Metabolite measurements
Major intermediates for glycolysis, TCA cycle, and
oxidative-phosphorylation were analyzed with the assistance of Applied
Protein Technology Co., Ltd. (Shanghai, China), as previously described
(Mitsuishi et al., 2012). Briefly, 10
samples of untreated and treated cells (H9c2 and HUVECs) were stored in
liquid nitrogen and centrifuged at 14,000 rpm for 20 min to collect the
supernatants, which were dried by vacuum. Then, cold
acetonitrile/H2O (1:1, v/v) was added to each sample and
centrifuged at 14,000 rpm for 20 min to obtain the supernatants, which
were separated and analyzed using a UHPLC (1290 Infinity LC system;
Agilent Technologies) coupled to the Triple Quad 5500 Mass Spectrometer
(QTRAP/MS; AB SCIEX, Framingham, MA, USA). The raw MS data were
processed using Multiquant software based on chromatographic peak area
and retention time to identify different energy metabolism-related
metabolites.