THE INFLUENCE OF ANTHOCYANIN TREATMENT ON THE
MESENTERIC ADIPOSE TISSUE EXPRESSION OF eNAMPT
IN ANIMAL MODELS OF OBESITY
MARIA MARGARIDA NOBRE PROJECTO
A disse a ion submi ed in pa ial ul illmen o he equi emen s o he Deg ee o Mas e s in
Human Nu i ion and Me abolism
Disse ação pa a ob enção do g au de Mes e em Nu ição Humana e Me abolismo
a Faculdade de Ciências Médicas | NOVA Medical School o Uni e sidade NOVA de Lisboa
Sep embe , 2022
ii
THE INFLUENCE OF ANTHOCYANIN TREATMENT ON THE MESENTERIC
ADIPOSE TISSUE EXPRESSION OF eNAMPT IN ANIMAL MODELS OF OBESITY
Ma ia Ma ga ida Nob e P ojec o
Supe iso s: Ma a P Sil es e, In i ed Assis an P o esso a Faculdade de Ciências Médicas |
NOVA Medical School o Uni e sidade NOVA de Lisboa
Ana Fa ia, Assis an P o esso a Faculdade de Ciências Médicas | NOVA Medical
School o Uni e sidade NOVA de Lisboa
A disse a ion submi ed in pa ial ul illmen o he equi emen s o he Deg ee o
Mas e s in Human Nu i ion and Me abolism
Sep embe , 2022
iii
This s udy was suppo ed by Na ional Funds h ough FCT – Fundação pa a a Ciência e a
Tecnologia (FCT) wi hin he p ojec PTDC/BAA-AGR/7419/2020.
i
ACKNOWLEDGEMENTS
Fi s ly, I would like o hank my supe iso s, P o esso a Dou o a Ma a Sil es e, and P o esso a
Dou o a Ana Fa ia, o hei p o essionalism, guidance, and a ailabili y. Sha ing hei knowledge
and as expe ience was a g ea asse o me and he elabo a ion o his hesis.
I also wan o hank and acknowledge he en i e acul y membe s o his Mas e o he quali y
o eaching and he encou agemen o scien i ic in es iga ion, igh om he beginning. In
pa icula , o P o esso a Dou o a Diana Teixei a and P o esso Dou o João A aújo o he essen ial
suppo p o ided in his wo k.
Jus as impo an is my g a i ude o Ped o, Shámila, and especially Ma ia João o he
undamen al suppo , collabo a ion, and company du ing he labo a o y expe ience.
Finally, a big hank you o my amily o hei suppo and wo ds o encou agemen h oughou
my academic jou ney, which allowed me o ge he e oday.
ABBREVIATIONS
AT Adipose Tissue
ANKO Adipose Tissue-Speci ic Namp Knockou
AUC A ea Unde he Cu e
BAT B own Adipose Tissue
BE S anda d Die + Blackbe y Ex ac
BMI Body Mass Index
C S anda d Die
Cy-3-G Cyanidin-3-O-β-Glucoside
eNAMPT Ex acellula Nico inamide Phospho ibosyl ans e ase
FRD F uc ose Rich Die
GSIS Glucose-S imula ed Insulin Sec e ion
GTT Glucose Tole ance Tes
HFD High-Fa Die
HFDBE High-Fa Die + Blackbe y Ex ac
HRP Ho se-Radish Pe oxidase
iNAMPT In acellula Nico inamide Phospho ibosyl ans e ase
IL-6 In e leukin 6
IR Insulin Resis ance
IS Insulin Sensi i i y
ITT Insulin Tole ance Tes
mAT Mesen e ic Adipose Tissue
NAD Nico inamide Adenine Dinucleo ide
NAMPT Nico inamide Phospho ibosyl ans e ase
NAMPT +/− NAMPT-de icien He e ozygous
NF-KB Nuclea Fac o Kappa B
NMN Nico inamide Mononucleo ide
NMNAT Nico inic Acid Mononucleo ide Adenylyl ans e ase
NMS|FCM NOVA Medical School | Faculdade de Ciências Médicas
PBS Phospha e-bu e ed saline
PVDF Poly inylidene di luo ide

i
RIPA Radio-Immunop ecipi a ion
scAT Subcu aneous Adipose Tissue
SD S anda d De ia ion
SEM S anda d E o o The Mean
SDS-PAGE Sodium dodecyl-sul a e polyac ylamide gel elec opho esis
STAT3 Signal T ansduce and Ac i a o o T ansc ip ion 3
T2D Type 2 Diabe es
TPBS PBS con aining 0,1% ( ol/ ol) Tween-20
TLR4 Toll-like Recep o 4
WAT Whi e Adipose Tissue
WB Wes e n Blo
ii
ABSTRACT
In oduc ion: Nico inamide Phospho ibosyl ans e ase (NAMPT) is he a e-limi ing enzyme o
NAD biosyn hesis. I s ex acellula o m (eNAMPT), mainly sec e ed by isce al a , has been
shown o ha e bimodal, concen a ion- and s uc u e- unc ional-dependen e ec s in impo an
me abolic pa hways and has been connec ed o a wide a ie y o diseases. Da a sugges s ha as
se um eNAMPT concen a ion ises o pa hophysiological le els, as in obesi y and ype 2 diabe es
(T2D), eNAMPT adop s a monome ic o m capable o p oin lamma o y NAD-independen e ec s.
S a egies o block he ac ions o he eNAMPT monome could ep esen p omising he apeu ic
app oaches o obesi y- ela ed me abolic diso de s. Consump ion o an hocyanin ich oods
appea s o p e en o ea obesi y- ela ed consequences, such as T2D, in lamma ion and
oxida i e s ess, bu he mechanism behind his is unknown. Mo eo e , an hocyanins ha e been
shown o inhibi he sec e ion o eNAMPT in animal models. Wi h his s udy, we aim o
unde s and i ea ing a a model o obesi y wi h an hocyanins could ab oga e he impac o a
high a die in he exp ession o monome ic eNAMPT.
Me hods: Mesen e ic adipose issue (mAT) was ob ained om ou g oups o male Wis a a s,
ea ed wi h di e en die s: (C) s anda d die ; (BE) s anda d die + blackbe y ex ac ; (HFD) high-
a die ; (HFDBE) high- a die + blackbe y ex ac . eNAMPT monome ’s p o ein exp ession was
measu ed by Wes e n Blo ing, a e p o ein ex ac ion and quan i ica ion om mAT, o access
he di e ences be ween he animals ed a s anda d die and hose o inc eased me abolic isk –
HFD, wi h and wi hou ea men wi h an hocyanins.
