A local glucagon-like peptide 1 (GLP-1) system in human pancreatic islets

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1 Dietologi (212) 55: DOI 1.17/s ARTICLE A locl glucgon-like peptide 1 (GLP-1) system in humn pncretic islets P. Mrchetti & R. Lupi & M. Buglini & C. L. Kirkptrick & G. Sestini & F. A. Grieco & S. Del Guerr & V. D Aleo & S. Piro & L. Mrselli & U. Boggi & F. Filipponi & L. Tinti & L. Slvini & C. B. Wollheim & F. Purrello & F. Dott Received: 27 Octoer 211 / Accepted: 8 August 212 / Pulished online: 11 Septemer 212 # Springer-Verlg 212 Astrct Aims/hypothesis Glucgon-like peptide 1 (GLP-1) is mjor incretin, minly produced y the intestinl L cells, with eneficil ctions on pncretic et cells. However, while in vivo only very smll mounts of GLP-1 rech the pncres in ioctive form, some oservtions indicte tht GLP-1 my lso e produced in the islets. We performed comprehensive morphologicl, functionl nd moleculr studies to evlute the presence nd vrious fetures of locl GLP-1 system in humn pncretic islet cells, including those from type 2 dietic ptients. Methods The presence of insulin, glucgon, GLP-1, proconvertse (PC) 1/3 nd PC2 ws determined in humn pncres y immunohistochemistry with confocl microscopy. Islets were isolted from non-dietic nd type 2 dietic donors. GLP-1 protein undnce ws evluted y immunolotting nd mtrix-ssisted lser desorption ionistion-time of flight (MALDI TOF) mss spectrometry. Single lph nd et cell suspensions were otined y enzymtic dissocition nd FACS sorting. Glucgon nd GLP-1 relese were mesured in response to nutrients. 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. P. Mrchetti () : M. Buglini : S. Del Guerr : V. D Aleo : L. Mrselli Deprtment of Endocrinology nd Metolism, University of Pis, Vi Prdis 2, Pis, Itly e-mil: piero.mrchetti@med.unipi.it P. Mrchetti Unit of Endocrinology nd Metolism of Trnsplnttion, Aziend Ospedlier Universitri Pisn (AOUP), Pis, Itly R. Lupi Deprtment of Medicine, Aziend Ospedlier Universitri Pisn (AOUP), Pis, Itly C. L. Kirkptrick : C. B. Wollheim Deprtment of Cell Physiology nd Metolism, University of Genev, Genev, Switzerlnd G. Sestini : F. A. Grieco : F. Dott Dietes Unit, Deprtment of Internl Medicine, Endocrine nd Metolic Sciences nd Biochemistry, University of Sien, Sien, Itly G. Sestini : F. A. Grieco : F. Dott Fondzione Umerto Di Mrio, Rome, Itly S. Piro : F. Purrello Deprtment of Internl Medicine, University of Ctni, Ctni, Itly U. Boggi Unit of Generl Surgery nd Trnsplnttion in Uremic nd Dietic Ptients, Aziend Ospedlier Universitri Pisn (AOUP), Pis, Itly F. Filipponi Unit of Generl Surgery nd Liver Trnsplnttion, Aziend Ospedlier Universitri Pisn (AOUP), Pis, Itly L. Tinti : L. Slvini Toscn Life Sciences Foundtion, Sien, Itly

2 Dietologi (212) 55: Results Confocl microscopy showed the presence of GLP-1- like nd PC1/3 immunorectivity in susets of lph cells, wheres GLP-1 ws not oserved in et cells. The presence of GLP-1 in isolted islets ws confirmed y immunolotting, followed y mss spectrometry. Isolted islets nd lph (ut not et) cell frctions relesed GLP-1, which ws regulted y glucose nd rginine. PC1/3 (lso known s PCSK1) gene expression ws shown in lph cells. GLP-1 relese ws significntly higher from type 2 dietic thn from nondietic isolted islets. Conclusions/interprettion We hve shown the presence of functionlly competent GLP-1 system in humn pncretic islets, which resides in lph cells nd might e modulted y type 2 dietes. Keywords Alph cells. GLP-1. Glucgon. Pncretic islets Arevitions DPP-4 Dipeptidyl peptidse IV GLP-1 Glucgon-like peptide 1 MALDI TOF Mtrix-ssisted lser desorption/ionistion time of flight PC Proconvertse Introduction Glucgon-like peptide 1 (GLP-1) is encoded in the proglucgon gene in the long rm of chromosome 2, nd is minly expressed in pncretic lph cells, intestinl L cells nd the cudl rin stem [1 4]. The primry trnsltion product is processed differently, so tht proconvertse (PC)2 leds to glucgon production in lph cells, wheres PC1/3 cleves proglucgon to GLP-1 in L cells nd the rin, with GLP-1 (7 37) nd GLP-1(7 36)mide representing the ioctive peptides [1 4]. As n incretin, GLP-1 lowers glucose levels y modulting glucose-stimulted insulin secretion from pncretic et cells [1 3]. It lso hs or my hve roles in the mintennce of et cell mss, regultion of gstric motility, reduction of plsm glucgon, protection of endothelium, promotion of stiety nd stimultion of glucose disposl [1 3, 5, 6]. The eneficil effects of GLP-1 nd its mimetics on et cells hve mde them potentil therpeutic gents for type 2 dietes [1 3, 5 7]. However, the ction of GLP-1 is limited in durtion due to clevge y dipeptidyl peptidse IV (DPP-4), resulting in GLP-1 hlf-life of pproximtely 2 min [1 3, 5, 6]. Indeed, more thn 5% of GLP-1 is inctivted on its relese from L cells, nd lrge mount of the remining intct peptide is inctivted s it psses through the liver, with only very smll mounts of ioctive GLP-1 reching the pncres [8]. Some experimentl evidence suggests tht GLP-1 my lso e produced in pncretic islets. In the mid-198s, investigtors using ntiodies ginst GLP-1 nd electron