GSH Biosynthesis Research Articles

Improved S-adenosylmethionine and glutathione biosynthesis by heterologous expression of an ATP6 gene in Candida utilis.

ATP is indispensable to the biosynthesis of both S-adenosylmethionine (SAM) and glutathione (GSH) in yeast cells. To improve ATP supply for overproduction of SAM and GSH in Candida utilis CCTCC M 209298, an exogenous ATP6 gene from Arabidopsis thaliana was expressed in the parental strain to construct the mutant C. utilis ATP6 by genomic integration. The maximal production of SAM and GSH in the mutant increased by 46.6 and 28.7%, respectively, when compared with those obtained in the parental strain. The mechanism underlying improved SAM and GSH biosynthesis by exogenous ATP6 gene expression revealed that the mutant had higher activities of key enzymes involved in SAM and GSH biosynthesis as well as energy metabolism. Increased NADH availability and F0 F1 -ATPase activity subsequently resulted in improved ATP regeneration and intracellular ATP supply for SAM and GSH overproduction. The present study not only developed an effective method for improving SAM and GSH biosynthesis by energy metabolism regulation, but also offered a novel approach for efficient production of similar energy-consuming products in eukaryotic cells.

Abstract 3999: Intracellular redox status determines self-renewal activity of breast cancer stem-like cells through activation of Nrf2-mediated FoxO3a-Bmi-1 signaling

Abstract A subpopulation of cancer cells, termed cancer stem cells (CSCs), has a self-renewal property, which drives cancer recurrence and tumor resistance. It has been reported that CSCs possess enhanced capabilities to protect against oxidative stress caused by reactive oxygen species (ROS) compared with non-stem-like cancer cells. In the present study, we found that the redox-sensitive transcription factor NF-E2-related factor 2 (Nrf2) plays a role in self-renewal activity of human breast cancer cells, and that this was mediated by Bmi-1. The manifestation of stemness in breast CSC-like cells was associated with an elevated ratio of reduced to oxidized glutathione (GSH) and consequently, lowered ROS levels compared with parent breast cancer (MCF-7) cells. We observed that there was a concurrent increase in the expression of CD44, glutamate cysteine ligase (GCLC) and Bmi-1 in CD24low/CD44high breast cancer stem-like cells sorted from MCF-7 mammospheres. Treatment with buthionine sulfoximine (BSO), an inhibitor of GSH biosynthesis, led to a decrease in the number and the size of mammospheres, which was accompanied by the reduction of Bmi-1 expression. Conversely, treatment with a membrane permeable GSH ethyl ester (GSH-MEE) enhanced the size and the number of mammospheres. In addition, phosphorylation of AMPK (Thr 172) and FoxO3a (Ser 7) as well as expression of Bmi-1 was increased in mammosphere cells treated with GSH-MEE. When GSH-MEE-treated mammospheres were challenged with hydrogen peroxide, the phosphorylation of AMPK was abolished. Notably, FoxO3a directly bound to the Bmi-1 promoter as assessed by the chromatin immunoprecipitation assay. Moreover, elevated nuclear accumulation of Nrf2 protein was evident in cells growing under sphere-forming conditions. Under physiologic conditions, Nrf2 is sequestered in the cytoplasm as an inactive complex with Keap1. Sphere-forming breast cancer cells exhibited an enhanced interaction between Keap1 and CD44, which may account for an increased translocation of Nrf2 in nucleus. We found that expression of GCLC, FoxO3a and Bmi-1 was inhibited following introduction of Nrf2 siRNA into MCF-7 mammsophere cells. Knockdown of Nrf2 suppressed the xenograft growth of orthotopically injected breast cancer stem-like cells in athymic nude mice, and this was accompanied by decreased expression of GCLC, FoxO3a and Bmi-1. In conclusion, CD44 promotes Nrf2 accumulation in breast cancer stem-like cells, which, in turn, upregulates GCLC expression and consequently enhances GSH biosynthesis with concomitant reduction in intracellular ROS levels. The consequent upregulation of nuclear FoxO3a and its binding to the Bmi-1 promoter may stimulate self-renewal and growth of breast cancer stem-like cells. Citation Format: Do-Hee Kim, Jeong-Hoon Jang, Young-Joon Surh. Intracellular redox status determines self-renewal activity of breast cancer stem-like cells through activation of Nrf2-mediated FoxO3a-Bmi-1 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3999.

