Abstract
The distinctive colour of brown adipose tissue (BAT) is attributed to its high content of haem-rich mitochondria. However, the mechanisms by which BAT regulates intracellular haem levels remain largely unexplored. Here we demonstrate that haem biosynthesis is the primary source of haem in brown adipocytes. Inhibiting haem biosynthesis results in an accumulation of the branched-chain amino acids (BCAAs) valine and isoleucine, owing to a haem-associated metabolon that channels BCAA-derived carbons into haem biosynthesis. Haem synthesis-deficient brown adipocytes display reduced mitochondrial respiration and lower UCP1 levels than wild-type cells. Although exogenous haem supplementation can restore intracellular haem levels and mitochondrial function, UCP1 downregulation persists. This sustained UCP1 suppression is linked to epigenetic regulation induced by the accumulation of propionyl-CoA, a byproduct of disrupted haem synthesis. Finally, disruption of haem biosynthesis in BAT impairs thermogenic response and, in female but not male mice, hinders the cold-induced clearance of circulating BCAAs in a sex-hormone-dependent manner. These findings establish adipose haem biosynthesis as a key regulator of thermogenesis and sex-dependent BCAA homeostasis.
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Data availability
All data and uncropped scans for all western blots are included in the Source Data files. RNA-seq data have been deposited in GEO under accession number GSE289295. Proteomics data have been deposited in PRIDE under accession number PXD060812. Primers and gRNA sequences are provided in the Supplementary Information. Source data are provided with this paper.
Code availability
All coding packages used in this study are described in the Methods and are available from the corresponding author upon request.
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Acknowledgements
We thank V. Lo Sardo, C. Alexander, D. Harris, D. Lamming, M. Merrins, E. Saez and L. Kazak for critical input and discussion; J. Simcox at the University of Wisconsin–Madison for sharing immortalized brown preadipocytes; V. Lo Sardo at the University of Wisconsin–Madison School of Medicine and Public Health for sharing reagents and resources; the University of Wisconsin–Madison Genome Editing and Animal Model core for assistance with the generation of the Alas1-floxed mouse model; N. Rosenthal at The Jackson Laboratory for sharing the RNA-seq data of the Diversity Outbred mice; and A. Konopka at the University of Wisconsin–Madison School of Medicine and Public Health for support with Oroboros instrumentation. This work was supported by the National Institutes of Health (NIH) grants 1R35GM150899 (A.G.), P41GM108538 and R35MG118110 (J.J.C.), R35GM147014 (J.F.), the Wisconsin Partnership Program at the University of Wisconsin School of Medicine and Public Health grant WPP5451 (A.G.) and the DRC at Washington University P30 DK020579 (A.G.). The University of Utah Metabolomics Core is supported by U54 DK110858 (J.E.C.). D.J.D. is supported by the National Institute On Aging training grant T32AG000213; H.B. is supported by NIH NCATS awards to UW-ICTR TL1TR002375 and UL1TR002373. M.F. is supported by a postdoctoral fellowship (PF-23-1070297-01-TBE; https://doi.org/10.53354/ACS.PF-23-1070297-01-TBE.pc.gr.175370) from the American Cancer Society. Y.B. is supported by the American Heart Association predoctoral fellowship 25PRE1374479. The model in Fig. 7j was created using NIAID NIH BIOART (https://bioart.niaid.nih.gov/bioart/77) (NIAID Visual and Medical Arts, 10/7/2024, Chromatin).
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D.J.D. and A.G. conceived the project, designed research and analysed data. D.J.D., J.K.H. and M.F. performed in vivo experiments. D.J.D., J.K.H., N.D.C. and Y.B. carried out cell-based assays. D.J.D. and H.B. performed RNA-seq analyses. D.J.D. and S.V.J. performed metabolomic analyses. D.J.D., A.J., L.Y.C., K.A.O. and E.S. prepared samples and carried out proteomic studies. M.P.K. and A.G. performed correlation expression analysis. D.J.D., J.L.C., Q.P., A.G. and J.E.C. designed and performed isotope tracing experiments. R.A.A., V.L.C., A.D.A., J.J.C. and J.F. provided advice and reagents. D.J.D. and A.G. wrote the paper and integrated comments from the other authors.
