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Steviolio glikozidai pagerina kasos beta ląstelių funkciją ir skonio pojūčius, sustiprindami trpm5 kanalo aktyvumą | gamtos komunikacijos
 Steviolio glikozidai (SG), tokie kaip steviosidas ir rebaudiosidas A, yra natūralios, nekaloringos saldaus skonio organinės molekulės, esančios šveitimo augalo Stevia rebaudiana ekstraktuose, kurie plačiai naudojami kaip saldikliai vartojimo maistuose ir gėrimuose. TRPM5 yra Ca 2+ aktyvuotas katijonų kanalas, ekspresuojamas II tipo skonio receptorių ląstelėse ir kasos β ląstelėse. Čia parodyta, kad steviosidas, rebaudiosidas A ir jų aglikono steviolis sustiprina TRPM5 aktyvumą. Mes pastebime, kad SG sustiprina kartaus, saldaus ir umami skonio suvokimą ir sustiprina gliukozės sukeltą insulino sekreciją priklausomai nuo Trpm5. Kasdien vartojant steviosidą , laukinio tipo pelėms neleidžiama išsivystyti riebios dietos sukelta diabetinė hiperglikemija, bet ne Trpm5 - / - pelėms. Šie rezultatai išaiškina molekulinį SG veikimo mechanizmą ir identifikuoja TRPM5 kaip galimą taikinį 2 tipo diabeto prevencijai ir gydymui.
 Steviolio glikozidai (SG) randami šveituojančio augalo lapuose, augančiuose Pietų Amerikos subtropiniuose regionuose, Stevia rebaudiana Bertoni sp. Stevijos ekstraktas šimtmečius buvo naudojamas kaip saldinamasis maisto priedas Pietų Amerikoje 1, todėl neseniai buvo patvirtintas šiam tikslui ir Europos Sąjungoje bei JAV. Šios molekulės turi bendrą aglikono šerdies struktūrą steviolį ( ent -13-hidroksikaur-16-en-18-oo rūgštį) ir skiriasi savo glikozilinimo modeliu. Du pagrindiniai lapuose esantys glikozidai yra steviosidas ir rebaudiosidas A 2 . Abu skonis yra 200–300 kartų saldesnis nei sacharozė. Nekaloringi saldikliai vis dažniau naudojami įprastuose maisto produktuose, pavyzdžiui, soduose, grūduose ir desertuose, kaip cukraus pakaitalas. Dėl šios priežasties jie yra rekomenduojami mažinant svorį ir asmenims, kenčiantiems nuo gliukozės netoleravimo ir diabeto 3 . Tačiau stevijos ekstraktai gali turėti tiesioginį insulinotropinį ir antihiperglikeminį poveikį 2 tipo diabeto gyvūnų modeliuose4. Trūksta mechaninės įžvalgos apie Stevijos ekstrakto ir SG biologinį poveikį sveikatai.
 Skonio suvokimas II tipo skonio receptorių ląstelėse priklauso nuo pereinamojo receptorių potencialo katijonų kanalo pošeimio melastatino 5 nario (TRPM5), monovalentinio katijono kanalo, aktyvumo, kurį padidina tarpląstelinis kalcis 5 . Trumpai tariant, II tipo skonio receptorių ląstelės nustato liežuvyje saldžius, karčius ir umaminius skonius. Ląstelės specifiškumas pagal skonį užtikrinamas specifinio receptoriaus, sujungto su G-baltymu, ekspresija. Saldaus, kartaus ir umamio skonio receptoriai turi bendrą pasroviui perduodamą signalo perdavimo kaskadą, kurią sudaro fosfolipazės C aktyvinimas, Ca 2+ išsiskyrimas iš IP 3 jautrių Ca 2+ atsargų ir TRPM5 aktyvacija (nuoroda 6). TRPM5 aktyvumas ląstelę depoliarizuoja, o tai sukelia 1 įtampos kanalo kalcio homeostazės moduliatorių 1, kuris tarpininkauja ATP neurotransmiterio išsiskyrimui, kuris savo ruožtu suaktyvina aferentinį signalą į smegenis 7 . TRPM5 taip pat yra insuliną gaminančiose β ląstelėse kasoje ir periferinėse enteroendokrininėse ląstelėse 5, 8, 9, 10, 11, 12 . Neseniai parodėme, kad TRPM5 nustato gliukozės sukeltų kalcio virpesių dažnį insuliną atpalaiduojančiose kasos β ląstelėse10. Atsižvelgiant į TRPM5 raišką ir veikimą, sisteminis Trpm5 trūkumų turinčių pelių fenotipas apima saldaus, kartaus ir umami skonio reakcijos praradimą 13 ir sumažėjusį gliukozės toleranciją dėl sutrikusios gliukozės sukeltos insulino išsiskyrimo 10, 14 . Taip pat įdomu pastebėti, kad diabetu sergančių pacientų Trpm5 raiška plonojoje žarnoje yra neigiamai koreliuojama su gliukozės koncentracija kraujyje 15 . Panašiai WFS-1 diabetinės kasos salelės rodo sumažintą Trpm5 išraišką 16 . Be to, TRPM5 variantai žmonėms yra susiję su prediabetiniu fenotipu. TRPM5 vieno nukleotido polimorfizmai dažniau pasireiškė pacientams, kuriems sutrikusi insulino sekrecija, padidėjusi gliukozės koncentracija plazmoje, mažesnis į gliukagoną panašaus 1 peptido (GLP-1) lygis ir sumažėjęs jautrumas insulinui 17 . Specifiniai TRPM5 polimorfizmai parodė padidėjusį paplitimą pacientams, sergantiems su nutukimu susijusiu metaboliniu sindromu 18 . Remiantis šiais pastebėjimais, buvo iškelta hipotezė, kad TRPM5 gali būti farmakologinis taikinys naujo tipo insulino sekreciją skatinančio faktoriaus 19, 20, 21 sukūrimui . Tačiau kadangi šiuo metu TRPM5 farmakologija yra apribota gana selektyviai blokuojančiomis medžiagomis, ši hipotezė liko nepatikrinta 22, 23, 24, 25 .
 Dabartiniai mūsų rezultatai rodo, kad SG sustiprina TRPM5 jonų kanalo aktyvumą. Tokiu būdu jie sustiprina gliukozės sukeltą insulino sekreciją (GIIS) iš kasos salelių ir padidina periferinio skonio reakciją II tipo skonio receptorių ląstelėse. Lėtinis steviozido vartojimas nuo TRPM5 priklausomai nuo pelių efektyviai apsaugo nuo riebios dietos (HFD) sukelto diabeto.
 Steviolis ir jo glikozidai stiprina TRPM5
 Norint patikrinti, ar SG neturi įtakos TRPM5 tarpininkaujamo signalo perdavimo keliams, HEK293T ląstelėse buvo išmatuotos TRPM5 srovės, naudojant visos ląstelės patch-clamp metodą. TRPM5 srovės lengvai suaktyvinamos įsilaužus į pipetės tirpalą su 1 μM laisvojo Ca 2+ ir greitai inaktyvinamos keičiant laiko intervalą 25 . Vietoj to, kai inaktyvinimo fazėje ląstelės buvo perfuzuojamos su steviozidu, pastebėtas stiprus vidinės ir išorinės srovės padidėjimas, kuris išnyko išplovimo metu (1a – d pav.). Esant stipriam buferiniam [Ca 2+ ] int, steviosidas nesuaktyvina TRPM5 (1 pav. D). Panašūs rezultatai buvo gauti naudojant rebaudiosidą A, kitą SG, kuris skiriasi nuo steviozido vienu papildomu glikozilinimu (1e – h pav.). Taip pat mes ištyrėme steviolį, cheminę pagrindinę steviolio junginių grupę ir cirkuliuojančią metabolitą kraujo plazmoje išgėrus SGs 1 (1i pav.). Kaip parodyta 1j – l pav., Steviolis, palyginti su jo glikozilintais dariniais, davė panašius rezultatus, tai rodo, kad steviolio grupės pakanka SGs ir TRPM5 sąveikai.
 a ) Steviozido struktūra. b ) TRPM5 vidinių ir išorinių srovių, per daug ekspresuojančių HEK ląsteles, laiko trukmė. Norėdami suaktyvinti TRPM5, pipetės tirpale buvo 1 μM laisvo Ca 2+ . Laikas 0 rodo įsilaužimą į ląstelę. Steviosidas buvo naudojamas kaip nurodyta. c ) tipiniai srovės pėdsakai nuo b punkte nurodytų laiko momentų; d ) Kairė: vidutinė ± sem ( n = 13 ląstelių, suporuota t- test, P <0, 01) didžiausia vidinė ir išorinė srovės stipris prieš naudojant steviosidą ir jo metu su 1 μM tarpląsteliniu Ca 2+ . Dešinė: vidutinė ± sem į vidinę ir išorinę srovę prieš naudojant steviosidą ir jo metu, kai nėra laisvo viduląstelinio Ca 2+ ( n = 10 ląstelių, P > 0, 2, suporuotas t- testas). e ) Rebaudiosido A struktūra. f ) Laiko pėdsakas su vidinėmis ir išorinėmis srovėmis iš Trpm5, per daug ekspresuojančių HEK ląsteles. Rebaudiosidas A buvo naudojamas kaip nurodyta. g ) srovės pėdsakai, naudojant rebaudiosidą A, kaip nurodyta f punkte . h ) Vidutinė ± sem ( n = 14 ląstelių, suporuota t- test P <0, 05), nukreipta į vidinę ir išorinę srovę prieš rebaudiosido A taikymą ir jo metu. i ) Steviolio struktūra. ( j ) Laiko pėdsakas su vidinėmis ir išorinėmis Trpm5 srovėmis, per daug ekspresuojančiomis HEK ląsteles. Steviolis buvo naudojamas kaip nurodyta. k ) srovės pėdsakai, naudojant steviolį, kaip nurodyta j punkte . l ) Vidutinė ± sem ( n = 12 ląstelių, suporuotų t- testų P <0, 001) vidinė ir išorinė srovė prieš steviolio naudojimą ir jo metu. Taip pat žiūrėkite papildomus 1 ir 2 pav. A, rebaudiosidas A; Stev., Steviosidas; NS, nereikšminga.