Resul s: The mAT om HFD a s displayed a highe exp ession o eNAMPT monome , compa ed
o C a s (138,6% ± 11,4% HFBE s 100% C, p=0,01). The an hocyanin’s ea men in luence on mAT
eNAMPT monome exp ession was also assessed. mAT eNAMPT monome exp ession was
signi ican ly dec eased in he HFDBE g oup compa ed o HFD (-54,1 ± 15,3 % [-89.4, -18.8], p<0,01).
Conclusions: An hocyanin consump ion migh be an in e es ing die a y app oach o ab oga e
he impac o a high a die on he ise o monome ic eNAMPT in mesen e ic adipose issue.
Ul ima ely, ou esul s sugges ha long- e m an hocyanin ea men /supplemen a ion migh
be e ec i e o sus aining lowe le els o monome ic eNAMPT in he con ex o die -induced
obesi y, po en ially p e en ing o delaying he consequen me abolic impai men s such as he
ala ming epidemic o T2D.
Keywo ds: Adipose issue, An hocyanins, eNAMPT, Nico inamide Phospho ibosyl ans e ase,
Obesi y, Type 2 Diabe es
iii
RESUMO
In odução: A Nico inamida Fos o ibosil an e ase (NAMPT) é a enzima limi an e no p ocesso de
biossín ese de NAD. A sua o ma ex acelula (eNAMPT), sec e ada p incipalmen e pelo ecido
adiposo isce al, demons ou e e ei os bimodais, dependen es da concen ação e da es u u a
uncional, em impo an es ias me abólicas e em sido associada a uma ampla a iedade de
doenças. Dados suge em que à medida que a concen ação sé ica de eNAMPT aumen a pa a
ní eis isiopa ológicos, como na obesidade e diabe es ipo 2 (T2D), a eNAMPT ado a uma o ma
monomé ica capaz de e ei os p ó-in lama ó ios independen es do NAD. Possí eis es a égias
que bloqueiem as ações do eNAMPT monomé ico podem ep esen a abo dagens e apêu icas
p omisso as pa a os dis ú bios me abólicos elacionados com a obesidade. O consumo de
alimen os icos em an ocianinas pa ece p e eni ou a a consequências elacionadas com a
obesidade, como T2D, in lamação e s ess oxida i o, mas o mecanismo subjacen e é ainda
desconhecido. Pa a além disso, as an ocianinas demons a am inibi a sec eção de eNAMPT em
modelos animais. Com es e es udo, p e endemos comp eende se o a amen o de um modelo
animal de obesidade com an ocianinas pode ia anula o impac o de uma die a ica em go du a
na exp essão de eNAMPT monomé ico.
Mé odos: O ecido adiposo mesen é ico (mAT) oi ob ido a pa i de qua o g upos de a os Wis a
machos, a ados com di e en es die as: (C) die a s anda d; (BE) die a s anda d + ex a o de
amo a; (HFD) die a ica em go du a; (HFDBE) die a ica em go du a + ex a o de amo a. A
exp essão p o eica do eNAMPT monomé ico oi medida po Wes e n Blo ing, após a ex ação e
quan i icação das p o eínas do mAT, pa a a alia as di e enças en e os animais alimen ados com
die a s anda d e os que ap esen am isco me abólico aumen ado – HFD, com e sem a amen o
com an ocianinas.
Resul ados: No mAT de a os HFD, a exp essão do monóme o eNAMPT oi 38,6% mais ele ada
quando compa ado com C (138,6% ± 11,4% HFBE s 100% C, p=0,01). A in luência do a amen o
com an ocianinas na exp essão do eNAMPT monomé ico no mAT ambém oi a aliada em a os
alimen ados com HFD e C. O eNAMPT monomé ico diminuiu signi ica i amen e em a os
alimen ados com HFDBE em compa ação com HFD (-54,1 ± 15,3 % [-89.4, -18.8], p<0,01).
Conclusão: O consumo de an ocianinas pa ece se uma abo dagem die é ica in e essan e pa a
anula o impac o de uma die a ica em go du a no aumen o de eNAMPT monomé ico no ecido
adiposo mesen é ico. Em úl ima análise, os nossos esul ados suge em que o
a amen o/suplemen ação com an ocianinas, a longo p azo, pode se e icaz pa a sus en a
ní eis mais baixos de eNAMPT monomé ico no con ex o de uma obesidade induzida pela die a,
con ibuindo pa a comba e ou as al e ações me abólicas, como a ala man e epidemia de T2D.
Pala as-cha e: An ocianinas, Diabe es ipo 2, eNAMPT, Nico inamida Fos o ibosil an e ase,
Obesidade, Tecido Adiposo
ix
3
When su eying he issue dis ibu ion o iNAMPT in mice, Re ollo e al. demons a ed ha
iNAMPT p o ein exp ession le els we e he highes in BAT, li e , and kidney. Hea , WAT, lung,
spleen, es is, and skele al muscle showed in e media e o e y low le els o iNAMPT, while in he
b ain and panc eas i was unde ec able. These indings sugges ha iNAMPT-media ed NAD
biosyn hesis migh signi ican ly di e amids di e en issues (15).
The ex acellula o m o NAMPT (eNAMPT), also known as p e-B cell colony enhancing ac o o
is a in, exhibi s obus , e en highe NAD biosyn he ic ac i i y han iNAMPT (15,17). The exac
mechanism o eNAMPT’s sec e ion is s ill unde in es iga ion, bu i seems o be sec e ed mainly
by adipocy es, h ough a non-classical pa hway (15,18), being la ely one o he adipokines wi h
g ea e esea ch in e es .
The physiological ole o eNAMPT, o he han he enzyma ic one, has been a ma e o much
deba e. I has been shown ha eNAMPT leads o di e en biological eac ions by di e se ypes o
cells, possibly h ough he ac i a ion o in acellula signalling pa hways (19). Resul s om
di e en s udies ha e been con o e sial and he mechanisms by which eNAMPT exe s
p oin lamma o y p ope ies o is p o ec i e agains cellula s ess o oxici y is s ill subjec o
s udy. Indeed, he ela ionship be ween NAMPT and cellula homeos asis is highly complex (19).
