Translationally controlled tumour protein (TCTP) is a novel glucose-regulated protein that is important for survival of pancreatic beta cells

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1 Dietologi (11) 54:368 9 DOI 1.7/s ARTICLE Trnsltionlly controlled tumour protein () is novel glucose-regulted protein tht is importnt for survivl of pncretic et cells F. Dirison & K. Hywrd & K. L. Snders & F. Brozzi & S. Ljus & J. Hncock & J. E. Frncis & E. Ainscow & U. A. Bommer & E. Molnr & N. D. Avent & A. Vrdi Received: 21 July 1 / Accepted: 27 Septemer 1 / Pulished online: 1 Novemer 1 # Springer-Verlg 1 Astrct Aims/hypothesis This study used proteomics nd iochemicl pproches to identify novel glucose-regulted proteins nd to unveil their role in pncretic et cell function. Trnsltionlly controlled tumour protein () ws identified to e one such protein, nd further investigtions into its function nd regultion were crried out. Methods Glol protein profiling of et cell homogentes following glucose stimultion ws performed using twodimensionl gel electrophoresis. Proteins were identified y mss spectroscopy nlysis. Immunolotting ws used to investigte ltertions in protein levels in response to glucose stimultion or cell stress induced y plmitte. To investigte the iologicl function of, immunoloclistion, gene knockdown nd overexpression of Tctp (lso known s Tpt1) were performed. Apoptosis ws mesured in Tctp knockdown or Tctp-overexpressing cells. Glucosestimulted insulin secretion ws crried out in Tctp knockdown cells. Results ws identified s novel glucose-regulted protein, the level of which is incresed t stimultory glucose concentrtion. Glucose lso induced dephosphoryltion nd its prtil trnsloction to the mitochondri nd the nucleus. protein levels were downregulted in response to cell stress induced y plmitte or thpsigrgin tretments. Gene knockdown y smll interfering RNA led to incresed poptosis, wheres overproduction of prevented plmitte-induced cell deth. Conclusions/interprettion Regultion of protein levels y glucose is likely to e n importnt cyto-protective F. Dirison, K. Hywrd nd K. L. Snders contriuted eqully to this study. Electronic supplementry mteril The online version of this rticle (doi:1.7/s ) contins supplementry mteril, which is ville to uthorised users. F. Dirison : K. Hywrd : K. L. Snders : F. Brozzi : S. Ljus : J. Hncock : N. D. Avent : A. Vrdi (*) Centre for Reserch in Biomedicine, Fculty of Helth nd Life Sciences, University of the West of Englnd, Bristol BS16 1QY, UK e-mil: Aniko.Vrdi@uwe.c.uk J. E. Frncis : E. Ainscow Advnced Science nd Technology Lortory, AstrZenec, Loughorough, UK U. A. Bommer Grdute School of Medicine, University of Wollongong, Wollongong, NSW, Austrli E. Molnr MRC Centre for Synptic Plsticity, School of Physiology nd Phrmcology, University of Bristol, Bristol, UK Present Address: K. L. Snders Sir Jmes Blck Centre, College of Life Sciences, University of Dundee, Dundee, UK Present Address: N. D. Avent School of Biomedicl nd Biologicl Science, University of Plymouth, Plymouth, UK

2 Dietologi (11) 54: mechnism for pncretic et cells ginst dmge cused y hyperglycemi. In contrst, high concentrtion of plmitte cuses cell stress, reduction in levels nd consequently reduced cell viility. Our results imply tht levels influence the sensitivity of et cells to poptosis. Keywords Apoptosis. Ftty cid plmitte. Insulin secretion. Proteomics nlysis.. Trnsltionlly controlled tumour protein Arevitions BAX BCL2-ssocited X protein BCL2 B cell lymphom 2 2DGE Two-dimensionl gel electrophoresis ER Endoplsmic reticulum ERK1/2 Extrcellulr signl regulted kinse GAPDH Glycerldehyde 3-phosphte dehydrogense IPG Immoilised ph grdient MCL-1 Myeloid cell leukemi sequence 1 PERK PKR-like ER kinse PKR Doule-strnded RNA-dependent protein kinse sirna Smll interfering RNA Trnsltionlly controlled tumour protein UPR Unfolded protein response Introduction Using proteomics-sed pproch, we identified nd chrcterised glucose-regulted proteins in pncretic et cells. Glol nlysis of protein levels using proteomics cn unveil iologiclly importnt chnges in protein production, which my not e identified y cdna microrry nlysis. The ltter method is limited y the fct tht mrna levels nd not their protein products re investigted. Gene expression studies sed on cdna microrrys do not lwys correlte with chnges found t the protein level [1 3]. Furthermore, mny proteins re lso regulted t post-trnscriptionl levels, effecting chnges tht trnscriptome nlysis would fil to identify. Our proteomics-sed nlysis identified trnsltionlly controlled tumour protein () s glucose-regulted protein in pncretic et cells. is highly conserved protein of 23 kd [4, 5], which shows no sequence similrity with ny other known proteins. hs een identified in wide rnge of eukryotic orgnisms nd hs een ssocited with diverse cellulr processes, s reviewed y others [6]. However, the physiologicl function of in humns is still not fully elucidted. hs een suggested to function s n ntipoptotic protein, since overproduction of the protein inhiits, wheres knockdown of its gene promotes poptosis [7 12]. Gene knockout