Endogenous GIP ameliorates impairment of insulin secretion in proglucagon-deficient mice under moderate beta cell damage induced by streptozotocin

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1 Dietologi (216) 59: DOI 1.17/s ARTICLE Endogenous GIP meliortes impirment of insulin seretion in proglugon-defiient mie under moderte et ell dmge indued y streptozotoin Atsushi Iid 1 & Yusuke Seino 1,2 & Ayko Fukmi 1 & Ryuy Mekw 1 & Disuke Ye 3,4 & Shinou Shimizu 3 & Keit Kinoshit 5,6 & Yusuke Tkgi 5,6 & Tkko Izumoto 7 & Hidetd Ogt 1 & Kot Ishikw 1 & Nouki Ozki 5 & Shin Tsunekw 1 & Yoji Hmd 1,2 & Yutk Oiso 1 & Hiroshi Arim 1 & Yoshitk Hyshi 6 Reeived: 17 Deemer 215 /Aepted: 2 Mrh 216 /Pulished online: 6 April 216 # The Author(s) 216. This rtile is pulished with open ess t Springerlink.om Astrt Aims/hypothesis The tion of inretin hormones inluding gluose-dependent insulinotropi polypeptide (GIP) nd glugon-like peptide-1 (GLP-1) is potentited in niml models defetive in glugon tion. It hs een reported tht suh niml models mintin normoglyemi under streptozotoin (STZ)-indued et ell dmge. However, the role of GIP in regultion of gluose metolism under omintion of glugon defiieny nd STZ-indued et ell dmge hs not een fully explored. Methods In this study, we investigted gluose metolism in mie defiient in proglugon-derived peptides (PGDPs) nmely glugon gene knokout (GgKO) mie dministered with STZ. Single high-dose STZ (2 mg/kg, hstz) or moderte-dose STZ for five onseutive dys (5 mg/kg 5, mstz) ws dministered to GgKO mie. The ontriution of GIP to gluose metolism in GgKO mie ws lso investigted y experiments employing dipeptidyl peptidse IV (DPP4) inhiitor (DPP4i) or Gg Gipr doule knokout (DKO) mie. Results GgKO mie developed severe dietes y hstz dministrtion despite the sene of glugon. Administrtion of mstz deresed pnreti insulin ontent to 18.8 ± 3.4 (%) in GgKO mie, ut d liitum-fed lood gluose levels did not signifintly inrese. Gluose-indued insulin seretion ws mrginlly impired in mstz-treted GgKO mie ut ws olished in mstz-treted DKO mie. Although GgKO mie lk GLP-1, tretment with DPP4i potentited gluose-indued insulin seretion nd meliorted gluose intolerne in mstz-treted GgKO mie, ut did not inrese et ell re or signifintly redue poptoti ells in islets. Eletroni supplementry mteril The online version of this rtile (doi:1.17/s ) ontins peer-reviewed ut unedited supplementry mteril, whih is ville to uthorised users. Yusuke Seino yusuke@med.ngoy-u..jp Yoshitk Hyshi hyshiy@riem.ngoy-u..jp Deprtment of Endorinology nd Dietes, Ngoy University Grdute Shool of Mediine, 65 Tsurumi-ho, Show-ku, Ngoy , Jpn Deprtment of Metoli Mediine, Ngoy University Grdute Shool of Mediine, Ngoy, Jpn Yutk Seino Distinguished Center for Dietes Reserh, Knsi Eletri Power Medil Reserh Institute, Koe, Jpn Division of Moleulr nd Metoli Mediine, Koe University Grdute Shool of Mediine, Koe, Jpn Reserh Center of Helth, Physil Fitness nd Sports, Reserh Institute of Environmentl Mediine, Ngoy University, Ngoy, Jpn Deprtment of Genetis, Division of Stress Adpttion nd Reognition, Reserh Institute of Environmentl Mediine, Ngoy University, Furo-ho, Chikus-ku, Ngoy , Jpn Deprtment of Orl nd Mxillofil Surgery, Ngoy University Grdute Shool of Mediine, Ngoy, Jpn

