Role of β-Adrenergic Receptor Regulation of TNF-α and Insulin Signaling in Retinal Muller Cells

PURPOSE. The goal of this study was to determine the relationship of TNF-alpha and the downregulation of insulin receptor signaling in retinal Muller cells cultured under hyperglycemic conditions and the role of beta-adrenergic receptors in regulating these responses. METHODS. Retinal Muller 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 mu M salmeterol followed by Western blot analysis or ELISA. For TNF-alpha inhibitory studies, the cells were treated with 5 ng/mL of TNF-alpha for 30 minutes or by a 30-minute pretreatment with TNF-alpha followed by salmeterol for 6 hours. In the TNF-alpha short hairpin (sh) RNA experiments, the cells were cultured until 90% confluent, followed by transfection with TNF-alpha shRNA for 18 hours. RESULTS. TNF-alpha-only treatments of Muller cells resulted in significant decreases of tyrosine phosphorylation of the insulin receptor and Akt in high-glucose conditions. Salmeterol (10 mu M), a beta-2-adrenergic receptor agonist, significantly increased phosphorylation of both insulin receptor and Akt. TNF-alpha shRNA significantly decreased phosphorylation of IRS-1(Ser307), which was further decreased after salmeterol + TNF-alpha shRNA. Both TNF-alpha shRNA and salmeterol significantly reduced death of the retinal Muller cells. CONCLUSIONS. These studies demonstrate that beta-adrenergic receptor agonists in vitro can restore the loss of insulin receptor activity noted in diabetes. By decreasing the levels of TNF-alpha and decreasing the phosphorylation of IRS-1(Ser307) while increasing tyrosine phosphorylation of insulin receptor, these results suggest a possible mechanism by which restoration of beta-adrenergic receptor signaling may protect the retina against diabetes-induced damage. (Invest Ophthalmol Vis Sci. 2011; 52:9527-9533) DOI: 10.1167/iovs.11-8631