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Protein-Caloric Food Restriction Affects Insulin-Like
Growth Factor System in Fetal Wistar Rat
M. A Martı´n, P. Serradas, S. Ramos, E. Ferna´ndez, L. Goya, M. N. Gangnerau, M. Lacorne,
A. M. Pascual-Leone, F. Escriva´, Bernard Portha, and C. A
´
lvarez
Instituto de Bioquı´mica, Consejo Superior de Investigaciones Cientificas Universidad Complutense de Madrid (M.A.M., S.R.,
E.F., L.G., A.M.P.-L., F.E., C.A.), Facultad de Farmacia, Universidad Complutense, Ciudad Universitaria, 28040 Madrid,
Spain; and the Laboratory of Physiopathology of Nutrition (P.S., M.N.G., M.L., B.P.), Centre National de la Recherche
Scientifique Unite´ Mixte de Recherche 7059, Universite´ Paris 7/D, Denis Diderot, 7525 Paris Cedex 05, France
We have previously shown that fetuses from protein-caloric
undernourished pregnant rats (35% of control diet during the
last week of pregnancy) at 21.5 d post coitum exhibit increased

-cell mass. This alteration is correlated with increased insu-
linemia and total pancreatic insulin content, a pattern similar
to that reported in infants of mild diabetic mothers. In this
work, we investigated in undernourished fetuses: 1) whether
availability of growth factors such as insulin, GH, and IGFs
and their binding proteins (IGFBPs) could be implicated in
this alteration, and 2) the

-cell mitogenic response to IGFs in
vitro. The results show that maternal undernutrition in-
creases pancreatic IGF-I expression and islet IGF-I receptor
content in undernourished fetuses, whereas hepatic IGF-I ex-
pression and serum IGF-I levels were decreased. No changes
were observed in serum IGF-II, and its expression was dimin-
ished in undernourished pancreases and unchanged in the
liver, compared with control fetuses. Serum levels and liver
and pancreatic mRNA expression of IGFBP-1 were found to be
normal in undernourished fetuses, whereas the serum con-
centration and abundance of IGFBP-2 mRNA in pancreas
were increased. Finally, the

-cell mitogenic response to IGFs
in vitro was significantly increased in undernourished fetal
islets, compared with controls. In conclusion, in undernour-
ished fetuses the increased

-cell mass can be related to the
stimulation of replicative

-cell response due to locally in-
creased pancreatic IGF-I mRNA; this effect is perhaps poten-
tiated or favored by the enhanced islet IGF-I receptor content
and pancreatic IGFBP-2 gene expression. (Endocrinology 146:
1364 –1371, 2005)
U
NDERNUTRITION LEADS TO an impairment of glu-
cose homeostasis, which in the mother has clear ef-
fects on the development of the fetus, especially on the fetal
pancreas. As indicated by the thrifty phenotype hypothesis
(1), the endocrine pancreas may be particularly susceptible
to the effects of poor maternal nutrition because fetal and
postnatal periods are critical for

-cell development and
maturation of pancreatic function. Several studies in this area
in experimental models with rats submitted to different pat-
terns of malnutrition have reported that maternal undernu-
trition significantly affects the pancreatic insulin stores and
the

-cell mass in the fetuses (2–4) and offspring neonates at
d1(5)andd4ofpostnatal life (6). In previous studies in our
model of general food restriction (65% restriction of ad libitum
food intake) (7), we have shown that fetuses from protein-
caloric undernourished pregnant rats (U) during the last
trimester of gestation at 21 d post coitum (dpc) exhibit in-
creased

-cell mass. This alteration is correlated with in-
creased insulinemia and total pancreatic insulin content (2),
a pattern similar to that reported in infants of both mild
diabetic mothers (8) and the poorly controlled diabetic moth-
ers (9).
The IGF system includes two ligands (IGF-I and IGF-II),
two cell surface receptors, the IGF binding proteins (IGFBPs)
and IGFBP proteases (10) and is involved in normal growth,
and especially in fetal pancreas

-cell development (11).
IGF-I and -II are essential cell growth regulators, as demon-
strated by null mutation experiments (12). IGFs are synthe-
sized primarily by the liver, but they are also produced
locally by many tissues, including the pancreas in which they
act in an autocrine/paracrine manner. In the rat, IGF-II is
expressed at high levels during embryonic development, but
its expression progressively disappears in most tissues after
birth, except in brain (9). The IGF-I gene is also expressed in
a variety of fetal rat tissues, although at lower levels than the
IGF-II gene. Whereas IGF-II is the primary growth factor
involved in embryonic growth, the dominant fetal growth
regulator in late gestation is IGF-I produced by the fetal liver
and other tissues (13). IGFs have insulin-like metabolic ef-
fects and stimulate cell proliferation and differentiation, and
these mitogenic effects are mediated through interaction
with the IGF receptor (IGF-IR) or insulin receptor (14, 15).
The IGF-IR, which activates mitogenesis via pathways par-
tially identical with insulin signaling, can be triggered by
IGF-I, IGF-II, and supraphysiological concentrations of in-
sulin (16). In vitro, both IGF-I and IGF-II enhance

