Involvement of thioredoxin-interacting protein (TXNIP) in glucocorticoid-mediated beta cell death

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1 Dietologi (12) 55: DOI 1.7/s z ARTICLE Involvement of thioredoxin-interting protein (TXNIP) in gluoortioid-medited et ell deth E. Reih & A. Tmry & R. Vogt Sionov & D. Melloul Reeived: 27 June 11 / Aepted: 7 Novemer 11 / Pulished online: 14 Jnury 12 # Springer-Verlg 12 Astrt Aim/hypothesis Gluoortioid hormones (GCs) re widely used to tret vriety of inflmmtory nd immune diseses. However, their long-term dministrtion is ssoited with dverse metoli effets, inluding gluose intolerne nd dietes. Our ojetive ws to eluidte the mehnisms y whih GCs ffet et ell survivl with speifi emphsis on the role of the thioredoxin-interting protein (TXNIP) in et ell poptosis. Methods Humn nd mouse islets, together with MIN6 et ells, were exposed to dexmethsone () nd poptosis ws ssessed y mesuring the perentge of su-g1 ells, the pperne of leved spse-3 or y using TUNEL ssy. -upregulted expression of Txnip mrna ws nlysed y rel-time PCR, nd GC-modulted prodution nd modifition of proteins were determined y western lotting. Results We provide evidene tht TXNIP, negtive regultor of the ntioxidnt thioredoxin (TRX), is strongly indued in et ells y GCs nd tht its indution is dependent on p38 mitogen-tivted protein kinse (MAPK) tivtion. TXNIP downregultion y RNA interferene, overexpression of the rdil svenger TRX1 or elevtion of intrellulr AMP levels ttenuted the -medited poptosis. -indued Txnip expression nd et ell poptosis re medited y the gluoortioid reeptor (GR), s the GR ntgonist RU486 fully olishes these effets. E. Reih nd A. Tmry ontriuted eqully to this study. Eletroni supplementry mteril The online version of this rtile (doi:1.7/s z) ontins peer-reviewed ut unedited supplementry mteril, whih is ville to uthorised users. E. Reih : A. Tmry : R. V. Sionov : D. Melloul (*) Deprtment of Endorinology, Hdssh University Hospitl, PO Box 12, 91 Jeruslem, Isrel e-mil: dniellem@ekmd.huji..il Conlusions/interprettion Altogether, our dt suggest TXNIP s novel meditor of GC-indued poptosis in et ells nd further ontriute to our understnding of et ell deth pthwys. Keywords Apoptosis. Gluoortioids. Insulin produing et ells Arevitions Akt Protein kinse B ChREBP Crohydrte response element inding protein methsone GCs Gluoortioids GR Gluoortioid reeptor MAPK Mitogen-tivted protein kinse PDE Phosphodiesterse TXNIP Thioredoxin-interting protein TRX Thioredoxin VDUP1 Vitmin D 3 -upregulted protein 1 VitD3 1α,25-Dihydroxyvitmin D 3 Introdution Gluoortioids (GCs) re widely used to tret vriety of inflmmtory nd immune diseses [1]. However, they re lso ssoited with dverse metoli effets, inluding gluose intolerne nd dietes [2, 3]. Cumultive evidene indites tht lne exists etween insulin resistne nd n effetive et ell mss. Bet ells dequtely dpt to hnges in insulin sensitivity y vrying insulin seretion, thus mintining euglyemi. However, in suseptile individuls, when the et ell mss fils to ompenste for insulin resistne, type 2 dietes ensues. Indeed, et ell dysfuntion hs een knowledged s key defet underlying the

2 Dietologi (12) 55: development of type 2 dietes [4]. In GC-indued impired gluose tolerne, the ompenstory inrese in plsm insulin ppers to e ttenuted y the diret inhiitory effet of GCs on insulin seretion, s suggested oth y in vivo nd in vitro studies [5 8]. Severl reports indite tht the effets of GCs on et ell funtion re highly dependent on durtion of exposure, dosge nd suseptiility of the popultion exposed [9]. Helthy volunteers treted with high-doses of orl prednisolone showed utely impired insulin seretion [1, 11]. A few studies hve lso reported the role of these GCs during pnreti development [12]. In normlly nourished rt fetuses, inresed et ell mss ws ssoited with low ortiosterone levels, while deresed et ell mss ws oserved under onditions of fetl overexposure to GCs [13]. In