Sohag University
Sohag Medical Journal
Faculty of Medicine
Effect of regular Exercise and iron injection on iron status of
anemic obese male rats.
Hoda Mostafa Ahmed1, Ahmed Mostafa Mahmoud1, Seham AbdAlrahman Abd-allatif1, Ahmed Roshdy Hamed2
1 Department of Physiology, Faculty of Medicine, Sohag University, Sohag, Egypt.
2 Department of patholology.Faculty of Medicine. Sohag University, Sohag, Egypt
Correspondant Author: Seham Abd-Alrahman Abd-allatif
Correspondant Address: Department of Physiology, Faculty of Medicine, Sohag University,
Sohag, Egypt
E-mail:www.seham_emad89@yahoo.com
Tel: 01096872578
Abstract
Many animal studies have reported an association between obesity and reduced iron
storage. Systemic chronic inflammation induced by obesity and higher hepcidin levels
has been suggested as a reason for the iron deficiency that occurs with obesity.
Hepcidin is a master regulator of systemic iron homeostasis. Hepcidin is a peptide
hormone, produced mainly from the liver. It inhibits intestinal iron absorption. The
hepatic production of hepcidin is up-regulated by pro-inflammatory cytokines. An
exercise is an effective approach for controlling obesity. Several studies showed that
exercise reduced body weight. So this study hypothesized that regular exercises may
be used as an adjuvant line in treating iron deficiency associated with obesity via
reducing serum hepcidin. Materials and methods: 75 young male albino rats were
categorized into 5 groups. Obesity was induced by high-fat diet (HFD) for 3 months
in Group II, III, IV, and V. After 12 weeks, Rats of GIII and GV were injected once
every 2 days with iron-III poly-maltose 50 ml/kg for 2 weeks. treadmill regular
exercise was performed for 8 weeks for rats of GIV and GV. Measurement of body
weight was done after 3 months of HFD and at the end of the experiment.
Biochemical analysis was done for serum iron and serum hepcidin. Results:
Measurement of body weight showed that exercise has a decreasing effect on body
weight in obese rats. Exercise decreased serum hepcidin levels in obese rats and
increased serum iron. Conclusion: Exercise decreases serum hepcidin level also
improves the serum iron level in obese rats.
Keywords:Exercise, hepcidin, obesity, serum iron.
Introduction:
Obesity is a common disease defined
by an excess of body fat to the extent
that health is negatively affected (19).
Within only a few years, obesity had
become a serious health problem (19).
Also; Iron deficiency is another
common problem in many countries.
Many animal and clinical studies have
showed an association between
overweigh and reduced iron storage
(24). Systemic low-grade inflammation
resulted from obesity and increase
hepcidin levels have been suggested as
a
reason for
iron
deficiency
(ID) that occurs with obesity
(21).
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SOHAG MEDICAL JOURNAL
Vol.24 No.1 Jan 2020
Effect of regular Exercise and iron injection on iron status
Seham Abd-Alrahman Abd-allatif1
There
are
minimally
three
mechanisms of (ID) that occur with
obesity have been proposed and
include: (i) decrease iron intake (29);
(ii) increased body mass leading to
increase iron demand (2); and (iii)
systemic
chronic
low-grade
inflammation that occurs with obesity
(6).
Hepcidin is the
main hormone that regulates systemic
iron homeostasis. Hepcidin is a peptide
hormone, produced mainly from the
hepatocytes. It inhibits iron absorption
in the intestine and reduces the iron
release from macrophages, the hepatic
production of hepcidin is induced by
pro-inflammatory cytokines and it is
inhibited by (ID) (12).
In overweighed individuals, fatty cells
are a major source that releases proinflammatory cytokines (17). Fatty
tissue, particularly visceral fatty tissue,
is the main source of pro-inflammatory
cytokines secretion and a possible
reservoir
for
pro-inflammatory
molecules that contribute many
diseases. The production of cytokines
by fatty tissue is elevated in the
presence of total and abdominal
adiposity (15).
Endothelial
abnormalities
is
associated with obesity and result in
vascular
damage
with
the
accumulation of phagocytes in the
subintimal space and release of many
inflammatory cytokines (22).
Many studies reported that exercise
reduced body weight and adipose mass
(4). So this work hypothesized that
regular exercises may be used as an
adjuvant line in treating iron deficiency
associated with obesity via reducing
serum hepcidin.
Aim of the work
-To evaluate the effect of obesity on an
iron level and hepcidin level.
