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

nu ien s A icle Hypoglycaemic and An ioxidan P ope ies o Ac ocomia aculea a (Jacq.) Lodd Ex Ma . Ex ac A e Associa ed wi h Be e Vascula Func ion o Type 2 Diabe ic Ra s Tamaeh Mon ei o-Al edo 1,2,3,4 , Sa a Oli ei a 1,2,3 , And eia Ama o 1,2,3, Daniela Rosendo-Sil a 1,2,3 , Ka ia An unes 4, Ana SaloméPi es 2,3,5 , Rica do Teixo 2,3,5, Ana Ma ga ida Ab an es 2,3,5, Ma ia Filomena Bo elho 2,3,5 , Miguel Cas elo-B anco 6,7,8,9, Raquel Seiça 1, Sónia Sil a 2,10, Kely de Picoli Souza 4and Paulo Ma a ome 1,2,3,11,*   Ci a ion: Mon ei o-Al edo, T.; Oli ei a, S.; Ama o, A.; Rosendo-Sil a, D.; An unes, K.; Pi es, A.S.; Teixo, R.; Ab an es, A.M.; Bo elho, M.F.; Cas elo-B anco, M.; e al. Hypoglycaemic and An ioxidan P ope ies o Ac ocomia aculea a (Jacq.) Lodd Ex Ma . Ex ac A e Associa ed wi h Be e Vascula Func ion o Type 2 Diabe ic Ra s. Nu ien s 2021,13, 2856. h ps:// doi.o g/10.3390/nu13082856 Academic Edi o : A igo Cice o Recei ed: 14 July 2021 Accep ed: 17 Augus 2021 Published: 20 Augus 2021 Publishe ’s No e: MDPI s ays neu al wi h ega d o ju isdic ional claims in published maps and ins i u ional a il- ia ions. Copy igh : © 2021 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). 1Ins i u e o Physiology, Facul y o Medicine, Uni e si y o Coimb a, 3000-548 Coimb a, Po ugal; [email p o ec ed] (T.M.-A.); [email p o ec ed] (S.O.); and eia.ama [email p o ec ed] (A.A.); [email p o ec ed] (D.R.-S.); [email p o ec ed] (R.S.) 2Coimb a Ins i u e o Clinical and Biomedical Resea ch (iCBR), Facul y o Medicine, Cen e o Inno a i e Biomedicine and Bio echnology (CIBB), Uni e si y o Coimb a, 3000-548 Coimb a, Po ugal; [email p o ec ed] (A.S.P.); ica [email p o ec ed] (R.T.); [email p o ec ed] (A.M.A.); [email p o ec ed] (M.F.B.); [email p o ec ed] (S.S.) 3Clinical Academic Cen e o Coimb a, 3000-548 Coimb a, Po ugal 4 Resea ch G oup o Bio echnology and Biop ospec ing Applied o Me abolism (GEBBAM), Fede al Uni e si y o G ande Dou ados, Dou ados 79825-070, MS, B azil; [email p o ec ed] (K.A.); [email p o ec ed] (K.d.P.S.) 5Ins i u e o Biophysics, Facul y o Medicine, Uni e si y o Coimb a, 3000-548 Coimb a, Po ugal 6Visual Neu oscience Labo a o y, Ins i u e o Biomedical Imaging and Li e Sciences (IBILI), Facul y o Medicine, Uni e si y o Coimb a, 3000-548 Coimb a, Po ugal; [email p o ec ed] 7Cen e o Neu oscience and Cell Biology (CNC), IBILI, Uni e si y o Coimb a, 3000-548 Coimb a, Po ugal 8 Ins i u e o Nuclea Sciences Applied o Heal h (ICNAS), Uni e si y o Coimb a, 3000-548 Coimb a, Po ugal 9 Labo a ó io de Bioes a ís ica Médica, Facul y o Medicine, Uni e si y o Coimb a, 3000-548 Coimb a, Po ugal 10 Ins i u e o Pha macology and Expe imen al The apeu ics, Facul y o Medicine, Uni e si y o Coimb a, 3000-548 Coimb a, Po ugal 11 Ins i u o Poli écnico de Coimb a, Coimb a Heal h School (ESTeSC), Depa men o Complemen a y Sciences, 3000-548 Coimb a, Po ugal *Co espondence: [email p o ec ed] Abs ac : Oxida i e s ess is in ol ed in he me abolic dys egula ion o ype 2 diabe es (DM2). Ac ocomia aculea a (Aa) ui pulp has been desc ibed o he ea men o se e al diseases, and ecen ly we ha e p o ed ha i s lea es ha e phenolic compounds wi h a ma ked an ioxidan e ec . We aimed o assess whe he hey can imp o e me abolic, edox and ascula unc ions in DM2. Con ol Wis a (W-C l) and non-obese ype 2 diabe ic Go o–Kakizaki (GK-C l) a s we e ea ed o 30 days wi h 200 mg.kg −1 aqueous ex ac o Aa (EA-Aa) (Wis a , W-EA-Aa/GK, GK-EA-Aa). EA-Aa