined the expression levels of the pathway proteins by Western blot. In ALI animal model, we evaluated the therapeutic effect of the overexpressing GCLc BMSCs by H&E staining, in vitro imaging, wet/dry weight ratio of lung tissue, and extraction of bronchoalveolar lavage fluid from mice to analyze protein concentrations, neutrophil, leukocyte and macrophage counts and ELISA for inflammatory factors. Results We demonstrated that overexpression of GCLc reduced MDA and ROS and increased GSH and SOD, while GCLc reduced the expression of pro-apoptotic proteins (bax, cleaved-caspase 3, caspase 3, cleaved-caspase 9, caspase 9) and elevated the expression of anti-apoptotic proteins (bcl-2) in BMSCs. We verified that it acts through the PI3K/AKT/Foxo1 pathway. In ALI vivo, overexpression of GCLc BMSCs had a longer retention time in the lung compared to vector BMSC and improved pulmonary edema, decreased alveolar protein concentration and reduced TNF-α, IL-1β, IL-6 levels and increased IL-10 levels in the lung. Conclusions These results show that GCLc overexpressing BMSCs with anti-apoptotic effects significantly improve acute lung injury.
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Aging is a natural process accompanied by inflammation and oxidative stress and is closely associated with age-related diseases. As a direct precursor of glutathione, γ-glutamylcysteine (γ-GC) possesses antioxidant and anti-inflammatory properties; however, whether γ-GC plays an important role in anti-aging remains unknown. Here, we investigated the protective effects and mechanisms of γ-GC in D-galactose (D-gal)-induced senescence in PC12 cells and aging mice. Our results showed that γ-GC treatment significantly reduced the percentage of senescence-associated-β-galactosidase (SA-β-Gal)-positive cells and inhibited D-gal-induced cell cycle arrest in PC12 cells. The results of Nissl and hematoxylin and eosin (H&E) staining in mouse brain showed that γ-GC treatment markedly reversed the damage in the hippocampus of D-gal-induced aging mice. Moreover, γ-GC increased the phosphorylation of AMP-activated protein kinase (AMPK) to promote the nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) while inhibiting the nuclear translocation of deleted in breast cancer 1 (DBC1), which leads to the activation of sirtuin 1 (SIRT1) and deacetylation of p53 in the nucleus. Therefore, γ-GC may be a potential therapeutic candidate compound for the prevention and treatment of age-related diseases.
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Alzheimer's disease (AD) is the most prevalent neurogenerative disease, characterized by progressive memory loss and cognitive deficits. Intracellular neurofibrillary tangles (NFTs) and amyloid-β (Aβ)-formed neuritic plaques are major pathological features of AD. Aβ evokes activation of microglia to release inflammatory mediators and ROS to induce neurotoxicity, leading to neurodegeneration. γ-Glutamylcysteine (γ-GC), an intermediate dipeptide of the GSH-synthesis pathway with anti-inflammatory and anti-oxidative properties, represents a relatively unexplored option for AD treatment. In the present study, we investigated the anti-inflammatory effect of γ-GC on Aβ oligomer (AβO)-induced neuroinflammation and the associated molecular mechanism in microglia. The results showed that γ-GC reduced AβO-induced release of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and nitric oxide (NO), and the expression of inducible NO synthase (iNOS) and cyclooxygenase 2 (COX-2). γ-GC decreased ROS and MDA production and increased the GSH level, GSH/GSSG ratio, and SOD activity in AβO-treated microglia. Mechanistically, γ-GC inhibited activation of nuclear factor kappa B (NF-κB), and upregulated the nuclear receptor-related 1 (Nurr1) protein expression to suppress the transcriptional effect of NF-κB on the inflammatory genes. Besides, γ-GC suppressed the AβO-induced neuroinflammation in mice. These findings suggested that γ-GC might represent a potential therapeutic agent for anti-neuroinflammation.
Article
Background: Ultraviolet (UV) radiation-induced oxidative stress is the main cause of photodamage to the skin. Glutathione (GSH) serves important physiological functions, including scavenging oxygen free radicals and maintaining intracellular redox balance. γ-glutamylcysteine (γ-GC), as an immediate precursor of GSH and harboring antioxidant and anti-inflammatory properties, represents an unexplored option for skin photodamage treatment. Purpose: The purpose of this study was to investigate whether γ-GC can reduce UVB-induced NIH-3T3 cell damage. Methods: The experimental groups were as follows: control, UVB radiation, UVB radiation after pretreatment with γ-GC. Cell counting kit-8 (CCK-8) assays were used to measure cell proliferation, flow cytometry, and immunoblotting to detect the apoptosis rate and apoptosis-associated proteins. The levels of Reactive Oxygen Species (ROS), Superoxide Dismutase (SOD) and GSH/GSSG (oxidized GSH) were measured to assess oxidative stress. Immunoblotting and immunofluorescence were used to detect DNA damage. The members of the MAPK signaling pathways were detected by immunoblotting. Results: UVB irradiation significantly reduced cell viability, destroyed the oxidative defense system. Pretreatment with γ-GC reduced UVB-induced cytotoxicity, restored the oxidation defense system, and inhibited activation of the MAPK pathway. It also reduced the apoptosis rate, downregulated the levels of cleaved caspase 3 and cleaved PARP. Furthermore, pretreatment with γ-GC reduced the accumulation of γH2AX after UVB radiation exposure, indicating that γ-GC could protect cells from DNA damage. Conclusion: γ-GC protected NIH-3T3 from damage caused by UVB irradiation. The photoprotective effect of γ-GC is mediated via strengthening the endogenous antioxidant defense system, which prevents DNA damage and inhibits the activation of the MAPK pathway.
Article
Gamma-glutamyl-cysteine (γ-EC) is a precursor of glutathione (GSH) biosynthesis. We investigated whether it functions as a substrate for three intracellular and one extracellular GSH metabolic enzymes, which mediate the antioxidant defense function of GSH. Among them, glutathione peroxidase, glutathione S-transferase, and γ-glutamyl transferase (GGT) exhibited substrate specificity for γ-EC, whereas glutathione reductase did not. The specificities of γ-EC and its disulfide form to GGT were comparable to GSH and its oxidized form, GSSG, respectively. These result indicate that they can supply GSH constituent amino acids, glutamate, cysteine, and cystine through degradation by GGT. γ-EC may contribute valuable antioxidant defense properties as a food and cosmetic additive.