studies reveled tht deficient mice [1, 13] nd -deficient mutnts of Drosophil [14] die erly during emryogenesis, presumly due to unregulted poptosis t criticl stge. It hs een shown tht inds to the nti-poptotic memers of the B cell lymphom 2 (BCL2) fmily of proteins, myeloid cell leukemi sequence 1 (MCL-1) [7, 8] nd B cell lymphom extr lrge (BCL-XL) [9]. It hs recently een proposed tht ntgonises poptosis y enhncing the nti-poptotic ctions of MCL-1 nd BCL-XL, nd y nchoring into the mitochondril memrne in wy tht inhiits dimeristion of the propoptotic protein BCL2- ssocited X protein (BAX) [1]. Thus, the ovementioned studies clerly indicte tht plys criticl role in the control of cell survivl in vivo. Besides its nti-poptotic function, is required for cell growth nd prolifertion. For exmple, Drosophil ws shown to control cell growth nd prolifertion y regulting GTPse ctivity of Rs homologue, Rhe [14], lthough this point remins controversil, s previously discussed [11]. In ddition, regultes cell growth through its gunine nucleotide dissocition inhiitor ctivity for the elongtion fctors eukryotic elongtion fctor 1A (eef-1a) nd elongtion fctor 1Bβ (EF1Bβ []. is lso tuulin- [16] nd clcium-inding [17] protein, serves s sustrte of polo-like kinse [18] nd hs properties of histmine-relesing fctor [19] or growth fctor []. These diverse functions of re likely to result from its specific ssocition with vrious protein prtners. levels my vry considerly etween vrious tissues nd its synthesis is regulted t the trnscriptionl nd posttrnscriptionl levels [21, 22], indicting involvement of tissue-specific fctors. Anlysis of the mouse Tctp (lso known s Tpt1) gene promoter reveled tht its expression is lso regulted y camp[23]. levels re, therefore, highly regulted in response to wide rnge of extrcellulr signls nd cellulr conditions. Vrious stress conditions, such s strvtion, het shock, hevy metls, clcium stress or propoptotic/cytotoxic signls cn up- or downregulte levels, s previously reviewed [6]. Furthermore, levels re regulted y the doule-strnded RNA-dependent protein kinse (PKR) [24]. In this context, we recently reveled tht ctivtion of PKR y pro-poptotic stimuli results in downregultion of protein levels [11]. ws first identified from tumour cells, ut it hs since een recognised tht is not tumour-specific protein, lthough its levels tend to e higher in tumours thn in the corresponding norml tissue []. There lso seems to e link etween cncer nd, since inhiition of results in suppression of the mlignnt phenotype []. Intriguingly, reduced levels of hve een detected in post-mortem rins from ptients with Down s syndrome nd Alzheimer s disese [26]. It is

3 Dietologi (11) 54:368 9 plusile tht diminished nti-poptotic protection y is involved in these disorders. Despite the mny importnt functions ttriuted to, its role in pncretic et cells is not known to dte. The current study identified s novel glucoseregulted protein nd investigted its role in glucoseregulted insulin secretion nd cell survivl. Methods Mterils All moleculr iologicls were from Sigm (Poole, UK). Two-dimensionl gel electrophoresis (2DGE) regents nd ll secondry ntiodies were from GE Helthcre (Chlfont St Giles, UK) nd Invitrogen (Pisley, UK). Anti-mouse monoclonl ntiody ws from Strtech Scientific (Newmrket, UK). Monoclonl ntiphosphotyrosine nd nti-phosphoserine ntiodies were from Acm (Cmridge, UK). -specific smll interfering RNA (sirna; sc-434) nd control scrmled RNAs were from Snt Cruz (Heidelerg, Germny). Rit polyclonl nti-extrcellulr signl regulted kinse (ERK)1/2 ntiody ws purchsed from Cell Signling (Hitchin, UK). Cell culture MIN6 nd HIT-T et cells were cultured s previously descried [27]. For glucose stimultion experiments, MIN6 cells were cultured for 12 h in DMEM contining 3 mmol/l glucose, which ws then replced y DMEM contining 3 or mmol/l glucose for 24 h. Isoelectric focusing nd 2DGE Cell homogente ws prepred y lysing the cells in RIPA uffer (138 mmol/l NCl, 2.6 mmol/l KCl, 1.5 mmol/l KH 2 PO 4, 6.3 mmol/l N 2 HPO 4, ph 7) contining 1% (vol./vol.) Igepl CA-63,.5% (wt/ vol.) sodium deoxycholte,.1% (wt/vol.) SDS. Immoilised ph grdient (IPG) strips (24 cm) were rehydrted overnight nd smple cup-loded. Isoelectric focusing ws performed for 1 h t V with voltge incresing step-wise from to 1, V, nd finlly to 8, V for 8 h. Strips were equilirted for min in LDS smple uffer (NuPAGE, Invitrogen, Pisley, UK) in the presence of 1% (vol./vol.) NuPAGE smple reducing gent. The smples were equilirted for min in LDS smple uffer (NuPAGE) contining 1 mmol/l iodocetmide. Strips were trnsferred on to gels (Novex 4 12% Bis Tris ZOOM gels; NuPAGE). The strips were overlid with.5% (wt/vol.) grose in running uffer (NuPAGE MOPS SDS) nd gels run in mini-cell device (XCell Surelock; Invitrogen). Immunolotting, immunocytochemistry nd sucellulr frctiontion These were performed s descried erlier [27]. To quntify nucler loclistion of in response to glucose, imging ws performed using the sme confocl settings for ll conditions nd the intensity of nucler stining ws quntified y softwre tool (Volocity; Perkin Elmer, Wlthm, MA, USA). Three