Multiple effects and stimulation of insulin secretion by the tyrosine kinase inhibitor genistein in normal mouse islets

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1 British Journl of Phrmcology (1995) rf Stockton Press All rights reserved /95 $9.00 Multiple effects nd stimultion of insulin secretion y the tyrosine kinse inhiitor genistein in norml mouse islets J.C. Jons, *T.D. Plnt, P. Gilon, P. Detimry, M. Nenquin & 'J.C. Henquin Unite d'endocrinologie et Metolisme, Fculty of Medicine, University of Louvin, B-1200 Brussels, Belgium nd *I. Physiologisches Institut, University of Srlnd, D Homurg/Sr, Germny 1 Islets from norml mice were used to test the cute effects of genistein, potent tyrosine kinse inhiitor, on stimulus-secretion coupling in pncretic P-cells. 2 produced concentrtion-dependent (10-100I1M), reversile, increse of insulin relese. This effect ws mrginl on sl relese or in the presence of non-metolized secretgogues, nd much lrger in the presence of glucose or other nutrients. The increse in insulin relese cused y 100 pm genistein ws olished y drenline or omission of extrcellulr C2". It ws not ccompnied y ny rise of cyclic AMP, inositol phosphte or denine nucleotide levels. 3 Although genistein slightly inhiited ATP-sensitive K+ chnnels, s shown y 'R efflux nd ptch-clmp experiments, this effect could not explin the ction of the drug on insulin relese ecuse the ltter persisted when ATP-sensitive K+ chnnels were ll locked y mximlly effective concentrtions of glucose nd tolutmide. ws lso effective when ATP-sensitive K+ chnnels were opened y dizoxide nd the P-cell memrne depolrized y 30 mm K, ut ineffective in the presence of dizoxide nd norml extrcellulr K. 4 prdoxiclly decresed C2` influx in P-cells, s shown y the inhiition of glucoseinduced electricl ctivity, y the inhiition of C2+ currents (perforted ptches) nd y the lowering of cytosolic [C2+], (fur-2 technique). thus increses insulin relese in spite of lowering of [C2+]i in P-cells. 5 Didzein, n nlogue of genistein reported not to ffect tyrosine kinses, ws slightly less potent thn genistein on K'nd C2" chnnels, ut incresed insulin secretion in similr wy. Three other tyrosine kinse inhiitors, tyrphostin A47, herimycin A nd n nlogue of ersttin vrily ffected insulin secretion. 6 exerts numer of heretofore unrecognized effects. The unusul mechnisms, y which genistein increses insulin relese in spite of decrese in P-cell [C2J]i nd without ctivting known signlling pthwys, do not seem to result from n inhiition of tyrosine kinses. Keywords: ; didzein; tyrosine kinse inhiitors; pncretic P3-cells; insulin relese; K' chnnels; C2" chnnels; stimulus-secretion coupling Introduction Protein phosphoryltion y protein kinses is widespred mechnism of signl trnsduction in eukryotic cells. Among serine/threonine kinses, the denosine 3': 5'-cyclic monophosphte (cyclic AMP)-dependent protein kinse A nd the dicylglycerol-dependent protein kinse C re involved in the regultion of insulin relese y hormones nd neurotrnsmitters, while C2+-clmodulin dependent kinses ply centrl role in the coupling etween the increse in cytoplsmic [C2+]i nd exocytosis (Prentki & Mtschinsky, 1987; Jones et l., 1991; Hughes & Ashcroft, 1992; Wenhm et l., 1994). On the other hnd, it is not known whether tyrosine kinses re involved in stimulus-secretion coupling in pncretic P- cells. IGF-1 receptors, which possess n intrinsic tyrosine kinse ctivity, re expressed y rt pncretic A- nd B-cells (Vn Schrvendijk et l., 1987), nd their ctivtion y physiologicl concentrtions of IGF-1 hs een reported to inhiit (Lehy & Vndekerkhove, 1990; Vn Schrvendijk et l., 199 or increse (Clrk et l., 199 glucose-induced insulin relese in vitro. Vndte, phosphotyrosine phosphtse inhiitor (Swrup et l., 1982) tht increses the numer of phosphotyrosine residues in mny cell types, potentites insulin relese from mouse islets in dose- nd ' Author for correspondence t Unite d'endocrinologie et Metolisme, UCL 55.30, Avenue Hippocrte 55, B-1200 Brussels, Belgium. glucose-dependent mnner (Zhng et l., 1991). This effect ws minly ttriuted to complex interply etween chnges in P-cell memrne potentil, C2" hndling nd n increse in phosphoinositide metolism (Zhng et l., 1991). All these dt indirectly suggest tht modultion of islet phosphotyrosine content could e implicted in the regultion of insulin relese. This hypothesis ws lso recently supported y two rief reports showing tht genistein, puttively selective inhiitor of tyrosine kinses, incresed insulin relese from the MIN 6 cell line (Ohno et l., 1993) nd from neontl rt islets (Sorenson et l., 1994). However, circumstntil evidence indictes tht, in other systems, the effects of genistein might not lwys e relted to tyrosine kinse inhiition (Aler et l., 1992; Ozki et l., 1993; Young et l., 1993). In this study, norml mouse islets were used to chrcterize the effects of genistein on stimulus-secretion coupling. Methods Preprtion Most experiments were performed t 37'C, with islets isolted y collgense digestion of the pncres of fed femle NMRI mice (25-30 g). Ptch-clmp experiments were crried out t room temperture, with P-cells otined y dispersion of the isolted islets.

