Transcription factor Ets-1 links glucotoxicity to pancreatic beta cell dysfunction through inhibiting PDX-1 expression in rodent models

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1 Dietologi () 9: DOI.7/s ARTICLE Trnsription ftor Ets links gluotoxiity to pnreti et ell ysfuntion through inhiiting PDX expression in roent moels Fng Chen & Min Sh & Ynyng Wng & Tijun Wu & Wei Shn & Ji Liu & Weno Zhou & Yunxi Zhu & Yujie Sun & Yugung Shi & Dvi Bleih & Xio Hn Reeive: June /Aepte: Otoer /Pulishe online: Novemer # SpringerVerlg Berlin Heielerg Astrt Aims/hypothesis Gluotoxiity is term use to onvey the negtive effet of hyperglyemi on et ell funtion; however, the unerlying moleulr mehnisms tht impir insulin seretion n gene expression re poorly efine. Our ojetive ws to efine the role of trnsription ftor vets vin erythrolstosis virus E onogene homologue (Ets) in et ell gluotoxiity. Methos Primry islets n Min ells were expose to high gluose n Ets expression ws mesure. Reominnt enovirus n trnsgeni mie were use to upregulte Ets expression in et ells in vitro n in vivo, n insulin seretion ws ssesse. The ining tivity of H/H histone on the Ets promoter, n tht of forkhe ox (FOX)A, FOXO n Ets on the Px promoter ws mesure y hromtin immunopreipittion n quntittive reltime PCR ssy. Results High gluose inue upregultion of Ets expression n hyperetyltion of histone H n H t the Ets gene promoter in et ells. Ets overexpression rmtilly suppresse insulin seretion n iosynthesis oth in vivo n in vitro. Besies, Ets overexpression inrese the tivity of Eletroni supplementry mteril The online version of this rtile (oi:.7/s) ontins peerreviewe ut uneite supplementry mteril, whih is ville to uthorise users. Xio Hn hnxio@njmu.eu.n Key Lortory of Humn Funtionl Genomis of Jingsu Provine, Nnjing Meil University, Hnzhong Ro, Nnjing 9, People s RepuliofChin Brshop Institute for Longevity n Aging Stuies, University of Texs Helth Siene Center, Sn Antonio, TX, USA Rutgers New Jersey Meil Shool, Newrk, NJ, USA FOXO ut erese tht of FOXA ining to the pnreti n uoenl homeoox (PDX) homology region (PH), resulting in inhiition of Px promoter tivity n ownregultion of PDX expression n tivity. In ition, high gluose promote the intertion of Ets n FOXO, n the tivity of Ets ining to the Px promoter. Importntly, PDX overexpression reverse the efet in pnreti et ells inue y Ets exess, while knokown of Ets prevente hyperglyemiinue ysfuntion of pnreti et ells. Conlusions/interprettion Our oservtions suggest tht Ets links gluotoxiity to pnreti et ell ysfuntion through inhiiting PDX expression in type ietes. Keywors Bet ell. Dysfuntion. Ets. Gluotoxiity. PDX Arevitions AEts Ets overexpression enovirus BW Boy weight ChIP Chromtin immunopreipittion ChIPqPCR Chromtin immunopreipittion n quntittive reltime PCR Ets Vets vin erythrolstosis virus E onogene homologue FOX Forkhe ox GCK Gluokinse Gluosestimulte inex S Gluosestimulte insulin seretion IP Immunopreipittion Neuro D Neurogeni ifferentition PDX Pnreti n uoenl homeoox PH PDX homology region siets Smll interfering (RNA)Ets enovirus TSA Trihosttin A

