Signaling in Insulin-Secreting MIN6 Pseudoislets and Monolayer Cells

Technical Note pubs.acs.org/jpr

Signaling in Insulin-Secreting MIN6 Pseudoislets and Monolayer Cells Azazul 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 Avenue des Hauts-Fourneaux, L-4362 Esch-sur-Alzette, Luxembourg ∥ Analytical Chemistry, Department of Chemistry−Biomedical Center and SciLifeLab, Uppsala University, Box 599, SE-75124 Uppsala, Sweden ⊥ Department of Chemistry, State Key Laboratory of Synthetic Chemistry, and Open Laboratory of Chemical Biology of the Institute of 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 importance for cellular function. In islets of Langerhans, which control blood glucose levels by secreting insulin in response to the blood glucose concentration, the secretory response of intact islets is higher than that of insulin-producing beta-cells not arranged in the islet architecture. The objective was to define mechanisms by which cellular performance is enhanced when cells are arranged in three-dimensional space. The task was addressed by making a comprehensive analysis based on protein expression patterns generated from insulin-secreting MIN6 cells grown as islet-like clusters, so-called pseudoislets, and in monolayers. After culture, glucose-stimulated insulin secretion (GSIS) was measured from monolayers and pseudoislets. GSIS rose 6-fold in pseudoislets but only 3-fold in monolayers when the glucose concentration was increased from 2 to 20 mmol/L. Proteins from pseudoislets and monolayers were extracted and analyzed by liquid-chromatography mass spectrometry, and differentially expressed proteins were mapped 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 of proteins belonging to the pathways in pseudoislets and monolayers, predictions of differential pathway activation were performed. Mechanisms enhancing insulin secretory capacity of the beta-cell, when situated in the islet, include pathways regulating glucose metabolism, cell interaction, and translational regulation. KEYWORDS: glucose-stimulated insulin secretion (GSIS), beta-cells, MIN6 cells, pseudoislets

■

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

INTRODUCTION Beta-cells from the pancreatic islet release insulin in response to elevated blood glucose levels. Proper insulin secretion depends on the architecture of the islets. As with most cell types, the cell-to-cell contacts within the islets have been shown to be crucial for the normal function.1−3 This view is supported by work demonstrating that dispersed islet cells respond poorly to glucose compared to intact islets.4 When cell-to-cell contact is re-established, such as in dispersed beta-cells that reaggregate to form structures so-called ‘pseudoislets’ with similar size and cellular organization as in primary islets, function is essentially regained.5,6 The mouse-derived insulinoma MIN6 cell line is glucose-responsive. 7 When MIN6 cells are allowed to reaggregate, they form cell clusters, which mimic the size and morphology of primary islets.3,8−11 MIN6 pseudoislets show © XXXX American Chemical Society

Received: May 3, 2013

A

dx.doi.org/10.1021/pr400864w | J. Proteome Res. XXXX, XXX, XXX−XXX

Journal of Proteome Research

Technical Note

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

their mixed cell populations are overcome. In this study, we addressed the issue of how these cell-to-cell contacts influence cellular signaling by comprehensively analyzing differential protein expression patterns obtained from cells that are arranged in three-dimensional structures, the MIN6 pseudoislets (PIs) and cells that do not enjoy this arrangement, the MIN6 monolayer cells (MOs).

■

MATERIALS AND METHODS

Protein Expression Profiles

Digested samples were redissolved in 0.1% trifluoroacetic acid to yield an approximate tryptic peptide concentration of 0.3 μg/μL prior to nanoLC−MS/MS analysis. The protein identification experiments were performed using a 7 T hybrid Linear ion trap Fourier Transform (LTQ-FT) mass spectrometer (ThermoFisher Scientific, Bremen, Germany) fitted with a nanoelectrospray ionization (ESI) ion source (ThermoFischer Scientific). Online nanoLC separations were performed using Agilent 1100 nanoflow system (Agilent Technologies, Waldbronn, Germany). The peptide separations were performed on in-house packed 15 cm fused silica emitters (75 μm inner diameter, 375 μm outer diameter). The emitters were packed with a methanol slurry of reversed-phase, fully endcapped Reprosil-Pur C18-AQ 3 μm resin (Dr. Maisch GmbH, Ammerbuch-Entringen, Germany) using a pressurized packing device operated at 50−60 bar (Proxeon Biosystems, Odense, Denmark). The separations were performed at a flow of 200 nL/min with mobile phases A (water with 0.5% acetic acid) and B (89.5% acetonitrile, 10% water, and 0.5% acetic acid). A 100 min gradient from 2% B to 50% B followed by a washing step with 98% B for 5 min was used. Mass spectrometric analysis was performed using unattended data-dependent acquisition mode, in which the mass spectrometer automatically switched between acquiring a high resolution survey mass spectrum in the FTMS (resolving power 100 000 full width at halfmaximum) and consecutive low-resolution, collision-induced dissociation fragmentation of up to five of the most abundant ions in the ion trap.

