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Endocrinology, doi:10.1210/en.2004-0665
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Endocrinology Vol. 146, No. 3 1364-1371
Copyright © 2005 by The Endocrine Society

Protein-Caloric Food Restriction Affects Insulin-Like Growth Factor System in Fetal Wistar Rat

M. A Martín, P. Serradas, S. Ramos, E. Fernández, L. Goya, M. N. Gangnerau, M. Lacorne, A. M. Pascual-Leone, F. Escrivá, Bernard Portha and C. Álvarez

Instituto de Bioquímica, 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 Unité Mixte de Recherche 7059, Université Paris 7/D, Denis Diderot, 7525 Paris Cedex 05, France

Address all correspondence and requests for reprints to: Dr. Carmen Alvarez, Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad Complutense, Ciudad Universitaria, 28040 Madrid, Spain. E-mail: calvarez{at}farm.ucm.es.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 ß-cell mass. This alteration is correlated with increased insulinemia 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 ß-cell mitogenic response to IGFs in vitro. The results show that maternal undernutrition increases pancreatic IGF-I expression and islet IGF-I receptor content in undernourished fetuses, whereas hepatic IGF-I expression and serum IGF-I levels were decreased. No changes were observed in serum IGF-II, and its expression was diminished 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 concentration and abundance of IGFBP-2 mRNA in pancreas were increased. Finally, the ß-cell mitogenic response to IGFs in vitro was significantly increased in undernourished fetal islets, compared with controls. In conclusion, in undernourished fetuses the increased ß-cell mass can be related to the stimulation of replicative ß-cell response due to locally increased pancreatic IGF-I mRNA; this effect is perhaps potentiated or favored by the enhanced islet IGF-I receptor content and pancreatic IGFBP-2 gene expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
UNDERNUTRITION LEADS TO an impairment of glucose homeostasis, which in the mother has clear effects 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 ß-cell development and maturation of pancreatic function. Several studies in this area in experimental models with rats submitted to different patterns of malnutrition have reported that maternal undernutrition significantly affects the pancreatic insulin stores and the ß-cell mass in the fetuses (2, 3, 4) and offspring neonates at d 1 (5) and d 4 of postnatal 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 increased ß-cell mass. This alteration is correlated with increased 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 mothers (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 ß-cell development (11). IGF-I and -II are essential cell growth regulators, as demonstrated by null mutation experiments (12). IGFs are synthesized 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 effects 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 partially identical with insulin signaling, can be triggered by IGF-I, IGF-II, and supraphysiological concentrations of insulin (16). In vitro, both IGF-I and IGF-II enhance ß-cell replication, 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).

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 modulate IGFs biological actions (17). Apart from this modulation, IGFBPs, mostly produced in the liver (17), may exert intrinsic bioactivity in either the absence of IGFs (IGF-independent 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, 20, 21). During the fetal period, insulin also regulates growth, and IGF regulation is GH independent (9).

Because there is considerable evidence that endocrine factors such as insulin, GH, and IGFs contribute to ß-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 nutritional 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
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
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 established. 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) pregnant 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 pentobarbital 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 procedures 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 Research, 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-µl serum sample. The coefficients of variation within and between assays were 10%. Aliquots of 10 µl obtained from 30 µl Ba (OH)2-ZnSO4 deproteinized blood were used to determine glucose by a glucose oxidase method (Boehringer-Mannheim, Mannheim, Germany). GH was determined in the plasma of fetuses with a rat GH 125I 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, Webster, 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 µCi/µg. Before IGF-II determination, 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 performed 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 chemiluminescence; 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 (Promega Biotech, Madison, WI) was linearized with HindIII, and an antisense 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 incubated with SP6 RNA polymerase to generate a 550-base antisense riboprobe 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. (32P)-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 C against a Hyperfilm MP film between intensifying screens. Bands representing protected probe fragments were quantified using a scanning densitometer (Molecular Dynamics, Sunnyvale, CA) and accompanying software. RNase-A and -T1 were purchased from Roche diagnostics (Barcelona, Spain).

Fetal rat islet preparation and islet culture with IGFs
Fetal islets from undernourished and control rats were prepared according to Hellerström 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 for 2 d in RPMI 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% CO2 in air. The complete culture medium 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 homogenization buffer [10 µM leupeptin, 2 mM O-vanadate, 2 mM benzamidine, 10 µM 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 µg) were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel. Proteins were then electrophoretically transferred to PVDF filters and probed with the antibodies against the IGF-IR ß-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.

