Abstract
Thermogenesis by brown and beige adipose tissue, which requires activation by external stimuli, can counter metabolic disease1. Thermogenic respiration is initiated by adipocyte lipolysis through cyclic AMP–protein kinase A signalling; this pathway has been subject to longstanding clinical investigation2,3,4. Here we apply a comparative metabolomics approach and identify an independent metabolic pathway that controls acute activation of adipose tissue thermogenesis in vivo. We show that substantial and selective accumulation of the tricarboxylic acid cycle intermediate succinate is a metabolic signature of adipose tissue thermogenesis upon activation by exposure to cold. Succinate accumulation occurs independently of adrenergic signalling, and is sufficient to elevate thermogenic respiration in brown adipocytes. Selective accumulation of succinate may be driven by a capacity of brown adipocytes to sequester elevated circulating succinate. Furthermore, brown adipose tissue thermogenesis can be initiated by systemic administration of succinate in mice. Succinate from the extracellular milieu is rapidly metabolized by brown adipocytes, and its oxidation by succinate dehydrogenase is required for activation of thermogenesis. We identify a mechanism whereby succinate dehydrogenase-mediated oxidation of succinate initiates production of reactive oxygen species, and drives thermogenic respiration, whereas inhibition of succinate dehydrogenase supresses thermogenesis. Finally, we show that pharmacological elevation of circulating succinate drives UCP1-dependent thermogenesis by brown adipose tissue in vivo, which stimulates robust protection against diet-induced obesity and improves glucose tolerance. These findings reveal an unexpected mechanism for control of thermogenesis, using succinate as a systemically-derived thermogenic molecule.
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Acknowledgements
This work is supported by the Claudia Adams Barr Program (E.T.C.), EMBO (E.L.M.), Novo Nordisk Foundation NFF18OC0032380 (S.W.), CIHR (L.K.), NIH-DK103295 (M.C.H), NIH-DK97441 and DK112268 (S.K.), MRC-MC_U105663142, Wellcome Trust 110159/Z/15/Z (M.P.M.), and NIH-GM067945 (S.P.G.). We thank B. Spiegelman for discussions, R. Goncalves for assistance with reagents, the Nikon Imaging Center at Harvard Medical School for assistance with microscopy, and Dana-Farber/Harvard Cancer Center (NIH-5-P30-CA06516) for preparing histology slides.
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Nature thanks N. Chandel, J. Rabinowitz and the other anonymous reviewer(s) for their contribution to the peer review of this work.
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E.L.M. designed research, carried out experiments, and analysed data. K.A.P., M.P.J. and J.B.S. carried out and analysed data from mass spectrometry experiments. R.G., S.W., S.V., T.Y. and G.Z.L. carried out cellular experiments. L.K. assisted with design of in vivo experiments. A.S.B. and S.K. oversaw calorimetry and cell experiments. M.P.M. provided advice and reagents. M.C.H., S.P.G. and C.B.C. oversaw mass spectrometry experiments. E.T.C. directed research and co-wrote the paper with assistance from the other authors.
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Extended data figures and tables
Extended Data Fig. 1 Quality control for mass-spectrometry analysis of succinate in thermogenic adipose tissue.
a, Because of its unusual abundance in BAT, special consideration is required to determine the linearity of the relationship between LC–MS succinate peak intensity and succinate concentration for quantitative analysis. Succinate abundance is measured in extraction solution as described in the methods section. Absolute determination of succinate concentration is compared between succinate extracted from BAT (red) and the same samples following 100-fold dilution (green). Samples are analysed in parallel with defined amounts of [13C]succinate (black) used at concentrations that are within the established linear range of the mass spectrometer. Following 100-fold dilution of BAT extracts, succinate signals are within the linear range of detection. However, undiluted extracts are at concentrations that result in a nonlinear relationship and are therefore not appropriate for quantitative analysis. b, Calculation of the apparent dilution factor reveals the effect of nonlinearity with apparent dilutions ranging from ~11–28-fold that are in fact 100-fold.
Extended Data Fig. 2 Metabolite analysis of the acute response of BAT to β-adrenergic stimulus in vivo.
a, b, Rapid modulation of BAT triacylglycerol (TAG; a) and diacylglycerol (DAG; b) species following intravenous injection of 1 mg kg−1 CL. c, Accumulation of free fatty acid species and acyl-carnitine species in BAT following intravenous injection of CL. d, Abundance of TCA cycle metabolites in BAT following intravenous β-adrenoreceptor agonism with 1 mg kg−1 isoproterenol or 1 mg kg−1 CL (n = 5; CL, iso, n = 4). c, One-way ANOVA; d, two-sided t-test; data are mean ± s.e.m. of biologically independent samples.
