Iron Imports. V. Transport of iron through the intestinal epithelium

Iron absorption across the brush-border membrane requires divalent metal transporter 1 (DMT1), whereas ferroportin (FPN) and hephaestin are required for exit across the basolateral membrane. However, how iron passes across the enterocyte is poorly understood. Both chaperones and transcytosis have been postulated to account for intracellular iron transport. With iron feeding, DMT1 undergoes endocytosis and FPN translocates from the apical cytosol to the basolateral membrane. The fluorescent metallosensor calcein offered to the basolateral surface of enterocytes is found in endosomes in the apical compartment, and its fluorescence is quenched when iron is offered to the apical surface. These experiments are consistent with vesicular iron transport as a possible pathway for intracellular iron transport.     

Different responses of Fe transporters in Caco-2/HT29-MTX cocultures than in independent Caco-2 cell cultures

The human intestinal epithelium is composed of several cell types, mainly enterocytes and goblet (mucin-secreting) cells. This study compares the cellular response of Fe transporters in Caco-2, HT29-MTX, and Caco-2/HT29-MTX co-culture models for Fe bioavailability. Caco-2 cells in vitro differentiate into enterocyte-like cells and HT29-MTX cell lineage into a mucin-secreting cellular population. Cell cultures were exposed to digests of Fe⁺³, Fe⁺³/ascorbic acid, cooked fish (high-available Fe) or white beans (low-available Fe). Cell responses as shown by mRNA expression of the main Fe transporters, DMT1 and DcytB, and cell ferritin formation were monitored. In Caco-2/HT29-MTX co-cultures, the mucin layer lowered the pool of free Fe to diffuse towards the cell brush border membrane of enterocytes, which was accompanied of an upregulation of DMT1 mRNA expression. In contrast, cultures exposed to digests of fish or white beans showed no significant differences in the regulation of Fe transporters.     

Iron uptake and Nramp2/DMT1/DCT1 in human bronchial epithelial cells

The capacity of natural resistance-associated macrophage protein-2 [Nramp2; also called divalent metal transporter-1 (DMT1) and divalent cation transporter-1 (DCT1)] to transport iron and its ubiquitous expression make it a likely candidate for transferrin-independent uptake of iron in peripheral tissues. We tested the hypothesis that non-transferrin-bound iron uptake by airway epithelial cells is associated with Nramp2/DMT1/DCT1 and that exposure to iron can increase Nramp2/DMT1/DCT1 mRNA and protein expression and transport of this metal. Exposure of BEAS-2B cells to ferric ammonium citrate (FAC) resulted in a decrease in Fe(3+) concentration in the supernatant that was dependent on time and initial iron concentration. In the presence of internalized calcein, FAC quenched the fluorescent signal, indicating intracellular transport of the metal. The Nramp2/DMT1/DCT1 mRNA isoform without an iron-response element (IRE) increased with exposure of BEAS-2B cells to FAC. RT-PCR demonstrated no change in the mRNA for the isoform with an IRE. Similarly, Western blot analysis for the isoform without an IRE confirmed an increased expression of this protein after FAC exposure, whereas the isoform with an IRE exhibited no change. Finally, immunohistochemistry revealed an increase in the isoform without an IRE in the rat lung epithelium after instillation of FAC. Comparable to mRNA and protein increases, iron transport was elevated after pretreatment of BEAS-2B cells with iron-containing compounds. We conclude that airway epithelial cells increase mRNA and expression of the Nramp2/DMT1/DCT1 without an IRE after exposure to iron. The increase results in an elevated transport of iron and its probable detoxification by these cells.     

Iron absorption by Belgrade rat pups during lactation

Divalent metal transporter-1 (DMT1) mediates dietary nonheme iron absorption. Belgrade (b) rats have defective iron metabolism due to a mutation in the DMT1 gene. To examine the role of DMT1 in neonatal iron assimilation, b/b and b/+ pups were cross-fostered to F344 Fischer dams injected with (59)FeCl(3) twice weekly during lactation. Tissue distribution of the radioisotope in the pups was determined at weaning (day 21). The b/b pups had blood (59)Fe levels significantly lower than b/+ controls but significantly higher (59)Fe tissue levels in heart, bone marrow, skeletal muscle, kidney, liver, spleen, stomach, and intestines. To study the pharmacokinetics of nonheme iron absorption at the time of weaning, (59)FeCl(3) was administered to 21-day-old b/b and b/+ rats by intragastric gavage. Blood (59)Fe levels measured 5 min to 4 h postgavage were significantly lower in b/b rats, consistent with impaired DMT1 function in intestinal iron absorption. Tissue (59)Fe levels were also lower in b/b rats postgavage. Combined, these data suggest that DMT1 function is not essential for iron assimilation from milk during early development in the rat.     

