Li elong ea men wi h a enolol dec eases memb ane a y
acid unsa u a ion and oxida i e s ess in hea and skele al
muscle mi ochond ia and imp o es immuni y and beha io ,
wi hou changing mice longe i y
Alexia G
omez,
1
Ines S
anchez-Roman,
1
Jose Gomez,
1
Julia C uces,
1
Iani e Ma e,
1
M
onica Lopez-To es,
1
Alba Naudi,
2
Manuel Po e o-O in,
2
Reinald Pamplona,
2
Monica De la Fuen e
1
and Gus a o Ba ja
1
1
Depa men o Animal Physiology-II, Facul y o Biological Sciences,
Complu ense Uni e si y o Mad id (UCM), Mad id, Spain
2
Depa men o Expe imen al Medicine, Facul y o Medicine, Uni e si y o
Lleida-IRBLLEIDA, Lleida, Spain
Summa y
The memb ane a y acid unsa u a ion hypo hesis o aging and
longe i y is expe imen ally es ed o he i s ime in mammals.
Li elong ea men o mice wi h he b1-blocke a enolol inc eased
he amoun o he ex acellula -signal- egula ed kinase signaling
p o ein and success ully dec eased one o he wo ai s app o-
p ia ely co ela ing wi h animal longe i y, he memb ane a y
acid unsa u a ion deg ee o ca diac and skele al muscle mi ochon-
d ia, changing hei lipid p o ile owa d ha p esen in much mo e
longe -li ed mammals. This was mainly due o dec eases in 22:6n-3
and inc eases in 18:1n-9 a y acids. The a enolol ea men also
lowe ed isce al adiposi y (by 24%), dec eased mi ochond ial
p o ein oxida i e, glycoxida i e, and lipoxida i e damage in bo h
o gans, and lowe ed oxida i e damage in hea mi ochond ial
DNA. A enolol also imp o ed a ious immune (chemo axis and
na u al kille ac i i ies) and beha io al unc ions (equilib ium,
mo o coo dina ion, and muscula igo ). I also o ally o pa ially
p e en ed he aging- ela ed de imen al changes obse ed in
mi ochond ial memb ane unsa u a ion, p o ein oxida i e modi i-
ca ions, and immune and beha io al unc ions, wi hou changing
longe i y. The con ols eached 3.93 yea s o age, a subs an ially
highe maximum longe i y han he bes p e iously desc ibed o
his s ain (3.0 yea s). Side e ec s o he d ug could ha e masked a
likely lowe ing o he endogenous aging a e induced by he
dec ease in memb ane a y acid unsa u a ion. We conclude ha i
is a enolol ha ailed o inc ease longe i y, and likely no he
dec ease in memb ane unsa u a ion induced by he d ug.
Key wo ds: aging; a enolol; a y acid unsa u a ion; hea
a e; longe i y; oxida i e s ess.
Abb e ia ions
AASA aminoadipic semialdehydes
AIF apop osis-inducing ac o
AT a enolol
CEL ca boxye hyl-lysine
CML ca boxyme hyl-lysine
DBI double bond index
GSA glu amic semialdehydes
MDAL malondialdehyde-lysine
mi ROS mi ochond ial eac i e oxygen species
m DNA mi ochond ial DNA
p-ERK phospho yla ed ex acellula -signal- egula ed kinase
PI pe oxidizabili y index
8-oxodG 8-oxo-7,8-dihyd o-2′deoxyguanosine
In oduc ion
Only wo known pa ame e s app op ia ely co ela e wi h animal
longe i y ac oss species – he a e o mi ochond ial eac i e oxygen
species p oduc ion (mi ROSp) and he memb ane a y acid unsa u a ion
deg ee. Long-li ed mammals and bi ds show a low a e o mi ROS
gene a ion ( e iewed in Ba ja, 2013) and ha e cellula memb anes wi h a
low deg ee o a y acid unsa u a ion (Pamplona e al., 1996, 2002;
Hulbe e al., 2007; Naudi e al., 2011). These wo ai s a e unda-
men al o he mi ochond ial ee adical heo y o aging (Ha man, 1972;
Ba ja, 2013). The low mi ROSp o long-li ed species dec eases oxida i e
damage o key molecules such as m DNA (Ba ja & He e o, 2000), and
hei low memb ane a y acid unsa u a ion diminishes he in ensi y o
lipid pe oxida ion and hus memb ane and p o ein damage (Pamplona
e al., 2002) and he seconda y gene a ion o oxic chemicals and ee
adicals (Pamplona, 2011; Zimniak, 2011).
Ne e heless, co ela ion does no imply causa ion. Expe imen al
app oaches ha can e eal causal connec ions o hose wo main ai s
wi h aging and longe i y a e needed. In he case o mi ROSp, i is
modi ied by die a y manipula ions ha inc ease mean and maximum
longe i y. Die a y (G edilla & Ba ja, 2005), p o ein (Sanz e al., 2004),
and me hionine es ic ion (Sanz e al., 2006) dec ease mi ROSp
(Sanchez-Rom
an and Ba ja, 2013) and hese h ee manipula ions also
inc ease longe i y in a s and mice and imp o e many heal h- ela ed
pa ame e s (Pe one e al., 2013), suppo ing he idea ha he low
mi ROSp o long-li ed animals causally con ibu es o dec eased aging
a e in long-li ed animal species.
We hypo hesize he e ha expe imen ally dec easing memb ane a y
acid unsa u a ion can p e en de imen al changes wi h age and
inc ease longe i y in mammals. The e a e no in es iga ions abou he
li elong e ec o dec easing issue memb ane a y acid unsa u a ion on
mammalian longe i y and egula ion o aging- ela ed pa ame e s.
Sho - e m –2-week –in es iga ions ha e shown ha a enolol, a
Co espondence
D . Gus a o Ba ja, Depa amen o de Fisiolog
ıa Animal II, Facul ad de Ciencias
Biol
ogicas, Uni e sidad Complu ense, c/ Jose An onio No ais 2, Mad id 28040,
Spain. Tel.: +34 91 3944919; ax: +34 91 3944935; e-mail: [email p o ec ed]
Accep ed o publica ion 12 Janua y 2014
ª2014 The Au ho s. Aging Cell published by he Ana omical Socie y and John Wiley & Sons L d.
This is an open access a icle unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s use,
dis ibu ion and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed.
551
Aging Cell (2014) 13, pp551–560 Doi: 10.1111/acel.12205
Aging
Cell
b1-ad ene gic ecep o blocke , gi en in d inking wa e , dec eases
memb ane a y acid unsa u a ion in he hea o C57BL/6 mice
(Sanchez-Roman e al., 2010) and a s (Sanchez-Roman e al., 2014).
The ex en o such dec ease was almos equal o he di e ence in
unsa u a ion be ween mammals di e ing by one o de o magni ude in
longe i y, such as mice and cows, and i lowe ed ca diac p o ein
oxida i e damage and lipoxida ion (Sanchez-Roman e al., 2010). This is
in e es ing also because knocked-ou mice o he adenylyl cyclase ype 5
gene (AC5KO mice), which blocks b-ad ene gic ecep o signaling and
inc eases he Ra /MEK/ERK signaling pa hway, showed inc eased
longe i y oge he wi h lowe ed ca dio ascula , hea , and bone aging
(Yan e al., 2007).
