ap2 cre mice  (Shanghai Model Organisms Center)

Journal: bioRxiv

Article Title: BCAA catabolism drives adipogenesis via an intermediate metabolite and promotes subcutaneous adipose tissue expansion during obesity

doi: 10.1101/2022.08.18.504380

Figure Lengend Snippet: (A) Illustration of partial BCAA catabolic pathway with enzymes, intermediates, and derivatives. (B) Schematic of conditional Bckdk deletion in adipose tissue with Fabp4 ( Ap2 )-Cre. (C) Western blot analysis of BCKDK, p-BCKDE1α, BCKDE1α in the indicated tissues from f/f ( Bckdk f/f ) and AKO ( Ap2-Bckdk -AKO) mice. (D) Metabolomic analysis of BCAA catabolites in the sWAT of Bckdk f/f and Ap2 - Bckdk -AKO mice. (E) Body weight (n=15- 18 per group) of male Bckdk f/f and Ap2-Bckdk - AKO mice on normal chow diet (NCD) for 12 weeks. (F-I) Body composition (n=9-12 per group) (F), organ weight (n=15-16 per group) (G), glucose tolerance test (GTT) (n=15 per group) and the area under the curve (AUC) (H), insulin tolerance test (ITT) (n=8-12 per group) and the area under the curve (AUC) (I) of male Bckdk f/f and Ap2-Bckdk -AKO mice on normal chow diet (NCD) for 6 weeks. (J-M) Oxygen consumption (VO2) (J), carbon dioxide production (VCO2) (K), heat (M), and locomotor activity (L) from 2 weeks HFD-fed male Bckdk f/f and Ap2-Bckdk -AKO mice (n=3- 4 per group). (N) Food intake in Bckdk f/f and Ap2-Bckdk -AKO mice fed NCD (n=12-15 per group) and HFD (n=13 per group). Schematic of conditional Bckdk deletion in adipose tissue with Adipoq -Cre. (P) Western blot analysis of BCKDK, p-BCKDE1α, BCKDE1α in the indicated tissues from f/f ( Bckdk f/f ) and AKO ( Adq-Bckdk -AKO) mice. (Q-S) Body weight (n=12-15 per group) (Q), body composition (n=12-15 per group) (R), and organ weight (n=11-13 per group) (S) of male Bckdk f/f and Adq-Bckdk -AKO mice on high fat diet (HFD) for 12 weeks. Data are represented as mean ± s.e.m. Statistical significance was determined by unpaired two-tailed Student’s t-test or two-way analysis of variance (ANOVA). *p<0.05, ***p<0.001.

Article Snippet: Fatty acid-binding protein 4–Cre recombinase ( Ap2- cre) transgenic mice expressing Cre recombinase under the control of mouse Fabp4 promoter (C57BL/6 background) were from Cyagen Biosciences (Suzhou, China).

Techniques: Western Blot, Activity Assay, Two Tailed Test

Journal: bioRxiv

Article Title: BCAA catabolism drives adipogenesis via an intermediate metabolite and promotes subcutaneous adipose tissue expansion during obesity

doi: 10.1101/2022.08.18.504380

Figure Lengend Snippet: (A-F) Bckdk f/f and Ap2 - Bckdk -AKO mice were fed with 60% HFD for 12 weeks (A) and for 6 weeks (B-F). A, Body weight (n=17-26 per group). B, Body composition (n=17-20 per group). C, Organ weight (n=12 per group). D, Images of iWAT and eWAT. Scale bars, 50 μm. Quantification of adipocytes surface areas (right, n=1025-1567 cells per group). E, Glucose tolerance test (GTT) (n=6-7 per group) and the area under the curve (AUC). F, Insulin tolerance test (ITT) (n=8-9 per group) and the AUC. (G-L) Bckdha f/f and Ap2 - Bckdha -AKO mice were fed with 60% HFD for 12 weeks (G) and for 6 weeks (H-J). G, Body weight (n=20-21 per group). H, Body composition (n=10-12 per group). I, Organ weight (n=15-17 per group). J, Images of iWAT and eWAT. Quantification of adipocytes surface areas (right, n=1025-1567 cells per group). (K-L) GTT (n=6-7 per group) and AUC (K), ITT (n=13-14 per group) and AUC (L) from mice on HFD for 2 weeks. Data are represented as mean ± s.e.m.; unpaired t-test or two-way ANOVA; * p<0.05, ** p<0.01, *** p<0.001.

