BCAA catabolism in brown fat controls energy homeostasis through SLC25A44

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.

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

a. Representative surface electromyogram (EMG) in adult humans at 27°C and following cold exposure at 19°C for 2 hours. 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 27°C 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/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 (TN, 30°C) or cold exposure (15°C) in diet-induced obese mice. n = 8 (TN), n = 7 (cold). b-f, biologically independent samples. 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/unpaired t-tests with Bonferroni’s correction (f). Pearson’s (r) or Spearman’s rank correlation coefficient (rs) was calculated, as appropriate (d,e).

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/group. b. Valine oxidation (per mg tissue) in indicated tissues of mice acclimated to 23°C or 12°C for one week. n = 5/group. c. Total valine oxidation in (b). Total Val oxidation was calculated by multiplying Val oxidation per mg tissue (cpm/mg tissue) and tissue mass of the depot (mg). d. Valine oxidation normalized by total protein (μg) in human brown adipocytes and white adipocytes following 2-hour-treatment with NE or vehicle. n = 5 (Veh), n =6 (NE). e. Expression profile of BCAA catabolic enzymes enriched in brown/beige fat relative to white fat of humans (left) and mouse (middle, right). Data were obtained from previous RNA-seq dataset in humans and microarray dataset in mice. The profiles were mapped onto the KEGG BCAA catabolic pathway. The number of brown/beige-enriched enzymes among total BCAA catabolic enzymes is shown. n = 3/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 weeks. n = 4/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 (TN, at 27°C) and after cold exposure at 19°C⁵. Color 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 previous RNA-seq dataset. 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 Figure 1. a-h, biologically independent samples. 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).

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

a. mRNA expression of Bckdha in BAT of Bckdha^UCP1 KO and littermate control mice. n = 5/group for all groups except n = 3 for control-gastrocnemius. b. Valine oxidation normalized by tissue weight (mg) in indicated tissues of mice in (a). n = 5/group. c. Enzymatic activity of BCKDH complex (KIV oxidation) in BAT of control and Bckdha^UCP1 KO mice acclimated to 23°C (n = 3/group) or 12°C (n = 5/group) for one week. d. Tissue weights of mice in (a) on a normal chow at ambient temperature. n = 4/group. e. mRNA expression of indicated genes in BAT of mice in (a). n = 5/group. f. EMG of muscle shivering in control (n = 7) and Bckdha^UCP1 KO mice (n = 9) at 30 ºC or 8 ºC. The right graph shows quantitative root mean square (RMS) of EMG. g. Liver temperature of control and Bckdha^UCP1 KO mice following NE treatment. n = 4/group. h. Plasma amino acid levels after three hours BCAA oral gavage. n = 5/group. i. Plasma BCAA concentration of control (n = 7) and Bckdha^UCP1 KO mice (n = 9) following cold exposure at 8ºC. a-i, biologically independent samples. 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/unpaired t-tests with Bonferroni’s correction (f,i).

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

a. Scheme of the metabolic tracer experiment in human brown adipocytes. Cells were treated with vehicle or NE for one hour in the presence of [¹³C₆, ¹⁵N₁] Leu. CE-TOFMS, capillary electrophoresis time-of-flight mass spectrometry. b. Isotopologue distributions of TCA intermediates from [¹³C₆, ¹⁵N₁] Leu in (a). n = 6/group. c. Protein expression of indicated BCAA catabolic enzymes at indicated time points of cold acclimation. The expression profile is analyzed in the proteomics dataset. n = 4 (TN, cold 3 weeks), n = 3 (cold 8 hours, 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 based on the results in (c). Enzymes whose protein expression was gradually upregulated following chronic cold adaptation were highlighted in blue. e. OCR normalized by total protein (μg) in human brown adipocytes. Differentiated adipocytes in the BCAA-free medium were supplemented with Val or vehicle, and subsequently stimulated with NE. n = 10/group. f. Schematics of the mitochondrial Val catabolic pathway. Vanadate and malonate inhibit succinyl coenzyme A synthetase (SCS) and succinate dehydrogenase (SDH), respectively. g. NE-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 NE. n = 9 (vehicle), n = 8 (Val), n = 4 (vehicle+vanadate, Val+vanadate), n = 5 (vehicle+malonate, Val+malonate). h. NE-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 NE. Brown adipocytes: n = 10 (Val−, Val+, Ile+), 9 (Leu−), 5 (Leu+), and 11 (Ile−). White adipocytes: n = 9 (Val−), and 10 (Val+). i. NE-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 prior to NE 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 by 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 AA (5 μM). Wild type: n = 5/group. Bckdha KO: n = 5/group. b,c,e,g-j, biologically independent samples. 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,i).

