Effects of elevated dietary iron on the gastrointestinalexpression of Nramp genes and iron homeostasis in rainbowtrout (Oncorhynchus mykiss)
Raymond W. M. Kwong •
Charmain D. Hamilton • Som Niyogi
Received: 22 May 2012 / Accepted: 6 August 2012 / Published online: 15 August 2012
� Springer Science+Business Media B.V. 2012
Abstract Diet is the primary source of iron (Fe) for
freshwater fish, and the absorption of Fe is believed to
occur via the Nramp family of divalent metal trans-
porters (also called DMT1). Presently, the homeostatic
regulation of dietary Fe absorption in fish is poorly
understood. This study examined the gastrointestinal
mRNA expression of two Nramp isoforms, Nramp-band Nramp-c, in the freshwater rainbow trout (On-
corhynchus mykiss), following exposure to elevated
dietary Fe [1,256 mg Fe/kg food vs. 136 mg Fe/kg
food (control)] for 14 days. The physiological perfor-
mance, plasma Fe status and tissue-specific accumu-
lation of Fe were also evaluated. In general, the mRNA
expression level of Nramp was higher in the intestine
relative to the stomach. Interestingly, fish fed on a
high-Fe diet exhibited a significant induction in
Nramp expression after 7 days, followed by a decrease
to the level observed in control fish on day 14. The
increase in Nramp expression correlated with the
elevated gastrointestinal and plasma Fe concentra-
tions. However, the hepatic Fe concentration remained
unchanged during the entire exposure period, indicat-
ing strong homeostatic regulation of hepatic Fe level
in fish. Fish appeared to handle increased systemic Fe
level by elevating the plasma transferrin level, thereby
enhancing the Fe-binding capacity in the plasma.
Overall, our study provides new interesting insights
into the homeostatic regulation of dietary Fe uptake
and handling in freshwater fish.
Keywords Diet � Homeostasis � Iron � Nramp �Rainbow trout � Transferrin
Introduction
Iron (Fe) is an essential micronutrient for vertebrates
including fish, and it is a component of many vital
proteins and enzymes such as hemoglobin, myoglobin
and mitochondrial electron transport chain enzymes.
However, excess Fe can be toxic because it can
generate free radical species and cause oxidative
damage (Crichton et al. 2002). Therefore, the main-
tenance of Fe homeostasis in the body is critical.
Teleost fish can acquire Fe from the water as well as
from the food; however, diet is believed to be the
major source of Fe (Bury and Grosell 2003). The
precise daily dietary Fe requirement for most fish is
R. W. M. Kwong
Toxicology Centre, University of Saskatchewan,
Saskatoon, SK S7N 5B3, Canada
Present Address:R. W. M. Kwong
Department of Biology, University of Ottawa,
30 Marie Curie, Ottawa, ON K1N 6N5, Canada
C. D. Hamilton � S. Niyogi (&)
Department of Biology, University of Saskatchewan,
Saskatoon, SK S7N 5E2, Canada
e-mail: [email protected]
123
Fish Physiol Biochem (2013) 39:363–372
DOI 10.1007/s10695-012-9705-2
not known. It has been suggested that a range of
30–170 mg Fe/kg in the diet is required for fish to
prevent Fe deficiency syndromes (Watanabe et al.
1997).
The mechanisms regulating the absorption of
dietary Fe in fish have not been fully elucidated.
Previous studies have suggested that ferric Fe (Fe3?),
the major form of inorganic Fe in the diet, is first
reduced to ferrous Fe (Fe2?) (Carriquiriborde et al.
2004; Kwong et al. 2010), and the absorption of Fe2?
into enterocytes is mediated by the Nramp family of
divalent metal transporters (also called DMT1)
(Kwong et al. 2010; Kwong and Niyogi 2008). In
freshwater rainbow trout (Oncorhynchus mykiss),
three different Nramp mRNA isoforms (Nramp-a,
Nramp-b and Nramp-c) have been identified to date
(Cooper et al. 2007; Dorschner and Phillips 1999;
Kwong et al. 2010). However, when expressed in
Xenopus oocytes, only Nramp-b and Nramp-c proteins
were found to mediate Fe2? uptake (Cooper et al.
