Effects of immunosuppression on alpha and beta cell renewal in transplanted mouse islets

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1 Dietologi () :9 DOI.7/s--89-z ARTICLE Effects of immunosuppression on lph nd et cell renewl in trnsplnted mouse islets C. Krutz & S. Wolk & A. Steffen & K.-P. Knoch & U. Ceglrek & J. Thiery & S. Bornstein & H.-D. Seger & M. Solimen & S. Kersting Received: 8 Septemer / Accepted: 7 Mrch / Pulished online: 7 Mrch # Springer-Verlg Berlin Heidelerg Astrct Aims/hypothesis Immunosuppressive drugs used in humn islet trnsplnttion interfere with the lnce etween et cell renewl nd deth, nd thus my contriute to progressive grft dysfunction. We nlysed the influence of immunosuppressnts on the prolifertion of trnsplnted lph nd et cells fter syngeneic islet trnsplnttion in streptozotocin-induced dietic mice. Methods C7BL/ dietic mice were trnsplnted with syngeneic islets in the liver nd simultneously dominlly implnted with mini-osmotic pump delivering BrdU lone or together with n immunosuppressnt (tcrolimus, C. Krutz : S. Wolk : H.-D. Seger : S. Kersting () Deprtment of Generl, Thorcic nd Vsculr Surgery, University Hospitl Crl Gustv Crus, Dresden University of Technology, Fetscherstrsse 7, 7 Dresden, Germny e-mil: stephn.kersting@uniklinikum-dresden.de C. Krutz : S. Wolk : A. Steffen : K.-P. Knoch : H.-D. Seger : M. Solimen () : S. Kersting Deprtment of Moleculr Dietology, Pul Lngerhns Institute, University Hospitl Crl Gustv Crus, Dresden University of Technology, Fetscherstrsse 7, 7 Dresden, Germny e-mil: michele.solimen@tu-dresden.de A. Steffen : S. Bornstein Deprtment of Internl Medicine III, University Hospitl Crl Gustv Crus, Dresden University of Technology, Dresden, Germny U. Ceglrek : J. Thiery Institute of Lortory Medicine, Clinicl Chemistry nd Moleculr Dignostics, University Hospitl Leipzig, Leipzig, Germny M. Solimen Mx Plnck Institute of Moleculr Cell Biology nd Genetics, Dresden, Germny sirolimus, everolimus or mycophenolte mofetil [MMF]). Glycemic control ws ssessed for weeks. The re nd prolifertion of trnsplnted lph nd et cells were susequently quntified. Results After weeks, glycemi ws significntly higher in treted mice thn in controls. Insulinemi ws significntly lower in mice treted with everolimus, tcrolimus nd sirolimus. MMF ws the only immunosuppressnt tht did not significntly reduce et cell re or prolifertion, leit its levels were in lower rnge thn those used in clinicl settings. Conclusions/interprettion After trnsplnttion in dietic mice, syngeneic et cells hve strong cpcity for selfrenewl. In contrst to other immunosuppressnts, MMF neither impired et cell prolifertion nor dversely ffected the frctionl et cell re. Although humn et cells re less prone to proliferte compred with rodent et cells, the use of MMF my improve the long-term outcome of islet trnsplnttion. Keywords Cell prolifertion. Drug monitoring. Glucgon-secreting cells. Immunosuppression. Insulin-secreting cells. Islets trnsplnttion. Mice Arevitions IMPDH Inosine--monophosphte dehydrogense LSD Lest significnt difference MMF Mycophenolte mofetil mtor Mmmlin trget of rpmycin STZ Streptozotocin Introduction Humn islet trnsplnttion is n estlished tretment option for et cell replcement in type dietic ptients with poor

2 Dietologi () :9 97 glycemic control nd frequent episodes of hypoglycemi. Despite promising short-term results, grdul impirment in glycemic control is commonly oserved in recipients of islet llogrfts []. This progressive grft dysfunction cn result from severl fctors, including inflmmtion, llorejection nd metolic stress, s well s toxic nd ntiprolifertive effects of the immunosuppressive therpy []. Dt from severl studies hve suggested tht the pncretic et cell mss chnges dynmiclly ccording to metolic demnds. In ddition, linege-trcing experiments hve shown tht pre-existing et cells re the mjor source of new et cells in dult mice [, ]. Recent studies hve suggested tht the re-differentition of lph cells into et cells is n dditionl mechnism for restortion of the et cell mss [, ]. Common immunosuppressnts used in humn