Reciprocal modulation of adult beta cell maturity by activin A and follistatin

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1 Dietologi (21) 53: DOI 1.17/s ARTICLE Reciprocl modultion of dult et cell mturity y ctivin A nd follisttin M. Szt & J. D. Johnson & J. M. Piret Received: 31 Jnury 21 /Accepted: 22 Mrch 21 /Pulished online: 4 My 21 # Springer-Verlg 21 Astrct Aims/hypothesis The functionl mturity of pncretic et cells is impired in dietes mellitus. We sought to define fctors tht cn influence dult et cell mturtion sttus nd function. Methods MIN6 cells lelled with Pdx1 monomeric red fluorescent protein Ins1 enhnced green fluorescent protein dul reporter lentivirus were used to screen cndidte growth nd/or differentition fctors using imge-sed pproches with confirmtion y rel-time RT-PCR nd ssys of et cell function using primry mouse islets. Electronic supplementry mteril The online version of this rticle (doi:1.17/s ) contins supplementry mteril, which is ville to uthorised users. M. Szt : J. M. Piret Michel Smith Lortories, University of British Columi, Vncouver, BC, Cnd M. Szt Genetics Grdute Progrm, University of British Columi, Vncouver, BC, Cnd J. D. Johnson () Deprtment of Cellulr nd Physiologicl Sciences, University of British Columi, 5358 Life Sciences Building, 235 Helth Sciences Mll, Vncouver, BC, Cnd V6T 1Z3 e-mil: jimjohn@interchnge.uc.c J. D. Johnson Deprtment of Surgery, University of British Columi, Vncouver, BC, Cnd J. M. Piret Deprtment of Chemicl nd Biologicl Engineering, University of British Columi, Vncouver, BC, Cnd Results Activin A strikingly decresed the numer of mture et cells nd incresed the numer of immture et cells. While ctivins re criticl for pncretic morphogenesis, their role in dult et cells remins controversil. In primry islets nd MIN6 cells, ctivin A significntly decresed the expression of insulin nd severl genes ssocited with et cell mturity (e.g. Pdx1, Mf, Glut2 [lso known s Slc22]). Genes found in immture et cells (e.g. Mf) tended to e upregulted y ctivin A. Insulin secretion ws lso reduced y ctivin A. In ddition, ctivin A-treted MIN6 cells proliferted fster thn non-treted cells. The effects of endogenous ctivin A on et cells were completely reversed y exogenous follisttin. Conclusions/interprettion These results suggest tht utocrine nd/or prcrine ctivin A signlling exerts suppressive effect on dult et cell mturtion nd function. Thus, the mturtion stte of dult et cells cn e modulted y externl fctors in culture. Interventions inhiiting ctivin or its signlling pthwys my improve et cell function. Understnding of mturtion nd plsticity of dult pncretic tissue hs significnt implictions for islet regenertion nd for in vitro genertion of functionl et cells. Keywords Bet cells. Gene expression. Humn nd mouse islets. Insulin. Differentition. MIN6. PDX1. Reporter lentivirus Arevitions BMP Bone morphogenetic protein egfp Enhnced green fluorescent protein mrfp Monomeric red fluorescent protein NEUROG3 Neurogenin 3

2 Dietologi (21) 53: Introduction The loss of functionl pncretic et cell mss is hllmrk of dietes. A fundmentl understnding of pncretic et cell fte decisions nd the process of et cell mturtion is impertive to correct this defect. Using dul reporter lentivirl system to perform single-cell nlysis of et cell differentition, we recently chrcterised dynmic immture et cell stte in dult islet cells from humns nd mice, distinguished y Pdx1 promoter ctivity prior to Ins1 promoter ctivity (Pdx1 + /Ins low )[1]. MIN6 et cell mturtion is mrked y the cquisition of Ins1 promoter ctivity nd tkes less thn 12 h [1]. Functionl et cell mss cn dpt to chnges in metolic demnd resulting from oesity or pregnncy [2], suggesting tht physiologicl nd pthophysiologicl fctors modulte the differentition sttus of dult et cells. In vitro nd in vivo, pncretic et cells hve lso een stimulted to proliferte, dedifferentite nd trnsdifferentite [3 5]. The plsticity of dult humn pncretic tissue hs significnt implictions for islet regenertion nd for in vitro genertion of functionl et cells. However, the specific conditions nd moleculr cues tht drive these mechnisms remin to e elucidted. Here, our im ws to define fctors tht modulte the mturtion stte of dult et cells. Fctoril design of experiments [6] ws