GLP-1 oestrogen attenuates hyperphagia and protects from beta cell failure in diabetes-prone New Zealand obese (NZO) mice

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1 Dietologi () 8: DOI.7/s ARTICLE GLP-1 oestrogen ttenutes hyperphgi nd protets from et ell filure in dietes-prone New Zelnd oese (NZO) mie Roert W. Shwenk & Christin Bumeier & Brin Finn & Oliver Kluth & Christine Bruer & Hns-Georg Joost & Rihrd D. DiMrhi & Mtthis H. Tshöp & Annette Shürmnn Reeived: 13 Otoer 14 /Aepted: 1 Deemer 14 /Pulished online: Deemer 14 # The Author(s) 14. This rtile is pulished with open ess t Springerlink.om Astrt Aims/hypothesis Oestrogens hve previously een shown to exert et ell protetive, gluose-lowering effets in mouse models. Therefore, the reent development of glugon-like peptide-1 (GLP-1) oestrogen onjugte, whih trgets oestrogen into ells expressing GLP-1 reeptors, offers n opportunity for ell-speifi nd enhned et ell protetion y oestrogen. The purpose of this study ws to ompre the effets of GLP-1 nd GLP-1 oestrogen during et ell filure under gluolipotoxi onditions. Methods Mle New Zelnd oese (NZO) mie were treted with dily s.. injetions of GLP-1 nd GLP-1 oestrogen, respetively. Susequently, the effets on energy homeostsis nd et ell integrity were mesured. In order to lrify the trgeting of GLP-1 oestrogen, trnsription nlyses of oestrogen-responsive genes in distint tissues s well s mirorry nlyses in pnreti islets were performed. Eletroni supplementry mteril The online version of this rtile (doi:.7/s ) ontins peer-reviewed ut unedited supplementry mteril, whih is ville to uthorised users. R. W. Shwenk (): C. Bumeier : O. Kluth : C. Bruer : H.<G. Joost: A. Shürmnn Deprtment of Experimentl Dietology, Germn Institute of Humn Nutrition Potsdm-Rehrueke, Arthur-Sheunert-Allee , 148 Nuthetl, Germny e-mil: roert.shwenk@dife.de R. W. Shwenk: C. Bumeier : B. Finn : O. Kluth : C. Bruer : H.<G. Joost: M. H. Tshöp : A. Shürmnn Germn Center for Dietes Reserh (DZD), Neuhererg, Germny B. Finn: M. H. Tshöp Institute for Dietes nd Oesity, Helmholtz Center Munih, Germn Reserh Center for Environmentl Helth (GmH) nd Tehnil University Munih, Munih, Germny R. D. DiMrhi Deprtment of Chemistry, Indin University, Bloomington, IN, USA Results In ontrst to GLP-1, GLP-1 oestrogen signifintly deresed food intke resulting in sustntil weight redution, preserved normoglyemi, inresed gluose tolerne nd enhned et ell protetion. Anlysis of hypothlmi mrna profiles reveled elevted expression of Pom nd Lepr. In livers from GLP-1 oestrogen-treted mie, expression of lipogeni genes ws ttenuted nd hepti triylglyerol levels were deresed. In pnreti islets, GLP-1 oestrogen ltered the mrna expression to pttern tht ws similr to tht of dietes-resistnt NZO femles. However, onventionl oestrogen-responsive genes were not different, inditing rther indiret protetion of pnreti et ells. Conlusions/interprettion GLP-1 oestrogen effiiently protets NZO mie ginst rohydrte-indued et ell filure y ttenution of hyperphgi. In this regrd, trgeted delivery of oestrogen to the hypothlmus y fr exeeds the norexigeni pity of GLP-1 lone. Keywords Bet ells. GLP-1. Liver ft. NZO. Oestrogen. Pom Arevitions CH Crohydrte-free high-ft diet +CH Crohydrte-ontining high-ft diet E 17β-Oestrdiol ERα Oestrogen reeptor α GLP-1 Glugon-like peptide-1 ISI Insulin sensitivity index NZO New Zelnd oese Introdution To ompenste for peripherl insulin resistne nd gluose intolerne, pnreti et ells strt to proliferte nd

