In Western society the consumption of high fat diet (HFD) as well as a sedentary lifestyle has been associated with metabolic derangements and impaired brain plasticity (Schrauwen and Westerterp, 2000; Unger and Orci, 2001; McGarry, 2002; Molteni et al., 2002, 2004). While it is generally accepted that HFD-induced obesity per se is associated with adverse health outcomes, some studies have also shown that maternal obesity is specifically associated with a variety of pregnancy complications, fetal and neonatal death, maternal hypertension and gestational diabetes (Lu et al., 2001; Bhattacharya and Sood, 2007; Thompson, 2008). Moreover, it is possible to hypothesize that maternal obesity might increase in the offspring, vulnerability for cardiovascular or metabolic pathological conditions, in addition to the often comorbid mood-disorders. Indeed, evidence suggests that epigenetic events initiated by HFD exposure during the prenatal period can result in persistent adaptations in physiological and metabolic regulations of the developing organism, predisposing the offspring towards disease (Barker, 1995; Lucas, 1998). Furthermore, HFD may negatively impact on metabolic regulation resulting in excess energy intake and increased adiposity (Woods et al., 2004; Zhang et al., 2009) also associated to low-grade inflammation and increased production of inflammatory cytokines (Chandalia and Abate, 2007).

Several studies have shown that administration of high fat or high calorie diets to rodents increases the generation of reactive oxygen species (ROS; Zhang et al., 2005) and protein oxidation (Souza et al., 2007), thus resulting in increased oxidative damage in the brain (Cecarini et al., 2007; Bruce-Keller et al., 2009; Zhang et al., 2009) and, more in general, in oxidative cell injury (Gutteridge and Halliwell, 2000). This evidence is consistent with the hypothesis that increased oxidative stress (OS) may mediate the effects of HFD consumption both on brain health and mood disorders and may as well exacerbate the HFD-related metabolic impairment.

In a previous work performed in mice (Bellisario et al., 2014), we found that the detrimental effect of prenatal exposure to a maternal HFD was counteracted by the lack of the p66Shc gene that results, among other several features, in reduced OS and resistance to diet-induced obesity. More in detail, p66Shc^−/− mice showed blunted changes in glycaemia levels in response to glucose or insulin challenges and were protected from the disrupting effect of prenatal HFD on neuroendocrine activation in response to a psychophysical stress (Bellisario et al., 2014). These findings suggested that reduced OS might increase resistance to impaired physiological adaptation driven by maternal HFD in the offspring during adult life. In light of this evidence, this study was aimed at developing a pharmacological model of reduced OS able to cope with metabolic challenges occurring during early life by means of prenatal administration of N-acetyl-cysteine (NAC). This compound is the rate-limiting substrate in the biosynthesis of glutathione, is a powerful antioxidant and a ROS scavenger; its clinical efficacy and safety as a mucolytic drug have been widely established in the last decades, both as a therapy for respiratory diseases and as a remedy towards several acute intoxications including the most common acetaminophen drug overdose (Crystal and Bast, 1991; Samuni et al., 2013). NAC is “a safe and well-tolerated supplementary drug without any considerable side effects commonly used in clinical practice for a variety of different disorders ranging from polycystic ovary syndrome, preterm-birth, recurrent pregnancy loss, chronic bronchitis, ulcerative colitis, liver cancer, muscle performance, asthma, Alzheimer and Parkinson” (see Mokhtari et al., 2017 and references therein for a complete review). Furthermore, there is evidence showing that NAC administration during pregnancy might counteract apoptosis and ROS-related genotoxicity by increasing glutathione levels and decreasing mitochondrial membrane depolarization within the cells (Amin et al., 2008).

Our working hypothesis was that administration of the antioxidant NAC during prenatal life might increase glutathione levels, preventing the negative effects exerted by increased ROS resulting from maternal HFD administration on metabolic and behavioral endpoints in the offspring. In addition, a growing body of evidence suggests a direct link between redox imbalance and emotional disturbance, particularly anxiety (Hovatta et al., 2005 and maternal obesity has been also independently related to increased incidence of mood disorders in the offspring (Bouayed et al., 2009; Contu and Hawkes, 2017; Edlow, 2017). Thus, another hypothesis to be tested was that a metabolic stressor, such as prenatal HFD, might lead to increased anxiety in the offspring and that NAC would prevent this effect.

To test such hypothesis female mice were fed HFD or control diet (CD) for 13 weeks (from 5 weeks of age until right before delivery) and were exposed to NAC for 8 weeks (from 10 weeks of age until right before delivery). The offspring was subsequently phenotyped for metabolic and behavioral responses at adulthood.

Overall, results from this study show that prenatal NAC administration counteracts some of the short- and long-term negative effects observed following in utero exposure to HFD. As an example, in females, NAC administration was able to reduce 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 a reduced plasma corticosterone levels in response to restraint stress. Analysis of glutathione—an endogenous substance with antioxidant properties—in the hypothalamus and in brown adipose tissue indicates that, while HFD administration to pregnant dams led to lower levels of glutathione in the male hypothalamus, NAC was able to revert this effect both in the periphery (BAT, both males and females) and in the CNS (males). This explains some of the positive long-term and sex-dependent effects of NAC administration per se. These data are in line with previous results from our group suggesting that the oxidative milieu during pregnancy can have important health outcomes on the offspring (Berry and Cirulli, 2013; Bellisario et al., 2014).

Exposure to HFD feeding during pregnancy represents a metabolic stressful challenge negatively affecting physiological adaptation to pregnancy. In this study maternal HFD led to an overall increase in body weight in all dams that was apparent in the offspring on PND 30, an effect possibly potentiated by weaning as previously shown with an equivalent animal model (Bellisario et al., 2014). NAC administration alone resulted in increased birth weight of the offspring on PND 3 and at adulthood PND 90.

