Critical role of c-kit in beta cell function: increased insulin secretion and protection against diabetes in a mouse model

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1 Dietologi (1) 55:14 5 DOI 1.17/s ARTICLE Criticl role of c-kit in et cell function: incresed insulin secretion nd protection ginst dietes in mouse model Z. C. Feng & J. Li & B. A. Turco & M. Riopel & S. P. Yee & R. Wng Received: 5 Ferury 1 /Accepted: 3 Mrch 1 /Pulished online: 14 My 1 # Springer-Verlg 1 Astrct Aims/hypothesis The receptor tyrosine kinse, c-kit, nd its lignd, stem cell fctor, control vriety of cellulr processes, including pncretic et cell survivl nd differentition s reveled in c-kit Wv mice, which hve point muttion in the c-kit llele leding to loss of kinse ctivity nd develop dietes. The present study further investigted the intrinsic role of c-kit in et cells, especilly the underlying mechnisms tht influence et cell function. Methods We generted novel trnsgenic mouse model with c-kit overexpression specificlly in et cells () to Z. C. Feng nd J. Li contriuted eqully to this work. 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. Z. C. Feng : J. Li : B. A. Turco : M. Riopel : R. Wng () Victori Reserch Lortories, Room A5-14, 8 Commissioners Rod Est, London, ON, Cnd NC V5 e-mil: rwng@uwo.c Z. C. Feng : J. Li : B. A. Turco : R. Wng Deprtments of Physiology nd Phrmcology, University of Western Ontrio, London, ON, Cnd R. Wng Deprtment of Medicine, University of Western Ontrio, London, ON, Cnd M. Riopel Deprtment of Pthology, University of Western Ontrio, London, ON, Cnd S. P. Yee Deprtment of Genetics nd Developmentl Biology, University of Connecticut Helth Center, Frmington, CT, USA further exmine the physiologicl nd functionl roles of c-kit in et cells. Isolted islets from these mice were used to investigte the underlying moleculr pthwy of c-kit in et cells. We lso chrcterised the ility of c-kit to protect nimls from high-ft-diet-induced dietes, s well s to rescue c-kit Wv mice from erly onset of dietes. Results mice exhiited improved et cell function, with significntly improved insulin secretion, nd incresed et cell mss nd prolifertion in response to high-ft-dietinduced dietes. islets exhiited upregultion of: (1) insulin receptor nd IRSs; () Akt nd glycogen synthse kinse 3β phosphoryltion; nd (3) trnscription fctors importnt for islet function. c-kit overexpression in et cells lso rescued dietes oserved in c-kit Wv mice. Conclusions/interprettion These findings demonstrte tht c-kit plys direct protective role in et cells, y regulting glucose metolism nd et cell function. c-kit my therefore represent novel trget for treting dietes. Keywords Bet cell function. Bet cell-specific c-kit trnsgenic mice. c-kit Wv muttion. High-ft-diet-induced dietes. Insulin secretion Arevitions mice Trnsgenic mouse model with c-kit overexpression specificlly in et cells egfp Enhnced green fluorescent protein GSIS Glucose-stimulted insulin secretion GSK3β Glycogen synthse kinse 3β HFD High-ft diet IPGTT Intrperitonel glucose tolernce test IPITT Intrperitonel insulin tolernce test MAFA v-mf musculoponeurotic firosrcom oncogene fmily, protein A (vin) PDX1 Pncretic nd duodenl homeoox 1

2 Dietologi (1) 55: PI3K RIP SCF Introduction Phosphoinositide-3-kinse Rt insulin promoter Stem cell fctor present study, we descrie novel trnsgenic mouse model with c-kit overexpression specificlly in et cells (), which we used to investigte the underlying mechnism of c-kit ctivity in et cells, nd to further delinete the physiologicl nd functionl role of c-kit in norml, high-ft diet (HFD)-induced dietic nd c-kit Wv mice. Therpeutic strtegies imed t repopulting insulinproducing cells show gret potentil for restoring glycemi in dietes. Extensive studies hve focused on wys to fcilitte the differentition of progenitor cells into et cells [1 3], nd to mintin their viility nd function [4, 5]. It hs een shown tht the hemtopoietic stem cell mrker c-kit is importnt in the development nd function of islets of Lngerhns, especilly in support of et cell prolifertion, mturtion nd survivl [ 1]. Upon inding to the stem cell fctor (SCF), c-kit undergoes dimeristion nd utophosphoryltion, followed y the recruitment of downstrem