Abstract
Brown adipose tissue (BAT) engages futile fatty acid synthesis–oxidation cycling, the purpose of which has remained elusive. Here, we show that ATP-citrate lyase (ACLY), which generates acetyl-CoA for fatty acid synthesis, promotes thermogenesis by mitigating metabolic stress. Without ACLY, BAT overloads the tricarboxylic acid cycle, activates the integrated stress response (ISR) and suppresses thermogenesis. ACLY’s role in preventing BAT stress becomes critical when mice are weaned onto a carbohydrate-plentiful diet, while removing dietary carbohydrates prevents stress induction in ACLY-deficient BAT. ACLY loss also upregulates fatty acid synthase (Fasn); yet while ISR activation is not caused by impaired fatty acid synthesis per se, deleting Fasn and Acly unlocks an alternative metabolic programme that overcomes tricarboxylic acid cycle overload, prevents ISR activation and rescues thermogenesis. Overall, we uncover a previously unappreciated role for ACLY in mitigating mitochondrial stress that links dietary carbohydrates to uncoupling protein 1-dependent thermogenesis and provides fundamental insight into the fatty acid synthesis–oxidation paradox in BAT.
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Data availability
All data are available in the main text or the Supplementary Information. RNA-seq data are available at the NCBI BioSample Project: PRJNA1151645.
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Acknowledgements
We thank the UMass Chan Morphology Core, Mouse Facility, EM Facility, and members of the laboratories of D.A.G., K.E.W., N.W.S. and C.J. for helpful discussions. This work was funded by R01DK116005, R01DK127175 and R01DK094004 to D.A.G. M.D.L. was supported by National Institutes of Health fellowship K01DK111714.
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Conceptualization: D.A.G., E.D.K., C.M.C. and K.E.W. Methodology: E.D.K., C.M.C., J.A.H., J.R.P., J.B.S., O.A., C.M.M., M.D.L., C.J. and N.W.S. Investigation: E.D.K., C.M.C., J.A.H., M.E.K., H.L., M.G., Q.C., H.L.P., H.A., A.B., S.M.F., T.-Y.L., A.L., J.P., S.T., C.R.G., P.V., C.F.S., O.A. and M.D.L. Visualization: E.D.K. and D.A.G. Supervision: D.A.G., K.E.W., N.W.S. and C.J. Writing: E.D.K. and D.A.G.
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Extended data
Extended Data Fig. 1 ACLY loss in Brown Fat Causes Tissue Whitening and Cold Intolerance in Male Mice.
a. Body weight of the AclyBATKO male mice. n=21 C, 16 KO. b. Adipose tissue weights of the AclyBATKO male mice. *p= 0.0246, *p= 0.0234. n=11 C, 8 KO. c. Lean tissue weights of the AclyBATKO male mice. n=11 C, 8 KO. d. ‘Survival curve’ for cold exposure experiment, where mice maintain body temperature above 30 °C, remain in the cold over the time course of experiment. n=C, 6 KO. e. Dorsal view representative infrared thermography image of heat signature of the iBAT from AclyBATKO mouse and littermate control. f. Body weights of the Acss2BATKO male mice. n=6 C, 6 KO. g. Adipose tissue weights of the Acss2BATKO male mice. n=6 C, 6 KO. h. Lean tissue weights of the Acss2BATKO male mice. n=6 C, 6 KO. i. ‘Survival curve’, how many mice remain in the cold over the time course of experiment. n=6 C, 6 KO. j. Acyl CoA levels measurements in the Acss2BATKO male mice BAT. **p= 0.0052, *p= 0.0170. n=9 C, 10 KO. Data are mean ± s.e.m. Statistical analysis unpaired two-tailed Student’s t-test.