Sulfur nutrition stimulates lead accumulation and alleviates its toxicity in Populus deltoides.

Sulfur (S) can modulate plant responses to toxic heavy metals, but the underlying physiological and transcriptional regulation mechanisms remain largely unknown. To investigate the effects of S supply on lead (Pb)-induced toxicity in poplars, Populus deltoides monilifera (Aiton) Eckenw. saplings were exposed to 0 or 50 μM Pb together with one of the three S concentrations (0 (low S), 100 (moderate S) or 1500 (high S) μM Na2SO4). Populus deltoides roots absorbed Pb and it was partially translocated to the aerial organs, thereby decreasing the CO2 assimilation rate and leaf growth. Lead accumulation in poplars caused the overproduction of O2- and H2O2 to induce higher levels of total thiols (T-SH) and glutathione (GSH). Lead uptake by the roots and its accumulation in the aerial organs were repressed by low S application, but stimulated by high S supply. Lead-induced O2- and H2O2 production were exacerbated by S limitation, but alleviated by high S supply. Moreover, the concentrations of S-containing antioxidants including T-SH and GSH were reduced in S-deficient poplars, but increased in high S-treated plants, which corresponded well to the changes in the activities of enzymes involved in S assimilation and GSH biosynthesis. The transcript levels of both genes encoding sulfate transporters, i.e., SULTR1.1 and SULTR2.2, were elevated by low S application or high S supply in the roots, and the transcriptional upregulation of both genes was more pronounced under Pb exposure. Furthermore, the mRNA levels of several genes involved in S assimilation and the biosynthesis of GSH and phytochelatins, i.e., ATPS1, ATPS3, GSHS1, GSHS2 and PCS1, were upregulated in poplar roots with high S supply, particularly under Pb exposure. These results indicate that a high S supply can stimulate Pb accumulation and reduce its toxicity in poplars by improving S assimilation and stimulating the biosynthesis of S-containing compounds including T-SH and GSH.

Glutathione Deficiency Induces Epigenetic Alterations of Vitamin D Metabolism Genes in the Liver of High-Fat Diet–Induced Type 2 Diabetic Mice

Vitamin D (VD) deficiency is an epidemic health problem. Over a billion people worldwide are VD-deficient or insufficient. VD-regulates the functions of over 200 genes and is essential for growth and development. VD-deficiency can result in diabetes, hypertension, heart disease and stroke. VD levels correlate positively with GSH and negatively with insulin resistance in type 2 diabetic patients. HFD-fed mice exhibited elevated blood glucose, and decreased blood GSH and 25(OH)VD with reduced GSH and increased oxidative stress in the liver. We investigated whether GSH deficiency induces epigenetic alterations in VD-metabolism genes in the high-fat diet (HFD) fed mice. The levels of VD-metabolism genes (CYP2R1, CYP27A1, CYP27B1, and VDR) and GSH biosynthesis pathway genes (GCLC, GCLM, GSS, and GSR) were significantly (p<0.005) down-regulated in the liver of HFD-fed mice. Conversely, CYP24A1 which metabolizes the active VD was significantly (p<0.005) increased. In vitro mouse hepatocyte GCLC knockdown decreases GSH and alters VD-regulatory genes in the similar pattern of HFD-fed mice. Further, significant increased DNMTs (1, 3a and 3b) and global DNA methylation levels with decreased TET1 (p<0.005) but not TET2/3 were observed in the cultured hepatocytes and liver. Notably, significant (p<0.005) gene-specific hypermethylation of CYP2R1 (1.71, 2.55; Control vs. HFD), CYP27A1 (7.58, 8.68), CYP27B1 (0.11, 0.77), VDR (0.16, 0.46) and hypomethylation of CYP24A1 (0.59, 0.33) was observed in the liver of HFD-fed (16 weeks) mice compared with control (normal diet) group. GSH deficiency-induced epigenetic alterations (methylation status) of VD-metabolism enzyme genes and the VDR gene appear to play a significant role in the circulating VD. These findings indicate that GSH epigenetically regulates VD-metabolism genes in obesity/T2DM and a novel adjuvant therapeutic target for restoring VD-deficiency in susceptible populations. Disclosure R. Parsanathan: None. S.K. Jain: None.