Extended Data Fig. 3 13C-isotopologue labelling of glucose and TCA cycle metabolites in mouse BAT following intravenous [13C]glucose at 29 °C or 4 °C.
a, Potential inputs to succinate-directed flux by conventional BAT metabolism and 13C-metabolite labelling strategy. Mice were administered [U-13C]glucose (b–h) or [U-13C]palmitic acid (i–m) intravenously as a bolus at 29 °C or 4 °C followed by BAT harvesting and snap freezing for LC–MS analysis at indicated time points. b, Proportional isotopic labelling of BAT glucose. c, d, Proportional isotopic labelling profile of glycolytic metabolites 3-phosphoglycerate (c) and lactate (d) in mouse BAT. e–h, Proportional isotopic labelling profile of TCA cycle metabolites citrate (e), succinate (f), fumarate (g), and malate (h) in mouse BAT. i, Proportional isotopic labelling of BAT palmitate. j–m, Proportional isotopic labelling profile of TCA cycle metabolites citrate (j), succinate (k), fumarate (l), and malate (m) in BAT (n = 5). Missing values or N.D. indicate value not determined. Data are mean ± s.e.m. of biologically independent samples.
Extended Data Fig. 4 Analysis of succinate levels in isolated brown adipocytes and effect of muscle shivering on BAT succinate accumulation.
a, Abundance of succinate in mouse plasma comparing 29 °C to acute 4 °C exposure (n = 6). b, Comparison of succinate abundance in BAT in vivo (n = 8) versus brown adipocytes (n = 7). c–f, Full 13C-isotopologue profile of fumarate (c), malate (d), citrate (e), and aspartate (f) in brown adipocytes following extracellular addition of [13C]succinate (n = 5). g, 13C-isotopologue (m + 4) profile of TCA metabolites downstream of mitochondrial succinate oxidation in BAT following intravenous administration of 100 mg kg−1 [13C]succinate as a bolus (n = 3). h, Time course of abundance of (m + 4) [13C]succinate in plasma following 100 mg kg−1 intravenous [13C]succinate (n = 3, except 15 min, n = 4). i, Representative mouse nuchal muscle EMG traces at 29 °C and after acute cold exposure with or without curare (0.1 mg kg−1). j, Quantification of succinate in BAT at 29 °C and after acute cold exposure with or without inhibition of muscle shivering with curare (0.1 mg kg−1; n = 5). k, Effect of acute addition of cellular and mitochondrial respiratory substrates on brown adipocyte respiration. Pyruvate: vehicle, n = 7; 1 mM, n = 6; 10 mM, n = 7; glucose: vehicle, n = 7; 1 mM, n = 6; 10 mM, n = 7; glutamine: vehicle, n = 6; 1 mM, n = 6; 10 mM, n = 7; G3P: vehicle, n = 7; 1 mM, n = 6; 10 mM, n = 6; αKG: vehicle, n = 6; 1 mM, n = 6; 10 mM, n = 5; fumarate: vehicle, n = 6; 1 mM, n = 6; 10 mM, n = 5; malate: vehicle, n = 6; 1 mM, n = 6; 10 mM, n = 7. l, Effect of acute addition of palmitic acid on brown adipocyte respiration (vehicle, n = 18; palmitic acid, n = 16). Effects on respiration were determined by acute addition of oligomycin (oli) to determine leak respiration, 2,4-dinitrophenol (DNP) to determine chemically uncoupled maximal respiration, and rotenone plus antimycin (r/a) to determine non-mitochondrial respiration. In all cases basal respiration in these cells is measured in the presence of 1 mM pyruvate. One-way ANOVA (a, j); data are mean ± s.e.m. of biologically independent samples.
