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Maternal exercise via exerkine apelin enhances brown adipogenesis and prevents metabolic dysfunction in offspring mice

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Science Advances  17 Apr 2020:
Vol. 6, no. 16, eaaz0359
DOI: 10.1126/sciadv.aaz0359
  • Fig. 1 Maternal exercise intervention markedly induces the expression of thermogenic markers in iBAT and ingWAT of offspring at weaning.

    (A and B) Body weight (A) and relative wet weight of iBAT and ingWAT (B) normalized to body weight of female and male M-Ctrl and M-Ex offspring at weaning (n = 6 mice per group). N.S., not significant. (C) Z scores of mRNA levels of different brown fat markers in the iBAT and ingWAT of M-Ctrl and M-Ex offspring at weaning (n = 6 mice per group). Expression was normalized by ΔCt values. (D) Cropped Western blots of UCP1, PRDM16, and PGC-1α in the iBAT (β-tubulin is the loading control) from the E18.5 fetuses of CON and EX maternal mice (n = 6 fetuses per group). (E and F) Cropped Western blots of UCP1 and PRDM16 (β-tubulin is the loading control) from iBAT (E) and ingWAT (F) isolated from female and male mice of M-Ctrl and M-Ex at weaning (n = 6 mice per group). Data are mean ± SEM, and each dot represents one litter. *P < 0.05, **P < 0.01, and ***P < 0.001 by two-tailed unpaired Student’s t test (A to C, E, and F) or one-way analysis of variance (ANOVA) (D).

  • Fig. 2 Maternal exercise intervention facilitates thermogenesis in iBAT and ingWAT of offspring following CE.

    (A) iBAT wet weight of female and male M-Ctrl and M-Ex offspring by 2 days of CE (n = 5 mice per group). (B) Representative thermographic images (left) and calculated averages of intrascapular temperature (right) of female and male M-Ctrl and M-Ex offspring at room temperature (RT) and 2 days of CE (n = 6 litters for RT and n = 5 litters for CE). (C and D) Cropped Western blots of UCP1 and PRDM16 (β-tubulin is the loading control) from iBAT (C) and ingWAT (D) of female and male M-Ctrl and M-Ex offspring followed by 2 days of CE (n = 5 mice per group). (E) Representative images of UCP1 immunocytochemical staining of iBAT and ingWAT from M-Ctrl or M-Ex offspring at RT or CE. Scale bars, 100 μm. DAPI, 4′,6-diamidino-2-phenylindole. (F) Cropped Western blots of GLUT4 in the iBAT (β-actin was used as the loading control) isolated from M-Ctrl or M-Ex offspring at RT and after 2 days of CE (n = 6 litters for RT and n = 5 litters for CE). Data are mean ± SEM, and each dot represents one litter. *P < 0.05, **P < 0.01, and ***P < 0.001 in M-Ctrl versus M-Ex, and ###P < 0.001 in RT versus CE by two-tailed unpaired Student’s t test (A, C, and D) or two-way ANOVA (B and F).

  • Fig. 3 Maternal exercise protects offspring from HFD-induced obesity.

    (A to C) Daily food intake (A), body weight gain (B), and ingWAT wet weight (C) of female and male M-Ctrl and M-Ex offspring mice that were fed an HFD for 8 weeks (n = 6 mice per group). (D to H) Glucose tolerance test (E and F), blood insulin (D), insulin resistance (G), and pancreatic β-cell function (H) of female and male M-Ctrl and M-Ex offspring that were fed an HFD for 8 weeks with 5-hour fasting (n = 6). AUC, area under the curve. (I) Hepatic Oil Red O staining of female and male M-Ctrl and M-Ex offspring. Scale bar, 100 μm. (J and K) Time-resolved oxygen consumption and analysis of female and male M-Ctrl or M-Ex offspring that were fed an HFD for 8 weeks. Graph depicts calculated means of each litter (n = 5 litters). Data are mean ± SEM, and each dot represents one litter. *P < 0.05, **P < 0.01, and ***P < 0.001 in M-Ctrl versus M-Ex, and ###P < 0.001 in female versus male by two-tailed unpaired Student’s t test (A, C to H, J, and K) or two-way ANOVA (B).