Article Snippet: Fatty acid-binding protein 4–Cre recombinase ( Ap2- cre) transgenic mice expressing Cre recombinase under the control of mouse Fabp4 promoter (C57BL/6 background) were from Cyagen Biosciences (Suzhou, China).

Techniques:

Journal: bioRxiv

Article Title: BCAA catabolism drives adipogenesis via an intermediate metabolite and promotes subcutaneous adipose tissue expansion during obesity

doi: 10.1101/2022.08.18.504380

Figure Lengend Snippet: (A) Representative photograph of mice and fat pads, body composition (n=17-19 per group), and organ weight (n=12 per group) of Bckdk f/f and Ap2 - Bckdk -AKO mice on HFD for 12 weeks. (B) Ratio of tissue mass to body weight in Bckdk f/f and Adq - Bckdk -AKO mice on HFD for 6 weeks (n=11-13 per group). (C) mRNA expression of genes in the iWAT (n=12 per group) and eWAT (n=6 per group) from Bckdk f/f and Ap2 - Bckdk -AKO mice on HFD for 12 weeks. (D) mRNA expression of genes in the iWAT (n=11-12 per group) (A) and eWAT (n=9-12 per group) (B) from Bckdk f/f and Ap2 - Bckdk -AKO mice on HFD for 6 weeks. (E) qPCR analysis of the expression of indicated genes in the iWAT (n=10-11 per group) and eWAT (n=10-11 per group) of male Bckdk f/f and Ap2-Bckdk -AKO mice on high fat diet (HFD) for 2 weeks. (F) qPCR analysis of the expression of indicated genes in the iWAT (n=6-8 per group) and eWAT (n=5-6 per group) of male Bckdha f/f and Ap2-Bckdha -AKO mice on high fat diet (HFD) for 6 weeks. Data are represented as mean ± s.e.m.; unpaired t-test; * p<0.05, ** p<0.01, *** p<0.001.

Article Snippet: Fatty acid-binding protein 4–Cre recombinase ( Ap2- cre) transgenic mice expressing Cre recombinase under the control of mouse Fabp4 promoter (C57BL/6 background) were from Cyagen Biosciences (Suzhou, China).

Techniques: Expressing

Journal: bioRxiv

Article Title: BCAA catabolism drives adipogenesis via an intermediate metabolite and promotes subcutaneous adipose tissue expansion during obesity

doi: 10.1101/2022.08.18.504380

Figure Lengend Snippet: (A) Schematic of conditional Bckdha deletion in adipose tissue with Fabp4 ( Ap2 )-Cre. (B) Western blot analysis of BCKDE1α in the indicated tissues from f/f ( Bckdha f/f ) and AKO ( Ap2-Bckdha -AKO) mice. (C) Body weight (n=15-19 per group) of male Bckdha f/f and Ap2-Bckdha -AKO mice on normal chow diet (NCD) for 12 weeks. (D-E) Body composition (n=16-18 per group) (D), and organ weight (n=13-14 per group) (E) of male Bckdha f/f and Ap2-Bckdha -AKO mice on normal chow diet (NCD) for 6 weeks. (F-G) Glucose tolerance test (GTT) (n=15 per group) and the area under the curve (AUC) (F), insulin tolerance test (ITT) (n=11-18 per group) and the area under the curve (AUC) (G) of male Bckdha f/f and Ap2-Bckdha -AKO mice on normal chow diet (NCD) for 2 weeks. (H) Food intake in Bckdha f/f and Ap2-Bckdha -AKO mice fed NCD (n=12 per group) and HFD (n=12 per group). (I-L) Oxygen consumption (VO2) (I), carbon dioxide production (VCO2) (J), locomotor activity (K), and heat (L) from 4 weeks HFD-fed male Bckdha f/f and Ap2-Bckdha -AKO mice (n=6 per group). (M) Schematic of conditional Bckdha deletion in adipose tissue with Adipoq -Cre. (N) Western blot analysis of BCKDE1α in the indicated tissues from f/f ( Bckdha f/f ) and AKO ( Adq-Bckdha -AKO) mice. (O) Body weight (n=12-13 per group) of male Bckdha f/f and Adq-Bckdha -AKO mice on high fat diet (HFD) for 10 weeks. (P-Q) Body composition: fat mass (P), lean mass (Q) (n=7-9 per group male) of Bckdha f/f and Adq-Bckdha -AKO mice on HFD for 10 weeks. (R-V) Organ weight: iWAT (R), eWAT (S), BAT (T), heart (U), and lung (V) (n=7-10 per group male) of Bckdha f/f and Adq-Bckdha -AKO mice on HFD for 12 weeks. Data are represented as mean ± s.e.m. Statistical significance was determined by unpaired two-tailed Student’s t-test or two-way analysis of variance (ANOVA).

