Hepcidin regulation of ferroportin 1 expression in the liver and intestine of the rat

Hepcidin has been implicated as the iron stores regulator: a hepatic signaling molecule that regulates intestinal iron absorption by undefined mechanisms. The possibility that hepcidin regulates the expression of ferroportin 1 (FPT1), the basolateral iron transporter, was examined in rats after administration of LPS, an iron chelator, or His-tagged recombinant hepcidin (His-rHepc). In the liver, LPS stimulated a biphasic increase of hepcidin mRNA with peaks of mRNA at 6 and 36 h. Concurrently, hepatic FPT1 mRNA expression decreased to minimal level at 6 h and then increased with a peak at 24-36 h. LPS also induced biphasic changes in intestinal FPT1 mRNA expression, with decreased levels at 6 h and increased expression at 48 h. Whereas the initial decrease of FPT1 coincides with an LPS-induced decrease in serum iron, both intestinal and hepatic FPT1 expression recovered, whereas serum iron concentration continued to decrease for at least 24 h. Dietary iron ingestion increased intestinal ferritin protein production but did not reduce intestinal FPT1 mRNA expression. The iron chelator pyrrolidinedithiocarbamate (PDTC) stimulated hepatic hepcidin without suppressing intestinal FPT1 expression. In PDTC-treated rats, LPS stimulated no additional hepatic hepcidin expression but did increase intestinal FPT1 expression. Administration of HisrHepc induced significant reduction of intestinal FPT1 expression. Taken together, these data suggest that hepcidin mediates LPS-induced downregulation of intestinal FPT1 expression and that the hepcidin signaling pathway involves a PDTC-sensitive step.     

Systemic regulation of intestinal iron absorption

The intestinal absorption of the essential trace element iron and its mobilization from storage sites in the body are controlled by systemic signals that reflect tissue iron requirements. Recent advances have indicated that the liver-derived peptide hepcidin plays a central role in this process by repressing iron release from intestinal enterocytes, macrophages and other body cells. When iron requirements are increased, hepcidin levels decline and more iron enters the plasma. It has been proposed that the level of circulating diferric transferrin, which reflects tissue iron levels, acts as a signal to alter hepcidin expression. In the liver, the proteins HFE, transferrin receptor 2 and hemojuvelin may be involved in mediating this signal as disruption of each of these molecules decreases hepcidin expression. Patients carrying mutations in these molecules or in hepcidin itself develop systemic iron loading (or hemochromatosis) due to their inability to down regulate iron absorption. Hepcidin is also responsible for the decreased plasma iron or hypoferremia that accompanies inflammation and various chronic diseases as its expression is stimulated by pro-inflammatory cytokines such as interleukin 6. The mechanisms underlying the regulation of hepcidin expression and how it acts on cells to control iron release are key areas of ongoing research.     

Hypoxia-inducible factor-2α and iron absorptive gene expression in Belgrade rat intestine

The divalent metal transporter (DMT1, Slc11a2) is an important molecule for intestinal iron absorption. In the Belgrade (b/b) rat, the DMT1 G185R mutation markedly decreases intestinal iron absorption. We used b/b rats as a model to examine the genes that could be compensatory for decreased iron absorption. When tissue hypoxia was assayed by detecting pimonidazole HCl adducts, the b/b liver and intestine exhibited more adducts than the +/+ rats, suggesting that hypoxia might signal altered gene expression. Total RNA in the crypt-villus bottom (C-pole) and villus top (V-pole) of +/+, b/b, and iron-fed b/b rats was isolated for gene array analyses. In addition, hepatic hepcidin and intestinal hypoxia-inducible factor-α (Hifα) expression were examined. The results showed that expression of hepatic hepcidin was significantly decreased and intestinal Hif2α was significantly increased in b/b and iron-fed b/b than +/+ rats. In b/b rats, the expression of Tfrc mRNA in the C-pole and of DMT1, Dcytb, FPN1, Heph, Hmox1, and ZIP14 mRNAs in the V-pole were markedly enhanced with increases occurring even in the C-pole. After iron feeding, the increased expression found in b/b rats persisted, except for Heph and ZIP14, which returned to normal levels. Thus in b/b rats depressed liver hepcidin production and activated intestinal Hif2α starting at the C-pole resulted in increasing expression of iron transport genes, including DMT1 G185R, in an attempt to compensate for the anemia in Belgrade rats.     

