Fatty acid transport and metabolism in the feto-placental unit and the role of fatty acid-binding proteins

Fatty acid transport and metabolism in the feto-placental unit and the role of fatty acid-binding proteins

Review Fatty acid transport and metabolism in the feto-placental unit and the role of fatty acid-binding proteins Asim K. Dutta-Roy Rowett Research ...

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Review

Fatty acid transport and metabolism in the feto-placental unit and the role of fatty acid-binding proteins Asim K. Dutta-Roy Rowett

Research

Institute,

Aberdeen,

Scotland,

UK

It is generally accepted that essential fatty acids (EFA) and their long-chain polyunsaturated fatty acid (LCPUFA) derivatives play a crucial role in fetal development and pregnancy outcome. Surprisingly, however, little is known about the transport and metabolism of EFA and LCPUFA in the feto-placental unit. The critical importance of maternal LCPUFA synthesis and the subsequent preferential transport of these by the placenta to the fetus is now well recognized, however underlying mechanisms of these processes are poorly understood. Increasing evidence suggests that the cytosolic and plasma membrane-associated fatty acid-binding proteins (FABP and FABP,,,, respectively) are involved in cellularfatty acid uptake, transport, and metabolism in several tissues: however, no information is available for the placenta. These proteins may also function in the fine-tuning of cellular events by modulating the metabolism of LCPUFA implicated in the regulation of cell growth and differentiation. In this review recent developments in the understanding of the mechanisms of EFA/LCPUFA transport by the human placenta and the role of the fatty acid-binding proteins in the sequestration of maternal EFA/LCPUFA for fetal delivery are discussed. (J. Nutr. Biochem.8548-557, 1997) 0 Elsevier Science Inc. 1997

Keywords: placenta; acid uptake; essential

plasma membrane fatty acid-binding protein; cytosolic fatty acid-binding protein; free fatty acid; fatty fatty acids; long-chain polyunsaturated fatty acids; fetal development; BeWo cells

Introduction Nutrition during pregnancy and early childhood has been shown to affect the risk of neurodevelopmentaldisordersas well as psychomotor and cognitive development in infancy.‘,’ In addition, evidence is growing that fetal and neonatal nutrition may have a programming effect on the risk of cardiovascular and certain metabolic diseasesin later life.3,4 Essential fatty acids (EFA) and their long-chain polyunsaturated fatty acids (LCPUFA) are of critical importance in fetal and infant development.5x6These fatty acids are the precursors of eicosanoids, and are essential constituents of the membrane lipids that maintain cellular

Address reprint requests to: Dr. Asim K. Dutta-Roy at Rowett Research Institute, Aberdeen AB21 9SB. Scotland, UK. This work was supported by the Scottish Office Agriculture, Environment, and Fisheries Department. Received February 24, 1997; accepted May 29, 1997.

Nutritional Biochemistry 8:548-557, 1997 0 Elsevier Science Inc. 1997 655 Avenue of the Americas. New York, NY 10010

and organelle integrity. 7p’oThey are also increasingly being recognized as important intracellular mediators of gene expression.” In fact, dietary EFA are part of the lipid supply necessaryfor energy, growth, cellular metabolism, and muscle activity. 7,“,‘2 Becauseof the fundamental role of EFA and LCPUFA as structural elementsand functional modulators,7*‘2it was hypothesized that the maternal, fetal, and neonatal EFA/LCPUFA statusis an important determinant of health and diseasein infancy and later life. Intrauterine life constitutes a vulnerable period in brain development, becausethe fetus is totally dependent on the maternal supply of specific nutrients for its growth. Fetal brain and retina are very rich in the LCPUFA, arachidonic acid,20:4n-6 (ARA) and docosahexaenoic acid,22:6n-3 (DHA), and a sufficient supply of these fatty acids during pregnancy and the neonatal period is of great importance.5,” The deposit of these LCPUFA in the fetus is rapid during growth, and it is suggestedthat a failure to accomplish a specific component of brain growth becauseof inadequacy

0955-2863/97/$17.00 PII SO955-2863(97)00087-9

fatty acid transport of LCPUFA of critical membrane lipids may lead to irrevocable damage.5 Evidence from various studies suggests that retinal function and learning ability may be permanently impaired if there is a reduction in the accumulation of sufficient DHA during intrauterine life.5.6 The fetal brain acquires approximately 2 1g of DHA per week during the last trimester of pregnancyI and yet overall EFA status of the developing fetus is marginal, if not sufficient.‘4,‘5 This is particularly true at a relatively high maternal intake of trufzs unsaturated fatty acids, as well as in preterm neonates.16 In early as well as late pregnancy, significant negative correlations were observed between the amounts of fetal trarw fatty acid and the fetal EFA/LCPUFA status.” The maternal DHA status is significantly lower in multipara as compared with primipara, suggesting that under the present dietary conditions, pregnancy is associated with mobilization of DHA from a store that is not fully replenished after delivery. ‘s Studies in primates also show that an insufficiency of these fatty acids at a critical stage in retinal development results in a functional impairment of the retina, which can be normalized by postnatal LCPUFA supplementation. I9 Comparable results of supplementation have been obtained in newborn infants.20 Because the overall maternal EFA/LCPUFA status steadily declines during pregnancy,‘s supplementation of pregnant mothers with EFA may improve neonatal EFA/LCPUFA status.” This is particularly important for premature babies, because it has been shown that premature infants born during the last trimester of pregnancy have low levels of DHA in the blood, which thus affects their eye and brain functions as measured by electroretinogram, cortical visual-evoked potential, and behavioral testing of visual acuity.20-‘3 In fact, the potential for dietary EFA deficiency has become a significant issue for the nutrition of preterm-born infants, because they do not receive the third trimester intrauterine supply of DHA and ARA. Therefore, a better understanding of the biochemical processes involved in placental EFA/LCPUFA transfer is required for optimum and safe supplementation of EFA during pregnancy. The delivery of fatty acids from the maternal circulation to the developing fetus has been studied using whole perfused placenta (see Reviews 5 and 24), but many of the details of the transport mechanisms are still poorly understood. For example, these studies failed to explain why the levels of LCPUFA are always higher in the fetal circulation than in the maternal circulation.5.24 The preferential accumulation of LCPUFA is crucial in the fetal circulation to support its rapid growth, but the underlying biochemical mechanisms responsible for the selective concentration of these fatty acids in fetal tissues are not fully understood yet. The critical importance of placental transfer of maternal LCPUFA rather than fetal synthesis by desaturation and elongation of transferred LA and ALA to the accumulation of LCPUFA to the fetus is now increasingly recognized, however. Therefore, it is pertinent to investigate whether the placenta is capable of preferentially transporting LCPUFA from the maternal circulation, and if so, what are the biochemical processes involved in preferential fatty acid transfer between mother and fetus. To understand the placental role in these processes we have been studying the interaction of EFA/LCPUFA with isolated human placental