2 1534 Dietologi (216) 59: Conlusions/interprettion These results indite tht GIP hs the potentil to meliorte gluose intolerne even under STZ-indued et ell dmge y inresing insulin seretion rther thn y promoting et ell survivl. Keywords Dipeptidyl peptidse IV. GIP. GLP-1. Glugon. Hyperglyemi. Insulin. Insulin seretion. Streptozotoin Arevitions BW Body weight DKO Gg Gipr doule knokout DPP4 Dipeptidyl peptidse IV DPP4i DPP4 inhiitor GgKO Glugon gene knokout mie, whih lk ll the PGDPs inluding glugon nd GLP-1 GIP Gluose-dependent insulinotropi polypeptide GiprKO GIP reeptor knokout GLP-1 Glugon-like peptide-1 Glu-DTR Mie with diphtheri toxin medited-ltion of lph nd L ells hstz High-dose streptozotoin IPGTT Intrperitonel gluose tolerne test mstz Moderte-dose streptozotoin PGDPs Proglugon-derived peptides STZ Streptozotoin Introdution Glugon is sereted from pnreti lph ells nd ontriutes to promoting hepti gluose prodution [1]. Dieti ptients show prdoxil seretion of glugon in response to mel test [2] nd suh dieti hyperglugonemi is thought to e due to the reltive defiieny of insulin tion [3]. Thus, lokde of glugon tion is onsidered to e novel trget for gluose-lowering drug development [3]. Severl niml models defiient in glugon tion hve een reported, inluding prohormone onvertse 2 knokout mie [4, 5], glugon reeptor knokout (Ggr / )mie[6], mie treted with glugon reeptor ntisense oligonuleotide [7] nd mie hving pnres-speifi Arx ltion [8]. All of these niml models show lower lood gluose levels, suggesting tht glugon plys mjor role in hepti gluose prodution nd the mintenne of lood gluose levels. Moreover, severl studies demonstrted tht suh niml models do not develop hyperglyemi fter et ell destrution y streptozotoin (STZ) tretment [8 11], suggesting tht glugon plys n indispensle role in hyperglyemi used y et ell destrution. Gluose-dependent insulinotropi polypeptide (GIP) nd glugon-like peptide-1 (GLP-1) re inretins relesed from intestinl K- nd L ells, respetively, nd potentite insulin seretion from et ells in gluose-dependent mnner [12, 13]. GLP-1 is produed from proglugon, whih lso serves s preursor of glugon. Severl niml models defiient in glugon tion show mrkedly elevted plsm GLP-1 levels [5 7, 14], suggesting tht GLP-1 might ontriute to normoglyemi under STZ-indued et ell destrution vi extr-pnreti effets. We previously generted mie lking proglugon-derived peptides (PGDPs), inluding glugon nd GLP-1 (GgKO mie) [15]. GgKO mie disply inresed insulin sensitivity due to glugon defiieny nd enhned erly-phse insulin seretion in GIP-dependent mnner [16]. In the present study, we investigted gluose metolism in GgKO mie dministered with STZ. We found tht GgKO mie developed mrked hyperglyemi under the severe insulin defiieny used y STZ-indued et ell destrution despite the sene of glugon. However, GgKO mie displyed normoglyemi under moderte insulin defiieny used y moderte et ell dmge. We lso investigted involvement of GIP in resistne to et ell dmge in GgKO mie. Methods Mterils Aetminophen, STZ nd BSA were otined from Sigm-Aldrih (St Louis, MO, USA). Angliptin, dipeptidyl peptidse IV (DPP4) inhiitor (DPP4i) ws provided from Snw Kgku Kenkyusho Co. (Ngoy, Aihi, Jpn). Animls GgKO mie in C57BL/6 kground were estlished nd mintined s previously reported (Gg tm1yhys )[15]. GgKO heterozygous nd wild-type mie were used s ontrols. GgKO nd heterozygous mie express green fluoresent protein (GFP) in ells expressing the glugon gene. GIP reeptor knokout (GiprKO) mie, originlly generted in the C57BL/6 kground (Gipr tm1yse )[17], were otined from RIKEN BRC (Tsuku, Jpn) through the Ntionl Bio-Resoure Projet of the Ministry of Edution, Culture, Sports, Siene nd Tehnology, Jpn. GgKO nd GiprKO mie were interrossed to otin Gg Gipr doule knokout (DKO) mie [16]. All mie used in this study were mle. All proedures were onduted ording to protools nd regultions pproved y the Ngoy University Animl Experiment Committee. STZ tretment To indue moderte dmge to et ells in the mie, STZ ws dministered i.p. t dose of 5 mg (kg ody weight [BW]) 1 for five onseutive dys (moderte-dose STZ [mstz]) [18]. To indue et ell

