A decrease in DKK1, a WNT inhibitor, contributes to placental lipid accumulation in an obesity-prone rat model

Abstract

Placenta, as the sole transport mechanism between mother and fetus, links the maternal physical state and the immediate as well as lifelong outcomes of the offspring. The present study examined the consequences of maternal obesity on placental lipid accumulation and metabolism. Pregnant obesity-prone (OP) and obesity-resistant (OR) rat strains were fed a control diet throughout gestation. Placentas were collected on Gestational Day 21 for mRNA and oxidative stress analysis, and frozen placental sections were analyzed for fat accumulation as well as beta-catenin and Dickkopf homolog 1 (Xenopus laevis) (DKK1) localization. JEG3 trophoblast cells were cultured in vitro to determine the relationship between DKK1 and lipid accumulation. Maternal plasma and placental nonesterified fatty acids and triglycerides (TG) were elevated in OP dams. Placental Dkk1 mRNA content was 4-fold lower in OP placentas, and a significant increase was noted in beta-catenin accumulation as well as in mRNA content of fat transport and TG synthesis genes, including Ppard (peroxisome proliferator-activated receptor delta), Slc27a1 (fatty acid transport protein 1; also known as Fatp1), Cd36 (cluster of differentiation 36; also known as fatty acid translocation [Fat]), Lipin1, and Lipin3. Significant lipid accumulation was found within the decidual zones in OP, but not OR, placentas, and thickness of the decidual and junctional zones was significantly smaller in OP than in OR placentas. Overexpression of DKK1 in JEG3 cells decreased lipid accumulation and mRNA content of PPARD, SLC27A1, CD36, LIPIN1, and LIPIN3. Our results demonstrate that DKK1 is regulating certain aspects of placental lipid metabolism through the WNT signaling pathway.

Fig. 1

Gestational food intake (A), body weight (B), and food intake:body weight ratio (C) curves in OR and OP dams throughout pregnancy. Results are reported as the mean ± SEM (n = 5 litters). *P < 0.01 when compared to OR dams after determining an overall difference throughout gestation by repeated-measures ANOVA.

Fig. 2

Fasting plasma TG (A) and NEFA (B), as well as placental TG (C) and NEFA (D), in OR and OP dams at the time of cesarean delivery on Gestational Day 21. Results are reported as the mean ± SEM (n = 5 litters). *P < 0.05 when compared to OR dams, ^#P < 0.01 when compared to OR dams.

Fig. 3

Representative image of Oil Red O staining of placental fat accumulation in placentas of OR (A) and OP (B) dams (Dz, decidual zone; Jz, junctional zone; Lz, labyrinthine zone). Arrows indicate areas of significant red staining. Measurement of the decidual/junctional thickness (C), mRNA analysis (D), and relative TBARS measurement (E) were performed in placentas of five dams (n = 2 from each dam) from each strain, with from five to seven measurements per placenta for thickness measurement. Results are reported as the mean ± SEM. *P < 0.01 when compared to OR dams.

Fig. 4

Whole-tissue Dkk1 mRNA content (A) in placentas of OR and OP dams. Representative images of DKK1 protein localization in the decidual/junctional zones in placentas of OR (B) and OP (C) dams with quantification of red fluorescence intensity relative to OR dams (D), as well as in the labyrinthine zone of placentas of OR (E) and OP (F) dams with quantification of red fluorescence intensity relative to OR dams (G), are shown. Red represents DKK1 protein, and blue represents nuclear staining (n = 5 [with 10 samples total] for mRNA analysis, n = 5 for staining, and n = 3 placentas, with eight images per placenta for fluorescence quantification). Results are reported as the mean ± SEM. *P < 0.01 when compared to OR dams. Original magnification ×20 (B, C, E, and F).

Fig. 5

Representative blot image of nuclear β-catenin and LaminA protein content in whole placental homogenates from OR and OP dams (A) as well as quantification of normalized nuclear β-catenin protein content (B). Representative image of β-catenin protein localization in the decidual/junctional zones in placentas of OR (C) and OP (D) dams with quantification of red fluorescence intensity relative to OR dams (E), as well as in the labyrinthine zone of placentas of OR (F) and OP (F) dams with quantification of red fluorescence intensity relative to OR dams (H), are also shown. Red represents β-catenin, and blue represents nuclear staining (n = 4 for nuclear protein blotting, n = 5 for staining, and n = 3 placentas, with eight images per placenta for fluorescence quantification). Results are reported as the mean ± SEM. *P < 0.01 when compared to OR dams. Original magnification ×20 (C, D, F, and G).

Fig. 6

DKK1 mRNA content in JEG3 cells following treatment with 400 μmol/L of NEFA (A), with representative images of Oil Red O staining in cells following 0 μM (B) and 400 μM (C) NEFA treatment and quantification of the Oil Red O staining (D), as well as the relative TBARS measurement from the same experiment (E) (n = 3 for mRNA and staining analyses). Results are reported as the mean ± SEM. *P < 0.01 when compared to the 0 μM treatment.

Fig. 7

DKK1 mRNA content following DKK1 overexpression in JEG3 cells treated with 400 μM NEFA (A) as well as representative images of β-catenin protein localization in WT (following transfection of empty pCMV; B) and +DKK1 (C) JEG3 cells following treatment with 400 μM NEFA with quantification of red fluorescence intensity relative to WT cells (D). Red represents β-catenin protein, and blue represents nuclear staining (n = 3 each for mRNA analysis, staining, Oil Red O quantification, and fluorescence quantification, with eight images per triplicate). Results are reported as the mean ± SEM. *P < 0.05 when compared to WT cells, ^#P < 0.01 when compared to WT cells.

Fig. 8

Representative image of Oil Red O staining in WT (A) and +DKK1 (B) JEG3 cells following treatment with 400 μM NEFA and quantification of the Oil Red O staining (C) and mRNA analysis of +DKK1 JEG3 cells following NEFA treatment (D) (n = 3 each for mRNA analysis and Oil Red O quantification). Results are reported as the mean ± SEM. *P < 0.05 when compared to WT cells, ^#P < 0.01 when compared to WT cells. Bar = 80 μm.