Transport of fatty acids across the human placenta: A review

Progress in Lipid Research 48 (2009) 52–61

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Review

Transport of fatty acids across the human placenta: A review Asim K. Duttaroy * Department of Nutrition, Institute for Basic Medical Sciences, Faculty of Medicine, University of Oslo, POB 1046 Blindern, N-0316 Oslo, Norway

a r t i c l e

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Keywords: Placenta Fatty acids Plasma membrane fatty acid-binding proteins Fatty acid transporter protein Fatty acid translocase Long chain polyunsaturated fatty acids Fetus Brain Trophoblast Human placenta EFA Nuclear transcription factors

a b s t r a c t Essential fatty acids and their long chain polyunsaturated fatty acid derivatives (20 C) such as docosahexaenoic and arachidonic acids are critical for proper fetal growth and development. Dietary intake as well as metabolism of these fatty acids, and their subsequent transfer from the mother to the fetus are therefore important requisites for developing fetus. The placenta is the key organ through which nutrients such as these fatty acids flow from the mother to the fetus. Cellular uptake and translocation of long chain fatty acids (LCFAs) in different tissues is achieved by a concert of co-existing mechanism. Although LCFA can enter the cell via passive diffusion, emerging reports indicate that LCFA uptake is tightly regulated by several plasma membrane-located transport/binding proteins such as fatty acid translocase (FAT/CD36), plasma membrane fatty acid binding protein (FABPpm), fatty acid transport protein (FATP) and intracellular FABPs in several tissues including human placenta. Fatty acid activated transcription factors (PPARs, LXR, RXR, and SREBP-1) have been demonstrated to regulate these fatty acid transport/binding proteins, and placental functions. Maternal fatty acids therefore may regulate their own placental transport as well as placental function via several fatty acid-activated transcription factors. This review summarizes recent developments on placental fatty acid transport and metabolisms, and the regulatory roles of these proteins in these processes. Ó 2008 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3. 4. 5.

6. 7.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Maternal metabolism and supply of essential fatty acids (EFA) and LCPUFA to the fetus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Placental metabolism of fatty acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Uptake of fatty acid by placental trophoblast cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Placental fatty acid uptake and transport: putative roles of fatty acid transport/binding proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Plasma membrane fatty acid binding protein in human placenta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Fatty acid translocase (FAT/CD36) in human placenta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Fatty acid transporter proteins (FATPs) in human placenta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Intracellular fatty acid binding proteins (FABPs). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nuclear transcription factors and their role in fatty acid transport and metabolism in placenta. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Effects of fatty acids on fatty acid transport system of the human placenta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

53 53 54 55 55 55 56 56 57 57 58 59 59 59

Abbreviations: ACSBG, acylCoA syntheatse bubblegum; ADRP, adipose differentiation-related protein; ARA, arachidonic acid; ALA, a linolenic acid; Cyclooxygenase, COX; cPLA2, cytosolic phospholipase A2; DHA, docosahexaneoic acid; EL, endothelial lipase; EFA, essential fatty acids; FABPs, fatty acid binding proteins; FABPpm, plasma membrane fatty acid-binding protein, FATP, fatty acid transporter protein, FAT, fatty acid translocase, hCG, human chorinnic gonadotropin; HETE, hydroxy-5Z,11Z,13Eeicosatetraenoic acid ; HODE, hydroxy-octadedienoic acid; IUGR, intrauterine growth retardation; IDDM, insulin dependent diabetes mellitus; LA, linoleic acid; LCFA, long chain fatty acids; LDL, low density lipoproteins; LPL, lipoprotein lipase; LCPUFA, long chain polyunsaturated fatty acids; LXR, liver X receptor; mAspAT, mitochondrial aspartate aminotransferase; OA, oleic acid; PA, palmitic acid, PPAR, peroxisome proliferator-activated nuclear receptors; PG, prostaglandins, RXR, retinoid X receptor; SREBP1, sterol regulatory element binding prtein-1; VLDL, very low density lipoproteins. * Tel.: +47 22 85 15 47; fax: +47 22 85 13 41. E-mail address: [email protected] 0163-7827/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.plipres.2008.11.001

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1. Introduction Essential fatty acids (EFA) and their long-chain polyunsaturated fatty acids (LCPUFA) are of critical importance in fetal growth and development [1–4]. These fatty acids are the precursors of eicosanoids and are essential constituents of the membrane lipids that maintain cellular and organelle integrity and important intracellular mediators of gene expression [5–9]. Because of these fundamental roles of EFA and LCPUFA, the maternal, fetal, and neonatal EFA/LCPUFA status is an important determinant of health and disease in infancy and later life. The deposit of these LCPUFA to the fetus is rapid during growth, and it is suggested that a failure to accomplish a specific component of brain growth because of inadequacy these fatty acids. In fact, fetal brain and retina are very rich in LCPUFA specially, arachidonic acid, 20:4n-6 (ARA) and docosahexaenoic acid, 22:6n-3 (DHA) [10–12]. Evidence from various studies suggests learning ability may be permanently impaired if there is a reduction in the accumulation of sufficient DHA during intrauterine life [13,14]. The premature babies, in particular, born during the last trimester of pregnancy have been shown to have low levels of DHA in the blood; this has been correlated with their abnormal eye and brain functions as measured by electroretinogram, cortical visual-evoked potential, and behavioral testing of visual acuity [9,14]. The critical importance of EFA/LCPUFA in fetoplacental unit demands an efficient uptake system for these fatty acids and their metabolism. Several plasma membrane fatty acid binding/transport proteins (FABPpm, FATP, and FAT) and intracellular fatty acid binding proteins (FABPs) have been suggested to be involved in cellular uptake and transport of fatty acids in human placenta [1,15]. In addition, involvement of several nuclear transcription factors (PPARs, LXR, RXR, and SREBP-1) in the expression of genes responsible for fatty acids uptake, placental trophoblast differentiation and human chorinnic gonadotropin (hCG) production indicating regulatory roles of fatty acid-activated transcriptions factors in placenta biology [16–21]. Maternal fatty acids thus may affect their placental transport as well as placental biology. Therefore, elucidating the pathways of placental fatty acid transport and metabolism, and the regulatory processes governing these pathways is critical for advancing our understanding the relationships between maternal fatty acid metabolism and the placental supply of EFA/LCPUFAs to the developing fetus and the potential implications on pregnancy and fetal outcome [20]. In this review, I would summarize recent development in the biochemical processes involved in placental fatty acid transfer to the fetus.