2 Dietologi () 9: 7 Introution Uner physiologil onitions, pnreti et ells moulte insulin seretion to mintin loo gluose in the norml rnge []. Dietes mellitus ours when et ells fil to mintin equte insulin proution to prevent hyperglyemi. Currently, intrinsi et ell ysfuntion n erese funtionl et ell mss re thought to e two importnt omponents of ieti et ell filure []. Results from stuies in / mie, the prtil pnretetomy rts n ZDF rts revele tht hroni hyperglyemi, ommonly lle gluotoxiity, is mjor eterminnt of using et ell filure [ ]. Severl omplex mehnisms re involve in et ell gluotoxiity, inluing et ell overstimultion, oxitive stress, enoplsmi retiulum stress, hypoxi stress n protein glytion []. Among these, OGlNA moifition of trnsription ftors suh s pnreti n uoenl homeoox (PDX), neurogeni ifferentition (NeuroD), SP n p might ply key role in gluotoxiity [7]. PDX, whih is expresse in et ells n few elt ells of the ult islet of Lngerhns, is mster regultor of et ell growth n funtion []. PDX regultes the expression of isletspeifi genes through its intertion with their promoter regions, whih inlue insulin, GLUT, gluokinse (GCK), islet myloi polypeptie (IAPP), somtosttin n PDX itself [9 ]. Clinilly, muttions in PDX use MODY n lteronset type ietes mellitus [, ]. In mie, seletive intivtion of PDX in et ells les to the evelopment of ietes with inresing ge []. In ition, hroni hyperglyemi uses loss of et ell funtion y reuing PDX expression n DNA ining tivities [7]. Forkhe ox (FOX)O is trnsription ftor tht negtively regultes PDX expression n hs lso een implite in et ell gluotoxiity [, 9]. FOXO inhiits PDX expression y ining to the PDX homology region (PH) of the PDX promoter, whih ontins FOXA ining site [, ]. As multifuntionl protein, FOXO regultes et ell funtion, prolifertion, ifferentition n poptosis []. As et l emonstrte tht FOXO oul intert with trnsription ftor vets vin erythrolstosis virus E onogene homologue (Ets) in ovine roti rtery enothelil ells, whih we hve lso emonstrte in pnreti et ells []. As result, Ets might e involve in regultion of et ell filure in ietes mellitus. In reent yers, eviene hs onfirme tht the etyltion sttus of histones hs een shown to e importnt in pnreti et ell funtion []. Histone etyltion promotes gene expression y inresing the pprohility of promoters to the trnsription mhinery []. We foun tht trnsription ftor Ets ws signifintly upregulte in high gluosetrete primry islets n Min ells s well s isolte islets from / mie. We lso foun the histone eetylse inhiitor oul upregulte Ets expression in pnreti et ells. We hypothesise tht epigeneti moifition might e involve in regultion of Ets expression in high gluosetrete et ells. Our reserh group teste this hypothesis using the pnreti et ell line Min, primry islets n trnsgeni mie tht overexpress Ets. Methos Regents See Eletroni Supplementry Mteril (ESM) Cell ulture Mouse Min n rt RINmF ell lines were estlishe s esrie previously [, ]. See ESM Islet isoltion n ulture Mle wiltype C7BL/BKS mie n mie funtionlly efiient for the longform leptin reeptor (/ mie) were otine from Nnjing University, Chin. Mle weekol SprgueDwley rts were purhse from Nnjing Meil University Lortory Animl Centre, Nnjing, Chin. Islet isoltion n ulturing tehniques were esrie previously [7]. See ESM RNA interferene, plsmi n reominnt enoviruses onstrution The Ets expression plsmi use ws s previously esrie []. Ets overexpression enovirus (A Ets), PDX overexpression enovirus (APx) n smll interfering (RNA)Ets enovirus (siets) were onstrute n purifie s previously esrie []. See ESM Gluosestimulte insulin seretion ssy n insulin ontent extrtion Gluosestimulte insulin seretion ssys n insulin ontent extrtion in Min ells n isolte islets were performe s previously reporte [7]. See ESM Reltime PCR Totl RNA ws extrte from Min ells, RINmF ells n isolte islets. Reltime PCR ws use to etermine the reltive expression levels of mrnas. See ESM Western lot nlysis Totl protein ws isolte from Min ells, RINmF ells n isolte islets. Western lotting ws performe s esrie [9]. See ESM Trnsient trnsfetion n luiferse reporter ssy The plsmis were trnsfete into RINmF ells. Trnsient trnsfetions n luiferse reporter ssys were performe s esrie []. See ESM

