東京医療保健大学紀要

研究報告第 13 巻第1号

2019. 3. 31

ラットを用いた鉄欠乏状態から惹起される
肝臓中の異所性脂肪蓄積の影響
Effect of hepatic lipotoxicity induced by iron deficiency in rats.

清水雅富

岡崎英規

グルーズ ピーター

池田昌代

山岸博之

Masatomi SHIMIZU, Hideki OKAZAKI, Petr GRÚZ, Masayo IKEDA and Hiroyuki YAMAGISHI

〈 研究報告 〉

ラットを用いた鉄欠乏状態から惹起される肝臓中の異所性脂肪蓄積の影響
Effect of hepatic lipotoxicity induced by iron deficiency in rats.

清水雅富

岡崎英規

1

1

2

グルーズ ピーター

3

池田昌代

4

東京医療保健大学

医療保健学部

医療栄養学科

武蔵丘短期大学

健康生活学科

健康栄養専攻2

3

国立医薬品食品衛生研究所
東京農業大学

4
5

1

2

栄養学部
3

5

変異遺伝部

応用生物科学部

関東学院大学

山岸博之

栄養科学科

管理栄養学科
4

Masatomi SHIMIZU , Hideki OKAZAKI , Petr GRÚZ , Masayo IKEDA and Hiroyuki YAMAGISHI

1Division of Medical Nutrition, Faculty of Healthcare, Tokyo Healthcare University2
3

4

Health Science, Musashigaoka Junior College

Division of Genetics and Mutagenesis, National Institute of Health Sciences

Department of Nutritional Science, Faculty of Applied Bioscience, Tokyo University of Agriculture5

要5College of Nutrition, Kanto-Gakuin University

旨：鉄は生体内において必須微量元素であり、重要な栄養素の一つである。鉄欠乏状態はヘモグロビン合成機能障害による酸素運搬能力の低下から、各種代謝異常を誘引することが知られており、特に脂質代謝異常による生体への影響は様々な疾病につながることが予想される。そこで本研究では、鉄欠乏時の代謝異常が生体内に及ぼす影響について検討した。Wistar 系雄ラットを用い AIN-93G を基本組成とし、ミネラル混合中に鉄を含む鉄充足食を与える群（control 群）、鉄を除いた鉄欠乏食を与える群（iron-deficiency群）、鉄欠乏食の飼料摂取量に合わせて鉄充足食を与える群（pair-fed 群）の 3 群を設け、5 週間の飼育観察を行った。飼育後、全血ならびに肝臓を摘出し各種実験に用いた。血中ヘモグロビン（Hb）、ヘマトクリット（Hct）、中性脂肪（TG）および肝臓 TG
含量を測定した結果、iron-deficiency 群で Hb、Hct の低下、血中および肝臓 TG 含量は高値を示した。また肝臓中の炎症関連因子の mRNA 発現量および脂質過酸化生成物であるマロンジアルデヒドを測定した結果、TNF- αにおいては iron-deficiency 群で高値傾向を示したものの大差はなかった。以上の結果から、鉄欠乏状態は肝臓への異所性脂肪蓄積を誘引するものの、その他の影響については確認できなかった。

A b s t r a c t：Iron deficiency remains a public health concern affecting the worldwide population.

The iron is an important element in a wide variety of biochemical reactions in humanand animals. Iron deficiency has a potent influence on various energy production
pathways particularly those of oxidative functions and capacity and lipotoxicity
causing the development of metabolic disorders, but the pathological mechanism is
not defined well. To further characterize the lipotoxicity response to iron-deficiency,
male Wister rats were fed either a control or iron-deficient diet or were pair-fed
the control diet to the level of intake of the iron-deficiency group for five weeks. In
addition to reductions in hemoglobin（Hb）and hematocrit（Hct）, the iron-deficientgroup also exhibited higher serum and hepatic triacylglycerol（TG）compared to the̶ 43 ̶

Masatomi SHIMIZU Hideki OKAZAKI Petr GRÚZ
Masayo IKEDA Hiroyuki YAMAGISHI

東京医療保健大学 紀要第1号

2018 年

pair-fed group. Accumulated hepatic TG stimulates malondialdehyde（MDA）andproinflammatory cytokines tumor necrosis factor α（TNF- α）production caused by 
lipid peroxidation, and these products are possible to hepatocellular injury. However,
our results did not confirm the alterations in MDA and TNF- α production.
Therefore, although iron deficiency increases serum and hepatic TG levels, we did
not found hepatic lipotoxicity in the case of lipid peroxidation and proinflammatory
cytokines.

