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Endocrinology, doi:10.1210/en.2007-0411
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Endocrinology Vol. 148, No. 9 4362-4370
Copyright © 2007 by The Endocrine Society

Early Pregnancy Maternal Endocrine Insulin-Like Growth Factor I Programs the Placenta for Increased Functional Capacity throughout Gestation

Amanda N. Sferruzzi-Perri, Julie A. Owens, Prue Standen, Robyn L. Taylor, Jeffrey S. Robinson and Claire T. Roberts

Research Centre for Reproductive Health, Discipline of Obstetrics and Gynaecology, School of Paediatrics and Reproductive Health, University of Adelaide, Adelaide, South Australia 5005, Australia

Address all correspondence and requests for reprints to: Claire T. Roberts, Research Centre for Reproductive Health, Discipline of Obstetrics and Gynaecology, School of Paediatrics and Reproductive Health, University of Adelaide, Adelaide, South Australia 5005, Australia. E-mail: claire.roberts{at}adelaide.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In early pregnancy, the concentrations of IGFs increase in maternal blood. Treatment of pregnant guinea pigs with IGFs in early to midpregnancy enhances placental glucose transport and fetal growth and viability near term. In the current study, we determined whether exogenous IGFs altered placental gene expression, transport, and nutrient partitioning during treatment, which may then persist. Guinea pigs were infused with IGF-I, IGF-II (both 1 mg/kg·d) or vehicle sc from d 20–35 of pregnancy and killed on d 35 (term is 70 d) after administration of [3H]methyl-D-glucose (MG) and [14C]amino-isobutyric acid (AIB). IGF-I increased placental and fetal weights (+15 and +17%, respectively) and MG and AIB uptake by the placenta (+42 and +68%, respectively) and fetus (+59 and +90%, respectively). IGF-I increased placental mRNA expression of the amino acid transporter gene Slc38a2 (+780%) and reduced that of Igf2 (–51%), without altering the glucose transporter Slc2a1 or Vegf and Igf1 genes. There were modest effects of IGF-I treatment on MG and AIB uptake by individual maternal tissues and no effect on plasma glucose, total amino acids, free fatty acids, triglycerides, and cholesterol concentrations. IGF-II treatment of the mother did not alter any maternal, fetal or placental parameter. In conclusion, exogenous IGF-I, but not IGF-II, in early pregnancy increases placental transport of MG and AIB, enhancing midgestational fetal nutrient uptake and growth. This suggests that early pregnancy rises in maternal circulating IGF-I play a major role in regulating placental growth and functional development and thus fetal growth throughout gestation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INTRAUTERINE GROWTH RESTRICTION (IUGR) is a major obstetric and neonatal condition that affects approximately 6% of births in developed nations (1) and up to 40% in developing countries (2, 3). IUGR is characterized by increased fetal and neonatal mortality and morbidity, neurological handicap in children (4, 5), and an increased risk of poor health in adult offspring, including the development of cardiovascular disease, diabetes, and obesity in later life (6). Many factors are implicated in the etiology of IUGR, but impaired transfer of nutrients and oxygen to the fetus and placental insufficiency are known to occur. Therefore, a better understanding of the regulation of nutrient partitioning and placental transport may provide insight into the pathophysiology of IUGR.

Fetal growth is dependent on the supply of nutrients and oxygen, which is determined by the mother’s ability to acquire these and direct them to the placenta and, in turn, its capacity to transfer these to the fetus. Maternal endocrine adaptation to pregnancy (7), coordinated in part by placental endocrine signals (8), is critical in ensuring adequate placental function and substrate partitioning between mother, placenta, and fetus. The IGFs may have a major role in placentation, because they are major determinants of fetal growth (9, 10, 11) and abundantly expressed by the mother and fetus in most species (12), with IGF-II also highly synthesized by the placenta (13). Furthermore, the concentrations of maternal circulating IGF-I and IGF-II increase during early pregnancy in several species (reviewed in Ref. 14), including humans (15). Because IGFs do not cross the placenta in physiologically significant quantities (16), this rise in maternal circulating IGFs during the first half of pregnancy may play a role in maternal adaptation to pregnancy and modulation of placental growth, functional development, and hence pregnancy outcome.

