Fig. 6: Lipolysis overrides the intrinsic rhythmicity of brown adipocytes in a cell-autonomous manner.

a Effect of lipolysis on mRNA expression of core clock genes derived from RNA-seq in unsynchronized mouse in vitro differentiated brown adipocytes. Cells were pre-treated with 10 µM Atglistatin (ATGL inhibitor) and 20 µM CAY10499 (HSL inhibitor) for 1 h and subsequently with 20 µM SR-3420 for 3 h before harvest. n = 3 biologically independent experiments examined, each carried out in a technical duplicate b Effect of lipolysis on mRNA expression of circadian clock genes quantified by qPCR in synchronized in vitro differentiated mouse-brown adipocytes. Cells were synchronized with serum and exposed to 20 µM SR-3420 (red line) or vehicle (black line) for the indicated times. n = 3 biologically independent experiments examined, each carried out in a technical duplicate c Experimental setup for d-h. Male C57BL/6 mice (12 weeks old) received 1.5 mg Atglistatin (red) or vehicle (black) by oral gavage at ZT0 and euthanized at ZT4, ZT8, ZT12, and ZT16. Created with BioRender.com. d Serum FAs levels at ZT4. n = 5 mice per condition. e Serum glycerol levels at ZT4. n = 5 mice per condition. f Correlations between serum FA levels and mRNA expression of core clock genes in BAT of vehicle-treated mice at ZT4, ZT8, ZT12, and ZT16. g Effect of Atglistatin on mRNA expression of core clock genes quantified by qPCR in brown adipose tissue at ZT4, ZT8, ZT12, and ZT16. n = 5 mice per condition per timepoint. h Effect of Atglistatin on core body temperature from ZT0-ZT16 (left), and quantification of core body temperature in the light phase from ZT2-ZT10 (right). n = 6 mice per condition. For cell culture experiments results, error bars represent + /- SEM of 3 independent biological experiments, each carried out in technical duplicates. For mice experiments, error bars represent SEM of 5-6 mice per treatment each time point. Statistical significance was determined by Pearson’s correlation coefficient test for f, one-way ANOVA with Tukey’s multiple comparisons test for a and unpaired Student’s t-test for d, e, g, h and area under the curve analysis for b (p ≤ 0.05 = *, p ≤ 0.01 = **, p ≤ 0.001 = ***). * versus Vehicle/Control, # versus SR-3420 (without Lipase inh.).