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IL-1 induces mitochondrial translocation of IRAK2 to suppress oxidative metabolism in adipocytes

Nature Immunology volume 21, pages 1219–1231 (2020)Cite this article

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Abstract

Chronic inflammation is a common feature of obesity, with elevated cytokines such as interleukin-1 (IL-1) in the circulation and tissues. Here, we report an unconventional IL-1R–MyD88–IRAK2–PHB/OPA1 signaling axis that reprograms mitochondrial metabolism in adipocytes to exacerbate obesity. IL-1 induced recruitment of IRAK2 Myddosome to mitochondria outer membranes via recognition by TOM20, followed by TIMM50-guided translocation of IRAK2 into mitochondria inner membranes, to suppress oxidative phosphorylation and fatty acid oxidation, thereby attenuating energy expenditure. Adipocyte-specific MyD88 or IRAK2 deficiency reduced high-fat-diet-induced weight gain, increased energy expenditure and ameliorated insulin resistance, associated with a smaller adipocyte size and increased cristae formation. IRAK2 kinase inactivation also reduced high-fat diet-induced metabolic diseases. Mechanistically, IRAK2 suppressed respiratory super-complex formation via interaction with PHB1 and OPA1 upon stimulation of IL-1. Taken together, our results suggest that the IRAK2 Myddosome functions as a critical link between inflammation and metabolism, representing a novel therapeutic target for patients with obesity.

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Fig. 1: IL-1β inhibits mitochondrial oxidative phosphorylation and super-complex formation in adipocytes.
Fig. 2: Adipocyte-specific MyD88 deficiency (Myd88AKO) improves HFD-induced obesity and energy expenditure.
Fig. 3: IL-1β induces translocation of IRAK2 into inner mitochondrial membranes to suppress oxidative phosphorylation and FAO.
Fig. 4: IRAK2 deficiency improves HFD-induced obesity and energy expenditure.
Fig. 5: IRAK2 deficiency prevents HFD-induced BAT dysfunction.
Fig. 6: IRAK2 disrupts mitochondrial respiratory chain super-complex formation via interaction with PHB–OPA1.
Fig. 7: IRAK2 kinase activity contributes to HFD-induced obesity and BAT dysfunction.
Fig. 8: Adipocyte-specific IRAK2 contributes to HFD-induced obesity and BAT dysfunction.

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Data availability

The primary data for analysis of all figures and supplementary figures are available upon request. Source data are provided with this paper.

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Acknowledgements

This work was supported by grants from the NIH (2P01HL029582, R01 AA023722, P01CA062220, R01 HL122283 and P50AA024333) and National Multiple Sclerosis Society (RG5130A2/1). H.Z. was supported by a Postdoctoral Research Fellowship Award (1-16-PDF-138) from the American Diabetes Association.

Author information

Author notes

  1. Julie A. Carman

    Present address: Immunology Discovery, Janssen Research and Development, Spring House, PA, USA

  2. These authors contributed equally: Hao Zhou, Han Wang, Minjia Yu.

Authors and Affiliations

  1. Department of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA

    Hao Zhou, Han Wang, Minjia Yu, Wen Qian, Fangqiang Tang, Weiwei Liu, Hui Yang, Ruth E. McDowell, Junjie Zhao & Xiaoxia Li

  2. School of Life Sciences, Lanzhou University, Lanzhou, China

    Han Wang

  3. Department of Medicine, Mount Auburn Hospital, Harvard Medical School, Cambridge, MA, USA

    Minjia Yu

  4. Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA

    Rebecca C. Schugar, Christopher Hine, Jonathan D. Smith, Paul L. Fox & J. Mark Brown

  5. Discovery Biology, Bristol Myers Squibb, Princeton, NJ, USA

    Ji Gao, Ashok Dongre & Julie A. Carman

  6. Imaging Core, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA

    Mei Yin & Judith A. Drazba

  7. University of Ottawa and Ottawa Hospital, Ottawa, Ontario, Canada

    Robert Dent

  8. Department of Integrative Medical Sciences, College of Medicine, Northeast Ohio Medical University, Rootstown, OH, USA

