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This is he pee e iewed e sion o he ollowing a icle: Rod iguez-Rod iguez
R, Jiménez-Al ayó F, Alsina L, One i Y, Rinaldi de Al a enga JF, Cla o C,
Ogalla E, Casals N, Lamuela-Ra en os RM. Medi e anean oma o-based
so i o p o ec s agains ascula al e a ions in obese Zucke a s by p ese ing
NO bioa ailabili y. Mol Nu Food Res. 2017 Sep;61(9). doi:
10.1002/mn .201601010. Epub 2017 Ap 5. PMID: 28319651.
h ps://doi.o g/10.1002/mn .201601010
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1
Medi e anean oma o-based so i o p o ec s agains ascula dis u bances in obese
Zucke a s by p ese ing NO bioa ailabili y
Rosalia Rod iguez-Rod iguez1, F ancesc Jiménez-Al ayó2, Laia Alsina2, Ya a One i2, Ca men
Cla o3, Elena Ogalla3, Nu ia Casals1,5, Rosa M. Lamuela-Ra en os4,5
1 Basic Sciences Depa men , Facul y o Medicine and Heal h Sciences, Uni e si a
In e nacional de Ca alunya, San Cuga del Vallès, Ba celona, Spain.
2 Depa amen de Fa macologia, de Te apèu ica i de Toxicologia, Ins i u de Neu ociències,
Facul a de Medicina, Uni e si a Au ònoma de Ba celona, Bella e a, Spain.
3 Depa amen o de Fa macología, Facul ad de Fa macia, Uni e sidad de Se illa, Se illa, Spain.
4 Nu i ion, Food Science Depa men and Gas onomy, XaRTA, INSA-UB Pha macy, Uni e si y
o Ba celona, Ba celona, Spain.
5 CIBER Fisiopa ología de la Obesidad y la Nu ición (CIBEROBN), Ins i u o de Salud Ca los III,
Mad id, Spain
2
Abs ac
Scope: So i o, a key componen o he Medi e anean die , p o ides nu i ional in e es due
o i s high con en in bioac i e compounds om oma o, onion and oli e oil, and especially o
he lipid ma ix in which hese compounds a e ound. In his s udy, he po en ial bene icial
e ec s o die a y in ake o so i o on obesi y- ela ed ascula dis u bances we e explo ed in
obese Zucke a s.
Me hods and esul s: Obese and lean a s we e ed a con ol die supplemen ed o no wi h
2% so i o o 8 weeks. Vascula unc ion was e alua ed in ao ic ings in o gan ba hs.
Dihyd oe hidium s aining and immuno luo escence was used o de e mine ao ic supe oxide
anion and pe oxyni i e p oduc ion, espec i ely. Despi e ood in ake was highe in so i o- ed
obese a s, no di e ences we e app ecia ed on body weigh gain, o gan weigh s and lipid
p o ile compa ed o obese con ol a s. In ao a, so i o in ake a enua ed phenyleph ine-
induced asocons ic ion in obese and lean a s. This e ec was associa ed wi h eco e y o
he ni ic oxide (NO) in luence on asocons ic ion in obese animals. Fu he mo e,
asop o ec ion was accompanied by no maliza ion o se um NO me aboli es and ascula
supe oxide anion and pe oxyni i e le els in obese a s.
Conclusion: This s udy demons a es o he i s ime ha oma o-based so i o p o ec s
agains ascula al e a ions ha could p ecede majo ca diome abolic complica ions
associa ed o obesi y by es o ing physiological ascula and ci cula ing NO le els. These
esul s could con ibu e o explain he he apeu ic p ope ies agains obesi y- ela ed diso de s
a ibu able o he Medi e anean die . The e o e, so i o is an a ac i e die a y app oach
agains obesi y and i s ca dio ascula heal h isks, a leas in he ea ly s ages o obesi y.
