lable at ScienceDirect

Placenta 30 (2009) 1037–1044

Contents lists avai

Placenta

journal homepage: www.elsevier .com/locate/placenta

Current Topic

Long-chain Polyunsaturated Fatty Acids Stimulate Cellular Fatty Acid Uptakein Human Placental Choriocarcinoma (BeWo) Cells

G.M. Johnsen a,b, M.S. Weedon-Fekjær a, K.A.R. Tobin a, A.C. Staff a,b, A.K. Duttaroy a,*

a Department of Nutrition, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, POB 1046 Blindern, N-316 Oslo, Norwayb Department of Obstetrics and Gynaecology, Oslo University Hospital, Ulleval, Faculty of Medicine, University of Oslo, Oslo, Norway

a r t i c l e i n f o

Article history:Accepted 10 October 2009

Keywords:PlacentaLCPUFAsDHAAAACSLEPAOAFatty acid uptakeBeWoTrophoblastPhospholipids

* Corresponding author. Tel.: þ47 22851547; fax: þE-mail address: a.k.duttaroy@medisin.uio.no (A.K.

0143-4004/$ – see front matter � 2009 Elsevier Ltd.doi:10.1016/j.placenta.2009.10.004

a b s t r a c t

Supplementation of long-chain polyunsaturated fatty acids (LCPUFAs) is advocated during pregnancy insome countries although very little information is available on their effects on placental ability to take upthese fatty acids for fetal supply to which the fetal growth and development are critically dependent. Toidentify the roles of LCPUFAs on placental fatty acid transport function, we examined the effects ofLCPUFAs on the uptake of fatty acids and expression of fatty acid transport/metabolic genes usingplacental trophoblast cells (BeWo). Following 24 h incubation of these cells with 100 mM of LCPUFAs(arachidonic acid, 20:4n-6, eicosapentaenoic acid, 20:5n-3, or docosahexaenoic acid, 22:6n-3), thecellular uptake of [14C] fatty acids was increased by 20–50%, and accumulated fatty acids were prefer-entially incorporated into phospholipid fractions. Oleic acid (OA, 18:1n-9), on the other hand, could notstimulate fatty acid uptake. LCPUFAs and OA increased the gene expression of ADRP whilst decreased theexpression of ASCL3, ACSL4, ACSL6, LPIN1, and FABP3 in these cells. However, LCPUFAs but not OAincreased expression of ACSL1 and ACSL5. Since acyl-CoA synthetases are involved in cellular uptake offatty acids via activation for their channelling to lipid metabolism and/or for storage, the increasedexpression of ACSL1 and ACLS5 by LCPUFAs may be responsible for the increased fatty acid uptake. Thesefindings demonstrate that LCPUFA may function as an important regulator of general fatty acid uptake introphoblast cells and may thus have impact on fetal growth and development.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

During intrauterine life, long-chain polyunsaturated fatty acids(LCPUFAs) such as docosahexaenoic acid, 22:6n-3 (DHA) andarachidonic acid, 20:4n-6 (AA) rapidly accumulate in fetal tissuesand play a critical role in the growth and development of the fetus[1,2]. Since human fetuses synthesize very low and insufficientamounts of AA and DHA, a considerable amount of these fatty acidsmust be obtained from the maternal circulation through theplacental fatty acid transport system [3]. Therefore, maintenance ofthe maternal status of LCPUFA during pregnancy is critical for theoptimal supply of LCPUFA to the fetus. LCPUFAs are synthesized denovo in the maternal organism from essential fatty acids such aslinoleic acid, 18:2n-6 and linolenic acid, 18:3n-3, acquired from thediet or mobilized from adipose tissue reserves [4]. Several investi-gators have reported that the intake of eicosapentaenoic acid,20:5n-3 (EPA) and DHA by pregnant women raises the content of

47 22851341.Duttaroy).

All rights reserved.

these fatty acids in fetal tissue [5]. Increased consumption ofLCPUFAs from fish or fish oils during pregnancy is suggested to bebeneficial for overall fetal development and to lower the risk ofearly delivery and preeclampsia [6,7]. Maternal plasma LCPUFAs aretaken up by placental trophoblasts via several fatty acid transportproteins (FAT/CD36, FATPs, FABPpm, and p-FABPpm) and intracel-lular fatty acid binding proteins (FABPs) [8,9]. In addition to theseproteins, involvement of long-chain acyl-CoA synthetases (ACSLs)in the fatty acid uptake has been demonstrated in different cellstypes [9–13]. Over-expression of ACSL1 [14,15], ACSL4 [16], andACSL5 [10] lead to enhanced cellular fatty acid uptake in differentcell types. ACSLs activate fatty acids by converting them intomembrane impermeable acyl-CoAs in an ATP-dependent reactionand thus facilitate the transport of exogenous fatty acids by trap-ping them inside the cells. The acyl-CoAs have numerous metabolicfates within cells, including incorporation into triacylglycerols(TAG) and membrane phospholipids (PL), as substrates forb-oxidation and protein acylation, and as ligands for transcriptionfactors. Long-chain fatty acids can be activated by three relatedmammalian protein families: ACSLs, FATPs and the ‘‘bubblegum’’family [17,18]. For FATPs, an additional independent transporter

G.M. Johnsen et al. / Placenta 30 (2009) 1037–10441038

function has been claimed [19], while ACSL and bubblegumproteins are generally assumed to be enzymes [17,20]. Similar toFATPs, the five ACSL isoforms (ACSL1, ACSL3, ACSL4, ACSL5, andACSL6) differ in their substrate preferences, enzyme kinetics, andintracellular locations [21–23], suggesting that each has a distinctfunction, and studies of the outcome of over-expression show thatthe fates of fatty acids diverge. However, very little information isavailable on regulation, roles and function of ACSLs on humanplacental fatty acid uptake and metabolism.

