Insulin resistance and adipose tissue inflammation induced by a high-fat diet are attenuated in the absence of hepcidin.

Increased body iron stores and inflammation in adipose tissue have been implicated in the pathogenesis of insulin resistance (IR) and type 2 diabetes mellitus. However, the underlying basis of these associations is unclear. To attempt to investigate this, we studied the development of IR and associated inflammation in adipose tissue in the presence of increased body iron stores. Male hepcidin knock-out (Hamp1-/-) mice, which have increased body iron stores, and wild-type (WT) mice were fed a high-fat diet (HFD) for 12 and 24 weeks. Development of IR and metabolic parameters linked to this, insulin signaling in various tissues, and inflammation and iron-related parameters in visceral adipose tissue were studied in these animals. HFD-feeding resulted in impaired glucose tolerance in both genotypes of mice. In response to the HFD for 24 weeks, Hamp1-/- mice gained less body weight and developed less systemic IR than corresponding WT mice. This was associated with less lipid accumulation in the liver and decreased inflammation and lipolysis in the adipose tissue in the knock-out mice, than in the WT animals. Fewer macrophages infiltrated the adipose tissue in the knockout mice than in wild-type mice, with these macrophages exhibiting a predominantly anti-inflammatory (M2-like) phenotype and indirect evidence of a possible lowered intracellular iron content. The absence of hepcidin was thus associated with attenuated inflammation in the adipose tissue and increased whole-body insulin sensitivity, suggesting a role for it in these processes.

Enhanced insulin signaling and its downstream effects in iron-overloaded primary hepatocytes from hepcidin knock-out mice.

BACKGROUND: Increased body iron stores have been implicated in the pathogenesis of diabetes mellitus. However, the molecular mechanisms involved are unclear. The liver plays a central role in homeostasis of iron and glucose in the body. Mice deficient in hepcidin (the central regulator of systemic iron homeostasis) (Hamp1-/- mice) accumulate iron in the liver in vivo. The effects of such iron loading on hepatic insulin signaling and glucose metabolism are not known. METHODS: Hepatocytes isolated from Hamp1-/- mice were studied for markers of insulin signaling (and its downstream effects), glucose production, expression of gluconeogenic and lipogenic enzymes, and markers of AMPK (AMP-activated protein kinase) activation and oxidative stress. These parameters were studied both in the absence and presence of insulin, and also with the use of an iron chelator. RESULTS: Akt in the insulin signaling pathway was found to be activated in the Hamp1-/- hepatocytes to a greater extent than wild-type (WT) cells, both under basal conditions and in response to insulin. Incubation of the Hamp1-/- hepatocytes with an iron chelator attenuated these effects. There was no evidence of oxidative stress or AMPK activation in the Hamp1-/- hepatocytes. Glucose production by these cells was similar to that by WT cells. Gene expression of key gluconeogenic enzymes was decreased in these cells. In addition, they showed evidence of increased lipogenesis. CONCLUSIONS: Hepatocytes from Hamp1-/- mice showed evidence of greater sensitivity to the effects of insulin than WT hepatocytes. This may explain the insulin-sensitive phenotype that has been reported in classical hemochromatosis.

Increased intracellular iron in mouse primary hepatocytes in vitro causes activation of the Akt pathway but decreases its response to insulin.

BACKGROUND: An iron-overloaded state has been reported to be associated with insulin resistance. On the other hand, conditions such as classical hemochromatosis (where iron overload occurs primarily in the liver) have been reported to be associated with increased insulin sensitivity. The reasons for these contradictory findings are unclear. In this context, the effects of increased intracellular iron per se on insulin signaling in hepatocytes are not known. METHODS: Mouse primary hepatocytes were loaded with iron in vitro by incubation with ferric ammonium citrate (FAC). Intracellular events related to insulin signaling, as well as changes in gene expression and hepatocyte glucose production (HGP), were studied in the presence and absence of insulin and/or forskolin (a glucagon mimetic). RESULTS: In vitro iron-loading of hepatocytes resulted in phosphorylation-mediated activation of Akt and AMP-activated protein kinase. This was associated with decreased basal and forskolin-stimulated HGP. Iron attenuated forskolin-mediated induction of the key gluconeogenic enzyme, glucose-6-phosphatase. It also attenuated activation of the Akt pathway in response to insulin, which was associated with decreased protein levels of insulin receptor substrates 1 and 2, constituting insulin resistance. CONCLUSIONS: Increased intracellular iron has dual effects on insulin sensitivity in hepatocytes. It increased basal activation of the Akt pathway, but decreased activation of this pathway in response to insulin. GENERAL SIGNIFICANCE: These findings may help explain why both insulin resistance and increased sensitivity have been observed in iron-overloaded states. They are of relevance to a variety of disease conditions characterized by hepatic iron overload and increased risk of diabetes.

