The nucleotide exchange factor SIL1 is required for glucose-stimulated insulin secretion from mouse pancreatic beta cells in vivo

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1 Dietologi (1) 7: DOI 1.17/s z ARTICLE The nuleotide exhnge ftor SIL1 is required for gluose-stimulted insulin seretion from mouse pnreti et ells in vivo Arne A. Ittner & Josefine Bertz & Tse Yn Beky Chn & Jnet vn Eersel & Ptsie Polly & Lrs M. Ittner Reeived: 1 Novemer 13 /Aepted: 13 Mrh 1 /Pulished online: 1 April 1 # Springer-Verlg Berlin Heidelerg 1 Astrt Aims/hypothesis Regultion of insulin seretion long the seretory pthwy is inompletely understood. We ddressed the expression of SIL1, nuleotide exhnge ftor for the endoplsmi retiulum (ER) hperone gluose-regulted protein 78 kd (GRP78), in pnreti et ells nd investigted whether or not SIL1 is involved in et ell funtion. Methods SIL1 expression ws nlysed y immunolotting nd immunofluoresene. Metoli nd islet vriles, inluding gluose tolerne, et ell mss, insulin seretion, islet ultrstruture, insulin ontent nd levels of ER stress mrker proteins, were ddressed in Sil1 knokout (Sil1 / ) mie., proinsulin nd C-peptide relese ws ddressed in Sil1 / islets, nd SIL1 overexpression or knokdown ws explored in MIN6 ells in vitro. Models of type 1 dietes nd insulin resistne were indued in Sil1 / mie y dministrtion of streptozotoin (STZ) nd high-ft diet (HFD), respetively. Results We show tht SIL1 is expressed in pnreti et ells nd is required for islet insulin ontent, islet sizing, gluose tolerne nd gluose-stimulted insulin seretion Eletroni supplementry mteril The online version of this rtile (doi:1.17/s z) ontins peer-reviewed ut unedited supplementry mteril, whih is ville to uthorised users. A. A. Ittner (): J. Bertz : J. vn Eersel : P. Polly : L. M. Ittner Shool of Medil Sienes, University of New South Wles, Botny Street, Kensington, Sydney, W, Austrli e-mil:.ittner@unsw.edu.u A. A. Ittner: J. Bertz : T. Y. B. Chn : J. vn Eersel : L. M. Ittner Fulty of Mediine, University of Sydney, Sydney, W, Austrli L. M. Ittner Neurosiene Reserh Austrli, Sydney, W, Austrli P. Polly Deprtment of Pthology, University of New South Wles, Sydney, W, Austrli in vivo. Levels of pnreti ER stress mrkers re inresed in Sil1 / mie, nd Sil1 / et ell ER is ultrstruturlly ompromised. Isolted Sil1 / islets show lower proinsulin nd insulin ontent nd impired gluose-stimulted insulin seretion. Modultion of SIL1 protein levels in MIN6 ells orreltes with hnges in insulin ontent nd sereted insulin. Furthermore, Sil1 / mie re more suseptile to STZindued type 1 dietes with inresed poptosis. Upon HFD feeding, Sil1 / mie show mrkedly lower insulin seretion nd exerted gluose intolerne ompred with ontrol mie. Surprisingly, however, HFD-fed Sil1 / mie disply pronouned islet hyperplsi with low mounts of insulin in totl pnres. Conlusions/interprettion These results revel novel role for the nuleotide exhnge ftor SIL1 in pnreti et ell funtion under physiologil nd disese onditions suh s dietes nd the metoli syndrome. Keywords Endoplsmi retiulum. Gluose-stimulted insulin seretion. High-ft diet. Islet size. Pnreti islets of Lngerhns. Proinsulin. Streptozotoin Arevitions BiP Binding immunogloulin protein CHOP CCAAT-enhner-inding protein homologous protein ER Endoplsmi retiulum GRP78 Gluose-regulted protein 78 kd HFD High-ft diet PERK Protein kinse RNA-like endoplsmi retiulum kinse shrna Short hirpin RNA shsil1 Short hirpin Sil1 STZ Streptozotoin TEM Trnsmission eletron mirosopy XBP-1 X-ox inding protein 1

