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Endocrinology Vol. 148, No. 8 3625-3634
Copyright © 2007 by The Endocrine Society

Ligand-Activated Peroxisome Proliferator Activated Receptor {gamma} Alters Placental Morphology and Placental Fatty Acid Uptake in Mice

W. Timothy Schaiff, F. F. (Russ) Knapp, Jr., Yaacov Barak, Tal Biron-Shental, D. Michael Nelson and Yoel Sadovsky

Departments of Obstetrics and Gynecology (W.T.S., T.B.-S., D.M.N., Y.S.), and Cell Biology and Physiology (W.T.S., Y.S.), Washington University School of Medicine, St. Louis, Missouri 63110; Nuclear Medicine Program (F.F.K.), Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831; and The Jackson Laboratory (Y.B.), Bar Harbor, Maine 04609-1500

Address all correspondence and requests for reprints to: Yoel Sadovsky, M.D., Washington University School of Medicine, Department of Obstetrics and Gynecology, Campus Box 8064, 4566 Scott Avenue, St. Louis, Missouri 63110. E-mail: ysadovsky{at}wustl.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The nuclear receptor peroxisome proliferator activated receptor {gamma} (PPAR{gamma}) is essential for murine placental development. We previously showed that activation of PPAR{gamma} in primary human trophoblasts enhances the uptake of fatty acids and alters the expression of several proteins associated with fatty acid trafficking. In this study we examined the effect of ligand-activated PPAR{gamma} on placental development and transplacental fatty acid transport in wild-type (wt) and PPAR{gamma}+/– embryos. We found that exposure of pregnant mice to the PPAR{gamma} agonist rosiglitazone for 8 d (embryonic d 10.5–18.5) reduced the weights of wt, but not PPAR{gamma}+/– placentas and embryos. Exposure to rosiglitazone reduced the thickness of the spongiotrophoblast layer and the surface area of labyrinthine vasculature, and altered expression of proteins implicated in placental development. The expression of fatty acid transport protein 1 (FATP1), FATP4, adipose differentiation related protein, S3-12, and myocardial lipid droplet protein was enhanced in placentas of rosiglitazone-treated wt embryos, whereas the expression of FATP-2, -3, and -6 was decreased. Additionally, rosiglitazone treatment was associated with enhanced accumulation of the fatty acid analog 15-(p-iodophenyl)-3-(R, S)-methyl pentadecanoic acid in the placenta, but not in the embryos. These results demonstrate that in vivo activation of PPAR{gamma} modulates placental morphology and fatty acid accumulation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TRANSPLACENTAL EXCHANGE OF nutrients and waste products is essential for fetal development and survival. Fetoplacental demand for long chain polyunsaturated fatty acids increases after mid pregnancy, resulting in enhanced fatty acid import from the maternal circulation. Notably, docosahexaenoic acid and arachidonic acid are essential for brain and retinal development, and are preferentially transported across the placenta (1, 2, 3). Little is currently known about the mechanism of fatty acid trafficking into the fetoplacental unit in vivo.

