doi:10.1111/j.1440-1746.2009.06173.

H E PAT O L O G Y

jgh_6173

1144..1150

Evidence for a critical role of ceruloplasmin oxidase activity

in iron metabolism of Wilson disease gene knockout mice
Uta Merle,* Sabine Tuma,* Thomas Herrmann,* Valer Muntean, Martin Volkmann,1 Sven G. Gehrke*1
and Wolfgang Stremmel*1
*Department of Gastroenterology, University Hospital, Heidelberg, and Labor Prof Dr H.-P. Seelig & Kollegen, Karlsruhe, Germany

Key words
copper, gene expression, iron, iron-related
genes, metabolic liver diseases.
Accepted for publication 27 October 2009.
Correspondence
Uta Merle, Department of Internal Medicine
IV, University Hospital Heidelberg, Im
Neuenheimer Feld 410, 69120 Heidelberg,
Germany. Email:
uta_merle@med.uni-heidelberg.de
1

Contributed equally.

Abstract
Background and Aim: Wilson disease is a genetic disorder associated with copper overload due to mutations within the ATP7B gene. Although copper and iron metabolism are
closely linked, the influence of mutations of the ATP7B gene on iron homeostasis is
unknown. Therefore, the present study was carried out to elucidate iron metabolism in
Atp7b(-/-) mice, an animal model of Wilson disease.
Methods: Hepatic iron content, serum iron parameters and blood hemoglobin levels of
Atp7b(-/-) mice and wild type mice were studied. Hepatic and duodenal expression of iron
metabolism-related genes was measured quantitatively by real-time reverse transcriptionpolymerase chain reaction and post-translational expression of Dmt1 was analyzed by
immunoblot.
Results: Atp7b(-/-) mice displayed copper accumulation (P < 0.001), slightly elevated
hepatic iron content (P = NS), and a low serum ceruloplasmin oxidase activity (1.5 1.9
U/L vs 18.9 4.0 U/L, P < 0.001) when compared with wild type mice. Serum iron, serum
transferrin saturation, and blood hemoglobin levels were significantly lower in Atp7b(-/-)
mice compared with controls (121.2 35.3 mg/dL vs 201.8 34.9 mg/dL (P < 0.001);
44.0 12.7% vs 68.0 8.2% (P < 0.001); and 12.7 0.2 g/dl vs 15.3 0.1 g/dl
(P < 0.001), respectively). Hepatic mRNA expression of hepcidin, TfR-1, TfR-2, hemojuvelin, and Dmt1 + IRE did not differ significantly between Atp7b(-/-) and wild type mice.
In the duodenum of Atp7b(-/-) mice Dmt1 + IRE and hephaestin did not show any
differences in their mRNA levels compared with wild type mice, while Dcytb mRNA
expression was 1.7-fold increased compared with wild type mice (P = 0.01).
Conclusion: Atp7b(-/-) mice demonstrated decreased serum iron parameters and hemoglobin levels most likely related to a low serum ceruloplasmin oxidase activity and not due
to total body iron deficiency.

Introduction
Hepatic copper overload is a key feature of Wilson disease (WD),
an autosomal recessive disorder of copper metabolism. The Wilson
disease gene ATP7B encodes a membrane-bound, P-type copper
transporting adenosine 5-triphosphatase (ATPase) expressed primarily in the liver.1 WD is characterized by a decreased biliary
copper excretion and a defective incorporation of copper into
ceruloplasmin leading to low serum ceruloplasmin ferroxidase
activity and copper accumulation in the liver and other peripheral
tissues.2 Established animal models for WD are the Long Evans
Cinnamon (LEC) rat,3 the toxic milk mouse,4 and the Atp7b(-/-)
mouse.5,6 All three animal models are characterized by hepatic
copper accumulation.
Copper and iron metabolism are closely linked.7 There is evidence for copper playing a role in intestinal iron absorption.8 In
1144

particular, ferric iron is reduced to ferrous iron by the copperdependent reductase Dcytb before it is taken up by enterocytes.
Ferrous iron is transported through the apical membrane of enterocytes by the divalent metal transporter Dmt1.9 Ferrous iron is then
exported through the basolateral membrane of intestinal enterocytes by the iron transporter ferroportin.10 Ferroportin is regulated
at the protein level by hepcidin, a peptide hormone secreted by the
liver in response to iron loading and inflammation.11 The iron
export via ferroportin requires an oxidation of ferrous to ferric
iron, mediated by hephaestin, a membrane bound copperdependent ferroxidase.10 Although a role for copper in iron transport and metabolism is well established, it is unclear if iron
homeostasis is disturbed in WD, as reports on disturbances in iron
metabolism of WD patients are contradictory.1214
Our study was carried out to elucidate iron-metabolism in
Atp7b(-/-) mice modeling the copper storage disease WD. We

