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RED CELLS

Nontransferrin-bound iron uptake by hepatocytes is increased in the Hfe knockout

mouse model of hereditary hemochromatosis
Anita C. G. Chua, John K. Olynyk, Peter J. Leedman, and Debbie Trinder

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. (Blood. 2004;104:
1519-1525)

2004 by The American Society of Hematology

Introduction
Hereditary 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 Ilike
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).6-8
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 surface before being transported into the cell by an Fe

carriermediated step.8,13
Recently, Zhou and colleagues14 produced an Hfe knockout (KO)
mouse model of HH. These Hfe KO mice display similar characteristics
of Fe overload observed in patients with HH. These animals have
elevated serum Tf saturation and increased hepatic Fe levels by 4 weeks
of age. Iron deposition occurs mainly in the hepatocytes, with little or no
Fe loading in the reticuloendothelial cells of the liver and the spleen.15
The Hfe KO mouse is a good animal model of HH and it provides the
opportunity to investigate the mechanisms underlying an increased
deposition of Fe in the liver in HH.
In previous studies, we and others have shown that the Fe absorbed
by the duodenum in vivo is increased in Hfe KO mice compared with
control mice.16,17 In addition, we found that the amount of absorbed Fe
deposited in the liver was increased. These results indicated that Fe
uptake by the liver from the Hfe KO mice is up-regulated. As NTBI
levels are elevated in the serum of HH patients,18,19 even when Tf is not
completely saturated with Fe, we hypothesize that NTBI uptake by
hepatocytes is up-regulated in HH.
In this study, we characterized the uptake of NTBI in the form
of ferric citrate by hepatocytes from Hfe KO and control C57BL/6J
mice. Results showed that the rate of NTBI uptake by hepatocytes
from Hfe KO mice was increased more than 2-fold compared with
control cells, demonstrating that NTBI contributes to Fe loading of
the liver in HH.

From the Department of Medicine, School of Medicine and Pharmacology,

University of Western Australia, Fremantle Hospital, Fremantle, Australia;
Western Australian Institute for Medical Research, Nedlands, Australia; and
Laboratory of Cancer Medicine, School of Medicine and Pharmacology,
Western Australian Institute for Medical Research, Royal Perth Hospital, The
University of Western Australia, Perth, Australia.

Biomedical Scholarship from the National Health and Medical Research

Council of Australia.

Submitted November 13, 2003; accepted May 4, 2004. Prepublished online as

Blood First Edition Paper, May 20, 2004; DOI 10.1182/blood-2003-11-3872.

The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked advertisement in accordance with 18 U.S.C. section 1734.

Supported by a grant from the National Health and Medical Research Council
of Australia (D.T., J.K.O., and P.J.L.). A.C.G.C. is a recipient of the Dora Lush

BLOOD, 1 SEPTEMBER 2004 VOLUME 104, NUMBER 5

Reprints: Debbie Trinder, School of Medicine and Pharmacology, The

University of Western Australia, Fremantle Hospital, PO Box 480, Fremantle
6959, WA, Australia; e-mail: dtrinder@cyllene.uwa.edu.au.

2004 by The American Society of Hematology

1519

From www.bloodjournal.org by guest on March 21, 2015. For personal use only.
1520

CHUA et al

BLOOD, 1 SEPTEMBER 2004 VOLUME 104, NUMBER 5

Serum iron concentration and Tf saturation in the Hfe KO and control mice
were determined using a colorimetric assay according to the manufacturers
instructions (Sigma Chemical).

