Hypoglycaemic and Antioxidant Properties of Acrocomia aculeata (Jacq.) Lodd Ex Mart. Extract Are Associated with Better Vascular Function of Type 2 Diabetic Rats

nutrients Article Hypoglycaemic and Antioxidant Properties of Acrocomia aculeata (Jacq.) Lodd Ex Mart. Extract Are Associated with Better Vascular Function of Type 2 Diabetic Rats Tamaeh Monteiro-Alfredo 1,2,3,4 , Sara Oliveira 1,2,3 , Andreia Amaro 1,2,3, Daniela Rosendo-Silva 1,2,3 , Katia Antunes 4, Ana SaloméPires 2,3,5 , Ricardo Teixo 2,3,5, Ana Margarida Abrantes 2,3,5, Maria Filomena Botelho 2,3,5 , Miguel Castelo-Branco 6,7,8,9, Raquel Seiça 1, Sónia Silva 2,10, Kely de Picoli Souza 4and Paulo Matafome 1,2,3,11,*   Citation: Monteiro-Alfredo, T.; Oliveira, S.; Amaro, A.; Rosendo-Silva, D.; Antunes, K.; Pires, A.S.; Teixo, R.; Abrantes, A.M.; Botelho, M.F.; Castelo-Branco, M.; et al. Hypoglycaemic and Antioxidant Properties of Acrocomia aculeata (Jacq.) Lodd Ex Mart. Extract Are Associated with Better Vascular Function of Type 2 Diabetic Rats. Nutrients 2021,13, 2856. https:// doi.org/10.3390/nu13082856 Academic Editor: Arrigo Cicero Received: 14 July 2021 Accepted: 17 August 2021 Published: 20 August 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1Institute of Physiology, Faculty of Medicine, University of Coimbra, 3000-548 Coimbra, Portugal; [email protected] (T.M.-A.); [email protected] (S.O.); andreia.amar[email protected] (A.A.); [email protected] (D.R.-S.); [email protected] (R.S.) 2Coimbra Institute of Clinical and Biomedical Research (iCBR), Faculty of Medicine, Center for Innovative Biomedicine and Biotechnology (CIBB), University of Coimbra, 3000-548 Coimbra, Portugal; [email protected] (A.S.P.); ricar[email protected] (R.T.); [email protected] (A.M.A.); [email protected] (M.F.B.); [email protected] (S.S.) 3Clinical Academic Center of Coimbra, 3000-548 Coimbra, Portugal 4 Research Group of Biotechnology and Bioprospecting Applied to Metabolism (GEBBAM), Federal University of Grande Dourados, Dourados 79825-070, MS, Brazil; [email protected] (K.A.); [email protected] (K.d.P.S.) 5Institute of Biophysics, Faculty of Medicine, University of Coimbra, 3000-548 Coimbra, Portugal 6Visual Neuroscience Laboratory, Institute for Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, 3000-548 Coimbra, Portugal; [email protected] 7Centre for Neuroscience and Cell Biology (CNC), IBILI, University of Coimbra, 3000-548 Coimbra, Portugal 8 Institute for Nuclear Sciences Applied to Health (ICNAS), University of Coimbra, 3000-548 Coimbra, Portugal 9 Laboratório de Bioestatística Médica, Faculty of Medicine, University of Coimbra, 3000-548 Coimbra, Portugal 10 Institute of Pharmacology and Experimental Therapeutics, Faculty of Medicine, University of Coimbra, 3000-548 Coimbra, Portugal 11 Instituto Politécnico de Coimbra, Coimbra Health School (ESTeSC), Department of Complementary Sciences, 3000-548 Coimbra, Portugal *Correspondence: [email protected] Abstract: Oxidative stress is involved in the metabolic dysregulation of type 2 diabetes (DM2). Acrocomia aculeata (Aa) fruit pulp has been described for the treatment of several diseases, and recently we have proved that its leaves have phenolic compounds with a marked antioxidant effect. We aimed to assess whether they can improve metabolic, redox and vascular functions in DM2. Control Wistar (W-Ctrl) and non-obese type 2 diabetic Goto–Kakizaki (GK-Ctrl) rats were treated for 30 days with 200 mg.kg −1 aqueous extract of Aa (EA-Aa) (Wistar, W-EA-Aa/GK, GK-EA-Aa). EA-Aa was able to reduce fasting glycaemia and triglycerides of GK-EA-Aa by improving proteins related to glucose and lipid metabolism, such as GLUT-4, PPAR γ , AMPK, and IR, when compared to GK-Ctrl. It also improved viability of 3T3-L1 pre-adipocytes exposed by H 2 O 2. EA-Aa also increased the levels of catalase in the aorta and kidney, reduced oxidative stress and increased relaxation of the aorta in GK-treated rats in relation to GK-Ctrl, in addition to the protective effect against oxidative stress in HMVec-D cells. We proved the direct antioxidant potential of the chemical compounds of EA-Aa, the increase in antioxidant defences in a tissue-specific manner and hypoglycaemic properties, improving vascular function in type 2 diabetes. EA-Aa and its constituents may have a therapeutic potential for the treatment of DM2 complications. Keywords: diabetes; macaúba;bocaiúva; vascular function; polyphenols Nutrients 2021,13, 2856. https://doi.org/10.3390/nu13082856 https://www.mdpi.com/journal/nutrients Nutrients 2021,13, 2856 2 of 17 1. Introduction Diabetes mellitus (DM) is a chronic disease characterized by hyperglycaemia, resulting from a deficiency in insulin production, desensitization of its action, or both [ 1 ]. According to the International Diabetes Federation, diabetes is one of the diseases with the highest incidence in the 21st century, having increased three-fold in the last two decades, and being estimated to affect 463 million individuals in 2019 [ 2 ]. As a consequence of hyperglycaemia, both DM1 and DM2 commonly have associated complications, which have significant morbidity and mortality and a considerable economic impact. These complications can be either micro (neuro, nephro, cardio and retinopathy) or macro vascular (stroke and cardiovascular diseases) [ 3 , 4 ]. One of the main factors for its cause and progression is oxidative stress, which is involved in the pathogenesis of complications through the overproduction of reactive oxygen species (ROS) and reactive nitrogen species (RNS), further impairing the redox balance [ 5 ]. The adoption of a better lifestyle (balanced diet and exercise) may delay the development of diabetes and its complications. On the other hand, several therapeutic options are available for DM2, most of them targeted for the reduction of the glycaemia [meglitinides, biguanides, sulfonylureas (SUs), thiazolidinedione (TZD), dipeptidyl peptidase 4 (DPP-4) inhibitors, GLP-1 receptor agonists, sodium glucose cotransporters inhibitors (SGLT2) and insulin] [ 6 ]. However, a considerable number of patients do not adhere to the treatment with allopathic medicine due to side effects [ 4 ], which affect mainly liver and kidney [ 7 ]. In this perspective, a considerable amount of research is focused on developing therapeutic alternatives, which could be both inexpensive and effective with fewer side effects [4]. Brazil has the greatest biodiversity in the world, with about 20% of the species distributed in its biomes. In particular, the Brazilian Cerrado, which occupies 22% of the national territory [ 8 ], has one of the world’s richest flora, where 35% of the species are endemic [ 9 ]. Among these species, many of them are considered medicinal due to their chemical composition [ 10 ]. Acrocomia aculeata (Jacq.) Lodd. ex Mart., commonly known as bocaiúva or macaúba, is a palm native from Cerrado with therapeutic (production of remedies based on ethnopharmacological knowledge—as antidiabetic, antioxidant, analgesic etc.) and economic importance (for cooking, biodiesel, and the cosmetic industry) [ 11 ]. Its carotenoid-rich fruit pulp was suggested to have beneficial effects on the treatment of respiratory diseases, as analgesic and laxative [ 12 ] and also in decreasing serum cholesterol and glucose levels [ 13 ]. Recently, our group proved the antioxidant potential of its leaves and the relevant chemical composition, mostly of vanillic, caffeic, ferulic and gallic acid, rutin and quercetin [ 11 ]. In addition to the new findings described by us about the potential of its leaves, it is only known that they are used for bovine nutritional supplementation and in the preparation of teas for human consumption. Therefore, our goal in this study was to assess whether the antioxidant potential of A. aculeata leaves can restore redox balance and improve metabolic and vascular function of type 2 diabetic rats, as well as to disclose the underlying mechanisms in tissues involved in glucose metabolism and its vascular complications. Our results show the antioxidant and hypoglycaemic potential of EA-Aa, observed through the tissue-dependent upregulation of pathways involved in antioxidant defences and glucose and lipid metabolism. Such effects were associated with the improvement of aortic relaxation and redox state. 2. Materials and Methods 2.1. Chemicals and Antibodies Salts and organic solvents used in this study were all purchased from Lonza, SigmaAldrich/Merck, Alfa-Aesar, Fischer Scientifics and Panreac. Antibodies used were targeted to Catalase, Glo-1, GLUT2 (ab76110, ab96032, ab54460 Abcam, Cambridge, UK), GLUT4, PPARgamma, Insulin Receptor, AMPK, phospho-AMPK-Thr-172, Sirt1, phospho-Sirt1Ser47 (#2213S, #2443S, #3025S, #2532S, #2535S, #9475S, #2314S, Cell Signaling Technology, Danvers, MA, USA) NRF2 (sc-518036, Santa Cruz Biotechnology, Dallas, TX, USA) phospho- Nutrients 2021,13, 2856 3 of 17 NRF2 (Ser40) (PA5-67520, Invitrogen, Waltham, MA, USA). Calnexin and GAPDH (AB0037, AB0049-20, Sicgen, Carcavelos, Portugal) were used as loading control. 2.2. Botanical Material and Isolation of Extract Fresh A. aculeata leaves were collected as before [ 11 ] in the region of Grande Dourados, Macaúba district, state of Mato Grosso do Sul (MS) (22 ◦ 0702.4 S 54 ◦ 2836.3 W), with the permission of the Brazilian Biodiversity Authorization and Information System (Sistema de Autorização e Informação sobre Biodiversidade, SISBIO; no. 50589). A plant taxonomist identified the species, and a specimen was deposited in the herbarium (DDMS-UFGD) of the Federal University of Grande Dourados, Dourados (MS), Brazil, registration number— 5103. The aqueous extract was prepared as previously described [11]. 