Signaling in Insulin-Secreting MIN6 Pseudoislets and Monolayer Cells

Signaling in Insulin-Secreting MIN6 Pseudoislets and Monolayer CellsAzazul Chowdhury,*,† Venkata P. Satagopam,‡,§ Levon Manukyan,† Konstantin A. Artemenko,∥

Yi Man Eva Fung,⊥ Reinhard Schneider,‡,§ Jonas Bergquist,∥ and Peter Bergsten†

†Department of Medical Cell Biology, Uppsala University, Box 571, SE-75123 Uppsala, Sweden‡Department of Structural and Computational Biology, EMBL, Meyerhofstrasse 1, D-69126 Heidelberg, Germany§Luxembourg Centre For Systems Biomedicine (LCSB), University of Luxembourg, Campus Belval, House of Biomedicine, 7 Avenuedes Hauts-Fourneaux, L-4362 Esch-sur-Alzette, Luxembourg∥Analytical Chemistry, Department of Chemistry−Biomedical Center and SciLifeLab, Uppsala University, Box 599, SE-75124Uppsala, Sweden⊥Department of Chemistry, State Key Laboratory of Synthetic Chemistry, and Open Laboratory of Chemical Biology of the Instituteof Molecular Technology for Drug Discovery and Synthesis, The University of Hong Kong, Pokfulam Road, Hong Kong, China

*S Supporting Information

ABSTRACT: Cell−cell interactions are of fundamental impor-tance for cellular function. In islets of Langerhans, which controlblood glucose levels by secreting insulin in response to the bloodglucose concentration, the secretory response of intact islets ishigher than that of insulin-producing beta-cells not arranged in theislet architecture. The objective was to define mechanisms bywhich cellular performance is enhanced when cells are arranged inthree-dimensional space. The task was addressed by making acomprehensive analysis based on protein expression patternsgenerated from insulin-secreting MIN6 cells grown as islet-likeclusters, so-called pseudoislets, and in monolayers. After culture,glucose-stimulated insulin secretion (GSIS) was measured frommonolayers and pseudoislets. GSIS rose 6-fold in pseudoislets butonly 3-fold in monolayers when the glucose concentration wasincreased from 2 to 20 mmol/L. Proteins from pseudoislets andmonolayers were extracted and analyzed by liquid-chromatography mass spectrometry, and differentially expressed proteins weremapped onto KEGG pathways. Protein profiling identified 1576 proteins, which were common to pseudoislets and monolayers.When mapped onto KEGG pathways, 11 highly enriched pathways were identified. On the basis of differences in expression ofproteins belonging to the pathways in pseudoislets and monolayers, predictions of differential pathway activation wereperformed. Mechanisms enhancing insulin secretory capacity of the beta-cell, when situated in the islet, include pathwaysregulating glucose metabolism, cell interaction, and translational regulation.

KEYWORDS: glucose-stimulated insulin secretion (GSIS), beta-cells, MIN6 cells, pseudoislets

■ INTRODUCTION

Beta-cells from the pancreatic islet release insulin in response toelevated blood glucose levels. Proper insulin secretion dependson the architecture of the islets. As with most cell types, thecell-to-cell contacts within the islets have been shown to becrucial for the normal function.1−3 This view is supported bywork demonstrating that dispersed islet cells respond poorly toglucose compared to intact islets.4 When cell-to-cell contact isre-established, such as in dispersed beta-cells that reaggregate toform structures so-called ‘pseudoislets’ with similar size andcellular organization as in primary islets, function is essentiallyregained.5,6 The mouse-derived insulinoma MIN6 cell line isglucose-responsive.7 When MIN6 cells are allowed toreaggregate, they form cell clusters, which mimic the size andmorphology of primary islets.3,8−11 MIN6 pseudoislets show

enhanced secretory capacity compared to MIN6 cells.12 Amongmechanisms reported to account for the difference betweenislets and dissociated beta-cells are the intercellular contactswithin the islet coordinating and synchronizing the beta-cellsthrough gap junctions.13 Other mechanisms responsible for thedifference between MIN6 pseudoislets and MIN6 cells grownin monolayers have also been reported including altered IRS-1signaling.3,8−12 Although primary islets are required to fullyunderstand the cellular interactions between the many celltypes involved in insulin secretion, insulin-producing cell linesare important tools in beta-cell research. With these cell lines,the difficulties to isolate large amounts of primary islets and

Received: May 3, 2013Published: September 6, 2013

Technical Note

pubs.acs.org/jpr

© 2013 American Chemical Society 5954 dx.doi.org/10.1021/pr400864w | J. Proteome Res. 2013, 12, 5954−5962

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their mixed cell populations are overcome. In this study, weaddressed the issue of how these cell-to-cell contacts influencecellular signaling by comprehensively analyzing differentialprotein expression patterns obtained from cells that arearranged in three-dimensional structures, the MIN6 pseudois-lets (PIs) and cells that do not enjoy this arrangement, theMIN6 monolayer cells (MOs).