2 Dietologi () 8: inrese the iosynthesis nd seretion of insulin [1]. The geneti kground nd environmentl ftors limit the pity of this ompenstion, however, nd et ells eventully fil, leding to type dietes. In order to prevent this progression, urrent reserh fouses on strtegies to protet et ells ginst the toxi miroenvironment produed y irulting rohydrtes nd lipids. Reent dt hve implited et ell protetive role of oestrogen (17β-oestrdiol, E). E hs een shown to inrese insulin iosynthesis vi tivtion of oestrogen reeptor α (ERα) [, 3] nd to protet et ells ginst toxi lipid intermedites y promoting ell prolifertion nd inhiition of lipogenesis nd poptosis [4, ]. Additionlly, systemi E- medited effets on food intke nd energy expenditure lso ontriute to et ell protetion [6]. In support of these findings, women generlly hve lower prevlene of type dietes thn ge-mthed mles, lthough this hnges fter menopuse [7]. In line with these oservtions, the EPIC-InterAt study reveled n inverse orreltion etween ge t menopuse onset with the risk of developing type dietes [8]. Despite ll these promising findings, oestrogen hs not een evluted s gluose-lowering drug due to its mitogeni effets in reprodutive tissue [9]. Similr to E, the inretin hormone GLP-1 inreses insulin iosynthesis nd survivl of et ells, lowers food intke nd inreses gluose uptke in dipose nd musle tissue []. In ontrst to more widespred tion of E, GLP-1 tion is restrited to tissues presenting the GLP-1 reeptor t its ell surfes. Reently, we showed tht hyrid moleules of E nd GLP-1 (GLP- 1 oestrogen) oost the weight lowering effets of GLP- 1 in C7BL/6 mie y trgeted delivery of oestrogen to the hypothlmus [11]. Consequently, lower doses of the steroid ould e used, nd the tumourigeni potentil of E ws msked. Although the ody weight lowering effets of GLP-1-oestrogen were ovious, the question remined whether the hyrid ompound would lso e suffiient to protet et ells under dietogeni onditions. Similr to humns, New Zelnd oese (NZO) mie develop oesity nd insulin resistne s result of hyperphgi, redued energy expenditure nd insuffiient physil tivity [1]. The progression from insulin resistne to type dietes in NZO mie is lrgely driven y dietry rohydrtes, s rohydrte-free diets fil to indue dietes in NZO mie [13]. Tking dvntge of this, we previously estlished dietry regimen of 13 weeks of rohydrte-free high-ft diet (to indue oesity nd insulin resistne) followed y rohydrte-ontining high-ft diet tht rpidly leds to hyperglyemi nd et ell destrution [14]. In this study, we used the sme model system to investigte the gluoselowering potentil of GLP-1 oestrogen under defined gluolipotoxi onditions. Methods Animls Mle NZO/HIBomDife mie (R. Kluge, Germn Institute of Humn Nutrition, Nuthetl, Germny) were housed in groups of five per ge (type II mrolon) t temperture of 1±1 C, with 1 h light drk yle (lights on t 6: hours). Animls hd free ess to food nd wter. All niml experiments were performed in ompline with the Germn niml protetion lw (TierShG). The mie were housed nd hndled in ordne with the Priniples of lortory niml re []. The niml welfre ommittees of the DIfE s well s the lol uthorities (LUGV, Brndenurg, Germny) pproved ll niml experiments. At the ge of weeks, nimls reeived rohydrte-free diet ( CH; % (wt/wt) protein nd 68% (wt/wt) ft, 9 kj/g). At the ge of 18 weeks, diets were swithed to rohydrteontining diet (+CH; % (wt/wt) protein, 8% (wt/wt) ft nd 4% (wt/wt) rohydrtes, 1 kj/g) for n dditionl 3 dys (Fig. 1).Tretmentwith9nmolGLP-1,GLP-1 oestrogen (supplied y R.D. DiMrhi, Indin University, Bloomington, IN, USA) or oestrogen per kg ody weight (dily s.. injetions) strted 4 dys efore the diet swith. Beuse of the soft texture of the diets, mie hd ess to wooden gnwing stiks in order to void exessive teeth growth. OGTT After n overnight 16 h fsting period, mie reeived mg gluose per g ody weight y orl gvge. At the indited time points (Fig. 3) lood gluose nd plsm insulin were mesured, s previously desried [16]. Immunohistohemistry of pnreti