Despite the critical importance of LCPUFAs in fetal growth anddevelopment, not much information is available as to how thesefatty acids affect fatty acid transport and expression of relevantlipid metabolic and transporter genes in human placenta. A recentstudy reported that the mRNA expression of fatty acid transporter-1(FATP1) and FATP4 in placental tissue was positively correlatedwith the uptake of maternal DHA into placental and cord bloodphospholipids [24]. Recently we also have shown that incubationwith AA, EPA, DHA but not oleic acid, 18:1–9 (OA) for 24 h increasedthe ability of a placental trophoblast cell line, BeWo, to accumulate[14C] OA [25]. However, it is important to know whether LCPUFAsalso modulate their own uptake in these cells as these fatty acidsare critically important for fetal growth and development. Inaddition, the mechanism responsible for LCPUFA stimulated fattyacid uptake in BeWo is not known. Therefore studying effects ofLPUFAs on fatty acid metabolic gene expression in trophoblast cellscould provide useful information for future practical applications.

In order to address these questions, we investigated first theeffects of LCPUFAs and OA on the uptake of OA, AA, EPA, and DHA inBeWo cells. In parallel, we also examined the expression of genesresponsible for fatty acid uptake and metabolism. In this paper wedemonstrate that incubation with LCPUFAs for 24 h, but not OA, ledto an increased ability of BeWo cells to accumulate exogenous [14C]fatty acids (OA, AA, EPA or DHA) by 20–50%. Enhanced uptake of[14C] fatty acids by LCPUFA was associated with increased incor-poration of these fatty acids into PL fractions as compared with TAGfractions in these cells. Stimulation of fatty acid uptake by LCPUFAcorresponds to the increased expression of ACSL1 and ACSL5,indicating that acyl-CoA synthetase activity may be responsible forstimulated fatty acid uptake by LCPUFAs in BeWo cells.

2. Materials and methods

2.1. Materials

[1-14C]OA (specific activity 54.6 mCi/mmol), [1-14C]EPA(specific activity 55.0 mCi/mmol), and [1-14C]DHA (specificactivity 55.5 mCi/mmol), were obtained from Perkin Elmer,Waltham, Massachusetts USA, American Radiolabelled Chemical,Inc, St. Louis, MO, USA. [1-14C]AA (specific activity 56 mCi/mmol)was obtained from Amersham, Piscataway, New Jersey USA.Trypsin–EDTA solution, penicillin–streptomycin solution, andHam’s F12 medium were obtained from Sigma–Aldrich NorwayAS, Oslo, Norway. Lipid standards were obtained from LarodanFine chemicals, Malmo, Sweden. Silica gel 60 plates were obtainedfrom Merck, Whitehouse Station, New Jersey, USA. Unlabelledfatty acids were from Cayman chemicals (Ann Arbor, Michigan,USA). All other chemicals and solvents were high puritycommercial materials obtained from Sigma–Aldrich.

2.2. Cell culture

BeWo cells (ATCC CCL-98, P194, Manassas, VA) were grown inHam’s F12 with 10% FBS supplemented with 2 mM L-glutamine and1% antibiotics (50 U/ml penicillin and 50 mg/ml streptomycin). Thecells were routinely maintained at 37 �C in a 5% CO2 atmosphere.

The cells were subcultured using a trypsin–EDTA solution tosuspend the cells.

2.3. Preparation of fatty acids

Stock solutions of 6 mM fatty acids were complexed with fattyacid free bovine serum albumin (BSA) (6%). The fatty acids weredissolved in 0.1 M NaOH for 10 min before adding pre-warmed(37 �C) 6% fatty acid free BSA in Ham’s F12. The fatty acid-BSAsolution was incubated at 37 �C for 10 min to allow fatty acid–albumin complex formation. For experiments, the fatty acid stocksolution was diluted to a final concentration of 100 mM in a 1%albumin solution (equals 150 mM of albumin). This results in a finalfatty acid:albumin ratio of 1:1.5.

2.4. Uptake of radiolabelled fatty acids

BeWo cells were seeded on six-well plates and grown until 80%confluence. The cells were incubated for 24 h with 100 mM of fattyacids (OA, AA, EPA and DHA) in serum-free F12 Ham’s medium with1% fatty acid free BSA. Concentrations up to 100 mM of fatty acidswere not toxic to these cells, as described before [25]. Control cellsreceived vehicle (1% fatty acid free BSA).

After the preincubation with fatty acids, the cells were washedwith F12 Ham’s and incubated with 100 mM of radiolabelled fattyacids ([14C] OA, [14C]AA, [14C]EPA or [14C]DHA) (specific activity1000–2000 cpm/nmol) for 2 h. Fatty acid uptake was stopped by theaddition of an ice-cold solution of 0.5% fatty acid free BSA, and thecells were washed twice with 0.5% fatty acid free BSA and twice withPBS to remove any surface-bound fatty acid. A whole cell homoge-nate was prepared by lysis of the cells in 0.2M NaOH and intracel-lular incorporation of [14C] fatty acids was determined byscintillation counting. Protein content of the cell lysate was analyzedusing the BC Assay Protein Quantitation Kit (Uptima, Cedex, France).

