Participation of mammary gland in long-chain polyunsaturated fatty acid synthesis during pregnancy and lactation in rats

Biochimica et Biophysica Acta 1811 (2011) 284–293

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Biochimica et Biophysica Acta

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Participation of mammary gland in long-chain polyunsaturated fatty acid synthesisduring pregnancy and lactation in rats

Maricela Rodriguez-Cruz a,⁎,1, Raúl Sánchez a,1, Apolos M. Sánchez a, Shannon L. Kelleher b,Fausto Sánchez-Muñoz c, Jorge Maldonado a, Mardia López-Alarcón a

a Laboratorio de Biología Molecular, Unidad de Investigación Médica en Nutrición, Hospital de Pediatría, Centro Medico Nacional Siglo XXI, IMSS, Mexico City, Mexicob Department of Nutritional Sciences, The Pennsylvania State University, University Park, PA, USAc Department of Gastroenterology, INCMNSZ, Tlalpan 14000, Department of Immunology, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City, Mexico

Abbreviations: LC-PUFAs, Long-chain polyunsaturatfatty acids; AA, arachidonic acid; EPA, eicosapentaenoiacid; LA, linoleic acid; ALA, α-linolenic acid; FADS1, FaFatty acid desaturase 2; Elovl, elongation of very long chsterol-regulatory element binding transcription factorMFA, monounsaturated fatty acids; FBS, fetal bovinediethylpyrocarbonate; VLDL, very low-density lipoproresponsive element-binding protein⁎ Corresponding author at: Apartado postal C-029 C. S

Col. Roma, 06703 México, D. F., Mexico. Tel.: +52 562756276944.

E-mail addresses: [email protected]@yahoo.com.mx (R. Sánchez), apolosm5@[email protected] (S.L. Kelleher), [email protected] ([email protected] (J. Maldonado), mardyalo@hotm

1 These authors contributed equally to this work.

1388-1981/$ – see front matter © 2011 Elsevier B.V. Adoi:10.1016/j.bbalip.2011.01.007

a b s t r a c t

Article history:Received 28 June 2010Received in revised form 21 January 2011Accepted 22 January 2011Available online 1 February 2011

Keywords:Fatty acid desaturase 1Fatty acid desaturase 2Elongase 5Sterol-regulatory element binding factorPregnancyLactation

Metabolic adaptations are triggered in the maternal organism to synthesize milk with an adequateconcentration of long-chain polyunsaturated fatty acids (LC-PUFAs) required to the newborn. They may be ahigh uptake of dietary linoleic acid and its conversion to LC-PUFAs by desaturases of fatty acids (FADS) 1 and 2in the mammary gland (MG). It is unknown if they also occur from onset of pregnancy. The aim of this studywas to explore the participation of the MG as a mechanism involved in LC-PUFAs synthesis to support theirdemand during pregnancy and lactation in rats. The expression of desaturases in MG was significantly(Pb0.05) higher (12.3-fold for FADS1 and 41.2-fold for FADS2) during the late pregnancy and throughoutlactation (31.7-fold for FADS1 and 67.1-fold higher for FADS2) than in nonpregnant rats. SREBF-1c showed asimilar pattern of increase during pregnancy but remained higher only during the early lactation (11.7-fold,Pb0.005). Transcript of ELOVL6 and FASN increased throughout pregnancy and lactation, respectively.ELOVL5 mRNA increased in MG only during lactation (2.8 to 5.3-fold, Pb0.005). Accordingly, a higher contentof LC-PUFAs was found in lactating MG than in nonpregnant rats. Results suggest that MG participates fromlate pregnancy and throughout lactation by expressing desaturases and elongases as a mechanism involved inLC-PUFAs synthesis, probably by SREBF-1c. Because desaturases and ELOVL5 were expressed in culturedlactocytes and such expression was downregulated by linoleic and arachidonic acid, these cells may be auseful model for understanding the regulatory mechanisms for LC-PUFAs synthesis in MG.

ed fatty acids; EFAs, essentialc acid; DHA, docosahexaenoictty acid desaturase 1; FADS2,ain fatty acids Elovl; SREBF-1c,1c; FASN, fatty acid synthase;serum; PRL, prolactin; DEPC,teins; CHREBP, carbohydrate-

.P.I. “Coahuila”, Coahuila No. 5,6900x22483, 22484; fax: +52

(M. Rodriguez-Cruz),o.com.mx (A.M. Sánchez),Sánchez-Muñoz),ail.com (M. López-Alarcón).

ll rights reserved.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Long-chain polyunsaturated fatty acids (LC-PUFAs) such asarachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahex-aenoic acid (DHA) acid are required for fetal and postnatal growth andneuronal development. They are derived from the essential fatty acids

linoleic acid (LA) and α-linolenic acid (ALA) [1,2]. Fetus and newbornobtain LC-PUFAs through placenta and milk, respectively [3,4]. Thus,AA and DHA are synthesized by fatty acid desaturases (FADS) 1 and 2and elongases ELOVL2 and ELOV5 in the maternal organism or aremobilized from maternal adipose tissue reserves [5–7].

We recently reported that FADS1, FADS2 [8] and ELOVL5 mRNAsare present in lactating mammary tissue [9]. Although there areseveral microarray reports aboutmurinemammary gland, they do notindicate if ELOVL2 is also expressed in mammary gland. It is wellknown that, in liver, FADS1 and FADS2 genes are regulated by sterol-regulatory element binding transcription factor 1c (SREBF-1c) [10].This regulation is probably similar in mammary gland [8]. However, ithas not been established whether this regulation also occurs inmammary tissue from the onset of pregnancy. In addition, liver is theprimary organ involved in the synthesis of LC-PUFAs [11], and adiposetissue, which is the main reservoir of such fatty acids and expressesFADS1 and FADS2, likely supplies LC-PUFAs during pregnancy.

Additionally, during the lactation a high proportion of dietary LA(35%) is taken up by the mammary gland. Because FADS1 and FADS2are expressed in mammary tissue, it is possible that this LA isconverted to AA, indicating that mammary gland together with liver

285M. Rodriguez-Cruz et al. / Biochimica et Biophysica Acta 1811 (2011) 284–293

participates in the endogenous synthesis of LC-PUFAs also duringlactation [8]. In particular, LC-PUFAs requirement increases during thelast period of pregnancy and the first stage of lactation for the braingrowth spurt of the fetus and newborn. This led us to hypothesize thatthere is an increase in expression of encoding enzymes and theregulator SREBF-1c involved in LC-PUFAs synthesis in the mammarygland during these physiological stages, providing these fatty acidstogether with liver. To test this hypothesis we first evaluated whetherthe transcript of elongase ELOVL2 is expressed in mammary tissue atthe same time that mRNA expression of ELOVL5 and desaturases wasdetermined during pregnancy and lactation. We then compared theirpattern of expression in mammary gland with that in liver andadipose tissue. Next, we analyzed the expression of SREBF-1c (and itstarget genes such as FASN and ELOVL6) [12] as a possible mechanismof regulation of desaturases and elongases in mammary tissue, liverand adipose tissue at different stages of pregnancy and lactation. Afterthat, because mammary tissue is composed of different types of cells[13], we evaluated whether lactocytes express desaturases, elongases,SREBF-1c, and FASN in nonsecreting undifferentiated cells andsecreting differentiated cells. Changes in basal expression levels inthese two states were also determined. Finally, we evaluated if suchexpression was regulated by LC-PUFAs possibly through down-regulation of SREBF-1c expression.

