Ferric Reductase Dcytb Research Articles

Iron Supplements Containing Lactobacillus plantarum 299v Increase Ferric Iron and Up-regulate the Ferric Reductase DCYTB in Human Caco-2/HT29 MTX Co-Cultures.

Several human interventions have indicated that Lactobacillus plantarum 299v (L. plantarum 299v) increases intestinal iron absorption. The aim of the present study was to investigate possible effects of L. plantarum 299v on the mechanisms of iron absorption on the cellular level. We have previously shown that lactic fermentation of vegetables increased iron absorption in humans. It was revealed that the level of ferric iron [Fe (H2O)5]2+ was increased after fermentation. Therefore, we used voltammetry to measure the oxidation state of iron in simulated gastrointestinal digested oat and mango drinks and capsule meals containing L. plantarum 299v. We also exposed human intestinal co-cultures of enterocytes and goblet cells (Caco-2/HT29 MTX) to the supplements in order to study the effect on proteins possibly involved (MUC5AC, DCYTB, DMT1, and ferritin). We detected an increase in ferric iron in the digested meals and drinks containing L. plantarum 299v. In the intestinal cell model, we observed that the ferric reductase DCYTB increased in the presence of L. plantarum 299v, while the production of mucin (MUC5AC) decreased independently of L. plantarum 299v. In conclusion, the data suggest that the effect of L. plantarum 299v on iron metabolism is mediated through driving the Fe3+/DCYTB axis.

An iron supplement containing L. plantarum increases ferric iron and up‐regulates the ferric reductase Dcytb in human Caco‐2/HT29 MTX co‐cultures

Iron absorption from a bread meal, added an encapsulated iron‐containing supplement, may be increased if the lactic acid bacteria L. plantarum (DSM 9843) is present. The aim of this study was to investigate the mechanism by which L. plantarum increases iron absorption. We have previously shown that lactic fermentation of mixed vegetables increased iron absorption in humans and voltammetric studies revealed that the level of ferric iron [Fe3+ (H2O)6] was increased in the fermented vegetables, compared to unfermented control. In this study, we detected a similar increase in ferric iron in the in vitro digested bread meal containing the supplement with L. plantarum. In a co‐culture of intestinal Caco‐2 cells and mucus‐producing goblet cells (HT29 MTX E12), we observed that the ferric reductase Dcytb increased in the presence of L. plantarum‐containing iron supplements while the production of mucin (muc5a1c) decreased independent of L. plantarum. This suggests that the L. plantarum‐effect on iron absorption is mediated through enhancing the Fe3+/Dcytb system.Support or Funding InformationThe work was financially supported by Probi ABThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Intestinal expression of genes implicated in iron absorption and their regulation by hepcidin

Through inhibition of iron absorption and iron mobilization from tissue stores, hepcidin exerts a negative control on iron homeostasis. Hepcidin, in fact, promotes the degradation of ferroportin (Fpn1), the iron exporter molecule expressed on the membrane of hepatocytes and macrophages, thus preventing iron release from cells to plasma. Hepcidin effects on enterocytes, however, are less clear. Aim of the present study was to further investigate the regulation of iron absorption by hepcidin. The transcriptional response of human duodenal mucosa to hepcidin was investigated using organ cultures of duodenal biopsies perendoscopically collected from healthy controls. Biopsies were cultured for 4h with or without hepcidin-25 and were then assayed for the expression of iron-related genes. In samples that had not been exposed to hepcidin, correlations were found between the expression of genes involved in iron absorption: DMT1, Fpn1, Dcytb and HCP1. In exvivo experiments hepcidin down-regulated mRNA levels of the iron transporters Fpn1, and DMT1, of the ferric reductase Dcytb, of the ferroxidase hephaestin, and of the putative heme carrier protein HCP1. Through the reported transcriptional changes hepcidin can modulate several steps of the iron absorption process, including the reduction of dietary iron by Dcytb, its uptake by enterocytes through DMT1, the mucosal uptake of heme iron by HCP1, and enterocyte iron release to plasma by Fpn1 in conjunction with hephaestin.

ChemInform Abstract: Brain Iron Metabolism and Its Perturbation in Neurological Diseases

AbstractReview: 101 refs.