Taking ad an age o he s iking capaci y o a enolol o s ongly
dec ease memb ane unsa u a ion in mice, we use i he e o expe imen-
ally es , o he i s ime, he hypo hesis ha lowe ing he deg ee o
memb ane a y acid unsa u a ion in a mammal causes bene icial
changes du ing aging and inc eases longe i y. A enolol has been widely
used in humans du ing he las 3–4 decades o ea a ious ca dio as-
cula pa hologies and has been classically conside ed a sa e d ug wi hou
signi ican side e ec s. A o al o 134 mice we e ea ed o no wi h
a enolol in hei d inking wa e du ing hei whole li espan, and he
e ec on mean and maximum longe i ies was s udied. A 18 mon hs o
age, subg oups o 48 sepa a ed ‘pilo ’ animals we e used o measu e
ca dio ascula and o he basic physiological pa ame e s: memb ane a y
acid composi ion and global unsa u a ion; mi ochond ial oxygen con-
sump ion and mi ROSp; amoun s o espi a o y complexes (I–IV) and he
complex I- ela ed apop osis-inducing ac o (AIF); oxida i e damage o
m DNA; oxida i e, glycoxida i e, and lipoxida i e p o ein modi ica ion in
hea and skele al muscle mi ochond ia; and he amoun o phospho -
yla ed ex acellula -signaling- egula ed kinase (p-ERK) o co obo a e
ac i a ion o he Ra /MEK/ERK signaling pa hway by he a enolol
ea men . Immunological pa ame e s and a ious beha io al es s we e
also pe o med o be e es ima e he gene al unc ional s a e o he
animals. Al hough i is gene ally hough ha immune cells ha e almos
exclusi ely b-2 ad ene gic ecep o s, he e a e esul s in ol ing b-1
ad ene gic ecep o signaling in he modula ion o immune esponses
(Emeny e al., 2007), bu hei immunological ele ance has been la gely
unexplo ed and he e ec o a enolol on he immune cell unc ions has
been sca cely s udied. Conce ning beha io , a ew epo s showed
dec eased anxie y (Dea y e al., 1991) and an idep essan e ec s
(Al-Tubuly e al., 2008) a e a enolol ea men , bu he impac o
a enolol on beha io al pa ame e s has no been in es iga ed. In he case
o he ca dio ascula pa ame e s, as an inc ease in mo ali y was
de ec ed only in e y old indi iduals, he measu emen s we e epea ed a
35 mon hs o age. A g oup o young bu ma u e adul con ol mice o
6 mon hs o age (a sac i ice) main ained in pa allel unde simila
condi ions o hose o he li elong con ol and ea ed animals was used
o check whe he a enolol can p e en age- ela ed changes. Hea and
skele al muscle we e selec ed as i al a ge o gans because hey a e
among he ones mos a ec ed by he endogenous aging p ocess and a e
almos ully composed o pos mi o ic cells.
Resul s
Body and o gan weigh s and physiological pa ame e s
A e 16 mon hs o expe imen a ion, he 18-mon h-old animals [bo h
Old con ols and Old a enolol (AT)] had highe o al weigh and highe
hea , kidney, li e , and o al isce al a weigh han Young ones,
whe eas he weigh s o he spleen, b ain, and hind limb skele al muscle
we e no modi ied by aging ( esul s no shown). Visce al a s ongly
inc eased om 0.441 0.051 g ams in Young o 2.31 0.20 g in Old
con ols (424% inc ease; P<0.00001). The long- e m a enolol ea -
men signi ican ly dec eased kidney weigh by 7.6% ( om 0.655
0.016 o 0.605 0.012 g; P<0.01) and isce al adipose issue mass by
23.8% ( om 2.31 0.20 o 1.76 0.18; P<0.02; by a mean o
23.2% when exp essed pe g ams o body mass), whe eas o al body
weigh and he weigh o he es o he o gans we e no a ec ed by he
ea men . No di e ences in ood o wa e in ake be ween Old and
Old-AT animals we e obse ed a e inspec ion once e e y 2 weeks.
The alues o a ious basic physiological pa ame e s a e shown in
Table S1 (Suppo ing In o ma ion). Old-AT animals had signi ican ly
lowe ou ine me abolic a e han Young animals bu showed no
signi ican di e ences compa ed o Old con ols. Nei he ec al empe -
a u e, hea a e, no he sys olic, mean, and dias olic a e ial p essu es
showed AT- o age- ela ed di e ences.
Long-li e su i al s udy
The su i al cu es o con ol and AT- ea ed animals a e shown in
Fig. 1. No signi ican di e ences in mean, median, maximum (90%)
longe i ies, o o al cu e su i al (Tables S2 and S3, Suppo ing
In o ma ion) we e obse ed compa ing con ol and AT- ea ed animals
(log- ank and Wilcoxon es s). The longe -li ed animal in he con ol
g oup eached 1433 days o age (3.93 yea s), and he longe -li ed AT
mouse eached 1310 days o age (3.6 yea s). A s a is ically signi ican
highe mo ali y in a enolol- ea ed compa ed o con ol animals was
obse ed be ween 1000 and 1275 days o age (Table S3, Suppo ing
In o ma ion). Due o his unexpec ed inding, he ca dio ascula
pa ame e s we e measu ed again, mo e han 1 yea a e he p e ious
measu emen s ( aken a 18 mon hs o age), in e y old animals. These
measu emen s showed ha , a 35 mon hs o age, hea a e and
sys olic, mean, and dias olic blood p essu es we e signi ican ly lowe in
Old-AT han in Old con ols (Table S4, Suppo ing In o ma ion).
Mi ochond ial oxygen consump ion and eac i e oxygen
species p oduc ion
Table S5 (Suppo ing In o ma ion) shows he mi ochond ial oxygen
consump ion alues. The a e o oxygen consump ion o hea
mi ochond ia in s a e 3 (phospho yla ing) wi h complex I-linked
subs a es (glu ama e/mala e) was signi ican ly highe in Old han in
Young con ols and signi ican ly dec eased in Old-AT o alues simila o
hose o Young mice. These di e ences we e no p esen wi h
succina e + o enone, a condi ion in which he inhibi o o enone a oids
elec ons o each complex I by e e se elec on low om complex II. No
signi ican di e ences due o age o a enolol ea men we e obse ed in
hea mi ochond ia in s a e 4 ( es ing) o in skele al muscle mi ochond ia
wi h any subs a e o mi ochond ial s a e.
The basal a e o ROS p oduc ion o hea mi ochond ia was no
modi ied by aging o he a enolol ea men wi h ei he glu ama e/
mala e o succina e + o enone (Table S6, Suppo ing In o ma ion). The
maximum a e o hea complex I mi ROS p oduc ion, which was
ob ained wi h glu ama e + o enone, was signi ican ly highe in Old
con ols (bu no in Old-AT) han in Young con ols. In he case o
skele al muscle mi ochond ia, no age- ela ed di e ences in he mi ROS
gene a ion o con ol mice we e obse ed wi h any subs a e. The
a enolol ea men signi ican ly dec eased skele al muscle maximum
complex I mi ROS gene a ion (measu ed wi h glu ama e/mala e + o e-
none) o Old-AT compa ed o Old con ols. Skele al muscle mi ROS
E ec o a enolol on mouse longe i y, A. G
omez e al.
