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Depletion of JunB increases adipocyte thermogenic capacity and ameliorates diet-induced insulin resistance

Nature Metabolism volume 6, pages 78–93 (2024)Cite this article

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Abstract

The coexistence of brown adipocytes with low and high thermogenic activity is a fundamental feature of brown adipose tissue heterogeneity and plasticity. However, the mechanisms that govern thermogenic adipocyte heterogeneity and its significance in obesity and metabolic disease remain poorly understood. Here we show that in male mice, a population of transcription factor jun-B (JunB)-enriched (JunB+) adipocytes within the brown adipose tissue exhibits lower thermogenic capacity compared to high-thermogenic adipocytes. The JunB+ adipocyte population expands in obesity. Depletion of JunB in adipocytes increases the fraction of adipocytes exhibiting high thermogenic capacity, leading to enhanced basal and cold-induced energy expenditure and protection against diet-induced obesity and insulin resistance. Mechanistically, JunB antagonizes the stimulatory effects of PPARγ coactivator-1α on high-thermogenic adipocyte formation by directly binding to the promoter of oestrogen-related receptor alpha, a PPARγ coactivator-1α downstream effector. Taken together, our study uncovers that JunB shapes thermogenic adipocyte heterogeneity, serving a critical role in maintaining systemic metabolic health.

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Fig. 1: JunB expression in thermogenic fat is positively correlated with obesity.
Fig. 2: A distinct adipocyte subset that expresses JunB is present in the brown and inguinal fat depots and increased by obesity in abundance.
Fig. 3: Adipose-specific JunB-deficient (JunB FKO) mice exhibit enhanced thermogenesis and cold adaptation.
Fig. 4: Specific depletion of JunB in UCP1+ adipocyte (JunB BKO) enhances energy expenditure and protects against diet-induced obesity and insulin resistance.
Fig. 5: JunB deficiency induces a shift of low-thermogenic to high-thermogenic adipocytes in BAT.
Fig. 6: JunB suppresses the formation of high-thermogenic adipocytes by binding to PGC-1α effector ERRα.
Fig. 7: JunB suppresses BAT mitochondrial biogenesis and energy expenditure through ERRα in vivo.

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Data availability

The data generated during the current study are shared with researchers. The snRNA-seq datasets have been deposited in the Gene Expression Omnibus and are publicly available under accession https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE244239. Source data are provided with this paper.

Code availability

No custom code was used.

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Acknowledgements

This work is supported by R01 Awards (DK132643 and DK110439 to M.L. from NIDDK, HL148337 to X.Y. from NHLBI, and CA163890 and CA194496 to E.P. from NCI); Innovative Basic Science Award (1-17-IBS-261 to M.L.) from the American Diabetes Association; Grant in Aid (15GRNT24940018 to M.L.) and Postdoc Fellowship Awards (20POST35120020 to X.Z.) from American Heart Association; P20 Award (GM121176, Project Director: V. Deretic, to Mentored Principal Investigators, M.L.); CoBRE Pilot Award associated with P30 (P30GM103400 principal investigator, J. Liu; to M.L.), CVMD Pilot Award (to M.L.), CTSC pilot Award associated with grant (UL1TR001449) (principal investigators, M. Campen and N. Pandhi; to M.L.) and UNMCCC pilot Award associated with P30 (CA118100) (principal investigator, Y. Sanchez; to M.L.) at the University of New Mexico Health Sciences Center (UNMHSC). This project was supported in part by the Dedicated Health Research Funds from the University of New Mexico School of Medicine. We thank the Autophagy, Inflammation and Metabolism Center at UNMHSC for providing the Cellomics HCS scanner for our present study and technical support. We want to thank UNM Comprehensive Cancer Center (UNMCCC) for the core support on snRNA-seq (completed in the ATG Core), bioinformatic analysis (performed by K. Brayer at the Cancer Research Facility) and flow cytometry.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University of New Mexico Health Sciences Center, Albuquerque, NM, USA

