Antioxidant Effects of Oral Ang-(1-7) Restore Insulin Pathway and RAS Components Ameliorating Cardiometabolic Disturbances in Rats

In prevention studies of metabolic syndrome (MetS), Ang-(1-7) has shown to improve the insulin signaling. We evaluated the HPβCD/Ang-(1-7) treatment on lipid metabolism, renin-angiotensin system (RAS) components, oxidative stress, and insulin pathway in the liver and gastrocnemius muscle and hepatic steatosis in rats with established MetS. After 7 weeks of high-fat (FAT) or control (CT) diets, rats were treated with cyclodextrin (HPβCD) or HPβCD/Ang-(1-7) in the last 6 weeks. FAT-HPβCD/empty rats showed increased adiposity index and body mass, gene expression of ACE/ANG II/AT1R axis, and oxidative stress. These results were accompanied by imbalances in the insulin pathway, worsening of liver function, hyperglycemia, and dyslipidemia. Oral HPβCD/Ang-(1-7) treatment decreased ACE and AT1R, increased ACE2 gene expression in the liver, and restored thiobarbituric acid reactive substances (TBARS), catalase (CAT), superoxide dismutase (SOD), insulin receptor substrate (Irs-1), glucose transporter type 4 (GLUT4), and serine/threonine kinase 2 (AKT-2) gene expression in the liver and gastrocnemius muscle improving hepatic function, cholesterol levels, and hyperglycemia in MetS rats. Overall, HPβCD/Ang-(1-7) treatment restored the RAS components, oxidative stress, and insulin signaling in the liver and gastrocnemius muscle contributing to the establishment of blood glucose and lipid homeostasis in MetS rats.


Introduction
The angiotensin-converting enzyme (ACE)2/angiotensin-(1-7)/Mas receptor axis belongs to the renin-angiotensin system (RAS) and has antiproliferative, antioxidant, and anti-inflammatory properties. This axis induces beneficial effects in hypertension, glucose intolerance, and insulin resistance (IR), and it acts in a counter-regulatory way to another important RAS axis, ACE/Ang II/AT1 receptor [1]. Imbalances in the actions of RAS components can trigger many pathological processes and disturbances in the meta-bolic functions of the liver and muscle, such as those observed in the metabolic syndrome (MetS) and diabetes mellitus type 2 (DM2) [2][3][4]. Previous studies have shown the correlation between increased oxidative stress and IR [5,6] and hyperactivity of the ACE/Ang II/AT1 axis [7,8]. Increased oxidative stress impairs glucose uptake in the muscle and in the liver, decreasing insulin secretion of pancreatic β cells in DM2 and MetS states [5,6,9].
A high-fat (FAT) diet has been reported as risk factor RAS components for the development of DM2 and MetS [10,11]. Ang-(1-7) prevents the damage in the insulin pathway in the gastrocnemius muscle and liver in the development of MetS [10,12]. However, there are no studies on the effect of an oral Ang-(1-7) treatment on the established MetS correlating oxidative stress, insulin signaling pathway, and RAS components balance. Thus, we evaluated whether Ang- (1)(2)(3)(4)(5)(6)(7) included in the oligosaccharide hydroxypropylβ-cyclodextrin (HPβCD) can be used as treatment in MetS already established in rats fed with FAT diet on lipid homeostasis, RAS axis components, oxidative stress, insulin pathway, and liver function. HPβCD has been reported to protect the Ang-(1-7) peptide during passage through the gastrointestinal tract after oral administration [13,14].

Materials and Methods
2.1. Ethical Approval. All procedures were performed in accordance with the Guidelines for Ethics in Care of Experimental Animals. The project was approved by the animal ethics committee of the Federal University of Ouro Preto protocol 2011/31.

