Diabetologia. I-2017; I-60 (8): 1502-1511.
Ipapashwe kwi-intanethi ye-2017 ngoMeyi 20. doi: 10.1007/s00125-017-4305-4
PMCID: PMC5491592
1Abstract
Iinjongo / hypothesis
Ukugqithiswa kweefisi zokutya kubangela ukutyeba kakhulu ebantwini nasezintanjeni. Izifundo zamva nje zabantu kunye neempuku zibonise ukuba umlutha kwi-fres uyabelana ngendlela eqhelekileyo yokulutha, i-nicotine kunye ne-narcotic ngokubhekisele ekusebenzeni kweenkqubo zembuyekezo yobuchopho. Kuvezwe ukuba indawo yokutya enamafutha aphezulu (HFD) ifumana i-dopamine D2 receptor (D2R) isayina kwi-striatum, umlawuli obalulekileyo wenkqubo yomvuzo wobuchopho, ekhokelela kwi-hedonic overeating. Besichaze ngaphambili ukuba umdaka o-brown wedreyini okhethekileyo we-γ-oryzanol ulwachongele ukhetho lwe-HFD ngolawulo lwe-hypothalamic. Sikhangele ke ukuba i γ-oryzanol iya kusebenzisa ukusebenza kwenkqubo yomvuzo wobuchopho ngeempuku.
tindlela
Amaduna C57BL / 6J iimpuku ezondliwe i-HFD zanyangwa ngomlomo nge-y-oryzanol, kunye ne-striatal amanqanaba eimolekyuli ezazibandakanyeka ekutyikityeni kwe-D2R zavavanywa. Impembelelo ye-γ-oryzanol kwi-DNA methylation ye-D2R yokunyusa kunye notshintsho olwalandelayo ngokuthanda kwamafutha okutya kwavavanywa. Ukongeza, iziphumo ze-5-aza-2'-deoxycytidine, inhibitor enobuchule ye-methyltransferases (DNMTs), kukhetho lokutya, ukusayinwa kwe-D2R kunye namanqanaba ee-DNMTs kwi-striatum ziphandwe. Iziphumo zokuthintela amandla of-oryzanol kwimisebenzi ye-DNMTs zavavanywa ngokubonakalayo kwi-vitro.
iziphumo
Kwi-striatum evela kwiigundane ezondliwe yi-HFD, ukuveliswa kwe-D2Rs kuncitshisiwe ngokwanda kwe-methylation ye-DNA yommandla wokukhuthaza we-D2R. Ukulawulwa ngomlomo kwe-γ-oryzanol kunciphisa ukubonakaliswa kunye nomsebenzi we-DNMTs, ngaloo ndlela ukubuyisela umgangatho we-D2Rs kwi-striatum. Inhibition ye-Pharmacological ye-DNMTs yi-5-aza-2'-deoxycytidine ikwanyuselwe ukuthanda kwamafutha okutya. Ngokuhambelana nezi ziphumo, i-enzymatic in vitro assows ibonise ukuba γ-oryzanol ithintele umsebenzi we-DNMTs.
Iziphetho / utoliko
Sibonisile ukuba i-γ-oryzanol i-ameliorates ye-HFD-eyenziweyo ye-DNA hypermethylation yommandla wokukhuthaza we-D2R kwi-striatum yeempuku. Uvavanyo lweparadigm yethu yokuvavanya iphakamisa i-γ-oryzanol njengento ethembisayo yokuchasana nezinto ezinepropathi engafaniyo yokuba yimodareyitha ye-epigenetic.
Izinto zokuncedisa ezisebenza ngombane
Inguqulelo ekwi-intanethi yeli nqaku (i-doi: 10.1007 / s00125-017-4305) inezinto eziqwalaselwe lontanga kodwa ezingezizo ezongeziweyo, ezifumanekayo kubasebenzisi abagunyazisiweyo.
intshayelelo
Ukuchitha ngaphezulu kwizabelo zabantu abatyebileyo, ubuncinci, indlela eqhelekileyo yokulutha i-alcohol, i-nicotine kunye ne-narcotic [1]. Ngokukodwa kunye ne-hypothalamic kunye ne-hormonal regulation yokutya, inkqubo yomvuzo wobuchopho, ngokukodwa ukuchaza i-dopamine receptor, inxulumene ngokusondeleyo nokuziphatha okuluthayo okanye i-hedonic feed [2]. Uphononongo oludlulileyo kwiigundane lubonise ukuba unkqonkqozi we-striatal dopamine D2 receptor (D2R) yi-lentivirus-mediated Shortpin ye-hairpin ephazamisa i-RNA ikhokelela ngokukhawuleza kwiintsilelo ezifana nokuvuzwa komvuzo kunye nokunyanzelwa-njengokufuna ukutya [3]. Ngenxa yokuncipha kwexinano le-D2R, i-drial striatum ayiphenduli kangako kumvuzo wokutya xa kuthelekiswa namaqela olawulo anqabileyo kubantu abatyebileyo kunye neentonga [3-5]. Ngokuhambelana nale mbono, TaqIAlele ANKK1 i-gene locus (encoding DRD2 / ankyrin ukuphindaphinda kunye ne-kinase domain equkethe i-1), enciphisa ukuveliswa kwe-D2R yemveliso, inxulunyaniswa nefenotype yobuntu ebantwini [6], ngelixa iimpembelelo zokulahleka kwesisindo emva kovavanyo lwe-bariatric zinxulunyaniswa nobunzima be-striatal D2R density [7]. Ezi datha zibonisa ukubaluleka kokubaluleka kwe-striatal D2R njengento ekujolise kuyo kwonyango lokunyanga ukukhuluphala. Nangona kunjalo, amanye amachiza athe aveliswa asebenza kwinkqubo yomvuzo wobuchopho abangela iziphumo ezibi kakhulu, kubandakanya neengxaki zengqondo ezinobuzaza, ezikhokelela ekuyekeni kwabo ezikliniki [8].