Fo ins ance, i has been shown ha eNAMPT binds o Toll-like ecep o 4 (TLR4) and ac i a es
he nuclea ac o kappa B (NF-KB) signalling pa hway (20,21). This causes he exp ession o
a ious ma ke s o in lamma ion and oxida i e s ess, as well as cell su i al and dea h pa hways
egula ion (22,23). In ac , he e a e se e al epo s o eNAMPT ha ing p oin lamma o y and
immunomodula ing p ope ies. A e mu ine eNAMPT adminis a ion in mice, ci cula ing le els
o in e leukin 6 (IL-6), a cy okine ha is s ongly implica ed in in lamma o y and immunological
p ocesses, a ose (3). Some au ho s obse ed ha ecombinan eNAMPT dose-dependen ly
induced mRNA exp ession le els o cy okines such as IL-1, IL-6, IL-10, and TNF-α and p o oked he
p oduc ion o hese cy okines in human lymphocy es and monocy es, in which IL-6 up- egula ion
and p oduc ion was he mos p onounced (3,21).
Fo he i s ime in 2008, in es iga o s ound ha eNAMPT could p omo e cell su i al in
mac ophages, by inhibi ing endoplasmic e iculum s ess-induced apop osis. This mechanism
occu s by ac i a ing an IL-6/Signal T ansduce and Ac i a o o T ansc ip ion 3 (STAT3 ) signalling
pa hway (24). Mac ophages ha e an impo an and p eponde an ole in he pa hophysiology o
como bidi ies associa ed wi h obesi y, such as a he oscle osis (25), in lamma ion (26), and cance
(27). The e o e, some hing ha changes he dynamics o su i al-apop osis o hese immune cells
will ha e an impac on he eme gence o diseases ela ed o obesi y. P olonged mac ophage
su i al was sugges ed o p op up he in lamma o y esponse, known as a c ucial ac o
unde lying obesi y- ela ed me abolic diseases (24). In e es ingly hese indings we e no
dependen on he enzyma ic ac i i y o eNAMPT, co obo a ed by he ac ha STAT3
phospho yla ion was no induced by NMN – NAMPT’s enzyma ic p oduc - and because he

4
inhibi o o NAMPT’s enzyma ic ac i i y - FK866 - also did no p e en eNAMPT's abili y o
s imula e phospho yla ion o STAT3 (24). As Y. Li e al. also epo , i seems ques ionable ha cells
elease an enzyme ha is capable o ac i a ing ce ain me abolic pa hways in a non-enzyma ic
way. La e , a possible jus i ica ion o his, ela ed o s uc u al and unc ional changes o he
eNAMPT p o ein, will be add essed.
1.3 NAMPT In The Con ex o Me abolic Disease – Obesi y and Diabe es
eNAMPT has ecen ly been shown o ha e an impo an connec ion o a wide a ie y o diseases.
The e is in ac e idence showing enhanced mRNA exp ession o NAMPT and highe ci cula ing
le els o eNAMPT in obesi y, T2D, cance , and o he in lamma o y condi ions (18,28,29), including
in lamma o y bowel diseases ((3,30)) heuma oid a h i is (31), a he oscle osis, myoca dial
in a c ion, and se e e gene alized pso iasis (3,13,30).
The biological ac ion o NAMPT in obesi y and obesi y- ela ed diso de s, such as T2D, has been
in e es ingly s udied since Fukuha a e al. demons a ed ha eNAMPT exe ed a glucose-
lowe ing e ec in a diabe ic mouse model. Howe e , he a icle ended up e ac ed due o he
con o e sy ega ding he insulin ecep o -binding ac i i y o eNAMPT ha could no be
ep oduced (32). Despi e he signi ican in e es , he e is a lo o con o e sy o e he associa ion
be ween ci cula ing le els o eNAMPT and obesi y- ela ed pa ame e s, and he e ec s o eNAMPT
in he con ex o T2D, pa icula ly ega ding he ole o his p o ein in be a cell unc ion.
The esul s om a me a-analysis o 13 obse a ional s udies in ol ing 644 subjec s showed a
signi ican inc ease in eNAMPT concen a ion in pa icipan s wi h o e weigh /obesi y compa ed
o no mal body mass index (BMI) con ols (28)). Mo eo e , Fukuha a e al. demons a ed ha
eNAMPT was mo e abundan ly sec e ed by AT in bo h humans and mice, han in scAT (13,32). As
men ioned, he a icle was e ac ed due o he lack o ep oducibili y o he main esul –
eNAMPT insulin-mime ic e ec – howe e o he au ho s co obo a ed he signi ican associa ion
be ween eNAMPT le els and isce al a (19,33,34).
To in es iga e he unc ion o eNAMPT speci ically sec e ed and egula ed by AT, Yoon e al.
c ea ed and analysed adipose issue-speci ic Namp knockou (ANKO) mice, which showed
educ ions in plasma eNAMPT le els and NAD le els, esul ing also in se e e impai men s in IS
and glucose-s imula ed insulin sec e ion (GSIS) (35). In line wi h his knowledge, AT may be
undamen al o egula e be a cell unc ion h ough sec e ion o eNAMPT and consequen
ex acellula biosyn hesis o NMN, as ully di e en ia ed adipocy es a e a na u al p oduce o
eNAMPT (13,15,35). Re ollo e al. showed ha NAMPT-de icien he e ozygous (NAMPT +/−) mice
had a de ec in NAD biosyn hesis and in GSIS in panc ea ic isle s. Adminis a ion o NMN could
comple ely amelio a e hese de ec s, s ongly indica ing ha hey we e due o a lack o NAMPT-
media ed NAD biosyn hesis (15). Adipocy e dys unc ion, wi h ul ima e dys egula ion o
adipokines, pa icula ly eNAMPT, can lead o al e a ions in IS and he de elopmen o T2D, wi h
simul aneously wo sening o in lamma ion and obesi y (20,36,37). Consis en e idence shows
5
ha T2D pa ien s p esen highe le els o eNAMPT compa ed o heal hy con ols (36–38)). In a
2011 me a-analysis, his e idence was ein o ced as i was shown ha eNAMPT was signi ican ly
inc eased in subjec s diagnosed wi h T2D (28). This ela ionship be ween NAMPT ac i i y and T2D
appea s o be ela ed o panc ea ic issue being mo e suscep ible o changes in NAMPT-
media ed NAD biosyn hesis, compa ed o o he issues. Because o he almos o al absence o
iNAMPT, isle s ely on ex acellula sou ces o NAD in e media es, such as NMN, o main ain hei
NAD le els adequa e o hei unc ions (14,15). Mo eo e , a ailable e idence sugges s ha he
ele a ed eNAMPT concen a ion in T2D may be a physiologic p o ec i e esponse o a
hype glycemic en i onmen (28). Al hough, a sup aphysiological le els, he e ec appa en ly
u ns dele e ious (20,39).