-cell rep-
lication, but IGF-I is a more potent mitogen on most cell types
because it is recognized by the IGF-IR with a binding affinity
of an order of magnitude greater than IGF-II (9).
First Published Online December 2, 2004
Abbreviations: BrdU, 5⬘-Bromo-2⬘-deoxyuridine; C, control pregnant
rat; dpc, days post coitum; IGFBP, IGF binding protein; IGF-IR, IGF
receptor; PVDF, polyvinylidene fluoride; TBS, Tris-buffered saline; U,
undernourished pregnant rat.
Endocrinology is published monthly by The Endocrine Society (http://
www.endo-society.org), the foremost professional society serving the
endocrine community.
0013-7227/05/$15.00/0 Endocrinology 146(3):1364–1371
Printed in U.S.A. Copyright © 2005 by The Endocrine Society
doi: 10.1210/en.2004-0665
1364
Both IGFs are present in serum and other extracellular
fluids associated with highly specific binding proteins,
IGFBPs, of which six have been characterized and can mod-
ulate IGFs biological actions (17). Apart from this modula-
tion, IGFBPs, mostly produced in the liver (17), may exert
intrinsic bioactivity in either the absence of IGFs (IGF-inde-
pendent effects) or the presence of IGFs without triggering
IGF-IR signaling (IGF-IR-independent effects) (18). In the
fetus, IGFs are predominantly complexed with IGFBP-1 and
-2 (19–21). During the fetal period, insulin also regulates
growth, and IGF regulation is GH independent (9).
Because there is considerable evidence that endocrine fac-
tors such as insulin, GH, and IGFs contribute to

