Oral iron therapy: Current concepts and future prospects for improving efficacy and outcomes.

Iron deficiency (ID) and iron-deficiency anaemia (IDA) are global public health concerns, most commonly afflicting children, pregnant women and women of childbearing age. Pathological outcomes of ID include delayed cognitive development in children, adverse pregnancy outcomes and decreased work capacity in adults. IDA is usually treated by oral iron supplementation, typically using iron salts (e.g. FeSO4 ); however, dosing at several-fold above the RDA may be required due to less efficient absorption. Excess enteral iron causes adverse gastrointestinal side effects, thus reducing compliance, and negatively impacts the gut microbiome. Recent research has sought to identify new iron formulations with better absorption so that lower effective dosing can be utilized. This article outlines emerging research on oral iron supplementation and focuses on molecular mechanisms by which different supplemental forms of iron are transported across the intestinal epithelium and whether these transport pathways are subject to regulation by the iron-regulatory hormone hepcidin.

Metal-ion transporter SLC39A8 is required for brain manganese uptake and accumulation.

Manganese (Mn) is an essential nutrient, but is toxic in excess. Whole-body Mn levels are regulated in part by the metal-ion influx transporter SLC39A8, which plays an essential role in the liver by reclaiming Mn from bile. Physiological roles of SLC39A8 in Mn homeostasis in other tissues, however, remain largely unknown. To screen for extrahepatic requirements for SLC39A8 in tissue Mn homeostasis, we crossed Slc39a8-inducible global-KO (Slc39a8 iKO) mice with Slc39a14 KO mice, which display markedly elevated blood and tissue Mn levels. Tissues were then analyzed by inductively coupled plasma-mass spectrometry to determine levels of Mn. Although Slc39a14 KO; Slc39a8 iKO mice exhibited systemic hypermanganesemia and increased Mn loading in the bone and kidney due to Slc39a14 deficiency, we show Mn loading was markedly decreased in the brains of these animals, suggesting a role for SLC39A8 in brain Mn accumulation. Levels of other divalent metals in the brain were unaffected, indicating a specific effect of SLC39A8 on Mn. In vivo radiotracer studies using 54Mn in Slc39a8 iKO mice revealed that SLC39A8 is required for Mn uptake by the brain, but not most other tissues. Furthermore, decreased 54Mn uptake in the brains of Slc39a8 iKO mice was associated with efficient inactivation of Slc39a8 in isolated brain microvessels but not in isolated choroid plexus, suggesting SLC39A8 mediates brain Mn uptake via the blood-brain barrier. These findings establish SLC39A8 as a candidate therapeutic target for mitigating Mn uptake and accumulation in the brain, the primary organ of Mn toxicity.

A mouse model characterizes the roles of ZIP8 in systemic iron recycling and lung inflammation and infection.

ZIP8 (SLC39A8) is a transmembrane divalent metal ion importer that is most highly expressed in the lung and is inducible by inflammatory stimuli. In addition to zinc and manganese, ZIP8 can transport iron, but its specific roles in iron regulation during homeostatic and pathologic processes remain poorly understood. Using a novel global inducible ZIP8 knockout (KO) mouse, we analyzed the role of ZIP8 in steady-state iron homeostasis and during inflammation and infection. We observed an unexpected phenotype of elevated spleen iron levels and decreased serum iron in ZIP8 KO mice, suggesting that ZIP8 plays a role in iron recycling. We also showed that ZIP8 is expressed on lung distal airspace epithelial cells and transports iron from the airway into lung tissue. LPS-induced inflammation induced ZIP8 expression in the lung, but ZIP8 deletion had no detrimental effect on the severity of LPS-induced acute lung injury or on the outcomes of Klebsiella pneumoniae lung infection. Thus, ZIP8 plays a role in systemic iron homeostasis but does not modulate the severity of inflammatory lung injury or the host defense against a common bacterial cause of pneumonia.

Iron and manganese transport in mammalian systems.