Cell Culture

Mouse insulinoma MIN6 cells were cultured under conditions previously published7 in 250 mL tissue culture flasks (Becton Dickson Labware, Franklin Lakes, NJ) at 37 °C (95% O2 and 5% CO2) in Dulbecco’s Modified Eagle medium (Invitrogen, Paisley, U.K.), containing 25 mmol/L glucose and supplemented with 10% fetal bovine serum, 100 units/mL penicillin, 100 μg/mL streptomycin (Invitrogen), and 50 μM βmercaptoethanol (Sigma, St. Louis, MO). Whereas monolayers of MIN6 cells were formed when MIN6 cells were cultured in negatively charged tissue culture plates (Becton Dickson Labware), MIN6 pseudoislets were formed when MIN6 cells were cultured in tissue culture dishes made of nonadherent plastic (Becton Dickson Labware), which allowed the aggregation of dispersed cells.3 For the latter purpose, 3 × 106 dispersed cells were cultured for 3−5 days using the same culture condition as for monolayer culture. All experiments were performed between passages 20 and 30. Insulin Secretion

After culture, insulin secretion was measured from MOs and PIs. For static incubation, monolayer cells or groups of 20 pseudoislets were preincubated for 60 min at 37 °C in KRBH buffer consisting of 130 mmol/L NaCl, 4.8 mmol/L KCl, 1.2 mmol/L MgSO4, 1.2 mmol/L KH2PO4, 1.2 mmol/L CaCl2, 5 mmol/L NaHCO3, and 5 mmol/L HEPES, titrated to pH 7.4 with NaOH, and supplemented with 1 mg/mL BSA and 2 mmol/L glucose. Subsequently, cells were incubated in KRBH buffer containing either 2 or 20 mmol/L glucose for 30 min at 37 °C. Aliquots (200 μL) of medium were collected for determination of insulin secretion. Monolayer cells and pseudoislets were washed three times with phosphate buffer saline (PBS) and lysed with buffer containing 0.1% Triton-X 100 and 25 mmol/L NaOH, and lysates were stored at −20 °C for measurement of total protein. Differences in insulin secretion among the groups were assessed using analysis of variance (ANOVA) followed by Bonferroni’s post hoc test. P < 0.05 was considered statistically significant.

Bioinformatics Analysis

(a). Label-Free Peptide and Protein Quantifications. The acquired spectra from 12 samples derived from pseudoislets (4 biological × 3 technical replicates) and 12 from monolayers (4 biological × 3 technical replicates) were uploaded to the Progenesis LC−MS Version 4.0 (Nonlinear Dynamics, Newcastle, England) after choosing Progenesis machine settings according to the respective acquisition instrument and analyzed the data as described previously.14 Profile data of the MS scans were transformed to peak lists with respective peak m/z values, intensities, abundances (areas under the peaks), and m/z width. In the alignment step, we manually selected a reference run and the retention times of the other samples were aligned by manual and automatic alignment to a maximal overlay of all features. Features with only one charge or more than seven charges are masked at this point and excluded from further analyses. After alignment and feature exclusion, samples were divided into the appropriate groups (PI and MO), and raw abundances of the remaining features were normalized to allow correction for factors resulting from experimental variation. Rank 1−10 MS/MS spectra were exported from the Progenesis software as Mascot generic file (mgf) and used for peptide identification with Mascot version 2.2 (Matrix Science, London, U.K.) in the International Protein Index (IPI)

Sample Preparation for Protein Profiling

Cells were washed twice with PBS and lysed with a buffer containing 8 mol/L urea, 1% octyl-β-D-glucopyranoside in 10 mmol/L Trizma Base, pH 8.0, for 20 min. After lysis, samples were centrifuged at 10 000 rpm for 10 min to pellet any remaining debris. Buffer in the samples was exchanged to 50 mmol/L ammonium bicarbonate using PD SpinTrap G-25 columns (GE Healthcare, Life Sciences, Uppsala, Sweden) according to the manufacturer’s protocol. Total protein content was 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 by iodoacetamide (20 mmol/L, room temperature in darkness for 30 min). Before digestion with trypsin, the Nanosep 3 Omega 3 kDa cutoff filters (Pall, Port Washington, NY) were B



Technical Note

database version 3.68 mouse containing a total of 56 729 protein sequences. Search parameters used were the following: 10 ppm peptide mass tolerance and 0.6 Da fragment mass tolerance, 0 missed cleavage allowed, carbamidomethylation was set as fixed modification and methionine oxidation was set as variable modification. A Mascot-integrated decoy database search calculated a false discovery of ≤1% when searching was performed on the concatenated mgf files with a significance threshold of p ≤ 0.01 and the resulting peptides were reimported into the Progenesis software. Calculations of the protein p-value (one-way ANOVA) were then performed on the sum of the normalized abundances across all runs. All protein identifications are listed in Supporting Information Table 1. (b). Differential Protein Expression and Pathway Enrichment Analysis. On the basis of the 12 technical replicates, the mean normalized abundance for each protein was calculated for both the samples (PI, MO). From these mean normalized abundances, the fold change (log 2(PI/MO)) was calculated for each protein. The proteins with ANOVA values of p ≤ 0.05 and additionally regulation of log 2 (fold) value ≥1 or ≤ −1 were regarded as significant and were selected for further bioinformatics analysis. The pathway enrichment analysis was done using KEGG database and bioCompendium tool developed at EMBL (available at http:// biocompendium.embl.de). In this tool, the Fisher’s exact test was used to calculate the P-values for enriched pathways, and the adjusted P-values were calculated by applying the Benjamini−Hochberg procedure in order to control the False Discovery Rate (FDR). Western Blot