-cell growth
as well as its maturation and function throughout life (9), that
IGF actions can be modulated by locally produced IGFBPs
(22), and that the IGF system is highly responsive to nutri-
tional status (23), the purpose of the present study was to
investigate in U fetuses at the end of fetal life (21.5 dpc): 1)
the circulating levels of insulin, GH, IGFs, and IGFBP-1 and
-2; 2) the expression of IGFs and IGFBP-1 and -2 mRNAs in
liver and pancreas; 3) the islet content of IGF-IR; and 4) the
in vitro mitogenic effect of IGFs in isolated fetal islets.
Materials and Methods
Animals and diets
Wistar rats bred in our laboratory under a controlled temperature and
artificial dark-light cycle (from 0700 to1900 h) were used throughout the
study. Females were caged with males, and mating was confirmed by
the presence of spermatozoa in a vaginal smear. Each dam was housed
individually from the 14th day, and maternal food restriction was es-
tablished. All animals were fed a standard laboratory diet (19 g protein,
56 g carbohydrate, 3.5 g lipid, and 4.5 g cellulose per 100 g, plus salt and
vitamin mixtures) and were divided into two groups. Control (C) preg-
nant dams were fed ad libitum, and the U group received 35% of the food
intake of a pregnant control during the third part of pregnancy, which
corresponds to the crucial period for fetal rat pancreas development.
Water was given ad libitum. Food intake of control and U rats was
previously reported (7). On 21.5 dpc dams were put under ip pento-
barbital anesthesia (4 mg/100 g body weight). The fetuses were obtained
from four to seven different litters per group. Fetal blood (pooled from
two to three fetuses) was obtained after axillary artery incision of fetuses
while still connected to the maternal circulation. Plasma or serum were
separated by centrifugation and stored frozen at ⫺20 C until analyzed.
Pancreases (pooled from five fetuses) and a piece of liver (from each
fetus) were rapidly excised, frozen in liquid nitrogen, and then stored
at ⫺70 C until RNA preparation. Isolated islets from fetal pancreases
were obtained from seven to nine different dams from each group,
frozen in liquid nitrogen, and stored at ⫺70 C until IGF-IR protein
determination. In this study the sex of the fetuses was not considered.
All studies were conducted according to the principles and proce-
dures outlined in the National Institutes of Health Guidelines for Care
and Use of Experimental Animals.
Determination of plasma insulin, glucose, and GH levels
Plasma insulin was determined with a rat insulin RIA (LINCO Re-
search, Inc., St. Louis, MO) with rat insulin used for the standard curve.
Sensitivity of 0.1 ng/ml was achieved with overnight equilibrium using
a 100-
l serum sample. The coefficients of variation within and between
assays were 10%. Aliquots of 10
l obtained from 30
l Ba (OH)
2
-ZnSO
4
deproteinized blood were used to determine glucose by a glucose ox-
idase method (Boehringer-Mannheim, Mannheim, Germany). GH was
determined in the plasma of fetuses with a rat GH
125
I assay system
(Biotrak; Amersham Life Science, Amersham, UK). The RIA was carried
out according to the kit protocol. The sensitivity of the assay was 1.6
ng/ml. The intra- and interassay variations were 3.0 and 10.5.%,
respectively.
Determination of serum IGF-I and -II
IGF-I in serum was measured by enzyme immunoassay using a rat
IGF-I enzyme immunoassay kit (Diagnostic Systems Laboratoires, Web-
ster, TX). The method incorporates a sample pretreatment to avoid
interference from IGFBPs. The intra- and interassay variations were 6.5
and 9.4%, respectively. For measurement of serum IGF-II, recombinant
human IGF-II was labeled by a modified chloramine T method (24). The
specific activity achieved was 90 –175
Ci/
g. Before IGF-II determi-
nation, serum IGFBPs were removed by standard acid gel filtration. This
method has proved to be the most reliable one for use with rat serum
in developing stages (24, 25). The rat liver membrane receptor assay for
IGF-II was carried out as previously described (24). The coefficients of
variation within and between assays were 8.4 and 9.9%. Recombinant
human IGF-II (R&D Systems, Abingdon, UK) was used for iodination.
Western immunoblotting and determination of serum
IGFBP-1 and -2
Western immunoblots for enhanced chemiluminescence were per-
formed in polyvinylidene fluoride (PVDF) Immobilon-P membranes
(Millipore, Madrid, Spain). PVDF membranes were blocked with 5%
(wt/vol) nonfat dry milk for 60 min in Tris-buffered saline [TBS; 0.01
mol/liter Tris and NaCl 0.15 mol/liter (pH 8)] with 0.05% Tween 20.
Membranes were then incubated with a 1:100 dilution (as suggested by
the manufacturer) of affinity-purified goat polyclonal antirat IGFBP-1 or
rat IGFBP-2 from Santa Cruz Biotechnology (Palo Alto, CA). In the same
buffer (TBS-Tween 20 plus 5% nonfat dry milk) at 4 C overnight, after
which the membrane was washed three times for 10 min in TBS-Tween
20. After a 1-h incubation at room temperature with a 1:1000 dilution of
antigoat Ig G-horseradish peroxidase in TBS-Tween 20 plus 5% nonfat
dry milk, the membrane was washed three times with TBS-Tween 20 and
finally once with TBS alone. Antigen-antibody complexes were detected
after an enhanced chemiluminescence (hyperfilm enhanced chemilu-
minescence; Amersham, Madrid, Spain).
Preparation of total RNA
Total RNA was isolated from fetal pancreases and livers with TRIzol
reagent according to the manufacturer’s instructions (Invitrogen Life
Technologies, Carlsbad, CA). RNA concentration was determined by
absorbance at 260 nm. Samples were electrophoresed through 1.1%
agarose and 2.2 mol/liter formaldehyde gels and then stained with
ethidium bromide to render the 28S and 18S ribosomal RNA visible and
thereby confirm the integrity of the RNA and normalize the quantity of
RNA in the different lanes. A pT7 RNA 18S antisense (Ambion, Austin,
TX) was used for lane loading control.
Riboprobes
Rat IGF-I and -II and IGFBP-1 and -2 cDNAs were kindly provided
by Drs. C. T. Roberts Jr. and D. LeRoith (National Institutes of Health,
Bethesda, MD). Rat IGF-I cDNA ligated into a pGEM-3 plasmid (Pro-
mega Biotech, Madison, WI) was linearized with HindIII, and an anti-
sense riboprobe was produced by T7 RNA polymerase. The size of the
protected fragment represented in the figures (IGF-Ib) was 386 bp. Rat
IGF-II cDNA ligated into a pGEM-3 plasmid was linearized with HindIII
and incubated with T7 RNA polymerase to generate a riboprobe that
recognized a fragment of 700 bp. Rat IGFBP-1 cDNA, ligated into a
pGEM-3 plasmid, was linearized with HindIII and incubated with T7
RNA polymerase to generate an antisense riboprobe that recognizes two
fragments of 300 and 700 bases. Rat IGFBP-2 cDNA, ligated into a
pGEM-4Z plasmid (Promega), was linearized with HindIII and incu-
bated with SP6 RNA polymerase to generate a 550-base antisense ribo-
probe devoid of pGEM-4Z complementary sequences. pT7 RNA 18S was
incubated with T7 RNA polymerase to produce a 109-nucleotide runoff
transcript, 80 nucleotides of which are complementary to human 18S
ribosomal RNA. (
32
P)-uridine 5-triphosphate was purchased from ICN
(Nuclear Iberica, Madrid, Spain). The Riboprobe Gemini II core system
(Promega) was used for the generation of RNA probes.
Solution hybridization/RNase protection assay
Solution hybridization/RNase protection assays were performed as
previously described (18, 19). Autoradiography was performed at ⫺70
Martı´n et al. • Undernutrition Affects IGF System in Fetal Rat Endocrinology, March 2005, 146(3):1364–1371 1365
C against a Hyperfilm MP film between intensifying screens. Bands
representing protected probe fragments were quantified using a scan-
ning densitometer (Molecular Dynamics, Sunnyvale, CA) and accom-
panying software. RNase-A and -T1 were purchased from Roche diag-
nostics (Barcelona, Spain).
Fetal rat islet preparation and islet culture with IGFs
Fetal islets from undernourished and control rats were prepared
according to Hellerstro¨m et al. (26) as previously described (27). At the
end of the 6-d culture period, 40 fetal islets in each group were collected
under a stereomicroscope and further cultured for2dinRPMI 1640
medium (BioWhittaker, Verviers, Belgium) supplemented with 2
mmol/liter glutamine (BioWhittaker), 1% heat-inactivated fetal bovine
serum (BioWhittaker), and 100 ng/ml IGF-I (R&D Systems) or 100
ng/ml IGF-II (R&D Systems). The culture dishes were kept at 37 C in
a humidified atmosphere of 5% CO
2
in air. The complete culture me
-
dium was changed every other day.
Determination of IGF-IR
The islet content of IGF-IR was analyzed by Western blot. Protein
extracts were obtained from islets cultured for 6 d sonicated in a ho-
mogenization buffer [10
m leupeptin, 2 mm O-vanadate, 2 mm ben-
zamidine, 10
m aprotinin, and 2 mm phenylmethylsulfonyl fluoride in
12.5 mm EGTA, 1.25 mm EDTA, and 0.25% Triton X-100 (pH 7.6)]. Equal
amounts of protein (70
g) were separated on a 10% sodium dodecyl
sulfate-polyacrylamide gel. Proteins were then electrophoretically trans-
ferred to PVDF filters and probed with the antibodies against the IGF-IR