Keywo ds: so i o; Medi e anean die ; obesi y; ascula unc ion; ni ic oxide
Lis o abb e ia ions:
3
1. In oduc ion
Obesi y is one o he leading public heal h p oblems in de eloping coun ies. Besides i s di ec
impac on li e quali y, obesi y is a majo isk ac o o highly le hal ch onic complica ions such
as insulin esis ance and ca dio ascula disease [1]. Pa icula ly, he pa hophysiology o
obesi y- ela ed ascula diso de s is an impo an a ge o de eloping new he apeu ic
s a egies aimed o p e en o amelio a e ca dio ascula complica ions in obesi y [2].
Al hough adi ional die a y app oaches ha e p o en success ul as pa o he ea men o
obesi y and ca dio ascula pa hologies in clinical ials, he exac ole and impo ance o he
indi idual componen s o he die a y pa e n on speci ic aspec s o hese diseases need u he
e alua ion [3, 4]. Resea ch has he e o e u ned i s a en ion o unc ional oods and
nu aceu ics as main componen s o a heal hy p o o ype die ha could ha e he po en ial
abili y o modula e physiological and pa hophysiological molecula mechanisms, hus esul ing
in a o able heal h ou comes [4]. In his sense, he Medi e anean die a y pa e n has been
shown o educe he bu den o e en p e en he de elopmen o ca dio ascula disease,
cance and obesi y, among o he pa hologies [3, 5, 6]. Wi hin he Medi e anean die , oma o
and oma o sauces a e ypical. In ac , oma o sauce is he mos commonly consumed oma o
p oduc wo ldwide and pa icula ly in Spain, whe e i ep esen s almos 40.8 % o all sauce
consump ion [7]. In addi ion, consump ion o oma o p oduc s has been consis en ly
associa ed wi h a lowe isk o se e al ypes o cance and co ona y hea disease, an e ec
mainly a ibu able o i s high con en in bioac i e compounds such as polyphenols,
ca o enoids and i amin C [8–11].
Toma o-based so i o, a key componen o he Medi e anean die , is pa icula ly in e es ing
o i s high con en in bioac i e compounds, no only om oma o bu also om onion and
i gin oli e oil, and especially o he lipid ma ix in which hese compounds a e ound [12].
Rega ding he la e , ecen in es iga ions ha e demons a ed ha mechanical and he mal
Comen ado [RR1]: Rosa, sólo he encon ado es e da o en la
e .7, no encuen o da o de consumo mundial compa ando con
o as sauces consump ion. Solo engo el da o de consumo de
oma e mundial pe capi a. ¿Tienes algún da o adicional?
4
ea men s, as well as oil ma ix addi ion du ing oma o and so i o sauce p ocessing, may
inc ease he bioaccessibili y, ex ac abili y and bioa ailabili y o phenolic compounds om
oma o [12, 13]. Once hey each he adequa e bioa ailabili y, p edominan phy ochemicals
p esen in so i o, especially polyphenols and ca o enoids, can exe hei heal h-p omo ing
e ec s [13–15]. Epidemiological and p eclinical s udies ha e demons a ed ha ui s,
ege ables and be e ages ich in ca o enoids (e.g. lycopene and be a-ca o ene) and
polyphenols (e.g. u in, que ce in and na ingenin) delay he onse o a he oscle osis and
exhibi an i-in lamma o y and an ipla ele ac i i ies [16, 17]. Addi ionally, hese
phy ochemicals a e able o educe neoin imal hickening by inhibi ing p oli e a ion o ascula
smoo h muscle cells, and can also p e en ascula dys unc ion by imp o ing ni ic oxide (NO)
a ailabili y [16]. In pa icula , ecen e idence sugges s ha ca o enoids and polyphenols a e
key playe s no only in he ea men o in lamma ion and ascula complica ions, bu also in
es o ing no mal adipocy e unc ion in obese subjec s [4, 18, 19]. In e es ingly, an in e se
associa ion has been es ablished be ween plasma ic le els o ca o enoids and in lamma o y
and oxida i e s ess ma ke s in obese pa ien s [20]. The ac ha obesi y is an in lamma o y
s a e ha con ibu es o oxida i e s ess and ascula complica ions [2], indica es ha a
die a y sou ce o ca o enoids and polyphenols could ep esen an a ac i e he apeu ic
s a egy o p e en o amelio a e he obesi y- ela ed complica ions.
