Available online at www.sciencedirect.com

ScienceDirect

Journal of Nutritional Biochemistry 24 (2013) 2119–2126

Grape skin extract protects against programmed changes in the adult ratoffspring caused by maternal high-fat diet during lactation☆,☆☆

Angela C. Resendea,⁎, Andréa F. Emilianoa, Viviane S.C. Cordeiroa, Graziele F. de Bema,Lenize C.R.M. de Cavalhoa, Paola Raquel B. de Oliveiraa, Miguel L. Netoa, Cristiane A. Costaa,

Gilson T. Boaventurab, Roberto S. de Mouraa

aDepartment of Pharmacology, Institute of Biology, Rio de Janeiro State University, Rio de Janeiro, BrazilbDepartment of Experimental Nutrition, Federal Fluminense University, Rio de Janeiro, Brazil

Received 17 December 2012; received in revised form 12 July 2013; accepted 5 August 2013

Abstract

Maternal overnutrition during suckling period is associated with increased risk of metabolic disorders in the offspring. We aimed to assess the effect of Vitisvinifera L. grape skin extract (ACH09) on cardiovascular and metabolic disorders in adult male offspring of rats fed a high-fat (HF) diet during lactation. Fourgroups of female rats were fed: control diet (7% fat), ACH09 (7% fat plus 200 mg kg−1 d−1 ACH09 orally), HF (24% fat), and HF+ACH09 (24% fat plus 200 mg kg−1

d−1 ACH09 orally) during lactation. After weaning, all male offspring were fed a control diet and sacrificed at 90 or 180 days old. Systolic blood pressure wasincreased in adult offspring of HF-fed dams and ACH09 prevented the hypertension. Increased adiposity, plasma triglyceride, glucose levels and insulin resistancewere observed in offspring from both ages, and those changes were reversed by ACH09. Expression of insulin cascade proteins IRS-1, AKT and GLUT4 in thesoleus muscle was reduced in the HF group of both ages and increased by ACH09. The plasma oxidative damage assessed by malondialdehyde levels wasincreased, and nitrite levels decreased in the HF group of both ages, which were reversed by ACH09. In addition, ACH09 restored the decreased plasma andmesenteric arteries antioxidant activities of superoxide dismutase, catalase and glutathione peroxidase in the HF group. In conclusion, the treatment of HF-feddams during lactation with ACH09 provides protection from later-life hypertension, body weight gain, insulin resistance and oxidative stress. The protectiveeffect ACH09 may involve NO synthesis, antioxidant action and activation of insulin-signaling pathways.© 2013 Elsevier Inc. All rights reserved.

Keywords: Grape skin extract; Hypertension; Insulin resistance; Oxidative stress; Developmental programming

1. Introduction

Metabolic syndrome (MS) including the presence of obesity,hypertension, dyslipidemia and insulin resistance that predisposestype 2 diabetes is becoming more prevalent in recent years [1,2].Epidemiological and experimental studies have described that theprogramming of energy balance already begins in very earlydevelopment. Indeed, particular conditions in the nutritional envi-ronment during the perinatal and or postnatal periods may lead to

☆ Sources of funding: This work was conducted with grants fromNational Council of Scientific and Technological Development (CNPq) andRio de Janeiro State Research Agency (FAPERJ).

☆☆ Conflicts of interest: Roberto Soares de Moura is the inventor of apatent (PCT/BR02/00038) that supported the development of a new patentapplication (PI0605693 A2-8). The other authors state no conflicts of interest.

⁎ Corresponding author. Departamento de Farmacologia e PsicobiologiaInstituto de Biologia, Universidade do Estado do Rio de Janeiro, Av. 28 deSetembro, 87, Rio de Janeiro 20 551-030, Brazil. Tel.: +55 21 28688299; fax:+55 21 28688629.

E-mail addresses: angelacr@uerj.br, angelacr@hotmail.com (A.C. Resende).

0955-2863/$ - see front matter © 2013 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.jnutbio.2013.08.003

adjustments in the physiology of humans and animals, with lastingeffects in adulthood [3–5].

Experimental studies have shown that adult offspring of rodentsfed fat (lard) or highly palatable (lard and sugar enriched) dietsduring gestation develop theMS phenotype, despite being reared on astandard chow diet [6–8]. Likewise, early postnatal nutrition may alsocause differential programming of energy homeostasis. However, fewhave investigated the role of the suckling period in inducingcardiovascular and metabolic disorders in offspring of dams fed afat-rich diet [9]. In this sense, we have recently described thatnormally fed adult female offspring of high fat-fed dams duringlactation developed hypertension, dyslipidemia and gain of weight. Inaddition, our findings have also suggested the oxidative stressprogramming in the adult female offspring [10]. This may reinforcesthe hypothesis that oxidative stress, the imbalance between proox-idant and antioxidants, can be an early event in the development ofchronic diseases related to metabolic and cardiovascular disordersassociated with MS [11–14].

Numerous studies have demonstrated a potential role forbioactive food components as an adjunct to the treatment of obesityand MS [15–17]. Natural polyphenols, obtained from many plants,

Table 1Composition of the experimental diet

Nutrient(U/kg diet)

Diets

Standard HF

Casein (g) 251.16 226.74Cornstarch (g) 478.5 330Sucrose (g) 100 100Corn oil (g) 70 43Lard (g) – 200Fiber (g) 50 50Mineral mix ⁎ (g) 35 35Vitamin mix ⁎ (g) 10 10L-Cystine (g) 3 3Choline (g) 2.5 2.5Energy (kcal) 3636.8 4614.9% as Carbohydrate 58.9 35.7% as Protein 23.8 16.9% as Fat 17.3 47.4

⁎ Vitamins and minerals mix following the AIN-93M recommendation for rodents.

2120 A.C. Resende et al. / Journal of Nutritional Biochemistry 24 (2013) 2119–2126

have been shown to exert important actions on the cardiovascularsystem [18].

