Dexamethasone exposure during pregnancy triggers metabolic syndrome in offspring via epigenetic alteration of IGF1.

BACKGROUND: Previous research has reported that prenatal exposure to dexamethasone (PDE) results in organ dysplasia and increased disease susceptibility in offspring. This study aimed to investigate the epigenetic mechanism of metabolic syndrome induced by PDE in offspring. METHODS: Pregnant Wistar rats were administered dexamethasone, and their offspring's serum and liver tissues were analyzed. The hepatocyte differentiation model was established to unveil the molecular mechanism. Neonatal cord blood samples were collected to validate the phenomenon and mechanism. RESULTS: The findings demonstrated that PDE leads to insulin resistance and typical metabolic syndrome traits in adult offspring rats, which originated from fetal liver dysplasia. Additionally, PDE reduced serum corticosterone level and inhibited hepatic insulin-like growth factor 1 (IGF1) signaling in fetal rats. It further revealed that liver dysplasia and functional impairment induced by PDE persist after birth, driven by the continuous downregulation of serum corticosterone and hepatic IGF1 signaling. Both in vitro and in vivo experiments confirmed that low endogenous corticosterone reduces the histone 3 lysine 9 acetylation (H3K27ac) level of IGF1 and its expression by blocking glucocorticoid receptor α, special protein 1, and P300 into the nucleus, resulting in hepatocyte differentiation inhibition and liver dysplasia. Intriguingly, neonatal cord blood samples validated the link between reduced liver function in neonates induced by PDE and decreased serum cortisol and IGF1 levels. CONCLUSIONS: This study demonstrated that low endogenous glucocorticoid level under PDE lead to liver dysplasia by downregulating the H3K27ac level of IGF1 and its expression, ultimately contributing to metabolic syndrome in adult offspring.

Prenatal dexamethasone exposure induced pancreatic ß-cell dysfunction and glucose intolerance of male offspring rats: Role of the epigenetic repression of ACE2.

The prevalence of diabetes in children and adolescents has been rising gradually, which is relevant to adverse environment during development, especially prepartum. We aimed to explore the effects of prenatal dexamethasone exposure (PDE) on ß-cell function and glucose homeostasis in juvenile offspring rats. Pregnant Wistar rats were subcutaneously administered with dexamethasone [0.1, 0.2, 0.4mg/(kg.d)] from gestational day 9 to 20. PDE impaired glucose tolerance in the male offspring rather than the females. In male offspring, PDE impaired the development and function of ß-cells, accompanied with lower H3K9ac, H3K14ac and H3K27ac levels in the promoter region of angiotensin-converting enzyme 2 (ACE2) as well as suppressed ACE2 expression. Meanwhile, PDE increased expression of glucocorticoid receptor (GR) and histone deacetylase 3 (HDAC3) in fetal pancreas. Dexamethasone also inhibited ACE2 expression and insulin production in vitro. Recombinant expression of ACE2 restored insulin production inhibited by dexamethasone. In addition, dexamethasone activated GR and HDAC3, increased protein interaction of GR with HDAC3, and promoted the binding of GR-HDAC3 complex to ACE2 promoter region. Both RU486 and TSA abolished dexamethasone-induced decline of histone acetylation and ACE2 expression. In summary, suppression of ACE2 is involved in PDE induced ß-cell dysfunction and glucose intolerance in juvenile male offspring rats.

Identification and validation of reference genes for RT-qPCR analysis in fetal rat pancreas.

The choice of reference gene is crucial for quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) assay. To screen and determine the suitable reference genes in fetal rat pancreas, we selected eight candidate reference genes (Gapdh, Actb, Rn18 s, B2m, Rpl13a, Tbp, Ywhaz and Ubc), and evaluated the constancy of gene expression from fetal rat pancreases in non-pathological situation and prenatal dexamethasone exposure (PDE) model, using four algorithms: GeNorm, NormFinder, Bestkeeper and Comparative ΔCt method. In addition, the alteration of mRNA levels of pancreatic insulin was compared between control and PDE groups to validate the reliability of selected reference genes for data normalization of RT-qPCR. The comprehensive ranking of reference genes under physiological condition was as follow: Gapdh > Actb > Ywhaz > Ubc > Rn18s > Rpl13a > B2m > Tbp (female); Actb > Ywhaz > Gapdh > Ubc > B2m > Rpl13a > Rn18 s | Tbp (male). The top ranking reference genes were also stably expressed in PDE fetal pancreas. The best reference gene combinations are: Ywhaz+Actb for female and Ywhaz+Gapdh for male fetal rat pancreas, respectively. Compared with low ranking or single reference gene, the change trend of insulin mRNA normalized by the best reference gene combination between control and PDE groups was more significant and consistent with that of serum insulin level. In conclusion, our results provided the optimal combination of stable reference genes for RT-qPCR assay in pancreatic developmental toxicity study.

