Figure 5.

Inhibition of TORC1 activity in TORC2^−/− DC leads to loss of their enhanced spare respiratory capacity (SRC) and glycolytic capacity. DC were generated from wild-type (WT) control (Ctrl) or TORC2^DC−/− mice (TORC2^−/− DC), then cultured with or without low concentration rapamycin (Rapa) (10 ng/mL) for 18 h. DC were analyzed using a Seahorse XFe96 Bioanalyzer for metabolic flux in real-time over 125 min with (1) oligomycin, (2) FCCP, (3) Rot/AA injected at the times indicated to obtain control values. (A) Representative mitochondria stress test showing oxygen consumption rate (OCR). (B) Quantification of basal OCR, maximal OCR and spare respiratory capacity (SRC). SRC was calculated as the difference in OCR after addition of FCCP (2) and OCR before the addition of oligomycin (1); n = 5 independent experiments with at least 2 mice per experiment; one-way ANOVA Tukey's multiple comparisons test, **p < 0.01, ***p < 0.001, ****p < 0.0001. (C) Representative glycolysis stress test showing responses of Ctrl or TORC2^−/− DC cultured with or without rapamycin and stimulated or not with LPS for 18 h; (D) Quantification of basal glycolysis (ECAR), glycolytic capacity and glycolytic reserve; n = 4 independent experiments with at least 2 mice per experiment; one-way ANOVA Tukey's multiple comparisons test, *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001. 2-DG, 2 deoxyglucose; ECAR, extracellular acidification rate; FCCP, carbonyl cyanide 4 p-(trifluoromethoxy) phenylhydrazone; NS, not significant; Rot/AA, rotenone/antimycin A.