BCAA catabolism in brown fat controls energy homeostasis through SLC25A44

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Abstract

Branched-chain amino acid (BCAA; valine, leucine and isoleucine) supplementation is often beneficial to energy expenditure; however, increased circulating levels of BCAA are linked to obesity and diabetes. The mechanisms of this paradox remain unclear. Here we report that, on cold exposure, brown adipose tissue (BAT) actively utilizes BCAA in the mitochondria for thermogenesis and promotes systemic BCAA clearance in mice and humans. In turn, a BAT-specific defect in BCAA catabolism attenuates systemic BCAA clearance, BAT fuel oxidation and thermogenesis, leading to diet-induced obesity and glucose intolerance. Mechanistically, active BCAA catabolism in BAT is mediated by SLC25A44, which transports BCAAs into mitochondria. Our results suggest that BAT serves as a key metabolic filter that controls BCAA clearance via SLC25A44, thereby contributing to the improvement of metabolic health.

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Fig. 1: Cold-induced BAT thermogenesis promotes systemic BCAA clearance in mice and humans.
Fig. 2: BCAA oxidation in BAT is required for BCAA clearance and energy homeostasis.
Fig. 3: Identification of SLC25A44 as a mitochondrial BCAA transporter.
Fig. 4: SLC25A44 is required for BAT thermogenesis and BCAA catabolism.

Data availability

The RNA-seq data generated in this study are available at Array Express under the accession code E-MTAB-7987. 13C-Leu tracing data are available in Supplementary Table 3. Uncropped immunoblot images are available in Supplementary Fig. 1. The other datasets that support the findings of this study are available in Supplementary Information and Source Data.

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Acknowledgements

We thank C. B. Newgard for the initial metabolomics analysis, E. Kunji for liposome study, Y. Seo and T. Huynh for the PET–CT scan, Y. Cheng and E. Green for HEK293S cells, E. T. Chouchani and E. Mills for cell respiration studies, and X. Lu and K. Shinoda for technical help. This work was supported by the NIH (DK97441 and DK112268) and the Edward Mallinckrodt Jr. Foundation to S.K., the American Diabetes Association Pathways Award (1-16-INI-17) to P.J.W., the AMED–CREST from the Japan Agency for Medical Research and Development to T.S., and the NIH (U19CA179513 and P30 DK063720) to M.T.M. T.Y. and M.K. are supported by the JSPS Fellowships.

Author information

T.Y. designed and carried out overall experiments and analysed data. Q.W. designed and performed cellular experiments and liposome assays and  interpreted data. K.T. and C.H.S. performed mouse experiments. M.M. and M.C. carried out human studies and analysed the data with M.S. and L.S. H. Maki, K. Igarashi, A.U. and M.O. performed BCAA-tracing studies and analysed the data with T.S. Z.D., M.K., H.L. and H. Majd performed liposome assays and analysed the data with F.C.S. P.J.W., R.W.M., O.R.I. and Y.D. measured amino acids in mice and BCKDH activity. Y.O., K. Ikeda, K.K., Y.C., M.Y. and Z.B. assisted with mouse experiments and cultured cell studies. R.N.P. conducted RNA-sequencing analysis. V.J.G. and M.T.M. developed dCas9–KRAB mice. H.T., T.G. and T.K. assisted with quantification of metabolites in human sera. S.K. conceived the project and directed the research. S.K. and T.Y wrote the paper with input from all the authors.

Correspondence to Shingo Kajimura.

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Competing interests

The authors declare no competing interests.

Additional information

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Peer review information Nature thanks Ferdinando Palmieri, Yibin Wang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Fig. 1 Cold-induced changes in circulating metabolites in mice and humans.