キーワード：鉄欠乏

貧血

脂質代謝異常MDATNF- αKeywords：Iron-deﬁcient, Anemia, Lipotoxicity, MDA, TNF- α

1．Introduction
Iron deficiency and iron - deficiency anemia are global health problems, and although the prevalence ofiron - deficiency anemia has recently declined somewhat, iron deficiency continues to be the top - ranking problem
worldwide ）. Iron is crucial in biological functions, including respiration, energy production, DNA synthesis, and1cell proliferation ）. Particularly, intracellular stable energy production, a chemical process in which oxygen is used2

to make energy from carbohydrates, uses the redox properties of iron. The most of iron utilization in eukaryotes
is focused on oxygen metabolism, either as an oxygen carrier or as an electron carrier that can facilitate oxygen based chemical reactions. In addition, iron is a cofactor for several key enzymes and its loss may negatively affectelectron transport systems that are responsible for energy metabolism in mitochondria. However, the biologicalrelevance of the effect of iron deficiency on energy metabolism is still not clear. Perhaps the various metabolic
pathway links between glucose, lipid, protein and anemia are complex and not yet fully elucidated.
The laboratory animal models of iron - deficiency have shown increased risk for developing metabolic
disease, such as accumulated ectopic fat

3,4 ）, hyperglycemia

5 -7 ）

hypertriglyceridemia is commonly reported in iron - deficient animals, and hyperlipidemia

5,8 ）. In particular,

9 - 11）, there are changes in hepatic TG content

in response to iron deficiency and an increased abundance of lipid droplets has been observed in the skeletal
muscles of iron - deficient rats

9, 12, 13）. This suggests iron - deficiency increases abundance of lipids in organs what is

an important common risk - factor for lipid peroxidation ）.  Moreover, peroxidation of membrane lipids may cause
14cell necrosis and megamitochondria and the aldehyde products of lipid peroxidation, 4- hydroxynonenal and MDA,
may contribute to inflammation by activating NF -κB that is a transcription factor regulating the expression ofseveral proinflammatory cytokines including tumor necrosis factor α（TNF -α）and interleukin 8 ）.
15

The purpose of this study was to clarify the effects of iron deficiency on lipotoxicity and lipid peroxidation,with special reference to rat liver. We investigated MDA levels and inflammation - related TNF - α mRNA
expression levels in rats fed an iron - deficient diet.

2．Materials and Methods
2.1．Animal care and diet

All experimental procedures on rats were approved by the Committee on the Ethics of Animal
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東京医療保健大学 紀要

ラットを用いた鉄欠乏状態から惹起される肝臓中の異所性脂肪蓄積の影響

第1号

2018 年

Experiments of Kanto - Gakuin University Institutional Animal Care and Use Committee. Three - week - oldmale Wistar rats（Japan Charles River Inc., Kanagawa, Japan）were categorized into three groups. First, we
obtained 16 rats for ad libitum feeding groups. All rats were allowed access to the control diet（ 50 mg Fe/
kg diet）, based on the AIN - 93G diet ）, for three days before starting dietary treatments. After the acclimation
16

period, rats were randomly assigned to one of two diet groups（n=8, respectively）for five weeks. The dietary
manipulations consisted of ad libitum feeding with control diet（control）or iron - deficiency diet（iron - deficiency,
8 mg Fe/kg diet）. After one week, eight rats were obtained and assigned to pair - feeding with the control dietgroup（pair - fed）. For pair feeding, each pair - fed rat was paired with an iron - deficiency rat in order of weight match. Accurate pair feeding was achieved by maintaining equal food intake between the iron - deficiency rat
and the pair - fed rat. All of the rats had free access to ion - exchanged water. The rats were fasted for 18 h before
evaluation. Body weights were measured at the completion of the feeding period, the rats then were killed, andliver and blood samples were taken. Blood was collected from the tail vein for Hb and Hct measurement. Hb
was measured using the Hemoglobin B - test Wako（FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan）.
Centrifugation in a capillary tube system determined Hct. Serum samples were obtained by centrifugation at 3000
rpm for 30 min at 4℃, and used for biochemical measurements.
2.2．Serum and hepatic TG measurements

Serum TG level was determined by the Nagahama Life - science Laboratory of Oriental Yeast Co., Ltd.
（Nagahama, Shiga, Japan）.
Liver lipids were extracted using Bligh and Dyer method ）. Frozen liver tissue（ 50 mg）was added to a17

homogenization tube containing 0.25 mL saline. The pellets were homogenized to form a suspension. Suspensionof 0.1 ml was mixed with 0.25 ml saline and was extracted with 3.6 ml of a chloroform/methanol（ 1：2 ）mixture
and centrifuged at 3000 rpm for 10 min at room temperature. After centrifugation, the lower liquid phase wasisolated and transferred to a separate vial. Hepatic TG concentration was then measured using the TriglycerideE - test Wako（FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan）.
2.3．RT-PCR analyses of TNF- α