2. Maternal metabolism and supply of essential fatty acids (EFA) and LCPUFA to the fetus Linoleic acid, 18:2n-6 (LA), and a-linolenic acid, 18:3n-3 (ALA) are the two main dietary EFA that are readily available from the dietary sources such as vegetable oils, but their LCPUFA derivatives (ARA, EPA, DHA) can be consumed in foods of animal origin. Dietary LA and ALA must be converted in the body to their further metabolites, LCPUFA to exert the full range of biologic actions. Therefore, maternal EFA metabolism is crucial for fetal growth and development, as the fetus depends on the maternal supply of LCPUFA such as ARA and DHA. ARA is a precursor for bioactive eicosanoids and leukotrienes. The different prostaglandin and leukotrienes, affect numerous immune inflammatory responses, including fetal thymic growth, whereas both DHA and ARA are an important structural component of the nervous system. A deficiency of these important fatty acids in the critical times of embryological organogenesis may be devastating, particularly in neurological development.

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Human brain growth is at peak velocity during the last three months of gestation and first few months after birth, leading to the concept that the third trimester fetus and newborn infant are particularly vulnerable to developmental deficits if DHA, an integral component of neuronal membrane is in an inadequate supply. Such an important role of DHA rests on its participation in maintaining membrane fluidity, impulse propagation, synaptic transmission, and its function as a cytosolic signal transducing factor for various gene expression during the critical period of brain development [22,23]. The critical role of DHA in neurogenesis, however, suggests that adverse affects of inadequate DHA in early gestation will be more to severe and more difficult to overcome than deficiencies occurring later on [24]. Infants born with higher blood levels of DHA, as well as ARA maintain this advantage for several weeks [25,26], as the fatty acids accumulated in fetal adipose tissue are readily available for release and uptake by other organs in the postnatal period. The breast fed infant may have an additional advantage – its mother’s milk, rich and readily available source of customized food components. Optimal maternal to fetal DHA and ARA transfer in gestation is, therefore, likely to have benefits both before birth and extending into the postnatal period and thus, mother’s physiological adaptation towards this feto-maternal cooperation must be remarkably coordinated. During early pregnancy, LCPUFA derived from the diet are stored in maternal adipose tissue. During late pregnancy, enhanced lipid catabolism as a consequence of the insulin-resistant condition, in part due to hyperestrogenism, causes the development of maternal hyperlipidemia, which plays a key role in the availability of LCPUFA to the fetus. Maternal body fat accumulation during early pregnancy allows the accumulation of an important store of LCPUFA derived from both the maternal diet and maternal metabolism. The importance of DHA during pregnancy in fetal development and its postnatal spill-over effect has been studied, by examining the relationships between maternal DHA intake during gestation and lactation on cognitive functioning in later childhood [14,27]. Although interventional studies to inquire into the importance of DHA in pregnancy and fetal development, are complicated by different variables, such as, food habits, placental functions, and transit to fetal circulation etc., several epidemiological and interventional studies do suggest that higher maternal intakes of n-6 and n-3 fatty acids, including DHA increase maternal to fetal transfer of the respective fatty acid. In the large ALSPAC cohort study the frequency of seafood consumption during pregnancy in 11,875 women, a marker of dietary n-3 LCPUFA intake, was compared to the developmental, behavioral and cognitive outcomes of the children up to school age [28]. The authors found a significantly increased risk for children of mothers who consumed less than 340 g fish/week to be in the lowest quartile for verbal intelligence at the age of 8 years. Low maternal fish intake was also associated with sub-optimal outcomes in fine motor skills and social developmental scores. In Europe the average dietary supply of LCPUFA and especially of DHA is rather low [29]. Large observational studies have shown that children of women with low fish intakes during pregnancy are at increased risk of poor cognitive and behavioural outcome [28,30]. Similarly, low blood levels of DHA in infants at birth or during breast-feeding are associated with lower visual and neural maturation in infancy, and later childhood [31–34]. Multivariate analyses showed that infant gender and maternal DHA supplementation were significantly related to visual acuity at 60d age; infants in the placebo group were more likely to have a visual acuity below the mean, by gender, than infants in the DHA supplemented group. Others have shown that infants with a higher DHA status at birth have more mature electroencephalography patterns at 2d of age, higher visual functional maturation in infancy, and less distraction and better attention in the second year of life [32–34].