As discussed ea lie , acco ding o some da a, he inhibi o o NAMPT’s ac i i y - FK866 - has been
shown o be ine ec i e, indica ing ha a leas pa o NAMPT’s e ec s is independen o i s
enzyma ic ac i i y ((18,24)). Dime iza ion o eNAMPT is essen ial o i s enzyma ic unc ion, and a
physiological le els, in heal hy indi iduals, eNAMPT is ound mainly in he dime ic o m (~100kDa).
Ye , Li e al. showed ha NAMPT mu an s, which we e unable o dime ize, main ained a cy okine-
like ac i i y (24). Fo example, as discussed p e iously, he an i-apop o ic e ec o NAMPT does
no equi e dime iza ion, sugges ing ha i is he monome ic o m (~50kDa) ha main ains his
p o-in lamma o y e ec (13).
A s udy deepened he cha ac e iza ion o his s uc u e- unc ional di e ence o eNAMPT in he
de e io a ion o panc ea ic be a cell unc ion and i s ela ionship wi h T2D, in o de o unde s and
he con adic o y e ec s o eNAMPT on be a cell unc ion (p o ec i e o dele e ious), p e iously
desc ibed in he li e a u e (40). The s udy concluded ha concen a ion played an impo an ole
in hese changes in s uc u e and unc ion, and ha dime ic eNAMPT, ound a low physiological
le els p omo es NAD biosyn hesis, while a a sup aphysiological s a e eNAMPT adop ed a
monome ic o m capable o p o-in lamma o y mechanisms, independen o NAD, dele e ious o
be a cell unc ion (40).
1.4 An hocyanins - Heal h P omo ion and Po en ial E ec on eNAMPT in Con ex o
Me abolic Disease
Despi e obesi y being he main isk ac o o T2D (37,41), die is an unques ionable modi iable
ac o ha may in luence he isk o bo h obesi y and diabe es (42). A high a die in he con ex
o o e nu i ion leads o inc eased lipid deposi ion in bo h adipose and non-adipose issues
(43,44). In i o s udies ha e shown ha animals ed high- a die s de elop obesi y and diabe es,
wi hin weeks (45–47)). Fu he mo e, a high a die has been shown o induce al e a ions in blood
lipid p o ile and in ci cula ing p oin lamma o y cy okines (48,49). In con as o he ypical
“wes e n die ”, high in suga and a , a ui and ege ables ich die has been associa ed wi h a
lowe isk o obesi y and ela ed co-mo bidi ies (50,51). In pa icula , obse a ional s udies show
ha a highe in ake o ui s and ege ables has been in e sely associa ed wi h he isk o T2D
6
(52–55). Howe e , he numbe o s udies is limi ed and indings ega ding hese associa ions ha e
no been consis en (56–58).
In ecen yea s, be ies ha e been a ield o esea ch ha has g own conside able in e es , due o
hei po en ial, as a na u al p oduc , o heal h p omo ion and disease p e en ion (59–61). Be ies
ha e been associa ed wi h cogni i e unc ion and an i-in lamma o y and an ioxidan esponses
(62,63), as well as wi h he educ ion o adipocy e dea h and lowe adipocy e size in mAT (46,61).
In addi ion, a g owing body o e idence, including p ospec i e coho s udies, sugges s ha
highe in akes o be ies a e in e sely associa ed wi h T2D isk (52–54). Conside ing his, in o he
indings, including hose om clinical ials, be ies ha e shown an imp o emen in glucose
esponse and in IS (52–54). These bene icial ou comes o ui , in pa icula be ies, seem o be
connec ed o hei composi ion in e ms o die a y ibe and polyphenols (64,65). Die a y
phy ochemicals (such as polyphenols) may esul in syne gis ic ac i i y ha can o e ad an ages
o e ea men s wi h single chemicals ha may comp ehend ad e se side e ec s (59,60).
An hocyanins a e a g oup o polyphenolic na u al pigmen s ha belong o a b oad class o
seconda y me aboli es called la onoids, abundan in a ious b igh ly colo ed ui and
ege ables (59–61). Despi e he limi ed numbe o clinical s udies, se e al in i o and in i o
s udies con i med ha he consump ion o an hocyanins ich ood p e en s o ea s obesi y-
ela ed consequences like T2D, in lamma ion and oxida i e s ess. (66,67). The s udies ha ha e
been conduc ed o e alua e he biological unc ions o die a y an hocyanins on in lamma ion
and obesi y ha e shown ha hei an i-obese p ope ies ely on hei abili y o con ol ood
consump ion and ene gy me abolism and imp o e in lamma o y esponse. Howe e , hese
a o able e ec s we e dependen on se e al physiological, immunological, neu ological, and
me abolic in e connec ed e en s (66,67). Die a y an hocyanins also appea o amelio a e IR
(42,46,61,64,65,68). Expe imen al s udies ha e demons a ed ha he bene i s in T2D associa ed
wi h an hocyanins may be ela ed o imp o emen s in IS (60), h ough di e se mechanisms
anging om inhibi ing ca bohyd a e diges ion in he gu , panc ea ic be a cell p o ec ion, and
insulin sec e ion o enhancing IS in i al o gans (59). Cyanidin-3-O-β-glucoside (Cy-3-G), an
abundan an hocyanin, was able o egula e he exp ession o adipokines in BAT, which we e
dis up ed by a high a die supplemen ed wi h choles e ol (8). Fu he mo e, supplemen a ion
wi h pu i ied an hocyanins dec eased eNAMPT in pa ien s wi h p ediabe es o newly diagnosed
diabe es and showed imp o emen s in glucolipid me abolic pa ame e s (69).
Taken oge he , hese s udies sugges a bene icial e ec o an hocyanins as p e en i e s a egy
o obesi y– ela ed me abolic abno mali ies, po en ially h ough a pa hway in ol ing NAMPT.
Howe e , he mechanism by which an hocyanins play a ole in obesi y h ough eNAMPT has no
been elucida ed no was he ela ionship be ween an hocyanin ea men and he speci ic
concen a ion o eNAMPT in i s dime ic/monome ic o m.