2 Dietologi (1) 7: Introdution prodution follows the seretory pthwy [1, ]. Preproinsulin is otrnsltionlly inserted into the endoplsmi retiulum (ER), where the signl peptide is removed forming proinsulin. After sorting vi Golgi pprtus into vesiles, proinsulin is leved y prohormone onvertses into mture insulin. As requirements for insulin prodution nd seretion n vry with hnges in lood gluose level, the et ell is physiologilly fed with high demnds of folding pity in the ER [3]. ER hperone tivity is tightly regulted in et ells y progrmmes reminisent of ER stress responses, inluding trnsriptionl responses medited y X-ox inding protein 1 (XBP-1) or trnsltionl regultion of insulin prodution y protein kinse RNA-like endoplsmi retiulum kinse (PERK) [3]. Bet ell-speifi ltion of XBP-1 results in impired gluose tolerne, redued insulin seretion nd defetive insulin prodution [], while deletion of PERK results in dietes nd erly mortlity []. In humns, PERK muttions re ssoited with neontl dietes [6]. A key trnsriptionl trget of ER stress pthwys is the ER hperone gluose-regulted protein 78kD (GRP78; lso known s inding immunogloulin protein [BiP]) [3, 7]. GRP78 inds nd stilises unfolded polypeptides, nd is key sensor for high ER folding demnd nd n indue ER stress progrmmes (e.g. vi tivtion of PERK or XBP-1). Consistent with this role, knokdown of GRP78 using Grp78 sirna in et ell line redues insulin ontent nd gluose-stimulted insulin seretion [8]. Overexpressing GRP78 inreses insulin seretion independent of insulin trnsription [8]. GRP78 expression orreltes with the demnd for insulin in stimulted et ells [9]. Ativity of GRP78 is dependent on effiient nuleotide exhnge [1]. Two nuleotide exhnge ftors hve een identified for GRP78 in mmmls, GRP17 nd SIL1 [11]. SIL1 ws disovered in Shromyes erevisie, nd its homologues re onserved etween mouse nd humn. Sil1 mrna is expressed in the entrl nervous system [1], nd Sil1 mutnt mie show txi nd ereellr dystrophy [13]. In humns, Sil1 muttions use Mrineso Sjögren syndrome [1, 1]. Funtions of SIL1 outside the entrl nervous system re, however, unknown. Here, we show novel role for the nuleotide exhnge ftor SIL1 in et ell funtion. Methods Further detils of the methods used in this study n e found in the Eletroni supplementry mteril (ESM) Methods, inluding methods for gluose nd insulin tolerne tests. Mie Animl experiments were pproved y the Animl Ethis Committee of the University of New South Wles (Sydney, W, Austrli). Sil1-mutnt mie (CXB/By-Sil1 wz /J; JAX mie 3777; Jkson Lortory, Br Hror, ME, USA) were desried previously [13]. Age- nd sex-mthed littermte mie red t the University of New South Wles were used in ll experiments t indited numers. Detils of STZ- nd high ft diet (HFD)-indued models of type 1 dietes re given in the ESM Methods. Gluose-stimulted insulin seretion in vitro The isoltion of pnreti islets hs een desried previously [16]. Isolted pnreti islets were wshed in Kres Ringer HEPES uffer (KRH) with.8 mmol/l gluose, ounted nd distriuted in equl numers in ulture pltes. KRH with either.8 mmol/l gluose, 16.8 mmol/l gluose or mmol/l KCl ws dded nd islets were inuted t 37 C under % CO.Seretion ws mesured y ELISA for insulin (ChrystlChem, Downers Grove, IL, USA), pro-insulin nd C-peptide (Alpo, Slem, NH, USA). Trnsmission eletron mirosopy Fixtion for trnsmission eletron mirosopy (TEM) ws rried out s previously desried [17] (seeesmmethods). Histologil nlysis Immunofluoresene stining ws performed s previously desried [18] (seeesmmethods). Western lotting Western lot nlysis ws performed s previously desried [19] (see ESM Methods). Lentivirus prodution Lentivirus ws produed s desried previously [] (see ESM Methods). Cell ulture MIN6 ells were desried previously [1] (see ESM Methods). Sttistil nlysis Student s t-test ws used for omprison of two dt sets, ANOVA for omprison of more thn two dt sets nd two-wy ANOVA for omprisons