 Ląstelėse be vidinių pleistrų TRPM5 gali būti suaktyvinamas tiesiogiai užtepant Ca 2+ į viduląstelinę membranos pusę 25 . Šiame nustatyme TRPM5 srovės greitai ir negrįžtamai išjungiamos 25 . Steviozidas ir steviolis uždelsia Ca 2+ aktyvuotų TRPM5 srovių inaktyvavimą, tiek naudojant vidinę, tiek tarpląstelinę membranos pusę (papildomas 1 pav.). Šie duomenys aiškiai rodo, kad steviolis tiesiogiai sąveikauja su TRPM5 baltymu.
 Mes anksčiau parodėme, kad TRPM5 silpnai priklauso nuo įtampos 25, 26 . Kaip parodyta papildomame 2a, b paveiksle, steviosidas sukelia nuo įtampos priklausančio TRPM5 aktyvavimo poslinkį link labiau neigiamos membranos potencialo. Įtampos pusė maksimalios aktyvacijos ( V 1/2 ) pasislinko nuo + 145, 9 ± 33, 9 iki + 42, 8 ± 30, 6 mV, esant steviosidui ( P = 5, 1 × 10 –4, n = 8, suporuotas t- testas, papildomas pav. 2c). Galiausiai steviosidas neturi įtakos neperkeltoms ląstelėms (papildomas 2d, e pav.), O steviosidas ir steviolis neturi įtakos ląstelėms, ekspresuojančioms TRPM4 ir kitus, labiau su nuotoliu susijusius, TRP kanalus (TRPA1, TRPV1, TRPM3 ir TRPM8; papildomas pav. 2f – h).
 Steviosidas padidina veikimo potencialą β ląstelėse
 Kasos β ląstelėse TRPM5 tarpininkauja depoliarizuojančioje srovėje, kuri sureguliuoja [Ca 2+ ] int ir V m svyravimo dažnį po gliukozės stimuliacijos 10 . Atsižvelgiant į tai, kasos β ląstelėse steviosidas sustiprina gliukozės sukeltą katijonų srovę. Ši srovė turi tam tikras TRPM5 savybes, įskaitant išoriškai ištaisomą I - V santykį ir atvirkštinį potencialą, esantį maždaug 0 mV (2a, b pav.). Mes išanalizavome steviosido poveikį aktyvinimo potencialo dažniui, kurį stimuliuoja didelė gliukozės (20 mM) arba tolbutamido (100 μM). Steviosidas padidino veikimo potencialo laukinio tipo (WT) β ląstelėse dažnį, kaip tai parodo sumažėjus ilgesnių intervalų tarp veikimo potencialų skaičiui (Wilcoxon sign-rank test, 2c pav., D, n = 5 ląstelės kiekvienoje) ). Priešingai, Trpm5 - / - β ląstelėse steviosidas smarkiai nepaveikė (tolbutamido sukeltos) veikimo potencialo (2e pav.).
 a ) Srovė, užfiksuota pirminėse WT β ląstelėse, kurią sukelia 20 mM gliukozės arba 20 mM gliukozės + 10 μM steviosidas (vidurkis ± sem; n = 5 ląstelės). ( b ) skirtumų pėdsakų srovė, paryškinanti steviozido dalį bendrosios srovės dalis. c ) Kairė: veikimo potencialai (AP), kuriuos WT β ląstelėse sukelia 20 mM gliukozės (viršuje) arba 20 mM gliukozės + 10 μM steviosido (apačia). Dešinė: tikimybės tankio funkcijos grafikas, vaizduojantis AP šaudymo dažnio savybių pasikeitimą. AP intervalas (-ai) parodo intervalo tarp AP trukmę. Tankis parodo intervalų tarp AP, esančių šiukšliadėžės trukmėje, dalį, normalizuotą atsižvelgiant į skirtingą šiukšliadėžės plotį. ( d ) Kaip c, bet su 100 μM tolbutamido, o ne 20 mM gliukozės. e ) AP, išprovokuojami Trpm5 - / - β ląstelėse, esant 100 μM tolbutamido arba 100 μM tolbutamido + 10 μM steviosido, kaip ir c ( n = 4 ląstelės). NS, nereikšminga.
 Steviolis ir steviosidas padidina kalcio aktyvumą salelėse
 Kai kasos salelės yra stimuliuojamos 10 mM gliukozės, jos reaguoja su [Ca 2+ ] int virpesiais (3 pav.). Steviosido vartojimas padidino gliukozės sukeltų Ca 2+ virpesių dažnį WT salelėse, bet ne Trpm5 - / - salelėse (nuo 3a – f pav.). Steviozidas daro šį efektą esant efektoriaus koncentracijai, kai pusės maksimalus atsakas yra 690 nM. Panašūs rezultatai buvo gauti naudojant steviolį (3h – j pav.). Atkreipkite dėmesį, kad steviosidas nesukėlė jokių pokyčių [Ca 2+ ], esant mažai gliukozės sąlygoms (3 mM; papildomas 3a pav.). Gliukozės sukeltų Ca 2+ virpesių dažnis yra stabilus iki 1 valandos, esant nuolatinei gliukozės koncentracijai (papildomas 3b, c pav.). Iš šių eksperimentų taip pat galima nustatyti, kad kasos salelių dydis, atspindintis β ląstelių masę, statistiškai nesiskiria nuo WT ir Trpm5 - / - pelių (3d pav., E, g).
 a ) Reprezentatyvus 10 mM gliukozės sukeltų Ca 2+ virpesių laiko raida kasos salelėse, išskirtose iš WT pelių, naudojant steviosidą, kaip nurodyta. ( b ) Tas pats kaip a punkte, bet iš Trpm5 - / - salelių. c ) WT vidutinis ± pus svyravimo dažnis ( P = 1, 6 × 10 –6, n = 71 salelė iš keturių pelių, suporuotas t- testas) ir Trpm5 - / - ( P = 0, 15, n = 68 salelės iš penkių pelių) salelių, kuriose yra tik 10 mM gliukozės arba 10 mM gliukozės, papildytos 10 μM steviosidu. Atkreipkite dėmesį į steviozido įtakos virpesių dažniui tarp WT ir Trpm5 - / - salelių skirtumą ( P = 2, 63 × 10 −4, dviejų mėginių t- testas). d ) WT kasos salelių plataus lauko vaizdas prieš matuojant fluorescenciją. e ) Trpm5 - / - salelių vaizdas prieš eksperimentą. f ) Skirtingų steviosido koncentracijų dozės ir atsako santykis stiprinant kalcio virpesių dažnį. Vidutiniai ± sem virpesiai per minutę, naudojant 0, 1 nM iki 100 μM steviosido 10 mM gliukozės (269 salelės iš devynių pelių). Juodoji linija parodo logistinį duomenų tinkamumą ir efektoriaus koncentraciją, kai atsakas yra maksimalus iki pusės (EC 50 ) = 690 nM. g ) WT ir Trpm5 - / - salelių salelių dydis ( n = 90 WT ir 102 Trpm5 - / - salelių). h ) Kalcio virpesių WT salelėse perfuzijant steviolį laikas. i ) Kalcio virpesiai salelėse iš Trpm5 - / - pelių perfuzijos būdu naudojant steviolį. j ) Vidutinis WT ir Trpm5 - / - salelių svyravimo dažnis ± 50 (WT: P = 5, 5 × 10 −15, suporuotas t- testas, n = 150 salelių iš keturių pelių; Trpm5 - / - : P = 0, 31, suporuotos) t- testas, n = 48 salelės iš dviejų pelių), esant 10 mM gliukozės arba 10 mM gliukozės, papildytos 10 μM steviolio. k ) Steviolio koncentracija pelių, veiktų 124 μM steviolio, koncentracijoje jų geriamajame vandenyje 3 savaites, kaip nustatyta efektyvios skysčių chromatografijos metodu. l ) WT salelės, veikiamos G10, ir fiziologinės 400 nM steviolio koncentracijos pavyzdys. m ) Kalcio virpesių dažnis WT salelėse žymiai sustiprėja vartojant 400 nM steviolio (porinis mėginio t- testas). Taip pat žiūrėkite papildomus 3 ir 4 paveikslus. NS, nereikšminga.
 Taikant Furjė analizę, steviolio ir steviosido poveikį WT salelėms galima aiškiai atskirti nuo poveikio Trpm5 - / - salelėms (papildomas 3g – k pav.). Furjė transformuoto (FT) dažnio spektras iš WT salelių pabrėžia dažnio skaičiaus poslinkį į aukštesnius dažnius, reaguojant į steviosidą, skirtingai nei Trpm5 - / - salelėse (Kolmogorovo – Smirnovo testas, P = 2, 96 × 10–4, papildoma fig.) .3g). Salų paskirstymas lėtai, greitai ar greitai besikeičiančiai svyruojančiai populiacijai (kaip aprašyta 10 nuorodoje) sąlygojo reikšmingą greitai svyruojančių salelių dalies padidėjimą esant steviosidui (Fišerio tikslus testas, P = 0, 009, Papildomas 3h pav.) Arba steviolis (papildomas 3i pav.). Pelėse Trpm5 - / - steviosidas ar steviolis nepakeitė greitai besisukančių salelių proporcijos (tikslus Fišerio testas, P = 0, 16, papildomas 3g pav., J, k). Šie radiniai rodo, kad SG padidina gliukozės sukeltų kalcio virpesių dažnį kasos salelėse priklausomai nuo TRPM5.