2.5. Incorporation of radiolabelled fatty acids into cellular lipidfractions

Incorporation of fatty acids into different lipid fractions wasdetermined by thin layer chromatography. BeWo cells were treatedin the same manner as in the uptake studies described above, butthe cells were harvested in 0.3 ml of 0.9% NaCl by scraping the baseof the cell culture plates with a cell scraper. Cellular lipids werethen extracted according to the method of Folch et al. [26]. Briefly;6 ml chloroform:methanol (2:1) containing 0.1% butyratedhydroxytoluene (BHT) was added and the samples were incubatedfor 30 min. Then 1.2 ml 0.88% KCl (pH 2) was added and the sampleswere incubated for 10 min before a 5 min centrifugation at2000 rpm at 4 �C. The water and protein phase was removed andthe chloroform phase was evaporated under a stream of nitrogenand resuspended in 0.2 ml hexane before 0.05 ml of each samplewas applied to thin layer silica gel plates (Silica gel 60, Cat.no.1.05748.0001, Merck, Darmstadt, Germany). Separation was ach-ieved using a hexane/diethylether/acetic acid (65:35:1 v/v) solventsystem. Lipid fractions were identified by co-chromatography withsuitable standards (90-3034 TLC Mix 34, 90-3040 TLC Mix 40,Larodan Fine Chemicals, Malmo, Sweden) and visualized by incu-bating the plate in an iodine chamber. Appropriate bands were cutdirectly into vials containing scintillation fluid (Ultima Gold, PerkinElmer, Waltham, Massachusetts, USA) and incubated for 24 hbefore counting. Percentage incorporation into each lipid fractionwas calculated by dividing the radioactivity found in that lipidfraction by the total radioactivity incorporated into cellular lipids.

G.M. Johnsen et al. / Placenta 30 (2009) 1037–1044 1039

2.6. Gene expression analysis

BeWo cells were incubated for 24 h with 100 mM of fatty acid(OA, AA, EPA or DHA) in F12 Ham’s medium with 1% fatty acid freeBSA. The cells were then washed with ice-cold PBS and lysed in RNAlysis buffer (Applied Biosystems, Foster City, CA) and total RNA wasisolated using ABI 6100 (Applied Biosystems) according to themanufacturer’s instruction.

For quantitative reverse transcription-PCR (qRT-PCR), cDNAswere produced from extracted total RNA using high-capacitycDNA Reverse Transcription kit according to manufacturersinstructions and analyzed using TaqMan Gene Expression Assays(listed in Table 1) and TaqMan Gene Expression Master Mix on the7900HT Real-Time PCR System (all Applied Biosystems). Briefly;a 20 ml reaction mix consisting of 1 ml cDNA, 10 ml 2� geneexpression master mix, 1 ml gene expression assay and 8 ml H2Owas amplified in a 96 well clear plate under the standard thermalcycling conditions as instructed by the manufacturer (50�C for2 min followed by an initial denaturation step at 95�C for 10 min,then 40 cycles at 95�C for 10 s and 60�C for 1 min). Cyclethreshold (Ct) values were calculated using the SDS software v.2.3using automatic baseline and threshold settings. Ct values above35 were considered to be below the detection level of the assays,therefore only genes with a Ct value at 35 or below were includedin the expression analysis. The Ct value of an endogenous controlgene (TBP or B2M) was subtracted from the corresponding Ctvalue for the target gene resulting in the delta Ct value which wasused for relative quantification of gene expression by thecomparative Ct method (2�DDCt method) [27].

2.7. Statistics

Statistical analyses were performed using one-way analysis ofvariance (ANOVA) or two-way ANOVA with Bonferroni’s post hoctest using Graph Pad Prism Version 5.0. Statistical significance wasdefined as a p value below 5%.

Table 1TaqMan assays used for gene expression analysis.

Symbol Gene name

ACSL1 long-chain acyl-CoA synthetase 1ACSL3 long-chain acyl-CoA synthetase 3ACSL4 long-chain acyl-CoA synthetase 4ACSL5 long-chain acyl-CoA synthetase 5ACSL6 long-chain acyl-CoA synthetase 6ADRP adipose differentiation-related proteinAGPAT 1-acylglycerol-3-phosphate-0-acyltransferase 1B2M beta-2-microglobulinCAV-1 caveolin-1CD36/FAT CD 36 molecule (thrombospondin receptor)/fatty acid tra

DGAT diacylglycerol-0-acyltransferaseFABP1 fatty acid binding protein 1FABP3 fatty acid binding protein 3FABP4 fatty acid binding protein 4

FABP5 fatty acid binding protein 5FABPpm Plasma membrane-associated fatty acid binding proteinFATP1 fatty acid transport protein 1FATP2 fatty acid transport protein 2FATP3 fatty acid transport protein 3FATP4 fatty acid transport protein 4FATP6 fatty acid transport protein 6GPAT/GPAM glycerol-3-phosphate acyltransferaseHSL hormone sensitive lipaseLPIN1 lipin-1PPARGC1A peroxisome proliferator-activated receptor gamma, coactTBP TATA binding protein

3. Results

3.1. [14C] fatty acid uptake after 24 h preincubation of BeWo cellswith OA, AA, EPA, and DHA

BeWo cells were preincubated with 100 mM fatty acids (OA, AA,EPA, or DHA) for 24 h before the uptake of 100 mM of the fattyacids ([14C] OA, [14C]EPA, [14C]AA, and [14C]DHA) was carried out.Fig. 1 shows the uptake of different [14C] fatty acids after 2 hincubation. With the exception of OA, preincubation of BeWo cellswith all LCPUFAs (AA, EPA and DHA) stimulated uptake of all fattyacids by 20–50% compared with their corresponding uptake bycontrol cells or OA pre-treated cells. Preincubation of these cellswith AA, EPA, or DHA stimulated the uptake of [14C] OA (Fig. 1A),[14C]AA (Fig. 1B) and [14C]DHA (Fig. 1D) by approximately 20–25%(p < 0.001) compared with OA treated or control cells. AA andDHA stimulated the uptake of [14C]EPA (Fig. 1C) by 34–38%,whereas EPA stimulated its own uptake by 53%, compared withthe control (p < 0.001).