used to compre multiple cndidte growth nd differentition fctors simultneously. Bsed on fctors reported to influence the development or differentition of et cells, we exmined the effects of glucose [7], nicotinmide [8], exendin 4 [9], insulin [1, 11], IGF-1 [12], etcellulin [12], lminin-1 [13], epiderml growth fctor [14], retinoic cid [15], gstrin17 [14], heptocyte growth fctor [16] nd ctivin A [17]. To dte, the effects of these fctors nd their interctions hve not een systemticlly compred in one study. Activins, memers of the TGFβ superfmily, elicit numerous context-dependent effects on growth nd differentition [18]. Activins control emryonic ptterning of foregut-derived orgns [19], hve importnt roles in pncretic development [2] nd hve een implicted in the control of insulin secretion [21, 22]. Activins nd their receptors re present in the developing pncres nd dult islet cells [23, 24]. Follisttin, potent endogenous ctivin ntgonist, is lso produced in dult islets [23, 24]. These oservtions suggest tht ctivins my ply dynmic, tightly controlled utocrine nd/or prcrine roles in dult islets. Here, using novel imge-sed screening pproch, ctivin A ws found to dedifferentite mture et cells. Activin A decresed expression of the insulin gene nd other mture et cell genes, while incresing et cell prolifertion. These effects were fully reversed y follisttin, which ugmented the mture et cell phenotype. Our dt point to powerful locl regultory system within islets, which controls the mturity of dult et cells. Methods Cell culture Humn islets were kindly provided y G. Wrnock nd the Ike Brer Humn Islet Trnsplnt Lortory (Vncouver Generl Hospitl, Vncouver, BC, Cnd) nd cultured s descried [11]. Mouse islets were isolted from 1- to 12-week-old C57BL/6J mice s descried [25] nd cultured overnight in RPMI 164 with 1% (vol./vol.) FBS. MIN6 cells were cultured s descried [1]. Activin A nd follisttin were purchsed from R&D Systems (Minnepolis, MN, USA). All doses of ctivin A used in this study were t sturting level (Electronic supplementry mteril [ESM] Fig. 1). Culture regents were from Invitrogen (Burlington, ON, Cnd), unless otherwise stted. Animl nd humn cell protocols were pproved y the University of British Columi, Cnd in ccordnce with ntionl guidelines. Lentivirl vector production nd infection The dul reporter ptiger Pdx1 monomeric red fluorescent protein (mrfp) Ins1 enhnced green fluorescent protein (egfp) nd control ptigercmvegfp nd ptigerins1egfp lentivirl vectors were used to lel MIN6 cells; detils on vector construction, virus genertion, infection protocols nd expression vlidtion hve een descried elsewhere [1]. Briefly, MIN6 cells were seeded in six-well pltes the dy efore infection. Lentivirl vectors were dded t multiplicity of infection of 1 in serum-free DMEM (with insulin trnsferrin selenium supplement nd.2% (wt/vol.) BSA) nd 8 μg/ml protmine sulphte. Pltes were centrifuged for 2 h t 1,5 g nd 3 C, then cultured overnight t 32 C. Medium ws chnged to complete DMEM nd expression ws monitored t lest 72 h post-infection. After infection with lentivirus, cells hve stle integrtion of the trnsgene(s), llowing long-term monitoring of reporter gene expression [26]. Infection efficiency rnged from 4% to 8%. However, popultions of infected MIN6 cells with prticulr infection efficiency were used for n individul iologicl replicte (i.e. treted with ctivin A or non-treted control) nd results were lwys normlised to the control within the sme lelled popultion of cells, therey controlling for differences in infection efficiency etween preprtions. Screening nd fctoril design of experiments JMP 7..2 softwre (SAS Institute, Cry, NC, USA) ws used to design two-level (i.e. zero dose nd fctor dded) frctionl fctoril experiments to screen the effects of fctors on Pdx1 nd Ins1 promoter ctivities. Initilly, 12 fctors were chosen t concentrtions sed on previous reports or preliminry single fctor experiments (dt not shown; ESM