 Žmonėms ar pelėms nurijus, steviosidas žarnyne metabolizuojamas į jo aglikono steviolį. Patekęs į kraują, steviolis cirkuliuoja kraujo plazmoje ir kepenyse galutinai metabolizuojamas į steviolio gliukuronidą, kuris išsiskiria su šlapimu 27 . Norėdami padidinti fiziologinį mūsų rezultatų svarbą, mes išmatuojome laisvo steviolio koncentraciją, cirkuliuojančią pelių, kurios 2 savaites gėrė 100 mg l –1 (124 μM) steviosidą, plazmoje, plazmoje. Cirkuliacinis steviolio kiekis plazmoje šiems gyvūnams sudarė 391 ± 58, 6 nM (3k pav.) 28 . Taikant tokią fiziologinę steviolio koncentraciją, žymiai padidėjo gliukozės sukeltas kalcio virpesių dažnis, panašus į steviosido (3l pav., M).
 Anksčiau buvo teigiama, kad steviosidas aktyvina saldaus skonio receptorius TAS1R2, TAS1R3 (nuorodos 29, 30). Kadangi kasos β ląstelės funkciškai ekspresuoja šiuos receptorius 31, mes išbandėme steviosido poveikį saldžiųjų receptorių nokauto pelėms ( Tas1r2, Tas1r3 (- / -) 2 ). Kokybiškai panašūs rezultatai buvo gauti lyginant su WT salelėmis perfuzijos būdu su steviosidu ar stevioliu (papildomas 3d – f pav.), Tai yra, reikšmingai padidėjęs (+ 29%) gliukozės sukeltas Ca 2+ virpesių dažnis (papildomas 3f pav.) ), tokios kaip WT salelės (+ 31%, 3c pav.). Kaip ir WT salelėse, dominuojančių salelių iš Tas1r2, Tas1r3 (- / -) 2 pelių virpesių dažnis pasiskirstė sparčiau, esant steviosidui ( P = 1, 07 × 10 −8, papildomas 3g pav.). mėlyna). Salų padalijimas į lėtas, mišrias ar greitas saleles parodė, kad esant steviosidui ar stevioliui, greitai atsiranda arba padidėja populiacija (papildomas 3l pav., M).
 Anksčiau buvo įrodyta, kad TRPM5 daro įtaką GLP-1 tarpininkaujamo insulino sekrecijai 32 . Tačiau stebėtas steviosido poveikis nepriklauso nuo GLP-1 receptorių (GLP-1R) tarpininkavimo. Steviozidas daro papildomą potencinį poveikį GLP-1 sustiprinto kalcio virpesių dažniui (papildomas 4a, d pav.). Kaip tikėtasi iš Shigeto ir kt . 32 parodyta , kad gliukozės sukeltų kalcio virpesių GLP-1 potencija buvo žymiai mažesnė Trpm5 - / - salelėse, o papildomos steviosidų sukeliamos potenciacijos nepastebėta (papildomas 4b, e pav.). GLP-1R antagonistas eksendinas-3 (100 nM) slopino GLP-1 sukeltą gliukozės sukeltų kalcio virpesių padidėjimą WT pelėms, tačiau netrukdė steviozido sukeltai kalcio virpesių potenciacijai (papildomas 4c pav., F). ).
 Steviosidas stimuliuoja insulino sekreciją in vitro ir in vivo
 Atsižvelgdami į SG poveikį gliukozės sukeltiems Ca 2+ virpesiams ir pastebėję, kad Ca 2+ virpesių dažnis koreliuoja su insulino išsiskyrimu 10, mes siekėme ištirti šių junginių poveikį GIIS. Kaip parodyta 4a, b pav., Steviozidas sustiprino GIIS WT salelėse, bet ne Trpm5 - / - salelėse (WT: P = 0, 001, Trpm5 - / - : P = 0, 35; dviejų mėginių t- testas). Tiek WT, tiek Trpm5 - / - salelėse gydymas steviosidu neturėjo įtakos insulino išsiskyrimui esant mažai gliukozės (3 mM). Suprafiziologinių sočiųjų sąlygų (30 mM KCl arba 20 mM gliukozės) metu steviolio papildo poveikis insulino sekrecijai nebebuvo akivaizdus (1 papildoma lentelė). Taip pat in vivo , atlikus intraperitoninį (ip) gliukozės arba gliukozės ir steviosido injekciją pelėms, nevalgiusiems anestezijos, pelėms, insulino koncentracijai plazmoje 30 min. Po WT pelių yra žymiai didesnis. Šio efekto lygiai nebuvo ir Trpm5 - / - pelėms (4c pav.).
 a ) Insulino išsiskyrimas in vitro išmatuotas atskirose izoliuotose WT salelėse ir b ) Trpm5 - / - pelėse (vidutiniškai ± sem iš n = 24–30 salelių kiekvienoje būklėje iš keturių pelių genotipui, dviejų mėginių t- testas). Horizontali punktyrinė linija žymi (10 mM) GIIS lygį. c ) Steviosido vartojimo poveikis gliukozės sukeltam insulino išsiskyrimui iš aštuonių WT ir devynių Trpm5 - / - pelių plazmoje (vidutiniškai ± sem). d ) Insulino sekrecijos pėdsako pavyzdys atliekant perifuzinius eksperimentus be steviolio (juodo) arba papildžius stevioliu 10 minučių 3 mM gliukozės (G3), 10 mM gliukozės (G10) ir 100 μM diazoksido (Dz) ir 30 mM KCl ( K30). e ) bendra insulino sekrecija skirtingomis sąlygomis, palyginti su K30 dirgikliu. Pirmoji GIIS yra pradinė insulino smailė nuo 20 iki 30 min. C, o antroji fazė - nuo 30 iki 50 min. Reikšmingas skirtumas tarp kontrolinių ( n = 6 eksperimentų) ir steviolio sąlygų ( n = 6) iš 866 salelių iš 22 WT pelių, naudojant dvipusę dispersijos analizę. Taip pat žiūrėkite papildomą 5 pav. Ir 1 papildomą lentelę. NS, nereikšminga.
 Perifuzijos eksperimentuose mes išanalizavome steviolio poveikį GIIS dinamikai (4d pav., E; papildomas 5 pav.). Steviolio papildas nedaro įtakos mažai gliukozės. 10 mM GIIS rodo greitą piko fazę (1 fazė) ir tvaresnį insulino atpalaidavimo lygį (2 fazė). Insulino sekrecija abiejose fazėse padidėja nuolat vartojant steviolį (4d, e pav.). Sekrecija buvo normalizuota iki sekrecijos maksimaliai stimuliuojant K30, kuri nesiskyrė tarp kontrolinės ir steviolio perfuzijos sąlygų (kontrolė: 0, 12 ± 0, 06 palyginti su stevioliu: 0, 15 ± 0, 04 pg kiekvienoje salelėje per minutę, P = 0, 46; dviejų mėginių t- testas) ). Šis poveikis nebuvo pastebėtas Trpm5 - / - salelėse (papildomas 5a, b pav.). Be to, esant vidutiniškai didelei gliukozės koncentracijai (15 mM), papildant 10 μM steviolio, grįžtamai sustiprėjo GIIS (papildomas 5c, d pav.).
 Steviolis ir steviosidas sustiprina skonio suvokimą
 Norėdami nustatyti SGs modifikacijos TRPM5 svarbą in vivo , pirmiausia apsvarstėme skonio suvokimą. TRPM5 yra svarbus tarpląstelinio signalo perdavimo perdavimo kaskados komponentas, kurį II tipo skonio receptorių ląstelėse iššaukia saldūs, kartūs ir umamio skoniai. Dviejų butelių pirmenybės testas yra gerai žinomas metodas pelėms 33 nustatyti, ir jis gali būti derinamas su laižymo nustatymu (lick-o-metras). Mes panaudojome šį testą steviolio ir SG poveikio WT, Trpm5 - / - ir Tas1r2, Tas1r3 (- / -) 2 pelėms įvairiomis laiko skalėmis įvertinimui. Atsižvelgiant į tai, kad SG yra ≈ 300 saldesnių nei sacharozė (žmonėms), kiekvienam SG ir stevioliui panaudojome 124 μM (ekvivalentinės 300 kartų <1% sacharozės) koncentraciją. Esant tokiai koncentracijai, WT pelėms 48 val. Dviejų butelių pirmenybės bandyme (palyginti su vandeniu; 5a pav., Mėlynos juostos), palyginti su vandeniu, reikšmingas yra steviosido ir rebaudiosido A pasirinkimas, kurį galima priskirti prie saldaus SG skonio. Kita vertus, Steviol WT pelėms nesukelia ypatingo pasirinkimo ar vengimo (5a, b pav.). Tačiau derinant su saldžiu (1% sacharozės), umamio (150 mM monokalio glutamato (MKG)) ar kartaus (100 μM chinino) skoniu, steviolis žymiai padidina sacharozės ir umami pasirinkimą ir vengia chinino (5a pav. ). Svarbu tai, kad padidėjęs skonio pasirinkimas ar jo vengimas pastebimas per 5 minutes atliekant vienkartinio matuoklio bandymą (5b – g pav.) Ir išlieka po 60 minučių (papildomi 6 ir 7 pav.). Steviolis nedaro jokios įtakos vengiant 10 mM citrinos rūgšties tiek trumpalaikio, tiek ilgalaikio poveikio bandymuose. Trpm5 - / - pelėms trūksta signalizacijos kelio pasroviui nuo skonio receptorių. Tai atsispindi nesuteikiant pirmenybės ar vengiant saldžių, kartaus ar umamio skonių (5a pav., Oranžinės spalvos juostos; papildomos 6 ir 8 pav.). Panašiai, Trpm5 - / - pelėms SG nėra teikiama pirmenybė. Įtraukus steviolį į tiriamąjį tirpalą, taip pat negalima pastebėti padidėjusio periferinio skonio reakcijų, tačiau citrinos rūgšties vengimas neturėjo įtakos.
