Maternal High-Fat Diet Impairs Placental Fatty Acid β-Oxidation and Metabolic Homeostasis in the Offspring

Abstract

Maternal overnutrition can affect fetal growth and development, thus increasing susceptibility to obesity and diabetes in later life of the offspring. Placenta is the central organ connecting the developing fetus with the maternal environment. It is indicated placental fatty acid metabolism plays an essential role in affecting the outcome of the pregnancy and fetus. However, the role of placental fatty acid β-oxidation (FAO) in maternal overnutrition affecting glucose metabolism in the offspring remains unclear. In this study, C57BL/6J female mice were fed with normal chow or high-fat diet before and during pregnancy and lactation. The placenta and fetal liver were collected at gestation day 18.5, and the offspring's liver was collected at weaning. FAO-related genes and AMP-activated protein kinase (AMPK) signaling pathway were examined both in the placenta and in the human JEG-3 trophoblast cells. FAO-related genes were further examined in the liver of the fetuses and in the offspring at weaning. We found that dams fed with high-fat diet showed higher fasting blood glucose, impaired glucose tolerance at gestation day 14.5 and higher serum total cholesterol (T-CHO) at gestation day 18.5. The placental weight and lipid deposition were significantly increased in maternal high-fat diet group. At weaning, the offspring mice of high-fat diet group exhibited higher body weight, impaired glucose tolerance, insulin resistance and increased serum T-CHO, compared with control group. We further found that maternal high-fat diet downregulated mRNA and protein expressions of carnitine palmitoyltransferase 2 (CPT2), a key enzyme in FAO, by suppressing the AMPK/Sirt1/PGC1α signaling pathway in the placenta. In JEG-3 cells, protein expressions of CPT2 and CPT1b were both downregulated by suppressing the AMPK/Sirt1/PGC1α signaling pathway under glucolipotoxic condition, but were later restored by the AMPK agonist 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR). However, there was no difference in CPT2 and CPT1 gene expression in the liver of fetuses and offspring at weaning age. In conclusion, maternal high-fat diet can impair gene expression involved in FAO in the placenta by downregulating the AMPK signaling pathway, and can cause glucose and lipid dysfunction of offspring at weaning, indicating that placental FAO may play a crucial role in regulating maternal overnutrition and metabolic health in the offspring.

Keywords: AMPK; fatty acid β-oxidation; maternal high-fat diet; offspring; placenta.

Figure 1

Maternal HF feeding impaired glucose and lipid metabolism in dams. (A) Body weight of dams during 4 weeks before mating; (B) FBG of dams before mating; (C) Body weight of dams at P14.5; (D) FBG of dams at P14.5; (E) OGTT of dams at P14.5; (F) AUC of dams at P14.5; (G) Serum insulin of dams at P18.5; (H) Serum T-CHO of dams at P18.5; (I) Serum TG of dams at P18.5; (J) Serum FFA of dams at P18.5. Data represented as the mean ± SEM. (F0-NC, n = 5–6; F0-HF, n = 6–7). *p < 0.05, **p < 0.01, ***p < 0.001 vs. F0-NC. NC, normal chow diet; HF, high-fat diet; FBG, fasting blood glucose; OGTT, oral glucose tolerances tests; AUC, the area under the glucose curve; T-CHO, total cholesterol; TG, triglyceride; FFA, free fatty acid.

Figure 2

Maternal HF feeding altered the development of the placenta and fetal, and induced lipid deposition in the placenta. (A) Representative images of H&E staining in the placenta. The black arrow indicates interstitial edema, magnification 400x. The scale bar indicates 100 μm; (B) Placental weight; (C) Fetal weight; (D) Fetal/ placental weight ratio; (E) Representative images and quantitative assessment of Oil Red O staining in the placenta, magnification 400x. The scale bar indicates 100 μm. Data represented as the mean ± SEM. (NC, n = 6; HF, n = 6–7). *p < 0.05, ***p < 0.001 vs. NC. NC, normal chow diet; HF, high-fat diet; H&E, hematoxylin and eosin; IOD, integrated optical density.