2. Materials and methods

2.1. Materials

Fatty acid methyl ester standards for chromatographic analysiswere obtained from PolySciences (Niles, IL), and fatty acids used formammary epithelial cell culture (LA and AA) were cell culture tested(≥99% purity, Sigma, St. Louis, MO). All other chemicals and solventswere reagent molecular grade from usual commercial sources.

2.2. Experimental animals and tissue collection

One hundred five female Sprague Dawley rats were obtained fromthe Animal Care Facility of the CentroMédico Nacional Siglo XXI of theMexican Institute of Social Security (IMSS) in Mexico City. Animalswere housed at 22±2 °C with a 12-h light/dark cycle and had freeaccess to water, with a maximum of five rats per cage. From weaninguntil 8 weeks of age, rats were fed a Chow 5008 commercial diet(Agribrands Purina, Mexico, D.F., Mexico) containing 24 g of protein,61 g of carbohydrates, and 5.0 g of fat per 100 g of dry weight. Whenrats reached 8 weeks of age, they were adapted to a purified diet for6 weeks with adequate nutrients (22.2 g of protein, 61.4 g ofcarbohydrates and 5% of fat) as previously reported [7,8]. At14 weeks of age, rats were randomly assigned to two groups: onegroup of nonpregnant rats (control, n=7) and another group of ratsthat were mated and randomly assigned to pregnant or lactatinggroups. Pregnant rats were housed individually. Rats were sacrificedat days 0, 1, 5, 10, 14, 16 and 20 of pregnancy; days 0, 1, 5, 10, 12, 20 oflactation; and at day 2 of postweaning (D2Pw). Seven rats from eachsubgroup of pregnancy or lactation were included. The day ofparturition was considered as day 0 of lactation. Litters were adjustedto eight pups per dam, and food intake and body weight of pups andtheir mothers were registered daily. A purified diet was administeredad libitum during pregnancy and lactation. No gender differentiationwas made. The protocol was approved by the Animal Care EthicsCommittee of the IMSS, Mexico.

Rats were euthanized according to the Mexican Official Norm(NOM-062-ZOO-1999). In this study, rats were decapitated afterdiethyl ether anesthesia, and whole mammary tissue, liver, andparametrial adipose tissues were removed. Tissues were harvested,rapidly frozen in liquid nitrogen, and stored at−70 °C for subsequentRNA isolation and total lipid extraction. Pups were euthanized under

anesthesia and their stomachs were then dissected. Milk clots wereremoved, weighed, mixed and stored at −70 °C for subsequent totallipid extraction and fatty acid composition.

2.3. Mammary epithelial cell culture

Mouse mammary epithelial cells (MECs) HC11 were a gift from JeffRosen (University of California, Davis, Davis, CA) and used withpermission from Dr. Bernd Groner (Institute for Biomedical Research,Frankfurt, Germany). HC11 cells, clonally derived from the mammaryepithelial COMMA-1D cell line, are an excellent model for studying theprogression of MEC differentiation to a secreting cell type and theregulation of mammary gland gene expression and milk proteinsecretion after lactogenic hormone exposure [14]. Although HC11 cellsare not functional in terms of protein and lipid secretion, it has recentlybeen demonstrated that in HC11 cells unsaturated fatty acids induce apronounced proliferation of cytoplasmic lipid droplets [15], indicatingthat HC11 cells express genes involved in lipid synthesis. Knowledgeabout the expression and regulation of enzymes involved in LC-PUFAs inthose types of cells will lead us to explore some of the molecularmechanisms of the synthesis of these fatty acids taking place in thenonsecreting and secreting epithelial cells from the mammary gland.

HC11 cells were cultured in growth medium (RPMI 1640 medium,Gibco Invitrogen Corporation, Grand Island, NY) supplemented withgentamycin (Sigma), sodium bicarbonate (Merck, México D.F.),insulin (Sigma), epidermal growth factor (EGF, JRH Biosciences, St.Louis, MO) and 10% fetal bovine serum (FBS, Sigma) to 100%confluence and allowed to expand at 37 °C with 5% CO2.

Upon confluence, cells were then cultured in differentiationmedium [growth medium without fetal bovine serum and epidermalgrowth factor containing prolactin (PRL; 1 μg/ml) and cortisol (1 μM)]for 48 h to differentiate HC11 cells into a secretory cell type.When thecells reached secretory phenotype, the culture mediumwas removed.Cells were then washed with PBS and incubated with freshdifferentiation medium containing 0, 50 or 200 μM of LA (cell culturetested, Sigma) or AA (for cell culture, from porcine liver, Sigma)according to previous studies carried out in liver and HC11 cells[16,17] at 37 °C with 5% CO2 for 24 h. Cellular shape by lightmicroscopy was evaluated to discard alterations due to the fattyacid concentration. Next, the medium was discarded, followed by cellaspiration, and the total RNA was extracted as described below. It isimportant to mention that HC11 cells remain viable when cultured for24 h in the presence of 300 μM of saturated or unsaturated fatty acidsand proliferation of cytoplasm lipid droplets are induced [15]. Twoindependent cell culture assays were done.

2.4. RNA isolation, cDNA synthesis and quantitative real-time (qRT)-PCR

Frozen tissues were pulverized and total RNA was isolated fromtissues (0.1−0.2 g) of the mammary gland, liver and adipose tissuefrom rats and from the HC11 epithelial cells using a TRIzol reagent(Invitrogen, Carlsbad, CA) according to the manufacturer's instruc-tions. RNA concentration was determined by absorbance at 260 nmand diluted to 1 μg/μl in RNAse-free water, and its integrity wasevaluated using electrophoresis with 1% agarose and ethidiumbromide staining (1.25 ng/μl, Sigma). First-strand cDNA from tissueswas synthesized from 2 μg of the total RNA with the MMLV reversetranscriptase. Total RNA was preincubated with random primers anddNTPs (100 mM) at 65 °C for 5 min. The product was then incubatedin reaction buffer (250 mM Tris–HCl, 375 mM KCl, and 15 mMMgCl2)and DTT (0.1 M) at 37 °C for 2 min. Finally, cDNA synthesis wascarried out with the MMLV reverse transcriptase. RNase-freedeionized, diethylpyrocarbonate (DEPC)-treated ultrapure MB gradewater (USB Corporation, Cleveland, OH) was used to bring the finalvolume to 20 μl. cDNA synthesis was done at 25 °C for 10 minfollowed by 37 °C for 50 min and 70 °C for 15 min. All reagents used

Table 1Primers for quantitative reverse transcriptase polymerase chain reaction.

