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Hepcidin is a liver‐derived peptide hormone that limits iron egress from tissues to the bloodstream. It operates by binding to the iron exporter ferroportin, which blocks iron transport and tags ferroportin for degradation. Genetic hepcidin inactivation leads to hereditary hemochromatosis, a disease of iron overload. We used wild‐type and Hjv‐/‐ mice, a model of hemochromatosis, to examine the expression of ferroportin and other proteins of iron metabolism in hepcidin target tissues. The animals were previously subjected to dietary iron manipulations. In Hjv‐/‐ mice, hepcidin messenger RNA correlated significantly with hepatic iron load (r = 0.8211, P, Ferroportin is differentially regulated by dietary iron in the spleen, liver and duodenum of wild type and Hjv‐/‐ mice.Overexpressed duodenal ferroportin does not respond to dietary iron in Hjv‐/‐ mice.Duodenal DMT1 is appropriately iron‐regulated in both wild‐type and Hjv‐/‐ mice.

The liver hormone hepcidin regulates systemic iron homeostasis. Hepcidin is also expressed by the kidney, but exclusively in distal nephron segments. Several studies suggest hepcidin protects against kidney damage involving Fe2+ overload. The nephrotoxic non-essential metal ion Cd2+ can displace Fe2+ from cellular biomolecules, causing oxidative stress and cell death. The role of hepcidin in Fe2+ and Cd2+ toxicity was assessed in mouse renal cortical [mCCD(cl.1)] and inner medullary [mIMCD3] collecting duct cell lines. Cells were exposed to equipotent Cd2+ (0.5–5 μmol/l) and/or Fe2+ (50–100 μmol/l) for 4–24 h. Hepcidin (Hamp1) was transiently silenced by RNAi or overexpressed by plasmid transfection. Hepcidin or catalase expression were evaluated by RT-PCR, qPCR, immunoblotting or immunofluorescence microscopy, and cell fate by MTT, apoptosis and necrosis assays. Reactive oxygen species (ROS) were detected using CellROX™ Green and catalase activity by fluorometry. Hepcidin upregulation protected against Fe2+-induced mIMCD3 cell death by increasing catalase activity and reducing ROS, but exacerbated Cd2+-induced catalase dysfunction, increasing ROS and cell death. Opposite effects were observed with Hamp1 siRNA. Similar to Hamp1 silencing, increased intracellular Fe2+ prevented Cd2+ damage, ROS formation and catalase disruption whereas chelation of intracellular Fe2+ with desferrioxamine augmented Cd2+ damage, corresponding to hepcidin upregulation. Comparable effects were observed in mCCD(cl.1) cells, indicating equivalent functions of renal hepcidin in different collecting duct segments. In conclusion, hepcidin likely binds Fe2+, but not Cd2+. Because Fe2+ and Cd2+ compete for functional binding sites in proteins, hepcidin affects their free metal ion pools and differentially impacts downstream processes and cell fate.

The SARS-CoV-2 virus that is the cause of coronavirus disease 2019 (COVID-19) affects not only peripheral organs such as the lungs and blood vessels, but also the central nervous system (CNS)—as seen by effects on smell, taste, seizures, stroke, neuropathological findings and possibly, loss of control of respiration resulting in silent hypoxemia. COVID-19 induces an inflammatory response and, in severe cases, a cytokine storm that can damage the CNS. Antimalarials have unique properties that distinguish them from other anti-inflammatory drugs. (A) They are very lipophilic, which enhances their ability to cross the blood-brain barrier (BBB). Hence, they have the potential to act not only in the periphery but also in the CNS, and could be a useful addition to our limited armamentarium against the SARS-CoV-2 virus. (B) They are non-selective inhibitors of phospholipase A2 isoforms, including cytosolic phospholipase A2 (cPLA2). The latter is not only activated by cytokines but itself generates arachidonic acid, which is metabolized by cyclooxygenase (COX) to pro-inflammatory eicosanoids. Free radicals are produced in this process, which can lead to oxidative damage to the CNS. There are at least 4 ways that antimalarials could be useful in combating COVID-19. (1) They inhibit PLA2. (2) They are basic molecules capable of affecting the pH of lysosomes and inhibiting the activity of lysosomal enzymes. (3) They may affect the expression and Fe2+/H+ symporter activity of iron transporters such as divalent metal transporter 1 (DMT1), hence reducing iron accumulation in tissues and iron-catalysed free radical formation. (4) They could affect viral replication. The latter may be related to their effect on inhibition of PLA2 isoforms. Inhibition of cPLA2 impairs an early step of coronavirus replication in cell culture. In addition, a secretory PLA2 (sPLA2) isoform, PLA2G2D, has been shown to be essential for the lethality of SARS-CoV in mice. It is important to take note of what ongoing clinical trials on chloroquine and hydroxychloroquine can eventually tell us about the use of antimalarials and other anti-inflammatory agents, not only for the treatment of COVID-19, but also for neurovascular disorders such as stroke and vascular dementia.

Objective Our previous research showed that Naotaifang (a compound traditional Chinese herbal medicine) extract (NTE) has clinically beneficial effects on neurological improvement of patients with acute cerebral ischemia. In this study, we investigated whether NTE protected acute brain injury in rats and whether its effects on ferroptosis could be linked to the dysfunction of glutathione peroxidase 4 (GPX4) and iron metabolism. Methods We established an acute brain injury model of middle cerebral artery occlusion (MCAO) in rats, in which we could observe the accumulation of iron in neurons, as detected by Perl’s staining. Using assay kits, we measured expression levels of ferroptosis biomarkers, such as iron, glutathione (GSH), reactive oxygen species (ROS) and malonaldehyde (MDA); further the expression levels of transferrin receptor 1 (TFR1), divalent metal transporter 1 (DMT1), solute carrier family 7 member 11 (SLC7A11) and GPX4 were determined using immunohistochemical analysis, real-time quantitative polymerase chain reaction and Western blot assays. Results We found that treatment with NTE reduced the expression levels of TFR1 and DMT1, reduced ROS, MDA and iron accumulation and reduced neurobehavioral scores, relative to untreated MCAO rats. Treatment with NTE increased the expression levels of SLC7A11, GPX4 and GSH, and the number of Nissl bodies in the MCAO rats. Conclusion Taken together, our data suggest that acute cerebral ischemia induces neuronal ferroptosis and the effects of treating MCAO rats with NTE involved inhibition of ferroptosis through the TFR1/DMT1 and SCL7A11/GPX4 pathways.

Background Hepcidin is a key regulator of iron homeostasis. Some studies showed that exogenous hepcidin decreased the expression of divalent metal transporter (DMT1) rather than ferroportin(FPN1) to regulate renal iron metabolism. This study explored the effects of hepcidin synthesized by the kidney and its mechanism of iron regulation. Methods In the in vivo experiments, mice were divided into a unilateral ureter obstruction (UUO) model group and a sham operation group, and mice in the UUO model group were sacrificed on days 1, 3, 5 and 7. The expression of renal hepcidin, FPN1, DMT1 and the retention of renal iron were studied. In the in vitro experiments, we overexpressed hepcidin in HK-2 cells. Then we tested the expression of renal hepcidin, FPN1, DMT1 and observed the production of intracellular ferrous ions. Results Renal hepcidin expression was consistently higher in the UUO group than in the sham group from the first day. The expression of FPN1 gradually decreased, and the expression of DMT1 gradually increased in the UUO model. Intracellular ferrous ions significantly increased on the first day of the UUO model. In hepcidin overexpressed HK-2 cells, the expression of FPN1 was decreased, while the expression of DMT1 has no significant change. In addition, production of intracellular ferrous ions increased. Conclusion: local hepcidin can regulate iron metabolism in the kidney by adjusting the expression of FPN1.

Iron deficiency anemia (IDA) is the most common nutrient-dependent health problem in the world and could be reversed by commercially available iron supplementation. The form of iron supplement is important due to its toxicity on the gastrointestinal system (GI), so the development of new dietary strategies might be important for the prevention of IDA. It has been shown that plant-based proteins bind to iron and might decrease the free form of iron before absorption and increase iron bioavailability. Thus, we aimed to form lentil derived protein-iron complexes and to test the functional properties of hydrolysed protein-iron complexes in anemic Caco-2 cell line. Our main findings were that (i) lentil derived proteins had the capacity to chelate iron minerals and (ii) hydrolysed protein-iron complexes significantly reduced the mRNA levels of iron regulated divalent metal transporter-1 (DMT1), transferrin receptor (TFR), and ankyrin repeat domain 37 (ANKRD37) marker genes that were induced by iron deficiency anemia. The current findings suggest that hydrolysed protein-iron complexes might have functional properties in iron deficiency anemia in vitro. Further in vivo studies are necessary to show lentil derived proteins and iron might be used as supplements or food additives to reduce the risk of iron deficiency anemia.