signlling molecules to induce susequent cell prolifertion, differentition, survivl nd migrtion [13, 14]. c-kit is found in fetl nd dult rodent pncretic islets [ 8, 1]. We hve demonstrted tht humn nd rt fetl pncretic ductl epithelil cells producing c-kit disply high prolifertion nd level of SCF [11, 1, 15]. After pncretic duct ligtion in the rt, c-kit is ctivted in ductl cells during islet cell neogenesis, long with n increse of pncretic nd duodenl homeoox 1 (PDX1) levels [1]. Incresed c-kit nd PDX1 undnce is lso oserved in islets from pncreses of streptozotocin-induced dietic rts, suggesting tht c-kit is involved in et cell regenertion [17]. Mnipultion of rt islets in cell culture hs further reveled tht c-kit-enriched cells cn give rise to new et cells tht secrete insulin in glucose-responsive fshion [18]. Fetl rt islets treted with SCF hd significnt increse in insulin levels nd DNA content [7]. Furthermore, INS-1 cells responded to SCF with incresed cell prolifertion [1]. Downregultion of c-kit (lso known s KIT) expression in humn islet epithelil clusters using smll interfering RNA leds to significntly reduced mrna nd protein levels of PDX1 nd insulin in conjunction with decresed cell prolifertion, s well s incresed cell deth [1]. These studies revel remrkle correltion etween the functions of c-kit nd enhnced et cell development nd function. Homozygous c-kit-null (c-kit W/W ) mutnt mice, disply reltively norml islet morphology, ut die shortly fter irth nd re not ville for further functionl studies [19]. We hve previously chrcterised c-kit Wv mice, which hve point muttion in the c-kit llele, disrupting receptor function. These mice exhiit loss of et cell mss nd prolifertion, resulting in erly onset of dietes []. In the Methods Genertion nd mintennce of mice Humn c-kit cdna (.9 k pirs) followed y the IRES-enhnced green fluorescence protein (egfp) (Clontech, Plo Alto, CA, USA) sequence ws inserted into the pks/rt insulin promoter (RIP) plsmid [1] to generte the trnsgene. Trnsgenic mice () were generted using C57BL/J emryos nd identified y PCR [] using the primers gloin 5 nd c-kit, nd further confirmed y second set of primers, egfp3 nd gloin 3 (Electronic supplementry mteril [ESM] Tle 1). Five RIP c-kit trnsgenic founders were otined nd red with C57BL/J mice (Jckson Lortory, Br Hror, ME, USA) to estlish independent mouse lines. Initil chrcteristion reveled tht the mice displyed similr, if not identicl, ptterns of trnsgene expression nd phenotype tht eliminte ny positionl effect due to the loction of trnsgene integrtion. We therefore used offspring from two independent trnsgenic lines for susequent detiled nlyses. All mice hd free ccess to stndrd diet. The protocol used ws pproved y the University of Western Ontrio Animl User Sucommittee in ccordnce with the guidelines of the Cndin Council of Animl Cre. Body weight, food intke nd in vivo metolic studies Body weight, lood glucose levels, intrperitonel glucose tolernce tests (IPGTT) nd intrperitonel insulin tolernce tests (IPITT) were performed using mice nd their wildtype littermtes from 4 to 4 weeks of ge s descried previously [, 3]. Food intke ws monitored t weeks of ge for week period. Blood glucose levels were exmined under non-fsting, nd fter 4 h nd overnight fsting (1 h) with free ccess to wter. For the IPGTT nd IPITT, n intrperitonel injection of glucose (D-(+)-glucose; dextrose; Sigm, St Louis, MO, USA) t dosge of mg/g ody weight or of humn insulin (Humlin; Eli Lilly, Toronto, ON, Cnd) t 1 U/kg ody weight ws dministered nd lood glucose levels were exmined. The AUC ws used to quntify responsiveness [, 3]. Genertion of HFD-induced dietes nd :Wv mouse models The HFD study ws initited t weeks of ge, with nd wild-type mle mice receiving HFD

