Systemic iron status

Iron is one of the essential micronutrients, and as such, is required for growth, development, and normal cellular functioning. In contrast to some other micronutrients such as water-soluble vitamins, there is a significant danger of toxicity if excessive amounts of iron accumulate in the body. A finely tuned feedback control system functions to limit this excessive accumulation by limiting absorption of iron. This chapter will discuss systemic and brain iron homeostasis.     

Cellular iron transport

Iron has a split personality as an essential nutrient that also has the potential to generate reactive oxygen species. We discuss how different cell types within specific tissues manage this schizophrenia. The emphasis in enterocytes is on regulating the body's supply of iron by regulating transport into the blood stream. In developing red blood cells, adaptations in transport manage the body's highest flux of iron. Hepatocytes buffer the body's stock of iron. Macrophage recycle the iron from effete red cells among other iron management tasks. Pneumocytes provide a barrier to prevent illicit entry that, when at risk of breaching, leads to a need to handle the dangers in a fashion essentially shared with macrophage. We also discuss or introduce cell types including renal cells, neurons, other brain cells, and more where our ignorance, currently still vast, needs to be removed by future research.     

Expression of iron transport proteins divalent metal transporter-1, Ferroportin-1, HFE and transferrin receptor-1 in human monocyte-derived dendritic cells

Iron is essential for cell survival and regulates many cell functions. In the context of the immune response, iron-related metabolism is tightly controlled in activated lymphocytes as well as in cells of the innate immunity. More precisely, for dendritic cells (DCs), which are the key cell type in the development of a specific immune response, the importance of iron absorption was recently unravelled by showing that depletion of iron inhibits the maturation of DCs. On this basis, we studied in detail the expression of iron transport proteins and HFE in DCs. We found that iron uptake in this cell type is mediated by divalent-metal transporter 1 (DMT1) and transferrin receptor-1 (TfR) whereas Ferroportin-1 is very weakly expressed. HFE that regulates TfR's activity is also detected at the mRNA level. The expression of DMT1 and HFE barely varies upon endotoxin-induced maturation but TfR is up-regulated and the iron export molecule Ferroportin-1 is down-regulated. As opposed to MHC class II molecules, the intracellular localization of TfR is not changed during maturation. Our results indicate that the uptake of iron during DCs development and maturation is mediated by a strong expression of iron-uptake molecules such as DMT1 and TfR as well as a down-regulation of iron export molecules such as Ferroportin-1.     

Iron limitation and its effect on membrane proteins and siderophore transport inNeurospora crassa

Summary Cells of the fungusNeurospora crassa were grown under iron-deficient and iron-sufficient conditions and their plasma membrane proteins were compared. Three strains were studied:N. crassa 74A (wild type), a siderophore-free mutantN. crassa (arg-5 ota aga) as well as a slime variant ofN. crassa which lacks a cell wall. Plasma membranes were purified, solubilized and analyzed by one-dimensional SDS/polyacrylamide gel electrophoresis yielding approximately 50 distinct protein bands with molecular masses in the range 14–160 kDa. Iron-sufficient and iron-deficient growth resulted in nearly identical plasma membrane protein profiles in all strains. Although minor alterations in the proportion of certain proteins could be detected, significant overproduction of certain membrane proteins during iron limitation could not be observed. Transport of⁵⁵ Fe-labeled siderophores seems to be correlated to the degree of iron limitation. For example, transport rates were enhanced five-fold after 16 h of growth in iron-deficient medium compared to growth in iron-sufficient medium. Extraction and HPLC measurement of siderophores from conidiospores yielded approximately 10^–15 mol/spore, indicating that germination tubes and young cells used for transport measurements are not iron-deficient. It is suggested that the putative transport systems for siderophores in fungal plasma membranes are constitutively expressed and enhanced uptake of siderophores during iron limitation is rather the result of cellular transport regulation mechanisms.     

Impaired Iron Transport Activity of Ferroportin 1 in Hereditary Iron Overload

To investigate the functional significance of mutations in Ferroportin that cause hereditary iron overload, we directly measured the iron efflux activity of the proteins expressed in Xenopus oocytes. We found that wild type and mutant Ferroportin molecules (A77D, N144H, Q248H and V162Δ) were all expressed at the plasma membrane at similar levels. All mutations caused significant reductions in ⁵⁹Fe efflux compared to wild type but all retained some residual transport activity. A77D had the strongest effect on ⁵⁹Fe efflux (remaining activity 9% of wild-type control), whereas the N144H mutation retained the highest efflux activity (42% of control). The Q248H and V162Δ mutations were intermediate between these values. Co-injection of mutant and wild-type mRNAs revealed that the A77D and N144H mutations had a dominant negative effect on the function of the WT protein.     