a, Representative EMG in adult humans at 27 °C and following cold exposure at 19 °C for 2 h. Voluntary muscle contraction as a positive control of EMG recording. b, c, Serum non-esterified fatty acids (NEFA) (b) and blood glucose (c) levels in high- (n = 9) and low-BAT subjects (n = 6) at thermoneutral 27 °C (TN) and following cold exposure at 19 °C. d, Correlation between BAT activity (SUV, log10) and cold-induced changes in serum amino acid levels of high- (red dots) and low-BAT subjects (blue dots). n = 33 per group (all amino acids) except n = 29 (Asp). e, Correlation between fat-free mass (kg) and changes in serum total BCAAs in d. n = 33. f, Changes in plasma BCAA levels at thermoneutral (30 °C) or cold exposure (15 °C) in diet-induced obese mice. n = 8 (TN), n = 7 (cold). bf, Biologically independent samples. Data are mean ± s.e.m.; two-sided P values by paired t-test (b, c) or two-way repeated-measures ANOVA followed by post hoc paired or unpaired t-tests with Bonferroni’s correction (f). Pearson’s (r) or Spearman’s rank correlation coefficient (rs) was calculated, as appropriate (d, e). Source Data

Extended Data Fig. 2 The BCAA catabolic pathway in human and mouse adipose tissues.

a, 18F-Fluciclovine-uptake into indicated organs determined by dynamic PET scanning. n = 5 per group. b, Val oxidation (per mg tissue) in indicated tissues of mice acclimatized to 23 °C or 12 °C for one week. n = 5 per group. c, Total Val oxidation in (b). Total Val oxidation was calculated by multiplying Val oxidation per mg tissue (cpm per mg tissue) and tissue mass of the depot (mg). d, Val oxidation normalized to total protein (μg) in human brown adipocytes and white adipocytes following 2-h treatment with noradrenaline or vehicle. n = 5 (Veh), n = 6 (noradrenaline). e, Expression profile of BCAA catabolic enzymes enriched in brown and beige fat relative to white fat of humans (left) and mouse (middle, right). Data were obtained from a previous RNA-seq dataset in humans15 and a microarray dataset in mice17. The profiles were mapped onto the KEGG BCAA catabolic pathway. The number of brown and beige-enriched enzymes among total BCAA catabolic enzymes is shown. n = 3 per group. f, Proteomic profile of indicated enzymes in the BCAA oxidation pathway and mitochondrial carriers (SLC25A families) in interscapular BAT of mice at thermoneutrality (29 °C) or 5 °C for 3 weeks16. n = 4 per group. g, Transcriptional profile of indicated genes in the glucose oxidation pathway (left) and the BCAA oxidation pathway (right) in the supraclavicular BAT and abdominal WAT from the identical subject under a thermoneutral condition (27 °C) and after cold exposure at 19 °C (ref. 5). The colour scale represents Z-scored FPKM (fragments per kilobase of exon per million fragments mapped). h, mRNA expression level (FPKM) of Bcat1 and Bcat2 in differentiated brown adipocytes, beige adipocytes and white adipocytes. The transcriptome data are from a previous RNA-seq dataset15. I, Immunoblotting of BCAT1 and BCAT2 in indicated tissues of mice kept at ambient temperature. GAPDH as a loading control. Representative result from two independent experiments. Gel source data are in Supplementary Fig. 1. ah, biologically independent samples. Data are mean ± s.e.m.; two-sided P values by unpaired Student’s t-test (b, c, f), two-way repeated measures ANOVA (a), or two-way factorial ANOVA followed by Tukey’s post hoc test (d). Source Data

Extended Data Fig. 3 Characterization of BAT-specific Bckdha-KO mice.

a, mRNA expression of Bckdha in BAT of BckdhaUCP1-KO and littermate control mice. n = 5 per group for all groups except n = 3 for control-gastrocnemius. b, Val oxidation normalized to tissue weight (in mg) in indicated tissues of mice in a. n = 4 per group. c, Enzymatic activity of BCKDH complex (KIV oxidation) in BAT of control and BckdhaUCP1-KO mice acclimatized to 23 °C (n = 3 per group) or 12 °C (control n = 5, KO n = 6) for one week. d, Tissue weights of mice in (a) on a normal chow at ambient temperature. n = 4 per group. e, mRNA expression of indicated genes in BAT of mice in a. n = 5 per group. f, EMG of muscle shivering in control (n = 7) and BckdhaUCP1-KO mice (n = 9) at 30 °C or 8 °C. The right graph shows quantitative root mean square (RMS) of EMG. g, Liver temperature of control and BckdhaUCP1-KO mice following noradrenaline treatment. n = 4 per group. h, Plasma amino acid levels after 3 h BCAA oral gavage. n = 5 per group. i, Plasma BCAA concentration of control (n = 7) and BckdhaUCP1-KO mice (n = 9) following cold exposure at 8 °C. ai, Biologically independent samples. Data are mean ± s.e.m.; two-sided P values by unpaired Student’s t-test (a, b, d, e, h), two-way factorial ANOVA followed by Tukey’s post hoc test (c), or two-way repeated measures ANOVA (f, g, i) followed by post hoc paired or unpaired t-tests with Bonferroni’s correction (f, i). Source Data

Extended Data Fig. 4 The effect of noradrenaline on BCAA metabolism in brown adipocytes.

