Fig. 2: Lipolysis activates gene expression independent of fatty acid activation or oxidation.

Mouse in vitro differentiated brown adipocytes were pre-treated with 5 µM triacsin C (TC; ACSL inhibitor) or 50 µM etomoxir (ETO; CPT1A inhibitor) for 1 h and subsequently with 20 µM SR-3420 (red bar) for 3 h before harvest. a Schematic overview of the experimental setup. Created with BioRender.com. b Effect of triacsin C (TC) or etomoxir (ETO) on FA release. n = 3 biologically independent experiments examined, each carried out in a technical duplicate. c Heatmap of RNA-seq data with K-means clustering. Only genes activated by SR-3420 (SR-3420 vs. vehicle Log2FC ≥ 1, pAdj < 0.05) are indicated. n = 2–3 biologically independent experiments examined, each carried out in a technical duplicate. d mRNA expression pattern of representative lipolysis-activated, FA activation-independent, and FA oxidation-dependent genes derived from RNA-seq. n = 2–3 biologically independent experiments examined, each carried out in a technical duplicate. e Intracellular ATP levels in mouse in vitro differentiated brown adipocytes pre-treated with 10 µM Atglistatin (ATGL inhibitor) and 20 µM CAY10499 (HSL inhibitor) for 1 h and subsequently stimulated with 100 nM isoproterenol (ISO) or 20 µM SR-3420 for 1 h before harvest. n = 7-8 biologically independent experiments examined. f Intracellular ADP levels in mouse in vitro differentiated brown adipocytes pre-treated with 10 µM Atglistatin (ATGL inhibitor) and 20 µM CAY10499 (HSL inhibitor) for 1 h and subsequently stimulated with 100 nM isoproterenol (ISO) or 20 µM SR-3420 for 1 h before harvest. n = 5 biologically independent experiments examined. g Intracellular ATP: ADP ratio in mouse in vitro differentiated brown adipocytes pre-treated with 10 µM Atglistatin (ATGL inhibitor) and 20 µM CAY10499 (HSL inhibitor) for 1 h and subsequently stimulated with 100 nM isoproterenol (ISO) or 20 µM SR-3420 for 1 h before harvest. n = 4 biologically independent experiments examined. h Intracellular ATP levels in mouse in vitro differentiated brown adipocytes pre-treated with 5 µM triacsin C for 1 h and subsequently stimulated with 100 nM isoproterenol (ISO) or 20 µM SR-3420 for 1 h before harvest. n = 4–5 biologically independent experiments examined. For all panels, error bars represent ± SEM of 2–7 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, h and by DESeq2 using FDR/Benjamini–Hochberg correction for d (p ≤ 0.05 = *, p ≤ 0.01 = **, p ≤ 0.001 = ***). * versus Vehicle/Control, # versus SR-3420/Control, ¤ versus Vehicle/Control.