Iron and copper metabolism - Miguel Arredondo a, Marco T. Nu n ez b
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Molecular Aspects of Medicine 26 (2005) 313–327
www.elsevier.com/locate/mam
Review
Iron and copper metabolism
Miguel Arredondo a, Marco T. Núñez b,*
a
Nutrition and Food Technology Institute, University of Chile, Casilla 13811, Santiago, Chile
b
Department of Biology, Faculty of Sciences, Cell Dynamics and Biotechnology Center,
University of Chile, Casilla 654, Santiago, Chile
Abstract
Iron and copper are essential nutrients, excesses or deficiencies of which cause impaired cel-
lular functions and eventually cell death. The metabolic fates of copper and iron are intimately
related. Systemic copper deficiency generates cellular iron deficiency, which in humans results
in diminished work capacity, reduced intellectual capacity, diminished growth, alterations in
bone mineralization, and diminished immune response. Copper is required for the function
of over 30 proteins, including superoxide dismutase, ceruloplasmin, lysyl oxidase, cytochrome
c oxidase, tyrosinase and dopamine-b-hydroxylase. Iron is similarly required in numerous
essential proteins, such as the heme-containing proteins, electron transport chain and micro-
somal electron transport proteins, and iron–sulfur proteins and enzymes such as ribonucleo-
tide reductase, prolyl hydroxylase phenylalanine hydroxylase, tyrosine hydroxylase and
aconitase. The essentiality of iron and copper resides in their capacity to participate in one-
electron exchange reactions. However, the same property that makes them essential also gen-
erates free radicals that can be seriously deleterious to cells. Thus, these seemingly paradoxical
properties of iron and copper demand a concerted regulation of cellular copper and iron levels.
Here we review the most salient characteristics of their homeostasis.
2005 Elsevier Ltd. All rights reserved.
Abbreviations: CP, ceruloplasmin; Ctr1, Cu transporter 1; Cu/Zn-SOD, superoxide dismutase 1; COX,
cytochrome c oxidase; IRE, iron-responsive elements; IRP, iron regulatory proteins; LIP, labile or reactive
iron pool; MNK, Menkes ATPase; MT, metallothionein; Tf, transferrin; WND, Wilson disease ATPase
*
Corresponding author.
E-mail address: mnunez@uchile.cl (M.T. Núñez).
0098-2997/$ - see front matter 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.mam.2005.07.010314 M. Arredondo, M.T. Núñez / Molecular Aspects of Medicine 26 (2005) 313–327
Keywords: Metal homeostasis; Labile iron pool; Iron and copper transport
Contents
1. Chemical properties of iron and copper . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314
2. Iron and copper essentiality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315
2.1. Physiological functions of iron . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315
2.2. Physiological functions of copper . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315
3. Iron and copper homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 316
3.1. The labile iron pool and cell iron homeostasis . . . . . . . . . . . . . . . . . . . . . 316
3.2. The labile iron pool. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 316
3.3. Iron regulatory proteins: translational regulation . . . . . . . . . . . . . . . . . . . 317
3.4. Iron transporters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318
3.5. Cell copper homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319
3.6. Competition between copper and iron during intestinal absorption . . . . . . 320
4. Body homeostasis of iron and copper . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321
4.1. Body iron homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321
4.2. Body copper homeostasis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322
5. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323
Acknowledgements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323
1. Chemical properties of iron and copper
Copper (Cu) and iron (Fe) belong to the sub-family of transition elements that
also includes Cr, Mn, Co, Ni and Zn. In living matter, iron exists in two stable oxi-
dative states: ferrous (Fe2+) and ferric (Fe3+). In aqueous media, Fe2+ is spontane-
ously oxidized by molecular oxygen to Fe3+ to form Fe(OH)3. Consequently, the
maximal solubility of Fe in an oxidative environment such as extracellular fluids is
limited by the product solubility constant of Fe(OH)3. At pH 7.0 the maximal solu-
bility of Fe3+ is very low at 1017 M, whereas Fe2+ solubility is much greater at
101 M. Because of the low solubility of Fe in the presence of oxygen, over time
organisms have been forced to evolve proteins that are able to bind Fe3+ and keep
it thermodynamically stable but, at the same time, make it kinetically available for
biological processes. In vertebrates, the function of extracellular Fe3+ binding and
transport is fulfilled by the plasma protein transferrin (Tf), which has two Fe3+ bind-
ing sites with affinity constants on the order of 1–6 · 1022 M1 for Fe3+ (Aisen and
Listwsky, 1980).
