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DIAB-3822; No of Pages 8

Study of iron metabolism disturbances in an animal model of

insulin resistance

Guillaume Le Guenno a,*, Emilie Chanseaume b, Marc Ruivard c,Beatrice Morio b, Andrzej Mazur a

a INRA, Equipe Stress Metabolique et Micronutriments, UNH, UMR 1019, Clermont-Ferrand/Theix,

St-Genes-Champanelle 63122, Franceb INRA, Equipe Metabolisme Lipidique et Energetique, UNH, UMR 1019, Clermont-Ferrand/Theix,

St-Genes-Champanelle 63122, Francec Service of Internal Medicine Hotel-dieu, C.H.U. Clermont Ferrand, France

Received 24 October 2006; accepted 10 February 2007

Abstract

The relationship between iron and insulin-resistance (IR) is documented by the positive correlation between iron stores and IR.

Moreover, some patients exhibited a hepatic iron overload associated with IR (HIO-IR) but the mechanism involved in this overload

is not known. Thus, we studied the iron metabolism disturbances in an animal model of IR and the influence of provoked

hyperglycemia/hyperinsulinemia on plasma iron parameters. Wistar rats were fed a control or a high-fat/high-energy (HF/HE) diet.

Plasma glucose, insulin, iron, transferrin and transferrin saturation (TS) were measured during intra-peritoneal glucose test

tolerance (IPGTT) compared to saline. Hemogram, tissue iron concentrations and hepatic hepcidin mRNA expression were

determined at the end of experiment. HF/HE rats exhibited higher body and liver weights, increased IR-index and hemoglobin

concentration. Iron content was lower in the spleen of HF/HE rats and tended to decrease in the liver as compared to controls.

Transferrin values were higher and these of TS lower in HF/HE group. The hepcidin mRNAwas 3.5-fold lower in HF/HE rats than in

controls. IPGTT had no effect on iron status parameters in both groups. As reflected by higher hemoglobin concentration, IR could

increase erythropoıesis which enhances iron requirement. Iron stores and TS value decreased leading to a down-regulation of

hepcidin expression which increased iron absorption. Hepcidin expression should be investigated in metabolic syndrome and

hepatic iron overload associated with IR.

# 2007 Elsevier Ireland Ltd. All rights reserved.

Keywords: Iron; Insulin resistance; High-fat/high-energy diet; IPGTT; Hepcidin; Erythropoıesis

www.elsevier.com/locate/diabres

Diabetes Research and Clinical Practice xxx (2007) xxx–xxx

1. Introduction

The relationship between iron metabolism and

metabolic disorders has recently gained interest in both

research and clinical practice. Indeed, body iron stores

* Corresponding author at: 8 impasse Chabrier 63540 Romagnat,

France. Tel.: +33 4 73 61 15 45; fax: +33 4 73 62 46 38.

E-mail address: G.leguenno@free.fr (G. Le Guenno).

0168-8227/$ – see front matter # 2007 Elsevier Ireland Ltd. All rights re

doi:10.1016/j.diabres.2007.02.004

Please cite this article in press as: G. Le Guenno et al., Study of

resistance, Diab. Res. Clin. Pract. (2007), doi:10.1016/j.diabres.2

are positively correlated with insulin-resistance (IR),

even in the absence of significant iron overload [1–4].

Ferritin, which reflects body iron stores, is closely

associated with IR and can be considered a marker for

metabolic syndrome [5]. It has been shown that

phlebotomy significantly improves insulin sensitivity

in type 2 diabetes [6]. In animals, iron deficiency

increases insulin sensitivity [7]. Moreover, a common

syndrome called insulin-resistance associated hepatic

iron overload (IR-HIO) was described in 1997 by

served.

iron metabolism disturbances in an animal model of insulin

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G. Le Guenno et al. / Diabetes Research and Clinical Practice xxx (2007) xxx–xxx2

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DIAB-3822; No of Pages 8

Moirand et al. [8]. This is the most frequent cause of

iron overload in France with an estimated prevalence of

1% of the population, 10 times more widespread than

haemochromatosis [9]. A study of 269 subjects with

metabolic syndrome showed a prevalence of 14.5% of

the IR-HIO [10]. IR-HIO combines an isolated

hyperferritinemia with a normal transferrin saturation,

steatohepatitis and insulin resistance. It represents the

most widespread indication for venesection in referral

care units for iron overload [9]. However, the

mechanism involved in this overload is presently

unknown.

A few studies of the perturbation of iron metabolism

during IR were focused on the genetic models of

obesity, the ob/ob mouse [11,12] and Zucker rats [13].