7
2. AIMS
This s udy seeks o help closing he gap in cu en li e a u e, by in es iga ing he di e ences in
eNAMPT’s exp ession, in i s monome ic o m, in he mesen e ic adipose issue o animal models
in he con ex o die -induced obesi y and inc eased me abolic isk ( ed a high a die ). Mo eo e ,
he s udy aims o unde s and how he e ec o such obesogenic die in he exp ession o
monome ic eNAMPT is impac ed by an an hocyanin ea men . Ul ima ely, he s udy aims o gi e
us an insigh in o ea ly mechanisms o disease p e en ion, po en ially con ibu ing o he
he apeu ic s a egies o me abolic diso de s.
8
3. METHODOLOGY
3.1 Expe imen al Design
This s udy p o ocol ob ained e hical app o al by he E hics Commi ee o NOVA Medical School |
Faculdade de Ciências Médicas (NMS|FCM), NOVA Uni e si y o Lisbon (nº113/2021/CEFCM). An
o e iew o he expe imen al p o ocol is ep esen ed in Figu e 2.
Figu e 2 - O e iew o expe imen al p o ocol and imeline. mAT - mesen e ic Adipose Tissue.
3.2 Animals and Sample Collec ion
The mAT samples used in his s udy we e p e iously collec ed du ing a esea ch p ojec in 2012
(62). Animal handling and housing p o ocols ollowed Eu opean Union guidelines (86/ 609/EEC)
and he Po uguese Ac (129/92) o he use o expe imen al animals. The s udy ob ained e hical
app o al om he E hical Commi ee o he Facul y o Medicine, Uni e si y o Po o and he São
João Hospi al Cen e (46). Twen y- ou male Wis a a s, acqui ed om Ha lan Labo a o ies
(San iga, Spain), we e di ided in o ou g oups (n = 6 a s pe g oup), a e wo weeks o
acclima iza ion, as ollows: (C) s anda d die ; (BE) s anda d die + blackbe y ex ac ; (HFD) high-
a die ; (HFDBE) high- a die + blackbe y ex ac . Animals we e ed ad libi um wi h “s anda d”
(Teklad 2014, Ha lan Labo a o ies, San iga, Spain) o “high- a ” die s (D1245 Resea ch Die s, New
B unswick, USA), acco ding o hei g oups, o 17 weeks. Blackbe y an hocyanin ich ex ac (25

9
mg/kg body weigh /day) was dissol ed daily in s e ile wa e and embedded in ood pelle s ha
animals had daily access o. Animals we e main ained a 23–25 °C wi h a 12/12 h ligh –da k cycle
and housed wo pe cage. Food inges ion was measu ed wice a week. Fas ing blood glucose,
and glucose and insulin ole ance es s we e also assessed hen, and he de ailed me hodology
is desc ibed in he pape om Mei eles e al. 2016 (46). This da a is p esen ed in his hesis o
con ex ualize u he esul s ob ained in his p ojec and o in e p e a ion pu poses. A e
sac i ice, mAT was collec ed, immedia ely ozen in liquid ni ogen, and s o ed a −80 °C un il use.
In he p esen s udy, p o ein ex ac ion and quan i ica ion we e pe o med om hese mAT
samples.
3.3 P o ein Ex ac ion
App oxima ely 300µg o each mAT sample we e g ounded in liquid ni ogen wi h a mo a and
pes le and ans e ed o new Eppendo ubes (1,5mL). Recons uc ed Radio-
Immunop ecipi a ion (RIPA) Lysis Bu e wi h p o eases inhibi o s (10μL PMSF solu ion, 10μL
sodium o ho anada e solu ion, and 10-20μL p o ease inhibi o cock ail solu ion pe mL o 1X
RIPA Lysis bu e ) (Ca #sc-24948A, San a C uz Bio echnology, San a C uz, CA, USA) was added o
he ubes, ollowing he supplie ecommenda ions, and homogenized using an elec ic
homogenize . RIPA Bu e enables e icien issue and cell lysis, and p o ein solubiliza ion while
a oiding p o ein deg ada ion. The samples we e e ige a ed a -20ºC, o a leas 2h and hen
cen i uged a 10000 x g, o 15 min a 4ºC. The ubes we e gen ly emo ed om he cen i uge
and placed on ice. The supe na an was aspi a ed om each sample (± 600 µL) and placed in
esh iden i ied Eppendo ubes kep on ice. The lipid uppe phase and he pelle we e disca ded.
3.4 P o ein Quan i ica ion
Fo p o ein quan i ica ion, an EZQ® P o ein Quan i a ion Ki (R33200, The mo Fishe Scien i ic,
USA) was used. A e p epa ing he mic opla e casse e wi h he assay pape , 1 well was le wi h
no hing o ensu e non-con amina ion o he assay pape (nega i e con ol 1), and 1µL o
econs uc ed RIPA Lysis Bu e was spo ed on o ano he well (nega i e con ol 2). The samples
we e spo ed on o assay pape in iplica e using 1µL o each p o ein ex ac ion sample pe well
(p o ein ex ac was no dilu ed o a oid loss o p o ein). The pape was d ied and ixed wi h
me hanol o 5 minu es wi h gen le agi a ion o emo e con amina ing subs ances. The EZQ®
p o ein quan i a ion eagen was added in o a s aining ay and he p o ein-spo ed assay pape
was added in o he s ain solu ion and agi a ed gen ly on an o bi al shake o 30 minu es. A e
s aining, he assay pape was washed wice o 1–2 minu es wi h a inse bu e solu ion (10%
me hanol, 7% ace ic acid) and a e comple ely d ied was eplaced in o he mic opla e. The
luo escence was measu ed in a Spec aMax i3x pla e eade (Molecula De ices; Sunny ale, CA)
using exci a ion/emission se ings o 450/618nm. The inal p o ein concen a ion was de e mined
using a s anda d cu e o o albumin (Appendix 1), and he samples we e ozen a -20ºC o la e
use. The samples had a e y low p o ein concen a ion, insu icien o p o ide enough p o ein o
10
pe o m he Wes e n blo s (WB). In o de o concen a e he samples, hey we e lyophilized
o e nigh and esuspended in a smalle olume o ul apu e wa e (Milli-Q® sys ems). P o ein
quan i ica ion was hen epea ed acco ding o he me hod desc ibed abo e.
3.4 eNamp Measu emen s - P o ein Gel Elec opho esis and Wes e n Blo ing
In o de o sepa a e p o eins, a sodium dodecyl-sul a e polyac ylamide gel elec opho esis (SDS-
PAGE) o 4-15% p ecas polyac ylamide gels (#4568034, Bio-Rad Labo a o ies Inc, USA) was used.
