Am J Physiol Cell Physiol 307: C738–C744, 2014. First published August 20, 2014; doi:10.1152/ajpcell.00196.2014.
Differential regulation of placental amino acid transport by saturated and unsaturated fatty acids Susanne Lager, Thomas Jansson, and Theresa L. Powell Center for Pregnancy and Newborn Research, Department of Obstetrics and Gynecology, University of Texas Health Science Center, San Antonio, Texas Submitted 18 June 2014; accepted in final form 19 August 2014
Lager S, Jansson T, Powell TL. Differential regulation of placental amino acid transport by saturated and unsaturated fatty acids. Am J Physiol Cell Physiol 307: C738 –C744, 2014. First published August 20, 2014; doi:10.1152/ajpcell.00196.2014.—Fatty acids are critical for normal fetal development but may also influence placental function. We have previously reported that oleic acid (OA) stimulates amino acid transport in primary human trophoblasts (PHTs). In other tissues, saturated and unsaturated fatty acids have distinct effects on cellular signaling, for instance, palmitic acid (PA) but not OA reduces IB␣ expression. We hypothesized that saturated and unsaturated fatty acids differentially affect trophoblast amino acid transport and cellular signaling. To test this hypothesis, PHTs were cultured in docosahexaenoic acid (DHA; 50 M), OA (100 M), or PA (100 M). DHA and OA were also combined to test whether DHA could counteract the OA stimulatory effect on amino acid transport. The effects of fatty acids were compared against a vehicle control. Amino acid transport was measured by isotope-labeled tracers. Activation of inflammatory-related signaling pathways and the mechanistic target of rapamycin (mTOR) pathway were determined by Western blot analysis. Exposure of PHTs to DHA for 24 h reduced amino acid transport and phosphorylation of p38 MAPK, STAT3, mTOR, eukaryotic initiation factor 4E-binding protein 1, and ribosomal protein (rp)S6. In contrast, OA increased amino acid transport and phosphorylation of ERK, mTOR, S6 kinase 1, and rpS6. The combination of DHA with OA increased amino acid transport and rpS6 phosphorylation. PA did not affect amino acid transport but reduced IB␣ expression. In conclusion, these fatty acids differentially regulated placental amino acid transport and cellular signaling. Taken together, these findings suggest that dietary fatty acids could alter the intrauterine environment by modifying placental function, thereby having long-lasting effects on the developing fetus. docosahexaenoic acid; oleic acid; palmitic acid; pregnancy; cell signaling FATTY ACIDS are critical for fetal development. They are essential structural components of cellular membranes and precursors of bioactive compounds, with particularly large amounts being deposited in the fetal brain (13). Fatty acids can also influence cellular signaling through a multitude of mechanisms, with effects dependent on their level of saturation. For instance, saturated and polyunsaturated fatty acids differentially modulate Toll-like receptor (TLR)4 signaling (38). The polyunsaturated -3 fatty acid docosahexaenoic acid (DHA; 22:6 n-3) has been shown to exert anti-inflammatory actions, partly mediated via the membrane-bound G protein-coupled receptor 120 (GPR120) (25). Both TLR4 (6) and GPR120 (21) are expressed on the syncytiotrophoblast microvillous plasma
Address for reprint requests and present address: S. Lager, Dept. of Obstetrics and Gynaecology, Univ. of Cambridge, Box 223, Robinson Way, The Rosie Hospital, Cambridge CB2 0SW, UK (e-mail: [email protected]. ac.uk). C738
membrane in the human-term placenta, consistent with the possibility that maternal fatty acids modulate placental function (10, 24, 26, 33) through interactions with these receptors. In a variety of tissues, it has been shown that the fatty acids DHA, oleic acid (OA; 18:1 n-9), and palmitic acid (PA; 16:0) have distinct effects on inflammatory signaling pathways. For instance, DHA inhibits, whereas PA stimulates, activity of the IB/NF-B signaling pathway (7, 25, 31). Other inflammatoryrelated pathways responding to fatty acids in other tissues include STAT3 (19) as well as MAPK pathways (ERK, JNK, and p38 MAPK) (2, 19, 25, 39). In addition to inflammatory pathways, fatty acids can also influence mechanistic target of rapamycin (mTOR) (2, 17, 31), a serine/threonine kinase regulating protein translation, cell growth, and metabolism (23). Activation of this kinase results in the phosphorylation of eukaryotic initiation factor 4E-binding protein (4EBP)1, S6 kinase (S6K)1, and ribosomal protein (rp)S6 (23). In the context of placental function, we have shown that the mTOR pathway is a positive regulator of trophoblast amino acid transport (27–29). At the end of pregnancy, OA and PA together constitute ⬃60% of the nonesterified fatty acid (NEFA) species in the maternal circulation (37), and NEFA levels correlate positively with maternal body mass index (19). We have previously reported that OA stimulates placental amino acid transport in primary human trophoblasts (PHTs) (19). This effect is dependent on TLR4 (19); however, the cellular signaling pathways mediating the activation of amino acid transport by OA remain to be fully established. In addition, regulation of trophoblast amino acid transport by DHA and PA has not previously been studied. Therefore, the aim of the present study was to determine the effects of DHA, OA, and PA on amino acid transport activity and cellular signaling in cultured PHTs. We tested the hypothesis that these fatty acids differentially regulate trophoblast amino acid transport and cellular signaling. MATERIALS AND METHODS
Ethics. Human placental tissue samples from term pregnancies (ⱖ37 ⫾ 0 wk of gestation) were collected after written informed consent. Placental samples and medical information were added to a tissue/data repository. The protocol was approved by the Institutional Review Board of the University of Texas Health Science Center (San Antonio, TX, HSC20100262H). De-identified tissue samples were made available for the present study. Isolation and maintenance of trophoblast cells. Villous cytotrophoblasts were isolated as previously described (4, 20). Trophoblast cells were separated by DNAse/trypsin digestion and purified on a Percoll gradient. Cells were plated at an approximate density of 150,000 cells/cm2. Cell culture media (equal volumes of Ham’s F-12 and high-glucose DMEM) contained 10% FBS (S11550, Atlanta Biologicals, Lawrenceville, GA) and antibiotics (gentamicin, penicillin, and
