ARTICLE. J. E. Bowe & A. Chander & B. Liu & S. J. Persaud & P. M. Jones

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1 Dietologi (23) 56: DOI.7/s ARTICLE The permissive effects of glucose on receptor-operted potentition of insulin secretion from mouse islets: role for ERK/2 ctivtion nd cytoskeletl remodelling J. E. Bowe & A. Chnder & B. Liu & S. J. Persud & P. M. Jones Received: 2 Septemer 22 / Accepted: 3 Decemer 22 / Pulished online: 24 Jnury 23 # Springer-Verlg Berlin Heidelerg 23 Astrct Aims/hypothesis Glucose plys two distinct roles in regulting insulin secretion from et cells n inititory role, nd permissive role enling receptor-operted secretgogues to potentite glucose-induced insulin secretion. The moleculr mechnisms underlying the permissive effects of glucose on receptor-operted insulin secretion remin uncertin. We hve investigted the role of extrcellulr signl-regulted kinse /2 (ERK/2) ctivtion nd consequent cytoskeletl remodelling in this process. Methods Insulin relese ws mesured from groups of isolted mouse islets using sttic incution experiments nd susequent rdioimmunossy of smples. ERK/2 ctivtion ws mesured y western lotting of islet protein smples for oth phosphorylted nd totl ERK/2. Rhodmine phlloidin stining ws used to mesure filmentous ctin in dispersed primry et cells. Results Inhiition of ERK/2 locked potentition of glucose-induced insulin relese y the receptor-operted secretgogues kisspeptin, A568, exendin-4 nd JWH5, lthough the gonists lone hd miniml effects on ERK/2 ctivtion, suggesting permissive rther thn cusl role for ERK/2 ctivtion in receptor-operted insulin relese. Following phrmcologicl ctivtion of ERK/2 ll gonists cused significnt increse in insulin relese from islets incuted with su-stimultory levels of glucose. ERK/2 inhiition significntly reduced the glucose-dependent decreses in filmentous ctin oserved in primry et cells, while phrmcologicl dissocition of ctin filments enled ll receptor-operted secretgogues J. E. Bowe () : A. Chnder : B. Liu : S. J. Persud : P. M. Jones Dietes Reserch Group, Division of Dietes nd Nutritionl Sciences, Hodgkin Building, King s College London, London SE UL, UK e-mil: jmes.owe@kcl.c.uk tested to significntly stimulte insulin relese from islets t su-stimultory glucose concentrtion. Conclusions/interprettion Glucose-induced ERK/2 ctivtion in et cells medites the permissive effects of stimultory glucose concentrtions on receptor-operted insulin secretgogues, t lest in prt through effects on ctin depolymeristion nd cytoskeletl remodelling. Keywords Actin. Bet cell. ERK/2. G-protein coupled receptor gonists. Islet. Insulin secretion Arevitions CB2R Cnninoid 2 receptor ERK/2 Extrcellulr signl-regulted kinse /2 GLP Glucgon-like peptide GPR54 G-protein coupled receptor 54 MAPK Mitogen-ctivted protein kinse MEK Mitogen-ctivted ERK kinse Introduction Secretion of insulin from pncretic et cells is the result of complex interctions etween nutritionl nd hormonl signls. Elevted plsm glucose levels comprise the primry physiologicl stimulus for inititing insulin secretory responses y inducing depolristion-dependent elevtions in intrcellulr C 2+. Insulin secretion is lso regulted y wide rnge of receptor-operted secretgogues, including neurotrnsmitters nd hormones, lthough the mjority of receptor-operted secretgogues potentite glucose-induced insulin relese rther thn inititing insulin relese per se, presumly to void inpproprite secretion of insulin under normoglycemic conditions. Glucose therefore plys two distinct roles in regulting insulin secretion from et cells: C 2+ -medited inititory ction, nd permissive role

2 784 Dietologi (23) 56: enling receptor-operted secretgogues to potentite insulin secretion []. The intrcellulr signlling pthwys through which glucose initites the exocytosis of insulin secretory vesicles from et cells re firly well understood, ut the mechnisms underlying the permissive effects of glucose on receptor-operted insulin secretion remin uncertin. Glucose-induced increses in intrcellulr C 2+ hve een reported to ctivte vrious kinses within et cells [2] including protein kinse C (PKC), protein kinse A (PKA), clmodulin kinse II (CAMKII) nd extrcellulr signl-relted kinse /2 (ERK/2) (lso clled p42/44 mitogen-ctivted protein kinse [p42/44 MAPK]) [3 5]. ERK/2 re serine/threonine kinses tht form n integrl component of n intrcellulr signlling cscde primrily involved in cell prolifertion nd growth-promoting effects vi upregultion of nucler trnscription fctors [6]. Physiologicl concentrtions of glucose ctivte ERK/2 in pncretic et cells [3, 7] nd glucose-ctivted ERK/2 hve een shown to upregulte insulin mrna levels [7] nd my ply role in regulting insulin secretion [8]. The potentil role of ERK/2 in receptor-operted insulin