nutrients
Communication

Omega-3 Polyunsaturated Fatty Acids Inhibit
the Function of Human URAT1, a Renal
Urate Re-absorber
Hiroki Saito 1,2,† , Yu Toyoda 2,† , Tappei Takada 2, *,† , Hiroshi Hirata 1 , Ami Ota-Kontani 1 ,Hiroshi Miyata 2 , Naoyuki Kobayashi 1 , Youichi Tsuchiya 1 and Hiroshi Suzuki 2
1

2

*
†

Frontier Laboratories for Value Creation, Sapporo Holdings Ltd., 10 Okatome, Yaizu, Shizuoka 425-0013,
Japan; hiroki.saito@sapporoholdings.co.jp (H.S.); hiroshi.hirata@sapporoholdings.co.jp (H.H.);
ami.ota@sapporoholdings.co.jp (A.O.-K.); naoyuki.kobayashi@sapporoholdings.co.jp (N.K.);
yoichi.tsuchiya@sapporoholdings.co.jp (Y.T.)
Department of Pharmacy, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655,
Japan; ytoyoda-tky@umin.ac.jp (Y.T.); hmiyata-tky@umin.ac.jp (H.M.); suzukihi-tky@umin.ac.jp (H.S.)
Correspondence: tappei-tky@umin.ac.jp
These authors contributed equally to this work.

Received: 2 May 2020; Accepted: 27 May 2020; Published: 29 May 2020


Abstract: The beneficial effects of fatty acids (FAs) on human health have attracted widespreadinterest. However, little is known about the impact of FAs on the handling of urate, the end-productof human purine metabolism, in the body. Increased serum urate levels occur in hyperuricemia,
a disease that can lead to gout. In humans, urate filtered by the glomerulus of the kidney is majorly
re-absorbed from primary urine into the blood via the urate transporter 1 (URAT1)-mediated pathway.
URAT1 inhibition, thus, contributes to decreasing serum urate concentration by increasing net renalurate excretion. Here, we investigated the URAT1-inhibitory effects of 25 FAs that are commonly
contained in foods or produced in the body. For this purpose, we conducted an in vitro transport
assay using cells transiently expressing URAT1. Our results showed that unsaturated FAs, especiallylong-chain unsaturated FAs, inhibited URAT1 more strongly than saturated FAs. Among thetested unsaturated FAs, eicosapentaenoic acid, α-linolenic acid, and docosahexaenoic acid exhibited
substantial URAT1-inhibitory activities, with half maximal inhibitory concentration values of 6.0,
14.2, and 15.2 µM, respectively. Although further studies are required to investigate whether the
ω-3 polyunsaturated FAs can be employed as uricosuric agents, our findings further confirm FAs as
nutritionally important substances influencing human health.
Keywords: Docosahexaenoic acid; eicosapentaenoic acid; functional food; gout; human health;hyperuricemia; PUFA; SLC22A12; transporter; uric acid; uricosuric activity

1. Introduction
Fatty acids (FAs) are physiologically important as energy sources and membrane constituents;
moreover, FAs have diverse biological activities that modulate numerous cell/tissue properties in livingorganisms [1]. Accumulating evidence suggests that via such actions, dietary FAs can influence humanhealth, well-being, and the risk of disease development. For instance, intake of polyunsaturated
fatty acids (PUFAs) of the ω-3 family, such as eicosapentaenoic acid (EPA) and docosahexaenoic
acid (DHA), has been considered to reduce the risk of chronic diseases including cardiovasculardiseases; however, other effects of FAs consumption on human health remain controversial and needfurther investigation [2–5]. In addition to cardiovascular diseases, it is currently acknowledged thatFAs influence a range of other diseases, such as metabolic and inflammatory diseases, as well as
Nutrients 2020, 12, 1601; doi:10.3390/nu12061601