Iron and cadmium uptake by duodenum of hypotransferrinaemic mice

Absorption from food is an important route for entry of the toxic metal, cadmium, into the body. Both cadmium and iron are believed to be taken up by duodenal enterocytes via the iron regulated, proton-coupled transporter, DMT1. This means that cadmium uptake could be enhanced in conditions where iron absorption is increased. We measured pH dependent uptake of ¹⁰⁹Cd and ⁵⁹Fe by duodenum from mice with an in vitro method. Mice with experimental (hypoxia, iron deficiency) or hereditary (hypotransferrinaemia) increased iron absorption were studied. All three groups of mice showed increased ⁵⁹Fe uptake (p<0.05) compared to their respective controls. Hypotransferrinaemic and iron deficient mice exhibited an increase in ¹⁰⁹Cd uptake (p<0.05). Cadmium uptake was not, however, increased by lowering the medium pH from 7.4 to 6. In contrast, ⁵⁹Fe uptake (from ⁵⁹FeNTA₂) and ferric reductase activity was increased by lowering medium pH in control and iron deficient mice (p<0.05). The data show that duodenal cadmium uptake can be increased by hereditary iron overload conditions. The uptake is not, however, altered by lowering medium pH suggesting that DMT1-independent uptake pathways may operate.     

Transport of divalent transition-metal ions is lost in small-intestinal tissue of b/b Belgrade rats

Belgrade rats exhibit microcytic, hypochromic anemia and systemic iron deficiency due to a glycine-to-arginine mutation at residue 185 in a metal ion transporter of a divalent metal transporter/divalent cation transporter/solute carrier 11 group A member 2 or 3 (DMT1/DCT1/SLC11A2), a member of the natural-resistance-associated macrophage protein (Nramp) family. By use of rabbit duodenal tissue, a calcein fluorescence assay has previously been developed to assess transport of divalent metal ions across the small-intestinal brush border membrane (BBM). The assay was readily applied here to rat BBM to learn if it detects DMT1 activity. The results demonstrate protein-mediated transport across the BBM of all tested ions: Mn(2+), Fe(2+), and Ni(2+). Transport into BBM vesicles (BBMV) from (b/b) Belgrade rats was below the detection limit. BBMV of +/b origin had substantial activity. The kinetic rate constant for Ni(2+) membrane transport for +/b BBMV was within the range for normal rabbit tissue. Vesicles from +/b basolateral membranes (BLM) showed similar activity to BBMV while b/b BLM vesicles (BLMV) lacked transport activity. Immunoblots using isoform-specific antibodies demonstrated that intestinal levels of b/b DMT1 were increased compared to +/b DMT1, reflecting iron deficiency. Immunoblots on BBMV indicated that lack of activity in b/b vesicles was not due to a failure of DMT1 to localize to the BBMV; an excess of specific isoforms was present compared to +/b BBMV or duodenal extracts. Immunoblots from BLMV also exhibited enrichment in DMT1 isoforms, despite their distinct origin. Immunofluorescent staining of thin sections of b/b and +/b proximal intestines confirmed that DMT1 localized similarly in mutant and control enterocytes and showed that DMT1 isoforms have distinct distributions within intestinal tissue.     

Enhanced autophagy from chronic toxicity of iron and mutant A53T α-synuclein: implications for neuronal cell death in Parkinson disease

