RESUMEN
An epidemic of obesity and type 2 diabetes is linked with the increase in consumption of fructose-containing sugars, such as sucrose and high-fructose corn syrup. In mammalian cells, fructose is metabolized predominantly via phosphorylation to fructose-1 phosphate by ketohexokinase (KHK) or by alternative pathways. Here we demonstrate that a KHK-dependent pathway mediates insulin resistance and inflammatory changes in the visceral fat in response to high fructose. We used mice (males, C57BL/6 background) including littermate wild-type control and mice lacking both isoforms of KHK (KHK-null). Fructose diet induced metabolic syndrome, including visceral obesity, insulin resistance, proinflammatory changes in the visceral fat (production of proinflammatory adipokines and macrophage infiltration), the endoplasmic reticulum stress signaling, and decrease of the high-molecular weight adiponectin followed by decrease in the downstream signaling. KHK-KO mice consuming the same high-fructose diet remained lean, with normal insulin sensitivity and healthy visceral adipose tissue with normal adiponectin function not distinguishable from the control by any of the tested parameters. This study demonstrates that blocking KHK and redirecting fructose metabolism to alternative pathways is an effective way to prevent visceral obesity and insulin resistance induced by high fructose, a widespread component of Western diets.
Asunto(s)
Adiponectina/metabolismo , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Fructoquinasas/metabolismo , Fructosa/farmacología , Regulación Enzimológica de la Expresión Génica/fisiología , Animales , Carbohidratos de la Dieta/farmacología , Fructoquinasas/genética , Resistencia a la Insulina , Riñón/metabolismo , Hígado/metabolismo , Ratones , Ratones Noqueados , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Aumento de PesoRESUMEN
Diabetes is associated with activation of the polyol pathway, in which glucose is converted to sorbitol by aldose reductase. Previous studies focused on the role of sorbitol in mediating diabetic complications. However, in the proximal tubule, sorbitol can be converted to fructose, which is then metabolized largely by fructokinase, also known as ketohexokinase, leading to ATP depletion, proinflammatory cytokine expression, and oxidative stress. We and others recently identified a potential deleterious role of dietary fructose in the generation of tubulointerstitial injury and the acceleration of CKD. In this study, we investigated the potential role of endogenous fructose production, as opposed to dietary fructose, and its metabolism through fructokinase in the development of diabetic nephropathy. Wild-type mice with streptozotocin-induced diabetes developed proteinuria, reduced GFR, and renal glomerular and proximal tubular injury. Increased renal expression of aldose reductase; elevated levels of renal sorbitol, fructose, and uric acid; and low levels of ATP confirmed activation of the fructokinase pathway. Furthermore, renal expression of inflammatory cytokines with macrophage infiltration was prominent. In contrast, diabetic fructokinase-deficient mice demonstrated significantly less proteinuria, renal dysfunction, renal injury, and inflammation. These studies identify fructokinase as a novel mediator of diabetic nephropathy and document a novel role for endogenous fructose production, or fructoneogenesis, in driving renal disease.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Nefropatías Diabéticas/metabolismo , Fructoquinasas/metabolismo , Fructosa/biosíntesis , Fructosa/metabolismo , Túbulos Renales Proximales/enzimología , Animales , Glucemia/metabolismo , Peso Corporal , Línea Celular Transformada , Quimiocinas/metabolismo , Citocinas/metabolismo , Diabetes Mellitus Experimental/patología , Nefropatías Diabéticas/patología , Humanos , Corteza Renal/enzimología , Corteza Renal/patología , Glomérulos Renales/citología , Glomérulos Renales/patología , Túbulos Renales Proximales/patología , Macrófagos/metabolismo , Macrófagos/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Polímeros/metabolismoRESUMEN
Reduced AMP kinase (AMPK) activity has been shown to play a key deleterious role in increased hepatic gluconeogenesis in diabetes, but the mechanism whereby this occurs remains unclear. In this article, we document that another AMP-dependent enzyme, AMP deaminase (AMPD) is activated in the liver of diabetic mice, which parallels with a significant reduction in AMPK activity and a significant increase in intracellular glucose accumulation in human HepG2 cells. AMPD activation is induced by a reduction in intracellular phosphate levels, which is characteristic of insulin resistance and diabetic states. Increased gluconeogenesis is mediated by reduced TORC2 phosphorylation at Ser171 by AMPK in these cells, as well as by the up-regulation of the rate-limiting enzymes PEPCK and G6Pc. The mechanism whereby AMPD controls AMPK activation depends on the production of a specific AMP downstream metabolite through AMPD, uric acid. In this regard, humans have higher uric acid levels than most mammals due to a mutation in uricase, the enzyme involved in uric acid degradation in most mammals, that developed during a period of famine in Europe 1.5 × 10(7) yr ago. Here, working with resurrected ancestral uricases obtained from early hominids, we show that their expression on HepG2 cells is enough to blunt gluconeogenesis in parallel with an up-regulation of AMPK activity. These studies identify a key role AMPD and uric acid in mediating hepatic gluconeogenesis in the diabetic state, via a mechanism involving AMPK down-regulation and overexpression of PEPCK and G6Pc. The uricase mutation in the Miocene likely provided a survival advantage to help maintain glucose levels under conditions of near starvation, but today likely has a role in the pathogenesis of diabetes.