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Several studies on fatty acid composition in fetal and maternal plasma have shown that at birth, LA represents about 10% of the total fatty acids in cord plasma compared with 30% in maternal plasma, but surprisingly, ARA concentration in cord plasma is twice (about 10%) that observed in the mother (around5%). Similarly, ALA concentration in the newborn is half that in the mother (0.3% vs. 0.6%), whereas DHA concentration is double (3% vs. 1.5%) and EPA levels are equally low in fetal and maternal plasma [35,36]. It is obviously an extremely favourable situation for the development of the new-born, especially at a time when high quantities of AA and particularly of DHA are needed by brain and retina. However, the biochemical mechanisms responsible for higher levels of LCPUFA in the fetal circulation compared with the maternal plasma are still not yet clear. Free fatty acids (FFA) in the maternal circulation are the major source of fatty acids for transport across the placenta, as triacylglycerols are not transported intact. An accelerated breakdown of fat depots occurs during the last trimester of pregnancy [37,38]. In this late stage of gestation, lipolytic activity in adipose tissue is increased. The fatty acids that are released, as well as fatty acids from dietary lipids and hepatic overproduction of triacylglycerol, are responsible for increasing the amount of triacylglycerol, in maternal circulating lipoproteins. Although maternal plasma lipoproteins do not directly cross the placental barrier, hypertriacylglycerolaemia in pregnancy plays a key role in the availability of fatty acids for the fetus. Placental lipase activities were dramatically increased during the last trimester of pregnancy, whereas there was an overall decrease in lipoprotein lipase activity, resulting in a subsequent decrease in triacylglycerol storage. Consequently, with decreased triacylglycerol hydrolysis for maternal storage, and an increase in placental lipolytic activity, the resulting availability of triacylglycerol is utilised by placental lipase for the provision of FFA for fetal transport. The placental lipoprotein lipase (LPL), however, hydrolyses triacyglycerols from post-hepatic low density lipoproteins (LDL) and very low density lipoproteins (VLDL) but not the triacylglycerols present in the chylomicrons [38–40]. Placental LPL activity was reduced by 47% in preterm intrauterine growth retardation, IUGR whereas in IDDM pregnancies the activity was increased by 39% compared with controls [41]. In contrast, placental LPL activity was up-regulated in pregnancies complicated by type 1 diabetes and associated with fetal overgrowth [42]. These data are compatible with the hypothesis that alterations in placental LPL activity contribute to changes in FFA delivery to the fetus and, as a consequence, fetal growth [41]. Placenta however expresses various lipase activities. Lipases associated with vascular endothelium or in the placenta may play an important role in providing FFA from circulating triacylglycerol. Triacylglycerol hydrolase activities of human placenta have been characterised [39,40]. Homogenates of placenta exhibited three distinct triacylglycerol hydrolase activities with pH optima 4.5, 6.0 and 8.0. On further fractionation, placental cytosol exhibited both acid cholesterol ester hydrolase (pH 4.5) and hormone-sensitive lipase (pH 6.0) activities, whereas purified placental microvillous membranes exhibited two distinct triacylyglycerol hydrolase activities: a minor activity at pH 8.0 and a second major activity at pH 6.0. Triacylglycerol hydrolase activity at pH 8.0 of microvillous membranes appeared to be LPL whereas at pH 6.0 the activity was unique in that it was almost abolished by serum but was not affected by high NaCl concentrations. Role of this unique triacylglycerol hydrolase activity at pH 6.0 not yet known, however it may be involved in the packaging and/or releasing FFAs from the placenta, whereas LPL, which is mostly present in placental macrophages, may be responsible for hydrolysis of maternal plasma lipoproteins. Endothelial lipase (EL) and lipoprotein lipase (LPL) were the only lipases expressed in key cells of the human placenta. In first trimester, EL and LPL were expressed in

trophoblasts [43]. At term, EL was detected in trophoblasts and endothelial cells, whereas LPL was absent in these cells. In total placental tissue EL expression prevails in first trimester and at term [43]. As gestation and placental development advance both lipases are significantly down-regulated at the maternal surface of the placenta. At the end of gestation, EL is still expressed, but LPL is virtually absent from the trophoblast. Indeed, substantial EL expression in endothelial cells and LPL in smooth muscle cells surrounding blood vessels may account for major lipase activity. Lipoprotein receptors have been detected both on placental macrophages and syncytiotropblasts [38]. These receptors may also be involved in fatty acid uptake by the placenta. Further work is required on the regulation of different lipases expressed in placenta for better understanding of their roles in fatty acid transport across the human placenta. 3. Placental metabolism of fatty acids The placenta requires a constant and abundant source of energy to supply the needs for its own rapid growth and maturation and to transport the nutrients, ions, vitamins, waste, and other molecules required for fetal growth and homeostasis from the maternal to the fetal circulation and vice versa. These nutrients, ions, vitamins, and other molecules required for fetal growth and homeostasis must pass from the maternal to the fetal circulation, the waste from fetal to maternal side and, bidirectionally, several hormones, cytokines and nutrients and even hematopoietic cells. Placental fatty acid metabolism may play a critical role in guiding pregnancy and fetal outcome. A selective cellular metabolism of certain fatty acids may contribute to the placental transfer process as would the conversion of certain proportion of ARA to prostaglandins, incorporation of some fatty acids into phospholipids, and triacylglycerols, the oxidation and synthesis of fatty acids in the placenta. Endogenous formation of cyclooxygenase and non-cyclooxygenase metabolites of ARA has been implicated in the expression of early response genes c-fos, Egr-1, c-my, or c-jun in different cell types after mitogenic stimulation, whereas EPA metabolites inhibit expression of these genes [5]. LCPUFAs can be metabolized to important cell signaling molecules in placenta by several major isoform families including: the cyclooxygenases, lipoxygenases, and cytochrome P450 subfamily 4A (CYP4A) (see the review, [20]). During pregnancy, placenta is the major source of prostaglandins (PGs) within intrauterine tissues. PGs play important roles in the processes of pregnancy and parturition [44]. Specifically, PGs have been linked to induction of fetal organ maturation, up-regulation of the fetal hypothalamic–pituitary–adrenal axis, stimulation of myometrial contractility and cervical ripening, maintenance of uterine and placental blood flow, and inhibition of fetal breathing and movement at the time of labor [45,46]. Therefore, regulating the concentration of PGs in uterus is crucial for controlling pregnancy and parturition [47–51]. PGD2 produced in human placenta can be converted to important PPAR ligand and may influence placental biology [52]. It has been demonstrated that human placenta is not only capable of synthesizing PGs but expresses high abundance of the oxidation of NAD-dependent 15-hydroxyprostaglandin dehydrogenase, the key enzyme for metabolism of active primary PGs to inactive forms. Thus, the output of bioactive PGs from placenta is controlled by both synthesis and metabolism of primary PGs within placenta. The enzymes necessary to convert PGD2 into PGJ2 are present and coexpressed with PPARc in placenta. 15-Deoxy-D12, 14-PGJ2 (15dPGJ2) and its precursor PGD2 are present in amniotic fluid [53]. The maternal blood may also be a source of PPAR ligands for the human placenta. Maternal serum heat stable components can activate the PPARc in human placenta [54,55]. The presence of ligands for PPARs in placenta may regulate expression of PPAR

