Caloric restriction, physical exercise, and CB1 receptor blockade as an efficient combined strategy for bodyweight control and cardiometabolic status improvement in male rats

1 Vol.:(0123456789) Scien i ic Repo s | (2021) 11:4286 | h ps://doi.o g/10.1038/s41598-021-83709-9 www.na u e.com/scien i ic epo s Calo ic es ic ion, physical exe cise, and CB1 ecep o blockade as an e icien combined s a egy o bodyweigh con ol and ca diome abolic s a us imp o emen in male a s Luisa M. Lopez T inidad1,3, Rosa io Ma inez1,3, Ga y allia Kap a elou1, Milag os Galis eo2, Pila A anda1, Jesus M. Po es1* & Ma ia Lopez‑Ju ado1 Obesi y is c i ically associa ed wi h he de elopmen o insulin esis ance and ela ed ca dio ascula and kidney diseases. Se e al s a egies o weigh loss ha e been de eloped bu mos o hem exhibi a pos ‑in e en ion ebound e ec . He e, we aimed o design combined weigh ‑loss s a egies o calo ic es ic ion, physical exe cise, and adminis a ion o a CB1 ecep o blocke o inhibi ood in ake ha also accomplish he objec i es o los ‑weigh main enance and imp o emen o ca dio ascula and enal unc ion. Die ‑induced obesi y (DIO) was gene a ed in Sp ague Dawley a s o 12 weeks o es he e ec s o single o combined s a egies (i.e. calo ic es ic ion, mixed aining p o ocol, and/o adminis a ion o appe i e supp essan ) on calo ic in ake, body weigh , ca dio ascula and enal unc ionali y esul ing om a weigh ‑loss in e en ion pe iod o 3 weeks ollowed by 6 weeks o weigh main enance. Consump ion o a high‑ a die (HFD) caused a signi ican inc ease in body weigh (5 h week o he expe imen al pe iod) and led o he de elopmen o insulin esis ance, ca dio ascula , and enal al e a ions. The di e en in e en ions es ed, esul ed in a signi ican body weigh loss and imp o ed glucose me abolism, ae obic capaci y, elec oca diog aphic pa ame e s, ascula exp ession o adhesion molecules and in lamma o y media o s, and enal unc ionali y, eaching alues simila o he con ol no mocalo ic g oup o e en imp o ing hem. Success ul main enance o los weigh was achie ed along a 6‑week main enance pe iod in addi ion o adequa e heal h s a us. In conclusion, he weigh ‑loss and main enance in e en ion s a egies es ed we e e icien a e e sing he obesi y‑ ela ed al e a ions in body weigh , glucose me abolism, ae obic capaci y, ca dio ascula and enal unc ionali y. The bene icial ac ion was e y consis en o calo ic es ic ion and physical exe cise, whe eas adminis a ion o a CB1 ecep o blocke complemen ed he e ec s o he p io in e en ions in some pa ame e s like body weigh o ae obic capaci y, and showed speci ic ac ions in enal s a us, inc easing glome ula il a ion a e and diu esis. O e all, he no el y o ou s udy elies on he easy implemen a ion o combined s a egies o e ec i e weigh managemen ha esul ed in signi ican heal h bene i s. O e weigh and obesi y a e de ined as abno mal o excessi e a accumula ion ha inc eases he isk o de elop mul iple pa hologies. They lead o ad e se me abolic e ec s on blood p essu e, choles e ol, iglyce ides, and insulin esis ance. This compila ion o ac o s is known as me abolic synd ome (Me S)1, which is di ec ly ela ed o ca dio ascula disease and he al e a ion in o he i al unc ions, such as he enal unc ion2. O e weigh , OPEN 1Depa men o Physiology, Ins i u e o Nu i ion and Food Technology (INyTA), Cen e o Biomedical Resea ch, Cen e o Resea ch in Spo and Heal h (IMUDS), Uni e sidad de G anada, A da. del Conocimien o S/N. A milla (18100), G anada, Spain. 2Depa men o Pha macology, School o Pha macy, Bioheal h Resea ch Ins i u e, Cen e o Biomedical Resea ch, Uni e sidad de G anada, G anada, Spain. 3 These au ho s con ibu ed equally: Luisa M. Lopez T inidad and Rosa io Ma inez. *email: jmpo es@ug .es 2 Vol:.(1234567890) Scien i ic Repo s | (2021) 11:4286 | h ps://doi.o g/10.1038/s41598-021-83709-9 www.na u e.com/scien i ic epo s/ obesi y and i s ela ed diseases a e la gely p e en able3. In his ega d, a comp ehensi e unde s anding o Me S may be impo an o he adequa e planning o p e en ion s a egies. Since i s componen s a e all e e sible, ea ly diagnosis and li es yle in e en ion s a egies o Me S o e an e ec i e ea men app oach, p ima ily a ge ing weigh managemen . The con ol and main enance o he body weigh a a s able le el a e achie ed when he e is a balance be ween ood in ake and ene gy expendi u e. A complex physiological con ol sys em is in ol ed in he main enance o he ene gy balance and includes a e en signals om he pe iphe y ega ding he s a e o ene gy s o es, as well as e e en signals a ec ing ene gy in ake and expendi u e4. This egula o y sys em includes mul iple in e ac ions be ween he gas oin es inal ac , adipose issue and cen al ne ous sys em, and is in luenced by beha io al, senso ial, au onomic, nu i ional and endoc ine mechanisms5. The i s