microscopic immunocytochemistry oserved moleculr forms of GLP-1 in the humn pncres [9, 1]. Lter, fully processed GLP-1 ws visulised in rt pncretic extrcts y chromtogrphic nlysis nd rdioimmunossys [11]. In nother study [12], monoclonl ntiodies to GLP-1(7 37) were produced for immunocytochemistry nd rdioimmunossy experiments, nd immunorective GLP-1 ws detected in rt lph cells, with incresed relese upon glucose stimultion [12]. Accordingly, GLP-1(7 36)mide ws found in the pncres of fetl nd neontl rts [13], nd fully processed GLP-1 ws produced y insulinom cell lines nd primry rt lph cells [14]. Furthermore, when prtil et cell loss ws induced in neontl rts y streptozotocin, islet cell regenertion ws ccompnied y lph cell hyperplsi, with n ltered phenotype of incresed GLP-1 synthesis [15]. More recently, study minly focused on the lph cell line, αtc1-6, showed the processing of proglucgon to GLP-1, with the incretin lso found in rt nd humn islets [16], while investigtors working on the Psmmomys oesus geril incidentlly oserved tht humn islets could relese GLP-1 [17]. Finlly, n extensive study in rts showed tht interleukin-6 could increse GLP-1 relese from L cells nd lph cells, with some of the results reproduced with humn islets [18]. With ll this in mind, we performed more thorough investigtion of the presence of locl GLP-1 system in humn islets, using morphologicl, moleculr nd functionl studies of intct islets nd humn lph nd et cellenriched frctions. Prt of the studies ws lso done on islets from type 2 dietic individuls. Methods Pncres nd islet smples In totl, 34 pncreses nd/or isolted islet preprtions from non-dietic (18 men, 16 women; ge 57±18 yers; BMI 24.7±2.7 kg/m 2 ) multiorgn donors nd 16 pncreses nd/or isolted islet preprtions from type 2 dietic (eight men, eight women; ge 6± 8. yers; BMI 26.1±2.5 kg/m 2 ) multiorgn donors were otined, with pprovl of the locl Ethics Committee. Immunocytochemistry Pncretic smples were tken from the neck of the glnd (the remining tissue eing processed for islet isoltion) nd hndled s descried previously [19]. Primry ntiodies were: guine pig polyclonl nti-swine insulin (A564; DAKO, Crpinteri, CA, USA); mouse monoclonl nti-humn/ mouse glucgon (MAB1249, clone 18142; R&D Systems, Aingdon, UK); rt monoclonl nti-somtosttin (3788; Acm, Cmridge, UK); rit polyclonl nti-glp-1 (22625; Acm), which rects with the mid to C terminl region of GLP-1 [1 19], enling immunorectivity with N terminl truncted nd C terminlly extended forms of GLP-1; mouse monoclonl nti-glp-1 (HYB 147-6; BioPorto Dignostic, Gentofte, Denmrk), which is specific for the midted

3 3264 Dietologi (212) 55: C-terminus of the peptide [2]; nd rit polyclonl ntiody to PC1/3 nd PC2 (Chemicon, Rosemont, IL, USA). Secondry ntiodies were: Alex Fluor 488: got nti-guine pig IgG, got nti-rit IgG, nd got nti-mouse IgG; nd Alex Fluor 594: got nti-mouse IgG nd got ntirit IgG (ll from Invitrogen, Moleculr Proes, Leiden, the Netherlnds). NCI-H716 cells, humn L cell model [21], were otined from Americn Type Culture Collection (Mnsss, VA, USA) nd used s GLP-1-like positive controls. Smples were nlysed y lser confocl microscopy (TCS SP5; Leic Microsystems, Wetzlr, Germny), s previously reported [22]. Islet isoltion nd culture Islets were prepred y enzymtic digestion nd grdient seprtion [22, 23], nd suspended in M199 culture medium (Sigm Chemicls, St Louis, MO, USA) supplemented s detiled elsewhere [22, 23]. Islets with dimeter 1 μm were hndpicked nd studied within 3 dys of isoltion. Immunolotting nd islet hormone content For isolted islet western lotting, 3 μg islet protein per lne were loded to 4 to 2% (wt/vol.) grdient SDS-PAGE gels nd sujected to electrophoresis. Proteins were then trnsferred to nitrocellulose memrnes nd proed for GLP-1 using monoclonl nti- GLP-1 ntiody (23472; Acm) or β-ctin (AC-15 ntiody, A5441; Sigm). The nti-glp-1 ntiody rects with the region corresponding to mino cids 7 to 36 of humn GLP-1 nd shows some cross-rectivity with GLP-1 precursors. Whole-cell lyste (A-431, SC-221; Snt Cruz Biotechnologies, Snt Cruz, CA, USA) ws used s positive control. Islet glucgon nd GLP-1 content ws mesured from four nondietic nd three type 2 dietic preprtions fter overnight cid ethnol (1.5% HCl, 79% ethnol [95% pure], 19.5% wter) extrction [22, 23]. Islet mtrix-ssisted lser desorption ionistion-time of flight mss spectrometry Similr to procedures previously descried for other cellulr models [24], 15 μg islet protein per lne ws loded to 4 to 2% grdient SDS-PAGE gel nd sujected to electrophoresis. After protein visulistion y Coomssie lue, gel nds were excised, de-stined nd sujected to overnight in-gel digestion y trypsin (Sigm) t 37 C, without reduction nd lkyltion. Of the resulting peptides, 2 μl ws mixed with 2 μl sturted di-hydroxyenzoic cid solution (Bruker Dltonics, Bremen, Germny) prepred in 5% cetonitrile nd 5% wter contining.1% (vol./vol.) trifluorcetic cid. Peptide solution (1 μl) nd di-hydroxy-enzoic cid-sturted mtrix were tken to smple plte of the mss spectrometry nlysis pprtus nd llowed to dry. The crystllised smples were nlysed using mtrix-ssisted