Fig. 5: Inhibitors of the PI3K-Akt-mTORC1 axis, LY294002 and Rapamycin, reduce mutation load, partially rescue mitochondrial function and reduce glucose dependence.

a, b Schematic depicting pharmacological inhibition of the PI3k-Akt-mTORC1 axis by LY290042 (LY), GDC0941 (GDC), MK2206 (MK) or Rapamycin (RP) (a; created with BioRender.com.). b Sustained treatment of cells over 6 or 12 weeks with LY (5 µM) GDC (1 µM), MK (1 µM) or RP (5 µM) caused a progressive decrease in relative mutant mtDNA load in A549 cybrid cells and fibroblasts of patient 1 and suppressed the progressive increase of mutant load with time in culture seen in patient 2 fibroblasts (n = 3 independent experiments; p < 0.0001 in all three cell lines). The concentrations here refer to all subsequent treatments. c, d The mitochondrial membrane potential (TMRM 25 nM) of patient 1 fibroblasts was significantly increased following treatment with LY, GDC, MK or RP for 12 weeks (n = 10 independent experiments; p < 0.0001). Scale bar = 50 μm. e The respiratory capacity of patient 1 fibroblasts treated with LY, GDC, MK or RP for 12 weeks showed a major increase in oxygen consumption under all conditions following the drug treatments cells (n = 14 of culture wells; p < 0.0001). f, g The NADH:NAD⁺ ratio of patient 1 fibroblasts treated with LY or RP for 12 weeks was measured under basal conditions (n = 243, 280 and 285 cells for Pat 1, RP and LY, respectively) and in the presence of pyruvate (200 μM, 30 min; n = 188, 272 and 342 cells for Pat 1, RP and LY, respectively; f) and quantified (g; p < 0.0001). The data showed a significant decrease compared to pretreatment levels (blue dash line, basal NADH:NAD⁺ ratio of controls; red dash line, NADH:NAD⁺ ratio of controls under pyruvate condition). Scale bar = 25 μm. h Acidification of the growth medium was significantly reduced following chronic drug treatments with RP and LY (n = 9 culture wells; p < 0.0001). Lactate production (n = 4 culture wells; p = 0.0070) was also significantly reduced. Blue dashed line, basal levels of controls. i Fibroblasts of patient 1 treated with LY or RP for 12 weeks were also cultured in media with a variety of glucose/galactose concentrations to further assess the glucose dependence, displayed a significant improvement of cell growth in all conditions from the drug-treated cells (n = 8 culture wells for the 25 and 10 mM glucose groups and n = 4 culture wells for the galactose + pyruvate group; p < 0.0001). j, k Respiratory chain proteins and supercomplex assembly of patient 1 fibroblasts treated with LY or RP (j) showed a major and significant increase in the assembly of almost all supercomplexes from the drug-treated cells (k, n = 3 independent experiments; p < 0.0001). l ROS production rates of patient 1 fibroblasts treated with LY or RP for 12 weeks were significantly reduced to the level that was lower than or no difference from controls (n = 6 wells for Ctrls and Pat 1; n = 3 wells for the RP and LY groups; p < 0.0001). Source data are provided as a Source Data file. All data represented as mean ± SD and were analysed by one/two-way ANOVA with Tukey’s multiple comparisons test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, vs patient/mutant control; #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001, vs WT control).