ross time. Results GRP78/BiP nuleotide exhnge ftor SIL1 is expressed in pnreti et ells Sil1 mrna ws found in the liver, plent nd kidney [1], ll tissues with seretory tivity. Therefore, we ddressed expression of SIL1 in pnres nd prtiulrly in islets. We deteted SIL1 in mouse pnres t muh higher levels thn in liver of wild-type ( )mie (Fig. 1). SIL1 ws not deteted in liver nd pnres extrts from Sil1 / mie. Musle displyed no detetle Sil1. Isolted islets showed high levels of Sil1 in ut not in Sil1 / islets (Fig. 1). In ddition, proinsulin levels in Sil1 /

3 11 Dietologi (1) 7: SIL1 Proinsulin Musle SIL1 Liver Proinsulin intensity normlised to were lower thn in islets (Fig. 1). We used immunofluoresene to determine whih islet ells express SIL1, nd found the exhnge ftor ws lrgely onfined to endorine pnreti islets ompred with exorine pnres (Fig. 1). SIL1 immunostining within insulin-positive ells presented s perinuler signl reminisent of ER lolistion (Fig. 1). SIL1 signls in insulin-negtive ells indited onomitnt expression in other islet ell types (e.g. lph or delt ells; Fig. 1). Speifiity is supported y lk of signl in Sil1 / islets (dt not shown). Tken together, SIL1 is expressed in pnreti et ells. SIL1 regultes pnreti islet mss nd et ell ER homeostsis in vivo To ddress the funtionl relevne of pnreti islet SIL1, we determined the mount nd morphology of islets in regulr how-fed Sil1 / mie. Pnres histology t months reveled lower islet numers nd smller size in Sil1 / mie (Fig. ). Totl islet mss in Sil1 / mie ws signifintly lower thn in mie (Fig. ). Individul islet size ws pproximtely % smller in Sil1 / mie thn in mie (Fig. ; men dimeter ± SEM 73± 6 μm, Sil1 / ± μm; p=.133; n=6 7). Pnreti insulin ontent ws mrkedly lower in Sil1 / thn in mie (Fig. d). Notly, no poptoti ells were detetle in Sil1 / SIL1 Pnres +/+ +/- -/- +/+ +/- -/- +/+ +/- -/ DAPI Sil1 DAPI 36 Fig. 1 SIL1 is expressed in pnreti et ells of mie. () Western lot of pnres, liver nd musle from, Sil1 +/- nd Sil1 / mie for SIL1 nd. (n=3). () Western lot of (white r) nd Sil1 / (lk r) islets for SIL1 nd proinsulin (n=3). () Pnreti immunofluoresene from mie for insulin nd SIL1; DAPI, ell nulei; Sle r, 1 μm. p<.., glyerldehyde 3-phosphte dehydrogense islets (dt not shown). Liver is lso involved in gluose homeostsis nd showed expression of SIL1 (Fig. 1). Yet, Sil1 / livers ppered norml (Fig. ). Islets in Sil1 / mie showed norml ptterns with entrl et ells nd with lph nd delt ells in periphery (Fig. e; dt not shown for delt ells). However, insulin stining in Sil1 / islets ppered less intense thn in islets. SIL1 regultes tivity of GRP78, ritil ER ftor involved in protein folding nd ER stress response [11]. We ddressed protein levels of ER stress mrkers in regulr howfed mie y immunolot. Sil1 / pnres showed inresed levels of ER stress mrkers XBP-1, phosphoryltion of PERK, GRP78 nd CCAAT-enhner-inding protein homologous protein (CHOP; Fig. f, g), suggesting ER stress nd distured of ER homeostsis. We ddressed ultrstruturl morphology y TEM. Mitohondri nd Golgi pprtus ppered well defined in nd Sil1 / et ells. ER showed typil memrnous struture studded with riosomes nd nrrow ER lumen. However, in Sil1 / et ells the ER ppered diffuse with undefined luminl spe (Fig. h). Eletron-dense vesiles were indistinguishle etween Sil1 / nd pnres (Fig. i). SIL1 is required for gluose tolerne nd insulin seretion in vivo Fsting gluose in Sil1 / nd mie ws similr (men ± SEM.3±.63 mmol/l, Sil1 /.7±.3 mmol/l; p=.98; n=6), s were ody weights (t 16 weeks men ± SEM 31.76±.9 g, Sil1 / 8.97±.71 g; p=.9; n=). However, Sil1 / mie showed impired gluose tolerne ompred with ontrols, with signifintly higher pek gluose levels (Fig. 3, ). Sil1 / nd mie