Systemic Iron Deficiency Research Articles

Vitamin A deficiency modulates iron metabolism independent of hemojuvelin (Hfe2) and bone morphogenetic protein 6 (Bmp6) transcript levels.

BackgroundConsidering that vitamin A deficiency modulates hepcidin expression and consequently affects iron metabolism, we evaluated the effect of vitamin A deficiency in the expression of genes involved in the hemojuvelin (HJV)-bone morphogenetic protein 6 (BMP6)-small mothers against decapentaplegic protein (SMAD) signaling pathway.MethodsMale Wistar rats were treated: control AIN-93G diet (CT), vitamin A-deficient diet (VAD), iron-deficient diet (FeD), vitamin A- and iron-deficient diet (VAFeD), or 12 mg all-trans retinoic acid (atRA)/kg diet.ResultsVitamin A deficiency (VAD) increased hepatic Bmp6 and Hfe2 mRNA levels and down-regulated hepatic Hamp, Smad7, Rarα, and intestinal Fpn1 mRNA levels compared with the control. The FeD rats showed lower hepatic Hamp, Bmp6, and Smad7 mRNA levels compared with those of the control, while in the VAFeD rats only Hamp and Smad7 mRNA levels were lower than those of the control. The VAFeD diet up-regulated intestinal Dmt1 mRNA levels in relation to those of the control. The replacement of retinyl ester by atRA did not restore hepatic Hamp mRNA levels; however, the hepatic Hfe2, Bmp6, and Smad7 mRNA levels were similar to the control. The atRA rats showed an increase of hepatic Rarα mRNA levels and a reduction of intestinal Dmt1 mRNA and Fpn1 levels compared with those of the control.ConclusionsThe HJV-BMP6-SMAD signaling pathway that normally activates the expression of hepcidin in iron deficiency is impaired by vitamin A deficiency despite increased expression of liver Bmp6 and Hfe2 mRNA levels and decreased expression of Smad7 mRNA. This response may be associated to the systemic iron deficiency and spleen iron retention promoted by vitamin A deficiency.Electronic supplementary materialThe online version of this article (doi:10.1186/s12263-016-0519-4) contains supplementary material, which is available to authorized users.

Iron Homeostasis and Inflammatory Status in Mice Deficient for the Cystic Fibrosis Transmembrane Regulator.

BackgroundCystic Fibrosis (CF) is a frequent and lethal autosomal recessive disease caused by mutations in the gene encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). Patients with CF suffer from chronic infections and severe inflammation, which lead to progressive pulmonary and gut diseases. Recently, an expanding body of evidence has suggested that iron homeostasis was abnormal in CF with, in particular, systemic iron deficiency and iron sequestration in the epithelium airway. The molecular mechanisms responsible for iron dysregulation and the relationship with inflammation in CF are unknown.Methods and ResultsWe assessed the impact of CFTR deficiency on systemic and tissue iron homeostasis as well as inflammation in wildtype and CFTR knockout (KO) mice. First, in contrast to the systemic and intestinal inflammation we observed in the CFTR KO mice, we reported the absence of lung phenotype with regards to both inflammation and iron status. Second, we showed a significant decrease of plasma ferritin levels in the KO mice, as in CF patients, likely caused by a decrease in spleen ferritin levels. However, we measured unchanged plasma iron levels in the KO mice that may be explained by increased intestinal iron absorption.ConclusionThese results indicate that in CF, the lung do not predominantly contributes to the systemic ferritin deficiency and we propose the spleen as the major organ responsible for hypoferritinemia in the KO mouse. These results should provide a better understanding of iron dysregulation in CF patients where treating or not iron deficiency remains a challenging question.

Degenerate DNA Binding By Mutant (E339D) KLF1 Dramatically Alters the Erythroid Transcriptome in the Nan Mouse Model

