Keywords ATP. GK rat. Na + /K + -ATPase. Pancreatic islet. ROS. Src

Transcription

1 Dietologi (28) 51: DOI 1.17/s x ARTICLE ctivtion genertes rective oxygen species nd impirs metolism secretion coupling in dietic Goto Kkizki nd ouin-treted rt pncretic islets R. Kominto & S. Fujimoto & E. Muki & Y. Nkmur & K. Ne & M. Shimodhir & Y. Nishi & S. Funkoshi & Y. Seino & N. Ingki Received: 18 Ferury 28 /Accepted: 16 Mrch 28 / Pulished online: 1 My 28 # Springer-Verlg 28 Astrct Aims/hypothesis N + /K + -ATPse inhiition y ouin suppresses ATP production y generting rective oxygen species (ROS) nd impirs glucose-induced insulin secretion from pncretic islets. To clrify the signl-trnsducing function of N + /K + -ATPse in decresing ATP production y the genertion of ROS in pncretic islets, the involvement of ws exmined. In ddition, the significnce of ctivtion in dietic islets ws exmined. Methods Isolted islets from Wistr rts nd dietic Goto Kkizki (GK) rts ( model for dietes) were used. ROS ws mesured y 5-(nd 6)-chloromethyl-2,7 -dichlorofluorescein fluorescence using dispersed islet cells. After lystes were immunoprecipitted y nti- ntiody, immunolotting ws performed. Results Ouin cused rpid Tyr 418 phosphoryltion, indicting ctivtion of in the presence of high glucose. The specific inhiitor 4-mino-5-(4-chlorophenyl)-7- (t-utyl)pyrzolo[3,4-d]pyrimidine (PP2) restored the ouin-induced decrese in ATP content nd the increse in ROS production. Both PP2 nd ROS scvenger restored the impired insulin relese nd impired ATP elevtion in GK islets, ut hd no such effect in control islets. PP2 reduced R. Kominto : S. Fujimoto () : E. Muki : Y. Nkmur : K. Ne : M. Shimodhir : Y. Nishi : S. Funkoshi : N. Ingki Deprtment of Dietes nd Clinicl Nutrition, Grdute School of Medicine, Kyoto University, 54 Shogoin Kwhr-cho, Skyo-ku, Kyoto , Jpn e-mil: fujimoto@met.kuhp.kyoto-u.c.jp Y. Seino Knsi Electric Power Hospitl, Osk, Jpn the high glucose-induced increse in ROS genertion in GK islet cells ut hd no effect on tht in control islet cells. Moreover, ouin hd no effect on ATP content nd ROS production in the presence of high glucose in GK islets. Conclusions/interprettion These results indicte tht plys role in the signl-trnsducing function of N + /K + - ATPse, in which ROS genertion decreses ATP production in control islets. Moreover, ROS generted y ctivtion plys n importnt role in impired glucoseinduced insulin secretion in GK islets, in which is endogenously ctivted independently of ouin. Keywords ATP. GK rt. N + /K + -ATPse. Pncretic islet. ROS. Arevitions ΔΨ m chnge in mitochondril memrne potentil CM-DCF 5-(nd 6)-chloromethyl-2,7 -dichlorofluorescein FCCP cronyl cynide p-trifluoromethoxyphenylhydrzone GK Goto Kkizki JC-1 5,5,6,6 -tetrchloro-1,1,3,3 -tetrethylenzimidzolcrocynine iodide KRBB Kres Ringer icronte uffer ROS rective oxygen species PP2 4-mino-5-(4-chlorophenyl)-7-(t-utyl)pyrzolo[3,4-d]pyrimidine Introduction In pncretic et cells, intrcellulr glucose metolism regultes exocytosis of insulin grnules ccording to metolism secretion coupling, in which glucose-induced

2 Dietologi (28) 51: mitochondril ATP production plys n essentil role [1]. Since depletion of mitochondril DNA olishes the glucose-induced ATP elevtion, mitochondri clerly re mjor source of ATP production in pncretic et cells [2, 3]. Glucose-induced insulin secretion from et cells is often impired y exposure to high concentrtions of fuels including glucose, NEFAs nd ketone odies, nd y dministrtion of dietogenic phrmcologicl gents, ll of which involve impired glucose-induced ATP elevtion in et cells [4 11]. Thus, reduced mitochondril ATP production plys n importnt role in impired glucoseinduced insulin secretion. Among the vrious gents tht impir metolism secretion coupling in et cells, the effects of rective oxygen species (ROS) on glucose-induced insulin secretion hve een extensively exmined. Exposure to exogenous hydrogen peroxide (H 2 O 2 ), the most undnt ROS, reduces glucose-induced insulin secretion y impiring mitochondril metolism in et cells. Trnsient exposure to H 2 O 2 suppresses the hyperpolristion of mitochondril memrne potentil [12], the increment in insulin secretion, nd the increse in ATP content induced y glucose in pncretic et cells [12, 13]. However, little is known of the role of endogenous ROS in impired glucose-induced insulin secretion. Recent studies hve shown tht mitochondri produce endogenous ROS in et cells under physiologicl nd pthophysiologicl conditions. Exposure to high glucose increses mitochondril ROS production [14, 15], nd the superoxide content of islets from Zucker dietic ftty rts is higher thn tht from Zucker len control islets under sl level of glucose ut re reltively insensitive to high glucose [14]. Ouin, well-known specific inhiitor of N + /K + - ATPse, decreses glucose-induced insulin relese in the second phse [16]. We hve found tht ouin decreses glucose-induced insulin relese y reducing ATP content [17]. In ddition, high glucose-induced hyperpolristion of mitochondril memrne potentil ws inhiited y ouin. Furthermore, ouin induced mitochondril ROS production tht ws locked y myxothizol, n inhiitor of site III of the mitochondril respirtory chin. Interestingly, these phenomen lso occurred in C 2+ -or N + -depleted conditions. An ntioxidnt, α-tocopherol, locked the ouin-induced ROS increse s well s the suppressive effect of ouin on ATP production nd insulin relese. However, ouin did not directly ffect ATP production from the mitochondril frction. These results suggest