膜铁转运蛋白1，铁调素的靶分子？

膜铁转运蛋白1是重要的跨膜铁输出分子,主要分布于十二指肠和单核巨噬系统的细胞膜上,参与机体的肠铁吸收和巨噬细胞对铁的再循环等过程。铁调素是调节机体铁代谢平衡的激素,机体通过肝脏分泌的铁调素对铁转运相关蛋白的表达进行调控,从而实现机体自身的铁稳态。最新研究显示,铁调素的靶分子可能是膜铁转运蛋白1,它通过直接的作用引起膜铁转运蛋白1的内化(internalization)、降解,从而调节其在细胞膜上的表达量,进而控制肠铁吸收和巨噬细胞对铁的再循环过程,以维持机体的铁稳态。     

Properties of carnitine transport in rat kidney cortex slices

The properties of carnitine transport were studied in rat kidney cortex slices. Tissue: medium concentration gradients of 7.9 for L-[methyl-¹⁴C]carnitine were attained after 60-min incubation at 37°C in 40 μM substrate. L- and D-carnitine uptake showed saturability. The concentration curves appeared to consist of (1) a high-affinity component, and (2) a lower affinity site. When corrected for the latter components, the estimated K_m for L-carnitine was 90 μM and $\text{V = 22}\text{nmol/min}$ per ml intracellular fluid; for D-carnitine, $\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 166}\text{μM}$ and $\text{V = 15}\text{nmol/min}$ per ml intracellular fluid. The system was stereospecific for L-carnitine. The uptake of L-carnitine was inhibited by (1) D-carnitine, γ-butyrobetaine, and (2) acetyl-L-carnitine. γ-Butyrobetaine and acetyl-L-carnitine were competitive inhibitors of L-carnitine uptake. Carnitine transport was not significantly reduced by choline, betaine, lysine or γ-aminobutyric acid. Carnitine uptake was inhibited by 2,4-dinitrophenol, carbonyl cyanide $\text{m-}\text{chlorophenylhydrazone}$, N₂ atmosphere, KCN, $\text{N-}\text{ethylmaleimide}$, low temperature (4°C) and ouabain. Complete replacement of Na⁺ in the medium by Li⁺ reduced L- and D-carnitine uptake by 75 and 60%, respectively. Complete replacement of K⁺ or Ca²⁺ in the medium also significantly reduces carnitine uptake. Two roles for the carnitine transport system in kidney are proposed: (1) a renal tubule reabsorption system for the steady-state maintenance of plasma carnitine; and (2) maintenance of normal carnitine levels in kidney cells, which is required for fatty acid oxidation.     

肥胖对小鼠十二指肠 DMT1和 FPN1表达的影响

目的：观察肥胖对小鼠十二指肠二价金属离子转运体（divalent metal transporter 1,DMT1）mRNA、膜铁转运蛋白（ferroportin1,FPN1）mRNA及蛋白表达的变化,探讨肥胖影响铁吸收的机制。方法 C57BL／6J小鼠随机分为正常对照组和肥胖模型组,每组6只,通过喂养高脂饲料喂养建立肥胖模型,对照组采用普通饲料饲养,实验干预期14周。建模完成后,采用实时荧光定量PCR方法检测小鼠十二指肠DMT1、FPN1 mRNA 的表达,用Western blot检测小鼠十二指肠FPN1蛋白表达。结果与对照组小鼠相比,肥胖模型组小鼠十二指肠DMT1、FPN1 mRNA表达以及FPN1蛋白表达水平降低,差异具有统计学意义（ P ＜0.05）。结论肥胖会下调机体十二指肠DMT1、FPN1的表达,导致铁吸收不良,为进一步研究肥胖引起铁缺乏机制提供理论和实验依据。     

A disease-causing mutation K240E disrupts ferroportin trafficking by SUMO (ferroportin SUMOylation)