islets Pnreti tissue exised immeditely fter exsnguintion ws fixed in 4% (wt/vol.) formldehyde nd emedded in prffin ording to stndrd proedures. For o-stining of insulin nd glugon, mouse monolonl nti-insulin (lone K36AC, Sigm- Aldrih, Munih, Germny) nd polylonl rit ntiglugon (Dko, Hmurg, Germny) ntiodies were used. Alex Fluor 46-lelled nti-rit (1:) nd Alex Fluor 488-lelled nti-mouse (1:; Invitrogen, Krlsruhe, Germny) were used s seondry ntiodies. Nulei were stined with DAPI. Pnreti insulin ontent For detetion of the pnreti insulin ontent, whole pnreses were homogenised in ieold idi ethnol (.1 mol/l HCl in 7% ethnol) nd inuted for 4 h t 4 C. After entrifugtion (16, g, min) insulin ws deteted in the superntnt frtion using the Mouse High Rnge Insulin ELISA (Alpo, Slem, USA). Insulin sensitivity index Whole ody insulin sensitivity ws lulted fter the method of Mtsud nd DeFronzo [17]. Briefly, fsting lood gluose (G ) nd insulin (I ) nd the

3 66 Dietologi () 8: Fig. 1 Crohydrte-indued hyperglyemi nd GLP-1 oestrogen tretment. () Study design. Rndom lood gluose (), ody weight development (), umultive energy intke (d), energy intke per dy (e), len mss (f) nd ft mss (g) were monitored throughout the study. White irles, CH ontrol; lk irles, +CH ontrol; white squres, +CH with GLP-1; lk squres, +CH with GLP-1 oestrogen. All dt re represented s mens±sem. Differenes ompred with the +CH vehile group were lulted y two-wy ANOVA. p<.,p<.1, p<.1 vs +CH ontrol group Rndom lood gluose (mmol/l) -CH Vehile (PBS) GLP-1 or GLP-1 oestrogen (9 nmol/kg ody weight, dily s.. injetions) -4 - Durtion of +CH diet (dys) Reltive ody weight (%) Time (dys) CH ogtt 8 - Durtion of +CH diet (dys) 16 d Cumultive energy intke (kj) 4, 3,, 1, End of experiment 3 - Durtion of +CH diet (dys) e f 1 g 14 Energy intke (kj/dy) - Durtion of +CH diet (dys) Reltive len mss (%) Durtion of +CH diet (dys) Reltive ft mss (%) Durtion of +CH diet (dys) men lood gluose nd insulin during OGTT (G nd I, respetively) were reorded. Insulin sensitivity index (ISI) ws lulted (,/squre root of [G I ] [G I]). Lser miro dissetion of hypothlmi nulei nd gene expression nlyses Disseted rins were immeditely frozen on dry ie, nd RNA ws extrted s desried previously [11]. Gene expression nlyses in dipose tissue nd liver Totl RNA from viserl dipose tissue nd liver tissue of mie ws extrted, nd DNA synthesis s well s TqMn gene expression ssys were performed s desried previously [18]. Liver histology nd triylglyerol determintion Histologil stining of liver onnetive tissue ws performed using Msson Goldner stining kit (Merk Millipore, Drmstdt, Germny). For the quntittive determintion of triylglyerol ontent, livers were homogenised in mmol/l sodium dihydrogen phosphte, 1 mmol/l EDTA, nd 1% (vol./vol.) polyoxyethylene--trideyl ether, inuted for min t 37 C, nd the triylglyerols in the superntnt frtion were deteted with ommeril kit (RndoxTR-, Crumlin, UK). Islet isoltion nd trnsriptome nlysis Isoltion of pnreti islets ws performed y modified protool of Gotoh et l [19]. Totl islet RNA preprtion ws performed with the RNAqueous Miro Kit (Life Tehnologies, Drmstdt, Germny). RNA integrity ws ssessed with the RNA6 nno kit (Agilent, Snt Clr, CA, USA). Mirorry nlyses were performed y OkLs (Hennigsdorf, Germny) on Agilent Mouse 8 6 K Chip. Sttistis Sttistil differenes during tretment were determined y two-wy ANOVA nd Bonferroni posttest. Differenesinendpointmesurementsweredeterminedy one-wy ANOVA nd Newmn Keuls Multiple Comprison Tests. Contingeny of the expression nlyses ws lulted y Fisher s Ext Test. Signifine levels were set t p<., p<.1 nd p<.1. Dt re presented s mens±sem. For sttistil nlysis nd for grphil presenttion GrphPd Prism (.; GrphPd Softwre, Sn Diego, CA, USA) ws used.