loding controls were used in this study. For the 2DGE nd the corresponding 1DGE, the density ws normlised to tht of the totl protein ecuse ERK1/2 nd glycerldehyde 3-phosphte dehydrogense (GAPDH) showed severl spots on the 2DGE. GAPDH levels incresed in response to glucose nd thus could not e used s loding control in experiments where the glucose level chnged. Thus, ERK1/2 ws used s loding control for islet proteins [28] nd lso for conditions where the glucose concentrtion ws ltered. Detection of the phosphoryltion stte of MIN6 et cells were treted nd proteins seprted s descried previously [27]. The gels were co-stined with gel stins (Pro-Q Dimond Phosphoprotein nd SYPRO Ruy; Invitrogen, Pisley, UK) ccording to the mnufcturer s instructions. The gels were imged using scnner (Typhoon 921; GE Helthcre, Chlfont St Giles, UK) with excittion of 532 or 4 nm, nd emission mximum of 58 or 61 nm for Pro-Q Dimond nd SYPRO Ruy, respectively. Immunolots were proed with phosphotyrosine- nd phosphoserine-specific ntiodies. sirna knockdown of Tctp expression nd mesurement of insulin secretion This ws crried out s descried previously [27]. For insulin secretion, cells t 96 h post-trnsfection were incuted for 1 h in KRB t 3 mmol/l glucose, followed y 3 min incution in KRB t low (3 mmol/l) or high (3 mmol/l) glucose concentrtions. Insulin ws mesured using kit (Ultrsensitive Mouse Insulin ELISA; Mercodi, Uppsl, Sweden). Mesurement of poptosis following Tctp sirna trnsfection Cells treted with sirna were collected from the medi nd plte, nd wshed three times with PBS. The cell pellet ws resuspended in μl miniml medium (DMEM without FCS) nd μg/ml propidium iodide ws dded immeditely efore nlysis. Fluorescence intensity of the propidium iodide ound to DNA ws mesured nd poptosis nlysed using FACS nlysis (FACSVntge SE FACS; Becton Dickinson, Oxford, UK) [29]. Isoltion of islets of Lngerhns nd RT-PCR of Tctp Islets were isolted s previously descried [3]. The study ws conducted in ccordnce with the Principles of Lortory Cre. Totl RNA ws isolted using regent (TRI regent, Sigm) ccording to the mnufcturer s protocol. RT-PCR ws performed using primers corresponding to nucleotides

4 Dietologi (11) 54: nd of the mouse Tctp (ccession numer NM_9429), respectively. Ftty cid incution MIN6 or HIT-T cells were seeded on six-well pltes nd grown overnight. Plmitte ws coupled to ftty cid-free BSA t 5:1 molr rtio nd dded to the culture medium to finl concentrtion of.5 mmol/l plmitte nd.68% (wt/vol.) BSA. Cells were incuted for 24 h efore use. Isolted islets were cultured for 16 h in DMEM contining 11 mmol/l glucose, hnd-picked nd incuted for 24 h in DMEM contining 3 or 17 mmol/l glucose in the presence or sence of.5 mmol/l plmitte. Overproduction of nd TUNEL ssy Cells were trnsfected with.5 μg pcdna3 contining the coding region of full-length mouse Tctp [16] or pcdna3 empty vector using lipofectmine (Invitrogen) s descried previously [27]. Cells t 24 h post-trnsfection were incuted with plmitte for 8 or 24 h. Cells were fixed nd the TUNEL ssy ws performed ccording to the mnufcturer s instructions (Click-iT TUNEL Alex Fluor 488; Invitrogen). The numer of -overproducing cells nd those positive for TUNEL stining were counted. Two controls were used: (1) cells trnsfected with pcdna3; nd (2) untrnsfected cells on the trnsfected pltes in which overproduction ws not visile. Mss spectroscopy Proteins were trypsin-digested using rootic digester (Ettn digester; GE Biosciences, UK). Peptide mss fingerprints were cquired using Mtrixssisted lser desorption/ionistion time-of-flight mss spectrometer (MALDI-TOF; Wters Micromss, Wterloo, ON, Cnd) with dt cquisition nd processing done with softwre pckge (MssLynx; Wters, Elstree, UK). Dtse serching ws performed y softwre tool (ProteinLynx, Wters) using peptide tolernce of ppm. Results levels re regulted y glucose Proteomic nlysis of MIN6 pncretic et cell homogentes identified 84 proteins (Fig. 1, Electronic supplementry mteril [ESM] Tles 1 3). Of the proteins identified y MALDI-TOF, 11 were found to e differentilly produced t vs 3 mmol/l glucose-contining medium (Fig. 1). The most gretly upregulted proteins were the cytoskeletl proteins tuulin nd ctin (2- to 4.5-fold). As one of the next in line, ws upregulted y ~1.8-fold. This protein hs not previously een identified s glucose-regulted protein nd thus we decided to further investigte levels in MIN6 cells. To confirm the proteomics dt, the two dimensionl gels were proed with monoclonl nti- ntiody nd levels compred etween non-stimultory (3 mmol/l) nd stimultory ( mmol/l) glucose concentrtions (Fig. 2). The sme nd tht ws identified y MALDI-TOF nd imge nlysis softwre (PDQuest, Bio-Rd Lortories, Hemel Hempsted, UK), nd hd moleculr mss of ~23 kd nd n isoelectric point of 4.3 ws lso recognised y the -specific ntiody t oth glucose concentrtions. The intensity of this protein nd incresed y 1.8- to 2.5-fold t stimultory glucose concentrtion (Fig. 2). The ntiody ppered to e very specific nd cross-rected with protein of ~21 to 23 kd on one dimensionl SDS-PAGE (Fig. 2c), where similr upregultion