**4. Discussion**

In this study, to the best of our knowledge, we revealed for the first time the inhibitory effects of FAs on URAT1-dependent urate transport in vitro. While further studies are highly warranted to address the pathophysiological impact of our findings in terms of the possible effect on SUA and renal urate excretion in hyperuricemia model animals as well as in humans, the obtained results will open a new avenue for FAs as nutritionally important substances influencing human health.

Our findings may extend the potentially beneficial effects of PUFAs of the ω-3 family on reducing the risk of hyperuricemia/gout. In fact, recent studies have shown that a dietary intake of certain ω-3 PUFAs, such as EPA and DHA can, at least partly, reduce a wide number of inflammation-related biological reactions [1,4,5,22]. With respect to gout, a small case–control study on patients with this inflammatory condition showed an association between high ω-3 FA levels in the blood and lower frequency of gou<sup>t</sup> attacks [23]. Additionally, ω-3 PUFA-rich fish consumption was reportedly associated

with lower risk of recurrent gou<sup>t</sup> attacks in a case-crossover study [24]. Moreover, the anti-inflammatory effects of ω-3 PUFAs were supported by the results obtained in animal models of acute inflammation induced by monosodium urate crystals [25,26]. Thus, in addition to the anti-inflammatory e ffects previously observed, it should be confirmed in future studies whether ω-3 PUFAs can exhibit uricosuric effects in hyperuricemia models or not.

The potential e ffects of the daily consumption of ω-3 PUFA-enriched foods on SUA will also be of interest. While the available information is limited and currently inconclusive, a randomized controlled trial in young healthy subjects showed that daily intake of fish oil (2 g; majorly consisting of DHA and EPA) resulted in a significant decrease of SUA after 4 and 8 weeks of supplementation [27]. A similar significant decline in SUA was also observed in healthy elderly men consuming daily supplement pills characterized by ω-3 FAs such as DHA and EPA for three months, although the change was not extensive [28]. Based on these pieces of evidence, increasing the daily intake of ω-3 FAs via eating pattern changes, such as appropriate choice of aliments and cooking oils, might be beneficial to health in terms of SUA management. On the other hand, the behavior of ω-3 FAs in the body has been hardly investigated in those clinical investigations, which warrants further studies focusing on the beneficial effects of dietary and/or endogenously produced ω-3 FAs on the renal urate handling in the body. Additionally, in such cases, not only the URAT1-inhibitory activity but also the disposition of target FAs should be considered.

There were some limitations to our study. First, the present study could not reveal how the FAs inhibited URAT1 function. Addressing this issue in the future will provide a deeper insight into the latent mechanistic features of URAT1. As an antiporter, URAT1 mediates urate transport in exchange for monocarboxylates such as lactate [12], which suggests that URAT1 must have at least two substrate recognition sites in its protein structure. Considering that FAs are carboxylic acids with a long aliphatic chain, they might a ffect the recognition and/or subsequent membrane transport of the counterpart substrates by URAT1 rather than the recognition of urate. Second, we could not exclude the possibility that the FAs a ffected the plasma membrane properties, which might result in the indirect decrease of URAT1 function. Nonetheless, given that the interaction of free FAs with cellular membranes occurs within minutes [21] and usually requires biochemical conversion of FAs into phospholipids, the experimental period we used in this study, 20 s incubation for urate uptake, was so short that the tested FAs must have had a negligible e ffect on the plasma membranes during the assay. Finally, the e ffects of FAs on other physiologically important urate transporters—GLUT9/SLC2A9 [29,30], OAT10/SLC22A13 [31], and ABCG2/BCRP [32–34]—remain to be elucidated. Since such urate transporters, including URAT1, coordinately regulate the behavior of urate in the human body, comprehensive understanding of the latent interaction between FAs and these transporters should be addressed in the future. Among them, GLUT9 expressed on the basal membrane of proximal tubular cells is involved in the urate transport from the cells to the blood as a counterpart of URAT1 [7]; *GLUT9* is the causative gene for renal hypouricemia type 2 [30]. Given these pieces of information, GLUT9 has the highest priority in the future investigation.
