Innervation of thermogenic adipose tissue via a calsyntenin 3β–S100b axis (original) (raw)

Data availability

Histone modification marker and transcription factor ChIP–seq datasets generated in this study are available at NIH Sequence Read Archive under the accession code PRJNA526243. Any other relevant data are available from the corresponding author upon reasonable request.

Change history

In Fig. 6a of this Article, the two dots corresponding to Cidea and S100b were erroneously moved to the top left of the volcano plot; this figure has been corrected online.
An amendment to this paper has been published and can be accessed via a link at the top of the paper

A Correction to this paper has been published: https://doi.org/10.1038/s41586-020-03161-z

References

  1. Cannon, B. & Nedergaard, J. Brown adipose tissue: function and physiological significance. Physiol. Rev. 84, 277–359 (2004).
    Article CAS Google Scholar
  2. Seale, P. & Lazar, M. A. Brown fat in humans: turning up the heat on obesity. Diabetes 58, 1482–1484 (2009).
    Article CAS Google Scholar
  3. Morrison, S. F. Central neural control of thermoregulation and brown adipose tissue. Auton. Neurosci. 196, 14–24 (2016).
    Article CAS Google Scholar
  4. Zeng, W. et al. Sympathetic neuro-adipose connections mediate leptin-driven lipolysis. Cell 163, 84–94 (2015).
    Article CAS Google Scholar
  5. Fedorenko, A., Lishko, P. V. & Kirichok, Y. Mechanism of fatty-acid-dependent UCP1 uncoupling in brown fat mitochondria. Cell 151, 400–413 (2012).
    Article CAS Google Scholar
  6. Kazak, L. et al. A creatine-driven substrate cycle enhances energy expenditure and thermogenesis in beige fat. Cell 163, 643–655 (2015).
    Article CAS Google Scholar
  7. Daniel, H. & Derry, D. M. Criteria for differentiation of brown and white fat in the rat. Can. J. Physiol. Pharmacol. 47, 941–945 (1969).
    Article CAS Google Scholar
  8. Seale, P. et al. Transcriptional control of brown fat determination by PRDM16. Cell Metab. 6, 38–54 (2007).
    Article CAS Google Scholar
  9. Cohen, P. et al. Ablation of PRDM16 and beige fat causes metabolic dysfunction and subcutaneous to visceral adipose switch. Cell 156, 304–316 (2013).
    Article Google Scholar
  10. Wang, W. & Seale, P. Control of brown and beige fat development. Nat. Rev. Mol. Cell Biol. 17, 691–702 (2016).
    Article CAS Google Scholar
  11. Seale, P. et al. Prdm16 determines the thermogenic program of subcutaneous white adipose tissue in mice. J. Clin. Invest. 121, 96–105 (2011).
    Article CAS Google Scholar
  12. Chi, J. et al. Three-dimensional adipose tissue imaging reveals regional variation in beige fat biogenesis and PRDM16-dependent sympathetic neurite density. Cell Metab. 27, 226–236 (2018).
    Article CAS Google Scholar
  13. Zeng, X. et al. Lysine-specific demethylase 1 promotes brown adipose tissue thermogenesis via repressing glucocorticoid activation. Genes Dev. 30, 1822–1836 (2016).
    Article CAS Google Scholar
  14. Pettem, K. L. et al. The specific α-neurexin interactor calsyntenin-3 promotes excitatory and inhibitory synapse development. Neuron 80, 113–128 (2013).
    Article CAS Google Scholar
  15. Saunders, A., Johnson, C. A. & Sabatini, B. L. Novel recombinant adeno-associated viruses for Cre activated and inactivated transgene expression in neurons. Front. Neural Circuits 6, 47 (2012).