3 Dietologi (216) 59: destrution,2mgstz(kgbw) 1 ws injeted fter 16 h-fst (high-dose STZ [hstz]) [19]. DPP4i tretment Angliptin ws dministered through feed wter t onentrtion of.625 mg/ml from 7 dys efore strting STZ tretment to the end of the experiment. Gluose tolerne tests OGTT nd i.p. gluose tolerne test (IPGTT) (2 g/kg BW) were performed s previously desried [16]. Gstri emptying Liquid nd solid phse of gstri emptying were ssessed s previously desried [2]. Biohemil nlyses Blood gluose levels were mesured using n Antsense Blood Gluose Meter (Hori, Kyoto, Jpn). Plsm GIP nd insulin were mesured using Rt/ Mouse GIP (totl) ELISA (Merk Millipore, Drmstdt, Germny) nd Mouse Insulin ELISA Kit (Moring Institute of Biologil Siene, Kngw, Jpn) s previously reported [21]. Blood smples for mesurements of iologilly intt GLP-1 nd GIP were olleted using BD P8 tues (Beton Dikinson, Frnklin Lkes, NJ, USA). Plsm levels of iologilly intt GLP-1 nd GIP were evluted using the following immunossys ording to mnufturers instrutions: GLP-1, Ative GLP-1 (ver. 2) Kit (Meso Sle Disovery, Rokville, MD, USA) nd GIP, Mouse GIP, Ative form Assy Kit (Immuno-Biologil Lortories, Gunm, Jpn). Pnreti insulin nd GIP ontent nlysis Pnreti tissue ws homogenised in Kres-Ringer uffer (ph 7.4) on ie. Tissue homogente ws extrted overnight in id-ethnol (1.5% (vol./vol.) HCl in 75% (vol./vol.) EtOH). Tissue extrts were diluted 1:1 or 1:2 for insulin mesurement. Diluted extrts were mesured y HTRF Insulin Kit (Cisio Biossys, Codolet, Frne). Pnreti insulin ontent ws orreted y tissue weight for nlysis. Tissue extrts were diluted 1:3 with Kres- Ringer uffer for GIP mesurement nd protein ontent ssy. Diluted extrts were mesured y Rt/Mouse GIP (totl) ELISA (Merk Millipore) nd BCA protein ssy (Sigm-Aldrih). Pnreti GIP ontent ws orreted y protein ontent for nlysis. Immunohistohemistry Tissue preprtion nd nlyses hve een desried in detil previously [16]. Pnres tissue ws olleted 1 dy fter the finl dministrtion of mstz to nlyse leved spse-3 nd t 9 weeks of ge to nlyse et ell mss. For nlysis of leved spse-3, setions were treted in itrte uffer-sed Trget Retrievl Solution (Dko, Glostrup, Denmrk) efore primry ntiody inution. Setions were inuted overnight with primry ntiodies to leved spse-3 (1:3; #9661; Cell Signling Tehnology, Dnvers, MA, USA) nd/or insulin (1:15; 7842; Am, Cmridge, MA, USA). Seondry ntiodies (Alex fluor 488, 594; 1:5; A-1173, A-1137 or A-1176; Moleulr Proes, Eugene, OR, USA) were pplied nd inuted for 9 min t room temperture. Antiodies used re ommerilly ville nd widely used for immunohistohemistry. Fluoresent imges were tken using BZ-9 Fluoresene Mirosope (Keyene, Osk, Jpn). The numer of islets used to nlyse leved spse-3 ws 355. The totl res of islets, insulin-positive ells (et ells) nd leved spse-3 were nlysed using BZ-X nlyser softwre (Keyene). Sttistil nlysis Results re presented s mens ± SEM. Sttistil signifine ws evluted y ANOVA or Student s t test using GrphPd Prism 6 for Windows (GrphPd Softwre, L Joll, CA, USA). Results Severe hyperglyemi