4 Dietologi () 8: Results GLP-1 oestrogen prevents rohydrte-indued hyperglyemi Mle NZO mie were kept on rohydrte-free, high-ft diet ( CH) until the ge of 18 weeks (Fig. 1). Due to this dietry regimen, NZO mie eome oese nd insulin resistnt, ut re proteted from developing dietes [14]. Afterwrds, the diet ws hnged to rohydrte-ontining high-ft diet (+CH), whih indues rpid hyperglyemi nd finlly et ell destrution in synhronised mnner [14] (Figs 1, ). GLP-1-treted nimls displyed the sme inrese in lood gluose upon rohydrte feeding s the vehile-treted +CH ontrol group (Fig. 1). In ontrst, GLP-1 oestrogen-treted nimls exhiited norml lood gluose levels similr to vehiletreted nimls tht ontinued with the CH diet. Tretment with GLP-1 led to modest redution in ody weight y 6.1± 3.4% from seline (7± g), while GLP-1 oestrogen redued ody weight y 1.8±1.8% (Fig. 1). The umultive energy intke of the GLP-1 group only differed t dys 19 nd 3; nimls of the GLP-1 oestrogen group lredy onsumed signifintly fewer lories fter dys of +CH feeding (Fig. 1d). This deresed totl mount of onsumed lories -CH +CH/GLP-1 Pnreti insulin ontent (μg/g wet weight) CH +CH/GLP-1 oestrogen -CH +CH G GE Fig. Pnreti islet integrity. () Stining of pnreti slies ginst insulin (green), glugon (red) nd nulei (lue). () Pnreti insulin ontent (n=3 nimls per group). All dt re represented s mens± SEM. Differenes ompred with the CH or +CH vehile group (s indited) were lulted y Student s t test. p<. vs CH ontrol nd +CH ontrol, respetively. G, GLP-1; GE, GLP-1 oestrogen ws lrgely due to trnsient nd roust redution in energy intke during the first dys of GLP-1 oestrogen tretment (Fig. 1e). Importntly, the derese in ody weight in the GLP-1 group ws due to loss of len mss (Fig. 1f, g). In ontrst, GLP-1 oestrogen-treted nimls minly lost ft mss (Fig. 1f, g). Pnreti islets re proteted y GLP-1 oestrogen ginst rohydrte-indued destrution Histology of the pnreti islets t the end of the study reveled sustntil islet destrution in +CH nimls (Fig. ). While GLP-1 tretment led to n intermedite phenotype with ler disruption of norml islet ytorhiteture, islets from GLP-1 oestrogentreted nimls displyed norml islet morphology (Fig. ). To tht end, pnreti insulin ontent ws mrkedly redued in response to the rohydrte intervention (+CH; Fig. ). This effet ws only prtilly prevented y GLP-1 (p=.); GLP-1 oestrogen resulted in signifint (p=.34) inrese in pnreti insulin ontent (Fig. ). While GLP-1 tretment lone filed to improve orl gluose tolerne, GLP-1 oestrogen-treted nimls displyed improved gluose tolerne (Fig. 3). Strikingly, fsting gluose levels were highest in CH ontrol mie, nd insulin levels were higher, lthough not signifintly, throughout the gluose tolerne test. This effet ould reflet the mrkedly insulin resistnt hepti gluose output in NZO mie on the CH diet [13]. Although not signifint, oth GLP-1 nd GLP-1 oestrogen nimls showed lower plsm insulin vlues during OGTT (Fig. 3). Insulin sensitivity, s mesured with the Mtsud index [17], ws improved upon tretment with the hyrid ompound (Fig. 3). Oestrogen improves islet funtion ut not gluose tolerne As effets of GLP-1 oestrogenondietesprevention were very muh different from tht of GLP-1, we performed dditionl experiments with oestrogen. In oestrogentreted nimls, the rohydrte-indued rise in lood gluose ws redued ut not fully olished (Fig. 4). Furthermore, oestrogen tretment did not redue ody weight or energy intke (Fig. 4, ) nd improved neither gluose tolerne nor insulin sensitivity (Fig. 4d f). Interestingly, pnreti islets of oestrogen-treted mie displyed lrgely preserved ytorhiteture nd unltered insulin ontent (Fig. 4g, h). Redued food intke is ssoited with inresed norexigeni signlling GLP-1 oestrogen tretment resulted in trnsient ut sustntil redution in food intke during the first week of tretment (Fig. 1e). Both GLP-1 oestrogen nd oestrogen tretment inresed the hypothlmi expression of oestrogen-responsive Trim [] (.±1.3- nd 18.±1.4- fold, respetively) in omprison with vehile-treted +CH ontrol nimls, inditing tht GLP-1-ound oestrogen is trgeted to the hypothlmus (Fig. ). Although to lesser