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streptomycin). Media were changed daily. Starting at 66 h after being plated, cells were cultured in media containing 1% FBS supplemented with BSA-bound fatty acids (DHA: 25 or 50 M, OA: 100 M, PA: 100 M), and each fatty acid treatment had its own control (containing cell culture media with an equal amount of fatty acid-free BSA stock buffer). Twenty-four hours later (at 90 h after cell plating), amino acid uptake was measured, cell viability was assessed, or samples were collected for protein expression analysis. Characterization and viability of isolated cells. As an indicator of biochemical differentiation, the release of human chorionic gonadotropin into the cell culture media was measured using a commercial ELISA (IBL-America, Minneapolis, MN). Cell viability was determined by the ability of the cultured cells to metabolize MTT (thiazolyl blue tetrazolium bromide, Sigma-Aldrich, St Louis, MO), and cleaved caspase-3 was used to measure apoptosis. Harvesting cells. Trophoblast cells were harvested by the addition of RIPA buffer containing phosphatase inhibitor cocktails 1 and 2 and protease inhibitor cocktail (final dilution: 1:100, Sigma-Aldrich) and collected after being scraped with a spatula. Cells were stored at ⫺80°C until further analysis. Protein concentrations were determined by the bicinchoninic acid assay method (Thermo Fisher Scientific, Waltham, MA). Measurement of amino acid uptake. System A amino acid transporter activity was measured as Na⫹-dependent [14C]methylaminoisobuturyric acid (final concentration: 20 M) uptake, and system L transporter activity was measured by 2-amino-2-norbornanecarboxylic acid (BCH)inhibitable uptake of [3H]leucine (final concentration: 12.5 nM), as previously described (4, 19). Transporter-mediated uptakes were calculated by subtracting uptake in Na⫹-free/BCH buffer (nonmediated uptake) from uptake in Na⫹-containing buffer (total uptake). Protein con-
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centrations were measured with the Lowry method. Uptakes were expressed as picomoles of amino acid taken up per milligram of protein per minute. The mean uptake of controls was arbitrarily assigned a value of 1.0 to facilitate comparisons between groups. Western blot analysis. Western blots were performed on precast gels from Bio-Rad (Hercules, CA). After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes (overnight, 4°C). Membranes were stained for total protein with amido black stain (Sigma-Aldrich) and blocked in 5% nonfat milk (1 h, room temperature). The following primary antibodies were purchased from Cell Signaling Technology (Davers, MA): 4EBP1 (no. 9452), phosphorylated (P)-4EBP1 (Thr37/46, no. 9459), AMP-activated protein kinase (AMPK; no. 2532), P-AMPK (Thr172, no. 2535), caspase-3 (no. 9662), ERK (no. 4695), P-ERK (Thr202/Tyr204, no. 4370), IB␣ (no. 4812), JNK (no. 9252), P-JNK (Thr183/Tyr185, no. 4668), mTOR (no. 2983), P-mTOR (Ser2448, no. 5536), p38 MAPK (no. 9212), S6K1 (no. 9202), P-S6K1 (Thr389, no. 9205), rpS6 (no. 2217), P-rpS6 (Ser235/236, no. 4858), STAT3 (no. 9139), and P-STAT3 (Tyr705, no. 9145). The primary antibody targeting P-p38 MAPK (Thr180/Tyr182) was purchased from Abcam (ab32557, Cambridge, UK). Primary antibodies were diluted in Tris-buffered saline containing 1–3% BSA, and incubations were carried out overnight (4°C). After incubation with the appropriate secondary peroxidase-labeled IgG antibody (1 h, room temperature), immunolabeling was detected using SuperSignal Dura West (Thermo Fisher Scientific) in a G:Box ChemiXL1.4 (Syngene, Cambridge, UK). The relative density of bands was measured by densitometry (GeneTools, Syngene), and protein expression was adjusted for amount of total protein (amido black stain). The mean value of controls was arbitrarily assigned a value of 1.0 to facilitate comparisons between groups.
Fig. 1. Characterization of cultured human trophoblast cells. A: release of human chorionic gonadotropin (hCG) into the cell culture media. *P ⬍ 0.05; **P ⬍ 0.01 vs. 18 and 42 h (repeated-measures ANOVA followed by Tukey’s post hoc test). B and C: cell viability measured by MTT assay [B; control as 100% (dotted line)] and apoptosis measured by the amount of cleaved caspase-3 (C) after 24-h exposure to fatty acids [docosahexaenoic acid (DHA): 50 M, oleic acid (OA): 100 M, palmitic acid (PA): 100 M] compared with vehicle controls (C). D: representative Western blot. AU, arbitrary units. Data are means ⫾ SE.
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Data presentation and statistics. The number of experiments (n) represents the number of placentas studied. Data are presented as means ⫾ SE. Statistical analysis was carried out with GraphPad Prism 5 (version 5.04, GraphPad Software, La Jolla, CA). Differences between two groups were evaluated statistically by paired t-test and for multiple groups by repeated-measures ANOVA followed by Tukey’s multiple-comparison test. P values of ⬍0.05 were considered significant. RESULTS
Characterization and viability of isolated trophoblast cells. The secretion of human chorionic gonadotropin into cell culture media was significantly higher after 66 and 90 h of culture compared with 18 and 42 h of culture, indicating biochemical differentiation (n ⫽ 12, P ⬍ 0.01; Fig. 1A). Twenty-four-hour treatments with fatty acids (DHA: 50 M, OA: 100 M, PA: 100 M) did not affect cell viability (n ⫽ 4; Fig. 1B), nor did it increase apoptosis (n ⫽ 4 –7; Fig. 1C). Amino acid transport. Mean amino acid transport activity in control PHTs (system A activity: 10 ⫾ 1.3 pmol·mg⫺1·min⫺1; system L activity: 0.1 ⫾ 0.01 pmol·mg⫺1·min⫺1) was similar to what we have previously reported (4). Activity of the system A amino acid transporter was markedly decreased after exposure to 25 or 50 M DHA (n ⫽ 10, P ⬍ 0.05; Fig. 2A). In contrast, incubation in 100 M OA stimulated system A activity (n ⫽ 7, P ⬍ 0.01; Fig. 2A). Cotreatment of trophoblast cells with DHA (50 M) and OA (100 M) did not prevent OA stimulation of system A activity (n ⫽ 7, P ⬍ 0.01; Fig. 2A). PA (100 M) did not alter trophoblast system A amino acid transport activity (n ⫽ 7; Fig. 2A). Uptake of amino acids by the system L amino acid transporter was significantly reduced by 25 or 50 M DHA (n ⫽ 10, P ⬍ 0.05; Fig. 2B). System L amino acid transport activity was not affected by 100 M OA or 100 M PA treatment (n ⫽ 7; Fig. 2B). The decrease in system L activity in response to 50 M DHA was prevented by cotreatment with 100 M OA (n ⫽ 7; Fig. 2B). Inflammatory signaling. DHA or PA alone did not alter the phosphorylation of ERK. In contrast, ERK phosphorylation was significantly increased by OA (n ⫽ 7, P ⬍ 0.05; Fig. 3A). Activation of the ERK signaling pathway by OA was prevented by coincubation with DHA (Fig. 3A). Expression of IB␣ was decreased by PA treatment (n ⫽ 7, P ⬍ 0.05; Fig. 3B), whereas it was unaffected by DHA and/or OA. JNK phosphorylation was not altered by any of the fatty acids studied (Fig. 3C). DHA inhibited p38 MAPK and STAT3 signaling (n ⫽ 9, P ⬍ 0.05; Fig. 3, D and E), whereas there was a trend toward increased STAT3 phosphorylation in response to OA (P ⫽ 0.07). PA or co-treatment with DHA and OA had no significant effect on p38 MAPK or STAT3 inflammatory signaling pathways. Total protein expression of ERK, JNK, and p38 MAPK was not affected by the fatty acid treatments, whereas total STAT3 was significantly decreased by exposure to DHA (P ⬍ 0.05; data not shown). AMPK and mTOR signaling. The cellular energy state, as measured by AMPK phosphorylation, was not affected by the fatty acid treatments (n ⫽ 6 –7; Fig. 4A). Phosphorylation of mTOR was reduced by DHA and increased by OA treatment (n ⫽ 9, P ⬍ 0.05; Fig. 4B). Downstream of mTOR, phosphorylation of 4EBP1 and rpS6 was lower in PHTs exposed to DHA (n ⫽ 9, P ⬍ 0.05; Fig. 4, C and E). OA increased the phosphorylation of S6K1 and rpS6 (n ⫽ 9, P ⬍ 0.05; Fig. 4, D and E) but not 4EBP1. Co-treatment of cells with DHA and
Fig. 2. Amino acid uptakes after fatty acid stimulation. A and B: system A (A) and system L (B) amino acid transport activity in cultured primary human trophoblast cells after exposure to fatty acids for 24 h (DHA: 25 or 50 M, OA: 100 M, PA: 100 M) compared with vehicle controls. Data are means ⫾ SE. *P ⬍ 0.05; **P ⬍ 0.01 (paired t-test).