We investigated the mechanism underlying intestinal cadmium (Cd) uptake based on the mediators (metal transporters) of essential elements, such as Fe, Zn, Cu, and Ca, under normal conditions in female rats. These elements interact with Cd uptake from the intestinal tract. Cd concentration at each site of the small intestine (duodenum, jejunum, and ileum) increased as Cd exposure increased. However, Cd concentration was the highest in the duodenum. The gene expression of ZIP14, DMT1, and ATP7A increased with increase in Cd concentration. Further, Cu concentration decreased as Cd concentration increased. In contrast, Fe concentration displayed a decreasing tendency with the increase in Cd concentration. The gene expression levels of ZIP14, DMT1, and ATP7A were positively correlated with Cd concentration. Immunohistochemical staining revealed the positive sites of ZIP14 and DMT1 scattered in the area adjacent to the goblet cells, resorbable epithelial cells, and lamina propria in the duodenum tissue, according to the increase in Cd concentration. Cd is induced to synthesize and bind to metallothionein (MT-I and -II) and accumulate in the intestinal tissues, mainly in the duodenum. Such findings suggest that Cd, a contaminant element, is taken up from the intestinal tract by multiple metal transporters such as Cu, Fe, and Zn, thereby involving in the intestinal Cd absorption.

Aluminum (Al) alters iron regulatory factors content and leads to the changes in iron-related proteins causing iron accumulation. But limited evidence ascertains this hypothesis. Therefore, our experiment was conducted and four groups of male Wistar rats were orally administrated of 0, 50, 150, and 450 mg/kg BW/d aluminum chloride (AlCl3) for 90 days by drinking water, respectively. The cognitive function, pathological lesion of hippocampus, oxidative stress, as well as iron-related proteins and iron regulatory factors expression were detected. The results showed that AlCl3 remarkably induced the oxidative stress and pathological lesion in the hippocampus and impaired the learning-memory ability. The contents of Al and iron increased in all AlCl3-exposed groups. Meanwhile, the increased divalent metal transporter 1 (DMT1) expression enhanced iron import and the decreased ferroportin 1 (Fpn1) expression reduced iron export in AlCl3-exposed groups. The iron accumulated and ferritin heavy chains (Fth) expression decreased in all AlCl3-exposed groups led to an increase in free iron. The study also showed that iron regulatory factor iron regulatory protein 2 (IRP2) was decreased and hepcidin was increased in AlCl3-exposed groups. The results indicated that AlCl3 induces iron dyshomeostasis presenting as iron accumulation, the disordered expression of iron import, export, store, and regulatory proteins in rat hippocampus accompanied with oxidative stress, pathological lesion, and impaired learning-memory ability.

Manganese supports numerous neuronal functions but in excess is neurotoxic. Consequently, regulation of manganese flux at the blood–brain barrier (BBB) is critical to brain homeostasis. However, the molecular pathways supporting the transcellular trafficking of divalent manganese ions within the microvascular capillary endothelial cells (BMVECs) that constitute the BBB have not been examined. In this study, we have determined that ZIP8 and ZIP14 (Zrt- and Irt-like proteins 8 and 14) support Mn(2+) uptake by BMVECs and that neither DMT1 nor an endocytosis-dependent pathway play any significant role in Mn(2+) uptake. Specifically, siRNA-mediated knockdown of ZIP8 and ZIP14 coincided with a decrease in manganese uptake, and kinetic analyses revealed that manganese uptake depends on pH and bicarbonate and is up-regulated by lipopolysaccharide, all biochemical markers of ZIP8 or ZIP14 activity. Mn(2+) uptake also was associated with cell-surface membrane presentation of ZIP8 and ZIP14, as indicated by membrane protein biotinylation. Importantly, surface ZIP8 and ZIP14 biotinylation and Mn(2+)-uptake experiments together revealed that these transporters support manganese uptake at both the apical, blood and basal, brain sides of BMVECs. This indicated that in the BMVECs of the BBB, these two transporters support a bidirectional Mn(2+) flux. We conclude that BMVECs play a critical role in controlling manganese homeostasis in the brain.

The aim of this study was to determine the iron (Fe) concentration profile within the lumen of the S2 renal proximal convoluted tubule (PCT) and to resolve whether this nephron segment transported Fe. To do this, we performed in vivo renal micropuncture on Wistar rats, collected PCT tubular fluid from superficial nephrons, and measured Fe concentration. The Fe concentration profile along the S2 PCT suggested significant Fe reabsorption. Proximal tubules were also microperfused in vivo with physiological solutions containing Fe and Zn, Cu, Mn, or Cd. PCTs perfused with 12μmol.l−1 55FeCl3 reabsorbed 105.2±12.7 fmol.mm−1.min−1 Fe, 435±52pmol.mm-1.min−1 Na, and 2.7±0.2nl.mm−1.min−1 water (mean ± SEM; n=19). Addition of ascorbate (1mmol.l−1) to the perfusate did not significantly alter Fe, Na, or water reabsorption. Supplementing the control perfusate with 60μmol.l−1 FeSO4 significantly decreased 55Fe uptake. Recalculating for the altered molar activity following addition of unlabeled Fe revealed a three-fold increase in Fe flux. Addition to the perfusate 12μmol.l−1 CuSO4, MnSO4, CdSO4, or ZnSO4 did not affect Fe, Na, or water flux. In conclusion, (1) in vivo, S2 PCTs of rat reabsorb Fe and (2) Fe is reabsorbed along the PCT via a pathway that is insensitive to Cu, Mn, Cd, or Zn. Together, these data demonstrate for the first time the hitherto speculated process of renal Fe filtration and subsequent tubular Fe reabsorption in a living mammal.

An experiment was conducted to investigate the effect of organic and inorganic Fe sources on Fe absorption and expression of related transporters in the small intestine of broilers. Iron-deficient intact broilers (7-day-old) were fed an Fe-unsupplemented corn-soybean meal basal diet or the basal diet supplemented with 60 mg Fe/kg as Fe sulfate (FeSO4•7H2O), Fe-Met with weak chelation strength (Fe-Met W), Fe-proteinate with moderate chelation strength (Fe-Prot M) or Fe-proteinate with extremely strong chelation strength (Fe-Prot ES) for 14 d. The plasma Fe contents were enhanced (P < 0.02) by Fe addition, and greater (P < 0.0002) in Fe-Prot M and Fe-Prot ES groups than in Fe-Met W and FeSO4 groups. Supplemental Fe decreased (P < 0.03) the divalent metal transporter 1 (DMT1) mRNA levels in the duodenum and jejunum, and ferroportin 1 (FPN1) mRNA levels in the duodenum on d 21, but no differences (P > 0.20) were detected among different Fe sources. Regardless of Fe source, the mRNA levels of DMT1 and FPN1 were higher (P < 0.02) in the duodenum than in the jejunum and ileum, and in the jejunum than in the ileum (P < 0.05). However, Fe addition did not affect (P > 0.10) the mRNA levels of amino acid transporters and protein levels of DMT1 and FPN1 in the small intestine of broilers. These results indicate that organic Fe sources with stronger chelation strength showed higher Fe absorption in broilers in vivo; the mRNA expression of Fe and amino acid transporters varied along with the extension of the small intestine; the absorption of Fe as organic Fe chelates was not mediated by the amino acid transporters in intact chicks in this study.

Recent researches suggested that iron dysregulation play an important role in the pathogenesis of vascular dementia (VD). Iron deposition had been found in hippocampus in vascular dementia model in recent research. Nevertheless, the underlying mechanisms of iron deposition and its neurotoxicity in vascular dementia was still unclear. Thus, our research was aimed at whether the neurotoxicity of iron was associated with autophagy regulation. We established a chronic cerebral hypoperfusion model in the rat brain in order to mimic the vascular dementia using permanent bilateral common carotid artery occlusion (2VO). The preparation of iron overloaded rats model by intraperitoneal injection of iron dextran. Following, we tested the learning and memory function of each group using Morris Water Maze. Consequently, we analyzed the iron content and iron transport related molecules (TFR1, DMT1) in hippocampus. Furthermore, we examined the effect of iron deposition on autophagy-related molecules including AMPK, Beclin1 and LC3 and the number of autophagosomes in hippocampus. Last, we tested the apoptosis of neurons in hippocampus. We found that iron deposition in hippocampus in model groups which accompanied the decline of learning and memory function. And the expression of TFR1 and DMT1 were up-regulated in model groups. Moreover, iron deposition up-regulated the expression of AMPK, Beclin1 and LC3 and increase the number of autophagosomes in hippocampus. And the expression of Bax was up-regulated and Bcl-2 was down-regulated in iron deposition groups. To sum up, our data suggested that iron deposition increased AMPK/autophagy pathway associated molecules in the hippocampus and promoted neuronal apoptosis, which might be a new pathogenesis in vascular dementia.