The C2H2 zinc finger transcription factor KLF1 regulates nearly all aspects of erythroid differentiation including iron trafficking, heme synthesis, globin production, membrane skeleton assembly and the cell cycle. KLF1 is primarily, but not exclusively, a transcriptional activator. Previously we showed that the inbred mouse model Nan (neonatal anemia) carries a missense mutation (E339D) in the second zinc finger of KLF1 that causes severe lifelong anemia in heterozygotes and embryonic lethality (E10-11) in homozygotes. We also showed that E339D confers an altered DNA binding specificity such that mutant Nan-KLF1 fails to properly bind numerous known KLF1 target genes. As a result, fetal liver (FL) expression of many genes including globin (Hbb, Hba), dematin (Dmtn/Epb4.9) and the cell cycle regulators E2F2 (E2f2) and p18 (Cdkn2c) are significantly decreased. To examine the Nan global erythroid transcriptome, we performed RNAseq on E14.5 FLs. Fetuses were genotyped for Y chromosome markers and 3 wild-type (WT) and 3 Nan/+ males were analyzed. Using a stringently filtered dataset (CPM > 1 in at least 3 of 6 samples, FDR 0.05, FC ≥ 2), 610 genes were upregulated and 200 downregulated in Nan vs. WT. Notably, only 18% of theupregulated and 52% of the downregulated genes overlapped the 1078 known KLF1 target genes (defined in prior studies as those differentially expressed in Klf1 -/- vs +/+ FL). Moreover, just 3% of the upregulated Nan genes overlapped genes normally activated by KLF1. Thus, significant ectopic gene expression occurs in Nan. Pathway analysis (IPA, PANTHER) identified Toll receptor signaling, acute phase response signaling, and response to interferon as among the top hits in the upregulated gene set in Nan, indicating an overall inflammatory state that would be expected to exacerbate anemia. Indeed, we confirmed significantly increased levels of serum IFNβ and IRF7 in Nan. In the Nan downregulated gene set, cytoskeletal regulation by Rho GTPase and cell cycle regulation were the top hits. Previous studies identified deficiencies of membrane skeleton proteins in Nan. To analyze cell cycle status, we sorted dividing adult spleen and bone marrow (BM) erythroid precursors (proerythroblasts, basophilic and polychromatophilic erythroblasts) using CD44, Ter119 and FSC. Polychromatophilic erythroblasts in Nan BM were arrested in G1 (significantly increased percentage of cells in G0/G1, significantly decreased percentage in S phase; p < 0.05) compared to saline-injected and phenylhydrazine treated WT controls. Surprisingly, however, no other cell cycle defects were seen among other BM compartments and none were seen in the spleen. We next analyzed gene ontology (GO) terms in both the up- and downregulated Nan gene sets (MGI, Mouse Genome Informatics). Ten cell cycle associated genes were downregulated (e.g., Rgcc, Cdc25b, E2f2/4), and 14 were upregulated (e.g., Gadd45b/g, Map3k8, Map3k6, Aim1) indicating a complicated pattern of gene expression differences and physiological outcomes in Nan. Similarly, expression of genes involved in iron regulation were among those most highly affected in Nan and did not predict the observed physiological outcome. The mitochondrial iron transporter SLC25A37 (Mitoferrin) and the transporter SLC25A38, both important in heme synthesis, and the ferrireductase required for transferrin-mediated iron uptake by red cells, STEAP3, are downregulated in Nan. Hamp encoding hepcidin, a negative regulator of the iron importer, ferroportin, is upregulated in Nan >14x. Ferroportin expression is not concomitantly downregulated, however. Despite these changes, no evidence of systemic iron deficiency has been identified in Nan; serum iron levels are normal. Moreover, the zinc protoporphyrin IX to heme ratio is markedly increased in Nan, indicating that a defect in heme biosynthesis up to and including ferrochelatase is unlikely, and uptake of radiolabeled iron by Nan reticulocytes and incorporation into heme are normal. From these studies we conclude: (1) Nan-KLF1 activates and represses a large number of non-KLF1 target genes; (2) Nan-KLF1 inappropriately increases and decreases expression of subsets of normal KLF1 genes resulting in (3) unpredictable physiogical outcomes in Nan. Overall, we conclude that new homeostatic set points arise in Nan as a result of the combined contributions of a large set of differentially expressed genes. DisclosuresNo relevant conflicts of interest to declare.

Abstract 15716: Tristetraprolin Protects the Heart Against Iron Deficiency by Reducing Expression of Iron-requiring Proteins and Optimizing Cellular Iron Utilization