s ep in he ea men o obesi y is ocused on losing he ex a-weigh and amelio a e he ela ed me abolic al e a ions. Ano he impo an issue o subjec s who comple e a weigh loss p og am is o a oid he pos -in e en ion ebound e ec . The bodyweigh egain usually akes place igh a e he end o weigh loss in e en ion as weigh loss p og ams a e jus ansien 6. A mul idisciplina y app oach is equi ed, including li es yle modi ica ions7 and, in some cases, he ein o cemen wi h pha macological ea men . Rela ed o li es yle in e en ions he e a e wo co e aspec s o co ec an al e ed ene gy balance: die and physical exe cise. Obesi y de elopmen is o en a o ed by he consump ion o unbalanced and hype calo ic die s, so he calo ic es ic- ion o a balanced die p o iding adequa e amoun s o nu ien s is highly ecommended. On he o he hand, physical ac i i y plays an essen ial ole in he p e en ion and ea men o obesi y. I con ibu es o gene a ing a nega i e ene gy balance, hus acili a ing weigh loss and a oiding he ebound e ec and subsequen body weigh egain8. I is well es ablished ha di e en aining p o ocols induce changes in a a ie y o molecula mechanisms in ol ed in nume ous in acellula pa hways ela ed o glucose and lipid me abolism, in lamma- ion, o an ioxidan s a us9. In addi ion o li es yle modi ica ions, he p esc ip ion o an app op ia e pha macological agen is some imes ecommended. The endocannabinoid sys em is comp ised by cannabinoid ecep o s 1 and 2 (CB1 and CB2), he wo endocannabinoids anandamide and 2-a achidonoylglyce ol, and endocannabinoid anabolic and ca abolic enzymes10. The endocannabinoid sys em (ECS) plays a c i ical ole in obesi y de elopmen in bo h cen al and pe iphe al unc ions ela ed o ene gy me abolism11. A he cen al le el, endocannabinoids ac as e og ade neu omodula o s o synap ic plas ici y, and pa icipa e in many physiological p ocesses including pain egula ion, lea ning and memo y, appe i e and ood in ake, lipogenesis, and c a ings12,13. A he pe iphe al le el, endocan- nabinoids exe a onic ac ion on lipogenesis and a accumula ion. The e o e, CB1 blockade may esul in a ood in ake-independen dec ease in a mass h ough lipolysis14. In ac , CB1 blockade has been shown o be e ec- i e in amelio a ing obesi y and ela ed me abolic diso de s15. In addi ion, he endocannabinoid 2-AG p e en myo ube o ma ion in a manne an agonized by CB1 knockdown and by CB1 an agonis s, which pe se, ins ead, s imula e di e en ia ion16. Mo eo e , an agonism o CB1 ecep o educed human sa elli e cell p oli e a ion and enhanced he o ma ion o myo ubes ep esen ing an adju an he apy o muscle dys ophies17. In ecen yea s, he disco e y o an expanded endocannabinoid sys em, he endocannabinoidome, which includes se e al media o s ha a e biochemically ela ed o he endocannabinoids, and hei ecep o s and me abolic enzymes, has co obo a ed i s complexi y and expanded he po en ial o de eloping new he apeu ic s a egies o ea mul iple ela ed pa hologies including neu ological, in lamma o y o me abolic al e a ions10. Gi en he abo e men ioned, we hypo hesized ha ou speci ic combined s a egy o li es yle and pha maco- logical in e en ions could p o ide in e es ing bene i s in he ea men o obesi y and i s ela ed ca dio ascula and enal al e a ions. The e o e, his s udy aimed o design hese new s a egies and es hem in an expe imen al model o DIO. Speci ically, we sough o (1) es he e ec s on body weigh , physical pe o mance, glycaemic and lipid p o ile, and di e en pa ame e s ela ed o ca dio ascula and enal heal h, o a combined p og am wi h calo ic es ic ion, mixed aining exe cise p o ocol, and pha macological ea men wi h he appe i e sup- p essan AM251, a well-known esea ch ool ha e ec i ely blocks CB1 ecep o s and exhibi s a s ong ood in ake inhibi ion ac ion combined wi h inc eased basal me abolic a e and dec eased plasma le els o glucose and LDL-choles e ol. (2) s udy he syne gies aking place be ween he h ee abo e-men ioned s a egies bo h on body weigh loss and in he main enance o weigh and me abolic, ca dio ascula and enal bene i s acqui ed. Figu e1. E ec s o DIO and weigh con ol in e en ions on calo ic in ake, body weigh , body weigh / emu leng h a io, and hypo halamic gene exp ession o ansc ip s in ol ed in he cen al egula ion o ood in ake and ene gy balance. Six con ol expe imen s we e ca ied ou du ing 21weeks using a s anda d a chow die (SD12, SD15, and SD21) o a high- a die o induce obesi y (HFD12, HFD15, and HFD21). Fo in e en ion ials, a s we e di ided in o 8 g oups ha we e ed he hype calo ic die o induce obesi y o 12weeks, ollowed by h ee weeks o in e en ion wi h a high p o ein die o weigh loss (WL15) combined o no wi h he aining p o ocol (e o s, espec i ely) and he pha macological ea men wi h CB1 ecep o blocke AM251 (AM). The in e en ion pe iod was ollowed