5 68 Dietologi () 8: Blood gluose (mmol/l) 3 Plsm insulin (pmol/l) 3, 3 6, 4 1, CH +CH G GE Time (min) Time (min) Fig. 3 OGTT. () Blood gluose exursion nd () plsm insulin vlues represented s mens±sem. Differenes ompred with the +CH vehile (n= nimls per group). () ISI mesured fter Mtsud [17]. White group were lulted y two-wy ANOVA (, ) nd one-wy ANOVA irles, CH ontrol; lk irles, +CH ontrol; white squres, +CH with (), respetively. p<., p<.1 vs +CH ontrol group GLP-1 (G); lk squres, +CH with GLP-1 oestrogen (GE). All dt re Mtsud ISI 8 extent, GLP-1 treted nimls lso displyed elevted Trim expression (3.8±.9-fold). The rohydrte hllenge suppressed Pom expression, wheres GLP-1 nd oestrogen prevented the drop in Pom mrna levels (Fig. ). GLP-1 oestrogen inresed Pom expression ompred with vehiletreted nd GLP-1-treted nimls (17.8±.4- nd.9±.9- fold, respetively; Fig. ). Leptin reeptor (Lepr) expression ws inresed to similr extent y ll tretments (1.8±.1-,.±.- nd.3±.-fold, respetively; Fig. ). Expression of orexigeni Crt (lso known s Crtpt) nd Fig. 4 Oestrogen tretment during rohydrte feeding. Mesurements of rndom lood gluose (), ody weight development () nd umultive energy intke (). Blood gluose exursion (d) nd plsm insulin (e) during OGTT. (f) ISI mesured fter Mtsud [17]. (g) Stining of pnreti slies ginst insulin (green), glugon (red) nd nulei (lue). (h) Pnreti insulin ontent. White irles, CH ontrol; lk irles, +CH ontrol; lk tringles, +CH with oestrogen (E). All dt re represented s mens±sem (n=6 nimls per group). Differenes ompred with the +CH vehile group were lulted y two-wy ANOVA ( e) nd one-wy ANOVA (f, h), respetively. p<.1 vs +CH ontrol group Rndom lood gluose (mmol/l) d Blood gluose (mmol/l) g Reltive ody weight (%) Durtion of +CH diet (dys) Durtion of +CH diet (dys) Durtion of +CH diet (dys) Time (min) Plsm insulin (pmol/l) 1,, 7,,, Time (min) -CH +CH +CH/oestrogen e Cumultive energy intke (kj) h f Mtsud ISI Pnreti insulin ontent (μg/g wet weight) 4, 3,, 1, 6 4 -CH +CH E -CH +CH E

6 Dietologi () 8: Fig. Hypothlmi gene expression. () Trim,() Pom, () Lepr, (d) Crt, (e) Npy nd (f) Agrp (n=7 11 nimls per group). All dt re represented s mens±sem. Differenes ompred with the +CH vehile group were lulted y onewy ANOVA. p<., p<.1, p<.1 vs +CH ontrol group. G, GLP-1; GE, GLP-1 oestrogen; E, oestrogen Trim mrna levels ( -ΔCt ) Pom mrna levels ( -ΔCt ) 3 3 Lepr mrna levels ( -ΔCt ) 3 1 d. e. f 8 Crt mrna levels ( -ΔCt ) Npy mrna levels ( -ΔCt ) Agrp mrna levels ( -ΔCt ) 6 4 Npy ws not different (Fig. d, e). However, expression of orexigeni Agrp ws elevted with GLP-1 nd GLP-1 oestrogen (4.4±.9- nd 3.±.6-fold, respetively; Fig. e). Attenution of lipogeni pthwys in GLP-1 oestrogen-treted mie Histologil nlyses reveled lrge vuoles, presumly lipid droplets, in livers of ontrol, GLP-1- nd oestrogentreted mie (Fig. 6). In ontrst, livers of mie tht reeived GLP-1 oestrogen showed sustntilly improved liver morphology. Aordingly, hepti triylglyerol umultion ws ttenuted in GLP-1 oestrogen-treted mie, ut lso oestrogen-treted mie reveled lower triylglyerol levels (Fig. 6). Crohydrte feeding sustntilly inresed hepti expression of lipogeni A, Fsn nd Sd1 (Fig. 6 e). GLP-1 oestrogen redued expression of A nd Fsn y 39±7.8 nd ±9.3%, respetively. Neither of the tretments ltered expression of Sd1 (Fig. 6). Importntly, hepti expression of Trim ws not different mong the tretment groups (eletroni supplementry mteril [ESM] Fig. 1). Beuse loss of ft mss ws mjor prt of the GLP-1 oestrogen phenotype (Fig. 1g), we lso investigted the expression of the ove mentioned genes in viserl dipose tissue. Although the dipose expression pttern ws similr to the hepti one, no signifint differenes mong the groups were oserved (Fig. 6f h). Also, in dipose tissue, expression of Trim ws not different (ESM Fig. 1), demonstrting tht liver nd dipose tissue re not diret trget site of tion of the GLP-1 oestrogen hyrid. GLP-1 nd GLP-1 oestrogen tretment ffets the trnsriptome of pnreti islets In order to investigte whether dietes protetion ould e due to diret effets of GLP-1 oestrogen on pnreti islets, we studied the islet trnsriptome dys fter diet swith (6 dys fter tretment initition). At this time point, the rise in lood gluose nd suppression of Akt signlling is oserved in untreted NZO mie [14]. In the islets of GLP-1-treted nimls, 1 mrnas were differentilly expressed ompred with the