    Article CAS Google Scholar
  16. Nakamura, K. et al. Identification of sympathetic premotor neurons in medullary raphe regions mediating fever and other thermoregulatory functions. J. Neurosci. 24, 5370–5380 (2004).
    Article CAS Google Scholar
  17. Cheng, L. et al. Identification of spinal circuits involved in touch-evoked dynamic mechanical pain. Nat. Neurosci. 20, 804–814 (2017).
    Article CAS Google Scholar
  18. Reeves, R. H. et al. Astrocytosis and axonal proliferation in the hippocampus of S100b transgenic mice. Proc. Natl Acad. Sci. USA 91, 5359–5363 (1994).
    Article ADS CAS Google Scholar
  19. Winningham-Major, F., Staecker, J. L., Barger, S. W., Coats, S. & Van Eldik, L. J. Neurite extension and neuronal survival activities of recombinant S100β proteins that differ in the content and position of cysteine residues. J. Cell Biol. 109, 3063–3071 (1989).
    Article CAS Google Scholar
  20. Nishiyama, H., Knöpfel, T., Endo, S. & Itohara, S. Glial protein S100B modulates long-term neuronal synaptic plasticity. Proc. Natl Acad. Sci. USA 99, 4037–4042 (2002).
    Article ADS CAS Google Scholar
  21. Steiner, G., Loveland, M. & Schonbaum, E. Effect of denervation on brown adipose tissue metabolism. Am. J. Physiol. 218, 566–570 (1970).
    Article CAS Google Scholar
  22. Bachman, E. S. et al. βAR signaling required for diet-induced thermogenesis and obesity resistance. Science 297, 843–845 (2002).
    Article ADS CAS Google Scholar
  23. Dulloo, A. G. & Miller, D. S. Energy balance following sympathetic denervation of brown adipose tissue. Can. J. Physiol. Pharmacol. 62, 235–240 (1984).
    Article CAS Google Scholar
  24. Cao, Y., Wang, H. & Zeng, W. Whole-tissue 3D imaging reveals intra-adipose sympathetic plasticity regulated by NGF-TrkA signal in cold-induced beiging. Protein Cell 9, 527–539 (2018).
    Article CAS Google Scholar
  25. Donato, R. et al. S100B’s double life: intracellular regulator and extracellular signal. Biochim. Biophys. Acta 1793, 1008–1022 (2009).
    Article CAS Google Scholar
  26. Nisoli, E., Tonello, C., Benarese, M., Liberini, P. & Carruba, M. O. Expression of nerve growth factor in brown adipose tissue: implications for thermogenesis and obesity. Endocrinology 137, 495–503 (1996).
    Article CAS Google Scholar
  27. Sornelli, F., Fiore, M., Chaldakov, G. N. & Aloe, L. Adipose tissue-derived nerve growth factor and brain-derived neurotrophic factor: results from experimental stress and diabetes. Gen. Physiol. Biophys. 28, 179–183 (2009).
    PubMed Google Scholar
  28. Chen, Y. et al. Crosstalk between KCNK3-mediated ion current and adrenergic signaling regulates adipose thermogenesis and obesity. Cell 171, 836–848.e13 (2017).
    Article CAS Google Scholar
  29. Long, J. Z. et al. A smooth muscle-like origin for beige adipocytes. Cell Metab. 19, 810–820 (2014).
    Article CAS Google Scholar
  30. Lam, S. S. et al. Directed evolution of APEX2 for electron microscopy and proximity labeling. Nat. Methods 12, 51–54 (2015).
    Article ADS CAS Google Scholar
  31. Simpson, I. A. et al. Insulin-stimulated translocation of glucose transporters in the isolated rat adipose cells: characterization of subcellular fractions. Biochim. Biophys. Acta 763, 393–407 (1983).
    Article CAS Google Scholar