Asunto(s)
AMP Desaminasa/fisiología , Gluconeogénesis/fisiología , Hígado/metabolismo , Inanición/fisiopatología , Ácido Úrico/metabolismo , AMP Desaminasa/antagonistas & inhibidores , AMP Desaminasa/genética , Proteínas Quinasas Activadas por AMP/fisiología , Animales , Diabetes Mellitus Experimental/metabolismo , Europa (Continente) , Regulación Enzimológica de la Expresión Génica , Gluconeogénesis/efectos de los fármacos , Glucosa-6-Fosfatasa/biosíntesis , Células Hep G2 , Historia Antigua , Hominidae/fisiología , Humanos , Insulina/metabolismo , Resistencia a la Insulina , Secreción de Insulina , Hígado/enzimología , Masculino , Diana Mecanicista del Complejo 2 de la Rapamicina , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Complejos Multiproteicos/fisiología , Fosfatos/metabolismo , Fosfatos/farmacología , Fosfoenolpiruvato Carboxiquinasa (ATP)/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Selección Genética , Organismos Libres de Patógenos Específicos , Inanición/historia , Serina-Treonina Quinasas TOR/fisiología , Transducción Genética , Urato Oxidasa/genética , Urato Oxidasa/historia , Urato Oxidasa/metabolismo , Ácido Úrico/farmacologíaRESUMEN
The intake of added sugars, such as from table sugar (sucrose) and high-fructose corn syrup has increased dramatically in the last hundred years and correlates closely with the rise in obesity, metabolic syndrome, and diabetes. Fructose is a major component of added sugars and is distinct from other sugars in its ability to cause intracellular ATP depletion, nucleotide turnover, and the generation of uric acid. In this article, we revisit the hypothesis that it is this unique aspect of fructose metabolism that accounts for why fructose intake increases the risk for metabolic syndrome. Recent studies show that fructose-induced uric acid generation causes mitochondrial oxidative stress that stimulates fat accumulation independent of excessive caloric intake. These studies challenge the long-standing dogma that "a calorie is just a calorie" and suggest that the metabolic effects of food may matter as much as its energy content. The discovery that fructose-mediated generation of uric acid may have a causal role in diabetes and obesity provides new insights into pathogenesis and therapies for this important disease.