 Nurodytos lengvatos matuojamos saldaus (1% sacharozės), umami (150 mM monokalio glutamato - MKG), kartaus (100 μM chinino - 25 μM chinino, skirto Tas1r2, Tas1r3 (- / -) 2 a ) ar rūgštaus (10), atžvilgiu. mM citrinos rūgšties) su geriamuoju tirpalu papildomu 124 μM steviolio arba SG arba be jo. a ) Stulpelių grafikai rodo nurodyto junginio pirmenybę ( viršutinės juostos) arba vengimą (apatinę juostą) pasirinkus dviejų butelių bandymą per 48 valandas. WT gyvūnams akivaizdžiai teikiama pirmenybė steviosidui ( P = 2 × 10 –26, n = 48 pelėms) ir rebaudiosidui A ( P = 2 × 10 –21, n = 24) ir abejingumui stevioliui ( P = 0, 24, n = 24, vieno pavyzdžio t- testas, palyginti su 0, 5). Steviolis padidina saldžiųjų ( P = 0, 037), umami ( P = 0, 001) ir karčiųjų ( P = 0, 003, n = 24, suporuotas t- testas) tirpalo suvokimą WT gyvūnams, bet ne Trpm5 - / - pelėms. Tas1r2, Tas1r3 (- / -) 2 pelių karčią skonį sustiprina steviolis ( P = 0, 018) ir steviosidas ( P = 0, 048, n = 24, suporuotas t- testas). Nurodytos reikšmės rodo skirtumą tarp skonio junginio ir skonio junginio kartu su stevioliu. ( b ) Pradinis skonio pasirinkimas pateikiamas kaip normalizuotas kaupiamo laižymo skaičius, palyginti su vandeniu, be vandens (kairėje) arba su stevioliu (dešinėje) per 5 minutes po 23 valandų vandens, kai 14 ir 7 WT pelės buvo atimtos. c ) Kaip b su sacharoze, ( d ) MKG ir e ) chininas. ( b - e ) Išsami informacija iš papildomo 7a, b, e – j pav. f ) Pirmenybė iš b - e atvaizduojama kaip vidutinis ± pusė santykinio laiškų skaičiaus tarp pelių buteliuke, kuriame yra skonys be steviolio ar be jo. Dviejų mėginių t- testas (steviolis / MKG) arba porinis mėginių t- testas (sacharozė / chininas). g ) Individualios pasirinktys, pateiktos f punkte, nurodant pelių kitimą. Dėžutė pateikia 25–75% duomenų intervalą su vidurkiu, nurodytu kaip kvadratas, o mediana - kaip linija. Taip pat žiūrėkite papildomus 6–9 pav. Citr. A, citrinos rūgštis; NS, nereikšmingas; Chin., Chininas; Stev., Steviosidas; Sacr., Sacharozė.
 Norėdami patikrinti, ar steviosido ir steviolio skonį gerinantis poveikis priklauso nuo saldaus skonio receptorių, mes panaudojome Tas1r2, Tas1r3 (- / -) 2 peles. Reikėtų pažymėti, kad šios pelės taip pat neaptinka umamio skonių, nes TAS1R3 yra svarbus umami receptoriaus 13 subvienetas. Karčiųjų suvokimas išsaugomas šioje pelės eilutėje. Įdomu tai, kad tiek steviolis, tiek steviosidas padidino chinino vengimą Tas1r2, Tas1r3 (- / -) 2 pelėse (5a pav., Žalios juostos; papildomos 6 ir 9 pav.). Vengiant rūgštaus skonio, švirkščiant steviolį, šitoje pelių linijoje jis nekinta.
 Vartojant per burną steviosidą, sumažėja hiperglikemija
 Norėdami išanalizuoti steviosido poveikį gliukozės metabolizmui sąmoningose pelėse, mes atlikome du ip gliukozės tolerancijos testus (GTT) toms pačioms pelėms, kartu su gydymu steviosidu ar nešikliu, su 2 savaičių pertrauka. Gliukozės kiekis kraujyje reikšmingai sumažėjo po gydymo steviosidu , bet ne pelių Trpm5 - / - pelėmis (6a, b, g pav.). Šis poveikis buvo dar ryškesnis nutukusioms WT pelėms po 20 savaičių HFD ir tokio pat nebuvimo Trpm5 - / - gyvūnams, sergantiems HFD (6c, d, g pav.). Pažymėtina, kad tiek WT, tiek Trpm5 - / - gyvūnams nepastebėta jokio staigaus steviosido vartojimo poveikio nevalgius gliukozės kiekiui (papildomas 10a pav.). Net ir vartojant labai dideles dozes į veną (iv), steviosidas ir steviolis nesukelia hipoglikemijos (papildomas 10b, c pav.).
 ( a - f ) Crossover tandemo GTT, atlikto su tomis pačiomis pelėmis, rezultatai su 2 savaičių intervalu. Pelėms buvo skiriama arba 0, 5 mg g – 1 steviosido, įlašinto per os 2 valandas prieš GTT, arba nešiklio. Atskiri taškai rodo vidurkį ± sem, o reikšmės yra lygiagretaus pavyzdžių t -verčių, gautų tame pačiame taške, rezultatas; nereikšmingi skirtumai nenurodyti. ( a ) WT pelės normalios dietos metu, b ) Trpm5 - / - pelės normalios dietos metu, ( c ) WT pelės 20 savaičių HFD, ( d ) Trpm5 - / - pelės 20 savaičių HFD, ( e) ) WT pelės su WT salelėmis, persodintomis po inksto kapsulę, ir ( f ) WT pelės su Trpm5 - / - salelėmis, persodintomis po inksto kapsulę. g ) Vidutinis ± sem plotas po kreive (AUC) iš eksperimentų a – f . Suporuotas mėginių t- testas grupėse, dviejų mėginių t -testas tarp grupių. Taip pat žiūrėkite papildomus 10 ir 11 paveikslus. NS, nereikšminga.
 Norėdami ištirti, ar steviosido gliukozės kiekį mažinantis poveikis priklauso nuo TRPM5 ekspresijos kasos salelėse, mes transplantavome kasos saleles iš Trpm5 - / - pelių ar WT pelių po WT gavusių pelių inkstų kapsulėmis, kurių selektyviai buvo išeikvota iš endogeninės kasos β. - ląstelės, įpurškiant aloksaną 34 (papildomas 11a pav.). Pelių gavėjų hiperglikemija buvo patvirtinta prieš implantavimą (0 diena). Po transplantacijos normoglikemija buvo atkurta (papildomas 11c pav., E, 1 diena). Po pasveikimo pelės-akceptoriai parodė normalų glikemijos profilį ir kūno masę (6e pav., F, vientisos linijos; papildomas 11b – e pav.). Tik pelėms, persodintoms su WT salelėmis, glikemija sumažėjo po gydymo steviosidu (6e – g pav.). Šis poveikis buvo panašus į WT pelių poveikį.
 Steviosidas apsaugo nuo gliukozės netoleravimo pelėms, sergančioms HFD
 Norėdami ištirti lėtinio gydymo steviosidais poveikį dietos sukelto diabeto vystymuisi, atsitiktinai suskirstėme WT pakratų peles į dvi grupes: viena grupė, gavusi HFD (kontrolinė), o kita, gavusi HFD ir steviosidas. Jų amžius, svoris ir glikeminis profilis eksperimento pradžioje nesiskyrė (7 pav.; Papildomas 12 pav.; 2 papildoma lentelė). Vėlesni ip GTT parodė HFD kontrolinių pelių gliukozės netoleravimo vystymąsi nuo laiko (7a, c pav.). Po 20 savaičių, sergant HFD, gliukozės kiekis kraujyje per 2 valandas po gliukozės vartojimo buvo 300 ± 53 mg dl −1, o tai rodo 2 tipo diabetą 35 . Mes pastebėjome stiprų apsauginį steviosido poveikį nuo HFD sukeltos gliukozės netoleravimo WT pelėms (7b, c pav.). Palyginti su kontrolinėmis pelėmis, gyvūnams po 20 savaičių HFD + steviosido koncentracijos plazmoje buvo 220 ± 70 mg dl – 1 praėjus 2 valandoms po gliukozės vartojimo ( P = 0, 04, dviejų mėginių t- testas, palyginti su negydytais gyvūnais). Šios pelės palaikė normalų glikemijos profilį per 20 HFD savaičių (7b pav.). Trpm5 - / - gyvuliuose mes nepastebėjome skirtumų tarp kontrolinės ir steviosidais apdorotos grupės (7d-f pav.). WT ir Trpm5 - / - pelės, tiek kontrolinės, tiek steviosidais gydomos grupės, turėjo panašų kūno svorį ir savaitinę kalorijų normą (papildomas 12a, b, f, g pav.). Pažymėtina, kad WT pelėms, sergančioms HFD, nustatyta reikšmingai mažesnė ramybės būsenos glikemija, kai buvo gydoma steviosidu, o taip nebuvo Trpm5 - / - pelėms (papildomas 12c pav., H).
 ( a ) ip GTT, atliktų aštuonioms WT pelėms, skirtingais laiko momentais po HFD pradžios, kontrolės sąlygomis. Nurodytas reikšmingumas yra poros mėginio t- testo rezultatas nuo 0 iki 20 savaitės. Reikšmės yra vidutinės ± sem ( b ) Rezultatų, gautų iš GTT aštuonioms WT pelėms, esant HFD, gydomiems dienos doze 25 mg kg –1 steviosido. Nurodyta reikšmė yra poros mėginio t- testo rezultatas nuo 0 iki 20 savaitės. Reikšmės yra vidutinės ± sem ( c ) Plotas po kreive (AUC) nuo 0 iki 120 min., Atliekant GTT WT pelėms nuo a, b . Kietos ir punktyrinės linijos parodo atitinkamų duomenų rinkinių logistinį tinkamumą. Pateiktos vertės yra vidurkis ± sem iš aštuonių kiekvienos grupės gyvūnų, atliekant du nepriklausomus eksperimentus, reikšmės yra dviejų mėginių t- bandymo rezultatas tarp neapdorotų ir steviosidais apdorotų grupių tą pačią savaitę HFD metu. ( d - f ) Kaip ir a - c, bet iš Trpm5 - / - pelių. g ) Glikemija (vidutiniškai ± sem) per hiperinsulineminę euglikeminę spaustuką iš WT ir Trpm5 - / - gyvūnų. Steviolio infuzija nepakeitė glikemijos euglikeminės gliukozės infuzijos metu. h ) Gliukozės infuzijos greitis per hiperinsulineminę euglikeminę spaustuką. Tarp WT ir Trpm5 - / - gyvūnų reikšmingų skirtumų nenustatyta. i ) WT pelių glikemija, sergantiems HFD su steviosidu arba be jo (124 μM geriamajame vandenyje) euglikeminės hiperinsulineminės gnybto metu. Pelėms, gaunančioms steviosidą, bazinė glikemija yra mažesnė, dviejų mėginių t- testas tarp gydymo grupių vidutiniškai –20 ir –10 min. Glikemijos vertės. j ) Gliukozės infuzijos greitis atliekant euglikeminės hiperinsulineminės spaustuko eksperimentus. Gliukozės infuzijos greitis ir todėl atsparumas insulinui nesiskyrė dėl gydymo steviosidais HFD metu. Ūminė 0, 8 μmol kg −1 steviolio infuzija iv nepakeitė glikemijos perfuzijos metu su euglikeminės gliukozės infuzijos greičiu. Taip pat žiūrėkite papildomus 12 ir 13 paveikslus bei 2 papildomą lentelę. NS, nereikšminga.