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target genes including fatty acid transporters in this tissue. In the PG synthesis pathways, the conversion of ARA into PGG2 and PGH2 by COX is currently known to be the rate-limiting step. The increased expression of COX-2 is believed to be associated with the PG synthesis at term and parturition. However, there are other enzymes, such as cytosolic phospholipase A2 (cPLA2), which are also potential regulating steps in PGs synthesis in addition to COX. The cPLA2 catalyzes the release of free ARA, an initial and rate-limiting substrate in PG synthesis, from the sn-2 position of membrane phospholipids. In addition, oxidized lipids including 9S-hydroxy-octadedienoic acid (HODE), 13S-HODE, 15S-hydroxy-5Z,11Z,13E-eicosatetraenoic acid (15S-HETE) were shown to activate PPARc in primary human villous trophoblasts [56]. Trophoblast has been shown to oxidize fatty acids in substantial amounts suggesting that the human placenta utilizes fatty acids as a significant metabolic fuel. In fact, placental oxidation of fatty acids is an important in fetal health determinant. Placenta expresses all the enzymes necessary for mitochondrial fatty acid oxidation [57]. Fatty acids are used as a major metabolic fuel by human placentas at all gestational ages, and any defect within this energy-producing pathway may hamper the growth, differentiation, and function of the placenta, thereby may compromise fetal growth and development [58]. Further work is required on the relationship between the fatty acid oxidative capacity of placenta and the fetal growth and development. 4. Uptake of fatty acid by placental trophoblast cells Underlying biochemical mechanisms responsible for the selective concentration of LCPUFA in fetal tissues are not fully understood yet. Evidence on the role of the placenta in preferential accumulation of LCPUFAs in fetal tissues has emerged from research in our laboratory in which the fatty acid binding characteristics of human placental membranes were determined by using 4 different fatty acids, LA, ALA, AA, and oleic acid, 18:1n-9 (OA) [59]. Placental plasma membrane binding sites have a strong preference for LCPUFAs: the order of competition was AA  LA > ALA  OA. Uptake and metabolism of oleic acid, LA, AA, and DHA were investigated using human placental choriocarcinoma (BeWo) cells.

Table 1 Uptake of oleic and linoleic acids by BeWo and HepG2 cells. Radiolabelled fatty acid + unlabelled fatty acid

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When fatty acid uptake was compared between BeWo cells and HepG2 cells, EFA/LCPUFA were taken up by BeWo cells preferentially over OA however, no such discrimination was observed in HepG2 cells (Table 1). Oleic acid was taken up least and DHA most by BeWo cells. Moreover, competitive studies indicated a preferential uptake compared with oleic acid in the order of DHA, ARA, and LA by these cells. Almost 60% of the total DHA taken up by cells was esterified into triacylglycerol whereas 37% was in phospholipid fractions. The reverse was true for AA; 60% of the total uptake was incorporated into phospholipid fractions whereas <35% was in triacyglycerol fractions [60]. The preferential uptake of LCPUFAs by placental membranes and BeWo cells was supported by the observation of Haggarty et al. [61] using a human placental perfusion system. They reported a selective preferential transport of DHA (order of preference DHA > AA > ALA > LA) in human placenta. In the in vitro perfused human placenta the concentration of nonesterified (free) fatty acids available to the maternal side regulated transfer to the fetal side – an 8-fold increase of in the DHA concentration on the maternal side resulted in a 13-fold increase in DHA transfer [35,61,62]. 5. Placental fatty acid uptake and transport: putative roles of fatty acid transport/binding proteins Most tissues rely heavily on fatty acids but, except for liver and adipose tissue, have limited or no capacity for fatty acid synthesis. Placenta requires maternal EFA/LCPUFA for fetal supply, the needed fatty acids are taken up from the maternal circulation. Therefore, placenta should be able to regulate fatty acid uptake to adapt to constant changes in demands for its own as well as for the developing fetus. The exact mechanism of how fatty acids are transferred across the plasma membrane are not yet fully known. Several studies however indicated that fatty acids are taken up across the cellular plasma membrane via a protein mediated mechanism [63]. Indeed, under physiological conditions, diffusion is quantitatively less important than the protein mediated transport of fatty acids [63,64]. There are several membrane proteins thought to be responsible for fatty acid uptake. These include, the 40-kDa plasma membrane associated fatty acid binding protein (FABPpm) [65], the heavily glycosylated 88-kDa fatty acid translocase (FAT), also known as CD36 [66] and a family of 63– 70-kDa fatty acid transport proteins (FATP 1–6) [67,68]. 5.1. Plasma membrane fatty acid binding protein in human placenta

Fatty acid uptake (nmol/mg protein) BeWo cells

HepG2 cells

[3H]Oleic acid, 18:1n-9

5.56 ± 0.16

22.89 ± 0.34

[3H]Oleic acid, 18:1n-9 + Linoleic acid, 18:2n-6 (-10 fold)

1.41 ± 0.15*

8.67 ± 0.48**

[14C]Linoleic acid, 18:2n-6

6.72 ± 0.50

27.78 ± 1.59

[14C]Linoleic acid, 18:2n-6 + oleic acid18:1n-9 (-10 fold)

5.40 ± 0.30

16.83 ± 1.89*

[14C]Linoleic acid, 18:2n-6+alinolenic acid, 18:3n-3 (-10 fold)

1.14 ± 0.09***

17.49 ± 0.36*

Competition between [3H]oleic acid and [14C]linoleic acid for uptake by BeWo and HepG2 cells was carried out by incubating these cells with the radiolabelled fatty acid in the presence or absence of a 10-fold excess of unlabelled fatty acid. Data represent the mean ± SEM of three separate experiments in which triplicate determinations were performed between uptake by controls and in the presence of 10-fold of excess unlabeled fatty acid for a particular cell type. Adapted from Campbell et al. [73]. * P > 0.05. ** P < 0.005 *** P < 0.001.

FABPpm is a peripheral membrane protein at the outer leaflet of the plasma membrane [69] and that was at first isolated from hepatic plasma membrane but later from major tissues with high transmembrane fatty acid fluxes. This protein is closely related, if not identical, to mitochondrial aspartate aminotransferase (mAspAT) [69]. FABPpm/mAspAt is a protein with distinct functions at different subcellular sites [69]. The presence of FABPpm both in sheep and human placental membranes was demonstrated [70,71]. A 40-kDa protein which binds only long-chain fatty acids was then isolated and purified from human placental membranes [71]. The human placental (p) FABPpm is different in several aspects (such as pI value and the amino acid composition, fatty acid binding activity and AspAT activity) from the previously described FABPpm from several tissues [71–73]. Table 2 summarises the different features of the p-FABPpm and ubiquitous FABPpm. This protein is only present in the microvillous membrane of the placenta facing fetal circulation [72]. Direct binding studies indicated that p-FABPpm bound only 15% of the total fatty acids when the protein was incubated with a mixture of fatty acids mimicking the composition maternal plasma FFA pool during the last trimester of pregnancy [74]. Almost 98% of the

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Table 2 Comparison between placenta FABPpm vs. Ubiquitous FABPpm.