2.3. Cell Culture and Viability Assays Mouse (Mus musculus) preadipocyte—3T3-L1 cells (cultured with Dulbecco’s Modified Eagle’s Medium—DMEM supplemented with 10% FBS and 1% penicillin/streptomycin) [ 14 ]; and human dermal microvascular endothelial Cells (HMVec-D, cultured with EGMTM-2, Endothelial Cell Growth Medium-2, BulletKitTM) [ 15 , 16 ], maintained at 37 ◦ C and 5% CO 2 were used in the assays. To evaluate cell viability, 1 × 10 5 HMVec-D cells and 3 × 10 4 3T3-L1 cells were seeded in 96-well microplates. After 24 h, cells were incubated with different concentrations (31,25– 500 µ g.mL −1 ) of EA-Aa for 24 h. After this period, cell viability was determined through the Alamar Blue assay. Absorbance was measured at 570 nm and 600 nm in a BioTek microplate reader (BioTek, Instruments, Inc., Winooski, VT, USA) and used to calculate cell viability, according to Equation (1) [11]. Cell viability =(Abs570−Abs600)of treated cells (Abs570−Abs600)of control cells×100 (1) To evaluate the antioxidant potential of EA-Aa, both cell lines 3T3-L1 and HMVec-D cells were treated with H 2 O 2 , the oxidative stress inductor. After 80% of confluence, cells were firstly incubated with the extract for 30 min followed by H 2 O 2 (IC 50 0.125 mM in 3T3-L1 cells and 0.25 mM in HMVec-D cells) for 2 h. Equation (1) was used to calculate the protective effect of EA-Aa in cell viability. Dependence of EA-Aa effects on NRF2 pathway was evaluated through the incubation of HMVec-D cells with the NRF2 inhibitor ML385 (20 µM). 2.4. Animal Maintenance and Treatment The study was performed according to good practices of animal handling, with the approval of the Institutional Animal Care and Use Committee (ORBEA 13/2018) and the procedures performed by licensed users by the Federation of Laboratory Animal Science Associations (FELASA), conformed to the guidelines from Directive 2010/63/EU of the European Parliament for the Protection of Animals Used for Science Purpose. Male 12week-old Wistar and non-obese type 2 diabetic Goto–Kakizaki (GK) rats from our breeding colonies (Faculty of Medicine, University of Coimbra), were randomly divided in 4 groups (n= 5–7), as presented in Figure 1A, which were: Wistar control (W); Wistar treated with EA-Aa (W-EA-Aa); GK control (GK) and GK treated with EA-Aa (GK-EA-Aa). Animals were kept under standard conditions—2 animals per cage, with temperature at 22–24 ◦ C, and 50–60% humidity, and standard light cycle (12 h light/12 h darkness), with water and food (standard diet A03, SAFE, France) ad libitum [ 17 ]. EA-Aa (200 mg.kg −1 ) was added in the daily water of the animals 28 days, which received the treatment during the night and normal water during the day. The weekly average of the rats’ weight was used to determine the daily dose of EA-Aa per cage. Nutrients 2021,13, 2856 4 of 17 Nutrients 2021, 13, x FOR PEER REVIEW 4 of 19 Figure 1. Glycaemic and lipid profile of Wistar and Goto–Kakizaki (GK) rats after 30-day treatment (n = 5–7). (A) Experimental design of in vivo study. (B) Body mass evolution of treated and non-treated rats represented every 7 days. (C) Initial and final body mass of treated animals. (D) Caloric intake. (E) Fasting blood glucose in percentage of the initial value; EA-Aa promoted a decrease in fasting glycaemia of GK-EA-Aa since the 7th day of treatment in relation to the initial glycaemia. (F) Fasting glycaemia; a restoration of values is found in the end of the treatment between GK rats and W-Ctrl group. (G) Area under the curve of glycaemia along 4-week treatment period; values are reduced by EA-Aa treatment in GK rats when comparing to the control group. (H) Area Under the curve of the glycaemia along all the treatment. (I) Area under the curve of ITT (insulin tolerance test). (J) Insulin; an increase in GK-EA-Aa is presented in relation to the W-Ctrl group. (K) Triglycerides; treatment with EA-Aa decreased plasma triglycerides levels in GK. (L) Free fatty acid levels. * vs. W-Ctrl at the same point; # vs. GK-Ctrl at the same point; *,# p < 0.05; ** p < 0.01; ***,### p < 0.001. Figure 1. Glycaemic and lipid profile of Wistar and Goto–Kakizaki (GK) rats after 30-day treatment (n= 5–7). ( A ) Experimental design of in vivo study. ( B ) Body mass evolution of treated and non-treated rats represented every 7 days. ( C ) Initial and final body mass of treated animals. ( D ) Caloric intake. ( E ) Fasting blood glucose in percentage of the initial value; EA-Aa promoted a decrease in fasting glycaemia of GK-EA-Aa since the 7th day of treatment in relation to the initial glycaemia. ( F ) Fasting glycaemia; a restoration of values is found in the end of the treatment between GK rats and W-Ctrl group. ( G ) Area under the curve of glycaemia along 4-week treatment period; values are reduced by EA-Aa treatment in GK rats when comparing to the control group. ( H ) Area Under the curve of the glycaemia along all the treatment. ( I ) Area under the curve of ITT (insulin tolerance test). ( J ) Insulin; an increase in GK-EA-Aa is presented in relation to the W-Ctrl group. ( K ) Triglycerides; treatment with EA-Aa decreased plasma triglycerides levels in GK. ( L ) Free fatty acid levels. * vs. W-Ctrl at the same point; # vs. GK-Ctrl at the same point; *,# p< 0.05; ** p< 0.01; ***,### p< 