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. 2021 Jun 9;12:3482. doi: 10.1038/s41467-021-23770-0

Fig. 4. Ablation of rhythmic feeding destroys the diurnal pattern of adipose stromal vascular cell proliferation in vivo.

Fig. 4

a Experimental model: 5-ethynyl-2ʹ-deoxyuridine (EdU) was intraperitoneally (IP) injected at zeitgeber (ZT)0 or ZT12 to mice fed ad libitum chow or fasted for 18 h. Epididymal fat (eWAT) and inguinal fat (iWAT) stromal vascular cell fractions (SVF) and pure adipocytes were isolated from fat harvested 4 h following EdU injection (see also Fig. S3). b Weight of control and fasted animals (n = 10 animals/group). c Weight of fat pads in control vs. fasted mice (n = 12 animals/group). d qPCR reveals expression of leptin (Lep) in pure adipocytes from eWAT and iWAT in control vs. fasted groups (n = 6 animals/group). mRNA levels from the iWAT for fasted mice at ZT4 were set to 1. e Representative fluorescence activated cell sorting of EdU-positive cells from eWAT-SVF (left panels) isolated at ZT4 and ZT16, 4 h following EdU injection. Quantification, right panels (n = 5 animals/group). In (b–d) and (e), circles and squares represent fed ad libitum and fasted mice, respectively. f Experimental model for multiple-meal paradigm: EdU was administered at ZT0 or ZT12 to mice fed ad libitum or with total food intake divided into four aliquots, 25% being administered every 6 h throughout the diurnal cycle for 48 h. eWAT and iWAT-derived SVF were isolated from fat pads harvested 4 h following EdU injection. g, h Quantification of fluorescence activated cell sorting of EdU-positive SVF from eWAT (g) and iWAT (h) (n = 5 animals/group). g, h Black and white circles represent mice fed ad libitum and in multi-doses, respectively. 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 (b) and for red asterisks; and two-way ANOVA followed by Tukey’s post hoc test in (c) [F fat pad(1, 44) = 36.08 / F group(1, 44) = 16.99], (d) [F group(1, 15) = 13.05 / F time(1, 15) = 20.35 (iWAT)] [F group(1, 31) = 37.19 / F time(3, 31) = 30.22 (eWAT)], (e) [F time(1, 16) = 2.735 / F group(1, 16) = 37.28], (g) [F time(1, 13) = 0.7485 / F group(1, 13) = 11.27], (h) [F time(1, 14) = 10.23 / F group(1, 14) = 0.06388].