Early deficits in insulin secretion, beta cell mass and islet blood perfusion precede onset of autoimmune type 1 diabetes in BioBreeding rats

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1 Dietologi (218) 61: ARTICLE Erly deficits in insulin secretion, et cell mss nd islet lood perfusion precede onset of utoimmune type 1 dietes in BioBreeding rts Any Medin 1 & S Prween 2 & Sr Ullsten 3 & Neelnjn Vishnu 1 & Yuk Ting Siu 1 & My Quch 3 & Hedvig Bennet 1 & Alexnder Blhuizen 1 & Lin Åkesson 1 & Nils Wierup 1 & Per Ol Crlsson 3 & Ulf Ahlgren 2 & Åke Lernmrk 1 & Mlin Fex 1 Received: 16 My 217 /Accepted: 18 Octoer 217 /Pulished online: 6 Decemer 217 # The Author(s) 217. This rticle is n open ccess puliction Astrct Aims/hypothesis Genetic studies show coupling of genes ffecting et cell function to type 1 dietes, ut hitherto no studies on whether et cell dysfunction could precede insulitis nd clinicl onset of type 1 dietes re ville. Methods We used 4-dy-old BioBreeding (BB) DRLyp/Lyp rts ( model of spontneous utoimmune type 1 dietes) nd dietes-resistnt DRLyp/+ nd DR+/+ littermtes (controls) to investigte et cell function in vivo, nd insulin nd glucgon secretion in vitro. Bet cell mss ws ssessed y opticl projection tomogrphy (OPT) nd morphometry. Additionlly, mesurements of intr-islet lood flow were performed using microsphere injections. We lso ssessed immune cell infiltrtion, cytokine expression in islets (y immunohistochemistry nd qpcr), s well s islet Glut2 expression nd ATP/ADP rtio to determine effects on glucose uptke nd metolism in et cells. Results DRLyp/Lyp rts were normoglycemic nd without trces of immune cell infiltrtes. However, IVGTTs reveled significnt decrese in the cute insulin response to glucose compred with control rts ( ± vs ± 148.7; p <.1). In greement, insulin secretion ws severely pertured in isolted islets, nd oth first- nd second-phse insulin relese were lowered compred with control rts, while glucgon secretion ws similr in oth groups. Interestingly, fter 5 7 dys of culture of islets from DRLyp/Lyp rts in norml medi, glucose-stimulted insulin secretion (GSIS) ws improved; lthough, significnt decrese in GSIS ws still evident compred with islets from control rts t this time ( ± vs ± pg islet 1 h 1 ; p <.1). Compred with controls, OPT of whole pncres from DRLyp/Lyp rts reveled significnt reductions in medium ( ± vs ± μm 3 ; p =.44) nd smll sized islets ( ± vs ± μm 3 ; p =.35). Finlly, we found lower intr-islet lood perfusion in vivo (113.1 ± 16.8 vs 76.9 ± 11.8 μl min 1 [g pncres] 1 ; p =.23) nd ltertions in the et cell ATP/ADP rtio in DRLyp/Lyp rts vs control rts. Conclusions/interprettion The present study identifies deteriortion of et cell function nd mss, nd intr-islet lood flow tht precedes insulitis nd dietes development in nimls prone to utoimmune type 1 dietes. These underlying chnges in islet function my e previously unrecognised fctors of importnce in type 1 dietes development. Keywords Bet cell dysfunction. Bet cell mss. Insulin secretion. Islet lood flow. Type 1 dietes S Prween nd Sr Ullsten contriuted eqully to this work. Electronic supplementry mteril The online version of this rticle ( contins peer-reviewed ut unedited supplementry mteril, which is ville to uthorised users. Any Medin ny_medin.envente@med.lu.se 1 Lund University Dietes Centre, Clinicl Reserch Centre, Skåne University Hospitl (SUS), Jn Wldentrömsgt 35, SE-252 Mlmö, Sweden 2 3 Umeå Centre for Moleculr Medicine, Umeå University, Umeå, Sweden Medicl Cell Biology, Uppsl Biomedicl Centre, Uppsl, Sweden

2 Dietologi (218) 61: Arevitions AIR Glucose BB GSIS OPT ROS SAB Introduction Acute insulin response to glucose BioBreeding Glucose-stimulted insulin secretion Opticl projection tomogrphy Rectiveoxygenspecies Secretion ssy uffer Type 1 dietes is ssocited with the immune-medited destruction of islet et cells. Studies in humn monozygotic twins, shring identicl genomes, demonstrte pirwise type 1dietesof13 52%, suggesting tht environmentl nd genetic cuses my contriute similrly to the disese [1]. Reserch pertining to the genetic contriution of type 1 dietes hve for the pst decdes focused on genetic loci implicted in regultion nd selection of utorective T lymphocytes [2], lthough single nucleotide polymorphisms within the humn insulin (INS) gene (minly present in et cells) remin one of the most importnt risk fctors for the development of type 1 dietes [3]. Recent studies hve reveled tht severl cndidte genes found in genome-wide ssocition studies of type 1 dietes susceptiility loci re expressed in et cells nd could thus influence et cell function [4]. The BioBreeding (BB; LEW.1WR1) rt cts s model of type 1 dietes, wherey type 1 dietes is suggested to originte from selective