Leptin regulates pancreatic beta-cell excitability through AMP-activated protein kinase (AMPK)-mediated ATP-sensitive potassium (K-ATP) channel trafficking. However, the signaling components connecting AMPK to K-ATP channel trafficking are not identified. In this study, we discovered that AMPK inhibits phosphatase and tensin homologue (PTEN) via glycogen synthase kinase 3 beta (GSK3 beta) and this signaling pathway is crucial for K-ATP channel trafficking in leptin-treated pancreatic beta-cells. Pharmacologic or genetic inhibition of AMPK or GSK3 beta, but not casein kinase 2 (CK2), impaired leptin-induced PTEN inactivation and thereby K-ATP channel trafficking. The PTEN mutant lacking both protein and lipid phosphatase activity is sufficient to induce K-ATP channel trafficking without leptin. These results present a novel signaling mechanism that underlies leptin regulation of K-ATP channel trafficking in pancreatic beta-cells. Our findings assist in gaining a broader perspective on the peripheral action of leptin on pancreatic beta-cell physiology and glucose homeostasis. (C) 2013 Elsevier Inc. All rights reserved.