3 1682 Dietologi (21) 53: Tle 1). We then chose eight fctors for the second screen. The fctoril design is presented in ESM Tles 2 nd 3. The dy efore tretment, lelled MIN6 cells were seeded t 1, cells/well (ViewPlte-96; Perkin Elmer, Wlthm, MA, USA) s heterogeneous unsorted popultion of cells contining Pdx1 + /Ins low immture cells, Pdx1 + /Ins + mture cells nd cells tht were not lelled (i.e. negtive for oth reporters). Cells were wshed with sl medium (DMEM contining 5.5 mmol/l glucose,.2% (wt/vol.) BSA, 4 mmol/l glutmine, 1 U/ml penicillin nd 172 μmol/l streptomycin) nd fctors were dded to sl medium t concentrtions nd comintions descried in ESM Tles 1, 2 nd 3. After 48 h of culture, the nucler stin Hoechst (.32 µmol/l; Invitrogen) ws dded 3 min prior to utomted imging using high-content screening instrument (ArryScn VTI; Cellomics, Pittsurgh, PA, USA). Hoechst-positive, GFP-positive nd RFP-positive cells were identified using fluorescence intensity cut-offs nd then utomticlly counted (Trget Activtion Bioppliction; Cellomics). Cell count nd intensity results for ech fctoril run were nlysed y JMP 7..2 sttisticl softwre to identify the significnt effects within ech experiment. This nlysis included nlysis of multiple internl replictes for ech fctor in vrious comintions. Grphs re presented s per cent effect of ech fctor on the given red-out reltive to no fctors dded (i.e. sl medium). Flow cytometry nd cell sorting For FACS nlysis nd sorting, stly infected MIN6 cells were lifted off pltes using trypsin EDTA, resuspended in PBS contining 5% vol./vol. FBS nd kept on ice. The influx sorter (BD Biosciences, Sn Jose, CA, USA) used ws equipped with tunle lser t 488 nm with filters 488LP nd 531/4 for GFP, nd solid-stte lser t 561 nm with filters 568LP nd 624/4 for RFP. Pdx1 + /Ins low nd Pdx1 + /Ins + cells were simultneously sorted into chilled medium efore seeding nd tretment with ctivin A. Anlysis nd sorting gtes re shown in smple FACS dot plot in ESM Fig. 2. Quntittive rel-time RT-PCR Totl RNA ws purified using kit (RNesy; Qigen, Mississug, ON, USA) nd used to prepre cdna using SuperScript III First-Strnd Synthesis SuperMix for quntittive RT-PCR (Invitrogen). Primers were designed to flnk n intron nd re listed in ESM Tle 4. Quntittive RT-PCR ws performed using SYBR GreenER qpcr SuperMix (Invitrogen) nd 75 Rel-Time PCR System (Applied Biosystems, Foster City, CA, USA). For reltive quntifiction of trnscripts, cycle threshold vlues for ech smple were normlised to Gpdh. BrdU lelling MIN6 cells were cultured for 48 h on 96- well micropltes (ViewPlte-96; Perkin Elmer) with nd without ctivin A in sl DMEM medium, fter which 1 μmol/l BrdU (Kit I; Roche Applied Science, Lvl, QC, Cnd) ws dded for 3 min prior to fixtion nd stining. Cells were imged using n inverted microscope (Axiovert 2 M) equipped with FLUAR 2 ojective (Crl Zeiss, Thornwood, NY, USA). Imges were nlysed nd quntified using softwre pckge (SlideBook; Intelligent Imging Innovtions, Boulder, CO, USA). A minimum of 5 cells per smple were imged nd quntified. Hormone secretion For sttic incution, cells in 24-well pltes were wshed once nd incuted for 1 h t 37 C in 3 mmol/l glucose Kre s uffer, which ws then replced for 2 h with 3 mmol/l glucose, 2 mmol/l glucose or 3 mmol/l KCl Kre s uffer t 37 C. Buffer smples were nlysed for insulin. Secreted insulin ws mesured using RIA (Linco/Millipore, Billeric, MA, USA). Secreted ctivin A ws quntified using n immunossy (Humn/ Mouse/Rt Activin A; R&D Systems). Dt nlysis Dt re presented s mens ± SEM. Differences etween mens were evluted y Student s pired or unpired t tests, s pproprite. A p vlue of 5 or less ws considered significnt. A minimum of three independent experiments ws performed, s noted. Results Screening for fctors tht modulte dult et cell mturity Two-level fctoril designs were used to screen for significnt single nd interction effects. Multiple growth fctors nd culture medium supplements were selected on the sis of previous reports of their mitogenic, differentiting or survivl effects on pncretic cells (ESM Tle 1). The mturtion stte of MIN6 cells ws monitored using the dul reporter Pdx1mRFP Ins1eGFP lentivirus descried y us previously [1] in the presence of chosen fctors. Chnges to mture Pdx1 + /Ins + cells (Fig. 1) nd immture Pdx1 + /Ins low cells (Fig. 1) re shown reltive to the negtive control (i.e. no fctors dded) from two fctoril experiments. All single-fctor effects nd only significnt interction effects re shown. After the first screen, fctors tht hd no significnt effect or hd significnt negtive effects on one cell type (without yielding positive effect on the other, e.g. FBS nd retinoic cid) were not considered for the second screen. For oth screens, 1 mmol/l glucose lone hd strong positive effect on the reltive numer of immture nd mture MIN6 cells. This effect ws proly due to increses in totl cell numer (dt not shown). Similrly, 1 mmol/

