Figure 4

Metabolic flexibility of HUVECs. (a) Inhibition of PDHA1 expression increased total ATP levels (measured as relative luminescence), whereas inhibition of PFKFB3 or LDHA expression did not show effects on total ATP levels at 72 h after siRNA transfection. Assessment of ECAR (b) and uptake levels of 2-NBDG (100 µM) (c) showed increased levels of glycolysis after inhibition of PDHA1 expression at 72 h after siRNA transfection. Inhibition of PFKFB3 expression decreased glucose-induced glycolysis (b), but did not have an effect on uptake levels of 2-NBDG (c). Inhibition of LDHA expression did not affect ECAR (b), but lowered 2-NBDG uptake levels (c) at 72 h after siRNA transfection. (d) siRNA against PDHA1 induced OCR-linked ATP production, but lowered spare capacity in HUVECs. Silencing of PFKFB3 gene expression increased OCR. Inhibition of LDHA expression lowered basal respiration levels, but increased OCR-linked ATP production, maximal respiration capacity, and spare capacity at 72 h after siRNA transfection. OCR and ECAR measurements were normalized for DNA content (RFU). mpH/min: milli-pH units per minute luminescence was normalized for cell numbers. Results are shown as means ± SEM of experiments with HUVECs of at least 3 donors. *P < 0.05, **P < 0.01, and ***P < 0.001 as compared to siNT (Unpaired Student’s t-test).