In living matter, Cu has two oxidation states: cuprous (Cu1+) and cupric (Cu2+).
Cu2+ is fairly soluble, whereas Cu1+ solubility is in the sub-micromolar range. In
biological systems, Cu is found mainly in the Cu2+form, since in the presence of oxy-
gen or other electron acceptors Cu1+ is readily oxidized to Cu2+. Cu oxidation isM. Arredondo, M.T. Núñez / Molecular Aspects of Medicine 26 (2005) 313–327 315
reversible since Cu2+ can accept an electron from strong reductants such as ascor-
bate and reduced glutathione (Galhardi et al., 2004). As in the case of iron, the abil-
ity to participate in one-electron reactions has allowed Cu to gain a strong foothold
in the redox reactions of living matter.
Through the Fenton reaction, Fe2+ and Cu1+ transform the weak oxidant hydro-
gen peroxide into hydroxyl radical (HO), one of the most reactive species in nature
(Symons and Gutteridge, 1998; Gutteridge and Halliwell, 2000). In the reductive
environment of the cell, Fe3+ or Cu2+ are non-enzymatically reduced back to
Fe2+ or Cu1+ by reductants such as ascorbate and reduced glutathione (Suzuki
and Maitani, 1981; Carter, 1995), giving rise to a vicious cycle of hydroxyl radical
production.
2. Iron and copper essentiality
2.1. Physiological functions of iron
The importance of optimal amounts of Fe for the survival of animals, plants and
microorganisms is well established, and human Fe deficiency is a worldwide prob-
lem. A report from the World Health Organization estimates that 46% of the worldÕs
5- to 14-year-old children are anemic. In addition, 48% of the worldÕs pregnant
women are anemic. The most common symptom of anemia is a general feeling of
lack of energy, which results in lack of productivity (Beard, 2001).
Fe has particularly relevant roles in neuronal and immune functions. Animal
studies have revealed that feeding rats low Fe diets early in life results in irreversible
alterations of brain functions related to insufficient myelination (Beard et al., 2003;
Ortiz et al., 2004) and defective establishment of the dopaminergic tracts (Agarwal,
2001; McGahan et al., 2005). In adult animals or humans, Fe deficiency affects the
immune response in several ways. Macrophages exhibit reduced bactericidal activity
(Hallquist et al., 1992), and neutrophils have reduced activity of the iron-containing
enzyme myeloperoxidase. Myeloperoxidase produces most of the reactive oxygen
intermediates responsible for intracellular killing of pathogens (Britigan et al.,
1996). Fe deficiency also results in decreased T-lymphocyte numbers and in de-
creased T-lymphocyte blastogenesis and mitogenesis in response to mitogens (Kuvi-
bidila et al., 1999; Spear and Sherman, 1992).
2.2. Physiological functions of copper
Cu is an essential nutrient. Infancy represents one of the most critical periods in
life in terms of Cu requirements because rapid growth increases Cu demands,
whereas diets based on milk provide low amounts of the element (Wright et al.,
1991; Lonnerdal, 1996). Although deficiency is the most prominent concern dur-
ing this stage of life, there is also a high risk of toxic effects associated with the inabil-
ity to handle higher Cu exposure because of immature liver function (Lonnerdal,
1996).316 M. Arredondo, M.T. Núñez / Molecular Aspects of Medicine 26 (2005) 313–327 Cu is an essential cofactor in a number of critical enzymes in metabolism, including superoxide dismutase 1 (Cu/Zn-SOD), cytochrome c oxidase (COX), lysyl oxidase and ceruloplasmin (CP) (Tainer et al., 1983; Linder and Hazegh-Azam, 1996; Kaplan and OÔHalloran, 1996; Rucker et al., 1998). Cu/Zn-SOD converts superoxide anions to hydrogen peroxide for further dismutation by catalase. Knock out mice for Cu/Zn- SOD show time-dependent damage to neuromuscular junctions in the hind limbs (Flood et al., 