Extended Data Fig. 2 ACLY loss in Brown Fat Causes Tissue Whitening and Cold Intolerance in Female Mice.
a. Representative H&E of the iBAT from AclyBATKO female mouse and littermate control. Scale bar 50um. Clear consistent phenotype across n=3 mice. b. Body weight of the AclyBATKO female mice. n=7 C, 11 KO. c. Adipose tissue weights of the AclyBATKO female mice. *p= 0.0232. n=7 C, 11 KO. d. Lean tissue weights of the AclyBATKO female mice. n=7 C, 11 KO. e. Rectal temperatures of the AclyBATKO female mice and littermate controls during cold exposure (4 °C). n=8 C, 8 KO. f. ‘Survival curve’, how many mice maintain body temperature above 30 °C, remain in the cold over the time course of experiment. n=8 C, 8 KO. g. Representative H&E of the iBAT from Acss2BATKO female mouse and littermate control. Scale bar 50um. Consistent across n=6 mice. h. Body weight of the Acss2BATKO female mice. n=5 C, 6 KO. i. Adipose tissue weights of the Acss2BATKO female mice. n=5 C, 6 KO. j. Lean tissue weights of the Acss2BATKO female mice. n=5 C, 6 KO. k. Rectal temperatures of the Acss2BATKO female mice and littermate controls during cold exposure (4 °C). n=4 C, 5 KO. l. ‘Survival curve’, how many mice maintain body temperature above 30 °C, remain in the cold over the time course of experiment. n=4 C, 5 KO. Data are mean ± s.e.m. Statistical analysis unpaired two-tailed Student’s t-test.
Extended Data Fig. 3 BAT ACLY Loss Broadly Impairs Metabolism.
a. Lipidomics analysis of DAG in the AclyBATKO mice BAT. ***p= 0.0007. n=9 C, 14 KO. b. Lipidomics analysis of TAG in the AclyBATKO mice BAT. n=9 C, 14 KO. c. Lipidomics analysis of CE in the AclyBATKO mice BAT. **p= 0.0035. n=9 C, 14 KO. d. Lipidomics analysis of DAG degree of saturation in the AclyBATKO mice BAT. ***p= 0.0001, **p= 0.0002. n=9 C, 14 KO. e. Lipidomics analysis of TAG degree of saturation in the AclyBATKO mice BAT. **** <0.0001, ***p= 0.0001, ***p= 0.0002. n=9 C, 14 KO. f. D2O labeling of newly synthesized palmitate in the AclyBATKO mice BAT and their littermates. *p= 0.0163. n=8 C, 8 KO. g. Palmitate abundance in BAT of AclyBATKO mice and littermate controls. **p= 0.0087. n=8 C, 8 KO. h. D2O enrichment in plasma of AclyBATKO mice and littermate controls. n=8 C, 8 KO. i. 3H-2-deoxy-glucose uptake assay into interscapular BAT, subscapular BAT, SAT, Quad and Liver of the AclyBATKO mice after 30 min at 4 °C. ***p= 0.0003, ***p= 0.0004. n=4 C, 5 KO. j. RT-PCR analysis of Glut1 and Glut4 in the AclyBATKO mice BAT. **p= 0.0015. n=6 C, 6 KO. k. RT-PCR analysis of cd36 and lpl in the AclyBATKO mice BAT. ***p= 0.0001, **p= 0.0072. n=6 C, 6 KO. Panels A-K: grey bar (C), orange bar (AclyBATKO). Data are mean ± s.e.m. Statistical analysis unpaired two-tailed Student’s t-test.
Extended Data Fig. 4 The Thermogenic Gene Program Requires ACLY.
a. Western blot of siACLY treated brown mature adipocytes. n=2 samples, result has been replicated. b. Seahorse Mito Stress Test OCR measurements of siACLY treated brown mature adipocytes. ****p <0.0001. n=30NT, n=30 siACLY wells, result has been replicated. c. Seahorse Mito Stress Test ECAR measurements of siACLY treated brown mature adipocytes. ****p <0.0001. n=30NT, n=30 siACLY wells, result has been replicated. d. Mitochondria/Nuclear DNA ratio in the siACLY treated brown mature adipocytes. n=6 C, 6 KO. e. RT-PCR analysis of Acly, Ucp1, Cycs and Pgc1a in the siRNA treated brown mature adipocytes.*p=0.0251, ***p= 0.0005, *p=0.0004, ***p=0.0373. n=4 C, 4 KO. f. Western blot of siACLY treated brown mature adipocytes, chromatin and WCL (whole cell lysate). n=3 samples. g. Western blot of ACLY inhibitor (BMS-303141) treated brown mature adipocytes, chromatin and WCL (whole cell lysate). n=2 samples. Panels B-E: grey bar (NT), orange bar (siAcly). Panels B, C: black line (NT), orange line (siAcly). Data are mean ± s.e.m. Statistical analysis unpaired two-tailed Student’s t-test.