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Cadmium (Cd) is a highly toxic environmental pollutant, leading to the occurrence and development of multiple neurological diseases. γ-glutamylcysteine (γ-GC) is a dipeptide formed by the condensation of L-glutamic acid and L-cysteine, which has antioxidant, anti-inflammatory, and chelating properties. The purpose of this study is to investigate the effect of γ-GC on Cd-induced apoptosis in PC12 cells. PC12 cells were pretreated with or without γ-GC (2 mM or 4 mM) for 2 h and exposed to Cd (10 μM) for 12 h, and survival, apoptosis, and oxidative stress of PC12 cells were detected after different treatments. The results showed that γ-GC significantly inhibited cell viability reduction, apoptosis, and depolarization of mitochondrial transmembrane potential in Cd-treated PC12 cells, as indicated by CCK-8 assay, flow cytometry, TUNEL staining, and JC-1 detection. Western blot showed that γ-GC down-regulated the ratio of Bax/Bcl-2 and the protein levels of cytosolic cytopigment c, cleaved-caspase-9, cleaved-caspase-3, and cleaved-PARP. Mechanistically, γ-GC suppressed Cd-induced ROS production, MDA accumulation, and GSH depletion, and increased the activity of antioxidant enzymes. Cd-induced activation of MAPK and PI3K/CAT signaling pathways were inhibited by γ-GC treatment, while sustained phosphorylation of JNK, p38, or Akt reversed anti-apoptotic effects of γ-GC. These results suggested that γ-GC inhibited Cd-induced apoptosis in PC12 cells through decreasing oxidative stress and inhibiting the activation of MAPK and PI3K/Akt signaling pathways. γ-GC could be used as a potential protective agent against Cd neurotoxicity.
Article
Recently, combination therapy has proven to be an effective strategy for treating polygenic/multifactorial/complex disorder such as Parkinson's disease (PD). Here, we hypothesized that dual up-regulation of glutamate cysteine ligase (GCL) catalytic subunit (GCLc) and GCL modifier subunit (GCLm) via nuclear factor E2-related factor (Nrf2) contribute to the antioxidant effect of paeoniflorin (PF) synergistically with glycyrrhetinic acid (GA) (henceforth called PF/GA) in the context of MPP+/MPTP neurotoxicity. Expectedly, CompuSyn synergism/antagonism analysis showed that PF/GA exerts synergistic neuroprotection. Moreover, the antioxidant effect of PF was significantly enhanced by the combined administration of GA, although GA alone did not confer the effect. Mechanistically, PF triggered extracellular signal-regulated kinase (ERK1/2) phosphorylation, resulting in Nrf2 nuclear translocation from cytoplasmic pool via de novo synthesis in MPP+-challenged SH-SY5Y cells. Concomitantly, GA activates Akt which in turn induces nuclear accumulation of Nrf2. Especially, PF/GA up-regulated glutamate-cysteine ligase catalytic subunit (Gclc) and glutamate-cysteine ligase modifier subunit (Gclm) are formed via two separate pathways. Furthermore, these results were confirmed through pathway blockade assays using PD98059 (ERK1/2 inhibitor), LY294002 (phosphatidylinositol-3-kinase inhibitor), and shRNA-induced Nrf2 knockdown. Additionally, using a mouse MPTP-induced model of PD, we demonstrated that PF/GA synergistically ameliorates both motor deficits and oxidative stress in the ventral midbrain. In parallel, PF/GA also up-regulated both GCLc and GCLm expression at levels of transcription and translation. Conversely, antiparkinsonism and antioxidant effects of PF/GA were not observed in Nrf2-knockout MPTP-mice. Collectively, these results show that ERK1/2 and Akt activation contribute to the synergistic antioxidant effect of PF/GA. Hence, PF/GA regimen warrants further preclinical and possible clinical study for PD.
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With the development of society, physical activity has come to be an effective means by which people pursue good health to improve the quality of life. However, with the increase of intensity and the passage of time, exercise injury has become a hazard that can no longer be ignored. It is imperative to find effective ways to inhibit or reduce the negative effects of exercise. Mitochondria are important organelles involved in exercise and play an important role in exercise injury and prevention. Studies have found that exercise preconditioning and increased mitochondrial nutrition can effectively decrease mitochondrial damage after exercise. Against this background, some of the newest developments in this important field are reviewed here. The results discussed indicate that exercise preconditioning and supplement mitochondrial nutrition need to be increased to prevent exercise-related injuries.
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Health is usually defined as the absence of pathology. Here, we endeavor to define health as a compendium of organizational and dynamic features that maintain physiology. The biological causes or hallmarks of health include features of spatial compartmentalization (integrity of barriers and containment of local perturbations), maintenance of homeostasis over time (recycling and turnover, integration of circuitries, and rhythmic oscillations), and an array of adequate responses to stress (homeostatic resilience, hormetic regulation, and repair and regeneration). Disruption of any of these interlocked features is broadly pathogenic, causing an acute or progressive derailment of the system coupled to the loss of numerous stigmata of health.
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Glutathione (GSH) is a tripeptide that is readily synthesized intracellularly in humans and other mammals. More than a century of research suggests that GSH has numerous biological functions, including protection from the potential adverse events associated with reactive oxygen species (ROS) and related redox reactions that may induce oxidative stress, and that may be linked to innate detoxification processes. Normal tissue and plasma levels of GSH decline through the aging process and decrease during various disease states. While the health value of dietary GSH remains controversial, there is evidence that some metabolic intermediates, such as γ-glutamylcysteine (GGC) may function to preserve adequate GSH levels when the synthetic pathways decline in activity, and the innate antioxidant system is challenged. It is also important to recognize that among the thousands of protein-coding human genes and their respective polymorphisms, at least two genes (Gclc and Gclm) are directly involved with GSH synthesis via glutamate-cysteine ligase. This commentary examines the classic biochemistry, toxicology, safety, and clinical value of GSH and its intermediates that may be modulated by dietary supplementation.
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Introduction The accumulation of oxidative stress, neuroinflammation and abnormal aggregation of amyloid β-peptide (Aβ) have been shown to induce synaptic dysfunction and memory deficits in Alzheimer’s disease (AD). Cellular depletion of the major endogenous antioxidant Glutathione (GSH) has been linked to cognitive decline and the development of AD pathology. Supplementation with γ-glutamylcysteine (γ-GC), the immediate precursor and the limiting substrate for GSH biosynthesis, can transiently augment cellular GSH levels by bypassing the regulation of GSH homeostasis. Methods In the present study, we investigated the effect of dietary supplementation of γ-GC on oxidative stress and Aβ pathology in the brains of APP/PS1 mice. The APP/PS1 mice were fed γ-GC from 3 months of age with biomarkers of apoptosis and cell death, oxidative stress, neuroinflammation and Aβ load being assessed at 6 months of age. Results Our data showed that supplementation with γ-GC lowered the levels of brain lipid peroxidation, protein carbonyls and apoptosis, increased both total GSH and the glutathione/glutathione disulphide (GSH/GSSG) ratio and replenished ATP and the activities of the antioxidant enzymes (superoxide dismutase (SOD), catalase, glutamine synthetase and glutathione peroxidase (GPX)), the latter being a key regulator of ferroptosis. Brain Aβ load was lower and acetylcholinesterase (AChE) activity was markedly improved compared to APP/PS1 mice fed a standard chow diet. Alteration in brain cytokine levels and matrix metalloproteinase enzymes MMP-2 and MMP-9 suggested that γ-GC may lower inflammation and enhance Aβ plaque clearance in vivo. Spatial memory was also improved by γ-GC as determined using the Morris water maze. Conclusion Our data collectively suggested that supplementation with γ-GC may represent a novel strategy for the treatment and/or prevention of cognitive impairment and neurodegeneration.
