*4.2. Diet*

Mice received a fat-rich diet (30 en%) containing either mixed fat with safflower oil and butter (control) or FO (n = 7 in each group). Diets were prepared as described previously [60,70], and the composition of the diet is listed in Table 3. Butter and safflower oil were purchased from Snow Brand Milk Corp. (Hokkaido, Japan) and Benibana Food (Tokyo, Japan), respectively. FO (containing 7% EPA and 24% DHA) was kindly provided by the NOF Corporation (Tokyo, Japan). The food was provided to the mice every day. To estimate daily food intake, the food weight of each day was subtracted from the initial food weight of the previous day. Mean food intake over the entire experimental period in the two groups of mice was calculated using these data. The diets were offered for 10 days.



Con: control; FO: fish oil; en%: energy %.

## *4.3. Measurement of O2 Consumption and CO2 Production to Calculate DIT and Energy Production*

Mice on the 9th day of the experimental diet administration were used for the experiment. The method for calculating DIT was described previously [37]. Briefly, mice were placed in the calorimeter without food 6 days before starting the experiment at 1700 h, and then energy metabolism was measured for the 11-h period from 0000–1100 h. Oxygen consumption (VO2) and carbon dioxide production (VCO2) were monitored with a system that measures O2/CO2 metabolism in small animals (MK-5000RQ; Muromachi Kikai Co., Ltd., Tokyo, Japan), and their values were used to calculate DIT and EE. The EE was calculated as follows: EE (kcal/min) = 3.9 VO2 + 1.1 VCO2 [71]. For the measurements made after feeding, the same mice used in the fasted measurements were placed in the calorimeter at 1600 h. The research diet was provided at 1700 h, and energy metabolism was measured over the 22-h period from 1700–1500 h. VO2, VCO2 and activity were monitored by the system at 3-min intervals, and every four data points were averaged. The average value

over the 12-min period was considered the mean value. The data were normalized to the square root of the activity count. Under the fasting conditions, 55 (5/h × 11 h) values each for EE and activity were selected from the measurements obtained over the 11-h period; we then plotted EE against the square root of activity and identified a linear regression equation by simple linear regression analysis. Under the fed conditions, 110 (5/h × 22 h) values each for EE and activity were selected from the measurements obtained over the 22-h period, and EE was then plotted against the square root of activity.

#### *4.4. Quantitative Real-Time PCR*

On the 10th day of the experimental diet, mice were sacrificed by cervical dislocation, and BAT, subWAT and liver were extracted from the mice. RNA was extracted from these tissues with TRIzol Reagent (Molecular Research Center, Inc., Cincinnati, OH, USA) following manufacturer's instructions. RNA was isolated and quantified with a NanoDrop ND-2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Total RNA isolated from BAT and subWAT was reverse transcribed, and quantitative real-time RT-PCR was performed as described previously [60,72]. The primers for quantitative real-time PCR are listed in Table 4.


