ARTICLE. Bo Liu & Zoheb Hassan & Stefan Amisten & Aileen J. King & James E. Bowe & Guo Cai Huang & Peter M. Jones & Shanta J.

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1 Dietologi (213) 56: DOI 1.17/s x ARTICLE The novel chemokine receptor, G-protein-coupled receptor 75, is expressed y islets nd is coupled to stimultion of insulin secretion nd improved glucose homeostsis Bo Liu & Zohe Hssn & Stefn Amisten & Aileen J. King & Jmes E. Bowe & Guo Ci Hung & Peter M. Jones & Shnt J. Persud Received: 1 April 213 /Accepted: 19 July 213 /Pulished online: 27 August 213 # Springer-Verlg Berlin Heidelerg 213 Astrct Aims/hypothesis Chemokine (C-C motif) lignd 5 () cts t C-C chemokine receptors (CCRs) to promote immune cell recruitment to sites of inflmmtion, ut is lso n gonist t G-protein-coupled receptor 75 (GPR75), which hs very limited homology with CCRs. GPR75 is coupled to Gq to elevte intrcellulr clcium, so we investigted whether islets express this receptor nd whether its ctivtion y increses et cell clcium levels nd insulin secretion. Methods Islet receptor mrna expression ws mesured y quntittive RT-PCR nd GPR75 ws detected in islets y western lotting nd immunohistochemistry. In some experiments GPR75 ws downregulted y trnsient trnsfection with smll interfering RNA. Rel-time chnges in intrcellulr clcium were determined y single-cell microfluorimetry. Dynmic insulin secretion from perifused islets ws quntified y rdioimmunossy. Glucose homeostsis in len nd oese mice ws determined y mesuring glucose nd insulin tolernce, nd insulin secretion in vivo. Results Mouse nd humn islets express GPR75 nd its lignd. Exogenous reversily incresed intrcellulr clcium in et cells vi GPR75, this phenomenon eing dependent on phospholipse C ctivtion nd clcium influx. lso stimulted insulin secretion from mouse nd humn islets in vitro, nd improved glucose tolernce in len mice nd in mouse model of hyperglycemi nd insulin resistnce (o/o). The improvement in glucose tolernce ws ssocited with enhnced insulin secretion in vivo, without chnges in insulin sensitivity. Conclusions/interprettion Although is implicted in the pthogenesis of dietes through ctivtion of CCRs, it hs eneficil effects on et cells through GPR75 ctivtion. Keywords Bet cell. Chemokine (C-C motif) lignd 5. GPR75. Insulin secretion. Intrcellulr clcium. Type 2 dietes Arevitions [C 2+ ] i Intrcellulr clcium Chemokine (C-C motif) lignd 5 CCR C-C chemokine receptor DPP4 Dipeptidyl peptidse 4 GPCR G-protein-coupled receptor GPR75 G-protein-coupled receptor 75 IP 3 Inositol 1,4,5-trisphosphte PLC Phospholipse C PTX Bordell pertussis toxin RANTES Regulted upon ctivtion, norml T cell expressed nd presumly secreted sirna Smll interfering RNA Introduction Bo Liu nd Zohe Hssn contriuted eqully to this study. B. Liu: Z. Hssn : S. Amisten : A. J. King : J. E. Bowe : G. C. Hung: P. M. Jones : S. J. Persud () Dietes Reserch Group, Division of Dietes & Nutritionl Sciences, School of Medicine, 2.9N Hodgkin Building, King s College London, Guy s Cmpus, London SE1 1UL, UK e-mil: shnt.persud@kcl.c.uk Chemokine (C-C motif) lignd 5 () is 68 mino cid protein, which ws formerly clled Regulted upon ctivtion, norml T cell expressed nd presumly secreted (RANTES) nd elongs to fmily of chemotctic cytokines tht ply key roles in lymphocyte trfficking during inflmmtion [1 3]. expression ws initilly thought to e T cell specific,

2 2468 Dietologi (213) 56: ut is now known to e produced y vriety of cells, including pltelets, endothelil cells, smooth muscle cells nd neurons [4], where it is thought to e involved in rnge of iologicl functions. promotes the recruitment of lymphocytes to sites of inflmmtion nd infection through promiscuous inding to the C-C chemokine receptor (CCR)1, CCR3 nd CCR5 memers of the C-C motif chemokine fmily of G-protein-coupled receptors (GPCRs) [5]. While trfficking of immune cells is normlly eneficil in reducing inflmmtion, in type 1 dietes cn promote the utoimmune destruction of et cells through lymphocyte recruitment nd the secretion of inflmmtory cytokines y ctivtion of lymphocyte CCRs. This scenrio is supported y the demonstrtion tht expression increses