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Polyphenols in red wine are bioactive compounds with positive effects on health and disease prevention. White grapes musts and wines have a lower concentration of phenolic compounds compared to the red ones and are therefore considered less beneficial to health. In Andalucia, a region located in the South West of Spain, the Pedro Ximenez white grapes are desiccated under the sun for a week before they are pressed and the juice (must) obtained. This ancient procedure increases the variety and content of polyphenols present in the Pedro Ximenez must (PXM). We have incorporated PXM to the daily diet of aged Mus spretus mice (24 months) and investigated their properties by comparing several parameters determined in these old mice with those measured in young mice (two months). Biochemical, histological and transcriptional analyses indicated that PXM exhibits potent antioxidant properties, promotes the normalization of the biotransforming ability of several cytochromes P450 in the liver, and regularizes the hepatic apoptosis, promoting proliferation instead. Our data indicate that PXM possesses a profound ability to promote liver regeneration of both the structure and the function, contributing to a healthy aging process.
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gamma-Glutamylcysteine synthetase (rat kidney; Mr approximately 104,000) is composed of 2 nonidentical subunits. In the present work, a procedure was developed for the reversible dissociation of the enzyme into its subunits (Mr = 73,000 and 27,700) under nondenaturing conditions. Students in which gel electrophoresis was used, in conjunction with an enzyme activity stain and elution and re-electrophoresis of protein bands, showed that the heavy subunit contains all of the structural requirements for enzymatic activity and also for feedback inhibition of the enzyme activity by glutathione. The light subunit, which may be formed from a precursor protein, has a significantly lower content of Trp, Phe, Tyr, Val, and Ala residues than the heavy subunit, while its content of Lys, His, Met, and Asx residues is higher.
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Nrf1 is a member of the CNC-basic leucine zipper (CNC-bZIP) family of transcription factors. CNC bZIP factors, together with small Maf proteins, bind as heterodimers to the NF-E2/AP-1 element. Similarity between the NF-E2/AP-1 element and the antioxidant response element identified in a number of promoters of genes involved in detoxification and antioxidant response raises the possibility that Nrf1 plays a role in mediating the antioxidant response element response. In this study, we exploited the availability of cells from Nrf1 knockout mice to study the role of Nrf1 transcription factor in the regulation of antioxidant gene expression and in cellular antioxidant response. Fibroblast cells derived from Nrf1 null embryos showed lower levels of glutathione and enhanced sensitivity to the toxic effects of oxidant compounds. Our results indicate that Nrf1 plays a role in the regulation of genes involved in glutathione synthesis and suggest a basis for a correspondingly low GSH concentration and reduced stress response.
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The aim of the present study was to evaluate the effect that a dietary intake of resveratrol (RSV) had on the expression of glutamate cysteine ligase (GCL) in the kidneys of aged rats. Young, middle-aged and aged rats were each randomly divided into two groups. The control groups were fed a controlled diet and the experimental groups received a controlled diet supplemented with RSV. GCL activity levels in the kidneys were determined. Protein content and relative gene expression levels of the two subunits of GCL were evaluated by western blot analysis and quantitative polymerase chain reaction, respectively. GCL activity levels significantly increased in the kidneys of aged rats fed the RSV-supplemented diet. In addition, RSV markedly increased the protein content and relative mRNA expression levels of the GCL subunits in the kidneys of aged rats. These observations have important implications for the development of therapeutic agents for the kidneys that may enable the elderly population to combat oxidative stress.
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Background Glutathione (GSH) is present in all mammalian tissues as the most abundant non-protein thiol that defends against oxidative stress. GSH is also a key determinant of redox signaling, vital in detoxification of xenobiotics, and regulates cell proliferation, apoptosis, immune function, and fibrogenesis. Biosynthesis of GSH occurs in the cytosol in a tightly regulated manner. Key determinants of GSH synthesis are the availability of the sulfur amino acid precursor, cysteine, and the activity of the rate-limiting enzyme, glutamate cysteine ligase (GCL), which is composed of a catalytic (GCLC) and a modifier (GCLM) subunit. The second enzyme of GSH synthesis is GSH synthetase (GS).Scope of reviewThis review summarizes key functions of GSH and focuses on factors that regulate the biosynthesis of GSH, including pathological conditions where GSH synthesis is dysregulated.Major conclusionsGCL subunits and GS are regulated at multiple levels and often in a coordinated manner. Key transcription factors that regulate the expression of these genes include NF-E2 related factor 2 (Nrf2) via the antioxidant response element (ARE), AP-1, and nuclear factor kappa B (NFκB). There is increasing evidence that dysregulation of GSH synthesis contributes to the pathogenesis of many pathological conditions. These include diabetes mellitus, pulmonary and liver fibrosis, alcoholic liver disease, cholestatic liver injury, endotoxemia and drug-resistant tumor cells.General significanceGSH is a key antioxidant that also modulates diverse cellular processes. A better understanding of how its synthesis is regulated and dysregulated in disease states may lead to improvement in the treatment of these disorders. This article is part of a Special Issue entitled Cellular functions of glutathione.
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Background X-linked adrenoleukodystrophy (X-ALD) is a genetic disorder of X-linked inheritance caused by a mutation in the ABCD1 gene which determines an accumulation of long-chain fatty acids in plasma and tissues. Recent evidence shows that oxidative stress may be a hallmark in the pathogenesis of X-ALD and glutathione plays an important role in the defense against free radicals. In this study we have analyzed glutathione homeostasis in lymphocytes of 14 patients with X-ALD and evaluated the balance between oxidized and reduced forms of glutathione, in order to define the role of this crucial redox marker in this condition. Methods Lymphocytes, plasma and erythrocytes were obtained from the whole blood of 14 subjects with X-ALD and in 30 healthy subjects. Total, reduced and protein-bound glutathione levels were measured in lymphocytes by HPLC analysis. Erythrocyte free glutathione and antioxidant enzyme activities, plasma thiols and carbonyl content were determined by spectrophotometric assays. Results A significant decrease of total and reduced glutathione was found in lymphocytes of patients, associated to high levels of all oxidized glutathione forms. A decline of free glutathione was particularly significant in erythrocytes. The increased oxidative stress in X-ALD was additionally confirmed by the decrease of plasma thiols and the high level of carbonyls. Conclusion Our results strongly support a role for oxidative stress in the pathophysiology of X-ALD and strengthen the importance of the balance among glutathione forms as a hallmark and a potential biomarker of the disease.
