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Mitochondrial ROS regulate thermogenic energy expenditure and sulfenylation of UCP1

A Corrigendum to this article was published on 18 May 2016

Abstract

Brown and beige adipose tissues can dissipate chemical energy as heat through thermogenic respiration, which requires uncoupling protein 1 (UCP1)1,2. Thermogenesis from these adipocytes can combat obesity and diabetes3, encouraging investigation of factors that control UCP1-dependent respiration in vivo. Here we show that acutely activated thermogenesis in brown adipose tissue is defined by a substantial increase in levels of mitochondrial reactive oxygen species (ROS). Remarkably, this process supports in vivo thermogenesis, as pharmacological depletion of mitochondrial ROS results in hypothermia upon cold exposure, and inhibits UCP1-dependent increases in whole-body energy expenditure. We further establish that thermogenic ROS alter the redox status of cysteine thiols in brown adipose tissue to drive increased respiration, and that Cys253 of UCP1 is a key target. UCP1 Cys253 is sulfenylated during thermogenesis, while mutation of this site desensitizes the purine-nucleotide-inhibited state of the carrier to adrenergic activation and uncoupling. These studies identify mitochondrial ROS induction in brown adipose tissue as a mechanism that supports UCP1-dependent thermogenesis and whole-body energy expenditure, which opens the way to improved therapeutic strategies for combating metabolic disorders.

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Figure 1: Increased BAT mitochondrial ROS levels support UCP1-dependent thermogenesis in vivo.
Figure 2: BAT mitochondrial ROS during thermogenesis drives oxidation of cellular and mitochondrial thiols.
Figure 3: BAT mitochondrial ROS oxidatively modify a cysteine residue on UCP1 and support UCP1-dependent leak respiration.
Figure 4: UCP1 Cys253 is sulfenylated during thermogenesis and sensitizes UCP1 to adrenergic activation.

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Acknowledgements

Supported by the JPB Foundation and National Institutes of Health (DK031405) to B.M.S. and by grants from Human Frontiers Science Program (to E.T.C.) and the Canadian Institutes for Health Research (to L.K.). We acknowledge M. Murphy, Y. Kirichok, and A. Bertholet for many discussions. We also thank M. Murphy for providing MitoQ.

Author information

Authors and Affiliations

Authors

Contributions

E.T.C. designed research, performed biochemical, cellular, and in vivo experiments, analysed data, and co-wrote the paper. L.K. designed and performed cellular and mutagenesis experiments. M.P.J. and K.A.P. performed and analysed data from mass spectrometric experiments. G.Z.L. performed cellular experiments. C.B.C. and S.P.G. oversaw mass spectrometric experiments. A.J.R. designed and performed structural modelling. E.T.C. and B.M.S. directed the research and co-wrote the paper, with assistance from all other authors.

Corresponding author

Correspondence to Bruce M. Spiegelman.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Assessing superoxide in brown adipocytes and effect of mitochondria-targeted compounds on shivering, body temperature, and movement.

a, Noradrenaline stimulates superoxide-dependent oxidation of DHE in primary brown adipocytes (n = 5). b, Effect of i.p. decyl-TPP on core body temperature after acute cold exposure (n = 10). c, d, Representative (c) raw and (d) root mean square mouse EMG traces at thermoneutrality and after acute cold exposure ± MitoQ, NAC, or curare (0.3 mg kg−1). e, Quantification of muscle burst frequency as determined by EMG at thermoneutrality and after acute cold exposure ± MitoQ or NAC (n = 3). f, Absolute oxygen consumed immediately before acute CL treatment described in Fig. 1f (n = 5). g, Effect of i.p. CL ± MitoQ on movement as assessed by number of beam breaks (n = 8). NS, not significant. Data are mean ± s.e.m. of at least three replicates. *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed Student’s t-test for pairwise comparisons, one-way ANOVA for multiple comparisons, two-way ANOVA for multiple comparisons involving two independent variables).

Extended Data Figure 2 Assessing thiol redox status in vivo and the effect of NAC on movement.

a, Mass spectrometric quantification of BAT-reduced and -oxidized glutathione at thermoneutrality and after acute cold exposure (n = 5). b, Scheme for quantitative assessment of protein thiol redox status by ratiometric labelling of BAT protein cysteine thiols. In vivo BAT thiol status is stabilized by protein precipitation in 20% TCA49. Unmodified cysteine thiols are labelled with ‘light’ iodoTMT tags (126, 127, 128). After removal of light iodoTMT, reversibly modified protein thiols are reduced with TCEP in the presence of ‘heavy’ iodoTMT (129, 130, 131). Samples are combined and subjected to trypsin digestion. Ratiometric assessment of iodoTMT labelled peptides provides a quantitative profile of overall protein cysteine thiol redox status. c, Average percentage oxidation status of total BAT and BAT mitochondrial protein thiols at thermoneutrality and after acute cold exposure (n = 3). d, Effect of i.p. NAC on movement as assessed by number of beam breaks (vehicle, n = 11; NAC, n = 7). Data are mean ± s.e.m. of at least three replicates. *P < 0.05, ***P < 0.001 (two-tailed Student’s t-test for pairwise comparisons, one-way ANOVA for multiple comparisons, two-way ANOVA for multiple comparisons involving two independent variables).