■ MATERIALS AND METHODS

Cell Culture

Mouse insulinoma MIN6 cells were cultured under conditionspreviously published7 in 250 mL tissue culture flasks (BectonDickson Labware, Franklin Lakes, NJ) at 37 °C (95% O2 and5% CO2) in Dulbecco’s Modified Eagle medium (Invitrogen,Paisley, U.K.), containing 25 mmol/L glucose and supple-mented with 10% fetal bovine serum, 100 units/mL penicillin,100 μg/mL streptomycin (Invitrogen), and 50 μM β-mercaptoethanol (Sigma, St. Louis, MO). Whereas monolayersof MIN6 cells were formed when MIN6 cells were cultured innegatively charged tissue culture plates (Becton DicksonLabware), MIN6 pseudoislets were formed when MIN6 cellswere cultured in tissue culture dishes made of nonadherentplastic (Becton Dickson Labware), which allowed theaggregation of dispersed cells.3 For the latter purpose, 3 ×106 dispersed cells were cultured for 3−5 days using the sameculture condition as for monolayer culture. All experimentswere performed between passages 20 and 30.

Insulin Secretion

After culture, insulin secretion was measured from MOs andPIs. For static incubation, monolayer cells or groups of 20pseudoislets were preincubated for 60 min at 37 °C in KRBHbuffer consisting of 130 mmol/L NaCl, 4.8 mmol/L KCl, 1.2mmol/L MgSO4, 1.2 mmol/L KH2PO4, 1.2 mmol/L CaCl2, 5mmol/L NaHCO3, and 5 mmol/L HEPES, titrated to pH 7.4with NaOH, and supplemented with 1 mg/mL BSA and 2mmol/L glucose. Subsequently, cells were incubated in KRBHbuffer containing either 2 or 20 mmol/L glucose for 30 min at37 °C. Aliquots (200 μL) of medium were collected fordetermination of insulin secretion. Monolayer cells andpseudoislets were washed three times with phosphate buffersaline (PBS) and lysed with buffer containing 0.1% Triton-X100 and 25 mmol/L NaOH, and lysates were stored at −20 °Cfor measurement of total protein. Differences in insulinsecretion among the groups were assessed using analysis ofvariance (ANOVA) followed by Bonferroni’s post hoc test. P <0.05 was considered statistically significant.

Sample Preparation for Protein Profiling

Cells were washed twice with PBS and lysed with a buffercontaining 8 mol/L urea, 1% octyl-β-D-glucopyranoside in 10mmol/L Trizma Base, pH 8.0, for 20 min. After lysis, sampleswere centrifuged at 10 000 rpm for 10 min to pellet anyremaining debris. Buffer in the samples was exchanged to 50mmol/L ammonium bicarbonate using PD SpinTrap G-25columns (GE Healthcare, Life Sciences, Uppsala, Sweden)according to the manufacturer’s protocol. Total protein contentwas determined by the Bradford Protein Assay (Bio-Rad,Hercules, CA). Samples were reduced by dithiothreitol (DTT)(10 mmol/L, 56 °C for 30 min) and alkylated byiodoacetamide (20 mmol/L, room temperature in darknessfor 30 min). Before digestion with trypsin, the Nanosep 3Omega 3 kDa cutoff filters (Pall, Port Washington, NY) were

washed once with 100 μL of 50% acetonitrile in 50 mmol/LNH4HCO3 by spinning them down. Samples were transferredonto the cutoff filters and spun. The membranes were washedonce with 100 μL of 2% acetonitrile in 50 mmol/L NaHCO3,spun, and then wash with 100 μL of 50% acetonitrile in 50mmol/L NH4HCO3. Samples were diluted in 50 μL ofNH4HCO3 and digested with trypsin at 37 °C for 18 h. Afterdigestion, samples were spun at 14 000 rpm for 30 min.