lser desorption ionistion-time of flight (MALDI TOF)/TOF system (Ultrflex III; Bruker Dltonics), operting in reflector mode. Typiclly, 1 to 2 lser shots were summed into ech mss spectrum. An externl clirtion of stndrd peptide mixture (Bruker Dltonics) ws used. For identifiction nd determintion of sequence coverge, the mesured mono-isotopic m/z vlues were serched ginst the SWISSPROT dtse using MASCOT (Mtrix Science, London, UK, ccessed 4 July 212) nd BIOTOOLS version 3. (Bruker Dltonics). Prmeters of missed clevge nd peptide tolernce were set to or 1 nd 15 ppm, respectively. Preprtion of lph nd et cells Methods previously developed nd vlidted were used [25, 26]. The dissocition of islets into single-cell suspensions ws chieved y incution with constnt gittion for 3 min t 37 C in.5% (wt/vol.) trypsin-edta (Invitrogen) supplemented with 3 mg/ml DNAse I (Roche Dignostics, Mnnheim, Germny), followed y pipetting vigorously to complete dissocition. Lelling nd sorting of lph nd et cell frctions were performed y Newport Green lelling [27] followed y FACS, s descried [25]. Quntittive RT-PCR studies The expression of genes encoding insulin, glucgon, PC1/3 nd PC2 ws ssessed y quntittive RT-PCR experiments in lph nd et cell frctions from eight non-dietic nd three type 2 dietic islet preprtions, ccording to methods detiled elsewhere [22, 23, 25]. Briefly, fter RNA extrction nd reverse trnscription, mrna levels of the genes of interest were quntified nd normlised to β-ctin in ionlyser (77; Applied Biosystems, Foster City, CA, USA). The oligonucleotides were otined from ssy-on-demnd gene expression products (Applied Biosystems). Functionl studies Non-dietic islets were incuted for 6 min in KRB solution with 5.5 mmol/l glucose; then, fter wshing, the following conditions were tested (t 6 min incution with KRB solution): 5.5 mmol/l glucose, 16.7 mmol/l glucose, nd 5.5 mmol/l glucose plus 2 mmol/l rginine. Purified cell preprtions were tested with 5.5 mmol/l glucose nd 5.5 mmol/l glucose plus 2 mmol/l rginine. In ddition, GLP-1 nd glucgon relese were mesured from tches of 5 islets of similr size from non-dietic nd type 2 dietic preprtions, kept in M199 culture medium for 4 h. Finlly, to test the ioctivity of islet-relesed GLP-1, experiments were performed similrly to methods previously reported [17, 18], y ssessing insulin relese from islets exposed to cell culture medium from untreted non-dietic humn islets; this ws done t 11 mmol/l glucose, nd in the sence or presence of the GLP-1 receptor ntgonist, exendin(9 39) (.5 μmol/l).

4 Dietologi (212) 55: GLP-1 relese ws mesured using GLP-1 (ctive) RIA kit (LINCO, Sint Chrles, MO, USA) nd n ELISA kit (Millipore, Billeric, MA, USA). The ntiodies used in the ssys re specific for the iologiclly ctive 7 36 mide nd 7 37 forms of GLP-1, nd do not rect with GLP-1(1 36)mide, GLP-1(1 37), GLP-1(9 36)mide or GLP-1(9 37). Glucgon levels were mesured using 125 I-lelled glucgon rdioimmunossy kit (Millipore). Sttisticl nlysis Results re expressed s men ± SD. Dt were compred y the two-tiled Student's t test for unpired dt. The ANOVA test followed y Bonferroni's correction ws used when more thn two groups were considered. Results Alph cells hve GLP-1-like nd PC1/3 immunorectivity Unsurprisingly, insulin co-loclised with PC1/3 (Fig. 1c, d) nd PC2 (Fig. 1g, h). PC1/3 nd PC2 cleve proinsulin t the B- chin/c-peptide nd C-peptide/A-chin junctions, respectively [28]. Glucgon co-loclised with PC2 (Fig. 1o, p) (PC2 cleves proglucgon to produce glucgon [29]). However, few glucgon-positive cells lso displyed PC1/3 immunorectivity (Fig. 1k, l) (PC1/3 leds to the formtion of GLP-1 from proglucgon [29]). Intriguingly, PC1/3 nd glucgon were present in different intrcellulr lph cell comprtments (Fig. 1k, l). This ws confirmed y superimposition nlysis of glucgon nd PC1/3 immunorectivity (electronic supplementry mteril [ESM] Fig. 1, ). GLP-1-like immunorectivity ws detected y polyclonl nd monoclonl nti-glp-1 ntiody recting, respectively, with the mid to C terminl region of GLP-1 [1 19] nd the midted C-terminus of the peptide (see the Methods). These ntiodies generted similr stining ptterns with NCI-H716 cells nd humn islets (ESM Figs 2 nd 3). We then exmined cells tht were doule-positive for GLP-1-like nd other hormone or PC immunorectivity, oserving superimposle results with oth nti-glp-1 ntiodies. As result, we decided to report only the dt cquired with the monoclonl ntiody. Figure 2 c shows tht no cells were doule-positive for insulin nd GLP-1- like immunorectivity. Co-loclistion of GLP-1-like nd glucgon immunorectivity ws found in some cells (Fig. 2f, i); however, some glucgon-contining cells did not show GLP-1-like immunorectivity (Fig. 2f, i). Fig. 1 Insulin, glucgon, PC1/ 3 nd PC2 immunorectivity in humn pncretic islet cells. Confocl microscopy nlysis showed the presence of () PC1/3 nd (f) PC2 in (, e) insulin-contining (INS) cells, s indicted y merged imges (c, g) nd enlrged detils (d, h). (n) PC2 positivity in glucgon-contining (GLN) cells (m), with merged imges (o) nd enlrged detils (p). (i) A suset of glucgonpositive cells lso produced PC1/3 (j), with merged imges (k) nd enlrged detils (l). Squres (c, g, k, o) indicte the res (d, h, l, p) shown t higher mgnifiction. Scle rs ( c, e g, m o) 3 μm; (i k) 4μm; (d, h, l, p) 8μm c d INS PC1/3 e f g h INS PC2 i j k l GLN m PC1/3 n o p GLN PC2