of production ws oserved to tht on two-dimensionl SDS-PAGE (Fig. 2d). Tctp mrna ws detected in primry islets of Lngerhns nd MIN6 cells (Fig. 2e). Moreover, protein levels were lso incresed in primry rt islets of Lngerhns t stimultory glucose concentrtion (Fig. 2d, f, g). Post-trnsltionl modifiction of is regulted y glucose We repetedly oserved slight smer of the nd to the cidic region or even clerly more cidic isoform t non-stimultory glucose concentrtion (Fig. 3). We therefore investigted whether the vrition of the spots (Fig. 3) my e cused y differentil post-trnsltionl modifictions. Potentil post-trnsltionl modifictions hd een previously predicted y our ioinformtic nlysis (NetPhos 2., ccessed Mrch 8). Three potentil phosphoryltion sites for serine, two for tyrosine nd one for threonine were predicted. In ddition, potentil glycosyltion sites (for N- nd O-linked glycosyltion) were lso predicted for. MIN6 whole-cell lystes were seprted y two dimensionl SDS-PAGE, using nrrow rnge (ph 3 5) IPG strips to increse the resolution nd improve the seprtion of the two proteins recognised y the ntiody. Using ntiphosphoserine nd nti-phosphotyrosine ntiodies, we oserved tht the 23 kd ws pprently dephosphorylted on these residues in response to glucose stimultion (Fig. 3). The presence of phosphorylted isoform ws confirmed y phosphostining (ProQ Dimond) (Fig. 3) nd susequent identifiction of the protein nd with qudrupole time-of-flight (Q-TOF) mss spectroscopy nlysis. We lso investigted the potentil glycosyltion of. However, the monoclonl ntiody ginst O-GlcNAc modifiction or the glycoprotein stin Emerld 488 (Pro-Q) produced very high ckground nd mny non-specific stining signls. Thus, the glycosyltion stte of proteins could not e resolved convincingly. Nevertheless, regultion of production y glucose nd dephosphoryltion on serine nd tyrosine residues were clerly demonstrted.

5 2 Dietologi (11) 54:368 9 Fig. 1 Proteomic nlysis of MIN6 pncretic et cells. MIN6 cell homogentes ( μg) were loded on to 24 cm non-liner IPG strip (ph 3 1), susequently seprted on 12% (vol./vol.) SDS- PAGE in the second dimension nd stined with colloidl Coomssie lue. The imge ws generted using softwre tool (PDQuest). The protein spots were excised from the gels, digested with trypsin nd identified y MALDI-TOF mss spectroscopy. The identified spots re designted with protein revition (for detils see ESM). List of differentilly produced proteins in stimulted vs non-stimulted conditions. Cells were incuted in 3 or mmol/l glucosecontining medi for 24 h nd difference in production given s rtio of :3 mmol/l. Cell homogentes were seprted on 2DGE. Differentil production ws nlysed using PDQuest. A minimum of six gels were nlysed for ech condition kd ph 3(+) RP1 EEF2 TRA1 VCP IMMT STF ACO2 GRP78 HSC7 GARS GRP75 ALB ALB GPD2 PDIA1 PC2 ATP6V1A TCP1 DDYSL2 PDK SERS TUBB7 TUBA CALR PDK TCP1 TCP1 HSP6 PD1A3 TCP1 ENO1 PDIA6 EEF1A ATP6V1B2 GRP54 CPA3 TUBB5 GLUD1 ATP5A1 ATP5B SCRN1 HNRNPF EEF1A PGK1 IDH2 PCBP1 ACAT1 GOT2 NPM1 ACT ALB GALE ANXA2 ALDO1 TXNl1 AKR1B1 HNRNPA2 MDH2 PCNA ANX5 ANXA4 ANX5 MDH1 GAPDH HNRNPA3 SCGN ETFB PHB2 PAFAH1B1 PRDX4 PRDX4 PGL5 ELA3B PSMA6 GSTM1 GDI1 PSMA2 GSTP1 Protein revition PRDX2 PBP Protein identity UMPCMPK SOD1 PSMB2 NME1 NME2 PRDX1 CFL1 DADF PP1A 1( ) Rtio (:3 mmol/l glucose) PDIA6 Protein disulphide isomerse A6 precursor.7 PC2 Prohormone convertse 2 1. ENO1 α-enolse 1.2 VCP Vlosin contining protein 1.4 HSC7 Het shock cognte 71 kd protein 1.6 SERS Seryl t-rna synthse 1.6 PBP Phosphtidylethnolmine inding protein 1.7 ATP6V1A Vcuolr ATP synthse ctlytic suunit A 1.8 Trnsltionlly controlled tumour protein 1.8 ACT Actin 2.2 TUBA Tuulin -6-chin 2.3 TUBB5 Tuulin -5-chin 4.4 Incresed nucler loclistion of in response to glucose Post-trnsltionl modifictions cn determine sucellulr loclistion nd ctivity of proteins. Mny proteins hve een reported to trnslocte to the nucleus upon dephosphoryltion [31 33]. Since is dephosphorylted t stimultory glucose concentrtion, we next investigted whether the sucellulr loclistion of is ltered y glucose. Immunocytochemistry reveled tht ws lrgely loclised to the cytosol. Due to the strong cytosolic stining, it ws impossile to estlish whether is ssocited with ny cellulr orgnelles in non-stimulted cells (Fig. 4, e). In contrst, in response to glucose stimultion, showed cler nucler loclistion (Fig. 4, d, f, g), while significnt proportion of the protein remined in the cytosol. Reduction of protein production results in enhnced et cell deth To investigte the physiologicl role of in pncretic et cells, gene knockdown experiments were performed using sirna. Vrious sirna trget sequences were tested to otin reduction in protein levels y t lest 4%. The lrgest decrese in level ws chieved t 96 h post-trnsfection (Fig. 5). This time point ws used for further poptosis nd insulin secretion experiments. During the sirna experiments, significnt numer of ded cells were oserved, which led us to investigte the potentil role

