Evaluation of the Effects of Fructose on Oxidative Stressand Inflammatory Parameters in Rat Brain

Abigail Lopes & Thais Ceresér Vilela & Luciane Taschetto & Franciele Vuolo &

Fabricia Petronilho & Felipe Dal-Pizzol & Emilio Luiz Streck & Gustavo Costa Ferreira &

Patrícia Fernanda Schuck

Received: 10 January 2014 /Accepted: 11 March 2014# Springer Science+Business Media New York 2014

Abstract Hereditary fructose intolerance is an autosomal reces-sive disorder characterized by the accumulation of fructose intissues and biological fluids of patients. The disease results froma deficiency of aldolase B, responsible for metabolizing fructosein the liver, kidney, and small intestine.We investigated the effectof acute fructose administration on oxidative stress andneuroinflammatory parameters in the cerebral cortex of 30-day-old Wistar rats. Animals received subcutaneous injection ofsodium chloride (0.9 %) (control group) or fructose solution(5 μmol/g) (fructose group). One hour later, the animals wereeuthanized and the cerebral cortex was isolated. Oxidativestress (levels of thiobarbituric acid-reactive substances

(TBA-RS), carbonyl content, nitrate and nitrite levels, 2′,7′-dihydrodichlorofluorescein (DCFH) oxidation, glutathione(GSH) levels, as well as the activities of catalase (CAT) andsuperoxide dismutase (SOD)) and neuroinflammatory parame-ters (TNF-α, IL-1β, and IL-6 levels and myeloperoxidase(MPO) activity) were investigated. Acute fructose administrationincreased levels of TBA-RS and carbonyl content, indicatinglipid peroxidation and protein damage. Furthermore, SOD activ-ity increased, whereas CATactivity was decreased. The levels ofGSH, nitrate, and nitrite andDCFHoxidationwere not altered byacute fructose administration. Finally, cytokines IL-1β, IL-6, andTNF-α levels, as well as MPO activity, were not altered. Ourpresent data indicate that fructose provokes oxidative stress in thecerebral cortex, which induces oxidation of lipids and proteinsand changes of CAT and SOD activities. It seems thereforereasonable to propose that antioxidants may serve as an adjuvanttherapy to diets or to other pharmacological agents used for thesepatients, to avoid oxidative damage to the brain.

Keywords Brain . Fructosemia . Oxidative damage .

Neuroinflammation . Hereditary fructose intolerance

Introduction

Hereditary fructose intolerance (HFI, OMIM 229600) is anautosomal-recessive disorder with an average incidence of1:40,000 newborns [1]. The disease arises from a deficiencyof aldolase B (EC 4.1.2.13), an enzyme responsible for me-tabolizing fructose in the liver, kidney, and small intestine, andit is biochemically characterized by the accumulation of fruc-tose and fructose-1-phosphate in biological fluids and tissuesof affected patients [2, 3]. The main clinical and biochemicalpresentation of patients includes hypoglycemia, vomiting,jaundice, liver failure, hepatomegaly, metabolic acidosis, sei-zures, coma, and eventually death [3–8]. Furthermore,

A. Lopes : T. C. Vilela : L. Taschetto : P. F. Schuck (*)Laboratório de Erros Inatos do Metabolismo, Programa dePós-Graduação em Ciências da Saúde, Unidade Acadêmica deCiências da Saúde, Universidade do Extremo Sul Catarinense,Avenida Universitária, 1105, Bloco S, Sala 6, 88806-000 Criciúma,SC, Brazile-mail: patischuck@gmail.com

F. Vuolo : F. Dal-PizzolLaboratório de Fisiopatologia Experimental, Programa dePós-Graduação em Ciências da Saúde, Unidade Acadêmica deCiências da Saúde, Universidade do Extremo Sul Catarinense,Criciúma, SC, Brazil

F. PetronilhoLaboratório de Fisiopatologia Clínica e Experimental, Programa dePós-Graduação em Ciências da Saúde, Universidade do Sul de SantaCatarina, Tubarão, SC, Brazil

E. L. StreckLaboratório de Bioenergética, Programa de Pós-Graduação emCiências da Saúde, Unidade Acadêmica de Ciências da Saúde,Universidade do Extremo Sul Catarinense, Criciúma, SC, Brazil

G. C. FerreiraUniversidade Federal do Rio de Janeiro, Instituto de Biofísica CarlosChagas Filho, Avenida Carlos Chagas Filho, 373, Bloco G, CidadeUniversitária, Ilha do Fundão 21941-902, RJ, Brazil

Mol NeurobiolDOI 10.1007/s12035-014-8676-y

neurological impairment may also be observed in HFI pa-tients, particularly during the acute phase of the disease [9].