Previously, we have demonstrated that an alcohol-free grape skinextract obtained from skins of Vitis labrusca (ACH09), a vinifera grapelargely used in Brazil to produce red wine, induced an endothelium-dependent vasodilation, antihypertensive and antioxidant effects[19,20]. The activation of the insulin-signaling cascade and reductionof hyperglycemia in alloxan-induced diabetic mice by the grape skinextract have also been demonstrated [21]. However, there is no reportabout the activity of ACH09 on cardiovascular and metabolicdisorders and oxidative stress of male offspring of an experimentalmodel of developmental programming. We have now furtherinvestigated the changes occurring at proteins expression of theinsulin-signaling cascade as a consequence of maternal food over-nutrition during suckling period that may be determinant of theinsulin resistance. Hence, this study aimed to test the hypothesis thatthe beneficial effects of ACH09 on metabolic control of dams wouldpass on to the male offspring as adults; that is, the metabolicderangements in offspring of high-fat (HF) diet fed dams could beameliorated if dams are supplemented with ACH09.

Therefore, experiments were undertaken to determine the actionof ACH09 on cardiovascular and metabolic programming with focuson insulin resistance and changes on oxidative status observed inadult male offspring of dams fed an HF diet during lactation.

2. Methods and materials

2.1. Preparation of the grape skin ACH09 extract

The dried and powdered skin fruits of Vitis vinifera L. (Vitaceae) were extracted inan aqueous solution at 100°C with occasional shaking for about 120 min. The solutionwas then introduced into an ion-exchange resin column (cationic) and washedsequentially with ethanol, ethanol/H2O (1:1) and H2O. The H2O fraction was discarded.The ethanolic and hydroalcoholic fractions were placed together and evaporated undervacuum at 60°C, followed by spray drying of the concentrated solution (inlettemperature 190°C and outlet temperature of 85°C). The extract obtained in theprocess was a fine powder, soluble in H2O, with about 30% of total polyphenolsaccording to the Folin–Ciocalteau procedure [22].

2.2. Analysis of grape skin ACH09 extract

To identify the active principles in grape skin, the extract was analyzed by LC/UV/MSwith an atmospheric pressure chemical ionization interface [21]. LC/UV analysis of thedried hydroalcoholic grape skin extractwas performed on aHewlett-Packard series 1100photodiode array detector (DAD) liquid chromatography system (Hewlett-Packard,Waldbronn, Germany). HPLC/UV/DAD analysis was performed with a Symmetry RP-18column (4 mm; 250 ¥ 3.9 mm i.d.; Waters, Milford, MA, USA), solvent system: A, MeOHwith 0.5% formic acid; B, H2Owith 0.5% formic acid; gradient mode 20% of A to 100% of Ain 25min; flow rate 1ml/min; injection volume10ml; sample concentration 10mg/ml inMeOH. DAD conditions were 210, 254 and 540 nm; UV data were recorded at 190–600nm (step 2 nm). LC/MSn was performed directly after UV-DAD measurements. AFinningan LCQ ion trap (FinninganMAT, San Jose, CA, USA)was usedwith anatmosphericpressure chemical ionization interface. MSn experiments were completed by program-ming-dependent scan events. The first event was a full MS scan Mr (150.0–1500.0)(MS1); during the second event, the main ion recorded was isolated and selectivelyfragmented in the ion trap (MS2).The collision energy was set to 15 eV. HPLC analysis ofthe dried hydroalcoholic grape skin extract involved dissolving 10 mg of the extract in1 ml methanol/H2O (1:1) with 0.5% formic acid (HPLC quality). A total of 20 ml wasanalyzed by HPLC. Standards of peonidin-3-O-glucoside (1), petunidin-3-O-glucoside(2), malvidin-3-O-glucoside (3) and malvidin-3-(6-O-trans-p-coumaryl)-5-O-diglico-side (4) were purchased from Polyphenols Laboratories (Sandnes, Norway).

2.3. Animals and diet

All procedures were carried out in accordance with The Ethics Committee forExperimental Animals Use and Care (CEA) of Instituto de Biologia Roberto AlcântaraGomes/Universidade do Estado do Rio de Janeiro. The CEA follow guidelines fromIntramural Animal Care and Use program of the National Institutes of Health (NIH).Virgin female Wistar rats 3 months aged were caged with one male rat at a proportionof 3:1. After mating, determined by the presence of a vaginal plug, each female wasplaced in an individual cage with free access to water and food until delivery. Within 24h of birth, excess pups were removed so that only eight offspring were kept per cage tostandardize milk availability during lactation. During the weaning period (21 days), thedams had free access to standard diet (AIN 93) or HF diet 24% (20% animal lard and 4%

corn oil) in a temperature-controlled room with 12-h light/dark cycle and allocatedinto four groups. The control group corresponding to male offspring from dams fed thestandard diet and allowed access to water (control group: 7% fat; 90.9±1.8 kcal) orACH09 (ACH09 group, 200 mg kg−1 d−1) during weaning. Two other groups were fedan HF diet with access to water (HF group: 24 % fat; 106.0±1.9 kcal) or ACH09(HF+ACH09 group: 24 % fat; 106±2.1 kcal) during weaning (Table 1). The dose ofACH09 was based on previous studies that showed antihypertensive and antioxidanteffects of the extract [10,19,23]. From weaning onward, all offspring were fed adlibitum the standard maintenance diet (AIN 93). Maternal food intake (g) wasrecorded daily in dams during weaning, and no changes were observed betweengroups (control: 25±1.8; control + ACH09: 24.3±2; HF: 24±1.9; HF+ACH09: 23.1±2.1). Food consumptionof damswas estimatedby subtracting the amount of food left on thegrid and amount of spilled food from the initial weight of food supplied. During lactation,offspring body weight (BW) was weekly recorded from 1 week of age (to avoid maternalrejection of the pups) until animals were fully grown at 90 or 180 days old.

