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As a novel discovered regulated cell death pattern, ferroptosis has been associated with the development of Parkinson’s disease (PD) and has attracted widespread attention. Nevertheless, the relationship between ferroptosis and PD pathogenesis is still unclear. This study aims to investigate the effect of iron overload on dopaminergic (DA) neurons and its correlation with ferroptosis. Here we use nerve growth factor (NGF) induced PC12 cells which are derived from pheochromocytoma of the rat adrenal to establish a classical PD in vitro model. We found significantly decreased cell viability in NGF-PC12 cell under ammonium ferric citrate (FAC) administration. Moreover, excessive intracellular iron ions induced the increase of (reactive oxygen species) ROS release as well as the decrease of mitochondrial membrane potential in PC12-NGF cells. In addition, we also found that overloaded iron can activate cell apoptosis and ferroptosis pathways, which led to cell death. Furthermore, MPP-induced PD cells were characterized by mitochondrial shrinkage, decreased expression of glutathione peroxidase 4 (Gpx4) and ferritin heavy chain (FTH1), and increased divalent metal transporter (DMT1) and transferrin receptor 1 (TfR1) expression level. In contrast, Lip-1 and DFO increased the expression level of GPX4 and FTH1 compared to MPP-induced PD cell. In conclusion, we indicated that overloaded intracellular iron contributes to neurons death via apoptosis and ferroptosis pathways, while DFO, an iron chelator, can inhibit ferroptosis in order to protect the neurons in vitro. Supplementary Information The online version contains supplementary material available at 10.1007/s11064-021-03262-9.

Cell iron uptake in mammals is commonly distinguished by whether the iron is presented to the cell as transferrin-bound or not: TBI or NTBI. This generic perspective conflates TBI with canonical transferrin receptor, endosomal iron uptake, and NTBI with uptake supported by a plasma membrane-localized divalent metal ion transporter, most often identified as DMT1. In fact, iron uptake by mammalian cells is far more nuanced than this somewhat proscribed view suggests. This view fails to accommodate the substantial role that ZIP8 and ZIP14 play in iron uptake, while adhering to the traditional premise that a relatively high endosomal [H+] is thermodynamically required for release of iron from holo-Tf. The canonical view of iron uptake also does not encompass the fact that plasma membrane electron transport – PMET – has long been linked to cell iron uptake. In fact, the known mammalian metallo-reductases – Dcytb and the STEAP proteins – are members of this cohort of cytochrome-dependent oxido-reductases that shuttle reducing equivalents across the plasma membrane. A not commonly appreciated fact is the reduction potential of ferric iron in holo-Tf is accessible to cytoplasmic reducing equivalents – reduced pyridine and flavin mono- and di-nucleotides and dihydroascorbic acid. This allows for the reductive release of Fe2+ at the extracellular surface of the PM and subsequent transport into the cytoplasm by a neutral pH transporter – a ZIP protein. What this perspective emphasizes is that there are two TfR-dependent uptake pathways, one which does and one which does not involve clathrin-dependent, endolysosomal trafficking. This raises the question as to the selective advantage of having two Tf, TfR-dependent routes of iron accumulation. This review of canonical and non-canonical iron uptake uses cerebral iron trafficking as a point of discussion, a focus that encourages inclusion also of the importance of ferritin as a circulating ‘chaperone’ of ferric iron.

In previous studies we demonstrated that zinc stimulates iron uptake in intestinal Caco-2 cells via Zinc-PI3K-IRP2-DMT1 axis. In the current study we investigated the effect of zinc on basolateral iron release and characterized the associated mechanisms. In Caco-2 cells grown on permeable supports, zinc induced iron transport and expression of DMT1, HEPH mRNA and protein, but not that of FPN1. LY294002, an inhibitor of PI3K, inhibited the zinc-induced iron transport, DMT1, HEPH mRNA and protein expression. In addition, LY294002 also inhibited the basal expression of HEPH and FPN1 resulting in blockade of iron egress from cells. In addition, siRNA-silencing of HEPH led to inhibition of both zinc-induced and basal iron transport. Conversely, TPEN, a chelator of zinc, inhibited iron uptake, DMT1, HEPH and FPN1 mRNA and protein expression. These results suggest that intestinal cell zinc status is a critical determinant of iron absorption and effects are mediated via activation of PI3K. Further, PI3K pathway appears to selectively modulate the expression of iron transporters and iron absorption, therefore this might serve as a therapeutic target in iron overload disorders.

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.

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.

Solute carrier proteins (SLCs) are membrane proteins controlling fluxes across biological membranes and represent an emerging class of drug targets. Here we searched for inhibitors of divalent metal transporters in a library of 1,676 commercially available 3D‐shaped fragment‐like molecules from the generated database GDB‐17, which lists all possible organic molecules up to 17 atoms of C, N, O, S and halogen following simple criteria for chemical stability and synthetic feasibility. While screening against DMT1 (SLC11A2), an iron transporter associated with hemochromatosis and for which only very few inhibitors are known, only yielded two weak inhibitors, our approach led to the discovery of the first inhibitor of ZIP8 (SLC39A8), a zinc transporter associated with manganese homeostasis and osteoarthritis but with no previously reported pharmacology, demonstrating that this target is druggable., Transport restrictions: Screening a library of 1,676 commercially available fragments from the database GDB‐17 revealed two weak inhibitors of DMT1, an iron transporter associated with hemochromatosis, and the first inhibitor of ZIP8, a zinc transporter associated with manganese homeostasis and osteoarthritis.

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.

