Uric acid stimulates fructokinase and accelerates fructose metabolism in the development of fatty liver - PubMed (original) (raw)

doi: 10.1371/journal.pone.0047948. Epub 2012 Oct 24.

Laura G Sanchez-Lozada, Christina Cicerchi, Nanxing Li, Carlos A Roncal-Jimenez, Takuji Ishimoto, Myphuong Le, Gabriela E Garcia, Jeffrey B Thomas, Christopher J Rivard, Ana Andres-Hernando, Brandi Hunter, George Schreiner, Bernardo Rodriguez-Iturbe, Yuri Y Sautin, Richard J Johnson

Affiliations

Uric acid stimulates fructokinase and accelerates fructose metabolism in the development of fatty liver

Miguel A Lanaspa et al. PLoS One. 2012.

Abstract

Excessive dietary fructose intake may have an important role in the current epidemics of fatty liver, obesity and diabetes as its intake parallels the development of these syndromes and because it can induce features of metabolic syndrome. The effects of fructose to induce fatty liver, hypertriglyceridemia and insulin resistance, however, vary dramatically among individuals. The first step in fructose metabolism is mediated by fructokinase (KHK), which phosphorylates fructose to fructose-1-phosphate; intracellular uric acid is also generated as a consequence of the transient ATP depletion that occurs during this reaction. Here we show in human hepatocytes that uric acid up-regulates KHK expression thus leading to the amplification of the lipogenic effects of fructose. Inhibition of uric acid production markedly blocked fructose-induced triglyceride accumulation in hepatocytes in vitro and in vivo. The mechanism whereby uric acid stimulates KHK expression involves the activation of the transcription factor ChREBP, which, in turn, results in the transcriptional activation of KHK by binding to a specific sequence within its promoter. Since subjects sensitive to fructose often develop phenotypes associated with hyperuricemia, uric acid may be an underlying factor in sensitizing hepatocytes to fructose metabolism during the development of fatty liver.

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Conflict of interest statement

Competing Interests: The authors have read the journal’s policy and have the following conflicts: MAL, TI, and RJJ are listed as inventors on a patent application from the University of Colorado related to developing isoform-specific fructokinase inhibitors in the treatment of disorders associated with obesity and insulin resistance. Patent international number PCT/US11/46938 filed on August 8, 2011. TI and RJJ are listed as inventors on several patent applications related to lowering uric acid as a means to prevent or treat metabolic syndrome, as follows: US Patent No. 6,352,975 B1, Issued March 5, 2002 (Application No. 09/392,932, filed 09/09/1999) “Methods of Treating Hypertension and Compositions for Use Therein.” US Patent No 6,677,300. Issued Jan 13, 2004. (Application No. 09/392, 931, filed 09/09/99) “Treatment of Microvascular Angiopathies.” US Patent No. 7,030,083 B2 Issued April 18, 2006 (Application No. 10/418,529, Filed 4/16/2003) Issued Nov 10, 2005 “Treatment of eclampsia and preeclampsia.” US Patent No 7,799,794 B2, Issued Sep 21, 2010, (Application 09/892,505; Filed Jun 28, 2001 Treatment for Cardiovascular Disease. RJJ also has a patent with the University of Washington and Merck for the use of allopurinol to treat hypertension. RJJ also discloses that he has consulted for Ardea, Astellas, Danone and Novartis, that he is on the scientific board of Amway, and that he has received grants from the National Institutes of Health and from Amway, Cardero, Danone, Questcor and the Sugar Foundation. GS is employed by a commercial company (Cardero Therapeutics). There are no further patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Figures

Figure 1

Figure 1. Allopurinol prevents the development of hepatic steatosis in adult male rats drinking fructose.

A) Quantity of UA in liver extracts from control, fructose-fed and fructose with allopurinol-fed rats. B) Liver weight normalized to body weight is expressed as percent body weight in control, fructose-fed and fructose with allopurinol-fed rats. Oil red O–stained section of fructose-fed liver. (C–H) Images of rat liver taken using an Aperio Scanscope at × 20 magnification. C–E) H&E-stained section of control (C) fructose (D) and allopurinol fed liver (E). F–H) Oil red O–stained section of control (F), fructose (G) and allopurinol (H) fed liver. I) Quantity of TG in liver extracts from control, fructose-fed and fructose with allopurinol-fed rats. J) Quantity of cholesterol (total and sterol esters) in liver extracts from control, fructose-fed and fructose with allopurinol-fed rats. (n = 5 for each group).