was able o educe as ing glycaemia and iglyce ides o GK-EA-Aa by imp o ing p o eins ela ed o glucose and lipid me abolism, such as GLUT-4, PPAR γ , AMPK, and IR, when compa ed o GK-C l. I also imp o ed iabili y o 3T3-L1 p e-adipocy es exposed by H 2 O 2. EA-Aa also inc eased he le els o ca alase in he ao a and kidney, educed oxida i e s ess and inc eased elaxa ion o he ao a in GK- ea ed a s in ela ion o GK-C l, in addi ion o he p o ec i e e ec agains oxida i e s ess in HMVec-D cells. We p o ed he di ec an ioxidan po en ial o he chemical compounds o EA-Aa, he inc ease in an ioxidan de ences in a issue-speci ic manne and hypoglycaemic p ope ies, imp o ing ascula unc ion in ype 2 diabe es. EA-Aa and i s cons i uen s may ha e a he apeu ic po en ial o he ea men o DM2 complica ions. Keywo ds: diabe es; macaúba;bocaiú a; ascula unc ion; polyphenols Nu ien s 2021,13, 2856. h ps://doi.o g/10.3390/nu13082856 h ps://www.mdpi.com/jou nal/nu ien s Nu ien s 2021,13, 2856 2 o 17 1. In oduc ion Diabe es melli us (DM) is a ch onic disease cha ac e ized by hype glycaemia, esul ing om a de iciency in insulin p oduc ion, desensi iza ion o i s ac ion, o bo h [ 1 ]. Acco ding o he In e na ional Diabe es Fede a ion, diabe es is one o he diseases wi h he highes incidence in he 21s cen u y, ha ing inc eased h ee- old in he las wo decades, and being es ima ed o a ec 463 million indi iduals in 2019 [ 2 ]. As a consequence o hype glycaemia, bo h DM1 and DM2 commonly ha e associa ed complica ions, which ha e signi ican mo bidi y and mo ali y and a conside able economic impac . These complica ions can be ei he mic o (neu o, neph o, ca dio and e inopa hy) o mac o ascula (s oke and ca dio ascula diseases) [ 3 , 4 ]. One o he main ac o s o i s cause and p og ession is oxida i e s ess, which is in ol ed in he pa hogenesis o complica ions h ough he o e - p oduc ion o eac i e oxygen species (ROS) and eac i e ni ogen species (RNS), u he impai ing he edox balance [ 5 ]. The adop ion o a be e li es yle (balanced die and exe cise) may delay he de elopmen o diabe es and i s complica ions. On he o he hand, se e al he apeu ic op ions a e a ailable o DM2, mos o hem a ge ed o he educ ion o he glycaemia [megli inides, biguanides, sul onylu eas (SUs), hiazolidinedione (TZD), dipep idyl pep idase 4 (DPP-4) inhibi o s, GLP-1 ecep o agonis s, sodium glucose co ans- po e s inhibi o s (SGLT2) and insulin] [ 6 ]. Howe e , a conside able numbe o pa ien s do no adhe e o he ea men wi h allopa hic medicine due o side e ec s [ 4 ], which a ec mainly li e and kidney [ 7 ]. In his pe spec i e, a conside able amoun o esea ch is ocused on de eloping he apeu ic al e na i es, which could be bo h inexpensi e and e ec i e wi h ewe side e ec s [4]. B azil has he g ea es biodi e si y in he wo ld, wi h abou 20% o he species dis- ibu ed in i s biomes. In pa icula , he B azilian Ce ado, which occupies 22% o he na ional e i o y [ 8 ], has one o he wo ld’s iches lo a, whe e 35% o he species a e endemic [ 9 ]. Among hese species, many o hem a e conside ed medicinal due o hei chemical composi ion [ 10 ]. Ac ocomia aculea a (Jacq.) Lodd. ex Ma ., commonly known as bocaiú a o macaúba, is a palm na i e om Ce ado wi h he apeu ic (p oduc ion o eme- dies based on e hnopha macological knowledge—as an idiabe ic, an ioxidan , analgesic e c.) and economic impo ance ( o cooking, biodiesel, and he cosme ic indus y) [ 11 ]. I s ca o enoid- ich ui pulp was sugges ed o ha e bene icial e ec s on he ea men