To evaluate the mRNA transcript level of TNF - α in the livers of each group, the total RNA was
isolated using TRIzol reagent（Thermo Fisher Scientific, Waltham, MA）and Direct - zol RNA MiniPrep kit（Zymo Research, Irvine, CA, USA）according to the manufacturer’s instructions. Then, total RNA was random
primed with hexamers and reverse - transcribed to cDNA using the SuperScrip III reverse transcription
kit（Thermo Fisher Scientific, Waltham, MA）. The cDNA product was PCR - amplified by TaKaRa EX
Taq（Takara Bio Inc., Shiga, Japan）. Primer sequences and PCR conditions are described below：The
primers were synthesized by Fasmac Co., Ltd.（Kanagawa, Japan）. The primers were TNF - α ,（sense）5’- CGTCGTAGCAAACCACCAAG - 3’ and（antisense）5’ - CACAGAGCAATGACTCCAAAG - 3’ and β - actin,（sense）5’ - GAAGAGCTATGAGCTGCCTG - 3’ and（antisense）5’ - AGCAATGCCTGGGTACATGG - 3’. PCR
amplification was performed with a program of 2 min incubation at 95℃ followed by 30 reaction cycles（denaturation at 95℃ for 1 min, annealing at 58℃ for 1 min, and extension at 72℃ for 1 min）.PCR products wereanalyzed by agarose gel electrophoresis in 3% agarose gels and visualized by GR Red staining. The gels were
scanned and band density was quantified using the Image J software（the U.S. National Institutes of Health）.
2.4．Measurement of hepatic MDA adducts.

The degree of lipid peroxidation was assessed by measuring MDA adducts in the homogenized liver tissue
using the OxiSelectTM MDA Adduct ELISA Kit（Cell Biolabs, San Diego, CA）.The MDA values were expressedas pmol/mg of protein.

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Masatomi SHIMIZU Hideki OKAZAKI Petr GRÚZ
Masayo IKEDA Hiroyuki YAMAGISHI

東京医療保健大学 紀要第1号

2018 年

2.5．Statistical Analysis

All analyses were showed as mean ± standard deviation（SD）. Comparisons between groups were madeby means of Tukey–Kramer test. When unequal variance occurred between groups, we used the Steel–Dwass
test. Software used in this study was JMP 12（JMP® 12 SAS Institute Inc., Cary, NC, USA）. Differences wereconsidered statistically significant at p < 0.05.

3．Results and Discussion
The body weight gain in the iron - deficiency compared to the control group was significantly lower（p <0.05 ）. There was no significant difference of body weight gain between the iron - deficient and pair - fed groups（Table 1 ）. Hb and Hct in the iron - deficient group were significantly lower（p < 0.05 ）than those in the control
group（Table 1 ）. An iron - deficient diet decreased Hb concentration and Hct value in this study, suggestingthat iron - deficient anemia occurred in rats fed the iron - deficient diet. Additionally, although the pair - fed rats
had restricted food intake, their iron levels were almost equal to that of control rats. The serum TG level was
significantly higher（p < 0.05 ）in the iron - deficient group than in the control and pair - fed groups（Table 1 ）.
Furthermore hepatic TG level was increased（p < 0.05 ）in the iron - deficient group than in the pair - fed group（Table 1 ）. Perhaps, iron deficiency induces low energy levels and protects against diet - induced metabolic
disorders by enhancing TG levels. These results suggest the hypothesis that iron deficiency induces impairmentof carbohydrate metabolism, which can cause adiposopathy and multiple metabolic disorders（to be submitted）.
Anemia results in an increase oxidative stress in red blood cells ）. Additionally, triacylglycerol over storage
18

in hepatocytes is the basis for hepatic lipotoxicity and further, oxidative stress from steatotic hepatocytes leads
to lipid peroxidation, impaired mitochondrial and peroxisomal oxidation of fatty acids, and cytokine release ）.
19

Endotoxin - inducible cytokines, particularly TNF - α, are associated with the pathogenesis of Nonalcoholic Fatty
Liver Disease（NAFLD）in experimental animals ） ）. Therefore we examined whether hepatic lipotoxicity
20 ,21

supported on the iron - deficiency treatment, was resulting in inflammatory responses and MDA accumulation in
rat liver. TNF-α mRNA expression indicated a slight increase in the iron - deficient group than in the control andpair - fed groups, hepatic MDA adducts were hardly any different in each group.（Fig. 1a,b）. Other studies havefound that the increase of lipid peroxidation and hepatic inflammation may be associated with in the degree of fat
infiltration in the liver ） ）. Our study suggests that MDA accumulation and TNF - α induced inflammatory effect
22 - 25