The absorption of iron is influenced by numerous dietary and physiological factors. We have previously demonstrated that zinc treatment of intestinal cells increases iron absorption via induction of the apical membrane iron transporter divalent metal ion transporter-1 (DMT1). To better understand the mechanisms of zinc-induced iron absorption, we have studied the effect of zinc on iron uptake, iron transporter and iron regulatory protein (IRP 1 and 2) expression and the impact of the PI3K pathway in differentiated Caco-2 cells, an intestinal cell culture model. We found that zinc induces DMT1 protein and mRNA expression. Zinc-induced DMT1 expression and iron absorption were inhibited by siRNA silencing of DMT1. Furthermore, zinc treatment led to increased abundance of IRP2 protein in cell lysates and in polysomal fractions, implying its binding to target mRNAs. Zinc treatment induced Akt phosphorylation, indicating the activation of the PI3K pathway. LY294002, a specific inhibitor of PI3K inhibited zinc-induced Akt phosphorylation, iron uptake, DMT1 and IRP2 expression. Furthermore, LY294002 also decreased the basal level of DMT1 mRNA but not protein expression. siRNA silencing of IRP2 led to down-regulation of both basal and zinc-induced DMT1 protein expression, implying possible involvement of post-transcriptional regulatory mechanisms. In agreement with these findings, zinc treatment stabilized DMT1 mRNA levels in actinomycin D-treated cells. Based on these findings, we conclude that zinc-induced iron absorption involves elevation of DMT1 expression by stabilization of its mRNA, by a PI3K/IRP2-dependent mechanism.

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.

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.

The nicotianamine-iron chelate [NA-Fe2+], which is found in many plant-based foods, has been recently described as a new form of bioavailable iron in mice and chickens. How NA-Fe2+ is assimilated from the diet, however, remains unclear. The current investigation by Murata et al. has identified the proton-coupled amino acid transporter 1 (PAT1) as the main mechanism by which NA-Fe2+ is absorbed in the mammalian intestine. Discovery of this new form of dietary iron and elucidation of its pathway of intestinal absorption may lead to the development of improved iron supplementation approaches.

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.

Alpha-synuclein plays a vital role in the pathology of Parkinson's disease (PD). Spreading of α-synuclein in neighboring cells was believed to contribute to progression in PD. How α-synuclein transmission affects adjacent cells is not full elucidated. Here, we used recombinant α-synuclein to mimic intercellular transmitted α-synuclein in MES23.5 dopaminergic cells, to investigate whether and how it could modulate iron metabolism. The results showed that α-synuclein treatment up-regulated divalent metal transporter 1 (DMT1) and down-regulated iron transporter (FPN), also up-regulated iron regulatory protein 1 (IRP1) protein levels and hepcidin mRNA levels. Endocytosis inhibitor dynasore pretreatment completely abolished and even reversed the upregulation of DMT1 and IRP1 induced by α-synuclein, however, FPN down-regulation was partially blocked by dynasore. Autophagy-inducing agent rapamycin reversed DMT1 up-regulation and FPN down-regulation, and fully blocked the upregulation of IRP1. Elevated hepcidin levels induced by α-synuclein was fully blocked by dynasore pretreatment, however, even higher with rapamycin pretreatment. Alpha-synuclein treatment triggered endoplasmic reticulum (ER) stress. ER stress inducer thapsigargin induced similar responses elicited by α-synuclein. ER stress inhibitor salubrinal blocked the up-regulation of IRP1 and hepcidin, as well as DMT1 up-regulation and FPN down-regulation, also dramatically abolished cAMP-response elements binding protein phosphorylation induced by α-synuclein. Taken together, these finding indicated that extracellular α-synuclein could regulate cellular iron metabolism, probably mediated by ER stress. It provides novel evidence to elucidate the relationships between transmitted α-synuclein and iron metabolism disturbance in PD.

Fumonisin B1 (FB1 ) is a common environmental mycotoxin produced by molds such as Fusarium verticillioides. The toxin poses health risks to domestic animals, including pigs, through FB1 -contaminanted feed. However, the cytotoxicity of FB1 to porcine intestines has not been fully analyzed. In the present study, the effects of FB1 on oxidative stress and nutrient transporter-associated genes of the porcine intestinal IPEC-J2 cells were explored. FB1 decreased IPEC-J2 proliferation but did not trigger reactive oxygen species (ROS) overproduction. Meanwhile, FB1 reduced the expression levels of the transporters l-type amino acid transporter-1 (y+ LAT1), solute carrier family 7 member 1 (SLC7A1), solute carrier family 1 member 5 (ASCT2), and excitatory amino acid carrier 1 (EAAC1); in addition, FB1 reduced the levels of the fatty acid transporters long-chain fatty acid transport protein 1 (FATP1) and long-chain fatty acid transport protein 4 (FATP4) as well as glucose transporters Na+ /glucose cotransporter 1 (SGLT1) and glucose transporter 2 (GLUT2). FB1 stimulation increased the expression levels of peptide transporter peptide transporter 1 (PepT1) and metal ion transport-related gene zinc transporter 1 (ZNT1). Moreover, metal ion transporter divalent metal transporter 1 (DMT1) expression was depressed by a higher dosage of FB1 . The data indicate that FB1 results in aberrant expression of nutrient transporters in IPEC-J2 cells, thereby exerting its toxicity even though it fails to exert ROS-dependent oxidative stress.