Ferroportin (Fpn/IREG1/MTP1) is the only known transporter mediating iron efflux from epithelial cells and macrophages, and thus regulates how much iron is released into the circulation. Consequently, Fpn mutations are associated with haemochromatosis. Fpn itself is post-translationally regulated by hepcidin (Hepc) which induces its redistribution and degradation in a ubiquitin-dependent process. Together, the two proteins appear to be the nexus for iron homeostasis. Here we show that a rare gain-of-function mutation (K240E) that is associated with iron overload, impedes Fpn binding and subcellular trafficking by the small ubiquitin-like modifier (SUMO). Whereas wild-type Fpn is ensconced within vesicular bodies, the FpnK240E mutant appeared diffused within the cell when co-expressed with SUMO. Furthermore, compared with wild type Fpn, the sumoylation-defective mutant was constitutively-active, resulting in a lower intracellular labile iron pool than the former. These findings suggest that SUMO may regulate iron homeostasis by controlling Fpn trafficking.     

Hypoxic preconditioning increases iron transport rate in astrocytes

The mechanisms involved in the neuroprotection induced by hypoxic preconditioning (HP) have not been fully elucidated. The involvement of hypoxia-inducible factor-1 alpha (HIF-1alpha) in such neuroprotection has been confirmed. There is also evidence showing that a series of genes with important functions in iron metabolism, including transferrin receptor (TfR1) and divalent metal transporter 1 (DMT1), are regulated by HIF-1alpha in response to hypoxia in extra-neural organs or cells. We therefore hypothesized that HP is able to affect the expression of iron metabolism proteins in the brain and that changes in these proteins induced by HP might be associated with the HP-induced neuroprotection. We herein demonstrated for the first time that HP could induce a significant increase in the expression of HIF-1alpha as well as iron uptake (TfR1 and DMT1) and release (ferroportin1) proteins, and thus increase tansferrin-bound iron (Tf-Fe) and non-transferrin-bound iron (NTBI) uptake and iron release in astrocytes. Moreover, HP could lead to a progressive increase in cellular iron content. We concluded that HP has the ability to increase iron transport speed in astrocytes. Based on our findings and the importance of astrocytes in neuronal survival in hypoxic/ischemic preconditioning, we proposed that the increase in iron transport rate and cellular iron in astocytes might be one of the mechanisms associated with the HP-induced neuroprotection. We also demonstrated that ferroportin1 expression was significantly affected by HIF-1alpha in astrocytes, implying that the gene encoding this iron efflux protein might be a hypoxia-inducible one.     

Characterization of three novel pathogenic SLC40A1 mutations and genotype/phenotype correlations in 7 Italian families with type 4 hereditary hemochromatosis

Mutations of SLC40A1 encoding ferroportin (Fpn), the unique cellular iron exporter, severely affect iron homeostasis causing type 4 hereditary hemochromatosis, an autosomal dominant iron overload condition with variable phenotypic manifestations. This disease can be classified as type 4A, better known as “ferroportin disease”, which is due to “loss of function” mutations that lead to decreased iron export from cells, or as type 4B hemochromatosis, which is caused by “gain of function” mutations, conferring partial or complete resistance to hepcidin-mediated Fpn degradation.In this work, we discuss clinical and molecular findings on a group of patients in whom a SLC40A1 single copy missense variant was identified. Three novel variants, p.D181N, p.G204R and p.R296Q were functionally characterized. Fpn D181N and R296Q mutants can be classified as full or partial loss of function, respectively. Replacement of G204 with arginine appears to cause a more complex defect with impact both on iron export function and hepcidin sensitivity. This finding confirms the difficulty of predicting the effect of a mutation on the molecular properties of Fpn in order to provide an exhaustive explanation to the wide variability of the phenotype in type 4 hereditary hemochromatosis.     

Iron Metabolism in the Reticuloendothelial System

Comprised mainly of monocytes and tissue macrophages, the reticuloendothelial system (RES) plays two major roles in iron metabolism: it recycles iron from senescent red blood cells and it serves as a large storage depot for excess iron. Although iron recycling by the RES represents the largest pathway of iron efflux in the body, the precise mechanisms involved have remained elusive. However, studies characterizing the function and regulation of Nramp1, DMT1, HFE, FPN1, CD163, and hepcidin are rapidly expanding our knowledge of the molecular aspects of RE iron handling. This review summarizes fundamental physiological and biochemical aspects of iron metabolism in the RES and focuses on how recent studies have advanced our understanding of these areas. Also discussed are novel insights into the molecular mechanisms contributing to the abnormal RE iron metabolism characteristic of hereditary hemochromatosis and the anemia of chronic disease.     