-To evaluate the effect of exercise
training and/or iron injection on the
iron status of obese male rats.
Materials and methods
Animals
Seventy-five male Wister albino rats,
average age about 3-4weeks and
weight 130 – 150 g, were housed in
groups of four in metal box cages (20
x32 X20 cm) at normal light-dark
cycle and room temperature. Rats were
obtained from the faculty of Science,
Sohag University.
Materials of the study
-Iron-III
poly-maltose
(Xiamen
Vastland Chemical Co., Ltd).
-Hepcidin Elisa Kits (Cat.No:
MBS103477 San Diego (USA)): to
measure the hepcidin concentration in
the serum.
Method of the study
This work was carried out underline
the guidelines of the University
Animal Ethics and approved by the
Research
Ethics
Committee
considering care and use of laboratory
animals.
The 75 rats were randomly divided
into five equal groups (n=15):
Group I (GI): were used as a control
group. Rats were fed a control diet (25
gram approximately of the ordinary
feeds for each rat per day) with water
all over the experiment (28). After 12
weeks, rats were injected intramuscular
(IM) once every 2 days with
physiological saline (0.9% NaCl) at a
dose
of
50
ml/kg
for
2
weeks.
Group II (GII): were fed a high-fat
diet (HFD) with water all over the
study (28). After 12 weeks, rats were
injected (i.m) once every 2 days with
physiological saline (0.9% NaCl) at a
dose
of
50
ml/kg
for
2
weeks.
Group III (GIII): were fed HFD
with water (28).After 12 weeks, Rats
were injected (i.m) once every 2 days
with iron-III poly-maltose 50 ml/kg for
2 weeks
(12).
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Effect of regular Exercise and iron injection on iron status
Seham Abd-Alrahman Abd-allatif1
Group IV (G IV): were fed HFD with
water daily during all the study(28)
then after 12weeks, treadmill regular
exercise was performed for 8
weeks.
Group (GV): were fed HFD with
water daily all over the study (28) then
after 12weeks, Rats were injected (i.m)
with
iron-III
poly-maltose
(50
ml/kg) every 2 days for 2 weeks(11)
with performing treadmill regular
exercise
for
8
weeks.
Measurement of body weight was done
after 3 months of high-fat diet and at
the
end
of
the
experiment.
Statistical analysis
All of the data are expressed as the
means ± standard deviation. Group
comparisons were performed using one
way ANOVA test and T-test. P-value
less than 5% was considered to be
significant.
Results:
Effect of iron injection on body
weight
IM iron injection to rats of GIII
resulted in a statistically nonsignificant change of body weight
when compared with GII (table1).
Effect of Exercise on body weight
Measuring the bodyweight of the rats
of GIV at the end of the study showed
that the exercise had a decreasing
effect on body weight. After 2 months
of exercise, there was a statistically
significant decrease in the bodyweight
of G IV when compared with G II
(p<0.05) (table 1).
Effect of combined exercise and iron
injection on body weight
The bodyweight measurement of GV
at the end of the study showed that the
combined exercise and iron injection
had a statistically significant decrease
in body weight when compared with G
II(p<0.05).(table1)
Results of biochemical analysis
Effect of exercise and/ or iron
injection on serum level of iron
The results of this study showed that
exercise and/or iron injection caused a
statistically significant increase in the
serum level of iron. GII showed a
statistically significant low level of
serum iron when compared with GI
(p<0.05). IM injection of iron to GIII
resulted in a statistically significant
elevated level of serum iron at the end
of the experiment when compared with
GII(p<0.05).
Interestingly,
also
exercise produced a statistically
significant high level of serum iron in
rats of GIV when compared with rats
of GII (p<0.05). Combined exercise
Exercise Program
During the period of 1st 4weeks of
the programme, exercise was done 5
days/week for 40 minutes /day with a 5
minute warm-up at a speed of 2 m/min,
and was followed by 30-minute main
exercise at a speed of 8 m/min, and
was ended by 5 minutes final cooldown session at a speed of 5
m/min. And then from 5 - 8 weeks,
exercise was done five days per week
for 60 minutes per day with a 10
minutes warm-up at a speed of 8
m/min, a 40-minute main exercise was
at a speed of 14 m /min, with a 10
minute-final cool-down at a speed of
11 m/min. Treadmill exercise was
performed where the slope was fixed
to zero degrees (28).