with the progression of dietes in islets of NOD mice, model of spontneous utoimmune dietes [6]. In ddition, is lso present in islets isolted from individuls with type 1 dietes, where it my ply role in ttrcting ctivted T cells. It is lso upregulted y cytokines [7, 8] nd the coxsckie B3 enterovirus [9] in islets from non-dietic donors. It hs recently ecome pprent tht is lso lignd t G-protein-coupled receptor 75 (GPR75), novel GPCR tht shres only 12 to 16% sequence homology with the chemokine receptor fmily memers [1]. GPR75 is 54 mino cid protein with seven trnsmemrne domins, typicl of previously chrcterised GPCRs [11], ut its C-terminl til is considerly longer thn tht of other chemokine receptors [12]; moreover, its expression hs een reported to e limited to the rin, spinl cord nd retinl pigment epithelium, rther thn the trditionl sites of chemokine receptors such s the thymus nd spleen [11]. In ddition, GPR75 lcks the sprtte rginine tyrosine motif of the trnsmemrne helix, which is thought to e importnt for CCR structure nd function [12]. Furthermore, while conventionl CCRs re thought to signl predominntly through the Bordetell pertussis toxin (PTX)-sensitive Gi fmily of G proteins tht inhiit camp production [13 16], ctivtion of GPR75 trnsfected into Chinese hmster ovry cells led to phospholipse C (PLC)-medited elevtions of inositol 1,4,5-trisphosphte (IP 3 ) formtion nd intrcellulr clcium ([C 2+ ] i ), oth of which were inhiited y the PLC inhiitor U73122, ut unffected y PTX [1]. Consistent with this, stimulted IP 3 formtion in GPR75-trnsfected humn emryonic kidney cells [1], confirming the Gq-dependent coupling of GPR75 ctivtion. Tretment of GPR75-overexpressing murine hippocmpl cells with hs lso een shown to increse cell viility, most likely vi PLC nd phosphtidylinositol 3-kinse ctivtion [1]. The exocytotic relese of insulin is potentited y incretins, ctivtion of the prsympthetic nervous system nd y numerous other secretgogues tht control the secretory process y ctivting islet GPCRs. This stimultory GPCR input occurs through coupling vi Gs to ctivte denylte cyclse nd elevte intrcellulr camp, or vi Gq to stimulte PLCmedited IP 3 nd dicylglycerol ccumultion. GPR75 ctivtion is coupled to elevtions of [C 2+ ] i, key messenger in et cell stimulus response coupling, so if this novel receptor is expressed y et cells its ctivtion my led to incresed insulin relese nd improved glucose homeostsis. This study therefore exmined the expression of nd GPR75 in islets of Lngerhns nd investigted the effects of nd GPR75 ctivtion upon [C 2+ ] i nd insulin secretion from mouse nd humn islets in vitro. The in vivo effects of on glucose nd insulin tolernce were lso investigted. Methods Mterils Culture medi nd supplements were purchsed from Sigm-Aldrich (Poole, UK) nd Invitrogen (Pisley, UK). Collgense XI, Fur-2 AM, U73122 nd ntiglucgon ntiody were lso provided y Sigm-Aldrich. ECL western lotting detection regents nd Rinow moleculr weight mrkers were from GE Helthcre (Little Chlfont, UK). RNesy mini kits nd RNse-free DNse sets were otined from Qigen (Mnchester, UK) s were QuntiTect primers for quntittive PCR (humn CCR1: QT4774; CCR3: QT ; CCR5: QT133661; GPR75: QT2189; GAPDH: QT ; mouse Ccr1: QT15658; Ccr3: QT262822; Ccr5: QT114569; Gpr75: QT253157; Gpdh: QT ). Primers for stndrd PCR were from Operon Biotech (Cologne, Germny). Antiodies ginst somtosttin nd GPR75 (13666) were purchsed from Acm (Cmridge, UK). The nti-insulin ntiody ws from Dko (Ely, UK), AlexFluor-conjugted secondry ntiodies were from Jckson ImmunoReserch (Suffolk, UK), non-interfering RNAs nd smll interfering RNAs (sirnas) ginst GPR75 were from Thermo Scientific (Loughorough, UK). The nti- ntiody (AF478) nd recominnt mouse nd humn were from R&D Systems (Aingdon, UK). Accu-Chek lood glucose meters nd strips were from Roche Dignostics (Burgess Hill, UK). Ultr-sensitive mouse insulin ELISA kits were purchsed from Mercodi (Uppsl, Sweden). ICR mice nd o/o mice were purchsed from Hrln (Bicester, UK). MIN6 et cells were kindly provided y Junichi I. Miyzki (University of Osk, Osk, Jpn). Isoltion of mouse nd humn islets Mouse islets were isolted from mle ICR mice y collgense digestion of the exocrine pncres [17]. Humn islets were isolted from nondietic donors t the King s College Hospitl Islet Trnsplnttion Unit (Kings College Hospitl, London, UK) with pproprite ethics pprovl [18]. Four seprte humn islet preprtions were used, with donor ge rnge of 22 to 6 yers nd BMI of 2 to 37 kg/m 2. Islets were mintined