Protein Expression Profiles

Digested samples were redissolved in 0.1% trifluoroacetic acidto yield an approximate tryptic peptide concentration of 0.3μg/μL prior to nanoLC−MS/MS analysis. The proteinidentification experiments were performed using a 7 T hybridLinear ion trap Fourier Transform (LTQ-FT) mass spec-trometer (ThermoFisher Scientific, Bremen, Germany) fittedwith a nanoelectrospray ionization (ESI) ion source (Thermo-Fischer Scientific). Online nanoLC separations were performedusing Agilent 1100 nanoflow system (Agilent Technologies,Waldbronn, Germany). The peptide separations were per-formed on in-house packed 15 cm fused silica emitters (75 μminner diameter, 375 μm outer diameter). The emitters werepacked with a methanol slurry of reversed-phase, fully end-capped Reprosil-Pur C18-AQ 3 μm resin (Dr. Maisch GmbH,Ammerbuch-Entringen, Germany) using a pressurized packingdevice operated at 50−60 bar (Proxeon Biosystems, Odense,Denmark). The separations were performed at a flow of 200nL/min with mobile phases A (water with 0.5% acetic acid) andB (89.5% acetonitrile, 10% water, and 0.5% acetic acid). A 100min gradient from 2% B to 50% B followed by a washing stepwith 98% B for 5 min was used. Mass spectrometric analysiswas performed using unattended data-dependent acquisitionmode, in which the mass spectrometer automatically switchedbetween acquiring a high resolution survey mass spectrum inthe FTMS (resolving power 100 000 full width at half-maximum) and consecutive low-resolution, collision-induceddissociation fragmentation of up to five of the most abundantions in the ion trap.

Bioinformatics Analysis

(a). Label-Free Peptide and Protein Quantifications.The acquired spectra from 12 samples derived frompseudoislets (4 biological × 3 technical replicates) and 12from monolayers (4 biological × 3 technical replicates) wereuploaded to the Progenesis LC−MS Version 4.0 (NonlinearDynamics, Newcastle, England) after choosing Progenesismachine settings according to the respective acquisitioninstrument and analyzed the data as described previously.14

Profile data of the MS scans were transformed to peak lists withrespective peak m/z values, intensities, abundances (areasunder the peaks), and m/z width. In the alignment step, wemanually selected a reference run and the retention times of theother samples were aligned by manual and automatic alignmentto a maximal overlay of all features. Features with only onecharge or more than seven charges are masked at this point andexcluded from further analyses. After alignment and featureexclusion, samples were divided into the appropriate groups (PIand MO), and raw abundances of the remaining features werenormalized to allow correction for factors resulting fromexperimental variation.Rank 1−10 MS/MS spectra were exported from the

Progenesis software as Mascot generic file (mgf) and used forpeptide identification with Mascot version 2.2 (Matrix Science,London, U.K.) in the International Protein Index (IPI)

Journal of Proteome Research Technical Note

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database version 3.68 mouse containing a total of 56 729protein sequences. Search parameters used were the following:10 ppm peptide mass tolerance and 0.6 Da fragment masstolerance, 0 missed cleavage allowed, carbamidomethylationwas set as fixed modification and methionine oxidation was setas variable modification. A Mascot-integrated decoy databasesearch calculated a false discovery of ≤1% when searching wasperformed on the concatenated mgf files with a significancethreshold of p ≤ 0.01 and the resulting peptides werereimported into the Progenesis software. Calculations of theprotein p-value (one-way ANOVA) were then performed onthe sum of the normalized abundances across all runs. Allprotein identifications are listed in Supporting InformationTable 1.(b). Differential Protein Expression and Pathway

Enrichment Analysis. On the basis of the 12 technicalreplicates, the mean normalized abundance for each proteinwas calculated for both the samples (PI, MO). From thesemean normalized abundances, the fold change (log 2(PI/MO))was calculated for each protein. The proteins with ANOVAvalues of p ≤ 0.05 and additionally regulation of log 2 (fold)value ≥1 or ≤ −1 were regarded as significant and wereselected for further bioinformatics analysis. The pathwayenrichment analysis was done using KEGG database andbioCompendium tool developed at EMBL (available at http://biocompendium.embl.de). In this tool, the Fisher’s exact testwas used to calculate the P-values for enriched pathways, andthe adjusted P-values were calculated by applying theBenjamini−Hochberg procedure in order to control the FalseDiscovery Rate (FDR).

Western Blot

For determination of levels of specific proteins, Westernblotting was performed on MIN6 monolayers and MIN6pseudoislets as described previously.15 Immunoblotting wasconducted with antibodies against Pfkl, Pkm2, ATP-citratesynthase (Cell Signaling, Danvers, MA), Sfrs4, Psmd7, Aga,Rpl35a (Santa Cruz, CA), Idh3g, Uba52 (Abcam, Cambridge,U.K.), and Snrpd1 (Proteintech Group, Inc., Chicago, IL). Theimmuno-reactive bands were imaged with ChemiDoc XRS+(Bio-Rad) and quantified with Image Lab software (Bio-Rad).Normalization was conducted for each protein by staining thePVDF membranes with Coomaasie and quantified with the Labsoftware as described previously.16

■ RESULTS

Formation of MIN6 Pseudoislets

MIN6 pseudoislets formed during culture of MIN6 cells for 3−5 days. After 1−2 days, pseudoislets started to form withdiameters ranging from 50 to 150 μm (Figure 1a,b). Thepseudoislets reached a diameter of approximately 150−250 μmafter 3−5 days (Figure 1c,d). Extended culture for up to 8−9days did not increase the diameter of the pseudoislets further(data not shown). Pseudoislets cultured for 3−5 days were usedfor the experiments. Monolayers of MIN6 cells reaching nearconfluency were used for experiments with monolayer cells(Figure 1e).