Article Snippet: Fatty acid-binding protein 4–Cre recombinase ( Ap2- cre) transgenic mice expressing Cre recombinase under the control of mouse Fabp4 promoter (C57BL/6 background) were from Cyagen Biosciences (Suzhou, China).

Techniques: Western Blot, Activity Assay, Two Tailed Test

Journal: bioRxiv

Article Title: BCAA catabolism drives adipogenesis via an intermediate metabolite and promotes subcutaneous adipose tissue expansion during obesity

doi: 10.1101/2022.08.18.504380

Figure Lengend Snippet: (A-B) mRNA levels of BCAA catabolic genes in the SVF from the iWAT (A) and eWAT (B) of Bckdk f/f and Ap2 - Bckdk -AKO mice (n=11 per group). (C-D) qPCR analysis of the expression of indicated genes in the SVF (n=11 per group) (C) and mature adipocytes (Mature) (n=4-5 per group) (D) of eWAT from male Bckdk f/f and Ap2-Bckdk -AKO mice on HFD for 2 weeks. (E-H) qPCR analysis of the expression of indicated genes in the SVF (n=7-9 per group) (E) and mature adipocytes (Mature) (n=6-9 per group) (F) of iWAT or SVF (n=5-9 per group) (G) and Mature (n=6-9 per group) (H) of eWAT from male Bckdha f/f and Ap2-Bckdha -AKO mice on HFD for 2 weeks. (I-S) Single cell sequencing analysis of SVF cells from iWAT of male Bckdk f/f and Ap2 - Bckdk -AKO mice on HFD for 2 weeks (n=3 per group). T-distributed stochastic neighbor embedding (tSNE) plot (I) and UMAPs plot (J) of SVF cells; expression of leukocyte marker Ptprc in different clusters of SVF cells shown in the integrated UMAP plot (K) and violin plots (L); violin plots of canonical markers Cd19 , Cd4 , Ms4a1 , Cd8a (M); 3, 4, 8 and 20 seurat clusters shown in the integrated UMAP plot (N); group 1-10 of CD45-SVF cells shown in the UMAP plot (O); feature plots of pseudotime analysis (P); histology(Q) and adipocyte size (R) and number (S) analysis in the eWAT of Bckdk f/f and Ap2-Bckdk -AKO mice on HFD for 2 weeks. Data are represented as mean ± s.e.m. Statistical significance was determined by unpaired two -tailed Student’s t-test. *p<0.05, **p<0.01.

Article Snippet: Fatty acid-binding protein 4–Cre recombinase ( Ap2- cre) transgenic mice expressing Cre recombinase under the control of mouse Fabp4 promoter (C57BL/6 background) were from Cyagen Biosciences (Suzhou, China).

