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The stimulation of erythrocyte formation increases the demand for iron by the bone marrow and this in turn may affect the levels of circulating diferric transferrin. As this molecule influences the production of the iron regulatory hormone hepcidin, we hypothesized that erythropoiesis-driven changes in diferric transferrin levels could contribute to the decrease in hepcidin observed following the administration of erythropoietin. To examine this, we treated mice with erythropoietin and examined diferric transferrin at various time points up to 18 hours. We also investigated the effect of altering diferric transferrin levels on erythropoietin-induced inhibition of Hamp1, the gene encoding hepcidin. We detected a decrease in diferric transferrin levels 5 hours after erythropoietin injection and prior to any inhibition of the hepatic Hamp1 message. Diferric transferrin returned to control levels 12 hours after erythropoietin injection and had increased beyond control levels by 18 hours. Increasing diferric transferrin levels via intravenous iron injection prevented the inhibition of Hamp1 expression by erythropoietin without altering hepatic iron concentration or the expression of Erfe, the gene encoding erythroferrone. These results suggest that diferric transferrin likely contributes to the inhibition of hepcidin production in the period shortly after injection of erythropoietin and that, under the conditions examined, increasing diferric transferrin levels can overcome the inhibitory effect of erythroferrone on hepcidin production. They also imply that the decrease in Hamp1 expression in response to an erythropoietic stimulus is likely to be mediated by multiple signals.

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Iron-loading disorders, such as hereditary hemochromatosis, are associated with inappropriately low expression of the iron regulatory hormone, hepcidin. A recent study has demonstrated that food deprivation can increase hepcidin production in mice. We have examined this effect in more detail to determine whether the pathway(s) that are responsible might provide novel targets for pharmaceutical intervention in disorders of iron homeostasis. C57BL/6 mice were deprived of food for 5, 10, 16, or 24 h before euthanasia, then blood and tissue samples were collected for analysis. The effect of food deprivation was also examined in Hfe-/- mice, a model of hereditary hemochromatosis, as well as mice that were maintained on an iron-deficient diet or injected with erythropoietin. Food deprivation increased the hepatic expression of the gene that encodes hepcidin, hepcidin antimicrobial peptide 1 ( Hamp1), with maximal expression observed after 16 h, and was able to overcome the reduction in Hamp1 expression associated with Hfe deficiency. Food deprivation also increased Hamp1 expression in response to stimuli that more strongly suppress the gene, such as iron deficiency and erythropoietin treatment, but the effects were not significant. These results indicate that Hamp1 induction by food deprivation is independent of HFE and suggest that targeting the pathway regulated by food deprivation could have clinical benefit in iron-loading conditions.-Mirciov, C. S. G., Wilkins, S. J., Anderson, G. J., Frazer, D. M. Food deprivation increases hepatic hepcidin expression and can overcome the effect of Hfe deletion in male mice.

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BACKGROUND & AIMS: Previous studies have suggested that iron absorption in suckling mammals is refractory to stimuli that normally would decrease absorption in adults. To better understand the regulation of iron absorption during suckling, we have characterized the relationship between hepcidin, ferroportin, and iron absorption at this crucial stage of life. METHODS: To determine whether ferroportin is involved in iron absorption during suckling, absorption was measured in intestine-specific ferroportin knockout mice. The effect of constitutive hepcidin overexpression on intestinal iron absorption also was investigated in suckling transmembrane serine protease 6 knockout mice. Finally, suckling mice were injected with lipopolysaccharide to induce hepcidin expression. Blood was collected for serum iron analysis, and liver tissue and duodenal enterocytes were collected for gene and protein expression profiles. RESULTS: Iron absorption was very low in suckling ferroportin knockout mice, indicating that ferroportin is responsible for the majority of the iron absorbed at this time. However, increases in hepcidin during suckling, as seen in transmembrane serine protease 6 knockout mice and in mice injected with lipopolysaccharide, did not affect enterocyte ferroportin levels. Immunofluorescent localization of ferroportin showed that the protein localized to the basolateral membrane of duodenal enterocytes in both suckling and weaned mice. CONCLUSIONS: These data show that the high iron absorption occurring during suckling is mediated by ferroportin. However, enterocyte ferroportin is hyporesponsive to hepcidin at this time, despite being expressed on the basolateral membrane. Alterations to ferroportin that prevent hepcidin binding during suckling may allow iron absorption to remain high regardless of hepcidin expression levels, reducing the likelihood of iron deficiency during development.

