ARTICLE

Maternal micronutrients, omega-3 fatty acids, and placentalPPAR� expressionAkshaya P. Meher, Asmita A. Joshi, and Sadhana R. Joshi

Abstract: An altered one-carbon cycle is known to influence placental and fetal development. We hypothesize that deficiency ofmaternal micronutrients such as folic acid and vitamin B12 will lead to increased oxidative stress, reduced long-chain polyun-saturated fatty acids, and altered expression of peroxisome proliferator activated receptor (PPAR�) in the placenta, and omega-3fatty acid supplementation to these diets will increase the expression of PPAR�. Female rats were divided into 5 groups: control,folic acid deficient, vitamin B12 deficient, folic acid deficient + omega-3 fatty acid supplemented, and vitamin B12 deficient +omega-3 fatty acid supplemented. Dams were dissected on gestational day 20. Maternal micronutrient deficiency leads to lower(p < 0.05) levels of placental docosahexaenoic acid, arachidonic acid, PPAR� expression and higher (p < 0.05) levels of plasmamalonidialdehyde, placental IL-6, and TNF-�. Omega-3 fatty acid supplementation to a vitamin B12 deficient diet normalized theexpression of PPAR� and lowered the levels of placental TNF-�. In the case of supplementation to a folic acid deficient diet itlowered the levels of malonidialdehyde and placental IL-6 and TNF-�. This study has implications for fetal growth as oxidativestress, inflammation, and PPAR� are known to play a key role in the placental development.

Key words: folic acid, vitamin B12, docosahexaenoic acid, PPAR�, arachidonic acid, IL-6, TNF-�.

Résumé : La modification du cycle monocarboné nuit, on le sait, au développement du placenta et du fœtus. Nous posonsl'hypothèse selon laquelle une carence maternelle en micronutriments tels que l'acide folique et la vitamine B12 aboutit a un plusgrand stress oxydatif, a moins d'acides gras polyinsaturés a longue chaîne et a une expression altérée des récepteurs activablespar les proliférateurs des peroxysomes (PPAR�) dans le placenta et qu'une supplémentation en acide gras oméga-3 augmentel'expression de PPAR�. On répartit les femelles du rat dans cinq groupes : contrôle, carencé en acide folique, carencé en vitamineB12, carencé en acide folique + supplémentation en acide gras oméga-3 et carencé en vitamine B12 + supplémentation en acidegras oméga-3. On dissèque les femelles enceintes au 20e jour de la gestation. La carence maternelle en micronutriments susciteun plus faible niveau placentaire (p < 0,05) d'acide docosahexanoïque, d'acide arachidonique, d'expression des PPAR� et un plushaut niveau (p < 0,05) de malonaldéhyde plasmatique, d'IL-6 placentaire et de TNF-� placentaire. La supplémentation en acidegras oméga-3 dans un régime carencé en vitamine B12 normalise l'expression de PPAR� et diminue le taux placentaire de TNF-�.Dans la condition de supplémentation dans un régime carencé en acide folique, on observe une diminution de malonaldéhydeet d'IL-6 et de TNF-� dans le placenta. Cette étude concerne aussi le développement du fœtus, car le stress oxydatif, l'inflammation etles PPAR� jouent, selon des études, un rôle important dans le développement du placenta. [Traduit par la Rédaction]

Mots-clés : acide folique, vitamine B12, acide docosahexanoïque, PPAR�, acide arachidonique, IL-6, TNF-�.

IntroductionIt is well established that an adequate maternal micronutrient

status during pregnancy is beneficial for a better pregnancy out-come (Mistry and Williams 2011). Deficiency of micronutrientsduring pregnancy is common among women in low-income coun-tries and adversely affects pregnancy outcome (Ronsmans et al.2010). Studies examining the micronutrient profile of the Indianpopulation have clearly shown the prevalence of folic acid defi-ciency in India (Kalaivani 2009). Additionally, a high prevalenceof vitamin B12 deficiency in Indian mothers has been reported(Yajnik and Deshmukh 2012; Yajnik et al. 2008), and it has beenmainly attributed to low dietary intake of animal foods that arerich in vitamin B12 (Gammon et al. 2012).

Maternal folate deficiency has been reported to result in fre-quent resorptions, neural tube defects, and a variety of malforma-tions in the developing embryos (De Castro et al. 2010; Pickell et al.2009; Godbole et al. 2011). Deficiencies in both folate and vitaminB12 are known to result in high homocysteine concentrations

(Cetin et al. 2010). A study has shown that low plasma vitamin B12

concentrations and high circulating concentrations of homocys-teine in the Indian mothers may predict risk of intrauterinegrowth retarded babies (Yajnik et al. 2008).