The obese animals exhibited a decreased iron con-

centration in the liver [11–13], whereas plasmatic iron,

transferrin and spleen iron stores were unchanged when

compared with controls [11]. In ob/ob mice, the rate of

iron absorption was two-fold greater and the haemo-

globin concentration was significantly higher than in

lean control mice [11]. The authors concluded that

increased erythropoıesis in the obese animals provoked

a higher iron requirement, which explains the improve-

ment in iron absorption and the decreased liver iron

stores.

An important advance in our knowledge of iron

metabolism was made with the discovery of hepcidin.

This peptide, initially described as an antimicrobial

peptide [14], is a key regulator of iron stores and it

inhibits duodenal iron absorption and iron release by

macrophages, thereby provoking the internalization of

ferroportin [15]. It has been shown that hepcidin

expression is upregulated during infection, inflamma-

tion and iron overload [16], whereas the expression is

down-regulated by hypoxia, anaemia, iron deficiency,

erythropoietin and erythropoietic stimulation [17].

However, even if hepcidin plays a key role in iron

absorption, it is unknown how hepcidin expression is

modulated in IR.

Studies of interactions between iron and glucose

metabolism have shown that insulin can cause a rapid

and pronounced stimulation of iron uptake by adipo-

cytes by redistributing the transferrin receptor from an

intracellular compartment to the cell surface [18].

Transferrin receptors co-localize with the glucose

transporters and insulin-like growth factor II receptor

in the microsomal membranes of cultured adipocytes,

this suggests that regulation of iron uptake by insulin

occurs in parallel with glucose uptake [19]. Moreover, a

study on the effect of extreme hyperinsulinemia,

obtained during a hyperinsulinemic euglycemic clamp

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resistance, Diab. Res. Clin. Pract. (2007), doi:10.1016/j.diabres.2

in five healthy women, showed a progressive improve-

ment of the plasmatic iron during the experiment [20].

In reviewing the current literature, there is no data to

support a mechanism for iron accumulation in IR and

the aims of the present work were:

� To study the perturbations of the iron status and

hepcidin expression in an IR animal model induced

by an experimental diet.

� To analyze changes in plasma iron parameters during

hyperglycemia/hyperinsulinemia, induced by intra-

peritoneal injection of glucose, in this animal model.

2. Materials and methods

2.1. Animals and experimental diets

Sixteen 3 months old male Wistar rats were randomly

divided into two groups. The groups consumed a control or a

high-fat/high-energy (HF/HE) diet for 6 weeks. Diets were

prepared in the experimental diet preparation unit of Jouy-en-

Josas (UE300, UPAE INRA Domaine de Vilvert 78352 Jouy-

en-Josas) and distributed in a semi-liquid form in individual

ramekins. Food was weighed daily and prepared for each

animal. Tap water was available ad libitum. Animals were

housed in individual cages with a normal light cycle (Day 8

a.m. to 8 p.m.), in a temperature-controlled room (22 8C). On a

caloric basis, the HF/HE diet consisted of 45% fat (6.7% from

groundnut oil, 6.7% from canola oil and 31.6% from lard),

37.6% carbohydrate (25.6% from starch and 12% from

sucrose), and 17.4% protein (total 4.68 kcal/g), whereas the

control diet contained 13.8% fat (6.7% from groundnut oil and

6.7% from canola oil), 68.8% carbohydrate from starch, and

25.8% protein (total 3.9 kcal/g). Iron was provided as iron

sulfate and the intake was 7.8 and 7.7 mg/g body weight in the

control and the HF/HE group, respectively.

The experimental protocol was approved by the institu-

tional animal care and use committee at INRA (Decree 87–

848 modified by decree 2001–464).

2.2. Experimental procedures

One week before the animals were sacrificed, an intra-

peritoneal glucose tolerance test (IPGTT) was performed at 9

a.m. After 12 h of starvation, four rats of each group received

an intraperitoneal injection of 1 g of glucose/kg body weight

(G50%, volume (ml) = weight (g) � 2) and the remaining

eight animals received an isotonic saline solution injection.

Blood samples were obtained by retro-orbital puncture at

0, 15, 30, 60 and 120 min. The plasma was collected by

centrifugation at 1000 � g for 10 min and stored at �80 8Cuntil further analyses.

After 6 weeks on the specific diets, the rats were anesthe-

tized by intraperitoneal injection of Imalgene (120 mg/kg;

Vetranquil, Merial, Lyon, France) and Diazepam (1.75 mg/kg;

iron metabolism disturbances in an animal model of insulin

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DIAB-3822; No of Pages 8

Valium, Roche, France) and sacrificed by decapitation. Blood

was collected after decapitation for hemogram measurements.

Liver, spleen and duodenum were collected, weighed and

immediately frozen in liquid nitrogen and stored at �80 8C.