Techniques: Expressing, Sequencing, Marker, Two Tailed Test

Journal: bioRxiv

Article Title: BCAA catabolism drives adipogenesis via an intermediate metabolite and promotes subcutaneous adipose tissue expansion during obesity

doi: 10.1101/2022.08.18.504380

Figure Lengend Snippet: (A-B) mRNA expression of genes in the stromal vascular fraction (SVF) (A, n=5-8 per group) and mature adipocytes (Mature) (B, n=5-7 per group) of iWAT from Bckdk f/f and Ap2 - Bckdk -AKO mice on HFD for 2 weeks. (C) tSNE plot of combined CD45-negative SVF cells isolated from the iWAT of Bckdk f/f and Ap2 - Bckdk -AKO mice (n=3 per group) on HFD for 2 weeks. (D) Violin plots for markers. (E-F) Trajectory analysis (E) and pseudotime analysis (F) of combined CD45-negative SVF cells. (G) Group distribution of CD45-negative SVF cells from the iWAT of Bckdk f/f and Ap2 - Bckdk -AKO mice on HFD for 2 weeks. (H-L) Flow cytometry analysis of CD45-SVF cells in the iWAT of Bckdk f/f and Ap2 - Bckdk -AKO mice (n=3 per group) on HFD for 2 weeks. (M-O) Histology (M), adipocyte size (N), and adipocyte number (O) analysis in the iWAT of Bckdk f/f and Ap2-Bckdk -AKO mice (n=6-8 per group) on HFD for 2 weeks. Data are represented as mean ± s.e.m.; unpaired t-test; * p<0.05, ** p<0.01.

Article Snippet: Fatty acid-binding protein 4–Cre recombinase ( Ap2- cre) transgenic mice expressing Cre recombinase under the control of mouse Fabp4 promoter (C57BL/6 background) were from Cyagen Biosciences (Suzhou, China).

Techniques: Expressing, Isolation, Flow Cytometry

Journal: bioRxiv

Article Title: BCAA catabolism drives adipogenesis via an intermediate metabolite and promotes subcutaneous adipose tissue expansion during obesity

doi: 10.1101/2022.08.18.504380

Figure Lengend Snippet: (A) Representative images of oil red staining of differentiated SVF cells from iWAT of Bckdk f/f and Ap2 - Bckdk -AKO mice. (B-C) Protein and mRNA expression during SVF cell differentiation. (D) Representative images of oil red staining of differentiated SVF cells from Bckdha f/f and Ap2 - Bckdha -AKO mice. (E-F) Protein and mRNA expression during SVF cell differentiation. (G-H) Representative images of oil red staining of differentiated 3T3-L1 adipocytes with Bcat2 silencing in the presence or absence of three BCKAs (500 μM each) (G) or individual BCKA (500 μM) (H) supplementation. (I) Leucine catabolic process. (J-M) Representative images of oil red staining of differentiated 3T3-L1 adipocytes with: shRNA silencing of Auh (J), Mccc 1 (K), Ivd (L), Bcat2 silencing with or without different leucine catabolite derivatives (M). (N) Abundances of leucine catabolite 3-methylcrotonyl-CoA in 3T3-L1 adipocytes on the 4th day of differentiation. (O) Metabolomics analysis of BCAA catabolites: isovaleryl-carnitine from leucine, 2-methylbutyryl-carnitine from isoleucine, and isobutyryl-carnitine from valine in 3T3-L1 adipocytes on the 4th day of differentiation. *p<0.05 vs Undifferentiated group; #p<0.05 vs Differentiated group; &p<0.05 vs Differentiated+shBCAT2 group. (P) Representative images of oil red staining of differentiated 3T3-L1 adipocytes with Bcat2 silencing, treated with DMAA (5 mM) during different periods of differentiation. (Q) Protein expression during 3T3-L1 differentiation. Data are represented as mean ± s.e.m.; unpaired t-test or one-way ANOVA; * p<0.05, ** p<0.01, *** p<0.001.

Article Snippet: Fatty acid-binding protein 4–Cre recombinase ( Ap2- cre) transgenic mice expressing Cre recombinase under the control of mouse Fabp4 promoter (C57BL/6 background) were from Cyagen Biosciences (Suzhou, China).

Techniques: Staining, Expressing, Cell Differentiation, shRNA