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ARTICLE Maternal micronutrients, omega-3 fatty acids, and placental PPAR␥ expression Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by YORK UNIV on 08/13/14 For personal use only.

Akshaya 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 of maternal micronutrients such as folic acid and vitamin B12 will lead to increased oxidative stress, reduced long-chain polyunsaturated fatty acids, and altered expression of peroxisome proliferator activated receptor (PPAR␥) in the placenta, and omega-3 fatty 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 plasma malonidialdehyde, placental IL-6, and TNF-␣. Omega-3 fatty acid supplementation to a vitamin B12 deficient diet normalized the expression of PPAR␥ and lowered the levels of placental TNF-␣. In the case of supplementation to a folic acid deficient diet it lowered the levels of malonidialdehyde and placental IL-6 and TNF-␣. This study has implications for fetal growth as oxidative stress, 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 posons l'hypothèse selon laquelle une carence maternelle en micronutriments tels que l'acide folique et la vitamine B12 aboutit a` un plus grand stress oxydatif, a` moins d'acides gras polyinsaturés a` longue chaîne et a` une expression altérée des récepteurs activables par les proliférateurs des peroxysomes (PPAR␥) dans le placenta et qu'une supplémentation en acide gras oméga-3 augmente l'expression de PPAR␥. On répartit les femelles du rat dans cinq groupes : contrôle, carencé en acide folique, carencé en vitamine B12, carencé en acide folique + supplémentation en acide gras oméga-3 et carencé en vitamine B12 + supplémentation en acide gras oméga-3. On dissèque les femelles enceintes au 20e jour de la gestation. La carence maternelle en micronutriments suscite un plus faible niveau placentaire (p < 0,05) d'acide docosahexanoïque, d'acide arachidonique, d'expression des PPAR␥ et un plus haut niveau (p < 0,05) de malonaldéhyde plasmatique, d'IL-6 placentaire et de TNF-␣ placentaire. La supplémentation en acide gras 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 malonaldéhyde et d'IL-6 et de TNF-␣ dans le placenta. Cette étude concerne aussi le développement du fœtus, car le stress oxydatif, l'inflammation et les 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-␣.