3.2. Incorporation of [14C] fatty acids in different lipid classes afterpreincubation with OA, AA, EPA, and DHA for 24 h

We then investigated the distribution of the [14C] fatty acids thatwere taken up by the BeWo cells into different intracellular lipidfractions. Table 2 shows the incorporation of [14C] fatty acids into PLand TAG fractions.

Incorporation of [14C]OA into PL fractions was significantlyincreased when BeWo cells were preincubated with AA, EPA orDHA(68–70%) compared with that in cells preincubated with OA orwithout fatty acid (control) (55–56%). Simultaneously there wasa decrease in the incorporation of [14C]OA into TAG fractions in theLCPUFA preincubated cells (16–21%) compared with OA treatedcells (30%) or control cells (28%).

Incorporation of [14C]AA in PL fractions was significantly higherin cells preincubated with LCPUFA (54–59%) compared with cells

Assay ID Amplicon length

Hs00960561_m1 100Hs00244853_m1 100Hs00244871_m1 86Hs00212106_m1 90Hs00362960_m1 74Hs00605340_m1 139Hs00262075_m1 58Hs99999907_m1 75Hs00971716_m1 66

nslocase Hs00169627_m1 77Hs00354519_m1 83Hs00201385_m1 64Hs00155026_m1 71Hs00269758_m1 92Hs00609791_m1 105Hs01086177_m1 96Hs02339439_g1 91Hs00905827_g1 67Hs01587917_m1 91Hs00186324_m1 90Hs00225680_m1 70Hs00192700_m1 75Hs00204034_m1 101Hs00326039_m1 69Hs00193510_m1 67Hs00299515_m1 78

ivator 1 alpha Hs01016724_m1 96Hs99999910_m1 127

Fig. 1. [14C] Fatty Acid uptake in BeWo cells after preincubation with Oleic Acid (OA), Arachidonic Acid (AA), Eicosapentaenoic Acid (EPA), or Docosahexaenoic Acid (DHA) for 24 h.Uptake of [14C] fatty acids was measured after 2 h in BeWo cells that had been previously incubated with 100 mM of different fatty acids (OA, AA, EPA, DHA) for 24 h. Control cellsreceived vehicle (1% fatty acid free BSA). Uptake of [14C] fatty acids was calculated as picomoles of [14C] fatty acid related to micrograms of protein per sample. A) [14C] OA B) [14C]AAC) [14C]EPA and D) [14C]DHA. The results are presented as percentage of Control (which was assigned as 100%) and represent means � SEM of 3–6 experiments run in triplicate.Statistical analysis was performed using one-way ANOVA with Bonferroni’s post hoc test. *p < 0.001. * denotes significant difference between controls and fatty acid treatments,while denotes significant difference between OA and other fatty acid treatments.

G.M. Johnsen et al. / Placenta 30 (2009) 1037–10441040

preincubated with OA (37%). The incorporation into TAG fractionswas significantly increased in cells preincubated with OA (50%)compared with controls (38%), whilst for LCPUFA preincubated cellsthere was a significant decrease in the incorporation of this fattyacid into TAG fraction (32–34%) compared with OA preincubatedcells (50%).

[14C]EPA incorporation into PL fractions was significantly higherin LCPUFA preincubated cells(60–67%) compared with that in OApreincubated cells (46%), and there was a concomitant decrease inincorporation of this fatty acid in TAG fractions (23–25%) comparedwith OA preincubated cells (38%). [14C]EPA incorporation wassignificantly higher into TAG fractions (38.3%) and lesser in PLfractions (46%) in cells preincubated with OA compared with thosein control cells (27.7%, and 56% respectively).

Incorporation of [14C]DHA in PL fractions was significantlyhigher in cells preincubated with LCPUFA(60–72%) comparedwith cells preincubated with OA (42%) or control cells (50%).The incorporation of this fatty acid into TAG fractions wassignificantly increased in cells preincubated with OA (38%)compared to controls (23%), while for LCPUFA preincubatedcells there was a significant decrease in the incorporation of thisfatty acid in TAG fractions (17–25%) compared with OA pre-incubated cells (38%).

No significant alteration was observed in incorporation ofthe [14C] fatty acids into other lipid fractions (diacylglycerols,monoacylglycerols, free fatty acids, and cholesterol ester) inLCPUFAs or OA treated cells compared with the control cells(data not shown).

3.3. Expression of genes involved fatty acid uptake and metabolismafter 24 h incubation of BeWo cells with different fatty acids

In order to explore the mechanisms responsible for LCPUFAstimulated [14C] fatty acid uptake, we examined the expression ofseveral genes involved in fatty acid uptake and metabolism inBeWo cells.

We investigated the effect of fatty acids on the expression of allthe known isoforms of ACSL (ACSL1, 3,4, 5 and 6) in these cells.Among these isoforms, expression of ACSL1 (Fig. 2A) and ACSL5(Fig. 2C) was significantly induced by all the LCPUFAs (AA, EPA andDHA) compared with OA, whereas expression of ACSL1 wasunaltered by OA. The expression of ACSL5 however was increasedby OA as compared with control cells, however did not attainstatistical significance. ACSL3 (Fig. 2B) expression was significantlydecreased by all fatty acids. Expression of ACSL4 and 6, on theother hand, was not altered after incubation with fatty acids (datanot shown). In contrast to ACSLs, the expression of any FATPs(FATP1, FATP2, FATP3, FATP4, and FATP6) was not regulated by anyof the fatty acids used in this study (data not shown).

