Fig 2: Identification of enymatically active AMPK on OMM in vivo. (A) Whole-cell lysates (WC), postnuclear lysates (PNL), and the corresponding enriched mitochondrial fractions of Percoll gradient isolation from mouse GA and Heart were probed for pan-AMPKa with Vdac, Catalase, and a-tubulin as loading and purity controls (n = 3). CS denotes mixed whole-tissue lysate comprised of mouse skeletal muscle, heart, and liver. (B) Immunofluorescence confocal microscopy of longitudinal sections of C57BL/6 mouse plantaris muscle probed by pan-AMPKa (red) and Cox4 (green) antibodies and DAPI for nuclear DNA (blue). Representative image of n = 3. (Scale bar, 20 µm.) (C) Enriched mitochondrial and cytosolic fractions isolated via differential centrifugation from mouse GA muscle and probed for each AMPK subunit isoform. n = 3. (D) Enriched mitochondrial fractions from frozen GA muscle of AMPKß1/ß2 knockout (KO) and wild-type littermate mice (WT). n = 3 per group. (E) Enriched mitochondrial fractions from mouse GA were treated with/without trypsin and probed for AMPKa1/a2/ß2/?1 (n = 2 per condition). An illustration of the physical association of AMPK with OMM is presented below. (F) Enriched mitochondrial fractions from human skeletal muscle biopsies (n = 2) and left ventricle biopsies (n = 3) were probed for AMPKa1/a2/ß2/?1. (G) AMPK activity in WC, cytosolic (Cyto), and enriched mitochondrial (Mito) factions with (+) and without (-) AMP (n = 3). All data presented as mean ± SEM. ***P < 0.001 by two-way ANOVA.

Fig 3: MBH FKBP51 regulates autophagy in an inversed U-shaped manner.FKBP51 deletion is depicted in green, and FKBP51 overexpression is depicted in blue. (A) Representative blots of autophagy and mTOR markers in FKBP51MBH-KO mice. (B) Quantification of FKBP51 deletion. (C) FKBP51 deletion reduced LKB1 and AMPK binding to WIPI4 as well as (D) AMPK phosphorylation at T172. (E) TSC2-WIPI3 binding was decreased in FKBP51MBH-KO animals. (F) Quantification of mTOR substrate pp70S6K (T389). (G) LC3B-II and (H) p62 levels in the MBH. (I) Representative blots of autophagy and mTOR marker in FKBP51MBH-OE mice. (J) Quantification of viral FKBP51 overexpression. (K) FKBP51 overexpression reduced LKB1 and AMPK binding to WIPI4. (L) Quantification of AMPK phosphorylation at T172. (M) TSC2-WIPI3 binding was decreased. (N) Quantification of pp70S6K phosphorylation at T389. (O) To assess autophagic flux FKBP51MBH-OE, animals were treated with chloroquine (50 mg/kg), and LC3B-II levels were analyzed 4 hours after treatment. (P) FKBP51 overexpression blocked autophagic flux and resulted in an accumulation of p62. (Q and R) Quantification of FKBP51, p62, and BECN1, while titrating AAV-HA-FKBP51 virus into mouse neuroblastoma cells. (S) MBH FKBP51 regulates autophagy and mTOR signaling in a dose-dependent manner. All data are shown as ±SEM. Data are shown as the relative protein expression compared to control; for (A) to (N), an unpaired Student’s t test was performed. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 4: Effects of Lusianthridin, AMP, or A-769662 on activation and protection from dephosphorylation of various AMPK mutant complexes. A–C, activation of various AMPK mutant complexes by Lusianthridin, AMP, or A-769662 determined using the HTRF KinEASE assay kit and displayed as the fold increase in AMPK activity (±SEM, n = 3). D–F, increasing concentrations of Lusianthridin (D), AMP (E), or A-769662 (F) were incubated with phosphorylated (Thr172) a2ß1?1 WT or a2ß1?1 S108A (S108A) in the presence of protein phosphatase 2Ca (PP2Ca) for 10 min at 30 °C. Phosphorylation of AMPKa was determined by Western blot analysis using the anti-phospho-Thr172 antibody. Phosphorylation was quantified and is displayed above a representative Western blot image and is the means ± SEM of at least three independent experiments. AMPK, AMP-activated protein kinase; HTRF, Homogenous Time-Resolved Fluorescence.

Fig 5: AMPK is activated in BAT, but not WAT, upon sustained cold exposure.(A) Angptl4 mRNA in differentiated mouse white adipocytes (primary adipocytes and 3T3F442a adipocytes) upon treatment with 10 µM isoproterenol (ISO) or control (CTRL) for 3 hr. (B) Angptl4 mRNA in differentiated brown adipocytes (primary adipocytes and T37i adipocytes) treated with 10 µM isoproterenol (ISO) orcontrol (CTRL) for 3 hr. (C) Immunoblot for ANGPTL4 and LPL protein in differentiated 3T3F422a cells treated with 10 µM isoproterenol (ISO) or control (CTRL) for 3 hr. (D) Lpl mRNA in differentiated mouse primary white or brown adipocytes upon treatment with 10 µM isoproterenol (ISO) or control (CTRL) for 3 hr. (E) Immunoblot for AMPKa1,2 and phospho-AMPK Thr172, AMPKa1, AMPKa2, AMPKß1 and AMPKß2 in tissue lysates of kidney, spleen, heart, muscle, liver, inguinal WAT, gonodal WAT and BAT. Homogenates are identical to the homogenates presented in Figure 1A. (F) Immunoblot for AMPKa1,2 and phospho-AMPK Thr172 in BAT and WAT lysates of wild-type mice exposed to 4°C or 28°C for 10 days. *Statistically significant compared to control samples or between indicated treatments according to Student’s t-test (p<0.05). Error bars represent ± SEM.DOI: http://dx.doi.org/10.7554/eLife.08428.012