phys-09-01247 Sep embe 4, 2018 Time: 9:42 # 1
BRIEF RESEARCH REPORT
published: 05 Sep embe 2018
doi: 10.3389/ phys.2018.01247
Edi ed by:
Ma ia Te esa C uz,
Uni e si y o Coimb a, Po ugal
Re iewed by:
Vicen e Lahe a,
Complu ense Uni e si y o Mad id,
Spain
Diana Ju ado Se a,
Uni e si y o Coimb a, Po ugal
*Co espondence:
Rica do Sil es e
[email p o ec ed]
Special y sec ion:
This a icle was submi ed o
In eg a i e Physiology,
a sec ion o he jou nal
F on ie s in Physiology
Recei ed: 09 Ma ch 2018
Accep ed: 17 Augus 2018
Published: 05 Sep embe 2018
Ci a ion:
Gai em J, Gonçal es LG,
Dinis-Oli ei a RJ, Cunha C,
Ca alho A, To ado E, Rod igues F,
Sa ai a M, Cas o AG and Sil es e R
(2018) L-Th eonine Supplemen a ion
Du ing Coli is Onse Delays Disease
Reco e y. F on . Physiol. 9:1247.
doi: 10.3389/ phys.2018.01247
L-Th eonine Supplemen a ion Du ing
Coli is Onse Delays Disease
Reco e y
Joana Gai em1,2, Luís G. Gonçal es3, Rica do J. Dinis-Oli ei a4,5,6, C is ina Cunha1,2,
Agos inho Ca alho1,2, Egídio To ado1,2, Fe nando Rod igues1,2, Ma ga ida Sa ai a7,8,
An ónio G. Cas o1,2 and Rica do Sil es e1,2*
1Li e and Heal h Sciences Resea ch Ins i u e, School o Heal h Sciences, Uni e si y o Minho, B aga, Po ugal,
2ICVS/3B’s – PT Go e nmen Associa e Labo a o y, Guima ães, Po ugal, 3Ins i u o de Tecnologia Química e Biológica
An ónio Xa ie , Uni e sidade NOVA de Lisboa, Oei as, Po ugal, 4IINFACTS – Ins i u e o Resea ch and Ad anced T aining
in Heal h Sciences and Technologies, Depa men o Sciences, CESPU, CRL, Uni e si y Ins i u e o Heal h Sciences,
Gand a, Po ugal, 5UCIBIO, REQUIMTE, Labo a o y o Toxicology, Depa men o Biological Sciences, Facul y o Pha macy,
Uni e si y o Po o, Po o, Po ugal, 6Depa men o Public Heal h and Fo ensic Sciences, and Medical Educa ion, Facul y
o Medicine, Uni e si y o Po o, Po o, Po ugal, 7Ins i u o de In es igação e Ino ação em Saúde, Uni e sidade do Po o,
Po o, Po ugal, 8Ins i u o de Biologia Molecula e Celula , Uni e sidade do Po o, Po o, Po ugal
Die a y nu ien s ha e eme ged as po en ial he apeu ic adjunc s o in lamma o y bowel
disease (IBD) gi en hei impac on in es inal homeos asis h ough he modula ion o
immune esponse, gu mic obio a composi ion and epi helial ba ie s abili y. Se e al
nu ien s ha e al eady been associa ed wi h a p o ec i e pheno ype. Ye , he e is a
lack o knowledge owa d he mos p omising ones as well as he mos adequa e
phase o ac ion. To un eil he mos p ominen he apy candida es we cha ac e ized
he colon me abolic p o ile du ing coli is de elopmen . We ha e obse ed a wo old
dec ease in h eonine le els in mice subjec ed o DSS-induced coli is. We hen assessed
he e ec o h eonine supplemen a ion in he beginning o he in lamma o y p ocess
(DSS +Th ) o when in lamma ion is al eady es ablished (DSS +Th D8). Coli is
p og ession was simila be ween he ea ed g oups and con ol coli ic mice, ye
h eonine had a su p isingly de imen al e ec when adminis e ed in he beginning
o he disease, wi h mice displaying a delayed eco e y when compa ed o con ol
mice and mice supplemen ed wi h h eonine a e day 8. Al hough no majo changes
we e ound in hei me abolic p o ile, DSS +Th mice displayed al e ed exp ession in
mucin-encoding genes, as well as in goble cell coun s, un eiling an impai ed abili y o
p oduce mucus. Mo eo e , IL-22 sec e ion was dec eased in DSS +Th mice when
compa ed o DSS +Th D8 mice. O e all, hese esul s sugges ha supplemen a ion
wi h h eonine du ing coli is induc ion impac goble cell numbe and delays he eco e y
pe iod. This ein o ces he impo ance o a deepe unde s anding ega ding h eonine
supplemen a ion in IBD.