3 Dietologi () 9: Eletron mirosopy nlysis Islets were fixe in.% (vol./ vol.) glutrlehye n emee in Lowiryl KM resin. See ESM Chromtin immunopreipittion n hromtin immunopreipittionquntittive PCR ssy Chromtin immunopreipittion (ChIP) n ChIPquntittivePCR (ChIPqPCR) ssys were performe using lystes of Min n RINmF ells s esrie previously []. See ESM Immunopreipittion ssy Immunopreipittion (IP) ssys were performe using extrts of RINmF ells s esrie previously []. See ESM Genertion of Ets trnsgeni mie Ets overexpression in et ells of trnsgeni mie (C7BL/J strin) ws onstrute (Moel Animl Reserh Centre, Nnjing University, Nnjing, Chin) s esrie previously [, ]. Founer mie were ientifie y PCR of til iopsies using two pirs of primers (ESM Tle ). Heterozygous trnsgeni mie were use throughout the present stuies n nontrnsgeni littermtes were use s ontrols. We stuie to weekol mle mie. All nimls h free ess to stnr mouse how n wter, n were house in tempertureontrolle fility with h light/rk yle. All niml stuies were performe oring to guielines estlishe y the Reserh Animl Cre Committee of Nnjing Meil University, Chin (Permit Numer: IACUCNJMU 7). The experimenters were not lin to group ssignment n outome ssessment. i.p. gluose tolerne test n insulin tolerne test in mie At weeks of ge, ontrol mie n trnsgeni mie were rnomise to perform i.p. gluose tolerne tests n insulin tolerne tests []. See ESM Sttistil nlysis Comprisons were performe using Stuent s t test etween two groups or ANOVA in multiple groups. Results re presente s mens±sd. A p vlue of <. ws onsiere to e sttistilly signifint. Results re representtive of three iniviul experiments. No t, smples or nimls were exlue or omitte from reporting. Results Ets expression is elevte in / mie islets n in pnreti et ells expose to hyperglyemi Although Ets ws reporte to e relte to pnreti evelopment [], few stuies hve investigte its expression in pnreti islets. We ompre Ets gene expression in pnreti islets from weekol / n ontrol C7BL/BKS mie. Our results showe tht Ets mrna n protein level ws signifintly inrese in islets from / mie ompre with ontrol C7BL/BKS mie (Fig.,). Moreover, high gluose inrese Ets expression n Ets protein level in Min ells, n isolte mouse n rt islets (Fig. f). High gluose meites hyperetyltion of histone H n H t the Ets gene promoter To explore whether high gluose inue upregultion of Ets expression y using hyperetyltion of histone, we trete Min ells with ifferent onentrtions of the histone eetylse inhiitor trihosttin A (TSA). We oserve tht TSA signifintly upregulte Ets expression oth in mrna n protein levels (Fig.,). Then, we performe ChIPqPCR ssy to mesure whether high gluose meite hyperetyltion of histone t the Ets gene promoter. Exposure of Min ells to high onentrtions of gluose for h le to.fol inrese in histone H etyltion n sixfol inrese in histone H etyltion (Fig.,). Overexpression of Ets impirs insulin seretion n synthesis in vitro n in vivo The fining tht Ets expression ws signifintly inrese y high gluose prompte us to investigte the effet of Ets on insulin seretion in Min ells n isolte primry mouse islets. Overexpression of Ets in Min ells n islets signifintly inhiite the gluosestimulte inex (), whih reflets the insulin seretion rtio of pnreti et ells stimulte y high n low gluose (Fig., n ESM Fig. ). Similr results for insulin ontent were otine (Fig.,). Moreover, insulin seretion uner