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membranes and human placental choriocarcinoma (BeWo) cells. The aim of this review is to bring together recent developments on placental EFA/LCPUFA transfer activity and to evolve concepts concerning the role of placental plasma membrane- and cytosolic fatty acid-binding proteins (FABP,, and FABP, respectively) in these processes.25,26

Maternal delivery

metabolism of EFALCPUFA to the feto-placental unit

and their

Linoleic acid (LA), 18:2n-6 and c-y-linolenic acid (ALA, 18:3n-3) are the two main dietary EFA.27,‘s LA and ALA are not interconvertible, but they can be further elongated and desaturated by the same enzyme systems to n-6 and n-3 LCPUFA in the body. Whereas LA and ALA are primarily present in the diet in vegetable oils, preformed LCPUFA may also be consumed in foods of animal origin. The importance of LCPUFA has been related to their structural action, their specific interaction with membrane proteins, or their ability to serve as precursors of second messengers.‘-“’ Therefore, LA and ALA must be converted to their further metabolites, LCPUFA to exert the full range of biologic actions. Thus, maternal EFA metabolism is crucial for fetal growth and development, as the fetus depends on the maternal supply of EFA and LCPUFA.5.‘5 Among LCPUFA, ARA, eicosapentaenoic acid,20:5n-3 (EPA), DHA. and dihomo gamma linolenic acid,20:3n-6 (DGLA) are present in tissues in different amounts. Except DHA, these LCPUFA compete for cyclooxygenase, and lipoxygenase, thus producing a variety of compounds collectively called eicosanoids.’ These compounds have diverse biologic function in cell growth and developments, inflammation, and in the cardiovascular system,7m’2 whereas DHA is mainly involved in retina and brain function.5,‘9 In addition, endogenous formation of cyclooxygenase and noncyclooxygenase 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 inhibits expression of these genes.” Therefore, supplementation of EPA not only reduces ARA availability, but also competes with ARA for cyclooxygenasellipoxygenase and thereby reduces formation of growth stimulatory eicosanoids of ARA.7-9 Because both n-3 and n-6 fatty acids owe their presence in vertebrate tissues to dietary intake, important physiologic consequences follow the inadvertent selection of different average daily dietary supplies of these two types of EFA.” It is, however. important to appreciate that each type of EFA can interfere with the metabolism of the other. Therefore, an excess of n-6 fatty acids will reduce the metabolism of ALA, possibly leading to a deficit of n-3 LCPUFA metabolites; this is a matter of concern in infant feeds that may contain an excess of LA, but no balancing n-3 fatty acids. There is therefore a real risk that high intake of either n-3 or n-6 fatty acids could lead to impairment of the metabolism of the other. Therefore, a proper balance between the n-6 and n-3 fatty acids in the diet is important to maintain optimum growth and development. There is leading consensus that infants should be supplemented with DHA rather than EPA and receive adequate amounts of ARA. However, it is not known how the pregnant mother J. Nutr. Biochem.,

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Review overcomes these complicated situations, especially during the last trimester of pregnancy when the fetus requires large amounts of both ARA (n-6 fatty acid) and DHA (n-3 fatty acid), but not EPA (n-3 fatty acid). Several studies’“-‘5.2s,30 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 (- 10%) that observed in the mother (-5%). 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. This situation in which the relative plasma concentration of the n-3 and n-6 LCPUFA exceeds that of their precursors is specific to the newborn and is never observed in adults. It is obviously an extremely favorable situation for the development of the newborn, especially at a time when high quantities of ARA and particularly of DHA are needed by the brain and retina. However, three very important questions arise: 1) what are the underlying mechanisms leading to this situation, at birth? 2) how does the situation evolve throughout gestation? 3) how are the n-3 and n-6 LCPUFA (DHA and ARA) preferentially transferred? Various suggestions have been proposed, but unfortunately none of these could answer the above questions satisfactorily. Because human placental tissue lacks both the delta 6 and delta 5 desaturase activities,2s,“m’” any LCPUFA in the fetal circulation are most probably derived from the maternal plasma. However, the situation has become more complicated by the discovery of desaturase and elongase activity in developing fetal brain and liver,32 although it is not certain to what extent desaturationelongation of transferred LA and ALA may contribute to fetal LCPUFA (Figure I). This indicates that the placental transport of maternally derived lipids may play a critical role in the fetal growth and development. Unesterified 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.s,3s.3” Placental lipoprotein lipase (PL) must be active to facilitate placental uptake of FFA from circulating tricylglycerol.‘,” In the guinea pig, PL activity increases approximately 1l-fold during the last stages of gestation and seems to be implicated in fatty acid transfer between mother and fetus. The presence of PL on the maternal side but not on the fetal side of the placenta3” may be important to facilitate transfer of esterified n-3 and n-6 fatty acids from the mother to the fetus and limiting transfer in the reverse direction. The PL, however, hydrolyses triglycerides from post-hepatic lipoproteins (LDL. VLDL) but not the tryglycerides present in the chylomicrons.s~‘s~36 Further characterization of this placental protein is required in order to understand its role in the feto-placental fatty acid transport and metabolism. Lipoprotein receptors, mainly responsible for the uptake of cholesterol required for fetal tissue synthesis. have been detected both on placental macrophages and syncytiotropblasts. These receptors may also be involved in fatty acid uptake by the placenta. However. the metabolic fate of fatty acids transported via lipoprotein 550

