Regulation of Glutamate Metabolism by Hydrocortisone and Branched Chain Keto Acids in Cultured Rat Retinal Müller Cells (TR–MUL)

Abstract

Purpose: : Excess glutamate in the retina may lead to diabetic retinopathy. We previously showed that de novo synthesis of glutamate is unchanged but the oxidative disposal of glutamate is reduced in diabetic retinas. By studying glutamate metabolism in the differentiated Müller cell culture we avoid the confounding cellular heterogeneity of the intact retina. Purpose of this study was first to investigate glutamate metabolism specific to Müller cells and then to investigate how hydrocortisone and /or branch chain amino acids influence glutamate metabolism in the Müller cells. These cells express glial cell specific marker proteins including glutamine synthetase (GS) and mitochondrial branch chain aminotransferase (BCATm).

Methods: : TR–MUL cells were grown in DMEM medium containing 5% serum. Serum was removed 2 hours before ± addition of hydrocortisone (1µM) for 12hours prior to experiments. Cells were incubated in a physiological Krebs bicarbonate buffer containing 3.5 mM glucose, 0.2 mM pyruvate, 0.03 mM NH₃ and 100 µM [U–¹⁴C]glutamate ± 0.6 mM branch chain keto acid (BCKA). Glutamate oxidation was measured as release of ¹⁴CO₂ and as production of ¹⁴C–lactate. ¹⁴C–labelled metabolites such as glutamine, glutamate, aspartate and lactate in the extracts were separated by anion exchange chromatography. BCATm and GS were determined by Western blotting.

Results: : Hydrocortisone increased GS protein but not BCATm protein expression. Active transport of glutamate was determined by the high glutamate concentration gradient across the cell membrane. Glutamine, aspartate, lactate and CO₂ were measured. Hydrocortisone increased the production of glutamine by almost 50% but did not alter the rate of lactate or aspartate production. Addition of branched chain keto acids (BCKA) increased the formation of lactate from glutamate (0.12± 0.03 to 0.22± 0.08 nmols/mg) and oxidation of glutamate to CO₂ from 0.58±0.05 to 0.72± 0.07 nmoles/mg.

Conclusions: : BCKA’s stimulated glutamate transamination and oxidation to CO₂ and lactate. Therefore a deficit in BCKA in diabetic retina could induce glutamate excitotoxicity. Studies have indicated that glutamine synthetase and BCATm promote opposite pathways of glutamate metabolism. TR–MUL cells will be useful in studies where expression of these enzymes can be manipulated.

Keywords: Muller cells • metabolism • retinal glia