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Uric Acid Induces Hepatic Steatosis by Generation of Mitochondrial Oxidative Stress

POTENTIAL ROLE IN FRUCTOSE-DEPENDENT AND -INDEPENDENT FATTY LIVER*
  • Miguel A. Lanaspa
    Correspondence
    To whom correspondence should be addressed. Tel.: 303-724-4898; Fax: 303-724-4868
    Affiliations
    Division of Renal Diseases and Hypertension, Department of Medicine, University of Colorado, Denver, Colorado 80045
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  • Laura G. Sanchez-Lozada
    Affiliations
    Division of Renal Diseases and Hypertension, Department of Medicine, University of Colorado, Denver, Colorado 80045

    Laboratory of Renal Physiopathology and Nephrology, Department of Medicine, Instituto Nacional de Cardiología-Ignacio Chavez, 14339 Mexico City, Mexico
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  • Yea-Jin Choi
    Affiliations
    Division of Nephrology, Department of Internal Medicine, EwhaWomans University School of Medicine, Ewha Medical Research Center, 120-750 Seoul, South Korea
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  • Christina Cicerchi
    Affiliations
    Division of Renal Diseases and Hypertension, Department of Medicine, University of Colorado, Denver, Colorado 80045
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  • Mehmet Kanbay
    Affiliations
    Department of Medicine, Division of Nephrology, Kayseri Training and Research Hospital, Kayseri 38039, Turkey
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  • Carlos A. Roncal-Jimenez
    Affiliations
    Division of Renal Diseases and Hypertension, Department of Medicine, University of Colorado, Denver, Colorado 80045
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  • Takuji Ishimoto
    Affiliations
    Division of Renal Diseases and Hypertension, Department of Medicine, University of Colorado, Denver, Colorado 80045
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  • Nanxing Li
    Affiliations
    Division of Renal Diseases and Hypertension, Department of Medicine, University of Colorado, Denver, Colorado 80045
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  • George Marek
    Affiliations
    Division of Nephrology, Hypertension, and Transplantation, Department of Medicine, University of Florida, Gainesville, Florida 32611
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  • Murat Duranay
    Affiliations
    Department of Medicine, Division of Nephrology, Kayseri Training and Research Hospital, Kayseri 38039, Turkey
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  • George Schreiner
    Affiliations
    Cardero Therapeutics, Inc., Menlo Park, California 94085
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  • Bernardo Rodriguez-Iturbe
    Affiliations
    Instituto Venezolano de Investigaciones Científicas-Zulia and Hospital Universitario y Universidad del Zulia, 4001-A Maracaibo, Venezuela
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  • Takahiko Nakagawa
    Affiliations
    Division of Renal Diseases and Hypertension, Department of Medicine, University of Colorado, Denver, Colorado 80045
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  • Duk-Hee Kang
    Affiliations
    Division of Nephrology, Department of Internal Medicine, EwhaWomans University School of Medicine, Ewha Medical Research Center, 120-750 Seoul, South Korea
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  • Yuri Y. Sautin
    Affiliations
    Division of Nephrology, Hypertension, and Transplantation, Department of Medicine, University of Florida, Gainesville, Florida 32611
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  • Richard J. Johnson
    Affiliations
    Division of Renal Diseases and Hypertension, Department of Medicine, University of Colorado, Denver, Colorado 80045
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grants HL-68607 and RC4 DK090869-01 (to R. J. J.). This work was also supported by Amway, Cardero, Danone, and Questcor (to R. J. .J.) and by startup funds from the University of Colorado (to R. J. J.) and Korea Healthcare Technology R&D Project, Ministry for Health, Welfare, and Family Affairs, Republic of Korea Grant A101742 (to D.-H. K.). M. A. L., T. I., and R. J. J. 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. T. N. and R. J. J. are listed as inventors on several patent applications related to lowering uric acid as a means to prevent or treat metabolic syndrome. Dr. Johnson also has a patent with the University of Washington and Merck for the use of allopurinol to treat hypertension. Dr. Johnson also discloses that he has consulted for Ardea, Astellas, Danone, and Novartis, that he is on the scientific board of Amway. R. J. J. and T. N. have patent applications related to lowering uric acid as a means to prevent fatty liver and metabolic syndrome. R. J. J., T. I., and M. L. also have patent applications related to blocking fructose metabolism as a means for preventing or treating metabolic syndrome. Dr. Johnson has also consulted for Novartis, Danone, Ardea, Mitsubishi Tanabe, and Astellas. Dr. Johnson also has two lay books on sugar, including the Sugar Fix (Rodale, 2008) and the Fat Switch (Mercola.com, 2012).