Collection of samples
After the experiment, the rats were
anesthetized with thiopental Na (40
mg/kg) intraperitoneally. Intracardiac
samples of blood were being collected,
centrifuged to se
h
h h
i
the
time
of
biochemical
analysis. Biochemical analysis was
done for serum iron and serum
hepcidin.
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Effect of regular Exercise and iron injection on iron status
Seham Abd-Alrahman Abd-allatif1
and iron injection resulted in a
statistically significant high level of
iron in rats of GV when compared with
rats of GII (p<0.05).
Results of one way ANOVA and
post hoc test done for GII, GIII, GIV,
and GV showed that there was a
statistically significant high level of
serum iron in GIII and GIV with
statistically significant more high level
of serum iron in GV(table 2). There
was a significant negative correlation
between serum iron and body weight
(r=-0.477). As correlation is significant
at 0.01.
Effect of exercise and/ or iron
injection on serum level of hepcidin.
Results of this study showed that
exercise and combined exercise and
iron injection caused a statistically
significant decrease in the serum level
of hepcidin. GII showed a statistically
significant high level of serum
hepcidin when compared with GI
(p<0.05). IM injection of iron to GIII
didn't have a decreasing effect on the
level of serum hepcidin at the end of
the experiment. Interestingly, exercise
produced a statistically significant low
level of serum hepcidin in rats of GIV
when compared with rats of GII
(p<0.05). Combined exercise and iron
injection resulted in a statistically
significant low level of hepcidin in rats
of GV when compared with rats of GII
(p<0.05). Results of one way ANOVA
and post hoc test done for GII, GIII,
GIV, and GV showed that there was a
statistically significant low level of
serum hepcidin in GIV and GV when
compared with rats of GII (table 2).
There was a significant positive
correlation between serum hepcidin
and body weight (r=0.981). Also,
There was a significant negative
correlation between serum iron and
serum
hepcidin
(r=-0.465). As
correlation is significant at 0.01.
Table (1): Average of body weight at the start, after 3months and at the end of the
experiment in all groups of the study.
Groups
GI
GII
GIII
Initial weight
147.85±9371
147.55±8.929
After 3 months
187.65±9.178
314.3±6.82
#
At the end of
experiment
223.95±5.633
542.75±18.761
#
GIV
GV
147.75± 9.324
148.10± 8.416
148.25± 9.210
#315.10±6.96
#314.85±7.27
314.50±7.186
#
247.45±10.475
*
261.50±8.859
*
528.55±13.953
#
-Data are expressed as mean ± standard deviation.
-P-Value was calculated sig (p<0.05).
#Significant when compared with GI done by t test
*significant when compared with GII done by ANOVA.
Table (2): Average of serum level of iron and hepcidin at the end of experiment in all
groups of the study
Groups
parameters
Serum level of iron
(μg/ )
Serum level of
hepcidin
(mg/dl)
GI
GII
GIII
GIV
GV
240.45±21.833
147.95±21.368
#
142.25±6.695
#
289.35±14.195
*
140.60±7.287
289.45±22.385
*
75.05±6.863
308.65±15.253
*
76.95±6.151
*
70.40±5.471
-Data are expressed as mean ± standard deviation.
-P- Value was calculated sig (p<0.05).
#Significant when compared with GI done by t test
*significant when compared with GII done by ANOVA.
111
#
*
SOHAG MEDICAL JOURNAL
Vol.24 No.1 Jan 2020
Effect of regular Exercise and iron injection on iron status
Seham Abd-Alrahman Abd-allatif1
Discussion
and suggest that production of hepcidin
by fatty cells may be higher in obesity.
In contrast, (26) showed that the
adipose tissue may produce very little
serum hepcidin levels compared to the
liver production of hepcidin
This investigation found a strong
positive relationship between BW and
hepcidin levels, also found a strong
negative relationship between hepcidin
levels and iron levels. This agreed with
(1) who confirmed that obese children
had high hepcidin levels and low
serum iron when compared to lean
children. In contrast, (23) supposed
that serum hepcidin does not contribute
to the development of (ID) anemia in
obese children.
Excess accumulation of fatty tissue
is characterized by a status of lowgrade chronic inflammation. This leads
to the production of inflammatory
cytokines, such as interleukin 6, which
is well known to stimulate hepatic
hepcidin
synthesis
(9). Hepcidin
decrease the release of iron from
macrophages and reduced intestinal
iron absorption resulted in less iron is
released and more iron stores,
explaining the status of low serum iron
in the over-weighted individuals (20).
This study investigated the effect of
exercise and\or iron injection on body
weight and the results showed that
exercise has a decreasing effect on
body weight.