Studies in recent years have significantly expanded, refined, and redefined the repertoire of transporters and other proteins involved in iron and manganese (Mn) transport and homeostasis. In this review, we discuss highlights of the recent literature on iron and Mn transport, focusing on the roles of membrane transporters and related proteins. Studies are considered from the vantage point of main organs, tissues, and cell types that actively control whole-body iron or Mn homeostasis, with emphasis on studies in which in vivo metal transport was measured directly or implicated by using knockout mouse models. Overviews of whole-body and cellular iron and Mn homeostasis are also provided to give physiological context for key transporters and to highlight how they participate in the uptake, intracellular trafficking, and efflux of each metal. Important similarities and differences in iron and Mn transport are noted, and future research opportunities and challenges are identified.

Non-transferrin-bound iron transporters.

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.

Metastatic cancers promote cachexia through ZIP14 upregulation in skeletal muscle.

Patients with metastatic cancer experience a severe loss of skeletal muscle mass and function known as cachexia. Cachexia is associated with poor prognosis and accelerated death in patients with cancer, yet its underlying mechanisms remain poorly understood. Here, we identify the metal-ion transporter ZRT- and IRT-like protein 14 (ZIP14) as a critical mediator of cancer-induced cachexia. ZIP14 is upregulated in cachectic muscles of mice and in patients with metastatic cancer and can be induced by TNF-α and TGF-ß cytokines. Strikingly, germline ablation or muscle-specific depletion of Zip14 markedly reduces muscle atrophy in metastatic cancer models. We find that ZIP14-mediated zinc uptake in muscle progenitor cells represses the expression of MyoD and Mef2c and blocks muscle-cell differentiation. Importantly, ZIP14-mediated zinc accumulation in differentiated muscle cells induces myosin heavy chain loss. These results highlight a previously unrecognized role for altered zinc homeostasis in metastatic cancer-induced muscle wasting and implicate ZIP14 as a therapeutic target for its treatment.

SLC39A14 deficiency alters manganese homeostasis and excretion resulting in brain manganese accumulation and motor deficits in mice.

Solute carrier family 39, member 14 (SLC39A14) is a transmembrane transporter that can mediate the cellular uptake of zinc, iron, and manganese (Mn). Studies of Slc39a14 knockout (Slc39a14-/-) mice have documented that SLC39A14 is required for systemic growth, hepatic zinc uptake during inflammation, and iron loading of the liver in iron overload. The normal physiological roles of SLC39A14, however, remain incompletely characterized. Here, we report that Slc39a14-/- mice spontaneously display dramatic alterations in tissue Mn concentrations, suggesting that Mn is a main physiological substrate for SLC39A14. Specifically, Slc39a14-/- mice have abnormally low Mn levels in the liver coupled with markedly elevated Mn concentrations in blood and most other organs, especially the brain and bone. Radiotracer studies using 54Mn reveal that Slc39a14-/- mice have impaired Mn uptake by the liver and pancreas and reduced gastrointestinal Mn excretion. In the brain of Slc39a14-/- mice, Mn accumulated in the pons and basal ganglia, including the globus pallidus, a region susceptible to Mn-related neurotoxicity. Brain Mn accumulation in Slc39a14-/- mice was associated with locomotor impairments, as assessed by various behavioral tests. Although a low-Mn diet started at weaning was able to reverse brain Mn accumulation in Slc39a14-/- mice, it did not correct their motor deficits. We conclude that SLC39A14 is essential for efficient Mn uptake by the liver and pancreas, and its deficiency results in impaired Mn excretion and accumulation of the metal in other tissues. The inability of Mn depletion to correct the motor deficits in Slc39a14-/- mice suggests that the motor impairments represent lasting effects of early-life Mn exposure.

Iron transport proteins: Gateways of cellular and systemic iron homeostasis.

Cellular iron homeostasis is maintained by iron and heme transport proteins that work in concert with ferrireductases, ferroxidases, and chaperones to direct the movement of iron into, within, and out of cells. Systemic iron homeostasis is regulated by the liver-derived peptide hormone, hepcidin. The interface between cellular and systemic iron homeostasis is readily observed in the highly dynamic iron handling of four main cell types: duodenal enterocytes, erythrocyte precursors, macrophages, and hepatocytes. This review provides an overview of how these cell types handle iron, highlighting how iron and heme transporters mediate the exchange and distribution of body iron in health and disease.