3 Dietologi (213) 56: t 37 C in RPMI (mouse) or CMRL (humn) medi supplemented with fetl clf serum (1%, vol./vol.), glutmine (2 mmol/l) nd penicillin-streptomycin (1 U/ml,.1 mg/ml). RT-PCR RNAs extrcted from MIN6 et cells, nd from mouse nd humn islets were reverse-trnscried into cdnas s descried previously [19]. cdnas were then mplified y stndrd PCR using primers specific for mouse Ccl5 (sense 5 -CCCTCACCATCATCCTCACT-3, ntisense 5 -CCTTC GAGTGACAAACACGA-3, spnning exons 1, 2 nd 3) nd humn (sense 5 -CGCTGTCATCCTCAT TGCTA-3, ntisense5 -GAGCACTTGCCACTGGTGTA-3, spnning exons 1 nd 2). For some nlyses, quntittive PCR ws performed using QuntiTect primers specific for mouse nd humn GPR75, CCR1, CCR3 nd CCR5. Reltive expression of mrnas ws determined fter normlistion ginst GAPDH (Gpdh) s n internl reference nd clculted y the 2 ΔΔC t method. Immunohistochemistry The ntiody-inding epitopes of nd GPR75 were retrieved y microwve tretment of mouse nd humn pncres sections for 1 min in oiling 1 mmol/l citric cid (ph 6). Sections were immunostined overnight t 4 C with ntiodies directed ginst, GPR75, insulin, glucgon or somtosttin (t 1:8, 1:1, 1:5, 1:5 nd 1:25 dilution, respectively). Immunorective nd GPR75 were detected with AlexFluor488-conjugted secondry ntiodies (1:1 dilution). Islet hormones were detected with AlexFluor594-conjugted secondry ntiodies, ll t 1:25 dilution. Some negtive control sections were lso proed with AlexFluor secondry ntiodies lone. Immunostined pncres sections were visulised under microscope (TE2; Nikon, Kngw, Jpn). Trnsient trnsfection GPR75 ws downregulted in MIN6 et cells y trnsient trnsfection with sirnas (15 nmol/l) directed ginst mouse GPR75 using previously descried protocols for efficient gene knockdown [2, 21]. MIN6 et cells trnsfected with non-interfering RNAs (15 nmol/l) were used s controls. The trnsfected cells were mintined in culture for 48 h efore eing collected for Gpr75 mrna quntifiction nd ssessment of GPR75 protein y western lotting using rit nti-gpr75 ntiody (1:5 dilution), with equl loding confirmed y immunoproing with n nti-β-ctin ntiody (1:2 dilution). Chnges in [C 2+ ] i in trnsfected cells were determined y clcium microfluorimetry. Single cell clcium microfluorimetry Ntive MIN6 et cells or MIN6 et cells tht hd een trnsiently trnsfected with non-interfering RNAs or Gpr75 sirnas were seeded on to cid-ethnol-wshed glss coverslips efore eing incuted for 3 min t 37 C with the clcium fluorophore Fur-2 AM (5 μmol/l). Cells were then perifused (1 ml/min) with physiologicl slt solution contining 2 mmol/l glucose [22] in the sence or presence of recominnt mouse (25 fmol/l to 25 nmol/l). Some of these experiments were performed in the presence of the PLC inhiitor U73122 (1 μmol/l) or in the sence of extrcellulr clcium (uffers supplemented with 1 mmol/l EGTA). Rel-time chnges in [C 2+ ] i were determined y illuminting cells lterntely t 34 nm nd 38 nm, with the emitted light eing filtered t 51 nm. Dynmic insulin secretion The effects of on insulin secretion from mouse nd humn islets were ssessed using temperture-controlled perifusion system [23, 24]. Briefly, isolted mouse or humn islets were trnsferred to chmers contining 1 μm pore-size nylon filters nd perifused t 37 C nd flow rte of.5 ml/min with physiologicl slt solution [22] contining 2 mmol/l or 2 mmol/l glucose in the sence or presence of recominnt mouse or humn. Perifuste smples were collected t 2 min intervls for the durtion of the experiments nd the secreted insulin mesured y rdioimmunossy [25]. Intrperitonel glucose nd insulin tolernce tests Glucose tolernce tests were performed on overnight fsted len mice or insulin-resistnt o/o mice following single i.p. dministrtion of glucose (2 g/kg ody weight) in the sence or presence of 65 pmol recominnt mouse. Til vein