Introduction It is well established that an adequate maternal micronutrient status during pregnancy is beneficial for a better pregnancy outcome (Mistry and Williams 2011). Deficiency of micronutrients during pregnancy is common among women in low-income countries and adversely affects pregnancy outcome (Ronsmans et al. 2010). Studies examining the micronutrient profile of the Indian population have clearly shown the prevalence of folic acid deficiency in India (Kalaivani 2009). Additionally, a high prevalence of vitamin B12 deficiency in Indian mothers has been reported (Yajnik and Deshmukh 2012; Yajnik et al. 2008), and it has been mainly attributed to low dietary intake of animal foods that are rich in vitamin B12 (Gammon et al. 2012). Maternal folate deficiency has been reported to result in frequent resorptions, neural tube defects, and a variety of malformations in the developing embryos (De Castro et al. 2010; Pickell et al. 2009; Godbole et al. 2011). Deficiencies in both folate and vitamin B12 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 homocysteine in the Indian mothers may predict risk of intrauterine growth retarded babies (Yajnik et al. 2008). It is known that the fetus meets its energy requirements with essential fatty acids, especially omega-3 fatty acids, which are constituents of biological membranes and the precursors to intraand inter-cellular signaling molecules (Coletta et al. 2011). These fatty acids regulate several cellular processes such as differentiation, development, and gene expression in tissues through peroxisome proliferator activated receptors (PPAR) (Jump 2004). PPARs play an important role in growth, development, and physiological functions of the feto-placental axis (Berry et al. 2003). PPARs are ligand activated nuclear transcription factors that regulate the expression of many genes involved in cell proliferation and differentiation (Yessoufou and Wahli 2010). The PPAR subfamily consists of three isotypes: PPAR␣, PPAR␤/␦, and PPAR␥. PPAR␥ is highly expressed in the trophoblastic layer of the placenta and thereby 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 Vidyapeeth University, Pune 411043, India. Corresponding author: Sadhana R. Joshi (e-mail: [email protected]). 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 endothelial growth factors in mice (Nadra et al. 2010). The natural ligands of PPAR include different polyunsaturated fatty acids and prostaglandin molecules. A review by Itoh and Yamamoto (2008) suggested that oxidized docosahexaenoic acid (DHA) may be a new class of ligands for PPAR␥. Upon activation by different longchain polyunsaturated fatty acids (LCPUFA), PPAR␥ molecules are reported to regulate the expression of genes as fatty acid synthase, mitochondrial acetyl-CoA carboxylase 2 responsible for several placental 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 our knowledge there are no studies examining the effect of maternal micronutrient deficiency on placental PPAR␥ expression. Recent animal studies in our department suggest that an imbalance in maternal folic acid and vitamin B12 results in reduced levels of LCPUFA especially DHA in the placenta and affects the placental global DNA methylation patterns, which could be ameliorated by omega-3 fatty acid supplementation (Kulkarni et al. 2011a). Beneficial effects of maternal omega-3 fatty acid supplementation on oxidative stress markers in the rat placenta have also been reported by others (Jones et al. 2013a, 2013b, 2013c). We hypothesize that maternal micronutrient deficiency results in increased plasma oxidative stress and reduced placental DHA levels that alter PPAR␥ regulation. Omega-3 fatty acid supplementation to these diets will help in normalizing the expression of PPAR␥. Both folic acid and vitamin B12 are important constituents of the one-carbon cycle and the deficiency of these components may independently result in adverse fetal outcomes. Therefore, for the first time, this study examines the independent effects of folic acid and vitamin B12 deficiency, starting from the preconception period and continuing throughout pregnancy, on the dam's oxidative stress, placental fatty acid levels, and the expression of PPAR␥. Further, the effect of maternal omega-3 fatty acid supplementation to the aforementioned micronutrient deficient diets is also examined.

Materials and methods All experimental procedures were in accordance with the guidelines of Institutional Animal Ethics Committee. The institute is recognized to undertake experiments on animals as per the Committee for the Purpose of Control and Supervision of Experiments on Animals (February 2011). All institutional and national guidelines for the care and use of laboratory animals were followed. Study design The study design and the 5 isocaloric formulations have been described in detail (Meher et al. 2013). Female pups were distributed randomly in 5 different groups (n = 8 per group) post weaning and throughout pregnancy. The composition of the control and the treatment diets was as per the guidelines of AIN-93G purified diets for laboratory rodents. In addition to the control group, there were 4 treatment diets: folic acid deficient (FD), vitamin B12 deficient (BD), folic acid deficient + omega-3 fatty acid supplemented (FDO), and vitamin B12 deficient + omega-3 fatty acid supplemented (BDO). Maxepa (fish oil, Merck Limited, Goa, India.) that contained a combination of DHA (120 mg) and eicosapentaenoic 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. To prevent coprophagy, trays were placed in the animal cages that separated the animals from their faeces and the bedding material. These cages were not used in the case of control animals. The fatty acid composition of the control, BD, and BDO groups was published previously in connection with another experiment (Wadhwani et al. 2012). The fatty acid composition (g/100 g fatty acids) in the FD group (myristic acid, 5.49; myristoleic acid, 0.76;

