Effects of TCDD on embryonic stem cells's (ESCS) differentiation towards pancreatic lineage and pancreatic beta cell function

Abstract

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an endocrine disrupting chemical postulated to exert diabetogenic effects. Most laboratory and epidemiological studies have demonstrated the association of persistent TCDD exposure to type 2 diabetes (T2D) susceptibility. Researchers commonly employ super high doses of TCDD treatments on adult cells/tissues to illustrate its diabetogenic effects which does not reflect physiological phenotypes. Data linking the epigenetic effects of TCDD exposure on embryonic cells / tissue to T2D susceptibility risks is very limited. It is therefore hypothesized that TCDD exposure during embryogenesis could influence development and epigenetic landscape of pancreatic islets, leading to T2D susceptibility later in life. The main objectives of this research were (i) to differentiate embryonic stem cells (ESCs) towards pancreatic lineage cells, (ii) to study the effects of TCDD treatment on human and mouse ESCs (hESCs and mESCs), (iii) to study the effects of TCDD on global DNA methylation pattern of ESCs and (iv) to conduct functional study on some TCDD induced differentially methylated target genes in functional beta cells. DMSO and physiological doses of TCDD (10pM, 100pM) treated hESCs and mESCs were differentiated towards pancreatic lineage. The results showed that TCDD treatment impaired the ESCs to pancreatic lineage differentiation potentials by suppressing some marker gene expressions in the derived definitive endoderm (DE) and pancreatic progenitors (PP) stages. The extracted DNA of the DMSO and TCDD treated hESCs were subjected to reduced representation bisulfite sequencing (RRBS) to generate methylome data and the results showed that TCDD treatments induced differential DNA methylation in the hESCs state. Bioinformatic analysis using Database for Annotation, Visualization and Integrated Discovery (DAVID) and Gene Set Enrichment Analysis (GSEA) of the methylome data revealed that biological pathways and processes related to pancreatic lineage cell development and functions (e.g. endoderm cell differentiation, insulin secretions, insulin signaling pathway) were enriched in the physiological doses of TCDD induced hypermethylated genes, while different pathways were induced by supraphysiological dose of TCDD. Among the pancreatic lineage enriched genes, PRKAG1, CAPN10, MAFA and HNF-1B were validated by bisulfite sequencing. The results showed that some TCDD induced hypomethylated genes were also reported to be hypomethylated in the islets of T2D patients. PRKAG1, one of the gamma regulatory subunits of AMP-activated protein kinase (AMPK) that was related to insulin resistance and metabolic syndrome-related diseases, was selected for downstream study. It was found that TCDD induced PRKAG1 hypermethylation at the hESCs state was maintained after differentiated into PP stage. In the functional studies, Prkag1 knockdown in INS-1E cells increased GSIS but decreased AMPKα and AMPKβ2 protein amounts. Prkag1 knockdown also induced the protein levels of mTORC1 and its downstream target, 4EBP1 in the transfected cells. In conclusion, this research is the first to demonstrate that the TCDD induced hypermethylation of the PRKAG1 was maintained during differentiation of hESCs to PP. TCDD induced Prkag1 hypermethylation might lead to hyperactivation of mTOR1 pathway and increase in GSIS. The current research data established that TCDD might exert diabetogenic effects during early prenatal development, alter processes of pancreatogenesis and increase the risk of T2D susceptibility later in life.