OA resulted in unaltered phosphorylation levels of 4EBP1 and S6K1, whereas rpS6 phosphorylation was increased compared with control (n ⫽ 9, P ⬍ 0.05; Fig. 4E). Total protein expression of AMPK, mTOR, 4EBP1, and S6K1 was not affected by the fatty acid treatments, whereas total rpS6 was significantly decreased by DHA and increased by OA (P ⬍ 0.05; data not shown). DISCUSSION
In the present report, we show, for the first time, that the fatty acids DHA, OA, and PA differentially regulate trophoblast amino acid transport. Similarly, these fatty acids had diverse effects on inflammatory and mTOR signaling pathways in cultured PHTs.
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Fig. 3. Inflammatory signaling pathways after fatty acid stimulation. A–E: phosphorylation of ERK (A), expression of IB␣ (B), phosphorylation of JNK (C), p38 MAPK (D), and STAT3 (E) in cultured primary human trophoblast cells after exposure to fatty acids for 24 h (DHA: 50 M, OA: 100 M, PA: 100 M) compared with vehicle controls. F: representative Western blot. P-, phosphorylated form. Data are means ⫾ SE. *P ⬍ 0.05 (paired t-test).
DHA inhibited both trophoblast cellular signaling (p38 MAPK, STAT3, and mTOR) and amino acid transport activity. This may reflect a cause-and-effect relationship. We have previously shown that p38 MAPK (3), STAT3 (18), and mTOR signaling (27, 28) are positive regulators of trophoblast amino acid transport. However, further studies are required to definitely establish through which of these pathways DHA affects amino acid transport. We have recently shown that placental p38 MAPK and STAT3 phosphorylation positively correlate with maternal body mass index (5), with obese mothers having an increased risk of delivering a large baby (34). Because placental amino acid transporter expression
positively correlates with birth weight (15), increasing DHA levels could represent a mechanism to prevent fetal overgrowth by alleviating excessive placental uptake of amino acids. Consistent with this hypothesis, preliminary data from our laboratory suggest that supplementation of DHA to obese pregnant women normalizes placental amino acid transport capacity (Lager et al., unpublished observations). We have previously reported that 400 M OA stimulates trophoblast system A amino acid transporter activity (19). In the present report, we extended these observations by showing that OA also increases system A activity at 100 M, which is a more physiological concentration (37). Furthermore, in our
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Fig. 4. Mechanistic target of rapamycin (mTOR) signaling after fatty acid stimulation. A–E: phosphorylation of AMP-activated protein kinase (AMPK; A), mTOR (B), eukaryotic initiation factor 4E-binding protein (4EBP)1 (C), S6 kinase (S6K)1 (D), and ribosomal protein (rp)S6 (E) in cultured primary human trophoblast cells after exposure to fatty acids for 24 h (DHA: 50 M, OA: 100 M, PA: 100 M) compared with vehicle controls. F: representative Western blot. Data are means ⫾ SE. *P ⬍ 0.05 (paired t-test).
previous study (19), we showed that OA (400 M) activated JNK and STAT3 while having no effect on the mTOR pathway. In the present study, OA (100 M) had a major impact on mTOR signaling, whereas STAT3 phosphorylation only tended to be increased. This discrepancy is likely due to the different fatty acid concentrations used and highlights that not only fatty acid species but also concentration will impact cellular signaling. A similar observation has previously been reported by Arous and coworkers (2); they showed that low and high doses of OA differentially regulated mTOR signaling in HepG2 cells. Individually, DHA and OA had distinct effects on PHT amino acid transport and cellular signaling. However, in vivo, the syncytiotrophoblast is exposed to a combination of
different fatty acids from maternal blood. The combination of DHA and OA resulted in increased system A activity and phosphorylation of rpS6. All other effects on cellular signaling by the fatty acids individually were counteracted in the combination experiments. Because mTOR signaling is a powerful regulator of trophoblast amino acid transporters (27–29), these findings suggest the effect of fatty acids on system A amino acid transport is mediated by the mTOR signaling pathway. Furthermore, when DHA and OA were combined, the reduction in system L amino acid transport activity observed in response to DHA alone was prevented. Taken together, these findings highlight the importance of not only studying fatty acids individually but also fatty acids in combination.
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PA has been reported to inhibit system A activity in rat muscle (14). In contrast, PA did not affect trophoblast amino acid transport in the present study. The effects of exposure to PA were limited to decreased expression of IB␣. Reduced IB␣ may suggest, but does not conclusively prove, increased activation of NF-B, a transcription factor associated with inflammation. Our findings are in agreement with previously published work demonstrating that PA reduces IB␣ expression in skeletal muscle (31). The activation of inflammatory pathways in PHTs by PA appears specific to IB␣ because no additional signaling pathways were affected. In agreement with our findings, PA does not alter the activity of ERK, JNK, or p38 MAPK signaling pathways in BeWo cells (a trophoblastlike choriocarcinoma cell line) (30). During pregnancy, maternal NEFA concentrations are less affected by fasting/postprandial cycles than in nonpregnant individuals, resulting in more constant levels of circulating NEFAs (13). Furthermore, maternal triglyceride and NEFA levels continue to increase as pregnancy progresses (1, 9). To mimic this chronic fatty acid exposure, PHTs were incubated in fatty acids for 24 h. However, it cannot be excluded that the effects of fatty acids on trophoblast cell signaling and amino acid transport may be different after acute exposure. In the present study, 100 M OA and 100 M PA were selected because they are within the span of observed concentrations in the maternal circulation at the end of pregnancy (37). DHA at 50 M is, however, above the physiological range, and the effect on trophoblast amino acid transport was therefore confirmed with a lower dose (25 M DHA). In the present report, we did not investigate the specific receptors or alternative mechanisms linking fatty acids to the signaling pathways and amino acid transporter systems under study. It is likely that TLR4 is involved in mediating the effects of OA, as we have previously shown (19). An alternative possibility for the effects of DHA could be activation of the GPR120 pathway. Membrane-bound GPR120 is expressed in the human placenta (21). GPR120 signaling interferes with inflammatory pathways by preventing formation of the complex between transforming growth factor--activated kinase (TAK)1 and TAK1-binding protein 1 (25), a complex that is upstream of IKB, JNK, and p38 MAPK (8). Hence, activation of GPR120 signaling may explain the reduced p38 MAPK phosphorylation seen with DHA exposure. Fatty acids can also affect signaling through intracellular receptors, such as peroxisome proliferator-activated receptors (11), and by affecting lipid rafts in the cell membranes (36). The precise mechanisms by which DHA, OA, and PA differentially modulate cellular signaling in PHTs remain to be established. Altered fatty acid content in the maternal diet has been shown to affect fetal growth (12) and placental efficiency (22) as well as resulting in developmental programming of offspring (32) in animal models. The possible role of the placenta in developmental programming has been highlighted in recent reviews (16, 35). In the present study, we show that the fatty acids DHA, OA, and PA have a differential effect on placental amino acid transport and cellular signaling pathways. Taken together, these findings suggest that fatty acids could alter the intrauterine environment by modifying placental function, thereby having long-term health effects upon the developing fetus.
