Methylglyoxal impairs insulin signalling and insulin action on glucose-induced insulin secretion in the pancreatic beta cell line INS-1E

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1 Diabetologia () 5:9 95 DOI.7/s ARTICLE Methylglyoxal impairs insulin signalling and insulin ation on gluose-indued insulin seretion in the panreati beta ell line INS-E F. Fiory & A. Lombardi & C. Miele & J. Giudielli & F. Beguinot & E. Van Obberghen Reeived: 8 Marh /Aepted: 8 July /Published online: August # Springer-Verlag Abstrat Aims/hypothesis Chroni hyperglyaemia aggravates insulin resistane, at least in part, by inreasing the formation of advaned glyation end-produts (AGEs). Methylglyoxal (MGO) is the most reative AGE preursor and its abnormal aumulation partiipates in damage in various tissues and organs. Here we investigated the ability of MGO to interfere with insulin signalling and to affet beta ell funtions in the INS-E beta ell line. Methods INS-E ells were inubated with MGO and then exposed to insulin or to gluose. Western blotting was used to study signalling pathways, and real-time PCR to analyse gene expression; insulin levels were determined by radioimmunoassay. Eletroni supplementary material The online version of this artile (doi:.7/s5--8-8) ontains peer-reviewed but unedited supplementary material, whih is available to authorised users. F. Fiory : A. Lombardi : C. Miele : F. Beguinot Dipartimento di Biologia e Patologia Cellulare e Moleolare and Istituto di Endorinologia ed Onologia Sperimentale del Consiglio Nazionale delle Rierhe, Università degli Studi di Napoli Federio II, Naples, Italy F. Fiory : J. Giudielli : E. Van Obberghen () Inserm U97, IFR5, Faulté de Médeine, Université de Nie Sophia Antipolis, 8 Avenue de Valombrose, 67 Nie Cedex, Frane Emmanuel.Van-Obberghen@unie.fr J. Giudielli : E. Van Obberghen Laboratoire de Biohimie, Hôpital Pasteur, CHU de Nie, Nie, Frane Results Non-ytotoxi MGO onentrations inhibited insulin-indued IRS tyrosine phosphorylation and phosphatidylinositol -kinase (PIK)/protein kinase B () pathway ativation independently from reative oxygen speies (ROS) prodution. Conomitantly, formation of AGE adduts on immunopreipitated IRS was observed. Aminoguanidine reversed MGO inhibitory effets and the formation of AGE adduts on IRS. Further, the insulin- and gluose-indued expression of Ins, Gk and Pdx mrna was abolished by MGO. Finally, MGO bloked gluoseindued insulin seretion and PIK/ pathway ativation. These MGO effets were abolished by LiCl, whih inhibits glyogen synthase kinase- (GSK-). Conlusions/interpretation MGO exerted major damaging effets on INS-E ells impairing both insulin ation and seretion. An important ator in these noxious MGO effets appears to be GSK-. In onlusion, MGO partiipates not only in the pathogenesis of the debilitating ompliations of type diabetes, but also in worsening of the diabeti state by favouring beta ell failure. Keywords Beta ell line. GSK-. INS-E. Insulin seretion. Insulin signalling. Methylglyoxal Abbreviations AGE Advaned glyation end-produt AG Aminoguanidine CEL N ε -(arboxyethyl)lysine CM-DCF Chloromethyl- 7 -dihlorofluoresein diaetate GLO Glyoxalase GSK- Glyogen synthase kinase- IR Insulin reeptor KRBH Krebs Ringer biarbonate HEPES MGO Methylglyoxal

2 9 Diabetologia () 5:9 95 MTT PDX- PIK p85 ROS Introdution -(,5-Dimethylthiazol--yl)-,5- diphenyltetrazolium bromide Panreati duodenal homeobox- Phosphatidylinositol -kinase Protein kinase B p85 Alpha regulatory subunit of PIK Reative oxygen speies The hallmark of type diabetes is insulin resistane oupled to panreati beta ell failure. In the proess leading to overt type diabetes, several key insulin target tissues, suh as the liver, musle and fat, beome resistant to the ations of the hormone. In the pathogenesis of insulin resistane dysregulation ours at multiple steps in the omplex network of the insulin signalling pathways []. Reent studies have shown that beta ells are targets of insulin ation. Indeed, insulin is thought to at in an autorine/pararine manner to regulate beta ell mass [] and funtion [], and