Certainly, we have recently shown that administration of IGF-I or -II to the mother during early to midgestation enhances late gestational fetal growth and survival near term in the guinea pig (17). Early pregnancy treatment with either IGF also increased placental glucose transport and the concentration of fetal circulating amino acids near term, without altering placental system A amino acid transport (17, 18), suggesting that other transporter systems may have been targeted by the exogenous IGFs. Furthermore, IGF-II, but not IGF-I, treatment in early pregnancy promoted development of the placental exchange region (labyrinth) in late gestation (17). These alterations in placental growth and function are consistent with enhanced placental substrate delivery (18) and suggest that IGF-II effects on fetal growth were likely to be secondary to impacts on placental development. In contrast, the mechanism by which maternal exogenous IGF-I enhanced fetal growth near term is less clear.

IGF-I administration to the mother reduced maternal adiposity in late gestation (17), suggesting repartitioning of nutrients to the conceptus, to promote fetal growth. Interestingly, however, both IGF treatments enhanced maternal tissue substrate uptake near term (18). Whether the effects of exogenous IGFs on the mother reflect altered maternal adaptation to the pregnant state or whether maternal substrate uptake was increased in response to greater growth of the conceptus to support it, remains to be determined. Perhaps the more important question is what effects are exogenous maternal IGFs exerting at the time of treatment in early pregnancy that may translate to the late gestational effects observed previously? Certainly, in a similar study, infusion of either IGF in guinea pigs from d 20–37 of pregnancy (term ~70 d) increased fetal and placental weights on d 40 of gestation (14).

Studies performed in vitro suggest that exogenous IGFs in early to midgestation may influence placental capacity to delivery substrate through effects on placental vascularity and/or the expression or activity of nutrient transporters. For instance, IGF-II promotes placental angiogenesis and vascular remodeling (19, 20) through induction of vascular endothelial growth factor (VEGF) expression (21, 22). Furthermore, exogenous IGFs stimulate placental trophoblast uptake of glucose and system A amino acids in vitro (23, 24, 25, 26). However, whether these effects are secondary to impacts on the expression of their transporter genes, Slc2 [encodes glucose transporters (GLUTs)] and Slc38 [encodes system A amino acid transporters (SNATs)], respectively, have yet to be determined.

The aims of this study were to investigate the effects of IGF-I or IGF-II supplementation of the mother from early to midpregnancy on placental uptake and transport of nonmetabolizable radioanalogs of glucose ([3H]methyl-D-glucose, MG) and amino acids ([14C]amino-isobutyric acid, AIB) and the partitioning of these substrates between the mother and conceptus in midgestation just before the cessation of treatment. We also determined the effect of such treatments on expression of the nutrient transporter genes Slc38a2 and Slc2a1 and Igf1, Igf2, and Vegf in the placenta.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
This study was approved by the University of Adelaide, Animal Ethics Committee. Pregnant female guinea pigs (IMVS colored strain, ~500 g, 3–4 months old) were housed individually in the University of Adelaide Medical School Animal House (12-h light, 12-h dark cycle) and provided with food and water ad libitum. Females were assigned to form three groups of similar mean weight at mating. On d 20 of pregnancy (d 1 is the day a copulatory plug was observed or the day after females were paired with a male), females were anesthetized with xylazine hydrochloride (4 mg/kg, im; Troy Laboratories, Sydney, Australia), ketamine hydrochloride (25 mg/kg, ip; Troy Laboratories), atropine sulfate (0.05 mg/kg, sc; Apex Laboratories, Sydney, Australia) and administered local analgesia with lignocaine hydrochloride (Troy Laboratories). A 200-µl mini osmotic pump (Alzet 2002; Alzet, San Francisco, CA) was surgically inserted beneath the skin on the back. Minipumps had previously been prepared to deliver vehicle (0.1 M acetic acid; n = 7) or 1 mg/kg·d IGF-I (n = 8) or IGF-II (n = 8) (human recombinant protein; GroPep Pty Ltd., Adelaide, Australia) for 18 d at a flow rate of 0.51 µl/h. This treatment was previously shown to increase the concentration of IGF-I and IGF-II in the maternal plasma at midpregnancy by 3.4-fold and 2.4-fold, respectively (17).