-subunit sc-713 (Santa Cruz Biotechnology). The rest of the Western
blot procedure was as described for IGFBP determinations, using a 1:500
dilution of anti-IGF-IR antibody.

-Cell replication
To measure

-cell replication in isolated fetal islets, 5⬘-bromo-2⬘-
deoxyuridine (BrdU) (Amersham International) was incorporated in
newly synthesized DNA and therefore labeled replicating cells. In each
group of fetal islets, 1 h before the end of islet cultures, BrdU was added
at 100
mol/liter final concentration. Thereafter, islets were collected
under stereomicroscope, fixed, and then processed for serial sections as
previously described (27). Islet sections were doubled stained for BrdU,
using a cell proliferation kit (Amersham International) and insulin.
Sections were incubated with a mouse monoclonal antibody anti-BrdU
diluted in a nuclease solution (according to the kit protocol) for1hat
room temperature and washed with Tris 0.05 mol/liter (pH 7.6). There-
after they were incubated with an affinity-purified peroxidase anti-
mouse IgG and stained with 3,3⬘-diaminobenzidine-tetra-hydrochloride
using a peroxidase substrate kit. Sections were then incubated with
guinea pig antiinsulin antibody for1hasdescribed above and then with
alkaline phosphatase-conjugated goat antiguinea pig IgG for 45 min
(Dako, Trappes, France). The activity of the antibody-alkaline phospha-
tase complex was revealed with an alkaline phosphatase substrate kit
(Valbiotech, Paris, France). Sections were mounted in Eukitt (Labonord,
Templemars, France). On these sections,

-cells showed red cytosol, and
BrdU-positive

-cells appeared with brown nuclei. A mean of 250

-cells
were counted per islet at a final magnification of ⫻1000. The proportion
of BrdU-positive