to modulate several ellular proesses suh as gene expression [] and Ca flux [5]. In a mouse model with beta ell speifi knokout of the insulin reeptor (IR), a derease in gluose-stimulated insulin release and a progressive impairment of gluose tolerane are observed, suggesting that defets in insulin signalling at the level of the beta ell may ontribute to alterations in hormone seretion found in type diabetes [6]. Moreover, disruption of insulin signalling in the beta ell at the level of the insulin reeptor substrates IRS- [7] or IRS- [8] leads, respetively, to altered seretion and beta ell proliferation. Finally, the IR appears to be involved in the modulation of the responses of beta ells to gluose as shown, for example, by studies in MIN-6 ells, in whih a derease in IRs using sirna inhibits the aumulation of preproinsulin, Pdx and Gk mrna normally seen at high gluose onentration [9]. To summarise, insulin resistane may not only affet the funtioning of the lassial insulin target tissues, suh as musle, fat and liver, but also that of beta ells. This organismal insulin resistane ould be a onsequential fator in the pathogenesis of type diabetes. The mehanisms underlying the deleterious effets of redued insulin signalling on hormone synthesis and seretion of beta ells are poorly defined. Geneti and aquired fators are responsible for insulin resistane in hormone target tissues, while hroni hyperglyaemia further impairs insulin ation by different mehanisms. In partiular, hroni hyperglyaemia promotes the endogenous nonenzymati glyoxidation of proteins, lipids and nulei aids, leading to the formation of advaned glyation end-produts (AGEs) []. Intraellularly formed α-ketoaldehydes, suh as methylglyoxal (MGO), are an essential soure of intraellular AGEs and abnormal MGO aumulation has been impliated in the development of damage in various tissues and organs []. MGO is formed non-enzymatially by dephosphorylation of triose phosphates and, under physiologial onditions, is degraded into D-latate by glyoxalase []. While evidene exists that AGEs interfere with the omplex pathways governing insulin signalling and seretion, it is not known whih speifi AGEs are the ulprits and whih pathways are affeted. We previously reported that human glyated albumin treatment of ells and dietary AGEs given to mie indue insulin resistane in vitro [] and in vivo [], respetively, via a seletive protein kinase C alpha ativation. We have also shown that MGO indues insulin resistane in skeletal musle ells through diret hemial modifiation of IRS proteins [5]. Finally, MGO is thought to exert deleterious effets on insulin seretion [6 ]. However, the moleular mehanisms involved in the effet of MGO on the regulation of insulin release remains to be larified. Given the mounting evidene for a major role of beta ells in the pathogenesis of type diabetes, we investigated the ability of MGO to interfere with insulin signalling in the INS-E beta ell line, and we evaluated its potential damaging impat on beta ell funtions. Methods Materials Media, sera and antibiotis for ell ulture were from Invitrogen (Paisley, UK). Protein eletrophoresis and western blot reagents were from Bio-Rad (Rihmond, VA, USA). Enhaned hemiluminesene reagents and protein-a sepharose were from Piere (Rokford, IL, USA). Insulin was from NovoNordisk (Bagsværd, Denmark). The antibodies used are listed in the eletroni supplementary material [ESM] Table. Chloromethyl- 7 -dihlorofluoresein diaetate (CM-DCF) was from Moleular Probes (Eugene, OR, USA). [- C] MGO (.5 GBq/mmol) was manufatured by Amerian Radiolabeled Chemials (St Louis, MO, USA). Insulin was measured by radioimmunoassay (Insulin Rat RIA kit; Lino Researh, St Louis, MO, USA). MGO, aminoguanidine (AG), and all other reagents were from Sigma (St Louis, MO, USA). INS-E ell ulture The insulin-sereting INS-E rat beta ells (Passages 9 ) were ultured as previously desribed []. Cells were starved for 6 h in serum-free medium ontaining.% (wt/vol.) BSA, pretreated or not with MGO, washed one with starvation medium and then exposed or not to nmol/l insulin.