Keywo ds: IBD, h eonine, DSS-induced coli is, goble cells, me abolomics, IL-22, mucin
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Gai em e al. Th eonine Supplemen a ion E ec in Coli is
INTRODUCTION
In lamma o y bowel disease (IBD) is a complex debili a ing
diso de o he gas oin es inal ac which comp ises bo h
C ohn’s disease and ulce a i e coli is. Despi e he unclea e iology
o IBD, se e al ac o s ha e been accoun ed as key o he
de elopmen o he disease, such as gene ics, immune sys em
and en i onmen al ac o s, namely die and gu mic obio a
composi ion (Kho e al., 2011).
Die a y supplemen a ion has eme ged as a p omising
he apeu ic p ac ice in he p e en ion and ea men o IBD
(Du chschein e al., 2016). Recen e idence has e ealed
ha ibe -en iched die s p omo e p o ec ion agains IBD
de elopmen , since die a y ibe is mainly e men ed by in es inal
mic obio a in o sho -chain a y acids (SCFAs), such as bu y a e,
ace a e and p opiona e (den Bes en e al., 2013). The p o ec i e
p ope ies o hese me aboli es a e widely desc ibed by hei
impac on immune cell ac i a ion and epi helial ba ie s abili y
(Kelly e al., 2015;Macia e al., 2015), wi h dec eased le els o
SCFAs being ound in colon samples om IBD pa ien s (Huda-
Faujan e al., 2010). O he s udies ha e also poin ed ou se e al
speci ic amino acids ha can imp o e in es inal homeos asis,
mainly by boos ing mucosal healing and egene a ion. Fo
ins ance, glu amine is known o p omo e p o ec ion in dex an
sul a e sodium (DSS)- and 2,4,6- ini obenzenesul onic acid
(TBNS)-induced in es inal in lamma ion, ac ing ia NF-κB
down egula ion (K e zmann e al., 2008). Coli ic mice o ally
adminis e ed wi h glu amine displayed supp essi e Th1/Th17
immune esponses and subsequen ly dec eased in lamma ion
when compa ed o mice ed wi h egula die (Hsiung e al., 2014).
O he amino acids ha e been also associa ed wi h a p o ec i e
pheno ype agains coli is. Using dis inc animal models o coli is,
die s en iched in h eonine, se ine, p oline and cys eine, gi en
be o e and h oughou disease de elopmen , ha e been shown
o es o e mucin syn hesis and s abiliza ion o gu mic obio a
(Fau e e al., 2006). Simila indings we e obse ed wi h he
adminis a ion o a mix u e o h eonine, me hionine and
monosodium glu ama e a e coli is induc ion (Liu e al., 2013).
Se e al s udies ha e so a add essed whe he adminis a ion
o speci ic nu ien s may a ise as a p ophylac ic and/o
he apeu ic app oach. Howe e , he e is a lack o knowledge
owa d he mos p omising and adequa e phase o ac ion. Thus,
we in es iga ed he me abolic p o ile o mice de eloping coli is,
aiming o iden i y a ia ion o me aboli es du ing in lamma ion.
The iden i ica ion o he mos a ac i e po en ial a ge s o
he apy and he de ini ion o a ime ange mo e p one o
po en ia e he e ec s o hei supplemen a ion may be ele an
o u u e applica ions in IBD p ophylaxis and he apy.
