Abstract
Purpose: :
Nuclear accumulation of the key glycolytic enzyme glyceraldehyder–3–phosphate dehydrogenase (GAPDH) is an early event in the apoptotic process. Previously, we have demonstrated that hyperglycemia leads to pro–apoptotic nuclear accumulation of GAPDH in retinal Müller cells in vitro and in vivo. Production of pro–inflammatory cytokines, such as interleukin–1beta (IL–1ß), precedes hyperglycemia–induced GAPDH nuclear accumulation in retinal Müller cells. Therefore, this study is focused on the role of IL–1ß on GAPDH nuclear accumulation and apoptosis in retinal Müller cells.
Methods: :
Transformed rat retinal Müller (rMC–1) cells were incubated in 5mM (low) glucose medium in the presence of 2ng/ml IL–1ß for 24 hours. GAPDH localization was assessed using immunocytochemistry. At 96 hours of IL–1ß treatment, caspase–3 activity was measured to assess apoptosis execution. To examine the role of IL–1ß in high glucose–induced GAPDH nuclear accumulation, rMC–1 cells were incubated in medium containing 5mM glucose, 25mM (high) glucose, 25mM glucose plus 100 µM of the caspase–1 (enzyme responsible for IL–1ß production) inhibitor YVAD–fmk, or 25mM glucose plus IL–1 receptor antagonist (50ng/ml) for 24 hours then GAPDH localization was assessed.
Results: :
In normal glucose, IL–1ß (2ng/mL) significantly increased the number of rMC–1 cells positive for nuclear GAPDH from 25 % ± 4.85 (untreated) to 41% ± 2.76 within the first 24 hours of treatment. 2ng/mL IL–1ß also increased the activity of the apoptosis executioner caspase–3 at 96 hours compared to untreated rMC–1 cells. 25 mM glucose led to a 1.5 fold increase in the number of rMC–1 cells positive for nuclear GAPDH compared to cells treated in low glucose. Inhibition of high glucose–induced IL–1ß production using a specific caspase–1 inhibitor or IL–1ß signaling using an IL–1 receptor antagonist decreased the number of rMC–1 cells positive for nuclear GAPDH following high glucose treatment.
Conclusions: :
These results indicate that the early, high glucose–induced IL–1ß production by retinal Müller cells and its autocrine signaling plays an important role in apoptosis induction mediated by GAPDH nuclear accumulation within these cells. Therefore, inhibition of IL–1ß production or signaling might represent a potential new strategy to prevent hyperglycemia–induced apoptosis of retinal cells and possibly the development of diabetic retinopathy.
Keywords: diabetic retinopathy • inflammation • Muller cells