The glutathione redox system is essential to prevent ferroptosis caused by impaired lipid metabolism in clear cell renal cell carcinoma.

Metabolic reprogramming is a prominent feature of clear cell renal cell carcinoma (ccRCC). Here we investigated metabolic dependencies in a panel of ccRCC cell lines using nutrient depletion, functional RNAi screening and inhibitor treatment. We found that ccRCC cells are highly sensitive to the depletion of glutamine or cystine, two amino acids required for glutathione (GSH) synthesis. Moreover, silencing of enzymes of the GSH biosynthesis pathway or glutathione peroxidases, which depend on GSH for the removal of cellular hydroperoxides, selectively reduced viability of ccRCC cells but did not affect the growth of non-malignant renal epithelial cells. Inhibition of GSH synthesis triggered ferroptosis, an iron-dependent form of cell death associated with enhanced lipid peroxidation. VHL is a major tumour suppressor in ccRCC and loss of VHL leads to stabilisation of hypoxia inducible factors HIF-1α and HIF-2α. Restoration of functional VHL via exogenous expression of pVHL reverted ccRCC cells to an oxidative metabolism and rendered them insensitive to the induction of ferroptosis. VHL reconstituted cells also exhibited reduced lipid storage and higher expression of genes associated with oxidiative phosphorylation and fatty acid metabolism. Importantly, inhibition of β-oxidation or mitochondrial ATP-synthesis restored ferroptosis sensitivity in VHL reconstituted cells. We also found that inhibition of GSH synthesis blocked tumour growth in a MYC-dependent mouse model of renal cancer. Together, our data suggest that reduced fatty acid metabolism due to inhibition of β-oxidation renders renal cancer cells highly dependent on the GSH/GPX pathway to prevent lipid peroxidation and ferroptotic cell death.

Sulfur Partitioning between Glutathione and Protein Synthesis Determines Plant Growth.

Photoautotrophic organisms must efficiently allocate their resources between stress-response pathways and growth-promoting pathways to be successful in a constantly changing environment. In this study, we addressed the coordination of sulfur flux between the biosynthesis of the reactive oxygen species scavenger glutathione (GSH) and protein translation as one example of a central resource allocation switch. We crossed the Arabidopsis (Arabidopsis thaliana) GSH synthesis-depleted cadmium-sensitive cad2-1 mutant, which lacks glutamate cysteine (Cys) ligase, into the sulfite reductase sir1-1 mutant, which suffers from a significantly decreased flux of sulfur into Cys and, consequently, is retarded in growth. Surprisingly, depletion of GSH synthesis promoted the growth of the sir1-1 cad2-1 double mutant (s1c2) when compared with sir1-1 Determination of GSH levels and in vivo live-cell imaging of the reduction-oxidation-sensitive green fluorescent protein sensor demonstrated significant oxidation of the plastidic GSH redox potential in cad2-1 and s1c2 This oxidized GSH redox potential aligned with significant activation of plastid-localized sulfate reduction and a significantly higher flux of sulfur into proteins. The specific activation of the serine/threonine sensor kinase Target of Rapamycin (TOR) in cad2-1 and s1c2 was the trigger for reallocation of Cys from GSH biosynthesis into protein translation. Activation of TOR in s1c2 enhanced ribosome abundance and partially rescued the decreased meristematic activity observed in sir1-1 mutants. Therefore, we found that the coordination of sulfur flux between GSH biosynthesis and protein translation determines growth via the regulation of TOR.

Functional analysis and heterologous expression of bifunctional glutathione synthetase from Lactobacillus

Bifunctional glutathione synthetase (GshF) has recently been reported to simultaneously catalyze the 2-step ATP-dependent biosynthesis of reduced glutathione (GSH). In this work, 19 putative gshF were mined from the complete sequenced genome of 20 representative Lactobacillus species. To functionally analyze these putative GshF, GshF from Lactobacillus plantarum and Lactobacillus casei were selected and successfully expressed in Escherichia coli. Compared with the control without expressing GshF, GSH titers were enhanced significantly in E. coli with overexpression of GshF, demonstrating that putative GshF from Lactobacillus have functional activities on GSH biosynthesis. Moreover, with the expression of GshF from L. plantarum in E. coli as a paradigm, GSH yield (286.5 μM) was strongly improved by 177.9% with optimized induced conditions and precursor concentration compared with the control under unoptimized conditions. Transcriptional analysis showed that key genes of endogenous GSH metabolism and precursor biosynthesis were remarkably suppressed by GshF expression, indicating that the increase of GSH titer was attributed to heterologous expression of GshF. Overall, our results suggested that gshF is enriched in Lactobacillus and that heterologous expression of GshF is an efficient strategy for improving GSH biosynthesis.