utoimmune destruction of et cells [5]. As in humns, the mjor histocomptiility complex holds genetic fctors tht predict disese in this model [6, 7]. This explins some, ut not ll, of the inherited predisposition to type 1 dietes. In the inred BB rt strin BBDRLyp/Lyp (herein referred to s DRLyp/Lyp), onset of type 1 dietes is linked to lymphopeni, which is cused y frmeshift muttion in the Gimp5 gene, while their littermtes DRLyp/+ nd DR+/+ re resistnt to dietes [8, 9]. Loss of T cells ecuse of lymphopeni ffects oth CD4 + nd CD8 + T cells, especilly ART2.1 + T cells [5]. In fct, depletion of the ART2.1 + T cells in dietes-resistnt BB rts induces type 1 dietes, suggesting tht loss of regultory T cells is ssocited with insulitis nd type 1 dietes [1]. Erly chnges in et cell function nd lood glucose hve not een elucidted in DRLyp/Lyp rts, lthough locl chnges in et cells in inred DRLyp/Lyp re reflected y production of eotxin (n eosinophil nd mst cell recruiting fctor) in islets t out 4 dys of ge, efore insulitis, hyperglycemi nd type 1 dietes [11, 12]. However, positive stining of infiltrting monocytes remins to e shown t this ge [11]. Additionlly, islets from 4-dy-old DRLyp/Lyp nimls express lower levels of genes involved in the metolism of rective oxygen species (ROS) [13] nd re more sensitive to chnges in redox lnce [14]. Over time, such n inherent sensitivity could contriute to ccumultion of the ROS tht diminish et cell function, rendering cells more sensitive to immune cell ttck. Islet function is lso dependent on functionl islet vsculture nd lood flow. In fct, inflmmtory chnges in vsculr endothelil cells, chrcterised y incresed expression of surfce receptors, fcilitte immune cell extrvstion into the inflmed tissue [15]. Additionlly, islet vsculture plys criticl role in mintining oxygen nd nutrient supply to the islets [16] nd poor intr-islet lood flow is ssocited with chnges in cute insulin response to glucose in vivo [17]. Interestingly, venulr defects were oserved in islets from BB (DP-BB/Wor) rts [18]. This, in comintion with n underlying et cell defect, could impir et cell function nd promote insulitis nd et cell destruction. Currently, evidence of chnges in et cell function prior to onset of type 1 dietes is limited. Therefore, we set out to explore whether insufficient et cell function, or chnges in et cell mss nd intr-islet lood flow, precede type 1 dietes using the DRLyp/Lyp rt s disese model. Methods Animls The BB rt ws originlly derived from Cndin colony of outred Wistr rts (originting from the Ottw Helth Reserch Institute, University of Ottw, Ottw, ON, Cnd) tht spontneously develop hyperglycemi nd ketocidosis, chrcteristics of clinicl onset of type 1 dietes. Heterozygous BB DRLyp/+ rts were used to otin congenic DRLyp/Lyp rts s previously descried [9, 19]. Briefly, the Lyp region from dietes-prone BB rts ws introgressed onto the dietes-resistnt BB rt nd kept in siling reeding for more thn 5 genertions y heterozygous reeders to yield 25% DRLyp/Lyp, 25% DR+/+ nd 5% DRLyp/+ rts. All DRLyp/Lyp rts developed dietes fter trnsferring the entire colony from University of Wshington, Settle to Lund University (including the Clinicl Reserch Centre in Mlmö, Sweden), in 28. Animls were red/kept in pthogen-free environment t the Clinicl Reserch Centre in Mlmö, Sweden. They were housed t C (12 h light/drk cycle) nd fed d liidum. All experiments were pproved y the Animl Ethicl Committee in Uppsl nd Lund. All nimls used in experiments were 4 dys old unless otherwise stted. Genotyping Til snips were otined from rt pups etween 25 3 dys of ge. DNAws isolted nd genotyped sed on microstellite nlysis, s previously descried [9, 2]. Blood glucose nd plsm insulin levels Blood glucose ws tested dily t 8: hours in DRLyp/Lyp (n=225, 129 mle [M]/96 femle [F]) nd control rts (DRLyp/+ nd DR+/+;

3 898 Dietologi (218) 61: n=1, 5M/5F) from dy 37 (ELTE XL glucometer; Byer Dietes Cre, Trrytown, NY, USA). Animls were considered to hve developed dietes when lood glucose levels were >11.1 mmol/l for two consecutive dys. Serum insulin wsmesuredinselinegroupt37 41 dys of ge (DRLyp/Lyp: n= 7, 4M/3F; control rts: n= 1, 5M/5F), t 5 dys (DRLyp/Lyp: n=6, 3M/3F; control rts: n=1, 5M/ 5F), t 6 dys (DRLyp/Lyp: n= 6, 3M/3F; control rts: n= 11, 6M/5F) nd t type 1 dietes onset (DRLyp/Lyp: n=7, 4M/3F; control rts: n=9, 5M/4F) in 1 μl of serum (rt insulin ELISA, Mercodi, Uppsl, Sweden). Blood ws otined from venipuncture of the til vein in the fed stte. IVGTT Glucose (1 g/kg) (Sigm Aldrich, Stockholm, Sweden) ws injected into the til vein of DRLyp/Lyp (n=1, 6M/4F) nd control (n= 1, 6M/4F) rts fter 6 h of fsting. Blood smples were collected from the sulingul vein t, 1, 5, 1, 2, 5 nd 75 min. Plsm glucose