Fructose enters hepatocytes and other cells (including tu-bular cells, adipocytes, and intestinal epithelial cells), where itis phosphorylated to fructose-1-phosphate by fructokinasewith the consumption of ATP [10]. A depletion of intracellularphosphate was observed in HFI patients after fructose admin-istration [11], which can inhibit the action of hepatic glycogenphosphorylase and, consequently, glycogenolysis [12]. Thatwould lead to hypoglycemia, which is considered the mostprominent symptom of IHF [13]. Moreover, the retroactiveinhibition of fructokinase by fructose-1-phosphate results inreduced uptake of fructose by the liver, leading to increasedfructose levels in the plasma of patients [13].

Studies have demonstrated that high-fructose intake caninduce weight gain, development of insulin resistance, andmetabolic syndrome [10, 14–19]. However, little is knownregarding the effects of fructose in the brain. Some early studiessuggested that fructose could not penetrate the blood–brainbarrier in significant amounts [20, 21]. In contrast, accumulat-ing evidence indicates that neural cells are able to metabolizefructose [22]. In this context, fructose intake was shown todisrupt plasma membranes of rat neurons impairing neuronalfunction [19]. It has been also demonstrated that a high-fructosediet has detrimental consequences for synaptic plasticity; im-pairs the cognitive function, memory, dendritic spine density,and neurogenesis in the hippocampus; as well as induces neu-ronal loss in the nucleus tractus solitaries of rats [23–28].

Therefore, considering that fructose is the main accumulatingmetabolite in HFI and that high-fructose diet can lead to oxidativestress in the heart and liver of rats [29–31], in the present work,weevaluated different oxidative stress parameters, namely thiobarbi-turic acid-reactive substances (TBA-RS), carbonyl content, nitriteand nitrate content, 2′,7′-dihydrodichlorofluorescein (DCFH) ox-idation, and reduced glutathione (GSH) levels, as well as theactivities of the antioxidant enzymes catalase (CAT) and super-oxide dismutase (SOD) in homogenates from the cerebral cortexof young rats in the hope to contribute to a better understanding ofpathomechanisms found in HFI patients. We also investigatedwhether inflammatory response is present following fructoseadministration, by evaluating TNF-α, IL-1β, and IL-6 levelsand myeloperoxidase (MPO) activity. To our knowledge, this isthe first study evaluating the effect of acute high fructose concen-trations on cell redox and inflammation in the brain of rats.

Methods

Reagents

All chemicals were purchased from Sigma-Aldrich (St. Louis,MO, USA), unless stated in the text. Fructose was dissolvedon the day of the experiments with its pH adjusted to 7.4.

Animals

For the experiments, a total of 36 30-day-old male Wistar ratsobtained from the Central Animal House of Universidade doExtremo Sul Catarinense were used. Rats were kept with damsuntil weaning at 21 days of age. The animals had free access towater and to a standard commercial chow and were main-tained on a 12:12-h light/dark cycle in an air-conditionedconstant temperature (22±1 °C) colony room. The Guide forthe Care and Use of Laboratory Animals (National ResearchCouncil, 2011) and the EC Directive 86/609/EEC werefollowed in all experiments. All efforts were made to mini-mize the number of animals used and their suffering. Thestudy was approved by the Local Ethical Committee on An-imal Use for Research under the protocol number 51/2012.

In Vivo Experiments

The animals were divided into two groups (six animals pergroup): control group, which received a single subcutaneousinjection of saline solution (0.9 %), and fructose group, whichreceived a single subcutaneous injection of 5 μmol/g of fruc-tose (body weight 0.9 mg/g) [32]. One hour after the admin-istration, the animals were euthanized by decapitation withoutanesthesia, and the brains were rapidly excised on a Petri dishplaced on ice and the cerebral cortex was isolated. The struc-ture was weighed and homogenized in 10 volumes (1:10,w/v)of 20 mM sodium phosphate buffer, pH 7.4 containing140 mM KCl. The homogenate was centrifuged at 750×gfor 10 min at 4 °C to discard nuclei and cell debris. Aliquotswere taken to measure the values of oxidative stress parame-ters (levels of TBA-RS, carbonyl content, nitrate and nitritelevels, DCFH oxidation, GSH levels, as well as the activitiesof CAT and SOD) and inflammatory parameters (TNF-α, IL-1β and IL-6 levels and MPO activity). Blood samples werealso collected in order to obtain serum aliquots for the deter-mination of fructose concentrations.