by an addi ional 6-week weigh -main enance pe iod o die a y ea men wi h a s anda d a chow die (WM21) combined o no wi h he aining p o ocol (e o s, espec i ely) and he pha macological ea men (AM) o main ain he weigh los du ing he p e ious in e en ion pe iod o h ee weeks. (A) Expe imen al design, (B) a e age daily calo ic in ake (kcal/day) along he di e en expe imen al s ages (DIO, weigh -loss in e en ion, and weigh -main enance), (C) a e age body weigh a he end o he di e en expe imen al s ages, (D) bodyweigh / emu leng h a io (g/cm2) a he end o he di e en expe imen al s ages, (E) hypo halamic gene exp ession a he end o he weigh -loss in e en ion and weigh -main enance s ages. Resul s a e means o eigh a s ± SEM depic ed by e ical ba s. ***P < 0.001 in - es (12weeks); A,B,C means wi h di e en le e s a e signi ican ly di e en (ANOVA ea men , P < 0.05; 15 and 21weeks). c os c-Fos, npy neu opep ide Y, lep lep in ecep o , hc o exin A, cn 1 cannabinoid ecep o . ▸ 3 Vol.:(0123456789) Scien i ic Repo s | (2021) 11:4286 | h ps://doi.o g/10.1038/s41598-021-83709-9 www.na u e.com/scien i ic epo s/ 4 Vol:.(1234567890) Scien i ic Repo s | (2021) 11:4286 | h ps://doi.o g/10.1038/s41598-021-83709-9 www.na u e.com/scien i ic epo s/ Figu e1. (con inued) 5 Vol.:(0123456789) Scien i ic Repo s | (2021) 11:4286 | h ps://doi.o g/10.1038/s41598-021-83709-9 www.na u e.com/scien i ic epo s/ Resul s Combined weigh con ol s a egies e icien ly achie e weigh loss and p e en body weigh ebound a e ea men . The e ec s o DIO and di e en weigh -loss and weigh -main enance in e en- ions on daily calo ic in ake and body weigh a he end o he di e en expe imen al s ages a e p esen ed in Fig.1A–C. Th oughou he expe imen al pe iod, and due o he highe calo ic con en o HFD, a signi ican ly highe calo ic in ake was exhibi ed by HFD- ea ed a s when compa ed o hei no mocalo ic con ols. Likewise, highe body weigh was gained by he o me animals. Du ing he weigh -loss in e en ion pe iod om week 12–15 o he expe imen al pe iod, he indi idual ac ion o calo ic es ic ion, physical exe cise, and AM251 adminis a ion caused a dec ease in calo ic in ake ha led o a concomi an dec ease in body weigh o ea ed a s. Howe e , s onge e ec s we e obse ed when he h ee in e en ions we e combined. Along he weigh -main enance s age, a s abiliza ion o calo ic in ake and main enance o bodyweigh among he di e en in e en ion g oups we e achie ed below he alues shown by he SD and HFD con ols. Besides, he aining p o ocol and he adminis a ion o he appe i e supp essan played a ole o a oid he ebound e ec on body weigh . These ben- e icial ac ions on body weigh we e e lec ed on a simila end in body weigh / emu leng h a io a he di e en expe imen al s ages, especially when exe cise was combined wi h AM251 adminis a ion. (Fig.1C). Such a io has been conside ed ep esen a i e o he body mass index and adequa e o es ablish he deg ee o obesi y and e olu ion o a s in a long expe imen al pe iod. The exp ession o c- os and Npy inc eased in animals ed he HFD on weeks 15 and 21 compa ed o he SD g oup, and dec eased as a esul o bodyweigh loss. Such dec ease was only obse able in Npy du ing he los - weigh main enance s age (Fig.1D). The adminis a ion o AM251 showed an inhibi o y ac ion on Npy and CB1 ecep o . Weigh ‑loss in e en ions cause a signi ican imp o emen in ae obic capaci y and glycemic p o ile. O e all, DIO caused a signi ican dec ease in ae obic capaci y and physical i ness pa ame e s (dis- ance and maximum speed) compa ed o he SD g oup a he end o week 21 o expe imen al pe iod (Fig.2A). The in e en ions assayed o bodyweigh con ol we e e icien a inc easing he abo e-men ioned pa ame e s s he HFD g oups and he combined e ec s o calo ic es ic ion and physical exe cise (WMe) we e o especial ele ance. Mo eo e , such e ec s we e po en ia ed by AM251 adminis a ion ha esul ed in he highes alues o all h ee pa ame e s in he WMeAM g oup (P < 0.05). Glycemic p o ile and AUC a e an o al glucose o e - load ca ied ou a he end o e e y expe imen al s age a e shown in Fig.2B,C. DIO esul ed in highe AUC, a highe pos p andial blood glucose peak a e 30min o adminis a ion, and highe alues o glycemia du ing a mo e p olonged pe iod a e glucose o e load, when compa ed o he SD g oup. These da a poin o a si ua ion o insulin esis ance, which was e e sed by he weigh -loss/main enance in e en ions assayed. Ca dio ascula unc ionali y is signi ican ly imp o ed by weigh ‑loss and emains s able du ‑ ing he main enance pe iod. The obesi y- ela ed inc ease in body weigh led o a hype ophied hea whe eas weigh -loss in e en ions dec eased hea weigh o simila alues as hose ound in he SD g oup in all expe imen al s ages (Table1). Simila ly, en icula elec oca diog aphic pa ame e s we e s ongly a ec ed in he HFD g oups