Purpose: To measure key proteins involved in insulin resistance in retinal Müller cells.
Methods: Cells known as retinal Müller cells were cultured in normal (5 mM) or high glucose (25 mM) to mimic a diabetic condition. Cells were treated with 50 nM Compound 49b, a novel β-adrenergic receptor agonist. Additional cells were treated with small interfering RNA (siRNA) against protein kinase A or cyclic adenosine monophosphate (cAMP) responsive element binding protein (CREB). Western blotting or enzyme-linked immunosorbent assay (ELISA) measurements were made for protein changes in TNFα, suppressor of cytokine signaling 3, insulin receptor substrate 1 (IRS-1), insulin receptor (IR), Akt, and cell death proteins (Fas, fas ligand, cytochrome C, Bax, cleaved caspase 3, and Bcl-xL).
Results: Hyperglycemia significantly increased TNFα and suppressor of cytokine signaling 3 levels. This was associated with increased phosphorylation of IRS-1(Ser307) and IR(Tyr960), with decreased phosphorylation of IR(Tyr1150/1151) and Akt(Ser473). The reduced insulin receptor and Akt phosphorylation led to a significant increase in proapoptotic proteins. Compound 49b reversed the loss of Akt and IR(Tyr1150/1151) phosphorylation, reducing Müller cell apoptosis.
Conclusions: Hyperglycemia-induced TNFα levels promote insulin resistance in retinal Müller cells, noted through increased phosphorylation of IRS-1(Ser307) and IR(Tyr960). The dysfunctional insulin signaling increases apoptosis of retinal Müller cells, which is blocked through treatment with Compound 49b. Taken together, β-adrenergic receptor agonists may protect retinal Müller cells through maintenance of normal insulin receptor signaling.