3 1 Dietologi (1) 55:14 5 chow (D149; Reserch Diets, New Brunswick, NJ, USA) for 4 weeks, followed y in vivo metolic studies [4, 5]. Breeding with c-kit Wv mice yielded four different mouse genotypes: (1) wild-type; () ; (3) c-kit Wv nd (4) :Wv. Presence of the c-kit Wv llele ws identified y the niml s chrcteristic fur pigmenttion []; the llele ws identified y PCR. In vivo metolic studies were performed on mle mice groups t 8 weeks of ge. INS-1 cell culture INS-1 (83/13) cells were cultured in RPMI 14 contining 1% FBS vol./vol., s descried previously []. For the exogenous SCF study, INS-1 cells were cultured in RPMI 14 plus 1% (wt/vol.) BSA, nd treted for 4 h with SCF (5 ng/ml; ID Lortory, London, ON, Cnd), SCF plus wortmnnin (1 nmol/l; Sigm) or non-scf (control) [15]. Cells were collected for immunofluorescence stining nd protein extrction. Cell prolifertion ws exmined using the 3-(4,5-dimethylthizol--yl)-,5- diphenyltetrzolium romide (MTT) ssy nd Ki7 stining [15]. Three different cell pssges were used for ech set of experiments, representing n3. In vivo nd ex vivo glucose-stimulted insulin secretion ssy nd insulin ELISA For the in vivo glucose-stimulted insulin secretion (GSIS) ssy, lood smples were collected following 4 h of fsting ( min), nd t 5 nd 35 min fter glucose loding [, 3]. For the ex vivo GSIS ssy, freshly isolted islets from mice were hnd-picked nd incuted for 1 h with RPMI 14 plus.5% (wt/vol.) BSA contining. or mmol/l glucose []. Insulin secretion ws mesured using n ultrsensitive (mouse) insulin ELISA (Alpco, Slem, NH, USA). A sttic GSIS index ws clculted [, ]. The insulin content of isolted islets ws mesured nd expressed s nnogrms per microgrm DNA. Immunofluorescence nd morphometric nlyses Pncreses were fixed in 4% (wt/vol.) prformldehyde, nd sections prepred from the entire length of the pncres nd stined with primry ntiodies of dilutions s listed (ESM Tle ). Quntittive evlutions of lph cell nd et cell mss were performed using Openl imge softwre (Improvision, Lexington, MA, USA) [, 3]. Bet cell prolifertion nd levels of different trnscription fctors were determined y doule immunofluorescence stining nd quntifiction from t lest 1 rndom islets per pncretic section [, 3]. RNA extrction nd rel-time RT-PCR RNA ws extrcted from isolted islets of nd wild-type mice with or without HFD using kit (mirnesy; Qigen, Germntown, MD, USA) [3]. Sequences of PCR primers re listed in ESM Tle 3. Rel-time PCR nlyses were performed using kit (iq SYBR Green Supermix; Bio-Rd Lortories, Mississug, ON, Cnd). Reltive levels of gene expression were clculted nd normlised to the internl gene, 18S rrna, with t lest four repets per ge per experimentl group [, 3]. Protein extrction nd Western lot nlysis Islet proteins from nd wild-type pncreses were extrcted in NP-4 lysis uffer [, ]. An equl mount of protein from ech group ws frctionted y 1% (wt/vol.) SDS- PAGE, trnsferred on to nitrocellulose memrne (Bio- Rd) nd incuted with primry ntiodies s listed (ESM Tle ). Proteins were detected using western lot detection regents (ECL-Plus; Perkin Elmer, Wellesley, MA, USA) nd imged using Versdoc Imging System (Bio-Rd). Bnds were densitometriclly quntified y Imge l softwre (Bio-Rd). Sttisticl nlysis Dt re expressed s mens±sem. Sttisticl significnce ws determined y unpired Student s t test or ANOVA followed y Fisher s lest significnt differences post-hoc test. Differences were considered to e sttisticlly significnt t p<.5. Results Genertion of mouse model We generted mice on C57BL/J ckground using the RIP to direct overexpression of humn c-kit specificlly in et cells; humn c-kit ws lso linked with n IRES-eGFP to fcilitte monitoring of trnsgene expression. Immunolot nlysis of egfp in pncres, liver, muscle, rin nd isolted islet protein lystes showed positive signls in the pncres nd islets of mice (Fig. 1). The RIP did not led to ny errnt trnsgene expression. Fluorescence microscopy showed tht egfp ws present in islets freshly isolted from, ut not in those from wildtype littermtes (Fig. 1). This ws further confirmed y immunofluorescence stining for egfp nd insulin (Fig. 1). Humn c-kit mrna ws only detected in mouse islets (Fig. 1c), finding corroorted y elevted islet c-kit levels (Fig. 1d) nd c-kit immunofluorescence stining (Fig. 1e). We lso exmined the undnce of SCF, lignd of c-kit, in mouse islets (ESM Fig. 1). A significnt increse of Scf (lso known s Kitl) mrna ws noted in islets compred with wild-type islets (ESM Fig. 1); however, protein levels of SCF were not sttisticlly different etween nd wild-type islets (ESM Fig. 1). Tken together, these results indicte tht overexpression of humn c-kit under the trnscriptionl control of the RIP in mice is specific to et cells.