Iron is an essential cofactor for many cellular enzymes involved in myelin synthesis, and iron homeostasis unbalance is a central component of peripheral neuropathies. However, iron absorption and management in the PNS are poorly understood. To study iron metabolism in Schwann cells (SCs), we have created 3 inducible conditional KO mice in which three essential proteins implicated in iron uptake and storage, the divalent metal transporter 1 (DMT1), the ferritin heavy chain (Fth), and the transferrin receptor 1 (Tfr1), were postnatally ablated specifically in SCs. Deleting DMT1, Fth, or Tfr1in vitrosignificantly reduce SC proliferation, maturation, and the myelination of DRG axons. This was accompanied by an important reduction in iron incorporation and storage. When these proteins were KOin vivoduring the first postnatal week, the sciatic nerve of all 3 conditional KO animals displayed a significant reduction in the synthesis of myelin proteins and in the percentage of myelinated axons. Knocking out Fth produced the most severe phenotype, followed by DMT1 and, last, Tfr1. Importantly, DMT1 as well as Fth KO mice showed substantial motor coordination deficits. In contrast, deleting these proteins in mature myelinating SCs results in milder phenotypes characterized by small reductions in the percentage of myelinated axons and minor changes in theg-ratio of myelinated axons. These results indicate that DMT1, Fth, and Tfr1 are critical proteins for early postnatal iron uptake and storage in SCs and, as a consequence, for the normal myelination of the PNS.SIGNIFICANCE STATEMENTTo determine the function of the divalent metal transporter 1, the transferrin receptor 1, and the ferritin heavy chain in Schwann cell (SC) maturation and myelination, we created 3 conditional KO mice in which these proteins were postnatally deleted in Sox10-positive SCs. We have established that these proteins are necessary for normal SC iron incorporation and storage, and, as a consequence, for an effective myelination of the PNS. Since iron is indispensable for SC maturation, understanding iron metabolism in SCs is an essential prerequisite for developing therapies for demyelinating diseases in the PNS.

Hinokitiol, a natural lipophilic chelator, appears capable of replacing several iron transporters after they have been genetically ablated. Divalent metal-ion transporter (DMT1) is the major iron importer in enterocytes and erythroblasts. We have compared DMT1 and hinokitiol in multiple fashions to learn if the smaller molecule is a suitable substitute using two HEK293 cell lines engineered to overexpress different isoforms of DMT1. Both the macromolecule and the lipophilic chelator enable import of ferrous ions into HEK293 cells. Hinokitiol also mediates ferric ion import but DMT1 cannot do so. While DMT1 can also import Mn2+ ions, hinokitiol lacks this ability. The Michaelis–Menten analysis for kinetics of macromolecular catalysis is also suitable for hinokitiol-supported iron import. To compare hinokitiol to DMT1 relative to other metal ions that DMT1 can transport, we employed an organic extraction procedure with which we initially matched the results obtained for Fe2+, Fe3+ and Mn2+, and then showed that multiple other cations were unlikely to enter via hinokitiol. The small chelator thus shares some functional properties with DMT1, but distinct difference were also noted. Electronic supplementary material The online version of this article (10.1007/s10534-019-00207-2) contains supplementary material, which is available to authorized users.

To investigate the effects of dietary iron (Fe) levels on manganese (Mn) utilization, 900 8-day-old broilers were randomly assigned to 1 of 6 treatments in a 3 (Fe level) × 2 (Mn level) factorial arrangement after feeding Mn- and Fe-unsupplemented diet for 7 days. The broilers were then fed with basal corn-soybean meal diets (approximately 28 mg Mn/kg and 60 mg Fe/kg) added with 0, 80, or 160 mg/kg Fe (L-Fe, M-Fe, or H-Fe), and 0 or 100 mg/kg Mn for 35 days. Body weight gain was lower for H-Fe broilers than that for L-Fe and M-Fe broilers. On day 42, H-Fe broilers had lower serum Mn concentration as compared with L-Fe and M-Fe broilers, and tibia Mn concentration decreased as dietary Fe increased. In Mn-supplemented broilers, liver Mn was lower in L-Fe and H-Fe treatments than that in M-Fe treatment. H-Fe treatment decreased Mn concentration and manganese-containing superoxide dismutase (MnSOD) activity in the heart when compared with L-Fe and M-Fe treatments. Dietary Fe did not significantly influence Mn concentrations in the liver and heart, and heart MnSOD activity in Mn-unsupplemented broilers. In the duodenum, L-Fe treatment decreased divalent metal transporter 1 (DMT1) mRNA abundance when compared with M-Fe and H-Fe treatments, and ferroportin 1 (FPN1) mRNA level was higher in M-Fe treatment than that in L-Fe and H-Fe treatments. These results suggested H-Fe diet decreased Mn status in broilers evaluated by Mn concentrations in serum and heart, and heart MnSOD activity. Dietary Fe influenced Mn absorption possibly through effects on duodenal DMT1 and FPN1 expression.

PLA2G6-associated neurodegeneration (PLAN, NBIA2) is the second most common type of neurodegeneration with brain iron accumulation (NBIA), caused by recessive mutations of PLA2G6 gene, which encodes Ca2+-independent phospholipase A2β (iPLA2β). In most PLAN cases, decreased iPLA2β activity and iron deposition was observed meanwhile, and researchers also identified a PLA2G6 mutation family without iron deposition shown by MRI images. This brought us the question of whether decreased iPLA2β activity was the cause of iron deposition in PLAN. In this study, we used S-BEL as the antagonist of iPLA2β to block its activity and used SH-SY5Y cells as the expression system. We incubated SH-SY5Y cells with different concentrations of S-BEL. The results showed that decreased iPLA2β activity led no obvious iron accumulation, while changes of cells state and activation of apoptosis were observed. To further investigate the cause of unchanged iron level, we examined the cellular iron regulatory proteins involved in iron uptake, storage and export. The results were as follows: TfR1 (iron uptake protein) expression was decreased, the expression of ferritin heavy chain and light chain (iron storage protein) was increased. There was no alteration of the expression of DMT1 (iron uptake protein) and FPN1 (iron export protein). Under the condition of decreased iPLA2β activity, there was no obvious iron accumulation but iron uptake activity decreased and iron storage activity increased. Therefore, we speculate that the decreased iPLA2β activity may not be the main reason for iron deposition in PLAN.

Background: Deferoxamine (DFO) is a commonly used iron chelator, which can reduce the iron levels in cells. DFO is normally used to treat iron-overload disease, including some types of cancer. However, our previous studies revealed that DFO treatment significantly increased the iron concentrations in triple-negative breast cancer cells (TNBCs) resulting in enhanced cell migration. But the mechanism of DFO-induced increasing iron uptake in aggressive TNBCs still remained unclear. Materials and methods: Iron metabolism-related proteins in aggressive breast cancer MDA-MB-231, HS578T and BT549 cells and nonaggressive breast cancer MCF-7 and T47D cells were examined by immunofluorescence and Western blotting. The possible regulatory mechanism was explored by Western blotting, co-incubation with neutralizing antibodies or inhibitors, and transwell assay. Results: In this study, we found that DFO treatment significantly increased the levels of iron uptake proteins, DMT1 and TfR1, in aggressive TNBCs. Moreover, both TfR1 and DMT1 expressed on cell membrane were involved in high iron uptake in TNBCs under DFO-induced iron deficient condition. For the possible regulatory mechanism, we found that DFO treatment could promote a high expression level of IL-6 in aggressive MDA-MB-231 cells. The activated IL-6/PI3K/AKT pathway upregulated the expression of iron-uptake related proteins, TfR1 and DMT1, leading to increased iron uptakes. Conclusion: We demonstrated that DFO could upregulate expression of TfR1 and DMT1 , which enhanced iron uptake via activating IL-6/PI3K/AKT signaling pathway in aggressive TNBCs.

Patients with chronic liver disease from different aetiologies show a light serum Cu isotopic composition compared to the reference population, with the enrichment in the 63Cu isotope correlating with the severity of the disease. However, the mechanisms underlying Cu isotope fractionation at the onset and during progression of the disease are still unclear. In this work, a common bile duct ligation (CBDL) murine model was used to investigate the effect of cholestasis-induced liver disease on the Cu isotopic composition. Wild type male and female mice underwent surgical ligation of the common bile duct and were sacrificed 2, 4 and 6 weeks, and 4, 6 and 8 weeks after the surgical intervention, respectively. The age- and gender-matched control mice underwent sham surgery. Disease progression was evaluated using serum bilirubin levels, hepatic pro-inflammatory chemokine levels and Metavir fibrosis score. CBDL-operated mice show an overall body enrichment in the light isotope 63Cu. The Cu isotopic composition of organs, bone and serum becomes gradually lighter compared to the sham-operated mice with increasing severity of the disease. The light Cu isotopic composition of the CBDL-operated mice might result from an altered Cu intake and/or excretion. As the intestinal uptake of dietary Cu is largely mediated by transporters of Cu(i), mRNA and protein expression levels of two major metal transporters (CTR1 and DMT1) and Cu reductases (STEAP proteins and duodenal cytochrome B) were examined in the duodenal tissues as potential factors inducing Cu isotope fractionation. However, no significant differences in protein expression levels were observed between the CBDL- and sham-operated mice.