4 Dietologi (21) 53: Chnge in %Pdx1 + /Ins low cells reltive to control (%) Chnge in %Pdx1 + /Ins + cells reltive to control (%) ND ND ND ND ND ND ND ND ND l nicotinmide hd positive effect on oth mturtion sttes in our second screen, ut only incresed the mture et cell numers in our first screen. Exendin-4 hd significnt negtive effect on the mture et cell popultion in oth fctoril experiments. Interestingly, ctivin A hd strong negtive effect on mture et cells, yet highly significnt positive effect on ND ND ND ND GLU ACTA NIC EX4 INS IGF1 BTC LAM1 EGF RA GAS17 FBS HGF NIC+FBS ACTA+GLU ACTA+NIC NIC+EX4 GLU ACTA NIC EX4 INS IGF1 BTC LAM1 EGF RA GAS17 FBS HGF GLU+NIC ACTA+NIC INS+NIC Fig. 1 Screening for fctors tht modulte dult et cell mturtion. Fctoril design of experiments ws used to screen for effects of vrious fctors on Pdx1 nd Ins1 promoter ctivity, nd mturtion stte of dul lelled MIN6 cells. Grphs re shown for effects on %Pdx1 + /Ins + mture cells () nd %Pdx1 + /Ins low immture cells () reltive to control (no fctors). Results re shown for two independent fctoril runs (fctoril 1, light grey rs; fctoril 2, drk grey rs). Sttisticl tests for significnt effects were ssessed within ech experiment, inherently y wy of severl internl replictes of ech fctor in vrious comintions, using sttisticl softwre. Similr results were otined when the grphs were represented using red-out of Pdx1 + /Ins low nd Pdx1 + /Ins + cell counts rther thn the per cent chnges (dt not shown). p<5. ND, not done. Fctors: GLU, glucose; ACTA, ctivin A; NIC, nicotinmide; EX4, exendin 4; INS, insulin; BTC, etcellulin; LAM1, lminin 1; EGF, epiderml growth fctor; RA, retinoic cid; GAS17, gstrin-17; FBS, fetl ovine serum; HGF, heptocyte growth fctor the reltive percentge of immture et cells (Fig. 1). Upon removl of five fctors in the second screen, this opposing effect of ctivin A ws ugmented twofold. In ddition, negtive nd positive interctions etween ctivin A nd glucose or nicotinmide, respectively, ecme significnt in the second screen. Similr fctoril design results were oserved using the INS-1 cell line lelled with the sme Pdx1mRFP Ins1eGFP dul reporter lentivirus (dt not shown). Significnt fctor effects, s well s fctor interctions, were uncovered using sttisticl design of experiments. For follow-up studies, we selected ctivin A s the most interesting cndidte involved in modulting the mturtion stte of dult et cells. Activin A reduces et cell mturity The effects of ctivin A on the reltive numers of immture nd mture et cells were exmined using lrger cultures of Pdx1mRFP Ins1eGFP dul lelled, unsorted MIN6 cells. Ins1 promoter ctivity (GFP intensity) ws significntly decresed y 4 nmol/l ctivin A fter 72 h of culture (Fig. 2). Similr results were otined using control single Ins1eGFP reporter vector, wheres ctivin A hd no effect on the ctivity of control promoter (CMVeGFP; ESM Fig. 3). Pdx1 promoter ctivity (RFP intensity) from the dul reporter vector ws lso decresed with ctivin A (dt not shown). Confirming the results of the fctoril screens, ctivin A tretment significntly incresed the reltive numer of Pdx1 + /Ins low immture cells (Fig. 2), wheres it significntly decresed the numer of Pdx1 + /Ins + mture cells (Fig. 2c). We next tested the effects of ctivin A on the spontneous mturtion of purified immture Pdx1 + /Ins low cells [1]. FACS-sorted immture Pdx1 + /Ins low MIN6 cells (>99% purity) were treted with ctivin A. This resulted in reduced Ins1 promoter ctivity (GFP levels; Fig. 2d), higher numer of immture cells nd fewer mture Pdx1 + / Ins + cells reltive to control (Fig. 2e). These results suggest tht ctivin A decreses insulin gene promoter ctivity nd reduces the rte of spontneous et cell mturtion. Activin A decreses expression of insulin nd mture et cell genes We nlysed ctivin A-treted primry mouse islets nd MIN6 cells for chnges in the expression of genes ssocited with et cell mturity nd function. Rel-time RT-PCR reveled tht severl key genes were differentilly expressed etween treted nd control cells (Fig. 3). Specificlly, Ins1, Ins2, Pdx1, Mf nd Glut2 (lso known s Slc22) were significntly decresed in ctivin A-treted primry mouse islets (Fig. 3). Mf is n importnt trnscription fctor for insulin gene expression [27]. Similrly, Ins1, Ins2, Egfp, Pdx1, Mrfp, Mf, Nkx6-1 nd Glut2 were lso downregulted y ctivin A tretment in MIN6 cells (Fig. 3). Conversely, importnt trnscription fctors involved in erly islet development, e.g. Mf nd