Background: Iron deficiency is the most common nutrient deficiency in the world, however, patients with severe iron deficiency generally have normal cardiac function. This suggests that a mechanism exists in the heart that maintains cardiac iron levels despite systemic iron deficiency, however, the mechanism for this adaptation of the heart to iron deficiency is not known. We recently discovered an mRNA-binding protein tristetraprolin (TTP) to mediate iron utilization within an iron-deprived cell independently of the canonical iron regulatory proteins (IRPs). Here, we hypothesized that TTP protects the heart against iron deficiency through intracellular conservation of iron, and that disruption of this pathway will make the heart vulnerable to low-iron states. Results: Consistent with the above hypothesis, we demonstrated that hearts of TTP knockout mice have normal baseline function, yet deteriorate when subjected to iron deficiency. On contrary, iron-deficient TTP WT mice show no decline in cardiac function in spite of significant reduction in iron levels. Since TTP is known to reduce protein expression through mRNA degradation, we hypothesized that it halts expression of iron-consuming proteins, reducing overall cardiomyocyte’s iron requirements. We identified three mitochondrial iron-requiring proteins to be negatively regulated by TTP: NDUFS1 (part of complex I), UQCRFS1 (part of complex III), and mitochondrial aconitase (part of the TCA cycle). Specifically, we showed that TTP binds to and degrades mRNAs of these targets. This is based on data that the steady state level of the mRNAs for these proteins are decreased with iron chelation, but do not change with TTP deletion. Additionally, mRNAs of these proteins are protected against after actinomycin-D treatment with TTP deletion. RNA co-immunoprecipitation studies confirmed TTP binding to the mRNAs of these proteins. Thus, in the absence of TTP, these proteins continue to be expressed and act as “iron sinks” in a cell that is already iron deficient. Conclusions: Our results, for the first time, provide the mechanism of cellular adaptation to iron deficiency, and demonstrate a major role for TTP in this process by regulating mitochondrial proteins.

Muscle specific iron deficiency has systemic consequences

Muscle specific iron deficiency has systemic consequences

Iron Distribution in Dietary‐Induced Obese Mice Tissues

Iron homeostasis is required to maintain normal physiological processes such as oxygen delivery, DNA synthesis, and enzymatic reactions. An overall disturbance of iron homeostasis, e.g. systemic iron deficiency, was reported in obese subjects. The objective of this study was to examine tissue iron distribution in the dietary‐induced obese mice. Thirty male C57BL/6J mice were randomly assigned into six dietary treatment groups with various levels of iron (control, high and low) and fat (control and high) for 24 weeks. At the end of 24 weeks, tissues including liver, brain, heart, spleen and kidney were harvested and embedded in paraffin. Tissues were then processed and stained for iron using the Prussian blue reaction. Images were recorded using the Motic BA310 compound light microscope at 1000x magnification. Results showed that brain and spleen were the most responsive organs to dietary fat and iron treatments. In both brain and spleen, iron deposits were observed in high iron groups at both control and high fat levels. In the brain, iron deposits were surprisingly observed in mice treated with high fat low iron diet, indicating the vulnerability of brain to possible iron‐induced oxidative stress. To further evaluate overall brain iron disturbance by high fat diet, the expression of iron‐related proteins (storage, transport, and export proteins) and oxidative stress markers will be measured.

Handling iron in restorative neuroscience.

The transition metal iron exerts essential roles in the central nervous system (CNS) for oxygen transport, myelin formation, and synthesis of neurotransmitters. Being redox active, iron switches between ferrous and ferric states. Switching between oxygen states also makes iron an important inducer of reactive oxygen species through the Fenton and Haber-Weiss reactions. Such reactive oxygen species are potentially damaging to nucleic acids, proteins, and fatty acids, which makes the handling of iron very important. Both of iron overload and iron deficiency are detrimental to cells of the nervous system. Iron overload increases the formation of oxidative species and thereby increasing risks for neuronal death, and regional iron accumulation in the brain is associated with neurodegenerative disorders like Alzheimer's disease and Parkinson's disease (Rouault, 2013; Andersen et al., 2014; Ward et al., 2014). Systemic iron overloading causes hemochromatosis with the surprising absence of brain impairment, which is attributed to down-regulation of the iron uptake and transport at the blood-brain barrier (BBB) (Andersen et al., 2014). Conversely, limited access to iron leads to systemic iron deficiency that may affect the iron levels in the CNS and peripheral nervous system, which can cause cell growth arrest or even cell death. This suggests that maternal iron deficiency seriously can affect the developing foetus, including the brain.

Measurement of total iron in Helicobacter pylori-infected gastric epithelial cells.