For determination of levels of specific proteins, Western blotting was performed on MIN6 monolayers and MIN6 pseudoislets as described previously.15 Immunoblotting was conducted with antibodies against Pfkl, Pkm2, ATP-citrate synthase (Cell Signaling, Danvers, MA), Sfrs4, Psmd7, Aga, Rpl35a (Santa Cruz, CA), Idh3g, Uba52 (Abcam, Cambridge, U.K.), and Snrpd1 (Proteintech Group, Inc., Chicago, IL). The immuno-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 the PVDF membranes with Coomaasie and quantified with the Lab software as described previously.16

■

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 (100 μm) in panel d is applicable to all pictures. Magnification was 20×.

was raised from 2 to 20 mmol/L glucose (Figure 2). In comparison, when MIN6 monolayer cells were exposed to the same glucose challenge, approximately 3-fold increase was observed.

RESULTS

Formation of MIN6 Pseudoislets

Proteomic Analysis

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

In MIN6 pseudoislets and monolayer cells, a total of 1792 proteins common to both pseudoislets and monolayers were identified. After discarding proteins with the abundances of zero appearing in both pseudoislets and monolayers, 1576 proteins common to both biological conditions remained (Supporting Information Table 1). For each protein, the mean normalized abundance in pseudoislets and monolayer cells was determined (Supporting Information Table 1). On the basis of the abundances obtained for pseudoislets and monolayer cells, 488 proteins were differentially expressed with ANOVA values of p ≤ 0.05 and additionally regulation of log2 (fold) value ≥1 or ≤ −1. Out of the 488 proteins, 475 proteins IPIs were matched with KEGG database. These 475 proteins were mapped to the bioCompendium tool and onto pathways using KEGG. Eleven pathways were significantly enriched (Table 1)

Glucose-Stimulated Insulin Secretion

The function of MIN6 pseudoislets and monolayer cells was determined by their insulin secretory activities in response to elevated glucose levels. Insulin release from pseudoislets was increased approximately 6-fold when the glucose concentration C



Technical Note

pathways and thus belong to pathways enriched in the MIN6 pseudoislets and monolayer cells (Table 1). When pyruvate kinase M2 isoform (Pkm2) and phosphofructokinase (Pfkl), members of the glycolytic pathway, were determined by Western blotting, they were both significantly up-regulated in pseudoislets (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 upregulated more than 5-fold in pseudoislets compared to monolayers (Figure 3e). Ribosomal protein L35a (Rpl35a), spliceosomal proteins small nuclear ribonucleoprotein D1 (Snrpd1), serine/arginine-rich splicing factor 4 (Sfrs4), and preoteosomal protein preoteosome 26s subunit (Psmd7) were also up-regulated in pseudoislets compared to monolayers (Figures 3f−i). In case of ribosomal protein ubiquitin A-52 residue ribosomal protein fusion product 1 (uba52), protein levels determined by Western blotting were not different between pseudoislets and monolayers (data not shown). Protein expression levels obtained by Western blotting of the 10 proteins were compared with protein levels obtained by LC LTQ-FT MS. All proteins with the exception of uba52 were consistently up-regulated by both approaches (Table 3). The MS-based approach showed higher fold change, however.

Figure 2. Glucose-stimulated insulin secretion from MIN6 monolayers (white bars) and MIN6 pseudoislets (black bars). Monolayer cells and pseudoislets 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 < 0.05 compared to low glucose and #p < 0.05 compared to monolayers.

Table 1. Pathways Enriched in MIN6 Pseudoislets and Monolayer Cellsa pathway Metabolic pathway

Junctional, adhesional and cyotoskeletal pathway

Protein degradation and translational pathway

pathway name

KEGG number

Glycolysis

mmu00010

Oxidative phosphorylation Citrate cycle (TCA cycle) Tight junction

mmu00190

mmu04530

Gap junction

mmu04540

Adherens junction

mmu04520

Regulation of actin cytoskeleton Lysosome

mmu04810

Ribosome

mmu03010

Spliceosome

mmu03040

Proteasome

mmu03050

mmu00020

mmu04142

■

p-value 3.44 × 10−36 4.18 × 10−13 8.93 × 10−6 1.51 × 10−3 1.01 × 10−2 1.87 × 10−2 2.76 × 10−2 2.38 × 10−9 7.41 × 10−6 5.73 × 10−4 1.51 × 10−3

DISCUSSION We found that in cells with extensive cell-to-cell contact several pathways were up-regulated compared to the cells with less cellto-cell contacts. Among these pathways, glycolysis, oxidative phosphorylation, and citric acid cycle were some of the most highly enriched pathways. In these pathways, a significant number of components had higher protein expression levels, measured by both MS- and immunometric-based approaches, in insulin-secreting MIN6 pseudoislets compared to monolayer cells. Among the identified proteins, phosphofructokinase showed up-regulation in pseudoislets compared to monolayers. This enzyme is one of the key regulatory enzymes in the glycolytic pathway and catalyzes phosphorylation of fructose-6phosphate to fructose 1,6-biphosphate. The enzyme has been implicated in the generation of rhythmic insulin oscillations.17,18 Indeed, we have recently demonstrated that insulin oscillations with similar amplitude and frequency as observed in primary islets are present in pseudoislets.12 In agreement with these observations, phosphofructokinase and pyruvate kinase, two key proteins of glycolysis, showed up-regulation in pseudoislets compared to monolayers using the two orthogonal methods, suggesting that glycolysis is enhanced in pseudoislets compared 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 carboxylated by pyruvate carboxylase generating oxaloacetate for anaplerosis.19 Both pathways have been shown to be important for proper glucose-stimulated insulin secretion as pyruvate enters into equal proportions.20,21 The importance of the latter ratio was evidenced by disruption of the gene encoding PDH specifically in beta-cells of mice, which had impaired GSIS.22 An equally important part of mitochondrial metabolism is the tricarboxylic acid cycle with its component enzymes, which enable the anaplerotic pathway in beta-cells to produce oxaloacetate and other metabolites and replenishment of the TCA cycle intermediates.23,24 The important shuttling path-