Gene Forward Reverse

FADS1 gCC TTg CTg CCT CTC TAC TT CgT CAC CCA CAC AAA CCAFADS2 gCC TTT gTC CTT gCT ACC TC TTg Tgg AAg ATg TTg ggC TELOVL2 ggA AgA AAT ACC TCA CgC Ag Tgg CTT TTT TCg gTA TgT CELOVL5 CTC ACC CTg CTg TCT CTC TA ATC Tgg Tgg TTg TTC TTA CgSREBF-1c

gAT TgC ACA TTT gAA gAC ATgCTT

gTC CCA ggA Agg CTT CCA gAg A

FASN CCA AgC Agg CAC ACA CAA Tg gAg TgA ggC Tgg gTT gAT ACELOVL6 CTA AgC AAA gCA CCC gAA CT ggC AAC CAT gTC TTT gTA ggAβ-actin Tgg AAT CCT gTg gCA TCC ATg TAA AAC gCA gCT CAg TAA CAgGAPDH TgC CAA gTA TgA TgA CAT CAA

gAA gAgC CCA ggA TgC CCT TTA gT

FADS1, FADS2, desaturases of fatty acids; ELOVL2, ELOVL5, ELOVL6 fatty acid elongase; SREBF-1c, sterol-regulatory element binding transcription factor 1c; FASN, fatty acid synthase.

286 M. Rodriguez-Cruz et al. / Biochimica et Biophysica Acta 1811 (2011) 284–293

were from Invitrogen. Before qRT-PCR analysis, cDNA quality wastested by final-point PCR amplifying the reference β-actin gene.

Relative mRNA levels of target genes and reference transcript β-actin (as previously reported for mammary tissue and HC11 cells[8,14,18]) were determined by qRT-PCR using cDNA preparation fortissues and HC11 cells. Synthesized cDNA from tissues was mixedwith LightCycler Fast Start DNA MasterPLUS SYBR Green I (Roche,Indianapolis, IN) and with various sets of gene-specific forward andreverse primers (Table 1) and then subjected to RT-PCR quantificationusing the Light Cycler 3.5 detection System (Roche). qRT-PCR wasperformed in a specially designed LightCycler Capillaries in a totalvolume of 20 μl containing 2 μl of the cDNA sample, 40 pmol of eachprimer, and 4 μl of MasterPLUS SYBER Green. For each reaction, thepolymerase was activated by preincubation at 95 °C for 10 min.Amplification was then performed with 35 cycles at 95 °C for 10 s and60 °C or 62 °C for 7 s. Relative amounts ofmRNAwere calculated usingthe comparative threshold cycle method (cycle method; LightCyclersoftware v.4.0, Roche). Expression values were normalized fromtissues of the nonpregnant group. Quantification of mRNA levels usingqRT-PCR from HC11 cells was developed in the Department ofNutrition, University of California, Davis as follows: first-strand cDNAfrom HC11 cells was synthesized from 1 μg of the total RNA using areverse transcription kit (TaqMan Reverse Transcription Reagents kit,Applied Biosystems, Carlsbad, CA) according to the manufacturer'sinstructions. The reaction was performed at 48 °C for 30 min followedby 95 °C for 5 min. qRT-PCR was performed using 1 μl of cDNA, 20 nMof each primer, and 10 μl of MasterPLUS SYBER Green (AppliedBiosystems). The following parameters were used: 50 °C for 2 min,95 °C for 10 min, 40 cycles of 95 °C for 15 s, and 60 °C for 1 min,followed by 95 °C for 15 s. Reactions from all samples including allanalyzed genes from undifferentiated or differentiated cells wereperformed in the same 384-well plate in an ABI 7900HT real-timethermocycler (PerkinElmer Applied Biosystems). Relative amounts ofmRNA were calculated using the ABI 7900HT software. The dynamicrange of amplification over which a reaction is linear (the highest tothe lowest quantifiable copy number [threshold cycle (Ct)]) wasdetermined by a calibration curve making serial dilutions (from 1:10to 1:1000) for the genes analyzed. On average, there were≤35 Ct cycles.

All reactions were performed in duplicate and fluorescent datawere acquired during each extension phase. Each sample wasnormalized to β-actin mRNA using the equation:

ΔCt = Ct target gen−Ct β�actin

Fold change in genes analyzed expression was calculated using theequation

2 ΔΔCtgeneð Þ

where ΔΔCt gene=mean ΔCt target gene nonpregnant−ΔCt targetgene of pregnancy, lactation or D2Pw. Values represent mean foldchanges±SE. We also evaluated the expression of ELOVL6 normal-izing with either β-actin or GAPDH (Supplementary Fig. 1). For eachgene, the mRNA level was expressed as a fold change relative to thecontrol (nonpregnant rats or HC11 cells cultured without fatty acid).

2.5. Assessment of fatty acid composition

Enzymatic activity of desaturases and elongases was indirectlymeasured by the fatty acid composition determination. First, totallipids were extracted from mammary gland and adipose tissuesamples (0.5 g) as well as liver and milk clot using the modifiedFolch method [19]. Next, fatty acids were methylated with 3N HCl inmethanol and hexane at 90 °C for 1 h, and fatty acid methyl esterswere extracted with hexane. Before methylation, heptadecanoic acidwas added as the internal standard. This solution was stored at−20 °C with 2 g butylhydroxytoluene/L (Sigma-Aldrich, Mexico, D.F.,Mexico) as the antioxidant for subsequent analysis. Finally, fatty acidswere quantified and analyzed by gas chromatography in an aliquot ofthis solution [7]. Individual fatty acid methyl esters were separated inan HP5890 gas chromatograph (serie II, Hewlett-Packard GC,Waldbronn, Germany) equipped with a flame ionization detectorand a 50 m×0.32 mm CP-Sil 88 Chrompack capillary column withhelium as the gas carrier. Identification of fatty acid methyl esters wasbased upon retention times obtained for methyl ester analyticalstandards (PolySciences). Each fatty acid was expressed as apercentage of total fatty acids in the sample.

2.6. Statistical analysis

Statistical analysis was performed using the Minitab statisticalsoftware (Minitab 14, State College, PA). Results are presented asmean±standard error of fold change. Fold change was calculated asexplained in Materials and methods section. Distributions of thestudied variables were identified as normal after examination withthe Shapiro–Wilk test; therefore, parametric analyses were applied.

To identify the precise point when the studied gene expressionchanges in each tissue, every point during pregnancy and lactationwas compared with the nonpregnant stage using one-way ANOVAand Dunnet's post-test. To identify differences among physiologicalstages (nonpregnant, pregnant and lactation) from mammary tissue,repeated measurement analyses were also conducted using theGeneral Linear Model procedure where each gene expression wasentered as a dependent variable and the physiological stage aspredictor.