Despite the evidence suggesting a role for Helicobacter pylori in the induction of systemic iron deficiency anaemia, little is known about the possibility of infection-associated changes in cellular iron homeostasis at the gastric epithelium. In this study we compared four different techniques for measuring iron in AGS cells, a gastric epithelial cell line that is widely used to model to H. pylori infection in vitro. Inductively coupled plasma-mass spectrometry proved to be an efficient method, but only when large numbers of cells were used. Two colorimetric assays that included the use of concentrated hydrochloric acid with or without potassium ferrocyanide detected iron in the micromolar but not the nanomolar range in cell-free standards. However, the third colorimetric assay that incorporated ferrozine proved to be highly accurate at detecting iron in the nanomolar range, and was able to detect iron in AGS cells, Moreover, using this assay, we were able to show that the level of iron in H. pylori-infected AGS cells is significantly increased when compared to uninfected cells.

The role of iron in the skin and cutaneous wound healing.

In this review article we discuss current knowledge about iron in the skin and the cutaneous wound healing process. Iron plays a key role in both oxidative stress and photo-induced skin damage. The main causes of oxidative stress in the skin include reactive oxygen species (ROS) generated in the skin by ultraviolet (UVA) 320–400 nm portion of the UVA spectrum and biologically available iron. We also discuss the relationships between iron deficiency, anemia and cutaneous wound healing. Studies looking at this fall into two distinct groups. Early studies investigated the effect of anemia on wound healing using a variety of experimental methodology to establish anemia or iron deficiency and focused on wound-strength rather than effect on macroscopic healing or re-epithelialization. More recent animal studies have investigated novel treatments aimed at correcting the effects of systemic iron deficiency and localized iron overload. Iron overload is associated with local cutaneous iron deposition, which has numerous deleterious effects in chronic venous disease and hereditary hemochromatosis. Iron plays a key role in chronic ulceration and conditions such as rheumatoid arthritis (RA) and Lupus Erythematosus are associated with both anemia of chronic disease and dysregulation of local cutaneous iron hemostasis. Iron is a potential therapeutic target in the skin by application of topical iron chelators and novel pharmacological agents, and in delayed cutaneous wound healing by treatment of iron deficiency or underlying systemic inflammation.

Prion Protein Regulates Iron Transport by Functioning as a Ferrireductase

Prion protein (PrPC) is implicated in the pathogenesis of prion disorders, but its normal function is unclear. We demonstrate that PrPC is a ferrireductase (FR), and its absence causes systemic iron deficiency in PrP knock-out mice (PrP-/-). When exposed to non-transferrin-bound (NTB) radioactive-iron (59FeCl3) by gastric-gavage, PrP-/- mice absorb significantly more 59Fe from the intestinal lumen relative to controls, indicating appropriate systemic response to the iron deficiency. Chronic exposure to excess dietary iron corrects this deficiency, but unlike wild-type (PrP+/+) controls that remain iron over-loaded, PrP-/- mice revert back to the iron deficient phenotype after 5 months of chase on normal diet. Bone marrow (BM) preparations of PrP-/- mice on normal diet show relatively less stainable iron, and this phenotype is only partially corrected by intraperitoneal administration of excess iron-dextran. Cultured PrP-/- BM-macrophages incorporate significantly less NTB-59Fe in the absence or presence of excess extracellular iron, indicating reduced uptake and/or storage of available iron in the absence of PrPC. When expressed in neuroblastoma cells, PrPC exhibits NAD(P)H-dependent cell-surface and intracellular FR activity that requires the copper-binding octa-peptide-repeat region and linkage to the plasma membrane for optimal function. Incorporation of NTB-59Fe by neuroblastoma cells correlates with FR activity of PrPC, implicating PrPC in cellular iron uptake and metabolism. These observations explain the correlation between PrPC expression and cellular iron levels, and the cause of iron imbalance in sporadic-Creutzfeldt-Jakob-disease brains where PrPC accumulates as insoluble aggregates.

PGC-1α Regulates Hepatic Hepcidin Expression and Iron Homeostasis in Response to inflammation

Systemic iron homeostasis is finely regulated by the liver through synthesis of the peptide hormone hepcidin (HAMP), which plays an important role in duodenal iron absorption and macrophage iron release. Clinical investigations have shown that chronic and low-grade inflammation leads to the increase of serum HAMP levels and the development of various diseases such as anemia of inflammation. However, gaps remain to fully elucidate the mechanism linking inflammation and iron dysregulation. Here we show that although inflammatory stimuli increase hepatic HAMP expression and cause systemic iron deficiency in mice, they inhibit the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a transcriptional coactivator actively involved in metabolic regulation. Liver-specific overexpression of PGC-1α antagonizes lipopolysaccharide-induced HAMP expression and alleviates various pathophysiological changes similar to anemia of inflammation. Consistently, overexpression of PGC-1α in HepG2 or HuH7 cells also suppresses HAMP expression and reduces iron accumulation. In contrast, knockdown of PGC-1α exaggerates LPS-induced HAMP expression and iron dysregulation. At the molecular level, PGC-1α suppresses HAMP transcription via the interaction with hepatocyte nuclear factor 4α. In addition, PGC-1α is present near hepatocyte nuclear factor 4α-binding site on the proximal HAMP promoter and turns the chromatin structure into an inactive state. Our data suggest a critical role for PGC-1α in the regulation of hepatic HAMP expression and iron homeostasis under inflammatory circumstances.