Inhibitory effect of unconjugated bilirubin on p-aminohippurate transport in rat kidney cortex slices

(1) The effects of unconjugated bilirubin on the accumulation of p-aminohippurate, kinetics of p-aminohippurate uptake, the efflux of pre-accumulated p-aminohippurate and water and electrolyte distribution were investigated in the rat kidney cortical slice. (2) The addition of unconjugated bilirubin to the incubation medium decreased the 60 min slice-to-medium concentration ratio of p-aminohippurate. (3) The decrease in p-aminohippurate accumulation by unconjugated bilirubin was found to be more pronounced by increasing the concentration of pigment in the medium. (4) The rate of uptake of p-aminohippurate as a function of p-aminohippurate concentration differed in aerobiosis and anaerobiosis, and unconjugated bilirubin decreased only the uptake of p-aminohippurate in aerobic conditions. (5) The efflux of pre-accumulated p-aminohippurate decreased when unconjugated bilirubin concentration in the medium was low (10–20 μM) but the efflux increased when the concentration of pigment was much higher (100 μM). (6) The addition of unconjugated bilirubin to the medium (40–100 μM) increased intracellular sodium and total tissue water content, and decreased intracellular potassium and oxygen consumption of tissue. However the slices incubated with low concentration of pigment (20 μM) did not exhibit significative changes in cellular functional parameters. (7) These findings suggest that unconjugated bilirubin impairs p-aminohippurate transport by a complex mechanism that might involve binding of pigment to sites necessary for anion transport, although effects related to pigment toxicity or to its oxidative decomposition are not excluded.     

Uptake of lead and iron by divalent metal transporter 1 in yeast and mammalian cells

Although the divalent metal transporter (DMT1) was suggested to transport a wide range of metals in Xenopus oocytes, recent studies in other models have provided contrasting results. Here, we provide direct evidence demonstrating that DMT1 expressed in yeast mutants defective for high affinity iron transport facilitates the transport of iron with an 'apparent K(m)' of approximately 1.2 microM, and transport of lead with an 'apparent K(m)' of approximately 1.8 microM. DMT1-dependent lead transport was H(+)-dependent and was inhibited by iron. Human embryonic kidney fibroblasts (HEK293 cells) overexpressing DMT1 also showed a higher uptake of lead than HEK293 cells without overexpressing DMT1. These results show that DMT1 transports lead and iron with similar affinity in a yeast model suggesting that DMT1 is a transporter for lead.     

Prion Protein Promotes Kidney Iron Uptake via Its Ferrireductase Activity

Brain iron-dyshomeostasis is an important cause of neurotoxicity in prion disorders, a group of neurodegenerative conditions associated with the conversion of prion protein (PrP^C) from its normal conformation to an aggregated, PrP-scrapie (PrP^Sc) isoform. Alteration of iron homeostasis is believed to result from impaired function of PrP^C in neuronal iron uptake via its ferrireductase activity. However, unequivocal evidence supporting the ferrireductase activity of PrP^C is lacking. Kidney provides a relevant model for this evaluation because PrP^C is expressed in the kidney, and ∼370 μg of iron are reabsorbed daily from the glomerular filtrate by kidney proximal tubule cells (PT), requiring ferrireductase activity. Here, we report that PrP^C promotes the uptake of transferrin (Tf) and non-Tf-bound iron (NTBI) by the kidney in vivo and mainly NTBI by PT cells in vitro. Thus, uptake of ⁵⁹Fe administered by gastric gavage, intravenously, or intraperitoneally was significantly lower in PrP-knock-out (PrP^−/−) mouse kidney relative to PrP^+/+ controls. Selective in vivo radiolabeling of plasma NTBI with ⁵⁹Fe revealed similar results. Expression of exogenous PrP^C in immortalized PT cells showed localization on the plasma membrane and intracellular vesicles and increased transepithelial transport of ⁵⁹Fe-NTBI and to a smaller extent ⁵⁹Fe-Tf from the apical to the basolateral domain. Notably, the ferrireductase-deficient mutant of PrP (PrP^Δ51–89) lacked this activity. Furthermore, excess NTBI and hemin caused aggregation of PrP^C to a detergent-insoluble form, limiting iron uptake. Together, these observations suggest that PrP^C promotes retrieval of iron from the glomerular filtrate via its ferrireductase activity and modulates kidney iron metabolism.     