ß-Cell replication
To measure ß-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 µmol/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) for 1 h at room temperature and washed with Tris 0.05 mol/liter (pH 7.6). Thereafter they were incubated with an affinity-purified peroxidase antimouse IgG and stained with 3,3'-diaminobenzidine-tetra-hydrochloride using a peroxidase substrate kit. Sections were then incubated with guinea pig antiinsulin antibody for 1 h as described above and then with alkaline phosphatase-conjugated goat antiguinea pig IgG for 45 min (Dako, Trappes, France). The activity of the antibody-alkaline phosphatase complex was revealed with an alkaline phosphatase substrate kit (Valbiotech, Paris, France). Sections were mounted in Eukitt (Labonord, Templemars, France). On these sections, ß-cells showed red cytosol, and BrdU-positive ß-cells appeared with brown nuclei. A mean of 250 ß-cells were counted per islet at a final magnification of x1000. The proportion of BrdU-positive ß-cell nuclei to total ß-cell nuclei was calculated. The result represents the percentage ß-cell replicative rate in a 1-h interval (BrdU labeling index of ß-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 statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
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 1Go). 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 pancreases and livers relative to their body weight were not different 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 in C vs. 56.1 ± 0.5 in U, respectively). No change in glycemia was found in undernourished fetuses, but a significant increase in plasma insulin was observed in this group, compared with controls. Plasma GH concentration was similar in the two groups of fetuses.


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  		TABLE 1. Biological characteristics of fetuses at 21 d gestation from control (C) or undernourished (U) mothers

 
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. 1Go). 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 expressed 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.



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  		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 measurements of bands from Western immunoblot are expressed as percent of the corresponding control values. White bars, control fetuses; black bars, undernourished fetuses. Values are means ± SE for 11–12 observations in each group. Fetuses were obtained from five to seven different litters. *, P < 0.05.

 
Liver IGF and IGFBP mRNA expression in undernourished and control fetuses
Densitometric measurements of protected probe fragments are expressed as percent of the corresponding control fetuses (Fig. 2Go). Liver IGF-I mRNA expression in U fetuses was significantly decreased as compared with control fetuses, 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.



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  		FIG. 2. A, RNase protection assay of liver IGF-I and -II mRNA transcripts in C and U fetuses at 21.5 dpc. Densitometric measurements of protected probe fragments are expressed as percent of the corresponding C fetuses. B, RNase protection assay of liver IGFBP-1 and -2 mRNA transcripts in control and U fetuses at 21.5 dpc. Densitometric measurements of protected probe fragments are expressed 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 five to eight observations in each group. Fetuses were obtained from four to six different litters. *, P < 0.05.

 
Pancreas IGF and IGFBP mRNA expression in undernourished and control fetuses
Densitometric measurements of protected probe fragments are expressed as percent of the corresponding control fetuses (Fig. 3Go). Pancreas IGF-II mRNA expression in U fetuses was significantly decreased, compared with control fetuses, whereas IGF-I mRNA expression was significantly increased in pancreas. IGFBP-1 mRNA expression was similar in the two groups of fetuses, but IGFBP-2 mRNA expression was significantly increased in U, compared with control fetuses.



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  		FIG. 3. A, RNase protection assay of pancreas IGF-I and -II mRNA transcripts in C and U fetuses at 21.5 dpc. Densitometric measurements 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 expressed 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.

 
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. 4Go, islet IGF-IR content was significantly increased (40%) in U fetuses, compared with control fetuses.



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  		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 contained 70 µg 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 obtained from seven to nine independent islet cultures. *, P < 0.05.

 
In vitro mitogenic effect of IGFs in isolated fetal islets
The number of ß-cells per isolated fetal islet from U rats was similar to that in control rats (361 ± 39 ß-cell/islet, n = 15, vs. 335 ± 24 ß-cell/islet, n = 14) (Fig. 5Go). BrdU labeling index of ß-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 ß-cell replication 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 ß-cell replication above the basal values (without IGF), but in this condition the maximal ß-cell mitogenic response to IGFs in vitro was significantly more elevated in U, compared with the response in control islets. Moreover, no difference was observed between the in vitro mitogenic effect of IGF-I and -II in U islets.