Asunto(s)
Diabetes Mellitus Tipo 2/etiología , Sacarosa en la Dieta/efectos adversos , Fructosa/efectos adversos , Hiperuricemia/complicaciones , Síndrome Metabólico/etiología , Obesidad/etiología , Edulcorantes/efectos adversos , Ácido Úrico/metabolismo , Animales , Enfermedades Cardiovasculares/etiología , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatología , Modelos Animales de Enfermedad , Ingestión de Energía , Hígado Graso/etiología , Conducta Alimentaria , Femenino , Fructosa/metabolismo , Humanos , Hiperuricemia/metabolismo , Hiperuricemia/fisiopatología , Resistencia a la Insulina , Masculino , Síndrome Metabólico/metabolismo , Síndrome Metabólico/fisiopatología , Obesidad/metabolismo , Obesidad/fisiopatología , Insuficiencia Renal Crónica/etiología , Edulcorantes/metabolismo , Ácido Úrico/efectos adversos , Aumento de PesoRESUMEN
Carbohydrates with high glycaemic index are proposed to promote the development of obesity, insulin resistance and fatty liver, but the mechanism by which this occurs remains unknown. High serum glucose concentrations are known to induce the polyol pathway and increase fructose generation in the liver. Here we show that this hepatic, endogenously produced fructose causes systemic metabolic changes. We demonstrate that mice unable to metabolize fructose are protected from an increase in energy intake and body weight, visceral obesity, fatty liver, elevated insulin levels and hyperleptinaemia after exposure to 10% glucose for 14 weeks. In normal mice, glucose consumption is accompanied by aldose reductase and polyol pathway activation in steatotic areas. In this regard, we show that aldose reductase-deficient mice are protected against glucose-induced fatty liver. We conclude that endogenous fructose generation and metabolism in the liver represents an important mechanism by which glucose promotes the development of metabolic syndrome.
Asunto(s)
Fructosa/biosíntesis , Fructosa/metabolismo , Hígado/metabolismo , Hígado/patología , Síndrome Metabólico/metabolismo , Síndrome Metabólico/patología , Aldehído Reductasa/metabolismo , Animales , Metabolismo Energético , Hígado Graso/metabolismo , Conducta Alimentaria , Fructoquinasas/deficiencia , Fructoquinasas/metabolismo , Glucosa/metabolismo , Células Hep G2 , Humanos , Hígado/enzimología , Hígado/fisiopatología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Polímeros/metabolismoRESUMEN
UNLABELLED: Fructose intake from added sugars has been implicated as a cause of nonalcoholic fatty liver disease. Here we tested the hypothesis that fructose may interact with a high-fat diet to induce fatty liver, and to determine if this was dependent on a key enzyme in fructose metabolism, fructokinase. Wild-type or fructokinase knockout mice were fed a low-fat (11%), high-fat (36%), or high-fat (36%) and high-sucrose (30%) diet for 15 weeks. Both wild-type and fructokinase knockout mice developed obesity with mild hepatic steatosis and no evidence of hepatic inflammation on a high-fat diet compared to a low-fat diet. In contrast, wild-type mice fed a high-fat and high-sucrose diet developed more severe hepatic steatosis with low-grade inflammation and fibrosis, as noted by increased CD68, tumor necrosis factor alpha, monocyte chemoattractant protein-1, alpha-smooth muscle actin, and collagen I and TIMP1 expression. These changes were prevented in the fructokinase knockout mice. CONCLUSION: An additive effect of high-fat and high-sucrose diet on the development of hepatic steatosis exists. Further, the combination of sucrose with high-fat diet may induce steatohepatitis. The protection in fructokinase knockout mice suggests a key role for fructose (from sucrose) in this development of steatohepatitis. These studies emphasize the important role of fructose in the development of fatty liver and nonalcoholic steatohepatitis.
Asunto(s)
Dieta Alta en Grasa , Hígado Graso/etiología , Fructoquinasas/fisiología , Sacarosa/administración & dosificación , Animales , Ingestión de Energía , Fructosa/metabolismo , Hígado/metabolismo , Hígado/patología , Ratones , Ratones Endogámicos C57BL , Aumento de PesoRESUMEN
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.
Asunto(s)
Hígado Graso/metabolismo , Fructoquinasas/metabolismo , Fructosa/metabolismo , Hepatocitos/metabolismo , Ácido Úrico/metabolismo , Alopurinol/farmacología , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Inhibidores Enzimáticos/farmacología , Hígado Graso/genética , Hígado Graso/patología , Fructoquinasas/genética , Células Hep G2 , Hepatocitos/efectos de los fármacos , Hepatocitos/patología , Humanos , Hígado/metabolismo , Hígado/patología , Masculino , Ratas , Ratas Sprague-Dawley , Activación Transcripcional , Regulación hacia Arriba/efectos de los fármacos , Ácido Úrico/antagonistas & inhibidoresRESUMEN
BACKGROUND: Uric acid is an independent risk factor in fructose-induced fatty liver, but whether it is a marker or a cause remains unknown. RESULTS: Hepatocytes exposed to uric acid developed mitochondrial dysfunction and increased de novo lipogenesis, and its blockade prevented fructose-induced lipogenesis. CONCLUSION: Rather than a consequence, uric acid induces fatty liver SIGNIFICANCE: Hyperuricemic people are more prone to develop fructose-induced fatty liver. 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.