Genetic Loss of Tmprss6 Increases Effective Erythropoiesis in a Mouse Model of β-Thalassemia

Abstract 482β-thalassemia is a disorder of ineffective erythropoiesis in which oxidative damage caused by unpaired α-globin chains leads to erythroid apoptosis, increased proliferation of erythroid precursors, and impaired erythroid differentiation. Patients develop systemic iron overload that is caused by red blood cell transfusions and by insufficient inhibition of gastrointestinal iron absorption by the iron regulatory hormone hepcidin. Previously we reported that homozygous genetic loss of Tmprss6, a hepatic transmembrane serine protease that inhibits hepcidin expression by the liver, led to hepcidin elevation and systemic iron deficiency in Hbbth3/+ mice, a model of β-thalassemia intermedia. Interestingly, we also found that while maintaining similar hemoglobin levels, Hbbth3/+mice with homozygous loss of Tmprss6 showed a significant reduction in splenomegaly and marked improvement in peripheral red blood cell (RBC) morphology.Here, we investigated the effects of genetic loss of Tmprss6 on erythropoiesis in Hbbth3/+ mice. In mice of different Tmprss6-Hbb genotypes, we used flow cytometry to quantify the proportion of total bone marrow cells of the erythroid lineage by measuring expression of TER119, an antigen expressed from the pro-erythroblast through the mature erythrocyte stage. Additionally, within the TER119+ population, we quantified the different erythroblast subpopulations by analyzing the intensity of forward scatter and CD44 expression. Compared to wild type (Tmprss6+/+Hbb+/+) controls, Hbbth3/+ mice with 2 wild-type Tmprss6 alleles (Tmprss6+/+Hbbth3/+) showed a significant increase in the proportion of total erythroid cells in the bone marrow, significant increases in the proportion of immature erythroid precursors (basophilic and polychromatic erythroblasts) within the erythroid population, and a significant decrease in the proportion of mature RBCs, resulting in a marrow profile consistent with ineffective erythropoiesis. In Hbbth3/+ mice with homozygous Tmprss6 disruption (Tmprss6−/−Hbbth3/+), the proportion of immature erythroid precursors (basophilic and polychromatic erythroblasts) within the erythroid population remained significantly elevated; however, the proportion of total erythroid cells in the bone marrow was no longer increased. Compared to Tmprss6+/+Hbbth3/+ mice, Tmprss6−/−Hbbth3/+ mice showed a significant increase in the proportion of mature RBCs; this was accompanied by a reduction in reactive oxygen species (ROS) production (as assessed by the indicator CM-H2DCFDA) and apoptotic cells (as assessed by annexin V binding) within both the orthochromatic erythroblast/reticulocyte and mature red cell subpopulations. Additionally, compared to Tmprss6+/+Hbbth3/+ mice, Tmprss6−/−Hbbth3/+ mice showed a marked reduction in α-globin precipitates in membrane fractions prepared from peripheral RBCs. Interestingly, when normalized to α-globin mRNA expression, bone marrow mRNA encoding α-hemoglobin stabilizing protein (AHSP), an α-globin chaperone, was significantly higher in Tmprss6−/−Hbbth3/+ mice compared to Tmprss6+/+Hbbth3/+ mice, compatible with the known stabilization of AHSP mRNA under low iron conditions. Together, these findings suggest a model in which genetic loss of Tmprss6 in Hbbth3/+ mice leads to a systemically iron-deficient state in which reduced iron availability to erythroid precursors leads to stabilization of free α-globin chains, a reduction in both ROS formation and erythroid apoptosis, and ultimately more effective erythropoiesis. In the context of previous findings, these results indicate that hepcidin-elevating strategies based on pharmacological inhibition of Tmprss6 might alter the clinical phenotype of β-thalassemia not only by reducing systemic iron loading but also by altering erythroid maturation. Disclosures:Rivella:Novartis Pharmaceuticals: Consultancy; Biomarin: Consultancy; Merganser Biotech: Consultancy, Equity Ownership, Research Funding; Isis Pharma: Consultancy, Research Funding.