Journal of Gastroenterology and Hepatology 25 (2010) 11441150 2010 The Authors

Journal compilation 2010 Journal of Gastroenterology and Hepatology Foundation and Blackwell Publishing Asia Pty Ltd

U Merle et al.

analyzed hepatic copper and iron content, serum iron parameters,

and expression of iron-metabolism-related genes in liver and
duodenum of Atp7b(-/-) mice and compared them with control
littermates.

Methods
Animals
The generation of Atp7b(-/-) mice has been described previously.5
The Atp7b(-/-) mice (with a genetic 129/Sv background) and the
wild type 129/Sv mice were housed at the University of Heidelberg according to the guidelines of the Institutional Animal Care
and Use Committees and in accordance with government guidelines. Atp7b(-/-) and wild type mice with an age of 13 weeks were
used for the experiments (10 animals per group). Mice were kept
on a 12 : 12 h light/dark cycle. Food (normal chow diet containing
176 mg/g iron and 16 mg/g copper) and water were provided ad
libitum.
Animals were anesthetized with ketamine/xylazine. Blood was
collected by cardiac puncture under anesthesia and afterwards
animals were systematically perfused with saline from the left
ventricle after thoracotomy to remove whole blood from organs.
Subsequently livers and duodenum were removed. Duodenal
tissue from the proximal 2 cm of the duodenum was excised,
washed in cold phosphate-buffered solution (PBS), divided longitudinally into three portions, and stored for further analysis.
Samples were either kept frozen at -80C, or stored in RNAlater
solution (Ambion, Austin TX, USA) at -20C.

Measurement of iron-metabolism parameters

Tissue iron and copper quantification using atomic absorption
spectroscopy was carried out as described previously.15 Iron and
copper concentrations of tissues (mg/g of wet tissue) were calculated from the results of this analysis.1618 The Ferrozin method
was used to quantify serum iron levels (AU 640, Olympus Diagnostics, Hamburg, Germany). In acidic environments, binding of
reduced iron to [2,4.6-Tri-(2-Pyridyl)-5-Triazin] results in a blue
colored complex, which is measured bichromatically at 600/
800 nm. Free iron binding capacity was measured in an AU 640
analyzer (Olympus Diagnostics) using reaction with NitrosoPSAP. Excess iron in the reagent forms a complex with NitrosoPSAP supplied in a second reagent. Upon the addition of serum,
binding of iron to free transferrin molecules diminishes the
amount of the colored Nitroso-PSAP-iron complex. The absorption difference of the sample with and without serum is a measure
of free iron binding capacity, which is expressed as an equivalent
of unbound iron molecules. Serum transferrin saturation (%) was
calculated from the data according to the equation: Serum iron/
(Serum iron + free iron binding capacity) 100.

Quantitative RT-PCR
Total RNA was isolated from liver and duodenum samples using
the RNAeasy Mini Kit (Qiagen, Hilden, Germany) including
DNAse digestion according to manufacturers instructions. As
previously described,19,20 real-time quantification of mouse mRNA
transcripts was carried outwith a two-step reverse transcription-

Iron metabolism of Atp7b(-/-) mice

polymerase chain reaction (RT-PCR) using the LightCycler

system and the Relative Quantification Software Version 1.0
(Roche Diagnostics, Mannheim, Germany). Quantification of
mRNA transcripts was carried out using the sense and the antisense primer for mouse Dcytb, Dmt1+IRE, Dmt1-IRE, hepcidin,
hephaestin, hemojuvelin, transferrin receptor-1 (TfR-1) and transferrin receptor-2 (TfR-2) as described previously.19,20 Calibrators
were generated from expressed sequence tag (EST) clones from
RZPD (Berlin, Germany) as described previously.19,20 Normalization to actin mRNA levels was carried out as previously described
and gene expression levels are reported relative to actin expression
levels.19,20 Mouse hepcidin primers were designed to amplify both
hepcidin variants (hepcidin 1 and 2).19,20

