**1. Introduction**

Obesity results when energy intake continuously exceeds energy expenditure (EE). Total daily energy expenditure (TEE) is comprised of multiple components such as basal metabolic rate, diet-induced thermogenesis (DIT) and physical activity-related EE [1]. DIT is defined as an increase in EE above that of the fasting state and is related to digestion, intestinal absorption of nutrients and storage of these nutrients [2]. One of the methods to prevent overweight and obesity is to increase energy consumption by upregulation of DIT [3].

Brown adipose tissue (BAT) is the main site for the induction of DIT and cold-induced thermogenesis, which significantly contributes to controlling body temperature and EE [4]. Although BAT is considered to be abundant in small rodents and human infants and decreases with aging in human [5], recent studies showed that functional BAT was identified in adult human [6,7]. The thermogenic ability of BAT is principally dependent on uncoupling protein 1 (UCP1) [8,9]. UCP1 facilitates uncoupling of mitochondrial substrate oxidation from ATP production, which leads to energy release as heat from free fatty acid oxidation [4].

UCP1-ablated mice consumed less oxygen than wild-type mice during the eating period, that is, DIT was UCP1-dependent [10]. UCP1-deficient mice maintained in a room at 23 ◦C developed obesity with age; therefore, UCP1 may play an important role against

**Citation:** Yamazaki, T.; Li, D.; Ikaga, R. Fish Oil Increases Diet-Induced Thermogenesis in Mice. *Mar. Drugs* **2021**, *19*, 278. https://doi.org/ 10.3390/md19050278

Academic Editors: Maria do Rosário Domingues and Philippe Soudant

Received: 26 March 2021 Accepted: 14 May 2021 Published: 17 May 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

obesity [11]. UCP1 gene polymorphism (−3826 A/G) showed lowered capacity of thermic effect in response to dietary intake in healthy boys aged 8–11 years [12]. Thus, the function of UCP1 and activity promoting the activation of BAT greatly contribute to the increase of DIT.

BAT is strongly activated by exposure to cold and by pharmacological effects, such as that of β3-adrenergic receptor agonist [6,13,14]. Moreover, it has been reported that BAT is activated by food ingredients such as capsinoids, thereby contributing to a reduction in body fat [15,16]. Fish oil (FO) also has anti-obesity effects in humans [17–19]. FO contains a high content of n-3 polyunsaturated fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which must be obtained from the diet or synthesized from alpha-linolenic acid in the body [20–22]. DHA and EPA bind to peroxisome proliferatoractivated receptor (PPAR) α and thereby activate PPARα [23,24], which is highly expressed in BAT [25]. PPARα binds to the PPAR response element of the *Ucp1* gene to increase mRNA expression of *Ucp1* [26].

Recently, beige adipose tissue, which is produced by the browning of white adipose tissue (WAT), has been reported as a third type of adipose tissue in addition to WAT and BAT [27–29]. Beige adipocytes are strongly induced by some environmental conditions and external cues such as exposure to cold and some pharmacological treatments, and they have potent thermogenic ability similar to that of classical brown adipocytes [30]. FO treatment leads to the browning of WAT, increases thermogenic genes such as *Ucp1* [31–33], stimulates thermogenesis, as measured by rectal temperature [34,35], and increases EE without changes in food intake [36]. These studies only suggest the possibility that FO influences DIT, however, and how much FO actually increases DIT is still unknown.

We recently developed a new technique to measure absolute DIT values in mice by applying a methodology used in the measurement of DIT in human to mice using a respiratory chamber [37]. In the present study, we showed how much FO increased DIT through activation of BAT and browning of WAT. An increase in DIT may have potential impact on anti-obesity and therapy for diabetes [6,7,38], and the evidence shown in this study indicates that FO might be a promising dietary fat.