Germany) in HBSS for 30 minutes at 4C to release membrane Fe.8 The cell

suspension was then centrifuged at 14 000g for 1 minute to separate into the
supernatant and cell pellet fractions that will be referred to as membrane Fe
and internalized Fe, respectively. The radioactivity in both fractions was
measured in a LKB Wallac 1282 CompuGamma gamma-counter (Wallac,
Turku, Finland). An aliquot of the cell suspension was then taken for protein
estimation using the bicinchoninic acid (BCA) protein method (Pierce,
Rockford, IL). All data were calculated as nmol Fe/mg protein to correct for
variation in cell density. The amount of protein/hepatocyte for Hfe KO mice
(921 29 pg protein/cell; mean SEM; n 5) and control (864 29 pg
protein/cell; mean SEM; n 11) was similar (P .25).
In other experiments, the effects of a 100-fold excess of various divalent
metals (iron [Fe], manganese [Mn], cobalt [Co], nickel [Ni], copper [Cu],
and zinc [Zn]) on 59Fe(III)citrate (ratio of 1 M Fe to 100 M citrate)
uptake by the Hfe KO and control hepatocytes were investigated. The cells
were incubated in the presence or absence of the metals for 1 hour at 37C.
Also, the effects of specific ferrous ion chelators (0-1000 M), bathophenanthroline disulfonate (BPS; Sigma Chemical) and 2,2-bipyridine (BP; BDH
AnalaR, Kilsyth, Australia), and diferric Tf (ratio of 0-10 M Tf to 0-20
M Fe) on 59Fe-citrate (ratio of 1 M Fe to 100 M citrate) by hepatocytes
from both types of mice were investigated. In these experiments, the
hepatocytes were incubated with up to 20-fold molar excess of diferric Tf
for 2 hours or with the chelators for 1 hour at 37C. The effect of
extracellular pH (range 5.5-8, buffered with 20 mM HEPES-Tris) on
59Fe-citrate (ratio of 10 M Fe to 1000 M citrate) uptake by hepatocytes
from Hfe KO and control mice was also examined.

Ferritin levels

RNA expression

The amount of ferritin protein in the hepatocytes from both types of mice
was measured using an immunoturbidometric method according to the
manufacturers instructions (Roche Diagnostics, Mannheim, Germany).

Total RNA was isolated from cultured hepatocytes using RNA-Bee

(Tel-Test, Friendswood, TX) and treated with DNAse I (Ambion, Austin,
TX). Reverse transcription was performed on 1 g of total RNA, 0.5 g
oligoDT15 (Promega, Sydney, Australia), and 1 mM each of deoxynucleotide triphosphate (Promega) and 12 units avian myeloblastosis virus
(AMV) reverse transcriptase (Promega) according to the manufacturers
instructions. Mouse DMT1 and -actin mRNA (endogenous control)
transcripts were quantified by real-time polymerase chain reaction (PCR)
using a Rotorgene (Corbett Research, Mortlake, Australia). The PCR
reaction mixture contained cDNA converted from 50 ng RNA, 0.25 M
each of the forward and reverse primers (Geneworks, Thebarton, Australia),
0.2 units Platinum Taq (Invitrogen), 0.5 SYBR Green (Molecular Probes,
Sydney, Australia), and 2 mM MgCl2. The cycling parameters were as
follows: an initial denaturation step at 95C for 300 seconds followed by 40
cycles of denaturation at 95C for 15 seconds, annealing at 55C (DMT1
and -actin) for 20 seconds and extension at 72C for 20 seconds, and a
final extension step at 72C for 30 seconds. Plasmids containing DMT1 and
-actin cDNA were made by PCR amplification using their primer sets
(DMT1 [GenBank accession no. NM_008732] forward primer 5TCTATCGCCATCATCCCCACCC-3 and reverse primer 5-TCCACAGTCCAGGAAAGACAGACCC-3; and -actin [GenBank accession
no. NM_007393] forward primer 5-CTGGCACCACACCTTCTA-3 and
reverse primer 5-GTCACCCACACTGTGCCC-3) and mouse duodenum
cDNA as a template. The PCR products were then inserted into pGEM-T
Easy vector (Promega) and cloned. Serial dilutions of known copy numbers
of the plasmids were used to generate standard curves to quantify gene
expression by control and Hfe KO mouse hepatocytes. DMT1 mRNA
expression was then normalized against -actin mRNA expression.