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 [13C6, 15N1]Leu. b, Isotopologue distributions of TCA intermediates from [13C6, 15N1]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

Extended Data Fig. 5 Metabolic characterization of BckdhaUCP1-KO mice.

a, Cumulative food intake of BckdhaUCP1-KO mice (n = 15) and littermate controls (n = 13) on high-fat diet. b, Fat mass and lean mass of mice in a at 10 weeks of high-fat diet. c, Tissue weights of mice in a. d, Triglyceride (TG) content in the liver of mice in a. n = 8 per group. e, Oleic acid oxidation normalized to tissue mass (mg) in the interscapular BAT of mice acclimatized to thermoneutral 30 °C or cold exposure at 12 °C. n = 4 per group. f, PDH activity in the inguinal WAT, gastrocnemius muscle and liver of BckdhaUCP1-KO mice and littermate controls that were exposed to cold at 12 °C for 1 week. Inguinal WAT (Ing-WAT): n = 5 (control) and 6 (BckdhaUCP1-KO). Gastrocnemius, liver: n = 4 per group. g, Immunoblotting for PDH-E1α(pSer232), PDH-E1α(pSer293), PDH-E1α(pSer300), and total PDH-E1α in the BAT of the control and BckdhaUCP1-KO mice. GAPDH as a loading control. n = 4 per group. Uncropped immunoblot images of are available in Supplementary Fig. 1. h, Quantification of phosphorylated PDH-E1α normalized to total PDH-E1α protein level in g. ah, Biologically independent samples. Data are mean ± s.e.m.; two-sided P values by unpaired Student’s t-test (b-d, f, h), two-way repeated measures ANOVA (a) or two-way factorial ANOVA followed by Tukey’s post hoc test (e). Source Data

Extended Data Fig. 6 Characterization of SLC25A44 in thermogenic adipocytes.

a, Expression profile of Slc25a family members in the inguinal WAT of mice acclimatized to 23 °C or 12 °C for 1 week. n = 3 per group. b, mRNA expression of UCP1, SLC25A44 and SLC25A39 normalized to TBP levels in the supraclavicular BAT from the same individuals (six pairs) at thermoneutrality (27 °C) and cold temperature (19 °C). c, Mitochondrial localization of SLC25A44 protein in differentiated mouse beige adipocytes. TOM20 was used as a mitochondrial marker. d, Immunoblotting for SLC25A44 in BAT and liver of control and Slc25a44-KD mice. GAPDH was used as a loading control. Red arrows indicate specific bands whose intensities were decreased in Slc25a44-KD mice. e, mRNA expression of Slc25a44 and indicated genes normalized to levels of 36B4 (also known as Rplp0) during mouse brown adipogenesis. n = 4 per group. f, Protein expression of SLC25A44 in mouse beige preadipocytes and differentiated adipocytes. β-actin was used as a loading control. g, Protein expression of UCP1 and SLC25A44 in immortalized human brown preadipocytes and differentiated adipocytes. β-actin was used as a loading control. a, b, e, Biologically independent samples. Data are mean ± s.e.m.; one-sided P values by paired t-test (b) and two-sided P values by unpaired Student’s t-test (a). c, d, f, g, Representative results from two independent experiments. Uncropped images are available in Supplementary Fig. 1. Source Data

Extended Data Fig. 7 Biochemical characterization of SLC25A44.