Most cells in the body acquire iron via receptor-mediated endocytosis of transferrin, the circulating iron transport protein. When cellular iron levels are sufficient, the uptake of transferrin decreases to limit further iron assimilation and prevent excessive iron accumulation. In iron overload conditions, such as hereditary hemochromatosis and thalassemia major, unregulated iron entry into the plasma overwhelms the carrying capacity of transferrin, resulting in non-transferrin-bound iron (NTBI), a redox-active, potentially toxic form of iron. Plasma NTBI is rapidly cleared from the circulation primarily by the liver and other organs (e.g., pancreas, heart, and pituitary) where it contributes significantly to tissue iron overload and related pathology. While NTBI is usually not detectable in the plasma of healthy individuals, it does appear to be a normal constituent of brain interstitial fluid and therefore likely serves as an important source of iron for most cell types in the CNS. A growing body of literature indicates that NTBI uptake is mediated by non-transferrin-bound iron transporters such as ZIP14, L-type and T-type calcium channels, DMT1, ZIP8, and TRPC6. This review provides an overview of NTBI uptake by various tissues and cells and summarizes the evidence for and against the roles of individual transporters in this process.

BACKGROUND Iron oversupplementation in healthy term infants may adversely affect growth and cognitive development. OBJECTIVE We hypothesized that early-life iron excess causes systemic and central nervous system iron overload, and compromises social behavior. METHODS The nursing pig was used as a translational model in a completely randomized study. On postnatal day (PD) 1, 24 pigs (1.57 ± 0.28 kg mean ± standard deviation body wt) were assigned to the following treatment groups: 1) nonsupplemented iron-deficient group (NON); 2) control group (CON), intramuscularly injected with iron dextran (100 mg Fe) on PD2; 3) moderate iron group (MOD), orally administered ferrous sulfate at 10 mg Fe · kg body wt-1 · d-1; and 4) high iron group (HIG), orally administered ferrous sulfate at 50 mg Fe · kg-1 · d-1. Piglets were nursed by sows during the study from PD1 to PD21. Tissue iron was analyzed by atomic absorption spectrophotometry. Messenger RNA and protein expression of iron regulator and transporters were analyzed by quantitative reverse transcriptase-polymerase chain reaction and Western blot. A sociability test was performed on PD19-20. RESULTS Both MOD and HIG treatments (5.51 and 9.85 µmol/g tissue), but not CON (0.54 µmol/g), increased hepatic iron as compared with NON (0.25 µmol/g, P

Inadequate iron levels during early life can have adverse consequences for the developing infant. Iron deficiency during this critical period of growth can affect brain development and cognitive function, problems that can be lifelong despite subsequent correction of the iron deficit. Therefore, it is critical that the suckling infant has sufficient iron for their developmental needs. Much of the iron used in the immediate post-natal period is stored iron that was acquired from the mother in the final trimester of pregnancy, however, despite having low iron levels, breast milk can also make a significant contribution to infant iron needs. This reflects the ability of the suckling infant to absorb dietary iron far more efficiently than is possible after weaning. The mechanisms underlying this enhanced iron absorption are poorly understood. The iron export protein ferroportin is essential for this process, as it is in adults, however, the role of other molecules normally involved in iron absorption following weaning is less clear. The composition and distribution of iron in breast milk may be important, as could the contribution of more distal parts of the gastrointestinal tract. This review discusses the potential role of each of the above components in intestinal iron absorption during suckling and highlights the need for further research into this important process.

Huntington’s disease (HD) is functionally linked to environmental factors including cigarette use and dyshomeostasis in the levels of metals. Interestingly, one of the most abundant heavy metals in cigarettes is cadmium (Cd), which also accumulates in the striatum and causes neurotoxicity upon exposure. Thus, we hypothesized that heterozygous huntingtin (HTT), responsible for the majority of cases of HD in patients, in combination with Cd exposure would cause neurotoxicity and neurodegeneration via increased intracellular accumulation of Cd and activation of oxidative stress signaling mechanisms in a mouse striatal cell line model of HD. We report that heterozygous HTT striatal cells are significantly more susceptible to Cd-induced cytotoxicity as compared to wild-type HTT cells upon exposure for 48 h. The heterozygous HTT and Cd-induced cytotoxicity led to a NADPH oxidase (NOX) mediated oxidative stress that was attenuated by exogenous antioxidants and a NOX inhibitor, apocynin. Heterozygous HTT coupled with Cd exposure caused increased expression of protein kinase C δ (PKCδ) and other key oxidative stress proteins levels, enhanced the activation of caspase-9 and caspase-3 mediated apoptosis, and blocked the overexpression of extracellular signal-regulated kinase (ERK). We observed significantly greater intracellular accumulation of Cd and reduced expression of divalent metal transporter 1 (DMT1) protein in the heterozygous HTT striatal cells upon Cd exposure. Treatment with zinc, manganese, and iron as well as exogenous antioxidants significantly attenuated the Cd-induced cytotoxicity. Collectively, these results demonstrate that heterozygous HTT exhibits greater neurotoxic properties when coupled with Cd exposure to cause cell death via caspase mediated apoptosis, altered metal transport, and modulation of ERK and PKCδ dependent oxidative signaling mechanisms.

This study was conducted to compare the effects of in ovo injection of different sources of manganese (Mn) on Divalent Metal Transporter 1 (DMT1) gene expression in the small intestine of broilers embryo and hatchling, and its subsequent effects on bone development. A total of 480 fertile eggs (ROSS 308) on embryonic day 17 were divided into 6 treatments with 3 replicates of 20 fertile eggs per replicate. Treatments were included: non-injected (NC) and diluent-injected controls (PC), manganese sulphate (MnS), manganese-methionine (Mn-Met), nano manganese (NMn) and nano manganese-methionine (NMn-Met) groups. Results showed that in ovo injection of different sources of Mn could change the DMT1 mRNA level of intestinal cells (p

It has been well established that glucotoxicity induces pancreatic β-cells dysfunction; however, the precise mechanism remains unclear. Our previous studies demonstrated that high glucose concentrations are associated with decreased hepcidin expression, which inhibits insulin synthesis. In this study, we focused on the role of low hepcidin level-induced increased iron deposition in β-cells and the relationship between abnormal iron metabolism and β-cell dysfunction. Decreased hepcidin expression increased iron absorption by upregulating transferrin receptor 1 (TfR1) and divalent metal transporter 1 (DMT1) expression, resulting in iron accumulation within cells. Prussia blue stain and calcein-AM assays revealed greater iron accumulation in the cytoplasm of pancreatic tissue isolated from db/db mice, cultured islets and Min6 cells in response to high glucose stimulation. Increased cytosolic iron deposition was associated with greater Fe2+ influx into the mitochondria, which depolarized the mitochondria membrane potential, inhibited ATP synthesis, generated excessive ROS and induced oxidative stress. The toxic effect of excessive iron on mitochondrial function eventually resulted in impaired insulin secretion. The restricted iron content in db/db mice via reduced iron intake or accelerated iron clearance improved blood glucose levels with decreased fasting blood glucose (FBG), fasting blood insulin (FIns), HbA1c level, as well as improved intraperitoneal glucose tolerance test (IPGTT) results. Thus, our study may reveal the mechanism involved in the role of hepcidin in the glucotoxcity impaired pancreatic β cell function pathway.

Iron is involved in numerous physiologically essential processes in our body. However, excessive iron is a pathogenic factor in neurodegenerative diseases, causing aberrant oxidative stress. Divalent metal transporter 1 (DMT1) acts as a primary transporter of Fe(II) ions. The intracellular delivery of DMT1 toward the cellular membrane via the trans-Golgi network during the endocytotic process is partially regulated by a retromer-mediated protein-sorting system comprising vacuolar protein-sorting proteins (VPSs). Thus, together with DMT1, the Golgi-apparatus acts as a hub organelle in the delivery system for intracellular Fe(II) ions. Dysfunction of the VPS-relevant protein sorting system can induce the abnormal delivery of DMT1 toward lysosomes concomitantly with Fe(II) ions. To explore this issue, we developed a fluorescent probe, Gol-SiRhoNox, for the Golgi-specific detection of Fe(II) ions by integrating our original N-oxide-based Fe(II)-specific chemical switch, a new Golgi-localizable chemical motif, and polarity-sensitive fluorogenic scaffold. Our synchronous imaging study using Gol-SiRhoNox and LysoRhoNox, a previously developed fluorescent probe for lysosomal Fe(II), revealed that the intracellular distribution balance of Fe(II) ions between the Golgi apparatus and lysosomes is normally Golgi-dominant, whereas the lysosome-specific elevation of Fe(II) ions was observed in cells with induced dysfunction of VPS35, a member of the retromer complex. Treatment of cells with dysfunctional VPS35 with R55, a molecular chaperone, resulted in the restoration of the subcellular distribution of Fe(II) ions to the Golgi-dominant state. These results indicate that the impairment of the DMT1 traffic machinery affects subcellular iron homeostasis, promoting Fe(II) leakage at the Golgi and lysosomal accumulation of Fe(II) through missorting of DMT1.