Western blot
For Western blot, an affinity-purified polyclonal rabbit antimouse DMT1 antibody NRAMP12-A (Alpha Diagnostics International, San Antonio, TX, USA) was used. For detection of
actin a monoclonal mouse anti-b-actin antibody (Sigma, St.
Louis, MO, USA) was used. Tissue specimens were ground to a
fine homogenate using a Potter-Elvehjem homogenizer with a
Teflon pestle at 1000 rpm for five strokes in three volumes of
buffer (for liver tissue: 0.25 M sucrose, 10 mM HEPES-NaOH,
0.5 mM ethylenediaminetetraacetic acid [EDTA], pH 7.4; for
duodenal tissue: 0.25 M sucrose, 20 mM HEPES-NaOH, 1 mM
EDTA, pH 7.4) to which Complete EDTA-Free Protease Inhibitor Cocktail (Roche) had been freshly added. The lysates were
centrifuged (100 000 g, 4C) to pellet tissue debris. Supernatants
were stored in aliquots at -80C. Protein concentrations were
determined by bicinchoninic acid (BCA) assay. Protein samples
were mixed with loading buffer (1% 2-mercaptoethanol, 2%
sodium dodecyl sulfate [SDS], 10% glycerol, 62 mM Tris-HCl,
pH 6.86, 0.01% bromophenol blue) and heated at 95C for 5 min
before they were electrophoretically separated on 7% SDSpolyacrylamide gels. The protein was transferred onto reinforced
nitrocellulose membrane in the presence of buffer containing
20% methanol. Membranes were blocked in a solution of 5%
skimmed milk in PBS with 0.1% Tween 20 for 1 h. Primary
antibody incubations (diluted 1:2000) to specifically detect the
mouse Dmt1 protein were carried out overnight at 4C in blocking solution. Membranes were then incubated with horseradish
peroxidase-conjugated for Dmt1-detection with goat anti-rabbit
IgG (Dianova, Hamburg, Germany) and for actin-detection with
goat anti-mouse IgG (Dianova), for 1 h in 5% skimmed milk in
PBS with 0.1% Tween 20 for 1 h at room temperature before
using the enhanced chemiluminescent western blotting detection
system (Amersham, Braunschweig, Germany). Quantification of
western blots was carried out by using ImageJ (NIH).

Measurement of serum ferroxidase activity

Serum ceruloplasmin ferroxidase activity was measured as previously described in sera of Atp7b(-/-) and wild type mice.21

Iron staining
For staining of hepatic non-heme ferrous iron, 3,3diaminobenzidine tetrahydrochloride (DAB)-enhanced Perls

Journal of Gastroenterology and Hepatology 25 (2010) 11441150 2010 The Authors

Journal compilation 2010 Journal of Gastroenterology and Hepatology Foundation and Blackwell Publishing Asia Pty Ltd

1145

Iron metabolism of Atp7b(-/-) mice

U Merle et al.

statistically significant. Statistical analyses were carried out with

SPSS for Windows, release 16.0 (SPSS, Chicago, IL, USA).

Prussian blue staining was applied. Snap-frozen livers of

Atp7b(-/-) mice and wild type mice were cut into 5-mm sections
and mounted on slides for staining with Perls solution (4% HCl
and 4% potassium ferrocyanide, 1:1) at room temperature for
60 min. Subsequently, slides were incubated for 15 min in DAB
and then immersed in 0.015% H2O2 in DAB. All microscopy was
carried out using an Olympus IX50 (Olympus Optical Co. GmbH,
Germany) microscope.

Results
Clinical phenotype of Atp7b(-/-) mice
The Atp7b(-/-) mouse is an established animal model of Wilson
disease.5,6 In our study Atp7b(-/-) mice appeared normal at birth
and developed and grew normally.
Hepatic copper content of 13-week-old Atp7b(-/-) mice was
50-fold higher compared with wild type mice (261.8 37.3 vs
5.2 0.7 mg/g of wet weight, P < 0.001) (Fig. 1A). Serum ceruloplasmin oxidase activity was significantly lower in Atp7b(-/-)
mice compared with wild type mice (1.5 1.9 U/L vs 18.9 4.0
U/L, P < 0.001) (Fig. 1B).

