Regulatory Peptides 178 (2012) 64–70

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Regulatory Peptides

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Increased circulating angiotensin-(1–7) protects white adipose tissue againstdevelopment of a proinflammatory state stimulated by a high-fat diet

Sérgio Henrique S. Santos a,c,1, Luciana Rodrigues Fernandes b, Camila Santos Pereira c,André L. Senna Guimarães c, Alfredo M.B. de Paula c, Maria José Campagnole-Santos a,Jacqueline Isaura Alvarez-Leite b, Michael Bader d, Robson Augusto S. Santos a,⁎a National Institute of Science and Technology (INCT-NanoBiofar), Physiology Department, Biological Sciences Institute (ICB), Federal University of Minas Gerais (UFMG),Belo Horizonte, MG, Brazilb Laboratory of Nutritional Biochemistry, Department of Biochemistry, Biological Sciences Institute, UFMG, Belo Horizonte, MG, Brazilc Laboratory of Health Science, Postgraduate Program in Health Sciences; UNIMONTES, Montes Claros, MG, Brazild Max Delbrück Center for Molecular Medicine (MDC), Berlin-Buch, Germany

⁎ Corresponding author at: Laborátorio de Hipertensãe Biofisica, Universidade Federal de Minas Gerais,31270‐901, Belo Horizonte, MG, Brazil. Tel./fax: +55 31

E-mail addresses: sergiosousas@hotmail.com (S.H.S.robsonsant@gmail.com (R.A.S. Santos).

1 Sérgio H.S. Santos' current address: DepartamentoHorizonte, MG, Brazil.

0167-0115/$ – see front matter © 2012 Elsevier B.V. Alldoi:10.1016/j.regpep.2012.06.009

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 20 March 2012Received in revised form 28 May 2012Accepted 22 June 2012Available online 29 June 2012

Keywords:Angiotensin-(1–7)MetabolismHyperlipidic dietObesityInflammation

Introduction: The aim of the present study was to evaluate the effect of a transgenic-induced chronic increaseof Ang-(1–7) on the expression of inflammatory markers in adipose tissue and the metabolic profile in ratstreated with high-fat diet.Research design and methods: Transgenic rats expressing an Ang-(1–7)-producing fusion protein (TGR L-3292)and Sprague Dawley (SD) control rats 4 weeks oldwere treated for 8 weekswith a high-fat diet. Food intake andbody weight were measured once a week. Glucose-tolerance and insulin sensitivity tests were performed oneweek before the sacrifice. At the end of the experiment plasma lipid concentrations were measured in TGRand SD rats. Adipose tissue were weighted and corrected by the body weight. Proinflammatory markers inadipose tissue were analyzed using Western-blotting, real time-PCR and immunohistochemistry.Results: High-fat diet TGR rats presented increased HDL cholesterol levels and decreased abdominal fat mass,without changes in food intake. In addition, rats with increased Ang-(1–7) levels had lower body weight.

Molecular analysis revealed decreased IL-1β and COX-2 in adipose tissue.Conclusions: Taken together, these results show that chronic high circulating angiotensin-(1–7) levels protectagainst metabolic stress induced by a high-fat diet decreasing the proinflammatory profile of adipose tissue.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

The prevalence of obesity is markedly rising in the world [1,2] andis of considerable concern given the strong association betweenobesity, dietary fat intake and cardiometabolic diseases [3,4]. Obesityis characterized by the accumulation of body fat. The body can adjustthe mix of metabolic fuels it oxidizes so that alcohol, carbohydrate,and protein intakes are tightly regulated [4,5]. In fact, the body canachieve carbohydrate and protein balances quickly. However, thebody has a poor autoregulatory system for fat and an almost unlimitedability to store it [4]. Ultimately, obesity is caused by a positive energybalance and presently dietary fat intake is considered an important con-tributor to energy balance [4]. In addition, several authors consider

o, Departamento de FisiologiaAv Antonio Carlos 6627-ICB,3499 2924/2956.Santos),

de Farmacologia UFMG/Belo

rights reserved.

obesity as a local and systemic inflammatory state associated with fattissue over-production of many cytokines [6,7].