In he p esen s udy, he po en ial bene icial e ec s o a sho - o middle- e m die a y in ake
o Medi e anean so i o we e explo ed o he i s ime in an animal model o obesi y.
Conc e ely, we used he Zucke a ha is an obesi y model ex emely use ul o explo e
ascula and me abolic complica ions associa ed o obesi y [21]. Thus, his s udy is an a emp
o e idence he po en ial con ibu ion o so i o consump ion in he heal hy p ope ies o he
Medi e anean die .
2. Ma e ials and me hods
2.1. Animals and die s

5
Six week-old male obese Zucke a s and hei lean li e ma e con ols we e pu chased om
Cha les Ri e (Cha les Ri e Labo a o ies, Ba celona, Spain). A 8 weeks o age, obese and lean
a s we e andomly assigned o he ollowing g oups (n=8): Lean a s ed con ol chow die
(LC), obese a s ed con ol chow die (OC), lean a s ed chow die supplemen ed in 2 % so i o
(LS), and obese a s ed chow die supplemen ed in 2 % so i o (OS). Con ol (s anda d) chow
die (Global Die 2014) was p o ided by Ha lan Labo a o ies (Milan, I aly). So i o (Gallina
Blanca-S a , Ba celona, Spain) was p e iously cha ac e ized [12] and con en o polyphenols
and ca o enoids is shown in Table I. 2% So i o supplemen was calcula ed acco ding o
p e ious s udies in humans in which 600-900 g so i o/week is adminis e ed.
Body weigh and ood in ake was weekly e alua ed. A e 8 weeks o adminis a ion o he
expe imen al die s, animals we e killed by decapi a ion. Blood samples we e immedia ely
collec ed and ho acic ao a, li e , isce al and epididymal adipose issues (VAT and EAT,
espec i ely) we e dissec ed.
The p o ocol o animal handling and expe imen a ion ag eed wi h he Eu opean Union
guidelines o he e hical managemen o animals and was app o ed by he Commi ee o
E hical Expe imen a ion o he Uni e si a o Ba celona.
2.2. Blood biochemical assays
Se um samples we e ob ained om blood by cen i uga ion o 5 minu es a 2000 g a oom
empe a u e. Glucose, o al choles e ol and iglyce ides le els in se um we e de e mined
using enzyma ic-based assays ollowing manu ac u e 's ins uc ions (Ca Bio Labo a o ies,
Ba celona, Spain). Se um le els o ni ic oxide me aboli es (NOx) we e de e mined by using a
me hodology based on he colo ime ic G iess eac ion. Abso bance was measu ed
spec opho ome ically a 540 nm [22].
2.3. Tissue p epa a ion
Comen ado [RR2]: He incluido la inges a caló ica en la abla I
6
Tho acic ao a was cleaned o adhe ing a and connec i e issue and placed in cold K ebs-
Henselei solu ion (KHS) (in mmol/L: NaCl 112; KCl 4.7; NaHCO3 25; MgSO4 1.2; CaCl2 2.5;
KH2PO4 1.2 and glucose 11.1; pH 7.4). Fo de ec ion o supe oxide anion (O2˙−), ao ic ings
we e main ained in 30 % suc ose-KHS o e nigh , placed in o c yomolds con aining Tissue-Tek
OCT embedding medium (Saku a Fine ek Eu ope, he Ne he lands), and immedia ely ozen in
liquid ni ogen o s o age a −80°C [23]. Fo immuno luo escence s udies, ao as we e ixed in
4 % phospha e-bu e ed pa a o maldehyde (pH 7.4) o 1 h and hen washed in phospha e-
bu e ed saline (PBS). A e wa ds, a e ial segmen s we e placed in 30% suc ose PBS o e nigh ,
ans e ed o c yomolds wi h embedding medium, ozen in liquid ni ogen, and kep a −80°C
un il analysis [23].