nd insulin levels were mesured (Infinity Glucose Oxidse Liquid Stle Regent, Thermo Scientific, Wlthm, MA, USA nd Rt Insulin ELISA, Mercodi, respectively). Perifusion of isolted islets Islets from DRLyp/Lyp (n = 14, 9M/5F) nd control rts (n=8, 4M/4F) were isolted using collgense digestion nd incuted in RPMI-164 medium contining 11.1 mmol/l glucose (Sigm Aldrich) + 1% FBS overnight t 37 C. Seventy islets per chmer were used in perifusion experiments (Suprfusion 1 System; Brndel, Glsgow, UK). Islets were perifused with secretion ssy uffer (SAB) contining: 114 mmol/l NCl, 4.7 mmol/l KCl, 1.2 mmol/l KH 2 PO 4, 1.16 mmol/l MgSO 4, 25.5 mmol/l NHCO 3, 2 mmol/l HEPES, 2.5 mmol/l CCl 2 nd.2% BSA (ftty cid free) (ph 7.2), supplemented with 2.8 mmol/l glucose for 2 h prior to smpling. Consecutive smples were tken t 2.8 mmol/l glucose to determine sl insulin relese efore chllenging islets with high glucose concentrtion (16.7 mmol/l). Experiments were concluded y estimting mximl insulin response y the ddition of SAB contining 35 mmol/l KCl. The flow rte ws.1 ml/min nd temperture ws kept t 37 C. Ech frction of perifuste ws collected t 4 min intervls nd stored t 2 C until nlysed (Rt Insulin ELISA, Mercodi). Btch incution of isolted islets of Lngerhns Isolted islets from DRLyp/Lyp nd control rts were cultured overnight (RPMI-164 medium, 11.1 mmol/l glucose, 1% FBS [Sigm Aldrich]; DRLyp/Lyp: n=6, 3M/3F; controls: n=6, 3M/3F), or for 5 7 dys (RPMI medium, 5.6 mmol/l glucose, 1% FBS + penicillin [1 units/ml] streptomycin [1 μg/ml]; DRLyp/Lyp: n=6, 3M/3F; controls: n=7, 3M/4F) t 37 C, 5% CO 2. Groups of three islets were plced in well of 96- well plte with SAB contining either 2.8 mmol/l or 16.7 mmol/l glucose t 37 C, 5% CO 2. Experiments were performed with 6 8 replictes for ech condition. Insulin nd glucgon levels were determined fter 1 h (Rt Insulin ELISA nd Glucgon ELISA, respectively; Mercodi). Insulin content Totl insulin ws extrcted from 5 islets per niml (DRLyp/Lyp: n= 6, 3M/3F; controls: n= 6, 3M/3F) using cid ethnol (.18 mmol/l HCl in 95% ethnol). Extrcted insulin ws diluted nd totl insulin ws mesured (Rt Insulin ELISA; Mercodi). qpcr of islets of Lngerhns Isolted islets from DRLyp/Lyp (n= 6, 3M/3F) nd control (n= 7, 3M/4F) rts were frozen ( 8 C) fter isoltion or fter 5 7 dys in culture (37 C, 5% CO 2 in RPMI medium, 5.6 mmol/l glucose, 1% FBS + penicillin [1 units/ml] streptomycin [1 μg/ml]). Totl RNA ws extrcted (RNAesy RNA purifiction kit; Qigen, Hilden, Germny) nd equl quntities of RNA were reverse trnscried (RevertAid First-Strnd cdna synthesis kit; Ferments, Vilnius, Lithuni). mrna levels were quntified (Mxim Proe/ROX qpcr Mster Mix; Ferments, Thermo Scientific, Helsingorg, Sweden) using n ABI PRISM 79 (Applied Biosystems ViiA Rel Time PCR System; Life Technologies, Foster City, CA, USA), using proes for Il1 (ID no. Rn58432), Tnf-α (lso known s Tnf) (ID no. Rn ), Ifng (ID no. Rn59478) nd Glut2 (lso known s Slc22) (ID no. Rn563565) (Applied Biosystems). Smples were run in triplicte nd the trnscript quntity ws normlised to the geometric men of mrna levels of the reference genes (Applied Biosystems) Ppi (ID no. Rn69933), Polr2 (ID no. Rn175226) nd Hprt (lso known s Hprt1) (ID no. Rn152784), using the formul 2 (minct smplect). Blood flow mesurements nd islet morphometry DRLyp/Lyp (n= 11, 4M/7F) nd control (n= 15, 6M/9F) rts were nesthetised (i.p. injection of thioutritl sodium; 12mg/kg;Inctin;SigmAldrich)ndplcedonheting pd to mintin ody temperture. The trche ws detched nd polyethylene ctheter ws inserted to secure free irwys. Ctheters were inserted into the right scending ort nd the left femorl rtery. A pressure trnsducer ws connected to the scending ort ctheter. A lood smple ws tken for lood glucose mesurement (Freestyle Lite; Aott, Clmed, CA, USA). When lood pressure hd stilised (1 15 min), nimls were injected with microspheres (dimeter: 1 μm) (E-Z Trc Ultrspheres; Stson Ls, Irwin, CA, USA) into the scending ort nd lood ws collected s descried [21]. Animls were then euthnised nd the pncres nd drenl glnds were dissected, weighed, cut in pieces nd plced etween oject glsses. Oject glsses contining