Download references

Acknowledgements

We thank Nikon Imaging Center at Harvard Medical School for all imaging studies; RIKEN Institute for sharing the S100b knockout strain; Z. Herbert and the Molecular Biology Core Facilities at Dana Farber Cancer Institute for sequencing studies; the Rodent Histology Core at Harvard Medical School for histology studies; the EM Core at Harvard Medical School for APEX2 imaging studies; the viral core at Children’s Hospital Boston for AAV production; the transgenic core at Beth Israel Deaconess Medical Center for generation of mouse models; Y. Zhu for advice on sequencing data analysis. X.Z. was supported by the American Heart Association postdoctoral fellowship. B.H. is a Cancer Research Institute/Leonard Kahn Foundation Fellow. D.D.G. is an investigator of the Howard Hughes Medical Institute. This study was supported by NIH grant DK31405 to B.M.S.

Author information

Authors and Affiliations

  1. Department of Cancer Biology, Dana Farber Cancer Institute, Boston, MA, USA
    Xing Zeng, Mark P. Jedrychowski, Bo Hu & Bruce M. Spiegelman
  2. Department of Cell Biology, Harvard Medical School, Boston, MA, USA
    Xing Zeng, Mark P. Jedrychowski, Bo Hu & Bruce M. Spiegelman
  3. Department of Neurobiology, Howard Hughes Medical Institute, Harvard Medical School, Boston, MA, USA
    Mengchen Ye & David D. Ginty
  4. Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
    Jon M. Resch & Bradford B. Lowell
  5. Program in Neuroscience, Harvard Medical School, Boston, MA, USA
    Bradford B. Lowell

Authors

  1. Xing Zeng
    You can also search for this author inPubMed Google Scholar
  2. Mengchen Ye
    You can also search for this author inPubMed Google Scholar
  3. Jon M. Resch
    You can also search for this author inPubMed Google Scholar
  4. Mark P. Jedrychowski
    You can also search for this author inPubMed Google Scholar
  5. Bo Hu
    You can also search for this author inPubMed Google Scholar
  6. Bradford B. Lowell
    You can also search for this author inPubMed Google Scholar
  7. David D. Ginty
    You can also search for this author inPubMed Google Scholar
  8. Bruce M. Spiegelman
    You can also search for this author inPubMed Google Scholar

Contributions

X.Z. conceived the project and designed experiments. X.Z., M.Y. and B.H. performed imaging experiments and data analysis. X.Z. and B.H. performed metabolic assays. J.M.R. performed stereotaxical surgeries, viral injections and post hoc histological analysis for the chemogenetic experiment. M.P.J. performed mass spectrometry analysis. B.B.L. supervised the chemogenetic experiments. D.D.G. supervised analysis of sympathetic innervation. B.M.S. supervised the entire project. X.Z. and B.M.S. wrote the manuscript with discussion and contributions from all authors.

Corresponding author

Correspondence toBruce M. Spiegelman.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Clstn3b encodes an adipocyte-specific protein.

a, Quantitative PCR analysis of Clstn3b expression in wild-type and _Lsd1_-knockout BAT (n = 3 mice). b, Histone marker and transcription regulator ChIP–seq at the Clstn3 locus from BAT. c, Quantitative PCR analysis of Clstn3b expression in inguinal subcutaneous WAT from mice acclimatized to room temperature or 4 °C (n = 4 mice). d, Mass spectrometry identification of CLSTN3β peptides. e, Conservation of CLSTN3β within the mammalian class. The red cross and green ticks indicates the absence and presence, respectively, of homologues of CLSTN3β in mammalian subclasses. f, Sequence alignment between the unique exon of Clstn3b from human, and a fragment, in an intron upstream of the penultimate exon of Clstn1, in the genome of Chinese softshell turtle. Note how the position of this fragment corresponds to the β-selective exon in Clstn3. All data are mean ± s.e.m. Statistical significance was calculated by unpaired Student’s two-sided _t_-test.

Source data

Extended Data Fig. 2 CLSTN3β localizes to the endoplasmic reticulum.

a, b, Electron microscopy analysis of primary brown adipocytes that express CLSTN3β–APEX2. In a, arrows denote the Golgi apparatus. In b, arrows denote peroxisomes. Scale bars, 100 nm. c, Western blot analysis of the fractionation pattern CLSTN3β. Asterisk denotes a nonspecific band. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 3 Ablation of Clstn3b impairs adipose thermogenesis.

a–d, Sanger sequencing (a), western blot (b), quantitative PCR (c) (n = 4 mice) and immunofluorescence (d) confirmation of CRISPR–Cas9 deletion of Clstn3b. Scale bars, 10 μm. e, Quantitative PCR analysis of Clstn3 expression in a panel of wild-type mouse tissues, and wild-type and _Clstn3b_-knockout brain (n = 2 mice for surveying tissue specificity in wild-type mouse; n = 3 mice for wild type and knockout). The primers target the junction between the third and the penultimate exons. f, g, Body weight curve (f) and body composition (g) of wild-type and _Clstn3b_-knockout mice on chow diet (n = 8 mice). h, Rates of CO2 production from indirect calorimetry analysis of wild-type and _Clstn3b_-knockout mice (n = 6 mice). i, j, Movement (i) and daily food intake (j) of wild-type and _Clstn3b_-knockout mice in metabolic chambers (n = 6 mice). k, Oxygen consumption response to acute β3 agonist injection, of wild-type and _Clstn3b_-knockout mice (n = 6 mice). All data are mean ± s.e.m. Statistical significance was calculated by unpaired Student’s two-sided _t_-test.