high gluose, normlise to insulin ontent, ws lso represse fter overexpressing Ets (Fig. e,f). To investigte whether Ets overexpression oul impir insulin seretion in vivo, we next mesure the effet of Ets on pnreti et ell funtion using trnsgeni mie (line 7) expressing the Ets gene irete y n insulin promoter [7]. Immunofluoresent stining of islets revele Ets ws speifilly overexpresse in pnreti et ells n there ws istint inverse reltionship etween Ets n insulin protein levels (ESM Fig. ). Islet eletron mirosopy showe erese in the numer of insulin seretory grnules in trnsgeni mie ompre with ontrol mie (Fig. g). In ition, Ets expression ws unffete in nonpnreti tissues, inluing spleen n liver (ESM Fig. ). The trnsgeni mie were orn t the expete Menelin rtio n mintine norml weight through our week oservtionl perio (ESM Fig. ); however, they isplye fsting hyperglyemi strting t 7 weeks of ge (Fig. h). Besies, loo gluose levels of trnsgeni mie were higher t every time point fter intrvenous gluose loing (. g/kg oy weight [BW]) ompre with wiltype ontrols (Fig. i). In ition, plsm insulin levels of trnsgeni mie were lower t every time point fter intrvenous gluose

4 Dietologi () 9: 9 Reltive Ets mrna level Reltive Ets mrna level e Reltive Ets mrna level Control Rt islets / Mouse islets... Gluose (mmol/l) Ets βatin Ets βatin f Ets βatin Control / Rt islets Mouse islets. mmol/l mmol/l... Gluose (mmol/l) loing ( g/kg BW) ompre with littermte ontrols (Fig. j). However, peripherl gluose uptke ws not signifintly ifferent etween trnsgeni mie n ontrol mie (Fig. k). This result inite tht the gluose intolerne of Ets trnsgeni mie ws ue to efetive insulin seretion ut not insulin resistne. It is importnt to note tht we lso otine three trnsgeni lines exhiiting high (line 7), meium (line ) n low (line 79) levels of Ets expression in islets. Both line n line 79 isplye efetive insulin seretion (ESM Fig. g). Ets overexpression exerts suppressive effet on PDX expression y inhiiting PDX promoter tivity To evlute the possile implitions of Ets overexpression for et ell ysfuntion, we exmine the effets of Ets on et ellssoite gene expression. As shown in Fig.,, the mount of Px mrna ws remrkly erese in the Ets. mmol/l mmol/l Reltive Ets protein level Reltive Ets protein level Reltive Ets protein level Control Rt islets / Mouse islets Gluose (mmol/l)... Fig. Ets expression is elevte in / mouse islets n in pnreti et ells expose to hyperglyemi. The mrna () n protein level () of Ets ws signifintly elevte in islets isolte from weekol / mie. (, ) Primry islets isolte from mie n rts were trete with high gluose ( mmol/l) for h, n the expression of Ets ws mesure. The mrna () n protein level () of Ets inrese in islets trete with mmol/l (lk rs) ompre with islets trete with. mmol/l (white rs). (e, f) Min ells were preulture in DMEM with. mmol/l gluose for h, n then ells were inute with ifferent onentrtions of gluose for n itionl h. The mrna (e) n protein level (f) of Ets ws upregulte in oseepenent mnner. Vlues re mens±sd n re representtive of three iniviul experiments. p<., p<. vs ontrol Reltive EtsmRNA level Fol enrihment of DNA H omplex level TSA (nmol/l). Gluose (mmol/l) Fol enrihment of DNA H omplex level overexpressing Min n RINmF ells, wheres the expression levels of Neuro, Mf n Fox were unhnge. Furthermore, Ets overexpression le to similr reution of PDX protein in Min n RINmF ells. The expression of PDX in islets of trnsgeni mie ws lso signifintly erese ompre with littermte ontrol islets (Fig. ). Therefore, the expression of PDX in vivo n in vitro