Open AccessPublished:October 03, 2012DOI:https://doi.org/10.1074/jbc.M112.399899
      Metabolic syndrome represents a collection of abnormalities that includes fatty liver, and it currently affects one-third of the United States population and has become a major health concern worldwide. Fructose intake, primarily from added sugars in soft drinks, can induce fatty liver in animals and is epidemiologically associated with nonalcoholic fatty liver disease in humans. Fructose is considered lipogenic due to its ability to generate triglycerides as a direct consequence of the metabolism of the fructose molecule. Here, we show that fructose also stimulates triglyceride synthesis via a purine-degrading pathway that is triggered from the rapid phosphorylation of fructose by fructokinase. Generated AMP enters into the purine degradation pathway through the activation of AMP deaminase resulting in uric acid production and the generation of mitochondrial oxidants. Mitochondrial oxidative stress results in the inhibition of aconitase in the Krebs cycle, resulting in the accumulation of citrate and the stimulation of ATP citrate lyase and fatty-acid synthase leading to de novo lipogeneis. These studies provide new insights into the pathogenesis of hepatic fat accumulation under normal and diseased states.

      Introduction

      Nonalcoholic fatty liver disease (NAFLD)
      The abbreviations used are: NAFLD
      nonalcoholic fatty liver disease
      KHK
      Ketohexokinase
      TG
      triglyceride
      AMPD
      AMP deaminase
      FAS
      fatty-acid synthase
      ACL
      ATP-citrate lyase
      DCF
      2′,7′-dichlorofluorescein
      AldoB
      aldolase B
      ACL
      ATP-citrate lyase
      BMI
      body mass index
      FAS
      fatty-acid synthase.
      is a common condition, present in 20–30% of adults in the United States, and is associated with increased mortality (
      • Adams L.A.
      • Lymp J.F.
      • St Sauver J.
      • Sanderson S.O.
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      • Angulo P.
      The natural history of nonalcoholic fatty liver disease. A population-based cohort study.
      ). A recently recognized risk factor of NAFLD is fructose, especially that present in soft drinks (
      • Ouyang X.
      • Cirillo P.
      • Sautin Y.
      • McCall S.
      • Bruchette J.L.
      • Diehl A.M.
      • Johnson R.J.
      • Abdelmalek M.F.
      Fructose consumption as a risk factor for nonalcoholic fatty liver disease.
      ,
      • Lim J.S.
      • Mietus-Snyder M.
      • Valente A.
      • Schwarz J.M.
      • Lustig R.H.
      The role of fructose in the pathogenesis of NAFLD and the metabolic syndrome.
      ,
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      • Diehl A.M.
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      Higher dietary fructose is associated with impaired hepatic ATP homeostasis in patients with nonalcoholic fatty liver disease.
      ,
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      • Suzuki A.
      • Guy C.
      • Johnson R.J.
      • Diehl A.M.
      Fructose-induced hyperuricemia as a causal mechanism for nonalcoholic fatty liver disease.
      ). Fructose is known to be lipogenic (
      • Havel P.J.
      Dietary fructose: implications for dysregulation of energy homeostasis and lipid/carbohydrate metabolism.
      ), and the feeding of fructose to rats causes the rapid development of fatty liver (
      • Ackerman Z.
      • Oron-Herman M.
      • Grozovski M.
      • Rosenthal T.
      • Pappo O.
      • Link G.
      • Sela B.A.
      Fructose-induced fatty liver disease. Hepatic effects of blood pressure and plasma triglyceride reduction.
      ). This is mediated by the initial rapid phosphorylation of fructose to fructose 1-phosphate by fructokinase (ketohexokinase, KHK) with paralleled ATP depletion and uric acid generation. Therefore, one unique aspect of fructose compared with glucose and other sugars is that it generates uric acid inside the hepatocyte during its metabolism (
      • van den Berghe G.