The diets were elaborated by the Department of Experimental Nutrition, FederalFluminense University (RJ, Brazil) in accordance with the standard recommendationsfor rodents in the maintenance state of American Institute of Nutrition (AIN-93M) [24].

2.4. Arterial pressure measurement and vascular perfusion study

Systolic blood pressure (SBP) was measured in conscious rats by use of tail-cuffplethysmopraphy (Letica 5000 device) once a week during 90 or 180 days.

Mesenteric arterial beds (MABs) of the rats were isolated in accordance with themethod previously described [25]. Briefly, rats were anesthetized, and the MAB wasrapidly removed, cannulated and perfused at a flow rate of 4 ml/min with physiologicalsalt solution (PSS), bubbledwith 95% O2/5% CO2, using a peristaltic pump (Lifecaremodel4; Abbott/Shaw, Chicago, IL, USA). The PSS (composition,mmol/l: NaCl 118, KCl 4.7, CaCl22.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25, EDTA 0.026 and glucose 6.0) was bubbled with95% O2/5% CO2 at 37°C. Perfusion pressurewasmeasured using a pressure transducer (HP1280) connected to a preamplifier (HP 8805B), and the chart was recorded (7754A;Hewlett-Packard, Lexington, MA, USA). The preparations were left to equilibrate for30 min. Then injections of 120 μmol of KCl were administered every 10 min untilconsistent responses were obtained. After the equilibration period, basal pressure (24±2mm Hg; n=48) was elevated (80–100 mm Hg) by adding norepinephrine (NE; 10–30μmol/l) to the perfusion solution. When the pressor effect of NE reached a plateau, dose–response curves to bolus injections of acetylcholine (ACh, 1–100 pmol) were injected assingle doses in order to assess the endothelium-dependent vasodilator response. Thevasodilator effect of AChwas expressed as a percentage decrease of the pressor effect of NE.

2.5. Plasma assays

Blood was collected from male rats with 90 or 180 days. The animals were fastedfor 6 h, and blood samples were then collected by cardiac punction in anesthetizedanimals. Glicemia was determined with a glucometer (Accu-Chek Active, Roche,Manhein, Germany), and insulin concentrations were determined with the Insulin 125Iradioimmunoassay Kit (MP Biomedicals, LLC-Orangeburg, NY, USA). Homeostaticmodel assessment of insulin resistance (HOMA-IR) was calculated from the real-timefasting serum glucose and fasting insulin concentrations of different groups of ratsusing the mathematical HOMA-IR formula: HOMA-IR=(fasting serum insulin in μU/ml×fasting serum glucose in mg/dl)/22.5. Plasma total cholesterol and triglyceridelevels were measured by a colorimetric assay (Analisa, Belo Horizonte, Brazil).

2.6. Malondialdehyde assay

As an index of lipid peroxidation, we used the thiobarbituric acid reactivesubstances method for analyzing malondialdehyde (MDA), as previously described

2121A.C. Resende et al. / Journal of Nutritional Biochemistry 24 (2013) 2119–2126

[26]. Briefly, the samples from plasma and mesentery homogenates (first- and second-order mesenteric arteries) were mixed with 1 ml of 10% trichloroacetic acid and 1ml of0.67% thiobarbituric acid. Subsequently, they were heated in a boiling water bath for30 min. The absorbance of the organic phase containing the pink chromogen wasmeasured spectrophotometrically at 532 nm. MDA equivalents were expressed innMol/mg protein.

2.7. Nitrite assay

Nitrite levels in plasma and mesentery homogenates were determined by amethod based on the Griess reaction [27]. A total of 100 μl of sample was mixed with100 μl of Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1%naphthylenediamide dihydrochloride in water) and incubated at room temperaturefor 10 min followed by measuring the absorbance in a plate reader at 550 nm (Bio-RadMicroplate Reader model 680, CA, USA). Nitrite concentrations in the sampleswere determined from a standard curve generated by different concentrations ofsodium nitrite.

2.8. Superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase(GPx) activities

Mesentery homogenates and plasma were used to determine SOD, CAT and GPxactivities. SOD activity was assayed by measuring the inhibition of adrenaline auto-oxidation as absorbance at 480 nm [28]. CAT activity was measured in terms of the rateof decrease in hydrogen peroxide at 240 nm [29]. GPx activity was measured bymonitoring the oxidation of NADPH at 340 nm in the presence of hydrogen peroxide[30]. The total protein content in each sample was determined using the Bradfordmethod [31].

2.9. Western blotting

The soleus muscle was rapidly removed from each anesthetized animal andimmediately frozen in liquid nitrogen. Tissue was further homogenized in HES buffer(0.3 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, 5 mM ethylenedia-mine-tetraacetic acid, 0.1 mM sodium orthovanadate, Triton X-100, 0.1 M sodiumfluoride, 1 M sodium pyrophosphate, 10 μl/ml aprotinin and 10 μg/ml leupeptin). Thelysate was centrifuged for 15 min at 42,000×g at 4°C. Then, the supernatant wascentrifuged for 90 min at 220,000×g at 4°C. The total protein content was determinedby the Lowry method (Bio-Rad, Hercules, CA, USA). Total protein (40 μg) wassubjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (10%). Thesamples were loaded with a protein standard (Rainbow, Sigma, USA), transferred topolyvinylidene difluoride filters (PVDF Hybond-P; Amersham Bioscience, Piscataway,NJ, USA), and blocked with Tween-TBS (20 mM Tris–HCl, pH 7.5; 500 mM NaCl; 0.01%Tween-20) containing 2% bovine serum albumin. Primary antibodies used in Westernblotting analysis were rabbit anti-GLUT-4, AKT, IRS-1 (1:1000; Santa CruzBiotechnology, Santa Cruz, CA, USA). PVDF filters were next incubated with theappropriate secondary antibody conjugated to biotin (1:10.000, Santa CruzBiotechnology), followed by 1-h incubation with horseradish peroxidase-conjugatedstreptavidin (1:1000; Caltag Laboratories, Burlingame, CA, USA). We also incubatedall membranes with β-tubulin antibody to avoid possible inconsistency in proteinloading and/or transfer. Immunoreactive proteins were visualized by 3,3′-diamino-benzidine (Sigma) staining. The bands were quantified by densitometry, using ImageJ Software (NIH, USA).