Materials and methods

Animals
The Hfe KO mouse, a mouse model of HH, was kindly provided by
Professor W. Sly (St Louis University, St Louis, MO). In these mice, the Hfe
gene has been disrupted and is a null allele.14 Control C57BL/6J and Hfe
KO mice were bred at the Animal Resource Centre (Murdoch, Australia).
The mice had access to a standard rodent chow and water ad libitum.
Plasma NTBI
NTBI concentration in the plasma from Hfe KO and control mice was measured
using a method similar to that described previously.18,19 Briefly, 100 L
Tris-carbanatocobaltate(III) trihydrate solution was added to 450 L plasma and
incubated at 37C for 1 hour. Nitriloacetic acid (Sigma Chemical, St Louis, MO)
was then added to give a final concentration of 80 mM. This mixture was left to
stand for 30 minutes at room temperature. The samples were ultrafiltered using
Centricon-30 (Millipore, North Ryde, Australia) at 3000g for 1 hour and assayed
for NTBI using a colorimetric method.
Serum Fe and Tf saturation

Isolation and culture of mouse hepatocytes

Mouse hepatocytes were isolated from livers of 11-week-old female Hfe
KO and control C57BL/6J mice using a modified method of Knight et al20
based on the method first described by Seglen.21 The liver was initially
perfused at a rate of 10 mL/min with a perfusion buffer (137 mM NaCl, 4.7
mM KCl, 1.2 mM KH2PO4, 0.65 mM MgSO4, and 10 mM HEPES
[N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid]) containing 0.5 mM
EGTA (ethyleneglycoltetraacetic acid; Sigma Chemical) for 5 minutes and
then with a second buffer at a rate of 12 mL/min (67 mM NaCl, 6.7 mM
KCl, 4.8 mM CaCl2, 100 mM HEPES, and 65 mM NaOH) containing
0.01% collagenase Type I (Worthington Biochem, Freehold, NJ) for 5
minutes. The hepatocytes were dispersed in Leibowitz (L15) medium
(Invitrogen, Melbourne, Australia) and 0.04% bovine serum albumin
(Sigma Chemical), filtered through gauze, and centrifuged at 200g. The
isolated hepatocytes were cultured in Dulbecco modified Eagle medium
(DMEM; Invitrogen) containing 2 mM L-glutamine (Invitrogen), 1 mM
sodium pyruvate (Invitrogen), 2.5 g/mL amphotericin B (ThermoTrace,
Sydney, Australia), 100 U/mL and 100 g/mL of penicillin/streptomycin
sulfate (Invitrogen), 10 nM dexamethasone (Sigma Chemical), and 10 nM
insulin (Sigma Chemical) supplemented with 10% (vol/vol) fetal bovine
serum (Invitrogen). The hepatocytes were maintained in a humidified atmosphere of 5% CO 2 for 24 hours before use. This procedure
was approved by the Animal Ethics Committee of The University of
Western Australia.

DMT1 Western blotting

NTBI uptake studies
Ferric (Fe(III)) citrate solution was labeled with 59FeCl3 (Perkin Elmer,
Melbourne, Australia) as previously described.6 Hepatocytes from both Hfe
KO and control mice were washed 3 times with Hanks buffer (HBSS) at
37C and the medium was replaced with incubation medium (DMEM
2% bovine serum albumin [pH 7.4] containing 59Fe-citrate [0-25 M Fe;
ratio of 1 Fe to 100 citrate]). The cells were incubated for 0 to 120 minutes
at 37C and then washed 5 times with HBSS at 4C. The hepatocytes were
incubated with 1 mg/mL Pronase (Boehringer Mannheim, Mannheim,

Hepatocytes from both types of mice were lysed in 150 mM NaCl, 25 mM

Tris-HCl (pH 7.5), and 0.5% Triton X-100 (BDH AnalaR) containing the
protein inhibitors phenylmethylsulfonyl fluoride (PMSF; 0.5 mM; Sigma
Chemical), leupeptin (10 g/mL; Sigma Chemical), and aprotinin (2
g/mL; Boehringer Mannheim), the reducing agent dithiothreitol (DTT; 1
mM; Sigma Chemical), and EDTA (ethylenediaminetetraacetic acid; 5 mM;
BDH AnalaR). Sodium dodecyl sulfatepolyacrylamide gel electrophoresis
was performed according to the method of Laemmli.22 Protein samples
were heated at 90C for 10 minutes in Laemmli buffer and separated by 7%

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BLOOD, 1 SEPTEMBER 2004 VOLUME 104, NUMBER 5

HEPATIC NTBI UPTAKE BY Hfe KNOCKOUT MICE

Table 1. Iron parameters in Hfe KO and control mice

Mouse types
Control

Hfe KO

Plasma NTBI, M; n 5

1.5 0.1

3.7 0.1

Serum iron, M; n 3

23 1

31 2

Serum transferrin saturation, %; n 3

40 1

77 5

Hepatocyte ferritin, ng/g protein; n 30

1.0 0.1

5.5 0.4

All values are mean SEM. P .05 Hfe KO versus control mice.

