David M. Frazer

Identification of an Intestinal Heme Transporter

Dietary heme iron is an important nutritional source of iron in carnivores and omnivores that is more readily absorbed than non-heme iron derived from vegetables and grain. Most heme is absorbed in the proximal intestine, with absorptive capacity decreasing distally. We utilized a subtractive hybridization approach to isolate a heme transporter from duodenum by taking advantage of the intestinal gradient for heme absorption. Here we show a membrane protein named HCP 1 (heme carrier protein 1), with homology to bacterial metal-tetracycline transporters, mediates heme uptake by cells in a temperature-dependent and saturable manner. HCP 1 mRNA was highly expressed in duodenum and regulated by hypoxia. HCP 1 protein was iron regulated and localized to the brush-border membrane of duodenal enterocytes in iron deficiency. Our data indicate that HCP 1 is the long-sought intestinal heme transporter.

The orchestration of body iron intake: how and where do enterocytes receive their cues?

Our understanding of how iron transverses the intestinal epithelium has improved greatly in recent years, although the mechanism by which body iron demands regulate this process remains poorly understood. By critically examining the earlier literature in this field and considering it in combination with recent advances we have formulated a model explaining how iron absorption could be regulated by body iron requirements. In particular, this analysis suggests that signals to alter absorption exert a direct effect on mature enterocytes rather than influencing the intestinal crypt cells. We propose that the liver plays a central role in the maintenance of iron homeostasis by regulating the expression of hepcidin in response to changes in the ratio of diferric transferrin in the circulation to the level of transferrin receptor 1. Such changes are detected by transferrin receptor 2 and the HFE/transferrin receptor 1 complex. Circulating hepcidin then directly influences the expression of Ireg1 in the mature villus enterocytes of the duodenum, thereby regulating iron absorption in response to body iron requirements. In this manner, the body can rapidly and appropriately respond to changes in iron demands by adjusting the release of iron from the duodenal enterocytes and, possibly, the macrophages of the reticuloendothelial system. This model can explain the regulation of iron absorption under normal conditions and also the inappropriate absorption seen in pathological states such as hemochromatosis and thalassemia.

A rapid decrease in the expression of DMT1 and Dcytb but not Ireg1 or hephaestin explains the mucosal block phenomenon of iron absorption

Background: A large oral dose of iron will reduce the absorption of a subsequent smaller dose of iron in a phenomenon known as mucosal block. Molecular analysis of this process may provide insights into the regulation of intestinal iron absorption. Aims: To determine the effect of an oral bolus of iron on duodenal expression of molecules associated with intestinal iron transport in rats and to relate this to changes in iron absorption. Methods: Rats were given an oral dose of iron and duodenal expression of divalent metal transporter 1 (DMT1), Dcytb, Ireg1, and hephaestin (Hp) was determined using the ribonuclease protection assay, western blotting, and immunofluorescence. Iron absorption was measured using radioactive 59Fe. Results: A decrease in intestinal iron absorption occurred following an oral dose of iron and this was associated with increased enterocyte iron levels, as assessed by iron regulatory protein activity and immunoblotting for ferritin. Reduced absorption was also accompanied by a rapid decrease in expression of the mRNAs encoding the brush border iron transport molecules Dcytb and the iron responsive element (IRE) containing the splice variant of DMT1. No such change was seen in expression of the non-IRE splice variant of DMT1 or the basolateral iron transport molecules Ireg1 and Hp. Similar changes were observed at the protein level. Conclusions: These data indicate that brush border, but not basolateral, iron transport components are regulated locally by enterocyte iron levels and support the hypothesis that systemic stimuli exert their primary effect on basolateral transport molecules.

Iron absorption and metabolism.

Purpose of review 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. Recent findings 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. Summary 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.

Delayed hepcidin response explains the lag period in iron absorption following a stimulus to increase erythropoiesis

Introduction: The delay of several days between an erythropoietic stimulus and the subsequent increase in intestinal iron absorption is commonly believed to represent the time required for body signals to programme the immature crypt enterocytes and for these cells to migrate to the villus. Recent data however suggest that signals from the body to alter absorption are mediated by circulating hepcidin and that this peptide exerts its effect on mature villus enterocytes. Methods: We have examined the delay in the absorptive response following stimulated erythropoiesis using phenylhydrazine induced haemolysis and correlated this with expression of hepcidin in the liver and iron transporters in the duodenum. Results: There was a delay of four days following haemolysis before a significant increase in iron absorption was observed. Hepatic hepcidin expression did not decrease until day 3, reaching almost undetectable levels by days 4 and 5. This coincided with the increase in duodenal expression of divalent metal transporter 1, duodenal cytochrome b, and Ireg1. Conclusion: These results suggest that the delayed increase in iron absorption following stimulated erythropoiesis is attributable to a lag in the hepcidin response rather than crypt programming, and are consistent with a direct effect of the hepcidin pathway on mature villus enterocytes.