After 2 months of regular exercise, it
was
a
statistically
significant
decreasing effect on BW in a group
(IV) and group (V) when compared
with the group (II). This agreed with
(14) who showed that regular exercise
is essential for weight control and
weight loss.
After 2 weeks of iron injection, iron
did not alter the (BW) in the GIII.
These results are following (11) who
showed
that
systemic
iron
Obesity is a major risk factor for
several depleting diseases such as
diabetes mellitus, cardiac problems,
and cancer that are now dramatically
increased in many countries. Obesity is
associated with an excessive increase
in visceral fatty tissue mass. White
fatty tissue secretes many proinflammatory and anti-inflammatory
factors (18).
An increase in body fat tissue may be
related not only to energy intake and
energy output in humans but also to the
quality of diet, especially (HFD),
which may lead to many metabolic
disturbances such as overfeeding in
humans, decreased lipolytic action in
fat tissue, decrease in leptin secretion
and/or
decrease
sensitivity,
hypothalamic neuron degeneration,
insulin resistance, and overweight (7).
In this present study, a model of
obesity was designed by using high-fat
diets (HFD)for3months
In this study, there was a statistically
significant decrease in the serum iron
level in GII when compared to group I.
This agreed with (2) who reported that
increases BMI affected the iron status
of obese patient by showing low iron
levels and high ferritin levels in overweighted patients than the normal
weight subjects. In contrast, (16) in
their study, there wasn't a clear
difference in iron status with different
levels of BMI among males and
females. Although, many studies have
reported that there was a link between
obesity and (ID), the mechanisms of
this are still confused.
Interestingly, the level of serum
hepcidin was significantly increased in
GII when compared to GI. These data
are following (23) who showed that
there was a statistically significantly
higher serum hepcidin level in
Overweight
individuals
when
compared to those with normal (BMI)
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Effect of regular Exercise and iron injection on iron status
Seham Abd-Alrahman Abd-allatif1
supplementation did not have a
decreasing effect on body weight and
adiposity.
This study investigated the effect of
exercise and\or iron injection on serum
level of iron. The results of this study
showed that exercise and/or iron
injection
caused
a
statistically
significant increase in the serum level
of iron.
After 2 months of regular exercise,
interestingly, exercise produced a
statistically significant high level of
serum iron in rats of a group (IV) when
compared with rats of a group (II).
These results are following (8) who
confirmed that physical exercise had
an increasing effect on iron profiles
and decreasing effect on levels of
inflammatory markers when compared
with the control group and the
mechanism by which regular exercise
increases iron levels occur by the
decreasing the adipose mass with a
significant decrease in inflammation
and reported that the decrease in IL-6
led to decrease in hepcidin level (8).
This study did not evaluate proinflammatory markers. In contrast, (5)
reported that (ID) anemia is likely to
be more in athletes than in sedentary
individuals.
After 2 weeks of iron injection, the
IM injection of iron to GIII resulted in
a statistically significant elevated level
of serum iron at the end of the
experiment when compared with GII.
These results are following (11) who
confirmed
that iron
supplementation increased
serum
levels of iron and decreased ironbinding capacity (IBC). Combined
exercise and iron injection resulted in a
statistically significant high level of
iron in rats of GV when compared with
rats of GII.
In this work, we investigated the
effect of exercise and/ or iron injection
on the serum level of hepcidin. The
results of this study showed that
exercise
caused
a
statistically
significant decrease in the serum level
of hepcidin.
After 2 months of regular
exercise, interestingly, exercise
produced a statistically significant low
level of serum hepcidin in rats of GIV
when compared with rats of GII. This
agreed with (3) who showed
that physical exercise reduces hepcidin
levels and increases iron levels. In
contrast,( 13 ) reported that high
intense exercise was associated with
marked high levels of hepcidin and
marked increase in Interleukin-6.
Moreover,(13) reported that acute high
intense exercise increases Interleukin-6
and hepcidin levels 3hours after the
exercise. The mechanism by which
physical regular exercise reduced
serum level of hepcidin is still unclear.
(8) showed that long term regular
exercise reduced BMI and improved
inflammatory status, particularly in IL6, which reduced hepcidin levels and
impairment
in
iron
status.
After 2 weeks of iron injection, the
IM injection of iron to GIII didn't have
a decreasing effect on the level of
serum hepcidin at the end of the
experiment. These results are the
following (11) who found that iron
injection did not affect systemic
hepcidin synthesis.
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