0.001. Nutrients 2021,13, 2856 5 of 17 2.4.1. In Vivo Procedures and Sample Collection Body weight, fasting glycaemia, water and food intake were evaluated weekly (calculated as the mean daily consumption per rat), and an insulin tolerance test (ITT) was performed at the beginning and at the end of the treatment. For the IIT, 250 mU.kg −1 insulin (Humulin, 1000 UI.mL −1 Lilly, Lisboa, Portugal) was injected (i.p.) after 6 h fasting, followed by glycaemia measurement in the tail vein with a glucometer (Precision Xtra Meter, Abbott Diabetes Care, Amadora, Portugal) and test strips (Abbott Diabetes Care, Portugal) at time 0, 15, 30, 60 and 120 min. Response to insulin was expressed by area under the curve (AUC) [ 17 ]. Serum triglycerides were measured in the same day before insulin administration. At the end of the treatment, animals were anesthetized (i.p.) with 2:1 (v/v) 50 mg.kg −1 ketamine (100 mg.mL −1 )/2.5% chlorpromazine (5 mg.mL −1 ) and samples of blood were collected by cardiac puncture followed by cervical dislocation. Epididymal adipose tissue (EAT), liver, kidney, heart and aorta were collected, blood samples were centrifuged (2200 × g, 4 ◦ C, 15 0 ) and serum and plasma were aliquoted and stored at − 80 ◦ C for further analysis. 2.4.2. Studies of Isometric Tension of Aorta Aorta rings were mounted on stainless steel hooks under 19.6 mN basal tension in organ baths filled with aerated (95% O 2 , 5% CO 2 ) Krebs–Henseleit solution (37 ◦ C, pH 7.4) (NaCl 118.67 mmol/L; KCl 5.36 mmol/L; CaCl 2 1.90 mmol/L; MgSO 4 0.57 mmol/L; NaHCO 3 25.00 mmol/L; KH 2 PO 4 .H 2 O 0.90 mmol/L; glucose 11.1 mmol/L). After an equilibration period of 60 min, aortic rings were precontracted with 10 µ M of noradrenaline and cumulative isometric concentration-response curves were performed in response to acetylcholine (ACh) (0.01 to 90 µ M) in the presence and absence of 100 µ M ascorbic acid. Cumulative curves were recorded with Letica Scientific Instruments isometric transducers connected to a four-channel polygraph (Polygraph 4006, Letica Scientific Instruments, Barcelona, Spain). 2.5. Biochemical Analyses Plasma insulin and free fatty acids (FFA) tests were determined through the Rat Insulin ELISA Kit (Mercodia, Uppsala, Sweden) and FFA Assay Kit (ZenBio, Research Triangle Park, NC, USA), according to the manufacturers’ instructions. Heart 8-Isoprostane levels were determined using an ELISA Kit according to the manufacturer’s instruction (Cayman Chemical, Ann Arbor, MI, USA). 2.6. Fluorescence Immunocytochemistry and Immunohistochemistry The evaluation of the antioxidant potential of EA-Aa was carried out in HMVec-D cells challenged with H 2 O 2 (same as the antioxidant assay described before), and in cryopreserved histological slices (4 µ m) of liver, kidney, and aorta of the animal models. Oxidative stress probes, 2,7-dichlorodihydrofluorescein diacetate (H 2 DCFDA) and dihydroethidium (DHE) were used and DAPI was used to stain the nucleus. In hydrated sections, probes were incubated for 30 min and the slices were mounted with mounting medium (Glycergel, DAKO, Carpinteria, CA, USA). Images were immediately obtained with a fluorescence microscope (Zeiss Axio Observer Z1) with an incorporated camera (Zeiss, Jena, Germany), detected with 504 nm of excitation and 525 nm of emission for DCF, 587 nm of excitation and 610 nm of emission for DHE, and 353 nm of excitation and 465 nm of emission for DAPI. The same settings were kept constant for all analysis and the entire image was used for quantification, which was performed with ImageJ software. 2.7. Western Blot The Western blot analysis were performed in both cells (3T3-L1 cells treated with EA-Aa 31.25–500 µ g.mL −1 for 24 h) and organs (EAT, heart, kidney, liver and aorta). Cells and organ samples were washed with PBS and disrupted in lysis buffer (0.25 M Tris-HCl, 125 mM NaCl, 1% Triton-X-100, 0.5% SDS, 1 mM EDTA, 1 mM EGTA, 20 mM NaF, 2 mM Nutrients 2021,13, 2856 6 of 17 Na 3 VO 4 , 10 mM β -glycerophosphate, 2.5 mM sodium pyrophosphate, 10 mM PMSF, 40 µ L of protease inhibitor) using the TissueLyser systems (Quiagen, Germany). The BCA Protein Assay Kit was carried out on the supernatant of the centrifugation of samples (14.000 rpm for 20 min at 4 ◦ C), followed by the addition of Laemmli buffer (62.5 mM Tris-HCl, 10% glycerol, 2% SDS, 5% β -mercaptoethanol, 0.01% bromophenol blue) [ 18 ]. Samples (20 µ g) were loaded into SDS-PAGE and electroblotted onto PVDF membrane (Advansta, San Jose, CA, USA). Membranes were blocked with TBS-T 0.01% and BSA 5%, then incubated with the primary (overnight, 4 ◦ C) and secondary antibodies (2 h, room temperature), following the dilutions suggested by the manufacturers. Immunoblots were detected with ECL substrate and the Versadoc system (Biorad, Hercules, CA, USA). 