Asunto(s)
Hígado Graso/metabolismo , Mitocondrias/metabolismo , Estrés Oxidativo , Ácido Úrico/metabolismo , Fructosa/metabolismo , Células Hep G2 , Humanos , Lipogénesis , Triglicéridos/metabolismo , Ácido Úrico/efectos adversosRESUMEN
Fructose intake from added sugars correlates with the epidemic rise in obesity, metabolic syndrome, and nonalcoholic fatty liver disease. Fructose intake also causes features of metabolic syndrome in laboratory animals and humans. The first enzyme in fructose metabolism is fructokinase, which exists as two isoforms, A and C. Here we show that fructose-induced metabolic syndrome is prevented in mice lacking both isoforms but is exacerbated in mice lacking fructokinase A. Fructokinase C is expressed primarily in liver, intestine, and kidney and has high affinity for fructose, resulting in rapid metabolism and marked ATP depletion. In contrast, fructokinase A is widely distributed, has low affinity for fructose, and has less dramatic effects on ATP levels. By reducing the amount of fructose for metabolism in the liver, fructokinase A protects against fructokinase C-mediated metabolic syndrome. These studies provide insights into the mechanisms by which fructose causes obesity and metabolic syndrome.
Asunto(s)
Fructoquinasas/metabolismo , Síndrome Metabólico/enzimología , Animales , Metabolismo Energético/efectos de los fármacos , Conducta Alimentaria/efectos de los fármacos , Fructosa/administración & dosificación , Fructosa/metabolismo , Fructosa/farmacología , Isoenzimas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Fructose induces metabolic syndrome in rats; but studies have been criticized for using high concentrations of fructose that are not physiologic, for using only pure fructose, and for not controlling for energy intake. We tested the hypothesis that a 40% sucrose diet (containing 20% fructose) might induce features of metabolic syndrome in male breeder rats independent of excess energy intake. Male Sprague-Dawley breeder rats were pair fed 40% sucrose or isocaloric starch diet for 4 months and evaluated for metabolic syndrome and diabetes. In vitro studies were performed in rat insulinoma cells (RIN-m5F) exposed to uric acid, and markers of inflammation were assessed. Rats fed a 40% sucrose diet developed accelerated features of metabolic syndrome with up-regulation of fructose-dependent transporter Glut5 and fructokinase. Fatty liver and low-grade pancreatic inflammation also occurred. Uric acid was found to stimulate inflammatory mediators and oxidative stress in islet cells in vitro. Sucrose, at concentrations ingested by a subset of Americans, can accelerate metabolic syndrome, fatty liver, and type 2 diabetes mellitus in male breeder rats; and the effects are independent of excess energy intake.