Statistical analysis
Data are presented as means standard deviations. The nonparametric MannWhitney test was used to evaluate the differences in hematological parameters, tissue metal concentration and
gene expression levels on real-time RT-PCR between Atp7b(-/-)
and wild type mice. A P-value less than 0.05 was considered

(b)

300
200
100

0
Atp7b/

30
20
10
0
Atp7b/

Atp7b+/+

Figure 1 Hepatic copper content and serum

ceruloplasmin oxidase activity of 13-week-old
Atp7b(-/-) mice (n = 10) and wild type mice
of the same age (n = 10). (a) Hepatic Cu
content of Atp7b(-/-) mice compared with
wild type mice. Hepatic Cu content (mg/g wet
weight) is significantly higher in Atp7b(-/-)
mice compared with wild type mice. (b)
Serum ceruloplasmin oxidase activity of
Atp7b(-/-) mice compared with wild type
mice. Serum ceruloplasmin oxidase activity is
significantly lower in Atp7b(-/-) mice compared with control mice.

(b)

150
Hemoglobin (g/dL)

Liver Fe (g/g wet tissue)

40

Atp7b+/+

(a)

100

50

0
Atp7b/

16
14
12
10
8
6
4
2
0

Atp7b/

Atp7b+/+

Atp7b+/+

(d)

250

Transferrin saturation (%)

(c)

Serum Fe (g/dL)

50

Oxidase activity (U/L)

Liver Cu (g/g wet tissue)

(a)

200
150
100
50
0
Atp7b/

1146

Atp7b+/+

80
70
60
50

40
30
20
10
0
Atp7b/

Atp7b+/+

Figure 2 Hepatic iron content, hemoglobin

concentration, serum iron concentration, and
serum transferrin saturation of 13-week-old
Atp7b(-/-) mice (n = 10) and wild type mice
of the same age (n = 10). (a) Hepatic iron
content of Atp7b(-/-) mice compared with
wild type mice. Hepatic iron content (mg/g
wet weight) shows no significant difference
between Atp7b(-/-) mice and wild type mice.
(b) Hemoglobin content of Atp7b(-/-) mice
compared with wild type mice. Hemoglobin
content is significantly lower in Atp7b(-/-)
mice compared with control mice. (c) Serum
iron concentration of Atp7b(-/-) mice compared with wild type mice. Serum iron concentration is significantly lower in Atp7b(-/-)
mice. (d) Serum transferrin saturation of
Atp7b(-/-) mice compared with wild type
mice. Serum transferrin saturation is significantly lower in Atp7b(-/-) mice. Data are
expressed as means standard error.
Asterisks (*) indicate that Atp7b(-/-)
mice differ significantly from the wild type
mice (P < 0.05).

Journal of Gastroenterology and Hepatology 25 (2010) 11441150 2010 The Authors

Journal compilation 2010 Journal of Gastroenterology and Hepatology Foundation and Blackwell Publishing Asia Pty Ltd

U Merle et al.

Iron metabolism of Atp7b(-/-) mice

Hepatic iron content and serum

iron-metabolism parameters

Hepatic and duodenal mRNA expression of

iron-metabolism related genes

Hepatic iron content was only slightly elevated in Atp7b(-/-) mice

compared with wild type mice (96.3 19.3 vs 81.8 29.4 mg/g
of wet weight, P = 0.212) (Fig. 2A). By Prussian blue iron staining, no relevant signal could be detected, neither in wild type mice
nor in Atp7b(-/-) mice (data not shown).
The mean hemoglobin level was significantly lower in
Atp7b(-/-) mice compared with wild type mice (12.7 0.2 vs
15.3 0.1 g/dl (P < 0.001)) (Fig. 2B). Serum iron concentration
and serum transferrin saturation were significantly lower in
Atp7b(-/-) mice compared with wild type mice (121.2
35.3 mg/dL vs 201.8 34.9 mg/dL (P < 0.001) and 44.0 12.7%
vs 68.0 8.2% (P < 0.001), respectively) (Fig. 2C,D).

The expression levels of iron metabolism-related genes were measured by means of quantitative real-time RT-PCR and expressed
relative to the expression levels of the housekeeping gene actin.
There was no significant difference between Atp7b(-/-) mice and
control mice in mRNA expression levels of the iron metabolismrelated genes hepcidin (P = 0.20) (Fig. 3A), TfR1 (P = 0.06)
(Fig. 3B), TfR2 (P = 0.50) (Fig. 3C), hemojuvelin (P = 0.27)
(Fig. 3D), and the Dmt1-isoform containing an iron-responsive
element (Dmt1+IRE, P = 0.31) (Fig. 3E). Expression of the Dmt1isoform lacking an iron-responsive element (Dmt1-IRE) was significantly lower in Atp7b(-/-) mice compared with control mice
(0.010 0.002 vs 0.019 vs 0.009, P = 0.005) (Fig. 3F).