Table 3 Effect of anti-human p-FABPpm antibody on fatty acid uptake by BeWo cells.

Features

Placental FABPpm

Ubiquitous FABPpm

Molecular mass pI value N-terminal Membrane location Tissue distribution Homology AspAT Activity Fatty acid binding activity

40 kDa 6.7 Blocked Extrinsic Placenta LAMP-2 Nil LCPUFA > EFA  OA

40 Kda 9.4 Open Extrinsic Broad distribution mAspAT Very high No preference

Data was adapted from [15,71,72,74,112,113].

ARA and 87% of DHA present in the mixture bound to the protein which was much higher than for OA and LA (21 and 13%, respectively) [74]. Radiolabelled fatty acid binding revealed that p-FABPpm had higher affinities (Kd) and binding capacities (Bmax) for LCPUFAs compared with other fatty acids. Furthermore, pretreatment of BeWo cells with anti-p-FABPpm antibodies inhibited most of the uptake of DHA (64%) and AA (68%), whereas OA uptake was inhibited only 32% compared with the controls treated with preimmune serum [1,21]. The order of fatty acid uptake inhibition by the antibodies was DHA > ARA  ALA > LA  OA, indicating that the p-FABPpm is involved in preferential uptake of LCPUFAs by these cells (Table 3). Similar inhibition of fatty acid uptake was observed with human placental membranes. Together, these results show the preferential uptake of LCPUFAs by BeWo cells, which is most probably mediated via p-FABPpm. The presence of p-FABPpm in the placental membrane facing maternal circulation may enforce unidirectional flow of the LCPUFA from the mother to the fetus. The uptake and subsequent esterification of DHA was the greatest and the esterification of DHA into triacylglycerol was more efficient than that of the other fatty acids. Incorporation of DHA into triacylglycerols in these cells may help to transport this fatty acid to the fetal circulation. However, at present it is not known how and in what form DHA is released from the placenta into the fetal circulation. Recent studies indicated the presence of ubiquitous FABPpm in human placenta [75] as well as in rat placenta [76]. 5.2. Fatty acid translocase (FAT/CD36) in human placenta FAT/CD36 a heavily glycosylated fatty acid translocase (FAT/ CD36), the sequence of which was 85% homologous with that of glycoprotein IV (CD36), is an integral membrane protein [66]. While FAT/CD36 is a class B scavenger receptor protein involved in angiogenesis, atherosclerosis, inflammation, and lipid metabolism, it is now also known that this protein is involved with facilitating fatty acid transport in heart and skeletal muscle. FAT is composed of a highly glycosylated polypeptide chain with an apparent molecular mass of 88 kDa which was cloned in adipocytes. This 472-amino-acid (53 kDa) protein is substantially glycosylated (10 predicted N-linked glycolsylated sites). Unlike

Fatty acid

% Inhibition of fatty acid uptake by the anti-p-FABPpm antibody

Oleic acid, 18:1n-9 Linoleic acid, 18:2n-6 Arachidonic acid, 20:4n-6 Docosahexaenoic acid, 22:6n-3

32 ± 3.7 50 ± 2.4** 68 ± 4.5** 64 ± 3.3**

Cultured BeWo cells pre-treated with p-FABPpm antiserum were compared with a control preparation with pre-immune serum. Data represent the mean ± SD of three separate experiments in which triplicate determinations were performed. Adapted from Campbell et al. [73]. ** P < 0.005 (Significantly different from antibody inhibition of oleic acid uptake).

other two fatty acid transporters (FABPpm, and FATP) FAT is a multifunctional protein and has a number of putative ligands including FFAs, collagen, thrombospondin and oxidized LDL [5,44–49]. The presence of FAT/CD36 was demonstrated in human placenta using both pure trophoblast cells and placental membrane preparations [1,15]. FAT is present in both the placental membranes, microvillous and basal membranes [15]. A series of studies have shown that CD36 is involved in FFA uptake. FAT is the prototype of a small family of single-polypeptide membrane glycoproteins that also includes LIMPII (a lysosomal membrane protein), rodent SR-B1 and CLA-1 (CD36 and LIMPII analogous receptor), the human homolog of SR-B. CD36 or FAT is predicted to have two trans-membrane domains at either ends of the molecule with short amino-terminal and carboxy-terminal cytoplasmic domains and a large, multiply N-glycosylated extracellular loop. CD36/FAT can associate with caveolae in transfected CHO cells or cultured Y1-BS1 adrenocortical cells [52]. Little is known about the regulation of FAT/CD36 function in placental cells but several lines point towards a translocational mechanism for increasing LCFA uptake by different cells [77,78]. Caveolin-1 may control FAT/CD36 mediated fatty acid uptake by increasing its surface availability [79]. Movement of caveolin-1 has been shown between the lipid droplet and the plasma membrane in response to fatty acid treatment [80]. However, further work is necessary to understand the mechanistic roles of these proteins in trophoblasts which may provide insight into lipid transport and metabolism in the placenta. 5.3. Fatty acid transporter proteins (FATPs) in human placenta The third protein which may act in concert with CD36 and FABPpm in the LCFA transport process is the fatty acid transport proteins (FATPs), expressed, in several tissues. Its importance has been confirmed with the finding of lipotoxicity in transgenic overexpressing mice. Targeted deletion of fatty acid transport protein-4 results in early embryonic lethality. FATP is the family of integral transmembrane proteins consisting so far of six (FATP 1–6) identified members which show different tissue expression patterns [81]. Table 4 shows the different FATPs in mammalian system.