-cell nuclei to total

-cell nuclei was calculated. The
result represents the percentage

-cell replicative rate in a 1-h interval
(BrdU labeling index of

-cells).
Statistical analysis
All data are presented as means ⫾ se. The difference between two
mean values was assessed using Student’s unpaired t test. For multiple
comparisons, significance was evaluated by ANOVA, followed by the
protected least significant difference test. P ⬍ 0.05 was considered sta-
tistically significant.
Results
Biological characteristics of undernourished and control
fetuses at 21 dpc
Food restriction of pregnant rats during the third week of
gestation provoked a significant decrease in body weight in
their fetuses, compared with those in controls (Table 1). U
pancreases and U livers used for RNase protection assay
(determination of IGF and IGFBP mRNA expression)
showed a significant lower weight than control pancreases
and livers, respectively. In contrast, the weights of pancre-
ases and livers relative to their body weight were not dif-
ferent between the two groups of fetuses (milligrams per
gram) (3.91 ⫾ 0.02 in C vs. 3.89 ⫾ 0.05 in U and 55.7 ⫾ 0.4
FIG. 1. A, Serum concentrations of
IGF-I and -II in C and U fetuses at 21.5
dpc. B, Serum IGFBP-1 and -2 levels in
C and U fetuses at 21.5 dpc. Left panel,
Representative Western immunoblot of
IGFBP-1 and -2 in C and U fetuses.
Right panel, Densitometric measure-
ments of bands from Western immuno-
blot are expressed as percent of the cor-
responding control values. White bars,
control fetuses; black bars, undernour-
ished fetuses. Values are means ⫾ SE for
11–12 observations in each group. Fe-
tuses were obtained from five to seven
different litters. *, P ⬍ 0.05.
TABLE 1. Biological characteristics of fetuses at 21 d gestation from control (C) or undernourished (U) mothers
Body weight
(g)
Pancreas weight
(mg)
Liver weight
(mg)
Plasma glucose
(mg/100 ml)
Plasma insulin
(
U/ml)
Plasma GH
(ng/ml)
C 4.9 ⫾ 0.3 20.1 ⫾ 0.4 268.3 ⫾ 22.4 50.9 ⫾ 2.8 166.0 ⫾ 10.9 92.2 ⫾ 5.5
U 4.1 ⫾ 0.2
a
17.5 ⫾ 0.5
a
231.6 ⫾ 21.7 51.5 ⫾ 1.9 209.4 ⫾ 11.5
a
99.2 ⫾ 5.3
Data are means ⫾ SE for 14–20 observations in each group. Fetuses were obtained from four to seven different litters.
a
P ⬍ 0.05 relative to control fetuses.
1366 Endocrinology, March 2005, 146(3):1364 –1371 Martı´n et al. • Undernutrition Affects IGF System in Fetal Rat
in C vs. 56.1 ⫾ 0.5 in U, respectively). No change in glycemia
was found in undernourished fetuses, but a significant in-
crease in plasma insulin was observed in this group, com-
pared with controls. Plasma GH concentration was similar in
the two groups of fetuses.
Serum levels of IGFs and IGFBPs in undernourished and
control fetuses
Serum levels of IGF-II in control fetuses at 21 dpc were
higher (P ⬍ 0.05) than those of IGF-I in the same rats (128 ⫾
11 ng/ml, n ⫽ 11, vs. 72.7 ⫾ 2.9 ng/ml, n ⫽ 12) (Fig. 1). Similar
serum IGF-II levels were observed in U and C fetuses at 21
dpc. However, serum IGF-I levels in U fetuses were reduced,
compared with control levels. Serum IGFBP levels were ex-
pressed as percent of corresponding control fetuses. Serum
IGFBP-2 levels in U fetuses were significantly increased,
compared with control fetuses. No change in serum IGFBP-1
was observed between the two groups of fetuses.
Liver IGF and IGFBP mRNA expression in undernourished
and control fetuses
Densitometric measurements of protected probe frag-
ments are expressed as percent of the corresponding control
fetuses (Fig. 2). Liver IGF-I mRNA expression in U fetuses
was significantly decreased as compared with control fe-
tuses, but no change in liver IGF-II expression was observed
between the two groups of fetuses. Finally, liver IGFBP-1 and
-2 mRNA expression were similar in U and control fetuses.
Pancreas IGF and IGFBP mRNA expression in
undernourished and control fetuses
Densitometric measurements of protected probe frag-
ments are expressed as percent of the corresponding control
fetuses (Fig. 3). Pancreas IGF-II mRNA expression in U fe-
tuses was significantly decreased, compared with control
fetuses, whereas IGF-I mRNA expression was significantly
increased in pancreas. IGFBP-1 mRNA expression was sim-
ilar in the two groups of fetuses, but IGFBP-2 mRNA ex-
FIG. 2. A, RNase protection assay of liver IGF-I and -II mRNA tran-
scripts in C and U fetuses at 21.5 dpc. Densitometric measurements
of protected probe fragments are expressed as percent of the corre-
sponding C fetuses. B, RNase protection assay of liver IGFBP-1 and
-2 mRNA transcripts in control and U fetuses at 21.5 dpc. Densito-
metric measurements of protected probe fragments are expressed as
percent of the corresponding C fetuses. 18S ribosomal antisense as-
sayed in the same samples is shown beneath the IGF and IGFBP
bands; ⫹ and ⫺ designate riboprobe lanes treated with or without
RNases, respectively. Representative experiments are shown in the
figure. White bars, C fetuses; black bars, U fetuses. Values are
means ⫾ SE for five to eight observations in each group. Fetuses were
obtained from four to six different litters. *, P ⬍ 0.05.
FIG. 3. A, RNase protection assay of pancreas IGF-I and -II mRNA
transcripts in C and U fetuses at 21.5 dpc. Densitometric measure-
ments of protected probe fragments are expressed as percent of the
corresponding C fetuses. B, RNase protection assay of pancreas
IGFBP-1 and -2 mRNA transcripts in C and U fetuses at 21.5 dpc.
Densitometric measurements of protected probe fragments are ex-
pressed as percent of the corresponding C fetuses. 18S ribosomal
antisense assayed in the same samples is shown beneath the IGF and
IGFBP bands; ⫹ and ⫺ designate riboprobe lanes treated with or
without RNases, respectively. Representative experiments are shown
in the figure. White bars, C fetuses; black bars, U fetuses. Values are
means ⫾ SE for six to eight observations in each group. Fetuses were
obtained from six to eight different litters. *, P ⬍ 0.05.
Martı´n et al. • Undernutrition Affects IGF System in Fetal Rat Endocrinology, March 2005, 146(3):1364–1371 1367
pression was significantly increased in U, compared with
control fetuses.
Islet content of IGF-IR
The abundance of IGF-IR protein was evaluated in islets
from control and undernourished fetal rats. As shown in Fig.
4, islet IGF-IR content was significantly increased (40%) in U
fetuses, compared with control fetuses.
In vitro mitogenic effect of IGFs in isolated fetal islets
The number of

-cells per isolated fetal islet from U rats
was similar to that in control rats (361 ⫾ 39

-cell/islet, n ⫽
15, vs. 335 ⫾ 24

-cell/islet, n ⫽ 14) (Fig. 5). BrdU labeling
index of

-cells in absence of IGF was similar in control and
U-isolated fetal islets (0.97 ⫾ 0.11%, n ⫽ 14, and 1.21 ⫾ 0.13%,
n ⫽ 12, respectively). Addition of IGF-I or -II to the control-
isolated fetal islets significantly increased (1.61 ⫾ 0.10%, n ⫽
12, and 1.67 ⫾ 0.12%, n ⫽ 12, respectively) the