2007). We have previously reported that both Nramp-
b and Nramp-c isoforms are expressed in the stomach
and intestinal tract of rainbow trout (Kwong et al.
2010). Using intestinal sac preparations from rainbow
trout, the absorption of Fe was shown to occur along
the entire (anterior, mid and posterior) intestinal tract
(Kwong and Niyogi 2008). However, the spatial
differences in Nramp expression between the stomach
and intestine, and the possible isoform-specific
responses to dietary Fe level, have not been investi-
gated. The precise mechanisms by which Fe2? trans-
ported across the basolateral membrane are also
poorly characterized, but the mammalian ortholog of
iron-regulated protein-1 (IREG1) has been suggested
to mediate the Fe2? extrusion (Donovan et al. 2000).
Following extrusion, Fe2? is oxidized to Fe3? by
hephaestin prior to its binding to transferrin in the
plasma, and Fe is subsequently delivered and stored in
the liver (Carriquiriborde et al. 2004; Walker and
Fromm 1976).
In vertebrates, Fe homeostasis is believed to be
regulated primarily by the absorption process, and
excretion plays only a minor role (Andrews 2000). In
mammals, the maintenance of Fe balance during high
or low dietary Fe exposure can be regulated by
modulating the Nramp2 (mammalian ortholog of
rainbow trout Nramp-b and Nramp-c) gene expression
in the gut, thereby regulating Fe acquisition (Gunshin
et al. 1997; Zoller et al. 2001). Changes in the Nramp
gene expression in response to dietary Fe level have
also been demonstrated in teleost fish. For example,
zebrafish (Danio rerio) and European sea bass
(Dicentrarchus labrax) fed with a low-Fe diet exhib-
ited an increase in the gene expression of Nramp2
ortholog in the intestine (Cooper et al. 2006; Neves
et al. 2011), presumably as an adaptive response to
up-regulate Fe uptake. However, Craig et al. (2009)
reported that treatment with a high-Fe diet also
induced the expression of Nramp gene in zebrafish
although Fe burden remained unchanged. Similarly,
Kwong et al. (2011) showed that rainbow trout fed on
an elevated Fe diet exhibited no marked changes in
tissue-specific Fe burden after 4 weeks. However, the
potential alterations in the temporal expression pattern
of Nramp genes in relation to systemic Fe regulation
have not been examined. In addition, it is yet to be
fully understood how fish are able to maintain Fe
homeostasis during exposure to high dietary Fe level
(Carriquiriborde et al. 2004; Kwong et al. 2011).
The present study was designed to examine the
Nramp gene expression and Fe homeostasis in fresh-
water fish following exposure to elevated dietary Fe,
using rainbow trout as a model species. The specific
objectives of this study were threefold: (1) to evaluate
the physiological status of fish fed with a high-Fe diet;
(2) to examine the expression of Nramp-b and Nramp-
c genes in the gastrointestinal tract (stomach and
intestine) of fish; and (3) to examine Fe homeostasis
by evaluating Fe handling in the plasma and tissue-
specific Fe accumulation in fish.
Materials and methods
Fish
Freshwater rainbow trout (O. mykiss; *200 g wet
weight) were obtained from the Fort Qu’Appelle Fish
Culture Station in Fort Qu’Appelle, Saskatchewan,
Canada. Fish were acclimated for at least 4 weeks in
the laboratory conditions and supplied with aerated,
dechlorinated water [hardness 157 mg/L, alkalinity
109 mg/L (both as CaCO3), pH 8.1–8.2, dissolved
organic carbon (DOC) 2.2 mg/L, Fe 5.5 lg/L] in a
flow-through system. The water temperature was
maintained at 15 �C with a photoperiod of 14:10 light
to dark cycle. Fish were fed daily with commercial
trout chow (Martin Mills Inc., Elmira, Canada;
364 Fish Physiol Biochem (2013) 39:363–372
123
comprised of 41.0 % crude protein, 11.0 % crude fat,
3.5 % crude fiber, 1 % calcium, 0.85 % total phos-
phate and 0.45 % total sodium). A week prior to the
beginning of the experiment, fish were transferred to a
control diet (see below for details) at 2.5 % daily
ration (dry feed weight/wet fish body weight).