ha showed a signi ican inc ease in he ampli udes o QRS complex and T wa e along he expe imen al pe iod. The inc eased ampli udes we e e e sed upon body weigh loss achie ed by he di e en in e en ions assayed, eaching alues ha we e equal o e en in e io o he SD g oup. Abou he QTc in e al, i was inc eased by obesi y and ended o dec ease by calo ic es ic ion and physical exe cise excep o g oup WMeAM on week 21. No ele an changes we e obse ed in any o he pa ame e s ela ed o a ial unc ionali y. Plasma ac i i y o CK-MB was inc eased in animals ha consumed he HFD on weeks 15 and 21 and dec eased by he di e en weigh -loss in e en ions in hese expe imen al s ages (Table2). Gene exp ession o ansc ip s ela ed o ascula adhesion and angiogenesis (Vcam, Sele, Veg a) we e highe in he ao a o HFD s SD con ols a e 21weeks o he expe imen al pe iod (Fig.3A). In con as , he di e en weigh -loss and main enance in e en ions caused a signi ican dec ease in exp ession le els o alues lowe han hose obse ed in SD. Such changes we e ma ched by signi ican imp o emen s in plasma a he ogenic index (Fig.3B). Likewise, gene exp ession o Nos2, ela ed o he in lamma o y s a us, was highe in he ao a o HFD- ed animals and his inc emen was e e sed by weigh con ol in e en ions ha also down- egula ed P gs2 exp ession. Obesi y‑induced al e a ions in enal unc ion and an ioxidan capaci y a e e e ed by he weigh ‑loss and main enance s a egies implemen ed. C ea inine clea ance a io was signi ican ly inc eased unde ou expe imen al condi ions by DIO and AM251 adminis a ion du ing he i s 2 s ages o he expe imen al pe iod (Table3). Also, he obesi y- ela ed inc ease was associa ed wi h incipien albuminu ia a 15weeks ha became signi ican a 21weeks (HFD s SD). Such was no he case wi h AM251 adminis a ion in which no albuminu ia was de ec ed. C ea inine clea ance a io also appea ed o be a ec ed by age o he animals and inc eased in SD a s a 21 s 12 o 15 weeks. In con as , calo ic es ic ion and physical exe cise ended o no malize all he abo e ma ke s o al e ed enal unc ionali y al hough esul s did no each s a is ical signi icance in all cases. Rega ding he u ina y olume and pa ame e s ela ed o kidney s one o ma ion, obesi y induc ion led o a conside able dec ease in u ina y olume and pH as well as inc eased phospha u ia a weeks 12 and 15, which un in pa allel o a dec eased calciu ia and inc eased calcemia (Table2). The me abolic s a us o bo h mine als ended o be no malized by he weigh con ol s a egies implemen ed. Renal unc ionali y was also a ec ed by 6 Vol:.(1234567890) Scien i ic Repo s | (2021) 11:4286 | h ps://doi.o g/10.1038/s41598-021-83709-9 www.na u e.com/scien i ic epo s/ age o he animals, and conside able changes in u ina y pH, phospha u ia, calciu ia, and enal clea ance we e e iden in he a s ed he SD die on week 21 compa ed o weeks 12 and 15. To assess whe he obesi y-induced changes in enal unc ionali y could be media ed h ough inc eased oxida- i e s ess, he kidney ac i i y o an ioxidan enzymes and lipid pe oxida ion we e assessed (Table4). Resul s a e complex and nume ous in e ac ions a e obse able among obesi y and weigh -loss in e en ions on he o me pa ame e s. Consump ion o HFD wo sened oxida i e s ess condi ions and esul ed in highe Mn-SOD and GPX ac i i ies in all he s ages o he expe imen al pe iod, whe eas Cu/Zn-SOD ac i i y exhibi ed an HFD-de i ed inc ease on week 15 and a educ ion on week 21. The e ec s o weigh con ol in e en ions di e ed based on hei implemen a ion du ing he 3weeks o weigh -loss o he 6weeks o weigh -main enance. Speci ically, on week 21 o expe imen al pe iod, he e was a signi ican dec ease in Mn-SOD ac i i y caused by he combined ac ion o exe cise and AM251 adminis a ion. Likewise, a ma ked dec ease was also ound in GPX ac i i y caused by he adminis a ion o AM251, alone o in combina ion wi h physical exe cise. Discussion The p esen s udy was ca ied ou o assess he changes p oduced by obesi y on some ca dio- enal unc ions and o demons a e he imp o emen in hese unc ions due o a dec ease in bodyweigh and main enance o los weigh . Calo ic es ic ion, physical exe cise, and blockade o CB1 ecep o s a egies we e es ed in an animal model o DIO. Unde ou expe imen al condi ions, obesi y was success ully es ablished (di e ence in body weigh be ween no mocalo ic and HFD- ed animals was equal o g ea e han 2 s anda d de ia ions) om he 5 h week o he Figu e2. E ec s o DIO and weigh con ol in e en ions on ae obic capaci y and glucose me abolism. (A) maximal oxygen consump ion, maximum speed, and dis ance un du ing an inc emen al es we e measu ed as ma ke s o long e m e ec s a e 21weeks o he expe imen al pe iod, (B,C) glycemic p o ile and a ea unde he cu e a e an o al glucose o e load measu ed a 12, 15 o 21weeks o he expe imen al