April 2011
Volume 52, Issue 14
ARVO Annual Meeting Abstract  |   April 2011
Silencing of IRS-1 Increases Cell Death in Retinal Müller Cells
Author Affiliations & Notes
  • Robert J. Walker
    Ophthalmology, Univ of Tennessee - Memphis, Memphis, Tennessee
  • Suleiman Bahouth
    Pharmacology, Univ of Tennessee Health Science Center, Memphis, Tennessee
  • Jena J. Steinle
    Ophthalmology, Univ of Tennessee Hlth Sci Ctr, Memphis, Tennessee
  • Footnotes
    Commercial Relationships  Robert J. Walker, None; Suleiman Bahouth, None; Jena J. Steinle, None
  • Footnotes
    Support  JDRF 2008-1044, JDRF 2006-144, RPB Special Scholar's Award, RPB Departmental Award, NIH Grant PHS 3P30 EY013080, NIH F31EY019240-02
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 3557. doi:
  • Views
  • Share
  • Tools
    • Alerts
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Robert J. Walker, Suleiman Bahouth, Jena J. Steinle; Silencing of IRS-1 Increases Cell Death in Retinal Müller Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3557.

      Download citation file:

      © ARVO (1962-2015); The Authors (2016-present)

  • Supplements

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 

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.