is indued y hstz-indued et ell ltion in GgKO mie To evlute gluose metolism under et ell dysfuntion in the GgKO nd ontrol mie, STZ ws given t high dose (2 mg/kg, one shot; Fig. 1). As shown in Fig. 1, othggko nd ontrol mie displyed severe hyperglyemi t 4, 7 nd 9 dys fter hstz dministrtion. In ord with the lood gluose levels, plsm insulin levels were signifintly deresed to <4 pmol/l in oth groups (Fig. 1). Pnreti insulin ontent ws nlysed 9 dys fter hstz dministrtion, nd showed nerly 9% of the et ells to e lted in oth GgKO nd ontrol mie (Fig. 1). These findings demonstrte tht severe destrution of et ells uses hyperglyemi even in GgKO mie, whih lk PGDPs inluding glugon, inditing tht glugon tion is not requisite for persistent hyperglyemi. Normoglyemi is mintined in GgKO mie fter mstz tretment We next nlysed gluose metolism in mstz (5 mg/kg one dily for 5 dys)-treted ontrol nd GgKO mie (Fig. 2). Bet ell dmge in mie treted with mstz ws milder thn tht in mie treted with hstz y nlysis of the fluoresent imges of leved spse-3 (Eletroni Supplementry Mteril [ESM] Fig. 1). In ontrst to hstz dministrtion, mstz tretment reveled differentil effets on lood gluose levels in ontrol nd GgKO mie. As shown in Fig. 2, lood gluose levels in mstz-treted GgKO () mie were not signifintly elevted ompred with those in sline (154 mmol/l NCl)-treted GgKO (sline-ggko) mie, wheres those in mstztreted ontrol (mstz-ontrol) mie were signifintly

4 1536 Dietologi (216) 59: Fig. 1 () Blood gluose levels, () plsm insulin levels nd () pnreti insulin ontent in ontrol nd GgKO mie fter hstz tretment (2 mg/kg). STZ ws i.p. injeted t dose of 2 mg/kg BW fter overnight (16 h) fst. () Blood gluose levels under d liitum-fed sttes were mesured in oth GgKO nd ontrol mie efore (seline) nd t 4, 7 nd 9 dys fter hstz dministrtion. () Plsm insulin levels under d liitum-fed sttes were mesured efore nd 9 dys fter hstz injetion. () Pnreti insulin ontent ws nlysed t 9 dys fter hstz injetion. p <.1, slinetreted vs hstz-treted. White rs, sline-ontrol; light grey rs, hstz-ontrol; lk rs, sline-ggko; drk grey rs, hstz-ggko. n =6 9 per group Bseline Time (dys) Insulin ontent (ng/mg of pnres) Bseline Dy 9 Sline-ontrol hstz-ontrol hstz-ggko elevted t 2 weeks nd therefter. After mstz dministrtion, plsm insulin levels in ontrol mie were signifintly deresed, ut they were not hnged in GgKO mie (Fig. 2). Plsm GIP levels were signifintly higher in GgKO thn in ontrol mie. To determine whether sene of GLP-1 nd inresed GIP modify motility of the gstrointestinl trt in GgKO mie, the gstri emptying rte ws evluted to ssess solid phse gstri emptying. Aetminophen sorption test lso ws performed to ssess liquid phse gstri emptying. There ws no signifint differene etween ontrol nd GgKO mie in either liquid (152.2 ± 6.79 μmol/l in ontrol; ± μmol/l in GgKO; p =.557) or solid phse (39.3 ± 11.3% in ontrol; 28.5 ± 12.2% in GgKO; p =.539) gstri emptying rte (ESM Fig. 2). GIP ontriutes to gluose homeostsis in GgKO mie To ssess the role of GIP in gluose homeostsis of mstztreted GgKO mie, we treted the DKO mie with mstz (mstz-dko) nd nlysed gluose tolerne. Gluose tolerne of DKO mie without STZ tretment ws omprle with tht of ontrol mie [16]. Blood gluose levels under d liitum-fed onditions in mstz-dko mie were not signifintly different from those in mie or non-dieti GiprKO mie; nd plsm insulin levels under d liitum-fed onditions in mstz-dko mie were not signifintly different from those in mie (Fig. 3, ). However, gluose