Uhlengahlengiso lwe-epigenetic lubalulekile hayi kuphela kuphuhliso kunye nokwahlula, kodwa nangenxa yokuba kuvela njengesiphumo sotshintsho lwendalo, kubandakanya ekudleni nasendleleni yokuphila [9]. I-methylation ye-DNA ngumsitho oyintloko we-epigenetic yokuzinza kwentetho ye-gene [9]. Kumagundane, ukubhencwa koomama kukutya okunamafutha aphezulu (i-HFD) kutshintsha-tshintsho kuguqulelo lwe-methylation ye-DNA ngaphakathi kwenkqubo yomvuzo oyintloko kwinzala, ekhokelela ekubonweni okungaphaya kwe-HFD ngabantwana [10]. Ngokukodwa, i-DNA methyltransferases (DNMTs) idlala indima ebalulekileyo kulawulo lokuziphatha kunye nokusebenza komzimba [10, 11], ndicebisa ukuba ii-DNMTs zinokuthembisa ekujolise kuko kunyango ngokunyangwa kwesifo sokutyeba kakhulu isifo seswekile. Ngokubalulekileyo, ezinye izinto zokutya ezikhutshwe yindalo, kubandakanya ne-caffeic acid kunye ne-epigallocatechin, ziyaziwa ngokuba zisebenza njengee-DNMT inhibitors [12, 13].
Kutshanje sibonisa ukuba icandelo le-io-oryzanol elingumdaka, umxube we-estulic acid ester kunye nee-phytosterols ezininzi, lixela ukhetho lwamafutha okutya ngokuncipha koxinzelelo lwe-hypothalamic endoplasmic reticulum (ER) [14]. Kwiimpuku kunye nemivundla, i-γ-oryzanol ekhutshwe ngomlomo yakhawuleza yanyuswa isuka emathunjini yaza yasasazwa ikakhulu kwingqondo [15, 16]. Ukuthatha ezi zinto zifunyanisiweyo, iimveliso zendalo ezenziwa kukutya kwinkqubo yeesistim ezisentloko zinokuba yenye indlela yokuziphilisa ngokuphelileyo kokuziphatha ngokutyeba kakhulu. Kule meko, sivavanye i-hypothesis ethi γ-oryzanol iyakutshintsha imeko ye-DNA methylation kwinkqubo yomvuzo wobuchopho, kukhokelela ekunikezelweni kokukhethwa kwe-HFD kumagundwane.
tindlela
izilwanyana
Iimpuku zeeveki ezisixhenxe ze-C57BL / 6J iimpuku ezifunyenwe kwiCharles River Laboratories eJapan (eKanagawa, eJapan) zagcinwa (3-4 kwikheyiji) kwiimeko ezithile ezingenazintsholongwane kwi-24 ° C phantsi kwe-12 h / 12 h ukukhanya / umjikelo omnyama. Emva kweveki ye-acclimatization, iimpuku zeeveki ezi-8 ubudala zazilinganiswa ngokomzimba zaza zahlulwa zangamaqela amabini okanye amathathu ukuya kuvavanyo ngalunye. Iimpuku zazivunyelwe ukufikelela simahla ekutyeni nasemanzini. Zonke iimvavanyo zezilwanyana zamkelwa yiKomiti yeeNqobo eziseSikweni zokuLinga kweZilwanyana kwiYunivesithi yaseRyukyus (Nombolo. 5352, 5718 kunye ne-5943).
Ulawulo lwe-γ-oryzanol kunye ne-5-aza-2'-deoxycytidine
Ukuvavanya ukuthanda i-HFD, i-γ-oryzanol (i-Wako Pure Chemical Industries, i-Osaka, Japan) yayilawulwa kwi-8-yegusha-yegundane endala nge-gavage ngexesha lovavanyo lokutya olukhethiweyo njengoko kuchaziwe ngaphambili [14, 17]. Olunye uvavanyo, i-HFD (D12079B; izidlo zoPhando, i-New Brunswick, NJ, e-USA) equlathe i-0.4% γ-oryzanol yenziwa njengepellets. Izixhobo zokutya ziboniswe kwitheyibhile yokuxhasa ngombane (ESM) iTafile 1. Emva kweeveki ezili-12 zokondla, izicubu zaqokelelwa kwi-striatum nakwi-hypothalamus. Ukutya kwemihla ngemihla kwe-γ-oryzanol, kuqikelelwa ukuba kuthetha ukutya kweempuku, kwakumalunga ne-320 μg / g yomzimba. Imithamo ye-γ-oryzanol yagqitywa njengoko bekuchaziwe ngaphambili14]. I-5-aza-2'-deoxycytidine (5-aza-dC; I-Sigma-Aldrich, iSt Louis, MO, e-USA) yatofwa ngaphakathi (0.25 μg / g ubunzima bomzimba) kathathu ngeveki kwiiveki ezili-12 [18].
Uqikelelo lokhetho lwamafutha okutya
Ukuvavanya ukukhethwa kwamafutha okutya, uvavanyo lokutya lubonelele ngokukhetha phakathi kwe-chow kunye ne-HFD (D12450B kunye ne-D12451; iZidlo zoPhando) njengoko kuchaziwe ngaphambili [14]. Izinto zokutya ziboniswe kwiTheyibhile ye-ESM 1. Ngokufutshane, iimpuku zazivunyelwe ukufikelela simahla kwi-chow kunye ne-HFD. Ukungeniswa kwe-chow kunye ne-HFD kwakulinganiswa ngeveki kwaye kwahlaziywa iinguqulelo ekukhethweni kwamanqatha okutya. Ukukhethwa kwe-HFD kwabalwa ngokwefomula: Ukukhetha kwe-HFD = [(ukutya kwe-HFD / ukutya okupheleleyo) × 100].