pe iod. Resul s a e means o eigh a s ± SEM depic ed by e ical ba s. *P < 0.05 in - es (12weeks); A,B,C means wi h di e en le e s a e signi ican ly di e en (ANOVA ea men , P < 0.05; 15 and 21weeks). AUC a ea unde he cu e (a bi a y uni s), SD no mocalo ic s anda d die g oup, HFD HFD- ea ed g oup. Weigh -loss in e en ions (WL) on weeks 13–15: WLs obese a s ea ed wi h calo ic es ic ion and no exe cise, WLe obese a s ea ed wi h calo ic es ic ion in combina ion wi h physical exe cise, WLsAM obese a s ea ed wi h calo ic es ic ion in combina ion wi h AM251 adminis a ion and no exe cise, WLeAM obese a s ea ed wi h calo ic es ic ion in combina ion wi h physical exe cise and AM251 adminis a ion. Weigh -main enance in e en ions (WM) on weeks 16–21: WMs a s ea ed wi h die SD and no exe cise, WMe a s ea ed wi h die SD in combina ion wi h physical exe cise, WMsAM a s ea ed wi h die SD in combina ion wi h AM251 adminis a ion and no exe cise, WMeAM a s ea ed wi h die SD in combina ion wi h physical exe cise and AM251 adminis a ion. 7 Vol.:(0123456789) Scien i ic Repo s | (2021) 11:4286 | h ps://doi.o g/10.1038/s41598-021-83709-9 www.na u e.com/scien i ic epo s/ DIO pe iod by consuming an obesogenic die compa ed o he no mocalo ic g oups. O he models o DIO ha e been desc ibed18,19. Howe e , i should be highligh ed ha his die a y combina ion led o apid weigh gain in he expe imen al animals. Once obesi y was es ablished, he HFD die con inued o be adminis e ed along all he s ages o he expe imen al pe iod o clea ly show he ela ed al e a ions. A e wa d, du ing he 12–15-week in e en ion pe iod, di e en weigh -loss s a egies we e implemen ed ha esul ed in subsequen body weigh loss. Such dec ease can be a consequence o se e al in e ac ing ac o s like he high p o ein con en o he die due o i s he mogenic ac ion and i s high le els o sa ia ing soluble die a y ibe , he po en ial ano exigenic ac ion o physical exe cise, he inhibi o y ac ion on ood in ake o AM251 a he hypo halamus and lep in sensi i i y which is usually inhibi ed by he consump ion o a high- a die 20–23. In his con ex , adminis a ion o AM251 has been shown o display dose-dependen dec eases in ood in ake and weigh gain, specially a doses o 2–5mg/ kg24. In addi ion, he inhibi o y ac ion o AM251 can be po en ia ed by co-adminis a ion o lep in. Recen ly, a c oss alk be ween CB1 and GLP1 ecep o s has been desc ibed ha p o ides new he apies o obesi y25. The coadminis a ion o a pe iphe al CB1 ecep o inhibi o wi h long-ac ing GLP1R agonis s achie es g ea e educ- ion in body weigh and a mass han mono he apies by p omo ing nega i e ene gy balance. Du ing 15–21weeks, weigh -main enance s a egies we e combined e ealing ha he combina ion o he h ee in e en ions assayed: a ce ain deg ee o calo ic es ic ion, physical exe cise, and adminis a ion o CB1 ecep o blocke was he mos e icien o success ully main ain body weigh and a oid he ebound e ec com- mon o o he weigh -loss ea men s. In ac , du ing his las s age o he expe imen al pe iod, he mixed aining p o ocol and, especially, AM251 adminis a ion, con ibu ed o main ain body weigh ia down- egula ion o hypo halamic ansc ip s coding o Npy. Adminis a ion o AM251 has been epo ed o p oduce a signi ican dec ease in he numbe o neu ons exp essing o exin A in he hypo halamus26, whe eas O exin-A ep esses sa ie y-inducing POMC neu ons and con ibu es o obesi y ia s imula ion o endocannabinoid signaling27. On Table 1. E ec s o DIO and weigh con ol in e en ions on pa ame e s o hea unc ionali y. SD s anda d a chow die , HFD hype calo ic die o die a y induc ion o obesi y, WLs high p o ein weigh -loss in e en ion die wi h a seden a y li es yle (weeks 12–15), WLe high p o ein weigh -loss in e en ion die wi h aining p o ocol, WLsAM high p o ein weigh -loss in e en ion die wi h a seden a y li es yle and pha macological ea men wi h AM251, WLeAM high p o ein weigh -loss in e en ion die wi h aining p o ocol and pha macological ea men wi h AM251. WMs high p o ein weigh -loss in e en ion die wi h a seden a y li es yle (weeks 12–15) ollowed by weigh -main enance s age (weeks 15–21) wi h SD die a y ea men and seden a y li es yle, WMe high p o ein weigh -loss in e en ion die wi h aining p o ocol ollowed by weigh -main enance s age wi h SD die a y ea men and aining p o ocol, WMsAM high p o ein weigh -loss in e en ion die wi h a seden a y li es yle and pha macological ea men wi h AM251 ollowed by weigh - main enance s age wi h SD die a y ea men , seden a y li es yle, and pha macological ea men wi h AM251, WMeAM high p o ein weigh -loss in e en ion die wi h aining p o ocol and pha macological ea men wi h AM251 ollowed by weigh -main enance s age wi h SD die a y ea men , aining p o ocol, and pha macological ea men wi h AM251, Bpm bi s pe minu e, QTc co ec ed