3 Diabetologia () 5: Inorporation of [- C]MGO The inorporation of [- C] MGO into INS-E ells was determined as previously desribed [5]. Details are provided in the ESM. Measurement of prodution of ROS The intraellular reative oxygen speies (ROS) were measured by CM- DCF fluoresene as previously desribed [5]. Details are provided in the ESM. Cell viability assay Cell viability was measured using the -(,5-dimethylthiazol--yl)-,5-diphenyltetrazolium bromide (MTT) assay as previously desribed []. Details are provided in the ESM. RNA isolation and real-time quantitative RT-PCR Total RNA extration, DNA synthesis and real-time PCR analysis were performed as desribed previously []. Proedures and primer sequenes used for amplifiation are provided in the ESM Methods and ESM Table, respetively. Immunoblot analysis INS-E ells were washed and solubilised as previously desribed []. Details for immunopreipitation, immunoblots and antibodies used are provided in the ESM. Measurement of insulin seretion Insulin seretion was assessed as previously desribed [5]. Details are provided in the ESM. Statistial methods Data were analysed with Statview software (Abaus Conepts, Pisataway, NJ, USA) by one-fator analysis of variane. p values.5 were onsidered as statistially signifiant. Results Inorporation of [- C]MGO into INS-E ells To determine whether the intraellular aumulation of MGO in INS-E ells is potentially involved in impairment of insulin ation, and to evaluate its impat on beta ell funtion, a time ourse of MGO inorporation into INS-E ells was performed. The ells were inubated from to min with [- C]MGO (7 kbq/assay) at onentrations ranging from.5 to. mmol/l, and thereafter extensively washed with PBS. We found that within min only.5±.5% of the labelled MGO was assoiated with the ells (Fig. a). Assuming that the ell-assoiated MGO is indeed intraellular, this would mean that at a onentration of.5 mmol/l, approximately 6 μmmol/l of this α-ketoaldehyde is within the ells. Similar observations have been reported onerning the inorporation of MGO into rat L6 skeletal musle ells [5], and into rat aorti smooth musle ells [6]. Role of MGO in prodution of ROS in INS-E ells To determine whether MGO leads to enhaned ROS prodution in INS-E ells, intraellular ROS levels were measured before and after treatment of ells with inreasing MGO onentrations (from.5 to. mmol/l) for different times (from to 6 min). As shown in Fig. b, treatment of INS-E ells with MGO at onentrations between.5 and. mmol/l does not lead to a marked ROS prodution. As a ontrol, INS-E ells were treated for min with 7 μmmol/l H O, whih indues approximately a 9.5-fold inrease in ROS prodution. Effets of MGO treatment on INS-E ell viability INS-E ells were exposed to MGO at onentrations ranging from.5 to. mmol/l, and for time periods of min to h. Thereafter, ell viability was measured using the MTT assay. As shown in Fig., under the onditions tested MGO failed to alter INS-E ell viability. Effets of MGO on insulin signalling in INS-E ells To investigate a possible role of MGO in the impairment of insulin ation, different proximal insulin signalling steps were investigated in MGO-treated ells. INS-E ells were exposed to different onentrations of MGO for min and then inubated or not with insulin for min. The insulinstimulated tyrosine phosphorylation of IR was unhanged in MGO-treated ells (data not shown). In ontrast, insulin-dependent IRS tyrosine phosphorylation was severely redued in ells preinubated for min with MGO (Fig. a). Indeed, MGO leads to a dose-dependent inhibition of insulin-stimulated IRS tyrosine phosphorylation. This derease in tyrosine phosphorylation was not aompanied by hanges in the level of IRS-/ protein, suggesting that a funtional defet is responsible for the redued phosphorylation. To evaluate the onsequenes of the defiient tyrosine phosphorylation of IRS, we first looked at the ability of IRS to dok with the PIK p85 subunit. To this end, we measured the levels of p85 subunits in IRS immunopreipitates obtained from INS-E ells treated with.5 mmol/l MGO for min, and in untreated ontrols. As shown in Fig. a insulin indued a marked inrease in the amount of p85 subunit o-immunopreipitating with IRS-/ (~threefold higher than the unstimulated level), and this p85 PIK IRS assoiation orrelated with the level of IRS- and IRS- tyrosine phosphorylation. As expeted from its inhibitory effet on IRS tyrosine phosphorylation, MGO virtually abolished omplex formation between p85 and IRS. Importantly, the redued p85 IRS-/ interation was not assoiated with hanges in the levels of the IRS proteins (Fig. a ). When the arbonyl savenger, AG, was added