Differences of gene expression between undifferentiated anddifferentiated HC11 cells and between HC11cells cultured with fattyacids and control (without fatty acid) were analyzed with Student t-test. Differences were considered significant when Pb0.05.

3. Results and discussion

3.1. Maternal food intake and body weight gain

Daily food intake throughout pregnancy ranged from 17.4±2.7 gat day 1 to 15.1±5.3 g at parturition. Food intake increasedprogressively during lactation, reaching a value of 77.5±8 g at theend of this period. After lactation, food intake decreased progressivelyto almost basal levels (25.7±2.1 g at day 1 postweaning). Contrary tofood intake, the body weight of dams increased progressivelythroughout pregnancy from 270±20 g to 395±26.5 g. Immediatelyafter parturition, body weight decreased to 312±20.8 g. Maternalbody weight did not vary during lactation.

287M. Rodriguez-Cruz et al. / Biochimica et Biophysica Acta 1811 (2011) 284–293

3.2. Expression of desaturases, elongases, SREBF-1c and FASNin mammary gland, liver and adipose tissue during pregnancyand lactation

LC-PUFAs (n-6 and n-3 family) are necessary for central nervoussystem and retina development in the fetus and newborn [20]. Thus,during pregnancy and lactation there is an augmented need for thesenutrients to meet the requirements for both mother and infant [21].We examined the expression of genes encoding enzymes involved inLC-PUFAs synthesis in mammary gland during pregnancy andlactation.

The mRNA expression of FADS1 and FADS2 desaturases inmammary gland gradually increased during pregnancy and lacta-

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Fig. 1. Gene expression of FADS1 (a), FADS2 (b), ELOVL5 (c), ELOVL2 (d), SREBF-1c (e) and ffrom rats throughout pregnancy and lactation as determined by real-time PCR assays. Whiteday 1, day 5 of pregnancy; L1, L5, day 1, day 5 of lactation. *Pb0.05 (ANOVA, Dunnett's methoof each gene obtained from animals (n=3) from assays performed in duplicate.

tion, returning to basal values 2 days after weaning (Fig. 1a and b,Table 2). Maximum expressions were 12.3-fold for FADS1 and 41.2-fold for FADS2, higher than in nonpregnant rats at day 16 ofpregnancy (Pb0.005). Similarly, the greatest expression at lactationwas 31.7-fold higher for FADS1 and 67.1-fold higher for FADS2 thanin nonpregnant rats at day 1 of lactation (Pb0.005). As in mammarytissue, the expression of desaturases in liver was significantly higherat day 16 of pregnancy, but the maximum expression was only 2.7-fold higher for FADS1 and 2.4-fold higher for FADS2 as compared tononpregnant rats. Expression of both desaturases in liver was alsoincreased during lactation. Maximum expression was 2.2- and 1.5-fold higher for FADS1 and FADS2, respectively (Pb0.005), returningto basal levels at the end of this period (day 20). In adipose tissue,

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NP P1 P5 P10 P14 P16 P20 L1 L5 L10 L12 L20 D2Pw

NP P1 P5 P10 P14 P16 P20 L1 L5 L10 L12 L20 D2Pw

NP P1 P5 P10 P14 P16 P20 L1 L5 L10 L12 L20 D2Pw

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atty acid synthase (FASN) (f) mRNAs in mammary gland (MG), liver and adipose tissuebars, MG; gray bars, liver; black bars, adipose tissue; NP, nonpregnant rats; P1, P5, etc.d) as compared to nonpregnant female rats. Bars indicate the mean±SE of fold change

Table 2Gene expression of FADS1, FADS2, ELOVL5, ELOVL2, SREBF-1c and FAS mRNAs in mammary gland, liver and adipose tissue from rats throughout pregnancy and lactation.

STAGE FADS1 FADS2 ELOVL2 ELOVL5 SREBF-1c FASN

Mammary glandP1 1.9±0.6 5.7±1.9 – 0.10±0.05 0.80±0.51 1.48±0.22P5 0.6±0.2 4.1±1.7 – 0.08±0.03 0.44±0.24 1.57±0.39P10 3.7±0.4 10.0±2.0 – 0.60±0.26 2.73±0.77 2.22±0.47P14 3.3±0.9 16.3±6.4 – 0.26±0.18 6.61±0.94* 1.98±0.51P16 12.4±0.3* 38.6±3.5* – 1.31±0.19 12.4±2.36* 2.13±0.48P20 10.1±1.0* 21.0±1.2* – 1.88±0.36 4.3±0.67 0.93±0.24L1 31.7±1.3* 67.1±8.7* – 2.81±0.97 6.2±1.25* 8.90±2.35*L5 22.7±3.2* 20.9±2.5* – 2.21±0.15 7.3±3.03* 3.96±0.40L10 21.1±0.7* 32.4±3.0* – 4.93±0.19* 7.8±2.58* 12.91±2.51*L12 22.4±2.1* 52.1±8.9* – 5.14±0.12* 10.2±1.78* 8.93±0.47*L20 16.5±1.0* 20.8±1.5* – 5.25±1.17* 1.6±0.11 15.49±2.09*D2Pw 1.0±0.3 4.0±0.3 – 0.65±0.15 0.77±0.22 1.04±0.04

LiverP1 1.56±0.16 0.59±0.05 1.46±0.01 2.43±0.15* 1.82±0.06* 1.97±0.56P5 1.45±0.17 1.02±0.11 1.37±0.28 2.17±0.09* 3.95±0.19* 4.52±0.88*P10 2.15±0.09* 0.83±0.18 1.85±0.08 2.13±0.58* 1.36±0.41 1.91±0.20P14 2.02±0.33 0.90±0.10 2.17±0.27* 2.53±0.70* 1.02±0.08 1.53±0.06P16 2.73±0.15* 2.46±0.32* 2.35±0.53* 1.77±0.14 0.71±0.17 0.52±0.14P20 1.59±0.12 1.41±0.02 8.27±1.18 * 2.73±0.52* 0.66±0.04 0.40±0.11L1 1.22±0.22 0.76±0.04 2.05±0.59 1.20±0.20 1.02±0.07 1.30±0.15L5 2.21±0.19* 1.50±0.09* 1.88±0.31 0.73±0.07 1.08±0.03 3.22±0.33*L10 2.04±0.10* 1.14±0.05 1.78±0.18 0.74±0.06 2.85±0.23* 1.53±0.35L12 2.10±0.15* 1.54±0.07* 2.60±0.40* 0.76±0.04 1.41±0.04 2.34±0.23*L20 1.08±0.13 0.79±0.04 0.75±0.26 0.77±0.11 1.37±0.07 2.15±0.01*D2Pw 0.75±0.05 0.65±0.06 0.64±0.06 0.61±0.05 1.45±0.13 0.80±0.06