Appl. Physiol. Nutr. Metab. Vol. 39, 2014

palmitic acid, 32.39; palmitoleic acid, 1.02; stearic acid, 5.94; oleic acid, 22.71; linoleic acid, 25.52; alpha linolenic acid, 3.32) was similar to that of the control group, whereas that of the FDO group (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; eicosapentaenoic acid, 6.04; DHA, 3.34) was similar to that of the BDO group. All dams were dissected by caesarean section on day 20 of gestation. Blood was collected and plasma was separated and stored at –80 °C until analysis. Placentas were removed and snap frozen in liquid nitrogen and stored at –80 °C until further use. One placenta per dam (8 per group) was randomly taken for the analysis. Malondialdehyde (MDA) was estimated from dam plasma. Erythrocyte and placental fatty acid levels Fatty acids were estimated from the dam erythrocytes and placenta using gas chromatography. We described this method previously in separate studies (Kulkarni et al. 2011a; Wadhwani et al. 2012). Fatty acids are expressed as g/100 g fatty acid. Dam plasma MDA levels MDA levels were estimated from dam plasma using the Bioxytech MDA-586 kit (Oxis International, Beverly Hills, CA, USA) and we described this method in Roy et al. (2012). Plasma MDA concentration is expressed as nmol mL−1. RNA isolation and cDNA synthesis Total RNA was isolated from placenta tissue using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and was quantified using the Biophotometer (Eppendorf, Germany). Reverse transcription was carried out with oligo(dT) primer and SuperScript II reverse transcriptase from 1000 ng of total RNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). Placental PPAR␥ expression Quantitative real-time polymerase chain reaction (PCR) for PPAR␥ and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was performed using the Applied Biosystems 7500 standard system. The relative expression level of the gene of interest was computed with respect to GAPDH mRNA to normalize for variation in the quality of RNA and the amount of input cDNA. Realtime PCR was performed with the TaqMan Universal PCR Master Mix (Applied Biosystems, USA) using cDNA equivalent to 100 ng total RNA. ⌬Ct (cycle threshold) values corresponded with the difference between the Ct values of the GAPDH (internal control) and those of the PPAR␥ gene. Relative expression level of the gene was calculated and expressed as 2⌬Ct. The following TaqMan assays (Applied Biosystems, USA) were used in this study: GAPDH (Rn99999916_S1) and PPAR␥ (Rn00440945_M1). Preparation of tissue lysates Whole placental tissue was weighed and centrifuged twice with 1× PBS at 4 °C. The supernatant was discarded and pellet was collected. The tissue pellet was lysed in chilled cell lysis buffer (50 mM TRIS HCl, 150 mM NaCl, 1 mM EDTA, 1 mM phenyl methane sulfonyl fluoride (PMSF), 10 mM Leupeptin, 0.1 mM Aprotinin) for 30 min on ice with intermittent vortexing. The extract was then centrifuged at 13000 rpm for 10 min at 4 °C. The clear supernatant (lysate) was then used for the assay. PPAR␥ protein levels in the placenta PPAR␥ protein levels in the placenta were determined using the ELISA kit (USCN Life, Wuhan EIAab Science Co. Ltd, China). The protein levels were expressed as ng/mL/gm of placenta. IL-6 and TNF-␣ levels in placenta Interleukin-6 (IL-6) levels were estimated by the standard sandwich enzyme linked immunosorbent assay (Abnova, Walnut, CA, Published by NRC Research Press

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

MYR (14:0) MYRO (14:1, n-5) PAL (16:0) PALO (16:1, n-7) STE (18:0) OLE (18:1, n-9) LA (18:2, n-6) ALA (18:3, n-3) GLA (18:3, n-6) DGLA (20:3, n-6) ARA (20:4, n-6) EPA (20:5, n-3) NA (24:1, n-9) DPA (22:5, n-6) DHA (22:6, n-3) Omega-3 fatty acids Omega-6 fatty acids MUFA SFA

Control

FD

BD

FDO

BDO

0.30±0.13 0.02±0.01 25.91±1.74 0.35±0.12 16.77±1.39 5.28±1.14 8.50±1.14 0.24±0.42 0.09±0.02 0.20±0.05 22.41±1.99 0.23±0.10 0.71±0.07 0.58±0.20 2.83±0.37 3.31±0.63 31.78±1.95 6.36±1.19 42.98±2.41

