Effect of hepcidin on intestinal iron absorption in mice

Abstract

The effect of the putative iron regulatory peptide hepcidin on iron absorption was investigated in mice. Hepcidin peptide was synthesized and injected into mice for up to 3 days, and in vivo iron absorption was measured with tied-off segments of duodenum. Liver hepcidin expression was measured by reverse transcriptase-polymerase chain reaction. Hepcidin significantly reduced mucosal iron uptake and transfer to the carcass at doses of at least 10 microg/mouse per day, the reduction in transfer to the carcass being proportional to the reduction in iron uptake. Synthetic hepcidin injections down-regulated endogenous liver hepcidin expression excluding the possibility that synthetic hepcidin was functioning by a secondary induction of endogenous hepcidin. The effect of hepcidin was significant at least 24 hours after injection of hepcidin. Liver iron stores and hemoglobin levels were unaffected by hepcidin injection. Similar effects of hepcidin on iron absorption were seen in iron-deficient and Hfe knockout mice. Hepcidin inhibited the uptake step of duodenal iron absorption but did not affect the proportion of iron transferred to the circulation. The effect was independent of iron status of mice and did not require Hfe gene product. The data support a key role for hepcidin in the regulation of intestinal iron uptake.

Full text

RED CELLS
Effect of hepcidin on intestinal iron absorption in mice
Abas H. Laftah, Bala Ramesh, Robert J. Simpson, Nita Solanky, Seiamak Bahram, Klaus Schu¨mann,
Edward S. Debnam, and Surjit K. S. Srai
The effect of the putative iron regulatory
peptide hepcidin on iron absorption was
investigated in mice. Hepcidin peptide
was synthesized and injected into mice
for up to 3 days, and in vivo iron absorp-
tion was measured with tied-off segments
of duodenum. Liver hepcidin expression
was measured by reverse transcriptase–
polymerase chain reaction. Hepcidin sig-
nificantly reduced mucosal iron uptake
and transfer to the carcass at doses of at
least 10 ␮g/mouse per day, the reduction
in transfer to the carcass being propor-
tional to the reduction in iron uptake.
Synthetic hepcidin injections down-regu-
lated endogenous liver hepcidin expres-
sion excluding the possibility that syn-
thetic hepcidin was functioning by a
secondary induction of endogenous hep-
cidin. The effect of hepcidin was signifi-
cant at least 24 hours after injection of
hepcidin. Liver iron stores and hemoglo-
bin levels were unaffected by hepcidin
injection. Similar effects of hepcidin on
iron absorption were seen in iron-
deficient and Hfe knockout mice. Hepci-
din inhibited the uptake step of duodenal
iron absorption but did not affect the
proportion of iron transferred to the circu-
lation. The effect was independent of iron
status of mice and did not require Hfe
gene product. The data support a key role
for hepcidin in the regulation of intestinal
iron uptake. (Blood. 2004;103:3940-3944)
©2004 by The American Society of Hematology
Introduction
Recent advances in molecular-level understanding of iron absorp-
tion regulation have implicated several genes as regulators of iron
absorption, 2 of which (Hfe and hepcidin) have received particular
attention.1-3 Hepcidin was originally identified as an antimicrobial
peptide synthesized in liver, but evidence from knockout mice
suggests this peptide is a negative regulator of iron absorption.3,4
Initial work implicated hepcidin as the long-sought “stores regula-
tor” of iron absorption proposed by Finch.5However, recent work
has suggested a wider role for this peptide as it also shows an
expression pattern consistent with the “erythroid regulator.”6A
mutation in hepcidin has recently been implicated as a cause of
juvenile hemochromatosis.7
Transgenic mice overexpressing hepcidin were found to
develop an iron-deficient phenotype, consistent with an effect
on placental iron transport and intestinal iron absorption.8
Frazer et al9provided data that quantitatively relates hepcidin
expression to iron absorption rates and expression of duodenal
transporters in an iron-deficient rat model. It can be deduced that
a similar inverse correlation between hepcidin expression and
iron absorption probably exists in humans, based on data
provided by Nemeth et al10 relating urinary hepcidin to serum
ferritin levels. Thus far, however, no data measuring the direct
effect of injecting hepcidin on iron absorption rates is available.