MATERIALS AND METHODS
Animals
Se en o nine-week old C57BL/6J male mice we e pu chased
om Cha les Ri e Labo a o ies and housed in i3S animal
acili ies, unde pa hogen ee condi ions, wi h ood and wa e
ad libi um. All expe imen al p ocedu es we e app o ed by he
i3S Animal E hics Commi ee and licensed by he Po uguese
Na ional Au ho i y o Animal Heal h (DGAV) wi h e e ence
014811/2016-07-13.
Coli is Induc ion
Dex an sul a e sodium (DSS; TdB Consul ancy; 2% (w/ ),
molecula weigh app oxima ely 40000 Da) was adminis e ed in
d inking wa e ad libi um o 5 days. Clinical signs o coli is
we e moni o ed daily and sco ed as a disease ac i i y index (DAI;
Supplemen a y Table S1).
L-Th eonine Adminis a ion
Mice we e di ided in o DSS (con ol), DSS wi h L-Th eonine
(DSS +Th ) and DSS ollowed by L-Th eonine adminis a ion a
day 8 (DSS +Th D8), as shown in Figu e 2A.L-Th eonine [Th ;
Sigma–Ald ich; 0.166% (w/ ) co esponding o 250 mg/Kg/day]
was gi en in he d inking wa e ad libi um. The dose was chosen
acco ding o he daily in ake in p e ious s udies wi h oden s
(Fau e e al., 2006;Liu e al., 2013) and o a high- h eonine
human supplemen a ion s udy (Pencha z e al., 2008). The sa e y
o ou p o ocol measu ing bioma ke s o enal and li e damage
was e alua ed o con i m he absence o oxici y (Supplemen a y
Figu e S1). Simila luid in ake was ound among all g oups.
Me abolomic Analysis by Nuclea
Magne ic Resonance (NMR)
Me hanol/wa e ex ac s o colon we e analyzed a an
Ul ashiedTM 800 Plus (B uke ) spec ome e as desc ibed
in G aça e al. (2017). Me aboli e concen a ions we e pe o med
by in eg a ion o 1H-NMR esonances using TSP as e e ence.
Quan i a i e Real-Time PCR (qPCR)
To al RNA was isola ed om colonic samples (T ipleX ac o ,
G isp). As DSS inhibi s bo h polyme ase and e e se ansc ip ase
ac i i ies, RNA was pu i ied wi h li hium chlo ide, as in Viennois
e al. (2013). qPCR was pe o med as desc ibed in Co eia e al.
(2017). The lis o p ime s used is in Supplemen a y Table S2.
His ology and Goble Cell Coun
Colons we e ixed in 10% bu e ed o malin (Sigma–Ald ich)
and embedded in pa a in. Sec ions o 5 µm we e s ained wi h
hema oxylin/eosin and Alcian Blue/Pe iodic acid-Schi . Goble
cell numbe was assessed o each expe imen al condi ion in
a blinded ashion. Only c yp s cu longi udinally om c yp
opening o bo om we e analyzed.
Cy okine Quan i ica ion
Colonic explan cul u es we e pe o med as p e iously desc ibed
(McNamee e al., 2011). Cy okine quan i ica ion was pe o med
in supe na an s by ELISA (Biolegend). Tissue explan s we e
homogenized and o al p o ein was measu ed using B ad o d
assay. The concen a ion o sec e ed cy okines in he supe na an
was no malized o o al issue p o ein and exp essed as picog am
o cy okine pe µg o o al issue p o ein.