1521

hepatocytes from both types of mice. The rate of Fe internalization

by the cells increased by over 2-fold by hepatocytes from the Hfe
KO compared with control mice. (28.0 2.4 pmol Fe/mg protein/
min vs 13.0 2.2 pmol Fe/mg protein/min; mean SEM, n 7;
P .001). However, no statistical difference was observed in the
rates of membrane uptake by hepatocytes from both Hfe KO and
control mice (7.13 1.11 vs 4.79 0.69 pmol Fe/mg protein/min;
mean SEM; n 7; P .10).
Effects of iron concentration on NTBI uptake

acrylamide gel electrophoresis. The proteins were then electroblotted onto a

polyvinylidene fluoride membrane (Millipore). For detection of DMT1
protein, the blot was incubated with rabbit anti-DMT1, which recognizes
both the iron responsive element (IRE) and non-IRE isoforms.23 After
incubation with the DMT1 antibody (1:1500 dilution), the blot was
incubated with goat antirabbit immunoglobulin Ghorseradish peroxidase
(IgG-HRP) antibody (1:2000 dilution; Santa Cruz Biotechnology, Santa
Cruz, CA). DMT1(total) protein expression was then detected using
enhanced chemiluminescence (ECL; Amersham Biosciences, Castle Hill,
Australia) chemiluminescence kit. Western blot analysis was quantified
using QuantityOne software (Bio-Rad, Regents Park, Australia).
Statistical analysis
Results are expressed as mean SD of triplicate measurements within each
experiment, unless otherwise stated. Each experiment was performed at
least 2 to 7 times. Student unpaired t test was used to determine differences
between groups and statistical significance was obtained for P values less
than .05.

Results
Iron parameters

In the Hfe KO mice, plasma NTBI concentration was approximately 2.5-fold higher compared with control C57BL/6J mice
(Table 1). Serum Fe levels and Tf saturation were also significantly
higher in the Hfe KO mice. Hepatocytes from Hfe KO mice
contained 5.5-fold more ferritin protein than hepatocytes from
control mice (Table 1).
Kinetics of NTBI uptake from 59Fe-citrate

Hepatocytes isolated from Hfe KO and control mice were cultured

for 24 hours prior to incubation with 59Fe-citrate (ratio of 10 M Fe
to 1000 M citrate) for 0 to 2 hours at 37C. Total (internalized and
membrane) Fe uptake by hepatocytes from both types of mice
increased over time (Figure 1). The rate of Fe internalization was
greater than the rate of Fe accumulation on the membrane of

As the extracellular concentration of 59Fe-citrate was increased, the

uptake of Fe by hepatocytes from both types of mice reached
saturation (Figure 2). The maximal velocity of uptake (Vmax) and
the affinity constant (Km) for iron uptake were estimated using the
software program GraphPad Prism (GraphPad Software, San
Diego, CA) based on the Michaelis-Menten equation. The Vmax was
greater in the hepatocytes from the Hfe KO mice (4823 pmol Fe/mg
protein/2 h) compared with the control mice (2842 pmol Fe/mg
protein/2 h; Figure 2), whereas Km values were 6.4 M and 6.8 M
for the Hfe KO and control mice, respectively. When this experiment was repeated (n 4), there was a significant increase in Vmax
values by 70% by the hepatocytes from Hfe KO mice compared
with the control mice (P .0001) but there was no statistically
significant difference in their Km values (P .13).
Effects of iron chelators on NTBI uptake

