The influence of anthocyanin treatment on the mesenteric adipose tissue expression of enampt in animal models of obesity

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 7. REFERENCES 1. Sun K, Kusminski CM, Sche e PE, Sun K, Kusminski CM, Sche e PE. Adipose issue emodeling and obesi y Find he la es e sion : Re iew se ies Adipose issue emodeling and obesi y. 2011;121(6):2094–101. 2. Cao H. Adipocy okines in obesi y and me abolic disease. Jou nal o Endoc inology [In e ne ]. 2014 Feb;220(2):T47–59. A ailable om: h ps://joe.bioscien i ica.com/ iew/jou nals/joe/220/2/T47.xml 3. Moschen AR, Kase A, En ich B, Mosheime B, Theu l M, Niede egge H, e al. Vis a in, an Adipocy okine wi h P oin lamma o y and Immunomodula ing P ope ies. The Jou nal o Immunology. 2007;178(3):1748–58. 4. Mohammadi M, Mianabadi F, Meh ad-Majd H. Ci cula ing is a in le els and cance s isk: A sys ema ic e iew and me a-analysis. J Cell Physiol. 2019;234(4):5011–22. 5. Flehmig G, Scholz M, Klö ing N, Fasshaue M, Tönjes A, S um oll M, e al. Iden i ica ion o adipokine clus e s ela ed o pa ame e s o a mass, insulin sensi i i y and in lamma ion. PLoS One. 2014;9(6). 6. Lago F, Gómez R, Gómez-Reino JJ, Dieguez C, Gualillo O. Adipokines as no el modula o s o lipid me abolism. T ends Biochem Sci. 2009;34(10):500–10. 7. Cannon B, Nede gaa d J. B own Adipose Tissue: Func ion and Physiological Signi icance. 2004; A ailable om: www.p .o g 8. Pei L, Wan T, Wang S, Ye M, Qiu Y, Jiang R, e al. Cyanidin-3-O-β-glucoside egula es he ac i a ion and he sec e ion o adipokines om b own adipose issue and alle ia es die induced a y li e . Biomedicine and Pha maco he apy [In e ne ]. 2018;105(74):625–32. A ailable om: h ps://doi.o g/10.1016/j.biopha.2018.06.018 9. Tilg H, Moschen AR. Role o adiponec in and PBEF/ is a in as egula o s o in lamma ion: In ol emen in obesi y-associa ed diseases. Clin Sci. 2008;114(3–4):275–88. 10. Longo M, Za e ale F, Nade i J, Pa illo L, Fo misano P, Raci i GA, e al. Adipose issue dys unc ion as de e minan o obesi y-associa ed me abolic complica ions. In J Mol Sci. 2019 May 1;20(9). 11. Kawai T, Au ie i M ., Scalia R. Adipose issue in lamma ion and me abolic dys unc ion in obesi y. Am J Physiol Cell Physiol. 2021 Ma 1;320(3):C375–91. 12. Zwick RK, Gue e o-Jua ez CF, Ho sley V, Plikus M . Ana omical, Physiological, and Func ional Di e si y o Adipose Tissue. Vol. 27, Cell Me abolism. Cell P ess; 2018. p. 68–83. 13. Imai S ichi o. Nico inamide Phospho ibosyl ans e ase (Namp ): A Link Be ween NAD Biology, Me abolism, and Diseases. Cu Pha m Des [In e ne ]. 2009 Jan 1;15(1):20–8. A ailable om: h ps://www.ncbi.nlm.nih.go /pmc/a icles/PMC3624763/pd /nihms412728.pd 14. Imai S, Yoshino J. The impo ance o NAMPT/NAD/SIRT1 in he sys emic egula ion o me abolism and ageing. Diabe es Obes Me ab [In e ne ]. 2013 Sep;15(s3):26–33. A ailable om: h ps://www.ncbi.nlm.nih.go /pmc/a icles/PMC3624763/pd /nihms412728.pd 15. Re ollo JR, Kö ne A, Mills KF, Sa oh A, Wang T, Ga en A, e al. Namp /PBEF/Vis a in Regula es Insulin Sec e ion in β Cells as a Sys emic NAD Biosyn he ic Enzyme. Cell Me ab [In e ne ]. 2007 No ;6(5):363–75. A ailable om: h ps://linkinghub.else ie .com/ e ie e/pii/S155041310700263X 16. Belenky P, Bogan KL, B enne C. NAD+ me abolism in heal h and disease. T ends Biochem Sci. 2007;32(1):12–9. 