a

Pathway analysis of protein expression data sets obtained from MIN6 pseudoislets and monolayer cells. Analysis was based on 12 samples (4 biological and 3 technical for each biological). The adjusted p-values were calculated by applying the Benjamini−Hochberg procedure in order to control the False Discovery Rate (FDR).

with p-value ≤ 0.05. For each of the pathways, the identified proteins belonging to the pathways were listed, a total of 221 proteins. No less than 98 of the 221 proteins belonging to the 11 significantly enriched pathways were found among the differentially expressed (1-fold up- or down-regulated; log2) proteins (Table 2). Validation

The 10 most up-regulated proteins when comparing pseudoislets and monolayers, based on the proteomic analysis (Table 2), were validated by the orthogonal methodology Western blotting. The proteins belonged to glycolysis, citrate cycle, lysosomal, ribosomal, spliceosomal and proteasomal D



Technical Note

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

Oxidative phosphorylation

Citrate cycle (TCA cycle)

Tight junction

Gap junction

Adherens junction

gene symbol

gene name Alpha-enolase Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, mitochondrial Gamma-enolase Glyceraldehyde 3-phosphate dehydrogenase Isoform M2 of Pyruvate kinase isozymes Lactate dehydrogenase A chain Phosphofructokinase, liver, B-type Phosphoglycerate kinase 1 Phosphoglycerate mutase 1 ATP synthase subunit beta, mitochondrial ATP synthase subunit epsilon, mitochondrial ATP synthase subunit f, mitochondrial ATP synthase subunit O, mitochondrial ATP synthase, H+ transporting, mitochondrial FO complex, subunit G2, pseudogene Cytochrome b-c1 complex subunit 1, mitochondrial Cytochrome b-c1 complex subunit 6, mitochondrial Cytochrome b-c1 complex subunit Rieske, mitochondrial Cytochrome b-c1 complex subunit Rieske, mitochondrial Cytochrome c oxidase subunit 6C Isoform 1 of NADH dehydrogenase [ubiquinone] flavoprotein 2, mitochondrial NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 5 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9, mitochondrial NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 10 NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 9 NADH dehydrogenase [ubiquinone] iron−sulfur protein 5 Succinate dehydrogenase [ubiquinone] iron−sulfur subunit, mitochondrial Ubiquinol-cytochrome c reductase binding protein V-type proton ATPase subunit d 1 V-type proton ATPase subunit F V-type proton ATPase subunit H V-type proton ATPase subunit S1 ATP-citrate synthase Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, mitochondrial Isocitrate dehydrogenase [NAD] subunit gamma, mitochondrial Isocitrate dehydrogenase [NADP], mitochondrial Malate dehydrogenase, cytoplasmic Malate dehydrogenase, mitochondrial Succinate dehydrogenase [ubiquinone] iron−sulfur subunit, mitochondrial Succinyl-CoA ligase [GDP-forming] subunit alpha, mitochondrial Casein kinase II subunit alpha Cingulin Epb4.1l3 protein Guanine nucleotide-binding protein G(k) subunit alpha Immunoglobulin superfamily, member 5 Isoform 1 of DNA-binding protein A Protein kinase C alpha type Putative uncharacterized protein Serine/threonine-protein phosphatase 2A catalytic subunit alpha isoform Guanine nucleotide-binding protein G(k) subunit alpha Isoform 2 of cAMP-dependent protein kinase catalytic subunit alpha Protein kinase C alpha type Tuba3a Tubulin alpha-3A chain Tuba3b Tubulin alpha-3B chain Tubulin alpha-1B chain Tubulin alpha-1C chain Casein kinase II subunit alpha Isoform 1 of Low molecular weight phosphotyrosine protein phosphatase Putative uncharacterized protein

E

Eno1 Dlat

log2 fold change 1.4 1.5

Eno2 Gapdh Pkm2 Ldha Pfkl Pgk1 Pgam1 Atp5b Atp5e Atp5j2 Atp5o Atp5l Uqcrc1 Uqcrh Ndufv1 Uqcrfs1 Cox6c Ndufv2 Ndufa5 Ndufa9 Ndufb10 Ndufb9 Ndufs5 Sdhb Uqcrb Atp6v0d1 Atp6v1f Atp6v1h Atp6ap1 Acly Dlat

2.4 1.1 4.0 1.9 2.0 1.0 2.4 1.0 1.2 −1.8 1.5 1.5 1.0 3.6 1.1 1.8 2.3 1.6 1.2 −1.8 1.9 2.1 2.5 1.9 2.0 2.0 −1.0 2.7 2.4 4.2 1.5