islet trnsplnttion, such s sirolimus nd tcrolimus, negtively ffect et cell repliction [, 7]. Consequently, long-term exposure to these drugs my hmper et cell turnover nd contriute to grft dysfunction in islet trnsplnt recipients. Immunosuppressnts tht re comptile with et cell regenertion my therefore prolong grft survivl nd function. In this study, we investigted the effects of severl immunosuppressive drugs on lph nd et cell prolifertion in dietic mice fter syngeneic heptic islet trnsplnttion. Methods Animls Helthy - to -week-old C7BL/ mice (Chrles River, Bd Sulzfeld, Germny) were housed in cges t constnt temperture nd with stndrd h light/ h drk cycle. All mice were fed % mouse chow diet (Altromin, Lge, Germny) nd hd free ccess to wter. The Institutionl Animl Cre nd Use Committee of the University of Dresden nd the stte government of Sxony pproved ll studies involving niml use. Tretment groups Thirty-one C7BL/ mice were ssigned to five groups, ech including five nimls, except for the everolimus-treted group, which included mice (see Results). A comintion of BrdU (Sigm, St Louis, MO, USA) nd n immunosuppressive drug ws dministered to the mice in ech tretment group; the control mice received BrdU only. The concentrtions of the immunosuppressnts in the pumps were s follows: mg (kg ody weight) dy everolimus (Sigm); mg (kg ody weight) dy tcrolimus (Astells Phrm, Tokyo, Jpn); mg (kg ody weight) dy mycophenolte mofetil (MMF; Roche Phrmceuticls, Bsel, Switzerlnd);. mg (kg ody weight) dy sirolimus (Sigm). Induction of dietes After overnight fsting, 8 mg/kg streptozotocin (STZ; Sigm) tht hd een freshly dissolved in μl citrte uffer ws dministered in single i.p. injection. Blood glucose nd serum insulin Glycemi ws mesured every third dy etween 9: nd : hours in nonfsting conditions using OneTouch Ultr glucometer (LifeScn, Milpits, CA, USA). Insulinemi ws mesured efore surgery nd t the end of weeks nd post trnsplnttion using n ultrsensitive mouse insulin ELISA kit (Crystl Chem, Downers Grove, IL, USA). Drug monitoring Whole lood otined from the til vein ws trnsferred onto filter pper (WS 9; Whtmn Interntionl, Midstone, UK) once per week fter trnsplnttion. The concentrtions of tcrolimus, sirolimus nd everolimus were determined in dried lood spots using HPLC-MS/MS (API ; AB SCIEX, Frminghm, MA, USA) s previously descried [8]. Islet trnsplnttion Mouse islets were isolted from 8- to -week-old C7BL/ littermtes s previously descried [9]. After overnight incution, islets were mnully hndpicked nd trnsferred into 7-guge utterfly needle ttched to ml syringe. The recipient mice were nesthetised y i.p. injection of mg/kg ketmine (Pfizer Interntionl, New York, NY, USA) nd mg/kg xylzine (Sigm). After clmping the left portl vein, the islets were selectively trnsplnted into the right liver loes y injection into the ileocecl vein. Osmotic mini-pumps (Alzet ; Plo Alto, CA, USA) were plced intrperitonelly to deliver μg/μl BrdU in % DMSO (Merck, Drmstdt, Germny) t the rte of. μl/h for 8 dys. At dys post surgery, the mice were killed y intrcrdil perfusion nd their right liver loes were hrvested. i.p. Glucose tolernce test Glucose tolernce ws ssessed t the end of weeks nd post trnsplnttion. Briefly, fter overnight fsting, the mice received n i.p. glucose olus ( g/kg ody weight), nd glycemi ws monitored in the conscious mice t selected time points. Glucose disposl, expressed s the AUC, ws used to clculte the chnge etween the two tests (ΔAUC). Immunohistochemistry nd et cell re mesurement After intrcrdil perfusion with PBS nd % prformldehyde (Merck), the right liver loes were removed, fixed overnight nd emedded in prffin. Ten μm thick prffin sections seprted μm from ech other were otined from ech specimen. After de-wxing nd microwve ntigen retrievl, the sections were first incuted in PBS with % got serum nd.% sponin (PAA Lortories, Psching, Austri) nd then incuted overnight with guine pig nti-insulin (:; Acm, Cmridge, MA, USA),

