Fig 2: (a–f) Muscle AMPK trimercomplex activities in acute (a, c, and e) and short‐term (b, d, and f) metformin exercise studies. Data are means ± SEM. n = 9 in acute study; n = 10 in short‐term study. Black bars shows results for the metformin trial in the working leg. White bars shows results for the placebo trial in the working leg. ∗∗p < .01 and ∗∗∗p < .001 versus “Pre”; † p < .05, †† p < .01, and ††† p < .001 versus preceding value. AMPK, 5´AMP–activated protein kinase

Fig 3: AMPK signaling in isolated mouse skeletal muscle after prior AICAR stimulation in serum-free incubation buffer. (A) Phosphorylation of AMPK Thr172; (B) ACC Ser212; and (C) TBC1D1 Ser231 in isolated EDL muscle from C57Bl/6 mice 6 h after prior AICAR stimulation in KRB; (D) Representative immunoblots. Data were analyzed by a two-way repeated-measures ANOVA. ** p < 0.01 and * p < 0.05 indicate main effect of AICAR. *** p < 0.001 indicates effect of AICAR within group. ### p < 0.001 indicates effect of insulin within AICAR. Values are means ± SEM. n = 8 and n = 12 in control and AICAR group, respectively. A.U., arbitrary units.

Fig 4: Schematic illustration of the molecular mechanisms downstream of ROS in PKD1 mutant kidneys.Mitochondrial ROS induce activation/phosphorylation of ERK, inhibition of AMPK, and the subsequent decreases (indicated by blue) in most enzymes involved in the fatty acid oxidation (FAO) and tricarboxylic acid (TCA) cycle. These enzymes were increased (indicated by red) by mCAT treatment of PKD1 mutant mice, suggesting a partial restoration of FAO and TCA, * through inhibition of acyl-CoA carboxylase and reduction of malonyl-CoA, an inhibitor of FAO (created by biorender).

Fig 5: Phosphorylation of serine residues at positions 131, 134, and 149 of cingulin regulates endothelial permeability. (A) AMPK target motifs (yellow) and cingulin amino acid sequence are shown. (B) Paracellular resistance was measured in CGN-overexpressing cells (green), phospho-dead (CGN mut S>A; blue) and phosphomimetic cingulin mutant cells (CGN mut S>D; black), and the control cells lacking cingulin (red). Cells were stimulated with 10 µg/ml histamine. The arrow denotes the time point of histamine stimulation. Changes in resistance were quantified after 15 min of stimulation. Data are presented as the mean±s.d. (n=4). *P<0.05, **P<0.01 (one-way ANOVA with Tukey's post-hoc test). (C) Immunoprecipitation (IP) results using cingulin or control antibodies in control cells (GFP), CGN-overexpressing cells (CGN-GFP), phospho-dead mutant cells (CGN mut S>A), and phosphomimetic mutant cells (CGN mut S>D) are shown. Cingulin phosphorylation was detected using a motif-specific antibody. (D) Representative western blots (of n=3)for the indicated proteins in control cells (GFP), CGN overexpressing cells (CGN-GFP), phospho-dead mutant cells (CGN mut S>A) and phosphomimetic mutant cells (CGN mut S>D) after histamine (10 µg/ml), VEGF-A (50 ng/ml) or thrombin (0.5 U/ml) stimulation are shown.