2.10. Statistical analyses

Data are expressed as the mean values of n observations±standard error of themean. One-way analysis of variance (ANOVA; plus Bonferroni tests were used tocompare differences between experimental groups. Two-way ANOVA plus Bonferronitests were used to compare different ages between groups. Values were taken to besignificantly different when Pb.05.

3. Results

3.1. Isolation and identification of the major constituents of theACH09 extract

All compounds showed the same UV spectra in the LC/UV/DADanalysis that are characteristic of anthocyanins. Compounds 1, 2, 3and 4 presented molecular ions at m/z 463 [M]+, 479 [M]+, 493 [M]+ and 801 [M]+, respectively (Fig. 1). All four showed a similarfragmentation pattern. Compounds 1, 2 and 3 presented two signalscorresponding to the molecular ion [M+] and the fragment resultingfrom the loss of a glucose molecule [M-162]+, corresponding to theaglicon. In the case of compound 4, the MS spectra showed the loss ofone glucose molecule and a fragment corresponding to the loss of a

p-coumaryl glucoside moiety [M-308]+ [32]. According to thesedata, the compounds were identified as peonidin-3-O-glucoside (1),petunidin-3-O-glucoside (2), malvidin-3-O-glucoside (3) and malvi-din-3-(6-O-trans-p-coumaryl)-5-O-diglicoside (4), all previously de-scribed in different Vitis spp. [32,33]. This conclusion was confirmedby comparison of retention time and UV and MS data in the LC/UV/MSanalysis using commercially available standards. Compounds 1–3 arealways present in grape extracts, generally in high concentrations,while 4 can be found in low concentrations [32].

3.2. Effect of ACH09 on BW

Initial BWs were compared between male offspring from thefour groups at the age of 7 days and during the period ofexperimental protocol until the ages of 90 or 180 days. The BW ofthe HF group was greater compared with controls (Pb.05, n=10) atthe beginning of the study (15 days) and from 120 to 150 days(Fig. 2). After 40 until 90 days, no difference was observed betweengroups. In male offspring of HF group, adiposity as assessed by thecombined wet weight of intra-abdominal and gonadal fat wasincreased in the HF group from both ages compared with controls.Treatment of fat-fed dams with ACH09 during lactation reduced(Pb.05) the BW and adiposity indicating a protective effect of ACH09against overweight (Fig. 2).

3.3. Effect of ACH09 on blood pressure and vascular function

Blood pressure was significantly increased in the HF group fromthe age of 60 to 180 days comparedwith the control group (Pb.05, n=6), and ACH09 prevented the hypertension throughout the durationof the experiment (Fig. 3).

Vasodilation in response to ACh was not significantly differentbetween the groups at the age of 90 and 180 days. However, thevasodilator response to ACh was lower (Pb.05, n=6) in MAB of 180-day-old offspring from the four groups as compared to those at 90days old (Fig. 4).

3.4. Effect of ACH09 on plasma lipid levels

There was a significant effect of maternal diet on the triglyceridelevels. At 90 and 180 days, HF diet was associated with highertriglyceride concentrations than other groups (Pb.05, Fig. 5). Treat-ment of HF-fed dams with ACH09 prevented the increase oftriglyceride concentrations of 90- and 180-day-old offspring (Pb.05,Fig. 5). There were no significant differences between the groups ontotal cholesterol levels (Fig. 5).

3.5. Effect of ACH09 on plasma levels of glucose and insulin

Plasma glucose levels were significantly higher in offspring fromHF diet than in control and ACH09 groups of both ages, which wasassociated with a significant increase in insulin levels only in 180-day-old rats (Pb.05, Fig. 6). The increased glucose levels in HF dietwere associatedwith increased insulin resistance measured by HOMAindex. Treatment of HF-fed dams with ACH09 prevented the increasein glycemia in offspring at both ages (Pb.05), which were associatedwith a decrease in insulin resistance to the same values as control,suggesting that ACH09 prevents insulin resistance in offspring fromHF-fed dams.

3.6. Effect of ACH09 on IRS-1, AKT and GLUT4 expression

Western blot analysis showed decreased (Pb.05) IRS-1, AKT andGLUT4 protein expressions in soleus muscle homogenates of the HFgroups (90 and 180 days) when compared to control groups (n=6,

Fig. 1. HPLC profile from grape skin ACH09 extract and the structures of the four identified anthocyanins.

2122 A.C. Resende et al. / Journal of Nutritional Biochemistry 24 (2013) 2119–2126

Fig. 6). Treatment of HF-fed dams with ACH09 during lactationincreased IRS-1, AKT and GLUT4 expression in HF+ACH09 groups ofboth ages (Pb.05; Fig. 7A–C). The IRS-1 and AKT expression wasincreased by aging in all groups (Pb.05).

3.7. Effect of ACH09 on plasma and mesentery MDA

The index of lipid peroxidation assessed by MDA was significantlyhigher (pb.05) in the plasma andmesentery of HF groups (90 and 180days) than in control groups (n=6, Table 2). The levels of MDA were

Fig. 2. Effects of ACH09 on BW evolution (A) inmale offspring during lactation and after weaninfed dams during lactation. Data are means±SEM, n=10 for all groups. *Significantly diffecorresponding HF group (pb.05).

increased by aging in all groups (pb.05). Treatment of HF-fed damswith ACH09 prevented the increase in the levels of MDA in HF+ACH09 groups of both ages (pb.05). Mesentery and plasma samplesfrom control treated groups showed no significant difference in MDAlevels from control animals.