1999). CP oxidizes Fe2+ prior to Tf binding (Miyajima, 2003). The ab- sence of CP does not produce marked changes in Cu metabolism. It does, however, produce a gradual accumulation of Fe in the liver and other tissues (Harris et al., 1995). During severe Cu deficiency, Fe transport within the body is adversely affected, and Fe tends to accumulate in many tissues. Generally, Cu deficiency is accompanied by a hypochromic microcytic anemia similar to that produced by Fe deficiency. Cu metabolism is altered in inflammation, infection, and cancer. In contrast to Fe levels, which decline in serum during infection and inflammation, Cu and CP levels rise. Synthesis and secretion of CP by hepatocytes is stimulated by interleukin-1 and interleukin-6 (Linder and Hazegh-Azam, 1996). Cu is necessary for an efficient im- mune response (Huang and Failla, 2000). In infection, Cu is essential for the produc- tion of interleukin-2 by activated lymphocytic cells (Percival, 1998). In cancer, plasma CP is positively correlated with disease stage (Linder et al., 1981; Senra- Varela et al., 1997), and malignant tumors have concentrations of Cu that are often higher than those of their tissue of origin (Eagon et al., 1999). 3. Iron and copper homeostasis 3.1. The labile iron pool and cell iron homeostasis The components of cell Fe homeostasis are shown in Fig. 1. Besides the transport- ers DMT1 and Ireg1, the scheme includes the Fe storage protein ferritin, Dcytb, the ferrireductase responsible for the reduction of Fe3+ prior to transport by DMT1 (McKie et al., 2001), and the ferroxidase hephaestin, responsible for the oxidation of Fe2+ after transport by Ireg1 and prior to the binding by apoTf (Frazer et al., 2001). Plasma Tf is present either in the apo form or with one or two Fe3+ atoms. Tf saturation in normal individuals is about 35%, which makes monoferric Tf the pre- dominant plasma Tf form (Williams and Moreton, 1980). Under physiological con- ditions, Fe enters the cell by Tf endocytosis. In the acidic pH of the endosome, Fe is released from Tf, reduced to Fe2+ by a ferric reductase, and transported to the cyto- sol (Núñez et al., 1990). Once in the cytosol, Fe2+ becomes part of the labile or reac- tive iron pool (LIP) (Breuer et al., 1995). Eventually, excess Fe is safely stored in ferritin, which can accept up to 4500 Fe3+ atoms in an oxy-hydroxyl form. 3.2. The labile iron pool The LIP is defined as a pool of weakly bound iron. When determined by calcein fluorescence quenching, the affinity constant of LIP complexes is operationally
M. Arredondo, M.T. Núñez / Molecular Aspects of Medicine 26 (2005) 313–327 317
+ TfR FeTf
IRE/IRP
IRE/IRP
- - - Fe-Tf TfR
Fe+3
Hephaestin
Fe3+
Fe2+ DMT1 Fe(LIP)
Fe(LIP) Dcytb Ireg1 Fe+2
DMT1 Fe2+
Dcytb
pH 6
Fe3+ apoTf TfR
FERRITIN TfR apoTf
Fig. 1. Model of cell iron homeostasis. Fe-Tf is internalized by endocytosis into an acidic, pH 6.0,
endosome where it releases its iron. Fe3+ is reduced by an endosomal ferric reductase that uses ascorbate
as the reductant (Núñez et al., 1990). The endosomal ferric reductase has not been positively identified but,
because of its kinetic characteristics, it is probably Dcytb, the duodenal cytochrome b reductase
responsible for Fe3+ reduction prior to absorption by the intestinal epithelia (McKie et al., 2001). Fe2+ is
transported to the cytoplasm by the Fe transporter DMT1. Alternatively, Fe2+ can enter the cell directly
through DMT1 located in the plasma membrane, by a process termed non-Tf-bound (NTB) Fe uptake.
NTB Fe uptake was initially limited to pathological situations in which the Tf Fe binding capacity was
exceeded, but this process may be more common than suspected since even normal individuals have a
fraction of their plasma Fe in the NTB form (I.Z. Cabantchik, personal communication).
