Abstract
Purpose: :
To determine if IRS-1 directly regulates apoptosis events in the insulin signaling pathway in retinal Müller cells.
Methods: :
Müller cells (rMC-1) were cultured in DMEM medium grown in high glucose (25mM) conditions. Medium was supplemented with 10% FBS and antibiotics. After cells were confluent, cells were serum-starved for 18-24 hours to eliminate any effects of insulin from the FBS. Cells were then transfected with 10ug of shRNA against IRS-1. Forty-eight hours following transfection, cells were lysed and harvested for protein analysis using Western blotting. Additional cells were treated with 10uM salmeterol 24 hours following knockdown of IRS-1. Following treatments, cells were lysed and harvested for various pro-apoptotic and anti-apoptotic markers of the Bcl-2 family in addition to Akt and cytochrome C.
Results: :
Silencing of IRS-1 in retinal Müller cells significantly decreased total levels of Akt, Bcl-xl and Bcl-2 when compared to control samples. Significant increases were obtained in pro-apoptotic markers Bad, Bax, and cytochrome c in cells with reduced IRS-1 levels vs. not treated samples. Treatment with a selective beta-2-adrenergic receptor agonist, salmeterol, following knockdown of IRS-1 showed significant increases in the anti-apoptotic markers, Akt, Bcl-xL, and Bcl-2 compared to both not treated samples and IRS-1 shRNA only samples. Protein levels of Bad, Bax, and cytochrome c were significantly decreased following salmeterol treatment IRS-1 knockdown.
Conclusions: :
The results suggest that silencing of IRS-1 plays a significant role in apoptosis in insulin receptor signaling. Pro-apoptotic markers were significantly increased in response to silencing of IRS-1. Treatment of cells with a selective beta-2-adrenergic receptor agonist in cells following IRS-1 knockdown showed that salmeterol can still prevent retinal Müller cell death, despite elimination of insulin signal transduction through knockdown of IRS-1. These results, combined with our other findings, strongly suggest that beta-adrenergic receptor stimulation can reduce apoptosis in retinal Müller cells through insulin-dependent and insulin-independent pathways in the retina.
Keywords: diabetic retinopathy • Muller cells • signal transduction: pharmacology/physiology