5 Dietologi (21) 53: GFP intensity geometric men (AU) d GFP intensity reltive to control Pdx1 + /Ins low cells (%) e Neurog3, tended to e incresed in ctivin A-treted MIN6 cells (Fig. 3) [28]. These results re similr to our previous dt compring sorted immture nd mture et cells, for instnce, with opposite chnges in expression of Mf nd Mf [1]. Similr differentil gene expression profiles were oserved with ctivin A-treted humn islet cells (M. Szt, unpulished dt). Together, these dt strongly suggest tht ctivin A negtively regultes dult et cell mturity. Activin A increses et cell prolifertion Since ctivin A decreses mture et cell gene expression, we investigted whether et cell prolifertion ws lso incresed in treted cells. Indeed, fter 3 min we sw 2.8-fold increse in BrdU incorportion in ctivin A-treted MIN6 cells (Fig. 4). Consistent with this, we oserved n 11±.5% increse in totl MIN6 cell numer with ctivin A tretment compred with controls in the fctoril experiments (dt not shown). We hd previously shown tht immture MIN6 cells proliferted fster thn mture cells [1]. Tken together, these results, long with the decresed mture gene expression ptterns, suggest tht ctivin A shifts the mturtion stte of et cells towrds the Pdx1 + /Ins low immture phenotype with n incresed prolifertion potentil. c Pdx1 + /Ins + cells (%) Pdx1 + /Ins low Pdx1 + /Ins + Cell type reltive to control Fig. 2 Activin A decreses insulin expression nd reduces mturtion of immture dult et cells. Dul lelled, unsorted MIN6 cells treted with ctivin A (4 nmol/l, grey rs) for 72 h were nlysed y FACS for () GFP (Ins1) expression nd for () chnges in Pdx1 + / Ins low immture vs (c) Pdx1 + /Ins + mture cell numers reltive to nontreted control (white rs). AU, ritrry units. d, e Sorted immture Pdx1 + /Ins low cells were llowed to mture into Pdx1 + /Ins + cells for 72 h with ctivin A (4 nmol/l, grey rs) to ssess effects on their mturtion reltive to non-treted control (white rs). Cells were then nlysed y FACS for (d) overll GFP levels nd (e) reltive chnges in resulting immture vs mture cell popultions (n=3). p<5 Reltive gene expression Reltive gene expression Ins1 Ins2 Pdx1 Mf Mf Nkx6-1 Nkx2-2 Glut2 Gck Neurog3 Activin A decreses insulin secretion The lrge decrese in Glut2 expression long with other genes importnt for et cell function in ctivin A-treted cells prompted us to exmine the effects of ctivin A on insulin secretion. Activin A significntly decresed insulin secretion in primry mouse islets (Fig. 5) nd MIN6 cells (Fig. 5). Insulin secretion ws lso 14.5% lower from ctivin A- treted humn islets (M. Szt, unpulished dt). Since previous studies hve reported tht cute ctivin A tretment of cultured rt nd humn islets incresed insulin secretion [21, 22], we lso exmined the cute effects of Ins1 Ins2 GFP Pdx1 Mrfp Mf Mf Nkx6-1 Nkx2-2 Glut2 Gck Neurog3 Fig. 3 Activin A decreses expression of mture et cell genes. Primry mouse islets (n=4) nd () dul lelled MIN6 cells (n=8) were cultured with ctivin A (4 nmol/l) for 72 h nd cdna smples were nlysed y rel-time RT-PCR. p<5 Fig. 4 Activin A increses et cell prolifertion. MIN6 cells were treted with ctivin A (4 nmol/l, grey r) or nontreted (white r) for 72 h nd 1 μmol/l BrdU ws dded for 3 min prior to fixing nd stining for BrdU incorportion (n=3). p<5 BrdU + cells (%)