The central role of the nuclear receptor peroxisome proliferator activated receptor {gamma} (PPAR{gamma}) in placental development and function has been well established (4, 5). PPAR{gamma} deficiency in mice results in embryonic death due to improper placental development (6, 7). Interestingly, PPAR{gamma} null placentas exhibit reduced lipid droplets in the labyrinthine trophoblasts, pointing to a role of PPAR{gamma} in placental fatty acid trafficking (6). We have recently shown that PPAR{gamma} and its heterodimeric nuclear receptor partner retinoid X receptor (RXR) enhance trophoblast fatty acid uptake and accumulation in vitro (8). We also showed (8, 9) that activation of PPAR{gamma} and/or RXR{alpha} in primary human trophoblasts resulted in increased expression of proteins known to be involved in fatty acid transport and accumulation, including fatty acid transport protein 1 (FATP1), FATP4, and adipophilin, a member of the perilipin adipophilin tail-interacting protein of 47 kDa (PAT) family of lipid droplet-associated proteins (10). Conversely, activation of RXR resulted in decreased expression of FATP2. In this study we sought to examine the influence of PPAR{gamma} activation in vivo on fetoplacental growth and transplacental fatty acid transport. We found that administration of the PPAR{gamma} agonist rosiglitazone to pregnant mice during the second half of pregnancy resulted in stunted fetal growth and placental development, associated with altered expression of transcripts that are implicated in placental development. Activation of PPAR{gamma} in vivo enhanced the expression of selected genes associated with fatty acid transport and accumulation in a manner similar to that observed in isolated human trophoblasts. These changes were associated with an increase in placental uptake of the fatty acid analog 15-(p-iodophenyl)-3-(R, S)-methyl pentadecanoic acid (BMIPP).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Rosiglitazone treatment of mice
All experiments were approved by the Washington University Animal Studies Committee. Mice were maintained in the Washington University barrier animal facility. Wild-type (wt) female C57Bl/6 mice were weighed, bred overnight with either wt or PPAR{gamma}+/– C57Bl/6 males, and then separated [defined as embryonic day (E) 0.5]. Unless stated otherwise, the mice were weighed at either E10.5 (8-d treatment group) or E16.5 (2-d treatment group) to determine pregnancy (3 and 10 g weight gain for E10.5 and E16.5, respectively). Pregnant mice were separated into two groups that received either standard powdered chow (LabDiet PicoLab Rodent Diet 20; Purina Mills, St. Louis, MO) or powdered chow-containing rosiglitazone (Biomol, Plymouth Meeting, PA) at a dose of 30 mg/kg·d. This dose was selected based on previous reports demonstrating that it effectively regulates PPAR{gamma} target genes in vivo (11, 12). The dose was within the dosage range used in these types of experiments (20–50 mg/kg·d) (11, 12, 13, 14, 15). Mice were weighed daily, and the amount of rosiglitazone was adjusted accordingly to maintain the desired dose until the mice were killed on E18.5. All mice were fed the same total amount of food per day starting with 6 g of food on E10.5 and gradually increasing to 8 g of food by E18.5.

Histology and stereology
Placentas were collected and fixed overnight at 4 C in fresh 4% paraformaldehyde in PBS, then dehydrated, and paraffin embedded. Sections (5 µm) were stained with either hematoxylin and eosin or periodic acid-Schiff (PAS) stain. The ratio of vasculature surface area to labyrinthine volume was determined using a cycloid arc grid, as previously described (16, 17). Briefly, a cycloid arc grid was overlaid high-magnification images of hematoxylin and eosin-stained sagittal placental sections from either control or rosiglitazone-treated pregnant mice. Cycloid line intersections with vascular walls were counted to determine vasculature surface area and points within the reference space counted to determine volume. Measurements were performed on systematic random samples of 10 sections per placenta using every fifth section for each placenta. Five placentas were used for each experimental group. The data are presented as combined area for maternal and fetal vessels, and expressed as cm2 vasculature surface area/cm3 of labyrinth.

Genotyping of embryos
In experiments involving breeding of wt female mice with PPAR{gamma}+/– male mice, PPAR{gamma} genotype was determined by PCR using 1 µl genomic DNA, extracted by a Gentra Puregene cell and tissue kit according to the manufacturer’s instructions (Gentra Systems, Minneapolis, MN). The primers used for PCR amplification were previously described (6).

RT-PCR and quantitative real-time PCR (RT-qPCR)
RNA was extracted from placenta and control tissues using the Ambion MELT Total Nucleic Acid isolation system (Ambion, Austin, TX), which included DNAse, according to the manufacturer’s instructions. Complementary DNA was prepared from 1 µg RNA using the TaqMan Gold RT-PCR kit with random hexamers (Applied Biosystems, Branchburg, NJ). PCR was performed on cDNA using primer pairs listed in Table 1Go. All sequences were checked for specificity using Basic Local Alignment Search Tool analysis.


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  		TABLE 1. Primer sequences used for standard PCR expression analysis

 
RT-qPCR was performed as previously described (8) with minor modifications. Briefly, RT-qPCR was performed in duplicate using 3-µl samples of cDNA and 12.5 µl SYBR Green PCR Master Mix (Applied Biosystems) in a total reaction volume of 25 µl that contained 300 nM each of forward and reverse primers. All PCR primer pairs were checked for specificity using Basic Local Alignment Search Tool analysis and are listed in Table 2Go. Reactions were run and analyzed using Applied Biosystems Geneamp 7300 and 5700 Sequence Detection Systems. Dissociation curves were run on all reactions to ensure amplification of a single product with the appropriate melting temperature. Samples were normalized to parallel reactions using primers specific for the L32 housekeeping gene (18). The fold change was calculated relative to control and was determined by the {Delta}{Delta}Ct method (19). The following primer set sequences were obtained using PrimerBank (http://pga.mgh.harvard.edu/primerbank/index.html) (20): myocardial lipid droplet protein (MLDP) (ID 21313202a2); CD36 (ID 31982474a1); Tpbpa (ID 31560633a1); Eomes (ID 5738950a3); Muc1 (ID 7305293a1); p57/Kip2 (ID 31981849a1); and long-chain acyl-coenzyme A dehydrogenase (LCAD) (ID 31982520a1). The primers for microsomal triglyceride transport protein (MTP) were as described by Ameen et al. (21).