SDS is an anionic de e gen , used o dena u e e ia y and seconda y p o ein s uc u es, hus
allowing a p o ein sepa a ion by size/molecula weigh depending on hei elec opho e ic
mobili y in a polyac ylamide gel. A e no malizing o 15μg o p o ein, adding 10μL o loading
bu e (6X, 400/L saccha ose, 2,5g/L b omophenol blue) and ensu ing an equi alen olume o
40μL o load each lane, samples we e hea ed a 100ºC o 10 minu es and cen i uged a 4,000
pm o 20 seconds. No educing agen was added o he p o ein samples p io o SDS-PAGE o
allow de ec ion o bo h monome ic and dime ic eNAMPT in biological samples. Be o e loading
he samples, 3μL o a p es ained p o ein ladde (R26619, PageRule ™Plus The mo Fishe
Scien i ic, USA) was loaded o one o he wells o each gel o se e as a size e e ence o which
samples could be compa ed la e . 40μL o he espec i e samples we e loaded o he emaining
gel wells, and elec opho esis was un a 100V o 90 minu es wi h mig a ion/ unning bu e (3g/L
TRIZMA® base, 14g/L glycine, 10mL/L SDS 10%) in mini-PROTEAN® Te a sys em gel anks (Bio-
Rad Labo a o ies Inc, USA). The e olu ion o elec opho esis was moni o ed by obse ing he
mig a ion o he b omophenol blue dye ha was added o he loading bu e . Due o i s low
molecula weigh and nega i e cha ge, i mig a es along wi h p o eins owa ds he posi i e
anode.
A e he p o eins ha e been sepa a ed by gel elec opho esis, he WB echnique equi es he
ans e o he p o eins o a memb ane, in his case a poly inylidene di luo ide (PVDF) memb ane,
o subsequen densi ome ic quan i ica ion. PVDF memb anes a e highly hyd ophobic, so p io
o hei u iliza ion hey we e p e-we ed wi h e hanol o 1 minu e, wa e o 10 minu es, and a
leas 10 minu es wi h ans e bu e (1.5g/L TRIZMA® base, 7.5g/L glycine). The ans e de ices,
wi h he black pa o he casse es acing bo om, we e p epa ed in he ollowing way o each
casse e: 1 sc a ch sponge, 2 Wha man® blo ing pape s, SDS-PAGE gel, PVDF memb ane, 2
Wha man® blo ing pape s, 1 sc a ch sponge, and e e y hing was embedded in ans e bu e .
The ans e casse es we e hen inse ed in o he Mini T ans-Blo Cell® bu e ank (Bio-Rad
Labo a o ies Inc, USA), which was illed wi h ans e bu e , and blo ing was pe o med a 50 V
o 95 minu es, so he nega i ely cha ged p o eins could ans e on o he memb ane, by
mig a ing owa ds he posi i ely cha ged elec ode o he blo ing chambe . The e iciency o
ans e was con olled by he p esence o he p o ein ladde on he memb ane a he end.
Following he ans e , memb anes we e washed wice wi h phospha e-bu e ed saline (PBS)
con aining 0,1% ( ol/ ol) Tween-20 (TPBS). Memb anes we e hen blocked wi h 5% (w / ol) d ied
11
skimmed milk (Molico®) dissol ed in TPBS (2,5 g pe 50mL) o 60 minu es a oom empe a u e
unde agi a ion, in o de o p e en non-speci ic binding du ing he ollowing an ibody
incuba ions. A e quickly insing he blocked memb anes h ee imes wi h TPBS, a mouse
p ima y an ibody agains NAMPT (Ca #sc-393444, San a C uz Bio echnology, San a C uz, CA,
USA) (1:500 dilu ion in TPBS con aining 5% BSA) was incuba ed wi h he memb anes o e nigh
a 4ºC on a ocking pla o m shake . Memb anes we e again washed h ee imes wi h TPBS, wi h
gen ly agi a ion on an o bi al shake o 10 minu es each ime, o elimina e unbound an ibody
esidues and pos e io ly incuba ed wi h seconda y an ibody an i-mouse conjuga ed o ho se-
adish pe oxidase (HRP) (Ca #sc-542741, San a C uz Bio echnology, San a C uz, CA, USA) (1:3000
dilu ion in TPBS con aining 5% BSA) o 60 minu es a oom empe a u e wi h gen le agi a ion.
Following one quick wash wi h TPBS, memb anes we e again washed h ee imes wi h TPBS,
wi h gen ly agi a ion on an o bi al shake o 10 minu es each ime.
The p o eins we e hen isualized in a ChemiDoc™ Touch Imaging Sys em (Bio-Rad Labo a o ies
Inc, USA) a e he memb anes we e washed o 1 minu e wi h a chemiluminescen WB de ec ion
eagen (RPN2235, Ame sham™ ECL Selec ™, cy i a), which allows he HRP o p oduce a
luminescen signal. The signal ob ained is p opo ional o he an ibody-bounded p o ein, which
appea s as bands, he e o e is used as he measu emen o p o ein quan i y. Densi ome y o
WB bands was calcula ed using Fiji so wa e e sion V1.52 E.
3.5 S a is ical Analysis
The no mali y o he da a was assessed by a Shapi o-Wilk es . Compa ison be ween C s HFD, C
s BE and HFD s HFDBE g oups was pe o med using unpai ed - es s. One way-ANOVA was
unde aken o analyse he a iance ac oss he g oups. Tukey’s pos hoc es was hen
implemen ed o e i y po en ial di e ences be ween he ea men g oups. Da a a e exp essed
as mean ± s anda d e o o he mean (SEM). Resul s we e conside ed s a is ically signi ican o
p≤0.05. All s a is ical analysis we e pe o med in G aphPad P ism 8 so wa e (G aphPad So wa e,
San Diego, USA).
12
4. RESULTS
4.1 Da a p e iously collec ed, analysed and desc ibed in Mei eles e al. 2016
The animal expe imen s o iginally ook place in 2012. All animals s a ed he die ea men a
simila body weigh s (Table 1). The high a die s - HFD and HFDBE - esul ed in o e weigh a s,
wi h inc eased body a pe cen age. The e ec o blackbe y an hocyanin ex ac
supplemen a ion on body weigh gain was no signi ican (62).