Adipose tissue is a complex population of cells that modulate notonly adipose tissue biology, but also insulin sensitivity, the reproductiveand endocrine systems, immunity, and inflammation [6]. There areseveral evidences suggesting that inflammation plays a major role inthe initiation and maintenance of obesity through adipogenesis[2,6–10]. White adipose tissue (WAT) becomes inflamed during adi-pose tissue hypertrophy due to infiltration bymacrophages that secreteproinflammatory cytokines, including tumor necrosis factor TNF-α andIL-1β [11]. Another important enzyme involved in the production ofseveral inflammatory mediators is cyclooxygenase 2 (COX2), whichparticipates in obesity-related disturbances [12,13].

There is an increasing body of evidence showing the involvementof the renin-angiotensin (ANG) system (RAS) in inflammatory dis-eases [14–16]. Indeed, inflammatory cells have several componentsof the RAS [15], which are upregulated in some diseases such as rheu-matoid arthritis [16,17].

The RAS is classically described as an important modulator ofblood pressure and hydroelectrolytic balance. More recent studies

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demonstrated the involvement of RAS with metabolic regulation[18,19]. Results from experimental animals and humans suggestthat obesity activates the RAS arm composed of ACE/Ang II/AT1[19,20]. It is well known that adipose tissue expresses all componentsof the RAS and is markedly involved in obesity in which augmentedexpression of angiotensinogen and increased tissue and circulatinglevels of Ang II are observed [19]. On the other hand, recent studiesindicate that the counterregulatory arm of RAS composed of theACE2/Ang-(1–7)/Mas axis is able to produce an improvement inlipid and glucose metabolism decreasing body fat [21–24]. The firststudy correlating Ang-(1–7) with metabolism showed that Mas re-ceptor deficiency in FVB/N mice presented a metabolic state similarto the metabolic syndrome with dyslipidemia, hypertension, lowerglucose tolerance and insulin sensitivity, hyperinsulinemia, hyper-leptinemia, decreased glucose uptake in white adipose cells, and anincreased adipose tissue mass [22]. A more recent study showedthat transgenic rats with increased plasma Ang-(1–7) caused by over-expression of an Ang-(1–7)‐producing fusion protein presentedenhanced glucose tolerance, insulin sensitivity, and insulin-stimulatedglucose uptake [25]. In addition, these transgenic rats presented de-creased triglycerides and cholesterol levels, as well as a significant de-crease in abdominal fat mass associated with augmented adiponectinproduction [25].

In the present study, we sought to evaluate the expression ofpro-inflammatory markers in adipose tissue and the metabolic profilein transgenic rats (TGR) presenting a significant increase in Ang-(1–7)plasma levels treated with high-fat diet. We used the model TGR(A1–7)3292 (TGR),which expresses anAng-(1–7)‐producing fusion protein,that results in a chronic elevation of circulating Ang-(1–7) levels[25,26].

2. Material and methods

2.1. Animals

Ten to twelve week-old male TGR and control Sprague Dawley(SD) rats were obtained from the transgenic animal facilities of theLaboratory of Hypertension, Federal University of Minas Gerais, BeloHorizonte, Brazil. All experimental protocols were performed inaccordance with the international guidelines for animal care andapproved by local authorities. The animals were maintained undercontrolled light and temperature conditions, and had free access towater and chow diet.

2.2. High-fat hypercaloric diet preparation

The high-fat diet was prepared according to the standards of theAssociation of Official Analytical Chemists used as described previ-ously [27,28]. Diet macronutrients were weighed and mixed homoge-neously to form a soft dough. The diet was stored separately in arefrigerator (4 °C) in hermetically sealed plastic boxes. The standarddiet (Purina–Labina®) used for the regular maintenance of our ratsis composed of 50.30% carbohydrates, 41.90% protein and 7.80% fatpresenting a total of 2.18 kcal per 1 g of the diet. The high-fat dietwas composed of 24.55% carbohydrates, 14.47% protein and 60.98%fat, presenting a total of 5.28 kcal per 1 g of the diet. All the high-fatdiet components were purchased from Rhoster® LTDA (São PauloSP; Brazil).

2.3. Measurements of body weight, food intake and tissue collection

The rats were individually housed and the food intake was mea-sured every other week, during two consecutive days in order to ob-tain food efficiency (food intake/body weight).

Overnight fasted rats were killed by decapitation and samples ofblood, epididymal and retroperitoneal white adipose tissues were

collected, weighed and immediately frozen in dry ice and stored at−80 °C for posterior analysis.

