High dietary salt decreases antioxidant defenses in the liver of fructose-fed insulin-resistant rats

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1 Aville online t ScienceDirect Journl of Nutritionl Biochemistry 24 (213) RESEARCH ARTICLES High dietry slt decreses ntioxidnt defenses in the liver of fructose-fed insulin-resistnt rts Wlesk Cludi Dorns, Wnderson Gerldo de Lim,, Rinldo Crdoso dos Sntos c, Joyce Ferreir d Cost Guerr, Melin Oliveir de Souz, Mís Silv, Loren Souz e Silv, Mirl Fiuz Diniz, Mrcelo Eustáquio Silv,c, Reserch in Biologicl Sciences - NUPEB, Federl University of Ouro Preto, Mins Geris, Brzil Deprtment of Biologicl Sciences, Institute of Exct nd Biologicl Sciences, Federl University of Ouro Preto, Mins Geris, Brzil c Deprtment of Foods, School of Nutrition, Federl University of Ouro Preto, Mins Geris, Brzil Received 23 Novemer 212; received in revised form 25 April 213; ccepted 14 June 213 Astrct In this study we investigted the hypothesis tht high-slt diet to hyperinsulinemic rts might impir ntioxidnt defense owing to its involvement in the ctivtion of sodium resorption to led to higher oxidtive stress. Rts were fed stndrd (CON), high-slt (HS), or high-fructose (HF) diet for 1 weeks fter which, 5% of the nimls elonging to the HF group were switched to regimen of high-fructose nd high-slt diet (HFS) for 1 more weeks, while the other groups were fed with their respective diets. Animls were then euthnized nd their lood nd liver were exmined. Fsting plsm glucose ws found to e significntly higher (pproximtely 5%) in fructose-fed rts thn in the control nd HS rts, wheres ft liver lso differed in these nimls, producing stetosis. Feeding fructose-fed rts with the high-slt diet triggered hyperinsulinemi nd lowered insulin sensitivity, which led to incresed levels of serum sodium compred to the HS group. This resulted in memrne perturtion, which in the presence of stetosis potentilly enhnced heptic lipid peroxidtion, therey decresing the level of ntioxidnt defenses, s shown y GSH/GSSG rtio (HFS rts, 7.98±2.1 versus CON rts, 13.2±6.1) nd superoxide dismutse (HFS rts, 2.1±.5 versus CON rts, 2.3±.1%), nd ctlse (HFS rts, 526.6±88.6 versus CON rts, 745.8±228.7 U/mg ptn) ctivities. Our results indicte tht consumption of slt-rich diet y insulin-resistnt rts my led to regultion of sodium resorption, worsening heptic lipid peroxidtion ssocited with impired ntioxidnt defenses. 213 Elsevier Inc. All rights reserved. Keywords: high-slt diet; fructose-fed rts; oxidtive stress; stetosis; ntioxidnt defenses 1. Introduction Incresing evidence suggests the involvement of oxidtive stress in insulin resistnce [1] nd hs generted high interest in the role of free rdicls in the mintennce of dequte levels of ntioxidnt defenses [2,3]. In type 2 dietes, significnt inverse correltion exists etween heptic ft lod nd the ntioxidnt defense system [4], which my prevent genertion of n dequte compenstory response for restortion of cellulr redox lnce [5]. In prticulr, Arevitions: AST, Asprtte minotrnsferse; ALT, Alnine minotrnsferse; CAT, Ctlse; GFR, Glomerulr filtrtion rte; GSH, Reduced glutthione; GSSG, Oxidized glutthione; GPx, Glutthione peroxidse (GPx); HOMA, Homeostsis model ssessment; ROS, Rective oxygen species; O 2, Superoxide nions; SOD, Superoxide dismutse; TBARS, Thiorituric cid rective sustnces. Corresponding uthor. Reserch in Biologicl Sciences - NUPEB, Federl University of Ouro Preto, Mins Geris, Brzil. E-mil ddresses: w.dorns@ig.com.r (W.C. Dorns), mesilv@enut.ufop.r (M.E. Silv). chnges hve een demonstrted in some components of the free rdicl defense system in different models [6 8]. We oserved tht the expression of genes encoding