Journal: iScience

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

doi: 10.1016/j.isci.2022.104547

Figure Lengend Snippet: 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 .

Article Snippet: For experiments with adeno-associated virus (AAV), F3 mouse cDNA (Origene MC200642) or F2r mouse cDNA (Origene MR206862) was subcloned into the pAAV-MCS expression vector (AAV-DJ Helper Free Expression System, Cell Biolabs Inc., VPK-410-DJ).

Techniques: Coagulation, Staining, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Sequencing

Journal: iScience

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

doi: 10.1016/j.isci.2022.104547

Figure Lengend Snippet: 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 +/− ; PAR1 flox/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 (PAR1 flox/flox ; Con), BAT PAR1 KO, and BAT PAR1 KO mice treated with an FXa inhibitor (BAT PAR1 KO + FXa-i). Right panels in Figure 4 A indicate the quantification of large lipid droplets (n = 4, 3, 4), and Figure 4 B indicates the quantification of dihydroethidium (DHE) staining (n = 5, 5, 5) of BAT in littermate control mice (PAR1 flox/flox ; Con), BAT PAR1 KO, and BAT PAR1 KO + FXa-i mice. Right panel in Figure 4 C 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 .

Article Snippet: For experiments with adeno-associated virus (AAV), F3 mouse cDNA (Origene MC200642) or F2r mouse cDNA (Origene MR206862) was subcloned into the pAAV-MCS expression vector (AAV-DJ Helper Free Expression System, Cell Biolabs Inc., VPK-410-DJ).

Techniques: Knock-Out, Staining, Transmission Assay, Electron Microscopy, Control, Comparison

Journal: iScience

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

doi: 10.1016/j.isci.2022.104547

Figure Lengend Snippet: 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 5 E 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 5 F 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 .

Article Snippet: For experiments with adeno-associated virus (AAV), F3 mouse cDNA (Origene MC200642) or F2r mouse cDNA (Origene MR206862) was subcloned into the pAAV-MCS expression vector (AAV-DJ Helper Free Expression System, Cell Biolabs Inc., VPK-410-DJ).

Techniques: Functional Assay, Virus, Injection, Enzyme-linked Immunosorbent Assay, Control, Western Blot, Staining, Transmission Assay, Electron Microscopy

Journal: iScience

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

doi: 10.1016/j.isci.2022.104547

Figure Lengend Snippet: 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 (Ca 2+ ; 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 .

Article Snippet: For experiments with adeno-associated virus (AAV), F3 mouse cDNA (Origene MC200642) or F2r mouse cDNA (Origene MR206862) was subcloned into the pAAV-MCS expression vector (AAV-DJ Helper Free Expression System, Cell Biolabs Inc., VPK-410-DJ).

Techniques: Membrane, Recombinant, Expressing, Coagulation, Incubation, Control, Enzyme-linked Immunosorbent Assay, Infection, Comparison

Journal: iScience

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

doi: 10.1016/j.isci.2022.104547

Figure Lengend Snippet:

Article Snippet: For experiments with adeno-associated virus (AAV), F3 mouse cDNA (Origene MC200642) or F2r mouse cDNA (Origene MR206862) was subcloned into the pAAV-MCS expression vector (AAV-DJ Helper Free Expression System, Cell Biolabs Inc., VPK-410-DJ).

Techniques: Virus, Expressing, Recombinant, Coagulation, Enzyme-linked Immunosorbent Assay, Microarray, Software

Journal: Frontiers in Genetics

Article Title: Whole Transcriptome Analysis Identifies the Taxonomic Status of a New Chinese Native Cattle Breed and Reveals Genes Related to Body Size

doi: 10.3389/fgene.2020.562855

Figure Lengend Snippet: 51 genes with P -values of less than 0.05 according to four independent gene-level LOF variant enrichment analysis methods.

Article Snippet: Additionally, three genes with a suggestive association were found: ESPNL (espin-like), PRCP (prolylcarboxypeptidase) and F2R (coagulation factor II thrombin receptor) (P_Chi < 1e-4).

Techniques: Variant Assay