Table 1 –Body weigh o a s om di e en g oups be o e (ini ial body weigh ) and a e he 17 weeks o die
and blackbe y an hocyanin ex ac supplemen a ion ( inal body weigh ). Values a e exp essed as
mean ± S anda d De ia ion (SD). Di e en supe sc ip le e s ep esen s a is ically signi ican di e ences
(p<0,05): C – s anda d die ; BE – s anda d die + blackbe y ex ac ; HFD – high- a die ; HFDBE – high- a die
+ blackbe y ex ac . Rep oduced om Mei eles e al., 2016 o con ex ualize u he esul s ob ained in he
p ojec he e p esen ed.
Fas ing blood glucose was assessed in hese animals, showing signi ican di e ences be ween
g oups (p<0,05). The HFDBE had highe as ing glucose le els compa ed o C a he 15 h week o
ea men . (*p<0,05) ( igu e 3).
Figu e 3 – Fas ing blood glucose le els o a s om he ou g oups (n = 6). Da a a e exp essed as means ±
SEM. S a is ical signi icance was es ed by one-way ANOVA, ollowed by a Tuckey’s pos hoc analysis o
compa ison be ween g oups (p<0,05). The HFDBE had highe as ing glucose le els compa ed o C (*p<0,05).
Rep oduced om Mei eles e al., 2016 o con ex ualize u he esul s ob ained in he p ojec he e p esen ed.
C
BE
HFD
HFDBE
Ini ial body weigh (g)
237,8±7,7
246,5±8.6
237,2±7.2
244,0±11,1
Final body weigh (g)
429,8±27,8a
432,7±28,4a
550,7±67,6b
581,0±63,1b
19
imp o ed all p o iles associa ed wi h he condi ion, a e ecei ing an an ibody agains he
monome ic eNAMPT, such as dec eased blood glucose, imp o ed glucose ole ance and IR,
imp o ed panc ea ic isle unc ion, and educed in lamma ion (73). On he o he hand, upon
adminis a ion o eNAMPT-monome o he non-diabe ic mouse model, hey de eloped a
diabe ic pheno ype, cha ac e ized by an inc ease in blood glucose, impai ed glucose ole ance
and insulin sec e ion and by he p esence o a gene alized in lamma ion, wi hou changes in NAD
le els (73). These esul s sugges ha hese p o-in lamma o y and diabe ic e ec s a e
independen o eNAMPT’s NAD biosyn he ic ac i i y, p o iding e idence ha his monome ic
o m no only does no p esen enzyma ic capaci y, bu is also esponsible o he p e iously
men ioned ad e se e ec s.
In ag eemen wi h he a o emen ioned s udy, o he analysis conduc ed in indi iduals wi h
obesi y, diabe ic and non-diabe ic, o de ec ion o eNAMPT’s dime ic and monome ic le els,
showed ha se um eNAMPT concen a ions inc eased wi h p og ession o T2D. eNAMPT
monome was ound o be signi ican ly highe in T2D (40). These inc easing concen a ions did
no co ela e wi h se um NMN le els. This s udy also showed ha eNAMPT concen a ion was a
c i ical ac o in hese changes in s uc u e and unc ion. A low and physiological concen a ions
(1 ng/ml) eNAMPT emained in i s dime ic o m, op imizing be a cell unc ion h ough i s NAD-
biosyn he ic capaci y. Howe e , a sup aphysiological le els (5 ng/ml), cha ac e is ic o obesi y
and T2D, eNAMPT adop ed a monome ic s uc u e and de e io a ion o be a cell unc ion was
obse ed, esul ing om p o-in lamma o y mechanisms independen o NAD (40).
Al hough is no ye clea how and why hese s uc u al di e ences occu du ing he ising
concen a ions, s udies p esen di e en hypo heses, being one o hem he possible necessi y
o a ligand-induced dime iza ion, ha begins o be de icien a highe eNAMPT concen a ions,
leading o he b eakup o he dime in o cons i u i e monome s (73). The o he possibili y
desc ibed in ol es an obesi y-media ed dys unc ional AT ha is cha ac e ized by poo ly
di e en ia ed adipocy es and immune cells, incapable o sec e ing eNAMPT dime , as his o m
is said o be sec e ed by ully di e en ia ed adipocy es (40). Ei he way, inding app oaches ha
can s abilize eNAMPT in dime o m, by con olling i s ising se um concen a ion, o ha can
block he ac ions o he monome o m, by neu alizing eNAMPT monome appea s o be a
p omising u u e o disease p e en ion and po en ially con ibu ing o he he apeu ic s a egies
o me abolic diso de s such as obesi y and T2D.
The main pu pose o his s udy was also o analyse he impac o an an hocyanin ea men in
eNAMPT’s exp ession on mAT o hese animal model o inc eased me abolic isk. Die is a key
ac o in e ms o managing he isk o obesi y and obesi y- ela ed dis unc ions. P e ious s udies
ha e shown ha a die a y in e en ion wi h an hocyanins is e ec i e in educing he isk o
obesi y and p e en ing o ea ing obesi y- ela ed co-mo bidi ies (66,67). The esul s in subjec s

20
wi h obesi y sugges ed ha daily in ake o die a y an hocyanins migh aid in main aining o
educing he body weigh and a enua ing some isk ac o s o me abolic synd ome (75,76).
The an hocyanin ex ac om blackbe y gi en o he animals in his s udy was able o a enua e
he aise o mAT eNAMPT monome in he con ex o an HFD. mAT eNAMPT monome exp ession
was signi ican ly dec eased in g oup HFDBE compa ed o HFD (-54,1 ± 15,3 % [-89.4, -18.8], p<0,01).
In suppo o hese esul s, a p e ious andomized placebo-con olled ial also aimed o examine
he e ec s o pu i ied an hocyanins supplemen a ion on eNAMPT and ano he adipokine in
Chinese adul s wi h p ediabe es and newly diagnosed, un ea ed T2D (69). Supplemen a ion wi h
pu i ied an hocyanins o 12 weeks dec eased se um eNAMPT in pa ien s wi h p ediabe es o
newly diagnosed diabe es and showed imp o emen s in glucolipid me abolic pa ame e s (69). A
signi ican di e ence in se um eNAMPT (-3,5 ng/mL [ -6,69, -0,31], p=0,03) was obse ed be ween
he an hocyanins and placebo g oups, howe e i is no men ioned i i is he monome ic o
dime ic o m ha has been e alua ed. Se um eNAMPT dec eased by 9,7% (-3,57 ng/mL [-5,98, -
1,15], p=0.004) a e an hocyanins ea men compa ed o he 0,2% dec ease (-0,07 ng/mL [-2,07,
1,94], p=0,947) a e placebo ea men (69). The au ho s had al eady demons a ed ha
an hocyanins inhibi ed he sec e ion o eNAMPT in a p e ious animal s udy (8), and an impo an
conclusion om ha expe imen was ha Cy-3-G, as he mos abundan monome o
an hocyanins, could egula e he sec e ion o monome ic eNAMPT om b own adipocy es. A e
being incuba ed wi h palmi a e, he elease o monome ic eNAMPT om hose cells was
inc eased and could be inhibi ed by Cy-3-G (8). The main an hocyanin in blackbe y is Cy-3-G
(abou 85%). As such, he an hocyanin ex ac om blackbe y used o ea ing he animals on
he p ojec ha led o ou s udy is ich in Cy-3-G.