Determination of Fructose Serum Concentrations

The determination of fructose serum concentrations was per-formed by a commercial kit. Levels of fructose are expressedas milligrams per milliliter and micromoles per milliliter.

Levels of TBA-RS

TBA-RS were determined according to the method ofEsterbauer and Cheeseman [33]. A calibration curve wasperformed using 1,1,3,3-tetramethoxypropane, and eachcurve point was subjected to the same treatment as superna-tants. Values of TBA-RS were calculated as nanomoles ofTBA-RS per milligram of protein.

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Determination of Protein Carbonyl Formation Content

Protein carbonyl content formation, a marker of oxidizedproteins, was measured spectrophotometrically according toReznick and Packer [34]. The results were calculated asnanomoles of carbonyls groups per milligram of protein, usingthe extinction coefficient of 22,000×106 nmol mL−1 for ali-phatic hydrazones.

Nitrate and Nitrite Determination

Nitrate and nitrite concentrations were determined accordingto Miranda et al. [35], using the Griess reagent. A calibrationcurve was performed using sodium nitrate, and each curvepoint was subjected to the same treatments as supernatants,and the concentrations were calculated nanomoles per milli-gram of protein.

DCFH Oxidation

Reactive species production was assessed according to Lebelet al. [36] by using 2′,7′-dihydrodichlorofluorescein diacetate(DCF-DA). A calibration curve was performed with standardDCF (0.25–10 mM), and the levels of reactive species werecalculated as picomoles of DCF formed per milligram ofprotein.

GSH Concentrations

GSH concentrations were measured according to Browne andArmstrong [37]. Calibration curve was prepared with standardGSH (0.01–1 mM) and the concentrations were calculated asnanomoles per milligram of protein.

CATActivity

CAT (EC 1.11.1.6) activity was assayed according to Aebi[38] by measuring the H2O2 absorbance decrease at 240 nm.The specific activity was expressed as nanomoles per minuteper milligram of protein.

SOD Activity

SOD (EC 1.15.1.1) activity was determined according toBannister and Calabrese [39] using a spectrophotometric as-say based on superoxide-dependent oxidation of epinephrineto adrenochrome at 32 °C. SOD specific activity is represent-ed as nanomoles per minute per milligram of protein.

Inflammatory Parameters

Determination of the dosage of TNF-α, IL-1β, and IL-6 wasperformed using enzyme-linked immunosorbent assay

(ELISA) by commercial kits. Levels of cytokines areexpressed as picograms per milliliter.

MPO (EC 1.11.2.2) activity was determined according toLiaudet et al. [40]. Results are expressed as milliunits permilligram of protein.

Statistical Analysis

Results are presented as mean ± standard error of the mean.Data were analyzed using Student’s t test for independentsamples. Differences between groups were rated significantat p<0.05. All analyses were carried out in an IBM-compatible PC computer using the Statistical Package forthe Social Sciences (SPSS) software for Windows 20.0.

Results

The serum fructose concentrations in the animals submitted tothe model of fructosemia described by Monteiro et al. [32]were first determined. It was found that these concentrationsreached approximately 3.05 μmol/mL (0.55 mg/mL, n=3animals).

Our next step was to evaluate whether acute fructose ad-ministration elicits oxidative damage to the biological com-pounds in the cerebral cortex (Fig. 1). It is observed in Fig. 1that animals receiving fructose acutely showed increased

Fig. 1 Effect of acute administration of fructose on the levels of TBA-RS(a) and carbonyl content (b) in the cerebral cortex of 30-day-old rats.Values are means ± standard deviation for six independent experimentsperformed in duplicate or triplicate and are expressed as nanomoles permilligram of protein. *p<0.05, **p<0.01 compared to control group(Student’s t test for independent samples)

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levels of TBA-RS [p=0.013] (Fig. 1a) and carbonyl content[p=0.021] (Fig. 1b) in the cerebral cortex.

Next, we assessed the influence of acute fructose adminis-tration on DCF production and observed that this carbohy-drate did not influence DCFH oxidation (Table 1). We thenevaluated the effects of acute fructose administration on ni-trate and nitrite production, in order to verify if this metabolitecould induce reactive nitrogen species generation, and thesame lack of interference was observed.