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The transcription factor Nrf2 (NF-E2-related factor 2) plays a vital role in maintaining cellular homeostasis, especially upon the exposure of cells to chemical or oxidative stress, through its ability to regulate the basal and inducible expression of a multitude of antioxidant proteins, detoxification enzymes and xenobiotic transporters. In addition, Nrf2 contributes to diverse cellular functions including differentiation, proliferation, inflammation and lipid synthesis and there is an increasing association of aberrant expression and/or function of Nrf2 with pathologies including cancer, neurodegeneration and cardiovascular disease. The activity of Nrf2 is primarily regulated via its interaction with Keap1 (Kelch-like ECH-associated protein 1), which directs the transcription factor for proteasomal degradation. Although it is generally accepted that modification (e.g. chemical adduction, oxidation, nitrosylation or glutathionylation) of one or more critical cysteine residues in Keap1 represents a likely chemico-biological trigger for the activation of Nrf2, unequivocal evidence for such a phenomenon remains elusive. An increasing body of literature has revealed alternative mechanisms of Nrf2 regulation, including phosphorylation of Nrf2 by various protein kinases (PKC, PI3K/Akt, GSK-3β, JNK), interaction with other protein partners (p21, caveolin-1) and epigenetic factors (micro-RNAs -144, -28 and -200a, and promoter methylation). These and other processes are potentially important determinants of Nrf2 activity, and therefore may contribute to the maintenance of cellular homeostasis. Here, we dissect evidence supporting these Keap1-dependent and -independent mechanisms of Nrf2 regulation. Furthermore, we highlight key knowledge gaps in this important field of biology, and suggest how these may be addressed experimentally.
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Dysregulation of glutathione homeostasis and alterations in glutathione-dependent enzyme activities are increasingly implicated in the induction and progression of neurodegenerative diseases, including Alzheimer's, Parkinson's and Huntington's diseases, amyotrophic lateral sclerosis, and Friedreich's ataxia. In this review background is provided on the steady-state synthesis, regulation, and transport of glutathione, with primary focus on the brain. A brief overview is presented on the distinct but vital roles of glutathione in cellular maintenance and survival, and on the functions of key glutathione-dependent enzymes. Major contributors to initiation and progression of neurodegenerative diseases are considered, including oxidative stress, protein misfolding, and protein aggregation. In each case examples of key regulatory mechanisms are identified that are sensitive to changes in glutathione redox status and/or in the activities of glutathione-dependent enzymes. Mechanisms of dysregulation of glutathione and/or glutathione-dependent enzymes are discussed that are implicated in pathogenesis of each neurodegenerative disease. Limitations in information or interpretation are identified, and possible avenues for further research are described with an aim to elucidating novel targets for therapeutic interventions. The pros and cons of administration of N-acetylcysteine or glutathione as therapeutic agents for neurodegenerative diseases, as well as the potential utility of serum glutathione as a biomarker, are critically evaluated.
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Reactive oxygen species regulate redox-signaling processes, but in excess they can cause cell damage, hence underlying the aetiology of several neurological diseases. Through its ability to down modulate reactive oxygen species, glutathione is considered an essential thiol-antioxidant derivative, yet under certain circumstances it is dispensable for cell growth and redox control. Here we show, by directing the biosynthesis of γ-glutamylcysteine-the immediate glutathione precursor-to mitochondria, that it efficiently detoxifies hydrogen peroxide and superoxide anion, regardless of cellular glutathione concentrations. Knocking down glutathione peroxidase-1 drastically increases superoxide anion in cells synthesizing mitochondrial γ-glutamylcysteine. In vitro, γ-glutamylcysteine is as efficient as glutathione in disposing of hydrogen peroxide by glutathione peroxidase-1. In primary neurons, endogenously synthesized γ-glutamylcysteine fully prevents apoptotic death in several neurotoxic paradigms and, in an in vivo mouse model of neurodegeneration, γ-glutamylcysteine protects against neuronal loss and motor impairment. Thus, γ-glutamylcysteine takes over the antioxidant and neuroprotective functions of glutathione by acting as glutathione peroxidase-1 cofactor.
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The tripeptide glutathione (GSH) is the most abundant free radical scavenger synthesized endogenously in humans. Increasing mechanistic, clinical, and epidemiological evidence demonstrates that GSH status is significant in acute and chronic diseases. Despite ease of delivery, little controlled clinical research data exist evaluating the effects of oral GSH supplementation. The study objectives were to determine the effect of oral GSH supplementation on biomarkers of systemic oxidative stress in human volunteers. This was a randomized, double-blind, placebo-controlled clinical trial. The study was conducted at Bastyr University Research Institute, Kenmore, WA and the Bastyr Center for Natural Health, Seattle, WA. Forty (40) adult volunteers without acute or chronic disease participated in this study. Intervention: Oral GSH supplementation (500 mg twice daily) was given to the volunteers for 4 weeks. Primary outcome measures included change in creatinine-standardized, urinary F2-isoprostanes (F2-isoP) and urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG). Changes in erythrocyte GSH concentrations, including total reduced glutathione (GSH), oxidized glutathione (GSSG), and their ratio (GSH:GSSG) were also measured by tandem liquid chromatography/mass spectrometry. Analysis of variance was used to evaluate differences between groups. There were no differences in oxidative stress biomarkers between treatment groups at baseline. Thirty-nine (39) participants completed the study per protocol. Changes in creatinine standardized F2-isoP (ng/mg creatinine) (0.0±0.1 versus 0.0±0.1, p=0.38) and 8-OHdG (μg/g creatinine) (-0.2±3.3 versus 1.0±3.2, p=0.27) were nonsignificant between groups at week 4. Total reduced, oxidized, and ratio measures of GSH status were also unchanged. No significant changes were observed in biomarkers of oxidative stress, including glutathione status, in this clinical trial of oral glutathione supplementation in healthy adults.
