Fig 2: 991 and contraction additively increase AMPK activity and glucose transport in mouse skeletal muscle. EDL muscles from C57BL/6 mice were isolated and incubated in the presence or absence of 991 (5 µM) for 45 min. Muscles were then either electrically stimulated to induce contraction or further incubated (no electrical stimulation) for 15 min. 2-DG transport was measured during the last 10 min of contraction or incubation as described in materials and methods. A: IB analysis was performed on 20 µg muscle lysate protein with the indicated antibodies. Representative blots are shown; n = 8. Shorter (20 s) and longer (2 min) exposure indicate the exposure time with X-ray film. B: 2-DG transport (n = 8). C and D: AMPK complexes were immunoprecipitated from 200 µg muscle lysate protein using either AMPK?1 (C) or AMPK?3 (D) antibody. AMPK activity was measured as described in materials and methods. Results are expressed as means ± SE. *Significance of contraction versus the respective control condition (0 or 5 µM 991); #significance of 991 versus the respective vehicle condition with or without 1-Hz contraction. P < 0.05.

Fig 3: 991 treatment enhances AMPK activity induced by 5-aminoimidazole-4-carboxamide riboside (AICAR) or C13 in hepatocytes. Hepatocytes were isolated from male C57BL/6NTac mice and cultured overnight as described in materials and methods. Hepatocytes were untreated (control) or treated with AICAR (0.1 mM) or C13 (10 µM) for 1 h in the presence of the indicated concentrations of 991. A: IB analysis was performed with 20 µg lysates using the indicated antibodies. Representative blots are shown; n = 3. Shorter (1 min) and longer (5 min) exposure indicate the exposure time with X-ray film. B--G: AMPK complexes were immunoprecipitated from 50 µg lysates with the indicated antibodies. AMPK activity (in duplicate) was measured in vitro as described in materials and methods. Results are expressed as means ± SE; n = 3. *Significance of AICAR or C13 versus the respective control condition (0, 0.01, or 0.1 µM 991); #significance of 991 versus the respective compound (AICAR or C13) or vehicle. P < 0.05.

Fig 4: Identification of Lusianthridin as a direct activator of AMPK.A, structure of Lusianthridin. B, AMPK activity of bacterially expressed recombinant human a2ß1?1 complex in the presence of increasing concentrations of AMP or Lusianthridin. C, comparison of Lusianthridin isolated from a natural source and Lusianthridin generated by chemical synthesis. AMPK activity was determined using the HTRF KinEASE assay kit, and results are presented as fold increase in AMPK activity (±SEM, n = 3) relative to the activity in the absence of activator. D–F, recombinant a2ß1?1 was incubated with increasing concentrations of AMP in the absence (black circles) or the presence of fixed concentrations of ZMP (D) (1.23–300 µM), Lusianthridin (E) (0.137–100 µM), or A-769662 (F) (0.014–10 µM). The increasing concentrations are indicated on the graph by the arrow and 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.

Fig 5: Allosteric activation of AMPK?1 and -?2 complexes by 991.AMPK a2ß2?1- and a2ß2?2-complexes (harbouring a FLAG tag at the N-terminus of the ?-isoform) were expressed in ß1ß2 dKO cells and isolated by immunoprecipitation using FLAG affinity resin. After extensive washing, proteins were eluted using FLAG peptide. (A) AMPK activity was measured using the SAMS peptide assay in the presence of varying concentrations of 991 (n = 5 independent experiments). (B) AMPK a2ß1?2, a2ß1(S108A)?2, a2ß2?2 and a2ß2(S108A)?2 were expressed in ß1ß2 dKO cells and isolated by immunoprecipitation using FLAG affinity resin (the ß-constructs contain a C-terminal FLAG tag). AMPK activity for each of the complexes was measured using the SAMS peptide assay in the presence of varying concentrations of 991 (n = 3 independent experiments). The data were fitted to the equation: v = b + {[(s × b) - b] × [991]}/(A0.5 + [991]), where s is the relative stimulation, b is the basal activity and A0.5 is the concentration of 991 giving half-maximal stimulation, using the GraphPad Prism software. In all cases, results are plotted as the means ± SEM and the theoretical curves were fitted using the curve-fitting programme.