The Journal of Nutrition. First published ahead of print August 13, 2014 as doi: 10.3945/jn.114.197202. The Journal of Nutrition Biochemical, Molecular, and Genetic Mechanisms

Fatty Acid Binding Protein 3 Is Involved in n–3 and n–6 PUFA Transport in Mouse Trophoblasts1–3 Ariful Islam,4 Yoshiteru Kagawa,4 Kazem Sharifi,4 Majid Ebrahimi,4 Hirofumi Miyazaki,4 Yuki Yasumoto,4 Saki Kawamura,4 Yui Yamamoto,4 Syuiti Sakaguti,7 Tomoo Sawada,4 Nobuko Tokuda,5 Norihiro Sugino,6 Ryoji Suzuki,8 and Yuji Owada4*

Abstract Low placental fatty acid (FA) transport during the embryonic period has been suggested to result in fetal developmental disorders and various adult metabolic diseases, but the molecular mechanism by which FAs are transported through the placental unit remains largely unknown. The aim of this study was to examine the distribution and functional relevance of FA binding protein (FABP), a cellular chaperone of FAs, in the mouse placenta. We clarified the localization of FABPs and sought to examine their function in placental FA transport through the phenotypic analysis of Fabp3-knockout mice. Four FABPs (FABP3, FABP4, FABP5, and FABP7) were expressed with spatial heterogeneity in placenta, and FABP3 was dominantly localized to the trophoblast cells. In placentas from the Fabp3-knockout mice (both sexes), the transportation coefficients for linoleic acid (LA) were significantly reduced compared with those from wild-type mice by 25% and 44% at embryonic day (E) 15.5 and E18.5, respectively, whereas those for a-linolenic acid (ALA) were reduced by 19% and 17%, respectively. The accumulation of LA (18% and 27% at E15.5 and E18.5) and ALA (16% at E15.5) was also significantly less in the Fabp3-knockout fetuses compared with wild-type fetuses. In contrast, transportation and accumulation of palmitic acid (PA) were unaffected and glucose uptake significantly increased by 23% in the gene-ablated mice compared with wild-type mice at E18.5. Incorporation of LA (51% and 52% at 1 and 60 min, respectively) and ALA (23% at 60 min), but not PA, was significantly less in FABP3-knockdown BeWo cells compared with controls, whereas glucose uptake was significantly upregulated by 51%, 50%, 31%, and 33% at 1, 20, 40, and 60 min, respectively. Collectively FABP3 regulates n–3 (v-3) and n–6 (v-6) polyunsaturated FA transport in trophoblasts and plays a pivotal role in fetal development.

J. Nutr. doi: 10.3945/jn.114.197202.

Introduction The placenta forms a selective barrier able to transport nutrients of critical importance to the fetus. Transplacental exchange of nutrients and waste products between mother and fetus is essential for fetal development and survival. In humans and rodents, the fully developed placenta is composed of 3 major layers: the outer maternal layer, which includes decidual cells of the uterus, as well as the maternal vasculature; a middle ‘‘junctional’’ region, which includes maternal spongiotrophoblast cells and attaches the fetal component of the placenta to the uterus; and an inner layer, 1 Supported by a grant-in-aid to Y.O. (no. 24390047) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan. 2 Author disclosures: A. Islam, Y. Kagawa, K. Sharifi, M. Ebrahimi, H. Miyazaki, Y. Yasumoto, S. Kawamura, Y.Yamamoto, S. Sakaguti, T. Sawada, N. Tokuda, N. Sugino, R. Suzuki, and Y. Owada, no conflicts of interest. 3 Supplemental Tables 1 and 2 and Supplemental Figures 1–3 are available from the ‘‘Online Supporting Material’’ link in the online posting of the article and from the same link in the online table of contents at http://jn.nutrition.org. * To whom correspondence should be addressed. E-mail: yowada@yamaguchi-u. ac.jp.