0.32±0.10 0.02±0.01 27.20±0.68 0.47±0.30 16.31±0.96 5.71±1.06 8.88±0.73 0.09±0.03 0.11±0.08 0.25±0.05* 21.68±1.10 0.35±0.43 0.82±0.09 0.78±0.22 2.40±0.37* 2.90±0.63 31.66±1.30 7.01±1.30 43.83±0.94

0.39±0.19 0.02±0.02 28.78±2.81** 0.46±0.38 16.51±2.34 6.12±1.42 8.57±1.78 0.08±0.04 0.09±0.05 0.21±0.04 20.66±3.31 0.73±0.85* 1.03±0.42** 0.59±0.21 2.21±0.42** 3.05±0.86 30.09±4.38 7.63±1.97* 45.69±4.35*

0.40±0.12 0.02±0.01 27.14±1.64 0.78±0.31**,§ 15.02±1.06* 5.61±0.73 6.71±0.61**,§§ 0.08±0.05 0.05±0.03**,§§ 0.37±0.04**,§§ 15.02±0.72**,§§ 4.92±0.79**,§§ 1.16±0.19**,§§ 3.02±0.22**,§§ 7.05±0.34**,§§ 12.06±0.83**,§§ 25.17±0.92**,§§ 7.56±0.93* 42.56±1.90

0.45±0.13* 0.03±0.03 26.01±2.20‡‡ 0.69±0.30* 15.39±1.71 5.82±1.02 6.48±0.44**,‡‡ 0.05±0.03* 0.04±0.02**,‡‡ 0.42±0.10**,‡‡ 13.77±1.52**,‡‡ 5.87±1.48**,‡‡ 1.08±0.12** 3.00±0.46**,‡‡ 7.32±1.12**,‡‡ 13.24±2.41**,‡‡ 23.71±1.48**,‡‡ 7.63±1.20* 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 < 0.05 compared with the FD group; §§p < 0.01 compared with the FD group; ‡p < 0.05 compared with the BD group; ‡‡p < 0.01 compared with the 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 linolenic acid; ALA, alpha linolenic acid; DGLA, di homo gamma linolenic acid; ARA, arachidonic acid; EPA, eicosapentaenoic acid; NA, nervonic acid; DPA, docosapentaenoic acid; DHA, docosahexaenoic acid; MUFA, mono unsaturated fatty acids; SFA, saturated fatty acids.)

USA). Tumor necrosis factor-␣ (TNF-␣) levels were also estimated by the in vitro enzyme linked immunosorbent assay (Abcam, Cambridge, MA, USA). 100 ␮L of placental lysate was used for analysis of IL-6 and TNF-␣. The detection limits for placenta IL-6 level was 62.5–4000 pg mL−1, whereas for placenta TNF-␣ levels it was 82.3–20000 pg mL−1. These cytokines are expressed as pg/mL/mg of total protein. Statistical analysis Values are expressed as mean ± SD. The data were analyzed using SPSS/PC+ package (IBM SPSS Statistics for Windows, Version 20.0. Armonk, NY). The treatment groups were compared with the control group by ANOVA and the post-hoc least significant difference test.