3.8. Effect of ACH09 on plasma and mesentery nitrite content

Rats from HF group showed a lower level of nitrite compared tothe control groups in plasma andmesentery samples (Table 2). Nitrite

g until 180 days old. Total fat mass (B) at 90- and 180-day-old offspring of control or HF-rent from the control and ACH09 groups (p≤.05). +Significantly different from the

Fig. 3. Effect of ACH09 on SBP (mm Hg) in male offspring of control or HF-fed damsduring lactation. Data are means±SEM, n=6 for all groups. *Significantly differentfrom the control and ACH09 groups (p≤.05). +Significantly different from thecorresponding HF group (pb.05).

Fig. 5. Effects of ACH09 on cholesterol (A) and triglycerides (B) at 90- and 180-day-oldoffspring of control or HF-fed dams during lactation. Data are means±SEM, n=10 forall groups. *Significantly different from the control and ACH09 groups (p≤.05).+Significantly different from the corresponding HF group (pb.05).

2123A.C. Resende et al. / Journal of Nutritional Biochemistry 24 (2013) 2119–2126

levels were not modified by aging between the groups. Treatment ofHF-fed dams with ACH09 during lactation prevented the decrease inthe levels of nitrite in HF+ACH09 groups of both ages (n=6, pb.05).Control-treated group showed no significant difference in nitritelevels from control animals.

3.9. Effect of ACH09 on plasma and tissue antioxidant enzymes (SOD,CAT and GPx)

As shown in Table 2, SOD, CAT and GPx activities are reduced(pb.05) in the plasma and mesentery homogenates of the HF groups(90 and 180 days) when compared to control groups (n=6 for eachgroup). Treatment with ACH09 recovered the SOD, CAT and GPxactivities in mesentery and plasma of HF group (pb.05) compared tonon-treated HF group. The SOD activity in mesentery and CAT activityin plasma were increased by aging in the different groups (pb.05).Control-treated groups showed no significant difference in antioxi-dant activities from control animals.

4. Discussion

Growing evidence has shown that maternal overnutrition duringsuckling period may program the development of their offspring

Fig. 4. Effect of ACH09 on vasodilator effect of acetylcholine (ACh) in the perfused MAB at 90-SEM, n=6 for all groups. +Significantly different from the corresponding HF group (pb.05).

toward different consequences [9,10,34]. Here, we show thatovernutrition during suckling period predisposes normally fedadult male offspring to MS. The supplementation of dams withACH09 during lactation significantly improved the blood pressurelevels, plasma and tissue oxidative stress and metabolic profiles ofadult male offspring to a condition that is comparable to offspringborn to dams fed a normal diet. Hence, the beneficial effects of ACH09on oxidative status and metabolic control of dams during lactationmay pass on to themale offspring as adults; that is, the oxidative stressand metabolic changes in adult offspring of HF-diet fed dams can beameliorated if dams are supplemented with ACH09.

and 180-day-old offspring of control or HF-fed dams during lactation. Data are means±

Fig. 6. Effects of ACH09 on glucose (A), insulin (B) and HOMA-IR (C) at 90 and 180 daysold offspring of control or HF-fed dams during lactation. Data are means±SEM, n=10for all groups. *Significantly different from the control and ACH09 groups (p≤.05).+Significantly different from the corresponding HF group (pb.05).

Fig. 7. Effects of ACH09 on IRS-1 (A), AKT (B) and GLUT4 (C) expressions in soleusmuscle at 90- and 180-day-old offspring of control or HF-fed dams during lactation.Data are means±SEM, n=6 for all groups. *Significantly different from the control andACH09 groups (p≤.05). +Significantly different from the corresponding HF group(pb.05).

2124 A.C. Resende et al. / Journal of Nutritional Biochemistry 24 (2013) 2119–2126

The antihypertensive effect of grape skin extract has previouslybeen demonstrated by our group in different models of hypertension[19,23]. In this study, treatment of fat-fed dams with ACH09 duringlactation prevented the increase in SBP of adult male offspring of bothages, as previously described for female offspring [10], suggesting thatconsumption of ACH09 early during lactation also confers protectionagainst the insult of a fat-rich diet in adult male offspring. Themechanism underling the prevention of hypertension by ACH09 inmale offspring may probably be mediated by endothelium-derivedrelaxing factors, since our previous findings showed that the grapeskin extract induces vasodilation of mesenteric arteries of the ratmediated by nitric oxide (NO) and the endothelium-derivedhyperpolarizing factor [20] and induces eNOS activation in porcinecoronary artery [35].

In this study, the vasodilation induced by ACh was not differentbetween the groups. However, an endothelial dysfunction wasobserved in 180-day-old offspring of all groups compared with

offspring at 90 days. This is in agreement with previous studies in themesenteric arteries from female offspring born to dams fed an HF diet,suggesting that these vascular responses are not gender specific [10].Further, the endothelial dysfunction was associated with increasedlevels of MDA in plasma and mesentery from 180-day-old offspring,suggesting that vascular oxidative damage may explain in part theendothelial dysfunction with aging.