Extended Data Figure 3 Assessing thermogenic gene expression, adrenergic response parameters, and strategy for determination of UCP1 cysteine thiol redox status.

a, b, Quantitative polymerase chain reaction (qPCR) analysis of mRNA expression of selected (a) BAT and (b) inguinal white adipose tissue (iWAT) genes ± cold exposure, ± MitoQ or NAC (n = 5). c, d, Immunoblot analysis of (c) PPAR-γ protein expression levels and (d) lipolytic phosphorylation cascade activation in BAT after cold exposure ± MitoQ. e, f, Raw OCR of primary brown adipocytes under basal conditions and after noradrenaline stimulation + oligomycin to determine leak respiration ± (e) MitoQ (n = 10) or (f) NAC (vehicle and 1 mM NAC, n = 8; 0.1 mM NAC, n = 7). g, h, OCR of primary brown adipocytes lacking UCP1 under basal conditions and after noradrenaline stimulation + oligomycin ± (g) MitoQ (n = 10) and (h) NAC (n = 10). i, Cys-redox mass shift strategy. After in vivo interventions, mouse BAT is excised and unmodified protein thiols are labelled with NEM, after which reversibly oxidized thiols are chemically reduced and labelled with polyethyleneglycol maleimide (PEG-Mal) allowing for determination of cysteine oxidation status on UCP1. Data are mean ± s.e.m. of at least five replicates. *P < 0.05, **P < 0.01 (two-tailed Student’s t-test for pairwise comparisons, one-way ANOVA for multiple comparisons, two-way ANOVA for multiple comparisons involving two independent variables).

Extended Data Figure 4 Assessing UCP1 reversible cysteine oxidation status in vivo by immunoblot and mass spectrometry.

a, Calibration of UCP1 cysteine gel shift immunoblot. Calibration of cysteine-dependent shifts by incubation of BAT protein with TCEP and different ratios of NEM and PEG-Mal indicates that a single PEG-mal labelling event shifts UCP1 by ~10 kDa above the native molecular mass. b, Calibration of UCP1 cysteine oxidation status indicates that the gel shift observed upon cold exposure (lane 1) is cysteine dependent, as TCEP pretreatment results in a loss of the shift (lane 2). In addition, the cysteine-dependent mass shift is due to a single oxidation event as determined by including the calibrating markers (lanes 4–6). c, Calibration of specificity of UCP1 antibody in BAT. d, Reducing and non-reducing SDS–PAGE analysis of UCP1 to monitor cysteine-dependent and -independent inter-protein interactions during acute cold exposure. e, Scheme for identification of sulfenylated cysteines on UCP1 by dimedone labelling and mass spectrometry. After acute cold exposure, BAT protein thiols are differentially alkylated with dimedone to selectively label sulfenylated thiols and NEM to label non-sulfenylated thiols. Samples are subjected to trypsin digestion, followed by Lys-TMT labelling, and MS quantification of UCP1 cysteine containing peptides in their dimedone and NEM alkylated forms. Two technical points should be noted in this strategy when interpreting relative quantitation of NEM and dimedone-alkylated peptides. First, these differently alkylated peptides may not necessarily ionize with the same efficiency. Second, NEM is reported to react with sulfenic acids albeit less efficiently than with free thiols50, which should be factored in when considering the order of addition of dimedone/NEM and potential underestimation of sulfenylation status. f, Top: amino-acid sequence alignment of UCP1 proteins highlighting the candidate cysteine residues contained within the mouse protein and their level of conservation across various species. Bottom: summary of MS determination of UCP1 cysteine sulfenylation status. Six of seven UCP1 cysteines were identified, with all but one being identified exclusively in the unmodified (NEM-alkylated state). Cys253 is identified as dimedone labelled, indicating that it is a site for sulfenylation.

Extended Data Figure 5 Structure of human UCP1 modelled on the AAC crystal structure including bound cardiolipin and sulfenylation of Cys253.

a, Entire UCP1 modelled structure including bound cardiolipin (green), and Cys253 in the oxidized sulfenic acid form. b, UCP1 region containing Cys253 in the oxidized sulfenic acid form. Cys253 localizes to a hydrophobic pocket between two matrix facing helices. Hydrophobic residues (pink) surround the Cys253 thiol, and a hydrogen bond between Arg238 and Glu261 (aqua) is proximal. These residues that stabilize interaction between the matrix facing helices are probably important for stabilization of the purine bound ‘c-state’ of the carrier. Two separate cardiolipin (green) binding domains are localized proximal to Cys253 within the UCP1 modelled structure.