Iron is the fundamental element for numerous physiological functions. Plasmalemmal divalent metal ion transporter 1 (DMT1) is responsible for cellular uptake of ferrous (Fe2+), whereas transferrin receptors (TFR) carry transferrin (TF)-bound ferric (Fe3+). In this study we performed detailed analysis of the action of Fe ions on cytoplasmic free calcium ion concentration ([Ca2+]i) in astrocytes. Administration of Fe2+ or Fe3+ in μM concentrations evoked [Ca2+]i in astrocytes in vitro and in vivo. Iron ions trigger increase in [Ca2+]i through two distinct molecular cascades. Uptake of Fe2+ by DMT1 inhibits astroglial Na+-K+-ATPase, which leads to elevation in cytoplasmic Na+ concentration, thus reversing Na+/Ca2+ exchanger and thereby generating Ca2+ influx. Uptake of Fe3+ by TF-TFR stimulates phospholipase C to produce inositol 1,4,5-trisphosphate (InsP3), thus triggering InsP3 receptor-mediated Ca2+ release from endoplasmic reticulum. In summary, these findings reveal the mechanisms of iron-induced astrocytic signalling operational in conditions of iron overload.

A plethora of studies indicate that iron metabolism is dysregulated in Parkinson's disease (PD). The literature reveals well-documented alterations consistent with established dogma, but also intriguing paradoxical observations requiring mechanistic dissection. An important fact is the iron loading in dopaminergic neurons of the substantia nigra pars compacta (SNpc), which are the cells primarily affected in PD. Assessment of these changes reveal increased expression of proteins critical for iron uptake, namely transferrin receptor 1 and the divalent metal transporter 1 (DMT1), and decreased expression of the iron exporter, ferroportin-1 (FPN1). Consistent with this is the activation of iron regulator protein (IRP) RNA-binding activity, which is an important regulator of iron homeostasis, with its activation indicating cytosolic iron deficiency. In fact, IRPs bind to iron-responsive elements (IREs) in the 3ꞌ untranslated region (UTR) of certain mRNAs to stabilize their half-life, while binding to the 5ꞌ UTR prevents translation. Iron loading of dopaminergic neurons in PD may occur through these mechanisms, leading to increased neuronal iron and iron-mediated reactive oxygen species (ROS) generation. The “gold standard” histological marker of PD, Lewy bodies, are mainly composed of α-synuclein, the expression of which is markedly increased in PD. Of note, an atypical IRE exists in the α-synuclein 5ꞌ UTR that may explain its up-regulation by increased iron. This dysregulation could be impacted by the unique autonomous pacemaking of dopaminergic neurons of the SNpc that engages L-type Ca+2 channels, which imparts a bioenergetic energy deficit and mitochondrial redox stress. This dysfunction could then drive alterations in iron trafficking that attempt to rescue energy deficits such as the increased iron uptake to provide iron for key electron transport proteins. Considering the increased iron-loading in PD brains, therapies utilizing limited iron chelation have shown success. Greater therapeutic advancements should be possible once the exact molecular pathways of iron processing are dissected.

Intestinal iron transport requires an iron importer (Dmt1) and an iron exporter (Fpn1). The hormone hepcidin regulates iron absorption by modulating Fpn1 protein levels on the basolateral surface of duodenal enterocytes. In the genetic, iron-loading disorder hereditary hemochromatosis (HH), hepcidin production is low and Fpn1 protein expression is elevated. High Fpn1-mediated iron export depletes intracellular iron, causing a paradoxical increase in Dmt1-mediated iron import. Increased activity of both transporters causes excessive iron absorption, thus initiating body iron loading. Logically then, silencing of intestinal Dmt1 or Fpn1 could be an effective therapeutic intervention in HH. It was previously established that Dmt1 knock down prevented iron-loading in weanling Hamp (encoding hepcidin) KO mice (modeling type 2B HH). Here, we tested the hypothesis that Dmt1 silencing combined with dietary iron restriction (which may be recommended for HH patients) will mitigate iron loading once already established. Accordingly, adult Hamp KO mice were switched to a low-iron (LFe) diet and (non-toxic) folic acid-coupled, ginger nanoparticle-derived lipid vectors (FA-GDLVs) were used to deliver negative-control (NC) or Dmt1 siRNA by oral, intragastric gavage daily for 21 days. The LFe diet reduced body iron burden, and experimental interventions potentiated iron losses. For example, Dmt1 siRNA treatment suppressed duodenal Dmt1 mRNA expression (by ~50%) and reduced serum and liver non-heme iron levels (by ~60% and &gt, 85%, respectively). Interestingly, some iron-related parameters were repressed similarly by FA-GDLVs carrying either siRNA, including 59Fe (as FeCl3) absorption (~20% lower), pancreatic non-heme iron (reduced by ~65%), and serum ferritin (decreased 40–50%). Ginger may thus contain bioactive lipids that also influence iron homeostasis. In conclusion, the combinatorial approach of FA-GDLV and Dmt1 siRNA treatment, with dietary iron restriction, mitigated pre-existing iron overload in a murine model of HH.

Iron is a critical metal for several vital biological processes. Most of the body’s iron is bound to hemoglobin in erythrocytes. Iron from senescent red blood cells is recycled by macrophages in the spleen, liver and bone marrow. Dietary iron is taken up by the divalent metal transporter 1 (DMT1) in enterocytes and transported to portal blood via ferroportin (FPN), where it is bound to transferrin and taken up by hepatocytes, macrophages and bone marrow cells via transferrin receptor 1 (TfR1). While most of the physiologically active iron is bound hemoglobin, the major storage of most iron occurs in the liver in a ferritin-bound fashion. In response to an increased iron load, hepatocytes secrete the peptide hormone hepcidin, which binds to and induces internalization and degradation of the iron transporter FPN, thus controlling the amount of iron released from the cells into the blood. This review summarizes the key mechanisms and players involved in cellular and systemic iron regulation.

Lead (Pb) is an environmental and public health toxicant. It affects various organ systems of the body, thereby disrupting their normal functions. To date, several genes that are known to influence the mechanism of action of lead and toxicity have been studied. Among them, the iron transporter gene, SLC11A2 (Solute Carrier 11 group A member 2) which codes for the transmembrane protein, DMT1 (Divalent Metal Transporter 1) has shown to transport other metals including zinc, copper, and lead. We investigated the influence of DMT1 polymorphism (rs224589) on blood lead (Pb-B) levels. In the present study, we enrolled 113 lead-exposed workers and performed a comprehensive biochemical analysis and genetic composition. The frequency of DMT1 variants observed in the total subjects (n = 113) was 42 % for homozygous CC wild type, 54 % for heterozygous CA, and 4 % for homozygous AA mutant. The heterozygous CA carriers presented higher Pb-B levels compared to wild type CC and mutant AA carriers. Further, a negative association was observed between Pb-B levels and hemoglobin in heterozygous CA carriers. Hence, C allele may be the risk allele that contributes to increased susceptibility to high Pb-B retention, and genotyping of DMT1 in lead exposed subjects might be used as a prognostic marker to impede organ damage due to lead toxicity.

The protein alpha-synuclein (α-syn) is unusual because, depending on its conformation and the type of cell in which it is expressed, it is pro-death or pro-survival, triggering neurodegeneration in Parkinson’s disease and enhancing cell survival of some melanomas. To probe the function of α-syn in melanoma, we used CRISPR/Cas9 to knockout SNCA, the gene that codes for α-syn, in SK-Mel-28 melanoma cells. The SNCA-knockout clones in culture exhibited a decrease in the transferrin receptor 1 (TfR1), an increase in ferritin, an increase of reactive oxygen species and proliferated slower than control cells. These SNCA-knockout clones grafted into SCID mice grew significantly slower than the SK-Mel-28 control cells that expressed α-syn. In the excised SNCA-knockout xenografts, TfR1 decreased 3.3-fold, ferritin increased 6.2-fold, the divalent metal ion transporter 1 (DMT1) increased threefold, and the iron exporter ferroportin (FPN1) decreased twofold relative to control xenografts. The excised SNCA-KO tumors exhibited significantly more ferric iron and TUNEL staining relative to the control melanoma xenografts. Collectively, depletion of α-syn in SK-Mel-28 cells dysregulates cellular iron metabolism, especially in xenografts, yielding melanoma cells that are deficient in TfR1 and FPN1, that accumulate ferric iron and ferritin, and that undergo apoptosis relative to control cells expressing α-syn.