There is increasing evidence supporting a role for the oxidativestress in the pathogenesis of MS [36,37]. We showed increased levelsof MDA in mesentery and plasma of adult male offspring at 90 and180 days from fat-fed dams. Furthermore, decreased antioxidantenzymatic activities (SOD, CAT and GPx) were also observed in maleoffspring showing that oxidative stress, in common with raised BP,can originate during suckling. Giving further support to thissuggestion, decreased levels of nitrite was detected in plasma andmesentery of male offspring indicating decreased NO bioavailability,

Table 2Effects of ACH09 on MDA, nitrite (NO2), SOD, CAT and GPx activities in plasma and mesentery at 90- and 180-day-old offspring of dams fed HF diet during suckling

MDA (nmol/mg protein) NO2 (μmol/mg protein) SOD (U/mg protein) CAT (U/mg protein) GPx (U/mg protein)

Plasma Mesentery Plasma Mesentery Plasma Mesentery Plasma Mesentery Plasma Mesentery

Control90 days 0.3±0.1 0.3±0.3 0.2±0.03 14.4±1.4 54.5±4.0 45.0±5 0.34±0.09 0.47±0.01 0.015±0.08 0.011±0.001180 days 1.3±0.1# 0.8±0.1# 0.2±0.03 14.0±0.2 55.0±3.8 66.0±6 0.38±0.05 0.78±0.01 0.016±0.02 0.016±0.003GSE90 days 0.4±0.2 0.4±0.2 0.2±0.09 13.5±1.2 54.5±4.5 46.0±0.5 0.30±0.06 0.49±0.03 0.02±0.002 ⁎ 0.012±0.001180 days 1.2±0.2# 0.78±0.1# 0.2±0.02 16.0±0.2 55.5±3.6 65.0±6.0 0.37±0.03 0.67±0.02 0.015±0.001 0.015±0.001HF90 days 0.65±0.01 ⁎ 0.65±0.01 ⁎ 0.06±0.05 ⁎ 7.3±0.8 ⁎ 7.0±0.2 ⁎ 17.0±0.1 ⁎ 0.16±0.01 ⁎ 0.31±0.02 ⁎ 0.01±0.001 ⁎ 0.002±0.001 ⁎180 days 3.3±0.1⁎,# 1.2±0.02⁎,# 0.08±0.06 ⁎ 7.8±0.4 ⁎ 10±1.5 ⁎ 35.0±6.0 ⁎ 0.14±0.02 ⁎ 0.49±0.07 ⁎ 0.006±0.001 ⁎ 0.0007±0.001 ⁎HF+GSE90 days 0.1±0.01⁎,+ 0.1±0.01⁎,+ 0.1±0.01⁎,+ 13.0±1.2+ 55.5±5.0+ 43.0±6.9+ 0.29±0.06⁎,+ 0.39±0.03⁎,+ 0.015±0.003+ 0.011±0.003+

180 days 2.3±0.09⁎,+,# 0.8±0.05+,# 0.2±0.01+ 14.0±0.8+ 54±5.0+ 46.0±3.0+ 0.31±0.04⁎,+ 0.62±0.06⁎,+ 0.013±0.003⁎,+ 0.01±0.003⁎,+

Data are means±SEM, n=6 for all groups.⁎ Significantly different from the control groups (pb.05).+ Significantly different from the corresponding control or HF groups (pb.05).# Significantly different from 90 days (pb.05).

2125A.C. Resende et al. / Journal of Nutritional Biochemistry 24 (2013) 2119–2126

which can explain in part the early increase in BP, since it is largelyknown that NO contributes to the maintenance of BP [38]. Theincreased MDA levels were reduced by ACH09, which corroboratesthe antioxidant action of grape skin extract, as previously demon-strated [10,19]. We believe that the mechanism underlying thiseffect may involve the increase in antioxidant defense by ACH09, asshown in mesentery and plasma, which may contribute to anincrease in NO bioavailability.

Control offspring suckled by fat-fed dams produces phenotypicsimilarities to the present model, such as increased adiposity,hyperglycemia and hyperinsulinemia in adulthood [9,10]. In thisstudy, the increase in BW and adiposity in male offspring of HF groupwas prevented by maternal supplementation of ACH09 duringlactation. The possible mechanisms behind this antiobesity effect ofACH09 may result from the observation of lower plasma triglyceridesand glucose, as reported by polyphenols from other sources [39].Alternatively, the improvement of adipocyte function and reductionof weight gain by polyphenols found in grape skin were described inC57BL/6 mice fed an HF diet [40]. More recently, an antiobesityactivity of green tea polyphenols has been demonstrated in obesemiddle-aged female rats that could be a result of polyphenol inducedalteration of obesity-related genes, anti-inflammation activity andantioxidative stress capacity [41].

Insulin resistance is the main pathophysiologic feature of the MS,since insulin represents the most determinant hormonal effect in themaintenance of metabolic balance of lipids, carbohydrates andproteins [42]. In the present model, hyperglycemia and hyperinsu-linemia appear to be induced in the suckling period, which is inagreement with previous study in the same model [10]. Remarkably,early exposure to the fat diet programmed the increased level ofinsulin at 180 days compared to 90 days, which probably prevented afurther increase in glycemia at 180 days and may explain similarlevels of glucose at both ages. These data suggest that increased levelof insulin in offspring may be an adaptive outcome of early lifeexposure to HF during critical developmental window [34]. We foundthat ACH09 prevented hyperglycemia and insulin resistance in adultmale offspring of HF group, suggesting that maternal HF diet andACH09 supplementation during suckling period may alter theoffspring environment to changes in metabolic processes. Thedecrease of BW and adiposity induced by ACH09 could explain inpart the improvement of insulin resistance by the extract, sincegrowing evidence suggests that obesity is associated with insulinresistance, impaired glucose tolerance and even diabetes [43].However, based on recent findings by our group, demonstratingthat ACH09 increases insulin sensitivity in alloxan-induced diabetic

mice, we hypothesized that the decreased insulin resistance found inthe present study could be explained by modulation of insulinsignaling and regulation of glucose transport [21].