Hepcidin suppression in ß-thalassemia is associated with the down-regulation of atonal homolog 8.

Atonal homolog 8 (ATOH8) is defined as a positive regulator of hepcidin transcription, which links erythropoietic activity with iron-sensing molecules. In the present study, we investigated the association between hepcidin and ATOH8 expression in ß-thalassemia. We found that inhibition of hepcidin expression in ß-thalassemia is correlated with reduced ATOH8 expression. Hepatic hepcidin 1 (Hamp1) and Atoh8 mRNA expression were down-regulated in ß-thalassemic mice. Hepcidin (HAMP) and ATOH8 mRNA expression were consistently suppressed in Huh7 cells cultured in medium supplemented with ß-thalassemia patient serum. The Huh7 cells, which were transfected with ATOH8-FLAG expression plasmid and cultured in the supplemented medium, exhibited increased levels of ATOH8 mRNA, ATOH8-FLAG protein, pSMAD1,5,8, and HAMP mRNA. Interestingly, over-expression of ATOH8 reversed the effects of hepcidin suppression induced by the ß-thalassemia patient sera. In conclusion, hepcidin suppression in ß-thalassemia is associated with the down-regulation of ATOH8 in response to anemia. We, therefore, suggest that ATOH8 is an important transcriptional regulator of hepcidin in ß-thalassemia.

Iron and oxygen sensing: a tale of 2 interacting elements?

Iron and oxygen metabolism are intimately linked with one another. A change in the level of either metabolite results in activation of common pathways. At the heart of these responses lies a group of iron and oxygen dependent enzymes called prolyl hydroxylases. Prolyl hydroxylases (PHDs) require both iron and oxygen for optimal activity and their biological activity is to carry out the critical post-translational modification of the addition of a hydroxyl group to specific proline residues within Hypoxia Inducible Factor (HIFs)-well known transcription factors originally thought to regulate responses to hypoxia but which are now known to regulate key iron metabolism proteins too. The addition of the hydroxyl group ultimately leads to the unbiquitylation and destruction of HIFs, thus PHDs control appropriate HIF transcriptional responses depending on cellular oxygen or iron levels. There are two major HIFs; HIF1α and HIF2α. In terms of responses to iron HIF2α is of major importance in key tissues such as the intestine where several iron transporters (Ferroportin, Dcytb) contain HREs within their promoters which bind HIF2α. Furthermore the recent discovery that HIF2α contains a 5' iron responsive element (IRE) has underlined the importance of HIF2α as a major player in iron metabolism. This review brings together recent findings with regard to the HIF2α/IRP network as well as other aspects of iron sensing in cells and tissues.

Expression of ABCG2 (BCRP) in mouse models with enhanced erythropoiesis.

Haem is a structural component of numerous cellular proteins which contributes significantly to iron metabolic processes in mammals but its toxicity demands that cellular levels must be tightly regulated. Breast Cancer Resistance Protein (BCRP/ABCG2), an ATP Binding Cassette G-member protein has been shown to possess porphyrin/haem efflux function. The current study evaluated the expression and regulation of Abcg2 mRNA and protein levels in mouse tissues involved in erythropoiesis. Abcg2 mRNA expression was enhanced in bone marrow hemopoietic progenitor cells from mice that were treated with phenylhydrazine (PHZ). Abcg2 mRNA expression was increased particularly in the extramedullary haematopoietic tissues from all the mice models with enhanced erythropoiesis. Haem oxygenase (ho1) levels tended to increase in the liver of mice with enhanced erythropoiesis and gene expression patterns differed from those observed in the spleen. Efflux of haem biosynthetic metabolites might be dependent on the relative abundance of Abcg2 or ho1 during erythropoiesis. Abcg2 appears to act principally as a safety valve regulating porphyrin levels during the early stages of erythropoiesis and its role in systemic haem metabolism and erythrophagocytosis, in particular, awaits further clarification.