The hepatocytes from Hfe KO and control mice were incubated

with 59Fe-citrate in the presence of the specific ferrous (Fe2) ion
chelators BPS and BP up to 1000-fold excess. These chelators
inhibited intracellular and membrane Fe uptake by hepatocytes
from both Hfe KO and control mice in a concentration-dependent
manner with Fe uptake decreasing as the concentration of the
chelators was increased (Figure 3). At 1 mM, both BPS and BP
almost abolished intracellular Fe uptake, reducing uptake to
approximately 5% of the control values in the absence of chelators.
At the same concentration, membrane Fe uptake was reduced by
BPS and BP to 40% and 15% of the control values, respectively. The
effects of the chelators BP and BPS on intracellular and membrane
uptake by the hepatocytes from both mouse types were similar.
Effects of divalent metals on NTBI uptake
59Fe-citrate

uptake was inhibited by most of the divalent metals

presented to the hepatocyte in culture in a 100-fold excess.
Divalent metals manganese (Mn), iron (Fe), zinc (Zn), and cobalt
(Co) significantly diminished intracellular Fe and membrane Fe

Figure 1. Kinetics of NTBI uptake from ferric citrate by hepatocytes. Hepatocytes from Hfe KO (filled symbols) and control (open symbols) mice were incubated for 0 to 90
minutes with 59Fe-citrate (ratio of 10 M Fe to 1000 M citrate) at 37C. (A) Internalized (F, E), (B) membrane (f, ), and (C) total (, ) uptake are shown. Results are
expressed as mean SD (n 3). This graph is a typical representation from 7 independent experiments performed.

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1522

CHUA et al

BLOOD, 1 SEPTEMBER 2004 VOLUME 104, NUMBER 5

2-fold more DMT1 than hepatocytes from the control mice (Figure
6A). DMT1(IRE) mRNA expression was up-regulated by 50% by
hepatocytes from the Hfe KO mice compared with control mice.
DMT1(non-IRE) mRNA was not detected in the hepatocytes from
either type of mice (data not shown). Expression of DMT1 protein
by the hepatocytes was detected by Western blot analysis. There
was an up-regulation of DMT1 protein expression by about 2-fold
in the hepatocytes from the Hfe KO mice compared with hepatocytes from control mice (Figure 6B).

Discussion
Figure 2. The effects of increasing ferric citrate concentration on Fe uptake.
Hepatocytes from Hfe KO (filled symbols) and control (open symbols) mice were
incubated with 0 to 20 M 59Fe-citrate (ratio of 1 Fe to 100 citrate) for 2 hours at 37C.
Internalized (F, E), membrane (f, ), and total (, ) uptakes are shown. Results
are expressed as mean SD (n 3). This graph is a typical representation from 4
independent experiments performed.

uptake from citrate by 40% to 80% of control values in the absence

of divalent metals, whereas divalent metals such as copper (Cu)
only decreased uptake by about 20% and nickel (Ni) had no effect.
Similar effects of the divalent metals on Fe uptake were observed
for hepatocytes from both types of mice (Figure 4).
Effects of pH on NTBI uptake
59Fe-citrate

uptake by hepatocytes from both Hfe KO and control

mice was pH dependent (data not shown). Iron uptake from citrate
increased as the pH of the incubation solution was raised from pH
5.5 to about 7 to 7.5 and then declined slightly as the pH was
further increased to 8.

Effects of diferric Tf on NTBI uptake

When the hepatocytes from Hfe KO and control mice were

incubated with 59Fe-citrate in the presence of increasing concentrations of diferric Tf, intracellular and membrane Fe uptake decreased as diferric Tf concentration increased. In the presence of
20-fold molar ratio of diferric Tf, internalized Fe uptake from citrate was
reduced by about 50% to 60%, as was membrane Fe uptake by
hepatocytes from both Hfe KO and control mice (Figure 5).
Expression of DMT1 by hepatocytes

The expression of DMT1 mRNA by hepatocytes from both Hfe KO

and C57BL/6J mice was determined by real-time PCR. Hepatocytes from the Hfe KO mice were shown to express approximately