secretion hs een less well documented [9 2]. Severl secretgogues, including the endogenous G-protein coupled receptor 54 (GPR54) lignd kisspeptin nd the clcimimetic ctivtor of the clcium-sensing receptor A568, hve een identified s requiring ERK/2 ctivtion to potentite glucose-induced insulin relese [, 2], while the potentiting effects of other secretgogues, including glucose-dependent insulinotropic peptide (GIP) nd oxytocin, do not pper to require ERK/2 ctivtion [9]. In this study we investigted whether ERK/2 ctivtion my e permissive for the potentition of glucose-induced insulin secretion y receptor-operted secretgogues. In other tissues, C 2+ -induced exocytosis hs een linked to ERK/2-dependent depolymeristion of the ctin skeleton, enling vesicles to ccess the exocytotic sites on the inner surfce of the plsm memrne [3]. In et cells, filmentous F-ctin forms we elow the plsm memrne tht is thought to restrict ccess of insulin secretory vesicles to the cell surfce [4]. It hs een suggested tht glucose-induced increses in intrcellulr C 2+ result in the trnsient depolymeristion of the F-ctin we, incresing exocytosis [3]. Little is known out the mechnism(s) through which ERK/2 ctivtion my influence receptoroperted insulin relese, lthough in other cell types, such s drenl chromffin cells, receptor-operted secretgogues hve een shown to increse exocytosis through ERK/2 ctivtion nd susequent F-ctin depolymeristion [5]. A link etween ctin depolristion nd exocytosis is therefore well estlished, s is link etween ERK/2 ctivtion nd cytoskeletl remodelling. The second im of the present study ws therefore to determine whether glucose-induced ERK/2-dependent cytoskeletl remodelling medites the permissive effects of glucose on receptor-medited insulin secretion. Methods Use of nimls All experiments involving nimls were pproved y the locl ethics committee. Mterils Mouse kisspeptin- ws synthesised y Alt Biosciences (Birminghm, UK). The clcimimetic A568 ws from Amgen (Thousnd Oks, CA, USA). The cnninoid receptor 2 gonist JWH5 ws from Tocris Bioscience (Bristol, UK). The inhiitors of ctin polymeristion, ltrunculin B nd cytochlsin B, nd the mitogen-ctivted ERK kinse (MEK) inhiitor PD9859 were from Cliochem (Nottinghm, UK). PD9859 ws dissolved in DMSO such tht the finl DMSO concentrtion in incution uffers ws.% (vol./vol.). The relevnt controls received.% DMSO lone. The glucgon-like peptide (GLP-) nlogue exendin-4, collgense, FCS, glutmine, penicillin/streptomycin, crchol, okdic cid, sodium pervndte nd guine pig nti-insulin ntiody were from Sigm (Poole, UK). RPMI nd DMEM were supplied y Invitrogen (Pisley, UK). The ntiodies ginst totl ERK/2 (:2,) nd ginst phosphorylted (phospho-)erk/2 (:2,5) were otined from Promeg (Southmpton, UK). Rhodmine phlloidin ws from Cmridge Bioscience (Cmridge, UK). The pca-mek plsmid nd the MEK pbepuro plsmid were kind gifts from T. Herert (Deprtment of Cell Physiology nd Phrmcology, University of Leicester, Leicester, UK) nd C. Mrshll (Division of Cncer Biology, Institute of Cncer Reserch, London, UK), respectively. Nucleofector II nd Lipofectmine 2 were from Amx (Cologne, Germny). Islet isoltion nd mintennce Islets of Lngerhns were isolted from mle ICR mice (Hrln, Bicester, UK) y collgense digestion of the exocrine pncres nd incuted t 37 C in RPMI (supplemented with % [vol./vol.] FCS, 2 mmol/l glutmine nd U/ml penicillin/. mg/ml streptomycin) for t lest 24 h efore use. Insulin secretion in vitro The effects of tretments on insulin secretion from mouse islets were ssessed using sttic incutions of islets. Islets were pre-incuted for h in RPMI contining 2 mmol/l glucose. Groups of three islets were trnsferred into.5 ml microcentrifuge tues nd incuted t 37 C for either 3 min or h in icronte-uffered physiologicl slt solution s descried y Gey nd Gey (.6 ml, 37 C, [6]) contining 2 mmol/l glucose, 2 mmol/l CCl 2 nd.5 mg/ml BSA supplemented with gents of interest. After 3 or 6 min smples of the incution medium

3 Dietologi (23) 56: were tken nd stored t 2 C until ssyed for insulin content using n in-house rdioimmunossy [7]. Mesurement of ERK/2 ctivtion Islets were cultured overnight in serum-free RPMI (supplemented with 2 mmol/l glucose, 2 mmol/l glutmine nd U/ml penicillin/. mg/ml streptomycin). Groups of 2 islets were incuted (37 C, 5 min) in physiologicl slt solution contining either 2 mmol/l or 2 mmol/l glucose in the presence or sence of gonists, cooled nd then pelleted y centrifugtion (, g, min). The superntnt frction ws discrded nd protein extrcts were prepred s descried [8]. Proteins were seprted y PAGE, trnsferred to memrnes nd immunoproed for ERK/2 nd for phospho-erk/2 (i.e. ctivted ERK/2), s descried previously [8]. Cell culture nd trnsfection These experiments hve een performed on mouse