OP-35 - Glutathione S-transferase P influences the Nrf2-dependent control of cellular glutathione metabolism

Glutathione-S-transferase P-1 (GSTP) was recently proposed to interact with Nrf2 thus regulating its transcriptional activity. The possible consequences of such functional interaction on the metabolism and redox of cellular GSH remain unexplored and were investigated for the first time in this study using both genetic and pharmacological manipulation models of GSTP in murine embryonic fibroblasts (MEFs). The GSTP-/- genotype was associated with a higher nuclear translocation of Nrf2 and apoptotic signaling of MEFs, and then with a marked increase of Cys uptake that resulted in a higher de novo biosynthesis and secretion of cellular GSH. Se-organic compounds (SOC) further stimulated these responses with a potency that correlated with the thiol peroxidase activity of the compounds. A defective expression of GSTP and SOC treatments also influenced the oxidation of GSH to GSSG and protein glutathionylation (PSSG) that was confirmed to be a GSTP-dependent process. The transient manipulation of Nrf2 gene interfered with the SOC-stimulated synthesis and secretion of GSH in the GSTP-/- aplotype, but not in GSTP+/+ cells. In conclusion, the GSTP-Nrf2 interaction strongly influences the metabolism and redox-signaling function of cellular GSH.

1331 Glutathione (GSH) distribution by quantitative MALDI imaging in reconstructed human skin upon activation of GSH biosynthesis by Nrf2 pathway activator

Skin is particularly exposed to oxidative stress, either from environmental insults such as sunlight or pollution or as a consequence of specific impairments in antioxidant status resulting from pathologies or aging. Traditionally, antioxidant products are exogenously provided to neutralize pro-oxidant species. However, another approach based on stimulation of endogenous antioxidant defense pathways is more original. Resveratrol (RSV) was reported to activate the Nrf2 pathway at nontoxic doses, from 20 μM up to 100μM, in primary culture of NHKs or in full-thickness reconstructed human skin. In parallel, a significant increase in glutathione (GSH) content, assessed by LC/MS analysis, was observed in both models. Following the development of a dedicated protocol for the reconstructed skin sectioning, a perfect visualization of the Stratum corneum, Epidermis was obtained on 10μm sections of some frozen samples. Thus MALDI-FTICR imaging at 20μm of spatial resolution was used to investigate the GSH distribution and relative quantification in both Stratum corneum and Epidermis of RSV treated reconstructed human skins at 20 and 100μM compared to a non-treated condition. GSH amount was confirmed to be significatively increased in the reconstructed tissues following a Resveratrol treatment at 20 μM and 100μM.

L-Glutamine Attenuates Apoptosis in Porcine Enterocytes by Regulating Glutathione-Related Redox Homeostasis

