Figure 3

Aging results in mast cell accumulation which impairs brown adipogenesis. (a) Toluidine blue staining of BAT-sections of young (2.5 months, left panel; n = 7) and aged mice (25 months; middle panel, n = 7) and subsequent cell count-based quantification (right panel) of stained mast cells (red arrows) normalized to total section area using sections of BAT. For each animal, 1–2 independent tissue sections per animal were assessed. Images were collected at 600-fold magnification (scale bar: 10 µm). (b) Immunofluorescence analysis of cells expressing mast cell marker carboxypeptidase A3 (CPA3) in BAT-sections of young (2.5 months, left panel; n = 8) and aged mice (25 months; middle panel, n = 8) and subsequent cell count-based quantification (right panel) of stained mast cells (green signal, white arrows) normalized to total section area using sections of BAT. For each animal, 1–2 independent tissue sections per animal were assessed. Images were collected at 600-fold magnification (scale bar: 10 µm). (c) mRNA levels of mast cell markers Cpa3 and Mcpt4 in BAT (left) and iWAT (right) of young mice maintained at room temperature (RT; white bars) or of young mice after 7 days of cold exposure (light blue) were compared to mice aged 25 months at RT (grey bars) or 25-month old mice after 7-day cold exposure (dark blue). Data are depicted as mean ± standard deviation (SD; n = 4 for 2.5 months BAT and iWAT; n = 8 for all cold exposed tissues and aged BAT)-8 mice; n = 7 for aged iWAT). (d) mRNA expression of Cpa3, Pparg and Ucp1 of primary brown (left, orange) and white (right, grey) adipose tissue-derived progenitors co-seeded with 0.05, 0.1 or 0.5% of p815-mast cells compared to control cultures without masts cells (control; C) after 10 days of differentiation (n = 15, from 3 independent repeat-experiments for BAT; n = 9 from 2 independent repeat experiments for iWAT). (e) mRNA expression of brown adipocyte marker genes Ucp1, Cidea, Ppargc1a, Pparg, Cebpa and of mast cell protease inhibitors Serpina3n and Serpin6b in in vitro differentiated brown adipocytes exposed to control conditioned media (white) or mast cell-conditioned media (light grey) for 24 h (n = 12 for Ppargc1a from 2 independent experiments; n = 15 for all other genes, from 3 independent repeat-experiments). All data (see Suppl. Table S5 for full gene names) are depicted as mean ± standard deviation (SD); *p < 0.05, **p < 0.01, ***p < 0.001 using a nonparametric Kruskal–Wallis test for multiple comparisons of panels with multiple BAT- or iWAT-samples, respectively, and non-parametric Mann–Whitney test for panels with pairwise comparisons.