2 J.C. Jons et l Solutions Except for ptch-clmp experiments (see elow), the medium used ws icronte-uffered solution contining (mm): NCl 120, KCl 4.8, CC12 2.5, MgCl2 1.2 nd NHCO3 24. It ws gssed with O2/CO2 (94/6) to mintin ph 7.4 nd ws supplemented with 1 mg ml-i ovine serum lumin frction V (Boehringer, Mnnheim, Germny). C2"-free solutions were prepred y replcing CCl2 with MgCl2. When the concentrtion of KCl ws incresed to 30 mm, tht of NCl ws reduced to 94.8 mm to keep the osmolrity of the medium unchnged. Insulin relese mesurements After isoltion, the islets were preincuted for 1 h in medium contining 15 mm glucose, concentrtion tht produces hlf-mximum stimultion of mouse,-cells. In one type of experiments, they were then incuted for 60 min, in tches of 3, in 1 ml of medium contining pproprite concentrtions of glucose nd test sustnces. At the end of the incution, portion of the medium ws withdrwn nd diluted efore insulin ssy. In nother type of experiment, preincuted islets were plced, in tches of 30, in prllel perifusion chmers. Effluent frctions were collected t 2 min intervls for insulin ssy. Insulin ws mesured y doule-ntiody rdioimmunossy with rt insulin s stndrd (Novo Reserch Institute, Bgsverd, Denmrk). Mesurements of86r efflux The islets were first loded with 86R (used s trcer for K) during 90 min of preincution in 1 ml of medium contining 15 mm glucose nd supplemented with 86RCl. The concentrtion of R never exceeded 0.4 mm. After eing wshed, the islets were then plced in perifusion chmers permitting 86R efflux to e monitored in the effluent frctions (Grrino & Henquin, 1988). Mesurements of cytosolic [C2/]i In the experiments imed t mesuring cytosolic C2, the islets were loded with fur-2 during preincution of 40 min-in the presence of 10 mm glucose. They were then wshed nd trnsferred to C2+ mesuring system in which the perifused tissue ws excited successively t 340 nd 380 nm, nd the fluorescence emitted t 510 nm ws cptured y CCD cmer (Photonic Science Ltd., Tunridge Wells, United Kingdom). The imges were nlysed y the system MgiCl (Applied Imging, Sunderlnd, United Kingdom). The technique hs een descried in detil previously (Gilon & Henquin, 1992). Electricl recordings The memrne potentil of -cells ws mesured with high resistnce microelectrode (Meissner & Schmelz, 1974). A piece of mouse pncres ws fixed in perifusion chmer, nd few islets were prtilly microdissected y hnd. After implement, P-cells were identified y the typicl electricl ctivity tht they disply in the presence of 15 mm glucose. The experiments were performed t 37 C nd the perifusion medium ws s descried ove, except for the sence of lumin. Ptch-clmp experiments were performed t room temperture on single P-cells mintined in culture for 1-2 dys. ATP-sensitive nd voltge-sensitive K+currents were studied using the norml whole-cell configurtion. C2e currents were recorded y the perforted ptch technique. Experimentl detils nd composition of the solutions used hve een descried previously (Plnt et l., 1991). In some experiments, the extrcellulr solution contined mg ml' BSA. Becuse BSA ws without effect lone nd did not J.C. Jons et l stimultion of insulin relese 873 influence the ction of the tested sustnces, ll results hve een pooled. Mesurements of cyclic AMP, inositol phosphtes nd denine nucleotides In the experiments imed t mesuring cyclic AMP, preincuted islets were incuted, in tches of 12, in 1 ml of medium contining 15 mm glucose, with or without genistein. The incution ws stopped, nd the cyclic AMP concentrtion of the islets mesured y rdioimmunossy s descried previously (Geml et l., 1993). In the experiments imed t mesuring inositol phosphtes, the islets were loded with myo-[2-3h]-inositol during preincution of 120 min in the presence of 15 mm glucose. After wshing, tches of 50 islets were incuted for 60 min in 1 ml of medium contining 15 mm glucose, 10 mm LiCl, 1 mm unlelled inositol, with or without genistein. The incution ws stopped y ddition of 3 ml of CHC13/CH30H/concentrted HCI (200:100:1) nd 100 gil of EDTA (100 mm). Free inositol nd inositol phosphtes were then seprted y nion exchnge chromtogrphy, s descried previously (Geml et l., 1993). In the experiments imed t mesuring denine nucleotides, preincuted islets were incuted, in tches of 5, in 0.4 ml of medium contining 15 mm glucose nd 4.8 mm K or 10 mm glucose nd 30 mm K, with or without genistein. The incution ws then stopped, ATP nd ADP were extrcted nd ssyed y luminometric method, s descried previously (Geml et l., 1993). Drugs, herimycin A nd the ersttin nlogue methyl 2,5-dihydroxycinnmte were otined from Cliochem- Behring (Sn Diego, CA, USA), didzein from either Crl Roth GmH (Krlsruhe, Germny) or LC Lortories (Wourn, MA, U.S.A.), tyrphostin A47 (RG 50864) from Cliochem-Behring or LC Lortories, nd genistin from Sigm (St. Louis, MO, U.S.A.). Tolutmide ws from Hoechst AG (Frnkfurt/Min, Germny) nd dizoxide from Schering-Plough Avondle (Rthdrum, Irelnd). Stock solutions of