MIN6 et cells s they re wellestlished physiologicl et cell model tht llows efficient trnsfection with constructs. MIN6 et cells were mintined in culture t 37 C in DMEM (25 mmol/l glucose) supplemented with 2 mmol/l glutmine, % FCS, U/ml penicillin nd μg/ml streptomycin. Trnsient trnsfection of MIN6 et cells ws chieved y electroportion using Nucleofector II electroportion mchine s previously descried [9], with trnsfection efficiency of 5 75% s determined through co-trnsfection with gfpexpressing plsmid. MIN6 et cells were trnsfected with two different plsmids in Lipofectmine 2 trnsfection regent, ech producing constitutively ctive MEK mutnt. Both plsmids hve een previously descried; the pca-mek plsmid (plsmid A) ws gift from T. Herert [2] nd the MEK pbepuro plsmid (plsmid B) ws gift from C. Mrshll [2, 22]. MIN6 et cells were then seeded into either 96-well pltes for insulin-relese experiments or tissue culture flsks for protein extrction, nd were mintined in culture for 48 h. Protein extrcts were prepred from cells mintined in flsks nd ctivtion of MEK determined y western lotting. For insulin-relese experiments, cells in 96-well pltes were pre-incuted for h in DMEM contining 2 mmol/l glucose. Cells were then incuted t 37 C for 3 min in icronte-uffered physiologicl slt solution (.6 ml, 37 C, [6]) contining 2 mmol/l glucose, 2 mmol/l CCl 2 nd.5 mg/ml BSA supplemented with gents of interest. Smples of the incution medium were tken, stored t 2 C nd ssyed for insulin content t lter dte. Actin immunostining Isolted mouse islets were dispersed into single cells using EDTA (37 C, 5 min) nd were then seeded onto poly-d-lysine treted coverslips nd cultured overnight in 2 mmol/l glucose RPMI (supplemented with % [vol./vol.] FCS, 2 mmol/l glutmine nd U/ml penicillin/. mg/ml streptomycin). Coverslips were incuted in physiologicl slt solution s descried ove (37 C, min) contining vrious tretments efore eing immeditely wshed with ice-cold PBS nd fixed with 4% (vol./vol.) prformldehyde for 5 min on ice. Cells were permeilised for min with.5% Triton X- efore incution in.5 U/ml rhodmine phlloidin for 3 min to stin ctin filments. Islet et cells were identified y insulin immunostining, s descried previously [23]. Imges of ctin stining in et cells were tken using Leic TCS SP2 confocl microscope. Sttisticl nlysis Numericl dt re expressed s mens± SEM. Differences etween two groups were nlysed y unpired Student s t test nd differences etween severl groups were nlysed y one-wy ANOVA followed y Tukey s honestly significnt differences test. A vlue of p<.5 ws considered significnt. Results The endogenous lignd for the GPR54 receptor, kisspeptin, clcimimetic ctivtor of the clcium-sensing receptor (CR), A568, the GLP- nlogue, exendin-4, nd the cnninoid receptor 2 gonist, JWH5, were used in this study to provide rnge of receptor-operted secretgogues. Kisspeptin nd exendin-4 re reported to hve no effect on insulin secretion t su-stimultory glucose concentrtions [, 24], oservtions tht were confirmed in the present study (Fig. ). JWH5 cused smll ut significnt stimultion of insulin relese t 2 mmol/l glucose (Fig. ), gin confirming previous reports [23, 25]. While A568 hs een previously reported to cuse smll increses in insulin relese t su-stimultory glucose in the MIN6 cell line [2, 8], no significnt effect of A568 on insulin secretion from mouse islets ws oserved t 2 mmol/l glucose. All of the secretgogues cused significnt potentition of insulin relese in the presence of 2 mmol/l glucose (Fig. c f), effects tht were completely locked y the presence of the MEK inhiitor PD9859 (5 μmol/l), which prevents the phosphoryltion nd ctivtion of ERK/2. Conversely, PD9859 hd no significnt effect on either sl insulin relese or glucosestimulted insulin relese (Fig. ). As expected, incuting islets in the presence of 2 mmol/l glucose induced rpid (5 min) increse in phospho-erk/2 compred with those mintined in 2 mmol/l glucose (Fig. 2). However, neither kisspeptin ( μmol/l) nor JWH5 hd ny detectle effect on ERK/2 ctivtion t 2 mmol/l or 2 mmol/l glucose (Fig. 2, c). Tretment with A568 cused n increse in ERK/2 phosphoryltion t 2 mmol/l glucose, ut hd no further effect t 2 mmol/l glucose (Fig. 2). Conversely,

