Role of ?2-Adrenergic Receptor Regulation of TNF-?± and Insulin Signaling in Retinal M??ller Cells

摘要

Purpose.: The goal of this study was to determine the relationship of TNF-?± and the downregulation of insulin receptor signaling in retinal M??ller cells cultured under hyperglycemic conditions and the role of ?2-adrenergic receptors in regulating these responses. Methods.: Retinal M??ller cells were cultured in normal (5 mM) or high (25 mM) glucose until 80% confluent and then were reduced to 2% serum for 18 to 24 hours. The cells were then treated with 10 ??M salmeterol followed by Western blot analysis or ELISA. For TNF-?± inhibitory studies, the cells were treated with 5 ng/mL of TNF-?± for 30 minutes or by a 30-minute pretreatment with TNF-?± followed by salmeterol for 6 hours. In the TNF-?± short hairpin (sh)RNA experiments, the cells were cultured until 90% confluent, followed by transfection with TNF-?± shRNA for 18 hours. Results.: TNF-?±-only treatments of M??ller cells resulted in significant decreases of tyrosine phosphorylation of the insulin receptor and Akt in high-glucose conditions. Salmeterol (10 ??M), a ?2-2-adrenergic receptor agonist, significantly increased phosphorylation of both insulin receptor and Akt. TNF-?± shRNA significantly decreased phosphorylation of IRS-1Ser307, which was further decreased after salmeterol+TNF-?± shRNA. Both TNF-?± shRNA and salmeterol significantly reduced death of the retinal M??ller cells. Conclusions.: These studies demonstrate that ?2-adrenergic receptor agonists in vitro can restore the loss of insulin receptor activity noted in diabetes. By decreasing the levels of TNF-?± and decreasing the phosphorylation of IRS-1Ser307 while increasing tyrosine phosphorylation of insulin receptor, these results suggest a possible mechanism by which restoration of ?2-adrenergic receptor signaling may protect the retina against diabetes-induced damage.