the ntioxidnt enzymes glutthione peroxidse (GPx), gmm-glutmylcysteine synthetse nd superoxide dismutse (SOD) decresed in the liver tissue of streptozotocin-induced dietic rts ecuse of incresed oxidtive stress [9] ssocited with overproduction of rective oxygen species (ROS)[1]. Under norml conditions, lmost ll of the produced superoxide nions (O 2 ) re converted to hydrogen peroxide (H 2 O 2 ) y the ction of SOD, which is further detoxified to wter y ctlse (CAT) or GPx [11]. However hyperglycemi induce the overproduction of O 2 [12] nd drmtic chnge in the oxidnt/ntioxidnt lnce hs een postulted to ply role in the pthogenesis of dietes. Incresing the sugr intke hs een reported to result in dyslipidemi, s indicted y elevted levels of serum triglycerides, cholesterol, nd low-density lipoproteins [13,14].These underlying metolic disturnces pper to induce insulin resistnce commonly oserved in high-fructose fed humn nd niml models [15] when fructose consumption cuses progressive liver disese stimulted /$ - see front mtter 213 Elsevier Inc. All rights reserved.

2 W.C. Dorns et l. / Journl of Nutritionl Biochemistry 24 (213) lipogenesis [16]. Furthermore, despite recent dvnces in elucidting the pthogenesis of relted conditions, studies hve shown tht presence of insulin resistnce nd compenstory hyperinsulinemi would led to sodium retention [17]. Therefore in the present study we exmined whether high-slt diet could impir ntioxidnt defenses in the liver of fructose-fed rts due ctivtion of enhnced renl sodium resorption potentiting oxidtive stress. 2. Mterils nd methods 2.1. Animl nd diets Forty-five mle 12-week-old Fischer rts, weighing pproximtely 3 g, were individully housed in temperture-nd humidity-controlled room under 12 h light/drk regimen. Initilly, the rts were rndomly ssigned to three experimentl groups (n=1 12) s follows: the control group (CON), fed with the AIN93M diet [18] nd wter; the high-slt group (HS), fed with the AIN93M diet plus 8% w/w NCl nd wter; nd the high-fructose group (HF), fed with the AIN93M diet nd 2% w/v fructose solution s drinking wter. After 1 weeks of tretment, the nimls elonging to the HF group were further divided into 2 groups: rts tht continued to e fed on the fructose solution (HF) nd rts tht were switched to high-fructose + high-slt regimen (HFS) for 1 more weeks. Detils of the experimentl diets re given in Tle 1. Food nd wter were provided d liitum nd their intke ws mesured. At the end of the experimentl period, the rts were fsted for 12 hours, nesthetized with isoflurne nd euthnized y totl lood collection from the rchil plexus. The lood ws centrifuged t 15g for 15 min. One liver loule from ech niml ws seprted for histologicl nlysis nd the rest ws frozen t 8 C until further nlysis. All the procedures were pproved y the Ethicl Committee for Animl Cre nd Use of the Federl University of Ouro Preto Biochemicl determintions Serum sprtte minotrnsferse (AST), lnine minotrnsferse (ALT) ctivities nd plsm glucose concentrtion were determined using commercil kits from Ltest Dignostic SA (Lgo Snt, MG, Brzil) # 18, 19 nd 84, respectively, y following the mnufcturer s instructions. ELISA ws utilized to quntify plsm insulin nd leptin levels using commercil kits from Ultr Sensitive Rt Insulin ELISA, Crystl Chem Downers Grove, IL, USA, nd Rt Leptin ELISA Kit, Linco Reserch, USA, (Ctlog #96 nd #94, respectively). The homeostsis model ssessment (HOMA), descried y Mtthews et l. [19] s mesure of insulin resistnce, ws clculted using the formul [insulin (μmol/ml) glucose (mm/l)/22.5]. Heptic ft ws extrcted using chloroform-methnol mixture (2:1, v/v) ccording to the method of Folch et l. [2] nd the totl lipids were quntified