Liver hepcidin mRNA correlates with iron stores, but not inflammation, in patients with chronic hepatitis C

PURPOSE: Liver iron is frequently elevated in chronic hepatitis C and may contribute to liver injury. The pathophysiology behind this phenomenon may involve hepcidin, a gene that is up-regulated in the liver by inflammation and iron. Inappropriately low hepcidin is important to the pathophysiology of hereditary hemochromatosis. However, the role of hepcidin in the iron loading of patients with hepatitis C is unknown. SUBJECTS AND METHODS: To determine whether liver hepcidin mRNA correlates with markers of hepatic inflammation and iron status in patients with hepatitis C, we extracted total RNA from liver biopsy specimens of patients with chronic hepatitis C and quantified hepcidin mRNA. Liver hepcidin mRNA levels were then correlated with aspartate aminotransferase, alanine aminotransferase, ferritin, viral load, fibrosis, hepatic iron concentration, and Hepatic Activity Index (HAI). RESULTS: Among patients with hepatitis C, there was a significant correlation of hepcidin mRNA expression in the liver with hepatic iron concentration and serum ferritin (r = 0.72, P = 0.006, and r = 0.60, P = 0.01, respectively). Hepcidin mRNA expression in the liver did not correlate with aspartate aminotransferase, alanine aminotransferase, HAI, or viral load. No differences in hepcidin mRNA were found based on viral genotype or the presence of fibrosis. CONCLUSION: In contrast to other inflammatory states, hepcidin mRNA expression in the liver was independent of markers of inflammation in hepatitis C. Instead, our results suggest that iron stores in patients with hepatitis C regulate hepcidin expression and that iron loading in chronic hepatitis C is not due to inappropriate hepcidin expression.     

Iron transferrin regulates hepcidin synthesis in primary hepatocyte culture through hemojuvelin and BMP2/4

The peptide hormone hepcidin is the principal regulator of systemic iron homeostasis. We examined the pathway by which iron stimulates the production of hepcidin. In humans who ingested 65 mg of iron, the increase in transferrin saturation preceded by hours the increase in urinary hepcidin excretion. Increases in urinary hepcidin concentrations were proportional to the increment in transferrin saturation. Paradoxically, in previous studies in primary hepatocytes and cell lines, hepcidin response to iron or iron transferrin was not observed. We now report that freshly isolated murine primary hepatocytes responded to holotransferrin but not apotransferrin by increasing hepcidin mRNA. Hepcidin increase was not due to contamination of the transferrin preparations by endotoxin, a potent pathologic stimulus of hepcidin synthesis. Using this culture system, we showed that holotransferrin concentrations regulate hepcidin mRNA concentrations through a hemojuvelin/BMP2/4-dependent pathway. Although BMP9 is known to be expressed in the liver and potently increased the basal concentrations of hepcidin mRNA, it did not interact with hemojuvelin, and interference with its signaling pathway did not affect iron regulation. Fresh primary hepatocytes constitute a sufficient system for the regulation of hepcidin by physiologic iron stimuli and will greatly facilitate studies of major disorders of iron homeostasis.     

Study of iron metabolism disturbances in an animal model of insulin resistance

The relationship between iron and insulin-resistance (IR) is documented by the positive correlation between iron stores and IR. Moreover, some patients exhibited a hepatic iron overload associated with IR (HIO-IR) but the mechanism involved in this overload is not known. Thus, we studied the iron metabolism disturbances in an animal model of IR and the influence of provoked hyperglycemia/hyperinsulinemia on plasma iron parameters. Wistar rats were fed a control or a high-fat/high-energy (HF/HE) diet. Plasma glucose, insulin, iron, transferrin and transferrin saturation (TS) were measured during intra-peritoneal glucose test tolerance (IPGTT) compared to saline. Hemogram, tissue iron concentrations and hepatic hepcidin mRNA expression were determined at the end of experiment. HF/HE rats exhibited higher body and liver weights, increased IR-index and hemoglobin concentration. Iron content was lower in the spleen of HF/HE rats and tended to decrease in the liver as compared to controls. Transferrin values were higher and these of TS lower in HF/HE group. The hepcidin mRNA was 3.5-fold lower in HF/HE rats than in controls. IPGTT had no effect on iron status parameters in both groups. As reflected by higher hemoglobin concentration, IR could increase erythropo?esis which enhances iron requirement. Iron stores and TS value decreased leading to a down-regulation of hepcidin expression which increased iron absorption. Hepcidin expression should be investigated in metabolic syndrome and hepatic iron overload associated with IR.     