5 3266 Dietologi (212) 55: c loclistion. Glucgon nd PC1/3 doule positivity ws found in 139 of 573 lph cells (24%) from non-dietic islets nd in 152 of 665 lph cells (23%) from dietic islets. INS d GLN g GLP-1-like e GLP-1-like h Accordingly, there were cells tht were doule-positive for GLP-1-like nd PC1/3 or PC2 immunorectivity (Fig. 3). GLP-1-like nd PC1/3 positivity were loclised in different su-cellulr comprtments (Fig. 3c, d). Somtosttinpositive cells did not stin for GLP-1 or for PC1/3 (ESM Fig. 4). The min results of the confocl microscopy studies re summrised in Tle 1. When lph cells tht were doule-positive for glucgon nd PC1/3 were counted, co-loclistion ws found in cells from 2 of 26 (76.9%) non-dietic islets nd from 21 of 26 (8.7%) type 2 dietic islets; conversely, round 2 to 25% of the exmined islets did not show glucgon nd PC1/3 co- f Fig. 2 Insulin, glucgon nd GLP-1-like immunorectivity in humn pncretic islet cells. () Confocl microscopy nlysis of insulin (INS), (d, g) glucgon (GLN) nd (, e, h) GLP-1-like immunorectivity showed GLP-1-like positivity in the merged imge of lph cells (f)with enlrged detil (i), ut not in the merged imge (c)ofetcells.blue(c, f, i), nucler stining with Hoechst. Squres (d, e, f) indicte the res (g, h, i) shown t higher mgnifiction. Scle rs ( f) 3μm; (g i) 15μm i Pncretic islets contin GLP-1 Non-dietic islets were used for GLP-1 immunolotting experiments nd showed nd corresponding to GLP-1 (moleculr mss pproximtely 4 kd), with the intensity vrying mong preprtions (Fig. 4). A nd corresponding to some GLP-1 precursors ws lso oserved in some cses, due to prtil cross-rectivity of the ntiody used (see the Methods). The moleculr mss of this nd (1 12 kd) is comptile with tht of proglucgonderived mjor proglucgon frgment. The nds of pproximtely 4 kd, ssigned s β-ctin, nd those of out 4 kd, ttriuted to GLP-1 peptides, were sumitted for identifiction. In oth spectr, kertin nd trypsin pek utolysis ws sutrcted. The experimentl pek lists were compred with the SWISSPROT dtse using MASCOT. The 4 kd nd ws identified s ACTB_HUMAN (P679) with MASCOT score of 143, in greement with the western lot dt. Figure 5 shows the results for the 4 kd nd. In this cse, the dtse serch showed, mong other species, the presence of glucgon (GLUC_HUMAN, P1275) with MASCOT score of 68; the sequence ws covered y five peptides, strting from the mino cids in position 98 of pre-proglucgon. This corresponds to position 78 of proglucgon, known to e the position of the first mino cid of GLP-1(7 37) nd GLP-1(7 36)mide. The corresponding spectr of the GLP-1(7 37) nd GLP-1 (7 36) tryptic sequences were mtched with the puttive digested sequences of the peptides using the sequence editor implemented on BIOTOOLS. GLP-1(7 37) nd GLP-1(7 36) peptides were identified y the species t m/z 115 ( 21-28) nd t m/z 298 ( 1-2). The presence of GLP-1 in isolted islets ws lso confirmed y islet extrction mesurements. Non-dietic islets contined 57.7±25.9 pg/islet GLP-1 (n4), with trend (p.5) towrds higher levels in the type 2 dietic smples (72.3±19. pg/ Fig. 3 PC1/3, PC2 nd GLP-1- like immunorectivity in humn pncretic islet cells. Confocl microscopy nlysis of PC1/3 (), PC2 (e) nd GLP-1-like (, f) immunorectivity, showing cells doule-positive for GLP- 1-like nd PC1/3 immunorectivity (c), with enlrged detil (d), nd (g) cells doulepositive for GLP-1-like nd PC2 immunorectivity, lso with enlrged detil (h). Squres (c, g) indicte the res shown t higher mgnifiction (d, h). Scle rs ( c, e g) 3 μm; (d) 5μm nd (h) 8μm PC1/3 e GLP-1-like f c g d h PC2 GLP-1-like

6 Dietologi (212) 55: Tle 1 Summry of the results of the immunohistochemistry studies performed with humn pncretic islets Vrile INS + GLN + GLP-1 + PC1/3 + PC2 + INS + No doule positivity No doule positivity Doule positivity Doule positivity GLN + No doule positivity Doule positivity Suset with doule positivity Doule positivity GLP-1 + No doule positivity Doule positivity Suset with doule positivity Doule positivity INS, insulin; GLN, glucgon; GLP-1, GLP-1-like islet; n3). In the sme preprtions, the glucgon content ws 255±119 nd 273±53 pg/islet (p.8), respectively. Alph cells express PC1/3 Quntittive RT-PCR experiments showed tht insulin gene expression in lph cell preprtions ws less thn 12% of glucgon gene expression, nd tht the expression of glucgon in et cells ws less thn 9% of tht of insulin (n3 preprtions ech). This is in line with protein undnce dt previously reported y our group [26]. Bet cells expressed PC1/3 (lso known s PCSK1) nd PC2 (lso known s PCSK2) (Fig. 6), with PC2 expression tending to e lower (not sttisticlly significnt). In type 2 dietic et cells, PC1/3 nd PC2 ppered to e upregulted, compred with non-dietic smples (Fig. 6). Alph cells lso expressed oth PC genes (Fig. 6); however, in non-dietic lph cells PC2 ws upregulted compred with PC1/3, wheres in type 2 dietic lph cells PC1/3 expression ws greter (Fig. 6). 