Chronic Glutathione Depletion Confers Protection against Alcohol-induced Steatosis: Implication for Redox Activation of AMP-activated Protein Kinase Pathway

Alcoholic liver disease (ALD) is a major cause of chronic liver disease, and accounts for over 30,000 deaths annually in the United States1. The majority of ingested ethanol is metabolized in the liver through two sequential steps2. Ethanol is oxidized to acetaldehyde by alcohol dehydrogenase I (ADH1), catalase (CAT) and/or cytochrome P450 2E1 (CYP2E1). Acetaldehyde is then oxidized by acetaldehyde dehydrogenases (ALDH2, ALDH1B1 and ALDH1A1) to acetate. During the development of ALD, numerous pathways in the liver are modulated by alcohol, many through mechanisms involving oxidative stress3. Ethanol metabolism, CYP2E1 induction, compromised antioxidant defenses, mitochondrial injury, activation of Kupffer and stellate cells, hypoxia, and iron overload can all contribute to the alcohol-induced oxidative environment. Accumulation of ethanol-associated reactive molecules, e.g., reactive oxygen (ROS) and nitrogen species (RNS), and electrophilic products, e.g., acetaldehyde and lipid peroxidation-derived products, can be harmful to biological systems due to their propensity to inactivate enzymes, leading to loss of protein function, DNA damage and cell death4. Manifestations of hepatic oxidative damage include dysregulation of lipid metabolism (leading to steatosis), hepatocyte degeneration and death, and activated immune response (leading to inflammation and fibrosis/cirrhosis). Importantly, these are all features of ALD depending on the disease stage5. Glutathione (GSH) is the most abundant cellular non-protein thiol, attaining millimolar concentrations in the liver6. It acts as an antioxidant by directly scavenging free radicals or serving as a cofactor for antioxidant enzymes6. Because of its abundance, GSH plays a key role in maintaining cellular redox homeostasis and in cellular mechanisms that protect against oxidative stress. The function of GSH in hepatocytes has been investigated by pharmacological inhibition or genetic ablation of the glutamate-cysteine ligase (GCL), the rate-limiting and regulatory enzyme in GSH biosynthesis. In higher eukaryotes GCL is a heterodimer comprising a catalytic (GCLC) and a modifier (GCLM) subunit7. Transgenic mice in which GCLC expression is abolished in hepatocytes have 5–8% of normal hepatic GSH levels and manifest liver pathologies characteristic of the various clinical stages of fatty liver disease8,9. Treatment of newborn rats with L-buthionine sulfoximine (BSO), an irreversible inhibitor of GCLC, leads to hepatic abnormalities including mitochondrial dysfunction10. These studies underscore the essential role of GSH in normal functioning of the liver. It has been proposed that depletion of hepatic GSH, particularly mitochondrial GSH, is one of the early changes associated with chronic alcohol consumption and is a critical contributor to ALD pathogenesis11. Nevertheless, the effect of ethanol consumption on the total hepatic GSH pool is equivocal in that studies have shown decreased, unchanged or increased levels12,13,14, most likely related to differences in ethanol exposure regimes and/or analytical assays. In one study, BSO treatment alleviated ethanol-induced elevations in serum ALT and liver TG content15. Given that BSO possesses non-specific pharmacological activities, the authors concluded that this effect was independent of the inhibition of GSH biosynthesis15. Collectively, the pathogenic role of GSH depletion in alcohol-induced liver injury remains to be defined. In the current study, we utilized a transgenic mouse model to elucidate the role of GSH in hepatic responses to chronic ethanol consumption and explored underlying mechanisms. Global disruption of Gclm results in mice (GCLM knockout) that have ≈15% of hepatic GSH levels seen in wild-type mice16. The 85% depletion of hepatic GSH results in a decreased GSH redox potential (ΔEGSH). Mitochondrial GSH is maintained at 40% of the wild-type level and is accompanied by increased H2O2 release. Nevertheless, in vitro mitochondrial function was indistinguishable from wild-type17. The viability and good health of naive GCLM knockout mice makes them a valuable model for studying the impact of chronic GSH deficiency18. Following 6 wk of ethanol administration, GCLM knockout mice were resistant to alcohol-induced steatosis and exhibited beneficial metabolic and stress responses to chronic ethanol consumption.