Abstract
Promoting brown adipose tissue (BAT) activity innovatively targets obesity and metabolic disease. While thermogenic activation of BAT is well understood, the rheostatic regulation of BAT to avoid excessive energy dissipation remains ill-defined. Here, we demonstrate that adenylyl cyclase 3 (AC3) is key for BAT function. We identified a cold-inducible promoter that generates a 5′ truncated AC3 mRNA isoform (Adcy3-at), whose expression is driven by a cold-induced, truncated isoform of PPARGC1A (PPARGC1A-AT). Male mice lacking Adcy3-at display increased energy expenditure and are resistant to obesity and ensuing metabolic imbalances. Mouse and human AC3-AT are retained in the endoplasmic reticulum, unable to translocate to the plasma membrane and lack enzymatic activity. AC3-AT interacts with AC3 and sequesters it in the endoplasmic reticulum, reducing the pool of adenylyl cyclases available for G-protein-mediated cAMP synthesis. Thus, AC3-AT acts as a cold-induced rheostat in BAT, limiting adverse consequences of cAMP activity during chronic BAT activation.
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Data availability
Illumina datasets from RT and cold-activated eWAT and iBAT are available at the Gene Expression Omnibus under accession GSE212574. H3K4me3 ChIP–seq data from BAT of chow diet-fed and HFD-fed male C57BL/6N mice housed at 22 °C or exposed to 5 °C for 24 h were downloaded from the Gene Expression Omnibus (GSE20065). snRNA-seq data from Adipoq-tdTomato-positive adipocyte nuclei were downloaded from ArrayExpress (E-MTAB-8562). All immunofluorescence images have been uploaded on figshare https://doi.org/10.6084/m9.figshare.c.6725808.v1Source data are provided with this paper.
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Acknowledgements
We thank J. Alber and A.-B. Marcher for indirect calorimetry (TSE Phenomaster) support. Sequencing was carried out at the Functional Genomics and Metabolism Research Unit, University of Southern Denmark. We thank T. P. Mortensen, M. Wishoff and R. Nielsen for sequencing assistance. We thank C. Baitzel and A. Lietzau, Max Planck Institute for Metabolism Research, for assistance in generating Adcy3∆AT mice. J.-W.K. and B.D.M.L. received funding from European Research Council Starting Grant (ERC StG) TransGenRNA (675014), Sygeforsikring Denmark Healthcall, the University of Southern Denmark, and the Danish Diabetes Academy, which is funded by the Novo Nordisk Foundation. J.-W.K., A.J. and A.R.T. received support from Challenge (33444) and Bioscience and Basic Biomedicine Programs of the NNF (28416). H.T. was supported by a research grant from the Danish Diabetes Academy, which is funded by the Novo Nordisk Foundation, grant number NNF17SA0031406. S.K. was supported by a German Academic Exchange Service (DAAD) PhD scholarship (A/12/97620). We thank the Microscopy Core Facility of the Medical Faculty at the University of Bonn for providing help, services, and devices funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation; project nos. 169331223 and 388159768). Research in the laboratory of D.W. was supported by grants from the DFG SFB 1454 (project number 432325352; TRR83 – 112927078, TRR333/1 – 450149205), under Germany’s Excellence Strategy (EXC2151 – 390873048), SPP1926, SPP1726, FOR2743, as well as intramural funding from the University of Bonn. A.B. appreciates funding from DFG SFB1123-B10 and ERC StG PROTEOFIT. A.P. and T.G. are funded by the DFG (450149205-TRR333/1; A.P.: P10; T.G.: P11).
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S.K., H.T., R.K., A.R.T., A.J., B.D.L.M., M.J.G., P.L., N.M.E., A.S.G., N.S., P.M.M.R., I.G., N.R.H., E.S., P.K., L.-M.V., R.S., F.D., J.-H.K., A.B., C.A.E., S.G., T.G. and M.P.-J. performed the experiments. H.T., S.K., P.F., A.R.-I., T.J.S., F.T.W., A.P., A.B. and M.J. contributed discussions and performed training. S.K., H.T., D.W. and J.-W.K. conceived the experiments. D.W. and J.-W.K. wrote the manuscript.
