Original articleGlutathione deficiency-elicited reprogramming of hepatic metabolism protects against alcohol-induced steatosis
Graphical abstract
Introduction
Excessive consumption of alcohol is a major cause of chronic liver disease. Globally, alcoholic liver disease (ALD) accounts for 0.9% of total mortality and 0.6% of disability-adjusted life years and remains a public health problem worldwide [1]. ALD initially manifests as simple fatty liver (steatosis), which may progress to steatohepatitis, liver fibrosis and cirrhosis, and eventually hepatocellular carcinoma. To date, the management of ALD remains challenging due to the lack of detailed understanding of determinants of its pathogenesis and progression.
Chronic alcohol consumption modulates numerous cellular pathways in the liver and other organs, among which alcohol-induced redox perturbation appears to play a critical role in the pathogenesis of ALD [2]. Over-production of reactive molecules, including electrophiles (e.g. acetaldehyde and lipid peroxidation byproducts), reactive oxygen species (ROS) and reactive nitrogen species (RNS), may arise from ethanol metabolism, CYP2E1 induction, mitochondrial dysfunction, and proinflammatory processes [2]. In addition, ethanol exposure was shown to reduce the antioxidant capacity of the liver through depleting antioxidants and inactivating antioxidant enzymes [2]. The pathophysiological consequences of oxidative insults include, among others, impaired lipid metabolism leading to steatosis, hepatocyte injury and activation of liver fibrosis, all of which are features of ALD [3].
It is well established that glutathione (GSH), the most abundant cellular non-protein thiol, plays a pivotal role in maintaining redox homeostasis and contributes significantly to protection against oxidative insults [4]. The rate-limiting step in the GSH biosynthesis pathway is catalyzed by the glutamate-cysteine ligase (GCL), a heterodimer comprising a catalytic (GCLC) and a modifier (GCLM) subunit [5]. Disruption of the mouse Gclc gene in hepatocytes results in >95% depletion of hepatic GSH and induces liver pathologies characteristic of various clinical stages of fatty liver disease [6,7]. Global disruption of mouse Gclm gene generates a mouse model (Gclm-null) that exhibits normal liver functioning in spite of the hepatic GSH concentration being only ~15% of normal [8]. Intriguingly, following 6 week of ethanol intake with the Lieber-DeCarli (LD) diet, Gclm-null mice were protected from alcohol-induced steatosis [9]. Such protection appeared to be associated with redox activation of nuclear factor erythroid 2-related factor 2 (NRF2) and AMP-activated protein kinase (AMPK), which are key regulators of cellular stress response, cellular metabolism and biogenesis [9]. Although these results revealed the hepatoprotective effect of low GSH, the mechanistic details remain largely unknown.
It was documented in ALD patients and experimental animals that alcohol consumption impacts multiple biochemical pathways [10]. In addition to aberrant lipid metabolism, alcohol-related dysregulation of other cellular metabolisms contributes to ALD pathogenesis [10]. In the current study, we aimed to gain insights into the mechanism(s) by which GSH deficiency (due to loss of GCLM) regulates hepatic metabolic homeostasis in response to chronic ethanol consumption. This was accomplished through global profiling of hepatic polar metabolome. Liver microarray analysis was conducted in parallel to explore molecular mechanisms underlying protection from steatosis through integrated pathway analysis.
Section snippets
Reagents
All chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified.
Animals and chronic ethanol feeding
All animal experiments were performed at the University of Colorado Anschutz Medical Campus, as reported previously [9]. All animal procedures were approved by and conducted in compliance with Institutional Animal Care and Use Committee (IACUC) of the same institution. Briefly, 10–12 week male C57BL/6J wild-type (WT) and Gclm-null (KO) mice were fed a modified LD diet (Bio-Serv,
Liver metabolic profiling reveals a distinct metabolic signature associated with loss of GCLM and ethanol consumption
During the 6-week feeding period, the daily intake of CON or EtOH diet by WT and Gclm-KO mice were no different (data not shown). The overall liver polar metabolic signature was analyzed by principal components analysis. The scores-scatter plot for liver weight-normalized metabolic signature recorded in positive (Fig. 1A, left panel) and negative (Fig. 1A, right panel) ESI modes showed that mice segregate along first principal component according to their genotypes, irrespective of EtOH
Discussion
There is a large body of evidence indicating that low hepatic GSH content is pathogenically involved in liver diseases of various etiologies [[27], [28], [29], [30]]. This is expected given the crucial function of GSH in maintaining cellular redox homeostasis and in detoxifying electrophiles. New knowledge derived from redox biology research, however, provides convincing evidence that chronic oxidative and nitrosative stress under physiological conditions trigger cellular mechanisms that
Conclusion
The present study has identified new hepatic metabolic signatures intrinsically linked to the loss of GCLM (and by extension low hepatic GSH) and modulated by alcohol consumption, the combination of which are, at least in part, causally associated with hepatic protection against alcohol-induced steatosis in Gclm-KO mice (Fig. 7). These metabolic changes cover multiple biochemical pathways and represent a metabolic network that appears to be reprogramed by both transcriptional and
Conflicts of interest
None.
Funding sources
This work was supported in part by the National Institutes of Health grants K01AA025093 (YC), R24AA022057 (VV), U01AA021724 (VV), the NIH Intramural Research Program (FJG) and the Department of Atomic Energy, Government of India (SKM).
Acknowledgement
We thank Dr. Peng Gang (Yale School of Public Health) for valuable input into our statistical analysis.
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- 1
Contributed equally.
- 2
Current address: Yale Center for Genome Analysis, Yale School of Medicine, Orange, CT 06477, USA.