Figure 2

Figure 2. Allopurinol prevents KHK up-regulation in adult male rats drinking fructose (n = 5 for each group).

(A–B) KHK and AldoB protein expression in liver extracts from control, fructose-fed and fructose with allopurinol-fed rats. C) mRNA expression of khk normalized to β-actin levels in liver extracts from control, fructose-fed and fructose with allopurinol-fed rats. D) Expression of lipogenic proteins FAS, ACC and ACL in liver extracts from control, fructose-fed and fructose with allopurinol-fed rats. E) Expression of the fat oxidation-related protein ECH1 in liver extracts from control, fructose-fed and fructose with allopurinol-fed rats. F) Beta-hydroxybutyrate levels in liver and serum normalized to triglyceride levels. G) Serum fructose levels in control, fructose-fed and fructose with allopurinol-fed rats. H) Beta-hydroxybutyrate levels in control, fructose-fed and fructose with allopurinol-fed rats.

Figure 3

Figure 3. Uric acid up-regulates KHK in human hepatocytes.

(A–B) KHK protein expression in cells exposed to increasing levels of uric acid for 72 hours in the presence or absence of the URAT1 inhibitor probenecid (2 mmol/L). (C–D) KHK protein expression in cells exposed to 750 µmol/L uric acid for different time points in the presence or absence of the URAT1 inhibitor probenecid. (E–F) KHK protein expression in cells exposed to 5 mmol/L fructose for 72 hours in the presence or absence of the XO inhibitor allopurinol (100 µmol/L). G) KHK activity in cells exposed to 5 mmol/L fructose for 72 hours in the presence or absence of the XO inhibitor allopurinol (100 µmol/L).

Figure 4

Figure 4. Allopurinol prevents fructose-induced CHREBP acetylation and nuclear translocation in human hepatocytes.

A) KHK mRNA expression in cells exposed to fructose (5 mmol/L) for different time points. B) Analysis of acetylation state in immunoprecipitated ChREBP in cells exposed to glucose, fructose or mannitiol in the presence or absence of allopurinol. (C–E) ChREBP and KHK expression in nuclear and cytoplasmic extracts of cells control and incubated with glucose (25 mM) and fructose (5 mM) in the presence or absence of allopurinol.

Figure 5

Figure 5. Identification in human hepatocytes of ChoRE sites in khk promoter that are activated by fructose and blocked with allopurinol.

A) mRNA expression of khk in cells exposed to fructose in the presence of ChREBP dominant negative (dnMLX). B) KHK activity in cells exposed to fructose in the presence of ChREBP dominant negative (dnMLX). C) ChIP analysis and khk promoter occupancy in distal and proximal ChoRE sites by ChREBP in cells exposed to fructose in the presence of ChREBP dominant negative (dnMLX) or allopurinol. D) Luciferase expression in human hepatocytes transfected with pGL3-_khk_proximal ChoRE and exposed to fructose in the presence of ChREBP dominant negative (dnMLX) E) Luciferase expression in human hepatocytes transfected with pGL3-_khk_distal ChoRE and exposed to fructose in the presence of ChREBP dominant negative (dnMLX) or allopurinol.

Figure 6

Figure 6. Allopurinol prevents fructose-induced CHREBP nuclear translocation in adult male rats drinking fructose.

(A–B) ChREBP protein expression in liver extracts from control, fructose-fed and fructose with allopurinol-fed rats. (C–D) ChREBP and KHK expression in nuclear and cytoplasmic extracts of liver extracts from control, fructose-fed and fructose with allopurinol-fed rats.

Figure 7

Figure 7. Uric acid sensitizes human hepatocytes to fructose.

(A–B) KHK expression in cells pre-exposed to different amounts of uric acid for 72 hours and further incubated with the same amount of fructose for 24 hours. C) Concentration of TG in liver extracts from cells pre-exposed to different amounts of uric acid for 72 hours and further incubated with the same amount of fructose for 24 hours. D) Adding back uric acid reverts the inhibitory effect of allopurinol on TG accumulation in fructose-exposed HepG2 cells.

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