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      • Vanneste R.
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      The mechanism of adenosine triphosphate depletion in the liver after a load of fructose. A kinetic study of liver adenylate deaminase.
      ). Serum uric acid rises acutely after the ingestion of fructose (
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      Fructose-induced hyperuricaemia.
      ). Chronic administration of diets enriched in fructose also raises both fasting and 24-h uric acid levels (
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      Consumption of fructose- but not glucose-sweetened beverages for 10 weeks increases circulating concentrations of uric acid, retinol-binding protein-4, and γ-glutamyl transferase activity in overweight/obese humans.
      ), and studies of the general population have linked soft drink intake with increased serum uric acid levels (
      • Choi J.W.
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      • Choi H.K.
      Sugar-sweetened soft drinks, diet soft drinks, and serum uric acid level. The third national health and nutrition examination survey.
      ). Given this information, it is not surprising that an elevated serum uric acid has been repeatedly shown to both predict as well as to be common in subjects with NAFLD (
      • Xu C.
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      • Miao M.
      • Li Y.
      High serum uric acid increases the risk for nonalcoholic fatty liver disease. A prospective observational study.
      ,
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      Gout and risk of nonalcoholic fatty liver disease.
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      Association of serum uric acid level with nonalcoholic fatty liver disease. A cross-sectional study.
      ,
      • Lee K.
      Relationship between uric acid and hepatic steatosis among Koreans.
      ,
      • Yamada T.
      • Suzuki S.
      • Fukatsu M.
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      • Joh T.
      Elevated serum uric acid is an independent risk factor for nonalcoholic fatty liver disease in Japanese undergoing a health checkup.
      ,
      • Petta S.
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      • Cabibi D.
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      Hyperuricemia is associated with histological liver damage in patients with nonalcoholic fatty liver disease.
      ,
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      Serum uric acid as a predictor for the development of nonalcoholic fatty liver disease in apparently healthy subjects. A 5-year retrospective cohort study.
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      Fasting insulin and uric acid levels but not indices of iron metabolism are independent predictors of nonalcoholic fatty liver disease. A case-control study.
      ,
      • Ryu S.
      • Chang Y.
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      • Guallar E.
      Serum uric acid levels predict incident nonalcoholic fatty liver disease in healthy Korean men.
      ,
      • Sirota J.C.
      • McFann K.
      • Targher G.
      • Johnson R.J.
      • Chonchol M.
      • Jalal D.
      Association between serum uric acid levels and ultrasound-diagnosed nonalcoholic fatty liver disease in the National Health and Nutrition Examination Survey.
      ).
      The possibility that uric acid may have a causal role in NAFLD has to our knowledge not been studied in detail. There is a report that lowering of uric acid can prevent hepatic steatosis in the Mongolian gerbil (
      • Xu C.F.
      • Yu C.H.
      • Xu L.
      • Sa X.Y.
      • Li Y.M.
      Hypouricemic therapy. A novel potential therapeutic option for nonalcoholic fatty liver disease.
      ), but the mechanism was not elucidated. However, recent studies have shown that uric acid can induce intracellular oxidative stress and proinflammatory effects in various cell types (
      • Sautin Y.Y.
      • Nakagawa T.
      • Zharikov S.
      • Johnson R.J.
      Adverse effects of the classic antioxidant uric acid in adipocytes. NADPH oxidase-mediated oxidative/nitrosative stress.
      ,
      • Yu M.A.
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      • Johnson R.J.
      • Kang D.H.
      Oxidative stress with an activation of the renin-angiotensin system in human vascular endothelial cells as a novel mechanism of uric acid-induced endothelial dysfunction.
      ,
      • Kang D.H.
      • Park S.K.
      • Lee I.K.
      • Johnson R.J.
      Uric acid-induced C-reactive protein expression. Implication on cell proliferation and nitric oxide production of human vascular cells.
      ,
      • Kanellis J.
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      • Li P.
      • Nakagawa T.
      • Wamsley A.
      • Sheikh-Hamad D.
      • Lan H.Y.