4 786 Dietologi (23) 56: c G 2 G + kisspeptin 2 G 2 G + PD G + A568 d G + exendin-4 2 G + JWH5 2 G 2 G + PD9859 Phospho- ERK /2 Totl ERK /2 d Active:totl ERK/2 c Phospho- ERK /2 Totl ERK / e Active:totl ERK/2.5.5 e 3 2 f f Active:totl ERK/ Fig. Effects of the MEK inhiitor PD9859 on receptor-operted stimultion of insulin relese. () At su-stimultory glucose level, μmol/l kisspeptin, μmol/l A568, CR gonist, nd nmol/l exendin-4, GLP- nlogue, hd no effect on insulin relese from mouse islets, while μmol/l JWH5, cnninoid receptor 2 gonist, cused smll ut significnt stimultion of insulin relese. p<.5 vs 2 mmol/l glucose controls, t test, n=9.() The MEK inhiitor PD9859, 5 μmol/l, hd no significnt effect on either sl insulin secretion t su-stimultory glucose level (2 mmol/l) or glucose-stimulted (2 mmol/l glucose) insulin secretion. p<.5 vs 2 mmol/l glucose controls, t test, n=9. (c f) At stimultory glucose levels (2 mmol/l) μmol/l kisspeptin (c), μmol/l A568 (d), nmol/l exendin-4 (e) nd μmol/l JWH5 (f) ll cused significnt potentition of glucosestimulted insulin relese. The MEK inhiitor PD9859, 5 μmol/l, completely locked the potentition of glucose-stimulted insulin relese of ll receptor-operted secretgogues tested. p<.5 vs 2 mmol/l glucose controls, t test, n=9. All dt shown re mens±sem. 2 G, 2 mmol/l glucose; 2 G, 2 mmol/l glucose exendin-4 cused smll increse in ERK/2 phosphoryltion of pproximtely 3% in the presence of 2 mmol/l Fig. 2 ERK/2 ctivtion in islets. ( c) Phospho-ERK/2 immunorectivities nd totl ERK/2 immunorectivities were detected y immunolots in extrcts of mouse islets incuted for 5 min t 37 C. Arrows show moleculr msses clculted from the gel-migrtion positions of proteins of known moleculr mss. The imges shown re representtive of lots reproduced three or four times using different smples. (d f)bnd densities were quntified for oth the ERK nd ERK2 proteins, nd expressed s rtio of ctive: totl ERK/2. Neither kisspeptin nor JWH5 hd ny effect on ERK/2 ctivtion t either 2 mmol/l or 2 mmol/l glucose. A568 tretment cused n increse in ERK/2 ctivtion t 2 mmol/l glucose. Exendin-4 tretment cused further ctivtion of ERK/2 in islets treted t 2 mmol/l glucose. All dt shown re mens±sem. White rs, ERK rtio; lck rs, ERK2 rtio. p<.5 vs 2 mmol/l glucose control; p<.5 vs 2 mmol/l glucose control, ANOVA; n= G, 2 mmol/l glucose; 2 G, 2 mmol/l glucose glucose, ut did not ctivte ERK/2 t su-stimultory glucose concentrtion (Fig. 2c).

5 Dietologi (23) 56: Trnsient trnsfection of MIN6 et cells with constitutively ctive MEK mutnt resulted in significntly incresed ERK/2 ctivity fter 48 h (Fig. 3, ). Constitutive ctivtion of MEK hd no significnt effect on sl insulin relese t 2 mmol/l glucose or on glucose-stimulted (2 mmol/l) insulin relese (dt not shown). In control cells A568, exendin-4 nd JWH5 hd no significnt effect on insulin secretion t 2 mmol/l glucose. However, following trnsfection with constitutively ctive MEK ll of the receptor-operted secretgogues tested cused significnt stimultion of insulin Phospho- ERK /2 Totl ERK /2 c Insulin relese (% of control 2 G) Active:totl ERK/ relese t 2 mmol/l glucose (Fig. 3c). Kisspeptin ws not used for these experiments s it hs previously een shown tht kisspeptin hs inhiitory effects on insulin relese in the MIN6 cell line [8]. Okdic cid nd sodium pervndte inhiit the dephosphoryltion of ERK/2, effectively mintining oth isoforms in their ctive phosphorylted stte, even t sustimultory glucose levels [26]. Okdic cid nd sodium pervndte ( μmol/l nd μmol/l, respectively) hd no significnt effect on insulin relese t 2 mmol/l glucose, s shown in Fig. 4, ut in their presence kisspeptin, A568 nd exendin-4 significntly stimulted insulin relese t 2 mmol/l glucose (Fig. 4). Similrly, the presence of okdic cid nd sodium pervndte gretly enhnced the smll insulin secretory response induced y JWH5 lone (Fig. 4). Incution of dispersed et cells in uffer contining 2 mmol/l glucose for min cused significnt reduction in rhodmine phlloidin stining intensity when compred with cells incuted in the presence of 2 mmol/l glucose (Fig. 5, ), indicting depolymeristion of F-ctin filments close to the inner surfce of the plsm memrne. The glucose-induced dissocition of ctin filments ws gretly reduced y the presence of the MEK inhiitor PD9859, s shown in Fig. 5c, with et cells displying levels of rhodmine phlloidin stining intensity similr to those of control cells treted with 2 mmol/l glucose (Fig. 5). Conversely, cells incuted in the presence of okdic cid nd sodium pervndte t 2 mmol/l glucose displyed reduced levels of rhodmine phlloidin stining intensity, similr to cells treted with 2 mmol/l glucose (Fig. 5d). 