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  		FIG. 5. BrdU labeling index of ß-cells in isolated fetal islets from C and U rats. Isolated fetal islets obtained after 6 d of culture 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.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In a previous work, we demonstrated that a 65% protein-caloric food restriction during the last trimester of gestation led to an increase in ß-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 submitted to a hypocaloric food restriction less severe than 65% of the diet (4). However, in accordance with the literature (4, 29), from 4 d of life, we observed decreased ß-cell mass and hypoinsulinemia, which persisted until adult age (6). Because in our model of general food restriction the ß-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 ß-cell response to IGFs in isolated undernourished fetal islets. IGFs are locally produced 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 ß-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 undernutrition 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 expression 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 abundance appeared reduced or unaffected by maternal malnutrition (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 expression 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 ß-cell mass observed 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 ß-cell mass observed at this stage. This pattern of reduced IGF-II expression and increased ß-cell mass differs from observations in fetuses from maternal protein restriction (35) or in fetuses from Goto-Kakizaki rats, which spontaneously develop 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 nutritional deficiency decreases IGF-I expression. Thus, the effect 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 fetuses, Calikoglu et al. (37) reported that undernutrition increased 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. Accordingly, the local expression of IGF-I may protect the endocrine 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 effects of IGF-I against cytokine-mediated ß-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 ß-cell mass growth in an autocrine/paracrine manner. This idea is consistent with the induction of ß-cell replication by IGF-I treatment in vitro (42) and the in vivo observations that signaling through IGF-IR promotes ß-cell development and proliferation (43). Moreover IGF-I is an effective stimulus for inducing differentiated pancreatic ß-cell growth (44). The mitogenic signaling is mediated 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 receptor substrate-2, resulting in the activation of MAPK and P70s6k. The present study shows that maternal undernutrition 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 ß-cell mass in U fetuses. In this line we have seen in our laboratory (Martín, M. A., E. Fernández, F. Escrivá, and C. Álvarez, unpublished data) that undernutrition evokes a higher phosphorylation of P70s6k.

Unlike the mitogenic effect of IGF-I on other mammalian cells (46), in the pancreatic ß-cell, an IGF-I-induced mitogenic response is glucose dependent (42). Glucose itself can stimulate ß-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 ß-cell is increased in U fetuses, compared with control fetuses (47). This is of particular importance because in pancreatic ß-cells glucose provides a permissive environment for IGF-I-induced ß-cell proliferation (42, 44) and may favor the mitogenic effect of locally expressed IGF-I in U fetuses. In addition, 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 ß-cell mass probably plays a relevant role in these effects. These observations suggest that local IGF-I mRNA expression in the pancreas might lead to increased ß-cell mass and hyperinsulinemia. Furthermore, insulin as well as IGF-I and -II also contribute to the regulation of ß-cell growth, function, and survival (9). It is possible that increased plasma insulin levels, acting via insulin receptor or IGF-IR, could also contribute to increased ß-cell mass in U fetuses. Thus, a cooperative action between insulin and IGF-I leading to increased ß-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 ß-cell mass, suggesting a negative role of glucocorticoids in fetal ß-cell development (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 ß-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 retardation 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 diabetic 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 related to its higher affinity for this peptide (12). Other than modulating IGF actions, IGFBPs may exert intrinsic bioactivity 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 osteosarcoma cells independently of IGF action (55, 56). In addition, several mechanisms of IGFBP-2 interaction with cells have been reported (18). The consequence of IGFBP-2 binding 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). Consistent with these observations, it is possible that the increased pancreatic mRNA expression of IGFBP-2 found in U fetuses could locally contribute to the increase of ß-cell mass through IGF-independent effects and/or favoring the mitogenic actions of locally produced IGF-I.

Finally, we tested the possibility that a direct biological action of IGFs on fetal U ß-cell was increased. Our in vitro results show that IGF-I and -II stimulate the ß-cell replication in fetal control islets in accordance with a previous demonstration (40). But addition of IGF-I or -II to the U-isolated islets significantly increased the ß-cell replication, compared with IGF-I- or IGF-II-exposed control fetal islets. These effects 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 receptors may favor or potentiate the mitogenic response to IGF-I and -II in U islets.

In summary, the increased ß-cell mass found in U fetuses at 21.5 dpc could be the result of the stimulation of ß-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 maternal undernutrition during the fetal period. However, increased ß-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.


   Footnotes
 
This work was supported by Grants BFI2001-2125 and BFI2002-00253 from Ministerio de Ciencia y Tecnología, Spain, and the France/Spain Program for Science (Cooperation Franco-Espagnole Centre National de la Recherche Scientifique/Consejo Superior Investigaciones Científicas; projects 5249 and 7963).

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.

Received May 25, 2004.

Accepted for publication November 19, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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