Fig 2: KUS121 and esculetin prevent mitochondrial dysfunction-induced ATP decrease, ER stress, and cell death in neuronally differentiated PC12 cells. (a) Effects of KUS121 and esculetin on the prevention of ATP decreases induced by mitochondrial respiratory chain complex inhibitors. Neuronally differentiated PC12 cells were incubated for 24 h in the absence (DMSO) or the presence of 50 μM KUS121 or 50 μM esculetin, and then further incubated with each of the mitochondrial respiratory chain complex inhibitors (75 μM MPP +; 10 nM rotenone; 3 mM metformin; 100 nM antimycin; 0.01 μg/ml oligomycin) for 24 h. Then, total ATP amounts and live cell numbers were determined. Mean values of relative ATP levels per cell were calculated and are shown, with values for DMSO alone set at 100%. Error bars indicate standard deviations. **P < 0.01, ***P < 0.001, ANOVA with Tukey's post-hoc test (n = 4), vs. MPP +, Rotenone, Metformin, Antimycin, or Oligomycin alone (-). (b) Effects of KUS121 and esculetin on the prevention of AMPK phosphorylation and ER stress induced by mitochondrial respiratory chain complex inhibitors. Neuronally differentiated PC12 cells were cultured with the conditions shown in (a), and were subjected to western blot analyses. Actin served as a loading control. (c) Effects of KUS121 and esculetin on the prevention of cell death induced by mitochondrial respiratory chain complex inhibitors. Neuronally differentiated PC12 cells were incubated for 24 h in the absence (DMSO) or the presence of 50 μM KUS121 or 50 μM esculetin, and then further incubated with each of the mitochondrial respiratory chain complex inhibitors shown in (a) for 28 h. Then, live cell numbers were counted by staining with trypan blue. Mean values of relative live cell numbers are shown, with values for DMSO alone set at 100%. Error bars indicate standard deviations. *P < 0.05, **P < 0.01, ANOVA with Games-Howell post-hoc test (n = 3), vs. MPP +, Rotenone, Metformin, Antimycin, or Oligomycin alone (-).

Fig 3: Deletion of Oct1 altered hepatic glucose metabolism.(A) Representative western blots of key proteins involved in energy metabolism in mouse liver. Quantification of the western blots is shown in the right panel (n = 4–10 mice per genotype). (B) Hepatic glucose-6-phosphate levels (n = 4 per genotype). (C) Western blot of GS-p641, GS, and loading control β-actin. Mice were fasted overnight for 16 hours for (A), (B), and (C). (D) GTT in mice fasted for 5 hours, adjusted for baseline, and associated glucose AUC (n = 10 per genotype). (E) PTT in mice fasted for 16 hours, adjusted for baseline, and associated glucose AUC (n = 6 per genotype). (F) ITT in mice fasted for 5 hours and associated glucose AUC (n = 6 per genotype). Data shown are mean ± SEM. Data were analyzed by unpaired two-tailed Student t test; *p < 0.05, **p < 0.01, and ***p < 0.001. Underlying data are provided in S1 Data. AMPK, AMP-activated protein kinase; AUC, area under the curve; Glut2, glucose transporter 2; GTT, glucose tolerance test; GS, glycogen synthase; GS-p641, phospho-glycogen synthase at S641; ITT, insulin tolerance test; Oct1, organic cation transporter 1; pACC, phosphorylate acetyl-CoA carboxylase; pAMPK, phosphorylate 5' adenosine monophosphate-activated protein kinase; PDH, pyruvate dehydrogenase; PDH-p, phospho-pyruvate dehydrogenase; PTT, pyruvate tolerance test; PYGL; glycogen phosphorylase.

Fig 4: ET1-induced downregulation of PGC-1α, and CPT-1β is attenuated by CB-13 in an AMPK-dependent manner. A, Exposure to CB-13 (1 µM) significantly increased phosphorylation of AMPKα at Thr172, which is an indicator of AMPK activation status. AMPK phosphorylation in the presence of CB-13, and ET1 is comparable to AMPK phosphorylation levels in the presence of CB-13 alone. n = 3–9. **P < 0.01 versus control (open bar). B, PGC-1α was reduced by ET1 (0.1 μM; 4 hours), and this was prevented by CB-13. Upregulation of PGC-1α by CB-13 in untreated myocytes was abolished by AMPKα1/α2 knockdown. Likewise, the ability of CB-13 to restore PGC-1α expression in ET1-treated myocytes was blocked by AMPKα1/α2 knockdown. n = 3–12. *P < 0.05 and **P < 0.01 versus control (open bars); †P < 0.05 versus ET1. C, ET1 decreased CPT-1β RNA expression, and this was partially attenuated by CB-13. AMPKα1/α2 knockdown blocked CB-13 effects. n = 5–8 (≥3 replicates per n value). *P < 0.05 versus control (open bars); ns = not significant versus control (open bars). Mean ± SEM.

Fig 5: Inhibition of mTOR reduces the increase in expression of nuclear p21Cip1/WAF1 in cardiomyocytes induced by H2O2 and promotes apoptosis. (A) Cardiomyocytes were unstimulated (Control) or exposed to 0.2 mM H2O2 (2 h) in the presence of 1 μM KU63794, 10 μM Rapamycin or vehicle only. (B and C) Nuclear or cytosolic samples were immunoblotted with antibodies to p21Cip1/WAF1, [(A) and (B)] or cleaved caspase 3 [(A) and (C)]. Data are means ± SEM (n = 3 independent myocyte preparations); *p < .05, **p < .01, ***p < .001 relative to vehicle control; #p < .05 relative to H2O2 (one-way ANOVA with Tukey post-test). (D) Schematic for regulation of signalling and gene expression under conditions of redox stress vs signalling. In conditions of low H2O2, immediate early genes (IEGs) are upregulated and mTOR is activated to promote protein synthesis, leading to production of antioxidant enzymes and cytoprotective proteins such as p21Cip1/WAF1 to alleviate the stress of increased oxidative burden. Under conditions of high H2O2, ATP levels fall dramatically causing activation of AMPK with phosphorylation of Raptor, inhibition of mTORC1 and decreased protein synthesis. Cells are unable to synthesise cytoprotective proteins such as p21Cip1/WAF1 and undergo apoptosis.