Reduced insulin receptor signaling in retinal Müller cells cultured in high glucose

To measure key proteins involved in insulin resistance in retinal Müller cells.

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).

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.

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.