Fasting Glucose and Serum Lipid Profile Analysis.
At the end of the experiment, after euthanasia by instant decapitation of animals fasted overnight, blood samples (2 to 3 mL) were collected and centrifuged (8000g, 4°C, 6 min) to separate the serum for determination of fasting glucose, total cholesterol, LDL, high-density lipoprotein (HDL), alanine aminotransferase (ALT), and aspartate aminotransferase (AST). The serum was aliquoted and stored at (−80°C) to conduct the biochemical analyses. The analyses were performed using individual commercial kits (Labtest, Lagoa Santa, MG, Brazil) according to the instructions provided by the manufacturer. Analyses were performed in the Pilot Laboratory of Clinical Analyses (LAPAC/UFOP).
2.5. Activity of Superoxide Dismutase and Catalase. Frozen liver and gastrocnemius muscle samples (100 mg) were homogenized in phosphate buffer (pH 7.4) and centrifuged at 12.000xg for 10 min at 4°C. Elisa reader at 570 nm was used to determine SOD activity as previously described [6,16]. CAT activity was measured by the rate of decrease of the hydrogen peroxide (H 2 O 2 ) at 240 nm. The total protein content in samples of organ homogenates was determined by using the Bradford method [17]. All results were expressed as activity per protein milligram.
2.6. Substance Reactive to Thiobarbituric Acid. The formation of TBARS during an acid warming reaction was used for lipid peroxidation index [6]. Frozen samples of the liver and gastrocnemius muscle (100 mg) were homogenized in KPE (potassium phosphate-EDTA) buffer (pH 7.4) and centrifuged (10.000xg, 10 min at 4°C). The supernatant was collected and mixed with 1 mL of 10% trichloroacetic acid and 1 mL of 0.67% thiobarbituric acid. After this, it was heated in a boiling water bath for 30 min. TBARS were determined from the absorbance at 532 nm. Data series were expressed in nmol/mg protein.

Histological Analyses.
Fragments of the liver were fixed in 10% formalin for 72 h, dehydrated, cleaned, and embedded in paraffin. The paraffin blocks were sectioned at 4 μm thick and stained with hematoxylin and eosin (H&E). The presence or absence of areas with nonalcoholic micro and macro steatoses (NAFLD) was observed by light microscopy in the hepatic tissue. Representative photomicrographs were obtained with a Leica BM5000 microscope coupled to a Leica DFC 300 FX camera in RGB mode, using a 40x magnification lens.
2.9. Statistical Analysis. Results are expressed as means ± SEM. Data were analyzed for Kolmogorov-Smirnov normality and followed the standard normal distribution. After, they were evaluated by two-way ANOVA, followed by Fisher's LSD post test. Statistical analyses were performed with GraphPad Prism software (version 6.0, San Diego, USA). Statistical significance was set at p < 0 05.

Evaluation of MetS Establishment.
In the seventh week of diets, the FAT-HPβCD/empty rats showed symptoms of MetS such as increased fasting blood glucose, MAP, HR, and body mass, compared to CT-HPβCD/empty rats (Table 1).
No difference was observed in SOD activity in the gastrocnemius muscle (Figure 1(f)).

mRNA Expression of the RAS Components and the
Insulin Signaling Pathway. FAT-HPβCD/empty rats showed increased angiotensinogen, ACE, AT1R, and Masr mRNA gene expression compared to CT-HPβCD/empty rats (Figures 2(a)-2(c) and 2(e)). However, FAT-HPβCD/Ang-(1-7) rats decreased ACE and AT1R increasing ACE2 gene expression compared to FAT-HPβCD/empty rats FAT-HPβCD/empty rats presented lower gene expression of the Irs-1 and Akt-2 components of the intracellular insulin pathway in the liver and gastrocnemius muscle compared to CT-HPβCD/empty rats (Figure 3(a)-(d)). In addition, FAT-HPβCD/empty group presented lower Glut-4 gene expression in the gastrocnemius muscle compared to CT-HPβCD/empty rats (Figure 3(f)). FAT-HPβCD/Ang-(1-7) rats presented increased Glut-4 gene expression in the liver compared to FAT-HPβCD/empty group (Figure 3(e)). In addition, in the FAT-HPβCD/Ang-(1-7) rats, the mRNA expression of Glut-4 became similar to CT-HPβCD/empty rats in the liver and gastrocnemius muscle (Figures 3(e) and (f)). No differences in mRNA expression of Inrs and Irs-2 were observed among all groups of rats in the liver and gastrocnemius muscle (data not shown).