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Extended data
Extended Data Fig. 1 Expression analysis of heme acquisition pathways in differentiating adipocytes and tissues.
a) Schematic of the eight-step mammalian heme biosynthesis pathway. ALAS1 serves as the rate limiting enzyme. Succinylacetone (SA) is a competitive inhibitor of ALAD/PBGS. Substrates (black), enzymes (red) and subcellular ___location (gray). Eight molecules each of succinyl-CoA and glycine are required for the synthesis of one molecule of heme. b) mRNA relative expression of Alas1 in various mouse tissues collected from 18-week-old wild type male (n = 3) and female (n = 3) mice. c) Log2 normalized counts of mRNA of genes within the heme biosynthesis pathway in differentiating primary white (yellow), brown (black), or immortalized brown (blue) adipocytes (n = 3 biological replicates per cell type per time point). d) Expression of Alas2 was not detected throughout differentiation in any adipocyte line (n = 3 biological replicates per cell type per time point). e) mRNA counts of putative heme transporters in differentiating adipocytes as described in c (n = 3 biological replicates per cell type per time point). Data shown as mean ± SD. * p < 0.05 vs day 0 of differentiation of pBAT (black stars), pWAT (orange stars), or immBAT (blue stars), respectively. # p < 0.05 for pBAT (black hashes) and immBAT (blue hashes) vs same time point of pWAT; one-way ANOVA with multiple comparisons and a Tukey’s post-test.
Extended Data Fig. 2 Chemical disruption of heme acquisition represses the thermogenic profile without impairing adipocyte differentiation.
a) mRNA relative expression of adipogenic genes and Alas1 in primary brown adipocytes differentiated in the presence of vehicle, succinylacetone (SA), heme-depleted serum (HDS), or SA + HDS (n = 3 biological replicates per condition). b) Primary brown adipocytes treated as described in a have comparable capacity for adipogenic differentiation. Nile red (red) and Hoechst (blue) used to stain lipid droplets and nuclei, respectively. Scale bar = 100 μm. c) Volcano plot of global protein abundance in primary brown adipocytes treated with SA relative to vehicle (n = 4 biological replicates per condition). d) Bubble plot of biological pathways enriched among significantly changed genes with a log2-fold change less than -1. Data shown as mean ± SD. p values vs. FBS, one-way ANOVA with multiple comparisons and a Tukey’s post-test.
Extended Data Fig. 3 Generation of Alas1-/- immortalized preadipocytes using CRISPR-Cas9.
a) mRNA levels of Alas1 in three representative Alas1 KO clones generated using three distinct gRNAs (n = 3 biological replicates per clone). b) Pparγ expression is not impacted by Alas1 deletion. c) Ucp1 levels are significantly reduced in all Alas1 KO clones (n = 3 biological replicates per clone for a-c). d) Protein levels of Alas1 and Ucp1 confirm loss of expression in screened clones. e) The relative abundance of numerous electron transport chain proteins is significantly reduced in Alas1 KO brown adipocytes compared to WT (n = 4 biological replicates per genotype). Data shown as mean ± SD. p values vs. WT; one-way ANOVA with multiple comparisons and a Tukey’s post-test.
Extended Data Fig. 4 Exogenous heme supplementation does not compensate for loss of heme synthesis.
a) Quantification of heme in isolated nuclei and mitochondria fractions derived from WT and Alas1 KO brown adipocytes following treatment with vehicle or 10 μM HA (n = 3 biological replicates per condition). b) Changes to the global proteome are mainly explained by genotype and active heme biosynthesis (WT vs. Alas1 KO) rather than HA supplementation and differences in the intracellular heme pool (n = 4 biological replicates per condition). c, d) Quantification of intracellular heme levels and mRNA relative expression (n = 4 biological replicates per condition) of Pparγ, Alas1, and Ucp1 in WT primary brown adipocytes treated with SA, HA, or both throughout differentiation. e) Alas1 and Ucp1 protein levels in primary brown adipocytes treated with SA, HA (1 or 10 μM), or both throughout differentiation. f) In contrast to inhibition of heme synthesis (SA), exogenous heme supplementation has limited impact on gene expression in primary brown adipocytes (n = 4 biological replicates per condition). g) ATP turnover is partially restored by 10 μM HA supplementation in Alas1 KO brown adipocytes (n = 8 biological replicates per condition). h) The contribution of the futile creatine cycle to maximal respiration is higher in Alas1 KO adipocytes treated with HA compared to WT (n = 4 biological replicates per condition). i, j) Pharmacological modulation of Rev-Erbα activity does not rescue Ucp1 expression in Alas1 KO adipocytes (n = 3 biological replicates). Data are shown as mean ± SD. p values vs. WT; one-way ANOVA with multiple comparisons and a Tukey’s post-test (a, c, d, g, h), two-tailed FDR-adjusted t-test (b), Wald test (f), and two-way ANOVA with multiple comparisons and a Tukey’s post-test (j).