We therefore synthesized hepcidin peptide and injected this into
normal, iron-deficient, and Hfe knockout mice and measured
iron absorption rates.
Materials and methods
Mice (129/Ola-C57BL/6 mixed background strain) with a 2-kb pgk-neor
gene flanked by loxP sites replacing a 2.5-kb BglII fragment (see Bahram et
al11 for details) were used as Hfe knockouts (KOs). Heterozygotes were
mated and wild-type and homozygote Hfe KO littermates were identified at
4 to 5 weeks of age. Mice were fed CRM diet (Scientific Diet Supplies
[SDS], Witham, Essex, United Kingdom) ad lib and a mixed group of males
and females (2 males and 4 females in each experimental group) was
studied at 8 to 12 weeks of age. Other experiments were performed with
male CD1 mice aged 6 to 10 weeks. Mice were injected with 0.15 M NaCl
or hepcidin dissolved in 0.15 M NaCl by the intraperitoneal route. Iron
deficiency was induced by feeding mice an iron-deficient purified diet (see
Bahram et al11 for details) for 3 weeks after weaning. Controls received an
iron-replete diet identical to the iron-deficient diet except for the addition of
180 mg/kg Fe.11 All animal experiments were performed under the
authority of a United Kingdom Home Office license.
Peptide synthesis
Hepcidin (human and mouse sequence, 25 amino acids) was synthesized on
a Wang alcohol resin with a loading of 1.30 mmol/g on a Rainin automatic
peptide synthesizer (Protein Technologies, Tuscon, AZ) using the standard
Fmoc chemistry. Cysteine sulphurs were protected with trityl groups and
all other side-chain functions were protected with trifluoroacetic acid
(TFA)–labile groups.All the cysteines were introduced as preformed symmetri-
cal anhydrides to prevent enantiomerization during assembly. The com-
pleted peptide was deprotected and cleaved from the resin with a mixture of
TFA, ethanedithiol, and water (94:3:3). The final product was precipitated
From the Department of Life Sciences, King’s College London, London, United
Kingdom; the Department of Biochemistry & Molecular Biology and the
Department of Physiology, Royal Free and University College School of
Medicine, London, United Kingdom; INSERM-CreS, Centre de Recherche
d’Immunologie et d’He´matologie, Strasbourg Cedex, France; and the Walther-
Straub-Institut fu¨r Pharmakologie und Toxikologie, Ludwig-Maximilians-
Universita¨t,Mu¨nchen, Germany.
Submitted March 27, 2003; accepted January 21, 2004. Prepublished
online as Blood First Edition Paper, January 29, 2004; DOI 10.1182/blood-
2003-03-0953.
Supported by the Wellcome Trust, Sir Jules Thorn CharitableTrust, and United
Kingdom Medical Research Council. S.B.’s laboratory is supported by the
Ministe`re de la Recherche and INSERM of France.
Reprints: S.K.S. Srai, Department of Biochemistry & Molecular Biology, Royal
Free and University College School of Medicine, Rowland Hill Street, London;
e-mail: k.srai@rfc.ucl.ac.uk.
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.
© 2004 by The American Society of Hematology
3940 BLOOD, 15 MAY 2004 䡠VOLUME 103, NUMBER 10

with cold diethyl ether, dried, and purified by reverse-phase high-
performance liquid chromatography (HPLC) on a Vydac C18 column
(Vydac Hessperia, Anaheim, CA).