www.mdpi.com/journal/nutrients

Nutrients 2020, 12, 1601

2 of 12cancer [1]. However, little is known about the association between FAs and hyperuricemia, a common
urate-related disease, especially regarding the effects of FAs on urate-handling machineries in the body.
Hyperuricemia, which is characterized by elevated serum uric acid (SUA) levels, is a lifestyle-relateddisease with high prevalence [6]. As hyperuricemia is a risk factor for gout, a very common form of
inflammatory arthritis, SUA management at appropriate levels is becoming increasingly important indaily life [7,8]. Due to the lack of functional uricase (urate-degrading enzyme) in humans [9], uric acid isthe end-product of human purine metabolism; urate excretion from the body is, therefore, necessary forthe maintenance of uric acid homeostasis. The kidney is responsible for elimination of approximately
two-thirds of urate [10]. However, only 3%–10% of the urate filtered by the glomerulus of the kidney issecreted to the urine [11] because most of it is re-absorbed from the primary urine into the blood in theproximal tubule by the urate transporter 1 (URAT1, also known as SLC22A12)-mediated pathway [12].
Thus, inhibition of this urate re-absorption pathway contributes to SUA lowering via the increase of
net renal urate excretion.
URAT1 is a physiologically important renal urate re-absorber expressed on the brush border
membrane of proximal tubular cells. Among the already identified urate re-absorbers expressed on
the apical side of the renal cells, URAT1 is most strongly associated with SUA levels in humans [7],as supported by the fact that URAT1 is the causative gene for renal hypouricemia type 1 [12], an inherited
disorder characterized by impaired urate re-absorption in the kidney that results in extremely low SUA
levels (SUA ≤ 2 mg/dL; normal range: 3.0–7.0 mg/dL). With hyperuricemia patients, this transporter isalso the pharmacological target of uricosuric agents, which promote the excretion of urate, such as
benzbromarone [12], lesinurad [13], and dotinurad [14]. In this context, daily consumption of nutrientswith URAT1-inhibitory activity may have a beneficial effect on SUA management in subjects with high
SUA levels. Actually, food ingredients that inhibit URAT1 function have attracted great interest; weand other groups identified some such natural ingredients from fruit flavonoids [15], coumarins [16],and wood pigments [17]. Nevertheless, despite the nutritional significance of FAs, their effects on
URAT1 activity remain to be elucidated.
In the present study, we examined the URAT1-inhibitory effects of 25 FAs using an in vitrotransport assay with mammalian cells transiently expressing URAT1. The cell-based assay revealed
that unsaturated FAs inhibited URAT1 more strongly than saturated FAs.
2. Materials and Methods
2.1. Materials
Critical materials and resources used in this study are summarized in Table 1. All other chemicals
used were commercially available and of analytical grade. The FAs were re-dissolved with dimethyl
sulfoxide (DMSO; Nacalai Tesque, Kyoto, Japan) after the solvents were gently evaporated withnitrogen gas on the heat block at 50 ◦ C. All experiments were conducted with the same lot of each
vector plasmid for URAT1 (URAT1 wild-type in pEGFP-C1) or mock (pEGFP-C1), which were derived
from our previous study [15].
2.2. Cell Culture
Human embryonic kidney 293-derived 293A cells were maintained in Dulbecco’s modified Eagle’s
medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (Biowest, Nuaillé,France), 1% penicillin–streptomycin (Nacalai Tesque, Kyoto, Japan), 2 mM L-glutamine (Nacalai
Tesque, Kyoto, Japan), and 1 × non-essential amino acid (Life Technologies, Carlsbad, CA, USA) at
37 ◦ C in a humidified atmosphere of 5% (v/v) CO2 in air.
URAT1-expressing or mock plasmids were transfected into 293A cells using polyethylenimine“MAX” (PEI-MAX) (Polysciences, Warrington, PA, USA) as described previously [18], with somemodifications. In brief, before transfection, 293A cells were seeded onto 12-well cell culture plates at aconcentration of 0.92 × 105 cells/cm2 . Then, 24 h after seeding, each plasmid vector was transientlyNutrients 2020, 12, 1601

3 of 12transfected into the cells using PEI-MAX (1 µg of plasmid/5 µL of PEI-MAX/well). The medium was
replaced with fresh medium 24 h after transfection.
Table 1. Key resources.
Reagent or Resource
Antibodies
Rabbit polyclonal anti-EGFP
Rabbit polyclonal anti-α-tubulin
Donkey anti-rabbit IgG-horseradish
peroxidase (HRP)-conjugate
Chemicals[8-14 C]-Uric acid (53 mCi/mmol)

