Abstract
Abstract: :
Purpose: The AMP–activated protein kinase (AMPK) is a metabolic–stress–sensing protein kinase that regulates metabolism in response to energy demand and supply. It has been reported recently that AMP–activated protein kinase (AMPK) mediates the effects of several anti–diabetic agents, which strongly suggests that it could be a therapeutic target to prevent diabetic complications. However, little is known about this novel signaling pathway in diabetic retinopathy. In the current study, we were exploring the possible role of the kinase in hyperglycemia–induced apoptosis in retinal cells using a pharmacological activator of AMPK and by overexpression of the constitutively active form of AMPK. Methods: Rats were made experimentally diabetic by injection of streptozotocin and retinas isolated from normal and diabetic animals after 8 weeks. Rat retinal Müller cells (rMC1) were cultured in the medium containing either 5.5 mM or 25 mM of glucose and treated with different concentrations of AICAR for 3–5 days. The cDNA encoding constitutively active form of AMPK (α312M) was transfected with rMC1 cells. Cells expressing α312M were selected with G418 and then cultured in either 5 mM or 25 mM of glucose for 72 h prior to assays. Phosphorylation state of AMPK was determined by Western blotting and enzyme activity assay. Apoptosis was determined by measuring caspase–3 activity and Annexin V binding assay. Results: AMPK activity was decreased by 45±5% in 8–week diabetic retinas compared to that in control animals, which was associated with a significant increase in caspase–3 activity (10±2%). Likewise, phosphorylated and activated AMPK was deceased by ∼5–fold in Müller cells cultured in 25 mM glucose for 5 days compared to that cultured in 5 mM glucose. Treatment of cells with 1 mM AICAR or overexpression of the constitutively active form of AMPK resulted in a several–fold increase in AMPK activity in cells cultured in 25 mM glucose compared to that transfected with empty vector. Activation of AMPK in these cells caused a significant decrease in caspase–3 activity (29±6%) and a significant decrease in the number of apoptotic cells. Conclusions: Our data showed that AMPK was inactivated in retinas in diabetes, associated with an increase in caspase–3 activity, and activation of AMPK by AICAR or overexpression of the constitutively active form of AMPK blocked hyperglycemia–induced apoptosis. These findings suggest that activation of caspases in retinal cells is mediated by AMPK, and inactivation of AMPK might play an important role in the development of diabetic retinopathy.
Keywords: diabetic retinopathy • apoptosis/cell death • signal transduction