TRANSFUSION SUPPRESSES ERYTHROPOIESIS AND INCREASES HEPCIDIN IN ADULT PATIENTS WITH BETA-THALASSEMIA MAJOR: A LONGITUDINAL STUDY

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 to 8 days after transfusion. Pretransfusion 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. Posttransfusion, hemoglobin and hepcidin increased, and erythropoietin and growth differentiation factor-15 decreased. By multiple regression, pre- and posttransfusion hepcidin concentrations were both associated positively with hemoglobin, inversely with erythropoiesis, and positively with ferritin. Although men and women had similar pretransfusion hemoglobin, men had significantly increased erythropoiesis and lower hepcidin, received a lower transfusion volume per liter blood volume, and experienced a smaller posttransfusion reduction in erythropoiesis and hepcidin rise. Age of blood was not associated with posttransfusion 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.

Regulation of systemic iron homeostasis: how the body responds to changes in iron demand

The iron that is required to meet the metabolic needs of cells and tissues is derived from the plasma. Plasma iron in turn reflects the release of iron from various body cells, principally the macrophages of the reticuloendothelial system, and the absorption of dietary iron by the proximal small intestine. This iron donation is highly regulated and the liver-derived peptide hepcidin has emerged as the key modulator of cellular iron export. Following its synthesis and secretion from the liver, circulating hepcidin reduces iron export into the plasma by binding to the iron efflux protein ferroportin1 on the surface of enterocytes, macrophages and other cell types and causing its internalization. The level of hepatic hepcidin expression is influenced by HFE, transferrin receptor 2 and hemojuvelin, and the signal transduction pathway(s) linking these proteins to hepcidin are only beginning to be revealed. Hemojuvelin has recently been shown to signal through the bone morphogenetic protein pathway, ultimately activating receptor SMAD/SMAD4 complexes to alter hepcidin transcription. Circulating differic transferrin has emerged as a possible upstream regulator of the liver-based hepcidin regulatory pathway. In addition to being regulated by body iron requirements, hepcidin expression can be modulated by pro-inflammatory cytokines such as interleukin-6. The continuing analysis of inherited disorders of iron metabolism combined with biochemical analysis of signal transduction pathways is essential to fully define this important regulatory system.

Changes in the expression of intestinal iron transport and hepatic regulatory molecules explain the enhanced iron absorption associated with pregnancy in the rat

Background: Iron absorption increases during pregnancy to cater for the increased iron requirements of the growing fetus. Aims: To investigate the role of the duodenal iron transport molecules and hepatic regulatory molecules in coordinating the changes in iron absorption observed during pregnancy. Methods: Rats at various days of gestation and 24–48 hours post-partum were examined for hepatic expression of hepcidin, transferrin receptors 1 and 2, and HFE (the gene mutated in the most prevalent form of hereditary haemochromatosis), and duodenal expression of divalent metal transporter 1 (DMT1), duodenal cytochrome b (Dcytb), iron regulated mRNA (Ireg1), and hephaestin (Hp) by ribonuclease protection assay, western blotting, and immunohistochemistry. Results: Decreased hepatic non-haem iron and transferrin saturation and increased expression of transferrin receptor 1 in the liver indicated a progressive reduction in maternal body iron stores during pregnancy. Duodenal expression of the iron transport molecules DMT1, Dcytb, and Ireg1 increased during pregnancy, and this corresponded with a reduction in hepcidin, HFE, and transferrin receptor 2 expression in the liver. Expression of all molecules returned towards control values by 24–48 hours post-partum. Conclusions: These data indicate that increased expression of key iron transport molecules is responsible for the elevated iron absorption associated with pregnancy, and implicate hepcidin, HFE, and transferrin receptor 2 in determining how the maternal iron homeostatic machinery responds to the increased iron demands accompanying gestation.

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, β2‐microglobulin knockout and C57BL/6J mice were injected with Freunds 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.

Mechanisms of Haem and Non-Haem Iron Absorption: Lessons from Inherited Disorders of Iron Metabolism

Our current state of knowledge of the mechanism and regulation of intestinal iron absorption has been critically dependent on the analysis of inherited disorders of iron homeostasis in both humans and other animal species. Mutations in DMT1 and Ireg1 have revealed that these molecules are major mediators of iron transport across the brush border and basolateral membranes of the enterocyte, respectively. Similarly, the iron oxidase hephaestin has been shown to play an important role in basolateral iron efflux. The analysis of a range of human iron loading disorders has provided very strong evidence that the products of the HFE, TfR2, hepcidin and hemojuvelin genes comprise integral components of the machinery that regulates iron absorption and iron traffic around the body. Engineered mouse strains have already proved very effective in helping to dissect pathways of iron homeostasis, and in the future they will continue to provide important insights into the absorption of both inorganic and haem iron by the gut.