Coagulation factors promote brown adipose tissue dysfunction and abnormal systemic metabolism in obesity

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  Figure 1. Coagulation factors and protease-activated receptors are upregulated in brown adipose tissue under metabolic stress (A) Hematoxylin and eosin (HE) staining of brown adipose tissue (BAT) from wild-type mice fed a normal chow (NC) or a high-fat diet (HFD). In the HFD group, mice were fed the diet from 4 weeks of age and were analyzed at 19 to 22 weeks of age. Scale bar = 100 μm. (B and C) Enzyme-linked immunosorbent assay (ELISA; B, n = 10, 10) or immunofluorescent staining (C) for tissue factor in BAT of the indicated mice aged 19-22weeks. Scale bar = 50 μm. (D-H) Results of quantitative polymerase chain reaction (PCR) for coagulation factor VII (F7; D, n = 8, 7) or coagulation factor X (F10; G, n = 8, 7) in mice aged 19-22weeks. Results of enzyme-linked immunosorbent assay (ELISA; E, H) or immunofluorescent staining (F) for coagulation factor VII (E and F, n = 10, 11) or activated coagulation factor X (Factor Xa; H, n = 9, 9) in BAT of the indicated mice aged 19-22weeks. Scale bar = 50 μm. (I) Western blot analysis of protease-activated receptor-1 (PAR-1) or PAR-2 expression in epididymal white adipose tissue (eWAT) or BAT from the indicated groups. Right panel indicates the…

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  Figure 2. Inhibition of FactorXa prevents whitening and dysfunction of brown adipose tissue Mice were fed a high-fat diet (HFD) from 4 weeks of age with or without the administration of an FXa inhibitor (FXa-i). Physiological studies were performed at age 13 to 19 weeks for FXa-i treatment, and samples were collected at age 18 to 22 weeks. (A) Enzyme-linked immunosorbent assay study of Factor Xa in brown adipose tissue (BAT) from mice aged 19-22 weeks fed a high-fat diet (HFD; Con HFD) or HFD + FXa inhibitor (FXa-i HFD; n = 8, 8). (B) Hematoxylin and eosin (HE) staining of BAT from the indicated mice. Scale bar = 100 μm. Right panel indicates the quantification of large lipid droplets in the indicated mice (n = 6, 9). Large lipid droplets were defined as droplets with a surface area >1000 μm2. (C) Staining with dihydroethidium (DHE) in brown adipose tissue (BAT) from mice fed the HFD (Con HFD) or HFD + FXa-i (FXa-i HFD). Scale bar = 50 μm. Right panels indicate quantification shown as relative dihydroethidium (DHE) level (DHE area of FXa-i HFD/Con HFD, n = 9, 6). (D) Findings on transmission electron microscopy in the indicated mice (scale bar = 2 μm). Right panels indicate mitochondrial…

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  Figure 3. Warfarin does not ameliorate brown adipose tissue dysfunction on dietary obesity Mice were fed a high-fat diet (HFD) from 4 weeks of age with or without the administration of warfarin. Physiological studies were performed at age 12 to 17 weeks for warfarin treatment, and samples were collected at age 17 weeks. (A) Enzyme-linked immunosorbent assay study analyzing Factor Xa in brown adipose tissue (BAT) from mice aged 17weeks fed a high-fat diet (HFD; Con HFD) or HFD + Warfarin (Warfarin HFD; n = 8, 8). (B) Hematoxylin and eosin (HE) staining of BAT from the indicated mice. Scale bar = 100 μm. Right panel indicates the number of large lipid droplets per field in BAT from the indicated mice. Large lipid droplets were defined as droplets with a surface area >1000 μm2 (n = 7, 7). (C) Staining with dihydroethidium (DHE) in brown adipose tissue (BAT) from mice fed the HFD (Con HFD) or HFD + warfarin (Warfarin HFD). Scale bar = 50 μm. Right panels indicate quantification shown as relative dihydroethidium (DHE) level (DHE area of Warfarin HFD/Con HFD; n = 4, 4). (D) Findings on transmission electron microscopy in the indicated mice (scale bar = 2 μm). Right panel indicate the…

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  Figure 4. Brown adipose tissue-specific protease-activated receptor-1 depletion ameliorates brown adipose tissue dysfunction Brown adipose tissue (BAT)-specific protease-activated receptor-1 (PAR1) knockout (KO) mice (UCP1-Cre+/−; PAR1flox/flox; BAT PAR1 KO) were fed a high-fat diet (HFD) from 4 weeks of age and physiological studies were performed after 12 to 13 weeks. Tissues were harvested at 18-21 weeks of age. (A, B, and C) Hematoxylin and eosin (HE) staining (A; scale bar = 100 μm), dihydroethidium (DHE) staining (B; scale bar = 50 μm), and transmission electron microscopy (C; scale bar = 2 μm) of BAT from littermate control mice (PAR1flox/flox; Con), BAT PAR1 KO, and BAT PAR1 KO mice treated with an FXa inhibitor (BAT PAR1 KO + FXa-i). Right panels in Figure 4A indicate the quantification of large lipid droplets (n = 4, 3, 4), and Figure 4B indicates the quantification of dihydroethidium (DHE) staining (n = 5, 5, 5) of BAT in littermate control mice (PAR1flox/flox; Con), BAT PAR1 KO, and BAT PAR1 KO + FXa-i mice. Right panel in Figure 4C indicates mitochondria area in % (analyzed as mitochondrial area/[non-capillary and non-lipid area]) in the pericapillary area of the respective…

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  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…

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  Figure 6. FactorXa/PAR1 signaling promotes the dysfunction of brown adipocytes (A-H) Nivo Multimode Microplate Reader study analyzing signal of mitochondrial ROS (MitoSox; A, n = 5, 5, 5; C, n = 8, 8, 8; G, n = 8, 8, 8), mitochondrial membrane potential (MitoRed; B, n = 8, 8, 8; D, n = 20, 20, 20; H, n = 8, 8, 8), mitochondrial calcium (Ca2+; Rhod2; E, n = 8, 8, 8; F, n = 8, 8, 8) in the indicated groups. For studies of MitoSox and MitoRed, recombinant FXa protein (10nM) was administered for a total of 3 h and other compounds were administered for 1 h before the administration of FXa at the following concentrations: PAR1 inhibitor (PAR1-i), 1μM; MitoTEMPO, 10μM; MCU inhibitor (250nM). For studies of Rhod2, recombinant FXa was administered for a total of 30 min, with or without the reagents described above. (I) Results of qPCR for expression of coagulation factor VII (F7; n = 5, 6), factor X (F10; n = 6, 6), tissue factor (F3; n = 4, 5), and F2r (PAR-1 transcript; n = 6, 6) in differentiated brown adipocytes incubated with control adenovirus (Mock) or adenovirus encoding constitutively activated Hif-1a (Ad-Hif-1a; 10 MOI, 48 h for F7, F10, and F2r; 30 MOI, 24 h for F3). (J) Enzyme-linked…