Extended Data Fig. 5 Induced ACLY loss triggers the Integrated Stress Response and Changes in Mitochondrial Processes.
a. Body weight of the AclyBATiKO male mice. n=8 C, 8 KO. b. Adipose tissue weights of the AclyBATiKO male mice. ****p=<0.0001. n=8 C, 8 KO. c. Lean tissue weights of the AclyBATiKO male mice. n=8 C, 8 KO. d. Gene Ontology Term analysis of the AclyBATiKO male mice RNAseq. e. RT-PCR analysis of iBAT from AclyBATiKO male mice and littermate controls. **p=0.0020, **p=0.0031, *p=0.0205, ***p<0.0001, *p=0.0380, *p=0.0254 n=5 C, 5 KO. f. Western blot of the AclyBATiKO male mice. N=4 individual mice.Panels A-D: grey bar (C), orange bar (AclyBATiKO). Data are mean ± s.e.m. Statistical analysis unpaired two-tailed Student’s t-test.
Extended Data Fig. 6 ACLY's Role in Mitigating Stress is Linked to Dietary Carbohydrates.
a. RT-PCR analysis of Atf4 in the AclyBATKO mice 2 and 4 week of age. *p=0.0496. 2W: n=5 C, 5 KO; 4W: n=6 C, 6 KO. b. RT-PCR analysis of Ucp1 in the AclyBATKO mice 2 and 4 week of age. *p=0.0106. c. RT-PCR analysis of Acss2 in the AclyBATKO mice 2 and 4 week of age. *p=0.0110, p*=0.0110, *p=0.0145. d. RT-PCR analysis of Fasn in the AclyBATKO mice 2 and 4 week of age.****p<0.0001, ***p=0.0001. e. RT-PCR analysis of Chrebpa in the AclyBATKO mice 2 and 4 week of age. ****p<0.0001, ****p<0.0001. f. RT-PCR analysis of Chrebpb in the AclyBATKO mice 2 and 4 week of age. *p=0.0434, *p=0.0434, ****p<0.0001, ****p<0.0001. Panels A-F: grey bar (C), orange bar (AclyBATKO). Data are mean ± s.e.m. Group differences determined via one-tailed ANOVA with Tukey’s post hoc.
Extended Data Fig. 7 ACLY Prevents TCA Cycle Overload During Thermogenesis.
a. Serum glucose fractional labeling AclyBATKO and Acly,FasnBATKO mice. b. Serum glucose relative abundance AclyBATKO and Acly,FasnBATKO mice. 15 minutes: AclyBATKO n=6C, 9 KO; Acly,FasnBATKO n=5C, 5 KO. 30 minutes: AclyBATKO n=6C, 9 KO; Acly,FasnBATKO n=4, 5 KO.
Extended Data Fig. 8 FasnBATKO male and female mice.
a. Representative H&E of the iBAT from FasnBATKO male and female mouse and littermate control. Scale bar 50um. Has been confirmed in n=3 males, n=5 females. b. Adipose tissue weights of the FasnBATKO male mice. n=6 C, 6 KO. Adipose tissue weights of the FasnBATKO female mice. n=6 C, 6 KO. c. Western blot of the FasnBATKO male and female mice. d. Rectal temperatures of the FasnBATKO male mice and littermate controls during cold exposure (4 °C). n=7 C, 7 KO. Rectal temperatures of the FasnBATKO female mice and littermate controls during cold exposure (4 °C). n=8 C, 6 KO. e. Mitochondria/Nuclear DNA ratio in the FasnBATKO mice BAT. n=6 C, 6 KO. f. CoQ levels in the FasnBATKO mice BAT. n=9 C, 8 KO. Panels B, E, F: grey bars (C), blue bars (FasnBATKO). Panel D: black lines (C), blue lines (FasnBATKO). Data are mean ± s.e.m. Statistical analysis unpaired two-tailed Student’s t-test.