Table 1 Body weight gain, Hb, Hct levels, serum TG and hepatic TG of control, pair-fed and ironTable 1 Body weight gain, Hb,
deficiency
rats.1weight gain, Hb, Hct levels, serum TG and hepatic TG of control, pair-fed and irondeficiency rats.1
Table 1 Body
deficiency rats.1

control

pair-fed

iron-deficiency

Body weight gain (g)280±30controla
189±10pair-fed

170±21 airon-deficiency
Body weight gain (g)Hb(g/dL)
Body
weight gain (g)15.8±2.7
280±3016.5±0.8a
189±104.0±0.5a a,b Hb (g/dL)
170±21Hct(%)
Hb (g/dL)

47.1±4.7
15.8±2.740.2±4.2
16.5±0.8a,b14.3±3.1
4.0±0.5 a,b Hct (%)

Serum
Hct
(%)TG (mg/dL)

87±2447.1±4.718±740.2±4.2

HepaticTG
TG(mg/dL)(mg/g liver)
Serum

70±36
87±24

33±21
18±7

a,b
255±11314.3±3.1 a,b Serum TG (mg/dL)

72±23 ca,b255±113

Hepatic TG (mg/g liver)

1

1
ValuesTG
are (mg/g
mean ±SD.
control, b p<0.05 versus
pair-fed by the Tukey–Kramer
Hepatic
liver) a p<0.05 versus
70±36
33±21
72±23 cValues are mean ±SD. a p<0.0

1test.

Values are mean ±SD. a p<0.05 versus control, b p<0.05 versus pair-fed by the Tukey–Kramer test.

cp<0.05 versus pair-fed by the Steel–Dwass test..
test.
c




c

p<0.05 versus pair-fed by the Steel–Dwass test..
̶ 46 ̶


p<0.05 versus pair-fed by the S

東京医療保健大学 紀要

Fig.1

ラットを用いた鉄欠乏状態から惹起される肝臓中の異所性脂肪蓄積の影響

第1号

2018 年

a1.5b-actin mRNA ratio
TNF-a/b-actin TNF-a/mRNA ratio(Fold change (Foldover control)change over control)

Fig.1
a



1
1.5

0.5
1

0

㼏㼛㼚㼠㼞㼛㼘

0.5

㼕㼞㼛㼚㻙㼐㼑㼒㼕㼏㼕㼑㼚㼠0㼏㼛㼚㼠㼞㼛㼘
㼜㼍㼕㼞㻙㼒㼑㼐

㼜㼍㼕㼞㻙㼒㼑㼐

㼕㼞㼛㼚㻙㼐㼑㼒㼕㼏㼕㼑㼚㼠b
250

MDA adducts
MDA adducts( pmol/ mg protein)( pmol/ mg protein)

b

200

250

150
200
150
100
100

50

50

0
0

control

control

pair-fed

pair-fed

iron-deficientiron-deficient





Fig.1
TheTNF-αTNF-αmRNA
expression
andadducts
MDA inadducts
Fig.1 ThemRNA
expression
and MDArat liver.in rat liver.
Valuesaremean±SD.
mean±SD.
a. RT-PCR
analysis.
TNF-αlevels
mRNA
levels fornormalizedfor β-actin mRNA
Values are
a. RT-PCR
analysis.
TNF-α mRNA
normalized
β-actin mRNAlevels wereas relative
ratio comparedwith the with
controlthegroup.
b. Levelof MDA
adducts.
levelswereexpressed
expressed
as relative
ratio compared
controlgroup.
b. Levelof MDA adducts.
have not occurred in hepatic cells after five weeks, but long - term continued monitoring is required to determine

this more precisely. In our study, serum TG and hepatic TG levels were significantly increased in rats treated with irondeficiency. The presumed mechanism for this effect is the reduced oxygen transport associated with anemia,
and energetic efficiency is affected at all levels of iron deficiency in human and animals. Our data suggest thatseveral fatty hepatic diseases may be due, in part, to inadequate dietary iron. Further, tissue iron deficiency may
also induce lipotoxicity through reduced cellular oxidative capacity. We analyzed mRNA expression of enzymes
associated with energy - metabolism isolated from each group. The results showed hardly any difference in eachenzyme mRNA expression（data not shown）.For all of the diseases that contribute to iron - deficiency anemia has
yet to be elucidated, it appears the possibility that iron deficiency anemia may extend to unexpected diseases.

4．Conclusion
Increased serum and hepatic TG levels in iron - deficient rats suggest the possibility of fatty liver diseases
as lipotoxicity. However, no alteration was found, at least in hepatic MDA levels and the expression levels of
hepatic TNF - α mRNA.

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Masatomi SHIMIZU Hideki OKAZAKI Petr GRÚZ
Masayo IKEDA Hiroyuki YAMAGISHI

東京医療保健大学 紀要第1号

2018 年

5．Acknowledgments      We thank the Tokyo Healthcare University Shimizu Laboratory students and the Kanto - Gakuin
University Yamagishi Laboratory students for their excellent technical assistance.

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2018 年

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