[Show abstract][Hide abstract] ABSTRACT: Iron deficiency is the most common nutritional disorder worldwide with substantial impact on human health and economy. Current treatments predominantly rely on soluble iron which adversely affects the gastrointestinal tract. We have developed organic acid-modified Fe(III) oxo-hydroxide nanomaterials, here termed nano Fe(III), as alternative safe iron delivery agents. Nano Fe(III) absorption in humans correlated with serum iron increase (p<0.0001) and direct in vitro cellular uptake (p=0.001), but not with solubility at simulated gastric pH. The most promising preparation (iron hydroxide adipate tartrate: IHAT) showed ~80% relative bioavailability to Fe(II) sulfate in humans and, in a rodent model, IHAT was equivalent to Fe(II) sulfate at repleting haemoglobin levels. Furthermore, IHAT did not accumulate in the intestinal mucosa and, unlike Fe(II) sulfate, promoted a beneficial microbiota. In cellular models, IHAT was 14-fold less toxic than Fe(II) sulfate/ascorbate. Nano Fe (III) manifests minimal acute intestinal toxicity in cellular and murine models and shows efficacy at treating iron deficiency anaemia.
Nanomedicine : nanotechnology, biology, and medicine. 06/2014;
[Show abstract][Hide abstract] ABSTRACT: The ferritin core is composed of fine nanoparticulate Fe(3+) oxohydroxide, and we have developed a synthetic mimetic, nanoparticulate Fe(3+) polyoxohydroxide (nanoFe(3+)). The aim of this study was to determine how dietary iron derived in this fashion is absorbed in the duodenum. Following a 4 wk run-in on an Fe-deficient diet, mice with intestinal-specific disruption of the Fpn-1 gene (Fpn-KO), or littermate wild-type (WT) controls, were supplemented with Fe(2+) sulfate (FeSO4), nanoFe(3+), or no added Fe for a further 4 wk. A control group was Fe sufficient throughout. Direct intestinal absorption of nanoFe(3+) was investigated using isolated duodenal loops. Our data show that FeSO4 and nanoFe(3+) are equally bioavailable in WT mice, and at wk 8 the mean ± sem hemoglobin increase was 18 ± 7 g/L in the FeSO4 group and 30 ± 5 g/L in the nanoFe(3+) group. Oral iron failed to be utilized by Fpn-KO mice and was retained in enterocytes, irrespective of the iron source. In summary, although nanoFe(3+) is taken up directly by the duodenum its homeostasis is under the normal regulatory control of dietary iron absorption, namely via ferroportin-dependent efflux from enterocytes, and thus offers potential as a novel oral iron supplement.-Aslam, M. F., Frazer, D. M., Faria, N., Bruggraber, S. F. A., Wilkins, S. J., Mirciov, C., Powell, J. J., Anderson, G. J., Pereira, D. I. A. Ferroportin mediates the intestinal absorption of iron from a nanoparticulate ferritin core mimetic in mice.
[Show abstract][Hide abstract] ABSTRACT: Hephaestin is a vertebrate multicopper ferroxidase important for the transfer of dietary iron from intestinal cells to the blood. Hephaestin is mutated in the sex-linked anemia mouse, resulting in iron deficiency. However, sex-linked anemia mice still retain some hephaestin ferroxidase activity. They survive, breed, and their anemia improves with age. To gain a better understanding of the role of hephaestin in iron homeostasis, we used the Cre-lox system to generate knockout mouse models with whole body or intestine-specific (Villin promoter) ablation of hephaestin. Both types of mice were viable, indicating that hephaestin is not essential and that other mechanisms, multicopper ferroxidase-dependent or not, must compensate for hephaestin deficiency. The knockout strains, however, both developed a microcytic, hypochromic anemia, suggesting severe iron deficiency and confirming that hephaestin plays an important role in body iron acquisition. Consistent with this, the knockout mice accumulated iron in duodenal enterocytes and had reduced intestinal iron absorption. In addition, the similarities of the phenotypes of the whole body and intestine-specific hephaestin knockout mice clarify the important role of hephaestin specifically in intestinal enterocytes in maintaining whole body iron homeostasis. These mouse models will serve as valuable tools to study the role of hephaestin and associated proteins in iron transport in the small intestine and other tissues.