Adipose tissueP1 1.23±0.07 0.77±0.16 0.46±0.02 1.40±0.10 0.74±0.13 2.04±0.17*P5 1.80±0.53 0.85±0.12 1.48±0.05* 3.09±0.92* 0.80±0.04 3.01±0.38*P10 1.01±0.12 0.49±0.26 0.25±0.03 0.76±0.09 0.60±0.11 1.76±0.28*P14 2.32±0.19* 0.56±0.05 0.91±0.19 1.72±0.19* 0.68±0.08 2.72±0.38*P16 0.43±0.08 0.72±0.10 0.34±0.01 0.66±0.17 0.31±0.09 0.14±0.01P20 0.71±0.08 0.61±0.12 0.59±0.03 1.56±0.15* 0.45±0.04 0.77±0.09L1 0.87±0.15 1.29±0.11 0.43±0.06 1.72±0.25* 0.62±0.09 0.06±0.01L5 1.98±0.15* 1.33±0.17 0.69±0.09 1.65±0.42 0.74±0.12 0.37±0.02L10 0.60±0.09 0.67±0.15 0.60±0.22 1.14±0.34 0.63±0.06 0.27±0.04L12 0.36±0.02 1.26±0.15 0.58±0.11 1.86±0.13* 0.87±0.14 0.17±0.01L20 0.64±0.16 0.97±0.07 0.37±0.10 2.19±0.53 1.44±0.03* 0.46±0.07D2Pw 1.43±0.11* 1.55±0.08 1.60±0.17* 1.82±0.46* 1.99±0.14* 2.15±0.30*

Values represent mean fold changes±SE. The fold change in genes analyzed expression was calculated using the equation 2(ΔΔCtgene), where ΔΔCt gene=mean ΔΔt target genenonpregnant−ΔCt target gene of pregnancy, lactation or D2Pw.*Pb0.05 compared to nonpregnant rats (ANOVA, Dunnett's method).P, pregnancy; L, lactation; D2Pw, day 2 post-weaning.

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an increase of FADS1 transcript was also observed at days 14 and 5of pregnancy and lactation, respectively (Pb0.005), but FADS2 didnot vary throughout the course of pregnancy and lactation. Adiposetissue expressing these genes encoding enzymes involved in LC-PUFAs synthesis does not appear to actively synthesize LC-PUFAsbecause they are present at very low concentrations. The magnitudeof the increase of both desaturases in liver and adipose tissue wasnot as high as in mammary gland.

ELOVL2 and ELOVL5 were actually expressed during pregnancyand lactation in mammary tissue. Although ELOVL5 mRNA wasexpressed in mammary tissue throughout pregnancy, no differenceswith nonpregnant rats were detected. However, expression pro-gressively increased during lactation (Pb0.005), returning to thebaseline at D2Pw. In liver, ELOVL5 mRNA was 2-fold higher than innonpregnant rats during almost all stages of pregnancy (Pb0.005),but no differences were found during lactation in nonpregnant rats.Expression of Elov15 in adipose tissue was higher during pregnancyand lactation than in nonpregnant rats (Pb0.005) (Fig. 1c, Table 2).It is noteworthy that, during pregnancy, the expression of ELOVL5was more important in liver than in other tissues, but during

lactation such expression was even higher in mammary tissue.These findings suggest that liver and mammary gland togetherpreferentially express this elongase in a stage-specific manner inresponse to the high demand of LC-PUFAs of the fetus andnewborn.

ELOVL2 mRNA was detected in mammary tissue, but suchexpression was negligible (average number Cts was 35). In contrast,expression of this elongase in liver increased gradually beginning atthe second period of pregnancy and reached 8.3-fold the concen-tration found in nonpregnant rats at day 20 of pregnancy(Pb0.005). During the middle of lactation (day 12) it was 2.6-foldhigher than in the nonpregnant group (Pb0.005). In adipose tissue,ELOVL2 mRNA increased only at day 5 (Pb0.005) and decreasedduring lactation (Pb0.005) (Fig. 1d, Table 2). It is worth mentioningthat although ELOVL2 mRNA was identified in mammary tissue, itsexpression was negligible during all the studied physiologicalstages. Additional research is needed to clarify the regulatorymechanism of ELOVL2 mRNA during pregnancy and lactation.

These findings show increase in expression of genes encodingenzymes involved in LC-PUFAs synthesis in mammary gland from

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the middle to the late period of pregnancy and throughoutlactation, providing, together with liver, fatty acids required bythe newborn. However, interpretation of gene expression profilesobtained in tissues is complicated by their characteristicallycomplex cellular environments. This results from the fact thatexpression measurements from heterogeneous tissues with distinctcellular compartments reflect weighted averages of expression levelswithin different cellular populations. Therefore, observed changes ingene expression may result from bona fide changes in the regulationwithin a given cellular compartment or from changes in theabundance of an expressing compartment within the tissue as awhole. As a consequence, changes in compartment size may bemistaken for the intracellular regulation of gene expression;conversely, genuine regulation within a given cell type may not bedetected due to changes in the abundance of cellular compartmentsthat mask its contribution to the tissue as a whole [22].

Our finding regarding the expression pattern of desaturases duringpregnancy and lactation in rats is novel. Its pattern is similar to othergenes reported by Lemay et al. who used microarray analysis anddescribed the tendency (50%) of 4832 genes from mammary tissuesamples indicative of an elevated gene expression during the latestage of pregnancy and throughout lactation but decreasing atinvolution [23]. Lemay et al. reported on some of these genesencoding proteins with known functions of mammary epithelialcells during lactation such as ion transport, calcium-mediatedsignaling, transferase activity, and cell proliferation. It is interestingthat genes associated with these known functions appear to betranscribed well in advance of the onset of lactation. It is somewhatintuitive that mammary gland transcriptomic adaptations occurduring these physiological stages and involve hundreds of genes.We also demonstrated here that mammary tissue has the ability toexpress elongases ELOVL2 and ELOVL5, which also participate in LC-PUFAs synthesis [24].

It is well known that SREBF-1c is a key factor of lipogenesis in liverand activates genes involved in fatty acid synthesis includingdesaturases. SREBF-1 associates and forms a complex with other ERmembrane proteins, the SREBF-cleavage activating protein (SCAP)and INSIG. SREBF-1 proteolytic activation is largely under the controlof hormonal or signal transduction systems [25]. For example, whenintracellular sterols are depleted, the SREBF/SCAP complex [26] isescorted from the ER to the Golgi for proteolytic processing. Proteasesin the Golgi, i.e., site 1 and site 2 protease, cleave the SREBF precursorto generate the mature nuclear form of SREBF (nSREBF). The N-terminal domain, a 68-kDa helix-loop-helix leucine zipper transcrip-tion factor (nSREBF), is transported to the nucleus as a dimer viaimportin β. In nuclei, SREBFs binds to sterol-regulatory elements(SRE) as dimers in promoters of target genes. Once bound, SREBFsrecruit co-activators to the promoter and stimulate gene transcription[25]. SREs have been identified in the promoter of desaturase FADS2,although this has not been clearly demonstrated in FADS1 [10].

