ThermoMouse: an in vivo model to identify modulators of UCP1 expression in brown adipose tissue

Abstract

Obesity develops when energy intake chronically exceeds energy expenditure. Because brown adipose tissue (BAT) dissipates energy in the form of heat, increasing energy expenditure by augmenting BAT-mediated thermogenesis may represent an approach to counter obesity and its complications. The ability of BAT to dissipate energy is dependent on expression of mitochondrial uncoupling protein 1 (UCP1). To facilitate the identification of pharmacological modulators of BAT UCP1 levels, which may have potential as antiobesity medications, we developed a transgenic model in which luciferase activity faithfully mimics endogenous UCP1 expression and its response to physiologic stimuli. Phenotypic screening of a library using cells derived from this model yielded a small molecule that increases UCP1 expression in brown fat cells and mice. Upon adrenergic stimulation, compound-treated mice showed increased energy expenditure. These tools offer an opportunity to identify pharmacologic modulators of UCP1 expression and uncover regulatory pathways that impact BAT-mediated thermogenesis.

Figure 1. Luciferase imaging of UCP1 expression in vivo

A: 3D reconstruction of luciferase signals in Ucp1-luciferase reporter mice. Adipose depots with specific luciferase signal are indicated. B: Quantification of luciferase in adipose depots, skeletal muscle, and liver of mice kept at room temperature. Values normalized to protein content. C: UCP1 protein expression in tissue lysates from the mouse in (B). D: Luciferase activity in Ucp1-luciferase reporter mice kept at 28°C and subsequently kept at 9°C for 24 hr. Representative mice are shown. E: Quantification of luciferase signal in interscapular BAT of Ucp1-luciferase reporter mice shown in (D) (n=9). *** P < 0.001. Data are expressed as mean ± SEM. F: Ucp1 mRNA expression in interscapular BAT of Ucp1-luciferase reporter mice kept at 28°C and 9°C for 24 hr. *** P < 0.001. Data are expressed as mean ± SEM. G: UCP1 protein expression in interscapular BAT of transgenic mice analyzed in (F). H: Representative image of luciferase signal in Ucp1-luciferase reporter mice treated with saline or CL-316,243 for 1 day (acute) or 4 days (subchronic) (n=3). I: UCP1 protein expression in BAT of transgenic mice analyzed in (H). J: Representative images of luciferase signal in inguinal WAT depots of Ucp1-luciferase reporter mice treated with saline or CL-316,243 for 4 days. K: UCP1 protein expression in inguinal WAT depots of Ucp1-luciferase mice treated with saline or CL-316,243 for 1 day or 4 days.

Figure 2. Cell-based system to monitor UCP1 expression

A: Luciferase activity in immortalized Ucp1-luciferase brown adipocytes. Differentiated adipocytes were treated with DMSO (control) or rosiglitazone for 5 days (n=3). B: UCP1 protein expression in cells analyzed in (A). C: Luciferase activity in differentiated Ucp1-luciferase brown adipocytes treated with DMSO (control), forskolin, or rosiglitazone (0.5 μM) for 5 days (n=3). D: UCP1 protein expression in cells analyzed in (C). E: Luciferase activity monitored at day 0, 1, 4, and 7 after the start of rosiglitazone treatment in mice implanted with Ucp1-luciferase immortalized preadipocytes. Saline (n=4) or rosiglitazone (n=6; 10 mg/kg) treatment started 6 days post-implantation. Representative mice are shown. F: Sequential changes in luciferase activity measured in fat transplants from mice in (E). G: Haematoxylin/Eosin and immunofluorescent stains for UCP1 or GFP (i.e., tdTomato) in transplants from mice treated with rosiglitazone. Merged UCP1 and GFP image counterstained with DAPI. Bar: 50 μm. *P <0.05, ** P <0.01, *** P < 0.001. Data are expressed as mean ± SEM.

