Gclm Null Mice Research Articles

CYP2E1 in 1,4-dioxane metabolism and liver toxicity: insights from CYP2E1 knockout mice study.

1,4-Dioxane (DX), an emerging water contaminant, is classified as a Group 2B liver carcinogen based on animal studies. Understanding of the mechanisms of action of DX liver carcinogenicity is important for the risk assessment and control of this environmental pollution. Previous studies demonstrate that high-dose DX exposure in mice through drinking water for up to 3 months caused liver mild cytotoxicity and oxidative DNA damage, a process correlating with hepatic CYP2E1 induction and elevated oxidative stress. To access the role of CYP2E1 in DX metabolism and liver toxicity, in the current study, male and female Cyp2e1-null mice were exposed to DX in drinking water (5000 ppm) for 1 week or 3 months. DX metabolism, redox and molecular investigations were subsequently performed on male Cyp2e1-null mice for cross-study comparisons to similarly treated male wildtype (WT) and glutathione (GSH)-deficient Gclm-null mice. Our results show that Cyp2e1-null mice of both genders were resistant to DX-induced hepatocellular cytotoxicity. In male Cyp2e1-null mice exposed to DX for 3 months, firstly, DX metabolism to β-hydroxyethoxyacetic acid was reduced to ~ 36% of WT levels; secondly, DX-induced hepatic redox dysregulation (lipid peroxidation, GSH oxidation, and activation of NRF2 antioxidant response) was substantially attenuated; thirdly, liver oxidative DNA damage was at a comparable level to DX-exposed WT mice, accompanied by suppression of DNA damage repair response; lastly, no aberrant proliferative or preneoplastic lesions were noted in DX-exposed livers. Overall, this study reveals, for the first time, that CYP2E1 is the main enzyme for DX metabolism at high dose and a primary contributor to DX-induced liver oxidative stress and associated cytotoxicity. High dose DX-induced genotoxicity may occur via CYP2E1-independent pathway(s), potentially involving impaired DNA damage repair.

Decreased glutathione synthesis in granulosa cells, but not oocytes, of growing follicles decreases fertility in mice.

Prior studies showed that mice deficient in the modifier subunit of glutamate cysteine ligase (Gclm), the rate-limiting enzyme in synthesis of the thiol antioxidant glutathione (GSH), have decreased ovarian GSH concentrations, chronic ovarian oxidative stress, poor oocyte quality resulting in early preimplantation embryonic mortality and decreased litter size, and accelerated age-related decline in ovarian follicle numbers. Global deficiency of the catalytic subunit of this enzyme, Gclc, is embryonic lethal. We tested the hypothesis that granulosa cell- or oocyte-specific deletion of Gclc recapitulates the female reproductive phenotype of global Gclm deficiency. We deleted Gclc in granulosa cells or oocytes of growing follicles using Gclc floxed transgenic mice paired with Amhr2-Cre or Zp3-Cre alleles respectively. We discovered that granulosa cell-specific deletion of Gclc in Amhr2Cre;Gclc(f/-) mice recapitulates the decreased litter size observed in Gclm-/- mice, but does not recapitulate the accelerated age-related decline in ovarian follicles observed in Gclm-/- mice. In addition to having lower GSH concentrations in granulosa cells, Amhr2Cre;Gclc(f/-) mice also had decreased GSH concentrations in oocytes. By contrast, oocyte-specific deletion of Gclc in Zp3Cre;Gclc(f/-) mice did not affect litter size or accelerate the age-related decline in follicle numbers, and these mice did not have decreased oocyte GSH concentrations, consistent with transport of GSH between cells via gap junctions. The results suggest that GSH deficiency at earlier stages of follicle development may be required to generate the accelerated follicle depletion phenotype observed in global Gclm null mice.