Our next step was to investigate the in vivo effects of acutefructose administration on important cellular antioxidant de-fenses (Fig. 2). It was observed that the levels of GSH, themain nonenzymatic antioxidant cellular defense, were notaltered by the administration of this carbohydrate (Fig. 2a).Regarding the enzymatic antioxidant defenses, it was verifiedthat acute fructose administration inhibited CAT activity [p=0.0449] (Fig. 2b), whereas SOD activity was increased by thistreatment [p=0.0165] in the cerebral cortex (Fig. 2c).

Finally, aiming to verify whether rats receiving fructoseacutely develop neuroinflammation, the levels of pro-inflammatory mediators (TNF-α, IL-1β, IL-6) and markerof neutrophil activation, MPO activity, in rat (Fig. 3) werealso investigated. It was observed that the levels of TNF-α,IL-1β, and IL-6, as well as MPO activity, were not altered inthe fructose group, as compared to the control group.

Discussion

Liver, kidney, and small intestine are the main sites of fructosemetabolism [41]. Toxicity of fructose, after intravenous ad-ministration at high doses, results in hyperuricemia,hyperlactatemia, and ultrastructural alterations in liver andintestinal cells [42–44]. Furthermore, animal studies haveshown that a high-fructose diet induces well-characterizedmetabolic syndrome resulting in hyperinsulinemia, insulinresistance, glucose tolerance reduction, hypertension,hypertriglyceridemia, and decreased high-density lipoproteincholesterol [45, 46].

Despite the number of studies investigating the peripheraleffects of fructose, the understanding of brain complications

related to this monosaccharide is still poor. Since oxidativestress and neuroinflammation are involved in the pathophys-iology of common neurodegenerative disorders [47, 48] andof some inborn errors of metabolism [49, 50] and that high-fructose diet can lead to oxidative stress in peripheral tissues[29–31], in the present study, we evaluated the in vivo influ-ence of acute fructose administration on several parameters ofoxidative stress, as well as neuroinflammation, in the cerebralcortex of young rats.

In this work, our first step was to reproduce the animalmodel described by Monteiro et al. [32], used for fructosemiastudies. We measured serum fructose concentrations in this ratmodel and found that this sugar reached a level as high as0.55 mg/mL (control values≈0.14 mg/mL). It is important tomention that brain fructose concentrations are approximatelyseven times lower than plasma levels [20]. These concentra-tions are in line with the fructose levels of patients [51],indicating that this animal model is suitable for preclinicalstudies of fructosemia effects.

Klein et al. [20] evaluated the distribution of intravenouslyinjected fructose between blood and brain in cats and observedthat the highest level of brain fructose was obtained about 1 hafter injection of this sugar and that appreciable levels wereobtained in some experiments about 30 min after injection.We then evaluate biochemical parameters 1 h after the fructoseadministration.

We chose 30-day-old rats in this study since this age in ratscorresponds to 2.5 years of life in humans [52], a life periodthat usually comprises the onset of clinical symptoms inpatients affected by fructosemia due to a higher content offructose in their diet. Herein, we demonstrate that after acutefructose administration, the levels of TBA-RS and carbonylcontent were increased. TBA-RS reflect the content ofmalondialdehyde, the most abundant individual aldehyderesulting from lipid breakdown due to lipid peroxidationprocess [53]. On the other hand, the level of carbonyl groupsof proteins is widely used as a marker of oxidative proteindamage [53]. Thus, our data indicate that acute fructose ad-ministration elicits oxidative damage to lipids and proteins inthe cerebral cortex of rats.

It was also observed in this study that the concentration offructose used was not able to increase the DCF levels. DCFHoxidation is widely used as an indicator of the formation ofreactive species, particularly H2O2, RO·, OH·, HOCl, andONOO− [54]. However, there are limitations to the interpre-tation of DCF production as a specific marker for quantitativeintracellular H2O2 formation. For example, the H2O2-depen-dent oxidation of DCFH to DCF occurs slowly, if at all, in theabsence of ferrous iron [55]. DCF formation is greatly en-hanced in the presence of heme-containing substances, suchas hematin, peroxidases, or cytochrome c [55, 56]. Regardingthe effects of fructose on reactive nitrogen species generation,it was observed that nitrate and nitrite production was

Table 1 Effects of the acute fructose administration on DCFH oxidationand nitrate and nitrite production

DCFH oxidation Nitrate and nitrite content

Control 170.56±20.29 15.05±1.87

Fructose 159.71±17.08 12.54±1.12

The experiments were performed in triplicate, and data represent mean ±standard deviation of the mean (n=6 animals/group). Any differencebetween groups (Student’s t test for independent samples) was not ob-served. DCFH oxidation is in picomoles per minute per milligram ofprotein; nitrate and nitrite content is nanomoles per milligram of protein

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unaffected by acute fructose administration, therefore indicat-ing that RNS production was not altered by fructose.