Glucose-Stimulated Insulin Secretion

The function of MIN6 pseudoislets and monolayer cells wasdetermined by their insulin secretory activities in response toelevated glucose levels. Insulin release from pseudoislets wasincreased approximately 6-fold when the glucose concentration

was raised from 2 to 20 mmol/L glucose (Figure 2). Incomparison, when MIN6 monolayer cells were exposed to thesame glucose challenge, approximately 3-fold increase wasobserved.Proteomic Analysis

In MIN6 pseudoislets and monolayer cells, a total of 1792proteins common to both pseudoislets and monolayers wereidentified. After discarding proteins with the abundances ofzero appearing in both pseudoislets and monolayers, 1576proteins common to both biological conditions remained(Supporting Information Table 1). For each protein, the meannormalized abundance in pseudoislets and monolayer cells wasdetermined (Supporting Information Table 1). On the basis ofthe abundances obtained for pseudoislets and monolayer cells,488 proteins were differentially expressed with ANOVA valuesof p ≤ 0.05 and additionally regulation of log2 (fold) value ≥1or ≤ −1. Out of the 488 proteins, 475 proteins IPIs werematched with KEGG database. These 475 proteins weremapped to the bioCompendium tool and onto pathways usingKEGG. Eleven pathways were significantly enriched (Table 1)

Figure 1. Micrographs of MIN6 pseudoislets after 1 (a), 2 (b), 3 (c),and 4 (d) days culture and MIN6 monolayer cells (e). Scale bar (100μm) in panel d is applicable to all pictures. Magnification was 20×.



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http://biocompendium.embl.de

http://biocompendium.embl.de

with p-value ≤ 0.05. For each of the pathways, the identifiedproteins belonging to the pathways were listed, a total of 221proteins. No less than 98 of the 221 proteins belonging to the11 significantly enriched pathways were found among thedifferentially expressed (1-fold up- or down-regulated; log2)proteins (Table 2).Validation

The 10 most up-regulated proteins when comparingpseudoislets and monolayers, based on the proteomic analysis(Table 2), were validated by the orthogonal methodologyWestern blotting. The proteins belonged to glycolysis, citratecycle, lysosomal, ribosomal, spliceosomal and proteasomal

pathways and thus belong to pathways enriched in the MIN6pseudoislets and monolayer cells (Table 1). When pyruvatekinase M2 isoform (Pkm2) and phosphofructokinase (Pfkl),members of the glycolytic pathway, were determined byWestern blotting, they were both significantly up-regulated inpseudoislets (Figure 3a,b). Proteins of the citrate pathway,ATP-citrate synthase (Acly), and isocitrate dehydrogenase 3(Idh3g), were also markedly up-regulated in pseudoislets(Figure 3c,d). N(4)-(β-N-acetylglucosaminyl)-L-asparaginase(Aga), which belongs to the lysosomal pathway, was up-regulated more than 5-fold in pseudoislets compared tomonolayers (Figure 3e). Ribosomal protein L35a (Rpl35a),spliceosomal proteins small nuclear ribonucleoprotein D1(Snrpd1), serine/arginine-rich splicing factor 4 (Sfrs4), andpreoteosomal protein preoteosome 26s subunit (Psmd7) werealso up-regulated in pseudoislets compared to monolayers(Figures 3f−i). In case of ribosomal protein ubiquitin A-52residue ribosomal protein fusion product 1 (uba52), proteinlevels determined by Western blotting were not differentbetween pseudoislets and monolayers (data not shown).Protein expression levels obtained by Western blotting of the10 proteins were compared with protein levels obtained by LCLTQ-FT MS. All proteins with the exception of uba52 wereconsistently up-regulated by both approaches (Table 3). TheMS-based approach showed higher fold change, however.