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ACKNOWLEDGMENTS The authors are grateful to the patients and staff at Labor & Delivery, University Hospital in San Antonio, for the donation of placentas and tissue collection, to E. Miller for logistics concerning placenta collection, and to W. G. D. Frederick II for valuable comments on the manuscript. GRANTS This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-089989 (to T. L. Powell) and grants from the Swedish Research Council (to S. Lager) and Swedish Society of Endocrinology (to S. Lager). DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS Author contributions: S.L., T.J., and T.L.P. conception and design of research; S.L. performed experiments; S.L. analyzed data; S.L., T.J., and T.L.P. interpreted results of experiments; S.L. prepared figures; S.L. drafted manuscript; S.L., T.J., and T.L.P. edited and revised manuscript; S.L., T.J., and T.L.P. approved final version of manuscript. REFERENCES 1. Alvarez JJ, Montelongo A, Iglesias A, Lasuncion MA, Herrera E. Longitudinal study on lipoprotein profile, high density lipoprotein subclass, and postheparin lipases during gestation in women. J Lipid Res 37: 299 –308, 1996. 2. Arous C, Naimi M, Van Obberghen E. Oleate-mediated activation of phospholipase D and mammalian target of rapamycin (mTOR) regulates proliferation and rapamycin sensitivity of hepatocarcinoma cells. Diabetologia 54: 954 –964, 2011. 3. Aye IL, Gao X, Weintraub ST, Jansson T, Powell TL. Adiponectin inhibits insulin function in primary trophoblasts by PPARalpha-mediated ceramide synthesis. Mol Endocrinol 28: 512–524, 2014. 4. Aye ILMH, Jansson T, Powell TL. Interleukin-1 inhibits insulin signaling and prevents insulin-stimulated system A amino acid transport in primary human trophoblasts. Mol Cell Endocrinol 381: 46 –55, 2013. 5. Aye ILMH, Lager S, Ramirez VI, Gaccioli F, Dudley DJ, Jansson T, Powell TL. Increasing maternal body mass index is associated with systemic inflammation in the mother and the activation of distinct placental inflammatory pathways. Biol Reprod 90: 129, 2014. 6. Beijar EC, Mallard C, Powell TL. Expression and subcellular localization of TLR-4 in term and first trimester human placenta. Placenta 27: 322–326, 2006. 7. Coll T, Jove M, Rodriguez-Calvo R, Eyre E, Palomer X, Sanchez RM, Merlos M, Laguna JC, Vazquez-Carrera M. Palmitate-mediated downregulation of peroxisome proliferator-activated receptor-␥ coactivator 1␣ in skeletal muscle cells involves MEK1/2 and nuclear factor-B activation. Diabetes 55: 2779 –2787, 2006. 8. Dai L, Thu CA, Liu XY, Xi JJ, Cheung PC. TAK1, more than just innate immunity. IUBMB Life 64: 825–834, 2012. 9. Damjanovic SS, Stojic RV, Lalic NM, Jotic AZ, Macut DP, Ognjanovic SI, Petakov MS, Popovic BM. Relationship between basal metabolic rate and cortisol secretion throughout pregnancy. Endocrine 35: 262–268, 2009. 10. Elchalal U, Schaiff WT, Smith SD, Rimon E, Bildirici I, Nelson DM, Sadovsky Y. Insulin and fatty acids regulate the expression of the fat droplet-associated protein adipophilin in primary human trophoblasts. Am J Obstet Gynecol 193: 1716 –1723, 2005. 11. Fournier T, Tsatsaris V, Handschuh K, Evain-Brion D. PPARs and the placenta. Placenta 28: 65–76, 2007. 12. Gaccioli F, White V, Capobianco E, Powell TL, Jawerbaum A, Jansson T. Maternal overweight induced by a diet with high content of saturated fat activates placental mtor and eIF2␣ signaling and increases fetal growth in rats. Biol Reprod 89: 96, 2013. 13. Haggarty P. Fatty acid supply to the human fetus. Annu Rev Nutr 30: 237–255, 2010. 14. Hyde R, Hajduch E, Powell DJ, Taylor PM, Hundal HS. Ceramide down-regulates system A amino acid transport and protein synthesis in rat skeletal muscle cells. FASEB J 18: 461–463, 2004.
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15. Jansson N, Rosario FJ, Gaccioli F, Lager S, Jones HN, Roos S, Jansson T, Powell TL. Activation of placental mTOR signaling and amino acid transporters in obese women giving birth to large babies. J Clin Endocrinol Metab 98: 105–113, 2013. 16. Jansson T, Powell TL. Role of the placenta in fetal programming: underlying mechanisms and potential interventional approaches. Clin Sci (Lond) 113: 1–13, 2007. 17. Jing K, Song KS, Shin S, Kim N, Jeong S, Oh HR, Park JH, Seo KS, Heo JY, Han J, Park JI, Han C, Wu T, Kweon GR, Park SK, Yoon WH, Hwang BD, Lim K. Docosahexaenoic acid induces autophagy through p53/AMPK/mTOR signaling and promotes apoptosis in human cancer cells harboring wild-type p53. Autophagy 7: 1348 –1358, 2011. 18. Jones HN, Jansson T, Powell TL. IL-6 stimulates system A amino acid transporter activity in trophoblast cells through STAT3 and increased expression of SNAT2. Am J Physiol Cell Physiol 297: C1228 –C1235, 2009. 19. Lager S, Gaccioli F, Ramirez VI, Jones HN, Jansson T, Powell TL. Oleic acid stimulates system A amino acid transport in primary human trophoblast cells mediated by toll-like receptor 4. J Lipid Res 54: 725–733, 2013. 20. Lager S, Jansson N, Olsson AL, Wennergren M, Jansson T, Powell TL. Effect of IL-6 and TNF-␣ on fatty acid uptake in cultured human primary trophoblast cells. Placenta 32: 121–127, 2011. 21. Lager S, Ramirez VI, Gaccioli F, Jansson T, Powell TL. Expression and localization of the omega-3 fatty acid receptor GPR120 in human term placenta. Placenta 35: 523–525, 2014. 22. Lager S, Samuelsson AM, Taylor PD, Poston L, Powell TL, Jansson T. Diet-induced obesity in mice reduces placental efficiency and inhibits placental mTOR signaling. Physiol Rep 2: e00242, 2014. 23. Laplante M, Sabatini DM. mTOR signaling in growth control and disease. Cell 149: 274 –293, 2012. 24. Magnusson-Olsson AL, Lager S, Jacobsson B, Jansson T, Powell TL. Effect of maternal triglycerides and free fatty acids on placental LPL in cultured primary trophoblast cells and in a case of maternal LPL deficiency. Am J Physiol Endocrinol Metab 293: E24 –E30, 2007. 25. Oh DY, Talukdar S, Bae EJ, Imamura T, Morinaga H, Fan WQ, Li PP, Lu WJ, Watkins SM, Olefsky JM. GPR120 is an omega-3 fatty acid receptor mediating potent anti-inflammatory and insulin-sensitizing effects. Cell 142: 687–698, 2010. 26. Pathmaperuma AN, Mana P, Cheung SN, Kugathas K, Josiah A, Koina ME, Broomfield A, Delghingaro-Augusto V, Ellwood DA, Dahlstrom JE, Nolan CJ. Fatty acids alter glycerolipid metabolism and induce lipid droplet formation, syncytialisation and cytokine production in human trophoblasts with minimal glucose effect or interaction. Placenta 31: 230 –239, 2010.