Ukulandelelana kweBisulphite ngokulandelelana kwe-DNA methylation
I-DNA yahlanjululwa kusetyenziswa i-DNeasy Igazi kunye neTisue Kit (QIAGEN, Tokyo, Japan). Isisombululo se-DNA sasixutywe ne-3 mol / l NaOH esandula kulungiswa, ifakwe kwi-37 ° C kangange-15 min kwaye yongezwa kwi-5.3 mol / l urea, 1.7 mol / l sodium bisulphite kunye ne-4.9 mmol / l hydroquinone. Isisombululo sasiphantsi kwemijikelo ye-15 ye-denaturation kwi-95 ° C ye-30 s kunye ne-50 ° C kwimizuzu eli-15 [19]. I-DNA yokunyanga i-baculphite yacocwa isebenzisa iMinElute PCR yokucocwa kweKhithi (QIAGEN) kwaye yandiswa yi-PCR isebenzisa i-KAPA HiFi HotStart Uracil + ReadyMix PCR Kit (i-KAPA Biosystems, Woburn, MA, USA) kunye ne-primers ejikeleze indawo ye-CpG ye-D2 . Ulandelelwano lwangaphambili lube ngolu hlobo lulandelayo: i-primer yangaphambili, i-5'-GTAAGAATTGGTTGGTTGGAGTTAAAA-3 '; iphinda iphinde ibuye umva, 5'-ACCCTACCCTCTAAACCACAACTAC-3 ′. Emva koko, ii-adaptha zongezwa kwaye zacocwa zisebenzisa iAgencourt AMPure XP (Beckman Coulter, Brea, CA, USA). Iisampulu zaxhonywa zaza zalayishwa kwi-GS Junior (Roche Diagnostics, Tokyo, Japan) ngokulandelelana ngokomgaqo wemveliso. Inqanaba le-methylation lacaciswa njengepesenti ye-methylated cytosines kuzo zonke iintsalela ze-cytosines.
Umsebenzi we-DNMT
I-assamatic ye-DNMT ye-enzymatic yomsebenzi yenziwa kusetyenziswa i-EpiQuik DNA Methyltransferase Activity / Inhibition Assay Kit (I-Epigentek Iqela, iBrooklyn, NY, iUSA) kunye ne-EPIgenible Methyltransferase Assay kit (Cisbio Japan, Chiba, Japan) ngokweeprotokholi zomvelisi.
Ukuvavanya umsebenzi we-inhibitory wecandelo ngalinye kwi-DNA methylation, ekubunjweni kwe S-adenosyl-l-homocysteine (SAH) yalinganiswa kubukho bekhompawundi nganye (i-20 μmol / l yovavanyo lokuhlola), S-adenosyl methionine (SAM; 10 μmol / l) kunye ne-DNMT substrate (4 ng / μl) kwi-37 ° C ye-90 min. Ukuvavanya iMichaelis-Menten kinetics, i-DNMT1 (20 μmol / l) yafakwa i-γ-oryzanol, i-SAM (5 μmol / l) kunye noxinzelelo lwe-poly dI-dC kwi-37 ° C ye-90 min. I-DNMT3a (100 μmol / l) kunye ne-DNMT3b (100 μmol / l) zafakwa i-γ-oryzanol, i-SAM (5 μmol / l) kunye noxinzelelo lwe-poly dG · dC kwi-37 ° C ye-120 min. Izilingo zenziwa ziphindwe kabini. Iprotheni ekhutshiweyo (0.75 mg / ml) ifakwe kwi-SAM (5 μmol / l), i-poly dI-dC (5 μg / ml), kunye ne-poly dG · dC (5 μg / ml) kwi-40 ° C ye-120 min, kunye Ukwenziwa kwe-SAH kulinganiswe.
I-assept--receptor-inxulumene nomsebenzi we-assay
Umsebenzi ochasene ne-γ-oryzanol kwi-receptor-γ enxulumene ne-estrogen (i-ERRγ) yavavanywa kusetyenziswa i-Human Estrogen-Related Receptor Gamma Reporter Assay System (INDIGO Bioscience, State College, PA, USA) ngokomgaqo womenzi. Ngokufutshane, iiseli zentatheli ezingezizo ezomntu ezibonisa ngokucacileyo ukuba i-ERRγ esebenzayo yavezwa kugxininiso olubonakalisiweyo lwekhompawundi nganye ye-24 h kathathu.
Ukucinywa kweNtshona
Oku kwenziwe njengoko bekuchaziwe ngaphambili [20] kunye nee-antibodies ezichasene ne-D2R (1: 500, umvundla), umthuthi we-dopamine (i-DAT; 1: 500, umvundla), i-tyrosine hydroxylase (TH; 1: 1000, umvundla) (AB5084P, AB1591P kunye no-AB152, Merck Millipore, Billerica, MA, I-USA), umqondiso wokuhambisa kunye ne-activator ye-transcript 3cy (STAT3α; 1: 1000, umvundla), i-DNMT1 (1: 1000, umvundla), i-DNMT3a (1: 1000, umvundla) (iinombolo 8768, 5032 kunye ne-3598; Itekhnoloji yokuTyikitya kweSeli, ITokyo, iJapan), i-DNMT3b (1 μg / ml, umvundla), ERRγ (1: 1000, umvundla) kunye ne-β-actin (1: 10,000, impuku) (ab16049, ab128930 kunye ne-ab6276; Abcam, Cambridge, MA, USA).
I-PCR yexesha langempela
Inkcazo ye-Gene yahlolwa njengoko kuchaziwe ngaphambili [14]. amanqanaba e-mRNA abaqhelekileyo I-Rn18s (I-18S rRNA). Iiseti zeprayimari ezisetyenziselwa ukuhlaziya ubungakanani bexesha le-PCR lushwankathelwe kwiTheyibhile ye-ESM 2.