3 98 Dietologi () :9 rit nti-glucgon (:; Millipore, Schwlch, Germny) nd mouse nti-brdu (:; Roche Dignostics, Mnnheim, Germny) or rit nti-ki7 (:; Acm) ntiodies followed y Alex 8 -got nti-guine pig, Alex -got nti-rit nd Alex 88 -got nti-mouse or Alex 88 -got nti-rit (:; Invitrogen, Crlsd, CA, USA) ntiodies. The nuclei were counterstined using DRAQ or DAPI (:,) (Enzo Life Sciences, Frmingdle, NY, USA). Imges were cquired with Zeiss LSM confocl microscope (Crl Zeiss, Oerkochen, Germny). The numers of BrdU + /Ki7 + et cells mong. et cells/mouse were mnully counted. The totl numers of lph cells nd BrdU + lph cells were recorded in prllel. Mistkes or counting of non-islet cells were ruled out y predefined protocol: mgnifiction ws used, nd only nuclei with strong, complete BrdU + stining nd completely surrounded y n insulin- or glucgon-positive cytosol were counted. To mesure the frctionl lph nd et cell re totl of rndomly selected insulin- nd glucgon-positive res per mouse were imged t the finl mgnifiction of. Ech re ws quntified s frction of the totl tissue re, corresponding to μm /imge. For ssessment of the men et cell size, we mnully trced the contour of rndomly selected et cells with clerly visile nuclei nd cytoplsm in five islets/mouse. All morphometric nlyses were performed using ImgeJ softwre (NIH, Bethesd, MD, USA). Sttistics nd grphics The vlues etween the control nd drug-treted mice t the sme time point fter islet trnsplnttion were compred using the Mnn Whitney U test, nd the vlues within the sme group t different time points were compred using the Wilcoxon signed-rnk test. Becuse of multiple testing, the significnce level (α) ws djusted ccording to the Holm Bonferroni method. For the sttisticl tests without multiple comprisons, the significnce level ws p<.. One-wy ANOVA ws clculted on dtsets of BrdU nd Ki7 lelling of et cells. Post hoc nlysis ws performed using Fisher s lest significnt difference test (LSD). The correltions etween pirs of vriles were clculted using Spermn s rnk correltion test. The results re presented s men±sd unless otherwise stted. The histogrms were prepred using Microsoft Excel. Results Mouse popultion nd drug monitoring The STZ-treted dietic mice in which pproximtely syngeneic islets were trnsplnted in the liver continuously received one of severl immunosuppressnts together with BrdU vi i.p. osmotic mini-pumps for or weeks, nd the mice were susequently killed (Fig. ). Doses of immunosuppressive drugs were chosen sed on the estlished serum concentrtion rnges Immunosuppressnt (ng/ml) STZ 7 Tx st week nd week rd week th week Pump implnttion Follow up Infusion: sline or immunosuppressnt + BrdU Time (weeks) used in humns undergoing islet or pncres trnsplnttion (Tle ). Becuse everolimus hs greter plsm protein inding nd shorter hlf-life in mice thn in humns, we imed for higher serum levels in this group. Moreover, it hs een shown tht lrger dosge of everolimus thn sirolimus is required to mintin the sme lood concentrtions in pncretic islettrnsplnted ptients []. Weekly monitoring indicted tht the serum concentrtions of MMF nd sirolimus were within the therpeutic levels, while the concentrtions of everolimus nd tcrolimus were ove the clinicl concentrtion rnges (Fig. nd Tle ). Notly, six of the islet-trnsplnted mice, ll of which were treted with everolimus, were excluded from ny further nlysis. Two of these mice hd very high everolimus levels (, ng/ml nd, ng/ml), conceivly ecuse of mlfunction of the pump, nd died 7 nd 9 dys, respectively, fter trnsplnttion. A third everolimus-treted mouse ws excluded ecuse the drug concentrtion ws 9 ng/ml. In the remining three mice, the everolimus levels were closer to the ccepted therpeutic rnge ( ng/ml), ut the mouse lood glucose levels remined > mmol/l during the first week post trnsplnttion, indicting filure of the islet engrftment. In ll other mice, the lood glucose levels dropped from > mmol/l (men.9±. mmol/l) fter STZ tretment to <. mmol/l (men 9.±. mmol/l) within week fter the islet trnsplnttion (Fig. ). Immunosuppressnts interfere with glycemic control fter trnsplnttion The verge ody weight of the trnsplnted Mouse deth Fig. () Experimentl timeline. () Averge serum concentrtion of immunosuppressnts (men±sd). Tx, tretment. Symols: dimonds, everolimus; squres, tcrolimus; crosses, sirolimus; tringles, MMF MMF (µg/ml)