showed similr levels of insulin sensitivity (Fig. 3, d). Impired insulin levels might ount for impired gluose tolerne in Sil1 / mie. Sil1 / mie showed signifintly lower plsm insulin upon gluose stimultion thn ontrols (Fig. 3e, f). In prtiulr, Sil1 / mie showed little inresed insulin seretion, while fsting insulin levels were omprle with ontrols. Fsting glugon levels were omprle etween Sil1 / nd mie (Fig. 3g). Thus, SIL1 is required for pek insulin seretion in vivo. SIL1 regultes insulin ontent nd seretion from et ells To ddress insulin relese from islets (i.e. independent of endorine or neuronl pthwys), we next isolted islets nd mesured insulin relese. Relesed insulin ws mrkedly lower from Sil1 / thn islets (Fig. ). The mount of C-peptide relesed from Sil1 / islets ws lower thn from islets (Fig. ). Although islets relesed minute mounts of proinsulin, Sil1 / showed signifintly more stimulted proinsulin relese thn islets (Fig. ). Individul islet insulin ontent ws pproximtely % lower in Sil1 / thn islets (Fig. d). Proinsulin ontent ws

4 Dietologi (1) 7: Fig. Sil1 / pnres shows redued islet mss, insulin ontent nd ER stress. () Pnreti nd hepti hemtoxylin nd eosin stining (1 μm) of how-fed Sil1 / nd mie; dshed lines show islets. Liver glyogen stining (PAS) on Sil1 / nd. Sle rs, 1 μm. () Islet mss in Sil1 / (lk r) nd (white r) mie. Vlues re men ± SEM (n=6 7 nimls). () IsletreinSil1 / (lk r) nd (white r) mie. Vlues re men ± SEM (n=6 7 nimls; 3 islets eh). (d) Pnreti insulin ontent in Sil1 / (lk r) nd (white r) mie. Vlues re men ±SEM(n=6 7 nimls).(e) Pnreti immunofluoresene (1 μm) of glugon (red) nd insulin (green). DAPI, ell nulei. Sle rs, 1 μm. (f) Immunolot of Sil1 / nd pnreti lystes for XBP-1, phospho-threonine98-perk, PERK, GRP78 (lso known s BiP), CHOP, nd SIL1. (g) Quntifition of (f). Blk rs, Sil1 / ; white rs,. Vlues re men ± SD (n= nimls). (h) TEM imges of Sil1 / nd et ells. Arrows, unstrutured ER in Sil1 / ells. Sle rs, 1 μm. (i) Vesile dimeter from (h). Blk r, Sil1 / ; white r,. Vlues re men ± SD (n=3 nimls); p<., p<.1. G, Golgi;, glyerldehyde 3-phosphte dehydrogense; M, mitohondrion; PAS, periodi id-shiff Pnres Liver Liver PAS f h SIL1 pt98-perk PERK GRP78/BiP XBP-1 CHOP ER M G 1 µm 1 µm M Islet mss (mg) e 8, 1 6 Men islet re (µm ) 3,, 1, Glugon Glugon ER G g Fold intensity 3 1 ontent (ng/mg) 1 Glugon Glugon XBP-1 p-perk PERK CHOP GRP78/BiP i Vesile dimeter (µm) d.6.. DAPI DAPI likewise lower (Fig. e), onfirming immunolotting dt (Fig. 1). Interestingly, reltive normlised insulin seretion ws still signifintly lower from Sil1 / ompred with islets (Fig. f), suggesting tht insulin seretion ws ompromised independent of lower insulin ontent. To determine the effets of SIL1 redution or overexpression in ellulr model, we used MIN6 ells []. Expression of Sil1-speifi short hirpin RNA (shrna) resulted in SIL1 knokdown (Fig. ). SIL1-knokdown MIN6 showed lower proinsulin levels (Fig. ), similr to isolted Sil1 / islets. Furthermore, mture insulin levels were lower in Sil1-speifi shrna thn in ontrol shrna-seleted MIN6 ells (Fig. ). Importntly, knokdown of SIL1 resulted in lower insulin nd C-peptide relese in response to either gluose or potssium hloride (KCl), with the ltter using depolristion nd vesile seretion (Fig., d). Next, we overexpressed SIL1 in MIN6 ells (Fig. f). levels in MIN6 expressing SIL1 were signifintly higher thn in ontrol-trnsfeted MIN6 ells (Fig. g). nd C-peptide relese from MIN6 expressing SIL1 ws signifintly higher thn in ontrol-trnsfeted ells (Fig. h, i). Neither SIL1 knokdown nor overexpression resulted in