ft, the onditionl intivtion of the gluoortioid reeptor (GR) in ll pnreti progenitors (GR-Pdx1-Cre mie) led to douling of et ell mss [14]. GCs hve lso een shown to suppress insulin [15] ndpdx1 [16] gene expression in et ells. Tken together, this reserh shows tht GCs ffet oth emryoni et ell development nd et ell funtion in the dult through interferene with vrious signlling pthwys, leding to impired et ell funtion nd ultimtely to poptosis [6 9, 17, 18]. The im of this study ws to etter understnd the mehnisms y whih GCs indue poptosis in et ells. Still little is known out the identity of the trget genes whose trnstivtion or trnsrepression re involved in GC-indued et ell poptosis. In n ttempt to further eluidte this proess, we tested the role of the TXNIP gene [19]. TXNIP, lso known s vitmin D 3 -upregulted protein 1 (VDUP-1) nd thioredoxininding protein-2 (TBP-2), is negtive regultor of the ntioxidnt thioredoxin (TRX) []. Cumultive evidene suggests link etween TRX nd mmmlin metolism through the tions of TXNIP. Txnip-null mie re hypoglyemi, hypoinsulinemi, nd show lunted gluose prodution following glugon hllenge, onsistent with entrl liver gluose-hndling defet [21]. Studies in pnreti et ells hve shown tht TXNIP funtions s n effetor of gluotoxiity, where high gluose stimultes [22], nd insulin represses its expression [23]. The gluose-indued inrese in Txnip trnsription is medited y rohydrte response element inding protein (ChREBP) [24]. Bet ell-speifi Txnip deletion (TKO) ws found to enhne et ell mss nd redue poptosis in streptozotoin-treted mie. Moreover, TXNIP defiieny leds to inresed protein kinse B (Akt)/poptosis regultor BCL-X (Bl-xL) signlling nd redued mitohondril et ell deth, suggesting tht these mehnisms my medite the et ell protetive effets in TXNIP-defiient ells [25, 26]. GCs re known to indue poptosis nd redue prolifertion in vriety of ell types y trgeting distint intrellulr signlling pthwys depending on the ell system nd on the responsiveness of the poptoti mhinery [27, 28]. The gene expression profile of dexmethsone ()-treted WEHI7.2 T ell lymphom ells reveled tht TXNIP mrna ws signifintly upregulted. It ws further demonstrted tht TXNIP is involved in mediting gluoortioid-indued poptosis in these ells nd in dult T ell leukemi [29]. In n ttempt to identify genes diretly ffeted y GCs nd involved in eliiting the poptoti proess in et ells, we present evidene tht GCs strongly indue the expression of the redox regultory protein in oth humn nd mouse islets, s well s in MIN6 et ells. While overexpression of Txnip ws suffiient to indue poptosis, its repression signifintly inhiited -indued poptosis. Both -indued Txnip expression nd poptosis in et ells re medited through the GR nd depend on the tivtion of the p38 MAPK pthwy. Together, our dt suggest TXNIP s one of the meditors of GC-indued poptosis in et ells. Methods Regents 3-[4,5-Dimethylthizol-2-yl]-2,5-diphenyltetrzolium romide; thizolyl lue (MTT), mifepristone (RU486), forskolin, IBMX (isoutylmethylxnthine) nd 1α,25-dihydroxyvitmin D 3 (VitD3) were purhsed from Sigm- Aldrih (St Louis, MO, USA); methylprednisolone sodium suinte ws purhsed from Pfizer, Belgium. Antiodies ginst were from Zymed (Invitrogen, Grnd Islnd, NY, USA), ntiodies to TRX1, leved spse-3 (Asp175), phospho-p38 MAPK (Thr1/Tyr182), nd Akt nd phospho-akt (Ser473) were from Cell Signling Tehnology (Dnvers, MA, USA) nd α-tuulin from Sigm-Aldrih. Sodium phosphte dexmethsone () solution ws from the Myln phrmeutil ompny, nd the p38 inhiitor SB35 ws purhsed from AG Sientifi (Sn Diego, CA, USA). Cell ulture nd islet tissue isoltion Mouse MIN6 et ells, kindly provided y J.-I. Miyzki (University of Osk, Jpn), nd rt INS1 ells were ultured in DMEM 2g/l nd RPMI 16 medium (Biologil Industries, Kiutz Beit Hemek, Isrel), with 13% nd 1% het intivted fetl ovine serum (FBS) respetively nd μmol/l et-merptoethnol. Mouse α-tc6.1 glugonom ells were mintined in DMEM 4.5g/l nd 1% FBS. Culture medi were supplemented with 2 mmol/l L-glutmine, peniillin ( U/ml) nd streptomyin ( μg/ml). Mouse islets were isolted from C57Bl/6 mie nd ultured s desried previously [3]. Humn islets were otined from the Europen Consortium for Islet Trnsplnttion (ECIT) t the Sn Rffele Hospitl (Miln, Itly). MTT ell viility ssy nd TUNEL ssy The viility of ells ws evluted using MIN6 ells ultured in 24-

