Nontransferrin-bound iron uptake by hepatocytes is increased in the Hfe knockout mouse model of hereditary hemochromatosis

Abstract

Hereditary hemochromatosis (HH) is an iron-overload disorder caused by a C282Y mutation in the HFE gene. In HH, plasma nontransferrin-bound iron (NTBI) levels are increased and NTBI is bound mainly by citrate. The aim of this study was to examine the importance of NTBI in the pathogenesis of hepatic iron loading in Hfe knockout mice. Plasma NTBI levels were increased 2.5-fold in Hfe knockout mice compared with control mice. Total ferric citrate uptake by hepatocytes isolated from Hfe knockout mice (34.1 ؎ 2.8 pmol Fe/mg protein/min) increased by 2-fold compared with control mice (17.8 ؎ 2.7 pmol Fe/mg protein/min; P < .001; mean ؎ SEM; n ‫؍‬ 7). Ferrous ion chelators, bathophenanthroline disulfonate, and 2,2-bipyridine inhibited ferric citrate uptake by hepatocytes from both mouse types. Divalent metal ions inhibited ferric citrate uptake by hepatocytes, as did diferric transferrin. Divalent metal transporter 1 (DMT1) mRNA and protein expression was increased approximately 2-fold by hepatocytes from Hfe knockout mice. We conclude that NTBI uptake by hepatocytes from Hfe knockout mice contributed to hepatic iron loading. Ferric ion was reduced to ferrous ion and taken up by hepatocytes by a pathway shared with diferric transferrin. Inhibition of uptake by divalent metals and up-regulation of DMT1 expression suggested that NTBI uptake was mediated by DMT1. IntroductionHereditary hemochromatosis (HH) is a common autosomal recessive disorder of iron (Fe) metabolism. This disease affects up to 1 in 200 individuals of North European descent. 1 Most HH patients are homozygous for a C282Y missense mutation in the HFE gene that encodes for a major histocompatibility complex class I-like protein. 2 HH is characterized by an increase in dietary Fe absorption, with a chronic rise in body Fe stores, leading to hepatic Fe overload, followed by the progressive loading of other parenchymal organs such as the heart, pancreas, and joints. The abnormally high levels of Fe in the liver can lead to hepatic fibrosis and cirrhosis, increasing the risk of hepatocellular carcinoma. 3,4 Hepatocytes of the liver are the main site of deposition of Fe in HH and hence play an important role in maintaining Fe homeostasis. It is well established that hepatocytes and hepatoma cells take up Fe from transferrin (Tf) 5 and nontransferrin-bound iron (NTBI). [6][7][8] The normal range of Tf saturation in plasma is usually 20% to 50%, but in Fe-overload diseases like HH, Tf saturation is elevated and can reach 100%. Under these conditions, a greater proportion of Fe in the plasma occurs as NTBI. It has been reported that NTBI can coexist with diferric Tf in the serum of patients with Fe overload. 9 NTBI in the plasma is directed to hepatocytes in the liver in vivo, 10 where it is rapidly cleared. The contribution of plasma NTBI to the toxicity associated with Fe overload is uncertain. Serum NTBI is found mainly in the form of ferric citrate. 9,11,12 The uptake of ferric (Fe(III))-citrate involves the dissociation of Fe from citrate at the cell surfa...

Zip14 (Slc39a14) mediates non-transferrin-bound iron uptake into cells

“…Although DMT1 is able to transport Zn 2ϩ as well, this cation is poorly transported relative to Fe 2ϩ (21). DMT1 expression has been detected at the plasma membrane of hepatocytes (23), and its levels increase in ironloaded Hfe Ϫ/Ϫ mice (24). However, the degree to which DMT1-mediated NTBI uptake can occur from the blood plasma remains to be determined, given that DMT1 transport activity proceeds optimally at pH 5.5 (22).…”

“…Further kinetic studies revealed that zinc increased the apparent K m for iron transport without changing the apparent V max , consistent with competitive inhibition. Similarly, in primary hepatocytes isolated from rats and mice, zinc has been shown to inhibit the uptake of iron when presented as ferric citrate, a physiologically relevant form of NTBI (24,28).…”

Cellular and molecular mechanisms regulating the hepatic erythropoietin expression during acute-phase response: a role for IL-6

“…Hepatocytes from C57BL/6J mouse livers were isolated by a two-step enzymatic dissociation as previously described, 23 with some modifications. Briefly, the liver was perfused at a rate of 10 ml/min with a perfusion buffer for 5 min and then with a second buffer containing 0.01% collagenase type I for 5 min at a rate of 12 ml/min.…”

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

“…Thus, it appears that iron loading per se increases ZIP14 levels, which could, in turn, increase the uptake of NTBI. Previous studies of HepG2 cells 48 and isolated rat and mouse hepatocytes 23,49,50 have shown that iron loading increases the uptake of NTBI, and that this process is mediated by a protein carrier. Our data in HepG2 cells suggest that ZIP14 may represent this ironresponsive NTBI transporter.…”