The lyophilized prepurified reduced hepcidin was dissolved in neat TFA
and rotary evaporated so as to give a thin film of peptide on the surface of a
quick-fitflask. The peptide was further dried under vacuum for 24 hours. To
this dried film, 0.1 M de-gassed ammonium bicarbonate was added and the
mixture was stirred for 48 hours while open to atmosphere. The reaction
was then analyzed by the Ellman reagent to ascertain complete oxidation.
The mixture was lyophilized and purified by reverse-phase HPLC on a
Vydac C18 column.
The mass of oxidized peptide was verified by mass spectrometry
(Maldi-Tof) and was found to be 2789. The oxidized hepcidin was further
analyzed by electrophoresis on a 16.5% tricine sodium dodecyl sulfate
(SDS)–polyacrylamide gel. It migrated as a narrow single band with an
apparent molecular weight of less than 10 000 in agreement with the
apparent molecular weight of native urinary hepcidin when electrophoresed
under identical conditions. We further compared our synthetic peptide with
standard hepcidin by electrophoresis on acid/urea gel12 (Figure 1); standard
hepcidin13 separated as a single band, whereas our synthetic peptide used in
this study separated as a broad band, like it was a mixture of multiple forms.
However, we found that both standard hepcidin and the synthetic hepcidin
were immunoreactive with an antibody raised against urinary hepcidin. We
concluded that our synthetic peptide is the correct molecular weight for
oxidized hepcidin, contains a form equivalent to urinary hepcidin, but also
has forms of hepcidin that migrate differently on acid/urea gel.
In vivo iron absorption was determined with tied-off duodenal segments
in anesthetized mice. 59FeNTA2medium (250 ␮M; 1:2 ferric nitrilotriac-
etate [Fe:NTA] in physiologic medium, 125 mM NaCl, 3.5 mM KCl, 10
mM MgCl2, 1 mM CaCl2, 16 mM Hepes, pH 7.4) was injected into
prewashed (0.5 mL warm 0.15 M NaCl) duodenal segments. After 10
minutes, the animal was killed and the duodenal segment removed, opened,
flushed with 10 mL 0.15 M NaCl, blotted, and weighed. Blood was drawn
from the heart and sampled (5 ␮L) for hemoglobin assay. The remainder
was allowed to clot, spun at 10 000g, and serum was separated from red
cells. Aliver sample was taken for nonheme iron assay. Radioactivity in the
duodenal segment and various samples was determined in a gamma counter
(LKB Wallac, Uppsala, Sweden). The carcass was counted in a large
volume sample counter.14 The activity of 59Fe present in the intestinal tissue
is referred to as mucosal retention (MR), whereas that in the carcass (sum of
activity in carcass and all samples taken for assays) is referred to as mucosal
transfer (MT). The sum of the mucosal retention and mucosal transfer
represents the total mucosal uptake (TMU).14 Hemoglobin and tissue
nonheme iron concentrations were determined as described previously.15
Serum iron, unsaturated iron binding capacity, and total iron binding
capacity were determined with a Sigma manual assay kit (Sigma Chemical,
Poole, United Kingdom).
Expression of specific transcripts for hepcidin were analyzed by
real-time PCR. Total liver RNAwas extracted using QIAamp RNA Blood
Mini Kits (Qiagen Crawley, West Sussex, United Kingdom) and was
reverse transcribed into cDNA by the avian myeloblastosis virus (AMV)
first-strand cDNA synthesis kit (Roche Diagnostic, Mannheim, Germany).
Amplification of messenger RNA was performed using LightCycler Fast
Start DNA master SYBR Green 1 (Roche Diagnostic) on a LightCycler
real-time PCR instrument (version 3.5; Roche Diagnostic) according to the
manufacturer’s protocol. The primer pairs used to quantify hepcidin 1 were
forward ACCACCTATCTCCATCAAC, reverse GGTCAGGATGTG-
GCTC. Quantification was obtained by comparing the crossing point (ie,
the cycle number at which fluorescence can be detected) of samples against
a standard curve constructed from known amounts of PCR product. Levels
Figure 2. Effect of hepcidin on iron absorption in mice. Mice were injected daily
for 3 days with 0.15 M NaCl (controls; ■) or hepcidin (10 ␮g[■]or100␮g[䊐])
dissolved in 0.15 M NaCl. Tied-off segments of duodenum were incubated with 250
␮M59FeNTA2for 10 minutes. Data show radioiron retained by the duodenal tissue
(MR), radioiron transferred to the carcass (MT), and total uptake of radioiron by
duodenum (TMU). *P⬍.05, **P⬍.01.