17. Yang L, Qiu Y, Ling W, Liu Z, Yang L, Wang C, e al. An hocyanins egula e se um adipsin and is a in in pa ien s wi h p ediabe es o newly diagnosed diabe es: a andomized con olled ial. Eu J Nu [In e ne ]. 2020; A ailable om: h ps://doi.o g/10.1007/s00394- 020-02379-x 18. G olla AA, T a elli C, Genazzani AA, Se hi JK. Ex acellula nico inamide phospho ibosyl ans e ase, a new cance me abokine. B J Pha macol [In e ne ]. 2016 Jul 2;173(14):2182–94. A ailable om: h ps://joe.bioscien i ica.com/ iew/jou nals/joe/220/2/T47.xml 19. Li H, Bai E, Zhang Y, Jia Z, He S, Fu J. Role o Namp and Visce al Adiposi y in Esophagogas ic Junc ion Adenoca cinoma. J Immunol Res. 2017;2017. 25 20. B own JEP, Onyango DJ, Ramanjaneya M, Conne AC, Pa el ST, Dunmo e SJ, e al. Vis a in egula es insulin sec e ion, insulin ecep o signalling and mRNA exp ession o diabe es- ela ed genes in mouse panc ea ic β-cells. J Mol Endoc inol. 2010;44(3):171–8. 21. R. Moschen A, R. Ge ne R, Tilg H. P e-B Cell Colony Enhancing Fac o /NAMPT/Vis a in in In lamma ion and Obesi y- Rela ed Diso de s. Cu Pha m Des. 2010;16(17):1913–20. 22. Camp SM, Ceco E, E enoski CL, Danilo SM, Zhou T, Chiang ET, e al. Unique Toll-Like Recep o 4 Ac i a ion by NAMPT/PBEF Induces NFκ B Signaling and In lamma o y Lung Inju y. Sci Rep [In e ne ]. 2015;5(July):1–14. A ailable om: h p://dx.doi.o g/10.1038/s ep13135 23. Oi a RC, Camp SM, Ma W, Ceco E, Ha beck M, Single on P, e al. No el mechanism o nico inamide phospho ibosyl ans e ase inhibi ion o TNF-α-media ed apop osis in human lung endo helial cells. Vol. 59, Ame ican Jou nal o Respi a o y Cell and Molecula Biology. 2018. p. 36–44. 24. Li Y, Zhang Y, Do weile B, Cui D, Wang T, Woo CW, e al. Ex acellula namp p omo es mac ophage su i al ia a nonenzyma ic in e leukin-6/STAT3 signaling mechanism. Jou nal o Biological Chemis y. 2008;283(50):34833–43. 25. Wilson PWF, D’Agos ino RB, Pa ise H, Sulli an L, Meigs JB. Me abolic synd ome as a p ecu so o ca dio ascula disease and ype 2 diabe es melli us. Ci cula ion. 2005 No ;112(20):3066–72. 26. Weisbe g SP, McCann D, Desai M, Rosenbaum M, Leibel RL, Fe an e AW. Obesi y is associa ed wi h mac ophage accumula ion in adipose issue. Jou nal o Clinical In es iga ion. 2003 Dec 15;112(12):1796–808. 27. Po a C, Subh a Kuma B, La ghi P, Rubino L, Mancino A, Sica A. Chap e 5 Tumo P omo ion by Tumo -Associa ed Mac ophages. 28. Chang YH, Chang DM, Lin KC, Shin SJ, Lee YJ. Vis a in in o e weigh /obesi y, ype 2 diabe es melli us, insulin esis ance, me abolic synd ome and ca dio ascula diseases: a me a- analysis and sys emic e iew. Diabe es Me ab Res Re [In e ne ]. 2011 Sep;27(6):515–27. A ailable om: h p://libweb.anglia.ac.uk/ 29. Mohammadi M, Mo adi A, Fa hadi J, Akba i A, Pou mandi S, Meh ad-Majd H. P ognos ic alue o is a in in a ious human malignancies: A sys ema ic e iew and me a-analysis. Cy okine [In e ne ]. 2020;127(No embe 2019):154964. A ailable om: h ps://doi.o g/10.1016/j.cy o.2019.154964 30. Luk T, Malam Z, Ma shall JC. P e-B cell colony-enhancing ac o (PBEF)/ is a in: a no el media o o inna e immuni y. J Leukoc Biol. 2008;83(4):804–16. 31. Lee YH, Bae SC. Ci cula ing adiponec in and is a in le els in heuma oid a h i is and hei co ela ion wi h disease ac i i y: A me a-analysis. In J Rheum Dis. 2018;21(3):664–72. 