Membrane proteins are involved in different physiological functions and are the target of pharmaceutical and abuse drugs. Xenopus laevis oocytes provide a powerful heterologous expression system for functional studies of these proteins. Typical experiments investigate transport using electrophysiology and radiolabeled uptake. A two-electrode voltage clamp is suitable only for electrogenic proteins, and uptake measurements require the existence of radiolabeled substrates and adequate laboratory facilities.Recently, Dictyostelium discoideum Nramp1 and NrampB were characterized using multidisciplinary approaches. NrampB showed no measurable electrogenic activity, and it was investigated in Xenopus oocytes by acquiring confocal images of the quenching of injected fluorophore calcein.This method is adequate to measure the variation in emitted fluorescence, and thus transporter activity indirectly, but requires long experimental procedures to collect statistically consistent data. Considering that optimal expression of heterologous proteins lasts for 48-72 h, a slow acquiring process requires the use of more than one batch of oocytes to complete the experiments. Here, a novel approach to measure substrate uptake is reported. Upon injection of a fluorophore, oocytes were incubated with the substrate and the transport activity measured, evaluating fluorescence quenching in a microplate reader. The technique permits the testing of tens of oocytes in different experimental conditions simultaneously, and thus the collection of significant statistical data for each batch, saving time and animals.The method was tested with different metal transporters (SLC11), DMT1, DdNramp1, and DdNrampB, and verified with the peptide transporter PepT1 (SLC15). Comparison with traditional methods (uptake, two-electrode voltage clamp) and with quenching images acquired by fluorescence microscopy confirmed its efficacy.

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.

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.

Divalent metal transporter 1 (DMT1) is essential for dietary iron assimilation and erythroid iron acquisition. The 3’ untranslated region of the murine DMT1 mRNA contains an iron responsive element (IRE) that is conserved in humans but whose functional role remains unclear. We generated and analyzed mice with targeted disruption of the DMT1 3’IRE. These animals display hypoferremia during the suckling period, associated with a reduction of DMT1 mRNA and protein in the intestine. In contrast, adult mice exhibit hyperferremia, accompanied by enlargement of hepatic and splenic iron stores. Intriguingly, disruption of the DMT1 3’IRE in adult animals augments intestinal DMT1 expression, in part due to increased mRNA translation. Hence, during postnatal growth, the DMT1 3’IRE promotes intestinal DMT1 expression and secures iron sufficiency; in adulthood, it suppresses DMT1 and prevents systemic iron loading. This work demonstrates that the 3’IRE of DMT1 plays a role in the control of DMT1 expression and systemic iron homeostasis, and reveals an age-dependent switch in its activity.Key pointsTargeted mutagenesis of the 3’IRE of DMT1 in mice reveals its importance for maintenance of systemic iron homeostasis.The 3’IRE stimulates intestinal DMT1 expression and prevents hypoferremia during early life, but exerts opposite effects in adulthood

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.

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).

In the light of hepatocyte growth factor (HGF) the inhibiting role on the expression of hepcidin, we hypothesized that HGF might be able to reduce cell and tissue iron by increasing ferroportin 1 (Fpn1) content and Fpn1-mediated iron release from cells and tissues. The hypothesized ability of HGF to reduce iron might be one of the mechanisms associated with its neuroprotective action under the conditions of ischemia/reperfusion (I/R). Here, we investigated the effects of HGF on the expression of hepcidin as well as transferrin receptor 1 (TfR1), divalent metal transporter 1 (DMT1), Fpn1, ferritin and iron regulatory proteins (IRPs) in oxygen-glucose deprivation and reoxygenation (OGD/R)-treated PC12 cells by real-time PCR and Western blot analysis. We demonstrated that HGF could completely reverse the OGD/R-induced reduction in Fpn1 and IRP1 expression and increase in ferritin light chain protein and hepcidin mRNA levels in PC12 cells. It was concluded that HGF protects PC12 cells against OGD/R-induced injury mainly by reducing cell iron contents via the up-regulation of Fpn1 and increased Fpn1-mediated iron export from cells. Our findings suggested that HGF may also be able to ameliorate OGD/R or I/R-induced overloading of brain iron by promoting Fpn1 expression.

Alzheimer’s disease (AD) is characterized by the accumulation of amyloid-beta (Aβ) senile plaques in patients’ brain tissues. Elevated levels of the pro-inflammatory cytokine interleukin-1beta (IL-1β) have been identified in cerebrospinal fluid of living AD patients and in animal models of AD. Increased expression of IL-1β and the accumulation of iron have been identified in microglial cells that cluster around amyloid plaques in AD mouse models and post-mortem brain tissues of AD patients. The goals of this study were to determine the effects of Aβ on the secretion of IL-1β by microglial cells and whether iron status influences this pro-inflammatory signaling cue. To accomplish this goal, immortalized microglial (IMG) cells were incubated with Aβ with or without the addition of iron. qRT-PCR and Western blot analyses showed that Aβ induces the expression and biosynthesis of IL-1β by IMG cells. These microglial cells secrete the mature form of IL-1β in a caspase-1 dependent manner as determined by ELISA. Incubation of IMG cells with iron provoked a greater pro-inflammatory response as measured by these assays. Inhibition of the iron transporter DMT1 protected IMG cells against Aβ-induced inflammation. Potentiation of Aβ-elicited IL-1β induction by FAC was also antagonized by ROS inhibitors, supporting the notion that DMT1-mediated iron loading and a subsequent increase in cellular ROS contribute to the inflammatory effects of Aβ in microglia. Immunoblots and immunofluorescence microscopy data indicated that the presence of iron enhances Aβ activation of NF-κB signaling to promote synthesis of IL-1β. These results support the hypothesis that Aβ stimulates IL-1β expression by activating NF-κB signaling in microglia cells. Most importantly, iron appears to exacerbate the pro-inflammatory effects of Aβ to increase IL-1β levels, most likely through ROS generation. Because IL-1β promotes neuronal degeneration, these results suggest that iron status can profoundly influence the pathogenesis of AD by altering production of this cytokine.