利用FRET技术研究Hepcidin 和Fpn 相互作用

铁是生命必需的微量元素,ferroportin(Fpn)是小肠吸收细胞铁释放的重要蛋白。新近发现肝脏分泌的抗菌多肽 hepcidin 具有调节肠铁吸收的重要作用,但目前尚缺少Fpn和hepcidin发生作用的实验依据。应用荧光共振能量转移技术(fluorescence resonance energy transfer ,FRET)对hepcidin和Fpn之间的作用关系进行了深入研究。首先进行了hepcidin-CF P融合蛋白表达载体的构建及表达鉴定;然后对含YFP,Fpn-YFP基因动物细胞表达载体的构建、表达和FRET检测。实验结果证实hepcidin和Fpn之间存在直接的相互作用,并发现两种蛋白发生相互作用后hepcidin也在细胞质中有分布。为临床治疗铁代谢紊乱性疾病提供了新的治疗策略和重要理论依据。     

Haemochromatosis protein is expressed on the terminal web of enterocytes in proximal small intestine of the rat

The haemochromatosis protein (HFE) is an important regulator of body iron stores. In the liver, HFE is required for appropriate expression of hepcidin, a humoral mediator of iron absorption. HFE is also present in enterocytes, though its function in the intestine is unknown; it is not intrinsically required for iron absorption, but can augment iron absorption when over-expressed—independent of hepcidin regulation by the liver. In this study, an antibody was raised against rat HFE and validated by enzyme-linked immunosorbent assay, Western blot and quenching of antibody function by the immunising peptide. The sub-cellular location of HFE in enterocytes of iron-deficient and control rats was determined by double-labelling experiments with markers for the microvillus membrane, terminal web, early endosomes, lysosomes and the transferrin receptor. Parallel studies were performed for the primary iron absorption protein, divalent metal transporter 1 (DMT1). HFE co-localised exclusively with the terminal web of intestinal enterocytes. HFE expression was increased in iron deficiency, consistent with a second regulatory role for HFE in iron absorption, independent of hepcidin from the liver. DMT1 was localised primarily on the microvillus membrane, but did partially co-localise with HFE raising the possibility that the two proteins may interact to regulate iron absorption.     

Hepcidin与疾病关系和其临床意义的研究进展

铁是人体必需的微量元素,是血红蛋白、肌红蛋白及多种酶的重要组成成分,广泛地参与氧气输运、氧化还原反应、细胞增殖与分化、基因表达调控等基本生命过程。机体铁稳态对生命体新陈代谢的平衡起着至关重要的作用。铁稳态依赖铁吸收、转运和储存、再循环利用等代谢过程共同调节。铁调素（Hepcidin）是铁代谢调节中最关键的调节分子,成熟的铁调素是一个由25个氨基酸组成的功能性小肽类激素,可以通过调节小肠上皮细胞和巨噬细胞表面的相关铁转运蛋白来调控机体内铁的储存和利用。铁调素同时受到机体铁水平的反馈,免疫应答和红细胞生成等因素的共同调节。许多铁代谢疾病、炎症和各种原因引起的贫血与铁调素的异常表达相关。因此,对于铁调素的检测不但可以反映机体的铁代谢状况,结合其他临床指标还能够辅助诊断和有针对性地检测相关疾病的治疗效果。     

Distinct requirements for Hfe in basal and induced hepcidin levels in iron overload and inflammation

Hepcidin is a negative regulator of iron absorption produced mainly by the liver in response to changes in iron stores and inflammation, and its levels have been shown to regulate the intestinal basolateral iron transporter ferroportin1 (Fp1). Hereditary hemochromatosis patients and Hfe-deficient mice show inappropriate expression of hepcidin but, in apparent contradiction, still retain the ability to regulate iron absorption in response to alterations of iron metabolism. To further understand the molecular relationships among Hfe, hepcidin, and Fp1, we investigated hepcidin and Fp1 regulation in Hfe-deficient mice (Hfe-/- and beta2m-/-) in response to iron deprivation, iron loading, and acute inflammation. We found that whereas basal hepcidin levels were manifestly dependent on the presence of Hfe and on the mouse background, all Hfe-deficient mice were still able to regulate hepcidin in situations of altered iron homeostasis. In the liver, Fp1 was modulated in opposite directions by iron and LPS, and its regulation in Hfe-deficient mice was similar to that observed in wild-type mice. In addition, we found that iron-deprived mice were able to mount a robust response after LPS challenge and that Toll-like receptor 4 (TLR-4)-deficient mice fail to regulate hepcidin expression in response to LPS. In conclusion, these results suggest that although Hfe is necessary for the establishment of hepcidin basal levels, it is dispensable for hepcidin regulation through both the iron-sensing and inflammatory pathways, and hepatic Fp1 regulation is largely independent of hepcidin and Hfe. The inflammatory pathway overrides the iron-sensing pathway and is TLR-4 dependent.     

