Epigenetic Modulation of GPER Expression in Gastric and Colonic Smooth Muscle of Male and Female Non-obese Diabetic (NOD) Mice: Insights into H3K4me3 and H3K27ac Modifications
Introduction: Type 1 diabetes (T1D) significantly affects gastrointestinal (GI) motility and contributes to complications, such as gastroparesis, constipation, and fecal incontinence. Notably, these complications have a higher prevalence in female patients, underscoring a critical knowledge gap in the understanding of the underlying mechanisms. Given the emerging role of the G-protein-coupled estrogen receptor (GPER) in GI motility regulation, our study investigated the potential contribution of diabetes-induced changes in GPER expression to pathophysiology. Hypothesis: We hypothesized that diabetes-induced perturbations in GPER expression contribute to pathophysiological mechanisms affecting GI motility in T1D, with notable sex-dependent variations. Methods: we assessed GPER mRNA and protein expression levels using quantitative reverse-transcription PCR and western blot analyses, and quantified the changes in nuclear DNA methyltransferases and histone modifications (H3K4Me3, H3Ac, and H3K27Ac) were quantified using ELISA kits. Targeted bisulfite and chromatin immunoprecipitation sequencing were used to evaluate DNA methylation and histone modifications around the GPER promoter by chromatin immunoprecipitation assays in gastric and colonic smooth muscle tissues of male and female control (CTR) and non-obese diabetic (NOD) mice. Results: Our results revealed significant downregulation of GPER in NOD mice, with marked sex-dependent variations. Mechanistically, this downregulation was associated with reduced H3K4me3, H3ac, and H3K27ac levels in NOD male gastric smooth muscle. In contrast, downregulation in female gastric smooth muscle cells has been linked to decreased H3K4me3 and H3ac levels. Male NOD colonic smooth muscle exhibited elevated H3ac and H3K27ac levels, whereas female NOD colonic smooth muscle demonstrated diminished enrichment of H3ac and H3K27ac at the GPER promoter. Additionally, DNA methylation is elevated in NOD male colonic smooth muscle compared to NOD females. Conclusion: Our findings elucidate sex-specific epigenetic mechanisms contributing to T1D-mediated suppression of GPER expression in the gastrointestinal tract. These insights not only advance our understanding of T1D complications but also suggest promising avenues for targeted therapeutic interventions. Overall design: To investigate the GPER expression in male and female gastric and colonic smooth muscle of Control and NOD mice, we performed DNA-methylation measurement at the GPER promoter region by targeted bisulfite sequencing assay. This was performed in the colonic and gastric tissue of 5 male Control, 5 male NOD, 5 female Control and 5 NOD female mice. The DNA methylation data was analyzed by comparative analysis, using ANOVA.