GLUT-4 is the major carrier involved in insulin-stimulatedglucose transport, so the amount of this transporter present onthe cell surface, at least in part, controls the rate of glucosetransport into muscle cells [44]. The present study is the first toreport that maternal ACH09 supplementation during sucklingperiod prevents hyperglycemia in adult male offspring, which wasaccompanied by an increase in GLUT-4, IRS-1 and AKT content inskeletal muscle, suggesting that the prevention of hyperglycemia byACH09 might result from activation of insulin signaling andregulation of glucose metabolism. These data are consistent withthe HOMA index results showing that ACH09 treatment signifi-cantly prevented the increase in insulin resistance observed in adultmale offspring. Because the decreased insulin resistance correlatedwith enhanced GLUT-4 content, we suggest that ACH09 action oninsulin sensitivity probably occurs through an increase in GLUT-4and the resulting increased glucose uptake. Our hypothesis isconsistent with the increased amount of GLUT-4 induced by grapeseed-derived polyphenols, suggesting an extrapancreatic mecha-nism for its antidiabetic action [45]. Furthermore, the increased IRS-1 and AKT content indicates that the prevention of hyperglycemiain adult male offspring by ACH09 may be the result not only of ahigher GLUT-4 content but also of increased sensitivity in theinsulin-signaling pathway.

The mechanisms underlying ACH09-induced insulin sensitivityare not yet established. Increasing evidence shows that oxidativestress and inflammation are the main causes of insulin resistance byinterfering with insulin signal transduction [46]. On the other hand,NO seems to positively modulate insulin sensitivity [47,48]. There-fore, we suggest that the antioxidant action of ACH09 demonstratedin this study, as well as the NO production, may also contribute tothe improvement of insulin resistance in adult male offspring of fat-fed dams.

In conclusion our results provide further support for the earlypostnatal environment playing an important role in programminglater life phenotype. ACH09 protected normally fed male offspring ofHF-fed dams from hypertension, gain of weight, hyperglycemia andinsulin resistance. The NO synthesis, antioxidant action andactivation of insulin-signaling pathways by ACH09 may contributeto these benefic effects of the grape skin extract. Our findingssuggest the metabolic disorders induced by overnourished damsduring lactation could be offset by supplementing ACH09 to thematernal diet.

2126 A.C. Resende et al. / Journal of Nutritional Biochemistry 24 (2013) 2119–2126

References

[1] Alberti KG, Zimmet P, Shaw J. Metabolic syndrome—a new world-wide definition.A consensus statement from the International Diabetes Federation. Diabet Med2006;23(5):469–80.

[2] Cascio G, Schiera G, Di Liegro I. Dietary fatty acids inmetabolic syndrome, diabetesand cardiovascular diseases. Curr Diabetes Rev 2012;8(1):2–17.

[3] Cripps RL, Martin-Gronert MS, Ozanne SE. Fetal and perinatal programming ofappetite. Clin Sci (Lond) 2005;109:1–11.

[4] Gluckman PD, HansonMA. The developmental origins of themetabolic syndrome.Trends Endocrinol Metab 2004;15:183–7.

[5] Palou M, Torrens JM, Priego T, Sánchez J, Palou A, Picó C. Moderate caloricrestriction in lactating rats programs their offspring for a better response to HFdiet feeding in a sex-dependent manner. J Nutr Biochem 2011;22:574–84.

[6] Bayol SA, Farrington SJ, Stickland NC. A maternal ‘junk food’ diet in pregnancy andlactation promotes an exacerbated taste for ‘junk food’ and a greater propensityfor obesity in rat offspring. Br J Nutr 2007;98:843–51.

[7] Samuelsson AM, Matthews PA, Argenton M, Christie MR, McConnel JM, et al. Diet-induced obesity in female mice leads to offspring hyperphagia, adiposity,hypertension, and insulin resistance: a novel murine model of developmentalprogramming. Hypertension 2008;51:383–92.

[8] Li S, Tse IMY, Li ETS. Maternal green tea extract supplementation to rats fed ahigh-fat diet ameliorates insulin resistance in adult male offspring. J Nutr Biochem2012;23(12):1655–60.

[9] Khan IY, Dekou V, Douglas G, Jensen R, Hanson MA, Poston L, et al. A high-fat dietduring rat pregnancy or suckling induces cardiovascular dysfunction in adultoffspring. Am J Physiol Regul Integr Comp Physiol 2005;288(1):R127–33.

[10] Emiliano AF, de Carvalho LCRM, Cordeiro VSC, da Costa CA, de Oliveira PRB,Queiroz EF, et al. Metabolic disorders and oxidative stress programming inoffspring of rats fed a high-fat diet during lactation: effects of a Vinifera grape skin(ACH 09) extract. J Cardiovasc Pharmacol 2011;58(3):319–28.

[11] Chang YC, Chuang LM. The role of oxidative stress in the pathogenesis of type 2diabetes: from molecular mechanism to clinical implication. Am J Transl Res2010;2(3):316–31.

[12] Archuleta TL, Lemieux AM, Saengsirisuwan V, Teachey MK, Lindborg KA, Kim JS,et al. Oxidant stress-induced loss of IRS-1 and IRS-2 proteins in rat skeletalmuscle: role of p38 MAPK. Free Radic Biol Med 2009;47(10):1486–93.

[13] Bruce KD, Cagampang FR, Argenton M, Zhang J, Ethirajan PL, Burdge GC, et al.Maternal high-fat feeding primes steatohepatitis in adult mice offspring,involving mitochondrial dysfunction and altered lipogenesis gene expression.Hepatology 2009;50(6):1796–808.

[14] Luo ZC, FraserWD, Julien P, Deal CL, Audibert F, Smith GN, et al. Tracing the originsof “fetal origens” of adult disease: programming by oxidative stress? MedicalHypothesis 2006;66:38–44.

[15] Tapsell LC, Hemphill I, Cobiac L, Patch CS, Sullivan DR, Fenech M, et al. Healthbenefits of herbs and spices: the past, the present, the future. Med J Aust 2006;185:S4–S24.

[16] Yin J, Zhang H, Ye J. Traditional Chinese medicine in treatment of metabolicsyndrome. Endocr Metab Immune Disord Drug Targets 2008;8:99–111.

[17] Sen S, Simmons RA. Maternal antioxidant supplementation prevents adiposity inthe offspring of Western diet-fed rats. Diabetes 2010;59:3058–65.

[18] Dohadwala MM, Vita JA. Grapes and cardiovascular disease. J Nutr 2009;139(9):1788S–93S.