Extended Data Figure 6 Assessing transduced UCP1 constructs, OCR, and sulfenylation in brown adipocytes.

a, Quantitative PCR analysis of UCP1 mRNA in WT, and UCP1−/− brown adipocytes transduced with WT and cysteine null UCP1 mutants (n = 4). b, Immunoblot of UCP1 protein in WT and UCP1−/− brown adipocytes transduced with WT and cysteine null UCP1 mutants. c, Immunoblot analysis of UCP1 protein in UCP1−/− brown adipocytes transduced with C224A/C253A double mutant compared with WT. d, Densitometry analysis of transduced UCP1 forms relative to WT across separate transduction experiments (n = 4; C224A/C253A n = 3). e, f, Summary of basal (e) and maximal (f) OCR of primary brown adipocytes containing cysteine-null UCP1 mutants. Raw OCR values provided in Extended Data Fig. 7. g, Immunodetection of protein sulfenic acid levels in primary brown adipocytes in the seconds after treatment with 100 nM noradrenaline. h, Time course of brown adipocyte OCR after stimulation with 100 nM noradrenaline (n = 12). Data are mean ± s.e.m. of at least three replicates.

Extended Data Figure 7 Assessing OCR under basal and FCCP-stimulated maximal respiration, and after noradrenaline stimulation + oligomycin in UCP1−/− primary brown adipocytes transduced with UCP1 cysteine null mutants.

a–g, Raw basal, maximal, and UCP1-dependent OCR from representative experiments of WT and UCP1−/− brown adipocytes transduced with (a) UCP1 C24A (WT n = 11; C24A n = 12), (b) C188A (n = 7), (c) UCP1 C213A (n = 19), (d) UCP1 C287A (WT n = 9; C287A n = 10), (e) UCP1 C304A (WT n = 7; C304A n = 10), (f) UCP1 C224A (WT n = 9; C224A n = 10), (g) UCP1 C224A/C253A (n = 8). Data are mean ± s.e.m. of at least seven replicates. **P < 0.01 (two-tailed Student’s t-test for pairwise comparisons).

Extended Data Figure 8 Assessing UCP1-dependent respiration and uncoupling following increasing degrees of adrenergic stimulus.

a, Glycerol release from brown adipocytes as an index of adrenergic stimulus and lipolysis in response to increasing concentrations of noradrenaline (n = 4; 0 and 2,000 nM noradrenaline n = 6). b, Representative raw noradrenaline + oligomycin leak OCR values in WT and UCP1−/− brown adipocytes after stimulation with a range of noradrenaline concentrations indicates that UCP1-dependent leak respiration is consistently ~25–35% of total leak OCR (50 nM n = 9; 100 nM n = 7; 500 nM n = 8; 2,000 nM n = 6). c, Assessment of UCP1-dependent leak respiration after stimulation by various concentrations of noradrenaline + oligomycin. Comparison of WT and UCP1−/− OCR (replotted data from Fig. 4 for comparison) indicates that UCP1-dependent respiration is consistently ~25–35% of leak respiration. Comparison of UCP1 WT and C224A indicates that the degree of UCP1 inhibition by C224A is relatively stable across various noradrenaline concentrations (n = 8; C224A 100 nM noradrenaline n = 9, 2,000 nM noradrenaline n = 14). d, Plasma-membrane-permeabilized OCR of brown adipocytes ± endogenous fatty-acid depletion with BSA (WT n = 30; KO n = 20; C224A n = 9; C253A n = 8; C224A/C253A n = 10). e, Comparison of UCP1-dependent uncoupling absent purine nucleotide inhibition in plasma-membrane-permeabilized brown adipocytes (n = 6; C224A/C253A, KO n = 4). f, Comparison of UCP1-dependent uncoupling after titration of GDP in permeabilized adipocytes containing WT UCP1, UCP1 C224A, C253A, and C224A/C253A (n = 6; C224A/C253A n = 12). Data are mean ± s.e.m. of at least four replicates. *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed Student’s t-test for pairwise comparisons, one-way ANOVA for multiple comparisons).

Supplementary information

Supplementary Figures

This file contains Supplementary Figures 1-3, which contain the uncropped scans with size markers indications for Figures 1-3 and Extended Data Figure 3 (1); Extended Data Figure 4 (2) and Extended Data Figure 6 (3). (PDF 512 kb)

Supplementary Data

This file contains the MS2 spectra of all UCP1 cysteine, containing peptides identified in the dimedone-MS experiments in either their unmodified (NEM alkylated form) or dimedone alkylated form. (PDF 3085 kb)

Supplementary Table 1

Iodo-TMT data file including summary of all identified proteins and peptides, those identified as substantially redox sensitive during thermogenesis, and pathway analysis. (XLSX 148 kb)

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Chouchani, E., Kazak, L., Jedrychowski, M. et al. Mitochondrial ROS regulate thermogenic energy expenditure and sulfenylation of UCP1. Nature 532, 112–116 (2016). https://doi.org/10.1038/nature17399

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