ws inhiite y Ets overexpression. Next, we etermine the mrna levels of insulin n Gk, whih re oth iretly regulte y PDX, to explore whether Ets h n effet on PDX trnsriptionl tivity. ChIPqPCR ssy onfirme tht DNA frgments pulle own y n ntipdx ntioy in the Ets overexpressing ells were reue to pproximtely.fol for the insulin promoter site n fol for the Gk promoter site (Fig. ). Further stuy showe tht Ets oul in to the PH region of the Px promoter, whih ws the ommon DNA ining site of FOXO n FOXA (Fig. e). To investigte whether Ets overexpression oul ffet the tivity of FOXA n FOXO on the Px promoters, ChIPqPCR ssy ws performe. As shown in Fig. f, DNA frgments pulle own y the ntifoxa ntioy in the Ets overexpression group were reue to pproximtely.fol for the PH region of the Px promoter. However, the effet of Ets βatin. Gluose (mmol/l) TSA (nmol/l) Fig. Gluose meites hyperetyltion of histones H n H t the Ets gene promoter. Min ells were trete with ifferent onentrtions of the histone eetylse inhiitor TSA for h, n the mrna () n protein () levels of Ets were mesure. Min ells were preulture in DMEM with. mmol/l gluose for h, n then ells were inute with. mmol/l or mmol/l gluose for n itionl h. ChIPqPCR nlysis of the pity of etyltion of histone H () n H () ining to the Ets promoter ws performe. Vlues re mens±sd n re representtive of three iniviul experiments. p<. vs ontrol

5 Dietologi () 9: Insulin ontent Insulin ontent AGfp AEts AGfp AEts AGfp AEts AGfp AEts.. e f g Insulin seretion (reltive to insulin ontent) h Fsting gluose (mmol/l) Insulin seretion (reltive to insulin ontent) AGfp AEts AGfp AEts i Bloo gluose (mmol/l) CON Totl grnule numer/ell setion Time (weeks) Time (min) CON TM TM j Serum insulin (pmol/) Time (min) Fig. Overexpression of Ets impire insulin seretion in vitro n in vivo. AEts ( multipliity of infetion [MOI]) or ontrol enovirus (AGfp, MOI) were infete into Min ells n mouse islets for h, when S ssys were rrie out n ws lulte. Infetion use n effetive erese of in Min ells () n islets (), insulin ontent in Min ells () n islets (), n insulin seretion normlise to insulin ontent in Min ells (e) ( mmol/l, white rs; mmol/l, lk rs) n islets (f) (. mmol/l, white rs;.7 mmol/l, lk rs). (g) Islets from weekol mle ontrol mie (CON) n trnsgeni mie (TM) were fixe, pellete n setione for EM (mgnifition,). Averge totl grnule numer per ell ross k Perentge of initil insulin (%) Time (min) setion is epite n t re represente s mens±sd from ells. (h) Hyperglyemi t fsting in CON (white tringles) n TM (lk tringles) t ifferent weeks of ge (n= from eh group). (i) Bloo gluose levels fter n i.p. injetion of gluose (. g/kg BW) in weekol mle CON n TM (n= from eh group). (j) Serum insulin onentrtions fter i.p. injetion of gluose ( g/kg BW) in weekol mle CON n TM (n= from eh group). (k) Insulin tolerne test (.7 U/kg BW) in weekol mle CON n TM (n= from eh group). Vlues re mens±sd n re representtive of three iniviul experiments. p<., p<. vs ontrol Ets overexpression on tivity of FOXO ining to the PH region ws elevte up to.fol (Fig. g). Aitionlly, in Ets overexpression groups, DNA frgments pulle own y the ntiets ntioy were inrese to twofol (Fig. h). More importntly, Ets overexpression signifintly erese Px promoter tivity (Fig. i). Overexpression of PDX restore et ell funtion impire y Ets To etermine the role of PDX in Ets inue impirment of et ell funtion, Min ells were infete with PDX reominnt enovirl vetors for h, n then infete with Ets reominnt enovirl vetors for nother h. Western lot emonstrte tht