lood glucose concentrtions were determined with glucose meter. Plsm insulin following dministrtion in vivo ws quntified using n ultr-sensitive ELISA. Insulin tolernce tests were crried out following i.p. dministrtion of insulin (.75 U/kg ody weight). All niml procedures were pproved y King s College London ethics Committee nd crried out under licence, in ccordnce with the UK Home Office Animls (Scientific Procedures) Act Sttisticl nlyses Numericl dt re expressed s mens ± SEM of multiple experiments. All sttisticl comprisons were mde y t tests or ANOVA, s pproprite. Vlues of p <.5 were considered sttisticlly significnt. Results Expression nd loclistion of in islets RT-PCR mplifiction using primers to detect mrna encoding the chemokine produced product of the pproprite size (185 p) from mouse islet cdna, ut not from MIN6 et cell cdna. As expected, no products were otined when the

4 247 Dietologi (213) 56: cdna templtes were replced y the pproprite RNAs (Fig. 1). Figure 1 shows tht primers for humn generted single product of 15 p from isolted humn islet cdna nd from cdna derived from et cell-enriched humn islet preprtion [26], in which more thn 97% of the cells express insulin nd only 1 to 2% express glucgon nd somtosttin. As with the mplifictions using mouse primers, no products were otined with the humn PCR primers when cdnas were replced y RNAs (Fig. 1). Sequencing of mplicons confirmed 1% homology etween predicted sequences nd those of the products mplified using mouse nd humn primers. The sence of Ccl5 mrna expression in MIN6 et cells suggests non-et cell loclistion of in mouse islets, which ws confirmed y immunostining of mouse pncres sections with got nti- ntiody. Immunostining in three individul mouse islets (Fig. 1c) shows tht immunorectivity ws confined to islet peripherl cells, suggesting non-et cell expression, s mouse et cells re clustered t the islet core, with lph cells nd delt cells loclised towrds the islet periphery. Doule stining of those mouse islets with ntiodies ginst islet hormones reveled tht co-loclised with glucgon, ut not with insulin or somtosttin. In humn islets the expression profile of ws wider, with doule stining of humn pncres sections with the ntiody nd ntiodies ginst insulin, glucgon nd somtosttin indicting tht some humn lph nd et, ut not delt cells express (Fig. 1d). Expression nd loclistion of GPR75 in islets ctivtesconventionlccrsswellsgpr75,soquntittive RT-PCR ws performed (1) to determine which receptor mrnas re expressed y mouse nd humn islets nd (2) to quntify their reltive undnce. Gpr75 ws the most undnt receptor mrna in mouse (Fig. 2) nd humn (Fig. 2) islets, with CCR1, CCR3 nd CCR5 mrnas eing expressed t significntly lower levels or eing undetectle. Gpr75 mrna ws lso present in MIN6 et cells, ut Ccr1, Ccr3 nd Ccr5 mrnas were not detectle. Prllel mplifictions using cdnas prepred from mouse cinr cells reveled tht the only receptor mrna identified ws tht encoding CCR3, leit t levels too low to quntify. No products were otined in ny of the rections when RNA rther thn cdna ws used. Immunohistochemicl nlysis of wx-emedded pncres sections ws conducted to exmine GPR75 cellulr loclistion within islets. GPR75 immunorective cells were dispersed throughout mouse (Fig. 2c) nd humn (Fig. 2d) islets. Immunostining of consecutive pncres sections with insulin, glucgon nd somtosttin ntiodies indicted tht GPR75 ws present in lph nd et cells, ut the lck of costining with the somtosttin ntiody suggests tht delt cells do not express this receptor. Some immunorective cells were lso oserved in the exocrine pncres, ut s immunopositive cinr cells were lso present in the sence of primry ntiody (dt not shown), this proly reflects non-specific inding of the secondry ntiody. mccl5 Mouse islet cdna MIN6 cell cdna Mouse islet RNA MIN6 cell RNA H 2 O h Humn islet cdna Humn et cell cdna Humn islet RNA Humn et cell RNA H 2 O c d Insulin Merged Insulin Merged Glucgon Merged Glucgon Merged Somtosttin Merged Somtosttin Merged Fig. 1 Expression nd loclistion of in mouse nd humn pncres. Products of RT-PCR mplifiction using mouse (m) Ccl5 () nd humn (h) () primers. Wx-emedded sections of mouse (c) nd humn (d) pncres showing expression of (green), nd insulin, glucgon nd somtosttin (red), with co-loclistion shown in yellow. Ech imge is representtive of four to seven replictes