2.4. Determination of blood parameters

Serum was obtained after centrifugation (3200 rpm for 10 min at4 °C). Total serum cholesterol and triglycerides were assayed usingenzymatic kits (Doles®, Goias, Brazil). ELISA kits were used to mea-sure serum adiponectin (AdipoGen®, Seoul, Korea), leptin (R&Dsystems®, Minneapolis, USA), and insulin (Linco Research Inc.®,USA) serum levels. Leptin and adiponectin secretion was calculatedas serum levels normalized to fat tissue weight.

2.5. Glucose tolerance and insulin sensitivity tests

For the glucose-tolerance test, D-glucose (2 mg/g of body weight)was intraperitoneally injected into overnight fasted rats. Glucoselevels from tail blood samples were monitored at 0, 15, 30, 60 and120 min after injection using an Accu-Check glucometer (RocheDiagnostics Corp.®, Indianapolis, Indiana, USA). Insulin sensitivitytest was performed on overnight fasted rats, after intraperitoneal in-jection of insulin (0.75 U/kg body weight—Sigma®, St Louis, MO,USA). Tail-blood samples were taken at time points 0, 15, 30 and60 min after injection for the measurement of blood glucose levels.

2.6. Western blotting analysis

Proteins were extracted from epididymal adipose tissue samples(~300 mg) of TGR and SD rats and 30 μg of protein were resolvedon SDS-PAGE gels (10%), transferred onto nitrocellulose membranesand blocked with 5% pure milk diluted in 1× TBS. The blot was probedwith COX2 enzyme antibody (Cell Signaling, USA; 1:1000), diluted in1× blocking buffer, washed in 1× TBS with 1% of Tween 20, and thenincubated with either IRDye 800 anti-goat secondary antibody(Rockland Immunochemicals®, Gilbertsville, Pa.) diluted to 1:10,000in blocking buffer followed by further washes with PBS-Tween 20and one wash in TBS. The blots were viewed using an infrared scannerfrom LICOR and analyzed using the Odyssey software.

2.7. Reverse transcription and real time PCR

The total RNA from the adipose tissues was prepared using theTRIzol reagent (Invitrogen Corp.®, San Diego, California, USA), treatedwith DNAse and reverse transcribed with M-MLV (Invitrogen Corp.®)using random hexamer primers. The levels of TNF-α, IL-1β mRNAwere determined by real-time PCR using SYBR Green reagent(Appllied Biosystems®—USA) in an ABI Prism 7000 platform (ApplliedBiosystems) with primers TNF-α‐FW: 5′-TGC CTC AGC CTC TTC TCATT-3′, and TNF-α–RV: 5′-CCC ATT TGG GAA CTT CTC CT-3′ andIL-1β-FW: 5′ATG GCA ACT GTC CCT GAA CTC ACC T‐3′ andIL-1β-RV: 5′CAG GAC AGG TAT AGA TTC AAC CCC T‐3′. Gene expres-sionwas normalized to the endogenous glyceraldehyde 3-phosphatedehydrogenase (GAPDH) FW: 5′‐AAC GAC CCC TTC ATT GAC CTC andRV: 5′-CTT CCC ATT CTC AGC CTT GAC T. The relative comparative CTmethod of Livak and Schmittgen was applied to compare gene ex-pression levels between groups, using the equation 2−ΔΔCT [29].

2.8. Hematoxylin and eosin staining

Epididymal fat tissues fromTGRand SD ratswerefixed in Bouin's so-lution and embedded in paraffin serially sectioned at 5 μm, stainedwithhematoxylin and eosin (HE), and evaluated under a conventional lightmicroscope using an Olympus BX50microscope (Tokyo, Japan). Imagesof fat tissue areas (×10 ocular and ×40 objective lenses) were capturedwith Evolution LC Color light camera (MediaCybernetics®, USA). A total

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area of 1.84 mm2, containing at least 100 fat cells for each sample, wasmeasured using the NIS-ELEMENTS BR 2.30 Software (ESTADO, USA®).