4 9 Diabetologia () 5:9 95 a MGO inorporation (% of labelled MGO added at time ) Inubation time (min) b ROS prodution (fold inrease) H O.5.5 MGO (mmol/l) MTT (% of ontrol) 8 6 MGO (mmol/l) Inubation time (h).5 6 Fig. Effets of MGO in INS-E ells. a After inubation of INS-E ells with different amounts of labelled MGO (from.5 to mmol/l) in starvation medium, the media were eliminated at the indiated times. The plated ells were washed three times with ie-old PBS to remove non-speifially bound MGO, and thereafter lysed in mmol/l NaOH. The radioativity inorporated into the ells was measured by liquid sintillation ounting (MGO: diamond,.5 mmol/l; square,.5 mmol/l; triangle,. mmol/l) (n=). b For the measurement of ROS prodution, INS-E ells were treated with different onentrations of MGO (from.5 to mmol/l) for min (white bars) or 6 min (blak bars). ROS prodution was determined with the fluoresent probe CM-DCF (n=). H O is inluded as a ontrol. INS-E ells were treated with different onentrations of MGO for min, h, 6 h and h and thereafter their viability was measured using an MTT olorimetri assay. The results represent the mean±sd of four independent experiments before and during the inubation with MGO, the inhibitory ations of the α-ketoaldehyde on these early insulin signalling steps were prevented (Fig. a ). Next we looked at events downstream of PIK. As shown in Fig. b, insulin-indued phosphorylation on Thr 8 was signifiantly redued (approximately.8-fold) after a min treatment with.5 mmol/l MGO, and was nearly absent after a.5 mmol/l MGO exposure. Note that, whilst low MGO onentrations (.5 and. mmol/l) had no effet on phosphorylation after min (Fig. ), inubation with the same onentrations for h bloked insulin-indued phosphorylation on Thr 8 (Fig. d). To further evaluate the MGO-indued defet in the PIK asade, the kinase downstream of was analysed. In beta ells, GSK- is present in α and β isoforms, whih are both inhibited by phosphorylation on the N-terminal serine residue (Ser in GSK-α and Ser 9 in GSK-β). As illustrated in Fig. e, -indued serine phosphorylation of GSK- was prevented by MGO. As was the ase for phosphorylation, the inhibitory effet of MGO on GSK- phosphorylation ourred without hanges in GSK- protein level. As expeted, AG added before and during the MGO inubation prevents the inhibition of and GSK- phosphorylation (Fig. d,e). Formation of MGO adduts on IRS in INS-E ells MGO is known to reat with free amino groups and thiols to form AGE protein adduts, thereby altering protein funtion. We investigated whether inubation of INS-E ells with MGO (.5 mmol/l for min, and.5 or. mmol/l for h) was assoiated with formation of AGE IRS adduts, more speifially N ε -(arboxyethyl)lysine (CEL) and argpyrimidine adduts. As shown by western blots of immunopreipitated IRS, inubation of INS-E ells with MGO resulted in the formation of CEL (Fig. a,b) and argpyrimidine IRS adduts (Fig.,d). To onfirm MGO-driven addut generation, we examined whether AG bloks IRS modifiation observed after MGO treatment. Figure a, shows that this is indeed the ase. Effets of MGO on insulin-indued gene expression in INS- E ells and on their gluose-indued insulin seretion To

5 Diabetologia () 5: a IP with anti-irs ptyr-irs IRS- IRS- b pthr 8 - Insulin ( nmol/l; min) Insulin ( nmol/l; min) MGO (mmol/l; min).5.5 MGO (mmol/l; min).5.5 ptyr-irs MGO (mmol/l; min) pthr 8 - MGO (mmol/l; min).5.5 d B pt Thr 8 -PK B/PK pthr 8 - Insulin ( nmol/l; min) MGO (mmol/l; min) 5.. Insulin ( nmol/l; min) MGO (mmol/l; h).5. pt Thr 8 -/ (fold over bassal) pt Thr 8 -/ (fold over bassal) 8 6 MGO (mmol/l; min) 5.5. MGO (mmol/l; h) 5.. Fig. Effet of MGO on insulin signalling in INS-E ells. INS-E ells were inubated for min without or with MGO at different onentrations (from.5 to mmol/l) and then stimulated for min with nmol/l insulin. a The ells were then solubilised and extrats were immunopreipitated with anti-irs-/ antibodies followed by western blot analysis with antibodies to phosphotyrosine, IRS- and IRS-. b, Aliquots of the ell extrats were subjeted to western blot analysis with antibodies to phospho- and. d INS-E ells were treated with.5 or. mmol/l MGO for h and aliquots of the extrats were subjeted to western blot analysis with antibodies to phospho- and. Results are representative of three separate experiments. White bars, without insulin treatment; blak bars, with insulin treatment; error bars show SD; p<.5, p<. gain further insight into the deleterious MGO ation, we evaluated the expression profile of different genes important for beta ell funtioning. Panreati duodenal homeobox- (PDX-) is a key transription fator for panreas development and beta ell funtions, whih regulates, for example, the expression of the genes enoding insulin and gluokinase. In addition, insulin modulates PDX- expression and ation [7]. Moreover, it was reently proposed that gluose regulates the levels of PDX- via the reiproal ations of GSK- and kinase [8]. To investigate whether MGO modulates gene expression, INS-E ells were inubated with.5 mmol/l MGO for min and then stimulated with insulin. Treatment of INS-E ells with insulin alone indued a signifiant inrease in Pdx, Ins and Gk mrna expression ompared with the basal ondition (approximately.8-,.6- and.