Western blotting has been widely used for investigation of protein expression, posttranslational modifications, and interactions. Because western blotting usually involves heat-denaturation of samples prior to gel loading, clarification of detailed procedures for sample preparation have been omitted or neglected in many publications. We show here the case that even excellent primary antibodies failed to detect a specific protein of interest due to a routine heating practice of protein samples. We performed western blotting for transmembrane iron transporter proteins; SLC11A2 (divalent metal transporter 1, DMT1), SLC40A1 (ferroportin 1, Fpn1), and transferrin receptor-1 (TfR1), along with cytoplasmic iron storage protein ferritin H. Our results in 12 human culture cell lysates indicated that only unheated samples prior to gel loading gave rise to clear resolution of DMT1 protein, while heated samples (95°C, 5min) caused the loss of resolution due to DMT1 protein aggregates. Unheated samples also resulted in better resolution for Fpn1 and TfR1 western blots. Conversely, only heated samples allowed to detect ferritin H, otherwise ferritin polymers failed to get into the gel. Neither different lysis/sample loading buffers nor sonication improved the resolution of DMT1 and Fpn1 western blots. Thus, heating samples most critically affected the outcome of western blotting, suggesting the similar cases for thousands of other transmembrane and heat-sensitive proteins.

Clinical studies suggest that pregnant women with elevated iron levels are more vulnerable to develop gestational diabetes mellitus (GDM), but the causes and underlying mechanisms are unknown. We hypothesized that hyperglycemia induces cellular stress responses leading to dysregulated placental iron homeostasis. Hence, we compared the expression of genes/proteins involved in iron homeostasis in placentae from GDM and healthy pregnancies (n = 11 each). RT-qPCR and LC-MS/MS analyses revealed differential regulation of iron transporters/receptors (DMT1/FPN1/ZIP8/TfR1), iron sensors (IRP1), iron regulators (HEPC), and iron oxidoreductases (HEPH/Zp). To identify the underlying mechanisms, we adapted BeWo trophoblast cells to normoglycemic (N), hyperglycemic (H), and hyperglycemic-hyperlipidemic (HL) conditions and assessed Fe3+ -uptake, expression patterns, and cellular pathways involving oxidative stress (OS), ER-stress, and autophagy. H and HL induced alterations in cellular morphology, differential iron transporter expression, and reduced Fe3+ -uptake confirming the impact of hyperglycemia on iron transport observed in GDM patients. Pathway analysis and rescue experiments indicated that dysregulated OS and disturbed autophagy processes contribute to the reduced placental iron transport under hyperglycemic conditions. These adaptations could represent a protective mechanism preventing the oxidative damage for both fetus and placenta caused by highly oxidative iron. In pregnancies with risk for GDM, antioxidant treatment, and controlled iron supplementation could help to balance placental OS levels protecting mother and fetus from impaired iron homeostasis.

Excessive iron increases the incidence of diabetes and worsens diabetic complications. Reciprocally, diabetes induces iron loading, partially attributable to elevated intestinal iron export according to a recent report. Herein, we show that iron uptake and the mRNA expression of iron importer divalent metal transporter 1 (DMT1) were significantly increased in the duodenum of streptozotocin-induced diabetic mice. Immunofluorescence staining of human intestinal biopsies revealed increased brush border membrane (BBM) and decreased cytoplasmic DMT1 expression in patients with diabetes, suggesting translocation of DMT1. This pattern of DMT1 regulation was corroborated by immunoblotting results in diabetic mice showing that BBM DMT1 expression was increased by 210%, in contrast to a 60% increase in total DMT1. PKC mediates many diabetic complications, and PKCα activity was increased in diabetic mouse intestine. Intriguingly, diabetic mice with PKCα deficiency did not show increases in iron uptake and BBM DMT1 expression. High-glucose treatment increased plasma membrane DMT1 expression via the activation of PKCα in cultured IECs. Inhibition of PKCα potentiated the ubiquitination and degradation of DMT1 protein. We further showed that high glucose suppressed membrane DMT1 internalization. These findings demonstrate that PKCα promotes microvillus membrane DMT1 expression and intestinal iron uptake, contributing to diabetic iron loading.—Zhao, L., Bartnikas, T., Chu, X., Klein, J., Yun, C., Srinivasan, S., He, P. Hyperglycemia promotes microvillus membrane expression of DMT1 in intestinal epithelial cells in a PKCα-dependent manner.

Microglia are immune cells of the central nervous system and are implicated in brain inflammation. However, how brain microglia modulate transport and metabolism of the essential metal iron in response to pro- and anti-inflammatory environmental cues is unclear. Here, we characterized uptake of transferrin (Tf)-bound iron (TBI) and non-Tf-bound iron (NTBI) by immortalized microglial (IMG) cells. We found that these cells preferentially take up NTBI in response to the proinflammatory stimulus lipopolysaccharide (LPS) or β-amyloid (Aβ). In contrast, the anti-inflammatory cytokine interleukin 4 (IL-4) promoted TBI uptake. Concordant with these functional data, levels of the Tf receptor (TfR) in IMG cells were up-regulated in response to IL-4, whereas divalent metal transporter-1 (DMT1) and ferritin levels increased in response to LPS or Aβ. Similar changes in expression were confirmed in isolated primary adult mouse microglia treated with pro- or anti-inflammatory inducers. LPS-induced changes in IMG cell iron metabolism were accompanied by notable metabolic changes, including increased glycolysis and decreased oxidative respiration. Under these conditions, the extracellular acidification rate was increased, compatible with changes in the cellular microenvironment that would support the pH-dependent function of DMT1. Moreover, LPS increased heme oxygenase-1 (HO1) expression in IMG cells, and iron released because of HO1 activity increased the intracellular labile free-iron pool. Together, this evidence indicates that brain microglia preferentially acquire iron from Tf or from non-Tf sources, depending on their polarization state; that NTBI uptake is enhanced by the proinflammatory response; and that under these conditions microglia sequester both extra- and intracellular iron.