BackgroundProgrammed cell death plays a fundamental role in intestinal development and mucosal homeostasis. Dysregulation of these processes is associated with an impaired intestinal-mucosal barrier, reduced nutrient absorption, and initiation and progression of intestinal diseases. 4-Hydroxy-2-nonenal (4-HNE), a product of lipid peroxidation, is commonly used to induce oxidative stress in cells. l-Glutamine is known to protect cells from apoptosis. However, the underlying mechanisms are largely unknown. ObjectiveThis study was conducted to test the hypothesis that l-glutamine attenuates 4-HNE–induced apoptosis by modulating glutathione (GSH) and thioredoxin (TXN) antioxidant systems and the expression of genes involved in 4-HNE metabolism in enterocytes. MethodsIntestinal porcine epithelial cell line 1 (IPEC-1) cells were cultured with or without 4-HNE (30 μmol/L) in the presence of 0.05 or 0.25 mmol l-glutamine/L (a physiological concentration in the lumen of the small intestine) for indicated time periods. Cell viability, abundances of apoptotic proteins, mitochondrial membrane depolarization, production of reactive oxygen species (ROS) and GSH, and expression of genes involved in the biosynthesis of GSH, thioredoxin, and 4-HNE metabolism were determined. ResultsCompared with basal medium containing 0.05 mmol l-glutamine/L, 4-HNE enhanced apoptosis by 19.6% (P < 0.05) in a caspase-3-dependent manner. This effect was accompanied by elevated intracellular ROS production (39.5% and 85.3% for 2- and 4-h treatment, respectively), increased mitochondrial depolarization by 80%, and decreased intracellular GSH concentrations by 17.7%. These effects of 4-HNE were reduced by 0.25 mmol l-glutamine/L. Further study showed that the protective effect of l-glutamine was associated with the enhanced expression of genes involved in GSH production (including GCLC, GCLM, GSR, CBS, and CTH) by 3.9–14-fold, as well as genes involved in 4-HNE metabolism [e.g., glutathione S-transferase A (GSTA)1 and GSTA4] by 1.9–7.2-fold. The mRNA levels for ADH5, AKR1C1, AKR1A1, and TXNRD1 were enhanced 1.4–8.8-fold by 4-HNE but were not changed in cells co-treated with 4-HNE and l-glutamine. ConclusionThese findings indicate that l-glutamine attenuates 4-HNE–induced apoptosis by regulating GSH-related redox homeostasis and enhancing GSTA-mediated metabolism in enterocytes.

Nitric oxide-induced salt stress tolerance in plants: ROS metabolism, signaling, and molecular interactions

Nitric oxide (NO), a non-charged, small, gaseous free-radical, is a signaling molecule in all plant cells. Several studies have proposed multifarious physiological roles for NO, from seed germination to plant maturation and senescence. Nitric oxide is thought to act as an antioxidant, quenching ROS during oxidative stress and reducing lipid peroxidation. NO also mediates photosynthesis and stomatal conductance and regulates programmed cell death, thus providing tolerance to abiotic stress. In mitochondria, NO participates in the electron transport pathway. Nitric oxide synthase and nitrate reductase are the key enzymes involved in NO-biosynthesis in aerobic plants, but non-enzymatic pathways have been reported as well. Nitric oxide can interact with a broad range of molecules, leading to the modification of protein activity, GSH biosynthesis, S-nitrosylation, peroxynitrite formation, proline accumulation, etc., to sustain stress tolerance. In addition to these interactions, NO interacts with fatty acids to form nitro-fatty acids as signals for antioxidant defense. Polyamines and NO interact positively to increase polyamine content and activity. A large number of genes are reprogrammed by NO; among these genes, proline metabolism genes are upregulated. Exogenous NO application is also shown to be involved in salinity tolerance and/or resistance via growth promotion, reversing oxidative damage and maintaining ion homeostasis. This review highlights NO-mediated salinity-stress tolerance in plants, including NO biosynthesis, regulation, and signaling. Nitric oxide-mediated ROS metabolism, antioxidant defense, and gene expression and the interactions of NO with other bioactive molecules are also discussed. We conclude the review with a discussion of unsolved issues and suggestions for future research.

Acrolein-stressed threshold adaptation alters the molecular and metabolic bases of an engineered Saccharomyces cerevisiae to improve glutathione production

Acrolein (Acr) was used as a selection agent to improve the glutathione (GSH) overproduction of the prototrophic strain W303-1b/FGPPT. After two rounds of adaptive laboratory evolution (ALE), an unexpected result was obtained wherein identical GSH production was observed in the selected isolates. Then, a threshold selection mechanism of Acr-stressed adaption was clarified based on the formation of an Acr-GSH adduct, and a diffusion coefficient (0.36 ± 0.02 μmol·min−1·OD600−1) was calculated. Metabolomic analysis was carried out to reveal the molecular bases that triggered GSH overproduction. The results indicated that all three precursors (glutamic acid (Glu), glycine (Gly) and cysteine (Cys)) needed for GSH synthesis were at a relativity higher concentration in the evolved strain and that the accumulation of homocysteine (Hcy) and cystathionine might promote Cys synthesis and then improve GSH production. In addition to GSH and Cys, it was observed that other non-protein thiols and molecules related to ATP generation were at obviously different levels. To divert the accumulated thiols to GSH biosynthesis, combinatorial strategies, including deletion of cystathionine β-lyase (STR3), overexpression of cystathionine γ-lyase (CYS3) and cystathionine β-synthase (CYS4), and reduction of the unfolded protein response (UPR) through up-regulation of protein disulphide isomerase (PDI), were also investigated.

