**3. Results**

#### *3.1. URAT1-Mediated Urate Uptake in 293A Cells*

Prior to screening the inhibitory effects of 25 FAs on URAT1, we verified our cell-based assay system—an in vitro urate transport assay with mammalian cells transiently expressing URAT1 (Figure 1). Expression of EGFP-tagged URAT1 (EGFP-URAT1) as a matured N-linked glycoprotein (Figure 1a) and its plasma membrane localization (Figure 1b) in 293A cells were confirmed 48 h after plasmid transfection by immunoblotting and confocal microscopy, respectively. Next, we successfully detected URAT1-mediated urate uptake into URAT1-expressing cells, which showed a much stronger transport activity compared to mock cells, representing background urate uptake, indicating that the assay was suitable for the screening (Figure 1c). As expected, URAT1-mediated urate uptake was almost completely inhibited by benzbromarone (30 μM), a URAT1 inhibitor employed as a uricosuric drug. These results were consistent with our previous study [15]. A schematic illustration of this urate transport assay is shown in Figure 1d.

**Figure 1.** Cell-based urate transport assay with 293A cells transiently expressing URAT1. (**a**) Immunoblot detection of URAT1 protein in whole-cell lysates prepared 48 h after the transfection. α-Tubulin, a loading control. (**b**) Intracellular localization of URAT1. Confocal microscopy images were obtained 48 h after the transfection. Nuclei were stained with TO-PRO-3 iodide (gray); plasma membrane was labeled with Alexa Fluor® 594-conjugated wheat germ agglutinin (red). Bars, 10 μm. (**c**) Urate transport activities. Urate uptake into cells treated with or without 30 μM of benzbromarone (Benz) was measured. Data are expressed as the mean ± SD; *n* = 3. \*\*, *p* < 0.01 (Tukey–Kramer multiple-comparison test). (**d**) Schematic illustration of URAT1-mediated urate transport examined using 293A cells transiently expressing URAT1.

#### *3.2. Unsaturated Fatty Acids Are Stronger Inhibitors of URAT1 Activity Than Saturated Fatty Acids*

Next, we examined the inhibitory effects of 25 FAs—8 saturated (Figure A1) and 17 unsaturated (Figure A2) FAs—at a concentration of 100 μM on URAT1 function (Figure 2). Despite some exceptions, in this study, almost all of the unsaturated FAs showed a stronger inhibitory effect on URAT1 than saturated FAs. Among the eight saturated FAs, relatively short FAs with chain lengths ranging from C4 to C8 had little effect on URAT1-mediated urate transport; instead, the others (C10 to C18) mildly inhibited URAT1 at the screening concentration. This result suggested that the length of FA could have a substantial effect on the URAT1-inhibitory activity of FAs. Among the 17 unsaturated FAs, 9 inhibited URAT1 activity by over 50%. We therefore focused on these candidates.

**Figure 2.** Inhibitory effects of each fatty acid on URAT1-mediated urate transport. The effects of each fatty acid (100 μM) on URAT1-mediated urate transport were investigated with the urate uptake assay. Control, vehicle (non-fatty acid treated) control. Data are expressed as the mean ± SD; *n* = 3–4. †, *p* < 0.05; ††, *p* < 0.01 vs. control (one-sample *t*-test). Red bars mean that the tested fatty acids inhibited URAT1-mediated urate transporter activity by over 50% compared to control.

#### *3.3.* ω*-3 Fatty Acids Are the Most E*ff*ective URAT1 Inhibitors*

Further investigation of the dose-dependent inhibitory effects of the nine unsaturated FAs on URAT1 determined the IC50 values that are illustrated in Figure 3. Based on the IC50 values, EPA was the strongest URAT1 inhibitor among the nine unsaturated FAs examined. Furthermore, EPA inhibited URAT1 activity more strongly than the other unsaturated FAs at low concentrations (≤1 μM) (Figure 3g). Second to EPA, its biosynthetic precursor α-linolenic acid (ALA) (Figure 3a) as well as its product DHA (Figure 3i) strongly inhibited URAT1, while ω-3 docosapentaenoic acid (DPA) showed an IC50 of > 100 μM (Figures 2 and A3). Additionally, contrary to ALA, linoleic acid (LA) had a high IC50 (133 μM); however, LA could inhibit URAT1 at low concentrations (≤1 μM) (Figure A3). Interestingly, considering the biosynthetic pathways of the above-described FAs distinguished by their structural feature (ω-3 or ω-6 family) (Figure A4), ω-3 FAs seem to inhibit URAT1 more effectively than ω-6 FAs. Given that elevated intake of ω-6 FAs may reportedly promote inflammation, while ω-3 FAs help reduce it [21], ω-3 FAs will be preferable to ω-6 FAs for the prevention of hyperuricemia/gout.

**Figure 3.** Concentration-dependent inhibition of URAT1-mediated urate transport by unsaturated fatty acids. The effects of each unsaturated fatty acid (0, 0.1, 0.3, 1, 3, 10, 30, 100, or 300 μM) on URAT1-mediated urate transport were investigated with the urate uptake assay. (**a**) α-Linolenic acid (ALA); (**b**) γ-linolenic acid; (**c**) eicosadienoic acid; (**d**) eicosatrienoic acid; (**e**) ω-3 eicosatetraenoic acid; (**f**) arachidonic acid (ω-6 eicosatetraenoic acid); (**g**) eicosapentaenoic acid (EPA); (**h**) henicosapentaenoic acid; (**i**) docosahexaenoic acid (DHA). Data are expressed as the mean ± SD; *n* = 4.