Objective: Iron overload is common in elderly people which is associated with an increased prevalence of osteoarthritis (OA), but the exact role of iron in the development of OA has not been established. The aim of the present study is to elucidate the connection between iron overload and OA using an iron overloaded mice model, as well as to explore the role of iron homeostasis, iron transporters dependent iron influx in OA pathogenesis.Methods: The iron overloaded mice model was established and OA was surgically induced. OA progression was assessed at 8 weeks after surgery. Next, primary chondrocytes were treated with pro-inflammatory cytokines and iron regulators mediated iron homeostasis were evaluated. Involvement of iron transporters was analyzed using chondrocytes mimicking an osteoarthritis-related phenotype in vitro.Results: Iron overloaded mice exhibited greater cartilage destruction and elevated ADAMTS5 as well as MMP13 expression along with increased iron accumulation and dysregulated iron regulators. Pro-inflammatory cytokines could disturb cellular iron homeostasis via upregulating iron import proteins, TFR1 and DMT1, downregulating iron efflux protein FPN, thus result in cellular iron overload. Among iron transporters, DMT1 was found to play pivotal roles in iron overload induced OA progress. Inhibition of DMT1 suppressed IL-1β induced inflammatory response and ECM degradation via blockade of MAPK and PI3K/AKT/NF-κB pathways.Conclusions: Our results suggest that iron takes parts in the development of OA and cutting iron influx via inhibiting DMT1 activity could be an attractive new target for OA treatment.

Previous studies have found that Salvia miltiorrhiza (SM) injection have a protective effect on the iron overloaded (IO) heart. However, the mechanisms are not completely known. In the present study, we investigated the underlying mechanisms based on the iron transport-related proteins. The rats were randomly divided into five groups: control, IO group, low-dose SM group, high-dose SM group, and deferoxamine control group. Iron dextran was injected to establish the IO model. After 14 days of treatment, cardiac histological changes were observed by hematoxylin and eosin (H&E) staining. Iron uptake-related proteins divalent metal transporter-1 (DMT-1), transferrin receptor-1 (TfR-1), and iron export-related proteins ferroportin1 (FP1) in the heart were detected by Western blotting. The results showed that SM injection decreased cardiac iron deposition, ameliorated cardiac function, and inhibited cardiac oxidation. Most important of all, SM injection downregulated the expression of DMT-1 and TfR-1 and upregulated FP1 protein levels compared with the IO group. Our results indicated that reducing cardiac iron uptake and increasing iron excretion may be one of the important mechanisms of SM injection reducing cardiac iron deposition and improving cardiac function under the conditions of IO.

Dithiolethiones are lipophilic, organosulfur compounds that activate the Nrf2 transcription factor causing an upregulation of various phase II antioxidant enzymes. A disubstituted dithiolethione 5-amino-3-thioxo-3H-(1,2) dithiole-4-carboxylic acid ethyl ester (ACDT) retains the functional pharmacophore while also containing modifiable functional groups. Neuroprotection against autoimmune encephalomyelitis in vivo and 6-hydroxy dopamine (a model for Parkinson's disease) in vitro have been previously reported with ACDT. Manganese (Mn) is a metal essential for metabolic processes at low concentrations. Overexposure and accumulation of Mn leads to a neurological condition called manganism which shares pathophysiological sequelae with parkinsonism. Here we hypothesized ACDT to be protective against manganese-induced cytotoxicity. SH-SY5Y human neuroblastoma cells exposed to 300 μM MnCl2 displayed approximately 50% cell death, and a 24-h pretreatment with 75 μM ACDT significantly reversed this cytotoxicity. ACDT pretreatment was also found to increase total GSH levels (2.18-fold) and the protein levels of NADPH:quinone oxidoreductase-1 (NQO1) enzyme (6.33-fold), indicating an overall increase in the cells' antioxidant defense stores. A corresponding 2.32-fold reduction in the level of Mn-induced reactive oxygen species was also observed in cells pretreated with ACDT. While no changes were observed in the protein levels of apoptotic markers Bax and Bcl-2, pretreatment with 75 μM ACDT led to a 2.09-fold downregulation of ZIP14 import transporter, indicating a potential reduction in the cellular uptake of Mn as an additional neuroprotective mechanism. These effects did not extend to other transporters like the divalent metal transporter 1 (DMT1) or ferroportin. Collectively, ACDT showed substantial neuroprotection against Mn-induced cytotoxicity, opening a path for dithiolethiones as a potential novel therapeutic option against heavy metal neurotoxicity.

Background/aim Altered iron metabolism is one of the pathophysiological mechanisms occurring during hypoxic injuries in the central nervous system. Proper homeostasis of cellular iron is regulated by iron import, storage, and export proteins that prevent excess iron overload or iron starvation in cells. Therapeutic hypothermia is an approved treatment for hypoxic ischemia in newborns, but the underlying molecular mechanism is still unknown. We studied the effects of hypoxia, preceded with preconditioning, on the iron homeostasis of glial cells, known as a major actor in the inflammatory process during perinatal brain injury. Materials and methods Primary microglia and astrocytes in culture were exposed to 12 h of hypoxia with or without mild hypothermic preconditioning. The mRNA expression was assessed using qPCR. Iron accumulation was visualized via modified Perl’s histochemistry. Cytokine levels in cell cultures were measured using ELISA. Results Hypothermic preconditioning enhanced microglial viability, which previously was decreased in both cell types due to hypoxia. Hypoxia increased iron accumulation in the mixed glial cells and in ferritin expression in both microglia and astrocytes. Hypotermic preconditioning decreased the elevated ferritin-light chain expression significantly in microglia. Iron importer proteins, DMT1 and TfR1, both increased their mRNA expression after hypoxia, and hypothermic preconditioning continued to support the elevation of DMT1 in both glial cell types. Ferroportin expression increased as a survival factor of the glial cell following hypoxia. Hypothermic preconditioning supported this increase in both cell types and was especially significant in astrocytes. IL-10 levels were prominently increased in cell culture after hypothermic preconditioning. Conclusion The data suggest that hypothermic preconditioning affects cellular iron homeostasis by regulating the storage and transfer proteins of iron. Regulation of the cellular iron traffic may prevent glial cells from experiencing the detrimental effects of hypoxia-related inflammation.

Fear memory is a pivotal biological function by which organisms can predict possible danger to avoid or reduce harm. However, dysregulation of fear memory processing may lead to pathological fear or anxiety and produce serious clinical symptoms, such as post-traumatic stress disorder (PTSD). Iron deficiency (ID) is reported to inhibit the initiation of fear memory. In our study, we found that ferroportin1 (FPN1), the only known cellular iron export protein in mammals, and ablation in neurons and astrocytes caused iron deficiency in the cortex and hippocampus. However, little is known about its role in the development of fear memory. Moreover, direct evidence of the role of FPN1, or the related molecular mechanisms of such a role, in balancing brain iron homeostasis, especially in neuronal cells, is lacking. Herein, we deleted Fpn1 in mouse neurons, using Nestin-cre transgenic mice, and explored the impact on neuronal iron recycling and brain iron homeostasis in the cortex and hippocampus. We investigated the response of the mice to contextual fear and found that formation of fear memory was impeded after neuronal FPN1 depletion. We also found that FPN1 ablation in neurons and astrocytes caused an atypical expression of iron metabolism-related proteins in these two regions: decreased expression of DMT1, Ft-H, and Ft-L, and increased TfR1 expression. In addition, the decreased FPN1 in brain microvascular endothelial cells (BMVECs) also shed light on the cause of the decreased iron delivery to the brain through the blood-brain barrier (BBB). Our research highlights the major role played by FPN1 in brain iron homeostasis and identifies a potential target for the treatment of PTSD.

Cadmium (Cd) is a global pollutant that accumulates in the placenta and can cause placental dysfunction. Although iron transporters have been suggested to participate in placental Cd uptake, it is still unknown which transporters are actually involved in this process. We specifically aimed to study the role of three iron transporters in the uptake of Cd into the placental cell line HTR-8/SVneo. For this purpose, Divalent Metal Transporter (DMT)1 and ZRT/IRT like protein (ZIP)8 and ZIP14 were downregulated and changes in cellular Cd levels analysed in relation to controls. As clearly shown by the reduction of the Cd content by ∼60% in DMT1- and ZIP14-downregulated cells, the two proteins are essential for Cd accumulation in HTR-8/SVneo cells. Using a validated antibody, we show DMT1 to be localised in situ in trophoblast and stromal cells. We further wanted to investigate how placental cells cope with Cd loading and which metallothionein (MT) isoforms they express. Cd-exposed cells accumulate Cd in a dose-dependent manner and upregulate MT2A accordingly (up to 15-fold induction upon 5 μM CdCl2 treatment for 72 h). 5 μM Cd exposure for 72 h decreased cell number to 60%, an effect that was aggravated by MT2A depletion (cell number reduced to 30%) indicating additive effects. In conclusion, our data suggest that DMT1 and ZIP14 are required for Cd uptake into human placental cells that upregulate MT2A to store and detoxify the metal. Cd storage in the placenta reduces Cd transport to the fetus, which, however, could impair placental functions and fetal development.

