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 ChemistryBiomedical 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

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Publication Date (Web): September 30, 2013 | doi: 10.1021/pr400864w

S Supporting Information
*

ABSTRACT: Cellcell 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 dene 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 dierentially expressed proteins were
mapped onto KEGG pathways. Protein proling identied 1576 proteins, which were common to pseudoislets and monolayers.
When mapped onto KEGG pathways, 11 highly enriched pathways were identied. On the basis of dierences in expression of
proteins belonging to the pathways in pseudoislets and monolayers, predictions of dierential 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 dierence 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
dierence between MIN6 pseudoislets and MIN6 cells grown
in monolayers have also been reported including altered IRS-1
signaling.3,812 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 diculties 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.13 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.3,811 MIN6 pseudoislets show
2013 American Chemical Society

Received: May 3, 2013

Published: September 6, 2013
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Technical Note

washed once with 100 L of 50% acetonitrile in 50 mmol/L

NH4HCO3 by spinning them down. Samples were transferred
onto the cuto lters and spun. The membranes were washed
once with 100 L of 2% acetonitrile in 50 mmol/L NaHCO3,
spun, and then wash with 100 L of 50% acetonitrile in 50
mmol/L NH4HCO3. Samples were diluted in 50 L 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 inuence
cellular signaling by comprehensively analyzing dierential
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 Proles

Digested samples were redissolved in 0.1% triuoroacetic acid

to yield an approximate tryptic peptide concentration of 0.3
g/L prior to nanoLCMS/MS analysis. The protein
identication experiments were performed using a 7 T hybrid
Linear ion trap Fourier Transform (LTQ-FT) mass spectrometer (ThermoFisher Scientic, Bremen, Germany) tted
with a nanoelectrospray ionization (ESI) ion source (ThermoFischer Scientic). Online nanoLC separations were performed
using Agilent 1100 nanoow system (Agilent Technologies,
Waldbronn, Germany). The peptide separations were performed on in-house packed 15 cm fused silica emitters (75 m
inner diameter, 375 m outer diameter). The emitters were
packed with a methanol slurry of reversed-phase, fully endcapped Reprosil-Pur C18-AQ 3 m resin (Dr. Maisch GmbH,
Ammerbuch-Entringen, Germany) using a pressurized packing
device operated at 5060 bar (Proxeon Biosystems, Odense,
Denmark). The separations were performed at a ow 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 ve of the most abundant
ions in the ion trap.

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Publication Date (Web): September 30, 2013 | doi: 10.1021/pr400864w

Cell Culture

Mouse insulinoma MIN6 cells were cultured under conditions

previously published7 in 250 mL tissue culture asks (Becton
Dickson Labware, Franklin Lakes, NJ) at 37 C (95% O2 and
5% CO2) in Dulbeccos Modied Eagle medium (Invitrogen,
Paisley, U.K.), containing 25 mmol/L glucose and supplemented with 10% fetal bovine serum, 100 units/mL penicillin,
100 g/mL streptomycin (Invitrogen), and 50 M mercaptoethanol (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 35 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
buer 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
buer containing either 2 or 20 mmol/L glucose for 30 min at
37 C. Aliquots (200 L) of medium were collected for
determination of insulin secretion. Monolayer cells and
pseudoislets were washed three times with phosphate buer
saline (PBS) and lysed with buer containing 0.1% Triton-X
100 and 25 mmol/L NaOH, and lysates were stored at 20 C
for measurement of total protein. Dierences in insulin
secretion among the groups were assessed using analysis of
variance (ANOVA) followed by Bonferronis post hoc test. P <
0.05 was considered statistically signicant.

Bioinformatics Analysis

(a). Label-Free Peptide and Protein Quantications.

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 LCMS 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
Prole 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 110 MS/MS spectra were exported from the
Progenesis software as Mascot generic le (mgf) and used for
peptide identication with Mascot version 2.2 (Matrix Science,
London, U.K.) in the International Protein Index (IPI)

Sample Preparation for Protein Proling

Cells were washed twice with PBS and lysed with a buer
containing 8 mol/L urea, 1% octyl--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. Buer 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 manufacturers 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 cuto lters (Pall, Port Washington, NY) were
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Journal of Proteome Research

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 xed modication and methionine oxidation was set
as variable modication. A Mascot-integrated decoy database
search calculated a false discovery of 1% when searching was
performed on the concatenated mgf les with a signicance
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 identications are listed in Supporting Information
Table 1.
(b). Dierential 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 signicant 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 Fishers exact test
was used to calculate the P-values for enriched pathways, and
the adjusted P-values were calculated by applying the
BenjaminiHochberg procedure in order to control the False
Discovery Rate (FDR).
Western Blot

For determination of levels of specic 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 quantied with Image Lab software (Bio-Rad).
Normalization was conducted for each protein by staining the
PVDF membranes with Coomaasie and quantied 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
m) in panel d is applicable to all pictures. Magnication 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 12 days, pseudoislets started to form with
diameters ranging from 50 to 150 m (Figure 1a,b). The
pseudoislets reached a diameter of approximately 150250 m
after 35 days (Figure 1c,d). Extended culture for up to 89
days did not increase the diameter of the pseudoislets further
(data not shown). Pseudoislets cultured for 35 days were used
for the experiments. Monolayers of MIN6 cells reaching near
conuency 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
identied. 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 dierentially 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 signicantly 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
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Journal of Proteome Research

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 signicantly 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)-(-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 3fi). In case of ribosomal protein ubiquitin A-52
residue ribosomal protein fusion product 1 (uba52), protein
levels determined by Western blotting were not dierent
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
1036
4.18
1013
8.93
106
1.51
103
1.01
102
1.87
102
2.76
102
2.38
109
7.41
106
5.73
104
1.51
103

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 signicant
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 identied 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
specically 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-

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 BenjaminiHochberg procedure in
order to control the False Discovery Rate (FDR).

with p-value 0.05. For each of the pathways, the identied

proteins belonging to the pathways were listed, a total of 221
proteins. No less than 98 of the 221 proteins belonging to the
11 signicantly enriched pathways were found among the
dierentially 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
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Table 2. Dierentially Expressed Proteins in MIN6 Pseudoislets and Monolayer Cellsa

pathway name
Glycolysis

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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] ironsulfur protein 5
Succinate dehydrogenase [ubiquinone] ironsulfur 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] ironsulfur 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

5958

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

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Table 2. continued
pathway name

Regulation of actin
cytoskeleton

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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
-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)-(-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.

ways are pyruvate/malate, pyruvate/citrate and pyruvate/

isocitrate, which were reported as playing important roles in
GSIS.2529 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
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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 ve separate
experiments. *P < 0.05 compared to monolayers.

GSIS.31 The oxidative phosphorylation pathway involved the

largest number of identied 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.3,811 Consistent with these ndings, 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 dierentially expressed in
pseudoislets were the lysosomal, ribosomal, spliceosome and

Table 3. Dierentially 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)-(-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
dierence
2.5
1.9
2.8

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 ve 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
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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)-(-N-acetylglucosaminyl)-L-asparaginase, a lysosomal enzyme cleaving N-acetylglucosamine at
aspargine. The accentuated level of this enzyme in pseudoislets
may reect 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 identied 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

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ASSOCIATED CONTENT

* Supporting Information
S

Table with all protein identications (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: azazul.chowdhury@mcb.uu.

se.
Notes

The authors declare no competing nancial 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).
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Journal of Proteome Research

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