Maternal High-Fat Feeding Increases Placental Lipoprotein Lipase Activity by Reducing SIRT1 Expression in Mice

Abstract

This study investigated how maternal overnutrition and obesity regulate expression and activation of proteins that facilitate lipid transport in the placenta. To create a maternal overnutrition and obesity model, primiparous C57BL/6 mice were fed a high-fat (HF) diet throughout gestation. Fetuses from HF-fed dams had significantly increased serum levels of free fatty acid and body fat. Despite no significant difference in placental weight, lipoprotein lipase (LPL) protein levels and activity were remarkably elevated in placentas from HF-fed dams. Increased triglyceride content and mRNA levels of CD36, VLDLr, FABP3, FABPpm, and GPAT2 and -3 were also found in placentas from HF-fed dams. Although both peroxisome proliferator-activated receptor-γ (PPARγ) and CCAAT/enhancer binding protein-α protein levels were significantly increased in placentas of the HF group, only PPARγ exhibited a stimulative effect on LPL expression in cultured JEG-3 human trophoblasts. Maternal HF feeding remarkably decreased SIRT1 expression in placentas. Through use of an SIRT1 activator and inhibitor and cultured trophoblasts, an inhibitory effect of SIRT1 on LPL expression was demonstrated. We also found that SIRT1 suppresses PPARγ expression in trophoblasts. Most importantly, inhibition of PPARγ abolished the SIRT1-mediated regulatory effect on LPL expression. Together, these results indicate that maternal overnutrition induces LPL expression in trophoblasts by reducing the inhibitory effect of SIRT1 on PPARγ.

© 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

Figures

Figure 1

Maternal HF feeding during gestation increased fetal body fat and placental TG content. Ten- to 12-week-old nulliparous C57BL/6 female mice were mated with chow-fed males. HF diet was provided to dams once the vaginal plug was detected. Fetuses, placentas, and other tissue samples were collected at E18.5 in the fed state through caesarean section. Increased body weight and body fat were assayed in HF-fed dams by EchoMRI scanning (n = 6–8) (A). Fetal body fat was measured using petroleum ether fat extraction. Although there were no remarkable differences in fetal body weight (B) and body fat (C), significantly elevated adiposity (D) was observed in fetuses from HF-fed dams (n = 20–36). Compared with fetuses of chow-fed dams, remarkably increased serum FFA levels (E) but not TG (F) and glucose (G) levels were detected in fetuses of HF-fed dams (n = 8). Comparing HF- and chow-fed dams, there were no significant differences in blood glucose (H) and FFA (I) levels; however, significantly decreased serum TG (J) and increased insulin (K) levels were found in HF-fed dams. Of note, fetal serum insulin concentrations were comparable between groups (L). There was no difference in placental weight (M), but placental TG content was significantly higher in the HF group (n = 6) (N). BW, body weight; Con, control; NS, not significant.

Figure 1

Maternal HF feeding during gestation increased fetal body fat and placental TG content. Ten- to 12-week-old nulliparous C57BL/6 female mice were mated with chow-fed males. HF diet was provided to dams once the vaginal plug was detected. Fetuses, placentas, and other tissue samples were collected at E18.5 in the fed state through caesarean section. Increased body weight and body fat were assayed in HF-fed dams by EchoMRI scanning (n = 6–8) (A). Fetal body fat was measured using petroleum ether fat extraction. Although there were no remarkable differences in fetal body weight (B) and body fat (C), significantly elevated adiposity (D) was observed in fetuses from HF-fed dams (n = 20–36). Compared with fetuses of chow-fed dams, remarkably increased serum FFA levels (E) but not TG (F) and glucose (G) levels were detected in fetuses of HF-fed dams (n = 8). Comparing HF- and chow-fed dams, there were no significant differences in blood glucose (H) and FFA (I) levels; however, significantly decreased serum TG (J) and increased insulin (K) levels were found in HF-fed dams. Of note, fetal serum insulin concentrations were comparable between groups (L). There was no difference in placental weight (M), but placental TG content was significantly higher in the HF group (n = 6) (N). BW, body weight; Con, control; NS, not significant.

Figure 2

Maternal HF feeding increased placental LPL expression and activity and genes that facilitate lipid transport. E18.5 placental samples were collected from chow- and HF-fed dams. A: Using real-time PCR, significantly increased mRNA of LPL and other key genes that facilitate lipid transport were detected in placentas from HF-fed dams (n = 6). B: Western blotting further revealed higher protein levels of LPL and CD36 in placentas from HF-fed dams (n = 7). C: Parallel with the increased gene expression, significantly elevated LPL activities were found in placentas of HF-fed dams (n = 8). *P < 0.05 vs. control. Con, control.

Figure 3

HF feeding during pregnancy increased PPARγ expression in placentas, and PPARγ stimulated LPL expression in trophoblasts. A: Significantly increased PPARγ and C/EBPα protein levels were revealed in placentas of HF-fed dams (n = 7–8). *P < 0.05 vs. control. B and C: Overnight (13 h) treatment with the PPARγ agonist ROSI (10 μmol/L) robustly increased LPL expression and activities in confluent JEG-3 trophoblasts (n = 6). D: However, Ad-C/EBPα overexpression (24 h) did not alter LPL protein levels in JEG-3 trophoblasts (n = 6). Con, control; NS, not significant.

Figure 4

Maternal HF feeding reduced expression of SIRT1, which suppresses LPL expression in trophoblasts. Western blotting and real-time PCR demonstrated remarkably reduced SIRT1 protein (A) and mRNA (B) levels in placental samples of E18.5 HF-fed dams (n = 6). Compared with WT MEFs, significantly higher LPL mRNA levels were found in Sirt1−/− MEFs (n = 6) (C). Confluent JEG-3 trophoblasts were treated with SIRT1 activator RSV (10 μmol/L) or inhibitor EX-527 (1 μmol/L) overnight (13 h). EX-527 treatment robustly increased LPL protein levels and activities (D and E). In contrast, significantly reduced LPL protein levels and activities were found in RSV-treated trophoblasts (n = 6) (D and E). *P < 0.05 vs. control. Con, control.

Figure 5

SIRT1 inhibits LPL expression by suppressing PPARγ expression in trophoblasts. To verify the relationship between SIRT1 and PPARγ in regulating LPL expression, Sirt1−/− MEFs (A) and JEG-3 trophoblasts (B) were treated with PPARγ agonist ROSI (10 μmol/L) for 13 h. ROSI treatment robustly increased LPL mRNA in Sirt1−/− MEFs (n = 6) (A) but showed no effect on SIRT1 protein expression in trophoblasts (n = 6) (B). By using viral vector–mediated small interfering RNA (siRNA) overexpression, Sirt1 was significantly knocked down in JEG-3 trophoblasts (C). However, increased LPL expression due to Sirt1 knockdown was abolished in PPARγ inhibitor GW9662-treated cells (3 μmol/L, 13 h) (n = 6) (C). *P < 0.05 vs. control. Con, control.