5 Dietologi (213) 56: Fig. 2 Expression of receptors in mouse nd humn islets. Quntifiction of receptor mrna in mouse ()nd humn () islets, expressed s percentge of GAPDH mrna levels in the sme smples; vlues re mens + SEM; n =3, p <.1 vs GPR75 in the corresponding species. Wxemedded sections of mouse (c) nd humn (d) pncres showing expression of GPR75 (green), nd insulin, glucgon nd somtosttin (red), with coloclistion shown in yellow. Ech imge is representtive of five to seven replictes mrna expression (% Gpdh) Gpr75 Ccr1 Ccr3 Ccr5 c d GPR75 Insulin Merged GPR75 Insulin Merged mrna expression (% GAPDH) Gpr75 Ccr1 Ccr3 Ccr5 GPR75 Glucgon Merged GPR75 Glucgon Merged GPR75 Somtosttin Merged GPR75 Somtosttin Merged increses [C 2+ ] i in et cells vi ctivtion of GPR75 Hving estlished tht GPR75 is the mjor receptor expressed y islets, we investigted the role of in regulting et cell function. stimultes [C 2+ ] i moilistion in emryonic dorsl root gngli cells [27], while kidney cells trnsfected with GPR75 plsmid show Gqdependent elevtions of [C 2+ ] i in response to [1]. We therefore exmined the ility of to stimulte increses in [C 2+ ] i in Fur-2-loded MIN6 et cells. As seen in Fig. 3, in the presence of 2 mmol/l glucose, mouse recominnt (.25, 2.5 nd 25 nmol/l) produced rpid nd reversile increses in [C 2+ ] i tht were not concentrtiondependent. The et cells then responded to the K ATP chnnel locker, tolutmide (1 μmol/l) y producing reversile increse in [C 2+ ] i,indictingthtetcellswerestillleto mintin memrne potentil fter exposure to. When et cells were exposed to lower concentrtions of, s little s 25 fmol/l induced significnt elevtions of [C 2+ ] i, with concentrtion-dependent effects oserved up to 25 pmol/l (Fig. 3). The requirement for PLC ctivtion nd extrcellulr C 2+ influx in the -induced increses in et cell [C 2+ ] i ws determined y recordings from Fur-2- loded MIN6 et cells in the presence of the PLC inhiitor U73122 nd in the sence of extrcellulr clcium, using uffers supplemented with 1 mmol/l EGTA. The response to (Fig. 3c) ws significntly reduced in the presence of U73122 nd olished in the sence of externl clcium, indicting tht the elevtions of clcium occur vi PLC ctivtion nd C 2+ influx. In some experiments MIN6 et cells were exposed for 48 h to GPR75-trgeted sirnas, which led to significnt reductions of Gpr75 mrna expression (Fig. 3d). Western lotting (Fig. 3e) indicted tht GPR75 protein undnce in sirna-treted cells ws lso gretly reduced fter 48 h. In those cells in which GPR75 hd een downregulted, the stimultory effect of ll concentrtions of on [C 2+ ] i ws sent (Fig. 3g), lthough it ws seen in MIN6 et cells tht hd een trnsiently trnsfected with non-interfering RNAs (Fig. 3f), with similr response profiles to those otined in ntive MIN6 et cells (Fig. 3). Smll nd trnsient fluctutions of fluorescence fter withdrwl of t.25 nd 2.5 nmol/l, ut not 25 nmol/l were oserved in non-interfering RNA- nd Gpr75 sirna-trnsfected cells. Cells treted with sirnas, s well s those exposed to noninterfering RNAs, showed roust responses to tolutmide, confirming the viility of et cells fter exposure to sirnas. increses insulin secretion in vitro The effect of exogenous on dynmic insulin secretion ws exmined y

6 2472 Dietologi (213) 56: Fig. 3 increses [C 2+ ] i in et cells vi ctivtion of GPR75. Stimultion of intrcellulr C 2+ y in non-trnsfected MIN6 et cells ( c) or MIN6 cells tht hd een trnsiently trnsfected with 15 nmol/l non-interfering RNA (f) or with 15 nmol/l sirna designed to downregulte Gpr75 (g). (, f, g) Responsesto 1 μmol/l tolutmide (Tol). (c) MIN6 cells were perifused with stndrd medium (continuous line), medium supplemented with 1 μmol/l U73122 (dshed line), or without C 2+ nd supplemented with 1 mmol/l EGTA (dotted line). Clcium dt re expressed s mens ± SEM 34/38 fluorescence rtios (, c, f, g) or s the pek mplitude response (); n =34 37 cells, three experiments. Quntifiction of Gpr75 mrna (d) nd protein (e) fter exposure