4 Dietologi (1) 55: Fig. 1 Genertion of C57BL/J trnsgenic mice with c-kit overexpression specificlly in et cells. Western lot nlysis of egfp undnce in tissues s indicted nd isolted islets (representtive lots shown). Aundnce of egfp on freshly isolted islets nd pncretic sections of 4-weekold mle wild-type () nd mice y doule immunofluorescence stining for egfp (green) with insulin (red). c RT-PCR nlysis of humn c-kit mrna in isolted islets of wild-type nd mice t 4 weeks of ge. d Western lot nlysis of c-kit levels in isolted islets of wild-type nd mice t 8 weeks of ge; vlues re men±sem; n3; p<.1 vs wild-type. e Immunofluorescence stining for c-kit (green) on pncretic sections from 4-week-old wild-type nd mle mice. Nuclei were stined with DAPI (lue). Representtive imges re shown. Scle rs (, e), 5 μm d Reltive c-kit protein level (normlised to clnexin) c-kit Clnexin c c-kit e egfp β-actin c-kit egfp expression in freshly isolted islets c-kit βtg Insulin c-kit βtg egfp/insulin/dapi c-kit/insulin/dapi Improved glucose tolernce nd insulin secretion in mice There were no significnt differences in ody weight during the 4 weeks of oservtion, with no chnges in food intke, etween nd wild-type mice (ESM Fig. ). No significnt differences in 4 h fsting plsm insulin nd lood glucose levels (Fig.,, ESM Fig. 3, ) were detected etween nd wild-type mice t 8 weeks of ge; however, the overnight fsting lood glucose levels were significntly lower in mice thn in wild-type littermtes (Fig., ESM Fig. 3). The IPGTT showed reltively similr response cpcity in nd wild-type mle mice t 4 weeks (ESM Fig. 4). However, significntly improved glucose tolernce ws oserved in mle mice t 8 nd weeks of ge, long with significnt decreses in the AUC during the IPGTT (Fig. c, ESM Fig. 4); no significnt chnge ws oserved in femle mice (ESM Fig. 3c, ESM Fig. 4). No chnges were oserved in insulin tolernce etween the experimentl groups (Fig. d, ESM Fig. 3d). Furthermore, the effect of c-kit overexpression on et cell insulin secretion ws significnt, with isolted islets from mle nd femle mice relesing significntly more insulin thn wild-type controls in response to mmol/ l glucose (Fig. e, ESM Fig. 3e). This ws ssocited with higher insulin content in islets (Fig. f). Since femle mice did not exhiit significnt ltertion in the IPGTT, we focused on mle mice for detiled chrcteristion. Incresed islet trnscription fctors, et cell mss nd prolifertion in mice To further chrcterise the functionl role of c-kit in et cells, we exmined levels of trnscription fctors essentil for islet growth, function nd morphology in nd wild-type mice. At 8 weeks of ge, we oserved slightly incresed islet numer (Fig. 3), with significnt increse in the numer of smll (<5 μm ) nd lrge islets (>1, μm ; Fig. 3) in the pncres of mice. No significnt ltertions in lph cell mss were detected etween wild-type nd mice (Fig. 3c). However, et cell mss in

5 18 Dietologi (1) 55:14 5 Fig. Glucose tolernce nd insulin secretion in 8-week-old mle mice. Fsting plsm insulin () nd lood glucose () in wild-type (, white rs) nd (lck rs) mice. c IPGTT nd (d) IPITT in wild-type (lck circles) nd (lck squres) mice. The glucose responsiveness of the corresponding experimentl groups is shown s the AUC of the IPGTT (c, insert) or IPITT (d, insert) grphs, with units of (mmol/l x min). Dt ( d) re expressed s men±sem; n5 18; p<.5 nd p<.1 vs wild-type mice. e Insulin secretion of isolted islets from wild-type nd mice in response to mmol/l glucose chllenge; dt re expressed s fold chnge normlised to sl (. mmol/l glucose) secretion; n3 4. f Insulin content in isolted islets; dt re normlised to DNA content; n7; p<.5 nd p<.1 vs wild-type mice c 3 1 Fsting plsm insulin (pmol/l) e GSIS in isolted islets (fold chnge normlised to sl) AUC (mmol/l min) non-fbg 4h-FBG ON-FBG,5, 1,5 1, Time fter glucose i.p. injection (min).5 8 d f Insulin content (ng/µg DNA) Time fter insulin i.p. injection (min) 3 1 AUC (mmol/l min) 8 4 mice ws incresed y 1.-fold compred with wild-type (Fig. 3d). Incresed et cell mss ws ssocited with n increse in et cell prolifertion in mice (Fig. 3e, ESM Fig. 5). Quntittive rel-time RT-PCR nlysis of Pdx1, Neurod1, Mf, Px, Nkx- nd Nkx-1 showed significntly incresed mrna levels in mice (Fig. 3f), with elevted intensity of the corresponding signls in the islets of mice (ESM Fig. ). The expression of Glut (lso known s Slc), Ins1 nd Ins, Gcg nd Glp1r mrna in isolted islets ws lso significntly incresed (Fig. 3g) with reltively enhnced Glut nd glucgon-like peptide 1 receptor (Glp1R) stining (ESM Fig. ). Incresed undnce of insulin receptor, phosphorylted IRS1/ nd their downstrem signlling molecules in the islets of mice Our previous study showed tht the c-kit Wv muttion cused et cell dysfunction [] nd tht this muttion ws ssocited with downregultion of the Akt glycogen synthse kinse 3β (GSK3β) cyclin D1 pthwy [7]. We therefore exmined whether signlling molecules up- nd downstrem of the phosphoinositide-3- kinse (PI3K) Akt pthwy or cell survivl signls re ltered in islets. Interestingly, significnt upregultion of Insr nd Irs1 mrna ws oserved in islets (Fig. 4), with n increse of insulin receptor nd phosphorylted IRS1/ protein levels (Fig. 4, c) compred with wild-type mice. Protein levels of phospho-akt (Fig. 4d), phospho-gsk3β (Fig. 4e), cyclin D1 (Fig. 4f) nd PDX1 (Fig. 4g) were lso significntly incresed in islets compred with islets isolted from wildtype littermtes. This oservtion ws further chrcterised using the INS-1 cell line. When treted with exogenous SCF, INS-1 cells exhiited significntly incresed cell prolifertion (ESM Fig. 7, ), with upregultion of the phospho-akt GSK3β pthwy (ESM Fig. 7c), together with incresed insulin receptor nd phosphorylted IRS1/ protein levels (ESM Fig. 7d). The increse in insulin receptor nd phospho-irs1/ protein levels ws regulted y the PI3K Akt signlling pthwy.