Much evidence indicates that iron stored in ferritin is mobilized through protein degradation in lysosomes, but concerns about this process have lingered, and the mechanistic details of its aspects are lacking. In the studies presented here, 59Fe-labeled ferritin was induced by preloading hepatic (HepG2) cells with radiolabeled Fe. Placing these cells in a medium containing desferrioxamine resulted in the loss of ferritin-59Fe, but adding high concentrations of reducing agents or modulating the internal GSH concentration failed to alter the rates of ferritin-59Fe release. Confocal microscopy showed that Fe deprivation increased the movement of ferritin into lysosomes and hyperaccumulation was observed when lysosomal proteolysis was inhibited. It also resulted in the rapid movement of DMT1 to lysosomes, which was inhibited by bafilomycin. Ferrihydrite crystals isolated from purified rat liver/spleen ferritin were solubilized at pH 5 and 7 by GSH, ascorbate, citrate and lysosomal fluids obtained from livers and J774a.1 macrophages. The inhibition of DMT1/Nramp2 and siRNA knockdown of Nramp1 each reduced the transfer of 59Fe from lysosomes to the cytosol; and hepatocyte-specific knockout of DMT1 in mice prevented the release of Fe from the liver responding to EPO treatment, but did not inhibit lysosomal ferritin degradation. We conclude that ferritin-Fe mobilization does not occur through changes in cellular concentrations of reducing/chelating agents but by the coordinated movement of ferritin and DMT1 to lysosomes, where the ferrihydrite crystals exposed by ferritin degradation dissolve in the lysosomal fluid, and the reduced iron is transported back to the cytosol via DMT1 in hepatocytes, and by both DMT1 and Nramp1 in macrophages, prior to release into the blood or storage in ferritin.

Objective The impact of multistrain probiotics on iron (Fe) metabolism under Fe-deficient diet conditions remains unknown. The study aimed to compare the effect of 6 weeks simultaneous and exclusive oral multistrain probiotic and iron supplementation on selected parameters of Fe metabolism in rats on an Fe-deficient diet. Methods Forty rats were assigned to five groups, with eight animals in each, and for 6 weeks received: the CC group- a standard diet, the DD group- an Fe-deficient diet, the DPB group- an Fe-deficient with a multispecies probiotic, the DFE group- an Fe-deficient diet supplemented with iron, the DPBFE group- an Fe-deficient diet with iron and a multispecies probiotic. The Fe content in blood and tissues; serum concentration of erythroferrone, ferritin (Ft), homocysteine, hepcidin (HEPC) and lactoferrin; liver content of divalent metal transporter 1 (DMT1), transferrin receptor protein 1 (TfR1) and 2 (TfR2) and ZRT/IRT-like protein 14 (ZIP14) and faecal microbiota were assessed. Results In DPBFE group, unlike in DPB and DFE groups, duodenal Fe content was higher compared to DD group. Similarly, serum Ft level was higher in DPBFE group, but not in DPB and DFE groups, compared to DD group. Conclusions Six weeks simultaneous oral multistrain probiotic and Fe supplementation, but not exclusive probiotic or Fe intake, increases duodenal Fe absorption in rats and presents higher effectiveness in increasing tissue Fe stores.

Many critical metabolic functions in the brain require adequate and timely delivery of iron. However, most studies when considering brain iron uptake have ignored the iron requirements of the endothelial cells that form the blood-brain barrier (BBB). Moreover, current models of BBB iron transport do not address regional regulation of brain iron uptake or how neurons, when adapting to metabolic demands, can acquire more iron. In this study, we demonstrate that both iron-poor transferrin (apo-Tf) and the iron chelator, deferoxamine, stimulate release of iron from iron-loaded endothelial cells in an in vitro BBB model. The role of the endosomal divalent metal transporter 1 (DMT1) in BBB iron acquisition and transport has been questioned. Here, we show that inhibition of DMT1 alters the transport of iron and Tf across the endothelial cells. These data support an endosome-mediated model of Tf-bound iron uptake into the brain and identifies mechanisms for local regional regulation of brain iron uptake. Moreover, our data provide an explanation for the disparity in the ratio of Tf to iron transport into the brain that has confounded the field.

