Deletion of Fas protects islet beta cells from cytotoxic effects of human islet amyloid polypeptide

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1 Dietologi (212) 55: DOI 1.17/s ARTICLE Deletion of Fs protects islet et cells from cytotoxic effects of humn islet myloid polypeptide Y. J. Prk & S. Lee & T. J. Kieffer & G. L. Wrnock & N. Sfikhn & M. Speck & Z. Ho & M. Woo & L. Mrzn Received: 15 Septemer 211 / Accepted: 9 Decemer 211 / Pulished online: 1 Ferury 212 # Springer-Verlg 212 Astrct Aims/hypothesis Islet myloid, which is minly composed of humn islet myloid polypeptide (hiapp), is pthologicl chrcteristic of type 2 dietes nd lso forms in cultured nd trnsplnted islets. We used islet et cells s well s two ex vivo models of islet myloid formtion, cultured humn islets nd hiappexpressing trnsgenic mouse islets with or without et cell Fs deletion, to test whether: (1) the ggregtion of endogenous hiapp induces Fs upregultion in et cells; nd (2) deletion or locking of Fs protects et cells from myloid toxicity. Electronic supplementry mteril The online version of this rticle (doi:1.17/s ) contins peer-reviewed ut unedited supplementry mteril, which is ville to uthorised users. Y. J. Prk : S. Lee : T. J. Kieffer : G. L. Wrnock : N. Sfikhn : M. Speck : L. Mrzn () Deprtment of Surgery, Fculty of Medicine, University of British Columi, Jim Pttison Pvilion, Vncouver Generl Hospitl, 91 W 1th Ave, Vncouver, BC, Cnd V5Z 4E3 e-mil: lucy.mrzn@uc.c T. J. Kieffer Deprtment of Cellulr nd Physiologicl Sciences, Fculty of Medicine, Life Sciences Institute, University of British Columi, Vncouver, BC, Cnd Z. Ho : M. Woo Deprtment of Medicine, University of Toronto, Ontrio Cncer Institute, St Michel s Hospitl, Toronto, ON, Cnd Methods INS-1, mouse or humn islet cells were cultured with hiapp lone, or with myloid inhiitor or Fs ntgonist. Non-trnsduced islets, nd humn islets or hiappexpressing mouse islets trnsduced with n denovirus tht delivers humn proiapp-specific smll interfering RNA (sirna) (Ad-ProhIAPP-siRNA) were cultured to form myloid. Mouse islets expressing hiapp with or without Fs were similrly cultured. Bet cell Fs upregultion, cspse- 3 ctivtion, poptosis nd function, nd islet IL-1β levels were ssessed. Results hiapp tretment induced Fs upregultion, cspse- 3 ctivtion nd poptosis in INS-1 nd islet cells. The myloid inhiitor or Fs ntgonist reduced poptosis in hiapp-treted et cells. Islet cells with Fs deletion hd lower hiapp-induced et cell poptosis thn those expressing Fs. Ad-ProhIAPP-siRNA-medited myloid inhiition reduced Fs upregultion nd IL-1β immunorectivity in humn nd hiapp-expressing mouse islets. Cultured hiapp-expressing mouse islets with Fs deletion hd similr myloid levels, ut lower cspse-3 ctivtion nd et cell poptosis, nd higher islet et:lph cell rtio nd insulin response to glucose, compred with islets expressing Fs nd hiapp. Conclusions/interprettion The ggregtion of iosynthetic hiapp produced in islets induces et cell poptosis, t lest prtilly, vi Fs upregultion nd the Fs-medited poptotic pthwy. Deletion of Fs protects islet et cells from the cytotoxic effects of endogenously secreted (nd exogenously pplied) hiapp. Keywords Amylin. Amyloid. Bet cell poptosis. Fs receptor. Islet myloid polypeptide. Type 2 dietes

2 136 Dietologi (212) 55: Arevitions Ad-Cont-siRNA Ad-ProhIAPP-siRNA HFIP hiapp riapp Introduction An denovirus tht delivers rndom non-specific smll interfering RNA An denovirus tht delivers humn proiapp-specific smll interfering RNA 1,1,1,3,3,3-Hexfluoro-2-propnol Humn islet myloid polypeptide Rt islet myloid polypeptide Islet myloid formtion is pthologicl chrcteristic of the pncres in type 2 dietes nd contriutes to progressive et cell dysfunction nd deth in this disese [1 3]. Islet myloid lso forms in humn islets during culture [4] nd following trnsplnttion into mouse models of type 1 dietes [5 7]. Importntly, widespred myloid deposition ws reported in humn islets tht hd een trnsplnted into ptient with type 1 dietes [8]. Amyloid formtion in trnsplnted islets is ssocited with et cell dysfunction nd recurrence of hyperglycemi in niml models of type 1 dietes [7, 9]. Thus, in ddition to its role in the pthogenesis of type 2 dietes, islet myloid formtion my lso ply role in islet grft filure in ptients with type 1 dietes. Islet myloid is minly formed y the ggregtion of islet myloid polypeptide (mylin) [1, 11], 37-mino cid hormone tht is co-loclised nd co-secreted with insulin in response to et cell secretgogues [12, 13]. Despite considerle study during the pst decde, it is still not cler why normlly solule humn islet myloid polypeptide (hiapp) molecules form toxic ggregtes in type 2 dietes. The following hve een proposed to contriute to the ggregtion of hiapp molecules in type 2 dietes: (1) presence of n myloidogenic sequence in the hiapp molecule [2, 14]; (2) elevted hiapp production nd secretion from et cells, ssocited with n incresed demnd for insulin [13, 15]; nd (3) defects in trfficking nd processing of proiapp, ssocited with et cell dysfunction [13, 15 17]. The rpid formtion of myloid in cultured nd trnsplnted humn islets my e relted to poor clernce of relesed hiapp from et cells due to the disrupted vsculture [18] nd/or to the impired proiapp processing nd secretion tht re ssocited with et cell dysfunction induced y islet isoltion, culture nd trnsplnttion [13, 19]. Aggregtes of hiapp, including smll oligomeric species, re toxic to et cells nd hve een shown to induce et cell dysfunction nd poptosis in isolted humn islets [4, 2]. Previous in vitro studies hve suggested different mechnisms for the et cell poptosis medited y hiapp ggregtes, including disruption of memrne integrity nd formtion of non-selective ion-chnnel-like structures [21 23], ctivtion of the cspse pthwys [24 27] nd interction of hiapp firils with components of et cell memrnes such s heprn sulphte proteoglycns [28, 29] or touch receptors [3]. It is, however, not cler which of these mechnism(s) contriute(s) to hiapp-induced et cell deth in primry islets, in which the level of endogenously produced hiapp (pmol/l) is severl fold lower [1, 31] thn tht used in vitro (μmol/l). Recent studies suggest tht endoplsmic reticulum stress [32 34], oxidtive stress [35] nd disruption in the utophgy lysosoml pthwy [36] due to formtion of intrcellulr hiapp ggregtes lso contriute to the cytotoxic effects of hiapp. It therefore ppers tht, t lest in vitro, hiapp-induced et cell poptosis involves severl mechnisms, some of which my shre the sme poptotic signlling pthwys. Fs (CD95/APO-1) is trnsmemrne receptor protein elonging to the tumour necrosis fctor superfmily of receptors [37 39]. The extrinsic poptotic pthwy initited y the interction etween Fs nd Fs lignd (FsL/CD95L) hs een implicted in utoimmune-medited et cell deth in type 1 dietes [4 42] nd non-immune-medited et cell deth in type 2 dietes [43, 44]. Islet et cells constitutively produce Fs lignd, ut do not normlly produce Fs t detectle levels [4, 43]. However, conditions ssocited with et cell stress, such s exposure to elevted glucose, leptin or cytokines (e.g. IL-1β), cn induce upregultion of Fs nd poptosis in isolted humn islets [43 45]. In the present study, we tested whether hiapp ggregtes derived from iosynthetic hiapp secreted from islets cn induce et cell poptosis vi upregultion of Fs. We used islets from humns nd trnsgenic mice with et cellspecific hiapp expression nd Fs deletion, s well s trnsformed nd primry islet et cells, to investigte the role of Fs in mediting islet myloid-induced et cell toxicity nd to exmine whether deletion of Fs cn protect et cells from hiapp ggregtes derived from endogenously secreted nd exogenously pplied hiapp. Methods Mterils Synthetic hiapp nd rt islet myloid polypeptide (riapp) (1-37) were from Bchem (Torrnce, CA, USA) nd Fs ntgonist (Kp7-6) ws from EMD Chemicls (Gistown, NJ, USA). Thioflvin S, dithizone, BSA, Congo red, HEPES uffer, 2-mercptoethnol, sodium icronte, poly-l-lysine, Triton X-1, DMSO, 1,1,1,3,3,3- hexfluoro-2-propnol (HFIP), triromoethnol nd collgense (Type XI) were otined from Sigm-Aldrich (Okville, ON, Cnd). RPMI-164, FBS, penicillin, streptomycin, gentmycin, cell dissocition uffer enzyme-free Hnks -sed solution, Hm s-f1, trypsin-edta nd

3 Dietologi (212) 55: glutmx were from Invitrogen Cnd (Burlington, ON, Cnd). CMRL culture medium ws from Meditech (Herndon, VA, USA). Humn islets Humn islets isolted from decesed pncres donors were provided y the Ike Brer Humn Islet Trnsplnt Lortory (Vncouver, BC, Cnd) in ccordnce with pproved procedures nd guidelines of the Clinicl Reserch Ethics Bord of the University of British Columi. Hnd-picked humn islets (purity >9%) were cultured for 7 dys t 37 C in CMRL supplemented with 11.1 mmol/l glucose, 1% (vol./vol.) FBS, 5 U/ml penicillin, 5 μg/ml streptomycin nd 5 μg/ml gentmycin. Culture procedures were in humidified 5% CO 2 /95% ir. Animl models Hemizygous C57BL/6 hiapp trnsgenic mice with et cell-specific hiapp expression (hiapp / ) were kindly provided y S. Khn (Deprtment of Medicine, University of Wshington, Settle, WA, USA) nd mintined y reeding with DBA/2J mice (Jckson Lortory, Br Hror, ME, USA). Mle hiapp-expressing mice form islet myloid in vivo nd develop dietes in the presence of predisposing fctor such s high-ft diet [46]. Mice with et cell-specific Fs deletion (RIPcre Fs fl/fl ) were generted using the Cre/loxP recominse system s previously descried [47]. RIPcre Fs fl/fl mice were mintined y reeding RIPcre Fs /fl mice from the Ontrio Cncer Institute. To generte mice with et cell-specific hiapp expression nd Fs deletion, hiapp / mice were cross-red with RIPcre Fs fl/fl mice to produce RIPcre Fs /fl /hiapp / mice, which were then red together to generte RIPcre Fs fl/fl / hiapp nd RIPcre Fs / /hiapp mice. The first genertion of these offspring ws used for the studies. Islets from RIPcre Fs fl/fl nimls hve norml islet mss nd enhnced insulin secretion [47]. All mice were fed mouse chow contining 9% (wt/wt) ft (Purin 521; LDiet, Richmond, IN, USA). Animls were cred for in ccordnce with the Guidelines nd Principles of Lortory Animl Cre nd the stndrd procedures estlished y the Cndin Council on Animl Cre nd the University of British Columi s Animl Policy nd Welfre Committee. Mouse islet isoltion nd culture Islets from wild-type (hiapp / ) nd hiapp / mice (8 12 weeks old) or different genotypes of RIPcre Fs/hIAPP mice (8 18 weeks) were isolted s previously descried [27] with some modifictions. Briefly, nimls were nesthetised with triromoethnol (.2 ml/g ody weight, i.p.) nd killed y cervicl disloction. The dominl cvity ws opened nd ice-cold collgense (Type XI; Sigm) in 2.5 ml clcium-free Hnks solution (finl concentrtion 1, U/ml) ws injected vi the common ile duct. The removed pncres ws incuted with collgense nd Hnks solution (1, U/ml) in shker wter th (14 min, 37 C, 12 rev/min). Digestion ws stopped y ddition of ice-cold Hnks solution contining 1 mmol/l CCl 2. Digested pncretic tissues were rinsed with the sme solution, resuspended in Hm s-f1 nd filtered through 7 μm mesh cell striner (BD Biosciences, Okville, ON, Cnd). Hnd-picked islets (purity >95%) were cultured in Hm s-f1 supplemented with 16.7 mmol/l glucose,.5% (wt/vol.) BSA nd ntiiotics s descried for humn islets. For ll islet studies, the culture medium ws chnged every 2 dys. Adenovirl trnsduction of humn nd mouse islets As previously descried [4], we used humn islets or hiappexpressing mouse islets trnsduced overnight with n denovirus tht delivers humn proiapp-specific smll interfering RNA (Ad-ProhIAPP-siRNA) (multiplicity of infection 2), or trnsduced with n denovirus tht delivers rndom non-specific smll interfering RNA (Ad-ContsiRNA) (multiplicity of infection 2). Adenovirl-siRNA trnsduced humn or hiapp-expressing mouse islets were then rinsed nd cultured in CMRL (11.1 mmol/l glucose, 7 dys) or Hm s-f1 (16.7 mmol/l, 6 dys), respectively. Trnsformed et cells nd islet cell culture INS-1 (832/13) cells, trnsformed rt et cell line, were gift from C. Newgrd (Duke University Medicl Center, NC, USA). Cells were grown in RPMI-164 contining 11.1 mmol/l glucose supplemented with 1% (vol./vol.) FBS, 5 U/ml penicillin, 5 μg/ml streptomycin nd 5 μmol/l 2-mercptoethnol. Humn or mouse islets (~2) were dissocited s previously descried [27] nd cell viility ssessed y Trypn lue. Islet cells were cultured in 8-well chmer slides (BD Biosciences) pre-coted with poly-l-lysine; the culture process ws in CMRL (humn) or Hm s-f1 (mouse) contining 5.5 nd 1 mmol/l glucose, respectively. Tretment studies with hiapp, Fs ntgonist nd myloid inding dye Lyophilised hiapp or riapp ws dissolved in HFIP nd incuted t room temperture (1 h); liquots were frozen (8 C) nd lyophilised. INS-1 or dispersed islet cells were treted with hiapp or riapp tht hd een prepred freshly y dissolving lyophilised peptide in medium; they were then cultured for different times s detiled. The myloid-inding dye Congo red or Fs ntgonist Kp7-6 ws dded to the culture medium 3 min efore ddition of hiapp t finl concentrtions of 25 μmol/l nd 1 mmol/ l, respectively. Assessment of et cell function in cultured islets Islets (25 islets per mouse; duplicte, four to six mice per group) were pre-incuted(1h)t37 Cin3μl KRB contining 1 mmol/l HEPES (ph 7.4),.25% (wt/vol.) BSA nd 1.67 mmol/l glucose, followed y 1 h of incution in 15 μl

4 138 Dietologi (212) 55: KRB contining 1.67 mmol/l glucose (sl insulin relese) ndy1hofincutioninkrbwith16.7mmol/lglucose (stimulted insulin relese). Islets were lysed in 1 μl of 1 mol/l cetic cid nd.1% (wt/vol.) BSA lysis uffer, followed y 1 min incution t 1 C. Incution medi nd islet lystes were centrifuged nd superntnt frctions frozen (2 C) until ssyed. Insulin nd proinsulin levels were mesured using mouse-specific insulin nd proinsulin ELISA kits (ALPCO Dignostics, Slem, NH, USA). Immunolelling, TUNEL nd thioflvin S stining Prffinemedded islet sections (5 μm) were de-wxed, rehydrted nd locked in 2% (vol./vol.) norml got nd/or donkey serum (Vector Lortories, Burlingme, CA, USA). Fixed islet sections (following ntigen retrievl with citrte uffer) or cells were incuted overnight with guine pig nti-insulin lone (islets 1:75, cells 1:2; Dko, Crpinteri, CA, USA) or with ech of rit nti-il-1β (1:1; Snt Cruz, Snt Cruz, CA, USA), nti-glucgon (1:75; Dko), nti-cleved cspse-3 (1:1; Cell Signling, Pickering, ON, Cnd) nd nti-fs (mouse islets 1:5, cells 1:1, Snt Cruz; humn islets 1:1, Cell Signling). Islet sections or cells were then incuted for 1 h with Texs red-conjugted nti-guine pig (1:75; Jckson Lortories, West Grove, PA, USA) nd Alex 488-conjugted nti-rit (1:2; Moleculr Proes, Eugene, OR, USA). For triple immunostining, islet cells were immunolelled overnight for insulin (1:2) nd Fs (1:1), followed y incution with minomethylcoumrin cette-conjugted nti-guine pig (1:2; Jckson) nd Texs red-conjugted nti-rit (1:1; Jckson), nd then with Alex 488-conjugted cleved cspse-3 ntiody (1:1; Cell Signling). The specificity of the Fs ntiody ws evluted y immunolelling of IL-1β- or hiapp-treted INS-1 cells fter ntiody locking using recominnt Fs (electronic supplementry mteril [ESM] Fig. 1). For doule insulin nd TUNEL or thioflvin S stining, fter immunolelling Control riapp hiapp hiapp 8h 12 h hiapp hiapp c 8 h Doule insulin/tunelpositive islet cells (%) hiapp hiapp 12 h d h Bet cell/totl islet cell (%) hiapp hiapp Fig. 1 Exposure to synthetic hiapp induces Fs upregultion, ssocited with cspse-3 ctivtion nd poptosis in INS-1 nd mouse islet et cells. INS-1 cells nd dispersed mouse islet cells were treted with 1 μmol/l synthetic hiapp or non-firillogenic riapp (s control) t different time points. Immunolelling of INS-1 cells for insulin (red) nd Fs (green) fter 8 nd 12 h of exposure to hiapp or riapp. Immunolelling of dispersed mouse islet cells for insulin (red) nd Fs (green), or cleved (ctive) cspse-3 (green) following exposure to hiapp for 8, 12 or 16 h. The squres (dshed white lines) denote regions enlrged nd depicted s insets t ottom right of the relevnt imges. c The proportion of TUNEL-positive islet cells nd (d) et cell:totl islet cell rtio in dispersed mouse islets following culture with or without hiapp for 24 h. The proportion of poptotic et cells (c) ws quntified y mnul counting of doule insulin- nd TUNEL-positive islet cells in minimum of ten microscopic fields ech contining 1 to 15 dispersed islet cells. The et cell:totl islet cell rtio (d) represents the men of insulin-positive islet cells divided y totl islet cells in ech microscopic field. Results (c, d) re expressed s mens ± SEM of three independent studies performed in triplicte. p<.5ystudent s t test vs non-treted group

5 Dietologi (212) 55: for insulin, islet sections (or cells) were incuted for 3 min t 37 C with TUNEL rection mixture (Roche Dignostics, Lvl, QC, Cnd) or for 5 min t room temperture with.5% (wt/vol.) thioflvin S solution. Sttisticl nlysis Dt re expressed s mens±sem. Sttisticl nlyses were performed using one-wy ANOVA followed y Newmn Keuls test or y Student s t test s pproprite. A vlue of p<.5 ws tken s significnt. All experiments were performed in duplicte or triplicte nd repeted t lest three times. Results Exogenously pplied hiapp induces Fs upregultion, cspse-3 ctivtion nd poptosis in trnsformed INS-1 cells nd in primry humn nd mouse islet et cells Trnsformed INS-1 cells nd dispersed mouse or humn islet cells were treted with synthetic hiapp (1 μmol/l) for 4, 8 nd 12 h. Fs ws not detectle in non-treted INS-1 cells y immunolelling, ut tretment with hiapp induced Fs upregultion in time-dependent mnner, with low levels of Fs present fter 8 h nd higher levels detectle fter 12 h Insulin Fs CASP-3 Merge Merge hiapp hiapp hiapp hiapp Fig. 2 Co-loclistion of Fs nd ctive cspse-3 in humn nd mouse islet cells treted with synthetic hiapp. Dispersed humn or () mouse islet cells cultured without (control) or with synthetic hiapp (1 μmol/l, 12 h) s indicted were immunolelled for insulin (lue), Fs (red) nd cleved (ctive) cspse-3 (CASP-3) (green). The squres (white outline) denote regions enlrged in ech imge. Note the higher numer of doule Fs- nd cleved cspse-3-positive islet et cells in humn nd mouse islet cells treted with hiapp compred with non-treted islet et cells. Imges represent three independent humn or mouse studies performed in duplicte

