In this study, we investigated the effects of fish oil intake on energy expenditure induced by UCP1. Consistent with previous studies, fish oil intake prevented the development of obesity; however, the mechanisms underlying the possible induction of thermogenesis and decreased fat accumulation remained unclear32,33. As shown previously in mice, the anti-obesity effect of fish oil could be dependent on lipid metabolism in the liver34 and fatty acid oxidation in the intestines35. Furthermore, as shown in this study, fish oil intake enhances oxygen consumption and rectal temperature. Thus, fish oil intake decreases in body weight gain and fat accumulation by increasing energy expenditure, suggesting that fish oil intake enhances thermogenesis.

UCP1-mediated thermogenesis in BAT plays an important role in the regulation of energy expenditure. Furthermore, UCP1 is a major determinant of BAT thermogenic activity36. Our data indicate that UCP1 expression in interscapular BAT (classical brown adipocytes) and inguinal WAT (beige/brite adipocytes) was increased by fish oil intake. Interscapular BAT and inguinal WAT share a number of BAT specific genes, such as UCP1, Pgc1α, Cpt1b, Cidea, Prdm16 and Fgf21; however, the two types of adipose tissue express these mRNAs at different levels. Interestingly, the inguinal WAT of fish oil-fed mice expressed beige adipocyte specific genes, such as Tbx137. Furthermore, human BAT isolated from multiple locations, including the supraclavicular and retroperitoneal regions, abundantly express beige adipocyte-specific genes, indicating that human BAT is similar to beige adipocytes38. Recently, it was revealed that inducible beige adipocytes have potent thermogenic activity that is comparable to classical brown adipocytes39. Furthermore, immunochemical analysis of UCP1 reveals the presence of UCP1-positive multilocular adipocytes, a sign of beige adipocytes, in inguinal WAT after fish oil intake. Taken together, fish oil intake induced thermogenesis in both interscapular BAT and inguinal WAT.

Interscapular BAT is heavily innervated by the SNS and NA released from the activated sympathetic nerves promotes thermogenesis by activating the β3AR40. Moreover, β3AR regulates the thermogenic functions of both brown and white adipocytes40. Fish oil intake increased catecholamine levels in the urine. In addition, fish oil intake increased NA turnover and rate constant in interscapular BAT and inguinal WAT. In particular, enhanced NA turnover and rate constant are considered a direct indicator of sympathetic activity in organs under sympathetic control41. The NA released from the SNS stimulates β3AR in interscapular BAT and inguinal WAT. Stimulation of β3AR leads to the induction of UCP1. Interestingly, β adrenergic blocker-treated and vagotomized mice showed the enhancement of UCP1 expression induced by EPA-enriched fish oil was canceled, suggesting that afferent vagal nerve in gastrointestinal tract mediates the stimulatory actions of fish oil. Taken together, our data indicate that fish oil intake can induce UCP1 in adipose tissues via the SNS.

TRPV4, a member of the TRPV family, is abundant in adipocytes and adipose tissues42,43. Ye et al. reported that mice that were intraperitoneally administered TRPV1 and TRPV4 antagonists showed increased browning (thermogenesis) of adipose tissues and were protected from diet-induced obesity43. However, our study suggested that dietary fish oil intake stimulated thermogenesis through the activation of TRPV1. This difference might have been a result of the different functions of TRPVs in different organs. Our additional experiments showed that both subdiaphragmatic vagotomy surgeries and treatment with a β-adrenergic blocker prevented the increase in UCP1 expression that was induced by the oral administration of fish oil. Moreover, TRPV1 expression in the gastrointestinal tract has been shown to play an important role in the activation of the sympathetic nervous system that is induced by capsinoids, which are TRPV1 agonists. Therefore, we speculated that the expression of TRPV1 in the gastrointestinal tract also plays an important role in the activation of the sympathetic nervous system that results from the enhancement of UCP1 expression that was induced by fish oil intake. However, Ye et al. reported that intraperitoneal injections of TRPV1 and TRPV4 antagonists directly inhibited Ca2 + influx in adipocytes, which resulted in the induction of adipocyte browning43. These results indicated that organs in which TRPV1 is activated are important for the browning of adipocytes.