2.3. Glycemia and insulinemia measurements

Glycemia was measured immediately using fresh blood

(Glucometer Gluco Touch, LifeScan, Inc.). Insulinemia was

measured using plasma by ELISA (Rat/Mouse Insulin ELISA

kit EZRMI-13K, LINCO research, Inc, USA).

Insulin-resistance (IR) was calculated by the IR-index

[AUC glycemia (mg/dl) � AUC insulinemia (ng/ml)], the

Homeostasis Model Assessment score [HOMA = glycemia

(mmol/l) � insulinemia (mUI/ml)/22.5] and the Quantitative

Insulin Sensitivity Check Index [QUICKI = 1/log insulinemia

(mUI/l) + log glycemia (mg/dl)].

2.4. Iron status parameters

Haemograms were determined with a Scil Vet ABC coun-

ter (Animal Blood Counter, Strasbourg, France). Plasma iron

and TIBC were measured using the ‘‘ferrimat kit’’ (bioMer-

ieux SA, Marcy-l’Etoile, France) and the ‘‘TIBC additif’’ kit

(bioMerieux SA,) in combination with a Progress Plus Chem-

istry Analyser automat (Kone, Evry, France). The transferrin

saturation (TS) was calculated as fasting plasmatic iron/TIBC

and transferrin as TIBC/25.

Nonheme iron was determined by a modification of the

method of Foy et al. [21] as described by Simpson and Peters

[22]. For the duodenum, nonheme iron concentrations were

expressed relative to the protein concentration (mg/mg of

protein). Tissue protein concentration was estimated using

the ‘‘protein BCA Uptima kit’’ (Interchim, Montlucon,

France).

2.5. Hepcidin mRNA measurements

Total RNA were extracted from the liver using the Qiagen

RNeasy Mini kit (Coutaboeuf, France) according to the

manufacturer’s instructions. We used 3 mg of total RNA for

cDNA synthesis, using the Ready To Go Your First Strand

Bead kit (Amersham Pharmacia Biotech, Orsay, France).

Polymerase chain reaction (PCR) was carried out using the

Pure Taq Ready To Go PCR Beads kit (Amersham Pharmacia

Biotech, Orsay, France) and a TC-512 Techne Thermal Cycler

Please cite this article in press as: G. Le Guenno et al., Study of

resistance, Diab. Res. Clin. Pract. (2007), doi:10.1016/j.diabres.2

Table 1

Effect of diet on body and organ weight, and hemoglobin concentration in

Control

Body weight (g) 514.2 �Liver weight (g) 15.9 �Spleen weight (g) 1.02 �Hemoglobin concentration (g/dl) 14.5 �

Values are expressed as means � S.E.M. for groups of eight rats.* Significantly different from controls ( p < 0.05).

(MIDSCI, USA). We performed quantitative RT-PCR using

the LightCycler Fast Start DNA Master SYBR Green I kit

(Roche Diagnostics, Meylan, France) and a LightCycler

(Roche Diagnostics). The hepcidin gene expression was nor-

malized to the GAPDH expression in the same sample. The

following primers were used for PCR amplification: GAPDH

(50-CAT GAC CAC AGT CCATGC CAT CAC-30 and 50-CAT

GTA GGC CAT GAG GTC CAC CAC-30), hepcidin (50-ACA

GAA GGC AAG ATG GCA CT-30 and 50-GAA GTT GGT

GTC TCG CTT CC-30). These primer pairs produced 458- and

201-bp amplification products, respectively.

2.6. Statistical analysis

Results were expressed as means � S.E.M. Statistical

analysis were performed using the Statview software (SAS

Institute Inc., SAS campus drive, Cary, NC, USA). If a

significant variance difference was observed between the

two groups, a log transformation was performed. The statis-

tical significance of differences between means from two

studied groups was assessed by the Student’s t-test. A two-

way repeated measures ANOVA, followed by PLSD Fisher’s

test, was performed to estimate the effect of group and

injection on values obtained during IPGTT. Differences were

considered as significant at p < 0.05.

3. Results

3.1. Effect of diet on haematological parameters,

body and organ weight

The effect of diet on the haematological parameters,

body and organ weight are shown in Table 1. After 6

weeks, the HF/HE group had significantly higher body

( p < 0.01) and liver weight ( p < 0.01), whereas spleen

weight was lower ( p = 0.03), when compared with the

control group. Expressing the organ weight relative to

body weight, the difference in the liver weight remained

at the edge of significance ( p = 0.04), but the spleen

weight was more pronounced ( p < 0.01). The haemo-

globin concentration was significantly higher in the HF/

HE group when compared with the control group

( p = 0.03). The other haematological parameters (white

iron metabolism disturbances in an animal model of insulin

007.02.004

the control and high-fat/high-energy diet fed groups

(n = 8) High-fat/high-energy (n = 8)