PLoS ONE 01/2014; 9(6):e98792. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Current oral treatment of iron deficiency anaemia is based upon soluble iron compounds, all of which have undesirable gastrointestinal effects. However, natural dietary iron (III) may be nano-formed ligand-modified iron hydroxide particles that are acquired endocytically and thus not soluble or available for adverse effects. Here, a series of five different nano Fe(III) hydroxide particles was developed: absorption in humans matched endocytic uptake by gut epithelial cells but not solubility in (stomach) acid. In murine and cellular models nano iron(III) treated iron deficiency anaemia as well as ferrous sulfate but, unlike the latter, was safe to the gut environment.Figure optionsDownload full-size imageDownload high-quality image (101 K)Download as PowerPoint slide
Nanomedicine: Nanotechnology, Biology and Medicine. 01/2014;
[Show abstract][Hide abstract] ABSTRACT: β-thalassemia major causes ineffective erythropoiesis and chronic anemia, and is associated with iron overload due to both transfused iron and increased iron absorption, the latter mediated by suppression of the iron-regulatory hormone hepcidin. We sought to determine whether, in β-thalassemia major, transfusion-mediated inhibition of erythropoiesis dynamically affects hepcidin. We recruited 31 chronically transfused patients with β-thalassemia major and collected samples immediately before and 4-8 days post-transfusion. Pre-transfusion hepcidin was positively correlated with hemoglobin and ferritin, and inversely with erythropoiesis. The hepcidin-ferritin ratio indicated hepcidin was relatively suppressed given the degree of iron loading. Post-transfusion, hemoglobin increased, erythropoietin and growth differentiation factor-15 (GDF-15) fell, and hepcidin rose. By multiple regression, pre- and post-transfusion hepcidin concentrations were both associated positively with hemoglobin, inversely with erythropoiesis, and positively with ferritin. Although males and females had similar pre-transfusion hemoglobin, males had significantly increased erythropoiesis and lower hepcidin, received a lower transfusion volume per liter blood volume, and experienced a smaller post-transfusion reduction in erythropoiesis and hepcidin rise. Age of blood was not associated with post-transfusion hemoglobin or ferritin change. Hepcidin levels in patients with β-thalassemia major dynamically reflect competing influences from erythropoiesis, anemia and iron overload. Measurement of these indices could assist clinical monitoring.
[Show abstract][Hide abstract] ABSTRACT: Heme oxygenases initiate the catabolism of heme, releasing carbon monoxide, iron, and biliverdin. Sustained induction of heme oxygenase-1 (HO-1) in nonerythroid cells plays a key role in many pathological processes, yet the effect of long-term HO-1 expression on cellular iron metabolism in the absence of exogenous heme is poorly understood. Here we report that in a model nonerythroid cell, both transient and stable HO-1 expression increased heme oxygenase activity, but total cellular heme content was decreased only with transient enzyme expression. Sustained HO-1 activity increased the expression of both the mitochondrial iron importer mitoferrin-2 and the rate-limiting enzyme in heme synthesis, aminolevulinate synthase-1, and it augmented the mitochondrial content of heme. Also, the expression of transferrin receptor-1 and the activities of iron-regulatory proteins 1 and 2 decreased, whereas total labile iron and the regulatory activity of the heme-binding transcription factor Bach1 were unaltered. In addition, stable, but not transient, HO-1 expression decreased the activities of aconitase, as well as increasing proteasomal degradation of ferritin. Together, our results reveal a novel and coordinated adaptive response of nonerythroid cells to sustained HO-1 induction that has an impact on cellular iron homeostasis.
[Show abstract][Hide abstract] ABSTRACT: The BMP/SMAD signalling pathway plays an important role in iron homeostasis, regulating hepcidin expression in response to body iron levels. However, the role of this pathway in the reduction in hepcidin associated with increased erythropoiesis (and secondary iron loading) is unclear. To investigate this, we established a mouse model of chronic stimulated erythropoiesis with secondary iron loading using the haemolytic agent phenylhydrazine. We then examined the expression of components of the BMP6/SMAD signalling pathway in these animals. We also examined this pathway in the Hbb(th3/+) mouse, a model of the iron loading anaemia β-thalassaemia intermedia. Increasing doses of phenylhydrazine led to a progressive increase in both liver iron levels and Bmp6 mRNA expression, but, in contrast, hepatic Hamp expression declined. The increase in Bmp6 expression was not associated with a corresponding change in the phosphorylation of hepatic SMAD1/5/8, indicating that stimulated erythropoiesis decreases the ability of BMP6 to alter SMAD phosphorylation. Increased erythropoiesis also reduces the capacity of phosphorylated SMAD (pSMAD) to induce hepcidin, as Hamp levels declined despite no changes in pSMAD1/5/8. Similar results were seen in Hbb(th3/+) mice. Thus the erythroid signal probably affects some components of BMP/SMAD signalling, but also may exert some independent effects.