      • Feng L.
      • Johnson R.J.
      Uric acid stimulates monocyte chemoattractant protein-1 production in vascular smooth muscle cells via mitogen-activated protein kinase and cyclooxygenase-2.
      ,
      • Corry D.B.
      • Eslami P.
      • Yamamoto K.
      • Nyby M.D.
      • Makino H.
      • Tuck M.L.
      Uric acid stimulates vascular smooth muscle cell proliferation and oxidative stress via the vascular renin-angiotensin system.
      ). This led us to hypothesize that uric acid may have a role in fructose-induced fatty liver disease and possibly in other models of NAFLD. In this study, we identify a mechanism by which uric acid may mediate fatty liver, involving a pathway in which uric acid alters mitochondrial function with the alteration in fat synthesis.

      DISCUSSION

      Increased fatty acid synthesis, through the lipogenic pathway in liver, results in the development of hepatic steatosis (NAFLD) that contributes to the development of chronic hepatic inflammation and insulin resistance. The fructose component of added sugars has been particularly implicated in the etiology of these syndromes as the administration of fructose (or sucrose) to rats results in fatty liver as well as all features of the metabolic syndrome (
      • Havel P.J.
      Dietary fructose: implications for dysregulation of energy homeostasis and lipid/carbohydrate metabolism.
      ,
      • Johnson R.J.
      • Perez-Pozo S.E.
      • Sautin Y.Y.
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      • Roncal C.
      • Nakagawa T.
      Hypothesis. Could excessive fructose intake and uric acid cause type 2 diabetes?.
      ,
      • Tappy L.
      • Lê K.A.
      Metabolic effects of fructose and the worldwide increase in obesity.
      ,
      • Roncal-Jimenez C.A.
      • Lanaspa M.A.
      • Rivard C.J.
      • Nakagawa T.
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      • Jalal D.
      • Andres-Hernando A.
      • Tanabe K.
      • Madero M.
      • Li N.
      • Cicerchi C.
      • Mc Fann K.
      • Sautin Y.Y.
      • Johnson R.J.
      Sucrose induces fatty liver and pancreatic inflammation in male breeder rats independent of excess energy intake.
      ).
      Although the lipogenic ability of fructose has been known for years, the molecular mechanism whereby fructose potentiates fat accumulation in the liver remains poorly understood. It is classically accepted that fructose can be directly metabolized to triglycerides in a mechanism involving the action of three major enzymes as follows: fructokinase (KHK), which phosphorylates fructose to fructose 1-phosphate; AldoB, which breaks fructose 1-phosphate into dihydroxyacetone phosphate and glyceraldehyde; and FAS, which metabolizes acetyl-CoA produced from dihydroxyacetone phosphate and glyceraldehydes into triglycerides. Nevertheless, studies in which fructose has been labeled have shown that only 3% of the fructose is incorporated into triglycerides (
      • Nikkilä E.A.
      Control of plasma and liver triglyceride kinetics by carbohydrate metabolism and insulin.
      ,
      • Nikkilä E.A.
      • Kekki M.
      Effects of dietary fructose and sucrose on plasma triglyceride metabolism in patients with endogenous hypertriglyceridemia.
      ), and this cannot explain why fructose is so lipogenic (
      • Van den Berghe G.
      Fructose. Metabolism and short term effects on carbohydrate and purine metabolic pathways.
      ).
      In this study, we have identified an additional pathway whereby fructose induces its lipogenic effects. We show that uric acid, either alone or as a by-product generated by the initial phosphorylation of fructose by KHK, directly regulates hepatic lipogenesis through the generation of mitochondrial oxidative stress. First, we found that exposure of HepG2 cells to uric acid at levels considered to be hyperuricemic in humans led to the development of hepatic steatosis characterized by an increase in de novo lipogenesis and triglyceride accumulation. This was due to enhanced mitochondrial translocation of the NADPH oxidase isoform, NOX4, which increased the generation of superoxide by the mitochondria. We found that aconitase, which is present in the mitochondrial matrix and is sensitive to changes in the mitochondrial oxidative state, was reduced in activity, and this led to citrate accumulation in the mitochondria with release into the cytosol where it acted as the substrate for de novo lipogenesis. Specifically, increased cytoplasmic citrate levels activated the ATP-sensitive enzyme, ACL by phosphorylation at Ser455, which converted it to acetyl-CoA for de novo lipogenesis through FAS. These findings suggest that uric acid-dependent regulation of mitochondrial function could be critical for the modulation of lipid homeostasis in fatty liver and that the lipogenic ability of fructose may be mediated at least partially by the generation of uric acid. In the settings of fructose, a condition associated with elevated uric acid production, we confirmed that blockade of uric acid generation with allopurinol prevented fructose-induced lipogenesis.