552
ª2014 The Au ho s. Aging Cell published by he Ana omical Socie y and John Wiley & Sons L d.
p oduc ion wi h glu ama e/mala e o succina e (which can come om
complex I o III) was signi ican ly lowe in Old-AT han in Young con ols,
whe eas mi ROSp wi h succina e + o enone (coming only om com-
plex III) did no change. All hese esul s, aken oge he , indica e ha
he dec ease in mi ROSp induced by a enolol akes place only a complex I.
Respi a o y chain complexes and apop osis-inducing ac o
The amoun s o he ou espi a o y chain p o ein complexes and AIF a e
shown in Table S7 (Suppo ing In o ma ion). In he hea , no di e ences
be ween Old and Young con ols we e de ec ed o any espi a o y
complex. The a enolol ea men signi ican ly inc eased he amoun o
complex II in hea mi ochond ia. Hea complex I (30kDa subuni )
amoun was signi ican ly highe in Old-AT han in Young con ols, bu
no di e ences we e obse ed be ween Old-AT and Old con ols, and
hea complexes III and IV did no show AT- ela ed di e ences. In skele al
muscle, complexes II and IV dec eased and complex III (FeS p o ein)
inc eased bo h in Old con ols and in Old-AT when compa ed o Young
animals, whe eas a enolol- ela ed di e ences in old animals we e no
obse ed o any complex. The amoun o AIF p o ein did no show
aging- o AT- ela ed a ia ions ei he in hea o in skele al muscle.
Memb ane a y acid unsa u a ion
The changes induced by aging and AT ea men on a y acid composi ion
o hea mi ochond ia a e shown in Table S8 (Suppo ing In o ma ion). As
a esul o hese changes, he global indexes o a y acid unsa u a ion o
hea mi ochond ia we e changed (Fig. 2). Thus, aging inc eased he hea
mi ochond ia double bond index (DBI) and pe oxidizabili y index (PI), and
a enolol dec eased he DBI and PI o Old-AT animals o alues simila o
hose o Young con ols (Fig. 2C). Quan i a i ely, he main a y acids
esponsible o he change in global unsa u a ion we e 18:1n-9 and
especially 22:6n-3. The dec ease in 18:1n-9 in Old con ols was o ally
p e en ed by a enolol, and he inc ease in 22:6n-3 induced by aging was
also o ally p e en ed by he AT ea men (Fig. 2).
The changes induced by aging and AT ea men on a y acid
composi ion o skele al muscle mi ochond ia a e shown in Table S9
(Suppo ing In o ma ion). These changes led o aging- ela ed inc eases
in a y acid unsa u a ion o skele al muscle mi ochond ia (s a is ically
signi ican o PI) and o AT- ela ed dec eases in bo h DBI and PI
(Fig. 2D). In his issue, he AT-induced dec ease was so s ong ha he
DBI and PI dec eased in Old-AT o alues lowe han hose o Young
con ols. Quan i a i ely, he main a y acids esponsible o he change
in global unsa u a ion we e again 18:1n-9 and, especially, 22:6n-3
(Fig. 2). The dec ease in 18:1n-9 in Old con ols was o ally p e en ed by
a enolol (Fig. 2A). The inc ease in 22:6n-3 induced by aging was o ally
abolished in Old-AT animals, which showed le els e en somewha below
hose o Young con ols (Fig. 2B).
Fig. 1 Li elong su i al cu es (Kaplan–Meye su i al plo s) du ing he whole
li espan o con ol and ch onically a enolol- ea ed animals. No signi ican
di e ences in su i al we e obse ed be ween he wo g oups wi h he log- ank
and he Wilcoxon es s, while he highe mo ali y obse ed wi h a enolol be ween
1000 and 1275 days o age was s a is ically signi ican (see Table S3). The longes -
li ing con ol animal eached 3.93 yea s o age (47,1 mon hs). Eigh y-six animals
(43 pe g oup) we e used in he su i al s udy. Raw da a ables o con ol and
a enolol- ea ed animals a e included in he online Suppo ing In o ma ion.
Hea SK. Muscle HEART Sk. Muscle
Mol %
0
5
10
15
20
25
30
35
a***
a*
a***
a***
b***
b***
b** b**
Young Old Old-AT
(A) 18:1n-9 (B) 22:6n-3
DBI PI DBI PI
% o con ol alues
0
20
40
60
80
100
120
140
a*
a**
a*
b*
a**
b***
b*
a**
b***
(C) Hea (D) Skele al muscle
Fig. 2 Global a y acid unsa u a ion indexes (C, D) and he main a y acids
esponsible o hei aging- and a enolol-induced modi ica ion (A, B: oleic,
18:1n-9; and docosahexaenoic, 22:6n-3 acids) in hea and skele al muscle
mi ochond ia om Young con ol, Old con ol, and Old a enolol- ea ed mice.
Values a e means SEM om 5 o 6 (hea ) o 6 (skele al muscle) animals and a e
exp essed as absolu e alues (A, B: mol% o 18:1n-9 and 22:6n-3) o as
pe cen age o hose in he Young con ols [C, D: double bond index (DBI) and
pe oxidizabili y index (PI)]. Con ol alues o unsa u a ion indexes: 200.9 5.0
(DBI, hea ); 213.6 6.7 (PI, hea ); 186.9 5.2 (DBI, skele al muscle);
185.1 6.2 (PI, skele al muscle). Fo he calcula ion o DBI and PI alues, see
he Ma e ials and Me hods sec ion in he online Suppo ing In o ma ion. a*:
signi ican ly di e en om Young con ols; b*: signi ican ly di e en om Old
con ols; *P<0.05; **P<0.01; ***P<0.001.
E ec o a enolol on mouse longe i y, A. G
omez e al. 553
ª2014 The Au ho s. Aging Cell published by he Ana omical Socie y and John Wiley & Sons L d.
Oxida i e damage ma ke s
Oxida i e damage o hea m DNA, measu ed as 8-oxodG (8-oxo-7,8-
dihyd o-2′deoxyguanosine), ended o inc ease wi h aging (nonsigni i-
can di e ence) and was signi ican ly dec eased by he ch onic a enolol
ea men (Table S10, Suppo ing In o ma ion). In he case o skele al
muscle, nonsigni ican ends o highe 8-oxodG alues in aged animals
we e also obse ed, in his case bo h in Old con ol and in Old-AT when
compa ed wi h Young con ols.
The alues o he di e en kinds o p o ein modi ica ion ma ke s
(p o ein oxida ion, glycoxida ion, and lipoxida ion) a e shown in Figs S1
and 3. In hea mi ochond ia, all he i e ma ke s measu ed, glu amic
semialdehydes (GSA), aminoadipic semialdehydes (AASA), ca boxye hyl-
lysine (CEL), ca boxyme hyl-lysine (CML), and malondialdehyde-lysine
(MDAL), we e signi ican ly highe in Old han in Young con ols (Fig. 3).