British Journal of Haematology 03/2012; 157(5):615-26. · 4.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Suckling mammals absorb high levels of iron to support their rapid growth. In adults, iron absorption is controlled by systemic signals that alter expression of the iron-regulatory hormone hepcidin. We investigated whether hepcidin and absorption respond appropriately to systemic stimuli during suckling.
In Sprague-Dawley rats, iron levels increased following administration of iron dextran, and inflammation was induced with lipopolysaccharide. Gene expression was measured by quantitative reverse-transcription polymerase chain reaction; protein levels were measured by immunoblot analyses. Iron absorption was determined based on retention of an oral dose of 59Fe.
Iron absorption was high during suckling and reduced to adult levels upon weaning. In response to iron dextran or lipopolysaccharide, iron absorption in adults decreased substantially, but, in suckling animals, the changes were minimal. Despite this, expression of hepcidin messenger RNA was strongly induced by each agent, before and after weaning. The hyporesponsiveness of iron absorption to increased levels of hepcidin during suckling correlated with reduced or absent duodenal expression of ferroportin 1 (Fpn1), normally a hepcidin target. Fpn1 expression was robust in adults. Predominance of the Fpn1A splice variant, which is under iron-dependent translational control, accounts for the low level of Fpn1 in the iron-deficient intestine of suckling rats.
Iron absorption during suckling is largely refractory to changes in expression of the systemic iron regulator hepcidin, and this in turn reflects limited expression of Fpn1 protein in the small intestine. Iron absorption is therefore not always controlled by hepcidin.
[Show abstract][Hide abstract] ABSTRACT: The maintenance of appropriate iron levels is important for mammalian health, particularly during the rapid growth period following birth. Too little iron can lead to irreversible damage to the developing central nervous system and too much iron at this point can have adverse long term consequences, possibly due to excessive free radical production. In order to maintain iron levels, intestinal iron absorption is very efficient in young mammals, such that almost all of the iron in breast milk is utilized. However this high level of absorption is unable to be down regulated in response to excess iron as it can be in adults, implying that different regulatory processes are involved during suckling. Various mechanisms have been proposed to explain this high absorption, including enhanced expression of the proteins involved in iron absorption in adults (particularly DMT1 and ferroportin), non-specific uptake via pinocytosis, and the uptake of lactoferrin bound iron by the lactoferrin receptor. However, at present the precise mechanism is unclear. It is possible that all of these components contribute to the high intestinal iron absorption seen during suckling, or a novel, as yet undescribed, mechanism could be involved. This review summarises the evidence for and against each of the mechanisms described above and highlights how little is known about iron homeostasis in this vital stage of development.
Biology of Metals 02/2011; 24(3):567-74. · 3.17 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Expression of the key iron regulatory hormone hepcidin is increased by some stimuli (iron loading, inflammation) but decreased by others (increased erythropoiesis, iron deficiency). We investigated the response of hepcidin to increased erythropoiesis and iron deficiency in the presence of an acute inflammation to assess the relative strengths of these stimuli.
Sprague-Dawley rats were maintained on control or iron-deficient diets and treated with lipopolysaccharide to induce inflammation or phenylhydrazine to stimulate erythropoiesis. The levels of Hamp, IL-6 and α2m mRNA were determined by qualitative real-time polymerase chain reaction and those of serum interleukin-6 and tumor necrosis factor-α were measured by enzyme-linked immunosorbent assay. Cultured RAW264.7 and HuH7 cells were used in associated studies.
The increase in hepatic hepcidin levels induced by lipopolysaccharide was not affected by phenylhydrazine treatment but was blunted by iron deficiency. Lipopolysaccharide-treated iron-deficient animals also showed lower liver α2m mRNA and reduced serum interleukin-6 and tumor necrosis factor-α, suggesting a more generalized effect of iron deficiency. Similarly, RAW 264.7 cells treated with iron chelators and then stimulated with lipopolysaccharide showed lower IL-6 mRNA than cells treated with lipopolysaccharide alone. Huh7 cells treated with an iron chelator showed a blunted hepcidin response to interleukin-6, suggesting that the response of hepatic parenchymal cells to inflammatory cytokines may also be iron-dependent.
In any one physiological situation, net hepcidin levels are determined by the relative strengths of competing stimuli. The ability of severe iron deficiency to blunt the response to lipopolysaccharide of both hepcidin and other markers of inflammation suggests that adequate iron levels are necessary for a full acute phase response.
