Tbx15 is required for adipocyte browning induced by adrenergic signaling pathway

Abstract

Objective: The T-box gene Tbx15 is abundantly expressed in adipose tissues, especially subcutaneous and brown fat. Although its expression is correlated with obesity, its precise biological role in adipose tissue is poorly understood in vivo. Here we investigated the function of Tbx15 in brown adipose thermogenesis and white adipose browning in vivo.

Methods: In the present study, we generated adipose-specific Tbx15 knockout (AKO) mice by crossing Tbx15 floxed mice with adiponectin-Cre mice to delineate Tbx15 function in adipose tissues. We systematically investigated the influence of Tbx15 on brown adipose thermogenesis and white adipose browning in mice, as well as the possible underlying molecular mechanism.

Results: Upon cold exposure, adipocyte browning in inguinal adipose tissue was significantly impaired in Tbx15 AKO mice. Furthermore, ablation of Tbx15 blocked adipocyte browning induced by β3 adrenergic agonist CL 316243, which did not appear to alter the expression of Tbx15. Analysis of DNA binding sites using chromatin-immunoprecipitation (ChIP) revealed that TBX15 bound directly to a key region in the Prdm16 promoter, indicating it regulates transcription of Prdm16, the master gene for adipocyte thermogenesis and browning. Compared to control mice, Tbx15 AKO mice displayed increased body weight gain and decreased whole body energy expenditure in response to high fat diets.

Conclusion: Taken together, these findings suggest that Tbx15 regulates adipocyte browning and might be a potential target for the treatment of obesity.

Keywords: Adipocyte; Browning; Obesity; Tbx15; Thermogenesis.

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.

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.

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.

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.

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.

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.