tht ouin suppresses mitochondril ATP production y generting mitochondril ROS vi signl trnsduction, independently of the intrcellulr ctionic lterntion, nd hs suppressive effect on insulin secretion. However, the detils of N + /K + -ATPse-medited signl trnsduction in suppressing ATP production y the genertion of mitochondril ROS in pncretic islets remin unknown. The inding of ouin to N + /K + -ATPse hs een shown to ctivte, non-receptor protein-tyrosine kinse, susequently enhncing mitochondril ROS production in crdic myocytes [18 2]. In the present study, we investigted the involvement of in the signltrnsducing function of N + /K + -ATPse tht reduces ATP production y generting mitochondril ROS in pncretic islets. In ddition, the role of ctivtion in impired glucose-induced insulin secretion from dietic islets ws exmined. Methods Animls Mle Wistr nd Goto Kkizki (GK) rts were otined from Shimizu (Kyoto, Jpn). The nimls were fed stndrd lortory chow d liitum nd llowed free ccess to wter in n ir-conditioned room with 12 h light:12 h drkness cycle until used in the experiments. All experiments were crried out with rts ged 8 12 weeks. The nimls were mintined nd used in ccordnce with the Guidelines for Animl Experiments of Kyoto University. Islet isoltion nd culture Islets of Lngerhns were isolted from Wistr nd GK rts y collgense digestion s descried previously [21]. Isolted islets were cultured for 12 h in RPMI 164 medium contining 1% (vol./vol.) FCS, 1 U/ml penicillin, 1 μg/ml streptomycin nd 5.5 mmol/l glucose, t 37 C in humidified ir contining 5% CO 2. Solutions The medium used for islet isoltion nd preincution of intct islets ws Kres Ringer icronte uffer contining (in mmol/l) NCl, 3.7 KCl, 2.7 CCl 2, 1.3 KH 2 PO 4, 1.3 MgSO 4, 24.8 NHCO 3 (equilirted with 5% CO 2 95% O 2, ph 7.4), nd.2% (vol./vol.) BSA, herefter referred to s KRBB. C 2+ -free medi were prepred with C 2+ -free KRBB plus 1 mmol/l EGTA nd 1 mmol/l HEPES (C 2+ -free KRBB). Mesurement of ATP content After groups of ten islets were preincuted in KRBB with 2.8 mmol/l glucose for 3 min, they were tch-incuted for the indicted times in.5 ml C 2+ -free KRBB with 2.8 or 16.7 mmol/l glucose with or without test mterils. 4-mino-5-(4-chlorophenyl)-7- (t-utyl)pyrzolo[3, 4-d]pyrimidine (PP2) nd α-tocopherol plus scorte were lso included during preincution. After immedite ddition of HClO 4,sonictioninice-cold wter for 3 min, nd centrifugtion, prt of the superntnt frction ws mixed with HEPES nd N 2 CO 3 nd the ATP

3 1228 Dietologi (28) 51: content in islets ws determined y luminometry s previously descried [22]. Fluorescence mesurement of ROS production nd chnge in mitochondril memrne potentil ROS production nd chnge in mitochondril memrne potentil (ΔΨ m ) in dispersed islet cells under C 2+ -free conditions were mesured y 5-(nd 6)-chloromethyl-2,7 -dichlorofluorescein (CM-DCF) fluorescence nd 5,5,6,6 -tetrchloro- 1,1,3,3 -tetrethylenzimidzolcrocynine iodide (JC-1) fluorescence, respectively, s previously reported [17]. Fluorescence ws corrected y sutrcting prllel lnks in which islet cells were not loded with proes, nd is presented s rtio with respect to the vlue t time zero. Mesurement of phosphoryltion of Activtion of in islets ws determined y Western lotting fter immunoprecipittion. After preincution in KRBB contining 2.8 mmol/l glucose, islets were exposed to ouin in C 2+ -free KRBB or KRBB with 16.7 mmol/l glucose for the indicted times. After wshing with ice-cold PBS, the islets were soluilised in ice-cold lysis uffer contining 1 mmol/l Tris HCl (ph 7.2), 1 mmol/l NCl, 1 mmol/l EDTA, 5 mmol/l sodium pyrophosphte,.5% sodium deoxycholte, 1% Nonidet P-4, protese inhiitor cocktil tlet (Roche, Penzerg, Germny) nd phosphtse inhiitor cocktil set II (Cliochem, Drmstdt, Germny) nd sonicted. Cell lystes were centrifuged (56, g for 1 min t 4 C) to otin crude cell extrcts. Protein content of the superntnt ws mesured nd djusted y the Brdford method. The superntnt ws mixed with 4 μg monoclonl nti- ntiody (mouse monoclonl IgG 1, clone GD11; Upstte, Lke Plcid, NY, USA) nd 3 μl of wshed Protein-G grose eds, nd gently rotted for 4 h t 4 C. After wshing three times with ice-cold lysis uffer, immunoprecipittes were dissolved in 3 μl SDS-PAGE smple uffer (5 mmol/l Tris HCl [ph 6.8], 2% SDS, 6% 2-mercptoethnol, 1% glycerol, 1% romophenol lue) nd oiled for 5 min t 95 C. The smples were sujected to electrophoresis on 1% SDS-polycrylmide gels nd trnsferred onto nitrocellulose memrne (Schleicher nd Schuell, Keene, NH, USA). After locking with PBS contining.1% Tween 2 nd 5% BSA (locking uffer) overnight t 4 C, lotted memrnes were incuted overnight with rit polyclonl nti- ntiody (Snt Cruz Biotechnology, Snt Cruz, CA, USA) t 4 C in locking uffer, nd susequently with nti-rit IgG horserdish peroxidse-conjugted secondry ntiody (Amershm Biosciences, Tokyo, Jpn) for 1 h prior to detection using ECL Plus (Amershm Biosciences). In the sme memrne, the process ws repeted for the following primry phosphospecific ntiodies: rit polyclonl ntiody to phosphorylted t Tyr 418 (py 418 ) or rit polyclonl ntiody to phosphorylted t Tyr 529 (py 529 ; Biosource, Cmrillo, CA, USA) nd mouse polyclonl nti-phosphotyrosine ntiody (py; clone 4G1; Upstte). Anti-mouse IgG horserdish peroxidse-conjugted secondry ntiody (Amershm Biosciences) ws used to detect the mouse primry ntiody. Mesurement of glucose oxidtion Glucose oxidtion ws mesured s previously descried [8]. Cultured islets were preincuted in KRBB with 2.8 mmol/l glucose in the presence or