Lack of hepcidin expression attenuates steatosis and causes fibrosis in the liver.

AIM: To investigate the role of key iron-regulatory protein, hepcidin in non-alcoholic fatty liver disease (NAFLD). METHODS: Hepcidin (Hamp1) knockout and floxed control mice were administered a high fat and high sucrose (HFS) or a regular control diet for 3 or 7 mo. Steatosis, triglycerides, fibrosis, protein and gene expression in mice livers were determined by histological and biochemical techniques, western blotting and real-time polymerase chain reaction. RESULTS: Knockout mice exhibited hepatic iron accumulation. Despite similar weight gains, HFS feeding induced hepatomegaly in floxed, but not knockout, mice. The livers of floxed mice exhibited higher levels of steatosis, triglycerides and c-Jun N-terminal kinase (JNK) phosphorylation than knockout mice. In contrast, a significant increase in fibrosis was observed in knockout mice livers within 3 mo of HFS administration. The hepatic gene expression levels of sterol regulatory element-binding protein-1c and fat-specific protein-27, but not peroxisome proliferator-activated receptor-alpha or microsomal triglyceride transfer protein, were attenuated in HFS-fed knockout mice. Knockout mice fed with regular diet displayed increased carnitine palmitoyltransferase-1a and phosphoenolpyruvate carboxykinase-1 but decreased glucose-6-phosphatase expression in the liver. In summary, attenuated steatosis correlated with decreased expression of lipogenic and lipid storage genes, and JNK phosphorylation. Deletion of Hamp1 alleles per se modulated hepatic expression of beta-oxidation and gluconeogenic genes. CONCLUSION: Lack of hepcidin expression inhibits hepatic lipid accumulation and induces early development of fibrosis following high fat intake. Hepcidin and iron may play a role in the regulation of metabolic pathways in the liver, which has implications for NAFLD pathogenesis.

Ceramide Induces Human Hepcidin Gene Transcription through JAK/STAT3 Pathway.

Changes in lipid metabolism and iron content are observed in the livers of patients with fatty liver disease. The expression of hepcidin, an iron-regulatory and acute phase protein synthesized by the liver, is also modulated. The potential interaction of lipid and iron metabolism is largely unknown. We investigated the role of lipid intermediate, ceramide in the regulation of human hepcidin gene, HAMP. Human hepatoma HepG2 cells were treated with cell-permeable ceramide analogs. Ceramide induced significant up-regulation of HAMP mRNA expression in HepG2 cells. The effect of ceramide on HAMP expression was mediated through transcriptional mechanisms because it was completely blocked with actinomycin D treatment. Reporter assays also confirmed the activation of 0.6 kb HAMP promoter by ceramide. HepG2 cells treated with ceramide displayed increased phosphorylation of STAT3, JNK, and NF-κB proteins. However, ceramide induced the binding of STAT3, but not NF-κB or c-Jun, to HAMP promoter, as shown by the chromatin immunoprecipitation assays. The mutation of STAT3 response element within 0.6 kb HAMP promoter region significantly inhibited the stimulatory effect of ceramide on HAMP promoter activity. Similarly, the inhibition of STAT3 with a pan-JAK kinase inhibitor and STAT3 siRNA pool also diminished the induction of both HAMP promoter activity and mRNA expression by ceramide. In conclusion, we have shown a direct role for ceramide in the activation of hepatic HAMP transcription via STAT3. Our findings suggest a crosstalk between lipid and iron metabolism in the liver, which may contribute to the pathogenesis of obesity-related fatty liver disease.

Saturated fatty acids induce post-transcriptional regulation of HAMP mRNA via AU-rich element-binding protein, human antigen R (HuR).