3 15 Dietologi (12) 55: well pltes. On the following dy, the ells were inuted with the indited tretments. During the lst 3 min of inution, MTT solution ws dded t finl onentrtion of.15 g/l. After removing the medium, 5 μl of dimethylsulfoxide ws dded to eh well to dissolve the formzn. The sorne ws red in n ELISA reder t 55 nm. Viility ws lulted in omprison with ontrol ells. Eh tretment ws performed in triplite. The TUNEL ssy ws performed s previously desried [3]. Cell yle nlysis MIN6 ells were seeded in six-well pltes nd, the following dy, the medium ws hnged to fresh medium in the presene or sene of. At the end of inution, the floting ells, s well s the trypsinised dherent ells, were olleted. Following entrifugtion, the ells were fixed in ie-old methnol, rehydrted in PBS, treted with 5 μg/ml DNse-free RNse, nd inuted in 5 μg/ml propidium iodide for 3 min prior to ell yle nlysis y flow ytometry (FACStr, Beton Dikinson, Frnklin Lkes, NJ, USA). Ten thousnd events were ounted for eh smple, where su-g1 ells re onsidered poptoti ells. In trnsfetion studies with GFP fusion proteins, seprte ell yle nlysis ws done on the GFP positive ( trnsfetnt ) nd the GFP negtive ( non-trnsfetnt ) ell popultions of the sme smple. Anlysis ws performed using Cellquest Softwre. Western lotting Cells were lysed in Lemmli protein smple uffer, seprted on SDS-polyrylmide gel, nd trnsferred to.2 μm nitroellulose memrnes (Shleiher nd Shuell, Dssel, Germny). After loking in 5% dried skimmed milk, the memrnes were first inuted with the pproprite ntiodies, nd then with horserdish peroxidse onjugted ntirit or nti-mouse IgG, followed y nlysis with n enhned hemiluminesene detetion kit (Biologil Industries). sirna eletroportion MIN6 ells were suspended in μl of Amx nuleofetor solution nd eletroported with 1 μmol/l Txnip sirna or ontrol sirna (Invitrogen) using Amx Nuleoportor (Lonz, Wlkersville, MA, USA). The ells were trnsferred into fresh medium immeditely fter eletroportion nd ultured for 24 h prior to tretments. Cell trnsfetion MIN6 ells were seeded in 12-well pltes nd, on the following dy, ells were trnsfeted in Opti-MEM medium (GIBCO, Invitrogen) with either humn TRX1-Gfp or mouse Txnip-Gfp using Lipofetmine (Invitrogen). Twenty hours lter, fresh medium ws dded in the presene or sene of. Rel-time PCR RNA ws isolted from ells y using TRIzol regent (Invitrogen) nd reverse trnsried into DNA using the Mstersript kit (5 PRIME, Hmurg, Germny) nd rndom primers (Applied Biosystems, Crlsd, CA, USA). Rel-time PCR ws performed using Pltinum SYBR Green qpcr SuperMix-UDG with ROX referene dye (Invitrogen). The mouse primers used re presented s eletroni supplementry mteril (ESM) Tle 1. Sttistil nlysis The dt re presented s men±sd. Student s t test ws used to ompre treted smples with untreted ontrol smples. A p vlue <.5 ws onsidered sttistilly signifint. Results GCs indue poptosis of et ells MIN6 et ells were exposed to vrious onentrtions of (1-5 nmol/l) for h, nd the ell viility ws determined y MTT ssy. Figure 1 shows tht signifintly deresed et ell survivl with time nd mximl effet ws oserved with Viility (%) Viility (%) Viility (%) 167 Pred (nmol/l) d Viility (%) (nmol/l) Fig. 1 Dose- nd time-dependent effet of GCs on ell viility. MIN6 ells were exposed for 24 h (white rs), 48 h (lk rs) or 72 h (hthed rs) to inresing onentrtions of. MIN6 ells were exposed for 48 h (lk rs) nd 72 h (hthed rs) to 167 nmol/l methylprednisolone (Pred). MIN6, d INS1 insulinom ells nd e α-tc6.1 glugonom ells were treted for 48 h with nmol/l (). Cell viility ws evluted y using the MTT ssy. Vlues re the verge of t lest three experiments ± SD, p<.1 vs unexposed ontrol e Viility (%)

