There are various chemical modifications of RNA, including m6A, m1A, m5C, and other methylation modifications, among which m6A modification is the most prevalent, abundant, and reversible methylation modification of both mammalian mRNA and non-coding RNA(Pinello et al., 2018b, Cai et al., 2018, Roundtree et al., 2017). m6A modification plays an important role in many biological processes, such as regulation of mRNA stability(Huang et al., 2018), pre-mRNA splicing(Bartosovic et al., 2017),mRNA decay and translation(Shi et al., 2017), and DNA damage repair(Xiang et al., 2017). A series of studies have shown that m6A RNA modification is associated with various human diseases, including cancer, cardiovascular diseases(Paramasivam et al., 2020, Dorn et al., 2019), diabetes(De Jesus et al., 2019), preeclampsia(Gu et al., 2021b), and recurrent abortion(Li et al., 2019). In this study, our data showed that m6A modification was increased in liver tissues of offspring born out of GDM, suggesting that m6A modification might develop insulin resistance and metabolic syndrome of the fetus.
Over the past decade, Lepr/+ mice have been used as a model of GDM because of accordance with the phenotype of GDM during pregnancy, such as reduced glucose tolerance, insulin tolerance, and elevated weight gain. However, some researches using this model have found that this phenotype cannot be simulated(Talton et al., 2016, Plows et al., 2017). The literature review found that mice fed with HFD before pregnancy showed phenotypes such as hyperglycemia and insulin resistance at late-stage of pregnancy. Thus, in our study, mice fed with HFD were used to establish the GDM model. Significant differences of glucose intolerance and insulin sensitivity were exhibited at the middle and late stage of pregnancy, suggesting the successful establishment of HFD-induced GDM model.
Interestingly, the weight of fetus from GDM group was slightly higher than the control group, nonetheless, the weight of the offspring in both groups exhibited indifferent as the week-age increased, suggesting that changes in the intrauterine microenvironment may contribute to neonatal macrosomia, but did not appear to be decisive for fetal growth and development. The GDM offspring mice exhibited poor glucose intolerance and insulin sensitivity, combined with plasma insulin concentration and HOMA-index, intrauterine hyperglycemia probably has a long-term impact on the offspring's blood glucose level and metabolism, resulting in susceptibility to develop insulin resistance.
RBM15 belongs to the SPEN(Split-end) protein family involved in cell fate decision(Hiriart et al., 2005). It is mainly located in the nucleus, which might interact with spliceosome components. RBM15 was initially identified as a leukemic ectopic which participates in pediatric acute megakaryocytic leukemia(Xiao et al., 2012, Ayllon et al., 2017). Notably, RBM15 may play different roles in different diseases. A previous study has shown that RBM15 regulates the alternative splicing of Mpl-TR, which may impair the hematopoietic stem cells engraftment(Xiao et al., 2015). Another study has shown that RBM15 mediated the degradation of BAF mRNA via the mechanism of mRNA methylation in the developing cortex(Xie et al., 2019). Moreover, RBM15 has been reported to promote the occurrence and development of laryngeal squamous cell carcinoma by maintaining the stability of TMBIM6(Wang et al., 2021).In this study, our data revealed that RBM15 might contribute to the development of metabolic syndrome based on insulin resistance through the m6A-mediated regulation of CLDN4 in GDM offspring.
It is worth noting that the downstream of RBM15 was vastly studied over the years, but its upstream roles remain poorly understood. A recent study showed that AS-RBM1, AS and lncRNA, can regulate the differentiation of megakaryocytes by regulating the expression level of RBM15(Tran et al., 2016). Similarly, microribonucleic acid (miRNA) can inhibit the activity of several m6A methylation enzymes (Cai et al., 2018b); thus, whether miRNA affects the expression of RBM15 in this study is still worthy of further exploration.
Most studies have shown that m6A modification plays a role in metabolism, obesity (Sun and Zhang, 2021), type Ⅱ diabetes (Yang et al., 2019), and cardiovascular disease (Zhong et al., 2020) by regulating glucose/lipid metabolism and inflammatory/immune response. Furthermore, according to Wang et al., m6A modification plays an important role in driving functional maturation of neonatal islet beta cells (Wang et al., 2020). According to our results, although there was no abnormality of the fetus at infant stage, the metabolic organ of GDM offspring mice at adult stage lost its normal structure with clearly visible vacuoles and exhibited obvious glycose deposition, indicating that intrauterine hyperglycemia might have an effect on the metabolic level of the progeny at adult stages. Despite the phenotype of metabolic change, parts of metabolites still showed difference including saturated fatty acids, which implied that the ability of glucolipid metabolism in offspring mice might be defective to some extent. Combined with above results, intrauterine hyperglycemia might affect fetal epigenomic modification; however, due to immature fetal body development, the regulated genes may not temporarily play a role, or short-term changes will not have a significant impact on fetal metabolism. As the offspring grows, epigenetic genes may become more functional, leading to metabolic disorders in adolescence and adulthood. Further exploring the specific mechanism of how m6A regulates the metabolic changes in GDM offspring mice is the future direction of our research team.