of MIN6 et cells to non-interfering RNAs (control) or sirnas, with (e) β-ctin expression in the sme smples. Dt for mrna quntifiction (d) re men + SEM, n =3, p <.5. The lot (e) is representtive of three seprte experiments Fluorescence (34/38 nm) c Fluorescence (34/38 nm) f Fluorescence (34/38) nmol/l 2.5 nmol/l nmol/l.25 nmol/l 2.5 nmol/l 25 nmol/l 25 nmol/l d Gpr75 mrna (% β-ctin) Tol g Fluorescence (34/38 nm) Control Tol sirna.25 nmol/l Bsl to pek response (34/38 nm) e Control 2.5 nmol/l (pmol/l) sirna 25 nmol/l GPR75 β-actin Tol perifusing mouse nd humn islets in temperture-controlled pprtus. Figure 4 demonstrtes tht recominnt mouse (2 nmol/l) induced smll, sustined increse in insulin secretion t 2 mmol/l glucose (2.1±.6-fold pek stimultion, p <.5). These islets were then le to mount significnt (p <.1) increse in insulin secretion in response to 2 mmol/l glucose, indicting tht the islets were metoliclly ctive nd exposure to hd not compromised their insulin secretory function. In prllel experiments, incresing the glucose concentrtion from 2 to 2 mmol/l initited iphsic secretory response from mouse islets (Fig. 4), while dministrtion of mouse in the continued presence of 2 mmol/l glucose induced further increse in secretion over the stle second phse of glucose-stimulted insulin secretion. This potentition of insulin secretion y ws rpid in onset, ut not redily reversile, s the response ws mintined for 2 min fter removl of. In similr experiments with isolted humn islets, recominnt humn (1 nmol/l) cused trnsient, yet pronounced 5.4±.7-fold increse in insulin secretion t 2 mmol/l glucose, with islets responding to susequent exposure to 2 mmol/l glucose with typicl iphsic secretory response (Fig. 5). Figure 5 shows tht lso potentited glucose-induced insulin relese from humn islets, ut in contrst to the secretory profile otined with mouse islets (Fig. 4), the potentition of insulin secretion from humn islets y ws trnsient nd insulin secretion returned to

7 Dietologi (213) 56: Insulin secretion (% of 2 mmol/l glucose) Insulin secretion (% of 2 mmol/l glucose) 1, nmol/l 2 mmol/l glucose 2 mmol/l glucose mmol/l glucose 2 nmol/l 2 mmol/l glucose Fig. 4 stimultes insulin secretion from mouse islets. Isolted mouse islets were perifused with uffers supplemented with 2 nmol/l recominnt mouse (lck circles) for the periods shown t 2 mmol/l () nd 2 mmol/l () glucose. In ll experiments islets were initilly perifused with uffer contining 2 mmol/l glucose. () Islets were lso exposed to 2 mmol/l glucose lone for 6 min (white circles). Dt, representtive of three seprte experiments, re percentges of insulin secretion t 2 mmol/l glucose nd re expressed s mens ± SEM, n =4 eing sujected to glucose tolernce tests following single intrperitonel dministrtion of glucose (2 g/kg) in the sence or presence of 65 pmol mouse. This dose of ws selected to replicte circulting concentrtion of pproximtely 3 to 4 nmol/l, ssuming mouse lood volume of 6 μl per g ody weight. We chose slightly higher concentrtion for in vivo studies s the N-terminl dipeptide of is cleved y dipeptidyl peptidse 4 (DPP4) [28, 29], potentilly reducing levels of io-ville in vivo. There were no significnt differences (p >.2) in the weights of the control mice or those treted with (Fig. 6), or in the fsting glucose concentrtions (Fig. 6), lthough, s expected, the o/o mice were significntly hevier nd hd higher fsting plsm glucose concentrtions thn the len mice (p <.1). improved glucose tolernce in len (Fig. 6c) nd oese (Fig. 6d) mice, with reductions in verge lood glucose concentrtions seen s erly s 15 min fter dministrtion (p <.5) nd enhnced glucose clernce eing sustined for t lest 6 min fter dministrtion. Clcultion of the AUCs for the glucose tolernce tests confirmed tht reduced lood glucose levels in len nd oese mice (p <.5 nd p =.8 respectively). In some glucose tolernce tests with len mice, lood ws retrieved 3 min fter dministrtion for quntifiction of plsm insulin levels. From Fig. 6g it cn e seen tht the glucose-induced elevtion of insulin ws enhnced y, lthough this ws not sttisticlly significnt. Insulin tolernce tests indicted tht dministrtion of 65 