    Yeong-Renn Chen

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Contributions

H.Z., H.W., M. Yu, R.C.S., W.Q., F.T., W.L., H.Y., R.E.M. and J.Z. conducted the experiments. J.A.C., J.G. and A.D. performed the proteomics analysis. R.D. collected human adipose tissue samples. M. Yin and J.A.D. performed the electron microscopy analysis. H.Z. and X.L. wrote the manuscript. C.H., Y.-R.C., J.D.S., P.L.F., J.M.B. and X.L. supervised the study.

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Correspondence to Xiaoxia Li.

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Extended data

Extended Data Fig. 1 Extended information related to Fig. 1.

a. Characteristics of patients, related to Fig. 1a. b. Extracellular acidification rate (ECAR), related to Fig. 1d. Data represent mean ± SEM. Data represent one of five independent experiments with similar results. c. Coomassie blue staining of mitochondrial proteins, related to Fig. 1e. d. Mitochondrial proteins were extracted from WT and Il1r1 KO; WT and Irak2 KO newly differentiated primary adipocytes treated with or without IL-1β for indicated time points and analyzed by SDS-PAGE, followed by western blot analysis with indicated antibodies. Data represent one of five independent experiments with similar results. e. Octanoyl-carnitine oxidation-rate in isolated mitochondria from WT and Il1r1 KO primary adipocytes treated with or without IL-1β (n=4). Student’s t-test (two-tailed) was performed. Data represent mean ± SEM.

Source data

Extended Data Fig. 2 IL-1 stimulation did not lead to mitochondria dysfunction.

Newly differentiated primary adipocytes were treated with IL-1β for indicated time points. Cytosolic mtDNA (n=3) (a) and cytosolic Ca2+ (n=4) (b) levels were measured. c. Western blot analysis of cytoplasmic and mitochondrial proteins from IL-1β or TNFα + cycloheximide (CHX) treated newly differentiated primary adipocytes. Data represent one of five independent experiments with similar results. d. Newly differentiated primary adipocytes were treated with IL-1β or trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP) for indicated time points. Cellular reactive oxygen species (ROS) were measured using fluorescent microscopy and microplate reader (n=5). e. Western blot analysis of cytoplasmic and mitochondrial proteins from oligomycin + antimycin (OA) treated newly differentiated WT and Irak2 KO primary adipocytes (pretreated with IL-1β or PBS). Data represent one of five independent experiments with similar results. Densitometric analysis of LC3 I/LC3 III were listed a bar graph (n=3). a, b, e: Student’s t-test (two-tailed) was performed. Data represent mean ± SEM. d: One-way ANOVA was performed. Data represent mean ± SEM.

Source data

Extended Data Fig. 3 Extended information related to Fig. 2.

a. Total lysates from iWAT, BAT and liver from Myd88FF and Myd88AKO mice were subjected to western blot analysis with indicated antibodies. Data represent one of five independent experiments with similar results. b. Transmission electron microscopy analysis of iWAT sections from HFD-fed mice. Scale bars, 1 μm. Morphometric analysis of cristae area versus mitochondria area in 40 randomly selected mitochondria per group. c. Coomassie blue staining of mitochondrial proteins, related to Fig. 2i. d. Octanoyl-carnitine oxidation-rate in isolated mitochondria from BAT of HFD-fed mice with indicated genotypes (n=5). e. Rectal temperatures were measured for HFD-fed mice with indicated genotypes (n=5). b, d, e: Student’s t-test (two-tailed) was performed. Data represent mean ± SEM.