defined as318 M. Arredondo, M.T. Núñez / Molecular Aspects of Medicine 26 (2005) 313–327 homeostasis: the transferrin receptor, involved in plasma-to-cell Fe transport, and the iron-storage protein ferritin (reviewed in Eisenstein and Ross, 2003). The activi- ties of both IRP1 and IRP2 respond to changes in cellular Fe through different mechanisms. In iron-replete conditions, IRP1 has a 4S–4Fe cubane structure that renders the protein active as a cytosolic aconitase but inactive for IRE-binding. Low levels of intracellular Fe induce disassembling of the 4S–4Fe cluster, which causes IRP1 to bind and stabilize TfR mRNA. Furthermore, IRP1 binds to ferritin mRNA, thus diminishing its translation. Besides iron, effectors such as nitric oxide (Bouton et al., 1997; Kim and Ponka, 2002), hydrogen peroxide (Martins et al., 1995), hypoxia (Hanson et al., 1999), and phosphorylation (Schalinske and Eisen- stein, 1996) also regulate IRP1. In contrast to IRP1, IRP2 activity is down-regulated through iron-induced oxidative damage followed by ubiquitination and proteasome degradation (Guo et al., 1995). IRP2/ mice are born normal but in adulthood de- velop a movement disorder characterized by ataxia, bradykinesia and tremor (LaV- aute et al., 2001). IRP1/ mice are normal with only slight misregulation of Fe metabolism in the kidney and brown fat (Meyron-Holtz et al., 2004). Thus, IRP2 seems to dominate the physiological regulation of Fe metabolism, whereas IRP1 seems to predominate in pathophysiological conditions. 3.4. Iron transporters The flux of Fe into or out of cells is determined by the activities of the transporters DMT1 and Ireg1 (also called ferroportin 1 and MTP1). DMT1 transports Fe into cells by an electrogenic mechanism that involves the co-transport of Fe2+ and one proton (Gunshin et al., 1997). Four isoforms are generated by alternative splicing of the 5 0 -end exons (exons 1A or 1B) and of the 3 0 -end exons (exons 16 (or +IRE) or 17 (or IRE)) (Hubert and Hentze, 2002). Concurrent with the presence of an IRE element in the 3 0 flanking region, DMT1 expression is down-regulated by high cell Fe content. Nevertheless, there is no agreement about the specific mechanisms causing this regulation. Expression of the +1A/+IRE isoform makes cells particu- larly sensitive to cell Fe levels; expression of the 1A/IRE isoform yields cells that also respond to Fe changes, whereas cells expressing the 1B/+IRE or the 1B/IRE isoforms do not respond. Thus, it is possible that the regulation of DMT1 expression involves two regulatory regions, one contained in exon 1A and another in the IRE- containing 3 0 exon. Ireg1 is the only member of the SLC40 family of transporters and the first re- ported protein that mediates the exit of Fe from cells (McKie et al., 2000). The pro- tein is expressed mainly in enterocytes and macrophages, but the presence of both DMT1 and Ireg1 has also been described in neurons and astrocytes (Burdo et al., 2001). In enterocytes, Ireg1 is responsible for Fe efflux during the process of intesti- nal Fe absorption, while in Kupffer cells, Ireg1 mediates Fe export for reutilization by the bone marrow (Devalia et al., 2002). The regulation of Ireg1 expression is un- known. In enterocytes, Fe deficiency induces Ireg1 expression (McKie et al., 2000), whereas in macrophages, Fe deficiency decreases it (Yang et al., 2002). In a recent study, we reported that SHSY5Y neuroblastoma cells and hippocampal neurons that
M. Arredondo, M.T. Núñez / Molecular Aspects of Medicine 26 (2005) 313–327 319
survive an Fe accumulation protocol evoke an adaptive response consisting of
decreased synthesis of DMT1 and increased synthesis of Ireg1 (Aguirre et al.,
2005). Thus, the concerted regulation of Fe transporters is clearly cell-specific and
adjusts to the particular functions of the cells.
