253. Toxic effects of hyperglycaemia arise from induced O-linked glycosylation in early mouse embryos

Abstract

Glucose flux through the hexosamine biosynthetic pathway (HBP) which is essential for preimplantation development (1) produces uridine 5′-diphospho-N-acetylglucosamine, a donor substrate for multiple glycosylation reactions including O-linked glycosylation. This novel signalling arm of the HBP, known as the hexosamine signalling pathway (HSP) operates via reversible addition of an O-linked β-N-acetylglucosamine (O-GlcNAc) unit to serine and threonine residues of proteins including transcription factors, cytoskeletal components, metabolic enzymes and cellular signalling components. O-linked glycosylation is functionally reciprocal to phosphorylation at the same residues, altering the activity and/or stability of targeted proteins, thus providing a mechanism for modulating cellular physiology in response to glucose availability. The enzymes regulating this O-GlcNAcylation are the β-linked-O-GlcNAc transferase (OGT) and an O-GlcNAc-selective β-N-acetylglucosaminidase (O-GlcNAcase). We hypothesised that the toxicity of hyperglycemia on early embryos arises from increased flux through HBP and increased O-GlcNAcylation of key proteins. Mouse zygotes (18 h post hCG) were cultured under conditions of modified flux through the HSP including hypoglycemia, hyperglycemia or supplemented with glucosamine which feeds exclusively into the HBP to increase downstream O-GlcNAcylation. BADGP was used to inhibit OGT and O-GlcNAcylation. Blastocyst formation, cell proliferation and apoptosis were assessed. Treatments that perturb levels of intracellular protein O-GlcNAcylation inhibited embryo development. Whilst some flux through HBP is required to activate embryonic differentiation (1), excess flux arising from a hyperglycemic environment or glucosamine supplementation reduced cell proliferation and blastocyst formation, confirming the criticality of this novel post-translational signalling pathway. Inhibition of OGT using 2 mM BADGP blocked the negative impact of hyperglycemia on blastocyst formation, cell number and apoptosis supporting our hypothesis that O-GlcNAcylation is a key mechanism used by the embryo to sense and respond to perturbations of glucose in its environment.

(1) Pantaleon M, Scott J and Kaye PL (2008) Biol Reprod, 78(4):595–600