When circulating adipokines were measured, prenatal HFD resulted in increased leptin levels in females, a phenomenon we have previously described (Bellisario et al., 2014). Here we show that this effect can be prevented by maternal NAC administration. In addition, NAC led to an overall increase in adiponectin levels, particularly in males. Leptin and adiponectin are two major adipokines involved in the control of metabolic homeostasis. Leptin affects food consumption and energy expenditure (Friedman and Halaas, 1998) acting directly on the hypothalamus (a brain region involved in energy homeostasis) and hyperleptinemia has been associated to obesity and the related disorders (Considine et al., 1996). By contrast, adiponectin increases tissue fat oxidation, leading to reduced levels of fatty acids and tissue triglyceride content, overall increasing insulin sensitivity (Matsuzawa et al., 2004). Thus, at least as far as adipokines are concerned, we can hypothesize an increased developmental flexibility of females offspring resulting both in an elevated vulnerability to a suboptimal metabolic intrauterine environment as well as in a more efficient response to NAC treatment, while males might be characterized by a higher metabolic resilience.

The interpretation of metabolic assessments at adulthood is more complex. In particular, HFD did not affect, per se, glucose tolerance or insulin sensitivity. While in males NAC alone (NAC-CD group) led to a more effective glycemic profile, as assessed both in the GTT and IST, this was not observed in females. In the case of insulin sensitivity, it appears that subjects experiencing prenatal NAC in combination with HFD were characterized by decreased insulin sensitivity. A potential explanation for this result comes from the consideration that, although the excessive production of ROS is associated with many human diseases, in recent years it has become apparent that low levels of H₂O₂ may in fact be required for normal cellular functioning and intracellular signaling (Veal et al., 2007; see also further in the “Discussion” section).

Assessment of HPA axis reactivity showed that, while HFD did not affect, per se, the neuroendocrine response to a psychophysical stress (restraint), NAC, alone (males) or in combination with HFD (females), overall led to a blunted rise in CORT levels in response to stress. Glucocorticoid hormones (GC) show a very broad variety of actions inducing alert responses through the activation of metabolic and behavioral processes (Oitzl et al., 1997). Interestingly, NAC treated subjects were overall characterized by a decreased emotionality as—differently from HFD-mice—they spent more time in the center of the arena, were less immobile and more explorative in the OF test, in agreement with the reduced activation of the HPA axis. In addition, they showed reduced emotionality in the Elevated Plus-Maze test spending more time in the open (anxiogenic) arms of the maze. Previous authors have found a link between genes involved in the regulation of OS and emotionality suggesting a tight relationship between OS metabolic pathways and normal anxiety (Hovatta et al., 2005). When compared with previous data obtained in other animal model, such as the p66Shc^−/− mouse (Berry et al., 2007, 2008; Berry and Cirulli, 2013) these results strengthen the notion of a link among OS, metabolism and emotional behavior, indicating a potential role for antioxidants in the management of anxiety and/or psychiatric disorders (Giorgio et al., 2012; Berry and Cirulli, 2013).

Overall, in this study, while prenatal HFD did not show pervasive effects on the metabolic or emotional profile of the experimental subjects, NAC-treated mice showed improved glucose tolerance in addition to a reduced activation of the HPA axis upon restraint stress. Moreover, when tested in the OF and in the Elevated Plus-Maze, they were characterized by a reduced emotional profile. Intriguingly, the observed effects of prenatal NAC mirror the overall phenotype of p66Shc knock-out mice previously described (Berry and Cirulli, 2013; Bellisario et al., 2014). Since this is an animal model of reduced OS and healthy aging, these data might suggest that the prenatal-NAC-phenotypic features might be protective upon further metabolic or stressful challenges that could be experienced at adult age. This hypothesis is strengthened by data showing meaningful effects of NAC on glutathione levels, a powerful antioxidant and a ROS scavenger, in central (hypothalamus) and peripheral (brown fat) tissues. Data indicate that, while HFD administration to pregnant dams leads to reduced levels of glutathione in male hypothalamus, NAC is able to revert this effect and to increase levels of glutathione in the periphery (BAT, both males and females). Females appear overall more resistant to changes in glutathione levels in the CNS compared to males, while the opposite holds true in the periphery.

Tight regulation of the redox balance, particularly in a developing organism is crucial for the maintenance of proper cell function (Ristow et al., 2009). Although antioxidant therapy is currently emerging as a promising strategy to counteract the prevalence of diabetes and other metabolism-related pathologies, in addition to anxiety and psychiatric disorders, unfortunately, major randomized clinical trials have yielded conflicting results (Falach-Malik et al., 2016). Results from this study, performed in an animal model, indicate positive effects of NAC given during pregnancy on the developing offspring although they also raise some questions concerning safety. A wide variety of stimuli including growth factors and hormones can promote the transient generation of H₂O₂, and recent data provide clear evidence for the enhancement of insulin signaling by ROS in vivo (Loh et al., 2009). This might suggest that lowering excessively ROS might endanger signal transduction mechanisms they regulate.

Overall, these data indicate that a major challenge for future investigations will be to determine the optimal dose of compounds capable to keep the redox balance within an optimal range, taking into account physiological functions involving redox-sensitive pathways, age- and sex/gender differences.

AB and FC designed the experiments, supervised all the experimental procedures and drafted the article. VB and PP performed all the behavioral and neuroendocrine assessments. CR performed all the biochemical assays, carried-out the statistical analysis and drafted the article. MA and MCM performed glutathione biochemical assay and carried-out statistical analysis and data interpretation on this assay.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.