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  		TABLE 2. Primer sequences used for RT-qPCR analysis

 
Preparation of radioiodinated BMIPP and 125I-BMIPP/BSA solution and measurement of tissue 125I-BMIPP content
Radioiodination of the long chain fatty acid analog BMIPP was prepared by electrophilic iodination of the 15-phenyl-3-R,S-methylpentadecanoic acid substrate, treating the thallated product with sodium-125iodide, as described (22, 23). 125I-BMIPP was dissolved in hot ethanol at approximately 4 mCi/ml and added drop-wise to a solution of 6% fatty acid-free BSA (Sigma, St. Louis, MO) in saline at 50 C with stirring to yield an activity of approximately 5 µCi/150 µl. The solution was filtered through a 0.45 µm sterile filter (Corning, Inc., Corning, NY) and allowed to equilibrate to room temperature before use. Pregnant mice were injected with 150 µl (3–5 µCi) 125I-BMIPP/BSA solution through the tail vein and killed by carbon dioxide 30 min after injection, unless otherwise indicated. The shortest time point for sample collection after 125I-BMIPP injection was 15 min, reflecting the time necessary for multiple tissue collection and processing. Embryos and placentas, as well as maternal blood, heart, adipose, and muscle samples were collected and 125I-cpm measured using a Packard Cobra II {gamma} counter (Packard Instrument Co., Meriden, CT). Samples were normalized to radioactivity per gram of maternal blood and expressed relative to blood radioactivity from a reference control mouse.

Lipids from maternal tissues, placentas, and embryos were analyzed as previously described (24). Briefly, lipids were Folch extracted by homogenization in 2:1 chloroform-methanol, followed by filter paper filtration, dilution with saline, and centrifugation to separate the organic (lower) and aqueous (upper) layers. The organic layer was evaporated and the fractions counted to determine the relative distribution of activity. After resuspension in chloroform, the lipid fractions were initially analyzed using aluminum-backed silica gel PF 254 thin-layer plates (Merck, Darmstadt, Germany) developed in petroleum ether:ether:acetic acid, 70:30:1. BMIPP and a 1,2-palmitoyl-BMIPP triglyceride were run on the plates as migration standards. After development, the plates were air dried, the standard positions detected under UV light, and the plates cut into 10 equal sections and counted in a {gamma} counter. The relative activity detected in each of the 10 regions was expressed as the percent total on the plate. The mean ± SD values for the sections corresponding to lipid extracts from the animals from each group were then calculated.

Statistics
Statistical significance was determined by ANOVA for placental and fetal weight, and for placental 125I-BMIPP uptake and transport. Statistical significance for gene expression folds, which were not normally distributed, was calculated by the Mann-Whitney U test. Statistical significance of changes in labyrinth surface area was determined by a t test. Significance of BMIPP distribution and transport was determined by a t test on samples normalized to counts per minute per gram of blood then further normalized to either grams of tissue (maternal tissues) or grams of placenta (embryos and placentas).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Rosiglitazone treatment of pregnant mice reduces fetoplacental weight
We previously showed that activation of PPAR{gamma} influenced trophoblast differentiation as well as the expression of fatty acid transporters in primary human trophoblasts in vitro (8, 25). Here we initially sought to examine the effect of PPAR{gamma} activation on placental and embryonic development in pregnant mice fed with the PPAR{gamma} agonist rosiglitazone. As shown in Fig. 1AGo, treatment of pregnant mice with rosiglitazone for 8 d in the second half of pregnancy (E10.5–E18.5) reduced embryonic weight by E18.5. This reduction was partially reversed in PPAR{gamma}+/– embryos, indicating that the effect of rosiglitazone was mediated through PPAR{gamma}. Treatment with rosiglitazone for 2 d (E16.5–E18.5) did not affect embryonic weight in the two mouse genotypes (Fig. 1AGo). A decrease in placental weight was evident within 2 d of rosiglitazone treatment and more pronounced after 8 d of treatment (Fig. 1BGo). PPAR{gamma}+/– embryos were resistant to rosiglitazone-induced decrease in placental weight, indicating that the effect was mediated by PPAR{gamma}. Interestingly, administration of rosiglitazone resulted in a slight reduction of maternal weight (Fig. 1CGo). This was associated with increased fetal mortality, where the frequency of dead or resorbing embryos was 20 of 95 (12 pregnancies) in rosiglitazone-treated mice vs. 1 of 84 (13 pregnancies) in control mice (P < 0.001). Genotype analysis of resorbing embryos revealed a 3:1 ratio of wt to PPAR{gamma}+/– embryos, correlating lethality to enhanced PPAR{gamma} activity.