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Aging is associated with oxidative stress, but underlying mechanisms remain poorly understood. We tested whether glutathione deficiency occurs because of diminished synthesis and contributes to oxidative stress in aging and whether stimulating glutathione synthesis with its precursors cysteine and glycine could alleviate oxidative stress. Eight elderly and 8 younger subjects received stable-isotope infusions of [2H(2)]glycine, after which red blood cell (RBC) glutathione synthesis and concentrations, plasma oxidative stress, and markers of oxidant damage (eg, F(2)-isoprostanes) were measured. Elderly subjects were restudied after 2 wk of glutathione precursor supplementation. Compared with younger control subjects, elderly subjects had markedly lower RBC concentrations of glycine (486.7 ± 28.3 compared with 218.0 ± 23.7 μmol/L; P < 0.01), cysteine (26.2 ± 1.4 compared with 19.8 ± 1.3 μmol/L; P < 0.05), and glutathione (2.08 ± 0.12 compared with 1.12 ± 0.18 mmol/L RBCs; P < 0.05); lower glutathione fractional (83.14 ± 6.43% compared with 45.80 ± 5.69%/d; P < 0.01) and absolute (1.73 ± 0.16 compared with 0.55 ± 0.12 mmol/L RBCs per day; P < 0.01) synthesis rates; and higher plasma oxidative stress (304 ± 16 compared with 346 ± 20 Carratelli units; P < 0.05) and plasma F(2)-isoprostanes (97.7 ± 8.3 compared with 136.3 ± 11.3 pg/mL; P < 0.05). Precursor supplementation in elderly subjects led to a 94.6% higher glutathione concentration, a 78.8% higher fractional synthesis rate, a 230.9% higher absolute synthesis rate, and significantly lower plasma oxidative stress and F(2)-isoprostanes. No differences in these measures were observed between younger subjects and supplemented elderly subjects. Glutathione deficiency in elderly humans occurs because of a marked reduction in synthesis. Dietary supplementation with the glutathione precursors cysteine and glycine fully restores glutathione synthesis and concentrations and lowers levels of oxidative stress and oxidant damages. These findings suggest a practical and effective approach to decreasing oxidative stress in aging.
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Modulation of monocyte function is a critical factor in the resolution of inflammatory responses. This role is mediated mainly by the production of TNF-α. Investigations of the actions of TNF have mostly focused on acute activation of other cell types such as fibroblasts and endothelial cells. Less is known about the effects of TNF on monocytes themselves, and little is known about the regulation of cell responses to TNF beyond the activation of NF-κB. In this study, we investigated the regulation of NF-E2-related factor 2 (Nrf2) cyctoprotective responses to TNF in human monocytes. We found that in monocytes TNF induces sustained Nrf2 activation and Nrf2 cytoprotective gene induction in a TNFR1-dependent manner. Under TNF activation, monocytes increased their expression of Nrf2-dependent genes, including NAD(P)H:quinone oxidoreductase 1 and glutamyl cysteine ligase modulatory, but not heme oxygenase-1. We also showed that autocrine TNF secretion was responsible for this sustained Nrf2 response and that Nrf2 activation by TNF was mediated by the generation of reactive oxygen species. Moreover, we showed that Nrf2-mediated gene induction can modulate TNF-induced NF-κB activation. These results show for the first time, to our knowledge, that TNF modulates prolonged Nrf2-induced gene expression, which in turn regulates TNF-induced inflammatory responses.
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Lymphocytes are an important immunological cell and have been played a significant role in acquired immune system; hence, may play in pivotal role in immunosenescence. Oxidative stress has been reported to increase in elderly subjects, possibly arising from an uncontrolled production of free radicals with aging and decreased antioxidant defenses. This study was aimed to evaluate the level of lipid-protein damage, and antioxidant status in lymphocytes of healthy individuals to correlate between oxidative damage with the aging process. Twenty healthy individuals of each age group (11 - 20; 21 - 30; 31 - 40; 41 - 50; and 51 - 60 years) were selected randomly. Blood samples were drawn by medical practitioner and lymphocytes were isolated from blood samples. Malondialdehyde (MDA), protein carbonyls (PC) level were evaluated to determine the lipid, and protein damage in lymphocytes. Superoxide dismutase (SOD), catalase (CAT), glutathione and glutathione dependent enzymes were estimated to evaluate the antioxidant status in the lymphocytes. Increased MDA and PC levels strongly support the increased oxidative damage in elderly subject than young subjects. The results indicated that, balance of oxidant and antioxidant systems in lymphocytes shifts in favor of accelerated oxidative damage during aging. Thus oxidative stress in lymphocytes may particular interest in aging, and may play important role in immunosenescence.
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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.
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Reduced glutathione (GSH) is the most abundant non-protein thiol in mammalian cells and the preferred substrate for several enzymes in xenobiotic metabolism and antioxidant defense. It plays an important role in many cellular processes, such as cell differentiation, proliferation and apoptosis. GSH deficiency has been observed in aging and in a wide range of pathologies, including neurodegenerative disorders and cystic fibrosis (CF), as well as in several viral infections. Use of GSH as a therapeutic agent is limited because of its unfavorable biochemical and pharmacokinetic properties. Several reports have provided evidence for the use of GSH prodrugs able to replenish intracellular GSH levels. This review discusses different strategies for increasing GSH levels by supplying reversible bioconjugates able to cross the cellular membrane more easily than GSH and to provide a source of thiols for GSH synthesis.
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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.
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Gamma-Glutamyl-cysteine synthetase is inhibited by glutathione under conditions similar to those which prevail in vivo, thus strongly suggesting a physiologically significant feedback mechanism. Inhibition by glutathione, which is not allosteric, appears to involve the binding of glutathione to the glutamate site of the enzyme as well as to another enzyme site; the latter binding appears to require a sulfhydryl group since ophthalmic acid (gamma-glutamyl-alpha-aminobutyryl-glycine) is only a weak inhibitor. The finding that glutathione regulates its own synthesis by inhibiting synthesis of gamma-glutamyl-cysteine appears to explain observations on patients with 5-oxoprolinuria, who were shown to have a block in the gamma-glutamyl cycle consisting of a marked deficiency of glutathione synthetase and consequently of glutathione. These patients produce greater than normal amounts of gamma-glutamyl-cysteine, which is converted by the action of gamma-glutamyl cyclotransferase to 5-oxoproline; production of the latter compound exceeds the capacity of 5-oxoprolinase to convert it to glutamate. The apparent Km value for L-cysteine for gamma-glutamyl-cysteine synthetase (0.35 mM) is not far from intracellular concentrations of L-cysteine suggesting that the availability of L-cysteine may also play a role in the regulation of glutathione synthesis.
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We reported that glucagon and phenylephrine decrease hepatocyte GSH by inhibiting gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in GSH synthesis (Lu, S.C., J. Kuhlenkamp, C. Garcia-Ruiz, and N. Kaplowitz. 1991. J. Clin. Invest. 88:260-269). In contrast, we have found that insulin (In, 1 microgram/ml) and hydrocortisone (HC, 50 nM) increased GSH of cultured hepatocytes up to 50-70% (earliest significant change at 6 h) with either methionine or cystine alone as the sole sulfur amino acid in the medium. The effect of In occurred independent of glucose concentration in the medium. Changes in steady-state cellular cysteine levels, cell volume, GSH efflux, or expression of gamma-glutamyl transpeptidase were excluded as possible mechanisms. Both hormones are known to induce cystine/glutamate transport, but this was excluded as the predominant mechanism since the induction in cystine uptake required a lag period of greater than 6 h, and the increase in cell GSH still occurred when cystine uptake was blocked. Assay of GSH synthesis in extracts of detergent-treated cells revealed that In and HC increased the activity of GCS by 45-65% (earliest significant change at 4 h) but not GSH synthetase. In and HC treatment increased the Vmax of GCS by 31-43% with no change in Km. Both the hormone-mediated increase in cell GSH and GCS activity were blocked with either cycloheximide or actinomycin D. Finally, when studied in vivo, streptozotocin-treated diabetic and adrenalectomized rats exhibited lower hepatic GSH levels and GCS activities than respective controls. Both of these abnormalities were prevented with hormone replacement. Thus, both in vitro and in vivo, In and glucocorticoids are required for normal expression of GCS.
