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 ␣ (C/EBP␣) but not ␤. Interestingly, the antioxidants vitamin E and N-acetylcysteine abolished both the alcohol-mediated down-regulation of C/EBP␣ 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/EBP␣, which in turn leads to increased duodenal iron transport.

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. (HEPATOLOGY 2007;46:1979-1985 H epcidin is a circulatory peptide that is synthesized in the liver and acts as the key regulator of iron metabolism by modulating iron absorption through the duodenum and the release of iron from macrophages. 1,2 We have recently demonstrated that acute alcohol-induced oxidative stress down-regulates liver hepcidin transcripts in mice by altering the DNA-binding activity of the transcription factor CCAAT/enhancer binding protein (C/EBP) alpha. 3 Alcohol-mediated down-regulation of liver hepcidin mRNA expression occurred without associated changes in liver histology or triglycerides. 3 Mice exposed to ethanol for 7 days exhibited elevated iron transporter protein expression in the duodenum. This was abolished by injecting mice with hepcidin peptide. 3 Bridle et al. also have reported reduced hepcidin mRNA expression in rats with chronic alcohol exposure. 4 These results demonstrated a role for alcohol in the regulation of iron metabolism by regulating the expression of hepcidin in the liver.Liver hepcidin messenger RNA (mRNA) expression is regulated inversely by iron and alcohol. 2-4 Namely, iron up-regulates, whereas alcohol down-regulates, liver hepcidin expression. However, the combined effect of alcohol and iron in the regulation of hepcidin expres...

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.

The regulation of iron metabolism involves multiple organs including the duodenum, liver and bone marrow. The recent discoveries of novel iron-regulatory proteins have brought the liver to the forefront of iron homeostasis. The iron overload disorder, genetic hemochromatosis, is one of the most prevalent genetic diseases in individuals of Caucasian origin. Furthermore, patients with non-hemochromatotic liver diseases, such as alcoholic liver disease, chronic hepatitis C or nonalcoholic steatohepatitis, often exhibit elevated serum iron indices (ferritin, transferrin saturation) and mild to moderate hepatic iron overload. Clinical data indicate significant differences between men and women regarding liver injury in patients with alcoholic liver disease, chronic hepatitis C or nonalcoholic steatohepatitis. The penetrance of genetic hemochromatosis also varies between men and women. Hepcidin has been suggested to act as a modifier gene in genetic hemochromatosis. Hepcidin is a circulatory antimicrobial peptide synthesized by the liver. It plays a pivotal role in the regulation of iron homeostasis. Hepcidin has been shown to be regulated by iron, inflammation, oxidative stress, hypoxia, alcohol, hepatitis C and obesity. Sex and genetic background have also been shown to modulate hepcidin expression in mice. The role of gender in the regulation of human hepcidin gene expression in the liver is unknown. However, hepcidin may play a role in gender-based differences in iron metabolism and liver diseases. Better understanding of the mechanisms associated with gender-related differences in iron metabolism and chronic liver diseases may enable the development of new treatment strategies.

Despite heavy consumption over a long period of time,

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.

Nonalcoholic steatohepatitis (NASH) patients have elevated plasma saturated free fatty acid levels. These toxic fatty acids can induce liver cell death and our recent results demonstrated that the biliary epithelium may be susceptible to lipotoxicity. Here, we explored the molecular mechanisms of cholangiocyte lipoapoptosis in cell culture and in an animal model of NASH. Treatment of cholangiocytes with palmitate (PA) showed increased caspase 3/7 activity and increased levels of cleaved poly (ADP-ribose) polymerase and cleaved caspase 3, demonstrating cholangiocyte lipoapoptosis. Interestingly, treatment with PA significantly increased the levels of microRNA miR-34a, a pro-apoptotic microRNA known to be elevated in NASH. PA induction of miR-34a was abolished in cholangiocytes transduced with forkhead family of transcription factor class O (FoxO)3 shRNA, demonstrating that FoxO3 activation is upstream of miR-34a and suggesting that FoxO3 is a novel transcriptional regulator of miR-34a. Further, anti-miR-34a protected cholangiocytes from PA-induced lipoapoptosis. Direct and indirect targets of miR-34a, such as SIRT1, receptor tyrosine kinase (MET), Kruppel-like factor 4, fibroblast growth factor receptor (FGFR)1, and FGFR4, were all decreased in PA-treated cholangiocytes. SIRT1 and MET were partially rescued by a miR-34a antagonist. Cholangiocyte apoptosis and miR-34a were dramatically increased in the liver of mice with early histologic features of NASH. Our study provides evidence for the pro-apoptotic role of miR-34a in PA-induced cholangiocyte lipoapoptosis in culture and in the liver.

Previously we described activating mutations of h ␤ c , the common signaling subunit of the receptors for the hematopoietic and inflammatory cytokines, GM-CSF, IL-3, and IL-5. The activated mutant, h ␤ c FI ⌬ , is able to confer growth factor-independent proliferation on the murine myeloid cell line FDC-P1, and on primary committed myeloid progenitors. We have used this activating mutation to study the effects of chronic cytokine receptor stimulation. Transgenic mice were produced carrying the h ␤ c FI ⌬ cDNA linked to the constitutive promoter derived from the phosphoglycerate kinase gene, PGK-1 . Transgene expression was demonstrated in several tissues and functional activity of the mutant receptor was confirmed in hematopoietic tissues by the presence of granulocyte macrophage and macrophage colony-forming cells (CFU-GM and CFU-M) in the absence of added cytokines. All transgenic mice display a myeloproliferative disorder characterized by splenomegaly, erythrocytosis, and granulocytic and megakaryocytic hyperplasia. This disorder resembles the human disease polycythemia vera, suggesting that activating mutations in h ␤ c may play a role in the pathogenesis of this myeloproliferative disorder. In addition, these transgenic mice develop a sporadic, progressive neurological disease and display bilateral, symmetrical foci of necrosis in the white matter of brain stem associated with an accumulation of macrophages. Thus, chronic h ␤ c activation has the potential to contribute to pathological events in the central nervous system. ( J. Clin. Invest. 1998.