Influence of O-GlcNAcylation on KGN cell function

O-GlcNAcylation is a unique form of post-translational glycosylation that affects a variety of cytoplasmic and nuclear proteins of cells. Aberrant O-GlcNAcylation is characteristic of many cancers, and impacts cell proliferation, tumorigenicity and metabolism. O-GlcNAcylation occurs in granulosa cells of ovarian follicles, its expression differs between small (3-5 mm) and large (>8.5 mm) antral follicles, and its manipulation in vitro alters granulosa cell proliferation and metabolism. Here, the aim was to assess whether O-GlcNAcylation similarly occurs in cells from a type of granulosa cell tumor, specifically KGN cells, knowing these cells share functional features of granulosa cells of mature, preovulatory follicles (e.g., FSH-responsiveness and estradiol production). The immortal KGN cell line was utilized to conduct cell culture experiments for the detection and manipulation of O-GlcNAcylation. The cells were grown to confluency in serum containing medium and then sub-cultured in serum-free conditions for immunodetection of O-GlcNAcylation ( n = 8 expts.), for cell proliferation ( n = 3 expts) and for metabolism assays ( n = 12 expts.). The KGN cells were also treated without or with small molecule inhibitors to directly enhance or impair O-GlcNAcylation. Immunoblotting confirmed O-GlcNAc expression in KGN cells, as well as the efficacy of Thiamet-G and OSMI-1 to augment ( P < 0.05) and inhibit O-GlcNAcylation (P < 0.05), respectively. Only the inhibition of O-GlcNAcylation compromised KGN cell proliferation ( P < 0.05), resulting in a 25 % reduction in proliferation compared to control conditions over a 72 h culture period. Seahorse XFe96 analysis measured effects of O-GlcNAcylation on cellular respiration in the KGN cells. Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) provided indices of glycolysis and oxidative phosphorylation, respectively. During a glycolysis stress test, high glucose increased ECAR and decreased OCR ( P < 0.05); oligomycin did not further affect ECAR ( P > 0.05), but impaired OCR ( P < 0.05); and 2-deoxy- d -glucose decreased ECAR (P < 0.05) without affecting OCR ( P > 0.05). Comparatively, a mitochondrial stress test revealed oligomycin increased ECAR ( P < 0.05) with a compensatory decrease in OCR (P < 0.05); FCCP increased both ECAR and OCR ( P < 0.05); and rotenone + antimycin A decreased both ECAR and OCR (P < 0.05). Manipulation of O-GlcNAcylation in the KGN cells had no effect on ECAR ( P > 0.05), but inhibited OCR ( P < 0.05). Collectively, the results indicate O-GlcNAcylation occurs in KGN cells, its inhibition impairs cell proliferation, and while KGN cells rely upon both glycolysis and oxidative phosphorylation for cellular respiration, manipulation of O-GlcNAcylation acutely perturbs only oxidative phosphorylation, an effect observed previously with granulosa cells of large antral follicles. • O-GlcNAcylation occurs in KGN cells and its inhibition impairs cell proliferation. • KGN cells rely on both glycolysis and oxidative phosphorylation for cellular respiration. • Manipulation of O-GlcNAcylation perturbs only oxidative phosphorylation. • Effects of O-GlcNAcylation on KGN cells are similar to those of granulosa cells of large antral follicles.