Figure 5.

Introduction of tissue factor and PAR-1 in brown adipose tissue promotes functional decline of this organ

All experiments with Adeno-associated virus (AAV) were performed in mice fed normal chow (NC). AAVs were injected at 10 weeks of age. Physiological studies were performed 10 to 14 days after AAV injection, and tissues were harvested at 14 weeks of age.

(A-C) Enzyme-linked immunosorbent assay (ELISA) for tissue factor (A, n = 10, 9) or FactorXa (C, n = 8, 8) in BAT from mice injected with control AAV (Mock) or with both AAV encoding F3 and AAV encoding F2r (AAV F3+F2r) aged 14 weeks. (B) Western blot analysis of PAR-1 in BAT from the indicated groups. Right panel indicates the quantification of PAR1 relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH; n = 9,9).

(D, E, and F) Hematoxylin and eosin (HE) staining (D; scale bar = 100μm), dihydroethidium (DHE) staining (E; scale bar = 50 μm), and transmission electron microscopy (F; scale bar = 10 μm for low magnification [Low Mag] and 2 μm for high magnification [High Mag]) of BAT from the indicated mice. Right panel in Figure 5E indicates the quantification of dihydroethidium (DHE) analyzed as relative DHE signal (DHE area of AAV F3+F2r/Mock) of indicated mice (n = 4, 5). Right panel in Figure 5F indicates mitochondria area (%; analyzed as mitochondrial area/[non-capillary and non-lipid area]) in the pericapillary area of the respective groups (n = 4, 4).

(G and H) Cold tolerance test (CTT; G, n = 8, 8) and glucose tolerance test (GTT; H, n = 6, 7) in the indicated mice aged 13 weeks for CTT, and 12 weeks for GTT. Data were analyzed by a 2-tailed Student’s t test (A, B, C, E, F) or by 2-way repeated measures ANOVA (G and H). ∗p < 0.05, ∗∗p < 0.01. Values represent the mean ± SEM NS = not significant. All data are from different biological replicates. See also Figure S5.