BCAA Metabolism and Ischemia-Reperfusion Injury

All animal studies were carried out in accordance with the National Institutes of Health Guidelines on the Use of Laboratory Animals and were approved by the Animal Care Committee of Air Force Medical University. PP2Cm global knockout (KO) mice are widely used as animal models with BCAA catabolic defects 10 (link). KO mice were obtained and maintained as we previously described 13 (link). Both KO and their wild-type (WT) littermates (aged from 10-12 weeks) were housed in a constant-temperature vivarium at 22°C with a 12-h light/dark cycle. Food and water were available ad libitum. BCAA mixture (weight ratio, leucine: valine: isoleucine=2:1:1; Sigma-Aldrich, St. Louis, MO, USA) were given into mice by oral gavage (1.5 mg/g/day) for 7 d before these mice received sham or I/R operation, as described by Li et al 8 (link). Vehicle or Etomoxir (Eto) (5 mg/kg body weight, Sigma-Aldrich) was intraperitoneally injected at 15 min before I/R procedure 16 (link). BCKA, including α-ketoisovaleric acid (αKIV), α-ketoisocaproate (αKIC) and α-keto-β-methylvalerate (αKMV), were commercially obtained from Sigma-Aldrich. A customized BCAA-free DMEM was used to exclude the impact of culture medium-contained BCAA. The detailed composition of BCAA-free DMEM is showed in Table S4.