Although the above results show oxidant activity, fructosedid not affect GSH concentrations in the brain. Since thisparameter is used to evaluate a tissue’s capacity to prevent orrespond to damage associated to reactive species [57]. Inter-estingly, similar findings were previously reported by Kundeet al. [31] in the liver of mice receiving a high-fructose diet,which showed increased levels of TBA-RS without howeveraltering GSH levels.

On the other hand, we observed that CAT activity wasinhibited in the cerebral cortex of rats receiving fructoseacutely, while SOD activity was increased. CAT is a ferricheme protein that directly catalyzes the decomposition ofhydrogen peroxide. It has been suggested that CAT activitymay be decreased secondarily to CAT saturation during thebreakdown of free radicals and hydrogen peroxide or theinhibition of CAT by these radicals [58]. SOD removes the

anion superoxide by accelerating the rate of its dismutation tohydrogen peroxide [53]. In this context, elevated SOD/CATratio may result in an increase of H2O2 concentration orincreased rate of H2O2 production and may lead to lipid andprotein oxidation, resulting in increased neuronal damage[59]. This finding corroborates the increased levels of TBA-RS and carbonyl content, however, without affecting DCFformation.

Considering that fructose may activate the inflammatorycascade in hepatocytes of mice [60] and hamsters [61] byincreasing the concentrations of TNF-α and Jun amino termi-nal kinase (JNK) [18], we assessed whether neuroinflamma-tion could also play a role in the brain following fructoseexposure. We did not observe alterations in the levels ofinflammatory markers (TNF-α, IL-1β, and IL-6 levels andMPO activity) in the cerebral cortex of rats receiving fructoseacutely, as compared to control animals. Exposure of rathepatocytes to fructose for 2 to 4 h caused the activation of

Fig. 2 Effect of acuteadministration of fructose onGSH levels (a), catalase activity(b), and superoxide dismutaseactivity (c) in the cerebral cortexof 30-day-old rats. Values aremeans ± standard deviation for sixindependent experimentsperformed in duplicate and areexpressed as nanomoles permilligram of protein (GSH levels)and nanomoles per minute permilligram of protein (catalase andsuperoxide dismutase activities).*p<0.05 compared to controlgroup (Student’s t test forindependent samples)

Fig. 3 Effect of acuteadministration of fructose on IL-1β (a), IL-6 (b), and TNF-α (c)levels and myeloperoxidaseactivity (d) in the cerebral cortexof 30-day-old rats. Values aremeans ± standard deviation for sixindependent experimentsperformed in duplicate and areexpressed as picomoles permilliliter (a–c) and milliunits permilligram of protein (d).Difference between groups wasnot detected (Student’s t test forindependent samples)

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JNK [18]. Then, we suggest that only 1 h after administrationof fructose was not sufficient to activate inflammatory path-ways and further studies should be undertaken to investigatethe effects of sustained exposition to fructose in the brain.

Since oxidative stress results from an imbalance betweenthe total antioxidant defense of the tissue and the reactivespecies generated, our present data strongly indicate that fruc-tose provokes oxidative stress in vivo in the cerebral cortex,which induces oxidation of lipids and proteins and changes inthe CAT and SOD activity. It should be emphasized that thebrain has low antioxidant defenses as compared to othertissues, rendering this tissue more vulnerable to oxidativedamage [62].

At present, we cannot establish whether our data have apathophysiological significance, since brain concentrations offructose in HFI patients are unknown. It should however benoted that the significant alterations of the oxidative stressparameters elicited by fructose occurred at high micromolarlevels (5 μmol/g), and during metabolic crises in patients, theconcentrations of the accumulating metabolites dramaticallyincrease [8].

Conclusion

In conclusion, the present data indicate that oxidative stress isinduced by fructose in the brain of young rats. It seemstherefore reasonable to propose that antioxidants may serveas an adjuvant therapy to specific diets or to other pharmaco-logical agents used for these patients, especially during crises,to avoid oxidative damage to the brain.

Acknowledgments This study was supported by grants from ConselhoNacional de Pesquisa e Desenvolvimento (CNPq), Universidade doExtremo Sul Catarinense (UNESC), and Núcleo de Excelência emNeurociências Aplicadas de Santa Catarina (NENASC project,PRONEX). We thank Soliany Grassi Maravai for the determination offructose levels in the serum of the animals.

Conflict of Interest The authors declare that they have no conflict ofinterest.

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