(b)

50

-2

TfR1/actin ratio (10 )

Hepcidin/actin ratio (10 )

(a)

40
30
20
10

Atp7b+/+

(c)

Atp7b/

3
Hjv/actin ratio (10 )

-1

-1

TfR2/actin ratio (10 )

3
2
1
0

Atp7b/

Atp7b/

Atp7b+/+

Atp7b+/+

(f)
-3

Dmt1-IRE/actin ratio (10 )

(e)
-3

Atp7b+/+

(d)

Dmt1+IRE/actin ratio (10 )

0
Atp7b/

Figure 3 Hepatic expression of iron-related

genes. (af) Hepatic mRNA levels of ironrelated gene (a, hepcidin; b, TfR1; c, TfR2; d,
hemojuvelin; e, Dmt1 + IRE; f, Dmt1-IRE) in
Atp7b(-/-) (n = 10) and wild type mice
(n = 10) quantified by real-time reverse
transcription-polymerase chain reaction (RTPCR) and expressed in relation to actin. Data
are expressed as means standard error.
Asterisks (*) indicate that Atp7b(-/-) mice
differ significantly from the wild type mice
(P < 0.05).

25
20
15
10
5
0

Atp7b/

30

20

10

Atp7b+/+

Journal of Gastroenterology and Hepatology 25 (2010) 11441150 2010 The Authors

Journal compilation 2010 Journal of Gastroenterology and Hepatology Foundation and Blackwell Publishing Asia Pty Ltd

Atp7b/

Atp7b+/+

1147

Iron metabolism of Atp7b(-/-) mice

U Merle et al.

-3

Dmt1+IRE/actin ratio (10 )

(b)

25
20
15

10
5
0
Atp7b/

(c)

50

Atp7b/

Atp7b+/+

(d)

20

-2

-3

Dmt1-IRE/actin ratio (10 )

100

Atp7b+/+

Hephaestin/actin ratio (10 )

-2

Dcytb/actin ratio (10 )

(a)

15
10
5
0
Atp7b/

Atp7b+/+

15

10

0
Atp7b/

Duodenal Dcytb mRNA-levels were 1.7-fold higher in

Atp7b(-/-) mice than in wild type mice (0.149 0.052 vs
0.087 0.040, P = 0.01) (Fig. 4A). Duodenal expression levels of
Dmt1+IRE (Fig. 4B), Dmt1-IRE (Fig. 4C), and hephaestin
(Fig. 4D) were not significantly different between Atp7b(-/-) and
wild type mice (P = 0.11, P = 0.26, and P = 0.23, respectively).

Hepatic and duodenal expression of Dmt1 on

protein level
Previously published in vitro data suggested a regulation of Dmt1
by copper.22 Therefore, we analyzed Dmt1 expression in liver and
duodenum additionally on the protein level. Western blot analysis
was carried out using a polyclonal antibody raised against Dmt1
protein. As shown in Figure 5A,B, there were no significant differences in the hepatic and duodenal expression levels between
Atp7b(-/-) mice and wild type mice. Densitometric analyses confirmed this observation (Fig. 5C,D).

Discussion
On the molecular level, copper and iron metabolism are closely
linked.8 However, it is still unclear whether this intimate relationship is of any pathological or clinical relevance. In Atp7b(-/-)
mice, an animal model of WD, we investigated whether WD is
associated with abnormalities in iron metabolism including the
expression profiles of iron-related genes.
As expected,6 the Atp7b(-/-) mice displayed typical features of
WD. Atp7b(-/-) mice demonstrated a 50-fold higher hepatic
1148

Atp7b+/+

Figure 4 Duodenal expression of ironrelated genes. (ad) Duodenal mRNA levels of

iron-related genes (a, Dcytb; b, Dmt1+IRE;
c, Dmt1-IRE; d, hephaestin) in Atp7b(-/-)
(n = 10) and wild type mice (n = 10) quantified
by real-time reverse transcription-polymerase
chain reaction (RT-PCR) and expressed in
relation to actin. Data are expressed as
means standard error. Asterisks (*) indicate
that Atp7b(-/-) mice differ significantly from
the wild type mice (P < 0.05).