  • Fig. 4 Maternal exercise protects the impairment of thermogenic markers and formation in iBAT and ingWAT of the offspring challenged with HFD.

    (A and B) Relative iBAT wet weight (A) and representative thermographic images (left) and calculated averages of intrascapular temperature (right) (B) of female and male M-Ctrl and M-Ex offspring challenged with HFD (n = 6). (C and F) Cropped Western blots of UCP1 and PRDM16 protein levels in the iBAT (C) and ingWAT (β-tubulin or β-actin were used as the loading control) (F) from the female and male M-Ctrl and M-Ex offspring challenged with HFD (n = 6). (D and G) Hematoxylin and eosin (H&E) staining (D) and transmission electron microscopic images (G) of iBAT of female and male offspring challenged with HFD. Scale bars, 100 μm (D) and 1 μm (G); m.: mitochondria; l.: lipid droplets. (E and H to J) H&E staining (E), mean cross-sectional areas (CSAs) (H), and percent distribution of lipid droplets (I and J) in the ingWAT of female and male offspring challenged with HFD. Scale bar, 100 μm. Data are mean ± SEM, and each dot represents one litter. *P < 0.05, **P < 0.01, and ***P < 0.001 in M-Ctrl versus M-Ex by two-tailed Student’s t test (A to C, F, and H).

  • Fig. 5 Apelin supplementation induces brown adipogenesis in the fetal BAT at E18.5.

    (A) Cropped Western blots of apelin protein levels in the BAT (β-tubulin was used as the loading control) of fetal and offspring mice (n = 6). apelin (APLN) (B) Serum apelin (APLN) concentrations in maternal mice at E18.5 (n = 6). (C) Protocol of apelin supplementation (i.p.). (D) Serum apelin concentrations in female and male fetuses of maternal exercise (ME) and APN studies (n = 6). (E) BAT wet weight of female and male fetuses maternally treated with apelin (0.5 μmol/kg per day) or phosphate-buffered saline (PBS) (n = 6). (F and G) Z scores of mRNA level of different brown fat markers in the female (D) and male (E) fetal BAT (n = 6). Expression was normalized by ΔCt values. (H) Cropped Western blots of UCP1, PGC-1α, PRDM16, and AMPK phosphorylation (β-tubulin was used as the loading control) in the female and male fetal BAT maternally treated with apelin or PBS (n = 6). Data are mean ± SEM, and each dot represents one litter. *P < 0.05, **P < 0.01, and ***P < 0.001 in CON versus EX, M-Ctrl versus M-Ex, or PBS versus APN, and ##P < 0.01 in female versus male by two-tailed Student’s t test (A, B, and D to H) or Z score (F and G). F0, initial filial generation; F1, first filial generation.

  • Fig. 6 Maternal exercise induces DNA demethylation of the Prdm16 promoter through apelin/α-KG axis.

    (A) mRNA expression of Prdm16 of fetal BAT from M-Ctrl and M-Ex (n = 6). Expression was normalized by ΔCt values. (B) Diagram showing three regions in the Prdm16 promoter. (C, D, and H) 5mC and 5hmC enrichment fold of fetal BAT (ME study, C; APN study, H) and adult offspring challenged with HFD (D) (n = 6). (E) α-KG to 2-hydroxyglutarate (2-HG) ratio of fetal iBAT from ME or APN study (n = 6). (F, G, I, and J) Z scores of mRNA level of ten-eleven translocation (Tet) and IDH families in the fetal iBAT of APN study (I and J) or ME study (F and G) (n = 6). Expression was normalized by ΔCt values. (K) Working model: DNA demethylation stimulated by α-KG/AMPK/maternal exercise–induced apelin. Data are mean ± SEM, and each dot represents one litter. *P < 0.05, **P < 0.01, and ***P < 0.001 in M-Ctrl versus M-Ex or PBS versus APN by two-tailed Student’s t test (A to J).

Supplementary Materials

  • Supplementary Materials

    Maternal exercise via exerkine apelin enhances brown adipogenesis and prevents metabolic dysfunction in offspring mice

    Jun Seok Son, Liang Zhao, Yanting Chen, Ke Chen, Song Ah Chae, Jeanene M. de Avila, Hongyang Wang, Mei-Jun Zhu, Zhihua Jiang, Min Du

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