-cell repli-
cation above the basal values (without IGF). In control islets,
no difference was observed between the in vitro mitogenic
effect of IGF-I or -II. Similarly, addition of IGF-I or -II to the
U-isolated fetal islets significantly enhanced (2.68 ⫾ 0.14%,
n ⫽ 12, and 3.00 ⫾ 0.22%, n ⫽ 12) the

-cell replication above
the basal values (without IGF), but in this condition the
maximal

-cell mitogenic response to IGFs in vitro was sig-
nificantly more elevated in U, compared with the response
in control islets. Moreover, no difference was observed be-
tween the in vitro mitogenic effect of IGF-I and -II in U islets.
Discussion
In a previous work, we demonstrated that a 65% protein-
caloric food restriction during the last trimester of gestation
led to an increase in

-cell mass and hyperinsulinemia in the
fetuses at 21.5 d of gestation (2). These results differ from
those described in fetuses from pregnant rats submitted to a
low-protein food intake (3, 28) as well as from those sub-
mitted to a hypocaloric food restriction less severe than 65%
of the diet (4). However, in accordance with the literature (4,
29), from4doflife, we observed decreased

-cell mass and
hypoinsulinemia, which persisted until adult age (6). Be-
cause in our model of general food restriction the

-cell mass
is increased in undernourished fetuses, the aim of this work
was to investigate whether availability of growth factors
such as insulin, GH, and IGFs and their IGFBPs could be
implicated in this alteration. In this study we found that
maternal undernutrition increased both pancreatic IGF-I
mRNA expression and islet IGF-IR protein content in U
fetuses and enhanced replicative

-cell response to IGFs in
isolated undernourished fetal islets. IGFs are locally pro-
duced by pancreas, in which they act in an autocrine or
paracrine manner and are involved in the regulation of islet
growth and differentiation (9). Thus, increased expression of
IGF-I in pancreas and IGF-IR protein content in islets could
play a role in the increased

-cell mass in U fetuses.
The IGF axis is highly responsive to nutritional status (23).
Most studies on nutritional regulation of IGF-I have focused
on the liver, and all such studies, including the relatively few
that investigated nonhepatic tissues, have shown that un-
dernutrition decreased IGF-I mRNA expression and protein
abundance in the neonatal and adult period (23, 30) as well
as in the fetal period (19, 31). The decrease of hepatic IGF-I
mRNA expression observed in U fetuses is in accordance
with the above-mentioned studies. In addition, in U fetuses
serum IGF-I levels are reduced, probably the result of the
decreased liver IGF-I mRNA expression. This is in agreement
with previous studies in which nutrient restriction reduced
the circulating levels of IGF-I (19, 31, 32). Furthermore, in U
fetuses the reduced IGF-I serum levels is GH independent
because serum concentration of GH is normal. By contrast,
we observed that serum IGF-II and liver IGF-II mRNA ex-
pression were both unaffected by general food restriction.
This is also in agreement with previous reports in which the
concentration of circulating IGF-II as well as its mRNA abun-
dance appeared reduced or unaffected by maternal malnu-
trition (19, 24, 32). These data along with other findings (33)
indicated that IGF-I is more affected by changes in maternal
nutrition than IGF-II, irrespective of the cause or nature of the
nutrient deficit.
In the present study, we found that pancreatic IGF-I ex-
pression is increased in U fetuses, and it is known that IGF-I
is produced by fetal and neonatal rat pancreatic islets (34).
Therefore, the elevated IGF-I expression in pancreas of U
fetuses could be the result of the increased

-cell mass ob-
served in these fetuses at this stage (2). However, in this work
we also observed that pancreatic IGF-II expression is reduced
in U fetuses. Thus, the increased pancreatic IGF-I expression
in U fetuses cannot be attributed solely to the increased

-cell
mass observed at this stage. This pattern of reduced IGF-II
FIG. 4. Islet content of IGF-IR protein in C and U fetuses at 21.5 dpc.
Results show a representative Western blot in which each lane con-
tained 70
g of protein extracted from isolated islet. Densitometric
measurements of bands from Western immunoblot are expressed as
percent of the corresponding control values. White bars, C fetuses;
black bars, U fetuses. Values are means ⫾ SE. Fetal islets were ob-
tained from seven to nine independent islet cultures. *, P ⬍ 0.05.
FIG. 5. BrdU labeling index of

-cells in isolated fetal islets from C
and U rats. Isolated fetal islets obtained after6dofculture were
further cultured for 2 d without (white bars) or with 100 ng/ml IGF-I
(striped bars) or 100 ng/ml IGF-II (black bars). Values are means ⫾
SE. BrdU labeling index was determined in each condition in 12–17
isolated fetal islets. Fetal islets were obtained from three to seven
independent islet cultures.
a
, P ⬍ 0.05 relative to fetal C and U islets
without IGFs;
b
, P ⬍ 0.05 relative to fetal control islets.
1368 Endocrinology, March 2005, 146(3):1364 –1371 Martı´n et al. • Undernutrition Affects IGF System in Fetal Rat
expression and increased