The dual role of mitochondrial superoxide in arsenite toxicity: Signaling at the boundary between apoptotic commitment and cytoprotection

Arsenite toxicity is in numerous cellular systems dependent on the formation of reactive oxygen and or nitrogen species. This is also true in U937 cells in which the metalloid selectively promotes the formation of mitochondrial superoxide (mitoO2−) rapidly converted to diffusible H2O2. We tested the hypothesis that, under the same conditions, mitoO2− also mediates the triggering of a parallel survival signaling. We found that a low concentration of the metalloid causes an early activation of nuclear factor erythroid 2 p45-related factor 2 (Nrf2), and a downstream signaling leading to enhanced GSH biosynthesis, via a mechanism sensitive to various treatments/strategies selectively preventing mitoO2− formation. Under the same conditions, the toxic effects mediated by arsenite, leading to delayed mitochondrial permeability transition (MPT)-dependent apoptosis, were also prevented. Additional studies revealed remarkable similarities in the kinetics of mitoO2− formation, MPT induction, Nrf2 activation and GSH biosynthesis, prior to the onset of apoptosis in a small portion of the cells. Importantly, mitoO2− formation, as well as the ensuing toxic events, were significantly potentiated and anticipated under conditions associated with inhibition of de novo GSH biosynthesis triggered by the metalloid through Nrf2 activation.We conclude that, in the arsenite toxicity paradigm under investigation, mitoO2− represents the only trigger of two opposite pathways leading to activation of the Nrf2 signaling and/or to a MPT-dependent apoptotic death. The first pathway, through enhanced GSH biosynthesis, mitigates the extent of further mitoO2− formation, thereby limiting and delaying an otherwise rapid and massive apoptotic death.

Ophthalmic acid is a marker of oxidative stress in plants as in animals

BackgroundOphthalmic acid (OPH), γ-glutamyl-L-2-aminobutyryl-glycine, a tripeptide analogue of glutathione (GSH), has recently captured considerable attention as a biomarker of oxidative stress in animals. The OPH and GSH biosynthesis, as well as some biochemical behaviors, are very similar. Here, we sought to investigate the presence of OPH in plants and its possible relationship with GSH, known to possess multiple functions in the plant development, growth and response to environmental changes. MethodsHPLC-ESI-MS/MS analysis was used to examine the occurrence of OPH in leaves from various plant species, and flours from several plant seeds. Different types of oxidative stress, i.e., water, dark, paraquat, and cadmium stress, were induced in rye, barley, oat, and winter wheat leaves to evaluate the effects on the levels of OPH and its metabolic precursors. ResultsOPH and its dipeptide precursor, γ-glutamyl-2-aminobutyric acid, were found to occur in phylogenetically distant plants. Interestingly, the levels of OPH were tightly associated with the oxidative stress tested. Levels of OPH precursors, γ-glutamyl-2-aminobutyric acid and 2-aminobutyric acid, the latter efficiently formed in plants via biosynthetic pathways absent in the animal kingdom, were also found to increase during oxidative stress. ConclusionsOPH occurs in plants and its levels are tightly associated with oxidative stress. General significanceOPH behaves as an oxidative stress marker and its biogenesis might occur through a biochemical pathway common to many living organisms.

Hydrogen sulfide increases glutathione biosynthesis, and glucose uptake and utilisation in C2C12 mouse myotubes

Diabetic patients have lower blood concentrations of hydrogen sulfide (H2S), L-cysteine (LC), and glutathione (GSH). Using C2C12 mouse myotubes as a model, this study investigates the hypothesis that the beneficial effects of LC supplementation are mediated by upregulation of the H2S status under diabetic conditions. Results show that exogenous administration of sodium hydrosulfide (NaHS, 10 or 20 µM; 6 hours), a H2S donor, significantly (p < .05) upregulates the gene expression of cystathionine-γ-lyase (CSE), LC transporter (Slc7a11/xCT), and the genes involved in GSH biosynthesis. Additionally, it reduces homocysteine (HCys), reactive oxygen species (ROS) production, and enhances cellular LC, H2S, and glucose uptake and utilisation in myoblasts. The use of CSE siRNA to induce deficient endogenous H2S production causes an increase in H2O2, ROS, HCys levels, and downregulation of GSH biosynthesis pathway enzymes. In additional, CSE knockdown downregulates glucose transporter type 4 (GLUT4) and gene expression of its key transcription factors, and reduces glucose uptake in C2C12 myotubes. CSE knockdown cells showed specific increases in the protein S-glutathionylation of LC transporter and GLUT4 along with increased total protein S-glutathionylation. Taken together, evidence from this study provides molecular insights into the importance of the CSE/H2S system in maintaining the cellular glutathione and glucose homeostasis in C2C12 myotubes.