2.4. Reac i i y expe imen s
Ao ic ings we e disposed in o gan ba hs, and ascula unc ion was measu ed as p e iously
desc ibed [23]. Con ac ile capaci y o he essels was assessed wi h KCl 100 mmol/L solu ion.
A e 30 minu es o s abiliza ion, endo helial-dependen asodila a ions we e s udied by
e alua ing he elaxa ion induced by ace ylcholine (ACh; 0.001–10 μmol/L) pe o med in
essels con ac ed o 60-80% o KCl con ac ion wi h phenyleph ine (Phe). To in es iga e
con ac ile esponses media ed by α1-ad enocep o s imula ion, concen a ion– esponse
cu es o Phe (0.001–100 μmol/L) we e pe o med. Concen a ion– esponse cu es o Phe
we e cons uc ed in he absence o p esence o he nonselec i e NO syn hase (NOS) inhibi o
N-ni o-L-a ginine me hyl es e (L-NAME, 300 μmol/L).
2.5. In si u de ec ion o supe oxide anion
The oxida i e luo escen dye dihyd oe hidium (DHE) was used o e alua e p oduc ion o
a e ial O2˙− in si u, as p e iously desc ibed [23]. B ie ly, 14-µm hick ao ic sec ions placed on
gela in-coa ed slides we e incuba ed wi h DHE (2 mmol/L) in K ebs-HEPES bu e . P epa a ions
we e iewed by lase scanning con ocal mic oscope (TCS SP2; Leica, Heidelbe g, Ge many; 20x
7
objec i e). DHE luo escence was isualized by exci a ion a 546 nm and emission a 610 nm. In
hese expe imen s, DHE luo escence was abolished by he O2˙− sca enge , Mn(III) e akis(1-
me hyl-4-py idyl)po phy in (0.1434 mg/ml, 30 min, 37°C), indica ing he speci ici y o his
eac ion. In eg a ed op ical densi ies we e quan i ied using Me aMo ph Image Analysis
So wa e (Molecula De ices, Sunny ale, CA, USA). The luo escence signal pe a ea was
measu ed in a leas wo ings o each animal, and he esul s we e exp essed as a bi a y
uni s.
2.6. Immuno luo escence
The in si u p oduc ion o pe oxyni i e (ONOO-) was measu ed indi ec ly by analyzing
ni o y osine le els, as p e iously desc ibed [24]. F ozen ans e se sec ions (14-µm hick) cu
on o gela in-coa ed slides we e incuba ed wi h abbi polyclonal an ibody agains ni o y osine
(1:100; Me ck Millipo e, Da ms ad , Ge many) in PBS con aining 2% bo ine se um albumin o
1 h a 37°C in a humidi ied chambe . A e washing, a e ial p epa a ions we e incuba ed wi h
he seconda y an ibody IgG conjuga ed o cyanine 3 (1:200; Jackson ImmunoResea ch
Labo a o ies Inc, Wes G o e, PA, USA) o 1 h a 37°C in a humidi ied chambe . Signals we e
iewed using a lase scanning con ocal mic oscope (TCS SP2; Leica, Heidelbe g, Ge many; 20x
objec i e). Cyanine 3-labeled an ibody was isualized by exci a ion a 561 nm and de ec ion a
600 o 700 nm. The speci ici y o he immunos aining was e alua ed by omission o he
p ima y an ibody and p ocessed as desc ibed abo e. Unde hese condi ions, no s aining was
obse ed in he a e ial wall in any expe imen al si ua ion. Op ical densi y o
immuno luo escence was assayed wi h Me aMo ph Image Analysis So wa e (Molecula
De ices, Sunny ale, CA, USA). The luo escence signal pe a ea was measu ed in a leas wo
ings o each animal, and he esul s we e exp essed as a bi a y uni s.
2.7. Da a analysis and s a is ics
Resul s we e shown as mean ± SEM o he numbe o a s (n) indica ed in he igu e legends.