Brain iron metabolism and its perturbation in neurological diseases

Enormous advances have been made in the last decade in understanding iron metabolism and iron homeostasis at both the cellular and the systemic level. This includes the identification of genes and proteins involved in iron transport, such as the ferric reductase DcytB, the proton-coupled ferrous (divalent) iron transporter DMT1, the iron exporter ferroportin and the membrane-bound ferroxidase hephaestin. The modulation of their translation by the iron regulatory protein (IRP) system has also been identified together with the impressive signalling cascades involved in regulating the chef d’orchestre of systemic iron homeostasis, hepcidin. However, exactly how the brain regulates fluxes and storage of iron between neurons, oligodendrocytes, astrocytes and microglial cells remains an enigma. In this review we discuss the possible mechanisms which may be involved in the transfer of iron across the blood–brain barrier, together with the possible role played by astrocytes. The consequences of iron deficiency and iron excess on brain function are described. Finally, various neurodegenerative diseases, where accumulation of iron may be important in the pathogenesis, are presented as well as the possible use of iron chelators to diminish disease progression.

Ascorbic acid uptake affects ferritin, Dcytb and Nramp2 expression in Caco-2 cells

Ascorbic acid (vitamin C) enhances iron uptake in human intestinal cells. It is commonly believed that the enhancement is due to the capacity of ascorbic acid to reduce ferric iron to ferrous iron. Other suggestions have recently been made about the effects of ascorbic acid on the cellular metabolism of iron. These effects must be investigated for several reasons. One important issue is to study whether ascorbic acid has effects on iron metabolism in the absence of extracellular iron in the intestinal lumen. The aim of this investigation was to determine whether cellular uptake of ascorbic acid affects iron acquisition in the Caco-2 cell line. The possible event was investigated by studying the expression of the iron storage protein ferritin, the iron uptake protein Nramp2 and a duodenal ferric reductase Dcytb after incubating ascorbic acid deficient or ascorbic acid fed cells with iron and/or ascorbic acid. The above stated interactions were studied in the human Caco-2 cell model. Cell lysates were collected and subjected to SDS-PAGE and Western blotting. The blotted samples were stained with specific antibodies (Rabbit alpha-human-Nramp2 and Goat alpha-human Dcytb) against the respective proteins and the bands achieved were analysed by reflective density measurements. The cellular ferritin content was analysed with a commercial kit and the intracellular ascorbic acid concentration was measured by HPLC. The results indicate that ascorbic acid uptake induces both iron independent and iron dependent ferritin formation, but the effect on iron dependent ferritin expression was significantly greater (470% compared to 19%). Western Blot analyses revealed a long term down-regulating effect of ascorbic acid on iron independent and iron dependent Nramp2 and Dcytb expression. However, the down-regulation of Dcytb was in general more extensive than that of Nramp2 (31-50% compared to 8-29%). In a second study of short term Nramp2 and Dcytb expression, the results suggested that both proteins were significantly up-regulated by ascorbic acid, regardless of intracellular ascorbic acid status. However, the impact of iron alone on Nramp2 up-regulation seems to be greater in the absence of ascorbic acid. The influence of intracellular ascorbic acid status on ferritin formation must be considered in iron uptake studies in Caco-2 cells. This could be a cause of diverging inter-laboratory results. The long term down-regulation of Nramp2 and Dcytb seems to correlate with results of human studies, where long term ascorbic acid supplementation does not affect iron status. Similarly, the short term up-regulation of Nramp2 and Dcytb seems to agree with the improvement in iron uptake shown in humans when single doses of ascorbic acid were administrated. These results are important for the understanding of the impact of ascorbic acid on iron status and will hopefully lead to further investigations on the matter.

Recombinant Expression and Initial Characterization of the Putative Human Enteric Ferric Reductase Dcytb

Recent studies of mutant mice with compromised ability to absorb dietary iron have identified involvement of two integral membrane proteins in the intestinal epithelial lining in iron uptake, a divalent metal ion transporter and a ferric reductase. The current study concerns the recombinant expression, purification, and initial spectroscopic characterization of a recombinant form of the human ferric reductase that was expressed and purified as the apoprotein from Escherichia coli. Reconstitution of the recombinant protein with ferriprotoporphyrin IX produced a red product with Soret (Fe3+, lambdamax 413.5 nm; Fe2+, lambdamax = 426 nm) and visible absorption maxima indicative of bisimidazole axial coordination. This observation was confirmed by electron paramagnetic resonance and magnetic circular dichroism spectroscopy. Titration of apo-Dcytb with ferriprotoporphyrin IX was consistent with the binding of two heme groups to the protein as predicted by the phylogenetic relationship of this protein to the cytochrome b561 family. Similar titrations and spectroscopic studies of two double variants of Dcytb, each lacking a pair of histidyl residues (H50 and H120 or H86 and H159) proposed on the basis of sequence alignment with other members of the cytochrome b561 family to provide axial ligands to bound heme, indicated that these variants were able to bind just one heme group each.