o espi a o y diseases, as analgesic and laxa i e [ 12 ] and also in dec easing se um choles e ol and glucose le els [ 13 ]. Recen ly, ou g oup p o ed he an ioxidan po en ial o i s lea es and he ele an chemical composi ion, mos ly o anillic, ca eic, e ulic and gallic acid, u in and que ce in [ 11 ]. In addi ion o he new indings desc ibed by us abou he po en ial o i s lea es, i is only known ha hey a e used o bo ine nu i ional supplemen a ion and in he p epa a ion o eas o human consump ion. The e o e, ou goal in his s udy was o assess whe he he an ioxidan po en ial o A. aculea a lea es can es o e edox balance and imp o e me abolic and ascula unc ion o ype 2 diabe ic a s, as well as o disclose he unde lying mechanisms in issues in ol ed in glucose me abolism and i s ascula complica ions. Ou esul s show he an ioxidan and hypoglycaemic po en ial o EA-Aa, obse ed h ough he issue-dependen up egula ion o pa hways in ol ed in an ioxidan de ences and glucose and lipid me abolism. Such e ec s we e associa ed wi h he imp o emen o ao ic elaxa ion and edox s a e. 2. Ma e ials and Me hods 2.1. Chemicals and An ibodies Sal s and o ganic sol en s used in his s udy we e all pu chased om Lonza, Sigma- Ald ich/Me ck, Al a-Aesa , Fische Scien i ics and Pan eac. An ibodies used we e a ge ed o Ca alase, Glo-1, GLUT2 (ab76110, ab96032, ab54460 Abcam, Camb idge, UK), GLUT4, PPARgamma, Insulin Recep o , AMPK, phospho-AMPK-Th -172, Si 1, phospho-Si 1- Se 47 (#2213S, #2443S, #3025S, #2532S, #2535S, #9475S, #2314S, Cell Signaling Technology, Dan e s, MA, USA) NRF2 (sc-518036, San a C uz Bio echnology, Dallas, TX, USA) phospho- Nu ien s 2021,13, 2856 3 o 17 NRF2 (Se 40) (PA5-67520, In i ogen, Wal ham, MA, USA). Calnexin and GAPDH (AB0037, AB0049-20, Sicgen, Ca ca elos, Po ugal) we e used as loading con ol. 2.2. Bo anical Ma e ial and Isola ion o Ex ac F esh A. aculea a lea es we e collec ed as be o e [ 11 ] in he egion o G ande Dou ados, Macaúba dis ic , s a e o Ma o G osso do Sul (MS) (22 ◦ 0702.4 S 54 ◦ 2836.3 W), wi h he pe mission o he B azilian Biodi e si y Au ho iza ion and In o ma ion Sys em (Sis ema de Au o ização e In o mação sob e Biodi e sidade, SISBIO; no. 50589). A plan axonomis iden i ied he species, and a specimen was deposi ed in he he ba ium (DDMS-UFGD) o he Fede al Uni e si y o G ande Dou ados, Dou ados (MS), B azil, egis a ion numbe — 5103. The aqueous ex ac was p epa ed as p e iously desc ibed [11]. 2.3. Cell Cul u e and Viabili y Assays Mouse (Mus musculus) p eadipocy e—3T3-L1 cells (cul u ed wi h Dulbecco’s Modi ied Eagle’s Medium—DMEM supplemen ed wi h 10% FBS and 1% penicillin/s ep omycin) [ 14 ]; and human de mal mic o ascula endo helial Cells (HMVec-D, cul u ed wi h EGMTM-2, Endo helial Cell G ow h Medium-2, Bulle Ki TM) [ 15 , 16 ], main ained a 37 ◦ C and 5% CO 2 we e used in he assays. To e alua e cell iabili y, 1 × 10 5 HMVec-D cells and 3 × 10 4 3T3-L1 cells we e seeded in 96-well mic opla es. A e 24 h, cells we e incuba ed wi h di e en concen a ions (31,25– 500 µ g.mL −1 ) o EA-Aa o 24 h. A e his pe iod, cell iabili y was de e mined h ough he Alama Blue assay. Abso bance was measu ed a 570 nm and 600 nm in a BioTek mic opla e eade (BioTek, Ins umen s, Inc., Winooski, VT, USA) and used o calcula e cell iabili y, acco ding o Equa ion (1) [11]. Cell iabili y =(Abs570−Abs600)o ea ed cells (Abs570−Abs600)o con ol cells×100 (1) To e alua e he an ioxidan po en ial o EA-Aa, bo h cell lines 3T3-L1 and HMVec-D cells we e ea ed wi h H 2 O 2 , he oxida i e s ess induc o . A e 80% o con luence, cells we e i s ly incuba ed wi h he ex ac o 30 min ollowed by H 2 O 2 (IC 50 0.125 mM in 3T3-L1 cells and 0.25 mM in HMVec-D cells) o 2 h. Equa ion (1) was used o calcula e he p o ec i e e ec o EA-Aa in cell iabili y. Dependence o EA-Aa e ec s on NRF2 pa hway was e alua ed h ough he incuba ion o HMVec-D cells wi h he NRF2 inhibi o ML385 (20 µM). 