4 Dietologi (12) 55: nmol/l. This effet ws lso oserved when MIN6 ells were exposed to methylprednisolone (Fig. 1). To vlidte whether the deleterious effet of on ell survivl ws speifi to et ells, MIN6 nd INS1 insulinoms, s well s the αtc6.1 glugonom ell line, were exposed to nmol/l for 48 h nd ell viility ws mesured. The results presented in Fig. 1 e onfirm tht signifintly deresed the viility of MIN6 (3±6 %) nd INS1 (25±4 %) et ells, ut hd rely ny effet on the viility of αtc6.1 or tht of the humn PANC1 pnreti dutl rinom ells (dt not shown). To eluidte whether the derese in ell viility is, t lest in prt, due to et ell poptosis, we nlysed the DNA ontent of -treted MIN6 ells for 48 or 72 h y flow ytometry nd mesured the su-g1 DNA pek ssoited with poptoti ells rrying hypodiploid DNA ontent. The results presented in Fig. 2 demonstrte tht tretment leds to n inrese in the perentge of ells undergoing poptosis. Further evidene for -medited poptosis omes from western lot nlysis demonstrting the tivtion of spse-3 in MIN6 ells exposed to the hormone for 16, 48 or 72 h (Fig. 2). The effets of dexmethsone on islet et ell dysfuntion depend on the durtion nd dose of exposure [3, 9, 31]. Sine -indued poptosis of pnreti islet et ells is deteted t signifint levels following long-term exposure, we inuted isolted mouse islets with nmol/l for 9 dys nd the prevlene of DNA dmge in et ells ws evluted y lelling DNA reks, whih re hrteristi event ourring in the lte stges of poptosis. Histologil nlysis of poptoti nulei o-stined for insulin onfirmed tht et ells re more suseptile to -indued poptosis (13.5% poptoti et ells/islet 4.8% in untreted islets; Fig. 2). methsone ffets nd TRX expression In n ttempt to lrify the mehnisms involved in -indued et ell deth, we investigted the potentil role of TXNIP in this poptoti proess. TXNIP, initilly identified s VDUP-1 [19], ws lter shown to e upregulted in mouse lymphom ell line [32] y. To study whether ould lso upregulte TXNIP in MIN6 ells, these ells were treted with either inresing onentrtions of or VitD3 for 48 h or with nmol/l for vrious time periods. VitD3 ws used here s positive ontrol for TXNIP prodution. The TXNIP/VDUP- 1 protein levels were nlysed y western lot nd the results presented in Fig. 3 show n umultion of TXNIP protein oth with inresing onentrtions of (Fig. 3) or VitD3 (Fig. 3) nd with time (Fig. 3). The indution of Txnip gene expression with time ws onfirmedyrel-timepcrnlysis of mrna levels in MIN6 ells treted with nmol/l for vrious time periods (Fig. 3d). TXNIP ws lso signifintly indued in humn nd mouse islets y nmol/l (Fig. 3 e, f). Altogether, our dt show strong tivtion of Txnip gene expression y, similr to tht otined with VitD3. As gluose is known to stimulte Txnip expression in et ells [24], we ompred the ility of to indue TXNIP in mouse islets with tht of high gluose (25 mmol/l). A roust indution ws oserved with oth tretments (Fig. 3f). -indued Txnip expression nd poptosis re medited through the GR To test the diret involvement of the GR in et ell deth, MIN6 ells were exposed to nmol/l of in the presene or sene of 1 μmol/l RU486, GR ntgonist. RU486 fully prevented et ell deth s mesured y MTT ssy (Fig. 4) nd the level of leved spse-3 (Fig. 4). Furthermore, RU486 loked the indution of TXNIP protein synthesis y (Fig. 4), inditing tht -stimulted TXNIP prodution depends on GR tivtion. RU486 lso signifintly inhiited the -medited redution in TRX1 levels oserved in MIN6 ells (Fig. 4). Of note, the redution of TRX1 protein y ourred despite no signifint ltertions in Trx1 mrna levels (dt not shown). Su-G1 (%) Time (h) Cleved spse 3 α-tuulin TUNEL positive/islet (%) * Fig. 2 indues poptosis in MIN6 nd mouse islet et ells. MIN6 ells were exposed to nmol/l for 48 h (lk rs) or 72 h (hthed rs) nd nlysed y flow ytometry. The perentge of the su-g1 ell popultion represents poptoti ells. The dt represent n verge ± SD of three independent smples. p<.7. MIN6 ells were treted with nmol/l for the indited time periods nd proessed for western lot nlysis using ntiodies speifi to leved spse-3 or α-tuulin. Isolted mouse islets were treted with nmol/l () for 9 dys nd the perentge of poptoti nulei ws evluted using TUNEL ssy in ells o-stined for insulin. *p<.5