sence of inhiitor nd ntioxidnts t 37 C for 3 min. Twenty-five islets in smll tue were incuted t 37 C for 9 min in 15 μl C 2+ -free KRBB contining 2.8 or 16.7 mmol/l glucose, test mterils, nd [U- 14 C]glucose ( Bq per tue) (Amershm, Buckinghmshire, UK). After 9 min incution, the rection ws stopped, nd the dpm of trpped 14 CO 2 in the hydroxide of hymine 1-X (Pckrd, Meriden, CT, USA) ws counted. Mesurement of insulin relese, insulin content nd DNA content Insulin relese from cultured islets ws monitored using sttic incution s descried previously [17]. After n liquot of incution medium for insulin ssy ws tken, the islets remining were lysed to determine insulin nd DNA contents s descried previously [22]. Mterils RPMI 164 medium, cronyl cynide p-trifluoromethoxyphenylhydrzone (FCCP), α-tocopherol nd L- scoric cid were purchsed from Sigm (St Louis, MO, USA). Luciferin-luciferse ws otined from Turner Designs (Sunnyvle, CA, USA). CM-DCFH dicette nd JC-1 were purchsed from Invitrogen (Eugene, OR, USA). PP2, herimycin A nd SU6656 were purchsed from Cliochem (L Joll, CA, USA). All other gents including ouin were otined from Ncli Tesque (Kyoto, Jpn). Sttisticl nlysis Results re expressed s mens±se. Sttisticl significnce ws evluted y n unpired Student s t test. p<.5 ws considered significnt. Results Effect of ouin on ATP content Exposure to 16.7 mmol/l glucose for 15 min incresed ATP content compred with tht in the presence of 2.8 mmol/l glucose (t 15 min, 16.7 mmol/l glucose: 17.1±.9 vs 2.8 mmol/l glucose: 8.5±.2 pmol/islet; p<.1; Fig. 1). For the 6 min incution, ATP content remined high in the presence of 16.7 mmo/l glucose compred with tht in the presence of 2.8 mmol/l glucose. Exposure to 1 mmol/l ouin for

4 Dietologi (28) 51: ATP (pmol/islet) c ATP (pmol/islet) Time (min) min did not suppress ATP content in the presence of 16.7 mmo/l glucose (t 15 min, 16.7 mmol/l glucose plus ouin: 15.6±.2 pmol per islet vs 16.7 mmol/l glucose; p= NS), ut such exposure for 3 min decresed ATP content in the presence of 16.7 mmo/l glucose (t 3 min, 16.7 mmol/l glucose plus ouin: 14.9±.4 vs 16.7 mmol/l glucose: 18.±.4 pmol per islet; p<.1; Fig. 1). Furthermore, n exposure for 6 min profoundly suppressed ATP content t high glucose (t 6 min, 16.7 mmol/l glucose plus ouin: 1.6±.6 vs 16.7 mmol/l glucose: 18.5±.6 pmol per islet; p<.1; Fig. 1). In the presence of 1 μmol/l PP2, inhiitor, 1 mmol/l ouin filed to suppress ATP content in the presence of 16.7 mmol/l glucose (16.7 mmol/l glucose plus ouin with PP2: 17.2±.9 vs 16.7 mmol/l glucose with ATP (pmol/islet) ATP (pmol/islet) 2 1 PP2 Her SU Fig. 1 Effects of inhiitors on ATP contents t high glucose fter exposure to ouin. Time-course of ouin-induced decrese of ATP contents. After preincution with 2.8 mmol/l glucose, islets were incuted t 2.8 mmol/l glucose (white circles) or 16.7 mmol/ l glucose with (lck squres) or without (lck circles) 1 mmol/ l ouin for the indicted times in C 2+ -depleted condition, nd ATP contents were determined. Vlues re mens±se (n=5). p<.1 vs 16.7 mmol/l glucose. d Effects of inhiitors on ouin-induced decrese of ATP contents in high glucose. After preincution with 2.8 mmol/l glucose with or without inhiitors, islets were incuted for 6 min t 2.8 mmol/l glucose (white rs) or 16.7 mmol/l glucose with (htched rs) or without (lck rs) ouin in the presence or sence of inhiitors under C 2+ - depleted condition, nd ATP contents were determined. Effect of 1 μmol/l PP2. c Effect of 1 μmol/l herimycin A (Her). d Effect of 5 μmol/l SU6656 (SU). Vlues re mens±se of n=8 (), n=1 (c) nd n=8 (d) determintions. p<.1 vs 2.8 mmol/l glucose; p<.1 vs 16.7 mmol/l glucose; p<.1 vs 16.7 mmol/l glucose plus ouin without inhiitors d PP2: 16.2±.9 pmol per islet; p=ns) (Fig. 1). ATP content in ouin-treted islets t high glucose in the presence of PP2 ws lrger thn tht in the sence of PP2 (16.7 mmol/ l glucose plus ouin with PP2 vs 16.7 mmol/l glucose plus ouin: 12.3±.5 pmol per islet; p<.1). Similr results were oserved in experiments using other inhiitors (Fig. 1c,d). Effect of ouin on ROS production Exposure to 1 mmol/ l ouin for 15 min did not increse CM-DCF fluorescence, which represents ROS production, in the presence of 16.7 mmo/l glucose (t 15 min, 16.7 mmol/l glucose plus ouin: 1.23±.11 vs 16.7 mmol/l glucose: 1.8±.13 reltive units; p=ns; Fig. 2). However, such exposure for 3 or 6 min ugmented CM-DCF fluorescence in the presence of 16.7 mmo/l glucose (t 3 min, 16.7 mmol/ l glucose plus ouin: 1.58±.1 vs 16.7 mmol/l glucose: 1.24±.7 reltive units; p<.5; t 6 min, 16.7 mmol/ l glucose plus ouin: 1.71±.12 vs 16.7 mmol/l glucose: 1.29±.4 reltive units; p<.5; Fig. 2). In the presence of 1 μmol/l PP2, 1 mmol/l ouin did not increse CM- DCF fluorescence in the presence of 16.7 mmol/l glucose (16.7 mmol/l glucose plus ouin with PP2: 1.31±.7 vs 16.7 mmol/l glucose with PP2: 1.33±.5 reltive units; p= NS) (Fig. 2). PP2 reduced CM-DCF fluorescence of islet cells in the presence of 16.7 mmol/l glucose nd 1 mmol/ l ouin (16.7 mmol/l glucose plus ouin with PP2 vs 16.7 mmol/l glucose plus ouin: 1.63±.8 reltive units; p<.5; Fig. 2). CM-DCF fluorescence (ritrry units) Time (min) CM-DCF fluorescence (ritrry units) PP2 + + Fig. 2 Effects of inhiitor on ROS production t high glucose fter exposure to ouin. Time-course of ouin-induced increse of ROS production. After fluorescence mesurements t time zero, the dispersed islet cells were incuted for the indicted times, with (squres) or without (circles) 1 mmol/l ouin in the presence of 16.7 mmol/l glucose under C 2+ -depleted conditions. Vlues re mens±se (n=4) s rtio of vlues t time zero. p<.5 vs 16.7 mmol/l glucose. Effects of inhiitor (PP2) on ouininduced increse of ROS production t high glucose. After CM-DCF fluorescence ws determined t time zero, islet cells were incuted for 6 min with 16.7 mmol/l glucose with (htched rs) or without (lck rs) 1 mmol/l ouin in the presence or sence of 1 μmol/ l PP2 under C 2+ -depleted conditions, nd fluorescence ws mesured t 6 min. Vlues re mens±se (n=4) s rtio of vlues t time zero. p<.5 vs 16.7 mmol/l glucose. p<.5 vs 16.7 mmol/ l glucose plus ouin without PP2