The present working hypothesis is that absorption of dietary Cu is related to mRNA expressions of genes involved in Cu uptake and transport of the intestine in fish. To this end, the full-length cDNA sequences of eight Cu uptake related genes, including two isoforms of copper transporter genes (ctr1 and ctr2), three copper chaperone genes (atox1, ccs and cox17), two Cu-ATPase genes (atp7a and atp7b) and divalent metal ion transporter 1 (dmt1), were cloned and characterized in yellow catfish P. fulvidraco, respectively. Their mRNA tissue expression and transcriptional responses to dietborne Cu exposure were investigated. Compared to the corresponding members of mammals, all of these members in P. fulvidraco shared the similar conserved domain structures. Their mRNAs were expressed in a wide range of tissues (including liver, muscle, spleen, brain, gill, intestine, heart and kidney), but at variable levels. In anterior intestine, mRNA levels of ctr1, cox17, dmt1 and atp7a declined with increasing dietary Cu levels. The mRNA levels of ctr2 and mt were the highest for excess dietary Cu group and showed no significant differences between other two treatments. Atox1 mRNA levels were the highest for Cu-deficient group and showed no significant differences between other two treatments. The mRNA levels of ccs were the highest for Cu-deficient group, followed by Cu-excess group and the lowest for adequate-Cu group. In contrast, atp7b mRNA levels were the highest for Cu-excess group and the lowest for adequate Cu group. In the mid-intestine, mRNA levels of ctr1, ctr2, atox1, ccs, cox17, dmt1 and atp7a declined with increasing dietary Cu levels. Atp7b mRNA levels were the lowest for adequate Cu group and showed no significant differences between other two treatments. Mt mRNA levels were the lowest for adequate Cu group and highest for Cu-excess group. For the first time, our study cloned and characterized ctr1, ctr2, atox1, ccs, cox17, atp7a, atp7b and dmt1 genes in P. fulvidraco and determined their tissue-specific expression, and transcriptional responses in the anterior and mid-intestine of yellow catfish under dietborne Cu exposure, which shed new light on the Cu uptake system and help to understand the molecular mechanisms of Cu homeostasis in fish.

Iron is an essential micronutrient that participates in a number of vital reactions and its absorption may be altered by various nutritional factors such as other micronutrients. Our hypothesis is that iron absorption is decreased because of the interactions with zinc and calcium. We evaluated the interaction between calcium and zinc on iron uptake and transport, intracellular Fe and Zn levels and mRNA expression of DMT1, ferroportin, Zip4 and ZnT1 in an in vitro model. Caco-2 cells were cultivated with 1 mM Ca; 10 or 30 µM Zn and/or 10, 20 or 30 µM Fe for 24 h. Intracellular Fe decreased in cells incubated with 30 µM Zn or with the mix Ca/10 µM Zn/Fe. Zn mostly increased under Ca, Zn and Fe treatment. DMT1 mRNA expression decreased when intracellular Fe increased. Ferroportin expression displayed no change in cells cultured with different Fe concentrations. The mix of Ca, Zn and Fe increased DMT1 and ferroportin expression mainly under high Zn concentration. Zip4 expression was mostly augmented by Ca and Fe; however, ZnT1 showed no change in all conditions studied. Fe uptake was higher in all the conditions studied compared to control cells; however, Fe transport increased only in cells incubated with Fe alone. In all the other conditions, Fe transport was lower than that in control cells. The present findings suggest that Ca and Zn interfere with iron metabolism. This interference is through an increase in ferroportin activity, which results in a diminished net iron absorption.

Diurnal variations in serum iron levels have been well documented in clinical studies, and serum iron is an important diagnostic index for iron-deficiency anemia. However, the underlying mechanism of dynamic iron regulation in response to the circadian rhythm is still unclear. In this study, we investigated daily variations in iron status in the plasma and liver of pigs. The transcripts encoding key factors involved in iron uptake and homeostasis were evaluated. The results showed that iron levels in the plasma and liver exhibited diurnal rhythms. Diurnal variations were also observed in transcript levels of divalent metal transporter 1 (DMT1), membrane-associated ferric reductase 1 (DCYTB), and transferrin receptor (TfR) in the duodenum and jejunum, as well as hepcidin (HAMP) and TfR in the liver. Moreover, the results showed a network in which diurnal variations in systemic iron levels were tightly regulated by hepcidin and Tf/TfR via DCYTB and DMT1. These findings provide new insights into circadian iron homeostasis regulation. The diurnal variations in serum iron levels may also have pathophysiological implications for clinical diagnostics related to iron deficiency anemia in pigs.

Exposure to divalent metals such as iron and manganese is thought to increase the risk for Parkinson’s disease (PD). Under normal circumstances, cellular iron and manganese uptake is regulated by the divalent metal transporter 1 (DMT1). Accordingly, alterations in DMT1 levels may underlie the abnormal accumulation of metal ions and thereby disease pathogenesis. Here, we have generated transgenic mice overexpressing DMT1 under the direction of a mouse prion promoter and demonstrated its robust expression in several regions of the brain. When fed with iron-supplemented diet, DMT1-expressing mice exhibit rather selective accumulation of iron in the substantia nigra, which is the principal region affected in human PD cases, but otherwise appear normal. Alongside this, the expression of Parkin is also enhanced, likely as a neuroprotective response, which may explain the lack of phenotype in these mice. When DMT1 is overexpressed against a Parkin null background, the double-mutant mice similarly resisted a disease phenotype even when fed with iron- or manganese-supplemented diet. However, these mice exhibit greater vulnerability toward 6-hydroxydopamine-induced neurotoxicity. Taken together, our results suggest that iron accumulation alone is not sufficient to cause neurodegeneration and that multiple hits are required to promote PD. Electronic supplementary material The online version of this article (doi:10.1007/s12017-017-8451-0) contains supplementary material, which is available to authorized users.

