S., N. we performed a high-throughput screen with natural Nutlin-3 plant compounds to identify such direct allosteric activators of AMPK. We identified a natural plant dihydrophenathrene, Lusianthridin, which allosterically activates and protects AMPK from dephosphorylation by binding to the ADaM site. Similar to Nutlin-3 other ADaM site activators, Lusianthridin showed preferential activation of AMPK1-containing complexes in intact cells and was unable to activate an AMPK1 S108A mutant. Lusianthridin dose-dependently increased phosphorylation of acetyl-CoA carboxylase in mouse primary hepatocytes, which led to a corresponding decrease in lipogenesis. This ability of Lusianthridin to inhibit lipogenesis was impaired in hepatocytes from 1 S108A knock-in mice and mice bearing a mutation at the AMPK phosphorylation site of acetyl-CoA carboxylase 1/2. Finally, we show that activation of AMPK by natural compounds extends to several analogs of Lusianthridin and the related chemical series, phenanthrenes. The emergence of natural plant compounds that regulate AMPK through the ADaM site raises the distinct possibility that other natural compounds share a common mechanism of regulation. efficacy and display beneficial effects in rodent and nonhuman primate models of type 2 diabetes (33, 34) and more recently in rodent models with nonalcoholic steatohepatitis (35). These studies suggest that direct activation of AMPK by targeting the ADaM-binding site is a viable approach for treating metabolic disorders. The identification of the ADaM site as a novel regulatory site on AMPK has raised the possibility that endogenous natural metabolite(s) may regulate AMPK. A recent publication reported that long-chain fatty acyl-CoAs may act as endogenous direct AMPK activators (36). In addition, the demonstration that salicylic acid binds and regulates AMPK through the ADaM site (20, 31) raises an intriguing possibility that other naturally occurring compounds may also regulate AMPK through this site. In this study, we identified natural pure compounds from two chemical classes, phenanthrenes and Rabbit Polyclonal to ERN2 dihydrophenanthrenes, which directly activate AMPK through the ADaM-binding pocket. These compounds increase AMPK activity in intact cells and show dose-dependent inhibition of lipogenesis in primary hepatocytes. The ability of these compounds to inhibit hepatic lipogenesis was impaired by the AMPK1 S108A knock-in (KI) mutation in the ADaM-binding site and mutation of the AMPK phosphorylation site in the substrate acetyl-CoA carboxylase 1/2 (ACC1/2). The identification of dihydrophenanthrenes and phenanthrenes, in addition to the previously reported salicylic acid, suggests that activation of Nutlin-3 AMPK through the ADaM site could represent a common mechanism for natural plant compounds to regulate AMPK. This study provides growing evidence that natural compounds can regulate AMPK and supports the concept that an endogenous mammalian natural metabolite may bind to the ADaM site and activate AMPK. Results High-throughput screening of a natural bioactive library identifies Lusianthridin as a direct activator of AMPK To identify pure natural compounds or extracts that directly activate AMPK, we performed a high-throughput screen of a panel of natural bioactives (around 42,000 pure compounds, extracts, and fractions) using the Homogenous Time-Resolved Fluorescence (HTRF) KinEASE STK Nutlin-3 assay (37) and monitored Nutlin-3 allosteric activation of recombinant human AMPK211 complex. We?identified that Lusianthridin (Chemical Abstracts Service?[CAS] registry number: 87530-30-1, Fig.?1and represented by a different color. AMPK activity was determined using the HTRF KinEASE assay kit, and results are presented as the ratio of 665/620?nm (STK1 substrate peptide phosphorylation). AMPK, AMP-activated protein kinase; HTRF, Homogenous Time-Resolved Fluorescence; ZMP, 5-aminoimidazole-4-carboxamide-1-D-ribofuranosyl-5′-monophosphate. To determine whether Lusianthridin mediated its effects through the same binding site as AMP, recombinant AMPK211 was incubated with varying concentrations of AMP in the absence or the presence of fixed concentrations of Lusianthridin. As shown in Figure?1and data demonstrate that Lusianthridin regulates AMPK through the ADaM-binding pocket similar to synthetic direct activators, including A-769662. Lusianthridin increases AMPK activity in intact cells To examine the effects of Lusianthridin in intact cells, we used the human osteosarcoma U2OS cell line to monitor activation of AMPK itself by determining the level of phosphorylation on AMPK Thr172 and phosphorylation of an established substrate, ACC. These effects were routinely quantified using Western blot analysis using the LI-COR Odyssey system. Furthermore, we also used a phospho-ACC (Ser79) cellular HTRF assay kit (96-well plate reader assay format), which we optimized for this cell line to measure activation of AMPK in a linear range across a number of.