All hese age- ela ed inc eases we e o ally p e en ed by he a enolol
ea men , as Old-AT animals always showed lowe le els han Old
con ols and did no show signi ican di e ences when compa ed wi h
Young con ols, excep o GSA alues ha we e e en lowe in Old-AT
han in Young animals.
In skele al muscle mi ochond ia, he i e p o ein ma ke s also showed
signi ican ly highe le els in Old han in Young con ol animals (Fig. S1,
Suppo ing In o ma ion). The age- ela ed inc eases in GSA, AASA, CML,
and MDAL we e o ally p e en ed by he a enolol ea men because he
alues in Old-AT we e s a is ically lowe han in Old con ols and simila
o hose o Young animals in all cases. Only in he case o CEL did he
dec ease shown by Old-AT compa ed o Old con ols no each s a is ical
signi icance. Howe e , he CEL alue o Old-AT animals was s a is ically
simila o ha o Young animals.
Ex acellula -signal- egula ed kinase signaling p o eins
The amoun s o ERK p o eins a e shown in Fig. 4. The a enolol ea men
signi ican ly inc eased he amoun o p-ERK/ o al ERK in hea and
skele al muscle o Old-AT mice when compa ed wi h Old and Young
con ols.
Immune unc ion
Lymphop oli e a ion o spleen leukocy es (basal, cpm, and pe cen age o
s imula ion in p oli e a ion in esponse o he mi ogens conca alin A and
LPS) was measu ed in Young and Old con ols and Old-AT g oups, and
s a is ically signi ican di e ences be ween g oups we e no obse ed
( esul s no shown). Mig a ion o spleen leukocy es is ep esen ed in
Fig. 5A as he chemo axis index. Chemo axis signi ican ly dec eased
om Young o Old con ols, and his change was o ally p e en ed in
Old-AT mice, which showed alues simila o hose o Young con ols.
Analogously, na u al kille (NK) ac i i y o spleen leukocy es dec eased
om Young o Old con ols, and his change was o ally p e en ed in
Old-AT, which showed alues s a is ically simila o hose o Young
con ol mice (Fig. 5B).
Beha io
In ela ion o senso imo o abili ies, a signi ican dec ease in equilib ium
was obse ed in Old compa ed o Young con ols, which was o ally
co ec ed in Old-AT animals (Fig. 6A). Mo o coo dina ion and muscula
igo (Fig. 6B,C) we e s ongly dec eased in Old compa ed o Young
con ols. Old-AT mice showed le els o mo o coo dina ion and muscula
igo highe han hose o Old con ols and lowe han hose o Young
con ols. Old-AT mice showed signi ican ly highe pe o mance o
cen al ea ing in he open- ield es and lowe de ec ion and u ina ion
beha io in he open- ield and holeboa d es s han Old con ols, while
no signi ican di e ences we e de ec ed o ho izon al ac i i y, sel -
g ooming beha io , and goal- o non-goal-di ec ed beha io ( esul s no
shown).
Discussion
In his longe i y expe imen , li elong ea men o mice wi h he
b1-blocke a enolol lowe ed one o he wo ai s app op ia ely
co ela ing wi h animal longe i y, he memb ane a y acid unsa u a ion
Fig. 3 P o ein oxida ion, glycoxida ion, and lipoxida ion indica o s in hea
mi ochond ia om Young con ol, Old con ol, and Old a enolol- ea ed mice.
Values a e means SEM om six animals and a e exp essed as pe cen age o
hose in he Young con ols o each p o ein modi ica ion ma ke . Con ol alues:
3172 87 (glu amic semialdehyde, GSA); 295 25 AASA, aminoadipic
semialdehyde, AASA); 455 22 (ca boxye hyl-lysine, CEL); 692 20
(ca boxyme hyl-lysine, CML); 805 60 (malondialdehyde-lysine, MDAL). Uni s:
lmol/mol lysine. a*: signi ican ly di e en om Young con ols; b*: signi ican ly
di e en om Old con ols; **P<0.01; ***P<0.001.
(A) (B)
Fig. 4 Amoun s o ex acellula -signal- egula ed kinase signaling p o eins in hea
(A) and skele al muscle (B) o Young con ol, Old con ol, and Old a enolol- ea ed
mice. Values a e means SEM om 4 animals. Uni s a e a io o p o ein con en /
ubulin. a* :signi ican ly di e en om Young con ol; b*:signi ican ly di e en
om Old con ol; *P<0.05; ** P<0.01.
E ec o a enolol on mouse longe i y, A. G
omez e al.
554
ª2014 The Au ho s. Aging Cell published by he Ana omical Socie y and John Wiley & Sons L d.
deg ee o hea and skele al muscle mi ochond ia, changing hei lipid
unsa u a ion p o ile owa d ha p esen in much longe -li ed mammals.
The a enolol ea men also lowe ed isce al adiposi y, dec eased
maximum mi ROSp om complex I and oxida i e, glycoxida i e, and
lipoxida i e p o ein damage in bo h o gans, and oxida i e damage in
hea m DNA. I also imp o ed chemo axis and na u al kille ac i i ies,
equilib ium, mo o coo dina ion, and muscula igo and o ally o
pa ially p e en ed he aging- ela ed changes obse ed o hese
pa ame e s. Howe e , nei he mean, median, no maximum longe i ies
we e modi ied by he ea men when he whole li espan was
conside ed. A enolol inc eased somewha he mo ali y a e only in e y
old animals, be ween 1000 and 1275 days o age. This is in e es ing
because his is gene ally consis en wi h he esul s o cu en me a-
analyses in hype ensi e human subjec s.
In spi e o he lowe ing o oxida i e s ess and he imp o emen in
immuni y and beha io , he gene al su i al and he median, mean, and
maximum longe i y we e no modi ied by he a enolol ea men . A
eason could be he high longe i y o he con ols o ou s udy, which
eached 3.93 yea s o maximum and 31 and 32 mon hs o mean and
median longe i y, espec i ely; hese alues a e highe han he bes
p e iously desc ibed o he B6D2F1 (C57BL/6xDBA/2N)F
1
s ain:
25.7 mon hs o median longe i y and 3.0 yea s o he maximum
longe i y o he longes -li ed mouse (Yama e e al., 1990). The median
(32 mon hs) and maximum (47.1 mon hs) longe i ies o ou con ol
mice a e simila o highe (maximum longe i y) han hose o he long-
li ed mu an AC5KO (C57BL/6Jx129S J) mice (33 and 37 mon hs,
espec i ely), which a ec he same Ra /MEK/ERK signaling pa hway
ac i a ed by a enolol (Yan e al., 2007); he e o e, hey a e also much
longe -li ed han hei co esponding con ols, 25 and 33 mon hs,
espec i ely, o he con ols o AC5KO. In li elong su i al expe imen s,
inc eases in longe i y a e mo e equen ly ob ained when he longe i y
o he con ols is on he low ange, bu a ely when i is nea e o he
op imum (see Table 1 in Ba ja, 2013). The di icul y in inc easing he
mean li espan o e y long-li ed con ols is logical because i is di icul o
make mo e ‘squa ed’ a su i al cu e ha is al eady e y ec angula ,
and in ou case, he shape o he con ol su i al cu e (Fig. 1)
app oaches ha si ua ion.