2.8. Statistical Analysis Data were expressed as the mean ± standard error of the mean (SEM) and compared by analysis of variance using the Kruskal–Wallis test or ANOVA followed by the Tukey post hoc test, according to normality evaluation. Student’s t-test was used to determine the differences between two groups. Values of p< 0.05 were considered significant. Statistical tests were performed with GraphPad Prism 5.0 and IBM SPSS Statistics Software. 3. Results 3.1. EA-Aa Improves the Metabolic Profile of Diabetic Rats After the 30-day treatment (presented in the experimental design, Figure 1A), animals did not show any alterations in body weight and food/caloric intake among groups (Figure 1B–D). However, the treatment with EA-Aa decreased the fasting hyperglycaemia of diabetic rats (GK-EA-Aa) by 30–40% along the treatment in comparison to both controls at all timepoints (Figure 1E). Similar data were observed for fasting glycaemia, where the initial difference between GK-Ctrl and W-Ctrl groups becomes non-significant after the treatment in GK-EA-Aa (Figure 1F), and for the AUC of glycaemia along the 4-week treatment period, which was also significantly reduced in GK-EA-Aa when compared to GK-Ctrl (Figure 1G). No significant effects of EA-Aa were observed in the AUC of the insulin tolerance test (Figure 1H). On the other hand, a significant increase of plasma insulin levels was observed in GK-EA-Aa in relation to W-Ctrl rats (Figure 1I). Regarding lipid metabolism, GK-Ctrl presented higher levels of triglycerides, which were reduced in the GK-EA-Aa group (Figure 1J), whereas the values of FFA showed no significant difference (Figure 1K). The epididymal adipose tissue and the liver were analysed to understand the mechanisms of EA-Aa-induced metabolic improvement. The haematoxylin-eosin staining presented no morphological alterations and no significant weight changes were observed after EA-Aa treatment in the EAT and liver of Wistar and diabetic rats, as shown in Figure 2A,B,G–H . In addition, GLUT4 and PPAR γ levels in EAT were increased in the diabetic rats treated with EA-Aa (Figure 2C,F). No changes were observed for the insulin receptor levels nor AMPK (Figure 2D,E), although a trend to higher AMPK levels was observed in diabetic rats after EA-Aa treatment. In the liver, EA-Aa partially restored the levels of the insulin receptor in GK rats (64% vs 38% of %W-Ctrl/Calnexin). No significant differences were observed in hepatic GLUT2 and AMPK levels (Figure 2I,K). Nutrients 2021,13, 2856 7 of 17 Nutrients 2021, 13, x FOR PEER REVIEW 7 of 19 Figure 2. Regulation of metabolic pathways in epididymal adipose tissue (EAT) and liver. (A) Epididymal adipose tissue weight (n = 5–7). (B) Haematoxylin-eosin staining in EAT (n = 4). (C) GLUT4 levels in EAT; treatment with EA-Aa increased the levels of the protein in the treated diabetic group. (D) Insulin receptor levels in EAT. (E) AMPK levels in EAT; an increase in AMPK levels is presented in EA-Aa GK-treated group in relation to both controls. (F) PPARγ levels in EAT; GK-EA-Aa shows an increase in relation to W-Ctrl group (n = 5 for Western blots). (G) Liver weight (n = 5–7). (H) Haematoxylin-eosin staining in liver (n = 4). (I) GLUT2 levels in liver. (J) Insulin receptor levels in liver; treatment with EA-Aa promoted a restoration in IR levels in relation to the normal control group. (K) AMPK levels in liver (n = 5 for Western blots). * vs. W-Ctrl; # vs. GK-Ctrl; *,# p < 0.05; **,## p < 0.01. Figure 2. Regulation of metabolic pathways in epididymal adipose tissue (EAT) and liver. ( A ) Epididymal adipose tissue weight (n= 5–7). ( B ) Haematoxylin-eosin staining in EAT (n= 4). ( C ) GLUT4 levels in EAT; treatment with EA-Aa increased the levels of the protein in the treated diabetic group. ( D ) Insulin receptor levels in EAT. ( E ) AMPK levels in EAT; an increase in AMPK levels is presented in EA-Aa GK-treated group in relation to both controls. ( F ) PPAR γ levels in EAT; GK-EA-Aa shows an increase in relation to W-Ctrl group (n= 5 for Western blots). ( G ) Liver weight (n= 5–7). ( H ) Haematoxylin-eosin staining in liver (n= 4). ( I ) GLUT2 levels in liver. ( J ) Insulin receptor levels in liver; treatment with EA-Aa promoted a restoration in IR levels in relation to the normal control group. ( K ) AMPK levels in liver (n= 5 for Western blots). * vs. W-Ctrl; # vs. GK-Ctrl; *,# p< 0.05; **,## p< 0.01. Nutrients 2021,13, 2856 8 of 17 3.2. EA-Aa Has Tissue-Specific Protective Antioxidant Effects To determine the antioxidant potential of EA-Aa in vivo , Sirt1, NRF2, catalase, SOD-1 and GLO-1 levels were evaluated in EAT, liver, heart and kidney of control and diabetic rats. Markers of oxidative stress were evaluated and, given the metabolic alterations in adipose tissue caused by EA-Aa, the protective antioxidant effects were evaluated in a 3T3-L1 preadipocyte cell line. In the adipose tissue (Figure 3A,C,D) and liver (Figure 3I–L), no significant differences were observed between control and diabetic rats in Sirt1, catalase, GLO-1 and SOD-1 levels, whereas a partially restoration of NRF2 levels was observed in EAT of GK-EA-Aa group (Figure 3B). In the liver, the histological staining