Extended Data Fig. 5 Alas1 substrates and their precursors accumulate upon heme biosynthesis blockade.
a, b) Relative abundance of Alas1 substrates and their metabolic precursors in differentiated WT and Alas1 KO brown adipocytes (a; n = 4 biological replicates per genotype) and primary brown adipocytes treated with vehicle or SA throughout differentiation (b; n = 3 biological replicates). Data shown as mean ± SD. p values vs. WT or vehicle; multiple two-tailed Student’s t-test.
Extended Data Fig. 6 BCAAs fuel heme synthesis in brown adipocytes.
a) Adipose, liver, and skeletal muscle correlation of Alas1 expression with genes involved in BCAA catabolism. b) Enrichment of propionate-derived carbon labeling of TCA metabolites (n = 3 biological replicates). c) Representative images and quantification of proximity ligation assay (PLA) foci for ALAS1 and PCCA in Alas1 KO brown adipocytes (n = 30 individual cells per genotype). Scale bar = 5 μm. Data are shown as mean ± SD. p values vs. WT, one-tailed Student’s t-test.
Extended Data Fig. 7 Alas2 does not compensate for the loss of Alas1 in BAKO BAT.
a) mRNA relative expression of Alas2 in red blood cells (RBC) relative to BAT collected from female WT (n = 4) and BAKO (n = 4) mice housed at thermoneutrality. b) Protein levels of ALAS1 and ALAS2 in protein lysates from red blood cells (RBC) and PBS-perfused BAT collected from female WT and BAKO mice as described in a. Data are shown as mean ± SEM. p values vs. WT; one-way ANOVA with multiple comparisons and a Dunnett’s post-test.
Extended Data Fig. 8 BAKO mice have normal development and metabolic physiology at thermoneutrality.
a) Female (n = 16 WT, 18 BAKO) and male (n = 7 WT, 8 BAKO) BAKO mice show no significant differences in body weight compared to WT when fed standard chow and housed at thermoneutrality. b) Tissue mass of brown (BAT), inguinal (iWAT), epididymal (eWAT) white adipose, and liver are similar in WT and BAKO females (n = 7 per genotype) and males (n = 5 WT, 7 BAKO). c) Oral glucose tolerance test (OGTT) reveals no differences between WT and BAKO females (n = 6 WT, 11 BAKO) and males (n = 7 WT, 8 BAKO). d) Insulin tolerance test (ITT) shows no significant differences between WT and BAKO females (n = 20 WT, 24 BAKO) or males (n = 6 WT, 8 BAKO). e, f) Female and male indirect calorimetry measurements of O2 consumption (VO2), CO2 production (VCO2), respiratory exchange ratio (RER), and energy expenditure (EE) in WT and BAKO males and females (n = 6 per group) housed at thermoneutrality. Data are shown as mean ± SEM. Mixed effects analysis with Šídák’s multiple comparisons test (a), unpaired two-tailed t-test for tissue weights and reverse AUC, two-way ANOVA followed by Šídák’s multiple comparisons test for OGTT and IGTT.
Extended Data Fig. 9 Altered molecular signatures of BAKO BAT at thermoneutrality.
a) Volcano plot of differentially expressed genes in BAT collected from chow-fed female WT and BAKO (n = 4 per group) mice housed at thermoneutrality. b) Volcano plot of differentially abundant proteins in BAT collected from female WT and BAKO (n = 7 per group) mice. c) Heatmap of differentially expressed genes (p < 0.05; |log2-FC | > 0.5) in BAT from WT and BAKO mice (n = 4 per group) as described in a.
Extended Data Fig. 10 Diet-induced thermogenesis is not impaired in BAKO mice housed at thermoneutrality.
a, b) Indirect calorimetry in female (n = 10 WT, 10 KO) and male (n = 7 WT, 8 BAKO) mice reveals no significant differences in diet-induced thermogenesis between WT and BAKO mice. Data are shown as mean ± SEM or Min to Max values (RER).
Supplementary information
Supplementary Information
Supplementary Fig. 1
Supplementary Tables 1–4
Table 1: Reagent/chemical information. Table 2: Antibody-based assay information including dilution factors used for western blots. Table 3: Oligo sequences used for cloning into PX459 plasmid backbone for CRISPR targeting of Alas1 gene. Table 4: PCR and qPCR primer sequences.
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Duerre, D.J., Hansen, J.K., John, S.V. et al. Haem biosynthesis regulates BCAA catabolism and thermogenesis in brown adipose tissue. Nat Metab 7, 1018–1033 (2025). https://doi.org/10.1038/s42255-025-01253-6
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DOI: https://doi.org/10.1038/s42255-025-01253-6