4 Dietologi (218) 61: pncretic tissue were freeze thwed to visulise islets [21]. The percentge of islet volume ws determined y pointcounting [22], nd the numer of microspheres in the exocrine nd endocrine pncres, drenl glnds nd reference smple ws counted in right nd drk field illumintion microscope. Opticl projection tomogrphy imging nd quntifiction of isletetcelldistriutionfollowing euthnistion using CO 2, pncreses from DRLyp/Lyp (n = 6, 4M/2F) nd control (n= 4, 2M/2F) rts were excised nd processed for opticl projection tomogrphy (OPT) imging [23]. Antiodies used for whole mount immunohistochemistry were: guine pig nti-insulin (1:5; A564; DAKO Denmrk, Glostrup, Denmrk) nd IRDye 68 got nti-guine pig (1:25; ; LI-COR Biosciences, Lincoln, NE, USA). Pncretic loes were scnned individully using ner-infrred OPT setup equipped with 665/45 excittion nd 725/5 emission filter (Chrom). Bet cell volumes were reconstructed sed on the signl from insulin-specific ntiodies nd pseudo-coloured to highlight the distriution of smll <1 1 6 μm 3 (white), medium μm 3 to μm 3 (yellow) nd lrge >5 1 6 μm 3 (red) islets [23, 24]. Live single cell ATP/ADP rtio mesurements Single cell ATP/ ADP rtio mesurements in islets from DRLyp/Lyp (n = 91 islets) nd control rts (n=7 islets) were performed using the ATP iosensor, Percevl (Addgene, Cmridge, MA, USA). Islets were trnsduced [25, 26], plted nd incuted on poly- D-lysine coted 8-well chmered cover glsses (Thermo Scientific, Wlthm, MA, USA) for 2 h with RPMI medium + penicillin(1 units/ml) streptomycin (1 μg/ml) contining the Percevl denovirus. Fresh medium ws dded nd cells were incuted overnight. The following dy, cells were pre-incuted t 37 C in 4 μl uffer P (135 mmol/l NCl, 3.6 mmol/l KCl, 1.5 mmol/l CCl 2,.5 mmol/l MgSO 4,.5 mmol/l N 2 HPO 4, 1 mmol/l HEPES, 5 mmol/l NHCO 3, ph 7.4) contining 2.8 mmol/l glucose for 1.5 h. After this, cells were first imged in the presence of low (2.8 mmol/l) glucose nd then in the presence of high glucose (16.7 mmol/l) to investigte the sl nd stimulted ATP/ ADP rtio. Therefter, ATP synthesis ws inhiited y the ddition of the ATP synthse inhiitor oligomycin (.2 mg/ml) nd n ionophore tht uncouples ATP synthesis, cronyl cynide-p-trifluoromethoxyphenylhydrzone (FCCP;.5 mmol/l). Cells were imged using 49 nm excittion nd 52 nm emission filter settings on Zeiss LSM51 inverted confocl fluorescence microscope (Zeiss, Oerkocken, Germny). Immunohistochemicl nlysis of islets of Lngerhns Pncretic sections from DRLyp/Lyp (n = 1, 5M/5F) nd control (n= 1, 5M/5F) rts were collected on slides nd ir-dried overnight t 37 C. Slides were deprffinised [27] nd sections incuted with the following primry ntiodies overnight t 4 C in moisturising chmers: mouse ntiglucgon (1:9; G-2654, Sigm Aldrich), guine pig ntiproinsulin (1:25; 93; EuroDignostic) nd rit nti- CD3 (1:2; C793; Sigm Aldrich). Sections were rinsed in PBS with Triton X-1 for 2 1 min. Antiodies for insulin nd glucgon ws crefully vlidted s detiled [27, 28]. CD3 specificity ws tested using primry ntiser presored with homologous ntigen (1 μg/ml ntiserum). Pncretic sections were incuted with the following secondry ntiodies with specificity for mouse, guine pig, or rit IgG: got nti-mouse Alex Fluor 568, (1:4; A21124; Invitrogen, Thermo Scientific, Helsingorg, Sweden), got nti-guine Pig, Alex Fluor 594, (1:4; A1176; Thermo Scientific) nd got nti-rit, Alex Fluor 594, (1:4; A1112; Thermo Scientific) [27]. Immunofluorescence ws exmined in n epi-fluorescence microscope (Olympus, BX6, Tokyo, Jpn). By chnging filters, doule stining ws used to determine the loction of the different secondry ntiodies in one smple. Imges were cptured with digitl cmer (Nikon DS-2Mv, Tokyo, Jpn). Sttisticl nlysis Dt re expressed s men ± SEM. IVGTTs, AUC nd cute insulin response to glucose (AIR Glucose ) were clculted s descried [6, 29, 3]. Mnn Whitney non-prmetricl testing ws employed in ll experiments, except for nlysis of islet size (OPT), lood flow mesurements, 1 h tch experiments, insulin content, qpcr nd ATP/ADP mesurements, which were nlysed with Student s t tests, nd plsm insulin levels, which were ssessed using two-wy ANOVA. Sttisticl nlyses were performed using GrphPd Prism 6 softwre (GrphPd Softwre, L Joll, CA, USA). p <.5 ws considered to e sttisticlly significnt. All experiments were performed nd nlysed in rndomised nd linded fshion when possile. Outliers were identified using Grus test for outliers. Results Dignosis of dietes DRLyp/Lyp nd control (DRLyp/+ nd DR+/+) rts were followed y dily lood glucose mesurements until dignosis of type 1 dietes (Fig. 1). Cumultive incidence reveled tht ll DRLyp/Lyp rts hd developed dietes y 8 dys of ge (Fig. 1). Men ge t onset of type 1 dietes ws 6 dys rnging from 47 to 8 dys (Fig. 1d). Femle rts developed dietes erlier thn mles (Fig. 1c; p=.4). Serum insulin prior to type 1 dietes onset Bsl insulin levels were evluted in DRLyp/Lyp nd control rts over time. Despite normoglycemi prior to onset of type 1