SourceIdentifierLife Technologies

Cat# A11122; RRID: AB_221569; 1:1,000 dilution 1

Abcam

Cat# ab15246; RRID: AB_301787; 1:1,000 dilution 1

GE Healthcare

Cat# NA934V; RRID: AB_772206; 1:3,000 dilution 1

American Radiolabeled Chemicals

Cat# ARC0513

Arachidonic acid

Cayman Chemical

Cat# 90010; CAS: 506-32-1; Purity: ≥98%

Benzbromarone

FUJIFILM Wako Pure Chemical

Cat# 028-1585; CAS: 3562-84-3; Purity: >98%

Butyric acid

SIGMA-ALDRICH

Cat# B103500-5ML; CAS: 107-92-6; Purity: ≥99%
Cat# 033-01073; CAS: 334-48-5; Purity: ≥98%

Decanoic acid

FUJIFILM Wako Pure Chemical

Dimethyl sulfoxide

Nacalai Tesque

Cat# 13445-74; CAS: 67-68-5

Docosadienoic acid

Cayman Chemical

Cat# 20749; CAS: 17735-98-7; Purity: ≥98%

Docosahexaenoic acid

Cayman Chemical

Cat# 90310; CAS: 6217-54-5; Purity: ≥98%

Docosatetraenoic acid

Cayman Chemical

Cat# 90300; CAS: 28874-58-0; Purity: ≥98%
Cat# L556-25G; CAS: 143-07-7; Purity: ≥98%

Dodecanoic acid

SIGMA-ALDRICH

Eicosadienoic acid

Cayman Chemical

Cat# 90330; CAS: 2091-39-6; Purity: ≥98%

Eicosapentaenoic acid

Cayman Chemical

Cat# 90110; CAS: 10417-94-4; Purity: ≥98%
Cat# 90192; CAS: 20590-32-3; Purity: ≥98%

Eicosatrienoic acid

Cayman Chemical

Henicosapentaenoic acid

Cayman Chemical

Cat# 10670; CAS: 24257-10-1; Purity: ≥95%

Hexanoic acid

FUJIFILM Wako Pure Chemical

Cat# 081-06292; CAS: 142-62-1; Purity: ≥99%

Linoleic acid

Cayman Chemical

Cat# 90150; CAS: 60-33-3; Purity: ≥98%

Myristic acid

FUJIFILM Wako Pure Chemical

Cat# 130-03432; CAS: 544-63-8; Purity: ≥98%

Octanoic acid

SIGMA-ALDRICH

Cat# C2875-10ML; CAS: 124-07-2; Purity: ≥99%

Oleic acid

Cayman Chemical

Cat# 90260; CAS: 112-80-1; Purity: ≥98%

Palmitic acid

Cayman Chemical

Cat# 10006627; CAS: 57-10-3; Purity: ≥98%
Cat# 10009871; CAS: 373-49-9; Purity: ≥99%

Palmitoleic acid

Cayman Chemical

Polyethelenimine “MAX”

Polysciences

Cat# 24765; CAS: 49553-93-7

Stearic acid

SIGMA-ALDRICH

Cat# S4751-1G; CAS: 57-11-4; Purity: ≥98.5%

α-Eleostearic acid

Cayman Chemical

Cat# 10008349; CAS: 506-23-0; Purity: ≥95%

α-Linolenic acid

Cayman Chemical

Cat# 90210; CAS: 463-40-1; Purity: ≥98%

γ-Linolenic acid

Cayman Chemical

Cat# 90220; CAS: 506-26-3; Purity: ≥98%

ω-3 Eicosatetraenoic acid

Larodan Fine Chemicals

Cat# 10-2024; CAS: 24880-40-8; Purity: ≥98%

ω-3 Docosapentaenoic acid
Critical Commercial Assays
PierceTM BCA Protein Assay Reagent A & B

Cayman Chemical

Cat# 90165; CAS: 24880-45-3; Purity: ≥98%

Thermo Fisher Scientific

Cat# 23223, Cat# 23224

Thermo Fisher Scientific

Cat# K210015

Miyata et al., 2016 [18]

