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. (
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. H2O2; ** p< 0.01; ***,### p< 0.001.
Nu ien s 2021,13, 2856 16 o 17
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