grvimetriclly y evporting the solvents in the extrct. Sodium concentrtions were mesured y flme photometry (Olidef model C-71 pprtus; São Pulo, Brzil) Antioxidnt defenses nd oxidtive stress Liver SOD ctivity ws mesured y the method of Mrklund nd Mrklund [21]. One unit of SOD ctivity ws defined s the mount of enzyme tht inhiited the rte of utoxidtion of pyrogllol y 5%, which ws determined t 57 nm. Ctlse ctivity ws mesured ccording to Aei [22] nd ws expressed in units per milligrm of protein using the extinction coefficient of.394 L/mmol/L/cm. The rte of H 2 O 2 decomposition ws followed y monitoring sorption t 24 nm in 5 mm phosphte uffer, ph 7., contining 5 mm H 2 O 2. Tissue protein content ws determined ccording to the method developed y Lowry et l. [23] using ovine serum lumin s the stndrd. The totl glutthione (GSH + GSSG) ws mesured fter precipittion of Tle 1 Diets composition Ingredient Composition (g/kg diet) CON HS HF 3 HFS 3 Strch Csein Sucrose Slt Cellulose Ft Minerls Vitmins Choline Energy content (kcl/kg) Minerl mixture for AIN93M; 2 Vitmin mixture for AIN93M; 3 D-Fructose (SynthLsynth, São Pulo, Brzil). proteins with n equl volume of 4% sulfoslicylic cid using the enzymtic method previously descried [24]. Oxidized glutthione (GSSG) ws determined fter derivtiztion of totl GSH with 2-vinylpiridine. Oxidtive stress index ws clculted from the GSH/GSSG rtio nd y lipid peroxidtion sttus through of levels of thiorituric cid rective sustnces (TBARS) s descried y Buege nd Aust [25] Liver histology After removl from ech niml, the livers were immeditely fixed in 1% uffered formldehyde, emedded in prffin, cut (4-μm thickness) nd mounted on glss slides. The sections were deprffinized in xylene, stined with hemtoxylin nd eosin (H&E) using the stndrd technique nd then exmined. Histologicl exmintion of the slides ws performed y using concentrted light microscope equipped with photogrphic digitl cmer (DM5; Leic) with softwre Qwin Plus. Scoring of the slides ws performed using semi-quntittive method reported y Brunt et l. [26]. Ft degenertion ws grded ccording to the percentge of ft-contining heptocytes. Grde of vesiculr stetosis ccording to the originl system involved 1 grdes, wheres in this system, stetosis ws grded from 4 sed on the percentge of heptocytes involved in the iopsy (, none; 1, 1%; 2, 1 33%; 3, 33 66% nd 4, N66%) Sttisticl nlysis Normlity of the smple distriution for ech continuous prmeter ws tested with the Kolmogorov Smirnov test. The significnce of ny differences in proportions of medins ws tested with Kruskl Wllis test nd in mens y one-wy nlysis of vrince (ANOVA), followed y Dunns nd Tukey tests, respectively. Correltion nlysis ws used to mesure the degree to which 2 vriles were relted. Significnce for ll mesures ws defined when P.5. GrphPd Prism version 5. for Windows (Sn Diego, CA, USA) ws used for sttisticl nlyses. 3. Results Notly, the men food intke ws significntly different mongst different dietry groups. In reltion to the control group, high dietry NCl resulted in lower cloric vlue leding to higher food intke in HS rts (P.1). On the other hnd, fructose supplementtion led to lower food intke (P.1) due to its energy content (Fig. 1A). Men vlues for liquid intke were significntly higher in the high-slt groups (P.1) thn in the CON nd HF groups (Fig. 1B). Higher fructose intke lowered the energetic demnd for solid food (P.1) in HFS rts thn in other groups (Fig. 1C). The high-slt diet led to incresed liquid intke, thus compelling HS nd HFS rts to drink pproximtely 4. nd 3. times the liquid volumes consumed y the CON nimls, respectively, resulting in the corresponding differences in the liquid clorie intke of HFS