Abstract
The post-translational protein modification O-linked β-N-acetylglucosamine (O-GlcNAc) is a proposed nutrient sensor that has been shown to regulate multiple biological pathways. This dynamic and inducible enzymatic modification to intracellular proteins utilizes the end product of the nutrient sensing hexosamine biosynthetic pathway, UDP-GlcNAc, as its substrate donor. Type II diabetic patients have elevated O-GlcNAc-modified proteins within pancreatic beta cells due to chronic hyperglycemia-induced glucose overload, but a molecular role for O-GlcNAc within beta cells remains unclear. Using directed pharmacological approaches in the mouse insulinoma-6 (Min6) cell line, we demonstrate that elevating nuclear O-GlcNAc increases intracellular insulin levels and preserves glucose-stimulated insulin secretion during chronic hyperglycemia. The molecular mechanism for these observed changes appears to be, at least in part, due to elevated O-GlcNAc-dependent increases in Ins1 and Ins2 mRNA levels via elevations in histone H3 transcriptional activation marks. Furthermore, RNA deep sequencing reveals that this mechanism of altered gene transcription is restricted and that the majority of genes regulated by elevated O-GlcNAc levels are similarly regulated by a shift from euglycemic to hyperglycemic conditions. These findings implicate the O-GlcNAc modification as a potential mechanism for hyperglycemic-regulated gene expression in the beta cell.
Highlights
Type II diabetes-related complications continue to be a leading cause of death in the United States [1]
We show that O-GlcNAc increases insulin 1 (Ins1) and insulin 2 (Ins2) mRNA levels in mouse insulinoma 6 (Min6) cells above physiological conditions in a glucose-independent manner
O-GlcNAc appears to modulate the expression of an abundance of genes in a parallel manner to high glucose. Together these results suggest that the O-GlcNAc modification serves as a glucose sensor in Min6 cells, regulating glucosemediated gene expression in the pancreatic  cell
Summary
O-GlcNAc, O-linked -N-acetylglucosamine; Min, mouse insulinoma 6; HBP, hexosamine biosynthetic pathway; OGA, O-GlcNAcase; PUG, O-(2-acetamido-2-deoxy-D-glucopyranosylidine)amino-N-phenylcarbamate; GNS, GlcNAcstatin; LG, low glucose; HG, high glucose; qPCR, quantitative PCR; ANOVA, analysis of variance; H3K4me, histone H3 trimethylated lysine 4; H3K9,14Ac, histone H3 bi-acetylated lysine 9 and 14; GalNT7, UDP-N-acetyl-␣-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 7; Fbxo, F-box protein 43. We show that O-GlcNAc increases insulin 1 (Ins1) and insulin 2 (Ins2) mRNA levels in Min cells above physiological conditions in a glucose-independent manner. O-GlcNAc appears to modulate the expression of an abundance of genes in a parallel manner to high glucose. Together these results suggest that the O-GlcNAc modification serves as a glucose sensor in Min cells, regulating glucosemediated gene expression in the pancreatic  cell
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