Table 4 Highest mRNA expression of fatty acid transport proteins/very long chain fatty acyl-CoA synthetases/ACSBG in tissues, and their intracellular locations. Isoforms

Tissue expression

Intracellular location

Accession numbers (human)

FATP1 (slc27a1) FATP2 (Sic27a2) FATP3 (Sic27a3) FATP4 (Sic27a4) FATP5 (Sic27a5) FATP6(Sic27a6) ACSBG1 ACSBG2

Heart, WAT, placenta, skeletal muscle Kidney cortex, liver Broad distribution Small intestine, placenta, liver, kidney Liver Heart Brain, adrenal, gonads, spleen Testis

Plasma membrane, Golgi Microsome, peroxisome Mitochondria, MAM, ER Plasma membrane, ER, mitochondria, nuclei, MAM, peroxisome Plasma membrane Sarcolemma Cytoplasm, mitochondria, microsomes Cytoplasm, mitochondria

NP_940982 NP_003636 NP_077306 NP_005085 NP_036836 NP_054750 NP_055977 NP_112186

Adapted from Mashek et al. [112].

A.K. Duttaroy / Progress in Lipid Research 48 (2009) 52–61

FATP-1 was first identified in human placental membranes [15]. Later other isoforms of FATPs have been detected in human placenta [75,82]. Consistent with the role of FATP1 in fatty acid internalization, a significant portion of FATP1 is localized at the plasma membrane. FATPs were classified as fatty acid transport proteins because, when overexpressed, they increase the rate of fatty acid internalization, most notably at low concentrations when diffusion may not be sufficient. FATP is suggested to act in concert with fatty acylCoA synthetase (ACS), an enzyme that prevents efflux of the incorporated fatty acids by their conversion into acylCoA derivatives and, hence, rendering fatty acid uptake unidirectional. These long-chain fatty acylCoA esters act both as substrates and intermediates in various intracellular functions. FATP-mediated uptake of LCFA was diminished in the face of cellular depletion of ATP. Since FATP is likely to be responsible for the increased LCFA necessary to sustain this increased b-oxidation, the tissue-selective effects of various PPAR activators and ligands on FATP and ACS provide insight in the relationship between FFA uptake and triacylglycerol synthesis and b-oxidation of fatty acids. The expression of FATP 1 and FATP 4 mRNAs in the brain [68,83,84] suggests that FATPs may be responsible for the activation and/or internalization of LCPUFAs such as DHA that are important for brain function. However, FATP1 overexpression does not increase LCPUFA internalization, suggesting that the primary function of FATP1 does not concern LCPUFA internalization [85]. Additionally, whereas FATP 1 possesses ACS activity toward long-chain fatty acids (16–22 carbons), compared to that of Acsl1, the ACS activity of FATP 1 is very low [85]. Although its structure has not been established, a series of molecular and cell biology experiments suggest that FATP 1 has only one membrane-spanning region and several membraneassociated regions [86]. Because this arrangement is not typical for a channel or transporter, FATP may increase fatty acid internalization by increasing the rate of ‘‘flip-flop”, trapping the fatty acids in the inner leaflet of the plasma membrane, or activating the fatty acid to its CoA species. Disruption of the FATP 4 gene in mice established that it performs an essential function in normal mouse development [87]. Surprisingly little is known on the specificity of FATP enzymes for different fatty acids. Overexpression of FATP 1 in 3T3-L1 cells results in 2.6-fold increases in the internalization of PA, OA, and AA, suggesting that FATP 1 does not have specific preference for any of these fatty acids [83]. Similarly, overexpression of FATP 4 in 293 cells has little effect on ARA internalization [88]. Schaiff et al. have reported that PPAR-c and RXR regulate fatty acid transport in primary human trophoblasts [89]. The treatment of human trophoblast cells with both PPAR-c and RXR agonists resulted in elevated mRNA expression of FATP-1 and FATP-4 but not FATP-2, -3 and -6 in these cells. Similar results were obtained by this group in vivo in mice by using PPARc agonist, reporting also an increase in the placental expression of FABPpm and FAT/CD36 [90]. The PPAR/RXR heterodimer may have roles in regulating placental fatty acid uptake. However, exposure to rosiglitazone reduced feto-placental weight and the thickness of the spongiotrophoblast layer and labyrinthine vasculature. 5.4. Intracellular fatty acid binding proteins (FABPs) The presence of cytoplasmic H-FABP and L-FABP was demonstrated in human placental trophoblasts [15]. Later the presence of several other FABPs such as adipose FABP, and epidermal FABP subtypes in trophoblasts have been reported [91]. The significance of the presence of several cytoplasmic FABPs in trophoblasts is not known but indicates that complex interactions of these proteins may be essential for effective fatty acid transport and metsbolism in the placenta. Although there are differences in the function and binding activity of these proteins [92,93], their individual and coor-

57

dinated roles and expression in trophoblasts are yet to be established. Increase in the expression of liver and heart type FABPs upon differentiation of trophoblast cells was observed [18]. They reported that there was no correlation between the expression of H- and L-FABP and the of uptake of LA by human trophoblast cells. However, expression of placental L-FABP was increased by 112% in IDDM and 64% in gestational diabetes but no change was observed in the expression of H-FABP [41]. Exposure to hypoxia markedly increased the expression of L-FABP, H-FABP, and A-FABP with the accumulation of lipid droplet in trophoblasts [91]. Ligands for PPARc enhanced the expression of these proteins in trophoblasts. Hypoxia induced increased expression of FABPs in human trophoblasts, suggesting that FABPs support fat accumulation in the hypoxic placenta [82,91].