Hepcidin mRNA levels in mouse liver respond to inhibition of erythropoiesis

Hepcidin, a key regulator of iron metabolism, decreases intestinal absorption of iron and its release from macrophages. Iron, anemia, hypoxia, and inflammation were reported to influence hepcidin expression. To investigate regulation of the expression of hepcidin and other iron-related genes, we manipulated erythropoietic activity in mice. Erythropoiesis was inhibited by irradiation or posttransfusion polycythemia and stimulated by phenylhydrazine administration and erythropoietin. Gene expression of hepcidin and other iron-related genes (hemojuvelin, DMT1, ferroportin, transferrin receptors, ferritin) in the liver was measured by the real-time polymerase chain reaction. Hepcidin expression increased despite severe anemia when hematopoiesis was inhibited by irradiation. Suppression of erythropoiesis by posttransfusion polycythemia or irradiation also increased hepcidin mRNA levels. Compensated hemolysis induced by repeated phenylhydrazine administration did not change hepcidin expression. The decrease caused by exogenous erythropoeitin was blocked by postirradiation bone marrow suppression. The hemolysis and anemia decrease hepcidin expression only when erythropoiesis is functional; on the other hand, if erythropoiesis is blocked, even severe anemia does not lead to a decrease of hepcidin expression, which is indeed increased. We propose that hepcidin is exclusively sensitive to iron utilization for erythropoiesis and hepatocyte iron balance, and these changes are not sensed by other genes involved in the control of iron metabolism in the liver.     

Effect of zinc depletion/repletion on intestinal iron absorption and iron status in rats

Iron and zinc deficiencies likely coexist in general population. We have previously demonstrated that zinc treatment induces while zinc deficiency inhibits iron absorption in intestinal cell culture models, but this needs to be tested in vivo. In the present study we assessed intestinal iron absorption, iron status (haemoglobin), red blood cell number, plasma ferritin, transferrin receptor, hepcidin) and tissue iron levels in zinc depleted, replete and pair fed control rats. Zinc depletion led to reduction in body weight, tissue zinc levels, intestinal iron absorption, protein and mRNA expression of iron transporters, the divalent metal ion transporter-1, hephaestin and ferroportin, but elevated the intestinal and liver tissue iron levels compared with the pair fed control rats. Zinc repletion led to a significant weight gain compared to zinc deficient rats and normalized the iron absorption, iron transporter expression, tissue iron levels to that of pair fed control rats. Surprisingly, haemoglobin levels and red blood cell number reduced significantly in zinc repleted rats, which could be due to rapid weight gain. Together, these results indicate that whole body zinc status has profound influence on growth, intestinal absorption and systemic utilization of iron, mediated via modulation of iron transporter expression.     

Inflammation neither increases hepatic hepcidin nor affects intestinal 59Fe-absorption in two murine models of bowel inflammation,hemizygous TNFΔARE/+ and homozygous IL-10−/− mice

Hepcidin-synthesis was reported to be stimulated by inflammation. In contrast, hepcidin synthesis was inhibited by TNFα and serum hepcidin was low. To elucidate these contradictions, we compare data on hepcidin expression, on iron absorption and homoeostasis and markers of inflammation between two murine models of intestinal inflammation and corresponding wild-types as determined by standard methods.In TNF^ΔARE/+ and IL-10^−/−-mice hepatic hepcidin expression and protein content was significantly lower than in corresponding wild-types. However, ⁵⁹Fe whole-body retention showed no difference between knock-outs and corresponding wild-types 7d after gavage, in neither strain. Compared to wild-types, body weight, hepatic non-haem iron content, hemoglobin and hematocrit were significantly decreased in TNF^ΔARE/+ mice, while erythropoiesis increased. These differences were not seen in IL-10^−/− mice. Duodenal IL-6 and TNFα content increased significantly in TNF^ΔARE/+ mice, while ferritin-H decreased along with hepatic hepcidin expression, ferritin L, and non-haem iron. In IL-10^−/− mice, these changes were less marked or missing for non-haem iron. Duodenal ferritin-L and ferroportin increased significantly, while HFE decreased.Our results corroborate the conflicting combination of low hepcidin with inflammation and without increased intestinal iron absorption. Speculating on underlying mechanism, decreased hepcidin may result from stimulated erythropoiesis. Unaltered intestinal iron-absorption may compromise between the stimulation by increased erythropoiesis and inhibition by local and systemic inflammation. The findings suggest intense interaction between counterproductive mechanisms and ask for further research.     