 Padidėjęs gliukozės tolerancija steviosidais gydomiems WT gyvūnams neatsiranda dėl padidėjusio salelės dydžio (papildomas 12e pav.). Pažymėtina, kad pelių, gydomų steviosidais, salelėse nebuvo hipertrofijos, kuri akivaizdi WT HFD pelėms (palyginkite papildomą 12e pav. (HFD ir HFD + steviosidas) su 3g pav., Juoda (kontrolinė dieta)). Mes taip pat išbandėme šių pelių išskirtų kasos salelių funkcinius atsakus. Gliukozės sukeliami Ca 2+ virpesiai, išmatuoti salelėse iš steviosidais apdorotų WT pelių, parodė tą patį jautrumą steviosido panaudojimui, kaip aprašyta aukščiau negydytoms pelėms, tuo tarpu Trpm5 - / - pelių salelėse šio poveikio lygiai taip pat nebuvo (papildomas 12d pav. i). Svorio padidėjimas per 20 HFD savaičių iš esmės buvo vienodas tarp kontrolinių ir steviosidais gydytų pelių (papildomas 12a, f pav.). Norėdami ištirti tai išsamiau, 20 WT pelių buvo dedamos ant HFD be reguliaraus GTT ir su tuo susijusio badavimo, o tai sumažina kūno svorio svyravimus. Pradinis svoris (20, 7 ± 0, 2 g (kontrolinis) ir 20, 9 ± 0, 4 g (steviosidas); P = 0, 69, n = 10 pelių kiekvienoje grupėje, dviejų mėginių t- testas) ir svoris po 10 savaičių HFD (35, 9 ± 0, 9 g ( kontrolinis) ir 34, 3 ± 0, 8 g (steviozidas) P = 0, 20, dviejų mėginių t- testas) nesiskyrė.
 Periferinio audinio atsparumas insulinui yra svarbus 2 tipo diabeto aspektas36. Norėdami patikrinti atsparumą insulinui, mes atlikome insulino tolerancijos testą kontrolinėms WT pelėms ir WT pelėms, kurios 2 savaites vartojo 124 μM steviolio per geriamąjį vandenį (∼ 0, 5 mg per dieną vienai pelei). Glikemijos pokyčiai po insulino injekcijų abiejose grupėse nesiskyrė (papildomas 13a pav.). Pelių, lėtiniu būdu maitinamų HFD su steviosidu ar be jo geriamojo vandenyje, jautrumas insulinui buvo įvertintas atliekant euglikeminį hiperinsulineminį spaustuko testą. Trpm5 - / - gyvūnai, palyginti su WT gyvūnais, jautrumo insulinui skirtumo nesiskyrė (7g pav., H; papildomas 13b, c pav.). Gliukozės infuzijos greitis, norint pasiekti HFD turinčių gyvūnų euglikemiją, nesiskyrė nuo gyvūnų, kuriems buvo skirtas HFD + steviosidas, ir tai rodo, kad steviosido vartojimas nekeičia jautrumo insulinui (7 pav., J). Tačiau abi HFD grupės buvo reikšmingai atsparios insulinui, palyginti su normaliais WT gyvūnais (7i pav., J, palyginti su 7g pav., H; papildomas 13b, c pav.). Atkreipkite dėmesį į gliukoemijos skirtumą prieš infuzuodami gliukozę tarp steviosidu gydytų pelių ir kontrolinių pelių (7i pav.). Norėdami ištirti ūminį steviolio poveikį periferiniam jautrumui insulinui, euglikeminėmis sąlygomis infuzėme 0, 8 nmol g- 1 steviolio iv, išlaikydami pastovų gliukozės infuzijos greitį. Dėl to glikemija nepasikeitė (7g pav., I; papildomas 13c, d pav.).
 Norėdami patikrinti, ar steviosidas daro įtaką inkretino keliui, palyginome glikemijos profilį geriamojo ir ip GTT metu. Stebėjome panašų gliukozės lygio sumažėjimą steviosidais gydomoje grupėje geriamojo GTT ir ip GTT metu (papildomas 14a, b, e pav.). Inkretino efektas, išmatuotas kaip dviejų GTT ploto skirtumas kreivėje, buvo toks pats, lyginant kontrolinę ir su steviosidais apdorotą grupę (papildomas 14d pav., Kairėje, P = 0, 16, n = 10 pelių kiekvienoje grupėje). dviejų mėginių t- testas). Tai rodo, kad steviosidas neturi įtakos inkretino sekrecijai iš K ir L ląstelių žarnyne.
 Galiausiai žarnyno mikrobiomas yra svarbus medžiagų apykaitos ligų, tokių kaip diabetas, vystymosi veiksnys37. Mes ištyrėme šį poveikį gydydami peles plataus veikimo spektro antibiotikais (ABX) jų geriamajame vandenyje, o tai sukelia mikrobų kolonijų disbiozę žarnyne 38 . Po 6 savaičių ABX gydytų gyvūnų steviosido poveikis ip GTT buvo visiškai išsaugotas. Glikemijos reikšmių padidėjimas po gydymo ABX buvo toks pat steviosidais gydytoje grupėje kaip ir kontrolinėje grupėje (papildomas 14c, d, e pav.).
 Nutraukus steviosido vartojimą, sumažėja gliukozės tolerancija
 Mes išbandėme steviosido poveikio grįžtamąjį poveikį sisteminei gliukozės homeostazei. Mes įvertinome gliukozės toleranciją WT pelėms, gavusioms HFD, pagal tris scenarijus: nenutrūkstamą (15 savaičių) gydymą steviosidu (1 grupė), 10 savaičių HFD + steviosidu, po to 5 savaites HFD be steviosido (2 grupė) ir grupę, sergančią HFD. be steviozido 15 savaičių (3 kontrolinė grupė; 8a pav.; 3 papildomoji lentelė). Pelės į geriamąjį vandenį pateko 124 μM steviosido, kaip aprašyta aukščiau. Šis eksperimentas patvirtina, kad steviosidas pagerina gliukozės toleranciją HFD gydytoms pelėms (palyginkite 1 ir 3 grupes; 8b, d pav.). Ryškiausias rezultatas pastebimas 2 grupėje, kur gliukozės tolerancija pablogėja per 5 savaites po to, kai steviosidas pašalinamas iš dietos iki nevalytos grupės lygio. Be to, praėjus 5 savaitėms po gydymo steviosidu, 2 grupės ramybės glikemija yra žymiai didesnė, palyginti su lygiu, kai buvo skiriama steviosido (8c pav., E, f; 3 papildomoji lentelė). Galiausiai, atliekant šį eksperimentinį modelį, gydymas steviosidu šiek tiek sumažina HFD sukeltą svorio padidėjimą (8g pav., Palyginkite 1, 2 ir 3 grupes po 10 savaičių). 2 grupės pelėms buvo nustatytas HFD sukeltas kūno svorio padidėjimas nutraukus gydymą steviosidu, todėl eksperimento pabaigoje (po 15 savaičių) rezultatas buvo panašus kaip ir negydytos grupės svorio (8 g pav .; 3 papildoma lentelė).
 a ) Trijų pelių grupių dietos ir gydymo steviozidu eiga. GTT laiko taškai yra tokie, kokie nurodyti. ( b ) GTT rezultatai po 10 savaičių HFD (juodos - 17 savaičių amžiaus) ir dar po 5 savaičių (žalios - 22 savaičių amžiaus) pelių su HFD + steviosidu (100 mg l –1 geriamajame vandenyje). ( c ) Pelių GTT tuo pačiu metu kaip ir b punkte, gydymas steviosidu buvo nutrauktas sulaukus 17 savaičių. Individual points indicate the average±sem and the significances are the result of a paired sample t -test at the same time point from the two GTTs. ( d ) GTTs of mice in the untreated control group at the same time points. All values are averages±sem of n =8–10 mice. ( e ) Average±sem area under the curve (AUC) from the experiments in b – d, paired sample t -test, only significant differences are indicated. ( f ) The weekly average±sem resting glycaemia (spheres) and the average over 5 weeks (lines) of the mice in group 2 during (black) and after (green) the stevioside treatment. Two-sample t -test between the average glycaemia during and after stevioside treatment (lines). ( g ) Average±sem Body mass of the three groups at the age of 17 weeks (black) and 22 weeks (green) before the fasting for the GTT (two-sample t -test). See also Supplementary Fig. 14 and Supplementary Table 3. NS, not significant.
 Steviol, stevioside and rebaudioside A are not direct agonists of TRPM5, but potentiate Ca 2+ -dependent activity of the channel. The steviol group of SGs is sufficient for the interaction, but the interaction site on the TRPM5 protein remains unknown. Inside-out patch-clamp experiments indicate that steviol can interact with TRPM5 directly, independent of an intracellular signal transduction pathway. On the other hand, our data do not exclude other receptors for SGs, but, as outlined below, our data strongly suggest that TRPM5 is essential for the effect of SGs on taste sensation and glucose-induced insulin secretion.