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Mother

Placenta

Fetus

Neonate

2 t ( Delta

Linoleic

y-Linolenic

Dihomo

y-linolenic

acid,

Arachidonic

acid,

6 desaturase

activity

a-Linolenic

delta

)

acid,

18:3n-3

6 desaturase 1

16~311-6

Stearidonic

acid,

18:4n-3

Elongase 1

acid,

1

delta

16:2n-6

1 1 acid,

5- and

20:3n-6

delta

20:4n-3

5 desaturase 1

20:4n-6

delta

Eicosapentaenoic

Elongase 6 desaturase P-oxidation

acid,

20:5n-3

acid,

22:6n-3

4 +

Docosahexaenoic

of the metabolism of Linoleic acid (18:‘2n-6) and Figure 1 Outline a-Linolenic acid (18:3n-3) by maternal, and the feto-placental unit. Presence (+) and absence (-) of delta 5 and delta 6 desaturase activities in respective tissues are indicated.

receptors is uncertain and therefore may not account for preferential accumulation of LCPUFA in the fetal tissues.

Preferential uptake of LCPUFA by placental membranes: Identification and characterization of a plasma membrane fatty acid-binding protein (FABP,,) The multiple roles of LCPUFA suggestthat careful regulation of all aspectsof their disposition, including cellular uptake, is critical for the maintenance of cellular integrity.7-'0.78.37 Th e very property that makeslong-chain fatty acids well suited to be componentsof membranes,i.e., their acyl chain hydrophobicity, complicates for the organismthe task of transporting them from sitesof intestinal absorption, hepatic synthesis, and lipolysis to sites of utilization. The intracellular metabolism of free fatty acids (FFA) and their transport in plasma as complexes with albumin have been

Fatty acid transport studied extensively, 38,39 but little attention has been paid (until recently) to the mechanism by which FFA enter and leave cells. Since 1981, several groups have clarified the influences of albumin binding on the uptake kinetics of FFA and other bound ligands, making more detailed studies of the uptake processes feasible.“8m4’ Once it became clear that FFA uptake occurred principally from the very small unbound ligand pool in plasma and not from the albuminbound compartment, it was then quickly reported that a major component of FFA uptake in certain cell types, including hepatocytes, adipocytes, and cardiac and skeletal myocytes. exhibited all the kinetic properties of facilitated transport processes (i.e., [runs-stimulation, &-inhibition, and counter transport).. ‘sm4’ All of these observations suggested the presence of a membrane fatty acid uptake protein. This was further pursued by isolation of such a membrane fatty acid binding protein from rat liver membranes.38-“’ However, the involvement of these plasma membrane proteins in fatty acid uptake does not exclude the possibility that the diffusion of the ligand into the plasma membrane phospholipids plays an important role. For evaluation of whether this uptake occurs by simple diffusion or facilitated transport, it was necessary to establish whether the fatty acids interact specifically with the plasma membrane. Therefore, the binding characteristics of a representative long-chain fatty acid, oleate, to isolated rat liver membranes were analyzed. There are now several reports providing evidence of the involvement of FABP,, in the uptake of FFA into a variety of mammalian tissues: hepatocytes, adipocytes, cardiomyocytes, and jejunal mucosal cells.39.“‘-‘h Experimental evidence has also recently been provided on the FABP,, mediated FFA uptake by the heartA FABP,, expression and the kinetics of FFA uptake are now known to be affected by the physiologic status of the cells46 suggesting the importance of this protein in cellular growth and differentiation. Various groups described the presence of the plasma membrane fatty acid-binding protein (FABP,,) or transport protein (FAR, FAT, and FATP) in various cell types.“‘-‘” The molecular mass of the protein was determined to be 22, 40, or 88 kDa.“‘-“” Fujii et a/ 48 described another membrane protein of about 60 kDa with high affinity for FFA, which was designatedfatty acid receptor (FAR). The third membrane protein (88 kDa) putatively involved in FFA uptake, has been identified in adipocyte”’ and the protein was recently cloned,“” and is termed as fatty acid translocase (FAT). FAT from rat was found to be highly homologous (85%) to the human leukocyte differentiation antigen CD36,“’ a receptor protein present among others in monocytes and platelets and thought to be involved in adhesion phenomenaand intracellular signaling. Recently, we have shown that CD36 is involved in arachidonic acid uptake by human platelets.“’ Comparison of rat FAT and recently cloned CD36 revealed an amino acid identity of 93%, which strongly suggeststhat these proteins are species homologues. In adipocytes, another membraneprotein (fatty acid transporter protein, FATP) of molecular mass 22kDa was implicated in the transmembranetransport of FFA.” The precise functions of these different plasma membranefatty acid binding proteins in the transport of FFA across the ceilular membranesare yet to be understood. They may