4 Dietologi () :9 99 Tle Metolic profiling nd morphometric islet nlysis of control nd drug-treted mice Vrile Controls Everolimus Tcrolimus MMF Sirolimus (n=) (n=) (n=) (n=) (n=) Drug concentrtion (ng/ml).±9.9.±.8.89±..89±. Body weight (g) Week.7±. 8.8±..87±.97.±.97 9.±. Week.±..±.8.8±.9.7±.9.±. Blood glucose (mmol/l) Week 8.±..±. 9.9±.8.89±.8 9.9±.7 Week.±.8.7±.77 8.±. 9.78±. 8.±. Serum insulin (pmol/l) Week 87.±9. 7.±..±8.9.±8. 8.±.9 Week.±. 8.±. 7.8±. 7.±.8 7.7±.9 Glucose tolernce (ΔAUC) 7.±..8±..±.7.±..7±.9 BrdU + et cells (%).9±..±8..±..79±.7.±. Ki7 + et cells (%).98±..±.7.±..7±..8±. Bet cell re (%).±..±..8±..7±.8.±. Bet cell size (μm ).±..± ±.9 9.±9..±. BrdU + lph cells (%).8±..±.7.78±.79.±..7±. Alph cell re (%).8±..±..±.8.9±.8.±. Clinicl concentrtion rnges: everolimus ng/ml; tcrolimus 8 ng/ml; MMF. μg/ml; sirolimus ng/ml μg/ml p<.; p<., Holm-djusted etween treted nd control nimls p<.; p<., etween week nd week post trnsplnttion mice did not differ significntly etween the control nd drugtreted mice (Tle ). Throughout the follow-up period, glycemi ws significntly improved in the control nimls (p=.;fig.). At week of follow-up, the verge lood glucose levels of the mice receiving MMF were significntly higher compred with the control nimls (p=.). However, t week, ll drug-treted mice displyed significntly higher lood glucose levels (Fig. nd Tle ). As expected, in ll groups, the serum insulin concentrtions incresed fter trnsplnttion, lthough they did not rech pre- STZ tretment levels (Fig. ). Insulinemi ws further incresed etween weeks nd post trnsplnttion in the control mice (87. vs. pmol/l; p=.), suggesting the recovery of et cell function (Tle ). Although the insulin levels of treted nimls did not differ significntly from those of untreted controls t week post trnsplnttion, t week they were significntly lower in the mice treted with everolimus (p=.), tcrolimus (p=.9) or sirolimus (p=.). Glucose tolernce ws ssessed using i.p. glucose tolernce tests t the end of weeks nd post trnsplnttion. We susequently clculted the ΔAUC within ech group. The ΔAUC ws significntly reduced in ll drug-treted mice compred with the control mice (Fig. c). Trnsplnted et nd lph cells replicte The prolifertive cpcities of the trnsplnted et nd lph cells tht Blood glucose (mmol/l) Serum insulin (pmol/l) c Glucose tolernce ΔAUC mmol/l min Fig. Averge lood glucose levels () nd serum insulin concentrtions () t the indicted time points. The grey horizontl line indictes the threshold vlue of mmol/l glucose; the mice ove this threshold were regrded s dietic. (c) Glucose tolernce t weeks nd post trnsplnttion represented y ΔAUC. Indictes significnt difference (p<.) etween week nd week post trnsplnttion; indictes significnt difference (p<., Holm-djusted) etween the treted nd control nimls. EVE, everolimus; SIR, sirolimus; TAC, tcrolimus. Mid-grey rs, pre-stz; lck rs, STZ; drk grey rs, week ; light grey rs, week

5 Dietologi () :9 were not sujected to immunosuppressive therpy were exmined fter nd weeks post trnsplnttion (Fig. ). After weeks,.8±.9% of et cells were BrdU + compred with.±.% fter weeks (p=.) (Fig., ). In the cse of the lph cells, the increse in cumultive BrdU + lelling ws not significnt, chnging from.±.9% fter weeks to.±.% fter weeks (p=.8). Not ll immunosuppressnts inhiit et cell prolifertion In the mice treted with MMF for weeks, the BrdU + et nd lph cells ccounted for.8±.% nd.7±.% of ll et nd lph cells, respectively, which ws comprle to those of the control mice (p=.9 nd p=., respectively) (Fig. c, d). In the tcrolimus-treted mice,.±. of et cells (p=., Holm-djusted significnce level α=.) nd.8±.8% of lph cells (p=.) were BrdU +. However, this result ws not significntly different from tht of untreted controls. The numer of BrdU + et cells ws significntly reduced in the mice treted with the mmmlin trget of rpmycin (mtor) inhiitors everolimus (.±8.