■ DISCUSSIONWe found that in cells with extensive cell-to-cell contact severalpathways were up-regulated compared to the cells with less cell-to-cell contacts. Among these pathways, glycolysis, oxidativephosphorylation, and citric acid cycle were some of the mosthighly enriched pathways. In these pathways, a significantnumber of components had higher protein expression levels,measured by both MS- and immunometric-based approaches,in insulin-secreting MIN6 pseudoislets compared to monolayercells. Among the identified proteins, phosphofructokinaseshowed up-regulation in pseudoislets compared to monolayers.This enzyme is one of the key regulatory enzymes in theglycolytic pathway and catalyzes phosphorylation of fructose-6-phosphate to fructose 1,6-biphosphate. The enzyme has beenimplicated in the generation of rhythmic insulin oscilla-tions.17,18 Indeed, we have recently demonstrated that insulinoscillations with similar amplitude and frequency as observed inprimary islets are present in pseudoislets.12 In agreement withthese observations, phosphofructokinase and pyruvate kinase,two key proteins of glycolysis, showed up-regulation inpseudoislets compared to monolayers using the two orthogonalmethods, suggesting that glycolysis is enhanced in pseudoisletscompared to monolayers.Pyruvate is produced from the glycolytic pathway and enters

into the mitochondrial matrix either by pyruvate dehydrogenase(PDH) to form acetyl-CoA (oxidative pathway) or carboxy-lated by pyruvate carboxylase generating oxaloacetate foranaplerosis.19 Both pathways have been shown to be importantfor proper glucose-stimulated insulin secretion as pyruvateenters into equal proportions.20,21 The importance of the latterratio was evidenced by disruption of the gene encoding PDHspecifically in beta-cells of mice, which had impaired GSIS.22 Anequally important part of mitochondrial metabolism is thetricarboxylic acid cycle with its component enzymes, whichenable the anaplerotic pathway in beta-cells to produceoxaloacetate and other metabolites and replenishment of theTCA cycle intermediates.23,24 The important shuttling path-

Figure 2. Glucose-stimulated insulin secretion from MIN6 monolayers(white bars) and MIN6 pseudoislets (black bars). Monolayer cells andpseudoislets were exposed to 2 or 20 mmol/L glucose for 30 min.Insulin released to the media was measured and normalized to protein.Results are means ± SEM of four separate experiments. *P < 0.05compared to low glucose and #p < 0.05 compared to monolayers.

Table 1. Pathways Enriched in MIN6 Pseudoislets andMonolayer Cellsa

pathway pathway nameKEGGnumber p-value

Metabolic pathway Glycolysis mmu00010 3.44 ×10−36

Oxidativephosphorylation

mmu00190 4.18 ×10−13

Citrate cycle (TCAcycle)

mmu00020 8.93 ×10−6

Junctional, adhesional andcyotoskeletal pathway

Tight junction mmu04530 1.51 ×10−3

Gap junction mmu04540 1.01 ×10−2

Adherens junction mmu04520 1.87 ×10−2

Regulation of actincytoskeleton

mmu04810 2.76 ×10−2

Protein degradation andtranslational pathway

Lysosome mmu04142 2.38 ×10−9

Ribosome mmu03010 7.41 ×10−6

Spliceosome mmu03040 5.73 ×10−4

Proteasome mmu03050 1.51 ×10−3

aPathway analysis of protein expression data sets obtained from MIN6pseudoislets and monolayer cells. Analysis was based on 12 samples (4biological and 3 technical for each biological). The adjusted p-valueswere calculated by applying the Benjamini−Hochberg procedure inorder to control the False Discovery Rate (FDR).



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Table 2. Differentially Expressed Proteins in MIN6 Pseudoislets and Monolayer Cellsa

pathway name gene namegenesymbol

log2 foldchange

Glycolysis Alpha-enolase Eno1 1.4Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex,mitochondrial

Dlat 1.5

Gamma-enolase Eno2 2.4Glyceraldehyde 3-phosphate dehydrogenase Gapdh 1.1Isoform M2 of Pyruvate kinase isozymes Pkm2 4.0Lactate dehydrogenase A chain Ldha 1.9Phosphofructokinase, liver, B-type Pfkl 2.0Phosphoglycerate kinase 1 Pgk1 1.0Phosphoglycerate mutase 1 Pgam1 2.4