On d 35 of pregnancy (term ~70 d) after an overnight fast, mothers were anesthetized according to weight on d 20 of pregnancy, as described above. Dams were then administered a mixture of [3H]MG and methyl [14C]AIB (both from Amersham, Little Chalfont, UK) in physiological saline in a single bolus under anesthesia via cardiac puncture. To each dam, we aimed to administer 100 µCi/kg of MG and 10 µCi/kg of AIB but determined the precise dose actually administered by weighing syringes containing each isotope before and after the preparation of the isotopic mixture. The total administered volume of the isotope mixture was 500 µl for each animal. Maternal blood was collected in heparinized tubes 20 min after administration of radioactive analog administration, and animals were then killed by overdose of sodium pentobarbitone (Lethobarb; Virbac, Sydney, Australia). Viable and resorbing implantation sites were counted, and the uterus and its contents and viable fetuses and placentas were weighed. Maternal blood was centrifuged at 2500 rpm for 15 min at 4 C, and plasma was recovered and stored at –20 C. Fetuses and maternal kidneys, liver, spleen, heart, brain, lungs, triceps and gastrocnemius muscles, and retroperitoneal, perirenal, and interscapular adipose tissues were weighed and snap frozen in liquid nitrogen for determination of MG and AIB content. Two pieces of each placenta were snap frozen in liquid nitrogen immediately after dissection from the uterus for gene expression analyses and determination of MG and AIB content.

MG and AIB content in plasma and tissues
Standards were prepared from tritiated water (3H2O) and [14C]AIB (both from Amersham, UK), respectively, with variable quenching using carbon tetrachloride. The content [disintegrations per minute (DPM)] of MG and AIB in plasma and tissues were determined by dual isotope ß-scintillation counting (LS 6500 Beckman) at 0–400 and 400–670 MeV for 3H and 14C, respectively, as previously described (18).

Briefly, plasma (50 µl) was deproteinized with 100 µl of 0.3 N Ba(OH)2 (Sigma Diagnostics, St. Louis, MO) and 100 µl of 0.3 N ZnSO4 (Sigma Diagnostics) and added to scintillant (Ready Safe; Beckman Coulter, Fullerton, CA) for counting as described above.

Frozen whole fetuses were ground (using liquid nitrogen) into a homogeneous powder with a mortar and pestle. Fetal tissue homogenate and maternal tissues (~100 mg) were solubilized with 0.7 ml 1 M NaOH, and deproteinized sample was recovered after a protein precipitation with 2.1 ml 6% perchloric acid and mixed with scintillant (Ready Safe; Beckman Coulter) for counting as described above.

The sample DPM was corrected for background and then adjusted for the actual dose of isotope administered to the mother. DPM are represented as DPM per milliliter for plasma samples. For tissues, DPM are represented as DPM per gram or total tissue uptake by multiplying the DPM per gram by the weight of the tissue.

Quantification of gene expression
Total RNA was isolated from about 100 mg placental tissue using Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. Contaminating DNA was removed by DNase I treatment (DNA-free kit; Ambion, Austin, TX) following the manufacturer’s instructions. The quantity of RNA extracted was determined using a NanoDrop spectrophotometer (absorbance 260–280 nm, NanoDrop Technologies, Inc., Wilmington, DE). 2 µg of total RNA was reverse-transcribed to cDNA using Superscript III reverse transcriptase (Invitrogen) and random sequence oligohexamers (100 µg/ml; GeneWorks, Adelaide, Australia) for priming according to manufacturer’s instructions using a Geneamp PCR System 9700 thermocycler (Applied Biosystems, Foster City, CA). A nontemplate control was performed for every sample, establishing the absence of genomic contamination.

Random oligonucleotide primers were designed for Igf1, Igf2, and Vegf using published guinea pig sequences using Primer Express software (Applied Biosystems). The primer sequences were as follows: Igf1 forward 5'-gttcgtgtgcggagataggg-3' and reverse 5'-cggaaacagcactcgtcca-3', Igf2 forward 5'-gaccgcggcttctatttcag-3' and reverse 5'-cactcttcaacgatgccacg-3', and Vegf forward 5'-tcaccatgcagatcatgcg-3' and reverse 5'-cacatttgctgtgctggagg-3', designed from the following GenBank accession nos. X52951.1, S59899.1, and M84230.1, respectively.

Because there are no published sequences for the glucose and system A amino acid transporters in the guinea pig, the latter partly responsible for AIB transport, we designed and tested primers for other species (human, mouse, and sheep) specific for Slc2a1, Slc2a3, Slc2a8, Slc38a2, and Slc38a4 using Primer Express software (Applied Biosystems). Standard desalted primers were constructed by Sigma Genosys (Sigma Genosys, Sydney, Australia). Primers designed specific to mouse Slc2a1 and Slc38a2, which encode Glut-1 and Snat-2, respectively, and bovine 18s rRNA were found to generate products of predicted amplicon size and, when sequenced, generated sequences that were 92, 98, and 95% homologous to the published sequences, respectively. As a consequence, these primers were subsequently employed. The primer sequences were as follows: Slc2a1 forward 5'-ccagctgggaatcgtcgtt-3' and reverse 5'-atcatgggcaatgcagacttg-3', Slc38a2 forward 5'-gaagaccgaaatgggaaggtt-3' and reverse 5'-gttacagctccaacagtgacttcaa-3', and 18s forward 5'-agaacggctaccacatccaa-3' and reverse 5'-cctgtattgttatttttcgtcactacct-3', designed from the following GenBank accession nos. BC055340, BC041108, and DQ222453, respectively.

