Maternal High-Fat Diet Leads to Non-alcoholic Fatty Liver Disease Through Upregulating Hepatic SCD1 Expression in Neonate Rats

Abstract

Non-alcoholic fatty liver disease (NAFLD) has become the leading cause of liver disease in children, with evidence that the maternal diet and the early life nutritional environment are potential risk for such disease. This study was aimed to investigate the effects of maternal high-fat diet (HFD) on the occurrence of NAFLD in offspring rats and the underlying mechanisms. In this study, the incidence of NAFLD was compared in F1 offspring rats between the maternal HFD group and standard chow (SC) group. In addition, the expression levels of inflammatory cytokines in the placenta, in the umbilical cord blood, and in the livers of neonate offsprings were compared between two groups. HepG2 cells were treated with recombinant IL6 (rIL6) to assess stearoyl-CoA desaturase 1 (SCD1) expression and lipid synthesis in an inflammatory condition. Lipid accumulation was assayed in both SCD1 overexpression and interference HepG2 cells as well as in neonatal rats. Our results showed that HFD exposure before and throughout the pregnancy induced the elevated hepatic TG content of F1 neonates. The levels of inflammatory cytokines in the placenta, umbilical cord blood, and the livers of HFD F1 neonates were significantly higher than those of the SC group. In addition, rIL6 treatment led to TG accumulation accompanied by the upregulation of SCD1 in HepG2 cell lines. Overexpression of SCD1 led to the accumulation of TG contents in HepG2 cells, whereas Scd1 knockdown attenuated the effects of rIL6 treatment. Overexpression of SCD1 in F1 neonatal rats led to hepatic lipid accumulation. Our study indicated that maternal HFD led to intrauterine inflammation, which subsequently caused transgenerationally abnormal hepatic lipid metabolism of F1 neonates. This modulation might be mediated by upregulating SCD1 expression in hepatic cells.

Keywords: HepG2 cells; SCD1 expression; inflammation; lipid metabolism; maternal high fat diet.

Figure 1

Pathological phenotypes and gene expression of lipid metabolism and inflammatory genes in neonate livers. (A) Hematoxylin and eosin (H&E) staining of neonate livers from F0-CON dams [(a): 200X; (b): 400X] and F0-HFD dams [(c): 200X; (d): 400X]. (B) Oil red O staining of neonate livers from F0-CON dams [(a): 200X; (b): 400X] and F0-HFD dams [(c): 200X; (d):400X]. (C) Comparison of hepatic lipid levels between the neonates from F0-CON and F0-HFD dams. (D) Comparison of gene expression levels related to lipid synthesis between the neonates from F0-CON and F0-HFD dams. (E) Comparison of gene expression levels related to lipid oxidation between the neonates from F0-CON and F0-HFD dams. (F) Protein levels in lipid metabolism between the neonates from F0-CON and F0-HFD dams. (G) Quantitative comparison of proteins in lipid metabolism between the neonates from F0-CON and F0-HFD dams. (H) Comparison of hepatic cytokine gene expression levels between the neonates from F0-CON and F0-HFD dams. (I) Protein levels of hepatic cytokine between the neonates from F0-CON and F0-HFD dams. (J) Quantitative comparison of hepatic cytokine protein levels between the neonates from F0-CON and F0-HFD dams. **P < 0.01; *P < 0.05.

Figure 2

Liver pathology and inflammation status in the placental and umbilical cord in maternity dams. (A) Hematoxylin and eosin (H&E) staining of livers from F0-CON dams [(a): 200X; (b): 400X] and F0-HFD dams [(c): 200X; (d): 400X] after full-term pregnancy. (B) Oil red O staining of livers from F0-CON dams [(a): 200X; (b): 400X] and F0-HFD dams [(c): 200X; (d): 400X] after full-term pregnancy. (C) Comparison of hepatic TG and TCHO levels between F0-CON and F0-HFD dams after full-term pregnancy. (D) Comparison of cytokines in umbilical cord blood between F0-CON and F0-HFD dams by ELISA. (E) Cytokine protein levels in placental tissues between F0-CON and F0-HFD dams. (F) Comparison of cytokine protein levels in placental tissues between F0-CON and F0-HFD dams. **P < 0.01; *P < 0.05.

Figure 3

Recombinant interleukin-6 (rIL6) treatment induces an increase in SCD1 expression and lipid metabolism in HepG2 cells. (A) Scd1 mRNA expression levels in HepG2 cells upon rIL6 treatment at different concentrations. (B) Scd1 mRNA expression levels in HepG2 cells upon rIL6 treatment at different time points. (C) SCD1 protein levels in HepG2 cells with the treatment of rIL6 at 40 ng/μl for 12 h. (D) The relative protein levels of SCD1 in HepG2 cells with the treatment of rIL6 at 40 ng/μl for 12 h. (E) TG levels in HepG2 cells with the treatment of rIL6 at 40 ng/μl for 12 h. (F) Oil red O staining of HepG2 cells. a: Control group; b: HepG2 cells upon rIL6 treatment at 40 ng/μl for 12 h. **P < 0.01; *P < 0.05.

Figure 4

SCD1 overexpression promotes the intracellular lipid accumulation in HepG2 cells. HepG2 cells were transfected with pEX3-NC or pEX3-Scd1 vectors. SCD1 protein levels were determined by western blot (A) and quantitatively compared (B) between two group cells. Meanwhile, mRNA levels of lipid synthesis-related genes (C), lipid oxidation-related genes (D), and intracellular TG levels (E) were compared as well between HepG2 cells transfected with pEX3-NC and pEX3-Scd1 vectors. Oil red O staining was performed (F) to observe the lipid droplets in HepG2 cells transfected with either pEX3-NC (a) or pEX3-Scd1 vectors (b). **P < 0.01; *P < 0.05.

Figure 5

Scd1 knockdown alleviates the lipid accumulation in HepG2 cells exposed to rIL6. (A) Determination of SCD1 protein levels upon siRNA interference in HepG2 cells with or without rIL6 treatment in vitro. (B) Quantitative comparison of SCD1 protein levels in HepG2 cells upon siRNA knockdown with or without rIL6 treatment in vitro. (C–E) Quantitative comparison of mRNA levels of genes related to lipid synthesis (C) and lipid oxidation (D) and intracellular TG levels (E) in HepG2 cells upon siRNA knockdown with or without rIL6 treatment in vitro. (F) Oil red O staining of HepG2 cells. (a): Negative control (NC) group; (b): NC+IL6 group; (c): siRNA group; (d): siRNA+IL6 group. **P < 0.01; *P < 0.05.

Figure 6

Liver pathology and gene expression in the rats at 3 weeks old after rAAV injection after birth. H&E staining (A) and oil red O staining (B) were performed in the livers from the rats of blank [(a): 200X; (b): 400X], rAAV-NC-injected [(c): 200X; (d): 400X], and rAAV-Scd1-injected groups [(e): 200X; (f): 400X] at 3 weeks old. SCD1 protein levels in livers of 3-week-old rats were detected by western blot (C) and compared among blank, rAAV-NC-injected, and rAAV-Scd1-injected groups. Quantitative comparison of Scd1 protein levels in livers of 3-week-old rats were also compared (D). Comparison of mRNA levels of genes related to lipid synthesis (E) and lipid oxidation (F) in the livers of 3-week-old rats was determined in blank, rAAV-NC, and rAAV-Scd1 groups at the same time. **P < 0.01; *P < 0.05.