Oxidative phosphorylation ATP synthase subunit beta, mitochondrial Atp5b 1.0ATP synthase subunit epsilon, mitochondrial Atp5e 1.2ATP synthase subunit f, mitochondrial Atp5j2 −1.8ATP synthase subunit O, mitochondrial Atp5o 1.5ATP synthase, H+ transporting, mitochondrial FO complex, subunit G2, pseudogene Atp5l 1.5Cytochrome b-c1 complex subunit 1, mitochondrial Uqcrc1 1.0Cytochrome b-c1 complex subunit 6, mitochondrial Uqcrh 3.6Cytochrome b-c1 complex subunit Rieske, mitochondrial Ndufv1 1.1Cytochrome b-c1 complex subunit Rieske, mitochondrial Uqcrfs1 1.8Cytochrome c oxidase subunit 6C Cox6c 2.3Isoform 1 of NADH dehydrogenase [ubiquinone] flavoprotein 2, mitochondrial Ndufv2 1.6NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 5 Ndufa5 1.2NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9, mitochondrial Ndufa9 −1.8NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 10 Ndufb10 1.9NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 9 Ndufb9 2.1NADH dehydrogenase [ubiquinone] iron−sulfur protein 5 Ndufs5 2.5Succinate dehydrogenase [ubiquinone] iron−sulfur subunit, mitochondrial Sdhb 1.9Ubiquinol-cytochrome c reductase binding protein Uqcrb 2.0V-type proton ATPase subunit d 1 Atp6v0d1 2.0V-type proton ATPase subunit F Atp6v1f −1.0V-type proton ATPase subunit H Atp6v1h 2.7V-type proton ATPase subunit S1 Atp6ap1 2.4

Citrate cycle (TCA cycle) ATP-citrate synthase Acly 4.2Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex,mitochondrial

Dlat 1.5

Isocitrate dehydrogenase [NAD] subunit gamma, mitochondrial Idh3g 3.7Isocitrate dehydrogenase [NADP], mitochondrial Idh2 1.1Malate dehydrogenase, cytoplasmic Mdh1 1.0Malate dehydrogenase, mitochondrial Mdh2 1.5Succinate dehydrogenase [ubiquinone] iron−sulfur subunit, mitochondrial Sdhb 1.9Succinyl-CoA ligase [GDP-forming] subunit alpha, mitochondrial Suclg1 1.4

Tight junction Casein kinase II subunit alpha Csnk2a1 1.6Cingulin Cgn −1.6Epb4.1l3 protein Epb4.1l3 1.2Guanine nucleotide-binding protein G(k) subunit alpha Gnai3 −3.4Immunoglobulin superfamily, member 5 Igsf5 3.5Isoform 1 of DNA-binding protein A Csda 1.4Protein kinase C alpha type Prkca 1.5Putative uncharacterized protein Ctnnb1 −3.2Serine/threonine-protein phosphatase 2A catalytic subunit alpha isoform Ppp2ca 7.2

Gap junction Guanine nucleotide-binding protein G(k) subunit alpha Gnai3 −3.4Isoform 2 of cAMP-dependent protein kinase catalytic subunit alpha Prkaca 2.2Protein kinase C alpha type Prkca 1.5Tuba3a Tubulin alpha-3A chain Tuba3a 1.8Tuba3b Tubulin alpha-3B chain Tuba3b 1.8Tubulin alpha-1B chain Tuba1b 2.0Tubulin alpha-1C chain Tuba1c 1.9

Adherens junction Casein kinase II subunit alpha Csnk2a1 1.6Isoform 1 of Low molecular weight phosphotyrosine protein phosphatase Acp1 1.2Putative uncharacterized protein Ctnnb1 −3.2



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ways are ‘pyruvate/malate’, ‘pyruvate/citrate’ and ‘pyruvate/isocitrate’, which were reported as playing important roles inGSIS.25−29 Thus, observed up-regulation of Dlat (dihydroli-poamide S-acetyltransferase (E2 component of pyruvatedehydrogenase complex)), Idh3g (isocitrate dehydrogenase),

Mdh1, Mdh2 (Malate dehydrogenase) and Acly (ATP citratesynthase) in pseudoislets may activate the TCA cycle pathwaymore considerably compared to that in monolayers andcontribute to the enhanced insulin secretion. The results arein agreement with previous reports demonstrating enhanced

Table 2. continued

pathway name gene namegenesymbol

log2 foldchange

Ras GTPase-activating-like protein IQGAP1 Iqgap1 −1.7RAS-related C3 botulinum substrate 1, isoform CRA_a Rac1 −3.1

Regulation of actincytoskeleton

Actin-related protein 2/3 complex subunit 1A Arpc1a 1.7Cofilin-1 Cfl1 1.2Insulin-2 Ins2 2.1Isoform 2 of Gelsolin Gsn 1.9Profilin-1 Pfn1 1.5Ras GTPase-activating-like protein IQGAP1 Iqgap1 −1.7RAS-related C3 botulinum substrate 1, isoform CRA_a Rac1 −3.1Serine/threonine-protein phosphatase PP1-beta catalytic subunit Ppp1cb 1.1