Idh3g Idh2 Mdh1 Mdh2 Sdhb Suclg1 Csnk2a1 Cgn Epb4.1l3 Gnai3 Igsf5 Csda Prkca Ctnnb1 Ppp2ca Gnai3 Prkaca Prkca Tuba3a Tuba3b Tuba1b Tuba1c Csnk2a1 Acp1 Ctnnb1

3.7 1.1 1.0 1.5 1.9 1.4 1.6 −1.6 1.2 −3.4 3.5 1.4 1.5 −3.2 7.2 −3.4 2.2 1.5 1.8 1.8 2.0 1.9 1.6 1.2 −3.2



Technical Note

Table 2. continued pathway name

Regulation of actin cytoskeleton

Lysosome

Ribosome

Spliceosome

Proteasome

gene name Ras GTPase-activating-like protein IQGAP1 RAS-related C3 botulinum substrate 1, isoform CRA_a Actin-related protein 2/3 complex subunit 1A Cofilin-1 Insulin-2 Isoform 2 of Gelsolin Profilin-1 Ras GTPase-activating-like protein IQGAP1 RAS-related C3 botulinum substrate 1, isoform CRA_a Serine/threonine-protein phosphatase PP1-beta catalytic subunit Acid ceramidase α-N-acetylgalactosaminidase AP-3 complex subunit delta-1 Cathepsin B Cathepsin D Cathepsin L1 Cation-dependent mannose-6-phosphate receptor Cation-independent mannose-6-phosphate receptor clathrin, light polypeptide A isoform d Epididymal secretory protein E1 Lysosomal protective protein N(4)-(β-N-acetylglucosaminyl)-L-asparaginase Palmitoyl-protein thioesterase Sulfated glycoprotein 1 V-type proton ATPase subunit d1 V-type proton ATPase subunit H V-type proton ATPase subunit S1 40S ribosomal protein S16 40S ribosomal protein S27 40S ribosomal protein S8 60S ribosomal protein L30 60S ribosomal protein L35a 60S ribosomal protein L38 60S ribosomal protein L40 60S ribosomal protein L8 Heat shock-related 70 kDa protein 2 Isoform 1 of Splicing factor, arginine/serine-rich 1 Nuclear cap-binding protein subunit 2 Small nuclear ribonucleoprotein Sm D1 Splicing factor 3A subunit 3 Splicing factor 3B subunit 1 Splicing factor arginine/serine-rich 4 Splicing factor U2AF 65 kDa subunit THO complex subunit 1 WD repeat domain 57 (U5 snRNP specific) 26S proteasome non-ATPase regulatory subunit 3 26S proteasome non-ATPase regulatory subunit 7 Isoform Rpn10A of 26S proteasome non-ATPase regulatory subunit 4 Proteasome (Prosome, macropain) 26S subunit ATPase 3 Proteasome subunit alpha type-7 Proteasome subunit beta type-4

gene symbol

log2 fold change

Iqgap1 Rac1 Arpc1a Cfl1 Ins2 Gsn Pfn1 Iqgap1 Rac1 Ppp1cb Asah1 Naga Ap3d1 Ctsb Ctsd Ctsl M6pr Igf2r Clta Npc2 Ctsa Aga Ppt1 Psap Atp6v0d1 Atp6v1h Atp6ap1 Rps16 Rps27 Rps8 Rpl30 Rpl35a Rpl38 Uba52 Rpl8 Hspa2 Sfrs1 Ncbp2 Snrpd1 Sf3a3 Sf3b1 Sfrs4 U2af2 Thoc1 Wdr57 Psmd3 Psmd7 Psmd4 Psmc3 Psma7 Psmb4

−1.7 −3.1 1.7 1.2 2.1 1.9 1.5 −1.7 −3.1 1.1 2.3 1.2 1.4 3.4 3.3 3.1 −1.8 4.3 1.8 1.4 1.1 7.7 1.2 1.0 2.0 2.7 2.4 1.3 −1.3 1.4 −1.5 3.8 1.0 2.0 −1.1 1.6 1.5 1.5 3.3 1.3 −1.3 2.8 2.6 1.5 1.7 1.1 5.6 1.3 1.2 1.2 2.1

Proteins 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 25 mmol/L glucose. Results were from four separate experiments.

a

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

Mdh1, Mdh2 (Malate dehydrogenase) and Acly (ATP citrate synthase) in pseudoislets may activate the TCA cycle pathway more considerably compared to that in monolayers and contribute to the enhanced insulin secretion. The results are in agreement with previous reports demonstrating enhanced F



Technical Note

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) were measured in monolayers (MO; white column) and pseudoislets (PI; black column) by Western blot. Results are means ± SEM of five separate experiments. *P < 0.05 compared to monolayers.