27. Roos S, Kanai Y, Prasad PD, Powell TL, Jansson T. Regulation of placental amino acid transporter activity by mammalian target of rapamycin. Am J Physiol Cell Physiol 296: C142–C150, 2009. 28. Roos S, Lagerlof O, Wennergren M, Powell TL, Jansson T. Regulation of amino acid transporters by glucose and growth factors in cultured primary human trophoblast cells is mediated by mTOR signaling. Am J Physiol Cell Physiol 297: C723–C731, 2009. 29. Rosario FJ, Kanai Y, Powell TL, Jansson T. Mammalian target of rapamycin signalling modulates amino acid uptake by regulating transporter cell surface abundance in primary human trophoblast cells. J Physiol 591: 609 –625, 2013. 30. Saben J, Zhong Y, Gomez-Acevedo H, Thakali KM, Borengasser SJ, Andres A, Shankar K. Early growth response protein-1 mediates lipotoxicity-associated placental inflammation: role in maternal obesity. Am J Physiol Endocrinol Metab 305: E1–E14, 2013. 31. Salvado L, Coll T, Gomez-Foix AM, Salmeron E, Barroso E, Palomer X, Vazquez-Carrera M. Oleate prevents saturated-fatty-acid-induced ER stress, inflammation and insulin resistance in skeletal muscle cells through an AMPK-dependent mechanism. Diabetologia 56: 1372–1382, 2013. 32. Samuelsson AM, Matthews PA, Argenton M, Christie MR, McConnell JM, Jansen EHJM, Piersma AH, Ozanne SE, Twinn DF, Remacle C, Rowlerson A, Poston L, Taylor PD. Diet-induced obesity in female mice leads to offspring hyperphagia, adiposity, hypertension, and insulin resistance–a novel murine model of developmental programming. Hypertension 51: 383–392, 2008. 33. Scifres CM, Chen B, Nelson DM, Sadovsky Y. Fatty acid binding protein 4 regulates intracellular lipid accumulation in human trophoblasts. J Clin Endocrinol Metab 96: E1083–E1091, 2011. 34. Sebire NJ, Jolly M, Harris JP, Wadsworth J, Joffe M, Beard RW, Regan L, Robinson S. Maternal obesity and pregnancy outcome: a study of 287,213 pregnancies in London. Int J Obes Relat Metab Disord 25: 1175–1182, 2001. 35. Sibley CP, Brownbill P, Dilworth M, Glazier JD. Review: Adaptation in placental nutrient supply to meet fetal growth demand: implications for programming. Placenta 31, Suppl: S70 –S74, 2010. 36. Turk HF, Chapkin RS. Membrane lipid raft organization is uniquely modified by n-3 polyunsaturated fatty acids. Prostag Leukotr Ess 88: 43–47, 2013. 37. Villa PM, Laivuori H, Kajantie E, Kaaja R. Free fatty acid profiles in preeclampsia. Prostaglandins Leukot Essent Fatty Acids 81: 17–21, 2009. 38. Weatherill AR, Lee JY, Zhao L, Lemay DG, Youn HS, Hwang DH. Saturated and polyunsaturated fatty acids reciprocally modulate dendritic cell functions mediated through TLR4. J Immunol 174: 5390 –5397, 2005. 39. Yuan HP, Zhang XY, Huang XQ, Lu YG, Tang WQ, Man Y, Wang S, Xi JZ, Li JA. NADPH oxidase 2-derived reactive oxygen species mediate FFAs-induced dysfunction and apoptosis of -cells via JNK, p38 MAPK and p53 pathways. PloS one 5: e15726, 2010.
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Differential regulation of placental amino acid transport by saturated and unsaturated fatty acids Susanne Lager, Thomas Jansson, and Theresa L. Powell Center for Pregnancy and Newborn Research, Department of Obstetrics and Gynecology, University of Texas Health Science Center, San Antonio, Texas Submitted 18 June 2014; accepted in final form 19 August 2014
Lager S, Jansson T, Powell TL. Differential regulation of placental amino acid transport by saturated and unsaturated fatty acids. Am J Physiol Cell Physiol 307: C738 –C744, 2014. First published August 20, 2014; doi:10.1152/ajpcell.00196.2014.—Fatty acids are critical for normal fetal development but may also influence placental function. We have previously reported that oleic acid (OA) stimulates amino acid transport in primary human trophoblasts (PHTs). In other tissues, saturated and unsaturated fatty acids have distinct effects on cellular signaling, for instance, palmitic acid (PA) but not OA reduces IB␣ expression. We hypothesized that saturated and unsaturated fatty acids differentially affect trophoblast amino acid transport and cellular signaling. To test this hypothesis, PHTs were cultured in docosahexaenoic acid (DHA; 50 M), OA (100 M), or PA (100 M). DHA and OA were also combined to test whether DHA could counteract the OA stimulatory effect on amino acid transport. The effects of fatty acids were compared against a vehicle control. Amino acid transport was measured by isotope-labeled tracers. Activation of inflammatory-related signaling pathways and the mechanistic target of rapamycin (mTOR) pathway were determined by Western blot analysis. Exposure of PHTs to DHA for 24 h reduced amino acid transport and phosphorylation of p38 MAPK, STAT3, mTOR, eukaryotic initiation factor 4E-binding protein 1, and ribosomal protein (rp)S6. In contrast, OA increased amino acid transport and phosphorylation of ERK, mTOR, S6 kinase 1, and rpS6. The combination of DHA with OA increased amino acid transport and rpS6 phosphorylation. PA did not affect amino acid transport but reduced IB␣ expression. In conclusion, these fatty acids differentially regulated placental amino acid transport and cellular signaling. Taken together, these findings suggest that dietary fatty acids could alter the intrauterine environment by modifying placental function, thereby having long-lasting effects on the developing fetus. docosahexaenoic acid; oleic acid; palmitic acid; pregnancy; cell signaling FATTY ACIDS are critical for fetal development. They are essential structural components of cellular membranes and precursors of bioactive compounds, with particularly large amounts being deposited in the fetal brain (13). Fatty acids can also influence cellular signaling through a multitude of mechanisms, with effects dependent on their level of saturation. For instance, saturated and polyunsaturated fatty acids differentially modulate Toll-like receptor (TLR)4 signaling (38). The polyunsaturated -3 fatty acid docosahexaenoic acid (DHA; 22:6 n-3) has been shown to exert anti-inflammatory actions, partly mediated via the membrane-bound G protein-coupled receptor 120 (GPR120) (25). Both TLR4 (6) and GPR120 (21) are expressed on the syncytiotrophoblast microvillous plasma
Address for reprint requests and present address: S. Lager, Dept. of Obstetrics and Gynaecology, Univ. of Cambridge, Box 223, Robinson Way, The Rosie Hospital, Cambridge CB2 0SW, UK (e-mail: [email protected]. ac.uk). C738