Uhlalutyo lwesatisatisti
Idatha ichazwe njengentsingiselo ± SEM. I-ANOVA yendlela enye kunye namanyathelo aphindaphindiweyo i-ANOVA elandelwa ziimvavanyo zokuthelekisa ezininzi (indlela yeBonferroni-Dunn) zisetyenzisiwe apho kufanelekileyo. Umfundi t uvavanyo lwalusetyenziselwa ukuhlaziya umahluko phakathi kwamaqela amabini. Umahluko uthathwe njengento ebalulekileyo p <0.05.
iziphumo
Isithinteli se-Pharmacological ye-DNMTs yi-5-aza-dC ichongele ukhetho lwamafutha okutya kwiimpuku.
Kwiimpuku zondla i-HFD, i-methylation ye-DNA kwindawo yokukhuthaza ye-D2R kwi-striatum yandiswa kakhulu xa ithelekiswa neempuku ezityiswe kukutya okutyiwayo (Fig. (Fig.1a) .1a). Ngakolunye uhlangothi, i-hypothalamic ye-methylation ye-DNA kwindawo yokukhuthaza ye-D2R ngokucacileyo yayiphezulu kunaleyo striatum phantsi kwesidlo se-chow (p <0.01) (Ikhiwane. (Fig.1a, 1a, f) kwaye ayitshintshwanga yi-HFD (Fig. (Fig.1f) .1f). Kwiimpuku zondla i-HFD, i-methylation ye-DNA eyandisiweyo kwindawo yokuxhasayo ye-D2R kwi-striatum yenziwa eqhelekileyo yonyango nge-5-aza-dC, inhibitor enamandla ye-DNMT (Fig. (Fig.1a) .1a). Ngokwahlukileyo, i-DNA methylation kwingingqi yokukhuthaza ye-D2R kwi-hypothalamus ayitshintshanga kangako kunyango kunye ne-5-aza-dC (Fig. (Fig.1f) .1f). Kwi-striatum yeeveki ezingama-20 zeeveki ezindala ezondla i-HFD kwiiveki ze-12, i-mRNA kunye neeprotein zamanqanaba e-D2R zehla kakhulu (Ikhiwane. (Fig.1b, 1b, k, l). Ngokwahlukileyo, amanqanaba e-dopamine D1 receptors (D1Rs, encoded by I-Drd1), esebenza ngendlela eyahlukileyo kwi-D2Rs kwi-adenylyl cyclase kunye ne-cAMP-Mediated intracellular signaling, yayingaguqukanga (Fig. (Fig.1c) .1c). Ngapha koko, kwakungekho lutshintsho kumanqanaba ezinye iimolekyuli ezinxulumene nokusayina kwe-D2R, njenge-TH kunye ne-DAT kwinqanaba le-mRNA kunye / okanye kwinqanaba leprotein (Fig. (Fig.1d, 1d, e, k, m). Ngakolunye uhlangothi, akukho tshintsho lubonakalayo lwabonwa kwi-hypothalamus, kubandakanya i-D2R (Fig. (Fig.1g-m) .1g-m). Ngokukodwa, amanqanaba eprotheyini e-D2R kunye ne-TH kwi-hypothalamus ayephantsi kakhulu kunalawo e-striatum (Fig. (Fig.1l, 1m, m), ukubonakalisa ukubaluleka kokulinganisa kwe-dopamine receptor isibonakaliso kwinkqubo yomvuzo wobuchopho ngokuthelekiswa ne-hypothalamus.
Ukujonga ukuba ngaba i-methylation ye-DNA kwindawo yokukhuthaza ye-D2R ingaguqula ukuthanda kukhetho lwamafutha okutya, indlela yokunyanga yeempuku ze-5-aza-dC eziphathisiweyo zahlalutywa. Njengoko kulindelekile, i-5-aza-dC inyuse kakhulu i-mRNA kunye namanqanaba eprotein ye-D2R kwi-striatum yeempuku ezityiswe yi-HFD (Fig. (Fig.1b, 1b, k, l). Kwelinye icala, kwakungekho siphumo kumanqanaba I-Drd1, Th kwaye I-Slc6a3 (encoding DAT) kwi-striatum, okanye kumanqanaba I-Drd2, I-Drd1, Th kwaye I-Slc6a3 kwi-hypothalamus (Fig. (Fig.1c-e, 1c-e, g-m). Ngelixa iigundane eziphathwe kwisithuthi zikhetha i-HFD, ukuthanda i-HFD kuncitshiswe kakhulu kwiimpuku eziphathwe nge-5-aza-dC (88% yamaxabiso eempuku eziphathwe zithuthi) (Fig. (Fig.1n) .1n). Ngenxa yoko, unyango nge-5-aza-dC linciphise ukufunyanwa kwesisindo somzimba (Fig. (Fig.11o).
I-y-oryzanol inciphisa amanqanaba ee-DNMTs kwi-striatum yeempuku ezityisiweyo ze-HFD
Njengoko besikhe sachaza ngaphambili [14], Ulawulo ngomlomo lwe-γ-oryzanol kwiimpuku zamadoda ngesavage ilwandise kakhulu ukhetho lwe-HFD (93% yamaxabiso ezimpuku eziphathwe kwisithuthi) (Fig. (Fig.2a), 2a), okukhokelela kwibonakalo ebonakalayo yokufumana ubunzima bomzimba (Fig. (Fig.2b) .2b). Sakhangela ke iimpembelelo ezinokubakho ze-γ-oryzanol module ye-epigenetic ye-D2Rs kwi-striatum.