Extended Data Fig. 5.. Metabolic characterization of Bckdha^UCP1 knockout mice.

a. Cumulative food intake of Bckdha^UCP1 KO (n = 15) and littermate controls (n = 13) on HFD. b. Fat mass and lean mass of mice in (a) at 10 weeks of HFD. c. Tissue weights of in (a). d. Triglyceride (TG) content in the liver of mice in (a). n = 8/group. e. Oleic acid oxidation normalized by tissue mass (mg) in the interscapular BAT of mice acclimated to thermoneutral 30°C or cold exposure at 12°C. n = 4/group. f. PDH activity in the inguinal WAT, gastrocnemius muscle, and liver of Bckdha^UCP1 KO mice and littermate controls that were exposed to cold at 12 °C for one week. Ing-WAT: n = 5 (control) and 6 (Bckdha^UCP1 KO). Gastrocnemius, liver: n = 4/group. g. Immunoblotting for PDH-E1α(pSer232), PDH-E1α(pSer293), PDH-E1α(pSer300), and total PDH-E1α in the BAT of the control and Bckdha^UCP1 KO mice. GAPDH as a loading control. n = 4/group. Uncropped immunoblot images of are available in Supplementary Figure 1. h. Quantification of phosphorylated PDH-E1α normalized by total PDH-E1α protein level in (g). a-h, biologically independent samples. 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).

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

a. Expression profile of Slc25a family members in the inguinal WAT of mice acclimated to 23°C or 12°C for one week. n = 3/group. b. mRNA expression of UCP1, SLC25A44, and SLC25A39 normalized to TBP levels in the supraclavicular BAT from the same individuals (6 pairs) at thermoneutrality (TN, 27°C) and cold temperature (19°C). c. Mitochondrial localization of SLC25A44 protein in differentiated mouse beige adipocytes. TOM20 as a mitochondrial marker. d. Immunoblotting for SLC25A44 in BAT and liver of control and Slc25a44 KD mice. GAPDH 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 by 36B4 levels during mouse brown adipogenesis. n = 4/group. f. Protein expression of SLC25A44 in mouse beige preadipocytes and differentiated adipocytes. β-actin as a loading control. g. Protein expression of UCP1 and SLC25A44 in immortalized human brown preadipocytes and differentiated adipocytes. β-actin as a loading control. a,b,e, biologically independent samples. Means ± 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 Figure 1.

Extended Data Fig. 7.. Biochemical characterization of SLC25A44.

a. Genomic Slc25a44 sequence and amino acid sequence of Slc25a44 KO brown cell line. 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-labeled 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) and shRNAs targeting Slc25a44 (shRNA #1, #2), Slc25a39, or both Slc25a44 shRNA #1 and Slc25a39 shRNA (double knockdown). n = 4/group for all group except n = 6 for Scr control. e. Mitochondria uptake of [U-¹⁴C₅] Val (left) and [U-¹⁴C₆] Leu (right) in brown adipocytes in (d). n = 3/group. f. mRNA and protein expression of Slc25a44 in mitochondria of Neuro2a cells expressing an empty vector or Slc25a44. COX-IV as a loading control. n = 3/group. g. Immunoblotting for SLC25A44 in the isolated mitochondria from differentiated Slc25a44 KO brown adipocytes expressing an empty vector or Slc25a44. TOM20 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 as a loading control. i. [U-¹⁴C₆] Leu uptake rate in the liposome in (h). n = 3/group. j. [U-¹⁴C₅] Glutamate (Glu) uptake rate in the liposome in (h). n = 3/group. k. Coomassie-blue staining of purified SLC25A44 protein from HEK293S cells overexpressing Slc25a44. l. Immunoblotting of SLC25A44 in liposomes reconstituted with purified SLC25A44 (proteo-liposome) and liposomes reconstituted without SLC25A44 (empty liposome). m. Left: [U-¹⁴C₆] Leu transport into proteo-liposome in (l). Right: Leucine uptake rate. n = 3/group. d-f, biologically independent samples. i,j,m, technically independent samples. f-m, representative result from two independent experiments. Means ± 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). f-h,k-l, uncropped images are available in Supplementary Figure 1.

Extended Data Fig. 8.. Generation of Slc25a44^BAT knockdown 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. The right graph: Slc25a44 knockdown efficiency for six independent gRNAs in the dCas9-KRAB-derived MEFs (n = 2/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 (Slc25a44^BAT 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 by 36B4 in the indicated tissues of dCas9-KRAB mice in (c). n = 4/group. e. H&E staining of inguinal WAT and liver of control and Slc25a44^BAT KD mice. b,d, biologically independent samples. Means ± s.e.m.; two-sided P-values by unpaired Student’s t-test (d).