6 Dietologi (1) 55: Fig. 3 Islet morphology in mle mice. Morphometric nlysis of islet numer (), islet size (), lph cell mss (c), et cell mss (d) nd the percentge of Ki7 + in et cells (e) in wild-type (, white rs) nd (lck rs) mice t 8 weeks of ge. f Trnscription fctor nd (g) islet gene expression in nd wild-type mice t 8 weeks of ge, s nlysed y quntittive RT-PCR. Dt re expressed s mens±sem; n4 8; p<.5, p<.1 nd p<.1 vs wild-type mice f c Alph cell mss (mg) Reltive mrna expression (normlised to 18S) Islet numer per mm Bet cell mss (mg) Islet size/totl islets (%) <5 µm 1. 5-,5 µm,5-1, µm >1, µm.. d Pdx1 Neurod1 Mf Px4 Px Nkx- Nkx-1 Glut Ins Gcg Glp1r g Reltive mrna expression (normlised to 18S) e Ki7 + /Insulin + cells (%) mice tolerted HFD-induced dietes To investigte how et cell-specific c-kit overexpression would ffect glucose homeostsis of mice under dietic conditions, mle nd wild-type littermtes were sujected to HFD. Similr food intke nd ody weight gins were oserved in oth experimentl groups (Fig. 5). Interestingly, fter 4 weeks of HFD, the ft pd mss ws significntly smller (Fig. 5) nd the 4 h fsting glucose level ws lower in HFD mice thn in wild-type HFD mice (Fig. 5c). Glucose intolernce ws noted in wildtype HFD littermtes, ut ws significntly improved in c- KitβTg HFD mice (Fig. 5d), with similr results for insulin response in wild-type HFD s determined y the IPITT (Fig. 5e). In vivo GSIS ssys reveled slightly higher sl nd 5 min plsm insulin levels in HFD mice (Fig. 5f); moreover, t 35 min fter glucose stimultion, the plsm insulin relese ws significntly reduced in wildtype HFD compred with HFD mice (Fig. 5f). GSIS on isolted islets further demonstrted tht HFD islet insulin secretion ws significntly incresed in response to mmol/l glucose compred with wild-type HFD islets (Fig. 5g). The insulin content ws lso significntly incresed in HFD islets (Fig. 5h). Improvements in glucose tolernce in HFD mice were ssocited with significntly incresed Pdx1, Mf, Ins, Insr nd Irs1 mrna, s well s phospho-irs1/ levels in HFD islets (Fig. ). The improved metolic phenotype of HFD mice ws ssocited with significnt increse in the numer of pncretic islets nd et cell mss compred with wild-type HFD mice (Fig., d). A slightly incresed lph cell mss ws oserved in HFD mice; however, the increse ws not sttisticlly significnt (Fig. c). Nevertheless, significnt increse in et cell mss ws detected, with incresed et cell prolifertion in HFD mice compred with the wild-type HFD group (Fig. e). c-kit Wv mice with specific overexpression of c-kit in et cells displyed norml glucose metolism c-kit Wv mice were red with mice to determine whether c-kit overexpression in et cells could rescue nimls with the c-kit Wv muttion from erly onset of dietes. Wild-type, nd :Wv mice exhiited similr fsting lood glucose levels, with significnt improvement in fsting lood glucose levels eing noted in :Wv compred with c-kit Wv mice (Fig. 7). :Wv mice lso hd similr glucose tolernce cpcity to tht of the wild-type nd groups, nd lso displyed significnt decreses in the AUC (Fig. 7). The IPITT reveled no differences etween the experimentl groups (Fig. 7c).