tolerne nd insulin seretion during OGTT in mstz-dko mie were impired ompred with those in nd sline-treted mie (Fig. 3, d nd dt not shown), inditing signifint role of GIP in et ell funtion under mstz-indued dmge. On the other hnd, no signifint differene in gluose levels ws oserved etween nd mstz-dko mie during IPGTT (Fig. 3e). Signifint inrese in insulin level in response to i.p. gluose lod ws oserved in ut not in mstz-dko mie (Fig. 3f). The pprently differentil insulin sensitivity oserved etween these two models might e due to presene or sene of the GIP reeptor. Previous studies hve shown tht mie defiient in GIP tion re more sensitive to insulin thn ontrol mie [22, 23]. DPP4i enhnes gluose-indued insulin seretion only in mstz-treted GgKO mie Beuse GIP nd GLP-1 re rpidly intivted y DPP4 [12], DPP4 inhiitors re widely used for linil tretment of dietes s n insulin seretgogue. In ddition, GIP nd GLP-1 re onsidered to ply ritil role in gluose-indued insulin seretion from pnreti et ells nd nti-poptoti tion for et ell survivl medited y DPP4 inhiition [24, 25]. To investigte whether enhnement of GIP signlling y DPP4i improves gluose tolerne nd/or protets et ells in mie, DPP4i ws dministered s shown in Fig. 4. Thistretment improved gluose tolerne y inresing insulin seretion during OGTT in non-dieti wild-type mie (ESM Fig. 3, ), ut did not during IPGTT (ESM Fig 3, d). DPP4i tretment lso signifintly inresed tive GIP nd GLP-1 levels

5 Dietologi (216) 59: STZ Time (weeks) Plsm GIP (pmol/l) Sline-ontrol mstz-ontrol during OGTT s well s under d liitum-fed sttus (ESM Fig 3e h). Tretment with DPP4i did not ffet lood gluose levels under d liitum-fed sttes in either ontrol or GgKO mie fter mstz tretment (Fig. 4). Pnreti insulin ontent ws deresed to 18.1 ±.2 (%) in ontrol mie nd to 18.8 ± 3.4 (%) in GgKO mie 9 weeks fter mstz tretment, nd DPP4i did not inrese pnreti insulin ontent in either mstz-ontrol mie or mie (Fig. 4). On IPGTT nd OGTT, oth mstz-ontrol nd mie showed impired gluose intolerne reltive to the orresponding sline-treted nimls (Fig. 4, e, g, i). However, tretment with DPP4i showed differentil effets on mstzontrol nd mie. Tretment y DPP4i filed to improve gluose intolerne nd insulin seretory response in mstz-ontrol mie during IPGTT (Fig. 4, d) nd OGTT (Fig. 4g, h). On the other hnd, with the sme tretment in GgKO mie, lood gluose levels were signifintly redued t 3, 6 nd 12 min during IPGTT (Fig. 4e) nd t 3 nd 6 min during OGTT (Fig. 4i). In ddition, gluose-indued insulin seretion ws inresed during IPGTT nd OGTT y hroni DPP4 inhiition in mie (Fig. 4f, j) Sline-ontrol mstz-ontrol Fig. 2 () Blood gluose levels, () plsm insulin levels nd () plsm GIP levels in ontrol nd GgKO mie fter mstz tretment in d liitum-fed sttes. STZ or sline ws i.p. injeted one dily t dose of 5 mg/kg BW for five onseutive dys. p <.5,p <.1;, not signifint. Signifine of lood gluose levels is expressed vs slinetreted mie (). Plsm insulin levels were evluted t 5 weeks (). Signifine of plsm GIP levels ws tested for ontrol vs GgKO mie (). White irles nd solid line, sline-ontrol; lk irles nd solid line, mstz-ontrol; white squres nd dshed line, sline-ggko; lk squres nd dshed line, (). White rs, sline-ontrol; light grey rs, mstz-ontrol; lk rs, sline-ggko; drk grey rs, (, ). n =6 12, sline-ontrol; n =12 15, mstz-ontrol; n =4 1, sline-ggko; n =5 12, e STZ Time (weeks) sline-giprko mstz-giprko mstz-dko DPP4i tretment did not improve gluose tolerne or insulin