%; p=.8, Holm-djusted significnce level BrdU + et cells (%) in control mice c BrdU + et cells (%) e Frctionl et cell re (%) weeks weeks 7 BrdU + lph cells (%) in control mice d BrdU + lph cells (%) f Frctionl lph cell re (%) weeks weeks Insulin/glucgon/BrdU/DRAQ Control ( weeks fter Tx) Control ( weeks fter Tx) Fig. Percentge of BrdU + () et nd () lph cells in the control mice nd weeks post trnsplnttion. Indictes significnt difference (p<.) etween week nd week post trnsplnttion. Percentge of BrdU + (c) et nd (d) lph cells weeks post trnsplnttion nd tretment with immunosuppressnts. Frctionl re of (e) et nd (f) lph cells. Indictes significnt difference (p<., Holm-djusted) etween the treted nd control nimls. EVE, everolimus; SIR, sirolimus; TAC, tcrolimus c e EVE MMF d f TAC SIR Fig. Representtive confocl imges of liver-trnsplnted islets in control mice () nd () weeks post trnsplnttion, nd in mice treted with the indicted drugs (c f) weeks post trnsplnttion. BrdU lelling ppers in yellow, insulin in red, glucgon in green nd DRAQ (nuclei) in lue. Scle rs, μm. EVE, everolimus; SIR, sirolimus; TAC, tcrolimus; Tx, trnsplnttion α=.) nd sirolimus (.±.%; p=., Holmdjusted significnce level α=.7), while the frction of BrdU + lph cells did not differ significntly (everolimus.±.7%, p=.; sirolimus.±.%, p =., Holm-djusted significnce level α=.). One-wy ANOVA ws clculted on BrdU lelling of et cells. This nlysis ws significnt: F(/)=., p=.. Fisher s LSD test of BrdU lelling found tht MMF ws the only drug tht ws comprle reltive to control ut differed significntly from ech of the other immunosuppressnts (Tle ). To verify differences found y BrdU ccumultion, we determined the frequency of prolifertion y immunohistochemicl detection of Ki7. Consistent with the BrdU lelling, the numer of Ki7 + et cells in the mice treted with MMF nd the untreted controls did not differ significntly (MMF.7±.%, controls.98±.; p=.8) (Fig. ). Likewise, the numer of Ki7 + et cells ws significntly reduced in the mice treted with the mtor inhiitors everolimus (.±.7%; p=.9, Holm-djusted significnce level α=.) nd sirolimus (.8±.; p=.8, Holm-djusted significnce level α=.). However, in tcrolimus-treted mice.±.% of et cells were Ki7 +, which ws significntly different from the untreted

6 Dietologi () :9 Tle Results of ANOVA with post hoc test for multiple comprisons (Fisher s LSD test) of BrdU nd Ki7 lelling Dependent vrile Ki7 lelling of et cells BrdU lelling of et cells Group (I) Group (J) Men difference (I J) Significnce Controls Everolimus.. Tcrolimus.. MMF.7.9 Sirolimus.. Everolimus Controls.. Tcrolimus.9.97 MMF.99. Sirolimus..7 Tcrolimus Controls.. Everolimus.9.97 MMF..8 Sirolimus.. MMF Controls.7.9 Everolimus.99. Tcrolimus..8 Sirolimus.9.8 Sirolimus Controls.. Everolimus..7 Tcrolimus.. MMF.9.8 Controls Everolimus 8.. Tcrolimus 8..9 MMF..8 Sirolimus 8.9. Everolimus Controls 8.. Tcrolimus 9..8 MMF.. Sirolimus.8.88 Tcrolimus Controls 8..9 Everolimus 9..8 MMF.8. Sirolimus.8.8 MMF Controls..8 Everolimus.. Tcrolimus.8. Sirolimus.. Sirolimus Controls 8.9. Everolimus.8.88 Tcrolimus.8.8 MMF.. Indictes significnt difference etween groups (I) nd (J) controls (p =.8, Holm-djusted significnce level α=.7). One-wy ANOVA of Ki7 lelling of et cells ws significnt: F(/9)=., p=.. Fisher s LSD test of Ki7 lelling found tht MMF ws the only drug tht ws comprle to control mice. However, MMF significntly differed only from sirolimus (Tle ). Frctionl et nd lph cell re nd et cell size The cumultive et cell frction of the liver res nd the cumultive prolifertion rtes of et cells were positively correlted (R=.7; p=.), indicting direct influence of et cell prolifertion on the et cell mss of the islet grft (Fig. ). There ws no significnt correltion etween the frctionl re nd BrdU ccumultion of lph cells (R=.; p>.). The frctionl et nd lph cell res of MMF-treted nimls were comprle to those of the control mice (p=.8 nd p=.9) (Fig. e, f). Likewise, the frctionl et nd lph cell res of the tcrolimustreted nimls did not differ significntly from those of the control mice (p=.8 nd p=., Holm-djusted significnce level α=.7). In the everolimus- nd sirolimus-treted mice, the frctionl et cell res decresed significntly (p=. nd