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When the plasma glutathione concentration is low, such as in patients with HIV infection, alcoholics, and patients with cirrhosis, increasing the availability of circulating glutathione by oral administration might be of therapeutic benefit. To assess the feasibility of supplementing oral glutathione we have determined the systemic availability of glutathione in 7 healthy volunteers. The basal concentrations of glutathione, cysteine, and glutamate in plasma were 6.2, 8.3, and 54 μmol·l−1 respectively. During the 270 min after the administration of glutathione in a dose of 0.15 mmol·kg−1 the concentrations of glutathione, cysteine, and glutamate in plasma did not increase significantly, suggesting that the systemic availability of glutathione is negligible in man. Because of hydrolysis of glutathione by intestinal and hepatic γ-glutamyltransferase, dietary glutathione is not a major determinant of circulating glutathione, and it is not possible to increase circulating glutathione to a clinically beneficial extent by the oral administration of a single dose of 3 g of glutathione.
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Our present work characterized the role of hormone-mediated signal transduction pathways in regulating hepatic reduced glutathione (GSH) synthesis. Cholera toxin, dibutyryl cAMP (DBcAMP), and glucagon inhibited GSH synthesis in cultured hepatocytes by 25-43%. Cellular cAMP levels exhibited a lower threshold for stimulation of the GSH efflux than inhibition of its synthesis. The effect of DBcAMP was independent of the type of sulfur amino acid precursor and cellular ATP levels and unassociated with increased GSH mixed disulfide formation or altered GSH/oxidized glutathione ratio. In liver cytosols, addition of DBcAMP and cAMP-dependent protein kinase (A-kinase) inhibited GSH synthesis from substrates (cysteine, ATP, glutamate, and glycine) by approximately 20% which was prevented by the A-kinase inhibitor. However, if only substrates of the second step in GSH synthesis were used (gamma-glutamylcysteine, glycine, and ATP), DBcAMP and A-kinase exerted no inhibitory effect. Phenylephrine, vasopressin, and phorbol ester also inhibited GSH synthesis in cultured cells by approximately 20%, and depleted cell GSH independent of the type of sulfur amino acid precursor. Cellular cysteine level was unchanged despite the significant fall in GSH after glucagon or phenylephrine treatment. Pretreatment with either staurosporine, C-kinase inhibitor, or calmidazolium, a calmodulin inhibitor, partially prevented but, together, completely prevented the inhibitory effect of phenylephrine. The same combination had no effect on the inhibitory effect of glucagon. The effects of hormones were confirmed in both the intact perfused liver and after in vivo administration. Thus, two classes of hormones acting through distinct signal transduction pathways may down-regulate hepatic GSH synthesis by phosphorylation of gamma-glutamylcysteine synthetase.
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gamma-Glutamylcysteine synthetase catalyzes the first step in the synthesis of glutathione. The enzyme isolated from rat kidney has two subunits (heavy, Mr 73,000; and light, Mr 27,700) which may be dissociated by treatment with dithiothreitol. The heavy subunit exhibits all of the catalytic activity of the isolated enzyme and also feedback inhibition by glutathione. The light subunit has no known function and may not be an integral part of the enzyme. cDNA clones encoding rat kidney gamma-glutamylcysteine synthetase were isolated from a lambda gt11 cDNA library by immunoscreening with antibody against the isolated enzyme and further screening with oligonucleotide probes derived from several peptides whose sequences were determined by the Edman method. The nucleotide sequence of the mRNA for the heavy subunit was deduced from the sequences of the cDNA of three such clones. The sequence, which codes for 637 residues (Mr 72,614), contains all four of the independently determined peptide sequences (approximately 100 residues). This amino acid sequence shows extremely low overall similarity to that of gamma-glutamylcysteine synthetase isolated from Escherichia coli.
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Rushworth, S. A., S. Shah, and D. J. MacEwan. 2011. TNF mediates the sustained activation of Nrf2 in human monocytes. J. Immunol. 187: [702–707][1]. In [Fig. 4 D ][2], the incorrect immunoblot for the loading control was mistakenly used. The correct immunoblot has now been used in the updated [
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Glutathione (GSH) is the one of the most abundant intracellular antioxidants. Mice lacking the modifier subunit of glutamate cysteine ligase (Gclm), the rate-limiting enzyme in GSH synthesis, have decreased GSH. Our prior work showed that GSH plays antiapoptotic roles in ovarian follicles. We hypothesized that Gclm-/- mice have accelerated ovarian aging due to ovarian oxidative stress. We found significantly decreased ovarian GSH concentrations and oxidized GSH/GSSG redox potential in Gclm-/- versus Gclm+/+ ovaries. Prepubertal Gclm-/- and Gclm+/+ mice had similar numbers of ovarian follicles, and as expected, the total number of ovarian follicles declined with age in both genotypes. However, the rate of decline in follicles was significantly more rapid in Gclm-/- mice, and this was driven by accelerated declines in primordial follicles, which constitute the ovarian reserve. We found significantly increased 4-hydroxynonenal immunostaining (oxidative lipid damage marker) and significantly increased nitrotyrosine immunostaining (oxidative protein damage marker) in prepubertal and adult Gclm-/- ovaries compared to controls. The percentage of small ovarian follicles with increased granulosa cell proliferation was significantly higher in prepubertal and 2 month old Gclm-/- versus Gclm+/+ ovaries, indicating accelerated recruitment of primordial follicles into the growing pool. The percentages of growing follicles with apoptotic granulosa cells were increased in young adult ovaries. Our results demonstrate increased ovarian oxidative stress and oxidative damage in young Gclm-/- mice, associated with an accelerated decline in ovarian follicles that appears to be mediated by increased recruitment of follicles into the growing pool, followed by apoptosis at later stages of follicular development.
Article
Increasing oxidative stress, a major characteristic of aging, has been implicated in variety of age-related pathologies. In aging, oxidant production from several sources is increased while antioxidant enzymes, the primary lines of defense, are decreased. Repair systems, including the proteasomal degradation of damaged proteins also declines. Importantly, the adaptive response to oxidative stress declines with aging. Nrf2/EpRE signaling regulates the basal and inducible expression of many antioxidant enzymes and the proteasome. Nrf2/EpRE activity is regulated at several levels including transcription, post-translation, and interaction with other proteins. This review summarizes current studies on age-related impairment of Nrf2/EpRE function and discusses the change of Nrf2 regulatory mechanisms with aging. Copyright © 2015. Published by Elsevier Inc.