5 11 Dietologi (1) 7: Blood gluose (mmol/l) 1 1 d e f g tolerne (mmol/l x min) Time (min) Plsm insulin (pmol/l) 3 1 Gluose tolerne (mmol/l x min) Fig. 3 Sil1 / mie hve impired gluose tolerne due to impired insulin seretion. () GTTinSil1 / (lk irles) nd (white irles) mie (n=6).() AUC during GTT in (); lk rs, Sil1 / ; white rs, (n=6). () ITTin (white irles) nd Sil1 / (lk irles) mie (n=6). (d) AUC during ITT in (); lk r, Sil1 / ; white r, (n=6). (e) Plsm insulin during gluose stimultion (1 mg/g 3 1, 1, 3,, 1, Time (min) Blood gluose (mmol/l) Plsm insulin (pmol/l x min) Time (min) Fsting glugon (pg/ml) ody weight) in Sil1 / (lk irles) nd (white irles) (n=7 9) (f) AUC of insulin in (e); lk r, Sil1 / ; white r, (n=7 9; p<.1). (g) Fsting glugon in Sil1 / (lk r) nd (white r) mie (n=7 9). Vlues re men ± SEM; p<.1, p<.1. ITT, insulin tolerne test;, not signifint signifint ltertion of relesed proinsulin (Fig. e, j). Hene, ltering SIL1 levels modultes insulin prodution nd insulin seretion in MIN6 ells. SIL1 protets from STZ- nd diet-indued dietes Although Sil1 / mie do not disply overt dietes, their et ells might e more vulnerle to stressors. We used STZ to indue relese (ng/µg protein) 6 C-peptide relese (ng/µg protein) Time (min) Time (min) Time (min) Proinsulin relese (ng/µg protein)...3 d e f Islet insulin (ng) Islet proinsulin (ng) 1 Fold insulin relese (reltive to ontent) Fig. SIL1 ffets islet insulin ontent nd stimulted seretion in vitro. () seretion from Sil1 / (lk symols) nd (white symols) islets during gluose stimultion (16.8 mmol/l; squres) or sl gluose (.8 mmol/l; irles), (n=). () C-peptide seretion from Sil1 / (lk symols) nd (white symols) islets (n=). () Proinsulin seretion from Sil1 / (lk symols) nd (white symols) islets 1 1 Time (min) (n=). (d) Sil1 / (lk r) nd (white r) islet insulin ontent (n=6). (e) Sil1 / (lk r) nd (white r) islet proinsulin ontent (n=6). (f) Stimulted insulin seretion reltive to islet insulin ontent nd sl seretion from Sil1 / (lk irles) nd (white irles) islets (n=). Vlues re men ± SEM; p<., p<.1

6 Dietologi (1) 7: SIL1 Proinsulin s shsil SIL1 level normlised to Proinsulin level normlised to (ng/µg protein) 7 relese (ng [mg protein] -1 [min] -1 ) KCI Gluose (mmol/l) d e f g C-peptide relese (ng [mg protein] -1 [min] -1 ) 1, 1, Proinsulin relese (ng [mg protein] -1 [min] -1 ) KCI KCI Gluose (mmol/l) Gluose (mmol/l) 3 1 GFPMOCK SIL1 SIL1 Proinsulin (ng/µg protein) h i j relese (ng [mg protein] -1 [min] -1 ), 1, 1, C-peptide relese (ng [mg protein] -1 [min] -1 ) 1, 1, Proinsulin relese (ng [mg protein] -1 [min] -1 ) KCl KCl KCl Gluose (mmol/l) Gluose (mmol/l) Gluose (mmol/l) Fig. SIL1 regultes insulin ontent nd seretion from MIN6 ells. () Western lot of MIN6 ells trnsfeted with Sil1-speifi shrna (shsil1) or srmled shrna s for Sil1, proinsulin nd (n=3). Quntifition: 7.7±.6% nd 9.1±.3% SIL1 nd proinsulin redution, respetively. () levels in shsil1 (lk r) nd s (white r) MIN6 (n=3). () seretion from shsil1 (lk rs) nd s (white rs) MIN6 during gluose or KCl ( mmol/l) stimultion (n=3). (d) C-peptide seretion from shsil1 (lk rs) nd s (white rs) MIN6 during gluose or KCl ( mmol/l) stimultion (n=3). (e) Proinsulin seretion from shsil1 (lk rs) nd s (white rs) MIN6 during gluose or KCl ( mmol/l) stimultion (n=3). (f) Western lot of MIN6 ells trnsfeted with GFP- or Sil1-expressing vetor, or MOCK 3 1 (n=3). (g) ontent in Sil1-expressing (lk r) MIN6 ells nd MOCK ontrols (white r) or GFP ontrols (grey r; n=3). (h) seretion in Sil1-expressing (lk rs) MIN6 ells nd MOCK ontrols (white rs) or GFP ontrols (grey rs) with gluose or KCl ( mmol/l; n=3). (i) C-peptide seretion in Sil1-expressing (lk rs) MIN6 ells nd MOCK ontrols (white rs) or GFP ontrols (grey rs) with gluose or KCl ( mmol/l; n=3).