64 kd 49 kd 37 kd 26 kd 19 kd 15 kd 6 kd 64 kd 49 kd 37 kd 26 kd 19 kd 15 kd 6 kd MM Cse 1 Cse 2 Cse 3 MM Cse 1 Cse 2 Cse 3 43 kd 12 kd 4 kd Fig. 4 Western lot nlysis of GLP-1 undnce in isolted humn pncretic islets. () β-actin (pproximtely 43 kd) ws used s control. () Imge showing the undnce of GLP-1 (pproximtely 4 kd) nd in two cses (cse 2 nd 3) lso the presence of GLP-1 precursor (pproximtely 12 kd). Findings for three different isolted humn islet smples (cse 1, cse 2 nd cse 3) re shown. Moleculr mss (MM) mrkers re indicted y lck rs representing the different moleculr mss vlues reported from nitrocellulose memrne loded with Benchmrk Pre-stined protein ldder (Invitrogen) Pncretic islets relese GLP-1 Glucgon relese from non-dietic islets decresed significntly t glucose concentrtions tht stimulte insulin relese nd incresed y pproximtely twofold in response to rginine (Fig. 7). In turn, GLP-1 secretion incresed significntly in response to high glucose nd rginine chllenge (Fig. 7). In these three different conditions, the glucgon:glp-1 molr rtio ws pproximtely 4.7,.8 nd 2.6, showing tht the two hormones were relesed with some degree of independence of ech other. Islets from type 2 dietic donors tended to relese more glucgon (.72±.16 vs.55±.11 pmol islet 1 h 1 ) nd secreted significntly higher mount of GLP- 1 (.44±.11 vs.17±.1 pmol islet 1 h 1, p<.1) thn non-dietic islets (Fig. 7). The respective glucgon:glp- 1 rtios were 1.6 nd 3.2, suggesting tht, t lest in sl conditions, the dietic islet phenotype is more oriented to the relese of GLP-1. We lso evluted the ioctivity of islet cell-relesed GLP-1 y mesuring insulin secretion from islets exposed to cell culture medium from nondietic humn islets; this ws done t 11 mmol/l glucose, nd in the sence or presence of the GLP-1 receptor ntgonist, exendin(9 39) (n3 experiments ech). Figure 8 shows tht the conditioned medium elicited stronger insulin relese in response to glucose, nd tht this ws prevented y locking GLP-1 receptors with exendin(9 39). Alph cells relese GLP-1 When GLP-1 relese from nondietic lph nd et cell preprtions ws studied, we found tht glucgon nd GLP-1 secretion from lph cells incresed significntly upon rginine stimultion (Fig. 9). The respective glucgon:glp-1 secretion rtios were 3.1 (sl) nd 6.8 (stimulted), gin suggesting tht the relese of the two hormones ws modulted differently under the tested conditions. Conversely, glucgon nd GLP-1 secretion from purified et cells ws undetectle, with et cell glucgon nd GLP-1 content elow the limits of detection of the ssys used (not shown). Discussion The present study shows tht locl GLP-1 system is present in humn pncretic islets, residing in lph cells, modulted y nutrients nd ffected y type 2 dietes. GLP-1-like

7 3268 Dietologi (212) 55: Fig. 5 MALDI mss spectrum of tryptic digestion of the 4 kd nd ove (Fig. 4). The corresponding peks mtched to GLP-1 (7 36) nd GLP-1 (7 37) prtil sequences re mrked y the downwrd rrows indicting pek vlue nd length of mino cid sequences, i.e.: m/z (21 EFIAWLVK 28) nd m/z (1 HAEGTFTSDVSSYLE GQAAK 2) Asornce intensity 1, , 1,2 1,4 1,6 1,8 2, 2,2 2,4 2,6 2,8 3, 3,2 3,4 3,6 3,8 m/z immunorectivity ws detected y immunohistochemistry, similrly to findings previously oserved y others [1, 12, 17]. However, the complex processing of proglucgon y PC1/3 leds to severl peptides, of which GLP-1(7 37) nd GLP-1(7 36)mide re the ioctive GLP-1 forms [1 4]. Moreover, the different commercilly or not commercilly Gene expression (2 -ΔΔC t) in et cells Gene expression (2 -ΔΔC t) in lph cells ND et cells ND lph cells T2D et cells T2D lph cells Fig. 6 () PC1/3 nd PC2 expression in et cell frctions from nondietic (ND) nd type 2 dietic (T2D) islets. Quntittive RT-PCR experiments reveled the expression of PC1/3 (lck rs) nd PC2 (white rs) in non-dietic (n8) nd type 2 dietic (n3) et cells, with higher expression in the ltter. p<.5 vs corresponding gene in non-dietic. () PC1/3 nd PC2 expression in lph cell frctions from non-dietic nd type 2 dietic islets. PC2 (white rs) ws expressed t higher levels thn PC1/3 (lck rs) in non-dietic lph cells, wheres PC1/3 ws upregulted in type 2 dietic lph cells, compred with non-dietic smples. p<.5 vs non-dietic; p<.5 vs non-dietic PC1/3 ville [17] ntiodies used y us nd in previous studies hve vrile specificities. In ddition, nti-glucgon ntiodies my not specificlly identify peptides derived from Secretion (pmol islet -1 h -1 ) Secretion (pmol islet -1 h -1 ) Bsl High glucose Arginine Non-dietic Type 2 dietic Fig. 7 () Glucgon nd GLP-1 secretion from non-dietic isolted humn islets. Glucgon (lck rs) nd GLP-1 (white rs) secretion ws mesured in isolted islet incution medi s descried. Conditions were: 5.5 mmol/l glucose (sl), 16.7 mmol/l glucose (high glucose) nd 5.5 mmol/l glucose plus 2 mmol/l rginine (rginine). Glucgon relese decresed in response to high glucose nd incresed in response to rginine; GLP-1 incresed with high glucose nd with rginine. p<.5 vs the respective sl vlues (Bonferroni's correction). () Glucgon nd GLP-1 secretion from non-dietic nd type 2 dietic islets. Glucgon nd GLP-1 secretion ws mesured in isolted islet incution medi s descried, following incution t 5.5 mmol/l glucose. p<.1 vs non-dietic (Student's t test)