genistein, didzein nd genistin (50 mm), of tyrphostin nd ersttin (100 mm), nd of herimycin A (2 mm) were prepred in dimethyl sulphoxide. Rdiochemicls were purchsed from the Rdiochemicl Centre (Amershm, Bucks, United Kingdom). Sttisticl nlysis Mesurements of intrcellulr C2", memrne potentil nd ion chnnel currents re illustrted y recordings which re representtive of the indicted numer of experiments. Otherwise, results re expressed s mens ± s.e.men for the indicted numer of experiments (different nimls or islet preprtions) or tches of islets. The sttisticl significnce of differences etween mens ws ssessed y unpired t test, or y nlysis of vrince followed y Dunnett's test when more thn two groups were compred. Differences were considered significnt t P<0.05. Results Chrcteristics of the effects of genistein on insulin relese The glucose- nd concentrtion-dependence of the effects of genistein on insulin relese were studied with incuted islets (Figure 1). In the presence of non-stimultory concentrtion of glucose (3 mm), genistein incresed sl relese only slightly t 100 plm. On the other hnd, genistein potentited the relesing effect of higher glucose concentrtions, including mximlly effective one (30 mm) (Figure l). This effect

3 874 J -C. Jons et l stimultion of insulin relese 874 JO. Jons et l stimultion of insulin relese~~~~~~~~~~~~~~~~~~~ ws sttisticlly significnt (P <0.05 or less) t 1OIM of the drug. As shown in Figure I, the sigmoidl reltionship etween glucose concentrtion nd insulin relese ws not mrkedly ltered y 100 glm genistein. The glucose concentrtions cusing hlf-mximl stimultion of relese were 17 nd._ U) cn ) C (0._ n mm without nd with genistein, respectively. Adrenline (1 pm) inhiited insulin relese induced y 15 mm glucose lone (1.25 ± 0.10 ng per islet h') or with 100 pm genistein (1.04 ± 0.08 ng per islet h-'). The dynmics of the potentition of glucose-induced insulin relese y genistein ws tested with perifused islets (Figure 2). In the presence of 15 mm glucose, the effect of genistein ws rpid in onset, reched plteu within out 15 min, nd ws completely reversile upon withdrwl of the drug. Figure 2 lso shows tht omission of C2+ from the perifusion medium prevented oth glucose-induced insulin relese nd its potentition y genistein. The effects of genistein were then tested in the presence of nutrient nd non-nutrient insulin secretgogues (Tle 1). A mixture of 1O mm leucine nd glutmine stimulted insulin relese s did 10 mm glucose. similrly potentited oth responses. On the other hnd, genistein hd little or no significnt effect on insulin relese induced y rium sustitution for C2+ or y 30 mm K (in the presence of dizoxide which opens K+ATP chnnels) when the medium did not contin nutrient. However, when high K ws comined with glucose or with mixture of leucine nd glutmine, (gm) Glucose (mm) Figure 1 Concentrtion- nd glucose-dependence of genistein effects on insulin relese from incuted mouse islets. Btches of 3 islets were incuted for 1 h in 1 ml of medium. () Effects of incresing concentrtions of genistein in the presence of different glucose concentrtions: (A) 30; ( 20; (U) 15; ( 10 nd (-) 3 mm. () Effects of incresing concentrtions of glucose in the sence ( or presence of 1I00 jam genistein (@). Vlues re mens ± s.e.men for tches of islets from three seprte experiments. 800 r 600 F _ 400- E 4) G 15 mm C 2.5 mm f- f Tle 1 Effects of genistein on insulin relese y mouse islets incuted under vrious conditions Experimentl conditions (mm) Glucose 0 Glucose 10 Glucose 15 Leu 10 + Gln 10 G 0 + B+ 2.5 G O+K 30 G 10+K 30 G 0+K 30+Leu 10+Gln 10 Insulin relese (ng per islet h-') Control ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.74 Btches of 3 islets were incuted for 1 h in 1 ml of medium contining the indicted concentrtion of glucose (G), leucine (Leu) nd glutmine (Gln), in the sence (control) or presence of 100 jam genistein. CCl2 ws omitted from the medium contining BCl2. Dizoxide (0.25 mm) ws present in the solutions contining 30 mm K. Vlues re mens ± s.e.men for tches of islets from three to five seprte experiments. p < 0.05; p <0.01 versus controls, y unpired t test. 0._ 'US 0 CO s _ ~~~~.. da 3 G 3 mm Ch X 600- ci G 7 mm K+ 30 mm Dz 250 gm 2.5 -n ' x Q cc 2 co 1% Figure 2 Effects of genistein on insulin relese from perifused mouse islets. Groups of 30 islets were perifused with medium contining: () 15 mm glucose (G) nd 2.5 mm C2l or 0 C, or () 7mM glucose, 30 mm K+ nd 250 jam dizoxide (Dz). In ech series, genistein (100 1AM) ws dded from 40 to 70 min to one group of islets (@, A), while the other group, perifused in prllel, served s control. Vlues re mens ± s.e. men for five experiments. 1.5' Figure 3 Effects of genistein on 86R efflux from perifused mouse islets. After loding with the trcer, groups of 30 islets were perifused with medium contining 3 mm glucose (G). (100 g1m) ws dded from 40 to 70 min. Control experiments without genistein re shown y the roken line. Vlues re mens ± s.e.men for five experiments.