32. Fukuha a A, Ma suda M, Nishizawa M, Segawa K, Tanaka M, Kishimo o K, e al. Vis a in : A P o ein Sec e ed by Visce al Fa Tha Mimics he E ec s o Insulin Published by : Ame ican Associa ion o he Ad ancemen o Science S able URL : h p://www.js o .o g/s able/3840062 REFERENCES Linked e e ences a e a ailable on JSTOR o h. Science (1979). 2005;307(5708):426–30. 33. Sandeep S, Velmu ugan K, Deepa R, Mohan V. Se um is a in in ela ion o isce al a , obesi y, and ype 2 diabe es melli us in Asian Indians. Me abolism. 2007;56(4):565–70. 34. Ba h S, Klein P, Ho bach T, Dö sch J, Rauh M, Rasche W, e al. Exp ession o neu opep ide Y, omen in and is a in in isce al and subcu aneous adipose issues in humans: Rela ion o endoc ine and clinical pa ame e s. Obes Fac s. 2010;3(4):245–51. 35. Yoon MJ, Yoshida M, Johnson S, Takikawa A, Usui I, Tobe K, e al. SIRT1-Media ed eNAMPT Sec e ion om Adipose Tissue Regula es Hypo halamic NAD+ and Func ion in Mice. Cell Me ab [In e ne ]. 2015 May;21(5):706–17. A ailable om: h ps://linkinghub.else ie .com/ e ie e/pii/S1550413115001576 36. Es eghama i A, Alamda i A, Zandieh A, Elahi S, Khalilzadeh O, Nakhja ani M, e al. Se um is a in is associa ed wi h ype 2 diabe es melli us independen o insulin esis ance and obesi y. Diabe es Res Clin P ac [In e ne ]. 2011;91(2):154–8. A ailable om: h p://dx.doi.o g/10.1016/j.diab es.2010.11.003 37. Chen MP, Chung FM, Chang DM, Tsai JCR, Huang HF, Shin SJ, e al. Ele a ed plasma le el o is a in/p e-B cell colony-enhancing ac o in pa ien s wi h ype 2 diabe es melli us. Jou nal o Clinical Endoc inology and Me abolism. 2006;91(1):295–9. 26 38. Re naka an R, Youn BS, Liu Y, Hanley AJG, Lee NS, Pa k JW, e al. Co ela ion o ci cula ing ull-leng h is a in (PBEF/NAMPT) wi h me abolic pa ame e s in subjec s wi h and wi hou diabe es: A c oss-sec ional s udy. Clin Endoc inol (Ox ). 2008;69(6):885–93. 39. López-Be mejo A, Chico-Julià B, Fe nàndez-Balsells M, Recasens M, Es e e E, Casami jana R, e al. Se um is a in inc eases wi h p og essi e β-cell de e io a ion. Diabe es. 2006;55(10):2871–5. 40. Saye s SR, Bea il RL, Fine NHF, Huang GC, Choudha y P, Pachola z KJ, e al. S uc u e- unc ional changes in eNAMPT a high concen a ions media e mouse and human be a cell dys unc ion in ype 2 diabe es. Diabe ologia. 2020;63(2):313–23. 41. Bloomga den ZT. Adiposi y and diabe es. Diabe es Ca e. 2002;25(12):2342–9. 42. Mu su J, Vi anen JK, Tuomainen TP, Nu mi T, Vou ilainen S. In ake o ui , be ies, and ege ables and isk o ype 2 diabe es in Finnish men: he Kuopio Ischaemic Hea Disease Risk Fac o S udy. Am J Clin Nu [In e ne ]. 2014 Feb 1;99(2):328–33. A ailable om: h ps://academic.oup.com/ajcn/a icle/99/2/328/4571480 43. Wu LLY, Dunning KR, Yang X, Russell DL, Lane M, No man RJ, e al. High- a die causes lipo oxici y esponses in cumulus - Oocy e complexes and dec eased e iliza ion a es. Endoc inology. 2010;151(11):5438–45. 44. Scha. Lipo oxici y: when issues o e ea . Cu Opin Lipidol. 2003;281–7. 45. Yoshino J, Mills KF, Yoon MJ, Imai S ichi o. Nico inamide Mononucleo ide, a Key NAD+ In e media e, T ea s he Pa hophysiology o Die - and Age-Induced Diabe es in Mice. Cell Me ab [In e ne ]. 2011 Oc ;14(4):528–36. A ailable om: h ps://linkinghub.else ie .com/ e ie e/pii/S1550413111003469 46. Mei eles M, Rod íguez-Alcalá LM, Ma ques C, No be o S, F ei as J, Fe nandes I, e al. E ec o ch onic consump ion o blackbe y ex ac on high- a induced obesi y in a s and i s co ela ion wi h me abolic and b ain ou comes. Food Func . 