6 14 Dietologi (212) 55: (Fig. 1). Tretment with non-firillogenic riapp did not hve ny noticele effect on Fs undnce. Similrly, exogenously pplied hiapp induced Fs upregultion in primry islet cells, minly in et cells, this eing detectle fter 8 nd 12 h (Fig. 1). Upregultion of Fs ws ssocited with n incresed numer of ctive cspse-3 nd TUNEL-positive (poptotic) et cells (Fig. 1, c), resulting in lower islet et:totl cell rtio in hiapp-treted thn in non-treted islet cells (Fig. 1d). The time point of Fs upregultion preceded tht of cspse-3 ctivtion (16 h) nd poptosis (24 h). Moreover, immunolelling studies showed co-loclistion of Fs nd ctive cspse-3 in hiapp-treted humn nd mouse islet et cells (Fig. 2). Unlike INS-1 cells, low numer of Fs-positive et nd non-et islet cells ws present in non-treted dispersed mouse nd humn islets (Figs 1 nd 2). Preventing the interction etween hiapp ggregtes nd et cells mrkedly reduces et cell poptosis in hiapptreted INS-1 cells We exmined whether the interction etween hiapp firils nd islet et cells is required for induction of Fs upregultion. INS-1 et cells were cultured without or with hiapp or non-firillogenic riapp (s control) in the presence or sence of the myloid inding dye, Congo red, for 12 h. We hve previously shown tht Congo red t this concentrtion does not hve ny dverse effects on et cell survivl [27]. Tretment with Congo red mrkedly reduced Fs upregultion, cspse-3 ctivtion nd poptosis in hiapp-treted INS-1 cells (Fig. 3 c). Tretment with non-firillogenic riapp did not hve ny detectle effect on et cell poptosis (Fig. 3c). Blocking or deletion of Fs reduces the et cell poptosis induced y exogenously pplied hiapp INS-1 cells or primry islet cells were cultured with or without synthetic hiapp in the sence or presence of Fs ntgonist (Kp7-6) for 12 h. INS-1 nd primry mouse islet cells treted with hiapp nd the Fs ntgonist hd significntly lower et cell poptosis thn cells treted with hiapp in the sence of the Fs ntgonist (Fig. 3 d). Tretment with the Fs ntgonist lone did not hve ny effect on et cell poptosis in non-treted cells (Fig. 3d). Furthermore, dispersed wild-type nd RIPcre Fs fl/fl mouse islet cells were treted with synthetic hiapp. There ws no significnt difference in sl et cell poptosis etween wild-type nd RIPcre Fs fl/fl mouse islet cells (Fig. 3e, f). Interestingly, hiapptreted RIPcre Fs fl/fl islets hd mrkedly lower et cell deth thn hiapp-treted wild-type islet cells, suggesting tht deletion of Fs reduces et cell deth induced y the ggregtion of exogenously pplied hiapp. Inhiition of islet myloid formtion reduces IL-1β levels, Fs upregultion nd et cell poptosis in humn nd hiapp-expressing trnsgenic mouse islets Humn or hiapp-expressing mouse islets were trnsduced with Ad- ProhIAPP-siRNA to suppress hiapp production, therey preventing myloid formtion [4]. Non-trnsduced nd trnsduced humn nd mouse islets were then cultured in 11.1 nd 16.7 mmol/l glucose, respectively, to potentite myloid formtion. Islets trnsduced with Ad-Cont-siRNA, which hd non-specific sirna, were used s control to detect ny potentil dverse effects of denovirl trnsduction on et cells. As expected [16, 48], hiapp-expressing mouse islets formed myloid during culture nd hd higher rte of et cell poptosis thn wild-type islets (Fig. 4). Interestingly, the ggregtion of endogenously produced hiapp in cultured hiapp-expressing mouse islets ws ssocited with Fs upregultion in et cells (Fig. 4) nd incresed islet IL-1β levels (Fig. 4). Adenovirl sirna-medited suppression of hiapp expression mrkedly reduced myloid formtion, islet IL-1β levels, et cell Fs upregultion nd poptosis. Similrly, the formtion of hiapp ggregtes in humn islets during culture ws ssocited with the upregultion of Fs in et cells nd incresed islet IL-1β levels (Fig. 5), oth of which were reduced y denovirl-sirna-medited suppression of myloid formtion. Trnsduction with control denovirus (Ad-Cont-siRNA) hd no noticele effect on myloid formtion or et cell survivl in humn or hiapp-expressing mouse islets. Fig. 3 Culture with islet myloid inhiitor or Fs ntgonist, or deletion of Fs in islet et cells mrkedly reduces poptosis induced y exogenously pplied hiapp. INS-1 cells treted with hiapp (1 μmol/l, 12 h) without or with Congo red (CR; 25 μmol/l) were immunolelled for insulin (red) nd Fs (green). INS-1 nd mouse islet cells were treted with hiapp lone (24 h), or with CR or Kp7-6 (1 mmol/l), nd immunolelled for insulin (green) nd TUNEL (red). The squres (dshed white lines) denote regions enlrged nd depicted s insets t ottom right of the relevnt imges. c The proportion of cleved cspse-3 (CASP-3) nd TUNEL-positive INS- 1 cells treted with hiapp (or riapp) in the sence nd presence of CR or Kp7-6. Blck rs, non-treted; white rs, riapp; grey rs, hiapp; dotted rs, hiappcr; htched rs, hiappkp7-6. d The proportion of poptotic mouse islet cells treted with hiapp lone or with Kp7-6 for 24 h. e hiapp-treted RIPcre Fs fl/fl nd wild-type (RIPcre Fs / ) mouse islet cells immunolelled for insulin (red) nd Fs (green). The squres (white outline) denote regions enlrged in ech imge. Fs-positive et cells were present in hiapp-treted wildtype islet cells, ut not in mouse islet cells with et cell Fs deletion. A low numer of Fs-positive non-et cells ws detected in wild-type nd Fs fl/fl islet cells. f The proportion of poptotic et cells in mouse islet cells with nd without Fs deletion fter tretment with hiapp (24 h). The proportion of poptotic et cells ws quntified y mnul counting of doule insulin- nd cleved cspse-3- or TUNEL-positive INS-1 or islet cells in minimum of ten microscopic fields, ech contining 2 to 3 INS-1 cells or 1 to 15 dispersed islet cells. Results (c, d, f) re expressed s mens ± SEM of three independent studies performed in triplicte; p<.5 y one-wy ANOVA vs nontreted groups; p<.5 y one-wy ANOVA vs hiapp lone group; p<.5 y one-wy ANOVA vs Fs-expressing hiapp-treted group