pmol hd no significnt effect on insulin sensitivity (Fig. 6h). tht seen in the 2 mmol/l glucose plteu during the period of exposure to. improves glucose tolernce in vivo Len ICR mice nd insulin-resistnt o/o mice were fsted overnight efore Discussion ws originlly chrcterised s T cell-specific protein [3] so most erlier studies focused on its pro-inflmmtory Insulin secretion (% of 2 mmol/l glucose) nmol/l 2 mmol/l glucose 2 mmol/l glucose Insulin secretion (% of 2 mmol/l glucosel) mmol/l glucose 1 nmol/l 2 mmol/l glucose Fig. 5 stimultes insulin secretion from humn islets. Isolted humn islets were perifused with uffers supplemented with 1 nmol/l recominnt humn t 2 mmol/l () nd 2 mmol/l ()glucose.in ll experiments islets were initilly perifused with uffer contining 2 mmol/l glucose. Dt, representtive of three seprte experiments, re percentges of insulin secretion t 2 mmol/l glucose nd re expressed s mens ± SEM, n =4

8 2474 Dietologi (213) 56: Weight (g) Len o/o Len o/o Fsting glucose (mmol/l) Blood glucose (mmol/l) e f g h Blood glucose AUC (mmol/l min) 1,4 1,2 1, Control Blood glucose AUC (mmol/l min) 1,8 1,6 1,4 1,2 1, c Control Fig. 6 improves glucose tolernce in vivo. Body weight () nd fsting lood glucose concentrtions () in len nd o/o mice dministered 2 g/kg glucose lone (lck rs) or 65 pmol nd 2 g/kg glucose (white rs). Blood glucose concentrtions in len (c) ndo/o (d) mice fter n i.p. glucose chllenge in the sence (circles) or presence (dimonds) of. AUCs were clculted from the glucose tolernce dt for len (e) nd o/o (f) mice. (g) Increse in plsm Increse in plsm insulin (pmol/l) Control d Blood glucose (mmol/l) Blood glucose (% of initil) insulin in response to i.p. delivery of glucose lone (control) or glucose in the presence of. (h) Blood glucose concentrtions in len mice fter n i.p. insulin chllenge in the sence (circles) or presence (dimonds) of. Dt re mens ± SEM of repeted mesures, n =4 6, p <.5 vs verge lood glucose concentrtions in mice fter n i.p. glucose chllenge in the sence of nd p <.1 vs corresponding conditions in len mice role. However, here we present evidence of n lterntive role for, nmely to influence norml et cell function vi GPR75 ctivtion. Ccl5 mrna nd protein hve een identified in islets from non-dietic BALB/c [6] nd C57BL/6 mice [31], nd we hve confirmed expression of this chemokine in islets isolted from ICR mice nd from helthy humn donors. We found tht ws constitutively expressed y mouse nd humn islet cells, lthough cellulr loclistion vried etween the two species, with eing expressed primrily y lph cells in mouse islets, while eing detected in lph nd in et cells in humn islets. The expression of receptors y helthy islets hs not een investigted previously. In the current study, the quntifiction of CCR1, CCR3 nd CCR5 mrna in non-dietic humn nd mouse islets indictes tht they were either undetectle or present t levels <1% of tht of the novel receptor, GPR75. These low levels of CCR chemokine receptors suggest tht they re unlikely to hve mjor function in norml islet physiology, ut do support the notion tht endogenous or circulting regultes islet function in non-immune environment through ctivtion of GPR75. The identifiction of GPR75 s the most undnt receptor in islets, its loclistion to et cells nd previous reports tht this receptor couples vi Gq to elevte [C 2+ ] i [1] led us to investigte whether similr signl trnsduction occurred in et cells. Determining the optiml exogenous concentrtion needed to identify whether this chemokine cts vi GPR75 to regulte et cell signlling is chllenging. In studies investigting the effects of in GPR75-trnsfected Humn emryonic kidney cells nd Chinese hmster ovry cells, the EC 5 for effects on IP 3 genertion nd C 2+ moilistion rnged from.1 to.3 nmol/l [1], while 1 nmol/l hs een reported to significntly elevte [C 2+ ] i in spinl cord synptosomes [32]. In ddition, the circulting concentrtion in helthy humns is 5.8 to 7.8 nmol/l [33, 34], yet s is expressed y islet cells, its locl concentrtion my e higher thn tht reching islets vi the circultion. We therefore exmined the effects of on chnges in [C 2+ ] i in MIN6 et cells over the rnge.25 to 25 nmol/l. In these experiments, t concentrtions s low s.25 nmol/l cused rpid nd reversile increses in [C 2+ ] i, with similr stimultory effects eing oserved in the presence of 2.5 nd 25 nmol/l. We therefore used lower concentrtions of nd found tht concentrtiondependent elevtions of [C 2+ ] i were otined using.25 to 25 pmol/l, suggesting high sensitivity of et cells to exogenous. The PLC inhiitor U73122 reduced the - induced elevtion of [C 2+ ] i, suggesting signlling vi Gq-