6 Dietologi (11) 54: kd 1 75 ph 3(+) 1( ) Reltive density c kd 75 3 d Reltive density e kp MIN6 Tctp Islets f kd ERK1/2 g Reltive density /ERK * 3.2 Act Glucose (mmol/l) Fig. 2 protein levels re regulted y glucose. Cells were incuted for 24 h in 3 or mmol/l glucose-contining medi. Cell homogentes were seprted on 2DGE using 7 cm IPG strips (ph 3 1). Proteins were then trnsferred to nitrocellulose memrnes. Memrnes were proed with -specific monoclonl ntiody (1:1, dilution). The resulting nd is highlighted y rectngles. Quntifiction of protein levels in vs 3 mmol/l glucosecontining medi. nds were scnned nd their density mesured using softwre pckge (ImgeQunt, GE Helthcre). Equl mounts of proteins were loded on ech gel nd minimum of six gels were nlysed for ech condition. Quntifiction of levels on one-dimensionl SDS-PAGE. c The nitrocellulose memrnes were stined for totl protein with Ponceu red prior to immunostining with n nti- ntiody. d Memrnes were scnned nd the density of the signl in ech protein lne mesured nd normlised to tht of the totl protein. A minimum of six gels were nlysed for ech condition. e Expression of Tctp mrna in MIN6 cells nd primry rt islets of Lngerhns. PCR ws performed with specific primers for Tctp nd Act (encoding β-ctin). f Rt islets of Lngerhns were incuted for 24 h in medium contining either 3 or 17 mmol/l glucose. Homogentes were seprted on SDS-PAGE nd immunolotting ws performed s descried ove () (except ntiody dilution 1:). Equl mounts of proteins were loded on ech lne ( μg). The sme immunolot ws proed with rit polyclonl nti-erk1/2 ntiody (1:1, dilution) [28]. The signl density of ech nd ws mesured nd (g) : ERK1/2 rtio clculted. Three independent experiments were crried out nd ech smple ws loded in duplictes. The films were exposed for vrious periods of time. For density mesurements n exposure time ws selected, where the chemiluminescence rection ws still in the liner phse. Error rs (, d, g) represent stndrd error; *p<.5 nd p<.1 of in poptosis. The extent of poptosis ws quntified y propidium iodide stining nd flow cytometry [29]. MIN6 et cells trnsfected with sirna specific to showed significntly higher cell deth thn cells trnsfected with the scrmled RNA (Fig. 5). These dt suggest tht decresed production reduces the viility of et cells. We next investigted the effect of reduced protein levels on glucose-induced insulin secretion. Glucose elicited n pproximtely threefold increse in insulin secretion; however, no significnt difference etween the vrious conditions ws oserved (Fig. 5c). Plmitte reduces protein production through mechnisms involving endoplsmic reticulum stress Hving

7 4 Dietologi (11) 54:368 9 kd 2 3 ph 4 5 ph Anti- Glucose (mmol/l) 3 A CB c e g 3 Fig. 3 phosphoryltion is regulted y glucose. MIN6 cells were incuted for 24 h in the presence of 3 (, c, e, g) or(, d, f, h)mmol/l glucose-contining medium s lelled nd the cell homogentes were seprted y 2DGE on 7 cm IPG strips (ph 3 5), followed y 4 12% (vol./vol.) SDS-PAGE in the second dimension. The proteins were trnsferred to nitrocellulose memrnes nd proed, s lelled, with n nti- monoclonl ntiody (1:1,;, ), n nti-phosphotyrosine ntiody (1:; c, d) or n ntiphosphoserine ntiody (1:2; e, f). A gel ws lso stined for phosphoproteins using Pro-Q Dimond (g, h) nd counterstined for totl proteins with SYPRO Ruy. The phosphorylted protein corresponding to (rectngles) ws excised nd its identity confirmed y Q-TOF mss spectroscopy. Antiodies or stins used re indicted on the right. A minimum of three gels were nlysed for ech condition demonstrted tht in norml et cells glucose stimultion resulted in upregultion of protein (Fig. 2), we next investigted whether levels re reduced when the cells were sujected to stress conditions. For this purpose, the cells were incuted with.5 mmol/l plmitte for 24 h. protein levels were significntly reduced (y out 4%) following ftty cid tretment (Fig. 6). In prllel, poptosis ws mesured nd significnt deth ws oserved in cells incuted with plmitte compred with controls (Fig. 6). The sme effect of plmitte on protein levels ws oserved in primry rt islets of Lngerhns d f h Anti-pTyr Anti-pSer Pro-Q Dimond DAPI Cc e g Reltive fluorescence intensity of in the nucleus Dd f 3 Fig. 4 Glucose stimultion induces incresed nucler loclistion of., c, e Immunoloclistion of in MIN6 cells t low (3 mmol/l) nd (, d, f) high ( mmol/l) glucose. Cells were grown on poly-l-lysine-coted glss coverslips nd incuted with glucose for 24 h. Cells were fixed with prformldehyde, wshed nd proed with mouse monoclonl nti- ntiody (1:) nd visulised with got nti-mouse Alex-488 secondry ntiody (1: dilution) (,, e, f). Nuclei were identified y DAPI stining (c, d). Arrowheds show nuclei of the cells on nd DAPI imges. Dotted outlines ( d) show cells seen (e, f) on n expnded scle. Scle rs 1 μm ( d) or 2.5 μm (e, f). g Quntifiction of nucler loclistion. The imging ws performed using sme confocl settings for ll conditions nd the intensity of nucler stining ws quntified with softwre pckge (Volicity). Three regions were rndomly selected on ech coverslip in three independent experiments. The totl cell numer nlysed t 3 nd mmol/l glucose ws 94 nd 65, respectively. Error rs represent stndrd error; p<.1