Inhibition of Mrp2- and Ycf1p-mediated transport by reducing agents: evidence for GSH transport on rat Mrp2

Mammalian Mrp2 and its yeast orthologue, Ycf1p, mediate the ATP-dependent cellular export of a variety of organic anions. Ycf1p also appears to transport the endogenous tripeptide glutathione (GSH), whereas no ATP-dependent GSH transport has been detected in Mrp2-containing mammalian plasma membrane vesicles. Because GSH uptake measurements in isolated membrane vesicles are normally carried out in the presence of 5-10 mM dithiothreitol (DTT) to maintain the tripeptide in the reduced form, the present study examined the effects of DTT and other sulfhydryl-reducing agents on Ycf1p- and Mrp2-mediated transport activity. Uptake of S-dinitrophenyl glutathione (DNP-SG), a prototypic substrate of both proteins, was measured in Ycf1p-containing Saccharomyces cerevisiae vacuolar membrane vesicles and in Mrp2-containing rat liver canalicular plasma membrane vesicles. Uptake was inhibited in both vesicle systems in a concentration-dependent manner by DTT, dithioerythritol, and β-mercaptoethanol, with concentrations of 10 mM inhibiting by ∼40%. DTT’s inhibition of DNP-SG transport was noncompetitive. In contrast, ATP-dependent transport of [³H]taurocholate, a substrate for yeast Bat1p and mammalian Bsep bile acid transporters, was not significantly affected by DTT. DTT also inhibited the ATP-dependent uptake of GSH by Ycf1p. As the DTT concentration in incubation solutions containing rat liver canalicular plasma membrane vesicles was gradually decreased, ATP-dependent GSH transport was now detected. These results demonstrate that Ycf1p and Mrp2 are inhibited by concentrations of reducing agents that are normally employed in studies of GSH transport. When this inhibition was partially relieved, ATP-dependent GSH transport was detected in rat liver canalicular plasma membranes, indicating that both Mrp2 and Ycf1p are able to transport GSH by an ATP-dependent mechanism.     

A simple method for the isolation of basolateral plasma membrane vesicles from rat kidney cortex Enzyme activities and some properties of glucose transport

A procedure for preparing basolateral membrane vesicles from rat renal cortex was developed by differential centrifugation and Percoll density gradient centrifugation, and the uptake of d-[³H]glucose into these vesicles was studied by a rapid filtration technique. (Na⁺ + K⁺)-ATPase, the marker enzyme for basolateral membranes, was enriched 22-fold compared with that found in the homogenate. The rate of d-glucose uptake was almost unaffected by Na⁺ gradient (no overshoot).     

Regulation of L-leucine transport in rat kidney by dexamethasone and triiodothyronine

We have investigated the transport mechanisms involved in the stimulation of renal tubular reabsorption of large amino acids by glucocorticoids in vivo through the examination of activity and expression of specific transport systems L and y(+)L for L-leucine in membrane preparations of rat kidneys. Kidneys were removed from adult female Wistar rats treated with dexamethasone or triiodothyronine, and the fractions of brush-border and basolateral membranes were isolated by density gradient centrifugation. Functional analysis of L-leucine uptake using rapid filtration technique revealed induction of a sodium-dependent, arginine-inhibitable system y(+)L transport component in the basolateral membrane in the dexamethasone-treated group. A minor sodium-independent, BCH-inhibitable, system L transport component was unaffected by glucocorticoids. L-leucine uptake remained unaffected in the triiodothyronine-treated group. Expression of both subunits of the system y(+)L transporter was increased in dexamethasone-treated rat kidneys: Western blot analysis showed a significant (46%) increase of 4F2hc protein abundance in the basolateral membrane fraction and competitive RT-PCR revealed an almost 4-times induced expression of y(+)LAT1 mRNA. Our results indicate that system y(+)L in rat kidney is regulated by glucocorticoids. We suggest that enhancement of both 4F2 heavy chain and y(+)LAT1 light chain is necessary for induction of this transport system in the kidney.