Article
Accumulating evidence has suggested the involvement of oxidative stress in the pathogenesis of Alzheimer's disease (AD). The main endogenous antioxidant, glutathione (GSH), has been shown to decline with ageing and in several age-related degenerative diseases, including AD. Potential options for replenishing GSH levels as a therapeutic target to treat these conditions include the administration of GSH itself, and low toxicity forms of the limiting amino acid for GSH synthesis; cysteine However, passive GSH uptake is limited due to an unfavourable concentration gradient between the plasma and cytosol. Similarly, cysteine prodrugs have demonstrated limited efficacy to elevate depleted GSH levels in several in vivo and in vitro models of disease. It has been suggested that the decline in GSH levels in AD, may be associated with down regulation of GSH homeostasis rather than substrate limitation. Cellular GSH homeostasis is regulated by non-allosteric feedback inhibition exerted by GSH on glutamate cysteine ligase (GCL), which is responsible for the synthesis of the GSH precursor γ-glutamylcysteine (GGC). In conditions involving down regulated GSH homeostasis, GGC serves as a crucial rate-limiting substrate for GSH synthetase, the main enzyme responsible for condensing glycine with GGC to form the final thiol tripeptide, GSH. In this review, we focus on the therapeutic potential of GGC to elevate cellular GSH levels. We also discuss the efficacy of GGC prodrugs which would be taken up and converted by the unregulated GS to GSH, and the administration of modified GSH compounds, such as GSH esters that could potentially overcome the concentration gradient that prohibits passive GSH uptake, in AD.
Article
γ-Glutamylcysteine synthetase (GCS; also referred to as glutamate-cysteine ligase, GLCL) catalyzes the rate-limiting reaction in glutathione (GSH) biosynthesis. The GCS holoenzyme is composed of a catalytic and regulatory subunit, each encoded by a unique gene. In addition to some conditions which specifically upregulate the catalytic subunit gene, expression of both genes is increased in response to many Phase II enzyme inducers including oxidants, heavy metals, phenolic antioxidants and GSH-conjugating agents. Electrophile Response Elements (EpREs), located in 5′-flanking sequences of both the GCSh and GCSl subunit genes, are hypothesized to at least partially mediate gene induction following xenobiotic exposure. Recent experiments indicate that the bZip transcription factor Nrf2 participates in EpRE-mediated GCS subunit gene activation in combination with other bZip proteins. An AP-1-like binding sequence and an NF-κB site have also been implicated in regulation of the catalytic subunit gene following exposure to certain pro-oxidants. Potential signaling mechanisms mediating GCS gene induction by the diverse families of Phase II enzyme inducers include thiol modification of critical regulatory sensor protein(s) and the generation of the reactive oxygen species. This review summarizes recent progress in defining the molecular mechanisms operative in transcriptional control of the genes encoding the two GCS subunits, identifying areas of agreement and controversy. The mechanisms involved in GCS regulation might also be relevant to the transcriptional control of other components of the antioxidant defense battery.
Article
Glutamate-cysteine ligase (EC 6.3.2.2, GLCL), formerly called γ-glutamylcysteine synthetase (GCS), is the rate-limiting enzyme in the de novo synthesis of the antioxidant tripeptide glutathione. GLCL consists of a heavy subunit, which possesses catalytic activity and is the site of glutathione feedback inhibition, and a light subunit, which has a regulatory function. Glutathione is ubiquitous in mammalian tissues and performs a variety of functions, including protection from reactive oxygen species through antioxidant properties; detoxification of xenobiotics, organic peroxides, and heavy metals; and maintenance of sulfhydryl groups of other molecules. Increased intracellular levels of glutathione have also been found in tumor cells resistant to chemotherapeutic agents. Increased expression of GLCL in melphalan-resistant myeloma and prostate carcinoma cells and cisplatinum-resistant ovarian carcinoma cells suggests that this enzyme may be involved in glutathione-associated drug resistance. Moreover, GLCL has been shown to be induced by phenolic antioxidants and heavy metals. Recently, Mulcahy and Gipp have shown that the GLCL catalytic subunit gene (GLCLC) contains a putative antioxidant regulatory element, which may explain the responsiveness of this gene to agents that induce oxidative stress. To further our understanding of GLCL, which is linked to such a wide variety of metabolic and physiological functions through its role in glutathione synthesis, we have mapped both the catalytic and regulatory subunit genes (GLCLC and GLCLR) to human and mouse chromosomes by fluorescence in situ hybridization (FISH). 16 refs., 1 fig.
Article
Erythrocytes endure constant exposure to oxidative stress. The major oxidative stress scavenger in erythrocytes is glutathione. The rate-limiting enzyme for glutathione synthesis is glutamate-cysteine ligase, which consists of a catalytic subunit (GCLC) and a modifier subunit (GCLM). Here, we examined erythrocyte survival in GCLM-deficient (gclm -/-) mice. Erythrocytes from gclm -/- mice showed greatly reduced intracellular glutathione. Prolonged incubation resulted in complete lysis of gclm -/- erythrocytes, which could be reversed by exogenous delivery of the antioxidant Trolox. To test the importance of GCLM in vivo, mice were treated with phenylhydrazine (PHZ; 0.07 mg/g b.w.) to induce oxidative stress. Gclm -/- mice showed dramatically increased hemolysis compared with gclm +/+ controls. In addition, PHZ-treated gclm -/- mice displayed markedly larger accumulations of injured erythrocytes in the spleen than gclm +/+ mice within 24 h of treatment. Iron staining indicated precipitations of the erythrocyte-derived pigment hemosiderin in kidney tubules of gclm -/- mice and none in gclm +/+ controls. In fact, 24 h after treatment, kidney function began to diminish in gclm -/- mice as evident from increased serum creatinine and urea. Consequently, while all PHZ-treated gclm +/+ mice survived, 90% of PHZ-treated gclm -/- mice died within 5 days of treatment. In vitro, upon incubation in the absence or presence of additional oxidative stress, gclm -/- erythrocytes exposed significantly more phosphatidylserine, a cell death marker, than gclm +/+ erythrocytes, an effect at least partially due to increased cytosolic Ca 2+ concentration. Under resting conditions, gclm -/- mice exhibited reticulocytosis, indicating that the enhanced erythrocyte death was offset by accelerated erythrocyte generation. GCLM is thus indispensable for erythrocyte survival, in vitro and in vivo, during oxidative stress.
Article
Alzheimer's disease (AD) is characterized by progressive loss of memory and cognition. Our laboratory and many others have shown that the AD brain is under extensive oxidative stress. Numerous therapeutic approaches to AD therapy have been hypothesized. Among these are exogenous antioxidants. We suggest that the body's own endogenous antioxidant systems should be mobilized against the oxidative stress inherent in AD brain. One of the most versatile antioxidants in the brain is glutathione. Glutathione is capable of protecting the cell against reactive oxygen species, redox metal ions, and reactive lipid peroxidation products and other electrophiles associated with AD. There are many ways to increase glutathione levels, and one or more of these ways to increase glutathione in the brain may be a promising therapeutic strategy for AD. Drug Dev. Res. 56:428–437, 2002. © 2002 Wiley-Liss, Inc.
