URAT1-selective inhibition ameliorates insulin resistance by attenuating diet-induced hepatic steatosis and brown adipose tissue whitening in mice

Abstract

Objective: Accumulating evidence indicates that high uric acid (UA) is strongly associated with obesity and metabolic syndrome and drives the development of nonalcoholic fatty liver disease (NAFLD) and insulin resistance. Although urate transporter-1 (URAT1), which is primarily expressed in the kidneys, plays a critical role in the development of hyperuricemia, its pathophysiological implication in NAFLD and insulin resistance remains unclear. We herein investigated the role and functional significance of URAT1 in diet-induced obese mice.

Methods: Mice fed a high-fat diet (HFD) for 16-18 weeks or a normal-fat diet (NFD) were treated with or without a novel oral URAT1-selective inhibitor (dotinurad [50 mg/kg/day]) for another 4 weeks.

Results: We found that URAT1 was also expressed in the liver and brown adipose tissue (BAT) other than the kidneys. Dotinurad administration significantly ameliorated HFD-induced obesity and insulin resistance. HFD markedly induced NAFLD, which was characterized by severe hepatic steatosis as well as the elevation of serum ALT activity and tissue inflammatory cytokine genes (chemokine ligand 2 (Ccl2) and tissue necrosis factor α (TNFα)), all of which were attenuated by dotinurad. Similarly, HFD significantly increased URAT1 expression in BAT, resulting in lipid accumulation (whitening of BAT), and increased the production of tissue reactive oxygen species (ROS), which were reduced by dotinurad via UCP1 activation.

Conclusions: In conclusion, a novel URAT1-selective inhibitor, dotinurad, ameliorates insulin resistance by attenuating hepatic steatosis and promoting rebrowning of lipid-rich BAT in HFD-induced obese mice. URAT1 serves as a key regulator of the pathophysiology of metabolic syndrome and may be a new therapeutic target for insulin-resistant individuals, particularly those with concomitant NAFLD.

Keywords: Insulin resistance; Metabolic syndrome; NAFLD; URAT1; Uric acid.

Graphical abstract

Figure 1

Effects of URAT1-selective inhibitor treatment on body weight and plasma uric acid. (A) A schematic diagram of the experimental protocol. (B) Body weight changes during the protocol (NFD, n = 10; NFD + Doti, n = 12; HFD, n = 13; HFD + Doti, n = 13). (C) The degree of body weight gain from baseline (NFD, n = 15; NFD + Doti, n = 15; HFD, n = 16; HFD + Doti, n = 16). (D) The dietary intake per day (NFD, n = 7; NFD + Doti, n = 9; HFD, n = 8; HFD + Doti, n = 9). Data are expressed as the mean ± SEM. (B) ∗∗∗P < 0.001, ∗∗P < 0.01, and ∗P < 0.05 versus NFD at each time point; ^†P < 0.05 versus HFD at each time point. (C) ∗∗P < 0.01, NFD versus HFD; ^††P < 0.01, HFD versus HFD + Doti. NFD, normal-fat diet; HFD, high-fat diet; Doti, dotinurad; IPGTT, intraperitoneal glucose tolerance test; ITT, insulin tolerance test.

Figure 2

Effects of URAT1-selective inhibitor treatment on HFD-induced insulin resistance. (A) Plasma glucose levels during IPGTT and the area under the curve (AUC) (NFD, n = 7; NFD + Doti, n = 7; HFD, n = 7; HFD + Doti, n = 9). (B) Plasma glucose levels during ITT and the AUC (NFD, n = 6; NFD + Doti, n = 8; HFD, n = 9; HFD + Doti, n = 8). Data are expressed as the mean ± SEM. (Line graphs) ∗∗∗P < 0.001, ∗∗P < 0.01, and ∗P < 0.05 versus NFD at each time point; ^††P < 0.01 and ^†P < 0.05 versus HFD at each time point. (AUC) ∗∗∗P < 0.001 and ∗P < 0.05, NFD versus HFD; ^††P < 0.01 and ^†P < 0.05, HFD versus HFD + Doti.