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Gai em e al. Th eonine Supplemen a ion E ec in Coli is
FIGURE 1 | Th eonine le els in he colon dec ease wi h in es inal in lamma ion. (A) Mice we e ea ed wi h DSS o induce coli is and colon me aboli es we e
measu ed a day 0 and day 5 by NMR. (B) Pa ial leas squa es – disc iminan analysis (PLS-DA) was ob ained o samples o bo h days. (C) Top- i e ea u es
iden i ied by he combina ion o he p- alue om - es analysis and magni ude o he change ( old change om day 5 e sus day 0). (D) Impo an me aboli es
selec ed by olcano plo wi h old change h eshold (x) 1.8 and - es h eshold (y) o 0.05. The pu ple ci cles ep esen ea u es abo e he h eshold. Bo h old
changes and p- alues a e log ans o med. (E) Th eonine le els in he colon a day 0 and day 5. Da a is shown as mean ±SD; n= 5 mice/g oup. One ep esen a i e
expe imen is shown ou o wo. ∗p<0.05.
S a is ical Analysis
Me aboli e di e ences we e e alua ed by ANOVA in R s a is ical
so wa e. Pa ial leas squa es – disc iminan analysis (PLS-DA)
models we e pe o med in SIMCA so wa e. O he s a is ical
analyses we e ca ied ou wi h G aphPad P ism ( e sion 6.01).
Fo mul iple g oup compa isons one-way ANOVA es wi h
a Tukey mul iple-compa ison pos es was pe o med, while
o mul iple g oup compa isons wi h epea ed measu es wo-
way ANOVA es wi h a Tukey mul iple-compa ison pos es
was applied. Da a a e p esen ed as mean ±s anda d de ia ion
(SD). S a is ically signi ican alues a e: ∗p<0.05; ∗∗p<0.01;
∗∗∗p<0.001.
RESULTS AND DISCUSSION
To cha ac e ize he colon me abolic p o ile du ing coli is
de elopmen , me aboli es p esen on colonic ex ac s o mice
p io (day 0) and a e 5 days o DSS-induced coli is we e
analyzed by NMR (Figu e 1A). These ime poin s we e selec ed
since i allows he compa ison o a homeos a ic p o ile (day
0) agains a pe iod wi h es ablished in lamma ion and lesion,
ye e e sible and ea able (day 5). Mul i a ia e analyses o
he me abolomic da a only demons a ed mino al e a ions
be ween p o iles o day 0 and day 5 o coli is de elopmen
(Figu e 1B). Howe e , uni a ia e analyses pe o med on he
me aboli es allow o disc imina e ADP and pa icula ly h eonine
as signi ican ly al e ed om day 0 o day 5 (Figu es 1C,D).
Among essen ial amino acids, h eonine has a p ominen ole
in main aining a heal hy gu . Th eonine is able o gene a e
he main h ee SCFAs, namely ace a e, bu y a e and p opiona e
(Neis e al., 2015). In ac , i has been p e iously iden i ied
se e al biosyn he ic genes o h eonine me abolism in he
human gu mic obio a, sugges ing he ele ance o his amino
acid o mic obio a biology (Abubucke e al., 2012). SCFAs a e
desc ibed as impo an modula o s o immune esponse, since
hey a e ligands o G-p o ein-coupled ecep o 43 (GPR43)
ha is exp essed by immune cells on he lamina p op ia,
such as egula o y T cells, egula ing he p oin lamma o y
esponses in he in es ine (Boll a h and Pow ie, 2013). Mo eo e ,
h eonine is as ly me abolized in he in es ine o mucin
syn hesis (Fau e e al., 2005). These p o eins a e pa amoun in
in es inal s abili y, since he mucus laye in he colonic ou e
laye p e en s he di ec con ac o luminal mic oo ganisms
wi h he epi helium (Johansson e al., 2014). I has also been
sugges ed ha h eonine equi emen s a e inc eased unde
pa hological se ings o main ain p ope in es inal unc ion,
such as p oduc ion and o ma ion o he mucus laye (Remond
e al., 2009). The e o e, by pa icipa ing in he mucus laye
syn hesis and p oduc ion o an i-in lamma o y SCFAs, h eonine
me abolism by gu mic obio a p o es o be essen ial o gu
ba ie in eg i y and unc ion. Ou da a show ha h eonine
le els d op 2- old du ing coli is de elopmen un il day 5
(Figu es 1C–E). The e o e, we hypo hesized ha h eonine
supplemen a ion du ing ac i e in lamma o y disease could help
o es o e he in es inal homeos asis and hus p esen some
he apeu ic po en ial.