It is well recognized that, in liver, FADS1 and FADS2 genes areregulated by SREBF-1c [10]. Because SREBF-1c mRNA has beenidentified in lactating mammary gland [8], in this study we analyzedthe expression of SREBF-1c as a possible mechanism of the regulationof desaturases and elongases during pregnancy and lactation. Ourresults show that the expression of SREBF-1c mRNA in mammarytissue increased from days 14 and 16 of pregnancy (Fig. 1e, Table 2).During lactation, the expression remained high until the early middleperiod (days 5, 10 and 12), returning to basal levels at the end oflactation. The maximum expression value of SREBF-1c was at day 5 oflactation (11.7-fold higher than that of nonpregnant rats, Pb0.005). Inliver, its expression increased only at the beginning of pregnancy(Pb0.005) and was higher only at day 10 of lactation (Pb0.005). Incontrast, expression of SREBF-1c decreased in adipose tissue through-out the course of pregnancy, reaching its minimum value at day 16and corresponding to a third of those values found in adipose tissue of

nonpregnant rats (Pb0.005) (Fig. 1e, Table 2). An expected increase(Pb0.005) at the end of lactation and after weaning was detected(Fig. 1e, Table 2). As expected for FASN, no increase in mammaryglandwas shown throughout pregnancy, but it was higher throughoutthe course of lactation (Table 2). In contrast, the expression of thisenzyme increased in liver, but only at day 5 of pregnancy (Pb0.005).During lactation, abundance of FASN mRNA was increased (Pb0.005),but such increase was not as elevated as in mammary tissue. Inadipose tissue, the FASN transcript increased during pregnancy atdays 1, 5, 10 and 14, reaching a maximum value of 3.0-fold higherthan in nonpregnant rats (Pb0.005). However, it was lower in theearly middle period of lactation than in nonpregnant rats andincreased at D2Pw (Fig. 1f, Table 2).

In the present study we suggest that SREBF-1c may participate inthe regulation of lipogenesis in mammary tissue because itsexpression is increased during the periods of high lipid demand(late pregnancy and early lactation). However, additional research isrequired to elucidate the true role of SREBF-1c in that tissue.Additionally, this hypothesis is supported because such increasecorresponded to increased expression of target gene ELOVL6(Supplementary Fig. 1), FASN, as well as FADS1 and FADS2. Thus,when LC-PUFAs requirements increase during the shift frompregnancy to lactation, there is an adaptive mechanism tosynthesize these fatty acids, probably through SREBF-1c in mam-mary tissue. We also observed that changes in basal expression ofSREBF-1c in mammary gland were higher than in liver. In thiscontext, Anderson et al. hypothesized that SREBF-1 is a criticalregulator of secretory activation with regard to lipid biosynthesis inmammary gland because they found that SREBF-1c expression iselevated during pregnancy in mice [27]. However, it is important toconsider that, as in liver, in mammary gland SREBF-1c cannot be thesole regulator of lipogenesis during lactation. In addition to SREBF-1c, carbohydrate-responsive element-binding protein (CHREBP) isknown to coregulate the expression of lipogenic genes FASN andACACA. Therefore, additional research is necessary to determine ifother key regulators such as the transcription factor CHREBP plays arole in the control of lipogenesis in the lactating mammary gland[28]. It has recently been demonstrated that CHREBP may be aregulator of lipogenesis in mammary gland because conjugatedlinoleic acid decreased the lipogenesis, possibly through reductionof CHREBP mRNA expression [29].

It is necessary to consider that SREBF-1 can be regulated bydifferent factors. Recently, it has been proposed that PPARγupregulates the expression of genes involved in de novo fatty acidsynthesis such as SREBF-1. This hypothesis may be explainedbecause there are evidences of a concerted action of PPARγ andINSIG1, where the latter was demonstrated to be a PPARγresponsive gene, suggesting that PPARγ in bovine mammary tissuemay serve as a regulator of SREBF-1 activity [30]. Also, INSIG1 andSCAP genes increased mRNA expressions 12- and 1.5-fold, respec-tively, during lactation. If this pattern of INSIG1 mRNA expressionextended to the protein level, more SREBF-1 may be retained in theER via SCAP-INSIG1 binding, maintaining SREBF in an inactive state[31]. These data clearly underscore a central role for SREBF-1 andPPARγ in controlling transcription of genes that regulate milk fatsynthesis. However, more functional studies are needed to deter-mine if there is a specific stage where both transcription factors areacting to induce and maintain fat synthesis during lactation in acoordinate manner [30].

In an attempt to determine which physiological state – pregnancyor lactation – in mammary gland is more important in the expressionof encoding enzymes and whether the regulator SREBF-1c is involvedin LC-PUFAs we compared fold changes by repeated measureanalyses.

We observed that in mammary gland only mRNA expression ofFADS1 and SREBF-1c was higher during pregnancy than in

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nonpregnant rats (P≤0.05). In contrast, desaturases FADS1 andFADS2, elongase ELOVL5, and SREBF-1c and FASN mRNA were higherduring lactation than throughout pregnancy in rats (P≤0.005). Theseresults, together with the high expression of desaturases, mayindicate a significant role for mammary gland in the synthesis of LC-PUFAs mainly during lactation.

3.3. Expression of desaturases, elongases and SREBF-1c in mammaryepithelial cells

During mammary gland development, the proportions of cellularcompartments change dramatically, relative to each other. Forexample, the epithelial compartment proliferates rapidly and expandsduring middle to late pregnancy (from ~35% to ~70%, respectively)and lactation (~78%), and adipose tissue compartments decrease from~68% during pregnancy to ~15% during lactation [22]. Consideringthese findings and in an effort to determine if epithelial cells frommammary gland may be partially responsible for LC-PUFAs synthesis,we determined whether these cells express genes encoding enzymesinvolved in that synthesis.

The expression of all the studied genes was detected in bothnonsecreting and secreting cells. Interestingly, the expression ofFADS1, FADS2, ELOVL5, ELOVL2 and SREBF-1c increased whennonsecreting cells were differentiated into a secreting state(Pb0.005). It is important to highlight that expression of SREBF-1ctranscript augmented 53-fold in secreting cells as opposed tononsecreting cells. In contrast, FASN expression was similar in bothcell types (Fig. 2). These data are in accordance with the expression inmammary tissue where expression of encoding enzymes and theregulator SREBF-1c involved in LC-PUFAs synthesis was higher duringlactation (secretory state) than throughout pregnancy (nonsecretorystate) in rats (P≤0.005).