4 J.C. Jons et l stimultion of insulin relese c d TL0 F-L Tolut 100 gm 100 glm 400 c d 0~~. X2001 ki L r'ns K+ current in some cells, nd decresed it in others. Blockde of K+ATP chnnels is expected to depolrize the Figure 4 Effects of genistein on whole-cell ATP-seinsitive K+ cur- P-cell memrne. The resting memrne potentil of P-cells, rents in mouse P-cells. These experiments were performed t room mesured with n intrcellulr microelectrode, during temperture on cultured single P-cells. Current vlue s were recorded perifusion of the islets with medium contining 3 mm t 15 s intervls, t -60, -70 nd -80 mv (uppher, middle nd glucose verged -63 ± 2 mv. (100 gm) lower trces of the lower pnel, respectively). Toluttmide (100 gtm) depolrized it y 7.8 ± 1.3 mv (n = 5), ut did not induce the nd genistein (100 gm) were pplied s indicted y the rs. In the upper pnel re shown currents recorded during 100 ppernce of electricl ctivity. ms pulses from -70 to -60 mv t the times indicted y the lettc r pnel. 600T 100 gm I, c 100 gm I, strong potentiting effect of genistein on irisulin relese Effects ofgenistein on K+ chnnels in 13-cells ecme pprent (Tle 1). The dynmics of thie potentition of insulin relese produced y genistein in th( e presence of Hypoglycemic sulphonylures nd mny other drugs in- 7 mm glucose, high K nd dizoxide is shown The increse in the secretory rte reched out 15 min nd returned to control vlues withdrwl of genistein. in Figure 2. crese insulin relese y closing ATP-sensitive K'chnnels plteu fter (K'ATP chnnels); this closure depolrizes the 0 cell mem- 20 min fter rne nd stimultes C2+ influx (Ashford, 1990; Henquin, 199. Closure of K'ATP chnnels is redily detected y decrese in the rte of "6R efflux from islets perifused with low concentrtion (3 mm) of glucose (Henquin et l., 1992). As shown in Figure 3, genistein inhiited 86R efflux under these conditions. The inhiition reched mximum fter out 15 min, nd ws completely reversile upon with- _J L. drwl of the drug. The whole cell mode of the ptch clmp technique ws then used to determine whether the drug directly ffects the K+ATP chnnels (Figure 4). At the eginning of the trce, ATP-sensitive K+ currents re lrge ecuse the cell interior hs equilirted with the pipette solution contining low * concentrtion of ATP (Plnt et l., 1991). Tolutmide (100 jtm) rpidly nd reversily inhiited the ATP-sensitive K+ current. Addition of 100 LM genistein to the th pro- _ duced slightly smller nd less rpidly reversile inhiition (Figure 4). The stedy-stte inhiition ws % (n = 9) _ ,... for genistein. Under similr conditions, 100 LM tolutmide inhiited the current y 93 ± 1% (Plnt et l., 1991). At the lower concentrtion of 25 fm, genistein hd inconsistent effects; it slightly nd trnsiently incresed the ATP-sensitive Io100 pa 80 ms Voltge-dependent K+ chnnels were studied in the whole cell mode, y depolrizing the P cell memrne from -70 to 0 mv. As shown in Figure 5, 100 AM genistein lso reversily inhiited these chnnels. The inhiition mounted to 82±1% (n=5). Role of the lockde of KATp chnnels in the potentition of insulin relese y genistein The increse in insulin relese rought out y tolutmide is entirely ttriutle to lockde of K'ATP chnnels 40 Glucose 15 mm Glucose 15 mm + dizoxide 250 gm t o i- o Figure 5 Effects of genistein on voltge-dependent K+ currents in mouse P-cells. These experiments were performed t room temperture on cultured single P-cells. The currents (,,c) shown in the upper pnel were recorded during 150ms voltge steps from -70 to 0 mv, t the times shown in the lower pnel. This pnel shows vlues of the mximum K+ current mesured during pulses to 0 mv, pplied t 15 s intervls. 1I00 JM ws pplied s indicted y the rs. The th solution contined 100 1LM tolutmide to inhiit ATP-sensitive K+ currents nd 100 IM Cd2+ to lock voltge-dependent C2l currents nd C2l-ctivted K+ currents.._ " 30 CL co 20 co = 10D en I. I- I-[ Tolutmide Figure 6 Comprison of the effects of genistein nd tolutmide on insulin relese from incuted mouse islets. Btches of 3 islets were incuted for I h in I ml of medium contining 15 mm glucose nd supplemented with 100 gm genistein nd/or 2501M tolutmide s indicted. In the experiments shown y the 4 right-hnd columns, 250 rm dizoxide ws lso present in the medium. Vlues re mens ± s.e.men for tches of islets from four seprte experiments.

