Sympathetic activation during cold exposure increases adipocyte thermogenesis via the expression of mitochondrial protein uncoupling protein 1 (UCP1)1. The propensity of adipocytes to express UCP1 is under a critical influence of the adipose microenvironment and varies between sexes and among various fat depots2,3,4,5,6,7. Here we report that mammary gland ductal epithelial cells in the adipose niche regulate cold-induced adipocyte UCP1 expression in female mouse subcutaneous white adipose tissue (scWAT). Single-cell RNA sequencing shows that glandular luminal epithelium subtypes express transcripts that encode secretory factors controlling adipocyte UCP1 expression under cold conditions. We term these luminal epithelium secretory factors ‘mammokines’. Using 3D visualization of whole-tissue immunofluorescence, we reveal sympathetic nerve–ductal contact points. We show that mammary ducts activated by sympathetic nerves limit adipocyte UCP1 expression via the mammokine lipocalin 2. In vivo and ex vivo ablation of mammary duct epithelium enhance the cold-induced adipocyte thermogenic gene programme in scWAT. Since the mammary duct network extends throughout most of the scWAT in female mice, females show markedly less scWAT UCP1 expression, fat oxidation, energy expenditure and subcutaneous fat mass loss compared with male mice, implicating sex-specific roles of mammokines in adipose thermogenesis. These results reveal a role of sympathetic nerve-activated glandular epithelium in adipocyte UCP1 expression and suggest that mammary duct luminal epithelium has an important role in controlling glandular adiposity.
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Source data for all figures are provided with the paper. The scRNA-seq dataset (Fig. 1) generated for this paper has been uploaded to the Gene Expression Omnibus under accession number GSE231394. The bulk RNA-sequencing dataset (Fig. 4) generated for this paper has been uploaded to the GEO under accession number GSE121098 (GSM7287577–GSM7287584). Source data are provided with this paper.
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The authors thank former P.R. laboratory members S. Sadeh and S. Hart for technical assistance with metabolic chambers and RNAscope; P. Cohen and C. H. J. Choi at The Rockefeller University for the Lrg1-KO mice. LSFM and confocal microscopy were performed at the Microscopy and Advances Bioimaging Core at ISMMS. A.A. is supported by senior postdoctoral fellowship from the Charles H. Revson Foundation (grant no. 18-25), a fellowship from Sweden–America Foundation (Ernst O. Eks fond), and a postdoctoral scholarship from the Swedish Society for Medical Research (SSMF). S.A.S. is supported by American Diabetes Association Pathway to Stop Diabetes Grant ADA no. 1-17-ACE-31, NIH (R01NS097184, OT2OD024912 and R01DK124461) and Department of Defense (W81XWH-20-1-0345, W81XWH-20-1-0156). L.G. is supported by R00HL150234. A.J.L. is supported by NIH U01 AG070959 and U54 DK120342. X.Y. is supported by NIH R01 DK117850. P.R. is supported by R00DK114571, NIDDK-supported Einstein-Sinai Diabetes Research Center (DRC) Pilot and Feasibility Award, and Diabetes Action Research and Education Foundation (DREF) grant no. 501 (PR). P.W. is a member of the Human Islet and Adenovirus Core of the Einstein-Sinai Diabetes Research Center (ES-DRC) supported by NIHP30DK020541. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
The authors declare no competing interests.
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Extended data figures and tables
a) UMAP plots of integrated single cell data from this study and 8 external datasets (see Methods and Supplementary Table 1). Each point represents a single cell and are colored by dataset. b) UMAP plots of integrated single cell data from this study and 8 external datasets (see Methods). Each point represents a single cell and clusters are colored by cell type. c) Expression of known canonical markers for cell types in the SVF and mammary gland. Color corresponds to average expression level and size corresponds to percentage of cells which express the gene within the cluster. d) Differentially expressed genes between COLD (24 hr) treated mice and RT animals across all cell types. Significant DEGs (adjusted p-value < 0.05) are highlighted with the average log fold change between 4 degree and RT indicated on the y-axis. Cell types are ordered on the x-axis based on the number of significant DEGs. e) Relative fractions of cell types within each sample. Dots indicate average relative fractions across all samples. f) Aggregated UMAP plot of subclustering of luminal single cells from RT and cold-exposed mice. g) Differentially expressed genes between COLD treated mice and RT animals across Luminal-HS-AV. Select significant DEGs (adjusted p-value < 0.05) are highlighted with the average log fold change between 4 degree and RT indicated on the y-axis. Genes indicated by red arrows encode for secreted factors. h) UMAP and violin plots of normalized gene expression levels for genes of interest in luminal cells. Violin plots also show expression levels of Adrb1, Adrb2, and Adrb3 in luminal subtypes. i–k) tSNE plots of cell type clusters and normalized gene expression levels for genes of interest across multiple datasets including female mgWAT SVFs (i), male iWAT SVFs (j), and mature mgWAT adipocytes (k). Statistics for scRNA-seq data (d,g) was derived using Wilcoxon rank sum test between cold vs RT cells for cells and the adjusted p-value is based on Bonferroni correction.