Lysosome Acid ceramidase Asah1 2.3α-N-acetylgalactosaminidase Naga 1.2AP-3 complex subunit delta-1 Ap3d1 1.4Cathepsin B Ctsb 3.4Cathepsin D Ctsd 3.3Cathepsin L1 Ctsl 3.1Cation-dependent mannose-6-phosphate receptor M6pr −1.8Cation-independent mannose-6-phosphate receptor Igf2r 4.3clathrin, light polypeptide A isoform d Clta 1.8Epididymal secretory protein E1 Npc2 1.4Lysosomal protective protein Ctsa 1.1N(4)-(β-N-acetylglucosaminyl)-L-asparaginase Aga 7.7Palmitoyl-protein thioesterase Ppt1 1.2Sulfated glycoprotein 1 Psap 1.0V-type proton ATPase subunit d1 Atp6v0d1 2.0V-type proton ATPase subunit H Atp6v1h 2.7V-type proton ATPase subunit S1 Atp6ap1 2.4

Ribosome 40S ribosomal protein S16 Rps16 1.340S ribosomal protein S27 Rps27 −1.340S ribosomal protein S8 Rps8 1.460S ribosomal protein L30 Rpl30 −1.560S ribosomal protein L35a Rpl35a 3.860S ribosomal protein L38 Rpl38 1.060S ribosomal protein L40 Uba52 2.060S ribosomal protein L8 Rpl8 −1.1

Spliceosome Heat shock-related 70 kDa protein 2 Hspa2 1.6Isoform 1 of Splicing factor, arginine/serine-rich 1 Sfrs1 1.5Nuclear cap-binding protein subunit 2 Ncbp2 1.5Small nuclear ribonucleoprotein Sm D1 Snrpd1 3.3Splicing factor 3A subunit 3 Sf3a3 1.3Splicing factor 3B subunit 1 Sf3b1 −1.3Splicing factor arginine/serine-rich 4 Sfrs4 2.8Splicing factor U2AF 65 kDa subunit U2af2 2.6THO complex subunit 1 Thoc1 1.5WD repeat domain 57 (U5 snRNP specific) Wdr57 1.7

Proteasome 26S proteasome non-ATPase regulatory subunit 3 Psmd3 1.126S proteasome non-ATPase regulatory subunit 7 Psmd7 5.6Isoform Rpn10A of 26S proteasome non-ATPase regulatory subunit 4 Psmd4 1.3Proteasome (Prosome, macropain) 26S subunit ATPase 3 Psmc3 1.2Proteasome subunit alpha type-7 Psma7 1.2Proteasome subunit beta type-4 Psmb4 2.1

aProteins in MIN6 pseudoislets and monolayer cells belonging to pathways listed in Table 1 and with regulation of log2 (fold) value ≥1 or ≤ −1.Protein levels were calculated from the mean normalized abundances and the fold change was calculated for each protein. Cells were cultured in 25mmol/L glucose. Results were from four separate experiments.



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insulin secretion from MIN6 pseudoislets compared to thatfrom monolayer cells in the presence of substrates ofmitochondrial metabolism.12

The metabolites from the TCA cycle enter into themitochondrial respiratory chain to generate ATP. Mitochon-drial DNA encodes 13 protein subunits of the mitochondrialrespiratory chain complexes and ATP synthase.30 Theimportance of these proteins was illustrated by knockout ofmitochondrial DNA in MIN6, which also caused impaired

GSIS.31 The oxidative phosphorylation pathway involved thelargest number of identified proteins among the 11 pathwaysexamined in the present study. A majority (19 out of 22)showed up-regulation in pseudoislets compared to monolayercells suggesting enhanced activity in this pathway inpseudoislets. In a recent study, we compared the transcriptlevels of 84 genes of the pathway in pseudoislets and monolayercells. We found that 76% of genes were at least 1.4-fold up-regulated in pseudoislets reinforcing the view of enhancedoxidative phosphorylation in the pseudoislets. Indeed, 14proteins which showed up-regulation in pseudoislets comparedto monolayer cells in the present study were among the genesexamined at the transcript level.12

The next top three pathways enriched in our proteomicanalysis were related to tight, gap and adherens junctions. It waspreviously reported that pseudoislets exchange small moleculesthrough gap junctions.11 Further, calcium-dependent celladhesion molecule E-cadherin and the gap junction proteinconnexin 36, which are required for proper insulin secretion,were up-regulated in pseudoislets compared to monolayercells.3,8−11 Consistent with these findings, it was demonstratedthat a majority of the levels of proteins in these pathways wereup-regulated in pseudoislets compared to monolayer cells.Proteins involved in the regulation of the actin cytoskeleton

were also enriched in our proteomic analysis. Microtubuleshave been shown to participate in GSIS and glucose stimulatestubulin synthesis, where tubulin participates in the transport ofnewly synthesized hormone from the endoplasmic reticulum tothe plasma membrane.32,33 The proteomics results showed thatmost of the proteins were up-regulated in pseudoislets, whichmay indicate that the pathway is more active in pseudoisletscompared to monolayers.Another four pathways that were differentially expressed in

pseudoislets were the lysosomal, ribosomal, spliceosome and

Figure 3. Protein levels of Pkm2 (a), Pfkl (b), ATP citrate synthase (c), Idh3g (d), Aga (e), Rpl35a (f), Snrpd1 (g), Sfrs4 (h), and Psmd7 (i) weremeasured in monolayers (MO; white column) and pseudoislets (PI; black column) by Western blot. Results are means ± SEM of five separateexperiments. *P < 0.05 compared to monolayers.