9 Dietologi (213) 56: coupled GPR75. A clcium influx component ws lso identified, since the stimultory effects of on [C 2+ ] i were olished in the sence of extrcellulr C 2+. The involvement of GPR75 in -stimulted increses of [C 2+ ] i ws confirmed y the loss of stimultory effects in et cells in which GPR75 hd een downregulted y exposure to Gpr75 sirnas. These oservtions, together with the low undnce of CCRs in islets, re consistent with the notion tht ctivtion of et cell GPR75 receptors stimultes Gq-medited IP 3 production nd C 2+ moilistion, s occurs in GPR75-trnsfected cells [1]. Elevtions of [C 2+ ] i ply key roles in et cell stimulus secretion coupling [35], so perifusions were crried out to identify whether stimulted insulin secretion. These dynmic secretion experiments reveled tht stimultory effects of were oserved t su-stimultory (2 mmol/l) nd mximl stimultory (2 mmol/l) glucose concentrtions in mouse nd humn islets, ut secretory profiles were species-specific. Thus, induced sustined elevtions of insulin relese from mouse islets tht were reversile t 2 mmol/l glucose, ut still evident upon withdrwl t 2 mmol/l glucose. In contrst, in humn islets the rpid responses to were not sustined for the durtion of exposure to the gonist, with only short-term elevtions of insulin relese, indicting tht -stimulted secretory profiles differ etween species. Agents tht stimulte insulin secretion in vivo cn reduce hyperglycemi y promoting glucose uptke nd storge, so the effects of in vivo dministrtion of on glucose nd insulin tolernce were exmined in mice. is sustrte for DPP4 [28, 29]. This protese is known to rpidly inctivte the insulin secretgogue glucgon-like peptide 1, nd indeed, DPP4 inhiitors re used cliniclly in the tretment of type 2 dietes to mintin glucgon-like peptide 1 levels in vivo [36, 37]. It hs een reported tht DPP4-medited clevge of the N-terminl serine nd proline in ffects its inding ffinity to CCR1, -3 nd -5 [28, 29], ut it is not known whether clevge of reduces its iologicl ctivity or ffects its interction with GPR75. Our in vivo experiments were crried out y dministering glucose solutions supplemented with, rther thn pre-dministering ; this ws done to limit degrdtion nd possile chnges in its receptor-inding chrcteristics. We lso used t dose of 65 pmol per mouse to chieve higher concentrtions in vivo thn those used in vitro; our im ws to counterct the reduced vilility cused y DPP4-induced clevge in vivo. Our results indicte tht improved glucose tolernce in len nd insulin-resistnt o/o mice, nd tht it lso incresed insulin secretion in vivo, without hving ny effects on insulin sensitivity. In conclusion, we hve shown here tht islet cells express the chemokine nd its recently identified receptor GPR75, while mrnas encoding the conventionl chemokine receptors CCR1, -3 nd -5 re undetectle or expressed t very low levels in norml islets. Our results lso indicte tht exogenous dministrtion of increses et cell [C 2+ ] i vi GPR75 nd stimultes insulin secretion in vitro nd in vivo; it lso improves glucose tolernce in vivo. Acknowledgements We re grteful to the reltives of orgn donors for donting humn pncreses for islet isoltion nd to Junichi I. Miyzki (University of Osk, Osk, Jpn) for providing the MIN6 et cells. Funding This study ws supported y Dietes UK (equipment grnt BDA:RD7/351 nd RD Lwrence Fellowship BDA:11/4172 to Stefn Amisten) nd y The Society for Endocrinology (Erly Creer grnt to Bo Liu). Dulity of interest The uthors declre tht there is no dulity of interest ssocited with this mnuscript. Contriution sttement This study ws conceived nd designed y BL, SA, PMJ nd SJP. 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