α ketoisocaproate

After the 15-min equilibration perfusion, male Wistar rat hearts were perfused for 30 min with Krebs Ringer bicarbonate buffer with the following additions: 3% BSA (fatty acid free; Fisher Scientific), 100 µU/mL insulin, 0.4 mM palmitate (Sigma) bound to BSA, physiologic concentrations of amino acids (Alanine 416 µM, Arginine 137 µM, Asparagine 33 µM, Aspartic Acid 10 µM, Cysteine 15 µM, Glutamic Acid 89 µM, Glutamine 483 µM, Glycine 220 µM, Histidine 50 µM, Isoleucine 88 µM, Leucine 134 µM, Lysine 287 µM, Methionine 40 µM, Phenylalanine 60 µM, Proline 112 µM, Serine 142 µM, Threonine 164 µM, Tryptophan 55 µM, Tyrosine 70 µM, Valine 179 µM), 1 μM puromycin (Sigma) and either no branched-chain α-ketoacids (BCKA), concentrations of BCKA found in the plasma of lean rats [α-ketoisovalerate (KIV) 12 μM; α-ketoisocaproate (KIC) 15 μM; α-keto-β-methylvalerate (KMV) 7 μM; all purchased from Sigma] or concentrations of BCKA found in the plasma of obese rats (KIV 24 μM; KIC 30 μM; KMV 14 μM)^{5 (link)}.

The plasmid pET28a-lad with wild-type L-AAD gene and the recombinant strain E. coli BL21-lad were constructed in a previous study (Song et al., 2015 (link)). The restriction endonucleases, PCR reagents, blunting kit used for saturation mutagenesis were purchased from Takara (Dalian, China). The standard of α-ketoisocaproate was purchased from Sig-ma-Aldrich (St. Louis, MO, USA), and other reagents were purchased from Sangon Biotech Co. (Shanghai, China).

L. reuteri (F 275^T = ATCC 23272^T = DSM 20016^T = JCM 1112^T = LMG 9213^T = LMG 13557^T) bacteria were obtained from the American Type Culture Collection (Manassas, VA, USA). ³H-L-kynurenine (³H-KYN) (16 Ci/mmol) was purchased from Amersham (Buckinghamshire, UK). Aminooxyacetic acid (AOAA), aspartate, α-aminoadipate, glutamine, pyruvate, α-ketoglutarate, α-ketoisocaproate, α-ketoisovalerate, oxaloacetate, 2-amino-2-norbornanecarboxylic acid (BCH) and PF-04859989 were obtained from Sigma-Aldrich (St. Louis, MO, USA). L-Kynurenine sulfate (L-KYN) was acquired from Sai Advantium (Hyderabad, India) and D-kynurenine sulfate (D-KYN) from Shanghai Hanhong Chemical Co. (Shanghai, China). BFF-122 [(S)-(-)-9-(4-aminopiperazine-1-yl)-8-fluoro-3-methyl-6-oxo-2,3,5,6-tetrahydro-4H-1-oxa-3a-azaphenalene-5-carboxylic acid] was kindly provided by Dr. Y. Kajii (Mitsubishi-Tanabe Pharma Corp., Yokohama, Japan). All other chemicals used were purchased from commercial suppliers and were of the highest available purity.