7 Dietologi (212) 55: Bet cell-specific deletion of Fs in hiapp-expressing trnsgenic mouse islets mrkedly reduces poptosis induced y the ggregtion of endogenously produced hiapp during culture We generted mouse model with et cell-specific expression of hiapp nd deletion of Fs (RIPcre Fs fl/fl / hiapp ) y cross-reeding hiapp-expressing mice with RIPcre Fs fl/fl mice. Our im ws to further exmine the role of Fs in et cell poptosis induced y the hiapp Control hlapp CR CR CR CR Control hiapp hiappcr hiappfa Mouse INS-1 c Cleved CASP-3-positive INS-1 cells (%) h 24 h 1 TUNEL-positive INS-1 cells (%) d Doule insulin/tunelpositive islet cells (%) hiapp Kp7-6 e Insulin Wild-type Fs fl/fl f Fs Merge Doule insulin/tunelpositive islet cells (%) hiapp Wild-type Fs fl/fl

8 142 Dietologi (212) 55: Fig. 4 Suppression of islet myloid formtion in cultured hiapp-expressing mouse islets reduces IL-1β levels, Fs upregultion nd et cell poptosis. Islets isolted from hiapp trnsgenic mice (8 to 12 weeks old) were trnsduced overnight with Ad-ProhIAPPsiRNA or Ad-Cont-siRNA (s control). Non-trnsduced nd trnsduced islets were cultured in Hm s-f1 (16.7 mmol/l glucose) for 6 dys to llow myloid formtion. Prffin-emedded islet sections were immunolelled for: () insulin (Ins; red) nd thioflvin S (Thio; lue), or insulin (red) nd Fs (green) s indicted; nd () insulin (red) nd thioflvin S (lue), or insulin (red) nd IL-1β (green) s indicted. The squres (dshed white outline) denote regions enlrged nd depicted s insets t ottom right of the relevnt imges. c The percentge of thioflvin S (myloid)- positive islets in non-trnsduced nd trnsduced islets with Ad- ProhIAPP-siRNA or Ad-ContsiRNA fter 6 dys of culture. d The proportion of poptotic et cells in islets s ove (c). The numer of doule insulin- nd TUNEL-positive islet cells ws counted in ech islet in totl of 15 to 2 islets from ech condition. Results re expressed s mens ± SEM of three independent studies; p<.5 y one-wy ANOVA vs corresponding non-trnsduced group Ins/Fs Ins/Thio Ins/IL-1β Ins/Thio Wild-type c Thioflvin S-positive islets (%) Control d Reduced myloid-induced et cell deth in hiapp-expressing trnsgenic mouse islets with Fs deletion is ssocited with enhnced et cell function Amyloid formtion in hiapphiapp Ad-Cont-siRNA Doule insulin/tunelpositive islet cells (%) Control Ad-Cont sirna Ad-ProhlAPP sirna Ad-ProhlAPP-siRNA Ad-ProhIAPP-siRNA Ad-Cont-siRNA Wild-type hiapp ggregtes tht re formed y endogenously produced hiapp in islets. Islets from RIPcre Fs / /hiapp / nd RIPcre Fs fl/fl /hiapp / mice were used s controls to detect ny hiapp-independent effects of Fs deletion on et cell poptosis. Cultured islets from RIPcre Fs fl/fl /hiapp nd RIPcre Fs / /hiapp mice hd comprle levels of myloid formtion (Fig. 6, ). Bsl et cell deth in RIPcre Fs fl/fl mice ws somewht lower thn in wild-type mice, ut this difference ws not sttisticlly significnt (Fig. 6c). Despite similr levels of myloid formtion, islets from RIPcre Fs fl/fl /hiapp mice hd mrkedly lower numer of ctive cspse-3 nd TUNEL-positive et cells thn RIPcre Fs / /hiapp islets, ut comprle numer of lph cells (Fig. 6, c), resulting in higher islet et:lph cell rtio in RIPcre Fs fl/fl /hiapp islets (Fig. 6d). Islet IL-1β immunorectivity ws mrkedly higher in hiapp-expressing mouse islets with myloid formtion thn in wildtype islets (Fig. 6).

9 Dietologi (212) 55: Ad-ProhIAPP-siRNA Ad-Cont-siRNA Ad-Cont-siRNA Ad-ProhIAPP-siRNA Ins/Thio Ins/Fs Ins/Thio/Fs Ins/Thio Ins/IL-1β Ins/Thio/IL-1β Fig. 5 Adenovirl-siRNA-medited suppression of islet myloid formtion in cultured humn islets reduces IL-1β levels nd Fs upregultion, nd enhnces et cell survivl. Isolted humn islets were trnsduced overnight with Ad-ProhIAPP-siRNA or Ad-Cont-siRNA (s control). Trnsduced islets were cultured for 7 dys in CMRL contining 11.1 mmol/l glucose to llow myloid formtion. Prffinemedded islet sections from cultured islets trnsduced s indicted were immunolelled for () insulin (Ins; red), Fs (green) nd thioflvin S (Thio; lue), nd () for insulin (Ins; red), IL-1β (green) nd thioflvin S (Thio; lue). Note the higher Fs nd islet IL-1β immunorectivity in thioflvin S (myloid)-positive islets compred with islets in which myloid formtion ws suppressed. Imges re representtive of studies performed on three humn islet preprtions expressing mouse islets during culture ws ssocited with lower islet insulin content nd glucose-stimulted insulin relese, nd higher proinsulin relese thn in wild-type islets (Fig. 7). This resulted in higher proinsulin:insulin secretion rtio from hiapp-expressing islets thn from wild-type islets. Glucose-stimulted insulin relese ws incresed y 28% nd proinsulin relese ws reduced y 18% in hiappexpressing mouse islets with et cell Fs deletion compred with islets expressing Fs. These findings suggest tht Fs deletion in hiapp-expressing mouse islets enhnces insulin secretion nd improves the rtio of proinsulin:insulin relesed from islets. Deletion of Fs in hiapp-expressing mouse islets did not hve ny significnt effect on islet insulin or proinsulin content during 7 dys of culture. Finlly, glucose-stimulted insulin relese ws somewht higher in non-trnsgenic (hiapp / ) islets with Fs deletion thn in non-trnsgenic islets expressing Fs, ut this difference ws not sttisticlly significnt. Discussion Growing evidence suggests tht the formtion of hiapp ggregtes in pncretic islets of ptients with type 2 dietes, cultured humn islets nd humn islet grfts is ssocited with progressive et cell dysfunction nd deth [1 3]. Bet cell-toxic effects of hiapp ggregtes re likely to e medited through similr mechnisms in ll conditions ssocited with islet myloid formtion. As yet, however, the moleculr mechnisms y which the ggregtion of iosynthetic hiapp induces et cell poptosis re not well understood. Previous studies hve shown tht myloid formtion in humn [4] ndhiapp-expressing mouse islets [16] increses et cell poptosis, nd tht prevention of myloid formtion enhnces survivl nd function of humn islets [4]. We further demonstrted tht prevention of cspse-3 ctivtion protects islet et cells from poptosis induced y firillogenesis of hiapp produced nd secreted from islet et cells [27]. In the present study, using two ex vivo models of islet myloid formtion, cultured humn islets nd hiappexpressing mouse islets with et cell-specific Fs deletion, we show tht Fs hs key role in the et cell poptosis induced y ggregtion of the iosynthetic hiapp produced y islet et cells. We lso show tht prevention of