a, Genomic Slc25a44 sequence of Slc25a44-KO brown cell line (upper panel). Predicted amino acid sequence of SLC25A44 is shown in lower panel. Homozygous mutation in the Slc25a44 gene by CRISPR-Cas9 results in a premature stop codon in KO cells. b, Scheme of mitochondrial BCAA uptake assay. Isolated mitochondria from differentiated brown adipocytes were incubated with [U-14C5]Val. Mitochondrial uptake was quantified by a scintillation counter. c, Validation of mitochondrial Val uptake assay in differentiated brown adipocytes. Note that addition of excess non-labelled Val (20 mM) abolished [U-14C5]Val uptake into the mitochondria. d, mRNA expression of Slc25a44 and Slc25a39 in differentiated mouse brown adipocytes expressing a scrambled control shRNA (Scr, n = 6) and shRNAs targeting Slc25a44 (shRNA #1, #2, n = 4 per group), Slc25a39 (n = 4) or both Slc25a44 shRNA #1 and Slc25a39 shRNA (double knockdown, n = 5). e, Mitochondrial uptake of [U-14C5]Val (left) and [U-14C6]Leu (right) in brown adipocytes in (d). n = 3 per group. f, mRNA and protein expression of Slc25a44 in mitochondria of Neuro2a cells expressing an empty vector or Slc25a44. COX-IV was used as a loading control. n = 3 per group. g, Immunoblotting for SLC25A44 in the isolated mitochondria from differentiated Slc25a44-KO brown adipocytes expressing an empty vector or Slc25a44. TOM20 was used as a loading control. h, Immunoblotting of SLC25A44 in the mitochondria-fused liposome. Mitochondrial membrane isolated from Slc25a44-KO brown adipocytes expressing an empty vector or Slc25a44 was fused with liposome. TOM20 was used as a loading control. i, [U-14C6]Leu uptake rate in the liposome in h. n = 3 per group. j, [U-14C5]Glu uptake rate in the liposome in h. n = 3 per group. k, Coomassie blue staining of purified SLC25A44 protein from HEK293S cells overexpressing Slc25a44. l, Immunoblotting of SLC25A44 in liposomes reconstituted with purified SLC25A44 (proteoliposome) and liposomes reconstituted without SLC25A44 (empty liposome). m, Left, [U-14C6]Leu transport into proteoliposomes in l. Right, Leu uptake rate. n = 3 per group. df, Biologically independent samples. i, j, m, Technically independent samples. fm, Representative result from two independent experiments. Data are mean ± s.e.m.; two-sided P values by unpaired Student’s t-test (f, i, j, m) or one-way ANOVA followed by Tukey’s post hoc test (d, e). fh, k, l, Uncropped images are available in Supplementary Fig. 1. Source Data

Extended Data Fig. 8 Generation of Slc25a44BAT-KD mice.

a, DNA constructs used in the generation of dCas9-KRAB mice. The dCas9-KRAB construct was inserted into the Hipp11 (H11) gene locus by the site-specific PhiC31 integrase. b, Experimental procedure of gRNA screening. MEFs from dCas9-KRAB mice were used to identify gRNA that effectively deplete Slc25a44. Graph shows Slc25a44-knockdown efficiency for six independent gRNAs in the dCas9-KRAB-derived MEFs (n = 2 per group). gRNA-Slc25a44 #1 (indicated by a red arrow) was used for generation of gRNA Tg mouse. c, Schematics of BAT-specific Slc25a44-KD mice (Slc25a44BAT-KD) by using the dCas9-KRAB system. AAV8-CAG-eGFP-U6-gRNA-tracr targeting Slc25a44 was injected into the interscapular BAT of mice expressing dCas9-KRAB on the H11 locus (dCas9-KRAB mouse). AAV8-CAG-eGFP without gRNA was used as a control. d, mRNA expression of Gfp normalized to 36B4 in the indicated tissues of dCas9-KRAB mice in c. n = 4 per group. e, H&E staining of inguinal WAT and liver of control and Slc25a44BAT-KD mice. b, d, biologically independent samples. Data are mean ± s.e.m.; two-sided P values by unpaired Student’s t-test (d). Source Data

Extended Data Fig. 9 Characterization of Slc25a44-KD mice.