3.5. Cell copper homeostasis
The main components of cell Cu homeostasis are shown in Fig. 2. Two homolo-
gous cation transporting P-type ATPases are the main gene products related to cell
Cu homeostasis, the Menkes and the Wilson ATPases, MNK and WND, respec-
tively (Mercer et al., 1993; Bull et al., 1993). They have similar structures and parallel
roles in regulating the Cu status of cells and tissues and in supplying Cu to secreted
cuproproteins. In most cells, MNK is responsible for excreting Cu when levels be-
come high. In hepatocytes, this role is carried out by WND, disposing excess Cu
plasma
WND
CP
CP
CP
bilis
GSH
TGN
MT
Cu/Zn
SOD
HAH1
Ccs
Cox 17
hCtr1 /
DMT1
Cu1+
Fig. 2. Model of copper uptake and metabolism in hepatocytes. Crossing the plasma membrane through
either Ctr1 or DMT1, most of the Cu is shuttled to the trans Golgi network (TGN) by chaperone Hah1/
Atox1, which delivers Cu to the P-type ATPase located in the TGN. In the case of hepatocytes, it is
ATP7B or WND, the protein defective in Wilson disease. In enterocytes, it is ATP7A or MNK, the protein
defective in Menkes disease. The chaperone protein, Ccs, delivers Cu to cytosolic Cu/Zn superoxide
dismutase (SOD), which dismutates superoxide into hydrogen peroxide. Cox17 delivers Cu to the
mitochondria, where it is required for cytochrome c oxidase. Glutathione (GSH) may also be a chaperone
by binding Cu1+ and delivering it to metallothionein (MT) and to some copper-dependent apoenzymes,
such as SOD (Carroll et al., 2004).320 M. Arredondo, M.T. Núñez / Molecular Aspects of Medicine 26 (2005) 313–327 through the bile. Both proteins supply Cu to the enzymes that are secreted by the cell by pumping the metal ion into the trans Golgi network, where the metal is incorpo- rated into the apoenzyme. The regulation of Cu efflux by the cell is achieved by copper-induced relocalization from the trans Golgi network to the plasma mem- brane in the case of MNK or to an intracellular vesicular compartment in the case of WND. Cu transporter 1 (Ctr1) was identified by using sequence information from anal- ogous genes in yeasts, then cloned in humans and more recently in mice. Ctr1 is homologous to, and can substitute for, yeast Ctr1 in complementation experiments (Zhou and Gitschier, 1997; Moller et al., 2000). Ctr1 is also the candidate Cu trans- porter in the brush border of intestinal cells (Kuo et al., 2001; Lee et al., 2001). Transfection experiments have confirmed that Ctr1 promotes Cu uptake into mam- malian cells. Ctr1 might thus provide for the facilitated diffusion of Cu across the brush border, even at low Cu concentrations. However, it seems likely that Ctr1 is also present in the basolateral membrane, since Cu can enter the enterocyte from the blood. It may even function in both directions, facilitating as well the release of excess Cu into the gastrointestinal tract. Another likely Cu transporter in the brush border is DMT1, already described as a transporter of Fe2+ (Gunshin et al., 1997), Cd2+ (Tallkvist et al., 2001), Mn2+ (Garrick et al., 2003) and Cu1+ (Arre- dondo et al., 2003). Several other genes and gene products involved in Cu transport within mamma- lian cells and across their cell membranes have been identified, most of which are ubiquitously expressed. These are Cu chaperone proteins that carry Cu to specific intracellular sites and enzymes (Markossian and Kurganov, 2003). These chaperones ‘‘help’’ to prevent the presence of free Cu ions by binding them and releasing them directly to their target proteins. Cu entering cells is carried to the trans Golgi net- work by HAH1/ATOX1, which deliver it to the P-type ATPase located there. In the case of the enterocyte, it is ATP7A, or MNK (the protein defective in Menkes disease); in the liver it is ATP7B, or WND (the protein defective in Wilson disease). MNK and WND are thought to then transfer the Cu into the trans Golgi network. Another chaperone, Ccs, delivers Cu to the Cu/Zn-SOD in the cytoplasm; a third, Cox17, takes it to the mitochondria, where it is required for cytochrome c oxidase. Some of the entering Cu will also associate with cytosolic metallothioneins (MTs). Additionally, glutathione could play the role of a general chaperone for Cu ions, delivering it to CTR1 in the plasma membrane, but this has been difficult to docu- ment and remains to be further explored. 3.6. Competition between copper and iron during intestinal absorption Cu and Fe are essential mineral elements that exhibit important interactions and possible competitive inhibitions in transport and bioavailability (Arredondo et al., in press). In Caco-2 cells, Cu1+ inhibits Fe2+ uptake and vice versa (Fig. 3). Moreover, treatment of cells with DMT1 antisense oligonucleotides diminishes DMT1 expres- sion and both Fe and Cu transport (Arredondo et al., 2003). These results suggest that DMT1 mediates both Fe and Cu transport.