Modulation of O-GlcNAc Levels in the Liver Impacts Acetaminophen-Induced Liver Injury by Affecting Protein Adduct Formation and Glutathione Synthesis.

Overdose of acetaminophen (APAP) results in acute liver failure. We have investigated the role of a posttranslational modification of proteins called O-GlcNAcylation, where the O-GlcNAc transferase (OGT) adds and O-GlcNAcase (OGA) removes a single β-D-N-acetylglucosamine (O-GlcNAc) moiety, in the pathogenesis of APAP-induced liver injury. Hepatocyte-specific OGT knockout mice (OGT KO), which have reduced O-GlcNAcylation, and wild-type (WT) controls were treated with 300 mg/kg APAP and the development of injury was studied over a time course from 0 to 24 h. OGT KO mice developed significantly lower liver injury as compared with WT mice. Hepatic CYP2E1 activity and glutathione (GSH) depletion following APAP treatment were not different between WT and OGT KO mice. However, replenishment of GSH and induction of GSH biosynthesis genes were significantly faster in the OGT KO mice. Next, male C57BL/6 J mice were treated Thiamet-G (TMG), a specific inhibitor of OGA to induce O-GlcNAcylation, 1.5 h after APAP administration and the development of liver injury was studied over a time course of 0-24 h. TMG-treated mice exhibited significantly higher APAP-induced liver injury. Treatment with TMG did not affect hepatic CYP2E1 levels, GSH depletion, APAP-protein adducts, and APAP-induced mitochondrial damage. However, GSH replenishment and GSH biosynthesis genes were lower in TMG-treated mice after APAP overdose. Taken together, these data indicate that induction in cellular O-GlcNAcylation exacerbates APAP-induced liver injury via dysregulation of hepatic GSH replenishment response.

Glutathione and Transsulfuration in Alcohol-Associated Tissue Injury and Carcinogenesis.

Glutathione (GSH) is the most abundant non-protein thiol, attaining cellular concentrations in the millimolar range. GSH functions to protect cells against endogenous and exogenous electrophiles. In addition, GSH serves as a cofactor for the GSH peroxidase family of enzymes which metabolize H2O2 as well as lipid peroxides. Through the action of glutathione S-transferase family of enzymes, GSH is conjugated to a variety of electrophilic endogenous compounds and exogenous chemicals, and thereby facilitates their efficient and safe elimination. Through the transsulfuration pathway, GSH biosynthesis is metabolically linked with cellular methylation, which is pivotal for epigenetic gene regulation. Accumulating evidence suggests that the underlying mechanisms of alcohol-associated tissue injury and carcinogenesis involve: (i) generation of the electrophilic metabolite acetaldehyde, (ii) induction of CYP2E1 leading to the formation of reactive oxygen species and pro-carcinogen activation, and (iii) nutritional deficiencies, such as methyl groups, resulting in enhanced susceptibility to cancer development. In this context, clinical and experimental investigations suggest an intimate involvement of GSH and related enzymes in the development of alcohol-induced pathological conditions. The aim of this review is to provide an overview of the GSH biosynthesis, cellular transsulfuration/transmethylation pathways, and their implications in the pathogenesis and treatment of alcohol-related disease and cancer.

Rice phytochelatin synthases OsPCS1 and OsPCS2 make different contributions to cadmium and arsenic tolerance.