Ferritin is the key to dietary iron absorption and tissue iron detoxification inDrosophila melanogaster

Mammalian ferritin is predominantly in the cytosol, with a minor portion found in plasma. In most insects, including Drosophila melanogaster, ferritin belongs to the secretory type. The functional role of secretory ferritin in iron homeostasis remains poorly understood in insects as well as in mammalians. Here we used Drosophila to dissect the involvement of ferritin in insect iron metabolism. Midgut-specific knockdown of ferritin resulted in iron accumulation in the gut but systemic iron deficiency (37% control), accompanied by retarded development and reduced survival (3% survival), and was rescued by dietary iron supplementation (50% survival) or exacerbated by iron depletion (0% survival). These results suggest an essential role of ferritin in removing iron from enterocytes across the basolateral membrane. Expression of wild-type ferritin in the midgut, especially in the iron cell region, could significantly rescue ferritin-null mutants (first-instar larvae rescued up to early adults), indicating iron deficiency as the major cause of early death for ferritin flies. In many nonintestinal tissues, tissue-specific ferritin knockdown also caused local iron accumulation (100% increase) and resulted in severe tissue damage, as evidenced by cell loss. Overall, our study demonstrated Drosophila ferritin is essential to two key aspects of iron homeostasis: dietary iron absorption and tissue iron detoxification.

Treatment of Dyssomnias and Parasomnias in Childhood

Dyssomnias are sleep disorders associated with complaints of insomnia or hypersomnia. The daytime sleepiness of narcolepsy is treated by a combination of planned daytime naps, regular exercise medications such as modafinil, or salts of methylphenidate, or amphetamine. Cataplexy that accompanies narcolepsy is treated with anticholinergic agents, selective serotonin reuptake inhibitors, or sodium oxybate. Children with neurodevelopmental disabilities such as autism have sleep initiation and maintenance difficulties on a multifactorial basis, with favorable response to melatonin in some patients. Childhood onset restless legs syndrome is often familial, associated with systemic iron deficiency, and responsive to iron supplementation and gabapentin. Parasomnias are episodic phenomena events which occur at the sleep -- wake transition or by intrusion on to sleep. Arousal parasomnias such as confusional arousals and sleep walking can sometimes be confused with seizures. A scheme for differentiating arousal parasomnias from nocturnal seizures is provided. Since arousal parasomnias are often triggered by sleep apnea, restless legs syndrome, or acid reflux, treatment measures directed specifically at these disorders often helps in resolution. Clonazepam provided in a low dose at bedtime can also alleviate sleep walking and confusional arousals. Obstructive sleep apnea affects about 2 percent of children. Adeno-tonsillar hypertrophy, cranio-facial anomalies, and obesity are common predisposing factors. Mild obstructive sleep apnea can be treated using a combination of nasal corticosteroids and a leukotriene antagonist. Moderate to severe obstructive sleep apnea are treated with adeno-tonsillectomy, positive airway pressure breathing devices, or weight reduction as indicated. This paper provides an overview of the topic, with an emphasis on management steps. Where possible, the level of evidence for treatment recommendations is indicated.

Restless Legs Syndrome (Willis–Ekbom Disease) in the Older Person

Restless legs syndrome (RLS) is a common condition characterized by uncomfortable sensations deep in the legs, developing at rest, that compel the person to move. Symptoms are worst at night and sleep disturbance is common. RLS occurs in 7–11% of the population in western countries, and is increasingly common in older people. Primary RLS is familial in up to two-thirds of patients. RLS may also be secondary to a number of conditions, including iron deficiency, pregnancy and end-stage renal failure and, perhaps, to neuropathy. Secondary RLS is most common in those presenting for the first time in later life. The pathogenesis of RLS probably involves the interplay between systemic or brain iron deficiency and impaired dopaminergic neurotransmission in the subcortex of the brain. Dopamine agonists are the first-line treatment for those requiring pharmacotherapy; however, correction of underlying problems, in particular iron deficiency, is also important.