2.4. Animal Main enance and T ea men The s udy was pe o med acco ding o good p ac ices o animal handling, wi h he app o al o he Ins i u ional Animal Ca e and Use Commi ee (ORBEA 13/2018) and he p ocedu es pe o med by licensed use s by he Fede a ion o Labo a o y Animal Science Associa ions (FELASA), con o med o he guidelines om Di ec i e 2010/63/EU o he Eu opean Pa liamen o he P o ec ion o Animals Used o Science Pu pose. Male 12- week-old Wis a and non-obese ype 2 diabe ic Go o–Kakizaki (GK) a s om ou b eeding colonies (Facul y o Medicine, Uni e si y o Coimb a), we e andomly di ided in 4 g oups (n= 5–7), as p esen ed in Figu e 1A, which we e: Wis a con ol (W); Wis a ea ed wi h EA-Aa (W-EA-Aa); GK con ol (GK) and GK ea ed wi h EA-Aa (GK-EA-Aa). Animals we e kep unde s anda d condi ions—2 animals pe cage, wi h empe a u e a 22–24 ◦ C, and 50–60% humidi y, and s anda d ligh cycle (12 h ligh /12 h da kness), wi h wa e and ood (s anda d die A03, SAFE, F ance) ad libi um [ 17 ]. EA-Aa (200 mg.kg −1 ) was added in he daily wa e o he animals 28 days, which ecei ed he ea men du ing he nigh and no mal wa e du ing he day. The weekly a e age o he a s’ weigh was used o de e mine he daily dose o EA-Aa pe cage. Nu ien s 2021,13, 2856 4 o 17 Nu ien s 2021, 13, x FOR PEER REVIEW 4 o 19 Figu e 1. Glycaemic and lipid p o ile o Wis a and Go o–Kakizaki (GK) a s a e 30-day ea men (n = 5–7). (A) Expe i- men al design o in i o s udy. (B) Body mass e olu ion o ea ed and non- ea ed a s ep esen ed e e y 7 days. (C) Ini ial and inal body mass o ea ed animals. (D) Calo ic in ake. (E) Fas ing blood glucose in pe cen age o he ini ial alue; EA-Aa p omo ed a dec ease in as ing glycaemia o GK-EA-Aa since he 7 h day o ea men in ela ion o he ini ial glycaemia. (F) Fas ing glycaemia; a es o a ion o alues is ound in he end o he ea men be ween GK a s and W-C l g oup. (G) A ea unde he cu e o glycaemia along 4-week ea men pe iod; alues a e educed by EA-Aa ea - men in GK a s when compa ing o he con ol g oup. (H) A ea Unde he cu e o he glycaemia along all he ea men . (I) A ea unde he cu e o ITT (insulin ole ance es ). (J) Insulin; an inc ease in GK-EA-Aa is p esen ed in ela ion o he W-C l g oup. (K) T iglyce ides; ea men wi h EA-Aa dec eased plasma iglyce ides le els in GK. (L) F ee a y acid le els. * s. W-C l a he same poin ; # s. GK-C l a he same poin ; *,# p < 0.05; ** p < 0.01; ***,### p < 0.001. Figu e 1. Glycaemic and lipid p o ile o Wis a and Go o–Kakizaki (GK) a s a e 30-day ea men (n= 5–7). ( A ) Expe imen al design o in i o s udy. ( B ) Body mass e olu ion o ea ed and non- ea ed a s ep esen ed e e y 7 days. ( C ) Ini ial and inal body mass o ea ed animals. ( D ) Calo ic in ake. ( E ) Fas ing blood glucose in pe cen age o he ini ial alue; EA-Aa p omo ed a dec ease in as ing glycaemia o GK-EA-Aa since he 7 h day o ea men in ela ion o he ini ial glycaemia. ( F ) Fas ing glycaemia; a es o a ion o alues is ound in he end o he ea men be ween GK a s and W-C l g oup. ( G ) A ea unde he cu e o glycaemia along 4-week ea men pe iod; alues a e educed by EA-Aa ea men in