Table 1. Effect of hepcidin on body weights, iron stores,
and hemoglobin in mice
Treatment n Body weight,
gHemoglobin,
g/L Liver nonheme
iron, nmol/mg
Control 10 38.8 ⫾0.6 156 ⫾05 1.82 ⫾0.19
10 ␮g hepcidin 6 37.2 ⫾1.4 154 ⫾07 1.67 ⫾0.28
100 ␮g hepcidin 6 36.4 ⫾1.0* 156 ⫾04 1.59 ⫾0.15
Mice were injected daily for 3 days with 0.15 M NaCl (controls) or hepcidin
dissolved in 0.15 M NaCl.
Data are expressed as means ⫾SEM for the number of mice per group.
*P⬍.05 compared with appropriate control.
Table 2. Effect of different hepcidin dose regimens
on iron absorption in mice
Treatment n
Mucosal
retention,
pmol/mg
Mucosal
transfer,
pmol/mg
Total
mucosal
uptake,
pmol/mg
Intraperitoneal injection*
Control 4 17.7 ⫾3.2 30.4 ⫾5.9 48.1 ⫾5.6
50 ␮g hepcidin ⫻1 4 17.7 ⫾2.4 20.4 ⫾3.8 38.1 ⫾6.1
Intraperitoneal injection†
Control 4 25.4 ⫾2.0 21.9 ⫾3.4 47.4 ⫾3.1
50 ␮g hepcidin ⫻1 4 19.1 ⫾2.9 18.8 ⫾1.6 37.8 ⫾3.7
Control 4 21.2 ⫾3.1 27.3 ⫾3.3 48.5 ⫾4.1
50 ␮g hepcidin ⫻2 4 13.2 ⫾2.2 19.3 ⫾1.9 32.5 ⫾3.6‡
Subcutaneous injection†
Control 4 20.4 ⫾0.8 30.1 ⫾2.6 50.6 ⫾2.5
50 ␮g hepcidin ⫻1 6 16.6 ⫾0.9‡16.5 ⫾0.6§33.1 ⫾1.2§
Mice were injected with 0.15 M NaCl (controls) or the indicated dose of hepcidin
dissolved in 0.15 M NaCl either once or twice with an 8-hour gap between the 2
injections.
Data are expressed as means ⫾SEM for the number of mice per group.
*Iron absorption was studied 4 hours after injections.
†Iron absorption was studied 24 hours after injections.
‡P⬍.05 compared with appropriate control.
§P⬍.001 compared with appropriate control.
Figure 1. Analysis of synthetic hepcidin by 12.5% acid-urea polyacrylamide gel
electrophoresis (PAGE). The gel was loaded with the indicated amounts of peptide
and after electrophoresis it was stained with Coomassie blue. The standard is 1 ␮g
hepcidin produced synthetically and validated as identical to urinary hepcidin-25 by
mass spectrometry, reverse-phase high-performance liquid chromatography on a
C18 column, and acid-urea PAGE (courtesy of E. Nemeth and T. Ganz, University of
California, Los Angeles).
HEPCIDIN AND IRONABSORPTION 3941BLOOD, 15 MAY 2004 䡠VOLUME 103, NUMBER 10

of mRNA for hepcidin were normalized to the housekeeping gene
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using LightCycler
Relative Quantification Software version 1.0.
