Dietary γ-Glutamylcysteine: Its Impact on Glutathione Status and Potential Health Outcomes.

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.

Supplementation with γ-glutamylcysteine (γ-GC) lessens oxidative stress, brain inflammation and amyloid pathology and improves spatial memory in a murine model of AD.

INTRODUCTION: The accumulation of oxidative stress, neuroinflammation and abnormal aggregation of amyloid ß-peptide (Aß) 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 Aß 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 Aß 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 Aß 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 Aß 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.

The Precursor to Glutathione (GSH), γ-Glutamylcysteine (GGC), Can Ameliorate Oxidative Damage and Neuroinflammation Induced by Aß40 Oligomers in Human Astrocytes.

Glutathione (GSH) is one of the most abundant thiol antioxidants in cells. Many chronic and age-related diseases are associated with a decline in cellular GSH levels or impairment in the catalytic activity of the GSH biosynthetic enzyme glutamate cysteine ligase (GCL). γ-glutamylcysteine (GGC), a precursor to glutathione (GSH), can replenish depleted GSH levels under oxidative stress conditions, by circumventing the regulation of GSH biosynthesis and providing the limiting substrate. Soluble amyloid-ß (Aß) oligomers have been shown to induce oxidative stress, synaptic dysfunction and memory deficits which have been reported in Alzheimer's disease (AD). Calcium ions, which are increased with age and in AD, have been previously reported to enhance the formation of Aß40 oligomers, which have been casually associated with the pathogenesis of the underlying neurodegenerative condition. In this study, we examined the potential beneficial effects of GGC against exogenous Aß40 oligomers on biomarkers of apoptosis and cell death, oxidative stress, and neuroinflammation, in human astrocytes. Treatment with Aß40 oligomers significantly reduced the cell viability and apoptosis of astrocyte brain cultures and increased oxidative modifications of DNA, lipids, and protein, enhanced pro-inflammatory cytokine release and increased the activity of the proteolytic matrix metalloproteinase enzyme, matric metalloproteinase (MMP)-2 and reduced the activity of MMP-9 after 24 h. Co-treatment of Aß40 oligomers with GGC at 200 µM increased the activity of the antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx) and led to significant increases in the levels of the total antioxidant capacity (TAC) and GSH and reduced the GSSG/GSH ratio. GGC also upregulated the level of the anti-inflammatory cytokine IL-10 and reduced the levels of the pro-inflammatory cytokines (TNF-α, IL-6, and IL-1ß) and attenuated the changes in metalloproteinase activity in oligomeric Aß40-treated astrocytes. Our data provides renewed insight on the beneficial effects of increased GSH levels by GGC in human astrocytes, and identifies yet another potential therapeutic strategy to attenuate the cytotoxic effects of Aß oligomers in AD.

Oral administration of γ-glutamylcysteine increases intracellular glutathione levels above homeostasis in a randomised human trial pilot study.

OBJECTIVE: To determine if orally dosed γ-glutamylcysteine (γ-GC) can increase cellular glutathione (GSH) levels above homeostasis. Many chronic and age-related disorders are associated with down-regulation, or impairment, of glutamate cysteine ligase (GCL). This suggests that γ-GC supply may become limiting for the maintenance of cellular GSH at the normal levels required to effectively protect against oxidative stress and any resulting physiological damage. METHODS: GSH levels were measured in lymphocytes of healthy, non-fasting participants before and after single oral doses (2 and 4g) of γ-GC. Blood samples were immediately processed using high speed fluorescence-activated cell sorting to isolate 106 lymphocytes that were then assayed for GSH content. RESULTS: A single 2g dose of γ-GC increased lymphocyte GSH content above basal levels (53±47%, p<0.01, n=14) within 90min of administration. A randomized dosage (2 and 4g γ-GC) crossover design was used to explore the pharmacokinetics of this GSH increase. In general, for both dose levels (n=9), GSH increased from initial basal levels over 3h (tmax) before reaching maximum GSH concentrations (Cmax) that were near two (2g γ-GC) to three (4g γ-GC) fold basal levels (0.4 nmol/106 lymphocytes). Beyond tmax, GSH levels progressively declined reaching near basal levels by 5h. The GSH half-life was between 2 and 3h with exposure (AUC) to increased GSH levels of 0.7 (2g γ-GC) and 1.8 (4g γ-GC) nmol.h/106 lymphocytes. CONCLUSIONS: Oral γ-GC is a non-toxic form of cysteine that can be directly taken up by cells and transiently increase lymphocyte GSH above homeostatic levels. Our findings that γ-GC can increase GSH levels in healthy subjects suggests that it may have potential as an adjunct for treating diseases associated with chronic GSH depletion. This trial was registered at anzctr.org.au as ACTRN12612000952842.

Therapeutic approaches to modulating glutathione levels as a pharmacological strategy in Alzheimer's disease.

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 crucialrate-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.