Results Intake The feed intake of rats was recorded during the preconception and pregnancy period and was previously reported by us (Meher et al. 2013). The intake of rats during pregnancy in the FD, BD, and FDO groups was comparable, whereas in the BDO group it was higher (p < 0.01) compared with the control group. Reproductive performance The total weight gain of dams during pregnancy was similar in the FD, BD, and BDO groups, whereas it was higher (p < 0.05) in the FDO group compared with the control group as previously reported by us (Meher et al. 2013). There was no difference in the litter size of pups in the different groups, whereas litter weights of pups were lower (p < 0.05) only in the BD group compared with the control group as previously reported by us (Meher et al. 2013). Placental weights There 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 the control group (0.36 ± 0.06 g). Omega-3 fatty acid supplementation increased 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 acids Table 1 shows the fatty acid composition in the dam erythrocytes from different groups. DHA levels were lower in both FD (p < 0.05) and BD (p < 0.01) groups compared with the control group. Supplementation of omega-3 fatty acids to these micronutrient deficient diets increased (p < 0.01) the levels of DHA in the FDO and BDO groups compared with the FD and BD groups, respectively, as well as when compared with the control group. Arachidonic acid (ARA) levels in the FD and BD groups were lower than that of the control group; however, they did not reach the level of statistical significance. Omega-3 fatty acid supplementation reduced (p < 0.01) the levels of ARA both in the FDO group and the BDO group compared with their respective deficiency groups as well as when compared with the control group. Table 2 shows the fatty acid composition of placenta in different groups. The levels of DHA in both the FD and BD groups were lower (p < 0.01 for both) compared with the control group. Omega-3 fatty acid supplementation to these micronutrient deficient diets increased (p < 0.01) the levels of DHA in the FDO and the BDO groups. The ARA levels were significantly lower (p < 0.05) in the BD group, whereas there was no significant difference observed in the FD group compared with the control group. However, omega-3 fatty acid supplementation significantly reduced (p < 0.01) the levels of ARA in both the FDO and BDO groups compared with the control group as well as their respective deficiency groups. Dam plasma MDA levels Dam 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 fatty acids to vitamin B12 deficient (BDO) (9.67 ± 0.3 nmol mL−1) diet Published by NRC Research Press

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

MYR (14:0) MYRO (14:1, n-5) PAL (16:0) PALO (16:1, n-7) STE (18:0) OLE (18:1, n-9) LA (18:2, n-6) ALA (18:3, n-3) GLA (18:3, n-6) DGLA (20:3, n-6) ARA (20:4, n-6) EPA (20:5, n-3) NA (24:1, n-9) DPA (22:5, n-6) DHA (22:6, n-3) Omega-3 fatty acids Omega-6 fatty acids MUFA SFA

Control

FD

BD

FDO

BDO

0.16±0.07 0.10±0.04 19.21±3.83 0.38±0.31 21.59±1.74 8.89±0.48 12.71±1.06 0.68±0.11 0.01±0.02 0.56±0.23 18.53±1.68 0.09±0.03 3.99±1.05 0.52±0.17 3.91±0.83 4.68±0.89 32.34±2.70 13.36±0.95 40.95±4.82

0.38±0.19** 0.01±0.0** 24.21±1.38** 0.38±0.04 19.97±1.37 9.80±1.07 13.34±1.31 0.56±0.23 0.01±0.0 0.38±0.16 18.69±1.93 0.41±0.64** 2.49±0.98** 0.55±0.15 2.08±0.49** 3.05±0.77** 32.97±1.73 12.69±1.51 44.55±1.40*

0.39±0.13** 0.01±0.0** 24.00±2.16** 0.48±0.34 19.93±2.35* 11.92±2.70** 14.31±1.50** 0.48±0.08** 0.05±0.11 0.42±0.13 16.80±1.86* 0.43±0.28** 1.70±0.49** 0.57±0.08 2.03±0.32** 2.94±0.28** 32.14±1.70 14.10±3.12 44.33±3.99*

0.51±0.10**,§ 0.01±0.0** 26.85±1.13**,§ 0.80±0.19**,§§ 18.63±0.65** 12.12±1.20**,§§ 9.44±0.55**,§§ 0.33±0.04**,§§ 0.01±0.0 0.63±0.14§ 11.03±0.97**,§§ 2.54±0.35**,§§ 0.31±0.07**,§§ 2.39±0.09**,§§ 7.98±1.21**,§§ 10.86±1.27**,§§ 23.50±1.07**,§§ 13.24±1.26 45.99±1.31**