Asunto(s)
Ingestión de Energía , Hígado Graso/etiología , Pancreatitis/etiología , Sacarosa/toxicidad , Animales , Proteínas de Transporte de Anión/genética , Línea Celular Tumoral , Fructosa/metabolismo , Masculino , Síndrome Metabólico/etiología , Ratas , Ratas Sprague-Dawley , Almidón/administración & dosificación , Sacarosa/administración & dosificaciónRESUMEN
OBJECTIVE: Hyperuricemia is strongly associated with obesity and metabolic syndrome and can predict visceral obesity and insulin resistance. Previously, we showed that soluble uric acid directly stimulated the redox-dependent proinflammatory signaling in adipocytes. In this study we demonstrate the role of hyperuricemia in the production of key adipokines. RESEARCH DESIGN AND METHODS: We used mouse 3T3-L1 adipocytes, human primary adipocytes, and a mouse model of metabolic syndrome and hyperuricemia. RESULTS: Uric acid induced in vitro an increase in the production (mRNA and secreted protein) of monocyte chemotactic protein-1 (MCP-1), an adipokine playing an essential role in inducing the proinflammatory state in adipocytes in obesity. In addition, uric acid caused a decrease in the production of adiponectin, an adipocyte-specific insulin sensitizer and anti-inflammatory agent. Uric acid-induced increase in MCP-1 production was blocked by scavenging superoxide or by inhibiting NADPH oxidase and by stimulating peroxisome-proliferator-activated receptor-γ with rosiglitazone. Downregulation of the adiponectin production was prevented by rosiglitazone but not by antioxidants. In obese mice with metabolic syndrome, we observed hyperuricemia. Lowering uric acid in these mice by inhibiting xanthine oxidoreductase with allopurinol could improve the proinflammatory endocrine imbalance in the adipose tissue by reducing production of MCP-1 and increasing production of adiponectin. In addition, lowering uric acid in obese mice decreased macrophage infiltration in the adipose tissue and reduced insulin resistance. CONCLUSIONS: Hyperuricemia might be partially responsible for the proinflammatory endocrine imbalance in the adipose tissue, which is an underlying mechanism of the low-grade inflammation and insulin resistance in subjects with the metabolic syndrome.
Asunto(s)
Tejido Adiposo/metabolismo , Hiperuricemia/metabolismo , Síndrome Metabólico/metabolismo , Células 3T3-L1 , Adipocitos/metabolismo , Adiponectina/sangre , Tejido Adiposo/fisiopatología , Animales , Células Cultivadas , Quimiocina CCL2/sangre , Humanos , Hiperuricemia/sangre , Inmunohistoquímica , Síndrome Metabólico/sangre , Ratones , Ratones Obesos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tiobarbitúricos/metabolismo , Ácido Úrico/metabolismoRESUMEN
BACKGROUND: There is evidence that disaccharide sucrose produce a greater increase in serum fructose and triglycerides (TGs) than the effect produced by their equivalent monosaccharides, suggesting that long-term exposure to sucrose or fructose + glucose could potentially result in different effects. AIM OF THE STUDY: We studied the chronic effects of a combination of free fructose and glucose relative to sucrose on rat liver. METHODS: Rats were fed either a combination of 30% fructose and 30% glucose (FG) or 60% sucrose (S). Control rats were fed normal rat chow (C). All rats were pair fed and were followed for 4 months. After killing, blood chemistries and liver tissue were examined. RESULTS: Both FG-fed- and S-fed rats developed early features of metabolic syndrome when compared with C. In addition, both diets induced hepatic alterations, including variable increases in hepatic TG accumulation and fatty liver, an increase in uric acid content in the liver, as well as an increase in hepatic levels of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-alpha (TNF-alpha) measured in liver homogenates. CONCLUSIONS: Diets containing 30% of fructose either as free fructose and glucose, or as sucrose, induce metabolic syndrome, intrahepatic accumulation of uric acid and TGs, increased MCP-1 and TNF-alpha as well as fatty liver in rats. It will be relevant to determine clinically whether pharmacological reduction in uric acid levels might have a therapeutic advantage in the treatment of non-alcoholic fatty liver disease.
Asunto(s)
Carbohidratos de la Dieta/administración & dosificación , Sacarosa en la Dieta/administración & dosificación , Hígado Graso/etiología , Fructosa/administración & dosificación , Glucosa/administración & dosificación , Animales , Distribución de la Grasa Corporal , Quimiocina CCL2/análisis , Ingestión de Energía , Hígado Graso/metabolismo , Hígado Graso/patología , Hígado/química , Masculino , Síndrome Metabólico/etiología , Ratas , Ratas Sprague-Dawley , Triglicéridos/análisis , Triglicéridos/sangre , Factor de Necrosis Tumoral alfa/análisis , Ácido Úrico/análisis , Ácido Úrico/sangreRESUMEN
BACKGROUND: Both ACE inhibitors and allopurinol have been shown to partially prevent metabolic syndrome induced by fructose. We tested the hypothesis that combined therapy might be more effective at blocking the metabolic syndrome induced with fructose. METHODS: Male Sprague-Dawley rats were fed a high fructose diet with or without allopurinol, captopril, or the combination for 20 weeks. A control group received a normal diet. All groups were pair-fed to assure equivalent caloric intake. RESULTS: Despite reduced energy intake, the fructose-fed rats developed features of metabolic syndrome including elevated blood pressure, abdominal obesity, hypertriglyceridemia, hyperuricemia and hyperinsulinemia. While both allopurinol and captopril alone tended to reduce features of the metabolic syndrome, the combined therapy was synergistic, with significant reduction in blood pressure, less accumulation of abdominal fat, an improvement in the dyslipidemia and a complete prevention of insulin resistance. CONCLUSION: A high fructose diet can induce metabolic syndrome even in the setting of caloric restriction. Captopril and allopurinol synergistically reduce features of the metabolic syndrome, especially hypertension, insulin resistance and dyslipidemia. Combination allopurinol and ACE inhibitor therapy might provide a superior means to prevent diabetes and cardiovascular disease.