NCBI Reference Sequence: NM_144585.3

Invitrogen

R70507Microsoft

https://products.office.com/ja-jp/home

OMS Publishing

http://www.oms-publ.co.jp/

PureLinkTM HiPure Plasmid Filter
Midiprep Kit
Recombinant DNA
The complete URAT1 cDNA
Experimental Models: Cell Lines
293A
Software and Algorithms
Excel 2019
Statcel4 add-in software

1 All antibodies were used at indicated dilutions in Tris-buffered saline containing 0.05% Tween 20 and 1% bovine

serum albumin for 1 h at room temperature.

2.3. Preparation of Protein Lysates and Immunoblotting
Whole-cell lysates were prepared with cell lysis buffer A containing 50 mM Tris/HCl (pH 7.4),
1 mM dithiothreitol, 1% (w/v) Triton X-100, and protease inhibitor cOmplete, EDTA free (Roche, Basel,
Switzerland), and were treated with peptide N-glycosidase F (PNGase F) (New England Biolabs,
Ipswich, MA, USA) as described previously [19]. Protein concentration was determined using theNutrients 2020, 12, 1601

4 of 12

PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific, Kanagawa, Japan) with BSA as a standard,
according to the manufacturer’s protocol.
Whole-cell lysate samples were separated by SDS-PAGE and transferred to an Immobilon-P
PVDF membrane (Millipore, Bedford, MA, USA) by electroblotting at 15 V for 60 min, as described
previously [15]. Blots were probed with appropriate antibodies (Table 1), and the signals were visualized
by chemiluminescence and detected using a multi-imaging Analyzer Fusion Solo 4TM system (Vilber
Lourmat, Eberhardzell, Germany).
2.4. Confocal Microscopy
For confocal laser scanning microscopic observation, 48 h after the transfection, 293A cells were
fixed with 4% paraformaldehyde for 15 min at room temperature, and further processed according
to previous studies [15,20]. In brief, the cells were treated with a fluorescent wheat germ agglutinin
conjugate (WGA, Alexa Fluor® 594 conjugate; Thermo Fisher Scientific) to visualize plasma membranes,
followed by nuclear staining using TO-PRO-3 Iodide (Molecular Probes, Eugene, OR, USA). Then,the cells were mounted in VECTASHIELD Mounting Medium (Vector Laboratories, Burlingame, CA,
USA). To analyze the localization of EGFP-fused URAT1 protein, fluorescence was observed using the
FV10i Confocal Laser Scanning Microscope (Olympus, Tokyo, Japan).
2.5. Urate Uptake Assay Using URAT1-Expressing 293A Cells
The urate uptake assay using URAT1-expressing 293A cells was conducted according to ourprevious studies [15,18] with minor modifications. In brief, 48 h after plasmid transfection, cells were
washed twice with Cl− -free transport buffer (Buffer T2: 125 mM Na-gluconate, 4.8 mM K-gluconate,
1.2 mM KH2 PO4 , 1.2 mM MgSO4 , 1.3 mM Ca-gluconate, 25 mM HEPES, 5.6 mM D-glucose, and pH 7.4)
and pre-incubated in Buffer T2 for 15 min at 37 ◦ C. The buffer was then exchanged with pre-warmed fresh
Buffer T2 containing 5 µM [8-14 C]-urate with or without test compound at the indicated concentrations(0, 0.1, 0.3, 1, 3, 10, 30, 100, or 300 µM), and the cells were further incubated for 20 sec; 1% DMSO
was used as a vehicle control. The cells were subsequently washed five times with ice-cold Buffer
T2 and then lysed with 500 µL of 0.2 M NaOH on ice with gentle shaking for 1 h. The lysates were
neutralized with 100 µL of 1 M HCl. Then, the radioactivity in the lysate was measured using aliquid scintillator (Tri-Carb 3110TR; PerkinElmer, Waltham, MA, USA). The protein concentration wasdetermined using the PierceTM BCA Protein Assay Kit. The urate transport activity was calculated
as the incorporated clearance (µL/mg protein/min): (incorporated level of urate [disintegrations per
minute (DPM)/mg protein/min]/urate level in the incubation mixture [DPM/µL]). URAT1-dependenturate transport activity was calculated by subtracting the urate transport activity of mock cells fromthat of URAT1-expressing cells.
Urate uptake was measured in the presence of several concentrations of each test compound toaddress their half maximal inhibitory concentration (IC50 ) values. URAT1-mediated transport activities
were then expressed as a percentage of control (100%). Based on the calculated values, fitting curves
were obtained according to the following formula using the least-squares method with Excel 2019
(Microsoft, Redmond, WA, USA):
Predicted value [%] = 100 −Emax × Cn
ECn50 + Cn!