p=.8, respectively, Holm-djusted significnce level α=.7), while the frctionl lph cell res were comprle to those of the control mice (p=. nd p=., respectively). Four weeks fter trnsplnttion, the et cells in ll groups exhiited comprle sizes (Tle ), which is consistent with previous findings []. Discussion Our dt indicte tht trnsplnted mouse pncretic et cells tht re not exposed to toxic insult or to uto- or llorectivity hve strong cpcity for repliction. We found.8% BrdU + et cells in the control group weeks fter trnsplnttion, which ws less thn Nir et l [] found weeks fter et cell ltion (% of et cells were BrdU + ). Zhr et l [7] reported tht in pregnnt mice receiving BrdU vi drinking wter for week % of the et cells were BrdU + t gesttionl dy. Previously, we found tht continuous BrdU dministrtion to mice for week fter 7 8% pncretectomy led to the lelling of 8% of the et cells []. The lower percentge of BrdU + et cells reported in the current study my reflect the reduced ility of the et cells within trnsplnted islets to replicte compred with et cells in the ntive pncres. In humn islet trnsplnttion the numer of donor islets is usully insufficient to restore the et cell mss in recipients. In our study, we decided to trnsplnt n lmost mrginl islet mss in order to mimic the clinicl setting of islet trnsplnttion s closely s possile. The use of lrger islet mss would reduce the stimulus for prolifertion ecuse of smller metolic demnd per islet. Consequently, differences in prolifertion rtes would e hrder to detect nd interference with et cell function would

7 Dietologi () :9 Fig. Representtive confocl imges of liver-trnsplnted islets in () control mice nd in mice treted with the indicted drugs ( e) weeks post trnsplnttion. Ki7 lelling ppers in yellow, insulin in red nd DAPI (nuclei) in lue. The r chrt shows the percentge of Ki7 + et cells weeks post trnsplnttion. Indictes significnt difference (p<., Holm-djusted) etween the treted nd control nimls. Scle r, μm. EVE, everolimus; SIR, sirolimus; TAC, tcrolimus Insulin/Ki7/DAPI Control EVE d MMF e SIR F c Ki7 + et cells (%) TAC Control EVE TAC MMF SIR proly e ttenuted. Therefore, in the control mice treted with STZ nd trnsplnted with islets, the non-fsting glucose levels were slightly elevted compred with the untreted control mice; their verge insulinemi ws significntly reduced nd their glucose tolernce ws impired. Conceivly, the reduction in the endogenous et cell mss stimulted the repliction of the trnsplnted et cells. Notly, the BrdU lelling of the et cells positively correlted with the frctionl et cell re. In our model, however, we cnnot exclude tht other pncretic (endocrine, cinr or ductl) or progenitor cells contriuted to et cell regenertion nd the restortion of glycemic control []. The reltive importnce of et cell genertion vs cell deth is difficult to discern using histologicl nlysis []. No relile procedures for the cumultive in vivo mesurement of poptosis or necrosis exist. Morphologicl methods, such s TUNEL or the detection of highly condensed nd/or frgmented nuclei, cn e pplied to determine the frequency of poptosis t given time point. However, these methods re not suitle for mesuring the cumultive rte of poptosis, especilly under conditions such s islet trnsplnttion, in which the frequency of poptosis is likely to vry gretly over time. Interestingly, some dt imply tht spontneous et cell regenertion results from ccelerted formtion rther thn the reduced poptosis of et cells [, ]. Moreover, the frequency of et cell poptosis does not increse following the comined dministrtion of sirolimus nd tcrolimus in vivo [, 7]. In our study, we consistently found no increse in et cell or heptocyte poptosis in rndom smples of liver sections of mice treted with immunosuppressnts nd untreted controls weeks post trnsplnttion using tetrmethylrhodmine (TMR) red lelling (dt not shown). The mtor inhiitors sirolimus nd everolimus hve negtive effect on et cell repliction nd glucose homeostsis [,, ]. In this study, their detrimentl effect on et cell repliction cn e explined y considering tht the inhiition of mtor decreses