6. Nuclear transcription factors and their role in fatty acid transport and metabolism in placenta The primary focus of the peroxisome proliferator-activated nuclear receptors (PPAR) field is cellular and systemic metabolism, PPARs may play at least equally essential roles in placental development and function [94,95]. PPARc and PPARd are essential for multiple physiological functions of the trophoblastic and amniotic parts, leading to major involvement of PPARs in the pathophysiology of gestational diseases. One of the first functions described for PPARc in other tissues, trophoblastic lipid uptake and accumulation are also regulated in part by this factor. The PPARc ligands seem to increase the uptake and accumulation of the fatty acids in human placenta [89]. This regulation is associated with an enhanced expression of adipophilin (fat droplet-associated protein) and FATPs (1 and 4) in human trophoblasts [21,89,90,96]. These results were confirmed recently by the in vivo activation of PPARc by its agonist rosiglitazone in mice, which also leads to the enhancement of the previously described genes plus two new ones involved in the lipid transport: S3-12 and myocardial lipid droplet protein/MLDP [90]. PPARs have been demonstrated to regulate a number of placental fatty acid/lipid homeostasis-related proteins (e.g., metabolizing enzymes and transporters) [21,89,97]. The molecular and functional relevance of fatty acid metabolizing enzymes and the role of PPARs in regulating their expression in the mammalian placenta has been recently reviewed [19–21]. Besides fatty acid uptake, denovo synthesis of long-chain saturated and monounsaturated fatty acids significantly contributes to the production of membrane phospholipids [98]. The up-regulated of transcription factor SREBP1 and fatty acid synthase are most likely to provide membrane phospholipids. SREBP1 controls endogenous synthesis of saturated and monounsaturated fatty acids, involving fatty acid synthase, long chain fatty acyl elongase and stearoyl-CoA desaturase. The two LXR isoforms, LXRa and LXRb, are well known to act as key regulators in sterol and fatty acid metabolism are present in placental trophoblasts [99]. Exposure of trophoblasts to a synthetic LXR agonist resulted in an increase in the amount of SREBP-1 and fatty acid synthase activity, also increased the synthesis of lipids [99]. LXR reduced secretion of hCG during differentiation of these cells although the implication of these observations are yet to understood. LXR also is suggested to regulate trophoblast invasion via endoglin synthesis [100]. Oxidized metabolites of linoleic acid, such as 9- and 13-HODE are found in oxidized LDLs and identified as PPARc-specific ligands [101]. Analysis of oxLDL content revealed that only oxLDL containing a high proportion of oxysterols and phosphatidylcholine hydroperoxide derivatives reduced trophoblast invasion. Phosphatidylcholine hydroperoxide derivatives provide PPARc ligands such as HETE and HODE, whereas oxysterols such as 7-ketocholesterol provide ligands to LXR. Activation of

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PPARc/RXRa or RXRa/LXRb heterodimers with specific natural and synthetic PPARc or LXR ligands markedly inhibit trophoblast invasion in vitro [102]. 6.1. Effects of fatty acids on fatty acid transport system of the human placenta Pregnancy is accompanied by a broad variety of physiological changes and complex metabolic processes that occur in the mother. During pregnancy, maternal and nutritional requirements are increased by the needs of the growing fetus and placenta. It has been indicated that increased consumption of n-3 LCPUFA from fish or fish oils may increase birth weight and lower the risk of early delivery and preeclampsia (see the review, [62]). However only limited evidence exists to support the notion that supplementation of n-3 LCPUFA favours fetal developmental outcome [77,103]. In vitro perfused human placenta studies demonstrate that the concentration of unesterified DHA available to the maternal side regulated its transfer to the fetal side [35,61,62]. However, very little is known as to whether the transit across the placenta by these LCPUFA in excess elicits any metabolic or physiological changes as may happen when supplementing these fatty acids during pregnancy. Fish-oil supplementation during the second half of pregnancy did not significantly modify the gene expression of FATP-1, FATP-4, FATP-6, FAT/CD36, FABPpm, H-FABP, A-FABP, or B-FABP in human placenta at the time of delivery [82]. However, the mRNA expression of FATP-1, and particularly of FATP-4, in placental tissue was positively correlated with the uptake of maternal DHA into placental and cord blood phospholipids [82]. The implications of this observation are not yet known. Increase in FATP 4 may lead to general fatty acid transport or uptake. FATP-1 and FATP-4 activate a wide range of fatty acids and show no preference for LCPUFA [104,105]. Moreover, evidence indicates that FATP 4 is localized in the endoplasmic reticulum and not the plasma membrane [106]. FATP 4 drives fatty acid uptake directly or indirectly by esterification as it is not a transporter protein involved in fatty acid translocation at the plasma membrane. FATPs are indirectly involved in the translocation process across the plasma membrane by providing LCFA synthetase activity [80,107] and thereby diminish the intracellular FFA pool so that less fatty acids are available for efflux [108]. Both FAT/CD36 and FATPs interact with lipid rafts [79,80]. Lipid rafts are dynamic assemblies of proteins and lipids, that float freely within the two dimensional matrix of the membrane bilayer. Lipid rafts are receiving increasing attention as de-

vices that regulate membrane function in vivo and play an important role in membrane trafficking and signal transduction. More information is required as to whether and how lipid rafts are involved in fatty acid trafficking across the placenta. The influence of insulin and fatty acids on the expression of FATPs in cultured trophoblasts was investigated [109]. The enhancement of adipose differentiation-related protein (ADRP) expression by combined insulin and fatty acids was associated with fat accumulation in trophoblasts but not with increased uptake of fatty acid. Similar data on the regulation of fatty acid uptake into trophoblasts by insulin and leptin was also reported [109,110]. ADRP is a component of lipid droplets in cells and involved in lipid storage and homeostasis, cell signaling and vesicle trafficking. In the BeWo cell line, ADRP mRNA was induced in a dose-dependent manner by exposure to EPA, ARA, DHA and OA with a concomitant increase of general fatty acid uptake [111]. Fig. 1 shows the effect of fatty acids and PPAR ligands on oleic acid uptake by BeWo cells. Incubation of BeWo cells with OA results in more triacylglycerol accumulation than incubation with DHA does (data not shown). This might reflect the different fates of these two fatty acids in their metabolism and subsequent transport across the placental trophoblasts to the fetus. Transport of maternally derived OA to the fetal circulation thus may be restricted by the placenta by directing this non-essential fatty acid to the lipid droplet. Induction of ADRP protein expression as well as induced triacylglycerol accumulation by fatty acids was abolished by co-incubation with triacsin C. Triacsin C is an inhibitor of long chain acylCoA synthetase 1, 3, and 4 [112]. When BeWo cells were co-incubated with triacsin C and fatty acids it resulted in a larger inhibition of incorporation of [1-14C]–OA in the cells compared with [1-14C]–DHA (unpublished data). OA preferentially is incorporated into triacylglycerol and forms relatively small amounts of FFA within the cell although DHA also incorporated into triacylglycerols, but form a larger pool of a FFA. OA may be preferentially become esterified and incorporated in the form of triacylglycerol and stored in lipid droplets within the cell. DHA therefore may circumvent the esterification step (bypassing CoA formation step by FATP mediated uptake avoiding acylCoA formation) to allow efflux from the placenta to the fetal circulation. Therefore, it is difficult to envisage that FATP transports LCPUFA across the placenta from the stand point of its non-specific fatty acid binding affinity, associated ACS activity and, in addition, its location on both sides of the placental membranes. But p-FABPpm which is exclusively located on the apical (maternal facing) side of the placental membrane may be the