8 Dietologi (212) 55: Insulin relese (% of content) mmol/l glucose 11.1 mmol/l glucose Fig. 8 Insulin relese from non-dietic islets exposed to unconditioned (lck rs) or conditioned (light grey rs) (from islets previously incuted with 11.1 mmol/l glucose) medium without exendin (9 39) (n ntgonist of GLP-1 receptors), or from islets exposed to unconditioned medium+exendin(9 39) (white rs) or to conditioned medium + exendin(9 39) (drk grey rs). Glucose-stimulted insulin relese ws higher thn tht t the sl (3.3 mmol/l glucose) condition, with further significnt potentition y the conditioned medium, which ws prevented y exendin(9 39). p<.5 vs the respective vlues t 3.3 mmol/l glucose (Student's t test); p<.5 vs the other vlues t 11.1 mmol/l glucose (Bonferroni's correction) PC2 processing. Nevertheless, we did consistently find tht the distriution of GLP-1 immunorectivity follows tht of glucgon in susets of glucgon-contining cells, s oserved y others [1, 12, 17]. In greement with previous studies [3, 31], we lso oserved tht suset of lph cells contined PC1/3, which cleves proglucgontoioctiveglp-1[29]. Finlly, no co-loclistion of GLP-1-like nd PC1/3 immunorectivity with somtosttin ws oserved. Therefore, despite the specific rrngement of endocrine humn islet cells, which my lso surround ech nother [32], the current evidence shows tht the immunorectivity to different sustnces descried ove occurs in seprte cells. In our experiments, GLP-1 nd PC1/3 immunorectivity ppered in different su-cellulr comprtments. Wheres our dt were unle to fully clrify this point, it cnnot e ruled out tht proglucgon might rech distinct comprtments in the Golgi pprtus contining either PC2 (yielding glucgon) or PC1/3 (generting GLP-1). Islet GLP-1 peptides were detected in the present study y immunolotting, MALDI TOF mss spectrometry nd mesurements in cell extrcts. A nd corresponding to 1 to 12 kd moleculr mss ws oserved y western lotting in some, ut not ll, islet preprtions, possily due to prtil cross-rectivity of the ntiody used. This suggests tht islets my e heterogeneous in the wy they process proglucgon to GLP-1, which is further confirmed y the oservtion tht pproximtely 2 to 25% of islets did not show cells tht were doule-positive for glucgon nd PC1/3. As for the precursor, it could e the mjor proglucgon frgment, which hs moleculr mss comptile with tht of the oserved nd [1, 8, 28].Thepresenceof GLP-1 nd n intermedite form ws lso found in rt pncres [13]. In seminl rticle [4], Holst et l reported tht GLP-1 (1 37) or GLP-1(1 36)mide is present in pncretic extrcts from humn nd porcine pncreses. In our study, mss spectr generted y tryptic sequences of GLP-1 peptides contined prtil segments scrile to GLP-1(7 37) nd GLP-1(7 36)mide. Although the mide form of GLP-1 (7 36) could not e identified (proly ecuse the peptide contining rginine midtion tht is generted y trypsin digestion hs very low mss [m/z]), islet production of ioctive GLP-1 ws confirmed y the GLP-1 receptordependent potentition of glucose-stimulted insulin relese, which ws oserved with medium deriving from previous islet cultures. This is in greement with the oservtion tht GLP-1 secreted from isolted humn islets could indeed potentite insulin relese in vitro [18]. Our results show tht glucose nd rginine incresed GLP-1 relese. This supports the concept tht GLP-1- secreting cells possess the mchinery needed to recognise sustrtes nd relese the hormone [1 4, 8]. Accordingly, Whlley et l found tht high glucose (25 mmol/l) could increse GLP-1 production in αtc1-6 cells nd rt islets exposed to streptozotocin [16]. Furthermore, Ellingsgrd et l reported tht rginine incresed GLP-1 secretion from humn islets nd tht this ws potentited y interleukin-6 [18]. We, moreover, oserved here tht purified lph cells express PC1/3 nd secrete GLP-1, nd tht this secretion is regulted y rginine. All this is in greement with experimentl evidence tht lph cells hve vrile functionlity nd cn, indeed, produce GLP-1, s oserved with the lph cell line, αtc-1-6, the glucgonom MSL-G-AN cell line, primry rt lph cells nd primry humn lph cells [16, 18, 33, 34]. Humn lph cells cn lso produce glucose-dependent insulinotropic peptide, the Secretion (pmol [1, cells] -1 h -1 ) Bsl Arginine Fig. 9 Glucgon nd GLP-1 secretion from purified humn lph cells. Glucgon (lck rs) nd GLP-1 (open rs) secretion ws mesured in lph cell incution medium s descried. Conditions were: 5.5 mmol/l glucose (sl) nd 5.5 mmol/l glucose plus 2 mmol/l rginine (rginine). Both hormones incresed significntly in response to rginine. p<.5 nd p<.1 vs the respective sl vlues (Student's t test)