P e ious s udies ha e in es iga ed h eonine in combina ion
wi h o he amino acids as a po en ial candida e o he apy
agains coli is (Fau e e al., 2006;Liu e al., 2013). Ne e heless,
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Gai em e al. Th eonine Supplemen a ion E ec in Coli is
FIGURE 2 | Th eonine supplemen a ion on he onse o coli is de elopmen delays eco e y and dec eases goble cell numbe . (A) Mice we e supplemen ed wi h
h eonine in he beginning o coli is induc ion (DSS + Th ) o a e es ablished in lamma ion (DSS + Th D8). Mice ea ed only o coli is induc ion we e used as
con ol (DSS). (B) Disease p og ession was assessed by sco ing he disease ac i i y index (DAI) h oughou he expe imen . (C) Quan i ica ion o sho -chain a y
acids (ace a e, bu y a e, p opiona e) and succina e by NMR was pe o med a day 13 o expe imen . (D) Exp ession o mucin-encoding genes Muc1 and Muc2 was
pe o med by qPCR a day 13. (E) Colonic issue sec ions we e s ained wi h Alcian Blue/pe iodic acid-Schi . (F) Quan i ica ion o goble cell numbe pe c yp . Scale
ba = 50 µm. (G) IL-22 quan i ica ion on colonic explan cul u es by ELISA. Da a is shown as mean ±SD. n= 5 mice/g oup. One ep esen a i e expe imen is
shown ou o wo. The symbol ∗co esponds o s a is ical di e ences be ween DSS + Th and DSS; while # is ela ed o s a is ical di e ences be ween DSS + Th
and DSS + Th D8. ∗p<0.05; ∗∗p<0.01; ∗∗∗p<0.001.
no only single h eonine supplemen a ion was no e alua ed
be o e, bu also he e is sca ce e idence ega ding he mos
adequa e ime ame o i s supplemen a ion in coli is ea men .
Acco dingly, we add essed h eonine supplemen a ion in
wo dis inc phases: (1) in pa allel wi h he ini ia ion o
he in lamma o y p ocess, i.e., simul aneously wi h DSS
adminis a ion (DSS +Th ), and (2) on day 8, when
in lamma ion is al eady es ablished (DSS +Th D8). Mice
subjec ed o DSS-induced coli is bu wi hou h eonine supply
we e used as con ol (DSS) (Figu e 2A). We obse ed ha
he h ee g oups de eloped coli is wi h a simila p og ession
p o ile. Ne e heless, a e his ime poin , he eco e y
p o ile o coli ic mice supplemen ed wi h h eonine in
he d inking wa e (DSS +Th ) was slowe han ha
o he o he wo g oups, showing s a is ically signi ican
di e ences om day 10 o he inal day o expe imen when
compa ed o con ol g oup (DSS). Besides, DSS +Th mice
had also a dis inc i e DAI sco e a day 11 and 12 when
compa ed wi h mice ha only ecei ed h eonine a e
day 8 (DSS +Th D8) (Figu e 2B). Despi e he di e gen
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Gai em e al. Th eonine Supplemen a ion E ec in Coli is
pheno ype, no majo di e ences we e ound in colon
leng h nei he in he in es inal pe meabili y (Supplemen a y
Figu e S1).
Th eonine adminis a ion was su p isingly de imen al o
disease eco e y when gi en a he se ing o he in lamma o y
p ocess. To unde s and his pheno ype, we i s e alua ed he
po en ial al e a ions in he colon me abolic p o ile among
he di e en g oups. No signi ican di e ences we e ound
be ween mos o he me aboli es. Pa icula ly, he le els o he
majo SCFAs (i.e., ace a e, bu y a e, and p opiona e), no mally
associa ed wi h a p o ec i e pheno ype, we e simila among
g oups. Only succina e le els, which is as an impo an ma ke
o in lamma ion p omo ing IL-1βinduc ion in in lamma o y
con ex s (Tannahill e al., 2013), we e ound o be ma kedly
dec eased in DSS +Th D8 mice (Figu e 2C). No majo
al e a ions we e obse ed in he in lamma o y in il a e p o ile,
issue o ganiza ion and hepa ic and enal oxici y se um
bioma ke s (Supplemen a y Figu e S1).