      Although the direct metabolism of fructose to triglycerides partially explains why fructose is lipogenic, the uric acid-dependent pathway may explain two paradoxes in the lipogenic effects of fructose. First, when the fructose molecule is radiolabeled, only 1–3% of the fructose is directly converted into triglycerides (
      • Van den Berghe G.
      Fructose. Metabolism and short term effects on carbohydrate and purine metabolic pathways.
      ). Second, people with hereditary fructose intolerance, a rare disorder in which AldoB is nonfunctional, develop fatty liver in response to dietary fructose even though it cannot be fully metabolized to triglycerides (
      • Odièvre M.
      • Gentil C.
      • Gautier M.
      • Alagille D.
      Hereditary fructose intolerance in childhood. Diagnosis, management, and course in 55 patients.
      ). Because fructose is distinct from glucose in its ability to increase intracellular uric acid, our studies provide an explanation for why fructose (or sucrose) can induce fatty liver much easier than glucose (or starch) in animals (
      • Ackerman Z.
      • Oron-Herman M.
      • Grozovski M.
      • Rosenthal T.
      • Pappo O.
      • Link G.
      • Sela B.A.
      Fructose-induced fatty liver disease. Hepatic effects of blood pressure and plasma triglyceride reduction.
      ,
      • Roncal-Jimenez C.A.
      • Lanaspa M.A.
      • Rivard C.J.
      • Nakagawa T.
      • Sanchez-Lozada L.G.
      • Jalal D.
      • Andres-Hernando A.
      • Tanabe K.
      • Madero M.
      • Li N.
      • Cicerchi C.
      • Mc Fann K.
      • Sautin Y.Y.
      • Johnson R.J.
      Sucrose induces fatty liver and pancreatic inflammation in male breeder rats independent of excess energy intake.
      ).
      Hyperuricemia is also a prevalent finding in patients presenting with NAFLD and metabolic syndrome, although its clinical meaning is still controversial and often underestimated. Numerous studies have consistently demonstrated that the presence of metabolic syndrome and its individual components is associated with serum uric acid levels (
      • Kodama S.
      • Saito K.
      • Yachi Y.
      • Asumi M.
      • Sugawara A.
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      • Saito A.
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      Association between serum uric acid and development of type 2 diabetes.
      ,
      • Masuo K.
      • Kawaguchi H.
      • Mikami H.
      • Ogihara T.
      • Tuck M.L.
      Serum uric acid and plasma norepinephrine concentrations predict subsequent weight gain and blood pressure elevation.
      ,
      • Dehghan A.
      • van Hoek M.
      • Sijbrands E.J.
      • Hofman A.
      • Witteman J.C.
      High serum uric acid as a novel risk factor for type 2 diabetes.
      ), but the importance of hyperuricemia still remains a matter of debate. In this context, our group and others have reported that lowering uric acid can block both fructose-induced metabolic syndrome as well as improve features of metabolic syndrome in other animal models (
      • Baldwin W.
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      ,
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      A causal role for uric acid in fructose-induced metabolic syndrome.
      ). Xu et al. (
      • Xu C.F.
      • Yu C.H.
      • Xu L.
      • Sa X.Y.
      • Li Y.M.
      Hypouricemic therapy. A novel potential therapeutic option for nonalcoholic fatty liver disease.
      ) also reported that lowering uric acid can improve fatty liver in the Mongolian gerbil. Pilot clinical studies have also reported benefits of lowering uric acid on inflammation, endothelial function, blood pressure, and insulin resistance (
      • Feig D.I.
      • Soletsky B.
      • Johnson R.J.