 Previously, it was shown that TRPM5 is essential for sweet, bitter and umami taste detection, which are type II taste receptor cell-dependent taste modalities 6 . Stevioside and steviol specifically enhance these modalities in WT mice: avoidance of a bitter compound (quinine) and preference for sweet (sucrose) and umami (MKG). Sour taste (which is TRPM5-independent 6 ) is not affected. The effect of steviol and SGs on taste is divergent, in a sense that avoidance and preference are intensified depending on the tastant. The observation that taste preference is markedly affected as short as 5 min into the preference test, excludes a significant contribution of post-ingestive effects independent of acute tasting. Taken together, our data are consistent with the model that TRPM5-mediated depolarization of type II taste receptor cells serves as the link between taste receptor activation and initiation of afferent signalling via ATP release from these cells, and that SG-mediated potentiation of TRPM5 activity potentiates this signalling pathway (Fig. 9). Our data identify steviol as a novel, potent and specific modulator of bitter, sweet and umami taste, which is taste neutral on itself. It is tempting to speculate that the extreme sweetness of SGs originates likely from a dual interaction with the sweet taste receptor but also via potentiation of TRPM5 channel activity, via its steviol core.
 Important factors in the regulation of taste perception and insulin secretion, relevant for this work, are highlighted in this figure. The effects of stevioside through TRPM5 are highlighted with red arrows. In type II taste receptor cells, positive modulation of TRPM5 increases ATP release and afferent signalling from the receptor cell. In the pancreatic β-cell, upon glucose application β-cells display parallel V m and [Ca 2+ ] cyt oscillations, which drive insulin secretion. TRPM5 is active during the lag phase in between bursts of action potentials, and determines the frequency of V m and Ca 2+ oscillations, which modulates insulin secretion: enhanced TRPM5 activity results in a higher oscillation frequency, which results in more insulin secretion. Note that steviol and its derivatives (for example, stevioside) only enhance TRPM5 activity and insulin secretion, but that glucose is the trigger for cell signalling. Instead, sulfonylureas such as glibenclamide (not depicted) block K ATP channels directly and trigger Ca 2+ signalling and insulin secretion independent of glucose transport.
 Steviol and SGs augment glucose-induced Ca 2+ oscillations and insulin release in WT pancreatic islets, but not Trpm5 −/− islets. These data are consistent with the idea that TRPM5 serves as a facilitator of fast glucose-induced Ca 2+ oscillations, which trigger insulin release (Fig. 9). This model was originally proposed from our analysis of Trpm5 −/− mice 10, and SGs are the first compounds that can validate this hypothesis. It is worth noting that steviol and SGs yield a very similar effect as GLP-1 on glucose-induced Ca 2+ oscillations in islets. Interestingly, Shigeto et al . 32 could show that TRPM5 is directly involved in the signal transduction in the pancreatic β-cell elicited by GLP-1. This further supports the similar action of TRPM5 activators and GLP-1R agonists. Our data exclude that the GLP-1R is involved in the functional effect of steviol and SGs on pancreatic islets, as the effect of stevioside and GLP-1 is additive, and the stevioside effect is not affected by a GLP-1R blocker.
 The dosage of stevioside used in the drinking water of mice in both our drinking test and during the HFD experiments was 124 μM or 100 mg l −1 . This concentration is half of the maximum allowed dosage of SGs in soft drinks allowed in the European Union, and <25% of the suggested maximal dose by the World Health Organization and Food and Agriculture Organization 39, 40 . Consumption of this dosage of stevioside in the drinking water resulted in a plasma concentration of 396 nM steviol in mice. We show that also at these physiological concentrations steviol has clear effect on glucose-induced Ca 2+ signalling in pancreatic islets, indicating that the dosages of stevioside that we use are realistic and physiologically relevant.
 It has been suggested that SGs interact with the mammalian sweet taste receptor, TAS1R2/TAS1R3 (refs 29, 30) and ATP-sensitive K + channels in pancreatic β-cells 41, which might explain some of the data we observe. However, our results exclude that the potentiating effect of steviol and stevioside on pancreatic islet function is mediated via the TAS1R2/TAS1R3 receptor. We observed essentially the same effect of steviol and stevioside on Ca 2+ signalling in Tas1r2, Tas1r3 (−/−)2 , as in WT islets. Our observation that stevioside does not induce Ca 2+ signals and insulin release in pancreatic islets in low-glucose conditions (3 mM) argues against the idea that stevioside would be a direct blocker of ATP-sensitive K + channels.
 Acute oral administration of stevioside reduces hyperglycaemia in a GTT in WT, but not in Trpm5 −/− mice. Importantly, the same effect is present in alloxan-diabetic WT-recipient mice that received WT islets, but not in WT-recipient mice that received Trpm5 −/− islets. This shows that the glucose-lowering effect of stevioside is dependent on the expression of TRPM5 in pancreatic islets, and not from a TRPM5-dependent action in peripheral tissue outside of the pancreatic islet.
 Long-term oral intake of stevioside improves the development of plasma hyperglycaemia in WT mice on HFD, but not in Trpm5 −/− mice. There was no difference in the development of insulin resistance in the control and stevioside-treated group. HFD-induced obesity in mice is commonly used for the development of insulin resistance and IGT. Because obesity is induced via food-intake/composition and not by genetic manipulation, this model is considered to be relevant for the human condition. Interestingly, two studies suggest that Trpm5 −/− mice are resistant to diet-induced obesity; in Larsson et al . 42 on a HFD and in Glendinning et al . 43 on a carbohydrate-based diet. However, in both studies, Trpm5 −/− mice had a significantly lower caloric intake, which could explain the apparent resistance to obesity. In our study, WT and Trpm5 −/− mice had the same caloric intake and gained the same amount of weight on HFD.
 Similar as GLP-1, stevioside treatment is weight neutral or even slightly limits weight gain during HFD. Furthermore, stevioside and steviol do not induce hypoglycaemia. Stevioside enhances the physiological profile of insulin secretion, instead of inducing increased plasma insulin levels independent of plasma-glucose concentrations, as sulfonylureas and exogenous insulin. In this respect, we propose that TRPM5-potentiating compounds might be functionally equivalent to GLP-1 mimetics. GLP-1R agonists are highly successful in diabetes care. The drawback of current GLP-1R agonists is the way of administration, as they are often injectables, the high price and the short time of action 44 . Although advances are being made in improving GLP-1R agonists, the identification of TRPM5 as a new pharmacological target with an analogous mode of action broadens the possibilities for drug development.
 HEK293T cells were obtained from American Type Culture Collection and split twice weekly. Cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 2 U ml −l penicillin and 2 mg ml −1 streptomycin, and 2 mM L -glutamine. Cells were transfected with Trpm3 , Trpm8 , Trpa1 or Trpv1 using Mirius transIT-293 transfection reagent.
 All animal experiments were approved by the ethical committee for animal welfare of the KU Leuven. Sample size was determined using a power analysis based on the results of preliminary experiments. Mouse strains were homozygously bred in a conventional animal facility. We used C57Bl6/J (WT) and B6;129- Trpm5 tm1Csz /J ( Trpm5 −/−, obtained from C. Zuker, NY, USA) animals. The Trpm5 − allele was backcrossed for 12 generations in C57Bl6/J background. Sweet receptor knockout ( Tas1r2, Tas1r3 (−/−)2 ) mice were derived by crossing B6;129- Tas1r2 tm1Csz /J and B6;129 -Tas1r3 tm1Csz /J mice (obtained from Jax Labs).
 Islets for Ca 2+ imaging were isolated from male and female mice aged 10–18 weeks, or after the diet study. Action potential recordings, static insulin secretion, insulin secretion under dynamic perfusion and in vivo insulin secretion (Fig. 4; Supplementary Fig. 5) was assessed in age-matched male mice. For the insulin secretion experiments in supraphysiological conditions (Supplementary Table 1), we used age-matched female mice. Drinking and licking experiments were performed on male mice aged 10–20 weeks. Donor pups for the transplantation experiments were used before visual sexing is possible and were whole-litter mixed gender. Receptor animals were 6-week-old littermate WT mice. Diet experiments were started with 7-week-old male mice. Male, 8-12 week old, WT or Trpm5 −/− mice or mice after the diet test were used for the euglycaemic clamp or insulin tolerance test.
 HEK293T cells stably transfected with Trpm5 or Trpm4 were developed using the Flp-In system (Life Technologies). The cells were seeded onto poly- L -lysine-coated coverslips and incubated for 2 h. The coverslips were mounted onto an inverted microscope equipped with a multichannel perfusion system. We used the whole-cell patch-clamp configuration. A ramp protocol from −125 to +125 mV in 300 ms, from V hold =0 mV, was applied to the cells at 1 Hz. The current at +100 and −100 mV was extracted to analyse time-dependent current changes. We measured steady-state control conditions and peak currents during the application of stevioside, rebaudioside A or steviol. Voltage dependency of TRPM5 was analysed with a voltage-step protocol, from V hold =+25 mV, consisting of a 75 ms test-pulse from −175 to +150 mV in 25 mV increments. The membrane potential was kept at −125 mV for 50 ms before returning to holding potential. The analysed current is the steady-state current at the end of the test-pulse. This was fitted with the Boltzmann equation and V 1/2 was calculated in the presence of stevioside and in control conditions, of the same cells. The standard external solution contained (mM) 150 NaCl, 6 KCl, 1.5 CaCl 2, 1 MgCl 2, 10 glucose and 10 HEPES titrated with 1 M NaOH to a pH of 7.4. The appropriate amount of stevioside was added to this solution. Our internal solution contained (mM) 50 NaCl, 100 N -methyl- D -glucamine, 10 HEPES, 4 K 2 ATP, 10 ethylene glycol tetra-acetic acid and 8.62 CaCl 2 (resulting in 1 μM of free Ca 2+ ), titrated to pH 7.2 with 1 M HCl. The internal solution for measuring in the absence of intracellular calcium contained (mM) 50 NaCl, 100 N -methyl- D -glucamine, 10 HEPES, 4 K 2 ATP and 10 1, 2-Bis(2-aminophenoxy)-ethane- N , N , N ′, N ′-tetra-acetic acid, titrated to pH 7.2 with 1 M HCl.