Kwizilwanyana ezanyisayo, kukho ezintathu ze-DNMTs-DNMT1, 3a kunye 3b. I-DNMT1 isebenza ukugcina i-methylation ye-DNA, ngelixa i-DNMT3a kunye ne-3b zidlala indima ekuququzeleleni de novo DNA methylation [21]. Ukujonga impembelelo enokubakho ye-γ-oryzanol kwi-DNMTs kwi-vivo, savavanya amanqanaba e-DNMTs kwiipilisi zeempuku ezityiswe yi-HFD. Nangona i-HFD kwi-se nganye ingenampembelelo kwi-mRNA kunye namanqanaba eprotein ye-DNMTs nokuba kwi-striatum okanye kwi-hypothalamus, ukuxhaswa kunye ne-γ-oryzanol kunciphisa kakhulu amanqanaba e-DNMTs kwi-striatum kodwa hayi kwi-hypothalamus (Fig. (Fig.2c-e, 2c-e, g-i, k-n). Ezi datha ziphakamisa ukuba i-γ-oryzanol inokulawula amanqanaba ee-DNMTs ngendlela ekhethekileyo ye-striatum. Kwindlela efanayo, i-5-aza-dC yehlise kakhulu amanqanaba e-mRNA e-DNMT3a kunye ne-3b ngokukhethekileyo kwi-striatum (ESM Fig. 1a. d.
Kwisiseko sophononongo oludlulileyo olubonisa ukuba inqanaba le-mRNA ye-DNMT1 yayilawulwa kakuhle, okungenani ngokuyinxalenye, nge-receptor yenyukliya i-ERRγ [22], sivavanye umphumo onokubakho we-γ-oryzanol kwimisebenzi ye-ERRγ. Kwiiseli ezingezomntu ezingezomntu ezichaza i-ERRγ esebenzayo, i-4-hydroxy tamoxifen, i-agonist enamandla eguquguqukayo ye-ERRγ, icekeceke kakhulu imisebenzi ye-ERRγ. Qaphela, i-γ-oryzanol inciphile umsebenzi we-ERRγ (umda we40% wexabiso langaphakathi) (Fig. (Fig.3a) .3a). Ngokubalulekileyo, i-ERRγ ibonakaliswe kakhulu kwi-striatum kodwa hayi kwi-hypothalamus (Fig. (Fig.3b-d) .3b-d). Ngokuchasene nemeko ye-striatum, i-γ-oryzanol yandise ngokubonakalayo amanqanaba eprotheyini e-DNMT1 kuphela kwi-hypothalamus (Fig. (Fig.2k, 2k, l). Ezi ziphumo zinokuchazwa, ubuncinci ngenxalenye, ngokufumanisa kwethu ukuba i-STAT3cy, ilawula inqanaba le-DNMT1 [23], wabonakaliswa kakhulu kwi-hypothalamus kodwa hayi kwi-striatum (Fig. (Fig.33e-g).
Ukuvavanya ngakumbi impembelelo ye-γ-oryzanol yomsebenzi we-DNMTs kwi-vivo, ukwakhiwa kwe-SAH, into edlulileyo edibanisayo ye-methylation ye-DNA kunye ne-inhibitor enamandla e-DNMTs, kwavavanywa kwimigundane ephathwa nge-γ-oryzanol okanye i-HFD. Akukho zinguqu zibalulekileyo kulwakhiwo lwe-SAH nokuba kukwimo ye-striatum okanye i-hypothalamus phakathi kweempuku ze-HFD-ezityisiweyo kunye neempuku-ezityiweyo (Fig. (Fig.2f, 2f, j). Kuyaphawuleka, i-γ-oryzanol inciphise kakhulu ukwenziwa kwe-SAH kwi-striatum (Fig. (Fig.2f) 2f) kodwa hayi kwi-hypothalamus (Fig. (Fig.2j), 2j), ucebisa ukuba i-γ-oryzanol inokucinezela umsebenzi we-DNMTs ngendlela ekhethekileyo yesitayile kwiigundane ezityiswe nge-HFD.
Uhlalutyo lwe-Enzymatic kwiipropati ze-γ-oryzanol ze-DNMTs kwi-vitro
Siphinde sayivavanya impembelelo ye-γ-oryzanol yomsebenzi we-DNMTs kwi-vitro. I-pothibitory potency ye-γ-oryzanol, i-ferulic acid, i-5-aza-dC, i-haloperidol (ummeli we-D2R ye-antagonist), i-quinpirole (ummeli we-agonist ye-D2R) kunye ne-SAH ngokuchasene ne-DNMTs. Njengolawulo oluqinisekileyo, i-SAH ilubambe ngamandla imisebenzi ye-DNMTs ngendlela exhomekeke kwidosi (Fig. (Fig.4a-f) .4a-f). Njengoko kulindelwe, i-haloperidol kunye ne-quinpirole azibonisanga mpembelelo kwimisebenzi ye-DNMTs (ESM Fig. 2). Kuyaphawuleka, i-γ-oryzanol ithintele kakhulu imisebenzi ye-DNMT1 (IC50 = 3.2 μmol / l), 3a (IC50 = 22.3 μmol / l) kunye ne-3b (inhibition ephezulu ye-57%) (Ikhiwane. (Fig.4d-f) .4d-f). Ngokwahlukileyo, umsebenzi we-inhibitory we-ferulic acid, i-metabolite ye-γ-oryzanol, yayingaphantsi kakhulu kune-that-oryzanol (Fig. (Fig.44d-f).