Extended Data Fig. 5 Representative OCR experiments for brown adipocytes and other cell types with or without acute addition of succinate and related substrates.
a, Representative OCR trace monitoring effect of acute addition of succinate or noradrenaline (NE) in the De2.3 immortalized brown adipocyte cell line32 (vehicle, n = 7; succinate, n = 6; NE, n = 7). b, Representative OCR trace monitoring dose-dependent effect of acute addition of succinate in the De2.3 immortalized brown adipocyte cell line (vehicle, n = 7; 1 mM succinate, n = 6; 5 mM succinate, n = 7). c, Representative OCR trace monitoring dose-dependent effect of acute addition of succinate in various cell types (subcutaneous white adipocytes: vehicle, n = 6; 1 mM, n = 6; 5 mM, n = 5; myoblasts: vehicle, n = 7; 1 mM, n = 6; 5 mM, n = 7; HEK: vehicle, n = 7; 1 mM, n = 6; 5 mM, n = 7; hepatocytes: vehicle, n = 7; 1 mM, n = 6; 5 mM, n = 7; osteocytes: vehicle, n = 7; 1 mM, n = 6; 5 mM, n = 7; A549 lung: vehicle, n = 7; 1 mM, n = 6; 5 mM, n = 7; brown pre-adipocytes: vehicle, n = 7; 1 mM, n = 6; 5 mM, n = 7). d, Effect of acute addition of succinate on cellular OCR in human brown adipocytes (basal, n = 6; 1 mM, n = 6; 5 mM, n = 7). e, Representative OCR trace monitoring dose-dependent effect of acute addition of succinate in human immortalized brown adipocytes (vehicle, n = 7; 1 mM succinate, n = 6; 5 mM succinate, n = 7). f, Inhibition of succinate-stimulated OCR in brown adipocytes by DTNB. g, Representative OCR experiment (n = 7; 0.1 mM DTNB, n = 6). h, i, Inhibition of succinate-stimulated OCR in brown adipocytes by DIDS. n = 12, except 1 mM DIDS, n = 8). j, k, Inhibition of succinate-stimulated OCR in brown adipocytes by treatment with the Na+/K+ ATPase inhibitor ouabain (n = 5). Effects on respiration were determined by acute addition of oligomycin (oli) to determine leak respiration, 2,4-dinitrophenol (DNP) to determine chemically uncoupled maximal respiration, or rotenone plus antimycin (r/a) to determine non-mitochondrial respiration. In all cases basal respiration in these cells is measured in the presence of 1 mM pyruvate. Two-sided t-test (d); two-way ANOVA (f, h, j); data are mean ± s.e.m. of biologically independent samples.
Extended Data Fig. 6 Examining mechanisms of succinate-driven thermogenesis in brown adipocytes.
a, Succinate-induced respiration is intact in brown adipocytes lacking SUCNR1 (n = 10, except 1 mM succinate, n = 9). b, Measurement of cAMP in brown adipocytes 10 min following addition of succinate. c, Immunoblot analysis of PKA substrate phosphorylation following addition of succinate (30 min) or NE (5 min). d, Glycerol release rate from brown adipocytes as an index of lipolysis in response to succinate or NE (n = 6). e–g, Effect of acute addition of succinate in mitochondria isolated from BAT, monitoring effects on basal respiration rate (f), leak respiration (g), and chemically uncoupled maximal respiration (g). n = 7, except succinate, n = 8). h, Quantitation of SLC25A10 protein levels in mouse liver, brain, heart, and BAT (n = 3). i, j, Inhibition of succinate-stimulated OCR in brown adipocytes by treatment with the SLC25A10 inhibitor diethyl butylmalonate (DEBM; n = 11). Data are mean ± s.e.m. of at least three replicates. k, Effect of succinate treatment on ROS levels in brown adipocyte assessed by DHE oxidation (n = 15). l, Acute addition of succinate drives rapid DHE oxidation in brown adipocytes (n = 15). m, Representative high resolution microscopy images illustrating effect of acute (10 min) addition of succinate on DHE oxidation in brown adipocytes. Scale bars, 20 μm. Two-sided t-test (a, e, f, g); one-way ANOVA (d, l); two-way ANOVA (k); data are mean ± s.e.m. of biologically independent samples.
Extended Data Fig. 7 Mechanisms of succinate-driven thermogenic ROS and respiration in brown adipocytes.
