Fig. 1: Lipolysis is sufficient and necessary for activation of a large fraction of the β-adrenergic gene program in brown adipocytes.

Mouse in vitro differentiated brown adipocytes were pre-treated with 10 µM Atglistatin (ATGL inhibitor) and 20 µM CAY10499 (HSL inhibitor) for 1 h and subsequently with 100 nM isoproterenol (ISO) (blue bar) or 20 µM SR-3420 (red bar) for 3 h before harvesting. a Schematic overview of the experimental setup. Created with BioRender.com. b FA release to medium in the different conditions. n = 3 biologically independent experiments examined, each carried out in a technical duplicate. c PCA plot of RNA-seq data. d Heatmap of RNA-seq data with K-means clustering. Only ISO-activated (ISO vs. vehicle: Log2FC ≥ 1, FDR/Benjamini-Hochberg≤0.05) genes are shown. e Models indicating the role of lipolysis in transcriptional activation of genes in C1–C4 by β-adrenergic signals. C1, C3, and C4 constitute lipolysis-activated genes, however, whereas C1 genes are strictly dependent on lipolysis for activation, C3 and C4 genes can also be activated by cAMP-independent of lipolysis. Created with BioRender.com. f mRNA expression pattern of representative genes from C1–C4 derived from RNA-seq. n = 3 biologically independent experiments examined, each carried out in a technical duplicate. g Heatmap indicating the fold enrichment of gene pathways (FDR/Benjamini-Hochberg ≤ 0.05) in C1–C4. For all panels, error bars represent +/- SEM of 3 independent biological experiments, each carried out in technical duplicates. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons test for b and by DESeq2 using FDR/Benjamini-Hochberg correction for Fig. 1f (p ≤ 0.05 = *, p ≤ 0.01 = **, p ≤ 0.001 = ***). * versus Vehicle, # versus ISO or SR-3420 (without Lipase inh), ¤ versus ISO (with/without Lipase inh.).