The transcription factor ATOH8 is regulated by erythropoietic activity and regulates HAMP transcription and cellular pSMAD1,5,8 levels.

ATOH8 has previously been shown to be an iron-regulated transcription factor, however its role in iron metabolism is not known. ATOH8 expression in HEK293 cells resulted in increased endogenous HAMP mRNA levels as well as HAMP promoter activity. Mutation of the E-box or SMAD response elements within the HAMP promoter significantly reduced the effects of ATOH8, indicating that ATOH8 activates HAMP transcription directly as well as through bone morphogenic protein (BMP) signalling. In support of the former, Chromatin immunoprecipitation assays provided evidence that ATOH8 binds to E-box regions within the HAMP promoter while the latter was supported by the finding that ATOH8 expression in HEK293 cells led to increased phosphorylated SMAD1,5,8 levels. Liver Atoh8 levels were reduced in mice under conditions associated with increased erythropoietic activity such as hypoxia, haemolytic anaemia, hypotransferrinaemia and erythropoietin treatment and increased by inhibitors of erythropoiesis. Hepatic Atoh8 mRNA levels increased in mice treated with holo transferrin, suggesting that Atoh8 responds to changes in plasma iron. ATOH8 is therefore a novel transcriptional regulator of HAMP, which is responsive to changes in plasma iron and erythroid activity and could explain how changes in erythroid activity lead to regulation of HAMP.

Regulation of iron metabolism in Hamp (-/-) mice in response to iron-deficient diet.

BACKGROUND: Hepcidin, the liver-secreted iron regulatory peptide, maintains systemic iron homeostasis in response to several stimuli including dietary iron levels and body iron status. In addition, iron metabolism is controlled by several local regulatory mechanisms including IRP and Hif-2α activities independently of hepcidin. However, the roles of these mechanisms and their interaction particularly in hepcidin-deficient individuals are not yet fully understood. We, therefore, aimed to explore whether Hamp disruption affects iron homeostatic responses to dietary iron deficiency. METHODS: Hepcidin1 knockout (Hamp (-/-)) mice and heterozygous littermates were fed with control or iron-deficient diet for 2 weeks. The expression of iron-related genes and proteins were determined by quantitative PCR and Western blot, respectively. RESULTS: Two-week iron-deficient diet feeding in Hamp (-/-) mice did not alter serum iron but significantly reduced liver non-heme iron levels. This was also associated with increased ferroportin protein expression in the duodenum and spleen, whereas decreased expression was found in the liver. In addition, significant inductive effects of iron-deficient diet on Dcytb and DMT1 mRNA expression in the duodenum were noted with more pronounced effects in Hamp (-/-) mice compared with controls. CONCLUSIONS: Hamp (-/-) mice exhibited a more dramatic increase in the expression of iron transport machinery, which may be responsible for the unaltered serum iron levels upon iron-deficient diet feeding in these mice. Despite the lack of hepcidin, Hamp (-/-) mice can maintain a degree of iron homeostasis in response to altered dietary iron through several hepcidin-independent mechanisms.

Duodenal reductase activity and spleen iron stores are reduced and erythropoiesis is abnormal in Dcytb knockout mice exposed to hypoxic conditions.

