Placental superoxide dismutase 3 mediates benefits of maternal exercise on offspring health

Abstract

Poor maternal diet increases the risk of obesity and type 2 diabetes in offspring, adding to the ever-increasing prevalence of these diseases. In contrast, we find that maternal exercise improves the metabolic health of offspring, and here, we demonstrate that this occurs through a vitamin D receptor-mediated increase in placental superoxide dismutase 3 (SOD3) expression and secretion. SOD3 activates an AMPK/TET signaling axis in fetal offspring liver, resulting in DNA demethylation at the promoters of glucose metabolic genes, enhancing liver function, and improving glucose tolerance. In humans, SOD3 is upregulated in serum and placenta from physically active pregnant women. The discovery of maternal exercise-induced cross talk between placenta-derived SOD3 and offspring liver provides a central mechanism for improved offspring metabolic health. These findings may lead to novel therapeutic approaches to limit the transmission of metabolic disease to the next generation.

Keywords: AMPK; DNA methylation; TET; glucose metabolism; maternal exercise; placenta; pregnancy; superoxide dismutase 3; vitamin D.

Figure 1:. Maternal Exercise Induces DNA Demethylation in the Promoter of Hepatic Genes in Offspring.

A, B, I, J) Total 5-mC (A, I) and 5-hmC (B, J) in genomic DNA from 4-week-old (A, B) and E13.5 (I, J) offspring livers. C-H, K) The relative 5-mC (C, E-H) and 5-hmC levels (D, K) at the promoter of specific liver metabolic genes in 4-week-old offspring hepatocytes (C, D), Day 0 (E) and E13.5 (F, K) offspring livers, epiblasts (G), and blastocysts (H) from dams that were either sedentary or trained and fed a chow HFD. (n=6, **P < 0.01 vs Sed-Chow, #P < 0.05 vs Sed-High Fat, $P < 0.01 vs Sed-High Fat)

Figure 2:. Maternal Exercise Activates the TET Signaling in Liver and Hepatoblasts of Offspring.

A-B) mRNA expression of Tet and Dnmt (A) and protein expression of TET1 and TET2 (B) in E 13.5 offspring livers from sedentary or trained dams. (n=6, *P < 0.05, **P < 0.01) C) The binding of TET1 and TET2 to the promoter region of liver metabolic genes in E 13.5 offspring liver from sedentary or trained dams. D) The amount of αKG in E13.5 offspring liver from sedentary or trained dams. E-G) The relative protein expression of IDH1 and IDH2 (E) and the phosphorylation of AMPK-α (F) and ACC (G) in the liver of E13.5 offspring from sedentary or trained dams. H-J) The amount of αKG (H) and mRNA expression of Tet, Idh (I), and glucose metabolic genes (J) in 100 μM AICAR or 100 μM metformin-treated hepatoblasts. (n=5, *P < 0.05, **P < 0.01)

Figure 3:. Serum Derived from Trained Dams Activates AMPK-TET Signaling in Primary Hepatoblasts via SOD3.

A-B) The mRNA expression of Tet, Idh, and liver metabolic genes (A) and the phosphorylation level of AMPKα (B) in hepatoblasts treated with 10% serum from sedentary or trained pregnant females at 13.5 d.p.c or trained non-pregnant females for 24h. (n=6, **P < 0.01 vs Sed serum) C) The expression of Tet, Idh, and liver metabolic genes in hepatoblasts treated with serum from trained dams after heat treatment at 37 or 80 °C. (n=3, **P < 0.01 vs 80 °C) D) The serum SOD3 in sedentary or trained, chow or HFD fed, pregnant or non-pregnant female at 13.5 d.p.c. (n=6, **P < 0.01 vs Sed-Chow-Pregnant) E) The correlation between serum SOD3 and total running distance in trained dams. (n=14) F-G) Sod3 mRNA expression in various types of tissues and E13.5 offspring livers from sedentary or trained pregnant (F) or non-pregnant (G) females. (CD; chow diet, HFD; high fat diet, S; sedentary, T; Train, n=6, *P < 0.05 vs S-CD, **P < 0.01 vs S-CD) H-I) RNA-seq from the placenta of sedentary or trained dam. (n=5) J) The protein levels of SOD3 in trophoblasts from chow or HFD fed and sedentary or trained dams (n=3, **P < 0.01 vs Sed-Chow, #P <0.01 vs Sed-High Fat) K) The mRNA expression of Sod3 in the placenta from sedentary or trained dams plotted throughout gestation (n=6, **P < 0.01 vs sed) L) Maternal and fetal serum levels of SOD3 in sedentary or trained dams at 13.5 dp.c. (n=7, **P < 0.01 vs Maternal Sed, $P < 0.01 vs Fetal Sed)

Figure 4:. SOD3 Treatment Mimics the Effects of Maternal Exercise in Primary Hepatoblasts.