9.2 570.3 � 9.9*

0.5 19.1 � 0.9*

0.2 0.89 � 0.4*

0.9 18.5 � 1.6*

G. Le Guenno et al. / Diabetes Research and Clinical Practice xxx (2007) xxx–xxx4

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DIAB-3822; No of Pages 8

Fig. 1. Effect of intraperitoneal injection of glucose or saline serum on glycemia and insulinemia. Values were measured in control and HF/HE diet

fed group at T0, T15, T30, T60 and T120 min. (A) Effect of isotonic saline solution injection on glycemia. (B) Effect of glucose solution injection on

glycemia. (C) Effect of isotonic saline solution injection on insulinemia. (D) Effect of glucose solution injection on insulinemia. Values are expressed

as means � S.E.M. for groups of four rats. Using a two-ways ANOVA, there is no significant effect of group’s factor on insulinemia and glycemia

values whereas injection’s factor has significant effect on insulinemia ( p < 0.01) and glycemia values ( p < 0.01). Black circles: HF/HE diet fed

group. Empty circles: control diet fed group.

blood cells and platelet counts) were not significantly

different (data not shown).

3.2. Effect of diet on IPGTT and evaluation of

insulin-resistance

Fasting glycemia was significantly higher in HF/HE

group ( p < 0.01). The effect of intraperitoneal injection

of isotonic saline and glucose solution on glycemia and

insulinemia values at T0, T15, T30, T60, T120 min in

the control and HF/HE groups are shown in Fig. 1.

Using a two-way ANOVA, diet factor had no significant

effect on glycemia ( p = 0.06) and insulinemia values

( p = 0.09) at the different times of the IPGTT. Insulin-

resistance was higher in HF/HE group whether

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resistance, Diab. Res. Clin. Pract. (2007), doi:10.1016/j.diabres.2

Table 2

Effect of diet on fasting glycemia, fasting insulinemia, IR index, QUICKI

Control (n =

Fasting glycemia (mg/dl) 72.12 � 1

Fasting insulinemia (ng/ml) 0.90 � 0

IR index (glucose injection)a 2.83.106 � 9

IR index (saline injection)a 9.09.105 � 2

QUICKIb 0.44 � 0

HOMAc 13.28 � 4

Values are expressed as means � S.E.M. for groups of eight rats, except th

injection of isotonic saline or glucose solution.* Significantly different from controls ( p < 0.05).a IR index = AUC glycemia (mg/dl) � AUC insulinemia (ng/ml).b QUICKI = 1/(log glycemia (mg/dl) + log insulinemia (mUI/l)).c HOMA = insulinemia (mUI/l) � glycemia (mmol/l)/22.5.

comparing IR index between groups receiving glucose

( p = 0.04) or isotonic saline injection ( p = 0.01), or the

QUICKI index ( p < 0.001). The HOMA index did not

differ significantly between the two groups ( p = 0.10).

These data are shown in Table 2.

3.3. Effect of diet on tissue nonheme iron

concentration

The tissue iron concentrations are shown in Table 3.

In HF/HE diet group, the nonheme iron content was

significantly lower in the spleen ( p < 0.01), tended to

decrease in the liver ( p = 0.06), but was unchanged in

the duodenum ( p = 0.92), when compared with the

control group.

iron metabolism disturbances in an animal model of insulin

007.02.004

and HOMA indexes

8) High-fat/high-energy (n = 8)

.02 77.87 � 1.88*

.29 1.35 � 0.25

.76.105 5.65.106 � 9.85.105*

.06.105 1.81.106 � 2.15.105*

.02 0.30 � 0.01*

.41 21.44 � 4.212

e IR index where four rats of each group received an intraperitoneal

G. Le Guenno et al. / Diabetes Research and Clinical Practice xxx (2007) xxx–xxx 5

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DIAB-3822; No of Pages 8

Table 3

Tissue non-heminic iron concentration in control and HF/HE diet fed groups

Control (n = 8) High-fat/high-energy (n = 8)

Liver iron (mg/g tissue) 66.76 � 8.14 51.51 � 4.20

Spleen iron (mg/g tissue) 424.29 � 45.10 304.62 � 18.04*

Duodenal iron (mg/mg protein) 0.21 � 0.06 0.22 � 0.09

Values are expressed as means � S.E.M. for groups of eight rats.* Significantly different from controls ( p < 0.05).