Vasocons ic o esponses we e exp essed as a pe cen age o one gene a ed by KCl 100
8
mmol/L. Vasodila o esponses o ACh we e exp essed as a pe cen age o he p e ious one
gene a ed by Phe. A ea unde he cu e (AUC) was calcula ed om each indi idual
concen a ion- esponse cu e o ACh and Phe, and illus a ed as a bi a y uni s. The
dependence o asocons ic o and asodila o esponse on s ain and die o on die and
essel ea men was assessed by a wo-way ANOVA wi h epea ed measu emen s on he
concen a ion ac o . Fo NOx, supe oxide and ni o y osine luo escence le els, di e ences
be ween g oups we e analyzed by unpai ed - es . Di e ences we e conside ed signi ican
when P< 0.05. Da a analysis was ca ied ou using G aphPad P ism So wa e 5.0 (San Diego,
CA, USA).
3. Resul s and discussion
3.1. E ec o so i o-supplemen ed die on body weigh gain, ood in ake, o gan weigh s and
se um biochemical de e mina ions
As illus a ed in Table II, obese a s gained mo e body weigh , and li e and whi e adipose
issue weigh s we e highe compa ed o lean a s. Eigh -weeks adminis a ion o so i o-
supplemen ed die was no able o modi y body weigh gain and o gan weigh s in obese a s.
Mo eo e , i is impo an o men ion ha , despi e ood and calo ic in ake in OS g oup was
signi ican ly highe han in OC a s, his hype phagia did no imply highe body weigh gain o
highe alues o li e and whi e adipose issue weigh s in so i o- ed a s (Table II). These
esul s a e in ag eemen wi h a ecen s udy published om he PREDIMED coho ha shows
ha obese o o e weigh people ollowing a Medi e anean die a y pa e n wi h heal hy a
in ake gain less body weigh han he con ol g oup [25].
Acco ding o he obese pheno ype o Zucke a s, se um analysis e ealed highe le els o o al
choles e ol and iglyce ides in obese compa ed wi h lean a s, and so i o- ed g oups showed
simila le els o bo h pa ame e s compa ed o hei espec i e lean o obese con ol g oups
(Table II). I is known ha ch onic adminis a ion o oma o-based die s and polyphenols- o
15
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21
Figu e legends:
Figu e 1. Concen a ion- esponse cu es o ace ylcholine (ACh) (A-B) and phenyleph ine (Phe)
(C-D) in ao ic ings om lean and obese Zucke a s. LC: lean a s ed a con ol die , LS: lean
a s ed a so i o-supplemen ed die , OC: obese a s ed a con ol die , OS: obese a s ed a
so i o-supplemen ed die . Da a a e mean ± SEM (n=5-6). *P<0.05 s LC; #P<0.05 s OC.
Figu e 2. Concen a ion- esponse cu es o phenyleph ine (Phe) in he absence o p esence o
he NO syn hesis inhibi o L-NAME in ao ic ings om lean (A-B) and obese Zucke (C-D) a s.
LC: lean a s ed a con ol die , LS: lean a s ed a so i o-supplemen ed die , OC: obese a s ed
a con ol die , OS: obese a s ed a so i o-supplemen ed die . Da a a e mean ± SEM (n=5-6).
*P<0.05 s LC; +P<0.05 s LS; #P<0.05 s OS.
Figu e 3. Se um NO me aboli es (ni a es and ni i es, NOx) le els a e so i o-supplemen ed
die adminis a ion in lean and obese Zucke a s. LC: lean a s ed a con ol die , LS: lean a s
ed a so i o-supplemen ed die , OC: obese a s ed a con ol die , OS: obese a s ed a so i o-
supplemen ed die . Da a a e mean ± SEM (n=5-6). *P<0.05 s LC.