Figure 3

HF feeding reduced placental FAO gene expression by suppressing the AMPK signaling pathway. (A–J) Placental mRNA expression of genes. (A) CPT2; (B) CPT1a; (C) CPT1b; (D) LCAD; (E) LCHAD; (F) AMPKα; (G) Sirt1; (H) PGC1α; (I) PPARγ; (J) TFAM; (K) Immunohistological analysis of CPT2 and CPT1b in the placenta, magnification 200x. The scale bar indicates 200 μm. Data represented as the mean ± SEM. (NC, n = 5–6; HF, n = 7). *p < 0.05, **p < 0.01, vs. NC. NC, normal chow diet; HF, high-fat diet; CPT2, carnitine palmitoyltransferase 2; CPT1a, carnitine palmitoyltransferase 1a; CPT1b, carnitine palmitoyltransferase 1b; LCAHD, long-chain 3-hydroxyacyl-coa dehydrogenase; LCAD, long chain acyl-CoA dehydrogenase; AMPKα, AMP-activated protein kinase α; Sirt1, sirtuin 1; PGC1α, peroxisome proliferator-activated receptor gamma coactivator 1-alpha; PPARγ, peroxisome proliferator-activated receptor γ; TFAM, mitochondrial transcription factor A; IOD, integrated optical density.

Figure 4

HF feeding reduced protein expression of placental FAO by suppressing the AMPK signaling pathway. (A) Bands' representative images of (B–G). (B) CPT2; (C) CPT1b; (D) p-AMPKα/t-AMPKα; (E) Sirt1; (F) PGC1α; (G) PPARγ; (H) TFAM. Data represented as the mean ± SEM. (NC, n = 5–6; HF, n = 6–7). *p < 0.05, **p < 0.01, vs. NC. NC, normal chow diet; HF, high-fat diet; AMPKα, AMP-activated protein kinase α; Sirt1, sirtuin 1; PGC1α, peroxisome proliferator-activated receptor gamma coactivator 1-alpha; PPARγ, Peroxisome proliferator-activated receptor γ; CPT1a, carnitine palmitoyltransferase 1a; CPT1b, carnitine palmitoyltransferase 1b; CPT2, carnitine palmitoyltransferase 2; TFAM, mitochondrial transcription factor A.

Figure 5

Glucolipotoxicity reduced FAO-related protein expression by suppressing the AMPK signaling pathway and reversed by AICAR. (A–E) JEG-3 cells were treated with BSA (Con) or glucose/FFA (GF), which were treated for 10 min for AMPKα and treated for 24h for other proteins. (A) CPT2; (B) CPT1b; (C) p-AMPKα/t-AMPKα protein ratio; (D) Sirt1; (E) PGC1α. (F–I) JEG-3 cells were treated with BSA (Con) or glucose/FFA (GF) or glucose/FFA/AICAR (GFA) for 24 h. (F) CPT2; (G) CPT1b; (H) Sirt1; (I) PGC1α. The experiment was repeated three-five times. Data represented as the mean ± SEM. *p < 0.05, vs. Con. ^#p < 0.05, vs. GF. GF, glucose/FFA; GFA, glucolipotoxicity plus AICAR; AICAR, 5-aminoimidazole-4-carboxamide ribonucleotide; AMPKα, AMP-activated protein kinase α; Sirt1, sirtuin 1; PGC1α, peroxisome proliferator-activated receptor gamma coactivator 1-alpha; CPT1b, carnitine palmitoyltransferase 1b; CPT2, carnitine palmitoyltransferase 2.

Figure 6

Maternal HF feeding impaired glucose and lipid metabolism of offspring at weaning. (A) Body weight of male offspring from birth to weaning; (B) FBG of male offspring at weaning; (C) IPGTT and (D) AUC of male offspring at weaning; (E) Serum insulin; (F) Serum T-CHO; (G) Serum TG; (H) Serum FFA of male offspring at weaning. Data represented as the mean ± SEM. (NC-3w, n = 8; HF-3w, n = 6–8). *p < 0.05, **p < 0.01 vs. NC-3w. NC, normal chow diet; HF, high-fat diet; 3w, the third week; FBG, fasting blood glucose; IPGTT, intraperitoneal glucose tolerance test; AUC, the area under the glucose curve; T-CHO, total cholesterol; TG, triglyceride; FFA, free fatty acid.

Figure 7

Maternal HF feeding did not affect FAO-related gene mRNA expression in offspring at fetal and weaning age. (A–D) hepatic mRNA expression in the fetus. (A) CPT2; (B) CPT1a; (C) CPT1b; (D) LCAD; (E–H) hepatic mRNA expression in the offspring at weaning. (E) CPT2, (F) CPT1a, (G) CPT1b, (H) LCAD. Data represented as the mean ± SEM. (NC-fetus, n = 6; HF-fetus, n = 7; NC-3w, n = 8; HF-3w, n = 8). NC, normal chow diet; HF, high-fat diet; 3w, the third week. CPT2, carnitine palmitoyltransferase 2; CPT1a, carnitine palmitoyltransferase 1a; CPT1b, carnitine palmitoyltransferase 1b; LCAD, long chain acyl-CoA dehydrogenase.