Dietary factors regulate the development and function of brown and beige adipocytes. Capsaicin in chili peppers, has been shown to enhance catecholamine secretion from the adrenal medulla through activation of the SNS44. Recently, capsinoids, a group of capsaicin analogues, were shown to activate gastrointestinal TRPV1 and induce BAT thermogenesis in humans45 and rodents27. Capsinoids-induced thermogenic sympathetic responses in BAT seem to require the activation of extrinsic nerves connected to the gastrointestinal tract27,31. Moreover, TRPV1 expressing afferent nerves were observed within gastrointestinal tracts46. However, TRPV1 has been reported to express in adipocytes47 and suggested to play roles in the regulation of energy metabolism47,48. Thus, we could not rule out the possibility of the contribution of TRPV1 to the fish oil-induced UCP1 expression in adipose tissues. Further studies are needed to clarify critical TRPV1-expressing tissues in which contribute to this phenomenon. Taken together, fish oil containing EPA and DHA, might activate SNS via the activation of TRPV1 expressing on the afferent nerves in the gastrointestinal tract, leading to the upregulation of UCP1 expression in interscapular BAT and inguinal WAT.

Body weight gain and fat accumulation were not decreased after fish oil intake in TRPV1 KO mice. Furthermore, fish oil intake did not increase oxygen consumption in TRPV1 KO mice. Additionally, UCP1 expression was significantly increased in WT mice following fish oil treatment, but not in TRPV1 KO mice. These findings indicate that TRPV1 plays an important role for upregulation of energy expenditure in response to fish oil intake. Interestingly, EPA and DHA can modulate TRPV1 activity directly and indirectly29. DHA and EPA displace TRPV1 ligand binding and evoke TRPV1 currents28. On the other hand, activation of TRPV1 by EPA and DHA requires activation of protein kinase C (PKC)28. PKC-dependent phosphorylation is known to increase the sensitivity of TRPV149,50. In addition, thermal and chemical stimuli have been reported to activate TRPV1 synergistically51,52,53 and this synergistic activation was also suggested in the case of EPA and DHA29. Thus, it can be postulated that fish oil containing EPA and DHA may have a potential for activating TRPV1 through both direct and indirect manners. Taken together, fatty acids such as EPA and DHA or food ingredients, which activate TRPV1, may elicit sympathetic nerve activation, leading to UCP1-dependent thermogenesis in both interscapular BAT and inguinal WAT.

Alternatively, UCP1 upregulation by fish oil may arise via its anti-inflammatory activity. Indeed, EPA and DHA suppress obesity-induced adipose inflammatory responses by reduction of inflammatory cytokines production in co-cultured adipocytes/macrophage54,55, which is associated with anti-inflammatory macrophage M2 phenotype switching55. More importantly, we previously demonstrated that macrophage-mediated inflammatory cytokine such as tumor necrosis factor α suppresses the induction of UCP1 expression in white adipocytes via extracellular signal-regulated kinase activation in obese and diabetic conditions56. Hence, fish oil-induced upregulation of UCP1 in our observation may be at least in part attributed to its anti-inflammatory action in inguinal WAT.

In summary, fish oil activates and recruits interscapular BAT and inguinal WAT via activation of TRPV1, thereby increasing energy expenditure and decreasing body weight gain and fat accumulation. Based on these results, we propose that the SNS and BAT mediate the thermogenic effect of fish oil. Furthermore, fish oil-mediated thermogenesis via the SNS enhanced energy expenditure and reduced fat accumulation. A schematic model of this unique mechanism is shown in Fig. 6D. Thus, fish oil intake can induce UCP1 expression in classical brown and beige adipocytes via the SNS and TRPV1 and may contribute to an effective treatment for obesity.