*2.2. Body Weight and Tissue Weights of Con- and FO-Fed Mice*

The mean energy intake was similar between the Con- and the FO-fed mice during the 10-day administration period (Con, 17.6 ± 0.8 kcal/day; FO, 17.7 ± 0.6 kcal/day, *p* = 0.97). Although final body weight (BW) was not different between the Con- and FO-fed mice, the BW gain in the FO-fed mice was significantly lower than that in the Con-fed mice during the 10-day period (Con, 10.3 ± 1.4%; FO, 6.6 ± 1.0%, *p* < 0.05). The weights of subcutaneous WAT (subWAT) in the FO-fed mice were not different from those in the

FO-fed mice (*p* = 0.07, Table 1). However, the weights of epididymal WAT and mesenteric WAT in the FO-fed mice were lower than those in the Con-fed mice. The weight of BAT was not affected by FO supplementation.

**Table 1.** BW and weights of tissues in Con- and FO-fed mice.


Values are mean ± SEM (n = 7). \* *p* < 0.05 vs. Con-fed mice. Significant differences between two groups were tested by Student *t*-test. BW: body weight; Con: control; FO: fish oil; BAT: brown adipose tissue; WAT: white adipose tissue.

#### *2.3. Serum Chemicals of Con- and FO-Fed Mice*

Because the weights of the epididymal WAT and mesenteric WAT in the FO-fed mice were lower than those in the Con-fed mice, we analyzed serum concentrations of glucose, non-esterified fatty acid (NEFA), triglyceride (TG) and total cholesterol (TC). The concentrations of serum glucose in the Con- and the FO-fed mice were the same (Table 2). However, the serum concentrations of NEFA, TG and TC in the FO-fed mice were significantly lower than those in the Con-fed mice (Table 2).

**Table 2.** Serum chemicals of Con- and FO-fed mice.


Values are mean ± SEM (n = 7). \*\* *p* < 0.01, \*\*\* *p* < 0.001 vs. Con-fed mice. Significant differences between two groups were tested by Student *t*-test. Con: control; FO: fish oil; NEFA: non-esterified fatty acid; TG: triglyceride; TC: total cholesterol.

#### *2.4. Effects of FO Supplementation on BAT*

To confirm the mechanism of increase of DIT in the FO-fed mice, we examined expression profiling of the *Ucp1* gene and UCP1 protein. FO supplementation resulted in a 1.5-fold increase in *Ucp1* mRNA in BAT (Figure 3a). UCP1 protein expression was also analyzed (n = 7 in each group), and representative data (n = 2 in each group) indicating a 1.2-fold increase in expression are shown in Figure 3b. The mRNA expression of *Pparα*, which is one of the nuclear transcription factors whose activation leads to increased fatty acid β-oxidation [39], was significantly increased by FO supplementation (Figure 3a). However, FO supplementation did not affect the mRNA expressions of target genes carnitine palmitoyltransferase I (*Cpt I*), acyl-CoA oxidase (*Aco*) and medium-chain acyl-CoA dehydrogenase (*Mcad*) (Figure 3a). Fibroblast growth factor 21 (*Fgf21*) expression was also not increased by FO administration (Figure 3a). The expression of the mitochondria biogenesis marker peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) and that of crucial thermogenesis biomarker type 2 iodothyronine deiodinase (Dio2) in the mice was not different between the two groups (Figure 3a). No difference in β3-adrenergic receptor (β3-AR) mRNA was observed in BAT (Con, 100.0 ± 7.0%; FO, 93.0 ± 11.4%).

**Figure 3.** Effect of fish oil (FO) supplementation on gene expression and UCP1 protein levels in brown adipose tissue. mRNA levels of *Ucp1* and *Pparα* and its target genes (**a**) and UCP1 protein (**b**) were assessed by quantitative RT-PCR or western blotting. β-actin was used as the normalization control. The percent of mRNA and protein levels relative to those of Con-fed mice are indicated. White and gray columns represent data from the Con- and FO-fed mice, respectively. Values are mean ± SEM (n = 7). \* *p* < 0.05, \*\*\* *p* < 0.001 vs. Con-fed mice. Significant differences between two groups were tested by Student *t*-test.