5 152 Dietologi (12) 55: (nmol/l) Vitmin D 3 (nmol/l) α-tuulin α-tuulin α-tuulin 4 h 8 h 12 h 24 h 48 h d Txnip/Gpdh mrna (fold) * Time (h) * * * e Humn islets α-tuulin f Gluose (mmol/l) Mouse islets α-tuulin Fig. 3 indues TXNIP prodution in MIN6 et ells, nd mouse nd humn islets. nd MIN6 ells were exposed to vrious onentrtions of or VitD3 s indited for 48 h prior to western lot nlysis using ntiodies to TXNIP or α-tuulin. Time-dependent prodution of TXNIP in MIN6 ells exposed to nmol/l () for the indited time periods. d Rel-time PCR nlysis of Txnip mrna expression in MIN6 ells exposed to nmol/l (lk rs) for the indited time periods. Gpdh mrna ws used s internl stndrd. The verge of three seprte experiments±sd is presented s fold inrese reltive to untreted ells (ontrol, white rs). *p<.5. e Humn islets were exposed to nmol/l () for 4 dys prior to western lot nlysis using ntiodies to TXNIP or α-tuulin. f Mouse islets were exposed to 11 mmol/l gluose in the sene ( ) or presene () of nmol/l or to 25 mmol/l gluose for 3 dys s indited, prior to western lot nlysis using ntiodies to TXNIP or α-tuulin Altogether, these dt indite tht GCs hve potentilly negtive effet on the redox regultory mehnisms of MIN6 ells. On the one hnd, indues the expression of TXNIP whih inhiits TRX1 tivity [14,, 21], nd on the other hnd, dereses TRX1 protein levels, therey further reduing the redox-regulting ility of the ell. The GR ntgonist RU486 prevented these effets, thus resuing et ells from -indued poptosis. TXNIP potentites nd TRX1 ttenutes poptosis in MIN6 ells To further determine the ontriution of TXNIP in -indued et ell poptosis, n expression vetor enoding the mouse Txnip gene fused to tht of enhned green fluoresent protein (Txnip-Gfp) ws trnsiently expressed in MIN6 ells. Twenty hours post-trnsfetion, the ells were inuted for n dditionl 48 h in the presene or sene of nmol/l. DNA ontent nd TXNIP-GFP levels were simultneously nlysed y flow ytometry. The perentge of su-g1 ells in the GFP positive supopultion ws ompred with tht of the GFP negtive ells within the sme ell ulture. DNA frgmenttion (su-g1 DNA ontent) inditive of poptoti ells ws deteted in.4±3.42% of ells produing TXNIP- GFP, in omprison with only 8.2±1.39% in the GFP negtive supopultion (Fig. 5, p<.5). These vlues were further inresed in the presene of. Sine TXNIP ssoites with redued TRX [], it ould exert its pro-poptoti effet t lest in prt y inhiiting TRX tivity. To test this hypothesis in -indued et ell deth, MIN6 ells were trnsiently trnsfeted with TRX1-GFP. Following trnsfetion, the ells were inuted with or without nmol/l. Figure 5 shows tht ells produing TRX1-GFP prtilly resued MIN6 ells from -indued poptosis (11.9±3.9% vs 24.3±4.8%; p<.2). These findings indite tht TXNIP lone is suffiient to indue poptosis nd tht overexpression of the rdil svenger TRX1 n, to ertin degree, protet et ells from -indued poptosis. Downregultion of endogenous Txnip expression redues the sensitivity of MIN6 ells to -medited poptosis To further investigte the involvement of endogenous TXNIP in -indued poptosis of et ells, RNA interferene ws used to repress Txnip expression. To this end, MIN6 ells were eletroported with Txnip sirna or ontrol sirna prior to tretment for 48 h. Knokdown of Txnip ttenuted the -indued poptosis of MIN6 ells s demonstrted y redued tivtion of spse-3 on western lot (Fig. 5). In ddition, ell viility ws determined y the MTT ssy nd Fig. 5d shows mild inrese in the survivl of -treted et ells produing lower TXNIP levels (64% vs 77% p<.7). These results