-fold, respetively). Interestingly, insulinindued expression of these mrnas was abolished by MGO (Fig. 5a). As expeted, AG present before and during the inubation with MGO restores insulin stimulatory ation on expression of these genes (Fig. 5a). Importantly, inubation of INS-E ells with low MGO onentrations (.5 and. mmol/l) for h also inhibited insulin's stimulatory ation on gene expression (Fig. 5b). Next we investigated the effets of MGO on insulin seretion. As shown in Fig. 5, insulin seretion by INS-E ells was signifiantly inreased upon exposure to mmol/l gluose, but this gluose ation was abolished when ells were treated with.5 mmol/l MGO for min. A similar inhibition of gluose-indued insulin seretion was seen

6 96 Diabetologia () 5:9 95 a IP with anti-irs ptyr-irs p85 IRS- IRS- Insulin ( nmo/; min) MGO (.5 mmol/l; min) AG ( mmol/l) b ptyr/irs MGO (.5 mmol/l; min) AG ( mmol/l) p85/irs MGO (.5 mmol/l; min) AG ( mmol/l) d pthr 8 - Insulin ( nmol/l; min) MGO (.5 mmol/l; min) AG ( mmol/l) pthr 8 -/ MGO (.5 mmol/l; min) AG ( mmol/l) Fig. Effet of MGO on insulin-indued ativation of the PIK pathway in INS-E ells. a INS-E ells were inubated for min without or with.5 mmol/l MGO in the absene or presene of mmol/l AG and then stimulated for min with nmol/l insulin. Thereafter, the ells were solubilised, the extrats were immunopreipitated with anti-irs-/ antibodies and subjeted to western blot analysis with antibodies to phosphotyrosine (ptyr), p85, IRS- and IRS-. IRS- and tyrosine phosphorylation (b) and their e pser-gsk- ( / ) GSK- ( / ) Insulin ( nmol/l; min) MGO (.5 mmol/l; min) AG ( mmol/l) pser-gsk-/gsk- MGO (.5 mmol/l; min) AG ( mmol/l) assoiation with p85 () were analysed by densitometry. Aliquots of the extrats were subjeted to western blot analysis with antibodies (d) to phospho- (pthr 8 -) and or (e) to phospho-gsk- (pser-gsk-) and GSK-. The western blots were analysed by densitometry. Results shown are representative of three separate experiments. White bars, without insulin treatment; blak bars, with insulin treatment; error bars show SD; p<.5, p<. with ells treated with.5 and. mmol/l MGO for h (Fig. 5d). As expeted, AG bloks MGO inhibition of a b IP with anti-irs IP with anti-irs Anti-CEL IRS- MGO (.5 mmol/l; min) AG ( mmol/l) Anti-argpyr IRS- IP with anti-irs MGO (.5 mmol/l; min) AG ( mmol/l) 7 kda 7 kda Anti-CEL IRS- MGO (mmol/l; h).5. d Anti-argpyr IRS- IP with anti-irs MGO (mmol/l; h).5. Fig. Detetion of CEL adduts on IRS immunopreipitated from INS-E. INS-E ells were treated with.5 mmol/l MGO for min without or with AG (a,) or treated with.5 or. mmol/l MGO for h (b,d). Thereafter, the ells were solubilised, the extrats were immunopreipitated with anti-irs-/ antibodies and subjeted to western blot analysis with antibodies to IRS-, CEL (a,b) and to argpyrimidine (argpyr) (,d) gluose-stimulated insulin seretion (Fig. 5). Taken together, our data demonstrate that MGO treatment of INS-E ells hampers their gluose responsiveness. Effets of MGO on gluose-indued signalling and gene expression in INS-E ells To determine whether MGO treatment influenes gluose signalling and interferes with gluose ation on gene expression, INS-E ells were pretreated with.5 mmol/l MGO for min or with.5 and. mmol/l MGO for h, and thereafter exposed to.8 or mmol/l gluose for min. In the absene of MGO, inubation of INS-E ells with mmol/l gluose inreased phosphorylation of the Thr 8 residue of. Pretreatment with MGO inhibits gluose-stimulated phosphorylation without a detetable hange in protein level (Fig. 6a,b), thus demonstrating that MGO restrains gluose-indued ativation of the PIK/ pathway. The MGO-produed impairment of gluose stimulatory ation on the PIK/ asade led us to investigate the effet of the α-ketoaldehyde on gluose-indued mrna

7 Diabetologia () 5: a mrna expression Insulin ( nmol/l; min) MGO (.5 mmol/l; min) AG ( mmol/l) Pdx Ins Gk b mrna expression Insulin ( nmol/l; min) MGO (.5 mmol/l; h) MGO (. mmol/l; h) Pdx Ins Gk Insulin seretion d Insulin seretion.. Gluose (mmol/l; min).8.8 MGO (.5 mmol/l; min) AG ( mmol/l) Fig. 5 Effet of MGO on insulin-indued gene expression in INS-E ells and on their gluose-indued insulin seretion. INS-E ells were inubated (a) for min without or with.5 mmol/l MGO in the absene or presene of mmol/l AG, or (b) for h with.5 or. mmol/l MGO. Thereafter they were stimulated for min with nmol/l insulin. Total RNA was extrated and Pdx, Ins and Gk mrna was measured by quantitative RT-PCR using 8S as internal Gluose (mmol/l; min) MGO (mmol/l; h).5. standard. INS-E ells were inubated () for min without or with.5 mmol/l MGO or (d) for h with.5 or. mmol/l MGO, and thereafter stimulated for min with.8 or mmol/l gluose. Released insulin