6 Reltive mrna level Reltive mrna level Dietologi () 9: PDX Ets βatin e f Fol enrihment of DNA FOXA omplex level i.. Luiferse tivity/ βgl tivity AGfp AEts Vetor Ets Min RINmF Mrker Input Ets IgG, 7,,,,,,,... Px Mf Neuro Fox Px Mf Neuro Fox Vetor Vetor Ets Ets g Fol enrihment of DNA FOXO omplex level CON TM Vetor... Fol enrihment of DNA PDX omplex level Ets Fol enrihment of DNA Ets omplex level Ins..... Vetor Fig. Ets exerts suppressive effet on PDX expression y inhiiting Px promoter tivity. Min ells were infete with A Ets (or AGfp) n RINmF ells were trnsfete with Ets expression plsmi (or vetor) for h, when reltime PCR ws rrie out. InfetioninMinells() or trnsfetion in RINmF ells () use n effetive erese in Px mrna level ut h no effet on Mf, Neuro n Fox mrna levels (AGfp or vetor, white rs; A Ets or Ets expression plsmi, lk rs). () Infetion or trnsfetion use n effetive erese in PDX protein level. The PDX protein level ws signifintly erese in trnsgeni mie (TM) ompre with ontrol mie (CON). () ChIPqPCR nlysis of effet of Ets on the pity of PDX ining to the Ins, Ins n Gk promoters ws performe in RINmF ells trnsfete with Ets expression plsmi for h (vetor, white rs; Ets expression plsmi, lk rs). (e) Ets oun iretly to the Px promoter in ChIP nlysis in RINmF ells trnsfete with Ets expression plsmi for h. ChIPqPCR nlysis of the pity of FOXA (f), FOXO (g)nets(h)ining to the PH omin of the Px promoter ws performe in RINmF ells trnsfete with Ets expression plsmi for h. (i) RINmF ells were trnsiently trnsfete with Ets expression plsmi (or vetor) n pglpdxph luiferse reporter onstrut. Luiferse tivity ws ssye h fter trnsfetion. Vlues re mens±sd n re representtive of three iniviul experiments. p<. vs ontrol h Ins Ets Gk PDX n Ets were oth overexpresse (Fig. ). Insulin seretion ws reue to % in Min ells trete with Ets overexpression lone, while restore to % y ooverexpression of Ets n PDX (Fig. ). Besies, PDX overexpression reverse the erese of insulin ontent, n insulin seretion, uner high gluose tht ws normlise to insulin ontent in Min ells overexpressing Ets (Fig.,). Hyperglyemi promotes the intertion of Ets n FOXO, n the tivity of Ets ining to the Px promoter To explore whether high gluose promotes the intertion of Ets n FOXO, we performe n IP ssy. As showninfig., enogenous intertion of Ets n FOXO ws signifintly inrese in RINmF ells trete with high gluose for h. Next, we performe ChIPqPCR ssy to mesure the tivity of Ets ining to the Px promoter in high gluosetrete RINmF ells. DNA frgments pulle own y the ntiets ntioy in the high gluosetrete group were inrese to pproximtely. fol for the Px promoter (Fig. ). Inhiition of Ets reverses hyperglyemiinue et ell filure Using the reominnt enoviruses for knokown of Ets expression (siets), we next nlyse the effet of Ets ownregultion on gluosestimulte insulin seretion (S) n insulin ontent in isolte islets, n Min ells trete with high gluose for 7 h. As emonstrte, siets effetively silene Ets gene expression n oorintely inrese PDX protein level in high gluose (Fig. 7). Knokown of Ets effetively restore in islets n Min ells previously expose to high gluose over 7 h perio (Fig. 7,), n inrese insulin ontent (Fig. 7e,f) n insulin seretion uner high gluose tht ws normlise to insulin ontent (Fig. 7g,h). Together, these results ientifie Ets s meitor of gluotoxiity, s epite in Fig. 7. Chroni high gluose tretment stimulte Ets expression, whih iretly ownregulte PDX expression n tivity. Following the inhiition of PDX expression n tivity, insulin expression ws ownregulte, whih resulte in pnreti et ell ysfuntion. Disussion Gluotoxiity is mjor eterminnt of using et ell filure in type ietes. The urrent stuies emonstrte tht Ets meites pnreti et ell ysfuntion, linking gluotoxiity to type ietes. In / mie islets s well s in high gluosetrete Min ells n primry islets, Ets expression ws signifintly inrese. Using newly generte trnsgeni mie with overexpression of Ets in pnreti