Figure 3. Small-molecule screen to identify regulators of UCP1 expression

A: Screen scheme. Ucp1-luciferase brown preadipocytes were differentiated in 96 well plates and treated at day 8 with compounds for 16 hr (n=3). B: Screen performance. Mean luciferase activity of compounds (blue circles) plotted relative to the value of DMSO-treated Ucp1-luciferase adipocytes (100%; green circles). Rosiglitazone served as positive control (red circles). Ucp1-luciferase preadipocytes (purple circles) were used to determine background and induction of signal upon differentiation. Hits selected for evaluation (blue circles, red outline) are numbered according to the scheme used in validation experiments. C: Luciferase activity in differentiated Ucp1-luciferase brown adipocytes treated with compounds for 5 days. Rosiglitazone (0.5 μM) and CL-316,243 (10 nM) served as positive controls (n=3). * P <0.05, ** P <0.01, *** P < 0.001 vs. control. Data expressed as means ± SEM. D: UCP1 protein expression in cells from (C).

Figure 4. Effect of WWL113 on UCP1 expression and cellular respiration

A: UCP1 protein expression in differentiated Ucp1-luciferase brown adipocytes treated with WWL113 for 5 days. Rosiglitazone (0.5 μM) served as positive control. B: Expression of thermogenic genes in differentiated Ucp1-luciferase brown adipocytes treated with WWL113 (10 μM) for 5 days. Rosiglitazone (0.5 μM) was served as positive control (n=3). * P <0.05, *** P < 0.001 vs. control. C: Ucp1 mRNA expression in primary differentiated brown adipocytes treated with WWL113 (10 μM) or rosiglitazone (5 μM) for 24 or 48 hours (n=3). *** P < 0.001 vs. control. D: Ucp1 mRNA expression in primary differentiated brown adipocytes treated with WWL113 (10 μM) or rosiglitazone (5 μM) for 48 hr. Norepinephrine (0.1 μM) was added 8 hr prior to harvest (n=3). ** P <0.01, *** P <0.001 vs. control. E: Total and uncoupled (oligomycin-insensitive) respiration of differentiated brown adipocytes (5 × 10⁵ cells/sample) treated with WWL113 (10 μM) in the presence or absence of norepinephrine (0.1 μM) (n=3–4). * P <0.05 vs. control. F: Ucp1 mRNA expression in differentiated brown adipocytes treated with vehicle or WWL113 (10 μM) for 24 hr with or without 30 min pre-treatment with a PPARα-selective antagonist (GW6471; 3 μM) and/or a PPARγ-selective antagonist (GW9662; 10 μM) (n=3). *** P < 0.001 vs. control. ### P< 0.001 vs. WWL113-treated cells. G. Ucp1 mRNA expression in differentiated brown adipocytes treated with vehicle, WWL113 (10 μM), a PPARα-selective agonist (GW9578), or the combination for 24 hr (n=4). * P < 0.05, *** P < 0.001.

Figure 5. WWL113 increases UCP1 expression in mice

A: Luciferase activity in Ucp1-luciferase reporter mice treated daily with WWL113 (50 mg/kg) or vehicle for 5 days (n=5). Representative mice are shown. B: Quantification of luciferase signal in interscapular BAT of mice treated as in (A). Values normalized to protein content and shown as fold change relative to vehicle. C: Expression of thermogenic genes in interscapular BAT of C57BL/6 mice treated daily with WWL113 (50 mg/kg) or vehicle for 5 days (n=5). * P <0.05, ** P <0.01 D: UCP1 protein expression in interscapular BAT of C57BL/6 mice analyzed in (C). E: VO₂ of wild-type mice treated daily with WWL113 (50 mg/kg) or vehicle for 7 days (n=6). CL316,243 (1 mg/kg) was injected to examine the response to adrenergic stimulation. ** P <0.01. Data expressed as means ± SEM. F: Locomotor activity of mice in (E). G: Food intake of mice in (E). H: Heart rate of mice in (E).