Source data

Extended Data Fig. 4 Extended information related to Fig. 3.

a. Western blot analysis of immune-precipitated IRAK2 from IL-1β treated mitochondria and subjected to indicated amount of Lambda phosphatase (Lambda PP) treatment for 10 min. b. Wild-type and HA-tagged IRAK2 were restored in Irak2 KO adipocytes. Western blot analysis of cytoplasmic and mitochondrial proteins from IL-1β treated cells. c. Immunogold staining of HA-tagged IRAK2 which was overexpressed in Irak2 KO, Myd88 KO, Il1r1 KO and Phb KD cells with or without IL-1 treatment for 24h. Scale bars, 50 nm. d. Immunogold staining of HA-tagged MyD88 in newly differentiated primary adipocytes from Myd88-HA reporter mice. Mito: mitochondrion; Cyto: cytosol. Scale bar, 200 nm. e. Co-immunoprecipitation (IP) analysis of TIMM50 and TOM20 in mitochondria of in newly differentiated primary adipocytes from Irak2-HA reporter mice treated with IL-1β for indicated time points and followed by western blot analysis. a, b, e: Data represent one of five independent experiments with similar results.

Source data

Extended Data Fig. 5 Extended information related to Fig. 3.

a. Coomassie blue staining of mitochondrial proteins, related to Fig. 3d. b. Expression of indicated mRNAs in WT and Irak2 KO newly differentiated primary adipocytes treated with or without IL-1β for indicated time points (n=3). c. Extracellular acidification rate (ECAR), related to Fig. 3f. Data represent mean ± SEM. Data represent one of five independent experiments with similar results. d. Octanoyl-carnitine oxidation-rate in isolated mitochondria from WT and Irak2 KO primary adipocytes treated with or without IL-1β for 24h (n=5). e. Secondary helical wheel structure of IRAK2 peptide (amino acid: 39–52) which contains mitochondrial localization signal (MLS). The picture was generated by http://lbqp.unb.br/NetWheels/. f. Coomassie blue staining of mitochondrial proteins, related to Fig. 3k. g. Octanoyl-carnitine oxidation-rate in isolated mitochondria from Flag-tagged wild-type and IRAK2 mito-mutant restored Irak2 KO adipocytes treated with or without IL-1β for 24h (n=5). b, d, g: Student’s t-test (two-tailed) was performed. Data represent mean ± SEM.

Source data

Extended Data Fig. 6 Extended information related to Fig. 4.

a. Body weight of WT and Irak2 KO mice on HFD feeding (n=6 females per group). b. Glucose tolerance test (GTT) and insulin tolerance test (ITT) were performed on HFD-fed of WT and Irak2 KO mice (n=5 females per group). c. H&E staining of iWAT sections from HFD-fed of WT and Irak2 KO mice. Cell size was quantified (3 views per slide, 3 sections per mouse, n=5). d. H&E staining of BAT sections from HFD-fed of WT and Irak2 KO mice. c, d: scale bars, 150 μm. a, b: Two-way ANOVA, followed by post hoc analysis was performed. Data represent mean ± SEM. c: Student’s t-test (two-tailed) was performed. Data represent mean ± SEM.

Source data

Extended Data Fig. 7 Extended information related to Fig. 5.

a. Octanoyl-carnitine oxidation-rate in isolated mitochondria from BAT of HFD-fed mice with indicated genotypes (n=5). b. Various pictures of immunogold staining of HA-tagged IRAK2 in BAT sections of HFD-fed Irak2-HA reporter mice, related to Fig. 5c Scale bars, 200nm. c. Coomassie blue staining of mitochondrial proteins, related to Fig. 5e. d-e. Immunohistochemical staining of RFP in BAT sections of HFD-fed WT and Irak2 KO Ucp1-luc/tdTomato reporter mice. Luciferase enzymatic activity in lysates from BAT (d) and iWAT (e) (n=8). f, g. HFD-fed WT and Irak2 KO Ucp1-luc/tdTomato reporter mice injected with CL-316,243 for 3 days. Immunohistochemical staining of RFP in BAT sections and Luciferase enzymatic activity in lysates from BAT (L) and iWAT (M) (n=6). d, f: Scale bars, 150 nm. a, d–g: Student’s t-test (two-tailed) was performed. Data represent mean ± SEM.