The Tumor Suppressor, P53, Decreases the Metal Transporter, ZIP14.

Loss of p53's proper function accounts for over half of identified human cancers. We identified the metal transporter ZIP14 (Zinc-regulated transporter (ZRT) and Iron-regulated transporter (IRT)-like Protein 14) as a p53-regulated protein. ZIP14 protein levels were upregulated by lack of p53 and downregulated by increased p53 expression. This regulation did not fully depend on the changes in ZIP14's mRNA expression. Co-precipitation studies indicated that p53 interacts with ZIP14 and increases its ubiquitination and degradation. Moreover, knockdown of p53 resulted in higher non-transferrin-bound iron uptake, which was mediated by increased ZIP14 levels. Our study highlights a role for p53 in regulating nutrient metabolism and provides insight into how iron and possibly other metals such as zinc and manganese could be regulated in p53-inactivated tumor cells.

The plasma membrane metal-ion transporter ZIP14 contributes to nontransferrin-bound iron uptake by human ß-cells.

The relationship between iron and ß-cell dysfunction has long been recognized as individuals with iron overload display an increased incidence of diabetes. This link is usually attributed to the accumulation of excess iron in ß-cells leading to cellular damage and impaired function. Yet, the molecular mechanism(s) by which human ß-cells take up iron has not been determined. In the present study, we assessed the contribution of the metal-ion transporters ZRT/IRT-like protein 14 and 8 (ZIP14 and ZIP8) and divalent metal-ion transporter-1 (DMT1) to iron uptake by human ß-cells. Iron was provided to the cells as nontransferrin-bound iron (NTBI), which appears in the plasma during iron overload and is a major contributor to tissue iron loading. We found that overexpression of ZIP14 and ZIP8, but not DMT1, resulted in increased NTBI uptake by ßlox5 cells, a human ß-cell line. Conversely, siRNA-mediated knockdown of ZIP14, but not ZIP8, resulted in 50% lower NTBI uptake in ßlox5 cells. In primary human islets, knockdown of ZIP14 also reduced NTBI uptake by 50%. Immunofluorescence analysis of islets from human pancreatic sections localized ZIP14 and DMT1 nearly exclusively to ß-cells. Studies in primary human islets suggest that ZIP14 protein levels do not vary with iron status or treatment with IL-1ß. Collectively, these observations identify ZIP14 as a major contributor to NTBI uptake by ß-cells and suggest differential regulation of ZIP14 in primary human islets compared with other cell types such as hepatocytes.

Measurement of Transferrin- and Non-transferrin-bound Iron Uptake by Mouse Tissues.

Iron in blood plasma is bound to its transport protein transferrin, which delivers iron to most tissues. In iron overload and certain pathological conditions, the carrying capacity of transferrin can become exceeded, giving rise to non-transferrin-bound iron, which is taken up preferentially by the liver, kidney, pancreas, and heart. The measurement of tissue transferrin- and non-transferrin-bound iron (TBI and NTBI, respectively) uptake in vivo can be achieved via intravenous administration of 59Fe-labeled TBI or NTBI followed by gamma counting of various organs. Here we describe a detailed protocol for the measurement of TBI and NTBI uptake by mouse tissues.

SLC39A14 Is Required for the Development of Hepatocellular Iron Overload in Murine Models of Hereditary Hemochromatosis.