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function as a translocator, but it also possiblethat they act as acceptors for FFA releasedfrom albumin and that FFA subsequently cross the plasma membrane by diffusion through the phospholipid bilayer. The role of the placenta on the preferential uptake of LCPUFA was first examined in our lab by determining the fatty acid binding characteristics of human placental membranes using four different radiolabeled fatty acids (LA, ALA, ARA and a nonessentialfatty acid, oleic acid, 18:ln9 (OA).s4 In this study, molar ratio between albumin and fatty acid was 1:1. The total FFA concentration in the pregnant mother during the last trimester of pregnancy is approximately 0.75 mM, of which saturated fatty acids are 35%; monounsaturated fatty acids, 41%; EFA (LA and ALA), 20%; and LCPUFA, 4%? whereasthe albumin concentration is 34.2 g per liter. 56This gives an approximate molar ratio of FFA to albumin of 1:1. The binding of EFA/ LCPUFA to humanplacental membraneswas highly reversible compared with that of OA.54 In addition, OA binding was inhibited very strongly by LCPUFA (ARA, gamma linolenic acid (18:3n-6,GLA), and EPA), followed by the relevant parent EFA (LA and ALA). The lack of strong inhibition of the binding of EFA/LCPUFA to placental membranes by OA suggests the existence of stronger affinities for EFA/LCPUFA compared with OA.54 As described earlier, the placenta hassignificant requirementsfor EFNLCPUFA in the growth and development of the feto-placental unit and this could be met by the preferential uptake of EFA/LCPUFA. EPA and its eicosanoid metabolites are growth inhibitory asthey reduce the availability of ARA and its metabolites by competing at cyclooxygenase/ lipoxygenase and desaturationlelongationpathways of EFA metabolism.’ However, EPA has very little inhibitory effect on ARA transport to placental membranes.In contrast to EPA, its parent fatty acid, ALA inhibited the binding of both ARA and LA strongly.s4 The competition experiments also suggestedthat the binding sites have heterogeneous binding affinities for different fatty acids. Binding sites seemto have a strong preference for LCPUFA: the order of competition was AA>>>LA>ALA>>>>OA. Our data also suggestedthat tram fatty acids competed with the EFA/LCPUFA for fatty acid binding sites in human placental membranes, thereby inhibiting the transport of EFA/ LCPUFA to the placenta.54Tralzs fatty acids are not synthesized in situ, but they do occur in both fetal and placental tissues,which suggeststhat they are transportedthrough the placenta from the maternal circulation.‘6.‘7 Our study support these observations16,” that maternal tram fatty acids can be transported to the fetal circulation via the placenta. Once trms fatty acidsare in the feto-placental unit they may exert detrimental effects on fetal growth and development by disturbing LCPUFA metabolism(eicosanoidproduction) and membrane structure and function.‘6.‘7 Fatty acid binding data from our laboratory have suggested that the membrane protein may be involved in placental fatty acid uptake. The presenceof the FABP,, in the placenta was investigated. We have purified and characterized FABP,, both in human and sheep placental membranes.‘6.s7A 40 kDa protein that binds only longchain fatty acids, was isolated and purified from human placental membranes.The apparent molecular massof the J. Nutr. Biochem., 1997, vol. 8, October

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Review placental-FABP,, (P-FABP,,) was determined by gel permeation chromatography and by SDS-polyacrylamide gel electrophoresis (PAGE).‘6 The p1 value and the amino acid composition of human placental protein are different from those of hepatic or gut FABP,,, 26.38.39indicating that the placental protein is different from ubiquitous FABP,,. The PAGE/autoradioblotting technique that was initially developed to detect and estimate cellular retinol-binding proteins was used to investigate the [‘“Cloleate binding activity of purified membrane protein and also to detect FABP,, in solubilized membranes.16 Evidence for the involvement of FABP,,,, in fatty acid binding came from trypsin-treated placental membranes that had decreased specific [ “C]oleate binding compared with that of untreated membranes.‘” Furthermore, pre-incubation of membranes with polyclonal antiserum against the human p-FABP,, inhibited the binding of fatty acids but the degree of inhibition varied depending on the type of fatty acid.5” Antibody mediated inhibition was much more acute for the EFA/LCPUFA binding than the OA binding, suggesting again the heterogeneity of fatty acid binding and that p-FABP,, may be preferentially involved in the placental EFA/LCPUFA uptake. Because studies have shown asymmetric distribution of various receptors and enzymes in the membranes of the trophoblast, we then examined the location of the p-FABP,, in these cells by using highly purified microvillous and basal membranes of the human placenta. Fatty acid binding activity, PAGE radiobinding assay, and Western blot analysis of these membranes clearly demonstrated that the p-FABP,, is located exclusively in the microvillous membranes of the human placenta.58 Because p-FABP,, may be responsible for FFA uptake, its location in the maternal facing side of the placenta favors the unidirectional flow of maternal FFA to the fetus. Lafond et al.“” also have also suggested a unidirectional transport of linoleic acid from mother to the fetus via syncytiotrophoblast membranes.