Figure 3

Relative mRNA profiling and the protein expression of URAT1 in NFD- and HFD-fed mice. (A) URAT1 mRNA expression in the liver, eWAT, iWAT, BAT, and kidney as assessed by qRT-PCR (n = 3 each). (B) URAT1 mRNA expression in either NFD- or HFD-fed mice with or without dotinurad treatment. ([Liver] n = 6 each; [eWAT] NFD, n = 6; NFD + Doti, n = 6; HFD, n = 6; HFD + Doti, n = 5; [iWAT] NFD, n = 5; NFD + Doti, n = 6; HFD, n = 5; HFD + Doti, n = 5; [BAT] NFD, n = 5; NFD + Doti, n = 4; HFD, n = 6; HFD + Doti, n = 4). The protein expression of URAT1 in liver (C), eWAT (D), and BAT (E) (n = 3 each). Data are expressed as the mean ± SEM. ∗∗P < 0.01 and ∗P < 0.05 between the indicated groups. eWAT, epididymal white adipose tissue; iWAT, inguinal white adipose tissue; BAT, brown adipose tissue.

Figure 4

Effects of URAT1-selective inhibitor treatment on HFD-induced NAFLD. (A) A representative image of liver harvested from the indicated mice. (B) Liver weight at 4 weeks after treatment with or without dotinurad (NFD, n = 7; NFD + Doti, n = 8; HFD, n = 9; HFD + Doti, n = 8). (C) Hematoxylin and eosin staining of liver sections of mice that received the indicated treatments. (D) Steatosis score (NFD, n = 4; NFD + Doti, n = 4; HFD, n = 5; HFD + Doti, n = 5). (E) NAFLD score (NFD, n = 4; NFD + Doti, n = 4; HFD, n = 5; HFD + Doti, n = 5). (E) Serum ALT activity (NFD, n = 6; NFD + Doti, n = 7; HFD, n = 8; HFD + Doti, n = 6). (F) Relative mRNA expression of Ccl2 and TNFα (n = 5 each). Data are expressed as the mean ± SEM. ∗∗∗P < 0.001, ∗∗P < 0.01, and ∗P < 0.05 between the indicated groups. NAFLD, nonalcoholic fatty liver disease; TL, tail length.

Figure 5

Effects of URAT1-selective inhibitor treatment on HFD-induced lipid-rich BAT. (A) A representative image of BAT harvested from the indicated mice. (B) BAT weight (NFD, n = 12; NFD + Doti, n = 11; HFD, n = 12; HFD + Doti, n = 11). (C) Hematoxylin and eosin staining of the BAT section of mice with the indicated treatments. (D) Lipid droplet area and brown adipocyte diameter were measured in the indicated mice (n = 3 each) ([Lipid droplet area] NFD, n = 607; NFD + Doti, n = 609; HFD, n = 624; HFD + Doti, n = 610; [Brown adipocyte diameter] NFD, n = 297; NFD + Doti, n = 305; HFD, n = 335; HFD + Doti, n = 353). (E) UCP1 immunostaining in BAT. (F) Relative mRNA expressions of UCP1, PGC1α, and Dio2 ([UCP1] NFD, n = 6; NFD + Doti, n = 6; HFD, n = 6; HFD + Doti, n = 4; [PGC1α and Dio2] NFD, n = 5; NFD + Doti, n = 6; HFD, n = 6; HFD + Doti, n = 4). (G) The protein expression of UCP1 (n = 3 each). (H) Total ROS was measured by determining the fluorescence of DCF (DCF RFU: Dichlorofluorescein Relative Fluorescence Units) (NFD, n = 8; NFD + Doti, n = 9; HFD, n = 10; HFD + Doti, n = 8). (I) The URAT1 expression in primary brown adipocytes exposed to the indicated treatments for 24 h. (J) The intracellular UA levels in primary brown adipocytes exposed to the indicated treatments for 30 min and 60 min (n = 4 each). Data are expressed as the mean ± SEM. ∗∗∗P < 0.001, ∗∗P < 0.01, and ∗P < 0.05 between the indicated groups. UA, uric acid; ROS, reactive oxygen species.