GK a s when compa ing o he con ol g oup. ( H ) A ea Unde he cu e o he glycaemia along all he ea men . ( I ) A ea unde he cu e o ITT (insulin ole ance es ). ( J ) Insulin; an inc ease in GK-EA-Aa is p esen ed in ela ion o he W-C l g oup. ( K ) T iglyce ides; ea men wi h EA-Aa dec eased plasma iglyce ides le els in GK. ( L ) F ee a y acid le els. * s. W-C l a he same poin ; # s. GK-C l a he same poin ; *,# p< 0.05; ** p< 0.01; ***,### p< 0.001. Nu ien s 2021,13, 2856 5 o 17 2.4.1. In Vi o P ocedu es and Sample Collec ion Body weigh , as ing glycaemia, wa e and ood in ake we e e alua ed weekly (cal- cula ed as he mean daily consump ion pe a ), and an insulin ole ance es (ITT) was pe o med a he beginning and a he end o he ea men . Fo he IIT, 250 mU.kg −1 insulin (Humulin, 1000 UI.mL −1 Lilly, Lisboa, Po ugal) was injec ed (i.p.) a e 6 h as ing, ollowed by glycaemia measu emen in he ail ein wi h a glucome e (P ecision X a Me e , Abbo Diabe es Ca e, Amado a, Po ugal) and es s ips (Abbo Diabe es Ca e, Po ugal) a ime 0, 15, 30, 60 and 120 min. Response o insulin was exp essed by a ea unde he cu e (AUC) [ 17 ]. Se um iglyce ides we e measu ed in he same day be o e insulin adminis a ion. A he end o he ea men , animals we e anes he ized (i.p.) wi h 2:1 ( / ) 50 mg.kg −1 ke amine (100 mg.mL −1 )/2.5% chlo p omazine (5 mg.mL −1 ) and samples o blood we e collec ed by ca diac punc u e ollowed by ce ical disloca ion. Epididymal adipose issue (EAT), li e , kidney, hea and ao a we e collec ed, blood samples we e cen i uged (2200 × g, 4 ◦ C, 15 0 ) and se um and plasma we e aliquo ed and s o ed a − 80 ◦ C o u he analysis. 2.4.2. S udies o Isome ic Tension o Ao a Ao a ings we e moun ed on s ainless s eel hooks unde 19.6 mN basal ension in o gan ba hs illed wi h ae a ed (95% O 2 , 5% CO 2 ) K ebs–Henselei solu ion (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). A e an equilib a ion pe iod o 60 min, ao ic ings we e p econ ac ed wi h 10 µ M o no ad enaline and cumula i e isome ic concen a ion- esponse cu es we e pe o med in esponse o ace ylcholine (ACh) (0.01 o 90 µ M) in he p esence and absence o 100 µ M asco bic acid. Cumula i e cu es we e eco ded wi h Le ica Scien i ic Ins umen s isome ic ansduce s connec ed o a ou -channel polyg aph (Polyg aph 4006, Le ica Scien i ic Ins umen s, Ba celona, Spain). 2.5. Biochemical Analyses Plasma insulin and ee a y acids (FFA) es s we e de e mined h ough he Ra Insulin ELISA Ki (Me codia, Uppsala, Sweden) and FFA Assay Ki (ZenBio, Resea ch T iangle Pa k, NC, USA), acco ding o he manu ac u e s’ ins uc ions. Hea 8-Isop os ane le els we e de e mined using an ELISA Ki acco ding o he manu ac u e ’s ins uc ion (Cayman Chemical, Ann A bo , MI, USA). 2.6. Fluo escence Immunocy ochemis y and Immunohis ochemis y The e alua ion o he an ioxidan po en ial o EA-Aa was ca ied ou in HMVec-D cells challenged wi h H 2 O 2 (same as he an ioxidan assay desc ibed be o e), and in c yop e- se ed his ological slices (4 µ m) o li e , kidney, and ao a o he animal models. Oxida i e s ess p obes, 2,7-dichlo odihyd o luo escein diace a e (H 2 DCFDA) and dihyd oe hidium (DHE) we e used and DAPI was used o s ain he nucleus. In hyd a ed sec ions, p obes we e incuba ed o 30 min and he slices we e moun ed wi h moun ing medium (Glyce gel, DAKO, Ca pin e ia, CA, USA). Images we e immedia ely ob ained wi h a luo escence mic oscope (Zeiss Axio Obse e Z1) wi h an inco po a ed came a (Zeiss, Jena, Ge many), de ec ed wi h 504 nm o exci a ion