Uptake and metabolism of fatty acids by the human placental cboriocarcinoma (BeWo) cell line All the above studies were performed using isolated human placental membranes and therefore no information is available on intact trophoblast cells with metabolic activity. To understand the uptake and metabolism of EFA/LCPUFA and role of FABP,,, in placental cells, we have therefore utilized the human placental choriocarcinoma (BeWo) cell line as a model of the human trophoblast.“’ The BeWo cell line displays some placental differentiation markers including the production of placental specific proteins and has been used extensively to study lipoprotein metabolism.6’ We examined the uptake and metabolism of LA, ARA, DHA, and OA in BeWo cells and also the role of the p-FABP pm in these processes. We also compared the fatty acid uptake by BeWo cells and Hep G2 cells. Although fatty acid uptake by Hep G2 is known to be, at least in part, carrier-mediated.62 no such information was available on the placental cell line, BeWo. These two cells types are derived from organs with specialized roles in lipid metabolism; i.e., liver is involved in an multiplicity of lipid 552

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metabolic roles including synthesis, esterification, lipoprotein export, and oxidation for energy, and the placenta is primarily concerned with sequestration of the maternal EFA/LCPUFA and transporting these to the fetus. The kinetic data and temperature-dependency of long-chain fatty acid uptake by BeWo cells indicated a saturable process. The involvement of cell metabolism with the uptake process is shown by the marked enhancement of the uptake with temperature that takes place when the temperature exceeds 26°C. The presence of competitive inhibition by fatty acids demonstrated the presence of a carrier-mediated uptake mechanism with, in the case of BeWo cells, a higher affinity for the EFA than for OA. In contrast, Hep G2 cells do not exhibit a differential affinity for the fatty acids studied.60 In addition the preference for EFA/LCPUFA especially DHA, by BeWo cells was observed and supported previous studies on the placental transport of DHA which involved measuring its level in umbilical cord at the time of delivery.6” The demonstration of competitive inhibition of fatty acid uptake further supports the saturable nature of this process in BeWo and Hep G2 cells, but there is a preferential EFA/ LCPUFA uptake system in BeWo cells that is not present in Hep G2 cells. This is in agreement with the notion that placental cells need to sequester adequate amounts of n-3 and n-6 LCPUFA from the maternal plasma, especially during the last trimester of pregnancy. Attempts were also made to demonstrate more directly the presence and involvement of p-FABP,, in fatty acid uptake in BeWo. Using a polyclonal antibody to the human p-FABP pm, Western blot analyses demonstrated the presence of p-FABP,, in BeWo cells that was absent from Hep G2 cells, confirming our previous observation that the placental protein is different from the hepatic protein.‘” This antibody also blocked the uptake of fatty acids by BeWo cells, the order of inhibition was DHA>ARA>>>ALA>LA>>>OA, indicating that the p-FABP,, is involved in preferential uptake of LCPUFA by these cells similar to what was observed with human placental membranes.5” Initial studies have suggested marked differences between the metabolism of the four fatty acids (DHA, ARA, LA, and OA) in BeWo cells. Both the uptake and subsequent esterification of DHA was the greatest among all these fatty acids. Second, the esterification of DHA into triacylglycerol was more efficient than that of the other three fatty acids. Because one of the main functions of the placenta is to deliver DHA into the fetal circulation it is possible the triacylglycerol form may favor the transport of DHA to the fetal circulation. However, at present it is not known how and in which form of DHA is released from the placenta into the fetal circulation. ARA was found to be distributed almost equivalently both in the triacylglycerol and phospholipid fractions. Given the absence of delta 5 and delta 6 desaturation and elongation activities in the human placenta,25.3’m34 the radioactivity incorporated into the cellular lipids cannot reflect desaturated and/or elongated metabolites of the exogeneously added fatty acids but other metabolites of these fatty acids (such as after monohydroxylation of fatty acids, etc.) could be incorporated into cellular lipid fractions.

Fatty acid transport

Cytosolic fatty acid-binding their role in feto-placental and metabolism

proteins (FABP) and fatty acid transport

Investigation of the mechanisms of intestinal absorption of long-chain fatty acids led to the discovery, in the enterocyte and several other tissues, of what was perceived to be a family of low molecular mass (14-15 kDa) cytosolic fatty acid-binding proteins (FABP).4’ Besides their abundance the FABPs differ also by their large heterogeneity of types and isofotms. Several distinct types of FABP have been characterized. They are denominated by the first tissue of isolation and show a different tissue distribution. Generally, FABPs are different from other cellular lipid-binding proteins such as acyl CoA-binding protein, phospholipidtransfer proteins, glycolipid-transfer proteins, sterol carrier protein, retinol-binding proteins, and ol-tocopherol-binding proteins.4’,64-70 All types of FABPs bind long-chain fatty acids and, depending on type, also other hydrophobic ligands.4’@.65 Liver, heart, and intestinal FABP types bind long-chain fatty acids. 4’ The measured values of the dissociation constants (Kd) ranged from approximately 2 to 1000 nM, depending on the tissue of origin of the FABP and the fatty acids.” The stoichiometry of binding is 1 to 3 mol fatty acid per mol protein for all FABP types, except for the liver FABP, which binds 1 to 3 mol fatty acids. The binding of ligands other than the fatty acids has not been fully investigated for all FABP types. The heart-type FABP (H-FABP) and intestinal-type (I-FABP) only bind longchain fatty acids,4’.64,65 whereas the liver-type FABP (LFABP) bind heterogeneous ligands.4’,64.65 Studies with exogenous ligands showed that many hydrophobic compounds bind or compete with fatty acids for L-FABP; these include bile salts, peroxisome proliferators, lysophosphatidic acid, and inhibitors of carnitine palmitoyl transferase and have generally lower affinity than fatty acids.4’@.65 Prostaglandin (PC) E, and haem bind L-FABP with higher affinity than fatty acids4’,” as do the lipoxygenase metabolites of arachidonic acid, 15 hydroperoxyeicosatetraenoic acid ( 15 HPETE), 5 HPETE, and 5 hydroxyeicosatetraenoic acid (5-HETE).‘” Other lipoxygenase metabolites including 12-HPETE, SHETE and leukotrienes C, bind to liver FABP to a lesser degree.73 The cyclooxygenase metabolites PGE,, TxB,, and LTB, did not bind L-FABP. Cyclopentenone prostaglandins (PGA,, PGA,, PGJ,, and ‘I2-PGJ,) bind more avidly than oleic acid to L-FABP. PGD,, PGE,, and PGF,, are poor competitors and PGE, is intermediate.69-74 L-FABP binds these cyclopentenone prostaglandins avidly and apparently transports them to the nuclear membranes. These prostaglandins induce gadd 153 mRNA, a member of novel class of genes associated with growth arrest and DNA damage. 75 Based on their tissue expression, binding affinities, regulation, in vitro effects on fatty acid transfer, and the enzymes involved in fatty acid and carbohydrate metabolism, it has been postulated that these proteins play an important controlling role in the cytoplasmic transport and metabolism of long-chain fatty acids.4’,64.65 Once taken up in the cell, the intracellular transport of long-chain fatty acids is also thought to be mediated by FABP within the cytosol. Fatty acid uptake and internalization by living cells