GSIS.31 The oxidative phosphorylation pathway involved the largest number of identified proteins among the 11 pathways examined in the present study. A majority (19 out of 22) showed up-regulation in pseudoislets compared to monolayer cells suggesting enhanced activity in this pathway in pseudoislets. In a recent study, we compared the transcript levels of 84 genes of the pathway in pseudoislets and monolayer cells. We found that 76% of genes were at least 1.4-fold upregulated in pseudoislets reinforcing the view of enhanced oxidative phosphorylation in the pseudoislets. Indeed, 14 proteins which showed up-regulation in pseudoislets compared to monolayer cells in the present study were among the genes examined at the transcript level.12 The next top three pathways enriched in our proteomic analysis were related to tight, gap and adherens junctions. It was previously reported that pseudoislets exchange small molecules through gap junctions.11 Further, calcium-dependent cell adhesion molecule E-cadherin and the gap junction protein connexin 36, which are required for proper insulin secretion, were up-regulated in pseudoislets compared to monolayer cells.3,8−11 Consistent with these findings, it was demonstrated that a majority of the levels of proteins in these pathways were up-regulated in pseudoislets compared to monolayer cells. Proteins involved in the regulation of the actin cytoskeleton were also enriched in our proteomic analysis. Microtubules have been shown to participate in GSIS and glucose stimulates tubulin synthesis, where tubulin participates in the transport of newly synthesized hormone from the endoplasmic reticulum to the plasma membrane.32,33 The proteomics results showed that most of the proteins were up-regulated in pseudoislets, which may indicate that the pathway is more active in pseudoislets compared to monolayers. Another four pathways that were differentially expressed in pseudoislets were the lysosomal, ribosomal, spliceosome and

Table 3. Differentially Expressed Proteins in MIN6 Pseudoislets and Monolayersa fold change PIs vs MOs gene name Isoform M2 of Pyruvate kinase isozymes Phosphofructokinase, liver, B-type ATP-citrate synthase Isocitrate dehydrogenase [NAD] subunit gamma, mitochondrial N(4)-(β-N-acetylglucosaminyl)-Lasparaginase 60S ribosomal protein L35a 60S ribosomal protein L40 Small nuclear ribonucleoprotein Sm D1 Splicing factor arginine/serine-rich 4 26S proteasome non-ATPase regulatory subunit 7

gene symbol

MS analysis

Pkm2 Pfkl Acly Idh3g

16.0 4.0 18.4 13.0

3.2 2.6 5.0 3.9

207.9

6.0

Aga Rpl35a Uba52

13.9 4.0

Snrpd1 Sfrs4 Psmd7

9.8 7.0 48.5

Western blot

1.9 No difference 2.5 1.9 2.8

a

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

insulin secretion from MIN6 pseudoislets compared to that from monolayer cells in the presence of substrates of mitochondrial metabolism.12 The metabolites from the TCA cycle enter into the mitochondrial respiratory chain to generate ATP. Mitochondrial DNA encodes 13 protein subunits of the mitochondrial respiratory chain complexes and ATP synthase.30 The importance of these proteins was illustrated by knockout of mitochondrial DNA in MIN6, which also caused impaired G


Journal of Proteome Research proteosome. Previously, it has been reported that GSIS was related to increased activities of lysosomal enzyme.34 The ribosome and the splicesome play important roles in translation. It has been reported that Ins1 mRNA levels were higher in pseudoislets compared to those in monolayers.35 The present observation of higher protein expression of Ins2 in pseudoislets compared to that in monolayer cells supports that not only transcription but also translation is enhanced. We found up-regulation in pseudoislets of clathrin, which belongs to the lysosomal pathway. This protein was reported to play an important role in the Golgi network for proper processing of proinsulin to insulin.36 The proteosomal pathway plays an important role to remove misfolded proteins from the cellular system and inhibition of proteosomal activity impairs insulin secretion from MIN6-m9 cells.37 The up-regulation of most of the proteins belonging to these pathways in pseudoislets may indicate enhanced protein turnover in pseudoislets compared to that in monolayers. The most up-regulated protein in pseudoislets was N(4)-(β-N-acetylglucosaminyl)-L-asparaginase, a lysosomal enzyme cleaving N-acetylglucosamine at aspargine. The accentuated level of this enzyme in pseudoislets may reflect that the formation of glycoproteins is an important event during culture of MIN6 cells, possibly due to the high ambient glucose concentration. To what extent MIN6 monolayer cells have elevated glycoproteins levels, and potentially display aspartylglucosaminuria, needs to be determined. In conclusion, the proteomic approach identified metabolism, cell coupling, gene translation and protein turnover as key features discriminating cells with extensive cell-to-cell contacts from cells with less cell-to-cell contacts. These cellular pathways help to explain how an elaborate cellular architecture, such as that found in islets of Langerhans, contributes to cellular function.