2 G A568 Exendin-4 JWH5 Fig. 3 Constitutive ctivtion of MEK in the MIN6 et cell line. () Phospho-ERK/2 immunorectivities nd totl ERK/2 immunorectivities were detected y immunolots of extrcts of MIN6 cells trnsfected with one of two different constitutively ctive MEK mutnts (plsmids A nd B). Control cells went through the trnsfection process in the sence of construct. Arrows show moleculr msses clculted from the gel-migrtion positions of proteins of known moleculr mss. The imges shown re representtive of lots reproduced three or four times using different smples. () Bnd densities were quntified for oth the ERK nd ERK2 proteins nd expressed s rtio of ctive:totl ERK/2. Both constructs ctivted MEK, nd consequently ERK/2 fter 48 h compred with controls. White rs, ERK rtio; lck rs, ERK2 rtio. p<.5 vs control, t test, n=3 4. (c) At su-stimultory glucose levels (2 mmol/l glucose), μmol/l A568, nmol/l exendin-4 nd μmol/l JWH5 hd no significnt effect on insulin relese from control MIN6 cells (white rs). In MIN6 cells trnsfected with either of the constitutively ctive MEK constructs, A568, exendin-4 nd JWH5 ll cused significnt increse in insulin secretion when compred with control cells treted with the sme secretgogues (lck rs, plsmid A; grey rs, plsmid B). All dt shown re mens±sem, p<.5 vs relevnt control cells, t test, n=8. 2 G, 2 mmol/l glucose Control Kisspeptin A568 Exendin-4 JWH5 Fig. 4 Phrmcologicl ctivtion of ERK/2 llows receptoroperted stimultion of insulin relese t su-stimultory glucose concentrtion. Kisspeptin, A568 nd exendin-4 hd no significnt effect on insulin relese from mouse islets in the presence of 2 mmol/l glucose (white rs), while JWH5 hd smll ut significnt stimultory effect. In the presence of μmol/l okdic cid nd μmol/l sodium pervndte (lck rs) kisspeptin, A568 nd exendin-4 ll significntly stimulted insulin relese t 2 mmol/l glucose. The stimultion of insulin relese cused y JWH5 t 2 mmol/l glucose ws lso significntly incresed in the presence of okdic cid/sodium pervndte compred with the effects of JWH5 lone. Dt shown re mens±sem, p<.5 vs relevnt 2 mmol/l glucose nd secretgogue control, p<.5 vs 2 mmol/l glucose lone, t test, n=9

6 788 Dietologi (23) 56: Incution of dispersed islet cells with ltrunculin, nturl toxin tht inhiits ctin polymeristion thus cusing the disruption of ctin filments, resulted in n lmost complete lck of rhodmine phlloidin stining in cells incuted t 2 mmol/l glucose, indicting extensive ctin depolymeristion (Fig. 5e). The effects of receptor-operted secretgogues on insulin secretion were tested t su-stimultory glucose levels in the presence of phrmcologicl inhiitors of ctin polymeristion, ltrunculin or cytochlsin B, mycotoxin tht lso inhiits ctin polymeristion. As in previous experiments (Figs, 3, 4) kisspeptin, A568 nd exendin-4 hd no effect on insulin relese t 2 mmol/l glucose, while JWH5 cused smll increse in insulin secretion (Fig. 6). Addition of either cytochlsin B (Fig. 6) or ltrunculin (Fig. 6) llowed kisspeptin, A568 nd exendin-4 to significntly stimulte insulin relese t 2 mmol/l glucose nd significntly incresed the JWH5-induced stimultion of insulin relese. c 2 G 2 G + PD9859 d 2 G 2 G + OA/SP Discussion An importnt feture of mny receptor-operted secretgogues is tht they re cple of only potentiting glucose-induced insulin relese, rther thn inititing insulin relese t su-stimultory glucose concentrtions. This is vitl physiologicl mechnism to void hypoglycemi y preventing inpproprite gonist-induced insulin relese e f. g 2 G + ltrunculin Control Kisspeptin A568 Exendin-4 JWH5 Stining intensity (ritrry units) G 2 G 2 G + 2 G + 2 G + PD9859 OA/SP ltrunculin Control Kisspeptin A568 Exendin-4 JWH5 Fig. 5 Glucose-induced ctin dissocition is medited through ERK/2 ctivtion. (, ) Compred with et cells treted with 2 mmol/l glucose (), et cells incuted for min in uffer contining 2 mmol/l glucose showed reduced levels of ctin stining (). (c e) Tretment with PD9859 locked the effects of 2 mmol/l glucose on ctin stining (c), while tretment with either okdic cid nd sodium pervndte (d) or ltrunculin (e) t 2 mmol/l glucose cused significnt dissocition of ctin filments within et cells. (f) Bet cells were identified through doule lelling for insulin. Br, 2 μm. (g) Actin stining intensity ws quntified for et cells from ech tretment group for sttisticl comprison. All dt shown re men±sem. p<.5 vs 2 mmol/l glucose lone, p<.5 vs 2 mmol/l glucose lone, t test, n= G, 2 mmol/l glucose; 2 G, 2 mmol/l glucose; OA, okdic cid; SP, sodium pervndte Fig. 6 Phrmcologicl dissocition of ctin llows receptor-operted stimultion of insulin relese t su-stimultory glucose concentrtions. Kisspeptin, A568 nd exendin-4 hd no significnt effect on insulin relese from mouse islets in the presence of 2 mmol/l glucose (white rs), while JWH5 hd smll ut significnt stimultory effect. In the presence of either μmol/l cytochlsin B (; lck rs) or μmol/l ltrunculin (; lck rs) kisspeptin, A568 nd exendin-4 significntly stimulted insulin relese t 2 mmol/l glucose. The stimultion of insulin relese cused y JWH5 t 2 mmol/l glucose ws lso significntly incresed in the presence of cytochlsin B or ltrunculin compred with the stimultion of insulin relese cused y JWH5 lone. All dt shown re men±sem. p<.5 vs relevnt 2 mmol/l glucose nd secretgogue control; p<.5 vs 2 mmol/l glucose lone, t test, n=9

