Fig. 6: Clock-deficiency induces proliferative markers in epididymal fat (eWAT).

a Models of Clock-deficiency: eWAT and inguinal fat (iWAT) was collected every 6 h or 5-ethynyl-2ʹ-deoxyuridine (EdU) was administered at zeitgeber time (ZT) 0 and eWAT and iWAT stromal vascular cell fractions (SVF) were isolated from fat 4 h later. b Energy intake of Clock KO and WT littermates under 12 h light/12 h dark and ad libitum feeding (n = 6 animals/genotype). c Home cage locomotion of Clock KO and WT littermates measured by infrared sensors. Bottom white and black lines indicate the light or dark periods, respectively (b, c). d Weights of Clock KO (n = 19 animals) and WT littermates (n = 10 animals). e Fat pad weights of Clock KO (n = 29 animals) and WT littermates (n = 18 animals). f Representative H&E staining of eWAT and iWAT from Clock KO and WT littermates (20X magnification, scale = 100 µm) and quantification (n = 3 animals/genotype, eWAT and n = 4 animals/genotype, iWAT examined over two independent experiments) (g). h–j RT-PCR reveals expression of clock (h), proliferation (i), and adipogenesis (j) markers in iWAT (blue = WT, orange = KO) (see also Fig. S7) and eWAT (gray = WT, yellow = KO), from Clock KO and WT littermate mice over 24 h (n = 3 animals/ ZT). mRNA levels in iWAT for WT at ZT3 set to 1 for all genes. k Western blot reveals expression of phosphorylated hormone-sensitive lipase (P-HSL), total HSL (T-HSL) and β-Tubulin throughout the circadian cycle (n = 3 animals/ZT) in eWAT (left panel). Quantification, right panel (gray = WT; yellow = KO). Protein levels from WT mice at ZT3 set to 1. l Representative in vivo fluorescence activated cell sorting of EdU-positive cells from eWAT (left panel) SVF isolated at ZT4, 4 h following a single EdU injection. Right panel: respective quantification (n = 6 animals for Clock KO and n = 4 for WT littermate animals). Data are represented as mean ± SEM. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001. Significance (p < 0.05) determined by two-tailed, unpaired t-test in (d, e) and for red asterisks; two-tailed Mann–Whitney U test in (g, l); and two-way ANOVA followed by Sidak’s post hoc test in (h–k).