Siphinde saqhuba uphando nge-inhibitory ye-γ-oryzanol kwi-DNMTs. Ukusekwa kwe-SAH kwakulinganiswa ukuvavanya umsebenzi we-inhibitory we-γ-oryzanol kwi-DNMTs kwi-vitro. Idatha ekwakhiweni kwe-SAH ngexesha le-DNMT-mediated DNA methylation ibonisa iphethini ehleliyo ye-Michaelis-Menten kinetics yobukho kunye nokungabikho kwe-γ-oryzanol (Fig. (Fig.4g-i) .4g-i). Kwi-DNMT1-Mediated DNA methylation, Uhlalutyo lwe-Eadie-Hofstee lubonise ukuba i-γ-oryzanol ayiboniswanga ziphumo V max Ukwenziwa kweSAH (isithuthi, i-597 pmol / min; or-oryzanol 2 μmol / l, 619 pmol / min; γ-oryzanol 20 μmol / l, 608 pmol / min), ngelixa i-γ-oryzanol ibonakala inyusa K m (isithuthi, 0.47 μg / ml; γ-oryzanol 2 μmol / l, 0.67 μg / ml; γ-oryzanol 20 μmol / l, 0.89 μg / ml) (Ikhiwane. (Fig.4j) .4j). Ezi ziphumo zibonisa ukuba i-γ-oryzanol inhibits DNMT1 ubuncinci ngenxalenye yokhuphiswano. Kwelinye icala, ye-DNMT3a- kunye ne-3b-mediated DNA methylation, γ-oryzanol iyinciphisile V max Yokwakhiwa kweSAH (DNMT3a: isithuthi, 85.3 pmol / min; γ-oryzanol 2 μmol / l, 63.1 pmol / min; γ-oryzanol 20 μmol / l, 42.5 pmol / min; DNMT3b: isithuthi, 42.3 pmol / min; γ -oryzanol 2 μmol / l; 28.0 pmol / min, γ-oryzanol 20 μmol / l, 15.0 pmol / min) kwaye, ngokufanayo, K m yokuphendula (DNMT3a: isithuthi, 0.0086 μg / ml; γ-oryzanol 2 μmol / l, 0.0080 μg / ml; γ-oryzanol 20 μmol / l, 0.0058 μg / ml; DNMT3b: isithuthi, 0.0122 μg / ml; i-oryzanol 2 μmol / l, 0.0097 μg / ml; γ-oryzanol 20 μmol / l, 0.0060 μg / ml) (Ikhiwane. (Fig.4k, 4k, l). Ezi ziphumo zibonisa ukuba i-γ-oryzanol inhibits i-DNMT3a kunye ne-3b ubuncinci ngokuyinxalenye ngendlela yokungakhuphisani.
I-oryzanol inyusa amanqanaba e-D2R kwi-striatum yeempuku ezityiswe yi-HFD
Sivavanye ngokulandelayo ukuba i-γ-oryzanol inokunyusa umxholo we-striatal D2R ngokuthintela i-DNMTs. Kwiigundane ezityiswe nge-HFD, ukulawulwa komlomo kwe-γ-oryzanol kunciphise kakhulu i-striatal ye-methylation ye-DNA kwindawo yokukhuthaza ye-D2Rs (Fig. (Fig.5a), 5a), kanti ayenzanga le nto kwi-hypothalamus (Fig. (Fig.5f) .5f). Ngokuhambelana nezi zinto zifunyanisiweyo, i-mRNA kunye namanqanaba eprotheyini ye-D2R anyuswe ngokwenyani (Fig. (Fig.5b, 5b, g, k, l). Ifana nedatha kunyango ene-5-aza-dC (Fig. (Fig.1), 1), kwakungekho miphumo ebonakalayo kwi-RNA kunye namanqanaba eprotheni I-Drd1, Th kwaye I-Slc6a3 (DAT) kwi-striatum, kwaye akukho miphumo kumanqanaba I-Drd1, Th kwaye I-Slc6a3 kwi-hypothalamus (Fig. (Fig.5c-e, 5c-e, h-k, m).
Izifundo zangaphambili zibonise ukuba amanqanaba e-D2R kunye ne-DNMT1 alawulwa ngoxinzelelo lwe-ER kunye nokuvuvukala ubuncinci ngenxalenye ye-NF-κB [17, 24, 25]. Saye ke savavanya amanqanaba e-ER enxulumene noxinzelelo kunye nohlobo olunxulumene nosulelo. Njengoko kubonisiwe ngaphambili [26], i-HFD yonyusa inkcazo ye-gene encoding TNF-α (Tnfa), i-monocyte chemoattractant protein-1 (MCP-1) (Ccl2), I-protein ye-C / EBP ye-Homologous (Chop), I-ER-yenzelwe indawo ye-DnaJ 4 (ERdj4) (I-Dnajb9) kunye ne-X-bhokisi ebunjiweyo ye-X-box binding protein 1 (I-Xbp1s) kwi-hypothalamus kodwa hayi kwi-striatum (Fig. (Fig.6) .6). Ngokubalaseleyo, ukongezwa kwe-HFD nge-γ-oryzanol kunciphisa kakhulu ukubonakaliswa okwandisiweyo kwegama Ccl2, Chop, I-Dnajb9 kwaye I-Xbp1s ngokukodwa kwi-hypothalamus kodwa hayi kwisistriatum (Fig. (Fig.66).
ingxoxo
Iziphumo eziphambili kwisifundo esikhoyo kukuba i-γ-oryzanol isebenza njenge-inhibitor enamandla ye-DNMT kwisimo seempuku, ngaloo ndlela kuthathwa, okungenani, kukhethwa i-HFD ngokusebenzisa i-epigenetic moduleo ye-striatal D2R. Kwi-striatum evela kwiigundane ezityiswe nge-HFD, amanqanaba e-D2R ancitshiswe kakhulu, ngelixa ezo ze-D1R, TH kunye ne-DAT azitshintshiwe (Fig. (Fig.1b-e, 1b-e, k-m). Ezi datha ziyahambelana nembono yokuba ukungasebenzi kakuhle kwe-striatal D2R kudlala indima ebalulekileyo ekuboneni umvuzo wokutya xa ukwi-HFD, ekhokelela ekuboneni ngokungaphaya kwe-HFD kwizilwanyana ezityebileyo [3]. Kwisifundo esikhoyo, unyango lweempuku ezondla-hFD ezine-5-aza-dC zonyusa kakhulu amanqanaba e-striatal D2R (Fig. (Fig.1b, 1b, k, l) ngokunokwenzeka ngokuncitshiswa kwenqanaba le-methylation ye-DNA kwindawo yokukhuthaza ye-D2R (Fig. (Fig.1a), 1a), kwaye ngenxa yoko bahambelana nokukhethwa kwamafutha okutya (Fig. (Fig.1n) .1n). Oku kufumanisa kukwaxhasa indima ebalulekileyo ye-striatal D2Rs kwimbono yomvuzo wokutya xa ukwi-HFD.