Discussion
In the present study, we showed that the treatment with oral formulation of Ang- (1-7), HPβCD/Ang- (1-7), in the last six  weeks in rats fed with FAT diet for 13 weeks was efficient in restoring RAS components and biometric (body mass and adiposity index) and biochemical (triglycerides, LDL, total cholesterol, fasting glucose, and ALT) parameters, reducing oxidative stress, and improving the insulin signaling pathway in the liver and gastrocnemius muscle and macrovesicular liver damage.
In previous studies [6,11,15] using the same diet protocol, we showed that rats fed with FAT diet for 7 weeks had disorders related to human MetS, such as increased fat deposits (epididymal, retroperitoneal, and inguinal), MAP, HR, fasting glucose, ALT, and total cholesterol levels. In the present study on the seventh week, the FAT-HPβCD/empty rats already had disorders characteristic of MetS such as elevated fasting glucose, body mass, MAP, and HR, so the present study refers to treatment and not prevention to the development of MetS.
This present study showed that oral Ang-(1-7) treatment in established MetS rats normalized body mass, adiposity index, LDL cholesterol, and triglycerides compared to untreated MetS rats. These data are in agreement with studies of prevention in transgenic rat model of inducible DM2 (Tet29) [18] insulin resistance and mice (FVB/N) fed with FAT diet [4] that oral Ang-(1-7) induced reductions on fat deposits, total cholesterol, ALT levels [4], and glucose levels [18].
Studies have shown that the ACE/Ang II/AT1 receptor axis is involved with oxidative stress and IR in the DM2 and MetS states [7,8,[18][19][20]. The unbalance between reactive oxygen species (ROS) and detoxification by antioxidant enzymes can affect intracellular signaling pathways being an important mechanism for metabolic diseases [5,21]. Oxi-dative damage is related to IR in the liver and gastrocnemius muscle [21,22]. In addition, the hepatic CAT enzyme activity that is responsible for the part of H 2 O 2 elimination is reduced in the liver in situations of MetS in the rat model [6,21]. It has already been established in the literature that Ang II participates in these cardiometabolic diseases due to its prooxidant actions, while Ang-(1-7) presents regulatory actions against Ang II actions [1,20,23,24]. In fact, Cao et al.'s study [12] showed in knockout mice for ACE2 (ACE2 KO) presented IR and elevated levels of ROS in the liver. Additionally, in the prevention study in FVB/N mice fed with FAT diet [19] or in high-fructose obese mouse model [25], Ang-(1-7) improved superoxide production in epididymal fat and improved lipid metabolism associated with increased Ace2 and decreased Ace expression in the liver [25]. The present study is in agreement with these studies, where FAT-HPβCD/empty rats showed increased expression of angiotensinogen, ACE, and AT1R in the liver, increased TBARS concentration, and decreased CAT activity in the liver and in the gastrocnemius muscle. The oral treatment with Ang-(1-7) was effective in decreasing hepatic ACE and AT1R, increasing ACE2 gene expression, and restoring CAT activity and TBARS concentration in FAT-HPβCD/Ang-(1-7) rats. In addition, our data showed that Ang-(1-7) treatment increased the SOD activity in the liver, thus increasing the ability to dismutase the superoxide anion to H 2 O 2 and reducing the oxidation products by reducing TBARS concentration in FAT-HPβCD/Ang-(1-7) rats.
In prevention studies, using oral administration of HPβCD/Ang-(1-7) in transgenic DM2 mice [18] and FVB/N mice fed with FAT diet [4] showed, in epididymal adipose tissue, normalization of glucose tolerance, insulin sensitivity, Table 2: Nutritional, biometric, and biochemical parameters of rats fed with a control diet or high-fat diet for 13 weeks and treated with HPβCD or HPβCD/Ang-(1-7) during the last 6 weeks of the diet.