8 Dietologi (11) 54: sirna Cont. 1.2 Reltive density compred with control.8.4 kd Time (h) * Cont Time (h) Cell deth (%) (-) Cont. sirna Cont. sirna (+) Cont. c Insulin secretion (% totl) * Cont. sirna Cont. * * sirna Fig. 5 Suppressed production of leds to reduced cell survivl, ut does not ffect glucose-stimulted insulin secretion. sirna suppression of protein production. MIN6 cells were kept in norml growth medium contining mmol/l glucose nd trnsfected with 8 pmol/l commercil sirna for. Totl protein ws extrcted 24, 48, 72 nd 96 h fter trnsfection. Protein ( μg) ws seprted y SDS-PAGE, trnsferred to nitrocellulose memrnes nd proed with mouse nti- ntiody (1:1, dilution). Cont., control, i.e. scrmled RNA. Protein nds were scnned nd their density mesured using softwre tool (ImgeQunt). Reltive densities compring the trnsfected smples t ech time point with the scrmled RNA control re shown. Four independent experiments were performed nd ech smple ws loded in triplicte. Reduced levels of following sirna tretment led to incresed cell deth. Cell deth ws determined y FACS nlysis using propidium iodide stining in cells trnsfected with sirna or scrmled RNA t 96 h post trnsfection. ( ) Cont., cells cultured for 96 h without ny tretment; sirna Cont., cells trnsfected with scrmled RNA; sirna, cells trnsfected with sirna for Tctp; (+) Cont., cells treted with methnol overnight t C nd used s positive control for cell deth. The grph represents three independent experiments with ech mesurement performed in triplictes. Note: cells were cultured for 144 h in totl, hence the high sl cell deth shown here. c Glucosestimulted insulin secretion in non-trnsfected cells nd cells trnsfected with scrmled RNA or sirna. Insulin secretion ws quntified 96 h post trnsfection. White rs, insulin secretion t 3 mmol/l glucose; lck rs, insulin secretion t mmol/l glucose. The grph represents three independent experiments with ech mesurement performed in triplictes. Error rs represent stndrd error; *p<.5, p<.1 (Fig. 6c), where protein levels were reduced y ~% (Fig. 6d). Since plmitte induces endoplsmic reticulum (ER) stress in pncretic et cells [34 36], we next investigted whether thpsigrgin, chemicl ER stress inducer, ffected protein level. We oserved significnt reduction (out ~%) of protein production fter 8 h thpsigrgin tretment (Fig. 6e, f). Incresed protein production protects cells from poptosis To test whether the incresed protein level seen fter glucose stimultion (Fig. 2) cn improve cell viility nd prtilly protect cells from plmitte-induced cell deth, we overproduced. The trnsfection efficiency ws out %. Therefore poptotic cells were counted in the -overproducing cells (n=1 nd 23 cells following 8 nd 24 h incution with plmitte, respectively; Fig. 7). The proportion of cells positive for TUNEL stining ws significntly lower in cells visily overproducing thn in neighouring untrnsfected cells on the sme plte or in the control plsmid-trnsfected cells (Fig. 7). A similr proportion of poptotic cells (~4%) ws otined in plmitte-treted cells trnsfected with the empty control vector (n=397 cells; Fig. 7) or in untrnsfected cells (n=476 cells; Fig. 7), s oserved previously following 24 h incution with plmitte (Fig. 6). Our dt thus indicte tht overproduction significntly reduces plmitte-induced cell deth (Fig. 7). To etter understnd the possile mechnism y which incresed protein levels reduce cell deth, we next tested whether higher proportion of ecomes ssocited with the mitochondri under this condition. In non-stimultory glucose condition only very smll mount of ws detectle in the mitochondril frction, wheres t stimultory glucose levels the mount of found in this sucellulr frction ws clerly incresed (Fig. 7c). This supports model, ccording to which nchors to the mitochondril memrne nd ntgonises the ction of the pro-poptotic protein BAX, which is involved in regulting the memrne permeility [1]. Discussion To contriute to the understnding of glucose regultion in pncretic et cells, we investigted the differentil regultion of proteins in response to high vs low glucose

9 6 Dietologi (11) 54:368 9 Fig. 6 Plmitte tretment nd ER stress led to reduced protein levels. Plmitte reduces protein level. Cells were incuted with.68% (wt/vol.) BSA with or without.5 mmol/l plmitte for 24 h. Equl mounts of protein ( μg) were loded on ech lne. Reltive protein levels were ssessed y immunolotting using mouse monoclonl nti- ntiody (1:1, dilution). The sme immunolot ws proed with mouse monoclonl nti- GAPDH ntiody. The signl density of ech nd ws mesured nd /GAPDH rtio clculted, which in plmitte-incuted cells ws compred with cells cultured with BSA only. Five independent experiments were crried out nd ech smple ws loded in triplicte. Apoptosis in cells incuted in the sence or presence of plmitte ws determined s descried (Fig. 5). Note: cells were cultured for 48 h in totl for experiments shown here (), hence the decresed sl cell deth compred with Fig. 5. The grph represents three independent experiments with ech mesurement performed in triplictes. c Plmitte reduces protein level in primry rt islets of Lngerhns. ws detected