7 Dietologi () 9: Insulin ontent..... AGfp AEts APx Ets PDX βatin AGfp AEts APx Insulin seretion (reltive to insulin ontent) AGfp et ells, we emonstrte tht Ets exess inues gluose intolerne with efetive insulin seretion. It is importnt to note tht we otine three trnsgeni lines exhiiting high (line 7), meium (line ) n low (line 79) levels of Ets expression in islets, whih ll isplye efetive insulin seretion n PDX expression (ESM Fig. h). Thus, the oserve effet in trnsgeni mie ws ue to expression levels of Ets n ws not expline y insertionl mutgenesis, i.e. rnom inorportion of the trnsgene leing to intivtion of nother gene. Histone moifition hs een shown to e importnt in pnreti et ell funtion []. Histone H etyltion is AEts APx AGfp AEts APx Fig. Overexpression of PDX restore et ell funtion impire y Ets. After infetion with APx for h, Min ells were infete with AEts for nother h. The expression of Ets n PDX (), (), insulin ontent () n insulin seretion normlise to insulin ontent () ( mmol/l, white rs; mmol/l, lk rs) ws mesure. Vlues re mens±sd n re representtive of three iniviul experiments. p<., p<. vs ontrol IB: Ets IB: βatin Input (%). mmol/l mmol/l IP FOXO. mmol/l mmol/l. mmol/l IgG mmol/l Fol enrihment of DNA Ets omplex level..... mmol/l mmol/l Fig. Hyperglyemi promotes the intertion of Ets n FOXO, n the tivity of Ets ining to the Px promoter. RINmF ells were trete with. or mmol/l gluose for h. IP for intertion of Ets n FOXO () n ChIPqPCR nlysis for the pity of Ets ining to the Px promoter () ws performe. Vlues re mens±sd n re representtive of three iniviul experiments. p<. vsontrol. IB, immunolot siontrol siets e Insulin ontent g siontrol siets Insulin seretion (reltive to insulin ontent) Ets PDX βatin siontrol siets Min siontrol siets Mouse islets ritil for tivtion of insulin gene expression []. However, it is unler whether epigeneti moifition is involve in mnipulting Ets expression in pnreti et ells trete with high gluose. We first nlyse the promoter region of Ets, n foun there were mny CpG islns. However, AzCR, speifi inhiitor of DNA methyltion, h no effet on Ets expression (t not shown). Interestingly, the histone eetylse inhiitor oul upregulte Ets expression in pnreti et ells. Menwhile, high gluose inrese histone H n H etyltion t the to regions of the Ets promoter. These results lerly Insulin ontent h f Insulin seretion (reltive to insulin ontent) siontrol siets siontrol siets Insulin synthesis Hyperglyemi Ets PDX Histone etyltion Insulin seretion siontrol siets Fig. 7 Inhiition of Ets reverse hyperglyemiinue impirment in Min ells n mouse islets. Min ells n mouse islets were infete with enovirusontrolsirna (siontrol) or siets for h n then trete with high gluose (; mmol/l) for 7 h. Ets n PDX protein levels (), in Min ells (), in mouse islets (), insulin ontent in Min ells (e), insulin ontent in mouse islets (f), insulin seretion normlise to insulin ontent in Min ells (g) ( mmol/l, white rs; mmol/l, lk rs), n insulin seretion normlise to insulin ontent in mouse islets (h) (. mmol/l, white rs;.7 mmol/l, lk rs) were mesure. () Digrm epiting the role of Ets in et ell gluose toxiity n signlling pthwy. Vlues re mens±sd n re representtive of three iniviul experiments. p<.