Persistence of improved glucose homeostasis in Gclm null mice with age and cadmium treatment

Antioxidant signaling/communication is among the most important cellular defense and survival pathways, and the importance of redox signaling and homeostasis in aging has been well-documented. Intracellular levels of glutathione (GSH), a very important endogenous antioxidant, both govern and are governed by the Nrf2 pathway through expression of genes involved in its biosynthesis, including the subunits of the rate-limiting enzyme (glutamate cysteine ligase, GCL) in GSH production, GCLC and GCLM. Mice homozygous null for the Gclm gene are severely deficient in GSH compared to wild-type controls, expressing approximately 10% of normal GSH levels. To compensate for GSH deficiency, Gclm null mice have upregulated redox-regulated genes, and, surprisingly, are less susceptible to certain types of oxidative damage. Furthermore, young Gclm null mice display an interesting lean phenotype, resistance to high fat diet-induced diabetes and obesity, improved insulin and glucose tolerance, and decreased expression of genes involved in lipogenesis. However, the persistence of this phenotype has not been investigated into old age, which is important in light of studies which suggest aging attenuates antioxidant signaling, particularly in response to exogenous stimuli. In this work, we addressed whether aging compromises the favorable phenotype of increased antioxidant activity and improved glucose homeostasis observed in younger Gclm null mice. We present data showing that under basal conditions and in response to cadmium exposure (2 mg/kg, dosed once via intraperitoneal injection), the phenotype previously described in young (<6 months) Gclm null mice persists into old age (24+ months). We also provide evidence that transcriptional activation of the Nrf2, AMPK, and PPARγ pathways underlie the favorable metabolic phenotype observed previously in young Gclm null mice.

Hepatic metabolic adaptation in a murine model of glutathione deficiency

Glutathione (GSH), the most abundant cellular non-protein thiol, plays a pivotal role in hepatic defense mechanisms against oxidative damage. Despite a strong association between disrupted GSH homeostasis and liver diseases of various etiologies, it was shown that GSH-deficient glutamate-cysteine ligase modifier subunit (Gclm)-null mice are protected against fatty liver development induced by a variety of dietary and environmental insults. The biochemical mechanisms underpinning this protective phenotype have not been clearly defined. The purpose of the current study was to characterize the intrinsic metabolic signature in the livers from GSH deficient Gclm-null mice. Global profiling of hepatic polar metabolites revealed a spectrum of changes in amino acids and metabolites derived from fatty acids, glucose and nucleic acids due to the loss of GCLM. Overall, the observed low GSH-driven metabolic changes represent metabolic adaptations, including elevations in glutamate, aspartate, acetyl-CoA and gluconate, which are beneficial for the maintenance of cellular redox and metabolic homeostasis.

329 - Hepatic epigenomic changes associated with chronic oxidative stress in a mouse model of glutathione deficiency

Purpose Oxidative stress has been shown to be intimately involved in the pathogenesis of fatty liver disease (FLD). A major factor contributing to oxidative stress is the depletion of glutathione (GSH). Our previous studies in glutamate cysteine ligase modifier subunit (Gclm)-null mice demonstrate that ~85% deficiency in hepatic GSH renders mice protected from fatty liver injuries induced by varieties of hepatic insults, including alcohol exposure. It is well-known that epigenetic mechanisms play active roles in modulating gene expression and disease outcome in FLD. The purpose of the current study is to utilize the Gclm-null mouse model to examine in vivo interplay between chronic oxidative stress and DNA methylation at the epigenome level and whether these epigenetic changes are functionally involved in FLD. Methods we performed DNA methylation profiling in the liver and whole blood using the Mouse Promoter Methylation Array. Genes with CpG sites that were significantly affected by GSH deficiency were further investigated for network and functional enrichment. Results Among 22,327 gene promoter regions (from -1300bp to +500bp of the Transcription Start Sites) that were analyzed, in the liver, only 52 and 6 gene promoters were hypo- and hyper-methylated, respectively, in Gclm-null mice relative to wild-type mice, indicating an overall hypo-methylated genome associated with low GSH. Interestingly, the DNA methylation profiles in the liver and blood appeared to be comparable irrespective of the genotype, which leads to the possibility of potential circulating biomarker(s) for hepatic epigenetic fingerprints related to chronic oxidative stress. Ingenuity Pathway Analysis (IPA) revealed that hypo-methylated genes in the KO liver are enriched in canonical pathways of protein phosphorylation, RNA transcription, cytoplasm organization and organismal death. Conclusion our preliminary data indicate that chronic GSH deficiency induces global DNA hypomethylation. The functional significance of observed epigenomic changes is under investigation.