3.3.1. Effect of diet on plasma iron, transferrin and

transferrin saturation during IPGTT

The effect of group (control or HF/HE) and injection

nature (glucose or saline) on these parameters are

shown in Fig. 2 and were assessed using two-way

ANOVA. In both groups, plasma iron progressively

decreased during the test because of circadian cycle

effect. Injection had no effect on plasma iron ( p = 0.87),

transferrin ( p = 0.89) and transferrin saturation

( p = 0.92). Group had no effect on plasma iron

( p = 0.42), but had a very significant effect on the

transferrin values ( p < 0.01). Comparing the mean

values measured during the IPGTT, the HF/HE group

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resistance, Diab. Res. Clin. Pract. (2007), doi:10.1016/j.diabres.2

Fig. 2. Effect of intraperitoneal injection of isotonic saline or glucose solut

HF/HE diet fed group at T0, T15, T30, T60 and T120 min. (A) Effect of injec

iron in HF/HE group. (C) Effect of injection on transferrin in control group

injection on transferrin saturation in control group. (F) Effect of injection

means � S.E.M. for groups of four rats. Using a two-ways ANOVA, there i

whereas group’s factor has significant effect on transferrin values ( p < 0.

glucose solution injection.

had significantly higher transferrin value ( p < 0.01),

lower transferrin saturation ( p = 0.04) and no difference

in plasma iron values ( p = 0.19), when compared with

the control group. These data are shown in Table 4.

3.4. Effect of diet on hepatic hepcidin expression

Liver hepcidin mRNA levels in the two groups were

determined by RT-PCR as shown in Fig. 3A. The qRT-

PCR measurements of the hepatic mRNA levels are

shown in Fig. 3B and demonstrate that the hepcidin

expression was 3.5-fold lower in HF/HE group

( p = 0.03).

iron metabolism disturbances in an animal model of insulin

007.02.004

ion on plasma iron parameters. Values were measured in control and

tion on plasma iron in control group. (B) Effect of injection on plasma

. (D) Effect of injection on transferrin in HF/HE group. (E) Effect of

on transferrin saturation in HF/HE group. Values are expressed as

s no significant effect of injection’s factor on plasma iron parameters

01). Black circles: isotonic saline solution injection. Empty circles:

G. Le Guenno et al. / Diabetes Research and Clinical Practice xxx (2007) xxx–xxx6

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DIAB-3822; No of Pages 8

Table 4

Average values for plasma iron parameters obtained in control and HF/

HE diet fed groups during intraperitoneal injection

Control

(n = 8)

High-fat/high-

energy (n = 8)

Plasma iron (mmol/l) 28.67 � 1.33 26.16 � 2.47

Plasma transferrin (g/l) 4.01 � 0.11 4.59 � 0.18*

Transferrin saturation (%) 29.61 � 2.04 23.29 � 2.76*

Values are expressed as means � S.E.M. for groups of eight rats.* Significantly different from controls ( p < 0.05).

4. Discussion

The relationship between iron metabolism and IR is

well-illustrated in clinical practice by the strong

correlation between iron stores and IR [1–5]. Moreover,

some of the patients with metabolic syndrome or type 2

diabetes exhibit an insulin-resistance which is asso-

ciated with hepatic iron overload (IR-HIO). However,

the mechanisms behind this overload are unknown and

the data from animal studies of genetic obesity models

are inconclusive. In this study, we have characterized

perturbations in iron metabolism using an animal model

rendered IR by an experimental HF/HE diet. Previous

studies had shown that 3–4 weeks on a high fat or high

energy/high fat diet induces IR as judged by the

euglycemic hyperinsulinemic clamp [23–25]. In these

studies, the animals exhibited the characteristic

abnormalities described in patients with the metabolic

syndrome or type 2 diabetes, including increased

visceral and muscle fat content, overweight, hepatic

steatosis, high fasting glycemia with or without high

fasting insulinemia, low HDL values and high plasma

fatty acid levels. The HF/HE diet produced similar

abnormalities in our study: the animals demonstrated

overweight, increased liver weight with macroscopic

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resistance, Diab. Res. Clin. Pract. (2007), doi:10.1016/j.diabres.2

Fig. 3. (A) Hepcidin mRNA abundance analyzed by RT-PCR (23 cycles) u

Products of RT-PCR amplification were separated by agarose gel electr

transillumination. GAPDH mRNA serves as reference. (B) qRT-PCR for liv

of hepcidin mRNA were normalized to that of GAPDH mRNA. Values are

different from controls ( p < 0.05).

steatosis and high fasting glycemia. The development of

IR was confirmed by a significant increase in the IR-

index and a significant decrease in the QUICKI index,

when compared with the control group.

