Maternal exercise conveys protection against NAFLD in the offspring via hepatic metabolic programming

Abstract

Maternal exercise (ME) during pregnancy has been shown to improve metabolic health in offspring and confers protection against the development of non-alcoholic fatty liver disease (NAFLD). However, its underlying mechanism are still poorly understood, and it remains unclear whether protective effects on hepatic metabolism are already seen in the offspring early life. This study aimed at determining the effects of ME during pregnancy on offspring body composition and development of NAFLD while focusing on proteomic-based analysis of the hepatic energy metabolism during developmental organ programming in early life. Under an obesogenic high-fat diet (HFD), male offspring of exercised C57BL/6J-mouse dams were protected from body weight gain and NAFLD in adulthood (postnatal day (P) 112). This was associated with a significant activation of hepatic AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptor alpha (PPARα) and PPAR coactivator-1 alpha (PGC1α) signaling with reduced hepatic lipogenesis and increased hepatic β-oxidation at organ programming peak in early life (P21). Concomitant proteomic analysis revealed a characteristic hepatic expression pattern in offspring as a result of ME with the most prominent impact on Cholesterol 7 alpha-hydroxylase (CYP7A1). Thus, ME may offer protection against offspring HFD-induced NAFLD by shaping hepatic proteomics signature and metabolism in early life. The results highlight the potential of exercise during pregnancy for preventing the early origins of NAFLD.

Figure 1

Maternal gestational exercise protects offspring from HFD-induced body weight gain (A) Experimental design. (B) Offspring body weight gain (§ Differences between CO-HFD and INT-HFD, * Differences between CO-HFD and CO, £ Differences between CO-HFD and INT, + Difference between CO and INT-HFD). (C) Total body weight at P112. (D) Epigonadal fat pad weight at P112. [CO (n = 28), INT (n = 14), CO-HFD (n = 33), INT-HFD (n = 9)]. Mean ± SEM;*^/+/§/£p < 0.05, **^{/++/§§/££}p < 0.01, ***^{/+++/§§§/£££}p < 0.001, ****^{/++++/§§§§/££££}p < 0.0001. CO control, INT intervention, HFD high fat diet, SD standard diet, g gram, P postnatal day.

Figure 2

Maternal exercise during pregnancy protects offspring from NAFLD in later life. (A) Histological hepatic steatosis in 4-month old male offspring (P112) by H&E staining. Magnification: 20×. (B) Percentage of steatosis, scored as 0 (< 5%); 1 (5–33%); 2 (34%-66%) and 3 (> 66%) percentage of hepatocytes containing lipid droplets. (C) Liver-biopsy assessment of NAFLD-activity score with average score. CO (n = 6), INT (n = 5), CO-HFD (n = 12), INT-HFD (n = 6). CO control, INT intervention, NAFLD non -alcoholic fatty liver disease, HFD high fat diet, H&E hematoxylin and eosin staining.

Figure 3

Maternal gestational exercise induces a distinct metabolic signature in the liver proteome of INT offspring at P21. (A) Volcano plot of univariate statistical analysis results from liver tissue of CO- and INT-offspring. A volcano plot based on fold change (Log₂) and P value (− Log₁₀) of all proteins identified in both groups. Red dots indicate proteins that showed statistically significant changes (N = 118). (B) Heatmap of unsupervised 2-dimensional hierarchical clustering of the proteome profile of significantly altered proteins. The normalized Z-Score of protein abundance is depicted by a pseudocolor scale with blue showing positive expression, yellow showing equal expression and red negative expression compared with the values of each protein. Visual inspection of the heatmap demonstrates the ability of these proteins to distinguish between offspring of exercised or sedentary dams. CO (n = 4), INT (n = 5). CO control, INT intervention, log logarithm.