58 - Plasticity of Antioxidant Defense Pathways in Response to Aging and Cadmium in Glutathione-Deficient Mice

Maintaining cellular homeostasis is an important determinant of health throughout the lifespan. The Nrf2 (NFE2L2) transcription factor is one of the most important for cellular defense and survival. Nrf2 and its orthologs have been shown to decrease aging associated pathology and increase longevity in a number of species. Two important Nrf2-regulated genes are glutamate cysteine ligase catalytic (GCLC) and modifier (GCLM) subunits, which are critical for the synthesis of the antioxidant glutathione (GSH). Gclm null mice are severely deficient in GSH compared to wild-type mice (~ 10% of WT). To compensate for low GSH, Gclm null mice have increased many Nrf2-regulated genes, and exhibit greater resistance to some but not all stressors. Young Gclm null mice also display a lean phenotype, resistance to high fat diet-induced diabetes and obesity, and improved glucose homeostasis. Since Nrf2 signaling has been reported to attenuate with aging, we hypothesized that older Gclm null mice would display greater susceptibility to exogenous stressors. We challenged Gclm WT, heterozygous and null mice with cadmium (Cd) because of its association with type 2 diabetes in several species, and its canonical role as an oxidative stress-inducing heavy metal. During aging and acute Cd administration, we unexpectedly found that Gclm null mice continue to maintain improved GSH redox, increased Nrf2 target gene expression and inducibility, and improved parameters of glucose homeostasis, relative to Gclm wild-type mice. These surprising findings suggest that chronic adaption to severely compromised GSH levels, likely due to continuous upregulation of Nrf2 mediated responses, persists throughout the lifespan in these mice. Dietary or other interventions that upregulate the Nrf2 pathway could thus improve late-life insulin sensitivity, glucose homeostasis, and promote an improved healthspan.

180 - Age and Cadmium as Modifiers of Metabolic and Thiol-Based Redox Homeostasis in Glutathione-Deficient Mice

The Nrf2 signaling pathway is one of the most important cellular defense and survival pathways, and the importance of Nrf2 signaling in aging has been well-documented. Intracellular levels of glutathione (GSH), a very important endogenous antioxidant, both governs and is governed by the Nrf2 pathway through expression of genes involved in its biosynthesis, including the subunits of the rate-limiting enzyme (glutamate cysteine ligase, GCL) in GSH production, GCLC and GCLM. Mice homozygous for the Gclm gene are severely deficient in GSH compared to wild-type controls, expressing approximately 10% of normal GSH levels (McConnachie et al., 2007). To compensate for reduced GSH, Gclm null mice have upregulated Nrf2-regulated genes, and, surprisingly, are less susceptible to certain types of oxidative damage. Young (

Glutathione Deficiency in Gclm Null Mice Results in Complex I Inhibition and Dopamine Depletion Following Paraquat Administration

Depletion of glutathione has been shown to occur in autopsied brains of patients with Parkinson's disease (PD) and in animal models of PD. The goal of this study was to determine whether chronic glutathione (GSH) deficiency per se resulted in complex I inhibition and/or dopamine depletion and whether these indices were further potentiated by aging or administration of paraquat, a redox-cycling herbicide that produces a PD-like neurodegeneration model in rodents (Brooks, A. I., Chadwick, C. A., Gelbard, H. A., Cory-Slechta, D. A., and Federoff, H. J. [1999]. Paraquat elicited neurobehavioral syndrome caused by dopaminergic neuron loss. Brain Res. 823, 1-10; McCormack, A. L., Thiruchelvam, M., Manning-Bog, A. B., Thiffault, C., Langston, J. W., Cory-Slechta, D. A., and Di Monte, D. A. [2002]. Environmental risk factors and Parkinson's disease: Selective degeneration of nigral dopaminergic neurons caused by the herbicide paraquat. Neurobiol. Dis. 10, 119-127.) Deletion of the rate-limiting GSH synthesis gene, glutamate-cysteine ligase modifier subunit (Gclm), leads to significantly lower GSH concentrations in all tissues including brain. Gclm null (Gclm (-/-)) mice provide a model of prolonged GSH depletion to explore the relationship between GSH, complex I inhibition, and dopamine loss in vivo. Despite ~60% depletion of brain GSH in Gclm (-/-) mice of ages 3-5 or 14-16 months, striatal complex I activity, dopamine levels, 3-nitrotyroine/tyrosine ratios, aconitase activity, and CoASH remained unchanged. Administration of paraquat (10mg/kg, twice/week, 3 weeks) to 3- to 5-month-old Gclm (-/-) mice resulted in significantly decreased aconitase activity, complex I activity, and dopamine levels but not in 3- to 5-month-old Gclm (+/+) mice. Furthermore, paraquat-induced inhibition of complex I and aconitase activities in Gclm (-/-) mice was observed in the striatum but not in the cortex. The results suggest that chronic deficiency of GSH in Gclm (-/-) mice was not sufficient to result in complex I inhibition or dopamine depletion perhaps due to homeostatic mechanisms but required an additional oxidative stress insult as shown with paraquat exposure.