2016;7(1):127–39. 47. Zhang XY, Guo CC, Yu YX, Xie L, Chang CQ. Es ablishmen o high- a die -induced obesi y and insulin esis ance model in a s. Beijing da xue xue bao Yi xue ban = Jou nal o Peking Uni e si y Heal h sciences. 2020;52(3):557–63. 48. Schwingshackl L, Ho mann G. Compa ison o E ec s o Long-Te m Low-Fa s High-Fa Die s on Blood Lipid Le els in O e weigh o Obese Pa ien s: A Sys ema ic Re iew and Me a-Analysis. J Acad Nu Die [In e ne ]. 2013;113(12):1640–61. A ailable om: h p://dx.doi.o g/10.1016/j.jand.2013.07.010 49. Tan BL, No haizan ME. E ec o High-Fa Die s on Oxida i e S ess, Cellula In lamma o y Response and Cogni i e Func ion. Nu ien s [In e ne ]. 2019 Oc 25;11(11):2579. A ailable om: h ps://www.mdpi.com/2072-6643/11/11/2579 50. Buijsse B, Feskens EJM, Schulze MB, Fo ouhi NG, Wa eham NJ, Sha p S, e al. F ui and ege able in akes and subsequen changes in body weigh in Eu opean popula ions: Resul s om he p ojec on Die , Obesi y, and Genes (DiOGenes). Ame ican Jou nal o Clinical Nu i ion. 2009;90(1):202–9. 51. González-Cas ejón M, Rod iguez-Casado A. Die a y phy ochemicals and hei po en ial e ec s on obesi y: A e iew. Pha macol Res. 2011;64(5):438–55. 52. Knek P, Kumpulainen J, Jä inen R, Rissanen H, Heliö aa a M, Reunanen A, e al. Fla onoid in ake and isk o ch onic diseases. Ame ican Jou nal o Clinical Nu i ion. 2002;76(3):560– 8. 53. Mon onen J, Jä inen R, Heliö aa a M, Reunanen A, A omaa A, Knek P. Food consump ion and he incidence o ype II diabe es melli us. Eu J Clin Nu . 2005;59(3):441–8. 54. Wedick NM, Pan A, Cassidy A, Rimm EB, Sampson L, Rosne B, e al. Die a y la onoid in akes and isk o ype 2 diabe es in US men and women. Ame ican Jou nal o Clinical Nu i ion. 2012;95(4):925–33. 55. Ca e P, G ay LJ, T ough on J, Khun i K, Da ies MJ. F ui and ege able in ake and incidence o ype 2 diabe es melli us: Sys ema ic e iew and me a-analysis. BMJ (Online). 2010;341(7772):543. 56. Villegas R, Xiao OS, Gao YT, Yang G, Elasy T, Li H, e al. Vege able bu no ui consump ion educes he isk o ype 2 diabe es in Chinese women. Jou nal o Nu i ion. 2008;138(3):574– 80. 27 57. Coope AJ, Khaw KT, Sha p SJ, Wa eham NJ, Len jes MAH, Fo ouhi NG, e al. A p ospec i e s udy o he associa ion be ween quan i y and a ie y o ui and ege able in ake and inciden ype 2 diabe es. Diabe es Ca e. 2012;35(6):1293–300. 58. Liu S, Se dula M, Janke SJ, Cook NR, Sesso HD, Wille WC, e al. A p ospec i e s udy o ui and ege able in ake and he isk o ype 2 diabe es in women. Diabe es Ca e. 2004;27(12):2993–6. 59. Belwal T, Naba i S, Naba i S, Hab ema iam S. Die a y An hocyanins and Insulin Resis ance: When Food Becomes a Medicine. Nu ien s [In e ne ]. 2017 Oc 12;9(10):1111. A ailable om: h p://www.mdpi.com/2072-6643/9/10/1111 60. Sasaki R, Nishimu a N, Hoshino H, Isa Y, Kadowaki M, Ichi T, e al. Cyanidin 3-glucoside amelio a es hype glycemia and insulin sensi i i y due o down egula ion o e inol binding p o ein 4 exp ession in diabe ic mice. Biochem Pha macol. 2007;74(11):1619–27. 61. No be o S, Sil a S, Mei eles M, Fa ia A, Pin ado M, Calhau C. Bluebe y an hocyanins in heal h p omo ion: A me abolic o e iew. J Func Foods. 2013;5(4):1518–28. 62. Mei eles M, Ma ques C, No be o S, Fe nandes I, Ma eus N, Rendei o C, e al. The impac o ch onic blackbe y in ake on he neu oin lamma o y s a us o a s ed a s anda d o high- a die . Jou nal o Nu i ional Biochemis y [In e ne ]. 