■

ABBREVIATIONS

■

REFERENCES

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

(1) Bergsten, P.; Grapengiesser, E.; Gylfe, E.; Tengholm, A.; Hellman, B. Synchronous oscillations of cytoplasmic Ca2+ and insulin release in glucose-stimulated pancreatic islets. J. Biol. Chem. 1994, 269 (12), 8749−53. (2) Pedersen, M. G.; Bertram, R.; Sherman, A. Intra- and inter-islet synchronization of metabolically driven insulin secretion. Biophys. J. 2005, 89 (1), 107−119. (3) Hauge-Evans, A. C.; Squires, P. E.; Persaud, S. J.; Jones, P. M. Pancreatic beta-cell-to-beta-cell interactions are required for integrated responses to nutrient stimuli: enhanced Ca2+ and insulin secretory responses of MIN6 pseudoislets. Diabetes 1999, 48 (7), 1402−1408. (4) Salomon, D.; Meda, P. Heterogeneity and contact-dependent regulation of hormone secretion by individual B cells. Exp. Cell Res. 1986, 162 (2), 507−520. (5) Hopcroft, D. W.; Mason, D. R.; Scott, R. S. Insulin secretion from perifused rat pancreatic pseudoislets. In Vitro Cell. Dev. Biol. 1985, 21 (8), 421−427. (6) Hopcroft, D. W.; Mason, D. R.; Scott, R. S. Structure-function relationships in pancreatic islets: support for intraislet modulation of insulin secretion. Endocrinology 1985, 117 (5), 2073−2080. (7) Miyazaki, J. I.; Araki, K.; Yamato, E.; Ikegami, H.; Asano, T.; Shibasaki, Y.; Oka, Y.; Yamamura, K. I. Establishment of a pancreatic beta-cell line that retains glucose-inducible insulin-secretion: special reference to expression of glucose transporter isoforms. Endocrinology 1990, 127 (1), 126−132. (8) Rogers, G. J.; Hodgkin, M. N.; Squires, P. E. E-cadherin and cell adhesion: a role in architecture and function in the pancreatic islet. Cell. Physiol. Biochem. 2007, 20 (6), 987−994. (9) Hauge-Evans, A. C.; Squires, P. E.; Belin, V. D.; Roderigo-Milne, H.; Ramracheya, R. D.; Persaud, S. J.; Jones, P. M. Role of adenine nucleotides in insulin secretion from MIN6 pseudoislets. Mol. Cell. Endocrinol. 2002, 191 (2), 167−176. (10) Brenner, M. B.; Mest, H. J. A buffer temperature controlled perifusion system to study temperature dependence and kinetics of insulin secretion in MIN6 pseudoislets. J. Pharmacol. Toxicol. Methods 2004, 50 (1), 53−57. (11) Kelly, C.; Guo, H.; McCluskey, J. T.; Flatt, P. R.; McClenaghan, N. H. Comparison of insulin release from MIN6 pseudoislets and pancreatic islets of Langerhans reveals importance of homotypic cell interactions. Pancreas 2010, 39 (7), 1016−1023. (12) Chowdhury, A.; Dyachok, O.; Tengholm, A.; Sandler, S.; Bergsten, P. Functional differences between aggregated and dispersed insulin-producing cells. Diabetologia 2013, 56 (7), 1557−1568. (13) Meda, P.; Chanson, M.; Pepper, M.; Giordano, E.; Bosco, D.; Traub, O.; Willecke, K.; el Aoumari, A.; Gros, D.; Beyer, E. C.; et al. In vivo modulation of connexin 43 gene expression and junctional coupling of pancreatic B-cells. Exp. Cell Res. 1991, 192 (2), 469−480. (14) Hauck, S. M.; Dietter, J.; Kramer, R. L.; Hofmaier, F.; Zipplies, J. K.; Amann, B.; Feuchtinger, A.; Deeg, C. A.; Ueffing, M. Deciphering membrane-associated molecular processes in target tissue of autoimmune uveitis by label-free quantitative mass spectrometry. Mol. Cell. Proteomics 2010, 9 (10), 2292−2305. (15) Thörn, K.; Bergsten, P. Fatty acid-induced oxidation and triglyceride formation is higher in insulin-producing MIN6 cells exposed to oleate compared to palmitate. J. Cell. Biochem. 2010, 111 (2), 497−507. (16) Sargsyan, E.; Bergsten, P. Lipotoxicity is glucose-dependent in INS-1E cells but not in human islets and MIN6 cells. Lipids Health Dis. 2011, 10, 115. (17) Tornheim, K. Are metabolic oscillations responsible for normal oscillatory insulin secretion? Diabetes 1997, 46 (9), 1375−1380.

ASSOCIATED CONTENT

* Supporting Information S

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

■

■

Technical Note

AUTHOR INFORMATION

Corresponding Author

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

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS Grants from the Swedish Medical Research Council (72X14019), Swedish Diabetes Association, Family Ernfors Foundation, and Uppsala University supported the study. The authors are grateful to Proteomics core facility, European Molecular Biology Laboratory (EMBL) especially Jenny Hansson, Satoshi Okawa and Jeroen Krijgsveld for providing the tools to analyze the data presented in this paper. The Special Equipment Grant from the University Grants Committee of the Hong Kong Special Administrative Region, China (Project Code: SEG_HKU02). H