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Extended data
Extended Data Fig. 1 cAMP biosynthesis in adipose tissue after cold.
Transcriptional regulation of cAMP biosynthesis in adipose tissue after cold exposure. a–c, Principal component analysis (PCA) of transcriptomic changes in BAT (a), iWAT (b), and eWAT (c) after 24 h (a,c) or 72 h (b) of 5 °C cold exposure (n = 3–5 per condition). Dots represent individual, chow diet fed, male C57BL/6N animals with n = 3–5 animals per condition. d–f, mRNA expression of phosphodiesterases (Pde) in BAT (d), iWAT (e) and eWAT (f) as determined by mRNA-seq and depicted in transcripts per million (TPM). Bar graphs represent mean + SEM with all data points plotted. Unpaired, two-tailed, and non-parametric Mann-Whitney tests were performed to assess statistical significance.
Extended Data Fig. 2 mAC3 is required for cAMP biosynthesis in BAT.
mAC3 is required for cAMP biosynthesis in BAT and required for cold adaptation in cold. a,b, mRNA expression of transcriptional BAT regulators (a) and BAT-specific genes (b) in BAT from LoxP and Adcy3-AdcKO mice (at 22 °C or after 24 h of 5 °C cold exposure). Expression has been determined by mRNA-seq and depicted in transcripts per million (TPM); n = 4. The upper/lower whiskers represent largest/smallest observation less/greater than upper/lower hinge+1.5 × interquartile range (IQR). Central median represents 50% quantile. c,d, qPCR analysis of indicated BAT mRNA markers measured in 1°BA derived from SVF of LoxP or Adcy3-AdcKO mice, injected with 100,00 multiplicity of infection (MOI) of AAV8-CMV-Cre (AAV8-Cre) or AAV8-CMV-GFP (AAV-GFP), and stimulated for 6 h with 10 µM CL316,243 (CL), n = 4. Bar graphs represent mean + SEM with all data points plotted. To test for statistical significance, non-parametric (ranked) Kruskal–Wallis one-way analysis of variance (ANOVA) tests with Dunn’s correction for multiple testing was performed.
Extended Data Fig. 3 mAC3 is required for cAMP biosynthesis in BAT.
mAC3 is required for cAMP biosynthesis in BAT and required for cold adaptation in cold. a–d, Relative (body-weight corrected) tissue weights from indicated adipose tissue depots and liver of LoxP (n = 4 for 22 °C, n = 8 for 5 °C) and Adcy3-AdcKO (n = 4 for 22 °C, n = 8 for 5 °C) mice either fed chow diet (a,b) or HFD (c,d) at 22 °C (a,c) or exposed for 24 h to 5 °C (b,d). Bar graphs represent mean + SEM with all data points plotted. Unpaired, two-tailed, and non-parametric Mann-Whitney tests were performed to assess statistical significance between both genotypes within each tissue. e, Western blot analysis of BAT from chow-diet fed, male LoxP (n = 3 for 22 °C, n = 4 for 5 °C) and Adcy3-AdcKO mice (n = 4 for 22 °C, n = 5 for 5 °C) at 22 °C or exposed to 5 °C for 6 days. Anti-phospho-PKA substrate antibodies were used for detection of PKA phosphorylation substrates. Anti-UCP1 antibodies probed for UCP1 protein levels. anti-HSC70 antibody served as loading control. f–h, Analysis of STK signaling activity in mouse BAT. Kinome trees depict the mean kinase statistic of differentially activated STKs measured by Pam Gene Array technology in LoxP at 22 °C compared to 5 °C for 24 h (f), LoxP at 22 °C compared to 5 °C for 6 days (g) and Adcy3-AdcKO compared to LoxP at 22 °C (h). i, Inserts show zoom-ins of relevant areas of the kinome tree (blue box: down-regulated kinases; red box: up-regulated kinases). ii, Mean kinase statistic of 15 most significantly differentially activated kinases is represented in bar graphs. iii, Venn diagram depicting differences in differentially activated kinases of the respective comparison. Data represents the mean of n = 4 biological replicates for all conditions.