Nearly all forms of hereditary hemochromatosis are characterized by pathological iron accumulation in the liver, pancreas, and heart. These tissues preferentially load iron because they take up non-transferrin-bound iron (NTBI), which appears in the plasma during iron overload. Yet, how tissues take up NTBI is largely unknown. We report that ablation of Slc39a14, the gene coding for solute carrier SLC39A14 (also called ZIP14), in mice markedly reduced the uptake of plasma NTBI by the liver and pancreas. To test the role of SLC39A14 in tissue iron loading, we crossed Slc39a14(-/-) mice with Hfe(-/-) and Hfe2(-/-) mice, animal models of type 1 and type 2 (juvenile) hemochromatosis, respectively. Slc39a14 deficiency in hemochromatotic mice greatly diminished iron loading of the liver and prevented iron deposition in hepatocytes and pancreatic acinar cells. The data suggest that inhibition of SLC39A14 may mitigate hepatic and pancreatic iron loading and associated pathologies in iron overload disorders.

Prion protein functions as a ferrireductase partner for ZIP14 and DMT1.

Excess circulating iron is stored in the liver, and requires reduction of non-Tf-bound iron (NTBI) and transferrin (Tf) iron at the plasma membrane and endosomes, respectively, by ferrireductase (FR) proteins for transport across biological membranes through divalent metal transporters. Here, we report that prion protein (PrP(C)), a ubiquitously expressed glycoprotein most abundant on neuronal cells, functions as a FR partner for divalent-metal transporter-1 (DMT1) and ZIP14. Thus, absence of PrP(C) in PrP-knock-out (PrP(-/-)) mice resulted in markedly reduced liver iron stores, a deficiency that was not corrected by chronic or acute administration of iron by the oral or intraperitoneal routes. Likewise, preferential radiolabeling of circulating NTBI with (59)Fe revealed significantly reduced uptake and storage of NTBI by the liver of PrP(-/-) mice relative to matched PrP(+/+) controls. However, uptake, storage, and utilization of ferritin-bound iron that does not require reduction for uptake were increased in PrP(-/-) mice, indicating a compensatory response to the iron deficiency. Expression of exogenous PrP(C) in HepG2 cells increased uptake and storage of ferric iron (Fe(3+)), not ferrous iron (Fe(2+)), from the medium, supporting the function of PrP(C) as a plasma membrane FR. Coexpression of PrP(C) with ZIP14 and DMT1 in HepG2 cells increased uptake of Fe(3+) significantly, and surprisingly, increased the ratio of N-terminally truncated PrP(C) forms lacking the FR domain relative to full-length PrP(C). Together, these observations indicate that PrP(C) promotes, and possibly regulates, the uptake of NTBI through DMT1 and Zip14 via its FR activity. Implications of these observations for neuronal iron homeostasis under physiological and pathological conditions are discussed.

An iron-regulated and glycosylation-dependent proteasomal degradation pathway for the plasma membrane metal transporter ZIP14.

Protein degradation is instrumental in regulating cellular function. Plasma membrane proteins targeted for degradation are internalized and sorted to multivesicular bodies, which fuse with lysosomes, where they are degraded. ZIP14 is a newly identified iron transporter with multitransmembrane domains. In an attempt to dissect the molecular mechanisms by which iron regulates ZIP14 levels, we found that ZIP14 is endocytosed, extracted from membranes, deglycosylated, and degraded by proteasomes. This pathway did not depend on the retrograde trafficking to the endoplasmic reticulum and thus did not involve the well-defined endoplasmic reticulum-associated protein degradation pathway. Iron inhibited membrane extraction of internalized ZIP14, resulting in higher steady-state levels of ZIP14. Asparagine-linked (N-linked) glycosylation of ZIP14, particularly the glycosylation at N102, was required for efficient membrane extraction of ZIP14 and therefore is necessary for its iron sensitivity. These findings highlight the importance of proteasomes in the degradation of endocytosed plasma membrane proteins.

Microarray analysis of rat pancreas reveals altered expression of Alox15 and regenerating islet-derived genes in response to iron deficiency and overload.