hiappinduced Fs upregultion or deletion of Fs protects islet et cells from the hiapp ggregtes formed during in situ islet culture. These findings suggestthtggregtionof iosynthetic hiapp induces et cell poptosis, t lest prtilly, through the Fs receptor nd the Fs-medited poptotic pthwy. Consistent with previous in vitro study performed on et cells [26], we found tht micromolr concentrtions of synthetic hiapp induces Fs upregultion in INS-1 cells, s well s in humn nd mouse islet et cells, in timedependent mnner. The mjority of Fs-positive islet cells were et cells, suggesting tht hiapp-induced upregultion of Fs is et cell-specific. Furthermore, our immunolelling studies showed cler co-loclistion of Fs nd ctive cspse-3 in INS-1 nd dispersed islet et cells. Finlly, culture in the presence of Fs ntgonist or deletion of Fs in islet et cells significntly reduced hiapp-induced poptosis. Tken together, these findings suggest tht in trnsformed nd primry islet et cells, exposure to hiapp ggregtes derived from exogenously pplied hiapp induces upregultion of Fs nd tht hiapp-induced Fs upregultion

10 144 Dietologi (212) 55: hiapp /Fs / hiapp /Fs fl/fl hiapp /Fs / hiapp /Fs fl/fl Ins/Glucgon Ins/TUNEL Ins/IL-1β/Thio Ins/CASP-3 Ins/Fs/Thio c d Thioflvin S-positive islets (%) hiapp Fs Doule insulin/tunelpositive islet cells hiapp Fs Islet et:lph cell rtio hiapp Fs Fig. 6 Bet cell-specific deletion of Fs in hiapp-expressing trnsgenic mouse islets mrkedly reduces poptosis induced y the ggregtion of endogenously produced hiapp during culture. Freshly isolted islets from wild-type (RIPcre Fs / ), RIPcre Fs fl/fl,nd hiapp trnsgenic mice expressing (RIPcre Fs / /hiapp ) or lcking (RIPcre Fs fl/fl /hiapp ) Fs were cultured for 7 dys. Prffinemedded sections of cultured islets from wild-type nd hiappexpressing mice (8 to 18 weeks old) with or without Fs deletion were doule immunolelled s indicted for: insulin (Ins; red), Fs (green), nd thioflvin S (Thio; lue); insulin (red) nd cleved (ctive) cspse-3 (CASP-3; green); insulin (red), IL-1β (green) nd thioflvin S (lue); insulin (green) nd TUNEL (red); nd insulin (red) nd glucgon (Glu; green). The squres (dshed white outline) denote regions enlrged nd depicted s insets t ottom right of the relevnt imges. The percentge of thioflvin S (myloid)-positive hiappexpressing islets with or without Fs deletion fter 7 dys of culture. c The proportion of TUNEL-positive et cells nd (d) the islet et: lph cell rtio were quntified following triple insulin/tunel/dapi or insulin/glucgon/dapi stining, respectively. The numer of doule insulin- nd TUNEL-positive islet cells (c) ws counted in ech islet in totl of 15 to 25 islets per niml from ech genotype (six to eight nimls per group). Islet et:lph cell rtio (d) represents the men of insulin-positive cells divided y glucgon-positive cells in ech islet. Results ( d) re presented s men ± SEM; p<.5 y one-wy ANOVA vs wild-type group; p<.5 y one-wy ANOVA vs corresponding Fs-expressing group

11 Dietologi (212) 55: Glucose-stimulted insulin secretion (% sl) c Glucose-stimulted proinsulin secretion (% sl) hiapp Fs hiapp Fs is ssocited with cspse-3 ctivtion nd susequent et cell poptosis. Under norml conditions, INS-1 cells did not produce Fs t levels detectle with the immunolelling techniques used y us. Unlike INS-1 cells, however, low numers of et nd non-et Fs-positive islet cells were present in dispersed humn nd mouse islets. It is possile tht cytokines or other Fs-inducing fctors tht re relesed from islet cells nd other cells in the digested pncretic tissue during islet isoltion nd culture contriute to the low levels of Fs detected in non-treted islet cells. Consistent with our findings, IL-1β nd elevted glucose hve een reported to induce Fs upregultion in islet et cells [43, 44]. Finlly, islet myloid nd elevted glucose, oth of which contriute to et cell deth in type 2 dietes, my induce Fs upregultion in n dditive mnner. Tretment of INS-1 cells with the myloid inding dye, Congo red, which prevents myloid formtion nd its interction with et cell memrnes, mrkedly reduced hiappinduced Fs upregultion nd the proportion of ctive Islet insulin content (% control) d Islet proinsulin content (% control) hiapp Fs hiapp Fs Fig. 7 Reduced myloid-induced et cell deth in hiapp-expressing trnsgenic mouse islets with Fs deletion is ssocited with enhnced et cell function. Islet insulin response to glucose stimultion (16.7 mmol/l), () insulin content, (c) glucose-stimulted proinsulin relese nd (d) proinsulin content in 7-dy cultured wild-type nd hiapp-expressing mouse islets with or without Fs deletion were ssessed y the glucose-stimulted insulin secretion test s descried (Methods). Islet insulin nd proinsulin content re reported s percentge of insulin or proinsulin content in cultured wild-type islets tken s 1%. Glucose-stimulted insulin or proinsulin relese is depicted s fold increse over sl relese (1.67 mmol/l glucose). Results re expressed s mens ± SEM of four to six nimls per genotype; p<.5 y one-wy ANOVA vs wild-type group; p<.5 y one-wy ANOVA vs corresponding Fs-expressing group cspse-3 nd poptotic et cells. These findings support the notion tht interction of extrcellulr hiapp ggregtes with et cell memrnes is importnt for Fs upregultion nd susequent et cell poptosis, nd tht inhiitors of islet myloid my provide wy of preventing myloidinduced Fs upregultion. To exmine whether the hiapp ggregtes formed y iosynthetic hiapp produced nd secreted from islet et cells cn induce Fs upregultion, we used humn nd trnsgenic mouse islets with et cell hiapp expression s two ex vivo models of islet myloid formtion [4, 16, 48]. Humn nd hiapp-expressing mouse islets, ut not wildtype mouse islets formed hiapp ggregtes during culture, this eing ssocited with upregultion of Fs. Immunolelling studies showed tht Fs-positive islet res coloclised with thioflvin S (myloid)-positive islet res, indicting the correltion etween myloid formtion nd Fs upregultion. Furthermore, the prevention of myloid formtion in humn nd hiapp-expressing mouse islets y Ad-ProhIAPP-siRNA mrkedly reduced Fs upregultion nd susequent et cell poptosis. We lso generted