Diet preparation
To prepare the high-Fe diet, the commercial trout
chow was first rehydrated with approximately 25 %
(v/w) Nanopure water and was ground to powder using
a blender. A known amount of Fe (as FeSO4�7H2O)
(dissolved in Nanopure water; EM Science, USA) was
added to the mix and blended for about 10 min to
ensure homogenous mixing. The mix was then
extruded through a pasta maker and cut into small
pellets. The pellets were kept at -20 �C until frozen
and was subsequently dried in a freeze dryer (Lab-
conco Freezone, USA). The dried pellet was stored at
4 �C until use. The control diet (normal Fe diet) was
prepared in the same fashion without the addition of
Fe. The Fe concentration in the food was verified using
a flame atomic absorption spectrometry (see below for
details) (Analyst 800, PerkinElmer, USA), and their
concentrations and daily doses are presented in
Table 1.
Experimental treatment and sampling
A total of 30 fish were divided equally into two tanks,
and each tank received continuous aeration and water
flow at a rate of 1 L/min. Fish in each tank were fed
with a different diet (control and Fe-enriched diet, see
Table 1) for 14 days at a daily ration of 2.5 % (dry
feed weight/wet fish weight), divided equally into two
rations per day. Fecal matters were removed daily 1 h
post-feeding using a siphon. No fish mortality
occurred in any treatments during the exposure period.
Water samples were collected from each treatment
tank three times a week 1 h post-feeding for the
measurement of Fe (see below for details), and no
elevated Fe concentration was recorded in any of the
water samples.
An initial six fish were randomly sampled on the
day the experiment was started (day 0), and six fish
from each tank were sampled on days 7 and 14. Fish
were immediately euthanized with an overdose of
MS-222 (Syndel Laboratories Ltd., Canada) and
measured individually for weight and length. Blood
samples were taken immediately by caudal puncture
with a 5-mL syringe pre-rinsed with heparin (Sigma-
Aldrich, USA) and centrifuged for 4 min at 10,000 g to
collect plasma. The stomach and intestine were
dissected out, cut open and rinsed in 0.9 % NaCl to
remove any food remains in the lumen. The mucosal
epithelium of the stomach and intestine was collected
by scraping using a glass slide and were stored in
RNAlater (Ambion, Inc., USA) at -80 �C until
further processing. The remaining tissues from the
stomach and intestine, as well as tissue sample from
the liver, were collected, weighed and stored at
-80 �C for later Fe measurements.
Real-time PCR
Total RNA extraction, DNase treatment and cDNA
synthesis were carried out as described in Kwong et al.
(2010). First-strand cDNA was used as a template to
examine the transcript expression of Nramp-b and
Nramp-c isoforms and the housekeeping gene b-actin,
using real-time quantitative polymerase chain reaction
(RT-qPCR). All RT-qPCR assays were performed on a
real-time thermocycler (IQ5; Bio-Rad, USA), using
SYBR Master Mix (Fermentas, Canada). Specific
primers were designed for Nramp-b (accession num-
ber AF048761; forward: 50-CCT CCC CTC CGG CTT
CAG AC-30, reverse: 50-CCC CAA CAA CAC CCA
CAG GAG-30) and Nramp-c (accession number
EF495162; forward: 50-ACC CGC TCC ATC GCC
ATC TT-30, reverse: 50-GAC CTG CCG CCC ATC
TCT G-30). RT-qPCR was performed in triplicate
using the following conditions: 95 �C for 10 min, 45
cycles of 95 �C for 15 s, 64 �C for 30 s and 72 �C for
25 s. The specificity of primers of PCR was checked at
the end of each amplification using a DNA melt curve
analysis with a ramping rate of 1 �C/min over a
temperature range of 60–95 �C. All expression data
were normalized to the expression of b-actin from the
Table 1 Measured iron (Fe) concentration (mg Fe/kg dry
weight of food) in the diet and the associated daily dose (mg
Fe/kg fish wet weight/day) to rainbow trout
Fe level Daily dose
Control 136 ± 11 2.7
High iron 1,256 ± 25 25.1
Values are mean ± SEM, n = 5 for Fe level in the diets
Fish Physiol Biochem (2013) 39:363–372 365
123
same tissue. Relative expression was calculated based
on the method described by Pfaffl (2001), where the
expression levels of each gene from the high-dietary-
Fe-treated fish were calculated relative to the control
on the same day.