membrane in the human-term placenta, consistent with the possibility that maternal fatty acids modulate placental function (10, 24, 26, 33) through interactions with these receptors. In a variety of tissues, it has been shown that the fatty acids DHA, oleic acid (OA; 18:1 n-9), and palmitic acid (PA; 16:0) have distinct effects on inflammatory signaling pathways. For instance, DHA inhibits, whereas PA stimulates, activity of the IB/NF-B signaling pathway (7, 25, 31). Other inflammatoryrelated pathways responding to fatty acids in other tissues include STAT3 (19) as well as MAPK pathways (ERK, JNK, and p38 MAPK) (2, 19, 25, 39). In addition to inflammatory pathways, fatty acids can also influence mechanistic target of rapamycin (mTOR) (2, 17, 31), a serine/threonine kinase regulating protein translation, cell growth, and metabolism (23). Activation of this kinase results in the phosphorylation of eukaryotic initiation factor 4E-binding protein (4EBP)1, S6 kinase (S6K)1, and ribosomal protein (rp)S6 (23). In the context of placental function, we have shown that the mTOR pathway is a positive regulator of trophoblast amino acid transport (27–29). At the end of pregnancy, OA and PA together constitute ⬃60% of the nonesterified fatty acid (NEFA) species in the maternal circulation (37), and NEFA levels correlate positively with maternal body mass index (19). We have previously reported that OA stimulates placental amino acid transport in primary human trophoblasts (PHTs) (19). This effect is dependent on TLR4 (19); however, the cellular signaling pathways mediating the activation of amino acid transport by OA remain to be fully established. In addition, regulation of trophoblast amino acid transport by DHA and PA has not previously been studied. Therefore, the aim of the present study was to determine the effects of DHA, OA, and PA on amino acid transport activity and cellular signaling in cultured PHTs. We tested the hypothesis that these fatty acids differentially regulate trophoblast amino acid transport and cellular signaling. MATERIALS AND METHODS
Ethics. Human placental tissue samples from term pregnancies (ⱖ37 ⫾ 0 wk of gestation) were collected after written informed consent. Placental samples and medical information were added to a tissue/data repository. The protocol was approved by the Institutional Review Board of the University of Texas Health Science Center (San Antonio, TX, HSC20100262H). De-identified tissue samples were made available for the present study. Isolation and maintenance of trophoblast cells. Villous cytotrophoblasts were isolated as previously described (4, 20). Trophoblast cells were separated by DNAse/trypsin digestion and purified on a Percoll gradient. Cells were plated at an approximate density of 150,000 cells/cm2. Cell culture media (equal volumes of Ham’s F-12 and high-glucose DMEM) contained 10% FBS (S11550, Atlanta Biologicals, Lawrenceville, GA) and antibiotics (gentamicin, penicillin, and
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streptomycin). Media were changed daily. Starting at 66 h after being plated, cells were cultured in media containing 1% FBS supplemented with BSA-bound fatty acids (DHA: 25 or 50 M, OA: 100 M, PA: 100 M), and each fatty acid treatment had its own control (containing cell culture media with an equal amount of fatty acid-free BSA stock buffer). Twenty-four hours later (at 90 h after cell plating), amino acid uptake was measured, cell viability was assessed, or samples were collected for protein expression analysis. Characterization and viability of isolated cells. As an indicator of biochemical differentiation, the release of human chorionic gonadotropin into the cell culture media was measured using a commercial ELISA (IBL-America, Minneapolis, MN). Cell viability was determined by the ability of the cultured cells to metabolize MTT (thiazolyl blue tetrazolium bromide, Sigma-Aldrich, St Louis, MO), and cleaved caspase-3 was used to measure apoptosis. Harvesting cells. Trophoblast cells were harvested by the addition of RIPA buffer containing phosphatase inhibitor cocktails 1 and 2 and protease inhibitor cocktail (final dilution: 1:100, Sigma-Aldrich) and collected after being scraped with a spatula. Cells were stored at ⫺80°C until further analysis. Protein concentrations were determined by the bicinchoninic acid assay method (Thermo Fisher Scientific, Waltham, MA). Measurement of amino acid uptake. System A amino acid transporter activity was measured as Na⫹-dependent [14C]methylaminoisobuturyric acid (final concentration: 20 M) uptake, and system L transporter activity was measured by 2-amino-2-norbornanecarboxylic acid (BCH)inhibitable uptake of [3H]leucine (final concentration: 12.5 nM), as previously described (4, 19). Transporter-mediated uptakes were calculated by subtracting uptake in Na⫹-free/BCH buffer (nonmediated uptake) from uptake in Na⫹-containing buffer (total uptake). Protein con-
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centrations were measured with the Lowry method. Uptakes were expressed as picomoles of amino acid taken up per milligram of protein per minute. The mean uptake of controls was arbitrarily assigned a value of 1.0 to facilitate comparisons between groups. Western blot analysis. Western blots were performed on precast gels from Bio-Rad (Hercules, CA). After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes (overnight, 4°C). Membranes were stained for total protein with amido black stain (Sigma-Aldrich) and blocked in 5% nonfat milk (1 h, room temperature). The following primary antibodies were purchased from Cell Signaling Technology (Davers, MA): 4EBP1 (no. 9452), phosphorylated (P)-4EBP1 (Thr37/46, no. 9459), AMP-activated protein kinase (AMPK; no. 2532), P-AMPK (Thr172, no. 2535), caspase-3 (no. 9662), ERK (no. 4695), P-ERK (Thr202/Tyr204, no. 4370), IB␣ (no. 4812), JNK (no. 9252), P-JNK (Thr183/Tyr185, no. 4668), mTOR (no. 2983), P-mTOR (Ser2448, no. 5536), p38 MAPK (no. 9212), S6K1 (no. 9202), P-S6K1 (Thr389, no. 9205), rpS6 (no. 2217), P-rpS6 (Ser235/236, no. 4858), STAT3 (no. 9139), and P-STAT3 (Tyr705, no. 9145). The primary antibody targeting P-p38 MAPK (Thr180/Tyr182) was purchased from Abcam (ab32557, Cambridge, UK). Primary antibodies were diluted in Tris-buffered saline containing 1–3% BSA, and incubations were carried out overnight (4°C). After incubation with the appropriate secondary peroxidase-labeled IgG antibody (1 h, room temperature), immunolabeling was detected using SuperSignal Dura West (Thermo Fisher Scientific) in a G:Box ChemiXL1.4 (Syngene, Cambridge, UK). The relative density of bands was measured by densitometry (GeneTools, Syngene), and protein expression was adjusted for amount of total protein (amido black stain). The mean value of controls was arbitrarily assigned a value of 1.0 to facilitate comparisons between groups.
Fig. 1. Characterization of cultured human trophoblast cells. A: release of human chorionic gonadotropin (hCG) into the cell culture media. *P ⬍ 0.05; **P ⬍ 0.01 vs. 18 and 42 h (repeated-measures ANOVA followed by Tukey’s post hoc test). B and C: cell viability measured by MTT assay [B; control as 100% (dotted line)] and apoptosis measured by the amount of cleaved caspase-3 (C) after 24-h exposure to fatty acids [docosahexaenoic acid (DHA): 50 M, oleic acid (OA): 100 M, palmitic acid (PA): 100 M] compared with vehicle controls (C). D: representative Western blot. AU, arbitrary units. Data are means ⫾ SE.