Th eonine plays a majo ole in mucin syn hesis and
consequen ly in he o ma ion o he mucus laye . Indeed, lack
o h eonine is also known o impai in es inal pa acellula
pe meabili y and is associa ed wi h ewe goble cells and mucus
syn hesis (Fau e e al., 2005;Mao e al., 2011). The mucus
laye se es as a ba ie agains mic obial ansloca ion o
he lamina p op ia and he e o e i s in eg i y is pa amoun
o in es inal homeos asis. When we analyzed he exp ession
o mucin-encoding genes, we ound ha Muc2 exp ession is
dec eased in DSS +Th mice when compa ed o mice ha
only ecei ed h eonine a day 8 (DSS +Th D8) (Figu e 2D).
Muc2 encodes o he oligome ic mucus gel- o ming mucin 2
p o ein ha is he majo esponsible o mucus syn hesis. Indeed,
impai men o o al absence o he mucus laye is associa ed wi h
se e e coli is, as obse ed in Muc2-de icien mice (Van de Sluis
e al., 2006). We also ound ha DSS +Th mice display highe
exp ession o Muc1, which has shown o con ibu e o in es inal
in lamma ion and colon cance p og ession (Baldus e al., 2004;
Takahashi e al., 2015). We nex quan i ied he numbe o goble
cells o he colonic mucosa. These a e a sec e o y epi helial
cell lineage ound in bo h he small and he la ge in es ines,
whose majo unc ion is he p oduc ion o mucus. By analyzing
colon slices s ained wi h Alcian Blue/Pe iodic acid-Schi , we
obse ed ha DSS +Th mice displayed signi ican ly ewe
goble cells when compa ed o bo h DSS and DSS +Th D8
(Figu es 2E,F). The e o e, ou esul s sugges ha an al e a ion
in mucus syn hesis due o h eonine adminis a ion du ing he
onse o disease may impac in es inal in eg i y, by delaying he
eco e y o disease. Fau e e al. (2006) ha e demons a ed ha
supplemen a ion wi h die con aining highe doses o amino
acids, including L- h eonine, lead o an inc ease in goble
cell numbe , egula ed mucin p oduc ion in he colon and
es o ed mic obio a composi ion a e DSS ea men in a s.
No wi hs anding, no only he animal model is di e en , bu also
L- h eonine was gi en be o e coli is induc ion, which may be
unde lying he dis inc i e ou come.
P e ious s udies ha e linked se e al cy okines o mucus
p oduc ion in he in es ine (Pa ks e al., 2015). To examine
he immunological p o ile o he h ee g oups, cy okine le els
in he colon we e quan i ied. No majo changes we e obse ed
be ween he g oups o in e leukin (IL)-1β, IL-12p70, IL-10,
IL-17A/F and g anulocy e-mac ophage colony-s imula ing ac o
(GM-CSF) le els (Supplemen a y Figu e S2). No wi hs anding,
he amoun o IL-22 was signi ican ly dec eased in DSS +Th
mice when compa ed o DSS +Th D8 mice (Figu e 2G).
IL-22 is a membe o he IL-10 amily o cy okines and has
been as ly s udied in he con ex o in es inal homeos asis.
I can be p oduced by se e al cell ypes, such as T helpe
(Th1) 1, Th17, Th22 and inna e lymphoid cells (ILCs), and
p esen se e al oles in he gas oin es inal ac , such as
issue egene a ion and main enance o he in es inal epi helial
ba ie (Ru z e al., 2013). Thus, he dec eased IL-22 le els
may be associa ed wi h delayed eco e y o he in es inal
balance.