Iron measurement
The experimental diets and tissue samples were
digested in 5 volumes of 1 N HNO3 at 60 �C for
48 h and then centrifuged at 15,000 g for 4 min. The
supernatant was collected, diluted appropriately with
0.2 % HNO3 and analyzed for Fe concentrations using
a flame atomic absorption spectrometry (AA800,
PerkinElmer, USA). The quality control and assurance
of Fe analysis were maintained using appropriate
method blanks and a certified standard for Fe (Fisher
Scientific, Canada) and were validated with certified
reference materials (DOLT-4; National Research
Council, Canada).
Plasma iron status
Total plasma Fe concentrations and unsaturated
Fe-binding capacity (UIBC) were measured using a
commercial kit (Pointe Scientific, Inc., USA). The
protocol was adapted for measurement in a 96-well
plate using a microplate reader (Varioskan Flash,
Thermo Scientific, USA) at 560 nm. The total plasma
Fe concentrations and UIBC values were used to
determine total Fe-binding capacity (TIBC) and
transferrin saturation (%) according to the manufac-
turer’s instructions.
Calculations and statistical analysis
Condition factor [K = 100 9 weight (g)/length (cm)3],
gastro-somatic index (GSI), intestinal somatic index
(ISI) and hepato-somatic index (HSI) [GSI, ISI or HSI
(%) = weight of each tissue 9 100/total body weight]
were calculated from individual fish sampled on days 0
and 14 (n = 6 per treatment).
Statistical analysis was performed using Sigma-
Plot� (version 11.0; Systat Software, Inc., Point
Richmond, CA, USA). Data were either analyzed
using one-way or two-way ANOVA (with exposure
time and dietary Fe as two independent variables),
followed by a post hoc Holm–Sidak test. Data were
either log-transformed or square-root-transformed
when the data did not meet the assumptions of equal
variance or normal distribution. Data are reported as
the mean ± SEM, and differences were considered
significant at p \ 0.05.
Results
Physiological status
The weight, condition factor and GSI values of fish
were not significantly different between the two
treatment groups during the exposure (Table 2). On
days 7 and 14, the HSI values of fish fed with a high-Fe
diet were significantly higher than day 0 (p \ 0.05).
Similarly, a significant increase in HSI (p \ 0.05) was
also recorded on day 7 in fish exposed to elevated
dietary Fe when compared with fish fed with normal
Fe diet (control) although the difference did not persist
on day 14. A significant increase in the ISI value was
also observed in fish exposed to the high-Fe diet
Table 2 Physiological status of rainbow trout exposed to
control and high-Fe diet
Days Control High Fe
Weight (g) 0 195.0 ± 17.8A
7 183.4 ± 17.5Aa 192.9 ± 25.5Aa
14 191.0 ± 28.9Aa 222.1 ± 33.6Aa
Condition factor 0 1.22 ± 0.04A
7 1.25 ± 0.04Aa 1.26 ± 0.04Aa
14 1.22 ± 0.06Aa 1.19 ± 0.04Aa
HSI (%) 0 0.95 ± 0.06A
7 1.09 ± 0.05Aa 1.32 ± 0.08Bb
14 1.10 ± 0.10Aa 1.18 ± 0.06ABa
GSI (%) 0 1.89 ± 0.11A
7 1.96 ± 0.18Aa 2.16 ± 0.16Aa
14 1.40 ± 0.12Ba 1.69 ± 0.09Aa
ISI (%) 0 5.25 ± 0.23A
7 5.31 ± 0.10Aa 6.59 ± 0.37Bb
14 4.73 ± 0.22Aa 5.62 ± 0.48ABb
HSI Hepato-somatic index, GSI Gastro-somatic index, ISIIntestinal somatic index. Values labeled with different letters
indicate statistical difference (two-way ANOVA followed by a
post hoc Holm–Sidak test, p \ 0.05). Upper case: among days
in the same diet treatment (including comparison with day 0 of
the control for the high-Fe diet treatment); lower case: between
two treatments on the same day. Values are mean ± SEM,
n = 6
366 Fish Physiol Biochem (2013) 39:363–372
123
compared with that in control fish on days 7 and 14
(p \ 0.05).