Duodenal cytochrome b (Dcytb, Cybrd1) is a ferric reductase localized in the duodenum that is highly upregulated in circumstances of increased iron absorption. To address the contribution of Dcytb to total duodenal ferric reductase activity as well as its wider role in iron metabolism, we first measured duodenal ferric reductase activity in wild-type (WT) and Dcytb knockout (Dcytb(-/-)) mice under 3 conditions known to induce gut ferric reductase: dietary iron deficiency, hypoxia, and pregnancy. Dcytb(-/-) and WT mice were randomly assigned to control (iron deficiency experiment, 48 mg/kg dietary iron; hypoxia experiment, normal atmospheric pressure; pregnancy experiment, nonpregnant animals) or treatment (iron deficiency experiment, 2-3 mg/kg dietary iron; hypoxia experiment, 53.3 kPa pressure; pregnancy experiment, d 20 of pregnancy) groups and duodenal reductase activity measured. We found no induction of ferric reductase activity in Dcytb(-/-) mice under any of these conditions, indicating there are no other inducible ferric reductases present in the duodenum. To test whether Dcytb was required for iron absorption in conditions with increased erythropoietic demand, we also measured tissue nonheme iron levels and hematological indices in WT and Dcytb(-/-) mice exposed to hypoxia. There was no evidence of gross alterations in iron absorption, hemoglobin, or total liver nonheme iron in Dcytb(-/-) mice exposed to hypoxia compared with WT mice. However, spleen nonheme iron was significantly less (6.7 ± 1.0 vs. 12.7 ± 0.9 nmol · mg tissue(-1); P < 0.01, n = 7-8) in hypoxic Dcytb(-/-) compared with hypoxic WT mice and there was evidence of impaired reticulocyte hemoglobinization with a lower reticulocyte mean corpuscular hemoglobin (276 ± 1 vs. 283 ± 2 g · L(-1); P < 0.05, n = 7-8) in normoxic Dcytb(-/-) compared with normoxic WT mice. We therefore conclude that DCYTB is the primary iron-regulated duodenal ferric reductase in the gut and that Dcytb is necessary for optimal iron metabolism.

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.

Using the arts and humanities to promote a liberal nursing education: strengths and weaknesses.

BACKGROUND: The requirement that all student nurses in the United Kingdom will be educated to degree level from 2013 permits a review of the current state of nursing education in university contexts. Recent educational standards for these new programmes (NMC, 2010) allow a liberal, or broad-based, education, with its features of breadth of knowledge, formativity, critical thinking and working with others, to be considered. OBJECTIVE: Select narratives from a PhD study featuring student nurses and nurse teachers exploring the relationship between reading literature and poetry and ethical practice are presented to critically support the place of liberal education within these programmes. DESIGN: These narratives are drawn from a research study based upon the use of a narrative methodology. SETTINGS: The study was set within the educational context of a school of nursing and midwifery in one Scottish university. PARTICIPANTS: Eight student nurses and four nurse teachers participated in the study. METHODS: These narratives were constructed from data derived from focus groups and individual interviews. RESULTS: These narratives suggest that liberal education can be promoted within international curricula via careful positioning of, and student nurse engagement with, the arts and humanities. A liberal education can influence student nurses' sense of discernment, enhance their own responsibility for learning, support ethical regard for others, provide different perspectives on human experience and contribute to a balanced curriculum. Although a liberal education cannot guarantee fully skilled and ethically sensitive practitioners, it can contribute towards its achievement. CONCLUSION: The current university education climate presents obstacles to the promotion of liberal education. Nevertheless, the considerable professional and personal challenges of nursing practice in global terms make such an educational preparation essential. If nursing education to degree level is to commence from 2013, these principal features of liberal education, via these educational standards, must be embedded prominently into new programmes.

Exploring clinical wisdom in nursing education.

The recent interest in wisdom in professional health care practice is explored in this article. Key features of wisdom are identified via consideration of certain classical, ancient and modern sources. Common themes are discussed in terms of their contribution to 'clinical wisdom' itself and this is reviewed against the nature of contemporary nursing education. The distinctive features of wisdom (recognition of contextual factors, the place of the person and timeliness) may enable their significance for practice to be promoted in more coherent ways in nursing education. Wisdom as practical knowledge (phronesis) is offered as a complementary perspective within the educational preparation and practice of students of nursing. Certain limitations within contemporary UK nursing education are identified that may inhibit development of clinical wisdom. These are: the modularization of programmes in higher education institutions, the division of pastoral and academic support and the relationship between theory and practice.

Effects of acute and chronic inflammation on proteins involved in duodenal iron absorption in mice: a time-course study.