Article
In our prior studies (Orr et al., 2005) we achieved a 30-50% increase in the life span of Drosophila by manipulating glutathione (GSH) production in neuronal tissues, through over-expression of glutamate-cysteine ligase (GCL), a key enzyme in glutathione biosynthesis. In the present study, we identified gene response patterns from which plausible mechanisms responsible for the observed effects on life span might be inferred. Functional clustering analysis of the transcriptome data revealed that biological processes affected by GCLc in young flies (10 days) were generally related to cell morphogenesis and differentiation, while those in older flies were associated with nucleosome organization and detoxification processes. Notably, in older flies there was considerable reduction in the expression of genes related to humoral immunity in the GCLc over-expressors and this was observed in flies of the same chronological age (∼40 days old flies) and in flies of equivalent physiological age (10% dead for both experimentals and controls). Our study demonstrates that most of the GSH-mediated processes and targets are relatively distinct in young and old flies. Nevertheless there exists a restricted number of related processes affected by GCLc in both young and old flies and prominent among them are those associated with proteolysis and metabolism.
Article
Individually, γ-glutamylcysteine (GGC), a dipeptide and precursor of glutathione (GSH), and conjugated linoleic acid (CLA), a trans-fatty acid, exhibit antioxidant properties. The objective of this study was to compare effects of co-administration of GGC and CLA to treatment with GGC alone on oxidative stress and GSH synthesis in human endothelial cells. Changes in levels of 8-epi-PGF2α, thiobarbituric acid reactive substances (TBARS), GSH, total antioxidants, GSH synthetase (GSS) expression, and transcription factor DNA binding were assessed in human umbilical vein endothelial cells (HUVEC) treated with GGC alone (100 μmol/L) or combined with CLA isomer mixture (10, 50, 100 μmol/L) for 24h. Significantly higher levels of TBARS, 8-epi-PGF2α, GSH, and GSS protein were found in cells treated with GGC and 10 μmol/L CLA, compared to cells treated with GGC alone, indicative of prooxidant effects of CLA. Approximately 40% cell death was microscopically observed in cells incubated with GGC and 100 μmol/L CLA. Despite lower levels of GSH, treatment with GGC and 50 μmol/L CLA appeared to be protective from oxidative stress similar to treatment with GGC alone, as indicated by lower levels of TBARS, compared to control cells not treated with GGC and CLA.
Article
Although glutathione (GSH) concentration has been reported to diminish with age, the mechanism underlying such age-associated decline in the GSH content is not well understood. In this study, we compared the gene expression of both subunits of γ-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in de novo GSH synthesis, in young, adult, and old Fisher 344 rats. It was found that GCS activity was significantly decreased with increased age in liver, kidney, lung, and red blood cells (RBC). Parallel with the decreased enzyme activity, the protein and mRNA contents of both GCS subunits also changed inversely with age in liver, kidney, and lung, implying a decreased GCS gene expression during aging. Such a reduced GCS gene expression was accompanied by a decline in total GSH content without any change in cysteine concentration. Furthermore, the decreased GCS gene expression in old rats was not associated with a decline in the plasma insulin or cortisol level. This study showed, for the first time, that the expression of both GCS subunit genes was decreased in some organs of old rats, which would result in a reduced rate of GSH biosynthesis. Such decline in GSH synthetic capacity may underlie the observed decrease in GSH content during aging.
Article
Many xenobiotic detoxifying (phase II) enzymes are induced by sublethal doses of environmental toxicants. However, these adaptive mechanisms have not been studied in response to vehicular-derived airborne nano-sized particulate matter (nPM). Because aging is associated with increased susceptibility to environmental toxicants, we also examined the expression of Nrf2-regulated phase II genes in middle-aged mice and their inducibility by chronic nPM. The nPM from vehicular traffic was collected in urban Los Angeles and reaerosolized for exposure of C57BL/6J male mice (3 and 18 months old) for 150 h over 10 weeks. Brain (cerebellum), liver, and lung were assayed by RT-PCR and/or Western blots for the expression of phase II enzymes, glutamate cysteine ligase (catalytic GCLC, and modifier GCLM subunits), NAD(P)H:quinone oxidoreductase 1 (NQO1), heme oxygenase 1 (HO-1), and relevant transcription factors, NF-E2-related factor 2 (Nrf2), c-Myc, Bach1. Chronic nPM exposure induced GCLC, GCLM, HO-1, NQO1 mRNA, and protein similarly in cerebellum, liver, and lung of young mice. Middle-aged mice had elevated basal levels, but showed impaired further induction by nPM. Similarly, Nrf2 increased with age and was induced by nPM in young but not old. c-Myc showed the same age and induction profile while the age increase in Bach1 was further induced by nPM. Chronic exposure to nanoparticles induced Nrf2-regulated detoxifying enzymes in brain (cerebellum), liver, and lung of young adult mice, indicating a systemic impact of nPM. In contrast, middle-aged mice did not respond above their elevated basal levels except for Bach1. The lack of induction of phase II enzymes in aging mice may be a model for the vulnerability of elderly to air pollution.
Article
γ-Glutamylcysteine (GGC) is a dipeptide and substrate for synthesis of the antioxidant glutathione (GSH), whose health promoting properties include reducing risks of oxidative stress-related injuries and diseases. The objective of this study was to investigate the efficacy of GGC on GSH synthesis and oxidative stress in human endothelial cells. We assessed oxidative stress, GSH, GSH synthetase (GSS) expression, and transcription factor DNA binding levels in human umbilical vein endothelial cells (HUVEC). We found significantly higher levels of PPARγ DNA binding and lower levels of GSH, GSS protein, NF-κB p65 DNA binding, thiobarbituric acid reactive substances (TBARS), and 8-epi-PGF(2α) in a concentration-dependent manner, compared with the control. GSH and GSS protein levels showed a negative correlation with PPARγ DNA binding levels and positive correlation trends with NF-κB p65 DNA binding, TBARS, and 8-epi-PGF(2α) levels. A putative binding site for NF-κB was found at 4 227 bases upstream from the transcription start site of GSS gene, but none for PPARs. These findings suggest the involvement of NF-κB in regulation of GSS expression. Subsequent GSH synthesis might be affected by the suppression of GSS expression in tested conditions. Besides its substrate role in GSH synthesis, GGC may play a role in protection against oxidative stress by serving as an antioxidant and modulating the expression of protein(s) related to antioxidant defense. Thus, we speculate that GGC may serve as a novel intra- and intercellular therapeutic dipeptide for oxidative stress-related injuries and diseases.