7 Dietologi (1) 55:14 5 Reltive mrna expression (normlised to18s) c Insr Irs1 Irs Reltive insulin receptor (normlised to β-ctin)..4.. InsR β-actin Significntly improved GSIS ws oserved t 5 nd 35 min in :Wv mice compred with c-kit Wv mice (Fig. 7d). To further confirm islet function, n ex vivo GSIS study ws conducted, in which insulin secretion in response to mmol/l glucose chllenge in :Wv islets ws similr to tht in wild-type nd islets (Fig. 7e), ut significntly higher thn tht of c-kit Wv islets (Fig. 7e). Insulincontentinisolted:Wv islets ws lso higher thn in c-kit Wv islets, ut significntly lower thn in controls (Fig. 7f). These results indicte tht the c-kit point muttion is directly responsile for defective et cell function in c-kit Wv mice nd tht c-kit overexpression ws le to preserve et cell function in c-kit Wv mice. Discussion Reltive phospho-akt S473 (normlised to totl Akt) Reltive cyclin D1 protein (normlised to β-ctin) d f pirs1/ pakt takt Insulin pirs1//insulin/dapi e Cyclin D1 β-actin Reltive phospho-gsk3β S9 (normlised to totl GSK3β) Reltive PDX1 protein (normlised to β-ctin) g pgsk3β tgsk3β PDX1 β-actin Fig. 4 Levels of phosphorylted Akt/GSK3β signlling nd downstrem signlling molecules in mle mice. Quntittive RT- PCR nlysis of Insr, Irs1 nd Irs in isolted islets of wild-type (, white rs) nd (lck rs) mice t 8 weeks of ge. Dt re expressed s men±sem; n3 4. Western lot nlysis for insulin receptor (InsR) nd (c) doule immunofluorescence stining for phospho-irs1/ (green) nd insulin (red) in pncretic sections from 8-week-old wild-type nd mice. Nuclei were stined y DAPI (lue); representtive imges shown; scle rs 5 μm. d Western lot nlysis of phosphorylted (p) nd totl (t) Akt, (e) pgsk3β nd tgsk3β, (f) cyclin D1 nd (g) PDX-1 undnce in isolted islets of wild-type nd mice t 8 weeks of ge. Representtive lots re shown. Dt re normlised to totl protein or loding control nd expressed s mens±sem; n4 5. p<.5, p<.1 nd p<.1 vs wild-type mice Here, we demonstrted tht c-kit overexpression in et cells confers improved glucose metolism y enhncing insulin secretion, nd incresing et cell mss nd prolifertion, proly through ctivtion of the PI3K Akt signlling pthwy. When mice were sujected to HFD, they displyed resistnce to HFD-induced glucose intolernce nd preserved et cell function reltive to wild-type littermtes. Moreover, mice were protected ginst erly onset of dietes. These results clerly indicte tht c-kit is intrinsic to et cell function nd prolifertion. This effect is medited y the regultion of key et cell trnscription fctors (e.g. PDX1 nd v-mf musculoponeurotic firosrcom oncogene fmily, protein A (vin) [MAFA]) nd possily through interction with the insulin receptor to ctivte downstrem PI3K Akt signlling. Compred with wild-type mice, mice hd significntly lower overnight fsting lood glucose levels, improved glucose tolernce nd enhnced GSIS. The improvement in glucose metolism of mice ws ssocited with n increse in et cell mss nd prolifertion, s well s in Glut nd Glp1r expression. These results corroorte our previous finding of glucose intolernce in c-kit Wv mice []. We monitored the mice up to 4 weeks of ge nd did not detect ny norml cell growth or islet tumours, indicting tht et cell-specific c-kit overexpression does not led to mlignncy. We detected significnt increse in Scf mrna, ut only modest increse in the corresponding protein. This is likely to e due to our western lotting technique, which could only detect memrne-ssocited, not solule, SCF. Nevertheless, the concentrtion of SCF my e in excess nd is sufficient to interct with the incresed numer of c-kit receptors ( 5%) to trnsduce mrkedly enhnced intrcellulr signls in mice. Our dt indicte tht c-kit overundnce in et cells enhnces glucose tolernce nd et