and 525 nm o emission o DCF, 587 nm o exci a ion and 610 nm o emission o DHE, and 353 nm o exci a ion and 465 nm o emission o DAPI. The same se ings we e kep cons an o all analysis and he en i e image was used o quan i ica ion, which was pe o med wi h ImageJ so wa e. 2.7. Wes e n Blo The Wes e n blo analysis we e pe o med in bo h cells (3T3-L1 cells ea ed wi h EA-Aa 31.25–500 µ g.mL −1 o 24 h) and o gans (EAT, hea , kidney, li e and ao a). Cells and o gan samples we e washed wi h PBS and dis up ed in lysis bu e (0.25 M T is-HCl, 125 mM NaCl, 1% T i on-X-100, 0.5% SDS, 1 mM EDTA, 1 mM EGTA, 20 mM NaF, 2 mM Nu ien s 2021,13, 2856 6 o 17 Na 3 VO 4 , 10 mM β -glyce ophospha e, 2.5 mM sodium py ophospha e, 10 mM PMSF, 40 µ L o p o ease inhibi o ) using he TissueLyse sys ems (Quiagen, Ge many). The BCA P o ein Assay Ki was ca ied ou on he supe na an o he cen i uga ion o samples (14.000 pm o 20 min a 4 ◦ C), ollowed by he addi ion o Laemmli bu e (62.5 mM T is-HCl, 10% glyce ol, 2% SDS, 5% β -me cap oe hanol, 0.01% b omophenol blue) [ 18 ]. Samples (20 µ g) we e loaded in o SDS-PAGE and elec oblo ed on o PVDF memb ane (Ad ans a, San Jose, CA, USA). Memb anes we e blocked wi h TBS-T 0.01% and BSA 5%, hen incuba ed wi h he p ima y (o e nigh , 4 ◦ C) and seconda y an ibodies (2 h, oom empe a u e), ollowing he dilu ions sugges ed by he manu ac u e s. Immunoblo s we e de ec ed wi h ECL subs a e and he Ve sadoc sys em (Bio ad, He cules, CA, USA). 2.8. S a is ical Analysis Da a we e exp essed as he mean ± s anda d e o o he mean (SEM) and compa ed by analysis o a iance using he K uskal–Wallis es o ANOVA ollowed by he Tukey pos hoc es , acco ding o no mali y e alua ion. S uden ’s - es was used o de e mine he di e ences be ween wo g oups. Values o p< 0.05 we e conside ed signi ican . S a is ical es s we e pe o med wi h G aphPad P ism 5.0 and IBM SPSS S a is ics So wa e. 3. Resul s 3.1. EA-Aa Imp o es he Me abolic P o ile o Diabe ic Ra s A e he 30-day ea men (p esen ed in he expe imen al design, Figu e 1A), animals did no show any al e a ions in body weigh and ood/calo ic in ake among g oups (Figu e 1B–D). Howe e , he ea men wi h EA-Aa dec eased he as ing hype glycaemia o diabe ic a s (GK-EA-Aa) by 30–40% along he ea men in compa ison o bo h con ols a all imepoin s (Figu e 1E). Simila da a we e obse ed o as ing glycaemia, whe e he ini ial di e ence be ween GK-C l and W-C l g oups becomes non-signi ican a e he ea men in GK-EA-Aa (Figu e 1F), and o he AUC o glycaemia along he 4-week ea men pe iod, which was also signi ican ly educed in GK-EA-Aa when compa ed o GK-C l (Figu e 1G). No signi ican e ec s o EA-Aa we e obse ed in he AUC o he insulin ole ance es (Figu e 1H). On he o he hand, a signi ican inc ease o plasma insulin le els was obse ed in GK-EA-Aa in ela ion o W-C l a s (Figu e 1I). Rega ding lipid me abolism, GK-C l p esen ed highe le els o iglyce ides, which we e educed in he GK-EA-Aa g oup (Figu e 1J), whe eas he alues o FFA showed no signi ican di e ence (Figu e 1K). The epididymal adipose issue and he li e we e analysed o unde s and he mecha- nisms o EA-Aa-induced me abolic imp o emen . The haema oxylin-eosin s aining p e- sen ed no mo phological al e a ions and no signi ican weigh changes we e obse ed a e EA-Aa ea men in he EAT and li e o Wis a and diabe ic a s, as shown in Figu e 2A,B,G–H . In addi ion, GLUT4 and PPAR γ le els in EAT we e inc eased in he diabe ic a s ea ed wi h EA-Aa (Figu e 2C,F). No changes we e obse ed o he insulin