6 Dietologi (21) 53: Insulin secretion reltive to control c Insulin (% pretretment) ,2 2, 1,8 1,6 1,4 1,2 1, Insulin secretion reltive to control 1 4 nmol/l ctivin A in perifused mouse islets (Fig. 5c). We did not oserve n increse in insulin secretion y ctivin A in the presence of 3 mmol/l or 2 mmol/l glucose, nor did we oserve n increse in insulin relese fter depolristion y 3 mmol/l KCl. In fct, we oserved tht ctivin A tended to decrese glucose-stimulted insulin secretion, lthough the AUC ws not significntly different. Follisttin reverses the effects of ctivin A Pncretic islets produce mny secreted fctors, which cn ct in n utocrine or prcrine mnner. Studies hve shown tht mouse, rt nd humn pncretic islet cells produce ctivin A nd follisttin [23, 24, 29]. We lso detected the ctivin βa suunit, ctivin type II receptors (ctrii nd ctriib) nd ctivin type I receptor (ctrib/lk4) in humn islets (dt not shown). Mouse islets nd MIN6 cells secreted 1 to 2 pmol/l ctivin A per dy per 2, cells into the culture medi (Fig. 6). These dt suggest tht ctivin A is relesed from pncretic islets, where it cn ct loclly to regulte mturtion stte of dult et cells. Follisttin is specific nd potent ntgonist of ctivin A, preventing inding to its receptor [3]. Indeed, follisttin Time (min) Fig. 5 Activin A decreses insulin secretion. Primry mouse islets nd () MIN6 cells were treted with ctivin A (4 nmol/l, grey rs) or non-treted (white rs) for 72 h; n=4, p<5. c A perifusion experiment ws performed using primry mouse islets to ssess cute effects of ctivin A (4 nmol/l, lck tringles) vs non-treted (white circles) on glucose-stimulted insulin secretion. Blck r, 4 mmol/l ctivin A; htched r, 2 mmol/l glucose; verticlly htched r, 3 mmol/l KCl. n=4; AUC not significnt completely reversed the effects of ctivin A on Ins1 promoter ctivity, MIN6 mturtion stte, insulin gene expression nd the expression of mturity-ssocited genes (Fig. 6). In some cses, follisttin not only reversed the effects of ctivin, ut ppered to hve significnt effects of its own, suggesting the reversl of endogenous locl ctivin signlling (Fig. 6 e). Since ctivin A decresed insulin secretion in primry mouse nd humn islets, nd in MIN6 cells, we investigted whether follisttin could increse insulin secretion. Indeed, ctivin A significntly decresed, wheres follisttin incresed insulin secretion from MIN6 cells over 72 h, leit t p vlue of 7 (Fig. 7). Co-tretment with ctivin A nd follisttin significntly incresed insulin secretion. Furthermore, glucose nd KCl-stimulted insulin secretion ws significntly e Reltive gene expression Activin A over 72 h (pmol/l) c Pdx1 + /Ins low cells (%) d Pdx1 + /Ins + cells (%) GFP intensity geometric men (AU) Ins1 Ins2 Gfp Pdx1 Mrfp Mf Mf Nkx6-1 Nkx2-2 Glut2 Gck Neurog3 Fig. 6 Follisttin increses expression of insulin gene nd other mture et cell genes. Rte of ctivin A secretion from MIN6 cells (white r) nd primry mouse islets (grey r) ws quntified. d Dul lelled MIN6 cells were treted for 72 h with ctivin A (2 nmol/l, drk grey rs), follisttin (1 nmol/l, light grey rs), oth (lck rs) or non-treted (white rs) nd nlysed y FACS for () GFP(Ins1) expression nd for chnges in (c) immture Pdx1 + /Ins low vs (d) mture Pdx1 + /Ins + cell numers. e cdna smples were nlysed for chnges in gene expression y rel-time RT-PCR; n=3, p<

7 1686 Dietologi (21) 53: Insulin reltive to 3 mmol/l glucose reduced y ctivin A nd incresed y follisttin in MIN6 cells (Fig. 7, c). Collectively, these results suggest tht ctivin A dedifferentites dult et cells, nd follisttin locks this effect nd enhnces et cell mturtion. Discussion Insulin (nmol/l) Glucose (2 mmol/l) The present study sought to identify fctors tht modulte dult et cell mturity. Using dul lelled MIN6 cells [1], cndidte fctors were systemticlly screened for their effects on Pdx1 nd Ins1 promoter ctivities. The mjor finding of our study ws tht ctivin A nd follisttin hd powerful reciprocl effects on et cell mturity. These results contriute to the understnding of mturtion nd plsticity in dult et cells. Fctoril design offers insight into the complexity of interction of vrious in vitro culture conditions, fctors nd nutrients, while sustntilly decresing the numer of experiments required [31]. For exmple, our frctionl fctoril design for fctoril 1 required = 64, insted of 2 12 = 4,96 individul tretments, if ll comintions of 12 fctors were screened. Thus, the sttisticl design of experiments, which is used much more frequently in engineering science, cn e vlule tool in screening experiments designed to elucidte importnt interction c KCl (3 mmol/l) Fig. 7 Follisttin reverses the effects of ctivin A on insulin secretion. MIN6 cells were cultured for 72 h with ctivin A (2 nmol/l, drk grey rs), follisttin (1 