Nramp mRNA expression
In both the stomach and intestine, the mRNA expres-
sion level of Nramp-c was about three- to fourfold
higher than that of Nramp-b on day 0 (p \ 0.05)
(Fig. 1a). The higher expression level of Nramp-c than
Nramp-b was also recorded on days 7 and 14 (data not
shown). In addition, the expression level of Nramp-c
was significantly higher in the intestine relative to that
in the stomach (p \ 0.001), whereas the expression
level of Nramp-b was comparable between the two
tissues (Fig. 1b). Similar spatial expression pattern of
Nramp-b and Nramp-c in the stomach and intestine
was also observed on days 7 and 14 (data not shown).
In the stomach, elevated dietary Fe did not signif-
icantly affect the expression of Nramp-b (Fig. 2a).
However, fish fed with a high-Fe diet exhibited a
significant increase in Nramp-c expression in the
stomach on day 7 (p \ 0.05), followed by a decrease
to the level comparable with that in control fish on day
14 (Fig. 2b). In the intestine, both Nramp-b and
Nramp-c mRNA expression levels were significantly
increased in fish fed with a high-Fe diet on day 7
(p \ 0.001–0.01), followed by a decrease to the level
similar to that in control fish on day 14 (Fig. 2c and d).
A two-way ANOVA revealed no interactive effects
between exposure time and dietary Fe treatments.
Tissue iron concentration
Exposure to elevated dietary Fe resulted in a significant
increase (p\0.001) in Fe concentration of the gastroin-
testinal tissue of fish during the entire exposure period
except in stomach at day 14 (Fig. 3a and b). However, Fe
concentration in the liver remained unchanged between
the two treatment groups over the entire exposure period
(Fig. 3c). A two-way ANOVA revealed a significant
interaction between exposure time and dietary Fe treat-
ments for both the stomach and intestine (p\0.001).
Plasma iron status
The plasma Fe concentration, TIBC and transferrin
saturation (%) were significantly increased in fish
exposed to high-Fe diet (p \ 0.001-0.05) during the
entire exposure period (Fig. 4a, c and d). However, no
significant change was observed in UIBC between the
two treatment groups on either day 7 or day 14
(Fig. 4b). A two-way ANOVA revealed no significant
interaction between exposure time and dietary Fe
treatments in any of the above measurements.
Discussion
To the best of our knowledge, this is the first study
to report the linkage between the expression of
(a)F
old
diffe
renc
e
0
1
2
3
4
5Nramp-βNramp-γ
(b)
Nramp isoforms
Stomach Intestine
Beta Gamma
Fol
d di
ffere
nce
0
1
2
3
4
5
20
40
60
80StomachIntestine
a
a
a
b
b
b
a a
Fig. 1 a Relative difference in Nramp-b and Nramp-c mRNA
expression level in the stomach and intestine of rainbow trout.
