Silencing of insulin receptor substrate–1 increases cell death in retinal Müller cells

PURPOSE To determine whether β-adrenergic receptors require insulin receptor substrate (IRS)-1 activity to regulate apoptosis in retinal Müller cells. METHODS Müller cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) medium grown in normal (5 mm) or high glucose (25 mM) conditions. The medium was supplemented with 10% fetal bovine serum and antibiotics. Cells were allowed to reach 80%-90% confluence. After becoming appropriately confluent, cells were placed in medium with reduced serum (2%) for 18-24 h to eliminate any effects of fetal bovine serum. Cells were then transfected with 10 ug of IRS-1 small hairpin RNA (shRNA). Forty-eight hours following transfection, cells were lysed and harvested for protein analysis using western blotting. In additional experiments, some cells were treated with 10 uM salmeterol for 24 h following transfection with IRS-1 shRNA. To determine whether IRS-1 directly regulates apoptotic events in the insulin-signaling pathway in retinal Müller cells, a cell death assay kit was used. In tumor necrosis factor (TNF)α inhibitory studies, cells were treated with 5 ng/ml of TNFα alone for 30 min or 30 min pretreatment with TNFα followed by salmeterol for 4 h. RESULTS Müller cells treated with 5 ng/ml TNFα in 25 mM glucose significantly increased phosphorylation of IRS-1(Ser307). Treatment with the selective beta-2-adrenergic receptor agonist, salmeterol, significantly decreased phosphorylation of IRS-1(Ser307). Following IRS-1 shRNA transfection+salmeterol treatment, Bcl-2-associated X protein (Bax) and cytochrome c levels were significantly decreased. Salmeterol+IRS-1 shRNA also decreased cell death and increased protein levels of B-cell lymphoma-extra large (Bcl-xL), an anti-apoptotic factor. CONCLUSIONS In these studies, we show for the first time that salmeterol, a beta-2-adrenergic receptor agonist, can reduce retinal Müller cell death through IRS-1 actions. These findings also suggest the importance of IRS-1 in beta-adrenergic receptor signaling in the prevention of cell death in retinal Müller cells.