High-fat diet causes iron deficiency via hepcidin-independent reduction of duodenal iron absorption

Obesity is often associated with disorders of iron homeostasis; however, the underlying mechanisms are not fully understood. Hepcidin is a key regulator of iron metabolism and may be responsible for obesity-driven iron deficiency. Herein, we used an animal model of diet-induced obesity to study high-fat-diet-induced changes in iron homeostasis. C57BL/6 mice were fed a standard (SD) or high-fat diet (HFD) for 8 weeks, and in addition, half of the mice received high dietary iron (Fe+) for the last 2 weeks. Surprisingly, HFD led to systemic iron deficiency which was traced back to reduced duodenal iron absorption. The mRNA and protein expressions of the duodenal iron transporters Dmt1 and Tfr1 were significantly higher in HFD- than in SD-fed mice, indicating enterocyte iron deficiency, whereas the mRNA levels of the duodenal iron oxidoreductases Dcytb and hephaestin were lower in HFD-fed mice. Neither hepatic and adipose tissue nor serum hepcidin concentrations differed significantly between SD- and HFD-fed mice, whereas dietary iron supplementation resulted in increased hepatic hepcidin mRNA expression and serum hepcidin levels in SD as compared to HFD mice. Our study suggests that HFD results in iron deficiency which is neither due to intake of energy-dense nutrient poor food nor due to increased sequestration in the reticulo-endothelial system but is the consequence of diminished intestinal iron uptake. We found that impaired iron absorption is independent of hepcidin but rather results from reduced metal uptake into the mucosa and discordant oxidoreductases expressions despite enterocyte iron deficiency.     

Role of duodenal iron transporters and hepcidin in patients with alcoholic liver disease

Patients with alcoholic liver disease (ALD) often display disturbed iron indices. Hepcidin, a key regulator of iron metabolism, has been shown to be down‐regulated by alcohol in cell lines and animal models. This down‐regulation led to increased duodenal iron transport and absorption in animals. In this study, we investigated gene expression of duodenal iron transport molecules and hepcidin in three groups of patients with ALD (with anaemia, with iron overload and without iron overload) and controls. Expression of DMT1, FPN1, DCYTB, HEPH, HFE and TFR1 was measured in duodenal biopsies by using real‐time PCR and Western blot. Serum hepcidin levels were measured by using ELISA. Serum hepcidin was decreased in patients with ALD. At the mRNA level, expressions of DMT1, FPN1 and TFR1 genes were significantly increased in ALD. This pattern was even more pronounced in the subgroups of patients without iron overload and with anaemia. Protein expression of FPN1 paralleled the increase at the mRNA level in the group of patients with ALD. Serum ferritin was negatively correlated with DMT1 mRNA. The down‐regulation of hepcidin expression leading to up‐regulation of iron transporters expression in the duodenum seems to explain iron metabolism disturbances in ALD. Alcohol consumption very probably causes suppression of hepcidin expression in patients with ALD.     

铁代谢红细胞系调节因子与铁稳态

红细胞合成是人类和其他脊椎动物最耗铁的生理过程,对机体铁稳态具有重要调节作用。Erythroferrone（ERFE）是红细胞系来源的调节铁调素的主要激素。当机体存在应激性红细胞合成时,ERFE合成增加,铁调素表达受抑,可促进机体铁吸收和储铁动员,满足红细胞合成对铁的需求,但在无效红细胞生成疾病中,通过此作用也导致了铁过载的发生。ERFE抑制肝细胞合成铁调素的作用机制尚不清楚,但至少部分地依赖BMP/SMAD信号通路。ERFE对铁代谢障碍性疾病和红细胞生成紊乱性贫血有重要的诊断及治疗价值。     

