Silencing of insulin receptor substrate-1 increases cell death in retinal müller cells

Robert J. Walker, Nancy M. Anderson, Suleiman Bahouth, Jena J. Steinle

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

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 insulinsignaling 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 +salmetrol treatment, Bcl-2-associated X protein (Bax) and cytochrome c levels were significantly decreased. Salmeterol+IRS-I 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.

Original languageEnglish (US)
Pages (from-to)271-279
Number of pages9
JournalMolecular Vision
Volume18
StatePublished - 2012

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Insulin Receptor Substrate Proteins
Cell Death
Small Interfering RNA
Adrenergic beta-2 Receptor Agonists
Tumor Necrosis Factor-alpha
Transfection
Serum
Phosphorylation
Glucose
bcl-2-Associated X Protein
Eagles
Receptors, Adrenergic, beta
Insulin Receptor
B-Cell Lymphoma
Cytochromes c
Adrenergic Receptors
Cultured Cells
Western Blotting
Salmeterol Xinafoate
Apoptosis

All Science Journal Classification (ASJC) codes

  • Ophthalmology

Cite this

Silencing of insulin receptor substrate-1 increases cell death in retinal müller cells. / Walker, Robert J.; Anderson, Nancy M.; Bahouth, Suleiman; Steinle, Jena J.

In: Molecular Vision, Vol. 18, 2012, p. 271-279.

Research output: Contribution to journalArticle

Walker, Robert J. ; Anderson, Nancy M. ; Bahouth, Suleiman ; Steinle, Jena J. / Silencing of insulin receptor substrate-1 increases cell death in retinal müller cells. In: Molecular Vision. 2012 ; Vol. 18. pp. 271-279.
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abstract = "Purpose: To determine whether β-adrenergic receptors require insulin receptor substrate (IRS)-1 activity to regulate apoptosis in retinal M{\"u}ller cells. Methods: M{\"u}ller 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 insulinsignaling pathway in retinal M{\"u}ller 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: M{\"u}ller 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 +salmetrol treatment, Bcl-2-associated X protein (Bax) and cytochrome c levels were significantly decreased. Salmeterol+IRS-I 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 M{\"u}ller 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 M{\"u}ller cells.",
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