It is known that the fetus meets its energy requirements withessential fatty acids, especially omega-3 fatty acids, which are con-stituents of biological membranes and the precursors to intra-and inter-cellular signaling molecules (Coletta et al. 2011). Thesefatty acids regulate several cellular processes such as differentia-tion, development, and gene expression in tissues through perox-isome proliferator activated receptors (PPAR) (Jump 2004). PPARsplay an important role in growth, development, and physiologicalfunctions of the feto-placental axis (Berry et al. 2003). PPARs areligand activated nuclear transcription factors that regulate theexpression of many genes involved in cell proliferation and differ-entiation (Yessoufou and Wahli 2010). The PPAR subfamily con-sists of three isotypes: PPAR�, PPAR�/�, and PPAR�. PPAR� ishighly expressed in the trophoblastic layer of the placenta andthereby plays an important role in the placental vasculature by

Received 6 November 2013. Accepted 20 December 2013.

A.P. Meher, A.A. Joshi, and S.R. Joshi. Department of Nutritional Medicine, Interactive Research School for Health Affairs, Bharati VidyapeethUniversity, Pune 411043, India.Corresponding author: Sadhana R. Joshi (e-mail: srjoshi62@gmail.com).

793

Appl. Physiol. Nutr. Metab. 39: 793–800 (2014) dx.doi.org/10.1139/apnm-2013-0518 Published at www.nrcresearchpress.com/apnm on 13 January 2014.

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regulating the expression of pro-angiogenic genes as vascular en-dothelial growth factors in mice (Nadra et al. 2010). The naturalligands of PPAR include different polyunsaturated fatty acids andprostaglandin molecules. A review by Itoh and Yamamoto (2008)suggested that oxidized docosahexaenoic acid (DHA) may be anew class of ligands for PPAR�. Upon activation by different long-chain polyunsaturated fatty acids (LCPUFA), PPAR� molecules arereported to regulate the expression of genes as fatty acid synthase,mitochondrial acetyl-CoA carboxylase 2 responsible for severalplacental functions as trophoblast invasion, nutrient transport,and hormone synthesis (Duttaroy 2004).

Reports indicate that maternal malnutrition influences the PPAR�expression in the fetal adipose tissue and placenta (Yiallourides et al.2009; Muhlhausler et al. 2009; Bagley et al. 2013). To the best of ourknowledge there are no studies examining the effect of maternalmicronutrient deficiency on placental PPAR� expression. Recentanimal studies in our department suggest that an imbalance inmaternal folic acid and vitamin B12 results in reduced levels ofLCPUFA especially DHA in the placenta and affects the placentalglobal DNA methylation patterns, which could be ameliorated byomega-3 fatty acid supplementation (Kulkarni et al. 2011a). Bene-ficial effects of maternal omega-3 fatty acid supplementation onoxidative stress markers in the rat placenta have also been re-ported by others (Jones et al. 2013a, 2013b, 2013c).

We hypothesize that maternal micronutrient deficiency resultsin increased plasma oxidative stress and reduced placental DHAlevels that alter PPAR� regulation. Omega-3 fatty acid supplemen-tation to these diets will help in normalizing the expression ofPPAR�. Both folic acid and vitamin B12 are important constituentsof the one-carbon cycle and the deficiency of these componentsmay independently result in adverse fetal outcomes. Therefore,for the first time, this study examines the independent effects offolic acid and vitamin B12 deficiency, starting from the preconcep-tion period and continuing throughout pregnancy, on the dam'soxidative stress, placental fatty acid levels, and the expression ofPPAR�. Further, the effect of maternal omega-3 fatty acid supple-mentation to the aforementioned micronutrient deficient diets isalso examined.

Materials and methodsAll experimental procedures were in accordance with the guide-

lines of Institutional Animal Ethics Committee. The institute is rec-ognized to undertake experiments on animals as per the Committeefor the Purpose of Control and Supervision of Experiments on Ani-mals (February 2011). All institutional and national guidelines for thecare and use of laboratory animals were followed.

Study designThe study design and the 5 isocaloric formulations have been

described in detail (Meher et al. 2013). Female pups were distrib-uted randomly in 5 different groups (n = 8 per group) post weaningand throughout pregnancy. The composition of the control andthe treatment diets was as per the guidelines of AIN-93G purifieddiets for laboratory rodents. In addition to the control group,there were 4 treatment diets: folic acid deficient (FD), vitamin B12deficient (BD), folic acid deficient + omega-3 fatty acid supple-mented (FDO), and vitamin B12 deficient + omega-3 fatty acidsupplemented (BDO). Maxepa (fish oil, Merck Limited, Goa, India.)that contained a combination of DHA (120 mg) and eicosapenta-enoic acid (180 mg) was used as the source of omega-3 fatty acids.The control group had normal levels of folic acid and vitamin B12. Toprevent coprophagy, trays were placed in the animal cages that sepa-rated the animals from their faeces and the bedding material. Thesecages were not used in the case of control animals.