[Show abstract][Hide abstract] ABSTRACT: Iron-loading disorders (haemochromatosis) represent an important class of human diseases. Primary iron loading results from inherited disturbances in the mechanisms regulating intestinal iron absorption, such that excess iron is taken up from the diet. Body iron load can also be increased by repeated blood transfusions (secondary iron loading), usually as part of the treatment for various haematological disorders. In these syndromes, an element of enhanced iron absorption is also often involved. The central regulator of body iron trafficking is the liver-derived peptide hepcidin. Hepcidin limits iron entry into the plasma from macrophages, intestinal enterocytes and other cells by binding to the sole iron-export protein ferroportin, and facilitating its removal from the plasma membrane. Mutations in hepcidin or its upstream regulators (HFE, TFR2, HFE2 and BMP6) lead to reduced or absent hepcidin expression and a concomitant increase in iron absorption. Mutations in ferroportin that prevent hepcidin binding produce a similar result. Increased ineffective erythropoiesis, which often characterises erythrocyte disorders, also leads to reduced hepcidin expression and increased absorption. Recent advances in our understanding of hepcidin and body iron homeostasis provide the potential for a range of new diagnostic and therapeutic tools for haemochromatosis and related conditions.
Expert Reviews in Molecular Medicine 01/2010; 12:e36. · 6.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hepcidin is a central regulator of iron homeostasis. HFE and transferrin receptor 2 (TFR2) are mutated in adult-onset forms of hereditary hemochromatosis and regulate the expression of hepcidin in response to iron. Whether they act through the same or parallel pathways is unclear. To investigate this, we generated a mouse model with deletion of both Hfe and Tfr2 genes by crossing Hfe and Tfr2 null mice on a genetically identical background. Tissue and serum from wildtype, single-, and double-null mice were analyzed. Serum transferrin saturation and hepatic iron concentrations were determined. The expression of iron-related messenger RNA (mRNA) transcripts was analyzed by real-time polymerase chain reaction (PCR). Levels of the iron-related proteins Tfr1, Tfr2, ferritin, and prohepcidin, and the phosphorylation status of the cell signaling proteins extracellular signal-regulated kinase 1/2 (Erk1/2) and Smad1/5/8, were analyzed by immunoblotting. Double-null mice had more severe iron loading than mice lacking either Hfe or Tfr2; Tfr2 null mice had a greater iron burden than Hfe-null mice. Hepcidin expression relative to iron stores was reduced in the Hfe-null mice, with significantly lower values in the Tfr2-null mice. In the absence of both Hfe and Tfr2, hepcidin expression was reduced even further. A significant decrease in phospho-Erk1/2 in the livers of null mice and a reduction in phospho-Smad1/5/8 suggest that both the mitogen-activated protein kinase (MAPK) and bone morphogenetic protein / mothers against decapentaplegic homolog (BMP/SMAD) signaling pathways may be involved in Hfe- and Tfr2-mediated regulation of hepcidin. CONCLUSION: These studies demonstrate that iron overload due to deletion of Tfr2 is more severe than that due to Hfe, and that loss of both molecules results in pronounced iron overload. Analysis of Hfe/Tfr2 double-null mice suggests that Hfe and Tfr2 regulate hepcidin through parallel pathways involving Erk1/2 and Smad1/5/8.
[Show abstract][Hide abstract] ABSTRACT: Intestinal iron absorption is an essential physiological process that is regulated by the liver-derived peptide hepcidin. This review will describe recent advances in hepcidin biology and enterocyte iron transport.
Hepcidin acts as a repressor of iron absorption and its expression in turn reflects a range of systemic cues, including iron status, hypoxia, erythropoiesis and inflammation. These act through proteins on the hepatocyte plasma membrane such as HFE, hemojuvelin and transferrin receptor 2 to alter transcription of the hepcidin gene. Bone morphogenetic protein-SMAD signaling provides a key pathway of hepcidin activation, whereas the membrane-bound serine protease matriptase-2 and the erythroid factor growth differentiation factor 15 have emerged as important negative regulators of hepcidin expression. At the enterocyte itself, the recent demonstration of a chaperone for delivering iron to ferritin and new data on iron release from the hepcidin target ferroportin are helping to define the pathway of iron movement across the intestinal epithelium.
Disturbances in the hepcidin regulatory pathway underlie a range of iron metabolism disorders, from iron deficiency to iron loading, and there is considerable promise that the exciting recent advances in understanding hepcidin action will be translated into improved diagnostic and therapeutic modalities in the near future.
Current opinion in gastroenterology 04/2009; 25(2):129-35. · 4.33 Impact Factor