Parkinson disease (PD), a prevalent neurodegenerative motor disorder, is characterized by the rather selective loss of dopaminergic neurons and the presence of α-synuclein-enriched Lewy body inclusions in the substantia nigra of the midbrain. Although the etiology of PD remains incompletely understood, emerging evidence suggests that dysregulated iron homeostasis may be involved. Notably, nigral dopaminergic neurons are enriched in iron, the uptake of which is facilitated by the divalent metal ion transporter DMT1. To clarify the role of iron in PD, we generated SH-SY5Y cells stably expressing DMT1 either singly or in combination with wild type or mutant α-synuclein. We found that DMT1 overexpression dramatically enhances Fe(2+) uptake, which concomitantly promotes cell death. This Fe(2+)-mediated toxicity is aggravated by the presence of mutant α-synuclein expression, resulting in increased oxidative stress and DNA damage. Curiously, Fe(2+)-mediated cell death does not appear to involve apoptosis. Instead, the phenomenon seems to occur as a result of excessive autophagic activity. Accordingly, pharmacological inhibition of autophagy reverses cell death mediated by Fe(2+) overloading. Taken together, our results suggest a role for iron in PD pathogenesis and provide a mechanism underlying Fe(2+)-mediated cell death.     

The effects of pH and the iron redox state on iron uptake in the intestine of a marine teleost fish, gulf toadfish (Opsanus beta)

In the marine teleost intestine the secretion of bicarbonate increases pH of the lumen (pH 8.4 -9.0) and importantly reduces Ca2+ and Mg2+ concentrations by the formation of insoluble divalent ion carbonates. The alkaline intestinal environment could potentially also cause essential metal carbonate formation reducing bioavailability. Iron accumulation was assessed in the Gulf toadfish (Opsanus beta) gut by mounting intestine segments in modified Ussing chambers fitted to a pH-stat titration system. This system titrates to maintain lumen pH constant and in the process prevents bicarbonate accumulation. The luminal saline pH was clamped to pH 5.5 or 7.0 to investigate the effect of proton concentrations on iron uptake. In addition, redox state was altered (gassing with N2, addition of dithiothreitol (DTT) and ascorbate) to evaluate Fe3+ versus Fe2+ uptake, enabling us to compare a marine teleost intestine model for iron uptake to the mammalian system for non-haem bound iron uptake that occurs via a ferrous/proton (Fe2+/H+) symporter called Divalent Metal Transporter 1 (DMT1). None of the redox altering strategies affected iron (Fe3+ or Fe2+) binding to mucus, but the addition of ascorbate resulted in a 4.6-fold increase in epithelium iron accumulation. This indicates that mucus iron binding is irrespective of valency and suggests that ferrous iron is preferentially transported across the apical surface. Altering luminal saline pH from 7.0 to 5.5 did not affect ferric or ferrous iron uptake, suggesting that if iron is entering via DMT1 in marine fish intestine this transporter works efficiently under circumneutral conditions.     

Cadmium transport in isolated enterocytes of freshwater rainbow trout: interactions with zinc and iron, effects of complexation with cysteine, and an ATPase-coupled efflux

The present study investigated the mechanisms of intestinal cadmium (Cd) uptake and efflux, using isolated enterocytes of freshwater rainbow trout (Oncorhynchus mykiss) as the experimental model. The apical uptake of free Cd(2+) in the enterocytes was a saturable and high-affinity transport process. Both zinc (Zn(2+)) and iron (Fe(2+)) inhibited cellular Cd(2+) uptake through a competitive interaction, suggesting that Cd(2+) enters enterocytes via both Zn(2+) (e.g., ZIP8) and Fe(2+) (e.g., DMT1) transport pathways. Cellular Cd(2+) uptake increased in the presence of HCO(3)(-), which resembled the function of mammalian ZIP8. Cellular Cd(2+) uptake was unaffected by Ca(2+), indicating that Cd(2+) does not compete with Ca(2+) for apical uptake. Interestingly, Cd uptake was influenced by the presence of l-cysteine, and under the exposure condition where Cd(Cys)(+) was the predominant Cd species, cellular Cd uptake rate increased with the increased concentration of Cd(Cys)(+). The kinetic analysis indicated that the uptake of Cd(Cys)(+) occurs through a low capacity transport mechanism relative to that of free Cd(2+). In addition, Cd efflux from the enterocytes decreased in the presence of an ATPase inhibitor (orthovanadate), suggesting the existence of an ATPase-coupled extrusion process. Overall, our findings provide new mechanistic insights into the intestinal Cd transport in freshwater fish.     