5 876 J.C. Jons et l (Ashford, 1990; Henquin, 199. At mximlly effective concentrtion of 250ILM, tolutmide potentited glucoseinduced insulin relese to the sme extent s did 100ILM genistein (Figure 6). The comintion of oth gents produced much lrger effect thn either gent lone (P <0.01). When K'ATP chnnels were opened y 250;LM dizoxide, glucose-induced insulin relese ws inhiited y 87 ± 1% (Figure 6). This inhiition ws not significntly reversed y 100 ItM genistein, wheres 250 IM tolutmide incresed insulin relese even more thn did glucose in the sence of dizoxide. Agin, the comintion of oth gents produced lrger effect thn either gent lone. These results, therefore, indicte tht mechnism other thn the inhiition of K'ATP chnnels y genistein plys role in the increse in insulin relese tht the drug produces. Effects ofgenistein on P-cell cytoplsmic [C2+]/ nd C2+ chnnels In the presence of 15 mm glucose, P-cells disply rhythmic electricl ctivity tht reflects the influx of C2` through voltge-dependent C2` chnnels (Figure 7). Agents tht close K+ATP chnnels increse this electricl ctivity nd stimulte C2" influx. Unexpectedly, genistein inhiited glucose-induced electricl ctivity to vrile degree from cell to cell. Even when the inhiition ws strong, smll oscilltions of the memrne potentil persisted nd the memrne never repolrized to the resting level (Figure 7 nd ). Figure 7c shows one cell in which the inhiition ws less pronounced. The intermittent influx of C2` (during the phses of electricl ctivity) cuses oscilltions of [C2+]i in P-cells (Gilon & Henquin, 1992; Sntos et l., 1991). Addition of genistein to medium contining 15mM glucose ws followed y progressive disppernce of these oscilltions (Figure 8) nd stiliztion of [C2+]i t level tht remined out 80nM higher thn sl [C2+]i. When K'ATP chnnels re open y dizoxide, n increse of extrcellulr K to 30 mm cuses sustined depolriztion -10 G 15mM -j stimultion of insulin relese -z C4 i C G 15 mm 2 min 2 min C G 7 mm K+30 mm Dz 250jM g CII min Figure 8 Effects of genistein on the concentrtion of cytoplsmic C2" ([C2"]) in islet cells. After loding with fur-2, islets were perifused with medium contining 15 mm glucose (,) or 7mM glucose, 30mM K+ nd 250 ftm dizoxide (c). 100 AM ws dded s indicted y the rrows. Pnel () is the continution of (); they show n experiment tht is representtive of results otined with 15 islets. Pnel (c) shows mens ± s.e.men for 9 islets. -60, I -10 mv min c G ) min Figure 7 Effects of genistein on the memrne potentil of mouse -cell perifused with 15 mm glucose (G). (100!M) ws dded nd removed s indicted y the rrows. Pnels () nd () show single experiment without interruption. Pnel (c) illustrtes results otined in nother cell. These recordings re representtive of results otined in five experiments with different mice.