islets of the + CH group (signl intensity>, log fold hnge> 1. nd p<.; ESM Tle 1). Similrly, in islets of GLP-1 oestrogen mie, 43 mrnas were differentilly expressed (ESM Tle ). Of these mrnas, 1 were upregulted (Tle 1) nd 13 were downregulted (Tle ) y GLP-1 oestrogen tretment only, inditing effets speifi for the hyrid ompound. Oestrogen-speifi effets on pnreti islets ould lso e the reson for lower dietes prevlene in femle NZO mie [16], whih do not disply hyperglyemi upon diet swith (Fig. 7). In order to nrrow down oestrogen-speifi effets of the hyrid ompound, we performed trnsriptome nlyses ompring pnreti islets from mle vs femle NZO mie dys fter diet swith. In islets of NZO femles, 73 genes were differentilly expressed ompred with tht of mle mie (signl intensity>, log fold hnge> 1. nd p<.; ESM Tle 3). Twelve (9%) of the genes ltered y GLP-1 oestrogen were lso differentilly expressed in femle NZO mie. Contingeny nlyses exlude n overlp y hne (Fisher s Ext Test, OR 6.49, p=. 16 ). However, there ws lso n overlp of 19 genes (16%) etween those genes tht were ltered with GLP-1 tretment nd those tht differed etween femles nd mles (OR 4.14, p=. 16 ). Three genes (Gt, Pst1 nd Up1l1) were enrihed in islets of GLP-1 oestrogen-treted mle mie nd

7 6 Dietologi () 8: Fig. 6 Effets on liver nd viserl dipose tissue. () Msson Goldner stining of liver setions with ytoskeletl elements nd ytoplsm (reddish), nulei (drk lue) nd firoti res (green to lue). () Hepti triylglyerol ontent. Gene expression of A, Fsn nd Sd1 in liver ( e) nd dipose tissue (f h) (n=3 6 nimls per group). All dt re represented s mens±sem. Differenes ompred with the +CH vehile group were lulted y one-wy ANOVA. p<.,p<.1, p<.1 vs CH ontrol nd +CH ontrol, respetively. G, GLP-1; GE, GLP-1 oestrogen; E, oestrogen A mrna levels ( -ΔCt ) -CH +CH/GLP-1 oestrogen CH +CH/oestrogen d Fsn mrna levels ( -ΔCt ) CH/GLP-1 Liver triylglyerols (mg/mgprotein) 3 1 e Sd1 mrna levels ( -ΔCt ) f A mrna levels ( -ΔCt ) g Fsn mrna levels ( -ΔCt ) h Sd1 mrna levels ( -ΔCt ) 3 femle mie (Fig. 7). Expression of Aqp4, Gprin3, Shd nd Txnip ws redued in islets of femles, s well s in islets of mle mie treted with oth GLP-1 nd GLP-1 oestrogen (Fig. 7). Expression of I11Rik, Arrd4, Gsto, Srf nd Sl ws redued only in islets from femles nd GLP-1 oestrogen-treted mle mie (Fig. 7). Disussion In this study, we evluted the potentil of oestrogen-oupled GLP-1 to protet et ell funtion under gluolipotoxi onditions in dietes-prone mle NZO mie. We show tht GLP- 1 oestrogen fully prevented the onset of hyperglyemi nd redued ody weight due to sustntilly deresed food intke, inditing the hypothlmus to e the min site of GLP-1 oestrogen tion. Susequently, GLP-1 oestrogen proteted the mie ginst rohydrte-indued et ell filure, inresed gluose tolerne nd insulin sensitivity nd ffeted the islet trnsriptome. Thus, ompred with GLP-1, low-dose GLP-1 oestrogen reveled superior effiy to preserve et ell integrity nd funtion under dietogeni onditions. The findings of the present study indite tht the omintion of GLP-1 nd oestrogen in hyrid moleule possesses gluose-lowering potentil tht exeeds the potentil of either one of the single moleules. Reently we showed tht oestrogen hs et ell protetive effets in femle NZO mie, s ovrietomised nimls displyed elevted lood gluose levels nd eventully n inresed prevlene of type dietes ompred with shm-operted ontrol mie [16]. Furthermore, tretment of oestrogen-defiient mie with oestrogen resulted in inresed protetion of pnreti et

8 Dietologi () 8: Tle 1 Genes seletively upregulted y GLP-1 oestrogen tretment Gene symol Gene nme Trget nme Log rtio p vlue n.d. n.d. ENSMUST Ddn Dendrin NM_ Up1l1 UDP-N-teylgluosmine pyrophosphorylse 1-like 1 NM_ Vmnr3 Vomeronsl, reeptor 3 NM_ Cd4 C lium-dependent domin ontining 4A NM_ Pst1 Phosphoserine minotrnsferse 1 NM_ Le1k Lte ornified envelope 1K NM_ Hemt1 Hemtopoieti ell trnsript 1 NM_ Olfr48 Olftory reeptor 48 NM_ Dpp1 Dul dptor for phosphotyrosine nd 3-phosphoinositides 1 NM_ Ar Androgen reeptor NM_ Pvl Prvlumin NM_ Gt Glyine C-etyltrnsferse (-mino-3-ketoutyrte-oenzyme A ligse) NM_ Chst11 Crohydrte sulfotrnsferse 11 NM_ Ankrd Ankyrin repet domin NM_ Olfr1 Olftory reeptor 1 NM_ Zfp846 Zin finger protein 846 NM_ Esyt3 Extended