a, Generation of Slc25a44-KD mice by the dCas9-KRAB system. The dCas9-KRAB mouse was crossed with transgenic mouse expressing gRNA targeting Slc25a44 to generate SLC25A44 deficient mice. b, Slc25a44 mRNA expression normalized to 36B4 levels and protein expression in the BAT of mice in a. β-actin was used as a loading control. n = 5 (control) and 4 (Slc25a44-KD). c, Expression profile of Slc25a family members in BAT in a by RNA-seq analysis. The colour scale shows log2-transformed fold change in TPM (Slc25a44-KD versus control). n = 3 per group. d, mRNA expression of Slc25a families normalized to 36B4 levels in Slc25a44-KO and control brown adipocytes. n = 6 per group. e, H&E staining of BAT, inguinal WAT, liver and gastrocnemius muscle from mice in a. f, Triglyceride content in the interscapular BAT of Slc25a44-KD and control mice. n = 4 per group. g, Expression profile of fatty acid synthesis- and oxidation-related genes in BAT of mice in a by RNA-seq analysis. The colour scale shows log2-transformed fold change in TPM (Slc25a44-KD versus control). n = 3 per group. h, Oleic acid oxidation normalized to tissue mass (mg) in BAT of Slc25a44-KD and control mice acclimatized to thermoneutral 30 °C or cold temperature (12 °C) for one week. n = 4 per group. i, EMG measurement of muscle shivering in control mice and Slc25a44-KD mice at 30 °C or 8 °C. The lower graph shows the RMS of the EMG. n = 6 per group. j, Tissue temperature in indicated tissues of control and Slc25a44-KD mice following noradrenaline treatment (indicated by red arrows). n = 4 per group. bd, fj, Biologically independent samples. Data are mean ± s.e.m.; two-sided P values by unpaired Student’s t-test (bd, f, g), two-way factorial ANOVA followed by Tukey’s post hoc test (h), or two-way repeated measures ANOVA (i, j) followed by post hoc paired or unpaired t-test with Bonferroni’s correction (i). b, e, Representative results from two independent experiments. Uncropped immunoblot images are available in Supplementary Fig. 1. Source Data

Extended Data Fig. 10 The cell-autonomous role of SLC25A44 in brown adipocytes.

a, Immunoblotting of SLC25A44 in human brown adipocytes expressing a scrambled control shRNA (Scr) and shRNAs targeting SLC25A44 (#1, #2). β-actin as a loading control. b, mRNA expression of SLC25A44 normalized to TBP levels in a. n = 3 per group. c, Noradrenaline-induced OCR normalized to total protein (μg) in the presence and absence of Val supplementation in a. Differentiated human brown adipocytes in the BCAA-free medium were supplemented with Val or vehicle, and subsequently treated with noradrenaline. n = 9 per group (Scr control, sh-Slc25a44 #1), n = 10 per group (sh-Slc25a44 #2). d, Mean noradrenaline-induced OCR in c. e, Illustration of Val metabolism in the mitochondria. f, Immunoblotting of mitochondrial proteins (as indicated) in the interscapular BAT of control and Slc25a44-KD mice. GAPDH as a loading control. g, ETC activity of BAT mitochondria. Isolated mitochondria from BAT of control mice and Slc25a44-KD mice were treated with rotenone (2 μM), succinate (10 mM), antimycin A (5 μM) and TMPD (100 μM) with ascorbate (Asc, 10mM). n = 5 per group. h, mRNA expression of Slc25a44 normalized to 36B4 in mouse beige adipocytes expressing an empty vector (n = 3) or Slc25a44 (n = 4). i, Mitochondrial Val uptake in beige adipocytes in h. n = 3 per group. j, Noradrenaline-induced OCR in h. Differentiated adipocytes in the BCAA-free medium were supplemented with Val or vehicle and subsequently stimulated with noradrenaline. Vector: n = 20 (vehicle) and 16 (Val). Slc25a44: n = 13 (vehicle) and 16 (Val). k, Immunoblotting of SLC25A44 in C2C12 myotubes expressing an empty vector or Slc25a44. β-actin as a loading control. l, Val oxidation normalized to total protein (μg) in C2C12 myotubes in k. n = 6 per group. m, OCR normalized to total protein (μg) in C2C12 myotubes in k. n = 9 per group. bd, gj, l, m, Biologically independent samples. Data are mean ± s.e.m.; two-sided P values by unpaired Student’s t-test (h, i, l, m), one-way (b) or two-way (d, j) factorial ANOVA followed by Tukey’s post hoc test, or two-way repeated measures ANOVA (c, g). a, f, k, Representative results from two independent experiments. Uncropped immunoblot images are available in Supplementary Fig. 1. Source Data

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