Hepcidin,a putative mediator of anemia of inflammation,is a type II acute-phase protein

Hepcidin is a liver-made peptide proposed to be a central regulator of intestinal iron absorption and iron recycling by macrophages. In animal models, hepcidin is induced by inflammation and iron loading, but its regulation in humans has not been studied. We report that urinary excretion of hepcidin was greatly increased in patients with iron overload, infections, or inflammatory diseases. Hepcidin excretion correlated well with serum ferritin levels, which are regulated by similar pathologic stimuli. In vitro iron loading of primary human hepatocytes, however, unexpectedly down-regulated hepcidin mRNA, suggesting that in vivo regulation of hepcidin expression by iron stores involves complex indirect effects. Hepcidin mRNA was dramatically induced by interleukin-6 (IL-6) in vitro, but not by IL-1 or tumor necrosis factor alpha (TNF-alpha), demonstrating that human hepcidin is a type II acute-phase reactant. The linkage of hepcidin induction to inflammation in humans supports its proposed role as a key mediator of anemia of inflammation.     

Increased hepcidin expression and hypoferraemia associated with an acute phase response are not affected by inactivation of HFE

The effect of HFE inactivation on iron homeostasis during an acute phase response was investigated in mice. HFE knockout, beta2-microglobulin knockout and C57BL/6J mice were injected with Freund's Complete Adjuvant to induce an acute phase response and hepatic hepcidin expression and serum transferrin saturation was determined 16 h later. Hepcidin mRNA increased in all strains in response to an acute phase stimulus when compared with untreated control animals. Hypoferraemia also occurred in all strains, indicating that both the upregulation of hepcidin and the decrease in transferrin saturation associated with an acute phase response is not dependent on HFE function.     

Inappropriate Expression of Hepcidin by Liver Congestion Contributes to Anemia and Relative Iron Deficiency

BackgroundAnemia and relative iron deficiency (RID) are prevalent in patients with heart failure (HF). The etiology of anemia and RID in HF patients is unclear. Hepcidin expression may be closely related to anemia and RID in HF patients. Although hepcidin is produced mainly by the liver, and the most frequent histologic appearance of liver in HF patients is congestion, the influence of liver congestion (LC) on hepcidin production has not yet been investigated. We investigated whether hepcidin contributed to anemia and RID in rats with LC.Methods and ResultsLC was induced in rats by ligating the inferior vena cava and compared with bleeding anemia (BA) model induced by phlebotomy and hemolytic anemia (HA) model induced by injection of phenylhydrazine. BA and HA strongly suppressed expression of hepcidin in liver and so did not cause decrease in serum iron and transferrin saturation. However, hepcidin expression did not decrease in LC rats, which resulted in anemia and lower transferrin saturation. In addition, many cells with hemosiderin deposits were observed in the liver and spleen and not in the bone marrow, and this appeared to be related to suppression of hepcidin expression. Iron accumulated in hepatocytes, and bone morphogenetic protein 6, which induces hepcidin, increased. Inflammation was observed in the congestive liver, and there was an increase in interleukin-6, which also induced hepcidin and was induced by free heme and hemoglobin via Toll-like receptor 4.ConclusionsWe conclude that LC contributes to RID and anemia, and it does so via inappropriate expression of hepcidin.     

Anemia in beta-thalassemia patients targets hepatic hepcidin transcript levels independently of iron metabolism genes controlling hepcidin expression

Thalassemia associates anemia and iron overload, two opposite stimuli regulating hepcidin gene expression. We characterized hepatic hepcidin expression in 10 thalassemia major and 13 thalassemia intermedia patients. Hepcidin mRNA levels were decreased in the thalassemia intermedia group which presented both lower hemoglobin and higher plasma soluble transferrin receptor levels. There was no relationship between hepcidin mRNA levels and those of genes controlling iron metabolism, including HFE, hemojuvelin, transferrin receptor-2 and ferroportin. These results underline the role of erythropoietic activity on hepcidin decrease in thalassemic patients and suggest that mRNA modulations of other studied genes do not have a significant impact.     

Gender-related variations in iron metabolism and liver diseases

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.     

Hepcidin expression inversely correlates with the expression of duodenal iron transporters and iron absorption in rats

BACKGROUND & AIMS: Hepcidin is an antimicrobial peptide thought to be involved in the regulation of intestinal iron absorption. To further investigate its role in this process, we examined hepatic and duodenal gene expression in rats after the switch from a control diet to an iron-deficient diet. METHODS: Adult rats on an iron-replete diet were switched to an iron-deficient diet and the expression of iron homeostasis molecules in duodenal and liver tissue was studied over 14 days. Intestinal iron absorption was determined at these same time-points by measuring the retention of an oral dose of (59)Fe. RESULTS: Iron absorption increased 2.7-fold within 6 days of switching to an iron-deficient diet and was accompanied by an increase in the duodenal expression of Dcytb, divalent metal transporter 1, and Ireg1. These changes precisely correlated with decreases in hepatic hepcidin expression and transferrin saturation. No change in iron stores or hematologic parameters was detected. CONCLUSIONS: This study showed a close relationship between the expression of hepcidin, duodenal iron transporters, and iron absorption. Both hepcidin expression and iron absorption can be regulated before iron stores and erythropoiesis are affected, and transferrin saturation may signal such changes.     

