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

Chen Y, Singh S, Matsumoto A, Manna SK, Abdelmegeed MA, Golla S, Murphy RC, Dong H, Song BJ, Gonzalez FJ, Thompson DC, Vasiliou V - Sci Rep (2016)

Bottom Line: The pathogenesis of alcoholic liver disease (ALD) is not well established.However, oxidative stress and associated decreases in levels of glutathione (GSH) are known to play a central role in ALD.We propose redox activation of the AMPK may represent a new therapeutic strategy for preventing ALD.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental Health Sciences, Yale University, New Haven, CT 06520, USA.

ABSTRACT
The pathogenesis of alcoholic liver disease (ALD) is not well established. However, oxidative stress and associated decreases in levels of glutathione (GSH) are known to play a central role in ALD. The present study examines the effect of GSH deficiency on alcohol-induced liver steatosis in Gclm knockout (KO) mice that constitutively have ≈15% normal hepatic levels of GSH. Following chronic (6 week) feeding with an ethanol-containing liquid diet, the Gclm KO mice were unexpectedly found to be protected against steatosis despite showing increased oxidative stress (as reflected in elevated levels of CYP2E1 and protein carbonyls). Gclm KO mice also exhibit constitutive activation of liver AMP-activated protein kinase (AMPK) pathway and nuclear factor-erythroid 2-related factor 2 target genes, and show enhanced ethanol clearance, altered hepatic lipid profiles in favor of increased levels of polyunsaturated fatty acids and concordant changes in expression of genes associated with lipogenesis and fatty acid oxidation. In summary, our data implicate a novel mechanism protecting against liver steatosis via an oxidative stress adaptive response that activates the AMPK pathway. We propose redox activation of the AMPK may represent a new therapeutic strategy for preventing ALD.

No MeSH data available.


Related in: MedlinePlus

Persistent oxidative stress and induction of NRF2 antioxidant response in GCLM KO livers.(a) liver cytosolic and mitochondrial GSH levels (nmol/mg protein) in livers from mice fed regular chow (REG), control (CON) or ethanol (EtOH) liquid diets for 6 wk. Data are mean ± SEM from 5–6 mice. (b), levels of carbonylated proteins in liver homogenates. C, Nuclear accumulation of NRF2 protein. Protein band intensity was quantified by densitometry and reported as fold of control (REG-fed WT mice) after normalization to β-actin (b) or lamin-A (c). Data are mean ± SEM from 4 mice. (d) Q-PCR analysis of liver mRNA for the oxidative stress responsive genes GCLC, metallothionein-1 (MT1) and heme oxygenase-1 (HMOX1). Relative mRNA levels were expressed as fold of control (REG-fed WT mice; dashed line), after normalization to the housekeeping gene β-2 microglobulin (B2M). Data are mean ± SEM from 4–6 mice. *P < 0.05, vs. diet-matched WT mice. #P < 0.05, vs. CON-fed mice of the same genotype.
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f3: Persistent oxidative stress and induction of NRF2 antioxidant response in GCLM KO livers.(a) liver cytosolic and mitochondrial GSH levels (nmol/mg protein) in livers from mice fed regular chow (REG), control (CON) or ethanol (EtOH) liquid diets for 6 wk. Data are mean ± SEM from 5–6 mice. (b), levels of carbonylated proteins in liver homogenates. C, Nuclear accumulation of NRF2 protein. Protein band intensity was quantified by densitometry and reported as fold of control (REG-fed WT mice) after normalization to β-actin (b) or lamin-A (c). Data are mean ± SEM from 4 mice. (d) Q-PCR analysis of liver mRNA for the oxidative stress responsive genes GCLC, metallothionein-1 (MT1) and heme oxygenase-1 (HMOX1). Relative mRNA levels were expressed as fold of control (REG-fed WT mice; dashed line), after normalization to the housekeeping gene β-2 microglobulin (B2M). Data are mean ± SEM from 4–6 mice. *P < 0.05, vs. diet-matched WT mice. #P < 0.05, vs. CON-fed mice of the same genotype.

Mentions: EtOH feeding decreased GSH levels specifically in the mitochondrial pool of livers of WT mice (Fig. 3a). Although KO mice had significantly lower levels of GSH in the cytosol and mitochondria, EtOH did not decrease it further (Fig. 3a). Consistent with ≈85% depletion of hepatic GSH, the KO liver showed much higher basal levels of protein carbonylation (Fig. 3b), suggesting increased oxidative stress. When compared with the respective CON-fed groups, EtOH dramatically induced hepatic protein carbonylation in both WT and KO mice, albeit the levels in the KO mice remained higher than those observed in WT mice. The sustained oxidative stress status in KO livers was paralleled by increased levels of transcription factor NRF2 in the nucleus (Fig. 3c). Transactivation of NRF2 in KO livers was further supported by constitutive induction of NRF2 target genes, including Gclc, metallothionein I (Mt1) and heme-oxgenase 1 (Hmox1) (Fig. 3d). Importantly, expression levels of these genes have been used in numerous studies as sensitive indices for oxidative stress81920.