Extended Data Fig. 4 Activated adipocytes express truncated Adcy3.
Activated thermogenic adipocytes express a truncated Adcy3 (Adcy-AT) transcript and protein isoform. a, UCSC genome browser tracks depicting changes in mRNA expression (grey) and H3K4me3 (magenta) in BAT from chow diet-fed, male C57BL/6N mice housed either at 22 °C or after 24 h of 5 °C cold exposure as determined by RNA-Seq (grey) and H3K4me3 ChiP-Seq (magenta). Red and blue transcripts depict contiguous Adcy3 and Adcy3-at transcripts, respectively, and Adcy3 isoform specific transcriptional start sites (TSS). b, ChIP-qPCR of H3K4me3 at the TSS of Adcy3 (blue), Adcy3-at (red) and exon 3, which is shared by Adcy3 and Adcy3-at (grey). Replicates represent H3K4me3 immunoprecipitations perform in individual animals (n = 3). Bar graphs represent mean + SEM with all data points plotted and unpaired, two-tailed, and non-parametric Mann-Whitney tests were performed to assess statistical significance. P values are indicated within the panel. c,d, Expression of Adcy3 (c) and Adcy3-at (d) in indicated tissues of chow diet fed, male C57BL/6N mice. Data points represent individual mice (n = 4 BAT, n = 2 iWAT, n = 3 eWAT, n = 3 pancreas, n = 3 kidneys, n = 2 skeletal muscle, n = 3 liver). e, Absolute expression (Cq value) of Adcy, Adcy3-at and housekeeping genes Hprt and Gapdh in hypothalamy of lean C57BL/6N male mice (n = 3 as determined by qPCR analysis. ND = Not detected. f–h, Absolute mRNA expression (Ct threshold levels) of Adcy3-at (f), Adcy3 (g) and Ucp1 (h) in BAT-derived SVF at indicated stages of brown adipogenesis and after stimulation with 1 mM db-cAMP or 10 µM CL316,243 as determined by qPCR. Replicates depict experiments performed in SVF isolated from individual mice (n = 4). Bar graphs represent mean + SEM with all data points. i,j, Expression of indicated BAT marker mRNAs in BAT (i) or iWAT (j) of chow diet or HFD-fed male C57BL/6N mice, each housed either at 22 °C or exposed to 24 h for 5 °C cold exposure as determined by qPCR. k, Expression of Adcy3-at in BAT of chow diet fed male C57BL/6N mice at 2.5 months or 25 months of age (n = 8). Bar graphs represent mean + SEM with all data points plotted. i–k, Bar graphs represent mean + SEM with all data points plotted. To test for statistical significance, non-parametric (ranked) Kruskal–Wallis one-way analysis of variance (ANOVA) tests with Dunn’s correction for multiple testing were performed. P values are indicated in the panel.
Extended Data Fig. 5 Activated adipocytes express truncated Adcy3.
Activated thermogenic adipocytes express a truncated Adcy3 (Adcy-AT) transcript and protein isoform. a–c, Relative expression of Adcy (blue) and Adcy3-at (red) as determined by qPCR analysis in BAT (a), iWAT (b) and eWAT (c) from chow diet fed, male C57BL/6N mice housed either at 28 °C (thermoneutrality) or exposed to 5 °C for 24 h and 7 days (n = 8). Bar graphs represent mean + SEM with all data points plotted. To test for statistical significance, non-parametric (ranked) Kruskal–Wallis one-way analysis of variance (ANOVA) tests with Dunn’s correction for multiple testing were performed. P values are indicated in the panel.