M. Arredondo, M.T. Núñez / Molecular Aspects of Medicine 26 (2005) 313–327 321
Cu uptake, pmol x mg protein-1
Fe uptake, pmol x mg protein-1
FeSO4 + AA
Fe:NTA + AA Cu-His + Ascorbate
Fe:NTA
20 FeSO4 30
20
10
10
59
64
1 10 100 1 10 100
A Fe in medium, µ M B Cu in medium, µM
Fig. 3. Competition studies between iron and copper uptake by Caco-2 cells. (A) Caco-2 cells were grown
in bicameral inserts and incubated from the apical side with 5 lM 64Cu–histidine with or without 250 lM
ascorbic acid and with the Fe species indicated. The presence of ascorbate was necessary to obtain
significant apical 64Cu uptake, providing an indication that in Caco-2 cells Cu is transported in the
reduced, +1, state. In the presence of ascorbate, Fe added either as FeSO4 (i.e., in a pure Fe2+ form) or as
Fe-NTA (i.e., in a Fe2+ form achieved after the reaction of Fe3+ with ascorbate) inhibited 64Cu uptake. (B)
Cells were incubated from the apical side with 5 lM 59Fe, 250 lM ascorbic acid and increasing
concentrations (0.5–250 lM) of Cu2+ as a Cu–histidine (Cu–His) complex. 59Fe uptake decreased when
extracellular Cu increased. Fifty percent inhibition of Fe uptake was achieved at 7.1 lM Cu that is at a
Cu:Fe ratio of 1:4. Inhibition increased to 79%, and to 92% when the ratio increased to 10, and to 100,
respectively. From Arredondo et al. (2003), with permission.
4. Body homeostasis of iron and copper
4.1. Body iron homeostasis
Because Fe losses are not regulated, body Fe homeostasis is achieved by its reg-
ulated intestinal absorption. Over the last several years, considerable progress has
been made in identifying key molecular components of the Fe absorption pathway
(Donovan and Andrews, 2004). Two kinds of signals have been proposed to regulate
Fe absorption: the ‘‘erythropoietic regulator’’ and the ‘‘stores regulator’’. The con-
cept of the ‘‘erythropoietic regulator’’ emerged from the observation that intestinal
Fe absorption increases when erythropoietic needs for Fe are not satisfied. Similarly,
the concept of ‘‘stores regulator’’ emerged from the observation that Fe absorption
responds inversely to the size of Fe stores. Hepcidin, a small peptide with antibacte-
rial activity originally identified as LEAP-1 (Krause et al., 2000), was recently recog-
nized as an important regulator of body Fe homeostasis. In accordance with a role
for hepcidin in Fe homeostasis, Pigeon et al. (2001) demonstrated that hepcidin gene
expression was strongly up-regulated in the liver of iron-overloaded mice. Moreover,
knock-out mice for the hepcidin gene showed marked Fe overload (Nicolas et al.,
2002). In contrast, transgenic mice overexpressing hepcidin experienced post-natal322 M. Arredondo, M.T. Núñez / Molecular Aspects of Medicine 26 (2005) 313–327 mortality due to severe microcytic hypochromic anemia (Nicolas et al., 2003). Recent evidence links increased liver hepcidin production to decreased Fe absorption. Apparently, circulating hepcidin levels adjust the amount of Fe absorbed by the duo- denum by regulating the expression of DMT1, Ireg1 and Dcytb (Frazer et al., 2002; Yeh et al., 2004). A model derived from the observed data proposes that alterations in body Fe levels are reflected in the saturation of circulating transferrin. The liver detects changes in transferrin saturation through a process mediated by HFE and TfR1 endocytosis and regulates hepcidin expression such that a reduction in trans- ferrin saturation decreases hepcidin production and vice versa (Frazer and Ander- son, 2003). Keeping with the importance of body Fe homeostasis, it is possible that regula- tion of Fe absorption does not rely on one single inhibitory mechanism. Evidence indicating that HFE may regulate the apical uptake phase comes from observations that HFE-knockout mice have increased DMT1-mediated intestinal Fe uptake (Levy et al., 2000), and that enterocytes from HH patients show increased intestinal DMT1 expression (Byrnes et al., 2002). In line with these studies, it was reported that HFE overexpression in intestinal Caco-2 cells results in inhibition of apical Fe uptake de- spite increased DMT1 content (Arredondo et al., 2001). It was concluded that wild type HFE is a negative regulator of apical Fe uptake by the enterocyte, possibly through the down-regulation of DMT1 activity. 