5 Amount reltive to control (fold) 123 Dietologi (28) 51: Amount reltive to control (fold) Time (min) 2 IB totl IB py 529 IB py 418 IB py IP Control Ouin d phospho- (Tyr 529) phospho- (Tyr 418) phospho-tyr IgG (hevy chin) c IP 2 IB totl 1 IB py 418 Scr Control Ouin 1 µmol/l Ouin 1 mmol/l Fig. 3 Ouin-induced tyrosine phosphoryltion in islets under C 2+ -deprived condition in the presence of 16.7 mmol/l glucose. Time-course of ouin-induced tyrosine phosphoryltion. After preincution with 2.8 mmol/l glucose, islets were incuted with or without 1 mmol/l ouin in the presence of 16.7 mmol/l glucose under C 2+ -depleted conditions for the indicted times. Islets were then lysed, immunoprecipitted with n nti- ntiody, nd ssyed for tyrosine phosphoryltion y Western lotting using Tyr 418 phosphospecific ntiody (py 418, white circles), Tyr 529 phosphospecific ntiody (py 529, white squres) or phosphotyrosine ntiody (py, lck squres) y repetition of stripping nd reproing for the sme lot. To ensure equl loding, totl ntiody (totl, lck circles) ws lso reproed. Dt re expressed reltive to control (16.7 mmol/l glucose without ouin) phospho- (Tyr 418) Totl py 529 py 418 Ouin 1 mmol/l py Totl py 418 Ouin 1 µmol/l vlue (mens±se, min: n=3, 1 min: n=3, 3 min: n=8, 5 min: n=4). p<.1 vs control (16.7 mmol/l glucose without ouin). Representtive immunolots (IB) for totl ntiody, Tyr 418 or Tyr 529 phosphospecific ntiodies (py 418 nd py 529 ) nd phosphotyrosine ntiody (py) t 3 min in the sme memrne. In the py immunolot, lots of IgG hevy chin derived from ntiody used during immunoprecipittion were lso oserved. c Dose-dependent effect of ouin on the level of tyrosine phosphoryltion in islets. Representtive immunolot (IB) for totl ntiody nd py 418 ntiody t 3 min in the sme memrne. d Quntifiction dt re expressed s mens±se of n=6 (1 μmol/l ouin), n=8 (1 mmol/ l ouin) determintions reltive to control (16.7 mmol/l glucose without ouin) vlues. p<.1 vs control (16.7 mmol/l glucose without ouin). IP, immunoprecipitted Effect of ouin on phosphoryltion ctivity is regulted y the phosphoryltion of Tyr 418 nd Tyr 529. Either decrese in phosphoryltion of Tyr 529 or n increse in phosphoryltion of Tyr 418 stimultes kinse ctivity. Ouin (1 mmol/l) cused rpid ctivtion of in the presence of 16.7 mmol/l glucose under C 2+ - deprived conditions. The mximum increse in Tyr 418 phospholyrtion ws oserved 3 min fter ouin exposure (Fig. 3). Ouin cused significnt increse in Try 418 nd totl tyrosine phosphoryltion, ut hd no effect on Try 529 phosphoryltion (t 3 min, fold increse reltive to control, py 418 : 1.79±.15, p<.1 vs control; totl tyrosine phosphoryltion (py): 2.17±.26, p<.1 vs control; py 529 : 1.9±.4, p=ns vs control; totl :.96±.3, p=ns vs control) (Fig. 3,d). A dose-dependent effect of ouin on Tyr 418 phosphoryltion ws lso oserved (1 μmol/l ouin, t 3 min, fold increse reltive to control, py 418 : 1.47±.5, p<.1 vs control; totl : 1.2±.4, p=ns vs control; Fig. 3c,d). Such effects of ouin on phosphoryltion were lso oserved in medium contining physiologicl concentrtion of C 2+ (fold increse reltive to control, py 418 : 1.59±.1, p<.1 vs control; py 529 : 1.7±.7, p=ns vs control; totl :.96±.6, p=ns vs control) (Fig. 4). Effect of ouin on ΔΨ m To evlute the effect of ouin on ΔΨ m, JC-1 fluorescence ws mesured in the presence of 16.7 mmol/l glucose without C 2+ (Fig. 5). After ddition of 16.7 mmol/l glucose to the medium, fluorescence incresed grdully, indicting hyperpolristion of mitochondril memrne potentil, wheres the sl level of fluorescence ws unchnged in the presence of 2.8 mmol/l glucose. Ouin (1 mmol/l) significntly inhiited glucose-induced hyperpolristion of mitochondril memrne potentil 3 min fter dministrtion (t 3 min, 16.7 mmol/l glucose plus ouin: 1.4±.3 vs 16.7 mmol/l glucose: 1.51±.5 reltive units; p<.1). However, in the presence of 1 mmol/l ouin with 16.7 mmol/l glucose, 1 μmol/l PP2 reversed the effect of ouin on ΔΨ m nd incresed JC-1 fluorescence 3 min

6 Dietologi (28) 51: IB totl IB py 529 IB py 418 Control IP Ouin Fig. 4 Ouin (1 mmol/l)-induced tyrosine phosphoryltion in islets in medium contining physiologicl concentrtion of C 2+ (2.8 mmol/l) in the presence of 16.7 mmol/l glucose. Representtive immunolot (IB) for totl ntiody nd py 418 or py 529 phospho- (Tyr 529) phospho- (Tyr 418) Amount reltive to control (fold) 2 1 Totl py 529 py 418 ntiodies t 3 min in the sme memrne. Quntifiction dt from n=5 independent experiments. Dt re expressed reltive to control vlues (mens±se). p<.1 vs control (16.7 mmol/l glucose without ouin). IP, immunoprecipitted fter dministrtion (16.7 mmol/l glucose plus ouin with PP2: 1.41±.7 vs 16.7 mmol/l glucose plus ouin: 1.4±.3 reltive units; p<.1). JC-1 fluorescence decresed to elow the sl level fter the ddition of 1 μmol/l FCCP. Effect of ouin on glucose oxidtion Glucose oxidtion in islets in the presence of 16.7 mmol/l glucose ws incresed compred with tht in the presence of 2.8 mmol/l glucose (Fig. 6). Glucose oxidtion with 2.8 mmol/l glucose ws not ffected y 1 mmol/l ouin (ouin plus 2.8 mmol/l glucose: 7.6±1. vs 2.8 mmol/l glucose: 5.6±.6 pmol islet 1 9 min 1 ; p=ns). However, glucose oxidtion with 16.7 mmol/l glucose ws suppressed y the gent (16.7 mmol/l glucose plus ouin: 3.