rts drinking the fructose solution (P.1) in reltion to HF rts (Fig. 1D). There were no significnt differences in the totl energy intke (Fig. 1E). Moreover, plsm leptin concentrtions in HF rts ws higher (P.1) compred to tht in other groups (Fig. 1F). The verge finl ody weight ws lower in HFS rts thn in HF nd CON rts (P.5). Reltive liver weights were higher (P.5) in fructose-fed rts (HF nd HFS) nd corresponding increse ws oserved in the liver lipid content nd plsm glucose in fructose-fed rts in reltion to CON (P.5) nd HS (P.1). The highest insulin concentrtion ws found in HFS rts (P.5) nd HOMA nlysis reveled tht this group hd significntly higher vlues thn the controls nimls (P.1), therey indicting tht the comintion of dietry fructose with NCl in HFS rts impired insulin response. For determintion of whether the dietry tretment induced liver injury, serum AST nd ALT ctivities were exmined, nd ALT ut not AST in the HF group were found to e significntly higher thn tht of the control group (P.5). Augmented ntriuretic response to high-slt diet significntly decresed serum sodium in HS rts compred to tht in other groups (P.5) nd prticulrly HFS showed sustntil increse in reltion to the HS group (Tle 2). Photomicrogrphs of heptic specimens stined with H&E re shown in Fig. 2A D, nd the scores of histologicl vriles re presented s medins in Fig. 2E. Mild or no heptic stetosis occurred in CON rts (Fig. 2A). HS rts did not show predominnt occurrence of stetosis, ut hyperemic vessels were oserved in the prenchym

3 218 W.C. Dorns et l. / Journl of Nutritionl Biochemistry 24 (213) A B C Food intke/dy (g) c d Liquid intke/dy (ml) c D E F Energy intke from liquid/dy (kcl) Totl energy intke/dy (kcl) c Energy intke from food/dy (kcl) Leptin (ng/ml) Fig. 1. Food, fluid, cloric intke nd serum leptin of experimentl rts. Symols represent the nimls in sctter plots to ech group. Different letters indicte significnt differences t P.5 y one-wy ANOVA followed y Tukey s test. Control diet (CON); high-slt diet (HS); high-fructose diet (HF); high-fructose nd slt (HFS) diet for 1 weeks fter previous tretment for period of 1 weeks with HF. cells (Fig. 2B). As expected, the high fructose tretment cused heptic lipid ccumultion, which ws evident in oth HF nd HFS rts (Fig. 2C nd 2D) with no signs of necroinflmmtion. Ft deposition in the HF group ws clssified s mcrovesiculr, while livers of HFS rts showed minly microvesiculr pttern with lesser grde of lipid ccumultion in reltion to HF rts. A sttisticlly significnt higher stetosis score (P.5) ws seen in livers from HF compred to CON nimls (Fig. 2E). Compred with the control group, the heptic levels of totl glutthione nd GSH were significntly lower in the HFS group (P.1), lthough no significnt difference ws oserved in the GSSG levels (Fig. 3). The GSH/GSSG rtio ws clculted to determine whether oxidtive stress hd een ugmented nd ws found to e lower in HFS rts thn in control rts (P.1). Assessment of lipid peroxidtion showed dmge in heptocytes, s verified through TBARS content of HFS in reltion to CON nimls (P.5). Furthermore, progressive functionl deficiency in ntioxidnt defenses ws lso evidenced in the HFS group, with significnt decrese in SOD nd CAT ctivities, (P.5; P.1, respectively) in reltion to the CON group. Additionlly, negtive correltion ws found etween TBARS Tle 2 Chrcteristics of experimentl rts Vrile Tretments Initil ody weight, g 295.± ± ± ±28.5 Finl ody weight, g 451.2± ± ± ±17.2 Asolute weight liver, g 12.6± ± ± ±1.3 Reltive liver weight, mg/g 27.8±3. 27.± ± ±1.3 Totl ft liver, mg/g 53.3± ± ± ±12.6 Plsm glucose, mmol/l 8.±.9 8.7±1.1 11,9± ±1.6 Plsm insulin, μmol/ml 2.9± ± ± ±25.8 HOMA-IR, score 7.6± ± ± ±16.4 Serum AST, U/L 56.8± ± ± ±16. Serum ALT, U/L 16.