Gene expression of divalent metal transporter 1 and transferrin receptor in duodenum of Belgrade rats

Regulation of iron absorption is thought to be mediated by the amount of iron taken up by duodenal crypt cells via the transferrin receptor (TfR)-transferrin cycle and the activity of the divalent metal transporter (DMT1), although DMT1 cannot be detected morphologically in crypt cells. We investigated the uptake of transferrin-bound iron by duodenal enterocytes in Wistar rats fed different levels of iron and Belgrade (b/b) rats in which iron uptake by the transferrin cycle is defective because of a mutation in DMT1. We showed that DMT1 in our colony of b/b rats contains the G185R mutation, which in enterocytes results in reduced cellular iron content and increased DMT1 gene expression similar to levels in iron deficiency of normal rats. In all groups the uptake of transferrin-bound iron by crypt cells was directly proportional to plasma iron concentration, being highest in iron-loaded Wistar rats and b/b rats. We conclude that the uptake of transferrin-bound iron by developing enterocytes is largely independent of DMT1.     

Rapid regulation of divalent metal transporter (DMT1) protein but not mRNA expression by non-haem iron in human intestinal Caco-2 cells.

A divalent metal transporter, DMT1, located on the apical membrane of intestinal enterocytes is the major pathway for the absorption of dietary non-haem iron. Using human intestinal Caco-2 TC7 cells, we have shown that iron uptake and DMT1 protein in the plasma membrane were significantly decreased by exposure to high iron for 24 h, in a concentration-dependent manner, whereas whole cell DMT1 protein abundance was unaltered. This suggests that part of the response to high iron involved redistribution of DMT1 between the cytosol and cell membrane. These events preceded changes in DMT1 mRNA, which was only decreased following 72 h exposure to high iron.     

Iron regulatory protein as an endogenous sensor of iron in rat intestinal mucosa. Possible implications for the regulation of iron absorption.

Duodenal enterocytes adjust intestinal iron absorption to the body's state of iron repletion. Here we tested how iron supply from the blood modulates the RNA-binding activity of iron regulatory proteins (IRP-1 and IRP-2) in immature duodenal rat enterocytes, and whether the modulation is compatible with the hypothesis that IRPs, in turn, may regulate the expression of iron transport proteins in maturating enterocytes during migration to the villus tips. Tissue uptake of parenterally applied 59Fe along the duodenal crypt-villus axis was compared to local IRP-1 and IRP-2 activity and to duodenal 59Fe transport capacity 12 h, 48 h, and 72 h after intravenous iron administration to iron-deficient rats. IRP-1 and IRP-2 activity was significantly increased in iron-deficiency. 59Fe administrated from the blood side was almost exclusively taken up by crypt enterocytes. Accordingly, the activity of IRP-1 decreased at this site 12 h after parenteral iron administration, but remained high at the villus tips. After 48 h the bulk of 59Fe containing enterocytes had migrated to the villus tips. Correspondingly, IRP-1 activity was decreased at duodenal villus tips after 48 h. IRP-2 activity also tended to decrease, though the change was statistically not significant. IRP-2 activity remained significantly higher at duodenal villus tips than in crypts, even after 72 h. Intestinal iron absorption capacity decreased with the same delay as IRP-1 activity after intravenous iron administration. In the ileum 59Fe uptake from the blood and IRP activity showed no significant difference between crypt and villus region. Luminal administration of iron decreased duodenal IRP-1 and IRP-2 activity at tips and crypts within 2 h. Thus, recently absorbed iron becomes available to cytosolic IRP during its passage through the enterocyte. Our results are compatible with a role of IRPs in gearing the expression of intestinal iron transporters in the duodenal brushborder to the body's state of iron repletion.     

Genotype and allele frequencies of divalent metal transporter 1 polymorphism in Turkish population

Divalent metal transporter 1 (DMT1 also known as DCT1, NRAMP2 or SCL11A2) is a membrane-bound divalent metal transporter which is conserved from prokaryotes to higher eukaryotes. It has been postulated to play important roles in intestinal iron absorption at the brush border of duodenal enterocytes, erythroid iron utilization, hepatic iron accumulation, placental iron transfer, and other processes. DMT1 gene which contains at least four isoforms (1A/+IRE, 1A/-IRE, 2/+IRE and 2/-IRE) is located on chromosome 12q13 in human. DMT1 mediates the transport of a wide range of metals, including the essential metals Fe2+, Zn2+, Mn2+, Cu2+, Co2+, Ni2+ and toxic metals such as Cd2+ and Pb2+. The intention of this study is to determine that IVS4+44C/A single nucleotide polymorphism in DMT1 gene of Turkish population. For this purpose blood samples from 192 female and 192 male volunteers were analyzed. DMT1 gene was amplified with the polymerase chain reaction-restriction fragment length polymorphism technique and 351 bp oligonucleotide was produced. The amplified oligonucleotides were cut with MnlI restriction enzyme according to their polymorphic characteristics. Digested and undigested products were separated on a 2% agarose gel electrophoresis, visualized by ethidium bromide staining under an ultraviolet illuminator. The genotype frequencies of DMT1 IVS4+44C/A polymorphism were determined as 47.9% for CC, 40.1% for AC and 12.0% for AA genotypes. The frequency of the C allele was found to as 68.0% and of the A allele as 32.0%. The genotype frequencies were consistent with Hardy-Weinberg equilibrium (χ2=2.394; Exact P=0.128).     

