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The effect of alcohol and hydrogen peroxide on liver hepcidin gene expression in mice lacking antioxidant enzymes, glutathione peroxidase-1 or catalase.

Harrison-Findik DD, Lu S - Biomolecules (2015)

Bottom Line: Gpx-1(-/-) displayed significantly higher hepatic H2O2 levels than catalase(-/-) compared to wild-type mice, as measured by 2'-7'-dichlorodihydrofluorescein diacetate (DCFH-DA).In conclusion, our findings strongly suggest that H2O2 inhibits hepcidin expression in vivo.Synergistic induction of CHOP by alcohol and H2O2, in the absence of gpx-1, stimulates liver hepcidin gene expression by ER stress independent of CREBH.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Division of Gastroenterology/Hepatology, University of Nebraska Medical Center, Omaha, NE 68198, USA. dharrisonfindik@unmc.edu.

ABSTRACT
This study investigates the regulation of hepcidin, the key iron-regulatory molecule, by alcohol and hydrogen peroxide (H2O2) in glutathione peroxidase-1 (gpx-1(-/-)) and catalase (catalase(-/-)) knockout mice. For alcohol studies, 10% ethanol was administered in the drinking water for 7 days. Gpx-1(-/-) displayed significantly higher hepatic H2O2 levels than catalase(-/-) compared to wild-type mice, as measured by 2'-7'-dichlorodihydrofluorescein diacetate (DCFH-DA). The basal level of liver hepcidin expression was attenuated in gpx-1(-/-) mice. Alcohol increased H2O2 production in catalase(-/-) and wild-type, but not gpx-1(-/-), mice. Hepcidin expression was inhibited in alcohol-fed catalase(-/-) and wild-type mice. In contrast, alcohol elevated hepcidin expression in gpx-1(-/-) mice. Gpx-1(-/-) mice also displayed higher level of basal liver CHOP protein expression than catalase(-/-) mice. Alcohol induced CHOP and to a lesser extent GRP78/BiP expression, but not XBP1 splicing or binding of CREBH to hepcidin gene promoter, in gpx-1(-/-) mice. The up-regulation of hepatic ATF4 mRNA levels, which was observed in gpx-1(-/-) mice, was attenuated by alcohol. In conclusion, our findings strongly suggest that H2O2 inhibits hepcidin expression in vivo. Synergistic induction of CHOP by alcohol and H2O2, in the absence of gpx-1, stimulates liver hepcidin gene expression by ER stress independent of CREBH.

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Liver C/EBPα protein expression. (A) Nuclear cell lysates isolated from the livers of H2O or ethanol (alc.)-fed wild-type and gpx-1−/− transgenic mice were employed to detect C/EBPα protein expression by western blotting. An anti-TATA-binding protein (TBP) antibody was used to demonstrate equal nuclear protein loading. (B) C/EBPα protein (30 kDa and 42 kDa) expression was quantified by densitometric analysis and normalized to TBP expression. Normalized expression in H2O or alcohol-fed gpx-1−/− and alcohol-fed wild-type mice was expressed as fold expression of that in H2O-fed wild-type mice.
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biomolecules-05-00793-f004: Liver C/EBPα protein expression. (A) Nuclear cell lysates isolated from the livers of H2O or ethanol (alc.)-fed wild-type and gpx-1−/− transgenic mice were employed to detect C/EBPα protein expression by western blotting. An anti-TATA-binding protein (TBP) antibody was used to demonstrate equal nuclear protein loading. (B) C/EBPα protein (30 kDa and 42 kDa) expression was quantified by densitometric analysis and normalized to TBP expression. Normalized expression in H2O or alcohol-fed gpx-1−/− and alcohol-fed wild-type mice was expressed as fold expression of that in H2O-fed wild-type mice.

Mentions: CHOP acts as a negative regulator of the C/EBP family of transcription factors. We have previously shown that alcohol inhibits C/EBPα in wild-type mice livers [8]. Since CHOP was induced by alcohol in gpx-1 mice, we determined nuclear C/EBPα expression in gpx-1−/− mice livers by western blotting. The absence of gpx-1 did not significantly alter the basal expression level of liver C/EBPα protein in gpx-1−/− mice compared to wild-type mice (Figure 4A,B). Alcohol inhibited C/EBPα expression to the same extent in gpx-1−/− and wild-type mice (Figure 4A,B).