In this study, the mechanisms of Fe loading of hepatocytes from the

Hfe KO mice were investigated. We demonstrated for the first time
that plasma NTBI levels were significantly increased in the Hfe KO
mice and that NTBI uptake by hepatocytes from the Hfe KO mice
was significantly increased compared with control mice. NTBI as
ferric citrate was reduced from 3 to 2 redox state at the cell
surface of the hepatocytes before being internalized. The inhibition
of ferric citrate uptake by divalent metals such as Mn, Zn, and Co is
consistent with a common transporter mediating the uptake of these
metals. Iron uptake from citrate was inhibited by Tf-Fe, suggesting
that both forms of Fe share a common uptake pathway. DMT1
mRNA and protein expression were up-regulated in hepatocytes
from the Hfe KO mice. These results suggest that NTBI uptake
contributed to hepatic Fe loading in the Hfe KO mice and that
DMT1 may mediate NTBI uptake by hepatocytes.
In patients with HH, serum Tf saturation is increased and NTBI
concentration is elevated, with levels in the range of 3 to 5
M,12,18,19 although higher levels of up to 22 M have been
reported.24,25 Indeed, we found an increase in plasma NTBI
concentration in the Hfe KO mice compared with control mice. The
elevated serum Fe levels and Tf saturation in the Hfe KO mice, as
well as the higher concentration of ferritin protein in hepatocytes
from the Hfe KO mice (Table 1), are consistent with the degree of
Fe overload observed in HH patients.26 The increase in ferritin
levels in hepatocytes from Hfe KO mice relative to hepatocytes
from control mice was similar to that reported previously for
nonheme iron levels in whole liver from these animals.14,17 This
indicates there was no loss of Fe from the hepatocytes in vitro.
Hepatocytes from the Hfe KO mice took up Fe from citrate at
twice the rate of control hepatocytes (Figure 1). Iron uptake from
citrate by both Hfe KO and control hepatocytes was saturable,
indicating that the uptake is mediated by an Fe transporter (Figure
2).7,8 The higher maximal rate of uptake by the Hfe KO hepatocytes

Figure 3. The effects of specific ferrous ion chelators on NTBI uptake.

Hepatocytes from Hfe KO (filled symbols) and control (open symbols)
mice were incubated with 59Fe-citrate (ratio of 1 M Fe to 100 M citrate)
in the presence of 0 to 1000 M BPS (A) and BP (B) for 1 hour at 37C.
Internalized (F, E) and membrane (f, ) uptakes are shown. Results are
expressed as mean SD (n 3). This graph is a typical representation
from 2 independent experiments performed.

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BLOOD, 1 SEPTEMBER 2004 VOLUME 104, NUMBER 5

HEPATIC NTBI UPTAKE BY Hfe KNOCKOUT MICE

1523

Figure 4. The effects of divalent metals on internalization and membrane ferric citrate uptake. (A) Internalized. (B) Membrane. Hepatocytes from Hfe KO mice (f)
and control () were incubated with 59Fe-citrate (ratio of
1 M Fe to 100 M citrate) in the absence and presence
of 100 M divalent metals for 1 hour at 37C. Results are
expressed as mean SD (n 3). *P .001; #P .05.
This graph is a typical representation from 3 independent
experiments performed.

compared with the control cells implies that there was an upregulation of the number of Fe transporters on the cell surface of
the hepatocytes. Although the rate of NTBI uptake by the hepatocytes from Hfe KO mice increased by 2-fold in vitro, the increased
concentration of NTBI in their circulation compared with control
mice suggests that hepatocytes from Hfe KO mice have the
capacity to take up more Fe from NTBI in vivo. It has been reported
that approximately 60% to 75% of NTBI in the serum is cleared by
rat liver in the first pass, possibly more in vivo.10,27 If so, the plasma
NTBI levels reported in HH patients and in the Hfe KO mice may
be an underestimation of the amount of NTBI that is present in the
portal blood and cleared by the liver in Fe-overload diseases such
as HH. These results indicate that NTBI is important in the
pathogenesis of hepatic Fe loading in HH.
Specific ferrous ion chelators, the membrane-impermeable BPS
and membrane-permeable BP, inhibited both internalized and
membrane Fe uptake by hepatocytes from Hfe KO and control mice
(Figure 3), indicating that ferric iron was reduced to the ferrous
form at the cell surface before being taken up by the hepatocytes.
Similarly, in other studies using hepatoma cells, it was shown that
NTBI uptake was inhibited by the ferrous chelators.8,28 Also the
membrane-impermeable ferricyanide competitively inhibited NTBI
uptake by both hepatoma and erythroleukemic cells, indicating
uptake was dependent on the reduction of Fe3 to Fe2 at the cell
surface.8,29 These observations provide strong evidence for the
presence of a ferrireductase at the cell surface that is coupled to
NTBI transport in both hepatocytes and hepatoma cells. NTBI
uptake by Hfe KO and control hepatocytes was also pH dependent,
confirming the previous findings of others using wild-type rat
hepatocytes.6,7
We found that divalent metals such as Fe, Mn, Zn, and, to a
lesser extent, Co and Cu inhibited ferric citrate uptake by hepato-