High-fat diet causes iron deficiency via hepcidin-independent reduction of duodenal iron absorption

Obesity is often associated with disorders of iron homeostasis; however, the underlying mechanisms are not fully understood. Hepcidin is a key regulator of iron metabolism and may be responsible for obesity-driven iron deficiency. Herein, we used an animal model of diet-induced obesity to study high-fat-diet-induced changes in iron homeostasis. C57BL/6 mice were fed a standard (SD) or high-fat diet (HFD) for 8 weeks, and in addition, half of the mice received high dietary iron (Fe+) for the last 2 weeks. Surprisingly, HFD led to systemic iron deficiency which was traced back to reduced duodenal iron absorption. The mRNA and protein expressions of the duodenal iron transporters Dmt1 and Tfr1 were significantly higher in HFD- than in SD-fed mice, indicating enterocyte iron deficiency, whereas the mRNA levels of the duodenal iron oxidoreductases Dcytb and hephaestin were lower in HFD-fed mice. Neither hepatic and adipose tissue nor serum hepcidin concentrations differed significantly between SD- and HFD-fed mice, whereas dietary iron supplementation resulted in increased hepatic hepcidin mRNA expression and serum hepcidin levels in SD as compared to HFD mice. Our study suggests that HFD results in iron deficiency which is neither due to intake of energy-dense nutrient poor food nor due to increased sequestration in the reticulo-endothelial system but is the consequence of diminished intestinal iron uptake. We found that impaired iron absorption is independent of hepcidin but rather results from reduced metal uptake into the mucosa and discordant oxidoreductases expressions despite enterocyte iron deficiency.

Rescuing iron‐overloaded macrophages by conservative relocation of the accumulated metal

Systemic iron deficiency concomitant with macrophage iron retention is characteristic of iron-refractory anaemias associated with chronic disease. The systemic misdistribution of iron, which is further exacerbated by parenteral iron supplementation, is mainly attributable to iron retention exerted on resident macrophages by hepcidin-mediated down-regulation of the iron exporter ferroportin. We aimed at developing an experimental macrophage-based cell model that recapitulates pathophysiological features of iron misdistribution found in chronic disorders and use it as a screening platform for identifying agents with the potential for relocating the accumulated metal and restoring affected functions. A raw macrophage subline was selected as cell model of iron retention based on their capacity to take up polymeric iron or aged erythrocytes excessively, resulting in a demonstrable increase of cell labile iron pools and oxidative damage that are aggravated by hepcidin. This model provided a three-stage high throughput screening platform for identifying agents with the combined ability to: (i) scavenge cell iron and thereby rescue macrophage cells damaged by iron-overload; (ii) bypass the ferroportin blockade by conveying the scavenged iron to other iron-starved cells in co-culture via transferrin but (iii) without promoting utilization of the scavenged iron by intracellular pathogens. As test agents we used chelators in clinical practice and found the oral chelator deferiprone fulfilled essentially all of the three criteria. We provide a proof of principle for conservative iron relocation as complementary therapeutic approach for correcting the misdistribution of iron associated with chronic disease and exacerbated by parenteral iron supplementation.

Tmprss6 is a genetic modifier of the Hfe-hemochromatosis phenotype in mice

AbstractThe hereditary hemochromatosis protein HFE promotes the expression of hepcidin, a circulating hormone produced by the liver that inhibits dietary iron absorption and macrophage iron release. HFE mutations are associated with impaired hepatic bone morphogenetic protein (BMP)/SMAD signaling for hepcidin production. TMPRSS6, a transmembrane serine protease mutated in iron-refractory iron deficiency anemia, inhibits hepcidin expression by dampening BMP/SMAD signaling. In the present study, we used genetic approaches in mice to examine the relationship between Hfe and Tmprss6 in the regulation of systemic iron homeostasis. Heterozygous loss of Tmprss6 in Hfe−/− mice reduced systemic iron overload, whereas homozygous loss caused systemic iron deficiency and elevated hepatic expression of hepcidin and other Bmp/Smad target genes. In contrast, neither genetic loss of Hfe nor hepatic Hfe overexpression modulated the hepcidin elevation and systemic iron deficiency of Tmprss6−/− mice. These results indicate that genetic loss of Tmprss6 increases Bmp/Smad signaling in an Hfe-independent manner that can restore Bmp/Smad signaling in Hfe−/− mice. Furthermore, these results suggest that natural genetic variation in the human ortholog TMPRSS6 might modify the clinical penetrance of HFE-associated hereditary hemochromatosis, raising the possibility that pharmacologic inhibition of TMPRSS6 could attenuate iron loading in this disorder.