In order to understand better the molecular mechanisms involved in the pathogenesis of anaemia of inflammation, we carried out a time-course study on the effects of turpentine-induced acute and chronic inflammation on duodenal proteins involved in Fe absorption in mice. Expression levels of these proteins and hepatic hepcidin and serum Fe levels were determined in inflamed mice. In acutely inflamed mice, significantly increased expression of ferritin was the earliest change observed, followed by decreased divalent metal transporter 1 expression in the duodenum and increased hepcidin expression in the liver. Ferroportin expression increased subsequently, despite high levels of hepcidin. Hypoferraemia, which developed at early time periods studied, was followed by increased serum Fe levels at later points. The present results thus show that acute inflammation induced several changes in the expression of proteins involved in duodenal Fe absorption, contributing to the development of hypoferraemia. Resolution of inflammation caused attenuation of many of these effects. Effects in chronically inflamed mice were less consistent. The present results also suggest that inflammation-induced increases in ferritin appeared to override the effects of hepcidin on the expression levels of ferroportin in enterocytes.

BMPER protein is a negative regulator of hepcidin and is up-regulated in hypotransferrinemic mice.

The BMP/SMAD4 pathway has major effects on liver hepcidin levels. Bone morphogenetic protein-binding endothelial cell precursor-derived regulator (Bmper), a known regulator of BMP signaling, was found to be overexpressed at the mRNA and protein levels in liver of genetically hypotransferrinemic mice (Trf(hpx/hpx)). Soluble BMPER peptide inhibited BMP2- and BMP6-dependent hepcidin promoter activity in both HepG2 and HuH7 cells. These effects correlated with reduced cellular levels of pSMAD1/5/8. Addition of BMPER peptide to primary human hepatocytes abolished the BMP2-dependent increase in hepcidin mRNA, whereas injection of Bmper peptide into mice resulted in reduced liver hepcidin and increased serum iron levels. Thus Bmper may play an important role in suppressing hepcidin production in hypotransferrinemic mice.

Duodenal cytochrome b (Cybrd 1) and HIF-2α expression during acute hypoxic exposure in mice.

BACKGROUND: Recent evidence suggests that the duodenum can regulate iron absorption independently of hepcidin via the transcription factor Hif-2α acting directly on the transcription of the proteins involved in the iron transport. The current study investigates the temporal relationship between Dcytb and Hif-2α during early hypoxic stimulus in the enterocyte in vivo. METHODS: Duodenal Dcytb and Hif-2α protein expression was analysed by Western blot technique while gene regulation was determined by quantitative PCR. RESULTS: Both Dcytb and Hif-2α protein expression were increased during the first hours of hypoxic duration. A change in hepcidin expression however, was significant only at 72 h hypoxia. Increased iron absorption reported in early hypoxia could be accounted for in part by the enhancement of Dcytb expression by Hif-2α in the duodenum. CONCLUSION: Modulation of Hif-2α predominates over hepcidin in the regulation of intestinal iron absorption during short hypoxic duration. The intestine exerts regulatory mechanisms in the dietary absorption of iron into systemic circulation.

Iron absorption in hepcidin1 knockout mice.

Hepcidin, the Fe-regulatory peptide, has been shown to inhibit Fe absorption and reticuloendothelial Fe recycling. The present study was conducted to explore the mechanism of in vivo Fe regulation through genetic disruption of hepcidin1 and acute effects of hepcidin treatment in hepcidin1 knockout (Hepc1-/-) and heterozygous mice. Hepcidin1 disruption resulted in significantly increased intestinal Fe uptake. Hepcidin injection inhibited Fe absorption in both genotypes, but the effects were more evident in the knockout mice. Hepcidin administration was also associated with decreased membrane localisation of ferroportin in the duodenum, liver and, most significantly, in the spleen of Hepc1-/- mice. Hypoferraemia was induced in heterozygous mice by hepcidin treatment, but not in Hepc1-/- mice, 4 h after injection. Interestingly, Fe absorption and serum Fe levels in Hepc1-/- and heterozygous mice fed a low-Fe diet were not affected by hepcidin injection. The present study demonstrates that hepcidin deficiency causes increased Fe absorption. The effects of hepcidin were abolished by dietary Fe deficiency, indicating that the response to hepcidin may be influenced by dietary Fe level or Fe status.