4.2. Body copper homeostasis Body Cu levels are the result of a balance between Cu absorption and Cu excre- tion by the bile (Linder and Hazegh-Azam, 1996). Although important advances have been made in the understanding of Cu excretion, the mechanisms that govern intestinal Cu absorption remain largely a mystery. In humans and other mammals, Cu is absorbed primarily by the small intestine. Although about the same net amounts of dietary Cu and Fe are absorbed daily by humans, the actual amount of Cu absorbed is about 4-times higher, since a great deal of Cu recycles between the intestinal tract and the organs that supply it with secretions. The mechanism by which Cu enters the cells of the intestinal mucosa and crosses into the interstitial fluid and blood is not well understood. Earlier studies indicated that uptake of Cu2+ across the brush border involved both a non-energy-dependent saturable carrier ac- tive at lower Cu concentrations, and diffusion at higher concentrations (Linder and Hazegh-Azam, 1996). Although Ctr1 is the default intestinal Cu transporter (Sharp, 2003), we demonstrated that Cu1+ is also transported by DMT1 (Arredondo et al., 2003). Intestinal (Caco-2 cells), hepatic (HepG2 cells) and kidney (Hek293 cells) cells regulate the uptake of Cu (Arredondo et al., 2000, 2004). In intestinal Caco-2 cells, it was found that pretreatment with excess Cu enhanced uptake and overall transport of 1 lM 64Cu, a response opposite to what would be expected for homeostasis (Arre- dondo et al., 2000; Zerounian and Linder, 2002) In contrast, Cu-depleted cells re- sponded by markedly increasing their uptake and overall transport of copper. These results suggest the possibility of a biphasic response to Cu concentration, with transport increasing at both low and high intracellular concentrations of copper.
M. Arredondo, M.T. Núñez / Molecular Aspects of Medicine 26 (2005) 313–327 323 Within the mucosal cell, most newly absorbed Cu (about 80%) is retained in the cyto- sol, the majority of which is bound to MTs and/or proteins of similar size. Since MTs have high affinity for copper, and their expression is largely increased under high Cu conditions (Sone et al., 1987), it is possible that under elevated Cu condi- tions inflow of Cu will be driven by cytosolic MT binding. 5. Concluding remarks A critical advance in understanding the mechanisms of Fe and Cu homeostasis has been the cloning and characterization of the main components of their homeo- stasis. There are still unknowns in the regulation of DMT1, Ireg1, Dcytb, Ctr1 and the Cu chaperones, but current evidence points to processes of transcriptional regu- lation. Cellular Cu homeostasis seems to be tightly controlled by the activity of metallothioneins and chaperones, with practically no free ionic Cu in the cytosol. In contrast to iron, Cu has an effective mechanism of excretion, which allows for ade- quate control of its body homeostasis. In contrast, the lack of mechanisms for cell secretion and body excretion causes Fe to accumulate in cells and the body. In cells this gives rise to the labile or reactive Fe pool that is probably involved in the induc- tion of oxidative damage to vital cellular components, whereas body Fe accumula- tion may give rise, in time, to neurodegenerative and aging processes. Acknowledgements This work was supported by project P99-031 from the Millennium Scientific Ini- tiative, Santiago, Chile and by grants from Fondo Nacional de Ciencia y Tecnologı´a, Chile, No. 1040448 and Dirección de Investigación, Universidad de Chile, Chile, TNAC 03/07. References Agarwal, K.N., 2001. Iron and the brain: neurotransmitter receptors and magnetic resonance spectroscopy. Br. J. Nutr. 85, S147–S150. Aguirre, P., Mena, N., Tapia, V., Arredondo, M., Núñez, M.T., 2005. Iron homeostasis in neuronal cells: a role for IREG1. BMC Neurosci. 6, 3. Aisen, P., Listwsky, I., 1980. Iron transport and storage proteins. Ann. Rev. Biochem. 49, 357– 393. Arredondo, M., Uauy, R., González, M., 2000. Regulation of copper uptake and transport in intestinal cell monolayers by acute and chronic copper exposure. Biochim. Biophys. Acta 1474, 169–176. Arredondo, M., Muñoz, P., Mura, C.V., Núñez, M.T., 2001. HFE inhibits apical iron uptake by intestinal epithelial (Caco-2) cells. FASEB J. 15, 1276–1278. Arredondo, M., Muñoz, P., Mura, C., Núñez, M.T., 2003. DMT1, a physiologically relevant apical Cu + 1 transporter of intestinal cells. Am. J. Physiol. 284, C1525–C1530. Arredondo, M., Cambiazo, V., Tapia, L., Núñez, M.T., Uauy, R., Gonzalez, M., 2004. Copper overload affects copper and iron metabolism in HepG2 cells. Am. J. Physiol. 287, G27–G32.
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