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Data presentation and statistics. The number of experiments (n) represents the number of placentas studied. Data are presented as means ⫾ SE. Statistical analysis was carried out with GraphPad Prism 5 (version 5.04, GraphPad Software, La Jolla, CA). Differences between two groups were evaluated statistically by paired t-test and for multiple groups by repeated-measures ANOVA followed by Tukey’s multiple-comparison test. P values of ⬍0.05 were considered significant. RESULTS
Characterization and viability of isolated trophoblast cells. The secretion of human chorionic gonadotropin into cell culture media was significantly higher after 66 and 90 h of culture compared with 18 and 42 h of culture, indicating biochemical differentiation (n ⫽ 12, P ⬍ 0.01; Fig. 1A). Twenty-four-hour treatments with fatty acids (DHA: 50 M, OA: 100 M, PA: 100 M) did not affect cell viability (n ⫽ 4; Fig. 1B), nor did it increase apoptosis (n ⫽ 4 –7; Fig. 1C). Amino acid transport. Mean amino acid transport activity in control PHTs (system A activity: 10 ⫾ 1.3 pmol·mg⫺1·min⫺1; system L activity: 0.1 ⫾ 0.01 pmol·mg⫺1·min⫺1) was similar to what we have previously reported (4). Activity of the system A amino acid transporter was markedly decreased after exposure to 25 or 50 M DHA (n ⫽ 10, P ⬍ 0.05; Fig. 2A). In contrast, incubation in 100 M OA stimulated system A activity (n ⫽ 7, P ⬍ 0.01; Fig. 2A). Cotreatment of trophoblast cells with DHA (50 M) and OA (100 M) did not prevent OA stimulation of system A activity (n ⫽ 7, P ⬍ 0.01; Fig. 2A). PA (100 M) did not alter trophoblast system A amino acid transport activity (n ⫽ 7; Fig. 2A). Uptake of amino acids by the system L amino acid transporter was significantly reduced by 25 or 50 M DHA (n ⫽ 10, P ⬍ 0.05; Fig. 2B). System L amino acid transport activity was not affected by 100 M OA or 100 M PA treatment (n ⫽ 7; Fig. 2B). The decrease in system L activity in response to 50 M DHA was prevented by cotreatment with 100 M OA (n ⫽ 7; Fig. 2B). Inflammatory signaling. DHA or PA alone did not alter the phosphorylation of ERK. In contrast, ERK phosphorylation was significantly increased by OA (n ⫽ 7, P ⬍ 0.05; Fig. 3A). Activation of the ERK signaling pathway by OA was prevented by coincubation with DHA (Fig. 3A). Expression of IB␣ was decreased by PA treatment (n ⫽ 7, P ⬍ 0.05; Fig. 3B), whereas it was unaffected by DHA and/or OA. JNK phosphorylation was not altered by any of the fatty acids studied (Fig. 3C). DHA inhibited p38 MAPK and STAT3 signaling (n ⫽ 9, P ⬍ 0.05; Fig. 3, D and E), whereas there was a trend toward increased STAT3 phosphorylation in response to OA (P ⫽ 0.07). PA or co-treatment with DHA and OA had no significant effect on p38 MAPK or STAT3 inflammatory signaling pathways. Total protein expression of ERK, JNK, and p38 MAPK was not affected by the fatty acid treatments, whereas total STAT3 was significantly decreased by exposure to DHA (P ⬍ 0.05; data not shown). AMPK and mTOR signaling. The cellular energy state, as measured by AMPK phosphorylation, was not affected by the fatty acid treatments (n ⫽ 6 –7; Fig. 4A). Phosphorylation of mTOR was reduced by DHA and increased by OA treatment (n ⫽ 9, P ⬍ 0.05; Fig. 4B). Downstream of mTOR, phosphorylation of 4EBP1 and rpS6 was lower in PHTs exposed to DHA (n ⫽ 9, P ⬍ 0.05; Fig. 4, C and E). OA increased the phosphorylation of S6K1 and rpS6 (n ⫽ 9, P ⬍ 0.05; Fig. 4, D and E) but not 4EBP1. Co-treatment of cells with DHA and
Fig. 2. Amino acid uptakes after fatty acid stimulation. A and B: system A (A) and system L (B) amino acid transport activity in cultured primary human trophoblast cells after exposure to fatty acids for 24 h (DHA: 25 or 50 M, OA: 100 M, PA: 100 M) compared with vehicle controls. Data are means ⫾ SE. *P ⬍ 0.05; **P ⬍ 0.01 (paired t-test).
OA resulted in unaltered phosphorylation levels of 4EBP1 and S6K1, whereas rpS6 phosphorylation was increased compared with control (n ⫽ 9, P ⬍ 0.05; Fig. 4E). Total protein expression of AMPK, mTOR, 4EBP1, and S6K1 was not affected by the fatty acid treatments, whereas total rpS6 was significantly decreased by DHA and increased by OA (P ⬍ 0.05; data not shown). DISCUSSION
In the present report, we show, for the first time, that the fatty acids DHA, OA, and PA differentially regulate trophoblast amino acid transport. Similarly, these fatty acids had diverse effects on inflammatory and mTOR signaling pathways in cultured PHTs.
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Fig. 3. Inflammatory signaling pathways after fatty acid stimulation. A–E: phosphorylation of ERK (A), expression of IB␣ (B), phosphorylation of JNK (C), p38 MAPK (D), and STAT3 (E) in cultured primary human trophoblast cells after exposure to fatty acids for 24 h (DHA: 50 M, OA: 100 M, PA: 100 M) compared with vehicle controls. F: representative Western blot. P-, phosphorylated form. Data are means ⫾ SE. *P ⬍ 0.05 (paired t-test).
DHA inhibited both trophoblast cellular signaling (p38 MAPK, STAT3, and mTOR) and amino acid transport activity. This may reflect a cause-and-effect relationship. We have previously shown that p38 MAPK (3), STAT3 (18), and mTOR signaling (27, 28) are positive regulators of trophoblast amino acid transport. However, further studies are required to definitely establish through which of these pathways DHA affects amino acid transport. We have recently shown that placental p38 MAPK and STAT3 phosphorylation positively correlate with maternal body mass index (5), with obese mothers having an increased risk of delivering a large baby (34). Because placental amino acid transporter expression
positively correlates with birth weight (15), increasing DHA levels could represent a mechanism to prevent fetal overgrowth by alleviating excessive placental uptake of amino acids. Consistent with this hypothesis, preliminary data from our laboratory suggest that supplementation of DHA to obese pregnant women normalizes placental amino acid transport capacity (Lager et al., unpublished observations). We have previously reported that 400 M OA stimulates trophoblast system A amino acid transporter activity (19). In the present report, we extended these observations by showing that OA also increases system A activity at 100 M, which is a more physiological concentration (37). Furthermore, in our
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Fig. 4. Mechanistic target of rapamycin (mTOR) signaling after fatty acid stimulation. A–E: phosphorylation of AMP-activated protein kinase (AMPK; A), mTOR (B), eukaryotic initiation factor 4E-binding protein (4EBP)1 (C), S6 kinase (S6K)1 (D), and ribosomal protein (rp)S6 (E) in cultured primary human trophoblast cells after exposure to fatty acids for 24 h (DHA: 50 M, OA: 100 M, PA: 100 M) compared with vehicle controls. F: representative Western blot. Data are means ⫾ SE. *P ⬍ 0.05 (paired t-test).
previous study (19), we showed that OA (400 M) activated JNK and STAT3 while having no effect on the mTOR pathway. In the present study, OA (100 M) had a major impact on mTOR signaling, whereas STAT3 phosphorylation only tended to be increased. This discrepancy is likely due to the different fatty acid concentrations used and highlights that not only fatty acid species but also concentration will impact cellular signaling. A similar observation has previously been reported by Arous and coworkers (2); they showed that low and high doses of OA differentially regulated mTOR signaling in HepG2 cells. Individually, DHA and OA had distinct effects on PHT amino acid transport and cellular signaling. However, in vivo, the syncytiotrophoblast is exposed to a combination of
different fatty acids from maternal blood. The combination of DHA and OA resulted in increased system A activity and phosphorylation of rpS6. All other effects on cellular signaling by the fatty acids individually were counteracted in the combination experiments. Because mTOR signaling is a powerful regulator of trophoblast amino acid transporters (27–29), these findings suggest the effect of fatty acids on system A amino acid transport is mediated by the mTOR signaling pathway. Furthermore, when DHA and OA were combined, the reduction in system L amino acid transport activity observed in response to DHA alone was prevented. Taken together, these findings highlight the importance of not only studying fatty acids individually but also fatty acids in combination.