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Extended Data Fig. 8 Extended information related to Fig. 6.

a. Co-immunoprecipitation (IP) analysis of HA-tagged IRAK2, in mitochondria of in newly differentiated primary adipocytes from Irak2-HA reporter mice treated with IL-1β for indicated time points and followed by western blot analysis. Data represent one of five independent experiments with similar results. b, c: Densitometric analysis of western blots in Fig. 6a, b. b. Signals corresponding to IP OPA1 were used and normalized to Mito input OPA1 in Fig. 6a (n=3). c. Signals corresponding to IP IRAK2 were used and normalized to Mito input IRAK2 in Fig. 6b (n=3). d, Mitochondrial proteins were extracted Ctrl and Phb KD primary adipocytes treated with IL-1β for indicated time points and analyzed by BN-PAGE, followed by Western blot analysis with anti-OxPhos cocktail antibodies. HMW SCs: high molecular weight super-complexes. e. [1-14C]-palmitic acid oxidation-rate f. Octanoyl-carnitine oxidation-rate in isolated mitochondria from WT and Phb KD primary adipocytes, treated with or without IL-1β for 24h (n=5). g. The activities of respiratory complexes h. Octanoyl-carnitine oxidation-rate in the isolated mitochondria in mitochondria of non-targeting siRNA (WT and Irak2 KO) and PHB siRNA transfected (WT/Phb KD, Irak2 / Phb KD) WT and Irak2 KO primary adipocytes treated with/without IL-1β (n=6). i. The activities of respiratory complexes j. Octanoyl-carnitine oxidation-rate in the isolated mitochondria in mitochondria of empty vector (WT and Irak2 KO) and PHB cDNA transfected (WT/PHB and Irak2 KO/PHB) WT and Irak2 KO primary adipocytes treated with/without IL-1β (n=6). b, c, e–j: Student’s t-test (two-tailed) was performed. Data represent mean ± SEM.

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Extended Data Fig. 9 Extended information related to Fig. 6.

a. Immunogold staining of HA-tagged IRAK2 and kinase-inactive (KI) mutant which were overexpressed in Irak2 KO cells with or without IL-1β treatment for 24h. Scale bars, 50 nm. b. Coomassie blue staining of mitochondrial proteins, related to Fig. 6d. c. Extracellular acidification rate (ECAR), related to Fig. 6g. Data represent mean ± SEM. Data represent one of five independent experiments with similar results. d. Octanoyl-carnitine oxidation-rate in isolated mitochondria from WT and Irak2 KI primary adipocytes, treated with or without IL-1β for 24h (n=5). Student’s t-test (two-tailed) was performed. Data represent mean ± SEM.

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Extended Data Fig. 10 Extended information related to Fig. 7 & 8.

a. Rectal temperatures were measured for HFD-fed mice with indicated genotypes (n=5) b. Coomassie blue staining of mitochondrial proteins, related to Fig. 7g. c. Targeting vector design for generation of a novel mouse strain with exon 1 of Irak2 flanked by loxP sites. d. Total lysates from iWAT, BAT and liver from Irak2FF and Irak2AKO mice were subjected to western blot analysis with indicated antibodies. Data represent one of five independent experiments with similar results. e. Rectal temperatures were measured for HFD-fed mice with indicated genotypes (n=5). f. Coomassie blue staining of mitochondrial proteins, related to Fig. 8g. a, c: Student’s t-test (two-tailed) was performed. Data represent mean ± SEM.

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Supplementary information

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Zhou, H., Wang, H., Yu, M. et al. IL-1 induces mitochondrial translocation of IRAK2 to suppress oxidative metabolism in adipocytes. Nat Immunol 21, 1219–1231 (2020). https://doi.org/10.1038/s41590-020-0750-1

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