Iron is implicated in fatty liver disease pathogenesis. The human hepcidin gene, HAMP, is the master switch of iron metabolism. The aim of this study is to investigate the regulation of HAMP expression by fatty acids in HepG2 cells. For these studies, both saturated fatty acids (palmitic acid (PA) and stearic acid (SA)) and unsaturated fatty acid (oleic acid (OA)) were used. PA and, to a lesser extent, SA, but not OA, up-regulated HAMP mRNA levels, as determined by real-time PCR. To understand whether PA regulates HAMP mRNA at the transcriptional or post-transcriptional level, the transcription inhibitor actinomycin D was employed. PA-mediated induction of HAMP mRNA expression was not blocked by actinomycin D. Furthermore, PA activated HAMP 3'-UTR, but not promoter, activity, as shown by reporter assays. HAMP 3'-UTR harbors a single AU-rich element (ARE). Mutation of this ARE abolished the effect of PA, suggesting the involvement of ARE-binding proteins. The ARE-binding protein human antigen R (HuR) stabilizes mRNA through direct interaction with AREs on 3'-UTR. HuR is regulated by phosphorylation-mediated nucleo-cytoplasmic shuttling. PA activated this process. The binding of HuR to HAMP mRNA was also induced by PA in HepG2 cells. Silencing of HuR by siRNA abolished PA-mediated up-regulation of HAMP mRNA levels. PKC is known to phosphorylate HuR. Staurosporine, a broad-spectrum PKC inhibitor, inhibited both PA-mediated translocation of HuR and induction of HAMP expression. Similarly, rottlerin, a novel class PKC inhibitor, abrogated PA-mediated up-regulation of HAMP expression. In conclusion, lipids mediate post-transcriptional regulation of HAMP throughPKC- and HuR-dependent mechanisms.

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

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.

Apoptosis induced by Fas signaling does not alter hepatic hepcidin expression.

AIM: To determine the regulation of human hepcidin (HAMP) and mouse hepcidin (hepcidin-1 and hepcidin-2) gene expression in the liver by apoptosis using in vivo and in vitro experimental models. METHODS: For the induction of the extrinsic apoptotic pathway, HepG2 cells were treated with various concentrations of CH11, an activating antibody for human Fas receptor, for 12 h. Male C57BL/6NCR and C57BL/6J strains of mice were injected intraperitoneally with sublethal doses of an activating antibody for mouse Fas receptor, Jo2. The mice were anesthetized and sacrificed 1 or 6 h after the injection. The level of apoptosis was quantified by caspase-3 activity assay. Liver injury was assessed by measuring the levels of ALT/AST enzymes in the serum. The acute phase reaction in the liver was examined by determining the expression levels of IL-6 and SAA3 genes by SYBR green quantitative real-time PCR (qPCR). The phosphorylation of transcription factors, Stat3, Smad4 and NF-κB was determined by western blotting. Hepcidin gene expression was determined by Taqman qPCR. The binding of transcription factors to hepcidin-1 promoter was studied using chromatin immunoprecipitation (ChIP) assays. RESULTS: The treatment of HepG2 cells with CH11 induced apoptosis, as shown by the significant activation of caspase-3 (P < 0.001), but did not cause any significant changes in HAMP expression. Short-term (1 h) Jo2 treatment (0.2 µg/g b.w.) neither induced apoptosis and acute phase reaction nor altered mRNA expression of mouse hepcidin-1 in the livers of C57BL/6NCR mice. In contrast, 6 h after Jo2 injection, the livers of C57BL/6NCR mice exhibited a significant level of apoptosis (P < 0.001) and an increase in SAA3 (P < 0.023) and IL-6 (P < 0.005) expression in the liver. However, mRNA expression of hepcidin-1 in the liver was not significantly altered. Despite the Jo2-induced phosphorylation of Stat3, no occupancy of hepcidin-1 promoter by Stat3 was observed, as shown by ChIP assays. Compared to C57BL/6NCR mice, Jo2 treatment (0.2 µg/g b.w.) of C57BL/6J strain mice for 6 h induced a more prominent activation of apoptosis, liver injury and acute phase reaction. Similar to C57BL/6NCR mice, the level of liver hepcidin-1 mRNA expression in the livers of C57BL/6J mice injected with a sublethal dose of Jo2 (0.2 µg/g b.w.) remained unchanged. The injection of C57BL/6J mice with a higher dose of Jo2 (0.32 µg/g b.w.) did not also alter hepatic hepcidin expression. CONCLUSION: Our findings suggest that human or mouse hepcidin gene expression is not regulated by apoptosis induced via Fas receptor activation in the liver.

TLR4 signaling and the inhibition of liver hepcidin expression by alcohol.