2.9. Immunohistochemical reactions

For immunohistochemical reactions, 3 μm-thick sections weremounted on organosilane‐coated slides. The primary mouse monoclo-nal antibodies against anti‐IL1-β (clone H-153, ABCAM®, Cambridge,UK) and TNF-α (clone ab66579, ABCAM®, Cambridge, UK) cytokineswere detected with the aid of an LSAB™ kit (product # K0690;Dako®, Glostrup, Denmark) employing chromogen diaminobenzidinefor color development. Slides were finally counterstained with Mayer'shematoxylin and were mounted. Negative controls were obtained bysubstituting normal whole rabbit serum (product # X0902; Dako) forthe primary antibodies. Positive controls were applied according tomanufacturer's instructions. Only cells that presented brown cytoplasmstaining were considered positive.

2.10. Statistical analysis

All data were transferred to the GraphPad Prism software (Version4.0®; San Diego, California, USA) and submitted to specific testswith a statistic confidence of 95% (pb0.05). Data are expressed as

Fig. 1. Body weight, fat weight, adipocyte area and hematoxylin/eosin staining in TGR, SD, TG(A), epididymal adipose tissue weight (B), retroperitoneal adipose tissue weight (C), adipocy(F). Curves in A are significantly different. * pb0.05 between SD and TGR; & pb0.05 betweTGRstd. The two-way ANOVA test was used in A, B and C.

the mean±SEM. The statistical significance of the differences inmean values between TGR and SD rats groups were assessed withthe unpaired Student's t-test or two-way ANOVA (body weight, glu-cose tolerance and insulin sensibility tests).

3. Results

3.1. Body composition and food intake

As shown in Fig. 1 the body weight of TGR rats (N=7) was lowerthan that of SD control rats (N=7) during the period of thehyperlipidic diet treatment. At the end of the experiment the bodyweight was 418.3±19.78 g and 355.8±12.34 g in SD and TGR respec-tively. Analysis of epididymal adipose tissue (0.01895±0.00153 g/gBW in SD vs 0.01303±0.000848 g/g BW in TGR) and retroperitonealadipose tissue (0.02024±0.00133 g/g BW in SD vs 0.01305±0.00146 g/g BW in TGR) demonstrated a reduced fat composition inTGR (Fig. 1). Despite of a decreased fatmass, adipocyte areawas not sig-nificantly diminished in TGR as demonstrated by the morphometricalassessment of the adipose tissue (Fig. 1).

In order to confirm obesity induced by the high-fat diet we mea-sured the body weight and adipose tissue weight of transgenic rats(TGRstd) and the Sprague Dawley rats (SDstd) treated with standard

R standard chow (TGRstd) and SD standard chow (SDstd) rats. Body weight over weekste area (D), SD hematoxylin and eosin staining (E), TGR hematoxylin and eosin stainingen SD and SDstd; # pb0.05 between TGR and TGRstd; + pb0.05 between SDstd and

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chow and we observed a significantly lower body weight in bothTGRstd and SDstd when compared with those of high-fat fed animalsas showed in Fig. 1.

Every other week, food efficiency (food intake corrected by bodyweight), was measured and no significant alteration in food con-sumption was observed as shown in the table. The measurementwas performed during two consecutive days and the result representsthe average value for food consumption of the entire treatmentperiod.

3.2. Lipid and glycemic parameters

Two weeks before the sacrifice, SD and TG presented similar insu-lin sensitivity and glucose tolerance (Fig. 2). Total cholesterol, HDLcholesterol and triglycerides were measured in serum after sacrifice.We observed a significant increase in HDL cholesterol (40.55±2.58 mg/dl in SD vs 55.49±3.16 mg/dl in TGR rats) without alter-ations in the other plasma lipids (Fig. 2).

3.3. Adipokines and insulin circulating levels

As shown in Table 1 serum circulating levels of insulin, adiponectinand leptin in TGR were similar to those of SD rats.

Fig. 2. Lipid and glycemic profile in TGR and SD rats. Insulin sensitivity test (A), glucose toleran(E). Data are presented as mean±SEM; * pb0.05.

3.4. Inflammatory markers in adipose tissue

Epididymal adipose tissue was used to perform Western blot forCOX2 and to determine the gene expression of proinflammatory cyto-kines. The results demonstrated a significant decrease in COX2 levelsin the fat tissue of TGR (0.08741±0.01877 AU) compared with thatof the SD (0.2541±0.0578 AU). An important reduction in IL-1βwas also observed (1.07±0.188 AU in SD vs 0.472±0.109 AU inTGR). TNF-α expression was similar in both groups (1.226±0.397 AU in SD vs 1.948±0.511 AU in TGR). Immunohistochemistryanalysis of adipose tissue illustrated the IL-1β and TNF-α results(Fig. 3).