As assessed by ou wo king g oup in he o iginal p ojec om Mei eles e al 2016, die was a
signi ican ac o ha in luenced he glucose sensibili y o he animals (46). The HFD showed a
endency o p omo e a highe as ing glucose in animals, an obse a ion ha was no a enua ed
by he an hocyanin ea men . The HFDBE had highe as ing glucose le els compa ed o C
(p<0,05). As hese animals gained weigh along he weeks o ea men , especially he HFD
g oups, he isk o g ea e insulin esis ance and glucose in ole ance was highe , as hese
condi ions a e highly associa ed wi h obesi y. As expec ed, animals om he HFD displayed a
lowe esponse o insulin as well as an impai ed glucose sensibili y (46). As discussed ea lie ,
educ ions in plasma biosyn hesis o NMN, esul in se e e impai men s in IS and GSIS (35). Wi h
his s udy we demons a ed ha an obesogenic condi ion, imposed by a HFD, led o an inc ease
in he monome ic eNAMPT in hese animal’s mAT. This se ing may ha e led o a g ea e sec e ion
o he less biosyn he ic monome ic eNAMPT in o he ci cula ion and consequen ly, o a dec ease
in se um NMN, being his he possible cause o he de angemen s in he a ge issues mos
suscep ible o hese al e a ions, such as be a cells (Figu e 9). Howe e , he dime ic o m o
eNAMPT should ha e been analysed o a be e comp ehensi e analysis o he esul s, including
he a io monome /dime ins ead o he absolu e quan i ica ion o he monome ic eNAMPT
21
alone. I he a io had inc eased, as would be expec ed a he ou se , his would lead o a dec ease
in he p oduc ion o NMN, as seen in o he s udies (40,73,74).
Figu e 9 - Schema ic p oposal o eNAMPT monome 's highe exp ession in obese adipose issue and he
consequen NMN de iciency e ec s on be a cell unc ion. An obesogenic condi ion, imposed by a HFD, led
o an inc ease in he monome ic eNAMPT in hese animal’s mAT. This se ing may ha e led o a g ea e
sec e ion o he less biosyn he ic monome ic eNAMPT in o he ci cula ion and consequen ly, o a dec ease
in se um NMN, being his he possible cause o he de angemen s in he a ge issues mos suscep ible o
hese al e a ions, such as be a cells.
None heless, o a ce ain ex en supplemen a ion wi h blackbe y an hocyanin ex ac was able
o a enua e he impai men o he glycemic esponse caused by he HFD, as seen in he AUC o
he HFDBE g oup, a e 8 weeks. A possible hypo hesis is ha as he an hocyanin’s ea men
was able o concu en ly a enua e he inc ease o he mAT monome ic o m o eNAMPT, as
demons a ed in his s udy, he dime ic o m may ha e been sec e ed in highe o no mal le els
om he AT in his g oup o animals, main aining se um NMN le els adequa e and as so
p o ec ing he be a cell om he dele e ious e ec s p e iously discussed, pa icula ly a enua ing
he impai men o he glucose sensibili y, and op imizing he insulin esponse. A e he 15 h week
o ea men HFBE g oup also had a be e esponse o insulin han HFD a s (p<0,05).
Toge he hese da a demons a e ha mAT le els o eNAMPT monome we e selec i ely ele a ed
in HFD- ed mice, bu he an hocyanin’s ea men was able o signi ican ly minimize/ab oga e
his abno mal inc ease.
Howe e , his ela ion equi es u he in es iga ion. I is necessa y o ca y ou s udies wi h a
la ge n and p e e ably a human s udy design o p o e he hypo hesis aised and deepen he
echnique ha allows he quan i ica ion o he dime ic o m, ha can p o ide aluable
22
in o ma ion. Being able o analyse bo h o ms o eNAMPT would b ing a mo e in-dep h
in es iga ion o he molecula mechanisms esul ing in he epe cussions o he al e a ions in
his adipokine. Fu he in es iga ion will also be equi ed o iden i y de ailed mechanisms o he
e icacy o he an hocyanins. Addi ional in e en ional s udies, p e e ably long- e m ials a e
needed o cla i y and con i m ou indings and es he po en ial o blackbe y supplemen a ion
as a he apeu ic in e en ion in humans.
23
6. CONCLUSIONS
Despi e NAMPT’s essen ial ole in me abolism h ough i s ca aly ic unc ion in he a e-limi ing
s ep in NAD biosyn hesis, he p o ein has become he cen e o a en ion due o he subsequen
associa ion o i s monome ic-non-biosyn he ic o m wi h me abolic and in lamma o y diseases.
This s udy demons a es ha an an hocyanin ea men ab oga es he ele a ion o monome ic
eNAMPT p o ein in mAT om animals in lic ed wi h a HFD - ha nega i ely impac ed hei
me abolism. He e we hypo hesize ha he an hocyanin ea men , by educing he mAT
monome ic eNAMPT le els, was able o a enua e some o he esul ed impai men s, such as
glucose in ole ance, and op imize he esponse o insulin.
Ul ima ely, ou esul s sugges ha long- e m an hocyanin ea men /supplemen a ion migh
be e ec i e o sus aining lowe le els o monome ic eNAMPT in he con ex o die -induced
obesi y, po en ially p e en ing o delaying he consequen me abolic impai men s as he
ala ming epidemic o T2D. Ne e heless, hese we e s udies pe o med in a p e-clinical model o
obesi y and clinical ials a e needed o cla i y such ela ionships. Howe e , he consump ion o
an hocyanins, h ough a die ich in ui s and ege ables, pa icula ly h ough ed ui s, can be
an in e es ing die a y app oach o implemen and/o emphasize in clinical p ac ice, gi en he
exis ing bene icial e idence ein o ced by his wo k.
24
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