 For inside-out patch recordings, the bath and pipette solution contains in (mM) 156 NaCl, 1.5 CaCl 2, 1 MgCl 2 and 10 HEPES at pH 7.4, supplemented with 10 μM steviol or 10 μM stevioside in the pipette as indicated. After excision, the patch is kept in a calcium-free bath solution containing (mM) 150 NaCl, 1 MgCl 2, 10 HEPES and 10 ethylene glycol tetra-acetic acid at pH 7.3. The calcium-containing solution was (mM) 150 NaCl, 1 MgCl 2, 10 HEPES and 0.5 CaCl 2 at pH 7.3 supplemented with 10 μM steviol in the indicated condition. The stimulation protocol consists of a 500 ms step at −100 mV immediately followed by a 250 ms step to +100 mV at 1 Hz. The analysed currents are the steady-state currents extracted at the end of the voltage step.
 Membrane potential recordings in β-cells were performed using the perforated-patch whole-cell configuration. The pipette solution contained (mM) 120 K-gluconate, 10 KCl, 10 NaCl, 1 MgCl 2, 5 HEPES (pH 7.2 with KOH) and 0.24 mg ml −l amphotericin B, while the bath solution contained (mM) 140 NaCl, 3.6 KCl, 0.5 MgSO 4, 1.5 CaCl 2 and 10 HEPES (pH 7.2 with NaOH). Action potential firing in isolated β-cells was induced by 100 μM tolbutamide or 20 mM glucose and recorded in the presence, or the absence, of 10 μM stevioside. The interval between subsequent action potentials was calculated and distribution was compared using a non-parametric Wilcoxon sign-rank test in Matlab (R2012a). To examine voltage dependency of the glucose-induced current, cells were held at −70 mV and stepped for 200 ms to voltages ranging from −80 to +50 mV.
 Cells were incubated with 1 μM FURA2-AM for 1 h and mounted on a fluorescence microscope equipped with a multichannel perfusion system. Transfected cells were identified by green fluorescent protein expression. FURA2 fluorescence was followed at 1 Hz sampling frequency. Extracellular solutions as indicated above were used. We applied 10 μM stevioside, 10 μM steviol and used specific activators of the channels of interest as positive control, that is, 2 μM capsaicin for TRPV1, 100 μM allyl isothiocyanate for TRPA1, 500 μM (+)menthol for TRPM8 and 20 μM pregnenolone sulphate for TRPM3.
 The Krebs solution for preparation contained (in mM) 119 NaCl, 4.75 KCl, 1.2 MgSO 4, 1.18 KH 2 PO 4, 2.54 CaCl 2, 5 NaHCO 3, 20 HEPES and 5 glucose (pH 7.4). Collagenase P (2 mg ml −l ) was injected into the pancreatic duct of C57Bl6/J WT, Trpm5 −/− or Tas1r2, Tas1r3 (−/−)2 mice, followed by dissection of the pancreas. Digestion of the whole pancreas was obtained by shaking for 8 min at 37 °C. After gravitational sedimentation, islets were isolated by three rounds of handpicking. Islets were cultured in advanced RPMI 1640 (Gibco) supplemented with 10% FCS, 100 U ml −1 penicillin/streptomycin and 4 mM glutamax at 37 °C and 5% CO 2 . Islets were dispersed into single cells by trypsin digestion. The cells were cultured at 37 °C and 5% CO 2 in RPMI 1640 medium supplemented with 10% FCS, 10 U ml −1 penicillin/streptomycin, and 10 mM glucose, for 24 h prior to experimentation.
 Pancreatic islets were incubated with 1 μM FURA2-AM for 1 h in a humidified incubator at 37 °C and 5% CO 2 . They were mounted on an Olympus BX51W1 inverted fluorescence microscope equipped with an UMPLANFL × 10 objective with a Bioptech objective heater set at 37 °C. We used a Warner Instruments CL-100 temperature controlled multichannel perfusion system, kept at 37 °C. Excitation was done with a Polychrome V lichtsource and image acquisition with an Andor iXon 888 camera, pre-amplifier gain was set to 1, EM gain to 160 with the TILL Photonics Live Acquisition 2.3.0.18 software. We measured the fluorescence of whole pancreatic islets. The islets were perfused with a solution containing (mM) 120 NaCl, 4.8 KCl, 1.2 MgCl 2, 2.5 CaCl 2 and 10 HEPES (pH 7.4) supplemented with the indicated amount of glucose and stevioside or steviol. Depolarization of the islets with 30 mM KCl was used as positive control at the end of the measurement. Ca 2+ oscillation frequency before or during the application of stevioside was analysed. The FT frequency spectrum of the first time derivative of the calcium oscillations was used in further analysis. The relevant oscillation frequencies between 0 and 0.05 Hz were determined. The highest peak represents the dominant oscillation frequency and all frequencies peaking at ≥60% of the maximum were considered relevant frequencies. The dominant oscillation frequency distribution during perfusion with 10 mM glucose was compared with the dominant oscillation frequency during perfusion with 10 mM glucose and 10 μM stevioside or steviol with the Kolmogorov–Smirnov test. On the basis of the oscillation frequency, the islets were divided into fast- (relevant frequencies only >0.015 Hz), slow- (relevant frequencies only <0.015 Hz) or mixed- (relevant frequencies in both parts of the FT spectrum) oscillating islets. The proportion of fast-oscillating islets in control and stevioside conditions was compared using Fisher's exact test.
 After isolation and overnight incubation, pancreatic islets from six mice were pooled and transferred from the culture medium to 3 mM glucose containing Krebs solution for 30 min. Individual islets were transferred to a well in a 96-well plate with the appropriate glucose and stevioside concentration 45 . After 60 min of incubation at 37 °C, the supernatant was collected. The experiment was repeated twice, and insulin concentration was determined on duplicate samples (Fig. 4a, b).
 For in vivo insulin release experiment, fasted WT or Trpm5 −/− mice were anaesthetised through an ip injection with 5 μl g –1 VDF (ventranquil:dormicum:fentanyl:water 1:2:10:3). The mice received 2.5 g kg –1 glucose (30% solution in H 2 O) or 2.5 g kg –1 glucose with 200 mg kg –1 stevioside at time point 0 min. Blood samples were taken at 0 and 30 min. Blood was sampled by filling heparinized capillaries through tail bleeding. The blood was centrifuged for 12 min at 5, 000 rpm and 4 °C, and the plasma was collected for further analysis.
 In all cases, the insulin concentration was determined using the Crystal Chem Inc, USA, ultra-sensitive mouse insulin ELISA kit, as per the manufacturer's protocol.
 After overnight culture in RPMI 1640 containing 10% heat-inactivated fetal calf serum, batches of 40–200 islets were perifused at 37 °C at a flow rate of 1 ml min −1 . The perifusion medium (pH 7.4) used for these experiments contained (in mM): 124 NaCl, 4.8 KCl, 2.5 CaCl 2, 1.2 MgCl 2, 20 NaHCO 3, 1 mg ml −l bovine serum albumin and various test agents as indicated. It was gassed with O 2 :CO 2 (95:5%). Effluent was collected every 2 min and insulin was measured by radioimmunoassay.
 Only male mice were used in the diet experiments. After weaning, the mice were randomly allocated to a test group per genotype and housed individual. At the age of 7 weeks, the diet was switched from normal chow (ssniff spezialdiäten, V1535-0) to pellets containing 30% ( w/w) saturated fat (ssniff spezialdiäten, E15116-34), given ad libitum. There was a weekly measuring of the body mass and the blood glucose. This resting glycaemia was taken during the light cycle of the mice with a standard glucometer by a drop of blood after tail puncturing in conscious unrestrained mice. The consumed food was measured by weighting the on the grid pellets every week. There was free access to drinking water and the supplementary drinking bottle from the indicated groups was filled daily with a stevioside containing solution (25 mg stevioside per kg in a 0.1% solution in water). The mice were monitored to consequently drink the stevioside solution. In the experiments decribed in Fig. 8 and Supplementary Fig. 14, five mice were housed in one cage. Stevioside (100 mg l −1 ) and antibiotics (ABX-1 g l −1 metronidazole and 200 mg l −1 ciprofloxacin) were added to the drinking water of the indicated groups. No animals were excluded from the analysis, outliers in resting glycaemia or food consumption at a distinct time point were attributed to experimental error and discarded from final analysis.
 The mice were fasted overnight in their home cage before the GTT with free access to water. Mice from different treatment groups were evenly distributed over technical replicates of the GTT. The investigator was blinded towards the treatment group the animal was allocated to. For the acute stevioside administration, the mice received either 500 mg kg −1 stevioside or the same volume of vehicle via oral gavage 2 h before the start of the GTT. At time 0 min, they receive an ip injection of 2.5 g kg −1 glucose in a 30% solution in water. In the case of the oral GTT, the glucose is administered through oral gavage. Glycaemia is followed at time points 0, 15, 30, 60 and 120 min using a standard glucometer (Aviva Accu-Check). The tip of the tail was punctured with a 26 G needle to initiate bleeding. After the GTT, the mice received ad libitum feeding of their appropriate diet. At individual time points, we show average±sem and the indicated significances are the result of a paired or a two-sample t -test. We excluded animals were the maximal glycaemia did not exceed 250 mg dl −1 from analysis. The results of the GTT are also presented as area under the curve of the glycaemia during the 120 min of the GTT.