Statistical analysis
Values are expressed as means plus or minus the standard error of the mean
(SEM). Significant differences between more than 2 corresponding groups
were analyzed by 2-way analysis of variance (ANOVA; 5% level). ANOVA
was performed with Statistical Package for Social Scientists (SPSS,
Chicago, IL).
Results
Injecting mice with hepcidin (10 ␮gor100␮g) daily for 3 days
was found to have no effect on hemoglobin levels or liver nonheme
iron levels (Table 1); however, a small decrease in body weight was
noted, the effect being significant at the higher dose.
Mucosal uptake of iron by duodenum was found to be decreased
(Figure 2) at both hepcidin doses, with a tendency for the effect to
increase with an increasing hepcidin dose. The quantity of iron
transferred to the carcass was also decreased, but this effect was not
statistically significant at the 10-␮g dose. To further investigate the
effects of dose and time, mice were injected with 50 ␮g hepcidin
once, and then iron absorption was measured after 4 hours or 24
hours (Table 2). Iron absorption was not significantly reduced by
either treatment. If mice were dosed twice with 25 ␮gor50␮g
hepcidin (8-hour gap between doses) then studied 24 hours later,
iron absorption was significantly reduced (Table 2).
The data suggest that there are dose and time dependencies of
the effect of hepcidin. The doses required to give significant effects
are, however, very high, suggesting that hepcidin may be rapidly
cleared from the circulation, thus providing an explanation for the
failure of earlier experiments to consistently find a humoral factor
controlling iron absorption.16
In order to test whether the injected synthetic hepcidin was
causing an inflammatory reaction resulting in enhanced endoge-
nous hepcidin production and consequent down-regulation of iron
absorption, we measured hepcidin mRNA levels in livers from
mice injected for 3 days with 100 ␮g hepcidin (Figure 3). The data
show that the synthetic hepcidin caused a decrease in endogenous
hepcidin gene expression. This finding resembles findings made
with transgenic mice with constitutive overexpression of hepcidin8
and may reflect developing iron deficiency.
As there are little data on endogenous levels of hepcidin or its
plasma kinetics, it is not possible to say how levels in the mice were
altered by the quantities we injected. We tried injecting mice by the
subcutaneous route to delay release into the blood, however, this
only slightly increased the effect of hepcidin (Table 2; intramuscu-
lar injection gave similar results, not shown). We also tried
injections of mouse hepcidin (this differs at amino acid positions
H3N, G12K, H15N, R16N, K18Q, and M21L compared with
human hepcidin); however, the effect was similar to the human
peptide (data not shown). Injection of hepcidin (10 ␮g/mL,
approximately 1 ␮g per segment) directly into the lumen of tied-off
segments of duodenum, together with the radioiron solutions used
for in vivo iron absorption measurements, did not affect iron
absorption (mucosal uptake: 37.0 ⫾5.7 compared with 40.6 ⫾10.2
[SEM; n ⫽3; pmol/mg over 10 minutes] for controls).
We then measured the effect of hepcidin injection on iron
absorption in iron-deficient mice (Figure 4). These mice are
expected to have decreased endogenous hepcidin levels.4,9 Figure 4
suggests that hepcidin decreased iron absorption by a similar
proportion in iron-deficient and control mice. This was tested by
investigating the statistical significance of the effects of diet and
hepcidin treatment on iron absorption parameters (Table 3).
Significant effects of iron deficiency and hepcidin treatment on iron
absorption were found. A significant interaction of iron deficiency
and hepcidin was found, suggesting that the 2 effects are not
additive. Logarithmic transformation of the data removed this
effect, confirming that hepcidin had decreased mucosal uptake or
transfer in both iron-deficient and control mice by a similar factor.
Figure 3. Effect of synthetic hepcidin injections on endogenous liver hepcidin
expression. Mice were injected daily for 3 days with 0.15 M NaCl (controls) or
hepcidin (100 ␮g) dissolved in 0.15 M NaCl, then liver samples were analyzed for
hepcidin and GAPDH mRNA levels. The ratio of hepcidin to GAPDH is shown
(means ⫾SEM). *P⬍.05.