I-in vitro assay yethu ibonise ukuba imisebenzi ye-hib-oryzanol ngokuchasene ne-DNMTs ngokucacileyo yayinamandla kune-metabolite Ferulic acid (Fig. (Fig.4d-f), 4d-f), ephakamisa ukubaluleka kwesakhiwo esipheleleyo se-γ-oryzanol kwisenzo sayo sokuthintela kwi-DNMTs. Kwigundane elondliweyo le-HFD, izifundo zethu ziphakamisa ukuba, emva kolawulo lomlomo, i-or-oryzanol ifikelela kwingqondo njengesakhiwo esipheleleyo kwaye inciphise amanqanaba kunye nemisebenzi ye-DNMTs ngokukhethekileyo kwi-striatum, kunye nesiphumo sokwehla kwe-methylation ye-DNA kwindawo yokukhuthaza I-D2R kwi-striatum. Ngapha koko, izifundo zethu ze-in vitro zibonise ukuba i-γ-oryzanol isebenza njengoyinxalenye yomchasi ngokuchasene ne-ERRγ, esebenza ikakhulu njengomlawuli olungileyo kwimveliso ye-DNMT1 [22], kwaye ngenxa yoko ukunciphisa umsebenzi we-DNMT1 (Fig. (Fig.3a) .3a). Qaphela, i-ERRγ yabonakaliswa kakhulu kwi-striatum kodwa hayi kwi-hypothalamus kwiimpuku (Fig. (Fig.3b) .3b). Ezi datha zibonisa ukuba i-γ-oryzanol inokubakho ukunciphisa inani le-mRNA ye-DNMT1, ubuncinci ngokuyinxalenye, ngokunqanda i-ERRγ. Ngokuchasene ne-striatum, i-γ-oryzanol ayiboniswanga siphumo kwinqanaba le-D2R kwi-hypothalamus evela kwiimpuku ezityiswe yi-HFD (Fig. (Fig.5g, 5g, k, l).
Kwelinye icala, sibonisa ukuba amanqanaba e-γ-oryzanol anyuke kakhulu kwi-DNMT1 kwi-hypothalamus kodwa hayi kwi-striatum (Fig. (Fig.2k, 2k, l). Kuboniswe ukuba i-STAT3 inyusa umxholo we-DNMT1 kwiiseli ezinobungozi zeT-lymphoma [23]. Ngokukodwa, ngaphambili sabonisa ukuba i-γ-oryzanol inyuse kakhulu iphosphorylation ye-leptin kwi-hypothalamus evela kwiimpuku ze-HFD [14]. Kufuneka iphawulwe ukuba i-STAT3cy yayivezwe kakhulu kwi-hypothalamus kodwa ingekho kwi-striatum kwimouse (Fig. (Fig.3e-g) .3e-g). Ezi datha zisilinga ukuba siqikelele ukuba umahluko obonakalayo kwisiphumo se-γ-oryzanol kumanqanaba e-DNMT1 phakathi kwe-hypothalamus kunye ne-striatum inokuthiwa, ubuncinci, kumxholo okhethekileyo wengingqi we-STAT3cy kunye ne-ERRγ kwingqondo yeempuku ( Ikhiwane. (Fig.3b-g) .3b-g). Ngokudibeneyo, kubonakala ngathi kukho indlela yokubuyisa yokubonisa ye-ERRγ kunye ne-STAT3cy phakathi kwe-striatum kunye ne-hypothalamus kwiimpuku. Kwisiphumo seziphumo zethu, ke kusengqiqweni ukuqikelela ukuba kwi-striatum, apho imveliso ye-ERRγ ininzi, i-or-oryzanol inokukhetha ngokukhethekileyo ukunciphisa izinga le-mRNA kunye nomsebenzi we-enzyme we-DNMT1 njengomlawuli ongekho mthethweni we-ERRγ. Ngokwahlukileyo, kwi-hypothalamus, apho imveliso ye-STAT3α igcweleyo, γ-oryzanol inokunyusa ngokukhethekileyo amanqanaba e-DNMT1.
Uphononongo lwakutsha nje lubonakalise ukuba ukubonwa kwe-striatal D2R kubonisa ukuba i-HFD dysregulates feed feed [3], ukucebisa ukubaluleka okunokubakho kokuthintelwa kwe-striatal DNMTs kunyango lokukhuluphala. Kwelinye icala, uphononongo oludlulileyo lubonise ukuba kungenzeka ukuba imeko ye-DNA methylation ye-melanocortin receptor 4 gene eboniswe kwi-hypothalamic nuclei inokutshintsha iindlela zokudluliselwa kweempawu zokutyeba kakhulu kwiimpuku ze-agouti ezisebenzisekayo zeempuku [27]. Nangona uphando olongezelelekileyo lucingelwa ukuba lucacisa iindlela ezisisiseko, olu phononongo lubonisa ukubaluleka kwezicubu-, gene- kunye nokulandelelana ngokuthe ngqo kwe-methylation ye-DNA kwiphathophologyology ye-HFD-indened koxinzelelo.