(1)

where Emax is the maximum effect, EC50 is the half maximal effective concentration, C is the concentrationof the test compound, and n is the sigmoid-fit factor. Finally, based on the results, the IC50 was calculated
as described previously [15].
2.6. Quantification and Statistical Analysis
All statistical analyses were performed using Excel 2019 with Statcel4 add-in software(OMS publishing, Saitama, Japan). Different statistical tests were used for different experiments

Nutrients 2020, 12, 1601

5 of 12as described in the figure legends, which include the number of biological replicates (n). Briefly, whenanalyzing multiple groups, the similarity of variance between groups was compared using Bartlett’s test.
When passing the test for homogeneity of variance, a parametric Tukey–Kramer multiple-comparison
test for all pairwise comparisons was used. To investigate the inhibitory effect of each FA on URAT1
function (vs. vehicle control indicated as 100%) in the screening stage, one-sample t-test (one-sided)
was conducted. Statistical significance was defined as p < 0.05 or 0.01.
Each experiment was designed to use samples required to obtain informative results and sufficientmaterial for subsequent studies. No specific statistical test was used to pre-determine the sample sizes
empirically determined in the current study. All experiments were monitored in a non-blinded fashion.
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 plasmidtransfection 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 uratetransport 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 waslabeled 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.

Nutrients 2020, 12, 1601

6 of 123.2. 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 thansaturated FAs. Among the eight saturated FAs, relatively short FAs with chain lengths ranging fromC4 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 ofeach 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 Effective 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 structuralfeature (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.

Nutrients 2020, 12, 1601

7 of 12Figure 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.

4. Discussion
In this study, to the best of our knowledge, we revealed for the first time the inhibitory effectsof FAs on URAT1-dependent urate transport in vitro. While further studies are highly warranted toaddress the pathophysiological impact of our findings in terms of the possible effect on SUA and renalurate 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 thisinflammatory condition showed an association between high ω-3 FA levels in the blood and lower
frequency of gout attacks [23]. Additionally, ω-3 PUFA-rich fish consumption was reportedly associatedNutrients 2020, 12, 1601