Extended Data Fig. 6 Generation of mice deficient for Adcy3-at.
Generation of transgenic mice deficient for Adcy3-at at exon 1 and TSS using CRISPR/CAS9 gene. a, Illustration of CRISPR-Cas9 mediated excision of Adcy3-at specific Exon 2b, including Adcy3-at TSS (light blue), yielding Adcy3-at specific knockout (Adcy3∆AT) mice. b, USCS genome browser track depicting mRNA (grey) and H3K4me3 signals (magenta) in the murine Adcy3 locus from chow diet fed, male C57BL/6N mice either housed at 22 °C (light grey) and exposed to 5 °C for 24 h (dark grey) and from HFD-fed, male C57BL/6N mice housed at 22 °C (light orange) and exposed to 5 °C for 24 h (dark orange) as determined by RNA-Seq and H3 K4me3 ChiP-Seq. Location and sequence of sgRNAs used for CRISPR/Cas9 mediated 978-nt deletion of Adcy3-at TSS are indicated. c, Southern blot of a control (C57BL/6N wildtype) and from Adcy3∆AT embryonic stem cell (ESC) genomic DNA. The asterisk depicts a heterozygous Adcy3∆AT/wild-type ESC clone. d, PCR from genomic DNA isolated from wild-type and Adcy3∆AT ESC genomic DNA. e,f, Relative expression of Adcy3-at (e) and Adcy3 (f) as determined by qPCR analysis in BAT, iWAT, and eWAT from chow diet fed, male LoxP (n = 4 for BAT and iWAT, n = 2 for eWAT) and Adcy3∆AT mice (n = 4) housed at 22 °C. Bar graphs represent mean + SEM with all data points plotted. An unpaired, two-tailed, and non-parametric Mann-Whitney test was performed to assess statistical significance. P values are indicated within the panel. Created with BioRender.com.
Extended Data Fig. 7 mAC3-AT inhibits oxidative metabolism and protects from obesity.
mAC3-AT inhibits oxidative metabolism in vitro and in vivo and protects from diet-induced obesity. a,b, Extracellular acidification rates in 1°iBA derived from SVF of LoxP (n=5) or Adcy3∆AT (n = 5) and stimulated with oligomycin (O), FCCP (F) and Antimycin A plus Rotenone (A/R) (a), or 10 µM CL316,243 (CL) (b) at the indicated time points. The numbers of measured wells are indicated in the panel. c,d, Oxygen consumption rates in 1°iWA derived from SVF of LoxP (n = 5) or Adcy3∆AT (n = 5) and stimulated with oligomycin (O), FCCP (F) and Antimycin A plus Rotenone (A/R) (c) or 10 µM CL316,243 (CL) (d) at the indicated time points. The numbers of measured wells are indicated in the panel. e,f, Extracellular acidification rates in 1°iWA derived from SVF of LoxP (n = 5) or Adcy3∆AT (n = 5) and stimulated with oligomycin (O), FCCP (F) and Antimycin A plus Rotenone (A/R) (e), or 10 µM CL316,243 (CL) (f) at the indicated time points. The numbers of measured wells are indicated in the panel. a–f, Bar graphs represent mean + SEM. Unpaired, two-tailed Student’s T-tests were performed to assess statistical significance. P values are indicated within the panel. Genotype P = 0.0072 (a), P = 0.218 (b), P = 0.007 (c), P = 0.1213 (d), P = 0.032 (e), P = 0.138 (f). g, Body weights in chow diet-fed, male LoxP (n = 7) and Adcy3∆AT (n = 9) mice. h, Blood glucose levels during intraperitoneal glucose tolerance tests in chow diet-fed, male LoxP (n = 7) and Adcy3∆AT (n = 9) mice. i, Blood glucose levels during intraperitoneal insulin tolerance tests in chow diet-fed, male LoxP (n = 7) and Adcy3∆AT (n = 9) mice. j, Indirect calorimetry mediated quantification of cumulative food intake in chow diet-fed, male LoxP (n = 7) and Adcy3∆AT (n = 9) mice. g–j, Graphs represent mean + SEM. Statistical significance was determined by performing Two-Way ANOVA with repeated measurements for x values (mixed models). Post-hoc P value correction to account for multiple testing was performed using Bonferroni adjustment. The source of variation, % of the variation and exact P values are given in the table insets. Genotype P = 0.749 (g), P = 0.48 (h), P = 0.66 (i), P = 0.003 (j). k, Indirect calorimetry measurement of oxygen consumption in chow-diet fed, male LoxP (n = 7) and Adcy3∆AT (n = 9) mice. l, Indirect calorimetry determination of mean daily oxygen consumption in chow diet fed, male LoxP (n = 7) and Adcy3-AdcKO (n = 8) mice. Bar graphs represent mean + SEM with all data points plotted and parametric, unpaired, two-tailed Student’s t-tests were performed to assess statistical significance. P values are indicated within the panel.