The fatty acid composition of the control, BD, and BDO groupswas published previously in connection with another experiment(Wadhwani et al. 2012). The fatty acid composition (g/100 g fattyacids) in the FD group (myristic acid, 5.49; myristoleic acid, 0.76;

palmitic acid, 32.39; palmitoleic acid, 1.02; stearic acid, 5.94; oleicacid, 22.71; linoleic acid, 25.52; alpha linolenic acid, 3.32) wassimilar to that of the control group, whereas that of the FDOgroup (myristic acid, 5.65, myristoleic acid, 0.76; palmitic acid,28.84; palmitoleic acid, 3.36; stearic acid, 4.06; oleic acid, 18.12;linoleic acid, 22.05; alpha linolenic acid, 1.69; eicosapentaenoicacid, 6.04; DHA, 3.34) was similar to that of the BDO group.

All dams were dissected by caesarean section on day 20 of ges-tation. Blood was collected and plasma was separated and storedat –80 °C until analysis. Placentas were removed and snap frozenin liquid nitrogen and stored at –80 °C until further use. Oneplacenta per dam (8 per group) was randomly taken for the anal-ysis. Malondialdehyde (MDA) was estimated from dam plasma.

Erythrocyte and placental fatty acid levelsFatty acids were estimated from the dam erythrocytes and pla-

centa using gas chromatography. We described this method pre-viously in separate studies (Kulkarni et al. 2011a; Wadhwani et al.2012). Fatty acids are expressed as g/100 g fatty acid.

Dam plasma MDA levelsMDA levels were estimated from dam plasma using the Bioxytech

MDA-586 kit (Oxis International, Beverly Hills, CA, USA) and wedescribed this method in Roy et al. (2012). Plasma MDA concentra-tion is expressed as nmol mL−1.

RNA isolation and cDNA synthesisTotal RNA was isolated from placenta tissue using Trizol re-

agent (Invitrogen, Carlsbad, CA, USA) and was quantified usingthe Biophotometer (Eppendorf, Germany). Reverse transcriptionwas carried out with oligo(dT) primer and SuperScript II reversetranscriptase from 1000 ng of total RNA using a high-capacitycDNA reverse transcription kit (Applied Biosystems, Foster City,CA, USA).

Placental PPAR� expressionQuantitative real-time polymerase chain reaction (PCR) for

PPAR� and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)was performed using the Applied Biosystems 7500 standardsystem. The relative expression level of the gene of interest wascomputed with respect to GAPDH mRNA to normalize for varia-tion in the quality of RNA and the amount of input cDNA. Real-time PCR was performed with the TaqMan Universal PCR MasterMix (Applied Biosystems, USA) using cDNA equivalent to 100 ngtotal RNA. �Ct (cycle threshold) values corresponded with thedifference between the Ct values of the GAPDH (internal control)and those of the PPAR� gene. Relative expression level of the genewas calculated and expressed as 2�Ct. The following TaqMan as-says (Applied Biosystems, USA) were used in this study: GAPDH(Rn99999916_S1) and PPAR� (Rn00440945_M1).

Preparation of tissue lysatesWhole placental tissue was weighed and centrifuged twice with

1× PBS at 4 °C. The supernatant was discarded and pellet wascollected. The tissue pellet was lysed in chilled cell lysis buffer(50 mM TRIS HCl, 150 mM NaCl, 1 mM EDTA, 1 mM phenyl meth-ane sulfonyl fluoride (PMSF), 10 mM Leupeptin, 0.1 mM Aprotinin)for 30 min on ice with intermittent vortexing. The extract wasthen centrifuged at 13000 rpm for 10 min at 4 °C. The clear super-natant (lysate) was then used for the assay.

PPAR� protein levels in the placentaPPAR� protein levels in the placenta were determined using the

ELISA kit (USCN Life, Wuhan EIAab Science Co. Ltd, China). Theprotein levels were expressed as ng/mL/gm of placenta.

IL-6 and TNF-� levels in placentaInterleukin-6 (IL-6) levels were estimated by the standard sand-

wich enzyme linked immunosorbent assay (Abnova, Walnut, CA,

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USA). Tumor necrosis factor-� (TNF-�) levels were also estimatedby the in vitro enzyme linked immunosorbent assay (Abcam,Cambridge, MA, USA). 100 �L of placental lysate was used foranalysis of IL-6 and TNF-�. The detection limits for placenta IL-6level was 62.5–4000 pg mL−1, whereas for placenta TNF-� levels itwas 82.3–20000 pg mL−1. These cytokines are expressed as pg/mL/mgof total protein.

Statistical analysisValues are expressed as mean ± SD. The data were analyzed

using SPSS/PC+ package (IBM SPSS Statistics for Windows, Version20.0. Armonk, NY). The treatment groups were compared with thecontrol group by ANOVA and the post-hoc least significant differ-ence test.

Results

IntakeThe feed intake of rats was recorded during the preconception

and pregnancy period and was previously reported by us (Meheret al. 2013). The intake of rats during pregnancy in the FD, BD, andFDO groups was comparable, whereas in the BDO group it washigher (p < 0.01) compared with the control group.

Reproductive performanceThe total weight gain of dams during pregnancy was similar in

the FD, BD, and BDO groups, whereas it was higher (p < 0.05) in theFDO group compared with the control group as previously re-ported by us (Meher et al. 2013). There was no difference in thelitter size of pups in the different groups, whereas litter weights ofpups were lower (p < 0.05) only in the BD group compared with thecontrol group as previously reported by us (Meher et al. 2013).