Background Protoporphyrin IX (PP IX), the immediate precursor to heme, combines with ferrous iron to make this product. The effects of exogenous PP IX on iron metabolism remain to be elucidated. Peripheral-type benzodiazepine receptor (PBR) is implicated in the transport of coproporphyrinogen into the mitochondria for conversion to PP IX. We have demonstrated that PBR-Associated Protein 7 (PAP7) bound to the Iron Responsive Element (IRE) isoform of divalent metal transporter 1 (DMT1). PP IX and PAP7 are ligands for PBR, thus, we hypothesized that PAP7 interact with PP IX via PBR. Methods We have examined in K562 cells, which can be induced to undergo erythroid differentiation by PP IX and hemin, the effects of PP IX on the expression of PAP7 and other proteins involved in cellular iron metabolism, transferrin receptor 1 (TfR1), DMT1, ferritin heavy chain (FTH), c-Myc and C/EBPα by western blot and quantitative real time PCR analyses. Results PP IX significantly decreased mRNA levels of DMT1 (IRE) and (non-IRE) from 4 h. PP IX markedly decreased protein levels of C/EBPα, PAP7 and DMT1. In contrast, hemin, which like PP IX also induces K562 cell differentiation, had no effect on PAP7 or DMT1 expression. Conclusion We hypothesize that PP IX binds to PBR displacing PAP7 protein, which is then degraded, decreasing the interaction of PAP7 with DMT1 (IRE) and resulting in increased turnover of DMT1. General significance These results suggest that exogenous PP IX disrupts iron metabolism by decreasing the protein expression levels of PAP7, DMT1 and C/EBPα., Highlights • Protoporphyrin IX (PP IX) decreased protein levels of PAP7 and DMT1 in K562. • PP IX decreased mRNA levels of DMT1 (IRE) and (non-IRE) isoforms in K562. • PP IX decreased protein level of C/EBPα, which transcribes DMT1 mRNA, in K562.

A high-mineral polysaccharide (LP) was extracted from Lycium barbarum L. leaves, and the absorption of endogenous calcium, iron, and zinc was evaluated in Caco-2 cells and rat models. The absorption of these minerals ranged from 2.7 to 5.9% in Caco-2 cells, compared with 60.5–81.9% in rat models. LP had a compact globular conformation at pH ∼ 7.2 when co-cultured with Caco-2 cells, and degraded to a loosely linear structure at pH ∼ 6.7 in rat cecum. The degraded polysaccharides didn’t change the mucosal morphometry but caused higher expression of calcium transporter 1 (CAT1) and zinc transporter 1 (ZIP1) proteins in rat cecum. Furthermore, fermentation of polysaccharides in cecum also caused elevated levels of serum cytokines, including TNF-α and IL-6. GL polysaccharides could increase the bioavailability of endogenous calcium via changes in the molecular conformation and through upregulation of the expression of mineral transporter proteins.

Multiple human diseases ensue from a hereditary or acquired deficiency of iron-transporting protein function that diminishes transmembrane iron flux in distinct sites and directions. Because other iron-transport proteins remain active, labile iron gradients build up across the corresponding protein-deficient membranes. Here we report that a small-molecule natural product, hinokitiol, can harness such gradients to restore iron transport into, within, and/or out of cells. The same compound promotes gut iron absorption in DMT1-deficient rats and ferroportin-deficient mice, as well as hemoglobinization in DMT1- and mitoferrin-deficient zebrafish. These findings illuminate a general mechanistic framework for small molecule-mediated site- and direction-selective restoration of iron transport. They also suggest that small molecules that partially mimic the function of missing protein transporters of iron, and possibly other ions, may have potential in treating human diseases.

Cadmium (Cd), a non-essential metal, stealthily enters the cells by utilizing the essential metal importing pathways. The zinc transporters Zip8, Zip14, and divalent metal transporter 1 (Dmt1) are now emerging as several important metal transporters involved in cellular Cd incorporation and their expressions have been shown to be down-regulated in several Cd-resistant (CdR) cell lines, however, the involvement of these transporters during the development of Cd-resistance in lung cells is unclear. In this study, we therefore check the expression of these metal transporters in our previously established rat lung epithelial cells (LECs) and show that the level of Zip8 is progressively silenced when LECs are adapted to increasing concentrations of CdCl2 (from 1 to 20 μM). Subsequent measurement of the cellular Cd content indicated that CdR LECs exhibit a marked decrease of Cd accumulation, possibly due to the loss of Zip8 expression. We investigate the possibility that epigenetic silencing of the Zip8 gene by DNA hypermethylation is involved in the down-regulation of Zip8 expression. CdR LECs show a higher mRNA level of DNA methyltransferase 3b (Dnmt3b) than parental cells. Treatment of CdR LECs with 5-aza-2'-deoxycytidine, an inhibitor of DNA methyltransferases, reverted the expression of Zip8 and sensitivity to Cd in these cells, indicating the critical role of Zip8 for Cd import. Taken together, our results demonstrate that the progressive silencing of Zip8 expression is involved in the acquisition of resistance against Cd in lung cells, representing an adaptive survival mechanism that resists Cd-induced cytotoxicity.

Intracellular retinal iron accumulation has been implicated in the pathogenesis of age-related macular degeneration (AMD), the leading cause of irreversible blindness among individuals over the age of 50. Ceruloplasmin/hephaestin double knockout mice (Cp/Heph DKO) and hepcidin knockout mice (Hepc KO) accumulate retinal iron and model some features of AMD. Two canonical pathways govern cellular iron import – transferrin-bound iron import and non-transferrin bound iron import. In Cp/Heph DKO and Hepc KO iron-loaded retinas, transferrin-bound iron import is downregulated. Despite this effort to reduce cellular iron burden, iron continues to accumulate in these retinas in an age-dependent manner. Quantitative RT-PCR and Western analysis were used to quantify the expression of three ferrous iron importers, Dmt1, Zip8, and Zip14, in wild-type (Wt), Cp/Heph DKO, and Hepc KO retinas. Zip8 and Zip14 protein levels were analyzed using Western analysis in mice injected intravitreally with either apo- or holo-transferrin to elucidate one possible mechanism of Zip14 regulation in the retina. Both zip8 and zip14 were expressed in the mouse retina. Paradoxically, protein levels of non-transferrin bound iron importers were upregulated in both Cp/Heph DKO and Hepc KO retinas. Intravitreal holo-transferrin injection decreased Zip 14 protein levels. These data indicate that Zip8 and Zip14 may take up increasing amounts of non-transferrin bound iron in these two mouse models of retinal iron accumulation. Their upregulation in these already iron-loaded retinas suggests a vicious cycle leading to toxicity.