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

Chen Y, Singh S, Matsumoto A, Manna SK, Abdelmegeed MA, Golla S, Murphy RC, Dong H, Song BJ, Gonzalez FJ, Thompson DC, Vasiliou V - Sci Rep (2016)

Persistent oxidative stress and induction of NRF2 antioxidant response in GCLM KO livers.(a) liver cytosolic and mitochondrial GSH levels (nmol/mg protein) in livers from mice fed regular chow (REG), control (CON) or ethanol (EtOH) liquid diets for 6 wk. Data are mean ± SEM from 5–6 mice. (b), levels of carbonylated proteins in liver homogenates. C, Nuclear accumulation of NRF2 protein. Protein band intensity was quantified by densitometry and reported as fold of control (REG-fed WT mice) after normalization to β-actin (b) or lamin-A (c). Data are mean ± SEM from 4 mice. (d) Q-PCR analysis of liver mRNA for the oxidative stress responsive genes GCLC, metallothionein-1 (MT1) and heme oxygenase-1 (HMOX1). Relative mRNA levels were expressed as fold of control (REG-fed WT mice; dashed line), after normalization to the housekeeping gene β-2 microglobulin (B2M). Data are mean ± SEM from 4–6 mice. *P < 0.05, vs. diet-matched WT mice. #P < 0.05, vs. CON-fed mice of the same genotype.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4940737&req=5

f3: Persistent oxidative stress and induction of NRF2 antioxidant response in GCLM KO livers.(a) liver cytosolic and mitochondrial GSH levels (nmol/mg protein) in livers from mice fed regular chow (REG), control (CON) or ethanol (EtOH) liquid diets for 6 wk. Data are mean ± SEM from 5–6 mice. (b), levels of carbonylated proteins in liver homogenates. C, Nuclear accumulation of NRF2 protein. Protein band intensity was quantified by densitometry and reported as fold of control (REG-fed WT mice) after normalization to β-actin (b) or lamin-A (c). Data are mean ± SEM from 4 mice. (d) Q-PCR analysis of liver mRNA for the oxidative stress responsive genes GCLC, metallothionein-1 (MT1) and heme oxygenase-1 (HMOX1). Relative mRNA levels were expressed as fold of control (REG-fed WT mice; dashed line), after normalization to the housekeeping gene β-2 microglobulin (B2M). Data are mean ± SEM from 4–6 mice. *P < 0.05, vs. diet-matched WT mice. #P < 0.05, vs. CON-fed mice of the same genotype.
Mentions: EtOH feeding decreased GSH levels specifically in the mitochondrial pool of livers of WT mice (Fig. 3a). Although KO mice had significantly lower levels of GSH in the cytosol and mitochondria, EtOH did not decrease it further (Fig. 3a). Consistent with ≈85% depletion of hepatic GSH, the KO liver showed much higher basal levels of protein carbonylation (Fig. 3b), suggesting increased oxidative stress. When compared with the respective CON-fed groups, EtOH dramatically induced hepatic protein carbonylation in both WT and KO mice, albeit the levels in the KO mice remained higher than those observed in WT mice. The sustained oxidative stress status in KO livers was paralleled by increased levels of transcription factor NRF2 in the nucleus (Fig. 3c). Transactivation of NRF2 in KO livers was further supported by constitutive induction of NRF2 target genes, including Gclc, metallothionein I (Mt1) and heme-oxgenase 1 (Hmox1) (Fig. 3d). Importantly, expression levels of these genes have been used in numerous studies as sensitive indices for oxidative stress81920.

Bottom Line: The pathogenesis of alcoholic liver disease (ALD) is not well established.However, oxidative stress and associated decreases in levels of glutathione (GSH) are known to play a central role in ALD.We propose redox activation of the AMPK may represent a new therapeutic strategy for preventing ALD.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental Health Sciences, Yale University, New Haven, CT 06520, USA.

ABSTRACT
The pathogenesis of alcoholic liver disease (ALD) is not well established. However, oxidative stress and associated decreases in levels of glutathione (GSH) are known to play a central role in ALD. The present study examines the effect of GSH deficiency on alcohol-induced liver steatosis in Gclm knockout (KO) mice that constitutively have ≈15% normal hepatic levels of GSH. Following chronic (6 week) feeding with an ethanol-containing liquid diet, the Gclm KO mice were unexpectedly found to be protected against steatosis despite showing increased oxidative stress (as reflected in elevated levels of CYP2E1 and protein carbonyls). Gclm KO mice also exhibit constitutive activation of liver AMP-activated protein kinase (AMPK) pathway and nuclear factor-erythroid 2-related factor 2 target genes, and show enhanced ethanol clearance, altered hepatic lipid profiles in favor of increased levels of polyunsaturated fatty acids and concordant changes in expression of genes associated with lipogenesis and fatty acid oxidation. In summary, our data implicate a novel mechanism protecting against liver steatosis via an oxidative stress adaptive response that activates the AMPK pathway. We propose redox activation of the AMPK may represent a new therapeutic strategy for preventing ALD.

No MeSH data available.


Related in: MedlinePlus