Panel of suitable reference genes and its gender differences of fetal rat liver under physiological conditions and exposure to dexamethasone during pregnancy.

The panel of suitable reference genes in the fetal liver have not been reported. In this study, five commonly used reference genes (GAPDH, ß-actin, Rn18 s, Rpl13a, and Rps29) were firstly selected as candidates. Bestkeeper, GeNorm, and NormFinder software were then used to screen out the panel of suitable reference genes of male and female fetal rat liver under physiological and prenatal dexamethasone exposure (PDE) conditions. Finally, we verified the reliability of the screened panel of reference genes by standardizing sterol regulatory element binding protein 1c (SREBP1c) expression with different reference genes. The results showed that GAPDH + Rn18 s and GAPDH + Rpl13a were respectively the panel of suitable reference genes in male and female rat fetal liver under the physiological model, while Rn18 s + Rps29 and GAPDH + Rn18 s were respectively under the PDE model. The results showed that different reference genes affected the statistical results of SREBP1c expression, and the screened panel of suitable reference genes under the PDE model had smaller intragroup differences, when compared with other reference genes under physiological and PDE models. In conclusion, we screened and determined that the panel of suitable reference genes were GAPDH + Rn18 s and Rn18 s + Rps29 in the male rat fetal liver under physiological and PDE models, while they were GAPDH + Rpl13a and GAPDH + Rn18 s in the females, and confirmed that the selection of the panel of suitable reference genes in the fetal liver had gender differences and pathological model specificity.

Prenatal dexamethasone exposure induces nonalcoholic fatty liver disease in male rat offspring via the miR-122/YY1/ACE2-MAS1 pathway.

Epidemiological studies have shown that nonalcoholic fatty liver disease (NAFLD) has an intrauterine developmental origin. We aimed to demonstrate that NAFLD is caused by prenatal dexamethasone exposure (PDE) in adult male rat offspring and to investigate the intrauterine programming mechanism. Liver samples were obtained on gestational day (GD) 21 and postnatal week (PW) 28. The effects and epigenetic mechanism of dexamethasone were studied with bone marrow mesenchymal stem cells (BMSCs) hepatoid differentiated cells and other cell models. In the PDE group, lipid accumulation increased, triglyceride synthesis-related gene expression increased, and oxidation-related gene expression decreased in livers of adult male rat offspring. In utero, hepatic triglyceride synthesis increased and oxidative function decreased in PDE fetal male rats. Moreover, low hepatic miR-122 expression, high Yin Yang-1 (YY1) expression and angiotensin-converting enzyme 2 (ACE2)-Mas receptor (MAS1) signaling pathway inhibition were observed before and after birth. At the cellular level, dexamethasone (100-2500 nM) elevated the intracellular triglyceride content, increased triglyceride synthesis-related gene expression and decreased oxidation-related gene expression. Dexamethasone treatment also decreased miR-122 expression, increased YY1 expression and inhibited the ACE2-MAS1 signaling pathway. Interference or overexpression of glucocorticoid receptor (GR), miR-122, YY1 and ACE2 could reverse the changes in downstream gene expression. In summary, PDE could induce NAFLD in adult male rat offspring. The programming mechanism included inhibition of miR-122 expression after GR activation, and dexamethasone increased hepatocyte YY1 expression; these effects resulted in ACE2-MAS1 signaling pathway inhibition, which led to increased hepatic triglyceride synthesis and decreased oxidative function. The increased triglyceride synthesis and decreased oxidative function of hepatocytes caused by low miR-122 expression due to dexamethasone could continue postnatally, eventually leading to NAFLD in adult rat offspring.