synptotgmin-like protein 3 NM_ Arl9 ADP-riosyltion ftor-like 9 NM_ Pin Pregnny indued nonoding RNA NR_ Gm1113 Predited gene 1113 NR_ n.d., not determined ells ginst streptozotoin-indued et ell poptosis nd the ollpse of insulin prodution []. In the present study, trnsriptome nlyses did not indite diret et ell protetion y GLP-1 oestrogen t the given dose, s neither Trim nor A, Fsn nd Sd1 were differentilly expressed in pnreti islets of GLP-1 oestrogen-treted mie. The lter three lipogeni genes mentioned hve previously een shown to e repressed in pnreti islets of Zuker dieti ftty rts upon oestrogen tretment [4]. Still, our trnsriptome nlyses indited severl ltertions of the pnreti expression pttern tht ould e protetive, even s seondry effets of hypothlmi GLP-1 oestrogen tion. Prtiulrly, the α- rrestin Txnip is well known s key plyer in pnreti et ell iology, s it is inresed in dieti islets nd indues et ell poptosis [1, ]. The GLP-1 oestrogen-medited suppression of Txnip ould e medited vi GLP-1 s ws Tle Genes seletively downregulted y GLP-1 oestrogen tretment Gene symol Gene nme Trget nme Log rtio p vlue Trpm1 Trnsient reeptor potentil tion hnnel, sufmily M, memer 1 NM_ Arrd4 Arrestin domin ontining 4 NM_ Fxo34 F-ox protein 34 NM_ Cd138 Coiled-oil domin ontining 138 NM_ Sl Solute rrier fmily (orgni tion trnsporter), memer NM_ Rmp1 Reeptor (litonin) tivity modifying protein 1 NM_ Gsto Glutthione S-trnsferse omeg NM_ Histh4 Histone luster, H4 NM_ Trim7 Triprtite motif-ontining 7 NM_ G13Rik RIKEN DNA G13 gene NM_ Olfr Olftory reeptor NM_ Srf Svenger reeptor lss F, memer NM_ Cox6 Cytohrome oxidse suunit VI polypeptide NM_

9 61 Dietologi () 8: Fig. 7 Gene expression in pnreti islets dys fter swith to +CH. () Blood gluose exursion in mle vs femle NZO mie upon swith to +CH diet. Genes eing upregulted () nd downregulted (), respetively, in NZO femles nd GLP-1 oestrogen-treted mles (n= 3 nimls per group). Blk irles, +CH mles; white dimonds, +CH femles. White rs, mles; light grey rs, femles; drk grey rs, mles with GLP-1; lk rs, mles with GLP-1 oestrogen. Differenes in lood gluose were lulted y twowy ANOVA. Expression differenes were lulted y Student s t test. p<., p<.1, p<.1 vs ontrol Rndom lood gluose (mmol/l) Expression reltive to mle ontrol (fold) Durtion of +CH diet (dys) I11Rik Aqp4 Expression reltive to mle ontrol (fold) Gt Pst1 Up1l1 Arrd4 Gprin3 Gsto Srf Shd Sl Txnip shown for exentide [3]. Additionlly, oestrogen-medited repression of Txnip ws demonstrted in vitro nd in vivo [4]. Still, inhiition of Txnip lone ws not suffiient to prevent et ell filure, s seen in GLP-1-treted nimls. Interestingly, seond α-rrestin, Arrd4, is suppressed in islets fter GLP-1 oestrogen tretment. This effet ppers to e medited y oestrogen, euse lso femles, ut not GLP-1-treted mles, exhiited this lower expression. Whether Arrd4 hs similr dverse effets s Txnip in et ells is not known; however, our dt suggest tht inhiition of oth genes in GLP-1 oestrogen-treted mie prtiiptes in et ell protetion. GLP-1-ound oestrogen stimulted norexigeni signlling tht ws fr more effetive thn GLP-1 lone. The hyrid ompound rehes the rin, s GLP-1 oestrogen tretment inresed hypothlmi expression of oestrogen-responsive Trim [] to similr extent s oestrogen lone. Nevertheless, indution of Pom expression ws lerly highest in GLP-1 oestrogen-treted nimls, inditing tht oestrogen only ffets ppetite in NZO mie when omined with GLP-1. Brin-trgeted oestrogen not only ffets Pom expression ut lso hs een shown to inrese the firing rte of Pom-expressing neurons, resulting in sustntil redution in food intke, nd susequently ody weight []. Therefore, our dt re in line with these pulished oservtions nd suggest tht redued lori intke vi pro-opiomelnoortin (POMC) tivtion is the mjor mehnism leding to improved glyemi in GLP-1 oestrogen-treted NZO mie. Indeed, severl studies hve proven tht lori restrition is suffiient to improve gluose tolerne nd insulin sensitivity in humns nd niml models [6, 7]. Espeilly, liver ft dereses within dys upon lori restrition nd ontriutes to improved gluose homeostsis [8]. In ontrst to Pom, expression of norexigeni Lepr ws elevted in GLP-1-, GLP-1 oestrogen- nd oestrogen-treted mie. In previous studies, NZO mie hve een shown to e severely