ecep o le els no AMPK (Figu e 2D,E), al hough a end o highe AMPK le els was obse ed in diabe ic a s a e EA-Aa ea men . In he li e , EA-Aa pa ially es o ed he le els o he insulin ecep o in GK a s (64% s 38% o %W-C l/Calnexin). No signi ican di e ences we e obse ed in hepa ic GLUT2 and AMPK le els (Figu e 2I,K). Nu ien s 2021,13, 2856 7 o 17 Nu ien s 2021, 13, x FOR PEER REVIEW 7 o 19 Figu e 2. Regula ion o me abolic pa hways in epididymal adipose issue (EAT) and li e . (A) Epididymal adipose issue weigh (n = 5–7). (B) Haema oxylin-eosin s aining in EAT (n = 4). (C) GLUT4 le els in EAT; ea men wi h EA-Aa inc eased he le els o he p o ein in he ea ed diabe ic g oup. (D) Insulin ecep o le els in EAT. (E) AMPK le els in EAT; an inc ease in AMPK le els is p esen ed in EA-Aa GK- ea ed g oup in ela ion o bo h con ols. (F) PPARγ le els in EAT; GK-EA-Aa shows an inc ease in ela ion o W-C l g oup (n = 5 o Wes e n blo s). (G) Li e weigh (n = 5–7). (H) Haema- oxylin-eosin s aining in li e (n = 4). (I) GLUT2 le els in li e . (J) Insulin ecep o le els in li e ; ea men wi h EA-Aa p omo ed a es o a ion in IR le els in ela ion o he no mal con ol g oup. (K) AMPK le els in li e (n = 5 o Wes e n blo s). * s. W-C l; # s. GK-C l; *,# p < 0.05; **,## p < 0.01. Figu e 2. Regula ion o me abolic pa hways in epididymal adipose issue (EAT) and li e . ( A ) Epididymal adipose issue weigh (n= 5–7). ( B ) Haema oxylin-eosin s aining in EAT (n= 4). ( C ) GLUT4 le els in EAT; ea men wi h EA-Aa inc eased he le els o he p o ein in he ea ed diabe ic g oup. ( D ) Insulin ecep o le els in EAT. ( E ) AMPK le els in EAT; an inc ease in AMPK le els is p esen ed in EA-Aa GK- ea ed g oup in ela ion o bo h con ols. ( F ) PPAR γ le els in EAT; GK-EA-Aa shows an inc ease in ela ion o W-C l g oup (n= 5 o Wes e n blo s). ( G ) Li e weigh (n= 5–7). ( H ) Haema oxylin-eosin s aining in li e (n= 4). ( I ) GLUT2 le els in li e . ( J ) Insulin ecep o le els in li e ; ea men wi h EA-Aa p omo ed a es o a ion in IR le els in ela ion o he no mal con ol g oup. ( K ) AMPK le els in li e (n= 5 o Wes e n blo s). * s. W-C l; # s. GK-C l; *,# p< 0.05; **,## p< 0.01. Nu ien s 2021,13, 2856 8 o 17 3.2. EA-Aa Has Tissue-Speci ic P o ec i e An ioxidan E ec s To de e mine he an ioxidan po en ial o EA-Aa in i o , Si 1, NRF2, ca alase, SOD-1 and GLO-1 le els we e e alua ed in EAT, li e , hea and kidney o con ol and diabe ic a s. Ma ke s o oxida i e s ess we e e alua ed and, gi en he me abolic al e a ions in adipose issue caused by EA-Aa, he p o ec i e an ioxidan e ec s we e e alua ed in a 3T3-L1 p eadipocy e cell line. In he adipose issue (Figu e 3A,C,D) and li e (Figu e 3I–L), no signi ican di e ences we e obse ed be ween con ol and diabe ic a s in Si 1, ca alase, GLO-1 and SOD-1 le els, whe eas a pa ially es o a ion o NRF2 le els was obse ed in EAT o GK-EA-Aa g oup (Figu e 3B). In he li e , he his ological s aining o a supe oxide anion p obe (DHE) did no show di e ences be ween he expe imen al g oups as well (Figu e 3H). Gi en ha he an ioxidan po en ial o EA-Aa was al eady con i med in Cos- 7 cells [ 11 ], we e alua ed such EA-Aa e ec in 3T3-L1 cells challenged wi h an oxidan s imulus. A e con i ming he absence o EA-Aa-induced oxici y (Figu e 3E), we incuba ed cells wi h H 2 O 2 (IC 50 –0.125 mM), which con i med he p o ec i e an ioxidan e ec o he ex ac (500 µ g./mL −1 ) agains H 2 O 2 -induced oxida i e s ess (Figu e 3F). No ac i a ion o Si 1 and NRF2 pa hways was obse ed sugges ing ha he e