Extended Data Fig. 9.. Characterization of Slc25a44 knockdown 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 by 36B4 levels and protein expression in the BAT of mice in (a). β-actin 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 color scale shows Z-scored fold change in FPKM (Slc25a44 KD vs control). n = 3/group. d. mRNA expression of Slc25a families normalized by 36B4 levels in Slc25a44 KO and control brown adipocytes. n = 6/group. e. H&E staining of BAT, inguinal WAT, liver, and gastrocnemius muscle from mice in (a). f. Triglyceride (TG) content in the interscapular BAT of Slc25a44 KD and control mice. n = 4/group. g. Expression profile of fatty acid synthesis- and oxidation-related genes in BAT of mice in (a) by RNA-seq analysis. The color scale shows Z-scored fold change in FPKM (Slc25a44 KD vs control). n = 3/group. h. Oleic acid oxidation normalized by tissue mass (mg) in BAT of Slc25a44 KD and control mice acclimated to thermoneutral 30°C or cold temperature (12°C) for one week. n = 4/group. i. EMG measurement of muscle shivering in control mice and Slc25a44 KD mice at 30 ºC or 8 ºC. The lower graph shows the quantitative root mean square (RMS) of the EMG. n = 6/group. j. Tissue temperature in indicated tissues of control and Slc25a44 KD mice following NE treatment (indicated by red arrows). n = 4/group. b-d,f-j, biologically independent samples. Means ± s.e.m.; two-sided P-values by unpaired Student’s t-test (b-d,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/unpaired t-test with Bonferroni’s correction (i). b,e, representative results from two independent experiments. Uncropped immunoblot images are available in Supplementary Figure 1.

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 by TBP levels in (a). n = 3/group. c. NE-induced OCR normalized by 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 NE. n = 9/group (Scr control, sh-Slc25a44 #1), n = 10/group (sh-Slc25a44 #2). d. Means NE-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), AA (5 μM), and TMPD (100 μM) with ascorbate (Asc, 10mM). n = 5/group. h. mRNA expression of Slc25a44 normalized by 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/group. j. NE-induced OCR in (h). Differentiated adipocytes in the BCAA-free medium were supplemented with Val or vehicle, and subsequently stimulated with NE. 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. Valine oxidation normalized by total protein (μg) in C2C12 myotubes in (k). n = 6/group. m. OCR normalized by total protein (μg) in C2C12 myotubes in (k). n = 9/group. b-d,g-j,l-m, biologically independent samples. Means ± 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 Figure 1.

Figure 1.. Cold-induced BAT thermogenesis promotes systemic BCAA clearance in mice and humans.

a. ¹⁸F-FDG-PET/CT images of subjects following cold exposure. Right graph: quantitative standardized uptake values (SUV) of ¹⁸F-FDG in the BAT deposits. n = 17 (High BAT), n = 16 (Low BAT). b. Circulating Val concentration in (a) at 27°C (TN) and at 19°C (cold). c. Correlation between BAT activity and cold-induced changes in serum Val concentration in (a). d. Correlation coefficient between cold-induced amino acid changes and BAT activity (y-axis) against the degree of BAT-dependent amino acid changes (x-axis) in (a). e. Cold-induced changes in plasma amino acids in diet-induced obese mice at 30°C (TN, n = 5) or 15°C (cold, n = 6). f. ¹⁸F-Fluciclovine-PET/CT images of mice acclimated to 30°C (TN) or 15°C (cold) for 2 weeks. Arrows indicate interscapular BAT. g. SUV of ¹⁸F-fluciclovine in BAT. n = 5/group. h. Morphology and H&E staining of interscapular BAT of Pparg^UCP1 KO and controls. Scale bar: 50 μm. Representative result from two independent experiments i. Plasma BCAA levels in (h) during cold temperature at 12°C. n = 7/group. a-i, biologically independent samples. Mean ± s.e.m.; two-sided P-values by paired t-test (b), unpaired Student’s t-test (e), or two-way repeated measures ANOVA (g) followed by post-hoc paired/unpaired t-tests with Bonferroni’s correction (i). Pearson’s or Spearman’s rank correlation coefficient was calculated, as appropriate (c,d).