The liver-specific microRNA miR-122 controls systemic iron homeostasis in mice

Systemic iron homeostasis is mainly controlled by the liver through synthesis of the peptide hormone hepcidin (encoded by Hamp), the key regulator of duodenal iron absorption and macrophage iron release. Here we show that the liver-specific microRNA miR-122 is important for regulating Hamp mRNA expression and tissue iron levels. Efficient and specific depletion of miR-122 by injection of a locked-nucleic-acid-modified (LNA-modified) anti-miR into WT mice caused systemic iron deficiency, characterized by reduced plasma and liver iron levels, mildly impaired hematopoiesis, and increased extramedullary erythropoiesis in the spleen. Moreover, miR-122 inhibition increased the amount of mRNA transcribed by genes that control systemic iron levels, such as hemochromatosis (Hfe), hemojuvelin (Hjv), bone morphogenetic protein receptor type 1A (Bmpr1a), and Hamp. Importantly, miR-122 directly targeted the 3′ untranslated region of 2 mRNAs that encode activators of hepcidin expression, Hfe and Hjv. These data help to explain the increased Hamp mRNA levels and subsequent iron deficiency in mice with reduced miR-122 levels and establish a direct mechanistic link between miR-122 and the regulation of systemic iron metabolism.

The Serine Protease Tmprss6 Regulates Hepcidin Expression, but Its Loss Does Not Cause Systemic Iron Deficiency In the Fetal and Neonatal Periods

AbstractAbstract 4258Mutations in TMPRSS6 (matriptase-2), a transmembrane serine protease expressed by the liver, result in the clinical phenotype of iron refractory iron deficiency anemia (IRIDA). Additionally, common polymorphisms in TMPRSS6 have been associated with variation in laboratory parameters of iron homeostasis in healthy populations. TMPRSS6 increases iron absorption by reducing expression of the hepatic hormone, hepcidin, via down-regulation of a BMP/SMAD signaling cascade. Hepcidin promotes the internalization and degradation of the duodenal iron transporter, ferroportin, thereby inhibiting iron absorption. Previous studies have demonstrated that adult mice with Tmprss6 deficiency exhibit elevated hepatic hepcidin mRNA levels that are associated with decreased hepatic iron stores. In one study, genetic loss of Tmprss6 was shown to result in significant elevation of hepatic hepcidin expression in mice at birth; however, whether this hepcidin elevation was associated with abnormalities in iron homeostasis was not reported. We therefore asked if the elevated hepcidin levels present in newborn Tmprss6-/- pups correlate with abnormal parameters of iron homeostasis in the fetal or neonatal periods. To answer this question, we intercrossed Tmprss6+/− mice to generate Tmprss6+/+, Tmprss6+/−, and Tmprss6-/- progeny for phenotypic characterization at either gestational day 17.5 (E17.5) or postnatal day 0 (P0). Consistent with prior observations, Tmprss6-/- pups at P0 showed a 4.6-fold increase in hepatic hepcidin mRNA compared to Tmprss6+/+ littermates (p=.006). However, despite this elevation in hepcidin expression, Tmprss6-/- pups were not pale, and they showed no significant differences in body mass or hepatic non-heme iron concentration compared to Tmprss6+/+ and Tmprss6+/− littermates. At E17.5, Tmprss6-/- fetuses showed a 50-fold increase in hepatic hepcidin mRNA compared to Tmprss6+/+ littermates (p=.005). However, Tmprss6-/- fetuses also were not pale, and they showed no significant difference in body mass compared to Tmprss6+/+ and Tmprss6+/− littermates. Surprisingly, hepatic non-heme iron concentration at E17.5 was significantly higher in Tmprss6-/- fetuses than in Tmprss6+/+ fetuses (p=.003). To determine if the increased hepcidin expression of Tmprss6-/- fetuses might affect iron homeostasis in their pregnant mothers, we measured iron parameters in Tmprss6+/− females gestating E17.5 litters that were enriched for either Tmprss6+/+ or Tmprss6-/- fetuses. No significant effects of fetal genotype on maternal iron parameters were observed. In summary, our results demonstrate that Tmprss6 regulates hepcidin expression in the fetal and neonatal periods in mice. However, Tmprss6 deficiency does not appear to be associated with systemic iron deficiency at these stages of development, and fetal Tmprss6 expression does not have a significant effect on maternal iron homeostasis in late gestation. These results may have implications for understanding the maintenance of iron homeostasis in early development, and may provide insight into the evolution of IRIDA as well as other disorders of iron homeostasis. Disclosures:No relevant conflicts of interest to declare.