Serum and liver iron differently regulate the bone morphogenetic protein 6 (BMP6)-SMAD signaling pathway in mice

The bone morphogenetic protein 6 (BMP6)-SMAD signaling pathway is a central regulator of hepcidin expression and systemic iron balance. However, the molecular mechanisms by which iron is sensed to regulate BMP6-SMAD signaling and hepcidin expression are unknown. Here we examined the effects of circulating and tissue iron on Bmp6-Smad pathway activation and hepcidin expression in vivo after acute and chronic enteral iron administration in mice. We demonstrated that both transferrin saturation and liver iron content independently influence hepcidin expression. Although liver iron content is independently positively correlated with hepatic Bmp6 messenger RNA (mRNA) expression and overall activation of the Smad1/5/8 signaling pathway, transferrin saturation activates the downstream Smad1/5/8 signaling cascade, but does not induce Bmp6 mRNA expression in the liver. Hepatic inhibitory Smad7 mRNA expression is increased by both acute and chronic iron administration and mirrors overall activation of the Smad1/5/8 signaling cascade. In contrast to the Smad pathway, the extracellular signal-regulated kinase 1 and 2 (Erk1/2) mitogen-activated protein kinase (Mapk) signaling pathway in the liver is not activated by acute or chronic iron administration in mice. CONCLUSION: Our data demonstrate that the hepatic Bmp6-Smad signaling pathway is differentially activated by circulating and tissue iron to induce hepcidin expression, whereas the hepatic Erk1/2 signaling pathway is not activated by iron in vivo.     

Iron metabolism in hepcidin1 knockout mice in response to phenylhydrazine-induced hemolysis

Hepcidin, an iron regulatory peptide, plays a central role in the maintenance of systemic iron homeostasis by inducing the internalization and degradation of the iron exporter, ferroportin. Hepcidin expression in the liver is regulated in response to several stimuli including iron status, erythropoietic activity, hypoxia and inflammation. Hepcidin expression has been shown to be reduced in phenylhydrazine-treated mice, a mouse model of acute hemolysis. In this mouse model, hepcidin suppression was associated with increased expression of molecules involved in iron transport and recycling. The present study aims to explore whether the response to phenylhydrazine treatment is affected by hepcidin deficiency and/or the subsequently altered iron metabolism. Hepcidin1 knockout (Hamp(-/-)) and wild type mice were treated with phenylhydrazine or saline and parameters of iron homeostasis were determined 3 days after the treatment. In wild type mice, phenylhydrazine administration resulted in significantly reduced serum iron, increased tissue non-heme iron levels and suppressed hepcidin expression. The treatment was also associated with increases in membrane ferroportin protein levels and spleen heme oxygenase 1 mRNA expression. In addition, trends toward increased mRNA expression of duodenal iron transporters were also observed. In contrast, serum iron and tissue non-heme iron levels in Hamp(-/-) mice were unaffected by the treatment. Moreover, the effects of phenylhydrazine on the expression of ferroportin and duodenal iron transporters were not observed in Hamp(-/-) mice. Interestingly, mRNA levels of molecules involved in splenic heme uptake and degradation were significantly induced by Hamp disruption. In summary, our study demonstrates that the response to phenylhydrazine-induced hemolysis differs between wild type and Hamp(-/-) mice. This observation may be caused by the absence of hepcidin per se or the altered iron homeostasis induced by the lack of hepcidin in these mice.     