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.     

Distinct targeting and recycling properties of two isoforms of the iron transporter DMT1 (NRAMP2, Slc11A2)

The metal transporter DMT1 (Slc11a2) plays a vital role in iron metabolism. Alternative splicing of the 3' exon generates two DMT1 isoforms with different C-terminal protein sequences and a 3' untranslated region harboring (isoform I, +IRE) or not (isoform II, -IRE), an iron-responsive element. Isoform I is expressed at the plasma membrane of certain epithelial cells including the duodenum brush border, where it is essential for the absorption of nutritional iron. Isoform II is expressed in many cells and is essential for the acquisiton of transferrin iron from acidified endosomes. The targeting and trafficking properties of DMT1 isoforms I and II were studied in transfected LLC-PK(1) kidney cells, with respect to isoform-specific differences in function, subcellular localization, endocytosis kinetics, and fate upon internalization. Isoform I showed higher surface expression and was internalized from the plasma membrane with slower kinetics than that of isoform II. As opposed to isoform II, which is efficiently sorted to recycling endosomes upon internalization, isoform I was not efficiently recycled and was targeted to lysosomes. Thus, alternative splicing of DMT1 critically regulates the subcellular localization and site of Fe(2+) transport.     

Effect of iron status on DMT1 expression in duodenal enterocytes from beta2-microglobulin knockout mice

Divalent metal transporter I (DMT1) is thought to be involved in transport of iron across the apical cell membrane of villus duodenal cells. To determine its role in hereditary hemochromatosis (HH), we used beta2-microglobulin knockout (B2M-/-) mice that accumulate iron as in HH. The B2M-/- and control C57BL/6 (B2M+/+) mice were fed diets with different iron contents. Increasing the iron availability increased plasma iron levels in both B2M+/+ and B2M-/- mice. Reducing the iron availability decreased the plasma iron concentration in B2M+/+ mice but was without effect on plasma iron in B2M-/- mice. DMT1 was not detectable in mice fed normal or iron-loaded diets when using immunohistochemistry. In Western blots, however, the protein was consistently observed regardless of the dietary regimen. DMT1 expression was increased to the same extent in B2M+/+ and B2M-/- mice when fed an iron-poor diet. In both strains of mice fed an iron-poor diet, DMT1 was evenly distributed in the differentiated enterocytes from the base to the tip of the villi but was absent from the crypts of Lieberkühn. These data suggest that the observed effects were due to the state of iron deficiency in mucosal cells rather than genetic defect.     

Divalent Metal Transporter 1 Expression and Regulation in Human Placenta

Divalent metal transporter 1 (DMT1) is likely responsible for the release of iron from endosomes to the cytoplasm in placental syncytiotrophoblasts (STB). To determine the localization and the regulation of DMT1 expression by iron directly in placenta, the expression of DMT1 in human term placental tissues and BeWo cells (human placental choriocarcinoma cell line) was detected and the change in expression in response to different iron treatments on BeWo cells was observed. DMT1 was shown to be most prominent near the maternal side in human term placenta and predominantly in the cytoplasm of BeWo cells. BeWo cells were treated with desferrioxamine (DFO) and human holotransferrin (hTf-2Fe) and it was found that both DMT1 mRNA and protein increased significantly with DFO treatment and decreased with hTf-2Fe treatment. Further, DMT1 mRNA responded more significantly to treatments if it possessed an iron-responsive element than mRNA without this element. This study indicated that DMT1 is likely involved in endosomal iron transport in placental STB and placental DMT1 + IRE expression was primarily regulated by the IRE/IRP mechanism.