Source data

Extended Data Fig. 4 Transgenic expression of Clstn3b increases adipose thermogenesis.

a, b, Western blot (a) and quantitative PCR (b) confirmation of transgenic overexpression of CLSTN3β in BAT (n = 5 mice). c, d, Body-weight curve (c) and body composition (d) of wild-type and _Clstn3b_-transgenic mice on chow diet (n = 6 mice). e, Rates of CO2 production from indirect calorimetry analysis of wild-type and _Clstn3b_-transgenic mice (n = 4 mice). f, g, Movement (f) and daily food intake (g) of wild-type and _Clstn3b_-transgenic mice in metabolic chambers (n = 4 mice). h, Oxygen consumption response to acute β3 agonist injection of wild-type and _Clstn3b_-transgenic mice (n = 4 mice). All data are mean ± s.e.m. Statistical significance was calculated by unpaired Student’s two-sided _t_-test.

Source data

Extended Data Fig. 5 CLSTN3β increases sympathetic innervation of thermogenic adipose tissue.

a, Gene expression analysis of wild-type and _Clstn3b_-knockout BAT upon 5 h of acute cold exposure, following mice being pre-acclimatized to thermoneutrality (n = 4 mice). Blue, wild-type; orange, knockout. b, Indirect calorimetry analysis of _Clstn3b_-knockout mice with or without Adipoq-cre, receiving AAV-DIO-Clstn3b injection (n = 4 mice). c, Whole-mount tyrosine hydroxylase staining of the inguinal region of the posterior subcutaneous WAT from wild-type and _Clstn3b_-knockout mice, acclimatized at 4 °C for 1 week. Scale bars, 50 μm. All data are mean ± s.e.m. Statistical significance was calculated by unpaired Student’s two-sided _t_-test.

Source data

Extended Data Fig. 6 CLSTN3β promotes secretion of S100b, an adipocyte-derived neurotrophic factor.

a, b, Quantitative PCR analysis of S100b expression in various fat depots (a) and in inguinal subcutaneous WAT (b), from mice acclimatized to room temperature or 4 °C (n = 4 mice). c, Quantitative PCR analysis of S100b expression in control or _Prdm16_-transgenic inguinal subcutaneous WAT (n = 4 mice). d, Quantitative PCR analysis of S100b expression in control or _Prdm16_-knockout inguinal subcutaneous WAT (n = 4 mice). e, PRDM16 ChIP–seq showing binding at the S100b locus. f, Indirect calorimetry analysis of _Clstn3b_-knockout mice with or without Adipoq-cre, receiving AAV-DIO-S100b injection (n = 4 mice). g, Tyrosine hydroxylase immunostaining of salivary gland from wild-type and _S100b_-knockout mice. h, Quantitative PCR analysis of S100b expression in wild-type and _Clstn3b_-knockout BAT from mice housed at room temperature (n = 4 mice). Note that this is a different housing condition from that used for experiments in Extended Data Fig. 5a. i, Western blot analysis of intracellular level of S100b in _Clstn3b_-knockout brown adipocytes that express S100b alone, or co-expressing S100b with CLSTN3β. j, Western blot analysis of S100b protein level in HEK293T cells transfected with various constructs as indicated. k, Western blot analysis of S100b and complement factor D secretion from HEK293T cells co-transfected with or without CLSTN3β. All data are mean ± s.e.m. Statistical significance was calculated by unpaired Student’s two-sided _t_-test.

Source data

Extended Data Fig. 7 Clstn3b is specifically expressed in human adipose tissue.

RNA sequencing in human tissues that shows adipose-specific expression of Clstn3b. RNA sequencing of 13 human tissue types was analysed for reads that uniquely map to the _Clstn3b_-specific exon.

Supplementary information

Source data

Rights and permissions

About this article

Cite this article

Zeng, X., Ye, M., Resch, J.M. et al. Innervation of thermogenic adipose tissue via a calsyntenin 3β–S100b axis.Nature 569, 229–235 (2019). https://doi.org/10.1038/s41586-019-1156-9

Download citation