leptin resistnt nd this leptin resistne might e due to the presene of severl polymorphisms in the Lepr gene [9, 3]. Therefore, the sene of ny norexigeni signlling in GLP-1- or oestrogen-treted NZO mie ould e illustrtive of n impired leptin signlling in NZO mie, despite inresed Lepr expression. GLP-1 oestrogen tretment did not lter Trim expression in liver or viserl dipose tissue. This is opposite to the findings in the hypothlmus nd similr to tht in pnreti islets, nd ould e explined y the lk of GLP-1 reeptor expression in these tissues [31, 3]. Interestingly, rohydrte feeding lso suppressed Trim expression in the hypothlmus, ut not in the periphery. The mehnism ehind this oservtion, s well s the tissue-speifi ounter regultion y oestrogen, is not known nd dt on metoli funtion of Trim re missing. Still, oth liver nd dipose tissue displyed sustntil redution in their lipid ontent, inditing seondry effet through entrl tions of GLP-1 oestrogen tht ultimtely influenes whole ody metolism. Furthermore, we oserved n inhiition of lipogeni genes in the liver. This is in line with previous studies showing tht oestrogen tretment of ovrietomised mie nd high-ft diet (HFD)-fed mie resulted in inhiition of lipogeni gene expression in liver nd dipose tissue [33, 34]. These pulished dt lso indited enhned lipolyti response nd β-oxidtion, whih ws not ssessed in our study nd would need

10 Dietologi () 8: further investigtion to lrify. However, oth studies ssumed diret effets of oestrogen on oth tissues. The dt in the present study suggest tht mjor prt of the oestrogen tion is vi the entrl nervous system, s diret tion of GLP-1 oestrogen on liver nd dipose tissue re more likely to e exluded. This onept is supported y the finding tht speifi deletion of hypothlmi ERα is suffiient to redue whole ody energy expenditure nd indue hyperphgi in femle mie, resulting in oesity nd impired gluose tolerne [3, 36]. In summry, oestrogen-oupled GLP-1 displys superior effiy in preventing the onset of diet-indued dietes thn GLP-1 lone. In NZO mie, this protetive effet is due to entrl ttenution of hyperphgi, resulting in systemi improvement of gluose tolerne nd insulin sensitivity. Therefore, hyrid ompounds like GLP-1 oestrogen might e the sis for novel therpeuti options for treting type dietes mellitus more effiiently. Aknowledgements The uthors thnk C. Gumz, A. Teihmnn nd K. Wrnke of the Germn Institute of Humn Nutrition Potsdm- Rehrueke for their skilful tehnil ssistne. Furthermore, we thnk S. Morin of the Institute for Dietes nd Oesity t the Helmholtz Center (Munih, Germny) for editing the mnusript, nd M. Jähnert nd G. Shulze of the Germn Institute of Humn Nutrition Potsdm- Rehrueke for dt nlysis. Funding This work ws supported y the Germn Ministry of Edution nd Reserh (BMBF, DZD, grnt 1GI9) nd the Germn Reserh Foundtion (GK18). Dulity of interest RDD ws ofounder of Mrdi Bioteh. All other uthors delre tht there is no dulity of interest ssoited with their ontriution to this mnusript. Contriution sttement RWS ws responsile for study oneption nd design, performed dt quisition nd nlysis, nd drfted the rtile. CB, BF, OK nd CBr performed dt quisition, dt nlysis nd ontriuted to the writing of the mnusript. HGJ dvised on the study onept nd ritilly revised the mnusript. RDD, MHT nd AS mde sustntil ontriutions to the study oneption nd ritilly revised the mnusript. RWS is the gurntor of this work nd, s suh, hd full ess to ll the dt in the study nd tkes responsiility for the integrity of the dt nd the ury of the dt nlysis. All listed uthors pproved the finl version of the mnusript. Open Aess This rtile is distriuted under the terms of the Cretive Commons Attriution Liense whih permits ny use, distriution, nd reprodution in ny medium, provided the originl uthor(s) nd the soure re redited. Referenes 1. Thorens B (13) The required et ell reserh for improving tretment of type dietes. J Intern Med 74:3 14. Wong WP, Tino JP, Liu S et l () Extrnuler estrogen reeptor-lph stimultes NeuroD1 inding to the insulin promoter nd fvors insulin synthesis. Pro Ntl Ad Si U S A 7: Alonso-Mgdlen P, Ropero AB, Crrer MP et l (8) Pnreti insulin ontent regultion y the estrogen reeptor ER lph. PLoS One 3:e69 4. 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