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may involve a sequence of steps: dissociation from albumin, transport across the plasma membranes (via FABP,,, FAR, FAT, and FATP), and transport within the cytoplasm and its metabolism via FABP.‘5*4’,64,65 They may also be involved in the modulation of the intracellular concentration of the fatty acids or their CoA or carnitine esters, and in this way regulate the activity of enzymes involved in lipid metabolism. Differential effects of I-FABP and FABPs on fatty acid uptake and esterification were demonstrated using transfected mouse L-cell fibroblasts.76 L-FABP increases fatty acid uptake and targets this fatty acid for esterification into phospholipids, whereas I-FABP does not increase fatty acid uptake but preferentially stimulates esterification into triacylglycerol. 76*77 They may also protect enzymes or membranes from deleterious effects of high fatty acid concentrations. FABPs are now reported to deliver fatty acids and acyl-CoA to mitochondrial and peroxisomes for oxidation.25v4’.64*65 FABPs may also be involved in both desaturationlelongation of EFA by virtue of binding and/or transporting substrates/products of these important pathways (e.g., for peroxisomal B oxidation of 24:6n-3 to produce DHA, 22:6n-3). In addition, these proteins may be involved in the cyclooxygenasellipoxygenase pathways. In addition, these proteins are thought to have more diverse functions, including, for example, a role in the modulation Studies of cell growth and differentiation. 25.4’,64.65,70~74,78 of low molecular weight amino-azo dye binding protein A, which is identical to L-FABP, suggested a possible relationship with neoplasia. 74.79L-FABP is also established to be an early target protein to which carcinogens form a covalent bond.74 Moreover expression of FABPs is greatly increased during hepatocyte mitosis and preadipocyte differentiation. L-FABP has also been identified as one of the cytosoiic selenium-binding proteins. *’ The molecular basis of selenium binding to L-FABP is not clear, but is considered to differ from the selenylation of glutathione peroxidase. Selenium has an inhibitory effect on tumorigenesis in mice, rats, and hamsters.” It has been shown to reversibly inhibit proliferation and prevent tumorigenesis in epithelial tissues, raising the possibility that L-FABP may modulate the effects of selenium on growth and proliferation. Finally, those eicosanoids that are most avidly bound to L-FABP are also those that are the most potent growth regulators. The nature of possible relationship of FABP to cell growth and differentiation is now more established, such as adipocyte FABP, which is phosphorylated by the insulin receptormediated tyrosine kinase; phosphorylated form of FABP may form part of the signal cascade responsible for activation of glucose transport in adipocytes.*’ There is evidence that &-unsaturated fatty acids can activate protein kinase C, which plays a vital role in signal transduction of several growth factors. W.65 Protein kinase C activation is associated with increased proliferation in many cells types. FABPs, by virtue of their affinity for long-chain fatty acids, could serve as potential modulators of cell growth. ARA and other fatty acids directly regulate KC, Ca’+, and Cll channels, but it is not known whether this is caused by the interaction with the ion channel protein or by modulation of its lipid environment 8,‘o A degree of similarity was found between a 13 1-residue domain of the N-methyl-D-aspartate (NMDA) receptor (residues 263-393 inclusive) and various members J. Nutr. Biochem.,