a, Effect of succinate treatment on Prx3 cysteine-thiol sulfonic acid formation (vehicle, n = 4; succinate, n = 3). b, Effect of succinate treatment on Prx family cysteine-thiol sulfonic acid (SO3−) status (vehicle, n = 4; succinate, n = 3). c, Representative OCR experiments on brown adipocytes with or without acute added succinate with or without MitoQ or NAC. Effects on respiration were determined by acute addition of oligomycin (oli) to determine leak respiration, DNP to determine chemically uncoupled maximal respiration, and rotenone plus antimycin (r/a) to determine non-mitochondrial respiration (MitoQ: vehicle, n = 6; 100 nM, n = 6; 500 nM, n = 5; NAC: vehicle, n = 7; 5 mM, n = 6; 10 mM, n = 7). d, e, Representative OCR experiments on brown adipocytes with and without acute addition of diamide. Vehicle, n = 6; 200 µM diamide, n = 5. f, Potential pathways for succinate-driven thermogenic ROS in brown adipocytes via SDH or electron transfer via ubiquinol (QH2): (1) Malonate inhibits succinate oxidation by SDH19; (2) atpenin A5 (AA5) inhibits electron transfer between SDH and the ubiquinone pool20; (3) S1Q2.2 inhibits ROS production from mitochondrial complex I21; (4) iGP1 inhibits electron transfer between αGPDH and the ubiquinone pool23; and (5) S3Q3 inhibits ROS production from mitochondrial complex III22. g, Representative OCR experiment demonstrating inhibition of succinate stimulated OCR by suppression of SDH oxidation with malonate (vehicle, n = 6; 1 mM malonate, n = 6; 5 mM malonate, n = 5). h, i, Treatment of brown adipocytes with malonate results in rapid intracellular accumulation (n = 4) (h) and prevents succinate-driven DHE oxidation (i) in brown adipocytes (n = 30). j–l, Representative OCR experiments in brown adipocytes with or without acute addition of succinate, with or without AA5 (j; vehicle, n = 6; 10 nM, n = 6; 100 nM, n = 5); S1Q2.2 (k, l; vehicle, n = 6; 10 μM, n = 6; 100 μM, n = 7); S3Q3 (m, n; vehicle, n = 13; 1 μM n = 12; 10 μM n = 13). o, Treatment of brown adipocytes with S3Q3 has no effect on succinate-driven DHE oxidation in brown adipocytes (n = 30, except 1 μM, n = 15). p, Succinate stimulation of brown adipocyte OCR with or without iGP(iGP vehicle, n = 21; 10 µM iGP, n = 17; 100 µM iGP, n = 26). q, Representative OCR experiments in brown adipocytes with or without acute addition of succinate and/or iGP1 (vehicle, n = 7; 1 μM, n = 6; 100 μM, n = 7). Two-sided t-test (a, e, i); one-way ANOVA (h, o); two-way ANOVA (k, m, p); data are mean ± s.e.m. of biologically independent samples.
Extended Data Fig. 8 Metabolic characterization of mice following systemic succinate administration.
a, Effect of acute intravenous administration of succinate on interscapular temperature (vehicle, n = 6; succinate, n = 5). b, Acute effect of intravenous succinate on whole body oxygen consumption in wild-type (WT) and UCP1(KO) mice. Basal O2 consumption rate determined as described in the Methods (vehicle, n = 10; succinate, n = 7; UCP1(KO), n = 9). c, Mouse interscapular temperature following acute exposure to 4 °C with or without acute intravenous administration of malonate (n = 8). Malonate was administered 10 min before transition to 4 °C. d, Acute oral administration of succinate by gavage drives elevation of circulating succinate (n = 4, except 10% 30 min, n = 6). e, Water consumption during high-fat feeding with or without intervention with 1% and 1.5% sodium succinate in drinking water indicates lack of aversion to succinate-containing water (vehicle, n = 35; 1%, n = 26; 1.5%, n = 18). f, Water consumption during high-fat feeding with or without intervention with 2% succinate in drinking water indicates lack of aversion to succinate-containing water (vehicle, n = 24; 2%, n = 22). g, Body weights of high-fat diet feeding mice before intervention with 1% and 1.5% sodium succinate in drinking water (vehicle, n = 35; 1%, n = 26; 1.5%, n = 18). h, Body weights of high-fat diet feeding mice before intervention with 2% sodium succinate in drinking water (vehicle, n = 24; 2%, n = 22; pair fed, n = 18). i, Caloric consumption during high-fat feeding with or without intervention with 1% or 1.5% sodium succinate in drinking water (vehicle, n = 35; 1%, n = 26; 1.5%, n = 18). j, Caloric consumption during high-fat feeding with or without 2% sodium succinate in drinking water, pair-fed mice in this experiment were fed the same number of calories as the 2% succinate group (vehicle, n = 24; 2%, n = 22; pair fed, n = 18). k, l, Caloric absorption and energy assimilation during high-fat feeding with or without 1.5% (k) or 2% (l) sodium succinate in drinking water. Proportion of energy assimilated from diet was determined by subtracting the total calories remaining in mouse faeces from the total calories consumed in the same period (n = 6). *P < 0.05, **P < 0.01; two-way ANOVA (a, b (left), c); one-way ANOVA (b (middle, right), d); two-sided t-test (k); data are mean ± s.e.m. of biologically independent samples.