±4.3 vs 16.7 mmol/l glucose: 53.9±6.1 pmol islet 1 9 min 1 ; JC-1 fluorescence (ritrry units) Time (min) Fig. 5 Time-course effects of inhiitor (PP2) on ouin-induced decrese of mitochondril memrne potentil t high glucose. After JC-1 ws loded, dispersed islet cells were preincuted for 3 min t 2.8 mmol/l glucose with or without 1 μmol/l PP2. At time zero, sl fluorescence ws determined, nd islet cells were incuted for the indicted time periods in C 2+ -depleted conditions t 2.8 mmol/ l glucose (white circles) or 16.7 mmol/l glucose with (lck squres) or without (lck circles) 1 mmol/l ouin, or with 16.7 mmol/ l glucose with 1 mmol/l ouin in the presence of 1 μmol/l PP2 (lck tringles). At 6 min, 1 μmol/l FCCP ws dded to the medium. Vlues re mens±se (n=6) s rtio of vlues t time zero. p<.1, 2.8 mmol/l vs 16.7 mmol/l glucose; p<.1, 16.7 mmol/ l glucose vs 16.7 mmol/l glucose + ouin; p<.1, 16.7 mmol/ l glucose + ouin vs 16.7 mmol/l glucose + ouin + PP2 p<.1). In the presence of PP2 or α-tocopherol plus scorte, ouin did not ffect glucose oxidtion t 16.7 mmol/l glucose. Glucose oxidtion with 16.7 mmol/l glucose nd ouin in the presence of PP2 or α-tocopherol plus scorte ws lrger thn tht in the sence of PP2 nd α-tocopherol plus scorte (16.7 mmol/l glucose plus ouin with PP2: 5.2±4.5 vs 16.7 mmol/l glucose plus ouin: 3.±4.3; p<.1; 16.7 mmol/l glucose plus ouin with α-tocopherol plus scorte: 45.6± 3.2 pmol islet 1 9 min 1 vs 16.7 mmol/l glucose plus ouin; p<.1). Chrcteristics of nimls nd islets Tle 1 shows the chrcteristics of the dietes model GK rts nd control Wistr rts used in this study. GK rts hd lower ody weight thn control Wistr rts. In the fed stte, GK rts hd higher plsm glucose concentrtion. DNA content nd Glucose oxidtion (pmol islet 1 9 min 1 ) Ou Ou Ou PP2 VE + VC Fig. 6 Effects of inhiitor nd ROS scvenger on ouininduced decrese of glucose oxidtion t high glucose. After preincution with 2.8 mmol/l glucose with or without inhiitor nd ROS scvenger, islets were incuted for 9 min t 2.8 mmol/ l glucose (white rs) or 16.7 mmol/l glucose (lck rs) with or without 1 mmol/l ouin (Ou) in the presence or sence of inhiitor (1 μmol/l PP2) nd ROS scvenger (1 μmol/l α- tocopherol plus 2 μmol/l scorte, VE+VC) under C 2+ -depleted conditions, nd glucose oxidtion ws determined. Vlues re mens ±SE of n=11 determintions. p<.1 vs 16.7 mmol/l glucose; p<.1 vs 16.7 mmol/l glucose plus ouin without PP2 nd α- tocopherol plus scorte

7 1232 Dietologi (28) 51: Tle 1 Chrcteristics of control Wistr nd dietic GK rts used in the experiments Chrcteristics Control Wistr GK Bodyweight (g) 24±1 (45) 163±1 (78) Non-fsting plsm glucose 5.83±.11 (45) 8.83±.11 (78) (mmol/l) Islet DNA content (ng/islet) 13.5±.6 (8) 13.5±.7 (8) Islet insulin content (ng/islet) 21.8±.9 (8) 24.2±1. (8) Dt re mens±se for the numer of oservtions shown in prentheses p<.1 vs control Wistr rt insulin content of islets derived from GK rts did not differ from those derived from control Wistr rts. Effect of inhiition nd ROS scvenger on insulin relese nd ATP content of GK islets In the presence of 16.7 mmol/l glucose, insulin relese from GK islets ws reduced compred with control Wistr rts (GK: 1.78±.25 vs Wistr: 4.36±.23 ng islet 1 3 min 1 ; p<.1) (Fig. 7). PP2 nd α-tocopherol plus scorte hd no effect on high glucose-induced insulin relese from Wistr islets (Fig. 7,). However, high glucose-induced insulin relese from GK islets ws restored to control levels y inhiitor (16.7 mmol/l glucose with PP2: 5.5±.43 vs 16.7 mmol/l glucose: 1.78±.25 ng islet 1 3 min 1 ; p<.1) nd ROS scvenger (16.7 mmol/l glucose with α- tocopherol plus scorte: 4.22±.6 vs 16.7 mmol/l glucose: 2.13±.42 ng islet 1 3 min 1 ; p<.1; Fig. 7,). The ATP content of GK islets in the presence of 2.8 mmol/ l glucose ws not different from tht in the presence of 16.7 mmol/l glucose (2.8 mmol/l glucose: 7.±.4 vs 16.7 mmol/l glucose: 8.3±.7 pmol/islet; p=ns; Fig. 7c). In GK islets, ouin did not suppress ATP content (16.7 mmol/l glucose plus ouin: 7.7±.6 pmol/islet vs 16.7 mmol/l glucose; p=ns), while PP2 nd α-tocopherol plus scorte incresed ATP content in the presence of 16.7 mmol/l glucose (16.7 mmol/l glucose with PP2: 12.3±.7 pmol/islet vs 16.7 mmol/l glucose, p<.1; 16.7 mmol/ l glucose with α-tocopherol plus scorte: 11.±.7 pmol/ islet vs 16.7 mmol/l glucose, p=.1; Fig. 7c). Effect of inhiition nd ROS scvenger on ROS production y GK islet cells Ouin hd no effect on ROS production in the presence of high glucose in GK islet cells (t 6 min, 16.7 mmol/l glucose plus ouin: 2.19±.18 vs 16.7 mmol/l glucose: 2.42±.27 reltive units; p=ns; Fig. 8). However, PP2 nd α-tocopherol plus scorte decresed ROS production in the presence of high glucose in GK islet cells (t 6 min, 16.7 mmol/l glucose with PP2: 1.53±.8 reltive units vs 16.7 mmol/l glucose, p<.5; 16.7 mmol/l glucose with α-tocopherol plus scorte: 1.46±.4 reltive units vs 16.7 mmol/l glucose, p<.5; Fig. 8). Discussion In the present study, we show tht plys role in the signl-trnsducing function of N + /K + -ATPse, y which ROS genertion decreses ATP production in control islets. Moreover, ROS generted y ctivtion plys n importnt role in impired glucose-induced insulin secretion in