AIM: To understand the role of toll-like receptor 4 (TLR4) signaling in the regulation of iron-regulatory hormone, hepcidin by chronic alcohol consumption. METHODS: For chronic alcohol intake studies, TLR4 mutant mice on C3H/HeJ background and wildtype counterpart on C3H/HeOuJ background were pair-fed with regular (control) and ethanol-containing Lieber De Carli liquids diets. Gene expression was determined by real-time quantitative PCR. Protein-protein interactions and protein expression were determined by co-immunoprecipitation and western blotting. The occupancy of hepcidin gene promoter was determined by chromatin immunoprecipitation assays. RESULTS: Chronic alcohol intake suppressed hepcidin mRNA expression in the livers of wildtype, but not TLR4 mutant, mice. The phosphorylation and nuclear translocation of nuclear factor (NF)-κB p65 subunit protein was observed in alcohol-fed wildtype, but not in alcohol-fed TLR4 mutant, mice. Similarly, alcohol induced the binding of NF-κB p50 subunit protein to hepcidin gene promoter in wildtype, but not in TLR4 mutant, mice. In contrast, the phosphorylation of Stat3 in the liver was stronger in alcohol-treated TLR4 mutant mice compared to alcohol-treated wildtype mice. The occupancy of hepcidin gene promoter by Stat3 was observed in alcohol-fed mutant, but not in wildtype, mice. An interaction between NF-κB p65 subunit protein and small heterodimer partner protein (SHP) was observed in the livers of both wildtype and TLR4 mutant mice fed with the control diet, as shown by co-immunoprecipitation studies. Alcohol intake elevated cytosolic SHP expression but attenuated its interaction with NF-κB in the liver, which was more prominent in the livers of wildtype compared to TLR4 mutant mice. CONCLUSION: Activation of TLR4 signaling and NF-кB are involved in the suppression of hepcidin gene transcription by alcohol in the presence of inflammation in the liver.

Effect of alcohol exposure on hepatic superoxide generation and hepcidin expression.

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.

Autophagy and cancer.

Autophagy is a homeostatic and evolutionarily conserved mechanism of self-digestion by which the cells degrade and recycle long-lived proteins and excess or damaged organelles. Autophagy is activated in response to both physiological and pathological stimuli including growth factor depletion, energy deficiency or the upregulation of Bcl-2 protein expression. A novel role of autophagy in various cancers has been proposed. Interestingly, evidence that supports both a positive and negative role of autophagy in the pathogenesis of cancer has been reported. As a tumor suppression mechanism, autophagy maintains genome stability, induces senescence and possibly autophagic cell death. On the other hand, autophagy participates in tumor growth and maintenance by supplying metabolic substrate, limiting oxidative stress, and maintaining cancer stem cell population. It has been proposed that the differential roles of autophagy in cancer are disease type and stage specific. In addition, substrate selectivity might be involved in carrying out the specific effect of autophagy in cancer, and represents one of the potential directions for future studies.

Alcohol Activates TGF-Beta but Inhibits BMP Receptor-Mediated Smad Signaling and Smad4 Binding to Hepcidin Promoter in the Liver.

Hepcidin, a key regulator of iron metabolism, is activated by bone morphogenetic proteins (BMPs). Mice pair-fed with regular and ethanol-containing L. De Carli diets were employed to study the effect of alcohol on BMP signaling and hepcidin transcription in the liver. Alcohol induced steatosis and TGF-beta expression. Liver BMP2, but not BMP4 or BMP6, expression was significantly elevated. Despite increased BMP expression, the BMP receptor, and transcription factors, Smad1 and Smad5, were not activated. In contrast, alcohol stimulated Smad2 phosphorylation. However, Smad4 DNA-binding activity and the binding of Smad4 to hepcidin promoter were attenuated. In summary, alcohol stimulates TGF-beta and BMP2 expression, and Smad2 phosphorylation but inhibits BMP receptor, and Smad1 and Smad5 activation. Smad signaling pathway in the liver may therefore be involved in the regulation of hepcidin transcription and iron metabolism by alcohol. These findings may help to further understand the mechanisms of alcohol and iron-induced liver injury.

Regulation of heme oxygenase expression by alcohol, hypoxia and oxidative stress.