The LCPUFAs increased the gene expression of the lipid dropletassociated protein ADRP compared with the control cells. DHAincreased the gene expression compared with OA (Fig. 2D). Incontrast, all the fatty acids decreased the expression of the enzymein TAG synthesis, phosphatidic acid phosphatase LPIN1 (Fig. 2E).The expression of FABP3 was significantly down-regulated by allfatty acids compared with controls (data not shown). The expres-sion of FABP5, FABPpm, CAV1, DGAT, AGPAT, GPAT/GPAM was not

Table 2Incorporation of [14C] fatty acids into phospholipid (PL) and triglyceride (TAG) fractions in BeWo cells after 24 h preincubation with different fatty acids.a

24 h preincubation with:

Control OA (100 mM) AA (100 mM) EPA (100 mM) DHA (100 mM)

[14C]OA

PL 56.1 � 2.8 55.1 � 2.1 68.9 � 1.8***/ 68.5 � 1.8***/ 70.2 � 1.3***/

TAG 28.7 � 3.5 30.4 � 2.8 21.5 � 3.2 20.2 � 2.7*/ 16.9 � 1.8**/

[14C]AA

PL 47.8 � 3.6 37.4 � 2.6* 54.9 � 2.0 57.4 � 5.2 59.5 � 3.4*/

TAG 38.8 � 3.6 50.0 � 3.7* 34.5 � 3.2 33.0 � 3.8 32.1 � 4.4

[14C]EPA

PL 56.4 � 3.0 46.0 � 2.9* 60.4 � 3.0 66.0 � 4.4*/ 67.7 � 2.8*/

TAG 27.7 � 1.8 38.3 � 2.7** 25.0 � 1.5 25.6 � 2.3 23.6 � 3.4

[14C]DHA

PL 50.4 � 2.9 42.7 � 3.6 60.5 � 2.2*/ 62.3 � 2.9**/ 72 � 3.2***/

TAG 23.7 � 2.9 38.3 � 2.3*** 25.3 � 1.6 25.2 � 1.7 17.5 � 1.6*/

*p < 0.05, **p < 0.01, ***p < 0.001. * denotes significant difference between controls and fatty acid preincubations, while denotes significant difference between OA andother fatty acid preincubations.

a The cells were first preincubated with 100 mM fatty acids for 24 h, followed by 2 h incubation with radiolabelled fatty acids. The data is given as percentage incorporation inthe different lipid fractions calculated by dividing the radioactivity found in each fraction by the total amount of radioactivity incorporated into cellular lipids. The data is givenas mean � SEM of 3 similar experiments. Statistical analysis was performed using one-way ANOVA with Bonferroni’s post hoc test.

G.M. Johnsen et al. / Placenta 30 (2009) 1037–1044 1041

altered by any of the fatty acids compared with control cells (datanot shown). Gene expression of fatty acid transporter (CD36/FAT),FABP4, HSL, and PPARGC1a was not determinable (Ct <35) in theBeWo cells.

3.4. Dose-dependent effect of fatty acid mediated ACSL1 and ACSL5expression in BeWo cells

To test the potency of the LCPUFAs on the gene expression ofACSL1 and ACSL5, BeWo cells were treated with increasingconcentrations of OA, AA, EPA, and DHA for 24 h as indicated inFig. 3A,B. The mRNA induction of ACSL1 and ACSL5 genes wasconcentration-dependent and increased steadily up to 100 mM withAA, EPA, and DHA. EPA and DHA significantly increased theexpression of both ACSL1 and ACLS5 at 50 mM and 100 mM(p < 0.001) whereas AA increased the expression of these twogenes significantly only at 100 mM (p < 0.001). OA did not signifi-cantly affect the expression of these two genes in these cells.

4. Discussion

Overall, our data highlights the complexity of LCPUFA effectssuch as metabolic adaptation (increased fatty acid uptake andtheir enhanced incorporation into PL fractions) and alteration ofexpression of several lipid metabolic genes in placental tropho-blastic cells, the BeWo cell line.

In this paper we report that LCPUFAs stimulate uptake of non-specific fatty acids (AA, EPA, DHA as well as a non-essential fattyacid, OA) to varying degrees (20%–50%) in BeWo cells. Fatty acidsare known to act in an autocrine manner to regulate their ownuptake, transport, and metabolism via different complex mecha-nisms [28]. Several studies revealed that the autocrine effect offatty acids might be facilitated via gene expression mediated byseveral nuclear receptors [28–30]. LCPUFAs mediated gene tran-scription exerts long-term regulation of metabolism and, as such,represents an important biological control point. Stimulation offatty acid uptake by LCPUFAs, but not OA, coincides with theincreased gene expression of ACSL1 and ACSL5 among other ACSLsor any fatty acid transporting/metabolic gene in BeWo cells.LCPUFAs and OA down-regulated the expression of ASCL3 in thesecells, while the expression of ACSL4 and ACSL6 remained unaltered.Therefore increased expression of ACSL1 and ACSL5 by LCPUFAsmay be responsible for increased accumulation of all types of fattyacids in these cells. However, further work on the protein expres-sion and enzyme activity of different ACSL isoforms is required fordefinitive conclusions under these experimental conditions, asseveral possibilities exist, including posttranslational modificationsthat regulate acute changes in enzyme activity or specificity andinteraction with proteins that dictate the function of particularACSL isoforms. Total long-chain acyl-CoA synthetase activity ina particular tissue is the sum of contributions from all ACSL iso-forms plus the activities of medium-chain and very-long-chainacyl-CoA synthetase activity whose substrate specificities overlap

Fig. 2. Expression of ACSL1, ACSL3, ACSL5, ADRP and LPIN1. BeWo cells were incubated with 100 mM of different fatty acids (OA, AA, EPA, DHA) for 24 h. Control cells receivedvehicle (1% fatty acid free BSA). The mRNA expression was analyzed using qRT-PCR normalized to the endogenous control B2M or TBP. A) ACSL1 B) ACSL3 C) ACSL5 D) ADRP andE) LPIN1. The results are presented as mean fold change � SEM relative to control. Statistical analysis was performed using one-way ANOVA with Bonferroni’s post hoc test.*p < 0.05, **p < 0.01, ***p < 0.001. * denotes significant difference between controls and fatty acid treatments, while denotes significant difference between OA and other fattyacid treatments.