O e all, ou da a demons a e ha supplemen a ion o
h eonine du ing coli is induc ion impai s goble cell numbe ,
wi h concomi an dec eased Muc2 exp ession and IL-22
p oduc ion. These a ia ions a e likely o be he cause o delayed
eco e y obse ed in his si ua ion. In e es ingly, hese e ec s
a e no seen when h eonine is adminis e ed once coli is is
es ablished. Acu e DSS-induced coli is is known o p omo e
gu mic obial dysbiosis (Munyaka e al., 2016). Th eonine is
me abolized by some in es inal commensal bac e ia, leading
o he p oduc ion o se e al me aboli es used o in es inal
main enance and o media e immune esponses (Neis e al.,
2015). Thus, h eonine supplemen a ion du ing induc ion o
coli is may impac di e en ly he colonic mic obio a popula ions
p esen du ing he onse and upon he es ablishmen o
in lamma ion, ha ing ul ima ely dis inc e ec s in in es inal
unc ion. Fu he unde s anding o he mechanisms unde lying
h eonine supplemen a ion may gi e new insigh s on how die a y
nu ien s modula e he dynamic balance be ween mic obiome,
immune esponse and ba ie unc ion.
ETHICS STATEMENT
This s udy was ca ied ou in acco dance wi h he
ecommenda ions o Eu opean Council Di ec i e (2010/63/EU)
guidelines ha whe e ansposed in o Po uguese law (Dec ee-
Law n.◦113/2013, Augus 7 h), i3S Animal E hics Commi ee
and licensed by he Po uguese Na ional Au ho i y o Animal
Heal h (DGAV). The p o ocol was app o ed by he i3S
Animal E hics Commi ee and licensed by he Po uguese
Na ional Au ho i y o Animal Heal h (DGAV) wi h e e ence
014811/2016-07-13.
AUTHOR CONTRIBUTIONS
JG, ET, CC, AgC, FR, MS, AnC, and RS designed he expe imen s.
JG, LG, RD-O, and RS pe o med he expe imen s. JG, LG, RD-O,
and RS analyzed he da a. JG, LG, RD-O, and RS in e p e ed
he esul s. JG and RS d a ed he manusc ip and p epa ed he
ables and igu es. JG, LG, RD-O, ET, CC, AgC, FR, MS, AnC,
and RS e ised he pape and app o ed he inal e sion o he
manusc ip .
F on ie s in Physiology | www. on ie sin.o g 5Sep embe 2018 | Volume 9 | A icle 1247

phys-09-01247 Sep embe 4, 2018 Time: 9:42 # 6
Gai em e al. Th eonine Supplemen a ion E ec in Coli is
FUNDING
This wo k was suppo ed by he No he n Po ugal Regional
Ope a ional P og amme (NORTE 2020), unde he Po ugal
2020 Pa ne ship Ag eemen , h ough he Eu opean Regional
De elopmen Fund (FEDER) (NORTE-01-0145-FEDER-
000013) and he Fundação pa a a Ciência e Tecnologia
(FCT) (con ac s PD/BD/106053/2015 o JG ia In e -
Uni e si y Doc o al P og amme in Ageing and Ch onic
Disease – PhDOC, IF/00021/2014 o RS, IF/01390/2014
o ET, IF/01147/2013 o RD-O, IF/00735/2014 o AgC,
SFRH/BPD/96176/2013 o CC, and SFRH/BPD/111100/2015
o LG). MS is a FCT in es iga o . The NMR da a was
acqui ed a CERMAX (Cen o de Ressonância Magné ica
An ónio Xa ie ) which is a membe o he Na ional NMR
ne wo k wi h he suppo o P ojec LISBOA-01-0145-FEDER-
007660.
SUPPLEMENTARY MATERIAL
The Supplemen a y Ma e ial o his a icle can be ound
online a : h ps://www. on ie sin.o g/a icles/10.3389/ phys.
2018.01247/ ull#supplemen a y-ma e ial
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he e iew.
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