    Xing Zhang, Xiaofeng Ding, Chunqing Wang, Que Le, Liping Luo, Yan Luo, Xin Yang, Aleyah E. Goins, Chengrui Qiu, Floyd D. Silva, Lily Elizabeth Feldman & Meilian Liu

  2. Department of Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center, Albuquerque, NM, USA

    Dandan Wu & Xuexian O. Yang

  3. Department of Molecular & Cellular Endocrinology, City of Hope Comprehensive Cancer Center, Duarte, CA, USA

    Anying Song & Qiong A. Wang

  4. The National and Local Joint Engineering Laboratory of Animal Peptide Drug Development, College of Life Sciences, Hunan Normal University, Changsha, People’s Republic of China

    Guixiang Huang & Jianlin Zhou

  5. Autophagy Inflammation and Metabolism Center for Biomedical Research Excellence, University of New Mexico Health Sciences Center, Albuquerque, NM, USA

    Sharina P. Desai, Eric R. Prossnitz, Xuexian O. Yang & Meilian Liu

  6. Department of Surgery, University of New Mexico Health Sciences Center, Albuquerque, NM, USA

    Michael F. Spafford & Nathan H. Boyd

  7. Department of Internal Medicine, University of New Mexico Health Sciences Center, Albuquerque, NM, USA

    Eric R. Prossnitz

  8. UNM Comprehensive Cancer Center (UNMCCC), University of New Mexico Health Sciences Center, Albuquerque, NM, USA

    Eric R. Prossnitz

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  1. Xing Zhang
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Contributions

M.L. and X.Z. designed the project. X.Z., X.D., C.W., Q.L., D.W., A.S., G.H., L.L., Y.L., X.Y., A.E.G. and C.Q. conducted the experiments, and X.Z., X.D., C.W., Q.L., D.W., G.H., L.L., Y.L., X.Y. and A.E.G. analysed the results. M.S., N.B., F.S. and L.E.F. completed the collection of human tissue samples. S.P.D. provided Cellomic and Seahorse technical support. Q.W. and A.S. provided the adipose tissue slides of AdipoChaser-GFP mice and completed the immunostaining of adipose tissue. J.Z. contributed to the transcriptional activity assays. X.O.Y. contributed to the design of JunB animal models and transcriptional activity assays. E.P. financially supported this project and edited the manuscript. X.Z. organized the data, and X.Z. and M.L. wrote the manuscript. L.E.F. edited the manuscript. All authors reviewed and approved the manuscript.

Corresponding author

Correspondence to Meilian Liu.

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Nature Metabolism thanks Camilla Scheele and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Isabella Samuelson and Yanina-Yasmin Pesch, in collaboration with the Nature Metabolism team.

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Extended data

Extended Data Fig. 1 The mRNA levels of JunB were upregulated by inhibition of mTORC1 and by obesity in BAT, related to Fig. 1.

a. The layout of fold changes (rapamycin: control) of 96 genes that are cAMP responsive in PCR array analysis. The reverse transcription was performed with total RNA extracted from BAT in mice treated with or without rapamycin (n = 3/group), and then the synthesized cDNAs was used for PCR array analysis. b. RNA-sequencing gene expression signatures of iWAT from 10-week-old male Raptor KO and control mice. n = 2–3/group. c. The protein levels of JunB were upregulated by HFD feeding in adipose tissue. d. Quantification of JunB expression in Extended Data Figure c. e. mRNA levels of JunB were induced in WAT of HFD-fed mice (6 mice/group). f. mRNA levels of Ucp1 and Pgc-1α in human deep neck fat were negatively correlated with BMI. g. The expression pattern of JunB in adipocyte subsets and APCs in human BAT. h. Quantification the protein level in Fig. 1k. i. JunB is expressed in Plin1-enriched primary differentiated adipocytes. j. JunB was highly enriched in primary preadipocytes which declined during differentiation (n = 3, independent experiments). Statistical analysis was performed using Pearson’s correlation coefficient in Extended Data Fig. 1f. Extended Data Fig. 1d, e, h, j were analyzed using unpaired two-sided T-Test.