8 Dietologi (1) 55: c Weight gined/food intke (g) HFD -HFD Ft pd/ody weight (%) 4 -HFD -HFD HFD -HFD d 3 1 AUC (mmol/l min),5, 1,5 1, Time fter glucose i.p. injection (min) -HFD -HFD e Percentge of seline lood glucose AUC (mmol/l min) Time fter insulin i.p. injection (min) -HFD -HFD f. 3 g h Insulin relese (ng/ml) Time fter i.p. glucose injection (min) GSIS in isolted islets (fold chnge normlised to sl) HFD -HFD Insulin content (ng/μg DNA) HFD -HFD Fig. 5 Effect of HFD on mice. Weight gined per food intke, () ft pd per ody weight nd (c) 4 h fsting lood glucose levels in HFD nd wild-type () HFD mice. d IPGTTs nd (e) IPITTs were performed on wild-type HFD (lck circles) nd HFD (lck squres) mice. The glucose responsiveness of the corresponding experimentl groups is shown s the AUC of the IPGTT (d, insert) or IPITT (e, insert) grphs, with units of (mmol/l x min). Dt ( e) re expressed s mens±sem; n7 8. f In vivo GSIS of wild-type HFD nd HFD mice (n). Lighted rs, - HFD insulin; drked rs, HFD insulin; lck circles, - HFD glucose; lck squres, HFD glucose. g GSIS is improved in isolted islets from HFD mice in response to mmol/l glucose chllenge; dt re expressed s fold chnge normlised to sl (. mmol/l glucose) secretion (n5). h Insulin content in isolted islets. Dt re normlised to DNA content (n1). p<.5, p<.1, p<.1 nd p<.5 vs wild-type HFD mice cell function in mles, while the effect is less significnt in femle mice. These sex-relted differences in glucose metolism re consistent with our previous results using c-kit Wv mice nd conditionl β1 integrin knockout mice [, 3], nd hve lso een descried in other mouse models [8, 9]. We oserved tht isolted islets from femle mice hd significntly enhnced insulin secretion in response to high glucose chllenge compred with wild-type femles, indicting tht the sex-relted differences my involve other pthwys, such s the contriution of oestrogen to glucose homeostsis in femle rodents. Significntly incresed Pdx1 mrna nd protein undnce ws oserved in mice. We previously reported tht SCF-stimulted c-kit receptor ctivity leds to incresed PDX1 mrna expression in humn fetl islet epithelil clusters [15], while c-kit Wv mice showed significnt reduction in Pdx1 expression in islets []. It is well documented tht PDX1 is integrl to norml pncres development nd et cell function [3]. In ddition to et cell prolifertion [31], Pdx1 expression is lso required for modultion of insulin gene expression nd glucose metolism [3]. Our results showed tht islets exhiited high PDX1 levels nd hd incresed et cell prolifertion nd mss, confirming correltion etween PDX1 nd islet et cell repliction. Indeed, the enhnced islet insulin secretion in response to high glucose chllenge nd the improved glucose tolernce oserved in mice my lso e due to incresed islet PDX1 undnce. Interestingly, significnt upregultion of

9 Dietologi (1) 55:14 5 Islet numers per mm d Bet cell mss (mg) Reltive mrna expression (normlised to 18S) HFD -HFD Pdx1 Mf Ins Insr Irs1 Irs c-kit βtg-hfd c-kit βtg-hfd Mf mrna ws oserved in oth sexes of mice. MAFA inds to the C1 element of the insulin gene to modulte insulin gene trnscription nd enhnce et cell mturtion [33]. Mf-null mice hd defect only in dult islet rchitecture nd et cell ctivity, while MAFA overproduction enhnced et cell insulin iosynthesis nd secretion through upregultion of importnt et cell genes, including Pdx1, Neurod1, Nkx-1 nd Glp1r [34]. Moreover, overexpression of MAFA in neontl rt et cells led to enhnced glucose-responsive insulin secretion nd et cell mturtion [35]. Thus, improved glucose metolism nd insulin secretion in mice my e due to incresed c-kit receptor stimultion of islet Pdx1 nd Mf expression. The moleculr mechnisms ssocited with the phenotypic chnges oserved in mice include significnt c Alph cell mss (mg) e Ki7 + /insulin + cells (%) HFD -HFD c-kit βtg-hfd c-kit βtg-hfd Fig. Effect of HFD on islet morphology of mice. Quntittive RT-PCR nlysis of genes s indicted on isolted islets of wild-type HFD (light rs) nd HFD (drk rs) mice. Dt re expressed s mens±sem; n3. Morphometric nlysis of islet numer, (c) lph cell mss, (d) et cell mss nd (e) percentge of Ki7 + in et cells from wild-type HFD nd HFD mice. Dt re expressed s mens±sem; n 7. e p<.5 nd p<.1 vs wild-type HFD mice upregultion of phospho-akt nd phospho-gsk3β in et cells. Our previous in vitro study on humn fetl islet epithelil clusters demonstrted tht incresed c-kit SCF interctions resulted in upregultion of PI3K Akt, ut not of mitogen-ctivted protein kinse (MAPK) extrcellulr signl-regulted kinse (ERK) pthwy signlling [15]. In the current study, we lso oserved significnt increses in insulin receptor nd phospho-irs1/ levels, in ddition to the increse in Akt nd GSK3β phosphoryltion together with upregultion of cyclin D1 in islets isolted from mice. These results suggest the following possile mechnisms, wherey c-kit might stimulte et cell function nd prolifertion: (1) vi direct ctivtion of the Akt GSK3β cyclin D1 pthwys [15, 3]; () vi direct or indirect interction etween c-kit