Asunto(s)
Alopurinol/farmacología , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Antimetabolitos/farmacología , Captopril/farmacología , Síndrome Metabólico/metabolismo , Síndrome Metabólico/prevención & control , Alimentación Animal , Animales , Peso Corporal , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Quimioterapia Combinada , Metabolismo Energético , Fructosa/farmacología , Resistencia a la Insulina , Masculino , Síndrome Metabólico/inducido químicamente , Ratas , Ratas Sprague-DawleyRESUMEN
Uric acid (UA) is known to be a major biological antioxidant in plasma. However, there is a strong correlation between UA levels and cardiovascular risk. Recent studies suggest that in the intracellular environment, UA can become a prooxidant that causes endothelial dysfunction. For conducting detailed studies of UA's role in human pathogenesis, there is a critical need for a sensitive and specific method for the determination of intracellular UA levels. We therefore developed a simple, sensitive method for determination of trace amounts of intracellular UA, as well as comparatively large amounts of UA in plasma and urine (for the determination of extracellular concentrations of UA), based on liquid chromatography and tandem mass spectrometry (LC-MS/MS). UA was separated from interferences by HPLC and quantified by mass spectrometry in the negative ESI mode using single reaction monitoring (SRM). For the identification and quantification of UA, the parent ions selected were m/z 167.0, which corresponds to the urate anion, and m/z 169.0, which corresponds to the 1,3-(15)N(2)-UA anion. 1,3-(15)N(2)-UA is used as an internal standard to ensure accuracy of the measurement. After precipitation of proteins with 10% TCA solution, UA was subjected to LC-MS/MS analysis. The correlation coefficient was 0.9998-1.0000 based on the calibration curve. The intra- and inter-day precision (C.V. %) ranged from 0.01 to 3.07 and 0.01 to 3.68 for in vivo and in vitro systems, respectively. Recovery tests of added standards have been successfully performed and the values ranged from 90.10 to 103.59% and 98.74 to 106.12% for in vivo and in vitro analyses, respectively. This study demonstrates that intracellular levels of UA can be measured using LC-MS/MS with isotope labeled UA as an internal standard.
Asunto(s)
Cromatografía Liquida/métodos , Espectrometría de Masas en Tándem/métodos , Ácido Úrico/análisis , Línea Celular , Humanos , Sensibilidad y Especificidad , Ácido Úrico/sangre , Ácido Úrico/orinaAsunto(s)
Inflamación/fisiopatología , Enfermedades Renales/fisiopatología , Síndrome Metabólico/fisiopatología , Proteína C-Reactiva/metabolismo , Quimiocina CCL2/metabolismo , Progresión de la Enfermedad , Fructosa/metabolismo , Humanos , Inflamación/metabolismo , Síndrome Metabólico/metabolismo , Ácido Úrico/metabolismoRESUMEN
Increased consumption of fructose may play an important role in the epidemic of metabolic syndrome and may presage the development of diabetes, cardiovascular disease, and chronic kidney disease. Once in the cell, fructose is phosphorylated by ketohexokinase (KHK), leading to consumption of ATP, formation of AMP, and generation of uric acid through xanthine oxidoreductase (XOR). This study aimed to examine the direct effects of fructose in human kidney proximal tubular cells (HK-2) and whether they are mediated by the fructose metabolism via KHK. At a similar concentration to that observed in peripheral blood after a meal, fructose induced production of monocyte chemotactic protein 1 (MCP-1) and reactive oxygen species in HK-2 cells. Knockdown of KHK by stable transfection with small hairpin RNA demonstrated that these processes were KHK dependent. Several antioxidants, including specific inhibitors of NADPH oxidase and XOR, prevented MCP-1 secretion. We detected XOR mRNA in HK-2 cells and confirmed its activity by identifying uric acid by mass spectrometry. Fructose increased intracellular uric acid, and uric acid induced production of MCP-1 as well. In summary, postprandial concentrations of fructose stimulate redox- and urate-dependent inflammatory mediators in proximal tubular cells.