Figure 4. Effect of hepcidin on iron absorption in iron-deficient mice. Mice were
fed an iron-replete (C) or iron-deficient (ID) diet for 3 weeks from weaning and
injected with 0.15 M NaCl (sal) or hepcidin (50 ␮g single injection) on each of 2
consecutive days, 24 hours before measuring iron absorption with tied-off duodenal
segments. Data (means ⫾SEM) shown are mucosal retention (f), transfer of
radioiron to the carcass (u), and total mucosal uptake of radioiron (䡺). Significance of
effects was analyzed by ANOVA and is shown in Table 3. *P⬍.05, **P⬍.01
compared with mice fed the same diet and injected with saline.
Table 3.Analysis of variance of effects of hepcidin and iron deficiency on iron absorption
Effect Mucosal
retention Mucosal
transfer Total mucosal
uptake % Mucosal
transfer Wt Hb
Iron deficiency .131 3.09 ⫻10⫺87.64 ⫻10⫺8.00019 .625 .00046
Hepcidin .00141 7.40 ⫻10⫺52.32 ⫻10⫺5.637 .995 .598
Interaction .0391 .00677 .00301 .304 .763 .613
Results shown are Pvalues for nontransformed data. Logarithmic transformation of data eliminated the significant interactions for mucosal retention, mucosal transfer, and
total mucosal uptake, but not the significant effects of iron deficiency or hepcidin.
Wt indicates body weight; and Hb, hemoglobin level.
3942 LAFTAH et al BLOOD, 15 MAY 2004 䡠VOLUME 103, NUMBER 10

It is also noteworthy that mucosal uptake and transfer were affected
by similar proportions so that the transfer of iron to the carcass,
expressed as a percentage of the iron taken up by the mucosa, was
not affected by hepcidin. In contrast, proportional mucosal transfer
of iron is affected by iron deficiency (Tables 3 and 4).
Finally, we tested whether the presence of the Hfe gene was
necessary for the effect of hepcidin on iron absorption by injecting
the peptide into Hfe KO and wild-type mice (Figure 5). Iron
absorption was increased in Hfe KO mice compared with wild-type
mice, but only to a small extent in the genetic background we used.
The effect of hepcidin was similar in Hfe KO and wild-type mice
(Table 5). As before, hepcidin had no effect on the percentage
transfer of iron from gut to carcass.
Discussion
The present data show that hepcidin injection causes reduced iron
absorption in mice. Our preparation of synthetic hepcidin was
shown to contain forms that are equivalent to native urinary
hepcidin; however, on acid urea gel it was found also to contain
additional forms of hepcidin. Despite this impurity, our preparation
of hepcidin evoked biologic responses and probably contained one
or more active forms. The nature of the active form of hepcidin will
only be known once the form in which it circulates and binds its
receptor has been identified. We found the effect was not secondary
to enhanced synthesis of endogenous hepcidin, suggesting that the
effect did not result from a proinflammatory response to the
injected peptide. The decrease occurs primarily at the mucosal
uptake (ie, presumably brush border membrane) step, with transfer
of iron from the duodenum to the animal being decreased in
proportion. This finding implies that hepcidin has little direct effect
on the basolateral transfer proteins for iron, with decreases in
transfer being a consequence of decreased uptake. This is in
agreement with previous kinetic findings on the adaptation of
uptake and transfer in iron-deficient rats, where the fraction of iron
available for transfer across the basolateral membrane increased in
parallel to the available fraction of iron when “uptake”and
“transfer”are in a steady state.17 It cannot however, be ruled out
that the decreased transfer of iron is due to a coordinated decrease
in the iron absorption pathway. The latter possibility is consistent
with the observations of Frazer et al9in iron-deficient rats.