copper content than wild type mice and a low serum ceruloplasmin
(Cp) oxidase activity. Evaluation of iron parameters revealed significantly lower serum levels of iron and a decreased serum transferrin saturation in Atp7b(-/-) mice compared with wild type
littermates. Moreover, hemoglobin levels were significantly
deceased and hepatic iron content was slightly elevated in
Atp7b(-/-) mice.
These findings demonstrate that disruption of Atp7b leads to
relevant changes in iron homeostasis in addition to the known
changes in copper metabolism. The phenotype resembles WD and,
on the other hand, suggests a defective iron homeostasis with a
reduced bioavailability of iron in erythropoetic cells leading to
mild anemia. Interestingly, such disturbances in iron metabolism
are also found in the rare autosomal recessive disorder aceruloplasminemia. Affected patients present with a unique combination
of neurovisceral iron overload and iron deficiency anemia.
Decreased serum iron and serum transferrin saturation are other
typical laboratory findings and hepatic iron overload is usually
moderate and not associated with advanced liver damage.23,24
The underlying genetic defect in aceruloplasminemia generally
leads to an inability for apo-Cp to bind copper. Compared with
copper-loaded holo-Cp, apo-Cp is unstable and rapidly degraded
in the serum of affected patients.25 It has been shown that ferroxidase activity of Cp is required to stabilize cell surface ferroportin,
the only known mammalian iron exporter.26 The exported ferrous
iron requires oxidation to ferric iron for incorporation into transferring, which is the crucial step for release of iron from cells and
allows the distribution of iron within the body, particularly to the
erythron for erythropoiesis. As a consequence, the loss of

Journal of Gastroenterology and Hepatology 25 (2010) 11441150 2010 The Authors

Journal compilation 2010 Journal of Gastroenterology and Hepatology Foundation and Blackwell Publishing Asia Pty Ltd

U Merle et al.

Iron metabolism of Atp7b(-/-) mice

(a)

Atp7b(/)
#1

#2

#3

Atp7b(+/+)
#1

#4

#2

#3

#4

120 kDa
100 kDa
70 kDa
55 kDa
40 kDa
(b)

Atp7b(/)
#1

#2

#3

Atp7b(+/+)
#4

#1

#2

#3

#4

120kDa
100kDa
70kDa
55 kDa
40 kDa
(c)

(d)

3000

1500
Arbitrary units

Arbitrary units

Figure 5 Hepatic and duodenal expression

of Dmt1 on protein level. (a,b) Western blots
of liver (a) and duodenum (b) homogenate
(80 mg protein) of Atp7b(-/-) mice and wild
type mice using a rabbit anti-DMT1 (upper
panel). As a gel loading control, the membranes were reprobed with anti-actin antibody (lower panel). Molecular weight
markers are indicated on the left. Four typical,
representative experiments for Atp7b(-/-)
mice and wild type mice are shown. (c,d)
Results of densitometric analysis of
Atp7b(-/-) mice (n = 10) and wild type mice
(n = 10). Hepatic (c) and duodenal (d) expression of Dmt1 is not significantly different
between Atp7b(-/-) mice and wild type mice.

2000

1000

1000

500

0
Atp7b/

ferroxidase activity leads to decreased intestinal iron absorption

and iron storage in visceral organs (e.g. liver).
Recently, di Patti et al.27 constituted the first evidence that
functional silencing of mammalian ATP7B due to a dominant Cp
mutation (leading to impaired ATP7B-mediated copper loading of
Cp) can result in a severe variant of aceruloplasminemia. Therefore,
in WD a low Cp oxidase activity due to defective ATP7B might lead
to changes in iron metabolism as found in aceruloplasminemia. Our
data in Atp7b(-/-) mice strongly support such a hypothesis. In our
Atp7b(-/-) mice, Cp oxidase activity was reduced to 7% of the
activity in wild type mice. The residual Cp oxidase activity might
explain why iron homeostasis is only moderately affected in
Atp7b-/- mice and, compared with aceruloplasminemia, is not
associated with statistically significant hepatic iron storage.
While aceruloplasminemic mice present with a reduced