seretion in mstz-dko mie (ESM Fig. 4). These results indite tht insulin seretion y GIP plys n essentil role in gluose metolism in mie under tretment with DPP4i. Pnreti GIP ontents were not redued in hstz- or mstz-ontrol mie. On the other hnd, pnreti GIP ontents were signifintly redued in hstz-ggko mie ut not in mie (ESM Fig. 5). GIP in GgKO mie did not enhne et ell survivl fter mstz tretment It ws reported tht endogenous GIP plys limited role in et ell survivl in STZ-indued d f Fig. 3 () Blood gluose levels, () plsm insulin levels, (, d) OGTT nd (e, f) IPGTT in sline-giprko, mstz-treted GiprKO mie (mstz- GiprKO), nd mstz-dko mie. OGTT nd IPGTT were performed 3 nd 4 weeks fter mstz dministrtion, respetively. Plsm insulin levels were evluted t 5 weeks (). White tringles nd solid line, sline-giprko; lk tringles nd solid line, mstz- GiprKO; lk squres nd dshed line, ; lk tringles nd dshed line, mstz-dko (,, e). Digonl-striped rs, sline- GiprKO; horizontl-striped rs, mstz-giprko; drk grey rs, ; horizontl-striped drk grey rs, mstz-dko (, d, f). n =8 11, sline-giprko; n =6 8, mstz-giprko; n =5, mstz- GgKO; n =5 6, mstz-dko. p <.5, p <.1 vs mstz-treted GgKO mie (,, e). p <.5,p <.1, p <.1;, not signifint (d, f)

6 1538 Dietologi (216) 59: Fig. 4 () Blood gluose levels, () pnreti insulin ontent, ( f) IPGTT nd (g j) OGTT in GgKO nd ontrol mie treted with DPP4i. IPGTT nd OGTT were performed t 6 nd 7 weeks fter mstz injetion, respetively. p <.5, p <.1, vs sline-treted (,, e, g, i). p <.5, p <.1;, not signifint (, d, f, h, j). White irles nd solid line, sline-ontrol; lk irles nd solid line, mstzontrol; lk tringles nd solid line, mstz-dpp4i-ontrol; white squres nd dshed line, sline- GgKO; lk squres nd dshed line, ; lk dimonds nd dshed line, mstz- DPP4i-GgKO (,, e, g, i). White rs, sline-ontrol; light grey rs, mstz-ontrol; horizontlstriped light grey rs, mstz- DPP4i-ontrol; lk rs, sline-ggko; drk grey rs, ; digonlstriped drk grey rs, mstz- DPP4i-GgKO (, d, f, h, j). n =4 7, sline-ontrol; n =3 7, mstz-ontrol; n =7 8, mstz- DPP4i-ontrol; n =4 9, sline- GgKO; n =5 9, mstz- GgKO; n =5 6, mstz- DPP4i-GgKO g DPP4i STZ Time (weeks) Insulin ontent (ng/mg of pnres) Sline-ontrol mstz-ontrol mstz-dpp4i-ontrol mstz-dpp4i-ggko d e f h 8 6 i 4 3 j 8 6 dietes models [25, 26]. To investigte the role of GIP in et ell survivl in the sene of PGDPs inluding GLP-1, we nlysed poptosis in islets nd et ell mss in the pnres of mie. The perentge of et ells positive for leved spse-3 ws inresed signifintly y mstz tretment. Tretment of with DPP4i did not signifintly redue spse-positive ells (Fig. 5, ) nd et ell mss in DPP4i- mie ws omprle with tht in mie (Fig. 5, d). Pnreti insulin ontent ws similr etween GgKO mie nd DKO mie 5 weeks fter mstz tretment (ESM Fig. 6). These results suggest tht improvement of insulin seretion nd gluose tolerne in mie y DPP4i-tretment is medited through mehnisms other thn promotion of et ell survivl. Disussion Glugon inreses hepti gluose prodution; insulin inhiits gluose prodution [27]. The reltive ontriution of dysregulted glugon seretion nd impirment of insulin seretion to hyperglyemi in dieti ptients hs een mtter of dete [28]. It hs een shown reently tht dministrtion of STZ to Ggr / mie disrupted 9% of the et ells nd olished gluose-indued insulin seretion yet filed to use hyperglyemi [1]. Bsed on this oservtion, it hs een proposed tht hyperglugonemi itself plys n essentil role in inresing lood gluose levels. However, in the present study