Cadmium (Cd) and arsenic (As) pollution in paddy soil and their accumulation in rice (Oryza sativa) pose serious threats to human health. Rice internally detoxifies these toxic metal and metalloid to some extent, resulting in their accumulation within the edible parts. However, the mechanisms of Cd and As detoxification in rice have been poorly elucidated. Plants synthesize thiol‐rich metal‐chelating peptides, termed phytochelatins (PCs). We characterized rice PC synthase (PCS) and investigated its contribution to Cd and As tolerance in rice. We identified two PCS homolog genes, OsPCS1 and OsPCS2, in the rice genome. The expression of OsPCS1 was upregulated by As(III) stress in the roots but that of OsPCS2 was not significantly affected. The expression level of OsPCS2 was higher than that of OsPCS1 in the shoots and roots. Recombinant OsPCS1 and OsPCS2 proteins differed in their metal activation. OsPCS1 was more strongly activated by As(III) than by Cd; however, OsPCS2 was more strongly activated by Cd than by As(III). Genetically engineered plants having their OsPCS2 expression silenced via RNA interference (OsPCS2 RNAi) contained less PCs and more glutathione (GSH), a substrate of PC synthesis, than wild‐type plants, although there was no significant difference in OsPCS1 RNAi plants. OsPCS2 RNAi plants were sensitive to As(III) stress, but Cd tolerance was little affected. On the other hand, treatment with buthionine sulfoximine, an inhibitor of GSH biosynthesis, significantly decreased Cd and As tolerance of rice seedlings. These findings indicate that OsPCS2 is a major isozyme controlling PC synthesis, and that PCs are important for As tolerance in rice. However, PC synthesis may make a smaller contribution to Cd tolerance in rice, and GSH plays crucial roles, not only as a substrate of PC synthesis.

Glycine Increases Insulin Sensitivity and Glutathione Biosynthesis and Protects against Oxidative Stress in a Model of Sucrose-Induced Insulin Resistance.

Oxidative stress and redox status play a central role in the link between insulin resistance (IR) and lipotoxicity in metabolic syndrome. This mechanistic link may involve alterations in the glutathione redox state. We examined the effect of glycine supplementation to diet on glutathione biosynthesis, oxidative stress, IR, and insulin cell signaling in liver from sucrose-fed (SF) rats characterized by IR and oxidative stress. Our hypothesis is that the correction of glutathione levels by glycine treatment leads to reduced oxidative stress, a mechanism associated with improved insulin signaling and IR. Glycine treatment decreases the levels of oxidative stress markers in liver from SF rats and increases the concentrations of glutathione (GSH) and γ-glutamylcysteine and the amount of γ-glutamylcysteine synthetase (γ-GCS), a key enzyme of GSH biosynthesis in liver from SF rats. In liver from SF rats, glycine also decreases the insulin-induced phosphorylation of insulin receptor substrate-1 (ISR-1) in serine residue and increases the phosphorylation of insulin receptor β-subunit (IR-β) in tyrosine residue. Thus, supplementing diets with glycine to correct GSH deficiency and to reduce oxidative stress provides significant metabolic benefits to SF rats by improving insulin sensitivity.

NMR-based metabolomic analysis of the effects of alanyl-glutamine supplementation on C2C12 myoblasts injured by energy deprivation.

The dipeptide alanyl-glutamine (Ala-Gln) is a well-known parenteral nutritional supplement. The Ala-Gln supplementation is a potential treatment for muscle-related diseases and injuries. However, molecular mechanisms underlying the polyphenic effects of Ala-Gln supplementation remain elusive. Here, we performed NMR-based metabolomic profiling to analyze the effects of Ala-Gln, and the free alanine (Ala) and glutamine (Gln) supplementations on the mouse myoblast cell line C2C12 injured by glucose and glutamine deprivation. All the three supplementations can promote the differentiation ability of the injured C2C12 cells, while only Ala-Gln supplementation can facilitate the proliferation of the injured cells. Ala-Gln supplementation can partially restore the metabolic profile of C2C12 myoblasts disturbed by glucose and glutamine deprivation, and exhibits more significant effects than Ala and Gln supplementations. Our results suggest that Ala-Gln supplementation can promote MyoD1 protein synthesis, upregulate the muscle ATP-storage phosphocreatine (PCr), maintain TCA cycle anaplerosis, enhance the antioxidant capacity through promoting GSH biosynthesis, and stabilize lipid membranes by suppressing glycerophospholipids metabolism. This work provides new insight into mechanistic understanding of the polyphenic effects of Ala-Gln supplementation on muscle cells injured by energy deprivation.