Figure 5. The effects of increasing concentrations of diferric transferrin on

NTBI uptake. Hepatocytes from Hfe KO (filled symbols) and control (open symbols)
mice were incubated with 59Fe-citrate (ratio of 1 M Fe to 100 M citrate) in the
presence of Fe 0 to 20 molar ratio for diferric transferrin to Fe-citrate for 2 hours at
37C. Internalized (F, E) and membrane (f, ) uptake are shown. Results are
expressed as mean SD (n 3). This graph is a typical representation from 2
independent experiments performed.

cytes from both Hfe KO and control mice. This suggests that uptake
of these metals by hepatocytes from both types of mice was
mediated by the same carrier. The varying degree of inhibition of
ferric citrate uptake by the different divalent metals is likely to be
due to the affinity that the carrier has for the different metals.
Similar results have been reported by other investigators that show
NTBI uptake was inhibited by the divalent metals Zn, Co, and Ni in
isolated rat hepatocytes6; Zn, Co, and Cd in hepatoma cells28; and
Zn, Mn, and Co in perfused rat liver.27
In the present study, internalized and membrane Fe uptake from
citrate by hepatocytes from both Hfe KO and control mice were
inhibited in the presence of increasing concentrations of Tf-Fe.
This implies that the uptake of Fe from both citrate and Tf shares a
common uptake mechanism. Similar results have been reported in
rat hepatocytes.7,30 We also showed the converse where Tf-Fe
uptake by hepatoma cells was reduced by the presence of excess
ferric citrate.31 In the latter study, we demonstrated that the
inhibition of ferric citrate uptake by Tf-Fe was not due to the
transfer or exchange of Fe between citrate and Tf. When radiolabeled diferric Tf was incubated with unlabeled ferric citrate for up
to 24 hours at 37C and Tf-bound and citrate-bound Fe were
separated using a 30-kDa molecular weight cutoff membrane, the
rate of exchange of 59Fe between Tf and citrate was less than
1.5%/hour, which was insufficient to account for the observed
reduction in Fe-citrate uptake.31
It is well established that hepatocytes and hepatoma cells
accumulate Tf-Fe by several pathways including high-affinity Tf
receptor 1mediated endocytic process and low-affinity Tf receptor
1independent processes, which may involve Tf receptor 2.32 The
mechanisms responsible for the low-affinity processes are unclear
but evidence suggests there may be 2 pathways, one involving the
endocytosis of Tf-Fe and the other, the release of Fe from
transferrin at the cell surface.33-35 Using hepatoma cells, we have
shown previously that NTBI had no effect on the uptake of Tf-Fe
by a high-affinity transferrin receptor-1mediated process. However, NTBI competitively inhibited the uptake of Tf-Fe released at
the surface by the low-affinity process, suggesting a common Fe
carrier mediates the uptake of both NTBI and Tf-Fe at the cell
surface. A similar mechanism is likely to be present in hepatocytes.31
In 1997, Gunshin et al36 cloned a duodenal NTBI transporter
called divalent metal transporter 1 (DMT1). Electrophysiologic
studies performed on Xenopus oocytes expressing DMT1 showed
that it transported not only Fe but also other divalent metals with
affinity decreasing in the order of Zn, Mn, Co, Cd, Cu, Ni, and Pb.
DMT1 is widely expressed in many other tissues including the
liver.23,36 DMT1 has also been identified as the endosomal Fe
carrier. Tf receptor 1mediated uptake of Tf-Fe involves the
internalization of Tf-Fe into an endosome where the Fe is released
from Tf and transported by DMT1 across the endosomal membrane

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1524

BLOOD, 1 SEPTEMBER 2004 VOLUME 104, NUMBER 5

CHUA et al

Figure 6. Expression of DMT1 mRNA and protein. (A) mRNA.