1997, vol. 8, October

553

Review of the FABP family.” Arachidonic acid potentates the NMDA receptor current when glutamate activates the receptor in isolated cerebeller granules cells. Receptor-mediated release of unsaturated fatty acids and diacylglycerol contribute synergystically to the activation of protein kinase C. FABP may modulate this process by binding fatty acids. The L-FABP and I-FABP genes exhibit distinct patterns of tissue-specific and developmental regulation. H-FABP is the most widely distributed and has a pattern of tissue specific expression is quite distinct from that of the other two FABPs, and is developmentally [email protected]’ The tissue-specific FABP have distinct ontogenic profiles, which are consistent with a role for FABP in fatty acid metabolism. L-FABP and I-FABP mRNA are detected in the small intestine at day 19 of the 21-day gestation period in rats. Their concentrations rise 3-fold within 24 hr of birth and continue to rise during the suckling phase, peaking to eight times the prepartum levels.83 Comparison of the developmental changes in L-FABP mRNA abundance with mRNA levels of a number of enzymes of cholesterol biosynthesis show that whereas L-FABP mRNA levels in rat liver rises after birth,” mRNA abundance of HMG-CoA synthase and prenyl transferase, which is highest in the fetal life, falls precipitously at birth.*5 Thus, the increases in L-FABP and I-FABP occur at a time when the neonatal intestine and liver are adapting to an increased influx of fatty acids, and when the metabolic apparatus in the liver is shifting from lipogenesis and cholesterol synthesis to ketogenesis. No significant H-FABP mRNA was detected in liver, intestine, or lung RNA prepared at any time in rat fetal life assayed. s5 In developing heart, H-FABP mRNA was detected at the earliest time point in rat (19-day gestation period). 85 Recently similar upregulation of the mRNAs of both H-FABP and FAT proteins was observed in the heart developmental during development.86 The observed changes are consistent with the hypothesis that these proteins participate in the metabolic targeting of fatty acids to beta oxidation. The rise in H-FABP level in the developing heart also parallels the beta oxidation capacity of the myocardium and correlates with carnitine palmitoyltransferase activity. *’ Developmental changes in kidney HFABP mRNA accumulation follow a pattern distinct from that observed in heart and brain with levels in fetal life. These experimental results concerning H-FABP mRNA tissue distribution and developmental regulation emphasize the need to consider the possibility that this protein may serve physiologic roles in addition to those related to beta oxidation. At the same time by documenting the unique patterns of L-FABP, I-FABP, and H-FABP mRNA accumulation throughout the development, the data support the notion that the FABPs represent a group of structurally distinct, yet homologous proteins, that have evolved to fulfill functions based on specific metabolic needs of a given cell types. Ontogenic profiles of human fetal liver FABPs show a gradual increase from an early stage to term to transport more fatty acids for lipid synthesis and to protect glucose 6-phosphate dehydrogenase.88 A significant rise in L-FABP concentration occurs in the maternal liver during pregnancy and lactation in rats,‘” possibly to target FFA toward esterification rather than oxidation during late pregnancy and lactation. 554

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A rapid increase in H-FABP mRNA concentrations was also observed in late gestation placenta. Human placenta however contains both L-FABP and H-FABP.90 The presence of two types of FABPs has also been reported in the kidney but in two different locations.64.65 However, no such information is available regarding FABP in the placenta. Regulation of the expression these two proteins (L-FABP and H-FABP) in the placenta is thought to be related to differential fatty acids transport and metabolism to meet necessarydemandsfor feto-placental growth and development. The different types of placental FABP may be involved in the channeling of specific fatty acids to Boxidation, in the synthesisof structural and storagelipid in the placenta, in the transfer to fetal circulation or in placental growth and regulation. H-FABP and L-FABP are also reported to differ in the rate at which bound fatty acids are transferred to a membraneacceptor. L-FABP has been implicated in cell growth and regulation by virtue of its binding to various growth stimulatory and inhibitory eicosanoids,aswell asto selenium.7o-73It is possiblethat these proteins have distinct roles in terms of intracellular trafficking of fatty acids as well as in the differentiation of trophoblast to syncytiotrophoblast. Figure 2 summarizesthe putative roles of the fatty acid-binding proteins in placental fatty acid uptake and metabolism. Increasedknowledge of the factors that modulate the expression of these proteins would give us a better understanding of their role in feto-placental growth and development.

Conclusions The preferential accumulation of maternal LCPUFA to fetal tissueswas thought to be the result of the combinedbiologic processesoccurring in the maternal and feto-placental unit. Studies from our lab have clearly demonstratedthe preferential uptake system (p-FABP,,) for LCPUFA in the placenta, which may be responsiblefor the accumulation of increased levels of these fatty acids in the fetal circulation compared with the maternal plasma. Subsequently, the p-FABP,,, hasbeen identified in the humanplacenta, which is exclusively present in the placental membranesfacing maternal circulation but absentfrom the basal membranes. Despite the similar size, p-FABP,, is different from the ubiquitous FABP,, as the former preferentially binds LCPUFA and also lacks glutamic oxaloacetic acid transmaninase activity. When fatty acid uptake was compared between BeWo cells and Hep G2 cells, EFA/LCPUFA are taken up by BeWo cells preferentially over OA; however, no such discrimination was observed HepG2 cells. Further, it was found that BeWo cells express a protein immunoreactive with the anti-human p-FABP,, antiserum. This antiserum also reduced fatty acid binding to both human placental membranesand BeWo cells, compared with preimmune serum. Both PL and p-FABP,, are located only in microvillous membranes.their location at the same side (microvillous membrane) of bipolar cells is necessaryfor these proteins to work in tandem for the sequestrationof maternal LCPUFA from circulating trigylcerides by the placenta for transport to the fetus. At the moment the roles of H-FABP and L-FABP are not well establishedin the placenta, but they could play important roles by controlling

Fatty acid transport FFA

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VLDL & LDL

Figure 2 Schematic presentations of the putative roles of the plasma membraneand cytoplasmic fatty acid-binding proteins (FABP,, and FABP, respectively) in fatty acid uptake by the human placenta. FABP,, may preferentially sequester maternal plasma LCPUFA and cytoplasmic FABPs may be responsible for transcytoplasmic movement of poorly soluble fatty acids to their sites of esterification, beta oxidation, or to the fetal circulation via placental basal membranes. Abbreviations; VLDL, very low density lipoproteins: LDL, low-density lipoproteins; PI, placental lipase.