Real-time PCR was performed using a Rotor-Gene 6000 thermocycler (Corbett Life Sciences, Sydney, Australia) and SYBR Green I chemistry (Applied Biosystems) to detect synthesized products. Thermocycling parameters were set according to the manufacturer’s instructions. The mRNA expression levels for all genes were determined using the 2–{Delta}{Delta}CT method for quantitation (27, 28). We employed the 18s rRNA gene as the internal control to normalize each sample and then expressed it relative to the vehicle treatment group.

Determination of circulating metabolite concentrations
Maternal plasma glucose (Glucose HK assay kit; Roche Diagnostics, Mannheim, Germany), free fatty acids (WAKO Nefa C free fatty acid kit; NovoChem, Nieuwegein, The Netherlands), cholesterol (cholesterol CHOD-PAP assay kit; Roche), and triglycerides (triglycerides assay kit; Roche) were quantified with enzymatic assay kits using a COBAS Mira automated centrifugal analyzer (Roche Diagnostic Systems). Maternal plasma {alpha}-amino nitrogen concentrations were determined using the ß-naphthoquinone sulfonate colorimetric assay as previously described (17).

Statistics
All data were analyzed using SPSS version 13 (SPSS, Chicago, IL). To determine the effect of IGF treatment in early pregnancy on maternal parameters, general linear model univariate ANOVA with Bonferroni post hoc tests were performed. To assess the impact of IGF treatment on placental and fetal parameters, linear mixed model repeated-measures ANOVA with Sidak post hoc tests were performed using the mother as the subject and the fetus or placenta as the repeated measure. The number of viable pups per litter was used as a covariate when required. Using Pearson’s two-tailed bivariate correlation analyses, associations between MG and AIB uptake and maternal circulating metabolites were performed. Igf gene expression by the placenta was averaged per mother and then correlated with previously determined maternal plasma IGF concentrations (17) using Pearson’s two-tailed bivariate correlation analyses. Data are expressed as means ± SEM or estimated marginal means ± SEM as required. Data were considered statistically significant when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of maternal IGF infusion on maternal, fetal, and placental weights
Exogenous IGF-I infused into the mother increased the absolute weights of the maternal kidneys (+18%, P = 0.01), heart (+13%, P = 0.029), and biceps (+14%, P = 0.042), compared with vehicle, without an effect on maternal weight gain during the treatment. There was a trend for an increase in the absolute mass of the maternal spleen with IGF-I treatment compared with vehicle (+44%, P = 0.066). Litter size and composition were not affected by exogenous maternal IGFs (Fig. 1AGo). Maternal IGF-I treatment increased fetal weight by 15% compared with vehicle (Fig. 1BGo, P = 0.024), and there was an effect of treatment on placental weight (vehicle, 1.2 ± 0.05 g; IGF-I, 1.4 ± 0.05 g; IGF-II, 1.2 ± 0.05 g; P = 0.006, with IGF-I vs. vehicle, P = 0.06; and IGF-I vs. IGF-II, P = 0.008).


Figure 1
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  		FIG. 1. Effect of maternal IGF infusion on litter composition (A) and fetal weights (B). Data are expressed as mean DPM ± SEM (A) and estimated marginal mean DPM ± SEM, controlling for the number of viable pups per litter (B). Different superscripts denote statistically significant differences between groups as determined by a linear mixed-model repeated-measures with Sidak post hoc test, using the mother as the subject and the fetus as a repeated measure: a vs. b, P < 0.05. Vehicle data represent 23 placentas from seven dams; IGF-I, 29 placentas from eight dams; and IGF-II, 20 placentas from eight dams.

 
Effect of maternal IGF infusion on net placental uptake of MG and AIB
Exogenous IGF-I increased total placental MG uptake (+42%, P = 0.036) and AIB uptake per gram and total placenta (+51%, P = 0.003, and +68%, P = 0.005, respectively), compared with vehicle (Fig. 1Go). IGF-I also increased total AIB uptake compared with IGF-II (+61%, P = 0.014) (Fig. 2Go).