AIM: To study the effect of both acute and chronic alcohol exposure on heme oxygenases (HOs) in the brain, liver and duodenum. METHODS: Wild-type C57BL/6 mice, heterozygous Sod2 knockout mice, which exhibit attenuated manganese superoxide dismutase activity, and liver-specific ARNT knockout mice were used to investigate the role of alcohol-induced oxidative stress and hypoxia. For acute alcohol exposure, ethanol was administered in the drinking water for 1 wk. Mice were pair-fed with regular or ethanol-containing Lieber De Carli liquid diets for 4 wk for chronic alcohol studies. HO expression was analyzed by real-time quantitative polymerase chain reaction and Western blotting. RESULTS: Chronic alcohol exposure downregulated HO-1 expression in the brain but upregulated it in the duodenum of wild-type mice. It did not alter liver HO-1 expression, nor HO-2 expression in the brain, liver or duodenum. In contrast, acute alcohol exposure decreased both liver HO-1 and HO-2 expression, and HO-2 expression in the duodenum of wild-type mice. The decrease in liver HO-1 expression was abolished in ARNT(+/-) mice. Sod2(+/-) mice with acute alcohol exposure did not exhibit any changes in liver HO-1 and HO-2 expression or in brain HO-2 expression. However, alcohol inhibited brain HO-1 and duodenal HO-2 but increased duodenal HO-1 expression in Sod2(+/-) mice. Collectively, these findings indicate that acute and chronic alcohol exposure regulates HO expression in a tissue-specific manner. Chronic alcohol exposure alters brain and duodenal, but not liver HO expression. However, acute alcohol exposure inhibits liver HO-1 and HO-2, and also duodenal HO-2 expression. CONCLUSION: The inhibition of liver HO expression by acute alcohol-induced hypoxia may play a role in the early phases of alcoholic liver disease progression.

Is the iron regulatory hormone hepcidin a risk factor for alcoholic liver disease?

Despite heavy consumption over a long period of time, only a small number of alcoholics develop alcoholic liver disease. This alludes to the possibility that other factors, besides alcohol, may be involved in the progression of the disease. Over the years, many such factors have indeed been identified, including iron. Despite being crucial for various important biological processes, iron can also be harmful due to its ability to catalyze Fenton chemistry. Alcohol and iron have been shown to interact synergistically to cause liver injury. Iron-mediated cell signaling has been reported to be involved in the pathogenesis of experimental alcoholic liver disease. Hepcidin is an iron-regulatory hormone synthesized by the liver, which plays a pivotal role in iron homeostasis. Both acute and chronic alcohol exposure suppress hepcidin expression in the liver. The sera of patients with alcoholic liver disease, particularly those exhibiting higher serum iron indices, have also been reported to display reduced prohepcidin levels. Alcohol-mediated oxidative stress is involved in the inhibition of hepcidin promoter activity and transcription in the liver. This in turn leads to an increase in intestinal iron transport and liver iron storage. Hepcidin is expressed primarily in hepatocytes. It is noteworthy that both hepatocytes and Kupffer cells are involved in the progression of alcoholic liver disease. However, the activation of Kupffer cells and TNF-alpha signaling has been reported not to be involved in the down-regulation of hepcidin expression by alcohol in the liver. Alcohol acts within the parenchymal cells of the liver to suppress the synthesis of hepcidin. Due to its crucial role in the regulation of body iron stores, hepcidin may act as a secondary risk factor in the progression of alcoholic liver disease. The clarification of the mechanisms by which alcohol disrupts iron homeostasis will allow for further understanding of the pathogenesis of alcoholic liver disease.

Regulation of liver hepcidin expression by alcohol in vivo does not involve Kupffer cell activation or TNF-alpha signaling.