Expression of FADS1 and FADS2, ELOVL5, and SREBF-1c mRNA innonsecreting cells decreased as LA or AA concentration increased(Pb0.05) in comparison with the control (without fatty acid) (Fig. 3aand b). ELOVL2 transcript was detected inmouseMECs (HC11), but itsexpression was lower as compared to the other genes studied underall conditions tested (on average, Cts were 31). However, the resultsof abundance of ELOVL2 mRNA demonstrated that high concentra-tions of LA or AA increased (Pb0.05) the concentration of its transcriptin nonsecreting cells. Regarding SREBF-1c, although our resultsindicate that it is downregulated by a high content of LA or AA, asimilar effect on FASN mRNA expression was not found as inpregnancy (nonsecreting physiological stage) under the studiedconditions.

Fig. 3c and d shows the expressions of FADS1, FADS2, ELOVL5,SREBF-1c and FASN mRNA in secreting cells. Such expression

Fig. 2. Gene expression of FADS1, FADS2, ELOVL5, ELOVL2 and SREBF-1c mRNAs innonsecreting (black bars) and secreting (white bars) lactocytes as determined by real-time PCR assays. *Pb0.005 (Student t-test) as compared to control nonsecretinglactocytes. Bars indicate mean±SE of fold change of each gene obtained from twoindependent cell culture experiments from assays performed in triplicate.

decreased as LA or AA concentration increased and in comparisonwith the non-fatty acid control (Pb0.05). In contrast, the concentra-tion of LA or AA did not affect the expression of ELOVL2 transcript(Fig. 3c and d).

Based on these data, we hypothesized that MECs may be mainlyresponsible for the increase in expression of enzymes involved in LC-PUFAs synthesis during late pregnancy and early lactation in themammary gland. Adipocyte cell population (~68% during pregnancyto ~15% during lactation) within the mammary gland would besufficient to mask these results. Supporting this hypothesis, ourresults demonstrate that desaturases, elongases and SREBF-1c areexpressed in epithelial cells (lactocytes) frommammary gland. In fact,this expression increased in secreting cells in vivo as in mammarygland and liver. That expression was negatively regulated by PUFAspossibly through downregulation of SREBF-1c. We proposed thathypothesis because there is abundant evidence about the key role ofSREBF-1 LC-PUFAs synthesis in the liver. It has been clearlydemonstrated that SREBF-1 activates the expression of desaturasesFADS1 and FADS2, and absence of SREBF-1 markedly impairs thehepatic expression of these enzymes [10]. Transgenic mice thatoverexpress nSREBF-1 or that have a deficiency of all SREBF isoformsas a result of deleting SCAP in liver show that the enzymes requiredfor synthesis of very long-chain polyunsaturated fatty acids (FADS1,FADS2, Elovl5, and Elovl2) were induced in liver from TgSREBF-1a butdecreased in SCAP-/- in livers [32]. These studies have been performedin hepatic tissue; however, to the best of our knowledge there is noevidence shown in mammary tissue or mammary epithelial cells.These findings indicate that LC-PUFAs synthesis by FADS1 and FADS2is not specific to the liver and may be nutritionally regulated bySREBF-1 in lactating mammary tissue (secreting physiological stage).However, additional research is needed to confirm that epithelial cellsfrom mammary tissue are mainly responsible for expression of genesinvolved in LC-PUFAs synthesis. In this context, the model ofexpression deconvolution from mammary gland may be a powerfultool for the study of complex cellular mixtures in higher organisms[22]. It is also important to consider that some components of theculture medium like prolactin and EGF may have some effect onSREBF-1c. There are studies to elucidate pathways involved in thecoordinate control of SREBF-1 signaling. Some of these studiesproposed that epidermal growth factor (EGF) receptor (EGFR)activated by EGF activates MAPK signaling, which has been shownto stimulate SREBF transcriptional activity [33]. EGF is also associatedwith a higher nuclear translocation of the active SREBF-1c [34].However, additional research is necessary to determine if this processoccurs in HC11 cells cultured in the presence of the EGF activator ofEGFR. Naylor et al. reported that lactogenic hormone prolactinstimulates the transcription of key regulatory molecules for fattyacid synthesis such as SREBF-1c during pregnancy [18].

3.4. Changes of FA composition in maternal tissues during pregnancyand lactation

Fatty acid composition of mammary gland and milk clotdemonstrated a predominance of LA (C18:2n-6), oleic acid (C18:1)and palmitic acid (C16:0). The proportion of C18:2n-6 in mammarytissue was lower than in nonpregnant rats during the last days ofpregnancy, in the early middle period of lactation, and after weaning(Pb0.005). In accordance with the decrease of LA, there was asignificant increase of its product (AA) (C20:4n-6) in the early middleperiod of lactation (Pb0.005), but it returned to basal level at D2Pw. Incontrast, ALA (C18:3n-3) was increased at day 12 of lactation andafter weaning (Pb0.005). DHA (C22:6n3) product of ALA followed thesame pattern as AA (Table 3). The increase of AA and DHAcorresponded with an elevated expression of desaturases FADS1 andFADS2 and ELOVL5 during lactation, suggesting an increase in theenzymatic activity of desaturases upregulated during these

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FADS2 FADS1 Elovl2 Elovl5 SREBF-1c FASN

FADS2 FADS1 Elovl2 Elovl5 SREBF-1c FASN

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Fig. 3. Effect of linoleic acid (a and c) and arachidonic acid (b and d) on gene expression of FADS1 and FADS2, ELOVL2, ELOVL5, SREBF-1c and FASN in nonsecreting (a and b) andsecreting (c and d) lactocytes as determined by real-time PCR assays. White bars, 0; gray bars, 50; black bars, 200 μMof each fatty acid. LA, linoleic acid; AA, arachidonic acid. *Pb0.05(ANOVA, Dunnett's method) as compared to control cell culture (0 μMof each fatty acid). Bars indicate themean±SE of fold change of each gene obtained from two independent cellculture experiments from assays performed in triplicate.

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physiological stages. This is proposed because many studies havedemonstrated that activity is always directly related to mRNA levels[6,10]. Regarding the proportion of monounsaturated fatty acids(MFA) such as palmitoleic acid (C16:1) and oleic acid, this wassignificantly (Pb0.005) lower in L12 and D2Pw than in mammarytissue from controls rats.

In the rat model, mammary gland becomes the major glucose-utilizing tissue in the body during lactation, and lipogenesis is

Table 3Percentage of fatty acids in the mammary gland of pregnant and lactating rats.