G.M. Johnsen et al. / Placenta 30 (2009) 1037–10441042

with those of ACSL. Therefore, the increased expression of ACSL1and ACSL5, may play important roles in the LCPUFA mediatedincreased uptake of fatty acids under these experimental condi-tions. This opposite regulation of expression of ACSL1 and ACSL5compared to ACSL3 by LCPUFAs is intriguing, and offers an inter-esting possibility of metabolic adaptation of these cells in thepresence of excess LCPUFAs.

In fact, fatty acids taken up via ACSL1 and ACSL5 can opt foreither anabolic (TAG or PL synthesis) or catabolic (beta oxidation)pathways [14]. The role of ACSL1 in fatty acid uptake and in TAG

synthesis was demonstrated in 3T3-L1 adipocytes during differ-entiation [31], and in adipose tissue [32]. However, ACSL1 in liver isa target of PPARa, which enhances the expression of genes involvedin b-oxidation [33]. ACSL1 channels fatty acids towards diac-ylglycerol and PL synthesis and increased reacylation of hydrolyzedfatty acids into TAG in rat primary hepatocytes [14]. Apart from itsplasma membrane location, ACSL1 is also located in endoplasmicreticulum and mitochondrial-associated membrane in different celltypes [34] but its location in BeWo cells is not known. By alteringfatty acid activation and trafficking, ACSL1 could contribute to the

Fig. 3. Concentration-dependent effects of fatty acid mediated mRNA expression ofACSL1 and ACSL5. BeWo cells were incubated with 0, 10, 50 or 100 mM of OA, AA, EPAor DHA for 24 h in serum-free medium containing 1% fatty acid free BSA. The mRNAexpression was analyzed using qRT-PCR normalized to the endogenous control B2M.The results are presented as mean fold change � SEM relative to control (1% fatty acidfree BSA). A) Incubation with EPA and DHA significantly increased the expression ofACSL1 at 50 mM and 100 mM (p < 0.001) and AA at 100 mM (p < 0.001); B) Incubationwith EPA and DHA significantly increased the expression of ACSL5 at 50 mM and100 mM (p < 0.001) and AA at 100 mM (p < 0.001). Statistical significance was calcu-lated using two-way ANOVA followed by Bonferroni’s post hoc test.

G.M. Johnsen et al. / Placenta 30 (2009) 1037–1044 1043

innate physiological differences between LCPUFAs and other non-essential fatty acids (OA) in regulating placental fatty acid traf-ficking. Thus, LCPUFAs may play an important role in fatty acidmetabolism as ACSLs are key determinants in regulating themetabolic fate of fatty acids within cells. Both ACSL5 and ACSL1have highest preference for saturated and unsaturated fatty acids of16–20 carbons [35]. Therefore increased expression of these twogenes, as observed in the present study, will increase fatty aciduptake without any preference for any particular fatty acids.Although it was initially suspected that ACSL5 activated fatty acidsdestined for b-oxidation (4), overexpressed ACSL5 in hepatomacells revealed that it activates exogenous fatty acids destined forTAG synthesis [10]. These discrepant findings suggest that thefunction of the ACSLs may differ in different tissues or cell types.

We reported earlier that LCPUFAs as well as OA increased theexpression of ADRP in BeWo cells [25]. Increased expression ofADRP in these cells by OA and LCPUFAs cannot therefore accountonly for LCPUFA-specific stimulation of fatty acid uptake in thesecells. However, increased expression of ADRP lead to an increasein the uptake of fatty acids in different cell types [36]. Theenhanced expression of ADRP by combined insulin and fatty acidswas associated with fat accumulation in BeWo cells but not withincreased uptake of fatty acids [37]. It supports our findingsthat increased expression of ADRP may not be associated withexogenous fatty acid accumulation in BeWo cells. In our recent

paper we showed that incubation of BeWo cells with 100 mM OAfor 24 h resulted in an increased accumulation of triglyceridesand lipid droplet formation compared with DHA [38]. Wemeasured total triglyceride content of the cells but not theaccumulation of exogenously added radioalbelled fatty acid inthese cells, hence this data did not represent the accumulation ofradiolabelled fatty acid after 24 h fatty acid preincubation.Triglycerides can be accumulated due to the redistribution ofintracellular fatty acids as it happened after insulin treatment incombination with OA in BeWo cells [37], hypoxia or otherconditions such as more production of lipid droplet proteins, asdescribed above. Despite an increased accumulation of triglycer-ides by OA, the relative transfer of radiolabelled DHA across theBeWo cell monolayer was 4 fold higher compared with that ofradiolabelled OA, indicating that transport or uptake of DHAacross the BeWo cells is favoured [38].

There is only one study so far published investigating theexpression of FATPs in human placenta after supplementation ofDHA in pregnant women [24]. This study support our findings,showing that LCPUFAs did not alter the expression of any of themembrane FATPs, FAT, FABPpm or FABPs in BeWo cells.