Transcriptional activity of iron homeostasis maintaining genes in HCV infection

Background and Objectives: In chronic hepatitis C, hepatic iron content is mildly elevated. An excess of hepatocyte iron is currently discussed as a cofactor for the development of HCC. The mechanisms leading to iron overload are not yet understood and may range simply from an iron shift from serum to tissue as it occurs e.g. in bacterial infections to a dysregulation of the complex iron homeostasis. To address this issue, we quantified the expression of several genes that are involved in iron metabolism both in liver tissue from chronic hepatitis C patients and in human Huh–7 hepatoma cells harbouring HCV replicons. Methods: Total cellular RNA was obtained from liver tissue or from hepatoma cells that had been treated with or without interferon-γ, and was reverse transcribed using random hexamer oligonucleotides. The amounts of transcripts were quantified by real-time RT-PCR using SYBR Green I dye. Data were normalized to two endogenous references, β-actin and GAPDH. Results: Liver tissue as well as hepatoma cells show high expression of hepcidin, ferritin, transferrin, transferrin receptor 2, ferroportin, and hemojuvelin, and less expression of the divalent metal transporter 1 (DMT1), the ferricreductase Dcytb, transferrin receptor 1, and hephaestin. The amount of transcripts, however, was not found to differ significantly when liver tissue from chronic hepatitis C patients (n=26) was compared to tissue obtained from patients with chronic hepatitis B (n=8), from those with liver disorders of non-viral etiology (n=24), or from healthy tissue (n=7). Gene expression in HCV RNA replicating Huh–7 cells was also not significantly different from naive Huh–7 or from mock-transfected cells. No significant changes in gene expression were observed when cells were treated with IFN-γ, although this cytokine induces IP–10. Hypothesis: HCV infection does not change hepatic expression of the iron-pathway genes neither directly nor indirectly through IFN-γ. However, other mediators of the inflammatory infiltrate may be responsible for increased iron uptake in hepatocytes and in non-parenchymal cells as well.

Recent advances in intestinal iron transport

Our understanding of intestinal iron absorption and its regulation has expanded enormously in recent years. Dietary iron crosses the enterocyte brush border membrane through the transporter DMT1 after first being reduced by the ferric reductase Dcytb. The subsequent movement of iron across the basolateral membrane and into the circulation is mediated by ferroportin1 in conjunction with the iron oxidase hephaestin. The activity of ferroportin1 is controlled by the liver-derived peptide hepcidin, and the expression of hepcidin in turn is influenced by plasma transferrin saturation via a pathway that involves HFE, TfR2, and hemojuvelin. Future studies investigating how these molecules interact will provide a comprehensive understanding of this essential physiologic process.

Tissue-specific changes in iron metabolism genes in mice following phenylhydrazine-induced haemolysis.

Iron metabolism in animals is altered by haemolytic anaemia induced by phenylhydrazine (PHZ). In common with a number of other modulators of iron metabolism, the mode and the mechanisms of this response are yet to be determined. However, recent studies have shown increased expression of the ferrous transporter DMT1 in the duodenum and other tissues of mice administered PHZ. We examined the expression of the ferric reductase Dcytb, DMT1 and some other genes involved in Fe metabolism in tissues of mice dosed with PHZ. The expression of iron-related genes in the duodenum, liver, and spleen of the mice were evaluated using Northern blot analyses, RT-PCR and immunocytochemistry. Dcytb, and DMT1 mRNA and protein increased markedly in the duodenum of mice given PHZ. The efflux protein Ireg1 also increased in the duodenum of the treated mice. These changes correlated with a decrease in hepatic hepcidin expression. Dcytb, DMT1, Ireg1 and transferrin receptor 1 mRNA expression in the spleen and liver of mice treated with PHZ responded to the enhanced iron demand associated with the resulting stimulation of erythropoiesis. Enhanced iron absorption observed in PHZ-treated animals is facilitated by the up-regulation of the genes involved in iron transport and recycling. The probable association of the erythroid and the store regulators of iron homeostasis and absorption in the mice is discussed.

Iron overload in adult Hfe-deficient mice independent of changes in the steady-state expression of the duodenal iron transporters DMT1 and Ireg1/ferroportin.