in rt islets of Lngerhns fter 24 h incution in the presence nd sence of.5 mmol/l plmitte. Equl mounts of protein ( μg) were loded on ech lne nd the mouse nti- ntiody ws used in 1: dilution. intensity ws normlised to ERK1/2, s the nti-erk1/2 ntiody worked etter in these smples thn the ntiody ginst GAPDH. Quntifiction (d) ws crried out s descried (Fig. 2d). e MIN6 cells were incuted for 8 h in the presence nd sence of the phrmcologicl ER stressor thpsigrgin (.4 μmol/l). Blots nd quntifiction (f) were performed s descried ove (, c, d). Error rs represent stndrd error; *p<.5, p<.1 concentrtions. Using proteomics pproch, we identified 11 out of 84 proteins tht re differentilly regulted (Fig. 1, ESM Tles 1 3). Of these proteins, we initilly selected for more detiled investigtion. Our study demonstrtes tht protein levels nd sucellulr distriution re regulted y glucose in pncretic et cells. Since incresed ftty cid circultion is mjor fctor in the development of type 2 dietes nd ftty cids hve een shown to e toxic to et cells [34 ], we lso investigted the effect of ftty cid on protein levels in et cells. Plmitte downregulted protein levels nd resulted in incresed cell deth (Fig. 6 d). Thus, the cytotoxic effect of plmitte is, t lest in prt, due to reduced production of this nti-poptotic protein. In contrst, overproduction of protein protected et cells from poptosis (Fig. 7, ). Our results thus imply tht levels influence the sensitivity of the et cell to poptosis. levels hve een shown to e highly regulted in response to wide rnge of extrcellulr signls, s reviewed y others [6, 21, 22]. However, to our knowledge, this is the first demonstrtion of regultion of levels in response to ltertions in glucose or ftty cid concentrtions. Protein synthesis in et cells is highly regulted in response to ltertions of glucose concentrtions. In prticulr, mechnisms of the unfolded protein response (UPR), physiologicl ER-stress response, re involved in this regultion. This includes ctivtion of the PKR-like ER kinse (PERK) nd susequent phosphoryltion of initition fctor Reltive density /GAPDH kd Plmitte (mmol/l).5 BSA (%) (wt/vol.) c e kd ERK1/ Plmitte (mmol/l).5 BSA (%) (wt/vol.) ERK1/2 Thpsigrgin (µmol/l).4 GAPDH Cell deth (%) Reltive density /ERK Reltive density /ERK eif2α, leding to (locl) inhiition of protein synthesis. At low glucose concentrtions, PERK is ctivted, resulting in incresed eif2α phosphoryltion nd inhiition of ER protein (nd therefore proinsulin) synthesis [38, 39]. We hve previously demonstrted tht synthesis is downregulted through phosphoryltion of eif2α y PKR in cellulr stress conditions [11, 24]. Although we did not specificlly ddress the role of PERK in these studies, oth kinses re proly involved in regultion, s oth re ctivted upon ER-stress [4]. We conclude tht the glucosedependent regultion of synthesis in et cells oserved in this study (Fig. 2) islsolikelytoemedited through mechnism involving eif2α phosphoryltion. While mechnisms of the UPR re involved in physiologicl regultion of et cells through glucose, they my lso e ctivted under chronic stress conditions, such s Plmitte (mmol/l) BSA (%) (wt/vol.) d Plmitte (mmol/l) BSA (%) (wt/vol.) f Thpsigrgin (µmol/l)

10 Dietologi (11) 54: Fig. 7 Incresed levels of leds to its trnsloction to the mitochondri nd enhnced protection from cell deth. Overproduction of protects MIN6 cells from plmitte-induced cell deth. Cells were trnsfected with plsmid encoding the full-length mouse Tctp or with the empty control vector nd incuted t 24 h post trnsfection with.5 mmol/l plmitte for 8 h (n=14) nd 24 h (n=18). The experiment ws crried out s descried in the Methods. Exmples of overproducing cells re indicted with rrows. Cell nuclei positive for TUNEL stining re lelled y rrowheds nd were lso visulised with DAPI stining. Scle r, 1 μm. The numers of poptotic cells in -trnsfected cells (white rs), in the neighouring un-trnsfected cells on the sme plte (grey rs) nd in cells trnsfected with the empty control vector pcdna3 (lck rs) were counted nd plotted. Three independent experiments were performed nd the numer of cells counted in ech condition is indicted elow grph. Regions on the pltes were rndomly selected nd ll cells were nlysed in field of view. Error rs represent stndrd error, which ws very smll for the pcdna3 24 h control (.42); p<.1. c prtilly trnsloctes to the mitochondri in response to glucose stimultion. MIN6 cells were incuted for 24 h in medium contining 3 mmol/l or mmol/l glucose. The mitochondril pellet (Mito.P) nd post-mitochondril superntnt frction (PMS) were otined from centrifugtion of the post-nucler superntnt frction (PNS) t 1, g for min. The Mito.P frction ws wshed severl times efore loding on to gels. The lots were proed with the nti- ntiody. PNS nd PMS ( μg), nd μg mitochondril pellets were loded. The sme lots were proed for ERK1/2 nd for the mitochondril mrker cytochrome c (cyt c) for equl loding. Note the incresed protein level t stimultory glucose concentrtion in the PNS smples. The result is representtive of four independent experiments elevted levels of NEFA, which contriute to et cell