the ctivity of the trnsltionl regultors EBP nd SK, which hs negtive effect on the expression of cyclins nd ssocited fctors, therey inhiiting the progression of the cell cycle from the G to the S phse [, 7]. The impirment of glucose homeostsis my e secondry to the reduction of the et cell mss [8] nd the inhiition of mtor downstrem of insulin signlling [9]. Fig. Sctter lots representing the reltionship etween the frctionl res nd cumultive percentges of BrdU + () et nd () lph cells. Circles, control; dimonds, everolimus; squres, tcrolimus; tringles, MMF; crosses, sirolimus BrdU + et cells (%) BrdU + lph cells (%) 7 Bet cell re (%).. Alph cell re (%)

8 Dietologi () :9 The negtive impct of tcrolimus on et cell prolifertion nd insulin secretion on the inhiition of the clcineurin/cytoplsmic nucler fctor of ctivted T cells complex hs lso een well descried in vitro []. In our study, tcrolimus interfered with insulin secretion. Surprisingly, the cumultive BrdU lelling nd frctionl re of et cells of the tcrolimus-treted mice did not significntly differ from those of the control mice, lthough, the Ki7 + et cells were significntly reduced weeks fter trnsplnttion. These dt imply only fint ntiprolifertive effects of tcrolimus tht did not led to significnt reduction of the cumultive BrdU lelling nd frctionl re of et cells. The lck of indirect drug ctions tht interfere with islet engrftment such s the inhiition of islet ngiogenesis my lso e n explntion. MMF strongly inhiits inosine--monophosphte dehydrogense (IMPDH), which converts IMP into xnthosine monophosphte, nd thus the de novo synthesis of gunine nucleotides. Gunine nucleotides re required for the iosynthesis of GTP nd GDP, whose rtio regultes oth et cell growth nd insulin secretion [, ]. In rodent islets, however, the mjor supply of gunine nucleotides is n IMPDH-independent slvge pthwy rther thn the de novo synthesis pthwy []. This my explin why MMF did not significntly ffect the prolifertion of trnsplnted et cells. However, it is uncler why MMF-treted mice lso presented with impired glycemic control. Blood glucose levels not only reflect et cell function ut lso my depend on the responsiveness of other cell types relevnt for glucose homeostsis. The design of the present study cnnot ddress why MMF-treted mice were s hyperglycemic s mice treted with immunosuppressnts tht inhiited et cell prolifertion. The present study, however, suggests tht hyperglycemi in MMF-treted mice ws not the result of impired et cell prolifertion. In recent clinicl trils, the tcrolimus MMF protocol ws shown to improve islet grft survivl when compred with tretments using tcrolimus sirolimus or sirolimus lone [,, ]. The uthors suggested tht llorectivity ws etter controlled using tcrolimus MMF. However, they could not exclude the possiility tht impired et cell repliction, poor islet engrftment or the induction of insulin resistnce ffected their outcomes. In our study, et cell repliction ws reduced y sirolimus ut not significntly ffected y MMF, which my prtly explin the superiority of the tcrolimus MMF protocol. In our opinion, it is of prticulr importnce to monitor serum drug concentrtions in order to drw conclusions on the possile effects of drug. In principle, we would hve preferred to use concentrtions tht re proven to e eqully effective nd well tolerted in mice s in humns, s phrmcokinetics nd phrmcodynmics differ mong species. In prticulr, it is known tht inding of everolimus to plsm proteins is greter in mice (99.9%) thn in humns (7%) [, 7]. Furthermore, the plsm hlf-life of everolimus is 8 hinmicecompredwith h in humns [, 8]. By contrst, the inding of sirolimus, tcrolimus nd MMF to erythrocytes nd plsm proteins is comprle in mice nd in humns [9, ]. Due to the pucity of this informtion, however, we imed t serum concentrtions of immunosuppressnts within the therpeutic rnges for humns, since these re well known nd estlished [,, 8, ]. To our knowledge, this is the first study in rodent islet trnsplnttion in which the levels of immunosuppressnts hve een mesured. Our dt indicte tht t defined serum concentrtions