Figure 2
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  		FIG. 2. Effect of maternal IGF infusion on placental uptake of substrate. A and B, Placental uptake of [3H]MG (A) and [14C]AIB (B). Data are expressed as estimated marginal mean DPM ± SEM, controlling for the number of viable pups per litter. Different superscripts denote statistically significant differences between groups as determined by a linear mixed-model repeated-measures with Sidak post hoc test, using the mother as the subject and the fetus as a repeated measure: a vs. b, P < 0.05. Vehicle data represent 23 placentas from seven dams; IGF-I, 29 placentas from eight dams; and IGF-II, 20 placentas from eight dams.

 
Effect of maternal IGF infusion on fetal uptake of MG and AIB
IGF-I infusion into the mother increased per gram and total fetal MG content (+48%, P = 0.012, and +59%, P = 0.004, respectively) and per gram and total fetal AIB uptake (+68%, P = 0.005, and +90%, P = 0.004, respectively), compared with vehicle-treated mothers in midgestation (Fig. 3Go). There was no acute effect of exogenous maternal IGF-II on uptake of either radiolabeled substrate by the fetus.


Figure 3
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  		FIG. 3. Effect of maternal IGF infusion on fetal utilization of substrate. A and B, Fetal uptake of [3H]MG (A) and [14C]AIB (B). Data are expressed as estimated marginal mean DPM ± SEM, controlling for the number of viable pups per litter. Different superscripts denote statistically significant differences between groups as determined by a linear mixed-model repeated-measures with Sidak post hoc test, using the mother as the subject and the fetus as a repeated measure: a vs. b, P < 0.05. Vehicle data represent 23 placentas from seven dams; IGF-I, 29 placentas from eight dams; and IGF-II, 20 placentas from eight dams.

 
Effect of maternal IGF infusion on uptake of MG by the mother
Uptake of MG by individual maternal organs and tissues are shown in Table 1Go. There was no significant effect of maternal IGF treatment on the uptake of MG per gram of tissue (Table 1Go). However, IGF-I infusion into the mother increased total spleen uptake of MG compared with vehicle and IGF-II-treated mothers (+97%, P = 0.012, and +65%, P = 0.043, respectively).


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  		TABLE 1. Effect of maternal IGF infusion on MG uptake in the mother

 
Effect of maternal IGF infusion on uptake of AIB by the mother
Uptake of AIB by individual maternal organs and tissues are shown in Table 2Go. Exogenous IGF-I increased per gram and total AIB uptake by the maternal brain (+77%, P = 0.007, and +85%, P = 0.004, respectively) and spleen (+52%, P = 0.035, and +222%, P = 0.004, respectively) (Table 2Go). Uptake (DPM per gram) of AIB by the maternal triceps was increased by maternal IGF-I and IGF-II (+35 and +40%, both P = 0.078), and maternal IGF-I increased total uptake by maternal gastrocnemius (+29%, P = 0.07), but these failed to reach statistical significance.


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  		TABLE 2. Effect of maternal IGF infusion on AIB uptake in the mother

 
Effect of maternal IGF infusion on circulating MG and AIB in the mother
There was no acute effect of maternal IGF infusion on the circulating concentrations of MG or AIB in the mother (Fig. 4AGo).


Figure 4
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  		FIG. 4. Effect of maternal IGF infusion on infused substrate and metabolite concentrations in maternal plasma. A, Maternal plasma [3H]MG and [14C]AIB content 20 min after bolus injection; B, concentrations of amino acids (AA), cholesterol (Chol), free fatty acids (FFA), glucose (Gluc), and triglycerides (Trig) (B). Data are expressed as mean DPM ± SEM from seven to eight mothers per treatment.

 
Effect of maternal IGF infusion on circulating metabolites in the mother
Exogenous IGF did not alter maternal plasma concentrations of glucose, free fatty acids, amino acids, triglycerides, or cholesterol (Fig. 4BGo). The concentrations of metabolites in maternal blood did not correlate with maternal body composition or with fetal or placental weights (data not shown).

Effect of maternal IGF infusion on placental gene expression
To determine the effect of maternal IGF infusion on the placental gene expression, all genes were normalized to 18s rRNA and expressed as a proportion of the mean value for the vehicle treatment group (Fig. 5Go). Exogenous IGF-I or IGF-II did not alter placental expression of Slc2a1 (Fig. 5AGo). However, IGF-I treatment increased placental Slc38a2 expression by 7.8- and 7.1-fold compared with vehicle and IGF-II, respectively (both P = 0.027) (Fig. 5AGo). Placental Slc38a2 gene expression was correlated positively with fetal weight (r = 0.50; P = 0.001) and placental (r > 0.35; P < 0.03) and fetal (r > 0.39; P < 0.017) uptake of AIB (Table 3Go).