7 Dietologi (23) 56: under normoglycemic conditions. In the present study, four different G-protein-coupled receptor lignds previously shown to potentite glucose-induced insulin secretion [, 2, 8, 23, 27] were chosen to investigte the role of ERK/2 in receptor-operted insulin relese. GPR54, CR nd cnninoid 2 receptor (CB2R) re ll Gq-proteincoupled receptors ssocited with incresed intrcellulr C 2+ levels, while GLP-R is Gs-protein-coupled receptor ssocited with incresing intrcellulr camp. Our experiments using mouse islets showed tht kisspeptin, A568 nd exendin-4 hd no significnt effects on insulin secretion t su-stimultory glucose concentrtion, lthough the CB2R gonist JWH5 did cuse smll stimultion of insulin relese t 2 mmol/l glucose, similr to tht oserved in previous studies in oth mouse islets nd MIN6 cells [23, 25]. As expected, ll the secretgogues used in our study cused significnt potentition of glucose-induced insulin relese t stimultory glucose concentrtion (2 mmol/l). Furthermore, in ll cses this potentition could e completely locked y co-dministrtion of the MEK inhiitor PD9859, suggesting tht these secretgogues require ERK/2 ctivtion to mplify glucose-induced insulin relese. We hve previously reported tht ERK/2 inhiition hs no effect on glucose-stimulted insulin secretion in sttic incution experiments [6], while other studies hve reported conflicting results [8, 9]. However, there is evidence tht ERK/2 my e involved specificlly in the first phse of glucose-induced insulin relese ut not the second phse [3]. This would correlte well with the time course of ERK/2 phosphoryltion in response to glucose, which is primrily within 5 min of glucose dministrtion, nd effects on the first phse of glucose-stimulted insulin relese my not e pprent in 3 or 6 min sttic incutions such s those in the present study. Regrdless, lthough the precise role of ERK/2 in mediting the insulin-relesing effects of glucose is uncler, it is pprent tht glucose-induced insulin secretion is not entirely dependent on ctivtion of the MAPK pthwy, ecuse even where ERK/2 inhiition ws reported to decrese glucose-induced insulin secretion the inhiition ws only prtil [8, 9]. In contrst, the receptor-operted secretgogues used in the present study provided evidence for requirement for ERK/2 ctivtion to potentite glucose-induced insulin secretion. Despite this requirement severl of the secretgogues tested did not ctivte ERK/2. Thus, stimultion with 2 mmol/l glucose cused lrge increse in islet ERK/2 phosphoryltion, wheres tretment with kisspeptin or JWH5 hd no significnt effect t either stimultory or su-stimultory glucose concentrtions. Exendin-4 hd no effect on ERK/2 phosphoryltion t 2 mmol/l glucose, ut did cuse smll increse in ERK/2 phosphoryltion t 2 mmol/l glucose, consistent with previous reports [28], while A568 ws the only secretgogue used in this study tht cused ERK/2 phosphoryltion t 2 mmol/l glucose. These dt indicte tht while the receptor-operted secretgogues require ERK/2 ctivity in order to potentite glucose-induced insulin secretion, they hve limited ility to ctivte ERK/2 themselves, suggesting tht ERK/2 ctivtion is permissive rther thn cusl for receptoroperted potentition of insulin secretion. These oservtions further suggest tht glucose-induced ERK/2 ctivtion is required for the potentiting effects of some receptoroperted secretgogues on insulin secretion, lthough ERK/2 ctivtion lone is insufficient to initite secretory response [29, 3]. In this model stimultory concentrtions of glucose enhnce ERK/2 ctivtion, thus enling receptoroperted secretgogues to potentite glucose-induced insulin secretion. This model lso predicts tht ctivtion of ERK/2 t low glucose concentrtions will hve permissive effects on receptor-operted secretgogues. Trnsient trnsfection of MIN6 et cells with constitutively ctive MEK mutnt results in incresed ERK/2 phosphoryltion fter 48 h. While this hs no effect on sl or glucose-stimulted insulin relese, constitutive MEK ctivtion llows receptoroperted insulin secretion t su-stimultory glucose concentrtions. The sme effect is oserved following phrmcologicl ctivtion of MEK. A comintion of okdic cid, serine/threonine phosphtse inhiitor, nd sodium pervndte, tyrosine phosphtse inhiitor, mintins islet ERK/2 in its phosphorylted ctivted form [26, 3], ut hs no direct effect on insulin secretion [3]. We here confirm tht phrmcologicl ctivtion of ERK/2 with okdic cid nd sodium pervndte did not stimulte insulin secretion t 2 mmol/l glucose, ut lso demonstrte tht this phrmcologicl ctivtion of ERK/2 hs glucose-like effects in enling receptoroperted secretgogues to stimulte insulin secretion. Together, these dt re consistent with glucose-induced ERK/2 ctivtion eing mechnism through which elevted glucose is permissive for receptor-dependent insulin secretory responses. ERK/2 my ply this permissive role through effects on the ctin cytoskeleton. It hs long een estlished tht ctin filments in et cells form corticl nd eneth the cell memrne tht plys n importnt role in restricting ccess of insulin secretory grnules to the cell memrne for exocytosis [4, 32]. Phrmcologicl disruption of this corticl ctin we is ssocited with enhnced insulin relese, nd severl studies hve confirmed link etween glucoseinduced ERK/2 ctivtion nd ctin remodelling [3, 33]. We therefore tested whether the permissive effect of glucoseinduced ERK/2 ctivtion on receptor-operted insulin relese involved remodelling of the corticl nd of F-ctin filments. At su-stimultory glucose concentrtions, mouse

