Alcohol intake and iron overload: Another role for hepcidin?


  • Potential conflict of interest: Nothing to report.

Harrison-Findik DD, Schafer D, Klein E, Timchenko NA, Kulaksiz H, Clemens D, Fein E, Andriopoulos B, Pantopoulos K, Gollan J. Alcohol metabolism-mediated oxidative stress down-regulates hepcidin transcription and leads to increased duodenal iron transporter expression. J Biol Chem 2006;281:22974-22982.


Patients with alcoholic liver disease frequently exhibit iron overload in association with increased hepatic fibrosis. Even moderate alcohol consumption elevates body iron stores; however, the underlying molecular mechanisms are unknown. Hepcidin, a circulatory peptide synthesized in the liver, is a key mediator of iron metabolism. Ethanol metabolism significantly down-regulated both in vitro and in vivo hepcidin mRNA and protein expression. 4-Methylpyrazole, a specific inhibitor of the alcohol-metabolizing enzymes, abolished the effects of ethanol on hepcidin. However, ethanol did not alter the expression of transferrin receptor1 and ferritin or the activation of iron regulatory RNA-binding proteins, IRP1 and IRP2. Mice maintained on 10-20% ethanol for 7 days displayed down-regulation of liver hepcidin expression without changes in liver triglycerides or histology. This was accompanied by elevated duodenal divalent metal transporter1 and ferroportin protein expression. Injection of hepcidin peptide negated the effect of ethanol on duodenal iron transporters. Ethanol down-regulated hepcidin promoter activity and the DNA binding activity of CCAAT/enhancer-binding protein alpha (C/EBPalpha) but not beta. Interestingly, the antioxidants vitamin E and N-acetylcysteine abolished both the alcohol-mediated down-regulation of C/EBPalpha binding activity and hepcidin expression in the liver and the up-regulation of duodenal divalent metal transporter 1. Collectively, these findings indicate that alcohol metabolism-mediated oxidative stress regulates hepcidin transcription via C/EBPalpha, which in turn leads to increased duodenal iron transport.


It has long been observed that chronic alcohol consumption in moderate to excess quantities results in increased serum ferritin, transferrin-iron saturation (TS), and may result in increased hepatic iron stores.1 Heavy alcohol consumption may lead to a false diagnosis of hemochromatosis because of markedly elevated serum TS and ferritin.2 A population-based study showed there is a dose-response relationship between daily alcohol consumption and body iron stores.3 The hepatic iron index (HII), in fact, was originally developed to differentiate putative hemochromatosis homozygotes from those with iron overload due to alcohol use.2 It was even argued by McDonald and others several decades ago that hemochromatosis may be a dietary disease, caused by excessive alcohol consumption and other dietary factors rather than a genetic predisposition to increased iron absorption from a normal diet, as is now believed to be the case with classical or HFE-associated hemochromatosis.4

The mechanism by which alcohol consumption leads to elevation in serum and tissue markers of iron overload has not been clear. One possible explanation is that this may be a nonspecific effect of cirrhosis. Several groups have shown that liver iron stores are increased among patients with alcoholic liver disease and hepatitis C in the setting of chronic liver disease.5, 6 Stuart and colleagues demonstrated that expression of duodenal iron transporters was increased at the transcriptional level in humans with cirrhosis of varying etiologies.7 Studies by Chapman and Sherlock in the 1970s suggested that alcohol may mediate increased absorption of iron.8 An interesting study from Norway showed that cessation of alcohol intake resulted in fairly prompt reduction in serum TS and ferritin, suggesting that the effect of alcohol was a direct one, rather than secondary to liver disease.9 However, it appears unlikely that alcohol was directly prompting increased iron absorption at the level of intestinal absorption, given that even 1-2 drinks per day are sufficient to lead to an increase in serum TS and ferritin.3

Hepcidin is a recently described circulating peptide with antimicrobial properties produced in the liver that appears to regulate iron absorption in the duodenum. Mice deficient in hepcidin develop iron overload;10 overexpression of the gene encoding for hepcidin results in iron deficiency anemia.11 In normal humans, there appears to be an inverse relationship between body iron stores and hepcidin production; among patients with HFE-associated hereditary hemochromatosis, hepcidin gene expression appears inappropriately low.12 Finally, cross-breeding of HFE knockout mice with those overexpressing the hepcidin gene has been shown to prevent the phenotype of iron overload.13 In sum, these data collectively suggest hepcidin is a negative regulator of body iron stores and exerts its effect via inhibition of iron absorption in the duodenum. Recent studies show that the mechanism appears to act via binding to and internalization of ferroportin 1 (FPN1), the basolateral iron transporter which is subsequently degraded.14

