The incidence of diabetes has increased in recent decades and by 2030, 366 million people worldwide will have the disease a significant proportion of these will be women of reproductive age. Diabetes is known to reduce fecundity and increase the likelihood of early pregnancy loss we propose that the high glucose levels seen in diabetes influence maternal fertility by impairing endometrial receptivity/function as a result of increased flux in the hexosamine biosynthetic pathway (HBP). The HBP integrates cellular nutrient, including glucose, metabolism to produce UDP-N-acetylglucosamine, which is used to O-GlcNAcylate proteins and regulate their function. Here we show that it is possible to manipulate the HBP in the Ishikawa endometrial epithelial cell line by increasing flux through the pathway using high glucose (25 mM) or the HBP intermediate, glucosamine (5 mM), leading to an increase in protein O-GlcNAcylation. In addition, we enhanced Ishikawa cell protein O-GlcNAcylation by inhibiting the enzyme, O-GlcNAcase (OGA), responsible for de-GlcNAcylation pharmacologically, or by siRNA knockdown (82% decrease in protein expression). However, glucosamine, but not high glucose or OGA inhibition, elicited an ER stress response, detected as an initial increase in expression of the transcription factor, XBP1S (5.4-fold), followed by increased GRP78/BiP expression (2.3-fold). The functional consequences of manipulating the HBP were investigated by assessing the attachment of blastocyst-sized (40100 μm) spheroids of human trophoblast (BeWo cells) to Ishikawa cells (n=8). Under control conditions, 75% of spheroids were attached after 30 minutes however, the ability of spheroids to attach to Ishikawa cells was not affected by any of the strategies used to increase endometrial cell protein O-GlcNAcylation. Ongoing experiments are testing the effect of altering HBP flux on embryo implantation competence. These studies will help understand the mechanisms underlying impaired fertility in women with diabetes and will contribute to developing interventions to improve endometrial receptivity and pregnancy success.
11 Jul 2016 - 11 Jul 2016