6 Dietologi (12) 55: Viility (%) RU-486 phosphoryltion, whih remined t reltively high levels up to 24 h fter stimultion (Fig. 7). To ssess whether p38 MAPK tivtion regultes Txnip expression, MIN6 ells were pretreted with μmol/l of the p38 inhiitor SB35 for min or with DMSO prior to tretment for 72 h. The p38 inhiitor redued the GC-indued TXNIP protein levels y 5% nd loked the tivtion of spse-3 y 7% (Fig. 7). Moreover, it prtilly lunted the -redued MIN6 ell viility, whih inresed from 71% to % (p<.4) nd from 48% to 61% (p<.2) following 48 or 72 h tretment, respetively (Fig. 7) s determined y the MTT ssy. The weker effet of p38 inhiition on MTT ssy indites tht -medited p38 MAPK is speifilly involved in GC-indued Txnip expression nd poptosis, while slightly relieving the metoli impirments ssoited with GC exess. RU-486 TRX-1 Cleved spse 3 α-tuulin Fig. 4 The GR ntgonist RU486 prevents TXNIP indution nd TRX downregultion nd protets MIN6 ells from -indued poptosis. MIN6 ells were treted with nmol/l nd/or with 1 μmol/l RU486 for 48 h nd ell viility ssessed y MTT ssy. Vlues represent the verge of t five experiments in qudruplite±sd, p<.1. Western lot nlysis of MIN6 ells treted with nmol/l nd/or 1 μmol/l RU486 for 48 h using ntiodies direted ginst the indited proteins support our hypothesis tht TXNIP minly ontriutes to -indued et ell deth. Elevted intrellulr AMP levels prevent the effets of -indued Txnip expression on et ell viility Inresed intrellulr AMP levels hve previously een shown to ttenute -indued et ell deth using exendin-4 nd forskolin [18, 33]. This urged us to study the effet of the phosphodiesterse inhiitor IBMX or the denylte ylse tivtor forskolin in MIN6 ells. The elevted AMP levels signifintly redued -stimulted Txnip expression oth t the protein (Fig. 6) nd t the RNA levels (Fig. 6,d). Under these onditions, inresed AMP lso prevented the redution in Akt tivtion (phospho-akt, S473) used y tretment (Fig. 6) nd lso proteted MIN6 ells from -redued ell viility (Fig. 6e, f). Indution of Txnip in GC-treted ells depends on the p38 MAPK pthwy Next, we investigted the signlling mehnisms involved in GC-indued TXNIP expression. MIN6 ells inutedwithshowedninreseinp38mapk Disussion Gluoortioids (GCs), whih tivte GR signlling nd thus modulte gene expression, re widely used to tret immune-medited diseses [1]. The role of GCs in et ell funtion hs een extensively studied in oth humns nd rodents [3]. In helthy men, it ws demonstrted tht ute nd hroni exposure to prednisolone led to impired et ell funtion in ddition to reduing insulin sensitivity [1, 11]. In trnsgeni mie speifilly overexpressing the GR in et ells, GCs inhiited insulin relese in vivo, suggesting tht the pnreti et ell is diret trget of the dietogeni effet of GCs [6]. Interestingly, these nimls develop dietes with ge [34]. In vitro, severl studies hve shown tht rodentderived islets deresed gluose-stimulted insulin relese following oth ute nd prolonged exposure to vrious GC nlogues [7, 9, 17, 31, 35 38]. In ddition to perturing the proess of insulin seretion, GCs were shown to derese et ell speifi gene expression nd indue poptosis [18, 33, 39]. However, it is still unler whih of the genes regulted y GCs ontriute to et ell deth. One of the GR-regulted genes is TXNIP, whih ws originlly found to e upregulted y gluose in humn islets using trnsription profiling (Shlev et l. []; L. Hvin nd L. Amior, unpulished dt). The effet of gluose on gene expression ws shown to e medited y ChREBP [24], nd inresed levels of TXNIP were ssoited with gluotoxiity [22 24, 26, 41]. In the present study, we provide new evidene imed t eluidting the mehnisms involved in GC-indued poptosis of et ells. We demonstrte tht strongly indues the expression of in oth MIN6 nd INS1 et ell lines s well s in norml humn nd mouse pnreti islets. In this study we show tht, in mouse islets nd in MIN6 ells, ppers to e slightly more potent induer of TXNIP thn high gluose (25 mm).