Placental weightsThere was no difference in the placental weights of the FD

(0.37 ± 0.07 g) and BD groups (0.37 ± 0.06 g) compared with thecontrol group (0.36 ± 0.06 g). Omega-3 fatty acid supplementationincreased the weight of the placenta in the FDO group (0.39 ±0.05 g) compared with the control (p < 0.01) and FD groups

(p < 0.05). However, there was no difference in the BDO group(0.37 ± 0.05 g).

Erythrocytes and placental fatty acidsTable 1 shows the fatty acid composition in the dam erythro-

cytes from different groups. DHA levels were lower in both FD(p < 0.05) and BD (p < 0.01) groups compared with the controlgroup. Supplementation of omega-3 fatty acids to these micronu-trient deficient diets increased (p < 0.01) the levels of DHA in theFDO and BDO groups compared with the FD and BD groups, re-spectively, as well as when compared with the control group.Arachidonic acid (ARA) levels in the FD and BD groups were lowerthan that of the control group; however, they did not reach thelevel of statistical significance. Omega-3 fatty acid supplementa-tion reduced (p < 0.01) the levels of ARA both in the FDO group andthe BDO group compared with their respective deficiency groupsas well as when compared with the control group.

Table 2 shows the fatty acid composition of placenta in differentgroups. The levels of DHA in both the FD and BD groups werelower (p < 0.01 for both) compared with the control group.Omega-3 fatty acid supplementation to these micronutrient defi-cient diets increased (p < 0.01) the levels of DHA in the FDO and theBDO groups. The ARA levels were significantly lower (p < 0.05) inthe BD group, whereas there was no significant difference ob-served in the FD group compared with the control group. How-ever, omega-3 fatty acid supplementation significantly reduced(p < 0.01) the levels of ARA in both the FDO and BDO groupscompared with the control group as well as their respective defi-ciency groups.

Dam plasma MDA levelsDam plasma MDA levels in both the micronutrient deficient

groups, i.e., FD (10.07 ± 0.35 nmol mL−1) and BD (9.96 ± 0.38 nmol mL−1),were higher (p < 0.01) compared with the control group (9.25 ±0.19 nmol mL−1). Omega-3 fatty acid supplementation reduced(p < 0.01) the MDA levels in the FDO group (9.65 ± 0.26 nmol mL−1)compared with the FD group. Supplementation of omega-3 fattyacids to vitamin B12 deficient (BDO) (9.67 ± 0.3 nmol mL−1) diet

Table 1. Dam erythrocytes fatty acid levels (g/100 g fatty acids) in different treatment groups.

Control FD BD FDO BDO

MYR (14:0) 0.30±0.13 0.32±0.10 0.39±0.19 0.40±0.12 0.45±0.13*MYRO (14:1, n-5) 0.02±0.01 0.02±0.01 0.02±0.02 0.02±0.01 0.03±0.03PAL (16:0) 25.91±1.74 27.20±0.68 28.78±2.81** 27.14±1.64 26.01±2.20‡‡

PALO (16:1, n-7) 0.35±0.12 0.47±0.30 0.46±0.38 0.78±0.31**,§ 0.69±0.30*STE (18:0) 16.77±1.39 16.31±0.96 16.51±2.34 15.02±1.06* 15.39±1.71OLE (18:1, n-9) 5.28±1.14 5.71±1.06 6.12±1.42 5.61±0.73 5.82±1.02LA (18:2, n-6) 8.50±1.14 8.88±0.73 8.57±1.78 6.71±0.61**,§§ 6.48±0.44**,‡‡

ALA (18:3, n-3) 0.24±0.42 0.09±0.03 0.08±0.04 0.08±0.05 0.05±0.03*GLA (18:3, n-6) 0.09±0.02 0.11±0.08 0.09±0.05 0.05±0.03**,§§ 0.04±0.02**,‡‡

DGLA (20:3, n-6) 0.20±0.05 0.25±0.05* 0.21±0.04 0.37±0.04**,§§ 0.42±0.10**,‡‡

ARA (20:4, n-6) 22.41±1.99 21.68±1.10 20.66±3.31 15.02±0.72**,§§ 13.77±1.52**,‡‡

EPA (20:5, n-3) 0.23±0.10 0.35±0.43 0.73±0.85* 4.92±0.79**,§§ 5.87±1.48**,‡‡

NA (24:1, n-9) 0.71±0.07 0.82±0.09 1.03±0.42** 1.16±0.19**,§§ 1.08±0.12**DPA (22:5, n-6) 0.58±0.20 0.78±0.22 0.59±0.21 3.02±0.22**,§§ 3.00±0.46**,‡‡

DHA (22:6, n-3) 2.83±0.37 2.40±0.37* 2.21±0.42** 7.05±0.34**,§§ 7.32±1.12**,‡‡

Omega-3 fatty acids 3.31±0.63 2.90±0.63 3.05±0.86 12.06±0.83**,§§ 13.24±2.41**,‡‡

