Animal Models of Normal and Disturbed Iron and Copper Metabolism

Abstract

Research on the interplay between iron and copper metabolism in humans began to flourish in the mid-20th century, and diseases associated with dysregulated homeostasis of these essential trace minerals are common even today. Iron deficiency is the most frequent cause of anemia worldwide, leading to significant morbidity, particularly in developing countries. Iron overload is also quite common, usually being the result of genetic mutations which lead to inappropriate expression of the iron-regulatory hormone hepcidin. Perturbations of copper homeostasis in humans have also been described, including rare genetic conditions which lead to severe copper deficiency (Menkes disease) or copper overload (Wilson disease). Historically, the common laboratory rat (Rattus norvegicus) was the most frequently utilized species to model human physiology and pathophysiology. Recently, however, the development of genetic-engineering technology combined with the worldwide availability of numerous genetically homogenous (i.e., inbred) mouse strains shifted most research on iron and copper metabolism to laboratory mice. This created new opportunities to understand the function of individual genes in the context of a living animal, but thoughtful consideration of whether mice are the most appropriate models of human pathophysiology was not necessarily involved. Given this background, this review is intended to provide a guide for future research on iron- and copper-related disorders in humans. Generation of complementary experimental models in rats, swine, and other mammals is now facile given the advent of newer genetic technologies, thus providing the opportunity to accelerate the identification of pathogenic mechanisms and expedite the development of new treatments to mitigate these important human disorders.

Keywords: Menkes disease; Wilson disease; anemia; copper deficiency; hepcidin; hereditary hemochromatosis; iron deficiency; iron-deficiency anemia; β-thalassemia.

FIGURE 1

Iron acquisition and metabolism in enterocytes and erythroid cells. Green stars highlight proteins associated with genetic defects that can cause IDA and other iron dyshomeostasis, as discussed in the text. (A) Duodenal enterocyte showing the main proteins involved in the absorption of dietary nonheme iron. A ferrireductase (possibly DCYTB) and DMT1 are depicted on the apical (upper) surface of the cell. Also shown is NHE3, which may directly provide protons for cotransport with ferrous iron via DMT1. The sodium gradient is maintained by the Na⁺/K⁺ ATPase, depicted on the basolateral surface. In enterocytes, iron is probably bound by chaperones, like poly-r(C)-binding proteins 1 and 2 (PCBP1/2) 1/2) (11, 12), and distributed into the cytosol (for cellular use), the mitochondria, or the nucleus, or stored in ferritin. Iron that transits the cell is exported by FPN1, oxidized by HEPH [and/or other (unknown) oxidases], and bound to apo-TF for distribution to the liver. (B) Mutations in SLC11A2 (encoding DMT1) also disrupt iron acquisition by developing erythroid cells; the defect here contributes further to the genetic iron deficiency. DMT1 is required for iron uptake by these cells via its role in the transferrin cycle. When DMT1 is dysfunctional, erythroid cells do not accumulate sufficient iron, impairing their ability to produce hemoglobin, and contributing to the development of IDA. In this cell type, DMT1 may also be required for iron import into mitochondria (as indicated by “?”), as recently suggested by Wolff et al. (13). MFRN1 and FXN also participate in mitochondrial iron metabolism, and heme iron produced in the mitochondrion may be utilized by cytochrome C oxidase. DCYTB, duodenal cytochrome B; DMT1, divalent metal-ion transporter 1; FPN1, ferroportin 1; FXN, frataxin; HEPH, hephaestin; IDA, iron-deficiency anemia; MFRN1, mitoferrin 1; NHE3, sodium-hydrogen exchanger 3; SLC, solute carrier; TF, transferrin.

FIGURE 2

Genetic disorders of iron metabolism in humans involving dysregulation of the HAMP/hepcidin/FPN1 axis. The molecular machinery that regulates transactivation of the HAMP gene is depicted. Under basal conditions, diferric-TF interacts with a complex of TFR1/HFE/β₂M on the surface of hepatocytes. When body iron amounts are elevated and transferrin saturation increases, a new complex forms between diferric-TF/TFR2/HFE/β₂M; then, this complex interacts with cell surface BMP ligands/BMP receptors and stimulates BMP/SMAD protein family signaling to increase HAMP expression. Mutations in the genes encoding HFE and TFR2 lead to inappropriately low hepcidin production and underlie type I and type III HH, respectively (see Table 1). HJV is required for proper BMP/BMPR signaling to HAMP, and mutations in the gene encoding HJV underlie type IIA HH. Other types of HH occur from mutations in HAMP (type IIB) that impair hepcidin production or lead to dysfunction of the hepcidin protein, and in SLC40A1 (encoding FPN1), leading to “ferroportin disease,” or type IV HH. Also indicated are the influences of inflammation on HAMP transcription, which underlie the AI, and mutations in TMPRSS6, which cause IRIDA because the ability to downregulate HAMP expression via cleaving HJV from the plasma membrane is abolished. Lastly, hepcidin, secreted by hepatocytes into the circulation, decreases FPN1 protein concentrations on the surface of enterocytes and RE macrophages (including hepatic Kupffer cells), thus inhibiting intestinal iron absorption and iron release from stores, to lower serum iron. AI, anemia of inflammation; BMP, bone morphogenetic protein; BMPR, bone morphogenetic protein receptor; BRE, BMP response element; FPN1, ferroportin 1; HAMP, hepcidin antimicrobial peptide gene; HFE, homeostatic iron regulator; HH, hereditary hemochromatosis; HJV, hemojuvelin; IRIDA, iron-refractory, iron-deficiency anemia; JAK, Janus kinase; RE, reticuloendothelial; SLC, solute carrier; SMAD, small mothers against decapentaplegic; SRE, SMAD response element; STAT, signal transducer and activator of transcription proteins; TF, transferrin; TFR, transferrin receptor; TMPRSS6, transmembrane protease, serine 6; β₂M, β₂-microglobulin.

FIGURE 3

Altered copper homeostasis in enterocytes and hepatocytes underlies MD and WD, respectively. (A, B) Normal copper homeostasis in enterocytes and hepatocytes; (C, D) perturbations that occur in MD and WD. The ATP7A protein in enterocytes is required to deliver copper to support cuproenzyme synthesis in the TGN, and when copper is in excess, it traffics to the basolateral membrane and functions in copper export. In MD, when mutations in the gene encoding ATP7A lead to the production of a dysfunctional protein, absorption of dietary copper is impaired, ultimately leading to severe systemic copper deficiency and notable pathophysiologic changes (C). An analogous protein expressed in hepatocytes, ATP7B, similarly functions in copper delivery to the TGN to support the production of CP, which is secreted into the circulation and functions mainly in iron metabolism. When hepatic copper is in excess, ATP7B traffics to the canalicular membrane where it excretes copper into the biliary tree. Excreted copper is presumably complexed with bile salts (and thus unavailable for reabsorption) and eliminated in the feces. In WD, the mutant ATP7B protein can no longer efficiently pump excess copper into the bile, so copper accumulates in hepatocytes, eventually exceeding the storage capacity of the liver and spilling out into the systemic circulation (leading to excess copper accumulation in some tissues) (D). CP production is also impaired, which disrupts iron homeostasis in WD patients (e.g., causing liver iron loading). ATP7A, ATPase copper transporting α; ATP7B, ATPase copper transporting β; CP, ceruloplasmin; CTR1, copper transporter 1; MD, Menkes disease; MT, metallothoinein; TGN, trans-Golgi network; WD, Wilson disease.