Iron is critical for many processes including oxygen transport and erythropoiesis. Transcriptomic analysis demonstrates that HIF-2α regulates over 90% of all transcripts induced following iron deficiency in the intestine. However, beyond divalent metal transporter 1 (DMT1), ferroportin 1 (Fpn1) and duodenal cytochrome b (Dcytb), no other genes/pathways have been critically assessed with respects to their importance in intestinal iron absorption. Ferritinophagy is associated with cargo specific autophagic breakdown of ferritin and subsequent release of iron. We show here that nuclear receptor co-activator 4 (NCOA4)-mediated intestinal ferritinophagy is integrated to systemic iron demand via HIF-2α. Duodenal NCOA4 expression is regulated by HIF-2α during high systemic iron demands. Moreover, overexpression of intestinal HIF-2α is sufficient to activate NCOA4 and promote lysosomal degradation of ferritin. Promoter analysis revealed NCOA4 as a direct HIF-2α target. To demonstrate the importance of intestinal HIF-2α/ferritinophagy axis in systemic iron homeostasis, whole body and intestine-specific NCOA4-null mouse lines were assessed. These analyses demonstrate an iron sequestration in the enterocytes, and significantly high tissue ferritin levels in the dietary iron deficiency and acute hemolytic anemia models. Together, our data suggests efficient ferritinophagy is critical for intestinal iron absorption and systemic iron homeostasis.

Emerging evidence has deemed vitamin D as a potential candidate for the intervention of type 2 diabetes (T2D). Herein, we explored the underlying mechanisms of T2D prevention by vitamin D, concentrating on pancreatic iron deposition reported recently. Zucker diabetic fatty (ZDF) rats were treated by vitamin D, with age-matched Zucker lean rats as control. As expected, vitamin D treatment for ZDF rats normalized islet morphology and β-cell function. Moreover, vitamin D alleviated iron accumulation and apoptosis in pancreatic cells of ZDF rats, accompanied by lowered divalent metal transporter 1 (DMT1) expression. Consistently, similar results were observed in high glucose-stimulated INS-1 cells treated with or without vitamin D. Nuclear factor-κB (NF-κB), a transcription factor involving DMT1 regulation, was activated in pancreases of ZDF rats and INS-1 cells exposed to high glucose, but inactivated by vitamin D or BAY 11-7082, a NF-κB inhibitor. Futhermore, IL-1β functioning as NF-κB activator abolished the suppression of NF-κB activation, DMT1 induction and the attenuation of apoptosis as a consequence of vitamin D incubation. Our study showed that iron overload in pancreas may contribute to T2D pathogenesis and uncovered a potentially protective role for vitamin D on iron deposition of diabetic pancreas through NF-κB- DMT1 signaling.

Iron is a key nutrient for normal central nervous system (CNS) development and function; thus, iron deficiency as well as iron excess may result in harmful effects in the CNS. Oligodendrocytes and astrocytes are crucial players in brain iron equilibrium. However, the mechanisms of iron uptake, storage, and efflux in oligodendrocytes and astrocytes during CNS development or under pathological situations such as demyelination are not completely understood. In the CNS, iron is directly required for myelin production as a cofactor for enzymes involved in ATP, cholesterol and lipid synthesis, and oligodendrocytes are the cells with the highest iron levels in the brain which is linked to their elevated metabolic needs associated with the process of myelination. Unlike oligodendrocytes, astrocytes do not have a high metabolic requirement for iron. However, these cells are in close contact with blood vessel and have a strong iron transport capacity. In several pathological situations, changes in iron homoeostasis result in altered cellular iron distribution and accumulation and oxidative stress. In inflammatory demyelinating diseases such as multiple sclerosis, reactive astrocytes accumulate iron and upregulate iron efflux and influx molecules, which suggest that they are outfitted to take up and safely recycle iron. In this review, we will discuss the participation of oligodendrocytes and astrocytes in CNS iron homeostasis. Understanding the molecular mechanisms of iron uptake, storage, and efflux in oligodendrocytes and astrocytes is necessary for planning effective strategies for iron management during CNS development as well as for the treatment of demyelinating diseases.

Iron is the fundamental element for numerous physiological functions. Reduced ferrous (Fe2+) and oxidized ferric (Fe3+) are the two ionized iron states in the living organisms. In the cell membrane, divalent metal ion transporter 1 (DMT1) is responsible for cellular uptake of Fe2+, whereas transferrin receptors (TFR) carry transferrin (TF)-bound Fe3+. In this study we performed, for the first time, detailed analysis of the action of Fe ions on cytoplasmic free calcium ion concentration ([Ca2+]i) in astrocytes. Using qPCR and immunocytochemistry we identified DMT1 and TFR in astrocytes in primary cultures, in acutely isolated astrocytes and in brain tissue preparations; in situ both DMT1 and TFR are concentrated in astroglial perivascular endfeet. Administration of Fe2+ or Fe3+ in low μM concentrations evoked Ca2+ signals in astrocytes in vitro and in vivo. Iron ions triggered increase in [Ca2+]i by acting through two distinct molecular cascades. Uptake of Fe2+ by DMT1 inhibited astroglial Na+-K+-ATPase (NKA), which led to an elevation in cytoplasmic Na+ concentration (as measured by SBFI probe), thus reversing Na+/Ca2+ exchanger (NCX) thereby generating Ca2+ influx. Uptake of Fe3+ by TF-TFR stimulated phospholipase C to produce inositol 1,4,5-trisphosphate (InsP3), thus trigering InsP3 receptor-mediated Ca2+ release from the endoplasmic reticulum. Iron-induced Ca2+ signals promote astroglial release of arachidonic acid and prostaglandin E2 cytokines by activating cytosolic phospholipase A2 (cPLA2) and NF-κB signalling cascade. In summary, these findings reveal new mechanisms of iron-induced astrocytic signalling operational in conditions of iron overload, in response to which astrocytes actively accumulate excessive iron and activate neuroprotective pathways.

Ischemic preconditioning (IP) reduces brain damage after subsequent ischemic strokes by activating endogenous protective mechanisms in rodents. Transient ischemic attack (TIA) induces tolerance in the human brain after ischemic strokes; defining mechanisms of IP effects may provide therapeutic targets to improve recovery of patients with ischemic strokes. Iron transported across the blood–brain barrier (BBB) is required for brain functions, including myelination, and its levels should be finely regulated to avoid harmful effects. This study aimed to determine whether IP enhances repair processes by modulating iron metabolism during the post-stroke chronic phase. Male mice were divided into sham and IP groups, and IP was induced 24 h before a transient focal ischemic stroke. Sensorimotor recovery was observed over 8 weeks after the stroke, and brain volumes and levels of proteins related to repair processes and iron metabolism in the ischemic brains were examined 8 weeks after the stroke. There was significantly less ischemic brain atrophy in the IP group than in the sham group, with no differences in sensorimotor recovery between the groups. Levels of tight junction proteins of BBB, neurites outgrowth markers, and myelin sheath proteins and markers for mature oligodendrocytes were significantly increased in the IP group. Iron import proteins, transferrin receptor 1 and DMT1, were also increased in the IP group. These results indicate that IP increases brain repair processes and iron uptake during the chronic phase after an ischemic stroke, and provide new insights to understand the molecular mechanisms of TIA effects on post-stroke recovery.

Iron metabolism imbalance plays a key role in the neurodegeneration of Parkinson's disease (PD), thus iron homeostasis should be tightly controlled by iron transporters. α-Synuclein (α-Syn) serves as a ferrireductase and iron-binding protein, which is supposed to be linked with iron metabolism, but little is known about how α-Syn affects iron homeostasis in PD. Our previous findings that up-regulation of divalent metal transporter 1 (DMT1) accounted for the nigral iron accumulation in PD raised the question whether α-Syn disturbed iron homeostasis by modulating DMT1 expression. Using α-Syn overexpressed SH-SY5Y cells and mutant human A53T α-Syn transgenic mice, we found that α-Syn could up-regulate DMT1 protein levels, followed by enhanced ferrous iron influx and subsequent aggravated oxidative stress injury. Mechanistic studies identified that α-Syn-induced p38 mitogen-activated protein kinase (MAPK) activation phosphorylated parkin at Ser131, which inactivated parkin's E3 ubiquitin ligase activity and further reduced DMT1 ubiquitylation level. Our findings revealed that α-Syn affected brain iron homeostasis through modulating DMT1 protein stability and altering cellular iron uptake, which might provide direct evidence for the involvement of α-Syn in iron metabolism dysfunction and provide insight into PD-associated nigral iron deposition.