6 J.C. Jons et l of the P-cell memrne nd rise in P-cell [C2J]i (Geml et l., 1993). Under these conditions, genistein ws without effect on the memrne potentil (not shown), ut decresed [C2+]i (Figure 8c). These prdoxicl effects of genistein led us to investigte possile ction of the drug on voltge-dependent C2" chnnels. These were studied y the perforted ptch technique (Plnt et l., 1991) during depolriztion steps from -70 to 0 mv. As shown in Figure 9, genistein reversily inhiited C2" currents. The effect of the drug on pek current verged 65 ± 4%, nd ws slowly reversile upon withdrwl. Effects of genistein on cyclic AMP, inositol phosphte nd denine nucleotide levels did not ffect cyclic AMP levels in islets incuted in the presence of 15 mm glucose (Tle 2). No effect could e detected either fter lockde of the cyclic AMP phosphodiesterse y 0.5 mm isoutylmethylxnthine (26.6 ± O ,- I 50 pa 20 ms gm Figure 9 Effects of genistein on C2l currents in mouse -cells. C2+ currents were recorded y the perforted ptch technique. The currents (,,c) shown in the upper pnel were recorded during 20 ms voltge steps from -70 to 0 mv, t the times shown in the lower pnel. This pnel shows pek C2+ currents recorded t 15 s intervls. 100 j1m ws pplied s indicted y the r. To lock K+ currents, the th solution contined tolutmide (100IM) nd tetrethylmmonium (20 mm), nd the pipette solution contined 140mM Cs'. To lock N+ currents, the th solution contined 0.511M tetrodotoxin. The concentrtion of C2` in the th ws 10mM. 20 c c stimultion of insulin relese nd 26.9 ± 2.6 fmol cyclic AMP per islet without nd with genistein, n = 9). did not ffect IP3 nd IP2 levels, nd slightly decresed IP, levels in islets incuted in the presence of 15 mm glucose (Tle 2). Neither ATP nor ADP levels were ffected y genistein in the presence of 15 mm glucose or of 10 mm glucose with high K nd dizoxide. The rtio ATP/ADP ws slightly decresed only in the ltter condition (Tle 2). Effects of didzein nd genistin Didzein is n nlogue of genistein reported to e without effect on tyrosine kinses (Akiym & Ogwr, 1991). At concentrtion of 50 LM, didzein incresed insulin relese from islets perifused with medium contining 15 mm glucose. This effect ws similr to tht produced y 501M genistein (10.7 ± 0.7 nd 11.5 ± 1.1 ng per islet 30min-', n = 4). Insulin relese induced y 7 mm glucose, 30 mm K nd 0.25 mm dizoxide (6.8 ± 0.2 ng per islet 30 min-', n = 4) ws lso similrly potentited y 100 tim didzein nd genistein (13.8 ± 1.1 nd 15.6 ± 2.0 ng per islet 30 min-', respectively). The effects of 100 1sM didzein on K+ATP chnnels were vrile s were those of 25 jlm genistein. Both slight ctivtion nd inhiition were oserved. The effects of didzein on cytoplsmic [C2+]i could not e tested ecuse the fluorescence of the drug interfered with the mesurements. C2' currents were inhiited y didzein, ut this inhiition ws smller thn tht produced y genistein (41 ± 11%, n = 4). In contrst to didzein, genistin, nother nlogue of genistein without effect on tyrosine kinses (Akiym & Ogwr, 1991), did not potentite glucoseinduced insulin relese (dt not shown). 30 Effects of tyrphostin A47, herimycin A nd n ersttin I nlogue Three tyrosine kinse inhiitors other thn genistein were tested on insulin relese t concentrtions selected ccording to their potency on the kinse (Levitzki et l., 1991; Uehr & Fukzw, 1991; Umezw & Imoto, 1991). In these experiments, the islets were incuted for 60 min in medium contining 15 mm glucose. As compred to controls insulin relese ws decresed to 48 ± 6% (n= 15, P<0.01) y 100 gtm tyrphostin A47, incresed to % (n = 15, P <0.05) y 2 gam herimycin A nd not ffected (93 ± 9%, n = 1 y 100 LM of the ersttin nlogue. Discussion In the present study we show tht the tyrosine kinse inhiitor, genistein, produces numer of sometimes prdoxicl effects in mouse islets, which result in mrked potentition of insulin relese. This potentition occurred over rnge of concentrtions (3-100,M) which encompsses the IC50 vlue (-25 LM) for the inhiition y genistein of tyrosine kinses (Akiym & Ogwr, 1991). Although Tle 2 Effects of genistein on cyclic AMP, inositol phosphte nd denine nucleotide levels in mouse incuted islets Experimentl conditions Cyclic AMP IPI IP2 IP3 ATJp ADP ATP/ADP (mm) (fmol per islet) (d.p.m. per islet) (pm ol per islet) Glucose 15 + genistein 0.1 G1O + K 30 +dizoxide genistein ± ± ±6 31 ±2 34± ± ± 5 30 ± 3 34 ± ± ± ± ± ± ± ± ± ± ± ± 0.05 The islets were incuted for 1 h in medium contining 15 mm glucose or 10 mm glucose (G), 30 mm K nd 0.25 mm dizoxide in the sence or presence of 100 gm genistein. At the end of the incution, cyclic AMP, inositol phosphtes nd denine nucleotides were mesured s descried in the Methods section. Vlues re mens ± s.e.men for tches of islets from 4-5 seprte experiments. p <0.05, p<0.01 versus controls without genistein, y unpired t test. 877