(B) Protein. Messenger RNA and protein expression of DMT1 in
the hepatocytes from Hfe KO (f) and control () mice were
quantified by real-time PCR and Western blotting, respectively,
and performed as described in the Materials and methods.
Results are expressed as mean SD (n 6-8) for real-time PCR
and mean SD (n 7) for Western blotting. *P .01.

to the cell cytosol.37 Immunofluorescence and immunohistochemical studies have localized DMT1 to both recycling endosomes and
plasma membranes,23,38-40 suggesting DMT1 cycles between the
endosomal membrane and the plasma membrane where it
may possibly mediate Fe uptake across both the endosome and
cell membrane.
There is evidence to suggest that DMT1 may be a putative
NTBI transporter in hepatocytes. In this study we found both
DMT1 mRNA and protein expression were increased in the
hepatocytes from the Hfe KO mice compared with cells from
control animals, and this increase parallels the increase in NTBI
uptake observed in the Hfe KO mice. Also the inhibitory effects of
the ferrous chelators and other divalent metals on NTBI uptake
suggest DMT1 may be involved. As Tf-Fe impairs the uptake of
NTBI across the hepatocyte cell surface, it suggests that one Fe
transporter, possibly DMT1 cycling between the endosome and the
cell membrane, may be responsible for the uptake of Fe from both
sources. Hence, further study to elucidate the site of NTBI and
Tf-Fe interaction is warranted. Nevertheless, others failed to see an
increase in liver DMT1 mRNA expression from Hfe KO mice.15,41
However, in both of these latter studies, only DMT1 mRNA and not
protein expression was measured in whole liver rather than in
isolated hepatocytes.
Iron loading of wild-type rat hepatocytes has been shown in at
least some previous studies to lead to increases in hepatocellular
DMT1 expression23,42 and/or uptake of NTBI.42,43 Thus, the
increases in NTBI uptake and DMT1 expression observed in the
Hfe KO mice in this study might reflect Fe loading rather than the
loss of HFE per se. The studies presented in this current body of
work cannot determine whether the increases in DMT1 expres-

sion and NTBI uptake by the Hfe KO mouse hepatocytes are the
primary cause of Fe loading or secondary to Fe loading by
another mechanism.
Hepcidin is synthesized by hepatocytes and has been postulated
to be a key player in the regulation of iron metabolism. Mice that
lack hepcidin develop Fe overload,44 whereas those with high
expression develop severe Fe deficiency anemia,45 indicating an
inverse relationship between dietary Fe absorption and hepatic
hepcidin expression. Recent studies showed that hepcidin expression is inappropriately low in Hfe KO mice and HH patients despite
Fe loading46,47 and this may be a key to the defective regulation of
Fe metabolism in HH. Whether hepcidin is important in the
up-regulation of NTBI transport or DMT1 expression by the
hepatocytes from Hfe KO mice is yet to be determined.
In conclusion, this study provides evidence supporting our
hypothesis that NTBI uptake by the liver is up-regulated in the Hfe
KO mice and contributes to increased Fe deposition in the liver.
These results demonstrate that Hfe KO mouse hepatocytes have
increased NTBI uptake even when hepatic Fe stores are high. These
findings provide new and important information on the mechanisms of hepatic Fe loading in HH.

Acknowledgments
We would like to thank S. Drake, C. Herbison, C. Smith, and T.
De-Jong for skillful technical assistance and the Department of
Biochemistry at Fremantle Hospital for performing the ferritin
measurements.

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2004 104: 1519-1525

doi:10.1182/blood-2003-11-3872 originally published online
May 20, 2004

Nontransferrin-bound iron uptake by hepatocytes is increased in the Hfe

knockout mouse model of hereditary hemochromatosis
Anita C. G. Chua, John K. Olynyk, Peter J. Leedman and Debbie Trinder

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