9 327 Dietologi (212) 55: other mjor incretin [35]. Therefore, lthough cell frctions from isolted islets usully show some degree of contmintion, s shown here nd y others [25, 26], the ville evidence suggests tht lph cells re the source of islet GLP-1. However, the quntifiction of GLP-1 nd glucgon relese from humn islet cells hs yielded conflicting results. Some uthors found tht islet GLP-1 undnce ws in the femtomolr rnge nd tht the hormone ws not mesurle in islet incution medium [16]. However, Hnsen et l were le to mesure GLP-1 in islet culture medium (t 5.5 mmol/l glucose), with the mount gin in the femtomolr rnge [17]. Another study [18] nd we, here, hve reported GLP-1 vlues in the picomolr rnge, suggesting intrinsic islet iologicl vriility nd/or differences in experimentl conditions. In ddition, in our study, glucgon relese from isolted islets ws similr to tht reported y some other uthors [36, 37], ut severl fold lower thn tht oserved during perifusion experiments [38]. All this led, in our hnds, to glucgon/glp-1 molr secretion rtio (sl condition) of pproximtely 5.7, similr to findings previously reported for αtc1-6 cells [16]. In ddition, when we mesured glucgon nd ctive GLP-1 in islet extrcts, the vlues nd proportions were close to those found y others [18, 37]. Interestingly, however, the i-hormone secretion rtio with lph cell frctions tended to decrese (pproximte vlue of 3), suggesting tht primry humn lph cells might rpidly chnge their phenotype. Accordingly, humn lph cells in culture relesed 5% more GLP-1 thn glucgon, slly nd upon stimultion [18]. We did not investigte the moleculr mechnisms tht cuse lph cells to modulte, to different degrees, their sensitivity to vrious stimuli, such tht glucose, for exmple, cuses reduced glucgon relese nd incresed GLP-1 secretion. However, this seems to e more generl phenomenon, s previous uthors hve lso oserved tht, with primry humn lph cells, incution with interleukin 6 cused twofold increse of GLP-1 secretion, while not ffecting tht of glucgon [18]. Somewht surprisingly, we oserved tht GLP-1 relese ws higher from type 2 dietic thn from non-dietic islets. We did not study the GLP-1 secretion of purified lph cells from type 2 dietic donors, due to the limited tissue vilility. However, when we counted islet cells tht were doule-positive for glucgon nd PC1/3, no mjor difference ws oserved etween non-dietic nd type 2 dietic smples. Insted, PC1/3 gene expression ws found to e higher in lph cells from type 2 dietic donors. This suggests functionl vritions in the two conditions, resemling wht hppens with glucgon, the relese of which is ltered in type 2 dietic islets, despite similr mounts of glucgon-contining cells [38 4]. Although type 2 dietic ptients my relese less GLP-1, which could contriute to decresed incretin effects in this ptient group [1, 41 43], it cnnot e ruled out tht the reduced concentrtions of ctive GLP-1 in such ptients might e prtly due to incresed DPP-4 ctivity. Incresed plsm DPP-4 ctivity in type 2 dietic ptients hs een reported [44], nd chronic hyperglycemi induced higher DPP-4 ctivity, with reductions of circulting ctive GLP-1 [45]. The incresed GLP-1 production from type 2 dietes islets could represent n ttempt to protect et cells in condition tending to led to et cell dysfunction [46, 47]. In line with this concept, the ctivtion of PC1/3 in lph cells hs een oserved in mouse models of insulin resistnce [34]. It hs lso een reported tht, following et cell injury, the iologicl function of lph cells switches from glucgon to GLP-1 production, promoting et cell function nd survivl [48]. In ddition, denovirus-medited production of PC1/3 in mouse lph cells incresed islet GLP-1 secretion, with improved glucose-stimulted insulin secretion nd enhnced survivl fter cytokine tretment [49], wheres the production of PC1/3, rther thn PC2, in αtc1-6 cells induced GLP-1 production nd converted lph cell function from hyperglycemi-promoting to lood glucoselowering fter trnsplnttion [5]. Finlly, GLP-1 relese from lph cells is upregulted in the Psmmomys oesus geril during the development of dietes, which hs een interpreted s n dptive response to incresed glucose levels [17]. This is in greement with the findings of the present study, which show enhnced relese of GLP-1 nd lower glucgon:glp-1 secretion rtio in islets from type 2 dietic donors. Overll, these results support the view tht locl nd functionlly competent GLP-1 system is present in humn islets, residing in lph cells nd possily modulted y type 2 dietes. This suggests tht lph cells my relese more thn one single hormone, depending on the needs of the islet microenvironment t given moment. These findings supplement current knowledge on the pthophysiology of incretins nd my open new pths for their use s therpeutic tools. Funding This study ws supported in prt y the Itlin Ministry of Helth nd the Itlin Ministry of Eduction. Dulity of interest The uthors declre tht there is no dulity of interest ssocited with this mnuscript Contriution sttement Ech uthor prticipted sufficiently in the work presented y this rticle to tke pulic responsiility for the content. Prticiption included sustntil contriutions to: the conception nd design of the study; the cquisition, nlysis nd interprettion of dt; the drfting or criticl revision of the rticle for importnt intellectul content; nd pprovl of the finl version.