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Iron is crucial for many biological functions, but quantitatively the most important use of iron is in the production of hemoglobin in red blood cell precursors. The amount of iron in the plasma, and hence its availability for hemoglobin synthesis, is determined by the liver-derived iron regulatory hormone hepcidin. When the iron supply to erythroid precursors is limited, as often occurs during stimulated erythropoiesis, these cells produce signals to inhibit hepatic hepcidin production, thereby increasing the amount of iron that enters the plasma. How stimulated erythropoiesis suppresses hepcidin production is incompletely understood, but erythroferrone, Gdf15 and Twsg1 have emerged as candidate regulatory molecules. To further examine the relationship between erythropoiesis and the candidate erythroid regulators, we have studied five mouse models of anemia, including two models of ß-thalassemia (Hbbth3/+ and RBC14), the hemoglobin deficit mouse (hbd), dietary iron deficient mice and mice treated with phenylhydrazine to induce acute hemolysis. Hematological parameters, iron status and the expression of Erfe (the gene encoding erythroferrone), Gdf15 and Twsg1 in the bone marrow and spleen were examined. Erfe expression was the most consistently upregulated of the candidate erythroid regulators in all of the mouse models examined. Gene expression was particularly high in the bone marrow and spleen of iron deficient animals, making erythroferrone an ideal candidate erythroid regulator, as its influence is strongest when iron supply to developing erythroid cells is limited. Gdf15 expression was also upregulated in most of the anemia models studied although the magnitude of the increase was generally less than that of Erfe. In contrast, very little regulation of Twsg1 was observed. These results support the prevailing hypothesis that erythroferrone is a promising erythroid regulator and demonstrate that Erfe expression is stimulated most strongly when the iron supply to developing erythroid cells is compromised.

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In conditions such as ß-thalassaemia, stimulated erythropoiesis can reduce the expression of the iron regulatory hormone hepcidin, increasing both macrophage iron release and intestinal iron absorption and leading to iron loading. However, in certain conditions, sustained elevation of erythropoiesis can occur without an increase in body iron load. To investigate this in more detail, we made use of a novel mouse strain (RBC14), which exhibits mild ß-thalassaemia intermedia with minimal iron loading. We compared iron homeostasis in RBC14 mice to that of Hbbth3/+ mice, a more severe model of ß-thalassaemia intermedia. Both mouse strains showed a decrease in plasma iron half-life, although the changes were less severe in RBC14 mice. Despite this, intestinal ferroportin and serum hepcidin levels were unaltered in RBC14 mice. In contrast, Hbbth3/+ mice exhibited reduced serum hepcidin and increased intestinal ferroportin. However, splenic ferroportin levels were increased in both mouse strains. These data suggest that in low-grade chronic haemolytic anaemia, such as that seen in RBC14 mice, the increased erythroid iron requirements can be met through enhanced macrophage iron release without the need to increase iron absorption, implying that hepcidin is not the sole regulator of macrophage iron release in vivo.

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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.

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Blocking the action of inhibitory molecules at sites of central nervous system injury has been proposed as a strategy to promote axonal regeneration and functional recovery. We have previously shown that genetic deletion or competitive antagonism of EphA4 receptor activity promotes axonal regeneration and functional recovery in a mouse model of lateral hemisection spinal cord injury. Here we have assessed the effect of blocking EphA4 activation using the competitive antagonist EphA4-Fc in a rat model of thoracic contusive spinal cord injury. Using a ledged tapered balance beam and open-field testing, we observed significant improvements in recovery of locomotor function after EphA4-Fc treatment. Consistent with functional improvement, using high-resolution ex vivo magnetic resonance imaging at 16.4T, we found that rats treated with EphA4-Fc had a significantly increased cross-sectional area of the dorsal funiculus caudal to the injury epicenter compared with controls. Our findings indicate that EphA4-Fc promotes functional recovery following contusive spinal cord injury and provides further support for the therapeutic benefit of treatment with the competitive antagonist in acute cases of spinal cord injury.

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