a) Light sheet microscopy fluorescence (LSFM) images of mgWAT isolated from female mice exposed to RT or COLD for 24 h and stained with TH antibody (SNS fibers) and EPCAM antibody (ductal cells). N = 6,6. Scale bar at 200 μm. b) Confocal images of mgWAT isolated from female mice exposed to RT or COLD for 24 h and stained for EPCAM and TH antibodies. Merged staining of EPCAM and TH represent neuroductal points. Representative images of 6 mice per condition. Scale bar at 100 μm. c-j) Quantification of indicated parameters of images from (b). N = 6,5. Unpaired Student’s t Test (j). Data are represented as mean ± S.E.M.
a) Representative FACS plot of CD49f and EPCAM expression in EPCAM bead selected EPCAM+ or EPCAM-ve epithelial cells from RT or cold mice. 2 mice per condition and 4 mammary fat pads per mouse. Representative data from 4 independent experiments. b) Real-time qPCR of indicated genes in beige differentiated SVFs from mgWAT treated with indicated concentration of Iso for 5 h. c) Real-time qPCR of indicated genes from beige differentiated primary mgWAT SVF (Parent) and EPCAM-ve (EPCAM-NEG) cells isolated from cold exposed mice treated with and without 10 µM isoproterenol (ISO) for 24 h ex vivo. d) Images showing cell morphology of D0-D9 beige differentiated 10T1/2 and NMuMG (0, 5, and 10%) mixture cells. Yellow enclosures show NMuMG epithelial cells surrounded by 10T1/2 cells. Scale bar at 100 μm. e) Real-time qPCR of indicated genes from beige differentiated and Iso treated 10T1/2 and 5-10% NMuMG mixture cells. f) Images showing cell morphology of D0-D8 beige differentiated 10T1/2 and NMuMG (0-10%) mixture cells. Yellow enclosures show NMuMG epithelial cells surrounded by 10T1/2 cells. Scale bar at 100 μm. g) Real-time qPCR of indicated genes from beige differentiated and Iso treated 10T1/2 and 2.5-10% NMuMG mixture cells. h–i) Real-time qPCR of indicated genes from a transwell system of beige differentiated norepinephrine (NE) treated (h) or Iso treated (APC) (i) APCs in the presence (APC:NMuMG) or absence (APC) of NMuMG. j) Average oxygen consumption rate (OCR) in APC and APC:NMuMG pretreated with (NE-APC and NE:APC:NMuMG) and without (Ctrl APC and Ctrl-APC:NMuMG) 10 μM norepinephrine (NE) followed by acute treatment with NE (10 μM), 5 μM oligomycin (Oligo), 1 μM FCCP, and 5 μM antimycin A (AA). Mean of N = 6,6. k) Fold increase in OCR from basal respiration (J) with and without acute NE injection. N = 6,6. l) Average OCR normalized to protein level (μg protein) in SVF and SVF:NMuMG with 10 μM NE, 5 μM Oligo, 1 μM FCCP, and 5 μM AA. N = 9,9. m) Real-time qPCR of indicated genes from a transwell system of beige differentiated (Ctrl 10T1/2) or Iso treated (Iso-10T1/2) 10T1/2 cells in the presence of NMuMG with (Iso-10T1/2:NMuMG) or without iso (Ctrl-10T1/2:NMuMG). Results are from three independent experiments. *, p < 0.05; **, p < 0.01, ***, p < 0.001 comparing Iso-10T1/2 and Iso-10T1/2:NMuMG. Unpaired Student’s t Test (E,G,H,I), 2way ANOVA Bonferroni posthoc test (k,l,m), *, p < 0.05, **, p < 0.01; ***, p < 0.001, ns, not significant. Data are represented as mean ± S.E.M. Cartoon models in h,i,m created using BioRender.