Figure 5
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  		FIG. 5. Effect of maternal IGF infusion on placental gene expression. Placental transcription of Slc2a1 and Slc38a2 (A), Igf1 and Igf2, and Vegf (B) were normalized to the internal control 18s rRNA and are expressed relative to the vehicle-treated group as mean ± SEM. Different superscripts denote statistically significant differences between groups as determined by a linear mixed-model repeated-measures with Sidak post hoc test, using the mother as the subject and the fetus as a repeated measure: a vs. b, P < 0.032. Data are from one to three placentas per mother, with seven to eight mothers per treatment.

 

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  		TABLE 3. The associations of placental Slc38a2 gene expression and fetal growth and AIB transport

 
IGF-I infusion into the mother reduced placental expression of Igf2 (–51%, P = 0.032). Placental Igf2 gene expression was positively correlated with previously determined maternal circulating IGF-II concentrations (17) (r = 0.32; P = 0.032), whereas there was no association of placental Igf1 transcription and maternal plasma IGF-I. Placental expression of Igf1 and Vegf transcription were unaltered by maternal IGF treatment (Fig. 5BGo).

Of all the genes surveyed, Igf2 and Vegf mRNA transcripts were most abundantly expressed in the placenta of vehicle-treated mothers, whereas that of Igf1 was least (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study shows for the first time that infusion of IGF-I into the mother from early pregnancy increases placental uptake and transfer of nutrients, in part by increasing placental transporter gene expression, enhancing placental and fetal growth by midpregnancy. By contrast, exogenous IGF-II did not affect midgestational placental transport and gene expression or partitioning of nutrients between the mother and fetus or placental and fetal weights. Exogenous IGF-I from early to midpregnancy in the mother has been previously shown to increase fetal growth and viability and placental substrate transfer after cessation of the treatment near term (17, 18). This study suggests that the rise in endogenous maternal IGF-I concentrations during early pregnancy may be critical in ensuring placental functional development throughout gestation.

System A transporters are responsible for the transfer of small, nonbranched amino acids such as AIB, proline, alanine, and glycine. Both IGFs are thought to regulate system A activity in vitro because IGF-I and IGF-II treatment of human placental trophoblasts stimulates amino acid uptake (23, 24, 25, 26). In the current study, treatment of the mother with IGF-I, but not IGF-II, from early to midpregnancy increased placental AIB uptake per gram and total placental weight and fetal AIB content, which suggests enhanced system A activity. Enhanced placental AIB transfer is expected to have contributed to the improved fetal growth, observed by midgestation, in the current study and near term (17) after infusion of IGF-I in early pregnancy. Certainly, functionality of placental system A amino acid transport is an important determinant of fetal growth, because system A activity is reduced in human pregnancies complicated by IUGR (29, 30), and inhibition of its activity in vivo results in IUGR in rats (31).

The system A amino acid transporters are encoded by the Slc38 gene family and give rise to three isoforms, SNAT1, SNAT2, and SNAT4, which are expressed by the placenta of humans (32, 33, 34, 35), rats (36), and mice (37), with SNAT-2 ubiquitously expressed (32). To our knowledge, no study has characterized or localized system A transporters in the guinea pig placenta. However, it has been reported that, like in humans (38, 39), system A activity is predominantly localized to the maternal-facing syncytial microvillous membrane of the guinea pig placenta (40, 41). This is thought to drive transfer of system A amino acids from mother to fetus. In the present investigation, primers specific for the murine Slc38a2 gene amplified a product in the guinea pig placenta that was the same molecular weight with 98% homology to that of the mouse. We also tested primers specific for the murine and human Slc38a4, but these did not generate a product, indicating that either the guinea pig placenta does not express this gene or the region to be amplified is not conserved between the two species. Nevertheless, exogenous IGF-I substantially increased placental transcription of Slc38a2 gene, by nearly 8-fold. Because a specific antibody against the product of Slc38a2, SNAT-2, is not commercially available for the guinea pig, it was not possible to localize or measure its abundance in the guinea pig placenta in response to maternal IGF treatment. Further investigation is needed to determine whether IGF-I infusion also resulted in increased SNAT-2 protein expression and translocation to the cell surface. This would be consistent with the observed increase in AIB uptake per gram of placenta in response to maternal IGF-I treatment and the positive correlation of placental expression of Slc38a2 with placental AIB transport in vivo reported herein. In accordance with our findings, it was recently demonstrated that restriction of protein intake by pregnant rats, which reduced maternal circulating IGF-I, decreased placental SNAT-2 protein expression and system A activity, resulting in IUGR (42).