GK islets, in which ctivtion is ouin independent. In pncretic et cells, ROS production vi nonmitochondril nd mitochondril pthwys hs een proposed. ROS production from non-mitochondril pthwys including the hexosmine pthwy [23], n unknown pthwy from D-glycerldehyde [24], nd NADPH oxidse [25] hve een reported. However, in most tissues, the mjor iologicl process leding to genertion of ROS is the electron trnsport chin ssocited with the mitochondril memrne [26, 27]. Recent studies hve shown tht et cells exposed to high glucose produce mitochondril ROS [14, 15]. Increse in ROS in the presence of high Insulin relese (ng islet 1 3 min 1 ) PP2 + + Wistr GK VE+VC Insulin relese (ng islet 1 3 min 1 ) Wistr Fig. 7 Effects of inhiitor nd ROS scvenger on insulin relese nd ATP contents in GK islets. After preincution with 2.8 mmol/ l glucose for 3 min, islets were incuted t 2.8 mmol/l glucose (white rs) or 16.7 mmol/l glucose (lck rs) with or without test mterils for 3 min (, ) or 6 min (c). PP2 nd α-tocopherol plus scorte were lso included during preincution. Effects of 1 μmol/l PP2 on insulin relese from control Wistr islets nd GK GK c ATP (pmol/islet) Control Ou PP2 VE+VC islets. Vlues re mens±se (n=1). p<.1 vs Wistr, 16.7 mmol/ l glucose; p<.1 vs GK, 16.7 mmol/l glucose without PP2. Effects of 1 μmol/l α-tocopherol plus 2 μmol/l scorte (VE+VC) on ATP contents in GK islets. After incution s indicted for 6 min in C 2+ -depleted conditions, ATP contents were determined. Vlues re mens±se (n=1). p<.1 vs 16.7 mmol/l glucose

8 Dietologi (28) 51: CM-DCF fluorescence (ritrry units) Time (min) glucose my e ttriutle to ΔΨ m -dependence of ROS formtion, in which n exponentil increse in ROS production is oserved ove 14 mv in mitochondril memrne potentil [28]. However, in the present study, we show for the first time tht there is n increse in mitochondril ROS production vi intrcellulr signl trnsduction in pncretic islets. Thus, ROS production vi the signl-trnsducing function of N + /K + -ATPse does not necessrily require hyperpolristion of mitochondril memrne potentil, s ouin increses ROS production while the gent simultneously inhiits hyperpolristion of mitochondril memrne potentil. is 6 kd memrne-ssocited non-receptor tyrosine kinse tht regultes vrious signl trnsduction pthwys. production is widespred nd hs een demonstrted in pncretic islets nd in et cell line [29 32]. Its ctlytic ctivity is controlled y tyrosine phosphoryltion nd protein protein interction. Phosphoryltion of Tyr 529 on holds the kinse in n inctive conformtion through n intrmoleculr interction with its homology 2 domin, wheres phosphoryltion of Tyr 418 ctivtes y disrupting the intrmoleculr interction nd creting the sustrte-inding site [33]. The inding of ouin to the N + /K + -ATPse cuses rpid ctivtion of in vrious cells including crdic myocytes [34], smooth muscle cells [34, 35] nd kidney epithelil cells [36] independently of the chnges in intrcellulr ion concentrtions. In the present study, ouin stimulted Tyr 418 phosphoryltion ut hd no effect on Tyr 529 phosphoryltion, phenomenon lso oserved in different types of cells [36]. Since ouin-induced direct interction CM-DCF fluorescence (ritrry units) Control Ou PP2 VE+VC Fig. 8 Effects of ouin, inhiitor nd ROS scvenger on ROS production t high glucose in GK islet cells. Effect of 1 mmol/l ouin on the time-course of high glucose-induced increse of ROS production. After fluorescence mesurements t time zero, the dispersed islet cells were incuted for the indicted times with (squres) or without (circles) 1 mmol/l ouin in the presence of 16.7 mmol/l glucose under C 2+ -depleted conditions. Vlues re mens±se (n=3) s rtio of vlues t time zero. Effects of 1 mmol/l ouin (Ou), 1 μmol/l PP2 nd 1 μmol/l α-tocopherol plus 2 μmol/l scorte (VE + VC) on ROS production in the presence of 16.7 mmol/l glucose in GK islet cells. After CM-DCF fluorescence ws determined t time zero, islet cells were incuted for 6 min with 16.7 mmol/l glucose in the presence or sence of test mterils under C 2+ -depleted conditions, nd fluorescence ws mesured t 6 min. Vlues re mens±se (n=3) s rtio of vlues t time zero. p<.5 vs 16.7 mmol/l glucose etween the N + /K + -ATPse α 1 suunit nd is oserved in kidney epithelil cells [36], ouin-induced direct interction etween N + /K + -ATPse nd my well e involved in ouin-induced phosphoryltion in pncretic et cells. A signl-trnsducing function of N + /K + -ATPse vi ctivtion hs een proposed recently in different types of cells including crdic myocytes, A7r5 cells nd HeL cells [37]. The inding of ouin to N + /K + -ATPse ctivtes, resulting in trnsctivtion of the EGF receptor nd incresed mitochondril production of ROS independently of chnges in intrcellulr ion concentrtions. In the present study, PP2, specific inhiitor tht reduces kinse ctivity nd Tyr 418 phosphoryltion in rt islets [32], ws found to decrese ouin-induced ROS production, indicting tht this signl-trnsducing function of N + /K + - ATPse plys role in regulting mitochondril ROS production in islets. However, the involvement of the trnsctivtion of the EGF receptor in this pthwy in islets remins unknown. In previous study, we found tht ouin reduces not only the increment in ATP content nd the hyperpolristion of mitochondril memrne potentil y glucose, ut lso the increment in O 2 consumption y glucose [17]. Since incresed O 2 consumption occurs in uncoupling [38], ouin-induced suppression of mitochondril ATP production clerly is not medited y uncoupling, nd the suppression my derive from direct