Recen ly, i was shown ha knocking down enzymes o he PUFA
biosyn hesis pa hways in C. elegans dec eases he DBI and PI due o he
same kind o a y acid di e ences obse ed in he in e species
compa a i e s udies (Naudi e al., 2011), and in ou long-li e a enolol
s udy, and ha change inc eases wo m longe i y (Shmookle Reis e al.,
2011). This demons a es he exis ence o a causal ela ionship be ween
desa u ase/elongase enzymes, memb ane unsa u a ion, and longe i y.
Ano he likely eason why longe i y was no inc eased in ou AT- ea ed
mice is ha a enolol ac s a ups eam om he inal a y acid changes
in he pa hway ‘b- ecep o -AC-Ra /MEK/ERK signaling o nucleus
changes in PUFA biosyn hesis pa hways –DBI modi ica ion’, compa ed
o he C. elegans RNAi expe imen s ha di ec ly cu ail he exp ession o
hese enzymes. The u he ups eam om he inal a ge s, he mo e
likely is ha hea y b anching and in e connec ion du ing cellula
signaling and hei me abolic e ec s will lead o de imen al side e ec s.
Nega i e side e ec s o a enolol du ing mos o he s udy could ha e
been compensa ed by i s mul iple posi i e e ec s obse ed in ou
in es iga ion, lea ing global su i al inally unchanged. We obse ed
ca dio ascula -linked nega i e e ec s o a enolol only a he end o he
li espan (be ween 1000 and 1275 days o age), while o he possible
nega i e e ec s a younge ages could ha e been unno iced due o he
pa icula selec ion o measu ed pa ame e s. Finally, i is gene ally
ag eed ha many ai s a he han a single one mus simul aneously and
coo dina ely change in he igh di ec ion in o de o (maximum)
longe i y o subs an ially inc ease (Ba ja, 2008). This can also help o
explain he lack o li espan ex ension obse ed in he p esen in es i-
ga ion.
(A) (B)
Fig. 5 Immune unc ion pa ame e s, chemo axis (A), and na u al kille ac i i y (B),
in Young con ol, Old con ol, and Old a enolol- ea ed mice. Values a e
means SEM om 8 animals. a*: signi ican ly di e en om Young con ols; b*:
signi ican ly di e en om Old con ols; **P<0.01; *** P<0.001.
(A)
(B)
(C)
Fig. 6 Beha io al senso imo o abili ies in Young con ol, Old con ol, and Old
a enolol- ea ed mice. Equilib ium (A) and mo o coo dina ion (B) we e assayed
wi h he wood od es , and muscula igo (C) wi h he igh ope es . Values a e
means SEM om 8 animals. a*: signi ican ly di e en om Young con ols; b*:
signi ican ly di e en om Old con ols; *P<0.05; *** P<0.001.
E ec o a enolol on mouse longe i y, A. G
omez e al. 555
ª2014 The Au ho s. Aging Cell published by he Ana omical Socie y and John Wiley & Sons L d.
The lack o changes in longe i y wi h a enolol ea men is in line wi h
almos all p e ious s udies a emp ing o e a d aging using d ugs and
o he small molecules. Thus, nei he aspi in, an idiabe ics including
me o min, es e a ol, and mela onin, no many o he d ugs ha e
consis en ly demons a ed inc eases in oden maximum longe i y
( e iewed in Spindle , 2012). Pe haps, he only excep ion o his could
be apamycin, which led o modes bu consis en 4–14% inc eases in
maximum longe i y (90 h pe cen ile su i al age) a e pa allel eplica-
ion o he longe i y expe imen a h ee ins i u ions (Ha ison e al.,
2009). In con as , many di e en single-gene mu an mice ha e shown
up o 40% inc eases in maximum longe i y. De imen al side e ec s o
he d ugs can be esponsible o such di e ence wi h he ou come o he
gene-modi ying in es iga ions. Pe haps, an analogous app oach o ha
used in C. elegans could inc ease longe i y in mammals by mo e di ec ly
down- egula ing PUFA biosyn hesis enzymes in mice. P e ious s udies
dis up ing he FADS2 gene coding o del a-6 desa u ase ha e esul ed
in / iable bu s e ile mice su e ing a ious pa hologies, including
ulce a i e de ma i is, splenomegaly, and in es inal ulce a ion (S o el
e al., 2008; S oud e al., 2009). Pe haps, al e na i e app oaches
leading o s ong bu no o al deple ion o he enzymes esponsible
o syn hesizing highly unsa u a ed PUFA could help o design use ul
es s o he unsa u a ion-longe i y hypo hesis in mammals (Pamplona
e al., 1996, 2002).
In e es ingly, while w i ing his manusc ip , we we e awa e ha a
e y simila s udy had ecen ly been published (Spindle e al., 2013). In
ha s udy, 36 B6C3F1 male mice ecei ed con inuously un il dea h o he
wo b1-blocke s, me op olol and nebi olol mixed in he die , beginning
he ea men a 12 mon hs o age. The inal e ec on su i al o hese
wo d ugs was app oxima ely 10% and 6.4% inc eases in median
li espan, espec i ely, wi h no inc ease in maximum li espan. Visual
inspec ion o he su i al cu es on ha s udy (Fig. S1a,b) sugges s o us
ha he maximum li espan o he b1-blocke - ea ed animals ended
(no signi ican ly) o be somewha smalle han ha o hei con ols. The
maximum longe i y in ha s udy (a ound 1400 days acco ding o he
igu e su i al plo s) was e y nea o ha ob ained in ou in es iga ion
(1433 days), and median li espan was also e y simila in bo h s udies,
983 days in he Spindle e al. (2013) s udy and 967 days in ou
in es iga ion. While we conside he esul s o bo h s udies e y simila ,
he di e ences conce ning median li espan and su i al could be due o
he a ious di e ences in design: use o di e en mice s ains, use o
di e en b1-blocke s, and di e en doses and ways o adminis a ion. In
addi ion, he d ug ea men s we e s a ed a 12 mon hs o age in he
Spindle e al. (2013) s udy and a 2 mon hs o age in ou case. Mos
in e es ingly, Spindle e al. (2013) ound signi ican ly lowe body weigh
and highe mo ali y wi h me op olol and nebi olol a 1245–1250 days
o age and la e and sugges ed ha his was due o mild d ug oxici y in
e y old mice due o li e aging. This esul is s ikingly simila o ou
de ec ion o a signi ican ly highe mo ali y wi h a enolol be ween 1000
and 1275 days o age, which would also ag ee wi h cu en me a-
analyses om hype ensi e human pa ien s (see below). In ou opinion,
he gene al simila i y in he ou come o bo h s udies inc eases hei
eliabili y, as i means ha hei esul s ha e been gene ally eplica ed
independen ly a wo si es in spi e o some di e ences in design.