we oserved tht GgKO mie lking oth glugon nd GLP-1 developed hyperglyemi upon hstz-indued et ell ltion. This differene etween Ggr / nd GgKO is most likely due to the presene or sene of extr-pnreti GLP-1 tion. Severl studies hve shown tht GLP-1 exhiits extrpnreti tion in inresing insulin sensitivity nd modulting gluose metolism [9, 2, 29]. This is supported y studies employing Ggr / Glp1r / doule knokout mie nd mie with diphtheri toxin medited-ltion of lph nd L ells (Glu-DTR). Both models re defiient in GLP-1 tion nd exhiit hyperglyemi on STZ tretment, undersoring the ritil importne of GLP-1 on glyemi ontrol under STZ-indued et ell ltion [3 32]. Nevertheless, there re unique hrteristis mong Ggr / Glp1r /, Glu-DTR nd GgKO mie. GgKO mie lk ll proglugon-derived peptides throughout life, while there is

7 Dietologi (216) 59: Fig. 5 (, ) Cleved spse-3 immunopositivity nd (, d) et ell re of islets from GgKO mie. () Representtive imges of leved spse-3-positive islets. Red, leved spse-3; green, insulin; lue, DAPI. Sle rs, 5 μm. (d) Representtive imges of pnres. Red, insulin; green, GFP (glugon); lue, DAPI. Sle rs, 3 μm. Blk rs, sline-ggko; drk grey rs, ; digonlstriped drk grey rs, mstz- DPP4i-GgKO (, ). p <.5, p <.1;, not signifint (p =.7for[]) Cleved spse-3-positive re (% of totl islet re) Bet ell re (% of totl pnres re) mstz-dpp4i-ggko mstz-dpp4i-ggko d DPP4i- Cleved spse-3 Insulin Merged (with DAPI) DPP4i- GFP/Insulin/DAPI derese in PGDPs in Glu-DTR mie: GLP-1 synthesis in Glu-DTR mie returns to norml levels 7 dys fter injetion of diphtheri toxin [33]. GLP-2 is present in Ggr / Glp1r / mie, ut is redued or sent in Glu-DTR nd GgKO mie, respetively. Thus, the presene or sene of GLP-2 nd residul glugon in Glu-DTR mie does not seem to ffet glyemi ontrol under et ell ltion. Nevertheless, GgKO mie, whih lk GLP-1, mintin normoglyemi fter mstz tretment, whih uses persistent hyperglyemi in the ontrol mie. Thus, the requirement for insulin to mintin norml lood gluose levels is lower in oth GgKO nd Ggr / mie [9, 15, 34, 35]. In the present study, plsm insulin levels under d liitum-fed sttes were omprle etween mstz-ontrol nd mie (Fig. 2). Insulin levels in GgKO mie efore STZ tretment were not signifintly different from those fter mstz tretment. These findings indite tht insulin plys ritil role in the mintenne of gluose levels in mie. Inretins regulte insulin seretion nd GIP is the mjor inretin in GgKO mie, whih lk GLP-1 [36]. Severl reports suggest tht GIP potentites the erly phse of gluose-indued insulin seretion to ontriute to improved glyemi ontrol [16, 37 39]. GIP lso hs een reported to ontriute to et ell survivl in vitro [4, 41], while GIP overexpression ws found to enhne the inrement of insulin ontent indued y high-ft diet feeding y deresing et ell poptosis [39]. We previously reported tht GIP ws expressed not only in the gstrointestinl trt ut lso in pnreti et ells in GgKO mie nd tht GIP hyperseretion ontriutes to the enhned gluose-indued insulin seretion nd improved gluose tolerne under nondieti sttes in GgKO mie [16]. These findings led us to investigte whether GIP ontriutes to resistne to developing dietes in mie. Moderte et ell dmge olished insulin seretion in DKO ut not in GgKO mie (Fig. 3).Tretment with DPP4i potentited gluose-indued insulin seretion nd meliorted