The stimulated uptake of fatty acids by LCPUFAs was associatedwith an increased incorporation of fatty acids into PL fractions anddecreased incorporation into TAG fractions. In most mammaliancell types, including adipocytes, TAG is synthesized via the glycerolphosphate pathway. Lipins are phosphatidic acid phosphatases[39] that convert phosphatidic acid to diacylglycerol during TAGsynthesis [40]. We found that LPIN1 expression was down-regu-lation by fatty acid treatment in BeWo cells, and this down-regulation suggests that the increased incorporation of fatty acidsinto phospholipid fractions found in our study can be explained bya decrease in TAG synthesis from phosphatidic acid. Our results aresupported by a study in which mice were fed a low-fat diet (4.8%fat), the saturated fatty acids dramatically increased hepatic LPIN1mRNA levels several-hundred-fold, whereas the expression wasnegatively regulated by LCPUFA [41]. These results implicate lipin1 levels as one of the factors that contribute to increased hepaticTAG levels in response to dietary fat. However, further work isrequired in order to understand the role of lipin 1 in placenta lipidmetabolism.

We have shown previously that BeWo cells preferentially takeup LCPUFAs compared with shorter fatty acids such as OA [8].Here, we show that treatment of BeWo cells with LCPUFAs, butnot OA, lead to an increased ability of the cells to accumulateexogenous fatty acids. However, a detailed study on effects ofLCPUFAs on the protein expression and activity of different ACSLswould give us much deeper understanding on the fatty aciduptake by the placental trophoblasts and its impact on fetalgrowth and development.

In conclusion, LCPUFAs stimulate fatty acid uptake in BeWocells. Enhanced uptake of fatty acids was associated with increasedincorporation into PL fractions. In addition, stimulation of fatty aciduptake coincides with increased expression of ACSL1 and ACSL5,indicating that acyl-CoA synthetase activities may be responsiblefor stimulated fatty acid uptake by LCPUFAs in BeWo cells. Becauseof renewed interest in the use of dietary lipids as management toolsto prevent metabolic and reproductive disorders, these types ofstudies can provide much-needed data that might be of practicalclinical application in the future.

Acknowledgements

The authors are grateful to Aud Jørgensen for her technicalassistance. This work was supported by grants from the Universityof Oslo, the Faculty of Medicine, the Johan Throne Holst Foundation,

G.M. Johnsen et al. / Placenta 30 (2009) 1037–10441044

the Regional Health Authority of South-Eastern Norway and OsloUniversity Hospital, Ulleval.

References

[1] Innis SM. Essential fatty acids in growth and development. Prog Lipid Res1991;30(1):39–103.

[2] Innis SM. Dietary omega 3 fatty acids and the developing brain. Brain Res2008;1237:35–43.

[3] Duttaroy AK. Transport mechanisms for long-chain polyunsaturated fattyacids in the human placenta. Am J Clin Nutr 2000;71(1 Suppl.):315S–22S.

[4] Herrera E. Implications of dietary fatty acids during pregnancy on placental,fetal and postnatal development–a review. Placenta 2002;23(Suppl. A):S9–19.

[5] Carlson SE. Docosahexaenoic acid supplementation in pregnancy and lacta-tion. Am J Clin Nutr 2009;89(2):678S–84S.

[6] Greenberg JA, Bell SJ, Ausdal WV. Omega-3 fatty acid supplementation duringpregnancy. Rev Obstet Gynecol 2008;1(4):162–9.

[7] Bourre JM. Dietary omega-3 fatty acids for women. Biomed Pharmacother2007;61(2–3):105–12.

[8] Campbell FM, Clohessy AM, Gordon MJ, Page KR, Duttaroy AK. Uptake of longchain fatty acids by human placental choriocarcinoma (BeWo) cells: role ofplasma membrane fatty acid-binding protein. J Lipid Res 1997;38(12):2558–68.

[9] Duttaroy AK. Transport of fatty acids across the human placenta: a review.Prog Lipid Res 2009;48(1):52–61.

[10] Mashek DG, McKenzie MA, Van Horn CG, Coleman RA. Rat long chain acyl-CoAsynthetase 5 increases fatty acid uptake and partitioning to cellular tri-acylglycerol in McArdle-RH7777 cells. J Biol Chem 2006;281(2):945–50.

[11] Tong F, Black PN, Coleman RA, DiRusso CC. Fatty acid transport by vectorialacylation in mammals: roles played by different isoforms of rat long-chainacyl-CoA synthetases. Arch Biochem Biophys 2006;447(1):46–52.

[12] Marszalek JR, Kitidis C, Dararutana A, Lodish HF. Acyl-CoA synthetase 2overexpression enhances fatty acid internalization and neurite outgrowth.J Biol Chem 2004;279(23):23882–91.

[13] Duttaroy AK. Cellular uptake of long-chain fatty acids: role of mem-brane-associated fatty-acid-binding/transport proteins. Cell Mol Life Sci 2000;57(10):1360–72.

[14] Li LO, Mashek DG, An J, Doughman SD, Newgard CB, Coleman RA. Over-expression of rat long chain acyl-coa synthetase 1 alters fatty acid metabolismin rat primary hepatocytes. J Biol Chem 2006;281(48):37246–55.

[15] Milger K, Herrmann T, Becker C, Gotthardt D, Zickwolf J, Ehehalt R, et al.Cellular uptake of fatty acids driven by the ER-localized acyl-CoA synthetaseFATP4. J Cell Sci 2006;119(Pt 22):4678–88.

[16] Heimli H, Hollung K, Drevon CA. Eicosapentaenoic acid-induced apoptosisdepends on acyl CoA-synthetase. Lipids 2003;38(3):263–8.