Atp7b+/+

Atp7b/

Atp7b+/+

hepcidin expression due to iron overload,28 our Atp7b(-/-) did not

demonstrate any significant changes in hepatic hepcidin expression compared with wild type mice. These data implicate that
hepcidin regulation is secondary to aceruloplasmina-induced
hepatic iron overload and does not play a significant role in the
iron-copper relationship. By contrast, the apical intestinal iron
transporter Dmt1 is also a physiologically-relevant copper transporter, suggesting that these two metals may compete with each
other for intestinal uptake.8 Accordingly, in our Atp7b(-/-) mice,
hepatic mRNA expression of the Dmt1 splicing variant missing an
iron responsive element (non-IRE variant) is significantly downregulated compared with wild type littermates. However, hepatic
mRNA expression of the IRE-containing Dmt1 variant as well as
duodenal mRNA expression of both Dmt1 variants did not show
any Atp7b-dependent regulation. In order to evaluate a post-

Journal of Gastroenterology and Hepatology 25 (2010) 11441150 2010 The Authors

Journal compilation 2010 Journal of Gastroenterology and Hepatology Foundation and Blackwell Publishing Asia Pty Ltd

1149

Iron metabolism of Atp7b(-/-) mice

U Merle et al.

translational regulation in response to copper overload, we also

analyzed the expression of Dmt1 on the protein level. Remarkably,
neither duodenal nor hepatic expression showed any significant
differences in Atp7b(-/-) mice compared with wild type mice.
Hepatic mRNA expression of other relevant iron metabolismrelated genes including transferrin receptor 1 and 2 (TfR1 and
TfR2) as well as hemojuvelin (Hjv) remained unchanged in copper
overloaded Atp7b(-/-) mice. Within the duodenum, there was no
evidence for a transcriptional regulation of the copper-containing
basolateral ferroxidase hephaestin. However, the apical ferrireductase Dcytb was significantly upregulated in Atb7b-/- mice compared with wild type mice. Wyman et al. recently identified Dcytb
as an in vitro cupric reductase indicating that it plays a physiological role in both, iron and copper uptake.29 Thus, it is conceivable
that Dcytb upregulation is also associated with the development of
pathological copper uptake leading to WD. Dcytb might reduce
copper in the intestine for uptake of Cu(I) by the apical transport
protein Ctr1 and might play an additional role in the copper accumulation process in WD.19
As previously shown in WD patients,30 our Atp7b(-/-) mice
showed a decreased Cp oxidase activity. In the Atp7b(-/-) mice
low Cp oxidase activity was associated with a slight, but not
significant increase in hepatic iron stores, low serum iron parameters and low hemoglobin values indicative of a misdistribution of
iron within the organism. Therefore, low serum iron parameters
and low hemoglobin levels in WD are not due to total body iron
deficiency but due to a misdistribution by low serum Cp oxidase
activity and, thus, should not be treated by iron supplementation.

Acknowledgments
We thank Professor T.C. Gilliam for kindly providing the
Atp7b(-/-) mice. The work was supported by grants to UM, TH,
SGG and WS from Deutsche Forschungsgemeinschaft and
Dietmar Hopp Foundation.

References
1 Bull PC, Thomas GR, Rommens JM, Forbes JR, Cox DW. The
Wilson disease gene is a putative copper transporting P-type ATPase
similar to the Menkes gene. Nat. Genet. 1993; 5: 32737.
2 Scheinberg IH. Wilsons disease. J. Rheumatol. Suppl. 1981; 7:
903.
3 Wu J, Forbes JR, Chen HS, Cox DW. The LEC rat has a deletion in
the copper transporting ATPase gene homologous to the Wilson
disease gene. Nat. Genet. 1994; 7: 5415.
4 Theophilos MB, Cox DW, Mercer JF. The toxic milk mouse is a
murine model of Wilson disease. Hum. Mol. Genet. 1996; 5:
161924.
5 Buiakova OI, Xu J, Lutsenko S et al. Null mutation of the murine
ATP7B (Wilson disease) gene results in intracellular copper
accumulation and late-onset hepatic nodular transformation. Hum.
Mol. Genet. 1999; 8: 166571.
6 Huster D, Finegold MJ, Morgan CT et al. Consequences of copper
accumulation in the livers of the Atp7b-/- (Wilson disease gene)
knockout mice. Am. J. Pathol. 2006; 168: 42334.
7 Fox PL. The copper-iron chronicles: the story of an intimate
relationship. Biometals 2003; 16: 940.
8 Sharp P. The molecular basis of copper and iron interactions. Proc.
Nutr. Soc. 2004; 63: 5639.