Background The mechanism underlying biological effects, including pro-inflammatory outcomes, of particles deposited in the lung has not been defined. Major conclusions A disruption in iron homeostasis follows exposure of cells to all particulate matter including air pollution particles. Following endocytosis, functional groups at the surface of retained particle complex iron available in the cell. In response to a reduction in concentrations of requisite iron, a functional deficiency can result intracellularly. Superoxide production by the cell exposed to a particle increases ferrireduction which facilitates import of iron with the objective being the reversal of the metal deficiency. Failure to resolve the functional iron deficiency following cell exposure to particles activates kinases and transcription factors resulting in a release of inflammatory mediators and inflammation. Tissue injury is the end product of this disruption in iron homeostasis initiated by the particle exposure. Elevation of available iron to the cell precludes deficiency of the metal and either diminishes or eliminates biological effects. General significance Recognition of the pathway for biological effects after particle exposure to involve a functional deficiency of iron suggests novel therapies such as metal supplementation (e.g. inhaled and oral). In addition, the demonstration of a shared mechanism of biological effects allows understanding the common clinical, physiological, and pathological presentation following exposure to disparate particles. This article is part of a Special Issue entitled Air Pollution, edited by Wenjun Ding, Andrew J. Ghio and Weidong Wu.

There is currently no effective treatment for neurological impairment caused by traumatic brain injury (TBI). It has been reported that excessive iron production in the brain may be a key factor in neurological impairment. In the present study, we investigated the effects of minocycline, a semi-synthetic tetracycline antibiotic, against TBI-induced neurological impairment and explored its underlying mechanism. Neurological impairment was assessed by foot-fault test, cylinder test, wire hang test, and Morris water maze. Nissl staining was performed to evaluate cell viability in the brain. The iron concentrations in cerebrospinal fluid (CSF), serum, and brain tissues were examined. The Fe2+- and Fe3+- chelating activity of minocycline was measured. Finally, the expression levels of important iron metabolism proteins ferritin, transferrin receptor 1 (TfR1), divalent metal transporter 1 (DMT1), ferroportin 1 (FPN1), and hepcidin in the hippocampus and cortex were measured by Western blot analysis. The results indicate that minocycline significantly attenuated the neurological impairment caused by TBI and increased neuronal viability. Minocycline showed a Fe2+- and Fe3+- chelating activity in vitro and reduced the iron concentration in CSF and brain tissues (cortex and hippocampus). Minocycline also inhibited the overexpression of ferritin and TfR1, but did not affect the expression of DMT1. Minocycline restored the expression of FPN1 by decreasing the expression of hepcidin. In conclusion, minocycline may attenuate neurological impairment caused by TBI and regulate iron metabolism.

The efficiency of maghemite nanoparticles for the treatment of anemia was sensibly higher when nanoparticles were incorporated onto the probiotic bacterium Lactobacillus fermentum (MNP-bacteria) than when administrated as uncoated nanoparticles (MNP). Plasma iron and hemoglobin, intestine expression of divalent metal transporter 1 (DMT1) and duodenal Cytochrome b (DcytB), as well as hepatic expression of the hormone hepcidin were fully restored to healthy levels after administration of MNP-bacteria but not of MNP. A magnetic study on biodistribution and biodegradation showed accumulation of maghemite nanoparticles in intestine lumen when MNP-bacteria were administrated. In contrast, MNP barely reached intestine. In vivo MRI studies suggested the internalization of MNP-bacteria into enterocytes, which did not occur with MNP. Transmission electronic microscopy confirmed this internalization. The collective analysis of results point out that L. fermentum is an excellent carrier to overcome the stomach medium and drive maghemite nanoparticles to intestine, where iron absorption occurs. Due the probiotic ability to adhere to the gut wall, MNP-bacteria internalize into the enterocyte, where maghemite nanoparticles are delivered, providing an adequate iron level into enterocyte. This paper advances a new route for effective iron absorption in the treatment of anemia. The efficiency of maghemite nanoparticles for the treatment of anemia was sensibly higher when nanoparticles were incorporated onto the probiotic bacterium Lactobacillus fermentum (MNP-bacteria) than when administrated as uncoated nanoparticles (MNP). Plasma iron and hemoglobin, intestine expression of divalent metal transporter 1 (DMT1) and duodenal Cytochrome b (DcytB), as well as hepatic expression of the hormone hepcidin were fully restored to healthy levels after administration of MNP-bacteria but not of MNP. A magnetic study on biodistribution and biodegradation showed accumulation of maghemite nanoparticles in intestine lumen when MNP-bacteria were administrated. In contrast, MNP barely reached intestine. In vivo MRI studies suggested the internalization of MNP-bacteria into enterocytes, which did not occur with MNP. Transmission electronic microscopy confirmed this internalization. The collective analysis of results point out that L. fermentum is an excellent carrier to overcome the stomach medium and drive maghemite nanoparticles to intestine, where iron absorption occurs. Due the probiotic ability to adhere to the gut wall, MNP-bacteria internalize into the enterocyte, where maghemite nanoparticles are delivered, providing an adequate iron level into enterocyte. This paper advances a new route for effective iron absorption in the treatment of anemia.