nd the insulin receptor nd IRSs to estlish cross-tlk; nd/or (3) y positive feedck of the insulin receptor vi PI3K Akt signlling in ssocition with PDX1- nd Mf-induced insulin secretion. Islets of et cell-specific Gsk3β lted mice hd incresed et cell mss with lower fsting lood glucose, long with improved glucose tolernce nd GSIS [37]. Furthermore, et cell-specific Gsk3β knockout mice hd incresed islet IRS-1 nd - levels with significntly improved et cell function [37], which is in greement with our oservtions. It hs een reported tht insulin secreted y pncretic et cells positively regultes its own iosynthesis y enhncing insulin gene trnscription in n utocrine mnner vi the et cell insulin receptor nd downstrem signlling pthwys [38, 39]. Specific knockout of Insr in pncretic et cells results in defective insulin secretion, similr to tht oserved in type dietes [4]. Tken together, these results suggest tht Akt GSK3β cyclin D1 signlling downstrem of c-kit is essentil for et cell function. HFD tretment is detrimentl to et cell function nd insulin sensitivity in mice [4, 41], nd leds to impired glucose tolernce due to insulin resistnce nd insufficient et cell insulin secretion [4, 41 43]. While nd wild-type mice mintined on HFD showed similr food intke nd weight gin, significntly less ft pd formtion ws oserved in the former fter 4 weeks on the HFD. Importntly, HFD mice exhiited significntly improved glucose tolernce nd GSIS, supporting the notion tht c-kit hs direct effect on et cell function. Islets of HFD mice lso showed significntly incresed levels of insulin receptor nd insulin signls, suggesting possile secondry mechnism, wherey c-kit stimultes et cell function y cross-tlking to the insulin receptor vi PI3K Akt signlling. Therefore, overundnce of c-kit in et cells plys primry role y incresing et cell mss nd prolifertion, s well s secondry role y incresing insulin secretion vi upregultion of the insulin receptor through the PI3K Akt signlling pthwy, which enles HFD mice to tolerte HFD-induced dietes.

10 Dietologi (1) 55: Blood glucose level (mmol/l) c-kit βtg c-kitwv c-kit βtg:wv AUC (mmol/l min),5, 1,5 1, Time fter glucose i.p. injection (min) c-kit βtg c-kitwv c-kit βtg:wv c Percentge of seline lood glucose AUC (mmol/l min) 3 Time fter insulin i.p. injection (min) c-kit βtg c-kitwv c-kit βtg:wv d Insulin relese (ng/ml) Time (min) 35 e GSIS in isolted islets (Fold chnge normlised to sl) f Insulin content (ng/μg DNA) 1.. c-kit βtg c-kitwv c-kit βtg:wv c-kit βtg c-kitwv.5..5 c-kit βtg:wv Fig. 7 Glucose tolernce nd insulin secretion in :Wv mice. Fsting lood glucose levels, () IPGTT nd (c) IPITT vlues in mle wild-type (),, c-kitwv nd :Wv mice t 8 weeks of ge. The glucose responsiveness of the corresponding experimentl groups is shown s the AUC of the (, insert) IPGTT or (c, insert) IPITT grphs with units of (mmol/l x min). Dt ( c) re expressed s mens±sem; n7 8. d In vivo GSIS of wild-type (white rs), c- KitβTg (lck rs), c-kitwv (grey rs) nd :Wv (dotted rs) mice (n3 4). e GSIS of isolted islets from :Wv mice is improved in response to mmol/l glucose chllenge; dt re expressed s fold chnge normlised to sl (. mmol/l glucose) secretion; n3. f Insulin content in isolted islets; dt re normlised to DNA content; n 8. p<.5, p<.1 nd p<.1 Finlly, we red with c-kit Wv mice to determine whether c-kit overexpression could prevent et cell dysfunction in c-kit Wv mice. Our results showed tht : Wv mice displyed norml fsting glycemi nd glucose tolernce, s well s enhnced glucose-induced insulin secretion. This mrked improvement in glucose metolism in :Wv mice provides direct evidence of primry effect of the c-kit receptor on et cell function. In summry, we showed tht c-kit overexpression in et cells led to improved et cell prolifertion nd function, nd protected mice from HFD-induced dietes. Furthermore, et cell-specific overexpression of c-kit ws le to prevent et cell defects in c-kit Wv mice. This study provides direct evidence to support the notion tht c-kit plys primry physiologicl role in et cells, nd thus my help efforts to develop gene nd cell therpeutic schemes for ptients with dietes. Funding This work ws supported y grnts from the Cndin Institutes of Helth Reserch (CIHR, grnt numer MOP 898). Dulity of interest The uthors declre tht there is no dulity of interest ssocited with this mnuscript.

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