nmol/l, light grey rs), oth (lck rs) or nontreted (white rs). Medi smples with ccumulted insulin over 72 h (n=4). Sttic incution uffer smples (n=3) were nlysed for insulin t 2 mmol/l glucose nd t (c) 3 mmol/l KCl. p<5 effects [6, 32]. The fctoril design method reveled numer of interesting effects on dult et cell mturity. For exmple, high glucose hd highly significnt positive effects on the reltive numers of immture nd mture et cells. Nicotinmide is generlly used in cell culture medi s vitmin supplement nd hs een shown to promote differentition of pncretic fetl cells into insulinpositive cells [8]. In our screens, nicotinmide lso hd positive effects. While synergistic or negtive interction effects cn e msked in conventionl screening experiments, our sttisticl design uncovered hidden interctions of fctors including synergy etween nicotinmide nd high glucose, FBS or exendin-4. However, upon removl of mny negtive or insignificnt first screen fctors (such s FBS, IGF-1, retinoic cid nd gstrin17) from the second screen, these synergies were lost, ut new synergies etween nicotinmide nd ctivin A or insulin were uncovered. While previous studies hve generlly shown eneficil effects of exendin-4 on et cell function nd survivl [33, 34], we oserved significnt negtive effect of exendin-4 on et cell mturity. Emerging evidence of pncretic tissue plsticity suggests tht normlly quiescent, terminlly differentited pncretic cells retin the potentil to dedifferentite, trnsdifferentite or increse their prolifertion fter specific moleculr cues in vitro nd in vivo [3 5, 35]. Our findings support the notion tht et cell plsticity cn e modified y externl moleculr cues. Activin A decresed Ins1 promoter ctivity, downregulted expression of severl mture et cell genes, incresed prolifertion nd dmpened insulin secretion in primry islets nd MIN6 cells. Conversely, follisttin completely reversed the dedifferentiting effects of ctivin A. Both ctivin A nd follisttin re produced in islets [23, 24], suggesting prcrine control of et cell mturity. Interestingly, preliminry experiment showed tht levels of the ctivin βa suunit were incresed in humn islets treted with ctivin A (M. Szt, unpulished dt), supporting positive feedck for utocrine or prcrine regultion of ctivin A expression. The mture et cell phenotype is commonly defined y the expression of genes such s the insulin gene, Pdx1, Glut2, Mf, Neurod1, glucokinse nd Nkx6-1 [36]. In our ctivin A-treted cells, genes ssocited with et cell mturity, specificlly Ins1, Ins2, Pdx1, Glut2, Nkx6-1 nd Mf were downregulted. Follisttin incresed the expression of these genes. Consistent with these negtive effects of ctivin A, the TGFβ/SMAD pthwy is known to restrict pncretic progenitor specifiction, in prt y restrining Pdx1 expression during erly emryonic development [37]. The notion tht ctivin A is tonic negtive regultor of et cell differentition nd function in the dult islet is supported y recent study showing tht TGFβ/SMAD3 signlling repressed insulin gene nd other mture et cell

8 Dietologi (21) 53: genes nd lso tht downregultion of Smd3 improved et cell function [38]. Bone morphogenetic protein (BMP) signlling, lso medited vi SMADs, prevented et cell differentition in zerfish [39]. Thus, oth the TGFβ nd BMP superfmilies pper to hve suppressive roles in dult et cell mturtion. Activin A incresed expression of Mf nd decresed tht of Mf in MIN6 cells, wheres follisttin hd the opposite effect. This pttern of Mf nd Mf expression in ctivin A-treted cells is consistent with dedifferentited, immture et cell phenotype, s it occurs during endocrine development. In murine pncres development, Mf is expressed efore Mf, followed y Mf downregultion in dult et cells [4]. Mf is required for et cell mturtion nd directly regultes expression of Mf, Pdx1, Nkx6-1, Glut2 nd insulin [28]. Activin A did not increse Mf expression in primry mouse islet cells, s it did in MIN6, proly ecuse islet lph cells express high levels of Mf [41] nd smll increse in et cell Mf expression with ctivin A tretment might not hve een detectle ove control. We previously found tht neurogenin 3 (NEUROG3) messge nd protein re present in dult humn islets, mouse islets nd MIN6 cells, where they re regulted y notch signlling [42]. Susequently, the presence of