Maternal Overnutrition During Gestation in Sheep Alters Autophagy Associated Pathways in Offspring Heart

Abstract

Poor maternal nutrition during gestation can negatively affect offspring growth, development, and health pre- and post-natally. Overfeeding during gestation or maternal obesity (MO) results in altered metabolism and imbalanced endocrine hormones in animals and humans which will have long-lasting and detrimental effects on offspring growth and health. In this study, we examined the effects of overnutrition during gestation on autophagy associated pathways in offspring heart muscles at two gestational and one early postnatal time point (n = 5 for treated and untreated male and female heart respectively at each time point). Two-way ANOVA was used to analyze the interaction between treatment and sex at each time point. Our results revealed significant interactions of maternal diet by developmental stages for offspring autophagy signaling. Overfeeding did not affect the autophagy signaling at mid-gestation day 90 (GD90) in both male and female offspring while the inflammatory cytokines were increased in GD90 MO male offsrping; however, overfeeding during gestation significantly increased autophagy signaling, but not inflammation level at a later developmental stage (GD135 and day 1 after birth) in both males and females. We also identified a sexual dimorphic response in which female progeny were more profoundly influenced by maternal diet than male progeny regardless of developmental stages. We also determined the cortisol concentrations in male and female hearts at three developmental stages. We did not observe cortisol changes between males and females or between overfeeding and control groups. Our exploratory studies imply that MO alters autophagy associated pathways in both male and female at later developmental stages with more profound effects in female. This finding need be confirmed with larger sample numbers in the future. Our results suggest that targeting on autophagy pathway could be a strategy for correction of adverse effects in offspring of over-fed ewes.

Keywords: autophagy; developmental programming; heart muscle; maternal obesity; poor maternal nutrition; sheep.

FIGURE 1

Alteration of MAPK/ERK signaling pathway in offspring heart of overfed ewes at two gestational and one early postnatal time point. (A–G). Relative expression and phosphorylation level of hallmark proteins in fetal heart at mid-gestation day 90; (H–N). Relative expression and phosphorylation level of hallmark proteins in fetal heart at late-gestation day 135; (O–U). Relative expression and phosphorylation level of hallmark proteins in neonatal heart at day 1 after birth; GAPDH, Protein loading control. CON, control, MO, maternal obesity, M, male, and F, female. Mean ± SEM (n = 5); *p < 0.05, **p < 0.01.

FIGURE 2

Alteration of AKT signaling pathway in offspring heart of overfed ewes at two gestational and one early postnatal time point. (A–C). Relative expression and phosphorylation level of the kinase Akt in fetal heart at mid-gestation day 90; (D–F). Relative expression and phosphorylation level of the kinase Akt in fetal heart at late-gestation day 135; (G–I). Relative expression and phosphorylation level of the kinase Akt in neonatal heart at day 1 after birth; GAPDH, Protein loading control. CON, control, MO, maternal obesity, M, male, and F, female. Mean ± SEM (n = 5); *p < 0.05, **p < 0.01.

FIGURE 3

Alteration of autophagy signaling pathway in offspring heart of overfed ewes at two gestational and one early postnatal time point. (A–E). Relative expression and phosphorylation level of hallmark proteins in fetal heart at mid-gestation day 90; (F–J). Relative expression and phosphorylation level of hallmark proteins in fetal heart at late-gestation day 135; (K–O). Relative expression and phosphorylation level of hallmark proteins in neonatal heart at day 1 after birth; GAPDH, Protein loading control. CON, control, MO, maternal obesity, M, male, and F, female. Mean ± SEM (n = 5); *p < 0.05, **p < 0.01.

FIGURE 4

Cortisol concentration in heart tissues of offspring of overfed ewes at two gestational and one early postnatal time point. (A) Cortisol concentration in fetal heart tissues at day 90 of gestation; (B) Cortisol concentration in fetal heart tissues at day 135 of gestation; (C) Cortisol concentration in neonatal heart tissues at day 1 after birth; CON, control, MO, maternal obesity, M, male, and F, female. Mean ± SEM (n = 5). No significance.

FIGURE 5

Alteration of inflammatory cytokine NF-κB and TNFα in offspring heart of overfed ewes at two gestational and one early postnatal time point. (A–C). Relative expression level of inflammatory cytokine in fetal heart at mid-gestation day 90; (D–F). Relative expression level of inflammatory cytokine in fetal heart at late-gestation day 135; (G–I). Relative expression level of inflammatory cytokine in neonatal heart at day 1 after birth; GAPDH, Protein loading control. CON, control, MO, maternal obesity, M, male, and F, female. Mean ± SEM (n = 5); *p < 0.05, **p < 0.01.

FIGURE 6

Schematic diagram of autophagy associated signaling pathways and potential mechanisms regulated by maternal overnutrition. Important hallmark proteins in the PI3K/Akt/mTOR and the MAPK/ERK signaling are indicated which were examined in this study. Alternation of these hallmark proteins in later gestation and early postnatal life increased autophagy. Cortisol level did not change in heart tissues and was not the cause for the increased autophagy in later gestation and early postnatal life indicated by red dash line. Inflammatory factors TNFα and NF-κB were altered by maternal overnutrition in early gestation but not late gestation or early postnatal life in which the possible mechanisms could be that increased inflammatory factors in early gestation inhibits autophagy (blue dash line and question mark) and unchanged inflammatory factors at late gestation and early postnatal life could be suppressed by increased autophagy (purple dash line and question mark).