Omega-6 fatty acids 31.78±1.95 31.66±1.30 30.09±4.38 25.17±0.92**,§§ 23.71±1.48**,‡‡

MUFA 6.36±1.19 7.01±1.30 7.63±1.97* 7.56±0.93* 7.63±1.20*SFA 42.98±2.41 43.83±0.94 45.69±4.35* 42.56±1.90 41.86±3.11‡

Note: All values are mean ± SD. *p < 0.05 compared with the control group; **p < 0.01 compared with the control group; §p < 0.05compared with the FD group; §§p < 0.01 compared with the FD group; ‡p < 0.05 compared with the BD group; ‡‡p < 0.01 compared withthe BD group. (Control, normal folic acid, normal vitamin B12; FD, folic acid deficient; BD, vitamin B12 deficient; FDO, folic acid deficient +omega-3 fatty acid supplementation; BDO, vitamin B12 deficient + omega-3 fatty acid supplementation; MYR, myristic acid; MYRO,myristoleic acid; PAL, palmitic acid; PALO, palmitoleic acid; STE, stearic acid; OLE, oleic acid; LA, linoleic acid; GLA, gamma linolenicacid; ALA, alpha linolenic acid; DGLA, di homo gamma linolenic acid; ARA, arachidonic acid; EPA, eicosapentaenoic acid; NA, nervonicacid; DPA, docosapentaenoic acid; DHA, docosahexaenoic acid; MUFA, mono unsaturated fatty acids; SFA, saturated fatty acids.)

Meher et al. 795

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reduced the MDA levels, although the reduction was not statisti-cally significant (p < 0.075) (Fig. 1).

PPAR� expression in placentaThe expression of the PPAR� gene in the FD and BD groups

was lower (p < 0.05) compared with the control group. Omega-3fatty acid supplementation increased the expression of PPAR�in the BDO group (p < 0.05) compared with the BD group(Fig. 2).

PPAR� protein levels in placentaThere was no change observed in the protein levels of PPAR�

across the treatment groups (control, 564.97 ± 182.47 ng/mL/gm ofplacenta; FD, 437.31 ± 133.87 ng/mL/gm of placenta; BD, 573.47 ±

232.95 ng/mL/gm of placenta; FDO, 498.32 ± 167.18 ng/mL/gm ofplacenta; and BDO, 518.72 ± 110.48 ng/mL/gm of placenta).

IL-6 and TNF-� levels in placentaIL-6 levels in the placenta in the FD group (441.26 ± 197.22 pg/

mL/mg of protein) and the BD group (463.33 ± 155.63 pg/mL/mg ofprotein) was higher (p < 0.05) compared with the control group(216.06 ± 77.13 pg/mL/mg of protein). Supplementation of omega-3fatty acid reduced (p < 0.05) the levels of IL-6 in the FDO group

Table 2. Placental fatty acid levels (g/100 g fatty acids) in different treatment groups.

Control FD BD FDO BDO

MYR (14:0) 0.16±0.07 0.38±0.19** 0.39±0.13** 0.51±0.10**,§ 0.55±0.08**,‡

MYRO (14:1, n-5) 0.10±0.04 0.01±0.0** 0.01±0.0** 0.01±0.0** 0.01±0.0**PAL (16:0) 19.21±3.83 24.21±1.38** 24.00±2.16** 26.85±1.13**,§ 25.79±0.98**PALO (16:1, n-7) 0.38±0.31 0.38±0.04 0.48±0.34 0.80±0.19**,§§ 0.77±0.14**,‡

STE (18:0) 21.59±1.74 19.97±1.37 19.93±2.35* 18.63±0.65** 19.93±1.38*OLE (18:1, n-9) 8.89±0.48 9.80±1.07 11.92±2.70** 12.12±1.20**,§§ 12.09±1.17**LA (18:2, n-6) 12.71±1.06 13.34±1.31 14.31±1.50** 9.44±0.55**,§§ 10.05±0.85**,‡‡

ALA (18:3, n-3) 0.68±0.11 0.56±0.23 0.48±0.08** 0.33±0.04**,§§ 0.37±0.05**GLA (18:3, n-6) 0.01±0.02 0.01±0.0 0.05±0.11 0.01±0.0 0.01±0.0DGLA (20:3, n-6) 0.56±0.23 0.38±0.16 0.42±0.13 0.63±0.14§ 0.79±0.23*,‡‡

ARA (20:4, n-6) 18.53±1.68 18.69±1.93 16.80±1.86* 11.03±0.97**,§§ 10.61±1.16**,‡‡