0.55±0.08**,‡ 0.01±0.0** 25.79±0.98** 0.77±0.14**,‡ 19.93±1.38* 12.09±1.17** 10.05±0.85**,‡‡ 0.37±0.05** 0.01±0.0 0.79±0.23*,‡‡ 10.61±1.16**,‡‡ 2.36±0.64**,‡‡ 0.43±0.32**,‡‡ 2.31±0.47**,‡‡ 7.55±0.86**,‡‡ 10.28±1.23**,‡‡ 23.77±1.81**,‡‡ 13.30±1.16 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 < 0.05 compared with the FD group; §§p < 0.01 compared with the FD group; ‡p < 0.05 compared with the BD group; ‡‡p < 0.01 compared with the 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 linolenic acid; ALA, alpha linolenic acid; DGLA, di homo gamma linolenic acid; ARA, arachidonic acid; EPA, eicosapentaenoic acid; NA, nervonic acid; 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 are expressed as mean (SD). ** indicates values significantly (p < 0.01) different from control group by one-way ANOVA and the post hoc least significant difference test. §§ indicates values significantly (p < 0.01) different from FD group by one-way ANOVA and the post hoc least significant difference test. (MDA, malonidialdehyde; control, normal folic acid, normal vitamin B12; FD, folic acid deficient; 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 from control group by one-way ANOVA and the post hoc least significant difference test. ‡ indicates values significantly (p < 0.05) different from BD group by one-way ANOVA and the post hoc least significant difference 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 acid supplementation; and BDO, vitamin B12 deficient + omega-3 fatty acid supplementation.)

reduced the MDA levels, although the reduction was not statistically significant (p < 0.075) (Fig. 1). PPAR␥ expression in placenta The expression of the PPAR␥ gene in the FD and BD groups was lower (p < 0.05) compared with the control group. Omega-3 fatty 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 placenta There was no change observed in the protein levels of PPAR␥ across the treatment groups (control, 564.97 ± 182.47 ng/mL/gm of placenta; 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 of placenta; and BDO, 518.72 ± 110.48 ng/mL/gm of placenta). IL-6 and TNF-␣ levels in placenta IL-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 of protein) was higher (p < 0.05) compared with the control group (216.06 ± 77.13 pg/mL/mg of protein). Supplementation of omega-3 fatty acid reduced (p < 0.05) the levels of IL-6 in the FDO group Published by NRC Research Press

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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) different from 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 acid supplementation; and BDO, vitamin B12 deficient + omega-3 fatty acid supplementation.)

(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 of protein) were also lower compared with BD group, although the difference 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) compared with 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. Supplementation 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) compared 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).

Discussion To the best of our knowledge this is the first report that has examined the effect of folic acid and vitamin B12 deficiency during preconception through pregnancy on placental PPAR␥ expression. The study also examined the effect of omega-3 fatty acid supplementation in these deficient diets. Our results indicate that 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-6 and TNF-␣ in the placenta. Omega-3 fatty acid supplementation ameliorated most of the effects observed as a result of maternal vitamin B12 and folic acid deficiency. In this study, levels of DHA in the placenta were lower in the folic acid and vitamin B12 deficient groups. We previously demonstrated that the folic acid, vitamin B12, and omega-3 fatty acids are interlinked in the one carbon cycle and a deficiency of maternal vitamin B12 exclusively during pregnancy leads to a reduction of placental DHA levels (Kulkarni et al. 2011a, 2011b; Wadhwani et al. 2012; Roy et al. 2012; Dangat et al 2011; Kale et al. 2010). In our earlier reports, we extensively discussed the possible mechanisms through which an imbalance or deficiency of micronutrients leads to increased oxidative stress that can be mediated through increased homocysteine (Roy et al. 2012). This increased oxidative stress is known to result in degradation of LCPUFA (Videla et al. 2004; Jaeschke et al. 2002; Martindale and Holbrook 2002). Alternatively, the reduced DHA can be a result of reduced expression of the phosphatidyl ethanolamine methyl transferase gene that catalyzes the conversion of phosphatidyl ethanolamine to phos-