Source data

Extended Data Fig. 2 The JunB Chaser mouse model was generated to show the presence of JunB-expressing adipocytes in fat, related to Fig. 2.

a. The generation strategy for JunBCreERT2 mice as described in the genomic structure. An internal ribosome entry site (IRES) fused to a CreERT2 fusion gene was inserted downstream of the internal stop codon of Junb gene. b. Representative tissue distribution of iCre and Junb mRNA in adult JunBCreERT2 transgenic mice as determined by qPCR. c. Immunofluorescence staining of YFP+ cells in liver and pancreas of JunBCreERT2 mice. Differentiated YFP+ brown adipocytes were present during primary adipogenesis by imaging (d) and flow cytometry (e) in Primary brown adipocytes of JunBCreERT2 mice. f. JunB+ adipocytes were increased by the treatment of 20 ng/mL TNFα or 20 ng/mL IL-6 for 24 hrs post primary adipogenesis. g. Quantification of JunB-expressing adipocytes in Figure f. h. JunB+ adipocytes were increased by the treatment of 20 ng/mL TNFα or 20 ng/mL IL-6 for 24 hrs post primary adipogenesis. Preadiocytes were isolated from AdipoChaser-YFP mice and differentiated into adipocytes. i. Quantification of JunB-expressing adipocytes in Figure h. All data in this Figure were analyzed by unpaired two-sided T-Test.

Source data

Extended Data Fig. 3 Both JunB FKO and BKO mice display enhanced energy expenditure and cold adaptation, related to Fig. 3.

10-week-old male were individually housed in Phenotyping Systems (Sable Systems International) coupled with a temperature controllable chamber. a. Respiratory exchange ratio (RER) was decreased in adipocyte-specific JunB KO (JunB FKO) mice compared to control littermates under cold stress conditions. Food intake (b) and motor activities (c) were little affected by JunB deficiency in fat. d. mRNA levels of JunB were significantly downregulated in BAT but not in gWAT of UCP1+ cell-specific JunB KO (JunB BKO) mice compared to the controls. e. JunB BKO mice exhibited enhanced basal and cold-induced O2 consumption throughout light and dark cycles compared with controls. f. The quantified data of Extended Data Fig. 3e using CaIR. There was little difference in food intake (g) despite slightly reduced motor activities under cold stress conditions (h) between JunB BKO and control mice. i. JunB deficiency significantly increased oxygen consumption in primary brown adipocytes (4 mice/group). j. The quantified data of Extended Data Fig. 3i using CaIR. k. Ablation of JunB-expressing adipocytes upregulated the expression levels of Ucp1, Ppargc1α, and Errα (7 mice/group). Extended Data Fig. 3i was analyzed by ANOVA analysis and the unpaired two-sided T-Test was used for the rest figures. Data are presented as the mean ± SEM. *P < 0.05 or **P < 0.01.

Source data

Extended Data Fig. 4 Both JunB FKO and BKO mice exhibit improved diet-induced insulin resistance, related to Fig. 4.

6-week-old male JunB FKO and control mice were fed with HFD for 16 weeks and used for the studies (4a-h). The percentage of larger adipocytes was decreased accompanied with an increase in smaller adipocytes in iWAT (a) and BAT (b) of HFD-fed JunB BKO mice compared to controls. Depletion of JunB in adipocytes improved glucose (c) and insulin (d) tolerance after 16-week HFD feeding. JunB FKO mice displayed improved hepatic steatosis (e) and triglyceride content in the liver (8 mice for NC and 6 mice for HFD). (f) of JunB FKO mice compared with control mice under HFD feeding conditions. g-h. O2 consumption was significantly increased in JunB FKO mice compared to controls after feeding with HFD for 12 weeks. Extended Data Fig. 4a–d were analyzed by ANOVA analysis. Extended Data Fig. 4f was analyzed with unpaired two-sided T-Test. Data are presented as the mean ± SEM. *P < 0.05 or **P < 0.01.