composed of highly branched villi that include fetal syncytiotrophoblast cells designed for efficient nutrient exchange (1). Fetal growth is closely related to the capacity of the placenta to transport nutrients, which is dependent on the expression and activity of specific transporter proteins on the syncytiotrophoblast (2,3). Abnormal embryonic or fetal nutrition can lead to deleterious consequences in adult life (4–6), and it has been suggested that there is a strong association between low birth weight and the risk of developing type 2 diabetes (7,8) and cardiovascular disease (9,10) in adulthood. Fetal demand for PUFAs increases after midpregnancy, leading to enhanced FA importation from the maternal circulation. Notably, DHA (22:6n–3) and arachidonic acid (AA)9 (20:4n–6) are essential for brain and retinal 9 Abbreviations used: AA, arachidonic acid; ALA, a-linolenic acid; CAG-EGFP, CAG promoter driven-enhanced green fluorescence protein; CD36, cluster of differentiation 36; E, embryonic day; FABP, fatty acid binding protein; Fatps, fatty acid transport protein; LA, linoleic acid; LCM, laser capture microdissection; MCT1, monocarboxylated transporter 1; PA, palmitic acid; p-Fabppm, placental plasma membrane fatty acid binding protein; RNAi, interfering RNA.

ã 2014 American Society for Nutrition. Manuscript received May 20, 2014. Initial review completed June 9, 2014. Revision accepted July 22, 2014. doi: 10.3945/jn.114.197202.

Copyright (C) 2014 by the American Society for Nutrition

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4 Department of Organ Anatomy, 5Department of Health Science, 6Department of Obstetrics and Gynecology, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan; 7Institute of Radioisotope Research and Education, Science Research Center, Organization for Research Initiative, Yamaguchi University, Yamaguchi, Japan; and 8Department of Anatomy, Akita University Graduate School of Medicine, Akita, Japan

Materials and Methods Mice and cells. In this study, we used the mouse strain C57BL/6J obtained from Yamaguchi University animal facilities. The generation of the Fabp3-knockout (Fabp32/2) mouse was previously described by Binas et al. (23). C57BL/6-Tg [CAG promoter driven–enhanced green fluorescence protein (CAG-EGFP)] mice (CAG-EGFP transgenic mice with C57BL/6 background) or ‘‘green’’ mice were kindly supplied by Dr. Masaru Okabe (Osaka University, Osaka, Japan). Mice were housed in a 12-h light/12-h dark cycle with ad libitum access to a standard rodent unpurified diet (composition: 5.1 g fat, 23.1 g protein, 2.8 g fiber, and 55.3 g carbohydrate per 7.9 g water and 5.8 g ash per 100 g; MF130912; Oriental Yeast). At 8 wk of age, both the Fabp3-knockout and wild-type female mice were mated with their male counterparts. For the preparation of chimeric placenta, male CAG-EGFP mice were mated with wild-type female mice. In these mice, the structures of placenta of fetal origin demonstrated green fluorescence, whereas those of maternal origin did not (Supplemental Fig. 1). The presence of a vaginal plug was designated as E1. All experimental protocols were reviewed by the Ethics Committee for Animal Experimentation of Yamaguchi University School of Medicine, Japan. BeWo cells (CCL-98; American Type Culture Collection) were cultured at 37°C in a 5% CO2 atmosphere in HamÕs F12 K medium (Wako) with 10% FBS (Hyclone), as described previously (24). PCR. RT-PCR and quantitative real-time RT-PCR (qRT-PCR) were performed as described previously (25). Briefly, total RNA was extracted from wild-type mouse placentas (at E11.5, E15.5, and E18.5) by using the Trizol method and reverse-transcribed to cDNA with the use of a high-fidelity reverse transcriptase cDNA synthesis kit (Roche). RT-PCR was performed under optimal conditions with gene-specific primers Fabp1, Fabp2, Fabp3, Fabp4, Fabp5, Fabp7 (26), Fabp8 (27), and Fabp9, with b-actin as the internal control (28), as listed in Supplemental Table 1. For qRT-PCR analysis, total RNA was extracted from wild-type and Fabp3-knockout mouse placentas (at E15.5 and E18.5), cDNA was prepared as described above, and reactions were performed by using the Step One Plus Real-Time PCR System (Applied Biosystems). Among the target genes (Supplemental Table 2), the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was used as an internal control. Western blotting. Western blotting was performed as described previously (25). Briefly, the protein was extracted from the wild-type 2 of 8

