Hormone-regulated and glucose-sensitive transport of dehydroascorbic acid in immature rat granulosa cells.

Abstract

Ascorbic acid is concentrated in granulosa cells of the follicle, and ascorbate deficiency causes follicular atresia. Dehydroascorbic acid (DHAA), the oxidized form of ascorbic acid, serves as an important source for the recycling of ascorbate. As we previously demonstrated endocrine up-regulation of ascorbic acid transport by granulosa cells, we investigated DHAA as an alternate source of ascorbate in the follicle. Granulosa cells were cultured for 24 h, and DHAA uptake was initiated by the addition of 14C-labeled ascorbic acid (300 microM) in the presence of ascorbic acid oxidase (2 U/ml), which catalyzes DHAA production. Almost 90% of accumulated DHAA was present as ascorbic acid within 2 h. Preculture of cells for 24 h with FSH (50 ng/ml) and IGF-I (30 ng/ml) significantly stimulated DHAA uptake compared with the control (158 +/- 16 vs. 43 +/- 8 pmol/10(6) cells, respectively). DHAA uptake by granulosa cells was inhibited by D-glucose (ID50, approximately 2.5 mM) and by the glucose transport inhibitors phloretin (200 microM) and cytochalasin B (10 microM), which reduced uptake to 13 +/- 2% and 8 +/- 3% of the control, respectively. Northern and Western analysis of GLUT1 in granulosa cells following 24 h coincubation with FSH and IGF-I revealed up-regulation of GLUT1 at both the messenger RNA and protein levels (1.6- and 1.3-fold of control, respectively), suggesting that the stimulatory effects of FSH and IGF-I on DHAA transport are mediated by the induction of GLUT1. GLUT4 protein was not detectable by Western analysis. Endocrine-regulated DHAA transport may represent an important mechanism for maintaining adequate antioxidant tone within the developing follicle.