QT in e al. Resul s a e means o 8 a s. SEM s anda d e o o he mean. *P < 0.05 in - es (12weeks); a, b, c, means wi hin he same column wi h di e en le e s a e signi ican ly di e en (ANOVA ea men , P < 0.05; 15 and 21weeks). Bodyweigh (g) Hea weigh (g) Hea a e (bpm) P wa eleng h (s) P wa e ampli ude (mV) PR in e al (s) QRS leng h (s) QRS ampli ude (mV) T wa e leng h (s) T wa e ampli ude (mV) QTc in e al (s) 12weeks SD 516.9 1.53 293.7 0.019 0.105 0.027 0.019 1.35 0.023 0.184 0.098 HFD 648.7*** 1.65 290.0 0.020 0.134*0.025 0.020 1.69*0.027 0.208*0.106 SEM 19.7 0.045 7.3 0.002 0.010 0.002 0.003 0.075 0.001 0.035 0.056 15weeks SD 503.3b 1.41a 263.5a 0.029b 0.112a 0.059b 0.021a 1.15a 0.031a 0.220b 0.103a HFD 704.1a 1.96b 242.4a 0.022b 0.066a 0.055b 0.021a 1.76b 0.040c 0.308b 0.128b WLs 566.7c 1.54a 265.3a 0.024ab 0.117a 0.064b 0.021a 0.911a 0.042ab 0.143a 0.121ab WLe 552.5bc 1.50a 260.9a 0.020b 0.130a 0.030a 0.021a 1.15a 0.041bc 0.192a 0.125b WLsAM 544.2bc 1.55a 268.0a 0.025ab 0.119a 0.065b 0.020a 0.885a 0.064d 0.188a 0.171c WLeAM 511.5bc 1.45a 254.4a 0.019b 0.106a 0.059b 0.020a 1.12a 0.041bc 0.161a 0.123b SEM 21.2 0.09 7.8 0.002 0.041 0.003 0.001 0.105 0.002 0.015 0.006 21weeks SD 631.7a 1.69a 267.9a 0.021ab 0.089a 0.061ab 0.023ab 1.64b 0.042a 0.176a 0.134ab HFD 803.8b 2.02b 274.5a 0.023b 0.129b 0.063ab 0.021a 1.97c 0.049a 0.291b 0.151b WMs 634.2a 1.73a 263.7a 0.020ab 0.103ab 0.061ab 0.021a 1.09a 0.039a 0.203a 0.124a WMe 574.4a 1.64a 255.9a 0.020ab 0.127a 0.067b 0.027b 1.14a 0.039a 0.206a 0.135ab WMsAM 546.4a 1.72a 257.1a 0.022b 0.076a 0.057a 0.022ab 1.32a 0.045a 0.186a 0.134ab WMeAM 590.9a 1.64a 273.9a 0.017c 0.095b 0.054a 0.021a 1.28a 0.049a 0.211a 0.152b SEM 41.8 0.093 6.5 0.001 0.010 0.002 0.002 0.102 0.004 0.011 0.008 8 Vol:.(1234567890) Scien i ic Repo s | (2021) 11:4286 | h ps://doi.o g/10.1038/s41598-021-83709-9 www.na u e.com/scien i ic epo s/ he o he hand, Di Ma zo e al.28 epo ed ha de ec i e lep in signalling is associa ed wi h ele a ed hypo halamic le els o endocannabinoids in obese db/db and ob/ob mice and Zucke a s, whe eas acu e lep in ea men o no mal a s and ob/ob mice educes he endocannabinoids anandamide and 2-a achidonoyl glyce ol in he hypo halamus. DIO a e consump ion o HFD du ing 12weeks esul ed in he ins au a ion o insulin esis ance, as i is shown by he highe AUC a e an o al glucose o e load compa ed o SD g oups Ne e heless, his pa hological si ua ion was e e sed by weigh loss in e en ions and AUC e u ned o alues simila o hose o SD animals. O he s a egies including legume-de i ed unc ional ing edien s and/o physical exe cise29 ha e also con i med his bene icial ac ion. Los weigh main enance u he imp o ed he glycemic p o ile o he animals a e he combined ac ion o physical exe cise and AM251 adminis a ion, enhancing me abolic ac i i ies ha p omo e insulin sensi i i y. The CB1 ecep o is highly exp essed in cen al and pe iphe al ne ous sys em, as well as a ious pe iphe al issues30,31. E idence sugges s ha endocannabinoids a e di ec ly in ol ed in he con ol o ood in ake and ene gy u iliza ion by a ge ing hese cen al and pe iphe al si es, including skele al muscle, li e , and adipose issue32. The e ec s o AM251 on glucose me abolism can be explained in a simila way o o he CB1 ecep o in e se agonis s33 ia me abolic “pe iphe al” ac ion in adipose issue in addi ion o i s known “cen al” e ec on ood in ake, hus p o iding an e ec i e s a egy o imp o ing insulin sensi i i y. The obse ed bene icial ac ion o AM251 is also in ag eemen wi h he esul s o Esposi o e al.34, who concluded ha modula ion o CB1 ecep o egula ed up ake a he le el o he PI3K signaling sys em in skele al muscle cells. The e o e, in e e ing wi h CB1 signaling could amelio a e gluco egula o y unc ions in pe iphe al issues. In a simila way, C espillo e al.35 sugges ed ha blockade o CB1 ecep o could play an impo an ole in he es o a ion o comp omised me abolic s a us caused by high a in ake and imp o e ca diome abolic isk ac o s. Fu he mo e, in a ecen Table 2. E ec s o DIO and weigh con ol in e en ions on plasma pa ame e s o hea and kidney unc ionali y. SD s anda d a chow die , HFD hype calo ic die o die a y induc ion o obesi y, WLs high p o ein weigh -loss in e en ion die wi h a seden a y li es yle (weeks 12–15), WLe high p o ein weigh -loss in e en ion die wi h aining p o ocol, WLsAM high p o ein weigh -loss in e en ion die wi h a seden a y li es yle and pha macological ea men wi h AM251, WLeAM high p o ein weigh -loss in e en ion die wi h aining p o ocol and pha macological ea men wi h AM251. WMs high p o ein weigh -loss in e en ion die wi h a seden a y li es yle (weeks 12–15) ollowed by weigh -main enance s age (weeks 15–21) wi h SD die a y ea men and seden a y li es yle, WMe high p o ein weigh -loss in e en ion die wi h