mouse model expressing hiapp with et cell-specific Fs deletion to further investigte the role of Fs in mediting the et cell-toxic effects of endogenously produced hiapp ggregtes in islets. Islets from hiapp-expressing mice with or without Fs deletion hd comprle levels of myloid formtion, confirming tht the sence of Fs s such does not hve ny significnt effect on myloid formtion in hiapp-expressing mouse islets. Interestingly, hiapp-expressing mouse islets lcking Fs hd significntly lower numers of ctive cspse-3 nd poptotic et cells, higher islet insulin response to glucose nd lower proinsulin relese thn islets expressing hiapp nd Fs, suggesting tht deletion of Fs protects islet et cells from endogenously produced hiapp ggregtes nd improves their survivl nd function. Enhnced insulin secretion in hiapp-expressing mouse islets with et cell Fs deletion is likely to e due to the reduction of hiapp-induced et cell poptosis medited y Fs upregultion nd to the effects of Fs deletion on et cell secretion [47]. Importntly, despite significnt increses in the proportion of ctive cspse-3 nd poptotic et cells, the numer of lph cells remined unchnged in cultured hiapp-trnsgenic mouse islets expressing Fs, resulting in lower et:lph cell rtio in hiapp-expressing mouse islets with Fs expression thn in those lcking Fs. The formtion of hiapp-ggregtes nd upregultion of Fs in humn nd hiapp-expressing mouse islets ws ssocited with incresed islet IL-1β immunorectivity, which ws reduced y denovirl-sirna-medited prevention of myloid formtion, suggesting tht IL-1β my contriute to hiappinduced Fs upregultion. The source of IL-1β detected y immunolelling in thioflvin S (myloid)-positive islets

12 146 Dietologi (212) 55: might e islet et cells, non-et cells or non-islet cells. In support of this notion, et cells hve een shown to produce IL-1β in conditions such s exposure to elevted glucose resulting in upregultion of Fs [44]. Furthermore, two recent studies hve demonstrted the relese of IL-1β from non-islet cells such s mcrophges ctivted y hiapp ggregtes [49, 5]. Tken together, these findings support role for IL-1β in hiapp-induced Fs upregultion, lthough further studies re required to identify the cell types tht relese IL-1β in conditions ssocited with islet myloid formtion. In summry, our studies show tht hiapp-induced et cell deth is medited, t lest prtilly, vi upregultion of Fs nd ctivtion of the Fs-medited poptotic pthwy. We lso show tht deletion of Fs protects islet et cells from the cytotoxic effects of endogenously secreted (nd exogenously pplied) hiapp. Preventing the interction etween hiapp ggregtes nd et cells, or locking Fs could constitute new wys to void myloid toxicity nd therey enhnce survivl nd function of humn islets in conditions ssocited with islet myloid formtion, such s type 2 dietes, islet culture, nd trnsplnttion. Acknowledgements Humn IAPP trnsgenic mice for these studies were kindly provided y S. Khn (Deprtment of Medicine, VA Puget Sound Helth Cre System nd University of Wshington, Settle, WA, USA). Humn islets were provided y the Ike Brer Humn Islet Trnsplnt Lortory (Vncouver, BC, Cnd). We grtefully cknowledge the outstnding technicl ssistnce of I. Brt, A. Asdi nd G. Soukhtchev to the completion of these studies. Funding This work ws supported y grnts from the Cndin Institutes of Helth Reserch (CIHR) to L. Mrzn (MOP-81375) nd M. Woo (MOP-21188). Y.J. Prk is supported y studentship from the CIHR Trnsplnttion Trining Progrm. L. Mrzn is Scholr of the Cndin Dietes Assocition nd T.J. Kieffer is Michel Smith Foundtion for Helth Reserch (MSFHR) Senior Scholr. Infrstructure support ws provided y grnts from the Cndin Foundtion for Innovtion (to L. Mrzn) nd the MSFHR to the Centre for Humn Islet Trnsplnttion nd Bet Cell Regenertion (G.L. Wrnock). Contriution sttement All uthors contriuted to the conception nd design, or nlysis nd interprettion of dt, nd to the drfting of the rticle or its criticl revision for importnt intellectul content. All uthors gve finl pprovl of the version to e pulished. Dulity of interest The uthors declre tht there is no dulity of interest ssocited with this mnuscript. References 1. Htj L, Gurlo T, Hung CJ, Butler PC (28) Islet myloid in type 2 dietes, nd the toxic oligomer hypothesis. Endocr Rev 29: Westermrk P, Andersson A, Westermrk GT (211) Islet myloid polypeptide, islet myloid, nd dietes mellitus. Physiol Rev 91: Jurgens CA, Touktly MN, Fligner CL et l (211) Bet-cell loss nd et-cell poptosis in humn type 2 dietes re relted to islet myloid deposition. Am J Pthol 178: Mrzn L, Toms A, Becker TC et l (28) Smll interfering RNA-medited suppression of proislet myloid polypeptide expression inhiits islet myloid formtion nd enhnces survivl of humn islets in culture. Dietes 57: Westermrk P, Eizirik DL, Pipeleers DG, Hellerstrom C, Andersson A (1995) Rpid deposition of myloid in humn islets trnsplnted into nude mice. Dietologi 38: Westermrk G, Westermrk P, Eizirik DL et l (1999) Differences in myloid deposition in islets of trnsgenic mice expressing humn islet myloid polypeptide versus humn islets implnted into nude mice. Metolism 48: Potter KJ, Aedini A, Mrek P et l (21) Islet myloid deposition limits the viility of humn islet grfts ut not porcine islet grfts. Proc Ntl Acd Sci USA 17: Westermrk GT, Westermrk P, Berne C, Korsgren O (28) Widespred myloid deposition in trnsplnted humn pncretic islets. N Engl J Med 359: Udysnkr J, Kodm K, Hull RL et l (29) Amyloid formtion results in recurrence of hyperglycemi following trnsplnttion of humn IAPP trnsgenic mouse islets. Dietologi 52: Westermrk P, Wernstedt C, Wilnder E, Hyden DW, O'Brien TD, Johnson KH (1987) Amyloid firils in humn insulinom nd islets of Lngerhns of the dietic ct re derived from neuropeptide-like protein lso present in norml islet cells. Proc Ntl Acd Sci USA 84: Cooper GJ, Willis AC, Clrk A, Turner RC, Sim RB, Reid KB (1987) Purifiction nd chrcteriztion of peptide from myloidrich pncreses of type 2 dietic ptients. Proc Ntl Acd Sci USA 84: Verchere CB, D Alessio DA, Prigeon RL, Hull RL, Khn SE (2) The constitutive secretory pthwy is mjor route for islet myloid polypeptide