Figure 4

Influences of maternal exercise on hepatic proteome analysis of CO and INT offspring in early life. (A) Analysis of the biological functions of differential expressed proteins via STRING database (https://string-db.org) to yield gene ontology annotation terms for KEGG pathways and biological processes; network nodes represent proteins. (B) KEGG Pathways and (C) biological process-based categories of liver proteins that displayed significantly changed levels among CO-and INT-offspring at P21. (D) CYP7A1 protein expression. (E) FAS protein expression at P21. CO (n = 4), INT (n = 5). CO control, INT intervention, KEGG Kyoto Encyclopedia of Genes and Genomes, P postnatal day.

Figure 5

Effects of maternal exercise during pregnancy on offspring liver energy sensing and on AMPK signaling in early life. (A) pAMPK/AMPK protein expression. (B) PPARα protein expression. (C) pACC/ACC protein expression. (D) Pgc1α mRNA expression. (E) Cpt1a mRNA expression. (F) Acc1 mRNA expression. (G) Acc2 mRNA expression. Representative immunoblots are presented above the respective graph. Immunoblots: CO (n = 5), INT (n = 5). Uncropped images of original blots are shown in Supplementary Fig. 6. mRNA Expression: CO (n = 5), INT (n = 7). Mean ± SEM; *p < 0.05, **p < 0.01. CO control, INT intervention, P postnatal day, AMPK adenosine monophosphate-activated protein kinase, PPARα peroxisome proliferator-activated receptor alpha, ACC acetyl-CoA carboxylase, GAPDH Glycerinaldehyd-3-phosphate-dehydrogenase, Pgc1α peroxisome proliferator-activated receptor gamma coactivator 1-alpha, Cpt1a carnitine palmitoyltransferase 1A.

Figure 6

Impact of maternal gestational exercise on hepatic glucose metabolism in offspring at P21. (A) Intraperitoneal glucose tolerance test (i.p. GTT) at P21 [CO (n = 12), INT (n = 8)]. (B) Assessment of regulators of hepatic glucose metabolism (Pgc1α, FoxO1, Pepck, G6Pase, Pparƴ) by qPCR [CO (n = 5), INT (n = 5–7)]. (C–F) Assessment of indicators of insulin signaling at P21 using immunoblots: (C) INS-R, (D) SOCS-3, (E) pAKT/AKT, (F) pERK/ERK [CO (n = 5), INT (n = 5)]. Representative immunoblots are presented above the respective graphs. Uncropped images of original blots are shown in Supplementary Fig. 6. Mean ± SEM; *p < 0.05, **p < 0.01. CO control, INT intervention, Ins-R Insulin receptor, SOCS-3 suppressor of cytokine signaling 3, erk extracellular signal-regulated kinases, GAPDH Glycerinaldehyd-3-phosphate-dehydrogenase.

Figure 7

Influences of maternal exercise on offspring cholesterol at P21. (A) CYP7A1 protein expression [CO (n = 4), INT (n = 5)]. (B) Srebp2 mRNA expression [CO (n = 11), INT (n = 7)]. (C) Ldl-R mRNA expression [CO (n = 11), INT (n = 8)]. (D) Hmgcr mRNA expression [CO (n = 5), INT (n = 7)]. (E) SREBP1 protein expression [CO (n = 5), INT (n = 5)]. Representative immunoblots are presented above the respective graph. Uncropped images of original blots are shown in Supplementary Fig. 6. Mean ± SEM; *p < 0.05. CO control, INT intervention, Srebp sterol regulatory element-binding protein, Ldl-R LDL-Receptor, Hmgcr 3-Hydroxy-3-Methylglutaryl-CoA Reductase, CYP7A1 cholesterol 7 alpha-hydroxylase, GAPDH Glycerinaldehyd-3-phosphate-dehydrogenase.

Figure 8

Hepatic metabolic programming—a working model depicting how maternal exercise during pregnancy conceivably affects hepatic metabolism of the offspring. Solid blue line: represent effects mediated by AMPK; dash blue line: represent effects mediated by PGC1α.