(j) Proinsulin seretion in Sil1-expressing (lk rs) MIN6 ells nd MOCK ontrols (white rs) or GFP ontrols (grey rs) with gluose or KCl ( mmol/l; n=3). Vlues re men ± SEM; p<., p<.1, p<.1., glyerldehyde 3-phosphte dehydrogense; GFP, green fluoresent protein; MOCK, empty vetor; s, ontrol vetor;, not signifint mie models of type 1 dietes. Sil1 / mie showed higher gluose levels within 36 h fter STZ dministrtion ompred with mie (Fig. 6). Overll, lood gluose levels inresed fster in STZ-treted Sil1 / thn in mie (Fig. 6). STZ indues et ell poptosis prior to rises in lood gluose. We nlysed islets in Sil1 / nd mie for poptosis y TUNEL stining 18 h post-stz dministrtion. Sil1 / islets showed more poptoti ells within islets (Fig. 6). Normlising for insulin-positive re to ount for smller Sil1 / islets reveled signifintly more TUNEL-positive ells in Sil1 / thn in islets (Fig. 6d). Thus, Sil1 / islets re more suseptile to STZ. Exess dietry intke imposes higher demnds for insulin, inresing islet size nd mount of sereted insulin [3]. Sil1 / mie fed HFD showed similr weight gin to HFD-fed (Fig. 7). However, gluose tolerne in Sil1 / mie ws signifintly lower thn in HFD-fed mie (Fig. 7, ). sensitivity in HFD Sil1 / mie ws similr to tht of HFD mie (Fig. 7d, e). Strikingly, however, mrkedly lower insulin levels nd insulin seretion were pprent in HFD Sil1 / ompred with HFD mie (Fig. 7f, g). Fsting glugon level ws similr in HFD Sil1 / nd HFD mie (Fig. 7h). Surprisingly, histologil pnreti setions from HFD-fed mie showed islets of lrger size in HFD Sil1 / ompred with HFD mie (Fig. 7i). Quntifition onfirmed ggrvted islet hyperplsi in HFD Sil1 / (Fig. 7j). Bet ell size ws indistinguishle etween HFD Sil1 / nd HFD islets (Fig. 7k).

7 116 Dietologi (1) 7: Blood gluose (mmol/l) 1 1 Strikingly, pnres of HFD Sil1 / mie showed mrkedly lower mounts of insulin thn those of HFD mie (Fig. 7l). HFD Sil1 / islets showed norml distriution nd intensity of glugon (Fig. 7m). Levels of GRP78 were mrkedly higher in HFD Sil1 / thn in HFD pnres, suggesting inresed ER stress (Fig. 7n, o). Furthermore, levels of ylin D1, G 1 mitoti phse mrker, were higher in HFD Sil1 / pnres. Thus, HFD Sil1 / mie hve pronouned islets hyperplsi, yet insuffiient insulin mounts nd defetive pek insulin seretion. Disussion Time (h) TUNEL TUNEL TUNEL TUNEL Our results present previously unknown role for nuleotide exhnge ftor SIL1 in et ells. Sil1 trnsript levels were nlysed in seleted humn tissues [1]. Neither protein levels nor expression in pnres or islets were ddressed. We found expression of SIL1 in islet ells, inluding et ells, using Sil1 / smples to ontrol for speifiity. Post-trnsriptionl mehnisms nd sensitivity might ount for differenes of Sil1 mrna [1] nd protein expression ptterns. SIL1 expression in liver is low nd Sil1 / mie present with norml liver histology nd glyogen storge. The liver mintins Gluose tolerne (mmol/l x min) DAPI DAPI 1, d TUNEL-positive ells (n/µm + re) Fig. 6 Sil1 / mie show inresed suseptiility to STZ-indued hyperglyemi nd et ell poptosis. () Blood gluose in Sil1 / (lk irles) nd (white irles) mie 36 h fter STZ dministrtion (n= ). () AUC of lood gluose in (); lk r, Sil1 / ; white r, (n= ). () Pnreti insulin immunofluoresene nd TUNEL stining of Sil1 / nd (1 μm) 18 h fter STZ dministrtion. DAPI, ell nulei. Sle r, 1 μm. (d) Quntifition of TUNEL-positive ells per insulin-positive re fter STZ dministrtion. Blk r, Sil1 / ; white r, (n=1 islets/mouse; four mie/group). Vlues re men ± SEM; p<., p<.1 fsting gluose level regulted y glugon from lph ells []. Sil1 / mie hve norml fsting gluose nd glugon levels, suggesting tht glugon relese nd liver funtions re unffeted y lk of SIL1. In the present study, Sil1 / mie show deresed gluose tolerne, pproximtely % redution in pnreti insulin ontent nd virtully no