Figure 2.. BCAA oxidation in BAT is required for BCAA clearance and energy homeostasis.

a. Immunoblotting of BCKDHA in BAT of Bckdha^UCP1 KO and controls. GAPDH as a loading control. Representative result from two independent experiments. Gel source data are in Supplementary Figure 1. b. Rectal core-body temperature following cold exposure at 8°C. n = 8 (control), n = 9 (Bckdha^UCP1 KO). c. Tissue temperature in BAT and muscle following NE treatment. n = 4/group. d. Plasma BCAA levels at indicated time points after a BCAA oral gavage at 12°C. n = 8/group. e. MPE of indicated metabolites derived from [U-¹³C₆] Leu in human brown adipocytes. Cells were treated with vehicle or NE for one hour. n = 6/group. f. Body weight of Bckdha^UCP1 KO (n = 15) and controls (n = 13) on HFD at ambient temperature. g. Glucose tolerance test in (f). h. insulin tolerance test in (f). i. Glucose oxidation in BAT normalized by tissue mass. n = 3/group. j. PDH activity in BAT of mice at 12°C for one week. n = 5 (control), n = 6 (Bckdha^UCP1 KO). b-j, biologically independent samples. Mean ± s.e.m.; two-sided P-values by unpaired Student’s t-test (e,i,j) or two-way repeated measures ANOVA (b-d,f) followed by post-hoc unpaired t-test (g,h).

Figure 3.. Identification of SLC25A44 as a mitochondrial BCAA transporter.

a. Expression profile of SLC25A family members in human supraclavicular BAT and abdominal subcutaneous WAT from the same individual at 27°C and 19°C⁵. b. Correlation of SLC25A44 mRNA expression with UCP1 or BCKDHA in human BAT. Expressions at TN (red) and cold (blue) from 6 biological independent subjects. Pearson’s correlation coefficient was calculated. c. Protein expression of SLC25A44 in indicated tissues of mice. GAPDH as a loading control. Representative result from two independent experiments. Gel source data are in Supplementary Figure 1. d-e. Mitochondrial uptakes of indicated molecules in control and Slc25a44 KO brown adipocytes (d) or in Neuro2a cells expressing Slc25a44 or an empty vector (e). n = 3/group, biologically independent samples. f. [U-¹⁴C₆] Leu transport into mitochondrial-liposomes from Slc25a44 KO brown adipocytes expressing an empty vector (KO+vector) or Slc25a44 (KO+Slc25a44). n = 3/group, technically independent samples. Representative result from two independent experiments. Mean ± s.e.m.; two-sided P-values by unpaired Student’s t-test (d, e) or two-way ANOVA (f).

Figure 4.. SLC25A44 is required for BAT thermogenesis and BCAA catabolism.

a. Slc25a44 mRNA expression in indicated tissues of Slc25a44^BAT KD and control mice. n = 4/group. b. Immunoblotting of SLC25A44 in BAT of mice in (a). β-actin as a loading control. Representative result from two independent experiments. Gel source data are in Supplementary Figure 1. c. Morphology, H&E staining, and immunofluorescent GFP staining of BAT in (a). DAPI was used for counter staining. Scale bar: 100 μm. Representative result from two independent mice. d. Tissue temperature of BAT and muscle in (a) following NE treatment (arrows). n = 5 (control), n = 7 (Slc25a44^BAT KD). e. Rectal core-body temperature of Slc25a44 KD (n = 6) and controls (n = 7) following cold exposure at 8°C. f. Valine oxidation in indicated tissues normalized by tissue mass. n = 4/group. g. Plasma BCAA levels in (e) following 8-hour cold temperature at 8°C. n = 6/group. h. NE-induced OCR normalized by total protein in control and Slc25a44 KO brown adipocytes. n = 9/group (Ctrl+Val, KO+Val+KIV), n = 10/group (KO+Val, KO+Val+succinate). i. Valine oxidation in inguinal WAT-derived white adipocytes expressing an empty vector or Slc25a44 after NE treatment. n = 5 (vehicle), n = 6 (NE). j. A proposed model of BCAA catabolism in thermogenic adipose cells. Cold stimuli activate BCAA uptake and oxidation in the mitochondria of thermogenic adipocytes. Mitochondrial BCAA oxidation promotes BAT thermogenesis. This process requires SCL25A44, the mitochondrial BCAA transporter. SLC7A5, L-amino acid transporter 1. Norepinephrine, NE. a,d-i, biologically independent samples. Mean ± s.e.m.; two-sided P-values by unpaired Student’s t-test (a,f), one-way factorial (h) or two-way repeated measures ANOVA (d,e,g) followed by post-hoc paired/unpaired t-test with Bonferroni’s correction (g) or Tukey’s test (h,i).