Extended Data Fig. 9 Assessment of morphologic effects of systemic succinate administration on mouse tissues.
a–f, Representative images of haematoxylin and eosin (a, b, d–f) or Masson’s trichrome (c) staining of indicated tissues harvested from mice following high-fat feeding with or without 4 weeks succinate supplementation in drinking water. (a, c, e top panels: 4× magnification; scale bars, 1 mm; a, c, e bottom panels, b, d, f: 40× magnification, scale bars, 50 μm). d, Cardiac morphometric analysis with or without 1.5% sodium succinate. Lower panels show representative images of cell width (40× magnification; scale bars, 50 μm). Bar charts show quantitative analysis of cardiomyocyte width and length and nuclear diameter (n = 15). g, Intraperitoneal glucose tolerance test in mice following high-fat feeding with or without 4 weeks succinate supplementation in drinking water, quantifying relative changes in glucose upon glucose challenge (n = 9). h, i, mRNA expression of inflammatory (h) and anti-inflammatory (i) markers in the indicated tissues with or without 1.5% sodium succinate in wild-type and Ucp1−/− mice (n = 3). Two-way ANOVA (g); one-way ANOVA (h); data are mean ± s.e.m. of biologically independent samples.
Extended Data Fig. 10 Metabolic characterization of UCP1-deficient mice following systemic succinate administration.
a, Change in body mass in Ucp1+/− (n = 9; 1.5% n = 9) and Ucp1−/− (UCP1KO, n = 7; 1.5% n = 8) male mice during high-fat feeding with or without 1.5% sodium succinate in drinking water. b, Change in body mass in Ucp1+/− (0%, n = 9; 1.5%, n = 8) and Ucp1−/− (0%, n = 6; 1.5% n = 7) female mice during high-fat feeding with or without 1.5% sodium succinate in drinking water. c, Body weights of high-fat diet feeding Ucp1+/− (vehicle, n = 18; 1.5%, n = 17) and Ucp1−/− (vehicle, n = 13; 1.5%, n = 15) mice prior to intervention with 1.5% sodium succinate in drinking water. d, Water consumption during high-fat feeding with or without intervention with 1.5% sodium succinate in drinking water in Ucp1+/− (0%, n = 18; 1.5%, n = 17) and Ucp1−/− (0%, n = 13; 1.5%, n = 15) mice indicates lack of aversion to succinate-containing water. e, Energy consumption during high-fat feeding with or without intervention with 1.5% sodium succinate in drinking water in Ucp1+/− (0%, n = 18; 1.5%, n = 17) and Ucp1−/− (0%, n = 13; 1.5%, n = 15) mice. f, Energy expenditure of Ucp1+/− and Ucp1−/− mice during 6 weeks high-fat feeding with or without 1.5% sodium succinate (Ucp1+/− 0%, n = 18; Ucp1+/− 1.5%, n = 17; Ucp1−/− 0%, n = 13; Ucp1−/− 1.5%, n = 15). Two-way ANOVA (a, b); one-way ANOVA (f); data are mean ± s.e.m. of biologically independent samples.
Supplementary information
Supplementary Figure 1
The uncropped blots from Extended Data Fig. 6.
Supplementary Table 1
Quantification of metabolites identified by targeted LC-MS metabolomics. Relative abundance is expressed as BAT abundance relative to subcutaneous white adipose tissue abundance. Log2FC = log2 of fold change between BAT and subcutaneous abundance. Negative log10(pvalue) = negative log10 of the P value.
Supplementary Table 2
Peak ion intensity of metabolites identified by targeted LC-MS metabolomics in BAT.
Supplementary Table 3
Quantification of metabolites identified by targeted LC-MS metabolomics. Relative abundance is expressed as BAT abundance following 3 Hrs exposure of mice to 4 degrees relative to BAT from mice exposed to thermoneutrality. Log2FC = log2 of fold change between BAT from cold exposed mice versus thermoneutrality. Negative log10(pvalue) = negative log10 of the P value.
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Mills, E.L., Pierce, K.A., Jedrychowski, M.P. et al. Accumulation of succinate controls activation of adipose tissue thermogenesis. Nature 560, 102–106 (2018). https://doi.org/10.1038/s41586-018-0353-2
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DOI: https://doi.org/10.1038/s41586-018-0353-2
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