      Effect of allopurinol on the blood pressure of adolescents with newly diagnosed essential hypertension: a randomized trial.
      ,
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      A randomized study of allopurinol on endothelial function and estimated glomerular filtration rate in asymptomatic hyperuricemic subjects with normal renal function.
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      ). These studies are reawakening the possibility that uric acid may have a causal role in metabolic syndrome and cardiovascular disease (
      • Feig D.I.
      • Kang D.H.
      • Johnson R.J.
      Uric acid and cardiovascular risk.
      ).
      The novel finding in this study is that uric acid can directly stimulate hepatic fat accumulation. Consistent with an independent deleterious role of uric acid in hepatic steatosis, we show that lowering hepatic uric acid could reduce fatty liver both in the fructose-exposed hepatocyte and in the pound mouse, an animal model of metabolic syndrome and hyperuricemia. These studies suggest that allopurinol could be helpful in the treatment of fatty liver in humans. However, the doses used in these animal studies are severalfold higher than we typically use in patients; the justification for doing this is because in rodents the xanthine oxidase activity is about 100 times higher compared with humans (
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      ).
      In conclusion, this study provides the first direct evidence that uric acid can stimulate fat synthesis in the hepatocyte. We identify a mechanism that involves the translocation of NADPH oxidase to the mitochondria with inactivation of aconitase, accumulation of citrate, and a stimulation of fat synthesis. We also show that this accounts for an important mechanism by which fructose induces hepatic fat accumulation. Together, our study may provide a mechanism for why soft drink intake is strongly linked with fatty liver (
      • Ouyang X.
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      Fructose consumption as a risk factor for nonalcoholic fatty liver disease.
      ,
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      ) and why uric acid is such a potent predictor of NAFLD (
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      High serum uric acid increases the risk for nonalcoholic fatty liver disease. A prospective observational study.
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      • Chen H.W.
      • See L.C.
      Gout and risk of nonalcoholic fatty liver disease.
      ,
      • Li Y.
      • Xu C.
      • Yu C.
      • Xu L.
      • Miao M.
      Association of serum uric acid level with nonalcoholic fatty liver disease. A cross-sectional study.
      ,
      • Lee K.
      Relationship between uric acid and hepatic steatosis among Koreans.
      ,
      • Yamada T.
      • Suzuki S.
      • Fukatsu M.
      • Wada T.
      • Yoshida T.
      • Joh T.
      Elevated serum uric acid is an independent risk factor for nonalcoholic fatty liver disease in Japanese undergoing a health checkup.
      ,
      • Petta S.
      • Cammà C.
      • Cabibi D.
      • Di Marco V.
      • Craxì A.
      Hyperuricemia is associated with histological liver damage in patients with nonalcoholic fatty liver disease.
      ,
      • Lee J.W.
      • Cho Y.K.
      • Ryan M.
      • Kim H.
      • Lee S.W.
      • Chang E.
      • Joo K.J.
      • Kim J.T.
      • Kim B.S.
      • Sung K.C.
      Serum uric acid as a predictor for the development of nonalcoholic fatty liver disease in apparently healthy subjects. A 5-year retrospective cohort study.
      ,
      • Lonardo A.
      • Loria P.
      • Leonardi F.
      • Borsatti A.
      • Neri P.
      • Pulvirenti M.
      • Verrone A.M.
      • Bagni A.
      • Bertolotti M.
      • Ganazzi D.
      • Carulli N.
      Fasting insulin and uric acid levels but not indices of iron metabolism are independent predictors of nonalcoholic fatty liver disease. A case-control study.
      ,
      • Ryu S.
      • Chang Y.
      • Kim S.G.
      • Cho J.
      • Guallar E.
      Serum uric acid levels predict incident nonalcoholic fatty liver disease in healthy Korean men.
      ,
      • Sirota J.C.
      • McFann K.
      • Targher G.
      • Johnson R.J.
      • Chonchol M.
      • Jalal D.
      Association between serum uric acid levels and ultrasound-diagnosed nonalcoholic fatty liver disease in the National Health and Nutrition Examination Survey.
      ). Further studies investigating the role of purine-degrading pathways and metabolites in metabolic syndrome seem warranted.

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