-cell mass differs from observa-
tions in fetuses from maternal protein restriction (35) or in
fetuses from Goto-Kakizaki rats, which spontaneously de-
velop type 2 diabetes without obesity (36). It seems that the
influence of maternal undernutrition in our conditions is
markedly different in the pancreas from liver, in which nu-
tritional deficiency decreases IGF-I expression. Thus, the ef-
fect of maternal undernutrition on the fetal IGF-I expression
may be tissue specific. Further investigation is necessary to
understand how nutritional regulation of IGF-I expression
differs between the liver and the developing pancreas.
There are few studies about the influence of nutritional
restriction on pancreatic IGF-I mRNA expression. Consistent
with our observation of increased pancreas IGF-I in U fe-
tuses, Calikoglu et al. (37) reported that undernutrition in-
creased brain IGF-I mRNA expression in mice during brain
development and that local expression of IGF-I may serve
partly to protect the brain from the nutritional insult. Ac-
cordingly, the local expression of IGF-I may protect the en-
docrine pancreas in U fetuses from deleterious effects of
maternal undernutrition during fetal period. Our result is
also consistent with findings that refer to the protective ef-
fects of IGF-I against cytokine-mediated

-cell death in vitro
(38, 39) or against the oxidative and apoptotic effects of
streptozotocin in vivo (40).
The actions of IGF-I are predominantly local during fetal
and early postnatal life (41). Thus, the locally expressed IGF-I
in U pancreases may stimulate

-cell mass growth in an
autocrine/paracrine manner. This idea is consistent with the
induction of

-cell replication by IGF-I treatment in vitro (42)
and the in vivo observations that signaling through IGF-IR
promotes

-cell development and proliferation (43). More-
over IGF-I is an effective stimulus for inducing differentiated
pancreatic

-cell growth (44). The mitogenic signaling is me-
diated by the IGF-IR present on pancreatic islet cells (44, 45)
and requires the recruitment of phosphatidylinositol 3-
kinase and growth factor binding protein 2 to insulin recep-
tor substrate-2, resulting in the activation of MAPK and
P70
s6k
. The present study shows that maternal undernutri
-
tion increased a 40% the islet content of IGF-IR in U fetuses,
compared with controls, and this may favor the mitogenic
action of locally expressed pancreatic IGF-I in U fetuses.
Thus, autocrine or paracrine interaction of IGF-I with IGF-IRs
in islets, and activation of IGF-I signaling pathway would
contribute to increase the

-cell mass in U fetuses. In this line
we have seen in our laboratory (Martı´n, M. A., E. Ferna´ndez,
F. Escriva´, and C. A
´
lvarez, unpublished data) that under-
nutrition evokes a higher phosphorylation of P70
s6k
.
Unlike the mitogenic effect of IGF-I on other mammalian
cells (46), in the pancreatic

-cell, an IGF-I-induced mitogenic
response is glucose dependent (42). Glucose itself can stim-
ulate

-cell mitogenesis in a manner dependent on glucose
metabolism (42, 44). In accordance with this, in our model of
maternal undernutrition, glucose oxidation in the

-cell is
increased in U fetuses, compared with control fetuses (47).
This is of particular importance because in pancreatic

-cells
glucose provides a permissive environment for IGF-I-
induced

-cell proliferation (42, 44) and may favor the mi-
togenic effect of locally expressed IGF-I in U fetuses. In ad-
dition, in our model of maternal undernutrition, fetal plasma
insulin is significantly increased in U fetuses, compared with
C fetuses, and islet insulin content and abundance of insulin
mRNA in the pancreas are increased and more insulin is
secreted in response to secretagogues (2, 47). The increased

-cell mass probably plays a relevant role in these effects.
These observations suggest that local IGF-I mRNA expres-
sion in the pancreas might lead to increased

-cell mass and
hyperinsulinemia. Furthermore, insulin as well as IGF-I and
-II also contribute to the regulation of

-cell growth, function,
and survival (9). It is possible that increased plasma insulin
levels, acting via insulin receptor or IGF-IR, could also con-
tribute to increased

-cell mass in U fetuses. Thus, a coop-
erative action between insulin and IGF-I leading to increased

-cell mass may have developed in U fetuses.
It is worth noting that, in other studies, maternal food
restriction (50%) increased fetal corticosterone levels and
decreased fetal pancreatic insulin and

-cell mass, suggest-
ing a negative role of glucocorticoids in fetal

-cell devel-
opment (48). Although the glucocorticoid status has not been
assessed in this study and a rise of glucocorticoids in U
fetuses cannot be ruled out, increase of both insulin levels
and pancreatic IGF-I mRNA expression at 21.5 dpc could
counteract the effect of high glucocorticoid levels on