It is well known that iron overload can result in pancreatic iron deposition, beta-cell destruction, and diabetes in humans. Recent studies in animals have extended the link between iron status and pancreatic function by showing that iron depletion confers protection against beta-cell dysfunction and diabetes. The aim of the present study was to identify genes in the pancreas that are differentially expressed in response to iron deficiency or overload. Weanling male Sprague-Dawley rats (n = 6/group) were fed iron-deficient, iron-adequate, or iron-overloaded diets for 3 weeks to alter their iron status. Total RNA was isolated from the pancreases and pooled within each group for microarray analyses in which gene expression levels were compared to those in iron-adequate controls. In iron-deficient pancreas, a total of 66 genes were found to be differentially regulated (10 up, 56 down), whereas in iron-overloaded pancreas, 164 genes were affected (82 up, 82 down). The most up-regulated transcript in iron-deficient pancreas was arachidonate 15-lipoxygenase (Alox15), which has been implicated in the development of diabetes. In iron-overloaded pancreas, the most upregulated transcripts were Reg1a, Reg3a, and Reg3b belonging to the regenerating islet-derived gene (Reg) family. Reg expression has been observed in response to pancreatic stress and is thought to facilitate pancreatic regeneration. Subsequent qRT-PCR validation indicated that Alox15 mRNA levels were 4 times higher in iron-deficient than in iron-adequate pancreas and that Reg1a, Reg3a, and Reg3b mRNA levels were 17-36 times higher in iron-overloaded pancreas. The elevated Alox15 mRNA levels in iron-deficient pancreas were associated with 8-fold higher levels of Alox15 protein as indicated by Western blotting. Overall, these data raise the possibility that Reg expression may serve as a biomarker for iron-related pancreatic stress, and that iron deficiency may adversely affect the risk of developing diabetes through up-regulation of Alox15.

Fine-mapping and genetic analysis of the loci affecting hepatic iron overload in mice.

The liver, as the major organ for iron storage and production of hepcidin, plays pivotal roles in maintaining mammalian iron homeostasis. A previous study showed that Quantitative Trait Loci (QTLs) on chromosome 7 (Chr7) and 16 (Chr16) may control hepatic non-heme iron overload in an F2 intercross derived from C57BL/6J (B6) and SWR/J (SWR) mice. In this study, we aimed to validate the existence of these loci and identify the genes responsible for the phenotypic variations by generating congenic mice carrying SWR chromosome segments expanding these QTLs (D7Mit68-D7Mit71 and D16Mit125-D16Mit185, respectively). We excluded involvement of Chr7 based on the lack of iron accumulation in congenic mice. In contrast, liver iron accumulation was observed in Chr16 congenic mice. Through use of a series of subcongenic murine lines the interval on Chr16 was further fine-mapped to a 0.8 Mb segment spanning 11 genes. We found that the mRNA expression pattern in the liver remained unchanged for all 11 genes tested. Most importantly, we detected 4 missense mutations in 3 candidate genes including Sidt1 (P172R), Spice1(R708S), Boc (Q1051R) and Boc (S450-insertion in B6 allele) in the liver of SWR homozygous congenic mice. To further delineate potential modifier gene(s), we reconstituted seven candidate genes, Sidt1, Boc, Zdhhc23, Gramd1c, Atp6v1a, Naa50 and Gtpbp8, in mouse liver through hydrodynamic transfection. However, we were unable to detect significant changes in liver iron levels upon reconstitution of these candidate genes. Taken together, our work provides strong genetic evidence of the existence of iron modifiers on Chr16. Moreover, we were able to delineate the phenotypically responsible region to a 0.8 Mb region containing 11 coding genes, 3 of which harbor missense mutations, using a series of congenic mice.

Hepatocyte divalent metal-ion transporter-1 is dispensable for hepatic iron accumulation and non-transferrin-bound iron uptake in mice.

