An improved method for the precise unravelment of non-shivering brown fat thermokinetics

Abstract

Since the discovery of functional brown adipose tissue (BAT) in adult humans, research on BAT gained a new popularity to combat obesity and related comorbidities. To date, however, methods to quantify BAT thermogenesis are often either highly invasive, require advanced equipment, are time consuming or of limited sensitivity. Here we present a simple yet highly effective and minimally invasive protocol for the Precise Unravelment of Non-shivering brown fat thermoKinetics (PUNK) in mice using infrared thermography in combination with Vaseline to brush up the fur between the shoulder blades. We also use physiological and molecular readouts including indirect calorimetry, qPCR and Western Blots to test our protocol. Our study demonstrates that Vaseline before thermography vastly advances the reproducibility and quality of BAT infrared pictures or videos, as it exposes the skin above the BAT for a direct line of sight for the infrared camera and thereby circumvents the well-known problems associated with shaving and anaesthesia. We subsequently validate that this approach does not affect physiological and molecular BAT function, but instead leads to more robust and less variable results when comparing for instance norepinephrine stimulation tests or knockout animals. Taken together, the PUNK protocol for BAT thermography quickly and effectively improves scientific outcomes of this method, and can be easily added to existing paradigms. Consequently, it safes money, time and experimental animals, thereby putting the 3R's principles of animal welfare into practice.

Figure 1

Vaseline enables more accurate and reproducible measurements of iBAT temperature using infrared (IR) thermography. (A) Mice were categorized according to their posture/position. Mouse position A: The mouse sits curled up or is grooming itself; no direct view on the skin. Mouse position B: The animal is moving or stretches forward a little; no direct view on the skin. Mouse position C: The animal stretches forward, but looks upward; no direct view on the skin. Mouse position D (including a closeup picture of the fur): The animal stretches forward and looks down; direct view on the skin. (B,C) Representative infrared pictures of three different mice per position without and with Vaseline. (D,E) iBAT surface temperature of all infrared pictures taken within two sessions (3 min per day) of the same animals without and with Vaseline (N = 12, n = 108–147 (without Vaseline), n = 118–165 (with Vaseline). (F,G) Mean of maximum iBAT temperature of the two sessions presented in (D,E), revealing more stable measurements after Vaseline application (N = 12, n = 24). Data are presented as mean (D,E) or box plot (min. to max. (F,G). *p < 0.05, **p < 0.01, ***p < 0.001 (1-way ANOVA with Tukey’s post-hoc test (D,E) and 1-way repeated measures ANOVA with Tukey’s post-hoc test (F,G)).

Figure 2

The thermogenic profile of BAT cells is not altered by Vaseline application. (A–C) The expression of important thermogenic genes (uncoupling protein 1 (Ucp1), β3-adrenergic receptor (Adrb3), deiodinase 2 (Dio2) and proteins (UCP1 and OxPhos) was unchanged 24 h after Vaseline treatment. (C–E) Furthermore, heat conductivity, heat diffusivity and heat capacity of the skin were normal 24 h after application, whereas acute application of Vaseline resulted in an increased heat conductivity. N = 6, data are presented as box plot (min. to max., C–E) or mean ± SEM (A,B), *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t-test with Welch’s correction (A,B (UCP1/OxPhos blot), C–E (groups no and 24 h Vaseline) or paired Student’s t-test (C – E (groups no and acute Vaseline)).

Figure 3

Vaseline application does not change energy expenditure. After acclimation to the climate chamber, Vaseline was applied to one group of animals (9:00 am, + Vaseline), whereas the other group received a sham treatment. (A) Oxygen consumption, as well as (B) daily energy expenditure (DEE), (C) resting metabolic rate at 23 °C (RMR) and resting metabolic rate at thermoneutrality (30 °C, basal metabolic rate, BMR) were not altered by Vaseline application. Furthermore, decreasing the thermal insulation of the fur did not result in changes in (E) fuel utilization (respiratory quotient, RQ), (F) food intake, (G) water intake or H) activity. N = 6, data are presented as mean ± SD (B–D,F–H) or mean ± SEM (A,E). *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t-test with Welch’s correction (B–D,F–H), 2-way repeated measures ANOVA with Bonferroni’s post-hoc test (A,E)).

Figure 4

Vaseline makes the quantification of non-shivering thermokinetics more precise. (A,B) Quantification and representative infrared pictures of wild-type (WT) and UCP1-KO mice without (−) and with (+) Vaseline, showing higher maximum iBAT temperatures and more stable results after Vaseline application (N = 7). (C) Correlation of maximum iBAT surface temperature and rectal body temperature of wild-type mice with and without Vaseline (N = 7). (D) Representative infrared pictures from video recordings after norepinephrine (NE) injection (1 mg/kg, left mouse: (−) Vaseline, right mouse: (+) Vaseline). (E) Maximum iBAT temperature of wild-type mice before and post NE injection. Of note, baseline measurements were not always obtained immediately before the NE injection, therefore a continuous time-axis before the injection was omitted. (F) Representative picture of a mouse without (−) Vaseline and with (+) Vaseline at the peak after NE injection. (G) Quantification of the maximum iBAT temperature of wild-type mice after before and post NE injection, showing only in the (+) Vaseline group a significant increase in iBAT temperature (N = 6). Data are presented as box plot (min. to max., A,B,G) or mean ± SD (E), *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t-test with Welch’s correction (A,B) or 2-way repeated measures ANOVA with Bonferroni’s post-hoc test (E,G)).