Overnutrition and maternal obesity in sheep pregnancy alter the JNK-IRS-1 signaling cascades and cardiac function in the fetal heart

Abstract

Maternal obesity in pregnancy predisposes offspring to insulin resistance and associated cardiovascular disease. Here, we used a well-established sheep model to investigate the effects of maternal obesity on cardiac functions. Multiparous ewes were assigned to a control (CON) diet [100% of National Research Council (NRC) recommendations] or an obesogenic (OB) diet (150% of NRC recommendations) from 60 d before conception to necropsy on d 135 of pregnancy. Fetal blood glucose and insulin were increased (P<0.01, n=8) in OB (35.09+/-2.03 mg/dl and 3.40+/-1.43 microU/ml, respectively) vs. CON ewes (23.80+/-1.38 mg/dl and 0.769+/-0.256 microU/ml). Phosphorylation of AMP-activated protein kinase (AMPK), a cardioprotective signaling pathway, was reduced (P<0.05), while the stress signaling pathway, p38 MAPK, was up-regulated (P<0.05) in OB maternal and fetal hearts. Phosphorylation of c-Jun N-terminal kinase (JNK) and insulin receptor substrate-1 (IRS-1) at Ser-307 were increased (P<0.05) in OB fetal heart associated with lower downstream PI3K-Akt activity (P<0.05), indicating impaired cardiac insulin signaling. Although OB fetal hearts exhibited a normal contractile function vs. CON fetal hearts during basal perfusion, they developed an impaired heart-rate-left-ventricular-developed pressure product in response to high workload stress. Taken together, fetuses of OB mothers demonstrate alterations in cardiac PI3K-Akt, AMPK, and JNK-IRS-1 signaling pathways that would predispose them to insulin resistance and cardiac dysfunction.

Figure 1.

Body weight (A), maternal plasma glucose (B), and maternal plasma insulin (C) in ewes fed a diet with either 100% of NRC recommendations (CON; n=8) or 150% of NRC recommendations (OB; n=8). Values are expressed as means ± se. *P < 0.05 vs. CON.

Figure 2.

Levels of activated JNK and p38/MAPK, GSH/GSSG ratio, and IRS-1-associated PI3K-Akt activity in heart tissue of CON and OB maternal sheep. A, B) Protein levels of JNK (A) and p38/MAPK (B), and their phosphorylation in the hearts of CON and OB pregnant sheep. Phosphorylated JNK and p38 were quantified relative to total JNK or p38 protein level, respectively. C) GSH/GSSG ratio in maternal sheep hearts. D) Phosphorylation of IRS-1 at Ser-307. E) Phosphorylation of Akt at Ser-473. F) AMPK at Thr¹⁷² in heart tissue of maternal sheep. Phosphorylated IRS-1 was quantified relative to total IRS-1 level from 500 μg total protein of sample homogenate. G) PP2Cα expression levels in CON and OB maternal sheep hearts. Values are means ± se (n=5). *P < 0.05 vs. CON.

Figure 3.

Body weight (BW), heart weight (HW), LV mass, plasma metabolic parameters of LV from CON (n=8) and OB (n=8) fetal sheep at d 135 of gestation. A) Fetal glucose. B) Fetal insulin. C) Ratio of fetal HW to BW. D) Ratio of fetal LV mass to HW (D). Values are means ± se. *P < 0.01 vs. CON.

Figure 4.

Fetal cardiac contractile function at d 135 of gestation, assessed by the Langendorff perfusion system. A) Cardiac contractile function estimated as heart rate (HR)-LVDP product. Fetal hearts were perfused for 15 min at baseline and then challenged with isoprenaline (ISO; 10 μM) at the high workload. *P < 0.05 vs. CON. B–D) Heart rate (B), LVDP (C), maximum derivative of diastolic pressure (D), and maximum derivative of systolic pressure (E). *P < 0.05 vs. basal; ^#P < 0.05 vs. CON ISO. Values are means ± se (n=3/group).

Figure 5.

Altered JNK-IRS-1 signaling cascades, increased oxidative stress, and impaired insulin signaling pathways in the OB fetal hearts. A, B) Protein levels of JNK (A) and p38/MAPK (B), and their phosphorylation in the hearts of CON and OB fetal hearts. Phosphorylated JNK and p38 were quantified relative to total JNK or p38 protein level, respectively. C) GSH/GSSG ratio in fetal hearts of CON and OB sheep. D) Immunoblots of phosphor-IRS1 at Ser-307. E) Phosphor-IRβ at Tyr^1150/1151 of fetal hearts. F, G) Phosphorylation of Akt at Ser-473 (F) and mTOR at Ser-2448 (G) in heart tissue of CON or OB fetus. Phosphorylated Akt and mTOR were quantified relative to total Akt or mTOR protein level, respectively. H) IRS-1-dependent PI3K activity. I) Akt kinase activity in fetal hearts, measured by immunoprecipitation kinase assays. Values are means ± se (n=5). *P < 0.05 vs. CON.

Figure 6.

Alteration in AMPK signaling pathways and the expression levels of fatty acid transporters in the fetal heart. A) Phosphorylation of AMPK at Thr¹⁷² in heart tissue of CON and OB fetus. Phosphor-AMPK was quantified relative to AMPKα protein level. B) Expression levels of PP2Cα in CON and OB fetal hearts. C) Immunoblots of FATP1, FATP4, and CD36 in fetal hearts. All immunoblots were quantified relative to β-actin protein level. Values are means ± se (n=5). *P < 0.05 vs. CON.