7 878 J.C. Jons et l cytotoxic effects of genistein hve sometimes een reported (Linssier et l., 199, we cn exclude the possiility tht the increse in insulin relese is due to dmge of P-cells for the following resons. The effect of genistein showed sturtion t high concentrtion, ws reversile upon removl of the drug, ws dependent on the presence of metolizle sustrte, nd ws inhiited y drenline or omission of extrcellulr C2+. ws prcticlly without effect in the presence of non-stimultory concentrtion of glucose. This my men tht the effect of genistein on insulin relese cn mnifest itself only if the concentrtion of glucose is high enough, or if insulin relese is lredy stimulted. The ltter condition is not sufficient ecuse genistein ws ineffective when insulin relese ws stimulted y rium or high K in the sence of glucose, nd ecme effective when glucose ws present in the high K medium. On the other hnd, glucose is not prerequisite, since it could e replced y mixture of leucine nd glutmine. The requirements for the cute effects of genistein re thus n lredy stimulted relese nd the vilility of fuel for P-cells. Blockde of K'ATP chnnels in the plsm memrne of P-cells is key step in the stimultion of insulin relese y glucose nd y mny phrmcologicl gents, in prticulr the hypoglycemic sulphonylures (Ashford, 1990; Henquin, 199. This lockde cuses depolriztion of the memrne, with susequent opening of voltge-dependent C2` chnnels, ccelertion of C2` influx nd rise of the cytosolic C2+. inhiited K+ATP chnnels in P-cells, s indicted y the decrese in 86R efflux tht the drug produced in the presence of low concentrtion of glucose, nd directly demonstrted y the ptch-clmp experiments. This lockde my explin the smll depolriztion of P-cells produced y genistein in the presence of 3 mm glucose. On the other hnd, it cnnot explin the potentition of insulin relese for severl resons. First, the effects of genistein nd tolutmide on insulin relese were dditive, even when tolutmide ws used t mximlly effective concentrtion tht locks ll K+ATP chnnels. Second, t concentrtion of 100 fim, genistein did not ntgonize the inhiition of insulin relese rought out y the opening of K+ATP chnnels with dizoxide. However, genistein incresed insulin relese induced y high K in the presence of dizoxide (provided fuel ws present). Third, in contrst to ll gents tht inhiit K+ATP chnnels, genistein did not increse cytoplsmic C2" in P cells. On the contrry, genistein decresed the concentrtion of cytoplsmic C2" in islet cells, oth in the presence of 15 mm glucose or when the memrne ws depolrized y high K in the presence of dizoxide. This decrese cn e scried to n inhiition of voltge-dependent C2` chnnels, s shown y the inhiition of the C2+-dependent electricl ctivity induced y glucose, nd y the lock of voltge-ctivted C2+ currents in P-cells. The potentition of insulin relese y genistein is thus very unusul, in tht it occurs in spite of decrese in cytoplsmic C2+. This mens tht the drug somehow sensitizes the relesing mchinery to the ction of [C2+]j. Three known mechnisms re possile. First, ctivtion of the cyclic AMP-dependent protein kinse A increses insulin relese prtly y incresing the efficcy of C2+ on the exocytotic process (Tmgw et l., 1985; Jones et l., 1988). In the MIN 6 cell line, genistein concomitntly incresed insulin relese nd cyclic AMP levels (Ohno et l., 1993). This ws not the cse in norml mouse islets, in which genistein did not ffect cyclic AMP levels in the presence or sence of isoutyl-methylxnthine. It is lso unlikely tht genistein ctivtes the kinse directly, since the drug (in contrst to cyclic AMP) ws without effect on insulin relese y permeilized rt islets (S.J. Persud nd P.M. Jones, personl communiction). Second, ctivtion of protein kinse C y phorol esters increses insulin relese while lowering cytoplsmic C2+ stimultion of insulin relese (Go et l., 1994). However, high concentrtions of genistein do not stimulte ut wekly inhiit the kinse in cell-free systems (Akiym & Ogwr, 1991). Moreover, in contrst to phorol esters (Tmgw et l., 1985; Jones et l., 1988) genistein does not increse insulin relese from permeilized islets (S.J. Persud nd P.M. Jones, personl communiction). This rgues ginst direct stimultion of PKC y the drug. It is lso cler tht phorol esters do not hve the effects of genistein on K+ or C2" chnnels nd on the memrne potentil of mouse P cells (Bozem et l., 1987; Henquin et l., 1989). The lck of effect of genistein on inositol phosphte production lso indictes tht genistein does not increse dicylglycerol production, t lest from phosphoinositides. Third, we nd others hve recently reported tht, esides its ction on K'ATP chnnels nd the P-cell memrne potentil, glucose cn increse insulin relese y ugmenting the effectiveness of C2+ on its intrcellulr trgets (Geml et l., 1992; Sto et l., 1992). The iochemicl nture of this novel effect