Source data

Extended Data Fig. 5 Depletion of JunB in adipocytes increased O2 consumption with insignificant anti-obesity effect under HFD condition, related to Fig. 4.

Under HFD feeding conditions, JunB FKO displayed similar RER (a) and food intake (b), while exhibited a significant decrease in motor activities (c) compared to control mice. There was no significant difference in body mass (d), fat mass, fat percentage (e), the image (f) and weight (g) of individual organs such as gWAT, iWAT, BAT and liver between JunB FKO and control mice when challenged with HFD.

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Extended Data Fig. 6 SnRNA-seq analysis of JunB KO BAT, related to Fig. 5.

a. The sorting strategy of isolated nuclei. The nuclei were first sorted based on forward scatter height (FSC) and side scatter height (SSC), and singlets were then sorted based on the combination of area and heights of FSC followed with sorting of DAPI-positive events used for the snRNA-Seq. b. Heat map of gene signature for each population of brown adipocyte nuclei (AD1-AD10). c. Feature plots for Adipoq, JunB, Ucp1, Ppargc1a, Cyp2e1, Fuca1, Slc12a2, Bmpr1a, Gm19951, Acly, Aco1, Flvcr1, Fam13a and Kcnd2.

Extended Data Fig. 7 JunB deficiency induces a shift of low to high-thermogenic adipocytes in BAT, related to Fig. 5.

a. JunB is mainly expressed in UCP1low adipocytes (expression levels <2 described in Fig. 5f). b-c. Majority of YFP+ (JunB+) differentiated adipocytes are Mitochondriallow indicated by the staining with MitoTracker.(4 mice/group) d. YFP+ (JunB+) differentiated adipocytes showed significantly upregulated expression of thermogenic genes compared to YFP− (JunB−) adipocytes(3 mice/group). The partition (e) and pseudotime (f) trajectory of adipocytes analyzed by Monocle. g. Violin plots for Adipoq, Plin1, JunB, Ucp1, Erra, and Ppargc1α for each sub-population of adipocytes in JunB KO and control BAT. h. Volcano plot showing the up regulation of Ucp1, Erra and Ppargc1a in JunB KO BAT compared to control. The unpaired two-sided T-Test was used to analyze Extended Data Fig. 4c, d.

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Extended Data Fig. 8 JunB deficiency enhances adipocyte thermogenic capacity in vivo, related to Fig. 5.

a. Feature plots for Adipoq, Junb, Ucp1 and Ppargc1a in JunB KO adipocytes and control cells. b. Mapping the present data sets of BAT snRNAseq with the published work by Sun et al (PMID: 33116305). c. UMAP of KO BAT adipocytes and controls after mapping with published work by Sun et al (PMID: 33116305).

Extended Data Fig. 9 JunB deficiency increases thermogenic capacity in primary brown adipocytes, related to Fig. 5.

a. JunB FKO mice displayed increased mitochondriahigh and decreased mitochondrialow adipocytes in BAT. LD, lipid droplet; N, nucleus. b. The quantified data of mitochondriahigh and mitochondrialow adipocytes from Figure a (n = 4, mice/group). c. JunB deficiency had little effect on the primary adipogenesis. d. Quantification of lipid droplet area of JunB FKO and control primary adipocytes in Figure c. e. Gene Set Enrichment Analysis (GSEA) of SnRNA-Seq in JunB FKO and Control mice. Extended Data Fig. 9b was analyzed by ANOVA analysis, Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01.

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Zhang, X., Ding, X., Wang, C. et al. Depletion of JunB increases adipocyte thermogenic capacity and ameliorates diet-induced insulin resistance. Nat Metab 6, 78–93 (2024). https://doi.org/10.1038/s42255-023-00945-1

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