EPA (20:5, n-3) 0.09±0.03 0.41±0.64** 0.43±0.28** 2.54±0.35**,§§ 2.36±0.64**,‡‡

NA (24:1, n-9) 3.99±1.05 2.49±0.98** 1.70±0.49** 0.31±0.07**,§§ 0.43±0.32**,‡‡

DPA (22:5, n-6) 0.52±0.17 0.55±0.15 0.57±0.08 2.39±0.09**,§§ 2.31±0.47**,‡‡

DHA (22:6, n-3) 3.91±0.83 2.08±0.49** 2.03±0.32** 7.98±1.21**,§§ 7.55±0.86**,‡‡

Omega-3 fatty acids 4.68±0.89 3.05±0.77** 2.94±0.28** 10.86±1.27**,§§ 10.28±1.23**,‡‡

Omega-6 fatty acids 32.34±2.70 32.97±1.73 32.14±1.70 23.50±1.07**,§§ 23.77±1.81**,‡‡

MUFA 13.36±0.95 12.69±1.51 14.10±3.12 13.24±1.26 13.30±1.16SFA 40.95±4.82 44.55±1.40* 44.33±3.99* 45.99±1.31** 46.27±2.08**

Note: All values are mean ± SD. *p < 0.05 compared with the control group; **p < 0.01 compared with the control group; §p < 0.05compared with the FD group; §§p < 0.01 compared with the FD group; ‡p < 0.05 compared with the BD group; ‡‡p < 0.01 compared withthe BD group. (Control, normal folic acid, normal vitamin B12; FD, folic acid deficient; BD, vitamin B12 deficient; FDO, folic acid deficient +omega-3 fatty acid supplementation; BDO, vitamin B12 deficient + omega-3 fatty acid supplementation; MYR, myristic acid; MYRO,myristoleic acid; PAL, palmitic acid; PALO, palmitoleic acid; STE, stearic acid; OLE, oleic acid; LA, linoleic acid; GLA, gamma linolenicacid; ALA, alpha linolenic acid; DGLA, di homo gamma linolenic acid; ARA, arachidonic acid; EPA, eicosapentaenoic acid; NA, nervonicacid; DPA, docosapentaenoic acid; DHA, docosahexaenoic acid; MUFA, mono unsaturated fatty acids; SFA, saturated fatty acids.)

Fig. 1. Dam plasma MDA levels in different groups. Data areexpressed as mean (SD). ** indicates values significantly (p < 0.01)different from control group by one-way ANOVA and the post hocleast significant difference test. §§ indicates values significantly(p < 0.01) different from FD group by one-way ANOVA and the posthoc least significant difference test. (MDA, malonidialdehyde;control, normal folic acid, normal vitamin B12; FD, folic aciddeficient; BD, vitamin B12 deficient; FDO, folic acid deficient +omega-3 fatty acid supplementation; and BDO, vitamin B12

deficient + omega-3 fatty acid supplementation.)

Fig. 2. Placental PPAR� expression in different groups. PPAR�mRNA expression was carried out in placenta using real-time PCR.Data are presented as the mean of 2�CT where �CT is CT GAPDH –CT PPAR�. * indicates values significantly (p < 0.05) different fromcontrol group by one-way ANOVA and the post hoc least significantdifference test. ‡ indicates values significantly (p < 0.05) different fromBD group by one-way ANOVA and the post hoc least significantdifference test. (PPAR�, peroxisome proliferator activated receptor;GAPDH, glyceraldehyde-3-phosphate dehydrogenase; control,normal folic acid, normal vitamin B12; FD, folic acid deficient;BD, vitamin B12 deficient; FDO, folic acid deficient + omega-3 fatty acidsupplementation; and BDO, vitamin B12 deficient + omega-3 fatty acidsupplementation.)

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(237.07 ± 128.54 pg/mL/mg of protein) compared with the FD group.The levels of IL-6 in the BDO group (326.95 ± 200.52 pg/mL/mg ofprotein) were also lower compared with BD group, although thedifference was not significant (Fig. 3).

TNF-� levels in the placenta of the BD group (14111.83 ±4035.78 pg/mL/mg of protein) were higher (p < 0.05) comparedwith the control group (9953.99 ± 2444.72 pg/mL/mg of protein),whereas in the FD group (11600.03 ± 5689.43 pg/mL/mg of protein)the levels were comparable with the control group. Supplemen-tation of omega-3 fatty acids reduced (p < 0.01) the levels of TNF-�in the BDO group (6067.56 ± 2066.40 pg/mL/mg of protein) com-pared with the BD group. The levels of TNF-� in the FDO group(6487.03 ± 1963.06 pg/mL/mg of protein) were also lower (p < 0.01)compared with the FD group (Fig. 3).

DiscussionTo the best of our knowledge this is the first report that has

examined the effect of folic acid and vitamin B12 deficiency duringpreconception through pregnancy on placental PPAR� expres-sion. The study also examined the effect of omega-3 fatty acidsupplementation in these deficient diets. Our results indicatethat micronutrient deficiency (vitamin B12, folic acid) resulted in(i) lower levels of DHA in the dam erythrocytes and placenta,(ii) lower levels of ARA in placenta, (iii) lower expression of PPAR�in the placenta, (iv) increased the levels of MDA in dam plasma,and (v) higher levels of pro-inflammatory cytokines such as IL-6and TNF-� in the placenta. Omega-3 fatty acid supplementationameliorated most of the effects observed as a result of maternalvitamin B12 and folic acid deficiency.