hs yet to e estlished, ut severl lines of evidence indicte tht chnges in the ATP/ADP rtio re involved (Geml et l., 1993). This is not the cse for the potentiting ction of genistein ecuse the drug ws either without effect or slightly decresed the ATP/ADP rtio in islet cells. nd severl other tyrosine kinse inhiitors lso inhiit lctte trnsport (Young et l., 1993). Inhiition of lctte efflux from islet cells hs sometimes een reported to increse insulin relese (Best et l., 1991), ut this oservtion hs not een explined. We do not elieve tht such n effect medites the ction of genistein ecuse the drug incresed insulin relese not only in the presence of glucose, ut lso in the presence of leucine nd glutmine which do not increse lctte production in islet cells (Mlisse et l., 1982). Severl recent studies hve reported tht genistein ffects the concentrtion of cytoplsmic [C2+], in other cell types. There is generl greement tht genistein inhiits the rise in [C2+], tht thromin produces in pltelets, ut the underlying mechnisms re still disputed. In one study, the inhiitory effect of genistein ws scried to n interference with inositol phosphte metolism (Ozki et l., 1993). In two other studies, genistein did not ffect the initil moiliztion of [C2+], (suggesting tht inositol phosphte formtion ws unltered) ut inhiited the delyed nd more sustined rise in C2+ requiring C2+ influx (Ashi et l., 1992; Srgent et l., 1993). A similr oservtion ws mde in firolsts stimulted with rdykinin (Lee et l., 1993). These studies, therefore, suggest tht genistein inhiits the voltge-independent C2+ influx pthwy tht is ctivted upon emptying of intrcellulr C2+ stores y gonists cting on memrne receptors. In the present study, voltgedependent C2+ chnnels were inhiited y genistein. A similr effect hs previously een oserved only in vsculr smooth muscle cells (Wijetunge et l., 1992). In pncretic P-cells, the inhiitory effects of genistein were not restricted to C2+ chnnels. The drug lso closed K'chnnels. Such n effect hs not een reported for other cell types. It is thus ecoming progressively evident tht genistein exerts numer of effects tht re not lwys clerly secondry to n inhiition of tyrosine kinses. Didzein nd genistin re often used s nlogues of genistein tht re devoid of ctivity on tyrosine kinses (Akiym & Ogwr, 1991). Didzein hs een reported not to mimic the effects of genistein in severl systems, e.g. C2+ chnnels on or C21 fluxes (Wijetunge et l., 1992; Srgent et l., 1993; Lee et l., 1993), nd this lck of effect of didzein ws interpreted s evidence in support of role of tyrosine kinses in the process ffected y genistein. On the other hnd, didzein reproduced the inhiitory effect of genistein on the responses of pltelets to thromoxne A2 (Nkshim et l., 1991). In P-cells, didzein ws slightly less potent thn genistein on K'nd C2+ chnnels, ut incresed

8 insulin relese in similr wy. Thus, lthough genistin ws without effect, we cnnot scrie the potentition of insulin relese y genistein to n inhiition of tyrosine kinses. The use of other tyrosine kinse inhiitors did not clrify the issue since tyrphostin A47 inhiited insulin secretion, herimycin A incresed it nd n ersttin nlogue ws without effect. In conclusion, genistein, which is currently widely used s selective inhiitor of tyrosine kinses, exerts numer of heretofore unrecognized effects. The present study hs severl importnt implictions. First, gret cution should e exercised efore concluding tht iologicl process inhiited y genistein is under direct control of tyrosine kinses. Second, the mode of ction of genistein in pncretic P-cells is rther J.C. Jons et l stimultion of insulin relese 879 unusul. The mechnisms y which the drug increses insulin relese in spite of decrese in [C2+]i nd without ctivting the known potentiting pthwys certinly clls for further explortion. Third, whether tyrosine kinses re involved in stimulus-secretion coupling in norml -cells remins to e estlished. This work ws supported y Grnts nd LN from the Fonds de l Recherche Scientifique Medicle, Brussels nd y Grnt SPPS-AC 89/ from the Ministry of Scientific Policy, Brussels. J.C.J. nd P.G. re 'Chrge de Recherches' of the Fonds Ntionl de l Recherche Scientifique, Brussels. References ABLER, A., SMITH, J.A., RANDAZZO, P.A., ROTHENBERG, P.L. & JARETT, L. (1992). differentilly inhiits postreceptor effects of insulin in rt dipocytes without inhiiting the insulin receptor kinse. J. Biol. Chem., 267, AKIYAMA, T. & OGAWARA, H. (1991). Use nd specificity of genistein s inhiitor of protein-tyrosine kinses. Methods Enzymol., 201, ASAHI, M., YANAGI, S., OHTA, S., INAZU, T., SAKAI, K., TAKEUCHI, F., TANIGUCHI, T. & YAMAMURA, H. (1992). Thromin-induced humn pltelet ggregtion is inhiited y protein-tyrosine kinse inhiitors, ST638 nd genistein. FEBS Lett., 309, ASHFORD, M.L.J. (199. 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