of a superoxide anion probe (DHE) did not show differences between the experimental groups as well (Figure 3H). Given that the antioxidant potential of EA-Aa was already confirmed in Cos7 cells [ 11 ], we evaluated such EA-Aa effect in 3T3-L1 cells challenged with an oxidant stimulus. After confirming the absence of EA-Aa-induced toxicity (Figure 3E), we incubated cells with H 2 O 2 (IC 50 –0.125 mM), which confirmed the protective antioxidant effect of the extract (500 µ g./mL −1 ) against H 2 O 2 -induced oxidative stress (Figure 3F). No activation of Sirt1 and NRF2 pathways was observed suggesting that the effect is at least partially independent of such pathways (representative Western Blots at Figure 3G). Heart and kidney were analysed given their high susceptibility to hyperglycaemiadriven complications. In both organs, no significant alterations were observed in Sirt1, NRF2, GLO-1 and SOD-1 levels (Figure 4A–D,I). Nevertheless, EA-Aa treatment resulted in a significant increase of kidney catalase levels of both normal and diabetic rats (Figure 4H). No significant alterations were observed in the heart levels of the lipid peroxidation marker 8-Isoprostane, as well as in kidney histological analysis of morphology (Figure 4E,G) and superoxide anion (DHE, Figure 4F). A trend to reduced DHE staining in the glomerulus was observed in some kidney regions after EA-Aa treatment, although quantification did not show significant differences (data not shown). 3.3. EA-Aa Improves Diabetic Endothelial Dysfunction Reducing Vascular Oxidative Stress Given that endothelial dysfunction is one of the major complications of hyperglycaemia, aorta relaxation was evaluated after EA-Aa treatment in Wistar and diabetic rats. Vascular relaxation was evaluated in NA-precontracted aorta rings in response to Ach in the presence or absence of ascorbic acid [ 19 ]. At 12-week-old, Wistar and GK rats had similar ACh-dependent relaxation (Figure 5A). In the rats treated with EA-Aa, a slight effect on aorta relaxation was observed, namely a 15% increment between W-EA-Aa and W-Ctrl (Figure 5B) and 10% between GKEA-Aa and GK-Ctrl (Figure 5C), although statistical significance was not reached. Pre-incubation of the rings with L-NAME (NOS inhibitor) practically abolished the relaxation of the aortic rings (data not shown), showing endothelial dependence. When the aorta was incubated with ascorbic acid, the maximum endothelial-dependent relaxation mediated by NA-precontracted rings in response to Ach reached 40% more in W-Ctrl and almost 74% more in W-EA-Aa (Figure 5D–E). Interestingly, no response to ascorbic acid was obtained in GK-Ctrl (Figure 5F), but it was restored after the treatment with the extract, which promoted a 30% increment of relaxation in GK-EA-Aa (Figure 5G). Such results are supported by the histological fluorescence staining of both oxidative stress probes, DHE and DCF, where 34% and 67% respectively lower signal intensity was observed in GK-EA-Aa in relation to the GK-Ctrl group (Figure 6A–D). The treatment also promoted an increase of catalase levels in the aorta of treated groups from both strains, mainly in diabetic rats (Figure 6E). Nutrients 2021,13, 2856 9 of 17 Nutrients 2021, 13, x FOR PEER REVIEW 9 of 19 Figure 3. Antioxidant effects of EA-Aa in EAT, liver and in 3T3-L1 pre-adipocytes (n = 3 independent experiments). (A) Sirt1 levels in EAT. (B) NRF2 levels in EAT, EA-Aa promotes partial restoration of NRF2 levels in the non-obese type 2 diabetic EA-Aa-treated group. (C) Catalase levels in EAT. (D) Representative images of EAT Western blot. Cell viability of pre-adipocytes 3T3-L1: (E) Treatment with EA-Aa for 24 h. (F) Treatment with EA-Aa (previously for 30 min) and induction to oxidative stress with H 2 O 2 (for 2 h). (G) Representative images of 3T3-L1 Western blot. (H) Dihydroethidium (DHE) staining in liver (n = 3). (I) Sirt1 levels in liver. (J) NRF2 levels in liver. (K) Catalase levels in liver (n = 5 for Western blots). (L) Representative images of liver Western blot. * vs. W-Ctrl/Ctrl; # vs. H 2 O 2 ; ** p < 0.01; ***,### p < 0.001. Figure 3. Antioxidant effects of EA-Aa in EAT, liver and in 3T3-L1 pre-adipocytes (n= 3 independent experiments). ( A ) Sirt1 levels in EAT. ( B ) NRF2 levels in EAT, EA-Aa promotes partial restoration of NRF2 levels in the non-obese type 2 diabetic EA-Aa-treated group. ( C ) Catalase levels in EAT. ( D ) Representative images of EAT Western blot. Cell viability of pre-adipocytes 3T3-L1: ( E ) Treatment with EA-Aa for 24 h. ( F ) Treatment with EA-Aa (previously for 30 min) and induction to oxidative stress with H 2 O 2 (for 2 h). ( G ) Representative images of 3T3-L1 Western blot. ( H ) Dihydroethidium (DHE) staining in liver (n= 3). (I) Sirt1 levels in liver. (J) NRF2 levels in liver. (K) Catalase levels in liver (n= 5 for Western blots). (L) Representative images of liver Western blot. * vs. W-Ctrl/Ctrl; # vs. H2O2; ** p< 0.01; ***,### p< 0.001. Nutrients 2021,13, 2856 16 of 17 18. Matafome, P.; Santos-Silva, D.; Crisóstomo, J.; Rodrigues, T.; Rodrigues, L.; Sena, C.M.; Pereira, P.; Seiça, R. Methylglyoxal causes structural and functional alterations in adipose tissue independently of obesity. Arch. Physiol. Biochem. 