Figure 1
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  		FIG. 1. The influence of rosiglitazone on embryonic, placental, and maternal weight. Pregnant mice were fed either chow alone (Ctrl) or chow containing rosiglitazone at 30 mg/kg·d for either 2 or 8 d starting on d E16.5 or E10.5, respectively. On E18.5, mice were killed, and embryonic (A) (n = 34–55) and placental (B) (n = 35–54) weights determined. Data are expressed as mean ± SD. *, P < 0.05 vs. respective control. **, P < 0.05 vs. all paradigms. C, Pregnant mice fed either chow alone (Control) or rosiglitazone for 8 d were weighed on the days indicated on the graph. *, P < 0.05. Control (n = 8–13) and rosiglitazone (n = 11–13) for each of the days on the graph.

 
Rosiglitazone treatment affects placental development
We next investigated the morphology of rosiglitazone-treated placentas. Microscopic examination of E18.5 placental sections revealed thinning of the spongiotrophoblast layer from rosiglitazone-treated pregnancies (Fig. 2Go, A and B). This was particularly evident in PAS-stained sections (Fig. 2Go, C and D), which delineates the glycogen-rich spongiotrophoblast (26). We also observed that the labyrinth of rosiglitazone-treated placentas was less dense than that of control animals. A closer examination revealed that the reduced density reflected dilated labyrinthine blood spaces (Fig. 2Go, A and B, inset). To quantify this finding, we used stereoscopic analysis of placental sections from control and rosiglitazone-treated embryos, and found that the ratio of surface area to volume of combined maternal and fetal labyrinthine blood spaces in control placentas was 897 ± 97 cm2/cm3, and was reduced by 25% to 671 ± 129 cm2/cm3 in placentas from rosiglitazone treated mice (P = 0.01). Together, these findings indicated that the reduced placental weight was likely secondary to thinner placental spongiotrophoblast and decreased surface area of the labyrinthine blood spaces.


Figure 2
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  		FIG. 2. The influence of rosiglitazone on placental morphology. Formaldehyde-fixed, paraffin-embedded sections of placentas from either control mice (A and C) or mice treated for 8 d with rosiglitazone (B and D) were sectioned and stained with either hematoxylin and eosin (A and B) or PAS stain (C and D). Inset shows higher magnification view of labyrinth (L) showing dilated vasculature in rosiglitazone-treated placentas. Scale bars, 0.5 (A–D) and 0.05 mm (A and B, inset). S, Spongiotrophoblast.

 
Because placentas from rosiglitazone-treated mice exhibited abnormal development of the spongiotrophoblast and labyrinth layers, we determined if the expression of genes that are expressed in these layers and implicated in their development was influenced by rosiglitazone. As shown in Fig. 3Go, the expression of spongiotrophoblast-associated genes Tpbpa and Mus musculus Achaete-Scute homolog 2 (Mash2) (27, 28, 29, 30) decreased in rosiglitazone-exposed mice by approximately 20- and 3-fold. In contrast, we observed an inconsistent pattern in expression of the trophectoderm and labyrinth Eomes and Esx1 (31, 32, 33, 34), with a 1.4-fold decrease in Eomes and 1.4-fold increase in Esx1 expression (Fig. 3Go). Interestingly, the expression of the maternally imprinted gene p57/Kip2, which is expressed in both spongiotrophoblast and labyrinth and limits placental growth (35, 36), was increased by approximately 1.8-fold, consistent with its reciprocal relationship with placental size (35). The expression of the previously identified placental PPAR{gamma}-target gene Muc1 (37) was increased by 2.5-fold, as expected.