8 154 Dietologi (216) 59: gluose intolerne in ut not in mstz-dko mie (Fig. 4, ESM Fig. 4). These results indite tht GIP plyed n importnt role in proteting mie defiient in PDGPs from dietes. However, tretment with DPP4i did not signifintly redue the numer of poptoti ells in islets (Fig. 5), nd loking GIP tions did not modify pnreti insulin ontent in mstz-treted mie (ESM Fig. 6). These results indite tht GIP does not ontriute to et ell protetion in mieutthtit does ontriute to inrese insulin seretion from eh of the et ells. It ws reported reently tht GIP nd GLP-1 re sereted not only from enteroendorine K- nd L ells ut lso from pnreti islets [16, 42 45]. In the present study, pnreti GIP ontent in ontrol mie deresed neither y hstz tretment nor y mstz tretment, most likely euse GIP is expressed in pnreti lph ells [16, 26, 45]. On the other hnd, GIP ontent in GgKO pnres ws deresed y hstz tretment, onfirming our previous results showing tht GIP is expressed in et ells in GgKO. However, pnreti GIP ontent ws not hnged y mstz tretment (ESM Fig. 5). The mehnism underlying the lk of hnge in pnreti GIP ontent under the mstz-indued et ell dmge, inluding tht of regenertion of GIPpositive ells, remins to e eluidted. Islet-derived GLP-1 hs een shown to enhne gluoseindued insulin seretion in vitro y using DPP4i in nondieti humn nd mouse islets [46]. In the present study, IPGTT nd OGTT nlyses suggested tht oth islet-derived GIP nd gut-derived GIP ontriutes to improving gluose metolism in mie. In, ddition, our results indite tht islet-derived GIP n exert n insulinotropi effet even when islet insulin ontents re deresed under glugon-defiient sttes. We therefore propose tht omintion therpy with glugon ntgonist nd DPP4i might e onsidered s therpeuti option to tret dietes. Aknowledgements The uthors re indeted to Snw Kgku Kenkyusho Co. Ltd for provision of ngliptin. The uthors ddress thnks to Yutk Seino (Knsi Eletri Power Medil Reserh Institute) for providing GiprKO mie nd suggestions. The uthors lso thnk H. Med (Koe University) nd M. Ktgiri, M. Ymd nd M. Ymmoto (Ngoy University) for tehnil ssistne, nd S. Seino (Koe University) for helpful support. Funding This study ws supported y grnts for young reserhers from the Jpn Assoition for Dietes Edution nd Cre to YS nd Grnts-in-Aid for Sientifi Reserh from the Jpn Soiety for the Promotion of Siene to YS ( ) nd YHyshi ( , 15K15356 nd 15H4681). Dulity of interest YS nd YHmd reeived reserh grnt from Snw Kgku Kenkyusho Co., Ltd. All remining uthors delre tht there is no dulity of interest ssoited with their ontriution to this mnusript. Contriution sttement AI, YS, NO, ST, YHmd, YO, HA nd YHyshi prtiipted in the reserh design; AI, YS, AF, RM, KK, YT, TI, HO nd KI rried out the experiments; AI, YS nd YHyshi wrote the mnusript; AI, YS, AF, RM, TI, HO, KI, NO, ST, YHmd, YO, HA nd YHyshi ontriuted to disussions; AI, YS, KK, YT nd YHyshi reviewed/revised the mnusript. DY nd SS ontriuted to quisition of dt, intelletul disussions nd writing of the mnusript. All uthors gve pprovl of the finl version to e pulished. YS is the gurntor of this work nd hd full ess to ll the dt in the study, nd tkes responsiility for the integrity of dt nd the ury of dt nlysis. Open Aess This rtile is distriuted under the terms of the Cretive Commons Attriution 4. Interntionl Liense ( retiveommons.org/lienses/y/4./), whih permits unrestrited use, distriution, nd reprodution in ny medium, provided you give pproprite redit to the originl uthor(s) nd the soure, provide link to the Cretive Commons liense, nd indite if hnges were mde. Referenes 1. 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