#### **2. Results**

## *2.1. Effects of Fish Oil Supplementation on DIT, EE, Activity and RER*

Energy metabolism of mice was measured after 9 days of feeding of each experimental diet. The measurements of O2 consumption, CO2 production and activity (defined as the count per minute of any movement made by mouse) of the mice were carried out over a 22-h period. The DIT of the control fat (Con)-fed mice began to increase as soon as they started eating, was maintained at a high level during the dark period, and then decreased toward the end of the dark period (Figure 1a). However, DIT increased again after the start of the light period. Similar changes were observed in the FO-fed mice (Figure 1a). When comparing the DIT in the Con- and FO-fed mice every hour, DIT in the FO-fed mice was significantly higher at 0000 and 0300. EE in the dark period and light period was not different in the Con- and FO-fed mice (Dark: Con, 7615 ± 76 cal/h/kg0.75; FO, <sup>7582</sup> ± 87 cal/h/kg0.75; Light: Con, 6712 ± 83 cal/h/kg0.75; FO, 6641 ± 92 cal/h/kg0.75). However, DIT in the dark period was higher in the FO-fed mice than that in the Con-fed mice (Dark: Con, 1275 ± 22 cal/h/kg0.75; FO, 1541 ± 32 cal/h/kg0.75, *<sup>p</sup>* < 0.01; Light: Con, <sup>1509</sup> ± 47 cal/h/kg0.75; FO, 1674 ± 46 cal/h/kg0.75). There was no difference in activity every hour between the two groups (Figure 1b). Activity in the dark period and light period was not different in the Con- and FO-fed mice (Dark: Con, 238.2 ± 13.2 count/min; FO, 221.2 ± 23.4 count/min; Light: Con, 95.4 ± 5.8 count/min; FO, 96.5 ± 11.7 count/min). The respiratory exchange ratio (RER) in the FO-fed mice was higher at 0600 and 1300 than that in the Con-fed mice, but there was no significant difference at other times (Figure 1c). RER in the dark period and light period was not different in the Con- and FO-fed mice (Dark: Con, 0.881 ± 0.010; FO, 0.901 ± 0.006; Light: Con, 0.860 ± 0.005; FO, 0.893 ± 0.009). The total energy intake during DIT measurement was almost the same in the Con- and

the FO-fed mice (Figure 2a). Total DIT over 22 h was calculated from the area under each curve. Total DIT in the FO-fed mice was 1.2-fold higher than that in the Con-fed mice (Figure 2b). TEE over 22 h was also calculated from the area under each curve. The values of activity and TEE between the two groups were not different (Figure 2c,d). DIT (%) versus calorie intake was calculated by dividing total DIT by total calorie intake and is indicated as DIT/intake in Figure 2e. DIT/intake was 11.2% for the FO-fed mice, which was 1.2-fold higher than that for the Con-fed mice. DIT (%) versus TEE was calculated by dividing total DIT by TEE and is indicated as DIT/TEE in Figure 2f. DIT/TEE for the FO-fed mice was 22.3%, which was also 1.2-fold higher than that for the Con-fed mice.

**Figure 1.** Time course of diet-induced thermogenesis (DIT), energy expenditure (EE), activity and respiratory exchange ratio (RER) in the control fat (Con)- and fish oil (FO)-fed male mice. The measurements were carried out over a 22-h period. The data of EE (upper lines), DIT (lower lines) (**a**), activity (**b**) and RER (**c**) are shown for every hour. White circles and gray squares represent data from the Con- and the FO-fed mice, respectively. The black and white bars on the x axis represent dark and light cycles, respectively. Values are mean ± SEM (n = 7). \* *p* < 0.05, \*\* *p* < 0.01 vs. Con-fed mice. Significant differences between two groups were tested by Student *t*-test.

**Figure 2.** Values of total energy intake, diet-induced thermogenesis (DIT), activity and total energy expenditure (TEE) during DIT measurement in the control fat (Con)- and fish oil (FO)-fed male mice. Total energy intake (**a**) at measurement of energy metabolism was estimated by subtracting the food weight at the completion of measurement from the initial food weight measurement. The values of total DIT (**b**), activity (**c**) and TEE (**d**) were calculated from measurements taken over 22 h under the fed condition. DIT/intake (**e**) and DIT/TEE (**f**) were calculated by dividing total DIT by total calorie intake and by TEE, respectively. White and gray columns represent data from the Con- and FO-fed mice, respectively. 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.