5 9 Dietologi (218) 61: Cumultive frequency (%) Glucose (mmol/l) Before onset (dys) Age t onset (dys) d Age t onset (dys) Dietes-free survivl (%) Age t onset (dys) dietes, insulin levels were lower t ll time points in DRLyp/ Lyp rts nd filed to increse with ge compred with control rts (Fig. 2; p=.4). In vivo insulin relese is pertured in DRLyp/Lyp rts In vivo glucose homeostsis nd et cell function were ssessed with n IVGTT in DRLyp/Lyp rts. DRLyp/Lyp rts remined glucose tolernt (Fig. 2). No difference in glucose clernce etween groups ws oserved, lso shown s AUC for glucose (Fig. 2d). However, DRLyp/Lyp rts secreted less insulin during the initil time points of the IVGTT vs controls (Fig. 2c) which ws further highlighted y reduction in AUC for insulinindrlyp/lyp rts (Fig. 2e; ± 16.2 vs ± pmol/l min; p=.4) nd decrese in the AIR Glucose (Fig. 2f; ± vs ± 148.7; p <.1). Insulin secretion is decresed in islets from DRLyp/Lyp rts To ssess differences in insulin relese (s evident y the IVGTT) etween DRLyp/Lyp nd control rts, we chrcterised the F c Fig. 1 () Dily glucose levels in 4-dy-old femle nd mle DRLyp/ Lyp (circles), control (DRLyp/+, tringles nd DR+/+, squres) rts presented s dys efore onset of type 1 dietes. () Cumultive increse in dietes incidence in mle (solid line, squres) nd femle (dotted line, circles) DRLyp/Lyp rts. (c) Dietes-free survivl in mle (solid line) nd femle (dotted line) DRLyp/Lyp rts. (d) Age t onset in femle (F) nd mle (M) DRLyp/Lyp rts. Dt shown s mens ± SEM. p <.1. DRLyp/Lyp: n = 225, 129M/96F; DRLyp/+ nd DR+/+: n = 1, 5M/ 5F M Insulin (pmol/l) c Insulin (pmol/l) e AUC (pmol/l min) Time (min) 25, 2, 15, 1, T1D onset Age (dys) Time (min) dynmics of insulin secretion in vitro using perifusion setup. Islets from DRLyp/Lyp nd control rts were first sujected to low concentrtion of glucose (2.8 mmol/l) (Fig. 3). Bsl insulin secretion ws similr etween the groups. When chllenging islets with stimultory concentrtion of glucose (16.7 mmol/l) during 4 min period, the mount of insulin secreted y islets from DRLyp/Lyp rts ws reduced. Control rts responded roustly to elevted glucose concentrtions (Fig. 3; control vs DR Lyp/Lyp AUC: ± 53.8 vs 26.1 ± 21.6 pmol/l min; p=.2). When islets were further chllenged with 35 mmol/l KCl nd 16.7 mmol/l glucose for 12 min, islets from DRLyp/Lyp rts continued to secrete less insulin thn those from control rts (Fig. 3c; control vs DR Lyp/Lyp AUC: ± 18.8 vs ± 14.9 pmol/l min; p=.2). Insulin content, however, ws similr in islets from DRLyp/Lyp nd control rts (Fig. 3d). Comprle results to those otined in perifused islets were oserved when islets were exposed to low (2.8 mmol/l) nd high (16.7 mmol/l) glucose concentrtions during 1 h sttic incution. A reduction oth in sl insulin secretion (282.5 ± 59.4 vs 186. ± 62.3 pg islet 1 h 1 ; p=.3) nd in glucose-stimulted insulin secretion (GSIS; ± vs 28.3 ± 64.4 pg islet 1 h 1 ; p <.1) from islets from Glucose (mmol/l) d AUC (mmol/l min) f AIR Glucose Fig. 2 () Serum insulin over time in DRLyp/Lyp (lck circles) nd control rts (white squres). At dys of ge: DRLyp/Lyp, n= 7 (4M/3F), control, n=1 (5M/5F); t 5 dys of ge: DRLyp/Lyp, n=6 (3M/3F), control, n=1 (5M/5F); t 6 dys of ge: DRLyp/Lyp, n=6 (3M/3F), control, n=11 (6M/5F; t type 1 dietes onset: DRLyp/Lyp, n= 7 (4M/3F), control n= 9 (5M/4F). ( f) IVGTTs in 4-dy-old DRLyp/Lyp (lck circles/rs; n = 1, 6M/4F) nd control rts (white squres/rs; n=1, 6M/4F). () Plsm glucose,(c) plsm insulin, (d) AUC for glucose nd (e)aucforinsulin.(f)air Glucose.Dtshowns mens ± SEM. p <.5,p <.1. T1D, type 1 dietes 5

6 Dietologi (218) 61: Insulin (pg/islet) c AUC (pg/islet min) e Insulin (pg islet 1 h 1 ) Time (min) G 16.7 G 2.8 G 16.7 G +K Glucose (mmol/l) DRLyp/Lyp rts vs control rts ws evident (Fig. 3e). Glucgon secretion ws similr in islets from oth groups when exposed to low nd high glucose concentrtions (ESM Fig. 1). Previous work suggests tht removing islets from n inflmmtory milieu cn restore GSIS [31]. Therefore, we cultured islets from DRLyp/Lyp nd control rts for 5 7 dys. Insulin secretion ws mesured fter exposure to low (2.8 mmol/l) nd high (16.7 mmol/l) glucose concentrtions in 1 h sttic incution. Overll insulin secretion ws improved, oth in DRLyp/Lyp nd control rt islets, ut significnt decrese in GSIS ws still evident in islets from DRLyp/ Lyp rts vs controls (Fig. 3f; ± vs ± pg islet 1 h 1 ; p <.1). Il1, Ifng nd Tnf-α expression in islets isolted from DRLyp/ Lyp rts Next we determined expression of cytokines in islets isolted from DRLyp/Lyp nd control rts. RNA ws extrcted either immeditely fter isoltion or fter culturing islets for AUC (pg/islet min) d Totl insulin (pg/islet) f , , Glucose (mmol/l) Fig. 3 () Isolted islets from 4-dy-old DRLyp/Lyp (lck circles; n= 14, 9M/5F) nd control rts (white squres; n= 8, 4M/4Fe) were perifused with 2.8 mmol/l nd 16.7 mmol/l glucose (G) with nd without 35 mmol/l KCl (K + ). ( c) AUC for secreted insulin () min t 16.7 mmol/l glucose nd (c)7 8 min t 16.7 mmol/l glucose + KCl. (d) Totl insulin content in islets from DRLyp/Lyp (n =6, 3M/3F) nd control rts (n= 6, 3M/3F). (e f) One-hour tch incution of isolted islets cultured (e) over night or(f) for 5 7 dys. Islets were stimulted with either 2.8 or 16.7 mmol/l glucose. Overnight incution: DRLyp/Lyp, n= 6, (3M/3F), control, n= 6 (3M/3F); 5 7 dy incution: DRLyp/ Lyp, n = 6 (3M/3F), control, n = 7 (3M/4F). White rs, control rts; lck rs, DRLyp/Lyp rts. Dt shown s mens ± SEM. p <.5, p <.1, p <.1 Insulin (pg islet 1 h 1 ) Pncretic lood flow (ml min 1 [g pncres] 1 ) Islet lood flow (µl min 1 [g pncres] 1 ) Fig. 4 () Whole pncretic lood flow nd () islet lood flow in 4- dy-old DRLyp/Lyp (n = 11, 4M/7F) nd control rts (n = 15, 6M/9F). White rs, control rts; lck rs, DRLyp/Lyp rts. Dt shown s mens ± SEM. p < dys.il1 ws present t similr levels in islets just fter isoltion (ESM Fig. 1). However, Tnf-α nd Ifng were undetectle. When islets where cultured over 5 7 dy period, detectle levels of ll cytokines were present (ESM Fig. 1c) ut did not differ etween groups. Islet lood perfusion To determine if reduced insulin secretion in vivo ws ssocited with microcircultory chnges [17, 32], we mesured islet lood perfusion. Men rteril lood pressure ws recorded in nimls prior to lood flow mesurements with no significnt difference etween the two groups (dt not shown). Whole pncretic lood flow did not differ etween DRLyp/Lyp nd control rts (Fig. 4). Interestingly, islet lood flow ws significntly reduced y 25% in the DRLyp/Lyp nimls vs controls (Fig. 4; 76.9 ± 11.8 vs ± 16.8 μl min 1 [g pncres] 1 ; p=.23). Smll nd medium sized islets re less common in the pncres of DRLyp/Lyp rts To understnd whether the oserved perturtion in insulin secretion in vivo ws ccompnied y differences in et cell mss, we performed OPT on the whole pncres from DRLyp/Lyp nd control rts. Overll, et cell mss did not differ etween groups (Fig. 5).However,there ws reduction in smll ( ± vs ± μm 3 ; p=.35) nd medium sized islets ( ± vs ± μm 3 ; p=.44) in the DRLyp/Lyp rts vs control rts (Fig. 5). Representtive imges from the OPT of splenic, duodenl nd gstric pncretic loes from heterozygote DRLyp/+ rt nd DRLyp/Lyp rt (Fig. 6) present size determintion y colour coding. Islets were stined with insulin: red depicts lrge islets, yellow depicts medium sized islets nd white depicts smll islets. Additionlly, we employed morphometricl method to ssess islet mss in our model [22]. We found no decrese in overll islet mss in the DRLyp/Lyp rts compred with controls (ESM Fig. 1d). ATP/ADP rtio is incresed in islets from DRLyp/Lyp rts GSIS is dependent on mitochondril metolism nd the resulting

7 92 Dietologi (218) 61: Bet cell volume (µm 3 ) 2. x x x x 1 9 Bet cell volume (µm 3 ) 1.5 x x x 1 9 Smll Medium Lrge Fig. 5 () Overll et cell volume in 4-dy-old DRLyp/Lyp (n = 6, 4M/ 2F) nd control rts (n=4, 2M/2F). () Islet volumes of ritrrily chosen islet size ctegories in DRLyp/Lyp nd control rts. White rs, control rts; lck rs, DRLyp/Lyp rts. Dt shown s mens ± SEM. p <.5 Percevl emission 52 nm 2.8 G 2 2 G Oligomycin Time (s) FCCP increse in intrcellulr rtio of ATP/ADP [33]. Therefore, we ssessed ATP/ADP rtio in et cells from DRLyp/Lyp nd control rts (Fig. 7). Interestingly, we oserved elevted sl ATP/ADP levels in et cells from DRLyp/Lyp vs control rts (Fig. 7; sl Percevl emission t 52 nm: ± 47.3 vs ± 36.4; p=.3). Addition of 2 mmol/l glucose rised the ATP/ADP rtio even further in DRLyp/Lyp vs control rts (visulised s Δ mx in Fig. 7c; ± 31.3 vs ±24.8; p=.3; nd slope-increse in Fig. 7d: 4.6 ±.5 vs 3.2 ±.4; p=.2). Moreover, AUC for the whole trce ws higher in et cells from DRLyp/Lyp rts (Fig. 7e; p=.3). Since mice lcking Glut2 lose the first phse of insulin secretion [34] nd disply similr secretory pttern s our model, we investigted Glut2 expression in islets from DRLyp/Lyp nd control rts. However, expression of Glut2 ws similr in islets from oth groups (ESM Fig. 1e). Islet morphology nd CD3 + cells re similr in DRLyp/Lyp nd control rts To determine chnges in islet morphology in Bsl Percevl emission 52 nm d Slope Percevl emission 52 nm e Percevl AUC (AU) c mx Percevl emission 52 nm ,2, 1,, 8, 6, 4, 2, Fig. 7 () ATP/ADP rtio in et cells from DRLyp/Lyp (lck circles; n= 91 islets) nd control rts (white squres; n= 7 islets). () Bsl ATP/ADP rtio, (c) Δ mx ATP/ADP rtio, (d) slope increse of ATP/ADP rtio nd (e) AUC for ATP/ADP mesurements in et cells from DRLyp/ Lyp (lck) nd control rts (white). Dt shown s mens ± SEM. p <.5, p <.1, p <.1. AU, ritrry units; G, glucose (mmol/l); FCCP, cronyl cynide-4-(trifluoromethoxy)phenylhydrzone; G, glucose (mmol/l) DRLyp/Lyp rts, we performed insulin nd glucgon stining. Islets in pncretic sections from oth DRLyp/Lyp nd control rts displyed norml islet rchitecture (core of et cells surrounded y lph cells; Fig. 8,c). To confirm previous findings tht 4-dy-old DRLyp/Lyp rts do not present immune cell infiltrtion, we performed stining using CD3 + specific ntiody comined with nucler DAPI. As expected, stining ws sprse, ut similr in DRLyp/Lyp nd control nimls (Fig. 8,d). Discussion Fig. 6 Representtive OPT imges from splenic, duodenl nd gstric pncretic loe from 4-dy-old heterozygote DRLyp/+ rt (control) nd DRLyp/Lyp rt. Scle r, 2 mm The present study demonstrtes tht GSIS is pertured in the DRLyp/Lyp rt s compred with dietes-resistnt

