Effects of Glucose and Insulin on GLAST and xc– Transporter Function in Primary Mouse Müller Cells

Abstract

Abstract: : Purpose: Glutamate is cleared from the extracellular milieu by a family of excitatory amino acid transporters (EAATs). In retina, Müller cells use GLAST (EAAT1) for this purpose. It has been reported that GLAST activity is decreased in Müller cells of diabetic rats and that antioxidants restore GLAST activity (Li and Puro, 2002). The contributions of glucose and insulin in the regulation of GLAST activity have not been assessed. Since hyperglycemia and reduced insulin are characteristic of diabetes, we hypothesized that glucose and insulin would play a role in regulating GLAST function. Additionally, we predicted that the glutamate–cystine exchanger, x_c^–, which supplies cystine for the production of the antioxidant, glutathione, would also be affected by these factors. Methods: Primary Müller cells were isolated from retinas of 9–10 day old C57BL/6 mice using the method of Hicks and Courtois. Culture purity was verified immunocytochemically using antibodies against GLAST, CRALBP, glutamine synthetase, and GFAP. Cells were maintained in DMEM (10% FBS) 5.5 mM glucose. For uptake assays 50,000 cells per well were seeded on 24 well plates. After 24 hours, media was replaced with DMEM (0.5% FBS). Uptake of radiolabeled glutamate was measured in cells treated for 15, 45, 90, 210 and 1200 min with 5 mM glucose or 35 mM glucose in the presence or absence of 20 ng/ml insulin. Results: There was a 20% increase in GLAST activity in cells treated for 15 min in 35 mM glucose in the presence or absence of 20 ng/ml insulin. Within 90 min, however, GLAST activity returned to control levels and remained similar to control values throughout the other time points studied. Activity of the x_c^– transporter increased transiently when cells were treated for 90 and 210 min with 35 mM glucose. Conclusions: These results suggest that short term rises in glucose levels, but not insulin levels, may actually stimulate GLAST and x_c^– activity in normal Müller cells. These factors alone do not account for the decreased GLAST activity reported in Müller cells of diabetic rats. Future studies will examine sensitivity to hyperglycemia and altered insulin levels in the presence of oxidative stress.

Keywords: Muller cells • diabetic retinopathy • excitatory neurotransmitters