-cell
mass. However, it cannot be excluded that a possible increase
of glucocorticoids in our maternal model of malnutrition
may affect the fetal programing of intrauterine development
inducing a predisposition to later dysfunctions and diseases
such as coronary heart disease and type 2 diabetes.
In view of the reported ability of IGFBPs to modulate IGF
bioactivity, we examined serum and tissue expression of
IGFBP-1 and -2 in U fetuses. IGFBP-1 can either inhibit or
potentiate the actions of IGF-I (12). In the present study, we
report normal serum concentration and liver and pancreatic
gene expression of IGFBP-1 in U fetuses. This result agrees
with a previous study by Muaku et al. (32), using protein
restriction. Instead, an increase in fetal serum IGFBP-1 and
liver IGFBP-1 mRNA levels has been reported in growth-
retarded fetuses after maternal fasting (20), maternal protein
malnutrition (31), caloric restriction (49), or fetal growth re-
tardation induced by dexamethasone (50). Interestingly,
plasma insulin was found reduced in these animal models.
Insulin appears to play a major role in regulating IGFBP-1
gene transcription, i.e. IGFBP-1 transcription is high in dia-
betic animals and rapidly reduced to normal values after
insulin treatment both in neonatal (51) and adult (52, 53) rats.
In our model of maternal undernutrition, insulin is increased
in the plasma of U fetuses (2). Thus, the hyperinsulinemic
status of U fetuses could counteract the IGFBP-1-reducing
effects of undernutrition and/or increased glucocorticoids, if
they were, and might help to normalize the IGFBP-1 levels.
In the case of glucocorticoids, a dominant effect of insulin vs.
dexamethasone on the regulation of IGFBPs has been noted
in cultured hepatocytes (54).
Unaltered liver mRNA expression of IGFBP-2 found in U
fetuses is consistent with the few changes in liver IGFBP-2
mRNA observed in fetuses from experimental diabetic (24)
or undernourished mothers (19, 31). In contrast, increased
serum levels and pancreatic mRNA expression of IGFBP-2
were found in U fetuses. In general, IGFBP-2 appears to
inhibit IGF actions, in particular those of IGF-II, possibly
Martı´n et al. • Undernutrition Affects IGF System in Fetal Rat Endocrinology, March 2005, 146(3):1364–1371 1369
related to its higher affinity for this peptide (12). Other than
modulating IGF actions, IGFBPs may exert intrinsic bioac-
tivity in either the absence of IGFs or the presence of IGFs
without triggering IGF-IR signaling. In particular, IGFBP-2 is
mitogenic for uterine endometrial epithelial cells and osteo-
sarcoma cells independently of IGF action (55, 56). In addi-
tion, several mechanisms of IGFBP-2 interaction with cells
have been reported (18). The consequence of IGFBP-2 bind-
ing for cell function is still unknown, but it may serve to
concentrate IGFs near IGF-IRs because IGFBP-2 can increase
IGF-stimulated proliferation in some cell types (57, 58). Con-
sistent with these observations, it is possible that the in-
creased pancreatic mRNA expression of IGFBP-2 found in U
fetuses could locally contribute to the increase of

-cell mass
through IGF-independent effects and/or favoring the mito-
genic actions of locally produced IGF-I.
Finally, we tested the possibility that a direct biological
action of IGFs on fetal U

-cell was increased. Our in vitro
results show that IGF-I and -II stimulate the

-cell replication
in fetal control islets in accordance with a previous demon-
stration (40). But addition of IGF-I or -II to the U-isolated
islets significantly increased the

-cell replication, compared
with IGF-I- or IGF-II-exposed control fetal islets. These ef-
fects were obtained with a submaximal IGF-II concentration
and a maximal IGF-I concentration based on our evaluation
of circulating levels and in vitro data, respectively (42, 59). It
is well established that the mitogenic effects of IGFs are
mediated mainly through interactions with the IGF-IR (12).
In this study we show that U fetuses expressed more IGF-IR
protein in islets. Thus, this increase in the number of recep-
tors may favor or potentiate the mitogenic response to IGF-I
and -II in U islets.
In summary, the increased

-cell mass found in U fetuses
at 21.5 dpc could be the result of the stimulation of

-cell
replication due to locally increased IGF-I in the pancreas, and
this effect is perhaps potentiated or favored by the elevated
number of IGF-IR and/or the enhanced pancreatic IGFBP-2
gene expression. Therefore, our study suggests that local
expression of IGF-I and IGF-IR may serve in part to protect
the endocrine pancreas in U fetuses from the impact of ma-
ternal undernutrition during the fetal period. However, in-
creased

-cell mass and hyperinsulinemia at an early stage
could be an initial event for diabetes onset in adult age. In this
context, our model of maternal undernutrition provides an
opportunity to assess early and long-term effects under
physiological conditions.
Acknowledgments
The authors thank Susana Fajardo for her invaluable technical help.
Received May 25, 2004. Accepted November 19, 2004.
Address all correspondence and requests for reprints to: Dr. Carmen
Alvarez, Departamento de Bioquı´mica y Biologı´a Molecular, Facultad de
Farmacia, Universidad Complutense, Ciudad Universitaria, 28040 Ma-
drid, Spain. E-mail: calvarez@farm.ucm.es.
This work was supported by Grants BFI2001-2125 and BFI2002-00253
from Ministerio de Ciencia y Tecnologı´a, Spain, and the France/Spain
Program for Science (Cooperation Franco-Espagnole Centre National de
la Recherche Scientifique/Consejo Superior Investigaciones Cientı´ficas;
projects 5249 and 7963).
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