I is known ha a enolol inc eases human su i al a e acu e
myoca dial in a c ion and p e en s p ema u e dea h in pa ien s wi h
co ona y hea diseases and sys olic hea ailu e. Howe e , me a-analyses
a e cu en ly aising he ques ion whe he b-blocke s (a enolol is he one
mos equen ly used) a e app op ia e o he ea men o hype ension
(Lindholm e al., 2005; Cockc o & Pede sen, 2012). Compa ed wi h
o he an ihype ensi e d ugs, b-blocke s showed highe all-cause
mo ali y, ca dio ascula mo ali y, myoca dial in a c ion, and hea
ailu e, especially in associa ion wi h a lowe hea a e. Conce ning aged
subjec s, he ea men o hype ension wi h b-blocke s has been
ques ioned in elde ly pa ien s (Lindholm e al., 2005), and he e is li le
in o ma ion on he possible di e en e ec s o a enolol as a unc ion o
age. The nega i e e ec s o a enolol can be due o i s lowe ing hea a e,
ine ec i e lowe ing o e en inc ease in cen al ao ic p essu e, lowe ed
dias olic p essu e, o nega i e me abolic e ec s on glucose and lipid
me abolism (Bangalo e e al., 2008). In pa ien s wi h slowe hea a e,
he e lec ed wa e om he pe iphe y eaches he nex wa e in sys ole
(ins ead o in dias ole) and hence may inc ease cen al ao ic p essu e.
When he su i al s a is ical analysis was es ic ed o he ime
window be ween 1000 and 1275 days o age, a enolol showed a
signi ican inc ease in mo ali y a e. This shows ha a enolol can
inc ease mo ali y o a mammal in e y old bu no in young o middle-
aged indi iduals, which would be in line wi h he me a-analyses in
human pa ien s, especially because hype ension is common in old
indi iduals. This is especially in e es ing because human hype ensi e
pa ien s a e usually no medica ed un il midli e diagnosis bu s ill seem o
show an inc eased a enolol-induced mo ali y. We ound ha 8–13%
dec eases in a e ial p essu es (mean, sys olic, and dias olic) and
especially in hea a e (by 20%) occu a he same ime window a
which mo ali y inc eases in he a enolol g oup, in e y old (35 mon hs
old) animals, bu no in young o mode a ely (18 mon hs) old mice in
which su i al is indis inguishable om ha o con ols. This ag ees wi h
he main change associa ed wi h he nega i e e ec s o a enolol on
human hype ensi e pa ien s, a dec ease in hea a e (Bangalo e e al.,
2008). A enolol-induced dec eases in hea a e and ca diac ou pu and
ansi o y peaks o a ia ion o a e ial p essu e due o poo e p essu e
egula ion in e y old animals, which ha e a myoca dium hea ily
degene a ed by aging- ela ed damage and he main sys emic a e ies
s i ened by a he oscle osis, can lead o hea ailu e and be a al.
Dec eased a e ial elas ici y would ampli y d ops in blood low o c i ical
main o gans including he hea and b ain when a e ial p essu e
a ia ions occu in he downwa d di ec ion. Such p ocesses a e absen
(hea a e and a e ial p essu es a e no mal a 18 mon hs o age) o a e
be e ole a ed in younge animals. They could be among he side
e ec s o he d ug coun e ac ing he bene icial e ec s on oxida i e
s ess, immune unc ion, and beha io ound in his long-li e s udy and
help o explain why longe i y was no changed in spi e o hose bene i s.
The ch onic a enolol ea men inc eased he amoun o p-ERK,
co obo a ing ac i a ion o he Ra /MEK/ERK signaling pa hway, as in
long-li ed mu an AC5KO mice (Yan e al., 2007). Nei he body weigh ,
ou ine me abolic a e, ec al empe a u e, ca dio ascula pa ame e s,
no mi ochond ial oxygen consump ion was changed by a enolol in 18-
mon h-old animals, excep o an inc ease in hea mi ochond ial oxygen
consump ion wi h glu ama e/mala e in s a e 3 (phospho yla ing) in he
Old con ol g oup, which was p e en ed by a enolol. This sugges s ha ,
a leas un il 18 mon hs o age, he ea men did no p omo e o e
nega i e side e ec s. The lack o modi ica ion o o al body and o gan
weigh s (excep o a dec ease in kidney weigh ) and he absence o
de ec ion o a ia ions in ood in ake indica e ha he many obse ed
bene icial e ec s o a enolol a e no due o calo ic es ic ion.
Inc eases in isce al a in old humans a e associa ed wi h degene -
a i e illnesses, including ype 2 diabe es, me abolic synd ome, and
a he oscle osis. A oiding such inc eases is conside ed heal hy. In ou
s udy, a enolol p o ided pa ial bu subs an ial p o ec ion agains
inc eases in isce al a . The s ong age- ela ed inc ease in isce al a
om Young o Old con ols (424% inc ease) was 29.5% smalle om
Young con ols o Old-AT (299% inc ease) and 32% smalle when
E ec o a enolol on mouse longe i y, A. G
omez e al.
556
ª2014 The Au ho s. Aging Cell published by he Ana omical Socie y and John Wiley & Sons L d.
exp essed as g ams o a pe uni body mass. The s ong dec ease in
isce al adiposi y induced by a enolol in Old animals (23.8% dec ease) is
also s iking because long- e m nu i ional in e en ions ex ending
li espan in oden s, such as 40% die a y es ic ion and 80% me hionine
es ic ion (Pe one e al., 2013), also s ongly dec ease isce al adiposi y
pe g am o body weigh (Malloy e al., 2006) while hey induce s ong
dec eases in g ow h and inal body size. In ou case, howe e , he
lowe ed isce al adiposi y was no accompanied by any de imen al
dec eases in body size, simila o wha happens in 40% me hionine
es ic ion (Sanchez-Roman & Ba ja, 2013), al hough he e ec o his
milde in e en ion on longe i y has no been in es iga ed.
The AT ea men did no modi y he a e o mi ROSp o hea
mi ochond ia, excep o a oidance o an age- ela ed inc ease in
maximum complex I ROS gene a ion. In he case o skele al muscle, no
age- ela ed di e ences in mi ROS gene a ion we e obse ed, and a enolol
ea men dec eased complex I mi ROSp in Old mice. This is he espi a o y
complex ha shows low o dec eased mi ROS gene a ion in long-li ed
species o in calo ic- es ic ed li e-ex ended animals (Ba ja, 2013). How-
e e , none o he a ia ions in mi ROSp obse ed in ei he o gan a e due o
changes in he amoun o complexI as e ealedby he Wes e n blo so he
espi a o y complexes. The e o e, hey mus be due o quali a i e complex
I modi ica ions such as hose leading o a dec ease in he educ ion s a e
and/o he educing midpoin po en ial o he complex I ROS gene a o /s.
In con as , m ROSp lowe ing h ough dec eases in complex I con en has
been desc ibed in die a y o me hionine- es ic ed animals (Sanchez-
Roman & Ba ja, 2013), al hough quali a i e complex I changes also seem
o be in ol ed in hese cases.