or indirect effects on the respirtory chin. Ouin (1 mmol/l) ws found to reduce glucose oxidtion in the presence of 16.7 mmol/l glucose in islets in medium contining physiologicl level of C 2+ [39]. In the present study, 1 mmol/l ouin lso suppressed glucose oxidtion in the presence of 16.7 mmol/l glucose in C 2+ -depleted conditions. Since ouin-induced suppression of glucose oxidtion ws restored y ROS scvenger nd y inhiitor, incresed ROS production derived from ctivtion my well suppress mitochondril metolism in the Kres cycle, in which CO 2 is relesed in the rection medited y dehydrogenses. This is supported y the fct tht dministrtion of 5 μmol/l H 2 O 2, concentrtion nerly equivlent to the 1 mmol/l ouininduced increse in ROS production [17], to mitochondri reduced ctivity of Kres cycle enzymes including conitse, α-ketoglutrte dehydrogense nd succinte dehydrogense, whose ctivities declined 96%, 39% nd 37%, respectively [4]. Considered together, these findings suggest tht ouin-induced mitochondril ROS suppresses mitochondril metolism in the Kres cycle, susequently reducing NADH supply to the respirtory chin, hyperpolristion of mitochondril memrne potentil, O 2 consumption nd ATP production. We then investigted the role of ROS generted y ctivtion in impired glucose-induced insulin secretion in

9 1234 Dietologi (28) 51: dietes. One of the chrcteristics of type 2 dietes is tht the insulin secretory response of et cells to glucose is selectively impired [41]. In the GK rt, genetic model of type 2 dietes mellitus [42], glucose-induced insulin secretion is selectively impired [43]. On single-chnnel recording, the glucose sensitivity of the et cell K ATP chnnel is remrkly reduced in GK rts, while the inhiitory effect of ATP on chnnel ctivity is not significntly different in control nd GK rts [5]. The intrcellulr ATP elevtion induced y high glucose is impired in GK rts [44] swellsinptientswithtype2 dietes [45]. Thus, the impired insulinotrophic ction of glucose in et cells of GK rts my e ttriutle to insufficient closure of the K ATP chnnel ecuse of deficient ATP production derived from impired glucose metolism. While there is evidence tht islets in GK rts ( dietes model) nd humn type 2 dietes re oxidtively stressed [46, 47], the ssocition etween oxidtive stress nd impired intrcellulr ATP elevtion in islets is uncler. In the present study, oth inhiitor nd ROS scvenger restored the impirment in high glucose-induced insulin relese nd ATP elevtion in GK islets ut hd no such effects in control islets. Moreover, inhiitor reduced the high glucose-induced increse in ROS genertion in GK islet cells ut hd no effect on tht in control islet cells. Ouin hd no effect on ATP content nd ROS production in the presence of high glucose despite the prominent recovery effect of inhiitor in GK islets, suggesting tht is endogenously ctivted independently of ouin. Tken together, these results indicte tht ROS generted y ctivtion plys n importnt role in impired glucoseinduced insulin secretion derived from impired glucose metolism in GK islets. Acknowledgements The uthors thnk T. Ymguchi for technicl ssistnce. This study ws supported y Scientific Reserch Grnts, Grnt for Leding Project for Biosimultion from the Ministry of Eduction, Culture, Sports, Science nd Technology of Jpn, nd grnt from Core Reserch for Evolutionl Science nd Technology (CREST) of Jpn Science nd Technology Coopertion. Dulity of interest The uthors declre tht there is no dulity of interest ssocited with this mnuscript. References 1. Mechler P, Wollheim CB (21) Mitochondril function in norml nd dietic β-cells. Nture 414: Kennedy ED, Mechler P, Wollheim CB (1998) Effects of depletion of mitochondril DNA in metolism secretion coupling in INS-1 cells. Dietes 47: Tsuruzoe K, Arki E, Furukw N et l (1998) Cretion nd chrcteriztion of mitochondril DNA-depleted pncretic β- cell line: impired insulin secretion induced y glucose, leucine, nd sulfonylures. Dietes 47: Tkehiro M, Fujimoto S, Shimodhir M et l (25) Chronic exposure to β-hydroxyutyrte inhiits glucose-induced insulin relese from pncretic islets y decresing NADH contents. Am J Physiol 288:E372 E38 5. Tsuur Y, Ishid H, Okmoto Y et l (1993) Glucose sensitivity of ATP-sensitive K + chnnels is impired in β-cells of the GK rt. A new genetic model of NIDDM. Dietes 42: Hughes SJ, Fehling M, Thorneley CW, Proks P, Ashcroft FM, Smith PA (1998) Electrophysiologicl nd metolic chrcteriztion of single β-cells nd islets from dietic GK rts. Dietes 47: Anello M, Lupi R, Spmpinto D et l (25) Functionl nd morphologicl ltertions of mitochondri in pncretic et cells from type 2 dietic ptients. Dietologi 48: Ne K, Fujimoto S, Shimodhir M et l (26) Diphenylhydntoin suppresses glucose-induced insulin relese y decresing cytoplsmic H + concentrtion in pncretic islets. Endocrinology 147: Rdu RG, Fujimoto S, Muki E et l (25) Tcrolimus suppresses glucose-induced insulin relese from pncretic islets y reducing glucokinse ctivity. Am J Physiol 288: E365 E Ptne G, Anello M, Piro S, Vigneri R, Purrello F, Ruzzo AM (22) Role of ATP production nd uncoupling protein-2 in the insulin secretory defect induced y chronic exposure to high glucose or free ftty cids nd effects of peroxisome prolifertorctivted receptor-γ inhiition. Dietes 51: Joseph JW, Koshkin V, Sleh MC et l (24) Free ftty cidinduced β-cell defects re dependent on uncoupling protein 2 expression. J Biol Chem 279: Mechler P, Jornot L, Wollheim CB (1999) Hydrogen peroxide lters mitochondril ctivtion nd insulin secretion in pncretic