aining p o ocol ollowed by weigh -main enance s age wi h SD die a y ea men and aining p o ocol, WMsAM high p o ein weigh -loss in e en ion die wi h a seden a y li es yle and pha macological ea men wi h AM251 ollowed by weigh -main enance s age wi h SD die a y ea men , seden a y li es yle, and pha macological ea men wi h AM251, WMeAM high p o ein weigh -loss in e en ion die wi h aining p o ocol and pha macological ea men wi h AM251 ollowed by weigh -main enance s age wi h SD die a y ea men , aining p o ocol, and pha macological ea men wi h AM251. CK-MB c ea ine kinase MB. Resul s a e means o 8 a s. SEM, s anda d e o o he mean. *P < 0.05, ***P < 0.001 in - es (12weeks); a, b, c, means wi hin he same column wi h di e en le e s a e signi ican ly di e en (ANOVA ea men , P < 0.05; 15 and 21weeks). CK-MB (U/dL) ACE (U/dL) To al P o eins (g/dL) Albumin (g/dL) U ea (mg/dL) U ic Acid (mg/ dL) C ea inine (mg/ dL) Phospho us (mg/dL) Calcium (mg/ dL) 12weeks SD 395.6 69.1 5.87 2.66 28.0 0.95 0.57 6.08 6.41 HFD 292.4 81.8 6.17* 2.92 32.7* 0.69 0.11 5.95 9.80*** SEM 0.16 0.67 0.14 0.15 0.16 0.16 0.08 0.41 0.67 15weeks SD 358.9a 73.4a 6.22c 3.33b 28.2a 0.94ab 0.71bc 6.13b 6.33a HFD 987.0b 73.6a 6.51c 3.08ab 31.0a 0.90ab 0.15a 5.40ab 10.1b WLs 335.6a 57.5b 5.57a 2.88ab 28.0a 0.72a 0.49abc 4.93a 6.59a WLe 362.1a 77.3a 5.85ab 2.86ab 29.7a 0.92ab 0.81c 5.45ab 6.73a WLsAM 213.6a 72.3a 5.34a 2.61a 34.0a 1.02ab 0.40ab 5.41ab 10.8b WLeAM 196.1a 79.8a 5.42a 2.73ab 26.2a 1.19b 0.40ab 6.16b 11.0b SEM 154.0 9.42 0.18 0.21 3.10 0.15 0.13 0.30 0.86 21weeks SD 166.7a 65.0a 6.39a 2.92ab 32.3b 0.78a 0.054a 6.28a 8.68a HFD 571.7b 70.8b 6.07a 2.94ab 24.3ab 1.07a 0.046a 5.93a 9.51ab WMs 206.4a 85.7bc 6.24a 3.09ab 24.0a 1.29ab 0.47a 5.59a 7.86a WMe 207.6a 96.7d 6.09a 3.37b 26.6ab 0.86a 0.55a 5.99a 7.79a WMsAM 283.0a 72.5b 6.00a 2.60a 25.6ab 1.29ab 0.54a 5.85a 11.1b WMeAM 254.0a 75.4b 5.74a 3.09ab 40.6c 1.24ab 0.55a 5.63a 10.1ab SEM 307.4 11.8 0.23 0.22 2.70 0.24 0.67 0.57 0.99 9 Vol.:(0123456789) Scien i ic Repo s | (2021) 11:4286 | h ps://doi.o g/10.1038/s41598-021-83709-9 www.na u e.com/scien i ic epo s/ s udy, Eid e al.36 ha e es ed he e ec s o wo neu al CB1 ecep o an agonis s (cen al and pe iphe al) wi h imp o ed sa e y p o iles on a p eclinical model o insulin esis ance. Bo h compounds alle ia ed insulin esis ance pe iphe ally, and exe ed simila e ec s on a s wi h me abolic synd ome. They also displayed an i-dyslipidemic, an i-hype u icemic and an i-in lamma o y e ec s. Consump ion o a die ich in sa u a ed a s has been associa ed wi h changes in hea weigh unning in pa allel o he de elopmen o obesi y and insulin esis ance and may cause en icula modi ica ions, inc easing le en icle mass ha will, in u n, lead o dias olic and sys olic al e a ions and modi ied le en icle ejec- ion a io37. Those changes a e e lec ed in elec oca diog aphic modi ica ions like he inc eased ampli ude o QRS complex o QTc in e al ela ed o ca diac pa hology and en icula a hy hmia38. He e, HFD-induced al e a ions in ca diac unc ionali y appea ed o be mos ly ela ed o he deg ee o ca diac hype ophy and ook place mainly a he en icula le el, esul ing in he highe ampli ude o QRS complex and T-wa e as well as leng hening o he co ec ed QT in e al. Pa hological al e a ion o hese pa ame e s indica es dis u bances in he elec ical ac i i y o he hea and, consequen ly, on he e iciency o pumping blood. He e, such changes began o show a end a 12weeks and we e signi ican a 15weeks. We ha e no obse ed any changes in ECG pa ame e s ela ed o a ial depola iza ion and conduc ion (P wa eleng h, P wa e ampli ude, o PR in e al), ein o cing he idea ha obesi y a ec ed mainly en icula unc ion. Modi ica ions in ECG (QRS ampli ude and T wa e ampli ude) we e co ec ed a e he weigh loss in e en ions, al hough no addi ional e ec o ha o calo ic es ic ion was achie ed by exe cise, CB1 ecep o blockade, o he combina ion o bo h in e en- ions. Fu he mo e, no signi ican e ec o exe cise was ound on QTc in e al in a simila way o wha has been epo ed in he obese Zucke a model39. The imp o emen in pa ame e s o ca dio ascula heal h achie ed by Figu e3. E ec s o DIO and weigh con ol in e en ions on ascula damage measu ed in he ao a and plasma. (A) gene exp ession o ansc ip s coding o ascula adhesion molecules and in lamma ion ma ke s a he end o he 21-week expe imen al pe iod, (B) plasma a he ogenic index a he end o he di e en expe imen al s ages. Resul s a e means o eigh a s ± SEM depic ed by e ical ba s. *P < 0.05 in - es (12weeks); A,B,C means wi h di e en le e s a e signi ican ly di e en (ANOVA ea men , P < 0.05; 15 and 21weeks). Vcam ascula cell adhesion molecule 1, Veg a Vascula endo helial g ow h ac o A, Sele Selec in E, Nos2 Ni ic oxide syn hase 2, P gs2 P os aglandin-endope oxide syn hase 2. A