The effect of alcohol and hydrogen peroxide on liver hepcidin gene expression in mice lacking antioxidant enzymes, glutathione peroxidase-1 or catalase.

Harrison-Findik DD, Lu S - Biomolecules (2015)

Liver C/EBPα protein expression. (A) Nuclear cell lysates isolated from the livers of H2O or ethanol (alc.)-fed wild-type and gpx-1−/− transgenic mice were employed to detect C/EBPα protein expression by western blotting. An anti-TATA-binding protein (TBP) antibody was used to demonstrate equal nuclear protein loading. (B) C/EBPα protein (30 kDa and 42 kDa) expression was quantified by densitometric analysis and normalized to TBP expression. Normalized expression in H2O or alcohol-fed gpx-1−/− and alcohol-fed wild-type mice was expressed as fold expression of that in H2O-fed wild-type mice.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496697&req=5

biomolecules-05-00793-f004: Liver C/EBPα protein expression. (A) Nuclear cell lysates isolated from the livers of H2O or ethanol (alc.)-fed wild-type and gpx-1−/− transgenic mice were employed to detect C/EBPα protein expression by western blotting. An anti-TATA-binding protein (TBP) antibody was used to demonstrate equal nuclear protein loading. (B) C/EBPα protein (30 kDa and 42 kDa) expression was quantified by densitometric analysis and normalized to TBP expression. Normalized expression in H2O or alcohol-fed gpx-1−/− and alcohol-fed wild-type mice was expressed as fold expression of that in H2O-fed wild-type mice.
Mentions: CHOP acts as a negative regulator of the C/EBP family of transcription factors. We have previously shown that alcohol inhibits C/EBPα in wild-type mice livers [8]. Since CHOP was induced by alcohol in gpx-1 mice, we determined nuclear C/EBPα expression in gpx-1−/− mice livers by western blotting. The absence of gpx-1 did not significantly alter the basal expression level of liver C/EBPα protein in gpx-1−/− mice compared to wild-type mice (Figure 4A,B). Alcohol inhibited C/EBPα expression to the same extent in gpx-1−/− and wild-type mice (Figure 4A,B).

Bottom Line: Gpx-1(-/-) displayed significantly higher hepatic H2O2 levels than catalase(-/-) compared to wild-type mice, as measured by 2'-7'-dichlorodihydrofluorescein diacetate (DCFH-DA).In conclusion, our findings strongly suggest that H2O2 inhibits hepcidin expression in vivo.Synergistic induction of CHOP by alcohol and H2O2, in the absence of gpx-1, stimulates liver hepcidin gene expression by ER stress independent of CREBH.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Division of Gastroenterology/Hepatology, University of Nebraska Medical Center, Omaha, NE 68198, USA. dharrisonfindik@unmc.edu.

ABSTRACT
This study investigates the regulation of hepcidin, the key iron-regulatory molecule, by alcohol and hydrogen peroxide (H2O2) in glutathione peroxidase-1 (gpx-1(-/-)) and catalase (catalase(-/-)) knockout mice. For alcohol studies, 10% ethanol was administered in the drinking water for 7 days. Gpx-1(-/-) displayed significantly higher hepatic H2O2 levels than catalase(-/-) compared to wild-type mice, as measured by 2'-7'-dichlorodihydrofluorescein diacetate (DCFH-DA). The basal level of liver hepcidin expression was attenuated in gpx-1(-/-) mice. Alcohol increased H2O2 production in catalase(-/-) and wild-type, but not gpx-1(-/-), mice. Hepcidin expression was inhibited in alcohol-fed catalase(-/-) and wild-type mice. In contrast, alcohol elevated hepcidin expression in gpx-1(-/-) mice. Gpx-1(-/-) mice also displayed higher level of basal liver CHOP protein expression than catalase(-/-) mice. Alcohol induced CHOP and to a lesser extent GRP78/BiP expression, but not XBP1 splicing or binding of CREBH to hepcidin gene promoter, in gpx-1(-/-) mice. The up-regulation of hepatic ATF4 mRNA levels, which was observed in gpx-1(-/-) mice, was attenuated by alcohol. In conclusion, our findings strongly suggest that H2O2 inhibits hepcidin expression in vivo. Synergistic induction of CHOP by alcohol and H2O2, in the absence of gpx-1, stimulates liver hepcidin gene expression by ER stress independent of CREBH.

Show MeSH
Related in: MedlinePlus