Kutshanje siye saxela ukuba i-HFD inyuse inqanaba le-D2R kwii-isanc pancreatic zegundane [17, 24]. Kungenzeka ukuba ukwandiswa okunjalo kuguqulwe, ubuncinci ngokuyinxalenye, ngoxinzelelo lwe-ER kunye nokuvuvukala nge-NF-κB, ngenxa yokuba kukho izinto eziphendulayo ze-NF-κB ezikwindawo yokukhuthaza ye-D2R [17, 24]. Ngapha koko, uphononongo lwakutsha nje lubonise ukuba i-TNF-α kunye ne-IL-1β yonyusa inqanaba nomsebenzi we-DNMT1 kwisidipose tisis ephuma kwiigundane ezityiswe nge-HFD [25]. Ngokubalulekileyo, uphononongo lwangoku lubonise ukuba i-HFD ibangela uxinzelelo lwe-ER kunye nokuvuvukala ngokukhethekileyo kwi-hypothalamus kodwa hayi kwi-striatum (Fig. (Fig.6) .6). Iindlela ezinzulu zezicubu-, umda-, kunye ne-methylation ye-DNA ekhethekileyo yendawo kunye nokwehliswa kwethala kwimo yethu yokuvavanya kufuneka zilindele uphando oluthe kratya.
Kunye nengxelo yethu yangaphambili ebonisa ukuba i-γ-oryzanol ifumana ukhetho lwe-HFD ngokusebenzisa umgaqo we-hypothalamic woxinzelelo lwe-ER kumagundane [14], I-γ-oryzanol ikwamele ipropathi ekhethekileyo yokuzihlaziya zombini i-hedonic kunye ne-metabolic dysregulation ye-feed yokuziphatha. Kuba ezinye iziyobisi ze-antiobesity eziye zaphuhliswa ziyaziwa ukuba zibangela iziphumo ezibi [8], indlela yokutya esekwe kwindalo kwinkqubo yomvuzo wobuchopho kulindeleke ukuba unyangwe ngokutyeba kakhulu kwisifo seswekile [16]. Kule paradigm, i-or-oryzanol ngumgqatswa othembisa ngokuchasene nobume obunepropathi engafaniyo yokuba yimodareyitha ye-epigenetic.
Imibulelo
Sinombulelo kuS. Okamoto (kwiYunivesithi yaseRyukyus, eJapan) ngokujonga kwakhona lo mbhalo-mbhalo. Sibulela uM. Hirata, H. Kaneshiro, I. Asato noC. Noguchi (kwiYunivesithi yaseRyukyus, eJapan) ngoncedo loonobhala.
izifinyezo
| I-5-aza-dC | I-5-aza-2'-deoxycytidine |
| D1R | I-Dopamine D1 receptor |
| D2R | I-Dopamine D2 receptor |
| DAT | Dopamine umthuthi |
| DNMT | IDNA methyltransferase |
| ER | I-endoplasmic reticulum |
| ERR | I-receptor enxulumene ne-estrogen |
| HFD | Ukutya okunamafutha aphezulu |
| SAH | S-Adenosyl-l-homocysteine |
| SAM | S-Adenosyl methionine |
| I-STAT3cy | Isigransidrali yomqondiso kunye ne-activator yokukhutshelwa kwe3cy |
| TH | ITyrosine hydroxylase |
amaNqaku
Ukufumaneka kwedatha
Iirekhodi ezivelisiweyo kunye / okanye ezihlalutywe ngexesha lofundo lwangoku ziyafumaneka kumbhali ohambelana nezicelo ezifanelekileyo.
Inkxaso
Lo msebenzi uxhaswe yinxalenye yeGranti-in-Aid evela eJapan Society yokuKhuthaza iSayensi (i-JSPS; KAKENHI Grant Numbers 15K19520 kunye ne-24591338), iBhunga lezeNzululwazi, iTekhnoloji kunye neNtengiso (CSTI), iCross-minister Strategic Innovation Development Programme. (SIP) 'Iitekhnoloji zokwenza ulwazelelo olulandelayo lwezolimo, amahlathi kunye nezokuloba', iLotte Foundation, iJapan Foundation yokuFakelwa i-Enzymology, iNational Energy and Development Technology Development Organisation (NEDO), iProjekthi yokuYilwa kweNtsebenzo yeNzululwazi yoBomi (Amachiza oMayeza ) (I-Okinawa Prefecture, iJapan) kunye neProjekthi yokuKhuthaza ukuHlanganiswa kwezoNyango kwiSithili sase-Okinawa, iJapan, kunye nesibonelelo esivela kwi-Okinawa Prefecture yokunyusa amayeza aphezulu (kwi-Okinawa Prefecture, eJapan).
Ubungqani bomdla
Ababhali baxela ukuba akukho bumdaka bumdla obunxibelelene nalo mbhalo wesandla.
Ingxelo yegalelo
I-CK kunye ne-HM benze uphando. I-CK kunye ne-TK zenze uvavanyo kwaye zahlalutya idatha. I-TK, i-CS-O, i-CT, i-MT, i-M kunye ne-KA ziye zanegalelo kutoliko lwedatha. I-CK kunye ne-HM babhala lo mbhalo-ngqangi. Bonke ababhali banegalelo kutoliko lweedatha. Bonke ababhali bajoyine ekuhlaziyeni lo mbhalo-ngqangi kwaye bayivuma inguqulelo yabo yokugqibela. I-HM iyisiqinisekiso salo msebenzi, yayinokufikelela ngokupheleleyo kuyo yonke idatha kwaye ithatha uxanduva olupheleleyo lokunyaniseka kwedatha kunye nokuchaneka kohlalutyo lwedatha.
Imihlathi
Izinto zokuncedisa ezisebenza ngombane
Inguqulelo ekwi-intanethi yeli nqaku (i-doi: 10.1007 / s00125-017-4305) inezinto eziqwalaselwe lontanga kodwa ezingezizo ezongeziweyo, ezifumanekayo kubasebenzisi abagunyazisiweyo.
Ucaphulo
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