Figure 3
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  		FIG. 3. The effect of rosiglitazone on spongiotrophoblast- and labyrinth-specific gene expression. RT-qPCR for genes expressed in placental spongiotrophoblast and/or labyrinth regions was performed on RNA prepared from E18.5 placentas from either control or pregnant mice treated with rosiglitazone from E10.5–E18.5. Each fold change was calculated as relative transcript expression in rosiglitazone-exposed placenta vs. control. Data are expressed as box plots showing median value, upper and lower quartiles, range, and outliers. Ctrl, Placentas from control mice (n = 30); Rosig, placentas from rosiglitazone-treated mice (n = 31). *, P < 0.01.

 
Placental expression of fatty acid transporters and transplacental transfer of BMIPP
To assess the influence of rosiglitazone administration in vivo on placental expression of fatty acid transporters, we initially interrogated the expression of FATP isoforms in the wt murine placenta. As shown in Fig. 4AGo, the transcripts for FATP1, 2, 3, 4, and 6, but not FATP5, are expressed in murine placenta. Among PAT proteins, we found that mRNA for adipose differentiation related protein (ADRP) (the murine ortholog of human adipophilin), tail-interacting protein of 47 kDa (Tip47), and the recently identified genes S3-12 (38) and MLDP [also known as OXPAT, PAT-1, or lipid storage droplet protein 5 (39, 40, 41, 42)] were expressed, but perilipin was not detected (Fig. 4BGo). These results were similar to the pattern of FATP and PAT expression in primary human trophoblasts (8, 9).


Figure 4
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  		FIG. 4. The expression of FATP, PAT, CD36, FABPpm, MTP, and LCAD in murine placenta. Standard PCR (left panels) or RT-qPCR (right panels) was performed on cDNA prepared from RNA purified from normal E18.5 placentas (or control tissues for standard PCR) using primers described in Tables 1Go and 2Go. Each fold change was calculated as relative transcript expression in rosiglitazone (Rosig)-exposed placenta vs. control. RT-qPCR data are expressed as box plots showing median value, upper and lower quartiles, range, and outliers. *, P < 0.05 (n = 22–48 for the different transcripts). No temp, No template control.

 
Changes in expression of genes involved in fatty acid transport were assessed by RT-qPCR using the {Delta}{Delta}Ct method (19). Samples were normalized to the housekeeping gene L32, which exhibited a stable expression in control or rosiglitazone treatment [mean Ct values: control, 19.79 ± 0.74; rosiglitazone treated, 19.98 ± 0.47 (n = 27); P = not significant by t test]. Importantly, treatment of pregnant mice with rosiglitazone for 8 d enhanced the expression of FATP1 and FATP4, and reduced the expression of FATP2 and FATP6, and to a lesser degree FATP3 (Fig. 4AGo). Notably, FATP2 was not detected in approximately one third of the examined placentas (41 of 125). This variability in placental FATP2 expression occurred within individual pregnancies and did not correlate with treatment or PPAR{gamma} genotype. Therefore, we confirmed the rosiglitazone-induced reduction in FATP2, even when placentas that did not express FATP2 were excluded. Among PAT proteins, placental expression of ADRP, S3-12, and MLDP was enhanced by rosiglitazone (Fig. 4BGo), whereas that of Tip47 was unchanged. We also determined that both CD36 (fatty acid translocase, FAT) and plasma membrane fatty acid binding protein (FABPpm), which are thought to be important in placental fatty acid transport (43, 44, 45), were expressed in the placenta, and the expression of both proteins was increased in placentas after rosiglitazone treatment (Fig. 4CGo). Finally, we examined the expression of MTP and LCAD, two genes that are expressed in the placenta (46, 47) and were previously up-regulated by PPAR{alpha}, but not PPAR{gamma} (21, 48). Interestingly, the expression of both MTP and LCAD was reduced in the placentas from rosiglitazone-exposed mice (Fig. 4DGo). This buttresses our notion that the dose of rosiglitazone given to the mice is unlikely to activate PPAR{alpha}.