8 79 Dietologi (23) 56: et cells contined extensive intense F-ctin stining tht rpidly depolymerised in the presence of stimultory concentrtion of glucose, confirming previous reports using the MIN6 cells [3]. Glucose-induced ctin depolymeristion ws sustntilly inhiited y PD9859, suggesting tht in primry et cells the glucose-induced remodelling of the F-ctin we to llow ccess of secretory vesicles to the cell memrne is medited, t lest in prt, y ERK/2. Phrmcologiclly induced F-ctin depolymeristion using ltrunculin or cytochlsin B did not initite n insulin secretory response ut enled receptor-operted gonists to stimulte insulin secretion t su-stimultory concentrtion of glucose, in similr mnner to tht induced y the phrmcologicl ctivtion of ERK/2. Agin, these oservtions suggest tht the glucoseinduced ERK/2-dependent effects on ctin depolymeristion re permissive for receptor-dependent insulin secretory responses. Our oservtions in et cells re similr to those reported in chromffin cells, in which ctin depolymeristion cn e locked y MEK inhiitors, suggesting tht ERK/2 my ct upstrem of ctin remodelling [5]. However, tretment of MIN6 et cells with ltrunculin increses ERK/2 phosphoryltion nd potentites glucose-stimulted insulin secretion, nd there is strong evidence tht ERK/2 my lso lie downstrem of glucose-dependent ctin rekdown [3]. These dt suggest potentil i-directionl interction etween ERK/2 nd ctin polymeristion in et cells tht hs yet to e fully chrcterised. In ny event, correltions etween the effects of glucose on ERK/2 ctivtion nd ctin remodelling hve een previously descried in the context of glucose-induced insulin relese [8, 34, 35]. The present study demonstrtes tht this interction etween glucose-induced ERK/2 ctivtion nd ctin my lso ply crucil role in permitting receptoroperted insulin relese, lthough the precise moleculr mechnisms involved re uncertin, nd my not necessrily e the sme s those involved in glucose-induced insulin relese. In summry, our dt re consistent with glucose-induced ERK/2 ctivtion in et cells eing key event in the permissive effects of stimultory glucose concentrtions on receptor-operted insulin secretgogues, t lest in prt through effects on ctin depolymeristion nd cytoskeletl remodelling. A full understnding of these events my ssist in the development of novel glucose-dependent therpeutic strtegies for type 2 dietes. Funding This work ws supported y the Dietes Reserch nd Wellness Foundtion nd Dietes UK (6/336 nd 7/35). Dulity of interest The uthors declre tht there is no dulity of interest ssocited with this mnuscript. Contriution sttement All uthors were involved in the conception nd design of the study. The dt were collected, nlysed nd interpreted y JEB, AC nd BL. The rticle ws drfted y JEB nd ll uthors revised the rticle criticlly for importnt intellectul content. All uthors gve their finl pprovl of the current version to e pulished. References. Henquin JC (2) The dul control of insulin secretion y glucose involves triggering nd mplifying pthwys in et-cells. Dietes Res Clin Prct 93(Suppl ):S27 S3 2. Jones PM, Persud SJ (998) Protein kinses, protein phosphoryltion, nd the regultion of insulin secretion from pncretic etcells. Endocr Rev 9: Briud I, Lingohr MK, Dickson LM, Wrede CE, Rhodes CJ (23) Differentil ctivtion mechnisms of Erk-/2 nd p7(s6k) y glucose in pncretic et-cells. Dietes 52: Knutson KL, Hoenig M (994) Identifiction nd sucellulr chrcteriztion of protein kinse-c isoforms in insulinom etcells nd whole islets. Endocrinology 35: Wenhm RM, Lndt M, Esom RA (994) Glucose ctivtes the multifunctionl C2+/clmodulin-dependent protein kinse II in isolted rt pncretic islets. J Biol Chem 269: Person G, Roinson F, Beers GT et l (2) Mitogen-ctivted protein (MAP) kinse pthwys: regultion nd physiologicl functions. Endocr Rev 22: Benes C, Poitout V, Mrie JC, Mrtin-Perez J, Roisin MP, Fgrd R (999) Mode of regultion of the extrcellulr signl-regulted kinses in the pncretic et-cell line MIN6 nd their impliction in the regultion of insulin gene trnscription. Biochem J 34: Longuet C, Broc C, Costes