Figu e 4. Rep esen a i e pho omic og aphs and quan i ica ion o dihyd oe hidium-de i ed (an
indi ec indica o o supe oxide anion) luo escence (A) and ni o y osine (an indi ec indica o
o pe oxyni i e) immuno luo escence (B) le els in con ocal mic oscopic ao ic sec ions om
lean and obese Zucke a s. LC: lean a s ed a con ol die , LS: lean a s ed a so i o-
supplemen ed die , OC: obese a s ed a con ol die , OS: obese a s ed a so i o-
supplemen ed die . Da a a e mean ± SEM (n=5-7). *P<0.05; **P<0.01 s LC.
22
Table I. Cha ac e iza ion o polyphenol and ca o enoid con en o he Medi e anean so i o
used in he s udy. The so i o is mainly composed by oma o (50%) (pulp and concen a ed),
onion (37%), i gin oli e oil (12%) and sal . Quan i ica ion o indi idual polyphenols o
ca o enoids (µg/g FW) and o al polyphenols by Folin-Ciocal eau (µg GAE/g FW) is exp essed as
mean ± SD.
Polyphenols
Ca o enoids
Fe ulic acid
Chlo ogenic acid
Ca eic acid
Sinapic acid
P o oca echuic acid
p-Couma ic acid
p-Hyd oxybenzoic acid
Ru in
Que ce in
Que ce in-3-O-glucoside
Na ingenin
Na ingenin-7-O-glucòsid
To al polyphenols (µg GAE/g
FW)
03,89 ± 0,16
05,50 ± 0,21
04,72 ± 0,12
03,54 ± 0,10
02,26 ± 0,07
04,01 ± 0,07
03,01 ± 0,08
16,66 ± 0,67
13,77 ± 0,25
04,20 ± 0,09
10,33 ± 0,44
04,21 ± 0,05
202 ± 2
ans-α-Ca o ene
ans-β-Ca o ene
ans-Lu ein
ans-Lycopene
5-cis-Lycopene
9-cis- Lycopene
13-cis- Lycopene
To al Ca o enoids
01,10 ± 0,03
29,69 ± 0,11
03,42 ± 0,04
40,50 ± 0,33
02,81 ± 0,07
01,88 ± 0,10
02,09 ± 0,07
81,49
Adap ed om [12]. SD: s anda d de ia ion; GAE: gallic acid equi alen ; FW: esh weigh .
23
Table II. Body weigh , ood in ake, o gan weigh s and se um biochemical de e mina ions
LC
LS
OC
OS
Body weigh (g)
383.6 ± 8.7
378.4 ± 6.5
518.3 ± 15.6*
517.3 ± 14.5+
Food in ake (g/day/ a )
20.00 ± 1.24
23.61 ± 1.48
28.64 ± 1.77*
34.44 ± 1.13+
Calo ic in ake (Kcal)
61.99 ± 3.86
73.71 ± 4.61
88.79 ± 5.50*
107.55 ± 3.52+ #
O gan weigh (g/ 100g b.w.)
Li e
3.78 ± 0.16
3.46 ± 0.08
5.11 ± 0.35*
5.44 ± 0.35+
VAT
0.78 ± 0.10
0.91 ± 0.10
1.58 ± 0.24*
1.35 ± 0.19+
EAT
0.75 ± 0.10
0.77 ± 0.15
1.94 ± 0.10*
0.89 ± 0.10+
Se um pa ame e s
Glucose (mmol/L)
7.1 ± 0.15
7.633 ± 0.12
8.433 ± 0.47*
9.4 ± 1.134+
T iglyce ides (mmol/L)
1.496 ± 0.15
1.327 ± 0.12
5.684 ± 0.54*
4.76 ± 0.28+
Choles e ol (mmol/L)
3.494 ± 0.10
3.523 ± 0.12
6.09 ± 0.47*
7.07 ± 0.39+
Da a a e mean ± SEM. *P<0.05 s LC; +P<0.05 s LS; #P<0.05 s OC. LC: lean a s ed a con ol die , LS: lean a s ed
a so i o-supplemen ed die , OC: obese a s ed a con ol die , OS: obese a s ed a so i o-supplemen ed die . VAT:
isce al adipose issue, EAT: epididymal adipose issue.