[17] Steinberg SJ, Morgenthaler J, Heinzer AK, Smith KD, Watkins PA. Very long-chain acyl-CoA synthetases. Human ‘‘bubblegum’’ represents a new family ofproteins capable of activating very long-chain fatty acids. J Biol Chem2000;275(45):35162–9.

[18] Mashek DG, Li LO, Coleman RA. Rat long-chain acyl-CoA synthetase mRNA,protein, and activity vary in tissue distribution and in response to diet. J LipidRes 2006;47(9):2004–10.

[19] Stremmel W, Pohl L, Ring A, Herrmann T. A new concept of cellular uptake andintracellular trafficking of long-chain fatty acids. Lipids 2001;36(9):981–9.

[20] Mashek DG, Coleman RA. Cellular fatty acid uptake: the contribution ofmetabolism. Curr Opin Lipidol 2006;17(3):274–8.

[21] Kim JH, Lewin TM, Coleman RA. Expression and characterization of recombi-nant rat Acyl-CoA synthetases 1, 4, and 5. Selective inhibition by triacsin C andthiazolidinediones. J Biol Chem 2001;276(27):24667–73.

[22] Van Horn CG, Caviglia JM, Li LO, Wang S, Granger DA, Coleman RA. Charac-terization of recombinant long-chain rat acyl-CoA synthetase isoforms 3 and6: identification of a novel variant of isoform 6. Biochemistry 2005;44(5):1635–42.

[23] Lewin TM, Wang S, Nagle CA, Van Horn CG, Coleman RA. Mitochondrialglycerol-3-phosphate acyltransferase-1 directs the metabolic fate of exoge-nous fatty acids in hepatocytes. Am J Physiol Endocrinol Metab 2005;288(5):E835–44.

[24] Larque E, Krauss-Etschmann S, Campoy C, Hartl D, Linde J, Klingler M, et al.Docosahexaenoic acid supply in pregnancy affects placental expression offatty acid transport proteins. Am J Clin Nutr 2006;84(4):853–61.

[25] Tobin KA, Harsem NK, Dalen KT, Staff AC, Nebb HI, Duttaroy AK. Regulation ofADRP expression by long-chain polyunsaturated fatty acids in BeWo cells,a human placental choriocarcinoma cell line. J Lipid Res 2006;47(4):815–23.

[26] Folch J, Lees M, Sloane Stanley GH. A simple method for the isolation and puri-fication of total lipides from animal tissues. J Biol Chem 1957;226(1):497–509.

[27] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods2001;25(4):402–8.

[28] Jump DB. Dietary polyunsaturated fatty acids and regulation of gene tran-scription. Curr Opin Lipidol 2002;13(2):155–64.

[29] Pawar A, Xu J, Jerks E, Mangelsdorf DJ, Jump DB. Fatty acid regulation of liver Xreceptors (LXR) and peroxisome proliferator-activated receptor alpha (PPAR-alpha) in HEK293 cells. J Biol Chem 2002;277(42):39243–50.

[30] Jump DB. The biochemistry of n-3 polyunsaturated fatty acids. J Biol Chem2002;277(11):8755–8.

[31] Chiu HC, Kovacs A, Ford DA, Hsu FF, Garcia R, Herrero P, et al. A novel mousemodel of lipotoxic cardiomyopathy. J Clin Invest 2001;107(7):813–22.

[32] Martin G, Schoonjans K, Lefebvre AM, Staels B, Auwerx J. Coordinate regula-tion of the expression of the fatty acid transport protein and acyl-CoAsynthetase genes by PPARalpha and PPARgamma activators. J Biol Chem1997;272(45):28210–7.

[33] Schoonjans K, Staels B, Auwerx J. Role of the peroxisome proliferator-activatedreceptor (PPAR) in mediating the effects of fibrates and fatty acids on geneexpression. J Lipid Res 1996;37(5):907–25.

[34] Lewin TM, Kim JH, Granger DA, Vance JE, Coleman RA. Acyl-CoA synthetaseisoforms 1, 4, and 5 are present in different subcellular membranes in rat liverand can be inhibited independently. J Biol Chem 2001;276(27):24674–9.

[35] Oikawa E, Iijima H, Suzuki T, Sasano H, Sato H, Kamataki A, et al. A novel acyl-CoA synthetase, ACS5, expressed in intestinal epithelial cells and proliferatingpreadipocytes. J Biochem 1998;124(3):679–85.

[36] Gao J, Serrero G. Adipose differentiation related protein (ADRP) expressed intransfected COS-7 cells selectively stimulates long chain fatty acid uptake. JBiol Chem 1999;274(24):16825–30.

[37] Elchalal U, Schaiff WT, Smith SD, Rimon E, Bildirici I, Nelson DM, et al. Insulinand fatty acids regulate the expression of the fat droplet-associated proteinadipophilin in primary human trophoblasts. Am J Obstet Gynecol2005;193(5):1716–23.

[38] Tobin KA, Johnsen GM, Staff AC, Duttaroy AK. Long-chain polyunsaturatedfatty acid transport across human placental choriocarcinoma (BeWo) cells.Placenta 2009;30(1):41–7.

[39] Donkor J, Sariahmetoglu M, Dewald J, Brindley DN, Reue K. Three mammalianlipins act as phosphatidate phosphatases with distinct tissue expressionpatterns. J Biol Chem 2007;282(6):3450–7.

[40] Coleman RA, Lee DP. Enzymes of triacylglycerol synthesis and their regulation.Prog Lipid Res 2004;43(2):134–76.

[41] Martin PG, Guillou H, Lasserre F, Dejean S, Lan A, Pascussi JM, et al. Novelaspects of PPARalpha-mediated regulation of lipid and xenobioticmetabolism revealed through a nutrigenomic study. Hepatology 2007;45(3):767–77.