S, Hni EH, Btille D, Dlle S (25) Extrcellulrly regulted kinses /2 (p44/42 mitogen-ctivted protein kinses) phosphorylte synpsin I nd regulte insulin secretion in the MIN6 et-cell line nd islets of Lngerhns. Endocrinology 46: Bocker D, Verspohl EJ (2) Role of protein kinse C, PI3-kinse nd tyrosine kinse in ctivtion of MAP kinse y glucose nd gonists of G-protein coupled receptors in INS- cells. Int J Exp Dietes Res 2: Bowe JE, King AJ, Kinsey-Jones JS et l (29) Kisspeptin stimultion of insulin secretion: mechnisms of ction in mouse islets nd rts. Dietologi 52: Frodin M, Sekine N, Roche E et l (995) Glucose, other secretgogues, nd nerve growth fctor stimulte mitogen-ctivted protein kinse in the insulin-secreting et-cell line, INS-. J Biol Chem 27: Kitsou-Mylon I, Burns CJ, Squires PE, Persud SJ, Jones PM (28) A role for the extrcellulr clcium-sensing receptor in cellcell communiction in pncretic islets of lngerhns. Cell Physiol Biochem 22: Toms A, Yermen B, Min L, Pessin JE, Hln PA (26) Regultion of pncretic et-cell insulin secretion y ctin cytoskeleton remodelling: role of gelsolin nd coopertion with the MAPK signlling pthwy. J Cell Sci 9: Orci L, Gy KH, Mlisse WJ (972) Pncretic et-cell we: its possile role in insulin secretion. Science 75: Prk YS, Jun DJ, Hur EM, Lee SK, Suh BS, Kim KT (26) Activity-dependent potentition of lrge dense-core vesicle relese modulted y mitogen-ctivted protein kinse/extrcellulrly regulted kinse signling. Endocrinology 47: Gey GO, Gey MK (936) Mintennce of humn norml cells in continuous culture: preliminry report; cultivtion of mesolstic

9 Dietologi (23) 56: tumours nd norml cells nd notes on methods of cultivtion. Am J Cnc 27: Jones PM, Slmon DM, Howell SL (988) Protein phosphoryltion in electriclly permeilized islets of Lngerhns. Effects of C2+, cyclic AMP, phorol ester nd nordrenline. Biochem J 254: Gry E, Muller D, Squires PE et l (26) Activtion of the extrcellulr clcium-sensing receptor initites insulin secretion from humn islets of Lngerhns: involvement of protein kinses. J Endocrinol 9: Persud SJ, Liu B, Smpio HB, Jones PM, Muller DS (2) Clcium/clmodulin-dependent kinse IV controls glucoseinduced Irs2 expression in mouse et cells vi ctivtion of camp response element-inding protein. Dietologi 54: Herert TP, Tee AR, Proud CG (22) The extrcellulr signlregulted kinse pthwy regultes the phosphoryltion of 4E-BP t multiple sites. J Biol Chem 277: Brros JC, Mrshll CJ (25) Activtion of either ERK/2 or ERK5 MAP kinse pthwys cn led to disruption of the ctin cytoskeleton. J Cell Sci 8: Cowley S, Pterson H, Kemp P, Mrshll CJ (994) Activtion of MAP kinse kinse is necessry nd sufficient for PC2 differentition nd for trnsformtion of NIH 3T3 cells. Cell 77: Li C, Bowe JE, Jones PM, Persud SJ (2) Expression nd function of cnninoid receptors in mouse islets. Islets 2: Peyot ML, Gry JP, Lmontgne J et l (29) Glucgon-like peptide- induced signling nd insulin secretion do not drive fuel nd energy metolism in primry rodent pncretic et-cells. PLoS One 4:e Li C, Jones PM, Persud SJ (2) Cnninoid receptors re coupled to stimultion of insulin secretion from mouse MIN6 et-cells. Cell Physiol Biochem 26: Burns CJ, Howell SL, Jones PM, Persud SJ (999) The p38 mitogen-ctivted protein kinse cscde is not required for the stimultion of insulin secretion from rt islets of Lngerhns. Mol Cell Endocrinol 48: Drucker DJ (26) The iology of incretin hormones. Cell Met 3: Kim MJ, Kng JH, Prk YG et l (26) Exendin-4 induction of cyclin D expression in INS- et-cells: involvement of campresponsive element. J Endocrinol 88: Arnette D, Gison TB, Lwrence MC et l (23) Regultion of ERK nd ERK2 y glucose nd peptide hormones in pncretic et cells. J Biol Chem 278: Burns CJ, Howell SL, Jones PM, Persud SJ (997) Glucosestimulted insulin secretion from rt islets of Lngerhns is independent of mitogen-ctivted protein kinse ctivtion. Biochem Biophys Res Commun 239: Persud SJ, Wheeler-Jones CP, Jones PM (996) The mitogenctivted protein kinse pthwy in rt islets of Lngerhns: studies on the regultion of insulin secretion. Biochem J 33: Vn OE, Somers G, Devis G et l (975) Dynmics of insulin relese nd microtuulr-microfilmentous system. VII. Do microfilments provide the motive force for the trnsloction nd extrusion of et grnules? Dietes 24: Ymmoto H, Mtsumoto K, Arki E, Miymoto E (23) New spects of neurotrnsmitter relese nd exocytosis: involvement of C2+/clmodulin-dependent phosphoryltion of synpsin I in insulin exocytosis. J Phrmcol Sci 93: Mtsumoto K, Eihr K, Ymmoto H et l (999) Cloning from insulinom cells of synpsin I ssocited with insulin secretory grnules. J Biol Chem 274: Wng Z, Thurmond DC (29) Mechnisms of iphsic insulingrnule exocytosis roles of the cytoskeleton, smll GTPses nd SNARE proteins. J Cell Sci 22:893 93