In this study, levels of DHA in the placenta were lower in thefolic acid and vitamin B12 deficient groups. We previously demon-strated that the folic acid, vitamin B12, and omega-3 fatty acids areinterlinked in the one carbon cycle and a deficiency of maternalvitamin B12 exclusively during pregnancy leads to a reduction ofplacental DHA levels (Kulkarni et al. 2011a, 2011b; Wadhwani et al.2012; Roy et al. 2012; Dangat et al 2011; Kale et al. 2010). In ourearlier reports, we extensively discussed the possible mechanismsthrough which an imbalance or deficiency of micronutrientsleads to increased oxidative stress that can be mediated throughincreased homocysteine (Roy et al. 2012). This increased oxidativestress is known to result in degradation of LCPUFA (Videla et al.2004; Jaeschke et al. 2002; Martindale and Holbrook 2002). Alter-natively, the reduced DHA can be a result of reduced expression ofthe phosphatidyl ethanolamine methyl transferase gene that cat-alyzes the conversion of phosphatidyl ethanolamine to phos-

phatidyl choline as previously reported (Kulkarni et al. 2011a; Kaleet al 2010).

Further, we also observed decreased levels of ARA in theomega-3 fatty acid supplemented groups in both dam erythro-cytes and placenta. This may be because an increase in one fattyacid (i.e., DHA) leads to a decrease in the levels of other fatty acidssuch as ARA as these fatty acids balance each other in membranephospholipids (Nevigato et al. 2012).

LCPUFA and their metabolites such as arachidonic acid, eicosa-pentaenoic acid, and prostaglandins are some of the ligands thatactivate PPAR� (Sampath and Ntambi 2004). Reports indicate thatDHA is the more preferred ligand as it is bulkier and fits into thelong and large hydrophobic ligand binding pocket of PPAR� (Itohand Yamamoto 2008). It is known that in the brain DHA binds toretinoid X receptors (a transcription factor) that heterodimerizewith PPARs and influences retinoid X receptor mediated tran-scription (Lengqvist et al. 2004). Further, maternal protein restric-tion during pregnancy in rats has also been shown to alter theexpression of PPAR� (Burdge et al. 2004; Lillycrop et al. 2005).

In the current study, we found that maternal vitamin B12 re-duces the expression of PPAR� in the placenta. A recent reportindicated that dams subjected to folate and vitamin B12 deficiencyduring gestation and lactation decrease PPAR� expression in themyocardium of weaning rats (Garcia et al. 2011). In contrast, damsfed a diet deficient in folic acid and associated methyl donorsduring the periconception and early preimplantation periods didnot alter the levels of PPAR� (Maloney et al. 2013). Further, in thecurrent study, supplementation of omega-3 fatty acids to the mi-cronutrient deficient diet increased the expression of PPAR� inthe vitamin B12 deficient groups. These results are similar to anearlier study where adipocytes when incubated with DHA in-creased the cellular adiponectin possibly by a mechanism thatinvolved PPAR� regulation (Oster et al. 2010).

We found no change in the PPAR� protein levels across thedifferent groups. It has been reported that there is a separate andpossibly independent regulation of protein translation and pro-tein stability (Murphy et al. 2004). Reports also suggest that RNAand protein have differences in synthesis time. (Pérez-Sepúlvedaet al. 2013). Similar variations in the mRNA and protein levels havebeen reported by others for hypoxia inducible factor-1�, vascularendothelial growth factor, and superoxide dismutase genes(Bruells et al. 2013; Poisson et al. 2013). In contrast, others havereported maternal DHA supplementation at normal protein levelsin the intrauterine growth retarded rat model increases the levels

Fig. 3. IL-6 and TNF-� levels in placenta of different groups. Data are expressed as mean (SD). * indicates values significantly (p < 0.05)different from control group; ** indicates values significantly (p < 0.01) different from control group; ‡‡ indicates values significantly (p < 0.01)different from BD group; § indicates values significantly (p < 0.05) different from FD group; §§ indicates values significantly (p < 0.01) differentfrom FD group by one-way ANOVA and the post hoc least significant difference test. (IL-6, Interleukin-6; TNF-a, tumor necrosis factor-�;control, normal folic acid, normal vitamin B12; FD, folic acid deficient; BD, vitamin B12 deficient; FDO, folic acid deficient + omega-3 fatty acidsupplementation; and BDO, vitamin B12 deficient + omega-3 fatty acid supplementation.)

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of PPAR� mRNA as well as protein in the neonatal rat lung (Joss-Moore et al. 2010).