et cells. J Biol Chem 274: Krippeit-Drews P, Krmer C, Welker S, Lng F, Ammon HP, Drews G (1999) Interference of H 2 O 2 with stimulus-secretion coupling in mouse pncretic β-cells. J Physiol 514: Bindoks VP, Kuznetsov A, Sreenn S, Polonsky KS, Roe MW, Philipson LH (23) Visulizing superoxide production in norml nd dietic rt islets of Lngerhns. J Biol Chem 278: Ski K, Mtsumoto K, Nishikw T et l (23) Mitochondril rective oxygen species reduce insulin secretion y pncretic β- cells. Biochem Biophys Res Commun 3: Lmert AE, Henquin JC, Mlvux P (1974) Ctionic environment nd dynmics of insulin secretion. IV. Effect of ouin. Horm Met Res 6: Kjikw M, Fujimoto S, Tsuur Y et l (22) Ouin suppresses glucose-induced mitochondril ATP production nd insulin relese y generting rective oxygen species in pncretic islets. Dietes 51: Kometini P, Li J, Gnudi L, Khn BB, Askri A, Xie Z (1998) Multiple signl trnsduction pthwys link N + /K + -ATPse to growth-relted genes in crdic myocytes. J Biol Chem 273: Xie Z, Kometini P, Liu J, Li J, Shpiro JI, Askri A (1999) Intrcellulr rective oxygen species medite the linkge of N + / K + -ATPse to hypertrophy nd its mrker genes in crdic myocytes. J Biol Chem 274: Liu J, Tin J, Hs M, Shpiro JI, Askri A, Xie Z (2) Ouin interction with crdic N + /K + -ATPse initites signl cscdes independent of chnges in intrcellulr N + nd C 2+ concentrtions. J Biol Chem 275: Fujimoto S, Ishid H, Kto S et l (1998) The novel insulinotropic mechnism of pimoendn: direct enhncement of the exocytotic process of insulin secretory grnules y incresed C 2+ sensitivity in β-cells. Endocrinology 139:

10 Dietologi (28) 51: Fujimoto S, Tsuur Y, Ishid H et l (2) Augmenttion of sl insulin relese from rt islets y preexposure to high concentrtion of glucose. Am J Physiol 279:E927 E Kneto H, Xu G, Song KH et l (21) Activtion of the hexosmine pthwy leds to deteriortion of pncretic β-cell function through the induction of oxidtive stress. J Biol Chem 276: Tkhshi H, Trn PO, LeRoy E, Hrmon JS, Tnk Y, Roertson RP (24) D-Glycerldehyde cuses production of intrcellulr peroxide in pncretic islets, oxidtive stress, nd defective et cell function vi non-mitochondril pthwys. J Biol Chem 279: Tsuouchi H, Inoguchi T, Inuo M et l (25) Sulfonylure s well s elevted glucose levels stimulte rective oxygen species production in the pncretic β-cell line, MIN6 role of NAD(P) H oxidse in β-cells. Biochem Biophys Res Commun 326: Yu BP (1994) Cellulr defenses ginst dmge from rective oxygen species. Physiol Rev 74: Turrens JF (23) Mitochondril formtion of rective oxygen species. J Physiol 552: Kdench B (23) Intrinsic nd extrinsic uncoupling of oxidtive phosphoryltion. Biochim Biophys Act 164: Ohnishi M, Tokud M, Mski T et l (1994) Chnges in nnexin I nd II levels during the postntl development of rt pncretic islets. J Cell Sci 17: Tejedo JR, Rmirez R, Chun GM, Rincon P, Sorino F, Bedoy FJ (21) Evidence for involvement of c- in the ntipoptotic ction of nitric oxide in serum-deprived RINm5F cells. Cell Signl 13: Tejedo JR, Chun GM, Rmirez R et l (24) Nitric oxide triggers the phosphtidylinositol 3-kinse/Akt survivl pthwy in insulin-producing RINm5F cells y rousing to ctivte insulin receptor sustrte-1. Endocrinology 145: Cheng H, Stru SG, Shrp GW (27) Inhiitory role of fmily tyrosine kinses on C 2+ -dependent insulin relese. Am J Physiol 292:E845 E Roskoski R Jr (24) protein-tyrosine kinse structure nd regultion. Biochem Biophys Res Commun 324: Hs M, Askri A, Xie Z (2) Involvement of nd epiderml growth fctor receptor in the signl-trnsducing function of N + /K + -ATPse. J Biol Chem 275: Aydemir-Koksoy A, Armowitz J, Allen JC (21) Ouininduced signling nd vsculr smooth muscle cell prolifertion. J Biol Chem 276: Hs M, Wng H, Tin J, Xie Z (22) -medited interreceptor cross-tlk etween the N + /K + -ATPse nd the epiderml growth fctor receptor relys the signl from ouin to mitogenctivted protein kinses. J Biol Chem 277: Xie Z, Ci T (23) N + -K + -ATPse-medited signl trnsduction: from protein interction to cellulr function. Mol Interv 3: Hutton JC, Mlisse WJ (198) Dynmics of O 2 consumption in rt pncretic islets. Dietologi 18: Sener A, Mlisse WJ (1991) Hexose metolism in pncretic islets. Regultion of D-[6 14 C]glucose oxidtion y non-nutrient secretgogues. Mol Cell Endocrinol 76: Nulton-Persson AC, Szwed LI (21) Modultion of mitochondril function y hydrogen peroxide. J Biol Chem 276: Lehy JL, Bonner-Weir S, Weir GC (1992) Bet-cell dysfunction induced y chronic hyperglycemi. Current ides on mechnism of impired glucose-induced insulin secretion. Dietes Cre 15: Kimur K, Toyot T, Kkizki M, Kudo M, Tkee K, Goto Y (1982) Impired insulin secretion in the spontneous dietes rts. Tohoku J Exp Med 137: Porth B, Serrds P, Bile D, Suzuki K, Goto Y, Giroix MH (1991) β-cell insensitivity to glucose in the GK rt, spontneous nonoese model for type II dietes. Dietes 4: Hughes SJ, Fehling M, Thorneley CW, Proks P, Ashcroft FM, Smith PA (1998) Electrophysiologicl nd metolic chrcteriztion of single β-cells nd islets from dietic GK rts. Dietes 47: Anello M, Lupi R, Spmpinto D et l (25) Functionl nd morphologicl ltertions of mitochondri in pncretic et cells from type 2 dietic ptients. Dietologi 48: Ihr Y, Toyokuni S, Uchid K et l (1999) Hyperglycemi cuses oxidtive stress in pncretic β-cells of GK rts, model of type 2 dietes. Dietes 48: Skur H, Mizukmi H, Ygihshi N, Wd R, Hnyu C, Ygihshi S (22) Reduced et-cell mss nd expression of oxidtive stress-relted DNA dmge in the islet of Jpnese type II dietic ptients. Dietologi 45:85 96