J.M.-F. was supported by two fellowships from the Ministry of Education, Culture and Sport (Spain), FPU and Traslados Temporales FPU (University of King´s College of London). J.M.-F. is grateful to the Excellence Program “Nutrición y Ciencias de los Alimentos” from the University of Granada. The authors are grateful to Susan Stevenson for her efficient support in the revision with the English language., Despite the crucial role of the liver as the central regulator of iron homeostasis, no studies have directly tested the modulation of liver gene and protein expression patterns during iron deficiency instauration and recovery with fermented milks. Fermented goat milk consumption improves the key proteins of intestinal iron metabolism during iron deficiency recovery, enhancing the digestive and metabolic utilization of iron. The aim of this study was to assess the influence of fermented goat or cow milk consumption on liver iron homeostasis during iron-deficiency anemia recovery with normal or iron-overload diets. Analysis included iron status biomarkers, gene and protein expression in hepatocytes. In general, fermented goat milk consumption either with normal or high iron content up-regulated liver DMT1, FPN1 and FTL1 gene expression and DMT1 and FPN1 protein expression. However, HAMP mRNA expression was lower in all groups of animals fed fermented goat milk. Additionally, hepcidin protein expression decreased in control and anemic animals fed fermented goat milk with normal iron content. In conclusion, fermented goat milk potentiates the up-regulation of key genes coding for proteins involved in iron metabolism, such as DMT1, and FPN1, FTL1 and down-regulation of HAMP, playing a key role in enhanced iron repletion during anemia recovery, inducing a physiological adaptation of the liver key genes and proteins coordinated with the fluctuation of the cellular iron levels, favoring whole-body iron homeostasis., Ministry of Education, Culture and Sport (Spain), Spanish Government, Traslados Temporales FPU (University of Kings College of London)

C57BL/6 (BL6) and Balb/c mice exhibit prototypical Th1‐ and Th2‐dominant immune predispositions, respectively. Iron is a proinflammatory metal ion; however, limited information is documented on the differences in iron homeostasis between BL6 and Balb/c strains. The objective of this study was to investigate the extent to which strain‐level differences in these mice dictates the regulation of iron homeostasis during physiologic and inflammatory conditions. At basal levels, Balb/c mice displayed significantly higher levels of iron in systemic circulation and tissue compared to BL6 mice. Moreover, Balb/c mice had greater iron absorption as indicated by higher gene expressions of duodenal DcytB, DMT1, Fpn, SFT, and Heph. Similarly, hepatic Tf, TfR1, TfR2, and DMT1 expressions were augmented in Balb/c mice. Interestingly, there was no change in hepatic Hamp expression between the two strains, suggesting that the disparity in their maintenance of iron is independent of hepcidin. Additionally, the basal levels of intracellular labile iron pool in Balb/c intestinal epithelial cells, and bone marrow‐derived macrophages and neutrophils, were higher compared to BL6 mice. When mice were challenged with lipopolysaccharide, the acute inflammatory response in BL6 mice was more pronounced than in Balb/c mice, as indicated by the more rapid development of hypoferremia and upregulation of serum IL‐6 and TNF‐α levels in BL6 mice. In conclusion, this study underscores that iron homeostasis is distinct between BL6 and Balb/c strains under both physiologic and inflammatory conditions., Relative to C57BL/6 mice (BL6), Balb/c mice harbor more circulating, tissue and labile iron pool (LIP), which correlates with their generally heightened expression of key iron‐regulatory proteins like divalent metal transporter 1 (DMT1) andferroportin. Accordingly, such strain‐level differences in iron homeostasis could explain the dissimilar degree of developing pro‐inflammatory responses, followed by hypoferremia, in response to inflammatory challenge like lipopolysaccharides (LPS), which occurs more rapidly in BL6 mice than Balb/c mice.

The Brazilian savanna fruit, tucum-do-cerrado (Bactris setosa Mart.) reduces hepatic hepcidin levels. Therefore, we investigated the effect of tucum-do-cerrado on the TfR/HFE and/or BMP/HJV/SMAD and JAK/STAT pathways, in normal and excess iron conditions. Rats were treated with: control diet (CT), control diet +15% tucum-do-cerrado (Tuc), iron-enriched diet (+Fe), or iron-enriched diet +15% tucum-do-cerrado (Tuc+Fe). Tucum-do-cerrado (Tuc) decreased hepatic Hamp and Hjv mRNA levels but did not alter Bmp6, Smad7, Tfr1, and Hfe mRNA levels, pSMAD1/5/8 and pSTAT3 protein levels, labile iron pool (LIP), and inflammatory biomarkers, compared to the CT group. The iron-enriched diet increased Hamp mRNA levels, as well as pSMAD1/5/8 and pSTAT3 protein levels, while no difference was observed in Hjv, Bmp6, Smad7, Tfr1, and Hfe mRNA levels and LIP compared to the CT group. The association of tucum-do-cerrado with the iron-enriched diet (Tuc+Fe) decreased Hamp, Hjv, Bmp6, and Hfe mRNA levels and pSTAT3 protein content compared to the +Fe group, while increased Hamp and decreased Hfe mRNA levels compared to the Tuc group. Therefore, the inhibition of hepatic hepcidin by tucum-do-cerrado consumption may involve the downregulation of intestinal Dmt1 and hepatic Hjv expression and deacetylation mediated by SIRT1 by a mechanism that is independent of tissue iron content. However, in excess iron conditions, the modulation of hepatic hepcidin expression by tucum-do-cerrado seems to be partially mediated by the inflammatory signaling pathway, as well as involves the chelating activity of tucum-do-cerrado.

Background Increasing evidence suggests that multiple or long-time exposure to general anaesthesia (GA) could be detrimental to cognitive development in young subjects and might also contribute to accelerated neurodegeneration in the elderly. Iron is essential for normal neuronal function, and excess iron in the brain is implicated in several neurodegenerative diseases. However, the role of iron in GA-induced neurotoxicity and cognitive deficits remains elusive. Methods We used the primary hippocampal neurons and rodents including young rats and aged mice to examine whether GA impacted iron metabolism and whether the impact contributed to neuronal outcomes. In addition, a pharmacological suppression of iron metabolism was performed to explore the molecular mechanism underlying GA-mediated iron overload in the brain. Results Our results demonstrated that GA, induced by intravenous ketamine or inhalational sevoflurane, disturbed iron homeostasis and caused iron overload in both in vitro hippocampal neuron culture and in vivo hippocampus. Interestingly, ketamine- or sevoflurane-induced cognitive deficits, very likely, resulted from a novel iron-dependent regulated cell death, ferroptosis. Notably, iron chelator deferiprone attenuated the GA-induced mitochondrial dysfunction, ferroptosis, and further cognitive deficits. Moreover, we found that GA-induced iron overload was activated by NMDAR-RASD1 signalling via DMT1 action in the brain. Conclusion We conclude that disturbed iron metabolism may be involved in the pathogenesis of GA-induced neurotoxicity and cognitive deficits. Our study provides new vision for consideration in GA-associated neurological disorders.

Iron deficiency anemia (IDA) is the most prevalent nutritional deficiency disorder in pregnant women. During pregnancy, placental transport protein Divalent metal transporter1 (DMT1) plays a crucial role in transit of iron across placenta. The developing fetus is observed to be immune to anemia despite presence of anemia in the mother. Hence, we planned the present study to explore the effect of maternal IDA on the expression of DMT1 in the placenta.Two hundred pregnant women recruited, were divided into anemic and nonanemic groups based on their predelivery hemoglobin levels (11 g/dL and ≥11 g/dL respectively). After delivery, placental expression of DMT1 was studied by immunohistochemistry and mRNA analysis and neonatal anthropometry was performed.Of the 200 women recruited, 58.8% were anemic with 60.35% having moderate anemia. Most of the red cell parameters were observed to be higher in cord blood than mothers. DMT1 protein immunohistochemical expression showed a statistically significant increase with increasing severity of anemia. Similarly, placental mRNA expression levels of DMT1 gene were observed to be higher in anemic mothers in comparison with nonanemic mothers.Our study thus demonstrated a definite increase in expression of DMT1 at both protein and mRNA levels in term placenta, in maternal IDA.

Cobalt (Co) is an essential trace element but may cause toxic effects upon occupational or environmental exposure. The present study is aimed to determine the urine concentrations of Co in four years-old children in the INMA-Asturias cohort (Spain) and to assess the factors determining the observed levels. This cohort is located in a heavily industrialized zone with strong potential for metal exposure. Some diet components such as consumption of sweets were meaningfully associated with higher urine Co concentrations. Traffic pollution also showed a noteworthy positive association with Co levels. Family tobacco consumption did not show substantial association with the urine concentrations of this metal in the INMA-Asturias children. A significant inverse association between urine Co and venous blood ferritin was found. Iron deficiency anemic children had significantly higher concentrations of Co than those with normal levels, e.g. median values 1.9 μg/g creatinine and 1.0 μg/g creatinine, respectively. This association could be explained by an increased expression of DMT1, a divalent metal transporter that captures higher levels of iron in deficiency states of this metal. This transporter is non-specific and not only captures iron but also other divalent metals such as Co. The presence of this metal in iron deficiency anemic children may represent an additional disturbing health factor that must be considered during treatment., The authors would particularly like to thank all the participants for their generous collaboration and the staff from Hospital San Agustin in Aviles for their effort. This study was funded by grants from CIBERESP, FIS-FEDER (PI04/2018, PI09/02311, PI13/02429, PI18/00909), Obra Social Cajastur/Fundación Liberbank and Oviedo University. This paper was also supported by funding from the European Union project: EDC-MET (H2020-HEALTH/0490–825762).