Isoleucine

Standards of amino acids (AA) namely phenylalanine (Phe), tryptophan (Trp), isoleucine (Ile), leucine (Leu), asparagine (Asn), methionine (Met), valine (Val), proline (Pro), tyrosine (Tyr), alanine (Ala), threonine (Thr), glycine (Gly), serine (Ser), glutamic acid (Glu), aspartic acid (Asp), arginine (Arg), glutamine (Gln), lysine (Lys), histidine (His), cysteine (Cys) and cystine (Cys2),were purchased from Sigma-Aldrich (Steinheim, Germany). Acetonitrile (ACN) and water (LC-MS grade) was purchased from Carlo Erba (Milan, Italy). Formic acid (LC-MS additive), TGA (thioglycolic acid; ≥98%), and ammonium formate (NH₄HCO₂) were purchased from Sigma-Aldrich (Steinheim, Germany). Analytical grade hydrochloric acid (HCl) 37% w/w was also supplied from Carlo Erba (Milan, Italy). The chromatographic column HILIC amide BEH, Acquity UPLC 1.7 m, 2 × 150 mm (Waters) were used.

Adult CMs were isolated and seeded at a density of 6,000 cells per well in a 96-well plate for Seahorse measurements (Agilent Seahorse XFe96 Analyzer). Cells were washed with PBS and Seahorse base medium after attachment to the plate. The following substrates were added as energy substrates to the medium 1 h before measurements of the oxygen consumption rate: glucose 5 mM (Sigma), pyruvate 0.2 mM (Sigma), glutamine 4 mM (Sigma), palmitate–BSA 0.2 mM (Agilent), BSA control (Agilent), carnitine 0.2 mM (Sigma), valine 1 mM (Sigma), isoleucine 1 mM (Sigma), leucine 1 mM (Sigma), sodium propionate 0.05 mM (Sigma), sodium acetate 0.05 mM (Sigma), sodium octanoate 0.1 mM (Sigma), sodium decanoate 0.1 mM (Sigma). The oxygen consumption rate was measured using the Mito Stress Test kit (Agilent). The following inhibitors were injected: oligomycin (2 µM), FCCP (2 µM), rotenone and antimycin A (1 µM).

Adult male Sprague-Dawley rats weighing 230 ± 10 g were obtained from the Animal Breeding Centre of Beijing Vital River Laboratories Company (Beijing, China). The project identification code was 20162005. All animals were housed at 22 ± 2°C with a relative humidity of 50 ± 10% and a 12 h light/12 h dark cycle. The animals had free access to water and fodder (Beijing Keaoxieli Co. Ltd.). All experimental animal procedures were approved by the China Academy of Chinese Medical Sciences' Administrative Panel on Laboratory Animal Care and performed in accordance with institutional guidelines and ethics of the committee as part of the China Academy of Chinese Medical Sciences (February 1^st, 2016).
Ten AA standards including alanine, valine, taurine, leucine, isoleucine, glutamine, glutamate (glutamic acid), phenylalanine, (S)-tyrosine, and tryptophan (purity > 99%, all) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile and methanol were provided from Fisher Scientific (Shanghai, China). Formic acid, ammonium formate, and phenylalanine-d5 (internal standard (IS)) were purchased from Toronto Research Chemicals (YTO, Canada). All chemicals used throughout this study were of HPLC grade unless stated otherwise. BNC (batch number: 140156) was provided from Buchang Pharma Co. Ltd.

HPLC-grade formic acid was obtained from Sigma-Aldrich (USA). HPLC/MS-grade formic acid, ammonium formate, and acetonitrile were acquired from Fisher Scientific (USA). The internal standards Labeled Carnitine Standards (L-carnitine (N-trimethyl-D₉), L-acetylcarnitine (N-methyl-D₃), L-propionylcarnitine (Nimethyl-D₃), L-butyrylcarnitine (N-methyl-D₃), L-isovalerylcarnitine (N-trimethyl-D₉), L-octanoylcarnitine (N-methyl-D₃), L-myristoylcarnitine (N-trimethyl-D₉), L-palmitoylcarnitine (N-methyl-D₃)) were attained from Cambridge Isotope Laboratories. Seventeen amino acid standards (alanine, valine, leucine, isoleucine, phenylalanine, methionine, proline, glycine, serine, threonine, cysteine, tyrosine, histidine, lysine, arginine, aspartic acid, glutamic acid) were purchased from Sigma-Aldrich. Eight amino acid standards (tryptophan, asparaginate, glutamine, ornithine, taurine, citrulline, cysteine, γ-aminobutyric acid) were gained from J&K Scientific. Ultrahigh purity water was prepared by Millipore-Q Water Purification System (Millipore, Germany).