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Regul Pept 146: Bosco D, Armnet M, Morel P et l (21) Unique rrngement of lph- nd et-cells in humn islets of Lngerhns. Dietes 59: Wilson ME, Klmrs JA, Germn MS (22) Expression pttern of IAPP nd prohormone convertse 1/3 revels distinctive set of endocrine cells in the emryonic pncres. Mech Dev 115: Kilimnik G, Kim A, Steiner DF, Friedmn TC, Hr M (21) Intrislet production of GLP-1 y ctivtion of prohormone convertse 1/3 in pncretic α-cells in mouse models of β-cell regenertion. Islets 2: Fujit Y, Widemn RD, Asdi A et l (21) Glucose-dependent insulinotropic polypeptide is expressed in pncretic islet lphcells nd promotes insulin secretion. Gstroenterology 138: De Mrinis YZ, Zhng E, Amisten S et l (21) Enhncement of glucgon secretion in mouse nd humn pncretic lph cells y protein kinse C (PKC) involves intrcellulr trfficking of PKClph nd PKCdelt. Dietologi 53: Wlker JN, Rmrchey R, Zhng Q, Johnson PR, Brun M, Rorsmn P (211) Regultion of glucgon secretion y glucose: prcrine, intrinsic or oth? Dietes Oes Met 13 (Suppl 1): Deng S, Vtmniuk M, Hung X et l (24) Structurl nd functionl normlities in the islets isolted from type 2 dietic sujects. Dietes 53: Henquin JC, Rhier J (211) Pncretic lph cell mss in Europen sujects with type 2 dietes. Dietologi 54: Meier JJ, Ueererg S, Kors S, Schneider S (211) Diminished glucgon suppression fter β-cell reduction is due to impired α-

11 3272 Dietologi (212) 55: cell function rther thn n expnsion of α-cell mss. Am J Physiol Endocrinol Met 3:E717 E Nuck MA, Vrdrli I, Decon CF, Holst JJ, Meier JJ (211) Secretion of glucgon-like peptide-1 (GLP-1) in type 2 dietes: wht is up, wht is down? Dietologi 54: Toft-Nielsen MB, Dmholt MB, Mdsd S et l (21) Determinnts of the impired secretion of glucgon-like peptide-1 in type 2 dietic ptients. J Clin Endocrinol Met 86: Dunning BE, Foley JE, Ahrén B (25) Alph cell function in helth nd disese: influence of glucgon-like peptide-1. Dietologi 48: Ryskjer J, Decon CF, Crr RD et l (26) Plsm dipeptidyl peptidse-iv ctivity in ptients with type-2 dietes mellitus correltes positively with HAlc levels, ut is not cutely ffected y food intke. Eur J Endocrinol 155: Mnnucci E, Pl L, Cini S et l (25) Hyperglycemi increses dipeptidyl peptidse IV ctivity in dietes mellitus. Dietologi 48: Cnop M, Foufelle F, Velloso LA (212) Endoplsmic reticulum stress, oesity nd dietes. Trends Mol Med 18: Mrchetti P, Lupi R, del Guerr S et l (29) Gols of tretment for type 2 dietes: et-cell preservtion for glycemic control. Dietes Cre 32(Suppl 2):S178 S Liu Z, Stnojevic V, Avdhni S, Yno T, Hener JF (211) Stroml cell-derived fctor-1 (SDF-1)/chemokine (C-X-C motif) receptor 4 (CXCR4) xis ctivtion induces intr-islet glucgonlike peptide-1 (GLP-1) production nd enhnces et cell survivl. Dietologi 54: Widemn RD, Yu IL, Weer TD et l (26) Improving function nd survivl of pncretic islets y endogenous production of glucgonlike peptide 1 (GLP-1). Proc Ntl Acd Sci U S A 13: Widemn RD, Covey SD, We GC, Drucker DJ, Kieffer TJ (27) A switch from prohormone convertse (PC)-2 to PC1/3 expression in trnsplnted lph-cells is ccompnied y differentil processing of proglucgon nd improved glucose homeostsis in mice. Dietes 56: