Highly recruited brown adipose tissue does not in itself protect against obesity

Figures

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  Figure 1. Large increase in total UCP1 protein amounts after recruitment. Adult mice (35 male C57Bl/6) were given high-fat diet and placed at two ambient temperatures (“non-recruited” at 29 °C and “recruited” at 4 °C) on day −33. At day 0, the recruited mice were transferred to 29 °C (thermoneutrality). At the indicated time-points, 5 mice from each group were euthanized and interscapular brown adipose tissue and inguinal white adipose tissue were excised, weighed and analyzed. A: Wet weight of IBAT. B: Total protein content in IBAT. C: Representative Western blot of UCP1 protein levels in IBAT. Non-recruited shown with red letters and recruited shown with blue letters, d0 = day 0 etc. The standard (Std) is here set to 1.00 AU (arbitrary units) of UCP1, as indicated. All other lanes were loaded with 2 μg protein per sample. D: UCP1 protein per mg protein. E: Total amount of UCP1 in IBAT, calculated as values from B multiplied with corresponding values from D. F: Wet weight of ingWAT. G: Total protein content in ingWAT. H: Representative Western blot of UCP1 protein levels in ingWAT. The standard (Std) loaded in this case was 0.15 AU (arbitrary units) UCP1 (compared to 1.00 AU UCP1 in the…

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  Figure 2. No obesity protection despite high UCP1 amounts. The mice from Figure 1 were monitored. Body composition was measured on the days indicated and food intake was measured from day −17 to day 18. Initial weights at day −33 were 23.2 ± 0.3 for non-recruited mice and 23.2 ± 0.4 for recruited mice (means ± SE). A: Body weight. B: Body composition; lean body mass and body fat mass. C: Body fat gain per day at different time periods (day 18 for non-recruited not shown for technical reasons). D: Food intake. E: Metabolic efficiency expressed as % of the energy in the ingested food stored as body fat. n = 15 (day -33 – 0); n = 10 (day 4); n = 5 (day 8–18). Significances as described in legend to Figure 1.

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  Figure 3. No difference in energy expenditure after transfer to warm. An independent group of mice were treated as the mice in Figure 1, Figure 2. At day 0, i.e. on transfer of recruited mice into thermoneutrality, the mice were placed for three nights and two days in metabolic chambers kept at 29 °C. Oxygen consumption and carbon dioxide production were measured. The first 3 h are not shown. Starting weights at day −33 were 24.3 g ± 0.3 for non-recruited and 24.8 g ± 0.5 for recruited (means ± SE). A: Energy expenditure, expressed as Watt per mouse. As the lean body weights of these mice were very similar (non-recruited: 20.0 ± 0.3 g; recruited 20.3 ± 0.3 g; means ± SE), the curves are unchanged if expressed per g lean body mass. B: Total energy expenditure while in the chambers (68 h). The data were further analysed by ANCOVA with body weight as additional variable (see also supplementary figure); this did not affect the statistical outcome. C: Total energy intake while in the chambers (68 h). The data were further analysed by ANCOVA with body weight as additional variable (see also supplementary figure); this did not affect the statistical outcome. D: Changes in body composition while…

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  Figure 4. Effects of BAT recruitment on food digestibility. Another independent group of mice were treated as the mice in Figure 1, Figure 2. A–C: At the end of the experiment (day 18), the intestine was dissected out and measured in its total length (A) or only the small intestine (B), and the total intestine was weighed (C). D–K: Effects on digestibility. D: food intake during 65 h from day 4 in the afternoon to day 7 in the morning. E: total amount of feces collected during this period. F: ratio between the E and D. G: total energy in food eaten (based on the information from the producers). H: energy in feces (determined as detailed in Methods). I: total energy in feces (E multiplied by H). J: total energy absorbed (G minus I). K: digestibility (% of food ingested not excreted; ((G minus I)/I). n = 6 for all panels; significances as described in in legend to Figure 1.

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  Figure 5. No qualitative difference in metabolic efficiency between wildtype and UCP1-ablated mice. A group of wild-type mice (ACEG) and a corresponding group of UCP1(−/−) mice (BDFH) were treated in the same way as in the previous studies, but food intake and body composition were measured every day; the two groups were not studied in parallel. Initial body weights at day −29 were for wild-type 28.1 ± 0.8 g for non-recruited and 27.7 ± 0.7 for recruited (means ± SE); for UCP1(−/−) 23.2 ± 0.8 for non-recruited and 23.0 ± 1.0 for recruited (means ± SE) (note that UCP1-ablated mice were smaller). A: Food intake in wild-type mice. B: Food intake in UCP1(−/−) mice. C: Body fat mass in wild-type mice. D: Body fat mass in UCP1(−/−) mice. E: Body fat gain per day in wild-type mice. F: Body fat gain per day in UCP1(−/−) mice. G: Metabolic efficiency in wild-type mice. H: Metabolic efficiency in UCP1(−/−) mice. In all panels, n = 5–8. Significances as described in legend to Figure 1. For E-H, the points represent the mean values of the single day measurements; the running curve is the smoothed curve calculated with 2 neighbours on each side.

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  Figure 6. Effect of BAT recruitment on blood glucose levels. ABC: Independent groups of mice (the same as in Figure 4) were treated as those described in Figure 1 and their body weight, fat mass and lean mass followed; n = 18/18. D: On days 1 and 18 after transfer of the recruited mice to 29 °C, a group of mice were fasted for 4.5 h and their blood glucose levels determined as described in Methods; n = 12/12. E: On the same days, another group of mice were fasted overnight and their blood glucose levels determined; n = 6/6. Error bars were smaller than the size of the symbols. Statistics as in Figure 1. # etc. indicates significant differences between day 1 and day 18 (colour-coded). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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  Figure 7. Effect of BAT recruitment on glucose-related metabolic parameters. The mice in Figure 4, Figure 7 were examined on the day they were transferred to thermoneutrality (AFK) and on day 18 after transfer (CHM). A–E: glucose tolerance tests. F–J: insulin tolerance tests. K–O: pyruvate tolerance tests. The compilation of the results in BDGILN are all incremental values, i.e. the initial blood glucose values were subtracted. Statistics as in Figure 1; # etc. indicates significant differences between day 1 and day 18 (colour-coded). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)