Alcohol downregulates hepcidin expression in the liver leading to an increase in intestinal iron transport and liver iron storage. We have previously demonstrated that alcohol-mediated oxidative stress is involved in the inhibition of hepcidin transcription by alcohol in vivo. Kupffer cells and TNF-alpha play a key role in alcohol-induced liver injury. The aim of this study was to define their involvement in the regulation of hepcidin expression by alcohol. Kupffer cells were inactivated or depleted by employing gadolinium chloride and liposomes containing clodronate, respectively. Rats pair fed with the alcohol-Lieber-DeCarli diet for 6 wk and mice fed with 20% ethanol in the drinking water for 1 wk were used as experimental models. Interestingly, alcohol downregulated hepcidin expression in the livers of rats and mice independent of gadolinium chloride or clodronate treatment. One week of alcohol treatment was sufficient to induce a significant increase in TNF-alpha levels and phosphorylation of NF-kappaB subunit p65. The neutralization of TNF-alpha by specific antibodies inhibited p65 phosphorylation. However, neither the neutralization of TNF-alpha nor the lack of TNF-alpha receptor expression reversed alcohol-induced suppression of liver hepcidin expression. The level of alcohol-induced ROS in the liver was also undiminished following Kupffer cell inactivation or depletion. Our results demonstrate that alcohol-induced Kupffer cell activation and TNF-alpha signaling are not involved in the suppression of liver hepcidin expression by alcohol-mediated oxidative stress in vivo. Therefore, these findings suggest that alcohol acts within hepatocytes to suppress hepcidin expression and thereby influences iron homeostasis.

Role of alcohol in the regulation of iron metabolism.

Patients with alcoholic liver disease frequently exhibit increased body iron stores, as reflected by elevated serum iron indices (transferrin saturation, ferritin) and hepatic iron concentration. Even mild to moderate alcohol consumption has been shown to increase the prevalence of iron overload. Moreover, increased hepatic iron content is associated with greater mortality from alcoholic cirrhosis, suggesting a pathogenic role for iron in alcoholic liver disease. Alcohol increases the severity of disease in patients with genetic hemochromatosis, an iron overload disorder common in the Caucasian population. Both iron and alcohol individually cause oxidative stress and lipid peroxidation, which culminates in liver injury. Despite these observations, the underlying mechanisms of iron accumulation and the source of the excess iron observed in alcoholic liver disease remain unclear. Over the last decade, several novel iron-regulatory proteins have been identified and these have greatly enhanced our understanding of iron metabolism. For example, hepcidin, a circulatory antimicrobial peptide synthesized by the hepatocytes of the liver is now known to play a central role in the regulation of iron homeostasis. This review attempts to describe the interaction of alcohol and iron-regulatory molecules. Understanding these molecular mechanisms is of considerable clinical importance because both alcoholic liver disease and genetic hemochromatosis are common diseases, in which alcohol and iron appear to act synergistically to cause liver injury.

Iron-mediated regulation of liver hepcidin expression in rats and mice is abolished by alcohol.

UNLABELLED: Alcohol reduces and iron increases liver hepcidin synthesis. This study investigates the interaction of alcohol and iron in the regulation of hepcidin messenger RNA (mRNA) expression in animal models. Mice were administered 10% ethanol for 7 days after an iron-overloaded diet. Rats were administered regular or ethanol-Lieber De Carli diets for 7 weeks with or without carbonyl iron. Hfe(-/-) mice were used as a model for genetic iron overload. Hepcidin mRNA expression was determined by real-time polymerase chain reaction (PCR) and northern blotting. Iron elevated and alcohol decreased liver hepcidin expression in mice and rats. Interestingly, despite iron overload, alcohol was capable of suppressing the up-regulation of hepcidin mRNA expression in both models. Liver iron and ferritin protein expression was elevated in alcohol-treated rats, but was not elevated further in rats treated with both iron and alcohol. Duodenal ferroportin protein expression was increased both in alcohol-treated mice and in mice treated with alcohol and iron. Hfe(-/-) mice treated with ethanol for 7 days exhibited a further decrease in hepcidin mRNA expression. The iron-induced increase in DNA-binding activity of the transcription factor CCAAT/enhancer binding protein alpha (C/EBP alpha) was also suppressed by alcohol. CONCLUSION: Alcohol abolishes the iron-induced up-regulation of both liver hepcidin transcription and the DNA-binding activity of C/EBP alpha. Of note, hepcidin protects the body from the harmful effects of iron overload. Our findings therefore suggest that alcohol negates the protective effect of hepcidin, which may have implications for the liver injury observed in alcoholic liver disease and genetic hemochromatosis in combination with alcohol.

Alcohol metabolism-mediated oxidative stress down-regulates hepcidin transcription and leads to increased duodenal iron transporter expression.

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.