A recent study tested the hypothesis that hepatic iron overload caused by alcohol is mediated by hepcidin.15 Harrison-Findik et al. treated 129/Sv mice with 10%-20% ethanol added to drinking water for 7 days. Hepatic hepcidin mRNA was measured in vivo at 3 and 7 days in male and female mice given 10% and 20% alcohol in comparison to control mice. There was no significant reduction in hepatic hepcidin gene expression between alcohol-treated and control mice at 3 days. However after 7 days, alcohol-treated mice had significantly decreased hepatic hepcidin mRNA expression compared to control mice; this effect was more pronounced among mice given 20% alcohol, and the latter group also showed a gender difference, with greater suppression of hepcidin gene expression among male mice. The difference in hepcidin mRNA expression between alcohol-treated and control mice was accounted for entirely by hepcidin 1 rather than hepcidin 2. These experiments were repeated in C57/BL6 mice and showed similar results; Northern blotting analysis confirmed the differential expression of hepcidin mRNA in alcohol-fed mice. The authors then showed that decreased hepcidin mRNA expression in alcohol-fed 129/Sv and C57/BL6 mice was associated with increased duodenal expression of DMT1 and FPN1, the mucosal and basolateral duodenal iron transporters, respectively, in the experimental mice. Finally, injection of synthetic hepcidin (1 μg/g of body weight in 0.9% saline between day 4 and 7) abrogated the up-regulation of DMT1 and FPN1 in the duodenum of alcohol-treated mice.

Harrison-Findik then further examined the mechanism whereby alcohol may lead to suppression of hepcidin mRNA expression. They used VL-17 cells, a HepG2 cell line capable of alcohol metabolism via transformation with alcohol dehydrogenase and cytochrome P450 2E1. After 2 days of exposure with 25 mM alcohol, there was significantly decreased hepcidin mRNA and protein (prohepcidin) production by the VL-17 cells; this effect was blocked by 4-methylpyrazole, a specific inhibitor of ADH and CYP 2E1, but not by isopropanol. These results suggest that the effect of alcohol on hepcidin mRNA expression is mediated via increased activity of alcohol-metabolizing enzymes. There was no effect in VL-17 cells treated with alcohol on expression of transferrin receptor-1, ferritin, and iron regulatory protein 2 (IRP2). Gel-shift assays also did not show any effect of IRP1 binding to RNA in these cells. Histologic evaluation did not demonstrate evidence of necrosis, inflammation, or lipid accumulation in livers of the 129/Sv mice treated with alcohol. Increased presence of reactive oxygen species (ROS) was shown in the liver of alcohol-fed mice by dihydroethidium staining. Dietary supplementation with high-dose vitamin E (2000 IU/kg body weight) reduced the production of alcohol-mediated ROS and also prevented reduction in hepcidin mRNA production in vivo; treatment with the antioxidant N-acetylcysteine also produced a similar effect.

Finally, using gel-shift assays and western blotting techniques, the authors showed that ROS produced by metabolism of alcohol resulted in decreased DNA binding of C/EBα, a transcription factor that has been shown to bind to the hepcidin promoter. This effect was associated with presence of decreased C/EBα protein and abrogated by vitamin E. Use of small interfering RNA inhibition confirmed that decreased C/EBα mRNA was associated with decreased hepcidin mRNA transcription.

In summary, this elegant work by Harrison-Findik and co-workers shows an important mechanism whereby alcohol could relate to iron overload. They show that increased production of ROS by alcohol via the ADH- and CYP2E1-metabolizing system appears to result in decreased production of hepcidin at the level of transcription via reduced activity of C/EBα. Furthermore, this effect could be blunted by administration of antioxidants. Given the many potentially adverse consequences of iron overload among patients who chronically use excess amounts of alcohol, it is intriguing to speculate that this study may provide a potential role of antioxidant supplementation to prevent these complications among patients with alcoholic disease.