4. Discussion

In this study we took advantage of TGR-L3292 rats, which presentelevated levels of circulating Ang-(1–7) in order to further addressthe metabolic role of this heptapeptide. The response to high-fatdiet of SD and TGR rats was evaluated. The main differences observedin the TGR rats in comparison with the SD rats were: decreased bodyweight and adipose tissue mass associated with increased circulatingHDL cholesterol and diminished pro-inflammatory markers in adi-pose tissue.

Despite an unchanged food intake and adipocyte diameter, TGRanimals exhibited an important reduction in abdominal fat mass as

ce test (B), plasma total cholesterol (C), plasma HDL cholesterol (D), plasma triglycerides

Table 1Food intake, adipokines and insulin in fasted SD and TGR rats.

Measurements SD TGR

Food intake (g/kg BW/day) 52.38±3.66 57.06±3.04Leptin (pg/ml) 1.359±0.272 1.046±0.323Adiponectin (ng/ml) 16,437±652 14,430±750Insulin (ng/ml) 0.950±0.131 1.014±0.126

Data are expressed as mean±SEM.

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compared with SD rats (epididymal and retroperitoneal adiposetissue) and decreased body weight gain. The absence of alterations infood intake indicates that the difference in fat mass was not inducedby decreased appetite and probably it was due to changes in metabolicregulation. These results point out to a decrease in adipocyte cell

Fig. 3. Adipose tissue inflammatory markers. Western blot for COX2-epididymal adipose tichemical reaction for IL-1β in epididymal adipose tissue (C), TGR immunohistochemicaepididymal adipose tissue (E), SD immunohistochemical reaction for TNF-α in epididymal atissue (G). Data are presented as mean±SEM; * pb0.05.

proliferation, which is commonly induced by a high-fat diet, justifyingthe lower fat mass. Jayasooriya et al. [30] reported that a mouse lackingthe ACE gene presented increased energy expenditure with reduced fatmass associated with increased Ang-(1–7) plasma levels [30]. Corrobo-rating these findings, it was demonstrated that FVB/N mice with thedeletion of the Ang-(1–7), Mas, receptor presented increased body fatwithmetabolic disturbance [22,31]. These observations suggest a pivot-al role of the RAS in fat mass control.

Recently it has been demonstrated that, at basal state, TGR ratspresent improved glycemic and lipid profiles [25]. However, in theprevious study it was not evaluated whether this profile could be al-tered under metabolic stress or in a metabolic disease state. In thepresent study, we observed an important decrease in both adiposetissue mass and body weight despite of the high-fat diet feeding, inthese rats. Nevertheless, the insulin sensitivity and glucose tolerance

ssue (A), RT-PCR expression of IL-1β‐epididymal adipose tissue (B), SD immunohisto-l reaction for IL-1β in epididymal adipose tissue (D), RT-PCR expression of TNF-α‐

dipose tissue (F), TGR immunohistochemical reaction for TNF-α in epididymal adipose

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test, as well as the total cholesterol and triglyceride levels, were notimproved in TGR rats when compared with those in SD control rats.The prevention of body weight and adiposity gain point to a potentialregulatory role of Ang-(1–7) in metabolism. However, the beneficialeffects can be attenuated by a continuous deleterious metabolic stim-ulus (high-fat diet) as evidenced by the non‐altered insulin sensitivi-ty and glucose tolerance in TGR. Previous studies have demonstratedthe prejudicial effects of high-fat diet on metabolic performance[32–34], which corroborate our results.

Several studies reported that TNF-α and IL-1β are increased in obe-sity and this increase is correlated with numerous metabolic disorders[35]. In patients with obesity, adipose tissue is characterized by low-intensity inflammation and increased secretion of cytokines [8,35,36].Tumor necrosis factor α (TNF-α), interleukin-6 and interleukin-1 βmay be themost pernicious, since they alter adipose tissue function, in-fluence adipogenesis, and are involved in the metabolic complicationsof obesity [8,36]. A recent work by da Silveira et al. [16] demonstratedthat the activation of the Ang-(1–7) Mas receptor exerts significantanti-inflammatory effects in two models of arthritis. Overall, geneticdeletion of the receptor was associated with slight worsening of someaspects of arthritis in mice [16]. A mechanism that can partially explainthis decreased pro-inflammatory profile is the regulation of thePPAR-gamma receptor by the Ang-(1–7)/Mas axis as described recentlyby Mario et.al using Mas-knokcoutmice [31].