Fig. 1. Effect of long-chain fatty acids on [14C]-oleic acid uptake by BeWo cells. Uptake of [14C]–OA was measured after BeWo cells had been stimulated with 100 lM fatty acids (OA, DHA, AA, EPA) or synthetic agonists for PPARc (BRL49653, 1 lM) PPARd (GW501516, 100 nM), PPARa (WY14643, 100 lM), LXR (T0901317, 1 lM), RXRa (LG100268, 100 nM) for 24 h. Uptake was measured 2 h after addition of 100 lM [14C]-OA. Uptake of OA was calculated as pmol OA and related to lg protein per well. Data represent the mean + SD obtained from 3 to 6 separate experiments, significantly different (<0.01) from control. (Reproduced with permission from Tobin et al. [111]).

A.K. Duttaroy / Progress in Lipid Research 48 (2009) 52–61

59

FFA-Albumin DHA/ARA

FA (c>14)

FAT

F A T P s

Complex lipids

Maternal facing membrane

FABP

ACBP

FFA FABP

Acs FA-CoA

Fetal facing membrane

p-FABPpm

FFA FFA PGs LTs

lipase

FABP

Transcription factors

FA oxidation

Complex lipids Lipid droplets

Other metabolic pathways

Complex lipids

F A T P s

Gene expression

FABP FFA

nucleus

FAT

FFA

? FFA α Fetoprotein Albumin

Fig. 2. Schematic diagram of the putative roles of fatty acid binding/transporter proteins in human placental fatty acid uptake and metabolism. Putative roles of the placentalmembrane-associated and cytoplasmic fatty acid binding proteins in placental fatty acid uptake and metabolism. The location of FAT and FATP on both sides of the bipolar placental cells and the lack of specificity for particular types of FFAs allows transport by all FFAs (non-essential, essential and long-chain polyunsaturated) bidirectionally, i.e. from the mother to the fetus and vice versa. However, by virtue of its exclusive location on both sides and preference for LCPUFAs, p-FABPpm sequesters maternal plasma LCPUFAs to the placenta. Cytoplasmic FABPs may be responsible for transcytoplasmic movement of FFAs to their sites of esterification, ß-oxidation, or to the fetal circulation via placental basal membranes. Adapted from Duttaroy [1].

candidate for LCPUFA uptake as it has a higher binding affinity for LCPUFAs. But further characterization of p-FABPpm is required for definitive conclusions (see Fig. 2). 7. Conclusions The human placenta plays a crucial role in mobilising the maternal adipose tissue fat stores and actively concentrating and channeling important LCPUFAs to the fetus via multiple mechanisms including selective uptake by the trophoblast, intracellular metabolic channeling and selective export to the fetal circulation. p-FABPpm, located exclusively on the maternal-facing membranes of the placenta, may be involved in the sequestration of maternal LCPUFAs. However, further studies are required on the fatty acidbinding domain of the p-FABPpm, which allows the preferential binding of LCPUFAs over non-essential fatty acids. A better understanding of the mechanisms involved in the placental transfer of LCPUFA to the fetus is required in order to improve fetal DHA status not only in uncomplicated pregnancies but also in disorders associated with poor DHA status, such as gestational diabetes mellitus or IUGR. Although limited evidence exists to support the notion that prenatal n-3 LCPUFA supplementation favours developmental outcome [103]. Supplementation of DHA during pregnancy was associated with FATP 4 expression in human placenta and this may result in general fatty acid uptake by the placenta. Our in vitro study also demonstrates that DHA increases general fatty acids uptake by placental trophoblasts. These observations suggests that FATP-4 (as expected) mediates general fatty acid uptake and these fatty acids are converted by ACS to acylCoA derivatives which can subsequently be used in both anabolic and catabolic pathways by the placental trophoblasts. Further work is required to understand the implication of supplementation of

DHA during pregnancy which may increase general fatty acid uptake by the placenta and its impact on feto-placental growth and development. A focus for future studies will be to further establish the complex interrelationships of the many multiple proteins influencing cellular LCFA uptake and retention by the placenta. Acknowledgements The author is grateful to Throne Holst foundation for financial support for the placenta work. References [1] Duttaroy AK. Transport mechanisms for long-chain polyunsaturated fatty acids in the human placenta. Am J Clin Nutr 2000;71(1):315S–22S. [2] Innis SM. Essential fatty acid transfer and fetal development. Placenta 2005;26(Suppl A):S70–5. [3] Innis SM. Perinatal biochemistry and physiology of long-chain polyunsaturated fatty acids. J Pediatr 2003;143(Suppl. 4):S1–8. [4] Innis SM. Fatty acids and early human development. Early Hum Dev 2007;83(12):761–6. [5] Schmitz G, Ecker J. The opposing effects of n-3 and n-6 fatty acids. Prog Lipid Res 2008;47(2):147–55. [6] Hulbert AJ, Turner N, Storlien LH, Else PL. Dietary fats and membrane function: implications for metabolism and disease. Biol Rev Camb Philos Soc 2005;80(1):155–69. [7] Sampath H, Ntambi JM. Polyunsaturated fatty acid regulation of gene expression. Nutr Rev 2004;62(9):333–9. [8] Jump DB. Fatty acid regulation of gene transcription. Crit Rev Clin Lab Sci 2004;41(1):41–78. [9] Uauy R, Hoffman DR, Peirano P, Birch DG, Birch EE. Essential fatty acids in visual and brain development. Lipids 2001;36(9):885–95. [10] Green P, Yavin E. Mechanisms of docosahexaenoic acid accretion in the fetal brain. J Neurosci Res 1998;52(2):129–36. [11] Williard DE, Harmon SD, Kaduce TL, Spector AA. Comparison of 20-, 22-, and 24-carbon n-3 and n-6 polyunsaturated fatty acid utilization in differentiated rat brain astrocytes. Prostaglandins Leukot Essent Fatty Acids 2002;67(2–3): 99–104.

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