everolimus, sirolimus nd tcrolimus reduce et cell repliction in vivo. The verge level of tcrolimus ws ove the clinicl concentrtion rnge, which could ccount for the oserved inhiition of insulin secretion nd et cell prolifertion. Similr considertions pply to everolimus, the levels of which were lso ove its clinicl concentrtion rnge. Notly, however, we found tht its derivtive sirolimus, whose levels were within the therpeutic rnge, comprly inhiited et cell prolifertion. Hence, the detrimentl impct of everolimus on et cell repliction my not only refer to suprtherpeutic levels. Finlly, the levels of MMF were t the lower end of its clinicl therpeutic rnge, which my hve ttenuted ntiprolifertive effects on et cells in these mice. Since MMF is commonly used t the upper therpeutic rnge, the results of our study cnnot e pplied to the clinicl setting. However, we cn conclude tht MMF t men serum concentrtion of.89±. μg/ml does not ffect prolifertion. In summry, our results demonstrte tht the et cells of trnsplnted islets hve strong cpcity for self-renewl when they re not ffected y immune or toxic insults. The incresed prolifertion nd concurrent improvement in glycemic control fter trnsplnttion support the proposition tht prolifertion is criticl for the preservtion of the trnsplnted et cell mss. This result emphsises the importnce of protecting islet grfts from fctors tht compromise cell prolifertion nd viility s erly s possile. In ddition, our dt indicte tht MMF is the only drug mong those we tested tht did not significntly reduce BrdU nd Ki7 lelling of et cells nd totl frctionl et cell re compred with control mice. Multiple comprisons of BrdU lelling lso point to MMF s the only drug tht ws comprle reltive to control ut differed significntly from ech of the other immunosuppressnts. These dt suggest tht mong the tested drugs MMF is less detrimentl for et cell prolifertion. This conclusion, however, should e tempered in view of the fct tht the concentrtions of MMF, unlike those of the other tested compounds, were t the lower end of its clinicl therpeutic rnge. Hence, we cnnot exclude the possiility tht in the

9 Dietologi () :9 clinicl setting MMF my lso ffect et cell prolifertion. Additionl studies with inter-drug comprisons nd longer follow-up periods should e conducted to verify these results. Acknowledgements We thnk C. Wegrod nd C. Münster (Deprtment of Moleculr Dietology, Pul Lngerhns Institute, Dresden University of Technology, Dresden, Germny) for technicl ssistnce, U. Rnge (Deprtment of Medicl Computer Science nd Biometry, Dresden University of Technology, Dresden, Germny) for help with sttisticl nlysis, E. Bonifcio (DFG-Centre for Regenertive Therpies, Dresden University of Technology, Dresden, Germny) for reding the mnuscript nd K. Pfriem (Deprtment of Moleculr Dietology, Pul Lngerhns Institute, Dresden University of Technology, Dresden, Germny) for dministrtive help. Funding This work ws prtilly supported y funds from the BMBF-Network of Competence on Dietes Mellitus nd the Germn Center for Dietes Reserch (DZD) to M. Solimen; the Germn Ministry for Eduction nd Reserch (BMBF) to the Germn Centre for Dietes Reserch (DZD; to M. Solimen, H.-D. Seger nd S. Kersting; nd y MeDDrive grnt from the Crl Gustv Crus Medicl Fculty t Dresden University of Technology to C. Krutz. Dulity of interest The uthors declre tht there is no dulity of interest ssocited with this mnuscript. Contriution sttement All uthors contriuted sustntilly to the conception nd design of the study, the cquisition of dt or the nlysis nd interprettion of dt; ll were involved in drfting the rticle or revising it criticlly for importnt intellectul content. All uthors hve given their pprovl of the finl version to e pulished. References. Roelen DL, Huurmn VA, Hilrnds R et l (9) Relevnce of cytotoxic llorectivity under different immunosuppressive regimens in clinicl islet cell trnsplnttion. Clin Exp Immunol : 8. Korsgren O, Lundgren T, Felldin M et l (8) Optimising islet engrftment is criticl for successful clinicl islet trnsplnttion. 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