In addition to effects on placental expression of the system A transporter gene, we cannot rule out an effect of IGF-I on the expression and activity of other transporter systems such as system L and ASC, which also transport AIB (43) and were not assessed in the present investigation. Interestingly, studies performed more recently in the BeWo choriocarcinoma cell line have demonstrated that IGF-I stimulation of AIB uptake is almost entirely dependant on system A transporter activity in vitro by little effect of inhibiting systems ASC and L on AIB transport (44). Certainly, employing methylated AIB, which is exclusively transported by system A, instead of AIB, would better clarify IGF regulation of this transporter system in vivo.

In addition to effects on amino acid transfer in the current study, infusion of IGF-I into the mother also increased placental uptake and supply of glucose to the fetus. This is consistent with IGF-I stimulation of human placental trophoblast glucose transporter activity in vitro (25) and our previous work demonstrating a sustained impact of maternal IGF treatment on placental uptake and transfer of glucose to the fetus in later gestation in the guinea pig (18). Glucose is the primary substrate for fetal oxidative metabolism and growth (8), and until late gestation, the fetus produces insignificant amounts (45). The fetus is, therefore, critically dependent on placental delivery of glucose from the mother (46). We speculate that enhanced midgestational placental glucose transport, like that of system A amino acids, is responsible for increased fetal growth observed in midgestation in the present study and in later gestation (17).

Uptake and transport of glucose across the placenta occurs via facilitated carrier-mediated diffusion that, in the human, mouse, rat, and ruminant placenta, primarily involves the glucose transporter protein (GLUT) isoforms-1 and -3 (47, 48, 49, 50, 51, 52, 53, 54). However, GLUT-4 and -12 placental expression have also been described in humans (55, 56) and GLUT-8 in sheep (57). Glucose transporters are transcribed from the Slc2 family of genes and are regulated by factors including glucose and glycolytic substrates (58), oxygen (59, 60), insulin, and IGF-I and -II (55, 61, 62). To our knowledge, characterization and localization of glucose transporters in the guinea pig placenta has yet to be performed. However, in the present study, primers designed for the murine Slc2a1 amplified a product from the guinea pig placenta that had 92% sequence identity with the murine amplicon. GLUT-1, encoded from Slc2a1, has been demonstrated to be the most abundantly expressed and primary mediator of placental glucose transfer in humans (63). Despite enhanced placental glucose transfer, infusion of IGF-I into the mother did not alter the transcription of Slc2a1. This is in contrast to findings in rats where exogenous maternal IGF-I increased placental glucose transporter mRNA expression of Slc2a1 and Slc2a3 (64) and the observation that GLUT-1 expression on the fetal-facing basal membrane of the placental barrier is positively regulated by IGF-I (46).

Alternatively, infusion of IGF-I into the mother may have increased GLUT-1 protein translation and and/or translocation to the cell surface, thus increasing placental supply of glucose to the fetus. Certainly, IGF-I induces GLUT-1 and -3 protein expression in vitro in bovine chromaffin cells (65) and equine articular chondrocytes (66). Exogenous IGF-I also promotes the translocation of GLUT-1, -3, and -4 from their intracellular storage sites to the cell surface in rat muscle cell lines, which correlate with increased glucose uptake and transfer (61, 67, 68). Future studies to determine the effect of IGF supplementation of the mother on placental GLUT expression and activity on the basal vs. microvillous trophoblast membranes would help to clarify the mechanisms by which IGF-I increases placental glucose transport. In addition, exogenous IGF-I may have increased the activity of the sodium-dependent active glucose transporters, encoded by the Slc5 gene family, which were recently identified in human (69) and rabbit placenta (70).

Placental vascularity is a major determinant of placental substrate transfer to the fetus; thus we determined whether maternal IGF treatment altered placental expression of Vegf, a potent stimulator of angiogenesis. However, in the present study, there was no effect of maternal IGF treatment on placental Vegf expression. Consistent with this, preliminary analyses suggest that there was no effect of maternal IGF treatment on the proportion and volume of fetal capillaries in the placenta in midgestation (Standen, P., unpublished observations). Interestingly, exogenous maternal IGF-I reduced placental expression of Igf2, which was correlated positively with maternal circulating IGF-II concentrations. This concurs with the previous demonstration that the placental synthesis of IGF-II contributes significantly to maternal plasma pools (71) and that IGF-I infusion into the mother reduced maternal circulating IGF-II (17). There was no effect of maternal IGF infusion on placental Igf1 gene expression in the present investigation, and abundance of this transcript was detectable but considerably lower than that of Igf2, which is consistent with the literature (13, 71).