phatidyl choline as previously reported (Kulkarni et al. 2011a; Kale et al 2010). Further, we also observed decreased levels of ARA in the omega-3 fatty acid supplemented groups in both dam erythrocytes and placenta. This may be because an increase in one fatty acid (i.e., DHA) leads to a decrease in the levels of other fatty acids such as ARA as these fatty acids balance each other in membrane phospholipids (Nevigato et al. 2012). LCPUFA and their metabolites such as arachidonic acid, eicosapentaenoic acid, and prostaglandins are some of the ligands that activate PPAR␥ (Sampath and Ntambi 2004). Reports indicate that DHA is the more preferred ligand as it is bulkier and fits into the long and large hydrophobic ligand binding pocket of PPAR␥ (Itoh and Yamamoto 2008). It is known that in the brain DHA binds to retinoid X receptors (a transcription factor) that heterodimerize with PPARs and influences retinoid X receptor mediated transcription (Lengqvist et al. 2004). Further, maternal protein restriction during pregnancy in rats has also been shown to alter the expression of PPAR␥ (Burdge et al. 2004; Lillycrop et al. 2005). In the current study, we found that maternal vitamin B12 reduces the expression of PPAR␥ in the placenta. A recent report indicated that dams subjected to folate and vitamin B12 deficiency during gestation and lactation decrease PPAR␣ expression in the myocardium of weaning rats (Garcia et al. 2011). In contrast, dams fed a diet deficient in folic acid and associated methyl donors during the periconception and early preimplantation periods did not alter the levels of PPAR␣ (Maloney et al. 2013). Further, in the current study, supplementation of omega-3 fatty acids to the micronutrient deficient diet increased the expression of PPAR␥ in the vitamin B12 deficient groups. These results are similar to an earlier study where adipocytes when incubated with DHA increased the cellular adiponectin possibly by a mechanism that involved PPAR␥ regulation (Oster et al. 2010). We found no change in the PPAR␥ protein levels across the different groups. It has been reported that there is a separate and possibly independent regulation of protein translation and protein stability (Murphy et al. 2004). Reports also suggest that RNA and protein have differences in synthesis time. (Pérez-Sepúlveda et al. 2013). Similar variations in the mRNA and protein levels have been reported by others for hypoxia inducible factor-1␣, vascular endothelial growth factor, and superoxide dismutase genes (Bruells et al. 2013; Poisson et al. 2013). In contrast, others have reported maternal DHA supplementation at normal protein levels in the intrauterine growth retarded rat model increases the levels Published by NRC Research Press