Bone-Marrow Derived Cells Can Reconstitute GCLM Function in the Sickle Lung

Abstract 4836Sickle cell disease (SCD) is a chronic hemolytic and inflammatory disorder characterized by repeated episodes of vaso-occlusion and hemolysis, resulting in oxidative stress and endothelial dysfunction. We have recently demonstrated that the heme scavenging capacity in SCD is severely impaired, highlighting the danger posed by excess heme in this disorder. Paradoxically, heme induces expression of several cyto-protective enzymes including the modifier subunit of glutamate cysteine ligase (GCLM), the rate-limiting enzyme in glutathione (GSH) synthesis, which is a crucial antioxidant in the lung. While the induction of cytoprotective enzymes is thought to attenuate the deleterious effects of heme in SCD the somatic origin of this protection has not previously been defined. Using transgenic mouse models we show for the first time that the level of GCLM in the sickle lung is markedly up-regulated due primarily to enhanced expression of the enzyme in the epithelium and blood mononuclear cells, but not in the endothelium. Based on these findings, we tested the hypothesis that leukocyte-derived GCLM was sufficient to protect the sickle lung from oxidative stress. Thus, bone marrow chimeric SCD mice with GCLM deficiency were generated by transplanting bone marrow from Berkeley SCD transgenic mice into GCLM null mice recipients. We confirmed that the chimeric GCLM-null-SCD mice had a SCD phenotype as determined by >95% engraftment of donor white blood cells, reticulocyte counts, urine osmolality and hemoglobin gel electrophoresis. Whole lung GCLM and total GSH levels in the chimeric mice were identical to the levels in the wild-type SCD mice. Moreover, lung function, as determined by oxygen saturation and breath rate, were identical in the two mouse strains. These results show that loss of GCLM expression in resident lung cells does not compromise production of GSH or the function of the lung in SCD. Disclosures:Ofori-Acquah:Emory University: Patents & Royalties.

Attenuated progression of diet-induced steatohepatitis in glutathione-deficient mice

In nonalcoholic fatty liver disease (NAFLD), depletion of hepatic antioxidants may contribute to the progression of steatosis to nonalcoholic steatohepatitis (NASH) by increasing oxidative stress that produces lipid peroxidation, inflammation, and fibrosis. We investigated whether depletion of glutathione (GSH) increases NASH-associated hepatic pathology in mice fed a diet deficient in methionine and choline (MCD diet). Wild-type (wt) mice and genetically GSH-deficient mice lacking the modifier subunit of glutamate cysteine ligase (Gclm null mice), the rate-limiting enzyme for de novo synthesis of GSH, were fed the MCD diet, a methionine/choline-sufficient diet, or standard chow for 21 days. We assessed NASH-associated hepatic pathology, including steatosis, fibrosis, inflammation, and hepatocyte ballooning, and used the NAFLD Scoring System to evaluate the extent of changes. We measured triglyceride levels, determined the level of lipid peroxidation products, and measured by qPCR the expression of mRNAs for several proteins associated with lipid metabolism, oxidative stress, and fibrosis. MCD-fed GSH-deficient Gclm null mice were to a large extent protected from MCD diet-induced excessive fat accumulation, hepatocyte injury, inflammation, and fibrosis. Compared with wt animals, MCD-fed Gclm null mice had much lower levels of F2-isoprostanes, lower expression of acyl-CoA oxidase, carnitine palmitoyltransferase 1a, uncoupling protein-2, stearoyl-coenzyme A desaturase-1, transforming growth factor-β, and plasminogen activator inhibitor-1 mRNAs, and higher activity of catalase, indicative of low oxidative stress, inhibition of triglyceride synthesis, and lower expression of profibrotic proteins. Global gene analysis of hepatic RNA showed that compared with wt mice, the livers of Gclm null mice have a high capacity to metabolize endogenous and exogenous compounds, have lower levels of lipogenic proteins, and increased antioxidant activity. Thus, metabolic adaptations resulting from severe GSH deficiency seem to protect against the development of steatohepatitis.