To determine if the observed change in transcript expression was functionally associated with altered placental fatty acid uptake and accumulation, we assessed fetoplacental uptake of the long chain fatty acid analog BMIPP, which has been previously used for studying in vivo fatty acid transport in other tissues (49, 50, 51). We first established that BMIPP is distributed and metabolized normally in placental and embryonic tissues. Using pregnant mice that were infused with 125I-BMIPP, we used thin-layer chromatography to analyze the distribution of extracted BMIPP metabolic products from whole embryos, placentas, and maternal tissues. We observed that the major BMIPP metabolite in the embryo was 2-(p-iodphenyl)acetic acid (PIPPA), the product of BMIPP oxidation (24), with a minor amount incorporated into phospholipids (Fig. 5AGo). In the placenta (Fig. 5BGo) we also observed the dominance of PIPPA, yet with enhanced incorporation of BMIPP into phospholipids, indicating that the free fatty acid pool in the placenta is likely required for the maintenance of membrane lipids. Additionally, a higher level of placental BMIPP was incorporated into triglycerides, indicating storage of free fatty acids. Maternal tissue distribution of BMIPP (Fig. 5CGo) followed the previously described pattern (49, 50).


Figure 5
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  		FIG. 5. Distribution of 125I-BMIPP. E15.5 pregnant mice were injected with 125I-BMIPP and killed at the times indicated in the figure. Lipids were extracted from the tissues indicated. Extracted lipids were analyzed as described in Materials and Methods. The data for embryos (A) and placentas (B) are expressed as mean fold ± SD of embryos or placentas from a representative pregnancy (representing duplicate experiments, with n = 4–8 in each experiment). For maternal tissues (C) the data represent a pregnant mouse from duplicate experiments. BMIPP, Unmetabolized BMIPP; DG, diglyceride; PL, phospholipid; TG, triglyceride.

 
To determine the optimal time for analysis of fetoplacental uptake of 125I-BMIPP, we injected pregnant mice (E15.5) with 125I-BMIPP, and killed at the time points indicated (Fig. 6AGo). The level of 125I-BMIPP in the placenta increased rapidly, as early as 15 min after injection, and slowly continued to increase for 60 min after injection, likely reflecting rapid filling of the maternal blood spaces with 125I-BMIPP-containing maternal blood, followed by slower uptake into placental trophoblasts. As expected, embryonic BMIPP uptake was slower, reflecting transport across the placenta. Therefore, in subsequent experiments we examined fetoplacental levels of 125I-BMIPP at 30 min after injection. Predictably, we found that rosiglitazone enhanced BMIPP uptake by the maternal liver and adipose tissue (Fig. 6BGo). We also found that 8-d exposure of pregnant mice to rosiglitazone caused a 15% increase in placental BMIPP accumulation (P < 0.05; Fig. 6CGo). In contrast, while rosiglitazone reduced the absolute level of fetal BMIPP, there was no effect of rosiglitazone on fetal BMIPP levels when normalized to placental weight. Furthermore, there was no effect of rosiglitazone on fetoplacental BMIPP accumulation in PPAR{gamma}+/– embryos (data not shown). Similarly, treatment of wt mice for 2 d with rosiglitazone had no effect on fetoplacental BMIPP accumulation (data not shown). Together, these results reflect the combined effect of rosiglitazone on placental development and function, with enhanced BMIPP accumulation in the placenta, but not in the fetus.