UNLABELLED: Divalent metal-ion transporter-1 (DMT1) is required for iron uptake by the intestine and developing erythroid cells. DMT1 is also present in the liver, where it has been implicated in the uptake of transferrin-bound iron (TBI) and non-transferrin-bound iron (NTBI), which appears in the plasma during iron overload. To test the hypothesis that DMT1 is required for hepatic iron uptake, we examined mice with the Dmt1 gene selectively inactivated in hepatocytes (Dmt1(liv/liv) ). We found that Dmt1(liv/liv) mice and controls (Dmt1(flox/flox) ) did not differ in terms of hepatic iron concentrations or other parameters of iron status. To determine whether hepatocyte DMT1 is required for hepatic iron accumulation, we crossed Dmt1(liv/liv) mice with Hfe(-) (/) (-) and hypotransferrinemic (Trf(hpx/hpx) ) mice that develop hepatic iron overload. Double-mutant Hfe(-) (/) (-) Dmt1(liv/liv) and Trf(hpx/hpx) ;Dmt1(liv/liv) mice were found to accumulate similar amounts of hepatic iron as did their respective controls. To directly assess the role of DMT1 in NTBI and TBI uptake, we injected (59) Fe-labeled ferric citrate (for NTBI) or (59) Fe-transferrin into plasma of Dmt1(liv/liv) and Dmt1(flox/flox) mice and measured uptake of (59) Fe by the liver. Dmt1(liv/liv) mice displayed no impairment of hepatic NTBI uptake, but TBI uptake was 40% lower. Hepatic levels of transferrin receptors 1 and 2 and ZRT/IRT-like protein 14, which may also participate in iron uptake, were unaffected in Dmt1(liv/liv) mice. Additionally, liver iron levels were unaffected in Dmt1(liv/liv) mice fed an iron-deficient diet. CONCLUSION: Hepatocyte DMT1 is dispensable for hepatic iron accumulation and NTBI uptake. Although hepatocyte DMT1 is partially required for hepatic TBI uptake, hepatic iron levels were unaffected in Dmt1(liv/liv) mice, suggesting that this pathway is a minor contributor to the iron economy of the liver.

ZIP14 and DMT1 in the liver, pancreas, and heart are differentially regulated by iron deficiency and overload: implications for tissue iron uptake in iron-related disorders.

The liver, pancreas, and heart are particularly susceptible to iron-related disorders. These tissues take up plasma iron from transferrin or non-transferrin-bound iron, which appears during iron overload. Here, we assessed the effect of iron status on the levels of the transmembrane transporters, ZRT/IRT-like protein 14 and divalent metal-ion transporter-1, which have both been implicated in transferrin- and non-transferrin-bound iron uptake. Weanling male rats (n=6/group) were fed an iron-deficient, iron-adequate, or iron-overloaded diet for 3 weeks. ZRT/IRT-like protein 14, divalent metal-ion transporter-1 protein and mRNA levels in liver, pancreas, and heart were determined by using immunoblotting and quantitative reverse transcriptase polymerase chain reaction analysis. Confocal immunofluorescence microscopy was used to localize ZRT/IRT-like protein 14 in the liver and pancreas. ZRT/IRT-like protein 14 and divalent metal-ion transporter-1 protein levels were also determined in hypotransferrinemic mice with genetic iron overload. Hepatic ZRT/IRT-like protein 14 levels were found to be 100% higher in iron-loaded rats than in iron-adequate controls. By contrast, hepatic divalent metal-ion transporter-1 protein levels were 70% lower in iron-overloaded animals and nearly 3-fold higher in iron-deficient ones. In the pancreas, ZRT/IRT-like protein 14 levels were 50% higher in iron-overloaded rats, and in the heart, divalent metal-ion transporter-1 protein levels were 4-fold higher in iron-deficient animals. At the mRNA level, ZRT/IRT-like protein 14 expression did not vary with iron status, whereas divalent metal-ion transporter-1 expression was found to be elevated in iron-deficient livers. Immunofluorescence staining localized ZRT/IRT-like protein 14 to the basolateral membrane of hepatocytes and to acinar cells of the pancreas. Hepatic ZRT/IRT-like protein 14, but not divalent metal-ion transporter-1, protein levels were elevated in iron-loaded hypotransferrinemic mice. In conclusion, ZRT/IRT-like protein 14 protein levels are up-regulated in iron-loaded rat liver and pancreas and in hypotransferrinemic mouse liver. Divalent metal-ion transporter-1 protein levels are down-regulated in iron-loaded rat liver, and up-regulated in iron-deficient liver and heart. Our results provide insight into the potential contributions of these transporters to tissue iron uptake during iron deficiency and overload.