2015;26(11):1166–73. A ailable om: h p://dx.doi.o g/10.1016/j.jnu bio.2015.05.008 63. DeFu ia J, Benne G, S issel KJ, Pe ield JW, Milbu y PE, G eenbe g AS, e al. Die a y bluebe y a enua es whole-body insulin esis ance in high a - ed mice by educing adipocy e dea h and i s in lamma o y sequelae. Jou nal o Nu i ion. 2009;139(8):1510–6. 64. Tö önen R, Kolehmainen M, Sa kkinen E, Mykkänen H, Niskanen L. Pos p andial glucose, insulin, and ee a y acid esponses o suc ose consumed wi h blackcu an s and lingonbe ies in heal hy women. Ame ican Jou nal o Clinical Nu i ion. 2012;96(3):527–33. 65. Tö önen R, Sa kkinen E, Niskanen T, Tapola N, Kilpi K, Niskanen L. Pos p andial glucose, insulin and glucagon-like pep ide 1 esponses o suc ose inges ed wi h be ies in heal hy subjec s. B J Nu . 2012;107(10):1445–51. 66. Si ama u hi BS, Kesika P, Chaiyasu C. The in luence o supplemen a ion o an hocyanins on obesi y-associa ed como bidi ies: A concise e iew. Vol. 9, Foods. MDPI Mul idisciplina y Digi al Publishing Ins i u e; 2020. 67. Lee YM, Yoon Y, Yoon H, Pa k HM, Song S, Yeum KJ. Die a y an hocyanins agains obesi y and in lamma ion. Vol. 9, Nu ien s. MDPI AG; 2017. 68. S ull AJ, Cash KC, Johnson WD, Champagne CM, Ce alu WT. Bioac i es in bluebe ies imp o e insulin sensi i i y in obese, insulin- esis an men and women. Jou nal o Nu i ion. 2010;140(10):1764–8. 69. Yang L, Qiu Y, Ling W, Liu Z, Yang L, Wang C, e al. An hocyanins egula e se um adipsin and is a in in pa ien s wi h p ediabe es o newly diagnosed diabe es: a andomized con olled ial. Eu J Nu [In e ne ]. 2021;60(4):1935–44. A ailable om: h ps://doi.o g/10.1007/s00394-020-02379-x 70. Be nd J, Klö ing N, K alisch S, Ko acs P, Fasshaue M, Schön MR, e al. Plasma is a in concen a ions and a depo -speci ic mRNA exp ession in humans. Diabe es. 2005;54(10):2911–6. 71. Haide DG, Holze G, Schalle G, Weghube D, Widhalm K, Wagne O, e al. The adipokine is a in is ma kedly ele a ed in obese child en. J Pedia Gas oen e ol Nu . 2006;43(4):548–9. 72. Owcza ek AJ, Olszanecka-Glinianowicz M, Kocełak P, Bożen owicz-Wika ek M, B zozowska A, Mossakowska M, e al. The ela ionship be ween ci cula ing is a in/nico inamide phospho ibosyl ans e ase, obesi y, in lamma ion and lipids p o ile in elde ly popula ion, de e mined by s uc u al equa ion modeling. Scand J Clin Lab In es [In e ne ]. 2016 No 16;76(8):632–40. A ailable om: h ps://www. and online.com/doi/ ull/10.1080/00365513.2016.1230884 73. Kieswich J, Saye s SR, Sil es e MF, Ha wood SM, Yaqoob MM, Ca on PW. Monome ic eNAMPT in he de elopmen o expe imen al diabe es in mice: a po en ial a ge o ype 2 diabe es ea men . Diabe ologia [In e ne ]. 2016;59(11):2477–86. A ailable om: h p://dx.doi.o g/10.1007/s00125-016-4076-3 28 74. Ca on PW, Kieswich J, Yaqoob MM, Holness MJ, Sugden MC. Nico inamide mononucleo ide p o ec s agains p o-in lamma o y cy okine-media ed impai men o mouse isle unc ion. Diabe ologia. 2011;54(12):3083–92. 75. Is ek N, Gu buz O. In es iga ion o he impac o bluebe ies on me abolic ac o s in luencing heal h. J Func Foods. 2017 No 1;38:298–307. 76. Bhaswan M, B own L, Ma hai ML. Queen Ga ne plum juice and aspbe y co dial in mildly hype ensi e obese o o e weigh subjec s: A andomized, double-blind s udy. J Func Foods. 2019 May 1;56:119–26.