dysfunction in type 2 dietes. NEFA, such s plmitte, trigger ER stress [34 ] nd eventully poptosis in cultured et cells in vitro, s reviewed y others [41, 42]. They lso induce poptosis in primry rodent nd humn islets of Lngerhns [34]. In et cells, plmitte hs een demonstrted to ctivte PERK nd phosphoryltion of eif2α, resulting in inhiition of trnsltion initition [35, 36, 43]. In recent study, we demonstrted downregultion of levels in mouse emryo firolsts under some, ut not ll ER stress conditions [11]. ws prticulrly downregulted in C 2+ stress conditions (including thpsigrgin tretment) in mnner dependent on ctive PKR nd eif2α phosphoryltion. This is consistent with our oservtion tht is downregulted in et cells under stress induced y plmitte or thpsigrgin (Fig. 6). Downregultion of nti-poptotic proteins is n importnt prerequisite for poptosis to occur, so it is not surprising tht [11] nd MCL-1 [44] hve een found to e downregulted in C 2+ /ER stress conditions in PKRdependent mnner. Since plmitte hs erlier een shown to result in ctivtion of PERK in et cells [35, 36, 43], we conclude tht downregultion of y plmitte (Fig. 6) is medited through PERK ctivtion. Our studies hve lso shown tht overproduction of cn dely poptosis induced y thpsigrgin, c 8 h 24 h Cell deth (%) kd Glucose (mmol/l) PNS 8 h 24 h PMS TUNEL Plsmid: pcdna3 pcdna3 No. of cells: Visile overproduction: tunicmycin nd etoposide [11] or plmitte (Fig. 7). Conversely, downregultion of protein production hs een reported to led to poptosis [1, 13, 14], consistent with the oservtions otined here in Tctp knockdown experiments on et cells (Fig. 5). Plmitte is known to e cytotoxic to et cells in oesity-ssocited niml models of dietes, s well s in norml et cells [34 ]. We propose tht these cytotoxic effects re t lest in prt medited y the inhiition of production of ntipoptotic proteins such s. hs een descried s cytosolic protein [6], ut nucler loclistion hs lso een reported [12, 45]. Under low glucose concentrtions, we oserved lrgely cytosolic stining in et cells, with very little stining in the nucleus. However, under high-glucose conditions we noticed significnt immunostining in the nucleus, in ddition to the cytosolic stining (Fig. 4). Similrly to our ERK1/2 1 DAPI Mito.P Plmitte (.5 mmol/l) Cyt c

11 8 Dietologi (11) 54:368 9 finding, other recent studies hve shown tht cn e trnsported to the nucleus under certin conditions [45]. Rid et l. [45] reported tht under oxidtive stress conditions trnsloctes to the nucleus. There re lso indictions tht might e involved in trnscriptionl regultion of specific genes in T lymphocytes [46] nd in erly development [47, 48]. Our current study indictes tht ecomes dephosphorylted on serine nd tyrosine residues in response to glucose stimultion, which might e importnt for this nucler trnsloction, s reported in other cses [31 33]. Since is n nti-poptotic protein, we lso ttempted to investigte its potentil co-loclistion with mitochondri in et cells. However, ecuse is still undntly present in the cytosol, giving rise to intense cytosolic stining, nd ecuse these cells were only poorly spred, it ws not possile to loclise to ny specific orgnelles using immunocytochemistry. Insted, we used sucellulr frctiontion, the results of which indicted tht proportion of ecomes ssocited with the mitochondri-enriched frction t high vs low glucose condition (Fig. 7c). It hs een demonstrted tht intercts with the nti-poptotic proteins MCL-1 nd BCL-XL in vitro [8, 9]. Immunofluorescence dt hve lso suggested tht co-loclises with BCL-XL [9] nd MCL-1 [8] in vivo, nd is therefore likely to ct, like other key regultors of poptosis, t the mitochondri. However, the interprettion of co-loclistion studies [8, 9] is rther complicted ecuse, s mentioned ove, is highly undnt in the cytosol. A more recent study hs demonstrted tht the nti-poptotic function of tkes plce, t lest in prt, t mitochondri, with inhiiting the function of BAX [1]. Our investigtions (Fig. 7c) suggest tht, in et cells, prtilly trnsloctes to the mitochondri in response to glucose, where it is likely to ct in similr fshion. In summry, we hve provided evidence tht, in pncretic et cells, is positively regulted y glucose nd negtively y pro-poptotic stimuli, such s plmitte. It is involved in the protection of et cells ginst poptosis. Further studies re required to determine the precise mechnisms of the glucose-dependent regultion of nd its trnsloction to the nucleus, s well s the significnce of this trnsloction for its nti-poptotic function. Further studies re lso required to elucidte regultion of y other propoptotic stimuli in et cells, s well s the importnce of in niml models of type 2 dietes. Acknowledgements We thnk J. Slinn nd M. Lewis (Centre for Reserch in Biomedicine, Fculty of Helth nd Life Sciences, University of the West of Englnd, Bristol, UK) for their technicl ssistnce nd dvice on mss spectroscopy nlysis. This study ws supported y grnts from the Wellcome Trust, the Biotechnology nd Biologicl Sciences Reserch Council nd the Medicl Reserch Council to A. Vrdi. S. 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