a, Scheme of the metabolic tracer experiment in human brown adipocytes. Cells were treated with vehicle or noradrenaline for 1 h in the presence of [13C 6 , 15N 1 ]Leu. b, Isotopologue distributions of TCA intermediates from [13C 6 , 15N 1 ]Leu in a. n = 6 per group. c, Protein expression of indicated BCAA catabolic enzymes at indicated time points of cold acclimatization. The expression profile is analysed in the proteomics dataset16. n = 4 (TN, cold 3 weeks), n = 3 (cold 8 h, 1 day, 3 days, 1 week). d, The BCAA catabolic pathway that indicates Val and Leu catabolic enzymes. Enzymes whose protein expression was transiently upregulated by acute cold exposure were highlighted in red on the basis of the results in c. Enzymes whose protein expression was gradually upregulated following chronic cold adaptation are highlighted in blue. e, OCR normalized to total protein (in μg) in human brown adipocytes. Differentiated adipocytes in the BCAA-free medium were supplemented with Val or vehicle, and subsequently stimulated with noradrenaline. n = 10 per group. f, Schematics of the mitochondrial Val catabolic pathway. Vanadate and malonate inhibit succinyl coenzyme A synthetase and succinate dehydrogenase, respectively. g, Noradrenaline-induced OCR in the presence and absence of Val in mouse brown adipocytes. Following pretreatment with vanadate (50 μM) or malonate (5 mM), differentiated cells in the BCAA-free medium were supplemented with Val or vehicle, and subsequently treated with noradrenaline. n = 9 (vehicle), n = 8 (Val), n = 4 (vehicle + vanadate, Val + vanadate), n = 5 (vehicle + malonate, Val + malonate). h, Noradrenaline-induced OCR in the presence and absence of BCAAs in mouse brown and white adipocytes. Differentiated cells were supplemented with indicated amino acids, and subsequently treated with 1 μM noradrenaline. Brown adipocytes: n = 10 (Val−, Val+, Ile+), 9 (Leu−), 5 (Leu+) and 11 (Ile−). White adipocytes: n = 9 (Val−) and 10 (Val+). i, Noradrenaline-induced OCR in the presence and absence of Val in wild-type, Ucp1-KO and Bckdha-KO brown adipocytes. Bckdha-KO brown adipocytes were treated with 2 mM KIV, 10 mM succinate or vehicle before noradrenaline stimulation. Wild type: n = 10 (Val−) and 9 (Val+). Ucp1-KO: n = 10 (Val−, Val+). Bckdha KO: n = 7 (Val+), 9 (Val+; KIV+) and 10 (Val+; succinate+). j, OCR normalized to total protein (μM) in wild-type (left) and Bcdkha-KO brown adipocytes (right). Differentiated adipocytes were pretreated with BCAT2 activator, clofibrate (300 μM), or vehicle. Following measurement of basal OCR, cells were treated with oligomycin (5 μM), FCCP (5 μM), and antimycin A (AA, 5 μM). Wild type: n = 5 per group. Bckdha KO: n = 7 per group. b, c, e, g–j, Biologically independent samples. Data are mean ± s.e.m.; two-sided P values by unpaired Student’s t-test (b, g, h), one-way factorial ANOVA followed by Tukey’s post hoc test (i) or two-way repeated measures ANOVA (e, j). Source Data