was measured by radioimmunoassay. Results shown are representative of three separate experiments. Error bars show SD; p<.5, p<., p<. expression of Pdx, Gk and Ins. In the absene of MGO, inubation with mmol/l gluose for min inreased mrna levels of these genes by 7%, 6% and 8%, respetively. In ontrast, the gluose effet is abolished by MGO treatment. It appears that gluose ation is mediated by the PIK pathway as the PIK inhibitor, LY9, bloks it (Fig. 6). Similarly, after a h inubation with.5 mmol/l MGO, the gluose effet on expression of Pdx and Ins mrna is redued by 5% and %, respetively, and is almost ompletely bloked after a h treatment with. mmol/l MGO (Fig. 6d). Role of GSK- in MGO inhibitory ation on biologial responses of INS-E ells Reently, it was reported that mie overexpressing a onstitutively ative form of Gsk-β (also known as Gskb) in beta ells showed a profound derease in PDX- levels ompared with ontrol mie [9]. To determine whether GSK- ativation is responsible for the deleterious effets of MGO, INS-E ells were inubated for min with.5 mmol/l MGO and then exposed to.8 or mmol/l gluose in the presene of mmol/l LiCl, a GSK- bloker []. In MGO-treated ells, GSK- phosphorylation in response to gluose is redued with the likely onurrent inrease in its ativation, but LiCl treatment reverses this MGO inhibitory effet on GSK- phosphorylation (Fig. 7a). To approah the potential role of MGO-stimulated GSK- ativation in the alteration of beta ell funtion, we investigated MGO effets on gluose-indued gene expression in the presene of LiCl. Gluose treatment leads to an inrease in Pdx, Ins and Gk mrna levels by approximately.-,.8- and.-fold, respetively (Fig. 7b). In ells exposed to MGO the stimulatory ation of gluose on gene expression is abolished. However, in MGO-treated ells LiCl inubation restores the inrease in Pdx, Ins and Gk mrna produed by mmol/l gluose. We then measured, in MGO-exposed ells, the effet of gluose on insulin seretion in the presene of LiCl. As shown in Fig. 7, gluose-indued insulin seretion is robustly dereased by MGO inubation. Pretreatment with LiCl partially restores gluose-indued insulin seretion in the presene of MGO, indiating that MGO ation on insulin seretion ours, at least in part, through ativation of GSK-.

8 98 Diabetologia () 5:9 95 a pthr 8 - Gluose (mmol/l; min) MGO (.5 mmol/l; min) pthr 8 -/.8.8 Gluose (mmol/l; min).8.8 MGO ( 5 mmol/l; min) b pthr 8 - Gluose (mmol/l; min) MGO (mmol/l; h) pthr 8 -/ Gluose (mmol/l; min) MGO (mmol/l; h).5. mrna expression d mrna expression Gluose (mmol/l; min) MGO (.5 mmol/l; min) LY9 (5 µmol/l) Pdx Ins Gk Fig. 6 Effet of MGO on gluose-indued phosphorylation of and on gene expression in INS-E ells. INS-E ells were inubated (a) for min with.5 mmol/l MGO or (b) for h with.5 or. mmol/l MGO, and thereafter stimulated for min with.8 or mmol/l gluose. Next the ells were solubilised and aliquots of the extrats were subjeted to western blot analysis with antibodies to phospho- or and analysed by densitometry. INS-E ells Gluose (mmol/l; min).8.8 MGO (.5 mmol/l; h) MGO (. mmol/l; h) Pdx Ins were inubated () for min without or with.5 mmol/l MGO in absene or presene of 5 μmol/l LY9 or (d) for h with.5 or. mmol/l MGO, and thereafter they were stimulated for min with.8 or mmol/l gluose. Total RNA was extrated and Pdx, Ins and Gk mrna was measured by quantitative RT-PCR using 8 s as internal standard. Results shown are representative of three separate experiments. Error bars show SD; p<. Disussion Evidene exists in favour of the idea that beta ells ould be targets for sereted insulin. Indeed, insulin is thought to stimulate beta ell proliferation and gene expression by ativating the IR signalling asade in an autorine/pararine fashion [, ]. Moreover, proinsulin synthesis and the beta ell responsiveness to gluose in terms of hormone seretion appear to be modulated by insulin signalling [, ]. Therefore, alterations of insulin signal transdution ould lead to beta ell dysfuntion ontributing to the pathogenesis of type diabetes. However, the moleular mehanisms through whih defets in insulin signalling affet insulin synthesis and seretion remain largely unknown. Inreased prodution of AGEs and MGO, a preursor of AGEs, has been shown to ontribute to insulin resistane in onditions assoiated to hroni hyperglyaemia [, 5]. Here we show that exposure of INS-E ells to MGO for short periods ( min at.5 mmol/l) or prolonged periods ( h at.5 and. mmol/l) impairs insulin-indued tyrosine phosphorylation of IRS. This ours together with a redution in p85 IRS assoiation, ativation and serine phosphorylation of GSK-. The derease in tyrosinephosphorylated IRS proteins is unlikely to be due to hanges in IRS synthesis/degradation, as IRS protein levels were unaltered after these MGO treatments. Generation of ROS is linked to MGO metabolism [6]. Further, oxidative stress has been impliated in the deterioration of insulin signalling in diabetes [], in dereased gluose-stimulated insulin seretion, and in beta ell apoptosis []. Oxidative stress thus represents a risk fator for the development of diabetes. Under our experimental onditions we did not detet a signifiant inrease in intraellular ROS aumulation and in ell death after a short or long exposure to MGO. Therefore, we would like to onlude that the MGO effets we observed in INS-E ells are not mediated by ROS prodution. Our findings in INS-E ells are onsistent with those we obtained in L6 skeletal musle ells, in whih MGO impairs insulin signalling not by produing ROS, but by binding to IRS-

