It is estimated that by 2035, about 592 million people worldwide will be affected by T2DM (Guariguata et al. 2014). It has been reported that obesity (body mass index > 30 kg/m2) is a major independent risk factor for T2DM and an important cause of NAFLD (Ganz et al. 2014). In the United States alone, 35.7 percent of adults and about 17 percent of children are obese (Rachdaoui 2020). Lipid metabolism disorders have a significant impact on morbidity and mortality of metabolic diseases, which have become major global public health challenges. EEDs capable of disrupting endocrine regulation have been recognized as Environmental Obesogens (Heindel et al. 2019). The study has shown that gestational urinary BPA concentration was associated with the prevalence of obesity in children and adolescents (Braun et al. 2019). Therefore, the present study focused on the effects of BPA exposure during pregnancy, a critical period of organ formation, and 0.05 mg/kg (Tolerable daily intake, TDI) and 5 mg/kg (NOAEL) were selected as the lowest and highest exposure doses respectively. 0.05 mg/kg was also recognized by FDA as the lifetime safe oral dose of BPA (Yu et al. 2020; Uchtmann et al. 2020).
In present study, the liver organ coefficient of male offspring at PND56 was higher than that in the control group, and was significant in the 0.05 and 0.5 mg/kg groups. Organ coefficient, as a common indicator in toxicology experiments, can not only reflect the toxicity of poisons but also observe the possibility of histopathological changes from the side, and help to find toxic target organs. In general, their elevation suggests that exposure to BPA may cause damage to offspring's organs. Relevant experiments have shown that BPA exposure can cause mild cellular swelling and steatosis in rat liver tissues (Huang et al. 2021), and similar results were obtained in two other studies on vertebrates (Tian et al. 2021; Mi et al. 2021).
TG is an important form of energy storage and oxidative energy supply in the body, and is often used as a key indicator to determine fatty acid biosynthesis in lipid metabolism. In this study, we measured the content of TG in serum and liver. The results showed that gestational BPA exposure increased the level of TG in male offspring at PND21 and 56, and the increase trend of TG in serum and liver was basically the same. Our results are consistent with those of several other epidemiological or animal studies (Liao et al. 2021; Meng et al. 2019; Wang et al. 2020), which demonstrated that intrauterine BPA exposure can lead to TG accumulation in male progeny, indicating the role of promoting adipogenesis. Two important pathways involved in the regulation of liver lipid metabolism are fatty acid synthesis and fatty acid oxidation, among which PPAR plays a very important role in fatty acid oxidation. PPARα is highly expressed in liver and promotes fatty acid oxidation by stimulating the transcription of rate-limiting enzyme CPT1 (Qin et al. 2021). In this study, we found that mRNA and protein expressions of PPARα and CPT1α were decreased in the liver at both stages of 5 mg/kg BPA exposure, suggesting that BPA inhibits liver fatty acid oxidation to increase TG accumulation, which is consistent with previous study (Grasselli et al. 2013).
In addition, to further investigate whether BPA-induced effects were related to the expression of key genes and proteins in lipid synthesis, SREBP-1 and its regulated genes SCD-1, ACC1, FAS were detected. Results showed that the mRNA expressions of these factors were significantly increased at both stages. SCD-1 is a key control point for lipid synthesis in the liver and can catalyze the formation of saturated fatty acids into monounsaturated fatty acids. During fat synthesis, monounsaturated fatty acids are more likely to become substrates of ACAT (ACYL-CoA cholesterol acyltransferase) and DGAT (Diacylglycerol acyltransferase) than saturated fatty acids, which produce cholesterol esters and TG (Ravaut et al. 2020). Therefore, we detected the protein levels of SCD-1 and its key regulatory factor SREBP-1, and found that they were consistent with the results of mRNA. These results suggest that BPA can induce SREBP-1 activation in male offspring, thereby increasing the expression of downstream key genes and leading to TG accumulation in vivo. Similar effects have been reported in the previous studies (Guan et al. 2019; Somm et al. 2009). Zhang et al also found that inhibition of SREBP-1 expression could effectively reduce TG accumulation (Zhang et al. 2019). At present, existing evidence fully shows that the expression of SREBP-1 is positively correlated with the content of TG, which is also an important target of BPA exposure affecting lipid metabolism.
Mammalian mTOR is an important regulator of cell proliferation, differentiation, apoptosis and protein synthesis, and also plays an important role in lipid metabolism. Our results suggest that 0.05 mg/kg BPA exposure increases the p-mTOR/mTOR ratio at PND56, which is consistent with the well-known pattern of mTOR regulating fatty acid synthesis (Lin et al. 2019). In the 0.5 and 5 mg/kg groups, the total protein level of mTOR increased, while the phosphorylation level did not change significantly. We also found that the phosphorylation level of CRTC2 at PND21 and 56 was significantly increased in 0.5 and 5 mg/kg groups, suggesting that mTOR may phosphorylates CRTC2 and then regulates the expression of SREBP-1. Previous studies on LO2 cells in vitro have also shown that phosphorylation of CRTC2 activates nuclear SREBP-1 activity and subsequent adipogenesis (Hu et al. 2019), but the difference is that phosphorylation of mTOR is not well defined. Zhang et al. (Zhang et al. 2018) found that in adipose tissue, mTORC1 plays a corresponding regulatory role through phosphorylation of CRTC2. Other studies also reported that CRTC2 knockdown or overexpression did not affect the phosphorylation of mTOR (Li et al. 2019), which strongly supported our research results. However, the effect of BPA on mTOR/CRTC2 pathway has not been reported. Our data demonstrate that gestational BPA exposure may increase the liver fatty acid synthesis by regulating mTOR/CRTC2/SREBP-1 pathway in male offspring. In addition, in this study, the expression results of mTOR and CRTC2 at mRNA and protein levels are inconsistent, which may be due to the influence of post-transcriptional regulation.
In summary, the present study proved that gestational BPA exposure could affect liver lipid levels by disturbing lipid metabolism, including inhibition of fatty acid oxidation and promotion of fatty acid synthesis. We first proposed that the mTOR/CRTC2/SREBP-1 pathway may play an important role in the effects of prenatal BPA exposure on liver fatty acid synthesis in male offspring. In this process, as a downstream mediator of mTOR, CRTC2 may play a potential role in the regulation of SREBP-1 and thus promoting lipid synthesis, which provides a novel insight into the established correlations between early-life BPA exposure and lipid metabolism disorders. Furthermore, these data might partially indicate that prenatal BPA exposure caused lasting effects to basal gene expression of lipid metabolism in adult rat liver. This will strengthen the evidence for prevention of exposure to environmental chemicals during pregnancy.