Extended Data Fig. 8 mAC3-AT inhibits oxidative metabolism and protects from obesity.
mAC3-AT inhibits oxidative metabolism in vitro and in vivo and protects from diet-induced obesity. a, relative expression of indicated thermogenic BAT marker mRNAs in chow diet-fed, male LoxP (n = 5 for 22 °C, n = 6 for 5 °C) and Adcy3∆AT (n = 3 for 22 °C, n = 4 for 5 °C) mice. Bar graphs represent mean + SEM with all data points plotted and unpaired, two-tailed, non-parametric Mann-Whitney tests were performed for indicated comparisons to assess statistical significance. P values are given within the panel. b,c, Relative expression of indicated thermogenic BAT marker mRNAs in 1°BA (b) and 1°iWA (c) from LoxP (n = 4 individual SVF preparations) and Adcy3∆AT (n = 4 individual SVF preparations). Primary adipocytes were stimulated with 10 μM CL316,243 for 6 h. Bar graphs represent mean + SEM with all data points plotted. To test for statistical significance, non-parametric, ranked Kruskal–Wallis one-way analysis of variance (ANOVA) tests with Dunn’s correction for multiple testing were performed. P values are indicated in the panel. d, Images of HE-stained adipose tisue, extracted from male LoxP and Adcy3ΔAT mice fed CD or HFD. Scale bars: 100 µm. e, Adipocyte size distribution quantified using images as shown in (d). iWAT adipocytes size distribution was analyzed for n = 7 LoxP and n = 3 Adcy3ΔAT animals. gWAT adipocyte size distribution was analyzed for n = 8 LoxP and n = 3 Adcy3ΔAT animals. Mean ± SD of the distributions from all mice per group; P values indicated for unpaired, two-sided t tests with Welch correction between LoxP and Adcy3ΔAT at the size range; *p ≤ 5 × 10–2; **p.
Extended Data Fig. 9 mAC3-AT alters mAC3 localization and limits cAMP.