Gclm Null Mice have Drastically Increased Angiogenic Potential

Previous studies have provided evidence that increased levels of glutathione (GSH) attenuate VEGF‐A induced cellular ROS production, thereby decreasing cell motility. The rate limiting step in GSH production is carried out by the holoenzyme glutamate‐cysteine ligase (GCL) which is composed of catalytic (Gclc) and modifier (Gclm) subunits. A mouse Gclm null model has been developed with tissue levels of GSH between 9 and 40% of wild type (WT) mice. This null model was employed to determine the role Gclm plays in redox regulation of angiogenesis following hind limb ischemia. The ischemic leg in the Gclm null mice had blood flows returning to pre‐ligation values by day 7, whereas WT blood flow had not recovered 21 days post‐ligation, indicating an increased cellular proliferation and migration in Gclm null mice. The angiogenic index in Gclm null mice was 2 fold higher in ischemic tissue compared with non‐ischemic tissue. The proliferative index was approximately 1 in ischemic tissue indicating that the proliferating cells were likely endothelial cells. Protein carbonyls were elevated, while GSH levels were depressed in the Gclm null mice. Together, these data provide strong evidence that Gclm null mice have a more intense angiogenic response to ischemia than WT mice. This may be due to increased VEGF‐A/NADPH oxidase signaling in these mice.

Modulating GSH Synthesis Using Glutamate Cysteine Ligase Transgenic and Gene-Targeted Mice

Glutathione (GSH) is an important antioxidant and cofactor for glutathione S-transferase conjugation. GSH synthesis is catalyzed by glutamate cysteine ligase (GCL), composed of catalytic (GCLC) and modifier (GCLM) subunits. Transgenic mice that conditionally over express GCL subunits are protected from acetaminophen induced liver injury. Gclm null mice exhibit low GSH levels and enhanced sensitivity to acetaminophen. When Gclm expression and GCL activity are restored in Gclm conditional transgenic X Gclm null mice, they become resistant to APAP-induced liver damage. These animal models are a valuable resource for investigating the role of GSH synthesis in modulating oxidative damage and drug-induced hepatotoxicity.