NEUROG3 protein hs een confirmed y others nd its role in the mintennce of dult et cell mturity ws suggested [43]. It is possile tht ctivin nd follisttin hve reciprocl effects on NEUROG3 levels in et cells, ut it remins to e determined whether NEUROG3 medites the effects of ctivin or follisttin on key et cell genes such s Mf nd Glut2. GLUT2 is required for glucose sensing, norml glucose homeostsis nd insulin secretion [44]. Glut2 ws one of the most highly regulted cndidte genes in ctivin A- or follisttin-treted islets nd MIN6 cells. Similrly, Glut2 ws significntly decresed in Pdx1 + /Ins low immture et cells [1]. Consistent with our findings, Glut2 ws elevted in Smd3 knockout islets [38]. A strong reduction in Glut2 expression is n erly indictor of et cell stress nd possily dedifferentition in mny mouse models of glucose intolernce or dietes [45], including mice with reduced Pdx1 [46]. It will e interesting in the future to exmine the chrcteristics nd plsticity of the popultion of dult pncretic cells with low Glut2 expression. Our prolifertion results with MIN6 cells re lso consistent with nother study tht showed n pproximtely threefold increse in prolifertion of primry rt et cells treted with ctivin A [47]. Collectively, ctivin A nd follisttin pper to modulte the mturtion stte of dult et cells, with ctivin A driving mture et cells to more progenitor-like phenotype with incresed prolifertion nd decresed differentited function. Reports on ctivin A effects on insulin secretion re conflicting. Acute ctivin A tretment of cultured rt nd humn islets ws reported to increse insulin secretion [21, 22]; however cutely treted MIN6 cells did not show incresed secretion [29]. In our study, ctivin A hd no cute stimultory effect on glucose-stimulted insulin secretion in perifused isolted mouse islets, while prolonged ctivin A tretment significntly decresed insulin secretion. It is possile tht ny cute ctivin A effects on insulin secretion my e medited through SMAD-independent, nontrnscriptionl signlling mechnism [48]. In ddition to its effects on et cell function, ctivin A hs een reported to e differentition fctor. In vitro, it ppered to direct pncretic fetl cells into insulin-positive cells [17]. Although ctivin A mintins pluripotency nd self-renewl of emryonic stem cells [49], it is lso required to induce stem cell differentition into insulin-positive cells [5]. Our results support dedifferentiting role for ctivin A in dult et cells. In this regrd, ctivin A receptors, ctivin type I receptor nd ctivin type II B receptor, were found to e expressed t higher level in dult vs neontl et cells (S. Bonner-Weir, Hrvrd, Boston, MA, USA; personl communiction), supporting differentil role for ctivin signlling t different stges of et cell development. In summry, our results demonstrte tht locl fctors, such s ctivin nd follisttin, control the mturtion sttus of dult et cells. Identifiction of modultors of et cell repliction nd mturtion will help in the development of therpies designed to increse functionl et cell mss in vivo, s well s helping to find lterntive sources of trnsplntle et cells in vitro. Acknowledgements We thnk T. Kieffer nd R. Ky for dvice nd regents, C. Hoesli nd D. Lucini for dvice, L. Mrmolejo nd X. Hu for technicl ssistnce, nd G. Wrnock for humn islets. Reserch ws supported y operting grnts to J.D. Johnson nd J. M. Piret from the Stem Cell Network, the Juvenile Dietes Reserch Foundtion (JDRF) nd the Cndin Institutes of Helth Reserch (CIHR). Infrstructure support of the Michel Smith Foundtion for Helth Reserch (MSFHR)-funded Centre for Humn Islet Trnsplnttion nd Bet-Cell Regenertion is cknowledged. J. D. Johnson ws supported y slry wrds from MSFHR, CIHR, JDRF nd the CDA. M. Szt ws supported y studentships from NSERC, MSFHR, CIHR nd the University of British Columi. Dulity of interest The uthors declre tht there is no dulity of interest ssocited with this mnuscript. References 1. Szt M, Lucini DS, Piret JM, Johnson JD (29) Mturtion of dult et-cells reveled using Pdx1/insulin dul-reporter lentivirus. Endocrinology 15: Schdev MM, Stoffers DA (29) Minireview: meeting the demnd for insulin: moleculr mechnisms of dptive postntl et-cell mss expnsion. Mol Endocrinol 23:

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