Hepcidin had no effect on iron stores or hemoglobin levels,
therefore the effect may be a direct action on the gut. There was no
immediate effect of coinjection of radioiron and hepcidin into the
lumen of tied-off segments of duodenum, however, suggesting an
endocrine effect of hepcidin, mediated via alterations in gene
expression, as shown by Frazer et al.9The effect of hepcidin was
independent of iron stores (and presumably endogenous hepcidin
levels) in keeping with our previous finding that hypoxia affects
iron absorption by a similar proportional factor in mice with normal
or decreased iron stores.18 The inhibition of iron absorption by
hepcidin was unaffected by knockout of the Hfe gene. Hepcidin
expression is reported to be decreased in adult Hfe KO mice,
despite the latter’s elevated iron stores.19,20 These findings are
consistent with a direct effect of hepcidin on the iron absorption
pathway and suggest that Hfe gene product is either (1) involved in
a distinct hepcidin-independent iron absorption regulation mecha-
nism, or (2) involved upstream of the interaction of hepcidin with
intestine, as suggested by Ahmad et al19 and Bridle et al.20 It is
noteworthy that dietary iron deficiency was found to significantly
alter proportional mucosal transfer of iron, whereas hepcidin
injection did not affect this parameter. This implies that some
additional factor, other than hepcidin, may also be involved in the
regulation of mucosal transfer of iron by low-iron diet feeding.
Our data support a role for hepcidin as a hormone that regulates
duodenal iron absorption, thereby controlling body iron levels.
Further work on the interaction of hepcidin with the duodenum is
necessary to elucidate the mechanism of action of this peptide.
Acknowledgments
We are grateful to Susan Gilfillan for supply of Hfe KO breeders.
We are grateful to Dr Tomas Ganz and Elizabeth Nemeth for
performing acid urea gel electrophoresis on our preparation of
synthetic hepcidin and for a gift of urinary hepcidin.
Figure 5. Effect of hepcidin on iron absorption in Hfe KO mice. Wild-type (wt) or
Hfe knockout (Hfe-KO) mice were injected with 0.15 M NaCl (sal) or hepcidin (50 ␮g
single intraperitoneal injection) on each of 2 consecutive days, 24 hours before
measuring iron absorption with tied-off duodenal segments. Data shown are mucosal
retention (f), transfer of radioiron to the carcass (u), and total mucosal uptake of
radioiron (䡺). Significance of effects was analyzed by ANOVA and is shown in Table
5. *P⬍.05, **P⬍.01 compared with mice of the same genotype injected with saline.
Table 4. Effect of dietary iron level and hepcidin on proportional
mucosal transfer of iron
Diet Treatment Mucosal transfer,
% of total uptake
Iron replete Control 63.4 ⫾3.9
Iron replete Hepcidin 57.2 ⫾6.8
Iron deficient Control 80.3 ⫾1.4
Iron deficient Hepcidin 82.6 ⫾0.7
Iron deficiency was induced by feeding the mice a low iron diet for 3 weeks.
Iron-replete controls received the same diet supplemented with iron. Mice were
injected with 0.15 M NaCl (controls) or hepcidin (50 ␮g single injection) on each of 2
consecutive days, 24 hours before they were killed. Statistical analysis is shown in
Table 3.
Table 5.Analysis of variance for effects of hepcidin and Hfe gene knockout on iron absorption
Effect Mucosal
retention Mucosal
transfer Total mucosal
uptake % Mucosal
transfer Wt Hb
Genotype .248 .981 .074 .335 .428 .588
Hepcidin .304 .013 9.65 ⫻10⫺6.700 .655 .167
Interaction .777 .680 .839 .956 .902 .569
Results shown are Pvalues for nontransformed data.
HEPCIDIN AND IRONABSORPTION 3943BLOOD, 15 MAY 2004 䡠VOLUME 103, NUMBER 10

References
1. Fleming RE, Sly WS. Mechanisms of iron accu-
mulation in hereditary hemochromatosis. Annu
Rev Physiol. 2002;64:663-680.