8 Dietologi () 9: emonstrte the role of epigenetis in regulting Ets trnsription inue y high gluose. It is importnt to reognise tht Ets overexpression use reution in insulin ontent in pnreti et ells oth in vivo n in ellse moels. It is therefore possile tht erese S inue y Ets ws ue to reue insulin grnule formtion. Another possiility is tht Ets might impir insulin seretion from pnreti et ells. We foun tht overexpression of Ets still erese insulin seretion when normlise to insulin ontent. We lso foun tht tht overexpression of Ets reue ATP ontent uner high gluose onitions (ESM Fig. ) n GCK expression (ESM Fig.,). Given ATP n GCK re mjor ontriuting ftors involve in S [9, ], reution of ATP ontent n GCK expression oul prtiipte in Ets impiring insulin seretion. We ientifie, for the first time, PDX s the ownstrem trget of Ets in meiting its regultory effets on pnreti et ell funtion. We emonstrte tht Ets oul in to the promoter of Px (the PH region) shre with FOXO n FOXA []. When Ets ws overexpresse in et ells, the tivity of FOXO ining to the PDX promoter inrese, ut tht of FOXA ining to the sme site erese, whih ws ompnie y the reution of tivity of the Px promoter. Previous stuies emonstrte tht FOXO negtively regulte PDX expression through eresing Px promoter tivity while FOXA positively regulte PDX expression through inresing Px promoter tivity [, ]. We lso foun tht FOXO oul intert with Ets in pnreti et ells (Fig. ). As shown here n previously, we propose tht overexpression of Ets enhne FOXO oupying the re left vnt y FOXA on the Px promoter, whih resulte in inhiiting Px promoter tivity. It shoul e notie tht Ets i not in to the Px promoter in the ontrol group, n interferene of sl Ets gene trnsription i not ffet PDX expression (Fig.,), whih inite tht Ets only inhiits PDX expression uner ertin pthologil situtions suh s hyperglyemi. Similrly, the FOXO trnsription ftor emonstrte lmost no ining to the Px gene promoter, whih ws onsistent with our previous results []. Thus, neither Ets nor FOXO regulte PDX expression y ining to the Px promoter uner sl sitution. The erese of PDX expression n DNA ining tivities ws implite previously in hroni hyperglyemiinue et ell ysfuntion [7]. However, the potentil mehnisms were unler. Here, we estlishe onnetion etween high gluose, Ets elevtion n reution of PDX expression. We esrie in etil the useneffet reltionship etween the inrese level of Ets n erese levels of PDX in vivo n in vitro. High gluose inrese the tivity of Ets ining to the Px promoter (Fig. ). On the sis of the existing investigtions, we speulte tht Ets ws responsile for the reution in PDX expression uner the hyperglyemi onition. In onlusion, our stuy emonstrtes tht hroni hyperglyemi ontriutes to upregultion of Ets expression y meiting hyperetyltion of histone H n H t the Ets gene promoter, leing to reution of PDX expression, n finlly to et ell ysfuntion. This proess my e one of the moleulr mehnisms responsile for gluotoxiity in et ells. Aknowlegements The uthors grtefully knowlege the generous support of the Collortive Innovtion Center for Criovsulr Disese Trnsltionl Meiine of Jingsu Provine (Nnjing, Chin). Funing This work ws supporte y grnts from: () the Speil Funs for Mjor Stte Bsi Reserh Progrm of Chin (97 Progrm, CB) n the Stte Key Progrm of Ntionl Nturl Siene of Chin () to XH; () the Ntionl Nturl Siene Fountion of Chin () to FC. Contriution sttement All uthors took prt in the oneption n esign of the stuy, s well s either rfting or ritilly revising the mnusript. All uthors hve pprove the finl version of the mnusript. MS, YW, TW, WS, JL n WZ ollete the t n rrie out the t nlysis. XH is responsile for the integrity of the work s whole. Dulity of interest The uthors elre tht there is no ulity of interest ssoite with this mnusript. Referenes. Henquin JC () Triggering n mplifying pthwys of regultion of insulin seretion y gluose. Dietes 9:7 7. NeutzskyWulff AV, Anressen KV, Hjuler ST et l () Future etetion n monitoring of ietes my entil nlysis of oth etell funtion n volume: how mrkers of etell loss my ssist. J Trnsl Me :. Ahlgren U, Jonsson J, Jonsson L, Simu K, Elun H (99) Betellspeifi intivtion of the mouse Ipf/Px gene results in loss of the etell phenotype n mturity onset ietes. Genes Dev :7 7. Jons JC, Shrm A, Hsenkmp W et l (999) Chroni hyperglyemi triggers loss of pnreti et ell ifferentition in n niml moel of ietes. J Biol Chem 7:. Tokuym Y, Sturis J, DePoli AM et l (99) Evolution of etell ysfuntion in the mle Zuker ieti ftty rt. Dietes :7 7. Brunner Y, Shvrtz D, PriegoCpote F, Coute Y, Snhez JC (9) Gluotoxiity n pnreti proteomis. J Proteomis 7: Poitout V, Amyot J, Semhe M, Zrrouki B, Hgmn D, Fontes G () Gluolipotoxiity of the pnreti et ell. Biohim Biophys At :9 9. Melloul D () Trnsription ftors in islet evelopment n physiology: role of PDX in etell funtion. Ann N Y A Si : 7

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