8 of 12with lower risk of recurrent gout 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 inflammationinduced by monosodium urate crystals [25,26]. Thus, in addition to the anti-inflammatory effects
previously observed, it should be confirmed in future studies whether ω-3 PUFAs can exhibit uricosuric
effects in hyperuricemia models or not.
The potential effects of the daily consumption of ω-3 PUFA-enriched foods on SUA will also be ofinterest. 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 similarsignificant 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 notextensive [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 healthin 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 theFAs 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 atleast two substrate recognition sites in its protein structure. Considering that FAs are carboxylic
acids with a long aliphatic chain, they might affect the recognition and/or subsequent membranetransport of the counterpart substrates by URAT1 rather than the recognition of urate. Second,
we could not exclude the possibility that the FAs affected the plasma membrane properties, whichmight result in the indirect decrease of URAT1 function. Nonetheless, given that the interactionof 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 effect on the plasma
membranes during the assay. Finally, the effects of FAs on other physiologically important uratetransporters—GLUT9/SLC2A9 [29,30], OAT10/SLC22A13 [31], and ABCG2/BCRP [32–34]—remain tobe elucidated. Since such urate transporters, including URAT1, coordinately regulate the behavior ofurate in the human body, comprehensive understanding of the latent interaction between FAs andthese 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 acounterpart of URAT1 [7]; GLUT9 is the causative gene for renal hypouricemia type 2 [30]. Given thesepieces of information, GLUT9 has the highest priority in the future investigation.
5. Conclusions
In conclusion, we herein found that FAs, especially ω-3 PUFAs such as EPA and DHA, could inhibit
URAT1. To gain insight into the potential SUA-lowering effects of certain FAs, further investigations
including human studies are required.
Author Contributions: Conceptualization, H.S. (Hiroki Saito), Y.T. (Yu Toyoda), and T.T.; methodology, H.S.
(Hiroki Saito), Y.T. (Yu Toyoda) and H.H.; validation, H.S. (Hiroki Saito), Y.T. (Yu Toyoda) and T.T.; formal
analysis, H.S. (Hiroki Saito) and Y.T. (Yu Toyoda); investigation, H.S. (Hiroki Saito), Y.T. (Yu Toyoda), H.H., A.O.-K.
and H.M.; data curation, H.S. (Hiroki Saito), Y.T. (Yu Toyoda), and T.T.; writing—original draft preparation,
H.S. (Hiroki Saito) and Y.T. (Yu Toyoda); writing—review and editing, Y.T. (Yu Toyoda) and T.T.; visualization,
H.S. (Hiroki Saito) and Y.T. (Yu Toyoda); supervision, Y.T. (Youichi Tsuchiya) and H.S. (Hiroshi Suzuki); project
administration, T.T. and N.K.; funding acquisition, T.T. All authors have read and agreed to the published versionof the manuscript.

Nutrients 2020, 12, 1601

9 of 12Funding: This study was supported by JSPS KAKENHI (grant numbers 16H01808 and 20H00568 to T.T.); T.T.
has received research grants from Gout and uric acid foundation of Japan, MSD Life Science Foundation, Public
Interest Incorporated Foundation, and Takeda Science Foundation. The funders had no role in the study design,
data collection and analysis, decision to publish, or preparation of the manuscript.
Acknowledgments: The authors thank Syuichi Segawa for his continuous encouragement and support for thisstudy. Also, we would like to thank Editage (www.editage.jp) for English language editing.
Conflicts of Interest: H. Saito, H.H., A.O.-K., N.K., and Y.Tsuchiya were the employees of Sapporo Holdings Ltd.
H.Saito, Y.Toyoda, T.T., H.H., A.O-K., and H. Suzuki have a patent pending related to the work reported in thisarticle. The remaining authors declare no competing interests.

Appendix A

Figure A1. Chemical structures of saturated fatty acids tested in this study. (a) Butyric acid; (b) hexanoicacid; (c) octanoic acid; (d) decanoic acid; (e) dodecanoic acid; (f) myristic acid; (g) palmitic acid; (h)stearic acid.

Figure A2. Chemical structures of unsaturated fatty acids tested in this study. (a) Palmitoleic acid;(b) oleic acid; (c) linoleic acid; (d) α-linolenic acid (ALA); (e) γ-linolenic acid; (f) α-eleostearic acid;(g) eicosadienoic acid; (h) eicosatrienoic acid; (i) ω-3 eicosatetraenoic acid; (j) arachidonic acid (ω-6eicosatetraenoic acid); (k) eicosapentaenoic acid (EPA); (l) henicosapentaenoic acid; (m) docosadienoic
acid; (n) docosatetraenoic acid; (o) ω-3 docosapentaenoic acid; (p) ω-6 docosapentaenoic acid;(q) docosahexaenoic acid (DHA).

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10 of 12Figure A3. Concentration-dependent inhibition of URAT1-mediated urate transport by each fatty acid.
Data are expressed as the mean ± SD; n = 4. (a) Linoleic acid; (b) ω-3 docosapentaenoic acid.

Figure A4. Biosynthetic route of fatty acids and half maximal inhibitory concentration (IC50 ) valuesof each tested fatty acid against the urate transport mediated by URAT1. The IC50 values are from
Figures 2, 3 and A3. Three polyunsaturated fatty acids with substantial URAT1-inhibitory activities
are indicated in red. N.D., not determined in this study; FAs, fatty acids; des, desaturase; elo, elongase.
The metabolic pathway is adapted from a previous report [4], with some modifications.

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