he ogenic index, ( o al- choles e ol (mg/dL)/HDL-choles e ol (mg/dL). SD no mocalo ic s anda d die g oup, HFD HFD- ea ed g oup. Weigh loss in e en ions (WL) on weeks 13–15: WLs obese a s ea ed wi h calo ic es ic ion and no exe cise, WLe obese a s ea ed wi h calo ic es ic ion in combina ion wi h physical exe cise, WLsAM obese a s ea ed wi h calo ic es ic ion in combina ion wi h AM251 adminis a ion and no exe cise, WLeAM obese a s ea ed wi h calo ic es ic ion in combina ion wi h physical exe cise and AM251 adminis a ion. Weigh -main enance in e en ions (WM) on weeks 16–21: WMs a s ea ed wi h die SD and no exe cise, WMe a s ea ed wi h die SD in combina ion wi h physical exe cise, WMsAM a s ea ed wi h die SD in combina ion wi h AM251 adminis a ion and no exe cise, WMeAM a s ea ed wi h die SD in combina ion wi h physical exe cise and AM251 adminis a ion. 16 Vol:.(1234567890) Scien i ic Repo s | (2021) 11:4286 | h ps://doi.o g/10.1038/s41598-021-83709-9 www.na u e.com/scien i ic epo s/ 60. Coll-Risco, I. e al. E ec s o in e al ae obic aining combined wi h s eng h exe cise on body composi ion, glycaemic and lipid p o ile and ae obic capaci y o obese a s. J. Spo s. Sci. 34(15), 1452–1460 (2016). 61. Ma ínez, R. e al. E ec s o a combined in e en ion wi h a len il p o ein hyd olysa e and a mixed aining p o ocol on he lipid me abolism and hepa ic ma ke s o NAFLD in Zucke a s. Food. Func . 9(2), 830–850 (2018). 62. Wislø , U., Helge ud, J., Kemi, O. J. & Ellingsen, Ø. In ensi y-con olled eadmill unning in a s: VO2 max and ca diac hype - ophy. Am. J. Physiol. Hea . Ci c. Physiol. 280(3), H1301–H1310 (2001). 63. Clemen e, V. J. C., Ma ín, S., Po es, J., Fuen es, S. & Ramí ez, P. A. E ec o de la suplemen ación de i amina en el endimien o de una p ueba inc emen al de consumo máximo de oxígeno en a as Wis a . A ch. Med. Dep. XXVIII, 168–173 (2011). 64. P ie o, P. G., Cancelas, J., Villanue a-Peñaca illo, M. L., Val e de, I. & Malaisse, W. J. Plasma D-glucose, D- uc ose and insulin esponses a e o al adminis a ion o D-glucose, D- uc ose and suc ose o no mal a s. J. Am. Coll. Nu . 23, 414–419 (2004). 65. P uessne , J. C., Ki schbaum, C., Meinlschmid, G. & Hellhamme , D. H. Two o mulas o compu a ion o he a ea unde he cu e ep esen measu es o o al ho mone concen a ion e sus ime-dependen change. Psychoneu oendoc inology. 28(7), 916–931 (2003). 66. Po es, J. M. e al. The combined ea men wi h len il p o ein hyd olysa e and a mixed aining p o ocol is an e icien li es yle in e en ion o manage ca dio ascula and enal al e a ions in obese Zucke a s. Eu . J. Nu . 59, 3473–3490 (2020). 67. Li ak, K. J. & Schmi gen, T. D. Analysis o ela i e gene exp ession da a using eal- ime quan i a i e PCR and he 2(-Del a Del a C(T))-DDC Me hod. Me hods 25, 402–408 (2001). 68. Cohen, G., Kim, M. & Ogwu, V. A modi ied ca alase assay sui able o a pla e eade and o he analysis o b ain cell cul u es. J. Neu osci. Me hods. 67(1), 53–56 (1996). 69. Law ence, R. A., Sunde, R. A., Schwa z, G. L. & Hoeks a, W. G. Glu a hione pe oxidase ac i i y in a lens and o he issues in ela ion o die a y selenium in ake. Exp. Eye. Res. 18(6), 563–569 (1974). 70. Ukeda, H., Maeda, S., Ishii, T. & Sawamu a, M. Spec opho ome ic assay o supe oxide dismu ase based on e azolium sal 3’-{1-[(phenylamino)-ca bonyl]-3, 4- e azolium}bis(4-me hoxy-6-ni o) benzenesul onic acid hyd a e educ ion by xan hine- xan hine oxidase. Anal. Biochem. 251(2), 206–209 (1997). 71. B ad o d, M. M. A apid and sensi i e me hod o he quan i a ion o mic og am quan i ies o p o ein u ilizing he p inciple o p o ein-dye binding. Anal. Biochem. 72(1–2), 248–254 (1976). 72. Ohkawa, H., Ohishi, N. & Yagi, K. Assay o lipid pe oxides in animal issues by hioba bi u ic acid eac ion. Anal. Biochem. 95, 351–358 (1979). Acknowledgemen s The au ho s acknowledge he Spanish Minis y o Science, Inno a ion and Uni e si ies and he Eu opean Union h ough p ojec s DEP2014-58296-R, RTC-2017-6540-1, RTI2018-100934-B-I00, and FEDER p og am, espec- i ely. The unde s had no ole in s udy design, da a collec ion and analysis, decision o publish, o p epa a ion o he manusc ip . Au ho con ibu ions L.M.L.T.: Me hodology, o mal analysis, in es iga ion, w i ing-o iginal d a . R.M.: Me hodology, o mal analysis, in es iga ion, w i ing-o iginal d a , isualiza ion. G.K.: Me hodology, o mal analysis, in es iga ion. M.G.: Me h- odology, o mal analysis, in es iga ion, isualiza ion. P.A.: Concep ualiza ion, unding acquisi ion, esou ces, me hodology, isualiza ion. J.M.P.: Concep ualiza ion, supe ision, p ojec adminis a ion, esou ces, o mal analysis, in es iga ion, w i ing— e iew and edi ing. M.L.J.: Concep ualiza ion, alida ion, o mal analysis, supe - ision, w i ing— e iew and edi ing. 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