2012 ,118, 58–68. [CrossRef] [PubMed] 19. Sena, C.M.; Matafome, P.; Louro, T.; Nunes, E.; Seiça, R.M. Effects of atorvastatin and insulin in vascular dysfunction associated with type 2 diabetes. Physiol. Res. 2014,63, 189–197. [CrossRef] [PubMed] 20. Shabab, S.; Gholamnezhad, Z.; Mahmoudabady, M. Protective effects of medicinal plant against diabetes induced cardiac disorder: A review. J. Ethnopharmacol. 2021,265, 113328. [CrossRef] [PubMed] 21. King, G.L.; Brownlee, M. The cellular and molecular mechanisms of diabetic complications. Endocrinol. Metab. Clin. North. Am. 1996,25, 255–270. [CrossRef] 22. Santos, R.X.; Cardoso, S.; Silva, S.; Correia, S.; Carvalho, C.; Crisóstomo, J.; Rodrigues, L.; Amaral, C.; Louro, T.; Matafome, P.; et al. Food deprivation promotes oxidative imbalance in rat brain. J. Food Sci. 2009,74, H8–H14. [CrossRef] 23. Da Silva, P.V.B.; Ramiro, M.M.; Iriguchi, E.K.K.; Corrêa, W.A.; Lowe, J.; Cardoso, C.A.L.; Arena, A.C.; Kassuya, C.A.L.; Muzzi, R.M. Antidiabetic, cytotoxic and antioxidant activities of oil extracted from Acrocomia aculeata pulp. Nat. Prod. Res. 2019 ,33, 2413–2416. [CrossRef] 24. Nonato, C.D.F.A.; Leite, D.O.D.; Pereira, R.C.; Boligon, A.A.; Ribeiro-Filho, J.; Rodrigues, F.F.G.; da Costa, J.G.M. Chemical analysis and evaluation of antioxidant and antimicrobial activities of fruit fractions of Mauritia flexuosa L. f. (Arecaceae). PeerJ 2018,6, e5991. [CrossRef] [PubMed] 25. Nunes, Â.A.; Buccini, D.F.; Jaques, J.A.S.; Portugal, L.C.; Guimarães, R.C.A.; Favaro, S.P.; Caldas, R.A.; Carvalho, C.M.E. Effect of Acrocomia aculeata kernel oil on adiposity in type 2 diabetic rats. Plant. Foods Hum. Nutr. 2018,73, 61–67. [CrossRef] 26. Nunes, Â.A.; Buccini, D.F.; dos Santos Jaques, J.A.; Portugal, L.C.; Guimarães, R.C.A.; Favaro, S.P.; de Araújo Caldas, R.; Carvalho, C.M.E. Effect of dietary Acrocomia aculeata kernel oil rich in medium chain fatty acids on type 2 diabetic rats. J. Funct. Foods 2020 , 75, 104295. [CrossRef] 27. Abdelaziz, D.H.A.; Ali, S.A.; Mostafa, M.M.A. Phoenix dactylifera seeds ameliorate early diabetic complications in streptozotocininduced diabetic rats. Pharm. Biol. 2015,53, 792–799. [CrossRef] [PubMed] 28. De Souza, F.G.; de Araújo, F.F.; de Paulo Farias, D.; Zanotto, A.W.; Neri-Numa, I.A.; Pastore, G.M. Brazilian fruits of Arecaceae family: An overview of some representatives with promising food, therapeutic and industrial applications. Food Res. Int. 2020 , 138, 109690. [CrossRef] 29. Preetha, P.P.; Girija Devi, V.; Rajamohan, T. Comparative effects of mature coconut water (Cocos Nucifera) and glibenclamide on some biochemical parameters in alloxan induced diabetic rats. Rev. Bras. De Farmacogn. 2013,23, 481–487. [CrossRef] 30. Renjith, R.S.; Chikku, A.M.; Rajamohan, T. Cytoprotective, antihyperglycemic and phytochemical properties of Cocos nucifera (L.) inflorescence. Asian Pac. J. Trop. Med. 2013,6, 804–810. [CrossRef] 31. Egawa, T.; Tsuda, S.; Oshima, R.; Goto, A.; Ma, X.; Goto, K.; Hayashi, T. Regulatory mechanism of skeletal muscle glucose transport by phenolic acids. Phenolic Compd.—Biol. Act. 2017. [CrossRef] 32. Chen, L.; Gnanaraj, C.; Arulselvan, P.; El-Seedi, H.; Teng, H. A review on advanced microencapsulation technology to enhance bioavailability of phenolic compounds: Based on its activity in the treatment of type 2 diabetes. Trends Food Sci. Technol. 2019 ,85, 149–162. [CrossRef] 33. Eid, H.M.; Vallerand, D.; Muhammad, A.; Durst, T.; Haddad, P.S.; Martineau, L.C. Structural constraints and the importance of lipophilicity for the mitochondrial uncoupling activity of naturally occurring caffeic acid esters with potential for the treatment of insulin resistance. Biochem. Pharmacol. 2010,79, 444–454. [CrossRef] 34. Tsuda, S.; Egawa, T.; Ma, X.; Oshima, R.; Kurogi, E.; Hayashi, T. Coffee polyphenol caffeic acid but not chlorogenic acid increases 5 0 AMP-activated protein kinase and insulin-independent glucose transport in rat skeletal muscle. J. Nutr. Biochem. 2012 ,23, 1403–1409. [CrossRef] 35. Huang, D.-W.; Chang, W.-C.; Wu, J.S.-B.; Shih, R.-W.; Shen, S.-C. Gallic acid ameliorates hyperglycemia and improves hepatic carbohydrate metabolism in rats fed a high-fructose diet. Nutr. Res. 2016,36, 150–160. [CrossRef] 36. Prasad, C.N.V.; Anjana, T.; Banerji, A.; Gopalakrishnapillai, A. Gallic acid induces GLUT4 translocation and glucose uptake activity in 3T3-L1 cells. FEBS Lett. 2010,584, 531–536. [CrossRef] 37. Shi, G.-J.; Li, Y.; Cao, Q.-H.; Wu, H.-X.; Tang, X.-Y.; Gao, X.-H.; Yu, J.-Q.; Chen, Z.; Yang, Y. In Vitro and In Vivo evidence that quercetin protects against diabetes and its complications: A systematic review of the literature. Biomed. Pharmacother. 