To our knowledge, iron metabolism has not been

assessed in this IR model. In the present study, we

assessed current biomarkers for the blood iron status and

the iron content within selected organs serving important

roles in iron metabolism, i.e. the liver (iron storage and

regulation), the spleen (iron recycling and erythropoıesis)

and the duodenum (iron absorption). Our data shows that

IR leads to a lower iron concentration in the liver and

spleen. This is consistent with data from genetic models

for IR, such as ob/ob mice [11] and Zucker rats [13],

except that in ob/ob mice, the spleen iron concentration is

unchanged. In addition, our data shows that there is no

difference in the duodenal iron concentration between the

two groups. Comparing the mean values of the blood iron

status parameters between the control and HF/HE

groups, a higher transferrin concentration is associated

with lower transferrin saturation in HF/HE group. These

changes in the blood parameters of the HF/HE group are

associated with lower tissue iron stores, which suggest an

increased iron need.

Erythropoıesis is the state where production of red

blood cells is sufficient to maintain a normal level of

haemoglobin. In the mammals, iron is mainly used for

haemoglobin synthesis. In the HF/HE group, the

haemoglobin concentration was higher than in the

control group. This is consistent with a study using ob/

ob mice [11] and observations from humans, where the

haemoglobin concentration is linked to IR [26,27]. This

physiological mechanism may involve insulin stimula-

tion of the sympathetic system and the erythropoietic

progenitors. However, the lower spleen weight in HF/

HE diet fed rats, also seen in ob/ob mice [11], is

iron metabolism disturbances in an animal model of insulin

007.02.004

sing 3 mg RNA from the liver of control and HF/HE diet fed groups.

ophoresis and visualized with ethidium bromide under ultraviolet

er hepcidin mRNA in control and HF/HE diet fed group. The values

expressed as means � S.E.M. for groups of eight rats. *Significantly

G. Le Guenno et al. / Diabetes Research and Clinical Practice xxx (2007) xxx–xxx 7

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DIAB-3822; No of Pages 8

contradicting this mechanism because spleen is a

haematopoiesis organ in rodents. Changes in haema-

topoiesis during IR in the rat models were not assessed.

However, a study on the role of leptin in haematopoiesis

using db/db mice [28] showed that lower spleen weight

was secondary to a significant decrease in erythrocytic

progenitors. The absence of a decrease in the

haemoglobin concentration was explained by a com-

pensatory increase in erythrocytic precursors at the

medullar level. As obesity is known to induce leptin-

resistance, the decrease in spleen weight in the HF/HE

group may result from a similar mechanism. Erythro-

poıesis and thus, haemoglobin synthesis, requires the

contribution of iron from erythrophagocytosis and

digestive absorption. The higher haemoglobin concen-

tration in HF/HE group suggests a higher erythropoı-

esis, which would rely on iron for haemoglobin

synthesis ‘‘drawn’’ from storage tissues. The increase

in plasma transferrin levels allows a more important

iron transport to the bone marrow.

To better understand the mechanisms behind the iron

metabolism changes in IR, we analyzed the changes in the

hepcidin expression, the major regulator of iron home-

ostasis. Hepcidin is a peptide synthesized by the liver [29]

and its role is crucial in iron metabolism. It can stimulate

the internalization of ferroportin [15] and thereby inhibit

digestive iron absorption and plasmatic release from

macrophages. Hepcidin liver expression is decreased by

hypoxia, iron deficiency, anaemia, erythropoietin, and

during stimulation of erythropoıesis [17]. In this study,

we show that the liver hepcidin mRNA level was 3.5-fold

lower in the HF/HE group when compared with the

control. This decrease could be explained by the lower

hepatic iron concentration and the increase in erythropoı-

esis in this group. Downregulation of hepcidin expression

would permit a more important digestive absorption of

iron to compensate for increased iron needs.

The short-term effects of variations in glycemia and

insulinemia on plasma iron parameters were evaluated.

In vitro studies, using cell cultures, suggest that insulin

can modulate iron uptake by recruiting the transferrin

receptors to the plasma membrane along with the

glucose transporters [19]. Another study, using rats,

showed that 3 days of high dose insulin treatment (4 UI/

kg) induced iron-loading of brown adipose tissue [30].

Moreover, a hyperinsulinemic euglycemic clamp study

in five healthy women showed a progressive improve-

ment of the plasma iron status during the experiment

[20]. Here, we show that the injection of glucose has no

effect on plasma iron, transferrin and TS. This suggests

that hyperinsulinemia has no influence on the iron status

parameters. The discrepancy between our data and

Please cite this article in press as: G. Le Guenno et al., Study of

resistance, Diab. Res. Clin. Pract. (2007), doi:10.1016/j.diabres.2

those of other studies [19,30] could be explained by the

use of different insulin concentrations and these reached

in our in vivo conditions.

The aim of this study was to characterize the changes

in iron metabolism in IR and the mechanisms involved.

