Maternal Exercise-Induced SOD3 Reverses the Deleterious Effects of Maternal High-Fat Diet on Offspring Metabolism Through Stabilization of H3K4me3 and Protection Against WDR82 Carbonylation

Abstract

Preclinical studies reveal maternal exercise as a promising intervention to reduce the transmission of multigenerational metabolic dysfunction caused by maternal obesity. The benefits of maternal exercise on offspring health may arise from multiple factors and have recently been shown to involve DNA demethylation of critical hepatic genes leading to enhanced glucose metabolism in offspring. Histone modification is another epigenetic regulator, yet the effects of maternal obesity and exercise on histone methylation in offspring are not known. Here, we find that maternal high-fat diet (HFD; 60% kcal from fat) induced dysregulation of offspring liver glucose metabolism in C57BL/6 mice through a mechanism involving increased reactive oxygen species, WD repeat-containing 82 (WDR82) carbonylation, and inactivation of histone H3 lysine 4 (H3K4) methyltransferase leading to decreased H3K4me3 at the promoters of glucose metabolic genes. Remarkably, the entire signal was restored if the HFD-fed dams had exercised during pregnancy. WDR82 overexpression in hepatoblasts mimicked the effects of maternal exercise on H3K4me3 levels. Placental superoxide dismutase 3 (SOD3), but not antioxidant treatment with N-acetylcysteine was necessary for the regulation of H3K4me3, gene expression, and glucose metabolism. Maternal exercise regulates a multicomponent epigenetic system in the fetal liver resulting in the transmission of the benefits of exercise to offspring.

Figure 1

Maternal exercise increases H3K4me3 at the promoters of offspring hepatic genes. A: Schematic illustration of the maternal exercise and diet program. B: H3K4me3, H3K9me3, H3K27me3, and H3K27ac levels in livers of 52-week-old offspring of chow- or HFD-fed and sedentary (Sed) or trained dams. C: H3K4me3 levels in livers of 4-week-old, day 0, and E13.5 offspring of chow- or HDS-fed and sedentary or trained dams. H3K4me3 levels at promoters of glucose metabolism genes in livers of E13.5 (D) and 52-week-old (E) offspring of chow- or HFD-fed and sedentary or trained dams (n = 6). All data are reported as means ± SEM. *P < 0.025 vs. Chow-Sed; **P < 0.01 vs. Chow-Sed; ***P < 0.001 vs. Chow-Sed; ****P < 0.0001 vs. Chow-Sed; †P < 0.025 High Fat-Sed vs. High Fat-Train; ‡P < 0.01 High Fat-Sed vs. High Fat-Train; ††P < 0.01 High Fat-Sed vs. High Fat-Train. Statistical significance was determined by one- or two-way ANOVA, with Tukey and Bonferroni post hoc analysis.

Figure 2

Maternal exercise promotes H3K4 methyltransferase activity and protects against ROS-induced protein carbonylation in offspring livers. Enzymatic activity of total H3K4 methyltransferase (A), mRNA expression of histone methylation-related gene expression (B), ROS level (C), Trolox equivalent capacity (D), and carbonylated protein content (E) in livers of E13.5 offspring of chow- or HFD-fed and sedentary (Sed) or trained dams (n = 6). All data are reported as means ± SEM. *P < 0.025 vs. Chow-Sed; **P < 0.01 vs. Chow-Sed; ****P < 0.0001 vs. Chow-Sed; ‡P < 0.01 High Fat-Sed vs. High Fat-Train; ††P < 0.01 High Fat-Sed vs. High Fat-Train. Statistical significance was determined by one- or two-way ANOVA, with Tukey and Bonferroni post hoc analysis.