Figure 6
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  		FIG. 6. Transport of 125I-BMIPP. A, Time course of embryonic and placental BMIPP uptake and transport. Pregnant mice were injected with 125I-BMIPP on E15.5, killed at the times indicated, and radioactivity determined (n = 11–19 for each time point). B, The impact of rosiglitazone-dependent activation of PPAR{gamma} on BMIPP uptake in maternal tissues. Pregnant controls (n = 9) or mice fed rosiglitazone (n = 13) for 8 d were injected with 125I-BMIPP, then killed 30 min after injection. Tissue counts per minute were determined as described in Materials and Methods. Data are expressed as normalized mean counts per minute/gram of tissue counted ± SD. *, P < 0.05. C, The effect of rosiglitazone-dependent activation of PPAR{gamma} on placental and embryonic BMIPP accumulation. Pregnant mice were fed either chow alone (Control) or chow containing rosiglitazone for 8 d. On E18.5, mice were injected with 125I-BMIPP, then killed after 30 min, and embryonic and placental counts per minute determined. Data are expressed as mean of normalized counts per minute/gram placenta tissue ± SD. *, P < 0.05 vs. control (n = 29–34 for each paradigm).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we examined the in vivo effects of ligand-activated PPAR{gamma} on fetoplacental development as well as transplacental fatty acid transport in mice. PPAR{gamma} activation elicited a reduction in placental and fetal size. Placental histology revealed thinner spongiotrophoblast and decreased labyrinth vascular surface area. Although the consequences of reduced spongiotrophoblast thickness are unclear, the changes in labyrinthine surface area in conjunction with smaller placental size likely contributed to the reduced size of rosiglitazone-exposed embryos. Interestingly, we also observed an increase in fetal mortality in rosiglitazone-treated wt mice. Furthermore, some of the live embryos from rosiglitazone-treated wt mice exhibited skin hemorrhages (unpublished data). The significance of this finding is presently unclear. Whether or not these changes reflect altered placental function remains to be established. Our results are consistent with the delayed fetal growth reported in the package inserts by the manufacturers of both Avandia (rosiglitazone; GlaxoSmithKline, Middlesex, UK) and Actos (pioglitazone; Eli Lilly and Co., Indianapolis, IN). In contrast to our studies and those reported in the Avandia and Actos package inserts, Klinkner et al. (52) reported that administration of up to 10 mg/kg·d rosiglitazone to pregnant mice for the entire pregnancy, which was designed to exceed a recommended human dose, had no effect on fetal development. However, Klinkner et al. based their dosage on a 25-g mouse and did not adjust the amount of rosiglitazone given to actual weight, especially as the mouse gained weight during pregnancy, thus effectively decreasing the dosage of rosiglitazone as pregnancy progressed. The dose in our studies is commonly used to study changes in gene expression induced by activation of PPAR{gamma} in vivo (11, 12, 13, 14, 15) and was adjusted to the daily weight gain during pregnancy to maintain a constant dosage. Sevillano et al. (53) reported a decrease in neonatal, but not fetal, mass in rats given englitazone (50 mg/kg·d) between E16 and E21. In contrast, exposure of pregnant rats to the PPAR{gamma} agonist troglitazone (20 mg/kg·d) on d E9–E11 was associated with reduced fetal mortality and no change in fetoplacental weight (54). The different outcome in our experiments likely reflects the different exposure period during pregnancy. It is likely that exposing the placenta to a PPAR{gamma} agonist for a limited time earlier in pregnancy could have affected placental development without depleting the trophoblastic stem cell pool, as might have occurred when the placenta was exposed to rosiglitazone for an extended period (discussed later). It should also be noted that the supra-therapeutic dose of rosiglitazone used in our study was chosen to give maximal effect. Importantly, the resistance of PPAR{gamma}+/– placentas and embryos to the effect of rosiglitazone and the lack of up-regulation of PPAR{alpha}-regulated genes strongly suggests that at the dosage used, this effect was mediated by PPAR{gamma}. Further studies may be needed to determine dosage threshold and timing of administration, which influence the results.

Consistent with the decrease in spongiotrophoblast thickness, we observed that the expression of genes that are crucial for spongiotrophoblast development, Tpbpa (27, 28) and Mash2 (29, 30), is reduced. Whereas this finding likely reflects reduced spongiotrophoblast expansion, the cause of the decrease in spongiotrophoblast development cannot be determined from our experiments. The effect of PPAR{gamma} activation on expression of labyrinthine gene expression was variable. While Esx1 (33, 34) expression was increased, the expression of Eomes (31, 32) was decreased. Embryos that lack a functional Esx1 gene exhibit an expanded spongiotrophoblast layer (34). These results are consistent with our observed increase in Esx1 expression coupled with the decrease in size of the spongiotrophoblast layer. In addition, Eomes is a marker of labyrinthine stem cells and is required for trophoblast development (31). Thus, the decrease in Eomes expression in placentas from rosiglitazone-treated mice might point to depletion of the trophoblast stem cell pool. The expression of p57/Kip2 was increased by rosiglitazone. Disruption of p57/Kip2 has caused overgrowth of the labyrinth, leading to a decrease in labyrinthine blood spaces (35, 36). These data suggest that P57/Kip2 functions by regulating expansion and promoting differentiation of trophoblastic stem cells within the labyrinth. Placentas that lack Rb, a downstream effector of p57/Kip2, exhibit expanded Eomes-expressing cells and failure of trophoblast differentiation in the labyrinth, accompanied by a decrease in placental transport of essential fatty acids (32). Our observation of rosiglitazone-induced decrease in Eomes expression and increase in p57/Kip2 expression is consistent with a reduction in trophoblastic stem cells, possibly due to premature differentiation. This possibility is supported by our previous observation that activation of PPAR{gamma} in primary human trophoblasts accelerates differentiation of cytotrophoblasts into syncytiotrophoblasts (25). Together, our results suggest that although basal PPAR{gamma} expression is required for proper placental development (6, 7), enhanced activation of placental PPAR{gamma} might lead to premature differentiation of trophoblast stem cells and depletion of the precursor pool, resulting in decreased placental size and abnormal labyrinthine development.