We show that a high fat/high energy diet lead to

overweight and IR in Wistar rats. The IR may increase the

plasma haemoglobin concentrations consistent with

stimulation of erythropoıesis, which in turn enhances

the needs for iron. In agreement with this prediction, we

observed a decrease in tissue iron stores and an increase

in transferrin. Erythropoıesis stimulation and hepatic iron

store reduction lead to downregulation of the hepatic

hepcidin expression which subsequently increases iron

absorption. Futures studies of this IR model, adjusting the

iron intake to achieve a similar liver iron concentration as

in the control, would clarify the influence of erythropoı-

esis on hepcidin expression during IR. Our results show a

decline in tissue iron stores in the IR group, the opposite is

observed in patients [1–5]. A long-term study of the HF/

HE animals would be interesting in order to assess if the

hepatic iron overload would appear latter. Interestingly,

we show for the first time that IR can lead to a

downregulation of hepcidin expression. Human studies

focused on iron absorption rates and hepcidin expression,

in the metabolic syndrome and HIO-IR, should be

performed to unravel the mechanisms behind this iron

metabolism disturbance. We also show that provoked

hyperglycemia/hyperinsulinemia during IPGTT has no

effect on plasma iron, transferrin and TS values. This data

suggests that under these conditions, there is no influence

of glycemia and insulinemia on plasma iron parameters.

Acknowledgments

We wish to thank Dominique Bayle, Severine Thien

and Alexandre Teynie for technical assistance. This

work was supported in part by Prix de Recherche du

Centre Evian pour l’Eau.

References

[1] M. Haap, A. Fritsche, H.J. Mensing, H.U. Haring, M. Stumvoll,

Association of high serum ferritin concentration with glucose

intolerance and insulin resistance in healthy people, Ann. Intern.

Med. 139 (2003) 869–871.

[2] M. Jehn, J.M. Clark, E. Guallar, Serum ferritin and risk of the

metabolic syndrome in U.S. adults, Diab. Care 27 (2004) 2422–

2428.

[3] T.P. Tuomainen, K. Nyyssonen, R. Salonen, A. Tervahauta, H.

Korpela, T. Lakka, et al., Body iron stores are associated with

serum insulin and blood glucose concentrations. Population

study in 1,013 eastern Finnish men, Diab. Care 20 (1997)

426–428.

iron metabolism disturbances in an animal model of insulin

007.02.004

G. Le Guenno et al. / Diabetes Research and Clinical Practice xxx (2007) xxx–xxx8

+ Models

DIAB-3822; No of Pages 8

[4] J.T. Salonen, T.P. Tuomainen, K. Nyyssonen, H.M. Lakka, K.

Punnonen, Relation between iron stores and non-insulin depen-

dent diabetes in men: case–control study, BMJ 317 (1998) 727.

[5] J.M. Fernandez-Real, W. Ricart-Engel, E. Arroyo, R. Balanca,

R. Casamitjana-Abella, D. Cabrero, et al., Serum ferritin as a

component of the insulin resistance syndrome, Diab. Care 21

(1998) 62–68.

[6] J.M. Fernandez-Real, G. Penarroja, A. Castro, F. Garcia-Brag-

ado, I. Hernandez-Aguado, W. Ricart, Blood letting in high-

ferritin type 2 diabetes: effects on insulin sensitivity and beta-cell

function, Diabetes 51 (2002) 1000–1004.

[7] M.J. Borel, J.L. Beard, P.A. Farrell, Hepatic glucose production

and insulin sensitivity and responsiveness in iron-deficient ane-

mic rats, Am. J. Physiol. 264 (1993) E380–E390.

[8] R. Moirand, A.M. Mortaji, O. Loreal, F. Paillard, P. Brissot, Y.

Deugnier, A new syndrome of liver iron overload with normal

transferrin saturation, Lancet 349 (1997) 95–97.

[9] A. Guillygomarc’h, M.H. Mendler, R. Moirand, F. Laine, V.

Quentin, V. David, et al., Venesection therapy of insulin resis-

tance-associated hepatic iron overload, J. Hepatol. 35 (2001)

344–349.

[10] C. Bozzini, D. Girelli, O. Olivieri, N. Martinelli, A. Bassi, G. De

Matteis, et al., Prevalence of body iron excess in the metabolic

syndrome, Diab. Care 28 (2005) 2061–2063.

[11] M.L. Failla, M.L. Kennedy, M.L. Chen, Iron metabolism in

genetically obese (ob/ob) mice, J. Nutr. 118 (1988) 46–51.

[12] M.L. Kennedy, M.L. Failla, J.C. Smith Jr., Influence of genetic

obesity on tissue concentrations of zinc, copper, manganese and

iron in mice, J. Nutr. 116 (1986) 1432–1441.

[13] R.E. Serfass, K.E. Park, M.L. Kaplan, Developmental changes of

selected minerals in Zucker rats, Proc. Soc. Exp. Biol. Med. 189

(1988) 229–239.

