Fig 1: Brown adipose tissue-specific protease-activated receptor-1 depletion ameliorates brown adipose tissue dysfunctionBrown 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 groups (n = 4, 4, 4). (D, E) Cold tolerance test (CTT; D, n = 16, 8, 5) and glucose tolerance test (GTT; E, n = 14, 11, 11) in the indicated mice aged 13 weeks for CTT, and 12 weeks for GTT. * indicates Con vs. BAT PAR1 KO, ## indicates Con vs. BAT PAR1 KO + FXa-i. Data were analyzed by 2-way ANOVA followed by Tukey’s multiple comparison test (A, B, C) or by 2-way repeated-measures ANOVA followed by Tukey’s multiple comparison test (D, E). *p < 0.05, **p < 0.01, ##p < 0.01. Values represent the mean ± SEM NS = not significant. All data are from different biological replicates. See also Figure S4.
Fig 2: 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 quantification of protease-activated receptor-1 (PAR-1) relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in BAT (n = 5, 5).(J) Immunofluorescent staining for PAR-1 in BAT from the indicated group. Scale bar = 50 µm.(K) RNA sequence data analyzing F2R in brown adipose tissue (BAT) from individuals with a body mass index (BMI) < 25 (n = 8) or BMI =25 (n = 23). Datasets were taken from Jespersen et al. (https://www.biorxiv.org/content/10.1101/2020.05.07.082057v1).(L) Enzyme-linked immunosorbent assay (ELISA) for Factor Xa in plasma from NC or HFD mice aged 19-22weeks (left panel; n = 3, 3), and plasma from lean (BMI <22) or obese (BMI >28) human volunteers (right panel; n = 13, 13). All data were analyzed by a 2-tailed Student’s t test. *p < 0.05, **p < 0.01. Values represent the mean ± SEM NS = not significant. All data are from different biological replicates. See also Figure S1.
Fig 3: Inhibition of FactorXa prevents whitening and dysfunction of brown adipose tissueMice 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 area (%; analyzed as mitochondrial area/[non-capillary and non-lipid area]) in the pericapillary area of respective groups (n = 4, 4).(E and F) Cold tolerance test (CTT; E, n = 7, 7) or glucose tolerance test (GTT; F, n = 14, 17) in the indicated mice aged 13 weeks for CTT, and 12 weeks for GTT.(G) Oxygen consumption (VO2), CO2 production (VCO2), energy expenditure (EE), and respiratory exchange ratio (RER) in the indicated groups aged 14-16 weeks (n = 12, 12, 12, 12). Data were analyzed by a 2-tailed Student’s t test (A, B, C, D, G) or 2-way repeated measures ANOVA (E, F). *p < 0.05, **p < 0.01. Values represent the mean ± SEM NS = not significant. All data are from different biological replicates. See also Figure S2.
Fig 4: 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 immunosorbent assay (ELISA) for tissue factor (n = 10, 10), coagulation factor VII (FVII; n = 9, 14), and FXa (n = 11, 9) in conditioned medium from differentiated brown adipocytes incubated with control adenovirus (Mock) or Ad-Hif-1a.(K and L) MitoSox/MitoGreen ratio (K, n = 8, 7, 7) and MitoRed/MitoGreen ratio (L, n = 8, 8, 8) in differentiated brown adipocytes incubated with conditioned medium (CM) obtained from brown adipocytes infected with Mock (Mock-CM), Ad-Hif-1a (Ad-Hif-1a-CM), or Ad-Hif-1a-CM + PAR1-i. Data were analyzed by a 2-tailed Student’s t test (I-L), or by 2-way ANOVA followed by Tukey’s multiple comparison test (A and C-H), or non-parametric Kruskal Wallis test (B). *p < 0.05, **p < 0.01. Values represent the mean ± SEM NS = not significant. All data are from different biological replicates. See also Figure S6.
Supplier Page from Abcam for Native human Factor Xa protein