insulin pek-seretion upon gluose stimultion. Sil1 / islets demonstrte little insulin nd C-peptide relese fter gluose stimultion. Sil1 / islets ontin pproximtely % less insulin thn islets. In reltion to insulin ontent, reltive normlised insulin seretion from Sil1 / islets remins signifintly lower thn from islets, suggesting tht esides insulin ontent, dditionl proesses suh s trffiking nd/or exoytosis re ffeted. Proinsulin levels re lower in Sil1 / islets thn in Si1l +/+ islets. Furthermore, Sil1 / islets show signifintly more proinsulin relese thn islets. This finding likely reflets ER stress in Sil1 / islets, sine elevted proinsulin relese hs een suggested s n dptive mehnism during ER stress []. Our dt from isolted islet nd MIN6 ells onfirm ellutonomous regultion of insulin ontent nd insulin seretion y SIL1. SIL1 is n ER-resident protein [6]. ER in Sil1 / et ells in vivo shows mrked ultrstruturl hnges, wheres Golgi pprtus nd vesiles pper norml. Ultrstruturl ER hnges re seen upon ER stress [, 7] nd proinsulin trffiking defets [8]. ER stress pthwys pper to e negtively ontrolled y SIL1, sine ER stress mrkers phospho- PERK, XBP-1, CHOP nd GRP78 re elevted in pnres of Sil1 / mie. ER stress pthwys ffet proinsulin trnsription, trnsltion nd trffiking [3, 8]. Furthermore, ER qulity ontrol my inhiit stimulted insulin seretion in Sil1 / et ells [3, ]. GRP78 knokdown redues insulin levels nd insulin relese [8]. GRP78 overexpression results in inresed insulin seretion independent of insulin trnsription [8]. Similrly, et ell knokout of Xp-1, trnsription ftor for GRP78, results in redued insulin prodution nd sene of insulin seretion in vivo []. Sine SIL1 is GRP78 nuleotide exhnge ftor [1], nd given the remrkly similr effets of modulting levels of GRP78 [8], XBP-1 [] or (s shown in the present study) SIL1, it is likely tht SIL1 regultes insulin iosynthesis nd pek-seretion through GRP78. Gluose inreses insulin trnsltion [9] nd ATP levels [3, 31]. ATP exhnge in GRP78 is limiting [1, 3, 33]. SIL1 might e ritil for effiient GRP78 funtion under onditions of gluose stimultion. GRP17, n lterntive exhnge ftor for GRP78 [3], seems not to ompenste for loss of SIL1, lthough how different exhnge ftors orhestrte GRP78 nd ER stress response is unknown. ER stress responses hve een implited in et ell survivl nd poptosis [3]. Sil1 / mie re more suseptile to STZ-indued dietes nd islet poptosis. We found ER

8 Dietologi (1) 7: Body weight (g) f Plsm insulin (pmol/l) m Time (weeks) Time (min) Time (min) Plsm insulin (pmol/l x min) DAPI Glugon Glugon DAPI Glugon Glugon Blood gluose (mmol/l) Fsting glugon (pg/ml) Gluose tolerne (mmol/l x min) d Reltive lood gluose e tolerne (mmol/l x min) g h i j k l 3 6,, 1 1, 1, , 1, 1, 1 3 Time (min) Isulin n SIL 1 GRP78/BiP Cylin D1 Fig. 7 Sil1 / mie show mrkedly lower insulin seretion ut more pronouned islet hyperplsi in HFD model. () Body weight of (white irles) nd Sil1 / (lk irles) mie during HFD (n= 6). () GTT fter 1 weeks of HFD in Sil1 / (lk irles) nd (white irles) mie (n=6).() AUC during GTT in (); lk r, Sil1 / ; white r, (n=6). (d) Reltive lood gluose during ITT in d liitum-fed HFD (white irles) nd Sil1 / (lk irles) mie (n=6). (e) AUC during ITT in (d). Blk r, Sil1 / ; white r, (n=6). (f) Plsm insulin during gluose stimultion (1 mg/g ody weight) in HFD Sil1 / (lk irles) nd (white irles) mie (n=6). (g) AUC of plsm insulin in (f); lk r, Sil1 / ; white r, (n=6). (h) Fsting glugon in HFD Sil1 / (lk r) nd (white r) mie (n=6). (i) Histology of HFD Sil1 / nd pnres (1 μm); dshed lines demrte islets; sle rs, 1 μm. (j) Men islet re (µm ) o Fold levels Grp78/BiP CylinD1 Men et ell size (µm) Quntifition of men islet re in HFD Sil1 / (lk r) nd mie from histology