Asunto(s)
Fructoquinasas/metabolismo , Fructosa/metabolismo , Mediadores de Inflamación/metabolismo , Túbulos Renales Proximales/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Secuencia de Bases , Línea Celular , Quimiocina CCL2/biosíntesis , Cartilla de ADN/genética , Fructoquinasas/antagonistas & inhibidores , Fructoquinasas/genética , Fructosa/farmacología , Humanos , Túbulos Renales Proximales/efectos de los fármacos , Masculino , Síndrome Metabólico/etiología , Síndrome Metabólico/metabolismo , Oxidación-Reducción , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Insuficiencia Renal Crónica/etiología , Insuficiencia Renal Crónica/metabolismo , Transfección , Ácido Úrico/farmacología , Xantina Deshidrogenasa/genética , Xantina Deshidrogenasa/metabolismoRESUMEN
We propose that excessive fructose intake (>50 g/d) may be one of the underlying etiologies of metabolic syndrome and type 2 diabetes. The primary sources of fructose are sugar (sucrose) and high fructose corn syrup. First, fructose intake correlates closely with the rate of diabetes worldwide. Second, unlike other sugars, the ingestion of excessive fructose induces features of metabolic syndrome in both laboratory animals and humans. Third, fructose appears to mediate the metabolic syndrome in part by raising uric acid, and there are now extensive experimental and clinical data supporting uric acid in the pathogenesis of metabolic syndrome. Fourth, environmental and genetic considerations provide a potential explanation of why certain groups might be more susceptible to developing diabetes. Finally, we discuss the counterarguments associated with the hypothesis and a potential explanation for these findings. If diabetes might result from excessive intake of fructose, then simple public health measures could have a major impact on improving the overall health of our populace.
Asunto(s)
Diabetes Mellitus Tipo 2/inducido químicamente , Fructosa/efectos adversos , Síndrome Metabólico/inducido químicamente , Edulcorantes/efectos adversos , Ácido Úrico/metabolismo , Animales , Diabetes Mellitus Tipo 2/epidemiología , Diabetes Mellitus Tipo 2/metabolismo , Sacarosa en la Dieta/efectos adversos , Fructosa/metabolismo , Humanos , Síndrome Metabólico/genética , Síndrome Metabólico/metabolismo , Edulcorantes/metabolismoRESUMEN
Uric acid has historically been viewed as a purine metabolic waste product excreted by the kidney and gut that is relatively unimportant other than its penchant to crystallize in joints to cause the disease gout. In recent years, however, there has been the realization that uric acid is not biologically inert but may have a wide range of actions, including being both a pro- and anti-oxidant, a neurostimulant, and an inducer of inflammation and activator of the innate immune response. In this paper, we present the hypothesis that uric acid has a key role in the foraging response associated with starvation and fasting. We further suggest that there is a complex interplay between fructose, uric acid and vitamin C, with fructose and uric acid stimulating the foraging response and vitamin C countering this response. Finally, we suggest that the mutations in ascorbate synthesis and uricase that characterized early primate evolution were likely in response to the need to stimulate the foraging "survival" response and might have inadvertently had a role in accelerating the development of bipedal locomotion and intellectual development. Unfortunately, due to marked changes in the diet, resulting in dramatic increases in fructose- and purine-rich foods, these identical genotypic changes may be largely responsible for the epidemic of obesity, diabetes and cardiovascular disease in today's society.