secretion in neontl ut not dult rt islet cells. Dietes 49: Mrzn L, Trigo-Gonzlez G, Verchere CB (25) Processing of pro-islet myloid polypeptide in the constitutive nd regulted secretory pthwys of et cells. Mol Endocrinol 19: Westermrk P, Engstrom U, Johnson KH, Westermrk GT, Betsholtz C (199) Islet myloid polypeptide: pinpointing mino cid residues linked to myloid firil formtion. Proc Ntl Acd Sci USA 87: Hou X, Ling Z, Qurtier E et l (1999) Prolonged exposure of pncretic et cells to rised glucose concentrtions results in incresed cellulr content of islet myloid polypeptide precursors. Dietologi 42: Mrzn L, Rhodes CJ, Steiner DF, Htj L, Hln PA, Verchere CB (26) Impired NH2-terminl processing of humn pro-islet myloid polypeptide y the prohormone convertse PC2 leds to myloid formtion nd cell deth. Dietes 55: Pulsson JF, Westermrk GT (25) Aerrnt processing of humn proislet myloid polypeptide results in incresed myloid formtion. Dietes 54: Nyqvist D, Kohler M, Whlstedt H, Berggren PO (25) Donor islet endothelil cells prticipte in formtion of functionl vessels within pncretic islet grfts. Dietes 54: Klimek AM, Soukhtchev G, Thompson DM et l (29) Impired proinsulin processing is chrcteristic of trnsplnted islets. Am J Trnsplnt 9: Ritzel RA, Meier JJ, Lin CY, Veldhuis JD, Butler PC (27) Humn islet myloid polypeptide oligomers disrupt cell coupling, induce poptosis, nd impir insulin secretion in isolted humn islets. Dietes 56:65 71

13 Dietologi (212) 55: Lst NB, Rhodes E, Mirnker AD (211) Islet myloid polypeptide demonstrtes persistent cpcity to disrupt memrne integrity. Proc Ntl Acd Sci USA 18: Engel MF, Khemtemourin L, Kleijer CC et l (28) Memrne dmge y humn islet myloid polypeptide through firil growth t the memrne. Proc Ntl Acd Sci USA 15: Quist A, Doudevski I, Lin H et l (25) Amyloid ion chnnels: common structurl link for protein-misfolding disese. Proc Ntl Acd Sci USA 12: Rumor L, Hdzij M, Brisic K, Mysinger D, Gruiic TZ (22) Amylin-induced cytotoxicity is ssocited with ctivtion of cspse-3 nd MAP kinses. Biol Chem 383: Zhng S, Liu J, Drgunow M, Cooper GJ (23) Firillogenic mylin evokes islet et-cell poptosis through linked ctivtion of cspse cscde nd JNK1. J Biol Chem 278: Zhng S, Liu H, Yu H, Cooper GJ (27) Fs-ssocited deth receptor signling evoked y humn mylin in islet et-cells. Dietes 57: Lw E, Lu S, Kieffer TJ et l (21) Differences etween myloid toxicity in lph nd et cells in humn nd mouse islets nd the role of cspse-3. Dietologi 53: Young ID, Ailles L, Nrindrsorsk S, Tn R, Kisilevsky R (1992) Locliztion of the sement memrne heprn sulfte proteoglycn in islet myloid deposits in type II dietes mellitus. Arch Pthol L Med 116: Prk K, Verchere CB (21) Identifiction of heprin inding domin in the N-terminl clevge site of pro-islet myloid polypeptide. Implictions for islet myloid formtion. J Biol Chem 276: Css S, Novils A, Reimnn F, Gomis R, Grile FM (28) Clcium elevtion in mouse pncretic et cells evoked y extrcellulr humn islet myloid polypeptide involves ctivtion of the mechnosensitive ion chnnel TRPV4. Dietologi 51: Zheng X, Ren W, Zhng S et l (21) Serum levels of promylin nd mylin in norml sujects nd ptients with impired glucose regultion nd type 2 dietes mellitus. Act Dietol 47: Hung CJ, Lin CY, Htj L et l (27) High expression rtes of humn islet myloid polypeptide induce endoplsmic reticulum stress medited et-cell poptosis, chrcteristic of humns with type 2 ut not type 1 dietes. Dietes 56: Mtveyenko AV, Gurlo T, Dvl M, Butler AE, Butler PC (29) Successful versus filed dpttion to high ft diet induced insulin resistnce; the role of IAPP induced et cell endoplsmic reticulum stress. Dietes 58: Costes S, Hung CJ, Gurlo T et l (211) Bet-cell dysfunctionl ERAD/uiquitin/protesome system in type 2 dietes medited y islet myloid polypeptide-induced UCH-L1 deficiency. Dietes 6: Zrik S, Hull RL, Udysnkr J et l (29) Oxidtive stress is induced y islet myloid formtion nd time-dependently medites myloid-induced et cell poptosis. Dietologi 52: River JF, Gurlo T, Dvl M et l (211) Humn-IAPP disrupts the utophgy/lysosoml pthwy in pncretic et-cells: protective role of p62-positive cytoplsmic inclusions. Cell Deth Differ 18: Ngt S, Golstein P (1995) The Fs deth fctor. Science 267: Perl-Yfe M, Yolcu ES, Yniv I, Stein J, Shirwn H, Askensy N (26) The dul role of Fs-lignd s n injury effector nd defense strtegy in dietes nd islet trnsplnttion. Bioessys 28: Gloire G, Chrlier E, Piette J (28) Regultion of CD95/APO-1/ Fs-induced poptosis y protein phosphtses. Biochem Phrmcol 76: Moriwki M, Itoh N, Miygw J et l (1999) Fs nd Fs lignd expression in inflmed islets in pncres sections of ptients with recent-onset type I dietes mellitus. Dietologi 42: Chervonsky AV, Wng Y, Wong FS et l (1997) The role of Fs in utoimmune dietes. Cell 89: Hnfus T, Imgw A (28) Insulitis in humn type 1 dietes. Ann N Y Acd Sci 115: Medler K, Spins GA, Lehmnn R et l (21) Glucose induces et-cell poptosis vi upregultion of the Fs receptor in humn islets. Dietes 5: Medler K, Sergeev P, Ris F et l (22) Glucose-induced et cell production of IL-1et contriutes to glucotoxicity in humn pncretic islets. J Clin Invest 11: Medler K, Sergeev P, Ehses JA et l (24) Leptin modultes et cell expression of IL-1 receptor ntgonist nd relese of IL-1et in humn islets. Proc Ntl Acd Sci USA 11: Verchere CB, D'Alessio DA, Plmiter RD et l (1996) Islet myloid formtion ssocited with hyperglycemi in trnsgenic mice with pncretic et cell expression of humn islet myloid polypeptide. Proc Ntl Acd Sci USA 93: Choi D, Rdziszewsk A, Schroer SA et l (29) Deletion of Fs in the pncretic et-cells leds to enhnced insulin secretion. Am J Physiol Endocrinol Met 297:E134 E Zrik S, Hull RL, Udysnkr J et l (27) Glucose- nd timedependence of islet myloid formtion in vitro. Biochem Biophys Res Commun 354: Westwell-Roper C, Di DL, Soukhtchev G et l (211) IL-1 lockde ttenutes islet myloid polypeptide-induced proinflmmtory cytokine relese nd pncretic islet grft dysfunction. J Immunol 187: Msters SL, Dunne A, Surmnin SL et l (21) Activtion of the NLRP3 inflmmsome y islet myloid polypeptide provides mechnism for enhnced IL-1et in type 2 dietes. Nt Immunol 11:897 94