Data were normalized with b-actin from the same tissue and
were expressed relative to the Nramp-b expression in the same
tissue. b Relative difference in Nramp-b and Nramp-c mRNA
expression level between the stomach and intestine of rainbow
trout. Data were normalized with b-actin from the same tissue
and were expressed relative to the expression of each respective
gene in the stomach. Bars labeled with different letters are
statistically different from each other (one-way ANOVA
followed by a post hoc Holm–Sidak test, p \ 0.05). Values
are mean ± SEM, n = 6
Fish Physiol Biochem (2013) 39:363–372 367
123
iron-transporting Nramp genes and systemic Fe reg-
ulation in teleost fish. Our study showed that the
expression of Nramp transcripts in the gastrointestinal
tract is modulated by dietary Fe level and subsequently
affects Fe accumulation in fish. In addition, we
demonstrated that regulation of Fe-binding pool in
the plasma is probably a key mechanism for regulating
Fe homeostasis in fish during exposure to elevated
dietary iron.
The Fe level in the control diet (136 mg Fe/kg dry
weight) used in this study was well within the range of
the dietary Fe level (100–250 mg Fe/kg dry weight)
suggested for normal physiological functioning in
salmonid fish (Andersen et al. 1996). The Fe concen-
tration in natural fish diet has been found to vary
between 160 and 12,500 mg/kg dry weight (Wint-
erbourn et al. 2000), and thus, the Fe level (1,256 mg
Fe/kg dry weight) in the Fe-enriched diet used in this
study was environmentally relevant. It should also be
noted here that no apparent toxicity (e.g., oxidative
stress) was reported in rainbow trout following
4 weeks of treatment with a diet containing a much
higher Fe level (1,975 mg Fe/kg dry weight) relative
to the Fe-enriched diet of the present study (Carri-
quiriborde et al. 2004). The present study indicated
that the exposure to the elevated dietary Fe does not
affect the condition factor, but significantly increases
the HSI value in rainbow trout. Kwong et al. (2011)
also reported similar findings in rainbow trout exposed
to elevated dietary Fe (1,108 mg Fe/kg dry weight) for
4 weeks. In contrast, Carriquiriborde et al. (2004) did
not observe any change in HSI value in rainbow trout
chronically treated with a high-Fe diet (high Fe:
1,975 mg Fe/kg dry weight). Nevertheless, they
recorded an enlargement of liver cells and suggested
that the metabolic activity in the liver was increased in
Nramp-β
Time (day)
Fol
d di
ffere
nce
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5 ControlHigh Fe
Nramp-γ
Time (day)
Fol
d di
ffere
nce
0
2
4
6
8
10
Nramp-β
Time (day)
Fol
d di
ffere
nce
0
1
2
3
4
5
6
7 ControlHigh Fe
Nramp-γ
Time (day)
7 14 7 14
7 14 7 14
Fol
d di
ffere
nce
0
2
4
6
8
10
12
Stomach
Intestine
b
aa
ab
a
b
a
ab
a
b
aab
(a) (b)
(c) (d)
a
a a
a
Fig. 2 Alterations in the mRNA expression level of Nramp-band Nramp-c in the stomach (a, b) and intestine (c, d) of rainbow
trout exposed to normal (control) and high-Fe diets. Data were
normalized with b-actin from the same tissue and were
expressed relative to the control at the same exposure time.
Bars labeled with different letters are statistically different from
each other (two-way ANOVA followed by a post hoc Holm–
Sidak test, p \ 0.05). Values are mean ± SEM, n = 6
368 Fish Physiol Biochem (2013) 39:363–372
123
fish fed with the high-Fe diet (Carriquiriborde et al.
2004).
In mammals, the Nramp2 is suggested to be
important in dietary Fe absorption (Gunshin et al.
1997; Mackenzie and Garrick 2005). In rainbow trout,
two different isoforms of Nramp protein (Nramp-band Nramp-c) are shown to mediate apical Fe uptake
when expressed in Xenopus oocytes (Cooper et al.