Extended Data Fig. 9 Acly,Acss2BATDKO male and female mice.
a. Representative H&E of the iBAT from Acly,Acss2BATKO male mouse and littermate control. Scale bar 50um. Clear consistent phenotype, confirmed n=6 mice. b. Western blot of the Acly,Acss2BATKO male mouse BAT. c. Adipose tissue weights of the Acly,Acss2BATKO male mice. *p=0.0291. n=5 C, 6 KO. d. Lean tissue weights of the Acly,Acss2BATKO male mice. n=5 C, 6 KO. e. Body weight of the Acly,Acss2BATKO male mice. n=5 C, 6 KO. f. Rectal temperatures of the Acly,Acss2BATKO male mice and littermate controls during cold exposure (4 °C), left. ‘Survival curve’, how many mice remain in the cold over the time course of experiment, right. n=7 C, 8 KO. g. Mitochondria/Nuclear DNA ratio in the Acly,Acss2BATKO male mice BAT. **p=0.0060. n=5 C, 6 KO. h. Representative H&E of the iBAT from Acly,Acss2BATKO female mouse and littermate control. Scale bar 50um. Clear consistent phenotype, confirmed in n=4 mice. i. Adipose tissue weights of the Acly,Acss2BATKO female mice. n=6 C, 5 KO. j. Lean tissue weights of the Acly,Acss2BATKO female mice. n=6 C, 5 KO. k. Body weight of the Acly, Acss2BATKO female mice. n=6 C, 5 KO. Panels C-E,G: grey bars (C), teal (Acly,Acss2BATKO) males. Panels I-K: grey bars (C), blue bars (Acly,Acss2BATKO) females. Panels F: black lines (C), teal lines (Acly,Acss2BATKO). Data are mean ± s.e.m. Statistical analysis unpaired two-tailed Student’s t-test.
Extended Data Fig. 10 BAT Acly;Fasn Double Knockout Rescues Thermogenesis Independent of DNL and Prevents ISR.
a. Body weight of the Acly,FasnBATKO male mice. n=5 C, 5 KO. b. Adipose tissue weights of the Acly,FasnBATKO male mice. n=5 C, 5 KO. c. Relative labeled abundance of palmitate in BAT of AclyBATKO,Acss2BATKO(n=11C, 5KO and 5KO), FasnBATKO(n=6C, 6KO),Acly/FasnBATKO(n=6C, 6KO)mice and littermate controls. *p=0.0404, ***p= 0.0007, **p=0.0035. d. Representative H&E of the iBAT from AclyBATiKO and Acly,FasnBATiKO male mouse and littermate control. Scale bar 50um. Has been confirmed in n=3 mice each. e. Body weight of the Acly,FasnBATiKO male mice. n=6 C, 6 KO. f. Adipose tissue and lean tissues weights of the Acly,FasnBATiKO male mice. n=6 C, 6 KO. g. Principal Component Analysis (PCA) plot of RNAseq from AclyBATiKO and Acly,FasnBATiKO male mouse and littermate controls. h. Heat map relative abundance of Immune Response genes in RNAseq of AclyBATiKO and Fasn,AclyBATiKO BAT. Panel G-H: light grey (C), orange bar (AclyBATiKO), dark grey (C), light purple bar (Fasn,AclyBATiKO). Data are mean ± s.e.m. Statistical analysis unpaired two-tailed Student’s t-test. Group differences determined via one-tailed ANOVA with Tukey’s post hoc.
Supplementary information
Supplementary Table 1
Primer sequences.
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GO terms for Figs. 4c–f, 7j and Extended Data Fig. 10h.
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Korobkina, E.D., Calejman, C.M., Haley, J.A. et al. Brown fat ATP-citrate lyase links carbohydrate availability to thermogenesis and guards against metabolic stress. Nat Metab 6, 2187–2202 (2024). https://doi.org/10.1038/s42255-024-01143-3
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DOI: https://doi.org/10.1038/s42255-024-01143-3
