No abstract

AMP-activated protein kinase (AMPK) serves as an energy sensor and is considered a promising drug target for treatment of type II diabetes and obesity. A previous report has shown that mammalian AMPK ␣1 catalytic subunit including autoinhibitory domain was inactive. To test the hypothesis that small molecules can activate AMPK through antagonizing the autoinhibition in ␣ subunits, we screened a chemical library with inactive human ␣1 394 (␣1, residues 1-394) and found a novel small-molecule activator, PT1, which dose-dependently activated AMPK ␣1 394 , ␣1 335 , ␣2 398 , and even heterotrimer ␣1␤1␥1. Based on PT1-docked AMPK ␣1 subunit structure model and different mutations, we found PT1 might interact with Glu-96 and Lys-156 residues near the autoinhibitory domain and directly relieve autoinhibition. Further studies using L6 myotubes showed that the phosphorylation of AMPK and its downstream substrate, acetyl-CoA carboxylase, were dose-dependently and time-dependently increased by PT1 without an increase in cellular AMP:ATP ratio. Moreover, in HeLa cells deficient in LKB1, PT1 enhanced AMPK phosphorylation, which can be inhibited by the calcium/calmodulin-dependent protein kinase kinases inhibitor STO-609 and AMPK inhibitor compound C. PT1 also lowered hepatic lipid content in a dose-dependent manner through AMPK activation in HepG2 cells, and this effect was diminished by compound C. Taken together, these data indicate that this small-molecule activator may directly activate AMPK via antagonizing the autoinhibition in vitro and in cells. This compound highlights the effort to discover novel AMPK activators and can be a useful tool for elucidating the mechanism responsible for conformational change and autoinhibitory regulation of AMPK.The AMP-activated protein kinase (AMPK) 3 is a highly conserved serine/threonine protein kinase that is widely expressed in higher eukaryotes, yeast, and plants and plays a unique and central role in the responses of cells to metabolic stresses such as nutrient starvation, heat shock, ischemia/hypoxia, and vigorous muscular exercise by depleting cellular ATP and elevating AMP levels (1, 2). Once activated, AMPK prevents depletion of ATP by increasing the rate of ATP generation, triggering changes in the rates of glucose transport, fatty acid oxidation, lipogenesis, sterol synthesis, and gluconeogenesis through direct regulation of key metabolic enzymes and transcriptional control of specific genes (1-4). There is mounting evidence of the involvement of AMPK in human physiological and pathological processes, especially type 2 diabetes and obesity. Previous studies indicate that several of the beneficial effects of rosiglitazone and metformin, two widely used antidiabetic drugs, are mediated by indirect activation of AMPK, suggesting the potential role of the AMPK pathway in the treatment of type 2 diabetes (5, 7). Two adipocyte-derived hormones, leptin and adiponectin, stimulate fatty acid oxidation and glucose uptake in peripheral tissues such as skeletal muscle and liver, whic...