NP P10 P16 P20

% Total fatty acids

SFAC12:0 0.14±0.02 ND 1.03±0.07 NDC14:0 1.67±0.04 1.83±0.03 1.80±0.04 1.72±0.05C16:0 23.99±0.4 24.71±0.3 25.10±0.5 24.09±1.0C18:0 3.52±0.1 3.38±0.07 3.64±0.02 3.78±0.1C20:0 0.41±0.02 0.47±0.02 0.44±0.04 0.35±0.04

MFAC16:1 6.09±0.4 6.78±0.3 6.36±0.4 6.51±0.3C18:1 37.56±0.3 36.64±0.3 36.94±0.6 36.66±1.3

PUFAsC18:2 n6 25.61±0.6 25.12±0.4 23.21±1.0 21.53±1.6C18:3n3 0.10±0.01 0.09±0.01 0.09±0.02 0.08±0.01C20:4n6 0.88±0.08 0.85±0.04 1.11±0.11 1.50±0.14C20:5n3 ND ND ND NDC22:6n3 0.14±0.02 0.14±0.02 0.29±0.06 0.17±0.02

Values are expressed as mean±standard error of seven rats.ND, not detected; NP, nonpregnant female rats; P, pregnancy; L, lactation; D2Pw, day 2polyunsaturated fatty acids.

a Pb0.05 compared to NP (ANOVA, Dunnett's method).

quantitatively the most important pathway in terms of glucoseutilization by the gland [35]. Synthesis of triacylglycerols (lipogene-sis) is a tightly controlled process in the lactatingmammary gland andtakes place in the epithelial cells through esterification of fatty acids,which are either exogenous fatty acids or de novo synthesized in themammary epithelial cell. The process of de novo lipogenesis requiresthe action of multiple enzymes including the key FASN [36,37]. Thisprocess also occurs in liver and adipose tissue, which contribute to the

L1 L5 L12 D2Pw

1.13±0.2 1.78±0.4a 2.58±0.4a 4.33±0.4a

1.81±0.08 3.23±0.4a 4.34±0.5a 5.36±0.4a

23.47±0.5 24.42±0.5 24.69±0.8a 28.89±1.0a

4.14±0.2 5.93±0.6a 7.62±0.6a 6.78±0.5a

0.37±0.05 0.22±0.02a 0.19±0.02a 0.22±0.01a

4.68±0.2 4.25±0.5 2.79±0.4a 3.24±0.1a

38.27±0.5 36.43±0.8 32.97±1.3a 29.87±0.9a

a 24.88±1.0 21.21±0.5a 21.75±0.8a 19.47±0.9a

0.11±0.01 0.12±0.02 0.15±0.01a 0.16±0.01a

1.27±0.13 2.07±0.48a 2.50±0.41a 1.47±0.11ND ND ND ND0.24±0.04 0.34±0.05a 0.41±0.04a 0.21±0.02

post-weaning, SFA, saturated fatty acid; MFA; monounsaturated fatty acid; PUFAs,

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fatty acid supply for mammary gland [38]. We observed that thepercentage of saturated fatty acids, e.g., lauric acid (12:0), myristicacid (C14:0), palmitic acid (C16:0) and stearic acid (C18:0), washigher (Pb0.005) in L5, L12 and D2Pw than in mammary gland fromthe nonpregnant group (Table 3). This is in accordance with the highFASN expression inmammary tissue throughout the course of lactation,indicativeof anelevated lipogenesis. Concerningmilk clot, percentage ofC18:2n-6 and C18:3n-3 was higher (Pb0.005) in L20 in comparisonwith L1. The proportion of LC-PUFAs (C20:4n-6 and C22:6n-3)decreased significantly throughout lactation in comparison with L1(supplementary Table 1). Similar to mammary gland, the proportion ofMFA (C16:1 and C18:1) was significantly lower (Pb0.005) during thecourse of lactation with respect to L1. The percentage of medium-chainsaturated fatty acids (C12:0 and C14:0) from milk clot increasedsignificantly during lactationwith respect to L1, whereas the proportionof long-chain saturated fatty acids (C16:0, C18:0 and C20:0)was similarin all stages studied (Supplementary Table 2).

More than90%of the fatty acid composition in the liverwas providedby C16:0, C18:0, C18:1, C18:2n-6 and C20:4n-6, and a small proportionof C18:3n-3, C20:5n-3 (EPA), C22:6n-3, C16:1, C14:0, and C20:0 wasdetected. Although therewere changes in expression of desaturases andELOVL5 (see Fig. 1a−c), the percentage of PUFAs such as C18:2n-6,C18:3n-3, C20:4n-6, C20:5 and C22:6n-3 identified in the liver wassimilar during the late and early middle period of pregnancy andlactation, respectively, in comparison with nonpregnant rats. However,content of these fatty acids was lower (Pb0.005) at D2Pw (Supplemen-tary Table 1). The high and constant concentration of AA in livermay bedue to the fact that this organ is the primary site for LC-PUFAs synthesisfor its ownneeds and for their secretion in very low-density lipoproteins(VLDL), as well as to be taken up by peripheral tissues, mainly heart,skeletal muscle and adipose tissue for supplying extrahepatic tissues[39,40]. Proportion of MFA (C16:1 and C18:1) in liver remained similarduring all analyzed stages of pregnancy and lactation. In contrast, it wassignificantly higher (Pb0.005) in D2Pw than in nonpregnant rats.Saturated fatty acid composition in the liver demonstrated that thepercentage of C12:0, C18:0 and C20:0 decreased significantly in D2Pw.In contrast, other saturated fatty acids (C14:0 and C16:0) were higher(Pb0.005) in D2Pw (Supplementary Table 2). Adipose tissue demon-strated a preponderance of C16:0, C18:1 and C18:2n-6. In this tissue weobserved a significantly higher percentage of C18:3 in L12 and C22:6n-3in L1andL12 as compared to nonpregnant rats. Significantdifferences inthe percentages of MFA within the stages of pregnancy and lactationstudied were not detected (Supplementary Tables 1 and 2).

Based on FASN expression and tissue fatty acid composition, ourfindings suggest that fatty acid synthesis during pregnancy is probablyconducted by the liver, but during lactation it was mainly accom-plished by mammary gland. It is well known that lipogenesis inlactating mammary gland is increased because it represents a periodof high lipid demand [23].

In this study we identified that mammary gland participates fromlate pregnancy expressing desaturases and elongases as one of thepossible mechanisms involved in LC-PUFAs synthesis for supportingpregnancy and lactation. This mechanism may be upregulated bySREBF-1c. It has been reported that cultured MECs induce apronounced proliferation of cytoplasmic lipid droplets. Because theyexpress genes encoding enzymes involved in the LC-PUFAs, they maybe a useful model for furthering our understanding of their expressionin mammary tissue.

Supplementarymaterials related to this article can be found onlineat doi:10.1016/j.bbalip.2011.01.007.

Acknowledgments

Sources of funding: This research was supported by the Coordina-ción de Investigación Médica en Salud, IMSS, México (Grant #FIS/IMSS/PROT/187). M.R.C designed the research, analyzed the data and

wrote the paper; R.S., A.M.S, S.L.K., F.S.M. and J.M. carried out theresearch and M.L.A. analyzed the data and reviewed the manuscript.M.R.C. was a recipient of training of University of California, Davis,Davis, California.

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