7 154 Dietologi (12) 55: Su-G1 (%) 3 1 Su-G1 (%) 3 1 sirna Cleved spse 3 α-tuulin on Txnip on Txnip d Viility (%) sirna on Txnip on Txnip Fig. 5 TXNIP potentites, nd TRX1 ttenutes poptosis in MIN6 et ells. MIN6 ells were trnsfeted with either Txnip-Gfp () or TRX1-Gfp (). Twenty hours post-trnsfetion, the ells were inuted for n dditionl 48 h in the presene or sene of. Cells were then olleted nd for eh smple, ell yle nlysis ws performed seprtely on TXNIP-GFP-positive ells (lk rs) nd TXNIP-GFPnegtive ells (white rs), nd the perentge of su-g1 ells determined y flow ytometry nlysis. The dt re the verge of three to five independent trnsfetions±sd, p<.5. Txnip downregultion in MIN6 ells ttenutes -indued poptosis. MIN6 ells were eletroported with Txnip sirna or ontrol sirna (on). Cells were exposed the following dy to nmol/l () for 48 h. Protein extrts were nlysed y western lot using ntiodies direted ginst the indited proteins. d Cell viility ws evluted y using the MTT ssy. Vlues re the verge of four to eight experiments±sd, p<.1 The inresed GC-medited Txnip expression with time ws ompnied y onomitnt indution of et ell deth. Apoptosis ws shown to e one of the mehnisms responsile for the oserved redued et ell viility, s determined y leved spse-3 nd the inresed perentge of su-g1 ells. Indeed, overexpression of Txnip in MIN6 ells inresed oth sl nd -indued poptosis. This is onsistent with previous findings regrding the role of TXNIP in GC-indued poptosis in T ells [29, 31] nd in et ells [22]. TXNIP is known to ssoite with redued TRX, one of the mjor pro-survivl thiol reduing systems in the ells [] nd ould exert its pro-poptoti effet t lest in prt y loking TRX tivity. Our study shows tht MIN6 ells overexpressing the rdil svenger TRX1 prtilly resued MIN6 ells from -indued poptosis. The role of TRX in proteting et ells in oth type 1 nd type 2 dietes hs een reported. Indeed, et ell speifi expression of Trx1 in NOD mie slows the development of the disese, without preventing insulitis [42]. In the d/d mouse model of type 2 dietes, overexpression of Trx1 signifintly suppressed the progression of hyperglyemi [43]. TRX1 prevented the redution of the pnreti nd duodenl homeoox 1 (PDX1) nd V-mf musuloponeuroti firosrom onogene homologue A (MfA) trnsription ftors s well s islet insulin ontent in these mie. In ontrst to the reiprol pttern of expression etween Txnip nd Trx1 mrna previously reported [44 46], the -redued TRX1 protein levels in GC-treted MIN6 ells ourred despite no signifint ltertions in Trx1 mrna (dt not shown). We further demonstrte the ontriution of TXNIP in GC-indued et ell poptosis y deresing its expression using sirna. MIN6 ells trnsfeted with Txnip sirna showed signifint derese in spse-3 tivtion. In greement with our results, TXNIP defiieny proteted pnreti et ells from gluose toxiity nd poptosis [22] nd resued mie from streptozotoin-indued dietes. Moreover, TXNIP defiieny prevented the development of type 2 dietes in o/o mie through preservtion of et ell mss nd funtion [25]. Cumultive evidene indites tht tivting AMP signlling ttenutes GC-indued et ell dysfuntion nd prevents poptosis [11, 18, 47, 48]. Here, we present evidene tht elevted intrellulr AMP levels lso resue MIN6 et ells from the negtive effets of on ell viility, onomitnt with derese in TXNIP protein levels. We further show tht elevted intrellulr AMP levels prevent the indued redution in phospho-akt levels. Our findings ord with previous studies showing tht the glugon-like peptide 1 (GLP-1) nlogue exendin-4, whih inreses AMP onentrtions, ntgonises -indued poptosis of INS1 et ells [18]. Gluoortioids were shown to inrese phosphodiesterse (PDE) tivity leding to redued intrellulr AMP levels [47]. More reently, this group reported tht AMP signlling stimultes protesome degrdtion of gluoseindued TXNIP in INS1 et ells [48]. It is therefore oneivle tht elevted intrellulr AMP levels (y either stimulting PDE tivity using the inhiitor IBMX, or denylte ylse using forskolin, exposing et ells to GLP-1 or to

