Tbx15 is required for adipocyte browning induced by adrenergic signaling pathway

Figures

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  Figure 1. Generation of adipose specific Tbx15 knockout mice. A. Schematic showing the cloning strategy for the adipose specific Tbx15 knockout mouse model. B. Determination of TBX15 protein expression in AKO and control mice by western blot analysis. Body weight (C), food intake (D), and tissue weights (E) of 8-week-old AKO and control mice on standard chow diets.

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  Figure 2. Tbx15 adipose-specific knockout impairs cold-induced adipocyte thermogenesis and browning. A-B. Representative H&E staining of brown adipose tissue (BAT, A) and inguinal white adipose tissue (IngWAT, B) from mice at room temperature (RT, upper panel) or following cold exposure (4 °C, lower panel). C-D. Western blot analysis of UCP1 in BAT (C) and IngWAT (D) normalized to GAPDH. E. Volcano map analysis of IngWAT RNA-seq data in separate cohorts of AKO and control mice after 24 h cold exposure. F. qPCR analysis of thermogenic and beige selective gene expression in inguinal adipose tissue from control and AKO mice following cold exposure at 4 °C for 24 h (n = 5). Data represent mean ± SEM, *p < 0.05.

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  Figure 3. Tbx15 is involved in adipocyte browning induced by beta3 adrenergic receptor agonism. A. Representative H&E staining of inguinal white adipose tissue (IngWAT) from control and AKO mice upon vehicle or CL 316243 treatment for 1 day. B. qPCR analysis of Ucp1 expression in inguinal adipose tissue from control and AKO mice upon vehicle or CL 316243 treatment for 1 day (n = 5). C. Western blot analysis of TBX15 in inguinal adipose tissue treated with CL 316243. D. qPCR analysis of Ucp1 and Tbx15 expression in primary inguinal differentiated adipocytes treated with DMSO or CL 316243 for 24 h (n = 3). Data represent mean ± SEM, *p < 0.05, unpaired student's t-test.

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  Figure 4. TBX15 directly regulates Prdm16 expression through promoter interactions. A. ChIP analysis of TBX15 with the promoter of Prdm16 from −5 kb to 0 kb. B. Agarose gel analysis of PCR products from TBX15 ChIP within the −2∼-1 kb promoter of Prdm16. Input refers to bead flow through. C. mRNA expression of Prdm16 in HA-tagged Tbx15 ASC's versus control cells (transfected with PMX empty vector) (n = 5). D. mRNA expression of Prdm16 in Tbx15 AKO primary inguinal adipocytes and control adipocytes (n = 5). E. Over-expression of Tbx15 promotes thermogenic gene expression in inguinal differentiated adipocytes (n = 5). Data represent mean ± SEM, *p < 0.05, unpaired student's t-test.

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  Figure 5. Adipose-specific Tbx15 knockout mice are predisposed toward obesity and display reduced energy expenditure on an obesity-promoting diet. Body weight (A) and food intake (B) of AKO and control mice on high fat diets (n = 10). C. Representative computed tomography (CT) images of AKO and control mice. D. Measurement of lean mass and fat mass in AKO and control mice. E. Tissue weights of adipose tissue in control and AKO mice. F. H&E staining of IngWAT and EpiWAT from control and AKO mouse. G. Glucose tolerant tests (n = 7). H–I Quantitative PCR analysis of thermogenic genes in BAT (H) and IngWAT (I) from control and AKO mice (n = 5). J. Energy expenditure (n = 5). Data represent mean ± SEM, *p < 0.05.

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  Figure 6. Schematic diagram showing role of Tbx15 on adipocyte browning and obesity. The transcriptional factor TBX15 is crucial for adipocyte browning induced by adrenergic signaling pathway in vivo through direct regulation of Prdm16 expression.