Published online Nov 26, 2013. doi: 10.4331/wjbc.v4.i4.119
Revised: November 5, 2013
Accepted: November 15, 2013
Published online: November 26, 2013
AIM: To understand the role of mitochondrial-produced superoxide (O2•-) in the regulation of iron-regulatory hormone, hepcidin by alcohol in the liver.
METHODS: For alcohol experiments, manganese superoxide dismutase knockout mice heterozygous for Sod2 gene expression (Sod2+/-) and age-matched littermate control mice (LMC), expressing Sod2 gene on both alleles, were exposed to either 10% (w/v) ethanol in the drinking water or plain water (control) for 7 d. Total cellular O2•- levels in hepatocytes isolated from the livers of mice were measured by electron paramagnetic resonance spectroscopy. The mitochondrial-targeted, O2•--sensitive fluorogenic probe, MitoSOX Red and flow cytometry were utilized to measure O2•- in mitochondria. Gene and protein expression were determined by Taqman Real-time quantitative PCR and Western blotting, respectively.
RESULTS: Sod2+/- mice expressed 40% less MnSOD protein (SOD2) in hepatocytes compared to LMC mice. The deletion of Sod2 allele did not alter the basal expression level of hepcidin in the liver. 10% ethanol exposure for 1 wk inhibited hepatic hepcidin mRNA expression three-fold both in Sod2+/- and LMC mice. O2•- levels in hepatocytes of untreated Sod2+/- mice were three-fold higher than in untreated LMC mice, as observed by electron paramagnetic resonance spectroscopy. O2•- levels in mitochondria of Sod2+/ mice were four-fold higher than in mitochondria of untreated LMC mice, as measured by MitoSOX Red fluorescence and flow cytometry. Alcohol induced a two-fold higher increase in O2•- levels in hepatocytes of LMC mice than in Sod2+/- mice compared to respective untreated counterparts. In contrast, 1 wk alcohol exposure did not alter mitochondrial O2•- levels in both Sod2+/- and control mice.
CONCLUSION: Mitochondrial O2•- is not involved in the inhibition of liver hepcidin transcription and thereby regulation of iron metabolism by alcohol. These findings also suggest that short-term alcohol consumption significantly elevates O2•- levels in hepatocytes, which appears not to originate from mitochondria.
Core tip: Patients with alcoholic liver disease frequently exhibit iron overload, which contributes to liver injury. Our previous research has shown that suppression of iron-regulatory hormone, hepcidin by alcohol-mediated oxidative stress in the liver is involved. This manuscript investigates the role of superoxide and mitochondria in this process by using MnSOD knockout mice.