Redox Imbalance in Diabetes

Antioxidants & Redox SignalingVol. 9, No. 7 Forum EditorialRedox Imbalance in DiabetesMelvin R. Hayden and James R. SowersMelvin R. HaydenSearch for more papers by this author and James R. SowersSearch for more papers by this authorPublished Online:17 May 2007https://doi.org/10.1089/ars.2007.1640AboutSectionsPDF/EPUB ToolsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail FiguresReferencesRelatedDetailsCited ByPersistence of improved glucose homeostasis in Gclm null mice with age and cadmium treatmentRedox Biology, Vol. 49Mitochondrial Regulation of Diabetic Kidney Disease27 September 2021 | Frontiers in Medicine, Vol. 8The Link between Type 2 Diabetes Mellitus and the Polymorphisms of Glutathione-Metabolizing Genes Suggests a New Hypothesis Explaining Disease Initiation and Progression28 August 2021 | Life, Vol. 11, No. 9Immunometabolic bases of type 2 diabetes in the severity of COVID-19World Journal of Diabetes, Vol. 12, No. 7NADH/NAD+ Redox Imbalance and Diabetic Kidney Disease14 May 2021 | Biomolecules, Vol. 11, No. 5Disruption of mitochondrial redox homeostasis by enzymatic activation of a trialkylphosphine probe1 January 2021 | Organic & Biomolecular Chemistry, Vol. 19, No. 12An Immediate and Long-Term Complication of COVID-19 May Be Type 2 Diabetes Mellitus: The Central Role of β-Cell Dysfunction, Apoptosis and Exploration of Possible Mechanisms13 November 2020 | Cells, Vol. 9, No. 11Type 2 Diabetes Mellitus Increases The Risk of Late-Onset Alzheimer’s Disease: Ultrastructural Remodeling of the Neurovascular Unit and Diabetic Gliopathy29 September 2019 | Brain Sciences, Vol. 9, No. 10Monitoring Dynamic Cellular Redox Homeostasis Using Fluorescence-Switchable Graphene Quantum Dots1 December 2016 | ACS Nano, Vol. 10, No. 12Store-operated calcium entry and diabetic complications14 October 2015 | Experimental Biology and Medicine, Vol. 241, No. 4Nuclear Factor κB Mediates Suppression of Canonical Transient Receptor Potential 6 Expression by Reactive Oxygen Species and Protein Kinase C in Kidney CellsJournal of Biological Chemistry, Vol. 288, No. 18Molecular and metabolic mechanisms of cardiac dysfunction in diabetesLife Sciences, Vol. 92, No. 11La variabilité glycémique en réanimationAnnales Françaises d'Anesthésie et de Réanimation, Vol. 31, No. 12The emerging role of cardiovascular risk factor-induced mitochondrial dysfunction in atherogenesis9 December 2009 | Journal of Biomedical Science, Vol. 16, No. 1QUANTITATIVE REDOX SCANNING OF TISSUE SAMPLES USING A CALIBRATION PROCEDURE21 November 2011 | Journal of Innovative Optical Health Sciences, Vol. 02, No. 04Attenuation of Endocrine-Exocrine Pancreatic Communication in Type 2 Diabetes: Pancreatic Extracellular Matrix Ultrastructural AbnormalitiesJournal of the CardioMetabolic Syndrome, Vol. 3, No. 4Ultrastructure of Islet Microcirculation, Pericytes and the Islet Exocrine Interface in the HIP Rat Model of Diabetes1 September 2008 | Experimental Biology and Medicine, Vol. 233, No. 9Treating hypertension while protecting the vulnerable islet in the cardiometabolic syndromeJournal of the American Society of Hypertension, Vol. 2, No. 4 Volume 9Issue 7Jul 2007 InformationCopyright 2007, Mary Ann Liebert, Inc.To cite this article:Melvin R. Hayden and James R. Sowers.Redox Imbalance in Diabetes.Antioxidants & Redox Signaling.Jul 2007.865-867.http://doi.org/10.1089/ars.2007.1640Published in Volume: 9 Issue 7: May 17, 2007PDF download

Glutamate Cysteine Ligase Modifier Subunit Deficiency and Gender as Determinants of Acetaminophen-Induced Hepatotoxicity in Mice

The analgesic and antipyretic drug acetaminophen (APAP) is bioactivated to the reactive intermediate N-acetyl-p-benzoquinoneimine, which is scavenged by glutathione (GSH). APAP overdose can deplete GSH leading to the accumulation of APAP-protein adducts and centrilobular necrosis in the liver. N-acetylcysteine (NAC), a cysteine prodrug and GSH precursor, is often given as a treatment for APAP overdose. The rate-limiting step in GSH biosynthesis is catalyzed by glutamate cysteine ligase (GCL) a heterodimer composed of catalytic and modifier (GCLM) subunits. Previous studies have indicated that GCL activity is likely to be an important determinant of APAP toxicity. In this study, we investigated APAP toxicity, and NAC or GSH ethyl ester (GSHee)-mediated rescue in mice with normal or compromised GCLM expression. Gclm wild-type, heterozygous, and null mice were administered APAP (500 mg/kg) alone, or immediately following NAC (800 mg/kg) or GSHee (168 mg/kg), and assessed for hepatotoxicity 6 h later. APAP caused GSH depletion in all mice. Gclm null and heterozygous mice exhibited more extensive hepatic damage compared to wild-type mice as assessed by serum alanine aminotransferase activity and histopathology. Additionally, male Gclm wild-type mice demonstrated greater APAP-induced hepatotoxicity than female wild-type mice. Cotreatment with either NAC or GSHee mitigated the effects of APAP in Gclm wild-type and heterozygous mice, but not in Gclm null mice. Collectively, these data reassert the importance of GSH in protection against APAP-induced hepatotoxicity, and indicate critical roles for GCL activity and gender in APAP-induced liver damage in mice.