[19] Soares De Moura R, Costa Viana FS, Souza MA, Kovary K, Guedes DC, Oliveira EP,et al. Antihypertensive, vasodilator and antioxidant effects of a vinifera grape skinextract. J Pharm Pharmacol 2002;54(11):1515–20.

[20] Madeira SV, Resende AC, Ognibene DT, de Sousa MA, Soares de Moura R.Mechanism of the endothelium-dependent vasodilator effect of an alcohol-freeextract obtained from a vinifera grape skin. Pharmacol Res 2005;52:321–7.

[21] Soares de Moura R, da Costa GF, Moreira AS, Queiroz EF, Moreira DD, Garcia-SouzaEP, et al. Vitis vinifera L. grape skin extract activates the insulin-signalling cascadeand reduces hyperglycaemia in alloxan-induced diabetic mice. J Pharm Pharmacol2012;64(2):268–76.

[22] Singleton VL, Rossi JA. Colorimetry of total phenolics with phosphomolybdic–phosphotungstic acid reagents. Am J Enol Vitic 1965;16:144–58.

[23] de Moura RS, Resende AC, Moura AS, Maradei MF. Protective action of ahydroalcoholic extract of a vinifera grape skin on experimental preeclampsia inrats. Hypertens Pregnancy 2007;26(1):89–100.

[24] Reeves PG, Nielsen FH, Fahey GC. AIN-93 purified diets for laboratory rodents:final report of the American Institute of Nutrition ad hoc writing committee on thereformulation of the AIN-76A rodent diet. J Nutr 1993;123:1939–51.

[25] McGregor DD. The effect of sympathetic nerve stimulation on vasoconstrictorresponse in perfusedmesenteric blood vessels of the rat. J Physiol 1965;177:21–30.

[26] Draper HH, Squires EJ, Mahmoodi H, Wu J, Agarwal S, Hadley M. A comparativeevaluation of thiobarbituric acid methods for the determination of malondialde-hyde in biological materials. Free Radic Biol Med 1993;15:353–63.

[27] Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR.Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem1982;126(1):131–8.

[28] Bannister JV, Calabrese L. Assays for superoxide dismutase. Methods BiochemAnal 1987;32:279–312.

[29] Aebi H. Catalase in vitro. Meth Enzymol 1984;105:121–6.[30] Flohe L, Gunzler WA. Assays of glutathione peroxidase. Methods Enzymol 1984;

105:114–21.[31] Bradford MM. A rapid and sensitive method for the quantitation of microgram

quantities of protein utilizing the principle of protein-dye binding. Anal Biochem1976;72:248–54.

[32] Beneytez EG, et al. Analysis of grape and wine anthocyanins by HPLCMS. J AgricFood Chem 2003;51:5622–9.

[33] Wang H, et al. Characterisation of anthocyanins in grape juices by ion trap liquidchromatography–mass spectrometry. J Agric Food Chem 2003;51:1839–44.

[34] Palou M, Priego T, Sanchez J, Torrens JM, Palou A, Pico C. Moderate caloricrestriction in lactating rats protects offspring against obesity and insulinresistance in later life. Endocrinology 2010;151:1030–41.

[35] Madeira SV, Auger C, Anselm E, Chataigneau M, Chataigneau T, Soares de Moura R,et al. eNOS activation induced by a polyphenol-rich grape skin extract in porcinecoronary arteries. J Vasc Res 2009;46(5):406–16.

[36] Otani H. Oxidative stress as pathogenesis of cardiovascular risk associated withmetabolic syndrome. Antioxid Redox Signal 2011;15(7):1911–26.

[37] James AM, Collins Y, Logan A, Murphy MP. Mitochondrial oxidative stress and themetabolic syndrome. Trends Endocrinol Metab 2012;23(9):429–34.

[38] Minami N, Imai Y, Hashimoto J, Abe K. Contribution of vascular nitric oxide tobasal blood pressure in conscious spontaneously hypertensive rats and normo-tensive Wistar Kyoto rats. Clin Sci (Lond) 1995;89(2):177–82.

[39] Kao YH, Hiipakka RA, Liao S. Modulation of endocrine systems and food intake bygreen tea epigallocatechin gallate. Endocrinology 2000;141(3):980–7.

[40] Tsuda T. Regulation of adipocyte function by anthocyanins; possibility ofpreventing the metabolic syndrome. J Agric Food Chem 2008;56:642–6.

[41] Lu C, ZhuW, Shen CL, GaoW. Green tea polyphenols reduce bodyweight in rats bymodulating obesity-related genes. PLoS One 2012;7(6):e38332.

[42] Devaskar SU, Thamotharan M. Metabolic programming in the pathogenesis ofinsulin resistance. Rev Endocr Metab Disord 2007;8(2):105–13.

[43] Bastard JP, Maachi M, Lagathu C, et al. Recent advances in the relationship betweenobesity, inflammation, and insulin resistance. Eur Cytokine Netw 2006;17:4–12.

[44] Zunino S. Type 2 diabetes and glycemic response to grapes or grape products. J Nutr2009;139:1794S–800S.

[45] Pinent M, Blay M, Bladé MC, Salvadó MJ, Arola L, Ardévol A. Grape seed-derivedprocyanidins have an antihyperglycemic effect in streptozotocin-induced diabeticrats and insulinomimetic activity in insulin-sensitive cell lines. Endocrinology2004;145(11):4985–90.

[46] Evans JL. Antioxidants: do they have a role in the treatment of insulin resistance?Indian J Med Res 2007;125(3):355–72.

[47] Roy D, Perreault M, Marette A. Insulin stimulation of glucose uptake in skeletalmuscles and adipose tissues in vivo is NO dependent. Am J Physiol 1998;274:E692–9.

[48] Balon TW, Nadler JL. Evidence that nitric oxide increases glucose transport inskeletal muscle. J Appl Physiol 1997;82:359–63.