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of PPAR␥ mRNA as well as protein in the neonatal rat lung (JossMoore et al. 2010). In the current study, micronutrient deficiency increased the levels of MDA, which is the end product of lipid peroxidation. Omega-3 fatty acid supplementation decreased the levels of lipid peroxidation in the folic acid deficient group. It is well demonstrated that a membrane rich in DHA should be exceptionally fluid and a DHA-deficient diet results in a brush border membrane with decreased fluidity (Stillwell and Wassall 2003). It is known that omega-3 fatty acids in membrane lipids make the double bonds less available for free radical attack (Applegate and Glomset 1986). Further, omega-3 fatty acids upregulate gene expression of antioxidant enzymes and downregulate genes associated with production of reactive oxygen species (Takahashi et al. 2002). Recent studies in rats have also shown that maternal omega-3 PUFA supplementation reduces isoprostanes, a marker of oxidative damage, and enhances placental and fetal growth (Jones et al. 2013a) and increases the labyrinth zone mRNA expression of antioxidant enzymes such as catalase and superoxide dismutase (Jones et al. 2013b). Thus, the increased maternal plasma oxidative stress results in reduced placental DHA levels that alter PPAR␥ regulation. In this study, deficiency of micronutrients such as folic acid and vitamin B12 from preconception through pregnancy results in increased levels of placental IL-6 and TNF-␣. Therefore, we propose that the increased maternal oxidative stress results in the reduced DHA levels in the placenta, further affecting the PPAR␥ regulation in the placenta and increasing the levels of pro-inflammatory cytokines. A recent report indicates that in myeloid cell lines, inhibition of PPAR␥ upregulates different pro-inflammatory cytokines such as IL-1, IL-6, and TNF-␣ (Wu et al. 2012). Studies in mice fed choline deficient diets during pregnancy have been shown to be associated with adverse reproductive outcomes because of a decrease in maternal PPAR␣ expression in the liver and an increase in inflammatory cytokines (Mikael et al. 2012). The current study examined how maternal micronutrient deficiency affects the pregnancy outcome and, therefore, measured the oxidative stress and inflammatory markers that are representative of abnormal physiological changes in the placenta. Further, reports indicate that there is no transfer of proinflammatory cytokines, TNF-␣, IL-1␤, and IL-6 across the placenta in either the maternal or fetal direction (Aaltonen et al. 2005). Therefore, it is likely that the levels of inflammatory markers in the placenta are possibly of placental origin. Nevertheless, future studies need to examine TNF-␣ and IL-6 mRNA levels from total placental RNA. Additionally, supplementation with omega-3 fatty acids was beneficial in bringing the IL-6 and TNF-␣ levels back to normalcy. DHA as well as PPAR␥ were extensively studied for their antiinflammatory activity and proresolving mechanisms in animals (Jones et al. 2013b; Rogers et al. 2011; Wang and Wan 2008). In the current study, although there was an increase in both proinflammatory cytokines and MDA, the magnitude of change was different. It may be possible that as MDA represents the oxidative stress contributed by lipid peroxidation and the inflammatory markers (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 have led to the activation of PPAR␥ in the omega-3 fatty acid supplemented groups which in turn decreased the levels of inflammatory cytokines such as IL-6 and TNF-␣. The current study was carried out in rats which, like human beings, exhibit a highly invasive type of placental development and is considered an appropriate model for studying the mechanisms of placentation (Fonseca et al. 2012). However, structurally the human and rat placenta differ in the number of trophoblastic layers that separate maternal and fetal endothelium (Ramsey 1982). Further, others suggest that the rat is not a good model to study placental blood flow in humans as it has lower number of

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Fig. 4. Possible mechanism of maternal micronutrient deficiency leading to reduced placental peroxisome proliferator activated receptor (PPAR␥) expression.

spiral arteries (Teklenburg et al. 2010). One limitation of this study is that the data for placenta were reported for whole placenta with no distinction between the placental zones. Therefore, future studies need to be carried out to examine the effect of maternal micronutrient deficiency and omega-3 fatty acid supplementation on the expression of PPAR␥ in the different zones of placenta and the subsequent implications in the fetal development.

Conclusion Our findings are unique as, for the first time, they demonstrate that maternal micronutrient deficiency leads to increased oxidative stress in the mother and results in lower levels of DHA, a lower expression of PPAR␥, and an increase of inflammatory cytokines (Fig. 4). Supplementation with omega-3 fatty acids ameliorated the aforementioned negative effects. We and others have demonstrated that oxidative stress and placental inflammation can lead to preterm or low birth weight babies (Dhobale and Joshi 2012; Joshi et al. 2008; Raunig et al. 2011). Our findings are of significance as the development of placenta plays a key role in maternal–fetal transfer and thereby determines fetal growth. This may have implications in countries such India where the majority of the population is vitamin B12 deficient due to vegetarian food habits and mothers are most likely to deliver low birth weight babies that are at a higher risk of developing noncommunicable diseases such as diabetes in later life (Yajnik et al. 2008). Further studies examining the epigenetic regulation of PPAR genes in the human placenta will throw more light into the mechanism leading to fetal programming of adult diseases.

Acknowledgements Akshaya P. Meher received the “INSPIRE fellowship” from the Department of Science and Technology, Government of India.

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