shown in (i) (n= nimls; 1 islets eh). (k) Islet et ell size from histology shown in (i) Blk r, Sil1 / ; white r, (n= nimls; 3 islets eh; 8 1 ells/islet). (l) Pnreti insulin ontent in HFD Sil1 / (lk r) nd (white r) mie (n=6). (m) Pnreti immunofluoresene (1 μm) for glugon (red) nd insulin (green) in HFD Sil1 / nd mie. DAPI, ell nulei; sle r, 1 μm. (n) Immunolot of HFD Sil1 / nd pnreti lystes for GRP78, ylin D1, nd SIL1. (o) Quntifition of (n). Blk rs, Sil1 / ; white rs, ;vluesremen±sd(n=3). Vlues re men ± SEM unless otherwise stted; p<.1, p<.1, p<.1., glyerldehyde 3-phosphte dehydrogense; ITT, insulin tolerne test;, not signifint ontent (ng/mg) 3 1 stress in the pnres of Sil1 / mie. While initil ER stress responses re protetive [3, 9], persistent ER stress n indue poptosis [3]. Persistent ER stress in Sil1 / et ells my render them suseptile to STZ y reduing poptosis thresholds. Rodent HFD models hve ompenstory islet hyperplsi [3, 36]. In our study, HFD Sil1 / islets were twie the size of HFD islets, suggesting pronouned hyperplsi. ontent nd levels were mrkedly lower in HFD Sil1 / mie, with sent pek insulin seretion in vivo. Thus, ompenstory hyperplsi under Sil1 / onditions produes lrge islets ut little insulin. Bet ells retin the pity of prolifertion unlike other differentited ells [37]. We found higher levels of ylin D1, mitoti mrker, in HFD Sil1 / ompred with pnres, suggesting inresed ompenstory prolifertion. signlling is required for ompenstory prolifertion [38]. SIL1 might influene ompenstory prolifertion y modulting insulin levels. We found higher levels of ER stress in HFD Sil1 / islets ompred with islet. HFD-indued ER stress pthwys lok trnsltion [3]. ER stress pthwys n ontriute to inresed prolifertion in the erly dieti stges [39]. Other ER-ssoited moleules my e involved in hyperprolifertion in HFD Sil1 / pnres. Inresed expression of endoplsmiretiulum-ssoited protein degrdtion omponent SEL1L promotes et ell prolifertion []. Our dt suggest role for SIL1 in ontrolling ompenstory prolifertion. Aknowledgements The uthors thnk Dr J.-I. Miyzki (Osk University, Osk, Jpn) for providing the MIN6 ell line nd S. Frser (Eletron Mirosope Unit, University of New South Wles, Sydney, W, Austrli) for tehnil ssistne.

9 118 Dietologi (1) 7: Dulity of interest The uthors delre tht there is no dulity of interest ssoited with this mnusript. Contriution sttement AI designed experiments, nlysed the dt nd wrote the mnusript. JB, BC nd JvE performed experiments, nlysed the dt nd drfted prts of mnusript. PP nd LMI designed study nd experiments nd wrote the mnusript. All uthors pproved the finl version. AI is responsile for the integrity of the work s whole. Funding The reserh ws supported y funding from the Ntionl Helth nd Medil Reserh Counil (NHMRC), the Austrlin Reserh Counil (ARC) nd the University of New South Wles. LMI is NHMRC Senior Reserh Fellow. Referenes 1. Rorsmn P, Brun M (13) Regultion of insulin seretion in humn pnreti islets. Annu Rev Physiol 7: Weiss MA (9) Proinsulin nd the genetis of dietes mellitus. J Biol Chem 8: Bk SH, Kufmn RJ (1) Endoplsmi retiulum stress nd type dietes. 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10 Dietologi (1) 7: Cozr-Cstellno I, Fishi-Tesh N, Bigtel TA et l (6) Moleulr ontrol of ell yle progression in the pnreti et-ell. Endor Rev 7: Okd T, Liew CW, Hu J et l (7) reeptors in et-ells re ritil for islet ompenstory growth response to insulin resistne. Pro Ntl Ad Si U S A 1: Ahren J, Ahren B, Wierup N (1) Inresed et-ell volume in mie fed high-ft diet: dynmi study over 1 months. Islets : Diferi GR, Cirulli V, Biunno I (13) SEL1L regultes dhesion, prolifertion nd seretion of insulin y ffeting integrin signling. PLoS One 8:e798