9 Diabetologia () 5: Fig. 7 Role of GSK- in MGO inhibitory ation on gluose effets in INS-E ells. INS-E ells were inubated for min without or with.5 mmol/l MGO in absene or presene of mmol/l LiCl, and thereafter stimulated for min with.8 or mmol/l gluose. a The ells were solubilised and phosphorylation levels of GSK- were analysed by western blot with speifi antibodies. The western blots were evaluated by densitometry. b Total RNA was extrated and Pdx, Ins and Gk mrna were measured by quantitative RT-PCR using 8 s as internal standard. Released insulin was measured by radioimmunoassay. Results shown are representative of three separate experiments. Error bars show SD; p<.5, p<., p<. and and leading to a derease in their tyrosine phosphorylation in response to insulin [5]. MGO is a reative diarbonyl moleule that an modify lysine and arginine residues in proteins. This also ours in INS-E ells, as inubation with MGO indues the formation of CEL and argpyrimidine IRS adduts. Interestingly, on rat IRS-, proximal to YMXM motifs, whih have been impliated in p85 binding, Lys 6 near Y 68 MPM and Arg 6 near Y 68 MPM are found [, 5]. It is likely that addut binding to Lys 6 and/or Arg 6 ould lead to redued tyrosine phosphorylation of IRS-/ resulting in dereased p85 anhoring by IRS. An alternative or additional senario ould be a similar modifiation of the Lys and/or Arg residues loated within the IRS PH or PTB domains, whih are essential for optimal IRS tyrosine phosphorylation by IR. As mentioned earlier, inreased degradation and/or dereased synthesis of IRS seems to be improbable as protein levels are unhanged. Taken as a whole, our data gather grounds for belief in MGO-indued alteration of IRS resulting in a derease in its doking funtion and hene perturbation of the downstream PIK/ signalling node. Several lines of evidene have been provided in favour of the idea that insulin modulates PDX- DNA-binding ativity and insulin promoter ativity [7]. PDX- is a key transription fator involved in panreas development and in modulation of the expression of several genes essential for beta ells, suh as the insulin and gluokinase genes (Ins and Gk). It has been shown that both gluose and insulin stimulates PDX- binding ativity to the insulin promoter via PIK and mitogen ativated protein kinase (SAPK/p8) [7, 6]. IR silening in MIN-6 ells robustly suppressed the expression of Pdx mrna, suggesting that insulin signalling plays a major role in the regulation of the expression and funtion of PDX- [9, 7]. PDX- also modulates the mrna expression of Gk, whih atalyses the first step of glyolysis, and hene regulates gluose responsiveness for insulin release [8]. Here we demonstrate in INS-E ells that a short and prolonged exposure to MGO leads to impaired insulin-indued expression of the genes oding for PDX-, insulin and gluokinase. Thus, MGO hampers the benefiial effet of insulin on the a pser-gsk ( α / β ) GSK ( α / β ) Gluose (mmol/l; min) MGO (.5 mmol/l; min) LiCl ( mmol/l) pser-gsk-/gsk- Gluose (mmol/l; min) MGO (.5 mmol/l; min) LiCl ( mmol/l) expression of genes involved in the preservation of the hief beta ell funtions. The MGO onentrations we have used are in the range of those applied by Brouwers et al. to mesenteri arteries b.5. mrna expression Insulin seretion.5..5 Gluose (mmol/l; min) MGO (.5 mmol/l; min) LiCl ( mmol/l) Pdx- Ins Gk Gluose (mmol/l; min) MGO (. 5 mmol/l; min ) LiCl ( mmol/l)

10 95 Diabetologia () 5:9 95 [9], by Cook et al. to rat beta ells [7], and by Bento et al. to human retinal pigment epithelium ells []. Generally speaking these onentrations are intended to mimi the aumulation of MGO seen after hroni hyperglyaemia found in diabetes. Using, in INS-E ells, either.5 mmol/ l MGO for min or between.5 and. mmol/l for h, similar inhibitory effets were observed on insulin signalling without hange in ell viability. Further, within min inubation with.5 mmol/l MGO approximately 6 μmmol/l MGO is found to be assoiated with INS-E ells. This is omparable with the results of a reent study in human retinal pigment epithelium ells []. Previous reports have shown that MGO alters insulin seretion [7, 9, ]. Our urrent study on INS-E ells further douments the inhibitory