a) Images showing cell morphology of beige differentiated SVFs isolated from male and female iWATs. Scale bar at 100 μm. b) Real-time qPCR of indicated genes from the scWATs of 24 h cold exposed male and female adrenalectomized mice. N = 6,6. c–e) Individual mice data for oxygen consumption (VO2 ml/hr), and energy expenditure (EE kcal/hr) (c), respiratory exchange ratio (RER) and generalized linear model (GLM)-based regression plots of RER with total body weight (Total), lean mass (Lean) and fat mass (Fat) as co-variates (Two-way ANCOVA) showing p-value for Mass effect, Group effect, and Interaction effect (d), and total food consumed (kcal), locomotor activity (beam breaks), and total distance in cage (m) (e) of male and female mice exposed to 22 °C for 21 h and 4 °C for 24 h in Sable Promethion metabolic chambers (12 h light/dark cycle, 45 h total duration, white bar represents light cycle and grey bar represents night cycle). N = 6,6. f and g) Body weight (f) and lean mass (g) of male and female mice before (RT) and after 24 h cold exposure (COLD). N = 6,6. h) Weights of indicated fat depots in cold exposed male and female mice. N = 8,8. i and j) Gross appearance, H&E staining, and immunostaining of UCP1 of male dorsolumbar iWATs and female dorsolumbar mgWATs exposed to 24 h cold (i) and Western blot of UCP1 in iWAT from 24 h cold-exposed males and females (j). Actin was used as a loading control and UCP1 bands were quantified and normalized to Actin intensity. N = 4,4. Scale bar at 200 μm. Unpaired Student’s t Test (b,f,h) and ANCOVA (d) *, p < 0.05, **, p < 0.01; ***, p < 0.001, n.s., not significant. Data are represented as mean ± S.E.M.
a) Whole tissue images and H&E sections of mgWATs of WT and Esr1KO cold exposed mice. Blue asterisks represent same samples at different magnifications. Scale bar at 100 μm. b and c) Cartoon showing intraductal injection into the 4th and 5th nipples of female mice (b) with adjacent cartoon showing injection of Evan’s blue or GFP expressing virus for confirmation ductal-specific injection (c). Scale bar at 100 μm d) Intraductal injection of Krt8-Dtr mice with contralateral injection of DTA or PBS and immunohistochemistry with DAPI or UCP1. White arrows indicated UCP1 signals in the stroma and yellow arrows indicated UCP1 signal in ducts. Scale bar at 50 μm e) H&E staining of mgWAT from mice intraductally injected with PBS, 70% EtOH, or 0.5% Trypsin. Yellow arrows indicate ablated ducts. Red asterixis indicate images used in Main Fig. 3j. Representative section from 7 mice. Scale bar at 50 μm. f) Immunofluorescence microscopy for UCP1 or DAPI in mice intraductally injected with PBS or EtOH. Dotted yellow outlines are ductal epithelium. Scale bar at 100 μm. g) Real-time qPCR analysis of indicated genes from mgWATs of mice intraductally injected with PBS or Trypsin. N = 9,9. Data are represented as mean ± S.E.M. Cartoon models in b,d,g created using BioRender.
Extended Data Fig. 6 LCN2 is a cold-induced mammokine involved in blocking cold-induced adipocyte UCP1 expression.