Oxidative stress plays an important role in decreasing insulin sensitivity in the liver. EROs are produced by many processes [9], and in skeletal muscle cells, the activation of NADPH oxidase induced by Ang II may worsen insulin signaling [26], 2006). The ACE2/Ang-(1-7)/Mas axis, that counterregulating effects of the ACE/Ang II/AT1R axis, is involved in the reduction of insulin resistance through antioxidant effects [8,12]. Cao et al. [12] demonstrated in HepG2 cells that Ang-(1-7) protects against oxidative stress by inhibiting the expression of NAPDH oxidase contributing to the increasing levels of ROS that can lead to the activation of Nterminal c-Jun kinases (JNKs), which can phosphorylate IRS proteins [27]. Cao et al. [12] showed that overexpression of ACE inhibited JNK phosphorylation, leading to decreased phosphorylation of the IRS-1 serine residue, which increased glucose uptake in HepG2 cells. In addition, Ang-(1-7) increases the protein expression of GLUT-4 in the skeletal muscle of ACE2 KO mice [27] and induces the phosphorylation of kinase that participates in the translocation of GLUT-4, PI3K-C2alpha, in human endothelial cells [28] in response to insulin stimulation [24,29]. Accordingly, data from the present study show that Ang-(1-7) treatment, despite not reversing the expression of all evaluated components of the insulin pathway, made the expression of IRS-1, AKT-2, and GLUT-4 in the liver and AKT-2 and GLUT-4 in the muscle similar to the levels of expression of the control rats (HPβCD/empty). Additionally, Williams et al. [30] showed that adult male C57BL/6J mice receiving a high lipid diet for 11 weeks and treated with Ang-(1-7) infused during the last 3 weeks of diet showed increased GLUT-4 in the skeletal muscle that were sufficient to improve insulin resistance and glucose uptake. All these data together suggest that the beneficial effect of Ang-(1-7) on improving the insulin signaling pathway is probably due to restoring RAS components and redox balance in the liver and gastrocnemius muscle in MetS rats.
Hepatic IR correlates with increased fat and steatosis in the liver [3,6,11]. In our present study, there is increased hepatic fat accumulation in FAT-HPβCD/empty rats which was attenuated by treatment with HPβCD/Ang-(1-7). These data are in accordance with the prevention study of Feltenberger et al. [19] in FVB/N mice fed with FAT diet in which HPβCD/Ang-(1-7) reduced total weight and steatosis in the liver. Our hypothesis, based on the present data, is that the composition of FAT diet increased the circulation of free fatty acids, which could be captured and undergo lipid peroxidation products by the ROS leading to a process of hepatic steatosis, probably by Ace and AT1R contribution [2,3,21]. Furthermore, the steatosis may be related to hepatic IR resulting from cell injury or hepatic inflammation [19]. In the present study, the treatment with HPβCD/Ang-(1-7) was effective in restoring this damage in the liver of FAT-HPβCD/Ang-(1-7) rats by normalizing body mass, adiposity index, LDL cholesterol, and triglycerides; decreasing ACE and AT1R; increasing Ace2 gene expression; and reversing lipid peroxidation in the liver and gastrocnemius muscle.
In conclusion, our data in rats with stablished MetSinduced FAT diet for 13 weeks and orally treated with HPβCD/Ang-(1-7) in the last six weeks show that Ang-(1-7) was efficient in normalizing lipid metabolism, improving the insulin signaling pathway in the liver and gastrocnemius muscle, function, and macrovesicular liver damage, probably by restoring the unbalance between RAS axis and reducing oxidative stress in the liver and gastrocnemius muscle. This protective effect of Ang-(1-7) in the liver and muscle can probably contribute in reversing the hyperglycemia and dyslipidemia in MetS-induced FAT diet rats.

Data Availability
The [DATA TYPE] data used to support the findings of this study are included within the article.

Conflicts of Interest
The authors declare no conflicting financial interests.