2007). The mRNA expression of both of these Nramp
isoforms has been recorded in the gastrointestinal tract
of rainbow trout, and the in vitro examination of Fe
uptake indicated their involvement in Fe transport in
trout intestine (Kwong et al. 2010; Kwong and Niyogi
2008). Interestingly, a considerably higher expression
of Nramp-c relative to Nramp-b was observed in both
stomach and intestine during the entire exposure
period. This finding suggested that Nramp-c may play
a more prominent role in Fe transport in trout. This
was also supported by our observation that the
increase in the expression of Nramp-c was much
higher in magnitude relative to that of Nramp-b in the
gastrointestinal tract of rainbow trout, following
7 days of treatment to elevated dietary iron. In
addition, the increased expression of Nramp-c gene
corresponded with the significant elevation of stomach
Fe level (discussed later) following exposure to high-
Fe diet, indicating that the stomach is an important site
of Fe absorption in trout. This is in line with the
previous studies that also reported stomach is an
important site for Cu absorption in trout (Nadella et al.
2011).
In mammals, it has been shown that exposure to an
elevated Fe diet led to the down-regulation of Nramp2
gene expression in the intestine, thereby reducing Fe
absorption (Frazer et al. 2003; Gunshin et al. 1997).
Interestingly, we observed that high dietary Fe expo-
sure increased the transcript expression of Nramp in
the gastrointestinal tract of fish on day 7, which was
followed by a decrease to the level similar to that in the
control fish (fed with a normal Fe diet) on day 14. The
induction of Nramp gene expression was also accom-
panied by an increase in gastrointestinal Fe accumu-
lation in the fish (discussed later). These findings
suggested an up-regulation of dietary Fe absorption
during the early phase of exposure to elevated dietary
Fe, while a reduction in Nramp gene expression during
the later phase might have been an adaptive response
to ameliorate systemic Fe burden. At present, it is not
clear why the exposure to elevated dietary Fe causes
(a) Stomach
0
5
10
15
20
25 Control
High Fe
(b) Intestine
0
10
20
30
40
50
(c) Liver
Time (day)
0 7 14
0 7 14
0 7 140
20
40
60
80
100
Fe
conc
entr
atio
ns (
μ g/g
wet
wt.)
120
a
a a
b
ab
a a a
b
b
a
a
aa
a
Fig. 3 Iron (Fe) concentration in the a stomach, b intestine and
c liver of rainbow trout exposed to normal (control) and high-Fe
diets. Bars labeled with different letters are statistically different
from each other (two-way ANOVA followed by a post hoc
Holm–Sidak test, p \ 0.05). Values are mean ± SEM, n = 6
Fish Physiol Biochem (2013) 39:363–372 369
123
an initial increase in the gastrointestinal expression of
Nramp genes in fish. We were not able to examine
whether the increased expression of Nramp genes
leads to corresponding increase in Nramp protein
levels, because of the lack of commercially available
antibodies specific to different Nramp proteins. It is
apparent that the regulation of Nramp proteins, not
only in the gut but in other major epithelia as well (e.g.,
gill, kidney), in relation to altered dietary Fe exposure
in fish requires further investigations. Overall, our
observations suggested that body Fe status probably
regulates the gastrointestinal expression of Nramp
genes in fish, at least in part.
The present study showed that Fe accumulation in the
stomach and intestine of fish was significantly increased
following exposure to high dietary Fe. However, Fe
level in the stomach appeared to reduce the control level
on day 14, which occurred likely due to the down-
regulation of the Nramp genes on day 14. In addition, we
found that fish exposed to the high-Fe diet had an
elevated plasma Fe level—a consequence of increased
dietary Fe absorption. The increase in the gastrointe-
stinal and plasma Fe levels correlated with the increase
in gastrointestinal Nramp transcript levels. In order to
handle elevated Fe level in the plasma, fish appeared to
up-regulate the plasma Fe-binding capacity by increas-
ing the plasma transferrin level (equivalent to TIBC), as
well as by increasing the percentage of Fe saturation in
the transferrin. These responses are physiologically
imperative since excess unbound Fe in the plasma may
cause oxidative damage to the vital cells/organs in the
body (Yamaji et al. 2004b). Transferrin is mainly
synthesized in the liver and regulated by the liver Fe
concentration (Anderson and Frazer 2005). Interest-
ingly though, we did not observe any apparent change in
the hepatic Fe level in fish exposed to elevated dietary
Fe. Since the first hepatic Fe measurement was
conducted on day 7 of the exposure, it is possible that
fish exposed to an elevated Fe diet exhibited a transient
increase in the hepatic Fe level, which was not recorded
in our study. In general, our results suggested that the
hepatic Fe status is tightly regulated in fish.
