Epac1 Requires AMPK Phosphorylation to Regulate HMGB1 in the Retinal Vasculature.

To investigate whether AMP-activated protein kinase (AMPK) is required for the reduction of high mobility group box 1 (HMGB1) by exchange proteins activated by cAMP 1 (Epac1) in the retinal vasculature. We measured AMPK phosphorylation in normal and diabetic Epac1 floxed and cdh5/Epac1 Cre mice. We also treated primary human retinal endothelial cells (RECs) in normal (5-mM) or high (25-mM) glucose with an Epac1 agonist and AMPK or insulin-like growth factor receptor binding protein 3 siRNA. We measured protein levels of AMPK, sirtuin 1 (SIRT1), and HMGB1. AMPK phosphorylation was reduced in cdh5/Epac1 Cre mice, suggesting that Epac1 regulated AMPK actions. High-glucose culturing conditions reduced AMPK levels in RECs, but the levels were increased by the Epac1 agonist, supporting the idea that Epac1 regulates AMPK. The Epac1 agonist was not able to reduce HMGB1 levels or increase SIRT1 when AMPK was blocked by AMPK siRNA, thus demonstrating that Epac1 requires AMPK to regulate SIRT1 and HMGB1. Epac1 requires AMPK to increase SIRT1 and reduce HMGB1 in the diabetic retinal vasculature. This finding provides another pathway by which Epac1 may protect the retina during diabetes.