Table 3. Differentially Expressed Proteins in MIN6Pseudoislets and Monolayersa

fold change PIs vsMOs

gene namegenesymbol

MSanalysis

Westernblot

Isoform M2 of Pyruvate kinase isozymes Pkm2 16.0 3.2Phosphofructokinase, liver, B-type Pfkl 4.0 2.6ATP-citrate synthase Acly 18.4 5.0Isocitrate dehydrogenase [NAD] subunitgamma, mitochondrial

Idh3g 13.0 3.9

N(4)-(β-N-acetylglucosaminyl)-L-asparaginase

Aga 207.9 6.0

60S ribosomal protein L35a Rpl35a 13.9 1.960S ribosomal protein L40 Uba52 4.0 No

differenceSmall nuclear ribonucleoprotein Sm D1 Snrpd1 9.8 2.5Splicing factor arginine/serine-rich 4 Sfrs4 7.0 1.926S proteasome non-ATPase regulatorysubunit 7

Psmd7 48.5 2.8

aFold change of proteins when comparing MIN6 pseudoislets (PIs)and monolayers (MOs) was calculated from protein levels obtained byMS (Table 2) and Western blot (Figure 2). Results were from fourseparate experiments for MS analyzed data and five separateexperiments for Western blot analysis.



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proteosome. Previously, it has been reported that GSIS wasrelated to increased activities of lysosomal enzyme.34 Theribosome and the splicesome play important roles in trans-lation. It has been reported that Ins1 mRNA levels were higherin pseudoislets compared to those in monolayers.35 Thepresent observation of higher protein expression of Ins2 inpseudoislets compared to that in monolayer cells supports thatnot only transcription but also translation is enhanced. Wefound up-regulation in pseudoislets of clathrin, which belongsto the lysosomal pathway. This protein was reported to play animportant role in the Golgi network for proper processing ofproinsulin to insulin.36 The proteosomal pathway plays animportant role to remove misfolded proteins from the cellularsystem and inhibition of proteosomal activity impairs insulinsecretion from MIN6-m9 cells.37 The up-regulation of most ofthe proteins belonging to these pathways in pseudoislets mayindicate enhanced protein turnover in pseudoislets compared tothat in monolayers. The most up-regulated protein inpseudoislets was N(4)-(β-N-acetylglucosaminyl)-L-asparagi-nase, a lysosomal enzyme cleaving N-acetylglucosamine ataspargine. The accentuated level of this enzyme in pseudoisletsmay reflect that the formation of glycoproteins is an importantevent during culture of MIN6 cells, possibly due to the highambient glucose concentration. To what extent MIN6monolayer cells have elevated glycoproteins levels, andpotentially display aspartylglucosaminuria, needs to bedetermined.In conclusion, the proteomic approach identified metabo-

lism, cell coupling, gene translation and protein turnover as keyfeatures discriminating cells with extensive cell-to-cell contactsfrom cells with less cell-to-cell contacts. These cellular pathwayshelp to explain how an elaborate cellular architecture, such asthat found in islets of Langerhans, contributes to cellularfunction.

■ ASSOCIATED CONTENT

*S Supporting Information

Table with all protein identifications (Excel). This material isavailable free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATION

Corresponding Author

*Azazul Islam Chowdhury. E-mail: [email protected].

Notes

The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

Grants from the Swedish Medical Research Council (72X-14019), Swedish Diabetes Association, Family ErnforsFoundation, and Uppsala University supported the study.The authors are grateful to Proteomics core facility, EuropeanMolecular Biology Laboratory (EMBL) especially JennyHansson, Satoshi Okawa and Jeroen Krijgsveld for providingthe tools to analyze the data presented in this paper. TheSpecial Equipment Grant from the University GrantsCommittee of the Hong Kong Special Administrative Region,China (Project Code: SEG_HKU02).

■ ABBREVIATIONS

ESI, nanoelectrospray ionization; FDR, false discovery rate;GSIS, glucose- stimulated insulin secretion; LTQ FT, linear iontrap Fourier Transform; MO, monolayers; PI, pseudoislets

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Documents

Signaling in Insulin-Secreting MIN6 Pseudoislets and Monolayer Cells