The e is a nega i e ela ionship be ween he issue and mi ochond ial
deg ee o memb ane a y acid unsa u a ion and longe i y in all he animal
species s udied o da e ( e iewed in Pamplona e al., 2002; Hulbe e al.,
2007; Naudi e al., 2011). This makes he memb anes o long-li ed
animals highly esis an o lipid pe oxida ion, because he sensi i i y o
a y acids o lipid pe oxida ion inc eases exponen ially as a unc ion o he
numbe o double bonds pe molecule. In ou s udy, he a enolol ea men
success ully and signi ican ly dec eased he global a y acid mi ochond ial
memb ane unsa u a ion measu ed as he DBI in hea and skele al muscle
(11% and 22.4% dec eases, espec i ely) and as he PI also in bo h issues
(16.8% and 30.8% dec eases). These esul s ag ee wi h ou sho - e m
s udy in o al hea issue o C57BL/6 mice (Sanchez-Roman e al., 2010).
Conce ning he in ensi y o he ob ained e ec , o example, in skele al
muscle, a enolol dec eased he PI o Old-AT mice o a alue simila o ha
o mammals wi h a i e old highe longe i y (14.7 yea s) acco ding o da a
in Fig. 7A om Hulbe e al. (2007). In e es ingly, he DBI and PI
signi ican ly inc eased wi h aging om Young o Old con ols (nonsigni -
ican end o hea DBI), whe eas he dec ease induced by a enolol
b ough back he DBI and PI o alues simila o (hea ) o e en lowe
(skele al muscle) han hose o Young mice. Thus, he a enolol ea men
was able o ully p e en all he age- ela ed changes in mi ochond ial
memb ane unsa u a ion. The dec eases in DBI and PI induced by a enolol
we e mainly due o dec eases in he mos highly unsa u a ed a y acid, he
PUFA docosahexaenoic acid (22:6n-3), aided by inc eases in he MUFA
oleic acid (18:1n-9) in bo h issues. Docosahexaenoic acid (22:6n-3) has six
double bonds and consequen ly has i e bis-allylic hyd ogens pe chain. I is
320 imes mo e suscep ible o ROS a ack han oleic acid (18:1n-9;
Holman, 1954; Hulbe e al., 2007).
I is expec ed ha changes in memb ane unsa u a ion would a ec
no only he memb ane lipids and p o eins bu also he le els o modi ied
p o eins in cells in gene al and he m DNA, which is si ua ed nea o
pe haps e en in con ac wi h he e y abundan inne mi ochond ial
memb anes. In ag eemen wi h ha , he i e ma ke s o p o ein
oxida ion, glycoxida ion, and lipoxida ion measu ed inc eased du ing
aging, and all hese changes (excep o CEL in skele al muscle) we e
o ally p e en ed in Old-AT mice back o he le els shown by Young
animals. I is gene ally accep ed ha aging inc eases p o ein oxida i e
modi ica ion, whe eas expe imen al p ocedu es ha ex end longe i y,
such as die a y (Ba ja, 2013) and me hionine es ic ion (Sanchez-Roman
& Ba ja, 2013), dec ease i . No ably, he s onges age- ela ed changes
we e obse ed o he lipoxida ion ma ke MDAL, which is in ag eemen
wi h ou p e ious s udies using o he longe i y- ela ed models.
Conce ning m DNA, in p e ious sho - e m s udies we ha e shown
ha e ec i ely inc easing issue a y acid unsa u a ion by eeding he
animals wi h he highly unsa u a ed (n-3) menhaden oil, compa ed o
die s p epa ed wi h sa u a ed a s, inc eased CML and MDAL in p o eins
and no ably he 8-oxodG le el in m DNA (and no in he nuclea DNA) o
a kidney and b ain (Pamplona e al., 2004). Nonsigni ican ends o 8-
oxodG inc eases in m DNA wi h aging (especially s ong in skele al
muscle) and a signi ican dec ease in 8-oxodG in he hea o Old-AT
mice o alues simila o hose o Young mice we e obse ed. This ag ees
wi h cu en models indica ing ha no only mi ROSp, bu he
memb ane unsa u a ion deg ee, can con ibu e o m DNA oxida i e
damage du ing aging, likely due o seconda y ee adicals eleased by
lipid pe oxida ion o he e y abundan mi ochond ial memb anes (Ba ja,
2013). These a e si ua ed e y nea o he si es o mi ROSp and o
m DNA i sel , while lipids a e sca ce in he nucleus, a oiding he
gene a ion o lipid pe oxida ion p oduc s in he icini y o nuclea DNA.
Age and a enolol ea men also a ec ed impo an physiological
unc ions, such as immuni y and beha io , simila ly o he changes
obse ed o memb ane a y acid unsa u a ion and oxida i e damage
ma ke s. The capaci y o chemo axis, which allows he mig a ion o
leukocy es owa d he in lamma o y ocus, cons i u es one o he i s
s eps o he immune esponse, and NK ac i i y is he i s line o immune
de ense agains i al in ec ions and neoplasia. P e ious s udies in CD1
and BALB/c mice showed ha he chemo ac ic esponse, he lympho-
p oli e a i e esponse, and he NK ac i i y we e p o oundly and
nega i ely a ec ed by aging in leukocy es om di e en loca ions, such
as he pe i oneum, hymus, lympha ic nodes, and spleen (De la Fuen e
e al., 2004; De la Fuen e, 2008). In ag eemen wi h hose s udies, in he
p esen in es iga ion chemo axis and NK ac i i y we e lowe in old
animals han in young ones. These age- ela ed dec eases we e
p e en ed by a enolol in Old-AT mice. A enolol inc eases a ious
ac i i ies o immune cells in i o (Guo e al., 2011), bu his had no
been desc ibed p e iously in i o.
The e ec s o a enolol on he beha io al capaci ies o mice we e
assessed h ough a wide ba e y o beha io al es s. P e ious s udies
ha e documen ed age- ela ed impai men s in senso imo o abili ies,
mo o coo dina ion, and muscula igo in ICR-CD1 mice (Baeza e al.,
2010). In ou li elong s udy, aging dec eased equilib ium, mo o
coo dina ion, and muscula igo in Old compa ed o Young con ols,
and all hese de imen al changes we e o ally (equilib ium) o pa ially
p e en ed by a enolol. In addi ion, a enolol inc eased he pe cen age o
Old mice ha pe o med cen al ea ing in he open- ield es . Rea ing is
conside ed he bes pa ame e o es ima e non-goal-di ec ed e ical
explo a o y ac i i y (Esco ihuela e al., 1999) and indica es he in e es o
abili y o he animal o in e ac wi h he en i onmen . Inc eases in
de eca ion and u ine incon inence also occu in old oden s. The
obse ed dec ease in de eca ion and u ina ion in Old-AT mice compa ed
o Old con ols indica es a lowe anxie y s a us in hese animals. These
esul s ag ee wi h he dec eases in anxie y (Dea y e al., 1991) and
dep ession (Al-Tubuly e al., 2008) epo ed a e ea men wi h
a enolol in human pa ien s.
E ec o a enolol on mouse longe i y, A. G
omez e al. 557
ª2014 The Au ho s. Aging Cell published by he Ana omical Socie y and John Wiley & Sons L d.
In summa y, in his s udy long- e m ea men wi h a enolol
e ec i ely dec eased memb ane a y acid unsa u a ion in mice owa d
alues ypical o much longe -li ed animals, lowe ing isce al adiposi y,
dec easing many ma ke s o oxida i e s ess, and imp o ing immune and
beha io al unc ions du ing aging wi hou changing longe i y. We
conclude ha i is a enolol ha ailed o inc ease longe i y, and likely no
he dec ease in memb ane unsa u a ion.
