Abstract 134: Activation of the Mineralocorticoid Receptor by O-glycosylation Through Hyperglycemia

Abstract

Introduction: Hypertension and diabetes are independent risk factors for cardiovascular disease, however why they frequently occur together is not clear. Inappropriate mineralocorticoid receptor (MR) activation is associated with diabetes and the metabolic syndrome, as well as hypertension and abnormal cardiovascular remodeling. MR antagonists are effective in reducing hypertension and delaying the onset of renal and cardiovascular complications in diabetes despite circulating aldosterone levels that are within normal limits. Glucose concentrations increase O-glycosylation of many proteins, thus alter their function. Hence, we hypothesized that increased O-GlcNac modification of the MR by high glucose enhances MR activation. Methods: MR transcriptional activity was studied in a mouse cortical collecting duct (M1) cell line stably transfected with a cDNA construct including the MR and one with a hormone-response element driving a Gaussia luciferase reporter gene. The cells were incubated for 48 h with low (5mM) or high (25mM) glucose media with and without Thiamet-G (TMG), an O-GlcNAcase inhibitor to inhibit deglycosylation, and 6-diazo-5-oxonorleucine (DON), a glucosamine-fructose-6-phosphate amidotransferase inhibitor (GFAT) to reduce O-GlcNAc levels. Additionally, MR and GR antagonists were used to identify receptor specificity under low and high glucose conditions. O-GlcNac-modified MR was co-immunoprecipitated with an MR antibody and detected with an O-GlcNAc antibody. Results: 1. Co-immunoprecipitation assays showed that high glucose and TMG increased O-GlcNac-MR by 3-fold. 2. Compared to low glucose, treatment with high glucose and with TMG increased the transcriptional activity of MR by 300%. 3. DON decreased MR-reporter activity by 75%. 4. High glucose alone had no significant basal effect but significantly increased MR activation by aldosterone. 5. MR reporter activity was increased similarly by aldosterone and corticosterone. Conclusion: High glucose increased glycosylation of the MR, augmenting its transcriptional activity. Enhancement of MR activation by hyperglycemia may explain how MRs play a significant role in the cardiorenal pathology in Diabetes.