Figure 3

Maternal exercise-induced H3K4me3 stabilization is mediated through protection against WDR82 carbonylation. A: DNP-labeled carbonylated proteins in livers of offspring of chow- or HFD-fed and sedentary (Sed) or trained dams. B: WDR82 protein expression in livers of offspring of chow- or HFD-fed and sedentary or trained dams. C: DNP immunoblotting of WDR82-immunoprecipitated proteins in livers of offspring of chow- or HDS-fed and sedentary or trained dams (n = 3). **P < 0.001 vs. Chow-Sed; ****P < 0.0001 vs. Chow-Sed; ††P < 0.001 High Fat-Sed vs. High Fat-Train. Effects of Wdr82 overexpression on H3K4me3 levels (D), H3K4 methyltransferase activity (E), and mRNA expression of glucose metabolism genes (F) in primary hepatoblasts of chow- or HFD-fed dams (n = 3). *P < 0.025 vs. Chow-control (Con) (Wdr82−); **P < 0.01 vs. Chow-Con (Wdr82−); ***P < 0.001 vs. Chow-Con (Wdr82−); ****P < 0.0001 vs. Chow-Con (Wdr82−); †P < 0.025 High Fat-Wdr82− vs. High Fat-Wdr82 overexpression (Wdr81+); ‡P < 0.01 High Fat-Con (Wdr82−) vs. High Fat-Wdr82+; ††P < 0.001 High Fat-Wdr82− vs. High Fat-Wdr82+). Effects of Wdr82 knockdown on H3K4me3 levels (G) and mRNA expression of glucose metabolism genes (H) in primary hepatoblasts (n = 3). All data are reported as means ± SEM. **P < 0.001; ***P < 0.001. Statistical significance was determined by one- or two-way ANOVA, with Tukey and Bonferroni post hoc analysis.

Figure 4

Beneficial effects of maternal exercise on glucose metabolism and WDR82 carbonylation in offspring of HFD-fed dams were blocked by placenta-specific Sod3 knockout. A and B: Glucose tolerance measured at 24 weeks in Sod3^f/f or Sod3^−/− offspring of dams that were sedentary or trained and fed chow or the HFD. Glucose area under the curve (AUC) of male (A) and female (B) offspring is shown. GTT, glucose tolerance test. Data are means ± SEM (n = 5–7/group). **P < 0.01 vs. Chow-Sod3^f/f-Sed; §P < 0.01 effect of genotype; ¶P < 0.01 effect of diet. Glucose production in hepatocytes of 16-week-old male (C) and female (D) Sod3^f/f or Sod3^−/− offspring of dams that were sedentary (Sed) or trained and fed the HFD. Data are means ± SEM (n = 3). **P < 0.01 vs. Sod3^f/f-HFD-Sedentary, §P < 0.01 effect of genotype. Effects of placenta-specific Sod3^−/− on mRNA expression of glucose metabolism genes (E), H3K4 methyltransferase activity (F), ROS levels (G), carbonylated protein content (H), and carbonylated WDR82 levels (I) in livers of E13.5 offspring of sedentary or trained HFD-fed dams (n = 3). HF, high-fat diet; IP, immunoprecipitation; Mat Treat. Maternal treatment. All data are reported as means ± SEM.**P < 0.01 vs. Sod3^f/f-HF-Sed; ***P < 0.001 vs. Sod3^f/f-HF-Sed; §P < 0.01 effect of genotype. Statistical significance was determined by one- or two-way ANOVA, with Tukey and Bonferroni post hoc analysis.

Figure 5

Effects of SOD3 on offspring glucose metabolism are distinct from NAC. A and D: Developmental system used to treat offspring livers with recombinant SOD3 or NAC exo utero. Offspring livers were collected at 4 weeks (A) or at E13.5 (D). Glucose production in primary hepatocytes of 4-week-old male (B) and female (C) offspring of HFD-fed, saline-, SOD3-, or diethyldithiocarbamate (DETCA)-treated dams (n = 3). **P < 0.01 vs. pCPT-saline. Effects of SOD3 or NAC treatment in utero on ROS levels (E), carbonylated protein content (F), WDR82 carbonylation levels (G), mRNA expression of glucose metabolism genes (H), AMPKα phosphorylation (pAMPKα) levels (I), and mRNA expression of Tet and Idh (J) in livers of E13.5 offspring of HFD-fed dams (n = 3). IP, immunoprecipitation. All data are reported as means ± SEM. **P < 0.01 vs. pCPT-saline; ***P < 0.01 vs. pCPT-saline; ****P < 0.01 vs. pCPT-saline. Statistical significance was determined by one- or two-way ANOVA, with Tukey and Bonferroni post hoc analysis.