Fig. 4.

PI5P4K inhibition perturbs energy metabolism. (A) Total cellular ATP measured using a luminescent ATP detection assay kit (Abcam) and normalized against total protein concentration. C2C12 myotubes were treated with 10 μM CC260 overnight before the assay (n = 3). (B) Levels of intracellular AMP, ADP, and ATP measured by LC-MS/MS. The elution profiles of the control and compound-treated samples are scaled according to total ATP determined by the luminescent assay. The AMP peak is shown as 10 times the actual height for easier visualization. C2C12 myotubes were treated with 10 μM CC260 overnight before nucleotide extraction (n = 3). AMP/ATP ratio was measured in triplicate. (C) After 16 h, glucose concentrations in the media were reduced by 2.3 and 2.9 mM for cultured C2C12 myotubes treated with DMSO or 10 μM CC260, respectively (n = 3). Lactate accumulated to 3.2 and 4.0 mM (∼70% of the consumed glucose was used for fermentation). The lactate in each well (∼300 μg) is several times greater in mass than the cells (total protein ∼50 μg), suggesting that it is mainly derived from glycolysis. (D) Metabolic flux analysis quantifying mitochondrial and glycolytic ATP production rates (n = 16). Results are shown as means ± SEM, * indicates P ≤ 0.01, and *** indicates P ≤ 0.0001 based on Student’s t test. N.S., nonstatistically significant.