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Innervation of thermogenic adipose tissue via a calsyntenin 3β–S100b axis

Abstract

The sympathetic nervous system drives brown and beige adipocyte thermogenesis through the release of noradrenaline from local axons. However, the molecular basis of higher levels of sympathetic innervation of thermogenic fat, compared to white fat, has remained unknown. Here we show that thermogenic adipocytes express a previously unknown, mammal-specific protein of the endoplasmic reticulum that we term calsyntenin 3β. Genetic loss or gain of expression of calsyntenin 3β in adipocytes reduces or enhances functional sympathetic innervation, respectively, in adipose tissue. Ablation of calsyntenin 3β predisposes mice on a high-fat diet to obesity. Mechanistically, calsyntenin 3β promotes endoplasmic-reticulum localization and secretion of S100b—a protein that lacks a signal peptide—from brown adipocytes. S100b stimulates neurite outgrowth from sympathetic neurons in vitro. A deficiency of S100b phenocopies deficiency of calsyntenin 3β, and forced expression of S100b in brown adipocytes rescues the defective sympathetic innervation that is caused by ablation of calsyntenin 3β. Our data reveal a mammal-specific mechanism of communication between thermogenic adipocytes and sympathetic neurons.

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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.

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

Change history

  • 22 May 2019

    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

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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

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.

Competing interests

The authors declare no competing interests.

Correspondence to Bruce M. Spiegelman.

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.

ad, 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

Supplementary Figure 1 Gel source data.

Reporting Summary

Supplementary Table 1 Proteomics analysis of WT and Clstn3β KO BAT from mice housed at RT (n=4 mice). Statistical significance was calculated by unpaired Student’s two-sided t-test.

Source data

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Fig. 1: Clstn3b encodes an adipocyte-specific protein.
Fig. 2: CLSTN3β localizes to the endoplasmic reticulum.
Fig. 3: Ablation of Clstn3b impairs adipose thermogenesis.
Fig. 4: Transgenic expression of CLSTN3β enhances adipose thermogenesis.
Fig. 5: CLSTN3β promotes functional sympathetic innervation of the adipose tissues.
Fig. 6: CLSTN3β promotes secretion of S100b, an adipocyte-derived neurotrophic factor.
Extended Data Fig. 1: Clstn3b encodes an adipocyte-specific protein.
Extended Data Fig. 2: CLSTN3β localizes to the endoplasmic reticulum.
Extended Data Fig. 3: Ablation of Clstn3b impairs adipose thermogenesis.
Extended Data Fig. 4: Transgenic expression of Clstn3b increases adipose thermogenesis.
Extended Data Fig. 5: CLSTN3β increases sympathetic innervation of thermogenic adipose tissue.
Extended Data Fig. 6: CLSTN3β promotes secretion of S100b, an adipocyte-derived neurotrophic factor.
Extended Data Fig. 7: Clstn3b is specifically expressed in human adipose tissue.

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