± ± ± ±7.2 Serum sodium, mmol/l 142.9± ± ± ±4.5 Vlues re expressed s mens±s.d. Different letters within the sme row indicte significnt differences t P.5 y one-wy ANOVA followed y Tukey s test. nd GSH/GSGG rtio (r=.4, P.1), SOD (r=.56, P.5) nd CAT ctivities (r=.5, P.2) (Tle 3). 4. Discussion The present study suggested tht the osmotic lod cused y ugmenting dietry NCl incresed plsm osmollity, stimulted thirst, nd enhnced liquid intke, s oserved y Mnesh et l. [27]. Studies in oth rts [28] nd humns [29] hve reported tht chronic fructose ingestion is ssocited with increse in plsm leptin levels nd leptin resistnce, which lters the informtion tht is relyed to the centrl nervous system on energy intke nd ody ft stores for regultion of food intke nd energy homeostsis [3]. Roglns et l. [31] reported tht this increse precedes oesity, suggesting tht the liver is key orgn in the development of metolic derngements induced y fructose consumption. Nevertheless, in our study this increse of leptin levels only in HF rts occurred in the sence of ugmented ody weight: HF nd control rts hd the sme ody weight, lthough leptin potently ctivtes cellulr fuel consumption y stimulting ftty cid oxidtion nd reducing lipogenesis [32]. On the other hnd, while fructose is more solule, sweeter, nd less glucogenic thn glucose or sucrose nd hs een recommended s replcement for these sugrs in the diets of dietic nd oese people, it is lipogenic nd usully cuses greter elevtion in triglyceride levels [3]. Significnt differences due to fructose dietinduced development of ftty liver were oserved in our experiments, s well s in other studies [33,34]. Fructose tretment-induced incresed ftty liver nd consequent heptomegly ws found in HF nd HFS nimls. Fructose-fed rts served s model for diet-induced insulin resistnce, suggesting tht pthophysiologicl mechnisms nd lipid retention in heptocytes (heptic stetosis) ws n importnt erly sign in the development of metolic normlities spectrum. Our model ws thus proved pproprite ecuse it reproduced histologiclly detectle stetosis resulting primrily from the deposition of ft in heptocytes of fructose-fed rts. However, our results demonstrted tht HFS rts consumed more fructose thn HF rts did, ut displyed lower degree of stetosis

4 W.C. Dorns et l. / Journl of Nutritionl Biochemistry 24 (213) Fig. 2. (A-D) Representtive photomicrogrphs H&E stining of liver sections of experimentl rts. Heptocytes normlities were not oserved in the CON group (A); note hyperemic vessels (rrowhed) nd norml prenchym ppernce in the HS group (B); spect of mcrovesiculr stetosis in the HF group (C). Note heptocytes with lrge negtive imge in the cytoplsm with nucleus displced into the periphery of the cell, lrge ft gloule in most cses (white rrow); spect of microvesiculr stetosis in the HFS group (D). Note cells with smll cytoplsmic vcuoles without displced nucleus (lck rrow) mgnifiction 44. Grde of liver stetosis of rts in experimentl groups (E). Vlues (medinne, n=8 11) (*P.5 vs. control rts). development. This my indicte tht the high-slt diet model, s in other studies, ttenuted gin in ody weight [35,36], which cn led to lower ccumultion of ft in heptocytes, s supported y our dt. Determintion of liver function prmeters lso reveled liver dysfunction due to fructose-feeding nd heptic dmge in fructose-fed rts. Fructose feeding ws found to significntly enhnce serum ALT ctivity, indicting considerle heptocellulr injury in HF rts. ALT hs een routinely mesured nd is considered surrogte mrker of liver ft ccumultion [37]. Injury to the heptocyte leds to disruption of the plsm memrne nd lekge of the enzyme to the extrcellulr