Fetal Plasma

a-fefoprotein

the metabolic fate of fatty acids (i.e., oxidation or esterification) and modulating cell growth and development. Because fatty acids play nutritional, metabolic, and functional roles in mammalian systems, their diverse effects may depend on their specific transport to the tissue and their subsequent metabolism. Both FABP,, and FABP may play crucial roles in this regard. From our work on placental cells. and others on keratinocytes,59 it can be concluded that uptake of FFA by the cells from the plasma FFA pools may be determined by these fatty acid-binding proteins.‘5 Recent studies in humans and animals suggest that a mild deficiency in EFA could limit fetal growth processes, whereas maternal dietary supplementation of EFA could prevent intrauterine growth retardation, indicating that EFA supplementation during pregnancy could affect outcomes.” However, it is not known whether placental fatty acid transport system or maternal EFA metabolism is impaired in this clinical condition. Therefore, to fully exploit the benefit of dietary supplementation of EFA/LCPUFA to pregnant mothers it is important to understand the respective role of maternal and feto-placental unit in EFAnCPUFA metabolism and transport. In fact, the discovery of a FABP,,, in the human placenta provides us with an unique opportunity to study the role of the placenta in supply of EFA/LCPUFA in both physiologic and clinical conditions such as intrauterine growth retardation, small-for-gestation age. diabetic pregnancies. and prematuritiesY’ Future studies on the coordinate expression and relationships among these proteins L-FABP. H-FABP. and PL) in the placenta are (P-FABP,,, required to understand their roles in the preferential uptake of maternal LCPUFA by the placenta.

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htematiorzal Conference on Highly Unsaturated Fatty Acids in Nutrition and Disease Prevention, p, 140 (abstract), Barcelona Gafvels. M.E.. Coukos, G.. Sayegh, R.. Coutifaris, C. Strickland D.K.. and Strauss. J.F. (1992). Regulated expression of the trophoblast alpha-2-macroglobulin receptor low-density lipoprotein receptor-related protein differentiation and CAMP modulate protein and messenger RNA levels. J. Biol. Chem. 267, 21230-21234 Schurer, N.Y.. Stremmel, W., Grundmann, J.-U.. Schliep, V., Kleinert. H.. Bass. N.M.. and Williams, M.L. (1994). Evidence for a novel keratinocyte fatty acid uptake mechanism with preference for linoleic acid: comparison of oleic acid uptake by cultured human keratinocytes, fibroblasts and a human hepatoma cell line. Biochim. Biophys. Acta 1211, 51-60 Ruyle, M.. Connor, W.E., Anderson G.J., and Lowensohn, RI. (I 990). Placental transfer of essential fatty acids in humans: venousarterial differences for docosahexaenoic acid in umbilical erythrocytes. Proc. Natl. Acad. Sci. USA 87, 7902-7906 Veerkamp. J.H., Peeters. R.A.. and Maatman, R.G.H.J. (1991). Structural and functional differences of cytoplasmic fatty acidbinding proteins. Biochim. Biophys. Acta 1081, 1-24 Glatz, J.F.C., Vork. M.M., Cistola, D.P., and van der Vusse, G.J. (1993). Cytoplasmic fatty acid-binding protein: significance for intracellular transport of fatty acids and putative role on signal transduction pathways. Prost. Leuk. Esstl. [email protected] Acids 48, 33-41 Dutta-Roy, A.K., Gordon. M.J., Campbell, F.M., Duthie, G.G., and James, W.P.T. ( 1994). Vitamin E requirements, transport and metabolism: role of a-tocopherol-binding proteins. J. Nutr. Biochem. 5, 562-570 Dutta-Roy. A.K., Leishman. D.J., Gordon, M.J., Campbell, F.M.. and Duthie, G.G. (1993). Identification of a low molecular mass (14.2 kDa) u-tocopherol-binding protein in the cytosol of rat liver and heart. Biochem. Biophys. Res. Commun. 196, 1108-l 112 Gordon, M.J., Campbell, F.M., Duthie, G.G., and Dutta-Roy, A.K. (1995 J. Characterization of a novel cu-tocopherol-binding protein from bovine heart cytosol. Arch. Biochem. Biophys. 318, 140-146 Chaudry. A.A., and Dutta-Roy, A.K. (1993). Purification and characterization of a fatty acid-binding protein from human prostatic tissue. Lipids 28, 383-388 Dutta-Roy, A.K.. Huang, Y., Dunbar. B., and Trayhum, P. (1993). Purification and characterization of fatty acid-binding proteins from brown adipose tissue of the rat. Biochim. Biophys. Acta 1169, 73-79 Richieri, G.V., Ogata, R.T., and Kleinfeld, A.M. (1994). Equilibrium constants of the binding of fatty acids with fatty acid-binding proteins from adipocyte, intestine, heart and liver measured with the fluorescent probe ADIFAB. J. Biol. Chem. 269, 23918-23930 Dutta-Roy. A.K., Gopalswamy, N., and Trulzsch, D.V. (1987). Prostaglandin E, binds to Z protein of rat liver. Eur. J. Biochem. 162, 615-619 Raza, H., Pogubala, J.R., and Sorof, S. (1989). Specific high affinity binding of lipooxygenase metabolites of arachdionic acid by liver fatty acid binding protein. Biochem. Biophys. Res. Commun. 161, 448-455 Khan, S.H.. and Sorof, S. (1990). Preferential binding of growth inhibitory prostaglandins by the target protein of a carcinogen. Proc. Natl. Acad. Sci. USA 87,940 I-9405 Choi. A.M.K.. Fargnoli, J.. Carlson, S.G.. and Holbrook, N.J. (1992). Cell-growth inhibition by prostaglandinA(2) results in elevated expression of gadd153 messenger RNA. Exptl. Cell Res. 199, 85-89 Prows, D.R.. Murphy, E.J.. and Schroeder, F. (1995). Intestinal and liver fatty acid binding proteins differentially affect fatty acid uptake and esterification in L-cells. Lipids 30, 907-910 Murphy. E.J.. Prows. D.R., Jefferson, J.R., and Schroeder, F. (1996).

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