IL-1β, a cytokine mainly produced by macrophages, endothelialcells, lymphocytes and epidermal cells, plays a role in the immune re-sponse and in inflammatory process, including obesity [37,38]. IL-1β,IL6 and TNF-α [6,39] are also produced by adipocytes and pre-adipocytes especially in unbalanced metabolic states [40,41]. Circulat-ing levels of IL-1β are correlated with the BMI of obese alcoholicsubjects [42] and are increased in overweight and obese subjectscompared with lean subjects [43]. In adipose tissue from obese sub-jects, the total release of IL1B was comparable with that of TNF-α[44]. The IL1 receptor is also overexpressed in the adipose tissue ofmice with diet-induced and genetic obesity, as it is also in the subcu-taneous adipose tissue of obese patients [45,46]. All these findingssupport the hypothesis that IL1 signaling pathways, more specificallyIL1B signaling in adipose tissue, may play an important role inobesity-linked insulin resistance. Silveira et al. showed in a micemodel of arthritis that treatment with Mas receptor agonist, AVE, in-duced the decreased expression of IL-1β [16]. Accordingly, our resultsdemonstrated an important decrease in IL-1B expression in adiposetissue of TGR animals pointing to an anti-inflammatory and anti‐obesogenic effect of Ang-(1–7). It remains to be determined whetherthis effect is due to an action of the peptide in adipocytes, in macro-phages [47] or both. In addition, further studies are necessary to clar-ify the absence of changes in TNF-α expression in the TGR rats treatedwith a high-fat diet. One possibility to be tested in future studies isthat plasma levels of Ang-(1–7) in our TGR (2–2.5‐fold increaseover the normal levels) may not be high enough to change TNF-αproduction in this metabolic condition.

Concerning the mechanisms involved in the prevention of aproinflammatory profile and the improvement of metabolism inTGR rats, despite the deleterious stimulus of high-fat diet, we hypoth-esize that decreased proinflammatory proteins in adipose tissue in-duced by Ang-(1–7) is able to improve metabolism. A recent studydemonstrated that the IL-1 receptor is a key mediator of high-fatdiet induced inflammation in adipose tissue and the deletion of theIL-1 receptor can partially protect against obesity-induced metabolicdysregulation in mice [48]. A similar mechanism can partially protectTGR rats against metabolic dysregulation.

A recent study suggested that cyclooxygenase (COX) 2-mediatedinflammation in fat may be crucially involved in obesity-related insu-lin resistance and fatty liver in high-fat-fed rats, increasing oxidativestress [12]. Accordingly, Hsieh et al. demonstrated that inhibition ofCOX2 activation in high fat-induced obese rats leads to attenuation

of adipose inflammation in both visceral and subcutaneous fat [13].The present results demonstrated that increased circulating Ang-(1–7)is able to decrease the expression of COX-2 in adipose tissue ofhigh-fat treated rats. Therefore, at least two pro-inflammatory mecha-nisms activated by high fat diet, increase in IL1B and COX-2, were atten-uated in TGR.

A limitation of the present study was not to have evaluated the di-rect effect of high-fat diet on plasma Ang-(1–7) levels. However, ourmain focus was to evaluate the ability of increased circulatingAng-(1–7), at the beginning of the diet treatment, to prevent WAT in-flammation. In future studies it would be important to test the effectof different diet compositions on the RAS balance and whether treat-ment with Ang-(1–7) could revert the pro-inflamatory changes in-duced by a high fat diet.

5. Conclusions

In conclusion, this study shows that increased circulating Ang-(1–7)at the beginning of the diet treatment presents a protective effectagainst the proinflammatory profile in the adipose tissue of an over-weight rat model induced by a high-fat diet. TGR animals presented de-creased body weight, increased HDL cholesterol and decreasedexpression of COX-2 and IL-1β in abdominal fat. These results supportthe putative use of Ang-(1–7) as a novel therapeutic agent for the pre-vention and treatment of obesity-related disorders.

Acknowledgments

Acknowledgments: This work was supported by a grant fromCNPq (INCT-NanoBiofar), Fapemig and CAPES.

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