[14] A. Krause, S. Neitz, H.J. Magert, A. Schulz, W.G. Forssmann, P.

Schulz-Knappe, et al., LEAP-1, a novel highly disulfide-bonded

human peptide, exhibits antimicrobial activity, FEBS Lett. 480

(2000) 147–150.

[15] E. Nemeth, M.S. Tuttle, J. Powelson, M.B. Vaughn, A. Donovan,

D.M. Ward, et al., Hepcidin regulates cellular iron efflux by

binding to ferroportin and inducing its internalization, Science

306 (2004) 2090–2093.

[16] G. Nicolas, C. Chauvet, L. Viatte, J.L. Danan, X. Bigard, I.

Devaux, et al., The gene encoding the iron regulatory peptide

hepcidin is regulated by anemia, hypoxia, and inflammation, J.

Clin. Invest. 110 (2002) 1037–1044.

[17] M. Vokurka, J. Krijt, K. Sulc, E. Necas, Hepcidin mRNA levels

in mouse liver respond to inhibition of erythropoıesis, Physiol.

Res. (2006).

Please cite this article in press as: G. Le Guenno et al., Study of

resistance, Diab. Res. Clin. Pract. (2007), doi:10.1016/j.diabres.2

[18] L.I. Tanner, G.E. Lienhard, Insulin elicits a redistribution of

transferrin receptors in 3T3-L1 adipocytes through an increase in

the rate constant for receptor externalization, J. Biol. Chem. 262

(1987) 8975–8980.

[19] L.I. Tanner, G.E. Lienhard, Localization of transferrin receptors

and insulin-like growth factor II receptors in vesicles from 3T3-

L1 adipocytes that contain intracellular glucose transporters, J.

Cell Biol. 108 (1989) 1537–1545.

[20] J.E. Nestler, J.N. Clore, M.L. Failla, W.G. Blackard, Effects of

extreme hyperinsulinaemia on serum levels of trace metals, trace

metal binding proteins, and electrolytes in normal females, Acta

Endocrinol. (Copenh.) 114 (1987) 235–242.

[21] A.L. Foy, H.L. Williams, S. Cortell, M.E. Conrad, A modified

procedure for the determination of nonheme iron in tissue, Anal.

Biochem. 18 (1967) 559–563.

[22] R.J. Simpson, M. Lombard, K.B. Raja, R. Thatcher, T.J. Peters,

Iron absorption by hypotransferrinaemic mice, Br. J. Haematol.

78 (1991) 565–570.

[23] B.D. Hegarty, G.J. Cooney, E.W. Kraegen, S.M. Furler,

Increased efficiency of fatty acid uptake contributes to lipid

accumulation in skeletal muscle of high fat-fed insulin-resistant

rats, Diabetes 51 (2002) 1477–1484.

[24] E.W. Kraegen, P.W. Clark, A.B. Jenkins, E.A. Daley, D.J.

Chisholm, L.H. Storlien, Development of muscle insulin resis-

tance after liver insulin resistance in high-fat-fed rats, Diabetes

40 (1991) 1397–1403.

[25] T. Akiyama, I. Tachibana, H. Shirohara, N. Watanabe, M. Otsuki,

High-fat hypercaloric diet induces obesity, glucose intolerance

and hyperlipidemia in normal adult male Wistar rat, Diab. Res.

Clin. Pract. 31 (1996) 27–35.

[26] F.S. Facchini, M. Carantoni, J. Jeppesen, G.M. Reaven, Hema-

tocrit, haemoglobin are independently related to insulin resis-

tance and compensatory hyperinsulinemia in healthy, non-obese

men and women, Metabolism 47 (1998) 831–835.

[27] M. Barbieri, E. Ragno, E. Benvenuti, G.A. Zito, A. Corsi, L.

Ferrucci, et al., New aspects of the insulin resistance syndrome:

impact on haematological parameters, Diabetologia 44 (2001)

1232–1237.

[28] B.D. Bennett, G.P. Solar, J.Q. Yuan, J. Mathias, G.R. Thomas, W.

Matthews, A role for leptin and its cognate receptor in hema-

topoiesis, Curr. Biol. 6 (1996) 1170–1180.

[29] C.H. Park, E.V. Valore, A.J. Waring, T. Ganz, Hepcidin, a urinary

antimicrobial peptide synthesized in the liver, J. Biol. Chem. 276

(2001) 7806–7810.

[30] A. Korac, M. Verec, V. Davidovic, Insulin-induced iron loading

in the rat brown adipose tissue: histochemical and electron-

microscopic study, Eur. J. Histochem. 47 (2003) 241–244.

iron metabolism disturbances in an animal model of insulin

007.02.004