Administration of the Antioxidant N-Acetyl-Cysteine in Pregnant Mice Has Long-Term Positive Effects on Metabolic and Behavioral Endpoints of Male and Female Offspring Prenatally Exposed to a High-Fat Diet

Abstract

A growing body of evidence suggests the consumption of high-fat diet (HFD) during pregnancy to model maternal obesity and the associated increase in oxidative stress (OS), might act as powerful prenatal stressors, leading to adult stress-related metabolic or behavioral disorders. We hypothesized that administration of antioxidants throughout gestation might counteract the negative effects of prenatal exposure to metabolic challenges (maternal HFD feeding during pregnancy) on the developing fetus. In this study, female C57BL/6J mice were fed HFD for 13 weeks (from 5-weeks of age until delivery) and were exposed to the N-acetyl-cysteine (NAC) antioxidant from 10-weeks of age until right before delivery. Body weight of the offspring was assessed following birth, up to weaning and at adulthood. The metabolic, neuroendocrine and emotional profile of the adult offspring was tested at 3-months of age. Prenatal HFD increased mother's body weight and offspring's weight at the time of weaning, when administered in conjunction with NAC. In females, NAC administration reduced high levels of leptin resulting from prenatal HFD. Prenatal NAC administration also resulted in greater glucose tolerance and insulin sensitivity while increasing adiponectin levels, as well as increasing exploratory behavior, an effect accompanied by reduced plasma corticosterone levels in response to restraint stress. Analysis of glutathione levels in the hypothalamus and in brown adipose tissue indicates that, while HFD administration to pregnant dams led to reduced levels of glutathione in the offspring, as in the male hypothalamus, NAC was able to revert this effect and to increase glutathione levels both in the periphery (Brown Adipose Tissue, both males and females) and in the central nervous system (males). Overall, results from this study indicate that the body redox milieu should be tightly regulated during fetal life and that buffering OS during pregnancy can have important long-term consequences on metabolic and behavioral endpoints.

Keywords: N-acetyl-cysteine; behavior; glutathione; high-fat diet; metabolism; mice; oxidative stress; pregnancy.

Figure 1

Graphical representation of the experimental timeline. HFD, high-fat diet; CD, control diet; SD, standard diet; NAC, N-acetyl-cysteine; PND, postnatal day; G, gestational day; GTT, Glucose tolerance test; IST, insulin sensitivity test; OF, open field; EPM, elevated plus maze.

Figure 2

Body weight of dams and offspring. (A) HFD increased dams’ body weight during both peri-conceptional time and pregnancy (gray shade) moreover, NAC increased dams’ body weight on week 5. (B) Prenatal NAC increased pups’ body weight on PND 3; (C) on PND 30 HFD overall increased pups’ body weight particularly when in combination with NAC (D) this effect was reverted on PND 90. Data are mean ± SEM (dams) and + SEM (pups); ^$p < 0.05, main effect of NAC; ^£p < 0.05, main effect of HFD; **p < 0.01 NAC-CD vs. NAC-HFD (A); *p < 0.05, NAC-HFD vs. WATER-HFD (A); NAC-HFD vs. NAC-CD (C); NAC-CD vs. WATER-CD (D).

Figure 3

Glucose tolerance test (GTT) and insulin sensitivity test (IST). Glucose tolerance: (A) NAC-CD males were characterized by improved glucose tolerance particularly 30 and 60 min following glucose loading. (B) No difference was found among treatment groups in females. Insulin sensitivity: (C) Interestingly, NAC-CD males showed improved insulin sensitivity 60 min following insulin injection. By contrast, the NAC-HFD subjects showed an overt worsening of the glycaemic profile. (D) Females showed a glycaemic profile comparable to that of NAC-HFD comparable males as a result of NAC alone. Data are mean ± SEM; **p < 0.01: (A) NAC-CD vs. all other treatment groups at time points 30 and 60. (C) NAC-HFD vs. all other treatment groups at time points 15 and 30; WATER-HFD and NAC-HFD vs. WATER-CD and NAC-CD at time point 120; (D) NAC-CD vs. WATER-CD at 15 and 120 min. *p < 0.05: (C), NAC-CD vs. all other treatment groups at time point 60; (D), NAC-CD and WATER-HFD vs. WATER-CD at time point 30; WATER-HFD vs. WATER-CD at time point 60.

Figure 4

Adiponectin and Leptin plasma levels. (A) NAC increased adiponectin in males while (B) no effect was observed in females. (C) An opposite effect of NAC was observed for leptin levels in males. (D) In females, this reduction of leptin levels was much stronger since they were more vulnerable to the effects of HFD. Data are mean + SEM. Main effect of NAC: ^$$p < 0.01 (A,C,D); **p < 0.01 (D), NAC-HFD vs. WATER-HFD (females).

Figure 5

Activation of the hypothalamus-pituitary-adrenal (HPA) axis after restraint stress. (A) In males, NAC led to overall decreased corticosterone (CORT) levels. This decrease was stronger in NAC-CD subjects, maternal HFD reducing this effect. (B) In females, the combination of prenatal NAC and HFD led to a reduction in CORT levels comparable to that observed in NAC-HFD males while NAC alone did not affect this parameter. Data are mean ± SEM. ***p < 0.001 (A) NAC-CD vs. all other groups and NAC-HFD vs. all other groups; *p < 0.05 (B) 30 min, NAC-HFD vs. all other groups.

Figure 6

Levels of glutathione in peripheral and central tissues. Hypothalamus: (A) glutathione levels were lowered by diet in male subjects (WATER-HFD). Independently from prenatal HFD, all males showed higher glutathione levels as a result of NAC administration. (B) Females mice showed reduced glutathione levels in this area and appeared to be more resilient to changes either upon HFD or NAC. Brown Adipose Tissue (BAT): (C) Both male and female subjects showed an increase in glutathione levels upon NAC administration. (D) In addition, females show a significant increase in glutathione (NAC-CD) that was reduced upon HFD administration (NAC-HFD). Data are mean + SEM. **p < 0.01 for WATER-HFD vs. WATER-CD in male (A) and NAC-CD vs. WATER-CD (D); *p < 0.05 for NAC-HFD vs. NAC-CD (D). ^$$p < 0.01 main effect of NAC (A,C,D).

Figure 7

Spontaneous behavior in the OF test. In general, NAC led to a reduced emotionality and increased exploration in the OF test (A). NAC-treated subject spent more time and were characterized by increased locomotion in the central part of the arena when compared to controls. In the plus-maze test, NAC mice showed an increased latency and a reduced frequency and duration of immobility (B). NAC increased rearing bouts, particularly in CD subjects while decreasing the overall duration of this behavior (B). In addition, the latency to the first risk-assessment episode (stretched-attend posture—SAP) was strongly reduced in NAC-treated mice while the frequency was increased; the combination of prenatal NAC and HFD increased the duration of the stretched-attend-posture behavior (B). Data are mean + SEM ^$$p < 0.01, NAC vs. WATER; **p < 0.01; NAC-CD vs. NAC-HFD.

Figure 8

Emotional behavior in the EPM test. All subjects spent more time in the closed arms of the maze although a significant interaction between zone and prenatal treatment revealed that NAC mice spent significantly more time in the open arms when compared to their controls (A) In addition, all NAC subjects were characterized by increased locomotion (n. of crossings, B). Data are mean + SEM. *p < 0.05, open arms: NAC vs. WATER; ^$$p < 0.01, NAC vs. WATER.