Our data point to altered expression of proteins involved in fatty acid transport and lipid droplet formation. The increased expression of FATP1, FATP4, and ADRP is similar to our observations in primary human trophoblasts exposed to the PPAR{gamma} agonist GW1929 (8). We also extended our previous findings and demonstrated that in addition to ADRP, the expression of placental S3-12 and MLDP, known targets of PPAR{gamma} in other tissues (40, 55), is increased by liganded PPAR{gamma}. Interestingly, activation of PPAR{gamma} resulted in decreased placental FATP2 expression, which was similarly decreased by RXR activation in primary human trophoblasts (8). The significance of the reciprocal relationship between the expression of FATP2 vs. FATP1 and FATP4 in placentas exposed to PPAR{gamma} ligands remains to be established.

FATP1 and FATP4 have been associated with increased cellular uptake of fatty acid (56, 57, 58), and knockdown of FATP4 in 3T3-L1 cells is associated with increased fatty acid efflux (59). ADRP, S3-12, and MLDP are associated with formation of cellular lipid droplets. The findings of enhanced expression of several FATPs, CD36, FABPpm, and PAT proteins, accumulation of BMIPP in the placenta, despite the reduced labyrinthine vasculature surface area, and reduced fetal size suggest that the up-regulated fatty acid transporters and PAT proteins might promote placental uptake and retention of fatty acids, but not their transport to the fetus. Consistent with this hypothesis was the intriguing rosiglitazone-induced decrease in MTP expression, which is involved in efflux of neutral lipids (60). Alternatively, the decrease in transplacental fatty acid transport could be due to reduced expression of other proteins involved in fatty acid transport. Although the study presented here focused on the expression of genes involved in fatty acid transport, the expression or activity of other proteins involved in fatty acid metabolism, such as lipoprotein lipase and the cytoplasmic FABPs, as well as those involved in inflammation, might be influenced by PPAR{gamma} activity and modulate transplacental fatty acid transport. Understanding the mechanism of PPAR{gamma} regulation of fetoplacental growth and fatty acid uptake may stimulate the development of treatment strategies to improve transplacental fatty acid uptake in pregnancies complicated by suboptimal intrauterine growth.


   Acknowledgments
 
We thank Lori Rideout for assistance in manuscript preparation, and John Cosgrove and Arnold Beets for technical assistance at Oak Ridge National Laboratory, which is managed by University of Tennessee Battelle, LLC, for the U.S. Department of Energy, under Contract No. DE-AC05-00OR22725.


   Footnotes
 
This work was supported by National Institutes of Health Grants R01HD044103 (to Y.B.) and R01ES11597 (to Y.S.).

Disclosure Statement: The authors have nothing to declare.

First Published Online April 26, 2007

Abbreviations: ADRP, Adipose differentiation related protein; BMIPP, 15-(p-iodophenyl)-3-(R, S)-methyl pentadecanoic acid; FABPpm, plasma membrane fatty acid binding protein; FATP, fatty acid transport protein; LCAD, long-chain acyl-coenzyme A dehydrogenase; Mash2, Mus musculus Achaete-Scute homolog 2; MLDP, myocardial lipid droplet protein; MTP, microsomal triglyceride transport protein; PAS, periodic acid-Schiff; PAT, perilipin adipophilin tail-interacting protein of 47 kDa; PIPPA, paraiodophenylacetic acid; PPAR{gamma}, peroxisome proliferator-activated receptor {gamma}; RT-qPCR, quantitative real-time PCR; RXR, retinoid X receptor; Tip47, tail-interacting protein of 47 kDa; Tpbpa, trophoblast-specific protein {alpha}; wt, wild type.

Received February 14, 2007.

Accepted for publication April 17, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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