Molecular mechanisms of iron homeostasis

Iron metabolism in mammals requires a complex and tightly regulated molecular network. The classical view of iron metabolism has been challenged over the past ten years by the discovery of several new proteins, mostly Fe (II) iron transporters, enzymes with ferro-oxydase (hephaestin or ceruloplasmin) or ferri-reductase (Dcytb) activity or regulatory proteins like HFE and hepcidin. Furthermore, a new transferrin receptor has been identified, mostly expressed in the liver, and the ability of the megalin-cubilin complex to internalise the urinary Fe (III)-transferrin complex in renal tubular cells has been highlighted. Intestinal iron absorption by mature duodenal enterocytes requires Fe (III) iron reduction by Dcytb and Fe (II) iron transport through apical membranes by the iron transporter Nramp2/DMT1. This is followed by iron transfer to the baso-lateral side, export by ferroportin and oxidation into Fe (III) by hephaestin prior to binding to plasma transferrin. Macrophages play also an important role in iron delivery to plasma transferrin through phagocytosis of senescent red blood cell, heme catabolism and recycling of iron. Iron egress from macrophages is probably also mediated by ferroportin and patients with heterozygous ferroportin mutations develop progressive iron overload in liver macrophages. Iron homeostasis at the level of the organism is based on a tight control of intestinal iron absorption and efficient recycling of iron by macrophages. Signalling between iron stores in the liver and both duodenal enterocytes and macrophages is mediated by hepcidin, a circulating peptide synthesized by the liver and secreted into the plasma. Hepcidin expression is stimulated in response to iron overload or inflammation, and down regulated by anemia and hypoxia. Hepcidin deficiency leads to iron overload and hepcidin overexpression to anemia. Hepcidin synthesis in response to iron overload seems to be controlled by the HFE molecule. Patients with hereditary hemochromatosis due to HFE mutation have impaired hepcidin synthesis and forced expression of an hepcidin transgene in HFE deficient mice prevents iron overload. These results open new therapeutic perspectives, especially with the possibility to use hepcidin or antagonists for the treatment of iron overload disorders.     

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.     

Hepcidin研究进展

Hepcidin是肝脏特异性表达的一种小分子抗菌肽,是铁代谢的负调节激素。与炎症性贫血、遗传性血色沉着病等疾病的发病机制密切相关。证据显示,Hepcidin直接抑制肠上皮细胞铁吸收和诱导单核巨噬细胞铁滞留。同时,Hepcidin还具有广谱抗菌活性,与固有免疫密切相关。铁超载、感染、炎症及细胞因子可诱导Hepcidin表达,而贫血和缺氧则抑制其表达。Hepcidin的发现及其相关的铁离子运输机制的研究,将为铁离子吸收及分配的铁稳态调节和炎症性贫血、遗传性血色沉着病中的铁代谢障碍的分子机制探索开辟新的途径。本文就Hepcidin的分子特征、表达调控及生物学功能等方面研究进展进行综述。     

Regulatory networks for the control of body iron homeostasis and their dysregulation in HFE mediated hemochromatosis

Although the recent identification of several genes has extended our knowledge on the maintenance of body iron homeostasis, their tissue specific expression patterns and the underlying regulatory networks are poorly understood. We studied C57black/Sv129 mice and HFE knockout (HFE -/-) variants thereof as a model for hemochromatosis, and investigated the expression of iron metabolism genes in the duodenum, liver, and kidney as a function of dietary iron challenge. In HFE +/+ mice dietary iron supplementation increased hepatic expression of hepcidin which was paralleled by decreased iron regulatory protein (IRP) activity, and reduced expression of divalent metal transporter-1 (DMT-1) and duodenal cytochrome b (Dcytb) in the enterocyte. In HFE -/- mice hepcidin formation was diminished upon iron challenge which was associated with decreased hepatic transferrin receptor (TfR)-2 levels. Accordingly, HFE -/- mice presented with high duodenal Dcytb and DMT-1 levels, and increased IRP and TfR expression, suggesting iron deficiency in the enterocyte and increased iron absorption. In parallel, HFE -/- resulted in reduced renal expression of Dcytb and DMT-1. Our data suggest that the feed back regulation of duodenal iron absorption by hepcidin is impaired in HFE -/- mice, a model for genetic hemochromatosis. This change may be linked to inappropriate iron sensing by the liver based on decreased TfR-2 expression, resulting in reduced circulating hepcidin levels and an inappropriate up-regulation of Dcytb and DMT-1 driven iron absorption. In addition, iron excretion/reabsorption by the kidneys may be altered, which may aggravate progressive iron overload.     

Iron Imports. VI. HFE and regulation of intestinal iron absorption

The majority of clinical cases of iron overload is caused by mutations in the HFE gene. However, the role that HFE plays in the physiology of intestinal iron absorption remains enigmatic. Two major models have been proposed: 1) HFE exerts its effects on iron homeostasis indirectly, by modulating the expression of hepcidin; and 2) HFE exerts its effects directly, by changing the iron status (and therefore the iron absorptive activity) of intestinal enterocytes. The first model places the primary role of HFE in the liver (hepatocytes and/or Kupffer cells). The second model places the primary role in the duodenum (crypt cells or villus enterocytes). These models are not mutually exclusive, and it is possible that HFE influences the iron status in each of these cell populations, leading to cell type-specific downstream effects on intestinal iron absorption and body iron distribution.