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PA has been reported to inhibit system A activity in rat muscle (14). In contrast, PA did not affect trophoblast amino acid transport in the present study. The effects of exposure to PA were limited to decreased expression of IB␣. Reduced IB␣ may suggest, but does not conclusively prove, increased activation of NF-B, a transcription factor associated with inflammation. Our findings are in agreement with previously published work demonstrating that PA reduces IB␣ expression in skeletal muscle (31). The activation of inflammatory pathways in PHTs by PA appears specific to IB␣ because no additional signaling pathways were affected. In agreement with our findings, PA does not alter the activity of ERK, JNK, or p38 MAPK signaling pathways in BeWo cells (a trophoblastlike choriocarcinoma cell line) (30). During pregnancy, maternal NEFA concentrations are less affected by fasting/postprandial cycles than in nonpregnant individuals, resulting in more constant levels of circulating NEFAs (13). Furthermore, maternal triglyceride and NEFA levels continue to increase as pregnancy progresses (1, 9). To mimic this chronic fatty acid exposure, PHTs were incubated in fatty acids for 24 h. However, it cannot be excluded that the effects of fatty acids on trophoblast cell signaling and amino acid transport may be different after acute exposure. In the present study, 100 M OA and 100 M PA were selected because they are within the span of observed concentrations in the maternal circulation at the end of pregnancy (37). DHA at 50 M is, however, above the physiological range, and the effect on trophoblast amino acid transport was therefore confirmed with a lower dose (25 M DHA). In the present report, we did not investigate the specific receptors or alternative mechanisms linking fatty acids to the signaling pathways and amino acid transporter systems under study. It is likely that TLR4 is involved in mediating the effects of OA, as we have previously shown (19). An alternative possibility for the effects of DHA could be activation of the GPR120 pathway. Membrane-bound GPR120 is expressed in the human placenta (21). GPR120 signaling interferes with inflammatory pathways by preventing formation of the complex between transforming growth factor--activated kinase (TAK)1 and TAK1-binding protein 1 (25), a complex that is upstream of IKB, JNK, and p38 MAPK (8). Hence, activation of GPR120 signaling may explain the reduced p38 MAPK phosphorylation seen with DHA exposure. Fatty acids can also affect signaling through intracellular receptors, such as peroxisome proliferator-activated receptors (11), and by affecting lipid rafts in the cell membranes (36). The precise mechanisms by which DHA, OA, and PA differentially modulate cellular signaling in PHTs remain to be established. Altered fatty acid content in the maternal diet has been shown to affect fetal growth (12) and placental efficiency (22) as well as resulting in developmental programming of offspring (32) in animal models. The possible role of the placenta in developmental programming has been highlighted in recent reviews (16, 35). In the present study, we show that the fatty acids DHA, OA, and PA have a differential effect on placental amino acid transport and cellular signaling pathways. Taken together, these findings suggest that fatty acids could alter the intrauterine environment by modifying placental function, thereby having long-term health effects upon the developing fetus.
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ACKNOWLEDGMENTS The authors are grateful to the patients and staff at Labor & Delivery, University Hospital in San Antonio, for the donation of placentas and tissue collection, to E. Miller for logistics concerning placenta collection, and to W. G. D. Frederick II for valuable comments on the manuscript. GRANTS This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-089989 (to T. L. Powell) and grants from the Swedish Research Council (to S. Lager) and Swedish Society of Endocrinology (to S. Lager). DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS Author contributions: S.L., T.J., and T.L.P. conception and design of research; S.L. performed experiments; S.L. analyzed data; S.L., T.J., and T.L.P. interpreted results of experiments; S.L. prepared figures; S.L. drafted manuscript; S.L., T.J., and T.L.P. edited and revised manuscript; S.L., T.J., and T.L.P. approved final version of manuscript. REFERENCES 1. Alvarez JJ, Montelongo A, Iglesias A, Lasuncion MA, Herrera E. Longitudinal study on lipoprotein profile, high density lipoprotein subclass, and postheparin lipases during gestation in women. J Lipid Res 37: 299 –308, 1996. 2. Arous C, Naimi M, Van Obberghen E. Oleate-mediated activation of phospholipase D and mammalian target of rapamycin (mTOR) regulates proliferation and rapamycin sensitivity of hepatocarcinoma cells. Diabetologia 54: 954 –964, 2011. 3. Aye IL, Gao X, Weintraub ST, Jansson T, Powell TL. Adiponectin inhibits insulin function in primary trophoblasts by PPARalpha-mediated ceramide synthesis. Mol Endocrinol 28: 512–524, 2014. 4. Aye ILMH, Jansson T, Powell TL. Interleukin-1 inhibits insulin signaling and prevents insulin-stimulated system A amino acid transport in primary human trophoblasts. Mol Cell Endocrinol 381: 46 –55, 2013. 5. Aye ILMH, Lager S, Ramirez VI, Gaccioli F, Dudley DJ, Jansson T, Powell TL. Increasing maternal body mass index is associated with systemic inflammation in the mother and the activation of distinct placental inflammatory pathways. Biol Reprod 90: 129, 2014. 6. Beijar EC, Mallard C, Powell TL. Expression and subcellular localization of TLR-4 in term and first trimester human placenta. Placenta 27: 322–326, 2006. 7. Coll T, Jove M, Rodriguez-Calvo R, Eyre E, Palomer X, Sanchez RM, Merlos M, Laguna JC, Vazquez-Carrera M. Palmitate-mediated downregulation of peroxisome proliferator-activated receptor-␥ coactivator 1␣ in skeletal muscle cells involves MEK1/2 and nuclear factor-B activation. Diabetes 55: 2779 –2787, 2006. 8. Dai L, Thu CA, Liu XY, Xi JJ, Cheung PC. TAK1, more than just innate immunity. IUBMB Life 64: 825–834, 2012. 9. Damjanovic SS, Stojic RV, Lalic NM, Jotic AZ, Macut DP, Ognjanovic SI, Petakov MS, Popovic BM. Relationship between basal metabolic rate and cortisol secretion throughout pregnancy. Endocrine 35: 262–268, 2009. 10. Elchalal U, Schaiff WT, Smith SD, Rimon E, Bildirici I, Nelson DM, Sadovsky Y. Insulin and fatty acids regulate the expression of the fat droplet-associated protein adipophilin in primary human trophoblasts. Am J Obstet Gynecol 193: 1716 –1723, 2005. 11. Fournier T, Tsatsaris V, Handschuh K, Evain-Brion D. PPARs and the placenta. Placenta 28: 65–76, 2007. 12. Gaccioli F, White V, Capobianco E, Powell TL, Jawerbaum A, Jansson T. Maternal overweight induced by a diet with high content of saturated fat activates placental mtor and eIF2␣ signaling and increases fetal growth in rats. Biol Reprod 89: 96, 2013. 13. Haggarty P. Fatty acid supply to the human fetus. Annu Rev Nutr 30: 237–255, 2010. 14. Hyde R, Hajduch E, Powell DJ, Taylor PM, Hundal HS. Ceramide down-regulates system A amino acid transport and protein synthesis in rat skeletal muscle cells. FASEB J 18: 461–463, 2004.
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