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mouse placentas (at E11.5, E15.5, and E18.5), electrophoresed by SDS PAGE (15% gel), and immunoblotted onto a polyvinylidene difluoride membrane (Millipore). The expression of FABP3, FABP4, and FABP5 was detected by using rabbit polyclonal antibodies anti-FABP3 [0.5 mg/mL dilution (29)], anti-FABP4 [0.5 mg/mL dilution (30)], and anti-FABP5 [0.5 mg/mL dilution (31)], with anti-GAPDH (1:500; Santa Cruz Biotech) as the internal control. Laser capture microdissection of the labyrinthine compartment. The localization of Fabp3, Fabp4, Fabp5, and Fabp7 at the mRNA level was also examined by RT-PCR in the placental labyrinthine compartment by using laser capture microdissection (LCM) at E18.5. LCM was performed following the protocol as described by Stemmer et al. (32) with a slight modification. Fresh-frozen placental specimens were prepared for cryosectioning (CM 1850; Leica) and stained with RNasefree staining solution (Histogene frozen section staining kit; Arcturus). The labyrinthine compartment was dissected by using the Laser Microdissection system (LMD 6500; Leica) under specific conditions, and total RNA was extracted from the microdissected sample and then prepared for RT-PCR as described above. Immunohistochemistry. Placental samples were collected from wildtype and Fabp3-knockout mice (at E11.5, E15.5, and E18.5), and immunohistochemical analysis was performed following the detailed protocol as described previously by Tokuda et al. (33). Briefly, after cryoprotection, placental sections were incubated with rabbit polyclonal primary antibodies for FABP3, FABP4, FABP5, or FABP7 (dilution of 0.5 mg/mL). For enzyme-based immunohistochemistry, biotinylated anti-rabbit IgG was used as a secondary antibody (Vector Laboratories) followed by reaction with 3,3#-diaminobenzidine tetrahydrochloride (Sigma-Aldrich). For immunofluorescence staining, sections were incubated with antiFABP3, anti-FABP5, and chicken anti-rat monocarboxylated transporter 1 (MCT1; 1:200; Chemicon) antisera followed by incubation with goat anti-rabbit IgG Alexa 488 (1:1000; Invitrogen) and goat anti-chicken IgG Alexa 594 (1:250; Invitrogen). For immunofluorescence staining of the chimeric placenta, the sections were incubated with FABP3, FABP5, and FABP7 antisera, followed by incubation with goat anti-rabbit IgG Alexa 568 (1:1000; Invitrogen). After counterstaining the nuclei with diamidino-2-phenylindole, the sections were observed by using a confocal laser microscope (LSM510 META; Carl Zeiss). Histological analysis. For microscopic comparison, placentas collected from wild-type or Fabp3-knockout mice at E18.5 were formalin fixed, and paraffin sections were prepared by microtome and stained with hematoxylin and eosin (Supplemental Fig. 2). Analysis of placental transport of radiolabeled FAs and glucose. Placental transport of radiolabeled FAs and glucose was examined as described earlier (34) with slight modifications. Briefly, Fabp3+/2 mice were intercrossed and pregnant dams (at E15.5 and E18.5) were anesthetized (isoflurane; Abbott) with the use of a small-animal anesthetizer (Muromachi). Then, the maternal jugular vein was exposed and PBS containing 3.5 mCi of [1-14C]-linoleic acid (LA,18:2n–6; NEC501, specific activity 58.2 mCi/mmol; Perkin Elmer), 9,12,15-[1-14C]alinolenic acid (ALA,18:3n–3; NEC-779, specific activity 51.7 mCi/mmol; Perkin Elmer), [1-14C]-palmitic acid (PA,16:0; NEC-075H, specific activity 60 mCi/mmol; Perkin Elmer), or D-[14C(U)]-glucose (NEC042X, specific activity 289 mCi/mmol; Perkin Elmer) was injected into the jugular vein. Fetuses and placentas were removed 4 min after radioisotope injection (35); studies show that there is minimal tracer backflux at this time (36). After weighing the fetus and placenta, the fetuses were minced and lysed in Solvable (Perkin Elmer). A small section of tail was collected from each fetus for genotyping. The liquid scintillation cocktail Ultima Gold (Perkin Elmer) was used for b-counting (liquid scintillation counter 5100; Aloka). The transport of radioactive material from dam to fetus was evaluated following the method previously described by Constaˆncia et al. (35). Briefly, radioactive counts, detected in each fetus, were used to calculate the amount of radioactive material transported or accumulated per gram of placenta

