Uric acid activates aldose reductase and the polyol pathway for endogenous fructose and fat production causing development of fatty liver in rats

Abstract

Dietary, fructose-containing sugars have been strongly associated with the development of nonalcoholic fatty liver disease (NAFLD). Recent studies suggest that fructose also can be produced via the polyol pathway in the liver, where it may induce hepatic fat accumulation. Moreover, fructose metabolism yields uric acid, which is highly associated with NAFLD. Here, using biochemical assays, reporter gene expression, and confocal fluorescence microscopy, we investigated whether uric acid regulates aldose reductase, a key enzyme in the polyol pathway. We evaluated whether soluble uric acid regulates aldose reductase expression both in cultured hepatocytes (HepG2 cells) and in the liver of hyperuricemic rats and whether this stimulation is associated with endogenous fructose production and fat accumulation. Uric acid dose-dependently stimulated aldose reductase expression in the HepG2 cells, and this stimulation was associated with endogenous fructose production and triglyceride accumulation. This stimulatory mechanism was mediated by uric acid-induced oxidative stress and stimulation of the transcription factor nuclear factor of activated T cells 5 (NFAT5). Uric acid also amplified the effects of elevated glucose levels to stimulate hepatocyte triglyceride accumulation. Hyperuricemic rats exhibited elevated hepatic aldose reductase expression, endogenous fructose accumulation, and fat buildup that was significantly reduced by co-administration of the xanthine oxidase inhibitor allopurinol. These results suggest that uric acid generated during fructose metabolism may act as a positive feedback mechanism that stimulates endogenous fructose production by stimulating aldose reductase in the polyol pathway. Our findings suggest an amplifying mechanism whereby soft drinks rich in glucose and fructose can induce NAFLD.

Keywords: aldose reductase; fatty acid; fructose; liver metabolism; metabolic syndrome; polyol pathway; sorbitol; uric acid.

Figure 1.

Uric acid stimulates aldose reductase and the polyol pathway in human hepatocytes. A, representative Western blotting and densitometry for AR, SDH, KHK, and actin in HepG2 cells exposed to increasing concentrations of uric acid for 72 h. A subset of cells exposed to 12 mg/dl uric acid was treated with the uric acid transporter URAT1 inhibitor probenecid (Prob). B, densitometry for AR expression in 12 mg/dl exposed cells in the absence (red) or presence (orange) of probenecid. C, correlation analysis between intracellular uric acid levels and AR expression in HepG2 cells. D, representative Western blotting and densitometry for AR and actin in HepG2 cells exposed to 12 mg/dl uric acid for different time points. E, intracellular sorbitol levels in the same groups as in D. F, intracellular fructose levels in the same groups as in D. G, representative Western blotting for AR in control (black) and silenced (white) HepG2 cells. Intracellular sorbitol and fructose levels in the same cells exposed to 12 mg/dl uric acid for 72 h. The data represent the results of three independent experiments. *, p < 0.05; **, p < 0.01 versus control; one-way ANOVA, Tukey post hoc analysis.

Figure 2.

Aldose reductase expression in hepatocytes is mediated by the transcription factor NFAT5. A, AR mRNA expression in 12 mg/dl uric acid–exposed HepG2 cells for different time points. B, representative Western blotting and densitometry for AR and actin in HepG2 cells exposed to 12 mg/dl uric acid for different time points in the presence or absence of the transcription inhibitor actinomycin D (ActD). C, representative Western blotting and densitometry for NFAT5, CREB (nuclear marker), and tubulin (cytosol marker) in HepG2 cells exposed to 12 mg/dl uric acid for different time points. D, representative confocal images for NFAT5 (green) and the nucleus marker, 4′,6′-diamino-2-phenylindole (DAPI, blue) in HepG2 cells control and exposed to 12 mg/dl uric acid for 6 h. E, quantification of NFAT5 nucleus/cytosol ratio signal in control and exposed to 12 mg/dl uric acid for 6 h. F, representative Western blotting and densitometry for AR, NFAT5, and actin in control (scramble, scr) and NFAT5-silenced (sNF or sNFAT5) HepG2 cells exposed to increasing concentrations of uric acid for 72 h. The data represent the results of three independent experiments. *, p < 0.05; **, p < 0.01 versus control; one-way ANOVA, Tukey post hoc analysis.

Figure 3.

Uric acid-dependent up-regulation of aldose reductase by NFAT5 is mediated by oxidative stress. A, intracellular TBARS (lipid peroxides) in HepG2 cells exposed to increasing levels of uric acid. B, intracellular TBARS in HepG2 cells control (scr) or deficient for AR expression (sAR) exposed to 12 mg/dl of uric acid. C, luciferase signal in HepG2 cells transfected with a luciferase reporter cassette containing a full AR promoter or a truncated version without the NFAT5 TonE site and exposed to 0 (control) or 12 mg/dl of uric acid for 3 h. D, luciferase signal in same experiment as in C in the presence or absence of the NADPH oxidase inhibitor apocynin. The data represent the results of three independent experiments. **, p < 0.01 versus control; one-way ANOVA, Tukey post hoc analysis.

Figure 4.

Up-regulation of AR activates the polyol pathway and induces fat accumulation in HepG2 cells. A, representative Western blotting and densitometry for AR and actin in HepG2 cells exposed to increased glucose or mannitol (equiosmolar control) concentrations for 72 h. B, medium osmolality of cells exposed to 5, 12.5, or 25 mm glucose or mannitol. C, intracellular sorbitol levels in HepG2 cells exposed to normal (5 mm) or high (25 mm) glucose levels in control (scr) and AR-deficient (sAR) cells. D, intracellular fructose levels in the same groups as in B. E, intracellular triglyceride levels in the same groups as in B. F, intracellular TBARS (lipid peroxides) in HepG2 cells exposed to normal (5 mm, gray) or high (25 mm, black) glucose in the presence of low (4 mg/dl) or high (12 mg/dl) uric acid. G, intracellular sorbitol levels in the same groups as in F. H, intracellular fructose levels in the same groups as in F. I, intracellular triglyceride levels in the same groups as in F. **, p < 0.01 versus control within the same uric acid; ##, p < 0.01 versus control within the same glucose; one-way ANOVA, Tukey post hoc analysis.

Figure 5.

Activation of the polyol pathway by uric acid induces hepatic fat accumulation in rats. A, plasma uric acid levels in control rats (white) and rats exposed to the uricase inhibitor, oxonic acid, in the absence (black) and presence of the xanthine oxidase inhibitor, allopurinol (gray). B, intrahepatic levels of uric acid in the same groups as in A. C, intrahepatic liver oxidized protein levels of in the same groups as in A. D, intrahepatic triglycerides in the same groups as in A. E, correlation between intracellular uric acid and triglyceride levels in all rats. F, representative Western blotting and densitometry for AR, fructokinase/KHK, and actin in the same groups as in A. G, intrahepatic sorbitol levels in the same groups as in A. H, intrahepatic fructose levels in the same groups as in A. I, representative Western blotting and densitometry for NFAT5 and the nuclear marker proliferating cell nuclear antigen (PCNA) in the same groups as in A; n = 4–7 rats/group. **, p < 0.01 versus the rest of groups; ##, p < 0.01 versus control one-way ANOVA, Tukey post hoc analysis.

Figure 6.

Schematic representing the effects of uric acid in the polyol pathway, oxidative stress, and fat accumulation. A, regular metabolism of glucose and fructose in normouricemic conditions. B, glucose is favored toward endogenous fructose production and metabolism, thus reducing glycolytic flux and promoting fat accumulation and oxidative stress.