effet of MGO on gluoseindued insulin seretion, but learly goes beyond by providing deisive insight into the mehanisms involved. While the issue remains ontroversial, fats have been presented suggesting that insulin exerts a positive effet on its exoytosis. Compatible with this notion, transfetion of IRs in beta ell lines inreases Ins mrna levels and gluose-indued insulin seretion []. In mie, both onditional and general knokout for proteins of the insulin signalling asade, suh as IR, IRS- and IRS-, led to impaired gluose-indued insulin release [6 8]. Irs transfetion in btc6-f7 ells inreases gluose-indued insulin seretion [], and isolated islets from Irs / mie present a redued insulin ontent and an impaired seretory response to gluose [7]. Therefore, inhibition of insulin signalling at the IRS and/or at level due to MGO exposure may be responsible for the defet in insulin seretion. Studies performed in mie with one Pdx allele inativated showed that PDX- defiieny alters gluoseindued insulin seretion []. Further, inreased AGE levels indued by hroni hyperglyaemia lead to islet dysfuntion assoiated with redued gluokinase levels in DBA/ mie []. We found that, after inubation of INS-E ells with MGO, high gluose failed to inrease expression of Pdx, Ins and Gk genes. This effet of gluose appears to be PIK-dependent, sine it is abolished by pretreatment with a PIK inhibitor. Further, in MGO-treated ells no inreased and GSK- phosphorylation was observed in response to high gluose, supporting the idea that ativation of the PIK/ asade has an important impat on the preservation of beta ell funtion. Indeed, our data dovetail with a previous work showing that the overexpression of a dominant-negative Pkb (also known as Akt) in beta ells leads to impaired insulin seretion [5]. Reent fats have been reported showing that GSK-β ativation is linked to beta ell failure in diabeti mouse models, and is involved in negative regulation of beta ell proliferation and mass [6, 7]. Moreover, GSK-β ativity is enhaned in insulin-resistant states, and in skeletal musle from type diabeti patients [8]. Studies in transgeni mie overexpressing a onstitutively ative form of Gsk-β in beta ells revealed that it leads to a profound derease in PDX- levels ompared with ontrol mie, probably due to redued stability [9]. In MGO-treated INS-E ells, GSK- phosphorylation in response to insulin is redued with a likely onurrent inrease in its ativation. Interestingly, the LiCl-indued restoration of GSK- phosphorylation and its ensuing inhibition reverses the repressive effet of MGO on gluose-indued insulin seretion in INS-E ells. This suggests that GSK- ativation may be responsible, at least in part, for the damaging MGO effets on beta ell funtion. The moleular mehanisms underlying GSK- ation on insulin seretion are poorly defined and we annot exlude the possibility that MGO ation is partially due also to the diret binding of MGO to beta ell transription fators and/or to proteins involved in the insulin seretion mahinery. To summarise, our present work strongly indiates that toxi reative aldehydes, suh as MGO, impat negatively on beta ell homeostasis. Hene, the deleterious ations of these moleules generated by metaboli dysregulation will worsen the diabetes disease proess by favouring beta ell failure. The development of new strategies to improve insulin transdution in beta ells will allow the preservation and/or improvement of beta ell funtion in type diabeti patients suffering from elevated irulating levels of toxi aldehydes due to hroni hyperglyaemia. Aknowledgements The researh in Nie was supported by Inserm, Université de Nie Sophia Antipolis, Conseil Régional PACA and Conseil Général des Alpes-Maritimes, by the European Community FP6 EUGENE (grant LSHM-CT--5). F. Fiory was supported by a grant-in-aid from Servier (Neuilly-Frane) to E. Van Obberghen. The researh in Naples was supported by the European Foundation for the Study of Diabetes (EFSD), the European Community s PREPOBEDIA(68) and by the Ministero dell Università e della Riera Sientifia. The finanial support of Telethon, Italy, to F. Beguinot is gratefully aknowledged. We thank A. Salvayre-Negre for antibodies to argpyrimidine. Contribution statement FF, AL, CM, JG and EVO were responsible for the oneption and design of the artile: FF, AL, CM, and JG performed the experiments. FF, CM, JG, FB and EVO analysed and interpreted data, and drafted the artile: FF, AL, CM, JG, FB and EVO revised the paper ritially for important intelletual ontent: All authors approved the final version. Duality of interest The authors delare that there is no duality of interest assoiated with this manusript. Referenes. Gallagher EJ, LeRoith D, Karnieli E (8) The metaboli syndrome from insulin resistane to obesity and diabetes. Endorinol Metab Clin North Am 7:

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