2019 ,109, 1085–1099. [CrossRef] 38. Akuodor, G.; Udia, P.; Bassey, A.; Chilaka, K.; Okezie, O. Antihyperglycemic and antihyperlipidemic properties of aqueous root extract of Icacina senegalensis in alloxan induced diabetic rats. J. Acute Dis. 2014,3, 99–103. [CrossRef] 39. AL-Ishaq, R.K.; Abotaleb, M.; Kubatka, P.; Kajo, K.; Büsselberg, D. Flavonoids and their anti-diabetic effects: Cellular mechanisms and effects to improve blood sugar levels. Biomolecules 2019,9, 430. [CrossRef] [PubMed] 40. Ginsberg, H.N.; Zhang, Y.-L.; Hernandez-Ono, A. Regulation of plasma triglycerides in insulin resistance and diabetes. Arch. Med. Res. 2005,36, 232–240. [CrossRef] [PubMed] 41. Koyama, K.; Chen, G.; Lee, Y.; Unger, R.H. Tissue triglycerides, insulin resistance, and insulin production: Implications for hyperinsulinemia of obesity. Am. J. Physiol. -Endocrinol. Metab. 1997,273, E708–E713. [CrossRef] [PubMed] 42. Kraegen, E.W.; Cooney, G.J.; Ye, J.; Thompson, A.L. Triglycerides, fatty acids and insulin resistance—Hyperinsulinemia. Exp. Clin. Endocrinol. Diabetes 2001,109, 516–526. [CrossRef] [PubMed] Nutrients 2021,13, 2856 17 of 17 43. Wu, W.; Feng, J.; Jiang, D.; Zhou, X.; Jiang, Q.; Cai, M.; Wang, X.; Shan, T.; Wang, Y. AMPK regulates lipid accumulation in skeletal muscle cells through FTO-dependent demethylation of N 6-methyladenosine. Sci. Rep. 2017,7, 41606. [CrossRef] 44. Zong, H.; Ren, J.M.; Young, L.H.; Pypaert, M.; Mu, J.; Birnbaum, M.J.; Shulman, G.I. AMP Kinase Is Required for Mitochondrial Biogenesis in Skeletal Muscle in Response to Chronic Energy Deprivation. Proc. Natl. Acad. Sci. USA 2002 ,99, 15983–15987. [CrossRef] 45. Fusi, F.; Saponara, S.; Pessina, F.; Gorelli, B.; Sgaragli, G. Effects of quercetin and rutin on vascular preparations: A comparison between mechanical and electrophysiological phenomena. Eur. J. Nutr. 2003,42, 10–17. [CrossRef] 46. Appeldoorn, M.M.; Venema, D.P.; Peters, T.H.F.; Koenen, M.E.; Arts, I.C.W.; Vincken, J.-P.; Gruppen, H.; Keijer, J.; Hollman, P.C.H. Some phenolic compounds increase the nitric oxide level in endothelial cells In Vitro. J. Agric. Food Chem. 2009 ,57, 7693–7699. [CrossRef] 47. Taubert, D.; Berkels, R.; Klaus, W.; Roesen, R. Nitric oxide formation and corresponding relaxation of porcine coronary arteries induced by plant phenols: Essential structural features. J. Cardiovasc. Pharmacol. 2002,40, 701–713. [CrossRef] 48. Choi, S.; Il Kim, H.; Hag Park, S.; Jung Lee, M.; Yeoul Jun, J.; Lee Kim, H.; Hoon Chung, J.; Ho Yeum, C. Endothelium-dependent vasodilation by ferulic acid in aorta from chronic renal hypertensive rats. Kidney Res. Clin. Pract. 2012,31, 227–233. [CrossRef] 49. Zhou, Z.-Y.; Xu, J.-Q.; Zhao, W.-R.; Chen, X.-L.; Jin, Y.; Tang, N.; Tang, J.-Y. Ferulic acid relaxed rat aortic, small mesenteric and coronary arteries by blocking voltage-gated calcium channel and calcium desensitization via dephosphorylation of ERK1/2 and MYPT1. Eur. J. Pharmacol. 2017,815, 26–32. [CrossRef] 50. De Oliveira, L.M.; de Oliveira, T.S.; da Costa, R.M.; de Souza Gil, E.; Costa, E.A.; de Cassia Aleixo Tostes Passaglia, R.; Filgueira, F.P.; Ghedini, P.C. The vasorelaxant effect of gallic acid involves endothelium-dependent and -independent mechanisms. Vasc. Pharmacol. 2016,81, 69–74. [CrossRef] 51. Incalza, M.A.; D’Oria, R.; Natalicchio, A.; Perrini, S.; Laviola, L.; Giorgino, F. Oxidative stress and reactive oxygen species in endothelial dysfunction associated with cardiovascular and metabolic diseases. Vasc. Pharmacol. 2018,100, 1–19. [CrossRef] 52. Traka, M.H.; Mithen, R.F. Plant science and human nutrition: Challenges in assessing health-promoting properties of phytochemicals. Plant Cell 2011,23, 2483–2497. [CrossRef] 53. Yang, K.; Zhang, L.; Liao, P.; Xiao, Z.; Zhang, F.; Sindaye, D.; Xin, Z.; Tan, C.; Deng, J.; Yin, Y.; et al. Impact of gallic acid on gut health: Focus on the gut microbiome, immune response, and mechanisms of action. Front. Immunol. 2020 ,11. [CrossRef] [PubMed] 54. Lafay, S.; Morand, C.; Manach, C.; Besson, C.; Scalbert, A. Absorption and metabolism of caffeic acid and chlorogenic acid in the small intestine of rats. Br. J. Nutr. 2006,96, 39–46. [CrossRef] [PubMed] 55. Sova, M.; Saso, L. Natural sources, pharmacokinetics, biological activities and health benefits of hydroxycinnamic acids and their metabolites. Nutrients 2020,12, 2190. [CrossRef] [PubMed] 56. Topakas, E.; Kalogeris, E.; Kekos, D.; Macris, B.J.; Christakopoulos, P. Bioconversion of ferulic acid into vanillic acid by the thermophilic fungus sporotrichum thermophile. LWT Food Sci. Technol. 2003,36, 561–565. [CrossRef] 57. Winterbone, M.S.; Tribolo, S.; Needs, P.W.; Kroon, P.A.; Hughes, D.A. Physiologically relevant metabolites of quercetin have no effect on adhesion molecule or chemokine expression in human vascular smooth muscle cells. Atherosclerosis 2009 ,202, 431–438. [CrossRef] 58. Pashikanti, S.; de Alba, D.R.; Boissonneault, G.A.; Cervantes-Laurean, D. Rutin metabolites: Novel inhibitors of nonoxidative advanced glycation end products. Free Radic. Biol. Med. 2010,48, 656–663. [CrossRef] [PubMed]