a and b) RNAScope FISH of indicated probes from mgWAT of RT or 24 h cold exposed (Cold) mice. Yellow asterisk indicate image used in Main Fig. 1f for Enho RNAScope. Scale bar at 50 μm. c) Real-time qPCR of indicated genes from mgWAT beige differentiated SVFs treated with 0-100 μm recombinant Adropin (Top) or 0-80 ng/ml recombinant LRG1 (Bottom) for 24 h. d) Real-time qPCR of indicated genes from mgWATs derived from 24 h cold-exposed WT or LRG1KO mice. N = 7,8. e) Gene correlation plots of Ucp1 and Lcn2 expression in WATs from male and female mice from the HMDP studies. f) Real-time qPCR of indicated genes from 24 h cold-exposed male and female iWATs. N = 6,6. g) tSNE plots of cell type clusters and normalized gene expression levels for Lcn2 across scRNA-seq datasets of female mgWAT SVFs and male iWAT SVFs. h) Real-time qPCR of Lcn2 in mgWAT EPCAM+ and EPCAM- cells from RT or 24 h cold-exposed mice. Inset shows violin plot of Lcn2 from scRNA-seq data. i) RNAscope FISH of indicated probes from mgWAT of 24 h cold exposed or Iso treated mice. Yellow asterisks indicate images used in Main Fig. 4b. Scale bar at 50 μm. j) Immunofluorescence microscopy for LCN2 or DAPI in mice exposed to 24 h cold or RT. Yellow outlines indicated ductal epithelium. Scale bar at 50 μm. k) The top five transcription factor regulons for luminal cell types at RT and COLD identified using SCENIC program. l) Subnetworks of transcription factor regulons of interest for Bhlhe41 in hormone sensing luminal cells (Up) and Elf5 in luminal alveolar cells (Down) derived using SCENIC. Transcription factors indicated in diamonds in the center and genes in the regulon in the surrounding circles. Genes in red were derived from the RT data, genes in blue were derived from the COLD data, and genes with both were present in the regulons in both conditions. Red arrow shows cold-induced mammokine Lcn2. m and n) Body weights (n) and scWAT weights (m) of 24 h cold exposed WT and Lcn2 KO male and female mice. N = 6,6 o) Real-time qPCR of indicated genes in organoids derived from K8rtTA-DTA mice treated with and without ISO and Doxycycline (Dox). Results from 3 organoid experiments. p-t) Confocal images of immunostaining of indicated antibodies in organoids derived from K8rtTA-DTA mice treated with and without Iso and Dox. Scale bar at 10 μm. Representative images from 3 organoid experiments. Each point represents three independent experiments (c, h, o). Unpaired two-tailed Student’s t test *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Data are represented as mean ± S.E.M.
Extended Data Fig. 7 LCN2 is a mammokine involved in blocking cold-induced adipocyte UCP1 expression.
a) Real-time qPCR of indicated genes from mgWAT of AAV-GFP or AAV-LCN2 treated Lcn2 KO mice. N = 4,4. b) Gross appearance and H&E section of mgWAT from RT and cold-exposed LCN2KO mice (Top) and WT or LCN2KO mice exposed to 24 h cold (Bottom). Scale bar at 100 μm. c) Real-time qPCR of indicated genes from 24 h cold exposed females (Top) and males (bottom) WT and LCN2KO scWATs. N = 6,6. d) Intraductal injection of Ad-GFP or Ad-LCN2 contralaterally in the 4th and 5th nipple and immunofluorescence microscopy of GFP or LCN2 from Ad-GFP or Ad-LCN2 injected mice with indicated viral concentrations and barplot showing intensity of GFP or LCN2 from microscopy. Scale bar at 100 μm. Yellow asterisks indicate images also used in Main Fig. 4g. Representative image of two independent injections. For bar plots, quantification of intensity was calculated as a mean from multiple ducts from 1 image except for 2X1010 PFU for Ad-GFP (N = 3) and 2X109 for Ad-LCN2. (N = 2). Unpaired two-tailed Student’s t test *, p < 0.05; **, p < 0.01; ***, p < 0.001. Data are represented as mean ± S.E.M. Cartoon model in D created using BioRender.
Gel source data for Extended Data Fig. 4j.
Cell selection and FACS sequential gating strategy. Middle gated panel in page 7 corresponds to Extended Data Fig. 2a.
Publicly available single-cell datasets used in this study
Curated set of canonical marker genes used in this study.
Real-time qPCR primer sequences.
LSFM video of mgWAT isolated from female mice exposed to RT for 24 h and stained with TH antibody (SNS fibres) and EPCAM antibody (ductal cells). Representative mgWAT video for 6 mice per condition.
LSFM video of mgWAT isolated from female mice exposed to cold for 24 h and stained with TH antibody (SNS fibres) and EPCAM antibody (ductal cells). Representative mgWAT video for 6 mice per condition.
Confocal video of mgWAT isolated from female mice exposed to RT for 24 h and stained for EPCAM and TH antibodies. Merged staining of EPCAM and TH represent neuroductal points. Representative video for 6 mice per condition
Confocal video of mgWAT isolated from female mice exposed to cold (24 h) and stained for EPCAM and TH antibodies. Merged staining of EPCAM and TH represent neuroductal points. Representative video for 6 mice per condition
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Patel, S., Sparman, N.Z.R., Arneson, D. et al. Mammary duct luminal epithelium controls adipocyte thermogenic programme. Nature 620, 192–199 (2023). https://doi.org/10.1038/s41586-023-06361-5
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