Decreased liver hepcidin expression in the Hfe knockout mouse

Hepcidin is a circulating antimicrobial peptide which has been proposed to regulate the uptake of dietary iron and its storage in reticuloendothelial macrophages. Transgenic mice lacking hepcidin expression demonstrate abnormalities of iron homeostasis similar to Hfe knockout mice and to patients with HFE-associated hereditary hemochromatosis (HH). To identify any association between liver hepcidin expression and the iron homeostasis abnormalities observed in HH, we compared liver hepcidin mRNA content in wild type and Hfe knockout mice. Because the iron homeostasis abnormalities in the Hfe knockout mice are greatest early in life, we analyzed mice at different ages. At four weeks of age, Hfe knockout mice had significantly decreased liver hepcidin mRNA expression compared to wild type mice. The decreased hepcidin expression was associated with hepatic iron deposition, elevated transferrin saturations, and decreased splenic iron concentrations. At 10 weeks of age, despite marked hepatic iron loading, Hfe knockout mice demonstrated liver hepcidin mRNA expression similar to that observed in wild type mice. Placing 8 week-old wild type and Hfe knockout mice on a 2% carbonyl iron diet for 2 weeks led to a similar degree of hepatic iron loading in each group. However, while the wild type mice demonstrated a mean five-fold increase in liver hepcidin mRNA, no change was observed in the Hfe knockout mice. The lack of an increase in liver hepcidin expression in these iron-loaded Hfe knockout mice was associated with sparing of iron deposition into the spleen. These data indicate that the normal relationship between body iron stores and liver hepcidin mRNA levels is altered in Hfe knockout mice, such that liver hepcidin expression is relatively decreased. We speculate that decreased hepcidin expression relative to body iron stores contributes to the iron homeostasis abnormalities characteristic of HH.     

The effects of erythropoetic activity and iron burden on hepcidin expression in patients with thalassemia major

Hepcidin production is homeostatically regulated by iron stores, anemia and hypoxia. We evaluated the effect of iron overload and of ineffective erythropoeisis on hepcidin expression in patients with thalassemia major. Liver hepcidin mRNA levels correlated with hemoglobin concentration and inversely correlated with serum transferrin receptor, erythropoietin and non-transferrin-bound iron. They did not correlate with indices of iron load. Urinary hepcidin levels were disproportionably suppressed in regards to iron burden. We conclude that hepcidin expression is regulated mainly by increased erythropoietic activity rather than by iron load and that hepcidin plays a central regulatory role in iron circulation and iron toxicity in patients with thalassemia.     

Hepcidin and iron-related gene expression in subjects with Dysmetabolic Hepatic Iron Overload

BACKGROUND/AIMS: Many patients with hepatic iron overload do not have identifiable mutations and often present with metabolic disorders and hepatic steatosis. Since the pathophysiology of Dysmetabolic Hepatic Iron Overload (DHIO) is still obscure, the aim of this study was to evaluate, in these patients, possible alterations in iron-related molecule expression. METHODS: Iron-related gene mRNA levels were determined by quantitative-PCR in liver biopsies of subjects with NAFLD without iron overload and patients with HFE-hemochromatosis, beta-thalassemia major and DHIO. Urinary hepcidin was measured by immunoblotting. RESULTS: No alterations in mRNA expression of either iron transporters or exporters were found in DHIO. mRNA and urinary hepcidin levels normalized for the amount of iron overload showed a significantly lower ratio than in controls, although not as low as in hemochromatosis or beta-thalassemia. Differently from what observed in hemochromatosis, hepcidin mRNA did not correlate with urinary hepcidin. CONCLUSIONS: Patients with DHIO show appropriate regulation of mRNAs encoding proteins involved in iron uptake and efflux but dysregulation of hepcidin production. The relatively elevated urinary hepcidin can explain the iron phenotype in DHIO (more macrophage iron retention and low/normal transferrin saturation).     

Maternal serum hepcidin is low at term and independent of cord blood iron status

Objectives: Hepcidin is the key regulator of iron homeostasis. The aims of this study were to determine serum hepcidin concentrations and reference ranges in pregnant women and cord blood of newborns at term and to evaluate the associations between hepcidin concentrations and iron status parameters. Methods: A total of 191 pregnant women–newborn pairs were studied in Kuopio University Hospital, Finland. The measured parameters were serum hepcidin, ferritin, transferrin receptor, transferrin saturation, red cell indices, and erythropoietin. Results: The hepcidin concentration in pregnant women was significantly lower than in cord blood at term [geometric mean concentration (GMC) (95% confidence intervals) in pregnant women 10.7 ng/mL (8.5–13.4 ng/mL) vs. GMC of cord blood hepcidin 69.3 ng/mL (55.3–86.8 ng/mL), P < 0.001, adjusted analysis of variance]. Hepcidin was undetectable in 12% of mothers. Hepcidin concentration in pregnant women was the lowest in those who had the lowest iron status. However, maternal hepcidin concentration was not associated with cord blood hepcidin or iron status markers. Hepcidin concentration in cord blood was associated with cord blood iron status, but not with maternal iron status. Conclusions: At term pregnancy, hepcidin concentrations are very low, allowing maximal availability of iron for the fetus. Maternal and cord blood hepcidin levels were independently associated with either maternal or cord blood iron status.