A-B) The expression of Tet, Idh, and liver metabolic genes in mouse hepatoblasts from chow and HFD fed dams (A), and human hepatoblasts (B) treated with 200 ng/ml SOD3 for 24h. (n=3, **P < 0.01 vs Control-Chow, #P < 0.05 vs Control-HFD, $P < 0.01 vs Control-HFD) C-E) TET1 activity (C), 5-mC (D) and 5-hmC levels (E) at the promoter of liver metabolic genes in SOD3-treated mouse hepatoblasts. (n=3, *P < 0.05, **P < 0.01) F) The effects of Tet1 and Tet2 knockdown on SOD3-induced glucose metabolic gene expression in mouse hepatoblasts. (n=3, *P < 0.05 vs Control si-Control, **P < 0.01 vs Control si-Control, $P <0.05 vs Control si-SOD3) G) The phosphorylation of AMPKα in SOD3, AICAR, or metformin-treated mouse and human hepatoblasts. (n=3, **P < 0.01 vs 0 min, #P < 0.01 vs Sod3 30 min) H) The effects of Tet1 and Tet2 knockdown or treament with Compound C on SOD3-induced Tet and Idh mRNA expression in mouse hepatoblasts. (n=3, *P < 0.05 vs Control si-Control, **P < 0.01 vs Control si-Control, $P <0.05 vs Control si-Sod3)

Figure 5:. The Effects of Maternal Exercise on the TET-AMPK Signaling Axis and Glucose Metabolism in Offspring Liver are Significantly Impaired by Trophoblast-Specific Sod3 Knockout.

A) SOD3 protein expression in Sod3^−/− or Sod3^f/f placenta from sedentary or trained dams. (n=6, **P < 0.01 vs Sed- Sod3^f/f $P < 0.01 vs Train- Sod3^f/f) B) Breeding method to generate both Sod3^−/− and Sod3^f/f placentae in the same dams. C) The effects of placenta-specific Sod3^−/− on the expression of Tet, Idh, and liver metabolic genes (C), the levels of 5-mC (D) and 5-hmC (E) at the promoter of liver metabolic genes, and the phosphorylation levels of AMPKα (F) in E13.5 offspring liver from sedentary or trained dams. (S; Sed, T; Train, n=6, **P < 0.01 vs Sed- Sod3^f/f, #P < 0.05 vs Sed-Sod3^−/−, $P < 0.01 vs Sed-Sod3^−/−) G-H) Glucose tolerance was measured at 24 and 52 weeks of age in Sod3^f/f or Sod3^−/− offspring from sedentary or trained dams. Glucose area under the curve (AUC) of male (G) and female (H) offspring are shown, respectively. I-J) Fasting serum insulin concentrations of male (I) and female (J) Sod3^f/f or Sod3^−/− offspring at 24 weeks. K-L) Glucose production in hepatocytes from 16-week-old male (K) and female (L) Sod3^f/f or Sod3^−/− offspring of sedentary or trained dams. Data are expressed as means ± SEM. (n=3, **P<0.01, §P<0.01 effect of genotype)

Figure 6:. Normal Vitamin D Levels are Necessary for the Beneficial Effects of Maternal Exercise on Offspring Liver.

A) Protein expression of VDR, RXRα and FoxP1 in the placenta of sedentary or trained dams at 13.5 day (n=3, **P < 0.01 vs Sed-Chow). B-C) SOD3 protein expression in mouse primary trophoblasts (B), HTR8, JAR, and JEG3 (C) with transient overexpression of FLAG-tagged VDR 10 nM vitamin D for 24h. (n=3, **P < 0.01 vs DMSO-Control). D-G) Serum levels of 25(OH) vitamin D (D), placental Vdr and Sod3 mRNA expression (E), serum levels of SOD3 (F), and Tet, Idh, and glucose metabolic gene expression of E13.5 offspring liver (G) from normal (1000 U), low (100 U), or high (10000 U) levels of vitamin D-diet-fed, sedentary or trained dams at 0 or 13.5 day of pregnancy. (n=6, *P < 0.05 vs Normal VD Sed, **P < 0.01 vs Normal VD Sed, $P < 0.01 vs Normal VD Train).

Figure 7:. The Effects of Pregnancy Stage and Physical Activity on the Level of SOD3 in Pregnant Women.

A) Serum SOD3 in 1st (1-13 weeks, n=395), 2nd (14-27 weeks, n=366), and 3rd trimesters (28-40 weeks, n=342) of pregnancy. (**P < 0.01) B) Correlation between the serum SOD3 in 3rd trimester and the placental weight at parturition. C) Relationship between the serum SOD3 at 1st, 2nd, and 3rd trimester and daily MVPA. D) Serum SOD3 in physically inactive or active pregnant women at 2nd trimester. E) Sod3 mRNA expression in placenta from physically inactive or active pregnant women at parturition. (n=9, *P < 0.05) F) Correlation between placental Sod3 expression and daily MVPA.