ec is a leas pa ially independen o such pa hways ( ep esen a i e Wes e n Blo s a Figu e 3G). Hea and kidney we e analysed gi en hei high suscep ibili y o hype glycaemia- d i en complica ions. In bo h o gans, no signi ican al e a ions we e obse ed in Si 1, NRF2, GLO-1 and SOD-1 le els (Figu e 4A–D,I). Ne e heless, EA-Aa ea men esul ed in a signi ican inc ease o kidney ca alase le els o bo h no mal and diabe ic a s (Figu e 4H). No signi ican al e a ions we e obse ed in he hea le els o he lipid pe oxida ion ma ke 8-Isop os ane, as well as in kidney his ological analysis o mo phology (Figu e 4E,G) and supe oxide anion (DHE, Figu e 4F). A end o educed DHE s aining in he glome ulus was obse ed in some kidney egions a e EA-Aa ea men , al hough quan i ica ion did no show signi ican di e ences (da a no shown). 3.3. EA-Aa Imp o es Diabe ic Endo helial Dys unc ion Reducing Vascula Oxida i e S ess Gi en ha endo helial dys unc ion is one o he majo complica ions o hype gly- caemia, ao a elaxa ion was e alua ed a e EA-Aa ea men in Wis a and diabe ic a s. Vascula elaxa ion was e alua ed in NA-p econ ac ed ao a ings in esponse o Ach in he p esence o absence o asco bic acid [ 19 ]. A 12-week-old, Wis a and GK a s had simila ACh-dependen elaxa ion (Figu e 5A). In he a s ea ed wi h EA-Aa, a sligh e ec on ao a elaxa ion was obse ed, namely a 15% inc emen be ween W-EA-Aa and W-C l (Figu e 5B) and 10% be ween GK- EA-Aa and GK-C l (Figu e 5C), al hough s a- is ical signi icance was no eached. P e-incuba ion o he ings wi h L-NAME (NOS inhibi o ) p ac ically abolished he elaxa ion o he ao ic ings (da a no shown), showing endo helial dependence. When he ao a was incuba ed wi h asco bic acid, he maximum endo helial-dependen elaxa ion media ed by NA-p econ ac ed ings in esponse o Ach eached 40% mo e in W-C l and almos 74% mo e in W-EA-Aa (Figu e 5D–E). In e es ingly, no esponse o asco bic acid was ob ained in GK-C l (Figu e 5F), bu i was es o ed a e he ea men wi h he ex ac , which p omo ed a 30% inc emen o elaxa ion in GK-EA-Aa (Figu e 5G). Such esul s a e suppo ed by he his ological luo escence s aining o bo h oxida i e s ess p obes, DHE and DCF, whe e 34% and 67% espec i ely lowe signal in ensi y was obse ed in GK-EA-Aa in ela ion o he GK-C l g oup (Figu e 6A–D). The ea men also p omo ed an inc ease o ca alase le els in he ao a o ea ed g oups om bo h s ains, mainly in diabe ic a s (Figu e 6E). Nu ien s 2021,13, 2856 9 o 17 Nu ien s 2021, 13, x FOR PEER REVIEW 9 o 19 Figu e 3. An ioxidan e ec s o EA-Aa in EAT, li e and in 3T3-L1 p e-adipocy es (n = 3 independen expe imen s). (A) Si 1 le els in EAT. (B) NRF2 le els in EAT, EA-Aa p omo es pa ial es o a ion o NRF2 le els in he non-obese ype 2 diabe ic EA-Aa- ea ed g oup. (C) Ca alase le els in EAT. (D) Rep esen a i e images o EAT Wes e n blo . Cell iabili y o p e-adipocy es 3T3-L1: (E) T ea men wi h EA-Aa o 24 h. (F) T ea men wi h EA-Aa (p e iously o 30 min) and induc ion o oxida i e s ess wi h H 2 O 2 ( o 2 h). (G) Rep esen a i e images o 3T3-L1 Wes e n blo . (H) Dihyd oe hidium (DHE) s aining in li e (n = 3). (I) Si 1 le els in li e . (J) NRF2 le els in li e . (K) Ca alase le els in li e (n = 5 o Wes e n blo s). (L) Rep esen a i e images o li e Wes e n blo . * s. W-C l/C l; # s. H 2 O 2 ; ** p < 0.01; ***,### p < 0.001. Figu e 3. An ioxidan e ec s o EA-Aa in EAT, li e and in 3T3-L1 p e-adipocy es (n= 3 independen expe imen s). ( A ) Si 1 le els in EAT. 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