Our data demonstrate modest effects on maternal organ weights and uptake of substrates in a few tissues, suggesting little effect on overall substrate availability for transfer to the fetus. Increased fetal growth in response to IGF-I treatment then is likely to be a consequence predominantly of actions within the placenta.

In contrast to IGF-I, exogenous IGF-II did not alter placental transporter transcription or activity, fetal or maternal substrate uptake, or fetal or placental growth on d 35 of pregnancy. It is possible that d 35 of gestation was too early to see an effect of infusion of IGF-II into the mother on the fetus and placenta, which was apparent by d 40 of pregnancy (14) and near term (17). Certainly, between d 35 and 40, fetal growth is rapid with about a 2.5-fold increase in fetal weight, whereas placental weight is less than doubled (Roberts, C. T., unpublished observations). Certainly, either IGF treatments may have modulated placental expression of molecules involved in metabolism, blood flow regulation, angiogenesis, trophoblast differentiation, and maternal adaptation to pregnancy, which were not assessed in the current investigation.

Indeed, in midgestational guinea pigs, maternal exogenous IGF-II tended to increase circulating estradiol and progesterone concentrations in the mother (17). This response may impact on maternal metabolic adaptation during pregnancy and placental perfusion and delivery of substrates to the fetus (8). Furthermore, exogenous IGF-II in early to midpregnancy increased the area and volume of the placental labyrinth, which is devoted to maternal-fetal exchange, as well as volumes of trophoblast and maternal blood spaces in the labyrinth and the total surface area of trophoblast for exchange in the placental labyrinth near term (17) but not in midgestation (Standen, P., A. N. Sferruzzi-Perri, R. L. Taylor, G. K. Heinemann, K. Kumarasamy, E. R. Lumbers, and C. T. Roberts, manuscript submitted). However, IGF-II treatment altered placental renin-angiotensin system gene expression and activity in midgestation (Standen, P., A. N. Sferruzzi-Perri, R. L. Taylor, G. K. Heinemann, K. Kumarasamy, E. R. Lumbers, and C. T. Roberts, manuscript submitted), which may in part explain the effects of IGF-II observed near term. We also speculate that exogenous maternal IGF-II may promote placental trophoblast invasion and differentiation in early to midpregnancy. However, the consequences for fetal and placental growth are not apparent until after treatment but are quite striking at term.

In conclusion, we have shown that increased IGF-I concentrations in maternal plasma from early pregnancy increases maternal substrate uptake and placental MG and AIB transfer to the fetus, enhancing fetal and placental growth, by midgestation. Although exogenous IGF-II administration to the mother does not induce immediate effects on the fetus, placenta, or mother on d 35 of gestation, this treatment increases fetal growth a few days later (14), which is sustained until near term (17). Although we have induced supraphysiological increases on circulating IGF concentrations with this treatment, the results are consistent with those observed in studies in which the converse was induced, that is, in the following reductions in circulating IGFs, for example, in food-restricted guinea pigs (72). Together, these suggest that the normal rise in maternal IGF-I in early pregnancy may play a major role in driving early placental growth and functional development, thus increasing its capacity to acquire substrates such as glucose and amino acids for itself and the fetus throughout gestation. This work also suggests that failure of this normal increase in endogenous IGF-I may result in placental insufficiency and IUGR. Conversely, exogenous IGF-I or indirectly promoting maternal IGF-I actions may help to prevent placental insufficiency.


   Acknowledgments
 
We thank GroPep Pty, Ltd., for supplying recombinant human IGFs and Mr. Gary Heinemann for his assistance in the guinea pig postmortems.


   Footnotes
 
This work was supported by a National Health and Medical Research Council project grant to C.T.R. and a Channel 7 Children’s Research Foundation grant to J.A.O. and C.T.R.

Disclosure Statement: Authors have nothing to disclose.

First Published Online May 24, 2007

Abbreviations: AIB, Amino-isobutyric acid; DPM, disintegrations per minute; GLUT, glucose transporter; IUGR, intrauterine growth restriction; MG, methyl-D-glucose; SNAT, system A amino acid transporter.

Received April 5, 2007.

Accepted for publication May 15, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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