Limits...
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

Constitutive activation of LKB1/AMPK pathway in GCLM KO livers.(a) Representative Western blotting of key players in LKB1-AMPK pathway in livers from WT and KO mice fed regular chow (REG), control (CON) or ethanol (EtOH) liquid diets for 6 wk. (b) Relative levels of phosphorylated proteins. Proteins were quantified by densitometric analysis of protein band intensity. Level of phosphorylated protein was calculated as the ratio to total proteins after normalization to β-actin (BACT). Relative levels are reported as ratios to control (CON-fed WT mice). Data represent mean ± SEM from 4 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

License
getmorefigures.php?uid=PMC4940737&req=5

f5: Constitutive activation of LKB1/AMPK pathway in GCLM KO livers.(a) Representative Western blotting of key players in LKB1-AMPK pathway in livers from WT and KO mice fed regular chow (REG), control (CON) or ethanol (EtOH) liquid diets for 6 wk. (b) Relative levels of phosphorylated proteins. Proteins were quantified by densitometric analysis of protein band intensity. Level of phosphorylated protein was calculated as the ratio to total proteins after normalization to β-actin (BACT). Relative levels are reported as ratios to control (CON-fed WT mice). Data represent mean ± SEM from 4 mice. #P < 0.05, vs. diet-matched WT mice. ^P < 0.05, vs. CON-fed mice of the same genotype.

Mentions: The resistance of KO mice to steatosis induced by chronic EtOH consumption suggests low hepatic GSH elicits a protective metabolic adaption in the liver. Previous studies suggest that the inhibitory actions of EtOH on the AMPK pathway are crucial for the development of alcoholic steatosis21. AMPK, a heterotrimeric kinase composed of catalytic α and regulatory β/γ subunits, is a master regulator of hepatic lipid metabolism22. Activation of AMPK by upstream kinases phosphorylates target enzymes, such as acetyl-coA carboxylase (ACC), leading to inhibition of lipogenic pathways and activation of FA oxidation22. AMPK also inhibits lipid biosynthesis by suppressing transcription factor sterol regulatory element-binding protein 1 (SREBP1) and promoting the action of peroxisome proliferator-activated receptor alpha (PPARα), a nuclear receptor that acts to enhance both mitochondrial and peroxisomal FA β-oxidation23. LKB1, a tumor suppressor, is a major mammalian AMPK kinase in the liver that activates AMPK by phosphorylating the Thr172 residues of AMPKα subunits; importantly, this action appears to be mediated by ROS/RNS24. In agreement with observed resistance in KO mice, levels of phosphorylated AMPKα subunit (at Thr172 residue) were higher in the livers of KO mice than in WT mice in all diet groups (Fig. 5). This may have resulted in higher levels of phosphorylation of ACC observed in livers of KO mice (Fig. 5b). Importantly, the observed inhibition of AMPKα phosphorylation by EtOH in WT mice was absent from KO mice (Fig. 5b). In addition, higher levels of phosphorylated (active) LKB1 were noted under these conditions in the livers of KO mice but not WT mice (Fig. 5b). This result is suggestive of the constitutive activation of LKB1/AMPKα pathway in the livers of KO mice. We measured mRNA levels of LKB1 and AMPKα for all diet groups. Our data (supplementary Fig. 2) revealed that expression of these genes were not different between WT and KO livers in any diet groups.


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)

Constitutive activation of LKB1/AMPK pathway in GCLM KO livers.(a) Representative Western blotting of key players in LKB1-AMPK pathway in livers from WT and KO mice fed regular chow (REG), control (CON) or ethanol (EtOH) liquid diets for 6 wk. (b) Relative levels of phosphorylated proteins. Proteins were quantified by densitometric analysis of protein band intensity. Level of phosphorylated protein was calculated as the ratio to total proteins after normalization to β-actin (BACT). Relative levels are reported as ratios to control (CON-fed WT mice). Data represent mean ± SEM from 4 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

License
Show All Figures
getmorefigures.php?uid=PMC4940737&req=5

f5: Constitutive activation of LKB1/AMPK pathway in GCLM KO livers.(a) Representative Western blotting of key players in LKB1-AMPK pathway in livers from WT and KO mice fed regular chow (REG), control (CON) or ethanol (EtOH) liquid diets for 6 wk. (b) Relative levels of phosphorylated proteins. Proteins were quantified by densitometric analysis of protein band intensity. Level of phosphorylated protein was calculated as the ratio to total proteins after normalization to β-actin (BACT). Relative levels are reported as ratios to control (CON-fed WT mice). Data represent mean ± SEM from 4 mice. #P < 0.05, vs. diet-matched WT mice. ^P < 0.05, vs. CON-fed mice of the same genotype.
Mentions: The resistance of KO mice to steatosis induced by chronic EtOH consumption suggests low hepatic GSH elicits a protective metabolic adaption in the liver. Previous studies suggest that the inhibitory actions of EtOH on the AMPK pathway are crucial for the development of alcoholic steatosis21. AMPK, a heterotrimeric kinase composed of catalytic α and regulatory β/γ subunits, is a master regulator of hepatic lipid metabolism22. Activation of AMPK by upstream kinases phosphorylates target enzymes, such as acetyl-coA carboxylase (ACC), leading to inhibition of lipogenic pathways and activation of FA oxidation22. AMPK also inhibits lipid biosynthesis by suppressing transcription factor sterol regulatory element-binding protein 1 (SREBP1) and promoting the action of peroxisome proliferator-activated receptor alpha (PPARα), a nuclear receptor that acts to enhance both mitochondrial and peroxisomal FA β-oxidation23. LKB1, a tumor suppressor, is a major mammalian AMPK kinase in the liver that activates AMPK by phosphorylating the Thr172 residues of AMPKα subunits; importantly, this action appears to be mediated by ROS/RNS24. In agreement with observed resistance in KO mice, levels of phosphorylated AMPKα subunit (at Thr172 residue) were higher in the livers of KO mice than in WT mice in all diet groups (Fig. 5). This may have resulted in higher levels of phosphorylation of ACC observed in livers of KO mice (Fig. 5b). Importantly, the observed inhibition of AMPKα phosphorylation by EtOH in WT mice was absent from KO mice (Fig. 5b). In addition, higher levels of phosphorylated (active) LKB1 were noted under these conditions in the livers of KO mice but not WT mice (Fig. 5b). This result is suggestive of the constitutive activation of LKB1/AMPKα pathway in the livers of KO mice. We measured mRNA levels of LKB1 and AMPKα for all diet groups. Our data (supplementary Fig. 2) revealed that expression of these genes were not different between WT and KO livers in any diet groups.

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