1150

9 Levy JE, Montross LK, Andrews NC. Genes that modify the
hemochromatosis phenotype in mice. J. Clin. Invest. 2000; 105:
120916.
10 Frazer DM, Anderson GJ. The orchestration of body iron intake:
how and where do enterocytes receive their cues? Blood. Cells. Mol.
Dis. 2003; 30: 28897.
11 Nemeth E, Tuttle MS, Powelson J et al. Hepcidin regulates cellular
iron efflux by binding to ferroportin and inducing its internalization.
Science 2004; 306: 20903.
12 Hafkemeyer P, Schupp M, Storch M, Gerok W, Haussinger D.
Excessive iron storage in a patient with Wilsons disease. Clin.
Investig. 1994; 72: 1346.
13 Shiono Y, Wakusawa S, Hayashi H et al. Iron accumulation in the
liver of male patients with Wilsons disease. Am. J. Gastroenterol.
2001; 96: 314751.
14 Hayashi H, Yano M, Fujita Y, Wakusawa S. Compound overload of
copper and iron in patients with Wilsons disease. Med. Mol.
Morphol. 2006; 39: 1216.
15 Nicholson JR, Savory MG, Savory J, Wills MR. Micro-quantity
tissue digestion for metal measurements by use of a microwave
acid-digestion bomb. Clin. Chem. 1989; 35: 48890.
16 Widdowson EM, Spray CM. Chemical development in utero. Arch.
Dis. Child. 1951; 26: 20514.
17 Widdowson EM, Chan H, Harrison GE, Milner RD. Accumulation
of Cu, Zn, Mn, Cr and Co in the human liver before birth. Biol.
Neonate 1972; 20: 3607.
18 Shah B, Belonje B. Liver storage iron in Canadians. Am. J. Clin.
Nutr. 1976; 29: 669.
19 Herrmann T, Muckenthaler M, van der Hoeven F et al. Iron overload
in adult Hfe-deficient mice independent of changes in the
steady-state expression of the duodenal iron transporters DMT1 and
Ireg1/ferroportin. J. Mol. Med. 2004; 82: 3948.
20 Gehrke SG, Herrmann T, Kulaksiz H et al. Iron stores modulate
hepatic hepcidin expression by an HFE-independent pathway.
Digestion 2005; 72: 2532.
21 Schosinsky KH, Lehmann HP, Beeler MF. Measurement of
ceruloplasmin from its oxidase activity in serum by use of
o-dianisidine dihydrochloride. Clin. Chem. 1974; 20: 155663.
22 Arredondo M, Cambiazo V, Tapia L et al. Copper overload affects
copper and iron metabolism in Hep-G2 cells. Am. J. Physiol.
Gastrointest. Liver Physiol. 2004; 287: G2732.
23 Kono S, Suzuki H, Takahashi K et al. Hepatic iron overload
associated with a decreased serum ceruloplasmin level in a novel
clinical type of aceruloplasminemia. Gastroenterology 2006; 131:
2405.
24 Xu X, Pin S, Gathinji M, Fuchs R, Harris ZL. Aceruloplasminemia:
an inherited neurodegenerative disease with impairment of iron
homeostasis. Ann. N. Y. Acad. Sci. 2004; 1012: 299305.
25 Gitlin JD. Wilson disease. Gastroenterology 2003; 125: 186877.
26 De Domenico I, Ward DM, di Patti MC et al. Ferroxidase activity is
required for the stability of cell surface ferroportin in cells
expressing GPI-ceruloplasmin. EMBO J. 2007; 26: 282331.
27 di Patti MC, Maio N, Rizzo G et al. Dominant mutants of
ceruloplasmin impair the copper loading machinery in
aceruloplasminemia. J. Biol. Chem. 2009; 284: 454554.
28 Guo P, Cui R, Chang YZ et al. Hepcidin, an antimicrobial peptide is
downregulated in ceruloplasmin-deficient mice. Peptides 2009; 30:
2626.
29 Wyman S, Simpson RJ, McKie AT, Sharp PA. Dcytb. Cybrd1)
functions as both a ferric and a cupric reductase in vitro. FEBS Lett.
2008; 582: 19016.
30 Merle U, Eisenbach C, Weiss K, Tuma S, Stremmel W. Serum
ceruloplasmin oxidase activity is a sensitive and highly specific
diagnostic marker for Wilson disease. J. Hepatol. 2009 51: 92530.

Journal of Gastroenterology and Hepatology 25 (2010) 11441150 2010 The Authors

Journal compilation 2010 Journal of Gastroenterology and Hepatology Foundation and Blackwell Publishing Asia Pty Ltd