2. Pietrangelo A. Physiology of iron transport and
the hemochromatosis gene. Am J Physiol. 2002;
282:403-414.
3. Nicolas G, Bennoun M, Devaux I, et al. Lack of
hepcidin gene expression and severe tissue iron
overload in upstream factor 2 (USF2) knockout
mice. Proc Natl Acad Sci U S A. 2001;98:8780-
8785.
4. Pigeon C, Ilyin G, Courselaud B, et al. A new
mouse liver-specific gene, encoding a protein ho-
mologous to human antimicrobial peptide hepci-
din, is overexpressed during iron overload. J Biol
Chem. 2001;276:7811-7819.
5. Finch C. Regulators of iron balance in humans.
Blood. 1994;84:1697-1702.
6. Nicolas G, Chauvet C, Viatte L, et al. The gene
encoding the iron regulatory peptide hepcidin is
regulated by anemia, hypoxia, and inflammation.
J Clin Invest. 2002;110:1037-1044.
7. Roetto A, Papanikolaou G, Politou M, et al. Mu-
tant antimicrobial peptide hepcidin is associated
with severe juvenile hemochromatosis. Nat
Genet. 2003;33:21-22.
8. Nicolas G, Bennoun M, Porteu A, et al. Severe
iron deficiency anemia in transgenic mice ex-
pressing liver hepcidin. Proc Natl Acad Sci U S A.
2002;99:4596-4601.
9. Frazer DM, Wilkins SJ, Becker EM, et al. Hepci-
din expression inversely correlates with the ex-
pression of duodenal iron transporters and iron
absorption in rats. Gastroenterology. 2002;123:
835-844.
10. Nemeth E, Valore EV, Territo M, Schiller G, Lich-
tenstein A, Ganz T. Hepcidin, a putative mediator
of anemia of inflammation, is a type II acute-
phase protein. Blood. 2003;101:2461-2463.
11. Bahram S, Gilfillan S, Kuhn LC, et al. Experimen-
tal hemochromatosis due to MHC class I HFE
deficiency: immune status and iron metabolism.
Proc Natl Acad Sci U S A. 1999;96:13312-13317.
12. Harwig SS, Ganz T, Lehrer RI. Neutrophil de-
fensins: purification, characterization, and antimi-
crobial testing. Methods Enzymol. 1994;236:160-
172.
13. Park CH, Valore EV, WaringAJ, Ganz T. Hepci-
din, a urinary antimicrobial peptide synthesized in
the liver. J Biol Chem. 2001;276:7806-7810.
14. Raja KB, Simpson RJ, Peters TJ. Comparison of
59Fe3⫹uptake in vitro and in vivo by mouse duo-
denum. Biochim Biophys Acta. 1987;901:52-60.
15. Simpson RJ, Lombard M, Raja KR, Thatcher R,
Peters TJ. Iron absorption by hypotransferrinae-
mic mice. Br J Haematol. 1991;78:565-570.
16. Conrad ME. Humoral regulation of iron absorp-
tion. Gastroenterology. 1969;57:225-228.
17. Schumann K, Elsenhans B, Forth W. Kinetic anal-
ysis of 59Fe movement across the intestinal wall
in duodenal rat segments ex vivo. Am J Physiol.
1999;276:G431-G444.
18. Simpson RJ. Dietary iron levels and hypoxia inde-
pendently affect iron absorption in mice. J Nutr.
1996;126:1858-1864.
19. Ahmad KA, Ahmann JR, Migas MC, et al. De-
creased liver hepcidin expression in the Hfe
knockout mouse. Blood Cells Mol Dis. 2002;29:
361-366.
20. Bridle KR, Frazer DM, Wilkins SJ, et al. Disrupted
hepcidin regulation in HFE-associated haemo-
chromatosis and the liver as a regulator of body
iron homoeostasis. Lancet. 2003;361:669-673.
3944 LAFTAH et al BLOOD, 15 MAY 2004 䡠VOLUME 103, NUMBER 10