 Mice were fasted overnight before the experiment. To obtain anaesthesia, mice under 40 g were ip injected with a mixture of 3.125 mg kg −1 acepromazine, 3.125 mg kg −1 midazolam and 0.156 mg kg −1 fentanyl while mice over 40 g received half this dose. A maintenance bolus of 18.75 μg acepromazine, 18.75 μg midazolam and 0.936 μg fentanyl was subcutaneous injected every 45 min. An iv catheter was placed in the tail vein and initially PBS was infused at 50 μl h −1 for 40 min using syringe pumps and the basal glycaemia was determined as the average measured at −20 and −10 min, before the start of the hyperinsulinemic clamp. The mice received a bolus of 2.7 mU insulin at 0 min and continuous infusion of 6.8 mU insulin per hour. In all, 12.5% glucose in PBS was iv infused at a variable rate to maintain euglycaemia, determined every 5–10 min by tail bleeding. After 2 h, we infused 0.8 μmol kg −1 steviol while maintaining the insulin and glucose infusions at the euglycaemic level.
 Islets from 2–3-week-old WT or Trpm5 −/− donor mice were isolated by collagenase digestion, washed and counted. Thereafter, islets were transferred to silicon microtubing (Becton Dickinson, Erembodegem, Belgium), and centrifuged for 5 min at 1, 500 rpm to create a compact pellet of islets for injection 46 . Endogenous β-cells from 7-week-old WT-recipient mice were selectively destroyed with 90 mg kg −1 alloxan 2 days before transplantation. Severe hyperglycaemia and glucosuria were confirmed in the recipient mice shortly before transplantation. During transplantation, the mice were anaesthetized with ketamine/xylazine ip (80–100 mg kg −1 ketamine and 5–10 mg kg −1 xylazine) and the left kidney was exposed by a lumbar incision. Recipient mice were given 500 islets under the kidney capsule. Non-fasting blood glucose levels from the tail vein of each recipient were measured to monitor functionality of the transplanted islets, 1 day, 1 week and 4 months after transplantation. Mice recovered for 14 days after surgery before a GTT was performed.
 Ten WT mice were given 124 μM steviol in their drinking water during 21 days. Blood was collected after anaesthesia by exsanguination through puncture of the maxillary vein. The plasma was separated by 10 min centrifugation at 10, 000 rpm A liquid–liquid extraction was employed as follows: to 50 μl of plasma, 1 μl (1 mg ml −1 in MeOH) ibuprofen was added as an internal standard. The mixture was vortexed and protein precipitation was carried out adding 50 μl acetonitrile while vortexing. After centrifugation, the upper layer was collected and 25 μl was injected. Liquid chromatography–mass spectrometryanalysis was carried out using a UFLC Shimadzu system consisting of a LC-20ADXR pump, a SIL-20ACXR autosampler, a DGU-20A3 degasser and a CTO-20A oven (Shimadzu Prominence, Antwerpen, Belgium) in combination with a 3200 QTRAP (ABSciex, Halle, Belgium) and Analyst software (version 1.5). The instrument was operated in atmospheric pressure chemical ionization, negative-single ion mode monitor m/z 317.1 (steviol) and m/z 205.2 (ibuprofen). The corona discharge needle voltage was −4, 500 V and heated nebulizer temperature was set at 500 °C for 0.5 ml min −1 flow rate. Nitrogen was set as nebulizer gas, auxiliary gas at 50 psi and curtain gas at 35 psi Other parameters were as follows: declustering potential −50 V, entrance potential −10 V. Liquid chromatography conditions: an Accucore C18 column (2.6 μm particle size, 2.1 mm × 100 mm) was used. Mobile phases were (A) 20 mM amoniumacetate at pH 4.8 and (B) acetonitrile. The used flow rate was 0.5 ml min −1 . The autosampler temperature was set at 10 °C, the column oven at 30 °C. The method had following gradient conditions 0–0.01 min: 20% B; 0.01–10 min: 70% B; 10–11: 20% B. Retention times were defined as 7.07 (steviol) and 6.21 (ibuprofen). Calibration curves were constructed using linear regression of the peak area ratios of steviol against internal standard (ibuprofen). Calibration standards were prepared by spiking blank plasma at concentrations 10, 50, 100, 250, 500, 1, 000, 2, 500, 5, 000 ng ml −1 ( R 2 =0.9926).
 WT, Trpm5 −/− or Tas1r2, Tas1r3 (−/−)2 mice were age-matched, individually caged and given two drinking bottles. After an adaptation period the animals drank 50% from each of the bottles containing deionized water. A test solution was placed in one of the bottles, and after 24 h, the position of the bottles was switched to control for a position preference. The bottles were weighted to measure the intake from each bottle. All tastants were given in an increasing stimulating order. The used solutions were 1% sucrose (sweet), the minimal concentration for which a preference can be detected 47 . A stimulatory concentration of 150 mM MKG; umami) 48, 100 μM quinine (bitter) or 25 μM in the Tas1r2, Tas1r3 (−/−)2 mice on the 48 h taste preference test and 10 mM citric acid (Sour). As stevioside tastes 300 times sweeter than sucrose and 3% sucrose is preferred by WT mice to a great extent, we used 0.01% or 124 μM stevioside, and similarly 124 μM rebaudioside A or steviol 29, 47 . The same animals were used in the control condition and the condition with steviol added and the indicated significance is the result of a paired t -test from two individual experiments with 24–48 animals per group each. For the brief access drinking tests, we used a custom-build lick-o-meter 49 . The mice were deprived from water for 23 h before the experiment. Individual mice were placed in a cage equipped with a copper grounding platform under two metal drinking spouts connected to an analogue/digital converter. The drinking spouts were isolated except for the tip so only tongue licks are recorded as a positive step of 25–150 mV. Custom software recorded the junction potential at 200 Hz. We analysed potentials exceeding 25 mV as a licking event. Licking events were recorded for 60 min. We analysed the total number of licks and the fraction of licks from each bottle (per mouse) over the complete time course to measure the 1 h preference. The first 5 min were analysed for initial taste preference. For each mouse, the total number of licks was normalized to avoid unequal contribution of extreme individuals. These incremental licks were pooled and divided by the number of animals in the group to yield the data in Fig. 5b–g and Supplementary Figs 7–9. Statistical significance was assessed with paired or two-sample t -tests on the normalized fractions that each mouse drank at the end of 5 min (Fig. 5f) or 60 min (Supplementary Fig. 6a, b). The variance between individual mice in the same condition is displayed with the data spread and the 25–75% data-interval boxplot, indicating the mean and median of each group. The amount of licks in each condition for each mouse is represented in Supplementary Fig. 6c–e for the 60 min lick-o-meter test and in Supplementary Fig. 6f for the first 5 min.
 Duomenų analizė ir statistika
 We used Matlab R2012a, OriginPro 8.6, Microsoft Excel and Igor Pro 6.34 for data analysis. Values deviating>3 × sd of the population mean were considered outliers. Normality was confirmed with the Shapiro–Wilk test (95% confidence level). For significance testing, we used two-tailed paired or unpaired t -test, unless otherwise indicated. For equivalence testing, we used the two one-sided tests approach and withheld the largest P -value. Data represent mean±sem Significance levels are indicated in the figures as P >0.05, not significant; * P <0, 05; ** P <0, 01; *** P <0, 001.
 Efektyvus Her2 specifinio priešnavikinio atsako indukcija dendritinėmis ląstelėmis, impulsinėmis Hsp70L1 – Her2341–456 sulietų baltymų pagalba
 Laminino E8 fragmentai palaiko efektyvų atskirtų žmogaus pluripotentinių kamieninių ląstelių adheziją ir plėtimąsi
 Senyvų ir silpnų pelių prieširdžių struktūra, funkcijos ir aritmogenezė
 Jautrumas išoriniams signalams ir sinchronizacijos savybės neizochroninio automatinio generatoriaus su atidėtu grįžtamuoju ryšiu
 Junginės epitelio ląstelių impresinė citologija pacientams, sergantiems cistine fibroze
 Seilių ir dubens baltymai: duomenų gavybos analizė
 Apoptozės signalą reguliuojanti 1 kinazė, reguliuodama C1q, tarpininkauja striatos degeneracijoje
 ARID1A / BAF250a ekspresijos praradimas endometriozėje: kancerogeninės transformacijos rizikos biomarkeris?


type: caselaw

jurisdiction: EU

text:
 20.2.2017
 MT
 Il-Ġurnal Uffiċjali tal-Unjoni Ewropea
 C 53/39
 Rikors ippreżentat fl-20 ta’ Diċembru 2016 – Puma vs EUIPO – Senator (TRINOMIC)
 (Kawża T-896/16)
 (2017/C 053/47)
 Lingwa tar-rikors: il-Ġermaniż
 Partijiet
 Rikorrenti: Puma SE (Herzogenaurach, il-Ġermanja) (rappreżentant: M. Schunke, avukat)
 Konvenut: L-Uffiċċju tal-Proprjetà Intellettwali tal-Unjoni Ewropea (EUIPO)
 Parti oħra quddiem il-Bord tal-Appell: Senator GmbH & Co. KGaA (Groß-Bieberau, il-Ġermanja)
 Informazzjoni dwar il-proċedimenti quddiem l-EUIPO
 Applikant għat-trade mark kontenzjuża: ir-rikorrenti
 Trade mark kontenzjuża kkonċernata: trade mark verbali tal-Unjoni Ewropea “TRINOMIC” – Applikazzjoni għal reġistrazzjoni Nru 12 697 074
 Proċedimenti quddiem l-EUIPO: proċedimenti ta’ oppożizzjoni
 Deċiżjoni kkontestata: deċiżjoni tar-Raba’ Bord tal-Appell tal-EUIPO tad-19 ta’ Ottubru 2016 fil-Każ R 70/2016-4
 Talbiet
 —
 tannulla d-deċiżjoni kkontestata;
 —
 tikkundanna lill-konvenut għall-ispejjeż tal-proċedimenti, inklużi dawk tal-proċedimenti tal-appell.
 Motivi invokati
 —
 Ksur tal-Artikolu 8(1)(b) tar-Regolament Nru 207/2009;
 Ksur tal-prinċipju ta’ trattament ugwali u tal-prinċipju ta’ patere legem quam ipse fecisti.