Patients suffering from hereditary hemochromatosis (HH) show progressive iron overload as a consequence of increased duodenal iron absorption. It has been hypothesized that mutations in the HH gene HFE cause misprogramming of the duodenal enterocytes towards a paradoxical iron-deficient state, resulting in increased iron transporter expression. Previous reports concerning gene expression levels of the duodenal iron transporters DMT1 and IREG1 in HH patients and animal models are controversial, however, and in many cases only mRNA expression levels were investigated. To analyze the duodenal expression of DMT1, Ireg1, Dcytb, and hephaestin and the association with iron overload in adult Hfe(-/-) mice, an Hfe(-/-) mouse line was generated. Duodenal DMT1 and Ireg1 protein levels, duodenal DMT1, Ireg1, Dcytb, hephaestin, and TfR1 mRNA levels, and hepatic hepcidin mRNA levels were quantified and the correlation to liver iron contents was calculated. We report that duodenal DMT1 and Ireg1 mRNA levels and DMT1 and Ireg1 protein levels remained unaffected by the Hfe deletion. Furthermore, duodenal hephaestin and TfR1 mRNA expression and hepatic hepcidin mRNA expression remained unaltered, while the duodenal mRNA expression of the brush border ferric reductase Dcytb was significantly increased in Hfe(-/-) mice. We found no correlation between the expression level of any of the analyzed transcripts and the liver iron content. In conclusion, the lack of correlation between DMT1 and Ireg1 protein expression and the liver iron content suggests that elevated duodenal iron transporter expression is not required for high liver iron overload. Hfe(-/-) mice do not necessarily display features of iron deficiency in the duodenum, indicated by an increase in mRNA and protein levels of DMT1 and Ireg1. Rather, the duodenal ferric reductase Dcytb may act as a possible mediator of iron overload in Hfe deficiency.

Mechanisms involved in the regulation of intestinal iron absorption.

Abstract Iron is one of the most abundant metals in the human body. However, it is only recently that many of the molecular mechanisms involved in the absorption and metabolism of this essential trace element have been elucidated. Dietary non-haem iron is absorbed via the divalent metal transporter (DMT1) following conversion from Fe(III) to Fe(II) by both dietary reducing agents and the endogenous ferric reductase Dcytb. Efflux of iron into the plasma is achieved by the IREG1 protein working in concert with a ferrioxidase, hephaestin to allow Fe(III) to be loaded onto transferrin for onward transport in the plasma. Current consensus suggests that the regulation of intestinal iron absorption relies on signals generated in the main sites of iron storage (the liver) and utilization (the bone marrow) to indicate body iron status, which together co-ordinate intestinal uptake with body requirement. This information, conveyed by factors such as transferrin saturation and serum levels of hepcidin, is integrated by the duodenal crypt cells, which act as iron sensors and pre-programme the level of expression of the iron transport proteins relative to body iron status. Recent data suggest that mature enterocytes also possess significant plasticity and can respond rapidly (within minutes to a few hours) to local changes in dietary iron levels, fine-tuning absorption to efficiently match the body's metabolic requirements. DMT1 expression is also down-regulated by other heavy metals thought to be substrates for this transporter. This may be an adaptive mechanism to avoid toxic build up of these metals in the body.

CHANGES IN DUODENAL IRON TRANSPORT GENE EXPRESSION IN RATS FOLLOWING A SWITCH TO AN IRON DEFICIENT DIET OCCUR BEFORE ALTERATIONS IN BODY IRON STORES

Introduction and aims The level of body iron stores is an important factor controlling the rate of intestinal iron absorption but how storage iron influences intestinal iron transport is unclear. A switch from a control diet to an iron deficient diet results in an increase in iron absorption within 5 days; long before any changes in body iron stores or haematological parameters. To investigate the mechanism by which this occurs, we have studied the adaptation in expression of iron transport genes in rat duodenum following the change to an iron deficient diet.Methods Adult Sprague–Dawley rats on a control diet were switched to an iron deficient diet and duodenal tissue was taken after 0, 2, 4, 6, 10 and 14 days. RNA was extracted and the expression of iron transport genes was determined by ribonuclease protection assay. Intestinal iron absorption was determined by measuring the retention of an oral dose of 59Fe.Results Intestinal iron absorption in rats on a control diet was approximately 10%, but by 6 days following a change to an iron deficient diet it had increased to near maximal levels of approximately 60%. No change in haematological status or hepatic iron stores was evident over this time period. Analysis of duodenal gene expression revealed a large increase in the expression of the brush border ferric reductase DcytB and iron transporter DMT1 (IRE form) within the first 6 days on a deficient diet. The expression of DMT1 (non‐IRE) and the basolateral transporter Ireg1 increased to a lesser extent, but the expression of hephaestin, which is also involved in basolateral iron transport, remained unchanged. The changes in intestinal iron transporter expression parallel the observed increase in iron absorption.Conclusions These results demonstrate that the expression of duodenal iron transport genes can be influenced by the amount of iron in the diet before body iron stores and red blood cell production are affected. Further studies will be required to determine whether the mechanism involves local changes in intestinal iron status or involves a systemic signal that is responsive to dietary iron content.