Fasting swiftly reduces the number of eWAT VAMs. (A–D) WT mice were fasted for 1 d, and the distribution of eWAT myeloid cells was evaluated by flow cytometry. (A) Experimental scheme. (B) Myeloid cell distribution of eWAT macrophages per gram of adipose tissue. (C) Weight of the epididymal adipose tissue. (D) Absolute numbers of cells from SVF and CD45⁺ cells in animals fed and fasted for 24 h. Representative data of three independent experiments (n = 3–5 mice per group). (E) t-SNE map displaying the eWAT CD45⁺ cell landscape from WT mice fed or fasted and analyzed by flow cytometry. Colors correspond to Cytobank-guided clustering of cell populations. Representative data of three independent experiments (n = 3–5 mice per group). (F–I) Recovery of eWAT VAM numbers after food was given back. WT mice were fasted for 1 d (fasted), fasted for 1 d followed by recovery in ND for 2 d (fasted+fed), or never deprived of food (fed), as schematically shown in F. (G) Myeloid cell population numbers in the eWAT. (H) Epididymal adipose tissue weight. (I) Absolute numbers of cells from SVF and CD45⁺ cells. Representative data of two independent experiments (n = 3–5 mice per group). (J) Representative confocal images of epididymal full-mount sections of WT mice fed, fasted, fasted+fed, or treated with the β3 adrenergic receptor agonist CL 316,243. Pictures show the distribution and morphology of VAMs alongside vasculature (labeled with Isolectin) and contouring adipocytes. Arrows, elongated CD206⁺DAPI⁺ VAMs in steady state; arrowheads, rounded CD206⁺DAPI⁺ VAMs in fasted or β3 agonist (CL 316,243)-treated mice. The far right panel shows an atypical crown-like structure formed by accumulation of neutrophils (Ly6G⁺) around an adipocyte. Bars, 10 µm. 25× magnification; n = 3, representative of three independent experiments. (K) Proliferation of the different myeloid cell populations shown in F–I, as determined by BrdU incorporation. 1 mg of BrdU was injected four times i.p. 12 h apart in the last 48 h of the experiment. Representative data from two independent experiments (n = 3–5 per group). (L and M) Lipolysis was induced in WT mice by i.v. injection of β3 adrenergic receptor agonist CL 316,243, without food withdrawal. 12 h after injection, the distribution of myeloid cells was assessed as indicated above. MAC, macrophages. Representative data from four independent experiments (n = 3–5 per group). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. Statistical analysis was performed using one-way ANOVA together with Tukey post hoc test or unpaired t test (only for pair comparisons). Animals were 12–14 wk old. Bar graphs are shown as mean ± SEM.