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development and are preferentially transported across the placenta (11–13). All of the n–3 and n–6 PUFAs accumulated in the fetus must be obtained from the mother by placental transport (14). However, little is currently known about the mechanism of PUFA trafficking into the feto-placental unit. FA-binding proteins (FABPs) constitute a multigene family of intracellular FA carrier molecules with low molecular masses of ;14–15 kDa. Multiple FABP isoforms (FABP1–12) have been isolated from distinct tissues. FABPs are generally believed to promote cellular FA uptake and transport toward specific metabolic pathways and to regulate gene transcription by delivering FAs as the ligands for specific nuclear receptors (15). Thus far, among the FABP family, FABP1, FABP3, FABP4, FABP5, and FABP7 were observed to occur in both the human and rodent placenta (16–22). However, the precise localization of each FABP in the placental units and their specific roles in feto-placental PUFA transport remain unknown. In the present study, we evaluated the cellular expression of FABP family members in the placenta during mid- to late gestation, i.e., at embryonic day (E) 11.5, E15.5, and E18.5, at both the mRNA and protein levels, and investigated the influence of Fabp3 ablation on PUFA transport from mouse dams to their fetuses through the placenta.

and fetus. Mean values for wild-type and Fabp3-knockout fetuses within a litter were then calculated and expressed as a ratio of Fabp3-knockout to Fabp3 wild-type. Stealth interfering RNA-mediated knockdown. FABP3-knockdown experiments were performed according to the manufacturerÕs instructions (Invitrogen). BeWo cells were maintained as described above. Stealth interfering RNA (RNAi) directed against the human FABP3 mRNA (Oligo ID: HSS103512; Invitrogen) was used for FABP3 knockdown. Nontargeting low guanine-cytosine content stealth RNAi (ID: 46–2002; Invitrogen) was used as a control (scramble control). After 48-h incubation with RNAi, FABP3 knockdown was confirmed by qRTPCR and Western blotting.

Calculations and statistical analyses. All values are expressed as means 6 SEMs. For comparison of the base pair strength within the embryonic stage, 1-factor ANOVAs followed by TukeyÕs comparison tests were performed, Fabp3 expression among the embryonic stages was also compared by same analysis. For compact and comprehensive presentation of placental in vivo transport studies, the 1-sample t test (35) was used; for the in vitro FA study and qRT-PCR, data were analyzed by using StudentÕs 2-tailed unpaired t test (SPSS software version 16.0). P values

Fatty Acid Binding Protein 3 Is Involved in n-3 and n-6 PUFA transport in mouse trophoblasts.

Low placental fatty acid (FA) transport during the embryonic period has been suggested to result in fetal developmental disorders and various adult me...
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