8 Dietologi (218) 61: Fig. 8 Pncretic sections from (,) 4-dy-old DRLyp/Lyp nd (c,d) control rts (DRLyp/+ nd DR+/+). Sections were stined for (,c) insulin (green) nd glucgon (red), nd (,d) CD3 + (red) with nucler DAPI (lue). Scle r, 5 μm littermtes. The secretory defect ws ccompnied y significnt reductions in the numer of medium nd smll sized islets, nd reduced intr-islet lood flow. Notly, these isletspecific derngements were oserved t 4 dys of ge efore hyperglycemi, insulitis nd onset of type 1 dietes. Type 1 dietes is ssocited with the immune-medited destruction of et cells, resulting in insulin deficiency. Recent dvnces hve highlighted genetic nd functionl chnges within the et cell s prt of type 1 dietes pthology [4, 29], suggesting tht et cells my hve n inherent sensitivity tht possily mkes them susceptile to utoimmune ttck. We oserved significnt reduction in insulin secretion oth in vivo nd in vitro in isolted islets from DRLyp/Lyp rts. Indeed, previous study showed tht non-inred BB rts (BB/ Hgedorn; model where lymphopeni is not present) displyed diminished relese of insulin during stimultion with 2 mmol/l glucose in perfused whole pncres t 5 dys of ge (efore onset of type 1 dietes) [35]. Similr oservtions hve een mde in islets from NOD mice, where insulin secretion immeditely fter isoltion ws pertured (due to insulitis). However, culture of islets from NOD mice over 5 7 dy period improved insulin secretion significntly [31]. Indeed, islets from DRLyp/Lyp rts displyed n improved response to glucose fter culturing period; however, secretory defect ws still evident. Similrly, islets removed from people with new-onset type 1 dietes show improved GSIS fter culture [36]. It is noteworthy, however, tht GSIS could not e fully restored in ll individuls. A mjor difference etween those studies nd ours is tht insulitis is not present in 4-dy-old DRLyp/Lyp rts. Islets from 4-dy-old DRLyp/ Lyp rts show reduced expression of the complement inhiitor protein CD59. CD59 is pivotl for norml et cell exocytosis [37], suggesting tht et cell exocytosis is compromised in DRLyp/Lyp rts. This corresponds to our perifusion dt, where islets from DRLyp/Lyp rts disply n improved response to 35 mmol/l KCl, suggesting tht insulin is not lost, rther tht exocytosis is compromised. A previous study highlighted similr findings where non-metolic secretgogues elicit insulin relese in predietic conditions nd in type 1 dietes [38]. Additionlly, insulin content is not ltered in isolted islets from 4-dy-old DRLyp/Lyp rts, which further supports this notion. In predietic NOD mice, et cell dysfunction is suggested to occur s consequence of erly immune cell infiltrtion nd ctivtion of inflmmtory cscdes [39]. However, the DRLyp/Lyp rts do not disply ny mjor infiltrtion y mononucler cells until few dys prior to clinicl onset of type 1 dietes [13]. We confirmed this, nd islets from DRLyp/Lyp rts did not show incresed infiltrtion of CD3 + cells in pncretic sections. Moreover, we were unle to detect elevted expression of Il1, Ifng nd Tnf-α in islets from DRLyp/Lyp rts; cytokines tht could e indictive of erly immune processes within the islets [4, 41]. Bet cell mss is tightly regulted during fetl life, time point representing criticl window when the pproprite numer of et cells re set in plce [42]. A potentil wekness in the present study is tht we hve not investigted neontl et cell growth nd postntl expnsion of et cells in our model. It my very well e tht DRLyp/Lyp rtsreornwithreduced numer of et cells, or fil to expnd their et cell mss during postntl stges. We oserve significnt reductions in smll nd medium sized islets in DRLyp/Lyp compred with control rts, leit overll islet mss ws not chnged. A previous study shows tht smller islets contin more insulin per islet volume in situ nd secrete insulin more efficiently in vitro [43]. In ddition, lrge islets my e sujected to oth hyperplsi nd hypoxi [44], resulting in impired et cell function. Thus, loss of smll nd medium sized islets my very well impct insulin secretion. Additionlly, OPT hs n dvntge over more conventionl methods, since it cn give informtion on sptil position nd volume of individul insulin-expressing islets