mAC3-AT alters mAC3 subcellular localization and, thereby, limits cAMP bio synthesis. a,b, analysis of STK activity in Adcy3∆AT BAT. Kinome trees depict the mean kinase statistic of differentially activated STKs in Adcy3 vAT versus LoxP (22 °C) (a) and Adcy3∆AT (22 °C) versus Adcy3∆AT (5 °C, 6 days) (b). c,d, Comparison of Adcy3∆AT versus LoxP at 22 °C (c) and Adcy3∆AT versus LoxP after 6 days at 5 °C (d). At 22 °C, a set of kinases is inversely regulated in BAT of Adcy3∆AT and Adcy3AdcKO mice compared to LoxP mice. The mean kinase statistic of these kinases is shown for 22 °C (c) and for 5 °C (d). Data includes n = 4 biological replicates for all conditions. e, CHO cells stably expressing AC3-HA and transiently transfected with mCherry-CAAX were sonicated and labeled with a Calnexin antibody (ER-marker, magenta) and an HA antibody (green). mCherryCAAX fluorescence is indicated in red. Single channels are shown at the top, merged images at the bottom row. White boxes are shown as magnified view at the bottom-right. Arrows point to endoplasmic reticulum-plasma membrane (ER-PM) contact sites, asterixis indicate contact-free parts within the membrane sheet. Scale bar: 10 μm. f, See (e) for CHO cells stably expressing AC3-AT-HA. The images represent at least four independent experiments, with 5-10 cells analysed per condition per experiment. g, Quantification of the average HA-signal intensity within the PM (co-localization with mCherry-CAAX) and the ER (co-localization with calnexin). Data shown as mean + SEM (empty vector: n = 4; AC3-AT: n = 8, AC3: n = 10). h, Ratio PM/ER distribution (empty vector: n = 4; mAC3-AT: n = 8, mAC3: n = 10). Bar graphs represent mean + SEM with all data points plotted. An unpaired, two-tailed, and non-parametric Mann-Whitney tests were performed to assess statistical significance; p-values are indicated within the panel. i,j, Quantification of intracellular cAMP levels. CHO cells stably expressing mAC3-AT or mAC3, or empty vector transfected CHO cells were stimulated with buffer or 2 μM Forskolin for 30 min (i) or buffer or 1 μM Isoproterenol for 15 min (j). Values were normalized to the respective buffer control and to the respective amount of total protein (empty vector: n = 4; AC3-AT: n = 5, AC3-FL: n = 5). g–j, Data is shown as mean + SEM with all data points plotted. To test for statistical significance, non-parametric (ranked) Kruskal–Wallis one-way analysis of variance (ANOVA) tests with Dunn’s correction for multiple testing were performed; p-values are indicated in the panel.
Extended Data Fig. 10 Conservation of Adcy3-at and Ppargc1a-at.
Evolutionary conservation of Adcy3-at and Ppargc1a-at isoforms. a, Mammalian phylogeny from Meredith et al.89 modified in iTOL88, with silhouettes in black indicating representative species that were selected for comparative analyses of PPARGC1A-AT. b, Genomic conservation among representative rodent species (rodentia) of Adcy3-at identified in the mouse. Color gradient indicates percent nucleotide identity of the Adcy3-at relative to the mouse. Phylogeny based on Swanson et al.91. c, Genomic conservation among representative primates (primates) of Adcy3-at identified in human. Color gradient indicates percent nucleotide identity of Adcy3-at relative to the human. Phylogeny based on Perelman et al.90. d, Nucleotide sequence alignment of Ppargc1a-at (Exon 1b) from various mammalian species. e, Percent identity matrix of Ppargc1a-at among 14 representative mammals (exon 1b). Reproduced from ref. 88 under a Creative Commons license CC BY 4.0.
Supplementary information
Supplementary Tables 1–6
Multi-tabs workbook with Supplementary Tables 1–6. Supplementary Table 1: Adipose depot-specific RNA-seq from cold-exposed C57BL/6 mice. Supplementary Table 2: Gene accession numbers for species conservation analyses. Supplementary Table 3: List of sgRNAs and primers for the generation of Adcy3∆AT mice. Supplementary Table 4: List of SYBR qPCR primers. Supplementary Table 5: List of LNA GapmeR inhibitors. Supplementary Table 6: Alignment mAC3/mAC3-AT.
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Khani, S., Topel, H., Kardinal, R. et al. Cold-induced expression of a truncated adenylyl cyclase 3 acts as rheostat to brown fat function. Nat Metab (2024). https://doi.org/10.1038/s42255-024-01033-8
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DOI: https://doi.org/10.1038/s42255-024-01033-8