In the current study, micronutrient deficiency increased thelevels of MDA, which is the end product of lipid peroxidation.Omega-3 fatty acid supplementation decreased the levels of lipidperoxidation in the folic acid deficient group. It is well demon-strated that a membrane rich in DHA should be exceptionallyfluid and a DHA-deficient diet results in a brush border membranewith decreased fluidity (Stillwell and Wassall 2003). It is knownthat omega-3 fatty acids in membrane lipids make the doublebonds less available for free radical attack (Applegate and Glomset1986). Further, omega-3 fatty acids upregulate gene expression ofantioxidant enzymes and downregulate genes associated withproduction of reactive oxygen species (Takahashi et al. 2002). Re-cent studies in rats have also shown that maternal omega-3 PUFAsupplementation reduces isoprostanes, a marker of oxidativedamage, and enhances placental and fetal growth (Jones et al.2013a) and increases the labyrinth zone mRNA expression of an-tioxidant enzymes such as catalase and superoxide dismutase(Jones et al. 2013b). Thus, the increased maternal plasma oxidativestress results in reduced placental DHA levels that alter PPAR�regulation.

In this study, deficiency of micronutrients such as folic acid andvitamin B12 from preconception through pregnancy results in in-creased levels of placental IL-6 and TNF-�. Therefore, we proposethat the increased maternal oxidative stress results in the reducedDHA levels in the placenta, further affecting the PPAR� regulationin the placenta and increasing the levels of pro-inflammatorycytokines. A recent report indicates that in myeloid cell lines,inhibition of PPAR� upregulates different pro-inflammatory cyto-kines such as IL-1, IL-6, and TNF-� (Wu et al. 2012). Studies in micefed choline deficient diets during pregnancy have been shown tobe associated with adverse reproductive outcomes because of adecrease in maternal PPAR� expression in the liver and an in-crease in inflammatory cytokines (Mikael et al. 2012).

The current study examined how maternal micronutrient defi-ciency affects the pregnancy outcome and, therefore, measuredthe oxidative stress and inflammatory markers that are represen-tative of abnormal physiological changes in the placenta. Further,reports indicate that there is no transfer of proinflammatory cy-tokines, TNF-�, IL-1�, and IL-6 across the placenta in either thematernal or fetal direction (Aaltonen et al. 2005). Therefore, it islikely that the levels of inflammatory markers in the placenta arepossibly of placental origin. Nevertheless, future studies need toexamine TNF-� and IL-6 mRNA levels from total placental RNA.

Additionally, supplementation with omega-3 fatty acids wasbeneficial in bringing the IL-6 and TNF-� levels back to normalcy.DHA as well as PPAR� were extensively studied for their anti-inflammatory activity and proresolving mechanisms in animals(Jones et al. 2013b; Rogers et al. 2011; Wang and Wan 2008). In thecurrent study, although there was an increase in both pro-inflammatory cytokines and MDA, the magnitude of change wasdifferent. It may be possible that as MDA represents the oxidativestress contributed by lipid peroxidation and the inflammatorymarkers (TNF-�, IL-6) are the markers of systemic inflammation,their magnitude of change may not correlate with each other.Thus, in the present study, an adequate amount of DHA may haveled to the activation of PPAR� in the omega-3 fatty acid supple-mented groups which in turn decreased the levels of inflamma-tory cytokines such as IL-6 and TNF-�.

The current study was carried out in rats which, like humanbeings, exhibit a highly invasive type of placental developmentand is considered an appropriate model for studying the mecha-nisms of placentation (Fonseca et al. 2012). However, structurallythe human and rat placenta differ in the number of trophoblasticlayers that separate maternal and fetal endothelium (Ramsey1982). Further, others suggest that the rat is not a good model tostudy placental blood flow in humans as it has lower number of

spiral arteries (Teklenburg et al. 2010). One limitation of this studyis that the data for placenta were reported for whole placenta withno distinction between the placental zones. Therefore, futurestudies need to be carried out to examine the effect of maternalmicronutrient deficiency and omega-3 fatty acid supplementationon the expression of PPAR� in the different zones of placenta andthe subsequent implications in the fetal development.

ConclusionOur findings are unique as, for the first time, they demonstrate

that maternal micronutrient deficiency leads to increased oxida-tive stress in the mother and results in lower levels of DHA, alower expression of PPAR�, and an increase of inflammatory cy-tokines (Fig. 4). Supplementation with omega-3 fatty acids amelio-rated the aforementioned negative effects. We and others havedemonstrated that oxidative stress and placental inflammationcan lead to preterm or low birth weight babies (Dhobale and Joshi2012; Joshi et al. 2008; Raunig et al. 2011). Our findings are ofsignificance as the development of placenta plays a key role inmaternal–fetal transfer and thereby determines fetal growth. Thismay have implications in countries such India where the majorityof the population is vitamin B12 deficient due to vegetarian foodhabits and mothers are most likely to deliver low birth weightbabies that are at a higher risk of developing noncommunicablediseases such as diabetes in later life (Yajnik et al. 2008). Furtherstudies examining the epigenetic regulation of PPAR genes in thehuman placenta will throw more light into the mechanism lead-ing to fetal programming of adult diseases.

AcknowledgementsAkshaya P. Meher received the “INSPIRE fellowship” from the

Department of Science and Technology, Government of India.

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