throughout the pncres, with high resolution nd the opportunity to ctegorise islets y size [23]. Another importnt fctor influencing et cell function is nutritionl lood sttus nd islet lood flow. This could e considered s the min venue y which et cells re kept informed of the ody s nutritionl stte [45]. We oserved reduced intr-islet lood flow in DRLyp/Lyp rts. The importnce of this finding for development of type 1 dietes remins to e determined, ut in generl lower lood perfusion in islets could compromise et cell function through hypoxi or limited dispersl of insulin into the systemic circultion [17, 32]. Moreover, decresed lood flow decreses sher stress, which increses the tendency for leucocyte dhesion in venules even in the sence of dditionl ctivtors [46]. This could promote islet immune cell infiltrtion. Indeed,

9 94 Dietologi (218) 61: previous study showed venulr defect in relted rt strin (BB/Wor rt), which supports our findings [18]. Currently, ny reltionship etween lood flow chnges nd lymphopeni in DRLyp/Lyp rts remins unknown. High sl islet lood flow in dietes-resistnt (nd/or wild-type) nimls is to lrge extent medited y loclly generted nitric oxide from endothelil cells nd inhiiting this system decreses lood perfusion [47]. It is noteworthy tht studies on islet endothelil cells from young normoglycemic dietesprone nd dietes-resistnt BB rts hve shown tht dietes-prone rts exhiit considerly lower endothelil cell nitric oxide synthse ctivity thn dietes-resistnt rts [48]. Insulin relese is to lrge extent dependent on mitochondril metolism of glucose nd the resulting increse in intrcellulr rtio of ATP/ADP [33]. Glucose uptke into et cells is the initil step in GSIS. In rodents this is medited y GLUT2 [49]. Mice lcking Glut2 lose the first phse of insulin secretion [34]. Thus, oth the ATP/ADP rtio nd Glut2 expression could influence GSIS in DRLyp/Lyp rts. We oserved no chnges in Glut2 expression. Intriguingly, however, ATP/ADP levels were elevted in islets from DRLyp/Lyp rts, which could signify compenstory mechnism s mitochondri re striving to mintin sufficient ATP/ADP rtio nd coupling fctors to ensure sufficient insulin relese. It my lso suggest tht the secretory deficiency lies distl of ATP genertion (i.e. depolristion of the plsm memrne/c 2+ influx or exocytosis). Clerly, more intense reserch efforts re required in this re. In summry, our results show tht DRLyp/Lyp rts disply secretory defect prior to utoimmune onset of type 1 dietes. This is mnifested y perturtions in insulin secretion in vivo nd in vitro, prtil loss of et cell mss nd reduced intr-islet lood flow; ll of which re fctors tht influence et cell function. These chnges my e of importnce for the development of type 1 dietes. Acknowledgements We thnk L. Fxius nd A-.H. T. Fischer for excellent technicl ssistnce. Dt vilility sttement The dtsets generted during nd/or nlysed during the current study re ville from the corresponding uthor on resonle request. Funding This study ws supported in prt y the Swedish Reserch Council (K213-99X nd K _3 [MF], K211-54X [ÅL], K213-55X [POC]), the Novo Nordisk Foundtion, the JDRF, The Gyllenstiernsk Krpperup Foundtion, The Crfoord Foundtion, SUS Funds, nd the Skåne County Council for Reserch nd Development. Dulity of interest uthors. No conflicts of interest re reported y ny of the Contriution sttement The study ws designed y MF nd ÅL. Blood smpling, glucose nlyses nd genotyping of BB rts ws performed y LÅ, AM nd YTS. Islet isoltion, dt cquisition, nlysis nd interprettion of perifusion studies nd tch incutions were performed y AM, YTS, HB nd AB. IVGTTs nd nlysis thereof ws performed y MF nd AM. NV performed ATP/ADP mesurements/imging nd dt nlysis. Pncretic lood flow nd intr-islet lood flow experiments nd nlysis ws performed y SU, MQ nd POC. Preprtion of pncres for OPT nd dt nlysis ws performed y AM, SP nd UA. Immunohistochemistry ws performed y AM nd NW, nd nlysis thereof ws performed y NW. Expression nd nlysis of genes ws performed y AM. The mnuscript ws drfted y AM nd MF. All uthors pproved the finl version of the mnuscript. MF is the gurntor of this work. Open Access This rticle is distriuted under the terms of the Cretive Commons Attriution 4. Interntionl License ( cretivecommons.org/licenses/y/4./), which permits unrestricted use, distriution, nd reproduction in ny medium, provided you give pproprite credit to the originl uthor(s) nd the source, provide link to the Cretive Commons license, nd indicte if chnges were mde. References 1. Hyttinen V, Kprio J, Kinnunen L, Koskenvuo M, Tuomilehto J (23) Genetic liility of type 1 dietes nd the onset ge mong 22,65 young Finnish twin pirs: ntionwide follow-up study. 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