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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
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
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
Technical Note
Cell Culture
Bioinformatics Analysis
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
5955
Technical Note
RESULTS
Proteomic Analysis
Technical Note
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-
Technical Note
Oxidative phosphorylation
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
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
-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.
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.
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
ABBREVIATIONS
REFERENCES
ASSOCIATED CONTENT
* Supporting Information
S
Technical Note
AUTHOR INFORMATION
Corresponding Author
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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Technical Note
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