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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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Hepcidin mRNA expression in the liver. cDNA synthesized from liver RNA of transgenic mice, lacking the expression of either glutathione peroxidase-1 (gpx-1−/−) or catalase (catalase−/−), and wild-type mice, fed with plain water (H2O) or ethanol (alc.), was employed to determine hepcidin mRNA expression by Taqman real-time PCR. (A) Hepcidin gene expression in water or alcohol-fed transgenic mice and alcohol-fed wild-type mice was expressed as-fold hepcidin expression of that in wild-type mice fed with water. (B) Hepcidin gene expression in alcohol-fed gpx-1−/− mice was expressed as-fold hepcidin expression of that in gpx-1−/− mice fed with water. Asterisks indicate statistical significance (p < 0.05).
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biomolecules-05-00793-f002: Hepcidin mRNA expression in the liver. cDNA synthesized from liver RNA of transgenic mice, lacking the expression of either glutathione peroxidase-1 (gpx-1−/−) or catalase (catalase−/−), and wild-type mice, fed with plain water (H2O) or ethanol (alc.), was employed to determine hepcidin mRNA expression by Taqman real-time PCR. (A) Hepcidin gene expression in water or alcohol-fed transgenic mice and alcohol-fed wild-type mice was expressed as-fold hepcidin expression of that in wild-type mice fed with water. (B) Hepcidin gene expression in alcohol-fed gpx-1−/− mice was expressed as-fold hepcidin expression of that in gpx-1−/− mice fed with water. Asterisks indicate statistical significance (p < 0.05).

Mentions: The combined effect of alcohol and H2O2 in the regulation of hepcidin gene expression was determined by real-time PCR, as described in Experimental Section. Alcohol inhibited hepcidin mRNA expression in the livers of wild-type mice (Figure 2A). The deletion of catalase gene in catalase−/− transgenic mice did not significantly alter the basal level of hepcidin expression in the liver (Figure 2A). Similar to wild-type mice, hepcidin expression in the livers of catalase−/− mice was also significantly inhibited by alcohol (Figure 2A). Contrary to catalase−/−, the basal level of hepcidin mRNA expression was decreased by two-fold in gpx-1−/− mice compared to wild-type mice (Figure 2A). Alcohol however up-regulated hepcidin gene expression in gpx-1−/− mice over two-fold compared to that in water-fed gpx-1−/− mice (Figure 2B).


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)

Hepcidin mRNA expression in the liver. cDNA synthesized from liver RNA of transgenic mice, lacking the expression of either glutathione peroxidase-1 (gpx-1−/−) or catalase (catalase−/−), and wild-type mice, fed with plain water (H2O) or ethanol (alc.), was employed to determine hepcidin mRNA expression by Taqman real-time PCR. (A) Hepcidin gene expression in water or alcohol-fed transgenic mice and alcohol-fed wild-type mice was expressed as-fold hepcidin expression of that in wild-type mice fed with water. (B) Hepcidin gene expression in alcohol-fed gpx-1−/− mice was expressed as-fold hepcidin expression of that in gpx-1−/− mice fed with water. Asterisks indicate statistical significance (p < 0.05).
© Copyright Policy
Related In: Results  -  Collection

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

biomolecules-05-00793-f002: Hepcidin mRNA expression in the liver. cDNA synthesized from liver RNA of transgenic mice, lacking the expression of either glutathione peroxidase-1 (gpx-1−/−) or catalase (catalase−/−), and wild-type mice, fed with plain water (H2O) or ethanol (alc.), was employed to determine hepcidin mRNA expression by Taqman real-time PCR. (A) Hepcidin gene expression in water or alcohol-fed transgenic mice and alcohol-fed wild-type mice was expressed as-fold hepcidin expression of that in wild-type mice fed with water. (B) Hepcidin gene expression in alcohol-fed gpx-1−/− mice was expressed as-fold hepcidin expression of that in gpx-1−/− mice fed with water. Asterisks indicate statistical significance (p < 0.05).
Mentions: The combined effect of alcohol and H2O2 in the regulation of hepcidin gene expression was determined by real-time PCR, as described in Experimental Section. Alcohol inhibited hepcidin mRNA expression in the livers of wild-type mice (Figure 2A). The deletion of catalase gene in catalase−/− transgenic mice did not significantly alter the basal level of hepcidin expression in the liver (Figure 2A). Similar to wild-type mice, hepcidin expression in the livers of catalase−/− mice was also significantly inhibited by alcohol (Figure 2A). Contrary to catalase−/−, the basal level of hepcidin mRNA expression was decreased by two-fold in gpx-1−/− mice compared to wild-type mice (Figure 2A). Alcohol however up-regulated hepcidin gene expression in gpx-1−/− mice over two-fold compared to that in water-fed gpx-1−/− mice (Figure 2B).

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