Expe imen al p ocedu es
Animals and s udy design
A o al o 134 B6D2F1 (C57BL/6 emale 9DBA/2 male)male mice we e
main ained unde SPF condi ions ecei ing ad libi um a s anda d oden
chow (Panlab, Spain) in indi idual mouse cages du ing hei whole
li espan. Eigh y-six o hese animals (43 Old con ol and 43 Old-AT-
ea ed) we e used only o ob ain he su i al cu es. The a enolol
ea men s a ed a 2 mon hs o age. Fo y-eigh sepa a ed ‘pilo ’
animals we e used o measu e he di e en physiological and biochem-
ical pa ame e s a 16 mon hs o age a e sac i ice by ce ical disloca-
ion. A young con ol g oup (6 mon hs o age) was also included.
A enolol (Sigma, A7655) was gi en in d inking wa e a 0.1 g L
1
, which
esul ed in a mean a enolol in ake o 0.559 mg pe mouse pe day. Jus
a e ce ical disloca ion, issues we e immedia ely p ocessed o isola e
unc ional mi ochond ia o espi a ion and mi ROS gene a ion mea-
su emen s. Tissue and excess mi ochond ial samples we e s o ed a 80
°C o he pos e io analyses. De ailed me hods o he physiological
pa ame e s and he measu emen s in unc ional mi ochond ia e e ed
o below a e desc ibed in he online Suppo ing In o ma ion.
Physiological pa ame e s
Rec al empe a u e was measu ed using a ec al p obe, he ou ine
me abolic a e was measu ed by closed sys em espi ome y, and hea
a e and blood p essu es we e measu ed wi h a ail-cu manome e .
Measu emen s in unc ional mi ochond ia
Hea and skele al muscle mi ochond ia we e ob ained a 4 °C om
esh issue by di e en ial cen i uga ion essen ially as desc ibed in Sanz
e al. (2006). The a e o mi ochond ial oxygen consump ion was
measu ed a 37 °C wi h a Cla k- ype O
2
elec ode in he absence (s a e 4
es ing) and in he p esence (s a e 3 phospho yla ing) o 500 lM ADP.
The a e o mi ochond ial ROS p oduc ion (O
2
+H
2
O
2
, as excess SOD
was added) was assayed by measu ing he inc ease in luo escence due
o oxida ion o homo anillic acid by H
2
O
2
in he p esence o ho se adish
pe oxidase (Sanz e al., 2006).
Measu emen o mi ochond ial complexes I o IV, apop osis-
inducing ac o , and ex acellula -signal- egula ed kinase
The amoun s o he mi ochond ial espi a o y chain complexes (I–IV), he
complex I egula o y ac o AIF, and ERK1/2 and phospho-ERK1/2 we e
es ima ed using Wes e n blo analyses. De ailed me hods a e desc ibed
in he online Suppo ing In o ma ion.
Fa y acid analyses, hei biosyn he ic enzymes, and global
a y acid unsa u a ion indexes
Fa y acids om mi ochond ial lipids we e analyzed as me hyl es e
de i a i es by gas ch oma og aphy (GC) as p e iously desc ibed
(Sanchez-Roman e al., 2010). Resul s a e exp essed as mol%.
Calcula ion o he densi y o double bonds in he mi ochond ial
memb anes (double bond index, DBI; and pe oxidizabili y index, PI) is
desc ibed in he online Suppo ing In o ma ion.
Oxida i e damage o m DNA (8-oxodG)
A e isola ion, m DNA was diges ed o deoxynucleoside le el. S eady-
s a e oxida i e damage o m DNA was es ima ed by measu ing he le el
o 8-oxodG e e ed o ha o he nonoxidized base (deoxyguanosine,
dG). 8-OxodG and dG we e analyzed by HPLC wi h online elec ochem-
ical and ul a iole de ec ion.
Oxida ion-de i ed p o ein damage ma ke s
Glu amic semialdehydes, AASA, CML, CEL, and MDAL in hea and
skele al muscle mi ochond ial p o eins we e de e mined by GC/MS wi h
selec ed ion moni o ing. De ailed me hods a e desc ibed in he online
Suppo ing In o ma ion.
Immune unc ion pa ame e s
The numbe o leukocy es was de e mined in cell suspensions ob ained
om he spleen. Cellula iabili y was highe han 95% in all cases.
Lymphop oli e a ion, chemo axis (di ec ed mig a ion), and na u al kille
(NK) ac i i y assays we e pe o med as e e ed in he online Suppo ing
In o ma ion.
Beha io al es s
The open- ield and holeboa d es s we e ca ied ou o analyze
explo a o y and anxie y-like beha io s. Beha io al es s we e pe o med
as e e ed in he online Suppo ing In o ma ion.
S a is ical me hods
Compa isons be ween he h ee g oups o animals we e pe o med by
one-way ANOVA. Compa ison o long-li e su i al o Old con ol and
Old-AT- ea ed animals was analyzed wi h he log- ank and Wilcoxon
es s. P<0.05 was selec ed as he minimum le el o s a is ical
signi icance.
Funding in o ma ion
This in es iga ion was suppo ed by I +D g an s om he Spanish
Minis y o Science and Inno a ion (BFU2011-23888 o G.Ba ja); UCM
G oups o Resea ch (910379ENEROINN), BFU2011-30336, and
RETICEF (RD 12/0043/0018) om ISCIII-FEDER o he Eu opean Union
o M. De la Fuen e; Spanish Minis y o Heal h (PI11/01532) o
M.Po e o O in; and Spanish Minis y o Science and Inno a ion
(BFU2009-11879) and he Gene ali a o Ca alonia (2009SGR735) o
R. Pamplona.
Au ho con ibu ions
AG, IS-R, JG, JC, and IM con ibu ed o gene a ing he s udy design,
sampling, pe o ming all he biochemical and physiological assays,
pe o ming da a analysis, in e p e ing da a, and d a ing he manusc ip .
MLT, AN, and MPO con ibu ed by helping wi h acquisi ion and
in e p e a ion o da a and c i ical eading o he manusc ip . RP, MDeF,
E ec o a enolol on mouse longe i y, A. G
omez e al.
558
ª2014 The Au ho s. Aging Cell published by he Ana omical Socie y and John Wiley & Sons L d.
and GB con ibu ed o gene a ing he s udy concep and design,
supe ising he esea ch, and w i ing he manusc ip .
Con lic o in e es
The au ho s ha e decla ed ha no con lic o in e es exis s.
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Suppo ing In o ma ion
Addi ional Suppo ing In o ma ion may be ound in he online e sion o his
a icle a he publishe ’s web-si e.
Fig. S1 P o ein oxida ion, glycoxida ion and lipoxida ion indica o s in skele al
muscle mi ochond ia om Young con ol, Old con ol and Old a enolol
ea ed mice.
Table S1 Basic physiological pa ame e s in Young con ol, Old*con ol, and
Old*a enolol ea ed mice a 16-18 mon hs o age.
E ec o a enolol on mouse longe i y, A. G
omez e al. 559
ª2014 The Au ho s. Aging Cell published by he Ana omical Socie y and John Wiley & Sons L d.
