Regina Schlichting

Type 2 Diabetes (T2D) is associated with both pancreatic beta-cell dysfunction and insulin resistance. Insulin transcription, beta-cell identity, and pancreas development are all regulated by the transcription factor pancreatic and duodenal homeobox 1 (Pdx1). Pdx1 is post-translationally modified at two residues by the addition of O-linked beta-N-acetylglucosamine (O-GlcNAc), by nutrient sensitive enzyme O-GlcNAc transferase (OGT). O-GlcNAcylation of Pdx1 is correlated with its nuclear localization, increased DNA binding activity, and increased insulin secretion. We first assessed the stability of Pdx1 by determining its half-life under OGT inhibited conditions by treating Min6 cells, a mouse beta-cell line, with Osmi-1, a drug that inhibits OGT. We observed that Pdx1 is less stable when OGT is inhibited. The impact of O-GlcNAcylation on localization of Pdx1 was assessed in mice with beta-cell specific deletion of OGT. We observed a weaker signal of Pdx1 and potentially less nuclear Pdx1 compared to the wild type mice. Together, these data suggest O-GlcNAcylation impacts both the stability and localization of Pdx1. Then, we identified putative O-GlcNAcylation residues on Pdx1 to be threonine 51 (T51), serine (S273), and serine (S274). We mutated these sites to alanine using site directed mutagenesis in a plasmid containing GFP-tagged mouse Pdx1, such that it couldn’t be O-GlcNAc modified. We confirmed the plasmid was functional by transfecting the plasmid in 293T cells, and observed the transfection efficiency using light microscopy. After immunoprecipitation, we observed a potential decrease of Pdx1 levels in cells transfected with the T51A/S274A mutant, suggesting O-GlcNAcylation may influence Pdx1 stability.