It is important to note that, in addition to Nramp and
transferrin, there are other proteins that are known to
Time (day)
Pla
sma
Fe
(μM
)
0
10
20
30
40
50
60ControlHigh Fe
Time (day)
UIB
C (
μ M)
0
20
40
60
80
100
Time (day)
TIB
C (
μ M)
0
25
50
75
100
125
150
175
Time (day)
7 14 7 14
7 14 7 14
Tran
sfer
rin s
atur
atio
n (%
)0
10
20
30
40
a
b
a
b
a a a a
a
b
ab
a
b
a
b
(b)(a)
(c) (d)
Fig. 4 a Plasma iron (Fe) level, b unsaturated Fe-binding
capacity (UIBC), c total Fe-binding capacity (TIBC), and
d transferrin saturation (%), in rainbow trout exposed to normal
(control) and high-Fe diets. Bars labeled with different letters
are statistically different from each other (two-way ANOVA
followed by a post hoc Holm–Sidak test, p \ 0.05). Values are
mean ± SEM, n = 6
370 Fish Physiol Biochem (2013) 39:363–372
123
play an important role in the regulation of Fe
homeostasis in vertebrates. For example, it has been
demonstrated that high-Fe diet decreases the intestinal
expression of IREG1, thereby reducing Fe extrusion
into the blood circulation (Chen et al. 2003). In
zebrafish, however, exposure to an elevated dietary Fe
was found to have no effect on the IREG1 levels in the
gut (Craig et al. 2009). On the other hand, hepcidin has
been suggested to play a critical role in the regulation
of systemic Fe balance in mammals (Ganz 2005).
Specifically, exposure to a high-Fe diet in mammals
has been shown to increase the level of hepcidin,
which mediates the degradation of Nramp2 (Brasse-
Lagnel et al. 2011) and IREG1 (Ganz 2005). Similarly,
Rodrigues et al. (2006) reported increased hepatic
mRNA expression of hepcidin in fish during Fe
overload. However, there is currently a debate about
whether hepcidin regulates systemic Fe level by acting
primarily on apical or basolateral Fe transport (Chung
et al. 2009; Yamaji et al. 2004a). Further studies are
required to understand the role and physiological
importance of IREG1 and hepcidin in systemic Fe
regulation in fish.
In conclusion, the present study demonstrated that
exposure to elevated dietary Fe induces the gastroin-
testinal expression of Nramp genes in fish, resulting in
increased Fe level in the gastrointestinal tissue and
plasma. Nevertheless, Nramp genes probably play an
important role in homeostatic regulation of Fe uptake,
particularly at the later phase of the exposure as the
systemic Fe level begins to increase. In addition, it
appears that fish maintain Fe homeostasis during
exposure to elevated dietary Fe, to a large degree, by
up-regulating the transferrin level and Fe-binding pool
in the plasma. Overall, our study provides fundamen-
tal insights into the homeostatic regulation of dietary
Fe absorption and systemic Fe handling in fish.
Acknowledgments We thank Drs Jose Andres and Ken
Wilson (University of Saskatchewan) for allowing us to use
their laboratory equipment. A Discovery Grant from the Natural
Sciences and Engineering Research Council of Canada
(NSERC) to S.N. supported this research. This work was also
supported (in part) by the Alexander Graham Bell Canada
Graduate Scholarship, as well as the Society of Environmental
Toxicology and Chemistry (SETAC)/ICA Chris Lee Award for
Metals Research, sponsored by the International Copper
Association, to R.W.M.K. This work was approved by the
University of Saskatchewan’s Animal Research Ethics Board
and adhered to the Canadian Council on Animal Care guidelines
for humane animal use.
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