Technical Note

(18) Yaney, G. C.; Schultz, V.; Cunningham, B. A.; Dunaway, G. A.; Corkey, B. E.; Tornheim, K. Phosphofructokinase isozymes in pancreatic islets and clonal beta-cells (INS-1). Diabetes 1995, 44 (11), 1285−1289. (19) Wiederkehr, A.; Wollheim, C. B. Mitochondrial signals drive insulin secretion in the pancreatic beta-cell. Mol. Cell. Endocrinol. 2012, 353 (1−2), 128−137. (20) Schuit, F.; De Vos, A.; Farfari, S.; Moens, K.; Pipeleers, D.; Brun, T.; Prentki, M. Metabolic fate of glucose in purified islet cells. Glucose-regulated anaplerosis in beta cells. J. Biol. Chem. 1997, 272 (30), 18572−18579. (21) Jitrapakdee, S.; Wutthisathapornchai, A.; Wallace, J. C.; MacDonald, M. J. Regulation of insulin secretion: role of mitochondrial signalling. Diabetologia 2010, 53 (6), 1019−1032. (22) Srinivasan, M.; Choi, C. S.; Ghoshal, P.; Pliss, L.; Pandya, J. D.; Hill, D.; Cline, G.; Patel, M. S. β-Cell-specific pyruvate dehydrogenase deficiency impairs glucose-stimulated insulin secretion. Am. J. Physiol.: Endocrinol. Metab. 2010, 299 (6), E910−917. (23) Lu, D.; Mulder, H.; Zhao, P.; Burgess, S. C.; Jensen, M. V.; Kamzolova, S.; Newgard, C. B.; Sherry, A. D. 13C NMR isotopomer analysis reveals a connection between pyruvate cycling and glucosestimulated insulin secretion (GSIS). Proc. Natl. Acad. Sci. U.S.A. 2002, 99 (5), 2708−2713. (24) MacDonald, M. J. Feasibility of a mitochondrial pyruvate malate shuttle in pancreatic islets. Further implication of cytosolic NADPH in insulin secretion. J. Biol. Chem. 1995, 270 (34), 20051−20058. (25) Liu, Y. Q.; Jetton, T. L.; Leahy, J. L. beta-Cell adaptation to insulin resistance. Increased pyruvate carboxylase and malate-pyruvate shuttle activity in islets of nondiabetic Zucker fatty rats. J. Biol. Chem. 2002, 277 (42), 39163−39168. (26) Jensen, M. V.; Joseph, J. W.; Ronnebaum, S. M.; Burgess, S. C.; Sherry, A. D.; Newgard, C. B. Metabolic cycling in control of glucosestimulated insulin secretion. Am. J. Physiol.: Endocrinol. Metab. 2008, 295 (6), E1287−1297. (27) Corkey, B. E.; Glennon, M. C.; Chen, K. S.; Deeney, J. T.; Matschinsky, F. M.; Prentki, M. A role for malonyl-CoA in glucosestimulated insulin secretion from clonal pancreatic beta-cells. J. Biol. Chem. 1989, 264 (36), 21608−21612. (28) Prentki, M.; Vischer, S.; Glennon, M. C.; Regazzi, R.; Deeney, J. T.; Corkey, B. E. Malonyl-CoA and long chain acyl-CoA esters as metabolic coupling factors in nutrient-induced insulin secretion. J. Biol. Chem. 1992, 267 (9), 5802−5810. (29) Ronnebaum, S. M.; Ilkayeva, O.; Burgess, S. C.; Joseph, J. W.; Lu, D.; Stevens, R. D.; Becker, T. C.; Sherry, A. D.; Newgard, C. B.; Jensen, M. V. A pyruvate cycling pathway involving cytosolic NADPdependent isocitrate dehydrogenase regulates glucose-stimulated insulin secretion. J. Biol. Chem. 2006, 281 (41), 30593−30602. (30) Breton, S.; Ghiselli, F.; Passamonti, M.; Milani, L.; Stewart, D. T.; Hoeh, W. R. Evidence for a fourteenth mtDNA-encoded protein in the female-transmitted mtDNA of marine Mussels (Bivalvia: Mytilidae). PLoS One 2011, 6 (4), e19365. (31) Soejima, A.; Inoue, K.; Takai, D.; Kaneko, M.; Ishihara, H.; Oka, Y.; Hayashi, J. Mitochondrial DNA is required for regulation of glucose-stimulated insulin secretion in a mouse pancreatic beta cell line, MIN6. J. Biol. Chem. 1996, 271 (42), 26194−26199. (32) Lacy, P. E.; Howell, S. L.; Young, D. A.; Fink, C. J. New hypothesis of insulin secretion. Nature 1968, 219 (5159), 1177−1179. (33) Pipeleers, D. G.; Pipeleers-Marichal, M. A.; Kipnis, D. M. Regulation of tubulin synthesis in islets of Langerhans. Proc. Natl. Acad. Sci. U.S.A. 1976, 73 (9), 3188−3191. (34) Skoglund, G.; Ahren, B.; Lundquist, I. Insulin secretion and lysosomal enzyme activities in isolated mouse islets. Effects of glucose, diazoxide and isobutylmethylxanthine. Diabetes Res. 1986, 3 (8), 411− 415. (35) Roderigo-Milne, H.; Hauge-Evans, A. C.; Persaud, S. J.; Jones, P. M. Differential expression of insulin genes 1 and 2 in MIN6 cells and pseudoislets. Biochem. Biophys. Res. Commun. 2002, 296 (3), 589−595. (36) Orci, L.; Halban, P.; Amherdt, M.; Ravazzola, M.; Vassalli, J. D.; Perrelet, A. A clathrin-coated, Golgi-related compartment of the

insulin secreting cell accumulates proinsulin in the presence of monensin. Cell 1984, 39 (1), 39−47. (37) Kawaguchi, M.; Minami, K.; Nagashima, K.; Seino, S. Essential role of ubiquitin-proteasome system in normal regulation of insulin secretion. J. Biol. Chem. 2006, 281 (19), 13015−13020.

I


Signaling in Insulin-Secreting MIN6 Pseudoislets and Monolayer Cells

Recommend Documents