Hypoxia inhibits hepcidin expression in HuH7 hepatoma cells via decreased SMAD4 signaling.

Hepcidin negatively regulates systemic iron homeostasis in response to inflammation and elevated serum iron. Conversely, hepcidin expression is diminished in response to hypoxia, oxidative stress, and increased erythropoietic demand, though the molecular intermediates involved are incompletely understood. To address this, we have investigated hypoxic hepcidin regulation in HuH7 hepatoma cells either cultured alone or cocultured with activated THP-1 macrophages. HuH7 hepcidin mRNA expression was determined using quantitative polymerase chain reaction (Q-PCR). Hepcidin promoter activity was measured using luciferase reporter constructs containing a 0.9 kb fragment of the wild-type human hepcidin promoter, and constructs containing mutations in bone morphogenetic protein (BMP)/SMAD4, signal transducer and activator of transcription 3 (STAT3), CCAAT/enhancer-binding protein (C/EBP), and E-box-responsive elements. Hepatic expression of bone morphogenetic proteins BMP2 and BMP6 and the BMP inhibitor noggin was determined using Q-PCR, and the protein expression of hemojuvelin (HJV), pSMAD 1/5/8, and SMAD4 was determined by western blotting. Following exposure to hypoxia or H(2)O(2), hepcidin mRNA expression and promoter activity increased in HuH7 cells monocultures but were decreased in HuH7 cells cocultured with THP-1 macrophages. This repression was attenuated by mutation of the BMP/SMAD4-response element, suggesting that modulation of SMAD signaling mediated the response to hypoxia. No changes in hepatocyte BMP2, BMP6 or noggin mRNA, or protein expression of HJV or pSMAD 1/5/8 were detected. However, treatment with hypoxia caused a marked decrease in nuclear and cytosolic SMAD4 protein and SMAD4 mRNA expression in cocultured HuH7 cells. Together these data indicate that hypoxia represses hepcidin expression through inhibition of BMP/SMAD signaling.

Are nurses empowered to make decisions about levels of patient observation in mental health?

BACKGROUND: The Clinical Resource and Audit Group (2002) guidelines emphasise that local policies should make the procedure for deciding and maintaining patient observation levels clear. AIM: To investigate the views of nurses, doctors and other members of the multidisciplinary team on patient observation. METHOD: A postal survey of all acute adultinpatient facilities and intensive patient care units in Scotland was carried out, followed by 38 interviews, 37 with staff members and one with a service user. RESULTS: We found a lack of multidisciplinary team involvement, limited pre agreed plans for nurses to reduce levels of observation and limited use of validated risk assessment tools. CONCLUSION: There is a need to ensure that local practice follows national policy recommendations more closely.

Characterization of the transition-metal-binding properties of hepcidin.

Accumulating evidence suggests that hepcidin, a 25-residue peptide hormone, is the master regulator of iron metabolism. Further evidence suggests that the five N-terminal amino acids are crucial for mediating its biological function. With a histidine residue at position 3, this region also has the potential to bind bivalent metal ions. To characterize this hepcidin-metal interaction in detail, the present study utilizes electrospray MS to measure the binding of a range of metal ions to wild-type and mutant human and murine hepcidins. In addition, the biological effects of these point mutations were tested on Caco-2 and HEK-293T human cell lines and in mice. Our results show that hepcidin-25 can form complexes with copper, nickel and zinc; however, we failed to detect any hepcidin-25 binding to either ferric or ferrous ions. The greatest affinity observed was between hepcidin-25 and copper with a dissociation constant <<1 microM. Substituting the histidine residue at position 3 in human hepcidin-25 and comparably the asparagine residue at position 3 in murine hepcidin-25 with an alanine residue markedly diminished the affinity for copper. The amino acid substitutions also decreased the biological activity of hepcidin-25; namely repression of ferroportin protein levels and hypoferraemia. In summary, the high affinity of hepcidin for copper suggests that hepcidin could bind copper in vivo and this may be of biological relevance.