fluid. Thus it cn e detected t norml levels in the serum nd this condition my e cuse of heptocyte deth. Rts re n excellent niml model to study the effects of fructose intke ecuse their fructose metolism closely resemles tht of humns [38] nd studies hve shown tht fructose induces hyperglycemi nd hyperinsulinemi [39,4]. The results demonstrted tht fructose dministrtion produces insulin resistnce in HFS rts consistently with previous studies crried out using different techniques to ssess insulin resistnce [4,41]. Insulin resistnce in fructose-fed rts hs een ttriuted to low level of insulin-stimulted glucose oxidtion due to modifictions in the post-receptor cscde of insulin ction [42]. Thus elevted plsm insulin concentrtions enhnce the synthesis of very-lowdensity lipoprotein, nd this my induce increse in ftty liver s oserved y us in HF nd HFS rts with decresed in response of HFS rts to glucose utiliztion, feturing lower insulin ction s indicted y higher HOMA vlues. In contrst, Nishimoto et l. [43] used fructose-fed insulin-resistnt rts with low or high-sodium diet nd found no significnt differences in plsm glucose or

5 22 W.C. Dorns et l. / Journl of Nutritionl Biochemistry 24 (213) nmol/ml Totl Gluthtione nmol/ml GSH nmol/ml GSSG GSH/GSSG U/mg de ptn TBARS. % inhiition SOD ctivity U/mg protein Ctlse ctivity. Fig. 3. Antioxidnt defenses nd oxidtive stress in the liver of experimentl rts. Different letters indicte significnt differences t P.5 y one-wy ANOVA followed y Tukey s test. Tle 3 Regression nlyses etween TBARS (U/mg ptn) nd ntioxidnt defenses of experimentl rts Vrile r P Glutthione totl, nmol/ml GSH, nmol/ml GSSG, nmol/ml GSH/GSSG, rtio SOD, % inhiition.56.5 Ctlse, U/mg ptn.5.24 insulin mong their groups, lthough they used lower dosge for oth slt nd fructose thn in our study. This discrepncy mong studies my e etter explined y the fct tht diet-induced modifictions in metolic nd hormonl profile re proly dependent on the durtion of diet tretment, on the mount of crohydrte nd slt in the diet esides interctions with other nutrients [44 46]. Our model ws sed on the dministrtion of high-fructose diet inducing insulin resistnce tht resemles the so-clled fst food tht is highly populr nowdys. This type of diet constitutes n importnt, typiclly westernized lifestyle, which includes consumption of processed foods tht re high in slt nd sugr. Fructose hs een rodly used in metolic studies, lthough there is no dt concerning liver normlities ssocited with high-slt regimens. In this study, the high-slt diet led to greter urinry excretion of sodium, ut hyperinsulinemi showed in HFS rts hs influence on sodium retention s demonstrted y higher serum sodium concentrtions in HFS when compred with the HS group. This ntintriuretic effect my e opposed y concomitnt decrese in proximl tuulr sodium resorption [47] or n increse in glomerulr filtrtion rte (GFR) [48]. Chronic hyperinsulinemi increses GFR in norml dogs [48], ut not in oese insulin-resistnt dogs [49] which suggest tht insulin could increse GFR, nd thus the filtered sodium lod, only in insulin-sensitive sujects. Insulin hs een known to enhnce sodium resorption in the proximl tuule nd stimultes not only sodium ut lso volume sorption in the rit proximl convoluted tuule. Thus, from these stimultory effects, it is cler tht insulin cts on proximl tuules to resor sodium filtered from the glomeruli nd yet, importnt regultory mechnisms exist susequently in the Henle s loop, distl tuule nd connecting tuule [17]. Besides, Vsdev et l. [5] showed tht intrcellulr sodium levels increse cytosolic free clcium, which cn increse oxidtive stress nd this condition chnges the memrne components compromising its integrity [51] ecuse incresed ROS genertion hs een shown to induce cell memrne lipid peroxidtion [52]. Therefore, it hs een suggested tht lipid ccumultion in the liver mkes heptocytes more sensitive to oxidtive stress [53], which in this study, potentilly ctivted lipid peroxidtion, s demonstrted y incresed TBARS in HFS rts. The oserved stetosis could hve ffected lipid composition nd fluidity of mitochondril memrnes, which incresed oxidtive stress in the liver. In ddition, chnges in liver glutthione redox sttus were monitored in this work y recording the GSH/GSSG rtio, ecuse severe oxidtive stress my deplete cellulr GSH, nd glutthione ply n importnt function in detoxifiction of free rdicls [54].Glutthione is the mjor intrcellulr non-protein ntioxidnt, nd GPx converts H 2 O 2 to H 2 O y oxidizing glutthione to glutthione disulfide [2]. We oserved decrese in liver GSH levels in HFS rts s well s in

6 W.C. Dorns et l. / Journl of Nutritionl Biochemistry 24 (213) the GSH/GSSG rtio, which could e consequence of dpttion of the ftty liver, s suggested y the negtive correltion of TBARS with GSH/GSSG rtio. It my represent consequence of the higher prooxidnt sttus developed in HFS rts, which is likely responsile for the high consumption of cellulr nd circulting ntioxidnts. Moreover, decresed SOD nd CAT ctivities were oserved in HFS rts tht could prticipte in heptic vulnerility to oxidtive stress. Decresed feedck regultory mechnisms involving these ntioxidnt enzymes, prevents the restortion to norml enzyme level. Thus, the susceptiility of the tissue to oxidtive stress ws dependent on the ltertion in lipid composition nd tissue dmge. SOD ply key role in cell protection ginst the deleterious effects of O 2, nd ctlse prevents dmge y rpidly converting H 2 O 2 to wter [55]. There re possile pthwys y which cellulr metolism in this model my e ltered, which in turn my ccelerte oxidtive stress. The incresed oxidtive stress could e due to production of oxygen free rdicls [3] nd H 2 O 2 resulting from SOD ctivity, which cn generte hydroxyl rdicls through the Fenton rection, nd thus, ROS cn themselves reduce the ctivity of ntioxidnt enzymes such s CAT nd GPx [56]. Consequently, lowering of these ctivities is suggestive of reduced scvenging potentil in the insulin-resistnt rts on high-slt diet. Another possiility is tht ccumultion of dvnced glyction products resulted in the production of free rdicls [57]. Therefore the high-slt diet might e regrded s n instigtor tht reduces ntioxidnt defenses, worsening insulin sensiility in the erly stge of experimentl dietes in rts; however, further reserch is needed to define the interdependencies/interctions mong fructose, slt nd oxidtive stress more clerly. In conclusion, the high-slt diet reduced heptic ntioxidnt defenses in the fructose-fed rts, providing evidence to support the ide tht incresed oxidtive stress is involved in memrne perturtion through importnt regultory mechnisms. Thus, our findings suggest tht hyperinsulinemi ply role in sodium retention nd it is therefore possile tht the deleterious effects of slt overlod on insulin- resistnt sujects my e ttriuted, in prt, to the development of sustntil pro-oxidnt condition tht impirs ntioxidnt defenses. Acknowledgments The uthors would like to thnk LAPAC (Pilot Lortory of Clinicl Anlysis, School of Phrmcy, Federl University of Ouro Preto) for technicl ssistnce. References [1] Evns JL, Goldfine ID, Mddux BA, Grodsky GM. Are oxidtive stress ctivted signling pthwys meditors of insulin resistnce nd et cell dysfunction? Dietes 23;52:1 8. [2] Sies H. Strtegies of ntioxidnt defense. Eur J Biochem 1993;215: [3] Myne ST. 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