8 Dietologi (12) 55: α-tuulin e p-akt (Ser473) 6 h 12 h 24 h IBMX Viility (%) Akt Forskolin Txnip/Gpdh mrna (fold) Forskolin its nlogue exendin-4) reverse the -inhiitory effet on Akt signlling nd indue the degrdtion of TXNIP, reduing its pro-poptoti effet, leding to inresed et ell survivl. Another importnt finding of our study is tht the upregultion of TXNIP y is dependent on p38 MAPK tivtion. Prevention of the sustined tivtion of the p38 pthwy ourring fter tretment signifintly redued oth Txnip expression nd spse-3 tivtion, nd restored, to ertin extent, MIN6 ell viility. The d * * * 4 * 2 IBMX Txnip/Gpdh mrna (fold) f Viility (%) IBMX Fig. 6 Inresed intrellulr AMP prevents -indued TXNIP prodution nd et ell deth nd restores Akt phosphoryltion., MIN6 ells were exposed to nmol/l () for 6, 12 or 24 h in the presene () or sene () of μmol/l IBMX prior to western lot nlysis using ntiodies to TXNIP or α-tuulin (); to phospho-akt (Ser 437) (p-akt) or totl Akt ()., d Rel-time PCR nlysis of Txnip mrna expression in MIN6 ells exposed to nmol/l (hthed nd dotted rs) for 24 h in the presene () or sene () of1μmol/l forskolin () or μmol/l IBMX (d). Gpdh mrnaws used s internl stndrd. The verge of three seprte experiments±sd is presented s fold inrese reltive to untreted ells. *p<.5. e, f MIN6 ells were exposed to nmol/l (hthed nd dotted rs) for 48 h inthepresene()orsene() of1μmol/l forskolin (e) or μmol/l IBMX (f). Cell viility ws evluted y using the MTT ssy. Vlues re the verge of t lest three experiments in duplite±sd, p<.3 Phospho-p38 α-tuulin Cleved spse 3 α-tuulin Viility (%) 4 h 8 h 12 h 24 h p38i p38i Fig. 7 -indued p38 tivtion ontriutes to TXNIP indution nd et ell deth. MIN6 ells were exposed to nmol/l () for 4, 8, 12 nd 24 h prior to western lot nlysis using ntiodies to phospho-p38 (Thr 1/Tyr 182), or α-tuulin. MIN6 ells were exposed to nmol/l () for 72 h in the presene () or sene () of the p38 MAPK inhiitor SB35 nd then nlysed y western lot using the indited ntiodies. MIN6 ells were exposed to nmol/l for 48 h (white rs) or 72 h (lk rs) in the presene () or sene () of the inhiitor SB35 nd ell viility ws evluted y MTT ssy. Vlues re the verge of t lest three experiments±sd, p<.2 weker effet of p38 inhiition on the MTT ssy indites tht -medited p38 MAPK is minly involved in GCindued Txnip expression nd poptosis, while slightly relieving the metoli impirments ssoited with GC exess. p38 MAPK hs lso een doumented to e involved in TXNIP upregultion y high gluose in endothelil ells [49] nd in mouse mesngil ells [5]. GCs led to the tivtion nd nuler trnslotion of the GR, where it regultes gene trnsription through trnstivtion nd trnsrepression [51]. Some of the genes ffeted y GCs hve een shown to e involved in GC-indued poptosis of lymphom ells [27, 28]. Our present dt suggest the involvement of TXNIP s one of the meditors of GC-indued poptosis in et ells nd its potentil ontriution to the development of steroid dietes. While oth GR nd p38 tivtion re required for TXNIP indution nd et ell

9 156 Dietologi (12) 55: deth, future studies re required to mp the pro-poptoti nd pro-survivl pthwys modified y nd/or TXNIP in islet et ells. Aknowledgements We re very grteful to L. Hvin for the mirorry nlysis, A. Mklkov for her ssistne, nd to D. Sever nd L. Amior for their tehnil help nd very helpful disussions. Our sinere thnks to L. Piemonti nd N. Rit, Europen Consortium for Islet Trnsplnttion t the Sn Rffele Hospitl (Miln, Itly) for the supply of humn islets. Mny thnks to D. Sever for helping with grphi design. Funding This work ws supported y grnts from the Europen Union (STREP Sveet, ontrt no. 3693) in the Frmework Progrmme 6 nd y the Isrel Siene Foundtion (grnt no ). Dulity of interest The uthors delre tht there is no dulity of interest ssoited with this mnusript. Contriution sttement ER, AT nd RS performed reserh, nlysed dt, ontriuted to the oneption nd design, nd nlysis nd interprettion of dt desried in this study. RS supervised nd ll uthors ritilly revised the mnusript. 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